regulaÇÃo do acÚmulo de mimosina em leucaena …
TRANSCRIPT
i
UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL
INSTITUTO DE BIOCIEcircNCIAS
PROGRAMA DE POacuteS-GRADUACcedilAtildeO EM BOTAcircNICA
REGULACcedilAtildeO DO ACUacuteMULO DE MIMOSINA EM
Leucaena leucocephala (Lam) de Wit e Mimosa bimucronata (DC) Kuntze
KELLY CRISTINE DA SILVA RODRIGUES CORREcircA
Tese submetida ao Programa de Poacutes-Graduaccedilatildeo em Botacircnica do Instituto de
Biociecircncias da Universidade Federal do Rio Grande do Sul como quesito parcial para a
obtenccedilatildeo do Tiacutetulo de Doutora
Orientador Prof Dr Arthur Germano Fett-Neto
Co-Orientador do periacuteodo sanduiacuteche Prof Dr Dulal Borthakur
Porto Alegre 2019
ii
O presente trabalho foi desenvolvido
No Laboratoacuterio de Fisiologia Vegetal (LFV) da
Universidade Federal do Rio Grande do Sul
No College of Tropical Agriculture amp Human Resources (CTAHR)
da University of Hawairsquoi at Manoa
Instituiccedilotildees Financiadoras
COORDENACcedilAtildeO DE APERFEICcedilOAMENTO
PESSOAL DE NIacuteVEL SUPERIOR
(CAPES)
CONSELHO NACIONAL DE DESENVOLVIMENTO
CIENTIacuteFICO E TECNOLOacuteGICO
(CNPq)
iii
Dedicatoacuteria
Agrave minha amada avoacute lsquode laacutersquo Doraliacutecia Marcelina Costa da Silva (aka
Dona Dora como preferia ser chamada) por ter sido minha maior referecircncia
de amor e zelo enquanto estava aqui por ser a mais habilidosa e diligente
lsquofazedora de hortasrsquo e a melhor lsquoBotanistersquo empiacuterica que jaacute conheci
Obrigada por fazer da tua horta meu fantaacutestico lsquoherbaacuterio vivorsquo e do teu
conhecimento etnobotacircnico meu primeiro referencial de respeito e admiraccedilatildeo
ao Reino Plantae Foi nesse lsquoJardim Secretorsquo que descobri e me encantei
irreversivelmente pelo lsquoextraordinaacuterio poder das plantasrsquo E a saudade tua soacute
aumenta nunca diminui
Ao meu muito querido e saudoso amigo Rafael Cortes Duarte cortecircs
(sempre) ateacute no nome Se ainda estivesses por aqui esse trabalho teria sido
teu
Dizem que o tempo aqui eacute relativo Logo a gente se vecirc
iv
AGRADECIMENTOS
Ao meu orientador Dr Arthur Germano Fett-Neto uma das melhores pessoas
que tive a honra de conhecer na Academia Um coraccedilatildeo imenso uma mente incrivelmente
brilhante integridade e empatia infinitas (e extremamente raras no meio cientiacutefico)
Muito obrigada pela confianccedila em mim depositada e sobretudo por ter cometido a
insanidade de aceitar me orientar novamente Obrigada por ter me possibilitado ir em
termos cientiacuteficos muito aleacutem do que eu ousaria imaginar dadas as minhas (inuacutemeras)
limitaccedilotildees (e por todo ATP e NADPH investidos nesse esforccedilo hercuacuteleo que constitui a
aacuterdua tarefa de me orientar de forma natildeo condescendente a despeito dessas) Minha
diacutevida contigo seraacute eterna sou uma pessoa duplamente aquinhoada pela tua orientaccedilatildeo
Lucky me
Aos Professores Janette Palma-Fett uma grande amiga e saacutebia conselheira
sempre especialmente na adversidade e Felipe Maraschin pelo pronto e inestimaacutevel
apoio teacutecnico-cientiacutefico sempre que solicitado
Aos colegas do Laboratoacuterio de Fisiologia Vegetal da UFRGS pela parceria e
auxiacutelio em todas as horas por formarem um grupo coeso alinhado e comprometido com
o bem maior da pesquisa e do bom funcionamento do lab Eacute gratificante trabalhar com
todos vocecircs
Aos amigos muito queridos que a UFRGS me trouxe Ana Paula Durand
Coelho Eudes Stiehl-Alves Johnatan Vilasboa Yohanna Miotto e as divas Juliana
Troleis Sofia Aumond Kuhn e Tamara Pastori Muito obrigada por estarem presentes
nas horas menos faacuteceis e por me auxiliarem de muitas maneiras sempre que precisei Toda
dificuldade eacute redimensionada quando se tem amigos
Agrave minha famiacutelia caucasoide Ana Cristina Stein Camila Junkes Camila e
Cassiano Busatta Carlos Eduardo Blanco Linares Daniela Sponchiado Jordana
Griebler Luft Karen Santos Karina Letiacutecia Lopes e Larissa Schemes Heinzelmann
O carinho o apoio e o encorajamento que recebo de vocecircs fazem qualquer lsquofardorsquo parecer
mais leve Muito lsquomercirsquo
I am very grateful to Dr Dulal Borthakur for generously having received me in
his lab and his loving and caring family I would also like to thank my lab mates at UH
Manoa James Carillo Maia Corpuz and Ahmed Bageel for being so helpful cheerful
and friendly with me during all my stay in Honolulu Most of all Irsquod like to thank my
dear friend Michael Honda for teaching very patiently and supporting me inside and
outside the lab by doing whatever was in his power to prevent my homesickness I am
also very grateful to Mariana de Souza and Fernanda Oliveira for all those amazing
places and hikes wersquove been together in Orsquoahu You guys are awesome Mahalo nui loa
for your kōkua Im now parsquou hana
Jaimerais bien remercier mes collegravegues et amis agrave lrsquoUniversiteacute de Montreacuteal
(Benjamin Mazin Marion Kretsch Yang Liu Fang Wen Raquel Parada et Micaela
Margutti) pour mavoir chaleureusement reccedilu chez vous speacutecialement agrave mon ami
Valentin Joly pour mavoir beaucoup appris sur lrsquoinconnu monde des bacteacuteries et des
v
levures (et surtout pour leur incroyable patience avec mon tregraves mauvais franccedilais) Crsquoeacutetait
vachement chouette Merci beaucoup agrave vous tous (et toutes) et agrave la prochaine
Agrave Coordenaccedilatildeo de Aperfeiccediloamento Pessoal de Niacutevel Superior (CAPES) pelo
financiamento da bolsa de pesquisa do PDSE
Aos meus pais (bioloacutegicos ou natildeo) Veacutera Maria da Silva Rodrigues Gilberto
Moraes Rodrigues Rosa Maria Lucas da Silva e Paulo Joseacute Costa da Silva pelo
exemplo de honestidade coragem trabalho forccedila e amor desde sempre
Aos meus irmatildeos Ana Paula da Silva Rodrigues Viniacutecius de Moraes da Silva
Rodrigues Marcello da Silva Rodrigues e Camila Stella Toledo Pereira por todas as
experiecircncias que dividimos e tudo o que me ensinaram ateacute hoje
Ao meu amor maior minha melhor amiga minha mais leal e extraordinaacuteria
parceria nessa grande (e agraves vezes tortuosa) jornada Maria Clara Rodrigues Correcirca Por
ser ela por ser imensa em generosidade amor e altruiacutesmo por despertar o melhor em
mim por ser minha forccedila motriz e sobretudo por ser a melhor das minhas metades
Minha vida soacute realmente comeccedilou quando eu tive a incriacutevel sorte de te conhecer
vi
SUMAacuteRIO
LISTA DE ABREVIATURASvii
RESUMO ix
INTRODUCcedilAtildeO GERAL1
HIPOacuteTESE E OBJETIVOS9
CAPIacuteTULO 1 Abiotic stresses and non-protein amino acids in plantshelliphellip10
CAPIacuteTULO 2 Mimosine accumulation in Leucaena leucocephala in response to
stress signaling molecules and acute UV exposurehelliphelliphelliphelliphelliphelliphelliphelliphelliphellip(432) 52
CAPIacuteTULO 3 Mimosine occurrence and accumulation in Mimosa bimucronata var
bimucronata (DC) Kuntze66
CONSIDERACcedilOtildeES FINAIS 84
PERSPECTIVAS85
REFEREcircNCIAS BIBLIOGRAacuteFICAS86
Artigos publicados no periacuteodo de doutoramento natildeo relacionados ao tema da
tese91
vii
LISTA DE ABREVIATURAS
24-D 24-dichlorophenoxyacetic acid
3H4P 3-hydroxy-4-pyridone (34-DHP 34-dihydroxypyridine)
ABA abscisic acid
Arg arginine
BABA β-aminobutyric acid
β-ODAP β-N-oxalyl-L-α β-diaminopropionic acid
BIA β-isoxazolinon-L-alanine
CAN canavanine
DAO diamine oxidase
DDC decarboxylase
ETH ethephon
FW fresh weight
GABA -aminobutyric acid
GABA-T GABA transaminase
GAD glutamate decarboxylase
GSM Global System for Mobile
HPLC High performance liquid chromatography
JA jasmonate
JA-Ile jasmonoyl-L-isoleucine
L-DOPA L-34- dihydroxyphenylalanine
MeJA methyl jasmonate
m-Tyr Meta-tyrosine
NO nitric oxide
NPAA non-protein amino acid
OAS o-acetylserine
OAS-TL o-acetylserine-thiol-lyase
PA polyamine
PAA protein amino acid
viii
PEG polyethylene glycol
PLP pyridoxal-5rsquo-phosphate
PPO polyphenol oxidase tyrosinase
qRT-PCR Reverse transcription polymerase chain reaction quantitative real time
RNS reactive nitrogen species
ROS reactive oxygen species
SA salicylic acid
SAR systemic acquired resistance
SNP sodium nitroprusside
UV ultraviolet radiation
ix
RESUMO
Ao longo de sua evoluccedilatildeo as plantas desenvolveram estrateacutegias estruturais e quiacutemicas de
defesa em resposta aos estresses bioacuteticos e abioacuteticos impostos pelo ambiente Dentre
essas satildeo reconhecidas moleacuteculas quimicamente especializadas denominadas
metaboacutelitos secundaacuterios produtos naturais ou metaboacutelitos especializados Aminoaacutecidos
natildeo proteicos (ANPs) satildeo compostos nitrogenados que constituem aleacutem de componentes
do arsenal de defesa quiacutemica vegetal uma importante fonte de reserva de carbono e
nitrogecircnio para diversos taxa especialmente aqueles pertencentes agrave famiacutelia Fabaceae de
Angiospermas Esse grupo de moleacuteculas quimicamente heterogecircneo eacute assim definido por
natildeo participar da formaccedilatildeo de estruturas proteicas funcionais sendo frequentemente
toacutexicos quando erroneamente incorporados nas cadeias polipeptiacutedicas em formaccedilatildeo em
funccedilatildeo da similaridade estrutural que apresentam com os aminoaacutecidos proteicos Sob o
ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados ANPs satildeo
em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou abioacutetica como
o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de elicitores
quiacutemicos por exemplo O objetivo da presente tese foi investigar o papel bioloacutegico da
mimosina endoacutegena em Leucaena leucocephala (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute) a partir da avaliaccedilatildeo do efeito de tratamentos
relacionados ao estresse abioacutetico (UV-C aacutecido saliciacutelico metil jasmonato e etileno)
Mimosina eacute um ANP aromaacutetico anaacutelogo da L-tirosina com atividade toacutexica para ceacutelulas
de procariotos e eucariotos Dentre as atividades descritas para esse ANP destacam-se a
atividade anti-mitoacutetica ou bloqueadora do ciclo celular atividade alelopaacutetica e
antioxidante Os resultados indicaram que em leucena a biossiacutentese e o acuacutemulo de
mimosina podem ser modulados por fatores causadores de estresses exibindo um padratildeo
de acumulaccedilatildeo similar ao das fitoalexinas Em maricaacute por outro lado a induccedilatildeo do
acuacutemulo dessa moleacutecula natildeo foi observada para os mesmos tratamentos testados para
leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas Aleacutem disso
o padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado
pela sua degradaccedilatildeo As respostas observadas nos testes que avaliaram a atividade de
mitigaccedilatildeo de espeacutecies reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode
agir como um agente antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de
estresse
1
Introduccedilatildeo
Na condiccedilatildeo de organismos seacutesseis ao longo de sua evoluccedilatildeo as plantas
desenvolveram estrateacutegias estruturais e quiacutemicas de defesa em resposta aos estresses bioacuteticos
e abioacuteticos impostos pelo ambiente Dentre essas satildeo reconhecidas moleacuteculas quimicamente
especializadas denominadas metaboacutelitos secundaacuterios produtos naturais (Kutchan et al 2015)
ou mais recentemente metaboacutelitos especializados
Entre as trecircs classes mais gerais de metaboacutelitos secundaacuterios (terpenos compostos
fenoacutelicos e compostos nitrogenados) aminoaacutecidos natildeo-proteicos (ANPs) satildeo incluiacutedos no
terceiro grupo e constituem aleacutem de componentes do arsenal de defesa quiacutemica uma
importante fonte de reserva de carbono e nitrogecircnio para diversos taxa especialmente aqueles
pertencentes agrave famiacutelia Fabaceae de Angiospermas (leguminosas sensu lato)
Aleacutem dos 20 aminoaacutecidos proteicos estima-se que existam entre 600 e 1000 ANPs
(Acamovic amp Brooker 2005 Rodgers et al 2015) Esse grupo de moleacuteculas quimicamente
heterogecircneo eacute assim definido por natildeo participar da formaccedilatildeo de estruturas proteicas
funcionais sendo frequentemente toacutexicos quando erroneamente incorporados nas cadeias
polipeptiacutedicas em formaccedilatildeo em funccedilatildeo da similaridade estrutural que apresentam com os
aminoaacutecidos proteicos (Taiz amp Zeiger 2010)
Conforme mencionado a ocorrecircncia de ANPs eacute comum em espeacutecies de leguminosas
e sua distribuiccedilatildeo pode ser restrita a alguns gecircneros de plantas circunscritos nessa famiacutelia
botacircnica (eg mimosina e canavanina) Por outro lado alguns ANPs como GABA por
exemplo podem apresentar distribuiccedilatildeo ubiacutequa no Reino Plantae assim como ocorrer em
outros tipos de organismos como animais por exemplo (Ramos-Ruiz et al 2018)
2
Apesar de representarem uma fonte nutricional importante sem tratamento preacutevio o
consumo de plantas que acumulam ANPs por animais eacute limitado Isso ocorre pois em longo
prazo a ingestatildeo prolongada de plantas (especialmente sementes) que acumulam ANPs pode
representar risco agrave sauacutede uma vez que estes comprometem o funcionamento de mecanismos
basais de manutenccedilatildeo da homeostase celular e podem tambeacutem em um quadro mais severo
desencadear doenccedilas neurotoacutexicas degenerativas como por exemplo o latirismo causado
por aacutecido β-N-oxalil-l-αβ-diaminopropiocircnico (β-ODAP) (Jiao et al 2011 Kusama-Eguchi
2019)
Sob o ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados
ANPs satildeo em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou
abioacutetica como o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de
elicitores quiacutemicos por exemplo No que concerne ao estudo dos efeitos da induccedilatildeo abioacutetica
sobre o acuacutemulo de ANPs em diferentes espeacutecies vegetais (Monocotiledocircneas e
Eudicotiledocircneas) as moleacuteculas mais amplamente investigadas ateacute o momento satildeo GABA
L-DOPA e mais recentemente mimosina (vide Tabela 1 do capiacutetulo primeiro) Em termos
de efeitos causados a partir da aplicaccedilatildeo exoacutegena de ANPs GABA tambeacutem figura como o
principal aminoaacutecido investigado seguido de L-DOPA e canavanina (vide Tabela 2 do
capiacutetulo primeiro)
No primeiro capiacutetulo da presente tese estatildeo descritas as caracteriacutesticas gerais dos
principais ANPs estudados seus possiacuteveis papeacuteis bioloacutegicos in planta e seus efeitos quando
aplicados exogenamente bem como os estresses abioacuteticos capazes de induzir seu(s)
acuacutemulo(s) nos diferentes tecidos vegetais Nos segundo e terceiro capiacutetulos
respectivamente satildeo elucidados os efeitos dos tratamentos de UV-C aacutecido saliciacutelico etileno
e jasmonato (claacutessicos elicitores do metabolismo secundaacuterio vegetal) sobre o acuacutemulo de
3
mimosina em Leucaena leucocephala var glabrata (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute)
Mimosina eacute um aminoaacutecido aromaacutetico natildeo-proteico anaacutelogo da L-tirosina com
atividade toacutexica para ceacutelulas de procariotos e eucariotos Embora em menor concentraccedilatildeo
mimosina foi primeiramente identificada em Mimosa pudica sendo posteriormente detectada
em outras espeacutecies do gecircnero como Mimosa pigra por exemplo (Soedarjo amp Borthakur
1998) Seu efeito toacutexico eacute atribuiacutedo agrave capacidade de quelar metais o que impede o
funcionamento adequado das metalo-proteiacutenas que dependem dos mesmos como co-fatores
(Negi et al 2014)
A concentraccedilatildeo basal de mimosina em espeacutecies de leucaena pode variar entre 1 e 12
do peso seco do oacutergatildeo (Soedarjo amp Borthakur 1998) Como eacute comum para outros ANPs
que ocorrem em espeacutecies de leguminosas em sementes de Leucaena spp eacute observada uma
maior concentraccedilatildeo de mimosina quando comparada aos demais oacutergatildeos da planta
(Rodrigues-Correcirca et al 2019) sendo esta a fonte de extraccedilatildeo comercial do padratildeo quiacutemico
de mimosina vendido por empresas de reagentes de pesquisa
Diversas atividades foram descritas para mimosina em outros organismos eou tipos
celulares Dentre essas destacam-se a atividade anti-mitoacutetica ou bloqueadora do ciclo
celular em ceacutelulas de eucariotos e procariotos Isto ocorre porque a mimosina impede a
formaccedilatildeo da forquilha de replicaccedilatildeo (e portanto a siacutentese de DNA) interrompendo assim o
avanccedilo do ciclo de divisatildeo celular na fase tardia G1 (Lalande 1990) Foram tambeacutem descritas
para mimosina atividade alelopaacutetica observada sobre o desenvolvimento de outras espeacutecies
de leguminosas e atividade antioxidante entre outras (Tabela 1)
A rota de biossiacutentese de mimosina eacute compartilhada em grande parte com a de cisteiacutena
um aminoaacutecido proteico sulfurado (Figura 1) A siacutentese da cisteiacutena se daacute a partir da conversatildeo
4
de serina e acetil-CoA em o-acetilserina pela enzima SAT (serina acetiltransferase) seguida
da conversatildeo de o-acetilserina e aacutecido sulfiacutedrico em cisteiacutena em uma reaccedilatildeo catalisada pela
OAS-TL (o-acetilserina tiol-liase) A siacutentese de mimosina por sua vez eacute compartilhada com
a da cisteiacutena ateacute esse ponto e acredita-se que pelo menos uma das isoformas de OAS-TL
catalise a conversatildeo de o-acetilserina e 3-hidroxi-4-piridona em mimosina
Tabela 1 Atividades descritas para mimosina de Leucaena leucocephala (Lam) de Wit
ATIVIDADE
ALVO AVALIADO
(organismo eou tecido tipo
celular)
REFEREcircNCIA
Bloqueio do complexo de ativaccedilatildeo
da preacute-replicaccedilatildeo do DNA
Ceacutelulas de mamiacuteferos
KUBOTA et al
(2014)
Alteraccedilatildeo no ciclo ovariano e
extensatildeo da duraccedilatildeo do corpo luacuteteo
bovino no periacuteodo poacutes-parto
Bovinos
(Bos taurus x
Bos indicus)
BOTTINI-
LUZARDO et al
(2015)
Supressatildeo do ciclo celular e reduccedilatildeo
da abundacircncia bacteriana em
mosquitos
Wolbachia pipientis
Aedes albopictus
FALLON
(2015)
Accedilatildeo inibitoacuteria da fibrose
pulmonar induzida
Ratos SD
LI et al
(2015)
Recuperaccedilatildeo da funccedilatildeo do
miocaacuterdio poacutes-isquemia
Miocaacuterdio de ratos (SD)
machos
CROWE et al
(2001)
Inseticida
Heteropsylla cubana
Crawford 1914 e Thrips tabaci
Lindemann 1889
AHMED et al
(2016)
Alelopaacutetica
Albizia procera Vigna
unguiculata Cicer arietinum
Cajanus cajan
AHMED et al
(2008)
Antioxidante
Sistemas modelo de oxidaccedilatildeo
lipiacutedica (β-caroteno - aacutecido
linolecircico e lecitina)
BENJAKUL et al
(2013)
Ateacute momento versotildees divergentes sobre a enzima responsaacutevel pela biossiacutentese de
mimosina (mimosina sintase) tecircm sido publicadas Em 1990 Ikegami e colaboradores
5
identificaram uma OAS-TL responsaacutevel pela formaccedilatildeo de cisteiacutena como sendo tambeacutem uma
mimosina sintase Mais tarde Yafuso et al (2014) realizaram a expressatildeo heteroacuteloga do gene
que codifica para OAS-TL em Escherichia coli e natildeo foi observada a formaccedilatildeo de mimosina
mesmo quando dadas as condiccedilotildees oacutetimas para tanto Mais recentemente Harun-Ur-Rashid
et al (2018) elucidaram a mimosina sintase como sendo uma isoforma da OAS-TL
corroborando o postulado por Ikegami e colaboradores em 1990
Figura 1 Rota de biossiacutentese da mimosina Fonte Ikegami et al (1990)
Espeacutecies estudadas
Leucaena leucocephala (Lam) de Wit (leucaena koa haole ou ldquoacaacutecia exoacuteticardquo na
liacutengua Hawairsquoiana) eacute uma espeacutecie de haacutebito arboacutereo ou arbustivo pertencente agrave famiacutelia
Fabaceae de Angiospermas e caracterizada pelo acuacutemulo de mimosina em todos os seus
oacutergatildeos Eacute nativa da Ameacuterica Central (especificamente da regiatildeo sudeste do Meacutexico) mas
irradiou-se atraveacutes de praticamente todas as zonas tropicais e subtropicais da Terra No
Brasil leucena eacute amplamente distribuiacuteda e classificada como naturalizada pelo REFLORA
(2019) ocorrendo em todo territoacuterio Nacional Satildeo reconhecidas no miacutenimo duas
6
subespeacutecies de leucena ocorrentes no Brasil L leucocephala var leucocephala e L
leucocephala var glabrata sendo a primeira a mais abundante
Leucaena apresenta atributos morfoloacutegicos caracteriacutesticos das leguminosas como o
fruto do tipo vagem deiscente no periacuteodo poacutes-maturaccedilatildeo folhas compostas e bipinadas As
flores satildeo seacutesseis actinomorfas e polistecircmones apresentam caacutelice sinseacutepala e corola
gamopeacutetala e satildeo dispostas em inflorescecircncias do tipo glomeacuterulo (Figura 2)
Figura 2 Oacutergatildeos vegetativos e reprodutivos de L leucocephala (Lam) de Wit Fonte Little Jr amp Skolmen
(1989)
Com base no conhecimento etnobotacircnico disponiacutevel acerca dessa espeacutecie em
diversas regiotildees tropicais e subtropicais leucena eacute utilizada para vaacuterios fins Extratos de
diferentes oacutergatildeos de leucena apresentam atividade anti-diabeacutetica (Kuppusamy et al 2014
Chowtivannakul et al 2016) antioxidante (Mohammed et al 2015 Chowtivannakul et al
2016 Zarin et al 2016) antimicrobiana (Zarin et al 2016) anti-helmiacutentica (Soares et al
2015 Jamous et al 2017) bactericida (Mohammed et al 2015) acaricida (Fernaacutendez-Salas
et al 2011) anti-tumoral (Chung et al 2017) e potencializadora da resposta imune em
peixes (Verma et al 2018) entre outras
7
Leucaena apresenta alta toleracircncia agrave seca sendo capaz de enfrentar estaccedilotildees sazonais
inteiras com deacuteficit hiacutedrico sem prejuiacutezo permanente de seus oacutergatildeos e de recuperar
vigorosamente sua biomassa vegetativa tatildeo logo o regime de precipitaccedilatildeo retome a
regularidade em frequecircncia Acredita-se que a toleracircncia agrave seca apresentada por essa espeacutecie
ocorra em funccedilatildeo do acuacutemulo de mimosina nos diferentes tecidos da planta a qual
funcionaria como um agente osmoregulador responsaacutevel pela preservaccedilatildeo da integridade das
membranas a das macromoleacuteculas intracelulares em periacuteodos de escassez de aacutegua no
ambiente
Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) eacute uma leguminosa
nativa natildeo endecircmica do Brasil amplamente distribuiacuteda nos domiacutenios fitogeograacuteficos da
Caatinga do Cerrado e da Mata Atlacircntica (Simon amp Proenccedila 2000 REFLORA 2019) Como
espeacutecie pioneira (Pilatti et al 2019) exerce importante papel ecoloacutegico na recuperaccedilatildeo de
aacutereas degradadas (Bitencourt et al 2007 Silva et al 2011) no estabelecimento de processos
de sucessatildeo vegetacional
Maricaacute eacute uma espeacutecie semi-deciacutedua a deciacutedua a qual atinge ateacute 15 m em altura (e
diacircmetro agrave altura do peito de ateacute 40 cm) na idade adulta com haacutebito arboacutereo ou arbustivo
(REFLORA 2019) e espinhos caracteriacutesticos desde os estaacutegios iniciais de desenvolvimento
(Carvalho 2004) Apresenta folhas compostas alternas e bipinadas (Figura 2) amplas
inflorescecircncias brancas com flores reunidas em glomeacuterulos esfeacutericos dispostos em grandes
paniacuteculas As flores satildeo diplostecircmones actinomorfas hipoacuteginas e unicarpelares (Silva et al
2011)
Assim como descrito para leucena maricaacute eacute considerado uma espeacutecie multifuncional
sendo comumente empregada para produccedilatildeo de mel como combustiacutevel (Olkoski amp
8
Wittmann 2011) em edificaccedilotildees na carpintaria e como lsquocerca-vivarsquo (Marchiori 1993
Lorenzi 1998) entre outras aplicaccedilotildees
Figura 2 Folhas e fruto de Mimosa bimucronata (DC) Kuntze Fonte Souza-Lima et al (2017)
Em contraste com a amplitude de habitats explorados por leucena (especialmente os
aacuteridos) no Sul do Brasil maricaacute ocorre preferencialmente em ambientes uacutemidos a alagadiccedilos
em aacutereas proacuteximas agraves margens de rios (Patreze amp Cordeiro 2004) embora possa tambeacutem
ocorrer em formaccedilotildees quase exclusivas dessa espeacutecie nas encostas de morros (Jacobi amp
Ferreira 1991)
Em relaccedilatildeo agraves atividades elucidadas para os extratos de maricaacute foram relatados os
efeitos alelopaacutetico (Jacobi amp Ferreira 1991 Ferreira et al 1992) diureacutetico natriureacutetico e
caliureacutetico (Schlickmann et al 2017)
9
Hipoacutetese
Mimosina apresenta perfil dinacircmico de acuacutemulo em Leucaena leucocephala e
Mimosa bimucronata frente a estresses associado a alteraccedilotildees significativas na expressatildeo de
genes relacionados ao metabolismo deste ANP o qual contribui para mitigar o desequiliacutebrio
oxidativo inerente a vaacuterios tipos de estresse
Objetivo geral
O objetivo da presente tese foi investigar o papel bioloacutegico da mimosina endoacutegena
em leucena e maricaacute a partir da avaliaccedilatildeo do efeito de tratamentos relacionados a estresses
ou sinalizadores de estresse
Objetivos especiacuteficos
- Analisar a concentraccedilatildeo constitutiva de mimosina nos diferentes oacutergatildeos de L leucocephala
(Lam) de Wit (leucena) e M bimucronata (DC) Kuntze (maricaacute)
- Verificar se apesar do seu alto teor constitutivo em plantas de leucena o acuacutemulo de
mimosina pode ser induzido com tratamentos que mimetizam diferentes estresses a partir da
avaliaccedilatildeo do efeito de moleacuteculas sinalizadoras (aacutecido saliciacutelico jasmonato etileno) e da
exposiccedilatildeo agrave radiaccedilatildeo UV-C na modulaccedilatildeo do acuacutemulo de mimosina em leucena bem como
em maricaacute
- Determinar se a expressatildeo de genes relacionados ao metabolismo de mimosina estaacute
associada agrave induccedilatildeo por estresses fisioloacutegicos
- Avaliar o potencial antioxidante da mimosina em experimentos realizados in situ
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry
journal homepage wwwelseviercomlocateplaphy
Research article
Mimosine accumulation in Leucaena leucocephala in response to stresssignaling molecules and acute UV exposure
Kelly Cristine da Silva Rodrigues-Correcircaab Michael DH Hondab Dulal BorthakurbArthur Germano Fett-Netoalowast
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970Porto Alegre Rio Grande do Sul BrazilbDepartment of Molecular Biosciences and Bioengineering University of Hawaii at Manoa Honolulu HI 96822 USA
A R T I C L E I N F O
KeywordsLeucaena leucocephalaMimosineMimosine amidohydrolaseJasmonic acidEthyleneSalicylic acidUV-C radiation
A B S T R A C T
Mimosine is a non-protein amino acid of Fabaceae such as Leucaena spp and Mimosa spp Several relevantbiological activities have been described for this molecule including cell cycle blocker anticancer antifungalantimicrobial herbivore deterrent and allelopathic activities raising increased economic interest in its pro-duction In addition information on mimosine dynamics in planta remains limited In order to address this topicand propose strategies to increase mimosine production aiming at economic uses the effects of several stress-related elicitors of secondary metabolism and UV acute exposure were examined on mimosine accumulation ingrowth room-cultivated seedlings of Leucaena leucocephala spp glabrata Mimosine concentration was not sig-nificantly affected by 10 ppm salicylic acid (SA) treatment but increased in roots and shoots of seedlings treatedwith 84 ppm jasmonic acid (JA) and 10 ppm Ethephon (an ethylene-releasing compound) and in shoots treatedwith UV-C radiation Quantification of mimosine amidohydrolase (mimosinase) gene expression showed thatethephon yielded variable effect over time whereas JA and UV-C did not show significant impact Consideringthe strong induction of mimosine accumulation by acute UV-C exposure additional in situ ROS localization aswell as in vitro antioxidant assays were performed suggesting that akin to several secondary metabolitesmimosine may be involved in general oxidative stress modulation acting as a hydrogen peroxide and superoxideanion quencher
1 Introduction
Different plant groups synthesize a large diversity of secondary orspecialized metabolites These molecules are generally produced inresponse to biotic and abiotic environmental stresses Indeed inductionof secondary metabolism usually involves stress-generating factorswhich have also been explored in biotechnological processes aiming atthe production of target metabolites of economic interest (Matsuuraet al 2018) Metabolic control of nitrogen-containing secondarycompounds (eg alkaloids and non-protein amino acids) has beenshown to be complex and influenced by phytohormones environmentalstresses (seasonality herbivory pathogen attack drought) UV radia-tion (Holloacutesy 2002) methyl jasmonate (MeJA) salicylic acid (SA)yeast extract (Cho et al 2008) abscisic acid (ABA) heavy metals os-motic stress (Nascimento et al 2013) and mechanical wounding (Portoet al 2014)
Due to their particular trait of associating with N-fixing micro-organisms Fabaceae species (leguminous sensu lato) are often proteinrich hence the relevance of several of these species as forage Fabaceaespecies are also known for accumulating nitrogen containing secondarymetabolites which play important roles as ecochemical molecules andat least for the case of non-protein amino acids potential cell reservoirsof nitrogen (Huang et al 2011)
High contents of mimosine a toxic aromatic non-protein aminoacid are found in species of two leguminous genera Leucaena spp andMimosa spp Leucaena leucocephala (Lam) de Wit (leucaena koa haole)is a fast-growing leguminous tree native from Central America (south-eastern Mexico) widely distributed in tropical and subtropical zonesThis species is also characterized by its high tolerance to droughtamong other environmental stresses (Honda et al 2018) Leucaena canbe divided into two subspecies (i) L leucocephala subsp leucocephala(common leucaena a bushy shrub) and (ii) L leucocephala subsp
httpsdoiorg101016jplaphy201811018Received 1 August 2018 Received in revised form 9 November 2018 Accepted 14 November 2018
lowast Corresponding authorE-mail addresses krodriguescbiotufrgsbr (KCdS Rodrigues-Correcirca) mhonda2hawaiiedu (MDH Honda) dulalhawaiiedu (D Borthakur)
fettnetocbiotufrgsbr (AG Fett-Neto)
Plant Physiology and Biochemistry 135 (2019) 432ndash440
Available online 19 November 20180981-9428 copy 2018 Elsevier Masson SAS All rights reserved
T
glabrata (giant leucaena a tree) The latter has been used as a fastgrowing tree for production of wood and paper pulp The foliage ofboth common and giant leucaena is used as a fodder because of its highprotein content and palatability to farm animals The foliage containsup to 18 protein 142 crude fiber and 64 ether extractcrude fat(Soedarjo and Borthakur 1996)
Production of nitrogen-containing secondary metabolites such asmimosine requires large amounts of carbon and nitrogen resourcesNegi et al (2014) estimated that up to 21 of the carbon-nitrogenresources may be used for production of mimosine in leucaenaBrewbaker et al (1972) determined the mimosine content of 96 Lleucocephala cultivars and 8 other Leucaena species collected from 38different countries by growing them in an observational nursery inHawaii and found that basal mimosine content varied from 189 to477 of the dry weight
Mimosine is biosynthesized from OAS (o-acetylserine) and 3H4P (3-hydroxy-4-pyridone or its tautoisomer 3-hydroxy-4-pyridine) A pre-vious analysis suggested that mimosine synthase is an OAS-TL (o-acetylserine-thiol-lyase) of the cysteine biosynthesis pathway (Ikegamiet al 1990) Later however recombinant enzyme tests did not supportan OAS-TL identity of mimosine synthase (Yafuso et al 2014) Recentfindings on mimosine biosynthesis revealed that a cytosolic cysteine-OAS-TL isoform can also catalyze the formation of mimosine underspecific conditions (Harun-Ur-Rashid et al 2018)
Mimosine toxicity is related to its ability of reducing the availabilityof divalent metal ions such as Fe(II) Zn(II) Cu(II) Co(II) and Mn(II)by chelating co-factors and preventing their association with metal-dependent enzymes Furthermore this non-protein amino acid is cap-able of forming a stable complex with pyridoxal-5prime-phosphate (PLP)leading to the inactivation of PLP-dependent enzymes (eg Asp-Glutransaminase and cystathionine synthetase) (Negi et al 2014)
Mimosine features several useful biological activities such as alle-lopathic antimicrobial insecticide cell cycle inhibitor agent antic-ancer phytoremediator (Nguyen and Tawata 2016) as well as anti-oxidant (Benjakul et al 2013) Despite the relatively well establishedbiological activities of purified mimosine on other organisms or celltypes little is known about its biological role in leguminous speciesHowever it has been suggested that at least in part its activity ismainly related to defense mechanisms against some biotic and abioticstresses and as nitrogen source during fast growth (Vestena et al2001)
Suda (1960) and Smith and Fowden (1966) identified enzymes in-volved in mimosine degradation in seedling extracts of L leucocephalaand Mimosa pudica A mimosine-degrading enzyme named mimosinase(mimosine amidohydrolase EC 35161 CAS registry number 104118-49-2) (IUBMB 2018) a carbon-nitrogen lyase which degrades mimo-sine into 3H4P was later purified by Tangendjaja et al (1986) Itsbiochemical characterization was described and the cDNA was isolatedby Negi et al (2014)
Although mimosinase has been described and isolated only fewstudies on the role played by biotic and abiotic factors on the dynamicmodulation of mimosine metabolism in leguminous species have beenconducted (Vestena et al 2001 Xu et al 2018) In aseptic cultures ofleucaena mechanical injury of shoots promoted local mimosine accu-mulation (Vestena et al 2001) In the same study cultivation in pre-sence of auxin or SA in culture medium also had a positive effect on
mimosine accumulation More recently the effect of drought treatmenton gene expression of leucaena was also evaluated by Honda et al(2018) However several potential factors regulating mimosine meta-bolism need to be further examined
To date there is a lack of information on the biological role ofmimosine in planta as well as on details of its metabolic dynamicsMoreover its overt potential for pharmaceutical applications and de-velopment of new drugs as well as the possible use as tool to addressheavy metal soil contamination or plant mineral nutrition improve-ment justify additional research The objective of this study was toinvestigate the effect of stress signaling molecules and acute UV ex-posure on modulation of mimosine accumulation and metabolism in Lleucocephala spp glabrata in order to better understand its biologicalrole and to identify strategies for yield improvement aiming at ex-ploring its useful bioactivities
2 Methods
21 Plant material
For the experiments carried out to evaluate the effects of elicitors onmimosine accumulation seeds of leucaena were kindly provided by DrJames Brewbaker and harvested at CTAHRs (College of TropicalAgriculture and Human Resources of the University of Hawaii atManoa) Waimanalo Research Station at Oahu Hawaii This plantmaterial was originated from the accession K636 of Leucaena leucoce-phala ssp glabrata (Brewbaker 2008)
22 Induced mimosine content in 5-week-old giant leucaena
221 Seed germinationIn order to overcome seed coat dormancy seeds were submitted to a
chemical scarification with sulfuric acid 95ndash98 for 20min and re-peatedly rinsed in distilled water to remove any residual trace of thisreagent Then seeds were distributed in 254 cmtimes508 cm plastictrays containing 11 vv of vermiculite and commercial soil watereduntil reaching substrate field capacity Three weeks after seed imbibi-tion seedlings displaying similar size and shape (eg number of com-pound leaves and leaflets) were transplanted to individual pots(250mL) in number of three plants per container
During the experimental period (except in the UV-C radiationtreatment) all tested seedlings were kept in a growth chamber andsubmitted to controlled conditions of temperature (circa 25 degC) and ir-radiance (approximately 100 μmol photons mminus2sdot s minus1) with a photo-period of 16 h light and 8 h dark
222 Treatments2221 JA Ethephon and SA Five-week-old giant leucaena seedlingswere treated with different solutions as described in Table 1 Idealconcentrations were defined in preliminary experiments under the sameconditions indicated above At the beginning of the experiments 30plants were sprayed with 84 ppm JA 10 ppm SA 10 or 100 ppmEthephon or Milli-Qreg water (control) until the point of imminent runoffPlant pots were kept closed inside transparent plastic bags for 24 h toavoid solution volatilization Fifteen plants arranged in 5 sets of 3 (5biological replicates) were harvested 48 h and 96 h after being treated
Table 1Treatments used to modulate mimosine biosynthesis in giant leucaena
ELICITOR CONCENTRATION UV FLUENCE EXPOSURE TIME RATIONALE FOR USE
Salicylic acid (SA) 10 ppm 24 h Pathogen signaling molecule (Shah 2003)Jasmonic acid (JA) 84 ppm 24 h Chemical elicitor of plant secondary metabolism (Dar et al 2015)Ethephon 10 ppm 24 h Ethylene releasing-compound (Kim et al 2016) elicitor of plant secondary metabolism (Wang
et al 2016)UV-C radiation 3 Jcmminus2 10min or 15min Elicitor of plant secondary metabolism (Kara 2013 Neelamegam and Sutha 2015)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
433
After collection shoots were separated from roots immediately frozenin liquid nitrogen and stored at ndash 80 degC prior to HPLC analyses
2222 UV-C Thirty seedlings of giant leucaena were exposed to UV-Cradiation (3 Jcmminus2) for 10 or 15min and kept in a growth chamberunder controlled conditions as described above until the end of theexperiments Fifteen plants arranged in groups of 3 were harvested at96 h and 120 h after UV-C exposure and processed as previouslydescribed
223 Mimosine extractionMimosine extraction was based on a modified version of the pro-
tocol published by Lalitha and Kulothungan (2006) as follows a knownweight of fresh tissue (shoots or roots) of giant leucaena was first addedto Milli-Qreg boiling water in a proportion of 110 (g of plant per mL ofsolvent) in test tubes Tubes were covered with foil to avoid solutionevaporation and placed on a hot stirrer at 100 degC for 10min A pro-portional volume of 01M HCl was added to the cooled suspensions andhomogenized using mortar and pestle The plant extracts were filteredthrough cotton and centrifuged twice for 7min in a bench top re-frigerated centrifuge at 4 degC and 13200 rpm Before being analyzed theextracts were diluted 13 with ondashphosphoric acid (OPA)
224 Mimosine detectionHPLC analyses were carried out as described by Negi and Borthakur
(2016) Pure mimosine (L-mimosine from koa haole seeds Sigma-Al-drich CAS number 500-44-7) was used as standard Separation andquantification of mimosine was done with a C18 column (PhenomenexC18 5 μm 46times250mm) under an isocratic solvent system of 002MOPA with a linear flow rate of 1mLsdotminminus1 Mimosine detection wasdone at 280 nm by photodiode array detection (200ndash400 nm) showingretention time of 277 plusmn 0042min Quantification was done using themethod of external standard curve Further confirmation of mimosineidentity was performed by co-chromatography with standard and peakpurity check Chromatograms were analyzed using the Waters Em-power 3 software
23 Quantitative real-time PCR analysis of mimosinase gene expression
Fifteen 8-week-old giant leucaena plants arranged in 4 sets of 3 (4biological replicates) were treated with either water (control) or10 ppm Ethephon 84 ppm JA acid or 15min of UV-C radiation ex-posure following the methods described above Following treatmentleucaena plants were harvested at 48 and 96 h or 72 and 144 h (UV-Ctreated plants only) after treatments Total RNA of samples was ex-tracted and purified from roots and shoots of giant leucaena by meansof a modified method using Qiagen RNeasy Plant Kit (Valencia CAUSA) and Fruit-mate (Takara Japan) according to the protocol de-scribed by Ishihara et al (2016a) The assessment of RNA quality andquantity was carried out at 230 260 and 280 nm by using a NanoDropSpectrophotometer ND-1000 (NanoDrop Technologies DE USA) Inorder to avoid genomic DNA contamination RNA samples were treatedwith TURBO DNAfree Kit (Invitrogen Carlsbad CA) Two microgramsof DNase-treated RNA were used to synthesize the first-strand cDNAusing M-MLV Reverse Transcriptase (Promega WI USA)
Quantitative real-time (qPCR) analysis was carried out to examinepossible differential expression of the mimosinase gene (GenBank ac-cession number AB2985971) in seedlings treated with 84 ppm JA10mM Ethephon or 15min of UV-C exposure Shoots and roots wereharvested 24 h before the time of mimosine concentration peak for eachtreatment previously observed as assessed by HPLC assays The 10 μLqPCR reaction consisted of 5 μL of PowerUpTM SYBRreg Green MasterMix (Applied Biosystems Foster City CA) 1 μL MgCl2 (50mM) 03 μLforward primer (10 μM) 03 μL reverse primer (10 μM) and 1 μL cDNAfirst-strand In the experimental validation through qPCR reactionconditions and melting curve analysis of the amplicon were performed
following the protocol published by Ishihara et al (2016b) for the sameleucaena variety qPCR analysis was conducted using StepOnetrade Real-Time PCR System (Applied Biosystems) Measurements were performedusing 4 biological and 3 technical replicates Relative expression wascalculated with the 2-ΔΔct method using OAS-TL as reference gene sinceits expression showed a consistently stable profile comparable to that ofUBQ-5 and ELF1α expressions Mimosinase primer sequences used forthese analyses were (FWD) 5prime- GAA AGG CAG GAA TCA CAG TGA AGAG ndash 3rsquo (REV) 5prime GGA GAC TCT AGC CAC ACC AAC TTA ndash 3rsquo
24 Antioxidant assays
241 Mimosine effect on hydrogen peroxide (H2O2) accumulationAs a follow up to the induction of mimosine accumulation profiles
under stress signals and conditions tests were conducted to verify mi-mosine antioxidant capacity In situ histological localization of hy-drogen peroxide (H2O2) accumulation was evaluated on foliar disks ofPhaseolus vulgaris L according to the protocol described by Shi et al(2010) Briefly the plant foliar tissue was exposed to 1 mgmiddotmLminus1 dia-minobenzidine (DAB) solution in 10 mM KH2PO4 (control) in presenceor absence of 10mM mimosine (equivalent to the average mimosineconcentration induced by UV-C radiation in giant leucaena) or 10mMascorbic acid (positive antioxidant control) Oxidative response wasidentified by the formation of a brown polymer on the injured leafareas indicating the presence of H2O2 and registered in a Leica M165FC stereomicroscope (Leica Microsystems)
242 Mimosine quenching of superoxide radicalsGeneration of superoxide radical and subsequent analysis was per-
formed by a modified protocol based on Zhishen et al (1999) Nitroblue tetrazolium (NBT) reduction was used to measure superoxide an-ions quenching activity Shortly a 50mM KH2PO4 pH 78 solutioncontaining 6 μM riboflavin 100mM methionine 1 mM NBT in pre-sence or absence of 5mM mimosine was exposed to white light(22 Jsdotcmminus2) for 25min on a white light transilluminator Five micro-molar rutin was used as positive control (Matsuura et al 2016) Theabsorbance was read at 560 nm before and after light exposure in aSpectraMaxreg M2 Microplate Reader (Molecular Devices LLC)
25 Statistical analyses
For HPLC and superoxide anions data simple analyses of variance(ANOVA) followed by Tukey or Welch ANOVA followed by Dunnetts Ctest were used as appropriate for data distribution characteristics InqPCR analysis results were analyzed by t-test In all cases at least fourbiological triplicates were used and experiments were repeated twiceindependently All data were analyzed using the statistical packageSPSS 200 for Windows (SPSS Inc USA) In all cases a ple 005 wasused
3 Results and discussion
31 Increased mimosine concentrations in giant leucaena treated withchemical elicitors
Leucaena produces high amounts of mimosine that accumulate in allparts of the plants including leaves stem flowers pods seeds rootsand root nodules (Soedarjo and Borthakur 1998) The highest con-centrations of mimosine can be found in the growing shoot tips andseeds (Wong and Devendra 1983) It is not known why leucaena pro-duces such high amounts of mimosine Negi et al (2014) estimated thatleucaena plants would be able to grow 21 larger if the nutrient re-sources spent on mimosine production were diverted for biomass in-crease In a previous analysis performed to quantify the basal con-centration of mimosine present in adult plants of common leucaena thehighest constitutive amount of mimosine per gram of fresh weight in
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
434
the analyzed organs was found in post-anthesis flowers (89448 μg)followed by green pods (82687 μg) leaves (67358 μg) and greenflower buds (51247 μg) which showed significantly less mimosineconcentration compared to the other reproductive structures(Supplementary Fig 1) Since mature seeds have very low moisturecontent (Wencomo et al 2017) its mimosine concentration was esti-mated as 338253 μgsdotgminus1 of dry weight Additionally it was also ob-served that the basal mimosine distribution in shoots of field-grownadult plants of leucaena is dependent on the variety type(Supplementary Table 1)
Phytohormones such as salicylic acid and jasmonic acid are knownto be produced by plants in response to various abiotic and bioticstresses These phytohormones trigger adaptive responses to stress byregulating major plant metabolic processes such as photosynthesisnitrogen metabolism defense systems and plant-water relationsthereby providing protection (for review see Khan et al 2015)
Secondary or specialized metabolite production and accumulationare also known to be controlled by biotic and abiotic stresses (Matsuuraet al 2018) In this study exposure of 5-week-old giant leucaenaseedlings to JA or Ethephon treatments significantly enhanced mimo-sine accumulation in shoots and roots in at least one of the two timepoints tested (48 and 96 h) albeit in a different way (Fig 1) Thehighest concentrations of mimosine in shoots were found in seedlingstreated with JA 84 ppm (43441 μgsdotgminus1) and Ethephon 100 ppm(38412 μgsdotgminus1) two days after application of the respective phyto-hormones Nevertheless after four days shoots yielded the highestconcentration of mimosine (approximately 460 μgsdotgminus1) upon treatmentwith 10 or 100 ppm Ethephon (Fig 1A) In roots after two and four
days JA 84 ppm and Ethephon 10 ppm resulted in highest mimosineaccumulation 18488 μgsdotgminus1 and 15801 μgsdotgminus1 respectively (Fig 1B)These observations show that mimosine accumulation response tospecific elicitors may vary over time after exposure
Although all treatments were applied exclusively on shoots of giantleucaena seedlings roots of some of them were also able to respond tothe different elicitors Overall shoots displayed higher basal and in-duced mimosine concentration compared to roots (Fig 1) which agreeswith previous observations in 1 to 3-week-old aseptic seedlings ofcommon leucaena (Vestena et al 2001) However as previouslymentioned significant post-induction increase of mimosine concentra-tion in roots and shoots simultaneously was only observed for JA andEthephon 10 ppm on day 02 and 04 respectively (Fig 1)
It is well established that perceived regulatory signals or elicitorsgenerate a transduction network mediated by secondary messengersresulting in changes in gene expression profiles that afford adaptiveresponses to environmental stimuli These modulation events are oftenmediated by transcription factors (TFs) which directly bind to specificgene promoters or act by forming complexes with repressor proteinslabeling them to degradation subsequently releasing other TFs toproceed with the gene expression program This is the case of the actionmechanism of JA and its active form jasmonoyl isoleucine for example(Kazan 2015 Wasternack and Strnad 2016)
JA ethylene and SA are known as important stress regulatory sig-nals in plants JA however is thought to be the most effective signal forinduction of plant secondary metabolism (Wasternack and Strnad2016) thereby contributing to mitigation of damage caused by severalstresses (Dar et al 2015) JA is mainly derived from linolenic acid
Fig 1 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings treated with differentelicitors CTRL=Milli-Q water SA = Salicylic AcidJA= Jasmonic Acid ETH=Ethephon Bars sharing a letterof same case do not differ by Tukey test (P le 005) Capitalletters (A B) compare treatments on day two and lowercaseletters (a b) compare treatments on day four Indicatessignificant statistical difference between day two and dayfour in the same treatment by t-test (Ple 005) The errorbars represent standard error of five replicates (each meanwas calculated with 15 individual seedlings organized in 5groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
435
(Wasternack and Strnad 2016) playing important roles in differentprocesses of plant growth and development such as plant defensemechanisms against herbivory pathogen attack fungal elicitation andsome abiotic factors such as osmotic temperature and salt stresses (Daret al 2015)
JA and its methyl ester MeJA have several different effects on le-guminous species MeJA exogenous application has increased iso-flavonoid content in cell suspension cultures of Pueraria candollei varcandollei and P candollei var mirifica (Korsangruang et al 2010) aswell as the production of the triterpenoid glycyrrhizin in Glycyrrhizaglabra roots Enhanced production of the triterpenoid however waspartly at the expense of root growth (Shabani et al 2009) MeJA ap-plication on shoots was observed to suppress root nodulation and lat-eral root formation in Lotus japonicus (Nakagawa and Kawaguchi2006) In grapevine a non-leguminous species proteinogenic aminoacids did not show an expressive increase under MeJA treatment(Gutieacuterrez-Gamboa et al 2017)
The effects of the application of four different jasmonate forms (JAMeJA jasmonoyl-L-isoleucine (JA-Ile) and 6-ethyl indanoyl glycineconjugate (2-[(6-ethyl-1-oxo-indane-4-carbonyl)-amino]-acetic acidmethyl ester - CGM) on leucaena metabolite profile has recently beenreported by Xu et al (2018) JA-Ile form was most effective althoughno major alteration was observed on monitored metabolite abundancesAlanine threonine and 34-dihydroxypyridine (34 DHP a metabolitederived from mimosine degradation) (Nguyen and Tawata 2016)among others were the major metabolites elicited by JA-Ile In contrastto the results described here mimosine concentration did not changesignificantly These divergent results on mimosine accumulation maybe due to a number of factors including mode of application jasmonateform used (JA-Ile x JA) and L leucocephala subspecies (common x giantleucaena)
Ethylene is also a phytohormone involved in plant response me-chanisms to different types of challenges such as mechanical damageand insect attack among others The integration mechanism betweenJA and ethylene signaling pathways is not completely understoodhowever it has been shown that they may work cooperatively in abioticstress tolerance (Kazan 2015) MeJA can induce ethylene production(Zhao et al 2004) and when applied simultaneously these moleculesseem to work in a synergic way by enhancing the magnitude of theplant response to external stimuli (Liu et al 2016)
Treatment with SA was able to significantly increase mimosine ac-cumulation in 12-week-old plants of common leucaena (SupplementaryFig 2) However no significant effect of SA treatment on mimosineconcentration was seen in 5-week-old seedlings of giant leucaena(Fig 1) suggesting some degree of genotype andor age dependency inelicitation by this phytohormone On the other hand several treat-ments including 90 ppm MeJA 10 and 100 ppm 2-chloroethylpho-sphonic acid (CEPA an ethylene-releasing compound) significantlyincreased mimosine accumulation (Supplementary Fig 2) in agree-ment with the data obtained for giant leucaena The lack of systemiceffects of externally applied SA on mimosine accumulation was alsoobserved when the phytohormone was supplied in the culture mediumof aseptically-grown seedlings in which case only roots had highercontent of mimosine (Vestena et al 2001) This could be due totransport limitations or to low methyl salicylate production from ap-plied SA since the former is recognized as the main systemic signalingform (Vlot et al 2009)
32 Increased mimosine concentrations in giant leucaena exposed to UV-Cradiation
UV-C treatment promoted increased concentration of the aminoacid in shoots but not in roots of giant leucaena (Fig 2) Increasedaccumulation of mimosine in shoots was also observed in 12-week-oldseedlings of common leucaena exposed to UV-C radiation for 10 and15min (Supplementary Fig 3) Similar to the SA treatment in giant
leucaena UV-C radiation did not induce mimosine biosynthesis in rootsregardless of time after exposure The absence of mimosine induction inroots by SA and UV indicates that these effectors do not cause a sys-temic response Moreover roots are shielded from irradiance by thepresence of substrate
UV radiation effects on different aspects of plant metabolism anddevelopment have been described However compared to UV-B (en-vironmentally relevant type of UV radiation) assays there are less re-ports related to the UV-C effects on secondary metabolites biosynthesisand accumulation (Cetin 2014) especially in leguminous (Fabaceae)plants They generally concern primary metabolism aspects such asgrowth and development For instance seedlings of Phaseolus vulgaris L(Fabaceae) exposed to low intensity UV-C radiation have displayeddecreased chlorophyll content and reduced height after 14 days of ex-posure (Kara 2013) Negative effects on growth parameters and ni-trogen metabolism were also observed in Vigna radiata L (Fabaceae)after UV-B radiation treatment in addition to adverse effects on JA SAand antioxidant compounds accumulation (Choudhary and Agrawal2014a) The same authors reported increased accumulation of flavo-noids SA and JA besides negative effects on growth biomass yieldnitrogen fixation and accumulation in 2 cultivars of Pisum sativum L(Fabaceae) under elevated UV-B treatment (Choudhary and Agrawal2014b) Despite the negative UV influence on growth reported for thepreviously mentioned leguminous UV-C radiation on groundnut plants(Arachis hypogaea L Fabaceae) increased seedling vigor and biomassand had no adverse effect on germination or other development para-meters (Neelamegam and Sutha 2015)
Besides its impact on growth and primary metabolism UV exposurecan cause important changes in secondary metabolism depending onintensity and time of exposure (Matsuura et al 2013) UV-B and UV-Cpre-treatments of Artemisia annua (Asteraceae) seedlings yielded in-creased biosynthesis of artemisinin a drug which displays anti-malarialproperties and activity against some others infectious diseases (egschistosomiasis leishmaniasis and hepatitis B) and several kinds oftumors (Rai et al 2011) The accumulation of nicotine in Nicotianarustica plants (Solanaceae) was also increased by UV-C treatment(Tiburcio et al 1985) Similar inducing effects on production of severalsecondary metabolites were observed in callus cultures of Vitis viniferaL Oumlkuumlzgoumlzuuml (grapevine Vitaceae) treated with a UV-C source for 5 or10min (Cetin 2014)
Regarding amino acid biosynthesis in response to UV radiationMartiacutenez-Luumlscher et al (2014) have found that in spite of not causingchanges in total amino acid content UV-B radiation exposure can affecttheir profile in grape berries Proteinogenic amino acids have beenknown to be important targets of the deleterious effects of UV radiation(Holloacutesy 2002) On the other hand in the present study acute UV-Ctreatment was found to increase mimosine accumulation in shoots byover twofold (Fig 2) which may suggest a possible participation of thismolecule as part of the antioxidant defense system in L leucocephalaThis possibility is further supported by the induction of the amino acidaccumulation by JA and Ethephon involved in abiotic and biotic stressresponses which are generally associated with oxidative imbalance andare signaling components in high UV stress (Matsuura et al 2013)
33 Mimosinase gene expression
In order to determine if increases in mimosine content upon ex-posure to JA CEPA or UV-C radiation were related to changes intranscription of mimosine metabolism-related genes RT-qPCR analysiswas carried out The complete pathway for mimosine biosynthesis hasnot yet been determined although the final step has been character-ized Based on transcription analysis (Ishihara et al 2016a) leucaenaappears to encode for multiple cysteine synthases one or more of whichmay be able to catalyze mimosine synthesis In addition a leucaenagene encoding a mimosinase (an enzyme responsible for mimosinedegradation) has been identified and characterized (Negi et al 2014)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
436
In addition to mimosinase gene expression several gene isoformsbelonging to the cysteine pathway [cysteine synthase (CYS SYN) serineacetyltransferase (SAT) and β-cyanoalanine synthase (CAS) Table 2 -supplementary material] were also tested in this study (data notshown) However expressions of these genes did not vary in giantleucaena throughout the experiments suggesting that the increasedcontent of mimosine observed in the treated plants might not be relatedto the expression of these genes but presumably to increased enzymeactivity andor release from conjugates such as mimoside a mimosineβ-D-glucoside (Murakoshi et al 1972)
Considering the time variation of mimosine accumulation observedin this work mimosinase gene expression in shoots and roots wasevaluated 24 h before the increase of mimosine concentration in giantleucaena seedlings (ie 24 h and 72 h after the chemical elicitorstreatments and 48 h and 120 h after UV-C exposure)
Ethylene signaling has been shown to up-regulate expression ofseveral genes related to secondary metabolism pathways as is the caseof phenolic compounds (Liu et al 2016) and terpenoid indole alkaloids(Wang et al 2016) Among all elicitors tested in the present workEthephon was the only one able to significantly change mimosinasegene expression Leucaena plants treated with Ethephon showed sig-nificant increases in mimosine concentration at both day 2 and 4 fol-lowing treatment which coincided with low-level expression of mi-mosinase Up-regulation of mimosinase gene expression was detected24 h before the increase of mimosine concentration in shoots treatedwith 10 ppm of Ethephon (Fig 3A) but not after JA or UV-C treatments(Fig 3C-D and 3E-F respectively) Nevertheless 72 h after treatment
application (24 h before the highest mimosine content measured inshoots) down regulation of mimosinase gene was seen in both shootsand roots treated with 10 ppm of Ethephon (Fig 3B) These data in-dicate that mimosine content in leucaena plants is at least partlyregulated by mimosinase expression in Ethephon exposed plants Onthe other hand the fact that mimosinase mRNA was not significantlyaffected by JA and UV-C treatments despite their stimulating effects onmimosine biosynthesis in giant leucaena may indicate that other levelsof regulation are at play or that the chosen harvesting time window wasunable to detect relevant changes
34 In situ and in vitro antioxidant assays
Considering the stimulation of mimosine accumulation byEthephon JA and UV all of which are often associated or known tocause oxidative imbalance the antioxidant capacity of mimosine wasevaluated Mimosine has been shown to have antioxidant activities oncultured cancer cells (Parmar et al 2015) In the present study it washypothesized that mimosine could confer radical scavenging propertieswhich would contribute to plant protection from possible damagecaused by reactive oxygen species generated during stress(Supplementary Fig 4)
Foliar disks of P vulgaris L were treated with 10mM mimosine for15min Treated disks showed less hydrogen peroxide accumulationinduced by wounding in contrast to untreated ones being comparableto those treated with ascorbic acid (a known hydrogen peroxide neu-tralizer) (Fig 4A) These observations support a possible antioxidant
Fig 2 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings exposed to UV-C lightCTRL= visible light (100 μmol photons mminus2 s minus1) UV-C 10primeand UV-C 15rsquo=UV-C exposure time (10 and 15min re-spectively) Bars sharing a letter of same case do not differ byTukey test (P le 005) Capital letters (A B) compare treat-ments on day three and lowercase letters (a b) comparetreatments on day six Indicates significant statistical dif-ference between day three and day six in the same treatmentby t-test (Ple 005) The error bars represent standard errorof five replicates (each mean was calculated with 15 in-dividual seedlings organized in 5 groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
437
role of mimosine as an in situ hydrogen peroxide scavengerMimosine was also able to quench superoxide anions generated by
light exposure Mimosine exhibited equivalent antioxidant effect com-pared to rutin (Fig 4B) a well-established effective superoxide anionquencher (Matsuura et al 2016) The radical scavenging activity ofmimosine may be due to the 3-OH group of the pyridine ring of mi-mosine (Fig 5) The pKa of the 3-OH of mimosine has been estimated tobe 88 (M Honda unpublished results) At physiological pH this OHgroup is expected to remain in a protonated state and therefore mayscavenge a radical by donating a proton and an electron In this processmimosine itself is converted to a stable radical form which is perhapsless toxic and less reactive than the reactive oxygen species generatedduring oxidative stress It is likely that the less toxic radical mimosineproduced may react with another radical or molecule and becomeconverted to a non-reactive indole molecule
In vivo antioxidant activity of mimosine has been previously eval-uated by means of its exogenous application on selenium-deficientseedlings of Vigna radiata In spite of its allelopathic properties (Ahmedet al 2008) the results showed mitigation of mitochondrial oxidativestress by treatment with 01mM mimosine (Lalitha and Kulothungan2007) DPPH radical scavenging activity was also reported for aqueous
seed extracts of leucaena rich in mimosine and phenolic compounds inin vitro assays (Benjakul et al 2014) Mimosine antioxidant activityshown in the present work is in good agreement with data reported forother non-protein amino acids such as L-DOPA (Dhanani et al 2015)and GABA (Malekzadeh et al 2014) for instance
4 Conclusion
Taken together results show that mimosine biosynthesis and ac-cumulation can be modulated by stress-related factors despite its re-latively high constitutive content in leucaena plants The pattern ofgene expression in stressed plants suggests mimosine steady-state con-trol may be regulated by its degradation in possible connection withdynamic changes in carbon and nitrogen metabolism of stressed plantsMimosine quenching activity against hydrogen peroxide and super-oxide anions in the in situ staining and in vitro assays respectivelyshowed that this non-protein amino acid can act as non-enzymaticantioxidant agent Increase in mimosine content in response to elicitorsmimicking environmental challenges in addition to its antiherbivoreand antimicrobial properties may be related to its activity as protectivemolecule against oxidative damage in line with other classes of plant
Fig 3 Relative expression of the mimosinase gene in shoots (A E and F) and shoots and roots (B C and D) of giant leucaena 24 h (A and C) 48 h (E) 72 h (B and D)and 120 h (F) after treatment with stress signaling molecules or UV-C exposure ETH = Ethephon JA = Jasmonic Acid Indicates significant statistical differencebetween control and treatment by t-test (Ple 005) The error bars represent standard error of four replicates
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
438
secondary metabolites
Funding
This work was funded by the National Council for Scientific andTechnological Development (CNPq-Brazil) grant 3060792013-5Coordenaccedilatildeo de Aperfeiccediloamento de Pessoal de Niacutevel Superior - Brazil(CAPES) - Finance Code 001 and the USDA NIFA Hatch projectHA05029-H managed by CTAHR
CRediT authorship contribution statement
Kelly Cristine da Silva Rodrigues-Correcirca InvestigationValidation Writing ndash original draft Michael DH HondaInvestigation Validation Dulal Borthakur Supervision Writing ndashreview amp editing Funding acquisition Arthur Germano Fett-NetoSupervision Funding acquisition Writing ndash review amp editing
Acknowledgements
The authors would like to thank Dr Jorge Ernesto Mariath fromLaVeg-UFRGS for kindly lending the Leica M165 FC stereomicroscopefor in situ analysis
Appendix A Supplementary data
Supplementary data to this article can be found online at httpsdoiorg101016jplaphy201811018
References
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leucocephala leaf litter on some forest and agricultural crops grown in nursery J ForRes 19 298 httpsdoi 101007s11676-008-0053-0
Benjakul S Kittiphattanabawon P Shahidi F Maqsood S 2013 Antioxidant activityand inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipidoxidation in model systems Food Sci Technol Int 19 (4) 365ndash376 httpsdoiorg1011771082013212455186
Benjakul S Kittiphattanabawon P Sumpavapol P Maqsood S 2014 Antioxidantactivities of lead (Leucaena leucocephala) seed as affected by extraction solvent priordechlorophyllisation and drying methods extracts against lipid oxidation in modelsystems Food Sci Technol 51 (11) 3026ndash3037 httpsdoiorg101007s13197-012-0846-1
Brewbaker JL Pluckett D Gonzalez V 1972 Varietal variation and yield trials ofLeucaena leucocephala (koa haole) in Hawaii Hawaii Agric Exp Stn Bull 166 26
Brewbaker JL 2008 Registration of KX2 ndash Hawaii interspecific-hybrid leucaena JPlant Registrations 1 (3) 190ndash193 httpsdoiorg103198jpr2007050298crc
Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitisvinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-6287-47-37
Cho H-Y Son SY Rhee HS Yoon S-YH Lee-Parsons CWT Park JM 2008Synergistic effects of sequential treatment with methyl jasmonate salicylic acid andyeast extract on benzophenanthridine alkaloid accumulation and protein expressionin Eschscholtzia californica suspension cultures J Biotechnol 135 117ndash122 httpsdoiorg101016jjbiotec200802020
Choudhary KK Agrawal SB 2014a Cultivar specificity of tropical mung bean (Vignaradiata L) to elevated ultraviolet-B changes in antioxidative defense system ni-trogen metabolism and accumulation of jasmonic and salicylic acids Environ ExpBot 99 122ndash132 httpsdoiorg101016jenvexpbot201311006
Choudhary KK Agrawal SB 2014b Ultraviolet-B induced changes in morphologicalphysiological and biochemical parameters of two cultivars of pea (Pisum sativum L)Ecotoxicol Environ Saf 100 178ndash187 httpsdoiorg101016jecoenv201310032
Dar TA Uddin M Khan MMA Hakeem KR Jaleel H 2015 Jasmonates counterplant stress a Review Environ Exp Bot 115 49ndash57 httpsdoiorg101016jenvexpbot201502010
Dhanani T Singh R Shah S Kumari P Kumar S 2015 Comparison of green ex-traction methods with conventional extraction method for extract yield L-DOPAconcentration and antioxidant activity of Mucuna pruriens seed Green Chem LettRev 8 (2) 43ndash48 httpsdoiorg1010801751825320151075070
Gutieacuterrez-Gamboa G Portu J Santamariacutea P Loacutepez R Garde-Cerdaacuten T 2017Effects on grape amino acid concentration through foliar application of three dif-ferent elicitors Food Res Int 99 688ndash692 httpsdoiorg101016jfoodres201706022
Fig 4 A In situ antioxidant assay Foliar disksof Phaseolus vulgaris L treated with (a) No an-tioxidant added (negative control) (b) 10 mMMimosine (c) 10mM ascorbic acid (positivecontrol) The oxidative damage can be seen bythe formation of a brown polymer in leaf veinsand injured areas B In vitro superoxidescavenging assay carried out with mimosineDifferent letters indicate significant differenceby Tukey test (Ple 005) The error bars re-present standard error of four replicates (Forinterpretation of the references to colour in thisfigure legend the reader is referred to the Webversion of this article)
Fig 5 Predicted mimosine radical formed followingquenching of hydroxyl radical Mimosine is first converted toa stable mimosine radical which may be then converted to anontoxic indole form
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
439
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad HossainMd Anai T Oku H 2018 Cytosolic cysteine synthase switch cysteine and mi-mosine production in Leucaena leucocephala Appl Biochem Biotechnol 186 (3)613ndash632 httpsdoiorg101007s12010-018-2745-z
Holloacutesy F 2002 Effects of ultraviolet radiation on plant cells Micron 33 (2) 179ndash197Honda MDH Ishihara KL Pham DT Borthakur D 2018 Identification of drought-
induced genes in giant leucaena (Leucaena leucocephala subsp glabrata) Trees 32571ndash585 httpsdoiorg101007s00468-018-1657-4
Huang T Jander G de Vos M 2011 Non-protein amino acids in plant defense againstinsect herbivores representative cases and opportunities for further functional ana-lysis Phytochemistry 72 1531ndash1537 httpsdoiorg101016jphytochem201103019
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of thethyrotoxic amino acid mimosine by cysteine synthase Phytochemistry 29 (11)3461ndash3465 httpsdoiorg1010160031-9422(90)85258-H
Ishihara K Lee EKW Borthakur D 2016a An improved method for RNA extractionfrom woody legume species Acacia koa A Gray and Leucaena leucocephala (Lam) deWit Int J For Wood Sci 3 (1) 031ndash035
Ishihara KL Honda MDH Pham DT Borthakur D 2016b Transcriptome analysisof Leucaena leucocephala and identification of highly expressed genes in roots andshoots Transcriptomics 4 135 httpsdoiorg1041722329-89361000135
IUBMB 2018 Enzyme Nomenclature EC 35161 httpwwwsbcsqmulacukiubmbenzymeEC35161html Accessed date 8 February 2018
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant(Phaseolus vulgaris) Biosci Res 10 (1) 29ndash32
Kazan K 2015 Diverse roles of jasmonates and ethylene in abiotic stress toleranceTrends Plant Sci 20 (4) 219ndash229 httpsdoiorg101016jtplants201502001
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect ofEthephon as an ethylene-releasing compound on the metabolic profile of Chlorellavulgaris J Agric Food Chem 64 (23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541
Khan MIR Fatma M Per TS Anjum NA Khan NA 2015 Salicylic acid-inducedabiotic stress tolerance and underlying mechanisms in plants Front Plant Sci 6 462httpsdoiorg103389fpls201500462
Korsangruang S Soonthornchareonnon N Chintapakorn Y Saralamp PPrathanturarug S 2010 Effects of abiotic and biotic elicitors on growth and iso-flavonoid accumulation in Pueraria candollei var candollei and P candollei var mir-ifica cell suspension cultures Plant Cell Tissue Organ Cult 103 (3) 333ndash342 httpsdoiorg101007s11240-010-9785-6
Lalitha K Kulothungan SR 2006 Selective determination of mimosine and its dihy-droxypyridinyl derivative in plant systems Amino Acids 31 (3) 279ndash287 httpsdoiorg101007s00726-005-0226-5
Lalitha K Kulothungan SR 2007 Mimosine mitigates oxidative stress in seleniumdeficient seedlings of Vigna radiata - Part I restoration of mitochondrial functionBiol Trace Elem Res 118 (1) 84ndash96 httpsdoiorg101007s12011-007-0013-0
Liu J Li Y Wang Y Zhang Z-H Zu Y-G Efferth T Tang Z-H 2016 Thecombined effects of ethylene and MeJA on metabolic profiling of phenolic com-pounds in Catharanthus roseus revealed by metabolomics analysis Front Physiol 71ndash11 httpsdoiorg103389fphys201600217 Article 217
Malekzadeh P Khara J Heydari R 2014 Alleviating effects of exogenous Gamma-aminobutiric acid on tomato seedling under chilling stress Physiol Mol Biol Plants20 (1) 133ndash137 httpsdoiorg101007s12298-013-0203-5
Martiacutenez-Luumlscher J Torres N Hilbert G Richard T Saacutenchez-Diacuteaz M Delrot SAguirreolea J Pascual I Gomegraves E 2014 Ultraviolet-B radiation modifies thequantitative and qualitative profile of flavonoids and amino acids in grape berriesPhytochemistry 102 106ndash114 httpsdoiorg101016jphytochem201403014
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation ofbioactive secondary metabolites by ultraviolet radiation mechanisms strategies andapplications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology forMedicinal Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash1902012
Matsuura HN Fragoso V Paranhos JT Rau MR Fett-Neto AG 2016 Thebioactive monoterpene indole alkaloid N szlig-D-glucopyranosylvincosamide is regu-lated by irradiance quality and development in Psychotria leiocarpa Ind Crop Prod86 210ndash218 httpsdoiorg101016jindcrop201603050
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo RBhambra AS Strnad M Bonfill M Fett-Neto AG 2018 Specialized plant me-tabolism characteristics and impact on target molecule biotechnological productionMol Biotechnol 60 (2) 169ndash183 httpsdoiorg101007s12033-017-0056-1
Murakoshi S Ohmiya S Haginiwa J 1972 Enzymic synthesis of mimoside a meta-bolite of mimosine in Mimosa pudica and Leucaena leucocephala Chem Pharm Bull20 (4) 855ndash857
Nakagawa T Kawaguchi M 2006 Shoot-applied MeJA suppresses root nodulation inLotus japonicus Plant Cell Physiol 47 (1) 176ndash180 httpsdoiorg101093pcppci222
Nascimento NC Menguer PK Henriques AT Fett-Neto AG 2013 Accumulation ofbrachycerine an antioxidant glucosidic indole alkaloid is induced by abscisic acidheavy metal and osmotic stress in leaves of Psychotria brachyceras Plant PhysiolBiochem 73 33ndash40 httpsdoiorg101016jplaphy201308007
Neelamegam R Sutha T 2015 UV-C irradiation effect on seed germination seedling
growth and productivity of groundnut (Arachis hypogaea L) Int J Curr MicrobiolApp Sci 4 (8) 430ndash443
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase fromLeucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol164 (2) 922ndash934 httpsdoiorg101104pp113230870
Negi VS Borthakur D 2016 Heterologous expression and characterization of mimo-sinase from Leucaena leucocephala In Fett-Neto Arthur Germano (Ed)Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods inMolecular Biology vol 1405 copySpringer Science+Business Media New York httpsdoiorg101007978-1-4939-3393-8_7 2016
Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636
Parmar F Kushawaha N Highland H George L-B 2015 In vitro antioxidant andanticancer activity of Mimosa pudica Linn extract and L-mimosine on lymphomaDaudi cells Int J Pharm Sci 12 100ndash104
Porto DD Matsuura HN Vargas LRB Henriques AT Fett-Neto AG 2014 Shootaccumulation kinetics and effects on herbivores of the wound-induced antioxidantindole alkaloid brachycerine of Psychotria brachyceras Nat Prod Commun 9 (5)629ndash632
Rai R Meena RP Smita SS Shukla A Rai SK Pandey-Rai S 2011 UV-B and UV-C pre-treatments induce physiological changes and artemisinin biosynthesis inArtemisia annua L ndash an antimalarial plant J Photochem Photobiol B Biol 105 (3)216ndash225 httpsdoiorg101016jjphotobiol201109004
Shabani L Ehsanpour AA Asghari G Emami J 2009 Glycyrrhizin production by invitro cultured Glycyrrhiza glabra elicited by methyl jasmonate and salicylic acid RussJ Plant Physiol 56 (5) 621ndash626 httpsdoiorg101134S1021443709050069
Shah J 2003 The salicylic acid loop in plant defense Curr Opin Plant Biol 6 (4)365ndash371
Shi J Fu XZ Peng T Huang XS Fan QJ Liu JH 2010 Spermine pretreatmentconfers dehydration tolerance of citrus in vitro plants via modulation of antioxidativecapacity and stomatal response Tree Physiol 30 (7) 914ndash922 httpsdoiorg101093treephystpq030
Smith IK Fowden L 1966 A study of mimosine toxicity in plants J Exp Bot 17750ndash761 httpsdoiorg101093jxb174750
Soedarjo M Borthakur D 1996 Simple procedures to remove mimosine from youngleaves pods and seeds of Leucaena leucocephala used as food Int J Food SciTechnol 31 (1) 97ndash103
Soedarjo M Borthakur D 1998 Mimosine a toxin produced by the tree-legumeLeucaena provides a nodulation competition advantage to mimosine-degradingRhizobium strains Soil Biol Biochem 30 1605ndash1613
Suda S 1960 On the physiological properties of mimosine Bot Mag Tokyo 73 (862)142ndash147 httpsdoiorg1015281jplantres188773142
Tangendjaja B Lowry JB Wills RBH 1986 Isolation of a mimosine degrading en-zyme from leucaena leaf J Sci Food Agric 37 523ndash526 httpsdoiorg101002jsfa2740370603
Tiburcio F Pintildeol MT Serrano M 1985 Effect of UV-C on growth soluble protein andalkaloids in Nicotiana rustica plants Environ Exp Bot 25 (3) 203ndash210 httpsdoiorg1010160098-8472(85)90004-8
Vestena S Fett-Neto AG Duarte RC Ferreira A 2001 Regulation of mimosineaccumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 httpsdoiorg101016S0168-9452(01)00448-4
Vlot AC Dempsey DMA Klessig DF 2009 Salicylic acid a multifaceted hormone tocombat disease Annu Rev Phytopathol 47 177ndash206 httpsdoiorg101146annurevphyto050908135202 2009
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene-induced vinblastine accumulation is related to activated expression of downstreamTIA pathway genes in Catharanthus roseus BioMed Res Int 2016 Article ID 3708187httpsdoiorg10115520163708187
Wasternack C Strnad M 2016 Jasmonate signaling in plant stress responses and de-velopment ndash active and inactive compounds N Biotech 33 (5B) 604ndash613 httpsdoiorg101016jnbt201511001
Wencomo HB Ortiz R Caacuteceres J 2017 Afr J Agric Res 12 (4) 279ndash285 httpsdoiorg105897AJAR201510604 26
Wong CC Devendra C 1983 Research on leucaena forage production in Malaysia InLeucaena Research in the Asian Pacific Region pp 55ndash60 Ottawa Ontario Canada
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX TsimKWK 2018 Jasmonate-elicited stress induces metabolic change in the leaves ofLeucaena leucocephala Molecules 23 (2) httpsdoiorg103390molecules23020188 E188
Yafuso JT Negi VS Bingham J-P Borthakur D 2014 An O-acetylserine (thiol)lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthaseAppl Biochem Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z
Zhao J Zheng S-H Fujita K Sakai K 2004 Jasmonate and ethylene signalling andtheir interaction are integral parts of the elicitor signalling pathway leading to b-thujaplicin biosynthesis in Cupressus lusitanica cell cultures J Exp Bot 55 (399)1003ndash1012 httpsdoiorg101093jxberh127
Zhishen J Mengcheng T Jianming W 1999 The determination of flavonoid contentsin mulberry and their scavenging effects on superoxide radicals Food Chem 64 (4)555ndash559 httpsdoiorg101016S0308-8146(98)00102-2
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
440
61
Supplementary Fig 1 Basal mimosine concentration in adult trees of common leucaena (L leucocephala
var leucocephala) Samples were collected from 10 field grown trees at Manoa Valley Honolulu Hawairsquoi
on June 25th 2017 Bars sharing a letter do not differ by Tukey test (P le 005) The error bars represent the
standard error
Supplementary Fig 2 Bar diagram showing mimosine concentration in shoots of 12-week-old common
leucaena seedlings treated with different elicitors CTRL = Milli-Q water SA = Salicylic Acid MeJA =
Methyl Jasmonate CEPA = 2-Chloroethylphosphonic acid (an ethylene releasing compound) Bars sharing a
letter of same case do not differ by Tukey test (P le 005) Capital letters (A B) compare treatments on day
two and lower-case letters (a b) compare treatments on day four Indicates significant statistical difference
ABB
A A
0
200
400
600
800
1000
1200
LEAVES GREEN FLOWERBUDS
POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
con
cen
trat
ion
(micro
gg
-1o
f FW
)
B AB AB AB B A
b
a
ab b
ab
0
2
4
6
8
10
12
14
16
18
20
CTRL SA 10 ppm SA 100 ppm CEPA 10 ppm CEPA 100 ppm MeJA 90 ppm
Mim
osi
ne
co
nce
ntr
atio
n (
gg
-1o
f FW
)
DAY 02 DAY 04
62
between day two and day four in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
Supplementary Fig 3 Bar diagram showing the effects of UV-C radiation exposure for 5 10 and 15 min on
mimosine accumulation in shoots of 12-week-old seedlings of common leucaena Bars sharing a letter of
same case do not differ by Tukey test (P le 005) Capital letters (A B C) compare treatments on day three
and lower-case letters (a b) compare treatments on day six Indicates significant statistical difference
between day three and day six in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
C BC AB A
bb
a
a
0
10
20
30
40
50
60
CTRL UV-C 5 UV-C 10 UV-C 15
Mim
osi
ne
co
nce
ntr
atio
n (
gg-1
of
FW)
DAY 03 DAY 06
63
Supplementary Fig 4 Model depicting induction of mimosine synthesis in leucaena following application of
stress elicitors such as CEPA and jasmonic acid or exposure to UV-C radiation The additional mimosine
synthesized may serve to alleviate oxidative stress induced by UV-C radiation
64
Supplementary Table 1 Mimosine contents in leaves of common and giant leucaena
Leucaena
type
Mimosine content
( FW)
Mimosine
content ( DW)
Dry matter
content ( FW)
Water content
( FW)
Common (1) 050 plusmn 009 245 plusmn 051 2011 plusmn 054 7989 plusmn 054
Common (2) 043 plusmn 006 214 plusmn 037 1998 plusmn 050 8002 plusmn 050
K636 (1) 070 plusmn 014 356 plusmn 077 1908 plusmn 052 8092 plusmn 052
K636 (2) 042 005 205 plusmn 033 2008plusmn 093 7992plusmn 093
KX2 (1) 122 plusmn 011 608 plusmn 082 1939 plusmn 123 8061 plusmn 123
KX2 (2) 134 plusmn 010 623 plusmn 056 2029 plusmn 114 7971 plusmn 114
KX3 (1) 044 plusmn 006 221 plusmn 030 1945 plusmn 073 8055 plusmn 073
KX3 (2) 054 plusmn 005 273 plusmn 023 1930 plusmn 038 8070 plusmn 038
KX4 (1) 086 plusmn 011 471 plusmn 065 1753 plusmn 084 8247 plusmn 084
KX4 (2) 089 plusmn 011 476 plusmn 065 180 plusmn 072 820 plusmn 072
KX5 (1) 099 plusmn 012 489 plusmn 048 1907 plusmn060 8093 plusmn 060
KX5 (2) 115 plusmn 015 548 plusmn080 1992 plusmn 053 8008 plusmn 053
Common leucaena variety koa haole grows widely on the island of Orsquoahu K636 is widely
grown variety of giant leucaena KX2 KX3 KX4 and KX5 are giant leucaena varieties
developed through interspecies hybridization (Brewbaker 2016) (1) and (2) indicate plants
from two separate locations within the University of Hawaii Waimanalo Research Center The
values are shown as mean plusmn standard error obtained from at least three biological replicates
65
Supplementary Table 2 GenBank accession numbers of the tested cysteine pathway genes isoforms
Gene name GenBank accession
OAS-TL (o-acetylserine-thiol-lyase) GDRZ01032940
GDRZ01061620
GDRZ01153117
GDSA01187555
GDSA01196891
GDSA01214467
Cys syn (cysteine synthase) GDRZ01015860
GDRZ01050898
GDRZ01086813
GDRZ01193515
GDRZ01202579
GDSA01180863
GDSA01215622
SAT (serine acetyltransferase) GDRZ01187456
GDRZ01189631
CAS (β-cyanoalanine synthase) GDRZ01054066
GDRZ01175418
GDSA01118400
66
SHORT COMMUNICATION 1
Mimosine occurrence and accumulation in Mimosa bimucronata var bimucronata (DC) 2
Kuntze 3
Kelly Cristine da Silva Rodrigues-Correcirca1 Lana Dorneles Pedroso2 Fernanda de Costa1 4
Arthur Germano Fett-Neto1 5
1Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal 6
University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970 7
Porto Alegre Rio Grande do Sul Brazil 2Department of Biological Sciences Unipampa ndash 8
Campus Satildeo Gabriel 9
Corresponding author 10
E-mail addresses krodriguescbiotufrgsbr (KCdaS Rodrigues-Correcirca) 11
lanalima2012gmailcom (LD Pedroso) fernandadecostayahoocombr (F de Costa) 12
fettnetocbiotufrgsbr (AG Fett-Neto) 13
14
15
16
17
18
19
20
21
22
67
ABSTRACT 23
Mimosine is a non-protein aromatic amino acid present in plants of Leucaena spp 24
and Mimosa spp Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) is a native 25
tree from Brazil which occurs as a pioneer species on plant succession processes In the 26
current study the presence of mimosine in M bimucronata was verified by HPLC analyses 27
Moreover mimosine accumulation upon exposure to UV-C and chemical elicitors of 28
specialized metabolism (salicylic acid - SA methyl jasmonate - MeJA sodium nitroprusside 29
- SNP and ethephon - ETH) most of which also known as promoters of the amino acid 30
production in leucaena plants was evaluated The results showed a lower concentration of 31
constitutive mimosine present in both maricaacute seedlings and mature trees when compared to 32
leucaena plants In spite of a trend towards increased mimosine accumulation observed in 33
MeJA and ETH treatments no statistical differences were found with the various stressors 34
used to induce its biosynthesis in maricaacute seedlings Data suggest that mimosine in M 35
bimucronata is probably a phytoanticipin-like metabolite or its accumulation is driven by 36
other types of stresses 37
38
39
Keywords Mimosine Mimosa bimucronata stress 40
41
42
43
44
45
46
68
Introduction 47
Mimosa bimucronata commonly known as maricaacute is a native tree from Brazil 48
(REFLORA 2019) ecologically important in plant succession and in processes of degraded 49
land recovery (Bitencourt et al 2007 Silva et al 2011) occurring as a pioneer species 50
(Pilatti et al 2019) Maricaacute is a deciduous or semi-deciduous plant which reaches up to 15 51
m in height and 40 cm of diameter at breast height (DBH) displays shrub or tree habit and 52
bears typical sharp thorns (Carvalho 2004) This species belongs to Fabaceae one of the 53
most economically important families of flowering plants due to its high diversity and 54
occurrence in different types of habitats (Gomes et al 2018) As well as several others 55
Mimosa spp maricaacute is usually referred to as a multipurpose tree (Olkoski and Wittmann 56
2011) employed for alternative medicinal uses (Champanerkar et al 2010 Silva et al 57
2011) honey production constructions and remodeling of landscape architecture (living 58
fences) for instance (Marchiori 1993 Lorenzi 1998) 59
In southern Brazil maricaacute is widely distributed and typically found either in wetland 60
areas close to river banks (Patreze and Cordeiro 2004) or composing large and almost pure 61
landscape formations on hillsides (Jacobi and Ferreira 1991) In dense populations this 62
species like several Mimosa spp (Simon and Proenccedila 2000) is considered an important and 63
highly invasive weed by preventing cattle to reach pasturesand water bodies as a result of its 64
thorny branches (Lorenzi 2008 Kestring et al 2009) Its dominant and nearly exclusive 65
pattern of distribution in those areas has led Jacobi and Ferreira (1991) to test its allelopathic 66
potential on cultivated species Indeed extracts of leaves and ripe fruits (but not the green 67
ones) of maricaacute showed phytotoxic effects on germination and initial radical growth of most 68
of the target species tested 69
69
Several investigations have been performed on maricaacute floristics (Silva et al 2011) 70
distribution (Simon and Proenccedila 2000) wood anatomy (Marchiori 1993) cytogenetic 71
parameters (Olkoski and Wittmann 2011) and allelopathic potential (Jacobi and Ferreira 72
1991 Ferreira et al 1992) However excluding two recent publications on maricaacute 73
constitutive chemical composition (Schlickmann et al 2017 Pilatti et al 2019) which 74
identified phenolic compounds (methyl gallate and water-soluble tannins) as its major 75
compounds little is known regarding this subject In other Mimosa species (eg M pudica 76
and M pigra) mimosine has been identified (Soedarjo and Borthakur 1998) as one of the 77
major specialized metabolites present in the different organs of the plant (Champanerkar et 78
al 2010) The presence of this molecule was also reported for M bimucronata in a thin layer 79
chromatography-based preliminary study performed by Ferreira et al (1992) showing co-80
chromatography of a leaf extract component with authentic mimosine The authors attributed 81
the allelopathic effect of maricaacute to the accumulation of this metabolite in its leaves 82
Mimosine is an aromatic non-protein amino acid initially found in plants of Mimosa 83
pudica and later in Leucaena leucocephala (Lam) de Wit (Soedarjo and Borthakur 1998) a 84
leguminous tree which biosynthesizes large amounts of this nitrogen-containing compound 85
(Rodrigues-Correcirca et al 2019) It is believed that the accumulation of high contents of 86
mimosine in L leucocephala tissues confers among other traits defense against herbivores 87
and pathogens (Vestena et al 2001) tolerance to drought (Negi et al 2014) as well as 88
general oxidative stress protection (Rodrigues-Correcirca et al 2019) Interestingly drought is 89
the opposite environmental and physiological condition to that observed in the wet habitats 90
occupied by native populations of M bimucronata in Brazil (Patreze and Cordeiro 2004 91
Kestring et al 2009) and Mimosa pudica Linn in India (Champanerkar et al 2010) 92
70
Nonetheless flooding is also associated with oxidative stress particularly as water levels 93
change (Fukao et al 2019) 94
In Leucaena leucocephala var leucocephala (common leucaena) and Leucaena 95
leucocephala var glabrata (giant leucaena) mimosine accumulation has been shown to be 96
both constitutive and inducible by stress-related phytohormones such as jasmonic acid (JA) 97
Ethephon (ETH an ethylene- releasing compound) salicylic acid (SA - only common 98
leucaena) (Vestena et al 2001) as well as by UV-C radiation (Xu et al 2018 Rodrigues-99
Correcirca et al 2019) On the other hand there is a lack of information regarding mimosine 100
content and elicitation effects in Mimosa spp plants 101
The aim of this study was to examine the presence of mimosine in Mimosa 102
bimucronata and examine the effects of stresses and stress-signaling molecules on its 103
accumulation in leaves 104
Material and Methods 105
Plant material 106
For all experiments the plant material was collected at Morro Santana campus do 107
Vale of UFRGS (Federal University of Rio Grande do Sul) Porto Alegre RS Brazil 108
(3004rsquoS 5108rsquoW) Authorization for access to genetic material was obtained from 109
SISGEN-Brazil (license number A845493) Constitutive mimosine content in adult plants of 110
M bimucronata var bimucronata (DC) Kuntze was determined in plant material (leaves 111
green flower buds post-anthesis flowers and green pods) harvested in January 2017 112
(summer) A voucher herbarium specimen (ICN 187953) was deposited in the ICN ndash UFRGS 113
herbarium (Herbaacuterio do Instituto de Biociecircncias of UFRGS) 114
71
For mimosine elicitation experiments legumes (fruits) of maricaacute were collected in 115
the end of June 2017 (winter) Seeds were then removed from the dry fruits and kept in the 116
dark until sowing and seedling development for use in the assays 117
Seed germination 118
To break the coat-imposed seed dormancy after surface sterilization dry seeds of 119
maricaacute were acid scarified by immersion in H2SO4 (95 ndash 98 ) for 2 min (see Correcirca et al 120
2008) and repeatedly washed in distilled water to remove any residue of the acid Then seeds 121
were distributed in 50 mL individual plastic tubes (dibble-tubes) (30 cm diameter x 120 cm 122
depth) filled up with 11 (vv) of commercial top soil and vermiculite Tubes were watered 123
every 2 days to avoid substrate dryness and were kept in a growth room under controlled 124
conditions of light (circa 75 μmol mminus2s minus1 photosynthetically active radiation photoperiod 125
of 16 h light and 8 h dark) and temperature (24plusmn2C) 126
127
Treatments 128
In order to verify inducibility of mimosine accumulation in M bimucronata fifty 12-129
week-old maricaacute seedlings (per treatment) exhibiting similar features were selected and 130
sprayed (saturated) with solutions of different chemical stressors (plant specialized 131
metabolism elicitors) as follows (for further details see Rodrigues-Correcirca et al 2019) 10 132
and 50 mM SA (pathogen-signaling molecule Shah 2003) 007 and 035 mM 2-133
chloroethylphosphonic acid (ETH ethylene releasing-compound Kim et al 2016 Wang et 134
al 2016) 100 and 200 mM MeJA (Dar et al 2015) 10 and 50 mM SNP (a nitric oxide 135
donor Perotti et al 2015) Alternatively maricaacute seedlings were also supplemented with UV-136
C radiation (13 minutes 105 kJ cm2) (elicitor of plant specialized metabolism Kara 2013) 137
72
After 2 and 4 days of exposure to the chemical treatments and 3 and 6 days of UV-138
C supplementation maricaacute shoots were harvested immediately frozen in liquid nitrogen and 139
stored at ndash 80 C until mimosine extraction and HPLC analyses 140
Mimosine extraction and detection 141
Mimosine extraction was conducted according to the modified protocol described by 142
Rodrigues-Correcirca et al (2019) for L leucocephala HPLC (Thermo Scientific Surveyor) 143
analyses (mimosine detection and quantification) were performed following previously 144
published procedures (Negi et al 2014) A C18 column (ACE C18 5 μm 46times250 mm) and 145
isocratic solvent system of 002M o-phosphoric acid with a linear flow rate of 1 mL min minus1 146
were used to separate and quantify the amino acid Mimosine detection was performed at 280 147
nm by photodiode array detection (200ndash400 nm) and retention time (229plusmn0024 min) 148
Mimosine quantification was done by means of the method of external standard curve 149
Additional confirmation of mimosine identity was performed by co-chromatography with 150
standard (Acros Organics authentic mimosine 99 used as reference) and peak purity check 151
The analyses of the chromatograms were done with the ChromQuest software 152
153
154
Results and Discussion 155
Constitutive accumulation of mimosine in M bimucronata 156
Mimosine was detected in all analyzed samples positively meeting all identification 157
criteria In agreement with what has been found for other Mimosa spp (Soedarjo and 158
Borthakur 1998) compared to L leucocephala adult plants (Rodrigues-Correcirca 2019) 159
mimosine content was lower in M bimucronata Of the adult plant tissues analyzed the 160
73
highest content of mimosine in maricaacute (per gram of fresh weight - FW) was found in post-161
anthesis flowers (36644 microg versus 89448 microg in common leucaena followed by leaves 162
(28838 microg x 67358 microg) green flower buds (28094 microg x 51247 microg) and green pods (19002 163
microg x 82687 microg) (Fig 1)The same pattern is observed for seedlings when both species are 164
compared In this study untreated 12-week-old maricaacute seedlings (control at day 2) showed a 165
shoot content of mimosine of 23029plusmn007 microg g-1 of (FW) Five-week-old untreated giant 166
leucaena seedlings cultivated in similar conditions exhibited between 83640 and 178736 167
microg g-1 of FW (Rodrigues-Correcirca et al 2019) In the same way mimosine concentration 168
percentage in dry matter of Mimosa pigra was found to be rather low (002 in nodules and 169
roots and 007 in leaves) (Soedarjo and Borthakur 1998) 170
In this investigation the lowest constitutive mimosine content was found in green 171
pods (Fig 1) This result may partly explain the absence of phytotoxic effect observed for 172
green pods on germination and growth of crop target plants tested by Jacobi and Ferreira 173
(1991) compared to the other maricaacute parts analyzed 174
Elicitation of mimosine biosynthesis in M bimucronata 175
Chemical stressors 176
Secondary metabolites (or natural products) are structural- and chemically 177
specialized compounds derived from primary metabolism These molecules are mainly 178
biosynthesized as part of a complex defense mechanism in response to biotic and abiotic 179
stresses such as pathogens herbivores water status metal toxicity and UV radiation for 180
example (Matsuura et al 2018) Ethephon SA SNP MeJA have been extensively used as 181
chemical elicitors of specialized metabolism (Wang et al 2016 Vestena et al 2001 Perotti 182
74
et al 2015 Zhang and Memelink 2009 Xu et al 2018) These phytohormonal signals can 183
simulate environmental challenges and modulate plant homeostasis often leading to 184
alterations in gene expression (Shinozaki et al 2015) Except SNP all treatments tested in 185
the present study showed positive effect on mimosine accumulation in common or giant 186
leucaena (Vestena et al 2001 Rodrigues-Correcirca 2019 Rodrigues-Correcirca unpublished 187
data) However in spite of the trend of increasing the mimosine content observed in seedlings 188
treated with 007 mM Ethephon (at day 2) and 100 mM MeJA (at day 4) no statistical 189
difference was confirmed for these treatments when compared to the control 190
On the other hand a within treatment difference on mimosine induction was seen 191
between day 2 and 4 in seedlings treated with 100 mM MeJA (Fig 2) In a lower 192
concentration (04 mM) jasmonic acid (JA)promoted a near threefold increase in mimosine 193
accumulation of giant leucaena seedlings after 2 days of application 194
UV-C radiation 195
Albeit UV-C radiation is not biologically active in natural environments it has been 196
widely used under controlled experimental conditions to generate acute responses of plant 197
specialized metabolism within a shorter period of time compared to that required to with UV-198
B radiation (Kara 2013 Cetin 2014) This fast response is due to the higher energy of UV-199
C photons that act as potent reactive oxygen species (ROS) generators causing extensive 200
damage to the cells either at the physiological level or on DNA structure (Gregianini et al 201
2003 Matsuura et al 2013) 202
Although divergent responses can be observed in plants exposed to UV-C radiation 203
the deleterious processes are usually reported on primary metabolism (decreasing of 204
chlorophyll content and plant height eg) (Kara 2013) In the present study no statistical 205
75
differences were observed in the mimosine concentration in maricaacute seedlings supplemented 206
with UV-C radiation However a decreasing in its content was found for both control and 207
treatment at day 6 post-treatment (Fig 03) Taking into account the lower constitutive 208
concentration of mimosine observed in maricaacute compared to the leucaena plants besides its 209
relative thermolability (Nguyen and Tawata 2016) it seems to be plausible to consider the 210
effect of the temperature inside the UV-C and the white light (control) chambers as an 211
additional abiotic factor contributing to the decrease of mimosine accumulation in both group 212
of plants 213
Besides mimosine identification the presence of 34-dihydroxypyridine (34-DHP or 214
3-hydroxy-4-pyridone - 3H4P) a mimosine degradation product (Negi et al 2014 Nguyen 215
and Tawata 2016) was also reported for maricaacute leaf extracts analyzed by TLC by Ferreira 216
et al (1992) In our chromatograms we detected a second large peak after that of mimosine 217
(229plusmn0024) and similar to that identified by Negi et al (2014) as 3H4P (data not shown) 218
Comparing the chromatogram profiles obtained from seedlings elicited with chemical 219
stressors and those supplemented with UV-C the largest area for this peak was found (in all 220
samples) in the latter treatment at day 6 It might indicate that the constitutive andor the 221
initially UV-C-induced mimosine was degraded into 3H4P to cope with the cellular damage 222
caused by this treatment associated with an increased temperature inside the chambers 223
Nevertheless it was not possible to determine 3H4P concentration (or confirm its identity) 224
in maricaacute plants since there is no commercial standard (pure 3H4P) available for purchase 225
to be used as a reference in calculations Establishment of improved protocols for obtaining 226
in house 3H4P reference substance by acid hydrolysis is ongoing 227
228
229
76
Conclusion 230
On the basis of the overall absence of effect of the treatments tested here on mimosine 231
concentration it is possible to suggest that its accumulation profile is similar to that of 232
phytoanticipins unlike what is observed for the same amino acid production in leucaena 233
which shows features of inducibility resembling phytoalexin-like metabolites Alternatively 234
a putative inducible pool of mimosine in maricaacute might be involved in other types of stress 235
such as extended drought periods If involved in protection against oxidative stress as 236
described for leucaena mimosine in maricaacute may act predominantly by physical quenching 237
of ROS as indicated by the lack of overt chemical degradation Nevertheless further 238
investigations are needed to assess these hypotheses 239
To sum up mimosine biosynthesis was not modulated by the treatments evaluated as 240
in L leucocephala (Lam) de Wit To the best of our knowledge this is the first work that 241
analytically identifies and quantifies mimosine accumulation in M bimucronata 242
243
REFERENCES 244
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Carvalho PER 2004 Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 248
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Jacobi US Ferreira AG 1991 Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK 272
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Kestring D Klein J Menezes LCCR Rossi MN 2009 Imbibition phases and 276
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Ethephon as an ethylene-releasing compound on the metabolic profile of Chlorella vulgaris 280
J Agric Food Chem 64(23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541 281
Lorenzi H 1998 Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 282
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 283
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metabolismcharacteristicsandimpactontargetmoleculebiotechnologicalproduction 294
Molecular Biotechnology 60(2) 169ndash183httpsdoiorg101007s12033-017-0056-1 295
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase from 296
Leucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 297
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Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine 299
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Olkoski D Wittmann MTS 2011 Cytogenetics of Mimosa bimucronata (DC) O Kuntze 301
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Appl Biotechnol 11 27-35 httpdxdoiorg101590S1984-70332011000100004 303
Patreze CM Cordeiro L 2004 Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 304
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285 httpdxdoiorg101016jforeco200403034 306
Perotti JC Rodrigues-Correcirca KCS Fett-Neto AG 2015 Control of resin production in 307
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Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG 2019 Mimosine 309
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UV exposure Plant Physiology and Biochemistry 135 432ndash440 311
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Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS 2011 Biologia da reproduccedilatildeo 315
deMimosa bimucronatandash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 316
Simon MF and Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 317
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 318
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Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 320
LM Andrade SF Cechinel-Filho V 2017 Chemical composition and diuretic natriuretic and 321
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kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its majority 322
constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash1624 323
Shah J 2003 The salicylic acid loop in plant defense Current Opinion Plant Biology6 (4) 324
365ndash371 325
Shinozaki K Uemura M Serres JB Bray EA Weretilnyk E 2015 Responses to Abiotic 326
Stress In Buchanan BB Gruissem W Jones RL (Eds) Biochemistry and Molecular 327
Biology of Plants Second Edition John Wiley and Sons Ltd 328
Soedarjo M and Borthakur D 1998 Mimosine a toxin produced by the tree-legume 329
Leucaena provides a nodulation competition advantage to mimosine-degrading Rhizobium 330
strains Soil Biology and Biochemistry 30(12)1605-1613 331
Vestena S Fett-Neto AG Duarte RC Ferreira AG 2001 Regulation of mimosine 332
accumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 333
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene induced 334
vinblastine accumulation is related to activated expression of downstream TIA pathway 335
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Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX Tsim KWK 2018 337
Jasmonate-elicited stress induces metabolic change in the leaves of Leucaena leucocephala 338
Molecules 23 (2) 339
Zhang H Memelink J 2009 Regulation of Secondary Metabolism by Jasmonate Hormones 340
In AE Osbourn and V Lanzotti (eds) Plant-derived Natural Products 3 DOI 101007978-341
0-387-85498-4_1 copy Springer Science + Business Media LLC 342
343
344
345
81
346
Figure 1 Constitutive concentration of mimosine in different plant organs of Mimosa 347
bimucronata Bars sharing the same letter do not differ statistically by Tukey test (Ple005) 348
The error bars denote standard error of 10 replicates 349
350
351
352
353
354
355
356
357
B B A C0
5
10
15
20
25
30
35
40
LEAVES GREEN FLOWER BUDS POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
co
nce
ntr
atio
n u
gg-1
Mimosine concentration in adult plants of Mimosa bimucronata (DC) Kuntze
82
C T R L S A
1 0 m M
S A
5 0 m M
E T H
0 0 7 m M
E T H
0 3 5 m M
M e J A
1 0 0 m M
M e J A
2 0 0 m M
S N P
1 0 m M
S N P
5 0 m M
0
1 0
2 0
3 0
T re a tm e n ts
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1) D A Y 2
D A Y 4
A B C C B C A B C C A B C A B C A
a b b b a a b a a b b a b
358
Figure 2 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 359
bimucronata treated with different signaling molecules SA = Salicylic Acid ETH = 360
Ethephon MeJA = Methyl Jasmonate SNP = Sodium Nitroprusside Uppercase and 361
lowercase letters indicate statistical differences among treatments in days 2 and 4 362
respectively Bars sharing a letter of the same case do not differ statistically by Tukey test 363
(Ple005) Indicates statistical difference in the same treatment between day 2 and 4 by t-364
test (Ple005) The error bars denote standard error of 5 replicates (25 individual seedlings 365
arranged in 5 groups of 5) 366
367
368
83
D AY 3 D AY 6
0
5
1 0
1 5
2 0
2 5
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1)
C O N TR O L
U V -C
369
Figure 3 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 370
bimucronata supplemented with UV-C radiation Indicates statistical difference in the same 371
treatment between day 3 and 6 by t-test (Ple005) The error bars denote standard error of 5 372
replicates (25 individual seedlings arranged in 5 groups of 5) 373
374
375
376
377
378
379
380
381
382
383
384
385
84
Consideraccedilotildees finais 386
- Experimentos que avaliam os efeitos da aplicaccedilatildeo exoacutegena de ANPs em diferentes espeacutecies 387
vegetais tecircm sido realizados principalmente com GABA Dentre os principais efeitos 388
conferidos pela aplicaccedilatildeo dessa moleacutecula em espeacutecies de mono e eudicotiledocircneas satildeo 389
relatados a toleracircncia agrave seca agrave salinidade e agraves temperaturas extremas 390
- Como metaboacutelitos especializados claacutessicos os ANPs podem ter sua concentraccedilatildeo basal 391
endoacutegena aumentada em resposta agrave induccedilatildeo mediada por uma vasta gama de tratamentos com 392
moleacuteculas sinalizadoras de estresse e fontes alternativas de estressores De um modo geral 393
observa-se o acuacutemulo das diferentes classes de ANPs em resposta agrave radiaccedilatildeo UV elicitores 394
quiacutemicos que mimetizam ataques por patoacutegenos dano mecacircnico agentes osmoacuteticos metais 395
pesados entre outros 396
- Especificamente em leucena a resposta observada em relaccedilatildeo aos diferentes tratamentos 397
testados indica que apesar do seu alto teor constitutivo nessa espeacutecie a biossiacutentese e o 398
acuacutemulo de mimosina podem ser modulados por fatores causadores de estresses exibindo -399
nessa espeacutecie - um padratildeo de acumulaccedilatildeo similar agrave fitoalexinas Em maricaacute por outro lado 400
aumento de acuacutemulo dessa moleacutecula natildeo foi observado para os mesmos tratamentos testados 401
para leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas 402
- O padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno 403
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado pela 404
sua degradaccedilatildeo 405
- As respostas observadas nos testes que avaliaram a atividade de mitigaccedilatildeo de espeacutecies 406
reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode agir como um agente 407
antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de estresse 408
85
Perspectivas 409
- Confirmaccedilatildeo em espectrocircmetro de massas eou ressonacircncia nuclear magneacutetica da natureza 410
quiacutemica da lsquomimosinarsquo presente em maricaacute 411
- Avaliaccedilatildeo do efeito de concentraccedilotildees mais elevadas e em diferentes periacuteodos de aplicaccedilatildeo 412
das moleacuteculas sinalizadoras testadas sobre o acuacutemulo de mimosina em leucena e maricaacute 413
- Ampliar a investigaccedilatildeo dos padrotildees de expressatildeo gecircnica dos genes que codificam para 414
mimosinase (em maricaacute) mimosina sintase (em ambas as espeacutecies testadas) bem como o 415
perfil de precursores e cataboacutelitos de mimosina em resposta aos tratamentos mencionados 416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
86
Referecircncias Bibliograacuteficas 436
437
Acamovic T Brooker JD (2005) Biochemistry of plant secondary metabolites and their 438
effects in animals P Nutr Soc 64 403ndash412 httpsdoiorg101079PNS2005449 439
Ahmed R Hoque ATMR Hossain MK (2008) Allelopathic effects of Leucaena 440
leucocephala leaf litter on some forest and agricultural crops grown in nursery J Forestry 441
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Ahmed AMM Saacutenchez FJS Bavileacutes LRY Mahdy REZ Camaal JBC (2016) Tannins and 443
mimosine in Leucaena genotypes and their relations to Leucaena resistance against 444
Leucaena Psyllid and Onion thrips Agroforestry Systems 1-8 445
Benjakul S Kittiphattanabawon P Shahidi F Maqsood S (2013) Antioxidant activity and 446
inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipid oxidation in 447
model systems Food Sci Technol Int 19(4)365-76 448
httpsdoiorg1011771082013212455186 449
Bitencourt F Zocche JJ Costa S Souza PZ Mendes AR (2007) Nucleaccedilatildeo de Mimosa 450
bimucronata (DC) O Kuntze em aacutereas degradadas pela mineraccedilatildeo de carvatildeo Revista 451
Brasileira de Biociecircncias 5 750-752 452
Bottini-Luzardo M Aguilar-Perez C Centurion-Castro F Solorio-Sanchez F Ayala-Burgos 453
A Montes-Perez R Muntildeoz-Rodriguez D Ku-Vera J (2015) Ovarian activity and estrus 454
behavior in early postpartum cows grazing Leucaena leucocephala in the tropics Trop Anim 455
Health Prod 47(8)1481-6 456
Carvalho PER (2004) Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 457
Teacutecnica 941-10 458
Chowtivannakul P Srichaikul B Talubmook C (2016) Antidiabetic and antioxidant activities 459
of seed extract from Leucaena leucocephala (Lam) de Wit Agriculture and Natural 460
Resources 50 (2016) 357e361 httpdxdoiorg101016janres201606007 461
Chung H-H Chen M-K Chang Y-C Yang S-F Lin C-C Lin C-W (2017) Inhibitory effects 462
of Leucaena leucocephala on the metastasis and invasion of human oral cancer cells 463
Environmental Toxicology 321765ndash1774 httpsdoiorg101002tox22399 464
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Crowe B Poynter JA Manukyan MC Wang Y Brewster BD Herrmann JL Abarbanell 465
AM Weil BR Meldrum DR (2001) Pretreatment with intracoronary mimosine improves 466
postischemic myocardial functional recovery Surgery 150(2) 191-106 467
Fallon (2015) Effects of mimosine on Wolbachia in mosquito cells cell cycle suppression 468
reduces bacterial abundance In Vitro Cell Dev Biol Anim 51(9)958-63 469
httpsdoiorg101007s11626-015-9918-7 Epub 2015 May 28 470
Fernaacutendez-Salas A Alonso-Diacuteaza MA Acosta-Rodriacuteguez A Torres-Acosta JFJ Sandoval-471
Castro CA Rodriacuteguez-Vivas RI (2011) In vitro acaricidal effect of tannin-rich plants against 472
the cattle tick Rhipicephalus (Boophilus) microplus (Acari Ixodidae) Veterinary 473
Parasitology 175113ndash118 2010 httpsdoiorg101016jvetpar201009016 474
Ferreira AG Aquila MEA Jacobi US Rizvi V (1992) Allelopathy in Brazil In Allelopathy 475
basic and applied aspects Rizvi V and Jacobi US (Eds) Chapman and Hall PP 243-250 476
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad Hossain Md Anai 477
T Oku H (2018) Cytosolic cysteine synthase switch cysteine and mimosine production in 478
Leucaena leucocephala Appl Biochem Biotechnol 186 (3) 613ndash632 479
httpsdoiorg101007s12010-018-2745-z 480
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of the thyrotoxic 481
amino acid mimosine by cysteine synthase Phytochemistry 29 (11) 3461ndash3465 482
httpsdoiorg1010160031-9422(90)85258-H 483
Jacobi US Ferreira AG (1991) Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK Sobre 484
espeacutecies cultivadas Pesquisa Agropecuaacuteria Brasileira 26(7) 935-943 485
Jamous RM Ali-Shtayeh MS Abu-Zaitoun SY Markovics A Azaizeh H (2017) Effects of 486
selected Palestinian plants on the in vitro exsheathment of the third stage larvae of 487
gastrointestinal nematodes BMC Veterinary Research 13308 488
httpdxdoiorg101186s12917-017-1237-7 489
Jiao CJ Jiang J-L Ke L-M Cheng W Li F-M Li Z-X Wang C-Y (2011) Factors affecting 490
β-ODAP content in Lathyrus sativus and their possible physiological mechanisms Food 491
Chem Toxicol 49 543ndash549 httpsdoiorg101016jfct201004050 492
Kubota S Fukumoto Y Ishibashi K Soeda S Kubota SS Yuki R Nakayama Y Aoyama K 493
Yamaguchi N (2014) Activation of the prereplication complex is blocked by mimosine 494
88
through reactive oxygen species-activated ataxia telangiectasia mutated (ATM) protein 495
without DNA damage J Biol Chem 28 289(9)5730-46 496
Kuppusamy UR Arumugam B Azaman N Wai CJ (2014) Leucaena leucocephala Fruit 497
Aqueous Extract Stimulates Adipogenesis Lipolysis and Glucose Uptake in Primary Rat 498
Adipocytes Hindawi Publishing Corporation e Scientific World Journal Article ID 737263 499
8 pages httpdxdoiorg1011552014737263 500
Kusama-Eguchi K (2019) Research in motor neuron diseases caused by natural substances 501
focus on pathological mechanisms of neurolathyrism Yakugaku Zasshi 139 (4) 609-502
615 httpsdoiorg101248yakushi18-00202 503
Kutchan TM Gershenzon J Moslashller BL Gang DR (2015) Natural Products In Buchanan 504
BB Gruissem W and Jones RL (eds) Biochemistry amp Molecular Biology of Plants 2nd edn 505
Wiley Blackwell Chichester pp 1135-1205 506
Lalande M (1990) A reversible arrest point in the late G1 phase of the mammalian cell cycle 507
Exp Cell Res 186 332ndash339 508
Li X-W Hu C-P Li Y-J Gao Y-X Wang XM Yang J-R (2015) Inhibitory effect of L-509
mimosine on bleomycin-induced pulmonary fibrosis in rats Role of eIF3a and p27 Int 510
Immunopharmacol 27(1) 53ndash64 511
Little Jr EL Skolmen RG (1989) Koa haole Agriculture Handbook 679 USDA 512
Lorenzi H (1998) Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 513
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 514
Marchiori JNC (1993) Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 515
O Kuntze Ciecircncia Florestal 3 85-106 516
Mohammed RS El Souda SS Taie HAA Moharam ME Shaker KH (2015) Antioxidant 517
antimicrobial activities of flavonoids glycoside from Leucaena leucocephala leaves Journal 518
of Applied Pharmaceutical Science 5(06)138-147 519
httpdxdoiorg107324JAPS201550623 520
Negi VS Bingham J-P Li QX Borthakur D (2014) A carbon-nitrogen lyase from Leucaena 521
leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 (2) 922ndash522
934 httpsdoiorg101104pp113230870 523
89
Olkoski D Wittmann MTS (2011) Cytogenetics of Mimosa bimucronata (DC) O Kuntze 524
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop 525
Breeding and Applied Biotechnology 11 27-35 526
Patreze CM Cordeiro L (2004) Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 527
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecology and Management 528
196275ndash285 529
Pilatti DM Fortes AMT Jorge TCM Boiago NP (2019) Comparison of the phytochemical 530
profiles of five native plant species in two different forest formations Brazilian Journal of 531
Biology 79(2) 233-242 532
Ramos-Ruiz R Poirot E Flores-Mosquera M (2018) GABA a non-protein amino acid 533
ubiquitous in food matrices Cogent Food Agric 41534323 534
httpsdoiorg1010802331193220181534323 535
REFLORA (2019) httpfloradobrasiljbrjgovbrreflora Acesso em agosto de 2019 536
Rodgers KJ Samardzic K Main BJ (2015) Toxic Nonprotein Amino Acids Plant Toxins 537
httpsdoiorg 101007978-94-007-6728-7_9-1 538
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG (2019) Mimosine 539
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 540
UV exposure Plant Physiology and Biochemistry 135 432ndash440 541
httpsdoiorg101016jplaphy201811018 542
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 543
LM Andrade SF Cechinel-Filho V (2017) Chemical composition and diuretic natriuretic 544
and kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its 545
majority constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash546
1624 547
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS (2011) Biologia da reproduccedilatildeo 548
de Mimosa bimucronata ndash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 549
Simon MF Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 550
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 551
endemism Biological Conservation 96 279-296 552
90
Soares AMS Arauacutejo SA Lopes SG Costa Junior LM (2015) Anthelmintic activity of 553
Leucaena leucocephala protein extracts on Haemonchus contortus Braz J Vet Parasitol 554
Jaboticabal 24(4) 396-401 httpdxdoiorg101590S1984-29612015072 555
Soerdajo M Borthakur D (1998) Mimosine a toxin produced by the tree-legume Leucaena 556
provides a nodulation competition advantage to mimosine-degrading Rhizobium strains Soil 557
Biol Biochem 30(12) 16051613 558
Souza-Lima ES Sinani TR Pott A Sartori ALB (2017) Mimosoideae (Leguminosae) in the 559
Brazilian Chaco of Porto Murtinho Mato Grosso do Sul Rodrigueacutesia 68(1) 263-290 2017 560
httpdxdoiorg1015902175-7860201768131 561
Taiz L amp Zeiger E (2010) Plant Physiology 5th edition Sinauer Associates Inc Sunderland 562
Verma VK Rani KV Kumara SR Prakash O (2018) Leucaena leucocephala pod seed 563
protein as an alternate to animal protein in fish feed and evaluation of its role to fight against 564
infection caused by Vibrio harveyi and Pseudomonas aeruginosa Fish and Shellfish 565
Immunology 76 (2018) 324ndash332 httpsdoiorg101016jfsi201803011 566
Yafuso JT Negi VS Bingham J-P Borthakur D (2014) An O-acetylserine (thiol) lyase from 567
Leucaena leucocephala is a cysteine synthase but not a mimosine synthase Appl Biochem 568
Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z 569
Zarin RMA Wan HY Isha A Armani N (2016) Antioxidant antimicrobial and cytotoxic 570
potential of condensed tannins from Leucaena leucocephala hybrid Food Science and 571
Human Wellness 5 65ndash75 httpdxdoiorg101016jfshw201602001 572
573
574
Contents lists available at ScienceDirect
Industrial Crops amp Productsjournal homepage wwwelseviercomlocateindcrop
Resin tapping transcriptome in adult slash pine (Pinus elliottii var elliottii)Camila Fernanda de Oliveira Junkes1 Artur Teixeira de Arauacutejo Juacutenior1 Juacutelio Ceacutesar de LimaFernanda de Costa Thanise Fuumlller Maacutercia Rodrigues de Almeida Franciele Antocircnia NeisKelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-NetoCenter for Biotechnology and Department of Botany Federal University of Rio Grande do Sul Porto Alegre PO Box 15005 91501-970 Brazil
A R T I C L E I N F O
KeywordsPinus elliottiResinResinosisTranscriptomeAdjuvant paste
A B S T R A C T
To better understand the bases of resin production a major source of terpenes for industry the transcriptome ofadult Pinus elliottii var elliottii (slash pine) trees under field commercial resinosis was obtained Samples werecollected from cambium after 5 and 15 days of treatment application which included tapping followed byapplication of commercial resin stimulant paste or control wounding without paste Overall mean number ofreads of all 16 libraries (2 treatments x 2 times x 4 replicated trees) was 34582048 Of these 89 were mappedagainst the reference sequence with a mismatch of 058 Using the Blast2Go 570 candidate genes were de-tected based on sequence annotation By comparing the expression profile between paste and control 310differentially expressed genes (DEGs) were identified at 5 days and 190 at 15 days with a significant fold changeof log2gt 12 Regarding changes in time comparisons within each treatment 210 and 105 DEGs were identifiedwithin control and paste treatment respectively Genes with different expression patterns in the times andtreatments examined included ethylene responsive transcription factors geranylgeranyl diphosphate synthasediterpene synthase cytochrome P450 and ABC transporters all of which may play important roles in resinproduction RT-qPCR analysis correlated well with the data obtained by RNAseq Resin composition changedover time This is the first transcriptomic investigation of resinosis of the main species used in the bioresinindustry and of molecular analyses of resinosis under field operations with implications for stand managementstimulant paste development genotype selection and breeding for high resinosis
1 Introduction
The adaptive success of conifers is largely due to the development ofa defense system based on the synthesis and secretion of terpenes in allmajor organs and different tissues (Miller et al 2005 Hall et al 2013Warren et al 2015) Conifer resin is a viscous fluid composed of acomplex mixture of terpenoids such as monoterpenes sesquiterpenesand diterpenes (Zulak and Bohlmann 2010) These terpenoids are se-creted from severed resin ducts when the tree is under biotic attack(Ralph et al 2006 Lange 2015 Geisler et al 2016) acting as pro-tectants (Schiebe et al 2012 Liu et al 2015)Biosynthesis of terpenes in conifers starts from isomerization of two
isoprenoid (C5) units dimethylallyl diphosphate (DMAPP) and iso-pentenyl diphosphate (IPP) These molecules can be biosynthesized viatwo separate routes in plants the methyl-erythritol 4-phosphate andmevalonate pathways IPP is synthesized and isomerized to DMAPP byisopentenyl diphosphate isomerase then prenyl transferases catalyze
the condensation of these two C5-units to geranyl diphosphate (Pazoukiand Niinemets 2016) Their elongation to prenyl diphosphates withaddition of IPP molecules leads to monoterpenes (C10) sesquiterpenes(C15) and diterpenes (C20) which are the substrates for terpene syn-thases (TPS) (Keeling and Bohlmann 2006b)TPSs are part of a large family of mechanistically related enzymes
involved in both primary and secondary metabolism (Keeling andBohlmann 2006b) The events of evolutionary diversification and ex-pansion of plant TPSs appear to have originated from gene duplicationsdomain losses and sub- or neofunctionalizations with subsequent di-vergence of an ancestral TPS gene of primary metabolism (Hall et al2013) Modification of TPS products changes their physical propertiesand may alter their biological activities (Chen et al 2011) TPSs of highsequence identity may have different functions even in closely relatedspecies Low sequence identity of TPSs in phylogenetically distantspecies does not preclude the possibility of independent evolution of thesame or related function of these enzymes (Zerbe and Bohlmann 2015)
httpsdoiorg101016jindcrop2019111545Received 4 January 2019 Received in revised form 10 June 2019 Accepted 4 July 2019
Corresponding authorE-mail address fettnetocbiotufrgsbr (AG Fett-Neto)1 These authors have equally contributed to this work
doi 1015900102-33062019abb0114
Acta Botanica Brasilica
Sustainable production of bioactive alkaloids in Psychotria L of
southern Brazil propagation and elicitation strategies
Yve Verocircnica da Silva Magedans1 Kelly Cristine da Silva Rodrigues-Correcirca1 Cibele Tesser da Costa1
Heacutelio Nitta Matsuura1 and Arthur Germano Fett-Neto1
Received April 1 2019Accepted June 28 2019
ABSTRACTPsychotria is the largest genus in Rubiaceae South American species of the genus are promising sources of natural
products mostly due to bioactive monoterpene indole alkaloids they accumulate ese alkaloids can have analgesic
antimutagenic and antioxidant activities in dierent experimental models among other pharmacological properties
of interest Propagation of genotypes with relevant pharmaceutical interest is important for obtaining natural
products in a sustainable and standardized fashion Besides the clonal propagation of elite individuals the alkaloid
content of Psychotria spp can also be increased by applying moderate stressors or stress-signaling molecules is
review explores advances in research on methods for plant propagation and elicitation techniques for obtaining
bioactive alkaloids from Psychotria spp of the South Region of Brazil
Keywords abiotic stress alkaloids elicitation monoterpenes plant propagation Psychotria southern Brazil
sustainability
Introduction
Psychotria belongs to Rubiaceae one of the major families
of $owering plants having economic interest e family
includes coee a few signicant poisonous plants to livestock
besides several important ornamental and medicinal species
(Souza amp Lorenzi 2012) Psychotria has captured researchersrsquo
attention mostly because of its medicinal properties
Psychotria colorata is an Amazonian species that produces
polyindolinic alkaloids with analgesic activity (Matsuura et
al 2013) e promising results obtained with P colorata
motivated the investigation of southern Brazilian Psychotria
species and the discovery of new bioactive alkaloids (Porto
et al 2009) Moreover leads on in planta alkaloid functions
were also topic of experimental evaluation
One of the key elements that needs to be addressed early
on during the process of developing new bioactive molecules
from plants is the capacity to generate catalytically active
biomass to support extraction and steady supply ere are a
number of ways through which these goals may be reached
including greenhouse rooting of cuttings (mini-cutting
1 Laboratoacuterio de Fisiologia Vegetal Departamento de Botacircnica Instituto de Biociecircncias e Centro de Biotecnologia Universidade Federal do Rio
Grande do Sul 91501-970 Porto Alegre RS Brazil
Corresponding author fettnetocbiotufrgsbr
Review
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Biomass yield of resin in adult Pinus elliottii Engelm trees is differentially
regulated by environmental factors and biochemical effectors
Franciele Antocircnia Neis Fernanda de Costa Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima
Kelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-Neto
Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) CP 15005 CEP 91501-970 Porto Alegre RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Biomass
Terpene resin
Seasonal
Benzoic acid
Regenerated forest
A B S T R A C T
Biomass of pine resin finds several applications in the chemical pharmaceutical biofuel and food industries
Resin exudation after injury is a key defense response in Pinaceae since this complex mixture of terpenes has
insecticidal antimicrobial and wound repair properties Resin yield is increased by effectors applied on the
wound area including phytohormones and metal cofactors of terpene synthases The interaction of resinosis
mechanism effectors is not fully understood particularly in adult forest setups under natural environmental
variations The aim of this work was to determine how resin exudation by wounded trunks of adult P elliottii
responded to combined chemical effectors involved in different regulatory pathways of resinosis (metal cofactors
of terpene synthases benzoic acid and plant growth regulators) and whether seasonal and tree distribution
variations affected these responses Symmetrically planted and scattered trees regenerated from the seed bank
had similar resin biomass yields suggesting that the homogeneity in development and spatial arrangement were
not significant factors in resin yield This new finding is of practical importance with the used tapping system
since costs of implanting forests by regeneration can be advantageous compared to planting In addition it was
shown for the first time that the salicylic acid precursor benzoic acid and the auxin naphthalene acetic acid
promoted resin exudation when individually applied to wound sites Both these adjuvants are two orders of
magnitude less costly compared to the conventionally used ethylene precursors besides facing less environ-
mental and health restrictions for use Most adjuvant-treated trees showed higher resin flow in the second year
indicating mechanisms of response build up Overall temperature was more important than rainfall as en-
vironmental parameter affecting resin biosynthesis which was higher in the warmer months of spring and
summer The combination of resinosis stimulant effectors from different signaling pathways showed no sig-
nificant synergistic or additive effect suggesting possible converging signaling pathways andor limitation of
common intermediate transducing molecules
1 Introduction
Pines occupy highly diverse environments over a range of tem-
peratures water and nutrient availabilities irradiance levels and pho-
toperiods being able to effectively face attacks from diverse herbivore
and pathogen guilds The success of conifers is linked to their complex
terpene biochemistry hosted by specialized secretory cells The terpe-
noid resin synthesized by Pinus spp is one of the main mechanisms of
defense of these trees particularly against bark beetles and the fungi
they carry (Fett-Neto and Rodrigues-Correcirca 2012) Pine resin biomass
is essentially composed of a monoterpene and sesquiterpene-rich tur-
pentine and diterpenoid-rich rosin fraction both finding numerous in-
dustrial applications as non-wood forest products (Rodrigues-Correcirca
et al 2012)
Molecules capable of modulating different signaling pathways have
been identified as resin yield stimulators including sulfuric acid (ex-
tends wound damage) 2-chloroethylphosphonic acid (CEPA a syn-
thetic ethylene precursor) paraquat (free radical generator) yeast ex-
tract (mimics attack by pathogens) salicylic acid (pathogen signaling
molecule) auxin (promotes ethylene biosynthesis and resin canal dif-
ferentiation) jasmonic acid (signals mechanical damage and promotes
secondary metabolism) and metal ions such as potassium iron and
manganese (cofactors of terpene synthases in conifers) and copper (a
component of ethylene receptors) (Clements 1970 Conrath et al
2002 Fett-Neto and Rodrigues-Correcirca 2012 Hudgins and Franceschi
2004 Lewinsohn et al 1994 Martin et al 2002 Popp et al 1995
httpsdoiorg101016jindcrop201803027
Received 12 December 2017 Received in revised form 9 March 2018 Accepted 13 March 2018
Corresponding author
E-mail addresses franci_neisyahoocombr (FA Neis) fernandadecostayahoocombr (F de Costa) thanisenfyahoocombr (TN Fuumlller)
jjuliocesarlimagmailcom (JC de Lima) krodriguescbiotufrgsbr (KC da Silva Rodrigues-Correcirca) jpfettcbiotufrgsbr (JP Fett) fettnetocbiotufrgsbr (AG Fett-Neto)
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Research Paper
Dual allelopathic effects of subtropical slash pine (Pinus elliottii Engelm)
needles Leads for using a large biomass reservoir
Kelly Cristine da Silva Rodrigues-Correcircaa Gelson Halmenschlagera Joseacuteli Schwambachb
Fernanda de Costaa Emili Mezzomo-Trevizana Arthur Germano Fett-Netoa
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 Brazilb University of Caxias do Sul Institute of Biotechnology Caxias do Sul RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Seasonality
Growth
Germination
Litter
Substrate
A B S T R A C T
Pinus elliottii Engelm (slash pine) is distributed along the maritime coast of Southern Brazil where it shows
invasive pattern and typical allelopathic features Large quantities of needle litter are produced by pine trees a
biomass that is little explored in areas where this species is alien Little is known about the dynamics of needle
and litter phytochemical interactions particularly in subtropical environments To elucidate the full range of
needle and litter allelopathic potential the effects of litter (superficial and deep) and seasonally harvested fresh
slash pine needles stored for different times were evaluated against lettuce tomato and cucumber seeds and
seedlings Increasing concentrations (0 1 2 4 and 8 wv) of hot and cold aqueous extracts of needles
and litter affected in different ways target plant development Growth and germination inhibition were directly
related to the highest extract concentrations (regardless of the season and mainly in hot water extracts) of
needles On the other hand stimulatory effects of litter extracts on lettuce growth were observed Growth and
germination of cucumber and tomato were not affected by pine litter as substrate when compared to rice husk
The presumable high polarity and thermal stability of slash pine leaf biomass allelochemicals and their transient
toxic effect or growth promoting impact suggest potential applications of this largely available biomass both as a
biological herbicide and growth substrate in plant propagation
1 Introduction
Native from the Northern Hemisphere Pinus is one of the most
widely distributed genera throughout different climate regions of the
globe growing either as native or alien species even in extreme habi-
tats (Rodrigues-Correcirca and Fett-Neto 2012) Despite the high economic
value currently attributed to pine wood and oleoresin (Rodrigues-
Correcirca et al 2012) there is increasing concern about the aggressive
potential of invasiveness displayed by Pinus species especially those
cultivated out of their native range of distribution (Richardson et al
2008 Rolon et al 2011) These species are dispersed by wind and there
is notably low plant diversity observed in most understories of pine
plantations (Kato-Noguchi et al 2009) This latter feature has been
considered an important trait of allelopathic interference
The term ldquoallelopathyrdquo was coined by Molisch in 1937 as a chemical
reciprocal interaction established among plants (including micro-
organisms) sharing the same site by means of the release of secondary
metabolites named allelochemicals (Rice 1984) For the most part
these metabolites are derived from the shikimic acid or isoprenoid
pathway and their biosynthesis can be modulated by biotic and abiotic
stresses (Nascimento and Fett-Neto 2010) including seasonal-related
changes (Sartor et al 2013) Allelopathy studies may range from sterile
assays (Aryakia et al 2015) to soil (Correcirca et al 2008 Sharma et al
2016) and field tests being a complex biological phenomenon to as-
certain in several circumstances due to issues of solubility release
mechanisms and stability of bioactive compounds (Scognamiglio et al
2013) Often the use of complementary methods provides more in-
formative data
The allelopathic effects of soil leachates green needles and litter
extracts of Pinus spp on germination and seedling growth aspects of
wild and crop species have been evaluated in natural and cultivated
pine stands and have proven to be stimulatory or inhibitory (Lodhi and
Killingbeck 1982 Kil and Yim 1983 Nektarios et al 2005 Akkaya
et al 2006 Machado 2007 Alrababah et al 2009 Sartor et al 2009
Kato-Noguchi et al 2011 Rolon et al 2011 Valera-Burgos et al
2012) exhibiting in some cases autotoxicity (Garnett et al 2004
Fernandez et al 2008 Zhu et al 2009 Monnier et al 2011) Studies
on potential dual allelopathic effects of Pinus elliottii Engelm (slash
httpdxdoiorg101016jindcrop201706019
Received 23 March 2017 Received in revised form 15 May 2017 Accepted 7 June 2017
Corresponding author
E-mail address fettnetocbiotufrgsbr (AG Fett-Neto)
ORIGINAL RESEARCHpublished 16 June 2016
doi 103389fpls201600849
Frontiers in Plant Science | wwwfrontiersinorg 1 June 2016 | Volume 7 | Article 849
Edited by
Juan Francisco Jimenez Bremont
Instituto Potosino de Investigacioacuten
Cientiacutefica y Tecnoloacutegica Mexico
Reviewed by
Mariacutea De La Luz Guerrero Gonzaacutelez
Universidad Autoacutenoma de San Luis
Potosiacute Mexico
Rosalia Cristina Paz
CIGEOBIO (CONICETFCEFN UNSJ)
Argentina
Correspondence
Arthur G Fett-Neto
fettnetocbiotufrgsbr
daggerThese authors have contributed
equally to this work
Specialty section
This article was submitted to
Plant Physiology
a section of the journal
Frontiers in Plant Science
Received 08 December 2015
Accepted 30 May 2016
Published 16 June 2016
Citation
de Lima JC de Costa F Fuumlller TN
Rodrigues-Correcirca KCdS Kerber MR
Lima MS Fett JP and Fett-Neto AG
(2016) Reference Genes for qPCR
Analysis in Resin-Tapped Adult Slash
Pine As a Tool to Address the
Molecular Basis of Commercial
Resinosis Front Plant Sci 7849
doi 103389fpls201600849
Reference Genes for qPCR Analysisin Resin-Tapped Adult Slash Pine Asa Tool to Address the MolecularBasis of Commercial Resinosis
Juacutelio C de Lima 1dagger Fernanda de Costa 1 dagger Thanise N Fuumlller 1
Kelly C da Silva Rodrigues-Correcirca 2 Magnus R Kerber 1 Mariano S Lima 1
Janette P Fett 1 and Arthur G Fett-Neto 1
1 Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul
Porto Alegre Brazil 2 Biological Sciences Department Regional Integrated University of Alto Uruguai and Missotildees (URI-FW)
Frederico Westphalen Brazil
Pine oleoresin is a major source of terpenes consisting of turpentine (mono- and
sesquiterpenes) and rosin (diterpenes) fractions Higher oleoresin yields are of economic
interest since oleoresin derivatives make up a valuable source of materials for chemical
industries Oleoresin can be extracted from living trees often by the bark streak method
in which bark removal is done periodically followed by application of stimulant paste
containing sulfuric acid and other chemicals on the freshly wounded exposed surface
To better understand the molecular basis of chemically-stimulated and wound induced
oleoresin production we evaluated the stability of 11 putative reference genes for the
purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis
Samples for RNA extraction were collected from field-grown adult trees under tapping
operations using stimulant pastes with different compositions and at various time points
after paste application Statistical methods established by geNorm NormFinder and
BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3
and UBI To confirm expression stability of the candidate reference genes expression
profiles of putative P elliottii orthologs of resin biosynthesis-related genes encoding Pinus
contorta β-pinene synthase [PcTPS-(minus)β-pin1] P contorta levopimaradieneabietadiene
synthase (PcLAS1) Pinus taeda α-pinene synthase [PtTPS-(+)αpin] and P taeda
α-farnesene synthase (PtαFS) were examined following stimulant paste application
Increased oleoresin yields observed in stimulated treatments using phytohormone-based
pastes were consistent with higher expression of pinene synthases Overall the
expression of all genes examined matched the expected profiles of oleoresin-related
transcript changes reported for previously examined conifers
Keywords resin Pinus gene expression normalizer genes terpene synthase
19
Chapter 2
Stimulant Paste Preparation and Bark Streak Tapping Technique for Pine Oleoresin Extraction
Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
Tapping technique comprises the extraction of pine oleoresin a non-wood forest product consisting of a
complex mixture of mono sesqui and diterpenes biosynthesized and exuded as a defense response to
wounding Oleoresin is used to produce gum rosin turpentine and their multiple derivatives Oleoresin
yield and quality are objects of interest in pine tree biotechnology both in terms of environmental and
genetic control Monitoring these parameters in individual trees grown in the fi eld provides a means to
examine the control of terpene production in resin canals as well as the identifi cation of genetic-based
differences in resinosis A typical method of tapping involves the removal of bark and application of a
chemical stimulant on the wounded area Here we describe the methods for preparing the resin-stimulant
paste with different adjuvants as well as the bark streaking process in adult pine trees
Key words Oleoresin Pine Tapping Chemical stimulant Wounding
1 Introduction
Several conifer species produce oleoresin a complex mixture of isoprenoid compounds relevant for defense against herbivores and pathogens Two major fractions can be recognized in oleoresin (a) turpentine the volatile fraction containing mono- and sesquiter-penes and (b) rosin the nonvolatile diterpene fraction Oleoresin is a forest commodity of global interest fi nding applications in diverse industry sectors Rosin is used in adhesives printing ink manufacture and paper sizing Turpentine can be used either as a solvent for paints and varnishes or as a raw material for fraction-ation of high-value chemicals used in the pharmaceutical agro-chemical and food industry [ 1 ndash 3 ]
During the extraction activity resin is obtained from the tree in a similar way as rubber tree tapping which generally involves the
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_2 copy Springer Science+Business Media New York 2016
These authors have equally contributed to this work
fettnetocbiotufrgsbr
27
Chapter 3
A Modifi ed Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines
Juacutelio Ceacutesar de Lima Thanise Nogueira Fuumlller Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes
through high-throughput sequencing technologies microarray and also northern blots RT-PCR and
RTqPCR Even though many specifi c protocols designed for plants with high content of secondary metab-
olites have been developed these are often expensive time consuming and not suitable for a wide range
of tissues Here we present a modifi cation of the method previously described using the commercially
available Concerttrade Plant RNA Reagent (Invitrogen) buffer for fi eld-grown adult pine trees with high
oleoresin content
Key words RNA Pines Concert plant RNA reagent Stem RNA extraction Oleoresin Conifers
1 Introduction
Several conifer species especially within the Pinaceae have tissues with high concentrations of phenolics terpenes and polysaccha-rides [ 1 ] Many specifi c protocols that are appropriate for plants rich in secondary metabolite s have been developed but the extrac-tion of high-quality RNA from these tissues using commercial kits is often diffi cult and usually not applicable to woody tissues [ 2 ndash 6 ] One of the major issues during RNA extraction concerns the pres-ence of phenolic compounds which oxidize and form quinones Aromatic compounds bind RNA which interferes in downstream steps and applications [ 3 7 ] Another point of concern is the har-vest of plant samples in the experimental fi eld which constitutes another obstacle in the efforts to avoid degradation of RNA [ 8 ] These problems often result in RNAs of low quality and insuffi -cient amounts especially for methodologies that normally require
These authors have equally contributed to this work
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_3 copy Springer Science+Business Media New York 2016
fettnetocbiotufrgsbr
RESEARCH PAPER
Control of resin production in Araucaria angustifolia an ancientSouth American coniferJ C Perotti1 K C da Silva Rodrigues-Correa123 amp A G Fett-Neto12
1 Plant Physiology Laboratory Department of Botany Federal University of Rio Grande do Sul (UFRGS) Porto Alegre RS Brazil
2 Center for Biotechnology UFRGS Porto Alegre RS Brazil
3 Present address Regional Integrated University of Alto Uruguai and Miss~oes (URI-FW) Frederico Westphalen RS Brazil
Keywords
Araucaria ethylene jasmonic acid nitric
oxide resin salicylic acid terpenes
Correspondence
A G Fett-Neto Plant Physiology Laboratory
Center for Biotechnology Federal University
of Rio Grande do Sul (UFRGS) PO Box 15005
Av Bento Goncalves 9500 91501-970 Porto
Alegre Brazil
E-mail fettnetocbiotufrgsbr
Editor
K Leiss
Received 22 July 2014 Accepted 11
December 2014
doi101111plb12298
ABSTRACT
Araucaria angustifolia is an ancient slow-growing conifer that characterises parts ofthe Southern Atlantic Forest biome currently listed as a critically endangered speciesThe species also produces bark resin although the factors controlling its resinosis arelargely unknown To better understand this defence-related process we examined theresin exudation response of A angustifolia upon treatment with well-known chemicalstimulators used in fast-growing conifers producing both bark and wood resin suchas Pinus elliottii The initial hypothesis was that A angustifolia would display signifi-cant differences in the regulation of resinosis The effect of Ethrel (ET ndash ethylene pre-cursor) salicylic acid (SA) jasmonic acid (JA) sulphuric acid (SuA) and sodiumnitroprusside (SNP ndash nitric oxide donor) on resin yield and composition in youngplants of A angustifolia was examined In at least one of the concentrations testedand frequently in more than one an aqueous glycerol solution applied on fresh woundsites of the stem with one or more of the adjuvants examined promoted an increase inresin yield as well as monoterpene concentration (a-pinene b-pinene camphene andlimonene) Higher yields and longer exudation periods were observed with JA and ETanother feature shared with Pinus resinosis The results suggest that resinosis controlis similar in Araucaria and Pinus In addition A angustifolia resin may be a relevantsource of valuable terpene chemicals whose production may be increased by usingstimulating pastes containing the identified adjuvants
INTRODUCTION
Many conifer species produce terpenoid-based resins that havelong been studied for their industrial importance and role indefence against attack by herbivores and pathogens The twomost important resin-producing families of conifers are Pina-ceae and Araucariaceae (Langenheim 1996) The viscous resinsecretion is generally composed of a complex mixture ofterpenoids consisting of roughly equal parts of volatile mono-(C10) and sesquiterpene (C15 turpentine) fractions and non-volatile diterpenic (C20 rosin) components (Rodrigues-Correaet al 2013) Terpenes act in a complex and multilayereddefence response providing toxicity against bark beetles andfungi bark wound sealing disruption of insect developmentand attraction of herbivore predators (Phillips amp Croteau1999)Most conifers rely on some combination of preformed and
inducible resin defences (Trapp amp Croteau 2001 Zulak amp Bohl-mann 2010) Resin defences are controlled by environmentaland genetic factors to various extents depending on species(Roberds et al 2003 Sampedro et al 2010 Moreira et al2013) Resin traits have been reported as highly variable havingmoderate heritability indicating that breeding efforts towardssuper-resinous forests are promising (Tadasse et al 2001Roberds et al 2003) Several chemicals are known as stimulantsof resin production Commercial extraction of resin from pine
trees uses periodic bark streaking and application of resin stim-ulant pastes to the wound
Resin-stimulant paste based on sulphuric acid (SuA) iswidely used for the commercial production of pine resin Cur-rent stimulant pastes usually have two chemically active com-ponents SuA to magnify the wounding and an ethyleneprecursor (2-chloroethylphosphonic acid CEPA or Ethrel ndash
ET) to stimulate resin flow (Rodrigues et al 2011 Rodrigues-Correa amp Fett-Neto 2013) Jasmonic acid (JA) and its methylester methyl jasmonate (MeJa) are among the most widelyused chemical elicitors of plant secondary metabolism It hasbeen shown that the exogenous application of MeJa or herbi-vore attack induce chemical and anatomical defence responsesin conifers such as the formation of traumatic resin ducts andresin accumulation in stems along with increased biosynthesisof terpenes and phenolics (Franceschi et al 2002 Martin et al2002 Heijari et al 2005 Zeneli et al 2006 Moreira et al 2008Gould et al 2009) JA commercial use however is limited byits high cost
The effects of exogenous salicylic acid (SA) on conifer ter-pene production have also been studied In Pinus elliottiiapplication of 10 molm3 of SA induced resin productionin wound panels but in Pseudotsuga menziesii and Sequoia-dendron giganteum it had no apparent effect on resinaccumulation (Hudgins amp Franceschi 2004 Rodrigues ampFett-Neto 2009) Nitric oxide (NO) has also emerged as an
Plant Biology 17 (2015) 852ndash859 copy 2014 German Botanical Society and The Royal Botanical Society of the Netherlands852
Plant Biology ISSN 1435-8603
ii
O presente trabalho foi desenvolvido
No Laboratoacuterio de Fisiologia Vegetal (LFV) da
Universidade Federal do Rio Grande do Sul
No College of Tropical Agriculture amp Human Resources (CTAHR)
da University of Hawairsquoi at Manoa
Instituiccedilotildees Financiadoras
COORDENACcedilAtildeO DE APERFEICcedilOAMENTO
PESSOAL DE NIacuteVEL SUPERIOR
(CAPES)
CONSELHO NACIONAL DE DESENVOLVIMENTO
CIENTIacuteFICO E TECNOLOacuteGICO
(CNPq)
iii
Dedicatoacuteria
Agrave minha amada avoacute lsquode laacutersquo Doraliacutecia Marcelina Costa da Silva (aka
Dona Dora como preferia ser chamada) por ter sido minha maior referecircncia
de amor e zelo enquanto estava aqui por ser a mais habilidosa e diligente
lsquofazedora de hortasrsquo e a melhor lsquoBotanistersquo empiacuterica que jaacute conheci
Obrigada por fazer da tua horta meu fantaacutestico lsquoherbaacuterio vivorsquo e do teu
conhecimento etnobotacircnico meu primeiro referencial de respeito e admiraccedilatildeo
ao Reino Plantae Foi nesse lsquoJardim Secretorsquo que descobri e me encantei
irreversivelmente pelo lsquoextraordinaacuterio poder das plantasrsquo E a saudade tua soacute
aumenta nunca diminui
Ao meu muito querido e saudoso amigo Rafael Cortes Duarte cortecircs
(sempre) ateacute no nome Se ainda estivesses por aqui esse trabalho teria sido
teu
Dizem que o tempo aqui eacute relativo Logo a gente se vecirc
iv
AGRADECIMENTOS
Ao meu orientador Dr Arthur Germano Fett-Neto uma das melhores pessoas
que tive a honra de conhecer na Academia Um coraccedilatildeo imenso uma mente incrivelmente
brilhante integridade e empatia infinitas (e extremamente raras no meio cientiacutefico)
Muito obrigada pela confianccedila em mim depositada e sobretudo por ter cometido a
insanidade de aceitar me orientar novamente Obrigada por ter me possibilitado ir em
termos cientiacuteficos muito aleacutem do que eu ousaria imaginar dadas as minhas (inuacutemeras)
limitaccedilotildees (e por todo ATP e NADPH investidos nesse esforccedilo hercuacuteleo que constitui a
aacuterdua tarefa de me orientar de forma natildeo condescendente a despeito dessas) Minha
diacutevida contigo seraacute eterna sou uma pessoa duplamente aquinhoada pela tua orientaccedilatildeo
Lucky me
Aos Professores Janette Palma-Fett uma grande amiga e saacutebia conselheira
sempre especialmente na adversidade e Felipe Maraschin pelo pronto e inestimaacutevel
apoio teacutecnico-cientiacutefico sempre que solicitado
Aos colegas do Laboratoacuterio de Fisiologia Vegetal da UFRGS pela parceria e
auxiacutelio em todas as horas por formarem um grupo coeso alinhado e comprometido com
o bem maior da pesquisa e do bom funcionamento do lab Eacute gratificante trabalhar com
todos vocecircs
Aos amigos muito queridos que a UFRGS me trouxe Ana Paula Durand
Coelho Eudes Stiehl-Alves Johnatan Vilasboa Yohanna Miotto e as divas Juliana
Troleis Sofia Aumond Kuhn e Tamara Pastori Muito obrigada por estarem presentes
nas horas menos faacuteceis e por me auxiliarem de muitas maneiras sempre que precisei Toda
dificuldade eacute redimensionada quando se tem amigos
Agrave minha famiacutelia caucasoide Ana Cristina Stein Camila Junkes Camila e
Cassiano Busatta Carlos Eduardo Blanco Linares Daniela Sponchiado Jordana
Griebler Luft Karen Santos Karina Letiacutecia Lopes e Larissa Schemes Heinzelmann
O carinho o apoio e o encorajamento que recebo de vocecircs fazem qualquer lsquofardorsquo parecer
mais leve Muito lsquomercirsquo
I am very grateful to Dr Dulal Borthakur for generously having received me in
his lab and his loving and caring family I would also like to thank my lab mates at UH
Manoa James Carillo Maia Corpuz and Ahmed Bageel for being so helpful cheerful
and friendly with me during all my stay in Honolulu Most of all Irsquod like to thank my
dear friend Michael Honda for teaching very patiently and supporting me inside and
outside the lab by doing whatever was in his power to prevent my homesickness I am
also very grateful to Mariana de Souza and Fernanda Oliveira for all those amazing
places and hikes wersquove been together in Orsquoahu You guys are awesome Mahalo nui loa
for your kōkua Im now parsquou hana
Jaimerais bien remercier mes collegravegues et amis agrave lrsquoUniversiteacute de Montreacuteal
(Benjamin Mazin Marion Kretsch Yang Liu Fang Wen Raquel Parada et Micaela
Margutti) pour mavoir chaleureusement reccedilu chez vous speacutecialement agrave mon ami
Valentin Joly pour mavoir beaucoup appris sur lrsquoinconnu monde des bacteacuteries et des
v
levures (et surtout pour leur incroyable patience avec mon tregraves mauvais franccedilais) Crsquoeacutetait
vachement chouette Merci beaucoup agrave vous tous (et toutes) et agrave la prochaine
Agrave Coordenaccedilatildeo de Aperfeiccediloamento Pessoal de Niacutevel Superior (CAPES) pelo
financiamento da bolsa de pesquisa do PDSE
Aos meus pais (bioloacutegicos ou natildeo) Veacutera Maria da Silva Rodrigues Gilberto
Moraes Rodrigues Rosa Maria Lucas da Silva e Paulo Joseacute Costa da Silva pelo
exemplo de honestidade coragem trabalho forccedila e amor desde sempre
Aos meus irmatildeos Ana Paula da Silva Rodrigues Viniacutecius de Moraes da Silva
Rodrigues Marcello da Silva Rodrigues e Camila Stella Toledo Pereira por todas as
experiecircncias que dividimos e tudo o que me ensinaram ateacute hoje
Ao meu amor maior minha melhor amiga minha mais leal e extraordinaacuteria
parceria nessa grande (e agraves vezes tortuosa) jornada Maria Clara Rodrigues Correcirca Por
ser ela por ser imensa em generosidade amor e altruiacutesmo por despertar o melhor em
mim por ser minha forccedila motriz e sobretudo por ser a melhor das minhas metades
Minha vida soacute realmente comeccedilou quando eu tive a incriacutevel sorte de te conhecer
vi
SUMAacuteRIO
LISTA DE ABREVIATURASvii
RESUMO ix
INTRODUCcedilAtildeO GERAL1
HIPOacuteTESE E OBJETIVOS9
CAPIacuteTULO 1 Abiotic stresses and non-protein amino acids in plantshelliphellip10
CAPIacuteTULO 2 Mimosine accumulation in Leucaena leucocephala in response to
stress signaling molecules and acute UV exposurehelliphelliphelliphelliphelliphelliphelliphelliphelliphellip(432) 52
CAPIacuteTULO 3 Mimosine occurrence and accumulation in Mimosa bimucronata var
bimucronata (DC) Kuntze66
CONSIDERACcedilOtildeES FINAIS 84
PERSPECTIVAS85
REFEREcircNCIAS BIBLIOGRAacuteFICAS86
Artigos publicados no periacuteodo de doutoramento natildeo relacionados ao tema da
tese91
vii
LISTA DE ABREVIATURAS
24-D 24-dichlorophenoxyacetic acid
3H4P 3-hydroxy-4-pyridone (34-DHP 34-dihydroxypyridine)
ABA abscisic acid
Arg arginine
BABA β-aminobutyric acid
β-ODAP β-N-oxalyl-L-α β-diaminopropionic acid
BIA β-isoxazolinon-L-alanine
CAN canavanine
DAO diamine oxidase
DDC decarboxylase
ETH ethephon
FW fresh weight
GABA -aminobutyric acid
GABA-T GABA transaminase
GAD glutamate decarboxylase
GSM Global System for Mobile
HPLC High performance liquid chromatography
JA jasmonate
JA-Ile jasmonoyl-L-isoleucine
L-DOPA L-34- dihydroxyphenylalanine
MeJA methyl jasmonate
m-Tyr Meta-tyrosine
NO nitric oxide
NPAA non-protein amino acid
OAS o-acetylserine
OAS-TL o-acetylserine-thiol-lyase
PA polyamine
PAA protein amino acid
viii
PEG polyethylene glycol
PLP pyridoxal-5rsquo-phosphate
PPO polyphenol oxidase tyrosinase
qRT-PCR Reverse transcription polymerase chain reaction quantitative real time
RNS reactive nitrogen species
ROS reactive oxygen species
SA salicylic acid
SAR systemic acquired resistance
SNP sodium nitroprusside
UV ultraviolet radiation
ix
RESUMO
Ao longo de sua evoluccedilatildeo as plantas desenvolveram estrateacutegias estruturais e quiacutemicas de
defesa em resposta aos estresses bioacuteticos e abioacuteticos impostos pelo ambiente Dentre
essas satildeo reconhecidas moleacuteculas quimicamente especializadas denominadas
metaboacutelitos secundaacuterios produtos naturais ou metaboacutelitos especializados Aminoaacutecidos
natildeo proteicos (ANPs) satildeo compostos nitrogenados que constituem aleacutem de componentes
do arsenal de defesa quiacutemica vegetal uma importante fonte de reserva de carbono e
nitrogecircnio para diversos taxa especialmente aqueles pertencentes agrave famiacutelia Fabaceae de
Angiospermas Esse grupo de moleacuteculas quimicamente heterogecircneo eacute assim definido por
natildeo participar da formaccedilatildeo de estruturas proteicas funcionais sendo frequentemente
toacutexicos quando erroneamente incorporados nas cadeias polipeptiacutedicas em formaccedilatildeo em
funccedilatildeo da similaridade estrutural que apresentam com os aminoaacutecidos proteicos Sob o
ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados ANPs satildeo
em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou abioacutetica como
o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de elicitores
quiacutemicos por exemplo O objetivo da presente tese foi investigar o papel bioloacutegico da
mimosina endoacutegena em Leucaena leucocephala (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute) a partir da avaliaccedilatildeo do efeito de tratamentos
relacionados ao estresse abioacutetico (UV-C aacutecido saliciacutelico metil jasmonato e etileno)
Mimosina eacute um ANP aromaacutetico anaacutelogo da L-tirosina com atividade toacutexica para ceacutelulas
de procariotos e eucariotos Dentre as atividades descritas para esse ANP destacam-se a
atividade anti-mitoacutetica ou bloqueadora do ciclo celular atividade alelopaacutetica e
antioxidante Os resultados indicaram que em leucena a biossiacutentese e o acuacutemulo de
mimosina podem ser modulados por fatores causadores de estresses exibindo um padratildeo
de acumulaccedilatildeo similar ao das fitoalexinas Em maricaacute por outro lado a induccedilatildeo do
acuacutemulo dessa moleacutecula natildeo foi observada para os mesmos tratamentos testados para
leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas Aleacutem disso
o padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado
pela sua degradaccedilatildeo As respostas observadas nos testes que avaliaram a atividade de
mitigaccedilatildeo de espeacutecies reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode
agir como um agente antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de
estresse
1
Introduccedilatildeo
Na condiccedilatildeo de organismos seacutesseis ao longo de sua evoluccedilatildeo as plantas
desenvolveram estrateacutegias estruturais e quiacutemicas de defesa em resposta aos estresses bioacuteticos
e abioacuteticos impostos pelo ambiente Dentre essas satildeo reconhecidas moleacuteculas quimicamente
especializadas denominadas metaboacutelitos secundaacuterios produtos naturais (Kutchan et al 2015)
ou mais recentemente metaboacutelitos especializados
Entre as trecircs classes mais gerais de metaboacutelitos secundaacuterios (terpenos compostos
fenoacutelicos e compostos nitrogenados) aminoaacutecidos natildeo-proteicos (ANPs) satildeo incluiacutedos no
terceiro grupo e constituem aleacutem de componentes do arsenal de defesa quiacutemica uma
importante fonte de reserva de carbono e nitrogecircnio para diversos taxa especialmente aqueles
pertencentes agrave famiacutelia Fabaceae de Angiospermas (leguminosas sensu lato)
Aleacutem dos 20 aminoaacutecidos proteicos estima-se que existam entre 600 e 1000 ANPs
(Acamovic amp Brooker 2005 Rodgers et al 2015) Esse grupo de moleacuteculas quimicamente
heterogecircneo eacute assim definido por natildeo participar da formaccedilatildeo de estruturas proteicas
funcionais sendo frequentemente toacutexicos quando erroneamente incorporados nas cadeias
polipeptiacutedicas em formaccedilatildeo em funccedilatildeo da similaridade estrutural que apresentam com os
aminoaacutecidos proteicos (Taiz amp Zeiger 2010)
Conforme mencionado a ocorrecircncia de ANPs eacute comum em espeacutecies de leguminosas
e sua distribuiccedilatildeo pode ser restrita a alguns gecircneros de plantas circunscritos nessa famiacutelia
botacircnica (eg mimosina e canavanina) Por outro lado alguns ANPs como GABA por
exemplo podem apresentar distribuiccedilatildeo ubiacutequa no Reino Plantae assim como ocorrer em
outros tipos de organismos como animais por exemplo (Ramos-Ruiz et al 2018)
2
Apesar de representarem uma fonte nutricional importante sem tratamento preacutevio o
consumo de plantas que acumulam ANPs por animais eacute limitado Isso ocorre pois em longo
prazo a ingestatildeo prolongada de plantas (especialmente sementes) que acumulam ANPs pode
representar risco agrave sauacutede uma vez que estes comprometem o funcionamento de mecanismos
basais de manutenccedilatildeo da homeostase celular e podem tambeacutem em um quadro mais severo
desencadear doenccedilas neurotoacutexicas degenerativas como por exemplo o latirismo causado
por aacutecido β-N-oxalil-l-αβ-diaminopropiocircnico (β-ODAP) (Jiao et al 2011 Kusama-Eguchi
2019)
Sob o ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados
ANPs satildeo em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou
abioacutetica como o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de
elicitores quiacutemicos por exemplo No que concerne ao estudo dos efeitos da induccedilatildeo abioacutetica
sobre o acuacutemulo de ANPs em diferentes espeacutecies vegetais (Monocotiledocircneas e
Eudicotiledocircneas) as moleacuteculas mais amplamente investigadas ateacute o momento satildeo GABA
L-DOPA e mais recentemente mimosina (vide Tabela 1 do capiacutetulo primeiro) Em termos
de efeitos causados a partir da aplicaccedilatildeo exoacutegena de ANPs GABA tambeacutem figura como o
principal aminoaacutecido investigado seguido de L-DOPA e canavanina (vide Tabela 2 do
capiacutetulo primeiro)
No primeiro capiacutetulo da presente tese estatildeo descritas as caracteriacutesticas gerais dos
principais ANPs estudados seus possiacuteveis papeacuteis bioloacutegicos in planta e seus efeitos quando
aplicados exogenamente bem como os estresses abioacuteticos capazes de induzir seu(s)
acuacutemulo(s) nos diferentes tecidos vegetais Nos segundo e terceiro capiacutetulos
respectivamente satildeo elucidados os efeitos dos tratamentos de UV-C aacutecido saliciacutelico etileno
e jasmonato (claacutessicos elicitores do metabolismo secundaacuterio vegetal) sobre o acuacutemulo de
3
mimosina em Leucaena leucocephala var glabrata (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute)
Mimosina eacute um aminoaacutecido aromaacutetico natildeo-proteico anaacutelogo da L-tirosina com
atividade toacutexica para ceacutelulas de procariotos e eucariotos Embora em menor concentraccedilatildeo
mimosina foi primeiramente identificada em Mimosa pudica sendo posteriormente detectada
em outras espeacutecies do gecircnero como Mimosa pigra por exemplo (Soedarjo amp Borthakur
1998) Seu efeito toacutexico eacute atribuiacutedo agrave capacidade de quelar metais o que impede o
funcionamento adequado das metalo-proteiacutenas que dependem dos mesmos como co-fatores
(Negi et al 2014)
A concentraccedilatildeo basal de mimosina em espeacutecies de leucaena pode variar entre 1 e 12
do peso seco do oacutergatildeo (Soedarjo amp Borthakur 1998) Como eacute comum para outros ANPs
que ocorrem em espeacutecies de leguminosas em sementes de Leucaena spp eacute observada uma
maior concentraccedilatildeo de mimosina quando comparada aos demais oacutergatildeos da planta
(Rodrigues-Correcirca et al 2019) sendo esta a fonte de extraccedilatildeo comercial do padratildeo quiacutemico
de mimosina vendido por empresas de reagentes de pesquisa
Diversas atividades foram descritas para mimosina em outros organismos eou tipos
celulares Dentre essas destacam-se a atividade anti-mitoacutetica ou bloqueadora do ciclo
celular em ceacutelulas de eucariotos e procariotos Isto ocorre porque a mimosina impede a
formaccedilatildeo da forquilha de replicaccedilatildeo (e portanto a siacutentese de DNA) interrompendo assim o
avanccedilo do ciclo de divisatildeo celular na fase tardia G1 (Lalande 1990) Foram tambeacutem descritas
para mimosina atividade alelopaacutetica observada sobre o desenvolvimento de outras espeacutecies
de leguminosas e atividade antioxidante entre outras (Tabela 1)
A rota de biossiacutentese de mimosina eacute compartilhada em grande parte com a de cisteiacutena
um aminoaacutecido proteico sulfurado (Figura 1) A siacutentese da cisteiacutena se daacute a partir da conversatildeo
4
de serina e acetil-CoA em o-acetilserina pela enzima SAT (serina acetiltransferase) seguida
da conversatildeo de o-acetilserina e aacutecido sulfiacutedrico em cisteiacutena em uma reaccedilatildeo catalisada pela
OAS-TL (o-acetilserina tiol-liase) A siacutentese de mimosina por sua vez eacute compartilhada com
a da cisteiacutena ateacute esse ponto e acredita-se que pelo menos uma das isoformas de OAS-TL
catalise a conversatildeo de o-acetilserina e 3-hidroxi-4-piridona em mimosina
Tabela 1 Atividades descritas para mimosina de Leucaena leucocephala (Lam) de Wit
ATIVIDADE
ALVO AVALIADO
(organismo eou tecido tipo
celular)
REFEREcircNCIA
Bloqueio do complexo de ativaccedilatildeo
da preacute-replicaccedilatildeo do DNA
Ceacutelulas de mamiacuteferos
KUBOTA et al
(2014)
Alteraccedilatildeo no ciclo ovariano e
extensatildeo da duraccedilatildeo do corpo luacuteteo
bovino no periacuteodo poacutes-parto
Bovinos
(Bos taurus x
Bos indicus)
BOTTINI-
LUZARDO et al
(2015)
Supressatildeo do ciclo celular e reduccedilatildeo
da abundacircncia bacteriana em
mosquitos
Wolbachia pipientis
Aedes albopictus
FALLON
(2015)
Accedilatildeo inibitoacuteria da fibrose
pulmonar induzida
Ratos SD
LI et al
(2015)
Recuperaccedilatildeo da funccedilatildeo do
miocaacuterdio poacutes-isquemia
Miocaacuterdio de ratos (SD)
machos
CROWE et al
(2001)
Inseticida
Heteropsylla cubana
Crawford 1914 e Thrips tabaci
Lindemann 1889
AHMED et al
(2016)
Alelopaacutetica
Albizia procera Vigna
unguiculata Cicer arietinum
Cajanus cajan
AHMED et al
(2008)
Antioxidante
Sistemas modelo de oxidaccedilatildeo
lipiacutedica (β-caroteno - aacutecido
linolecircico e lecitina)
BENJAKUL et al
(2013)
Ateacute momento versotildees divergentes sobre a enzima responsaacutevel pela biossiacutentese de
mimosina (mimosina sintase) tecircm sido publicadas Em 1990 Ikegami e colaboradores
5
identificaram uma OAS-TL responsaacutevel pela formaccedilatildeo de cisteiacutena como sendo tambeacutem uma
mimosina sintase Mais tarde Yafuso et al (2014) realizaram a expressatildeo heteroacuteloga do gene
que codifica para OAS-TL em Escherichia coli e natildeo foi observada a formaccedilatildeo de mimosina
mesmo quando dadas as condiccedilotildees oacutetimas para tanto Mais recentemente Harun-Ur-Rashid
et al (2018) elucidaram a mimosina sintase como sendo uma isoforma da OAS-TL
corroborando o postulado por Ikegami e colaboradores em 1990
Figura 1 Rota de biossiacutentese da mimosina Fonte Ikegami et al (1990)
Espeacutecies estudadas
Leucaena leucocephala (Lam) de Wit (leucaena koa haole ou ldquoacaacutecia exoacuteticardquo na
liacutengua Hawairsquoiana) eacute uma espeacutecie de haacutebito arboacutereo ou arbustivo pertencente agrave famiacutelia
Fabaceae de Angiospermas e caracterizada pelo acuacutemulo de mimosina em todos os seus
oacutergatildeos Eacute nativa da Ameacuterica Central (especificamente da regiatildeo sudeste do Meacutexico) mas
irradiou-se atraveacutes de praticamente todas as zonas tropicais e subtropicais da Terra No
Brasil leucena eacute amplamente distribuiacuteda e classificada como naturalizada pelo REFLORA
(2019) ocorrendo em todo territoacuterio Nacional Satildeo reconhecidas no miacutenimo duas
6
subespeacutecies de leucena ocorrentes no Brasil L leucocephala var leucocephala e L
leucocephala var glabrata sendo a primeira a mais abundante
Leucaena apresenta atributos morfoloacutegicos caracteriacutesticos das leguminosas como o
fruto do tipo vagem deiscente no periacuteodo poacutes-maturaccedilatildeo folhas compostas e bipinadas As
flores satildeo seacutesseis actinomorfas e polistecircmones apresentam caacutelice sinseacutepala e corola
gamopeacutetala e satildeo dispostas em inflorescecircncias do tipo glomeacuterulo (Figura 2)
Figura 2 Oacutergatildeos vegetativos e reprodutivos de L leucocephala (Lam) de Wit Fonte Little Jr amp Skolmen
(1989)
Com base no conhecimento etnobotacircnico disponiacutevel acerca dessa espeacutecie em
diversas regiotildees tropicais e subtropicais leucena eacute utilizada para vaacuterios fins Extratos de
diferentes oacutergatildeos de leucena apresentam atividade anti-diabeacutetica (Kuppusamy et al 2014
Chowtivannakul et al 2016) antioxidante (Mohammed et al 2015 Chowtivannakul et al
2016 Zarin et al 2016) antimicrobiana (Zarin et al 2016) anti-helmiacutentica (Soares et al
2015 Jamous et al 2017) bactericida (Mohammed et al 2015) acaricida (Fernaacutendez-Salas
et al 2011) anti-tumoral (Chung et al 2017) e potencializadora da resposta imune em
peixes (Verma et al 2018) entre outras
7
Leucaena apresenta alta toleracircncia agrave seca sendo capaz de enfrentar estaccedilotildees sazonais
inteiras com deacuteficit hiacutedrico sem prejuiacutezo permanente de seus oacutergatildeos e de recuperar
vigorosamente sua biomassa vegetativa tatildeo logo o regime de precipitaccedilatildeo retome a
regularidade em frequecircncia Acredita-se que a toleracircncia agrave seca apresentada por essa espeacutecie
ocorra em funccedilatildeo do acuacutemulo de mimosina nos diferentes tecidos da planta a qual
funcionaria como um agente osmoregulador responsaacutevel pela preservaccedilatildeo da integridade das
membranas a das macromoleacuteculas intracelulares em periacuteodos de escassez de aacutegua no
ambiente
Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) eacute uma leguminosa
nativa natildeo endecircmica do Brasil amplamente distribuiacuteda nos domiacutenios fitogeograacuteficos da
Caatinga do Cerrado e da Mata Atlacircntica (Simon amp Proenccedila 2000 REFLORA 2019) Como
espeacutecie pioneira (Pilatti et al 2019) exerce importante papel ecoloacutegico na recuperaccedilatildeo de
aacutereas degradadas (Bitencourt et al 2007 Silva et al 2011) no estabelecimento de processos
de sucessatildeo vegetacional
Maricaacute eacute uma espeacutecie semi-deciacutedua a deciacutedua a qual atinge ateacute 15 m em altura (e
diacircmetro agrave altura do peito de ateacute 40 cm) na idade adulta com haacutebito arboacutereo ou arbustivo
(REFLORA 2019) e espinhos caracteriacutesticos desde os estaacutegios iniciais de desenvolvimento
(Carvalho 2004) Apresenta folhas compostas alternas e bipinadas (Figura 2) amplas
inflorescecircncias brancas com flores reunidas em glomeacuterulos esfeacutericos dispostos em grandes
paniacuteculas As flores satildeo diplostecircmones actinomorfas hipoacuteginas e unicarpelares (Silva et al
2011)
Assim como descrito para leucena maricaacute eacute considerado uma espeacutecie multifuncional
sendo comumente empregada para produccedilatildeo de mel como combustiacutevel (Olkoski amp
8
Wittmann 2011) em edificaccedilotildees na carpintaria e como lsquocerca-vivarsquo (Marchiori 1993
Lorenzi 1998) entre outras aplicaccedilotildees
Figura 2 Folhas e fruto de Mimosa bimucronata (DC) Kuntze Fonte Souza-Lima et al (2017)
Em contraste com a amplitude de habitats explorados por leucena (especialmente os
aacuteridos) no Sul do Brasil maricaacute ocorre preferencialmente em ambientes uacutemidos a alagadiccedilos
em aacutereas proacuteximas agraves margens de rios (Patreze amp Cordeiro 2004) embora possa tambeacutem
ocorrer em formaccedilotildees quase exclusivas dessa espeacutecie nas encostas de morros (Jacobi amp
Ferreira 1991)
Em relaccedilatildeo agraves atividades elucidadas para os extratos de maricaacute foram relatados os
efeitos alelopaacutetico (Jacobi amp Ferreira 1991 Ferreira et al 1992) diureacutetico natriureacutetico e
caliureacutetico (Schlickmann et al 2017)
9
Hipoacutetese
Mimosina apresenta perfil dinacircmico de acuacutemulo em Leucaena leucocephala e
Mimosa bimucronata frente a estresses associado a alteraccedilotildees significativas na expressatildeo de
genes relacionados ao metabolismo deste ANP o qual contribui para mitigar o desequiliacutebrio
oxidativo inerente a vaacuterios tipos de estresse
Objetivo geral
O objetivo da presente tese foi investigar o papel bioloacutegico da mimosina endoacutegena
em leucena e maricaacute a partir da avaliaccedilatildeo do efeito de tratamentos relacionados a estresses
ou sinalizadores de estresse
Objetivos especiacuteficos
- Analisar a concentraccedilatildeo constitutiva de mimosina nos diferentes oacutergatildeos de L leucocephala
(Lam) de Wit (leucena) e M bimucronata (DC) Kuntze (maricaacute)
- Verificar se apesar do seu alto teor constitutivo em plantas de leucena o acuacutemulo de
mimosina pode ser induzido com tratamentos que mimetizam diferentes estresses a partir da
avaliaccedilatildeo do efeito de moleacuteculas sinalizadoras (aacutecido saliciacutelico jasmonato etileno) e da
exposiccedilatildeo agrave radiaccedilatildeo UV-C na modulaccedilatildeo do acuacutemulo de mimosina em leucena bem como
em maricaacute
- Determinar se a expressatildeo de genes relacionados ao metabolismo de mimosina estaacute
associada agrave induccedilatildeo por estresses fisioloacutegicos
- Avaliar o potencial antioxidante da mimosina em experimentos realizados in situ
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry
journal homepage wwwelseviercomlocateplaphy
Research article
Mimosine accumulation in Leucaena leucocephala in response to stresssignaling molecules and acute UV exposure
Kelly Cristine da Silva Rodrigues-Correcircaab Michael DH Hondab Dulal BorthakurbArthur Germano Fett-Netoalowast
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970Porto Alegre Rio Grande do Sul BrazilbDepartment of Molecular Biosciences and Bioengineering University of Hawaii at Manoa Honolulu HI 96822 USA
A R T I C L E I N F O
KeywordsLeucaena leucocephalaMimosineMimosine amidohydrolaseJasmonic acidEthyleneSalicylic acidUV-C radiation
A B S T R A C T
Mimosine is a non-protein amino acid of Fabaceae such as Leucaena spp and Mimosa spp Several relevantbiological activities have been described for this molecule including cell cycle blocker anticancer antifungalantimicrobial herbivore deterrent and allelopathic activities raising increased economic interest in its pro-duction In addition information on mimosine dynamics in planta remains limited In order to address this topicand propose strategies to increase mimosine production aiming at economic uses the effects of several stress-related elicitors of secondary metabolism and UV acute exposure were examined on mimosine accumulation ingrowth room-cultivated seedlings of Leucaena leucocephala spp glabrata Mimosine concentration was not sig-nificantly affected by 10 ppm salicylic acid (SA) treatment but increased in roots and shoots of seedlings treatedwith 84 ppm jasmonic acid (JA) and 10 ppm Ethephon (an ethylene-releasing compound) and in shoots treatedwith UV-C radiation Quantification of mimosine amidohydrolase (mimosinase) gene expression showed thatethephon yielded variable effect over time whereas JA and UV-C did not show significant impact Consideringthe strong induction of mimosine accumulation by acute UV-C exposure additional in situ ROS localization aswell as in vitro antioxidant assays were performed suggesting that akin to several secondary metabolitesmimosine may be involved in general oxidative stress modulation acting as a hydrogen peroxide and superoxideanion quencher
1 Introduction
Different plant groups synthesize a large diversity of secondary orspecialized metabolites These molecules are generally produced inresponse to biotic and abiotic environmental stresses Indeed inductionof secondary metabolism usually involves stress-generating factorswhich have also been explored in biotechnological processes aiming atthe production of target metabolites of economic interest (Matsuuraet al 2018) Metabolic control of nitrogen-containing secondarycompounds (eg alkaloids and non-protein amino acids) has beenshown to be complex and influenced by phytohormones environmentalstresses (seasonality herbivory pathogen attack drought) UV radia-tion (Holloacutesy 2002) methyl jasmonate (MeJA) salicylic acid (SA)yeast extract (Cho et al 2008) abscisic acid (ABA) heavy metals os-motic stress (Nascimento et al 2013) and mechanical wounding (Portoet al 2014)
Due to their particular trait of associating with N-fixing micro-organisms Fabaceae species (leguminous sensu lato) are often proteinrich hence the relevance of several of these species as forage Fabaceaespecies are also known for accumulating nitrogen containing secondarymetabolites which play important roles as ecochemical molecules andat least for the case of non-protein amino acids potential cell reservoirsof nitrogen (Huang et al 2011)
High contents of mimosine a toxic aromatic non-protein aminoacid are found in species of two leguminous genera Leucaena spp andMimosa spp Leucaena leucocephala (Lam) de Wit (leucaena koa haole)is a fast-growing leguminous tree native from Central America (south-eastern Mexico) widely distributed in tropical and subtropical zonesThis species is also characterized by its high tolerance to droughtamong other environmental stresses (Honda et al 2018) Leucaena canbe divided into two subspecies (i) L leucocephala subsp leucocephala(common leucaena a bushy shrub) and (ii) L leucocephala subsp
httpsdoiorg101016jplaphy201811018Received 1 August 2018 Received in revised form 9 November 2018 Accepted 14 November 2018
lowast Corresponding authorE-mail addresses krodriguescbiotufrgsbr (KCdS Rodrigues-Correcirca) mhonda2hawaiiedu (MDH Honda) dulalhawaiiedu (D Borthakur)
fettnetocbiotufrgsbr (AG Fett-Neto)
Plant Physiology and Biochemistry 135 (2019) 432ndash440
Available online 19 November 20180981-9428 copy 2018 Elsevier Masson SAS All rights reserved
T
glabrata (giant leucaena a tree) The latter has been used as a fastgrowing tree for production of wood and paper pulp The foliage ofboth common and giant leucaena is used as a fodder because of its highprotein content and palatability to farm animals The foliage containsup to 18 protein 142 crude fiber and 64 ether extractcrude fat(Soedarjo and Borthakur 1996)
Production of nitrogen-containing secondary metabolites such asmimosine requires large amounts of carbon and nitrogen resourcesNegi et al (2014) estimated that up to 21 of the carbon-nitrogenresources may be used for production of mimosine in leucaenaBrewbaker et al (1972) determined the mimosine content of 96 Lleucocephala cultivars and 8 other Leucaena species collected from 38different countries by growing them in an observational nursery inHawaii and found that basal mimosine content varied from 189 to477 of the dry weight
Mimosine is biosynthesized from OAS (o-acetylserine) and 3H4P (3-hydroxy-4-pyridone or its tautoisomer 3-hydroxy-4-pyridine) A pre-vious analysis suggested that mimosine synthase is an OAS-TL (o-acetylserine-thiol-lyase) of the cysteine biosynthesis pathway (Ikegamiet al 1990) Later however recombinant enzyme tests did not supportan OAS-TL identity of mimosine synthase (Yafuso et al 2014) Recentfindings on mimosine biosynthesis revealed that a cytosolic cysteine-OAS-TL isoform can also catalyze the formation of mimosine underspecific conditions (Harun-Ur-Rashid et al 2018)
Mimosine toxicity is related to its ability of reducing the availabilityof divalent metal ions such as Fe(II) Zn(II) Cu(II) Co(II) and Mn(II)by chelating co-factors and preventing their association with metal-dependent enzymes Furthermore this non-protein amino acid is cap-able of forming a stable complex with pyridoxal-5prime-phosphate (PLP)leading to the inactivation of PLP-dependent enzymes (eg Asp-Glutransaminase and cystathionine synthetase) (Negi et al 2014)
Mimosine features several useful biological activities such as alle-lopathic antimicrobial insecticide cell cycle inhibitor agent antic-ancer phytoremediator (Nguyen and Tawata 2016) as well as anti-oxidant (Benjakul et al 2013) Despite the relatively well establishedbiological activities of purified mimosine on other organisms or celltypes little is known about its biological role in leguminous speciesHowever it has been suggested that at least in part its activity ismainly related to defense mechanisms against some biotic and abioticstresses and as nitrogen source during fast growth (Vestena et al2001)
Suda (1960) and Smith and Fowden (1966) identified enzymes in-volved in mimosine degradation in seedling extracts of L leucocephalaand Mimosa pudica A mimosine-degrading enzyme named mimosinase(mimosine amidohydrolase EC 35161 CAS registry number 104118-49-2) (IUBMB 2018) a carbon-nitrogen lyase which degrades mimo-sine into 3H4P was later purified by Tangendjaja et al (1986) Itsbiochemical characterization was described and the cDNA was isolatedby Negi et al (2014)
Although mimosinase has been described and isolated only fewstudies on the role played by biotic and abiotic factors on the dynamicmodulation of mimosine metabolism in leguminous species have beenconducted (Vestena et al 2001 Xu et al 2018) In aseptic cultures ofleucaena mechanical injury of shoots promoted local mimosine accu-mulation (Vestena et al 2001) In the same study cultivation in pre-sence of auxin or SA in culture medium also had a positive effect on
mimosine accumulation More recently the effect of drought treatmenton gene expression of leucaena was also evaluated by Honda et al(2018) However several potential factors regulating mimosine meta-bolism need to be further examined
To date there is a lack of information on the biological role ofmimosine in planta as well as on details of its metabolic dynamicsMoreover its overt potential for pharmaceutical applications and de-velopment of new drugs as well as the possible use as tool to addressheavy metal soil contamination or plant mineral nutrition improve-ment justify additional research The objective of this study was toinvestigate the effect of stress signaling molecules and acute UV ex-posure on modulation of mimosine accumulation and metabolism in Lleucocephala spp glabrata in order to better understand its biologicalrole and to identify strategies for yield improvement aiming at ex-ploring its useful bioactivities
2 Methods
21 Plant material
For the experiments carried out to evaluate the effects of elicitors onmimosine accumulation seeds of leucaena were kindly provided by DrJames Brewbaker and harvested at CTAHRs (College of TropicalAgriculture and Human Resources of the University of Hawaii atManoa) Waimanalo Research Station at Oahu Hawaii This plantmaterial was originated from the accession K636 of Leucaena leucoce-phala ssp glabrata (Brewbaker 2008)
22 Induced mimosine content in 5-week-old giant leucaena
221 Seed germinationIn order to overcome seed coat dormancy seeds were submitted to a
chemical scarification with sulfuric acid 95ndash98 for 20min and re-peatedly rinsed in distilled water to remove any residual trace of thisreagent Then seeds were distributed in 254 cmtimes508 cm plastictrays containing 11 vv of vermiculite and commercial soil watereduntil reaching substrate field capacity Three weeks after seed imbibi-tion seedlings displaying similar size and shape (eg number of com-pound leaves and leaflets) were transplanted to individual pots(250mL) in number of three plants per container
During the experimental period (except in the UV-C radiationtreatment) all tested seedlings were kept in a growth chamber andsubmitted to controlled conditions of temperature (circa 25 degC) and ir-radiance (approximately 100 μmol photons mminus2sdot s minus1) with a photo-period of 16 h light and 8 h dark
222 Treatments2221 JA Ethephon and SA Five-week-old giant leucaena seedlingswere treated with different solutions as described in Table 1 Idealconcentrations were defined in preliminary experiments under the sameconditions indicated above At the beginning of the experiments 30plants were sprayed with 84 ppm JA 10 ppm SA 10 or 100 ppmEthephon or Milli-Qreg water (control) until the point of imminent runoffPlant pots were kept closed inside transparent plastic bags for 24 h toavoid solution volatilization Fifteen plants arranged in 5 sets of 3 (5biological replicates) were harvested 48 h and 96 h after being treated
Table 1Treatments used to modulate mimosine biosynthesis in giant leucaena
ELICITOR CONCENTRATION UV FLUENCE EXPOSURE TIME RATIONALE FOR USE
Salicylic acid (SA) 10 ppm 24 h Pathogen signaling molecule (Shah 2003)Jasmonic acid (JA) 84 ppm 24 h Chemical elicitor of plant secondary metabolism (Dar et al 2015)Ethephon 10 ppm 24 h Ethylene releasing-compound (Kim et al 2016) elicitor of plant secondary metabolism (Wang
et al 2016)UV-C radiation 3 Jcmminus2 10min or 15min Elicitor of plant secondary metabolism (Kara 2013 Neelamegam and Sutha 2015)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
433
After collection shoots were separated from roots immediately frozenin liquid nitrogen and stored at ndash 80 degC prior to HPLC analyses
2222 UV-C Thirty seedlings of giant leucaena were exposed to UV-Cradiation (3 Jcmminus2) for 10 or 15min and kept in a growth chamberunder controlled conditions as described above until the end of theexperiments Fifteen plants arranged in groups of 3 were harvested at96 h and 120 h after UV-C exposure and processed as previouslydescribed
223 Mimosine extractionMimosine extraction was based on a modified version of the pro-
tocol published by Lalitha and Kulothungan (2006) as follows a knownweight of fresh tissue (shoots or roots) of giant leucaena was first addedto Milli-Qreg boiling water in a proportion of 110 (g of plant per mL ofsolvent) in test tubes Tubes were covered with foil to avoid solutionevaporation and placed on a hot stirrer at 100 degC for 10min A pro-portional volume of 01M HCl was added to the cooled suspensions andhomogenized using mortar and pestle The plant extracts were filteredthrough cotton and centrifuged twice for 7min in a bench top re-frigerated centrifuge at 4 degC and 13200 rpm Before being analyzed theextracts were diluted 13 with ondashphosphoric acid (OPA)
224 Mimosine detectionHPLC analyses were carried out as described by Negi and Borthakur
(2016) Pure mimosine (L-mimosine from koa haole seeds Sigma-Al-drich CAS number 500-44-7) was used as standard Separation andquantification of mimosine was done with a C18 column (PhenomenexC18 5 μm 46times250mm) under an isocratic solvent system of 002MOPA with a linear flow rate of 1mLsdotminminus1 Mimosine detection wasdone at 280 nm by photodiode array detection (200ndash400 nm) showingretention time of 277 plusmn 0042min Quantification was done using themethod of external standard curve Further confirmation of mimosineidentity was performed by co-chromatography with standard and peakpurity check Chromatograms were analyzed using the Waters Em-power 3 software
23 Quantitative real-time PCR analysis of mimosinase gene expression
Fifteen 8-week-old giant leucaena plants arranged in 4 sets of 3 (4biological replicates) were treated with either water (control) or10 ppm Ethephon 84 ppm JA acid or 15min of UV-C radiation ex-posure following the methods described above Following treatmentleucaena plants were harvested at 48 and 96 h or 72 and 144 h (UV-Ctreated plants only) after treatments Total RNA of samples was ex-tracted and purified from roots and shoots of giant leucaena by meansof a modified method using Qiagen RNeasy Plant Kit (Valencia CAUSA) and Fruit-mate (Takara Japan) according to the protocol de-scribed by Ishihara et al (2016a) The assessment of RNA quality andquantity was carried out at 230 260 and 280 nm by using a NanoDropSpectrophotometer ND-1000 (NanoDrop Technologies DE USA) Inorder to avoid genomic DNA contamination RNA samples were treatedwith TURBO DNAfree Kit (Invitrogen Carlsbad CA) Two microgramsof DNase-treated RNA were used to synthesize the first-strand cDNAusing M-MLV Reverse Transcriptase (Promega WI USA)
Quantitative real-time (qPCR) analysis was carried out to examinepossible differential expression of the mimosinase gene (GenBank ac-cession number AB2985971) in seedlings treated with 84 ppm JA10mM Ethephon or 15min of UV-C exposure Shoots and roots wereharvested 24 h before the time of mimosine concentration peak for eachtreatment previously observed as assessed by HPLC assays The 10 μLqPCR reaction consisted of 5 μL of PowerUpTM SYBRreg Green MasterMix (Applied Biosystems Foster City CA) 1 μL MgCl2 (50mM) 03 μLforward primer (10 μM) 03 μL reverse primer (10 μM) and 1 μL cDNAfirst-strand In the experimental validation through qPCR reactionconditions and melting curve analysis of the amplicon were performed
following the protocol published by Ishihara et al (2016b) for the sameleucaena variety qPCR analysis was conducted using StepOnetrade Real-Time PCR System (Applied Biosystems) Measurements were performedusing 4 biological and 3 technical replicates Relative expression wascalculated with the 2-ΔΔct method using OAS-TL as reference gene sinceits expression showed a consistently stable profile comparable to that ofUBQ-5 and ELF1α expressions Mimosinase primer sequences used forthese analyses were (FWD) 5prime- GAA AGG CAG GAA TCA CAG TGA AGAG ndash 3rsquo (REV) 5prime GGA GAC TCT AGC CAC ACC AAC TTA ndash 3rsquo
24 Antioxidant assays
241 Mimosine effect on hydrogen peroxide (H2O2) accumulationAs a follow up to the induction of mimosine accumulation profiles
under stress signals and conditions tests were conducted to verify mi-mosine antioxidant capacity In situ histological localization of hy-drogen peroxide (H2O2) accumulation was evaluated on foliar disks ofPhaseolus vulgaris L according to the protocol described by Shi et al(2010) Briefly the plant foliar tissue was exposed to 1 mgmiddotmLminus1 dia-minobenzidine (DAB) solution in 10 mM KH2PO4 (control) in presenceor absence of 10mM mimosine (equivalent to the average mimosineconcentration induced by UV-C radiation in giant leucaena) or 10mMascorbic acid (positive antioxidant control) Oxidative response wasidentified by the formation of a brown polymer on the injured leafareas indicating the presence of H2O2 and registered in a Leica M165FC stereomicroscope (Leica Microsystems)
242 Mimosine quenching of superoxide radicalsGeneration of superoxide radical and subsequent analysis was per-
formed by a modified protocol based on Zhishen et al (1999) Nitroblue tetrazolium (NBT) reduction was used to measure superoxide an-ions quenching activity Shortly a 50mM KH2PO4 pH 78 solutioncontaining 6 μM riboflavin 100mM methionine 1 mM NBT in pre-sence or absence of 5mM mimosine was exposed to white light(22 Jsdotcmminus2) for 25min on a white light transilluminator Five micro-molar rutin was used as positive control (Matsuura et al 2016) Theabsorbance was read at 560 nm before and after light exposure in aSpectraMaxreg M2 Microplate Reader (Molecular Devices LLC)
25 Statistical analyses
For HPLC and superoxide anions data simple analyses of variance(ANOVA) followed by Tukey or Welch ANOVA followed by Dunnetts Ctest were used as appropriate for data distribution characteristics InqPCR analysis results were analyzed by t-test In all cases at least fourbiological triplicates were used and experiments were repeated twiceindependently All data were analyzed using the statistical packageSPSS 200 for Windows (SPSS Inc USA) In all cases a ple 005 wasused
3 Results and discussion
31 Increased mimosine concentrations in giant leucaena treated withchemical elicitors
Leucaena produces high amounts of mimosine that accumulate in allparts of the plants including leaves stem flowers pods seeds rootsand root nodules (Soedarjo and Borthakur 1998) The highest con-centrations of mimosine can be found in the growing shoot tips andseeds (Wong and Devendra 1983) It is not known why leucaena pro-duces such high amounts of mimosine Negi et al (2014) estimated thatleucaena plants would be able to grow 21 larger if the nutrient re-sources spent on mimosine production were diverted for biomass in-crease In a previous analysis performed to quantify the basal con-centration of mimosine present in adult plants of common leucaena thehighest constitutive amount of mimosine per gram of fresh weight in
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
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the analyzed organs was found in post-anthesis flowers (89448 μg)followed by green pods (82687 μg) leaves (67358 μg) and greenflower buds (51247 μg) which showed significantly less mimosineconcentration compared to the other reproductive structures(Supplementary Fig 1) Since mature seeds have very low moisturecontent (Wencomo et al 2017) its mimosine concentration was esti-mated as 338253 μgsdotgminus1 of dry weight Additionally it was also ob-served that the basal mimosine distribution in shoots of field-grownadult plants of leucaena is dependent on the variety type(Supplementary Table 1)
Phytohormones such as salicylic acid and jasmonic acid are knownto be produced by plants in response to various abiotic and bioticstresses These phytohormones trigger adaptive responses to stress byregulating major plant metabolic processes such as photosynthesisnitrogen metabolism defense systems and plant-water relationsthereby providing protection (for review see Khan et al 2015)
Secondary or specialized metabolite production and accumulationare also known to be controlled by biotic and abiotic stresses (Matsuuraet al 2018) In this study exposure of 5-week-old giant leucaenaseedlings to JA or Ethephon treatments significantly enhanced mimo-sine accumulation in shoots and roots in at least one of the two timepoints tested (48 and 96 h) albeit in a different way (Fig 1) Thehighest concentrations of mimosine in shoots were found in seedlingstreated with JA 84 ppm (43441 μgsdotgminus1) and Ethephon 100 ppm(38412 μgsdotgminus1) two days after application of the respective phyto-hormones Nevertheless after four days shoots yielded the highestconcentration of mimosine (approximately 460 μgsdotgminus1) upon treatmentwith 10 or 100 ppm Ethephon (Fig 1A) In roots after two and four
days JA 84 ppm and Ethephon 10 ppm resulted in highest mimosineaccumulation 18488 μgsdotgminus1 and 15801 μgsdotgminus1 respectively (Fig 1B)These observations show that mimosine accumulation response tospecific elicitors may vary over time after exposure
Although all treatments were applied exclusively on shoots of giantleucaena seedlings roots of some of them were also able to respond tothe different elicitors Overall shoots displayed higher basal and in-duced mimosine concentration compared to roots (Fig 1) which agreeswith previous observations in 1 to 3-week-old aseptic seedlings ofcommon leucaena (Vestena et al 2001) However as previouslymentioned significant post-induction increase of mimosine concentra-tion in roots and shoots simultaneously was only observed for JA andEthephon 10 ppm on day 02 and 04 respectively (Fig 1)
It is well established that perceived regulatory signals or elicitorsgenerate a transduction network mediated by secondary messengersresulting in changes in gene expression profiles that afford adaptiveresponses to environmental stimuli These modulation events are oftenmediated by transcription factors (TFs) which directly bind to specificgene promoters or act by forming complexes with repressor proteinslabeling them to degradation subsequently releasing other TFs toproceed with the gene expression program This is the case of the actionmechanism of JA and its active form jasmonoyl isoleucine for example(Kazan 2015 Wasternack and Strnad 2016)
JA ethylene and SA are known as important stress regulatory sig-nals in plants JA however is thought to be the most effective signal forinduction of plant secondary metabolism (Wasternack and Strnad2016) thereby contributing to mitigation of damage caused by severalstresses (Dar et al 2015) JA is mainly derived from linolenic acid
Fig 1 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings treated with differentelicitors CTRL=Milli-Q water SA = Salicylic AcidJA= Jasmonic Acid ETH=Ethephon Bars sharing a letterof same case do not differ by Tukey test (P le 005) Capitalletters (A B) compare treatments on day two and lowercaseletters (a b) compare treatments on day four Indicatessignificant statistical difference between day two and dayfour in the same treatment by t-test (Ple 005) The errorbars represent standard error of five replicates (each meanwas calculated with 15 individual seedlings organized in 5groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
435
(Wasternack and Strnad 2016) playing important roles in differentprocesses of plant growth and development such as plant defensemechanisms against herbivory pathogen attack fungal elicitation andsome abiotic factors such as osmotic temperature and salt stresses (Daret al 2015)
JA and its methyl ester MeJA have several different effects on le-guminous species MeJA exogenous application has increased iso-flavonoid content in cell suspension cultures of Pueraria candollei varcandollei and P candollei var mirifica (Korsangruang et al 2010) aswell as the production of the triterpenoid glycyrrhizin in Glycyrrhizaglabra roots Enhanced production of the triterpenoid however waspartly at the expense of root growth (Shabani et al 2009) MeJA ap-plication on shoots was observed to suppress root nodulation and lat-eral root formation in Lotus japonicus (Nakagawa and Kawaguchi2006) In grapevine a non-leguminous species proteinogenic aminoacids did not show an expressive increase under MeJA treatment(Gutieacuterrez-Gamboa et al 2017)
The effects of the application of four different jasmonate forms (JAMeJA jasmonoyl-L-isoleucine (JA-Ile) and 6-ethyl indanoyl glycineconjugate (2-[(6-ethyl-1-oxo-indane-4-carbonyl)-amino]-acetic acidmethyl ester - CGM) on leucaena metabolite profile has recently beenreported by Xu et al (2018) JA-Ile form was most effective althoughno major alteration was observed on monitored metabolite abundancesAlanine threonine and 34-dihydroxypyridine (34 DHP a metabolitederived from mimosine degradation) (Nguyen and Tawata 2016)among others were the major metabolites elicited by JA-Ile In contrastto the results described here mimosine concentration did not changesignificantly These divergent results on mimosine accumulation maybe due to a number of factors including mode of application jasmonateform used (JA-Ile x JA) and L leucocephala subspecies (common x giantleucaena)
Ethylene is also a phytohormone involved in plant response me-chanisms to different types of challenges such as mechanical damageand insect attack among others The integration mechanism betweenJA and ethylene signaling pathways is not completely understoodhowever it has been shown that they may work cooperatively in abioticstress tolerance (Kazan 2015) MeJA can induce ethylene production(Zhao et al 2004) and when applied simultaneously these moleculesseem to work in a synergic way by enhancing the magnitude of theplant response to external stimuli (Liu et al 2016)
Treatment with SA was able to significantly increase mimosine ac-cumulation in 12-week-old plants of common leucaena (SupplementaryFig 2) However no significant effect of SA treatment on mimosineconcentration was seen in 5-week-old seedlings of giant leucaena(Fig 1) suggesting some degree of genotype andor age dependency inelicitation by this phytohormone On the other hand several treat-ments including 90 ppm MeJA 10 and 100 ppm 2-chloroethylpho-sphonic acid (CEPA an ethylene-releasing compound) significantlyincreased mimosine accumulation (Supplementary Fig 2) in agree-ment with the data obtained for giant leucaena The lack of systemiceffects of externally applied SA on mimosine accumulation was alsoobserved when the phytohormone was supplied in the culture mediumof aseptically-grown seedlings in which case only roots had highercontent of mimosine (Vestena et al 2001) This could be due totransport limitations or to low methyl salicylate production from ap-plied SA since the former is recognized as the main systemic signalingform (Vlot et al 2009)
32 Increased mimosine concentrations in giant leucaena exposed to UV-Cradiation
UV-C treatment promoted increased concentration of the aminoacid in shoots but not in roots of giant leucaena (Fig 2) Increasedaccumulation of mimosine in shoots was also observed in 12-week-oldseedlings of common leucaena exposed to UV-C radiation for 10 and15min (Supplementary Fig 3) Similar to the SA treatment in giant
leucaena UV-C radiation did not induce mimosine biosynthesis in rootsregardless of time after exposure The absence of mimosine induction inroots by SA and UV indicates that these effectors do not cause a sys-temic response Moreover roots are shielded from irradiance by thepresence of substrate
UV radiation effects on different aspects of plant metabolism anddevelopment have been described However compared to UV-B (en-vironmentally relevant type of UV radiation) assays there are less re-ports related to the UV-C effects on secondary metabolites biosynthesisand accumulation (Cetin 2014) especially in leguminous (Fabaceae)plants They generally concern primary metabolism aspects such asgrowth and development For instance seedlings of Phaseolus vulgaris L(Fabaceae) exposed to low intensity UV-C radiation have displayeddecreased chlorophyll content and reduced height after 14 days of ex-posure (Kara 2013) Negative effects on growth parameters and ni-trogen metabolism were also observed in Vigna radiata L (Fabaceae)after UV-B radiation treatment in addition to adverse effects on JA SAand antioxidant compounds accumulation (Choudhary and Agrawal2014a) The same authors reported increased accumulation of flavo-noids SA and JA besides negative effects on growth biomass yieldnitrogen fixation and accumulation in 2 cultivars of Pisum sativum L(Fabaceae) under elevated UV-B treatment (Choudhary and Agrawal2014b) Despite the negative UV influence on growth reported for thepreviously mentioned leguminous UV-C radiation on groundnut plants(Arachis hypogaea L Fabaceae) increased seedling vigor and biomassand had no adverse effect on germination or other development para-meters (Neelamegam and Sutha 2015)
Besides its impact on growth and primary metabolism UV exposurecan cause important changes in secondary metabolism depending onintensity and time of exposure (Matsuura et al 2013) UV-B and UV-Cpre-treatments of Artemisia annua (Asteraceae) seedlings yielded in-creased biosynthesis of artemisinin a drug which displays anti-malarialproperties and activity against some others infectious diseases (egschistosomiasis leishmaniasis and hepatitis B) and several kinds oftumors (Rai et al 2011) The accumulation of nicotine in Nicotianarustica plants (Solanaceae) was also increased by UV-C treatment(Tiburcio et al 1985) Similar inducing effects on production of severalsecondary metabolites were observed in callus cultures of Vitis viniferaL Oumlkuumlzgoumlzuuml (grapevine Vitaceae) treated with a UV-C source for 5 or10min (Cetin 2014)
Regarding amino acid biosynthesis in response to UV radiationMartiacutenez-Luumlscher et al (2014) have found that in spite of not causingchanges in total amino acid content UV-B radiation exposure can affecttheir profile in grape berries Proteinogenic amino acids have beenknown to be important targets of the deleterious effects of UV radiation(Holloacutesy 2002) On the other hand in the present study acute UV-Ctreatment was found to increase mimosine accumulation in shoots byover twofold (Fig 2) which may suggest a possible participation of thismolecule as part of the antioxidant defense system in L leucocephalaThis possibility is further supported by the induction of the amino acidaccumulation by JA and Ethephon involved in abiotic and biotic stressresponses which are generally associated with oxidative imbalance andare signaling components in high UV stress (Matsuura et al 2013)
33 Mimosinase gene expression
In order to determine if increases in mimosine content upon ex-posure to JA CEPA or UV-C radiation were related to changes intranscription of mimosine metabolism-related genes RT-qPCR analysiswas carried out The complete pathway for mimosine biosynthesis hasnot yet been determined although the final step has been character-ized Based on transcription analysis (Ishihara et al 2016a) leucaenaappears to encode for multiple cysteine synthases one or more of whichmay be able to catalyze mimosine synthesis In addition a leucaenagene encoding a mimosinase (an enzyme responsible for mimosinedegradation) has been identified and characterized (Negi et al 2014)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
436
In addition to mimosinase gene expression several gene isoformsbelonging to the cysteine pathway [cysteine synthase (CYS SYN) serineacetyltransferase (SAT) and β-cyanoalanine synthase (CAS) Table 2 -supplementary material] were also tested in this study (data notshown) However expressions of these genes did not vary in giantleucaena throughout the experiments suggesting that the increasedcontent of mimosine observed in the treated plants might not be relatedto the expression of these genes but presumably to increased enzymeactivity andor release from conjugates such as mimoside a mimosineβ-D-glucoside (Murakoshi et al 1972)
Considering the time variation of mimosine accumulation observedin this work mimosinase gene expression in shoots and roots wasevaluated 24 h before the increase of mimosine concentration in giantleucaena seedlings (ie 24 h and 72 h after the chemical elicitorstreatments and 48 h and 120 h after UV-C exposure)
Ethylene signaling has been shown to up-regulate expression ofseveral genes related to secondary metabolism pathways as is the caseof phenolic compounds (Liu et al 2016) and terpenoid indole alkaloids(Wang et al 2016) Among all elicitors tested in the present workEthephon was the only one able to significantly change mimosinasegene expression Leucaena plants treated with Ethephon showed sig-nificant increases in mimosine concentration at both day 2 and 4 fol-lowing treatment which coincided with low-level expression of mi-mosinase Up-regulation of mimosinase gene expression was detected24 h before the increase of mimosine concentration in shoots treatedwith 10 ppm of Ethephon (Fig 3A) but not after JA or UV-C treatments(Fig 3C-D and 3E-F respectively) Nevertheless 72 h after treatment
application (24 h before the highest mimosine content measured inshoots) down regulation of mimosinase gene was seen in both shootsand roots treated with 10 ppm of Ethephon (Fig 3B) These data in-dicate that mimosine content in leucaena plants is at least partlyregulated by mimosinase expression in Ethephon exposed plants Onthe other hand the fact that mimosinase mRNA was not significantlyaffected by JA and UV-C treatments despite their stimulating effects onmimosine biosynthesis in giant leucaena may indicate that other levelsof regulation are at play or that the chosen harvesting time window wasunable to detect relevant changes
34 In situ and in vitro antioxidant assays
Considering the stimulation of mimosine accumulation byEthephon JA and UV all of which are often associated or known tocause oxidative imbalance the antioxidant capacity of mimosine wasevaluated Mimosine has been shown to have antioxidant activities oncultured cancer cells (Parmar et al 2015) In the present study it washypothesized that mimosine could confer radical scavenging propertieswhich would contribute to plant protection from possible damagecaused by reactive oxygen species generated during stress(Supplementary Fig 4)
Foliar disks of P vulgaris L were treated with 10mM mimosine for15min Treated disks showed less hydrogen peroxide accumulationinduced by wounding in contrast to untreated ones being comparableto those treated with ascorbic acid (a known hydrogen peroxide neu-tralizer) (Fig 4A) These observations support a possible antioxidant
Fig 2 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings exposed to UV-C lightCTRL= visible light (100 μmol photons mminus2 s minus1) UV-C 10primeand UV-C 15rsquo=UV-C exposure time (10 and 15min re-spectively) Bars sharing a letter of same case do not differ byTukey test (P le 005) Capital letters (A B) compare treat-ments on day three and lowercase letters (a b) comparetreatments on day six Indicates significant statistical dif-ference between day three and day six in the same treatmentby t-test (Ple 005) The error bars represent standard errorof five replicates (each mean was calculated with 15 in-dividual seedlings organized in 5 groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
437
role of mimosine as an in situ hydrogen peroxide scavengerMimosine was also able to quench superoxide anions generated by
light exposure Mimosine exhibited equivalent antioxidant effect com-pared to rutin (Fig 4B) a well-established effective superoxide anionquencher (Matsuura et al 2016) The radical scavenging activity ofmimosine may be due to the 3-OH group of the pyridine ring of mi-mosine (Fig 5) The pKa of the 3-OH of mimosine has been estimated tobe 88 (M Honda unpublished results) At physiological pH this OHgroup is expected to remain in a protonated state and therefore mayscavenge a radical by donating a proton and an electron In this processmimosine itself is converted to a stable radical form which is perhapsless toxic and less reactive than the reactive oxygen species generatedduring oxidative stress It is likely that the less toxic radical mimosineproduced may react with another radical or molecule and becomeconverted to a non-reactive indole molecule
In vivo antioxidant activity of mimosine has been previously eval-uated by means of its exogenous application on selenium-deficientseedlings of Vigna radiata In spite of its allelopathic properties (Ahmedet al 2008) the results showed mitigation of mitochondrial oxidativestress by treatment with 01mM mimosine (Lalitha and Kulothungan2007) DPPH radical scavenging activity was also reported for aqueous
seed extracts of leucaena rich in mimosine and phenolic compounds inin vitro assays (Benjakul et al 2014) Mimosine antioxidant activityshown in the present work is in good agreement with data reported forother non-protein amino acids such as L-DOPA (Dhanani et al 2015)and GABA (Malekzadeh et al 2014) for instance
4 Conclusion
Taken together results show that mimosine biosynthesis and ac-cumulation can be modulated by stress-related factors despite its re-latively high constitutive content in leucaena plants The pattern ofgene expression in stressed plants suggests mimosine steady-state con-trol may be regulated by its degradation in possible connection withdynamic changes in carbon and nitrogen metabolism of stressed plantsMimosine quenching activity against hydrogen peroxide and super-oxide anions in the in situ staining and in vitro assays respectivelyshowed that this non-protein amino acid can act as non-enzymaticantioxidant agent Increase in mimosine content in response to elicitorsmimicking environmental challenges in addition to its antiherbivoreand antimicrobial properties may be related to its activity as protectivemolecule against oxidative damage in line with other classes of plant
Fig 3 Relative expression of the mimosinase gene in shoots (A E and F) and shoots and roots (B C and D) of giant leucaena 24 h (A and C) 48 h (E) 72 h (B and D)and 120 h (F) after treatment with stress signaling molecules or UV-C exposure ETH = Ethephon JA = Jasmonic Acid Indicates significant statistical differencebetween control and treatment by t-test (Ple 005) The error bars represent standard error of four replicates
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
438
secondary metabolites
Funding
This work was funded by the National Council for Scientific andTechnological Development (CNPq-Brazil) grant 3060792013-5Coordenaccedilatildeo de Aperfeiccediloamento de Pessoal de Niacutevel Superior - Brazil(CAPES) - Finance Code 001 and the USDA NIFA Hatch projectHA05029-H managed by CTAHR
CRediT authorship contribution statement
Kelly Cristine da Silva Rodrigues-Correcirca InvestigationValidation Writing ndash original draft Michael DH HondaInvestigation Validation Dulal Borthakur Supervision Writing ndashreview amp editing Funding acquisition Arthur Germano Fett-NetoSupervision Funding acquisition Writing ndash review amp editing
Acknowledgements
The authors would like to thank Dr Jorge Ernesto Mariath fromLaVeg-UFRGS for kindly lending the Leica M165 FC stereomicroscopefor in situ analysis
Appendix A Supplementary data
Supplementary data to this article can be found online at httpsdoiorg101016jplaphy201811018
References
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Benjakul S Kittiphattanabawon P Shahidi F Maqsood S 2013 Antioxidant activityand inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipidoxidation in model systems Food Sci Technol Int 19 (4) 365ndash376 httpsdoiorg1011771082013212455186
Benjakul S Kittiphattanabawon P Sumpavapol P Maqsood S 2014 Antioxidantactivities of lead (Leucaena leucocephala) seed as affected by extraction solvent priordechlorophyllisation and drying methods extracts against lipid oxidation in modelsystems Food Sci Technol 51 (11) 3026ndash3037 httpsdoiorg101007s13197-012-0846-1
Brewbaker JL Pluckett D Gonzalez V 1972 Varietal variation and yield trials ofLeucaena leucocephala (koa haole) in Hawaii Hawaii Agric Exp Stn Bull 166 26
Brewbaker JL 2008 Registration of KX2 ndash Hawaii interspecific-hybrid leucaena JPlant Registrations 1 (3) 190ndash193 httpsdoiorg103198jpr2007050298crc
Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitisvinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-6287-47-37
Cho H-Y Son SY Rhee HS Yoon S-YH Lee-Parsons CWT Park JM 2008Synergistic effects of sequential treatment with methyl jasmonate salicylic acid andyeast extract on benzophenanthridine alkaloid accumulation and protein expressionin Eschscholtzia californica suspension cultures J Biotechnol 135 117ndash122 httpsdoiorg101016jjbiotec200802020
Choudhary KK Agrawal SB 2014a Cultivar specificity of tropical mung bean (Vignaradiata L) to elevated ultraviolet-B changes in antioxidative defense system ni-trogen metabolism and accumulation of jasmonic and salicylic acids Environ ExpBot 99 122ndash132 httpsdoiorg101016jenvexpbot201311006
Choudhary KK Agrawal SB 2014b Ultraviolet-B induced changes in morphologicalphysiological and biochemical parameters of two cultivars of pea (Pisum sativum L)Ecotoxicol Environ Saf 100 178ndash187 httpsdoiorg101016jecoenv201310032
Dar TA Uddin M Khan MMA Hakeem KR Jaleel H 2015 Jasmonates counterplant stress a Review Environ Exp Bot 115 49ndash57 httpsdoiorg101016jenvexpbot201502010
Dhanani T Singh R Shah S Kumari P Kumar S 2015 Comparison of green ex-traction methods with conventional extraction method for extract yield L-DOPAconcentration and antioxidant activity of Mucuna pruriens seed Green Chem LettRev 8 (2) 43ndash48 httpsdoiorg1010801751825320151075070
Gutieacuterrez-Gamboa G Portu J Santamariacutea P Loacutepez R Garde-Cerdaacuten T 2017Effects on grape amino acid concentration through foliar application of three dif-ferent elicitors Food Res Int 99 688ndash692 httpsdoiorg101016jfoodres201706022
Fig 4 A In situ antioxidant assay Foliar disksof Phaseolus vulgaris L treated with (a) No an-tioxidant added (negative control) (b) 10 mMMimosine (c) 10mM ascorbic acid (positivecontrol) The oxidative damage can be seen bythe formation of a brown polymer in leaf veinsand injured areas B In vitro superoxidescavenging assay carried out with mimosineDifferent letters indicate significant differenceby Tukey test (Ple 005) The error bars re-present standard error of four replicates (Forinterpretation of the references to colour in thisfigure legend the reader is referred to the Webversion of this article)
Fig 5 Predicted mimosine radical formed followingquenching of hydroxyl radical Mimosine is first converted toa stable mimosine radical which may be then converted to anontoxic indole form
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
439
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad HossainMd Anai T Oku H 2018 Cytosolic cysteine synthase switch cysteine and mi-mosine production in Leucaena leucocephala Appl Biochem Biotechnol 186 (3)613ndash632 httpsdoiorg101007s12010-018-2745-z
Holloacutesy F 2002 Effects of ultraviolet radiation on plant cells Micron 33 (2) 179ndash197Honda MDH Ishihara KL Pham DT Borthakur D 2018 Identification of drought-
induced genes in giant leucaena (Leucaena leucocephala subsp glabrata) Trees 32571ndash585 httpsdoiorg101007s00468-018-1657-4
Huang T Jander G de Vos M 2011 Non-protein amino acids in plant defense againstinsect herbivores representative cases and opportunities for further functional ana-lysis Phytochemistry 72 1531ndash1537 httpsdoiorg101016jphytochem201103019
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of thethyrotoxic amino acid mimosine by cysteine synthase Phytochemistry 29 (11)3461ndash3465 httpsdoiorg1010160031-9422(90)85258-H
Ishihara K Lee EKW Borthakur D 2016a An improved method for RNA extractionfrom woody legume species Acacia koa A Gray and Leucaena leucocephala (Lam) deWit Int J For Wood Sci 3 (1) 031ndash035
Ishihara KL Honda MDH Pham DT Borthakur D 2016b Transcriptome analysisof Leucaena leucocephala and identification of highly expressed genes in roots andshoots Transcriptomics 4 135 httpsdoiorg1041722329-89361000135
IUBMB 2018 Enzyme Nomenclature EC 35161 httpwwwsbcsqmulacukiubmbenzymeEC35161html Accessed date 8 February 2018
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant(Phaseolus vulgaris) Biosci Res 10 (1) 29ndash32
Kazan K 2015 Diverse roles of jasmonates and ethylene in abiotic stress toleranceTrends Plant Sci 20 (4) 219ndash229 httpsdoiorg101016jtplants201502001
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect ofEthephon as an ethylene-releasing compound on the metabolic profile of Chlorellavulgaris J Agric Food Chem 64 (23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541
Khan MIR Fatma M Per TS Anjum NA Khan NA 2015 Salicylic acid-inducedabiotic stress tolerance and underlying mechanisms in plants Front Plant Sci 6 462httpsdoiorg103389fpls201500462
Korsangruang S Soonthornchareonnon N Chintapakorn Y Saralamp PPrathanturarug S 2010 Effects of abiotic and biotic elicitors on growth and iso-flavonoid accumulation in Pueraria candollei var candollei and P candollei var mir-ifica cell suspension cultures Plant Cell Tissue Organ Cult 103 (3) 333ndash342 httpsdoiorg101007s11240-010-9785-6
Lalitha K Kulothungan SR 2006 Selective determination of mimosine and its dihy-droxypyridinyl derivative in plant systems Amino Acids 31 (3) 279ndash287 httpsdoiorg101007s00726-005-0226-5
Lalitha K Kulothungan SR 2007 Mimosine mitigates oxidative stress in seleniumdeficient seedlings of Vigna radiata - Part I restoration of mitochondrial functionBiol Trace Elem Res 118 (1) 84ndash96 httpsdoiorg101007s12011-007-0013-0
Liu J Li Y Wang Y Zhang Z-H Zu Y-G Efferth T Tang Z-H 2016 Thecombined effects of ethylene and MeJA on metabolic profiling of phenolic com-pounds in Catharanthus roseus revealed by metabolomics analysis Front Physiol 71ndash11 httpsdoiorg103389fphys201600217 Article 217
Malekzadeh P Khara J Heydari R 2014 Alleviating effects of exogenous Gamma-aminobutiric acid on tomato seedling under chilling stress Physiol Mol Biol Plants20 (1) 133ndash137 httpsdoiorg101007s12298-013-0203-5
Martiacutenez-Luumlscher J Torres N Hilbert G Richard T Saacutenchez-Diacuteaz M Delrot SAguirreolea J Pascual I Gomegraves E 2014 Ultraviolet-B radiation modifies thequantitative and qualitative profile of flavonoids and amino acids in grape berriesPhytochemistry 102 106ndash114 httpsdoiorg101016jphytochem201403014
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation ofbioactive secondary metabolites by ultraviolet radiation mechanisms strategies andapplications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology forMedicinal Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash1902012
Matsuura HN Fragoso V Paranhos JT Rau MR Fett-Neto AG 2016 Thebioactive monoterpene indole alkaloid N szlig-D-glucopyranosylvincosamide is regu-lated by irradiance quality and development in Psychotria leiocarpa Ind Crop Prod86 210ndash218 httpsdoiorg101016jindcrop201603050
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo RBhambra AS Strnad M Bonfill M Fett-Neto AG 2018 Specialized plant me-tabolism characteristics and impact on target molecule biotechnological productionMol Biotechnol 60 (2) 169ndash183 httpsdoiorg101007s12033-017-0056-1
Murakoshi S Ohmiya S Haginiwa J 1972 Enzymic synthesis of mimoside a meta-bolite of mimosine in Mimosa pudica and Leucaena leucocephala Chem Pharm Bull20 (4) 855ndash857
Nakagawa T Kawaguchi M 2006 Shoot-applied MeJA suppresses root nodulation inLotus japonicus Plant Cell Physiol 47 (1) 176ndash180 httpsdoiorg101093pcppci222
Nascimento NC Menguer PK Henriques AT Fett-Neto AG 2013 Accumulation ofbrachycerine an antioxidant glucosidic indole alkaloid is induced by abscisic acidheavy metal and osmotic stress in leaves of Psychotria brachyceras Plant PhysiolBiochem 73 33ndash40 httpsdoiorg101016jplaphy201308007
Neelamegam R Sutha T 2015 UV-C irradiation effect on seed germination seedling
growth and productivity of groundnut (Arachis hypogaea L) Int J Curr MicrobiolApp Sci 4 (8) 430ndash443
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase fromLeucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol164 (2) 922ndash934 httpsdoiorg101104pp113230870
Negi VS Borthakur D 2016 Heterologous expression and characterization of mimo-sinase from Leucaena leucocephala In Fett-Neto Arthur Germano (Ed)Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods inMolecular Biology vol 1405 copySpringer Science+Business Media New York httpsdoiorg101007978-1-4939-3393-8_7 2016
Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636
Parmar F Kushawaha N Highland H George L-B 2015 In vitro antioxidant andanticancer activity of Mimosa pudica Linn extract and L-mimosine on lymphomaDaudi cells Int J Pharm Sci 12 100ndash104
Porto DD Matsuura HN Vargas LRB Henriques AT Fett-Neto AG 2014 Shootaccumulation kinetics and effects on herbivores of the wound-induced antioxidantindole alkaloid brachycerine of Psychotria brachyceras Nat Prod Commun 9 (5)629ndash632
Rai R Meena RP Smita SS Shukla A Rai SK Pandey-Rai S 2011 UV-B and UV-C pre-treatments induce physiological changes and artemisinin biosynthesis inArtemisia annua L ndash an antimalarial plant J Photochem Photobiol B Biol 105 (3)216ndash225 httpsdoiorg101016jjphotobiol201109004
Shabani L Ehsanpour AA Asghari G Emami J 2009 Glycyrrhizin production by invitro cultured Glycyrrhiza glabra elicited by methyl jasmonate and salicylic acid RussJ Plant Physiol 56 (5) 621ndash626 httpsdoiorg101134S1021443709050069
Shah J 2003 The salicylic acid loop in plant defense Curr Opin Plant Biol 6 (4)365ndash371
Shi J Fu XZ Peng T Huang XS Fan QJ Liu JH 2010 Spermine pretreatmentconfers dehydration tolerance of citrus in vitro plants via modulation of antioxidativecapacity and stomatal response Tree Physiol 30 (7) 914ndash922 httpsdoiorg101093treephystpq030
Smith IK Fowden L 1966 A study of mimosine toxicity in plants J Exp Bot 17750ndash761 httpsdoiorg101093jxb174750
Soedarjo M Borthakur D 1996 Simple procedures to remove mimosine from youngleaves pods and seeds of Leucaena leucocephala used as food Int J Food SciTechnol 31 (1) 97ndash103
Soedarjo M Borthakur D 1998 Mimosine a toxin produced by the tree-legumeLeucaena provides a nodulation competition advantage to mimosine-degradingRhizobium strains Soil Biol Biochem 30 1605ndash1613
Suda S 1960 On the physiological properties of mimosine Bot Mag Tokyo 73 (862)142ndash147 httpsdoiorg1015281jplantres188773142
Tangendjaja B Lowry JB Wills RBH 1986 Isolation of a mimosine degrading en-zyme from leucaena leaf J Sci Food Agric 37 523ndash526 httpsdoiorg101002jsfa2740370603
Tiburcio F Pintildeol MT Serrano M 1985 Effect of UV-C on growth soluble protein andalkaloids in Nicotiana rustica plants Environ Exp Bot 25 (3) 203ndash210 httpsdoiorg1010160098-8472(85)90004-8
Vestena S Fett-Neto AG Duarte RC Ferreira A 2001 Regulation of mimosineaccumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 httpsdoiorg101016S0168-9452(01)00448-4
Vlot AC Dempsey DMA Klessig DF 2009 Salicylic acid a multifaceted hormone tocombat disease Annu Rev Phytopathol 47 177ndash206 httpsdoiorg101146annurevphyto050908135202 2009
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene-induced vinblastine accumulation is related to activated expression of downstreamTIA pathway genes in Catharanthus roseus BioMed Res Int 2016 Article ID 3708187httpsdoiorg10115520163708187
Wasternack C Strnad M 2016 Jasmonate signaling in plant stress responses and de-velopment ndash active and inactive compounds N Biotech 33 (5B) 604ndash613 httpsdoiorg101016jnbt201511001
Wencomo HB Ortiz R Caacuteceres J 2017 Afr J Agric Res 12 (4) 279ndash285 httpsdoiorg105897AJAR201510604 26
Wong CC Devendra C 1983 Research on leucaena forage production in Malaysia InLeucaena Research in the Asian Pacific Region pp 55ndash60 Ottawa Ontario Canada
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX TsimKWK 2018 Jasmonate-elicited stress induces metabolic change in the leaves ofLeucaena leucocephala Molecules 23 (2) httpsdoiorg103390molecules23020188 E188
Yafuso JT Negi VS Bingham J-P Borthakur D 2014 An O-acetylserine (thiol)lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthaseAppl Biochem Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z
Zhao J Zheng S-H Fujita K Sakai K 2004 Jasmonate and ethylene signalling andtheir interaction are integral parts of the elicitor signalling pathway leading to b-thujaplicin biosynthesis in Cupressus lusitanica cell cultures J Exp Bot 55 (399)1003ndash1012 httpsdoiorg101093jxberh127
Zhishen J Mengcheng T Jianming W 1999 The determination of flavonoid contentsin mulberry and their scavenging effects on superoxide radicals Food Chem 64 (4)555ndash559 httpsdoiorg101016S0308-8146(98)00102-2
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
440
61
Supplementary Fig 1 Basal mimosine concentration in adult trees of common leucaena (L leucocephala
var leucocephala) Samples were collected from 10 field grown trees at Manoa Valley Honolulu Hawairsquoi
on June 25th 2017 Bars sharing a letter do not differ by Tukey test (P le 005) The error bars represent the
standard error
Supplementary Fig 2 Bar diagram showing mimosine concentration in shoots of 12-week-old common
leucaena seedlings treated with different elicitors CTRL = Milli-Q water SA = Salicylic Acid MeJA =
Methyl Jasmonate CEPA = 2-Chloroethylphosphonic acid (an ethylene releasing compound) Bars sharing a
letter of same case do not differ by Tukey test (P le 005) Capital letters (A B) compare treatments on day
two and lower-case letters (a b) compare treatments on day four Indicates significant statistical difference
ABB
A A
0
200
400
600
800
1000
1200
LEAVES GREEN FLOWERBUDS
POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
con
cen
trat
ion
(micro
gg
-1o
f FW
)
B AB AB AB B A
b
a
ab b
ab
0
2
4
6
8
10
12
14
16
18
20
CTRL SA 10 ppm SA 100 ppm CEPA 10 ppm CEPA 100 ppm MeJA 90 ppm
Mim
osi
ne
co
nce
ntr
atio
n (
gg
-1o
f FW
)
DAY 02 DAY 04
62
between day two and day four in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
Supplementary Fig 3 Bar diagram showing the effects of UV-C radiation exposure for 5 10 and 15 min on
mimosine accumulation in shoots of 12-week-old seedlings of common leucaena Bars sharing a letter of
same case do not differ by Tukey test (P le 005) Capital letters (A B C) compare treatments on day three
and lower-case letters (a b) compare treatments on day six Indicates significant statistical difference
between day three and day six in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
C BC AB A
bb
a
a
0
10
20
30
40
50
60
CTRL UV-C 5 UV-C 10 UV-C 15
Mim
osi
ne
co
nce
ntr
atio
n (
gg-1
of
FW)
DAY 03 DAY 06
63
Supplementary Fig 4 Model depicting induction of mimosine synthesis in leucaena following application of
stress elicitors such as CEPA and jasmonic acid or exposure to UV-C radiation The additional mimosine
synthesized may serve to alleviate oxidative stress induced by UV-C radiation
64
Supplementary Table 1 Mimosine contents in leaves of common and giant leucaena
Leucaena
type
Mimosine content
( FW)
Mimosine
content ( DW)
Dry matter
content ( FW)
Water content
( FW)
Common (1) 050 plusmn 009 245 plusmn 051 2011 plusmn 054 7989 plusmn 054
Common (2) 043 plusmn 006 214 plusmn 037 1998 plusmn 050 8002 plusmn 050
K636 (1) 070 plusmn 014 356 plusmn 077 1908 plusmn 052 8092 plusmn 052
K636 (2) 042 005 205 plusmn 033 2008plusmn 093 7992plusmn 093
KX2 (1) 122 plusmn 011 608 plusmn 082 1939 plusmn 123 8061 plusmn 123
KX2 (2) 134 plusmn 010 623 plusmn 056 2029 plusmn 114 7971 plusmn 114
KX3 (1) 044 plusmn 006 221 plusmn 030 1945 plusmn 073 8055 plusmn 073
KX3 (2) 054 plusmn 005 273 plusmn 023 1930 plusmn 038 8070 plusmn 038
KX4 (1) 086 plusmn 011 471 plusmn 065 1753 plusmn 084 8247 plusmn 084
KX4 (2) 089 plusmn 011 476 plusmn 065 180 plusmn 072 820 plusmn 072
KX5 (1) 099 plusmn 012 489 plusmn 048 1907 plusmn060 8093 plusmn 060
KX5 (2) 115 plusmn 015 548 plusmn080 1992 plusmn 053 8008 plusmn 053
Common leucaena variety koa haole grows widely on the island of Orsquoahu K636 is widely
grown variety of giant leucaena KX2 KX3 KX4 and KX5 are giant leucaena varieties
developed through interspecies hybridization (Brewbaker 2016) (1) and (2) indicate plants
from two separate locations within the University of Hawaii Waimanalo Research Center The
values are shown as mean plusmn standard error obtained from at least three biological replicates
65
Supplementary Table 2 GenBank accession numbers of the tested cysteine pathway genes isoforms
Gene name GenBank accession
OAS-TL (o-acetylserine-thiol-lyase) GDRZ01032940
GDRZ01061620
GDRZ01153117
GDSA01187555
GDSA01196891
GDSA01214467
Cys syn (cysteine synthase) GDRZ01015860
GDRZ01050898
GDRZ01086813
GDRZ01193515
GDRZ01202579
GDSA01180863
GDSA01215622
SAT (serine acetyltransferase) GDRZ01187456
GDRZ01189631
CAS (β-cyanoalanine synthase) GDRZ01054066
GDRZ01175418
GDSA01118400
66
SHORT COMMUNICATION 1
Mimosine occurrence and accumulation in Mimosa bimucronata var bimucronata (DC) 2
Kuntze 3
Kelly Cristine da Silva Rodrigues-Correcirca1 Lana Dorneles Pedroso2 Fernanda de Costa1 4
Arthur Germano Fett-Neto1 5
1Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal 6
University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970 7
Porto Alegre Rio Grande do Sul Brazil 2Department of Biological Sciences Unipampa ndash 8
Campus Satildeo Gabriel 9
Corresponding author 10
E-mail addresses krodriguescbiotufrgsbr (KCdaS Rodrigues-Correcirca) 11
lanalima2012gmailcom (LD Pedroso) fernandadecostayahoocombr (F de Costa) 12
fettnetocbiotufrgsbr (AG Fett-Neto) 13
14
15
16
17
18
19
20
21
22
67
ABSTRACT 23
Mimosine is a non-protein aromatic amino acid present in plants of Leucaena spp 24
and Mimosa spp Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) is a native 25
tree from Brazil which occurs as a pioneer species on plant succession processes In the 26
current study the presence of mimosine in M bimucronata was verified by HPLC analyses 27
Moreover mimosine accumulation upon exposure to UV-C and chemical elicitors of 28
specialized metabolism (salicylic acid - SA methyl jasmonate - MeJA sodium nitroprusside 29
- SNP and ethephon - ETH) most of which also known as promoters of the amino acid 30
production in leucaena plants was evaluated The results showed a lower concentration of 31
constitutive mimosine present in both maricaacute seedlings and mature trees when compared to 32
leucaena plants In spite of a trend towards increased mimosine accumulation observed in 33
MeJA and ETH treatments no statistical differences were found with the various stressors 34
used to induce its biosynthesis in maricaacute seedlings Data suggest that mimosine in M 35
bimucronata is probably a phytoanticipin-like metabolite or its accumulation is driven by 36
other types of stresses 37
38
39
Keywords Mimosine Mimosa bimucronata stress 40
41
42
43
44
45
46
68
Introduction 47
Mimosa bimucronata commonly known as maricaacute is a native tree from Brazil 48
(REFLORA 2019) ecologically important in plant succession and in processes of degraded 49
land recovery (Bitencourt et al 2007 Silva et al 2011) occurring as a pioneer species 50
(Pilatti et al 2019) Maricaacute is a deciduous or semi-deciduous plant which reaches up to 15 51
m in height and 40 cm of diameter at breast height (DBH) displays shrub or tree habit and 52
bears typical sharp thorns (Carvalho 2004) This species belongs to Fabaceae one of the 53
most economically important families of flowering plants due to its high diversity and 54
occurrence in different types of habitats (Gomes et al 2018) As well as several others 55
Mimosa spp maricaacute is usually referred to as a multipurpose tree (Olkoski and Wittmann 56
2011) employed for alternative medicinal uses (Champanerkar et al 2010 Silva et al 57
2011) honey production constructions and remodeling of landscape architecture (living 58
fences) for instance (Marchiori 1993 Lorenzi 1998) 59
In southern Brazil maricaacute is widely distributed and typically found either in wetland 60
areas close to river banks (Patreze and Cordeiro 2004) or composing large and almost pure 61
landscape formations on hillsides (Jacobi and Ferreira 1991) In dense populations this 62
species like several Mimosa spp (Simon and Proenccedila 2000) is considered an important and 63
highly invasive weed by preventing cattle to reach pasturesand water bodies as a result of its 64
thorny branches (Lorenzi 2008 Kestring et al 2009) Its dominant and nearly exclusive 65
pattern of distribution in those areas has led Jacobi and Ferreira (1991) to test its allelopathic 66
potential on cultivated species Indeed extracts of leaves and ripe fruits (but not the green 67
ones) of maricaacute showed phytotoxic effects on germination and initial radical growth of most 68
of the target species tested 69
69
Several investigations have been performed on maricaacute floristics (Silva et al 2011) 70
distribution (Simon and Proenccedila 2000) wood anatomy (Marchiori 1993) cytogenetic 71
parameters (Olkoski and Wittmann 2011) and allelopathic potential (Jacobi and Ferreira 72
1991 Ferreira et al 1992) However excluding two recent publications on maricaacute 73
constitutive chemical composition (Schlickmann et al 2017 Pilatti et al 2019) which 74
identified phenolic compounds (methyl gallate and water-soluble tannins) as its major 75
compounds little is known regarding this subject In other Mimosa species (eg M pudica 76
and M pigra) mimosine has been identified (Soedarjo and Borthakur 1998) as one of the 77
major specialized metabolites present in the different organs of the plant (Champanerkar et 78
al 2010) The presence of this molecule was also reported for M bimucronata in a thin layer 79
chromatography-based preliminary study performed by Ferreira et al (1992) showing co-80
chromatography of a leaf extract component with authentic mimosine The authors attributed 81
the allelopathic effect of maricaacute to the accumulation of this metabolite in its leaves 82
Mimosine is an aromatic non-protein amino acid initially found in plants of Mimosa 83
pudica and later in Leucaena leucocephala (Lam) de Wit (Soedarjo and Borthakur 1998) a 84
leguminous tree which biosynthesizes large amounts of this nitrogen-containing compound 85
(Rodrigues-Correcirca et al 2019) It is believed that the accumulation of high contents of 86
mimosine in L leucocephala tissues confers among other traits defense against herbivores 87
and pathogens (Vestena et al 2001) tolerance to drought (Negi et al 2014) as well as 88
general oxidative stress protection (Rodrigues-Correcirca et al 2019) Interestingly drought is 89
the opposite environmental and physiological condition to that observed in the wet habitats 90
occupied by native populations of M bimucronata in Brazil (Patreze and Cordeiro 2004 91
Kestring et al 2009) and Mimosa pudica Linn in India (Champanerkar et al 2010) 92
70
Nonetheless flooding is also associated with oxidative stress particularly as water levels 93
change (Fukao et al 2019) 94
In Leucaena leucocephala var leucocephala (common leucaena) and Leucaena 95
leucocephala var glabrata (giant leucaena) mimosine accumulation has been shown to be 96
both constitutive and inducible by stress-related phytohormones such as jasmonic acid (JA) 97
Ethephon (ETH an ethylene- releasing compound) salicylic acid (SA - only common 98
leucaena) (Vestena et al 2001) as well as by UV-C radiation (Xu et al 2018 Rodrigues-99
Correcirca et al 2019) On the other hand there is a lack of information regarding mimosine 100
content and elicitation effects in Mimosa spp plants 101
The aim of this study was to examine the presence of mimosine in Mimosa 102
bimucronata and examine the effects of stresses and stress-signaling molecules on its 103
accumulation in leaves 104
Material and Methods 105
Plant material 106
For all experiments the plant material was collected at Morro Santana campus do 107
Vale of UFRGS (Federal University of Rio Grande do Sul) Porto Alegre RS Brazil 108
(3004rsquoS 5108rsquoW) Authorization for access to genetic material was obtained from 109
SISGEN-Brazil (license number A845493) Constitutive mimosine content in adult plants of 110
M bimucronata var bimucronata (DC) Kuntze was determined in plant material (leaves 111
green flower buds post-anthesis flowers and green pods) harvested in January 2017 112
(summer) A voucher herbarium specimen (ICN 187953) was deposited in the ICN ndash UFRGS 113
herbarium (Herbaacuterio do Instituto de Biociecircncias of UFRGS) 114
71
For mimosine elicitation experiments legumes (fruits) of maricaacute were collected in 115
the end of June 2017 (winter) Seeds were then removed from the dry fruits and kept in the 116
dark until sowing and seedling development for use in the assays 117
Seed germination 118
To break the coat-imposed seed dormancy after surface sterilization dry seeds of 119
maricaacute were acid scarified by immersion in H2SO4 (95 ndash 98 ) for 2 min (see Correcirca et al 120
2008) and repeatedly washed in distilled water to remove any residue of the acid Then seeds 121
were distributed in 50 mL individual plastic tubes (dibble-tubes) (30 cm diameter x 120 cm 122
depth) filled up with 11 (vv) of commercial top soil and vermiculite Tubes were watered 123
every 2 days to avoid substrate dryness and were kept in a growth room under controlled 124
conditions of light (circa 75 μmol mminus2s minus1 photosynthetically active radiation photoperiod 125
of 16 h light and 8 h dark) and temperature (24plusmn2C) 126
127
Treatments 128
In order to verify inducibility of mimosine accumulation in M bimucronata fifty 12-129
week-old maricaacute seedlings (per treatment) exhibiting similar features were selected and 130
sprayed (saturated) with solutions of different chemical stressors (plant specialized 131
metabolism elicitors) as follows (for further details see Rodrigues-Correcirca et al 2019) 10 132
and 50 mM SA (pathogen-signaling molecule Shah 2003) 007 and 035 mM 2-133
chloroethylphosphonic acid (ETH ethylene releasing-compound Kim et al 2016 Wang et 134
al 2016) 100 and 200 mM MeJA (Dar et al 2015) 10 and 50 mM SNP (a nitric oxide 135
donor Perotti et al 2015) Alternatively maricaacute seedlings were also supplemented with UV-136
C radiation (13 minutes 105 kJ cm2) (elicitor of plant specialized metabolism Kara 2013) 137
72
After 2 and 4 days of exposure to the chemical treatments and 3 and 6 days of UV-138
C supplementation maricaacute shoots were harvested immediately frozen in liquid nitrogen and 139
stored at ndash 80 C until mimosine extraction and HPLC analyses 140
Mimosine extraction and detection 141
Mimosine extraction was conducted according to the modified protocol described by 142
Rodrigues-Correcirca et al (2019) for L leucocephala HPLC (Thermo Scientific Surveyor) 143
analyses (mimosine detection and quantification) were performed following previously 144
published procedures (Negi et al 2014) A C18 column (ACE C18 5 μm 46times250 mm) and 145
isocratic solvent system of 002M o-phosphoric acid with a linear flow rate of 1 mL min minus1 146
were used to separate and quantify the amino acid Mimosine detection was performed at 280 147
nm by photodiode array detection (200ndash400 nm) and retention time (229plusmn0024 min) 148
Mimosine quantification was done by means of the method of external standard curve 149
Additional confirmation of mimosine identity was performed by co-chromatography with 150
standard (Acros Organics authentic mimosine 99 used as reference) and peak purity check 151
The analyses of the chromatograms were done with the ChromQuest software 152
153
154
Results and Discussion 155
Constitutive accumulation of mimosine in M bimucronata 156
Mimosine was detected in all analyzed samples positively meeting all identification 157
criteria In agreement with what has been found for other Mimosa spp (Soedarjo and 158
Borthakur 1998) compared to L leucocephala adult plants (Rodrigues-Correcirca 2019) 159
mimosine content was lower in M bimucronata Of the adult plant tissues analyzed the 160
73
highest content of mimosine in maricaacute (per gram of fresh weight - FW) was found in post-161
anthesis flowers (36644 microg versus 89448 microg in common leucaena followed by leaves 162
(28838 microg x 67358 microg) green flower buds (28094 microg x 51247 microg) and green pods (19002 163
microg x 82687 microg) (Fig 1)The same pattern is observed for seedlings when both species are 164
compared In this study untreated 12-week-old maricaacute seedlings (control at day 2) showed a 165
shoot content of mimosine of 23029plusmn007 microg g-1 of (FW) Five-week-old untreated giant 166
leucaena seedlings cultivated in similar conditions exhibited between 83640 and 178736 167
microg g-1 of FW (Rodrigues-Correcirca et al 2019) In the same way mimosine concentration 168
percentage in dry matter of Mimosa pigra was found to be rather low (002 in nodules and 169
roots and 007 in leaves) (Soedarjo and Borthakur 1998) 170
In this investigation the lowest constitutive mimosine content was found in green 171
pods (Fig 1) This result may partly explain the absence of phytotoxic effect observed for 172
green pods on germination and growth of crop target plants tested by Jacobi and Ferreira 173
(1991) compared to the other maricaacute parts analyzed 174
Elicitation of mimosine biosynthesis in M bimucronata 175
Chemical stressors 176
Secondary metabolites (or natural products) are structural- and chemically 177
specialized compounds derived from primary metabolism These molecules are mainly 178
biosynthesized as part of a complex defense mechanism in response to biotic and abiotic 179
stresses such as pathogens herbivores water status metal toxicity and UV radiation for 180
example (Matsuura et al 2018) Ethephon SA SNP MeJA have been extensively used as 181
chemical elicitors of specialized metabolism (Wang et al 2016 Vestena et al 2001 Perotti 182
74
et al 2015 Zhang and Memelink 2009 Xu et al 2018) These phytohormonal signals can 183
simulate environmental challenges and modulate plant homeostasis often leading to 184
alterations in gene expression (Shinozaki et al 2015) Except SNP all treatments tested in 185
the present study showed positive effect on mimosine accumulation in common or giant 186
leucaena (Vestena et al 2001 Rodrigues-Correcirca 2019 Rodrigues-Correcirca unpublished 187
data) However in spite of the trend of increasing the mimosine content observed in seedlings 188
treated with 007 mM Ethephon (at day 2) and 100 mM MeJA (at day 4) no statistical 189
difference was confirmed for these treatments when compared to the control 190
On the other hand a within treatment difference on mimosine induction was seen 191
between day 2 and 4 in seedlings treated with 100 mM MeJA (Fig 2) In a lower 192
concentration (04 mM) jasmonic acid (JA)promoted a near threefold increase in mimosine 193
accumulation of giant leucaena seedlings after 2 days of application 194
UV-C radiation 195
Albeit UV-C radiation is not biologically active in natural environments it has been 196
widely used under controlled experimental conditions to generate acute responses of plant 197
specialized metabolism within a shorter period of time compared to that required to with UV-198
B radiation (Kara 2013 Cetin 2014) This fast response is due to the higher energy of UV-199
C photons that act as potent reactive oxygen species (ROS) generators causing extensive 200
damage to the cells either at the physiological level or on DNA structure (Gregianini et al 201
2003 Matsuura et al 2013) 202
Although divergent responses can be observed in plants exposed to UV-C radiation 203
the deleterious processes are usually reported on primary metabolism (decreasing of 204
chlorophyll content and plant height eg) (Kara 2013) In the present study no statistical 205
75
differences were observed in the mimosine concentration in maricaacute seedlings supplemented 206
with UV-C radiation However a decreasing in its content was found for both control and 207
treatment at day 6 post-treatment (Fig 03) Taking into account the lower constitutive 208
concentration of mimosine observed in maricaacute compared to the leucaena plants besides its 209
relative thermolability (Nguyen and Tawata 2016) it seems to be plausible to consider the 210
effect of the temperature inside the UV-C and the white light (control) chambers as an 211
additional abiotic factor contributing to the decrease of mimosine accumulation in both group 212
of plants 213
Besides mimosine identification the presence of 34-dihydroxypyridine (34-DHP or 214
3-hydroxy-4-pyridone - 3H4P) a mimosine degradation product (Negi et al 2014 Nguyen 215
and Tawata 2016) was also reported for maricaacute leaf extracts analyzed by TLC by Ferreira 216
et al (1992) In our chromatograms we detected a second large peak after that of mimosine 217
(229plusmn0024) and similar to that identified by Negi et al (2014) as 3H4P (data not shown) 218
Comparing the chromatogram profiles obtained from seedlings elicited with chemical 219
stressors and those supplemented with UV-C the largest area for this peak was found (in all 220
samples) in the latter treatment at day 6 It might indicate that the constitutive andor the 221
initially UV-C-induced mimosine was degraded into 3H4P to cope with the cellular damage 222
caused by this treatment associated with an increased temperature inside the chambers 223
Nevertheless it was not possible to determine 3H4P concentration (or confirm its identity) 224
in maricaacute plants since there is no commercial standard (pure 3H4P) available for purchase 225
to be used as a reference in calculations Establishment of improved protocols for obtaining 226
in house 3H4P reference substance by acid hydrolysis is ongoing 227
228
229
76
Conclusion 230
On the basis of the overall absence of effect of the treatments tested here on mimosine 231
concentration it is possible to suggest that its accumulation profile is similar to that of 232
phytoanticipins unlike what is observed for the same amino acid production in leucaena 233
which shows features of inducibility resembling phytoalexin-like metabolites Alternatively 234
a putative inducible pool of mimosine in maricaacute might be involved in other types of stress 235
such as extended drought periods If involved in protection against oxidative stress as 236
described for leucaena mimosine in maricaacute may act predominantly by physical quenching 237
of ROS as indicated by the lack of overt chemical degradation Nevertheless further 238
investigations are needed to assess these hypotheses 239
To sum up mimosine biosynthesis was not modulated by the treatments evaluated as 240
in L leucocephala (Lam) de Wit To the best of our knowledge this is the first work that 241
analytically identifies and quantifies mimosine accumulation in M bimucronata 242
243
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Bitencourt F Zocche JJ Costa S Souza PZ Mendes AR 2007 Nucleaccedilatildeo de 245
Mimosa bimucronata (DC) O Kuntze em aacutereas degradadas pela mineraccedilatildeo de carvatildeo R 246
Bras Bioci 5 750-752 247
Carvalho PER 2004 Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 248
Teacutecnica 94 1-10 249
Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitis 250
vinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-251
6287-47-37 252
77
Champanerkar PA Vaidya VV Shailajan S Menon SN 2010 A sensitive rapid and 253
validated liquid chromatography ndash tandem mass spectrometry (LC-MS-MS) method for 254
determination of Mimosine in Mimosa pudica Linn Nat Sci 2 713-717 255
httpsdoiorg104236ns201027088 256
Gomes GS Silva GS Silva DLS Oliveira RR Conceiccedilatildeo GM 2018 Botanical 257
Composition of Fabaceae Family in the Brazilian Northeast Maranhatildeo Brazil Asian J 258
Environ Ecol 6(4) 1-10 httpsdoiorg109734AJEE201841207 259
Correcirca LR Soares GLG Fett-Neto AG 2008 Allelopathic potential of Psychotria 260
leiocarpa a dominant understorey species of subtropical forests S Afri J Bot 74 583ndash261
590 httpsdoiorg101016jsajb200802006 262
Ferreira AG Aquila MEA Jacobi US Rizvi V 1992 Allelopathy in Brazil In Allelopathy 263
basic and applied aspects Rizvi V and Jacobi US (Eds) Chapman and Hall pp 243-250 264
Fukao T Barrera-Figueroa BE Juntawong P Pentildea-Castro JM 2019 Submergence 265
and waterlogging stress in plants a review highlighting research opportunities and 266
understudied aspects Front Plant Sci 10 340 httpsdoiorg103389fpls201900340 267
Gregianini TS Silveira VC Porto DD Kerber VA Henriques AT Fett-Neto AG 268
2003 The alkaloid brachycerine is induced by ultraviolet radiation and is a singlet oxygen 269
quencher Photochem Photobiol 78(5) 470ndash474 httpsdoiorg1015620031-270
8655(2003)0784070TABIIB20CO2 271
Jacobi US Ferreira AG 1991 Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK 272
sobre espeacutecies cultivadas Pesq Agropec Bras 26(7) 935-943 273
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant 274
(Phaseolus vulgaris) Biosci Res 10(1) 29ndash32 275
78
Kestring D Klein J Menezes LCCR Rossi MN 2009 Imbibition phases and 276
germination response of Mimosa bimucronata (Fabaceae Mimosoideae) to water 277
submersion Aquat Bot 91 105ndash109 httpsdoiorg101016jaquabot200903004 278
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect of 279
Ethephon as an ethylene-releasing compound on the metabolic profile of Chlorella vulgaris 280
J Agric Food Chem 64(23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541 281
Lorenzi H 1998 Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 282
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 283
Lorenzi H 2008 Plantas daninhas do Brasil terrestres aquaacuteticas parasitas e toacutexicas 4 ed 284
Nova Odessa Instituto Plantarum 640 p 285
Marchiori JNC 1993 Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 286
O Kuntze Ciecircncia Florestal 3 85-106 287
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation of 288
bioactive secondary metabolites by ultraviolet radiation mechanisms strategies and 289
applications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology for Medicinal 290
Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash190= 291
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo R Bhambra AS 292
Strnad M Bonfill M Fett-Neto AG 2018 Specializedplant 293
metabolismcharacteristicsandimpactontargetmoleculebiotechnologicalproduction 294
Molecular Biotechnology 60(2) 169ndash183httpsdoiorg101007s12033-017-0056-1 295
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase from 296
Leucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 297
922ndash934 httpsdoiorg101104pp113230870 298
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Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine 299
areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636 300
Olkoski D Wittmann MTS 2011 Cytogenetics of Mimosa bimucronata (DC) O Kuntze 301
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop Breed 302
Appl Biotechnol 11 27-35 httpdxdoiorg101590S1984-70332011000100004 303
Patreze CM Cordeiro L 2004 Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 304
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecol Manag 196 275ndash305
285 httpdxdoiorg101016jforeco200403034 306
Perotti JC Rodrigues-Correcirca KCS Fett-Neto AG 2015 Control of resin production in 307
Araucaria angustifolia an ancient South American conifer Plant Biology 17 852ndash859 308
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG 2019 Mimosine 309
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 310
UV exposure Plant Physiology and Biochemistry 135 432ndash440 311
Pilatti DM Fortes AMT Jorge TCM Boiago NP 2019 Comparison of the phytochemical 312
profiles of five native plant species in two different forest formations Brazilian Journal of 313
Biology 79(2) 233-242 314
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS 2011 Biologia da reproduccedilatildeo 315
deMimosa bimucronatandash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 316
Simon MF and Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 317
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 318
endemism Biological Conservation 96 279-296 319
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 320
LM Andrade SF Cechinel-Filho V 2017 Chemical composition and diuretic natriuretic and 321
80
kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its majority 322
constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash1624 323
Shah J 2003 The salicylic acid loop in plant defense Current Opinion Plant Biology6 (4) 324
365ndash371 325
Shinozaki K Uemura M Serres JB Bray EA Weretilnyk E 2015 Responses to Abiotic 326
Stress In Buchanan BB Gruissem W Jones RL (Eds) Biochemistry and Molecular 327
Biology of Plants Second Edition John Wiley and Sons Ltd 328
Soedarjo M and Borthakur D 1998 Mimosine a toxin produced by the tree-legume 329
Leucaena provides a nodulation competition advantage to mimosine-degrading Rhizobium 330
strains Soil Biology and Biochemistry 30(12)1605-1613 331
Vestena S Fett-Neto AG Duarte RC Ferreira AG 2001 Regulation of mimosine 332
accumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 333
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene induced 334
vinblastine accumulation is related to activated expression of downstream TIA pathway 335
genes in Catharanthus roseus BioMed Research International Article ID 3708187 336
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX Tsim KWK 2018 337
Jasmonate-elicited stress induces metabolic change in the leaves of Leucaena leucocephala 338
Molecules 23 (2) 339
Zhang H Memelink J 2009 Regulation of Secondary Metabolism by Jasmonate Hormones 340
In AE Osbourn and V Lanzotti (eds) Plant-derived Natural Products 3 DOI 101007978-341
0-387-85498-4_1 copy Springer Science + Business Media LLC 342
343
344
345
81
346
Figure 1 Constitutive concentration of mimosine in different plant organs of Mimosa 347
bimucronata Bars sharing the same letter do not differ statistically by Tukey test (Ple005) 348
The error bars denote standard error of 10 replicates 349
350
351
352
353
354
355
356
357
B B A C0
5
10
15
20
25
30
35
40
LEAVES GREEN FLOWER BUDS POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
co
nce
ntr
atio
n u
gg-1
Mimosine concentration in adult plants of Mimosa bimucronata (DC) Kuntze
82
C T R L S A
1 0 m M
S A
5 0 m M
E T H
0 0 7 m M
E T H
0 3 5 m M
M e J A
1 0 0 m M
M e J A
2 0 0 m M
S N P
1 0 m M
S N P
5 0 m M
0
1 0
2 0
3 0
T re a tm e n ts
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1) D A Y 2
D A Y 4
A B C C B C A B C C A B C A B C A
a b b b a a b a a b b a b
358
Figure 2 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 359
bimucronata treated with different signaling molecules SA = Salicylic Acid ETH = 360
Ethephon MeJA = Methyl Jasmonate SNP = Sodium Nitroprusside Uppercase and 361
lowercase letters indicate statistical differences among treatments in days 2 and 4 362
respectively Bars sharing a letter of the same case do not differ statistically by Tukey test 363
(Ple005) Indicates statistical difference in the same treatment between day 2 and 4 by t-364
test (Ple005) The error bars denote standard error of 5 replicates (25 individual seedlings 365
arranged in 5 groups of 5) 366
367
368
83
D AY 3 D AY 6
0
5
1 0
1 5
2 0
2 5
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1)
C O N TR O L
U V -C
369
Figure 3 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 370
bimucronata supplemented with UV-C radiation Indicates statistical difference in the same 371
treatment between day 3 and 6 by t-test (Ple005) The error bars denote standard error of 5 372
replicates (25 individual seedlings arranged in 5 groups of 5) 373
374
375
376
377
378
379
380
381
382
383
384
385
84
Consideraccedilotildees finais 386
- Experimentos que avaliam os efeitos da aplicaccedilatildeo exoacutegena de ANPs em diferentes espeacutecies 387
vegetais tecircm sido realizados principalmente com GABA Dentre os principais efeitos 388
conferidos pela aplicaccedilatildeo dessa moleacutecula em espeacutecies de mono e eudicotiledocircneas satildeo 389
relatados a toleracircncia agrave seca agrave salinidade e agraves temperaturas extremas 390
- Como metaboacutelitos especializados claacutessicos os ANPs podem ter sua concentraccedilatildeo basal 391
endoacutegena aumentada em resposta agrave induccedilatildeo mediada por uma vasta gama de tratamentos com 392
moleacuteculas sinalizadoras de estresse e fontes alternativas de estressores De um modo geral 393
observa-se o acuacutemulo das diferentes classes de ANPs em resposta agrave radiaccedilatildeo UV elicitores 394
quiacutemicos que mimetizam ataques por patoacutegenos dano mecacircnico agentes osmoacuteticos metais 395
pesados entre outros 396
- Especificamente em leucena a resposta observada em relaccedilatildeo aos diferentes tratamentos 397
testados indica que apesar do seu alto teor constitutivo nessa espeacutecie a biossiacutentese e o 398
acuacutemulo de mimosina podem ser modulados por fatores causadores de estresses exibindo -399
nessa espeacutecie - um padratildeo de acumulaccedilatildeo similar agrave fitoalexinas Em maricaacute por outro lado 400
aumento de acuacutemulo dessa moleacutecula natildeo foi observado para os mesmos tratamentos testados 401
para leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas 402
- O padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno 403
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado pela 404
sua degradaccedilatildeo 405
- As respostas observadas nos testes que avaliaram a atividade de mitigaccedilatildeo de espeacutecies 406
reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode agir como um agente 407
antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de estresse 408
85
Perspectivas 409
- Confirmaccedilatildeo em espectrocircmetro de massas eou ressonacircncia nuclear magneacutetica da natureza 410
quiacutemica da lsquomimosinarsquo presente em maricaacute 411
- Avaliaccedilatildeo do efeito de concentraccedilotildees mais elevadas e em diferentes periacuteodos de aplicaccedilatildeo 412
das moleacuteculas sinalizadoras testadas sobre o acuacutemulo de mimosina em leucena e maricaacute 413
- Ampliar a investigaccedilatildeo dos padrotildees de expressatildeo gecircnica dos genes que codificam para 414
mimosinase (em maricaacute) mimosina sintase (em ambas as espeacutecies testadas) bem como o 415
perfil de precursores e cataboacutelitos de mimosina em resposta aos tratamentos mencionados 416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
86
Referecircncias Bibliograacuteficas 436
437
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Teacutecnica 941-10 458
Chowtivannakul P Srichaikul B Talubmook C (2016) Antidiabetic and antioxidant activities 459
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Environmental Toxicology 321765ndash1774 httpsdoiorg101002tox22399 464
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Crowe B Poynter JA Manukyan MC Wang Y Brewster BD Herrmann JL Abarbanell 465
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Ferreira AG Aquila MEA Jacobi US Rizvi V (1992) Allelopathy in Brazil In Allelopathy 475
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Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of the thyrotoxic 481
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Jacobi US Ferreira AG (1991) Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK Sobre 484
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Jiao CJ Jiang J-L Ke L-M Cheng W Li F-M Li Z-X Wang C-Y (2011) Factors affecting 490
β-ODAP content in Lathyrus sativus and their possible physiological mechanisms Food 491
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Kubota S Fukumoto Y Ishibashi K Soeda S Kubota SS Yuki R Nakayama Y Aoyama K 493
Yamaguchi N (2014) Activation of the prereplication complex is blocked by mimosine 494
88
through reactive oxygen species-activated ataxia telangiectasia mutated (ATM) protein 495
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Aqueous Extract Stimulates Adipogenesis Lipolysis and Glucose Uptake in Primary Rat 498
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Kusama-Eguchi K (2019) Research in motor neuron diseases caused by natural substances 501
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Kutchan TM Gershenzon J Moslashller BL Gang DR (2015) Natural Products In Buchanan 504
BB Gruissem W and Jones RL (eds) Biochemistry amp Molecular Biology of Plants 2nd edn 505
Wiley Blackwell Chichester pp 1135-1205 506
Lalande M (1990) A reversible arrest point in the late G1 phase of the mammalian cell cycle 507
Exp Cell Res 186 332ndash339 508
Li X-W Hu C-P Li Y-J Gao Y-X Wang XM Yang J-R (2015) Inhibitory effect of L-509
mimosine on bleomycin-induced pulmonary fibrosis in rats Role of eIF3a and p27 Int 510
Immunopharmacol 27(1) 53ndash64 511
Little Jr EL Skolmen RG (1989) Koa haole Agriculture Handbook 679 USDA 512
Lorenzi H (1998) Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 513
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 514
Marchiori JNC (1993) Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 515
O Kuntze Ciecircncia Florestal 3 85-106 516
Mohammed RS El Souda SS Taie HAA Moharam ME Shaker KH (2015) Antioxidant 517
antimicrobial activities of flavonoids glycoside from Leucaena leucocephala leaves Journal 518
of Applied Pharmaceutical Science 5(06)138-147 519
httpdxdoiorg107324JAPS201550623 520
Negi VS Bingham J-P Li QX Borthakur D (2014) A carbon-nitrogen lyase from Leucaena 521
leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 (2) 922ndash522
934 httpsdoiorg101104pp113230870 523
89
Olkoski D Wittmann MTS (2011) Cytogenetics of Mimosa bimucronata (DC) O Kuntze 524
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop 525
Breeding and Applied Biotechnology 11 27-35 526
Patreze CM Cordeiro L (2004) Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 527
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecology and Management 528
196275ndash285 529
Pilatti DM Fortes AMT Jorge TCM Boiago NP (2019) Comparison of the phytochemical 530
profiles of five native plant species in two different forest formations Brazilian Journal of 531
Biology 79(2) 233-242 532
Ramos-Ruiz R Poirot E Flores-Mosquera M (2018) GABA a non-protein amino acid 533
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REFLORA (2019) httpfloradobrasiljbrjgovbrreflora Acesso em agosto de 2019 536
Rodgers KJ Samardzic K Main BJ (2015) Toxic Nonprotein Amino Acids Plant Toxins 537
httpsdoiorg 101007978-94-007-6728-7_9-1 538
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG (2019) Mimosine 539
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 540
UV exposure Plant Physiology and Biochemistry 135 432ndash440 541
httpsdoiorg101016jplaphy201811018 542
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 543
LM Andrade SF Cechinel-Filho V (2017) Chemical composition and diuretic natriuretic 544
and kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its 545
majority constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash546
1624 547
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS (2011) Biologia da reproduccedilatildeo 548
de Mimosa bimucronata ndash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 549
Simon MF Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 550
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 551
endemism Biological Conservation 96 279-296 552
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Soares AMS Arauacutejo SA Lopes SG Costa Junior LM (2015) Anthelmintic activity of 553
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Soerdajo M Borthakur D (1998) Mimosine a toxin produced by the tree-legume Leucaena 556
provides a nodulation competition advantage to mimosine-degrading Rhizobium strains Soil 557
Biol Biochem 30(12) 16051613 558
Souza-Lima ES Sinani TR Pott A Sartori ALB (2017) Mimosoideae (Leguminosae) in the 559
Brazilian Chaco of Porto Murtinho Mato Grosso do Sul Rodrigueacutesia 68(1) 263-290 2017 560
httpdxdoiorg1015902175-7860201768131 561
Taiz L amp Zeiger E (2010) Plant Physiology 5th edition Sinauer Associates Inc Sunderland 562
Verma VK Rani KV Kumara SR Prakash O (2018) Leucaena leucocephala pod seed 563
protein as an alternate to animal protein in fish feed and evaluation of its role to fight against 564
infection caused by Vibrio harveyi and Pseudomonas aeruginosa Fish and Shellfish 565
Immunology 76 (2018) 324ndash332 httpsdoiorg101016jfsi201803011 566
Yafuso JT Negi VS Bingham J-P Borthakur D (2014) An O-acetylserine (thiol) lyase from 567
Leucaena leucocephala is a cysteine synthase but not a mimosine synthase Appl Biochem 568
Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z 569
Zarin RMA Wan HY Isha A Armani N (2016) Antioxidant antimicrobial and cytotoxic 570
potential of condensed tannins from Leucaena leucocephala hybrid Food Science and 571
Human Wellness 5 65ndash75 httpdxdoiorg101016jfshw201602001 572
573
574
Contents lists available at ScienceDirect
Industrial Crops amp Productsjournal homepage wwwelseviercomlocateindcrop
Resin tapping transcriptome in adult slash pine (Pinus elliottii var elliottii)Camila Fernanda de Oliveira Junkes1 Artur Teixeira de Arauacutejo Juacutenior1 Juacutelio Ceacutesar de LimaFernanda de Costa Thanise Fuumlller Maacutercia Rodrigues de Almeida Franciele Antocircnia NeisKelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-NetoCenter for Biotechnology and Department of Botany Federal University of Rio Grande do Sul Porto Alegre PO Box 15005 91501-970 Brazil
A R T I C L E I N F O
KeywordsPinus elliottiResinResinosisTranscriptomeAdjuvant paste
A B S T R A C T
To better understand the bases of resin production a major source of terpenes for industry the transcriptome ofadult Pinus elliottii var elliottii (slash pine) trees under field commercial resinosis was obtained Samples werecollected from cambium after 5 and 15 days of treatment application which included tapping followed byapplication of commercial resin stimulant paste or control wounding without paste Overall mean number ofreads of all 16 libraries (2 treatments x 2 times x 4 replicated trees) was 34582048 Of these 89 were mappedagainst the reference sequence with a mismatch of 058 Using the Blast2Go 570 candidate genes were de-tected based on sequence annotation By comparing the expression profile between paste and control 310differentially expressed genes (DEGs) were identified at 5 days and 190 at 15 days with a significant fold changeof log2gt 12 Regarding changes in time comparisons within each treatment 210 and 105 DEGs were identifiedwithin control and paste treatment respectively Genes with different expression patterns in the times andtreatments examined included ethylene responsive transcription factors geranylgeranyl diphosphate synthasediterpene synthase cytochrome P450 and ABC transporters all of which may play important roles in resinproduction RT-qPCR analysis correlated well with the data obtained by RNAseq Resin composition changedover time This is the first transcriptomic investigation of resinosis of the main species used in the bioresinindustry and of molecular analyses of resinosis under field operations with implications for stand managementstimulant paste development genotype selection and breeding for high resinosis
1 Introduction
The adaptive success of conifers is largely due to the development ofa defense system based on the synthesis and secretion of terpenes in allmajor organs and different tissues (Miller et al 2005 Hall et al 2013Warren et al 2015) Conifer resin is a viscous fluid composed of acomplex mixture of terpenoids such as monoterpenes sesquiterpenesand diterpenes (Zulak and Bohlmann 2010) These terpenoids are se-creted from severed resin ducts when the tree is under biotic attack(Ralph et al 2006 Lange 2015 Geisler et al 2016) acting as pro-tectants (Schiebe et al 2012 Liu et al 2015)Biosynthesis of terpenes in conifers starts from isomerization of two
isoprenoid (C5) units dimethylallyl diphosphate (DMAPP) and iso-pentenyl diphosphate (IPP) These molecules can be biosynthesized viatwo separate routes in plants the methyl-erythritol 4-phosphate andmevalonate pathways IPP is synthesized and isomerized to DMAPP byisopentenyl diphosphate isomerase then prenyl transferases catalyze
the condensation of these two C5-units to geranyl diphosphate (Pazoukiand Niinemets 2016) Their elongation to prenyl diphosphates withaddition of IPP molecules leads to monoterpenes (C10) sesquiterpenes(C15) and diterpenes (C20) which are the substrates for terpene syn-thases (TPS) (Keeling and Bohlmann 2006b)TPSs are part of a large family of mechanistically related enzymes
involved in both primary and secondary metabolism (Keeling andBohlmann 2006b) The events of evolutionary diversification and ex-pansion of plant TPSs appear to have originated from gene duplicationsdomain losses and sub- or neofunctionalizations with subsequent di-vergence of an ancestral TPS gene of primary metabolism (Hall et al2013) Modification of TPS products changes their physical propertiesand may alter their biological activities (Chen et al 2011) TPSs of highsequence identity may have different functions even in closely relatedspecies Low sequence identity of TPSs in phylogenetically distantspecies does not preclude the possibility of independent evolution of thesame or related function of these enzymes (Zerbe and Bohlmann 2015)
httpsdoiorg101016jindcrop2019111545Received 4 January 2019 Received in revised form 10 June 2019 Accepted 4 July 2019
Corresponding authorE-mail address fettnetocbiotufrgsbr (AG Fett-Neto)1 These authors have equally contributed to this work
doi 1015900102-33062019abb0114
Acta Botanica Brasilica
Sustainable production of bioactive alkaloids in Psychotria L of
southern Brazil propagation and elicitation strategies
Yve Verocircnica da Silva Magedans1 Kelly Cristine da Silva Rodrigues-Correcirca1 Cibele Tesser da Costa1
Heacutelio Nitta Matsuura1 and Arthur Germano Fett-Neto1
Received April 1 2019Accepted June 28 2019
ABSTRACTPsychotria is the largest genus in Rubiaceae South American species of the genus are promising sources of natural
products mostly due to bioactive monoterpene indole alkaloids they accumulate ese alkaloids can have analgesic
antimutagenic and antioxidant activities in dierent experimental models among other pharmacological properties
of interest Propagation of genotypes with relevant pharmaceutical interest is important for obtaining natural
products in a sustainable and standardized fashion Besides the clonal propagation of elite individuals the alkaloid
content of Psychotria spp can also be increased by applying moderate stressors or stress-signaling molecules is
review explores advances in research on methods for plant propagation and elicitation techniques for obtaining
bioactive alkaloids from Psychotria spp of the South Region of Brazil
Keywords abiotic stress alkaloids elicitation monoterpenes plant propagation Psychotria southern Brazil
sustainability
Introduction
Psychotria belongs to Rubiaceae one of the major families
of $owering plants having economic interest e family
includes coee a few signicant poisonous plants to livestock
besides several important ornamental and medicinal species
(Souza amp Lorenzi 2012) Psychotria has captured researchersrsquo
attention mostly because of its medicinal properties
Psychotria colorata is an Amazonian species that produces
polyindolinic alkaloids with analgesic activity (Matsuura et
al 2013) e promising results obtained with P colorata
motivated the investigation of southern Brazilian Psychotria
species and the discovery of new bioactive alkaloids (Porto
et al 2009) Moreover leads on in planta alkaloid functions
were also topic of experimental evaluation
One of the key elements that needs to be addressed early
on during the process of developing new bioactive molecules
from plants is the capacity to generate catalytically active
biomass to support extraction and steady supply ere are a
number of ways through which these goals may be reached
including greenhouse rooting of cuttings (mini-cutting
1 Laboratoacuterio de Fisiologia Vegetal Departamento de Botacircnica Instituto de Biociecircncias e Centro de Biotecnologia Universidade Federal do Rio
Grande do Sul 91501-970 Porto Alegre RS Brazil
Corresponding author fettnetocbiotufrgsbr
Review
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Biomass yield of resin in adult Pinus elliottii Engelm trees is differentially
regulated by environmental factors and biochemical effectors
Franciele Antocircnia Neis Fernanda de Costa Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima
Kelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-Neto
Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) CP 15005 CEP 91501-970 Porto Alegre RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Biomass
Terpene resin
Seasonal
Benzoic acid
Regenerated forest
A B S T R A C T
Biomass of pine resin finds several applications in the chemical pharmaceutical biofuel and food industries
Resin exudation after injury is a key defense response in Pinaceae since this complex mixture of terpenes has
insecticidal antimicrobial and wound repair properties Resin yield is increased by effectors applied on the
wound area including phytohormones and metal cofactors of terpene synthases The interaction of resinosis
mechanism effectors is not fully understood particularly in adult forest setups under natural environmental
variations The aim of this work was to determine how resin exudation by wounded trunks of adult P elliottii
responded to combined chemical effectors involved in different regulatory pathways of resinosis (metal cofactors
of terpene synthases benzoic acid and plant growth regulators) and whether seasonal and tree distribution
variations affected these responses Symmetrically planted and scattered trees regenerated from the seed bank
had similar resin biomass yields suggesting that the homogeneity in development and spatial arrangement were
not significant factors in resin yield This new finding is of practical importance with the used tapping system
since costs of implanting forests by regeneration can be advantageous compared to planting In addition it was
shown for the first time that the salicylic acid precursor benzoic acid and the auxin naphthalene acetic acid
promoted resin exudation when individually applied to wound sites Both these adjuvants are two orders of
magnitude less costly compared to the conventionally used ethylene precursors besides facing less environ-
mental and health restrictions for use Most adjuvant-treated trees showed higher resin flow in the second year
indicating mechanisms of response build up Overall temperature was more important than rainfall as en-
vironmental parameter affecting resin biosynthesis which was higher in the warmer months of spring and
summer The combination of resinosis stimulant effectors from different signaling pathways showed no sig-
nificant synergistic or additive effect suggesting possible converging signaling pathways andor limitation of
common intermediate transducing molecules
1 Introduction
Pines occupy highly diverse environments over a range of tem-
peratures water and nutrient availabilities irradiance levels and pho-
toperiods being able to effectively face attacks from diverse herbivore
and pathogen guilds The success of conifers is linked to their complex
terpene biochemistry hosted by specialized secretory cells The terpe-
noid resin synthesized by Pinus spp is one of the main mechanisms of
defense of these trees particularly against bark beetles and the fungi
they carry (Fett-Neto and Rodrigues-Correcirca 2012) Pine resin biomass
is essentially composed of a monoterpene and sesquiterpene-rich tur-
pentine and diterpenoid-rich rosin fraction both finding numerous in-
dustrial applications as non-wood forest products (Rodrigues-Correcirca
et al 2012)
Molecules capable of modulating different signaling pathways have
been identified as resin yield stimulators including sulfuric acid (ex-
tends wound damage) 2-chloroethylphosphonic acid (CEPA a syn-
thetic ethylene precursor) paraquat (free radical generator) yeast ex-
tract (mimics attack by pathogens) salicylic acid (pathogen signaling
molecule) auxin (promotes ethylene biosynthesis and resin canal dif-
ferentiation) jasmonic acid (signals mechanical damage and promotes
secondary metabolism) and metal ions such as potassium iron and
manganese (cofactors of terpene synthases in conifers) and copper (a
component of ethylene receptors) (Clements 1970 Conrath et al
2002 Fett-Neto and Rodrigues-Correcirca 2012 Hudgins and Franceschi
2004 Lewinsohn et al 1994 Martin et al 2002 Popp et al 1995
httpsdoiorg101016jindcrop201803027
Received 12 December 2017 Received in revised form 9 March 2018 Accepted 13 March 2018
Corresponding author
E-mail addresses franci_neisyahoocombr (FA Neis) fernandadecostayahoocombr (F de Costa) thanisenfyahoocombr (TN Fuumlller)
jjuliocesarlimagmailcom (JC de Lima) krodriguescbiotufrgsbr (KC da Silva Rodrigues-Correcirca) jpfettcbiotufrgsbr (JP Fett) fettnetocbiotufrgsbr (AG Fett-Neto)
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Research Paper
Dual allelopathic effects of subtropical slash pine (Pinus elliottii Engelm)
needles Leads for using a large biomass reservoir
Kelly Cristine da Silva Rodrigues-Correcircaa Gelson Halmenschlagera Joseacuteli Schwambachb
Fernanda de Costaa Emili Mezzomo-Trevizana Arthur Germano Fett-Netoa
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 Brazilb University of Caxias do Sul Institute of Biotechnology Caxias do Sul RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Seasonality
Growth
Germination
Litter
Substrate
A B S T R A C T
Pinus elliottii Engelm (slash pine) is distributed along the maritime coast of Southern Brazil where it shows
invasive pattern and typical allelopathic features Large quantities of needle litter are produced by pine trees a
biomass that is little explored in areas where this species is alien Little is known about the dynamics of needle
and litter phytochemical interactions particularly in subtropical environments To elucidate the full range of
needle and litter allelopathic potential the effects of litter (superficial and deep) and seasonally harvested fresh
slash pine needles stored for different times were evaluated against lettuce tomato and cucumber seeds and
seedlings Increasing concentrations (0 1 2 4 and 8 wv) of hot and cold aqueous extracts of needles
and litter affected in different ways target plant development Growth and germination inhibition were directly
related to the highest extract concentrations (regardless of the season and mainly in hot water extracts) of
needles On the other hand stimulatory effects of litter extracts on lettuce growth were observed Growth and
germination of cucumber and tomato were not affected by pine litter as substrate when compared to rice husk
The presumable high polarity and thermal stability of slash pine leaf biomass allelochemicals and their transient
toxic effect or growth promoting impact suggest potential applications of this largely available biomass both as a
biological herbicide and growth substrate in plant propagation
1 Introduction
Native from the Northern Hemisphere Pinus is one of the most
widely distributed genera throughout different climate regions of the
globe growing either as native or alien species even in extreme habi-
tats (Rodrigues-Correcirca and Fett-Neto 2012) Despite the high economic
value currently attributed to pine wood and oleoresin (Rodrigues-
Correcirca et al 2012) there is increasing concern about the aggressive
potential of invasiveness displayed by Pinus species especially those
cultivated out of their native range of distribution (Richardson et al
2008 Rolon et al 2011) These species are dispersed by wind and there
is notably low plant diversity observed in most understories of pine
plantations (Kato-Noguchi et al 2009) This latter feature has been
considered an important trait of allelopathic interference
The term ldquoallelopathyrdquo was coined by Molisch in 1937 as a chemical
reciprocal interaction established among plants (including micro-
organisms) sharing the same site by means of the release of secondary
metabolites named allelochemicals (Rice 1984) For the most part
these metabolites are derived from the shikimic acid or isoprenoid
pathway and their biosynthesis can be modulated by biotic and abiotic
stresses (Nascimento and Fett-Neto 2010) including seasonal-related
changes (Sartor et al 2013) Allelopathy studies may range from sterile
assays (Aryakia et al 2015) to soil (Correcirca et al 2008 Sharma et al
2016) and field tests being a complex biological phenomenon to as-
certain in several circumstances due to issues of solubility release
mechanisms and stability of bioactive compounds (Scognamiglio et al
2013) Often the use of complementary methods provides more in-
formative data
The allelopathic effects of soil leachates green needles and litter
extracts of Pinus spp on germination and seedling growth aspects of
wild and crop species have been evaluated in natural and cultivated
pine stands and have proven to be stimulatory or inhibitory (Lodhi and
Killingbeck 1982 Kil and Yim 1983 Nektarios et al 2005 Akkaya
et al 2006 Machado 2007 Alrababah et al 2009 Sartor et al 2009
Kato-Noguchi et al 2011 Rolon et al 2011 Valera-Burgos et al
2012) exhibiting in some cases autotoxicity (Garnett et al 2004
Fernandez et al 2008 Zhu et al 2009 Monnier et al 2011) Studies
on potential dual allelopathic effects of Pinus elliottii Engelm (slash
httpdxdoiorg101016jindcrop201706019
Received 23 March 2017 Received in revised form 15 May 2017 Accepted 7 June 2017
Corresponding author
E-mail address fettnetocbiotufrgsbr (AG Fett-Neto)
ORIGINAL RESEARCHpublished 16 June 2016
doi 103389fpls201600849
Frontiers in Plant Science | wwwfrontiersinorg 1 June 2016 | Volume 7 | Article 849
Edited by
Juan Francisco Jimenez Bremont
Instituto Potosino de Investigacioacuten
Cientiacutefica y Tecnoloacutegica Mexico
Reviewed by
Mariacutea De La Luz Guerrero Gonzaacutelez
Universidad Autoacutenoma de San Luis
Potosiacute Mexico
Rosalia Cristina Paz
CIGEOBIO (CONICETFCEFN UNSJ)
Argentina
Correspondence
Arthur G Fett-Neto
fettnetocbiotufrgsbr
daggerThese authors have contributed
equally to this work
Specialty section
This article was submitted to
Plant Physiology
a section of the journal
Frontiers in Plant Science
Received 08 December 2015
Accepted 30 May 2016
Published 16 June 2016
Citation
de Lima JC de Costa F Fuumlller TN
Rodrigues-Correcirca KCdS Kerber MR
Lima MS Fett JP and Fett-Neto AG
(2016) Reference Genes for qPCR
Analysis in Resin-Tapped Adult Slash
Pine As a Tool to Address the
Molecular Basis of Commercial
Resinosis Front Plant Sci 7849
doi 103389fpls201600849
Reference Genes for qPCR Analysisin Resin-Tapped Adult Slash Pine Asa Tool to Address the MolecularBasis of Commercial Resinosis
Juacutelio C de Lima 1dagger Fernanda de Costa 1 dagger Thanise N Fuumlller 1
Kelly C da Silva Rodrigues-Correcirca 2 Magnus R Kerber 1 Mariano S Lima 1
Janette P Fett 1 and Arthur G Fett-Neto 1
1 Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul
Porto Alegre Brazil 2 Biological Sciences Department Regional Integrated University of Alto Uruguai and Missotildees (URI-FW)
Frederico Westphalen Brazil
Pine oleoresin is a major source of terpenes consisting of turpentine (mono- and
sesquiterpenes) and rosin (diterpenes) fractions Higher oleoresin yields are of economic
interest since oleoresin derivatives make up a valuable source of materials for chemical
industries Oleoresin can be extracted from living trees often by the bark streak method
in which bark removal is done periodically followed by application of stimulant paste
containing sulfuric acid and other chemicals on the freshly wounded exposed surface
To better understand the molecular basis of chemically-stimulated and wound induced
oleoresin production we evaluated the stability of 11 putative reference genes for the
purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis
Samples for RNA extraction were collected from field-grown adult trees under tapping
operations using stimulant pastes with different compositions and at various time points
after paste application Statistical methods established by geNorm NormFinder and
BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3
and UBI To confirm expression stability of the candidate reference genes expression
profiles of putative P elliottii orthologs of resin biosynthesis-related genes encoding Pinus
contorta β-pinene synthase [PcTPS-(minus)β-pin1] P contorta levopimaradieneabietadiene
synthase (PcLAS1) Pinus taeda α-pinene synthase [PtTPS-(+)αpin] and P taeda
α-farnesene synthase (PtαFS) were examined following stimulant paste application
Increased oleoresin yields observed in stimulated treatments using phytohormone-based
pastes were consistent with higher expression of pinene synthases Overall the
expression of all genes examined matched the expected profiles of oleoresin-related
transcript changes reported for previously examined conifers
Keywords resin Pinus gene expression normalizer genes terpene synthase
19
Chapter 2
Stimulant Paste Preparation and Bark Streak Tapping Technique for Pine Oleoresin Extraction
Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
Tapping technique comprises the extraction of pine oleoresin a non-wood forest product consisting of a
complex mixture of mono sesqui and diterpenes biosynthesized and exuded as a defense response to
wounding Oleoresin is used to produce gum rosin turpentine and their multiple derivatives Oleoresin
yield and quality are objects of interest in pine tree biotechnology both in terms of environmental and
genetic control Monitoring these parameters in individual trees grown in the fi eld provides a means to
examine the control of terpene production in resin canals as well as the identifi cation of genetic-based
differences in resinosis A typical method of tapping involves the removal of bark and application of a
chemical stimulant on the wounded area Here we describe the methods for preparing the resin-stimulant
paste with different adjuvants as well as the bark streaking process in adult pine trees
Key words Oleoresin Pine Tapping Chemical stimulant Wounding
1 Introduction
Several conifer species produce oleoresin a complex mixture of isoprenoid compounds relevant for defense against herbivores and pathogens Two major fractions can be recognized in oleoresin (a) turpentine the volatile fraction containing mono- and sesquiter-penes and (b) rosin the nonvolatile diterpene fraction Oleoresin is a forest commodity of global interest fi nding applications in diverse industry sectors Rosin is used in adhesives printing ink manufacture and paper sizing Turpentine can be used either as a solvent for paints and varnishes or as a raw material for fraction-ation of high-value chemicals used in the pharmaceutical agro-chemical and food industry [ 1 ndash 3 ]
During the extraction activity resin is obtained from the tree in a similar way as rubber tree tapping which generally involves the
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_2 copy Springer Science+Business Media New York 2016
These authors have equally contributed to this work
fettnetocbiotufrgsbr
27
Chapter 3
A Modifi ed Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines
Juacutelio Ceacutesar de Lima Thanise Nogueira Fuumlller Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes
through high-throughput sequencing technologies microarray and also northern blots RT-PCR and
RTqPCR Even though many specifi c protocols designed for plants with high content of secondary metab-
olites have been developed these are often expensive time consuming and not suitable for a wide range
of tissues Here we present a modifi cation of the method previously described using the commercially
available Concerttrade Plant RNA Reagent (Invitrogen) buffer for fi eld-grown adult pine trees with high
oleoresin content
Key words RNA Pines Concert plant RNA reagent Stem RNA extraction Oleoresin Conifers
1 Introduction
Several conifer species especially within the Pinaceae have tissues with high concentrations of phenolics terpenes and polysaccha-rides [ 1 ] Many specifi c protocols that are appropriate for plants rich in secondary metabolite s have been developed but the extrac-tion of high-quality RNA from these tissues using commercial kits is often diffi cult and usually not applicable to woody tissues [ 2 ndash 6 ] One of the major issues during RNA extraction concerns the pres-ence of phenolic compounds which oxidize and form quinones Aromatic compounds bind RNA which interferes in downstream steps and applications [ 3 7 ] Another point of concern is the har-vest of plant samples in the experimental fi eld which constitutes another obstacle in the efforts to avoid degradation of RNA [ 8 ] These problems often result in RNAs of low quality and insuffi -cient amounts especially for methodologies that normally require
These authors have equally contributed to this work
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_3 copy Springer Science+Business Media New York 2016
fettnetocbiotufrgsbr
RESEARCH PAPER
Control of resin production in Araucaria angustifolia an ancientSouth American coniferJ C Perotti1 K C da Silva Rodrigues-Correa123 amp A G Fett-Neto12
1 Plant Physiology Laboratory Department of Botany Federal University of Rio Grande do Sul (UFRGS) Porto Alegre RS Brazil
2 Center for Biotechnology UFRGS Porto Alegre RS Brazil
3 Present address Regional Integrated University of Alto Uruguai and Miss~oes (URI-FW) Frederico Westphalen RS Brazil
Keywords
Araucaria ethylene jasmonic acid nitric
oxide resin salicylic acid terpenes
Correspondence
A G Fett-Neto Plant Physiology Laboratory
Center for Biotechnology Federal University
of Rio Grande do Sul (UFRGS) PO Box 15005
Av Bento Goncalves 9500 91501-970 Porto
Alegre Brazil
E-mail fettnetocbiotufrgsbr
Editor
K Leiss
Received 22 July 2014 Accepted 11
December 2014
doi101111plb12298
ABSTRACT
Araucaria angustifolia is an ancient slow-growing conifer that characterises parts ofthe Southern Atlantic Forest biome currently listed as a critically endangered speciesThe species also produces bark resin although the factors controlling its resinosis arelargely unknown To better understand this defence-related process we examined theresin exudation response of A angustifolia upon treatment with well-known chemicalstimulators used in fast-growing conifers producing both bark and wood resin suchas Pinus elliottii The initial hypothesis was that A angustifolia would display signifi-cant differences in the regulation of resinosis The effect of Ethrel (ET ndash ethylene pre-cursor) salicylic acid (SA) jasmonic acid (JA) sulphuric acid (SuA) and sodiumnitroprusside (SNP ndash nitric oxide donor) on resin yield and composition in youngplants of A angustifolia was examined In at least one of the concentrations testedand frequently in more than one an aqueous glycerol solution applied on fresh woundsites of the stem with one or more of the adjuvants examined promoted an increase inresin yield as well as monoterpene concentration (a-pinene b-pinene camphene andlimonene) Higher yields and longer exudation periods were observed with JA and ETanother feature shared with Pinus resinosis The results suggest that resinosis controlis similar in Araucaria and Pinus In addition A angustifolia resin may be a relevantsource of valuable terpene chemicals whose production may be increased by usingstimulating pastes containing the identified adjuvants
INTRODUCTION
Many conifer species produce terpenoid-based resins that havelong been studied for their industrial importance and role indefence against attack by herbivores and pathogens The twomost important resin-producing families of conifers are Pina-ceae and Araucariaceae (Langenheim 1996) The viscous resinsecretion is generally composed of a complex mixture ofterpenoids consisting of roughly equal parts of volatile mono-(C10) and sesquiterpene (C15 turpentine) fractions and non-volatile diterpenic (C20 rosin) components (Rodrigues-Correaet al 2013) Terpenes act in a complex and multilayereddefence response providing toxicity against bark beetles andfungi bark wound sealing disruption of insect developmentand attraction of herbivore predators (Phillips amp Croteau1999)Most conifers rely on some combination of preformed and
inducible resin defences (Trapp amp Croteau 2001 Zulak amp Bohl-mann 2010) Resin defences are controlled by environmentaland genetic factors to various extents depending on species(Roberds et al 2003 Sampedro et al 2010 Moreira et al2013) Resin traits have been reported as highly variable havingmoderate heritability indicating that breeding efforts towardssuper-resinous forests are promising (Tadasse et al 2001Roberds et al 2003) Several chemicals are known as stimulantsof resin production Commercial extraction of resin from pine
trees uses periodic bark streaking and application of resin stim-ulant pastes to the wound
Resin-stimulant paste based on sulphuric acid (SuA) iswidely used for the commercial production of pine resin Cur-rent stimulant pastes usually have two chemically active com-ponents SuA to magnify the wounding and an ethyleneprecursor (2-chloroethylphosphonic acid CEPA or Ethrel ndash
ET) to stimulate resin flow (Rodrigues et al 2011 Rodrigues-Correa amp Fett-Neto 2013) Jasmonic acid (JA) and its methylester methyl jasmonate (MeJa) are among the most widelyused chemical elicitors of plant secondary metabolism It hasbeen shown that the exogenous application of MeJa or herbi-vore attack induce chemical and anatomical defence responsesin conifers such as the formation of traumatic resin ducts andresin accumulation in stems along with increased biosynthesisof terpenes and phenolics (Franceschi et al 2002 Martin et al2002 Heijari et al 2005 Zeneli et al 2006 Moreira et al 2008Gould et al 2009) JA commercial use however is limited byits high cost
The effects of exogenous salicylic acid (SA) on conifer ter-pene production have also been studied In Pinus elliottiiapplication of 10 molm3 of SA induced resin productionin wound panels but in Pseudotsuga menziesii and Sequoia-dendron giganteum it had no apparent effect on resinaccumulation (Hudgins amp Franceschi 2004 Rodrigues ampFett-Neto 2009) Nitric oxide (NO) has also emerged as an
Plant Biology 17 (2015) 852ndash859 copy 2014 German Botanical Society and The Royal Botanical Society of the Netherlands852
Plant Biology ISSN 1435-8603
iii
Dedicatoacuteria
Agrave minha amada avoacute lsquode laacutersquo Doraliacutecia Marcelina Costa da Silva (aka
Dona Dora como preferia ser chamada) por ter sido minha maior referecircncia
de amor e zelo enquanto estava aqui por ser a mais habilidosa e diligente
lsquofazedora de hortasrsquo e a melhor lsquoBotanistersquo empiacuterica que jaacute conheci
Obrigada por fazer da tua horta meu fantaacutestico lsquoherbaacuterio vivorsquo e do teu
conhecimento etnobotacircnico meu primeiro referencial de respeito e admiraccedilatildeo
ao Reino Plantae Foi nesse lsquoJardim Secretorsquo que descobri e me encantei
irreversivelmente pelo lsquoextraordinaacuterio poder das plantasrsquo E a saudade tua soacute
aumenta nunca diminui
Ao meu muito querido e saudoso amigo Rafael Cortes Duarte cortecircs
(sempre) ateacute no nome Se ainda estivesses por aqui esse trabalho teria sido
teu
Dizem que o tempo aqui eacute relativo Logo a gente se vecirc
iv
AGRADECIMENTOS
Ao meu orientador Dr Arthur Germano Fett-Neto uma das melhores pessoas
que tive a honra de conhecer na Academia Um coraccedilatildeo imenso uma mente incrivelmente
brilhante integridade e empatia infinitas (e extremamente raras no meio cientiacutefico)
Muito obrigada pela confianccedila em mim depositada e sobretudo por ter cometido a
insanidade de aceitar me orientar novamente Obrigada por ter me possibilitado ir em
termos cientiacuteficos muito aleacutem do que eu ousaria imaginar dadas as minhas (inuacutemeras)
limitaccedilotildees (e por todo ATP e NADPH investidos nesse esforccedilo hercuacuteleo que constitui a
aacuterdua tarefa de me orientar de forma natildeo condescendente a despeito dessas) Minha
diacutevida contigo seraacute eterna sou uma pessoa duplamente aquinhoada pela tua orientaccedilatildeo
Lucky me
Aos Professores Janette Palma-Fett uma grande amiga e saacutebia conselheira
sempre especialmente na adversidade e Felipe Maraschin pelo pronto e inestimaacutevel
apoio teacutecnico-cientiacutefico sempre que solicitado
Aos colegas do Laboratoacuterio de Fisiologia Vegetal da UFRGS pela parceria e
auxiacutelio em todas as horas por formarem um grupo coeso alinhado e comprometido com
o bem maior da pesquisa e do bom funcionamento do lab Eacute gratificante trabalhar com
todos vocecircs
Aos amigos muito queridos que a UFRGS me trouxe Ana Paula Durand
Coelho Eudes Stiehl-Alves Johnatan Vilasboa Yohanna Miotto e as divas Juliana
Troleis Sofia Aumond Kuhn e Tamara Pastori Muito obrigada por estarem presentes
nas horas menos faacuteceis e por me auxiliarem de muitas maneiras sempre que precisei Toda
dificuldade eacute redimensionada quando se tem amigos
Agrave minha famiacutelia caucasoide Ana Cristina Stein Camila Junkes Camila e
Cassiano Busatta Carlos Eduardo Blanco Linares Daniela Sponchiado Jordana
Griebler Luft Karen Santos Karina Letiacutecia Lopes e Larissa Schemes Heinzelmann
O carinho o apoio e o encorajamento que recebo de vocecircs fazem qualquer lsquofardorsquo parecer
mais leve Muito lsquomercirsquo
I am very grateful to Dr Dulal Borthakur for generously having received me in
his lab and his loving and caring family I would also like to thank my lab mates at UH
Manoa James Carillo Maia Corpuz and Ahmed Bageel for being so helpful cheerful
and friendly with me during all my stay in Honolulu Most of all Irsquod like to thank my
dear friend Michael Honda for teaching very patiently and supporting me inside and
outside the lab by doing whatever was in his power to prevent my homesickness I am
also very grateful to Mariana de Souza and Fernanda Oliveira for all those amazing
places and hikes wersquove been together in Orsquoahu You guys are awesome Mahalo nui loa
for your kōkua Im now parsquou hana
Jaimerais bien remercier mes collegravegues et amis agrave lrsquoUniversiteacute de Montreacuteal
(Benjamin Mazin Marion Kretsch Yang Liu Fang Wen Raquel Parada et Micaela
Margutti) pour mavoir chaleureusement reccedilu chez vous speacutecialement agrave mon ami
Valentin Joly pour mavoir beaucoup appris sur lrsquoinconnu monde des bacteacuteries et des
v
levures (et surtout pour leur incroyable patience avec mon tregraves mauvais franccedilais) Crsquoeacutetait
vachement chouette Merci beaucoup agrave vous tous (et toutes) et agrave la prochaine
Agrave Coordenaccedilatildeo de Aperfeiccediloamento Pessoal de Niacutevel Superior (CAPES) pelo
financiamento da bolsa de pesquisa do PDSE
Aos meus pais (bioloacutegicos ou natildeo) Veacutera Maria da Silva Rodrigues Gilberto
Moraes Rodrigues Rosa Maria Lucas da Silva e Paulo Joseacute Costa da Silva pelo
exemplo de honestidade coragem trabalho forccedila e amor desde sempre
Aos meus irmatildeos Ana Paula da Silva Rodrigues Viniacutecius de Moraes da Silva
Rodrigues Marcello da Silva Rodrigues e Camila Stella Toledo Pereira por todas as
experiecircncias que dividimos e tudo o que me ensinaram ateacute hoje
Ao meu amor maior minha melhor amiga minha mais leal e extraordinaacuteria
parceria nessa grande (e agraves vezes tortuosa) jornada Maria Clara Rodrigues Correcirca Por
ser ela por ser imensa em generosidade amor e altruiacutesmo por despertar o melhor em
mim por ser minha forccedila motriz e sobretudo por ser a melhor das minhas metades
Minha vida soacute realmente comeccedilou quando eu tive a incriacutevel sorte de te conhecer
vi
SUMAacuteRIO
LISTA DE ABREVIATURASvii
RESUMO ix
INTRODUCcedilAtildeO GERAL1
HIPOacuteTESE E OBJETIVOS9
CAPIacuteTULO 1 Abiotic stresses and non-protein amino acids in plantshelliphellip10
CAPIacuteTULO 2 Mimosine accumulation in Leucaena leucocephala in response to
stress signaling molecules and acute UV exposurehelliphelliphelliphelliphelliphelliphelliphelliphelliphellip(432) 52
CAPIacuteTULO 3 Mimosine occurrence and accumulation in Mimosa bimucronata var
bimucronata (DC) Kuntze66
CONSIDERACcedilOtildeES FINAIS 84
PERSPECTIVAS85
REFEREcircNCIAS BIBLIOGRAacuteFICAS86
Artigos publicados no periacuteodo de doutoramento natildeo relacionados ao tema da
tese91
vii
LISTA DE ABREVIATURAS
24-D 24-dichlorophenoxyacetic acid
3H4P 3-hydroxy-4-pyridone (34-DHP 34-dihydroxypyridine)
ABA abscisic acid
Arg arginine
BABA β-aminobutyric acid
β-ODAP β-N-oxalyl-L-α β-diaminopropionic acid
BIA β-isoxazolinon-L-alanine
CAN canavanine
DAO diamine oxidase
DDC decarboxylase
ETH ethephon
FW fresh weight
GABA -aminobutyric acid
GABA-T GABA transaminase
GAD glutamate decarboxylase
GSM Global System for Mobile
HPLC High performance liquid chromatography
JA jasmonate
JA-Ile jasmonoyl-L-isoleucine
L-DOPA L-34- dihydroxyphenylalanine
MeJA methyl jasmonate
m-Tyr Meta-tyrosine
NO nitric oxide
NPAA non-protein amino acid
OAS o-acetylserine
OAS-TL o-acetylserine-thiol-lyase
PA polyamine
PAA protein amino acid
viii
PEG polyethylene glycol
PLP pyridoxal-5rsquo-phosphate
PPO polyphenol oxidase tyrosinase
qRT-PCR Reverse transcription polymerase chain reaction quantitative real time
RNS reactive nitrogen species
ROS reactive oxygen species
SA salicylic acid
SAR systemic acquired resistance
SNP sodium nitroprusside
UV ultraviolet radiation
ix
RESUMO
Ao longo de sua evoluccedilatildeo as plantas desenvolveram estrateacutegias estruturais e quiacutemicas de
defesa em resposta aos estresses bioacuteticos e abioacuteticos impostos pelo ambiente Dentre
essas satildeo reconhecidas moleacuteculas quimicamente especializadas denominadas
metaboacutelitos secundaacuterios produtos naturais ou metaboacutelitos especializados Aminoaacutecidos
natildeo proteicos (ANPs) satildeo compostos nitrogenados que constituem aleacutem de componentes
do arsenal de defesa quiacutemica vegetal uma importante fonte de reserva de carbono e
nitrogecircnio para diversos taxa especialmente aqueles pertencentes agrave famiacutelia Fabaceae de
Angiospermas Esse grupo de moleacuteculas quimicamente heterogecircneo eacute assim definido por
natildeo participar da formaccedilatildeo de estruturas proteicas funcionais sendo frequentemente
toacutexicos quando erroneamente incorporados nas cadeias polipeptiacutedicas em formaccedilatildeo em
funccedilatildeo da similaridade estrutural que apresentam com os aminoaacutecidos proteicos Sob o
ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados ANPs satildeo
em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou abioacutetica como
o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de elicitores
quiacutemicos por exemplo O objetivo da presente tese foi investigar o papel bioloacutegico da
mimosina endoacutegena em Leucaena leucocephala (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute) a partir da avaliaccedilatildeo do efeito de tratamentos
relacionados ao estresse abioacutetico (UV-C aacutecido saliciacutelico metil jasmonato e etileno)
Mimosina eacute um ANP aromaacutetico anaacutelogo da L-tirosina com atividade toacutexica para ceacutelulas
de procariotos e eucariotos Dentre as atividades descritas para esse ANP destacam-se a
atividade anti-mitoacutetica ou bloqueadora do ciclo celular atividade alelopaacutetica e
antioxidante Os resultados indicaram que em leucena a biossiacutentese e o acuacutemulo de
mimosina podem ser modulados por fatores causadores de estresses exibindo um padratildeo
de acumulaccedilatildeo similar ao das fitoalexinas Em maricaacute por outro lado a induccedilatildeo do
acuacutemulo dessa moleacutecula natildeo foi observada para os mesmos tratamentos testados para
leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas Aleacutem disso
o padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado
pela sua degradaccedilatildeo As respostas observadas nos testes que avaliaram a atividade de
mitigaccedilatildeo de espeacutecies reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode
agir como um agente antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de
estresse
1
Introduccedilatildeo
Na condiccedilatildeo de organismos seacutesseis ao longo de sua evoluccedilatildeo as plantas
desenvolveram estrateacutegias estruturais e quiacutemicas de defesa em resposta aos estresses bioacuteticos
e abioacuteticos impostos pelo ambiente Dentre essas satildeo reconhecidas moleacuteculas quimicamente
especializadas denominadas metaboacutelitos secundaacuterios produtos naturais (Kutchan et al 2015)
ou mais recentemente metaboacutelitos especializados
Entre as trecircs classes mais gerais de metaboacutelitos secundaacuterios (terpenos compostos
fenoacutelicos e compostos nitrogenados) aminoaacutecidos natildeo-proteicos (ANPs) satildeo incluiacutedos no
terceiro grupo e constituem aleacutem de componentes do arsenal de defesa quiacutemica uma
importante fonte de reserva de carbono e nitrogecircnio para diversos taxa especialmente aqueles
pertencentes agrave famiacutelia Fabaceae de Angiospermas (leguminosas sensu lato)
Aleacutem dos 20 aminoaacutecidos proteicos estima-se que existam entre 600 e 1000 ANPs
(Acamovic amp Brooker 2005 Rodgers et al 2015) Esse grupo de moleacuteculas quimicamente
heterogecircneo eacute assim definido por natildeo participar da formaccedilatildeo de estruturas proteicas
funcionais sendo frequentemente toacutexicos quando erroneamente incorporados nas cadeias
polipeptiacutedicas em formaccedilatildeo em funccedilatildeo da similaridade estrutural que apresentam com os
aminoaacutecidos proteicos (Taiz amp Zeiger 2010)
Conforme mencionado a ocorrecircncia de ANPs eacute comum em espeacutecies de leguminosas
e sua distribuiccedilatildeo pode ser restrita a alguns gecircneros de plantas circunscritos nessa famiacutelia
botacircnica (eg mimosina e canavanina) Por outro lado alguns ANPs como GABA por
exemplo podem apresentar distribuiccedilatildeo ubiacutequa no Reino Plantae assim como ocorrer em
outros tipos de organismos como animais por exemplo (Ramos-Ruiz et al 2018)
2
Apesar de representarem uma fonte nutricional importante sem tratamento preacutevio o
consumo de plantas que acumulam ANPs por animais eacute limitado Isso ocorre pois em longo
prazo a ingestatildeo prolongada de plantas (especialmente sementes) que acumulam ANPs pode
representar risco agrave sauacutede uma vez que estes comprometem o funcionamento de mecanismos
basais de manutenccedilatildeo da homeostase celular e podem tambeacutem em um quadro mais severo
desencadear doenccedilas neurotoacutexicas degenerativas como por exemplo o latirismo causado
por aacutecido β-N-oxalil-l-αβ-diaminopropiocircnico (β-ODAP) (Jiao et al 2011 Kusama-Eguchi
2019)
Sob o ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados
ANPs satildeo em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou
abioacutetica como o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de
elicitores quiacutemicos por exemplo No que concerne ao estudo dos efeitos da induccedilatildeo abioacutetica
sobre o acuacutemulo de ANPs em diferentes espeacutecies vegetais (Monocotiledocircneas e
Eudicotiledocircneas) as moleacuteculas mais amplamente investigadas ateacute o momento satildeo GABA
L-DOPA e mais recentemente mimosina (vide Tabela 1 do capiacutetulo primeiro) Em termos
de efeitos causados a partir da aplicaccedilatildeo exoacutegena de ANPs GABA tambeacutem figura como o
principal aminoaacutecido investigado seguido de L-DOPA e canavanina (vide Tabela 2 do
capiacutetulo primeiro)
No primeiro capiacutetulo da presente tese estatildeo descritas as caracteriacutesticas gerais dos
principais ANPs estudados seus possiacuteveis papeacuteis bioloacutegicos in planta e seus efeitos quando
aplicados exogenamente bem como os estresses abioacuteticos capazes de induzir seu(s)
acuacutemulo(s) nos diferentes tecidos vegetais Nos segundo e terceiro capiacutetulos
respectivamente satildeo elucidados os efeitos dos tratamentos de UV-C aacutecido saliciacutelico etileno
e jasmonato (claacutessicos elicitores do metabolismo secundaacuterio vegetal) sobre o acuacutemulo de
3
mimosina em Leucaena leucocephala var glabrata (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute)
Mimosina eacute um aminoaacutecido aromaacutetico natildeo-proteico anaacutelogo da L-tirosina com
atividade toacutexica para ceacutelulas de procariotos e eucariotos Embora em menor concentraccedilatildeo
mimosina foi primeiramente identificada em Mimosa pudica sendo posteriormente detectada
em outras espeacutecies do gecircnero como Mimosa pigra por exemplo (Soedarjo amp Borthakur
1998) Seu efeito toacutexico eacute atribuiacutedo agrave capacidade de quelar metais o que impede o
funcionamento adequado das metalo-proteiacutenas que dependem dos mesmos como co-fatores
(Negi et al 2014)
A concentraccedilatildeo basal de mimosina em espeacutecies de leucaena pode variar entre 1 e 12
do peso seco do oacutergatildeo (Soedarjo amp Borthakur 1998) Como eacute comum para outros ANPs
que ocorrem em espeacutecies de leguminosas em sementes de Leucaena spp eacute observada uma
maior concentraccedilatildeo de mimosina quando comparada aos demais oacutergatildeos da planta
(Rodrigues-Correcirca et al 2019) sendo esta a fonte de extraccedilatildeo comercial do padratildeo quiacutemico
de mimosina vendido por empresas de reagentes de pesquisa
Diversas atividades foram descritas para mimosina em outros organismos eou tipos
celulares Dentre essas destacam-se a atividade anti-mitoacutetica ou bloqueadora do ciclo
celular em ceacutelulas de eucariotos e procariotos Isto ocorre porque a mimosina impede a
formaccedilatildeo da forquilha de replicaccedilatildeo (e portanto a siacutentese de DNA) interrompendo assim o
avanccedilo do ciclo de divisatildeo celular na fase tardia G1 (Lalande 1990) Foram tambeacutem descritas
para mimosina atividade alelopaacutetica observada sobre o desenvolvimento de outras espeacutecies
de leguminosas e atividade antioxidante entre outras (Tabela 1)
A rota de biossiacutentese de mimosina eacute compartilhada em grande parte com a de cisteiacutena
um aminoaacutecido proteico sulfurado (Figura 1) A siacutentese da cisteiacutena se daacute a partir da conversatildeo
4
de serina e acetil-CoA em o-acetilserina pela enzima SAT (serina acetiltransferase) seguida
da conversatildeo de o-acetilserina e aacutecido sulfiacutedrico em cisteiacutena em uma reaccedilatildeo catalisada pela
OAS-TL (o-acetilserina tiol-liase) A siacutentese de mimosina por sua vez eacute compartilhada com
a da cisteiacutena ateacute esse ponto e acredita-se que pelo menos uma das isoformas de OAS-TL
catalise a conversatildeo de o-acetilserina e 3-hidroxi-4-piridona em mimosina
Tabela 1 Atividades descritas para mimosina de Leucaena leucocephala (Lam) de Wit
ATIVIDADE
ALVO AVALIADO
(organismo eou tecido tipo
celular)
REFEREcircNCIA
Bloqueio do complexo de ativaccedilatildeo
da preacute-replicaccedilatildeo do DNA
Ceacutelulas de mamiacuteferos
KUBOTA et al
(2014)
Alteraccedilatildeo no ciclo ovariano e
extensatildeo da duraccedilatildeo do corpo luacuteteo
bovino no periacuteodo poacutes-parto
Bovinos
(Bos taurus x
Bos indicus)
BOTTINI-
LUZARDO et al
(2015)
Supressatildeo do ciclo celular e reduccedilatildeo
da abundacircncia bacteriana em
mosquitos
Wolbachia pipientis
Aedes albopictus
FALLON
(2015)
Accedilatildeo inibitoacuteria da fibrose
pulmonar induzida
Ratos SD
LI et al
(2015)
Recuperaccedilatildeo da funccedilatildeo do
miocaacuterdio poacutes-isquemia
Miocaacuterdio de ratos (SD)
machos
CROWE et al
(2001)
Inseticida
Heteropsylla cubana
Crawford 1914 e Thrips tabaci
Lindemann 1889
AHMED et al
(2016)
Alelopaacutetica
Albizia procera Vigna
unguiculata Cicer arietinum
Cajanus cajan
AHMED et al
(2008)
Antioxidante
Sistemas modelo de oxidaccedilatildeo
lipiacutedica (β-caroteno - aacutecido
linolecircico e lecitina)
BENJAKUL et al
(2013)
Ateacute momento versotildees divergentes sobre a enzima responsaacutevel pela biossiacutentese de
mimosina (mimosina sintase) tecircm sido publicadas Em 1990 Ikegami e colaboradores
5
identificaram uma OAS-TL responsaacutevel pela formaccedilatildeo de cisteiacutena como sendo tambeacutem uma
mimosina sintase Mais tarde Yafuso et al (2014) realizaram a expressatildeo heteroacuteloga do gene
que codifica para OAS-TL em Escherichia coli e natildeo foi observada a formaccedilatildeo de mimosina
mesmo quando dadas as condiccedilotildees oacutetimas para tanto Mais recentemente Harun-Ur-Rashid
et al (2018) elucidaram a mimosina sintase como sendo uma isoforma da OAS-TL
corroborando o postulado por Ikegami e colaboradores em 1990
Figura 1 Rota de biossiacutentese da mimosina Fonte Ikegami et al (1990)
Espeacutecies estudadas
Leucaena leucocephala (Lam) de Wit (leucaena koa haole ou ldquoacaacutecia exoacuteticardquo na
liacutengua Hawairsquoiana) eacute uma espeacutecie de haacutebito arboacutereo ou arbustivo pertencente agrave famiacutelia
Fabaceae de Angiospermas e caracterizada pelo acuacutemulo de mimosina em todos os seus
oacutergatildeos Eacute nativa da Ameacuterica Central (especificamente da regiatildeo sudeste do Meacutexico) mas
irradiou-se atraveacutes de praticamente todas as zonas tropicais e subtropicais da Terra No
Brasil leucena eacute amplamente distribuiacuteda e classificada como naturalizada pelo REFLORA
(2019) ocorrendo em todo territoacuterio Nacional Satildeo reconhecidas no miacutenimo duas
6
subespeacutecies de leucena ocorrentes no Brasil L leucocephala var leucocephala e L
leucocephala var glabrata sendo a primeira a mais abundante
Leucaena apresenta atributos morfoloacutegicos caracteriacutesticos das leguminosas como o
fruto do tipo vagem deiscente no periacuteodo poacutes-maturaccedilatildeo folhas compostas e bipinadas As
flores satildeo seacutesseis actinomorfas e polistecircmones apresentam caacutelice sinseacutepala e corola
gamopeacutetala e satildeo dispostas em inflorescecircncias do tipo glomeacuterulo (Figura 2)
Figura 2 Oacutergatildeos vegetativos e reprodutivos de L leucocephala (Lam) de Wit Fonte Little Jr amp Skolmen
(1989)
Com base no conhecimento etnobotacircnico disponiacutevel acerca dessa espeacutecie em
diversas regiotildees tropicais e subtropicais leucena eacute utilizada para vaacuterios fins Extratos de
diferentes oacutergatildeos de leucena apresentam atividade anti-diabeacutetica (Kuppusamy et al 2014
Chowtivannakul et al 2016) antioxidante (Mohammed et al 2015 Chowtivannakul et al
2016 Zarin et al 2016) antimicrobiana (Zarin et al 2016) anti-helmiacutentica (Soares et al
2015 Jamous et al 2017) bactericida (Mohammed et al 2015) acaricida (Fernaacutendez-Salas
et al 2011) anti-tumoral (Chung et al 2017) e potencializadora da resposta imune em
peixes (Verma et al 2018) entre outras
7
Leucaena apresenta alta toleracircncia agrave seca sendo capaz de enfrentar estaccedilotildees sazonais
inteiras com deacuteficit hiacutedrico sem prejuiacutezo permanente de seus oacutergatildeos e de recuperar
vigorosamente sua biomassa vegetativa tatildeo logo o regime de precipitaccedilatildeo retome a
regularidade em frequecircncia Acredita-se que a toleracircncia agrave seca apresentada por essa espeacutecie
ocorra em funccedilatildeo do acuacutemulo de mimosina nos diferentes tecidos da planta a qual
funcionaria como um agente osmoregulador responsaacutevel pela preservaccedilatildeo da integridade das
membranas a das macromoleacuteculas intracelulares em periacuteodos de escassez de aacutegua no
ambiente
Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) eacute uma leguminosa
nativa natildeo endecircmica do Brasil amplamente distribuiacuteda nos domiacutenios fitogeograacuteficos da
Caatinga do Cerrado e da Mata Atlacircntica (Simon amp Proenccedila 2000 REFLORA 2019) Como
espeacutecie pioneira (Pilatti et al 2019) exerce importante papel ecoloacutegico na recuperaccedilatildeo de
aacutereas degradadas (Bitencourt et al 2007 Silva et al 2011) no estabelecimento de processos
de sucessatildeo vegetacional
Maricaacute eacute uma espeacutecie semi-deciacutedua a deciacutedua a qual atinge ateacute 15 m em altura (e
diacircmetro agrave altura do peito de ateacute 40 cm) na idade adulta com haacutebito arboacutereo ou arbustivo
(REFLORA 2019) e espinhos caracteriacutesticos desde os estaacutegios iniciais de desenvolvimento
(Carvalho 2004) Apresenta folhas compostas alternas e bipinadas (Figura 2) amplas
inflorescecircncias brancas com flores reunidas em glomeacuterulos esfeacutericos dispostos em grandes
paniacuteculas As flores satildeo diplostecircmones actinomorfas hipoacuteginas e unicarpelares (Silva et al
2011)
Assim como descrito para leucena maricaacute eacute considerado uma espeacutecie multifuncional
sendo comumente empregada para produccedilatildeo de mel como combustiacutevel (Olkoski amp
8
Wittmann 2011) em edificaccedilotildees na carpintaria e como lsquocerca-vivarsquo (Marchiori 1993
Lorenzi 1998) entre outras aplicaccedilotildees
Figura 2 Folhas e fruto de Mimosa bimucronata (DC) Kuntze Fonte Souza-Lima et al (2017)
Em contraste com a amplitude de habitats explorados por leucena (especialmente os
aacuteridos) no Sul do Brasil maricaacute ocorre preferencialmente em ambientes uacutemidos a alagadiccedilos
em aacutereas proacuteximas agraves margens de rios (Patreze amp Cordeiro 2004) embora possa tambeacutem
ocorrer em formaccedilotildees quase exclusivas dessa espeacutecie nas encostas de morros (Jacobi amp
Ferreira 1991)
Em relaccedilatildeo agraves atividades elucidadas para os extratos de maricaacute foram relatados os
efeitos alelopaacutetico (Jacobi amp Ferreira 1991 Ferreira et al 1992) diureacutetico natriureacutetico e
caliureacutetico (Schlickmann et al 2017)
9
Hipoacutetese
Mimosina apresenta perfil dinacircmico de acuacutemulo em Leucaena leucocephala e
Mimosa bimucronata frente a estresses associado a alteraccedilotildees significativas na expressatildeo de
genes relacionados ao metabolismo deste ANP o qual contribui para mitigar o desequiliacutebrio
oxidativo inerente a vaacuterios tipos de estresse
Objetivo geral
O objetivo da presente tese foi investigar o papel bioloacutegico da mimosina endoacutegena
em leucena e maricaacute a partir da avaliaccedilatildeo do efeito de tratamentos relacionados a estresses
ou sinalizadores de estresse
Objetivos especiacuteficos
- Analisar a concentraccedilatildeo constitutiva de mimosina nos diferentes oacutergatildeos de L leucocephala
(Lam) de Wit (leucena) e M bimucronata (DC) Kuntze (maricaacute)
- Verificar se apesar do seu alto teor constitutivo em plantas de leucena o acuacutemulo de
mimosina pode ser induzido com tratamentos que mimetizam diferentes estresses a partir da
avaliaccedilatildeo do efeito de moleacuteculas sinalizadoras (aacutecido saliciacutelico jasmonato etileno) e da
exposiccedilatildeo agrave radiaccedilatildeo UV-C na modulaccedilatildeo do acuacutemulo de mimosina em leucena bem como
em maricaacute
- Determinar se a expressatildeo de genes relacionados ao metabolismo de mimosina estaacute
associada agrave induccedilatildeo por estresses fisioloacutegicos
- Avaliar o potencial antioxidante da mimosina em experimentos realizados in situ
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry
journal homepage wwwelseviercomlocateplaphy
Research article
Mimosine accumulation in Leucaena leucocephala in response to stresssignaling molecules and acute UV exposure
Kelly Cristine da Silva Rodrigues-Correcircaab Michael DH Hondab Dulal BorthakurbArthur Germano Fett-Netoalowast
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970Porto Alegre Rio Grande do Sul BrazilbDepartment of Molecular Biosciences and Bioengineering University of Hawaii at Manoa Honolulu HI 96822 USA
A R T I C L E I N F O
KeywordsLeucaena leucocephalaMimosineMimosine amidohydrolaseJasmonic acidEthyleneSalicylic acidUV-C radiation
A B S T R A C T
Mimosine is a non-protein amino acid of Fabaceae such as Leucaena spp and Mimosa spp Several relevantbiological activities have been described for this molecule including cell cycle blocker anticancer antifungalantimicrobial herbivore deterrent and allelopathic activities raising increased economic interest in its pro-duction In addition information on mimosine dynamics in planta remains limited In order to address this topicand propose strategies to increase mimosine production aiming at economic uses the effects of several stress-related elicitors of secondary metabolism and UV acute exposure were examined on mimosine accumulation ingrowth room-cultivated seedlings of Leucaena leucocephala spp glabrata Mimosine concentration was not sig-nificantly affected by 10 ppm salicylic acid (SA) treatment but increased in roots and shoots of seedlings treatedwith 84 ppm jasmonic acid (JA) and 10 ppm Ethephon (an ethylene-releasing compound) and in shoots treatedwith UV-C radiation Quantification of mimosine amidohydrolase (mimosinase) gene expression showed thatethephon yielded variable effect over time whereas JA and UV-C did not show significant impact Consideringthe strong induction of mimosine accumulation by acute UV-C exposure additional in situ ROS localization aswell as in vitro antioxidant assays were performed suggesting that akin to several secondary metabolitesmimosine may be involved in general oxidative stress modulation acting as a hydrogen peroxide and superoxideanion quencher
1 Introduction
Different plant groups synthesize a large diversity of secondary orspecialized metabolites These molecules are generally produced inresponse to biotic and abiotic environmental stresses Indeed inductionof secondary metabolism usually involves stress-generating factorswhich have also been explored in biotechnological processes aiming atthe production of target metabolites of economic interest (Matsuuraet al 2018) Metabolic control of nitrogen-containing secondarycompounds (eg alkaloids and non-protein amino acids) has beenshown to be complex and influenced by phytohormones environmentalstresses (seasonality herbivory pathogen attack drought) UV radia-tion (Holloacutesy 2002) methyl jasmonate (MeJA) salicylic acid (SA)yeast extract (Cho et al 2008) abscisic acid (ABA) heavy metals os-motic stress (Nascimento et al 2013) and mechanical wounding (Portoet al 2014)
Due to their particular trait of associating with N-fixing micro-organisms Fabaceae species (leguminous sensu lato) are often proteinrich hence the relevance of several of these species as forage Fabaceaespecies are also known for accumulating nitrogen containing secondarymetabolites which play important roles as ecochemical molecules andat least for the case of non-protein amino acids potential cell reservoirsof nitrogen (Huang et al 2011)
High contents of mimosine a toxic aromatic non-protein aminoacid are found in species of two leguminous genera Leucaena spp andMimosa spp Leucaena leucocephala (Lam) de Wit (leucaena koa haole)is a fast-growing leguminous tree native from Central America (south-eastern Mexico) widely distributed in tropical and subtropical zonesThis species is also characterized by its high tolerance to droughtamong other environmental stresses (Honda et al 2018) Leucaena canbe divided into two subspecies (i) L leucocephala subsp leucocephala(common leucaena a bushy shrub) and (ii) L leucocephala subsp
httpsdoiorg101016jplaphy201811018Received 1 August 2018 Received in revised form 9 November 2018 Accepted 14 November 2018
lowast Corresponding authorE-mail addresses krodriguescbiotufrgsbr (KCdS Rodrigues-Correcirca) mhonda2hawaiiedu (MDH Honda) dulalhawaiiedu (D Borthakur)
fettnetocbiotufrgsbr (AG Fett-Neto)
Plant Physiology and Biochemistry 135 (2019) 432ndash440
Available online 19 November 20180981-9428 copy 2018 Elsevier Masson SAS All rights reserved
T
glabrata (giant leucaena a tree) The latter has been used as a fastgrowing tree for production of wood and paper pulp The foliage ofboth common and giant leucaena is used as a fodder because of its highprotein content and palatability to farm animals The foliage containsup to 18 protein 142 crude fiber and 64 ether extractcrude fat(Soedarjo and Borthakur 1996)
Production of nitrogen-containing secondary metabolites such asmimosine requires large amounts of carbon and nitrogen resourcesNegi et al (2014) estimated that up to 21 of the carbon-nitrogenresources may be used for production of mimosine in leucaenaBrewbaker et al (1972) determined the mimosine content of 96 Lleucocephala cultivars and 8 other Leucaena species collected from 38different countries by growing them in an observational nursery inHawaii and found that basal mimosine content varied from 189 to477 of the dry weight
Mimosine is biosynthesized from OAS (o-acetylserine) and 3H4P (3-hydroxy-4-pyridone or its tautoisomer 3-hydroxy-4-pyridine) A pre-vious analysis suggested that mimosine synthase is an OAS-TL (o-acetylserine-thiol-lyase) of the cysteine biosynthesis pathway (Ikegamiet al 1990) Later however recombinant enzyme tests did not supportan OAS-TL identity of mimosine synthase (Yafuso et al 2014) Recentfindings on mimosine biosynthesis revealed that a cytosolic cysteine-OAS-TL isoform can also catalyze the formation of mimosine underspecific conditions (Harun-Ur-Rashid et al 2018)
Mimosine toxicity is related to its ability of reducing the availabilityof divalent metal ions such as Fe(II) Zn(II) Cu(II) Co(II) and Mn(II)by chelating co-factors and preventing their association with metal-dependent enzymes Furthermore this non-protein amino acid is cap-able of forming a stable complex with pyridoxal-5prime-phosphate (PLP)leading to the inactivation of PLP-dependent enzymes (eg Asp-Glutransaminase and cystathionine synthetase) (Negi et al 2014)
Mimosine features several useful biological activities such as alle-lopathic antimicrobial insecticide cell cycle inhibitor agent antic-ancer phytoremediator (Nguyen and Tawata 2016) as well as anti-oxidant (Benjakul et al 2013) Despite the relatively well establishedbiological activities of purified mimosine on other organisms or celltypes little is known about its biological role in leguminous speciesHowever it has been suggested that at least in part its activity ismainly related to defense mechanisms against some biotic and abioticstresses and as nitrogen source during fast growth (Vestena et al2001)
Suda (1960) and Smith and Fowden (1966) identified enzymes in-volved in mimosine degradation in seedling extracts of L leucocephalaand Mimosa pudica A mimosine-degrading enzyme named mimosinase(mimosine amidohydrolase EC 35161 CAS registry number 104118-49-2) (IUBMB 2018) a carbon-nitrogen lyase which degrades mimo-sine into 3H4P was later purified by Tangendjaja et al (1986) Itsbiochemical characterization was described and the cDNA was isolatedby Negi et al (2014)
Although mimosinase has been described and isolated only fewstudies on the role played by biotic and abiotic factors on the dynamicmodulation of mimosine metabolism in leguminous species have beenconducted (Vestena et al 2001 Xu et al 2018) In aseptic cultures ofleucaena mechanical injury of shoots promoted local mimosine accu-mulation (Vestena et al 2001) In the same study cultivation in pre-sence of auxin or SA in culture medium also had a positive effect on
mimosine accumulation More recently the effect of drought treatmenton gene expression of leucaena was also evaluated by Honda et al(2018) However several potential factors regulating mimosine meta-bolism need to be further examined
To date there is a lack of information on the biological role ofmimosine in planta as well as on details of its metabolic dynamicsMoreover its overt potential for pharmaceutical applications and de-velopment of new drugs as well as the possible use as tool to addressheavy metal soil contamination or plant mineral nutrition improve-ment justify additional research The objective of this study was toinvestigate the effect of stress signaling molecules and acute UV ex-posure on modulation of mimosine accumulation and metabolism in Lleucocephala spp glabrata in order to better understand its biologicalrole and to identify strategies for yield improvement aiming at ex-ploring its useful bioactivities
2 Methods
21 Plant material
For the experiments carried out to evaluate the effects of elicitors onmimosine accumulation seeds of leucaena were kindly provided by DrJames Brewbaker and harvested at CTAHRs (College of TropicalAgriculture and Human Resources of the University of Hawaii atManoa) Waimanalo Research Station at Oahu Hawaii This plantmaterial was originated from the accession K636 of Leucaena leucoce-phala ssp glabrata (Brewbaker 2008)
22 Induced mimosine content in 5-week-old giant leucaena
221 Seed germinationIn order to overcome seed coat dormancy seeds were submitted to a
chemical scarification with sulfuric acid 95ndash98 for 20min and re-peatedly rinsed in distilled water to remove any residual trace of thisreagent Then seeds were distributed in 254 cmtimes508 cm plastictrays containing 11 vv of vermiculite and commercial soil watereduntil reaching substrate field capacity Three weeks after seed imbibi-tion seedlings displaying similar size and shape (eg number of com-pound leaves and leaflets) were transplanted to individual pots(250mL) in number of three plants per container
During the experimental period (except in the UV-C radiationtreatment) all tested seedlings were kept in a growth chamber andsubmitted to controlled conditions of temperature (circa 25 degC) and ir-radiance (approximately 100 μmol photons mminus2sdot s minus1) with a photo-period of 16 h light and 8 h dark
222 Treatments2221 JA Ethephon and SA Five-week-old giant leucaena seedlingswere treated with different solutions as described in Table 1 Idealconcentrations were defined in preliminary experiments under the sameconditions indicated above At the beginning of the experiments 30plants were sprayed with 84 ppm JA 10 ppm SA 10 or 100 ppmEthephon or Milli-Qreg water (control) until the point of imminent runoffPlant pots were kept closed inside transparent plastic bags for 24 h toavoid solution volatilization Fifteen plants arranged in 5 sets of 3 (5biological replicates) were harvested 48 h and 96 h after being treated
Table 1Treatments used to modulate mimosine biosynthesis in giant leucaena
ELICITOR CONCENTRATION UV FLUENCE EXPOSURE TIME RATIONALE FOR USE
Salicylic acid (SA) 10 ppm 24 h Pathogen signaling molecule (Shah 2003)Jasmonic acid (JA) 84 ppm 24 h Chemical elicitor of plant secondary metabolism (Dar et al 2015)Ethephon 10 ppm 24 h Ethylene releasing-compound (Kim et al 2016) elicitor of plant secondary metabolism (Wang
et al 2016)UV-C radiation 3 Jcmminus2 10min or 15min Elicitor of plant secondary metabolism (Kara 2013 Neelamegam and Sutha 2015)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
433
After collection shoots were separated from roots immediately frozenin liquid nitrogen and stored at ndash 80 degC prior to HPLC analyses
2222 UV-C Thirty seedlings of giant leucaena were exposed to UV-Cradiation (3 Jcmminus2) for 10 or 15min and kept in a growth chamberunder controlled conditions as described above until the end of theexperiments Fifteen plants arranged in groups of 3 were harvested at96 h and 120 h after UV-C exposure and processed as previouslydescribed
223 Mimosine extractionMimosine extraction was based on a modified version of the pro-
tocol published by Lalitha and Kulothungan (2006) as follows a knownweight of fresh tissue (shoots or roots) of giant leucaena was first addedto Milli-Qreg boiling water in a proportion of 110 (g of plant per mL ofsolvent) in test tubes Tubes were covered with foil to avoid solutionevaporation and placed on a hot stirrer at 100 degC for 10min A pro-portional volume of 01M HCl was added to the cooled suspensions andhomogenized using mortar and pestle The plant extracts were filteredthrough cotton and centrifuged twice for 7min in a bench top re-frigerated centrifuge at 4 degC and 13200 rpm Before being analyzed theextracts were diluted 13 with ondashphosphoric acid (OPA)
224 Mimosine detectionHPLC analyses were carried out as described by Negi and Borthakur
(2016) Pure mimosine (L-mimosine from koa haole seeds Sigma-Al-drich CAS number 500-44-7) was used as standard Separation andquantification of mimosine was done with a C18 column (PhenomenexC18 5 μm 46times250mm) under an isocratic solvent system of 002MOPA with a linear flow rate of 1mLsdotminminus1 Mimosine detection wasdone at 280 nm by photodiode array detection (200ndash400 nm) showingretention time of 277 plusmn 0042min Quantification was done using themethod of external standard curve Further confirmation of mimosineidentity was performed by co-chromatography with standard and peakpurity check Chromatograms were analyzed using the Waters Em-power 3 software
23 Quantitative real-time PCR analysis of mimosinase gene expression
Fifteen 8-week-old giant leucaena plants arranged in 4 sets of 3 (4biological replicates) were treated with either water (control) or10 ppm Ethephon 84 ppm JA acid or 15min of UV-C radiation ex-posure following the methods described above Following treatmentleucaena plants were harvested at 48 and 96 h or 72 and 144 h (UV-Ctreated plants only) after treatments Total RNA of samples was ex-tracted and purified from roots and shoots of giant leucaena by meansof a modified method using Qiagen RNeasy Plant Kit (Valencia CAUSA) and Fruit-mate (Takara Japan) according to the protocol de-scribed by Ishihara et al (2016a) The assessment of RNA quality andquantity was carried out at 230 260 and 280 nm by using a NanoDropSpectrophotometer ND-1000 (NanoDrop Technologies DE USA) Inorder to avoid genomic DNA contamination RNA samples were treatedwith TURBO DNAfree Kit (Invitrogen Carlsbad CA) Two microgramsof DNase-treated RNA were used to synthesize the first-strand cDNAusing M-MLV Reverse Transcriptase (Promega WI USA)
Quantitative real-time (qPCR) analysis was carried out to examinepossible differential expression of the mimosinase gene (GenBank ac-cession number AB2985971) in seedlings treated with 84 ppm JA10mM Ethephon or 15min of UV-C exposure Shoots and roots wereharvested 24 h before the time of mimosine concentration peak for eachtreatment previously observed as assessed by HPLC assays The 10 μLqPCR reaction consisted of 5 μL of PowerUpTM SYBRreg Green MasterMix (Applied Biosystems Foster City CA) 1 μL MgCl2 (50mM) 03 μLforward primer (10 μM) 03 μL reverse primer (10 μM) and 1 μL cDNAfirst-strand In the experimental validation through qPCR reactionconditions and melting curve analysis of the amplicon were performed
following the protocol published by Ishihara et al (2016b) for the sameleucaena variety qPCR analysis was conducted using StepOnetrade Real-Time PCR System (Applied Biosystems) Measurements were performedusing 4 biological and 3 technical replicates Relative expression wascalculated with the 2-ΔΔct method using OAS-TL as reference gene sinceits expression showed a consistently stable profile comparable to that ofUBQ-5 and ELF1α expressions Mimosinase primer sequences used forthese analyses were (FWD) 5prime- GAA AGG CAG GAA TCA CAG TGA AGAG ndash 3rsquo (REV) 5prime GGA GAC TCT AGC CAC ACC AAC TTA ndash 3rsquo
24 Antioxidant assays
241 Mimosine effect on hydrogen peroxide (H2O2) accumulationAs a follow up to the induction of mimosine accumulation profiles
under stress signals and conditions tests were conducted to verify mi-mosine antioxidant capacity In situ histological localization of hy-drogen peroxide (H2O2) accumulation was evaluated on foliar disks ofPhaseolus vulgaris L according to the protocol described by Shi et al(2010) Briefly the plant foliar tissue was exposed to 1 mgmiddotmLminus1 dia-minobenzidine (DAB) solution in 10 mM KH2PO4 (control) in presenceor absence of 10mM mimosine (equivalent to the average mimosineconcentration induced by UV-C radiation in giant leucaena) or 10mMascorbic acid (positive antioxidant control) Oxidative response wasidentified by the formation of a brown polymer on the injured leafareas indicating the presence of H2O2 and registered in a Leica M165FC stereomicroscope (Leica Microsystems)
242 Mimosine quenching of superoxide radicalsGeneration of superoxide radical and subsequent analysis was per-
formed by a modified protocol based on Zhishen et al (1999) Nitroblue tetrazolium (NBT) reduction was used to measure superoxide an-ions quenching activity Shortly a 50mM KH2PO4 pH 78 solutioncontaining 6 μM riboflavin 100mM methionine 1 mM NBT in pre-sence or absence of 5mM mimosine was exposed to white light(22 Jsdotcmminus2) for 25min on a white light transilluminator Five micro-molar rutin was used as positive control (Matsuura et al 2016) Theabsorbance was read at 560 nm before and after light exposure in aSpectraMaxreg M2 Microplate Reader (Molecular Devices LLC)
25 Statistical analyses
For HPLC and superoxide anions data simple analyses of variance(ANOVA) followed by Tukey or Welch ANOVA followed by Dunnetts Ctest were used as appropriate for data distribution characteristics InqPCR analysis results were analyzed by t-test In all cases at least fourbiological triplicates were used and experiments were repeated twiceindependently All data were analyzed using the statistical packageSPSS 200 for Windows (SPSS Inc USA) In all cases a ple 005 wasused
3 Results and discussion
31 Increased mimosine concentrations in giant leucaena treated withchemical elicitors
Leucaena produces high amounts of mimosine that accumulate in allparts of the plants including leaves stem flowers pods seeds rootsand root nodules (Soedarjo and Borthakur 1998) The highest con-centrations of mimosine can be found in the growing shoot tips andseeds (Wong and Devendra 1983) It is not known why leucaena pro-duces such high amounts of mimosine Negi et al (2014) estimated thatleucaena plants would be able to grow 21 larger if the nutrient re-sources spent on mimosine production were diverted for biomass in-crease In a previous analysis performed to quantify the basal con-centration of mimosine present in adult plants of common leucaena thehighest constitutive amount of mimosine per gram of fresh weight in
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
434
the analyzed organs was found in post-anthesis flowers (89448 μg)followed by green pods (82687 μg) leaves (67358 μg) and greenflower buds (51247 μg) which showed significantly less mimosineconcentration compared to the other reproductive structures(Supplementary Fig 1) Since mature seeds have very low moisturecontent (Wencomo et al 2017) its mimosine concentration was esti-mated as 338253 μgsdotgminus1 of dry weight Additionally it was also ob-served that the basal mimosine distribution in shoots of field-grownadult plants of leucaena is dependent on the variety type(Supplementary Table 1)
Phytohormones such as salicylic acid and jasmonic acid are knownto be produced by plants in response to various abiotic and bioticstresses These phytohormones trigger adaptive responses to stress byregulating major plant metabolic processes such as photosynthesisnitrogen metabolism defense systems and plant-water relationsthereby providing protection (for review see Khan et al 2015)
Secondary or specialized metabolite production and accumulationare also known to be controlled by biotic and abiotic stresses (Matsuuraet al 2018) In this study exposure of 5-week-old giant leucaenaseedlings to JA or Ethephon treatments significantly enhanced mimo-sine accumulation in shoots and roots in at least one of the two timepoints tested (48 and 96 h) albeit in a different way (Fig 1) Thehighest concentrations of mimosine in shoots were found in seedlingstreated with JA 84 ppm (43441 μgsdotgminus1) and Ethephon 100 ppm(38412 μgsdotgminus1) two days after application of the respective phyto-hormones Nevertheless after four days shoots yielded the highestconcentration of mimosine (approximately 460 μgsdotgminus1) upon treatmentwith 10 or 100 ppm Ethephon (Fig 1A) In roots after two and four
days JA 84 ppm and Ethephon 10 ppm resulted in highest mimosineaccumulation 18488 μgsdotgminus1 and 15801 μgsdotgminus1 respectively (Fig 1B)These observations show that mimosine accumulation response tospecific elicitors may vary over time after exposure
Although all treatments were applied exclusively on shoots of giantleucaena seedlings roots of some of them were also able to respond tothe different elicitors Overall shoots displayed higher basal and in-duced mimosine concentration compared to roots (Fig 1) which agreeswith previous observations in 1 to 3-week-old aseptic seedlings ofcommon leucaena (Vestena et al 2001) However as previouslymentioned significant post-induction increase of mimosine concentra-tion in roots and shoots simultaneously was only observed for JA andEthephon 10 ppm on day 02 and 04 respectively (Fig 1)
It is well established that perceived regulatory signals or elicitorsgenerate a transduction network mediated by secondary messengersresulting in changes in gene expression profiles that afford adaptiveresponses to environmental stimuli These modulation events are oftenmediated by transcription factors (TFs) which directly bind to specificgene promoters or act by forming complexes with repressor proteinslabeling them to degradation subsequently releasing other TFs toproceed with the gene expression program This is the case of the actionmechanism of JA and its active form jasmonoyl isoleucine for example(Kazan 2015 Wasternack and Strnad 2016)
JA ethylene and SA are known as important stress regulatory sig-nals in plants JA however is thought to be the most effective signal forinduction of plant secondary metabolism (Wasternack and Strnad2016) thereby contributing to mitigation of damage caused by severalstresses (Dar et al 2015) JA is mainly derived from linolenic acid
Fig 1 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings treated with differentelicitors CTRL=Milli-Q water SA = Salicylic AcidJA= Jasmonic Acid ETH=Ethephon Bars sharing a letterof same case do not differ by Tukey test (P le 005) Capitalletters (A B) compare treatments on day two and lowercaseletters (a b) compare treatments on day four Indicatessignificant statistical difference between day two and dayfour in the same treatment by t-test (Ple 005) The errorbars represent standard error of five replicates (each meanwas calculated with 15 individual seedlings organized in 5groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
435
(Wasternack and Strnad 2016) playing important roles in differentprocesses of plant growth and development such as plant defensemechanisms against herbivory pathogen attack fungal elicitation andsome abiotic factors such as osmotic temperature and salt stresses (Daret al 2015)
JA and its methyl ester MeJA have several different effects on le-guminous species MeJA exogenous application has increased iso-flavonoid content in cell suspension cultures of Pueraria candollei varcandollei and P candollei var mirifica (Korsangruang et al 2010) aswell as the production of the triterpenoid glycyrrhizin in Glycyrrhizaglabra roots Enhanced production of the triterpenoid however waspartly at the expense of root growth (Shabani et al 2009) MeJA ap-plication on shoots was observed to suppress root nodulation and lat-eral root formation in Lotus japonicus (Nakagawa and Kawaguchi2006) In grapevine a non-leguminous species proteinogenic aminoacids did not show an expressive increase under MeJA treatment(Gutieacuterrez-Gamboa et al 2017)
The effects of the application of four different jasmonate forms (JAMeJA jasmonoyl-L-isoleucine (JA-Ile) and 6-ethyl indanoyl glycineconjugate (2-[(6-ethyl-1-oxo-indane-4-carbonyl)-amino]-acetic acidmethyl ester - CGM) on leucaena metabolite profile has recently beenreported by Xu et al (2018) JA-Ile form was most effective althoughno major alteration was observed on monitored metabolite abundancesAlanine threonine and 34-dihydroxypyridine (34 DHP a metabolitederived from mimosine degradation) (Nguyen and Tawata 2016)among others were the major metabolites elicited by JA-Ile In contrastto the results described here mimosine concentration did not changesignificantly These divergent results on mimosine accumulation maybe due to a number of factors including mode of application jasmonateform used (JA-Ile x JA) and L leucocephala subspecies (common x giantleucaena)
Ethylene is also a phytohormone involved in plant response me-chanisms to different types of challenges such as mechanical damageand insect attack among others The integration mechanism betweenJA and ethylene signaling pathways is not completely understoodhowever it has been shown that they may work cooperatively in abioticstress tolerance (Kazan 2015) MeJA can induce ethylene production(Zhao et al 2004) and when applied simultaneously these moleculesseem to work in a synergic way by enhancing the magnitude of theplant response to external stimuli (Liu et al 2016)
Treatment with SA was able to significantly increase mimosine ac-cumulation in 12-week-old plants of common leucaena (SupplementaryFig 2) However no significant effect of SA treatment on mimosineconcentration was seen in 5-week-old seedlings of giant leucaena(Fig 1) suggesting some degree of genotype andor age dependency inelicitation by this phytohormone On the other hand several treat-ments including 90 ppm MeJA 10 and 100 ppm 2-chloroethylpho-sphonic acid (CEPA an ethylene-releasing compound) significantlyincreased mimosine accumulation (Supplementary Fig 2) in agree-ment with the data obtained for giant leucaena The lack of systemiceffects of externally applied SA on mimosine accumulation was alsoobserved when the phytohormone was supplied in the culture mediumof aseptically-grown seedlings in which case only roots had highercontent of mimosine (Vestena et al 2001) This could be due totransport limitations or to low methyl salicylate production from ap-plied SA since the former is recognized as the main systemic signalingform (Vlot et al 2009)
32 Increased mimosine concentrations in giant leucaena exposed to UV-Cradiation
UV-C treatment promoted increased concentration of the aminoacid in shoots but not in roots of giant leucaena (Fig 2) Increasedaccumulation of mimosine in shoots was also observed in 12-week-oldseedlings of common leucaena exposed to UV-C radiation for 10 and15min (Supplementary Fig 3) Similar to the SA treatment in giant
leucaena UV-C radiation did not induce mimosine biosynthesis in rootsregardless of time after exposure The absence of mimosine induction inroots by SA and UV indicates that these effectors do not cause a sys-temic response Moreover roots are shielded from irradiance by thepresence of substrate
UV radiation effects on different aspects of plant metabolism anddevelopment have been described However compared to UV-B (en-vironmentally relevant type of UV radiation) assays there are less re-ports related to the UV-C effects on secondary metabolites biosynthesisand accumulation (Cetin 2014) especially in leguminous (Fabaceae)plants They generally concern primary metabolism aspects such asgrowth and development For instance seedlings of Phaseolus vulgaris L(Fabaceae) exposed to low intensity UV-C radiation have displayeddecreased chlorophyll content and reduced height after 14 days of ex-posure (Kara 2013) Negative effects on growth parameters and ni-trogen metabolism were also observed in Vigna radiata L (Fabaceae)after UV-B radiation treatment in addition to adverse effects on JA SAand antioxidant compounds accumulation (Choudhary and Agrawal2014a) The same authors reported increased accumulation of flavo-noids SA and JA besides negative effects on growth biomass yieldnitrogen fixation and accumulation in 2 cultivars of Pisum sativum L(Fabaceae) under elevated UV-B treatment (Choudhary and Agrawal2014b) Despite the negative UV influence on growth reported for thepreviously mentioned leguminous UV-C radiation on groundnut plants(Arachis hypogaea L Fabaceae) increased seedling vigor and biomassand had no adverse effect on germination or other development para-meters (Neelamegam and Sutha 2015)
Besides its impact on growth and primary metabolism UV exposurecan cause important changes in secondary metabolism depending onintensity and time of exposure (Matsuura et al 2013) UV-B and UV-Cpre-treatments of Artemisia annua (Asteraceae) seedlings yielded in-creased biosynthesis of artemisinin a drug which displays anti-malarialproperties and activity against some others infectious diseases (egschistosomiasis leishmaniasis and hepatitis B) and several kinds oftumors (Rai et al 2011) The accumulation of nicotine in Nicotianarustica plants (Solanaceae) was also increased by UV-C treatment(Tiburcio et al 1985) Similar inducing effects on production of severalsecondary metabolites were observed in callus cultures of Vitis viniferaL Oumlkuumlzgoumlzuuml (grapevine Vitaceae) treated with a UV-C source for 5 or10min (Cetin 2014)
Regarding amino acid biosynthesis in response to UV radiationMartiacutenez-Luumlscher et al (2014) have found that in spite of not causingchanges in total amino acid content UV-B radiation exposure can affecttheir profile in grape berries Proteinogenic amino acids have beenknown to be important targets of the deleterious effects of UV radiation(Holloacutesy 2002) On the other hand in the present study acute UV-Ctreatment was found to increase mimosine accumulation in shoots byover twofold (Fig 2) which may suggest a possible participation of thismolecule as part of the antioxidant defense system in L leucocephalaThis possibility is further supported by the induction of the amino acidaccumulation by JA and Ethephon involved in abiotic and biotic stressresponses which are generally associated with oxidative imbalance andare signaling components in high UV stress (Matsuura et al 2013)
33 Mimosinase gene expression
In order to determine if increases in mimosine content upon ex-posure to JA CEPA or UV-C radiation were related to changes intranscription of mimosine metabolism-related genes RT-qPCR analysiswas carried out The complete pathway for mimosine biosynthesis hasnot yet been determined although the final step has been character-ized Based on transcription analysis (Ishihara et al 2016a) leucaenaappears to encode for multiple cysteine synthases one or more of whichmay be able to catalyze mimosine synthesis In addition a leucaenagene encoding a mimosinase (an enzyme responsible for mimosinedegradation) has been identified and characterized (Negi et al 2014)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
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In addition to mimosinase gene expression several gene isoformsbelonging to the cysteine pathway [cysteine synthase (CYS SYN) serineacetyltransferase (SAT) and β-cyanoalanine synthase (CAS) Table 2 -supplementary material] were also tested in this study (data notshown) However expressions of these genes did not vary in giantleucaena throughout the experiments suggesting that the increasedcontent of mimosine observed in the treated plants might not be relatedto the expression of these genes but presumably to increased enzymeactivity andor release from conjugates such as mimoside a mimosineβ-D-glucoside (Murakoshi et al 1972)
Considering the time variation of mimosine accumulation observedin this work mimosinase gene expression in shoots and roots wasevaluated 24 h before the increase of mimosine concentration in giantleucaena seedlings (ie 24 h and 72 h after the chemical elicitorstreatments and 48 h and 120 h after UV-C exposure)
Ethylene signaling has been shown to up-regulate expression ofseveral genes related to secondary metabolism pathways as is the caseof phenolic compounds (Liu et al 2016) and terpenoid indole alkaloids(Wang et al 2016) Among all elicitors tested in the present workEthephon was the only one able to significantly change mimosinasegene expression Leucaena plants treated with Ethephon showed sig-nificant increases in mimosine concentration at both day 2 and 4 fol-lowing treatment which coincided with low-level expression of mi-mosinase Up-regulation of mimosinase gene expression was detected24 h before the increase of mimosine concentration in shoots treatedwith 10 ppm of Ethephon (Fig 3A) but not after JA or UV-C treatments(Fig 3C-D and 3E-F respectively) Nevertheless 72 h after treatment
application (24 h before the highest mimosine content measured inshoots) down regulation of mimosinase gene was seen in both shootsand roots treated with 10 ppm of Ethephon (Fig 3B) These data in-dicate that mimosine content in leucaena plants is at least partlyregulated by mimosinase expression in Ethephon exposed plants Onthe other hand the fact that mimosinase mRNA was not significantlyaffected by JA and UV-C treatments despite their stimulating effects onmimosine biosynthesis in giant leucaena may indicate that other levelsof regulation are at play or that the chosen harvesting time window wasunable to detect relevant changes
34 In situ and in vitro antioxidant assays
Considering the stimulation of mimosine accumulation byEthephon JA and UV all of which are often associated or known tocause oxidative imbalance the antioxidant capacity of mimosine wasevaluated Mimosine has been shown to have antioxidant activities oncultured cancer cells (Parmar et al 2015) In the present study it washypothesized that mimosine could confer radical scavenging propertieswhich would contribute to plant protection from possible damagecaused by reactive oxygen species generated during stress(Supplementary Fig 4)
Foliar disks of P vulgaris L were treated with 10mM mimosine for15min Treated disks showed less hydrogen peroxide accumulationinduced by wounding in contrast to untreated ones being comparableto those treated with ascorbic acid (a known hydrogen peroxide neu-tralizer) (Fig 4A) These observations support a possible antioxidant
Fig 2 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings exposed to UV-C lightCTRL= visible light (100 μmol photons mminus2 s minus1) UV-C 10primeand UV-C 15rsquo=UV-C exposure time (10 and 15min re-spectively) Bars sharing a letter of same case do not differ byTukey test (P le 005) Capital letters (A B) compare treat-ments on day three and lowercase letters (a b) comparetreatments on day six Indicates significant statistical dif-ference between day three and day six in the same treatmentby t-test (Ple 005) The error bars represent standard errorof five replicates (each mean was calculated with 15 in-dividual seedlings organized in 5 groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
437
role of mimosine as an in situ hydrogen peroxide scavengerMimosine was also able to quench superoxide anions generated by
light exposure Mimosine exhibited equivalent antioxidant effect com-pared to rutin (Fig 4B) a well-established effective superoxide anionquencher (Matsuura et al 2016) The radical scavenging activity ofmimosine may be due to the 3-OH group of the pyridine ring of mi-mosine (Fig 5) The pKa of the 3-OH of mimosine has been estimated tobe 88 (M Honda unpublished results) At physiological pH this OHgroup is expected to remain in a protonated state and therefore mayscavenge a radical by donating a proton and an electron In this processmimosine itself is converted to a stable radical form which is perhapsless toxic and less reactive than the reactive oxygen species generatedduring oxidative stress It is likely that the less toxic radical mimosineproduced may react with another radical or molecule and becomeconverted to a non-reactive indole molecule
In vivo antioxidant activity of mimosine has been previously eval-uated by means of its exogenous application on selenium-deficientseedlings of Vigna radiata In spite of its allelopathic properties (Ahmedet al 2008) the results showed mitigation of mitochondrial oxidativestress by treatment with 01mM mimosine (Lalitha and Kulothungan2007) DPPH radical scavenging activity was also reported for aqueous
seed extracts of leucaena rich in mimosine and phenolic compounds inin vitro assays (Benjakul et al 2014) Mimosine antioxidant activityshown in the present work is in good agreement with data reported forother non-protein amino acids such as L-DOPA (Dhanani et al 2015)and GABA (Malekzadeh et al 2014) for instance
4 Conclusion
Taken together results show that mimosine biosynthesis and ac-cumulation can be modulated by stress-related factors despite its re-latively high constitutive content in leucaena plants The pattern ofgene expression in stressed plants suggests mimosine steady-state con-trol may be regulated by its degradation in possible connection withdynamic changes in carbon and nitrogen metabolism of stressed plantsMimosine quenching activity against hydrogen peroxide and super-oxide anions in the in situ staining and in vitro assays respectivelyshowed that this non-protein amino acid can act as non-enzymaticantioxidant agent Increase in mimosine content in response to elicitorsmimicking environmental challenges in addition to its antiherbivoreand antimicrobial properties may be related to its activity as protectivemolecule against oxidative damage in line with other classes of plant
Fig 3 Relative expression of the mimosinase gene in shoots (A E and F) and shoots and roots (B C and D) of giant leucaena 24 h (A and C) 48 h (E) 72 h (B and D)and 120 h (F) after treatment with stress signaling molecules or UV-C exposure ETH = Ethephon JA = Jasmonic Acid Indicates significant statistical differencebetween control and treatment by t-test (Ple 005) The error bars represent standard error of four replicates
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
438
secondary metabolites
Funding
This work was funded by the National Council for Scientific andTechnological Development (CNPq-Brazil) grant 3060792013-5Coordenaccedilatildeo de Aperfeiccediloamento de Pessoal de Niacutevel Superior - Brazil(CAPES) - Finance Code 001 and the USDA NIFA Hatch projectHA05029-H managed by CTAHR
CRediT authorship contribution statement
Kelly Cristine da Silva Rodrigues-Correcirca InvestigationValidation Writing ndash original draft Michael DH HondaInvestigation Validation Dulal Borthakur Supervision Writing ndashreview amp editing Funding acquisition Arthur Germano Fett-NetoSupervision Funding acquisition Writing ndash review amp editing
Acknowledgements
The authors would like to thank Dr Jorge Ernesto Mariath fromLaVeg-UFRGS for kindly lending the Leica M165 FC stereomicroscopefor in situ analysis
Appendix A Supplementary data
Supplementary data to this article can be found online at httpsdoiorg101016jplaphy201811018
References
Ahmed R Hoque ATMR Hossain MK 2008 Allelopathic effects of Leucaena
leucocephala leaf litter on some forest and agricultural crops grown in nursery J ForRes 19 298 httpsdoi 101007s11676-008-0053-0
Benjakul S Kittiphattanabawon P Shahidi F Maqsood S 2013 Antioxidant activityand inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipidoxidation in model systems Food Sci Technol Int 19 (4) 365ndash376 httpsdoiorg1011771082013212455186
Benjakul S Kittiphattanabawon P Sumpavapol P Maqsood S 2014 Antioxidantactivities of lead (Leucaena leucocephala) seed as affected by extraction solvent priordechlorophyllisation and drying methods extracts against lipid oxidation in modelsystems Food Sci Technol 51 (11) 3026ndash3037 httpsdoiorg101007s13197-012-0846-1
Brewbaker JL Pluckett D Gonzalez V 1972 Varietal variation and yield trials ofLeucaena leucocephala (koa haole) in Hawaii Hawaii Agric Exp Stn Bull 166 26
Brewbaker JL 2008 Registration of KX2 ndash Hawaii interspecific-hybrid leucaena JPlant Registrations 1 (3) 190ndash193 httpsdoiorg103198jpr2007050298crc
Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitisvinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-6287-47-37
Cho H-Y Son SY Rhee HS Yoon S-YH Lee-Parsons CWT Park JM 2008Synergistic effects of sequential treatment with methyl jasmonate salicylic acid andyeast extract on benzophenanthridine alkaloid accumulation and protein expressionin Eschscholtzia californica suspension cultures J Biotechnol 135 117ndash122 httpsdoiorg101016jjbiotec200802020
Choudhary KK Agrawal SB 2014a Cultivar specificity of tropical mung bean (Vignaradiata L) to elevated ultraviolet-B changes in antioxidative defense system ni-trogen metabolism and accumulation of jasmonic and salicylic acids Environ ExpBot 99 122ndash132 httpsdoiorg101016jenvexpbot201311006
Choudhary KK Agrawal SB 2014b Ultraviolet-B induced changes in morphologicalphysiological and biochemical parameters of two cultivars of pea (Pisum sativum L)Ecotoxicol Environ Saf 100 178ndash187 httpsdoiorg101016jecoenv201310032
Dar TA Uddin M Khan MMA Hakeem KR Jaleel H 2015 Jasmonates counterplant stress a Review Environ Exp Bot 115 49ndash57 httpsdoiorg101016jenvexpbot201502010
Dhanani T Singh R Shah S Kumari P Kumar S 2015 Comparison of green ex-traction methods with conventional extraction method for extract yield L-DOPAconcentration and antioxidant activity of Mucuna pruriens seed Green Chem LettRev 8 (2) 43ndash48 httpsdoiorg1010801751825320151075070
Gutieacuterrez-Gamboa G Portu J Santamariacutea P Loacutepez R Garde-Cerdaacuten T 2017Effects on grape amino acid concentration through foliar application of three dif-ferent elicitors Food Res Int 99 688ndash692 httpsdoiorg101016jfoodres201706022
Fig 4 A In situ antioxidant assay Foliar disksof Phaseolus vulgaris L treated with (a) No an-tioxidant added (negative control) (b) 10 mMMimosine (c) 10mM ascorbic acid (positivecontrol) The oxidative damage can be seen bythe formation of a brown polymer in leaf veinsand injured areas B In vitro superoxidescavenging assay carried out with mimosineDifferent letters indicate significant differenceby Tukey test (Ple 005) The error bars re-present standard error of four replicates (Forinterpretation of the references to colour in thisfigure legend the reader is referred to the Webversion of this article)
Fig 5 Predicted mimosine radical formed followingquenching of hydroxyl radical Mimosine is first converted toa stable mimosine radical which may be then converted to anontoxic indole form
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
439
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad HossainMd Anai T Oku H 2018 Cytosolic cysteine synthase switch cysteine and mi-mosine production in Leucaena leucocephala Appl Biochem Biotechnol 186 (3)613ndash632 httpsdoiorg101007s12010-018-2745-z
Holloacutesy F 2002 Effects of ultraviolet radiation on plant cells Micron 33 (2) 179ndash197Honda MDH Ishihara KL Pham DT Borthakur D 2018 Identification of drought-
induced genes in giant leucaena (Leucaena leucocephala subsp glabrata) Trees 32571ndash585 httpsdoiorg101007s00468-018-1657-4
Huang T Jander G de Vos M 2011 Non-protein amino acids in plant defense againstinsect herbivores representative cases and opportunities for further functional ana-lysis Phytochemistry 72 1531ndash1537 httpsdoiorg101016jphytochem201103019
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of thethyrotoxic amino acid mimosine by cysteine synthase Phytochemistry 29 (11)3461ndash3465 httpsdoiorg1010160031-9422(90)85258-H
Ishihara K Lee EKW Borthakur D 2016a An improved method for RNA extractionfrom woody legume species Acacia koa A Gray and Leucaena leucocephala (Lam) deWit Int J For Wood Sci 3 (1) 031ndash035
Ishihara KL Honda MDH Pham DT Borthakur D 2016b Transcriptome analysisof Leucaena leucocephala and identification of highly expressed genes in roots andshoots Transcriptomics 4 135 httpsdoiorg1041722329-89361000135
IUBMB 2018 Enzyme Nomenclature EC 35161 httpwwwsbcsqmulacukiubmbenzymeEC35161html Accessed date 8 February 2018
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant(Phaseolus vulgaris) Biosci Res 10 (1) 29ndash32
Kazan K 2015 Diverse roles of jasmonates and ethylene in abiotic stress toleranceTrends Plant Sci 20 (4) 219ndash229 httpsdoiorg101016jtplants201502001
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect ofEthephon as an ethylene-releasing compound on the metabolic profile of Chlorellavulgaris J Agric Food Chem 64 (23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541
Khan MIR Fatma M Per TS Anjum NA Khan NA 2015 Salicylic acid-inducedabiotic stress tolerance and underlying mechanisms in plants Front Plant Sci 6 462httpsdoiorg103389fpls201500462
Korsangruang S Soonthornchareonnon N Chintapakorn Y Saralamp PPrathanturarug S 2010 Effects of abiotic and biotic elicitors on growth and iso-flavonoid accumulation in Pueraria candollei var candollei and P candollei var mir-ifica cell suspension cultures Plant Cell Tissue Organ Cult 103 (3) 333ndash342 httpsdoiorg101007s11240-010-9785-6
Lalitha K Kulothungan SR 2006 Selective determination of mimosine and its dihy-droxypyridinyl derivative in plant systems Amino Acids 31 (3) 279ndash287 httpsdoiorg101007s00726-005-0226-5
Lalitha K Kulothungan SR 2007 Mimosine mitigates oxidative stress in seleniumdeficient seedlings of Vigna radiata - Part I restoration of mitochondrial functionBiol Trace Elem Res 118 (1) 84ndash96 httpsdoiorg101007s12011-007-0013-0
Liu J Li Y Wang Y Zhang Z-H Zu Y-G Efferth T Tang Z-H 2016 Thecombined effects of ethylene and MeJA on metabolic profiling of phenolic com-pounds in Catharanthus roseus revealed by metabolomics analysis Front Physiol 71ndash11 httpsdoiorg103389fphys201600217 Article 217
Malekzadeh P Khara J Heydari R 2014 Alleviating effects of exogenous Gamma-aminobutiric acid on tomato seedling under chilling stress Physiol Mol Biol Plants20 (1) 133ndash137 httpsdoiorg101007s12298-013-0203-5
Martiacutenez-Luumlscher J Torres N Hilbert G Richard T Saacutenchez-Diacuteaz M Delrot SAguirreolea J Pascual I Gomegraves E 2014 Ultraviolet-B radiation modifies thequantitative and qualitative profile of flavonoids and amino acids in grape berriesPhytochemistry 102 106ndash114 httpsdoiorg101016jphytochem201403014
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation ofbioactive secondary metabolites by ultraviolet radiation mechanisms strategies andapplications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology forMedicinal Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash1902012
Matsuura HN Fragoso V Paranhos JT Rau MR Fett-Neto AG 2016 Thebioactive monoterpene indole alkaloid N szlig-D-glucopyranosylvincosamide is regu-lated by irradiance quality and development in Psychotria leiocarpa Ind Crop Prod86 210ndash218 httpsdoiorg101016jindcrop201603050
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo RBhambra AS Strnad M Bonfill M Fett-Neto AG 2018 Specialized plant me-tabolism characteristics and impact on target molecule biotechnological productionMol Biotechnol 60 (2) 169ndash183 httpsdoiorg101007s12033-017-0056-1
Murakoshi S Ohmiya S Haginiwa J 1972 Enzymic synthesis of mimoside a meta-bolite of mimosine in Mimosa pudica and Leucaena leucocephala Chem Pharm Bull20 (4) 855ndash857
Nakagawa T Kawaguchi M 2006 Shoot-applied MeJA suppresses root nodulation inLotus japonicus Plant Cell Physiol 47 (1) 176ndash180 httpsdoiorg101093pcppci222
Nascimento NC Menguer PK Henriques AT Fett-Neto AG 2013 Accumulation ofbrachycerine an antioxidant glucosidic indole alkaloid is induced by abscisic acidheavy metal and osmotic stress in leaves of Psychotria brachyceras Plant PhysiolBiochem 73 33ndash40 httpsdoiorg101016jplaphy201308007
Neelamegam R Sutha T 2015 UV-C irradiation effect on seed germination seedling
growth and productivity of groundnut (Arachis hypogaea L) Int J Curr MicrobiolApp Sci 4 (8) 430ndash443
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase fromLeucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol164 (2) 922ndash934 httpsdoiorg101104pp113230870
Negi VS Borthakur D 2016 Heterologous expression and characterization of mimo-sinase from Leucaena leucocephala In Fett-Neto Arthur Germano (Ed)Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods inMolecular Biology vol 1405 copySpringer Science+Business Media New York httpsdoiorg101007978-1-4939-3393-8_7 2016
Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636
Parmar F Kushawaha N Highland H George L-B 2015 In vitro antioxidant andanticancer activity of Mimosa pudica Linn extract and L-mimosine on lymphomaDaudi cells Int J Pharm Sci 12 100ndash104
Porto DD Matsuura HN Vargas LRB Henriques AT Fett-Neto AG 2014 Shootaccumulation kinetics and effects on herbivores of the wound-induced antioxidantindole alkaloid brachycerine of Psychotria brachyceras Nat Prod Commun 9 (5)629ndash632
Rai R Meena RP Smita SS Shukla A Rai SK Pandey-Rai S 2011 UV-B and UV-C pre-treatments induce physiological changes and artemisinin biosynthesis inArtemisia annua L ndash an antimalarial plant J Photochem Photobiol B Biol 105 (3)216ndash225 httpsdoiorg101016jjphotobiol201109004
Shabani L Ehsanpour AA Asghari G Emami J 2009 Glycyrrhizin production by invitro cultured Glycyrrhiza glabra elicited by methyl jasmonate and salicylic acid RussJ Plant Physiol 56 (5) 621ndash626 httpsdoiorg101134S1021443709050069
Shah J 2003 The salicylic acid loop in plant defense Curr Opin Plant Biol 6 (4)365ndash371
Shi J Fu XZ Peng T Huang XS Fan QJ Liu JH 2010 Spermine pretreatmentconfers dehydration tolerance of citrus in vitro plants via modulation of antioxidativecapacity and stomatal response Tree Physiol 30 (7) 914ndash922 httpsdoiorg101093treephystpq030
Smith IK Fowden L 1966 A study of mimosine toxicity in plants J Exp Bot 17750ndash761 httpsdoiorg101093jxb174750
Soedarjo M Borthakur D 1996 Simple procedures to remove mimosine from youngleaves pods and seeds of Leucaena leucocephala used as food Int J Food SciTechnol 31 (1) 97ndash103
Soedarjo M Borthakur D 1998 Mimosine a toxin produced by the tree-legumeLeucaena provides a nodulation competition advantage to mimosine-degradingRhizobium strains Soil Biol Biochem 30 1605ndash1613
Suda S 1960 On the physiological properties of mimosine Bot Mag Tokyo 73 (862)142ndash147 httpsdoiorg1015281jplantres188773142
Tangendjaja B Lowry JB Wills RBH 1986 Isolation of a mimosine degrading en-zyme from leucaena leaf J Sci Food Agric 37 523ndash526 httpsdoiorg101002jsfa2740370603
Tiburcio F Pintildeol MT Serrano M 1985 Effect of UV-C on growth soluble protein andalkaloids in Nicotiana rustica plants Environ Exp Bot 25 (3) 203ndash210 httpsdoiorg1010160098-8472(85)90004-8
Vestena S Fett-Neto AG Duarte RC Ferreira A 2001 Regulation of mimosineaccumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 httpsdoiorg101016S0168-9452(01)00448-4
Vlot AC Dempsey DMA Klessig DF 2009 Salicylic acid a multifaceted hormone tocombat disease Annu Rev Phytopathol 47 177ndash206 httpsdoiorg101146annurevphyto050908135202 2009
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene-induced vinblastine accumulation is related to activated expression of downstreamTIA pathway genes in Catharanthus roseus BioMed Res Int 2016 Article ID 3708187httpsdoiorg10115520163708187
Wasternack C Strnad M 2016 Jasmonate signaling in plant stress responses and de-velopment ndash active and inactive compounds N Biotech 33 (5B) 604ndash613 httpsdoiorg101016jnbt201511001
Wencomo HB Ortiz R Caacuteceres J 2017 Afr J Agric Res 12 (4) 279ndash285 httpsdoiorg105897AJAR201510604 26
Wong CC Devendra C 1983 Research on leucaena forage production in Malaysia InLeucaena Research in the Asian Pacific Region pp 55ndash60 Ottawa Ontario Canada
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX TsimKWK 2018 Jasmonate-elicited stress induces metabolic change in the leaves ofLeucaena leucocephala Molecules 23 (2) httpsdoiorg103390molecules23020188 E188
Yafuso JT Negi VS Bingham J-P Borthakur D 2014 An O-acetylserine (thiol)lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthaseAppl Biochem Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z
Zhao J Zheng S-H Fujita K Sakai K 2004 Jasmonate and ethylene signalling andtheir interaction are integral parts of the elicitor signalling pathway leading to b-thujaplicin biosynthesis in Cupressus lusitanica cell cultures J Exp Bot 55 (399)1003ndash1012 httpsdoiorg101093jxberh127
Zhishen J Mengcheng T Jianming W 1999 The determination of flavonoid contentsin mulberry and their scavenging effects on superoxide radicals Food Chem 64 (4)555ndash559 httpsdoiorg101016S0308-8146(98)00102-2
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
440
61
Supplementary Fig 1 Basal mimosine concentration in adult trees of common leucaena (L leucocephala
var leucocephala) Samples were collected from 10 field grown trees at Manoa Valley Honolulu Hawairsquoi
on June 25th 2017 Bars sharing a letter do not differ by Tukey test (P le 005) The error bars represent the
standard error
Supplementary Fig 2 Bar diagram showing mimosine concentration in shoots of 12-week-old common
leucaena seedlings treated with different elicitors CTRL = Milli-Q water SA = Salicylic Acid MeJA =
Methyl Jasmonate CEPA = 2-Chloroethylphosphonic acid (an ethylene releasing compound) Bars sharing a
letter of same case do not differ by Tukey test (P le 005) Capital letters (A B) compare treatments on day
two and lower-case letters (a b) compare treatments on day four Indicates significant statistical difference
ABB
A A
0
200
400
600
800
1000
1200
LEAVES GREEN FLOWERBUDS
POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
con
cen
trat
ion
(micro
gg
-1o
f FW
)
B AB AB AB B A
b
a
ab b
ab
0
2
4
6
8
10
12
14
16
18
20
CTRL SA 10 ppm SA 100 ppm CEPA 10 ppm CEPA 100 ppm MeJA 90 ppm
Mim
osi
ne
co
nce
ntr
atio
n (
gg
-1o
f FW
)
DAY 02 DAY 04
62
between day two and day four in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
Supplementary Fig 3 Bar diagram showing the effects of UV-C radiation exposure for 5 10 and 15 min on
mimosine accumulation in shoots of 12-week-old seedlings of common leucaena Bars sharing a letter of
same case do not differ by Tukey test (P le 005) Capital letters (A B C) compare treatments on day three
and lower-case letters (a b) compare treatments on day six Indicates significant statistical difference
between day three and day six in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
C BC AB A
bb
a
a
0
10
20
30
40
50
60
CTRL UV-C 5 UV-C 10 UV-C 15
Mim
osi
ne
co
nce
ntr
atio
n (
gg-1
of
FW)
DAY 03 DAY 06
63
Supplementary Fig 4 Model depicting induction of mimosine synthesis in leucaena following application of
stress elicitors such as CEPA and jasmonic acid or exposure to UV-C radiation The additional mimosine
synthesized may serve to alleviate oxidative stress induced by UV-C radiation
64
Supplementary Table 1 Mimosine contents in leaves of common and giant leucaena
Leucaena
type
Mimosine content
( FW)
Mimosine
content ( DW)
Dry matter
content ( FW)
Water content
( FW)
Common (1) 050 plusmn 009 245 plusmn 051 2011 plusmn 054 7989 plusmn 054
Common (2) 043 plusmn 006 214 plusmn 037 1998 plusmn 050 8002 plusmn 050
K636 (1) 070 plusmn 014 356 plusmn 077 1908 plusmn 052 8092 plusmn 052
K636 (2) 042 005 205 plusmn 033 2008plusmn 093 7992plusmn 093
KX2 (1) 122 plusmn 011 608 plusmn 082 1939 plusmn 123 8061 plusmn 123
KX2 (2) 134 plusmn 010 623 plusmn 056 2029 plusmn 114 7971 plusmn 114
KX3 (1) 044 plusmn 006 221 plusmn 030 1945 plusmn 073 8055 plusmn 073
KX3 (2) 054 plusmn 005 273 plusmn 023 1930 plusmn 038 8070 plusmn 038
KX4 (1) 086 plusmn 011 471 plusmn 065 1753 plusmn 084 8247 plusmn 084
KX4 (2) 089 plusmn 011 476 plusmn 065 180 plusmn 072 820 plusmn 072
KX5 (1) 099 plusmn 012 489 plusmn 048 1907 plusmn060 8093 plusmn 060
KX5 (2) 115 plusmn 015 548 plusmn080 1992 plusmn 053 8008 plusmn 053
Common leucaena variety koa haole grows widely on the island of Orsquoahu K636 is widely
grown variety of giant leucaena KX2 KX3 KX4 and KX5 are giant leucaena varieties
developed through interspecies hybridization (Brewbaker 2016) (1) and (2) indicate plants
from two separate locations within the University of Hawaii Waimanalo Research Center The
values are shown as mean plusmn standard error obtained from at least three biological replicates
65
Supplementary Table 2 GenBank accession numbers of the tested cysteine pathway genes isoforms
Gene name GenBank accession
OAS-TL (o-acetylserine-thiol-lyase) GDRZ01032940
GDRZ01061620
GDRZ01153117
GDSA01187555
GDSA01196891
GDSA01214467
Cys syn (cysteine synthase) GDRZ01015860
GDRZ01050898
GDRZ01086813
GDRZ01193515
GDRZ01202579
GDSA01180863
GDSA01215622
SAT (serine acetyltransferase) GDRZ01187456
GDRZ01189631
CAS (β-cyanoalanine synthase) GDRZ01054066
GDRZ01175418
GDSA01118400
66
SHORT COMMUNICATION 1
Mimosine occurrence and accumulation in Mimosa bimucronata var bimucronata (DC) 2
Kuntze 3
Kelly Cristine da Silva Rodrigues-Correcirca1 Lana Dorneles Pedroso2 Fernanda de Costa1 4
Arthur Germano Fett-Neto1 5
1Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal 6
University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970 7
Porto Alegre Rio Grande do Sul Brazil 2Department of Biological Sciences Unipampa ndash 8
Campus Satildeo Gabriel 9
Corresponding author 10
E-mail addresses krodriguescbiotufrgsbr (KCdaS Rodrigues-Correcirca) 11
lanalima2012gmailcom (LD Pedroso) fernandadecostayahoocombr (F de Costa) 12
fettnetocbiotufrgsbr (AG Fett-Neto) 13
14
15
16
17
18
19
20
21
22
67
ABSTRACT 23
Mimosine is a non-protein aromatic amino acid present in plants of Leucaena spp 24
and Mimosa spp Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) is a native 25
tree from Brazil which occurs as a pioneer species on plant succession processes In the 26
current study the presence of mimosine in M bimucronata was verified by HPLC analyses 27
Moreover mimosine accumulation upon exposure to UV-C and chemical elicitors of 28
specialized metabolism (salicylic acid - SA methyl jasmonate - MeJA sodium nitroprusside 29
- SNP and ethephon - ETH) most of which also known as promoters of the amino acid 30
production in leucaena plants was evaluated The results showed a lower concentration of 31
constitutive mimosine present in both maricaacute seedlings and mature trees when compared to 32
leucaena plants In spite of a trend towards increased mimosine accumulation observed in 33
MeJA and ETH treatments no statistical differences were found with the various stressors 34
used to induce its biosynthesis in maricaacute seedlings Data suggest that mimosine in M 35
bimucronata is probably a phytoanticipin-like metabolite or its accumulation is driven by 36
other types of stresses 37
38
39
Keywords Mimosine Mimosa bimucronata stress 40
41
42
43
44
45
46
68
Introduction 47
Mimosa bimucronata commonly known as maricaacute is a native tree from Brazil 48
(REFLORA 2019) ecologically important in plant succession and in processes of degraded 49
land recovery (Bitencourt et al 2007 Silva et al 2011) occurring as a pioneer species 50
(Pilatti et al 2019) Maricaacute is a deciduous or semi-deciduous plant which reaches up to 15 51
m in height and 40 cm of diameter at breast height (DBH) displays shrub or tree habit and 52
bears typical sharp thorns (Carvalho 2004) This species belongs to Fabaceae one of the 53
most economically important families of flowering plants due to its high diversity and 54
occurrence in different types of habitats (Gomes et al 2018) As well as several others 55
Mimosa spp maricaacute is usually referred to as a multipurpose tree (Olkoski and Wittmann 56
2011) employed for alternative medicinal uses (Champanerkar et al 2010 Silva et al 57
2011) honey production constructions and remodeling of landscape architecture (living 58
fences) for instance (Marchiori 1993 Lorenzi 1998) 59
In southern Brazil maricaacute is widely distributed and typically found either in wetland 60
areas close to river banks (Patreze and Cordeiro 2004) or composing large and almost pure 61
landscape formations on hillsides (Jacobi and Ferreira 1991) In dense populations this 62
species like several Mimosa spp (Simon and Proenccedila 2000) is considered an important and 63
highly invasive weed by preventing cattle to reach pasturesand water bodies as a result of its 64
thorny branches (Lorenzi 2008 Kestring et al 2009) Its dominant and nearly exclusive 65
pattern of distribution in those areas has led Jacobi and Ferreira (1991) to test its allelopathic 66
potential on cultivated species Indeed extracts of leaves and ripe fruits (but not the green 67
ones) of maricaacute showed phytotoxic effects on germination and initial radical growth of most 68
of the target species tested 69
69
Several investigations have been performed on maricaacute floristics (Silva et al 2011) 70
distribution (Simon and Proenccedila 2000) wood anatomy (Marchiori 1993) cytogenetic 71
parameters (Olkoski and Wittmann 2011) and allelopathic potential (Jacobi and Ferreira 72
1991 Ferreira et al 1992) However excluding two recent publications on maricaacute 73
constitutive chemical composition (Schlickmann et al 2017 Pilatti et al 2019) which 74
identified phenolic compounds (methyl gallate and water-soluble tannins) as its major 75
compounds little is known regarding this subject In other Mimosa species (eg M pudica 76
and M pigra) mimosine has been identified (Soedarjo and Borthakur 1998) as one of the 77
major specialized metabolites present in the different organs of the plant (Champanerkar et 78
al 2010) The presence of this molecule was also reported for M bimucronata in a thin layer 79
chromatography-based preliminary study performed by Ferreira et al (1992) showing co-80
chromatography of a leaf extract component with authentic mimosine The authors attributed 81
the allelopathic effect of maricaacute to the accumulation of this metabolite in its leaves 82
Mimosine is an aromatic non-protein amino acid initially found in plants of Mimosa 83
pudica and later in Leucaena leucocephala (Lam) de Wit (Soedarjo and Borthakur 1998) a 84
leguminous tree which biosynthesizes large amounts of this nitrogen-containing compound 85
(Rodrigues-Correcirca et al 2019) It is believed that the accumulation of high contents of 86
mimosine in L leucocephala tissues confers among other traits defense against herbivores 87
and pathogens (Vestena et al 2001) tolerance to drought (Negi et al 2014) as well as 88
general oxidative stress protection (Rodrigues-Correcirca et al 2019) Interestingly drought is 89
the opposite environmental and physiological condition to that observed in the wet habitats 90
occupied by native populations of M bimucronata in Brazil (Patreze and Cordeiro 2004 91
Kestring et al 2009) and Mimosa pudica Linn in India (Champanerkar et al 2010) 92
70
Nonetheless flooding is also associated with oxidative stress particularly as water levels 93
change (Fukao et al 2019) 94
In Leucaena leucocephala var leucocephala (common leucaena) and Leucaena 95
leucocephala var glabrata (giant leucaena) mimosine accumulation has been shown to be 96
both constitutive and inducible by stress-related phytohormones such as jasmonic acid (JA) 97
Ethephon (ETH an ethylene- releasing compound) salicylic acid (SA - only common 98
leucaena) (Vestena et al 2001) as well as by UV-C radiation (Xu et al 2018 Rodrigues-99
Correcirca et al 2019) On the other hand there is a lack of information regarding mimosine 100
content and elicitation effects in Mimosa spp plants 101
The aim of this study was to examine the presence of mimosine in Mimosa 102
bimucronata and examine the effects of stresses and stress-signaling molecules on its 103
accumulation in leaves 104
Material and Methods 105
Plant material 106
For all experiments the plant material was collected at Morro Santana campus do 107
Vale of UFRGS (Federal University of Rio Grande do Sul) Porto Alegre RS Brazil 108
(3004rsquoS 5108rsquoW) Authorization for access to genetic material was obtained from 109
SISGEN-Brazil (license number A845493) Constitutive mimosine content in adult plants of 110
M bimucronata var bimucronata (DC) Kuntze was determined in plant material (leaves 111
green flower buds post-anthesis flowers and green pods) harvested in January 2017 112
(summer) A voucher herbarium specimen (ICN 187953) was deposited in the ICN ndash UFRGS 113
herbarium (Herbaacuterio do Instituto de Biociecircncias of UFRGS) 114
71
For mimosine elicitation experiments legumes (fruits) of maricaacute were collected in 115
the end of June 2017 (winter) Seeds were then removed from the dry fruits and kept in the 116
dark until sowing and seedling development for use in the assays 117
Seed germination 118
To break the coat-imposed seed dormancy after surface sterilization dry seeds of 119
maricaacute were acid scarified by immersion in H2SO4 (95 ndash 98 ) for 2 min (see Correcirca et al 120
2008) and repeatedly washed in distilled water to remove any residue of the acid Then seeds 121
were distributed in 50 mL individual plastic tubes (dibble-tubes) (30 cm diameter x 120 cm 122
depth) filled up with 11 (vv) of commercial top soil and vermiculite Tubes were watered 123
every 2 days to avoid substrate dryness and were kept in a growth room under controlled 124
conditions of light (circa 75 μmol mminus2s minus1 photosynthetically active radiation photoperiod 125
of 16 h light and 8 h dark) and temperature (24plusmn2C) 126
127
Treatments 128
In order to verify inducibility of mimosine accumulation in M bimucronata fifty 12-129
week-old maricaacute seedlings (per treatment) exhibiting similar features were selected and 130
sprayed (saturated) with solutions of different chemical stressors (plant specialized 131
metabolism elicitors) as follows (for further details see Rodrigues-Correcirca et al 2019) 10 132
and 50 mM SA (pathogen-signaling molecule Shah 2003) 007 and 035 mM 2-133
chloroethylphosphonic acid (ETH ethylene releasing-compound Kim et al 2016 Wang et 134
al 2016) 100 and 200 mM MeJA (Dar et al 2015) 10 and 50 mM SNP (a nitric oxide 135
donor Perotti et al 2015) Alternatively maricaacute seedlings were also supplemented with UV-136
C radiation (13 minutes 105 kJ cm2) (elicitor of plant specialized metabolism Kara 2013) 137
72
After 2 and 4 days of exposure to the chemical treatments and 3 and 6 days of UV-138
C supplementation maricaacute shoots were harvested immediately frozen in liquid nitrogen and 139
stored at ndash 80 C until mimosine extraction and HPLC analyses 140
Mimosine extraction and detection 141
Mimosine extraction was conducted according to the modified protocol described by 142
Rodrigues-Correcirca et al (2019) for L leucocephala HPLC (Thermo Scientific Surveyor) 143
analyses (mimosine detection and quantification) were performed following previously 144
published procedures (Negi et al 2014) A C18 column (ACE C18 5 μm 46times250 mm) and 145
isocratic solvent system of 002M o-phosphoric acid with a linear flow rate of 1 mL min minus1 146
were used to separate and quantify the amino acid Mimosine detection was performed at 280 147
nm by photodiode array detection (200ndash400 nm) and retention time (229plusmn0024 min) 148
Mimosine quantification was done by means of the method of external standard curve 149
Additional confirmation of mimosine identity was performed by co-chromatography with 150
standard (Acros Organics authentic mimosine 99 used as reference) and peak purity check 151
The analyses of the chromatograms were done with the ChromQuest software 152
153
154
Results and Discussion 155
Constitutive accumulation of mimosine in M bimucronata 156
Mimosine was detected in all analyzed samples positively meeting all identification 157
criteria In agreement with what has been found for other Mimosa spp (Soedarjo and 158
Borthakur 1998) compared to L leucocephala adult plants (Rodrigues-Correcirca 2019) 159
mimosine content was lower in M bimucronata Of the adult plant tissues analyzed the 160
73
highest content of mimosine in maricaacute (per gram of fresh weight - FW) was found in post-161
anthesis flowers (36644 microg versus 89448 microg in common leucaena followed by leaves 162
(28838 microg x 67358 microg) green flower buds (28094 microg x 51247 microg) and green pods (19002 163
microg x 82687 microg) (Fig 1)The same pattern is observed for seedlings when both species are 164
compared In this study untreated 12-week-old maricaacute seedlings (control at day 2) showed a 165
shoot content of mimosine of 23029plusmn007 microg g-1 of (FW) Five-week-old untreated giant 166
leucaena seedlings cultivated in similar conditions exhibited between 83640 and 178736 167
microg g-1 of FW (Rodrigues-Correcirca et al 2019) In the same way mimosine concentration 168
percentage in dry matter of Mimosa pigra was found to be rather low (002 in nodules and 169
roots and 007 in leaves) (Soedarjo and Borthakur 1998) 170
In this investigation the lowest constitutive mimosine content was found in green 171
pods (Fig 1) This result may partly explain the absence of phytotoxic effect observed for 172
green pods on germination and growth of crop target plants tested by Jacobi and Ferreira 173
(1991) compared to the other maricaacute parts analyzed 174
Elicitation of mimosine biosynthesis in M bimucronata 175
Chemical stressors 176
Secondary metabolites (or natural products) are structural- and chemically 177
specialized compounds derived from primary metabolism These molecules are mainly 178
biosynthesized as part of a complex defense mechanism in response to biotic and abiotic 179
stresses such as pathogens herbivores water status metal toxicity and UV radiation for 180
example (Matsuura et al 2018) Ethephon SA SNP MeJA have been extensively used as 181
chemical elicitors of specialized metabolism (Wang et al 2016 Vestena et al 2001 Perotti 182
74
et al 2015 Zhang and Memelink 2009 Xu et al 2018) These phytohormonal signals can 183
simulate environmental challenges and modulate plant homeostasis often leading to 184
alterations in gene expression (Shinozaki et al 2015) Except SNP all treatments tested in 185
the present study showed positive effect on mimosine accumulation in common or giant 186
leucaena (Vestena et al 2001 Rodrigues-Correcirca 2019 Rodrigues-Correcirca unpublished 187
data) However in spite of the trend of increasing the mimosine content observed in seedlings 188
treated with 007 mM Ethephon (at day 2) and 100 mM MeJA (at day 4) no statistical 189
difference was confirmed for these treatments when compared to the control 190
On the other hand a within treatment difference on mimosine induction was seen 191
between day 2 and 4 in seedlings treated with 100 mM MeJA (Fig 2) In a lower 192
concentration (04 mM) jasmonic acid (JA)promoted a near threefold increase in mimosine 193
accumulation of giant leucaena seedlings after 2 days of application 194
UV-C radiation 195
Albeit UV-C radiation is not biologically active in natural environments it has been 196
widely used under controlled experimental conditions to generate acute responses of plant 197
specialized metabolism within a shorter period of time compared to that required to with UV-198
B radiation (Kara 2013 Cetin 2014) This fast response is due to the higher energy of UV-199
C photons that act as potent reactive oxygen species (ROS) generators causing extensive 200
damage to the cells either at the physiological level or on DNA structure (Gregianini et al 201
2003 Matsuura et al 2013) 202
Although divergent responses can be observed in plants exposed to UV-C radiation 203
the deleterious processes are usually reported on primary metabolism (decreasing of 204
chlorophyll content and plant height eg) (Kara 2013) In the present study no statistical 205
75
differences were observed in the mimosine concentration in maricaacute seedlings supplemented 206
with UV-C radiation However a decreasing in its content was found for both control and 207
treatment at day 6 post-treatment (Fig 03) Taking into account the lower constitutive 208
concentration of mimosine observed in maricaacute compared to the leucaena plants besides its 209
relative thermolability (Nguyen and Tawata 2016) it seems to be plausible to consider the 210
effect of the temperature inside the UV-C and the white light (control) chambers as an 211
additional abiotic factor contributing to the decrease of mimosine accumulation in both group 212
of plants 213
Besides mimosine identification the presence of 34-dihydroxypyridine (34-DHP or 214
3-hydroxy-4-pyridone - 3H4P) a mimosine degradation product (Negi et al 2014 Nguyen 215
and Tawata 2016) was also reported for maricaacute leaf extracts analyzed by TLC by Ferreira 216
et al (1992) In our chromatograms we detected a second large peak after that of mimosine 217
(229plusmn0024) and similar to that identified by Negi et al (2014) as 3H4P (data not shown) 218
Comparing the chromatogram profiles obtained from seedlings elicited with chemical 219
stressors and those supplemented with UV-C the largest area for this peak was found (in all 220
samples) in the latter treatment at day 6 It might indicate that the constitutive andor the 221
initially UV-C-induced mimosine was degraded into 3H4P to cope with the cellular damage 222
caused by this treatment associated with an increased temperature inside the chambers 223
Nevertheless it was not possible to determine 3H4P concentration (or confirm its identity) 224
in maricaacute plants since there is no commercial standard (pure 3H4P) available for purchase 225
to be used as a reference in calculations Establishment of improved protocols for obtaining 226
in house 3H4P reference substance by acid hydrolysis is ongoing 227
228
229
76
Conclusion 230
On the basis of the overall absence of effect of the treatments tested here on mimosine 231
concentration it is possible to suggest that its accumulation profile is similar to that of 232
phytoanticipins unlike what is observed for the same amino acid production in leucaena 233
which shows features of inducibility resembling phytoalexin-like metabolites Alternatively 234
a putative inducible pool of mimosine in maricaacute might be involved in other types of stress 235
such as extended drought periods If involved in protection against oxidative stress as 236
described for leucaena mimosine in maricaacute may act predominantly by physical quenching 237
of ROS as indicated by the lack of overt chemical degradation Nevertheless further 238
investigations are needed to assess these hypotheses 239
To sum up mimosine biosynthesis was not modulated by the treatments evaluated as 240
in L leucocephala (Lam) de Wit To the best of our knowledge this is the first work that 241
analytically identifies and quantifies mimosine accumulation in M bimucronata 242
243
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Carvalho PER 2004 Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 248
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Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitis 250
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Champanerkar PA Vaidya VV Shailajan S Menon SN 2010 A sensitive rapid and 253
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determination of Mimosine in Mimosa pudica Linn Nat Sci 2 713-717 255
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Gomes GS Silva GS Silva DLS Oliveira RR Conceiccedilatildeo GM 2018 Botanical 257
Composition of Fabaceae Family in the Brazilian Northeast Maranhatildeo Brazil Asian J 258
Environ Ecol 6(4) 1-10 httpsdoiorg109734AJEE201841207 259
Correcirca LR Soares GLG Fett-Neto AG 2008 Allelopathic potential of Psychotria 260
leiocarpa a dominant understorey species of subtropical forests S Afri J Bot 74 583ndash261
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Ferreira AG Aquila MEA Jacobi US Rizvi V 1992 Allelopathy in Brazil In Allelopathy 263
basic and applied aspects Rizvi V and Jacobi US (Eds) Chapman and Hall pp 243-250 264
Fukao T Barrera-Figueroa BE Juntawong P Pentildea-Castro JM 2019 Submergence 265
and waterlogging stress in plants a review highlighting research opportunities and 266
understudied aspects Front Plant Sci 10 340 httpsdoiorg103389fpls201900340 267
Gregianini TS Silveira VC Porto DD Kerber VA Henriques AT Fett-Neto AG 268
2003 The alkaloid brachycerine is induced by ultraviolet radiation and is a singlet oxygen 269
quencher Photochem Photobiol 78(5) 470ndash474 httpsdoiorg1015620031-270
8655(2003)0784070TABIIB20CO2 271
Jacobi US Ferreira AG 1991 Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK 272
sobre espeacutecies cultivadas Pesq Agropec Bras 26(7) 935-943 273
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant 274
(Phaseolus vulgaris) Biosci Res 10(1) 29ndash32 275
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Kestring D Klein J Menezes LCCR Rossi MN 2009 Imbibition phases and 276
germination response of Mimosa bimucronata (Fabaceae Mimosoideae) to water 277
submersion Aquat Bot 91 105ndash109 httpsdoiorg101016jaquabot200903004 278
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect of 279
Ethephon as an ethylene-releasing compound on the metabolic profile of Chlorella vulgaris 280
J Agric Food Chem 64(23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541 281
Lorenzi H 1998 Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 282
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 283
Lorenzi H 2008 Plantas daninhas do Brasil terrestres aquaacuteticas parasitas e toacutexicas 4 ed 284
Nova Odessa Instituto Plantarum 640 p 285
Marchiori JNC 1993 Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 286
O Kuntze Ciecircncia Florestal 3 85-106 287
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation of 288
bioactive secondary metabolites by ultraviolet radiation mechanisms strategies and 289
applications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology for Medicinal 290
Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash190= 291
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo R Bhambra AS 292
Strnad M Bonfill M Fett-Neto AG 2018 Specializedplant 293
metabolismcharacteristicsandimpactontargetmoleculebiotechnologicalproduction 294
Molecular Biotechnology 60(2) 169ndash183httpsdoiorg101007s12033-017-0056-1 295
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase from 296
Leucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 297
922ndash934 httpsdoiorg101104pp113230870 298
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Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine 299
areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636 300
Olkoski D Wittmann MTS 2011 Cytogenetics of Mimosa bimucronata (DC) O Kuntze 301
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop Breed 302
Appl Biotechnol 11 27-35 httpdxdoiorg101590S1984-70332011000100004 303
Patreze CM Cordeiro L 2004 Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 304
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecol Manag 196 275ndash305
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Perotti JC Rodrigues-Correcirca KCS Fett-Neto AG 2015 Control of resin production in 307
Araucaria angustifolia an ancient South American conifer Plant Biology 17 852ndash859 308
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG 2019 Mimosine 309
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UV exposure Plant Physiology and Biochemistry 135 432ndash440 311
Pilatti DM Fortes AMT Jorge TCM Boiago NP 2019 Comparison of the phytochemical 312
profiles of five native plant species in two different forest formations Brazilian Journal of 313
Biology 79(2) 233-242 314
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS 2011 Biologia da reproduccedilatildeo 315
deMimosa bimucronatandash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 316
Simon MF and Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 317
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 318
endemism Biological Conservation 96 279-296 319
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 320
LM Andrade SF Cechinel-Filho V 2017 Chemical composition and diuretic natriuretic and 321
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kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its majority 322
constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash1624 323
Shah J 2003 The salicylic acid loop in plant defense Current Opinion Plant Biology6 (4) 324
365ndash371 325
Shinozaki K Uemura M Serres JB Bray EA Weretilnyk E 2015 Responses to Abiotic 326
Stress In Buchanan BB Gruissem W Jones RL (Eds) Biochemistry and Molecular 327
Biology of Plants Second Edition John Wiley and Sons Ltd 328
Soedarjo M and Borthakur D 1998 Mimosine a toxin produced by the tree-legume 329
Leucaena provides a nodulation competition advantage to mimosine-degrading Rhizobium 330
strains Soil Biology and Biochemistry 30(12)1605-1613 331
Vestena S Fett-Neto AG Duarte RC Ferreira AG 2001 Regulation of mimosine 332
accumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 333
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene induced 334
vinblastine accumulation is related to activated expression of downstream TIA pathway 335
genes in Catharanthus roseus BioMed Research International Article ID 3708187 336
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX Tsim KWK 2018 337
Jasmonate-elicited stress induces metabolic change in the leaves of Leucaena leucocephala 338
Molecules 23 (2) 339
Zhang H Memelink J 2009 Regulation of Secondary Metabolism by Jasmonate Hormones 340
In AE Osbourn and V Lanzotti (eds) Plant-derived Natural Products 3 DOI 101007978-341
0-387-85498-4_1 copy Springer Science + Business Media LLC 342
343
344
345
81
346
Figure 1 Constitutive concentration of mimosine in different plant organs of Mimosa 347
bimucronata Bars sharing the same letter do not differ statistically by Tukey test (Ple005) 348
The error bars denote standard error of 10 replicates 349
350
351
352
353
354
355
356
357
B B A C0
5
10
15
20
25
30
35
40
LEAVES GREEN FLOWER BUDS POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
co
nce
ntr
atio
n u
gg-1
Mimosine concentration in adult plants of Mimosa bimucronata (DC) Kuntze
82
C T R L S A
1 0 m M
S A
5 0 m M
E T H
0 0 7 m M
E T H
0 3 5 m M
M e J A
1 0 0 m M
M e J A
2 0 0 m M
S N P
1 0 m M
S N P
5 0 m M
0
1 0
2 0
3 0
T re a tm e n ts
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1) D A Y 2
D A Y 4
A B C C B C A B C C A B C A B C A
a b b b a a b a a b b a b
358
Figure 2 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 359
bimucronata treated with different signaling molecules SA = Salicylic Acid ETH = 360
Ethephon MeJA = Methyl Jasmonate SNP = Sodium Nitroprusside Uppercase and 361
lowercase letters indicate statistical differences among treatments in days 2 and 4 362
respectively Bars sharing a letter of the same case do not differ statistically by Tukey test 363
(Ple005) Indicates statistical difference in the same treatment between day 2 and 4 by t-364
test (Ple005) The error bars denote standard error of 5 replicates (25 individual seedlings 365
arranged in 5 groups of 5) 366
367
368
83
D AY 3 D AY 6
0
5
1 0
1 5
2 0
2 5
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1)
C O N TR O L
U V -C
369
Figure 3 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 370
bimucronata supplemented with UV-C radiation Indicates statistical difference in the same 371
treatment between day 3 and 6 by t-test (Ple005) The error bars denote standard error of 5 372
replicates (25 individual seedlings arranged in 5 groups of 5) 373
374
375
376
377
378
379
380
381
382
383
384
385
84
Consideraccedilotildees finais 386
- Experimentos que avaliam os efeitos da aplicaccedilatildeo exoacutegena de ANPs em diferentes espeacutecies 387
vegetais tecircm sido realizados principalmente com GABA Dentre os principais efeitos 388
conferidos pela aplicaccedilatildeo dessa moleacutecula em espeacutecies de mono e eudicotiledocircneas satildeo 389
relatados a toleracircncia agrave seca agrave salinidade e agraves temperaturas extremas 390
- Como metaboacutelitos especializados claacutessicos os ANPs podem ter sua concentraccedilatildeo basal 391
endoacutegena aumentada em resposta agrave induccedilatildeo mediada por uma vasta gama de tratamentos com 392
moleacuteculas sinalizadoras de estresse e fontes alternativas de estressores De um modo geral 393
observa-se o acuacutemulo das diferentes classes de ANPs em resposta agrave radiaccedilatildeo UV elicitores 394
quiacutemicos que mimetizam ataques por patoacutegenos dano mecacircnico agentes osmoacuteticos metais 395
pesados entre outros 396
- Especificamente em leucena a resposta observada em relaccedilatildeo aos diferentes tratamentos 397
testados indica que apesar do seu alto teor constitutivo nessa espeacutecie a biossiacutentese e o 398
acuacutemulo de mimosina podem ser modulados por fatores causadores de estresses exibindo -399
nessa espeacutecie - um padratildeo de acumulaccedilatildeo similar agrave fitoalexinas Em maricaacute por outro lado 400
aumento de acuacutemulo dessa moleacutecula natildeo foi observado para os mesmos tratamentos testados 401
para leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas 402
- O padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno 403
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado pela 404
sua degradaccedilatildeo 405
- As respostas observadas nos testes que avaliaram a atividade de mitigaccedilatildeo de espeacutecies 406
reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode agir como um agente 407
antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de estresse 408
85
Perspectivas 409
- Confirmaccedilatildeo em espectrocircmetro de massas eou ressonacircncia nuclear magneacutetica da natureza 410
quiacutemica da lsquomimosinarsquo presente em maricaacute 411
- Avaliaccedilatildeo do efeito de concentraccedilotildees mais elevadas e em diferentes periacuteodos de aplicaccedilatildeo 412
das moleacuteculas sinalizadoras testadas sobre o acuacutemulo de mimosina em leucena e maricaacute 413
- Ampliar a investigaccedilatildeo dos padrotildees de expressatildeo gecircnica dos genes que codificam para 414
mimosinase (em maricaacute) mimosina sintase (em ambas as espeacutecies testadas) bem como o 415
perfil de precursores e cataboacutelitos de mimosina em resposta aos tratamentos mencionados 416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
86
Referecircncias Bibliograacuteficas 436
437
Acamovic T Brooker JD (2005) Biochemistry of plant secondary metabolites and their 438
effects in animals P Nutr Soc 64 403ndash412 httpsdoiorg101079PNS2005449 439
Ahmed R Hoque ATMR Hossain MK (2008) Allelopathic effects of Leucaena 440
leucocephala leaf litter on some forest and agricultural crops grown in nursery J Forestry 441
Res (2008) 19 298 httpsdoiorg101007s11676-008-0053-0 442
Ahmed AMM Saacutenchez FJS Bavileacutes LRY Mahdy REZ Camaal JBC (2016) Tannins and 443
mimosine in Leucaena genotypes and their relations to Leucaena resistance against 444
Leucaena Psyllid and Onion thrips Agroforestry Systems 1-8 445
Benjakul S Kittiphattanabawon P Shahidi F Maqsood S (2013) Antioxidant activity and 446
inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipid oxidation in 447
model systems Food Sci Technol Int 19(4)365-76 448
httpsdoiorg1011771082013212455186 449
Bitencourt F Zocche JJ Costa S Souza PZ Mendes AR (2007) Nucleaccedilatildeo de Mimosa 450
bimucronata (DC) O Kuntze em aacutereas degradadas pela mineraccedilatildeo de carvatildeo Revista 451
Brasileira de Biociecircncias 5 750-752 452
Bottini-Luzardo M Aguilar-Perez C Centurion-Castro F Solorio-Sanchez F Ayala-Burgos 453
A Montes-Perez R Muntildeoz-Rodriguez D Ku-Vera J (2015) Ovarian activity and estrus 454
behavior in early postpartum cows grazing Leucaena leucocephala in the tropics Trop Anim 455
Health Prod 47(8)1481-6 456
Carvalho PER (2004) Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 457
Teacutecnica 941-10 458
Chowtivannakul P Srichaikul B Talubmook C (2016) Antidiabetic and antioxidant activities 459
of seed extract from Leucaena leucocephala (Lam) de Wit Agriculture and Natural 460
Resources 50 (2016) 357e361 httpdxdoiorg101016janres201606007 461
Chung H-H Chen M-K Chang Y-C Yang S-F Lin C-C Lin C-W (2017) Inhibitory effects 462
of Leucaena leucocephala on the metastasis and invasion of human oral cancer cells 463
Environmental Toxicology 321765ndash1774 httpsdoiorg101002tox22399 464
87
Crowe B Poynter JA Manukyan MC Wang Y Brewster BD Herrmann JL Abarbanell 465
AM Weil BR Meldrum DR (2001) Pretreatment with intracoronary mimosine improves 466
postischemic myocardial functional recovery Surgery 150(2) 191-106 467
Fallon (2015) Effects of mimosine on Wolbachia in mosquito cells cell cycle suppression 468
reduces bacterial abundance In Vitro Cell Dev Biol Anim 51(9)958-63 469
httpsdoiorg101007s11626-015-9918-7 Epub 2015 May 28 470
Fernaacutendez-Salas A Alonso-Diacuteaza MA Acosta-Rodriacuteguez A Torres-Acosta JFJ Sandoval-471
Castro CA Rodriacuteguez-Vivas RI (2011) In vitro acaricidal effect of tannin-rich plants against 472
the cattle tick Rhipicephalus (Boophilus) microplus (Acari Ixodidae) Veterinary 473
Parasitology 175113ndash118 2010 httpsdoiorg101016jvetpar201009016 474
Ferreira AG Aquila MEA Jacobi US Rizvi V (1992) Allelopathy in Brazil In Allelopathy 475
basic and applied aspects Rizvi V and Jacobi US (Eds) Chapman and Hall PP 243-250 476
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad Hossain Md Anai 477
T Oku H (2018) Cytosolic cysteine synthase switch cysteine and mimosine production in 478
Leucaena leucocephala Appl Biochem Biotechnol 186 (3) 613ndash632 479
httpsdoiorg101007s12010-018-2745-z 480
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of the thyrotoxic 481
amino acid mimosine by cysteine synthase Phytochemistry 29 (11) 3461ndash3465 482
httpsdoiorg1010160031-9422(90)85258-H 483
Jacobi US Ferreira AG (1991) Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK Sobre 484
espeacutecies cultivadas Pesquisa Agropecuaacuteria Brasileira 26(7) 935-943 485
Jamous RM Ali-Shtayeh MS Abu-Zaitoun SY Markovics A Azaizeh H (2017) Effects of 486
selected Palestinian plants on the in vitro exsheathment of the third stage larvae of 487
gastrointestinal nematodes BMC Veterinary Research 13308 488
httpdxdoiorg101186s12917-017-1237-7 489
Jiao CJ Jiang J-L Ke L-M Cheng W Li F-M Li Z-X Wang C-Y (2011) Factors affecting 490
β-ODAP content in Lathyrus sativus and their possible physiological mechanisms Food 491
Chem Toxicol 49 543ndash549 httpsdoiorg101016jfct201004050 492
Kubota S Fukumoto Y Ishibashi K Soeda S Kubota SS Yuki R Nakayama Y Aoyama K 493
Yamaguchi N (2014) Activation of the prereplication complex is blocked by mimosine 494
88
through reactive oxygen species-activated ataxia telangiectasia mutated (ATM) protein 495
without DNA damage J Biol Chem 28 289(9)5730-46 496
Kuppusamy UR Arumugam B Azaman N Wai CJ (2014) Leucaena leucocephala Fruit 497
Aqueous Extract Stimulates Adipogenesis Lipolysis and Glucose Uptake in Primary Rat 498
Adipocytes Hindawi Publishing Corporation e Scientific World Journal Article ID 737263 499
8 pages httpdxdoiorg1011552014737263 500
Kusama-Eguchi K (2019) Research in motor neuron diseases caused by natural substances 501
focus on pathological mechanisms of neurolathyrism Yakugaku Zasshi 139 (4) 609-502
615 httpsdoiorg101248yakushi18-00202 503
Kutchan TM Gershenzon J Moslashller BL Gang DR (2015) Natural Products In Buchanan 504
BB Gruissem W and Jones RL (eds) Biochemistry amp Molecular Biology of Plants 2nd edn 505
Wiley Blackwell Chichester pp 1135-1205 506
Lalande M (1990) A reversible arrest point in the late G1 phase of the mammalian cell cycle 507
Exp Cell Res 186 332ndash339 508
Li X-W Hu C-P Li Y-J Gao Y-X Wang XM Yang J-R (2015) Inhibitory effect of L-509
mimosine on bleomycin-induced pulmonary fibrosis in rats Role of eIF3a and p27 Int 510
Immunopharmacol 27(1) 53ndash64 511
Little Jr EL Skolmen RG (1989) Koa haole Agriculture Handbook 679 USDA 512
Lorenzi H (1998) Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 513
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 514
Marchiori JNC (1993) Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 515
O Kuntze Ciecircncia Florestal 3 85-106 516
Mohammed RS El Souda SS Taie HAA Moharam ME Shaker KH (2015) Antioxidant 517
antimicrobial activities of flavonoids glycoside from Leucaena leucocephala leaves Journal 518
of Applied Pharmaceutical Science 5(06)138-147 519
httpdxdoiorg107324JAPS201550623 520
Negi VS Bingham J-P Li QX Borthakur D (2014) A carbon-nitrogen lyase from Leucaena 521
leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 (2) 922ndash522
934 httpsdoiorg101104pp113230870 523
89
Olkoski D Wittmann MTS (2011) Cytogenetics of Mimosa bimucronata (DC) O Kuntze 524
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop 525
Breeding and Applied Biotechnology 11 27-35 526
Patreze CM Cordeiro L (2004) Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 527
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecology and Management 528
196275ndash285 529
Pilatti DM Fortes AMT Jorge TCM Boiago NP (2019) Comparison of the phytochemical 530
profiles of five native plant species in two different forest formations Brazilian Journal of 531
Biology 79(2) 233-242 532
Ramos-Ruiz R Poirot E Flores-Mosquera M (2018) GABA a non-protein amino acid 533
ubiquitous in food matrices Cogent Food Agric 41534323 534
httpsdoiorg1010802331193220181534323 535
REFLORA (2019) httpfloradobrasiljbrjgovbrreflora Acesso em agosto de 2019 536
Rodgers KJ Samardzic K Main BJ (2015) Toxic Nonprotein Amino Acids Plant Toxins 537
httpsdoiorg 101007978-94-007-6728-7_9-1 538
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG (2019) Mimosine 539
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 540
UV exposure Plant Physiology and Biochemistry 135 432ndash440 541
httpsdoiorg101016jplaphy201811018 542
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 543
LM Andrade SF Cechinel-Filho V (2017) Chemical composition and diuretic natriuretic 544
and kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its 545
majority constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash546
1624 547
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS (2011) Biologia da reproduccedilatildeo 548
de Mimosa bimucronata ndash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 549
Simon MF Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 550
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 551
endemism Biological Conservation 96 279-296 552
90
Soares AMS Arauacutejo SA Lopes SG Costa Junior LM (2015) Anthelmintic activity of 553
Leucaena leucocephala protein extracts on Haemonchus contortus Braz J Vet Parasitol 554
Jaboticabal 24(4) 396-401 httpdxdoiorg101590S1984-29612015072 555
Soerdajo M Borthakur D (1998) Mimosine a toxin produced by the tree-legume Leucaena 556
provides a nodulation competition advantage to mimosine-degrading Rhizobium strains Soil 557
Biol Biochem 30(12) 16051613 558
Souza-Lima ES Sinani TR Pott A Sartori ALB (2017) Mimosoideae (Leguminosae) in the 559
Brazilian Chaco of Porto Murtinho Mato Grosso do Sul Rodrigueacutesia 68(1) 263-290 2017 560
httpdxdoiorg1015902175-7860201768131 561
Taiz L amp Zeiger E (2010) Plant Physiology 5th edition Sinauer Associates Inc Sunderland 562
Verma VK Rani KV Kumara SR Prakash O (2018) Leucaena leucocephala pod seed 563
protein as an alternate to animal protein in fish feed and evaluation of its role to fight against 564
infection caused by Vibrio harveyi and Pseudomonas aeruginosa Fish and Shellfish 565
Immunology 76 (2018) 324ndash332 httpsdoiorg101016jfsi201803011 566
Yafuso JT Negi VS Bingham J-P Borthakur D (2014) An O-acetylserine (thiol) lyase from 567
Leucaena leucocephala is a cysteine synthase but not a mimosine synthase Appl Biochem 568
Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z 569
Zarin RMA Wan HY Isha A Armani N (2016) Antioxidant antimicrobial and cytotoxic 570
potential of condensed tannins from Leucaena leucocephala hybrid Food Science and 571
Human Wellness 5 65ndash75 httpdxdoiorg101016jfshw201602001 572
573
574
Contents lists available at ScienceDirect
Industrial Crops amp Productsjournal homepage wwwelseviercomlocateindcrop
Resin tapping transcriptome in adult slash pine (Pinus elliottii var elliottii)Camila Fernanda de Oliveira Junkes1 Artur Teixeira de Arauacutejo Juacutenior1 Juacutelio Ceacutesar de LimaFernanda de Costa Thanise Fuumlller Maacutercia Rodrigues de Almeida Franciele Antocircnia NeisKelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-NetoCenter for Biotechnology and Department of Botany Federal University of Rio Grande do Sul Porto Alegre PO Box 15005 91501-970 Brazil
A R T I C L E I N F O
KeywordsPinus elliottiResinResinosisTranscriptomeAdjuvant paste
A B S T R A C T
To better understand the bases of resin production a major source of terpenes for industry the transcriptome ofadult Pinus elliottii var elliottii (slash pine) trees under field commercial resinosis was obtained Samples werecollected from cambium after 5 and 15 days of treatment application which included tapping followed byapplication of commercial resin stimulant paste or control wounding without paste Overall mean number ofreads of all 16 libraries (2 treatments x 2 times x 4 replicated trees) was 34582048 Of these 89 were mappedagainst the reference sequence with a mismatch of 058 Using the Blast2Go 570 candidate genes were de-tected based on sequence annotation By comparing the expression profile between paste and control 310differentially expressed genes (DEGs) were identified at 5 days and 190 at 15 days with a significant fold changeof log2gt 12 Regarding changes in time comparisons within each treatment 210 and 105 DEGs were identifiedwithin control and paste treatment respectively Genes with different expression patterns in the times andtreatments examined included ethylene responsive transcription factors geranylgeranyl diphosphate synthasediterpene synthase cytochrome P450 and ABC transporters all of which may play important roles in resinproduction RT-qPCR analysis correlated well with the data obtained by RNAseq Resin composition changedover time This is the first transcriptomic investigation of resinosis of the main species used in the bioresinindustry and of molecular analyses of resinosis under field operations with implications for stand managementstimulant paste development genotype selection and breeding for high resinosis
1 Introduction
The adaptive success of conifers is largely due to the development ofa defense system based on the synthesis and secretion of terpenes in allmajor organs and different tissues (Miller et al 2005 Hall et al 2013Warren et al 2015) Conifer resin is a viscous fluid composed of acomplex mixture of terpenoids such as monoterpenes sesquiterpenesand diterpenes (Zulak and Bohlmann 2010) These terpenoids are se-creted from severed resin ducts when the tree is under biotic attack(Ralph et al 2006 Lange 2015 Geisler et al 2016) acting as pro-tectants (Schiebe et al 2012 Liu et al 2015)Biosynthesis of terpenes in conifers starts from isomerization of two
isoprenoid (C5) units dimethylallyl diphosphate (DMAPP) and iso-pentenyl diphosphate (IPP) These molecules can be biosynthesized viatwo separate routes in plants the methyl-erythritol 4-phosphate andmevalonate pathways IPP is synthesized and isomerized to DMAPP byisopentenyl diphosphate isomerase then prenyl transferases catalyze
the condensation of these two C5-units to geranyl diphosphate (Pazoukiand Niinemets 2016) Their elongation to prenyl diphosphates withaddition of IPP molecules leads to monoterpenes (C10) sesquiterpenes(C15) and diterpenes (C20) which are the substrates for terpene syn-thases (TPS) (Keeling and Bohlmann 2006b)TPSs are part of a large family of mechanistically related enzymes
involved in both primary and secondary metabolism (Keeling andBohlmann 2006b) The events of evolutionary diversification and ex-pansion of plant TPSs appear to have originated from gene duplicationsdomain losses and sub- or neofunctionalizations with subsequent di-vergence of an ancestral TPS gene of primary metabolism (Hall et al2013) Modification of TPS products changes their physical propertiesand may alter their biological activities (Chen et al 2011) TPSs of highsequence identity may have different functions even in closely relatedspecies Low sequence identity of TPSs in phylogenetically distantspecies does not preclude the possibility of independent evolution of thesame or related function of these enzymes (Zerbe and Bohlmann 2015)
httpsdoiorg101016jindcrop2019111545Received 4 January 2019 Received in revised form 10 June 2019 Accepted 4 July 2019
Corresponding authorE-mail address fettnetocbiotufrgsbr (AG Fett-Neto)1 These authors have equally contributed to this work
doi 1015900102-33062019abb0114
Acta Botanica Brasilica
Sustainable production of bioactive alkaloids in Psychotria L of
southern Brazil propagation and elicitation strategies
Yve Verocircnica da Silva Magedans1 Kelly Cristine da Silva Rodrigues-Correcirca1 Cibele Tesser da Costa1
Heacutelio Nitta Matsuura1 and Arthur Germano Fett-Neto1
Received April 1 2019Accepted June 28 2019
ABSTRACTPsychotria is the largest genus in Rubiaceae South American species of the genus are promising sources of natural
products mostly due to bioactive monoterpene indole alkaloids they accumulate ese alkaloids can have analgesic
antimutagenic and antioxidant activities in dierent experimental models among other pharmacological properties
of interest Propagation of genotypes with relevant pharmaceutical interest is important for obtaining natural
products in a sustainable and standardized fashion Besides the clonal propagation of elite individuals the alkaloid
content of Psychotria spp can also be increased by applying moderate stressors or stress-signaling molecules is
review explores advances in research on methods for plant propagation and elicitation techniques for obtaining
bioactive alkaloids from Psychotria spp of the South Region of Brazil
Keywords abiotic stress alkaloids elicitation monoterpenes plant propagation Psychotria southern Brazil
sustainability
Introduction
Psychotria belongs to Rubiaceae one of the major families
of $owering plants having economic interest e family
includes coee a few signicant poisonous plants to livestock
besides several important ornamental and medicinal species
(Souza amp Lorenzi 2012) Psychotria has captured researchersrsquo
attention mostly because of its medicinal properties
Psychotria colorata is an Amazonian species that produces
polyindolinic alkaloids with analgesic activity (Matsuura et
al 2013) e promising results obtained with P colorata
motivated the investigation of southern Brazilian Psychotria
species and the discovery of new bioactive alkaloids (Porto
et al 2009) Moreover leads on in planta alkaloid functions
were also topic of experimental evaluation
One of the key elements that needs to be addressed early
on during the process of developing new bioactive molecules
from plants is the capacity to generate catalytically active
biomass to support extraction and steady supply ere are a
number of ways through which these goals may be reached
including greenhouse rooting of cuttings (mini-cutting
1 Laboratoacuterio de Fisiologia Vegetal Departamento de Botacircnica Instituto de Biociecircncias e Centro de Biotecnologia Universidade Federal do Rio
Grande do Sul 91501-970 Porto Alegre RS Brazil
Corresponding author fettnetocbiotufrgsbr
Review
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Biomass yield of resin in adult Pinus elliottii Engelm trees is differentially
regulated by environmental factors and biochemical effectors
Franciele Antocircnia Neis Fernanda de Costa Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima
Kelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-Neto
Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) CP 15005 CEP 91501-970 Porto Alegre RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Biomass
Terpene resin
Seasonal
Benzoic acid
Regenerated forest
A B S T R A C T
Biomass of pine resin finds several applications in the chemical pharmaceutical biofuel and food industries
Resin exudation after injury is a key defense response in Pinaceae since this complex mixture of terpenes has
insecticidal antimicrobial and wound repair properties Resin yield is increased by effectors applied on the
wound area including phytohormones and metal cofactors of terpene synthases The interaction of resinosis
mechanism effectors is not fully understood particularly in adult forest setups under natural environmental
variations The aim of this work was to determine how resin exudation by wounded trunks of adult P elliottii
responded to combined chemical effectors involved in different regulatory pathways of resinosis (metal cofactors
of terpene synthases benzoic acid and plant growth regulators) and whether seasonal and tree distribution
variations affected these responses Symmetrically planted and scattered trees regenerated from the seed bank
had similar resin biomass yields suggesting that the homogeneity in development and spatial arrangement were
not significant factors in resin yield This new finding is of practical importance with the used tapping system
since costs of implanting forests by regeneration can be advantageous compared to planting In addition it was
shown for the first time that the salicylic acid precursor benzoic acid and the auxin naphthalene acetic acid
promoted resin exudation when individually applied to wound sites Both these adjuvants are two orders of
magnitude less costly compared to the conventionally used ethylene precursors besides facing less environ-
mental and health restrictions for use Most adjuvant-treated trees showed higher resin flow in the second year
indicating mechanisms of response build up Overall temperature was more important than rainfall as en-
vironmental parameter affecting resin biosynthesis which was higher in the warmer months of spring and
summer The combination of resinosis stimulant effectors from different signaling pathways showed no sig-
nificant synergistic or additive effect suggesting possible converging signaling pathways andor limitation of
common intermediate transducing molecules
1 Introduction
Pines occupy highly diverse environments over a range of tem-
peratures water and nutrient availabilities irradiance levels and pho-
toperiods being able to effectively face attacks from diverse herbivore
and pathogen guilds The success of conifers is linked to their complex
terpene biochemistry hosted by specialized secretory cells The terpe-
noid resin synthesized by Pinus spp is one of the main mechanisms of
defense of these trees particularly against bark beetles and the fungi
they carry (Fett-Neto and Rodrigues-Correcirca 2012) Pine resin biomass
is essentially composed of a monoterpene and sesquiterpene-rich tur-
pentine and diterpenoid-rich rosin fraction both finding numerous in-
dustrial applications as non-wood forest products (Rodrigues-Correcirca
et al 2012)
Molecules capable of modulating different signaling pathways have
been identified as resin yield stimulators including sulfuric acid (ex-
tends wound damage) 2-chloroethylphosphonic acid (CEPA a syn-
thetic ethylene precursor) paraquat (free radical generator) yeast ex-
tract (mimics attack by pathogens) salicylic acid (pathogen signaling
molecule) auxin (promotes ethylene biosynthesis and resin canal dif-
ferentiation) jasmonic acid (signals mechanical damage and promotes
secondary metabolism) and metal ions such as potassium iron and
manganese (cofactors of terpene synthases in conifers) and copper (a
component of ethylene receptors) (Clements 1970 Conrath et al
2002 Fett-Neto and Rodrigues-Correcirca 2012 Hudgins and Franceschi
2004 Lewinsohn et al 1994 Martin et al 2002 Popp et al 1995
httpsdoiorg101016jindcrop201803027
Received 12 December 2017 Received in revised form 9 March 2018 Accepted 13 March 2018
Corresponding author
E-mail addresses franci_neisyahoocombr (FA Neis) fernandadecostayahoocombr (F de Costa) thanisenfyahoocombr (TN Fuumlller)
jjuliocesarlimagmailcom (JC de Lima) krodriguescbiotufrgsbr (KC da Silva Rodrigues-Correcirca) jpfettcbiotufrgsbr (JP Fett) fettnetocbiotufrgsbr (AG Fett-Neto)
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Research Paper
Dual allelopathic effects of subtropical slash pine (Pinus elliottii Engelm)
needles Leads for using a large biomass reservoir
Kelly Cristine da Silva Rodrigues-Correcircaa Gelson Halmenschlagera Joseacuteli Schwambachb
Fernanda de Costaa Emili Mezzomo-Trevizana Arthur Germano Fett-Netoa
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 Brazilb University of Caxias do Sul Institute of Biotechnology Caxias do Sul RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Seasonality
Growth
Germination
Litter
Substrate
A B S T R A C T
Pinus elliottii Engelm (slash pine) is distributed along the maritime coast of Southern Brazil where it shows
invasive pattern and typical allelopathic features Large quantities of needle litter are produced by pine trees a
biomass that is little explored in areas where this species is alien Little is known about the dynamics of needle
and litter phytochemical interactions particularly in subtropical environments To elucidate the full range of
needle and litter allelopathic potential the effects of litter (superficial and deep) and seasonally harvested fresh
slash pine needles stored for different times were evaluated against lettuce tomato and cucumber seeds and
seedlings Increasing concentrations (0 1 2 4 and 8 wv) of hot and cold aqueous extracts of needles
and litter affected in different ways target plant development Growth and germination inhibition were directly
related to the highest extract concentrations (regardless of the season and mainly in hot water extracts) of
needles On the other hand stimulatory effects of litter extracts on lettuce growth were observed Growth and
germination of cucumber and tomato were not affected by pine litter as substrate when compared to rice husk
The presumable high polarity and thermal stability of slash pine leaf biomass allelochemicals and their transient
toxic effect or growth promoting impact suggest potential applications of this largely available biomass both as a
biological herbicide and growth substrate in plant propagation
1 Introduction
Native from the Northern Hemisphere Pinus is one of the most
widely distributed genera throughout different climate regions of the
globe growing either as native or alien species even in extreme habi-
tats (Rodrigues-Correcirca and Fett-Neto 2012) Despite the high economic
value currently attributed to pine wood and oleoresin (Rodrigues-
Correcirca et al 2012) there is increasing concern about the aggressive
potential of invasiveness displayed by Pinus species especially those
cultivated out of their native range of distribution (Richardson et al
2008 Rolon et al 2011) These species are dispersed by wind and there
is notably low plant diversity observed in most understories of pine
plantations (Kato-Noguchi et al 2009) This latter feature has been
considered an important trait of allelopathic interference
The term ldquoallelopathyrdquo was coined by Molisch in 1937 as a chemical
reciprocal interaction established among plants (including micro-
organisms) sharing the same site by means of the release of secondary
metabolites named allelochemicals (Rice 1984) For the most part
these metabolites are derived from the shikimic acid or isoprenoid
pathway and their biosynthesis can be modulated by biotic and abiotic
stresses (Nascimento and Fett-Neto 2010) including seasonal-related
changes (Sartor et al 2013) Allelopathy studies may range from sterile
assays (Aryakia et al 2015) to soil (Correcirca et al 2008 Sharma et al
2016) and field tests being a complex biological phenomenon to as-
certain in several circumstances due to issues of solubility release
mechanisms and stability of bioactive compounds (Scognamiglio et al
2013) Often the use of complementary methods provides more in-
formative data
The allelopathic effects of soil leachates green needles and litter
extracts of Pinus spp on germination and seedling growth aspects of
wild and crop species have been evaluated in natural and cultivated
pine stands and have proven to be stimulatory or inhibitory (Lodhi and
Killingbeck 1982 Kil and Yim 1983 Nektarios et al 2005 Akkaya
et al 2006 Machado 2007 Alrababah et al 2009 Sartor et al 2009
Kato-Noguchi et al 2011 Rolon et al 2011 Valera-Burgos et al
2012) exhibiting in some cases autotoxicity (Garnett et al 2004
Fernandez et al 2008 Zhu et al 2009 Monnier et al 2011) Studies
on potential dual allelopathic effects of Pinus elliottii Engelm (slash
httpdxdoiorg101016jindcrop201706019
Received 23 March 2017 Received in revised form 15 May 2017 Accepted 7 June 2017
Corresponding author
E-mail address fettnetocbiotufrgsbr (AG Fett-Neto)
ORIGINAL RESEARCHpublished 16 June 2016
doi 103389fpls201600849
Frontiers in Plant Science | wwwfrontiersinorg 1 June 2016 | Volume 7 | Article 849
Edited by
Juan Francisco Jimenez Bremont
Instituto Potosino de Investigacioacuten
Cientiacutefica y Tecnoloacutegica Mexico
Reviewed by
Mariacutea De La Luz Guerrero Gonzaacutelez
Universidad Autoacutenoma de San Luis
Potosiacute Mexico
Rosalia Cristina Paz
CIGEOBIO (CONICETFCEFN UNSJ)
Argentina
Correspondence
Arthur G Fett-Neto
fettnetocbiotufrgsbr
daggerThese authors have contributed
equally to this work
Specialty section
This article was submitted to
Plant Physiology
a section of the journal
Frontiers in Plant Science
Received 08 December 2015
Accepted 30 May 2016
Published 16 June 2016
Citation
de Lima JC de Costa F Fuumlller TN
Rodrigues-Correcirca KCdS Kerber MR
Lima MS Fett JP and Fett-Neto AG
(2016) Reference Genes for qPCR
Analysis in Resin-Tapped Adult Slash
Pine As a Tool to Address the
Molecular Basis of Commercial
Resinosis Front Plant Sci 7849
doi 103389fpls201600849
Reference Genes for qPCR Analysisin Resin-Tapped Adult Slash Pine Asa Tool to Address the MolecularBasis of Commercial Resinosis
Juacutelio C de Lima 1dagger Fernanda de Costa 1 dagger Thanise N Fuumlller 1
Kelly C da Silva Rodrigues-Correcirca 2 Magnus R Kerber 1 Mariano S Lima 1
Janette P Fett 1 and Arthur G Fett-Neto 1
1 Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul
Porto Alegre Brazil 2 Biological Sciences Department Regional Integrated University of Alto Uruguai and Missotildees (URI-FW)
Frederico Westphalen Brazil
Pine oleoresin is a major source of terpenes consisting of turpentine (mono- and
sesquiterpenes) and rosin (diterpenes) fractions Higher oleoresin yields are of economic
interest since oleoresin derivatives make up a valuable source of materials for chemical
industries Oleoresin can be extracted from living trees often by the bark streak method
in which bark removal is done periodically followed by application of stimulant paste
containing sulfuric acid and other chemicals on the freshly wounded exposed surface
To better understand the molecular basis of chemically-stimulated and wound induced
oleoresin production we evaluated the stability of 11 putative reference genes for the
purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis
Samples for RNA extraction were collected from field-grown adult trees under tapping
operations using stimulant pastes with different compositions and at various time points
after paste application Statistical methods established by geNorm NormFinder and
BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3
and UBI To confirm expression stability of the candidate reference genes expression
profiles of putative P elliottii orthologs of resin biosynthesis-related genes encoding Pinus
contorta β-pinene synthase [PcTPS-(minus)β-pin1] P contorta levopimaradieneabietadiene
synthase (PcLAS1) Pinus taeda α-pinene synthase [PtTPS-(+)αpin] and P taeda
α-farnesene synthase (PtαFS) were examined following stimulant paste application
Increased oleoresin yields observed in stimulated treatments using phytohormone-based
pastes were consistent with higher expression of pinene synthases Overall the
expression of all genes examined matched the expected profiles of oleoresin-related
transcript changes reported for previously examined conifers
Keywords resin Pinus gene expression normalizer genes terpene synthase
19
Chapter 2
Stimulant Paste Preparation and Bark Streak Tapping Technique for Pine Oleoresin Extraction
Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
Tapping technique comprises the extraction of pine oleoresin a non-wood forest product consisting of a
complex mixture of mono sesqui and diterpenes biosynthesized and exuded as a defense response to
wounding Oleoresin is used to produce gum rosin turpentine and their multiple derivatives Oleoresin
yield and quality are objects of interest in pine tree biotechnology both in terms of environmental and
genetic control Monitoring these parameters in individual trees grown in the fi eld provides a means to
examine the control of terpene production in resin canals as well as the identifi cation of genetic-based
differences in resinosis A typical method of tapping involves the removal of bark and application of a
chemical stimulant on the wounded area Here we describe the methods for preparing the resin-stimulant
paste with different adjuvants as well as the bark streaking process in adult pine trees
Key words Oleoresin Pine Tapping Chemical stimulant Wounding
1 Introduction
Several conifer species produce oleoresin a complex mixture of isoprenoid compounds relevant for defense against herbivores and pathogens Two major fractions can be recognized in oleoresin (a) turpentine the volatile fraction containing mono- and sesquiter-penes and (b) rosin the nonvolatile diterpene fraction Oleoresin is a forest commodity of global interest fi nding applications in diverse industry sectors Rosin is used in adhesives printing ink manufacture and paper sizing Turpentine can be used either as a solvent for paints and varnishes or as a raw material for fraction-ation of high-value chemicals used in the pharmaceutical agro-chemical and food industry [ 1 ndash 3 ]
During the extraction activity resin is obtained from the tree in a similar way as rubber tree tapping which generally involves the
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_2 copy Springer Science+Business Media New York 2016
These authors have equally contributed to this work
fettnetocbiotufrgsbr
27
Chapter 3
A Modifi ed Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines
Juacutelio Ceacutesar de Lima Thanise Nogueira Fuumlller Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes
through high-throughput sequencing technologies microarray and also northern blots RT-PCR and
RTqPCR Even though many specifi c protocols designed for plants with high content of secondary metab-
olites have been developed these are often expensive time consuming and not suitable for a wide range
of tissues Here we present a modifi cation of the method previously described using the commercially
available Concerttrade Plant RNA Reagent (Invitrogen) buffer for fi eld-grown adult pine trees with high
oleoresin content
Key words RNA Pines Concert plant RNA reagent Stem RNA extraction Oleoresin Conifers
1 Introduction
Several conifer species especially within the Pinaceae have tissues with high concentrations of phenolics terpenes and polysaccha-rides [ 1 ] Many specifi c protocols that are appropriate for plants rich in secondary metabolite s have been developed but the extrac-tion of high-quality RNA from these tissues using commercial kits is often diffi cult and usually not applicable to woody tissues [ 2 ndash 6 ] One of the major issues during RNA extraction concerns the pres-ence of phenolic compounds which oxidize and form quinones Aromatic compounds bind RNA which interferes in downstream steps and applications [ 3 7 ] Another point of concern is the har-vest of plant samples in the experimental fi eld which constitutes another obstacle in the efforts to avoid degradation of RNA [ 8 ] These problems often result in RNAs of low quality and insuffi -cient amounts especially for methodologies that normally require
These authors have equally contributed to this work
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_3 copy Springer Science+Business Media New York 2016
fettnetocbiotufrgsbr
RESEARCH PAPER
Control of resin production in Araucaria angustifolia an ancientSouth American coniferJ C Perotti1 K C da Silva Rodrigues-Correa123 amp A G Fett-Neto12
1 Plant Physiology Laboratory Department of Botany Federal University of Rio Grande do Sul (UFRGS) Porto Alegre RS Brazil
2 Center for Biotechnology UFRGS Porto Alegre RS Brazil
3 Present address Regional Integrated University of Alto Uruguai and Miss~oes (URI-FW) Frederico Westphalen RS Brazil
Keywords
Araucaria ethylene jasmonic acid nitric
oxide resin salicylic acid terpenes
Correspondence
A G Fett-Neto Plant Physiology Laboratory
Center for Biotechnology Federal University
of Rio Grande do Sul (UFRGS) PO Box 15005
Av Bento Goncalves 9500 91501-970 Porto
Alegre Brazil
E-mail fettnetocbiotufrgsbr
Editor
K Leiss
Received 22 July 2014 Accepted 11
December 2014
doi101111plb12298
ABSTRACT
Araucaria angustifolia is an ancient slow-growing conifer that characterises parts ofthe Southern Atlantic Forest biome currently listed as a critically endangered speciesThe species also produces bark resin although the factors controlling its resinosis arelargely unknown To better understand this defence-related process we examined theresin exudation response of A angustifolia upon treatment with well-known chemicalstimulators used in fast-growing conifers producing both bark and wood resin suchas Pinus elliottii The initial hypothesis was that A angustifolia would display signifi-cant differences in the regulation of resinosis The effect of Ethrel (ET ndash ethylene pre-cursor) salicylic acid (SA) jasmonic acid (JA) sulphuric acid (SuA) and sodiumnitroprusside (SNP ndash nitric oxide donor) on resin yield and composition in youngplants of A angustifolia was examined In at least one of the concentrations testedand frequently in more than one an aqueous glycerol solution applied on fresh woundsites of the stem with one or more of the adjuvants examined promoted an increase inresin yield as well as monoterpene concentration (a-pinene b-pinene camphene andlimonene) Higher yields and longer exudation periods were observed with JA and ETanother feature shared with Pinus resinosis The results suggest that resinosis controlis similar in Araucaria and Pinus In addition A angustifolia resin may be a relevantsource of valuable terpene chemicals whose production may be increased by usingstimulating pastes containing the identified adjuvants
INTRODUCTION
Many conifer species produce terpenoid-based resins that havelong been studied for their industrial importance and role indefence against attack by herbivores and pathogens The twomost important resin-producing families of conifers are Pina-ceae and Araucariaceae (Langenheim 1996) The viscous resinsecretion is generally composed of a complex mixture ofterpenoids consisting of roughly equal parts of volatile mono-(C10) and sesquiterpene (C15 turpentine) fractions and non-volatile diterpenic (C20 rosin) components (Rodrigues-Correaet al 2013) Terpenes act in a complex and multilayereddefence response providing toxicity against bark beetles andfungi bark wound sealing disruption of insect developmentand attraction of herbivore predators (Phillips amp Croteau1999)Most conifers rely on some combination of preformed and
inducible resin defences (Trapp amp Croteau 2001 Zulak amp Bohl-mann 2010) Resin defences are controlled by environmentaland genetic factors to various extents depending on species(Roberds et al 2003 Sampedro et al 2010 Moreira et al2013) Resin traits have been reported as highly variable havingmoderate heritability indicating that breeding efforts towardssuper-resinous forests are promising (Tadasse et al 2001Roberds et al 2003) Several chemicals are known as stimulantsof resin production Commercial extraction of resin from pine
trees uses periodic bark streaking and application of resin stim-ulant pastes to the wound
Resin-stimulant paste based on sulphuric acid (SuA) iswidely used for the commercial production of pine resin Cur-rent stimulant pastes usually have two chemically active com-ponents SuA to magnify the wounding and an ethyleneprecursor (2-chloroethylphosphonic acid CEPA or Ethrel ndash
ET) to stimulate resin flow (Rodrigues et al 2011 Rodrigues-Correa amp Fett-Neto 2013) Jasmonic acid (JA) and its methylester methyl jasmonate (MeJa) are among the most widelyused chemical elicitors of plant secondary metabolism It hasbeen shown that the exogenous application of MeJa or herbi-vore attack induce chemical and anatomical defence responsesin conifers such as the formation of traumatic resin ducts andresin accumulation in stems along with increased biosynthesisof terpenes and phenolics (Franceschi et al 2002 Martin et al2002 Heijari et al 2005 Zeneli et al 2006 Moreira et al 2008Gould et al 2009) JA commercial use however is limited byits high cost
The effects of exogenous salicylic acid (SA) on conifer ter-pene production have also been studied In Pinus elliottiiapplication of 10 molm3 of SA induced resin productionin wound panels but in Pseudotsuga menziesii and Sequoia-dendron giganteum it had no apparent effect on resinaccumulation (Hudgins amp Franceschi 2004 Rodrigues ampFett-Neto 2009) Nitric oxide (NO) has also emerged as an
Plant Biology 17 (2015) 852ndash859 copy 2014 German Botanical Society and The Royal Botanical Society of the Netherlands852
Plant Biology ISSN 1435-8603
iv
AGRADECIMENTOS
Ao meu orientador Dr Arthur Germano Fett-Neto uma das melhores pessoas
que tive a honra de conhecer na Academia Um coraccedilatildeo imenso uma mente incrivelmente
brilhante integridade e empatia infinitas (e extremamente raras no meio cientiacutefico)
Muito obrigada pela confianccedila em mim depositada e sobretudo por ter cometido a
insanidade de aceitar me orientar novamente Obrigada por ter me possibilitado ir em
termos cientiacuteficos muito aleacutem do que eu ousaria imaginar dadas as minhas (inuacutemeras)
limitaccedilotildees (e por todo ATP e NADPH investidos nesse esforccedilo hercuacuteleo que constitui a
aacuterdua tarefa de me orientar de forma natildeo condescendente a despeito dessas) Minha
diacutevida contigo seraacute eterna sou uma pessoa duplamente aquinhoada pela tua orientaccedilatildeo
Lucky me
Aos Professores Janette Palma-Fett uma grande amiga e saacutebia conselheira
sempre especialmente na adversidade e Felipe Maraschin pelo pronto e inestimaacutevel
apoio teacutecnico-cientiacutefico sempre que solicitado
Aos colegas do Laboratoacuterio de Fisiologia Vegetal da UFRGS pela parceria e
auxiacutelio em todas as horas por formarem um grupo coeso alinhado e comprometido com
o bem maior da pesquisa e do bom funcionamento do lab Eacute gratificante trabalhar com
todos vocecircs
Aos amigos muito queridos que a UFRGS me trouxe Ana Paula Durand
Coelho Eudes Stiehl-Alves Johnatan Vilasboa Yohanna Miotto e as divas Juliana
Troleis Sofia Aumond Kuhn e Tamara Pastori Muito obrigada por estarem presentes
nas horas menos faacuteceis e por me auxiliarem de muitas maneiras sempre que precisei Toda
dificuldade eacute redimensionada quando se tem amigos
Agrave minha famiacutelia caucasoide Ana Cristina Stein Camila Junkes Camila e
Cassiano Busatta Carlos Eduardo Blanco Linares Daniela Sponchiado Jordana
Griebler Luft Karen Santos Karina Letiacutecia Lopes e Larissa Schemes Heinzelmann
O carinho o apoio e o encorajamento que recebo de vocecircs fazem qualquer lsquofardorsquo parecer
mais leve Muito lsquomercirsquo
I am very grateful to Dr Dulal Borthakur for generously having received me in
his lab and his loving and caring family I would also like to thank my lab mates at UH
Manoa James Carillo Maia Corpuz and Ahmed Bageel for being so helpful cheerful
and friendly with me during all my stay in Honolulu Most of all Irsquod like to thank my
dear friend Michael Honda for teaching very patiently and supporting me inside and
outside the lab by doing whatever was in his power to prevent my homesickness I am
also very grateful to Mariana de Souza and Fernanda Oliveira for all those amazing
places and hikes wersquove been together in Orsquoahu You guys are awesome Mahalo nui loa
for your kōkua Im now parsquou hana
Jaimerais bien remercier mes collegravegues et amis agrave lrsquoUniversiteacute de Montreacuteal
(Benjamin Mazin Marion Kretsch Yang Liu Fang Wen Raquel Parada et Micaela
Margutti) pour mavoir chaleureusement reccedilu chez vous speacutecialement agrave mon ami
Valentin Joly pour mavoir beaucoup appris sur lrsquoinconnu monde des bacteacuteries et des
v
levures (et surtout pour leur incroyable patience avec mon tregraves mauvais franccedilais) Crsquoeacutetait
vachement chouette Merci beaucoup agrave vous tous (et toutes) et agrave la prochaine
Agrave Coordenaccedilatildeo de Aperfeiccediloamento Pessoal de Niacutevel Superior (CAPES) pelo
financiamento da bolsa de pesquisa do PDSE
Aos meus pais (bioloacutegicos ou natildeo) Veacutera Maria da Silva Rodrigues Gilberto
Moraes Rodrigues Rosa Maria Lucas da Silva e Paulo Joseacute Costa da Silva pelo
exemplo de honestidade coragem trabalho forccedila e amor desde sempre
Aos meus irmatildeos Ana Paula da Silva Rodrigues Viniacutecius de Moraes da Silva
Rodrigues Marcello da Silva Rodrigues e Camila Stella Toledo Pereira por todas as
experiecircncias que dividimos e tudo o que me ensinaram ateacute hoje
Ao meu amor maior minha melhor amiga minha mais leal e extraordinaacuteria
parceria nessa grande (e agraves vezes tortuosa) jornada Maria Clara Rodrigues Correcirca Por
ser ela por ser imensa em generosidade amor e altruiacutesmo por despertar o melhor em
mim por ser minha forccedila motriz e sobretudo por ser a melhor das minhas metades
Minha vida soacute realmente comeccedilou quando eu tive a incriacutevel sorte de te conhecer
vi
SUMAacuteRIO
LISTA DE ABREVIATURASvii
RESUMO ix
INTRODUCcedilAtildeO GERAL1
HIPOacuteTESE E OBJETIVOS9
CAPIacuteTULO 1 Abiotic stresses and non-protein amino acids in plantshelliphellip10
CAPIacuteTULO 2 Mimosine accumulation in Leucaena leucocephala in response to
stress signaling molecules and acute UV exposurehelliphelliphelliphelliphelliphelliphelliphelliphelliphellip(432) 52
CAPIacuteTULO 3 Mimosine occurrence and accumulation in Mimosa bimucronata var
bimucronata (DC) Kuntze66
CONSIDERACcedilOtildeES FINAIS 84
PERSPECTIVAS85
REFEREcircNCIAS BIBLIOGRAacuteFICAS86
Artigos publicados no periacuteodo de doutoramento natildeo relacionados ao tema da
tese91
vii
LISTA DE ABREVIATURAS
24-D 24-dichlorophenoxyacetic acid
3H4P 3-hydroxy-4-pyridone (34-DHP 34-dihydroxypyridine)
ABA abscisic acid
Arg arginine
BABA β-aminobutyric acid
β-ODAP β-N-oxalyl-L-α β-diaminopropionic acid
BIA β-isoxazolinon-L-alanine
CAN canavanine
DAO diamine oxidase
DDC decarboxylase
ETH ethephon
FW fresh weight
GABA -aminobutyric acid
GABA-T GABA transaminase
GAD glutamate decarboxylase
GSM Global System for Mobile
HPLC High performance liquid chromatography
JA jasmonate
JA-Ile jasmonoyl-L-isoleucine
L-DOPA L-34- dihydroxyphenylalanine
MeJA methyl jasmonate
m-Tyr Meta-tyrosine
NO nitric oxide
NPAA non-protein amino acid
OAS o-acetylserine
OAS-TL o-acetylserine-thiol-lyase
PA polyamine
PAA protein amino acid
viii
PEG polyethylene glycol
PLP pyridoxal-5rsquo-phosphate
PPO polyphenol oxidase tyrosinase
qRT-PCR Reverse transcription polymerase chain reaction quantitative real time
RNS reactive nitrogen species
ROS reactive oxygen species
SA salicylic acid
SAR systemic acquired resistance
SNP sodium nitroprusside
UV ultraviolet radiation
ix
RESUMO
Ao longo de sua evoluccedilatildeo as plantas desenvolveram estrateacutegias estruturais e quiacutemicas de
defesa em resposta aos estresses bioacuteticos e abioacuteticos impostos pelo ambiente Dentre
essas satildeo reconhecidas moleacuteculas quimicamente especializadas denominadas
metaboacutelitos secundaacuterios produtos naturais ou metaboacutelitos especializados Aminoaacutecidos
natildeo proteicos (ANPs) satildeo compostos nitrogenados que constituem aleacutem de componentes
do arsenal de defesa quiacutemica vegetal uma importante fonte de reserva de carbono e
nitrogecircnio para diversos taxa especialmente aqueles pertencentes agrave famiacutelia Fabaceae de
Angiospermas Esse grupo de moleacuteculas quimicamente heterogecircneo eacute assim definido por
natildeo participar da formaccedilatildeo de estruturas proteicas funcionais sendo frequentemente
toacutexicos quando erroneamente incorporados nas cadeias polipeptiacutedicas em formaccedilatildeo em
funccedilatildeo da similaridade estrutural que apresentam com os aminoaacutecidos proteicos Sob o
ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados ANPs satildeo
em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou abioacutetica como
o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de elicitores
quiacutemicos por exemplo O objetivo da presente tese foi investigar o papel bioloacutegico da
mimosina endoacutegena em Leucaena leucocephala (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute) a partir da avaliaccedilatildeo do efeito de tratamentos
relacionados ao estresse abioacutetico (UV-C aacutecido saliciacutelico metil jasmonato e etileno)
Mimosina eacute um ANP aromaacutetico anaacutelogo da L-tirosina com atividade toacutexica para ceacutelulas
de procariotos e eucariotos Dentre as atividades descritas para esse ANP destacam-se a
atividade anti-mitoacutetica ou bloqueadora do ciclo celular atividade alelopaacutetica e
antioxidante Os resultados indicaram que em leucena a biossiacutentese e o acuacutemulo de
mimosina podem ser modulados por fatores causadores de estresses exibindo um padratildeo
de acumulaccedilatildeo similar ao das fitoalexinas Em maricaacute por outro lado a induccedilatildeo do
acuacutemulo dessa moleacutecula natildeo foi observada para os mesmos tratamentos testados para
leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas Aleacutem disso
o padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado
pela sua degradaccedilatildeo As respostas observadas nos testes que avaliaram a atividade de
mitigaccedilatildeo de espeacutecies reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode
agir como um agente antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de
estresse
1
Introduccedilatildeo
Na condiccedilatildeo de organismos seacutesseis ao longo de sua evoluccedilatildeo as plantas
desenvolveram estrateacutegias estruturais e quiacutemicas de defesa em resposta aos estresses bioacuteticos
e abioacuteticos impostos pelo ambiente Dentre essas satildeo reconhecidas moleacuteculas quimicamente
especializadas denominadas metaboacutelitos secundaacuterios produtos naturais (Kutchan et al 2015)
ou mais recentemente metaboacutelitos especializados
Entre as trecircs classes mais gerais de metaboacutelitos secundaacuterios (terpenos compostos
fenoacutelicos e compostos nitrogenados) aminoaacutecidos natildeo-proteicos (ANPs) satildeo incluiacutedos no
terceiro grupo e constituem aleacutem de componentes do arsenal de defesa quiacutemica uma
importante fonte de reserva de carbono e nitrogecircnio para diversos taxa especialmente aqueles
pertencentes agrave famiacutelia Fabaceae de Angiospermas (leguminosas sensu lato)
Aleacutem dos 20 aminoaacutecidos proteicos estima-se que existam entre 600 e 1000 ANPs
(Acamovic amp Brooker 2005 Rodgers et al 2015) Esse grupo de moleacuteculas quimicamente
heterogecircneo eacute assim definido por natildeo participar da formaccedilatildeo de estruturas proteicas
funcionais sendo frequentemente toacutexicos quando erroneamente incorporados nas cadeias
polipeptiacutedicas em formaccedilatildeo em funccedilatildeo da similaridade estrutural que apresentam com os
aminoaacutecidos proteicos (Taiz amp Zeiger 2010)
Conforme mencionado a ocorrecircncia de ANPs eacute comum em espeacutecies de leguminosas
e sua distribuiccedilatildeo pode ser restrita a alguns gecircneros de plantas circunscritos nessa famiacutelia
botacircnica (eg mimosina e canavanina) Por outro lado alguns ANPs como GABA por
exemplo podem apresentar distribuiccedilatildeo ubiacutequa no Reino Plantae assim como ocorrer em
outros tipos de organismos como animais por exemplo (Ramos-Ruiz et al 2018)
2
Apesar de representarem uma fonte nutricional importante sem tratamento preacutevio o
consumo de plantas que acumulam ANPs por animais eacute limitado Isso ocorre pois em longo
prazo a ingestatildeo prolongada de plantas (especialmente sementes) que acumulam ANPs pode
representar risco agrave sauacutede uma vez que estes comprometem o funcionamento de mecanismos
basais de manutenccedilatildeo da homeostase celular e podem tambeacutem em um quadro mais severo
desencadear doenccedilas neurotoacutexicas degenerativas como por exemplo o latirismo causado
por aacutecido β-N-oxalil-l-αβ-diaminopropiocircnico (β-ODAP) (Jiao et al 2011 Kusama-Eguchi
2019)
Sob o ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados
ANPs satildeo em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou
abioacutetica como o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de
elicitores quiacutemicos por exemplo No que concerne ao estudo dos efeitos da induccedilatildeo abioacutetica
sobre o acuacutemulo de ANPs em diferentes espeacutecies vegetais (Monocotiledocircneas e
Eudicotiledocircneas) as moleacuteculas mais amplamente investigadas ateacute o momento satildeo GABA
L-DOPA e mais recentemente mimosina (vide Tabela 1 do capiacutetulo primeiro) Em termos
de efeitos causados a partir da aplicaccedilatildeo exoacutegena de ANPs GABA tambeacutem figura como o
principal aminoaacutecido investigado seguido de L-DOPA e canavanina (vide Tabela 2 do
capiacutetulo primeiro)
No primeiro capiacutetulo da presente tese estatildeo descritas as caracteriacutesticas gerais dos
principais ANPs estudados seus possiacuteveis papeacuteis bioloacutegicos in planta e seus efeitos quando
aplicados exogenamente bem como os estresses abioacuteticos capazes de induzir seu(s)
acuacutemulo(s) nos diferentes tecidos vegetais Nos segundo e terceiro capiacutetulos
respectivamente satildeo elucidados os efeitos dos tratamentos de UV-C aacutecido saliciacutelico etileno
e jasmonato (claacutessicos elicitores do metabolismo secundaacuterio vegetal) sobre o acuacutemulo de
3
mimosina em Leucaena leucocephala var glabrata (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute)
Mimosina eacute um aminoaacutecido aromaacutetico natildeo-proteico anaacutelogo da L-tirosina com
atividade toacutexica para ceacutelulas de procariotos e eucariotos Embora em menor concentraccedilatildeo
mimosina foi primeiramente identificada em Mimosa pudica sendo posteriormente detectada
em outras espeacutecies do gecircnero como Mimosa pigra por exemplo (Soedarjo amp Borthakur
1998) Seu efeito toacutexico eacute atribuiacutedo agrave capacidade de quelar metais o que impede o
funcionamento adequado das metalo-proteiacutenas que dependem dos mesmos como co-fatores
(Negi et al 2014)
A concentraccedilatildeo basal de mimosina em espeacutecies de leucaena pode variar entre 1 e 12
do peso seco do oacutergatildeo (Soedarjo amp Borthakur 1998) Como eacute comum para outros ANPs
que ocorrem em espeacutecies de leguminosas em sementes de Leucaena spp eacute observada uma
maior concentraccedilatildeo de mimosina quando comparada aos demais oacutergatildeos da planta
(Rodrigues-Correcirca et al 2019) sendo esta a fonte de extraccedilatildeo comercial do padratildeo quiacutemico
de mimosina vendido por empresas de reagentes de pesquisa
Diversas atividades foram descritas para mimosina em outros organismos eou tipos
celulares Dentre essas destacam-se a atividade anti-mitoacutetica ou bloqueadora do ciclo
celular em ceacutelulas de eucariotos e procariotos Isto ocorre porque a mimosina impede a
formaccedilatildeo da forquilha de replicaccedilatildeo (e portanto a siacutentese de DNA) interrompendo assim o
avanccedilo do ciclo de divisatildeo celular na fase tardia G1 (Lalande 1990) Foram tambeacutem descritas
para mimosina atividade alelopaacutetica observada sobre o desenvolvimento de outras espeacutecies
de leguminosas e atividade antioxidante entre outras (Tabela 1)
A rota de biossiacutentese de mimosina eacute compartilhada em grande parte com a de cisteiacutena
um aminoaacutecido proteico sulfurado (Figura 1) A siacutentese da cisteiacutena se daacute a partir da conversatildeo
4
de serina e acetil-CoA em o-acetilserina pela enzima SAT (serina acetiltransferase) seguida
da conversatildeo de o-acetilserina e aacutecido sulfiacutedrico em cisteiacutena em uma reaccedilatildeo catalisada pela
OAS-TL (o-acetilserina tiol-liase) A siacutentese de mimosina por sua vez eacute compartilhada com
a da cisteiacutena ateacute esse ponto e acredita-se que pelo menos uma das isoformas de OAS-TL
catalise a conversatildeo de o-acetilserina e 3-hidroxi-4-piridona em mimosina
Tabela 1 Atividades descritas para mimosina de Leucaena leucocephala (Lam) de Wit
ATIVIDADE
ALVO AVALIADO
(organismo eou tecido tipo
celular)
REFEREcircNCIA
Bloqueio do complexo de ativaccedilatildeo
da preacute-replicaccedilatildeo do DNA
Ceacutelulas de mamiacuteferos
KUBOTA et al
(2014)
Alteraccedilatildeo no ciclo ovariano e
extensatildeo da duraccedilatildeo do corpo luacuteteo
bovino no periacuteodo poacutes-parto
Bovinos
(Bos taurus x
Bos indicus)
BOTTINI-
LUZARDO et al
(2015)
Supressatildeo do ciclo celular e reduccedilatildeo
da abundacircncia bacteriana em
mosquitos
Wolbachia pipientis
Aedes albopictus
FALLON
(2015)
Accedilatildeo inibitoacuteria da fibrose
pulmonar induzida
Ratos SD
LI et al
(2015)
Recuperaccedilatildeo da funccedilatildeo do
miocaacuterdio poacutes-isquemia
Miocaacuterdio de ratos (SD)
machos
CROWE et al
(2001)
Inseticida
Heteropsylla cubana
Crawford 1914 e Thrips tabaci
Lindemann 1889
AHMED et al
(2016)
Alelopaacutetica
Albizia procera Vigna
unguiculata Cicer arietinum
Cajanus cajan
AHMED et al
(2008)
Antioxidante
Sistemas modelo de oxidaccedilatildeo
lipiacutedica (β-caroteno - aacutecido
linolecircico e lecitina)
BENJAKUL et al
(2013)
Ateacute momento versotildees divergentes sobre a enzima responsaacutevel pela biossiacutentese de
mimosina (mimosina sintase) tecircm sido publicadas Em 1990 Ikegami e colaboradores
5
identificaram uma OAS-TL responsaacutevel pela formaccedilatildeo de cisteiacutena como sendo tambeacutem uma
mimosina sintase Mais tarde Yafuso et al (2014) realizaram a expressatildeo heteroacuteloga do gene
que codifica para OAS-TL em Escherichia coli e natildeo foi observada a formaccedilatildeo de mimosina
mesmo quando dadas as condiccedilotildees oacutetimas para tanto Mais recentemente Harun-Ur-Rashid
et al (2018) elucidaram a mimosina sintase como sendo uma isoforma da OAS-TL
corroborando o postulado por Ikegami e colaboradores em 1990
Figura 1 Rota de biossiacutentese da mimosina Fonte Ikegami et al (1990)
Espeacutecies estudadas
Leucaena leucocephala (Lam) de Wit (leucaena koa haole ou ldquoacaacutecia exoacuteticardquo na
liacutengua Hawairsquoiana) eacute uma espeacutecie de haacutebito arboacutereo ou arbustivo pertencente agrave famiacutelia
Fabaceae de Angiospermas e caracterizada pelo acuacutemulo de mimosina em todos os seus
oacutergatildeos Eacute nativa da Ameacuterica Central (especificamente da regiatildeo sudeste do Meacutexico) mas
irradiou-se atraveacutes de praticamente todas as zonas tropicais e subtropicais da Terra No
Brasil leucena eacute amplamente distribuiacuteda e classificada como naturalizada pelo REFLORA
(2019) ocorrendo em todo territoacuterio Nacional Satildeo reconhecidas no miacutenimo duas
6
subespeacutecies de leucena ocorrentes no Brasil L leucocephala var leucocephala e L
leucocephala var glabrata sendo a primeira a mais abundante
Leucaena apresenta atributos morfoloacutegicos caracteriacutesticos das leguminosas como o
fruto do tipo vagem deiscente no periacuteodo poacutes-maturaccedilatildeo folhas compostas e bipinadas As
flores satildeo seacutesseis actinomorfas e polistecircmones apresentam caacutelice sinseacutepala e corola
gamopeacutetala e satildeo dispostas em inflorescecircncias do tipo glomeacuterulo (Figura 2)
Figura 2 Oacutergatildeos vegetativos e reprodutivos de L leucocephala (Lam) de Wit Fonte Little Jr amp Skolmen
(1989)
Com base no conhecimento etnobotacircnico disponiacutevel acerca dessa espeacutecie em
diversas regiotildees tropicais e subtropicais leucena eacute utilizada para vaacuterios fins Extratos de
diferentes oacutergatildeos de leucena apresentam atividade anti-diabeacutetica (Kuppusamy et al 2014
Chowtivannakul et al 2016) antioxidante (Mohammed et al 2015 Chowtivannakul et al
2016 Zarin et al 2016) antimicrobiana (Zarin et al 2016) anti-helmiacutentica (Soares et al
2015 Jamous et al 2017) bactericida (Mohammed et al 2015) acaricida (Fernaacutendez-Salas
et al 2011) anti-tumoral (Chung et al 2017) e potencializadora da resposta imune em
peixes (Verma et al 2018) entre outras
7
Leucaena apresenta alta toleracircncia agrave seca sendo capaz de enfrentar estaccedilotildees sazonais
inteiras com deacuteficit hiacutedrico sem prejuiacutezo permanente de seus oacutergatildeos e de recuperar
vigorosamente sua biomassa vegetativa tatildeo logo o regime de precipitaccedilatildeo retome a
regularidade em frequecircncia Acredita-se que a toleracircncia agrave seca apresentada por essa espeacutecie
ocorra em funccedilatildeo do acuacutemulo de mimosina nos diferentes tecidos da planta a qual
funcionaria como um agente osmoregulador responsaacutevel pela preservaccedilatildeo da integridade das
membranas a das macromoleacuteculas intracelulares em periacuteodos de escassez de aacutegua no
ambiente
Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) eacute uma leguminosa
nativa natildeo endecircmica do Brasil amplamente distribuiacuteda nos domiacutenios fitogeograacuteficos da
Caatinga do Cerrado e da Mata Atlacircntica (Simon amp Proenccedila 2000 REFLORA 2019) Como
espeacutecie pioneira (Pilatti et al 2019) exerce importante papel ecoloacutegico na recuperaccedilatildeo de
aacutereas degradadas (Bitencourt et al 2007 Silva et al 2011) no estabelecimento de processos
de sucessatildeo vegetacional
Maricaacute eacute uma espeacutecie semi-deciacutedua a deciacutedua a qual atinge ateacute 15 m em altura (e
diacircmetro agrave altura do peito de ateacute 40 cm) na idade adulta com haacutebito arboacutereo ou arbustivo
(REFLORA 2019) e espinhos caracteriacutesticos desde os estaacutegios iniciais de desenvolvimento
(Carvalho 2004) Apresenta folhas compostas alternas e bipinadas (Figura 2) amplas
inflorescecircncias brancas com flores reunidas em glomeacuterulos esfeacutericos dispostos em grandes
paniacuteculas As flores satildeo diplostecircmones actinomorfas hipoacuteginas e unicarpelares (Silva et al
2011)
Assim como descrito para leucena maricaacute eacute considerado uma espeacutecie multifuncional
sendo comumente empregada para produccedilatildeo de mel como combustiacutevel (Olkoski amp
8
Wittmann 2011) em edificaccedilotildees na carpintaria e como lsquocerca-vivarsquo (Marchiori 1993
Lorenzi 1998) entre outras aplicaccedilotildees
Figura 2 Folhas e fruto de Mimosa bimucronata (DC) Kuntze Fonte Souza-Lima et al (2017)
Em contraste com a amplitude de habitats explorados por leucena (especialmente os
aacuteridos) no Sul do Brasil maricaacute ocorre preferencialmente em ambientes uacutemidos a alagadiccedilos
em aacutereas proacuteximas agraves margens de rios (Patreze amp Cordeiro 2004) embora possa tambeacutem
ocorrer em formaccedilotildees quase exclusivas dessa espeacutecie nas encostas de morros (Jacobi amp
Ferreira 1991)
Em relaccedilatildeo agraves atividades elucidadas para os extratos de maricaacute foram relatados os
efeitos alelopaacutetico (Jacobi amp Ferreira 1991 Ferreira et al 1992) diureacutetico natriureacutetico e
caliureacutetico (Schlickmann et al 2017)
9
Hipoacutetese
Mimosina apresenta perfil dinacircmico de acuacutemulo em Leucaena leucocephala e
Mimosa bimucronata frente a estresses associado a alteraccedilotildees significativas na expressatildeo de
genes relacionados ao metabolismo deste ANP o qual contribui para mitigar o desequiliacutebrio
oxidativo inerente a vaacuterios tipos de estresse
Objetivo geral
O objetivo da presente tese foi investigar o papel bioloacutegico da mimosina endoacutegena
em leucena e maricaacute a partir da avaliaccedilatildeo do efeito de tratamentos relacionados a estresses
ou sinalizadores de estresse
Objetivos especiacuteficos
- Analisar a concentraccedilatildeo constitutiva de mimosina nos diferentes oacutergatildeos de L leucocephala
(Lam) de Wit (leucena) e M bimucronata (DC) Kuntze (maricaacute)
- Verificar se apesar do seu alto teor constitutivo em plantas de leucena o acuacutemulo de
mimosina pode ser induzido com tratamentos que mimetizam diferentes estresses a partir da
avaliaccedilatildeo do efeito de moleacuteculas sinalizadoras (aacutecido saliciacutelico jasmonato etileno) e da
exposiccedilatildeo agrave radiaccedilatildeo UV-C na modulaccedilatildeo do acuacutemulo de mimosina em leucena bem como
em maricaacute
- Determinar se a expressatildeo de genes relacionados ao metabolismo de mimosina estaacute
associada agrave induccedilatildeo por estresses fisioloacutegicos
- Avaliar o potencial antioxidante da mimosina em experimentos realizados in situ
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry
journal homepage wwwelseviercomlocateplaphy
Research article
Mimosine accumulation in Leucaena leucocephala in response to stresssignaling molecules and acute UV exposure
Kelly Cristine da Silva Rodrigues-Correcircaab Michael DH Hondab Dulal BorthakurbArthur Germano Fett-Netoalowast
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970Porto Alegre Rio Grande do Sul BrazilbDepartment of Molecular Biosciences and Bioengineering University of Hawaii at Manoa Honolulu HI 96822 USA
A R T I C L E I N F O
KeywordsLeucaena leucocephalaMimosineMimosine amidohydrolaseJasmonic acidEthyleneSalicylic acidUV-C radiation
A B S T R A C T
Mimosine is a non-protein amino acid of Fabaceae such as Leucaena spp and Mimosa spp Several relevantbiological activities have been described for this molecule including cell cycle blocker anticancer antifungalantimicrobial herbivore deterrent and allelopathic activities raising increased economic interest in its pro-duction In addition information on mimosine dynamics in planta remains limited In order to address this topicand propose strategies to increase mimosine production aiming at economic uses the effects of several stress-related elicitors of secondary metabolism and UV acute exposure were examined on mimosine accumulation ingrowth room-cultivated seedlings of Leucaena leucocephala spp glabrata Mimosine concentration was not sig-nificantly affected by 10 ppm salicylic acid (SA) treatment but increased in roots and shoots of seedlings treatedwith 84 ppm jasmonic acid (JA) and 10 ppm Ethephon (an ethylene-releasing compound) and in shoots treatedwith UV-C radiation Quantification of mimosine amidohydrolase (mimosinase) gene expression showed thatethephon yielded variable effect over time whereas JA and UV-C did not show significant impact Consideringthe strong induction of mimosine accumulation by acute UV-C exposure additional in situ ROS localization aswell as in vitro antioxidant assays were performed suggesting that akin to several secondary metabolitesmimosine may be involved in general oxidative stress modulation acting as a hydrogen peroxide and superoxideanion quencher
1 Introduction
Different plant groups synthesize a large diversity of secondary orspecialized metabolites These molecules are generally produced inresponse to biotic and abiotic environmental stresses Indeed inductionof secondary metabolism usually involves stress-generating factorswhich have also been explored in biotechnological processes aiming atthe production of target metabolites of economic interest (Matsuuraet al 2018) Metabolic control of nitrogen-containing secondarycompounds (eg alkaloids and non-protein amino acids) has beenshown to be complex and influenced by phytohormones environmentalstresses (seasonality herbivory pathogen attack drought) UV radia-tion (Holloacutesy 2002) methyl jasmonate (MeJA) salicylic acid (SA)yeast extract (Cho et al 2008) abscisic acid (ABA) heavy metals os-motic stress (Nascimento et al 2013) and mechanical wounding (Portoet al 2014)
Due to their particular trait of associating with N-fixing micro-organisms Fabaceae species (leguminous sensu lato) are often proteinrich hence the relevance of several of these species as forage Fabaceaespecies are also known for accumulating nitrogen containing secondarymetabolites which play important roles as ecochemical molecules andat least for the case of non-protein amino acids potential cell reservoirsof nitrogen (Huang et al 2011)
High contents of mimosine a toxic aromatic non-protein aminoacid are found in species of two leguminous genera Leucaena spp andMimosa spp Leucaena leucocephala (Lam) de Wit (leucaena koa haole)is a fast-growing leguminous tree native from Central America (south-eastern Mexico) widely distributed in tropical and subtropical zonesThis species is also characterized by its high tolerance to droughtamong other environmental stresses (Honda et al 2018) Leucaena canbe divided into two subspecies (i) L leucocephala subsp leucocephala(common leucaena a bushy shrub) and (ii) L leucocephala subsp
httpsdoiorg101016jplaphy201811018Received 1 August 2018 Received in revised form 9 November 2018 Accepted 14 November 2018
lowast Corresponding authorE-mail addresses krodriguescbiotufrgsbr (KCdS Rodrigues-Correcirca) mhonda2hawaiiedu (MDH Honda) dulalhawaiiedu (D Borthakur)
fettnetocbiotufrgsbr (AG Fett-Neto)
Plant Physiology and Biochemistry 135 (2019) 432ndash440
Available online 19 November 20180981-9428 copy 2018 Elsevier Masson SAS All rights reserved
T
glabrata (giant leucaena a tree) The latter has been used as a fastgrowing tree for production of wood and paper pulp The foliage ofboth common and giant leucaena is used as a fodder because of its highprotein content and palatability to farm animals The foliage containsup to 18 protein 142 crude fiber and 64 ether extractcrude fat(Soedarjo and Borthakur 1996)
Production of nitrogen-containing secondary metabolites such asmimosine requires large amounts of carbon and nitrogen resourcesNegi et al (2014) estimated that up to 21 of the carbon-nitrogenresources may be used for production of mimosine in leucaenaBrewbaker et al (1972) determined the mimosine content of 96 Lleucocephala cultivars and 8 other Leucaena species collected from 38different countries by growing them in an observational nursery inHawaii and found that basal mimosine content varied from 189 to477 of the dry weight
Mimosine is biosynthesized from OAS (o-acetylserine) and 3H4P (3-hydroxy-4-pyridone or its tautoisomer 3-hydroxy-4-pyridine) A pre-vious analysis suggested that mimosine synthase is an OAS-TL (o-acetylserine-thiol-lyase) of the cysteine biosynthesis pathway (Ikegamiet al 1990) Later however recombinant enzyme tests did not supportan OAS-TL identity of mimosine synthase (Yafuso et al 2014) Recentfindings on mimosine biosynthesis revealed that a cytosolic cysteine-OAS-TL isoform can also catalyze the formation of mimosine underspecific conditions (Harun-Ur-Rashid et al 2018)
Mimosine toxicity is related to its ability of reducing the availabilityof divalent metal ions such as Fe(II) Zn(II) Cu(II) Co(II) and Mn(II)by chelating co-factors and preventing their association with metal-dependent enzymes Furthermore this non-protein amino acid is cap-able of forming a stable complex with pyridoxal-5prime-phosphate (PLP)leading to the inactivation of PLP-dependent enzymes (eg Asp-Glutransaminase and cystathionine synthetase) (Negi et al 2014)
Mimosine features several useful biological activities such as alle-lopathic antimicrobial insecticide cell cycle inhibitor agent antic-ancer phytoremediator (Nguyen and Tawata 2016) as well as anti-oxidant (Benjakul et al 2013) Despite the relatively well establishedbiological activities of purified mimosine on other organisms or celltypes little is known about its biological role in leguminous speciesHowever it has been suggested that at least in part its activity ismainly related to defense mechanisms against some biotic and abioticstresses and as nitrogen source during fast growth (Vestena et al2001)
Suda (1960) and Smith and Fowden (1966) identified enzymes in-volved in mimosine degradation in seedling extracts of L leucocephalaand Mimosa pudica A mimosine-degrading enzyme named mimosinase(mimosine amidohydrolase EC 35161 CAS registry number 104118-49-2) (IUBMB 2018) a carbon-nitrogen lyase which degrades mimo-sine into 3H4P was later purified by Tangendjaja et al (1986) Itsbiochemical characterization was described and the cDNA was isolatedby Negi et al (2014)
Although mimosinase has been described and isolated only fewstudies on the role played by biotic and abiotic factors on the dynamicmodulation of mimosine metabolism in leguminous species have beenconducted (Vestena et al 2001 Xu et al 2018) In aseptic cultures ofleucaena mechanical injury of shoots promoted local mimosine accu-mulation (Vestena et al 2001) In the same study cultivation in pre-sence of auxin or SA in culture medium also had a positive effect on
mimosine accumulation More recently the effect of drought treatmenton gene expression of leucaena was also evaluated by Honda et al(2018) However several potential factors regulating mimosine meta-bolism need to be further examined
To date there is a lack of information on the biological role ofmimosine in planta as well as on details of its metabolic dynamicsMoreover its overt potential for pharmaceutical applications and de-velopment of new drugs as well as the possible use as tool to addressheavy metal soil contamination or plant mineral nutrition improve-ment justify additional research The objective of this study was toinvestigate the effect of stress signaling molecules and acute UV ex-posure on modulation of mimosine accumulation and metabolism in Lleucocephala spp glabrata in order to better understand its biologicalrole and to identify strategies for yield improvement aiming at ex-ploring its useful bioactivities
2 Methods
21 Plant material
For the experiments carried out to evaluate the effects of elicitors onmimosine accumulation seeds of leucaena were kindly provided by DrJames Brewbaker and harvested at CTAHRs (College of TropicalAgriculture and Human Resources of the University of Hawaii atManoa) Waimanalo Research Station at Oahu Hawaii This plantmaterial was originated from the accession K636 of Leucaena leucoce-phala ssp glabrata (Brewbaker 2008)
22 Induced mimosine content in 5-week-old giant leucaena
221 Seed germinationIn order to overcome seed coat dormancy seeds were submitted to a
chemical scarification with sulfuric acid 95ndash98 for 20min and re-peatedly rinsed in distilled water to remove any residual trace of thisreagent Then seeds were distributed in 254 cmtimes508 cm plastictrays containing 11 vv of vermiculite and commercial soil watereduntil reaching substrate field capacity Three weeks after seed imbibi-tion seedlings displaying similar size and shape (eg number of com-pound leaves and leaflets) were transplanted to individual pots(250mL) in number of three plants per container
During the experimental period (except in the UV-C radiationtreatment) all tested seedlings were kept in a growth chamber andsubmitted to controlled conditions of temperature (circa 25 degC) and ir-radiance (approximately 100 μmol photons mminus2sdot s minus1) with a photo-period of 16 h light and 8 h dark
222 Treatments2221 JA Ethephon and SA Five-week-old giant leucaena seedlingswere treated with different solutions as described in Table 1 Idealconcentrations were defined in preliminary experiments under the sameconditions indicated above At the beginning of the experiments 30plants were sprayed with 84 ppm JA 10 ppm SA 10 or 100 ppmEthephon or Milli-Qreg water (control) until the point of imminent runoffPlant pots were kept closed inside transparent plastic bags for 24 h toavoid solution volatilization Fifteen plants arranged in 5 sets of 3 (5biological replicates) were harvested 48 h and 96 h after being treated
Table 1Treatments used to modulate mimosine biosynthesis in giant leucaena
ELICITOR CONCENTRATION UV FLUENCE EXPOSURE TIME RATIONALE FOR USE
Salicylic acid (SA) 10 ppm 24 h Pathogen signaling molecule (Shah 2003)Jasmonic acid (JA) 84 ppm 24 h Chemical elicitor of plant secondary metabolism (Dar et al 2015)Ethephon 10 ppm 24 h Ethylene releasing-compound (Kim et al 2016) elicitor of plant secondary metabolism (Wang
et al 2016)UV-C radiation 3 Jcmminus2 10min or 15min Elicitor of plant secondary metabolism (Kara 2013 Neelamegam and Sutha 2015)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
433
After collection shoots were separated from roots immediately frozenin liquid nitrogen and stored at ndash 80 degC prior to HPLC analyses
2222 UV-C Thirty seedlings of giant leucaena were exposed to UV-Cradiation (3 Jcmminus2) for 10 or 15min and kept in a growth chamberunder controlled conditions as described above until the end of theexperiments Fifteen plants arranged in groups of 3 were harvested at96 h and 120 h after UV-C exposure and processed as previouslydescribed
223 Mimosine extractionMimosine extraction was based on a modified version of the pro-
tocol published by Lalitha and Kulothungan (2006) as follows a knownweight of fresh tissue (shoots or roots) of giant leucaena was first addedto Milli-Qreg boiling water in a proportion of 110 (g of plant per mL ofsolvent) in test tubes Tubes were covered with foil to avoid solutionevaporation and placed on a hot stirrer at 100 degC for 10min A pro-portional volume of 01M HCl was added to the cooled suspensions andhomogenized using mortar and pestle The plant extracts were filteredthrough cotton and centrifuged twice for 7min in a bench top re-frigerated centrifuge at 4 degC and 13200 rpm Before being analyzed theextracts were diluted 13 with ondashphosphoric acid (OPA)
224 Mimosine detectionHPLC analyses were carried out as described by Negi and Borthakur
(2016) Pure mimosine (L-mimosine from koa haole seeds Sigma-Al-drich CAS number 500-44-7) was used as standard Separation andquantification of mimosine was done with a C18 column (PhenomenexC18 5 μm 46times250mm) under an isocratic solvent system of 002MOPA with a linear flow rate of 1mLsdotminminus1 Mimosine detection wasdone at 280 nm by photodiode array detection (200ndash400 nm) showingretention time of 277 plusmn 0042min Quantification was done using themethod of external standard curve Further confirmation of mimosineidentity was performed by co-chromatography with standard and peakpurity check Chromatograms were analyzed using the Waters Em-power 3 software
23 Quantitative real-time PCR analysis of mimosinase gene expression
Fifteen 8-week-old giant leucaena plants arranged in 4 sets of 3 (4biological replicates) were treated with either water (control) or10 ppm Ethephon 84 ppm JA acid or 15min of UV-C radiation ex-posure following the methods described above Following treatmentleucaena plants were harvested at 48 and 96 h or 72 and 144 h (UV-Ctreated plants only) after treatments Total RNA of samples was ex-tracted and purified from roots and shoots of giant leucaena by meansof a modified method using Qiagen RNeasy Plant Kit (Valencia CAUSA) and Fruit-mate (Takara Japan) according to the protocol de-scribed by Ishihara et al (2016a) The assessment of RNA quality andquantity was carried out at 230 260 and 280 nm by using a NanoDropSpectrophotometer ND-1000 (NanoDrop Technologies DE USA) Inorder to avoid genomic DNA contamination RNA samples were treatedwith TURBO DNAfree Kit (Invitrogen Carlsbad CA) Two microgramsof DNase-treated RNA were used to synthesize the first-strand cDNAusing M-MLV Reverse Transcriptase (Promega WI USA)
Quantitative real-time (qPCR) analysis was carried out to examinepossible differential expression of the mimosinase gene (GenBank ac-cession number AB2985971) in seedlings treated with 84 ppm JA10mM Ethephon or 15min of UV-C exposure Shoots and roots wereharvested 24 h before the time of mimosine concentration peak for eachtreatment previously observed as assessed by HPLC assays The 10 μLqPCR reaction consisted of 5 μL of PowerUpTM SYBRreg Green MasterMix (Applied Biosystems Foster City CA) 1 μL MgCl2 (50mM) 03 μLforward primer (10 μM) 03 μL reverse primer (10 μM) and 1 μL cDNAfirst-strand In the experimental validation through qPCR reactionconditions and melting curve analysis of the amplicon were performed
following the protocol published by Ishihara et al (2016b) for the sameleucaena variety qPCR analysis was conducted using StepOnetrade Real-Time PCR System (Applied Biosystems) Measurements were performedusing 4 biological and 3 technical replicates Relative expression wascalculated with the 2-ΔΔct method using OAS-TL as reference gene sinceits expression showed a consistently stable profile comparable to that ofUBQ-5 and ELF1α expressions Mimosinase primer sequences used forthese analyses were (FWD) 5prime- GAA AGG CAG GAA TCA CAG TGA AGAG ndash 3rsquo (REV) 5prime GGA GAC TCT AGC CAC ACC AAC TTA ndash 3rsquo
24 Antioxidant assays
241 Mimosine effect on hydrogen peroxide (H2O2) accumulationAs a follow up to the induction of mimosine accumulation profiles
under stress signals and conditions tests were conducted to verify mi-mosine antioxidant capacity In situ histological localization of hy-drogen peroxide (H2O2) accumulation was evaluated on foliar disks ofPhaseolus vulgaris L according to the protocol described by Shi et al(2010) Briefly the plant foliar tissue was exposed to 1 mgmiddotmLminus1 dia-minobenzidine (DAB) solution in 10 mM KH2PO4 (control) in presenceor absence of 10mM mimosine (equivalent to the average mimosineconcentration induced by UV-C radiation in giant leucaena) or 10mMascorbic acid (positive antioxidant control) Oxidative response wasidentified by the formation of a brown polymer on the injured leafareas indicating the presence of H2O2 and registered in a Leica M165FC stereomicroscope (Leica Microsystems)
242 Mimosine quenching of superoxide radicalsGeneration of superoxide radical and subsequent analysis was per-
formed by a modified protocol based on Zhishen et al (1999) Nitroblue tetrazolium (NBT) reduction was used to measure superoxide an-ions quenching activity Shortly a 50mM KH2PO4 pH 78 solutioncontaining 6 μM riboflavin 100mM methionine 1 mM NBT in pre-sence or absence of 5mM mimosine was exposed to white light(22 Jsdotcmminus2) for 25min on a white light transilluminator Five micro-molar rutin was used as positive control (Matsuura et al 2016) Theabsorbance was read at 560 nm before and after light exposure in aSpectraMaxreg M2 Microplate Reader (Molecular Devices LLC)
25 Statistical analyses
For HPLC and superoxide anions data simple analyses of variance(ANOVA) followed by Tukey or Welch ANOVA followed by Dunnetts Ctest were used as appropriate for data distribution characteristics InqPCR analysis results were analyzed by t-test In all cases at least fourbiological triplicates were used and experiments were repeated twiceindependently All data were analyzed using the statistical packageSPSS 200 for Windows (SPSS Inc USA) In all cases a ple 005 wasused
3 Results and discussion
31 Increased mimosine concentrations in giant leucaena treated withchemical elicitors
Leucaena produces high amounts of mimosine that accumulate in allparts of the plants including leaves stem flowers pods seeds rootsand root nodules (Soedarjo and Borthakur 1998) The highest con-centrations of mimosine can be found in the growing shoot tips andseeds (Wong and Devendra 1983) It is not known why leucaena pro-duces such high amounts of mimosine Negi et al (2014) estimated thatleucaena plants would be able to grow 21 larger if the nutrient re-sources spent on mimosine production were diverted for biomass in-crease In a previous analysis performed to quantify the basal con-centration of mimosine present in adult plants of common leucaena thehighest constitutive amount of mimosine per gram of fresh weight in
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
434
the analyzed organs was found in post-anthesis flowers (89448 μg)followed by green pods (82687 μg) leaves (67358 μg) and greenflower buds (51247 μg) which showed significantly less mimosineconcentration compared to the other reproductive structures(Supplementary Fig 1) Since mature seeds have very low moisturecontent (Wencomo et al 2017) its mimosine concentration was esti-mated as 338253 μgsdotgminus1 of dry weight Additionally it was also ob-served that the basal mimosine distribution in shoots of field-grownadult plants of leucaena is dependent on the variety type(Supplementary Table 1)
Phytohormones such as salicylic acid and jasmonic acid are knownto be produced by plants in response to various abiotic and bioticstresses These phytohormones trigger adaptive responses to stress byregulating major plant metabolic processes such as photosynthesisnitrogen metabolism defense systems and plant-water relationsthereby providing protection (for review see Khan et al 2015)
Secondary or specialized metabolite production and accumulationare also known to be controlled by biotic and abiotic stresses (Matsuuraet al 2018) In this study exposure of 5-week-old giant leucaenaseedlings to JA or Ethephon treatments significantly enhanced mimo-sine accumulation in shoots and roots in at least one of the two timepoints tested (48 and 96 h) albeit in a different way (Fig 1) Thehighest concentrations of mimosine in shoots were found in seedlingstreated with JA 84 ppm (43441 μgsdotgminus1) and Ethephon 100 ppm(38412 μgsdotgminus1) two days after application of the respective phyto-hormones Nevertheless after four days shoots yielded the highestconcentration of mimosine (approximately 460 μgsdotgminus1) upon treatmentwith 10 or 100 ppm Ethephon (Fig 1A) In roots after two and four
days JA 84 ppm and Ethephon 10 ppm resulted in highest mimosineaccumulation 18488 μgsdotgminus1 and 15801 μgsdotgminus1 respectively (Fig 1B)These observations show that mimosine accumulation response tospecific elicitors may vary over time after exposure
Although all treatments were applied exclusively on shoots of giantleucaena seedlings roots of some of them were also able to respond tothe different elicitors Overall shoots displayed higher basal and in-duced mimosine concentration compared to roots (Fig 1) which agreeswith previous observations in 1 to 3-week-old aseptic seedlings ofcommon leucaena (Vestena et al 2001) However as previouslymentioned significant post-induction increase of mimosine concentra-tion in roots and shoots simultaneously was only observed for JA andEthephon 10 ppm on day 02 and 04 respectively (Fig 1)
It is well established that perceived regulatory signals or elicitorsgenerate a transduction network mediated by secondary messengersresulting in changes in gene expression profiles that afford adaptiveresponses to environmental stimuli These modulation events are oftenmediated by transcription factors (TFs) which directly bind to specificgene promoters or act by forming complexes with repressor proteinslabeling them to degradation subsequently releasing other TFs toproceed with the gene expression program This is the case of the actionmechanism of JA and its active form jasmonoyl isoleucine for example(Kazan 2015 Wasternack and Strnad 2016)
JA ethylene and SA are known as important stress regulatory sig-nals in plants JA however is thought to be the most effective signal forinduction of plant secondary metabolism (Wasternack and Strnad2016) thereby contributing to mitigation of damage caused by severalstresses (Dar et al 2015) JA is mainly derived from linolenic acid
Fig 1 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings treated with differentelicitors CTRL=Milli-Q water SA = Salicylic AcidJA= Jasmonic Acid ETH=Ethephon Bars sharing a letterof same case do not differ by Tukey test (P le 005) Capitalletters (A B) compare treatments on day two and lowercaseletters (a b) compare treatments on day four Indicatessignificant statistical difference between day two and dayfour in the same treatment by t-test (Ple 005) The errorbars represent standard error of five replicates (each meanwas calculated with 15 individual seedlings organized in 5groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
435
(Wasternack and Strnad 2016) playing important roles in differentprocesses of plant growth and development such as plant defensemechanisms against herbivory pathogen attack fungal elicitation andsome abiotic factors such as osmotic temperature and salt stresses (Daret al 2015)
JA and its methyl ester MeJA have several different effects on le-guminous species MeJA exogenous application has increased iso-flavonoid content in cell suspension cultures of Pueraria candollei varcandollei and P candollei var mirifica (Korsangruang et al 2010) aswell as the production of the triterpenoid glycyrrhizin in Glycyrrhizaglabra roots Enhanced production of the triterpenoid however waspartly at the expense of root growth (Shabani et al 2009) MeJA ap-plication on shoots was observed to suppress root nodulation and lat-eral root formation in Lotus japonicus (Nakagawa and Kawaguchi2006) In grapevine a non-leguminous species proteinogenic aminoacids did not show an expressive increase under MeJA treatment(Gutieacuterrez-Gamboa et al 2017)
The effects of the application of four different jasmonate forms (JAMeJA jasmonoyl-L-isoleucine (JA-Ile) and 6-ethyl indanoyl glycineconjugate (2-[(6-ethyl-1-oxo-indane-4-carbonyl)-amino]-acetic acidmethyl ester - CGM) on leucaena metabolite profile has recently beenreported by Xu et al (2018) JA-Ile form was most effective althoughno major alteration was observed on monitored metabolite abundancesAlanine threonine and 34-dihydroxypyridine (34 DHP a metabolitederived from mimosine degradation) (Nguyen and Tawata 2016)among others were the major metabolites elicited by JA-Ile In contrastto the results described here mimosine concentration did not changesignificantly These divergent results on mimosine accumulation maybe due to a number of factors including mode of application jasmonateform used (JA-Ile x JA) and L leucocephala subspecies (common x giantleucaena)
Ethylene is also a phytohormone involved in plant response me-chanisms to different types of challenges such as mechanical damageand insect attack among others The integration mechanism betweenJA and ethylene signaling pathways is not completely understoodhowever it has been shown that they may work cooperatively in abioticstress tolerance (Kazan 2015) MeJA can induce ethylene production(Zhao et al 2004) and when applied simultaneously these moleculesseem to work in a synergic way by enhancing the magnitude of theplant response to external stimuli (Liu et al 2016)
Treatment with SA was able to significantly increase mimosine ac-cumulation in 12-week-old plants of common leucaena (SupplementaryFig 2) However no significant effect of SA treatment on mimosineconcentration was seen in 5-week-old seedlings of giant leucaena(Fig 1) suggesting some degree of genotype andor age dependency inelicitation by this phytohormone On the other hand several treat-ments including 90 ppm MeJA 10 and 100 ppm 2-chloroethylpho-sphonic acid (CEPA an ethylene-releasing compound) significantlyincreased mimosine accumulation (Supplementary Fig 2) in agree-ment with the data obtained for giant leucaena The lack of systemiceffects of externally applied SA on mimosine accumulation was alsoobserved when the phytohormone was supplied in the culture mediumof aseptically-grown seedlings in which case only roots had highercontent of mimosine (Vestena et al 2001) This could be due totransport limitations or to low methyl salicylate production from ap-plied SA since the former is recognized as the main systemic signalingform (Vlot et al 2009)
32 Increased mimosine concentrations in giant leucaena exposed to UV-Cradiation
UV-C treatment promoted increased concentration of the aminoacid in shoots but not in roots of giant leucaena (Fig 2) Increasedaccumulation of mimosine in shoots was also observed in 12-week-oldseedlings of common leucaena exposed to UV-C radiation for 10 and15min (Supplementary Fig 3) Similar to the SA treatment in giant
leucaena UV-C radiation did not induce mimosine biosynthesis in rootsregardless of time after exposure The absence of mimosine induction inroots by SA and UV indicates that these effectors do not cause a sys-temic response Moreover roots are shielded from irradiance by thepresence of substrate
UV radiation effects on different aspects of plant metabolism anddevelopment have been described However compared to UV-B (en-vironmentally relevant type of UV radiation) assays there are less re-ports related to the UV-C effects on secondary metabolites biosynthesisand accumulation (Cetin 2014) especially in leguminous (Fabaceae)plants They generally concern primary metabolism aspects such asgrowth and development For instance seedlings of Phaseolus vulgaris L(Fabaceae) exposed to low intensity UV-C radiation have displayeddecreased chlorophyll content and reduced height after 14 days of ex-posure (Kara 2013) Negative effects on growth parameters and ni-trogen metabolism were also observed in Vigna radiata L (Fabaceae)after UV-B radiation treatment in addition to adverse effects on JA SAand antioxidant compounds accumulation (Choudhary and Agrawal2014a) The same authors reported increased accumulation of flavo-noids SA and JA besides negative effects on growth biomass yieldnitrogen fixation and accumulation in 2 cultivars of Pisum sativum L(Fabaceae) under elevated UV-B treatment (Choudhary and Agrawal2014b) Despite the negative UV influence on growth reported for thepreviously mentioned leguminous UV-C radiation on groundnut plants(Arachis hypogaea L Fabaceae) increased seedling vigor and biomassand had no adverse effect on germination or other development para-meters (Neelamegam and Sutha 2015)
Besides its impact on growth and primary metabolism UV exposurecan cause important changes in secondary metabolism depending onintensity and time of exposure (Matsuura et al 2013) UV-B and UV-Cpre-treatments of Artemisia annua (Asteraceae) seedlings yielded in-creased biosynthesis of artemisinin a drug which displays anti-malarialproperties and activity against some others infectious diseases (egschistosomiasis leishmaniasis and hepatitis B) and several kinds oftumors (Rai et al 2011) The accumulation of nicotine in Nicotianarustica plants (Solanaceae) was also increased by UV-C treatment(Tiburcio et al 1985) Similar inducing effects on production of severalsecondary metabolites were observed in callus cultures of Vitis viniferaL Oumlkuumlzgoumlzuuml (grapevine Vitaceae) treated with a UV-C source for 5 or10min (Cetin 2014)
Regarding amino acid biosynthesis in response to UV radiationMartiacutenez-Luumlscher et al (2014) have found that in spite of not causingchanges in total amino acid content UV-B radiation exposure can affecttheir profile in grape berries Proteinogenic amino acids have beenknown to be important targets of the deleterious effects of UV radiation(Holloacutesy 2002) On the other hand in the present study acute UV-Ctreatment was found to increase mimosine accumulation in shoots byover twofold (Fig 2) which may suggest a possible participation of thismolecule as part of the antioxidant defense system in L leucocephalaThis possibility is further supported by the induction of the amino acidaccumulation by JA and Ethephon involved in abiotic and biotic stressresponses which are generally associated with oxidative imbalance andare signaling components in high UV stress (Matsuura et al 2013)
33 Mimosinase gene expression
In order to determine if increases in mimosine content upon ex-posure to JA CEPA or UV-C radiation were related to changes intranscription of mimosine metabolism-related genes RT-qPCR analysiswas carried out The complete pathway for mimosine biosynthesis hasnot yet been determined although the final step has been character-ized Based on transcription analysis (Ishihara et al 2016a) leucaenaappears to encode for multiple cysteine synthases one or more of whichmay be able to catalyze mimosine synthesis In addition a leucaenagene encoding a mimosinase (an enzyme responsible for mimosinedegradation) has been identified and characterized (Negi et al 2014)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
436
In addition to mimosinase gene expression several gene isoformsbelonging to the cysteine pathway [cysteine synthase (CYS SYN) serineacetyltransferase (SAT) and β-cyanoalanine synthase (CAS) Table 2 -supplementary material] were also tested in this study (data notshown) However expressions of these genes did not vary in giantleucaena throughout the experiments suggesting that the increasedcontent of mimosine observed in the treated plants might not be relatedto the expression of these genes but presumably to increased enzymeactivity andor release from conjugates such as mimoside a mimosineβ-D-glucoside (Murakoshi et al 1972)
Considering the time variation of mimosine accumulation observedin this work mimosinase gene expression in shoots and roots wasevaluated 24 h before the increase of mimosine concentration in giantleucaena seedlings (ie 24 h and 72 h after the chemical elicitorstreatments and 48 h and 120 h after UV-C exposure)
Ethylene signaling has been shown to up-regulate expression ofseveral genes related to secondary metabolism pathways as is the caseof phenolic compounds (Liu et al 2016) and terpenoid indole alkaloids(Wang et al 2016) Among all elicitors tested in the present workEthephon was the only one able to significantly change mimosinasegene expression Leucaena plants treated with Ethephon showed sig-nificant increases in mimosine concentration at both day 2 and 4 fol-lowing treatment which coincided with low-level expression of mi-mosinase Up-regulation of mimosinase gene expression was detected24 h before the increase of mimosine concentration in shoots treatedwith 10 ppm of Ethephon (Fig 3A) but not after JA or UV-C treatments(Fig 3C-D and 3E-F respectively) Nevertheless 72 h after treatment
application (24 h before the highest mimosine content measured inshoots) down regulation of mimosinase gene was seen in both shootsand roots treated with 10 ppm of Ethephon (Fig 3B) These data in-dicate that mimosine content in leucaena plants is at least partlyregulated by mimosinase expression in Ethephon exposed plants Onthe other hand the fact that mimosinase mRNA was not significantlyaffected by JA and UV-C treatments despite their stimulating effects onmimosine biosynthesis in giant leucaena may indicate that other levelsof regulation are at play or that the chosen harvesting time window wasunable to detect relevant changes
34 In situ and in vitro antioxidant assays
Considering the stimulation of mimosine accumulation byEthephon JA and UV all of which are often associated or known tocause oxidative imbalance the antioxidant capacity of mimosine wasevaluated Mimosine has been shown to have antioxidant activities oncultured cancer cells (Parmar et al 2015) In the present study it washypothesized that mimosine could confer radical scavenging propertieswhich would contribute to plant protection from possible damagecaused by reactive oxygen species generated during stress(Supplementary Fig 4)
Foliar disks of P vulgaris L were treated with 10mM mimosine for15min Treated disks showed less hydrogen peroxide accumulationinduced by wounding in contrast to untreated ones being comparableto those treated with ascorbic acid (a known hydrogen peroxide neu-tralizer) (Fig 4A) These observations support a possible antioxidant
Fig 2 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings exposed to UV-C lightCTRL= visible light (100 μmol photons mminus2 s minus1) UV-C 10primeand UV-C 15rsquo=UV-C exposure time (10 and 15min re-spectively) Bars sharing a letter of same case do not differ byTukey test (P le 005) Capital letters (A B) compare treat-ments on day three and lowercase letters (a b) comparetreatments on day six Indicates significant statistical dif-ference between day three and day six in the same treatmentby t-test (Ple 005) The error bars represent standard errorof five replicates (each mean was calculated with 15 in-dividual seedlings organized in 5 groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
437
role of mimosine as an in situ hydrogen peroxide scavengerMimosine was also able to quench superoxide anions generated by
light exposure Mimosine exhibited equivalent antioxidant effect com-pared to rutin (Fig 4B) a well-established effective superoxide anionquencher (Matsuura et al 2016) The radical scavenging activity ofmimosine may be due to the 3-OH group of the pyridine ring of mi-mosine (Fig 5) The pKa of the 3-OH of mimosine has been estimated tobe 88 (M Honda unpublished results) At physiological pH this OHgroup is expected to remain in a protonated state and therefore mayscavenge a radical by donating a proton and an electron In this processmimosine itself is converted to a stable radical form which is perhapsless toxic and less reactive than the reactive oxygen species generatedduring oxidative stress It is likely that the less toxic radical mimosineproduced may react with another radical or molecule and becomeconverted to a non-reactive indole molecule
In vivo antioxidant activity of mimosine has been previously eval-uated by means of its exogenous application on selenium-deficientseedlings of Vigna radiata In spite of its allelopathic properties (Ahmedet al 2008) the results showed mitigation of mitochondrial oxidativestress by treatment with 01mM mimosine (Lalitha and Kulothungan2007) DPPH radical scavenging activity was also reported for aqueous
seed extracts of leucaena rich in mimosine and phenolic compounds inin vitro assays (Benjakul et al 2014) Mimosine antioxidant activityshown in the present work is in good agreement with data reported forother non-protein amino acids such as L-DOPA (Dhanani et al 2015)and GABA (Malekzadeh et al 2014) for instance
4 Conclusion
Taken together results show that mimosine biosynthesis and ac-cumulation can be modulated by stress-related factors despite its re-latively high constitutive content in leucaena plants The pattern ofgene expression in stressed plants suggests mimosine steady-state con-trol may be regulated by its degradation in possible connection withdynamic changes in carbon and nitrogen metabolism of stressed plantsMimosine quenching activity against hydrogen peroxide and super-oxide anions in the in situ staining and in vitro assays respectivelyshowed that this non-protein amino acid can act as non-enzymaticantioxidant agent Increase in mimosine content in response to elicitorsmimicking environmental challenges in addition to its antiherbivoreand antimicrobial properties may be related to its activity as protectivemolecule against oxidative damage in line with other classes of plant
Fig 3 Relative expression of the mimosinase gene in shoots (A E and F) and shoots and roots (B C and D) of giant leucaena 24 h (A and C) 48 h (E) 72 h (B and D)and 120 h (F) after treatment with stress signaling molecules or UV-C exposure ETH = Ethephon JA = Jasmonic Acid Indicates significant statistical differencebetween control and treatment by t-test (Ple 005) The error bars represent standard error of four replicates
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
438
secondary metabolites
Funding
This work was funded by the National Council for Scientific andTechnological Development (CNPq-Brazil) grant 3060792013-5Coordenaccedilatildeo de Aperfeiccediloamento de Pessoal de Niacutevel Superior - Brazil(CAPES) - Finance Code 001 and the USDA NIFA Hatch projectHA05029-H managed by CTAHR
CRediT authorship contribution statement
Kelly Cristine da Silva Rodrigues-Correcirca InvestigationValidation Writing ndash original draft Michael DH HondaInvestigation Validation Dulal Borthakur Supervision Writing ndashreview amp editing Funding acquisition Arthur Germano Fett-NetoSupervision Funding acquisition Writing ndash review amp editing
Acknowledgements
The authors would like to thank Dr Jorge Ernesto Mariath fromLaVeg-UFRGS for kindly lending the Leica M165 FC stereomicroscopefor in situ analysis
Appendix A Supplementary data
Supplementary data to this article can be found online at httpsdoiorg101016jplaphy201811018
References
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Benjakul S Kittiphattanabawon P Shahidi F Maqsood S 2013 Antioxidant activityand inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipidoxidation in model systems Food Sci Technol Int 19 (4) 365ndash376 httpsdoiorg1011771082013212455186
Benjakul S Kittiphattanabawon P Sumpavapol P Maqsood S 2014 Antioxidantactivities of lead (Leucaena leucocephala) seed as affected by extraction solvent priordechlorophyllisation and drying methods extracts against lipid oxidation in modelsystems Food Sci Technol 51 (11) 3026ndash3037 httpsdoiorg101007s13197-012-0846-1
Brewbaker JL Pluckett D Gonzalez V 1972 Varietal variation and yield trials ofLeucaena leucocephala (koa haole) in Hawaii Hawaii Agric Exp Stn Bull 166 26
Brewbaker JL 2008 Registration of KX2 ndash Hawaii interspecific-hybrid leucaena JPlant Registrations 1 (3) 190ndash193 httpsdoiorg103198jpr2007050298crc
Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitisvinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-6287-47-37
Cho H-Y Son SY Rhee HS Yoon S-YH Lee-Parsons CWT Park JM 2008Synergistic effects of sequential treatment with methyl jasmonate salicylic acid andyeast extract on benzophenanthridine alkaloid accumulation and protein expressionin Eschscholtzia californica suspension cultures J Biotechnol 135 117ndash122 httpsdoiorg101016jjbiotec200802020
Choudhary KK Agrawal SB 2014a Cultivar specificity of tropical mung bean (Vignaradiata L) to elevated ultraviolet-B changes in antioxidative defense system ni-trogen metabolism and accumulation of jasmonic and salicylic acids Environ ExpBot 99 122ndash132 httpsdoiorg101016jenvexpbot201311006
Choudhary KK Agrawal SB 2014b Ultraviolet-B induced changes in morphologicalphysiological and biochemical parameters of two cultivars of pea (Pisum sativum L)Ecotoxicol Environ Saf 100 178ndash187 httpsdoiorg101016jecoenv201310032
Dar TA Uddin M Khan MMA Hakeem KR Jaleel H 2015 Jasmonates counterplant stress a Review Environ Exp Bot 115 49ndash57 httpsdoiorg101016jenvexpbot201502010
Dhanani T Singh R Shah S Kumari P Kumar S 2015 Comparison of green ex-traction methods with conventional extraction method for extract yield L-DOPAconcentration and antioxidant activity of Mucuna pruriens seed Green Chem LettRev 8 (2) 43ndash48 httpsdoiorg1010801751825320151075070
Gutieacuterrez-Gamboa G Portu J Santamariacutea P Loacutepez R Garde-Cerdaacuten T 2017Effects on grape amino acid concentration through foliar application of three dif-ferent elicitors Food Res Int 99 688ndash692 httpsdoiorg101016jfoodres201706022
Fig 4 A In situ antioxidant assay Foliar disksof Phaseolus vulgaris L treated with (a) No an-tioxidant added (negative control) (b) 10 mMMimosine (c) 10mM ascorbic acid (positivecontrol) The oxidative damage can be seen bythe formation of a brown polymer in leaf veinsand injured areas B In vitro superoxidescavenging assay carried out with mimosineDifferent letters indicate significant differenceby Tukey test (Ple 005) The error bars re-present standard error of four replicates (Forinterpretation of the references to colour in thisfigure legend the reader is referred to the Webversion of this article)
Fig 5 Predicted mimosine radical formed followingquenching of hydroxyl radical Mimosine is first converted toa stable mimosine radical which may be then converted to anontoxic indole form
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
439
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad HossainMd Anai T Oku H 2018 Cytosolic cysteine synthase switch cysteine and mi-mosine production in Leucaena leucocephala Appl Biochem Biotechnol 186 (3)613ndash632 httpsdoiorg101007s12010-018-2745-z
Holloacutesy F 2002 Effects of ultraviolet radiation on plant cells Micron 33 (2) 179ndash197Honda MDH Ishihara KL Pham DT Borthakur D 2018 Identification of drought-
induced genes in giant leucaena (Leucaena leucocephala subsp glabrata) Trees 32571ndash585 httpsdoiorg101007s00468-018-1657-4
Huang T Jander G de Vos M 2011 Non-protein amino acids in plant defense againstinsect herbivores representative cases and opportunities for further functional ana-lysis Phytochemistry 72 1531ndash1537 httpsdoiorg101016jphytochem201103019
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of thethyrotoxic amino acid mimosine by cysteine synthase Phytochemistry 29 (11)3461ndash3465 httpsdoiorg1010160031-9422(90)85258-H
Ishihara K Lee EKW Borthakur D 2016a An improved method for RNA extractionfrom woody legume species Acacia koa A Gray and Leucaena leucocephala (Lam) deWit Int J For Wood Sci 3 (1) 031ndash035
Ishihara KL Honda MDH Pham DT Borthakur D 2016b Transcriptome analysisof Leucaena leucocephala and identification of highly expressed genes in roots andshoots Transcriptomics 4 135 httpsdoiorg1041722329-89361000135
IUBMB 2018 Enzyme Nomenclature EC 35161 httpwwwsbcsqmulacukiubmbenzymeEC35161html Accessed date 8 February 2018
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant(Phaseolus vulgaris) Biosci Res 10 (1) 29ndash32
Kazan K 2015 Diverse roles of jasmonates and ethylene in abiotic stress toleranceTrends Plant Sci 20 (4) 219ndash229 httpsdoiorg101016jtplants201502001
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect ofEthephon as an ethylene-releasing compound on the metabolic profile of Chlorellavulgaris J Agric Food Chem 64 (23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541
Khan MIR Fatma M Per TS Anjum NA Khan NA 2015 Salicylic acid-inducedabiotic stress tolerance and underlying mechanisms in plants Front Plant Sci 6 462httpsdoiorg103389fpls201500462
Korsangruang S Soonthornchareonnon N Chintapakorn Y Saralamp PPrathanturarug S 2010 Effects of abiotic and biotic elicitors on growth and iso-flavonoid accumulation in Pueraria candollei var candollei and P candollei var mir-ifica cell suspension cultures Plant Cell Tissue Organ Cult 103 (3) 333ndash342 httpsdoiorg101007s11240-010-9785-6
Lalitha K Kulothungan SR 2006 Selective determination of mimosine and its dihy-droxypyridinyl derivative in plant systems Amino Acids 31 (3) 279ndash287 httpsdoiorg101007s00726-005-0226-5
Lalitha K Kulothungan SR 2007 Mimosine mitigates oxidative stress in seleniumdeficient seedlings of Vigna radiata - Part I restoration of mitochondrial functionBiol Trace Elem Res 118 (1) 84ndash96 httpsdoiorg101007s12011-007-0013-0
Liu J Li Y Wang Y Zhang Z-H Zu Y-G Efferth T Tang Z-H 2016 Thecombined effects of ethylene and MeJA on metabolic profiling of phenolic com-pounds in Catharanthus roseus revealed by metabolomics analysis Front Physiol 71ndash11 httpsdoiorg103389fphys201600217 Article 217
Malekzadeh P Khara J Heydari R 2014 Alleviating effects of exogenous Gamma-aminobutiric acid on tomato seedling under chilling stress Physiol Mol Biol Plants20 (1) 133ndash137 httpsdoiorg101007s12298-013-0203-5
Martiacutenez-Luumlscher J Torres N Hilbert G Richard T Saacutenchez-Diacuteaz M Delrot SAguirreolea J Pascual I Gomegraves E 2014 Ultraviolet-B radiation modifies thequantitative and qualitative profile of flavonoids and amino acids in grape berriesPhytochemistry 102 106ndash114 httpsdoiorg101016jphytochem201403014
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation ofbioactive secondary metabolites by ultraviolet radiation mechanisms strategies andapplications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology forMedicinal Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash1902012
Matsuura HN Fragoso V Paranhos JT Rau MR Fett-Neto AG 2016 Thebioactive monoterpene indole alkaloid N szlig-D-glucopyranosylvincosamide is regu-lated by irradiance quality and development in Psychotria leiocarpa Ind Crop Prod86 210ndash218 httpsdoiorg101016jindcrop201603050
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo RBhambra AS Strnad M Bonfill M Fett-Neto AG 2018 Specialized plant me-tabolism characteristics and impact on target molecule biotechnological productionMol Biotechnol 60 (2) 169ndash183 httpsdoiorg101007s12033-017-0056-1
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Nakagawa T Kawaguchi M 2006 Shoot-applied MeJA suppresses root nodulation inLotus japonicus Plant Cell Physiol 47 (1) 176ndash180 httpsdoiorg101093pcppci222
Nascimento NC Menguer PK Henriques AT Fett-Neto AG 2013 Accumulation ofbrachycerine an antioxidant glucosidic indole alkaloid is induced by abscisic acidheavy metal and osmotic stress in leaves of Psychotria brachyceras Plant PhysiolBiochem 73 33ndash40 httpsdoiorg101016jplaphy201308007
Neelamegam R Sutha T 2015 UV-C irradiation effect on seed germination seedling
growth and productivity of groundnut (Arachis hypogaea L) Int J Curr MicrobiolApp Sci 4 (8) 430ndash443
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase fromLeucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol164 (2) 922ndash934 httpsdoiorg101104pp113230870
Negi VS Borthakur D 2016 Heterologous expression and characterization of mimo-sinase from Leucaena leucocephala In Fett-Neto Arthur Germano (Ed)Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods inMolecular Biology vol 1405 copySpringer Science+Business Media New York httpsdoiorg101007978-1-4939-3393-8_7 2016
Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636
Parmar F Kushawaha N Highland H George L-B 2015 In vitro antioxidant andanticancer activity of Mimosa pudica Linn extract and L-mimosine on lymphomaDaudi cells Int J Pharm Sci 12 100ndash104
Porto DD Matsuura HN Vargas LRB Henriques AT Fett-Neto AG 2014 Shootaccumulation kinetics and effects on herbivores of the wound-induced antioxidantindole alkaloid brachycerine of Psychotria brachyceras Nat Prod Commun 9 (5)629ndash632
Rai R Meena RP Smita SS Shukla A Rai SK Pandey-Rai S 2011 UV-B and UV-C pre-treatments induce physiological changes and artemisinin biosynthesis inArtemisia annua L ndash an antimalarial plant J Photochem Photobiol B Biol 105 (3)216ndash225 httpsdoiorg101016jjphotobiol201109004
Shabani L Ehsanpour AA Asghari G Emami J 2009 Glycyrrhizin production by invitro cultured Glycyrrhiza glabra elicited by methyl jasmonate and salicylic acid RussJ Plant Physiol 56 (5) 621ndash626 httpsdoiorg101134S1021443709050069
Shah J 2003 The salicylic acid loop in plant defense Curr Opin Plant Biol 6 (4)365ndash371
Shi J Fu XZ Peng T Huang XS Fan QJ Liu JH 2010 Spermine pretreatmentconfers dehydration tolerance of citrus in vitro plants via modulation of antioxidativecapacity and stomatal response Tree Physiol 30 (7) 914ndash922 httpsdoiorg101093treephystpq030
Smith IK Fowden L 1966 A study of mimosine toxicity in plants J Exp Bot 17750ndash761 httpsdoiorg101093jxb174750
Soedarjo M Borthakur D 1996 Simple procedures to remove mimosine from youngleaves pods and seeds of Leucaena leucocephala used as food Int J Food SciTechnol 31 (1) 97ndash103
Soedarjo M Borthakur D 1998 Mimosine a toxin produced by the tree-legumeLeucaena provides a nodulation competition advantage to mimosine-degradingRhizobium strains Soil Biol Biochem 30 1605ndash1613
Suda S 1960 On the physiological properties of mimosine Bot Mag Tokyo 73 (862)142ndash147 httpsdoiorg1015281jplantres188773142
Tangendjaja B Lowry JB Wills RBH 1986 Isolation of a mimosine degrading en-zyme from leucaena leaf J Sci Food Agric 37 523ndash526 httpsdoiorg101002jsfa2740370603
Tiburcio F Pintildeol MT Serrano M 1985 Effect of UV-C on growth soluble protein andalkaloids in Nicotiana rustica plants Environ Exp Bot 25 (3) 203ndash210 httpsdoiorg1010160098-8472(85)90004-8
Vestena S Fett-Neto AG Duarte RC Ferreira A 2001 Regulation of mimosineaccumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 httpsdoiorg101016S0168-9452(01)00448-4
Vlot AC Dempsey DMA Klessig DF 2009 Salicylic acid a multifaceted hormone tocombat disease Annu Rev Phytopathol 47 177ndash206 httpsdoiorg101146annurevphyto050908135202 2009
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene-induced vinblastine accumulation is related to activated expression of downstreamTIA pathway genes in Catharanthus roseus BioMed Res Int 2016 Article ID 3708187httpsdoiorg10115520163708187
Wasternack C Strnad M 2016 Jasmonate signaling in plant stress responses and de-velopment ndash active and inactive compounds N Biotech 33 (5B) 604ndash613 httpsdoiorg101016jnbt201511001
Wencomo HB Ortiz R Caacuteceres J 2017 Afr J Agric Res 12 (4) 279ndash285 httpsdoiorg105897AJAR201510604 26
Wong CC Devendra C 1983 Research on leucaena forage production in Malaysia InLeucaena Research in the Asian Pacific Region pp 55ndash60 Ottawa Ontario Canada
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX TsimKWK 2018 Jasmonate-elicited stress induces metabolic change in the leaves ofLeucaena leucocephala Molecules 23 (2) httpsdoiorg103390molecules23020188 E188
Yafuso JT Negi VS Bingham J-P Borthakur D 2014 An O-acetylserine (thiol)lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthaseAppl Biochem Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z
Zhao J Zheng S-H Fujita K Sakai K 2004 Jasmonate and ethylene signalling andtheir interaction are integral parts of the elicitor signalling pathway leading to b-thujaplicin biosynthesis in Cupressus lusitanica cell cultures J Exp Bot 55 (399)1003ndash1012 httpsdoiorg101093jxberh127
Zhishen J Mengcheng T Jianming W 1999 The determination of flavonoid contentsin mulberry and their scavenging effects on superoxide radicals Food Chem 64 (4)555ndash559 httpsdoiorg101016S0308-8146(98)00102-2
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
440
61
Supplementary Fig 1 Basal mimosine concentration in adult trees of common leucaena (L leucocephala
var leucocephala) Samples were collected from 10 field grown trees at Manoa Valley Honolulu Hawairsquoi
on June 25th 2017 Bars sharing a letter do not differ by Tukey test (P le 005) The error bars represent the
standard error
Supplementary Fig 2 Bar diagram showing mimosine concentration in shoots of 12-week-old common
leucaena seedlings treated with different elicitors CTRL = Milli-Q water SA = Salicylic Acid MeJA =
Methyl Jasmonate CEPA = 2-Chloroethylphosphonic acid (an ethylene releasing compound) Bars sharing a
letter of same case do not differ by Tukey test (P le 005) Capital letters (A B) compare treatments on day
two and lower-case letters (a b) compare treatments on day four Indicates significant statistical difference
ABB
A A
0
200
400
600
800
1000
1200
LEAVES GREEN FLOWERBUDS
POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
con
cen
trat
ion
(micro
gg
-1o
f FW
)
B AB AB AB B A
b
a
ab b
ab
0
2
4
6
8
10
12
14
16
18
20
CTRL SA 10 ppm SA 100 ppm CEPA 10 ppm CEPA 100 ppm MeJA 90 ppm
Mim
osi
ne
co
nce
ntr
atio
n (
gg
-1o
f FW
)
DAY 02 DAY 04
62
between day two and day four in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
Supplementary Fig 3 Bar diagram showing the effects of UV-C radiation exposure for 5 10 and 15 min on
mimosine accumulation in shoots of 12-week-old seedlings of common leucaena Bars sharing a letter of
same case do not differ by Tukey test (P le 005) Capital letters (A B C) compare treatments on day three
and lower-case letters (a b) compare treatments on day six Indicates significant statistical difference
between day three and day six in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
C BC AB A
bb
a
a
0
10
20
30
40
50
60
CTRL UV-C 5 UV-C 10 UV-C 15
Mim
osi
ne
co
nce
ntr
atio
n (
gg-1
of
FW)
DAY 03 DAY 06
63
Supplementary Fig 4 Model depicting induction of mimosine synthesis in leucaena following application of
stress elicitors such as CEPA and jasmonic acid or exposure to UV-C radiation The additional mimosine
synthesized may serve to alleviate oxidative stress induced by UV-C radiation
64
Supplementary Table 1 Mimosine contents in leaves of common and giant leucaena
Leucaena
type
Mimosine content
( FW)
Mimosine
content ( DW)
Dry matter
content ( FW)
Water content
( FW)
Common (1) 050 plusmn 009 245 plusmn 051 2011 plusmn 054 7989 plusmn 054
Common (2) 043 plusmn 006 214 plusmn 037 1998 plusmn 050 8002 plusmn 050
K636 (1) 070 plusmn 014 356 plusmn 077 1908 plusmn 052 8092 plusmn 052
K636 (2) 042 005 205 plusmn 033 2008plusmn 093 7992plusmn 093
KX2 (1) 122 plusmn 011 608 plusmn 082 1939 plusmn 123 8061 plusmn 123
KX2 (2) 134 plusmn 010 623 plusmn 056 2029 plusmn 114 7971 plusmn 114
KX3 (1) 044 plusmn 006 221 plusmn 030 1945 plusmn 073 8055 plusmn 073
KX3 (2) 054 plusmn 005 273 plusmn 023 1930 plusmn 038 8070 plusmn 038
KX4 (1) 086 plusmn 011 471 plusmn 065 1753 plusmn 084 8247 plusmn 084
KX4 (2) 089 plusmn 011 476 plusmn 065 180 plusmn 072 820 plusmn 072
KX5 (1) 099 plusmn 012 489 plusmn 048 1907 plusmn060 8093 plusmn 060
KX5 (2) 115 plusmn 015 548 plusmn080 1992 plusmn 053 8008 plusmn 053
Common leucaena variety koa haole grows widely on the island of Orsquoahu K636 is widely
grown variety of giant leucaena KX2 KX3 KX4 and KX5 are giant leucaena varieties
developed through interspecies hybridization (Brewbaker 2016) (1) and (2) indicate plants
from two separate locations within the University of Hawaii Waimanalo Research Center The
values are shown as mean plusmn standard error obtained from at least three biological replicates
65
Supplementary Table 2 GenBank accession numbers of the tested cysteine pathway genes isoforms
Gene name GenBank accession
OAS-TL (o-acetylserine-thiol-lyase) GDRZ01032940
GDRZ01061620
GDRZ01153117
GDSA01187555
GDSA01196891
GDSA01214467
Cys syn (cysteine synthase) GDRZ01015860
GDRZ01050898
GDRZ01086813
GDRZ01193515
GDRZ01202579
GDSA01180863
GDSA01215622
SAT (serine acetyltransferase) GDRZ01187456
GDRZ01189631
CAS (β-cyanoalanine synthase) GDRZ01054066
GDRZ01175418
GDSA01118400
66
SHORT COMMUNICATION 1
Mimosine occurrence and accumulation in Mimosa bimucronata var bimucronata (DC) 2
Kuntze 3
Kelly Cristine da Silva Rodrigues-Correcirca1 Lana Dorneles Pedroso2 Fernanda de Costa1 4
Arthur Germano Fett-Neto1 5
1Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal 6
University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970 7
Porto Alegre Rio Grande do Sul Brazil 2Department of Biological Sciences Unipampa ndash 8
Campus Satildeo Gabriel 9
Corresponding author 10
E-mail addresses krodriguescbiotufrgsbr (KCdaS Rodrigues-Correcirca) 11
lanalima2012gmailcom (LD Pedroso) fernandadecostayahoocombr (F de Costa) 12
fettnetocbiotufrgsbr (AG Fett-Neto) 13
14
15
16
17
18
19
20
21
22
67
ABSTRACT 23
Mimosine is a non-protein aromatic amino acid present in plants of Leucaena spp 24
and Mimosa spp Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) is a native 25
tree from Brazil which occurs as a pioneer species on plant succession processes In the 26
current study the presence of mimosine in M bimucronata was verified by HPLC analyses 27
Moreover mimosine accumulation upon exposure to UV-C and chemical elicitors of 28
specialized metabolism (salicylic acid - SA methyl jasmonate - MeJA sodium nitroprusside 29
- SNP and ethephon - ETH) most of which also known as promoters of the amino acid 30
production in leucaena plants was evaluated The results showed a lower concentration of 31
constitutive mimosine present in both maricaacute seedlings and mature trees when compared to 32
leucaena plants In spite of a trend towards increased mimosine accumulation observed in 33
MeJA and ETH treatments no statistical differences were found with the various stressors 34
used to induce its biosynthesis in maricaacute seedlings Data suggest that mimosine in M 35
bimucronata is probably a phytoanticipin-like metabolite or its accumulation is driven by 36
other types of stresses 37
38
39
Keywords Mimosine Mimosa bimucronata stress 40
41
42
43
44
45
46
68
Introduction 47
Mimosa bimucronata commonly known as maricaacute is a native tree from Brazil 48
(REFLORA 2019) ecologically important in plant succession and in processes of degraded 49
land recovery (Bitencourt et al 2007 Silva et al 2011) occurring as a pioneer species 50
(Pilatti et al 2019) Maricaacute is a deciduous or semi-deciduous plant which reaches up to 15 51
m in height and 40 cm of diameter at breast height (DBH) displays shrub or tree habit and 52
bears typical sharp thorns (Carvalho 2004) This species belongs to Fabaceae one of the 53
most economically important families of flowering plants due to its high diversity and 54
occurrence in different types of habitats (Gomes et al 2018) As well as several others 55
Mimosa spp maricaacute is usually referred to as a multipurpose tree (Olkoski and Wittmann 56
2011) employed for alternative medicinal uses (Champanerkar et al 2010 Silva et al 57
2011) honey production constructions and remodeling of landscape architecture (living 58
fences) for instance (Marchiori 1993 Lorenzi 1998) 59
In southern Brazil maricaacute is widely distributed and typically found either in wetland 60
areas close to river banks (Patreze and Cordeiro 2004) or composing large and almost pure 61
landscape formations on hillsides (Jacobi and Ferreira 1991) In dense populations this 62
species like several Mimosa spp (Simon and Proenccedila 2000) is considered an important and 63
highly invasive weed by preventing cattle to reach pasturesand water bodies as a result of its 64
thorny branches (Lorenzi 2008 Kestring et al 2009) Its dominant and nearly exclusive 65
pattern of distribution in those areas has led Jacobi and Ferreira (1991) to test its allelopathic 66
potential on cultivated species Indeed extracts of leaves and ripe fruits (but not the green 67
ones) of maricaacute showed phytotoxic effects on germination and initial radical growth of most 68
of the target species tested 69
69
Several investigations have been performed on maricaacute floristics (Silva et al 2011) 70
distribution (Simon and Proenccedila 2000) wood anatomy (Marchiori 1993) cytogenetic 71
parameters (Olkoski and Wittmann 2011) and allelopathic potential (Jacobi and Ferreira 72
1991 Ferreira et al 1992) However excluding two recent publications on maricaacute 73
constitutive chemical composition (Schlickmann et al 2017 Pilatti et al 2019) which 74
identified phenolic compounds (methyl gallate and water-soluble tannins) as its major 75
compounds little is known regarding this subject In other Mimosa species (eg M pudica 76
and M pigra) mimosine has been identified (Soedarjo and Borthakur 1998) as one of the 77
major specialized metabolites present in the different organs of the plant (Champanerkar et 78
al 2010) The presence of this molecule was also reported for M bimucronata in a thin layer 79
chromatography-based preliminary study performed by Ferreira et al (1992) showing co-80
chromatography of a leaf extract component with authentic mimosine The authors attributed 81
the allelopathic effect of maricaacute to the accumulation of this metabolite in its leaves 82
Mimosine is an aromatic non-protein amino acid initially found in plants of Mimosa 83
pudica and later in Leucaena leucocephala (Lam) de Wit (Soedarjo and Borthakur 1998) a 84
leguminous tree which biosynthesizes large amounts of this nitrogen-containing compound 85
(Rodrigues-Correcirca et al 2019) It is believed that the accumulation of high contents of 86
mimosine in L leucocephala tissues confers among other traits defense against herbivores 87
and pathogens (Vestena et al 2001) tolerance to drought (Negi et al 2014) as well as 88
general oxidative stress protection (Rodrigues-Correcirca et al 2019) Interestingly drought is 89
the opposite environmental and physiological condition to that observed in the wet habitats 90
occupied by native populations of M bimucronata in Brazil (Patreze and Cordeiro 2004 91
Kestring et al 2009) and Mimosa pudica Linn in India (Champanerkar et al 2010) 92
70
Nonetheless flooding is also associated with oxidative stress particularly as water levels 93
change (Fukao et al 2019) 94
In Leucaena leucocephala var leucocephala (common leucaena) and Leucaena 95
leucocephala var glabrata (giant leucaena) mimosine accumulation has been shown to be 96
both constitutive and inducible by stress-related phytohormones such as jasmonic acid (JA) 97
Ethephon (ETH an ethylene- releasing compound) salicylic acid (SA - only common 98
leucaena) (Vestena et al 2001) as well as by UV-C radiation (Xu et al 2018 Rodrigues-99
Correcirca et al 2019) On the other hand there is a lack of information regarding mimosine 100
content and elicitation effects in Mimosa spp plants 101
The aim of this study was to examine the presence of mimosine in Mimosa 102
bimucronata and examine the effects of stresses and stress-signaling molecules on its 103
accumulation in leaves 104
Material and Methods 105
Plant material 106
For all experiments the plant material was collected at Morro Santana campus do 107
Vale of UFRGS (Federal University of Rio Grande do Sul) Porto Alegre RS Brazil 108
(3004rsquoS 5108rsquoW) Authorization for access to genetic material was obtained from 109
SISGEN-Brazil (license number A845493) Constitutive mimosine content in adult plants of 110
M bimucronata var bimucronata (DC) Kuntze was determined in plant material (leaves 111
green flower buds post-anthesis flowers and green pods) harvested in January 2017 112
(summer) A voucher herbarium specimen (ICN 187953) was deposited in the ICN ndash UFRGS 113
herbarium (Herbaacuterio do Instituto de Biociecircncias of UFRGS) 114
71
For mimosine elicitation experiments legumes (fruits) of maricaacute were collected in 115
the end of June 2017 (winter) Seeds were then removed from the dry fruits and kept in the 116
dark until sowing and seedling development for use in the assays 117
Seed germination 118
To break the coat-imposed seed dormancy after surface sterilization dry seeds of 119
maricaacute were acid scarified by immersion in H2SO4 (95 ndash 98 ) for 2 min (see Correcirca et al 120
2008) and repeatedly washed in distilled water to remove any residue of the acid Then seeds 121
were distributed in 50 mL individual plastic tubes (dibble-tubes) (30 cm diameter x 120 cm 122
depth) filled up with 11 (vv) of commercial top soil and vermiculite Tubes were watered 123
every 2 days to avoid substrate dryness and were kept in a growth room under controlled 124
conditions of light (circa 75 μmol mminus2s minus1 photosynthetically active radiation photoperiod 125
of 16 h light and 8 h dark) and temperature (24plusmn2C) 126
127
Treatments 128
In order to verify inducibility of mimosine accumulation in M bimucronata fifty 12-129
week-old maricaacute seedlings (per treatment) exhibiting similar features were selected and 130
sprayed (saturated) with solutions of different chemical stressors (plant specialized 131
metabolism elicitors) as follows (for further details see Rodrigues-Correcirca et al 2019) 10 132
and 50 mM SA (pathogen-signaling molecule Shah 2003) 007 and 035 mM 2-133
chloroethylphosphonic acid (ETH ethylene releasing-compound Kim et al 2016 Wang et 134
al 2016) 100 and 200 mM MeJA (Dar et al 2015) 10 and 50 mM SNP (a nitric oxide 135
donor Perotti et al 2015) Alternatively maricaacute seedlings were also supplemented with UV-136
C radiation (13 minutes 105 kJ cm2) (elicitor of plant specialized metabolism Kara 2013) 137
72
After 2 and 4 days of exposure to the chemical treatments and 3 and 6 days of UV-138
C supplementation maricaacute shoots were harvested immediately frozen in liquid nitrogen and 139
stored at ndash 80 C until mimosine extraction and HPLC analyses 140
Mimosine extraction and detection 141
Mimosine extraction was conducted according to the modified protocol described by 142
Rodrigues-Correcirca et al (2019) for L leucocephala HPLC (Thermo Scientific Surveyor) 143
analyses (mimosine detection and quantification) were performed following previously 144
published procedures (Negi et al 2014) A C18 column (ACE C18 5 μm 46times250 mm) and 145
isocratic solvent system of 002M o-phosphoric acid with a linear flow rate of 1 mL min minus1 146
were used to separate and quantify the amino acid Mimosine detection was performed at 280 147
nm by photodiode array detection (200ndash400 nm) and retention time (229plusmn0024 min) 148
Mimosine quantification was done by means of the method of external standard curve 149
Additional confirmation of mimosine identity was performed by co-chromatography with 150
standard (Acros Organics authentic mimosine 99 used as reference) and peak purity check 151
The analyses of the chromatograms were done with the ChromQuest software 152
153
154
Results and Discussion 155
Constitutive accumulation of mimosine in M bimucronata 156
Mimosine was detected in all analyzed samples positively meeting all identification 157
criteria In agreement with what has been found for other Mimosa spp (Soedarjo and 158
Borthakur 1998) compared to L leucocephala adult plants (Rodrigues-Correcirca 2019) 159
mimosine content was lower in M bimucronata Of the adult plant tissues analyzed the 160
73
highest content of mimosine in maricaacute (per gram of fresh weight - FW) was found in post-161
anthesis flowers (36644 microg versus 89448 microg in common leucaena followed by leaves 162
(28838 microg x 67358 microg) green flower buds (28094 microg x 51247 microg) and green pods (19002 163
microg x 82687 microg) (Fig 1)The same pattern is observed for seedlings when both species are 164
compared In this study untreated 12-week-old maricaacute seedlings (control at day 2) showed a 165
shoot content of mimosine of 23029plusmn007 microg g-1 of (FW) Five-week-old untreated giant 166
leucaena seedlings cultivated in similar conditions exhibited between 83640 and 178736 167
microg g-1 of FW (Rodrigues-Correcirca et al 2019) In the same way mimosine concentration 168
percentage in dry matter of Mimosa pigra was found to be rather low (002 in nodules and 169
roots and 007 in leaves) (Soedarjo and Borthakur 1998) 170
In this investigation the lowest constitutive mimosine content was found in green 171
pods (Fig 1) This result may partly explain the absence of phytotoxic effect observed for 172
green pods on germination and growth of crop target plants tested by Jacobi and Ferreira 173
(1991) compared to the other maricaacute parts analyzed 174
Elicitation of mimosine biosynthesis in M bimucronata 175
Chemical stressors 176
Secondary metabolites (or natural products) are structural- and chemically 177
specialized compounds derived from primary metabolism These molecules are mainly 178
biosynthesized as part of a complex defense mechanism in response to biotic and abiotic 179
stresses such as pathogens herbivores water status metal toxicity and UV radiation for 180
example (Matsuura et al 2018) Ethephon SA SNP MeJA have been extensively used as 181
chemical elicitors of specialized metabolism (Wang et al 2016 Vestena et al 2001 Perotti 182
74
et al 2015 Zhang and Memelink 2009 Xu et al 2018) These phytohormonal signals can 183
simulate environmental challenges and modulate plant homeostasis often leading to 184
alterations in gene expression (Shinozaki et al 2015) Except SNP all treatments tested in 185
the present study showed positive effect on mimosine accumulation in common or giant 186
leucaena (Vestena et al 2001 Rodrigues-Correcirca 2019 Rodrigues-Correcirca unpublished 187
data) However in spite of the trend of increasing the mimosine content observed in seedlings 188
treated with 007 mM Ethephon (at day 2) and 100 mM MeJA (at day 4) no statistical 189
difference was confirmed for these treatments when compared to the control 190
On the other hand a within treatment difference on mimosine induction was seen 191
between day 2 and 4 in seedlings treated with 100 mM MeJA (Fig 2) In a lower 192
concentration (04 mM) jasmonic acid (JA)promoted a near threefold increase in mimosine 193
accumulation of giant leucaena seedlings after 2 days of application 194
UV-C radiation 195
Albeit UV-C radiation is not biologically active in natural environments it has been 196
widely used under controlled experimental conditions to generate acute responses of plant 197
specialized metabolism within a shorter period of time compared to that required to with UV-198
B radiation (Kara 2013 Cetin 2014) This fast response is due to the higher energy of UV-199
C photons that act as potent reactive oxygen species (ROS) generators causing extensive 200
damage to the cells either at the physiological level or on DNA structure (Gregianini et al 201
2003 Matsuura et al 2013) 202
Although divergent responses can be observed in plants exposed to UV-C radiation 203
the deleterious processes are usually reported on primary metabolism (decreasing of 204
chlorophyll content and plant height eg) (Kara 2013) In the present study no statistical 205
75
differences were observed in the mimosine concentration in maricaacute seedlings supplemented 206
with UV-C radiation However a decreasing in its content was found for both control and 207
treatment at day 6 post-treatment (Fig 03) Taking into account the lower constitutive 208
concentration of mimosine observed in maricaacute compared to the leucaena plants besides its 209
relative thermolability (Nguyen and Tawata 2016) it seems to be plausible to consider the 210
effect of the temperature inside the UV-C and the white light (control) chambers as an 211
additional abiotic factor contributing to the decrease of mimosine accumulation in both group 212
of plants 213
Besides mimosine identification the presence of 34-dihydroxypyridine (34-DHP or 214
3-hydroxy-4-pyridone - 3H4P) a mimosine degradation product (Negi et al 2014 Nguyen 215
and Tawata 2016) was also reported for maricaacute leaf extracts analyzed by TLC by Ferreira 216
et al (1992) In our chromatograms we detected a second large peak after that of mimosine 217
(229plusmn0024) and similar to that identified by Negi et al (2014) as 3H4P (data not shown) 218
Comparing the chromatogram profiles obtained from seedlings elicited with chemical 219
stressors and those supplemented with UV-C the largest area for this peak was found (in all 220
samples) in the latter treatment at day 6 It might indicate that the constitutive andor the 221
initially UV-C-induced mimosine was degraded into 3H4P to cope with the cellular damage 222
caused by this treatment associated with an increased temperature inside the chambers 223
Nevertheless it was not possible to determine 3H4P concentration (or confirm its identity) 224
in maricaacute plants since there is no commercial standard (pure 3H4P) available for purchase 225
to be used as a reference in calculations Establishment of improved protocols for obtaining 226
in house 3H4P reference substance by acid hydrolysis is ongoing 227
228
229
76
Conclusion 230
On the basis of the overall absence of effect of the treatments tested here on mimosine 231
concentration it is possible to suggest that its accumulation profile is similar to that of 232
phytoanticipins unlike what is observed for the same amino acid production in leucaena 233
which shows features of inducibility resembling phytoalexin-like metabolites Alternatively 234
a putative inducible pool of mimosine in maricaacute might be involved in other types of stress 235
such as extended drought periods If involved in protection against oxidative stress as 236
described for leucaena mimosine in maricaacute may act predominantly by physical quenching 237
of ROS as indicated by the lack of overt chemical degradation Nevertheless further 238
investigations are needed to assess these hypotheses 239
To sum up mimosine biosynthesis was not modulated by the treatments evaluated as 240
in L leucocephala (Lam) de Wit To the best of our knowledge this is the first work that 241
analytically identifies and quantifies mimosine accumulation in M bimucronata 242
243
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Bitencourt F Zocche JJ Costa S Souza PZ Mendes AR 2007 Nucleaccedilatildeo de 245
Mimosa bimucronata (DC) O Kuntze em aacutereas degradadas pela mineraccedilatildeo de carvatildeo R 246
Bras Bioci 5 750-752 247
Carvalho PER 2004 Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 248
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Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitis 250
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6287-47-37 252
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Champanerkar PA Vaidya VV Shailajan S Menon SN 2010 A sensitive rapid and 253
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httpsdoiorg104236ns201027088 256
Gomes GS Silva GS Silva DLS Oliveira RR Conceiccedilatildeo GM 2018 Botanical 257
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Correcirca LR Soares GLG Fett-Neto AG 2008 Allelopathic potential of Psychotria 260
leiocarpa a dominant understorey species of subtropical forests S Afri J Bot 74 583ndash261
590 httpsdoiorg101016jsajb200802006 262
Ferreira AG Aquila MEA Jacobi US Rizvi V 1992 Allelopathy in Brazil In Allelopathy 263
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Fukao T Barrera-Figueroa BE Juntawong P Pentildea-Castro JM 2019 Submergence 265
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Gregianini TS Silveira VC Porto DD Kerber VA Henriques AT Fett-Neto AG 268
2003 The alkaloid brachycerine is induced by ultraviolet radiation and is a singlet oxygen 269
quencher Photochem Photobiol 78(5) 470ndash474 httpsdoiorg1015620031-270
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Jacobi US Ferreira AG 1991 Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK 272
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Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant 274
(Phaseolus vulgaris) Biosci Res 10(1) 29ndash32 275
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Kestring D Klein J Menezes LCCR Rossi MN 2009 Imbibition phases and 276
germination response of Mimosa bimucronata (Fabaceae Mimosoideae) to water 277
submersion Aquat Bot 91 105ndash109 httpsdoiorg101016jaquabot200903004 278
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect of 279
Ethephon as an ethylene-releasing compound on the metabolic profile of Chlorella vulgaris 280
J Agric Food Chem 64(23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541 281
Lorenzi H 1998 Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 282
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 283
Lorenzi H 2008 Plantas daninhas do Brasil terrestres aquaacuteticas parasitas e toacutexicas 4 ed 284
Nova Odessa Instituto Plantarum 640 p 285
Marchiori JNC 1993 Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 286
O Kuntze Ciecircncia Florestal 3 85-106 287
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation of 288
bioactive secondary metabolites by ultraviolet radiation mechanisms strategies and 289
applications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology for Medicinal 290
Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash190= 291
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo R Bhambra AS 292
Strnad M Bonfill M Fett-Neto AG 2018 Specializedplant 293
metabolismcharacteristicsandimpactontargetmoleculebiotechnologicalproduction 294
Molecular Biotechnology 60(2) 169ndash183httpsdoiorg101007s12033-017-0056-1 295
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase from 296
Leucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 297
922ndash934 httpsdoiorg101104pp113230870 298
79
Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine 299
areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636 300
Olkoski D Wittmann MTS 2011 Cytogenetics of Mimosa bimucronata (DC) O Kuntze 301
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop Breed 302
Appl Biotechnol 11 27-35 httpdxdoiorg101590S1984-70332011000100004 303
Patreze CM Cordeiro L 2004 Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 304
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecol Manag 196 275ndash305
285 httpdxdoiorg101016jforeco200403034 306
Perotti JC Rodrigues-Correcirca KCS Fett-Neto AG 2015 Control of resin production in 307
Araucaria angustifolia an ancient South American conifer Plant Biology 17 852ndash859 308
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG 2019 Mimosine 309
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 310
UV exposure Plant Physiology and Biochemistry 135 432ndash440 311
Pilatti DM Fortes AMT Jorge TCM Boiago NP 2019 Comparison of the phytochemical 312
profiles of five native plant species in two different forest formations Brazilian Journal of 313
Biology 79(2) 233-242 314
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS 2011 Biologia da reproduccedilatildeo 315
deMimosa bimucronatandash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 316
Simon MF and Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 317
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 318
endemism Biological Conservation 96 279-296 319
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 320
LM Andrade SF Cechinel-Filho V 2017 Chemical composition and diuretic natriuretic and 321
80
kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its majority 322
constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash1624 323
Shah J 2003 The salicylic acid loop in plant defense Current Opinion Plant Biology6 (4) 324
365ndash371 325
Shinozaki K Uemura M Serres JB Bray EA Weretilnyk E 2015 Responses to Abiotic 326
Stress In Buchanan BB Gruissem W Jones RL (Eds) Biochemistry and Molecular 327
Biology of Plants Second Edition John Wiley and Sons Ltd 328
Soedarjo M and Borthakur D 1998 Mimosine a toxin produced by the tree-legume 329
Leucaena provides a nodulation competition advantage to mimosine-degrading Rhizobium 330
strains Soil Biology and Biochemistry 30(12)1605-1613 331
Vestena S Fett-Neto AG Duarte RC Ferreira AG 2001 Regulation of mimosine 332
accumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 333
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene induced 334
vinblastine accumulation is related to activated expression of downstream TIA pathway 335
genes in Catharanthus roseus BioMed Research International Article ID 3708187 336
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX Tsim KWK 2018 337
Jasmonate-elicited stress induces metabolic change in the leaves of Leucaena leucocephala 338
Molecules 23 (2) 339
Zhang H Memelink J 2009 Regulation of Secondary Metabolism by Jasmonate Hormones 340
In AE Osbourn and V Lanzotti (eds) Plant-derived Natural Products 3 DOI 101007978-341
0-387-85498-4_1 copy Springer Science + Business Media LLC 342
343
344
345
81
346
Figure 1 Constitutive concentration of mimosine in different plant organs of Mimosa 347
bimucronata Bars sharing the same letter do not differ statistically by Tukey test (Ple005) 348
The error bars denote standard error of 10 replicates 349
350
351
352
353
354
355
356
357
B B A C0
5
10
15
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30
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40
LEAVES GREEN FLOWER BUDS POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
co
nce
ntr
atio
n u
gg-1
Mimosine concentration in adult plants of Mimosa bimucronata (DC) Kuntze
82
C T R L S A
1 0 m M
S A
5 0 m M
E T H
0 0 7 m M
E T H
0 3 5 m M
M e J A
1 0 0 m M
M e J A
2 0 0 m M
S N P
1 0 m M
S N P
5 0 m M
0
1 0
2 0
3 0
T re a tm e n ts
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1) D A Y 2
D A Y 4
A B C C B C A B C C A B C A B C A
a b b b a a b a a b b a b
358
Figure 2 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 359
bimucronata treated with different signaling molecules SA = Salicylic Acid ETH = 360
Ethephon MeJA = Methyl Jasmonate SNP = Sodium Nitroprusside Uppercase and 361
lowercase letters indicate statistical differences among treatments in days 2 and 4 362
respectively Bars sharing a letter of the same case do not differ statistically by Tukey test 363
(Ple005) Indicates statistical difference in the same treatment between day 2 and 4 by t-364
test (Ple005) The error bars denote standard error of 5 replicates (25 individual seedlings 365
arranged in 5 groups of 5) 366
367
368
83
D AY 3 D AY 6
0
5
1 0
1 5
2 0
2 5
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1)
C O N TR O L
U V -C
369
Figure 3 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 370
bimucronata supplemented with UV-C radiation Indicates statistical difference in the same 371
treatment between day 3 and 6 by t-test (Ple005) The error bars denote standard error of 5 372
replicates (25 individual seedlings arranged in 5 groups of 5) 373
374
375
376
377
378
379
380
381
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84
Consideraccedilotildees finais 386
- Experimentos que avaliam os efeitos da aplicaccedilatildeo exoacutegena de ANPs em diferentes espeacutecies 387
vegetais tecircm sido realizados principalmente com GABA Dentre os principais efeitos 388
conferidos pela aplicaccedilatildeo dessa moleacutecula em espeacutecies de mono e eudicotiledocircneas satildeo 389
relatados a toleracircncia agrave seca agrave salinidade e agraves temperaturas extremas 390
- Como metaboacutelitos especializados claacutessicos os ANPs podem ter sua concentraccedilatildeo basal 391
endoacutegena aumentada em resposta agrave induccedilatildeo mediada por uma vasta gama de tratamentos com 392
moleacuteculas sinalizadoras de estresse e fontes alternativas de estressores De um modo geral 393
observa-se o acuacutemulo das diferentes classes de ANPs em resposta agrave radiaccedilatildeo UV elicitores 394
quiacutemicos que mimetizam ataques por patoacutegenos dano mecacircnico agentes osmoacuteticos metais 395
pesados entre outros 396
- Especificamente em leucena a resposta observada em relaccedilatildeo aos diferentes tratamentos 397
testados indica que apesar do seu alto teor constitutivo nessa espeacutecie a biossiacutentese e o 398
acuacutemulo de mimosina podem ser modulados por fatores causadores de estresses exibindo -399
nessa espeacutecie - um padratildeo de acumulaccedilatildeo similar agrave fitoalexinas Em maricaacute por outro lado 400
aumento de acuacutemulo dessa moleacutecula natildeo foi observado para os mesmos tratamentos testados 401
para leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas 402
- O padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno 403
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado pela 404
sua degradaccedilatildeo 405
- As respostas observadas nos testes que avaliaram a atividade de mitigaccedilatildeo de espeacutecies 406
reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode agir como um agente 407
antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de estresse 408
85
Perspectivas 409
- Confirmaccedilatildeo em espectrocircmetro de massas eou ressonacircncia nuclear magneacutetica da natureza 410
quiacutemica da lsquomimosinarsquo presente em maricaacute 411
- Avaliaccedilatildeo do efeito de concentraccedilotildees mais elevadas e em diferentes periacuteodos de aplicaccedilatildeo 412
das moleacuteculas sinalizadoras testadas sobre o acuacutemulo de mimosina em leucena e maricaacute 413
- Ampliar a investigaccedilatildeo dos padrotildees de expressatildeo gecircnica dos genes que codificam para 414
mimosinase (em maricaacute) mimosina sintase (em ambas as espeacutecies testadas) bem como o 415
perfil de precursores e cataboacutelitos de mimosina em resposta aos tratamentos mencionados 416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
86
Referecircncias Bibliograacuteficas 436
437
Acamovic T Brooker JD (2005) Biochemistry of plant secondary metabolites and their 438
effects in animals P Nutr Soc 64 403ndash412 httpsdoiorg101079PNS2005449 439
Ahmed R Hoque ATMR Hossain MK (2008) Allelopathic effects of Leucaena 440
leucocephala leaf litter on some forest and agricultural crops grown in nursery J Forestry 441
Res (2008) 19 298 httpsdoiorg101007s11676-008-0053-0 442
Ahmed AMM Saacutenchez FJS Bavileacutes LRY Mahdy REZ Camaal JBC (2016) Tannins and 443
mimosine in Leucaena genotypes and their relations to Leucaena resistance against 444
Leucaena Psyllid and Onion thrips Agroforestry Systems 1-8 445
Benjakul S Kittiphattanabawon P Shahidi F Maqsood S (2013) Antioxidant activity and 446
inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipid oxidation in 447
model systems Food Sci Technol Int 19(4)365-76 448
httpsdoiorg1011771082013212455186 449
Bitencourt F Zocche JJ Costa S Souza PZ Mendes AR (2007) Nucleaccedilatildeo de Mimosa 450
bimucronata (DC) O Kuntze em aacutereas degradadas pela mineraccedilatildeo de carvatildeo Revista 451
Brasileira de Biociecircncias 5 750-752 452
Bottini-Luzardo M Aguilar-Perez C Centurion-Castro F Solorio-Sanchez F Ayala-Burgos 453
A Montes-Perez R Muntildeoz-Rodriguez D Ku-Vera J (2015) Ovarian activity and estrus 454
behavior in early postpartum cows grazing Leucaena leucocephala in the tropics Trop Anim 455
Health Prod 47(8)1481-6 456
Carvalho PER (2004) Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 457
Teacutecnica 941-10 458
Chowtivannakul P Srichaikul B Talubmook C (2016) Antidiabetic and antioxidant activities 459
of seed extract from Leucaena leucocephala (Lam) de Wit Agriculture and Natural 460
Resources 50 (2016) 357e361 httpdxdoiorg101016janres201606007 461
Chung H-H Chen M-K Chang Y-C Yang S-F Lin C-C Lin C-W (2017) Inhibitory effects 462
of Leucaena leucocephala on the metastasis and invasion of human oral cancer cells 463
Environmental Toxicology 321765ndash1774 httpsdoiorg101002tox22399 464
87
Crowe B Poynter JA Manukyan MC Wang Y Brewster BD Herrmann JL Abarbanell 465
AM Weil BR Meldrum DR (2001) Pretreatment with intracoronary mimosine improves 466
postischemic myocardial functional recovery Surgery 150(2) 191-106 467
Fallon (2015) Effects of mimosine on Wolbachia in mosquito cells cell cycle suppression 468
reduces bacterial abundance In Vitro Cell Dev Biol Anim 51(9)958-63 469
httpsdoiorg101007s11626-015-9918-7 Epub 2015 May 28 470
Fernaacutendez-Salas A Alonso-Diacuteaza MA Acosta-Rodriacuteguez A Torres-Acosta JFJ Sandoval-471
Castro CA Rodriacuteguez-Vivas RI (2011) In vitro acaricidal effect of tannin-rich plants against 472
the cattle tick Rhipicephalus (Boophilus) microplus (Acari Ixodidae) Veterinary 473
Parasitology 175113ndash118 2010 httpsdoiorg101016jvetpar201009016 474
Ferreira AG Aquila MEA Jacobi US Rizvi V (1992) Allelopathy in Brazil In Allelopathy 475
basic and applied aspects Rizvi V and Jacobi US (Eds) Chapman and Hall PP 243-250 476
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad Hossain Md Anai 477
T Oku H (2018) Cytosolic cysteine synthase switch cysteine and mimosine production in 478
Leucaena leucocephala Appl Biochem Biotechnol 186 (3) 613ndash632 479
httpsdoiorg101007s12010-018-2745-z 480
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of the thyrotoxic 481
amino acid mimosine by cysteine synthase Phytochemistry 29 (11) 3461ndash3465 482
httpsdoiorg1010160031-9422(90)85258-H 483
Jacobi US Ferreira AG (1991) Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK Sobre 484
espeacutecies cultivadas Pesquisa Agropecuaacuteria Brasileira 26(7) 935-943 485
Jamous RM Ali-Shtayeh MS Abu-Zaitoun SY Markovics A Azaizeh H (2017) Effects of 486
selected Palestinian plants on the in vitro exsheathment of the third stage larvae of 487
gastrointestinal nematodes BMC Veterinary Research 13308 488
httpdxdoiorg101186s12917-017-1237-7 489
Jiao CJ Jiang J-L Ke L-M Cheng W Li F-M Li Z-X Wang C-Y (2011) Factors affecting 490
β-ODAP content in Lathyrus sativus and their possible physiological mechanisms Food 491
Chem Toxicol 49 543ndash549 httpsdoiorg101016jfct201004050 492
Kubota S Fukumoto Y Ishibashi K Soeda S Kubota SS Yuki R Nakayama Y Aoyama K 493
Yamaguchi N (2014) Activation of the prereplication complex is blocked by mimosine 494
88
through reactive oxygen species-activated ataxia telangiectasia mutated (ATM) protein 495
without DNA damage J Biol Chem 28 289(9)5730-46 496
Kuppusamy UR Arumugam B Azaman N Wai CJ (2014) Leucaena leucocephala Fruit 497
Aqueous Extract Stimulates Adipogenesis Lipolysis and Glucose Uptake in Primary Rat 498
Adipocytes Hindawi Publishing Corporation e Scientific World Journal Article ID 737263 499
8 pages httpdxdoiorg1011552014737263 500
Kusama-Eguchi K (2019) Research in motor neuron diseases caused by natural substances 501
focus on pathological mechanisms of neurolathyrism Yakugaku Zasshi 139 (4) 609-502
615 httpsdoiorg101248yakushi18-00202 503
Kutchan TM Gershenzon J Moslashller BL Gang DR (2015) Natural Products In Buchanan 504
BB Gruissem W and Jones RL (eds) Biochemistry amp Molecular Biology of Plants 2nd edn 505
Wiley Blackwell Chichester pp 1135-1205 506
Lalande M (1990) A reversible arrest point in the late G1 phase of the mammalian cell cycle 507
Exp Cell Res 186 332ndash339 508
Li X-W Hu C-P Li Y-J Gao Y-X Wang XM Yang J-R (2015) Inhibitory effect of L-509
mimosine on bleomycin-induced pulmonary fibrosis in rats Role of eIF3a and p27 Int 510
Immunopharmacol 27(1) 53ndash64 511
Little Jr EL Skolmen RG (1989) Koa haole Agriculture Handbook 679 USDA 512
Lorenzi H (1998) Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 513
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 514
Marchiori JNC (1993) Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 515
O Kuntze Ciecircncia Florestal 3 85-106 516
Mohammed RS El Souda SS Taie HAA Moharam ME Shaker KH (2015) Antioxidant 517
antimicrobial activities of flavonoids glycoside from Leucaena leucocephala leaves Journal 518
of Applied Pharmaceutical Science 5(06)138-147 519
httpdxdoiorg107324JAPS201550623 520
Negi VS Bingham J-P Li QX Borthakur D (2014) A carbon-nitrogen lyase from Leucaena 521
leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 (2) 922ndash522
934 httpsdoiorg101104pp113230870 523
89
Olkoski D Wittmann MTS (2011) Cytogenetics of Mimosa bimucronata (DC) O Kuntze 524
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop 525
Breeding and Applied Biotechnology 11 27-35 526
Patreze CM Cordeiro L (2004) Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 527
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecology and Management 528
196275ndash285 529
Pilatti DM Fortes AMT Jorge TCM Boiago NP (2019) Comparison of the phytochemical 530
profiles of five native plant species in two different forest formations Brazilian Journal of 531
Biology 79(2) 233-242 532
Ramos-Ruiz R Poirot E Flores-Mosquera M (2018) GABA a non-protein amino acid 533
ubiquitous in food matrices Cogent Food Agric 41534323 534
httpsdoiorg1010802331193220181534323 535
REFLORA (2019) httpfloradobrasiljbrjgovbrreflora Acesso em agosto de 2019 536
Rodgers KJ Samardzic K Main BJ (2015) Toxic Nonprotein Amino Acids Plant Toxins 537
httpsdoiorg 101007978-94-007-6728-7_9-1 538
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG (2019) Mimosine 539
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 540
UV exposure Plant Physiology and Biochemistry 135 432ndash440 541
httpsdoiorg101016jplaphy201811018 542
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 543
LM Andrade SF Cechinel-Filho V (2017) Chemical composition and diuretic natriuretic 544
and kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its 545
majority constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash546
1624 547
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS (2011) Biologia da reproduccedilatildeo 548
de Mimosa bimucronata ndash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 549
Simon MF Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 550
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 551
endemism Biological Conservation 96 279-296 552
90
Soares AMS Arauacutejo SA Lopes SG Costa Junior LM (2015) Anthelmintic activity of 553
Leucaena leucocephala protein extracts on Haemonchus contortus Braz J Vet Parasitol 554
Jaboticabal 24(4) 396-401 httpdxdoiorg101590S1984-29612015072 555
Soerdajo M Borthakur D (1998) Mimosine a toxin produced by the tree-legume Leucaena 556
provides a nodulation competition advantage to mimosine-degrading Rhizobium strains Soil 557
Biol Biochem 30(12) 16051613 558
Souza-Lima ES Sinani TR Pott A Sartori ALB (2017) Mimosoideae (Leguminosae) in the 559
Brazilian Chaco of Porto Murtinho Mato Grosso do Sul Rodrigueacutesia 68(1) 263-290 2017 560
httpdxdoiorg1015902175-7860201768131 561
Taiz L amp Zeiger E (2010) Plant Physiology 5th edition Sinauer Associates Inc Sunderland 562
Verma VK Rani KV Kumara SR Prakash O (2018) Leucaena leucocephala pod seed 563
protein as an alternate to animal protein in fish feed and evaluation of its role to fight against 564
infection caused by Vibrio harveyi and Pseudomonas aeruginosa Fish and Shellfish 565
Immunology 76 (2018) 324ndash332 httpsdoiorg101016jfsi201803011 566
Yafuso JT Negi VS Bingham J-P Borthakur D (2014) An O-acetylserine (thiol) lyase from 567
Leucaena leucocephala is a cysteine synthase but not a mimosine synthase Appl Biochem 568
Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z 569
Zarin RMA Wan HY Isha A Armani N (2016) Antioxidant antimicrobial and cytotoxic 570
potential of condensed tannins from Leucaena leucocephala hybrid Food Science and 571
Human Wellness 5 65ndash75 httpdxdoiorg101016jfshw201602001 572
573
574
Contents lists available at ScienceDirect
Industrial Crops amp Productsjournal homepage wwwelseviercomlocateindcrop
Resin tapping transcriptome in adult slash pine (Pinus elliottii var elliottii)Camila Fernanda de Oliveira Junkes1 Artur Teixeira de Arauacutejo Juacutenior1 Juacutelio Ceacutesar de LimaFernanda de Costa Thanise Fuumlller Maacutercia Rodrigues de Almeida Franciele Antocircnia NeisKelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-NetoCenter for Biotechnology and Department of Botany Federal University of Rio Grande do Sul Porto Alegre PO Box 15005 91501-970 Brazil
A R T I C L E I N F O
KeywordsPinus elliottiResinResinosisTranscriptomeAdjuvant paste
A B S T R A C T
To better understand the bases of resin production a major source of terpenes for industry the transcriptome ofadult Pinus elliottii var elliottii (slash pine) trees under field commercial resinosis was obtained Samples werecollected from cambium after 5 and 15 days of treatment application which included tapping followed byapplication of commercial resin stimulant paste or control wounding without paste Overall mean number ofreads of all 16 libraries (2 treatments x 2 times x 4 replicated trees) was 34582048 Of these 89 were mappedagainst the reference sequence with a mismatch of 058 Using the Blast2Go 570 candidate genes were de-tected based on sequence annotation By comparing the expression profile between paste and control 310differentially expressed genes (DEGs) were identified at 5 days and 190 at 15 days with a significant fold changeof log2gt 12 Regarding changes in time comparisons within each treatment 210 and 105 DEGs were identifiedwithin control and paste treatment respectively Genes with different expression patterns in the times andtreatments examined included ethylene responsive transcription factors geranylgeranyl diphosphate synthasediterpene synthase cytochrome P450 and ABC transporters all of which may play important roles in resinproduction RT-qPCR analysis correlated well with the data obtained by RNAseq Resin composition changedover time This is the first transcriptomic investigation of resinosis of the main species used in the bioresinindustry and of molecular analyses of resinosis under field operations with implications for stand managementstimulant paste development genotype selection and breeding for high resinosis
1 Introduction
The adaptive success of conifers is largely due to the development ofa defense system based on the synthesis and secretion of terpenes in allmajor organs and different tissues (Miller et al 2005 Hall et al 2013Warren et al 2015) Conifer resin is a viscous fluid composed of acomplex mixture of terpenoids such as monoterpenes sesquiterpenesand diterpenes (Zulak and Bohlmann 2010) These terpenoids are se-creted from severed resin ducts when the tree is under biotic attack(Ralph et al 2006 Lange 2015 Geisler et al 2016) acting as pro-tectants (Schiebe et al 2012 Liu et al 2015)Biosynthesis of terpenes in conifers starts from isomerization of two
isoprenoid (C5) units dimethylallyl diphosphate (DMAPP) and iso-pentenyl diphosphate (IPP) These molecules can be biosynthesized viatwo separate routes in plants the methyl-erythritol 4-phosphate andmevalonate pathways IPP is synthesized and isomerized to DMAPP byisopentenyl diphosphate isomerase then prenyl transferases catalyze
the condensation of these two C5-units to geranyl diphosphate (Pazoukiand Niinemets 2016) Their elongation to prenyl diphosphates withaddition of IPP molecules leads to monoterpenes (C10) sesquiterpenes(C15) and diterpenes (C20) which are the substrates for terpene syn-thases (TPS) (Keeling and Bohlmann 2006b)TPSs are part of a large family of mechanistically related enzymes
involved in both primary and secondary metabolism (Keeling andBohlmann 2006b) The events of evolutionary diversification and ex-pansion of plant TPSs appear to have originated from gene duplicationsdomain losses and sub- or neofunctionalizations with subsequent di-vergence of an ancestral TPS gene of primary metabolism (Hall et al2013) Modification of TPS products changes their physical propertiesand may alter their biological activities (Chen et al 2011) TPSs of highsequence identity may have different functions even in closely relatedspecies Low sequence identity of TPSs in phylogenetically distantspecies does not preclude the possibility of independent evolution of thesame or related function of these enzymes (Zerbe and Bohlmann 2015)
httpsdoiorg101016jindcrop2019111545Received 4 January 2019 Received in revised form 10 June 2019 Accepted 4 July 2019
Corresponding authorE-mail address fettnetocbiotufrgsbr (AG Fett-Neto)1 These authors have equally contributed to this work
doi 1015900102-33062019abb0114
Acta Botanica Brasilica
Sustainable production of bioactive alkaloids in Psychotria L of
southern Brazil propagation and elicitation strategies
Yve Verocircnica da Silva Magedans1 Kelly Cristine da Silva Rodrigues-Correcirca1 Cibele Tesser da Costa1
Heacutelio Nitta Matsuura1 and Arthur Germano Fett-Neto1
Received April 1 2019Accepted June 28 2019
ABSTRACTPsychotria is the largest genus in Rubiaceae South American species of the genus are promising sources of natural
products mostly due to bioactive monoterpene indole alkaloids they accumulate ese alkaloids can have analgesic
antimutagenic and antioxidant activities in dierent experimental models among other pharmacological properties
of interest Propagation of genotypes with relevant pharmaceutical interest is important for obtaining natural
products in a sustainable and standardized fashion Besides the clonal propagation of elite individuals the alkaloid
content of Psychotria spp can also be increased by applying moderate stressors or stress-signaling molecules is
review explores advances in research on methods for plant propagation and elicitation techniques for obtaining
bioactive alkaloids from Psychotria spp of the South Region of Brazil
Keywords abiotic stress alkaloids elicitation monoterpenes plant propagation Psychotria southern Brazil
sustainability
Introduction
Psychotria belongs to Rubiaceae one of the major families
of $owering plants having economic interest e family
includes coee a few signicant poisonous plants to livestock
besides several important ornamental and medicinal species
(Souza amp Lorenzi 2012) Psychotria has captured researchersrsquo
attention mostly because of its medicinal properties
Psychotria colorata is an Amazonian species that produces
polyindolinic alkaloids with analgesic activity (Matsuura et
al 2013) e promising results obtained with P colorata
motivated the investigation of southern Brazilian Psychotria
species and the discovery of new bioactive alkaloids (Porto
et al 2009) Moreover leads on in planta alkaloid functions
were also topic of experimental evaluation
One of the key elements that needs to be addressed early
on during the process of developing new bioactive molecules
from plants is the capacity to generate catalytically active
biomass to support extraction and steady supply ere are a
number of ways through which these goals may be reached
including greenhouse rooting of cuttings (mini-cutting
1 Laboratoacuterio de Fisiologia Vegetal Departamento de Botacircnica Instituto de Biociecircncias e Centro de Biotecnologia Universidade Federal do Rio
Grande do Sul 91501-970 Porto Alegre RS Brazil
Corresponding author fettnetocbiotufrgsbr
Review
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Biomass yield of resin in adult Pinus elliottii Engelm trees is differentially
regulated by environmental factors and biochemical effectors
Franciele Antocircnia Neis Fernanda de Costa Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima
Kelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-Neto
Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) CP 15005 CEP 91501-970 Porto Alegre RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Biomass
Terpene resin
Seasonal
Benzoic acid
Regenerated forest
A B S T R A C T
Biomass of pine resin finds several applications in the chemical pharmaceutical biofuel and food industries
Resin exudation after injury is a key defense response in Pinaceae since this complex mixture of terpenes has
insecticidal antimicrobial and wound repair properties Resin yield is increased by effectors applied on the
wound area including phytohormones and metal cofactors of terpene synthases The interaction of resinosis
mechanism effectors is not fully understood particularly in adult forest setups under natural environmental
variations The aim of this work was to determine how resin exudation by wounded trunks of adult P elliottii
responded to combined chemical effectors involved in different regulatory pathways of resinosis (metal cofactors
of terpene synthases benzoic acid and plant growth regulators) and whether seasonal and tree distribution
variations affected these responses Symmetrically planted and scattered trees regenerated from the seed bank
had similar resin biomass yields suggesting that the homogeneity in development and spatial arrangement were
not significant factors in resin yield This new finding is of practical importance with the used tapping system
since costs of implanting forests by regeneration can be advantageous compared to planting In addition it was
shown for the first time that the salicylic acid precursor benzoic acid and the auxin naphthalene acetic acid
promoted resin exudation when individually applied to wound sites Both these adjuvants are two orders of
magnitude less costly compared to the conventionally used ethylene precursors besides facing less environ-
mental and health restrictions for use Most adjuvant-treated trees showed higher resin flow in the second year
indicating mechanisms of response build up Overall temperature was more important than rainfall as en-
vironmental parameter affecting resin biosynthesis which was higher in the warmer months of spring and
summer The combination of resinosis stimulant effectors from different signaling pathways showed no sig-
nificant synergistic or additive effect suggesting possible converging signaling pathways andor limitation of
common intermediate transducing molecules
1 Introduction
Pines occupy highly diverse environments over a range of tem-
peratures water and nutrient availabilities irradiance levels and pho-
toperiods being able to effectively face attacks from diverse herbivore
and pathogen guilds The success of conifers is linked to their complex
terpene biochemistry hosted by specialized secretory cells The terpe-
noid resin synthesized by Pinus spp is one of the main mechanisms of
defense of these trees particularly against bark beetles and the fungi
they carry (Fett-Neto and Rodrigues-Correcirca 2012) Pine resin biomass
is essentially composed of a monoterpene and sesquiterpene-rich tur-
pentine and diterpenoid-rich rosin fraction both finding numerous in-
dustrial applications as non-wood forest products (Rodrigues-Correcirca
et al 2012)
Molecules capable of modulating different signaling pathways have
been identified as resin yield stimulators including sulfuric acid (ex-
tends wound damage) 2-chloroethylphosphonic acid (CEPA a syn-
thetic ethylene precursor) paraquat (free radical generator) yeast ex-
tract (mimics attack by pathogens) salicylic acid (pathogen signaling
molecule) auxin (promotes ethylene biosynthesis and resin canal dif-
ferentiation) jasmonic acid (signals mechanical damage and promotes
secondary metabolism) and metal ions such as potassium iron and
manganese (cofactors of terpene synthases in conifers) and copper (a
component of ethylene receptors) (Clements 1970 Conrath et al
2002 Fett-Neto and Rodrigues-Correcirca 2012 Hudgins and Franceschi
2004 Lewinsohn et al 1994 Martin et al 2002 Popp et al 1995
httpsdoiorg101016jindcrop201803027
Received 12 December 2017 Received in revised form 9 March 2018 Accepted 13 March 2018
Corresponding author
E-mail addresses franci_neisyahoocombr (FA Neis) fernandadecostayahoocombr (F de Costa) thanisenfyahoocombr (TN Fuumlller)
jjuliocesarlimagmailcom (JC de Lima) krodriguescbiotufrgsbr (KC da Silva Rodrigues-Correcirca) jpfettcbiotufrgsbr (JP Fett) fettnetocbiotufrgsbr (AG Fett-Neto)
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Research Paper
Dual allelopathic effects of subtropical slash pine (Pinus elliottii Engelm)
needles Leads for using a large biomass reservoir
Kelly Cristine da Silva Rodrigues-Correcircaa Gelson Halmenschlagera Joseacuteli Schwambachb
Fernanda de Costaa Emili Mezzomo-Trevizana Arthur Germano Fett-Netoa
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 Brazilb University of Caxias do Sul Institute of Biotechnology Caxias do Sul RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Seasonality
Growth
Germination
Litter
Substrate
A B S T R A C T
Pinus elliottii Engelm (slash pine) is distributed along the maritime coast of Southern Brazil where it shows
invasive pattern and typical allelopathic features Large quantities of needle litter are produced by pine trees a
biomass that is little explored in areas where this species is alien Little is known about the dynamics of needle
and litter phytochemical interactions particularly in subtropical environments To elucidate the full range of
needle and litter allelopathic potential the effects of litter (superficial and deep) and seasonally harvested fresh
slash pine needles stored for different times were evaluated against lettuce tomato and cucumber seeds and
seedlings Increasing concentrations (0 1 2 4 and 8 wv) of hot and cold aqueous extracts of needles
and litter affected in different ways target plant development Growth and germination inhibition were directly
related to the highest extract concentrations (regardless of the season and mainly in hot water extracts) of
needles On the other hand stimulatory effects of litter extracts on lettuce growth were observed Growth and
germination of cucumber and tomato were not affected by pine litter as substrate when compared to rice husk
The presumable high polarity and thermal stability of slash pine leaf biomass allelochemicals and their transient
toxic effect or growth promoting impact suggest potential applications of this largely available biomass both as a
biological herbicide and growth substrate in plant propagation
1 Introduction
Native from the Northern Hemisphere Pinus is one of the most
widely distributed genera throughout different climate regions of the
globe growing either as native or alien species even in extreme habi-
tats (Rodrigues-Correcirca and Fett-Neto 2012) Despite the high economic
value currently attributed to pine wood and oleoresin (Rodrigues-
Correcirca et al 2012) there is increasing concern about the aggressive
potential of invasiveness displayed by Pinus species especially those
cultivated out of their native range of distribution (Richardson et al
2008 Rolon et al 2011) These species are dispersed by wind and there
is notably low plant diversity observed in most understories of pine
plantations (Kato-Noguchi et al 2009) This latter feature has been
considered an important trait of allelopathic interference
The term ldquoallelopathyrdquo was coined by Molisch in 1937 as a chemical
reciprocal interaction established among plants (including micro-
organisms) sharing the same site by means of the release of secondary
metabolites named allelochemicals (Rice 1984) For the most part
these metabolites are derived from the shikimic acid or isoprenoid
pathway and their biosynthesis can be modulated by biotic and abiotic
stresses (Nascimento and Fett-Neto 2010) including seasonal-related
changes (Sartor et al 2013) Allelopathy studies may range from sterile
assays (Aryakia et al 2015) to soil (Correcirca et al 2008 Sharma et al
2016) and field tests being a complex biological phenomenon to as-
certain in several circumstances due to issues of solubility release
mechanisms and stability of bioactive compounds (Scognamiglio et al
2013) Often the use of complementary methods provides more in-
formative data
The allelopathic effects of soil leachates green needles and litter
extracts of Pinus spp on germination and seedling growth aspects of
wild and crop species have been evaluated in natural and cultivated
pine stands and have proven to be stimulatory or inhibitory (Lodhi and
Killingbeck 1982 Kil and Yim 1983 Nektarios et al 2005 Akkaya
et al 2006 Machado 2007 Alrababah et al 2009 Sartor et al 2009
Kato-Noguchi et al 2011 Rolon et al 2011 Valera-Burgos et al
2012) exhibiting in some cases autotoxicity (Garnett et al 2004
Fernandez et al 2008 Zhu et al 2009 Monnier et al 2011) Studies
on potential dual allelopathic effects of Pinus elliottii Engelm (slash
httpdxdoiorg101016jindcrop201706019
Received 23 March 2017 Received in revised form 15 May 2017 Accepted 7 June 2017
Corresponding author
E-mail address fettnetocbiotufrgsbr (AG Fett-Neto)
ORIGINAL RESEARCHpublished 16 June 2016
doi 103389fpls201600849
Frontiers in Plant Science | wwwfrontiersinorg 1 June 2016 | Volume 7 | Article 849
Edited by
Juan Francisco Jimenez Bremont
Instituto Potosino de Investigacioacuten
Cientiacutefica y Tecnoloacutegica Mexico
Reviewed by
Mariacutea De La Luz Guerrero Gonzaacutelez
Universidad Autoacutenoma de San Luis
Potosiacute Mexico
Rosalia Cristina Paz
CIGEOBIO (CONICETFCEFN UNSJ)
Argentina
Correspondence
Arthur G Fett-Neto
fettnetocbiotufrgsbr
daggerThese authors have contributed
equally to this work
Specialty section
This article was submitted to
Plant Physiology
a section of the journal
Frontiers in Plant Science
Received 08 December 2015
Accepted 30 May 2016
Published 16 June 2016
Citation
de Lima JC de Costa F Fuumlller TN
Rodrigues-Correcirca KCdS Kerber MR
Lima MS Fett JP and Fett-Neto AG
(2016) Reference Genes for qPCR
Analysis in Resin-Tapped Adult Slash
Pine As a Tool to Address the
Molecular Basis of Commercial
Resinosis Front Plant Sci 7849
doi 103389fpls201600849
Reference Genes for qPCR Analysisin Resin-Tapped Adult Slash Pine Asa Tool to Address the MolecularBasis of Commercial Resinosis
Juacutelio C de Lima 1dagger Fernanda de Costa 1 dagger Thanise N Fuumlller 1
Kelly C da Silva Rodrigues-Correcirca 2 Magnus R Kerber 1 Mariano S Lima 1
Janette P Fett 1 and Arthur G Fett-Neto 1
1 Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul
Porto Alegre Brazil 2 Biological Sciences Department Regional Integrated University of Alto Uruguai and Missotildees (URI-FW)
Frederico Westphalen Brazil
Pine oleoresin is a major source of terpenes consisting of turpentine (mono- and
sesquiterpenes) and rosin (diterpenes) fractions Higher oleoresin yields are of economic
interest since oleoresin derivatives make up a valuable source of materials for chemical
industries Oleoresin can be extracted from living trees often by the bark streak method
in which bark removal is done periodically followed by application of stimulant paste
containing sulfuric acid and other chemicals on the freshly wounded exposed surface
To better understand the molecular basis of chemically-stimulated and wound induced
oleoresin production we evaluated the stability of 11 putative reference genes for the
purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis
Samples for RNA extraction were collected from field-grown adult trees under tapping
operations using stimulant pastes with different compositions and at various time points
after paste application Statistical methods established by geNorm NormFinder and
BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3
and UBI To confirm expression stability of the candidate reference genes expression
profiles of putative P elliottii orthologs of resin biosynthesis-related genes encoding Pinus
contorta β-pinene synthase [PcTPS-(minus)β-pin1] P contorta levopimaradieneabietadiene
synthase (PcLAS1) Pinus taeda α-pinene synthase [PtTPS-(+)αpin] and P taeda
α-farnesene synthase (PtαFS) were examined following stimulant paste application
Increased oleoresin yields observed in stimulated treatments using phytohormone-based
pastes were consistent with higher expression of pinene synthases Overall the
expression of all genes examined matched the expected profiles of oleoresin-related
transcript changes reported for previously examined conifers
Keywords resin Pinus gene expression normalizer genes terpene synthase
19
Chapter 2
Stimulant Paste Preparation and Bark Streak Tapping Technique for Pine Oleoresin Extraction
Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
Tapping technique comprises the extraction of pine oleoresin a non-wood forest product consisting of a
complex mixture of mono sesqui and diterpenes biosynthesized and exuded as a defense response to
wounding Oleoresin is used to produce gum rosin turpentine and their multiple derivatives Oleoresin
yield and quality are objects of interest in pine tree biotechnology both in terms of environmental and
genetic control Monitoring these parameters in individual trees grown in the fi eld provides a means to
examine the control of terpene production in resin canals as well as the identifi cation of genetic-based
differences in resinosis A typical method of tapping involves the removal of bark and application of a
chemical stimulant on the wounded area Here we describe the methods for preparing the resin-stimulant
paste with different adjuvants as well as the bark streaking process in adult pine trees
Key words Oleoresin Pine Tapping Chemical stimulant Wounding
1 Introduction
Several conifer species produce oleoresin a complex mixture of isoprenoid compounds relevant for defense against herbivores and pathogens Two major fractions can be recognized in oleoresin (a) turpentine the volatile fraction containing mono- and sesquiter-penes and (b) rosin the nonvolatile diterpene fraction Oleoresin is a forest commodity of global interest fi nding applications in diverse industry sectors Rosin is used in adhesives printing ink manufacture and paper sizing Turpentine can be used either as a solvent for paints and varnishes or as a raw material for fraction-ation of high-value chemicals used in the pharmaceutical agro-chemical and food industry [ 1 ndash 3 ]
During the extraction activity resin is obtained from the tree in a similar way as rubber tree tapping which generally involves the
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_2 copy Springer Science+Business Media New York 2016
These authors have equally contributed to this work
fettnetocbiotufrgsbr
27
Chapter 3
A Modifi ed Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines
Juacutelio Ceacutesar de Lima Thanise Nogueira Fuumlller Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes
through high-throughput sequencing technologies microarray and also northern blots RT-PCR and
RTqPCR Even though many specifi c protocols designed for plants with high content of secondary metab-
olites have been developed these are often expensive time consuming and not suitable for a wide range
of tissues Here we present a modifi cation of the method previously described using the commercially
available Concerttrade Plant RNA Reagent (Invitrogen) buffer for fi eld-grown adult pine trees with high
oleoresin content
Key words RNA Pines Concert plant RNA reagent Stem RNA extraction Oleoresin Conifers
1 Introduction
Several conifer species especially within the Pinaceae have tissues with high concentrations of phenolics terpenes and polysaccha-rides [ 1 ] Many specifi c protocols that are appropriate for plants rich in secondary metabolite s have been developed but the extrac-tion of high-quality RNA from these tissues using commercial kits is often diffi cult and usually not applicable to woody tissues [ 2 ndash 6 ] One of the major issues during RNA extraction concerns the pres-ence of phenolic compounds which oxidize and form quinones Aromatic compounds bind RNA which interferes in downstream steps and applications [ 3 7 ] Another point of concern is the har-vest of plant samples in the experimental fi eld which constitutes another obstacle in the efforts to avoid degradation of RNA [ 8 ] These problems often result in RNAs of low quality and insuffi -cient amounts especially for methodologies that normally require
These authors have equally contributed to this work
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_3 copy Springer Science+Business Media New York 2016
fettnetocbiotufrgsbr
RESEARCH PAPER
Control of resin production in Araucaria angustifolia an ancientSouth American coniferJ C Perotti1 K C da Silva Rodrigues-Correa123 amp A G Fett-Neto12
1 Plant Physiology Laboratory Department of Botany Federal University of Rio Grande do Sul (UFRGS) Porto Alegre RS Brazil
2 Center for Biotechnology UFRGS Porto Alegre RS Brazil
3 Present address Regional Integrated University of Alto Uruguai and Miss~oes (URI-FW) Frederico Westphalen RS Brazil
Keywords
Araucaria ethylene jasmonic acid nitric
oxide resin salicylic acid terpenes
Correspondence
A G Fett-Neto Plant Physiology Laboratory
Center for Biotechnology Federal University
of Rio Grande do Sul (UFRGS) PO Box 15005
Av Bento Goncalves 9500 91501-970 Porto
Alegre Brazil
E-mail fettnetocbiotufrgsbr
Editor
K Leiss
Received 22 July 2014 Accepted 11
December 2014
doi101111plb12298
ABSTRACT
Araucaria angustifolia is an ancient slow-growing conifer that characterises parts ofthe Southern Atlantic Forest biome currently listed as a critically endangered speciesThe species also produces bark resin although the factors controlling its resinosis arelargely unknown To better understand this defence-related process we examined theresin exudation response of A angustifolia upon treatment with well-known chemicalstimulators used in fast-growing conifers producing both bark and wood resin suchas Pinus elliottii The initial hypothesis was that A angustifolia would display signifi-cant differences in the regulation of resinosis The effect of Ethrel (ET ndash ethylene pre-cursor) salicylic acid (SA) jasmonic acid (JA) sulphuric acid (SuA) and sodiumnitroprusside (SNP ndash nitric oxide donor) on resin yield and composition in youngplants of A angustifolia was examined In at least one of the concentrations testedand frequently in more than one an aqueous glycerol solution applied on fresh woundsites of the stem with one or more of the adjuvants examined promoted an increase inresin yield as well as monoterpene concentration (a-pinene b-pinene camphene andlimonene) Higher yields and longer exudation periods were observed with JA and ETanother feature shared with Pinus resinosis The results suggest that resinosis controlis similar in Araucaria and Pinus In addition A angustifolia resin may be a relevantsource of valuable terpene chemicals whose production may be increased by usingstimulating pastes containing the identified adjuvants
INTRODUCTION
Many conifer species produce terpenoid-based resins that havelong been studied for their industrial importance and role indefence against attack by herbivores and pathogens The twomost important resin-producing families of conifers are Pina-ceae and Araucariaceae (Langenheim 1996) The viscous resinsecretion is generally composed of a complex mixture ofterpenoids consisting of roughly equal parts of volatile mono-(C10) and sesquiterpene (C15 turpentine) fractions and non-volatile diterpenic (C20 rosin) components (Rodrigues-Correaet al 2013) Terpenes act in a complex and multilayereddefence response providing toxicity against bark beetles andfungi bark wound sealing disruption of insect developmentand attraction of herbivore predators (Phillips amp Croteau1999)Most conifers rely on some combination of preformed and
inducible resin defences (Trapp amp Croteau 2001 Zulak amp Bohl-mann 2010) Resin defences are controlled by environmentaland genetic factors to various extents depending on species(Roberds et al 2003 Sampedro et al 2010 Moreira et al2013) Resin traits have been reported as highly variable havingmoderate heritability indicating that breeding efforts towardssuper-resinous forests are promising (Tadasse et al 2001Roberds et al 2003) Several chemicals are known as stimulantsof resin production Commercial extraction of resin from pine
trees uses periodic bark streaking and application of resin stim-ulant pastes to the wound
Resin-stimulant paste based on sulphuric acid (SuA) iswidely used for the commercial production of pine resin Cur-rent stimulant pastes usually have two chemically active com-ponents SuA to magnify the wounding and an ethyleneprecursor (2-chloroethylphosphonic acid CEPA or Ethrel ndash
ET) to stimulate resin flow (Rodrigues et al 2011 Rodrigues-Correa amp Fett-Neto 2013) Jasmonic acid (JA) and its methylester methyl jasmonate (MeJa) are among the most widelyused chemical elicitors of plant secondary metabolism It hasbeen shown that the exogenous application of MeJa or herbi-vore attack induce chemical and anatomical defence responsesin conifers such as the formation of traumatic resin ducts andresin accumulation in stems along with increased biosynthesisof terpenes and phenolics (Franceschi et al 2002 Martin et al2002 Heijari et al 2005 Zeneli et al 2006 Moreira et al 2008Gould et al 2009) JA commercial use however is limited byits high cost
The effects of exogenous salicylic acid (SA) on conifer ter-pene production have also been studied In Pinus elliottiiapplication of 10 molm3 of SA induced resin productionin wound panels but in Pseudotsuga menziesii and Sequoia-dendron giganteum it had no apparent effect on resinaccumulation (Hudgins amp Franceschi 2004 Rodrigues ampFett-Neto 2009) Nitric oxide (NO) has also emerged as an
Plant Biology 17 (2015) 852ndash859 copy 2014 German Botanical Society and The Royal Botanical Society of the Netherlands852
Plant Biology ISSN 1435-8603
v
levures (et surtout pour leur incroyable patience avec mon tregraves mauvais franccedilais) Crsquoeacutetait
vachement chouette Merci beaucoup agrave vous tous (et toutes) et agrave la prochaine
Agrave Coordenaccedilatildeo de Aperfeiccediloamento Pessoal de Niacutevel Superior (CAPES) pelo
financiamento da bolsa de pesquisa do PDSE
Aos meus pais (bioloacutegicos ou natildeo) Veacutera Maria da Silva Rodrigues Gilberto
Moraes Rodrigues Rosa Maria Lucas da Silva e Paulo Joseacute Costa da Silva pelo
exemplo de honestidade coragem trabalho forccedila e amor desde sempre
Aos meus irmatildeos Ana Paula da Silva Rodrigues Viniacutecius de Moraes da Silva
Rodrigues Marcello da Silva Rodrigues e Camila Stella Toledo Pereira por todas as
experiecircncias que dividimos e tudo o que me ensinaram ateacute hoje
Ao meu amor maior minha melhor amiga minha mais leal e extraordinaacuteria
parceria nessa grande (e agraves vezes tortuosa) jornada Maria Clara Rodrigues Correcirca Por
ser ela por ser imensa em generosidade amor e altruiacutesmo por despertar o melhor em
mim por ser minha forccedila motriz e sobretudo por ser a melhor das minhas metades
Minha vida soacute realmente comeccedilou quando eu tive a incriacutevel sorte de te conhecer
vi
SUMAacuteRIO
LISTA DE ABREVIATURASvii
RESUMO ix
INTRODUCcedilAtildeO GERAL1
HIPOacuteTESE E OBJETIVOS9
CAPIacuteTULO 1 Abiotic stresses and non-protein amino acids in plantshelliphellip10
CAPIacuteTULO 2 Mimosine accumulation in Leucaena leucocephala in response to
stress signaling molecules and acute UV exposurehelliphelliphelliphelliphelliphelliphelliphelliphelliphellip(432) 52
CAPIacuteTULO 3 Mimosine occurrence and accumulation in Mimosa bimucronata var
bimucronata (DC) Kuntze66
CONSIDERACcedilOtildeES FINAIS 84
PERSPECTIVAS85
REFEREcircNCIAS BIBLIOGRAacuteFICAS86
Artigos publicados no periacuteodo de doutoramento natildeo relacionados ao tema da
tese91
vii
LISTA DE ABREVIATURAS
24-D 24-dichlorophenoxyacetic acid
3H4P 3-hydroxy-4-pyridone (34-DHP 34-dihydroxypyridine)
ABA abscisic acid
Arg arginine
BABA β-aminobutyric acid
β-ODAP β-N-oxalyl-L-α β-diaminopropionic acid
BIA β-isoxazolinon-L-alanine
CAN canavanine
DAO diamine oxidase
DDC decarboxylase
ETH ethephon
FW fresh weight
GABA -aminobutyric acid
GABA-T GABA transaminase
GAD glutamate decarboxylase
GSM Global System for Mobile
HPLC High performance liquid chromatography
JA jasmonate
JA-Ile jasmonoyl-L-isoleucine
L-DOPA L-34- dihydroxyphenylalanine
MeJA methyl jasmonate
m-Tyr Meta-tyrosine
NO nitric oxide
NPAA non-protein amino acid
OAS o-acetylserine
OAS-TL o-acetylserine-thiol-lyase
PA polyamine
PAA protein amino acid
viii
PEG polyethylene glycol
PLP pyridoxal-5rsquo-phosphate
PPO polyphenol oxidase tyrosinase
qRT-PCR Reverse transcription polymerase chain reaction quantitative real time
RNS reactive nitrogen species
ROS reactive oxygen species
SA salicylic acid
SAR systemic acquired resistance
SNP sodium nitroprusside
UV ultraviolet radiation
ix
RESUMO
Ao longo de sua evoluccedilatildeo as plantas desenvolveram estrateacutegias estruturais e quiacutemicas de
defesa em resposta aos estresses bioacuteticos e abioacuteticos impostos pelo ambiente Dentre
essas satildeo reconhecidas moleacuteculas quimicamente especializadas denominadas
metaboacutelitos secundaacuterios produtos naturais ou metaboacutelitos especializados Aminoaacutecidos
natildeo proteicos (ANPs) satildeo compostos nitrogenados que constituem aleacutem de componentes
do arsenal de defesa quiacutemica vegetal uma importante fonte de reserva de carbono e
nitrogecircnio para diversos taxa especialmente aqueles pertencentes agrave famiacutelia Fabaceae de
Angiospermas Esse grupo de moleacuteculas quimicamente heterogecircneo eacute assim definido por
natildeo participar da formaccedilatildeo de estruturas proteicas funcionais sendo frequentemente
toacutexicos quando erroneamente incorporados nas cadeias polipeptiacutedicas em formaccedilatildeo em
funccedilatildeo da similaridade estrutural que apresentam com os aminoaacutecidos proteicos Sob o
ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados ANPs satildeo
em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou abioacutetica como
o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de elicitores
quiacutemicos por exemplo O objetivo da presente tese foi investigar o papel bioloacutegico da
mimosina endoacutegena em Leucaena leucocephala (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute) a partir da avaliaccedilatildeo do efeito de tratamentos
relacionados ao estresse abioacutetico (UV-C aacutecido saliciacutelico metil jasmonato e etileno)
Mimosina eacute um ANP aromaacutetico anaacutelogo da L-tirosina com atividade toacutexica para ceacutelulas
de procariotos e eucariotos Dentre as atividades descritas para esse ANP destacam-se a
atividade anti-mitoacutetica ou bloqueadora do ciclo celular atividade alelopaacutetica e
antioxidante Os resultados indicaram que em leucena a biossiacutentese e o acuacutemulo de
mimosina podem ser modulados por fatores causadores de estresses exibindo um padratildeo
de acumulaccedilatildeo similar ao das fitoalexinas Em maricaacute por outro lado a induccedilatildeo do
acuacutemulo dessa moleacutecula natildeo foi observada para os mesmos tratamentos testados para
leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas Aleacutem disso
o padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado
pela sua degradaccedilatildeo As respostas observadas nos testes que avaliaram a atividade de
mitigaccedilatildeo de espeacutecies reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode
agir como um agente antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de
estresse
1
Introduccedilatildeo
Na condiccedilatildeo de organismos seacutesseis ao longo de sua evoluccedilatildeo as plantas
desenvolveram estrateacutegias estruturais e quiacutemicas de defesa em resposta aos estresses bioacuteticos
e abioacuteticos impostos pelo ambiente Dentre essas satildeo reconhecidas moleacuteculas quimicamente
especializadas denominadas metaboacutelitos secundaacuterios produtos naturais (Kutchan et al 2015)
ou mais recentemente metaboacutelitos especializados
Entre as trecircs classes mais gerais de metaboacutelitos secundaacuterios (terpenos compostos
fenoacutelicos e compostos nitrogenados) aminoaacutecidos natildeo-proteicos (ANPs) satildeo incluiacutedos no
terceiro grupo e constituem aleacutem de componentes do arsenal de defesa quiacutemica uma
importante fonte de reserva de carbono e nitrogecircnio para diversos taxa especialmente aqueles
pertencentes agrave famiacutelia Fabaceae de Angiospermas (leguminosas sensu lato)
Aleacutem dos 20 aminoaacutecidos proteicos estima-se que existam entre 600 e 1000 ANPs
(Acamovic amp Brooker 2005 Rodgers et al 2015) Esse grupo de moleacuteculas quimicamente
heterogecircneo eacute assim definido por natildeo participar da formaccedilatildeo de estruturas proteicas
funcionais sendo frequentemente toacutexicos quando erroneamente incorporados nas cadeias
polipeptiacutedicas em formaccedilatildeo em funccedilatildeo da similaridade estrutural que apresentam com os
aminoaacutecidos proteicos (Taiz amp Zeiger 2010)
Conforme mencionado a ocorrecircncia de ANPs eacute comum em espeacutecies de leguminosas
e sua distribuiccedilatildeo pode ser restrita a alguns gecircneros de plantas circunscritos nessa famiacutelia
botacircnica (eg mimosina e canavanina) Por outro lado alguns ANPs como GABA por
exemplo podem apresentar distribuiccedilatildeo ubiacutequa no Reino Plantae assim como ocorrer em
outros tipos de organismos como animais por exemplo (Ramos-Ruiz et al 2018)
2
Apesar de representarem uma fonte nutricional importante sem tratamento preacutevio o
consumo de plantas que acumulam ANPs por animais eacute limitado Isso ocorre pois em longo
prazo a ingestatildeo prolongada de plantas (especialmente sementes) que acumulam ANPs pode
representar risco agrave sauacutede uma vez que estes comprometem o funcionamento de mecanismos
basais de manutenccedilatildeo da homeostase celular e podem tambeacutem em um quadro mais severo
desencadear doenccedilas neurotoacutexicas degenerativas como por exemplo o latirismo causado
por aacutecido β-N-oxalil-l-αβ-diaminopropiocircnico (β-ODAP) (Jiao et al 2011 Kusama-Eguchi
2019)
Sob o ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados
ANPs satildeo em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou
abioacutetica como o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de
elicitores quiacutemicos por exemplo No que concerne ao estudo dos efeitos da induccedilatildeo abioacutetica
sobre o acuacutemulo de ANPs em diferentes espeacutecies vegetais (Monocotiledocircneas e
Eudicotiledocircneas) as moleacuteculas mais amplamente investigadas ateacute o momento satildeo GABA
L-DOPA e mais recentemente mimosina (vide Tabela 1 do capiacutetulo primeiro) Em termos
de efeitos causados a partir da aplicaccedilatildeo exoacutegena de ANPs GABA tambeacutem figura como o
principal aminoaacutecido investigado seguido de L-DOPA e canavanina (vide Tabela 2 do
capiacutetulo primeiro)
No primeiro capiacutetulo da presente tese estatildeo descritas as caracteriacutesticas gerais dos
principais ANPs estudados seus possiacuteveis papeacuteis bioloacutegicos in planta e seus efeitos quando
aplicados exogenamente bem como os estresses abioacuteticos capazes de induzir seu(s)
acuacutemulo(s) nos diferentes tecidos vegetais Nos segundo e terceiro capiacutetulos
respectivamente satildeo elucidados os efeitos dos tratamentos de UV-C aacutecido saliciacutelico etileno
e jasmonato (claacutessicos elicitores do metabolismo secundaacuterio vegetal) sobre o acuacutemulo de
3
mimosina em Leucaena leucocephala var glabrata (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute)
Mimosina eacute um aminoaacutecido aromaacutetico natildeo-proteico anaacutelogo da L-tirosina com
atividade toacutexica para ceacutelulas de procariotos e eucariotos Embora em menor concentraccedilatildeo
mimosina foi primeiramente identificada em Mimosa pudica sendo posteriormente detectada
em outras espeacutecies do gecircnero como Mimosa pigra por exemplo (Soedarjo amp Borthakur
1998) Seu efeito toacutexico eacute atribuiacutedo agrave capacidade de quelar metais o que impede o
funcionamento adequado das metalo-proteiacutenas que dependem dos mesmos como co-fatores
(Negi et al 2014)
A concentraccedilatildeo basal de mimosina em espeacutecies de leucaena pode variar entre 1 e 12
do peso seco do oacutergatildeo (Soedarjo amp Borthakur 1998) Como eacute comum para outros ANPs
que ocorrem em espeacutecies de leguminosas em sementes de Leucaena spp eacute observada uma
maior concentraccedilatildeo de mimosina quando comparada aos demais oacutergatildeos da planta
(Rodrigues-Correcirca et al 2019) sendo esta a fonte de extraccedilatildeo comercial do padratildeo quiacutemico
de mimosina vendido por empresas de reagentes de pesquisa
Diversas atividades foram descritas para mimosina em outros organismos eou tipos
celulares Dentre essas destacam-se a atividade anti-mitoacutetica ou bloqueadora do ciclo
celular em ceacutelulas de eucariotos e procariotos Isto ocorre porque a mimosina impede a
formaccedilatildeo da forquilha de replicaccedilatildeo (e portanto a siacutentese de DNA) interrompendo assim o
avanccedilo do ciclo de divisatildeo celular na fase tardia G1 (Lalande 1990) Foram tambeacutem descritas
para mimosina atividade alelopaacutetica observada sobre o desenvolvimento de outras espeacutecies
de leguminosas e atividade antioxidante entre outras (Tabela 1)
A rota de biossiacutentese de mimosina eacute compartilhada em grande parte com a de cisteiacutena
um aminoaacutecido proteico sulfurado (Figura 1) A siacutentese da cisteiacutena se daacute a partir da conversatildeo
4
de serina e acetil-CoA em o-acetilserina pela enzima SAT (serina acetiltransferase) seguida
da conversatildeo de o-acetilserina e aacutecido sulfiacutedrico em cisteiacutena em uma reaccedilatildeo catalisada pela
OAS-TL (o-acetilserina tiol-liase) A siacutentese de mimosina por sua vez eacute compartilhada com
a da cisteiacutena ateacute esse ponto e acredita-se que pelo menos uma das isoformas de OAS-TL
catalise a conversatildeo de o-acetilserina e 3-hidroxi-4-piridona em mimosina
Tabela 1 Atividades descritas para mimosina de Leucaena leucocephala (Lam) de Wit
ATIVIDADE
ALVO AVALIADO
(organismo eou tecido tipo
celular)
REFEREcircNCIA
Bloqueio do complexo de ativaccedilatildeo
da preacute-replicaccedilatildeo do DNA
Ceacutelulas de mamiacuteferos
KUBOTA et al
(2014)
Alteraccedilatildeo no ciclo ovariano e
extensatildeo da duraccedilatildeo do corpo luacuteteo
bovino no periacuteodo poacutes-parto
Bovinos
(Bos taurus x
Bos indicus)
BOTTINI-
LUZARDO et al
(2015)
Supressatildeo do ciclo celular e reduccedilatildeo
da abundacircncia bacteriana em
mosquitos
Wolbachia pipientis
Aedes albopictus
FALLON
(2015)
Accedilatildeo inibitoacuteria da fibrose
pulmonar induzida
Ratos SD
LI et al
(2015)
Recuperaccedilatildeo da funccedilatildeo do
miocaacuterdio poacutes-isquemia
Miocaacuterdio de ratos (SD)
machos
CROWE et al
(2001)
Inseticida
Heteropsylla cubana
Crawford 1914 e Thrips tabaci
Lindemann 1889
AHMED et al
(2016)
Alelopaacutetica
Albizia procera Vigna
unguiculata Cicer arietinum
Cajanus cajan
AHMED et al
(2008)
Antioxidante
Sistemas modelo de oxidaccedilatildeo
lipiacutedica (β-caroteno - aacutecido
linolecircico e lecitina)
BENJAKUL et al
(2013)
Ateacute momento versotildees divergentes sobre a enzima responsaacutevel pela biossiacutentese de
mimosina (mimosina sintase) tecircm sido publicadas Em 1990 Ikegami e colaboradores
5
identificaram uma OAS-TL responsaacutevel pela formaccedilatildeo de cisteiacutena como sendo tambeacutem uma
mimosina sintase Mais tarde Yafuso et al (2014) realizaram a expressatildeo heteroacuteloga do gene
que codifica para OAS-TL em Escherichia coli e natildeo foi observada a formaccedilatildeo de mimosina
mesmo quando dadas as condiccedilotildees oacutetimas para tanto Mais recentemente Harun-Ur-Rashid
et al (2018) elucidaram a mimosina sintase como sendo uma isoforma da OAS-TL
corroborando o postulado por Ikegami e colaboradores em 1990
Figura 1 Rota de biossiacutentese da mimosina Fonte Ikegami et al (1990)
Espeacutecies estudadas
Leucaena leucocephala (Lam) de Wit (leucaena koa haole ou ldquoacaacutecia exoacuteticardquo na
liacutengua Hawairsquoiana) eacute uma espeacutecie de haacutebito arboacutereo ou arbustivo pertencente agrave famiacutelia
Fabaceae de Angiospermas e caracterizada pelo acuacutemulo de mimosina em todos os seus
oacutergatildeos Eacute nativa da Ameacuterica Central (especificamente da regiatildeo sudeste do Meacutexico) mas
irradiou-se atraveacutes de praticamente todas as zonas tropicais e subtropicais da Terra No
Brasil leucena eacute amplamente distribuiacuteda e classificada como naturalizada pelo REFLORA
(2019) ocorrendo em todo territoacuterio Nacional Satildeo reconhecidas no miacutenimo duas
6
subespeacutecies de leucena ocorrentes no Brasil L leucocephala var leucocephala e L
leucocephala var glabrata sendo a primeira a mais abundante
Leucaena apresenta atributos morfoloacutegicos caracteriacutesticos das leguminosas como o
fruto do tipo vagem deiscente no periacuteodo poacutes-maturaccedilatildeo folhas compostas e bipinadas As
flores satildeo seacutesseis actinomorfas e polistecircmones apresentam caacutelice sinseacutepala e corola
gamopeacutetala e satildeo dispostas em inflorescecircncias do tipo glomeacuterulo (Figura 2)
Figura 2 Oacutergatildeos vegetativos e reprodutivos de L leucocephala (Lam) de Wit Fonte Little Jr amp Skolmen
(1989)
Com base no conhecimento etnobotacircnico disponiacutevel acerca dessa espeacutecie em
diversas regiotildees tropicais e subtropicais leucena eacute utilizada para vaacuterios fins Extratos de
diferentes oacutergatildeos de leucena apresentam atividade anti-diabeacutetica (Kuppusamy et al 2014
Chowtivannakul et al 2016) antioxidante (Mohammed et al 2015 Chowtivannakul et al
2016 Zarin et al 2016) antimicrobiana (Zarin et al 2016) anti-helmiacutentica (Soares et al
2015 Jamous et al 2017) bactericida (Mohammed et al 2015) acaricida (Fernaacutendez-Salas
et al 2011) anti-tumoral (Chung et al 2017) e potencializadora da resposta imune em
peixes (Verma et al 2018) entre outras
7
Leucaena apresenta alta toleracircncia agrave seca sendo capaz de enfrentar estaccedilotildees sazonais
inteiras com deacuteficit hiacutedrico sem prejuiacutezo permanente de seus oacutergatildeos e de recuperar
vigorosamente sua biomassa vegetativa tatildeo logo o regime de precipitaccedilatildeo retome a
regularidade em frequecircncia Acredita-se que a toleracircncia agrave seca apresentada por essa espeacutecie
ocorra em funccedilatildeo do acuacutemulo de mimosina nos diferentes tecidos da planta a qual
funcionaria como um agente osmoregulador responsaacutevel pela preservaccedilatildeo da integridade das
membranas a das macromoleacuteculas intracelulares em periacuteodos de escassez de aacutegua no
ambiente
Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) eacute uma leguminosa
nativa natildeo endecircmica do Brasil amplamente distribuiacuteda nos domiacutenios fitogeograacuteficos da
Caatinga do Cerrado e da Mata Atlacircntica (Simon amp Proenccedila 2000 REFLORA 2019) Como
espeacutecie pioneira (Pilatti et al 2019) exerce importante papel ecoloacutegico na recuperaccedilatildeo de
aacutereas degradadas (Bitencourt et al 2007 Silva et al 2011) no estabelecimento de processos
de sucessatildeo vegetacional
Maricaacute eacute uma espeacutecie semi-deciacutedua a deciacutedua a qual atinge ateacute 15 m em altura (e
diacircmetro agrave altura do peito de ateacute 40 cm) na idade adulta com haacutebito arboacutereo ou arbustivo
(REFLORA 2019) e espinhos caracteriacutesticos desde os estaacutegios iniciais de desenvolvimento
(Carvalho 2004) Apresenta folhas compostas alternas e bipinadas (Figura 2) amplas
inflorescecircncias brancas com flores reunidas em glomeacuterulos esfeacutericos dispostos em grandes
paniacuteculas As flores satildeo diplostecircmones actinomorfas hipoacuteginas e unicarpelares (Silva et al
2011)
Assim como descrito para leucena maricaacute eacute considerado uma espeacutecie multifuncional
sendo comumente empregada para produccedilatildeo de mel como combustiacutevel (Olkoski amp
8
Wittmann 2011) em edificaccedilotildees na carpintaria e como lsquocerca-vivarsquo (Marchiori 1993
Lorenzi 1998) entre outras aplicaccedilotildees
Figura 2 Folhas e fruto de Mimosa bimucronata (DC) Kuntze Fonte Souza-Lima et al (2017)
Em contraste com a amplitude de habitats explorados por leucena (especialmente os
aacuteridos) no Sul do Brasil maricaacute ocorre preferencialmente em ambientes uacutemidos a alagadiccedilos
em aacutereas proacuteximas agraves margens de rios (Patreze amp Cordeiro 2004) embora possa tambeacutem
ocorrer em formaccedilotildees quase exclusivas dessa espeacutecie nas encostas de morros (Jacobi amp
Ferreira 1991)
Em relaccedilatildeo agraves atividades elucidadas para os extratos de maricaacute foram relatados os
efeitos alelopaacutetico (Jacobi amp Ferreira 1991 Ferreira et al 1992) diureacutetico natriureacutetico e
caliureacutetico (Schlickmann et al 2017)
9
Hipoacutetese
Mimosina apresenta perfil dinacircmico de acuacutemulo em Leucaena leucocephala e
Mimosa bimucronata frente a estresses associado a alteraccedilotildees significativas na expressatildeo de
genes relacionados ao metabolismo deste ANP o qual contribui para mitigar o desequiliacutebrio
oxidativo inerente a vaacuterios tipos de estresse
Objetivo geral
O objetivo da presente tese foi investigar o papel bioloacutegico da mimosina endoacutegena
em leucena e maricaacute a partir da avaliaccedilatildeo do efeito de tratamentos relacionados a estresses
ou sinalizadores de estresse
Objetivos especiacuteficos
- Analisar a concentraccedilatildeo constitutiva de mimosina nos diferentes oacutergatildeos de L leucocephala
(Lam) de Wit (leucena) e M bimucronata (DC) Kuntze (maricaacute)
- Verificar se apesar do seu alto teor constitutivo em plantas de leucena o acuacutemulo de
mimosina pode ser induzido com tratamentos que mimetizam diferentes estresses a partir da
avaliaccedilatildeo do efeito de moleacuteculas sinalizadoras (aacutecido saliciacutelico jasmonato etileno) e da
exposiccedilatildeo agrave radiaccedilatildeo UV-C na modulaccedilatildeo do acuacutemulo de mimosina em leucena bem como
em maricaacute
- Determinar se a expressatildeo de genes relacionados ao metabolismo de mimosina estaacute
associada agrave induccedilatildeo por estresses fisioloacutegicos
- Avaliar o potencial antioxidante da mimosina em experimentos realizados in situ
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry
journal homepage wwwelseviercomlocateplaphy
Research article
Mimosine accumulation in Leucaena leucocephala in response to stresssignaling molecules and acute UV exposure
Kelly Cristine da Silva Rodrigues-Correcircaab Michael DH Hondab Dulal BorthakurbArthur Germano Fett-Netoalowast
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970Porto Alegre Rio Grande do Sul BrazilbDepartment of Molecular Biosciences and Bioengineering University of Hawaii at Manoa Honolulu HI 96822 USA
A R T I C L E I N F O
KeywordsLeucaena leucocephalaMimosineMimosine amidohydrolaseJasmonic acidEthyleneSalicylic acidUV-C radiation
A B S T R A C T
Mimosine is a non-protein amino acid of Fabaceae such as Leucaena spp and Mimosa spp Several relevantbiological activities have been described for this molecule including cell cycle blocker anticancer antifungalantimicrobial herbivore deterrent and allelopathic activities raising increased economic interest in its pro-duction In addition information on mimosine dynamics in planta remains limited In order to address this topicand propose strategies to increase mimosine production aiming at economic uses the effects of several stress-related elicitors of secondary metabolism and UV acute exposure were examined on mimosine accumulation ingrowth room-cultivated seedlings of Leucaena leucocephala spp glabrata Mimosine concentration was not sig-nificantly affected by 10 ppm salicylic acid (SA) treatment but increased in roots and shoots of seedlings treatedwith 84 ppm jasmonic acid (JA) and 10 ppm Ethephon (an ethylene-releasing compound) and in shoots treatedwith UV-C radiation Quantification of mimosine amidohydrolase (mimosinase) gene expression showed thatethephon yielded variable effect over time whereas JA and UV-C did not show significant impact Consideringthe strong induction of mimosine accumulation by acute UV-C exposure additional in situ ROS localization aswell as in vitro antioxidant assays were performed suggesting that akin to several secondary metabolitesmimosine may be involved in general oxidative stress modulation acting as a hydrogen peroxide and superoxideanion quencher
1 Introduction
Different plant groups synthesize a large diversity of secondary orspecialized metabolites These molecules are generally produced inresponse to biotic and abiotic environmental stresses Indeed inductionof secondary metabolism usually involves stress-generating factorswhich have also been explored in biotechnological processes aiming atthe production of target metabolites of economic interest (Matsuuraet al 2018) Metabolic control of nitrogen-containing secondarycompounds (eg alkaloids and non-protein amino acids) has beenshown to be complex and influenced by phytohormones environmentalstresses (seasonality herbivory pathogen attack drought) UV radia-tion (Holloacutesy 2002) methyl jasmonate (MeJA) salicylic acid (SA)yeast extract (Cho et al 2008) abscisic acid (ABA) heavy metals os-motic stress (Nascimento et al 2013) and mechanical wounding (Portoet al 2014)
Due to their particular trait of associating with N-fixing micro-organisms Fabaceae species (leguminous sensu lato) are often proteinrich hence the relevance of several of these species as forage Fabaceaespecies are also known for accumulating nitrogen containing secondarymetabolites which play important roles as ecochemical molecules andat least for the case of non-protein amino acids potential cell reservoirsof nitrogen (Huang et al 2011)
High contents of mimosine a toxic aromatic non-protein aminoacid are found in species of two leguminous genera Leucaena spp andMimosa spp Leucaena leucocephala (Lam) de Wit (leucaena koa haole)is a fast-growing leguminous tree native from Central America (south-eastern Mexico) widely distributed in tropical and subtropical zonesThis species is also characterized by its high tolerance to droughtamong other environmental stresses (Honda et al 2018) Leucaena canbe divided into two subspecies (i) L leucocephala subsp leucocephala(common leucaena a bushy shrub) and (ii) L leucocephala subsp
httpsdoiorg101016jplaphy201811018Received 1 August 2018 Received in revised form 9 November 2018 Accepted 14 November 2018
lowast Corresponding authorE-mail addresses krodriguescbiotufrgsbr (KCdS Rodrigues-Correcirca) mhonda2hawaiiedu (MDH Honda) dulalhawaiiedu (D Borthakur)
fettnetocbiotufrgsbr (AG Fett-Neto)
Plant Physiology and Biochemistry 135 (2019) 432ndash440
Available online 19 November 20180981-9428 copy 2018 Elsevier Masson SAS All rights reserved
T
glabrata (giant leucaena a tree) The latter has been used as a fastgrowing tree for production of wood and paper pulp The foliage ofboth common and giant leucaena is used as a fodder because of its highprotein content and palatability to farm animals The foliage containsup to 18 protein 142 crude fiber and 64 ether extractcrude fat(Soedarjo and Borthakur 1996)
Production of nitrogen-containing secondary metabolites such asmimosine requires large amounts of carbon and nitrogen resourcesNegi et al (2014) estimated that up to 21 of the carbon-nitrogenresources may be used for production of mimosine in leucaenaBrewbaker et al (1972) determined the mimosine content of 96 Lleucocephala cultivars and 8 other Leucaena species collected from 38different countries by growing them in an observational nursery inHawaii and found that basal mimosine content varied from 189 to477 of the dry weight
Mimosine is biosynthesized from OAS (o-acetylserine) and 3H4P (3-hydroxy-4-pyridone or its tautoisomer 3-hydroxy-4-pyridine) A pre-vious analysis suggested that mimosine synthase is an OAS-TL (o-acetylserine-thiol-lyase) of the cysteine biosynthesis pathway (Ikegamiet al 1990) Later however recombinant enzyme tests did not supportan OAS-TL identity of mimosine synthase (Yafuso et al 2014) Recentfindings on mimosine biosynthesis revealed that a cytosolic cysteine-OAS-TL isoform can also catalyze the formation of mimosine underspecific conditions (Harun-Ur-Rashid et al 2018)
Mimosine toxicity is related to its ability of reducing the availabilityof divalent metal ions such as Fe(II) Zn(II) Cu(II) Co(II) and Mn(II)by chelating co-factors and preventing their association with metal-dependent enzymes Furthermore this non-protein amino acid is cap-able of forming a stable complex with pyridoxal-5prime-phosphate (PLP)leading to the inactivation of PLP-dependent enzymes (eg Asp-Glutransaminase and cystathionine synthetase) (Negi et al 2014)
Mimosine features several useful biological activities such as alle-lopathic antimicrobial insecticide cell cycle inhibitor agent antic-ancer phytoremediator (Nguyen and Tawata 2016) as well as anti-oxidant (Benjakul et al 2013) Despite the relatively well establishedbiological activities of purified mimosine on other organisms or celltypes little is known about its biological role in leguminous speciesHowever it has been suggested that at least in part its activity ismainly related to defense mechanisms against some biotic and abioticstresses and as nitrogen source during fast growth (Vestena et al2001)
Suda (1960) and Smith and Fowden (1966) identified enzymes in-volved in mimosine degradation in seedling extracts of L leucocephalaand Mimosa pudica A mimosine-degrading enzyme named mimosinase(mimosine amidohydrolase EC 35161 CAS registry number 104118-49-2) (IUBMB 2018) a carbon-nitrogen lyase which degrades mimo-sine into 3H4P was later purified by Tangendjaja et al (1986) Itsbiochemical characterization was described and the cDNA was isolatedby Negi et al (2014)
Although mimosinase has been described and isolated only fewstudies on the role played by biotic and abiotic factors on the dynamicmodulation of mimosine metabolism in leguminous species have beenconducted (Vestena et al 2001 Xu et al 2018) In aseptic cultures ofleucaena mechanical injury of shoots promoted local mimosine accu-mulation (Vestena et al 2001) In the same study cultivation in pre-sence of auxin or SA in culture medium also had a positive effect on
mimosine accumulation More recently the effect of drought treatmenton gene expression of leucaena was also evaluated by Honda et al(2018) However several potential factors regulating mimosine meta-bolism need to be further examined
To date there is a lack of information on the biological role ofmimosine in planta as well as on details of its metabolic dynamicsMoreover its overt potential for pharmaceutical applications and de-velopment of new drugs as well as the possible use as tool to addressheavy metal soil contamination or plant mineral nutrition improve-ment justify additional research The objective of this study was toinvestigate the effect of stress signaling molecules and acute UV ex-posure on modulation of mimosine accumulation and metabolism in Lleucocephala spp glabrata in order to better understand its biologicalrole and to identify strategies for yield improvement aiming at ex-ploring its useful bioactivities
2 Methods
21 Plant material
For the experiments carried out to evaluate the effects of elicitors onmimosine accumulation seeds of leucaena were kindly provided by DrJames Brewbaker and harvested at CTAHRs (College of TropicalAgriculture and Human Resources of the University of Hawaii atManoa) Waimanalo Research Station at Oahu Hawaii This plantmaterial was originated from the accession K636 of Leucaena leucoce-phala ssp glabrata (Brewbaker 2008)
22 Induced mimosine content in 5-week-old giant leucaena
221 Seed germinationIn order to overcome seed coat dormancy seeds were submitted to a
chemical scarification with sulfuric acid 95ndash98 for 20min and re-peatedly rinsed in distilled water to remove any residual trace of thisreagent Then seeds were distributed in 254 cmtimes508 cm plastictrays containing 11 vv of vermiculite and commercial soil watereduntil reaching substrate field capacity Three weeks after seed imbibi-tion seedlings displaying similar size and shape (eg number of com-pound leaves and leaflets) were transplanted to individual pots(250mL) in number of three plants per container
During the experimental period (except in the UV-C radiationtreatment) all tested seedlings were kept in a growth chamber andsubmitted to controlled conditions of temperature (circa 25 degC) and ir-radiance (approximately 100 μmol photons mminus2sdot s minus1) with a photo-period of 16 h light and 8 h dark
222 Treatments2221 JA Ethephon and SA Five-week-old giant leucaena seedlingswere treated with different solutions as described in Table 1 Idealconcentrations were defined in preliminary experiments under the sameconditions indicated above At the beginning of the experiments 30plants were sprayed with 84 ppm JA 10 ppm SA 10 or 100 ppmEthephon or Milli-Qreg water (control) until the point of imminent runoffPlant pots were kept closed inside transparent plastic bags for 24 h toavoid solution volatilization Fifteen plants arranged in 5 sets of 3 (5biological replicates) were harvested 48 h and 96 h after being treated
Table 1Treatments used to modulate mimosine biosynthesis in giant leucaena
ELICITOR CONCENTRATION UV FLUENCE EXPOSURE TIME RATIONALE FOR USE
Salicylic acid (SA) 10 ppm 24 h Pathogen signaling molecule (Shah 2003)Jasmonic acid (JA) 84 ppm 24 h Chemical elicitor of plant secondary metabolism (Dar et al 2015)Ethephon 10 ppm 24 h Ethylene releasing-compound (Kim et al 2016) elicitor of plant secondary metabolism (Wang
et al 2016)UV-C radiation 3 Jcmminus2 10min or 15min Elicitor of plant secondary metabolism (Kara 2013 Neelamegam and Sutha 2015)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
433
After collection shoots were separated from roots immediately frozenin liquid nitrogen and stored at ndash 80 degC prior to HPLC analyses
2222 UV-C Thirty seedlings of giant leucaena were exposed to UV-Cradiation (3 Jcmminus2) for 10 or 15min and kept in a growth chamberunder controlled conditions as described above until the end of theexperiments Fifteen plants arranged in groups of 3 were harvested at96 h and 120 h after UV-C exposure and processed as previouslydescribed
223 Mimosine extractionMimosine extraction was based on a modified version of the pro-
tocol published by Lalitha and Kulothungan (2006) as follows a knownweight of fresh tissue (shoots or roots) of giant leucaena was first addedto Milli-Qreg boiling water in a proportion of 110 (g of plant per mL ofsolvent) in test tubes Tubes were covered with foil to avoid solutionevaporation and placed on a hot stirrer at 100 degC for 10min A pro-portional volume of 01M HCl was added to the cooled suspensions andhomogenized using mortar and pestle The plant extracts were filteredthrough cotton and centrifuged twice for 7min in a bench top re-frigerated centrifuge at 4 degC and 13200 rpm Before being analyzed theextracts were diluted 13 with ondashphosphoric acid (OPA)
224 Mimosine detectionHPLC analyses were carried out as described by Negi and Borthakur
(2016) Pure mimosine (L-mimosine from koa haole seeds Sigma-Al-drich CAS number 500-44-7) was used as standard Separation andquantification of mimosine was done with a C18 column (PhenomenexC18 5 μm 46times250mm) under an isocratic solvent system of 002MOPA with a linear flow rate of 1mLsdotminminus1 Mimosine detection wasdone at 280 nm by photodiode array detection (200ndash400 nm) showingretention time of 277 plusmn 0042min Quantification was done using themethod of external standard curve Further confirmation of mimosineidentity was performed by co-chromatography with standard and peakpurity check Chromatograms were analyzed using the Waters Em-power 3 software
23 Quantitative real-time PCR analysis of mimosinase gene expression
Fifteen 8-week-old giant leucaena plants arranged in 4 sets of 3 (4biological replicates) were treated with either water (control) or10 ppm Ethephon 84 ppm JA acid or 15min of UV-C radiation ex-posure following the methods described above Following treatmentleucaena plants were harvested at 48 and 96 h or 72 and 144 h (UV-Ctreated plants only) after treatments Total RNA of samples was ex-tracted and purified from roots and shoots of giant leucaena by meansof a modified method using Qiagen RNeasy Plant Kit (Valencia CAUSA) and Fruit-mate (Takara Japan) according to the protocol de-scribed by Ishihara et al (2016a) The assessment of RNA quality andquantity was carried out at 230 260 and 280 nm by using a NanoDropSpectrophotometer ND-1000 (NanoDrop Technologies DE USA) Inorder to avoid genomic DNA contamination RNA samples were treatedwith TURBO DNAfree Kit (Invitrogen Carlsbad CA) Two microgramsof DNase-treated RNA were used to synthesize the first-strand cDNAusing M-MLV Reverse Transcriptase (Promega WI USA)
Quantitative real-time (qPCR) analysis was carried out to examinepossible differential expression of the mimosinase gene (GenBank ac-cession number AB2985971) in seedlings treated with 84 ppm JA10mM Ethephon or 15min of UV-C exposure Shoots and roots wereharvested 24 h before the time of mimosine concentration peak for eachtreatment previously observed as assessed by HPLC assays The 10 μLqPCR reaction consisted of 5 μL of PowerUpTM SYBRreg Green MasterMix (Applied Biosystems Foster City CA) 1 μL MgCl2 (50mM) 03 μLforward primer (10 μM) 03 μL reverse primer (10 μM) and 1 μL cDNAfirst-strand In the experimental validation through qPCR reactionconditions and melting curve analysis of the amplicon were performed
following the protocol published by Ishihara et al (2016b) for the sameleucaena variety qPCR analysis was conducted using StepOnetrade Real-Time PCR System (Applied Biosystems) Measurements were performedusing 4 biological and 3 technical replicates Relative expression wascalculated with the 2-ΔΔct method using OAS-TL as reference gene sinceits expression showed a consistently stable profile comparable to that ofUBQ-5 and ELF1α expressions Mimosinase primer sequences used forthese analyses were (FWD) 5prime- GAA AGG CAG GAA TCA CAG TGA AGAG ndash 3rsquo (REV) 5prime GGA GAC TCT AGC CAC ACC AAC TTA ndash 3rsquo
24 Antioxidant assays
241 Mimosine effect on hydrogen peroxide (H2O2) accumulationAs a follow up to the induction of mimosine accumulation profiles
under stress signals and conditions tests were conducted to verify mi-mosine antioxidant capacity In situ histological localization of hy-drogen peroxide (H2O2) accumulation was evaluated on foliar disks ofPhaseolus vulgaris L according to the protocol described by Shi et al(2010) Briefly the plant foliar tissue was exposed to 1 mgmiddotmLminus1 dia-minobenzidine (DAB) solution in 10 mM KH2PO4 (control) in presenceor absence of 10mM mimosine (equivalent to the average mimosineconcentration induced by UV-C radiation in giant leucaena) or 10mMascorbic acid (positive antioxidant control) Oxidative response wasidentified by the formation of a brown polymer on the injured leafareas indicating the presence of H2O2 and registered in a Leica M165FC stereomicroscope (Leica Microsystems)
242 Mimosine quenching of superoxide radicalsGeneration of superoxide radical and subsequent analysis was per-
formed by a modified protocol based on Zhishen et al (1999) Nitroblue tetrazolium (NBT) reduction was used to measure superoxide an-ions quenching activity Shortly a 50mM KH2PO4 pH 78 solutioncontaining 6 μM riboflavin 100mM methionine 1 mM NBT in pre-sence or absence of 5mM mimosine was exposed to white light(22 Jsdotcmminus2) for 25min on a white light transilluminator Five micro-molar rutin was used as positive control (Matsuura et al 2016) Theabsorbance was read at 560 nm before and after light exposure in aSpectraMaxreg M2 Microplate Reader (Molecular Devices LLC)
25 Statistical analyses
For HPLC and superoxide anions data simple analyses of variance(ANOVA) followed by Tukey or Welch ANOVA followed by Dunnetts Ctest were used as appropriate for data distribution characteristics InqPCR analysis results were analyzed by t-test In all cases at least fourbiological triplicates were used and experiments were repeated twiceindependently All data were analyzed using the statistical packageSPSS 200 for Windows (SPSS Inc USA) In all cases a ple 005 wasused
3 Results and discussion
31 Increased mimosine concentrations in giant leucaena treated withchemical elicitors
Leucaena produces high amounts of mimosine that accumulate in allparts of the plants including leaves stem flowers pods seeds rootsand root nodules (Soedarjo and Borthakur 1998) The highest con-centrations of mimosine can be found in the growing shoot tips andseeds (Wong and Devendra 1983) It is not known why leucaena pro-duces such high amounts of mimosine Negi et al (2014) estimated thatleucaena plants would be able to grow 21 larger if the nutrient re-sources spent on mimosine production were diverted for biomass in-crease In a previous analysis performed to quantify the basal con-centration of mimosine present in adult plants of common leucaena thehighest constitutive amount of mimosine per gram of fresh weight in
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
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the analyzed organs was found in post-anthesis flowers (89448 μg)followed by green pods (82687 μg) leaves (67358 μg) and greenflower buds (51247 μg) which showed significantly less mimosineconcentration compared to the other reproductive structures(Supplementary Fig 1) Since mature seeds have very low moisturecontent (Wencomo et al 2017) its mimosine concentration was esti-mated as 338253 μgsdotgminus1 of dry weight Additionally it was also ob-served that the basal mimosine distribution in shoots of field-grownadult plants of leucaena is dependent on the variety type(Supplementary Table 1)
Phytohormones such as salicylic acid and jasmonic acid are knownto be produced by plants in response to various abiotic and bioticstresses These phytohormones trigger adaptive responses to stress byregulating major plant metabolic processes such as photosynthesisnitrogen metabolism defense systems and plant-water relationsthereby providing protection (for review see Khan et al 2015)
Secondary or specialized metabolite production and accumulationare also known to be controlled by biotic and abiotic stresses (Matsuuraet al 2018) In this study exposure of 5-week-old giant leucaenaseedlings to JA or Ethephon treatments significantly enhanced mimo-sine accumulation in shoots and roots in at least one of the two timepoints tested (48 and 96 h) albeit in a different way (Fig 1) Thehighest concentrations of mimosine in shoots were found in seedlingstreated with JA 84 ppm (43441 μgsdotgminus1) and Ethephon 100 ppm(38412 μgsdotgminus1) two days after application of the respective phyto-hormones Nevertheless after four days shoots yielded the highestconcentration of mimosine (approximately 460 μgsdotgminus1) upon treatmentwith 10 or 100 ppm Ethephon (Fig 1A) In roots after two and four
days JA 84 ppm and Ethephon 10 ppm resulted in highest mimosineaccumulation 18488 μgsdotgminus1 and 15801 μgsdotgminus1 respectively (Fig 1B)These observations show that mimosine accumulation response tospecific elicitors may vary over time after exposure
Although all treatments were applied exclusively on shoots of giantleucaena seedlings roots of some of them were also able to respond tothe different elicitors Overall shoots displayed higher basal and in-duced mimosine concentration compared to roots (Fig 1) which agreeswith previous observations in 1 to 3-week-old aseptic seedlings ofcommon leucaena (Vestena et al 2001) However as previouslymentioned significant post-induction increase of mimosine concentra-tion in roots and shoots simultaneously was only observed for JA andEthephon 10 ppm on day 02 and 04 respectively (Fig 1)
It is well established that perceived regulatory signals or elicitorsgenerate a transduction network mediated by secondary messengersresulting in changes in gene expression profiles that afford adaptiveresponses to environmental stimuli These modulation events are oftenmediated by transcription factors (TFs) which directly bind to specificgene promoters or act by forming complexes with repressor proteinslabeling them to degradation subsequently releasing other TFs toproceed with the gene expression program This is the case of the actionmechanism of JA and its active form jasmonoyl isoleucine for example(Kazan 2015 Wasternack and Strnad 2016)
JA ethylene and SA are known as important stress regulatory sig-nals in plants JA however is thought to be the most effective signal forinduction of plant secondary metabolism (Wasternack and Strnad2016) thereby contributing to mitigation of damage caused by severalstresses (Dar et al 2015) JA is mainly derived from linolenic acid
Fig 1 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings treated with differentelicitors CTRL=Milli-Q water SA = Salicylic AcidJA= Jasmonic Acid ETH=Ethephon Bars sharing a letterof same case do not differ by Tukey test (P le 005) Capitalletters (A B) compare treatments on day two and lowercaseletters (a b) compare treatments on day four Indicatessignificant statistical difference between day two and dayfour in the same treatment by t-test (Ple 005) The errorbars represent standard error of five replicates (each meanwas calculated with 15 individual seedlings organized in 5groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
435
(Wasternack and Strnad 2016) playing important roles in differentprocesses of plant growth and development such as plant defensemechanisms against herbivory pathogen attack fungal elicitation andsome abiotic factors such as osmotic temperature and salt stresses (Daret al 2015)
JA and its methyl ester MeJA have several different effects on le-guminous species MeJA exogenous application has increased iso-flavonoid content in cell suspension cultures of Pueraria candollei varcandollei and P candollei var mirifica (Korsangruang et al 2010) aswell as the production of the triterpenoid glycyrrhizin in Glycyrrhizaglabra roots Enhanced production of the triterpenoid however waspartly at the expense of root growth (Shabani et al 2009) MeJA ap-plication on shoots was observed to suppress root nodulation and lat-eral root formation in Lotus japonicus (Nakagawa and Kawaguchi2006) In grapevine a non-leguminous species proteinogenic aminoacids did not show an expressive increase under MeJA treatment(Gutieacuterrez-Gamboa et al 2017)
The effects of the application of four different jasmonate forms (JAMeJA jasmonoyl-L-isoleucine (JA-Ile) and 6-ethyl indanoyl glycineconjugate (2-[(6-ethyl-1-oxo-indane-4-carbonyl)-amino]-acetic acidmethyl ester - CGM) on leucaena metabolite profile has recently beenreported by Xu et al (2018) JA-Ile form was most effective althoughno major alteration was observed on monitored metabolite abundancesAlanine threonine and 34-dihydroxypyridine (34 DHP a metabolitederived from mimosine degradation) (Nguyen and Tawata 2016)among others were the major metabolites elicited by JA-Ile In contrastto the results described here mimosine concentration did not changesignificantly These divergent results on mimosine accumulation maybe due to a number of factors including mode of application jasmonateform used (JA-Ile x JA) and L leucocephala subspecies (common x giantleucaena)
Ethylene is also a phytohormone involved in plant response me-chanisms to different types of challenges such as mechanical damageand insect attack among others The integration mechanism betweenJA and ethylene signaling pathways is not completely understoodhowever it has been shown that they may work cooperatively in abioticstress tolerance (Kazan 2015) MeJA can induce ethylene production(Zhao et al 2004) and when applied simultaneously these moleculesseem to work in a synergic way by enhancing the magnitude of theplant response to external stimuli (Liu et al 2016)
Treatment with SA was able to significantly increase mimosine ac-cumulation in 12-week-old plants of common leucaena (SupplementaryFig 2) However no significant effect of SA treatment on mimosineconcentration was seen in 5-week-old seedlings of giant leucaena(Fig 1) suggesting some degree of genotype andor age dependency inelicitation by this phytohormone On the other hand several treat-ments including 90 ppm MeJA 10 and 100 ppm 2-chloroethylpho-sphonic acid (CEPA an ethylene-releasing compound) significantlyincreased mimosine accumulation (Supplementary Fig 2) in agree-ment with the data obtained for giant leucaena The lack of systemiceffects of externally applied SA on mimosine accumulation was alsoobserved when the phytohormone was supplied in the culture mediumof aseptically-grown seedlings in which case only roots had highercontent of mimosine (Vestena et al 2001) This could be due totransport limitations or to low methyl salicylate production from ap-plied SA since the former is recognized as the main systemic signalingform (Vlot et al 2009)
32 Increased mimosine concentrations in giant leucaena exposed to UV-Cradiation
UV-C treatment promoted increased concentration of the aminoacid in shoots but not in roots of giant leucaena (Fig 2) Increasedaccumulation of mimosine in shoots was also observed in 12-week-oldseedlings of common leucaena exposed to UV-C radiation for 10 and15min (Supplementary Fig 3) Similar to the SA treatment in giant
leucaena UV-C radiation did not induce mimosine biosynthesis in rootsregardless of time after exposure The absence of mimosine induction inroots by SA and UV indicates that these effectors do not cause a sys-temic response Moreover roots are shielded from irradiance by thepresence of substrate
UV radiation effects on different aspects of plant metabolism anddevelopment have been described However compared to UV-B (en-vironmentally relevant type of UV radiation) assays there are less re-ports related to the UV-C effects on secondary metabolites biosynthesisand accumulation (Cetin 2014) especially in leguminous (Fabaceae)plants They generally concern primary metabolism aspects such asgrowth and development For instance seedlings of Phaseolus vulgaris L(Fabaceae) exposed to low intensity UV-C radiation have displayeddecreased chlorophyll content and reduced height after 14 days of ex-posure (Kara 2013) Negative effects on growth parameters and ni-trogen metabolism were also observed in Vigna radiata L (Fabaceae)after UV-B radiation treatment in addition to adverse effects on JA SAand antioxidant compounds accumulation (Choudhary and Agrawal2014a) The same authors reported increased accumulation of flavo-noids SA and JA besides negative effects on growth biomass yieldnitrogen fixation and accumulation in 2 cultivars of Pisum sativum L(Fabaceae) under elevated UV-B treatment (Choudhary and Agrawal2014b) Despite the negative UV influence on growth reported for thepreviously mentioned leguminous UV-C radiation on groundnut plants(Arachis hypogaea L Fabaceae) increased seedling vigor and biomassand had no adverse effect on germination or other development para-meters (Neelamegam and Sutha 2015)
Besides its impact on growth and primary metabolism UV exposurecan cause important changes in secondary metabolism depending onintensity and time of exposure (Matsuura et al 2013) UV-B and UV-Cpre-treatments of Artemisia annua (Asteraceae) seedlings yielded in-creased biosynthesis of artemisinin a drug which displays anti-malarialproperties and activity against some others infectious diseases (egschistosomiasis leishmaniasis and hepatitis B) and several kinds oftumors (Rai et al 2011) The accumulation of nicotine in Nicotianarustica plants (Solanaceae) was also increased by UV-C treatment(Tiburcio et al 1985) Similar inducing effects on production of severalsecondary metabolites were observed in callus cultures of Vitis viniferaL Oumlkuumlzgoumlzuuml (grapevine Vitaceae) treated with a UV-C source for 5 or10min (Cetin 2014)
Regarding amino acid biosynthesis in response to UV radiationMartiacutenez-Luumlscher et al (2014) have found that in spite of not causingchanges in total amino acid content UV-B radiation exposure can affecttheir profile in grape berries Proteinogenic amino acids have beenknown to be important targets of the deleterious effects of UV radiation(Holloacutesy 2002) On the other hand in the present study acute UV-Ctreatment was found to increase mimosine accumulation in shoots byover twofold (Fig 2) which may suggest a possible participation of thismolecule as part of the antioxidant defense system in L leucocephalaThis possibility is further supported by the induction of the amino acidaccumulation by JA and Ethephon involved in abiotic and biotic stressresponses which are generally associated with oxidative imbalance andare signaling components in high UV stress (Matsuura et al 2013)
33 Mimosinase gene expression
In order to determine if increases in mimosine content upon ex-posure to JA CEPA or UV-C radiation were related to changes intranscription of mimosine metabolism-related genes RT-qPCR analysiswas carried out The complete pathway for mimosine biosynthesis hasnot yet been determined although the final step has been character-ized Based on transcription analysis (Ishihara et al 2016a) leucaenaappears to encode for multiple cysteine synthases one or more of whichmay be able to catalyze mimosine synthesis In addition a leucaenagene encoding a mimosinase (an enzyme responsible for mimosinedegradation) has been identified and characterized (Negi et al 2014)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
436
In addition to mimosinase gene expression several gene isoformsbelonging to the cysteine pathway [cysteine synthase (CYS SYN) serineacetyltransferase (SAT) and β-cyanoalanine synthase (CAS) Table 2 -supplementary material] were also tested in this study (data notshown) However expressions of these genes did not vary in giantleucaena throughout the experiments suggesting that the increasedcontent of mimosine observed in the treated plants might not be relatedto the expression of these genes but presumably to increased enzymeactivity andor release from conjugates such as mimoside a mimosineβ-D-glucoside (Murakoshi et al 1972)
Considering the time variation of mimosine accumulation observedin this work mimosinase gene expression in shoots and roots wasevaluated 24 h before the increase of mimosine concentration in giantleucaena seedlings (ie 24 h and 72 h after the chemical elicitorstreatments and 48 h and 120 h after UV-C exposure)
Ethylene signaling has been shown to up-regulate expression ofseveral genes related to secondary metabolism pathways as is the caseof phenolic compounds (Liu et al 2016) and terpenoid indole alkaloids(Wang et al 2016) Among all elicitors tested in the present workEthephon was the only one able to significantly change mimosinasegene expression Leucaena plants treated with Ethephon showed sig-nificant increases in mimosine concentration at both day 2 and 4 fol-lowing treatment which coincided with low-level expression of mi-mosinase Up-regulation of mimosinase gene expression was detected24 h before the increase of mimosine concentration in shoots treatedwith 10 ppm of Ethephon (Fig 3A) but not after JA or UV-C treatments(Fig 3C-D and 3E-F respectively) Nevertheless 72 h after treatment
application (24 h before the highest mimosine content measured inshoots) down regulation of mimosinase gene was seen in both shootsand roots treated with 10 ppm of Ethephon (Fig 3B) These data in-dicate that mimosine content in leucaena plants is at least partlyregulated by mimosinase expression in Ethephon exposed plants Onthe other hand the fact that mimosinase mRNA was not significantlyaffected by JA and UV-C treatments despite their stimulating effects onmimosine biosynthesis in giant leucaena may indicate that other levelsof regulation are at play or that the chosen harvesting time window wasunable to detect relevant changes
34 In situ and in vitro antioxidant assays
Considering the stimulation of mimosine accumulation byEthephon JA and UV all of which are often associated or known tocause oxidative imbalance the antioxidant capacity of mimosine wasevaluated Mimosine has been shown to have antioxidant activities oncultured cancer cells (Parmar et al 2015) In the present study it washypothesized that mimosine could confer radical scavenging propertieswhich would contribute to plant protection from possible damagecaused by reactive oxygen species generated during stress(Supplementary Fig 4)
Foliar disks of P vulgaris L were treated with 10mM mimosine for15min Treated disks showed less hydrogen peroxide accumulationinduced by wounding in contrast to untreated ones being comparableto those treated with ascorbic acid (a known hydrogen peroxide neu-tralizer) (Fig 4A) These observations support a possible antioxidant
Fig 2 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings exposed to UV-C lightCTRL= visible light (100 μmol photons mminus2 s minus1) UV-C 10primeand UV-C 15rsquo=UV-C exposure time (10 and 15min re-spectively) Bars sharing a letter of same case do not differ byTukey test (P le 005) Capital letters (A B) compare treat-ments on day three and lowercase letters (a b) comparetreatments on day six Indicates significant statistical dif-ference between day three and day six in the same treatmentby t-test (Ple 005) The error bars represent standard errorof five replicates (each mean was calculated with 15 in-dividual seedlings organized in 5 groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
437
role of mimosine as an in situ hydrogen peroxide scavengerMimosine was also able to quench superoxide anions generated by
light exposure Mimosine exhibited equivalent antioxidant effect com-pared to rutin (Fig 4B) a well-established effective superoxide anionquencher (Matsuura et al 2016) The radical scavenging activity ofmimosine may be due to the 3-OH group of the pyridine ring of mi-mosine (Fig 5) The pKa of the 3-OH of mimosine has been estimated tobe 88 (M Honda unpublished results) At physiological pH this OHgroup is expected to remain in a protonated state and therefore mayscavenge a radical by donating a proton and an electron In this processmimosine itself is converted to a stable radical form which is perhapsless toxic and less reactive than the reactive oxygen species generatedduring oxidative stress It is likely that the less toxic radical mimosineproduced may react with another radical or molecule and becomeconverted to a non-reactive indole molecule
In vivo antioxidant activity of mimosine has been previously eval-uated by means of its exogenous application on selenium-deficientseedlings of Vigna radiata In spite of its allelopathic properties (Ahmedet al 2008) the results showed mitigation of mitochondrial oxidativestress by treatment with 01mM mimosine (Lalitha and Kulothungan2007) DPPH radical scavenging activity was also reported for aqueous
seed extracts of leucaena rich in mimosine and phenolic compounds inin vitro assays (Benjakul et al 2014) Mimosine antioxidant activityshown in the present work is in good agreement with data reported forother non-protein amino acids such as L-DOPA (Dhanani et al 2015)and GABA (Malekzadeh et al 2014) for instance
4 Conclusion
Taken together results show that mimosine biosynthesis and ac-cumulation can be modulated by stress-related factors despite its re-latively high constitutive content in leucaena plants The pattern ofgene expression in stressed plants suggests mimosine steady-state con-trol may be regulated by its degradation in possible connection withdynamic changes in carbon and nitrogen metabolism of stressed plantsMimosine quenching activity against hydrogen peroxide and super-oxide anions in the in situ staining and in vitro assays respectivelyshowed that this non-protein amino acid can act as non-enzymaticantioxidant agent Increase in mimosine content in response to elicitorsmimicking environmental challenges in addition to its antiherbivoreand antimicrobial properties may be related to its activity as protectivemolecule against oxidative damage in line with other classes of plant
Fig 3 Relative expression of the mimosinase gene in shoots (A E and F) and shoots and roots (B C and D) of giant leucaena 24 h (A and C) 48 h (E) 72 h (B and D)and 120 h (F) after treatment with stress signaling molecules or UV-C exposure ETH = Ethephon JA = Jasmonic Acid Indicates significant statistical differencebetween control and treatment by t-test (Ple 005) The error bars represent standard error of four replicates
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
438
secondary metabolites
Funding
This work was funded by the National Council for Scientific andTechnological Development (CNPq-Brazil) grant 3060792013-5Coordenaccedilatildeo de Aperfeiccediloamento de Pessoal de Niacutevel Superior - Brazil(CAPES) - Finance Code 001 and the USDA NIFA Hatch projectHA05029-H managed by CTAHR
CRediT authorship contribution statement
Kelly Cristine da Silva Rodrigues-Correcirca InvestigationValidation Writing ndash original draft Michael DH HondaInvestigation Validation Dulal Borthakur Supervision Writing ndashreview amp editing Funding acquisition Arthur Germano Fett-NetoSupervision Funding acquisition Writing ndash review amp editing
Acknowledgements
The authors would like to thank Dr Jorge Ernesto Mariath fromLaVeg-UFRGS for kindly lending the Leica M165 FC stereomicroscopefor in situ analysis
Appendix A Supplementary data
Supplementary data to this article can be found online at httpsdoiorg101016jplaphy201811018
References
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Benjakul S Kittiphattanabawon P Shahidi F Maqsood S 2013 Antioxidant activityand inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipidoxidation in model systems Food Sci Technol Int 19 (4) 365ndash376 httpsdoiorg1011771082013212455186
Benjakul S Kittiphattanabawon P Sumpavapol P Maqsood S 2014 Antioxidantactivities of lead (Leucaena leucocephala) seed as affected by extraction solvent priordechlorophyllisation and drying methods extracts against lipid oxidation in modelsystems Food Sci Technol 51 (11) 3026ndash3037 httpsdoiorg101007s13197-012-0846-1
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Brewbaker JL 2008 Registration of KX2 ndash Hawaii interspecific-hybrid leucaena JPlant Registrations 1 (3) 190ndash193 httpsdoiorg103198jpr2007050298crc
Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitisvinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-6287-47-37
Cho H-Y Son SY Rhee HS Yoon S-YH Lee-Parsons CWT Park JM 2008Synergistic effects of sequential treatment with methyl jasmonate salicylic acid andyeast extract on benzophenanthridine alkaloid accumulation and protein expressionin Eschscholtzia californica suspension cultures J Biotechnol 135 117ndash122 httpsdoiorg101016jjbiotec200802020
Choudhary KK Agrawal SB 2014a Cultivar specificity of tropical mung bean (Vignaradiata L) to elevated ultraviolet-B changes in antioxidative defense system ni-trogen metabolism and accumulation of jasmonic and salicylic acids Environ ExpBot 99 122ndash132 httpsdoiorg101016jenvexpbot201311006
Choudhary KK Agrawal SB 2014b Ultraviolet-B induced changes in morphologicalphysiological and biochemical parameters of two cultivars of pea (Pisum sativum L)Ecotoxicol Environ Saf 100 178ndash187 httpsdoiorg101016jecoenv201310032
Dar TA Uddin M Khan MMA Hakeem KR Jaleel H 2015 Jasmonates counterplant stress a Review Environ Exp Bot 115 49ndash57 httpsdoiorg101016jenvexpbot201502010
Dhanani T Singh R Shah S Kumari P Kumar S 2015 Comparison of green ex-traction methods with conventional extraction method for extract yield L-DOPAconcentration and antioxidant activity of Mucuna pruriens seed Green Chem LettRev 8 (2) 43ndash48 httpsdoiorg1010801751825320151075070
Gutieacuterrez-Gamboa G Portu J Santamariacutea P Loacutepez R Garde-Cerdaacuten T 2017Effects on grape amino acid concentration through foliar application of three dif-ferent elicitors Food Res Int 99 688ndash692 httpsdoiorg101016jfoodres201706022
Fig 4 A In situ antioxidant assay Foliar disksof Phaseolus vulgaris L treated with (a) No an-tioxidant added (negative control) (b) 10 mMMimosine (c) 10mM ascorbic acid (positivecontrol) The oxidative damage can be seen bythe formation of a brown polymer in leaf veinsand injured areas B In vitro superoxidescavenging assay carried out with mimosineDifferent letters indicate significant differenceby Tukey test (Ple 005) The error bars re-present standard error of four replicates (Forinterpretation of the references to colour in thisfigure legend the reader is referred to the Webversion of this article)
Fig 5 Predicted mimosine radical formed followingquenching of hydroxyl radical Mimosine is first converted toa stable mimosine radical which may be then converted to anontoxic indole form
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
439
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad HossainMd Anai T Oku H 2018 Cytosolic cysteine synthase switch cysteine and mi-mosine production in Leucaena leucocephala Appl Biochem Biotechnol 186 (3)613ndash632 httpsdoiorg101007s12010-018-2745-z
Holloacutesy F 2002 Effects of ultraviolet radiation on plant cells Micron 33 (2) 179ndash197Honda MDH Ishihara KL Pham DT Borthakur D 2018 Identification of drought-
induced genes in giant leucaena (Leucaena leucocephala subsp glabrata) Trees 32571ndash585 httpsdoiorg101007s00468-018-1657-4
Huang T Jander G de Vos M 2011 Non-protein amino acids in plant defense againstinsect herbivores representative cases and opportunities for further functional ana-lysis Phytochemistry 72 1531ndash1537 httpsdoiorg101016jphytochem201103019
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of thethyrotoxic amino acid mimosine by cysteine synthase Phytochemistry 29 (11)3461ndash3465 httpsdoiorg1010160031-9422(90)85258-H
Ishihara K Lee EKW Borthakur D 2016a An improved method for RNA extractionfrom woody legume species Acacia koa A Gray and Leucaena leucocephala (Lam) deWit Int J For Wood Sci 3 (1) 031ndash035
Ishihara KL Honda MDH Pham DT Borthakur D 2016b Transcriptome analysisof Leucaena leucocephala and identification of highly expressed genes in roots andshoots Transcriptomics 4 135 httpsdoiorg1041722329-89361000135
IUBMB 2018 Enzyme Nomenclature EC 35161 httpwwwsbcsqmulacukiubmbenzymeEC35161html Accessed date 8 February 2018
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant(Phaseolus vulgaris) Biosci Res 10 (1) 29ndash32
Kazan K 2015 Diverse roles of jasmonates and ethylene in abiotic stress toleranceTrends Plant Sci 20 (4) 219ndash229 httpsdoiorg101016jtplants201502001
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect ofEthephon as an ethylene-releasing compound on the metabolic profile of Chlorellavulgaris J Agric Food Chem 64 (23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541
Khan MIR Fatma M Per TS Anjum NA Khan NA 2015 Salicylic acid-inducedabiotic stress tolerance and underlying mechanisms in plants Front Plant Sci 6 462httpsdoiorg103389fpls201500462
Korsangruang S Soonthornchareonnon N Chintapakorn Y Saralamp PPrathanturarug S 2010 Effects of abiotic and biotic elicitors on growth and iso-flavonoid accumulation in Pueraria candollei var candollei and P candollei var mir-ifica cell suspension cultures Plant Cell Tissue Organ Cult 103 (3) 333ndash342 httpsdoiorg101007s11240-010-9785-6
Lalitha K Kulothungan SR 2006 Selective determination of mimosine and its dihy-droxypyridinyl derivative in plant systems Amino Acids 31 (3) 279ndash287 httpsdoiorg101007s00726-005-0226-5
Lalitha K Kulothungan SR 2007 Mimosine mitigates oxidative stress in seleniumdeficient seedlings of Vigna radiata - Part I restoration of mitochondrial functionBiol Trace Elem Res 118 (1) 84ndash96 httpsdoiorg101007s12011-007-0013-0
Liu J Li Y Wang Y Zhang Z-H Zu Y-G Efferth T Tang Z-H 2016 Thecombined effects of ethylene and MeJA on metabolic profiling of phenolic com-pounds in Catharanthus roseus revealed by metabolomics analysis Front Physiol 71ndash11 httpsdoiorg103389fphys201600217 Article 217
Malekzadeh P Khara J Heydari R 2014 Alleviating effects of exogenous Gamma-aminobutiric acid on tomato seedling under chilling stress Physiol Mol Biol Plants20 (1) 133ndash137 httpsdoiorg101007s12298-013-0203-5
Martiacutenez-Luumlscher J Torres N Hilbert G Richard T Saacutenchez-Diacuteaz M Delrot SAguirreolea J Pascual I Gomegraves E 2014 Ultraviolet-B radiation modifies thequantitative and qualitative profile of flavonoids and amino acids in grape berriesPhytochemistry 102 106ndash114 httpsdoiorg101016jphytochem201403014
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation ofbioactive secondary metabolites by ultraviolet radiation mechanisms strategies andapplications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology forMedicinal Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash1902012
Matsuura HN Fragoso V Paranhos JT Rau MR Fett-Neto AG 2016 Thebioactive monoterpene indole alkaloid N szlig-D-glucopyranosylvincosamide is regu-lated by irradiance quality and development in Psychotria leiocarpa Ind Crop Prod86 210ndash218 httpsdoiorg101016jindcrop201603050
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo RBhambra AS Strnad M Bonfill M Fett-Neto AG 2018 Specialized plant me-tabolism characteristics and impact on target molecule biotechnological productionMol Biotechnol 60 (2) 169ndash183 httpsdoiorg101007s12033-017-0056-1
Murakoshi S Ohmiya S Haginiwa J 1972 Enzymic synthesis of mimoside a meta-bolite of mimosine in Mimosa pudica and Leucaena leucocephala Chem Pharm Bull20 (4) 855ndash857
Nakagawa T Kawaguchi M 2006 Shoot-applied MeJA suppresses root nodulation inLotus japonicus Plant Cell Physiol 47 (1) 176ndash180 httpsdoiorg101093pcppci222
Nascimento NC Menguer PK Henriques AT Fett-Neto AG 2013 Accumulation ofbrachycerine an antioxidant glucosidic indole alkaloid is induced by abscisic acidheavy metal and osmotic stress in leaves of Psychotria brachyceras Plant PhysiolBiochem 73 33ndash40 httpsdoiorg101016jplaphy201308007
Neelamegam R Sutha T 2015 UV-C irradiation effect on seed germination seedling
growth and productivity of groundnut (Arachis hypogaea L) Int J Curr MicrobiolApp Sci 4 (8) 430ndash443
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase fromLeucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol164 (2) 922ndash934 httpsdoiorg101104pp113230870
Negi VS Borthakur D 2016 Heterologous expression and characterization of mimo-sinase from Leucaena leucocephala In Fett-Neto Arthur Germano (Ed)Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods inMolecular Biology vol 1405 copySpringer Science+Business Media New York httpsdoiorg101007978-1-4939-3393-8_7 2016
Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636
Parmar F Kushawaha N Highland H George L-B 2015 In vitro antioxidant andanticancer activity of Mimosa pudica Linn extract and L-mimosine on lymphomaDaudi cells Int J Pharm Sci 12 100ndash104
Porto DD Matsuura HN Vargas LRB Henriques AT Fett-Neto AG 2014 Shootaccumulation kinetics and effects on herbivores of the wound-induced antioxidantindole alkaloid brachycerine of Psychotria brachyceras Nat Prod Commun 9 (5)629ndash632
Rai R Meena RP Smita SS Shukla A Rai SK Pandey-Rai S 2011 UV-B and UV-C pre-treatments induce physiological changes and artemisinin biosynthesis inArtemisia annua L ndash an antimalarial plant J Photochem Photobiol B Biol 105 (3)216ndash225 httpsdoiorg101016jjphotobiol201109004
Shabani L Ehsanpour AA Asghari G Emami J 2009 Glycyrrhizin production by invitro cultured Glycyrrhiza glabra elicited by methyl jasmonate and salicylic acid RussJ Plant Physiol 56 (5) 621ndash626 httpsdoiorg101134S1021443709050069
Shah J 2003 The salicylic acid loop in plant defense Curr Opin Plant Biol 6 (4)365ndash371
Shi J Fu XZ Peng T Huang XS Fan QJ Liu JH 2010 Spermine pretreatmentconfers dehydration tolerance of citrus in vitro plants via modulation of antioxidativecapacity and stomatal response Tree Physiol 30 (7) 914ndash922 httpsdoiorg101093treephystpq030
Smith IK Fowden L 1966 A study of mimosine toxicity in plants J Exp Bot 17750ndash761 httpsdoiorg101093jxb174750
Soedarjo M Borthakur D 1996 Simple procedures to remove mimosine from youngleaves pods and seeds of Leucaena leucocephala used as food Int J Food SciTechnol 31 (1) 97ndash103
Soedarjo M Borthakur D 1998 Mimosine a toxin produced by the tree-legumeLeucaena provides a nodulation competition advantage to mimosine-degradingRhizobium strains Soil Biol Biochem 30 1605ndash1613
Suda S 1960 On the physiological properties of mimosine Bot Mag Tokyo 73 (862)142ndash147 httpsdoiorg1015281jplantres188773142
Tangendjaja B Lowry JB Wills RBH 1986 Isolation of a mimosine degrading en-zyme from leucaena leaf J Sci Food Agric 37 523ndash526 httpsdoiorg101002jsfa2740370603
Tiburcio F Pintildeol MT Serrano M 1985 Effect of UV-C on growth soluble protein andalkaloids in Nicotiana rustica plants Environ Exp Bot 25 (3) 203ndash210 httpsdoiorg1010160098-8472(85)90004-8
Vestena S Fett-Neto AG Duarte RC Ferreira A 2001 Regulation of mimosineaccumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 httpsdoiorg101016S0168-9452(01)00448-4
Vlot AC Dempsey DMA Klessig DF 2009 Salicylic acid a multifaceted hormone tocombat disease Annu Rev Phytopathol 47 177ndash206 httpsdoiorg101146annurevphyto050908135202 2009
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene-induced vinblastine accumulation is related to activated expression of downstreamTIA pathway genes in Catharanthus roseus BioMed Res Int 2016 Article ID 3708187httpsdoiorg10115520163708187
Wasternack C Strnad M 2016 Jasmonate signaling in plant stress responses and de-velopment ndash active and inactive compounds N Biotech 33 (5B) 604ndash613 httpsdoiorg101016jnbt201511001
Wencomo HB Ortiz R Caacuteceres J 2017 Afr J Agric Res 12 (4) 279ndash285 httpsdoiorg105897AJAR201510604 26
Wong CC Devendra C 1983 Research on leucaena forage production in Malaysia InLeucaena Research in the Asian Pacific Region pp 55ndash60 Ottawa Ontario Canada
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX TsimKWK 2018 Jasmonate-elicited stress induces metabolic change in the leaves ofLeucaena leucocephala Molecules 23 (2) httpsdoiorg103390molecules23020188 E188
Yafuso JT Negi VS Bingham J-P Borthakur D 2014 An O-acetylserine (thiol)lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthaseAppl Biochem Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z
Zhao J Zheng S-H Fujita K Sakai K 2004 Jasmonate and ethylene signalling andtheir interaction are integral parts of the elicitor signalling pathway leading to b-thujaplicin biosynthesis in Cupressus lusitanica cell cultures J Exp Bot 55 (399)1003ndash1012 httpsdoiorg101093jxberh127
Zhishen J Mengcheng T Jianming W 1999 The determination of flavonoid contentsin mulberry and their scavenging effects on superoxide radicals Food Chem 64 (4)555ndash559 httpsdoiorg101016S0308-8146(98)00102-2
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
440
61
Supplementary Fig 1 Basal mimosine concentration in adult trees of common leucaena (L leucocephala
var leucocephala) Samples were collected from 10 field grown trees at Manoa Valley Honolulu Hawairsquoi
on June 25th 2017 Bars sharing a letter do not differ by Tukey test (P le 005) The error bars represent the
standard error
Supplementary Fig 2 Bar diagram showing mimosine concentration in shoots of 12-week-old common
leucaena seedlings treated with different elicitors CTRL = Milli-Q water SA = Salicylic Acid MeJA =
Methyl Jasmonate CEPA = 2-Chloroethylphosphonic acid (an ethylene releasing compound) Bars sharing a
letter of same case do not differ by Tukey test (P le 005) Capital letters (A B) compare treatments on day
two and lower-case letters (a b) compare treatments on day four Indicates significant statistical difference
ABB
A A
0
200
400
600
800
1000
1200
LEAVES GREEN FLOWERBUDS
POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
con
cen
trat
ion
(micro
gg
-1o
f FW
)
B AB AB AB B A
b
a
ab b
ab
0
2
4
6
8
10
12
14
16
18
20
CTRL SA 10 ppm SA 100 ppm CEPA 10 ppm CEPA 100 ppm MeJA 90 ppm
Mim
osi
ne
co
nce
ntr
atio
n (
gg
-1o
f FW
)
DAY 02 DAY 04
62
between day two and day four in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
Supplementary Fig 3 Bar diagram showing the effects of UV-C radiation exposure for 5 10 and 15 min on
mimosine accumulation in shoots of 12-week-old seedlings of common leucaena Bars sharing a letter of
same case do not differ by Tukey test (P le 005) Capital letters (A B C) compare treatments on day three
and lower-case letters (a b) compare treatments on day six Indicates significant statistical difference
between day three and day six in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
C BC AB A
bb
a
a
0
10
20
30
40
50
60
CTRL UV-C 5 UV-C 10 UV-C 15
Mim
osi
ne
co
nce
ntr
atio
n (
gg-1
of
FW)
DAY 03 DAY 06
63
Supplementary Fig 4 Model depicting induction of mimosine synthesis in leucaena following application of
stress elicitors such as CEPA and jasmonic acid or exposure to UV-C radiation The additional mimosine
synthesized may serve to alleviate oxidative stress induced by UV-C radiation
64
Supplementary Table 1 Mimosine contents in leaves of common and giant leucaena
Leucaena
type
Mimosine content
( FW)
Mimosine
content ( DW)
Dry matter
content ( FW)
Water content
( FW)
Common (1) 050 plusmn 009 245 plusmn 051 2011 plusmn 054 7989 plusmn 054
Common (2) 043 plusmn 006 214 plusmn 037 1998 plusmn 050 8002 plusmn 050
K636 (1) 070 plusmn 014 356 plusmn 077 1908 plusmn 052 8092 plusmn 052
K636 (2) 042 005 205 plusmn 033 2008plusmn 093 7992plusmn 093
KX2 (1) 122 plusmn 011 608 plusmn 082 1939 plusmn 123 8061 plusmn 123
KX2 (2) 134 plusmn 010 623 plusmn 056 2029 plusmn 114 7971 plusmn 114
KX3 (1) 044 plusmn 006 221 plusmn 030 1945 plusmn 073 8055 plusmn 073
KX3 (2) 054 plusmn 005 273 plusmn 023 1930 plusmn 038 8070 plusmn 038
KX4 (1) 086 plusmn 011 471 plusmn 065 1753 plusmn 084 8247 plusmn 084
KX4 (2) 089 plusmn 011 476 plusmn 065 180 plusmn 072 820 plusmn 072
KX5 (1) 099 plusmn 012 489 plusmn 048 1907 plusmn060 8093 plusmn 060
KX5 (2) 115 plusmn 015 548 plusmn080 1992 plusmn 053 8008 plusmn 053
Common leucaena variety koa haole grows widely on the island of Orsquoahu K636 is widely
grown variety of giant leucaena KX2 KX3 KX4 and KX5 are giant leucaena varieties
developed through interspecies hybridization (Brewbaker 2016) (1) and (2) indicate plants
from two separate locations within the University of Hawaii Waimanalo Research Center The
values are shown as mean plusmn standard error obtained from at least three biological replicates
65
Supplementary Table 2 GenBank accession numbers of the tested cysteine pathway genes isoforms
Gene name GenBank accession
OAS-TL (o-acetylserine-thiol-lyase) GDRZ01032940
GDRZ01061620
GDRZ01153117
GDSA01187555
GDSA01196891
GDSA01214467
Cys syn (cysteine synthase) GDRZ01015860
GDRZ01050898
GDRZ01086813
GDRZ01193515
GDRZ01202579
GDSA01180863
GDSA01215622
SAT (serine acetyltransferase) GDRZ01187456
GDRZ01189631
CAS (β-cyanoalanine synthase) GDRZ01054066
GDRZ01175418
GDSA01118400
66
SHORT COMMUNICATION 1
Mimosine occurrence and accumulation in Mimosa bimucronata var bimucronata (DC) 2
Kuntze 3
Kelly Cristine da Silva Rodrigues-Correcirca1 Lana Dorneles Pedroso2 Fernanda de Costa1 4
Arthur Germano Fett-Neto1 5
1Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal 6
University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970 7
Porto Alegre Rio Grande do Sul Brazil 2Department of Biological Sciences Unipampa ndash 8
Campus Satildeo Gabriel 9
Corresponding author 10
E-mail addresses krodriguescbiotufrgsbr (KCdaS Rodrigues-Correcirca) 11
lanalima2012gmailcom (LD Pedroso) fernandadecostayahoocombr (F de Costa) 12
fettnetocbiotufrgsbr (AG Fett-Neto) 13
14
15
16
17
18
19
20
21
22
67
ABSTRACT 23
Mimosine is a non-protein aromatic amino acid present in plants of Leucaena spp 24
and Mimosa spp Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) is a native 25
tree from Brazil which occurs as a pioneer species on plant succession processes In the 26
current study the presence of mimosine in M bimucronata was verified by HPLC analyses 27
Moreover mimosine accumulation upon exposure to UV-C and chemical elicitors of 28
specialized metabolism (salicylic acid - SA methyl jasmonate - MeJA sodium nitroprusside 29
- SNP and ethephon - ETH) most of which also known as promoters of the amino acid 30
production in leucaena plants was evaluated The results showed a lower concentration of 31
constitutive mimosine present in both maricaacute seedlings and mature trees when compared to 32
leucaena plants In spite of a trend towards increased mimosine accumulation observed in 33
MeJA and ETH treatments no statistical differences were found with the various stressors 34
used to induce its biosynthesis in maricaacute seedlings Data suggest that mimosine in M 35
bimucronata is probably a phytoanticipin-like metabolite or its accumulation is driven by 36
other types of stresses 37
38
39
Keywords Mimosine Mimosa bimucronata stress 40
41
42
43
44
45
46
68
Introduction 47
Mimosa bimucronata commonly known as maricaacute is a native tree from Brazil 48
(REFLORA 2019) ecologically important in plant succession and in processes of degraded 49
land recovery (Bitencourt et al 2007 Silva et al 2011) occurring as a pioneer species 50
(Pilatti et al 2019) Maricaacute is a deciduous or semi-deciduous plant which reaches up to 15 51
m in height and 40 cm of diameter at breast height (DBH) displays shrub or tree habit and 52
bears typical sharp thorns (Carvalho 2004) This species belongs to Fabaceae one of the 53
most economically important families of flowering plants due to its high diversity and 54
occurrence in different types of habitats (Gomes et al 2018) As well as several others 55
Mimosa spp maricaacute is usually referred to as a multipurpose tree (Olkoski and Wittmann 56
2011) employed for alternative medicinal uses (Champanerkar et al 2010 Silva et al 57
2011) honey production constructions and remodeling of landscape architecture (living 58
fences) for instance (Marchiori 1993 Lorenzi 1998) 59
In southern Brazil maricaacute is widely distributed and typically found either in wetland 60
areas close to river banks (Patreze and Cordeiro 2004) or composing large and almost pure 61
landscape formations on hillsides (Jacobi and Ferreira 1991) In dense populations this 62
species like several Mimosa spp (Simon and Proenccedila 2000) is considered an important and 63
highly invasive weed by preventing cattle to reach pasturesand water bodies as a result of its 64
thorny branches (Lorenzi 2008 Kestring et al 2009) Its dominant and nearly exclusive 65
pattern of distribution in those areas has led Jacobi and Ferreira (1991) to test its allelopathic 66
potential on cultivated species Indeed extracts of leaves and ripe fruits (but not the green 67
ones) of maricaacute showed phytotoxic effects on germination and initial radical growth of most 68
of the target species tested 69
69
Several investigations have been performed on maricaacute floristics (Silva et al 2011) 70
distribution (Simon and Proenccedila 2000) wood anatomy (Marchiori 1993) cytogenetic 71
parameters (Olkoski and Wittmann 2011) and allelopathic potential (Jacobi and Ferreira 72
1991 Ferreira et al 1992) However excluding two recent publications on maricaacute 73
constitutive chemical composition (Schlickmann et al 2017 Pilatti et al 2019) which 74
identified phenolic compounds (methyl gallate and water-soluble tannins) as its major 75
compounds little is known regarding this subject In other Mimosa species (eg M pudica 76
and M pigra) mimosine has been identified (Soedarjo and Borthakur 1998) as one of the 77
major specialized metabolites present in the different organs of the plant (Champanerkar et 78
al 2010) The presence of this molecule was also reported for M bimucronata in a thin layer 79
chromatography-based preliminary study performed by Ferreira et al (1992) showing co-80
chromatography of a leaf extract component with authentic mimosine The authors attributed 81
the allelopathic effect of maricaacute to the accumulation of this metabolite in its leaves 82
Mimosine is an aromatic non-protein amino acid initially found in plants of Mimosa 83
pudica and later in Leucaena leucocephala (Lam) de Wit (Soedarjo and Borthakur 1998) a 84
leguminous tree which biosynthesizes large amounts of this nitrogen-containing compound 85
(Rodrigues-Correcirca et al 2019) It is believed that the accumulation of high contents of 86
mimosine in L leucocephala tissues confers among other traits defense against herbivores 87
and pathogens (Vestena et al 2001) tolerance to drought (Negi et al 2014) as well as 88
general oxidative stress protection (Rodrigues-Correcirca et al 2019) Interestingly drought is 89
the opposite environmental and physiological condition to that observed in the wet habitats 90
occupied by native populations of M bimucronata in Brazil (Patreze and Cordeiro 2004 91
Kestring et al 2009) and Mimosa pudica Linn in India (Champanerkar et al 2010) 92
70
Nonetheless flooding is also associated with oxidative stress particularly as water levels 93
change (Fukao et al 2019) 94
In Leucaena leucocephala var leucocephala (common leucaena) and Leucaena 95
leucocephala var glabrata (giant leucaena) mimosine accumulation has been shown to be 96
both constitutive and inducible by stress-related phytohormones such as jasmonic acid (JA) 97
Ethephon (ETH an ethylene- releasing compound) salicylic acid (SA - only common 98
leucaena) (Vestena et al 2001) as well as by UV-C radiation (Xu et al 2018 Rodrigues-99
Correcirca et al 2019) On the other hand there is a lack of information regarding mimosine 100
content and elicitation effects in Mimosa spp plants 101
The aim of this study was to examine the presence of mimosine in Mimosa 102
bimucronata and examine the effects of stresses and stress-signaling molecules on its 103
accumulation in leaves 104
Material and Methods 105
Plant material 106
For all experiments the plant material was collected at Morro Santana campus do 107
Vale of UFRGS (Federal University of Rio Grande do Sul) Porto Alegre RS Brazil 108
(3004rsquoS 5108rsquoW) Authorization for access to genetic material was obtained from 109
SISGEN-Brazil (license number A845493) Constitutive mimosine content in adult plants of 110
M bimucronata var bimucronata (DC) Kuntze was determined in plant material (leaves 111
green flower buds post-anthesis flowers and green pods) harvested in January 2017 112
(summer) A voucher herbarium specimen (ICN 187953) was deposited in the ICN ndash UFRGS 113
herbarium (Herbaacuterio do Instituto de Biociecircncias of UFRGS) 114
71
For mimosine elicitation experiments legumes (fruits) of maricaacute were collected in 115
the end of June 2017 (winter) Seeds were then removed from the dry fruits and kept in the 116
dark until sowing and seedling development for use in the assays 117
Seed germination 118
To break the coat-imposed seed dormancy after surface sterilization dry seeds of 119
maricaacute were acid scarified by immersion in H2SO4 (95 ndash 98 ) for 2 min (see Correcirca et al 120
2008) and repeatedly washed in distilled water to remove any residue of the acid Then seeds 121
were distributed in 50 mL individual plastic tubes (dibble-tubes) (30 cm diameter x 120 cm 122
depth) filled up with 11 (vv) of commercial top soil and vermiculite Tubes were watered 123
every 2 days to avoid substrate dryness and were kept in a growth room under controlled 124
conditions of light (circa 75 μmol mminus2s minus1 photosynthetically active radiation photoperiod 125
of 16 h light and 8 h dark) and temperature (24plusmn2C) 126
127
Treatments 128
In order to verify inducibility of mimosine accumulation in M bimucronata fifty 12-129
week-old maricaacute seedlings (per treatment) exhibiting similar features were selected and 130
sprayed (saturated) with solutions of different chemical stressors (plant specialized 131
metabolism elicitors) as follows (for further details see Rodrigues-Correcirca et al 2019) 10 132
and 50 mM SA (pathogen-signaling molecule Shah 2003) 007 and 035 mM 2-133
chloroethylphosphonic acid (ETH ethylene releasing-compound Kim et al 2016 Wang et 134
al 2016) 100 and 200 mM MeJA (Dar et al 2015) 10 and 50 mM SNP (a nitric oxide 135
donor Perotti et al 2015) Alternatively maricaacute seedlings were also supplemented with UV-136
C radiation (13 minutes 105 kJ cm2) (elicitor of plant specialized metabolism Kara 2013) 137
72
After 2 and 4 days of exposure to the chemical treatments and 3 and 6 days of UV-138
C supplementation maricaacute shoots were harvested immediately frozen in liquid nitrogen and 139
stored at ndash 80 C until mimosine extraction and HPLC analyses 140
Mimosine extraction and detection 141
Mimosine extraction was conducted according to the modified protocol described by 142
Rodrigues-Correcirca et al (2019) for L leucocephala HPLC (Thermo Scientific Surveyor) 143
analyses (mimosine detection and quantification) were performed following previously 144
published procedures (Negi et al 2014) A C18 column (ACE C18 5 μm 46times250 mm) and 145
isocratic solvent system of 002M o-phosphoric acid with a linear flow rate of 1 mL min minus1 146
were used to separate and quantify the amino acid Mimosine detection was performed at 280 147
nm by photodiode array detection (200ndash400 nm) and retention time (229plusmn0024 min) 148
Mimosine quantification was done by means of the method of external standard curve 149
Additional confirmation of mimosine identity was performed by co-chromatography with 150
standard (Acros Organics authentic mimosine 99 used as reference) and peak purity check 151
The analyses of the chromatograms were done with the ChromQuest software 152
153
154
Results and Discussion 155
Constitutive accumulation of mimosine in M bimucronata 156
Mimosine was detected in all analyzed samples positively meeting all identification 157
criteria In agreement with what has been found for other Mimosa spp (Soedarjo and 158
Borthakur 1998) compared to L leucocephala adult plants (Rodrigues-Correcirca 2019) 159
mimosine content was lower in M bimucronata Of the adult plant tissues analyzed the 160
73
highest content of mimosine in maricaacute (per gram of fresh weight - FW) was found in post-161
anthesis flowers (36644 microg versus 89448 microg in common leucaena followed by leaves 162
(28838 microg x 67358 microg) green flower buds (28094 microg x 51247 microg) and green pods (19002 163
microg x 82687 microg) (Fig 1)The same pattern is observed for seedlings when both species are 164
compared In this study untreated 12-week-old maricaacute seedlings (control at day 2) showed a 165
shoot content of mimosine of 23029plusmn007 microg g-1 of (FW) Five-week-old untreated giant 166
leucaena seedlings cultivated in similar conditions exhibited between 83640 and 178736 167
microg g-1 of FW (Rodrigues-Correcirca et al 2019) In the same way mimosine concentration 168
percentage in dry matter of Mimosa pigra was found to be rather low (002 in nodules and 169
roots and 007 in leaves) (Soedarjo and Borthakur 1998) 170
In this investigation the lowest constitutive mimosine content was found in green 171
pods (Fig 1) This result may partly explain the absence of phytotoxic effect observed for 172
green pods on germination and growth of crop target plants tested by Jacobi and Ferreira 173
(1991) compared to the other maricaacute parts analyzed 174
Elicitation of mimosine biosynthesis in M bimucronata 175
Chemical stressors 176
Secondary metabolites (or natural products) are structural- and chemically 177
specialized compounds derived from primary metabolism These molecules are mainly 178
biosynthesized as part of a complex defense mechanism in response to biotic and abiotic 179
stresses such as pathogens herbivores water status metal toxicity and UV radiation for 180
example (Matsuura et al 2018) Ethephon SA SNP MeJA have been extensively used as 181
chemical elicitors of specialized metabolism (Wang et al 2016 Vestena et al 2001 Perotti 182
74
et al 2015 Zhang and Memelink 2009 Xu et al 2018) These phytohormonal signals can 183
simulate environmental challenges and modulate plant homeostasis often leading to 184
alterations in gene expression (Shinozaki et al 2015) Except SNP all treatments tested in 185
the present study showed positive effect on mimosine accumulation in common or giant 186
leucaena (Vestena et al 2001 Rodrigues-Correcirca 2019 Rodrigues-Correcirca unpublished 187
data) However in spite of the trend of increasing the mimosine content observed in seedlings 188
treated with 007 mM Ethephon (at day 2) and 100 mM MeJA (at day 4) no statistical 189
difference was confirmed for these treatments when compared to the control 190
On the other hand a within treatment difference on mimosine induction was seen 191
between day 2 and 4 in seedlings treated with 100 mM MeJA (Fig 2) In a lower 192
concentration (04 mM) jasmonic acid (JA)promoted a near threefold increase in mimosine 193
accumulation of giant leucaena seedlings after 2 days of application 194
UV-C radiation 195
Albeit UV-C radiation is not biologically active in natural environments it has been 196
widely used under controlled experimental conditions to generate acute responses of plant 197
specialized metabolism within a shorter period of time compared to that required to with UV-198
B radiation (Kara 2013 Cetin 2014) This fast response is due to the higher energy of UV-199
C photons that act as potent reactive oxygen species (ROS) generators causing extensive 200
damage to the cells either at the physiological level or on DNA structure (Gregianini et al 201
2003 Matsuura et al 2013) 202
Although divergent responses can be observed in plants exposed to UV-C radiation 203
the deleterious processes are usually reported on primary metabolism (decreasing of 204
chlorophyll content and plant height eg) (Kara 2013) In the present study no statistical 205
75
differences were observed in the mimosine concentration in maricaacute seedlings supplemented 206
with UV-C radiation However a decreasing in its content was found for both control and 207
treatment at day 6 post-treatment (Fig 03) Taking into account the lower constitutive 208
concentration of mimosine observed in maricaacute compared to the leucaena plants besides its 209
relative thermolability (Nguyen and Tawata 2016) it seems to be plausible to consider the 210
effect of the temperature inside the UV-C and the white light (control) chambers as an 211
additional abiotic factor contributing to the decrease of mimosine accumulation in both group 212
of plants 213
Besides mimosine identification the presence of 34-dihydroxypyridine (34-DHP or 214
3-hydroxy-4-pyridone - 3H4P) a mimosine degradation product (Negi et al 2014 Nguyen 215
and Tawata 2016) was also reported for maricaacute leaf extracts analyzed by TLC by Ferreira 216
et al (1992) In our chromatograms we detected a second large peak after that of mimosine 217
(229plusmn0024) and similar to that identified by Negi et al (2014) as 3H4P (data not shown) 218
Comparing the chromatogram profiles obtained from seedlings elicited with chemical 219
stressors and those supplemented with UV-C the largest area for this peak was found (in all 220
samples) in the latter treatment at day 6 It might indicate that the constitutive andor the 221
initially UV-C-induced mimosine was degraded into 3H4P to cope with the cellular damage 222
caused by this treatment associated with an increased temperature inside the chambers 223
Nevertheless it was not possible to determine 3H4P concentration (or confirm its identity) 224
in maricaacute plants since there is no commercial standard (pure 3H4P) available for purchase 225
to be used as a reference in calculations Establishment of improved protocols for obtaining 226
in house 3H4P reference substance by acid hydrolysis is ongoing 227
228
229
76
Conclusion 230
On the basis of the overall absence of effect of the treatments tested here on mimosine 231
concentration it is possible to suggest that its accumulation profile is similar to that of 232
phytoanticipins unlike what is observed for the same amino acid production in leucaena 233
which shows features of inducibility resembling phytoalexin-like metabolites Alternatively 234
a putative inducible pool of mimosine in maricaacute might be involved in other types of stress 235
such as extended drought periods If involved in protection against oxidative stress as 236
described for leucaena mimosine in maricaacute may act predominantly by physical quenching 237
of ROS as indicated by the lack of overt chemical degradation Nevertheless further 238
investigations are needed to assess these hypotheses 239
To sum up mimosine biosynthesis was not modulated by the treatments evaluated as 240
in L leucocephala (Lam) de Wit To the best of our knowledge this is the first work that 241
analytically identifies and quantifies mimosine accumulation in M bimucronata 242
243
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Bitencourt F Zocche JJ Costa S Souza PZ Mendes AR 2007 Nucleaccedilatildeo de 245
Mimosa bimucronata (DC) O Kuntze em aacutereas degradadas pela mineraccedilatildeo de carvatildeo R 246
Bras Bioci 5 750-752 247
Carvalho PER 2004 Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 248
Teacutecnica 94 1-10 249
Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitis 250
vinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-251
6287-47-37 252
77
Champanerkar PA Vaidya VV Shailajan S Menon SN 2010 A sensitive rapid and 253
validated liquid chromatography ndash tandem mass spectrometry (LC-MS-MS) method for 254
determination of Mimosine in Mimosa pudica Linn Nat Sci 2 713-717 255
httpsdoiorg104236ns201027088 256
Gomes GS Silva GS Silva DLS Oliveira RR Conceiccedilatildeo GM 2018 Botanical 257
Composition of Fabaceae Family in the Brazilian Northeast Maranhatildeo Brazil Asian J 258
Environ Ecol 6(4) 1-10 httpsdoiorg109734AJEE201841207 259
Correcirca LR Soares GLG Fett-Neto AG 2008 Allelopathic potential of Psychotria 260
leiocarpa a dominant understorey species of subtropical forests S Afri J Bot 74 583ndash261
590 httpsdoiorg101016jsajb200802006 262
Ferreira AG Aquila MEA Jacobi US Rizvi V 1992 Allelopathy in Brazil In Allelopathy 263
basic and applied aspects Rizvi V and Jacobi US (Eds) Chapman and Hall pp 243-250 264
Fukao T Barrera-Figueroa BE Juntawong P Pentildea-Castro JM 2019 Submergence 265
and waterlogging stress in plants a review highlighting research opportunities and 266
understudied aspects Front Plant Sci 10 340 httpsdoiorg103389fpls201900340 267
Gregianini TS Silveira VC Porto DD Kerber VA Henriques AT Fett-Neto AG 268
2003 The alkaloid brachycerine is induced by ultraviolet radiation and is a singlet oxygen 269
quencher Photochem Photobiol 78(5) 470ndash474 httpsdoiorg1015620031-270
8655(2003)0784070TABIIB20CO2 271
Jacobi US Ferreira AG 1991 Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK 272
sobre espeacutecies cultivadas Pesq Agropec Bras 26(7) 935-943 273
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant 274
(Phaseolus vulgaris) Biosci Res 10(1) 29ndash32 275
78
Kestring D Klein J Menezes LCCR Rossi MN 2009 Imbibition phases and 276
germination response of Mimosa bimucronata (Fabaceae Mimosoideae) to water 277
submersion Aquat Bot 91 105ndash109 httpsdoiorg101016jaquabot200903004 278
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect of 279
Ethephon as an ethylene-releasing compound on the metabolic profile of Chlorella vulgaris 280
J Agric Food Chem 64(23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541 281
Lorenzi H 1998 Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 282
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 283
Lorenzi H 2008 Plantas daninhas do Brasil terrestres aquaacuteticas parasitas e toacutexicas 4 ed 284
Nova Odessa Instituto Plantarum 640 p 285
Marchiori JNC 1993 Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 286
O Kuntze Ciecircncia Florestal 3 85-106 287
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation of 288
bioactive secondary metabolites by ultraviolet radiation mechanisms strategies and 289
applications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology for Medicinal 290
Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash190= 291
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo R Bhambra AS 292
Strnad M Bonfill M Fett-Neto AG 2018 Specializedplant 293
metabolismcharacteristicsandimpactontargetmoleculebiotechnologicalproduction 294
Molecular Biotechnology 60(2) 169ndash183httpsdoiorg101007s12033-017-0056-1 295
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase from 296
Leucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 297
922ndash934 httpsdoiorg101104pp113230870 298
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Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine 299
areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636 300
Olkoski D Wittmann MTS 2011 Cytogenetics of Mimosa bimucronata (DC) O Kuntze 301
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop Breed 302
Appl Biotechnol 11 27-35 httpdxdoiorg101590S1984-70332011000100004 303
Patreze CM Cordeiro L 2004 Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 304
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecol Manag 196 275ndash305
285 httpdxdoiorg101016jforeco200403034 306
Perotti JC Rodrigues-Correcirca KCS Fett-Neto AG 2015 Control of resin production in 307
Araucaria angustifolia an ancient South American conifer Plant Biology 17 852ndash859 308
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG 2019 Mimosine 309
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 310
UV exposure Plant Physiology and Biochemistry 135 432ndash440 311
Pilatti DM Fortes AMT Jorge TCM Boiago NP 2019 Comparison of the phytochemical 312
profiles of five native plant species in two different forest formations Brazilian Journal of 313
Biology 79(2) 233-242 314
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS 2011 Biologia da reproduccedilatildeo 315
deMimosa bimucronatandash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 316
Simon MF and Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 317
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 318
endemism Biological Conservation 96 279-296 319
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 320
LM Andrade SF Cechinel-Filho V 2017 Chemical composition and diuretic natriuretic and 321
80
kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its majority 322
constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash1624 323
Shah J 2003 The salicylic acid loop in plant defense Current Opinion Plant Biology6 (4) 324
365ndash371 325
Shinozaki K Uemura M Serres JB Bray EA Weretilnyk E 2015 Responses to Abiotic 326
Stress In Buchanan BB Gruissem W Jones RL (Eds) Biochemistry and Molecular 327
Biology of Plants Second Edition John Wiley and Sons Ltd 328
Soedarjo M and Borthakur D 1998 Mimosine a toxin produced by the tree-legume 329
Leucaena provides a nodulation competition advantage to mimosine-degrading Rhizobium 330
strains Soil Biology and Biochemistry 30(12)1605-1613 331
Vestena S Fett-Neto AG Duarte RC Ferreira AG 2001 Regulation of mimosine 332
accumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 333
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene induced 334
vinblastine accumulation is related to activated expression of downstream TIA pathway 335
genes in Catharanthus roseus BioMed Research International Article ID 3708187 336
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX Tsim KWK 2018 337
Jasmonate-elicited stress induces metabolic change in the leaves of Leucaena leucocephala 338
Molecules 23 (2) 339
Zhang H Memelink J 2009 Regulation of Secondary Metabolism by Jasmonate Hormones 340
In AE Osbourn and V Lanzotti (eds) Plant-derived Natural Products 3 DOI 101007978-341
0-387-85498-4_1 copy Springer Science + Business Media LLC 342
343
344
345
81
346
Figure 1 Constitutive concentration of mimosine in different plant organs of Mimosa 347
bimucronata Bars sharing the same letter do not differ statistically by Tukey test (Ple005) 348
The error bars denote standard error of 10 replicates 349
350
351
352
353
354
355
356
357
B B A C0
5
10
15
20
25
30
35
40
LEAVES GREEN FLOWER BUDS POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
co
nce
ntr
atio
n u
gg-1
Mimosine concentration in adult plants of Mimosa bimucronata (DC) Kuntze
82
C T R L S A
1 0 m M
S A
5 0 m M
E T H
0 0 7 m M
E T H
0 3 5 m M
M e J A
1 0 0 m M
M e J A
2 0 0 m M
S N P
1 0 m M
S N P
5 0 m M
0
1 0
2 0
3 0
T re a tm e n ts
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1) D A Y 2
D A Y 4
A B C C B C A B C C A B C A B C A
a b b b a a b a a b b a b
358
Figure 2 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 359
bimucronata treated with different signaling molecules SA = Salicylic Acid ETH = 360
Ethephon MeJA = Methyl Jasmonate SNP = Sodium Nitroprusside Uppercase and 361
lowercase letters indicate statistical differences among treatments in days 2 and 4 362
respectively Bars sharing a letter of the same case do not differ statistically by Tukey test 363
(Ple005) Indicates statistical difference in the same treatment between day 2 and 4 by t-364
test (Ple005) The error bars denote standard error of 5 replicates (25 individual seedlings 365
arranged in 5 groups of 5) 366
367
368
83
D AY 3 D AY 6
0
5
1 0
1 5
2 0
2 5
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1)
C O N TR O L
U V -C
369
Figure 3 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 370
bimucronata supplemented with UV-C radiation Indicates statistical difference in the same 371
treatment between day 3 and 6 by t-test (Ple005) The error bars denote standard error of 5 372
replicates (25 individual seedlings arranged in 5 groups of 5) 373
374
375
376
377
378
379
380
381
382
383
384
385
84
Consideraccedilotildees finais 386
- Experimentos que avaliam os efeitos da aplicaccedilatildeo exoacutegena de ANPs em diferentes espeacutecies 387
vegetais tecircm sido realizados principalmente com GABA Dentre os principais efeitos 388
conferidos pela aplicaccedilatildeo dessa moleacutecula em espeacutecies de mono e eudicotiledocircneas satildeo 389
relatados a toleracircncia agrave seca agrave salinidade e agraves temperaturas extremas 390
- Como metaboacutelitos especializados claacutessicos os ANPs podem ter sua concentraccedilatildeo basal 391
endoacutegena aumentada em resposta agrave induccedilatildeo mediada por uma vasta gama de tratamentos com 392
moleacuteculas sinalizadoras de estresse e fontes alternativas de estressores De um modo geral 393
observa-se o acuacutemulo das diferentes classes de ANPs em resposta agrave radiaccedilatildeo UV elicitores 394
quiacutemicos que mimetizam ataques por patoacutegenos dano mecacircnico agentes osmoacuteticos metais 395
pesados entre outros 396
- Especificamente em leucena a resposta observada em relaccedilatildeo aos diferentes tratamentos 397
testados indica que apesar do seu alto teor constitutivo nessa espeacutecie a biossiacutentese e o 398
acuacutemulo de mimosina podem ser modulados por fatores causadores de estresses exibindo -399
nessa espeacutecie - um padratildeo de acumulaccedilatildeo similar agrave fitoalexinas Em maricaacute por outro lado 400
aumento de acuacutemulo dessa moleacutecula natildeo foi observado para os mesmos tratamentos testados 401
para leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas 402
- O padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno 403
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado pela 404
sua degradaccedilatildeo 405
- As respostas observadas nos testes que avaliaram a atividade de mitigaccedilatildeo de espeacutecies 406
reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode agir como um agente 407
antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de estresse 408
85
Perspectivas 409
- Confirmaccedilatildeo em espectrocircmetro de massas eou ressonacircncia nuclear magneacutetica da natureza 410
quiacutemica da lsquomimosinarsquo presente em maricaacute 411
- Avaliaccedilatildeo do efeito de concentraccedilotildees mais elevadas e em diferentes periacuteodos de aplicaccedilatildeo 412
das moleacuteculas sinalizadoras testadas sobre o acuacutemulo de mimosina em leucena e maricaacute 413
- Ampliar a investigaccedilatildeo dos padrotildees de expressatildeo gecircnica dos genes que codificam para 414
mimosinase (em maricaacute) mimosina sintase (em ambas as espeacutecies testadas) bem como o 415
perfil de precursores e cataboacutelitos de mimosina em resposta aos tratamentos mencionados 416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
86
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437
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Teacutecnica 941-10 458
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Crowe B Poynter JA Manukyan MC Wang Y Brewster BD Herrmann JL Abarbanell 465
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Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of the thyrotoxic 481
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Jacobi US Ferreira AG (1991) Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK Sobre 484
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Jiao CJ Jiang J-L Ke L-M Cheng W Li F-M Li Z-X Wang C-Y (2011) Factors affecting 490
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Yamaguchi N (2014) Activation of the prereplication complex is blocked by mimosine 494
88
through reactive oxygen species-activated ataxia telangiectasia mutated (ATM) protein 495
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Aqueous Extract Stimulates Adipogenesis Lipolysis and Glucose Uptake in Primary Rat 498
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Kusama-Eguchi K (2019) Research in motor neuron diseases caused by natural substances 501
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Kutchan TM Gershenzon J Moslashller BL Gang DR (2015) Natural Products In Buchanan 504
BB Gruissem W and Jones RL (eds) Biochemistry amp Molecular Biology of Plants 2nd edn 505
Wiley Blackwell Chichester pp 1135-1205 506
Lalande M (1990) A reversible arrest point in the late G1 phase of the mammalian cell cycle 507
Exp Cell Res 186 332ndash339 508
Li X-W Hu C-P Li Y-J Gao Y-X Wang XM Yang J-R (2015) Inhibitory effect of L-509
mimosine on bleomycin-induced pulmonary fibrosis in rats Role of eIF3a and p27 Int 510
Immunopharmacol 27(1) 53ndash64 511
Little Jr EL Skolmen RG (1989) Koa haole Agriculture Handbook 679 USDA 512
Lorenzi H (1998) Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 513
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 514
Marchiori JNC (1993) Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 515
O Kuntze Ciecircncia Florestal 3 85-106 516
Mohammed RS El Souda SS Taie HAA Moharam ME Shaker KH (2015) Antioxidant 517
antimicrobial activities of flavonoids glycoside from Leucaena leucocephala leaves Journal 518
of Applied Pharmaceutical Science 5(06)138-147 519
httpdxdoiorg107324JAPS201550623 520
Negi VS Bingham J-P Li QX Borthakur D (2014) A carbon-nitrogen lyase from Leucaena 521
leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 (2) 922ndash522
934 httpsdoiorg101104pp113230870 523
89
Olkoski D Wittmann MTS (2011) Cytogenetics of Mimosa bimucronata (DC) O Kuntze 524
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop 525
Breeding and Applied Biotechnology 11 27-35 526
Patreze CM Cordeiro L (2004) Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 527
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecology and Management 528
196275ndash285 529
Pilatti DM Fortes AMT Jorge TCM Boiago NP (2019) Comparison of the phytochemical 530
profiles of five native plant species in two different forest formations Brazilian Journal of 531
Biology 79(2) 233-242 532
Ramos-Ruiz R Poirot E Flores-Mosquera M (2018) GABA a non-protein amino acid 533
ubiquitous in food matrices Cogent Food Agric 41534323 534
httpsdoiorg1010802331193220181534323 535
REFLORA (2019) httpfloradobrasiljbrjgovbrreflora Acesso em agosto de 2019 536
Rodgers KJ Samardzic K Main BJ (2015) Toxic Nonprotein Amino Acids Plant Toxins 537
httpsdoiorg 101007978-94-007-6728-7_9-1 538
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG (2019) Mimosine 539
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 540
UV exposure Plant Physiology and Biochemistry 135 432ndash440 541
httpsdoiorg101016jplaphy201811018 542
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 543
LM Andrade SF Cechinel-Filho V (2017) Chemical composition and diuretic natriuretic 544
and kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its 545
majority constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash546
1624 547
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS (2011) Biologia da reproduccedilatildeo 548
de Mimosa bimucronata ndash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 549
Simon MF Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 550
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 551
endemism Biological Conservation 96 279-296 552
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Soares AMS Arauacutejo SA Lopes SG Costa Junior LM (2015) Anthelmintic activity of 553
Leucaena leucocephala protein extracts on Haemonchus contortus Braz J Vet Parasitol 554
Jaboticabal 24(4) 396-401 httpdxdoiorg101590S1984-29612015072 555
Soerdajo M Borthakur D (1998) Mimosine a toxin produced by the tree-legume Leucaena 556
provides a nodulation competition advantage to mimosine-degrading Rhizobium strains Soil 557
Biol Biochem 30(12) 16051613 558
Souza-Lima ES Sinani TR Pott A Sartori ALB (2017) Mimosoideae (Leguminosae) in the 559
Brazilian Chaco of Porto Murtinho Mato Grosso do Sul Rodrigueacutesia 68(1) 263-290 2017 560
httpdxdoiorg1015902175-7860201768131 561
Taiz L amp Zeiger E (2010) Plant Physiology 5th edition Sinauer Associates Inc Sunderland 562
Verma VK Rani KV Kumara SR Prakash O (2018) Leucaena leucocephala pod seed 563
protein as an alternate to animal protein in fish feed and evaluation of its role to fight against 564
infection caused by Vibrio harveyi and Pseudomonas aeruginosa Fish and Shellfish 565
Immunology 76 (2018) 324ndash332 httpsdoiorg101016jfsi201803011 566
Yafuso JT Negi VS Bingham J-P Borthakur D (2014) An O-acetylserine (thiol) lyase from 567
Leucaena leucocephala is a cysteine synthase but not a mimosine synthase Appl Biochem 568
Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z 569
Zarin RMA Wan HY Isha A Armani N (2016) Antioxidant antimicrobial and cytotoxic 570
potential of condensed tannins from Leucaena leucocephala hybrid Food Science and 571
Human Wellness 5 65ndash75 httpdxdoiorg101016jfshw201602001 572
573
574
Contents lists available at ScienceDirect
Industrial Crops amp Productsjournal homepage wwwelseviercomlocateindcrop
Resin tapping transcriptome in adult slash pine (Pinus elliottii var elliottii)Camila Fernanda de Oliveira Junkes1 Artur Teixeira de Arauacutejo Juacutenior1 Juacutelio Ceacutesar de LimaFernanda de Costa Thanise Fuumlller Maacutercia Rodrigues de Almeida Franciele Antocircnia NeisKelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-NetoCenter for Biotechnology and Department of Botany Federal University of Rio Grande do Sul Porto Alegre PO Box 15005 91501-970 Brazil
A R T I C L E I N F O
KeywordsPinus elliottiResinResinosisTranscriptomeAdjuvant paste
A B S T R A C T
To better understand the bases of resin production a major source of terpenes for industry the transcriptome ofadult Pinus elliottii var elliottii (slash pine) trees under field commercial resinosis was obtained Samples werecollected from cambium after 5 and 15 days of treatment application which included tapping followed byapplication of commercial resin stimulant paste or control wounding without paste Overall mean number ofreads of all 16 libraries (2 treatments x 2 times x 4 replicated trees) was 34582048 Of these 89 were mappedagainst the reference sequence with a mismatch of 058 Using the Blast2Go 570 candidate genes were de-tected based on sequence annotation By comparing the expression profile between paste and control 310differentially expressed genes (DEGs) were identified at 5 days and 190 at 15 days with a significant fold changeof log2gt 12 Regarding changes in time comparisons within each treatment 210 and 105 DEGs were identifiedwithin control and paste treatment respectively Genes with different expression patterns in the times andtreatments examined included ethylene responsive transcription factors geranylgeranyl diphosphate synthasediterpene synthase cytochrome P450 and ABC transporters all of which may play important roles in resinproduction RT-qPCR analysis correlated well with the data obtained by RNAseq Resin composition changedover time This is the first transcriptomic investigation of resinosis of the main species used in the bioresinindustry and of molecular analyses of resinosis under field operations with implications for stand managementstimulant paste development genotype selection and breeding for high resinosis
1 Introduction
The adaptive success of conifers is largely due to the development ofa defense system based on the synthesis and secretion of terpenes in allmajor organs and different tissues (Miller et al 2005 Hall et al 2013Warren et al 2015) Conifer resin is a viscous fluid composed of acomplex mixture of terpenoids such as monoterpenes sesquiterpenesand diterpenes (Zulak and Bohlmann 2010) These terpenoids are se-creted from severed resin ducts when the tree is under biotic attack(Ralph et al 2006 Lange 2015 Geisler et al 2016) acting as pro-tectants (Schiebe et al 2012 Liu et al 2015)Biosynthesis of terpenes in conifers starts from isomerization of two
isoprenoid (C5) units dimethylallyl diphosphate (DMAPP) and iso-pentenyl diphosphate (IPP) These molecules can be biosynthesized viatwo separate routes in plants the methyl-erythritol 4-phosphate andmevalonate pathways IPP is synthesized and isomerized to DMAPP byisopentenyl diphosphate isomerase then prenyl transferases catalyze
the condensation of these two C5-units to geranyl diphosphate (Pazoukiand Niinemets 2016) Their elongation to prenyl diphosphates withaddition of IPP molecules leads to monoterpenes (C10) sesquiterpenes(C15) and diterpenes (C20) which are the substrates for terpene syn-thases (TPS) (Keeling and Bohlmann 2006b)TPSs are part of a large family of mechanistically related enzymes
involved in both primary and secondary metabolism (Keeling andBohlmann 2006b) The events of evolutionary diversification and ex-pansion of plant TPSs appear to have originated from gene duplicationsdomain losses and sub- or neofunctionalizations with subsequent di-vergence of an ancestral TPS gene of primary metabolism (Hall et al2013) Modification of TPS products changes their physical propertiesand may alter their biological activities (Chen et al 2011) TPSs of highsequence identity may have different functions even in closely relatedspecies Low sequence identity of TPSs in phylogenetically distantspecies does not preclude the possibility of independent evolution of thesame or related function of these enzymes (Zerbe and Bohlmann 2015)
httpsdoiorg101016jindcrop2019111545Received 4 January 2019 Received in revised form 10 June 2019 Accepted 4 July 2019
Corresponding authorE-mail address fettnetocbiotufrgsbr (AG Fett-Neto)1 These authors have equally contributed to this work
doi 1015900102-33062019abb0114
Acta Botanica Brasilica
Sustainable production of bioactive alkaloids in Psychotria L of
southern Brazil propagation and elicitation strategies
Yve Verocircnica da Silva Magedans1 Kelly Cristine da Silva Rodrigues-Correcirca1 Cibele Tesser da Costa1
Heacutelio Nitta Matsuura1 and Arthur Germano Fett-Neto1
Received April 1 2019Accepted June 28 2019
ABSTRACTPsychotria is the largest genus in Rubiaceae South American species of the genus are promising sources of natural
products mostly due to bioactive monoterpene indole alkaloids they accumulate ese alkaloids can have analgesic
antimutagenic and antioxidant activities in dierent experimental models among other pharmacological properties
of interest Propagation of genotypes with relevant pharmaceutical interest is important for obtaining natural
products in a sustainable and standardized fashion Besides the clonal propagation of elite individuals the alkaloid
content of Psychotria spp can also be increased by applying moderate stressors or stress-signaling molecules is
review explores advances in research on methods for plant propagation and elicitation techniques for obtaining
bioactive alkaloids from Psychotria spp of the South Region of Brazil
Keywords abiotic stress alkaloids elicitation monoterpenes plant propagation Psychotria southern Brazil
sustainability
Introduction
Psychotria belongs to Rubiaceae one of the major families
of $owering plants having economic interest e family
includes coee a few signicant poisonous plants to livestock
besides several important ornamental and medicinal species
(Souza amp Lorenzi 2012) Psychotria has captured researchersrsquo
attention mostly because of its medicinal properties
Psychotria colorata is an Amazonian species that produces
polyindolinic alkaloids with analgesic activity (Matsuura et
al 2013) e promising results obtained with P colorata
motivated the investigation of southern Brazilian Psychotria
species and the discovery of new bioactive alkaloids (Porto
et al 2009) Moreover leads on in planta alkaloid functions
were also topic of experimental evaluation
One of the key elements that needs to be addressed early
on during the process of developing new bioactive molecules
from plants is the capacity to generate catalytically active
biomass to support extraction and steady supply ere are a
number of ways through which these goals may be reached
including greenhouse rooting of cuttings (mini-cutting
1 Laboratoacuterio de Fisiologia Vegetal Departamento de Botacircnica Instituto de Biociecircncias e Centro de Biotecnologia Universidade Federal do Rio
Grande do Sul 91501-970 Porto Alegre RS Brazil
Corresponding author fettnetocbiotufrgsbr
Review
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Biomass yield of resin in adult Pinus elliottii Engelm trees is differentially
regulated by environmental factors and biochemical effectors
Franciele Antocircnia Neis Fernanda de Costa Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima
Kelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-Neto
Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) CP 15005 CEP 91501-970 Porto Alegre RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Biomass
Terpene resin
Seasonal
Benzoic acid
Regenerated forest
A B S T R A C T
Biomass of pine resin finds several applications in the chemical pharmaceutical biofuel and food industries
Resin exudation after injury is a key defense response in Pinaceae since this complex mixture of terpenes has
insecticidal antimicrobial and wound repair properties Resin yield is increased by effectors applied on the
wound area including phytohormones and metal cofactors of terpene synthases The interaction of resinosis
mechanism effectors is not fully understood particularly in adult forest setups under natural environmental
variations The aim of this work was to determine how resin exudation by wounded trunks of adult P elliottii
responded to combined chemical effectors involved in different regulatory pathways of resinosis (metal cofactors
of terpene synthases benzoic acid and plant growth regulators) and whether seasonal and tree distribution
variations affected these responses Symmetrically planted and scattered trees regenerated from the seed bank
had similar resin biomass yields suggesting that the homogeneity in development and spatial arrangement were
not significant factors in resin yield This new finding is of practical importance with the used tapping system
since costs of implanting forests by regeneration can be advantageous compared to planting In addition it was
shown for the first time that the salicylic acid precursor benzoic acid and the auxin naphthalene acetic acid
promoted resin exudation when individually applied to wound sites Both these adjuvants are two orders of
magnitude less costly compared to the conventionally used ethylene precursors besides facing less environ-
mental and health restrictions for use Most adjuvant-treated trees showed higher resin flow in the second year
indicating mechanisms of response build up Overall temperature was more important than rainfall as en-
vironmental parameter affecting resin biosynthesis which was higher in the warmer months of spring and
summer The combination of resinosis stimulant effectors from different signaling pathways showed no sig-
nificant synergistic or additive effect suggesting possible converging signaling pathways andor limitation of
common intermediate transducing molecules
1 Introduction
Pines occupy highly diverse environments over a range of tem-
peratures water and nutrient availabilities irradiance levels and pho-
toperiods being able to effectively face attacks from diverse herbivore
and pathogen guilds The success of conifers is linked to their complex
terpene biochemistry hosted by specialized secretory cells The terpe-
noid resin synthesized by Pinus spp is one of the main mechanisms of
defense of these trees particularly against bark beetles and the fungi
they carry (Fett-Neto and Rodrigues-Correcirca 2012) Pine resin biomass
is essentially composed of a monoterpene and sesquiterpene-rich tur-
pentine and diterpenoid-rich rosin fraction both finding numerous in-
dustrial applications as non-wood forest products (Rodrigues-Correcirca
et al 2012)
Molecules capable of modulating different signaling pathways have
been identified as resin yield stimulators including sulfuric acid (ex-
tends wound damage) 2-chloroethylphosphonic acid (CEPA a syn-
thetic ethylene precursor) paraquat (free radical generator) yeast ex-
tract (mimics attack by pathogens) salicylic acid (pathogen signaling
molecule) auxin (promotes ethylene biosynthesis and resin canal dif-
ferentiation) jasmonic acid (signals mechanical damage and promotes
secondary metabolism) and metal ions such as potassium iron and
manganese (cofactors of terpene synthases in conifers) and copper (a
component of ethylene receptors) (Clements 1970 Conrath et al
2002 Fett-Neto and Rodrigues-Correcirca 2012 Hudgins and Franceschi
2004 Lewinsohn et al 1994 Martin et al 2002 Popp et al 1995
httpsdoiorg101016jindcrop201803027
Received 12 December 2017 Received in revised form 9 March 2018 Accepted 13 March 2018
Corresponding author
E-mail addresses franci_neisyahoocombr (FA Neis) fernandadecostayahoocombr (F de Costa) thanisenfyahoocombr (TN Fuumlller)
jjuliocesarlimagmailcom (JC de Lima) krodriguescbiotufrgsbr (KC da Silva Rodrigues-Correcirca) jpfettcbiotufrgsbr (JP Fett) fettnetocbiotufrgsbr (AG Fett-Neto)
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Research Paper
Dual allelopathic effects of subtropical slash pine (Pinus elliottii Engelm)
needles Leads for using a large biomass reservoir
Kelly Cristine da Silva Rodrigues-Correcircaa Gelson Halmenschlagera Joseacuteli Schwambachb
Fernanda de Costaa Emili Mezzomo-Trevizana Arthur Germano Fett-Netoa
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 Brazilb University of Caxias do Sul Institute of Biotechnology Caxias do Sul RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Seasonality
Growth
Germination
Litter
Substrate
A B S T R A C T
Pinus elliottii Engelm (slash pine) is distributed along the maritime coast of Southern Brazil where it shows
invasive pattern and typical allelopathic features Large quantities of needle litter are produced by pine trees a
biomass that is little explored in areas where this species is alien Little is known about the dynamics of needle
and litter phytochemical interactions particularly in subtropical environments To elucidate the full range of
needle and litter allelopathic potential the effects of litter (superficial and deep) and seasonally harvested fresh
slash pine needles stored for different times were evaluated against lettuce tomato and cucumber seeds and
seedlings Increasing concentrations (0 1 2 4 and 8 wv) of hot and cold aqueous extracts of needles
and litter affected in different ways target plant development Growth and germination inhibition were directly
related to the highest extract concentrations (regardless of the season and mainly in hot water extracts) of
needles On the other hand stimulatory effects of litter extracts on lettuce growth were observed Growth and
germination of cucumber and tomato were not affected by pine litter as substrate when compared to rice husk
The presumable high polarity and thermal stability of slash pine leaf biomass allelochemicals and their transient
toxic effect or growth promoting impact suggest potential applications of this largely available biomass both as a
biological herbicide and growth substrate in plant propagation
1 Introduction
Native from the Northern Hemisphere Pinus is one of the most
widely distributed genera throughout different climate regions of the
globe growing either as native or alien species even in extreme habi-
tats (Rodrigues-Correcirca and Fett-Neto 2012) Despite the high economic
value currently attributed to pine wood and oleoresin (Rodrigues-
Correcirca et al 2012) there is increasing concern about the aggressive
potential of invasiveness displayed by Pinus species especially those
cultivated out of their native range of distribution (Richardson et al
2008 Rolon et al 2011) These species are dispersed by wind and there
is notably low plant diversity observed in most understories of pine
plantations (Kato-Noguchi et al 2009) This latter feature has been
considered an important trait of allelopathic interference
The term ldquoallelopathyrdquo was coined by Molisch in 1937 as a chemical
reciprocal interaction established among plants (including micro-
organisms) sharing the same site by means of the release of secondary
metabolites named allelochemicals (Rice 1984) For the most part
these metabolites are derived from the shikimic acid or isoprenoid
pathway and their biosynthesis can be modulated by biotic and abiotic
stresses (Nascimento and Fett-Neto 2010) including seasonal-related
changes (Sartor et al 2013) Allelopathy studies may range from sterile
assays (Aryakia et al 2015) to soil (Correcirca et al 2008 Sharma et al
2016) and field tests being a complex biological phenomenon to as-
certain in several circumstances due to issues of solubility release
mechanisms and stability of bioactive compounds (Scognamiglio et al
2013) Often the use of complementary methods provides more in-
formative data
The allelopathic effects of soil leachates green needles and litter
extracts of Pinus spp on germination and seedling growth aspects of
wild and crop species have been evaluated in natural and cultivated
pine stands and have proven to be stimulatory or inhibitory (Lodhi and
Killingbeck 1982 Kil and Yim 1983 Nektarios et al 2005 Akkaya
et al 2006 Machado 2007 Alrababah et al 2009 Sartor et al 2009
Kato-Noguchi et al 2011 Rolon et al 2011 Valera-Burgos et al
2012) exhibiting in some cases autotoxicity (Garnett et al 2004
Fernandez et al 2008 Zhu et al 2009 Monnier et al 2011) Studies
on potential dual allelopathic effects of Pinus elliottii Engelm (slash
httpdxdoiorg101016jindcrop201706019
Received 23 March 2017 Received in revised form 15 May 2017 Accepted 7 June 2017
Corresponding author
E-mail address fettnetocbiotufrgsbr (AG Fett-Neto)
ORIGINAL RESEARCHpublished 16 June 2016
doi 103389fpls201600849
Frontiers in Plant Science | wwwfrontiersinorg 1 June 2016 | Volume 7 | Article 849
Edited by
Juan Francisco Jimenez Bremont
Instituto Potosino de Investigacioacuten
Cientiacutefica y Tecnoloacutegica Mexico
Reviewed by
Mariacutea De La Luz Guerrero Gonzaacutelez
Universidad Autoacutenoma de San Luis
Potosiacute Mexico
Rosalia Cristina Paz
CIGEOBIO (CONICETFCEFN UNSJ)
Argentina
Correspondence
Arthur G Fett-Neto
fettnetocbiotufrgsbr
daggerThese authors have contributed
equally to this work
Specialty section
This article was submitted to
Plant Physiology
a section of the journal
Frontiers in Plant Science
Received 08 December 2015
Accepted 30 May 2016
Published 16 June 2016
Citation
de Lima JC de Costa F Fuumlller TN
Rodrigues-Correcirca KCdS Kerber MR
Lima MS Fett JP and Fett-Neto AG
(2016) Reference Genes for qPCR
Analysis in Resin-Tapped Adult Slash
Pine As a Tool to Address the
Molecular Basis of Commercial
Resinosis Front Plant Sci 7849
doi 103389fpls201600849
Reference Genes for qPCR Analysisin Resin-Tapped Adult Slash Pine Asa Tool to Address the MolecularBasis of Commercial Resinosis
Juacutelio C de Lima 1dagger Fernanda de Costa 1 dagger Thanise N Fuumlller 1
Kelly C da Silva Rodrigues-Correcirca 2 Magnus R Kerber 1 Mariano S Lima 1
Janette P Fett 1 and Arthur G Fett-Neto 1
1 Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul
Porto Alegre Brazil 2 Biological Sciences Department Regional Integrated University of Alto Uruguai and Missotildees (URI-FW)
Frederico Westphalen Brazil
Pine oleoresin is a major source of terpenes consisting of turpentine (mono- and
sesquiterpenes) and rosin (diterpenes) fractions Higher oleoresin yields are of economic
interest since oleoresin derivatives make up a valuable source of materials for chemical
industries Oleoresin can be extracted from living trees often by the bark streak method
in which bark removal is done periodically followed by application of stimulant paste
containing sulfuric acid and other chemicals on the freshly wounded exposed surface
To better understand the molecular basis of chemically-stimulated and wound induced
oleoresin production we evaluated the stability of 11 putative reference genes for the
purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis
Samples for RNA extraction were collected from field-grown adult trees under tapping
operations using stimulant pastes with different compositions and at various time points
after paste application Statistical methods established by geNorm NormFinder and
BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3
and UBI To confirm expression stability of the candidate reference genes expression
profiles of putative P elliottii orthologs of resin biosynthesis-related genes encoding Pinus
contorta β-pinene synthase [PcTPS-(minus)β-pin1] P contorta levopimaradieneabietadiene
synthase (PcLAS1) Pinus taeda α-pinene synthase [PtTPS-(+)αpin] and P taeda
α-farnesene synthase (PtαFS) were examined following stimulant paste application
Increased oleoresin yields observed in stimulated treatments using phytohormone-based
pastes were consistent with higher expression of pinene synthases Overall the
expression of all genes examined matched the expected profiles of oleoresin-related
transcript changes reported for previously examined conifers
Keywords resin Pinus gene expression normalizer genes terpene synthase
19
Chapter 2
Stimulant Paste Preparation and Bark Streak Tapping Technique for Pine Oleoresin Extraction
Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
Tapping technique comprises the extraction of pine oleoresin a non-wood forest product consisting of a
complex mixture of mono sesqui and diterpenes biosynthesized and exuded as a defense response to
wounding Oleoresin is used to produce gum rosin turpentine and their multiple derivatives Oleoresin
yield and quality are objects of interest in pine tree biotechnology both in terms of environmental and
genetic control Monitoring these parameters in individual trees grown in the fi eld provides a means to
examine the control of terpene production in resin canals as well as the identifi cation of genetic-based
differences in resinosis A typical method of tapping involves the removal of bark and application of a
chemical stimulant on the wounded area Here we describe the methods for preparing the resin-stimulant
paste with different adjuvants as well as the bark streaking process in adult pine trees
Key words Oleoresin Pine Tapping Chemical stimulant Wounding
1 Introduction
Several conifer species produce oleoresin a complex mixture of isoprenoid compounds relevant for defense against herbivores and pathogens Two major fractions can be recognized in oleoresin (a) turpentine the volatile fraction containing mono- and sesquiter-penes and (b) rosin the nonvolatile diterpene fraction Oleoresin is a forest commodity of global interest fi nding applications in diverse industry sectors Rosin is used in adhesives printing ink manufacture and paper sizing Turpentine can be used either as a solvent for paints and varnishes or as a raw material for fraction-ation of high-value chemicals used in the pharmaceutical agro-chemical and food industry [ 1 ndash 3 ]
During the extraction activity resin is obtained from the tree in a similar way as rubber tree tapping which generally involves the
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_2 copy Springer Science+Business Media New York 2016
These authors have equally contributed to this work
fettnetocbiotufrgsbr
27
Chapter 3
A Modifi ed Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines
Juacutelio Ceacutesar de Lima Thanise Nogueira Fuumlller Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes
through high-throughput sequencing technologies microarray and also northern blots RT-PCR and
RTqPCR Even though many specifi c protocols designed for plants with high content of secondary metab-
olites have been developed these are often expensive time consuming and not suitable for a wide range
of tissues Here we present a modifi cation of the method previously described using the commercially
available Concerttrade Plant RNA Reagent (Invitrogen) buffer for fi eld-grown adult pine trees with high
oleoresin content
Key words RNA Pines Concert plant RNA reagent Stem RNA extraction Oleoresin Conifers
1 Introduction
Several conifer species especially within the Pinaceae have tissues with high concentrations of phenolics terpenes and polysaccha-rides [ 1 ] Many specifi c protocols that are appropriate for plants rich in secondary metabolite s have been developed but the extrac-tion of high-quality RNA from these tissues using commercial kits is often diffi cult and usually not applicable to woody tissues [ 2 ndash 6 ] One of the major issues during RNA extraction concerns the pres-ence of phenolic compounds which oxidize and form quinones Aromatic compounds bind RNA which interferes in downstream steps and applications [ 3 7 ] Another point of concern is the har-vest of plant samples in the experimental fi eld which constitutes another obstacle in the efforts to avoid degradation of RNA [ 8 ] These problems often result in RNAs of low quality and insuffi -cient amounts especially for methodologies that normally require
These authors have equally contributed to this work
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_3 copy Springer Science+Business Media New York 2016
fettnetocbiotufrgsbr
RESEARCH PAPER
Control of resin production in Araucaria angustifolia an ancientSouth American coniferJ C Perotti1 K C da Silva Rodrigues-Correa123 amp A G Fett-Neto12
1 Plant Physiology Laboratory Department of Botany Federal University of Rio Grande do Sul (UFRGS) Porto Alegre RS Brazil
2 Center for Biotechnology UFRGS Porto Alegre RS Brazil
3 Present address Regional Integrated University of Alto Uruguai and Miss~oes (URI-FW) Frederico Westphalen RS Brazil
Keywords
Araucaria ethylene jasmonic acid nitric
oxide resin salicylic acid terpenes
Correspondence
A G Fett-Neto Plant Physiology Laboratory
Center for Biotechnology Federal University
of Rio Grande do Sul (UFRGS) PO Box 15005
Av Bento Goncalves 9500 91501-970 Porto
Alegre Brazil
E-mail fettnetocbiotufrgsbr
Editor
K Leiss
Received 22 July 2014 Accepted 11
December 2014
doi101111plb12298
ABSTRACT
Araucaria angustifolia is an ancient slow-growing conifer that characterises parts ofthe Southern Atlantic Forest biome currently listed as a critically endangered speciesThe species also produces bark resin although the factors controlling its resinosis arelargely unknown To better understand this defence-related process we examined theresin exudation response of A angustifolia upon treatment with well-known chemicalstimulators used in fast-growing conifers producing both bark and wood resin suchas Pinus elliottii The initial hypothesis was that A angustifolia would display signifi-cant differences in the regulation of resinosis The effect of Ethrel (ET ndash ethylene pre-cursor) salicylic acid (SA) jasmonic acid (JA) sulphuric acid (SuA) and sodiumnitroprusside (SNP ndash nitric oxide donor) on resin yield and composition in youngplants of A angustifolia was examined In at least one of the concentrations testedand frequently in more than one an aqueous glycerol solution applied on fresh woundsites of the stem with one or more of the adjuvants examined promoted an increase inresin yield as well as monoterpene concentration (a-pinene b-pinene camphene andlimonene) Higher yields and longer exudation periods were observed with JA and ETanother feature shared with Pinus resinosis The results suggest that resinosis controlis similar in Araucaria and Pinus In addition A angustifolia resin may be a relevantsource of valuable terpene chemicals whose production may be increased by usingstimulating pastes containing the identified adjuvants
INTRODUCTION
Many conifer species produce terpenoid-based resins that havelong been studied for their industrial importance and role indefence against attack by herbivores and pathogens The twomost important resin-producing families of conifers are Pina-ceae and Araucariaceae (Langenheim 1996) The viscous resinsecretion is generally composed of a complex mixture ofterpenoids consisting of roughly equal parts of volatile mono-(C10) and sesquiterpene (C15 turpentine) fractions and non-volatile diterpenic (C20 rosin) components (Rodrigues-Correaet al 2013) Terpenes act in a complex and multilayereddefence response providing toxicity against bark beetles andfungi bark wound sealing disruption of insect developmentand attraction of herbivore predators (Phillips amp Croteau1999)Most conifers rely on some combination of preformed and
inducible resin defences (Trapp amp Croteau 2001 Zulak amp Bohl-mann 2010) Resin defences are controlled by environmentaland genetic factors to various extents depending on species(Roberds et al 2003 Sampedro et al 2010 Moreira et al2013) Resin traits have been reported as highly variable havingmoderate heritability indicating that breeding efforts towardssuper-resinous forests are promising (Tadasse et al 2001Roberds et al 2003) Several chemicals are known as stimulantsof resin production Commercial extraction of resin from pine
trees uses periodic bark streaking and application of resin stim-ulant pastes to the wound
Resin-stimulant paste based on sulphuric acid (SuA) iswidely used for the commercial production of pine resin Cur-rent stimulant pastes usually have two chemically active com-ponents SuA to magnify the wounding and an ethyleneprecursor (2-chloroethylphosphonic acid CEPA or Ethrel ndash
ET) to stimulate resin flow (Rodrigues et al 2011 Rodrigues-Correa amp Fett-Neto 2013) Jasmonic acid (JA) and its methylester methyl jasmonate (MeJa) are among the most widelyused chemical elicitors of plant secondary metabolism It hasbeen shown that the exogenous application of MeJa or herbi-vore attack induce chemical and anatomical defence responsesin conifers such as the formation of traumatic resin ducts andresin accumulation in stems along with increased biosynthesisof terpenes and phenolics (Franceschi et al 2002 Martin et al2002 Heijari et al 2005 Zeneli et al 2006 Moreira et al 2008Gould et al 2009) JA commercial use however is limited byits high cost
The effects of exogenous salicylic acid (SA) on conifer ter-pene production have also been studied In Pinus elliottiiapplication of 10 molm3 of SA induced resin productionin wound panels but in Pseudotsuga menziesii and Sequoia-dendron giganteum it had no apparent effect on resinaccumulation (Hudgins amp Franceschi 2004 Rodrigues ampFett-Neto 2009) Nitric oxide (NO) has also emerged as an
Plant Biology 17 (2015) 852ndash859 copy 2014 German Botanical Society and The Royal Botanical Society of the Netherlands852
Plant Biology ISSN 1435-8603
vi
SUMAacuteRIO
LISTA DE ABREVIATURASvii
RESUMO ix
INTRODUCcedilAtildeO GERAL1
HIPOacuteTESE E OBJETIVOS9
CAPIacuteTULO 1 Abiotic stresses and non-protein amino acids in plantshelliphellip10
CAPIacuteTULO 2 Mimosine accumulation in Leucaena leucocephala in response to
stress signaling molecules and acute UV exposurehelliphelliphelliphelliphelliphelliphelliphelliphelliphellip(432) 52
CAPIacuteTULO 3 Mimosine occurrence and accumulation in Mimosa bimucronata var
bimucronata (DC) Kuntze66
CONSIDERACcedilOtildeES FINAIS 84
PERSPECTIVAS85
REFEREcircNCIAS BIBLIOGRAacuteFICAS86
Artigos publicados no periacuteodo de doutoramento natildeo relacionados ao tema da
tese91
vii
LISTA DE ABREVIATURAS
24-D 24-dichlorophenoxyacetic acid
3H4P 3-hydroxy-4-pyridone (34-DHP 34-dihydroxypyridine)
ABA abscisic acid
Arg arginine
BABA β-aminobutyric acid
β-ODAP β-N-oxalyl-L-α β-diaminopropionic acid
BIA β-isoxazolinon-L-alanine
CAN canavanine
DAO diamine oxidase
DDC decarboxylase
ETH ethephon
FW fresh weight
GABA -aminobutyric acid
GABA-T GABA transaminase
GAD glutamate decarboxylase
GSM Global System for Mobile
HPLC High performance liquid chromatography
JA jasmonate
JA-Ile jasmonoyl-L-isoleucine
L-DOPA L-34- dihydroxyphenylalanine
MeJA methyl jasmonate
m-Tyr Meta-tyrosine
NO nitric oxide
NPAA non-protein amino acid
OAS o-acetylserine
OAS-TL o-acetylserine-thiol-lyase
PA polyamine
PAA protein amino acid
viii
PEG polyethylene glycol
PLP pyridoxal-5rsquo-phosphate
PPO polyphenol oxidase tyrosinase
qRT-PCR Reverse transcription polymerase chain reaction quantitative real time
RNS reactive nitrogen species
ROS reactive oxygen species
SA salicylic acid
SAR systemic acquired resistance
SNP sodium nitroprusside
UV ultraviolet radiation
ix
RESUMO
Ao longo de sua evoluccedilatildeo as plantas desenvolveram estrateacutegias estruturais e quiacutemicas de
defesa em resposta aos estresses bioacuteticos e abioacuteticos impostos pelo ambiente Dentre
essas satildeo reconhecidas moleacuteculas quimicamente especializadas denominadas
metaboacutelitos secundaacuterios produtos naturais ou metaboacutelitos especializados Aminoaacutecidos
natildeo proteicos (ANPs) satildeo compostos nitrogenados que constituem aleacutem de componentes
do arsenal de defesa quiacutemica vegetal uma importante fonte de reserva de carbono e
nitrogecircnio para diversos taxa especialmente aqueles pertencentes agrave famiacutelia Fabaceae de
Angiospermas Esse grupo de moleacuteculas quimicamente heterogecircneo eacute assim definido por
natildeo participar da formaccedilatildeo de estruturas proteicas funcionais sendo frequentemente
toacutexicos quando erroneamente incorporados nas cadeias polipeptiacutedicas em formaccedilatildeo em
funccedilatildeo da similaridade estrutural que apresentam com os aminoaacutecidos proteicos Sob o
ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados ANPs satildeo
em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou abioacutetica como
o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de elicitores
quiacutemicos por exemplo O objetivo da presente tese foi investigar o papel bioloacutegico da
mimosina endoacutegena em Leucaena leucocephala (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute) a partir da avaliaccedilatildeo do efeito de tratamentos
relacionados ao estresse abioacutetico (UV-C aacutecido saliciacutelico metil jasmonato e etileno)
Mimosina eacute um ANP aromaacutetico anaacutelogo da L-tirosina com atividade toacutexica para ceacutelulas
de procariotos e eucariotos Dentre as atividades descritas para esse ANP destacam-se a
atividade anti-mitoacutetica ou bloqueadora do ciclo celular atividade alelopaacutetica e
antioxidante Os resultados indicaram que em leucena a biossiacutentese e o acuacutemulo de
mimosina podem ser modulados por fatores causadores de estresses exibindo um padratildeo
de acumulaccedilatildeo similar ao das fitoalexinas Em maricaacute por outro lado a induccedilatildeo do
acuacutemulo dessa moleacutecula natildeo foi observada para os mesmos tratamentos testados para
leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas Aleacutem disso
o padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado
pela sua degradaccedilatildeo As respostas observadas nos testes que avaliaram a atividade de
mitigaccedilatildeo de espeacutecies reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode
agir como um agente antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de
estresse
1
Introduccedilatildeo
Na condiccedilatildeo de organismos seacutesseis ao longo de sua evoluccedilatildeo as plantas
desenvolveram estrateacutegias estruturais e quiacutemicas de defesa em resposta aos estresses bioacuteticos
e abioacuteticos impostos pelo ambiente Dentre essas satildeo reconhecidas moleacuteculas quimicamente
especializadas denominadas metaboacutelitos secundaacuterios produtos naturais (Kutchan et al 2015)
ou mais recentemente metaboacutelitos especializados
Entre as trecircs classes mais gerais de metaboacutelitos secundaacuterios (terpenos compostos
fenoacutelicos e compostos nitrogenados) aminoaacutecidos natildeo-proteicos (ANPs) satildeo incluiacutedos no
terceiro grupo e constituem aleacutem de componentes do arsenal de defesa quiacutemica uma
importante fonte de reserva de carbono e nitrogecircnio para diversos taxa especialmente aqueles
pertencentes agrave famiacutelia Fabaceae de Angiospermas (leguminosas sensu lato)
Aleacutem dos 20 aminoaacutecidos proteicos estima-se que existam entre 600 e 1000 ANPs
(Acamovic amp Brooker 2005 Rodgers et al 2015) Esse grupo de moleacuteculas quimicamente
heterogecircneo eacute assim definido por natildeo participar da formaccedilatildeo de estruturas proteicas
funcionais sendo frequentemente toacutexicos quando erroneamente incorporados nas cadeias
polipeptiacutedicas em formaccedilatildeo em funccedilatildeo da similaridade estrutural que apresentam com os
aminoaacutecidos proteicos (Taiz amp Zeiger 2010)
Conforme mencionado a ocorrecircncia de ANPs eacute comum em espeacutecies de leguminosas
e sua distribuiccedilatildeo pode ser restrita a alguns gecircneros de plantas circunscritos nessa famiacutelia
botacircnica (eg mimosina e canavanina) Por outro lado alguns ANPs como GABA por
exemplo podem apresentar distribuiccedilatildeo ubiacutequa no Reino Plantae assim como ocorrer em
outros tipos de organismos como animais por exemplo (Ramos-Ruiz et al 2018)
2
Apesar de representarem uma fonte nutricional importante sem tratamento preacutevio o
consumo de plantas que acumulam ANPs por animais eacute limitado Isso ocorre pois em longo
prazo a ingestatildeo prolongada de plantas (especialmente sementes) que acumulam ANPs pode
representar risco agrave sauacutede uma vez que estes comprometem o funcionamento de mecanismos
basais de manutenccedilatildeo da homeostase celular e podem tambeacutem em um quadro mais severo
desencadear doenccedilas neurotoacutexicas degenerativas como por exemplo o latirismo causado
por aacutecido β-N-oxalil-l-αβ-diaminopropiocircnico (β-ODAP) (Jiao et al 2011 Kusama-Eguchi
2019)
Sob o ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados
ANPs satildeo em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou
abioacutetica como o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de
elicitores quiacutemicos por exemplo No que concerne ao estudo dos efeitos da induccedilatildeo abioacutetica
sobre o acuacutemulo de ANPs em diferentes espeacutecies vegetais (Monocotiledocircneas e
Eudicotiledocircneas) as moleacuteculas mais amplamente investigadas ateacute o momento satildeo GABA
L-DOPA e mais recentemente mimosina (vide Tabela 1 do capiacutetulo primeiro) Em termos
de efeitos causados a partir da aplicaccedilatildeo exoacutegena de ANPs GABA tambeacutem figura como o
principal aminoaacutecido investigado seguido de L-DOPA e canavanina (vide Tabela 2 do
capiacutetulo primeiro)
No primeiro capiacutetulo da presente tese estatildeo descritas as caracteriacutesticas gerais dos
principais ANPs estudados seus possiacuteveis papeacuteis bioloacutegicos in planta e seus efeitos quando
aplicados exogenamente bem como os estresses abioacuteticos capazes de induzir seu(s)
acuacutemulo(s) nos diferentes tecidos vegetais Nos segundo e terceiro capiacutetulos
respectivamente satildeo elucidados os efeitos dos tratamentos de UV-C aacutecido saliciacutelico etileno
e jasmonato (claacutessicos elicitores do metabolismo secundaacuterio vegetal) sobre o acuacutemulo de
3
mimosina em Leucaena leucocephala var glabrata (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute)
Mimosina eacute um aminoaacutecido aromaacutetico natildeo-proteico anaacutelogo da L-tirosina com
atividade toacutexica para ceacutelulas de procariotos e eucariotos Embora em menor concentraccedilatildeo
mimosina foi primeiramente identificada em Mimosa pudica sendo posteriormente detectada
em outras espeacutecies do gecircnero como Mimosa pigra por exemplo (Soedarjo amp Borthakur
1998) Seu efeito toacutexico eacute atribuiacutedo agrave capacidade de quelar metais o que impede o
funcionamento adequado das metalo-proteiacutenas que dependem dos mesmos como co-fatores
(Negi et al 2014)
A concentraccedilatildeo basal de mimosina em espeacutecies de leucaena pode variar entre 1 e 12
do peso seco do oacutergatildeo (Soedarjo amp Borthakur 1998) Como eacute comum para outros ANPs
que ocorrem em espeacutecies de leguminosas em sementes de Leucaena spp eacute observada uma
maior concentraccedilatildeo de mimosina quando comparada aos demais oacutergatildeos da planta
(Rodrigues-Correcirca et al 2019) sendo esta a fonte de extraccedilatildeo comercial do padratildeo quiacutemico
de mimosina vendido por empresas de reagentes de pesquisa
Diversas atividades foram descritas para mimosina em outros organismos eou tipos
celulares Dentre essas destacam-se a atividade anti-mitoacutetica ou bloqueadora do ciclo
celular em ceacutelulas de eucariotos e procariotos Isto ocorre porque a mimosina impede a
formaccedilatildeo da forquilha de replicaccedilatildeo (e portanto a siacutentese de DNA) interrompendo assim o
avanccedilo do ciclo de divisatildeo celular na fase tardia G1 (Lalande 1990) Foram tambeacutem descritas
para mimosina atividade alelopaacutetica observada sobre o desenvolvimento de outras espeacutecies
de leguminosas e atividade antioxidante entre outras (Tabela 1)
A rota de biossiacutentese de mimosina eacute compartilhada em grande parte com a de cisteiacutena
um aminoaacutecido proteico sulfurado (Figura 1) A siacutentese da cisteiacutena se daacute a partir da conversatildeo
4
de serina e acetil-CoA em o-acetilserina pela enzima SAT (serina acetiltransferase) seguida
da conversatildeo de o-acetilserina e aacutecido sulfiacutedrico em cisteiacutena em uma reaccedilatildeo catalisada pela
OAS-TL (o-acetilserina tiol-liase) A siacutentese de mimosina por sua vez eacute compartilhada com
a da cisteiacutena ateacute esse ponto e acredita-se que pelo menos uma das isoformas de OAS-TL
catalise a conversatildeo de o-acetilserina e 3-hidroxi-4-piridona em mimosina
Tabela 1 Atividades descritas para mimosina de Leucaena leucocephala (Lam) de Wit
ATIVIDADE
ALVO AVALIADO
(organismo eou tecido tipo
celular)
REFEREcircNCIA
Bloqueio do complexo de ativaccedilatildeo
da preacute-replicaccedilatildeo do DNA
Ceacutelulas de mamiacuteferos
KUBOTA et al
(2014)
Alteraccedilatildeo no ciclo ovariano e
extensatildeo da duraccedilatildeo do corpo luacuteteo
bovino no periacuteodo poacutes-parto
Bovinos
(Bos taurus x
Bos indicus)
BOTTINI-
LUZARDO et al
(2015)
Supressatildeo do ciclo celular e reduccedilatildeo
da abundacircncia bacteriana em
mosquitos
Wolbachia pipientis
Aedes albopictus
FALLON
(2015)
Accedilatildeo inibitoacuteria da fibrose
pulmonar induzida
Ratos SD
LI et al
(2015)
Recuperaccedilatildeo da funccedilatildeo do
miocaacuterdio poacutes-isquemia
Miocaacuterdio de ratos (SD)
machos
CROWE et al
(2001)
Inseticida
Heteropsylla cubana
Crawford 1914 e Thrips tabaci
Lindemann 1889
AHMED et al
(2016)
Alelopaacutetica
Albizia procera Vigna
unguiculata Cicer arietinum
Cajanus cajan
AHMED et al
(2008)
Antioxidante
Sistemas modelo de oxidaccedilatildeo
lipiacutedica (β-caroteno - aacutecido
linolecircico e lecitina)
BENJAKUL et al
(2013)
Ateacute momento versotildees divergentes sobre a enzima responsaacutevel pela biossiacutentese de
mimosina (mimosina sintase) tecircm sido publicadas Em 1990 Ikegami e colaboradores
5
identificaram uma OAS-TL responsaacutevel pela formaccedilatildeo de cisteiacutena como sendo tambeacutem uma
mimosina sintase Mais tarde Yafuso et al (2014) realizaram a expressatildeo heteroacuteloga do gene
que codifica para OAS-TL em Escherichia coli e natildeo foi observada a formaccedilatildeo de mimosina
mesmo quando dadas as condiccedilotildees oacutetimas para tanto Mais recentemente Harun-Ur-Rashid
et al (2018) elucidaram a mimosina sintase como sendo uma isoforma da OAS-TL
corroborando o postulado por Ikegami e colaboradores em 1990
Figura 1 Rota de biossiacutentese da mimosina Fonte Ikegami et al (1990)
Espeacutecies estudadas
Leucaena leucocephala (Lam) de Wit (leucaena koa haole ou ldquoacaacutecia exoacuteticardquo na
liacutengua Hawairsquoiana) eacute uma espeacutecie de haacutebito arboacutereo ou arbustivo pertencente agrave famiacutelia
Fabaceae de Angiospermas e caracterizada pelo acuacutemulo de mimosina em todos os seus
oacutergatildeos Eacute nativa da Ameacuterica Central (especificamente da regiatildeo sudeste do Meacutexico) mas
irradiou-se atraveacutes de praticamente todas as zonas tropicais e subtropicais da Terra No
Brasil leucena eacute amplamente distribuiacuteda e classificada como naturalizada pelo REFLORA
(2019) ocorrendo em todo territoacuterio Nacional Satildeo reconhecidas no miacutenimo duas
6
subespeacutecies de leucena ocorrentes no Brasil L leucocephala var leucocephala e L
leucocephala var glabrata sendo a primeira a mais abundante
Leucaena apresenta atributos morfoloacutegicos caracteriacutesticos das leguminosas como o
fruto do tipo vagem deiscente no periacuteodo poacutes-maturaccedilatildeo folhas compostas e bipinadas As
flores satildeo seacutesseis actinomorfas e polistecircmones apresentam caacutelice sinseacutepala e corola
gamopeacutetala e satildeo dispostas em inflorescecircncias do tipo glomeacuterulo (Figura 2)
Figura 2 Oacutergatildeos vegetativos e reprodutivos de L leucocephala (Lam) de Wit Fonte Little Jr amp Skolmen
(1989)
Com base no conhecimento etnobotacircnico disponiacutevel acerca dessa espeacutecie em
diversas regiotildees tropicais e subtropicais leucena eacute utilizada para vaacuterios fins Extratos de
diferentes oacutergatildeos de leucena apresentam atividade anti-diabeacutetica (Kuppusamy et al 2014
Chowtivannakul et al 2016) antioxidante (Mohammed et al 2015 Chowtivannakul et al
2016 Zarin et al 2016) antimicrobiana (Zarin et al 2016) anti-helmiacutentica (Soares et al
2015 Jamous et al 2017) bactericida (Mohammed et al 2015) acaricida (Fernaacutendez-Salas
et al 2011) anti-tumoral (Chung et al 2017) e potencializadora da resposta imune em
peixes (Verma et al 2018) entre outras
7
Leucaena apresenta alta toleracircncia agrave seca sendo capaz de enfrentar estaccedilotildees sazonais
inteiras com deacuteficit hiacutedrico sem prejuiacutezo permanente de seus oacutergatildeos e de recuperar
vigorosamente sua biomassa vegetativa tatildeo logo o regime de precipitaccedilatildeo retome a
regularidade em frequecircncia Acredita-se que a toleracircncia agrave seca apresentada por essa espeacutecie
ocorra em funccedilatildeo do acuacutemulo de mimosina nos diferentes tecidos da planta a qual
funcionaria como um agente osmoregulador responsaacutevel pela preservaccedilatildeo da integridade das
membranas a das macromoleacuteculas intracelulares em periacuteodos de escassez de aacutegua no
ambiente
Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) eacute uma leguminosa
nativa natildeo endecircmica do Brasil amplamente distribuiacuteda nos domiacutenios fitogeograacuteficos da
Caatinga do Cerrado e da Mata Atlacircntica (Simon amp Proenccedila 2000 REFLORA 2019) Como
espeacutecie pioneira (Pilatti et al 2019) exerce importante papel ecoloacutegico na recuperaccedilatildeo de
aacutereas degradadas (Bitencourt et al 2007 Silva et al 2011) no estabelecimento de processos
de sucessatildeo vegetacional
Maricaacute eacute uma espeacutecie semi-deciacutedua a deciacutedua a qual atinge ateacute 15 m em altura (e
diacircmetro agrave altura do peito de ateacute 40 cm) na idade adulta com haacutebito arboacutereo ou arbustivo
(REFLORA 2019) e espinhos caracteriacutesticos desde os estaacutegios iniciais de desenvolvimento
(Carvalho 2004) Apresenta folhas compostas alternas e bipinadas (Figura 2) amplas
inflorescecircncias brancas com flores reunidas em glomeacuterulos esfeacutericos dispostos em grandes
paniacuteculas As flores satildeo diplostecircmones actinomorfas hipoacuteginas e unicarpelares (Silva et al
2011)
Assim como descrito para leucena maricaacute eacute considerado uma espeacutecie multifuncional
sendo comumente empregada para produccedilatildeo de mel como combustiacutevel (Olkoski amp
8
Wittmann 2011) em edificaccedilotildees na carpintaria e como lsquocerca-vivarsquo (Marchiori 1993
Lorenzi 1998) entre outras aplicaccedilotildees
Figura 2 Folhas e fruto de Mimosa bimucronata (DC) Kuntze Fonte Souza-Lima et al (2017)
Em contraste com a amplitude de habitats explorados por leucena (especialmente os
aacuteridos) no Sul do Brasil maricaacute ocorre preferencialmente em ambientes uacutemidos a alagadiccedilos
em aacutereas proacuteximas agraves margens de rios (Patreze amp Cordeiro 2004) embora possa tambeacutem
ocorrer em formaccedilotildees quase exclusivas dessa espeacutecie nas encostas de morros (Jacobi amp
Ferreira 1991)
Em relaccedilatildeo agraves atividades elucidadas para os extratos de maricaacute foram relatados os
efeitos alelopaacutetico (Jacobi amp Ferreira 1991 Ferreira et al 1992) diureacutetico natriureacutetico e
caliureacutetico (Schlickmann et al 2017)
9
Hipoacutetese
Mimosina apresenta perfil dinacircmico de acuacutemulo em Leucaena leucocephala e
Mimosa bimucronata frente a estresses associado a alteraccedilotildees significativas na expressatildeo de
genes relacionados ao metabolismo deste ANP o qual contribui para mitigar o desequiliacutebrio
oxidativo inerente a vaacuterios tipos de estresse
Objetivo geral
O objetivo da presente tese foi investigar o papel bioloacutegico da mimosina endoacutegena
em leucena e maricaacute a partir da avaliaccedilatildeo do efeito de tratamentos relacionados a estresses
ou sinalizadores de estresse
Objetivos especiacuteficos
- Analisar a concentraccedilatildeo constitutiva de mimosina nos diferentes oacutergatildeos de L leucocephala
(Lam) de Wit (leucena) e M bimucronata (DC) Kuntze (maricaacute)
- Verificar se apesar do seu alto teor constitutivo em plantas de leucena o acuacutemulo de
mimosina pode ser induzido com tratamentos que mimetizam diferentes estresses a partir da
avaliaccedilatildeo do efeito de moleacuteculas sinalizadoras (aacutecido saliciacutelico jasmonato etileno) e da
exposiccedilatildeo agrave radiaccedilatildeo UV-C na modulaccedilatildeo do acuacutemulo de mimosina em leucena bem como
em maricaacute
- Determinar se a expressatildeo de genes relacionados ao metabolismo de mimosina estaacute
associada agrave induccedilatildeo por estresses fisioloacutegicos
- Avaliar o potencial antioxidante da mimosina em experimentos realizados in situ
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry
journal homepage wwwelseviercomlocateplaphy
Research article
Mimosine accumulation in Leucaena leucocephala in response to stresssignaling molecules and acute UV exposure
Kelly Cristine da Silva Rodrigues-Correcircaab Michael DH Hondab Dulal BorthakurbArthur Germano Fett-Netoalowast
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970Porto Alegre Rio Grande do Sul BrazilbDepartment of Molecular Biosciences and Bioengineering University of Hawaii at Manoa Honolulu HI 96822 USA
A R T I C L E I N F O
KeywordsLeucaena leucocephalaMimosineMimosine amidohydrolaseJasmonic acidEthyleneSalicylic acidUV-C radiation
A B S T R A C T
Mimosine is a non-protein amino acid of Fabaceae such as Leucaena spp and Mimosa spp Several relevantbiological activities have been described for this molecule including cell cycle blocker anticancer antifungalantimicrobial herbivore deterrent and allelopathic activities raising increased economic interest in its pro-duction In addition information on mimosine dynamics in planta remains limited In order to address this topicand propose strategies to increase mimosine production aiming at economic uses the effects of several stress-related elicitors of secondary metabolism and UV acute exposure were examined on mimosine accumulation ingrowth room-cultivated seedlings of Leucaena leucocephala spp glabrata Mimosine concentration was not sig-nificantly affected by 10 ppm salicylic acid (SA) treatment but increased in roots and shoots of seedlings treatedwith 84 ppm jasmonic acid (JA) and 10 ppm Ethephon (an ethylene-releasing compound) and in shoots treatedwith UV-C radiation Quantification of mimosine amidohydrolase (mimosinase) gene expression showed thatethephon yielded variable effect over time whereas JA and UV-C did not show significant impact Consideringthe strong induction of mimosine accumulation by acute UV-C exposure additional in situ ROS localization aswell as in vitro antioxidant assays were performed suggesting that akin to several secondary metabolitesmimosine may be involved in general oxidative stress modulation acting as a hydrogen peroxide and superoxideanion quencher
1 Introduction
Different plant groups synthesize a large diversity of secondary orspecialized metabolites These molecules are generally produced inresponse to biotic and abiotic environmental stresses Indeed inductionof secondary metabolism usually involves stress-generating factorswhich have also been explored in biotechnological processes aiming atthe production of target metabolites of economic interest (Matsuuraet al 2018) Metabolic control of nitrogen-containing secondarycompounds (eg alkaloids and non-protein amino acids) has beenshown to be complex and influenced by phytohormones environmentalstresses (seasonality herbivory pathogen attack drought) UV radia-tion (Holloacutesy 2002) methyl jasmonate (MeJA) salicylic acid (SA)yeast extract (Cho et al 2008) abscisic acid (ABA) heavy metals os-motic stress (Nascimento et al 2013) and mechanical wounding (Portoet al 2014)
Due to their particular trait of associating with N-fixing micro-organisms Fabaceae species (leguminous sensu lato) are often proteinrich hence the relevance of several of these species as forage Fabaceaespecies are also known for accumulating nitrogen containing secondarymetabolites which play important roles as ecochemical molecules andat least for the case of non-protein amino acids potential cell reservoirsof nitrogen (Huang et al 2011)
High contents of mimosine a toxic aromatic non-protein aminoacid are found in species of two leguminous genera Leucaena spp andMimosa spp Leucaena leucocephala (Lam) de Wit (leucaena koa haole)is a fast-growing leguminous tree native from Central America (south-eastern Mexico) widely distributed in tropical and subtropical zonesThis species is also characterized by its high tolerance to droughtamong other environmental stresses (Honda et al 2018) Leucaena canbe divided into two subspecies (i) L leucocephala subsp leucocephala(common leucaena a bushy shrub) and (ii) L leucocephala subsp
httpsdoiorg101016jplaphy201811018Received 1 August 2018 Received in revised form 9 November 2018 Accepted 14 November 2018
lowast Corresponding authorE-mail addresses krodriguescbiotufrgsbr (KCdS Rodrigues-Correcirca) mhonda2hawaiiedu (MDH Honda) dulalhawaiiedu (D Borthakur)
fettnetocbiotufrgsbr (AG Fett-Neto)
Plant Physiology and Biochemistry 135 (2019) 432ndash440
Available online 19 November 20180981-9428 copy 2018 Elsevier Masson SAS All rights reserved
T
glabrata (giant leucaena a tree) The latter has been used as a fastgrowing tree for production of wood and paper pulp The foliage ofboth common and giant leucaena is used as a fodder because of its highprotein content and palatability to farm animals The foliage containsup to 18 protein 142 crude fiber and 64 ether extractcrude fat(Soedarjo and Borthakur 1996)
Production of nitrogen-containing secondary metabolites such asmimosine requires large amounts of carbon and nitrogen resourcesNegi et al (2014) estimated that up to 21 of the carbon-nitrogenresources may be used for production of mimosine in leucaenaBrewbaker et al (1972) determined the mimosine content of 96 Lleucocephala cultivars and 8 other Leucaena species collected from 38different countries by growing them in an observational nursery inHawaii and found that basal mimosine content varied from 189 to477 of the dry weight
Mimosine is biosynthesized from OAS (o-acetylserine) and 3H4P (3-hydroxy-4-pyridone or its tautoisomer 3-hydroxy-4-pyridine) A pre-vious analysis suggested that mimosine synthase is an OAS-TL (o-acetylserine-thiol-lyase) of the cysteine biosynthesis pathway (Ikegamiet al 1990) Later however recombinant enzyme tests did not supportan OAS-TL identity of mimosine synthase (Yafuso et al 2014) Recentfindings on mimosine biosynthesis revealed that a cytosolic cysteine-OAS-TL isoform can also catalyze the formation of mimosine underspecific conditions (Harun-Ur-Rashid et al 2018)
Mimosine toxicity is related to its ability of reducing the availabilityof divalent metal ions such as Fe(II) Zn(II) Cu(II) Co(II) and Mn(II)by chelating co-factors and preventing their association with metal-dependent enzymes Furthermore this non-protein amino acid is cap-able of forming a stable complex with pyridoxal-5prime-phosphate (PLP)leading to the inactivation of PLP-dependent enzymes (eg Asp-Glutransaminase and cystathionine synthetase) (Negi et al 2014)
Mimosine features several useful biological activities such as alle-lopathic antimicrobial insecticide cell cycle inhibitor agent antic-ancer phytoremediator (Nguyen and Tawata 2016) as well as anti-oxidant (Benjakul et al 2013) Despite the relatively well establishedbiological activities of purified mimosine on other organisms or celltypes little is known about its biological role in leguminous speciesHowever it has been suggested that at least in part its activity ismainly related to defense mechanisms against some biotic and abioticstresses and as nitrogen source during fast growth (Vestena et al2001)
Suda (1960) and Smith and Fowden (1966) identified enzymes in-volved in mimosine degradation in seedling extracts of L leucocephalaand Mimosa pudica A mimosine-degrading enzyme named mimosinase(mimosine amidohydrolase EC 35161 CAS registry number 104118-49-2) (IUBMB 2018) a carbon-nitrogen lyase which degrades mimo-sine into 3H4P was later purified by Tangendjaja et al (1986) Itsbiochemical characterization was described and the cDNA was isolatedby Negi et al (2014)
Although mimosinase has been described and isolated only fewstudies on the role played by biotic and abiotic factors on the dynamicmodulation of mimosine metabolism in leguminous species have beenconducted (Vestena et al 2001 Xu et al 2018) In aseptic cultures ofleucaena mechanical injury of shoots promoted local mimosine accu-mulation (Vestena et al 2001) In the same study cultivation in pre-sence of auxin or SA in culture medium also had a positive effect on
mimosine accumulation More recently the effect of drought treatmenton gene expression of leucaena was also evaluated by Honda et al(2018) However several potential factors regulating mimosine meta-bolism need to be further examined
To date there is a lack of information on the biological role ofmimosine in planta as well as on details of its metabolic dynamicsMoreover its overt potential for pharmaceutical applications and de-velopment of new drugs as well as the possible use as tool to addressheavy metal soil contamination or plant mineral nutrition improve-ment justify additional research The objective of this study was toinvestigate the effect of stress signaling molecules and acute UV ex-posure on modulation of mimosine accumulation and metabolism in Lleucocephala spp glabrata in order to better understand its biologicalrole and to identify strategies for yield improvement aiming at ex-ploring its useful bioactivities
2 Methods
21 Plant material
For the experiments carried out to evaluate the effects of elicitors onmimosine accumulation seeds of leucaena were kindly provided by DrJames Brewbaker and harvested at CTAHRs (College of TropicalAgriculture and Human Resources of the University of Hawaii atManoa) Waimanalo Research Station at Oahu Hawaii This plantmaterial was originated from the accession K636 of Leucaena leucoce-phala ssp glabrata (Brewbaker 2008)
22 Induced mimosine content in 5-week-old giant leucaena
221 Seed germinationIn order to overcome seed coat dormancy seeds were submitted to a
chemical scarification with sulfuric acid 95ndash98 for 20min and re-peatedly rinsed in distilled water to remove any residual trace of thisreagent Then seeds were distributed in 254 cmtimes508 cm plastictrays containing 11 vv of vermiculite and commercial soil watereduntil reaching substrate field capacity Three weeks after seed imbibi-tion seedlings displaying similar size and shape (eg number of com-pound leaves and leaflets) were transplanted to individual pots(250mL) in number of three plants per container
During the experimental period (except in the UV-C radiationtreatment) all tested seedlings were kept in a growth chamber andsubmitted to controlled conditions of temperature (circa 25 degC) and ir-radiance (approximately 100 μmol photons mminus2sdot s minus1) with a photo-period of 16 h light and 8 h dark
222 Treatments2221 JA Ethephon and SA Five-week-old giant leucaena seedlingswere treated with different solutions as described in Table 1 Idealconcentrations were defined in preliminary experiments under the sameconditions indicated above At the beginning of the experiments 30plants were sprayed with 84 ppm JA 10 ppm SA 10 or 100 ppmEthephon or Milli-Qreg water (control) until the point of imminent runoffPlant pots were kept closed inside transparent plastic bags for 24 h toavoid solution volatilization Fifteen plants arranged in 5 sets of 3 (5biological replicates) were harvested 48 h and 96 h after being treated
Table 1Treatments used to modulate mimosine biosynthesis in giant leucaena
ELICITOR CONCENTRATION UV FLUENCE EXPOSURE TIME RATIONALE FOR USE
Salicylic acid (SA) 10 ppm 24 h Pathogen signaling molecule (Shah 2003)Jasmonic acid (JA) 84 ppm 24 h Chemical elicitor of plant secondary metabolism (Dar et al 2015)Ethephon 10 ppm 24 h Ethylene releasing-compound (Kim et al 2016) elicitor of plant secondary metabolism (Wang
et al 2016)UV-C radiation 3 Jcmminus2 10min or 15min Elicitor of plant secondary metabolism (Kara 2013 Neelamegam and Sutha 2015)
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After collection shoots were separated from roots immediately frozenin liquid nitrogen and stored at ndash 80 degC prior to HPLC analyses
2222 UV-C Thirty seedlings of giant leucaena were exposed to UV-Cradiation (3 Jcmminus2) for 10 or 15min and kept in a growth chamberunder controlled conditions as described above until the end of theexperiments Fifteen plants arranged in groups of 3 were harvested at96 h and 120 h after UV-C exposure and processed as previouslydescribed
223 Mimosine extractionMimosine extraction was based on a modified version of the pro-
tocol published by Lalitha and Kulothungan (2006) as follows a knownweight of fresh tissue (shoots or roots) of giant leucaena was first addedto Milli-Qreg boiling water in a proportion of 110 (g of plant per mL ofsolvent) in test tubes Tubes were covered with foil to avoid solutionevaporation and placed on a hot stirrer at 100 degC for 10min A pro-portional volume of 01M HCl was added to the cooled suspensions andhomogenized using mortar and pestle The plant extracts were filteredthrough cotton and centrifuged twice for 7min in a bench top re-frigerated centrifuge at 4 degC and 13200 rpm Before being analyzed theextracts were diluted 13 with ondashphosphoric acid (OPA)
224 Mimosine detectionHPLC analyses were carried out as described by Negi and Borthakur
(2016) Pure mimosine (L-mimosine from koa haole seeds Sigma-Al-drich CAS number 500-44-7) was used as standard Separation andquantification of mimosine was done with a C18 column (PhenomenexC18 5 μm 46times250mm) under an isocratic solvent system of 002MOPA with a linear flow rate of 1mLsdotminminus1 Mimosine detection wasdone at 280 nm by photodiode array detection (200ndash400 nm) showingretention time of 277 plusmn 0042min Quantification was done using themethod of external standard curve Further confirmation of mimosineidentity was performed by co-chromatography with standard and peakpurity check Chromatograms were analyzed using the Waters Em-power 3 software
23 Quantitative real-time PCR analysis of mimosinase gene expression
Fifteen 8-week-old giant leucaena plants arranged in 4 sets of 3 (4biological replicates) were treated with either water (control) or10 ppm Ethephon 84 ppm JA acid or 15min of UV-C radiation ex-posure following the methods described above Following treatmentleucaena plants were harvested at 48 and 96 h or 72 and 144 h (UV-Ctreated plants only) after treatments Total RNA of samples was ex-tracted and purified from roots and shoots of giant leucaena by meansof a modified method using Qiagen RNeasy Plant Kit (Valencia CAUSA) and Fruit-mate (Takara Japan) according to the protocol de-scribed by Ishihara et al (2016a) The assessment of RNA quality andquantity was carried out at 230 260 and 280 nm by using a NanoDropSpectrophotometer ND-1000 (NanoDrop Technologies DE USA) Inorder to avoid genomic DNA contamination RNA samples were treatedwith TURBO DNAfree Kit (Invitrogen Carlsbad CA) Two microgramsof DNase-treated RNA were used to synthesize the first-strand cDNAusing M-MLV Reverse Transcriptase (Promega WI USA)
Quantitative real-time (qPCR) analysis was carried out to examinepossible differential expression of the mimosinase gene (GenBank ac-cession number AB2985971) in seedlings treated with 84 ppm JA10mM Ethephon or 15min of UV-C exposure Shoots and roots wereharvested 24 h before the time of mimosine concentration peak for eachtreatment previously observed as assessed by HPLC assays The 10 μLqPCR reaction consisted of 5 μL of PowerUpTM SYBRreg Green MasterMix (Applied Biosystems Foster City CA) 1 μL MgCl2 (50mM) 03 μLforward primer (10 μM) 03 μL reverse primer (10 μM) and 1 μL cDNAfirst-strand In the experimental validation through qPCR reactionconditions and melting curve analysis of the amplicon were performed
following the protocol published by Ishihara et al (2016b) for the sameleucaena variety qPCR analysis was conducted using StepOnetrade Real-Time PCR System (Applied Biosystems) Measurements were performedusing 4 biological and 3 technical replicates Relative expression wascalculated with the 2-ΔΔct method using OAS-TL as reference gene sinceits expression showed a consistently stable profile comparable to that ofUBQ-5 and ELF1α expressions Mimosinase primer sequences used forthese analyses were (FWD) 5prime- GAA AGG CAG GAA TCA CAG TGA AGAG ndash 3rsquo (REV) 5prime GGA GAC TCT AGC CAC ACC AAC TTA ndash 3rsquo
24 Antioxidant assays
241 Mimosine effect on hydrogen peroxide (H2O2) accumulationAs a follow up to the induction of mimosine accumulation profiles
under stress signals and conditions tests were conducted to verify mi-mosine antioxidant capacity In situ histological localization of hy-drogen peroxide (H2O2) accumulation was evaluated on foliar disks ofPhaseolus vulgaris L according to the protocol described by Shi et al(2010) Briefly the plant foliar tissue was exposed to 1 mgmiddotmLminus1 dia-minobenzidine (DAB) solution in 10 mM KH2PO4 (control) in presenceor absence of 10mM mimosine (equivalent to the average mimosineconcentration induced by UV-C radiation in giant leucaena) or 10mMascorbic acid (positive antioxidant control) Oxidative response wasidentified by the formation of a brown polymer on the injured leafareas indicating the presence of H2O2 and registered in a Leica M165FC stereomicroscope (Leica Microsystems)
242 Mimosine quenching of superoxide radicalsGeneration of superoxide radical and subsequent analysis was per-
formed by a modified protocol based on Zhishen et al (1999) Nitroblue tetrazolium (NBT) reduction was used to measure superoxide an-ions quenching activity Shortly a 50mM KH2PO4 pH 78 solutioncontaining 6 μM riboflavin 100mM methionine 1 mM NBT in pre-sence or absence of 5mM mimosine was exposed to white light(22 Jsdotcmminus2) for 25min on a white light transilluminator Five micro-molar rutin was used as positive control (Matsuura et al 2016) Theabsorbance was read at 560 nm before and after light exposure in aSpectraMaxreg M2 Microplate Reader (Molecular Devices LLC)
25 Statistical analyses
For HPLC and superoxide anions data simple analyses of variance(ANOVA) followed by Tukey or Welch ANOVA followed by Dunnetts Ctest were used as appropriate for data distribution characteristics InqPCR analysis results were analyzed by t-test In all cases at least fourbiological triplicates were used and experiments were repeated twiceindependently All data were analyzed using the statistical packageSPSS 200 for Windows (SPSS Inc USA) In all cases a ple 005 wasused
3 Results and discussion
31 Increased mimosine concentrations in giant leucaena treated withchemical elicitors
Leucaena produces high amounts of mimosine that accumulate in allparts of the plants including leaves stem flowers pods seeds rootsand root nodules (Soedarjo and Borthakur 1998) The highest con-centrations of mimosine can be found in the growing shoot tips andseeds (Wong and Devendra 1983) It is not known why leucaena pro-duces such high amounts of mimosine Negi et al (2014) estimated thatleucaena plants would be able to grow 21 larger if the nutrient re-sources spent on mimosine production were diverted for biomass in-crease In a previous analysis performed to quantify the basal con-centration of mimosine present in adult plants of common leucaena thehighest constitutive amount of mimosine per gram of fresh weight in
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the analyzed organs was found in post-anthesis flowers (89448 μg)followed by green pods (82687 μg) leaves (67358 μg) and greenflower buds (51247 μg) which showed significantly less mimosineconcentration compared to the other reproductive structures(Supplementary Fig 1) Since mature seeds have very low moisturecontent (Wencomo et al 2017) its mimosine concentration was esti-mated as 338253 μgsdotgminus1 of dry weight Additionally it was also ob-served that the basal mimosine distribution in shoots of field-grownadult plants of leucaena is dependent on the variety type(Supplementary Table 1)
Phytohormones such as salicylic acid and jasmonic acid are knownto be produced by plants in response to various abiotic and bioticstresses These phytohormones trigger adaptive responses to stress byregulating major plant metabolic processes such as photosynthesisnitrogen metabolism defense systems and plant-water relationsthereby providing protection (for review see Khan et al 2015)
Secondary or specialized metabolite production and accumulationare also known to be controlled by biotic and abiotic stresses (Matsuuraet al 2018) In this study exposure of 5-week-old giant leucaenaseedlings to JA or Ethephon treatments significantly enhanced mimo-sine accumulation in shoots and roots in at least one of the two timepoints tested (48 and 96 h) albeit in a different way (Fig 1) Thehighest concentrations of mimosine in shoots were found in seedlingstreated with JA 84 ppm (43441 μgsdotgminus1) and Ethephon 100 ppm(38412 μgsdotgminus1) two days after application of the respective phyto-hormones Nevertheless after four days shoots yielded the highestconcentration of mimosine (approximately 460 μgsdotgminus1) upon treatmentwith 10 or 100 ppm Ethephon (Fig 1A) In roots after two and four
days JA 84 ppm and Ethephon 10 ppm resulted in highest mimosineaccumulation 18488 μgsdotgminus1 and 15801 μgsdotgminus1 respectively (Fig 1B)These observations show that mimosine accumulation response tospecific elicitors may vary over time after exposure
Although all treatments were applied exclusively on shoots of giantleucaena seedlings roots of some of them were also able to respond tothe different elicitors Overall shoots displayed higher basal and in-duced mimosine concentration compared to roots (Fig 1) which agreeswith previous observations in 1 to 3-week-old aseptic seedlings ofcommon leucaena (Vestena et al 2001) However as previouslymentioned significant post-induction increase of mimosine concentra-tion in roots and shoots simultaneously was only observed for JA andEthephon 10 ppm on day 02 and 04 respectively (Fig 1)
It is well established that perceived regulatory signals or elicitorsgenerate a transduction network mediated by secondary messengersresulting in changes in gene expression profiles that afford adaptiveresponses to environmental stimuli These modulation events are oftenmediated by transcription factors (TFs) which directly bind to specificgene promoters or act by forming complexes with repressor proteinslabeling them to degradation subsequently releasing other TFs toproceed with the gene expression program This is the case of the actionmechanism of JA and its active form jasmonoyl isoleucine for example(Kazan 2015 Wasternack and Strnad 2016)
JA ethylene and SA are known as important stress regulatory sig-nals in plants JA however is thought to be the most effective signal forinduction of plant secondary metabolism (Wasternack and Strnad2016) thereby contributing to mitigation of damage caused by severalstresses (Dar et al 2015) JA is mainly derived from linolenic acid
Fig 1 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings treated with differentelicitors CTRL=Milli-Q water SA = Salicylic AcidJA= Jasmonic Acid ETH=Ethephon Bars sharing a letterof same case do not differ by Tukey test (P le 005) Capitalletters (A B) compare treatments on day two and lowercaseletters (a b) compare treatments on day four Indicatessignificant statistical difference between day two and dayfour in the same treatment by t-test (Ple 005) The errorbars represent standard error of five replicates (each meanwas calculated with 15 individual seedlings organized in 5groups of three)
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435
(Wasternack and Strnad 2016) playing important roles in differentprocesses of plant growth and development such as plant defensemechanisms against herbivory pathogen attack fungal elicitation andsome abiotic factors such as osmotic temperature and salt stresses (Daret al 2015)
JA and its methyl ester MeJA have several different effects on le-guminous species MeJA exogenous application has increased iso-flavonoid content in cell suspension cultures of Pueraria candollei varcandollei and P candollei var mirifica (Korsangruang et al 2010) aswell as the production of the triterpenoid glycyrrhizin in Glycyrrhizaglabra roots Enhanced production of the triterpenoid however waspartly at the expense of root growth (Shabani et al 2009) MeJA ap-plication on shoots was observed to suppress root nodulation and lat-eral root formation in Lotus japonicus (Nakagawa and Kawaguchi2006) In grapevine a non-leguminous species proteinogenic aminoacids did not show an expressive increase under MeJA treatment(Gutieacuterrez-Gamboa et al 2017)
The effects of the application of four different jasmonate forms (JAMeJA jasmonoyl-L-isoleucine (JA-Ile) and 6-ethyl indanoyl glycineconjugate (2-[(6-ethyl-1-oxo-indane-4-carbonyl)-amino]-acetic acidmethyl ester - CGM) on leucaena metabolite profile has recently beenreported by Xu et al (2018) JA-Ile form was most effective althoughno major alteration was observed on monitored metabolite abundancesAlanine threonine and 34-dihydroxypyridine (34 DHP a metabolitederived from mimosine degradation) (Nguyen and Tawata 2016)among others were the major metabolites elicited by JA-Ile In contrastto the results described here mimosine concentration did not changesignificantly These divergent results on mimosine accumulation maybe due to a number of factors including mode of application jasmonateform used (JA-Ile x JA) and L leucocephala subspecies (common x giantleucaena)
Ethylene is also a phytohormone involved in plant response me-chanisms to different types of challenges such as mechanical damageand insect attack among others The integration mechanism betweenJA and ethylene signaling pathways is not completely understoodhowever it has been shown that they may work cooperatively in abioticstress tolerance (Kazan 2015) MeJA can induce ethylene production(Zhao et al 2004) and when applied simultaneously these moleculesseem to work in a synergic way by enhancing the magnitude of theplant response to external stimuli (Liu et al 2016)
Treatment with SA was able to significantly increase mimosine ac-cumulation in 12-week-old plants of common leucaena (SupplementaryFig 2) However no significant effect of SA treatment on mimosineconcentration was seen in 5-week-old seedlings of giant leucaena(Fig 1) suggesting some degree of genotype andor age dependency inelicitation by this phytohormone On the other hand several treat-ments including 90 ppm MeJA 10 and 100 ppm 2-chloroethylpho-sphonic acid (CEPA an ethylene-releasing compound) significantlyincreased mimosine accumulation (Supplementary Fig 2) in agree-ment with the data obtained for giant leucaena The lack of systemiceffects of externally applied SA on mimosine accumulation was alsoobserved when the phytohormone was supplied in the culture mediumof aseptically-grown seedlings in which case only roots had highercontent of mimosine (Vestena et al 2001) This could be due totransport limitations or to low methyl salicylate production from ap-plied SA since the former is recognized as the main systemic signalingform (Vlot et al 2009)
32 Increased mimosine concentrations in giant leucaena exposed to UV-Cradiation
UV-C treatment promoted increased concentration of the aminoacid in shoots but not in roots of giant leucaena (Fig 2) Increasedaccumulation of mimosine in shoots was also observed in 12-week-oldseedlings of common leucaena exposed to UV-C radiation for 10 and15min (Supplementary Fig 3) Similar to the SA treatment in giant
leucaena UV-C radiation did not induce mimosine biosynthesis in rootsregardless of time after exposure The absence of mimosine induction inroots by SA and UV indicates that these effectors do not cause a sys-temic response Moreover roots are shielded from irradiance by thepresence of substrate
UV radiation effects on different aspects of plant metabolism anddevelopment have been described However compared to UV-B (en-vironmentally relevant type of UV radiation) assays there are less re-ports related to the UV-C effects on secondary metabolites biosynthesisand accumulation (Cetin 2014) especially in leguminous (Fabaceae)plants They generally concern primary metabolism aspects such asgrowth and development For instance seedlings of Phaseolus vulgaris L(Fabaceae) exposed to low intensity UV-C radiation have displayeddecreased chlorophyll content and reduced height after 14 days of ex-posure (Kara 2013) Negative effects on growth parameters and ni-trogen metabolism were also observed in Vigna radiata L (Fabaceae)after UV-B radiation treatment in addition to adverse effects on JA SAand antioxidant compounds accumulation (Choudhary and Agrawal2014a) The same authors reported increased accumulation of flavo-noids SA and JA besides negative effects on growth biomass yieldnitrogen fixation and accumulation in 2 cultivars of Pisum sativum L(Fabaceae) under elevated UV-B treatment (Choudhary and Agrawal2014b) Despite the negative UV influence on growth reported for thepreviously mentioned leguminous UV-C radiation on groundnut plants(Arachis hypogaea L Fabaceae) increased seedling vigor and biomassand had no adverse effect on germination or other development para-meters (Neelamegam and Sutha 2015)
Besides its impact on growth and primary metabolism UV exposurecan cause important changes in secondary metabolism depending onintensity and time of exposure (Matsuura et al 2013) UV-B and UV-Cpre-treatments of Artemisia annua (Asteraceae) seedlings yielded in-creased biosynthesis of artemisinin a drug which displays anti-malarialproperties and activity against some others infectious diseases (egschistosomiasis leishmaniasis and hepatitis B) and several kinds oftumors (Rai et al 2011) The accumulation of nicotine in Nicotianarustica plants (Solanaceae) was also increased by UV-C treatment(Tiburcio et al 1985) Similar inducing effects on production of severalsecondary metabolites were observed in callus cultures of Vitis viniferaL Oumlkuumlzgoumlzuuml (grapevine Vitaceae) treated with a UV-C source for 5 or10min (Cetin 2014)
Regarding amino acid biosynthesis in response to UV radiationMartiacutenez-Luumlscher et al (2014) have found that in spite of not causingchanges in total amino acid content UV-B radiation exposure can affecttheir profile in grape berries Proteinogenic amino acids have beenknown to be important targets of the deleterious effects of UV radiation(Holloacutesy 2002) On the other hand in the present study acute UV-Ctreatment was found to increase mimosine accumulation in shoots byover twofold (Fig 2) which may suggest a possible participation of thismolecule as part of the antioxidant defense system in L leucocephalaThis possibility is further supported by the induction of the amino acidaccumulation by JA and Ethephon involved in abiotic and biotic stressresponses which are generally associated with oxidative imbalance andare signaling components in high UV stress (Matsuura et al 2013)
33 Mimosinase gene expression
In order to determine if increases in mimosine content upon ex-posure to JA CEPA or UV-C radiation were related to changes intranscription of mimosine metabolism-related genes RT-qPCR analysiswas carried out The complete pathway for mimosine biosynthesis hasnot yet been determined although the final step has been character-ized Based on transcription analysis (Ishihara et al 2016a) leucaenaappears to encode for multiple cysteine synthases one or more of whichmay be able to catalyze mimosine synthesis In addition a leucaenagene encoding a mimosinase (an enzyme responsible for mimosinedegradation) has been identified and characterized (Negi et al 2014)
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In addition to mimosinase gene expression several gene isoformsbelonging to the cysteine pathway [cysteine synthase (CYS SYN) serineacetyltransferase (SAT) and β-cyanoalanine synthase (CAS) Table 2 -supplementary material] were also tested in this study (data notshown) However expressions of these genes did not vary in giantleucaena throughout the experiments suggesting that the increasedcontent of mimosine observed in the treated plants might not be relatedto the expression of these genes but presumably to increased enzymeactivity andor release from conjugates such as mimoside a mimosineβ-D-glucoside (Murakoshi et al 1972)
Considering the time variation of mimosine accumulation observedin this work mimosinase gene expression in shoots and roots wasevaluated 24 h before the increase of mimosine concentration in giantleucaena seedlings (ie 24 h and 72 h after the chemical elicitorstreatments and 48 h and 120 h after UV-C exposure)
Ethylene signaling has been shown to up-regulate expression ofseveral genes related to secondary metabolism pathways as is the caseof phenolic compounds (Liu et al 2016) and terpenoid indole alkaloids(Wang et al 2016) Among all elicitors tested in the present workEthephon was the only one able to significantly change mimosinasegene expression Leucaena plants treated with Ethephon showed sig-nificant increases in mimosine concentration at both day 2 and 4 fol-lowing treatment which coincided with low-level expression of mi-mosinase Up-regulation of mimosinase gene expression was detected24 h before the increase of mimosine concentration in shoots treatedwith 10 ppm of Ethephon (Fig 3A) but not after JA or UV-C treatments(Fig 3C-D and 3E-F respectively) Nevertheless 72 h after treatment
application (24 h before the highest mimosine content measured inshoots) down regulation of mimosinase gene was seen in both shootsand roots treated with 10 ppm of Ethephon (Fig 3B) These data in-dicate that mimosine content in leucaena plants is at least partlyregulated by mimosinase expression in Ethephon exposed plants Onthe other hand the fact that mimosinase mRNA was not significantlyaffected by JA and UV-C treatments despite their stimulating effects onmimosine biosynthesis in giant leucaena may indicate that other levelsof regulation are at play or that the chosen harvesting time window wasunable to detect relevant changes
34 In situ and in vitro antioxidant assays
Considering the stimulation of mimosine accumulation byEthephon JA and UV all of which are often associated or known tocause oxidative imbalance the antioxidant capacity of mimosine wasevaluated Mimosine has been shown to have antioxidant activities oncultured cancer cells (Parmar et al 2015) In the present study it washypothesized that mimosine could confer radical scavenging propertieswhich would contribute to plant protection from possible damagecaused by reactive oxygen species generated during stress(Supplementary Fig 4)
Foliar disks of P vulgaris L were treated with 10mM mimosine for15min Treated disks showed less hydrogen peroxide accumulationinduced by wounding in contrast to untreated ones being comparableto those treated with ascorbic acid (a known hydrogen peroxide neu-tralizer) (Fig 4A) These observations support a possible antioxidant
Fig 2 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings exposed to UV-C lightCTRL= visible light (100 μmol photons mminus2 s minus1) UV-C 10primeand UV-C 15rsquo=UV-C exposure time (10 and 15min re-spectively) Bars sharing a letter of same case do not differ byTukey test (P le 005) Capital letters (A B) compare treat-ments on day three and lowercase letters (a b) comparetreatments on day six Indicates significant statistical dif-ference between day three and day six in the same treatmentby t-test (Ple 005) The error bars represent standard errorof five replicates (each mean was calculated with 15 in-dividual seedlings organized in 5 groups of three)
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437
role of mimosine as an in situ hydrogen peroxide scavengerMimosine was also able to quench superoxide anions generated by
light exposure Mimosine exhibited equivalent antioxidant effect com-pared to rutin (Fig 4B) a well-established effective superoxide anionquencher (Matsuura et al 2016) The radical scavenging activity ofmimosine may be due to the 3-OH group of the pyridine ring of mi-mosine (Fig 5) The pKa of the 3-OH of mimosine has been estimated tobe 88 (M Honda unpublished results) At physiological pH this OHgroup is expected to remain in a protonated state and therefore mayscavenge a radical by donating a proton and an electron In this processmimosine itself is converted to a stable radical form which is perhapsless toxic and less reactive than the reactive oxygen species generatedduring oxidative stress It is likely that the less toxic radical mimosineproduced may react with another radical or molecule and becomeconverted to a non-reactive indole molecule
In vivo antioxidant activity of mimosine has been previously eval-uated by means of its exogenous application on selenium-deficientseedlings of Vigna radiata In spite of its allelopathic properties (Ahmedet al 2008) the results showed mitigation of mitochondrial oxidativestress by treatment with 01mM mimosine (Lalitha and Kulothungan2007) DPPH radical scavenging activity was also reported for aqueous
seed extracts of leucaena rich in mimosine and phenolic compounds inin vitro assays (Benjakul et al 2014) Mimosine antioxidant activityshown in the present work is in good agreement with data reported forother non-protein amino acids such as L-DOPA (Dhanani et al 2015)and GABA (Malekzadeh et al 2014) for instance
4 Conclusion
Taken together results show that mimosine biosynthesis and ac-cumulation can be modulated by stress-related factors despite its re-latively high constitutive content in leucaena plants The pattern ofgene expression in stressed plants suggests mimosine steady-state con-trol may be regulated by its degradation in possible connection withdynamic changes in carbon and nitrogen metabolism of stressed plantsMimosine quenching activity against hydrogen peroxide and super-oxide anions in the in situ staining and in vitro assays respectivelyshowed that this non-protein amino acid can act as non-enzymaticantioxidant agent Increase in mimosine content in response to elicitorsmimicking environmental challenges in addition to its antiherbivoreand antimicrobial properties may be related to its activity as protectivemolecule against oxidative damage in line with other classes of plant
Fig 3 Relative expression of the mimosinase gene in shoots (A E and F) and shoots and roots (B C and D) of giant leucaena 24 h (A and C) 48 h (E) 72 h (B and D)and 120 h (F) after treatment with stress signaling molecules or UV-C exposure ETH = Ethephon JA = Jasmonic Acid Indicates significant statistical differencebetween control and treatment by t-test (Ple 005) The error bars represent standard error of four replicates
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secondary metabolites
Funding
This work was funded by the National Council for Scientific andTechnological Development (CNPq-Brazil) grant 3060792013-5Coordenaccedilatildeo de Aperfeiccediloamento de Pessoal de Niacutevel Superior - Brazil(CAPES) - Finance Code 001 and the USDA NIFA Hatch projectHA05029-H managed by CTAHR
CRediT authorship contribution statement
Kelly Cristine da Silva Rodrigues-Correcirca InvestigationValidation Writing ndash original draft Michael DH HondaInvestigation Validation Dulal Borthakur Supervision Writing ndashreview amp editing Funding acquisition Arthur Germano Fett-NetoSupervision Funding acquisition Writing ndash review amp editing
Acknowledgements
The authors would like to thank Dr Jorge Ernesto Mariath fromLaVeg-UFRGS for kindly lending the Leica M165 FC stereomicroscopefor in situ analysis
Appendix A Supplementary data
Supplementary data to this article can be found online at httpsdoiorg101016jplaphy201811018
References
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leucocephala leaf litter on some forest and agricultural crops grown in nursery J ForRes 19 298 httpsdoi 101007s11676-008-0053-0
Benjakul S Kittiphattanabawon P Shahidi F Maqsood S 2013 Antioxidant activityand inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipidoxidation in model systems Food Sci Technol Int 19 (4) 365ndash376 httpsdoiorg1011771082013212455186
Benjakul S Kittiphattanabawon P Sumpavapol P Maqsood S 2014 Antioxidantactivities of lead (Leucaena leucocephala) seed as affected by extraction solvent priordechlorophyllisation and drying methods extracts against lipid oxidation in modelsystems Food Sci Technol 51 (11) 3026ndash3037 httpsdoiorg101007s13197-012-0846-1
Brewbaker JL Pluckett D Gonzalez V 1972 Varietal variation and yield trials ofLeucaena leucocephala (koa haole) in Hawaii Hawaii Agric Exp Stn Bull 166 26
Brewbaker JL 2008 Registration of KX2 ndash Hawaii interspecific-hybrid leucaena JPlant Registrations 1 (3) 190ndash193 httpsdoiorg103198jpr2007050298crc
Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitisvinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-6287-47-37
Cho H-Y Son SY Rhee HS Yoon S-YH Lee-Parsons CWT Park JM 2008Synergistic effects of sequential treatment with methyl jasmonate salicylic acid andyeast extract on benzophenanthridine alkaloid accumulation and protein expressionin Eschscholtzia californica suspension cultures J Biotechnol 135 117ndash122 httpsdoiorg101016jjbiotec200802020
Choudhary KK Agrawal SB 2014a Cultivar specificity of tropical mung bean (Vignaradiata L) to elevated ultraviolet-B changes in antioxidative defense system ni-trogen metabolism and accumulation of jasmonic and salicylic acids Environ ExpBot 99 122ndash132 httpsdoiorg101016jenvexpbot201311006
Choudhary KK Agrawal SB 2014b Ultraviolet-B induced changes in morphologicalphysiological and biochemical parameters of two cultivars of pea (Pisum sativum L)Ecotoxicol Environ Saf 100 178ndash187 httpsdoiorg101016jecoenv201310032
Dar TA Uddin M Khan MMA Hakeem KR Jaleel H 2015 Jasmonates counterplant stress a Review Environ Exp Bot 115 49ndash57 httpsdoiorg101016jenvexpbot201502010
Dhanani T Singh R Shah S Kumari P Kumar S 2015 Comparison of green ex-traction methods with conventional extraction method for extract yield L-DOPAconcentration and antioxidant activity of Mucuna pruriens seed Green Chem LettRev 8 (2) 43ndash48 httpsdoiorg1010801751825320151075070
Gutieacuterrez-Gamboa G Portu J Santamariacutea P Loacutepez R Garde-Cerdaacuten T 2017Effects on grape amino acid concentration through foliar application of three dif-ferent elicitors Food Res Int 99 688ndash692 httpsdoiorg101016jfoodres201706022
Fig 4 A In situ antioxidant assay Foliar disksof Phaseolus vulgaris L treated with (a) No an-tioxidant added (negative control) (b) 10 mMMimosine (c) 10mM ascorbic acid (positivecontrol) The oxidative damage can be seen bythe formation of a brown polymer in leaf veinsand injured areas B In vitro superoxidescavenging assay carried out with mimosineDifferent letters indicate significant differenceby Tukey test (Ple 005) The error bars re-present standard error of four replicates (Forinterpretation of the references to colour in thisfigure legend the reader is referred to the Webversion of this article)
Fig 5 Predicted mimosine radical formed followingquenching of hydroxyl radical Mimosine is first converted toa stable mimosine radical which may be then converted to anontoxic indole form
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
439
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad HossainMd Anai T Oku H 2018 Cytosolic cysteine synthase switch cysteine and mi-mosine production in Leucaena leucocephala Appl Biochem Biotechnol 186 (3)613ndash632 httpsdoiorg101007s12010-018-2745-z
Holloacutesy F 2002 Effects of ultraviolet radiation on plant cells Micron 33 (2) 179ndash197Honda MDH Ishihara KL Pham DT Borthakur D 2018 Identification of drought-
induced genes in giant leucaena (Leucaena leucocephala subsp glabrata) Trees 32571ndash585 httpsdoiorg101007s00468-018-1657-4
Huang T Jander G de Vos M 2011 Non-protein amino acids in plant defense againstinsect herbivores representative cases and opportunities for further functional ana-lysis Phytochemistry 72 1531ndash1537 httpsdoiorg101016jphytochem201103019
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of thethyrotoxic amino acid mimosine by cysteine synthase Phytochemistry 29 (11)3461ndash3465 httpsdoiorg1010160031-9422(90)85258-H
Ishihara K Lee EKW Borthakur D 2016a An improved method for RNA extractionfrom woody legume species Acacia koa A Gray and Leucaena leucocephala (Lam) deWit Int J For Wood Sci 3 (1) 031ndash035
Ishihara KL Honda MDH Pham DT Borthakur D 2016b Transcriptome analysisof Leucaena leucocephala and identification of highly expressed genes in roots andshoots Transcriptomics 4 135 httpsdoiorg1041722329-89361000135
IUBMB 2018 Enzyme Nomenclature EC 35161 httpwwwsbcsqmulacukiubmbenzymeEC35161html Accessed date 8 February 2018
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant(Phaseolus vulgaris) Biosci Res 10 (1) 29ndash32
Kazan K 2015 Diverse roles of jasmonates and ethylene in abiotic stress toleranceTrends Plant Sci 20 (4) 219ndash229 httpsdoiorg101016jtplants201502001
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect ofEthephon as an ethylene-releasing compound on the metabolic profile of Chlorellavulgaris J Agric Food Chem 64 (23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541
Khan MIR Fatma M Per TS Anjum NA Khan NA 2015 Salicylic acid-inducedabiotic stress tolerance and underlying mechanisms in plants Front Plant Sci 6 462httpsdoiorg103389fpls201500462
Korsangruang S Soonthornchareonnon N Chintapakorn Y Saralamp PPrathanturarug S 2010 Effects of abiotic and biotic elicitors on growth and iso-flavonoid accumulation in Pueraria candollei var candollei and P candollei var mir-ifica cell suspension cultures Plant Cell Tissue Organ Cult 103 (3) 333ndash342 httpsdoiorg101007s11240-010-9785-6
Lalitha K Kulothungan SR 2006 Selective determination of mimosine and its dihy-droxypyridinyl derivative in plant systems Amino Acids 31 (3) 279ndash287 httpsdoiorg101007s00726-005-0226-5
Lalitha K Kulothungan SR 2007 Mimosine mitigates oxidative stress in seleniumdeficient seedlings of Vigna radiata - Part I restoration of mitochondrial functionBiol Trace Elem Res 118 (1) 84ndash96 httpsdoiorg101007s12011-007-0013-0
Liu J Li Y Wang Y Zhang Z-H Zu Y-G Efferth T Tang Z-H 2016 Thecombined effects of ethylene and MeJA on metabolic profiling of phenolic com-pounds in Catharanthus roseus revealed by metabolomics analysis Front Physiol 71ndash11 httpsdoiorg103389fphys201600217 Article 217
Malekzadeh P Khara J Heydari R 2014 Alleviating effects of exogenous Gamma-aminobutiric acid on tomato seedling under chilling stress Physiol Mol Biol Plants20 (1) 133ndash137 httpsdoiorg101007s12298-013-0203-5
Martiacutenez-Luumlscher J Torres N Hilbert G Richard T Saacutenchez-Diacuteaz M Delrot SAguirreolea J Pascual I Gomegraves E 2014 Ultraviolet-B radiation modifies thequantitative and qualitative profile of flavonoids and amino acids in grape berriesPhytochemistry 102 106ndash114 httpsdoiorg101016jphytochem201403014
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation ofbioactive secondary metabolites by ultraviolet radiation mechanisms strategies andapplications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology forMedicinal Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash1902012
Matsuura HN Fragoso V Paranhos JT Rau MR Fett-Neto AG 2016 Thebioactive monoterpene indole alkaloid N szlig-D-glucopyranosylvincosamide is regu-lated by irradiance quality and development in Psychotria leiocarpa Ind Crop Prod86 210ndash218 httpsdoiorg101016jindcrop201603050
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo RBhambra AS Strnad M Bonfill M Fett-Neto AG 2018 Specialized plant me-tabolism characteristics and impact on target molecule biotechnological productionMol Biotechnol 60 (2) 169ndash183 httpsdoiorg101007s12033-017-0056-1
Murakoshi S Ohmiya S Haginiwa J 1972 Enzymic synthesis of mimoside a meta-bolite of mimosine in Mimosa pudica and Leucaena leucocephala Chem Pharm Bull20 (4) 855ndash857
Nakagawa T Kawaguchi M 2006 Shoot-applied MeJA suppresses root nodulation inLotus japonicus Plant Cell Physiol 47 (1) 176ndash180 httpsdoiorg101093pcppci222
Nascimento NC Menguer PK Henriques AT Fett-Neto AG 2013 Accumulation ofbrachycerine an antioxidant glucosidic indole alkaloid is induced by abscisic acidheavy metal and osmotic stress in leaves of Psychotria brachyceras Plant PhysiolBiochem 73 33ndash40 httpsdoiorg101016jplaphy201308007
Neelamegam R Sutha T 2015 UV-C irradiation effect on seed germination seedling
growth and productivity of groundnut (Arachis hypogaea L) Int J Curr MicrobiolApp Sci 4 (8) 430ndash443
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase fromLeucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol164 (2) 922ndash934 httpsdoiorg101104pp113230870
Negi VS Borthakur D 2016 Heterologous expression and characterization of mimo-sinase from Leucaena leucocephala In Fett-Neto Arthur Germano (Ed)Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods inMolecular Biology vol 1405 copySpringer Science+Business Media New York httpsdoiorg101007978-1-4939-3393-8_7 2016
Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636
Parmar F Kushawaha N Highland H George L-B 2015 In vitro antioxidant andanticancer activity of Mimosa pudica Linn extract and L-mimosine on lymphomaDaudi cells Int J Pharm Sci 12 100ndash104
Porto DD Matsuura HN Vargas LRB Henriques AT Fett-Neto AG 2014 Shootaccumulation kinetics and effects on herbivores of the wound-induced antioxidantindole alkaloid brachycerine of Psychotria brachyceras Nat Prod Commun 9 (5)629ndash632
Rai R Meena RP Smita SS Shukla A Rai SK Pandey-Rai S 2011 UV-B and UV-C pre-treatments induce physiological changes and artemisinin biosynthesis inArtemisia annua L ndash an antimalarial plant J Photochem Photobiol B Biol 105 (3)216ndash225 httpsdoiorg101016jjphotobiol201109004
Shabani L Ehsanpour AA Asghari G Emami J 2009 Glycyrrhizin production by invitro cultured Glycyrrhiza glabra elicited by methyl jasmonate and salicylic acid RussJ Plant Physiol 56 (5) 621ndash626 httpsdoiorg101134S1021443709050069
Shah J 2003 The salicylic acid loop in plant defense Curr Opin Plant Biol 6 (4)365ndash371
Shi J Fu XZ Peng T Huang XS Fan QJ Liu JH 2010 Spermine pretreatmentconfers dehydration tolerance of citrus in vitro plants via modulation of antioxidativecapacity and stomatal response Tree Physiol 30 (7) 914ndash922 httpsdoiorg101093treephystpq030
Smith IK Fowden L 1966 A study of mimosine toxicity in plants J Exp Bot 17750ndash761 httpsdoiorg101093jxb174750
Soedarjo M Borthakur D 1996 Simple procedures to remove mimosine from youngleaves pods and seeds of Leucaena leucocephala used as food Int J Food SciTechnol 31 (1) 97ndash103
Soedarjo M Borthakur D 1998 Mimosine a toxin produced by the tree-legumeLeucaena provides a nodulation competition advantage to mimosine-degradingRhizobium strains Soil Biol Biochem 30 1605ndash1613
Suda S 1960 On the physiological properties of mimosine Bot Mag Tokyo 73 (862)142ndash147 httpsdoiorg1015281jplantres188773142
Tangendjaja B Lowry JB Wills RBH 1986 Isolation of a mimosine degrading en-zyme from leucaena leaf J Sci Food Agric 37 523ndash526 httpsdoiorg101002jsfa2740370603
Tiburcio F Pintildeol MT Serrano M 1985 Effect of UV-C on growth soluble protein andalkaloids in Nicotiana rustica plants Environ Exp Bot 25 (3) 203ndash210 httpsdoiorg1010160098-8472(85)90004-8
Vestena S Fett-Neto AG Duarte RC Ferreira A 2001 Regulation of mimosineaccumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 httpsdoiorg101016S0168-9452(01)00448-4
Vlot AC Dempsey DMA Klessig DF 2009 Salicylic acid a multifaceted hormone tocombat disease Annu Rev Phytopathol 47 177ndash206 httpsdoiorg101146annurevphyto050908135202 2009
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene-induced vinblastine accumulation is related to activated expression of downstreamTIA pathway genes in Catharanthus roseus BioMed Res Int 2016 Article ID 3708187httpsdoiorg10115520163708187
Wasternack C Strnad M 2016 Jasmonate signaling in plant stress responses and de-velopment ndash active and inactive compounds N Biotech 33 (5B) 604ndash613 httpsdoiorg101016jnbt201511001
Wencomo HB Ortiz R Caacuteceres J 2017 Afr J Agric Res 12 (4) 279ndash285 httpsdoiorg105897AJAR201510604 26
Wong CC Devendra C 1983 Research on leucaena forage production in Malaysia InLeucaena Research in the Asian Pacific Region pp 55ndash60 Ottawa Ontario Canada
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX TsimKWK 2018 Jasmonate-elicited stress induces metabolic change in the leaves ofLeucaena leucocephala Molecules 23 (2) httpsdoiorg103390molecules23020188 E188
Yafuso JT Negi VS Bingham J-P Borthakur D 2014 An O-acetylserine (thiol)lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthaseAppl Biochem Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z
Zhao J Zheng S-H Fujita K Sakai K 2004 Jasmonate and ethylene signalling andtheir interaction are integral parts of the elicitor signalling pathway leading to b-thujaplicin biosynthesis in Cupressus lusitanica cell cultures J Exp Bot 55 (399)1003ndash1012 httpsdoiorg101093jxberh127
Zhishen J Mengcheng T Jianming W 1999 The determination of flavonoid contentsin mulberry and their scavenging effects on superoxide radicals Food Chem 64 (4)555ndash559 httpsdoiorg101016S0308-8146(98)00102-2
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
440
61
Supplementary Fig 1 Basal mimosine concentration in adult trees of common leucaena (L leucocephala
var leucocephala) Samples were collected from 10 field grown trees at Manoa Valley Honolulu Hawairsquoi
on June 25th 2017 Bars sharing a letter do not differ by Tukey test (P le 005) The error bars represent the
standard error
Supplementary Fig 2 Bar diagram showing mimosine concentration in shoots of 12-week-old common
leucaena seedlings treated with different elicitors CTRL = Milli-Q water SA = Salicylic Acid MeJA =
Methyl Jasmonate CEPA = 2-Chloroethylphosphonic acid (an ethylene releasing compound) Bars sharing a
letter of same case do not differ by Tukey test (P le 005) Capital letters (A B) compare treatments on day
two and lower-case letters (a b) compare treatments on day four Indicates significant statistical difference
ABB
A A
0
200
400
600
800
1000
1200
LEAVES GREEN FLOWERBUDS
POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
con
cen
trat
ion
(micro
gg
-1o
f FW
)
B AB AB AB B A
b
a
ab b
ab
0
2
4
6
8
10
12
14
16
18
20
CTRL SA 10 ppm SA 100 ppm CEPA 10 ppm CEPA 100 ppm MeJA 90 ppm
Mim
osi
ne
co
nce
ntr
atio
n (
gg
-1o
f FW
)
DAY 02 DAY 04
62
between day two and day four in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
Supplementary Fig 3 Bar diagram showing the effects of UV-C radiation exposure for 5 10 and 15 min on
mimosine accumulation in shoots of 12-week-old seedlings of common leucaena Bars sharing a letter of
same case do not differ by Tukey test (P le 005) Capital letters (A B C) compare treatments on day three
and lower-case letters (a b) compare treatments on day six Indicates significant statistical difference
between day three and day six in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
C BC AB A
bb
a
a
0
10
20
30
40
50
60
CTRL UV-C 5 UV-C 10 UV-C 15
Mim
osi
ne
co
nce
ntr
atio
n (
gg-1
of
FW)
DAY 03 DAY 06
63
Supplementary Fig 4 Model depicting induction of mimosine synthesis in leucaena following application of
stress elicitors such as CEPA and jasmonic acid or exposure to UV-C radiation The additional mimosine
synthesized may serve to alleviate oxidative stress induced by UV-C radiation
64
Supplementary Table 1 Mimosine contents in leaves of common and giant leucaena
Leucaena
type
Mimosine content
( FW)
Mimosine
content ( DW)
Dry matter
content ( FW)
Water content
( FW)
Common (1) 050 plusmn 009 245 plusmn 051 2011 plusmn 054 7989 plusmn 054
Common (2) 043 plusmn 006 214 plusmn 037 1998 plusmn 050 8002 plusmn 050
K636 (1) 070 plusmn 014 356 plusmn 077 1908 plusmn 052 8092 plusmn 052
K636 (2) 042 005 205 plusmn 033 2008plusmn 093 7992plusmn 093
KX2 (1) 122 plusmn 011 608 plusmn 082 1939 plusmn 123 8061 plusmn 123
KX2 (2) 134 plusmn 010 623 plusmn 056 2029 plusmn 114 7971 plusmn 114
KX3 (1) 044 plusmn 006 221 plusmn 030 1945 plusmn 073 8055 plusmn 073
KX3 (2) 054 plusmn 005 273 plusmn 023 1930 plusmn 038 8070 plusmn 038
KX4 (1) 086 plusmn 011 471 plusmn 065 1753 plusmn 084 8247 plusmn 084
KX4 (2) 089 plusmn 011 476 plusmn 065 180 plusmn 072 820 plusmn 072
KX5 (1) 099 plusmn 012 489 plusmn 048 1907 plusmn060 8093 plusmn 060
KX5 (2) 115 plusmn 015 548 plusmn080 1992 plusmn 053 8008 plusmn 053
Common leucaena variety koa haole grows widely on the island of Orsquoahu K636 is widely
grown variety of giant leucaena KX2 KX3 KX4 and KX5 are giant leucaena varieties
developed through interspecies hybridization (Brewbaker 2016) (1) and (2) indicate plants
from two separate locations within the University of Hawaii Waimanalo Research Center The
values are shown as mean plusmn standard error obtained from at least three biological replicates
65
Supplementary Table 2 GenBank accession numbers of the tested cysteine pathway genes isoforms
Gene name GenBank accession
OAS-TL (o-acetylserine-thiol-lyase) GDRZ01032940
GDRZ01061620
GDRZ01153117
GDSA01187555
GDSA01196891
GDSA01214467
Cys syn (cysteine synthase) GDRZ01015860
GDRZ01050898
GDRZ01086813
GDRZ01193515
GDRZ01202579
GDSA01180863
GDSA01215622
SAT (serine acetyltransferase) GDRZ01187456
GDRZ01189631
CAS (β-cyanoalanine synthase) GDRZ01054066
GDRZ01175418
GDSA01118400
66
SHORT COMMUNICATION 1
Mimosine occurrence and accumulation in Mimosa bimucronata var bimucronata (DC) 2
Kuntze 3
Kelly Cristine da Silva Rodrigues-Correcirca1 Lana Dorneles Pedroso2 Fernanda de Costa1 4
Arthur Germano Fett-Neto1 5
1Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal 6
University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970 7
Porto Alegre Rio Grande do Sul Brazil 2Department of Biological Sciences Unipampa ndash 8
Campus Satildeo Gabriel 9
Corresponding author 10
E-mail addresses krodriguescbiotufrgsbr (KCdaS Rodrigues-Correcirca) 11
lanalima2012gmailcom (LD Pedroso) fernandadecostayahoocombr (F de Costa) 12
fettnetocbiotufrgsbr (AG Fett-Neto) 13
14
15
16
17
18
19
20
21
22
67
ABSTRACT 23
Mimosine is a non-protein aromatic amino acid present in plants of Leucaena spp 24
and Mimosa spp Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) is a native 25
tree from Brazil which occurs as a pioneer species on plant succession processes In the 26
current study the presence of mimosine in M bimucronata was verified by HPLC analyses 27
Moreover mimosine accumulation upon exposure to UV-C and chemical elicitors of 28
specialized metabolism (salicylic acid - SA methyl jasmonate - MeJA sodium nitroprusside 29
- SNP and ethephon - ETH) most of which also known as promoters of the amino acid 30
production in leucaena plants was evaluated The results showed a lower concentration of 31
constitutive mimosine present in both maricaacute seedlings and mature trees when compared to 32
leucaena plants In spite of a trend towards increased mimosine accumulation observed in 33
MeJA and ETH treatments no statistical differences were found with the various stressors 34
used to induce its biosynthesis in maricaacute seedlings Data suggest that mimosine in M 35
bimucronata is probably a phytoanticipin-like metabolite or its accumulation is driven by 36
other types of stresses 37
38
39
Keywords Mimosine Mimosa bimucronata stress 40
41
42
43
44
45
46
68
Introduction 47
Mimosa bimucronata commonly known as maricaacute is a native tree from Brazil 48
(REFLORA 2019) ecologically important in plant succession and in processes of degraded 49
land recovery (Bitencourt et al 2007 Silva et al 2011) occurring as a pioneer species 50
(Pilatti et al 2019) Maricaacute is a deciduous or semi-deciduous plant which reaches up to 15 51
m in height and 40 cm of diameter at breast height (DBH) displays shrub or tree habit and 52
bears typical sharp thorns (Carvalho 2004) This species belongs to Fabaceae one of the 53
most economically important families of flowering plants due to its high diversity and 54
occurrence in different types of habitats (Gomes et al 2018) As well as several others 55
Mimosa spp maricaacute is usually referred to as a multipurpose tree (Olkoski and Wittmann 56
2011) employed for alternative medicinal uses (Champanerkar et al 2010 Silva et al 57
2011) honey production constructions and remodeling of landscape architecture (living 58
fences) for instance (Marchiori 1993 Lorenzi 1998) 59
In southern Brazil maricaacute is widely distributed and typically found either in wetland 60
areas close to river banks (Patreze and Cordeiro 2004) or composing large and almost pure 61
landscape formations on hillsides (Jacobi and Ferreira 1991) In dense populations this 62
species like several Mimosa spp (Simon and Proenccedila 2000) is considered an important and 63
highly invasive weed by preventing cattle to reach pasturesand water bodies as a result of its 64
thorny branches (Lorenzi 2008 Kestring et al 2009) Its dominant and nearly exclusive 65
pattern of distribution in those areas has led Jacobi and Ferreira (1991) to test its allelopathic 66
potential on cultivated species Indeed extracts of leaves and ripe fruits (but not the green 67
ones) of maricaacute showed phytotoxic effects on germination and initial radical growth of most 68
of the target species tested 69
69
Several investigations have been performed on maricaacute floristics (Silva et al 2011) 70
distribution (Simon and Proenccedila 2000) wood anatomy (Marchiori 1993) cytogenetic 71
parameters (Olkoski and Wittmann 2011) and allelopathic potential (Jacobi and Ferreira 72
1991 Ferreira et al 1992) However excluding two recent publications on maricaacute 73
constitutive chemical composition (Schlickmann et al 2017 Pilatti et al 2019) which 74
identified phenolic compounds (methyl gallate and water-soluble tannins) as its major 75
compounds little is known regarding this subject In other Mimosa species (eg M pudica 76
and M pigra) mimosine has been identified (Soedarjo and Borthakur 1998) as one of the 77
major specialized metabolites present in the different organs of the plant (Champanerkar et 78
al 2010) The presence of this molecule was also reported for M bimucronata in a thin layer 79
chromatography-based preliminary study performed by Ferreira et al (1992) showing co-80
chromatography of a leaf extract component with authentic mimosine The authors attributed 81
the allelopathic effect of maricaacute to the accumulation of this metabolite in its leaves 82
Mimosine is an aromatic non-protein amino acid initially found in plants of Mimosa 83
pudica and later in Leucaena leucocephala (Lam) de Wit (Soedarjo and Borthakur 1998) a 84
leguminous tree which biosynthesizes large amounts of this nitrogen-containing compound 85
(Rodrigues-Correcirca et al 2019) It is believed that the accumulation of high contents of 86
mimosine in L leucocephala tissues confers among other traits defense against herbivores 87
and pathogens (Vestena et al 2001) tolerance to drought (Negi et al 2014) as well as 88
general oxidative stress protection (Rodrigues-Correcirca et al 2019) Interestingly drought is 89
the opposite environmental and physiological condition to that observed in the wet habitats 90
occupied by native populations of M bimucronata in Brazil (Patreze and Cordeiro 2004 91
Kestring et al 2009) and Mimosa pudica Linn in India (Champanerkar et al 2010) 92
70
Nonetheless flooding is also associated with oxidative stress particularly as water levels 93
change (Fukao et al 2019) 94
In Leucaena leucocephala var leucocephala (common leucaena) and Leucaena 95
leucocephala var glabrata (giant leucaena) mimosine accumulation has been shown to be 96
both constitutive and inducible by stress-related phytohormones such as jasmonic acid (JA) 97
Ethephon (ETH an ethylene- releasing compound) salicylic acid (SA - only common 98
leucaena) (Vestena et al 2001) as well as by UV-C radiation (Xu et al 2018 Rodrigues-99
Correcirca et al 2019) On the other hand there is a lack of information regarding mimosine 100
content and elicitation effects in Mimosa spp plants 101
The aim of this study was to examine the presence of mimosine in Mimosa 102
bimucronata and examine the effects of stresses and stress-signaling molecules on its 103
accumulation in leaves 104
Material and Methods 105
Plant material 106
For all experiments the plant material was collected at Morro Santana campus do 107
Vale of UFRGS (Federal University of Rio Grande do Sul) Porto Alegre RS Brazil 108
(3004rsquoS 5108rsquoW) Authorization for access to genetic material was obtained from 109
SISGEN-Brazil (license number A845493) Constitutive mimosine content in adult plants of 110
M bimucronata var bimucronata (DC) Kuntze was determined in plant material (leaves 111
green flower buds post-anthesis flowers and green pods) harvested in January 2017 112
(summer) A voucher herbarium specimen (ICN 187953) was deposited in the ICN ndash UFRGS 113
herbarium (Herbaacuterio do Instituto de Biociecircncias of UFRGS) 114
71
For mimosine elicitation experiments legumes (fruits) of maricaacute were collected in 115
the end of June 2017 (winter) Seeds were then removed from the dry fruits and kept in the 116
dark until sowing and seedling development for use in the assays 117
Seed germination 118
To break the coat-imposed seed dormancy after surface sterilization dry seeds of 119
maricaacute were acid scarified by immersion in H2SO4 (95 ndash 98 ) for 2 min (see Correcirca et al 120
2008) and repeatedly washed in distilled water to remove any residue of the acid Then seeds 121
were distributed in 50 mL individual plastic tubes (dibble-tubes) (30 cm diameter x 120 cm 122
depth) filled up with 11 (vv) of commercial top soil and vermiculite Tubes were watered 123
every 2 days to avoid substrate dryness and were kept in a growth room under controlled 124
conditions of light (circa 75 μmol mminus2s minus1 photosynthetically active radiation photoperiod 125
of 16 h light and 8 h dark) and temperature (24plusmn2C) 126
127
Treatments 128
In order to verify inducibility of mimosine accumulation in M bimucronata fifty 12-129
week-old maricaacute seedlings (per treatment) exhibiting similar features were selected and 130
sprayed (saturated) with solutions of different chemical stressors (plant specialized 131
metabolism elicitors) as follows (for further details see Rodrigues-Correcirca et al 2019) 10 132
and 50 mM SA (pathogen-signaling molecule Shah 2003) 007 and 035 mM 2-133
chloroethylphosphonic acid (ETH ethylene releasing-compound Kim et al 2016 Wang et 134
al 2016) 100 and 200 mM MeJA (Dar et al 2015) 10 and 50 mM SNP (a nitric oxide 135
donor Perotti et al 2015) Alternatively maricaacute seedlings were also supplemented with UV-136
C radiation (13 minutes 105 kJ cm2) (elicitor of plant specialized metabolism Kara 2013) 137
72
After 2 and 4 days of exposure to the chemical treatments and 3 and 6 days of UV-138
C supplementation maricaacute shoots were harvested immediately frozen in liquid nitrogen and 139
stored at ndash 80 C until mimosine extraction and HPLC analyses 140
Mimosine extraction and detection 141
Mimosine extraction was conducted according to the modified protocol described by 142
Rodrigues-Correcirca et al (2019) for L leucocephala HPLC (Thermo Scientific Surveyor) 143
analyses (mimosine detection and quantification) were performed following previously 144
published procedures (Negi et al 2014) A C18 column (ACE C18 5 μm 46times250 mm) and 145
isocratic solvent system of 002M o-phosphoric acid with a linear flow rate of 1 mL min minus1 146
were used to separate and quantify the amino acid Mimosine detection was performed at 280 147
nm by photodiode array detection (200ndash400 nm) and retention time (229plusmn0024 min) 148
Mimosine quantification was done by means of the method of external standard curve 149
Additional confirmation of mimosine identity was performed by co-chromatography with 150
standard (Acros Organics authentic mimosine 99 used as reference) and peak purity check 151
The analyses of the chromatograms were done with the ChromQuest software 152
153
154
Results and Discussion 155
Constitutive accumulation of mimosine in M bimucronata 156
Mimosine was detected in all analyzed samples positively meeting all identification 157
criteria In agreement with what has been found for other Mimosa spp (Soedarjo and 158
Borthakur 1998) compared to L leucocephala adult plants (Rodrigues-Correcirca 2019) 159
mimosine content was lower in M bimucronata Of the adult plant tissues analyzed the 160
73
highest content of mimosine in maricaacute (per gram of fresh weight - FW) was found in post-161
anthesis flowers (36644 microg versus 89448 microg in common leucaena followed by leaves 162
(28838 microg x 67358 microg) green flower buds (28094 microg x 51247 microg) and green pods (19002 163
microg x 82687 microg) (Fig 1)The same pattern is observed for seedlings when both species are 164
compared In this study untreated 12-week-old maricaacute seedlings (control at day 2) showed a 165
shoot content of mimosine of 23029plusmn007 microg g-1 of (FW) Five-week-old untreated giant 166
leucaena seedlings cultivated in similar conditions exhibited between 83640 and 178736 167
microg g-1 of FW (Rodrigues-Correcirca et al 2019) In the same way mimosine concentration 168
percentage in dry matter of Mimosa pigra was found to be rather low (002 in nodules and 169
roots and 007 in leaves) (Soedarjo and Borthakur 1998) 170
In this investigation the lowest constitutive mimosine content was found in green 171
pods (Fig 1) This result may partly explain the absence of phytotoxic effect observed for 172
green pods on germination and growth of crop target plants tested by Jacobi and Ferreira 173
(1991) compared to the other maricaacute parts analyzed 174
Elicitation of mimosine biosynthesis in M bimucronata 175
Chemical stressors 176
Secondary metabolites (or natural products) are structural- and chemically 177
specialized compounds derived from primary metabolism These molecules are mainly 178
biosynthesized as part of a complex defense mechanism in response to biotic and abiotic 179
stresses such as pathogens herbivores water status metal toxicity and UV radiation for 180
example (Matsuura et al 2018) Ethephon SA SNP MeJA have been extensively used as 181
chemical elicitors of specialized metabolism (Wang et al 2016 Vestena et al 2001 Perotti 182
74
et al 2015 Zhang and Memelink 2009 Xu et al 2018) These phytohormonal signals can 183
simulate environmental challenges and modulate plant homeostasis often leading to 184
alterations in gene expression (Shinozaki et al 2015) Except SNP all treatments tested in 185
the present study showed positive effect on mimosine accumulation in common or giant 186
leucaena (Vestena et al 2001 Rodrigues-Correcirca 2019 Rodrigues-Correcirca unpublished 187
data) However in spite of the trend of increasing the mimosine content observed in seedlings 188
treated with 007 mM Ethephon (at day 2) and 100 mM MeJA (at day 4) no statistical 189
difference was confirmed for these treatments when compared to the control 190
On the other hand a within treatment difference on mimosine induction was seen 191
between day 2 and 4 in seedlings treated with 100 mM MeJA (Fig 2) In a lower 192
concentration (04 mM) jasmonic acid (JA)promoted a near threefold increase in mimosine 193
accumulation of giant leucaena seedlings after 2 days of application 194
UV-C radiation 195
Albeit UV-C radiation is not biologically active in natural environments it has been 196
widely used under controlled experimental conditions to generate acute responses of plant 197
specialized metabolism within a shorter period of time compared to that required to with UV-198
B radiation (Kara 2013 Cetin 2014) This fast response is due to the higher energy of UV-199
C photons that act as potent reactive oxygen species (ROS) generators causing extensive 200
damage to the cells either at the physiological level or on DNA structure (Gregianini et al 201
2003 Matsuura et al 2013) 202
Although divergent responses can be observed in plants exposed to UV-C radiation 203
the deleterious processes are usually reported on primary metabolism (decreasing of 204
chlorophyll content and plant height eg) (Kara 2013) In the present study no statistical 205
75
differences were observed in the mimosine concentration in maricaacute seedlings supplemented 206
with UV-C radiation However a decreasing in its content was found for both control and 207
treatment at day 6 post-treatment (Fig 03) Taking into account the lower constitutive 208
concentration of mimosine observed in maricaacute compared to the leucaena plants besides its 209
relative thermolability (Nguyen and Tawata 2016) it seems to be plausible to consider the 210
effect of the temperature inside the UV-C and the white light (control) chambers as an 211
additional abiotic factor contributing to the decrease of mimosine accumulation in both group 212
of plants 213
Besides mimosine identification the presence of 34-dihydroxypyridine (34-DHP or 214
3-hydroxy-4-pyridone - 3H4P) a mimosine degradation product (Negi et al 2014 Nguyen 215
and Tawata 2016) was also reported for maricaacute leaf extracts analyzed by TLC by Ferreira 216
et al (1992) In our chromatograms we detected a second large peak after that of mimosine 217
(229plusmn0024) and similar to that identified by Negi et al (2014) as 3H4P (data not shown) 218
Comparing the chromatogram profiles obtained from seedlings elicited with chemical 219
stressors and those supplemented with UV-C the largest area for this peak was found (in all 220
samples) in the latter treatment at day 6 It might indicate that the constitutive andor the 221
initially UV-C-induced mimosine was degraded into 3H4P to cope with the cellular damage 222
caused by this treatment associated with an increased temperature inside the chambers 223
Nevertheless it was not possible to determine 3H4P concentration (or confirm its identity) 224
in maricaacute plants since there is no commercial standard (pure 3H4P) available for purchase 225
to be used as a reference in calculations Establishment of improved protocols for obtaining 226
in house 3H4P reference substance by acid hydrolysis is ongoing 227
228
229
76
Conclusion 230
On the basis of the overall absence of effect of the treatments tested here on mimosine 231
concentration it is possible to suggest that its accumulation profile is similar to that of 232
phytoanticipins unlike what is observed for the same amino acid production in leucaena 233
which shows features of inducibility resembling phytoalexin-like metabolites Alternatively 234
a putative inducible pool of mimosine in maricaacute might be involved in other types of stress 235
such as extended drought periods If involved in protection against oxidative stress as 236
described for leucaena mimosine in maricaacute may act predominantly by physical quenching 237
of ROS as indicated by the lack of overt chemical degradation Nevertheless further 238
investigations are needed to assess these hypotheses 239
To sum up mimosine biosynthesis was not modulated by the treatments evaluated as 240
in L leucocephala (Lam) de Wit To the best of our knowledge this is the first work that 241
analytically identifies and quantifies mimosine accumulation in M bimucronata 242
243
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Bitencourt F Zocche JJ Costa S Souza PZ Mendes AR 2007 Nucleaccedilatildeo de 245
Mimosa bimucronata (DC) O Kuntze em aacutereas degradadas pela mineraccedilatildeo de carvatildeo R 246
Bras Bioci 5 750-752 247
Carvalho PER 2004 Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 248
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Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitis 250
vinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-251
6287-47-37 252
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Champanerkar PA Vaidya VV Shailajan S Menon SN 2010 A sensitive rapid and 253
validated liquid chromatography ndash tandem mass spectrometry (LC-MS-MS) method for 254
determination of Mimosine in Mimosa pudica Linn Nat Sci 2 713-717 255
httpsdoiorg104236ns201027088 256
Gomes GS Silva GS Silva DLS Oliveira RR Conceiccedilatildeo GM 2018 Botanical 257
Composition of Fabaceae Family in the Brazilian Northeast Maranhatildeo Brazil Asian J 258
Environ Ecol 6(4) 1-10 httpsdoiorg109734AJEE201841207 259
Correcirca LR Soares GLG Fett-Neto AG 2008 Allelopathic potential of Psychotria 260
leiocarpa a dominant understorey species of subtropical forests S Afri J Bot 74 583ndash261
590 httpsdoiorg101016jsajb200802006 262
Ferreira AG Aquila MEA Jacobi US Rizvi V 1992 Allelopathy in Brazil In Allelopathy 263
basic and applied aspects Rizvi V and Jacobi US (Eds) Chapman and Hall pp 243-250 264
Fukao T Barrera-Figueroa BE Juntawong P Pentildea-Castro JM 2019 Submergence 265
and waterlogging stress in plants a review highlighting research opportunities and 266
understudied aspects Front Plant Sci 10 340 httpsdoiorg103389fpls201900340 267
Gregianini TS Silveira VC Porto DD Kerber VA Henriques AT Fett-Neto AG 268
2003 The alkaloid brachycerine is induced by ultraviolet radiation and is a singlet oxygen 269
quencher Photochem Photobiol 78(5) 470ndash474 httpsdoiorg1015620031-270
8655(2003)0784070TABIIB20CO2 271
Jacobi US Ferreira AG 1991 Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK 272
sobre espeacutecies cultivadas Pesq Agropec Bras 26(7) 935-943 273
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant 274
(Phaseolus vulgaris) Biosci Res 10(1) 29ndash32 275
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Kestring D Klein J Menezes LCCR Rossi MN 2009 Imbibition phases and 276
germination response of Mimosa bimucronata (Fabaceae Mimosoideae) to water 277
submersion Aquat Bot 91 105ndash109 httpsdoiorg101016jaquabot200903004 278
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect of 279
Ethephon as an ethylene-releasing compound on the metabolic profile of Chlorella vulgaris 280
J Agric Food Chem 64(23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541 281
Lorenzi H 1998 Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 282
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 283
Lorenzi H 2008 Plantas daninhas do Brasil terrestres aquaacuteticas parasitas e toacutexicas 4 ed 284
Nova Odessa Instituto Plantarum 640 p 285
Marchiori JNC 1993 Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 286
O Kuntze Ciecircncia Florestal 3 85-106 287
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation of 288
bioactive secondary metabolites by ultraviolet radiation mechanisms strategies and 289
applications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology for Medicinal 290
Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash190= 291
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo R Bhambra AS 292
Strnad M Bonfill M Fett-Neto AG 2018 Specializedplant 293
metabolismcharacteristicsandimpactontargetmoleculebiotechnologicalproduction 294
Molecular Biotechnology 60(2) 169ndash183httpsdoiorg101007s12033-017-0056-1 295
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase from 296
Leucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 297
922ndash934 httpsdoiorg101104pp113230870 298
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Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine 299
areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636 300
Olkoski D Wittmann MTS 2011 Cytogenetics of Mimosa bimucronata (DC) O Kuntze 301
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop Breed 302
Appl Biotechnol 11 27-35 httpdxdoiorg101590S1984-70332011000100004 303
Patreze CM Cordeiro L 2004 Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 304
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecol Manag 196 275ndash305
285 httpdxdoiorg101016jforeco200403034 306
Perotti JC Rodrigues-Correcirca KCS Fett-Neto AG 2015 Control of resin production in 307
Araucaria angustifolia an ancient South American conifer Plant Biology 17 852ndash859 308
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG 2019 Mimosine 309
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 310
UV exposure Plant Physiology and Biochemistry 135 432ndash440 311
Pilatti DM Fortes AMT Jorge TCM Boiago NP 2019 Comparison of the phytochemical 312
profiles of five native plant species in two different forest formations Brazilian Journal of 313
Biology 79(2) 233-242 314
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS 2011 Biologia da reproduccedilatildeo 315
deMimosa bimucronatandash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 316
Simon MF and Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 317
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 318
endemism Biological Conservation 96 279-296 319
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 320
LM Andrade SF Cechinel-Filho V 2017 Chemical composition and diuretic natriuretic and 321
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kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its majority 322
constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash1624 323
Shah J 2003 The salicylic acid loop in plant defense Current Opinion Plant Biology6 (4) 324
365ndash371 325
Shinozaki K Uemura M Serres JB Bray EA Weretilnyk E 2015 Responses to Abiotic 326
Stress In Buchanan BB Gruissem W Jones RL (Eds) Biochemistry and Molecular 327
Biology of Plants Second Edition John Wiley and Sons Ltd 328
Soedarjo M and Borthakur D 1998 Mimosine a toxin produced by the tree-legume 329
Leucaena provides a nodulation competition advantage to mimosine-degrading Rhizobium 330
strains Soil Biology and Biochemistry 30(12)1605-1613 331
Vestena S Fett-Neto AG Duarte RC Ferreira AG 2001 Regulation of mimosine 332
accumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 333
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene induced 334
vinblastine accumulation is related to activated expression of downstream TIA pathway 335
genes in Catharanthus roseus BioMed Research International Article ID 3708187 336
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX Tsim KWK 2018 337
Jasmonate-elicited stress induces metabolic change in the leaves of Leucaena leucocephala 338
Molecules 23 (2) 339
Zhang H Memelink J 2009 Regulation of Secondary Metabolism by Jasmonate Hormones 340
In AE Osbourn and V Lanzotti (eds) Plant-derived Natural Products 3 DOI 101007978-341
0-387-85498-4_1 copy Springer Science + Business Media LLC 342
343
344
345
81
346
Figure 1 Constitutive concentration of mimosine in different plant organs of Mimosa 347
bimucronata Bars sharing the same letter do not differ statistically by Tukey test (Ple005) 348
The error bars denote standard error of 10 replicates 349
350
351
352
353
354
355
356
357
B B A C0
5
10
15
20
25
30
35
40
LEAVES GREEN FLOWER BUDS POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
co
nce
ntr
atio
n u
gg-1
Mimosine concentration in adult plants of Mimosa bimucronata (DC) Kuntze
82
C T R L S A
1 0 m M
S A
5 0 m M
E T H
0 0 7 m M
E T H
0 3 5 m M
M e J A
1 0 0 m M
M e J A
2 0 0 m M
S N P
1 0 m M
S N P
5 0 m M
0
1 0
2 0
3 0
T re a tm e n ts
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1) D A Y 2
D A Y 4
A B C C B C A B C C A B C A B C A
a b b b a a b a a b b a b
358
Figure 2 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 359
bimucronata treated with different signaling molecules SA = Salicylic Acid ETH = 360
Ethephon MeJA = Methyl Jasmonate SNP = Sodium Nitroprusside Uppercase and 361
lowercase letters indicate statistical differences among treatments in days 2 and 4 362
respectively Bars sharing a letter of the same case do not differ statistically by Tukey test 363
(Ple005) Indicates statistical difference in the same treatment between day 2 and 4 by t-364
test (Ple005) The error bars denote standard error of 5 replicates (25 individual seedlings 365
arranged in 5 groups of 5) 366
367
368
83
D AY 3 D AY 6
0
5
1 0
1 5
2 0
2 5
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1)
C O N TR O L
U V -C
369
Figure 3 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 370
bimucronata supplemented with UV-C radiation Indicates statistical difference in the same 371
treatment between day 3 and 6 by t-test (Ple005) The error bars denote standard error of 5 372
replicates (25 individual seedlings arranged in 5 groups of 5) 373
374
375
376
377
378
379
380
381
382
383
384
385
84
Consideraccedilotildees finais 386
- Experimentos que avaliam os efeitos da aplicaccedilatildeo exoacutegena de ANPs em diferentes espeacutecies 387
vegetais tecircm sido realizados principalmente com GABA Dentre os principais efeitos 388
conferidos pela aplicaccedilatildeo dessa moleacutecula em espeacutecies de mono e eudicotiledocircneas satildeo 389
relatados a toleracircncia agrave seca agrave salinidade e agraves temperaturas extremas 390
- Como metaboacutelitos especializados claacutessicos os ANPs podem ter sua concentraccedilatildeo basal 391
endoacutegena aumentada em resposta agrave induccedilatildeo mediada por uma vasta gama de tratamentos com 392
moleacuteculas sinalizadoras de estresse e fontes alternativas de estressores De um modo geral 393
observa-se o acuacutemulo das diferentes classes de ANPs em resposta agrave radiaccedilatildeo UV elicitores 394
quiacutemicos que mimetizam ataques por patoacutegenos dano mecacircnico agentes osmoacuteticos metais 395
pesados entre outros 396
- Especificamente em leucena a resposta observada em relaccedilatildeo aos diferentes tratamentos 397
testados indica que apesar do seu alto teor constitutivo nessa espeacutecie a biossiacutentese e o 398
acuacutemulo de mimosina podem ser modulados por fatores causadores de estresses exibindo -399
nessa espeacutecie - um padratildeo de acumulaccedilatildeo similar agrave fitoalexinas Em maricaacute por outro lado 400
aumento de acuacutemulo dessa moleacutecula natildeo foi observado para os mesmos tratamentos testados 401
para leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas 402
- O padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno 403
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado pela 404
sua degradaccedilatildeo 405
- As respostas observadas nos testes que avaliaram a atividade de mitigaccedilatildeo de espeacutecies 406
reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode agir como um agente 407
antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de estresse 408
85
Perspectivas 409
- Confirmaccedilatildeo em espectrocircmetro de massas eou ressonacircncia nuclear magneacutetica da natureza 410
quiacutemica da lsquomimosinarsquo presente em maricaacute 411
- Avaliaccedilatildeo do efeito de concentraccedilotildees mais elevadas e em diferentes periacuteodos de aplicaccedilatildeo 412
das moleacuteculas sinalizadoras testadas sobre o acuacutemulo de mimosina em leucena e maricaacute 413
- Ampliar a investigaccedilatildeo dos padrotildees de expressatildeo gecircnica dos genes que codificam para 414
mimosinase (em maricaacute) mimosina sintase (em ambas as espeacutecies testadas) bem como o 415
perfil de precursores e cataboacutelitos de mimosina em resposta aos tratamentos mencionados 416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
86
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Bitencourt F Zocche JJ Costa S Souza PZ Mendes AR (2007) Nucleaccedilatildeo de Mimosa 450
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Crowe B Poynter JA Manukyan MC Wang Y Brewster BD Herrmann JL Abarbanell 465
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88
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Kutchan TM Gershenzon J Moslashller BL Gang DR (2015) Natural Products In Buchanan 504
BB Gruissem W and Jones RL (eds) Biochemistry amp Molecular Biology of Plants 2nd edn 505
Wiley Blackwell Chichester pp 1135-1205 506
Lalande M (1990) A reversible arrest point in the late G1 phase of the mammalian cell cycle 507
Exp Cell Res 186 332ndash339 508
Li X-W Hu C-P Li Y-J Gao Y-X Wang XM Yang J-R (2015) Inhibitory effect of L-509
mimosine on bleomycin-induced pulmonary fibrosis in rats Role of eIF3a and p27 Int 510
Immunopharmacol 27(1) 53ndash64 511
Little Jr EL Skolmen RG (1989) Koa haole Agriculture Handbook 679 USDA 512
Lorenzi H (1998) Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 513
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 514
Marchiori JNC (1993) Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 515
O Kuntze Ciecircncia Florestal 3 85-106 516
Mohammed RS El Souda SS Taie HAA Moharam ME Shaker KH (2015) Antioxidant 517
antimicrobial activities of flavonoids glycoside from Leucaena leucocephala leaves Journal 518
of Applied Pharmaceutical Science 5(06)138-147 519
httpdxdoiorg107324JAPS201550623 520
Negi VS Bingham J-P Li QX Borthakur D (2014) A carbon-nitrogen lyase from Leucaena 521
leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 (2) 922ndash522
934 httpsdoiorg101104pp113230870 523
89
Olkoski D Wittmann MTS (2011) Cytogenetics of Mimosa bimucronata (DC) O Kuntze 524
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop 525
Breeding and Applied Biotechnology 11 27-35 526
Patreze CM Cordeiro L (2004) Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 527
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecology and Management 528
196275ndash285 529
Pilatti DM Fortes AMT Jorge TCM Boiago NP (2019) Comparison of the phytochemical 530
profiles of five native plant species in two different forest formations Brazilian Journal of 531
Biology 79(2) 233-242 532
Ramos-Ruiz R Poirot E Flores-Mosquera M (2018) GABA a non-protein amino acid 533
ubiquitous in food matrices Cogent Food Agric 41534323 534
httpsdoiorg1010802331193220181534323 535
REFLORA (2019) httpfloradobrasiljbrjgovbrreflora Acesso em agosto de 2019 536
Rodgers KJ Samardzic K Main BJ (2015) Toxic Nonprotein Amino Acids Plant Toxins 537
httpsdoiorg 101007978-94-007-6728-7_9-1 538
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG (2019) Mimosine 539
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 540
UV exposure Plant Physiology and Biochemistry 135 432ndash440 541
httpsdoiorg101016jplaphy201811018 542
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 543
LM Andrade SF Cechinel-Filho V (2017) Chemical composition and diuretic natriuretic 544
and kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its 545
majority constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash546
1624 547
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS (2011) Biologia da reproduccedilatildeo 548
de Mimosa bimucronata ndash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 549
Simon MF Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 550
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 551
endemism Biological Conservation 96 279-296 552
90
Soares AMS Arauacutejo SA Lopes SG Costa Junior LM (2015) Anthelmintic activity of 553
Leucaena leucocephala protein extracts on Haemonchus contortus Braz J Vet Parasitol 554
Jaboticabal 24(4) 396-401 httpdxdoiorg101590S1984-29612015072 555
Soerdajo M Borthakur D (1998) Mimosine a toxin produced by the tree-legume Leucaena 556
provides a nodulation competition advantage to mimosine-degrading Rhizobium strains Soil 557
Biol Biochem 30(12) 16051613 558
Souza-Lima ES Sinani TR Pott A Sartori ALB (2017) Mimosoideae (Leguminosae) in the 559
Brazilian Chaco of Porto Murtinho Mato Grosso do Sul Rodrigueacutesia 68(1) 263-290 2017 560
httpdxdoiorg1015902175-7860201768131 561
Taiz L amp Zeiger E (2010) Plant Physiology 5th edition Sinauer Associates Inc Sunderland 562
Verma VK Rani KV Kumara SR Prakash O (2018) Leucaena leucocephala pod seed 563
protein as an alternate to animal protein in fish feed and evaluation of its role to fight against 564
infection caused by Vibrio harveyi and Pseudomonas aeruginosa Fish and Shellfish 565
Immunology 76 (2018) 324ndash332 httpsdoiorg101016jfsi201803011 566
Yafuso JT Negi VS Bingham J-P Borthakur D (2014) An O-acetylserine (thiol) lyase from 567
Leucaena leucocephala is a cysteine synthase but not a mimosine synthase Appl Biochem 568
Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z 569
Zarin RMA Wan HY Isha A Armani N (2016) Antioxidant antimicrobial and cytotoxic 570
potential of condensed tannins from Leucaena leucocephala hybrid Food Science and 571
Human Wellness 5 65ndash75 httpdxdoiorg101016jfshw201602001 572
573
574
Contents lists available at ScienceDirect
Industrial Crops amp Productsjournal homepage wwwelseviercomlocateindcrop
Resin tapping transcriptome in adult slash pine (Pinus elliottii var elliottii)Camila Fernanda de Oliveira Junkes1 Artur Teixeira de Arauacutejo Juacutenior1 Juacutelio Ceacutesar de LimaFernanda de Costa Thanise Fuumlller Maacutercia Rodrigues de Almeida Franciele Antocircnia NeisKelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-NetoCenter for Biotechnology and Department of Botany Federal University of Rio Grande do Sul Porto Alegre PO Box 15005 91501-970 Brazil
A R T I C L E I N F O
KeywordsPinus elliottiResinResinosisTranscriptomeAdjuvant paste
A B S T R A C T
To better understand the bases of resin production a major source of terpenes for industry the transcriptome ofadult Pinus elliottii var elliottii (slash pine) trees under field commercial resinosis was obtained Samples werecollected from cambium after 5 and 15 days of treatment application which included tapping followed byapplication of commercial resin stimulant paste or control wounding without paste Overall mean number ofreads of all 16 libraries (2 treatments x 2 times x 4 replicated trees) was 34582048 Of these 89 were mappedagainst the reference sequence with a mismatch of 058 Using the Blast2Go 570 candidate genes were de-tected based on sequence annotation By comparing the expression profile between paste and control 310differentially expressed genes (DEGs) were identified at 5 days and 190 at 15 days with a significant fold changeof log2gt 12 Regarding changes in time comparisons within each treatment 210 and 105 DEGs were identifiedwithin control and paste treatment respectively Genes with different expression patterns in the times andtreatments examined included ethylene responsive transcription factors geranylgeranyl diphosphate synthasediterpene synthase cytochrome P450 and ABC transporters all of which may play important roles in resinproduction RT-qPCR analysis correlated well with the data obtained by RNAseq Resin composition changedover time This is the first transcriptomic investigation of resinosis of the main species used in the bioresinindustry and of molecular analyses of resinosis under field operations with implications for stand managementstimulant paste development genotype selection and breeding for high resinosis
1 Introduction
The adaptive success of conifers is largely due to the development ofa defense system based on the synthesis and secretion of terpenes in allmajor organs and different tissues (Miller et al 2005 Hall et al 2013Warren et al 2015) Conifer resin is a viscous fluid composed of acomplex mixture of terpenoids such as monoterpenes sesquiterpenesand diterpenes (Zulak and Bohlmann 2010) These terpenoids are se-creted from severed resin ducts when the tree is under biotic attack(Ralph et al 2006 Lange 2015 Geisler et al 2016) acting as pro-tectants (Schiebe et al 2012 Liu et al 2015)Biosynthesis of terpenes in conifers starts from isomerization of two
isoprenoid (C5) units dimethylallyl diphosphate (DMAPP) and iso-pentenyl diphosphate (IPP) These molecules can be biosynthesized viatwo separate routes in plants the methyl-erythritol 4-phosphate andmevalonate pathways IPP is synthesized and isomerized to DMAPP byisopentenyl diphosphate isomerase then prenyl transferases catalyze
the condensation of these two C5-units to geranyl diphosphate (Pazoukiand Niinemets 2016) Their elongation to prenyl diphosphates withaddition of IPP molecules leads to monoterpenes (C10) sesquiterpenes(C15) and diterpenes (C20) which are the substrates for terpene syn-thases (TPS) (Keeling and Bohlmann 2006b)TPSs are part of a large family of mechanistically related enzymes
involved in both primary and secondary metabolism (Keeling andBohlmann 2006b) The events of evolutionary diversification and ex-pansion of plant TPSs appear to have originated from gene duplicationsdomain losses and sub- or neofunctionalizations with subsequent di-vergence of an ancestral TPS gene of primary metabolism (Hall et al2013) Modification of TPS products changes their physical propertiesand may alter their biological activities (Chen et al 2011) TPSs of highsequence identity may have different functions even in closely relatedspecies Low sequence identity of TPSs in phylogenetically distantspecies does not preclude the possibility of independent evolution of thesame or related function of these enzymes (Zerbe and Bohlmann 2015)
httpsdoiorg101016jindcrop2019111545Received 4 January 2019 Received in revised form 10 June 2019 Accepted 4 July 2019
Corresponding authorE-mail address fettnetocbiotufrgsbr (AG Fett-Neto)1 These authors have equally contributed to this work
doi 1015900102-33062019abb0114
Acta Botanica Brasilica
Sustainable production of bioactive alkaloids in Psychotria L of
southern Brazil propagation and elicitation strategies
Yve Verocircnica da Silva Magedans1 Kelly Cristine da Silva Rodrigues-Correcirca1 Cibele Tesser da Costa1
Heacutelio Nitta Matsuura1 and Arthur Germano Fett-Neto1
Received April 1 2019Accepted June 28 2019
ABSTRACTPsychotria is the largest genus in Rubiaceae South American species of the genus are promising sources of natural
products mostly due to bioactive monoterpene indole alkaloids they accumulate ese alkaloids can have analgesic
antimutagenic and antioxidant activities in dierent experimental models among other pharmacological properties
of interest Propagation of genotypes with relevant pharmaceutical interest is important for obtaining natural
products in a sustainable and standardized fashion Besides the clonal propagation of elite individuals the alkaloid
content of Psychotria spp can also be increased by applying moderate stressors or stress-signaling molecules is
review explores advances in research on methods for plant propagation and elicitation techniques for obtaining
bioactive alkaloids from Psychotria spp of the South Region of Brazil
Keywords abiotic stress alkaloids elicitation monoterpenes plant propagation Psychotria southern Brazil
sustainability
Introduction
Psychotria belongs to Rubiaceae one of the major families
of $owering plants having economic interest e family
includes coee a few signicant poisonous plants to livestock
besides several important ornamental and medicinal species
(Souza amp Lorenzi 2012) Psychotria has captured researchersrsquo
attention mostly because of its medicinal properties
Psychotria colorata is an Amazonian species that produces
polyindolinic alkaloids with analgesic activity (Matsuura et
al 2013) e promising results obtained with P colorata
motivated the investigation of southern Brazilian Psychotria
species and the discovery of new bioactive alkaloids (Porto
et al 2009) Moreover leads on in planta alkaloid functions
were also topic of experimental evaluation
One of the key elements that needs to be addressed early
on during the process of developing new bioactive molecules
from plants is the capacity to generate catalytically active
biomass to support extraction and steady supply ere are a
number of ways through which these goals may be reached
including greenhouse rooting of cuttings (mini-cutting
1 Laboratoacuterio de Fisiologia Vegetal Departamento de Botacircnica Instituto de Biociecircncias e Centro de Biotecnologia Universidade Federal do Rio
Grande do Sul 91501-970 Porto Alegre RS Brazil
Corresponding author fettnetocbiotufrgsbr
Review
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Biomass yield of resin in adult Pinus elliottii Engelm trees is differentially
regulated by environmental factors and biochemical effectors
Franciele Antocircnia Neis Fernanda de Costa Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima
Kelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-Neto
Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) CP 15005 CEP 91501-970 Porto Alegre RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Biomass
Terpene resin
Seasonal
Benzoic acid
Regenerated forest
A B S T R A C T
Biomass of pine resin finds several applications in the chemical pharmaceutical biofuel and food industries
Resin exudation after injury is a key defense response in Pinaceae since this complex mixture of terpenes has
insecticidal antimicrobial and wound repair properties Resin yield is increased by effectors applied on the
wound area including phytohormones and metal cofactors of terpene synthases The interaction of resinosis
mechanism effectors is not fully understood particularly in adult forest setups under natural environmental
variations The aim of this work was to determine how resin exudation by wounded trunks of adult P elliottii
responded to combined chemical effectors involved in different regulatory pathways of resinosis (metal cofactors
of terpene synthases benzoic acid and plant growth regulators) and whether seasonal and tree distribution
variations affected these responses Symmetrically planted and scattered trees regenerated from the seed bank
had similar resin biomass yields suggesting that the homogeneity in development and spatial arrangement were
not significant factors in resin yield This new finding is of practical importance with the used tapping system
since costs of implanting forests by regeneration can be advantageous compared to planting In addition it was
shown for the first time that the salicylic acid precursor benzoic acid and the auxin naphthalene acetic acid
promoted resin exudation when individually applied to wound sites Both these adjuvants are two orders of
magnitude less costly compared to the conventionally used ethylene precursors besides facing less environ-
mental and health restrictions for use Most adjuvant-treated trees showed higher resin flow in the second year
indicating mechanisms of response build up Overall temperature was more important than rainfall as en-
vironmental parameter affecting resin biosynthesis which was higher in the warmer months of spring and
summer The combination of resinosis stimulant effectors from different signaling pathways showed no sig-
nificant synergistic or additive effect suggesting possible converging signaling pathways andor limitation of
common intermediate transducing molecules
1 Introduction
Pines occupy highly diverse environments over a range of tem-
peratures water and nutrient availabilities irradiance levels and pho-
toperiods being able to effectively face attacks from diverse herbivore
and pathogen guilds The success of conifers is linked to their complex
terpene biochemistry hosted by specialized secretory cells The terpe-
noid resin synthesized by Pinus spp is one of the main mechanisms of
defense of these trees particularly against bark beetles and the fungi
they carry (Fett-Neto and Rodrigues-Correcirca 2012) Pine resin biomass
is essentially composed of a monoterpene and sesquiterpene-rich tur-
pentine and diterpenoid-rich rosin fraction both finding numerous in-
dustrial applications as non-wood forest products (Rodrigues-Correcirca
et al 2012)
Molecules capable of modulating different signaling pathways have
been identified as resin yield stimulators including sulfuric acid (ex-
tends wound damage) 2-chloroethylphosphonic acid (CEPA a syn-
thetic ethylene precursor) paraquat (free radical generator) yeast ex-
tract (mimics attack by pathogens) salicylic acid (pathogen signaling
molecule) auxin (promotes ethylene biosynthesis and resin canal dif-
ferentiation) jasmonic acid (signals mechanical damage and promotes
secondary metabolism) and metal ions such as potassium iron and
manganese (cofactors of terpene synthases in conifers) and copper (a
component of ethylene receptors) (Clements 1970 Conrath et al
2002 Fett-Neto and Rodrigues-Correcirca 2012 Hudgins and Franceschi
2004 Lewinsohn et al 1994 Martin et al 2002 Popp et al 1995
httpsdoiorg101016jindcrop201803027
Received 12 December 2017 Received in revised form 9 March 2018 Accepted 13 March 2018
Corresponding author
E-mail addresses franci_neisyahoocombr (FA Neis) fernandadecostayahoocombr (F de Costa) thanisenfyahoocombr (TN Fuumlller)
jjuliocesarlimagmailcom (JC de Lima) krodriguescbiotufrgsbr (KC da Silva Rodrigues-Correcirca) jpfettcbiotufrgsbr (JP Fett) fettnetocbiotufrgsbr (AG Fett-Neto)
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Research Paper
Dual allelopathic effects of subtropical slash pine (Pinus elliottii Engelm)
needles Leads for using a large biomass reservoir
Kelly Cristine da Silva Rodrigues-Correcircaa Gelson Halmenschlagera Joseacuteli Schwambachb
Fernanda de Costaa Emili Mezzomo-Trevizana Arthur Germano Fett-Netoa
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 Brazilb University of Caxias do Sul Institute of Biotechnology Caxias do Sul RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Seasonality
Growth
Germination
Litter
Substrate
A B S T R A C T
Pinus elliottii Engelm (slash pine) is distributed along the maritime coast of Southern Brazil where it shows
invasive pattern and typical allelopathic features Large quantities of needle litter are produced by pine trees a
biomass that is little explored in areas where this species is alien Little is known about the dynamics of needle
and litter phytochemical interactions particularly in subtropical environments To elucidate the full range of
needle and litter allelopathic potential the effects of litter (superficial and deep) and seasonally harvested fresh
slash pine needles stored for different times were evaluated against lettuce tomato and cucumber seeds and
seedlings Increasing concentrations (0 1 2 4 and 8 wv) of hot and cold aqueous extracts of needles
and litter affected in different ways target plant development Growth and germination inhibition were directly
related to the highest extract concentrations (regardless of the season and mainly in hot water extracts) of
needles On the other hand stimulatory effects of litter extracts on lettuce growth were observed Growth and
germination of cucumber and tomato were not affected by pine litter as substrate when compared to rice husk
The presumable high polarity and thermal stability of slash pine leaf biomass allelochemicals and their transient
toxic effect or growth promoting impact suggest potential applications of this largely available biomass both as a
biological herbicide and growth substrate in plant propagation
1 Introduction
Native from the Northern Hemisphere Pinus is one of the most
widely distributed genera throughout different climate regions of the
globe growing either as native or alien species even in extreme habi-
tats (Rodrigues-Correcirca and Fett-Neto 2012) Despite the high economic
value currently attributed to pine wood and oleoresin (Rodrigues-
Correcirca et al 2012) there is increasing concern about the aggressive
potential of invasiveness displayed by Pinus species especially those
cultivated out of their native range of distribution (Richardson et al
2008 Rolon et al 2011) These species are dispersed by wind and there
is notably low plant diversity observed in most understories of pine
plantations (Kato-Noguchi et al 2009) This latter feature has been
considered an important trait of allelopathic interference
The term ldquoallelopathyrdquo was coined by Molisch in 1937 as a chemical
reciprocal interaction established among plants (including micro-
organisms) sharing the same site by means of the release of secondary
metabolites named allelochemicals (Rice 1984) For the most part
these metabolites are derived from the shikimic acid or isoprenoid
pathway and their biosynthesis can be modulated by biotic and abiotic
stresses (Nascimento and Fett-Neto 2010) including seasonal-related
changes (Sartor et al 2013) Allelopathy studies may range from sterile
assays (Aryakia et al 2015) to soil (Correcirca et al 2008 Sharma et al
2016) and field tests being a complex biological phenomenon to as-
certain in several circumstances due to issues of solubility release
mechanisms and stability of bioactive compounds (Scognamiglio et al
2013) Often the use of complementary methods provides more in-
formative data
The allelopathic effects of soil leachates green needles and litter
extracts of Pinus spp on germination and seedling growth aspects of
wild and crop species have been evaluated in natural and cultivated
pine stands and have proven to be stimulatory or inhibitory (Lodhi and
Killingbeck 1982 Kil and Yim 1983 Nektarios et al 2005 Akkaya
et al 2006 Machado 2007 Alrababah et al 2009 Sartor et al 2009
Kato-Noguchi et al 2011 Rolon et al 2011 Valera-Burgos et al
2012) exhibiting in some cases autotoxicity (Garnett et al 2004
Fernandez et al 2008 Zhu et al 2009 Monnier et al 2011) Studies
on potential dual allelopathic effects of Pinus elliottii Engelm (slash
httpdxdoiorg101016jindcrop201706019
Received 23 March 2017 Received in revised form 15 May 2017 Accepted 7 June 2017
Corresponding author
E-mail address fettnetocbiotufrgsbr (AG Fett-Neto)
ORIGINAL RESEARCHpublished 16 June 2016
doi 103389fpls201600849
Frontiers in Plant Science | wwwfrontiersinorg 1 June 2016 | Volume 7 | Article 849
Edited by
Juan Francisco Jimenez Bremont
Instituto Potosino de Investigacioacuten
Cientiacutefica y Tecnoloacutegica Mexico
Reviewed by
Mariacutea De La Luz Guerrero Gonzaacutelez
Universidad Autoacutenoma de San Luis
Potosiacute Mexico
Rosalia Cristina Paz
CIGEOBIO (CONICETFCEFN UNSJ)
Argentina
Correspondence
Arthur G Fett-Neto
fettnetocbiotufrgsbr
daggerThese authors have contributed
equally to this work
Specialty section
This article was submitted to
Plant Physiology
a section of the journal
Frontiers in Plant Science
Received 08 December 2015
Accepted 30 May 2016
Published 16 June 2016
Citation
de Lima JC de Costa F Fuumlller TN
Rodrigues-Correcirca KCdS Kerber MR
Lima MS Fett JP and Fett-Neto AG
(2016) Reference Genes for qPCR
Analysis in Resin-Tapped Adult Slash
Pine As a Tool to Address the
Molecular Basis of Commercial
Resinosis Front Plant Sci 7849
doi 103389fpls201600849
Reference Genes for qPCR Analysisin Resin-Tapped Adult Slash Pine Asa Tool to Address the MolecularBasis of Commercial Resinosis
Juacutelio C de Lima 1dagger Fernanda de Costa 1 dagger Thanise N Fuumlller 1
Kelly C da Silva Rodrigues-Correcirca 2 Magnus R Kerber 1 Mariano S Lima 1
Janette P Fett 1 and Arthur G Fett-Neto 1
1 Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul
Porto Alegre Brazil 2 Biological Sciences Department Regional Integrated University of Alto Uruguai and Missotildees (URI-FW)
Frederico Westphalen Brazil
Pine oleoresin is a major source of terpenes consisting of turpentine (mono- and
sesquiterpenes) and rosin (diterpenes) fractions Higher oleoresin yields are of economic
interest since oleoresin derivatives make up a valuable source of materials for chemical
industries Oleoresin can be extracted from living trees often by the bark streak method
in which bark removal is done periodically followed by application of stimulant paste
containing sulfuric acid and other chemicals on the freshly wounded exposed surface
To better understand the molecular basis of chemically-stimulated and wound induced
oleoresin production we evaluated the stability of 11 putative reference genes for the
purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis
Samples for RNA extraction were collected from field-grown adult trees under tapping
operations using stimulant pastes with different compositions and at various time points
after paste application Statistical methods established by geNorm NormFinder and
BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3
and UBI To confirm expression stability of the candidate reference genes expression
profiles of putative P elliottii orthologs of resin biosynthesis-related genes encoding Pinus
contorta β-pinene synthase [PcTPS-(minus)β-pin1] P contorta levopimaradieneabietadiene
synthase (PcLAS1) Pinus taeda α-pinene synthase [PtTPS-(+)αpin] and P taeda
α-farnesene synthase (PtαFS) were examined following stimulant paste application
Increased oleoresin yields observed in stimulated treatments using phytohormone-based
pastes were consistent with higher expression of pinene synthases Overall the
expression of all genes examined matched the expected profiles of oleoresin-related
transcript changes reported for previously examined conifers
Keywords resin Pinus gene expression normalizer genes terpene synthase
19
Chapter 2
Stimulant Paste Preparation and Bark Streak Tapping Technique for Pine Oleoresin Extraction
Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
Tapping technique comprises the extraction of pine oleoresin a non-wood forest product consisting of a
complex mixture of mono sesqui and diterpenes biosynthesized and exuded as a defense response to
wounding Oleoresin is used to produce gum rosin turpentine and their multiple derivatives Oleoresin
yield and quality are objects of interest in pine tree biotechnology both in terms of environmental and
genetic control Monitoring these parameters in individual trees grown in the fi eld provides a means to
examine the control of terpene production in resin canals as well as the identifi cation of genetic-based
differences in resinosis A typical method of tapping involves the removal of bark and application of a
chemical stimulant on the wounded area Here we describe the methods for preparing the resin-stimulant
paste with different adjuvants as well as the bark streaking process in adult pine trees
Key words Oleoresin Pine Tapping Chemical stimulant Wounding
1 Introduction
Several conifer species produce oleoresin a complex mixture of isoprenoid compounds relevant for defense against herbivores and pathogens Two major fractions can be recognized in oleoresin (a) turpentine the volatile fraction containing mono- and sesquiter-penes and (b) rosin the nonvolatile diterpene fraction Oleoresin is a forest commodity of global interest fi nding applications in diverse industry sectors Rosin is used in adhesives printing ink manufacture and paper sizing Turpentine can be used either as a solvent for paints and varnishes or as a raw material for fraction-ation of high-value chemicals used in the pharmaceutical agro-chemical and food industry [ 1 ndash 3 ]
During the extraction activity resin is obtained from the tree in a similar way as rubber tree tapping which generally involves the
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_2 copy Springer Science+Business Media New York 2016
These authors have equally contributed to this work
fettnetocbiotufrgsbr
27
Chapter 3
A Modifi ed Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines
Juacutelio Ceacutesar de Lima Thanise Nogueira Fuumlller Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes
through high-throughput sequencing technologies microarray and also northern blots RT-PCR and
RTqPCR Even though many specifi c protocols designed for plants with high content of secondary metab-
olites have been developed these are often expensive time consuming and not suitable for a wide range
of tissues Here we present a modifi cation of the method previously described using the commercially
available Concerttrade Plant RNA Reagent (Invitrogen) buffer for fi eld-grown adult pine trees with high
oleoresin content
Key words RNA Pines Concert plant RNA reagent Stem RNA extraction Oleoresin Conifers
1 Introduction
Several conifer species especially within the Pinaceae have tissues with high concentrations of phenolics terpenes and polysaccha-rides [ 1 ] Many specifi c protocols that are appropriate for plants rich in secondary metabolite s have been developed but the extrac-tion of high-quality RNA from these tissues using commercial kits is often diffi cult and usually not applicable to woody tissues [ 2 ndash 6 ] One of the major issues during RNA extraction concerns the pres-ence of phenolic compounds which oxidize and form quinones Aromatic compounds bind RNA which interferes in downstream steps and applications [ 3 7 ] Another point of concern is the har-vest of plant samples in the experimental fi eld which constitutes another obstacle in the efforts to avoid degradation of RNA [ 8 ] These problems often result in RNAs of low quality and insuffi -cient amounts especially for methodologies that normally require
These authors have equally contributed to this work
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_3 copy Springer Science+Business Media New York 2016
fettnetocbiotufrgsbr
RESEARCH PAPER
Control of resin production in Araucaria angustifolia an ancientSouth American coniferJ C Perotti1 K C da Silva Rodrigues-Correa123 amp A G Fett-Neto12
1 Plant Physiology Laboratory Department of Botany Federal University of Rio Grande do Sul (UFRGS) Porto Alegre RS Brazil
2 Center for Biotechnology UFRGS Porto Alegre RS Brazil
3 Present address Regional Integrated University of Alto Uruguai and Miss~oes (URI-FW) Frederico Westphalen RS Brazil
Keywords
Araucaria ethylene jasmonic acid nitric
oxide resin salicylic acid terpenes
Correspondence
A G Fett-Neto Plant Physiology Laboratory
Center for Biotechnology Federal University
of Rio Grande do Sul (UFRGS) PO Box 15005
Av Bento Goncalves 9500 91501-970 Porto
Alegre Brazil
E-mail fettnetocbiotufrgsbr
Editor
K Leiss
Received 22 July 2014 Accepted 11
December 2014
doi101111plb12298
ABSTRACT
Araucaria angustifolia is an ancient slow-growing conifer that characterises parts ofthe Southern Atlantic Forest biome currently listed as a critically endangered speciesThe species also produces bark resin although the factors controlling its resinosis arelargely unknown To better understand this defence-related process we examined theresin exudation response of A angustifolia upon treatment with well-known chemicalstimulators used in fast-growing conifers producing both bark and wood resin suchas Pinus elliottii The initial hypothesis was that A angustifolia would display signifi-cant differences in the regulation of resinosis The effect of Ethrel (ET ndash ethylene pre-cursor) salicylic acid (SA) jasmonic acid (JA) sulphuric acid (SuA) and sodiumnitroprusside (SNP ndash nitric oxide donor) on resin yield and composition in youngplants of A angustifolia was examined In at least one of the concentrations testedand frequently in more than one an aqueous glycerol solution applied on fresh woundsites of the stem with one or more of the adjuvants examined promoted an increase inresin yield as well as monoterpene concentration (a-pinene b-pinene camphene andlimonene) Higher yields and longer exudation periods were observed with JA and ETanother feature shared with Pinus resinosis The results suggest that resinosis controlis similar in Araucaria and Pinus In addition A angustifolia resin may be a relevantsource of valuable terpene chemicals whose production may be increased by usingstimulating pastes containing the identified adjuvants
INTRODUCTION
Many conifer species produce terpenoid-based resins that havelong been studied for their industrial importance and role indefence against attack by herbivores and pathogens The twomost important resin-producing families of conifers are Pina-ceae and Araucariaceae (Langenheim 1996) The viscous resinsecretion is generally composed of a complex mixture ofterpenoids consisting of roughly equal parts of volatile mono-(C10) and sesquiterpene (C15 turpentine) fractions and non-volatile diterpenic (C20 rosin) components (Rodrigues-Correaet al 2013) Terpenes act in a complex and multilayereddefence response providing toxicity against bark beetles andfungi bark wound sealing disruption of insect developmentand attraction of herbivore predators (Phillips amp Croteau1999)Most conifers rely on some combination of preformed and
inducible resin defences (Trapp amp Croteau 2001 Zulak amp Bohl-mann 2010) Resin defences are controlled by environmentaland genetic factors to various extents depending on species(Roberds et al 2003 Sampedro et al 2010 Moreira et al2013) Resin traits have been reported as highly variable havingmoderate heritability indicating that breeding efforts towardssuper-resinous forests are promising (Tadasse et al 2001Roberds et al 2003) Several chemicals are known as stimulantsof resin production Commercial extraction of resin from pine
trees uses periodic bark streaking and application of resin stim-ulant pastes to the wound
Resin-stimulant paste based on sulphuric acid (SuA) iswidely used for the commercial production of pine resin Cur-rent stimulant pastes usually have two chemically active com-ponents SuA to magnify the wounding and an ethyleneprecursor (2-chloroethylphosphonic acid CEPA or Ethrel ndash
ET) to stimulate resin flow (Rodrigues et al 2011 Rodrigues-Correa amp Fett-Neto 2013) Jasmonic acid (JA) and its methylester methyl jasmonate (MeJa) are among the most widelyused chemical elicitors of plant secondary metabolism It hasbeen shown that the exogenous application of MeJa or herbi-vore attack induce chemical and anatomical defence responsesin conifers such as the formation of traumatic resin ducts andresin accumulation in stems along with increased biosynthesisof terpenes and phenolics (Franceschi et al 2002 Martin et al2002 Heijari et al 2005 Zeneli et al 2006 Moreira et al 2008Gould et al 2009) JA commercial use however is limited byits high cost
The effects of exogenous salicylic acid (SA) on conifer ter-pene production have also been studied In Pinus elliottiiapplication of 10 molm3 of SA induced resin productionin wound panels but in Pseudotsuga menziesii and Sequoia-dendron giganteum it had no apparent effect on resinaccumulation (Hudgins amp Franceschi 2004 Rodrigues ampFett-Neto 2009) Nitric oxide (NO) has also emerged as an
Plant Biology 17 (2015) 852ndash859 copy 2014 German Botanical Society and The Royal Botanical Society of the Netherlands852
Plant Biology ISSN 1435-8603
vii
LISTA DE ABREVIATURAS
24-D 24-dichlorophenoxyacetic acid
3H4P 3-hydroxy-4-pyridone (34-DHP 34-dihydroxypyridine)
ABA abscisic acid
Arg arginine
BABA β-aminobutyric acid
β-ODAP β-N-oxalyl-L-α β-diaminopropionic acid
BIA β-isoxazolinon-L-alanine
CAN canavanine
DAO diamine oxidase
DDC decarboxylase
ETH ethephon
FW fresh weight
GABA -aminobutyric acid
GABA-T GABA transaminase
GAD glutamate decarboxylase
GSM Global System for Mobile
HPLC High performance liquid chromatography
JA jasmonate
JA-Ile jasmonoyl-L-isoleucine
L-DOPA L-34- dihydroxyphenylalanine
MeJA methyl jasmonate
m-Tyr Meta-tyrosine
NO nitric oxide
NPAA non-protein amino acid
OAS o-acetylserine
OAS-TL o-acetylserine-thiol-lyase
PA polyamine
PAA protein amino acid
viii
PEG polyethylene glycol
PLP pyridoxal-5rsquo-phosphate
PPO polyphenol oxidase tyrosinase
qRT-PCR Reverse transcription polymerase chain reaction quantitative real time
RNS reactive nitrogen species
ROS reactive oxygen species
SA salicylic acid
SAR systemic acquired resistance
SNP sodium nitroprusside
UV ultraviolet radiation
ix
RESUMO
Ao longo de sua evoluccedilatildeo as plantas desenvolveram estrateacutegias estruturais e quiacutemicas de
defesa em resposta aos estresses bioacuteticos e abioacuteticos impostos pelo ambiente Dentre
essas satildeo reconhecidas moleacuteculas quimicamente especializadas denominadas
metaboacutelitos secundaacuterios produtos naturais ou metaboacutelitos especializados Aminoaacutecidos
natildeo proteicos (ANPs) satildeo compostos nitrogenados que constituem aleacutem de componentes
do arsenal de defesa quiacutemica vegetal uma importante fonte de reserva de carbono e
nitrogecircnio para diversos taxa especialmente aqueles pertencentes agrave famiacutelia Fabaceae de
Angiospermas Esse grupo de moleacuteculas quimicamente heterogecircneo eacute assim definido por
natildeo participar da formaccedilatildeo de estruturas proteicas funcionais sendo frequentemente
toacutexicos quando erroneamente incorporados nas cadeias polipeptiacutedicas em formaccedilatildeo em
funccedilatildeo da similaridade estrutural que apresentam com os aminoaacutecidos proteicos Sob o
ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados ANPs satildeo
em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou abioacutetica como
o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de elicitores
quiacutemicos por exemplo O objetivo da presente tese foi investigar o papel bioloacutegico da
mimosina endoacutegena em Leucaena leucocephala (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute) a partir da avaliaccedilatildeo do efeito de tratamentos
relacionados ao estresse abioacutetico (UV-C aacutecido saliciacutelico metil jasmonato e etileno)
Mimosina eacute um ANP aromaacutetico anaacutelogo da L-tirosina com atividade toacutexica para ceacutelulas
de procariotos e eucariotos Dentre as atividades descritas para esse ANP destacam-se a
atividade anti-mitoacutetica ou bloqueadora do ciclo celular atividade alelopaacutetica e
antioxidante Os resultados indicaram que em leucena a biossiacutentese e o acuacutemulo de
mimosina podem ser modulados por fatores causadores de estresses exibindo um padratildeo
de acumulaccedilatildeo similar ao das fitoalexinas Em maricaacute por outro lado a induccedilatildeo do
acuacutemulo dessa moleacutecula natildeo foi observada para os mesmos tratamentos testados para
leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas Aleacutem disso
o padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado
pela sua degradaccedilatildeo As respostas observadas nos testes que avaliaram a atividade de
mitigaccedilatildeo de espeacutecies reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode
agir como um agente antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de
estresse
1
Introduccedilatildeo
Na condiccedilatildeo de organismos seacutesseis ao longo de sua evoluccedilatildeo as plantas
desenvolveram estrateacutegias estruturais e quiacutemicas de defesa em resposta aos estresses bioacuteticos
e abioacuteticos impostos pelo ambiente Dentre essas satildeo reconhecidas moleacuteculas quimicamente
especializadas denominadas metaboacutelitos secundaacuterios produtos naturais (Kutchan et al 2015)
ou mais recentemente metaboacutelitos especializados
Entre as trecircs classes mais gerais de metaboacutelitos secundaacuterios (terpenos compostos
fenoacutelicos e compostos nitrogenados) aminoaacutecidos natildeo-proteicos (ANPs) satildeo incluiacutedos no
terceiro grupo e constituem aleacutem de componentes do arsenal de defesa quiacutemica uma
importante fonte de reserva de carbono e nitrogecircnio para diversos taxa especialmente aqueles
pertencentes agrave famiacutelia Fabaceae de Angiospermas (leguminosas sensu lato)
Aleacutem dos 20 aminoaacutecidos proteicos estima-se que existam entre 600 e 1000 ANPs
(Acamovic amp Brooker 2005 Rodgers et al 2015) Esse grupo de moleacuteculas quimicamente
heterogecircneo eacute assim definido por natildeo participar da formaccedilatildeo de estruturas proteicas
funcionais sendo frequentemente toacutexicos quando erroneamente incorporados nas cadeias
polipeptiacutedicas em formaccedilatildeo em funccedilatildeo da similaridade estrutural que apresentam com os
aminoaacutecidos proteicos (Taiz amp Zeiger 2010)
Conforme mencionado a ocorrecircncia de ANPs eacute comum em espeacutecies de leguminosas
e sua distribuiccedilatildeo pode ser restrita a alguns gecircneros de plantas circunscritos nessa famiacutelia
botacircnica (eg mimosina e canavanina) Por outro lado alguns ANPs como GABA por
exemplo podem apresentar distribuiccedilatildeo ubiacutequa no Reino Plantae assim como ocorrer em
outros tipos de organismos como animais por exemplo (Ramos-Ruiz et al 2018)
2
Apesar de representarem uma fonte nutricional importante sem tratamento preacutevio o
consumo de plantas que acumulam ANPs por animais eacute limitado Isso ocorre pois em longo
prazo a ingestatildeo prolongada de plantas (especialmente sementes) que acumulam ANPs pode
representar risco agrave sauacutede uma vez que estes comprometem o funcionamento de mecanismos
basais de manutenccedilatildeo da homeostase celular e podem tambeacutem em um quadro mais severo
desencadear doenccedilas neurotoacutexicas degenerativas como por exemplo o latirismo causado
por aacutecido β-N-oxalil-l-αβ-diaminopropiocircnico (β-ODAP) (Jiao et al 2011 Kusama-Eguchi
2019)
Sob o ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados
ANPs satildeo em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou
abioacutetica como o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de
elicitores quiacutemicos por exemplo No que concerne ao estudo dos efeitos da induccedilatildeo abioacutetica
sobre o acuacutemulo de ANPs em diferentes espeacutecies vegetais (Monocotiledocircneas e
Eudicotiledocircneas) as moleacuteculas mais amplamente investigadas ateacute o momento satildeo GABA
L-DOPA e mais recentemente mimosina (vide Tabela 1 do capiacutetulo primeiro) Em termos
de efeitos causados a partir da aplicaccedilatildeo exoacutegena de ANPs GABA tambeacutem figura como o
principal aminoaacutecido investigado seguido de L-DOPA e canavanina (vide Tabela 2 do
capiacutetulo primeiro)
No primeiro capiacutetulo da presente tese estatildeo descritas as caracteriacutesticas gerais dos
principais ANPs estudados seus possiacuteveis papeacuteis bioloacutegicos in planta e seus efeitos quando
aplicados exogenamente bem como os estresses abioacuteticos capazes de induzir seu(s)
acuacutemulo(s) nos diferentes tecidos vegetais Nos segundo e terceiro capiacutetulos
respectivamente satildeo elucidados os efeitos dos tratamentos de UV-C aacutecido saliciacutelico etileno
e jasmonato (claacutessicos elicitores do metabolismo secundaacuterio vegetal) sobre o acuacutemulo de
3
mimosina em Leucaena leucocephala var glabrata (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute)
Mimosina eacute um aminoaacutecido aromaacutetico natildeo-proteico anaacutelogo da L-tirosina com
atividade toacutexica para ceacutelulas de procariotos e eucariotos Embora em menor concentraccedilatildeo
mimosina foi primeiramente identificada em Mimosa pudica sendo posteriormente detectada
em outras espeacutecies do gecircnero como Mimosa pigra por exemplo (Soedarjo amp Borthakur
1998) Seu efeito toacutexico eacute atribuiacutedo agrave capacidade de quelar metais o que impede o
funcionamento adequado das metalo-proteiacutenas que dependem dos mesmos como co-fatores
(Negi et al 2014)
A concentraccedilatildeo basal de mimosina em espeacutecies de leucaena pode variar entre 1 e 12
do peso seco do oacutergatildeo (Soedarjo amp Borthakur 1998) Como eacute comum para outros ANPs
que ocorrem em espeacutecies de leguminosas em sementes de Leucaena spp eacute observada uma
maior concentraccedilatildeo de mimosina quando comparada aos demais oacutergatildeos da planta
(Rodrigues-Correcirca et al 2019) sendo esta a fonte de extraccedilatildeo comercial do padratildeo quiacutemico
de mimosina vendido por empresas de reagentes de pesquisa
Diversas atividades foram descritas para mimosina em outros organismos eou tipos
celulares Dentre essas destacam-se a atividade anti-mitoacutetica ou bloqueadora do ciclo
celular em ceacutelulas de eucariotos e procariotos Isto ocorre porque a mimosina impede a
formaccedilatildeo da forquilha de replicaccedilatildeo (e portanto a siacutentese de DNA) interrompendo assim o
avanccedilo do ciclo de divisatildeo celular na fase tardia G1 (Lalande 1990) Foram tambeacutem descritas
para mimosina atividade alelopaacutetica observada sobre o desenvolvimento de outras espeacutecies
de leguminosas e atividade antioxidante entre outras (Tabela 1)
A rota de biossiacutentese de mimosina eacute compartilhada em grande parte com a de cisteiacutena
um aminoaacutecido proteico sulfurado (Figura 1) A siacutentese da cisteiacutena se daacute a partir da conversatildeo
4
de serina e acetil-CoA em o-acetilserina pela enzima SAT (serina acetiltransferase) seguida
da conversatildeo de o-acetilserina e aacutecido sulfiacutedrico em cisteiacutena em uma reaccedilatildeo catalisada pela
OAS-TL (o-acetilserina tiol-liase) A siacutentese de mimosina por sua vez eacute compartilhada com
a da cisteiacutena ateacute esse ponto e acredita-se que pelo menos uma das isoformas de OAS-TL
catalise a conversatildeo de o-acetilserina e 3-hidroxi-4-piridona em mimosina
Tabela 1 Atividades descritas para mimosina de Leucaena leucocephala (Lam) de Wit
ATIVIDADE
ALVO AVALIADO
(organismo eou tecido tipo
celular)
REFEREcircNCIA
Bloqueio do complexo de ativaccedilatildeo
da preacute-replicaccedilatildeo do DNA
Ceacutelulas de mamiacuteferos
KUBOTA et al
(2014)
Alteraccedilatildeo no ciclo ovariano e
extensatildeo da duraccedilatildeo do corpo luacuteteo
bovino no periacuteodo poacutes-parto
Bovinos
(Bos taurus x
Bos indicus)
BOTTINI-
LUZARDO et al
(2015)
Supressatildeo do ciclo celular e reduccedilatildeo
da abundacircncia bacteriana em
mosquitos
Wolbachia pipientis
Aedes albopictus
FALLON
(2015)
Accedilatildeo inibitoacuteria da fibrose
pulmonar induzida
Ratos SD
LI et al
(2015)
Recuperaccedilatildeo da funccedilatildeo do
miocaacuterdio poacutes-isquemia
Miocaacuterdio de ratos (SD)
machos
CROWE et al
(2001)
Inseticida
Heteropsylla cubana
Crawford 1914 e Thrips tabaci
Lindemann 1889
AHMED et al
(2016)
Alelopaacutetica
Albizia procera Vigna
unguiculata Cicer arietinum
Cajanus cajan
AHMED et al
(2008)
Antioxidante
Sistemas modelo de oxidaccedilatildeo
lipiacutedica (β-caroteno - aacutecido
linolecircico e lecitina)
BENJAKUL et al
(2013)
Ateacute momento versotildees divergentes sobre a enzima responsaacutevel pela biossiacutentese de
mimosina (mimosina sintase) tecircm sido publicadas Em 1990 Ikegami e colaboradores
5
identificaram uma OAS-TL responsaacutevel pela formaccedilatildeo de cisteiacutena como sendo tambeacutem uma
mimosina sintase Mais tarde Yafuso et al (2014) realizaram a expressatildeo heteroacuteloga do gene
que codifica para OAS-TL em Escherichia coli e natildeo foi observada a formaccedilatildeo de mimosina
mesmo quando dadas as condiccedilotildees oacutetimas para tanto Mais recentemente Harun-Ur-Rashid
et al (2018) elucidaram a mimosina sintase como sendo uma isoforma da OAS-TL
corroborando o postulado por Ikegami e colaboradores em 1990
Figura 1 Rota de biossiacutentese da mimosina Fonte Ikegami et al (1990)
Espeacutecies estudadas
Leucaena leucocephala (Lam) de Wit (leucaena koa haole ou ldquoacaacutecia exoacuteticardquo na
liacutengua Hawairsquoiana) eacute uma espeacutecie de haacutebito arboacutereo ou arbustivo pertencente agrave famiacutelia
Fabaceae de Angiospermas e caracterizada pelo acuacutemulo de mimosina em todos os seus
oacutergatildeos Eacute nativa da Ameacuterica Central (especificamente da regiatildeo sudeste do Meacutexico) mas
irradiou-se atraveacutes de praticamente todas as zonas tropicais e subtropicais da Terra No
Brasil leucena eacute amplamente distribuiacuteda e classificada como naturalizada pelo REFLORA
(2019) ocorrendo em todo territoacuterio Nacional Satildeo reconhecidas no miacutenimo duas
6
subespeacutecies de leucena ocorrentes no Brasil L leucocephala var leucocephala e L
leucocephala var glabrata sendo a primeira a mais abundante
Leucaena apresenta atributos morfoloacutegicos caracteriacutesticos das leguminosas como o
fruto do tipo vagem deiscente no periacuteodo poacutes-maturaccedilatildeo folhas compostas e bipinadas As
flores satildeo seacutesseis actinomorfas e polistecircmones apresentam caacutelice sinseacutepala e corola
gamopeacutetala e satildeo dispostas em inflorescecircncias do tipo glomeacuterulo (Figura 2)
Figura 2 Oacutergatildeos vegetativos e reprodutivos de L leucocephala (Lam) de Wit Fonte Little Jr amp Skolmen
(1989)
Com base no conhecimento etnobotacircnico disponiacutevel acerca dessa espeacutecie em
diversas regiotildees tropicais e subtropicais leucena eacute utilizada para vaacuterios fins Extratos de
diferentes oacutergatildeos de leucena apresentam atividade anti-diabeacutetica (Kuppusamy et al 2014
Chowtivannakul et al 2016) antioxidante (Mohammed et al 2015 Chowtivannakul et al
2016 Zarin et al 2016) antimicrobiana (Zarin et al 2016) anti-helmiacutentica (Soares et al
2015 Jamous et al 2017) bactericida (Mohammed et al 2015) acaricida (Fernaacutendez-Salas
et al 2011) anti-tumoral (Chung et al 2017) e potencializadora da resposta imune em
peixes (Verma et al 2018) entre outras
7
Leucaena apresenta alta toleracircncia agrave seca sendo capaz de enfrentar estaccedilotildees sazonais
inteiras com deacuteficit hiacutedrico sem prejuiacutezo permanente de seus oacutergatildeos e de recuperar
vigorosamente sua biomassa vegetativa tatildeo logo o regime de precipitaccedilatildeo retome a
regularidade em frequecircncia Acredita-se que a toleracircncia agrave seca apresentada por essa espeacutecie
ocorra em funccedilatildeo do acuacutemulo de mimosina nos diferentes tecidos da planta a qual
funcionaria como um agente osmoregulador responsaacutevel pela preservaccedilatildeo da integridade das
membranas a das macromoleacuteculas intracelulares em periacuteodos de escassez de aacutegua no
ambiente
Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) eacute uma leguminosa
nativa natildeo endecircmica do Brasil amplamente distribuiacuteda nos domiacutenios fitogeograacuteficos da
Caatinga do Cerrado e da Mata Atlacircntica (Simon amp Proenccedila 2000 REFLORA 2019) Como
espeacutecie pioneira (Pilatti et al 2019) exerce importante papel ecoloacutegico na recuperaccedilatildeo de
aacutereas degradadas (Bitencourt et al 2007 Silva et al 2011) no estabelecimento de processos
de sucessatildeo vegetacional
Maricaacute eacute uma espeacutecie semi-deciacutedua a deciacutedua a qual atinge ateacute 15 m em altura (e
diacircmetro agrave altura do peito de ateacute 40 cm) na idade adulta com haacutebito arboacutereo ou arbustivo
(REFLORA 2019) e espinhos caracteriacutesticos desde os estaacutegios iniciais de desenvolvimento
(Carvalho 2004) Apresenta folhas compostas alternas e bipinadas (Figura 2) amplas
inflorescecircncias brancas com flores reunidas em glomeacuterulos esfeacutericos dispostos em grandes
paniacuteculas As flores satildeo diplostecircmones actinomorfas hipoacuteginas e unicarpelares (Silva et al
2011)
Assim como descrito para leucena maricaacute eacute considerado uma espeacutecie multifuncional
sendo comumente empregada para produccedilatildeo de mel como combustiacutevel (Olkoski amp
8
Wittmann 2011) em edificaccedilotildees na carpintaria e como lsquocerca-vivarsquo (Marchiori 1993
Lorenzi 1998) entre outras aplicaccedilotildees
Figura 2 Folhas e fruto de Mimosa bimucronata (DC) Kuntze Fonte Souza-Lima et al (2017)
Em contraste com a amplitude de habitats explorados por leucena (especialmente os
aacuteridos) no Sul do Brasil maricaacute ocorre preferencialmente em ambientes uacutemidos a alagadiccedilos
em aacutereas proacuteximas agraves margens de rios (Patreze amp Cordeiro 2004) embora possa tambeacutem
ocorrer em formaccedilotildees quase exclusivas dessa espeacutecie nas encostas de morros (Jacobi amp
Ferreira 1991)
Em relaccedilatildeo agraves atividades elucidadas para os extratos de maricaacute foram relatados os
efeitos alelopaacutetico (Jacobi amp Ferreira 1991 Ferreira et al 1992) diureacutetico natriureacutetico e
caliureacutetico (Schlickmann et al 2017)
9
Hipoacutetese
Mimosina apresenta perfil dinacircmico de acuacutemulo em Leucaena leucocephala e
Mimosa bimucronata frente a estresses associado a alteraccedilotildees significativas na expressatildeo de
genes relacionados ao metabolismo deste ANP o qual contribui para mitigar o desequiliacutebrio
oxidativo inerente a vaacuterios tipos de estresse
Objetivo geral
O objetivo da presente tese foi investigar o papel bioloacutegico da mimosina endoacutegena
em leucena e maricaacute a partir da avaliaccedilatildeo do efeito de tratamentos relacionados a estresses
ou sinalizadores de estresse
Objetivos especiacuteficos
- Analisar a concentraccedilatildeo constitutiva de mimosina nos diferentes oacutergatildeos de L leucocephala
(Lam) de Wit (leucena) e M bimucronata (DC) Kuntze (maricaacute)
- Verificar se apesar do seu alto teor constitutivo em plantas de leucena o acuacutemulo de
mimosina pode ser induzido com tratamentos que mimetizam diferentes estresses a partir da
avaliaccedilatildeo do efeito de moleacuteculas sinalizadoras (aacutecido saliciacutelico jasmonato etileno) e da
exposiccedilatildeo agrave radiaccedilatildeo UV-C na modulaccedilatildeo do acuacutemulo de mimosina em leucena bem como
em maricaacute
- Determinar se a expressatildeo de genes relacionados ao metabolismo de mimosina estaacute
associada agrave induccedilatildeo por estresses fisioloacutegicos
- Avaliar o potencial antioxidante da mimosina em experimentos realizados in situ
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry
journal homepage wwwelseviercomlocateplaphy
Research article
Mimosine accumulation in Leucaena leucocephala in response to stresssignaling molecules and acute UV exposure
Kelly Cristine da Silva Rodrigues-Correcircaab Michael DH Hondab Dulal BorthakurbArthur Germano Fett-Netoalowast
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970Porto Alegre Rio Grande do Sul BrazilbDepartment of Molecular Biosciences and Bioengineering University of Hawaii at Manoa Honolulu HI 96822 USA
A R T I C L E I N F O
KeywordsLeucaena leucocephalaMimosineMimosine amidohydrolaseJasmonic acidEthyleneSalicylic acidUV-C radiation
A B S T R A C T
Mimosine is a non-protein amino acid of Fabaceae such as Leucaena spp and Mimosa spp Several relevantbiological activities have been described for this molecule including cell cycle blocker anticancer antifungalantimicrobial herbivore deterrent and allelopathic activities raising increased economic interest in its pro-duction In addition information on mimosine dynamics in planta remains limited In order to address this topicand propose strategies to increase mimosine production aiming at economic uses the effects of several stress-related elicitors of secondary metabolism and UV acute exposure were examined on mimosine accumulation ingrowth room-cultivated seedlings of Leucaena leucocephala spp glabrata Mimosine concentration was not sig-nificantly affected by 10 ppm salicylic acid (SA) treatment but increased in roots and shoots of seedlings treatedwith 84 ppm jasmonic acid (JA) and 10 ppm Ethephon (an ethylene-releasing compound) and in shoots treatedwith UV-C radiation Quantification of mimosine amidohydrolase (mimosinase) gene expression showed thatethephon yielded variable effect over time whereas JA and UV-C did not show significant impact Consideringthe strong induction of mimosine accumulation by acute UV-C exposure additional in situ ROS localization aswell as in vitro antioxidant assays were performed suggesting that akin to several secondary metabolitesmimosine may be involved in general oxidative stress modulation acting as a hydrogen peroxide and superoxideanion quencher
1 Introduction
Different plant groups synthesize a large diversity of secondary orspecialized metabolites These molecules are generally produced inresponse to biotic and abiotic environmental stresses Indeed inductionof secondary metabolism usually involves stress-generating factorswhich have also been explored in biotechnological processes aiming atthe production of target metabolites of economic interest (Matsuuraet al 2018) Metabolic control of nitrogen-containing secondarycompounds (eg alkaloids and non-protein amino acids) has beenshown to be complex and influenced by phytohormones environmentalstresses (seasonality herbivory pathogen attack drought) UV radia-tion (Holloacutesy 2002) methyl jasmonate (MeJA) salicylic acid (SA)yeast extract (Cho et al 2008) abscisic acid (ABA) heavy metals os-motic stress (Nascimento et al 2013) and mechanical wounding (Portoet al 2014)
Due to their particular trait of associating with N-fixing micro-organisms Fabaceae species (leguminous sensu lato) are often proteinrich hence the relevance of several of these species as forage Fabaceaespecies are also known for accumulating nitrogen containing secondarymetabolites which play important roles as ecochemical molecules andat least for the case of non-protein amino acids potential cell reservoirsof nitrogen (Huang et al 2011)
High contents of mimosine a toxic aromatic non-protein aminoacid are found in species of two leguminous genera Leucaena spp andMimosa spp Leucaena leucocephala (Lam) de Wit (leucaena koa haole)is a fast-growing leguminous tree native from Central America (south-eastern Mexico) widely distributed in tropical and subtropical zonesThis species is also characterized by its high tolerance to droughtamong other environmental stresses (Honda et al 2018) Leucaena canbe divided into two subspecies (i) L leucocephala subsp leucocephala(common leucaena a bushy shrub) and (ii) L leucocephala subsp
httpsdoiorg101016jplaphy201811018Received 1 August 2018 Received in revised form 9 November 2018 Accepted 14 November 2018
lowast Corresponding authorE-mail addresses krodriguescbiotufrgsbr (KCdS Rodrigues-Correcirca) mhonda2hawaiiedu (MDH Honda) dulalhawaiiedu (D Borthakur)
fettnetocbiotufrgsbr (AG Fett-Neto)
Plant Physiology and Biochemistry 135 (2019) 432ndash440
Available online 19 November 20180981-9428 copy 2018 Elsevier Masson SAS All rights reserved
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glabrata (giant leucaena a tree) The latter has been used as a fastgrowing tree for production of wood and paper pulp The foliage ofboth common and giant leucaena is used as a fodder because of its highprotein content and palatability to farm animals The foliage containsup to 18 protein 142 crude fiber and 64 ether extractcrude fat(Soedarjo and Borthakur 1996)
Production of nitrogen-containing secondary metabolites such asmimosine requires large amounts of carbon and nitrogen resourcesNegi et al (2014) estimated that up to 21 of the carbon-nitrogenresources may be used for production of mimosine in leucaenaBrewbaker et al (1972) determined the mimosine content of 96 Lleucocephala cultivars and 8 other Leucaena species collected from 38different countries by growing them in an observational nursery inHawaii and found that basal mimosine content varied from 189 to477 of the dry weight
Mimosine is biosynthesized from OAS (o-acetylserine) and 3H4P (3-hydroxy-4-pyridone or its tautoisomer 3-hydroxy-4-pyridine) A pre-vious analysis suggested that mimosine synthase is an OAS-TL (o-acetylserine-thiol-lyase) of the cysteine biosynthesis pathway (Ikegamiet al 1990) Later however recombinant enzyme tests did not supportan OAS-TL identity of mimosine synthase (Yafuso et al 2014) Recentfindings on mimosine biosynthesis revealed that a cytosolic cysteine-OAS-TL isoform can also catalyze the formation of mimosine underspecific conditions (Harun-Ur-Rashid et al 2018)
Mimosine toxicity is related to its ability of reducing the availabilityof divalent metal ions such as Fe(II) Zn(II) Cu(II) Co(II) and Mn(II)by chelating co-factors and preventing their association with metal-dependent enzymes Furthermore this non-protein amino acid is cap-able of forming a stable complex with pyridoxal-5prime-phosphate (PLP)leading to the inactivation of PLP-dependent enzymes (eg Asp-Glutransaminase and cystathionine synthetase) (Negi et al 2014)
Mimosine features several useful biological activities such as alle-lopathic antimicrobial insecticide cell cycle inhibitor agent antic-ancer phytoremediator (Nguyen and Tawata 2016) as well as anti-oxidant (Benjakul et al 2013) Despite the relatively well establishedbiological activities of purified mimosine on other organisms or celltypes little is known about its biological role in leguminous speciesHowever it has been suggested that at least in part its activity ismainly related to defense mechanisms against some biotic and abioticstresses and as nitrogen source during fast growth (Vestena et al2001)
Suda (1960) and Smith and Fowden (1966) identified enzymes in-volved in mimosine degradation in seedling extracts of L leucocephalaand Mimosa pudica A mimosine-degrading enzyme named mimosinase(mimosine amidohydrolase EC 35161 CAS registry number 104118-49-2) (IUBMB 2018) a carbon-nitrogen lyase which degrades mimo-sine into 3H4P was later purified by Tangendjaja et al (1986) Itsbiochemical characterization was described and the cDNA was isolatedby Negi et al (2014)
Although mimosinase has been described and isolated only fewstudies on the role played by biotic and abiotic factors on the dynamicmodulation of mimosine metabolism in leguminous species have beenconducted (Vestena et al 2001 Xu et al 2018) In aseptic cultures ofleucaena mechanical injury of shoots promoted local mimosine accu-mulation (Vestena et al 2001) In the same study cultivation in pre-sence of auxin or SA in culture medium also had a positive effect on
mimosine accumulation More recently the effect of drought treatmenton gene expression of leucaena was also evaluated by Honda et al(2018) However several potential factors regulating mimosine meta-bolism need to be further examined
To date there is a lack of information on the biological role ofmimosine in planta as well as on details of its metabolic dynamicsMoreover its overt potential for pharmaceutical applications and de-velopment of new drugs as well as the possible use as tool to addressheavy metal soil contamination or plant mineral nutrition improve-ment justify additional research The objective of this study was toinvestigate the effect of stress signaling molecules and acute UV ex-posure on modulation of mimosine accumulation and metabolism in Lleucocephala spp glabrata in order to better understand its biologicalrole and to identify strategies for yield improvement aiming at ex-ploring its useful bioactivities
2 Methods
21 Plant material
For the experiments carried out to evaluate the effects of elicitors onmimosine accumulation seeds of leucaena were kindly provided by DrJames Brewbaker and harvested at CTAHRs (College of TropicalAgriculture and Human Resources of the University of Hawaii atManoa) Waimanalo Research Station at Oahu Hawaii This plantmaterial was originated from the accession K636 of Leucaena leucoce-phala ssp glabrata (Brewbaker 2008)
22 Induced mimosine content in 5-week-old giant leucaena
221 Seed germinationIn order to overcome seed coat dormancy seeds were submitted to a
chemical scarification with sulfuric acid 95ndash98 for 20min and re-peatedly rinsed in distilled water to remove any residual trace of thisreagent Then seeds were distributed in 254 cmtimes508 cm plastictrays containing 11 vv of vermiculite and commercial soil watereduntil reaching substrate field capacity Three weeks after seed imbibi-tion seedlings displaying similar size and shape (eg number of com-pound leaves and leaflets) were transplanted to individual pots(250mL) in number of three plants per container
During the experimental period (except in the UV-C radiationtreatment) all tested seedlings were kept in a growth chamber andsubmitted to controlled conditions of temperature (circa 25 degC) and ir-radiance (approximately 100 μmol photons mminus2sdot s minus1) with a photo-period of 16 h light and 8 h dark
222 Treatments2221 JA Ethephon and SA Five-week-old giant leucaena seedlingswere treated with different solutions as described in Table 1 Idealconcentrations were defined in preliminary experiments under the sameconditions indicated above At the beginning of the experiments 30plants were sprayed with 84 ppm JA 10 ppm SA 10 or 100 ppmEthephon or Milli-Qreg water (control) until the point of imminent runoffPlant pots were kept closed inside transparent plastic bags for 24 h toavoid solution volatilization Fifteen plants arranged in 5 sets of 3 (5biological replicates) were harvested 48 h and 96 h after being treated
Table 1Treatments used to modulate mimosine biosynthesis in giant leucaena
ELICITOR CONCENTRATION UV FLUENCE EXPOSURE TIME RATIONALE FOR USE
Salicylic acid (SA) 10 ppm 24 h Pathogen signaling molecule (Shah 2003)Jasmonic acid (JA) 84 ppm 24 h Chemical elicitor of plant secondary metabolism (Dar et al 2015)Ethephon 10 ppm 24 h Ethylene releasing-compound (Kim et al 2016) elicitor of plant secondary metabolism (Wang
et al 2016)UV-C radiation 3 Jcmminus2 10min or 15min Elicitor of plant secondary metabolism (Kara 2013 Neelamegam and Sutha 2015)
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433
After collection shoots were separated from roots immediately frozenin liquid nitrogen and stored at ndash 80 degC prior to HPLC analyses
2222 UV-C Thirty seedlings of giant leucaena were exposed to UV-Cradiation (3 Jcmminus2) for 10 or 15min and kept in a growth chamberunder controlled conditions as described above until the end of theexperiments Fifteen plants arranged in groups of 3 were harvested at96 h and 120 h after UV-C exposure and processed as previouslydescribed
223 Mimosine extractionMimosine extraction was based on a modified version of the pro-
tocol published by Lalitha and Kulothungan (2006) as follows a knownweight of fresh tissue (shoots or roots) of giant leucaena was first addedto Milli-Qreg boiling water in a proportion of 110 (g of plant per mL ofsolvent) in test tubes Tubes were covered with foil to avoid solutionevaporation and placed on a hot stirrer at 100 degC for 10min A pro-portional volume of 01M HCl was added to the cooled suspensions andhomogenized using mortar and pestle The plant extracts were filteredthrough cotton and centrifuged twice for 7min in a bench top re-frigerated centrifuge at 4 degC and 13200 rpm Before being analyzed theextracts were diluted 13 with ondashphosphoric acid (OPA)
224 Mimosine detectionHPLC analyses were carried out as described by Negi and Borthakur
(2016) Pure mimosine (L-mimosine from koa haole seeds Sigma-Al-drich CAS number 500-44-7) was used as standard Separation andquantification of mimosine was done with a C18 column (PhenomenexC18 5 μm 46times250mm) under an isocratic solvent system of 002MOPA with a linear flow rate of 1mLsdotminminus1 Mimosine detection wasdone at 280 nm by photodiode array detection (200ndash400 nm) showingretention time of 277 plusmn 0042min Quantification was done using themethod of external standard curve Further confirmation of mimosineidentity was performed by co-chromatography with standard and peakpurity check Chromatograms were analyzed using the Waters Em-power 3 software
23 Quantitative real-time PCR analysis of mimosinase gene expression
Fifteen 8-week-old giant leucaena plants arranged in 4 sets of 3 (4biological replicates) were treated with either water (control) or10 ppm Ethephon 84 ppm JA acid or 15min of UV-C radiation ex-posure following the methods described above Following treatmentleucaena plants were harvested at 48 and 96 h or 72 and 144 h (UV-Ctreated plants only) after treatments Total RNA of samples was ex-tracted and purified from roots and shoots of giant leucaena by meansof a modified method using Qiagen RNeasy Plant Kit (Valencia CAUSA) and Fruit-mate (Takara Japan) according to the protocol de-scribed by Ishihara et al (2016a) The assessment of RNA quality andquantity was carried out at 230 260 and 280 nm by using a NanoDropSpectrophotometer ND-1000 (NanoDrop Technologies DE USA) Inorder to avoid genomic DNA contamination RNA samples were treatedwith TURBO DNAfree Kit (Invitrogen Carlsbad CA) Two microgramsof DNase-treated RNA were used to synthesize the first-strand cDNAusing M-MLV Reverse Transcriptase (Promega WI USA)
Quantitative real-time (qPCR) analysis was carried out to examinepossible differential expression of the mimosinase gene (GenBank ac-cession number AB2985971) in seedlings treated with 84 ppm JA10mM Ethephon or 15min of UV-C exposure Shoots and roots wereharvested 24 h before the time of mimosine concentration peak for eachtreatment previously observed as assessed by HPLC assays The 10 μLqPCR reaction consisted of 5 μL of PowerUpTM SYBRreg Green MasterMix (Applied Biosystems Foster City CA) 1 μL MgCl2 (50mM) 03 μLforward primer (10 μM) 03 μL reverse primer (10 μM) and 1 μL cDNAfirst-strand In the experimental validation through qPCR reactionconditions and melting curve analysis of the amplicon were performed
following the protocol published by Ishihara et al (2016b) for the sameleucaena variety qPCR analysis was conducted using StepOnetrade Real-Time PCR System (Applied Biosystems) Measurements were performedusing 4 biological and 3 technical replicates Relative expression wascalculated with the 2-ΔΔct method using OAS-TL as reference gene sinceits expression showed a consistently stable profile comparable to that ofUBQ-5 and ELF1α expressions Mimosinase primer sequences used forthese analyses were (FWD) 5prime- GAA AGG CAG GAA TCA CAG TGA AGAG ndash 3rsquo (REV) 5prime GGA GAC TCT AGC CAC ACC AAC TTA ndash 3rsquo
24 Antioxidant assays
241 Mimosine effect on hydrogen peroxide (H2O2) accumulationAs a follow up to the induction of mimosine accumulation profiles
under stress signals and conditions tests were conducted to verify mi-mosine antioxidant capacity In situ histological localization of hy-drogen peroxide (H2O2) accumulation was evaluated on foliar disks ofPhaseolus vulgaris L according to the protocol described by Shi et al(2010) Briefly the plant foliar tissue was exposed to 1 mgmiddotmLminus1 dia-minobenzidine (DAB) solution in 10 mM KH2PO4 (control) in presenceor absence of 10mM mimosine (equivalent to the average mimosineconcentration induced by UV-C radiation in giant leucaena) or 10mMascorbic acid (positive antioxidant control) Oxidative response wasidentified by the formation of a brown polymer on the injured leafareas indicating the presence of H2O2 and registered in a Leica M165FC stereomicroscope (Leica Microsystems)
242 Mimosine quenching of superoxide radicalsGeneration of superoxide radical and subsequent analysis was per-
formed by a modified protocol based on Zhishen et al (1999) Nitroblue tetrazolium (NBT) reduction was used to measure superoxide an-ions quenching activity Shortly a 50mM KH2PO4 pH 78 solutioncontaining 6 μM riboflavin 100mM methionine 1 mM NBT in pre-sence or absence of 5mM mimosine was exposed to white light(22 Jsdotcmminus2) for 25min on a white light transilluminator Five micro-molar rutin was used as positive control (Matsuura et al 2016) Theabsorbance was read at 560 nm before and after light exposure in aSpectraMaxreg M2 Microplate Reader (Molecular Devices LLC)
25 Statistical analyses
For HPLC and superoxide anions data simple analyses of variance(ANOVA) followed by Tukey or Welch ANOVA followed by Dunnetts Ctest were used as appropriate for data distribution characteristics InqPCR analysis results were analyzed by t-test In all cases at least fourbiological triplicates were used and experiments were repeated twiceindependently All data were analyzed using the statistical packageSPSS 200 for Windows (SPSS Inc USA) In all cases a ple 005 wasused
3 Results and discussion
31 Increased mimosine concentrations in giant leucaena treated withchemical elicitors
Leucaena produces high amounts of mimosine that accumulate in allparts of the plants including leaves stem flowers pods seeds rootsand root nodules (Soedarjo and Borthakur 1998) The highest con-centrations of mimosine can be found in the growing shoot tips andseeds (Wong and Devendra 1983) It is not known why leucaena pro-duces such high amounts of mimosine Negi et al (2014) estimated thatleucaena plants would be able to grow 21 larger if the nutrient re-sources spent on mimosine production were diverted for biomass in-crease In a previous analysis performed to quantify the basal con-centration of mimosine present in adult plants of common leucaena thehighest constitutive amount of mimosine per gram of fresh weight in
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the analyzed organs was found in post-anthesis flowers (89448 μg)followed by green pods (82687 μg) leaves (67358 μg) and greenflower buds (51247 μg) which showed significantly less mimosineconcentration compared to the other reproductive structures(Supplementary Fig 1) Since mature seeds have very low moisturecontent (Wencomo et al 2017) its mimosine concentration was esti-mated as 338253 μgsdotgminus1 of dry weight Additionally it was also ob-served that the basal mimosine distribution in shoots of field-grownadult plants of leucaena is dependent on the variety type(Supplementary Table 1)
Phytohormones such as salicylic acid and jasmonic acid are knownto be produced by plants in response to various abiotic and bioticstresses These phytohormones trigger adaptive responses to stress byregulating major plant metabolic processes such as photosynthesisnitrogen metabolism defense systems and plant-water relationsthereby providing protection (for review see Khan et al 2015)
Secondary or specialized metabolite production and accumulationare also known to be controlled by biotic and abiotic stresses (Matsuuraet al 2018) In this study exposure of 5-week-old giant leucaenaseedlings to JA or Ethephon treatments significantly enhanced mimo-sine accumulation in shoots and roots in at least one of the two timepoints tested (48 and 96 h) albeit in a different way (Fig 1) Thehighest concentrations of mimosine in shoots were found in seedlingstreated with JA 84 ppm (43441 μgsdotgminus1) and Ethephon 100 ppm(38412 μgsdotgminus1) two days after application of the respective phyto-hormones Nevertheless after four days shoots yielded the highestconcentration of mimosine (approximately 460 μgsdotgminus1) upon treatmentwith 10 or 100 ppm Ethephon (Fig 1A) In roots after two and four
days JA 84 ppm and Ethephon 10 ppm resulted in highest mimosineaccumulation 18488 μgsdotgminus1 and 15801 μgsdotgminus1 respectively (Fig 1B)These observations show that mimosine accumulation response tospecific elicitors may vary over time after exposure
Although all treatments were applied exclusively on shoots of giantleucaena seedlings roots of some of them were also able to respond tothe different elicitors Overall shoots displayed higher basal and in-duced mimosine concentration compared to roots (Fig 1) which agreeswith previous observations in 1 to 3-week-old aseptic seedlings ofcommon leucaena (Vestena et al 2001) However as previouslymentioned significant post-induction increase of mimosine concentra-tion in roots and shoots simultaneously was only observed for JA andEthephon 10 ppm on day 02 and 04 respectively (Fig 1)
It is well established that perceived regulatory signals or elicitorsgenerate a transduction network mediated by secondary messengersresulting in changes in gene expression profiles that afford adaptiveresponses to environmental stimuli These modulation events are oftenmediated by transcription factors (TFs) which directly bind to specificgene promoters or act by forming complexes with repressor proteinslabeling them to degradation subsequently releasing other TFs toproceed with the gene expression program This is the case of the actionmechanism of JA and its active form jasmonoyl isoleucine for example(Kazan 2015 Wasternack and Strnad 2016)
JA ethylene and SA are known as important stress regulatory sig-nals in plants JA however is thought to be the most effective signal forinduction of plant secondary metabolism (Wasternack and Strnad2016) thereby contributing to mitigation of damage caused by severalstresses (Dar et al 2015) JA is mainly derived from linolenic acid
Fig 1 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings treated with differentelicitors CTRL=Milli-Q water SA = Salicylic AcidJA= Jasmonic Acid ETH=Ethephon Bars sharing a letterof same case do not differ by Tukey test (P le 005) Capitalletters (A B) compare treatments on day two and lowercaseletters (a b) compare treatments on day four Indicatessignificant statistical difference between day two and dayfour in the same treatment by t-test (Ple 005) The errorbars represent standard error of five replicates (each meanwas calculated with 15 individual seedlings organized in 5groups of three)
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(Wasternack and Strnad 2016) playing important roles in differentprocesses of plant growth and development such as plant defensemechanisms against herbivory pathogen attack fungal elicitation andsome abiotic factors such as osmotic temperature and salt stresses (Daret al 2015)
JA and its methyl ester MeJA have several different effects on le-guminous species MeJA exogenous application has increased iso-flavonoid content in cell suspension cultures of Pueraria candollei varcandollei and P candollei var mirifica (Korsangruang et al 2010) aswell as the production of the triterpenoid glycyrrhizin in Glycyrrhizaglabra roots Enhanced production of the triterpenoid however waspartly at the expense of root growth (Shabani et al 2009) MeJA ap-plication on shoots was observed to suppress root nodulation and lat-eral root formation in Lotus japonicus (Nakagawa and Kawaguchi2006) In grapevine a non-leguminous species proteinogenic aminoacids did not show an expressive increase under MeJA treatment(Gutieacuterrez-Gamboa et al 2017)
The effects of the application of four different jasmonate forms (JAMeJA jasmonoyl-L-isoleucine (JA-Ile) and 6-ethyl indanoyl glycineconjugate (2-[(6-ethyl-1-oxo-indane-4-carbonyl)-amino]-acetic acidmethyl ester - CGM) on leucaena metabolite profile has recently beenreported by Xu et al (2018) JA-Ile form was most effective althoughno major alteration was observed on monitored metabolite abundancesAlanine threonine and 34-dihydroxypyridine (34 DHP a metabolitederived from mimosine degradation) (Nguyen and Tawata 2016)among others were the major metabolites elicited by JA-Ile In contrastto the results described here mimosine concentration did not changesignificantly These divergent results on mimosine accumulation maybe due to a number of factors including mode of application jasmonateform used (JA-Ile x JA) and L leucocephala subspecies (common x giantleucaena)
Ethylene is also a phytohormone involved in plant response me-chanisms to different types of challenges such as mechanical damageand insect attack among others The integration mechanism betweenJA and ethylene signaling pathways is not completely understoodhowever it has been shown that they may work cooperatively in abioticstress tolerance (Kazan 2015) MeJA can induce ethylene production(Zhao et al 2004) and when applied simultaneously these moleculesseem to work in a synergic way by enhancing the magnitude of theplant response to external stimuli (Liu et al 2016)
Treatment with SA was able to significantly increase mimosine ac-cumulation in 12-week-old plants of common leucaena (SupplementaryFig 2) However no significant effect of SA treatment on mimosineconcentration was seen in 5-week-old seedlings of giant leucaena(Fig 1) suggesting some degree of genotype andor age dependency inelicitation by this phytohormone On the other hand several treat-ments including 90 ppm MeJA 10 and 100 ppm 2-chloroethylpho-sphonic acid (CEPA an ethylene-releasing compound) significantlyincreased mimosine accumulation (Supplementary Fig 2) in agree-ment with the data obtained for giant leucaena The lack of systemiceffects of externally applied SA on mimosine accumulation was alsoobserved when the phytohormone was supplied in the culture mediumof aseptically-grown seedlings in which case only roots had highercontent of mimosine (Vestena et al 2001) This could be due totransport limitations or to low methyl salicylate production from ap-plied SA since the former is recognized as the main systemic signalingform (Vlot et al 2009)
32 Increased mimosine concentrations in giant leucaena exposed to UV-Cradiation
UV-C treatment promoted increased concentration of the aminoacid in shoots but not in roots of giant leucaena (Fig 2) Increasedaccumulation of mimosine in shoots was also observed in 12-week-oldseedlings of common leucaena exposed to UV-C radiation for 10 and15min (Supplementary Fig 3) Similar to the SA treatment in giant
leucaena UV-C radiation did not induce mimosine biosynthesis in rootsregardless of time after exposure The absence of mimosine induction inroots by SA and UV indicates that these effectors do not cause a sys-temic response Moreover roots are shielded from irradiance by thepresence of substrate
UV radiation effects on different aspects of plant metabolism anddevelopment have been described However compared to UV-B (en-vironmentally relevant type of UV radiation) assays there are less re-ports related to the UV-C effects on secondary metabolites biosynthesisand accumulation (Cetin 2014) especially in leguminous (Fabaceae)plants They generally concern primary metabolism aspects such asgrowth and development For instance seedlings of Phaseolus vulgaris L(Fabaceae) exposed to low intensity UV-C radiation have displayeddecreased chlorophyll content and reduced height after 14 days of ex-posure (Kara 2013) Negative effects on growth parameters and ni-trogen metabolism were also observed in Vigna radiata L (Fabaceae)after UV-B radiation treatment in addition to adverse effects on JA SAand antioxidant compounds accumulation (Choudhary and Agrawal2014a) The same authors reported increased accumulation of flavo-noids SA and JA besides negative effects on growth biomass yieldnitrogen fixation and accumulation in 2 cultivars of Pisum sativum L(Fabaceae) under elevated UV-B treatment (Choudhary and Agrawal2014b) Despite the negative UV influence on growth reported for thepreviously mentioned leguminous UV-C radiation on groundnut plants(Arachis hypogaea L Fabaceae) increased seedling vigor and biomassand had no adverse effect on germination or other development para-meters (Neelamegam and Sutha 2015)
Besides its impact on growth and primary metabolism UV exposurecan cause important changes in secondary metabolism depending onintensity and time of exposure (Matsuura et al 2013) UV-B and UV-Cpre-treatments of Artemisia annua (Asteraceae) seedlings yielded in-creased biosynthesis of artemisinin a drug which displays anti-malarialproperties and activity against some others infectious diseases (egschistosomiasis leishmaniasis and hepatitis B) and several kinds oftumors (Rai et al 2011) The accumulation of nicotine in Nicotianarustica plants (Solanaceae) was also increased by UV-C treatment(Tiburcio et al 1985) Similar inducing effects on production of severalsecondary metabolites were observed in callus cultures of Vitis viniferaL Oumlkuumlzgoumlzuuml (grapevine Vitaceae) treated with a UV-C source for 5 or10min (Cetin 2014)
Regarding amino acid biosynthesis in response to UV radiationMartiacutenez-Luumlscher et al (2014) have found that in spite of not causingchanges in total amino acid content UV-B radiation exposure can affecttheir profile in grape berries Proteinogenic amino acids have beenknown to be important targets of the deleterious effects of UV radiation(Holloacutesy 2002) On the other hand in the present study acute UV-Ctreatment was found to increase mimosine accumulation in shoots byover twofold (Fig 2) which may suggest a possible participation of thismolecule as part of the antioxidant defense system in L leucocephalaThis possibility is further supported by the induction of the amino acidaccumulation by JA and Ethephon involved in abiotic and biotic stressresponses which are generally associated with oxidative imbalance andare signaling components in high UV stress (Matsuura et al 2013)
33 Mimosinase gene expression
In order to determine if increases in mimosine content upon ex-posure to JA CEPA or UV-C radiation were related to changes intranscription of mimosine metabolism-related genes RT-qPCR analysiswas carried out The complete pathway for mimosine biosynthesis hasnot yet been determined although the final step has been character-ized Based on transcription analysis (Ishihara et al 2016a) leucaenaappears to encode for multiple cysteine synthases one or more of whichmay be able to catalyze mimosine synthesis In addition a leucaenagene encoding a mimosinase (an enzyme responsible for mimosinedegradation) has been identified and characterized (Negi et al 2014)
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In addition to mimosinase gene expression several gene isoformsbelonging to the cysteine pathway [cysteine synthase (CYS SYN) serineacetyltransferase (SAT) and β-cyanoalanine synthase (CAS) Table 2 -supplementary material] were also tested in this study (data notshown) However expressions of these genes did not vary in giantleucaena throughout the experiments suggesting that the increasedcontent of mimosine observed in the treated plants might not be relatedto the expression of these genes but presumably to increased enzymeactivity andor release from conjugates such as mimoside a mimosineβ-D-glucoside (Murakoshi et al 1972)
Considering the time variation of mimosine accumulation observedin this work mimosinase gene expression in shoots and roots wasevaluated 24 h before the increase of mimosine concentration in giantleucaena seedlings (ie 24 h and 72 h after the chemical elicitorstreatments and 48 h and 120 h after UV-C exposure)
Ethylene signaling has been shown to up-regulate expression ofseveral genes related to secondary metabolism pathways as is the caseof phenolic compounds (Liu et al 2016) and terpenoid indole alkaloids(Wang et al 2016) Among all elicitors tested in the present workEthephon was the only one able to significantly change mimosinasegene expression Leucaena plants treated with Ethephon showed sig-nificant increases in mimosine concentration at both day 2 and 4 fol-lowing treatment which coincided with low-level expression of mi-mosinase Up-regulation of mimosinase gene expression was detected24 h before the increase of mimosine concentration in shoots treatedwith 10 ppm of Ethephon (Fig 3A) but not after JA or UV-C treatments(Fig 3C-D and 3E-F respectively) Nevertheless 72 h after treatment
application (24 h before the highest mimosine content measured inshoots) down regulation of mimosinase gene was seen in both shootsand roots treated with 10 ppm of Ethephon (Fig 3B) These data in-dicate that mimosine content in leucaena plants is at least partlyregulated by mimosinase expression in Ethephon exposed plants Onthe other hand the fact that mimosinase mRNA was not significantlyaffected by JA and UV-C treatments despite their stimulating effects onmimosine biosynthesis in giant leucaena may indicate that other levelsof regulation are at play or that the chosen harvesting time window wasunable to detect relevant changes
34 In situ and in vitro antioxidant assays
Considering the stimulation of mimosine accumulation byEthephon JA and UV all of which are often associated or known tocause oxidative imbalance the antioxidant capacity of mimosine wasevaluated Mimosine has been shown to have antioxidant activities oncultured cancer cells (Parmar et al 2015) In the present study it washypothesized that mimosine could confer radical scavenging propertieswhich would contribute to plant protection from possible damagecaused by reactive oxygen species generated during stress(Supplementary Fig 4)
Foliar disks of P vulgaris L were treated with 10mM mimosine for15min Treated disks showed less hydrogen peroxide accumulationinduced by wounding in contrast to untreated ones being comparableto those treated with ascorbic acid (a known hydrogen peroxide neu-tralizer) (Fig 4A) These observations support a possible antioxidant
Fig 2 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings exposed to UV-C lightCTRL= visible light (100 μmol photons mminus2 s minus1) UV-C 10primeand UV-C 15rsquo=UV-C exposure time (10 and 15min re-spectively) Bars sharing a letter of same case do not differ byTukey test (P le 005) Capital letters (A B) compare treat-ments on day three and lowercase letters (a b) comparetreatments on day six Indicates significant statistical dif-ference between day three and day six in the same treatmentby t-test (Ple 005) The error bars represent standard errorof five replicates (each mean was calculated with 15 in-dividual seedlings organized in 5 groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
437
role of mimosine as an in situ hydrogen peroxide scavengerMimosine was also able to quench superoxide anions generated by
light exposure Mimosine exhibited equivalent antioxidant effect com-pared to rutin (Fig 4B) a well-established effective superoxide anionquencher (Matsuura et al 2016) The radical scavenging activity ofmimosine may be due to the 3-OH group of the pyridine ring of mi-mosine (Fig 5) The pKa of the 3-OH of mimosine has been estimated tobe 88 (M Honda unpublished results) At physiological pH this OHgroup is expected to remain in a protonated state and therefore mayscavenge a radical by donating a proton and an electron In this processmimosine itself is converted to a stable radical form which is perhapsless toxic and less reactive than the reactive oxygen species generatedduring oxidative stress It is likely that the less toxic radical mimosineproduced may react with another radical or molecule and becomeconverted to a non-reactive indole molecule
In vivo antioxidant activity of mimosine has been previously eval-uated by means of its exogenous application on selenium-deficientseedlings of Vigna radiata In spite of its allelopathic properties (Ahmedet al 2008) the results showed mitigation of mitochondrial oxidativestress by treatment with 01mM mimosine (Lalitha and Kulothungan2007) DPPH radical scavenging activity was also reported for aqueous
seed extracts of leucaena rich in mimosine and phenolic compounds inin vitro assays (Benjakul et al 2014) Mimosine antioxidant activityshown in the present work is in good agreement with data reported forother non-protein amino acids such as L-DOPA (Dhanani et al 2015)and GABA (Malekzadeh et al 2014) for instance
4 Conclusion
Taken together results show that mimosine biosynthesis and ac-cumulation can be modulated by stress-related factors despite its re-latively high constitutive content in leucaena plants The pattern ofgene expression in stressed plants suggests mimosine steady-state con-trol may be regulated by its degradation in possible connection withdynamic changes in carbon and nitrogen metabolism of stressed plantsMimosine quenching activity against hydrogen peroxide and super-oxide anions in the in situ staining and in vitro assays respectivelyshowed that this non-protein amino acid can act as non-enzymaticantioxidant agent Increase in mimosine content in response to elicitorsmimicking environmental challenges in addition to its antiherbivoreand antimicrobial properties may be related to its activity as protectivemolecule against oxidative damage in line with other classes of plant
Fig 3 Relative expression of the mimosinase gene in shoots (A E and F) and shoots and roots (B C and D) of giant leucaena 24 h (A and C) 48 h (E) 72 h (B and D)and 120 h (F) after treatment with stress signaling molecules or UV-C exposure ETH = Ethephon JA = Jasmonic Acid Indicates significant statistical differencebetween control and treatment by t-test (Ple 005) The error bars represent standard error of four replicates
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
438
secondary metabolites
Funding
This work was funded by the National Council for Scientific andTechnological Development (CNPq-Brazil) grant 3060792013-5Coordenaccedilatildeo de Aperfeiccediloamento de Pessoal de Niacutevel Superior - Brazil(CAPES) - Finance Code 001 and the USDA NIFA Hatch projectHA05029-H managed by CTAHR
CRediT authorship contribution statement
Kelly Cristine da Silva Rodrigues-Correcirca InvestigationValidation Writing ndash original draft Michael DH HondaInvestigation Validation Dulal Borthakur Supervision Writing ndashreview amp editing Funding acquisition Arthur Germano Fett-NetoSupervision Funding acquisition Writing ndash review amp editing
Acknowledgements
The authors would like to thank Dr Jorge Ernesto Mariath fromLaVeg-UFRGS for kindly lending the Leica M165 FC stereomicroscopefor in situ analysis
Appendix A Supplementary data
Supplementary data to this article can be found online at httpsdoiorg101016jplaphy201811018
References
Ahmed R Hoque ATMR Hossain MK 2008 Allelopathic effects of Leucaena
leucocephala leaf litter on some forest and agricultural crops grown in nursery J ForRes 19 298 httpsdoi 101007s11676-008-0053-0
Benjakul S Kittiphattanabawon P Shahidi F Maqsood S 2013 Antioxidant activityand inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipidoxidation in model systems Food Sci Technol Int 19 (4) 365ndash376 httpsdoiorg1011771082013212455186
Benjakul S Kittiphattanabawon P Sumpavapol P Maqsood S 2014 Antioxidantactivities of lead (Leucaena leucocephala) seed as affected by extraction solvent priordechlorophyllisation and drying methods extracts against lipid oxidation in modelsystems Food Sci Technol 51 (11) 3026ndash3037 httpsdoiorg101007s13197-012-0846-1
Brewbaker JL Pluckett D Gonzalez V 1972 Varietal variation and yield trials ofLeucaena leucocephala (koa haole) in Hawaii Hawaii Agric Exp Stn Bull 166 26
Brewbaker JL 2008 Registration of KX2 ndash Hawaii interspecific-hybrid leucaena JPlant Registrations 1 (3) 190ndash193 httpsdoiorg103198jpr2007050298crc
Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitisvinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-6287-47-37
Cho H-Y Son SY Rhee HS Yoon S-YH Lee-Parsons CWT Park JM 2008Synergistic effects of sequential treatment with methyl jasmonate salicylic acid andyeast extract on benzophenanthridine alkaloid accumulation and protein expressionin Eschscholtzia californica suspension cultures J Biotechnol 135 117ndash122 httpsdoiorg101016jjbiotec200802020
Choudhary KK Agrawal SB 2014a Cultivar specificity of tropical mung bean (Vignaradiata L) to elevated ultraviolet-B changes in antioxidative defense system ni-trogen metabolism and accumulation of jasmonic and salicylic acids Environ ExpBot 99 122ndash132 httpsdoiorg101016jenvexpbot201311006
Choudhary KK Agrawal SB 2014b Ultraviolet-B induced changes in morphologicalphysiological and biochemical parameters of two cultivars of pea (Pisum sativum L)Ecotoxicol Environ Saf 100 178ndash187 httpsdoiorg101016jecoenv201310032
Dar TA Uddin M Khan MMA Hakeem KR Jaleel H 2015 Jasmonates counterplant stress a Review Environ Exp Bot 115 49ndash57 httpsdoiorg101016jenvexpbot201502010
Dhanani T Singh R Shah S Kumari P Kumar S 2015 Comparison of green ex-traction methods with conventional extraction method for extract yield L-DOPAconcentration and antioxidant activity of Mucuna pruriens seed Green Chem LettRev 8 (2) 43ndash48 httpsdoiorg1010801751825320151075070
Gutieacuterrez-Gamboa G Portu J Santamariacutea P Loacutepez R Garde-Cerdaacuten T 2017Effects on grape amino acid concentration through foliar application of three dif-ferent elicitors Food Res Int 99 688ndash692 httpsdoiorg101016jfoodres201706022
Fig 4 A In situ antioxidant assay Foliar disksof Phaseolus vulgaris L treated with (a) No an-tioxidant added (negative control) (b) 10 mMMimosine (c) 10mM ascorbic acid (positivecontrol) The oxidative damage can be seen bythe formation of a brown polymer in leaf veinsand injured areas B In vitro superoxidescavenging assay carried out with mimosineDifferent letters indicate significant differenceby Tukey test (Ple 005) The error bars re-present standard error of four replicates (Forinterpretation of the references to colour in thisfigure legend the reader is referred to the Webversion of this article)
Fig 5 Predicted mimosine radical formed followingquenching of hydroxyl radical Mimosine is first converted toa stable mimosine radical which may be then converted to anontoxic indole form
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
439
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad HossainMd Anai T Oku H 2018 Cytosolic cysteine synthase switch cysteine and mi-mosine production in Leucaena leucocephala Appl Biochem Biotechnol 186 (3)613ndash632 httpsdoiorg101007s12010-018-2745-z
Holloacutesy F 2002 Effects of ultraviolet radiation on plant cells Micron 33 (2) 179ndash197Honda MDH Ishihara KL Pham DT Borthakur D 2018 Identification of drought-
induced genes in giant leucaena (Leucaena leucocephala subsp glabrata) Trees 32571ndash585 httpsdoiorg101007s00468-018-1657-4
Huang T Jander G de Vos M 2011 Non-protein amino acids in plant defense againstinsect herbivores representative cases and opportunities for further functional ana-lysis Phytochemistry 72 1531ndash1537 httpsdoiorg101016jphytochem201103019
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of thethyrotoxic amino acid mimosine by cysteine synthase Phytochemistry 29 (11)3461ndash3465 httpsdoiorg1010160031-9422(90)85258-H
Ishihara K Lee EKW Borthakur D 2016a An improved method for RNA extractionfrom woody legume species Acacia koa A Gray and Leucaena leucocephala (Lam) deWit Int J For Wood Sci 3 (1) 031ndash035
Ishihara KL Honda MDH Pham DT Borthakur D 2016b Transcriptome analysisof Leucaena leucocephala and identification of highly expressed genes in roots andshoots Transcriptomics 4 135 httpsdoiorg1041722329-89361000135
IUBMB 2018 Enzyme Nomenclature EC 35161 httpwwwsbcsqmulacukiubmbenzymeEC35161html Accessed date 8 February 2018
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant(Phaseolus vulgaris) Biosci Res 10 (1) 29ndash32
Kazan K 2015 Diverse roles of jasmonates and ethylene in abiotic stress toleranceTrends Plant Sci 20 (4) 219ndash229 httpsdoiorg101016jtplants201502001
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect ofEthephon as an ethylene-releasing compound on the metabolic profile of Chlorellavulgaris J Agric Food Chem 64 (23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541
Khan MIR Fatma M Per TS Anjum NA Khan NA 2015 Salicylic acid-inducedabiotic stress tolerance and underlying mechanisms in plants Front Plant Sci 6 462httpsdoiorg103389fpls201500462
Korsangruang S Soonthornchareonnon N Chintapakorn Y Saralamp PPrathanturarug S 2010 Effects of abiotic and biotic elicitors on growth and iso-flavonoid accumulation in Pueraria candollei var candollei and P candollei var mir-ifica cell suspension cultures Plant Cell Tissue Organ Cult 103 (3) 333ndash342 httpsdoiorg101007s11240-010-9785-6
Lalitha K Kulothungan SR 2006 Selective determination of mimosine and its dihy-droxypyridinyl derivative in plant systems Amino Acids 31 (3) 279ndash287 httpsdoiorg101007s00726-005-0226-5
Lalitha K Kulothungan SR 2007 Mimosine mitigates oxidative stress in seleniumdeficient seedlings of Vigna radiata - Part I restoration of mitochondrial functionBiol Trace Elem Res 118 (1) 84ndash96 httpsdoiorg101007s12011-007-0013-0
Liu J Li Y Wang Y Zhang Z-H Zu Y-G Efferth T Tang Z-H 2016 Thecombined effects of ethylene and MeJA on metabolic profiling of phenolic com-pounds in Catharanthus roseus revealed by metabolomics analysis Front Physiol 71ndash11 httpsdoiorg103389fphys201600217 Article 217
Malekzadeh P Khara J Heydari R 2014 Alleviating effects of exogenous Gamma-aminobutiric acid on tomato seedling under chilling stress Physiol Mol Biol Plants20 (1) 133ndash137 httpsdoiorg101007s12298-013-0203-5
Martiacutenez-Luumlscher J Torres N Hilbert G Richard T Saacutenchez-Diacuteaz M Delrot SAguirreolea J Pascual I Gomegraves E 2014 Ultraviolet-B radiation modifies thequantitative and qualitative profile of flavonoids and amino acids in grape berriesPhytochemistry 102 106ndash114 httpsdoiorg101016jphytochem201403014
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation ofbioactive secondary metabolites by ultraviolet radiation mechanisms strategies andapplications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology forMedicinal Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash1902012
Matsuura HN Fragoso V Paranhos JT Rau MR Fett-Neto AG 2016 Thebioactive monoterpene indole alkaloid N szlig-D-glucopyranosylvincosamide is regu-lated by irradiance quality and development in Psychotria leiocarpa Ind Crop Prod86 210ndash218 httpsdoiorg101016jindcrop201603050
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo RBhambra AS Strnad M Bonfill M Fett-Neto AG 2018 Specialized plant me-tabolism characteristics and impact on target molecule biotechnological productionMol Biotechnol 60 (2) 169ndash183 httpsdoiorg101007s12033-017-0056-1
Murakoshi S Ohmiya S Haginiwa J 1972 Enzymic synthesis of mimoside a meta-bolite of mimosine in Mimosa pudica and Leucaena leucocephala Chem Pharm Bull20 (4) 855ndash857
Nakagawa T Kawaguchi M 2006 Shoot-applied MeJA suppresses root nodulation inLotus japonicus Plant Cell Physiol 47 (1) 176ndash180 httpsdoiorg101093pcppci222
Nascimento NC Menguer PK Henriques AT Fett-Neto AG 2013 Accumulation ofbrachycerine an antioxidant glucosidic indole alkaloid is induced by abscisic acidheavy metal and osmotic stress in leaves of Psychotria brachyceras Plant PhysiolBiochem 73 33ndash40 httpsdoiorg101016jplaphy201308007
Neelamegam R Sutha T 2015 UV-C irradiation effect on seed germination seedling
growth and productivity of groundnut (Arachis hypogaea L) Int J Curr MicrobiolApp Sci 4 (8) 430ndash443
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase fromLeucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol164 (2) 922ndash934 httpsdoiorg101104pp113230870
Negi VS Borthakur D 2016 Heterologous expression and characterization of mimo-sinase from Leucaena leucocephala In Fett-Neto Arthur Germano (Ed)Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods inMolecular Biology vol 1405 copySpringer Science+Business Media New York httpsdoiorg101007978-1-4939-3393-8_7 2016
Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636
Parmar F Kushawaha N Highland H George L-B 2015 In vitro antioxidant andanticancer activity of Mimosa pudica Linn extract and L-mimosine on lymphomaDaudi cells Int J Pharm Sci 12 100ndash104
Porto DD Matsuura HN Vargas LRB Henriques AT Fett-Neto AG 2014 Shootaccumulation kinetics and effects on herbivores of the wound-induced antioxidantindole alkaloid brachycerine of Psychotria brachyceras Nat Prod Commun 9 (5)629ndash632
Rai R Meena RP Smita SS Shukla A Rai SK Pandey-Rai S 2011 UV-B and UV-C pre-treatments induce physiological changes and artemisinin biosynthesis inArtemisia annua L ndash an antimalarial plant J Photochem Photobiol B Biol 105 (3)216ndash225 httpsdoiorg101016jjphotobiol201109004
Shabani L Ehsanpour AA Asghari G Emami J 2009 Glycyrrhizin production by invitro cultured Glycyrrhiza glabra elicited by methyl jasmonate and salicylic acid RussJ Plant Physiol 56 (5) 621ndash626 httpsdoiorg101134S1021443709050069
Shah J 2003 The salicylic acid loop in plant defense Curr Opin Plant Biol 6 (4)365ndash371
Shi J Fu XZ Peng T Huang XS Fan QJ Liu JH 2010 Spermine pretreatmentconfers dehydration tolerance of citrus in vitro plants via modulation of antioxidativecapacity and stomatal response Tree Physiol 30 (7) 914ndash922 httpsdoiorg101093treephystpq030
Smith IK Fowden L 1966 A study of mimosine toxicity in plants J Exp Bot 17750ndash761 httpsdoiorg101093jxb174750
Soedarjo M Borthakur D 1996 Simple procedures to remove mimosine from youngleaves pods and seeds of Leucaena leucocephala used as food Int J Food SciTechnol 31 (1) 97ndash103
Soedarjo M Borthakur D 1998 Mimosine a toxin produced by the tree-legumeLeucaena provides a nodulation competition advantage to mimosine-degradingRhizobium strains Soil Biol Biochem 30 1605ndash1613
Suda S 1960 On the physiological properties of mimosine Bot Mag Tokyo 73 (862)142ndash147 httpsdoiorg1015281jplantres188773142
Tangendjaja B Lowry JB Wills RBH 1986 Isolation of a mimosine degrading en-zyme from leucaena leaf J Sci Food Agric 37 523ndash526 httpsdoiorg101002jsfa2740370603
Tiburcio F Pintildeol MT Serrano M 1985 Effect of UV-C on growth soluble protein andalkaloids in Nicotiana rustica plants Environ Exp Bot 25 (3) 203ndash210 httpsdoiorg1010160098-8472(85)90004-8
Vestena S Fett-Neto AG Duarte RC Ferreira A 2001 Regulation of mimosineaccumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 httpsdoiorg101016S0168-9452(01)00448-4
Vlot AC Dempsey DMA Klessig DF 2009 Salicylic acid a multifaceted hormone tocombat disease Annu Rev Phytopathol 47 177ndash206 httpsdoiorg101146annurevphyto050908135202 2009
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene-induced vinblastine accumulation is related to activated expression of downstreamTIA pathway genes in Catharanthus roseus BioMed Res Int 2016 Article ID 3708187httpsdoiorg10115520163708187
Wasternack C Strnad M 2016 Jasmonate signaling in plant stress responses and de-velopment ndash active and inactive compounds N Biotech 33 (5B) 604ndash613 httpsdoiorg101016jnbt201511001
Wencomo HB Ortiz R Caacuteceres J 2017 Afr J Agric Res 12 (4) 279ndash285 httpsdoiorg105897AJAR201510604 26
Wong CC Devendra C 1983 Research on leucaena forage production in Malaysia InLeucaena Research in the Asian Pacific Region pp 55ndash60 Ottawa Ontario Canada
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX TsimKWK 2018 Jasmonate-elicited stress induces metabolic change in the leaves ofLeucaena leucocephala Molecules 23 (2) httpsdoiorg103390molecules23020188 E188
Yafuso JT Negi VS Bingham J-P Borthakur D 2014 An O-acetylserine (thiol)lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthaseAppl Biochem Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z
Zhao J Zheng S-H Fujita K Sakai K 2004 Jasmonate and ethylene signalling andtheir interaction are integral parts of the elicitor signalling pathway leading to b-thujaplicin biosynthesis in Cupressus lusitanica cell cultures J Exp Bot 55 (399)1003ndash1012 httpsdoiorg101093jxberh127
Zhishen J Mengcheng T Jianming W 1999 The determination of flavonoid contentsin mulberry and their scavenging effects on superoxide radicals Food Chem 64 (4)555ndash559 httpsdoiorg101016S0308-8146(98)00102-2
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
440
61
Supplementary Fig 1 Basal mimosine concentration in adult trees of common leucaena (L leucocephala
var leucocephala) Samples were collected from 10 field grown trees at Manoa Valley Honolulu Hawairsquoi
on June 25th 2017 Bars sharing a letter do not differ by Tukey test (P le 005) The error bars represent the
standard error
Supplementary Fig 2 Bar diagram showing mimosine concentration in shoots of 12-week-old common
leucaena seedlings treated with different elicitors CTRL = Milli-Q water SA = Salicylic Acid MeJA =
Methyl Jasmonate CEPA = 2-Chloroethylphosphonic acid (an ethylene releasing compound) Bars sharing a
letter of same case do not differ by Tukey test (P le 005) Capital letters (A B) compare treatments on day
two and lower-case letters (a b) compare treatments on day four Indicates significant statistical difference
ABB
A A
0
200
400
600
800
1000
1200
LEAVES GREEN FLOWERBUDS
POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
con
cen
trat
ion
(micro
gg
-1o
f FW
)
B AB AB AB B A
b
a
ab b
ab
0
2
4
6
8
10
12
14
16
18
20
CTRL SA 10 ppm SA 100 ppm CEPA 10 ppm CEPA 100 ppm MeJA 90 ppm
Mim
osi
ne
co
nce
ntr
atio
n (
gg
-1o
f FW
)
DAY 02 DAY 04
62
between day two and day four in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
Supplementary Fig 3 Bar diagram showing the effects of UV-C radiation exposure for 5 10 and 15 min on
mimosine accumulation in shoots of 12-week-old seedlings of common leucaena Bars sharing a letter of
same case do not differ by Tukey test (P le 005) Capital letters (A B C) compare treatments on day three
and lower-case letters (a b) compare treatments on day six Indicates significant statistical difference
between day three and day six in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
C BC AB A
bb
a
a
0
10
20
30
40
50
60
CTRL UV-C 5 UV-C 10 UV-C 15
Mim
osi
ne
co
nce
ntr
atio
n (
gg-1
of
FW)
DAY 03 DAY 06
63
Supplementary Fig 4 Model depicting induction of mimosine synthesis in leucaena following application of
stress elicitors such as CEPA and jasmonic acid or exposure to UV-C radiation The additional mimosine
synthesized may serve to alleviate oxidative stress induced by UV-C radiation
64
Supplementary Table 1 Mimosine contents in leaves of common and giant leucaena
Leucaena
type
Mimosine content
( FW)
Mimosine
content ( DW)
Dry matter
content ( FW)
Water content
( FW)
Common (1) 050 plusmn 009 245 plusmn 051 2011 plusmn 054 7989 plusmn 054
Common (2) 043 plusmn 006 214 plusmn 037 1998 plusmn 050 8002 plusmn 050
K636 (1) 070 plusmn 014 356 plusmn 077 1908 plusmn 052 8092 plusmn 052
K636 (2) 042 005 205 plusmn 033 2008plusmn 093 7992plusmn 093
KX2 (1) 122 plusmn 011 608 plusmn 082 1939 plusmn 123 8061 plusmn 123
KX2 (2) 134 plusmn 010 623 plusmn 056 2029 plusmn 114 7971 plusmn 114
KX3 (1) 044 plusmn 006 221 plusmn 030 1945 plusmn 073 8055 plusmn 073
KX3 (2) 054 plusmn 005 273 plusmn 023 1930 plusmn 038 8070 plusmn 038
KX4 (1) 086 plusmn 011 471 plusmn 065 1753 plusmn 084 8247 plusmn 084
KX4 (2) 089 plusmn 011 476 plusmn 065 180 plusmn 072 820 plusmn 072
KX5 (1) 099 plusmn 012 489 plusmn 048 1907 plusmn060 8093 plusmn 060
KX5 (2) 115 plusmn 015 548 plusmn080 1992 plusmn 053 8008 plusmn 053
Common leucaena variety koa haole grows widely on the island of Orsquoahu K636 is widely
grown variety of giant leucaena KX2 KX3 KX4 and KX5 are giant leucaena varieties
developed through interspecies hybridization (Brewbaker 2016) (1) and (2) indicate plants
from two separate locations within the University of Hawaii Waimanalo Research Center The
values are shown as mean plusmn standard error obtained from at least three biological replicates
65
Supplementary Table 2 GenBank accession numbers of the tested cysteine pathway genes isoforms
Gene name GenBank accession
OAS-TL (o-acetylserine-thiol-lyase) GDRZ01032940
GDRZ01061620
GDRZ01153117
GDSA01187555
GDSA01196891
GDSA01214467
Cys syn (cysteine synthase) GDRZ01015860
GDRZ01050898
GDRZ01086813
GDRZ01193515
GDRZ01202579
GDSA01180863
GDSA01215622
SAT (serine acetyltransferase) GDRZ01187456
GDRZ01189631
CAS (β-cyanoalanine synthase) GDRZ01054066
GDRZ01175418
GDSA01118400
66
SHORT COMMUNICATION 1
Mimosine occurrence and accumulation in Mimosa bimucronata var bimucronata (DC) 2
Kuntze 3
Kelly Cristine da Silva Rodrigues-Correcirca1 Lana Dorneles Pedroso2 Fernanda de Costa1 4
Arthur Germano Fett-Neto1 5
1Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal 6
University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970 7
Porto Alegre Rio Grande do Sul Brazil 2Department of Biological Sciences Unipampa ndash 8
Campus Satildeo Gabriel 9
Corresponding author 10
E-mail addresses krodriguescbiotufrgsbr (KCdaS Rodrigues-Correcirca) 11
lanalima2012gmailcom (LD Pedroso) fernandadecostayahoocombr (F de Costa) 12
fettnetocbiotufrgsbr (AG Fett-Neto) 13
14
15
16
17
18
19
20
21
22
67
ABSTRACT 23
Mimosine is a non-protein aromatic amino acid present in plants of Leucaena spp 24
and Mimosa spp Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) is a native 25
tree from Brazil which occurs as a pioneer species on plant succession processes In the 26
current study the presence of mimosine in M bimucronata was verified by HPLC analyses 27
Moreover mimosine accumulation upon exposure to UV-C and chemical elicitors of 28
specialized metabolism (salicylic acid - SA methyl jasmonate - MeJA sodium nitroprusside 29
- SNP and ethephon - ETH) most of which also known as promoters of the amino acid 30
production in leucaena plants was evaluated The results showed a lower concentration of 31
constitutive mimosine present in both maricaacute seedlings and mature trees when compared to 32
leucaena plants In spite of a trend towards increased mimosine accumulation observed in 33
MeJA and ETH treatments no statistical differences were found with the various stressors 34
used to induce its biosynthesis in maricaacute seedlings Data suggest that mimosine in M 35
bimucronata is probably a phytoanticipin-like metabolite or its accumulation is driven by 36
other types of stresses 37
38
39
Keywords Mimosine Mimosa bimucronata stress 40
41
42
43
44
45
46
68
Introduction 47
Mimosa bimucronata commonly known as maricaacute is a native tree from Brazil 48
(REFLORA 2019) ecologically important in plant succession and in processes of degraded 49
land recovery (Bitencourt et al 2007 Silva et al 2011) occurring as a pioneer species 50
(Pilatti et al 2019) Maricaacute is a deciduous or semi-deciduous plant which reaches up to 15 51
m in height and 40 cm of diameter at breast height (DBH) displays shrub or tree habit and 52
bears typical sharp thorns (Carvalho 2004) This species belongs to Fabaceae one of the 53
most economically important families of flowering plants due to its high diversity and 54
occurrence in different types of habitats (Gomes et al 2018) As well as several others 55
Mimosa spp maricaacute is usually referred to as a multipurpose tree (Olkoski and Wittmann 56
2011) employed for alternative medicinal uses (Champanerkar et al 2010 Silva et al 57
2011) honey production constructions and remodeling of landscape architecture (living 58
fences) for instance (Marchiori 1993 Lorenzi 1998) 59
In southern Brazil maricaacute is widely distributed and typically found either in wetland 60
areas close to river banks (Patreze and Cordeiro 2004) or composing large and almost pure 61
landscape formations on hillsides (Jacobi and Ferreira 1991) In dense populations this 62
species like several Mimosa spp (Simon and Proenccedila 2000) is considered an important and 63
highly invasive weed by preventing cattle to reach pasturesand water bodies as a result of its 64
thorny branches (Lorenzi 2008 Kestring et al 2009) Its dominant and nearly exclusive 65
pattern of distribution in those areas has led Jacobi and Ferreira (1991) to test its allelopathic 66
potential on cultivated species Indeed extracts of leaves and ripe fruits (but not the green 67
ones) of maricaacute showed phytotoxic effects on germination and initial radical growth of most 68
of the target species tested 69
69
Several investigations have been performed on maricaacute floristics (Silva et al 2011) 70
distribution (Simon and Proenccedila 2000) wood anatomy (Marchiori 1993) cytogenetic 71
parameters (Olkoski and Wittmann 2011) and allelopathic potential (Jacobi and Ferreira 72
1991 Ferreira et al 1992) However excluding two recent publications on maricaacute 73
constitutive chemical composition (Schlickmann et al 2017 Pilatti et al 2019) which 74
identified phenolic compounds (methyl gallate and water-soluble tannins) as its major 75
compounds little is known regarding this subject In other Mimosa species (eg M pudica 76
and M pigra) mimosine has been identified (Soedarjo and Borthakur 1998) as one of the 77
major specialized metabolites present in the different organs of the plant (Champanerkar et 78
al 2010) The presence of this molecule was also reported for M bimucronata in a thin layer 79
chromatography-based preliminary study performed by Ferreira et al (1992) showing co-80
chromatography of a leaf extract component with authentic mimosine The authors attributed 81
the allelopathic effect of maricaacute to the accumulation of this metabolite in its leaves 82
Mimosine is an aromatic non-protein amino acid initially found in plants of Mimosa 83
pudica and later in Leucaena leucocephala (Lam) de Wit (Soedarjo and Borthakur 1998) a 84
leguminous tree which biosynthesizes large amounts of this nitrogen-containing compound 85
(Rodrigues-Correcirca et al 2019) It is believed that the accumulation of high contents of 86
mimosine in L leucocephala tissues confers among other traits defense against herbivores 87
and pathogens (Vestena et al 2001) tolerance to drought (Negi et al 2014) as well as 88
general oxidative stress protection (Rodrigues-Correcirca et al 2019) Interestingly drought is 89
the opposite environmental and physiological condition to that observed in the wet habitats 90
occupied by native populations of M bimucronata in Brazil (Patreze and Cordeiro 2004 91
Kestring et al 2009) and Mimosa pudica Linn in India (Champanerkar et al 2010) 92
70
Nonetheless flooding is also associated with oxidative stress particularly as water levels 93
change (Fukao et al 2019) 94
In Leucaena leucocephala var leucocephala (common leucaena) and Leucaena 95
leucocephala var glabrata (giant leucaena) mimosine accumulation has been shown to be 96
both constitutive and inducible by stress-related phytohormones such as jasmonic acid (JA) 97
Ethephon (ETH an ethylene- releasing compound) salicylic acid (SA - only common 98
leucaena) (Vestena et al 2001) as well as by UV-C radiation (Xu et al 2018 Rodrigues-99
Correcirca et al 2019) On the other hand there is a lack of information regarding mimosine 100
content and elicitation effects in Mimosa spp plants 101
The aim of this study was to examine the presence of mimosine in Mimosa 102
bimucronata and examine the effects of stresses and stress-signaling molecules on its 103
accumulation in leaves 104
Material and Methods 105
Plant material 106
For all experiments the plant material was collected at Morro Santana campus do 107
Vale of UFRGS (Federal University of Rio Grande do Sul) Porto Alegre RS Brazil 108
(3004rsquoS 5108rsquoW) Authorization for access to genetic material was obtained from 109
SISGEN-Brazil (license number A845493) Constitutive mimosine content in adult plants of 110
M bimucronata var bimucronata (DC) Kuntze was determined in plant material (leaves 111
green flower buds post-anthesis flowers and green pods) harvested in January 2017 112
(summer) A voucher herbarium specimen (ICN 187953) was deposited in the ICN ndash UFRGS 113
herbarium (Herbaacuterio do Instituto de Biociecircncias of UFRGS) 114
71
For mimosine elicitation experiments legumes (fruits) of maricaacute were collected in 115
the end of June 2017 (winter) Seeds were then removed from the dry fruits and kept in the 116
dark until sowing and seedling development for use in the assays 117
Seed germination 118
To break the coat-imposed seed dormancy after surface sterilization dry seeds of 119
maricaacute were acid scarified by immersion in H2SO4 (95 ndash 98 ) for 2 min (see Correcirca et al 120
2008) and repeatedly washed in distilled water to remove any residue of the acid Then seeds 121
were distributed in 50 mL individual plastic tubes (dibble-tubes) (30 cm diameter x 120 cm 122
depth) filled up with 11 (vv) of commercial top soil and vermiculite Tubes were watered 123
every 2 days to avoid substrate dryness and were kept in a growth room under controlled 124
conditions of light (circa 75 μmol mminus2s minus1 photosynthetically active radiation photoperiod 125
of 16 h light and 8 h dark) and temperature (24plusmn2C) 126
127
Treatments 128
In order to verify inducibility of mimosine accumulation in M bimucronata fifty 12-129
week-old maricaacute seedlings (per treatment) exhibiting similar features were selected and 130
sprayed (saturated) with solutions of different chemical stressors (plant specialized 131
metabolism elicitors) as follows (for further details see Rodrigues-Correcirca et al 2019) 10 132
and 50 mM SA (pathogen-signaling molecule Shah 2003) 007 and 035 mM 2-133
chloroethylphosphonic acid (ETH ethylene releasing-compound Kim et al 2016 Wang et 134
al 2016) 100 and 200 mM MeJA (Dar et al 2015) 10 and 50 mM SNP (a nitric oxide 135
donor Perotti et al 2015) Alternatively maricaacute seedlings were also supplemented with UV-136
C radiation (13 minutes 105 kJ cm2) (elicitor of plant specialized metabolism Kara 2013) 137
72
After 2 and 4 days of exposure to the chemical treatments and 3 and 6 days of UV-138
C supplementation maricaacute shoots were harvested immediately frozen in liquid nitrogen and 139
stored at ndash 80 C until mimosine extraction and HPLC analyses 140
Mimosine extraction and detection 141
Mimosine extraction was conducted according to the modified protocol described by 142
Rodrigues-Correcirca et al (2019) for L leucocephala HPLC (Thermo Scientific Surveyor) 143
analyses (mimosine detection and quantification) were performed following previously 144
published procedures (Negi et al 2014) A C18 column (ACE C18 5 μm 46times250 mm) and 145
isocratic solvent system of 002M o-phosphoric acid with a linear flow rate of 1 mL min minus1 146
were used to separate and quantify the amino acid Mimosine detection was performed at 280 147
nm by photodiode array detection (200ndash400 nm) and retention time (229plusmn0024 min) 148
Mimosine quantification was done by means of the method of external standard curve 149
Additional confirmation of mimosine identity was performed by co-chromatography with 150
standard (Acros Organics authentic mimosine 99 used as reference) and peak purity check 151
The analyses of the chromatograms were done with the ChromQuest software 152
153
154
Results and Discussion 155
Constitutive accumulation of mimosine in M bimucronata 156
Mimosine was detected in all analyzed samples positively meeting all identification 157
criteria In agreement with what has been found for other Mimosa spp (Soedarjo and 158
Borthakur 1998) compared to L leucocephala adult plants (Rodrigues-Correcirca 2019) 159
mimosine content was lower in M bimucronata Of the adult plant tissues analyzed the 160
73
highest content of mimosine in maricaacute (per gram of fresh weight - FW) was found in post-161
anthesis flowers (36644 microg versus 89448 microg in common leucaena followed by leaves 162
(28838 microg x 67358 microg) green flower buds (28094 microg x 51247 microg) and green pods (19002 163
microg x 82687 microg) (Fig 1)The same pattern is observed for seedlings when both species are 164
compared In this study untreated 12-week-old maricaacute seedlings (control at day 2) showed a 165
shoot content of mimosine of 23029plusmn007 microg g-1 of (FW) Five-week-old untreated giant 166
leucaena seedlings cultivated in similar conditions exhibited between 83640 and 178736 167
microg g-1 of FW (Rodrigues-Correcirca et al 2019) In the same way mimosine concentration 168
percentage in dry matter of Mimosa pigra was found to be rather low (002 in nodules and 169
roots and 007 in leaves) (Soedarjo and Borthakur 1998) 170
In this investigation the lowest constitutive mimosine content was found in green 171
pods (Fig 1) This result may partly explain the absence of phytotoxic effect observed for 172
green pods on germination and growth of crop target plants tested by Jacobi and Ferreira 173
(1991) compared to the other maricaacute parts analyzed 174
Elicitation of mimosine biosynthesis in M bimucronata 175
Chemical stressors 176
Secondary metabolites (or natural products) are structural- and chemically 177
specialized compounds derived from primary metabolism These molecules are mainly 178
biosynthesized as part of a complex defense mechanism in response to biotic and abiotic 179
stresses such as pathogens herbivores water status metal toxicity and UV radiation for 180
example (Matsuura et al 2018) Ethephon SA SNP MeJA have been extensively used as 181
chemical elicitors of specialized metabolism (Wang et al 2016 Vestena et al 2001 Perotti 182
74
et al 2015 Zhang and Memelink 2009 Xu et al 2018) These phytohormonal signals can 183
simulate environmental challenges and modulate plant homeostasis often leading to 184
alterations in gene expression (Shinozaki et al 2015) Except SNP all treatments tested in 185
the present study showed positive effect on mimosine accumulation in common or giant 186
leucaena (Vestena et al 2001 Rodrigues-Correcirca 2019 Rodrigues-Correcirca unpublished 187
data) However in spite of the trend of increasing the mimosine content observed in seedlings 188
treated with 007 mM Ethephon (at day 2) and 100 mM MeJA (at day 4) no statistical 189
difference was confirmed for these treatments when compared to the control 190
On the other hand a within treatment difference on mimosine induction was seen 191
between day 2 and 4 in seedlings treated with 100 mM MeJA (Fig 2) In a lower 192
concentration (04 mM) jasmonic acid (JA)promoted a near threefold increase in mimosine 193
accumulation of giant leucaena seedlings after 2 days of application 194
UV-C radiation 195
Albeit UV-C radiation is not biologically active in natural environments it has been 196
widely used under controlled experimental conditions to generate acute responses of plant 197
specialized metabolism within a shorter period of time compared to that required to with UV-198
B radiation (Kara 2013 Cetin 2014) This fast response is due to the higher energy of UV-199
C photons that act as potent reactive oxygen species (ROS) generators causing extensive 200
damage to the cells either at the physiological level or on DNA structure (Gregianini et al 201
2003 Matsuura et al 2013) 202
Although divergent responses can be observed in plants exposed to UV-C radiation 203
the deleterious processes are usually reported on primary metabolism (decreasing of 204
chlorophyll content and plant height eg) (Kara 2013) In the present study no statistical 205
75
differences were observed in the mimosine concentration in maricaacute seedlings supplemented 206
with UV-C radiation However a decreasing in its content was found for both control and 207
treatment at day 6 post-treatment (Fig 03) Taking into account the lower constitutive 208
concentration of mimosine observed in maricaacute compared to the leucaena plants besides its 209
relative thermolability (Nguyen and Tawata 2016) it seems to be plausible to consider the 210
effect of the temperature inside the UV-C and the white light (control) chambers as an 211
additional abiotic factor contributing to the decrease of mimosine accumulation in both group 212
of plants 213
Besides mimosine identification the presence of 34-dihydroxypyridine (34-DHP or 214
3-hydroxy-4-pyridone - 3H4P) a mimosine degradation product (Negi et al 2014 Nguyen 215
and Tawata 2016) was also reported for maricaacute leaf extracts analyzed by TLC by Ferreira 216
et al (1992) In our chromatograms we detected a second large peak after that of mimosine 217
(229plusmn0024) and similar to that identified by Negi et al (2014) as 3H4P (data not shown) 218
Comparing the chromatogram profiles obtained from seedlings elicited with chemical 219
stressors and those supplemented with UV-C the largest area for this peak was found (in all 220
samples) in the latter treatment at day 6 It might indicate that the constitutive andor the 221
initially UV-C-induced mimosine was degraded into 3H4P to cope with the cellular damage 222
caused by this treatment associated with an increased temperature inside the chambers 223
Nevertheless it was not possible to determine 3H4P concentration (or confirm its identity) 224
in maricaacute plants since there is no commercial standard (pure 3H4P) available for purchase 225
to be used as a reference in calculations Establishment of improved protocols for obtaining 226
in house 3H4P reference substance by acid hydrolysis is ongoing 227
228
229
76
Conclusion 230
On the basis of the overall absence of effect of the treatments tested here on mimosine 231
concentration it is possible to suggest that its accumulation profile is similar to that of 232
phytoanticipins unlike what is observed for the same amino acid production in leucaena 233
which shows features of inducibility resembling phytoalexin-like metabolites Alternatively 234
a putative inducible pool of mimosine in maricaacute might be involved in other types of stress 235
such as extended drought periods If involved in protection against oxidative stress as 236
described for leucaena mimosine in maricaacute may act predominantly by physical quenching 237
of ROS as indicated by the lack of overt chemical degradation Nevertheless further 238
investigations are needed to assess these hypotheses 239
To sum up mimosine biosynthesis was not modulated by the treatments evaluated as 240
in L leucocephala (Lam) de Wit To the best of our knowledge this is the first work that 241
analytically identifies and quantifies mimosine accumulation in M bimucronata 242
243
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Bitencourt F Zocche JJ Costa S Souza PZ Mendes AR 2007 Nucleaccedilatildeo de 245
Mimosa bimucronata (DC) O Kuntze em aacutereas degradadas pela mineraccedilatildeo de carvatildeo R 246
Bras Bioci 5 750-752 247
Carvalho PER 2004 Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 248
Teacutecnica 94 1-10 249
Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitis 250
vinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-251
6287-47-37 252
77
Champanerkar PA Vaidya VV Shailajan S Menon SN 2010 A sensitive rapid and 253
validated liquid chromatography ndash tandem mass spectrometry (LC-MS-MS) method for 254
determination of Mimosine in Mimosa pudica Linn Nat Sci 2 713-717 255
httpsdoiorg104236ns201027088 256
Gomes GS Silva GS Silva DLS Oliveira RR Conceiccedilatildeo GM 2018 Botanical 257
Composition of Fabaceae Family in the Brazilian Northeast Maranhatildeo Brazil Asian J 258
Environ Ecol 6(4) 1-10 httpsdoiorg109734AJEE201841207 259
Correcirca LR Soares GLG Fett-Neto AG 2008 Allelopathic potential of Psychotria 260
leiocarpa a dominant understorey species of subtropical forests S Afri J Bot 74 583ndash261
590 httpsdoiorg101016jsajb200802006 262
Ferreira AG Aquila MEA Jacobi US Rizvi V 1992 Allelopathy in Brazil In Allelopathy 263
basic and applied aspects Rizvi V and Jacobi US (Eds) Chapman and Hall pp 243-250 264
Fukao T Barrera-Figueroa BE Juntawong P Pentildea-Castro JM 2019 Submergence 265
and waterlogging stress in plants a review highlighting research opportunities and 266
understudied aspects Front Plant Sci 10 340 httpsdoiorg103389fpls201900340 267
Gregianini TS Silveira VC Porto DD Kerber VA Henriques AT Fett-Neto AG 268
2003 The alkaloid brachycerine is induced by ultraviolet radiation and is a singlet oxygen 269
quencher Photochem Photobiol 78(5) 470ndash474 httpsdoiorg1015620031-270
8655(2003)0784070TABIIB20CO2 271
Jacobi US Ferreira AG 1991 Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK 272
sobre espeacutecies cultivadas Pesq Agropec Bras 26(7) 935-943 273
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant 274
(Phaseolus vulgaris) Biosci Res 10(1) 29ndash32 275
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Kestring D Klein J Menezes LCCR Rossi MN 2009 Imbibition phases and 276
germination response of Mimosa bimucronata (Fabaceae Mimosoideae) to water 277
submersion Aquat Bot 91 105ndash109 httpsdoiorg101016jaquabot200903004 278
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect of 279
Ethephon as an ethylene-releasing compound on the metabolic profile of Chlorella vulgaris 280
J Agric Food Chem 64(23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541 281
Lorenzi H 1998 Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 282
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 283
Lorenzi H 2008 Plantas daninhas do Brasil terrestres aquaacuteticas parasitas e toacutexicas 4 ed 284
Nova Odessa Instituto Plantarum 640 p 285
Marchiori JNC 1993 Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 286
O Kuntze Ciecircncia Florestal 3 85-106 287
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation of 288
bioactive secondary metabolites by ultraviolet radiation mechanisms strategies and 289
applications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology for Medicinal 290
Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash190= 291
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo R Bhambra AS 292
Strnad M Bonfill M Fett-Neto AG 2018 Specializedplant 293
metabolismcharacteristicsandimpactontargetmoleculebiotechnologicalproduction 294
Molecular Biotechnology 60(2) 169ndash183httpsdoiorg101007s12033-017-0056-1 295
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase from 296
Leucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 297
922ndash934 httpsdoiorg101104pp113230870 298
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Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine 299
areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636 300
Olkoski D Wittmann MTS 2011 Cytogenetics of Mimosa bimucronata (DC) O Kuntze 301
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop Breed 302
Appl Biotechnol 11 27-35 httpdxdoiorg101590S1984-70332011000100004 303
Patreze CM Cordeiro L 2004 Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 304
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecol Manag 196 275ndash305
285 httpdxdoiorg101016jforeco200403034 306
Perotti JC Rodrigues-Correcirca KCS Fett-Neto AG 2015 Control of resin production in 307
Araucaria angustifolia an ancient South American conifer Plant Biology 17 852ndash859 308
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG 2019 Mimosine 309
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UV exposure Plant Physiology and Biochemistry 135 432ndash440 311
Pilatti DM Fortes AMT Jorge TCM Boiago NP 2019 Comparison of the phytochemical 312
profiles of five native plant species in two different forest formations Brazilian Journal of 313
Biology 79(2) 233-242 314
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS 2011 Biologia da reproduccedilatildeo 315
deMimosa bimucronatandash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 316
Simon MF and Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 317
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 318
endemism Biological Conservation 96 279-296 319
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 320
LM Andrade SF Cechinel-Filho V 2017 Chemical composition and diuretic natriuretic and 321
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kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its majority 322
constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash1624 323
Shah J 2003 The salicylic acid loop in plant defense Current Opinion Plant Biology6 (4) 324
365ndash371 325
Shinozaki K Uemura M Serres JB Bray EA Weretilnyk E 2015 Responses to Abiotic 326
Stress In Buchanan BB Gruissem W Jones RL (Eds) Biochemistry and Molecular 327
Biology of Plants Second Edition John Wiley and Sons Ltd 328
Soedarjo M and Borthakur D 1998 Mimosine a toxin produced by the tree-legume 329
Leucaena provides a nodulation competition advantage to mimosine-degrading Rhizobium 330
strains Soil Biology and Biochemistry 30(12)1605-1613 331
Vestena S Fett-Neto AG Duarte RC Ferreira AG 2001 Regulation of mimosine 332
accumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 333
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene induced 334
vinblastine accumulation is related to activated expression of downstream TIA pathway 335
genes in Catharanthus roseus BioMed Research International Article ID 3708187 336
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX Tsim KWK 2018 337
Jasmonate-elicited stress induces metabolic change in the leaves of Leucaena leucocephala 338
Molecules 23 (2) 339
Zhang H Memelink J 2009 Regulation of Secondary Metabolism by Jasmonate Hormones 340
In AE Osbourn and V Lanzotti (eds) Plant-derived Natural Products 3 DOI 101007978-341
0-387-85498-4_1 copy Springer Science + Business Media LLC 342
343
344
345
81
346
Figure 1 Constitutive concentration of mimosine in different plant organs of Mimosa 347
bimucronata Bars sharing the same letter do not differ statistically by Tukey test (Ple005) 348
The error bars denote standard error of 10 replicates 349
350
351
352
353
354
355
356
357
B B A C0
5
10
15
20
25
30
35
40
LEAVES GREEN FLOWER BUDS POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
co
nce
ntr
atio
n u
gg-1
Mimosine concentration in adult plants of Mimosa bimucronata (DC) Kuntze
82
C T R L S A
1 0 m M
S A
5 0 m M
E T H
0 0 7 m M
E T H
0 3 5 m M
M e J A
1 0 0 m M
M e J A
2 0 0 m M
S N P
1 0 m M
S N P
5 0 m M
0
1 0
2 0
3 0
T re a tm e n ts
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1) D A Y 2
D A Y 4
A B C C B C A B C C A B C A B C A
a b b b a a b a a b b a b
358
Figure 2 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 359
bimucronata treated with different signaling molecules SA = Salicylic Acid ETH = 360
Ethephon MeJA = Methyl Jasmonate SNP = Sodium Nitroprusside Uppercase and 361
lowercase letters indicate statistical differences among treatments in days 2 and 4 362
respectively Bars sharing a letter of the same case do not differ statistically by Tukey test 363
(Ple005) Indicates statistical difference in the same treatment between day 2 and 4 by t-364
test (Ple005) The error bars denote standard error of 5 replicates (25 individual seedlings 365
arranged in 5 groups of 5) 366
367
368
83
D AY 3 D AY 6
0
5
1 0
1 5
2 0
2 5
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1)
C O N TR O L
U V -C
369
Figure 3 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 370
bimucronata supplemented with UV-C radiation Indicates statistical difference in the same 371
treatment between day 3 and 6 by t-test (Ple005) The error bars denote standard error of 5 372
replicates (25 individual seedlings arranged in 5 groups of 5) 373
374
375
376
377
378
379
380
381
382
383
384
385
84
Consideraccedilotildees finais 386
- Experimentos que avaliam os efeitos da aplicaccedilatildeo exoacutegena de ANPs em diferentes espeacutecies 387
vegetais tecircm sido realizados principalmente com GABA Dentre os principais efeitos 388
conferidos pela aplicaccedilatildeo dessa moleacutecula em espeacutecies de mono e eudicotiledocircneas satildeo 389
relatados a toleracircncia agrave seca agrave salinidade e agraves temperaturas extremas 390
- Como metaboacutelitos especializados claacutessicos os ANPs podem ter sua concentraccedilatildeo basal 391
endoacutegena aumentada em resposta agrave induccedilatildeo mediada por uma vasta gama de tratamentos com 392
moleacuteculas sinalizadoras de estresse e fontes alternativas de estressores De um modo geral 393
observa-se o acuacutemulo das diferentes classes de ANPs em resposta agrave radiaccedilatildeo UV elicitores 394
quiacutemicos que mimetizam ataques por patoacutegenos dano mecacircnico agentes osmoacuteticos metais 395
pesados entre outros 396
- Especificamente em leucena a resposta observada em relaccedilatildeo aos diferentes tratamentos 397
testados indica que apesar do seu alto teor constitutivo nessa espeacutecie a biossiacutentese e o 398
acuacutemulo de mimosina podem ser modulados por fatores causadores de estresses exibindo -399
nessa espeacutecie - um padratildeo de acumulaccedilatildeo similar agrave fitoalexinas Em maricaacute por outro lado 400
aumento de acuacutemulo dessa moleacutecula natildeo foi observado para os mesmos tratamentos testados 401
para leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas 402
- O padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno 403
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado pela 404
sua degradaccedilatildeo 405
- As respostas observadas nos testes que avaliaram a atividade de mitigaccedilatildeo de espeacutecies 406
reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode agir como um agente 407
antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de estresse 408
85
Perspectivas 409
- Confirmaccedilatildeo em espectrocircmetro de massas eou ressonacircncia nuclear magneacutetica da natureza 410
quiacutemica da lsquomimosinarsquo presente em maricaacute 411
- Avaliaccedilatildeo do efeito de concentraccedilotildees mais elevadas e em diferentes periacuteodos de aplicaccedilatildeo 412
das moleacuteculas sinalizadoras testadas sobre o acuacutemulo de mimosina em leucena e maricaacute 413
- Ampliar a investigaccedilatildeo dos padrotildees de expressatildeo gecircnica dos genes que codificam para 414
mimosinase (em maricaacute) mimosina sintase (em ambas as espeacutecies testadas) bem como o 415
perfil de precursores e cataboacutelitos de mimosina em resposta aos tratamentos mencionados 416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
86
Referecircncias Bibliograacuteficas 436
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Acamovic T Brooker JD (2005) Biochemistry of plant secondary metabolites and their 438
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Ahmed R Hoque ATMR Hossain MK (2008) Allelopathic effects of Leucaena 440
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Res (2008) 19 298 httpsdoiorg101007s11676-008-0053-0 442
Ahmed AMM Saacutenchez FJS Bavileacutes LRY Mahdy REZ Camaal JBC (2016) Tannins and 443
mimosine in Leucaena genotypes and their relations to Leucaena resistance against 444
Leucaena Psyllid and Onion thrips Agroforestry Systems 1-8 445
Benjakul S Kittiphattanabawon P Shahidi F Maqsood S (2013) Antioxidant activity and 446
inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipid oxidation in 447
model systems Food Sci Technol Int 19(4)365-76 448
httpsdoiorg1011771082013212455186 449
Bitencourt F Zocche JJ Costa S Souza PZ Mendes AR (2007) Nucleaccedilatildeo de Mimosa 450
bimucronata (DC) O Kuntze em aacutereas degradadas pela mineraccedilatildeo de carvatildeo Revista 451
Brasileira de Biociecircncias 5 750-752 452
Bottini-Luzardo M Aguilar-Perez C Centurion-Castro F Solorio-Sanchez F Ayala-Burgos 453
A Montes-Perez R Muntildeoz-Rodriguez D Ku-Vera J (2015) Ovarian activity and estrus 454
behavior in early postpartum cows grazing Leucaena leucocephala in the tropics Trop Anim 455
Health Prod 47(8)1481-6 456
Carvalho PER (2004) Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 457
Teacutecnica 941-10 458
Chowtivannakul P Srichaikul B Talubmook C (2016) Antidiabetic and antioxidant activities 459
of seed extract from Leucaena leucocephala (Lam) de Wit Agriculture and Natural 460
Resources 50 (2016) 357e361 httpdxdoiorg101016janres201606007 461
Chung H-H Chen M-K Chang Y-C Yang S-F Lin C-C Lin C-W (2017) Inhibitory effects 462
of Leucaena leucocephala on the metastasis and invasion of human oral cancer cells 463
Environmental Toxicology 321765ndash1774 httpsdoiorg101002tox22399 464
87
Crowe B Poynter JA Manukyan MC Wang Y Brewster BD Herrmann JL Abarbanell 465
AM Weil BR Meldrum DR (2001) Pretreatment with intracoronary mimosine improves 466
postischemic myocardial functional recovery Surgery 150(2) 191-106 467
Fallon (2015) Effects of mimosine on Wolbachia in mosquito cells cell cycle suppression 468
reduces bacterial abundance In Vitro Cell Dev Biol Anim 51(9)958-63 469
httpsdoiorg101007s11626-015-9918-7 Epub 2015 May 28 470
Fernaacutendez-Salas A Alonso-Diacuteaza MA Acosta-Rodriacuteguez A Torres-Acosta JFJ Sandoval-471
Castro CA Rodriacuteguez-Vivas RI (2011) In vitro acaricidal effect of tannin-rich plants against 472
the cattle tick Rhipicephalus (Boophilus) microplus (Acari Ixodidae) Veterinary 473
Parasitology 175113ndash118 2010 httpsdoiorg101016jvetpar201009016 474
Ferreira AG Aquila MEA Jacobi US Rizvi V (1992) Allelopathy in Brazil In Allelopathy 475
basic and applied aspects Rizvi V and Jacobi US (Eds) Chapman and Hall PP 243-250 476
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad Hossain Md Anai 477
T Oku H (2018) Cytosolic cysteine synthase switch cysteine and mimosine production in 478
Leucaena leucocephala Appl Biochem Biotechnol 186 (3) 613ndash632 479
httpsdoiorg101007s12010-018-2745-z 480
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of the thyrotoxic 481
amino acid mimosine by cysteine synthase Phytochemistry 29 (11) 3461ndash3465 482
httpsdoiorg1010160031-9422(90)85258-H 483
Jacobi US Ferreira AG (1991) Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK Sobre 484
espeacutecies cultivadas Pesquisa Agropecuaacuteria Brasileira 26(7) 935-943 485
Jamous RM Ali-Shtayeh MS Abu-Zaitoun SY Markovics A Azaizeh H (2017) Effects of 486
selected Palestinian plants on the in vitro exsheathment of the third stage larvae of 487
gastrointestinal nematodes BMC Veterinary Research 13308 488
httpdxdoiorg101186s12917-017-1237-7 489
Jiao CJ Jiang J-L Ke L-M Cheng W Li F-M Li Z-X Wang C-Y (2011) Factors affecting 490
β-ODAP content in Lathyrus sativus and their possible physiological mechanisms Food 491
Chem Toxicol 49 543ndash549 httpsdoiorg101016jfct201004050 492
Kubota S Fukumoto Y Ishibashi K Soeda S Kubota SS Yuki R Nakayama Y Aoyama K 493
Yamaguchi N (2014) Activation of the prereplication complex is blocked by mimosine 494
88
through reactive oxygen species-activated ataxia telangiectasia mutated (ATM) protein 495
without DNA damage J Biol Chem 28 289(9)5730-46 496
Kuppusamy UR Arumugam B Azaman N Wai CJ (2014) Leucaena leucocephala Fruit 497
Aqueous Extract Stimulates Adipogenesis Lipolysis and Glucose Uptake in Primary Rat 498
Adipocytes Hindawi Publishing Corporation e Scientific World Journal Article ID 737263 499
8 pages httpdxdoiorg1011552014737263 500
Kusama-Eguchi K (2019) Research in motor neuron diseases caused by natural substances 501
focus on pathological mechanisms of neurolathyrism Yakugaku Zasshi 139 (4) 609-502
615 httpsdoiorg101248yakushi18-00202 503
Kutchan TM Gershenzon J Moslashller BL Gang DR (2015) Natural Products In Buchanan 504
BB Gruissem W and Jones RL (eds) Biochemistry amp Molecular Biology of Plants 2nd edn 505
Wiley Blackwell Chichester pp 1135-1205 506
Lalande M (1990) A reversible arrest point in the late G1 phase of the mammalian cell cycle 507
Exp Cell Res 186 332ndash339 508
Li X-W Hu C-P Li Y-J Gao Y-X Wang XM Yang J-R (2015) Inhibitory effect of L-509
mimosine on bleomycin-induced pulmonary fibrosis in rats Role of eIF3a and p27 Int 510
Immunopharmacol 27(1) 53ndash64 511
Little Jr EL Skolmen RG (1989) Koa haole Agriculture Handbook 679 USDA 512
Lorenzi H (1998) Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 513
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 514
Marchiori JNC (1993) Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 515
O Kuntze Ciecircncia Florestal 3 85-106 516
Mohammed RS El Souda SS Taie HAA Moharam ME Shaker KH (2015) Antioxidant 517
antimicrobial activities of flavonoids glycoside from Leucaena leucocephala leaves Journal 518
of Applied Pharmaceutical Science 5(06)138-147 519
httpdxdoiorg107324JAPS201550623 520
Negi VS Bingham J-P Li QX Borthakur D (2014) A carbon-nitrogen lyase from Leucaena 521
leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 (2) 922ndash522
934 httpsdoiorg101104pp113230870 523
89
Olkoski D Wittmann MTS (2011) Cytogenetics of Mimosa bimucronata (DC) O Kuntze 524
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop 525
Breeding and Applied Biotechnology 11 27-35 526
Patreze CM Cordeiro L (2004) Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 527
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecology and Management 528
196275ndash285 529
Pilatti DM Fortes AMT Jorge TCM Boiago NP (2019) Comparison of the phytochemical 530
profiles of five native plant species in two different forest formations Brazilian Journal of 531
Biology 79(2) 233-242 532
Ramos-Ruiz R Poirot E Flores-Mosquera M (2018) GABA a non-protein amino acid 533
ubiquitous in food matrices Cogent Food Agric 41534323 534
httpsdoiorg1010802331193220181534323 535
REFLORA (2019) httpfloradobrasiljbrjgovbrreflora Acesso em agosto de 2019 536
Rodgers KJ Samardzic K Main BJ (2015) Toxic Nonprotein Amino Acids Plant Toxins 537
httpsdoiorg 101007978-94-007-6728-7_9-1 538
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG (2019) Mimosine 539
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 540
UV exposure Plant Physiology and Biochemistry 135 432ndash440 541
httpsdoiorg101016jplaphy201811018 542
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 543
LM Andrade SF Cechinel-Filho V (2017) Chemical composition and diuretic natriuretic 544
and kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its 545
majority constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash546
1624 547
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS (2011) Biologia da reproduccedilatildeo 548
de Mimosa bimucronata ndash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 549
Simon MF Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 550
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 551
endemism Biological Conservation 96 279-296 552
90
Soares AMS Arauacutejo SA Lopes SG Costa Junior LM (2015) Anthelmintic activity of 553
Leucaena leucocephala protein extracts on Haemonchus contortus Braz J Vet Parasitol 554
Jaboticabal 24(4) 396-401 httpdxdoiorg101590S1984-29612015072 555
Soerdajo M Borthakur D (1998) Mimosine a toxin produced by the tree-legume Leucaena 556
provides a nodulation competition advantage to mimosine-degrading Rhizobium strains Soil 557
Biol Biochem 30(12) 16051613 558
Souza-Lima ES Sinani TR Pott A Sartori ALB (2017) Mimosoideae (Leguminosae) in the 559
Brazilian Chaco of Porto Murtinho Mato Grosso do Sul Rodrigueacutesia 68(1) 263-290 2017 560
httpdxdoiorg1015902175-7860201768131 561
Taiz L amp Zeiger E (2010) Plant Physiology 5th edition Sinauer Associates Inc Sunderland 562
Verma VK Rani KV Kumara SR Prakash O (2018) Leucaena leucocephala pod seed 563
protein as an alternate to animal protein in fish feed and evaluation of its role to fight against 564
infection caused by Vibrio harveyi and Pseudomonas aeruginosa Fish and Shellfish 565
Immunology 76 (2018) 324ndash332 httpsdoiorg101016jfsi201803011 566
Yafuso JT Negi VS Bingham J-P Borthakur D (2014) An O-acetylserine (thiol) lyase from 567
Leucaena leucocephala is a cysteine synthase but not a mimosine synthase Appl Biochem 568
Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z 569
Zarin RMA Wan HY Isha A Armani N (2016) Antioxidant antimicrobial and cytotoxic 570
potential of condensed tannins from Leucaena leucocephala hybrid Food Science and 571
Human Wellness 5 65ndash75 httpdxdoiorg101016jfshw201602001 572
573
574
Contents lists available at ScienceDirect
Industrial Crops amp Productsjournal homepage wwwelseviercomlocateindcrop
Resin tapping transcriptome in adult slash pine (Pinus elliottii var elliottii)Camila Fernanda de Oliveira Junkes1 Artur Teixeira de Arauacutejo Juacutenior1 Juacutelio Ceacutesar de LimaFernanda de Costa Thanise Fuumlller Maacutercia Rodrigues de Almeida Franciele Antocircnia NeisKelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-NetoCenter for Biotechnology and Department of Botany Federal University of Rio Grande do Sul Porto Alegre PO Box 15005 91501-970 Brazil
A R T I C L E I N F O
KeywordsPinus elliottiResinResinosisTranscriptomeAdjuvant paste
A B S T R A C T
To better understand the bases of resin production a major source of terpenes for industry the transcriptome ofadult Pinus elliottii var elliottii (slash pine) trees under field commercial resinosis was obtained Samples werecollected from cambium after 5 and 15 days of treatment application which included tapping followed byapplication of commercial resin stimulant paste or control wounding without paste Overall mean number ofreads of all 16 libraries (2 treatments x 2 times x 4 replicated trees) was 34582048 Of these 89 were mappedagainst the reference sequence with a mismatch of 058 Using the Blast2Go 570 candidate genes were de-tected based on sequence annotation By comparing the expression profile between paste and control 310differentially expressed genes (DEGs) were identified at 5 days and 190 at 15 days with a significant fold changeof log2gt 12 Regarding changes in time comparisons within each treatment 210 and 105 DEGs were identifiedwithin control and paste treatment respectively Genes with different expression patterns in the times andtreatments examined included ethylene responsive transcription factors geranylgeranyl diphosphate synthasediterpene synthase cytochrome P450 and ABC transporters all of which may play important roles in resinproduction RT-qPCR analysis correlated well with the data obtained by RNAseq Resin composition changedover time This is the first transcriptomic investigation of resinosis of the main species used in the bioresinindustry and of molecular analyses of resinosis under field operations with implications for stand managementstimulant paste development genotype selection and breeding for high resinosis
1 Introduction
The adaptive success of conifers is largely due to the development ofa defense system based on the synthesis and secretion of terpenes in allmajor organs and different tissues (Miller et al 2005 Hall et al 2013Warren et al 2015) Conifer resin is a viscous fluid composed of acomplex mixture of terpenoids such as monoterpenes sesquiterpenesand diterpenes (Zulak and Bohlmann 2010) These terpenoids are se-creted from severed resin ducts when the tree is under biotic attack(Ralph et al 2006 Lange 2015 Geisler et al 2016) acting as pro-tectants (Schiebe et al 2012 Liu et al 2015)Biosynthesis of terpenes in conifers starts from isomerization of two
isoprenoid (C5) units dimethylallyl diphosphate (DMAPP) and iso-pentenyl diphosphate (IPP) These molecules can be biosynthesized viatwo separate routes in plants the methyl-erythritol 4-phosphate andmevalonate pathways IPP is synthesized and isomerized to DMAPP byisopentenyl diphosphate isomerase then prenyl transferases catalyze
the condensation of these two C5-units to geranyl diphosphate (Pazoukiand Niinemets 2016) Their elongation to prenyl diphosphates withaddition of IPP molecules leads to monoterpenes (C10) sesquiterpenes(C15) and diterpenes (C20) which are the substrates for terpene syn-thases (TPS) (Keeling and Bohlmann 2006b)TPSs are part of a large family of mechanistically related enzymes
involved in both primary and secondary metabolism (Keeling andBohlmann 2006b) The events of evolutionary diversification and ex-pansion of plant TPSs appear to have originated from gene duplicationsdomain losses and sub- or neofunctionalizations with subsequent di-vergence of an ancestral TPS gene of primary metabolism (Hall et al2013) Modification of TPS products changes their physical propertiesand may alter their biological activities (Chen et al 2011) TPSs of highsequence identity may have different functions even in closely relatedspecies Low sequence identity of TPSs in phylogenetically distantspecies does not preclude the possibility of independent evolution of thesame or related function of these enzymes (Zerbe and Bohlmann 2015)
httpsdoiorg101016jindcrop2019111545Received 4 January 2019 Received in revised form 10 June 2019 Accepted 4 July 2019
Corresponding authorE-mail address fettnetocbiotufrgsbr (AG Fett-Neto)1 These authors have equally contributed to this work
doi 1015900102-33062019abb0114
Acta Botanica Brasilica
Sustainable production of bioactive alkaloids in Psychotria L of
southern Brazil propagation and elicitation strategies
Yve Verocircnica da Silva Magedans1 Kelly Cristine da Silva Rodrigues-Correcirca1 Cibele Tesser da Costa1
Heacutelio Nitta Matsuura1 and Arthur Germano Fett-Neto1
Received April 1 2019Accepted June 28 2019
ABSTRACTPsychotria is the largest genus in Rubiaceae South American species of the genus are promising sources of natural
products mostly due to bioactive monoterpene indole alkaloids they accumulate ese alkaloids can have analgesic
antimutagenic and antioxidant activities in dierent experimental models among other pharmacological properties
of interest Propagation of genotypes with relevant pharmaceutical interest is important for obtaining natural
products in a sustainable and standardized fashion Besides the clonal propagation of elite individuals the alkaloid
content of Psychotria spp can also be increased by applying moderate stressors or stress-signaling molecules is
review explores advances in research on methods for plant propagation and elicitation techniques for obtaining
bioactive alkaloids from Psychotria spp of the South Region of Brazil
Keywords abiotic stress alkaloids elicitation monoterpenes plant propagation Psychotria southern Brazil
sustainability
Introduction
Psychotria belongs to Rubiaceae one of the major families
of $owering plants having economic interest e family
includes coee a few signicant poisonous plants to livestock
besides several important ornamental and medicinal species
(Souza amp Lorenzi 2012) Psychotria has captured researchersrsquo
attention mostly because of its medicinal properties
Psychotria colorata is an Amazonian species that produces
polyindolinic alkaloids with analgesic activity (Matsuura et
al 2013) e promising results obtained with P colorata
motivated the investigation of southern Brazilian Psychotria
species and the discovery of new bioactive alkaloids (Porto
et al 2009) Moreover leads on in planta alkaloid functions
were also topic of experimental evaluation
One of the key elements that needs to be addressed early
on during the process of developing new bioactive molecules
from plants is the capacity to generate catalytically active
biomass to support extraction and steady supply ere are a
number of ways through which these goals may be reached
including greenhouse rooting of cuttings (mini-cutting
1 Laboratoacuterio de Fisiologia Vegetal Departamento de Botacircnica Instituto de Biociecircncias e Centro de Biotecnologia Universidade Federal do Rio
Grande do Sul 91501-970 Porto Alegre RS Brazil
Corresponding author fettnetocbiotufrgsbr
Review
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Biomass yield of resin in adult Pinus elliottii Engelm trees is differentially
regulated by environmental factors and biochemical effectors
Franciele Antocircnia Neis Fernanda de Costa Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima
Kelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-Neto
Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) CP 15005 CEP 91501-970 Porto Alegre RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Biomass
Terpene resin
Seasonal
Benzoic acid
Regenerated forest
A B S T R A C T
Biomass of pine resin finds several applications in the chemical pharmaceutical biofuel and food industries
Resin exudation after injury is a key defense response in Pinaceae since this complex mixture of terpenes has
insecticidal antimicrobial and wound repair properties Resin yield is increased by effectors applied on the
wound area including phytohormones and metal cofactors of terpene synthases The interaction of resinosis
mechanism effectors is not fully understood particularly in adult forest setups under natural environmental
variations The aim of this work was to determine how resin exudation by wounded trunks of adult P elliottii
responded to combined chemical effectors involved in different regulatory pathways of resinosis (metal cofactors
of terpene synthases benzoic acid and plant growth regulators) and whether seasonal and tree distribution
variations affected these responses Symmetrically planted and scattered trees regenerated from the seed bank
had similar resin biomass yields suggesting that the homogeneity in development and spatial arrangement were
not significant factors in resin yield This new finding is of practical importance with the used tapping system
since costs of implanting forests by regeneration can be advantageous compared to planting In addition it was
shown for the first time that the salicylic acid precursor benzoic acid and the auxin naphthalene acetic acid
promoted resin exudation when individually applied to wound sites Both these adjuvants are two orders of
magnitude less costly compared to the conventionally used ethylene precursors besides facing less environ-
mental and health restrictions for use Most adjuvant-treated trees showed higher resin flow in the second year
indicating mechanisms of response build up Overall temperature was more important than rainfall as en-
vironmental parameter affecting resin biosynthesis which was higher in the warmer months of spring and
summer The combination of resinosis stimulant effectors from different signaling pathways showed no sig-
nificant synergistic or additive effect suggesting possible converging signaling pathways andor limitation of
common intermediate transducing molecules
1 Introduction
Pines occupy highly diverse environments over a range of tem-
peratures water and nutrient availabilities irradiance levels and pho-
toperiods being able to effectively face attacks from diverse herbivore
and pathogen guilds The success of conifers is linked to their complex
terpene biochemistry hosted by specialized secretory cells The terpe-
noid resin synthesized by Pinus spp is one of the main mechanisms of
defense of these trees particularly against bark beetles and the fungi
they carry (Fett-Neto and Rodrigues-Correcirca 2012) Pine resin biomass
is essentially composed of a monoterpene and sesquiterpene-rich tur-
pentine and diterpenoid-rich rosin fraction both finding numerous in-
dustrial applications as non-wood forest products (Rodrigues-Correcirca
et al 2012)
Molecules capable of modulating different signaling pathways have
been identified as resin yield stimulators including sulfuric acid (ex-
tends wound damage) 2-chloroethylphosphonic acid (CEPA a syn-
thetic ethylene precursor) paraquat (free radical generator) yeast ex-
tract (mimics attack by pathogens) salicylic acid (pathogen signaling
molecule) auxin (promotes ethylene biosynthesis and resin canal dif-
ferentiation) jasmonic acid (signals mechanical damage and promotes
secondary metabolism) and metal ions such as potassium iron and
manganese (cofactors of terpene synthases in conifers) and copper (a
component of ethylene receptors) (Clements 1970 Conrath et al
2002 Fett-Neto and Rodrigues-Correcirca 2012 Hudgins and Franceschi
2004 Lewinsohn et al 1994 Martin et al 2002 Popp et al 1995
httpsdoiorg101016jindcrop201803027
Received 12 December 2017 Received in revised form 9 March 2018 Accepted 13 March 2018
Corresponding author
E-mail addresses franci_neisyahoocombr (FA Neis) fernandadecostayahoocombr (F de Costa) thanisenfyahoocombr (TN Fuumlller)
jjuliocesarlimagmailcom (JC de Lima) krodriguescbiotufrgsbr (KC da Silva Rodrigues-Correcirca) jpfettcbiotufrgsbr (JP Fett) fettnetocbiotufrgsbr (AG Fett-Neto)
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Research Paper
Dual allelopathic effects of subtropical slash pine (Pinus elliottii Engelm)
needles Leads for using a large biomass reservoir
Kelly Cristine da Silva Rodrigues-Correcircaa Gelson Halmenschlagera Joseacuteli Schwambachb
Fernanda de Costaa Emili Mezzomo-Trevizana Arthur Germano Fett-Netoa
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 Brazilb University of Caxias do Sul Institute of Biotechnology Caxias do Sul RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Seasonality
Growth
Germination
Litter
Substrate
A B S T R A C T
Pinus elliottii Engelm (slash pine) is distributed along the maritime coast of Southern Brazil where it shows
invasive pattern and typical allelopathic features Large quantities of needle litter are produced by pine trees a
biomass that is little explored in areas where this species is alien Little is known about the dynamics of needle
and litter phytochemical interactions particularly in subtropical environments To elucidate the full range of
needle and litter allelopathic potential the effects of litter (superficial and deep) and seasonally harvested fresh
slash pine needles stored for different times were evaluated against lettuce tomato and cucumber seeds and
seedlings Increasing concentrations (0 1 2 4 and 8 wv) of hot and cold aqueous extracts of needles
and litter affected in different ways target plant development Growth and germination inhibition were directly
related to the highest extract concentrations (regardless of the season and mainly in hot water extracts) of
needles On the other hand stimulatory effects of litter extracts on lettuce growth were observed Growth and
germination of cucumber and tomato were not affected by pine litter as substrate when compared to rice husk
The presumable high polarity and thermal stability of slash pine leaf biomass allelochemicals and their transient
toxic effect or growth promoting impact suggest potential applications of this largely available biomass both as a
biological herbicide and growth substrate in plant propagation
1 Introduction
Native from the Northern Hemisphere Pinus is one of the most
widely distributed genera throughout different climate regions of the
globe growing either as native or alien species even in extreme habi-
tats (Rodrigues-Correcirca and Fett-Neto 2012) Despite the high economic
value currently attributed to pine wood and oleoresin (Rodrigues-
Correcirca et al 2012) there is increasing concern about the aggressive
potential of invasiveness displayed by Pinus species especially those
cultivated out of their native range of distribution (Richardson et al
2008 Rolon et al 2011) These species are dispersed by wind and there
is notably low plant diversity observed in most understories of pine
plantations (Kato-Noguchi et al 2009) This latter feature has been
considered an important trait of allelopathic interference
The term ldquoallelopathyrdquo was coined by Molisch in 1937 as a chemical
reciprocal interaction established among plants (including micro-
organisms) sharing the same site by means of the release of secondary
metabolites named allelochemicals (Rice 1984) For the most part
these metabolites are derived from the shikimic acid or isoprenoid
pathway and their biosynthesis can be modulated by biotic and abiotic
stresses (Nascimento and Fett-Neto 2010) including seasonal-related
changes (Sartor et al 2013) Allelopathy studies may range from sterile
assays (Aryakia et al 2015) to soil (Correcirca et al 2008 Sharma et al
2016) and field tests being a complex biological phenomenon to as-
certain in several circumstances due to issues of solubility release
mechanisms and stability of bioactive compounds (Scognamiglio et al
2013) Often the use of complementary methods provides more in-
formative data
The allelopathic effects of soil leachates green needles and litter
extracts of Pinus spp on germination and seedling growth aspects of
wild and crop species have been evaluated in natural and cultivated
pine stands and have proven to be stimulatory or inhibitory (Lodhi and
Killingbeck 1982 Kil and Yim 1983 Nektarios et al 2005 Akkaya
et al 2006 Machado 2007 Alrababah et al 2009 Sartor et al 2009
Kato-Noguchi et al 2011 Rolon et al 2011 Valera-Burgos et al
2012) exhibiting in some cases autotoxicity (Garnett et al 2004
Fernandez et al 2008 Zhu et al 2009 Monnier et al 2011) Studies
on potential dual allelopathic effects of Pinus elliottii Engelm (slash
httpdxdoiorg101016jindcrop201706019
Received 23 March 2017 Received in revised form 15 May 2017 Accepted 7 June 2017
Corresponding author
E-mail address fettnetocbiotufrgsbr (AG Fett-Neto)
ORIGINAL RESEARCHpublished 16 June 2016
doi 103389fpls201600849
Frontiers in Plant Science | wwwfrontiersinorg 1 June 2016 | Volume 7 | Article 849
Edited by
Juan Francisco Jimenez Bremont
Instituto Potosino de Investigacioacuten
Cientiacutefica y Tecnoloacutegica Mexico
Reviewed by
Mariacutea De La Luz Guerrero Gonzaacutelez
Universidad Autoacutenoma de San Luis
Potosiacute Mexico
Rosalia Cristina Paz
CIGEOBIO (CONICETFCEFN UNSJ)
Argentina
Correspondence
Arthur G Fett-Neto
fettnetocbiotufrgsbr
daggerThese authors have contributed
equally to this work
Specialty section
This article was submitted to
Plant Physiology
a section of the journal
Frontiers in Plant Science
Received 08 December 2015
Accepted 30 May 2016
Published 16 June 2016
Citation
de Lima JC de Costa F Fuumlller TN
Rodrigues-Correcirca KCdS Kerber MR
Lima MS Fett JP and Fett-Neto AG
(2016) Reference Genes for qPCR
Analysis in Resin-Tapped Adult Slash
Pine As a Tool to Address the
Molecular Basis of Commercial
Resinosis Front Plant Sci 7849
doi 103389fpls201600849
Reference Genes for qPCR Analysisin Resin-Tapped Adult Slash Pine Asa Tool to Address the MolecularBasis of Commercial Resinosis
Juacutelio C de Lima 1dagger Fernanda de Costa 1 dagger Thanise N Fuumlller 1
Kelly C da Silva Rodrigues-Correcirca 2 Magnus R Kerber 1 Mariano S Lima 1
Janette P Fett 1 and Arthur G Fett-Neto 1
1 Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul
Porto Alegre Brazil 2 Biological Sciences Department Regional Integrated University of Alto Uruguai and Missotildees (URI-FW)
Frederico Westphalen Brazil
Pine oleoresin is a major source of terpenes consisting of turpentine (mono- and
sesquiterpenes) and rosin (diterpenes) fractions Higher oleoresin yields are of economic
interest since oleoresin derivatives make up a valuable source of materials for chemical
industries Oleoresin can be extracted from living trees often by the bark streak method
in which bark removal is done periodically followed by application of stimulant paste
containing sulfuric acid and other chemicals on the freshly wounded exposed surface
To better understand the molecular basis of chemically-stimulated and wound induced
oleoresin production we evaluated the stability of 11 putative reference genes for the
purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis
Samples for RNA extraction were collected from field-grown adult trees under tapping
operations using stimulant pastes with different compositions and at various time points
after paste application Statistical methods established by geNorm NormFinder and
BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3
and UBI To confirm expression stability of the candidate reference genes expression
profiles of putative P elliottii orthologs of resin biosynthesis-related genes encoding Pinus
contorta β-pinene synthase [PcTPS-(minus)β-pin1] P contorta levopimaradieneabietadiene
synthase (PcLAS1) Pinus taeda α-pinene synthase [PtTPS-(+)αpin] and P taeda
α-farnesene synthase (PtαFS) were examined following stimulant paste application
Increased oleoresin yields observed in stimulated treatments using phytohormone-based
pastes were consistent with higher expression of pinene synthases Overall the
expression of all genes examined matched the expected profiles of oleoresin-related
transcript changes reported for previously examined conifers
Keywords resin Pinus gene expression normalizer genes terpene synthase
19
Chapter 2
Stimulant Paste Preparation and Bark Streak Tapping Technique for Pine Oleoresin Extraction
Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
Tapping technique comprises the extraction of pine oleoresin a non-wood forest product consisting of a
complex mixture of mono sesqui and diterpenes biosynthesized and exuded as a defense response to
wounding Oleoresin is used to produce gum rosin turpentine and their multiple derivatives Oleoresin
yield and quality are objects of interest in pine tree biotechnology both in terms of environmental and
genetic control Monitoring these parameters in individual trees grown in the fi eld provides a means to
examine the control of terpene production in resin canals as well as the identifi cation of genetic-based
differences in resinosis A typical method of tapping involves the removal of bark and application of a
chemical stimulant on the wounded area Here we describe the methods for preparing the resin-stimulant
paste with different adjuvants as well as the bark streaking process in adult pine trees
Key words Oleoresin Pine Tapping Chemical stimulant Wounding
1 Introduction
Several conifer species produce oleoresin a complex mixture of isoprenoid compounds relevant for defense against herbivores and pathogens Two major fractions can be recognized in oleoresin (a) turpentine the volatile fraction containing mono- and sesquiter-penes and (b) rosin the nonvolatile diterpene fraction Oleoresin is a forest commodity of global interest fi nding applications in diverse industry sectors Rosin is used in adhesives printing ink manufacture and paper sizing Turpentine can be used either as a solvent for paints and varnishes or as a raw material for fraction-ation of high-value chemicals used in the pharmaceutical agro-chemical and food industry [ 1 ndash 3 ]
During the extraction activity resin is obtained from the tree in a similar way as rubber tree tapping which generally involves the
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_2 copy Springer Science+Business Media New York 2016
These authors have equally contributed to this work
fettnetocbiotufrgsbr
27
Chapter 3
A Modifi ed Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines
Juacutelio Ceacutesar de Lima Thanise Nogueira Fuumlller Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes
through high-throughput sequencing technologies microarray and also northern blots RT-PCR and
RTqPCR Even though many specifi c protocols designed for plants with high content of secondary metab-
olites have been developed these are often expensive time consuming and not suitable for a wide range
of tissues Here we present a modifi cation of the method previously described using the commercially
available Concerttrade Plant RNA Reagent (Invitrogen) buffer for fi eld-grown adult pine trees with high
oleoresin content
Key words RNA Pines Concert plant RNA reagent Stem RNA extraction Oleoresin Conifers
1 Introduction
Several conifer species especially within the Pinaceae have tissues with high concentrations of phenolics terpenes and polysaccha-rides [ 1 ] Many specifi c protocols that are appropriate for plants rich in secondary metabolite s have been developed but the extrac-tion of high-quality RNA from these tissues using commercial kits is often diffi cult and usually not applicable to woody tissues [ 2 ndash 6 ] One of the major issues during RNA extraction concerns the pres-ence of phenolic compounds which oxidize and form quinones Aromatic compounds bind RNA which interferes in downstream steps and applications [ 3 7 ] Another point of concern is the har-vest of plant samples in the experimental fi eld which constitutes another obstacle in the efforts to avoid degradation of RNA [ 8 ] These problems often result in RNAs of low quality and insuffi -cient amounts especially for methodologies that normally require
These authors have equally contributed to this work
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_3 copy Springer Science+Business Media New York 2016
fettnetocbiotufrgsbr
RESEARCH PAPER
Control of resin production in Araucaria angustifolia an ancientSouth American coniferJ C Perotti1 K C da Silva Rodrigues-Correa123 amp A G Fett-Neto12
1 Plant Physiology Laboratory Department of Botany Federal University of Rio Grande do Sul (UFRGS) Porto Alegre RS Brazil
2 Center for Biotechnology UFRGS Porto Alegre RS Brazil
3 Present address Regional Integrated University of Alto Uruguai and Miss~oes (URI-FW) Frederico Westphalen RS Brazil
Keywords
Araucaria ethylene jasmonic acid nitric
oxide resin salicylic acid terpenes
Correspondence
A G Fett-Neto Plant Physiology Laboratory
Center for Biotechnology Federal University
of Rio Grande do Sul (UFRGS) PO Box 15005
Av Bento Goncalves 9500 91501-970 Porto
Alegre Brazil
E-mail fettnetocbiotufrgsbr
Editor
K Leiss
Received 22 July 2014 Accepted 11
December 2014
doi101111plb12298
ABSTRACT
Araucaria angustifolia is an ancient slow-growing conifer that characterises parts ofthe Southern Atlantic Forest biome currently listed as a critically endangered speciesThe species also produces bark resin although the factors controlling its resinosis arelargely unknown To better understand this defence-related process we examined theresin exudation response of A angustifolia upon treatment with well-known chemicalstimulators used in fast-growing conifers producing both bark and wood resin suchas Pinus elliottii The initial hypothesis was that A angustifolia would display signifi-cant differences in the regulation of resinosis The effect of Ethrel (ET ndash ethylene pre-cursor) salicylic acid (SA) jasmonic acid (JA) sulphuric acid (SuA) and sodiumnitroprusside (SNP ndash nitric oxide donor) on resin yield and composition in youngplants of A angustifolia was examined In at least one of the concentrations testedand frequently in more than one an aqueous glycerol solution applied on fresh woundsites of the stem with one or more of the adjuvants examined promoted an increase inresin yield as well as monoterpene concentration (a-pinene b-pinene camphene andlimonene) Higher yields and longer exudation periods were observed with JA and ETanother feature shared with Pinus resinosis The results suggest that resinosis controlis similar in Araucaria and Pinus In addition A angustifolia resin may be a relevantsource of valuable terpene chemicals whose production may be increased by usingstimulating pastes containing the identified adjuvants
INTRODUCTION
Many conifer species produce terpenoid-based resins that havelong been studied for their industrial importance and role indefence against attack by herbivores and pathogens The twomost important resin-producing families of conifers are Pina-ceae and Araucariaceae (Langenheim 1996) The viscous resinsecretion is generally composed of a complex mixture ofterpenoids consisting of roughly equal parts of volatile mono-(C10) and sesquiterpene (C15 turpentine) fractions and non-volatile diterpenic (C20 rosin) components (Rodrigues-Correaet al 2013) Terpenes act in a complex and multilayereddefence response providing toxicity against bark beetles andfungi bark wound sealing disruption of insect developmentand attraction of herbivore predators (Phillips amp Croteau1999)Most conifers rely on some combination of preformed and
inducible resin defences (Trapp amp Croteau 2001 Zulak amp Bohl-mann 2010) Resin defences are controlled by environmentaland genetic factors to various extents depending on species(Roberds et al 2003 Sampedro et al 2010 Moreira et al2013) Resin traits have been reported as highly variable havingmoderate heritability indicating that breeding efforts towardssuper-resinous forests are promising (Tadasse et al 2001Roberds et al 2003) Several chemicals are known as stimulantsof resin production Commercial extraction of resin from pine
trees uses periodic bark streaking and application of resin stim-ulant pastes to the wound
Resin-stimulant paste based on sulphuric acid (SuA) iswidely used for the commercial production of pine resin Cur-rent stimulant pastes usually have two chemically active com-ponents SuA to magnify the wounding and an ethyleneprecursor (2-chloroethylphosphonic acid CEPA or Ethrel ndash
ET) to stimulate resin flow (Rodrigues et al 2011 Rodrigues-Correa amp Fett-Neto 2013) Jasmonic acid (JA) and its methylester methyl jasmonate (MeJa) are among the most widelyused chemical elicitors of plant secondary metabolism It hasbeen shown that the exogenous application of MeJa or herbi-vore attack induce chemical and anatomical defence responsesin conifers such as the formation of traumatic resin ducts andresin accumulation in stems along with increased biosynthesisof terpenes and phenolics (Franceschi et al 2002 Martin et al2002 Heijari et al 2005 Zeneli et al 2006 Moreira et al 2008Gould et al 2009) JA commercial use however is limited byits high cost
The effects of exogenous salicylic acid (SA) on conifer ter-pene production have also been studied In Pinus elliottiiapplication of 10 molm3 of SA induced resin productionin wound panels but in Pseudotsuga menziesii and Sequoia-dendron giganteum it had no apparent effect on resinaccumulation (Hudgins amp Franceschi 2004 Rodrigues ampFett-Neto 2009) Nitric oxide (NO) has also emerged as an
Plant Biology 17 (2015) 852ndash859 copy 2014 German Botanical Society and The Royal Botanical Society of the Netherlands852
Plant Biology ISSN 1435-8603
viii
PEG polyethylene glycol
PLP pyridoxal-5rsquo-phosphate
PPO polyphenol oxidase tyrosinase
qRT-PCR Reverse transcription polymerase chain reaction quantitative real time
RNS reactive nitrogen species
ROS reactive oxygen species
SA salicylic acid
SAR systemic acquired resistance
SNP sodium nitroprusside
UV ultraviolet radiation
ix
RESUMO
Ao longo de sua evoluccedilatildeo as plantas desenvolveram estrateacutegias estruturais e quiacutemicas de
defesa em resposta aos estresses bioacuteticos e abioacuteticos impostos pelo ambiente Dentre
essas satildeo reconhecidas moleacuteculas quimicamente especializadas denominadas
metaboacutelitos secundaacuterios produtos naturais ou metaboacutelitos especializados Aminoaacutecidos
natildeo proteicos (ANPs) satildeo compostos nitrogenados que constituem aleacutem de componentes
do arsenal de defesa quiacutemica vegetal uma importante fonte de reserva de carbono e
nitrogecircnio para diversos taxa especialmente aqueles pertencentes agrave famiacutelia Fabaceae de
Angiospermas Esse grupo de moleacuteculas quimicamente heterogecircneo eacute assim definido por
natildeo participar da formaccedilatildeo de estruturas proteicas funcionais sendo frequentemente
toacutexicos quando erroneamente incorporados nas cadeias polipeptiacutedicas em formaccedilatildeo em
funccedilatildeo da similaridade estrutural que apresentam com os aminoaacutecidos proteicos Sob o
ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados ANPs satildeo
em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou abioacutetica como
o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de elicitores
quiacutemicos por exemplo O objetivo da presente tese foi investigar o papel bioloacutegico da
mimosina endoacutegena em Leucaena leucocephala (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute) a partir da avaliaccedilatildeo do efeito de tratamentos
relacionados ao estresse abioacutetico (UV-C aacutecido saliciacutelico metil jasmonato e etileno)
Mimosina eacute um ANP aromaacutetico anaacutelogo da L-tirosina com atividade toacutexica para ceacutelulas
de procariotos e eucariotos Dentre as atividades descritas para esse ANP destacam-se a
atividade anti-mitoacutetica ou bloqueadora do ciclo celular atividade alelopaacutetica e
antioxidante Os resultados indicaram que em leucena a biossiacutentese e o acuacutemulo de
mimosina podem ser modulados por fatores causadores de estresses exibindo um padratildeo
de acumulaccedilatildeo similar ao das fitoalexinas Em maricaacute por outro lado a induccedilatildeo do
acuacutemulo dessa moleacutecula natildeo foi observada para os mesmos tratamentos testados para
leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas Aleacutem disso
o padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado
pela sua degradaccedilatildeo As respostas observadas nos testes que avaliaram a atividade de
mitigaccedilatildeo de espeacutecies reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode
agir como um agente antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de
estresse
1
Introduccedilatildeo
Na condiccedilatildeo de organismos seacutesseis ao longo de sua evoluccedilatildeo as plantas
desenvolveram estrateacutegias estruturais e quiacutemicas de defesa em resposta aos estresses bioacuteticos
e abioacuteticos impostos pelo ambiente Dentre essas satildeo reconhecidas moleacuteculas quimicamente
especializadas denominadas metaboacutelitos secundaacuterios produtos naturais (Kutchan et al 2015)
ou mais recentemente metaboacutelitos especializados
Entre as trecircs classes mais gerais de metaboacutelitos secundaacuterios (terpenos compostos
fenoacutelicos e compostos nitrogenados) aminoaacutecidos natildeo-proteicos (ANPs) satildeo incluiacutedos no
terceiro grupo e constituem aleacutem de componentes do arsenal de defesa quiacutemica uma
importante fonte de reserva de carbono e nitrogecircnio para diversos taxa especialmente aqueles
pertencentes agrave famiacutelia Fabaceae de Angiospermas (leguminosas sensu lato)
Aleacutem dos 20 aminoaacutecidos proteicos estima-se que existam entre 600 e 1000 ANPs
(Acamovic amp Brooker 2005 Rodgers et al 2015) Esse grupo de moleacuteculas quimicamente
heterogecircneo eacute assim definido por natildeo participar da formaccedilatildeo de estruturas proteicas
funcionais sendo frequentemente toacutexicos quando erroneamente incorporados nas cadeias
polipeptiacutedicas em formaccedilatildeo em funccedilatildeo da similaridade estrutural que apresentam com os
aminoaacutecidos proteicos (Taiz amp Zeiger 2010)
Conforme mencionado a ocorrecircncia de ANPs eacute comum em espeacutecies de leguminosas
e sua distribuiccedilatildeo pode ser restrita a alguns gecircneros de plantas circunscritos nessa famiacutelia
botacircnica (eg mimosina e canavanina) Por outro lado alguns ANPs como GABA por
exemplo podem apresentar distribuiccedilatildeo ubiacutequa no Reino Plantae assim como ocorrer em
outros tipos de organismos como animais por exemplo (Ramos-Ruiz et al 2018)
2
Apesar de representarem uma fonte nutricional importante sem tratamento preacutevio o
consumo de plantas que acumulam ANPs por animais eacute limitado Isso ocorre pois em longo
prazo a ingestatildeo prolongada de plantas (especialmente sementes) que acumulam ANPs pode
representar risco agrave sauacutede uma vez que estes comprometem o funcionamento de mecanismos
basais de manutenccedilatildeo da homeostase celular e podem tambeacutem em um quadro mais severo
desencadear doenccedilas neurotoacutexicas degenerativas como por exemplo o latirismo causado
por aacutecido β-N-oxalil-l-αβ-diaminopropiocircnico (β-ODAP) (Jiao et al 2011 Kusama-Eguchi
2019)
Sob o ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados
ANPs satildeo em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou
abioacutetica como o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de
elicitores quiacutemicos por exemplo No que concerne ao estudo dos efeitos da induccedilatildeo abioacutetica
sobre o acuacutemulo de ANPs em diferentes espeacutecies vegetais (Monocotiledocircneas e
Eudicotiledocircneas) as moleacuteculas mais amplamente investigadas ateacute o momento satildeo GABA
L-DOPA e mais recentemente mimosina (vide Tabela 1 do capiacutetulo primeiro) Em termos
de efeitos causados a partir da aplicaccedilatildeo exoacutegena de ANPs GABA tambeacutem figura como o
principal aminoaacutecido investigado seguido de L-DOPA e canavanina (vide Tabela 2 do
capiacutetulo primeiro)
No primeiro capiacutetulo da presente tese estatildeo descritas as caracteriacutesticas gerais dos
principais ANPs estudados seus possiacuteveis papeacuteis bioloacutegicos in planta e seus efeitos quando
aplicados exogenamente bem como os estresses abioacuteticos capazes de induzir seu(s)
acuacutemulo(s) nos diferentes tecidos vegetais Nos segundo e terceiro capiacutetulos
respectivamente satildeo elucidados os efeitos dos tratamentos de UV-C aacutecido saliciacutelico etileno
e jasmonato (claacutessicos elicitores do metabolismo secundaacuterio vegetal) sobre o acuacutemulo de
3
mimosina em Leucaena leucocephala var glabrata (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute)
Mimosina eacute um aminoaacutecido aromaacutetico natildeo-proteico anaacutelogo da L-tirosina com
atividade toacutexica para ceacutelulas de procariotos e eucariotos Embora em menor concentraccedilatildeo
mimosina foi primeiramente identificada em Mimosa pudica sendo posteriormente detectada
em outras espeacutecies do gecircnero como Mimosa pigra por exemplo (Soedarjo amp Borthakur
1998) Seu efeito toacutexico eacute atribuiacutedo agrave capacidade de quelar metais o que impede o
funcionamento adequado das metalo-proteiacutenas que dependem dos mesmos como co-fatores
(Negi et al 2014)
A concentraccedilatildeo basal de mimosina em espeacutecies de leucaena pode variar entre 1 e 12
do peso seco do oacutergatildeo (Soedarjo amp Borthakur 1998) Como eacute comum para outros ANPs
que ocorrem em espeacutecies de leguminosas em sementes de Leucaena spp eacute observada uma
maior concentraccedilatildeo de mimosina quando comparada aos demais oacutergatildeos da planta
(Rodrigues-Correcirca et al 2019) sendo esta a fonte de extraccedilatildeo comercial do padratildeo quiacutemico
de mimosina vendido por empresas de reagentes de pesquisa
Diversas atividades foram descritas para mimosina em outros organismos eou tipos
celulares Dentre essas destacam-se a atividade anti-mitoacutetica ou bloqueadora do ciclo
celular em ceacutelulas de eucariotos e procariotos Isto ocorre porque a mimosina impede a
formaccedilatildeo da forquilha de replicaccedilatildeo (e portanto a siacutentese de DNA) interrompendo assim o
avanccedilo do ciclo de divisatildeo celular na fase tardia G1 (Lalande 1990) Foram tambeacutem descritas
para mimosina atividade alelopaacutetica observada sobre o desenvolvimento de outras espeacutecies
de leguminosas e atividade antioxidante entre outras (Tabela 1)
A rota de biossiacutentese de mimosina eacute compartilhada em grande parte com a de cisteiacutena
um aminoaacutecido proteico sulfurado (Figura 1) A siacutentese da cisteiacutena se daacute a partir da conversatildeo
4
de serina e acetil-CoA em o-acetilserina pela enzima SAT (serina acetiltransferase) seguida
da conversatildeo de o-acetilserina e aacutecido sulfiacutedrico em cisteiacutena em uma reaccedilatildeo catalisada pela
OAS-TL (o-acetilserina tiol-liase) A siacutentese de mimosina por sua vez eacute compartilhada com
a da cisteiacutena ateacute esse ponto e acredita-se que pelo menos uma das isoformas de OAS-TL
catalise a conversatildeo de o-acetilserina e 3-hidroxi-4-piridona em mimosina
Tabela 1 Atividades descritas para mimosina de Leucaena leucocephala (Lam) de Wit
ATIVIDADE
ALVO AVALIADO
(organismo eou tecido tipo
celular)
REFEREcircNCIA
Bloqueio do complexo de ativaccedilatildeo
da preacute-replicaccedilatildeo do DNA
Ceacutelulas de mamiacuteferos
KUBOTA et al
(2014)
Alteraccedilatildeo no ciclo ovariano e
extensatildeo da duraccedilatildeo do corpo luacuteteo
bovino no periacuteodo poacutes-parto
Bovinos
(Bos taurus x
Bos indicus)
BOTTINI-
LUZARDO et al
(2015)
Supressatildeo do ciclo celular e reduccedilatildeo
da abundacircncia bacteriana em
mosquitos
Wolbachia pipientis
Aedes albopictus
FALLON
(2015)
Accedilatildeo inibitoacuteria da fibrose
pulmonar induzida
Ratos SD
LI et al
(2015)
Recuperaccedilatildeo da funccedilatildeo do
miocaacuterdio poacutes-isquemia
Miocaacuterdio de ratos (SD)
machos
CROWE et al
(2001)
Inseticida
Heteropsylla cubana
Crawford 1914 e Thrips tabaci
Lindemann 1889
AHMED et al
(2016)
Alelopaacutetica
Albizia procera Vigna
unguiculata Cicer arietinum
Cajanus cajan
AHMED et al
(2008)
Antioxidante
Sistemas modelo de oxidaccedilatildeo
lipiacutedica (β-caroteno - aacutecido
linolecircico e lecitina)
BENJAKUL et al
(2013)
Ateacute momento versotildees divergentes sobre a enzima responsaacutevel pela biossiacutentese de
mimosina (mimosina sintase) tecircm sido publicadas Em 1990 Ikegami e colaboradores
5
identificaram uma OAS-TL responsaacutevel pela formaccedilatildeo de cisteiacutena como sendo tambeacutem uma
mimosina sintase Mais tarde Yafuso et al (2014) realizaram a expressatildeo heteroacuteloga do gene
que codifica para OAS-TL em Escherichia coli e natildeo foi observada a formaccedilatildeo de mimosina
mesmo quando dadas as condiccedilotildees oacutetimas para tanto Mais recentemente Harun-Ur-Rashid
et al (2018) elucidaram a mimosina sintase como sendo uma isoforma da OAS-TL
corroborando o postulado por Ikegami e colaboradores em 1990
Figura 1 Rota de biossiacutentese da mimosina Fonte Ikegami et al (1990)
Espeacutecies estudadas
Leucaena leucocephala (Lam) de Wit (leucaena koa haole ou ldquoacaacutecia exoacuteticardquo na
liacutengua Hawairsquoiana) eacute uma espeacutecie de haacutebito arboacutereo ou arbustivo pertencente agrave famiacutelia
Fabaceae de Angiospermas e caracterizada pelo acuacutemulo de mimosina em todos os seus
oacutergatildeos Eacute nativa da Ameacuterica Central (especificamente da regiatildeo sudeste do Meacutexico) mas
irradiou-se atraveacutes de praticamente todas as zonas tropicais e subtropicais da Terra No
Brasil leucena eacute amplamente distribuiacuteda e classificada como naturalizada pelo REFLORA
(2019) ocorrendo em todo territoacuterio Nacional Satildeo reconhecidas no miacutenimo duas
6
subespeacutecies de leucena ocorrentes no Brasil L leucocephala var leucocephala e L
leucocephala var glabrata sendo a primeira a mais abundante
Leucaena apresenta atributos morfoloacutegicos caracteriacutesticos das leguminosas como o
fruto do tipo vagem deiscente no periacuteodo poacutes-maturaccedilatildeo folhas compostas e bipinadas As
flores satildeo seacutesseis actinomorfas e polistecircmones apresentam caacutelice sinseacutepala e corola
gamopeacutetala e satildeo dispostas em inflorescecircncias do tipo glomeacuterulo (Figura 2)
Figura 2 Oacutergatildeos vegetativos e reprodutivos de L leucocephala (Lam) de Wit Fonte Little Jr amp Skolmen
(1989)
Com base no conhecimento etnobotacircnico disponiacutevel acerca dessa espeacutecie em
diversas regiotildees tropicais e subtropicais leucena eacute utilizada para vaacuterios fins Extratos de
diferentes oacutergatildeos de leucena apresentam atividade anti-diabeacutetica (Kuppusamy et al 2014
Chowtivannakul et al 2016) antioxidante (Mohammed et al 2015 Chowtivannakul et al
2016 Zarin et al 2016) antimicrobiana (Zarin et al 2016) anti-helmiacutentica (Soares et al
2015 Jamous et al 2017) bactericida (Mohammed et al 2015) acaricida (Fernaacutendez-Salas
et al 2011) anti-tumoral (Chung et al 2017) e potencializadora da resposta imune em
peixes (Verma et al 2018) entre outras
7
Leucaena apresenta alta toleracircncia agrave seca sendo capaz de enfrentar estaccedilotildees sazonais
inteiras com deacuteficit hiacutedrico sem prejuiacutezo permanente de seus oacutergatildeos e de recuperar
vigorosamente sua biomassa vegetativa tatildeo logo o regime de precipitaccedilatildeo retome a
regularidade em frequecircncia Acredita-se que a toleracircncia agrave seca apresentada por essa espeacutecie
ocorra em funccedilatildeo do acuacutemulo de mimosina nos diferentes tecidos da planta a qual
funcionaria como um agente osmoregulador responsaacutevel pela preservaccedilatildeo da integridade das
membranas a das macromoleacuteculas intracelulares em periacuteodos de escassez de aacutegua no
ambiente
Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) eacute uma leguminosa
nativa natildeo endecircmica do Brasil amplamente distribuiacuteda nos domiacutenios fitogeograacuteficos da
Caatinga do Cerrado e da Mata Atlacircntica (Simon amp Proenccedila 2000 REFLORA 2019) Como
espeacutecie pioneira (Pilatti et al 2019) exerce importante papel ecoloacutegico na recuperaccedilatildeo de
aacutereas degradadas (Bitencourt et al 2007 Silva et al 2011) no estabelecimento de processos
de sucessatildeo vegetacional
Maricaacute eacute uma espeacutecie semi-deciacutedua a deciacutedua a qual atinge ateacute 15 m em altura (e
diacircmetro agrave altura do peito de ateacute 40 cm) na idade adulta com haacutebito arboacutereo ou arbustivo
(REFLORA 2019) e espinhos caracteriacutesticos desde os estaacutegios iniciais de desenvolvimento
(Carvalho 2004) Apresenta folhas compostas alternas e bipinadas (Figura 2) amplas
inflorescecircncias brancas com flores reunidas em glomeacuterulos esfeacutericos dispostos em grandes
paniacuteculas As flores satildeo diplostecircmones actinomorfas hipoacuteginas e unicarpelares (Silva et al
2011)
Assim como descrito para leucena maricaacute eacute considerado uma espeacutecie multifuncional
sendo comumente empregada para produccedilatildeo de mel como combustiacutevel (Olkoski amp
8
Wittmann 2011) em edificaccedilotildees na carpintaria e como lsquocerca-vivarsquo (Marchiori 1993
Lorenzi 1998) entre outras aplicaccedilotildees
Figura 2 Folhas e fruto de Mimosa bimucronata (DC) Kuntze Fonte Souza-Lima et al (2017)
Em contraste com a amplitude de habitats explorados por leucena (especialmente os
aacuteridos) no Sul do Brasil maricaacute ocorre preferencialmente em ambientes uacutemidos a alagadiccedilos
em aacutereas proacuteximas agraves margens de rios (Patreze amp Cordeiro 2004) embora possa tambeacutem
ocorrer em formaccedilotildees quase exclusivas dessa espeacutecie nas encostas de morros (Jacobi amp
Ferreira 1991)
Em relaccedilatildeo agraves atividades elucidadas para os extratos de maricaacute foram relatados os
efeitos alelopaacutetico (Jacobi amp Ferreira 1991 Ferreira et al 1992) diureacutetico natriureacutetico e
caliureacutetico (Schlickmann et al 2017)
9
Hipoacutetese
Mimosina apresenta perfil dinacircmico de acuacutemulo em Leucaena leucocephala e
Mimosa bimucronata frente a estresses associado a alteraccedilotildees significativas na expressatildeo de
genes relacionados ao metabolismo deste ANP o qual contribui para mitigar o desequiliacutebrio
oxidativo inerente a vaacuterios tipos de estresse
Objetivo geral
O objetivo da presente tese foi investigar o papel bioloacutegico da mimosina endoacutegena
em leucena e maricaacute a partir da avaliaccedilatildeo do efeito de tratamentos relacionados a estresses
ou sinalizadores de estresse
Objetivos especiacuteficos
- Analisar a concentraccedilatildeo constitutiva de mimosina nos diferentes oacutergatildeos de L leucocephala
(Lam) de Wit (leucena) e M bimucronata (DC) Kuntze (maricaacute)
- Verificar se apesar do seu alto teor constitutivo em plantas de leucena o acuacutemulo de
mimosina pode ser induzido com tratamentos que mimetizam diferentes estresses a partir da
avaliaccedilatildeo do efeito de moleacuteculas sinalizadoras (aacutecido saliciacutelico jasmonato etileno) e da
exposiccedilatildeo agrave radiaccedilatildeo UV-C na modulaccedilatildeo do acuacutemulo de mimosina em leucena bem como
em maricaacute
- Determinar se a expressatildeo de genes relacionados ao metabolismo de mimosina estaacute
associada agrave induccedilatildeo por estresses fisioloacutegicos
- Avaliar o potencial antioxidante da mimosina em experimentos realizados in situ
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry
journal homepage wwwelseviercomlocateplaphy
Research article
Mimosine accumulation in Leucaena leucocephala in response to stresssignaling molecules and acute UV exposure
Kelly Cristine da Silva Rodrigues-Correcircaab Michael DH Hondab Dulal BorthakurbArthur Germano Fett-Netoalowast
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970Porto Alegre Rio Grande do Sul BrazilbDepartment of Molecular Biosciences and Bioengineering University of Hawaii at Manoa Honolulu HI 96822 USA
A R T I C L E I N F O
KeywordsLeucaena leucocephalaMimosineMimosine amidohydrolaseJasmonic acidEthyleneSalicylic acidUV-C radiation
A B S T R A C T
Mimosine is a non-protein amino acid of Fabaceae such as Leucaena spp and Mimosa spp Several relevantbiological activities have been described for this molecule including cell cycle blocker anticancer antifungalantimicrobial herbivore deterrent and allelopathic activities raising increased economic interest in its pro-duction In addition information on mimosine dynamics in planta remains limited In order to address this topicand propose strategies to increase mimosine production aiming at economic uses the effects of several stress-related elicitors of secondary metabolism and UV acute exposure were examined on mimosine accumulation ingrowth room-cultivated seedlings of Leucaena leucocephala spp glabrata Mimosine concentration was not sig-nificantly affected by 10 ppm salicylic acid (SA) treatment but increased in roots and shoots of seedlings treatedwith 84 ppm jasmonic acid (JA) and 10 ppm Ethephon (an ethylene-releasing compound) and in shoots treatedwith UV-C radiation Quantification of mimosine amidohydrolase (mimosinase) gene expression showed thatethephon yielded variable effect over time whereas JA and UV-C did not show significant impact Consideringthe strong induction of mimosine accumulation by acute UV-C exposure additional in situ ROS localization aswell as in vitro antioxidant assays were performed suggesting that akin to several secondary metabolitesmimosine may be involved in general oxidative stress modulation acting as a hydrogen peroxide and superoxideanion quencher
1 Introduction
Different plant groups synthesize a large diversity of secondary orspecialized metabolites These molecules are generally produced inresponse to biotic and abiotic environmental stresses Indeed inductionof secondary metabolism usually involves stress-generating factorswhich have also been explored in biotechnological processes aiming atthe production of target metabolites of economic interest (Matsuuraet al 2018) Metabolic control of nitrogen-containing secondarycompounds (eg alkaloids and non-protein amino acids) has beenshown to be complex and influenced by phytohormones environmentalstresses (seasonality herbivory pathogen attack drought) UV radia-tion (Holloacutesy 2002) methyl jasmonate (MeJA) salicylic acid (SA)yeast extract (Cho et al 2008) abscisic acid (ABA) heavy metals os-motic stress (Nascimento et al 2013) and mechanical wounding (Portoet al 2014)
Due to their particular trait of associating with N-fixing micro-organisms Fabaceae species (leguminous sensu lato) are often proteinrich hence the relevance of several of these species as forage Fabaceaespecies are also known for accumulating nitrogen containing secondarymetabolites which play important roles as ecochemical molecules andat least for the case of non-protein amino acids potential cell reservoirsof nitrogen (Huang et al 2011)
High contents of mimosine a toxic aromatic non-protein aminoacid are found in species of two leguminous genera Leucaena spp andMimosa spp Leucaena leucocephala (Lam) de Wit (leucaena koa haole)is a fast-growing leguminous tree native from Central America (south-eastern Mexico) widely distributed in tropical and subtropical zonesThis species is also characterized by its high tolerance to droughtamong other environmental stresses (Honda et al 2018) Leucaena canbe divided into two subspecies (i) L leucocephala subsp leucocephala(common leucaena a bushy shrub) and (ii) L leucocephala subsp
httpsdoiorg101016jplaphy201811018Received 1 August 2018 Received in revised form 9 November 2018 Accepted 14 November 2018
lowast Corresponding authorE-mail addresses krodriguescbiotufrgsbr (KCdS Rodrigues-Correcirca) mhonda2hawaiiedu (MDH Honda) dulalhawaiiedu (D Borthakur)
fettnetocbiotufrgsbr (AG Fett-Neto)
Plant Physiology and Biochemistry 135 (2019) 432ndash440
Available online 19 November 20180981-9428 copy 2018 Elsevier Masson SAS All rights reserved
T
glabrata (giant leucaena a tree) The latter has been used as a fastgrowing tree for production of wood and paper pulp The foliage ofboth common and giant leucaena is used as a fodder because of its highprotein content and palatability to farm animals The foliage containsup to 18 protein 142 crude fiber and 64 ether extractcrude fat(Soedarjo and Borthakur 1996)
Production of nitrogen-containing secondary metabolites such asmimosine requires large amounts of carbon and nitrogen resourcesNegi et al (2014) estimated that up to 21 of the carbon-nitrogenresources may be used for production of mimosine in leucaenaBrewbaker et al (1972) determined the mimosine content of 96 Lleucocephala cultivars and 8 other Leucaena species collected from 38different countries by growing them in an observational nursery inHawaii and found that basal mimosine content varied from 189 to477 of the dry weight
Mimosine is biosynthesized from OAS (o-acetylserine) and 3H4P (3-hydroxy-4-pyridone or its tautoisomer 3-hydroxy-4-pyridine) A pre-vious analysis suggested that mimosine synthase is an OAS-TL (o-acetylserine-thiol-lyase) of the cysteine biosynthesis pathway (Ikegamiet al 1990) Later however recombinant enzyme tests did not supportan OAS-TL identity of mimosine synthase (Yafuso et al 2014) Recentfindings on mimosine biosynthesis revealed that a cytosolic cysteine-OAS-TL isoform can also catalyze the formation of mimosine underspecific conditions (Harun-Ur-Rashid et al 2018)
Mimosine toxicity is related to its ability of reducing the availabilityof divalent metal ions such as Fe(II) Zn(II) Cu(II) Co(II) and Mn(II)by chelating co-factors and preventing their association with metal-dependent enzymes Furthermore this non-protein amino acid is cap-able of forming a stable complex with pyridoxal-5prime-phosphate (PLP)leading to the inactivation of PLP-dependent enzymes (eg Asp-Glutransaminase and cystathionine synthetase) (Negi et al 2014)
Mimosine features several useful biological activities such as alle-lopathic antimicrobial insecticide cell cycle inhibitor agent antic-ancer phytoremediator (Nguyen and Tawata 2016) as well as anti-oxidant (Benjakul et al 2013) Despite the relatively well establishedbiological activities of purified mimosine on other organisms or celltypes little is known about its biological role in leguminous speciesHowever it has been suggested that at least in part its activity ismainly related to defense mechanisms against some biotic and abioticstresses and as nitrogen source during fast growth (Vestena et al2001)
Suda (1960) and Smith and Fowden (1966) identified enzymes in-volved in mimosine degradation in seedling extracts of L leucocephalaand Mimosa pudica A mimosine-degrading enzyme named mimosinase(mimosine amidohydrolase EC 35161 CAS registry number 104118-49-2) (IUBMB 2018) a carbon-nitrogen lyase which degrades mimo-sine into 3H4P was later purified by Tangendjaja et al (1986) Itsbiochemical characterization was described and the cDNA was isolatedby Negi et al (2014)
Although mimosinase has been described and isolated only fewstudies on the role played by biotic and abiotic factors on the dynamicmodulation of mimosine metabolism in leguminous species have beenconducted (Vestena et al 2001 Xu et al 2018) In aseptic cultures ofleucaena mechanical injury of shoots promoted local mimosine accu-mulation (Vestena et al 2001) In the same study cultivation in pre-sence of auxin or SA in culture medium also had a positive effect on
mimosine accumulation More recently the effect of drought treatmenton gene expression of leucaena was also evaluated by Honda et al(2018) However several potential factors regulating mimosine meta-bolism need to be further examined
To date there is a lack of information on the biological role ofmimosine in planta as well as on details of its metabolic dynamicsMoreover its overt potential for pharmaceutical applications and de-velopment of new drugs as well as the possible use as tool to addressheavy metal soil contamination or plant mineral nutrition improve-ment justify additional research The objective of this study was toinvestigate the effect of stress signaling molecules and acute UV ex-posure on modulation of mimosine accumulation and metabolism in Lleucocephala spp glabrata in order to better understand its biologicalrole and to identify strategies for yield improvement aiming at ex-ploring its useful bioactivities
2 Methods
21 Plant material
For the experiments carried out to evaluate the effects of elicitors onmimosine accumulation seeds of leucaena were kindly provided by DrJames Brewbaker and harvested at CTAHRs (College of TropicalAgriculture and Human Resources of the University of Hawaii atManoa) Waimanalo Research Station at Oahu Hawaii This plantmaterial was originated from the accession K636 of Leucaena leucoce-phala ssp glabrata (Brewbaker 2008)
22 Induced mimosine content in 5-week-old giant leucaena
221 Seed germinationIn order to overcome seed coat dormancy seeds were submitted to a
chemical scarification with sulfuric acid 95ndash98 for 20min and re-peatedly rinsed in distilled water to remove any residual trace of thisreagent Then seeds were distributed in 254 cmtimes508 cm plastictrays containing 11 vv of vermiculite and commercial soil watereduntil reaching substrate field capacity Three weeks after seed imbibi-tion seedlings displaying similar size and shape (eg number of com-pound leaves and leaflets) were transplanted to individual pots(250mL) in number of three plants per container
During the experimental period (except in the UV-C radiationtreatment) all tested seedlings were kept in a growth chamber andsubmitted to controlled conditions of temperature (circa 25 degC) and ir-radiance (approximately 100 μmol photons mminus2sdot s minus1) with a photo-period of 16 h light and 8 h dark
222 Treatments2221 JA Ethephon and SA Five-week-old giant leucaena seedlingswere treated with different solutions as described in Table 1 Idealconcentrations were defined in preliminary experiments under the sameconditions indicated above At the beginning of the experiments 30plants were sprayed with 84 ppm JA 10 ppm SA 10 or 100 ppmEthephon or Milli-Qreg water (control) until the point of imminent runoffPlant pots were kept closed inside transparent plastic bags for 24 h toavoid solution volatilization Fifteen plants arranged in 5 sets of 3 (5biological replicates) were harvested 48 h and 96 h after being treated
Table 1Treatments used to modulate mimosine biosynthesis in giant leucaena
ELICITOR CONCENTRATION UV FLUENCE EXPOSURE TIME RATIONALE FOR USE
Salicylic acid (SA) 10 ppm 24 h Pathogen signaling molecule (Shah 2003)Jasmonic acid (JA) 84 ppm 24 h Chemical elicitor of plant secondary metabolism (Dar et al 2015)Ethephon 10 ppm 24 h Ethylene releasing-compound (Kim et al 2016) elicitor of plant secondary metabolism (Wang
et al 2016)UV-C radiation 3 Jcmminus2 10min or 15min Elicitor of plant secondary metabolism (Kara 2013 Neelamegam and Sutha 2015)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
433
After collection shoots were separated from roots immediately frozenin liquid nitrogen and stored at ndash 80 degC prior to HPLC analyses
2222 UV-C Thirty seedlings of giant leucaena were exposed to UV-Cradiation (3 Jcmminus2) for 10 or 15min and kept in a growth chamberunder controlled conditions as described above until the end of theexperiments Fifteen plants arranged in groups of 3 were harvested at96 h and 120 h after UV-C exposure and processed as previouslydescribed
223 Mimosine extractionMimosine extraction was based on a modified version of the pro-
tocol published by Lalitha and Kulothungan (2006) as follows a knownweight of fresh tissue (shoots or roots) of giant leucaena was first addedto Milli-Qreg boiling water in a proportion of 110 (g of plant per mL ofsolvent) in test tubes Tubes were covered with foil to avoid solutionevaporation and placed on a hot stirrer at 100 degC for 10min A pro-portional volume of 01M HCl was added to the cooled suspensions andhomogenized using mortar and pestle The plant extracts were filteredthrough cotton and centrifuged twice for 7min in a bench top re-frigerated centrifuge at 4 degC and 13200 rpm Before being analyzed theextracts were diluted 13 with ondashphosphoric acid (OPA)
224 Mimosine detectionHPLC analyses were carried out as described by Negi and Borthakur
(2016) Pure mimosine (L-mimosine from koa haole seeds Sigma-Al-drich CAS number 500-44-7) was used as standard Separation andquantification of mimosine was done with a C18 column (PhenomenexC18 5 μm 46times250mm) under an isocratic solvent system of 002MOPA with a linear flow rate of 1mLsdotminminus1 Mimosine detection wasdone at 280 nm by photodiode array detection (200ndash400 nm) showingretention time of 277 plusmn 0042min Quantification was done using themethod of external standard curve Further confirmation of mimosineidentity was performed by co-chromatography with standard and peakpurity check Chromatograms were analyzed using the Waters Em-power 3 software
23 Quantitative real-time PCR analysis of mimosinase gene expression
Fifteen 8-week-old giant leucaena plants arranged in 4 sets of 3 (4biological replicates) were treated with either water (control) or10 ppm Ethephon 84 ppm JA acid or 15min of UV-C radiation ex-posure following the methods described above Following treatmentleucaena plants were harvested at 48 and 96 h or 72 and 144 h (UV-Ctreated plants only) after treatments Total RNA of samples was ex-tracted and purified from roots and shoots of giant leucaena by meansof a modified method using Qiagen RNeasy Plant Kit (Valencia CAUSA) and Fruit-mate (Takara Japan) according to the protocol de-scribed by Ishihara et al (2016a) The assessment of RNA quality andquantity was carried out at 230 260 and 280 nm by using a NanoDropSpectrophotometer ND-1000 (NanoDrop Technologies DE USA) Inorder to avoid genomic DNA contamination RNA samples were treatedwith TURBO DNAfree Kit (Invitrogen Carlsbad CA) Two microgramsof DNase-treated RNA were used to synthesize the first-strand cDNAusing M-MLV Reverse Transcriptase (Promega WI USA)
Quantitative real-time (qPCR) analysis was carried out to examinepossible differential expression of the mimosinase gene (GenBank ac-cession number AB2985971) in seedlings treated with 84 ppm JA10mM Ethephon or 15min of UV-C exposure Shoots and roots wereharvested 24 h before the time of mimosine concentration peak for eachtreatment previously observed as assessed by HPLC assays The 10 μLqPCR reaction consisted of 5 μL of PowerUpTM SYBRreg Green MasterMix (Applied Biosystems Foster City CA) 1 μL MgCl2 (50mM) 03 μLforward primer (10 μM) 03 μL reverse primer (10 μM) and 1 μL cDNAfirst-strand In the experimental validation through qPCR reactionconditions and melting curve analysis of the amplicon were performed
following the protocol published by Ishihara et al (2016b) for the sameleucaena variety qPCR analysis was conducted using StepOnetrade Real-Time PCR System (Applied Biosystems) Measurements were performedusing 4 biological and 3 technical replicates Relative expression wascalculated with the 2-ΔΔct method using OAS-TL as reference gene sinceits expression showed a consistently stable profile comparable to that ofUBQ-5 and ELF1α expressions Mimosinase primer sequences used forthese analyses were (FWD) 5prime- GAA AGG CAG GAA TCA CAG TGA AGAG ndash 3rsquo (REV) 5prime GGA GAC TCT AGC CAC ACC AAC TTA ndash 3rsquo
24 Antioxidant assays
241 Mimosine effect on hydrogen peroxide (H2O2) accumulationAs a follow up to the induction of mimosine accumulation profiles
under stress signals and conditions tests were conducted to verify mi-mosine antioxidant capacity In situ histological localization of hy-drogen peroxide (H2O2) accumulation was evaluated on foliar disks ofPhaseolus vulgaris L according to the protocol described by Shi et al(2010) Briefly the plant foliar tissue was exposed to 1 mgmiddotmLminus1 dia-minobenzidine (DAB) solution in 10 mM KH2PO4 (control) in presenceor absence of 10mM mimosine (equivalent to the average mimosineconcentration induced by UV-C radiation in giant leucaena) or 10mMascorbic acid (positive antioxidant control) Oxidative response wasidentified by the formation of a brown polymer on the injured leafareas indicating the presence of H2O2 and registered in a Leica M165FC stereomicroscope (Leica Microsystems)
242 Mimosine quenching of superoxide radicalsGeneration of superoxide radical and subsequent analysis was per-
formed by a modified protocol based on Zhishen et al (1999) Nitroblue tetrazolium (NBT) reduction was used to measure superoxide an-ions quenching activity Shortly a 50mM KH2PO4 pH 78 solutioncontaining 6 μM riboflavin 100mM methionine 1 mM NBT in pre-sence or absence of 5mM mimosine was exposed to white light(22 Jsdotcmminus2) for 25min on a white light transilluminator Five micro-molar rutin was used as positive control (Matsuura et al 2016) Theabsorbance was read at 560 nm before and after light exposure in aSpectraMaxreg M2 Microplate Reader (Molecular Devices LLC)
25 Statistical analyses
For HPLC and superoxide anions data simple analyses of variance(ANOVA) followed by Tukey or Welch ANOVA followed by Dunnetts Ctest were used as appropriate for data distribution characteristics InqPCR analysis results were analyzed by t-test In all cases at least fourbiological triplicates were used and experiments were repeated twiceindependently All data were analyzed using the statistical packageSPSS 200 for Windows (SPSS Inc USA) In all cases a ple 005 wasused
3 Results and discussion
31 Increased mimosine concentrations in giant leucaena treated withchemical elicitors
Leucaena produces high amounts of mimosine that accumulate in allparts of the plants including leaves stem flowers pods seeds rootsand root nodules (Soedarjo and Borthakur 1998) The highest con-centrations of mimosine can be found in the growing shoot tips andseeds (Wong and Devendra 1983) It is not known why leucaena pro-duces such high amounts of mimosine Negi et al (2014) estimated thatleucaena plants would be able to grow 21 larger if the nutrient re-sources spent on mimosine production were diverted for biomass in-crease In a previous analysis performed to quantify the basal con-centration of mimosine present in adult plants of common leucaena thehighest constitutive amount of mimosine per gram of fresh weight in
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
434
the analyzed organs was found in post-anthesis flowers (89448 μg)followed by green pods (82687 μg) leaves (67358 μg) and greenflower buds (51247 μg) which showed significantly less mimosineconcentration compared to the other reproductive structures(Supplementary Fig 1) Since mature seeds have very low moisturecontent (Wencomo et al 2017) its mimosine concentration was esti-mated as 338253 μgsdotgminus1 of dry weight Additionally it was also ob-served that the basal mimosine distribution in shoots of field-grownadult plants of leucaena is dependent on the variety type(Supplementary Table 1)
Phytohormones such as salicylic acid and jasmonic acid are knownto be produced by plants in response to various abiotic and bioticstresses These phytohormones trigger adaptive responses to stress byregulating major plant metabolic processes such as photosynthesisnitrogen metabolism defense systems and plant-water relationsthereby providing protection (for review see Khan et al 2015)
Secondary or specialized metabolite production and accumulationare also known to be controlled by biotic and abiotic stresses (Matsuuraet al 2018) In this study exposure of 5-week-old giant leucaenaseedlings to JA or Ethephon treatments significantly enhanced mimo-sine accumulation in shoots and roots in at least one of the two timepoints tested (48 and 96 h) albeit in a different way (Fig 1) Thehighest concentrations of mimosine in shoots were found in seedlingstreated with JA 84 ppm (43441 μgsdotgminus1) and Ethephon 100 ppm(38412 μgsdotgminus1) two days after application of the respective phyto-hormones Nevertheless after four days shoots yielded the highestconcentration of mimosine (approximately 460 μgsdotgminus1) upon treatmentwith 10 or 100 ppm Ethephon (Fig 1A) In roots after two and four
days JA 84 ppm and Ethephon 10 ppm resulted in highest mimosineaccumulation 18488 μgsdotgminus1 and 15801 μgsdotgminus1 respectively (Fig 1B)These observations show that mimosine accumulation response tospecific elicitors may vary over time after exposure
Although all treatments were applied exclusively on shoots of giantleucaena seedlings roots of some of them were also able to respond tothe different elicitors Overall shoots displayed higher basal and in-duced mimosine concentration compared to roots (Fig 1) which agreeswith previous observations in 1 to 3-week-old aseptic seedlings ofcommon leucaena (Vestena et al 2001) However as previouslymentioned significant post-induction increase of mimosine concentra-tion in roots and shoots simultaneously was only observed for JA andEthephon 10 ppm on day 02 and 04 respectively (Fig 1)
It is well established that perceived regulatory signals or elicitorsgenerate a transduction network mediated by secondary messengersresulting in changes in gene expression profiles that afford adaptiveresponses to environmental stimuli These modulation events are oftenmediated by transcription factors (TFs) which directly bind to specificgene promoters or act by forming complexes with repressor proteinslabeling them to degradation subsequently releasing other TFs toproceed with the gene expression program This is the case of the actionmechanism of JA and its active form jasmonoyl isoleucine for example(Kazan 2015 Wasternack and Strnad 2016)
JA ethylene and SA are known as important stress regulatory sig-nals in plants JA however is thought to be the most effective signal forinduction of plant secondary metabolism (Wasternack and Strnad2016) thereby contributing to mitigation of damage caused by severalstresses (Dar et al 2015) JA is mainly derived from linolenic acid
Fig 1 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings treated with differentelicitors CTRL=Milli-Q water SA = Salicylic AcidJA= Jasmonic Acid ETH=Ethephon Bars sharing a letterof same case do not differ by Tukey test (P le 005) Capitalletters (A B) compare treatments on day two and lowercaseletters (a b) compare treatments on day four Indicatessignificant statistical difference between day two and dayfour in the same treatment by t-test (Ple 005) The errorbars represent standard error of five replicates (each meanwas calculated with 15 individual seedlings organized in 5groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
435
(Wasternack and Strnad 2016) playing important roles in differentprocesses of plant growth and development such as plant defensemechanisms against herbivory pathogen attack fungal elicitation andsome abiotic factors such as osmotic temperature and salt stresses (Daret al 2015)
JA and its methyl ester MeJA have several different effects on le-guminous species MeJA exogenous application has increased iso-flavonoid content in cell suspension cultures of Pueraria candollei varcandollei and P candollei var mirifica (Korsangruang et al 2010) aswell as the production of the triterpenoid glycyrrhizin in Glycyrrhizaglabra roots Enhanced production of the triterpenoid however waspartly at the expense of root growth (Shabani et al 2009) MeJA ap-plication on shoots was observed to suppress root nodulation and lat-eral root formation in Lotus japonicus (Nakagawa and Kawaguchi2006) In grapevine a non-leguminous species proteinogenic aminoacids did not show an expressive increase under MeJA treatment(Gutieacuterrez-Gamboa et al 2017)
The effects of the application of four different jasmonate forms (JAMeJA jasmonoyl-L-isoleucine (JA-Ile) and 6-ethyl indanoyl glycineconjugate (2-[(6-ethyl-1-oxo-indane-4-carbonyl)-amino]-acetic acidmethyl ester - CGM) on leucaena metabolite profile has recently beenreported by Xu et al (2018) JA-Ile form was most effective althoughno major alteration was observed on monitored metabolite abundancesAlanine threonine and 34-dihydroxypyridine (34 DHP a metabolitederived from mimosine degradation) (Nguyen and Tawata 2016)among others were the major metabolites elicited by JA-Ile In contrastto the results described here mimosine concentration did not changesignificantly These divergent results on mimosine accumulation maybe due to a number of factors including mode of application jasmonateform used (JA-Ile x JA) and L leucocephala subspecies (common x giantleucaena)
Ethylene is also a phytohormone involved in plant response me-chanisms to different types of challenges such as mechanical damageand insect attack among others The integration mechanism betweenJA and ethylene signaling pathways is not completely understoodhowever it has been shown that they may work cooperatively in abioticstress tolerance (Kazan 2015) MeJA can induce ethylene production(Zhao et al 2004) and when applied simultaneously these moleculesseem to work in a synergic way by enhancing the magnitude of theplant response to external stimuli (Liu et al 2016)
Treatment with SA was able to significantly increase mimosine ac-cumulation in 12-week-old plants of common leucaena (SupplementaryFig 2) However no significant effect of SA treatment on mimosineconcentration was seen in 5-week-old seedlings of giant leucaena(Fig 1) suggesting some degree of genotype andor age dependency inelicitation by this phytohormone On the other hand several treat-ments including 90 ppm MeJA 10 and 100 ppm 2-chloroethylpho-sphonic acid (CEPA an ethylene-releasing compound) significantlyincreased mimosine accumulation (Supplementary Fig 2) in agree-ment with the data obtained for giant leucaena The lack of systemiceffects of externally applied SA on mimosine accumulation was alsoobserved when the phytohormone was supplied in the culture mediumof aseptically-grown seedlings in which case only roots had highercontent of mimosine (Vestena et al 2001) This could be due totransport limitations or to low methyl salicylate production from ap-plied SA since the former is recognized as the main systemic signalingform (Vlot et al 2009)
32 Increased mimosine concentrations in giant leucaena exposed to UV-Cradiation
UV-C treatment promoted increased concentration of the aminoacid in shoots but not in roots of giant leucaena (Fig 2) Increasedaccumulation of mimosine in shoots was also observed in 12-week-oldseedlings of common leucaena exposed to UV-C radiation for 10 and15min (Supplementary Fig 3) Similar to the SA treatment in giant
leucaena UV-C radiation did not induce mimosine biosynthesis in rootsregardless of time after exposure The absence of mimosine induction inroots by SA and UV indicates that these effectors do not cause a sys-temic response Moreover roots are shielded from irradiance by thepresence of substrate
UV radiation effects on different aspects of plant metabolism anddevelopment have been described However compared to UV-B (en-vironmentally relevant type of UV radiation) assays there are less re-ports related to the UV-C effects on secondary metabolites biosynthesisand accumulation (Cetin 2014) especially in leguminous (Fabaceae)plants They generally concern primary metabolism aspects such asgrowth and development For instance seedlings of Phaseolus vulgaris L(Fabaceae) exposed to low intensity UV-C radiation have displayeddecreased chlorophyll content and reduced height after 14 days of ex-posure (Kara 2013) Negative effects on growth parameters and ni-trogen metabolism were also observed in Vigna radiata L (Fabaceae)after UV-B radiation treatment in addition to adverse effects on JA SAand antioxidant compounds accumulation (Choudhary and Agrawal2014a) The same authors reported increased accumulation of flavo-noids SA and JA besides negative effects on growth biomass yieldnitrogen fixation and accumulation in 2 cultivars of Pisum sativum L(Fabaceae) under elevated UV-B treatment (Choudhary and Agrawal2014b) Despite the negative UV influence on growth reported for thepreviously mentioned leguminous UV-C radiation on groundnut plants(Arachis hypogaea L Fabaceae) increased seedling vigor and biomassand had no adverse effect on germination or other development para-meters (Neelamegam and Sutha 2015)
Besides its impact on growth and primary metabolism UV exposurecan cause important changes in secondary metabolism depending onintensity and time of exposure (Matsuura et al 2013) UV-B and UV-Cpre-treatments of Artemisia annua (Asteraceae) seedlings yielded in-creased biosynthesis of artemisinin a drug which displays anti-malarialproperties and activity against some others infectious diseases (egschistosomiasis leishmaniasis and hepatitis B) and several kinds oftumors (Rai et al 2011) The accumulation of nicotine in Nicotianarustica plants (Solanaceae) was also increased by UV-C treatment(Tiburcio et al 1985) Similar inducing effects on production of severalsecondary metabolites were observed in callus cultures of Vitis viniferaL Oumlkuumlzgoumlzuuml (grapevine Vitaceae) treated with a UV-C source for 5 or10min (Cetin 2014)
Regarding amino acid biosynthesis in response to UV radiationMartiacutenez-Luumlscher et al (2014) have found that in spite of not causingchanges in total amino acid content UV-B radiation exposure can affecttheir profile in grape berries Proteinogenic amino acids have beenknown to be important targets of the deleterious effects of UV radiation(Holloacutesy 2002) On the other hand in the present study acute UV-Ctreatment was found to increase mimosine accumulation in shoots byover twofold (Fig 2) which may suggest a possible participation of thismolecule as part of the antioxidant defense system in L leucocephalaThis possibility is further supported by the induction of the amino acidaccumulation by JA and Ethephon involved in abiotic and biotic stressresponses which are generally associated with oxidative imbalance andare signaling components in high UV stress (Matsuura et al 2013)
33 Mimosinase gene expression
In order to determine if increases in mimosine content upon ex-posure to JA CEPA or UV-C radiation were related to changes intranscription of mimosine metabolism-related genes RT-qPCR analysiswas carried out The complete pathway for mimosine biosynthesis hasnot yet been determined although the final step has been character-ized Based on transcription analysis (Ishihara et al 2016a) leucaenaappears to encode for multiple cysteine synthases one or more of whichmay be able to catalyze mimosine synthesis In addition a leucaenagene encoding a mimosinase (an enzyme responsible for mimosinedegradation) has been identified and characterized (Negi et al 2014)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
436
In addition to mimosinase gene expression several gene isoformsbelonging to the cysteine pathway [cysteine synthase (CYS SYN) serineacetyltransferase (SAT) and β-cyanoalanine synthase (CAS) Table 2 -supplementary material] were also tested in this study (data notshown) However expressions of these genes did not vary in giantleucaena throughout the experiments suggesting that the increasedcontent of mimosine observed in the treated plants might not be relatedto the expression of these genes but presumably to increased enzymeactivity andor release from conjugates such as mimoside a mimosineβ-D-glucoside (Murakoshi et al 1972)
Considering the time variation of mimosine accumulation observedin this work mimosinase gene expression in shoots and roots wasevaluated 24 h before the increase of mimosine concentration in giantleucaena seedlings (ie 24 h and 72 h after the chemical elicitorstreatments and 48 h and 120 h after UV-C exposure)
Ethylene signaling has been shown to up-regulate expression ofseveral genes related to secondary metabolism pathways as is the caseof phenolic compounds (Liu et al 2016) and terpenoid indole alkaloids(Wang et al 2016) Among all elicitors tested in the present workEthephon was the only one able to significantly change mimosinasegene expression Leucaena plants treated with Ethephon showed sig-nificant increases in mimosine concentration at both day 2 and 4 fol-lowing treatment which coincided with low-level expression of mi-mosinase Up-regulation of mimosinase gene expression was detected24 h before the increase of mimosine concentration in shoots treatedwith 10 ppm of Ethephon (Fig 3A) but not after JA or UV-C treatments(Fig 3C-D and 3E-F respectively) Nevertheless 72 h after treatment
application (24 h before the highest mimosine content measured inshoots) down regulation of mimosinase gene was seen in both shootsand roots treated with 10 ppm of Ethephon (Fig 3B) These data in-dicate that mimosine content in leucaena plants is at least partlyregulated by mimosinase expression in Ethephon exposed plants Onthe other hand the fact that mimosinase mRNA was not significantlyaffected by JA and UV-C treatments despite their stimulating effects onmimosine biosynthesis in giant leucaena may indicate that other levelsof regulation are at play or that the chosen harvesting time window wasunable to detect relevant changes
34 In situ and in vitro antioxidant assays
Considering the stimulation of mimosine accumulation byEthephon JA and UV all of which are often associated or known tocause oxidative imbalance the antioxidant capacity of mimosine wasevaluated Mimosine has been shown to have antioxidant activities oncultured cancer cells (Parmar et al 2015) In the present study it washypothesized that mimosine could confer radical scavenging propertieswhich would contribute to plant protection from possible damagecaused by reactive oxygen species generated during stress(Supplementary Fig 4)
Foliar disks of P vulgaris L were treated with 10mM mimosine for15min Treated disks showed less hydrogen peroxide accumulationinduced by wounding in contrast to untreated ones being comparableto those treated with ascorbic acid (a known hydrogen peroxide neu-tralizer) (Fig 4A) These observations support a possible antioxidant
Fig 2 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings exposed to UV-C lightCTRL= visible light (100 μmol photons mminus2 s minus1) UV-C 10primeand UV-C 15rsquo=UV-C exposure time (10 and 15min re-spectively) Bars sharing a letter of same case do not differ byTukey test (P le 005) Capital letters (A B) compare treat-ments on day three and lowercase letters (a b) comparetreatments on day six Indicates significant statistical dif-ference between day three and day six in the same treatmentby t-test (Ple 005) The error bars represent standard errorof five replicates (each mean was calculated with 15 in-dividual seedlings organized in 5 groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
437
role of mimosine as an in situ hydrogen peroxide scavengerMimosine was also able to quench superoxide anions generated by
light exposure Mimosine exhibited equivalent antioxidant effect com-pared to rutin (Fig 4B) a well-established effective superoxide anionquencher (Matsuura et al 2016) The radical scavenging activity ofmimosine may be due to the 3-OH group of the pyridine ring of mi-mosine (Fig 5) The pKa of the 3-OH of mimosine has been estimated tobe 88 (M Honda unpublished results) At physiological pH this OHgroup is expected to remain in a protonated state and therefore mayscavenge a radical by donating a proton and an electron In this processmimosine itself is converted to a stable radical form which is perhapsless toxic and less reactive than the reactive oxygen species generatedduring oxidative stress It is likely that the less toxic radical mimosineproduced may react with another radical or molecule and becomeconverted to a non-reactive indole molecule
In vivo antioxidant activity of mimosine has been previously eval-uated by means of its exogenous application on selenium-deficientseedlings of Vigna radiata In spite of its allelopathic properties (Ahmedet al 2008) the results showed mitigation of mitochondrial oxidativestress by treatment with 01mM mimosine (Lalitha and Kulothungan2007) DPPH radical scavenging activity was also reported for aqueous
seed extracts of leucaena rich in mimosine and phenolic compounds inin vitro assays (Benjakul et al 2014) Mimosine antioxidant activityshown in the present work is in good agreement with data reported forother non-protein amino acids such as L-DOPA (Dhanani et al 2015)and GABA (Malekzadeh et al 2014) for instance
4 Conclusion
Taken together results show that mimosine biosynthesis and ac-cumulation can be modulated by stress-related factors despite its re-latively high constitutive content in leucaena plants The pattern ofgene expression in stressed plants suggests mimosine steady-state con-trol may be regulated by its degradation in possible connection withdynamic changes in carbon and nitrogen metabolism of stressed plantsMimosine quenching activity against hydrogen peroxide and super-oxide anions in the in situ staining and in vitro assays respectivelyshowed that this non-protein amino acid can act as non-enzymaticantioxidant agent Increase in mimosine content in response to elicitorsmimicking environmental challenges in addition to its antiherbivoreand antimicrobial properties may be related to its activity as protectivemolecule against oxidative damage in line with other classes of plant
Fig 3 Relative expression of the mimosinase gene in shoots (A E and F) and shoots and roots (B C and D) of giant leucaena 24 h (A and C) 48 h (E) 72 h (B and D)and 120 h (F) after treatment with stress signaling molecules or UV-C exposure ETH = Ethephon JA = Jasmonic Acid Indicates significant statistical differencebetween control and treatment by t-test (Ple 005) The error bars represent standard error of four replicates
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
438
secondary metabolites
Funding
This work was funded by the National Council for Scientific andTechnological Development (CNPq-Brazil) grant 3060792013-5Coordenaccedilatildeo de Aperfeiccediloamento de Pessoal de Niacutevel Superior - Brazil(CAPES) - Finance Code 001 and the USDA NIFA Hatch projectHA05029-H managed by CTAHR
CRediT authorship contribution statement
Kelly Cristine da Silva Rodrigues-Correcirca InvestigationValidation Writing ndash original draft Michael DH HondaInvestigation Validation Dulal Borthakur Supervision Writing ndashreview amp editing Funding acquisition Arthur Germano Fett-NetoSupervision Funding acquisition Writing ndash review amp editing
Acknowledgements
The authors would like to thank Dr Jorge Ernesto Mariath fromLaVeg-UFRGS for kindly lending the Leica M165 FC stereomicroscopefor in situ analysis
Appendix A Supplementary data
Supplementary data to this article can be found online at httpsdoiorg101016jplaphy201811018
References
Ahmed R Hoque ATMR Hossain MK 2008 Allelopathic effects of Leucaena
leucocephala leaf litter on some forest and agricultural crops grown in nursery J ForRes 19 298 httpsdoi 101007s11676-008-0053-0
Benjakul S Kittiphattanabawon P Shahidi F Maqsood S 2013 Antioxidant activityand inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipidoxidation in model systems Food Sci Technol Int 19 (4) 365ndash376 httpsdoiorg1011771082013212455186
Benjakul S Kittiphattanabawon P Sumpavapol P Maqsood S 2014 Antioxidantactivities of lead (Leucaena leucocephala) seed as affected by extraction solvent priordechlorophyllisation and drying methods extracts against lipid oxidation in modelsystems Food Sci Technol 51 (11) 3026ndash3037 httpsdoiorg101007s13197-012-0846-1
Brewbaker JL Pluckett D Gonzalez V 1972 Varietal variation and yield trials ofLeucaena leucocephala (koa haole) in Hawaii Hawaii Agric Exp Stn Bull 166 26
Brewbaker JL 2008 Registration of KX2 ndash Hawaii interspecific-hybrid leucaena JPlant Registrations 1 (3) 190ndash193 httpsdoiorg103198jpr2007050298crc
Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitisvinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-6287-47-37
Cho H-Y Son SY Rhee HS Yoon S-YH Lee-Parsons CWT Park JM 2008Synergistic effects of sequential treatment with methyl jasmonate salicylic acid andyeast extract on benzophenanthridine alkaloid accumulation and protein expressionin Eschscholtzia californica suspension cultures J Biotechnol 135 117ndash122 httpsdoiorg101016jjbiotec200802020
Choudhary KK Agrawal SB 2014a Cultivar specificity of tropical mung bean (Vignaradiata L) to elevated ultraviolet-B changes in antioxidative defense system ni-trogen metabolism and accumulation of jasmonic and salicylic acids Environ ExpBot 99 122ndash132 httpsdoiorg101016jenvexpbot201311006
Choudhary KK Agrawal SB 2014b Ultraviolet-B induced changes in morphologicalphysiological and biochemical parameters of two cultivars of pea (Pisum sativum L)Ecotoxicol Environ Saf 100 178ndash187 httpsdoiorg101016jecoenv201310032
Dar TA Uddin M Khan MMA Hakeem KR Jaleel H 2015 Jasmonates counterplant stress a Review Environ Exp Bot 115 49ndash57 httpsdoiorg101016jenvexpbot201502010
Dhanani T Singh R Shah S Kumari P Kumar S 2015 Comparison of green ex-traction methods with conventional extraction method for extract yield L-DOPAconcentration and antioxidant activity of Mucuna pruriens seed Green Chem LettRev 8 (2) 43ndash48 httpsdoiorg1010801751825320151075070
Gutieacuterrez-Gamboa G Portu J Santamariacutea P Loacutepez R Garde-Cerdaacuten T 2017Effects on grape amino acid concentration through foliar application of three dif-ferent elicitors Food Res Int 99 688ndash692 httpsdoiorg101016jfoodres201706022
Fig 4 A In situ antioxidant assay Foliar disksof Phaseolus vulgaris L treated with (a) No an-tioxidant added (negative control) (b) 10 mMMimosine (c) 10mM ascorbic acid (positivecontrol) The oxidative damage can be seen bythe formation of a brown polymer in leaf veinsand injured areas B In vitro superoxidescavenging assay carried out with mimosineDifferent letters indicate significant differenceby Tukey test (Ple 005) The error bars re-present standard error of four replicates (Forinterpretation of the references to colour in thisfigure legend the reader is referred to the Webversion of this article)
Fig 5 Predicted mimosine radical formed followingquenching of hydroxyl radical Mimosine is first converted toa stable mimosine radical which may be then converted to anontoxic indole form
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
439
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad HossainMd Anai T Oku H 2018 Cytosolic cysteine synthase switch cysteine and mi-mosine production in Leucaena leucocephala Appl Biochem Biotechnol 186 (3)613ndash632 httpsdoiorg101007s12010-018-2745-z
Holloacutesy F 2002 Effects of ultraviolet radiation on plant cells Micron 33 (2) 179ndash197Honda MDH Ishihara KL Pham DT Borthakur D 2018 Identification of drought-
induced genes in giant leucaena (Leucaena leucocephala subsp glabrata) Trees 32571ndash585 httpsdoiorg101007s00468-018-1657-4
Huang T Jander G de Vos M 2011 Non-protein amino acids in plant defense againstinsect herbivores representative cases and opportunities for further functional ana-lysis Phytochemistry 72 1531ndash1537 httpsdoiorg101016jphytochem201103019
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of thethyrotoxic amino acid mimosine by cysteine synthase Phytochemistry 29 (11)3461ndash3465 httpsdoiorg1010160031-9422(90)85258-H
Ishihara K Lee EKW Borthakur D 2016a An improved method for RNA extractionfrom woody legume species Acacia koa A Gray and Leucaena leucocephala (Lam) deWit Int J For Wood Sci 3 (1) 031ndash035
Ishihara KL Honda MDH Pham DT Borthakur D 2016b Transcriptome analysisof Leucaena leucocephala and identification of highly expressed genes in roots andshoots Transcriptomics 4 135 httpsdoiorg1041722329-89361000135
IUBMB 2018 Enzyme Nomenclature EC 35161 httpwwwsbcsqmulacukiubmbenzymeEC35161html Accessed date 8 February 2018
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant(Phaseolus vulgaris) Biosci Res 10 (1) 29ndash32
Kazan K 2015 Diverse roles of jasmonates and ethylene in abiotic stress toleranceTrends Plant Sci 20 (4) 219ndash229 httpsdoiorg101016jtplants201502001
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect ofEthephon as an ethylene-releasing compound on the metabolic profile of Chlorellavulgaris J Agric Food Chem 64 (23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541
Khan MIR Fatma M Per TS Anjum NA Khan NA 2015 Salicylic acid-inducedabiotic stress tolerance and underlying mechanisms in plants Front Plant Sci 6 462httpsdoiorg103389fpls201500462
Korsangruang S Soonthornchareonnon N Chintapakorn Y Saralamp PPrathanturarug S 2010 Effects of abiotic and biotic elicitors on growth and iso-flavonoid accumulation in Pueraria candollei var candollei and P candollei var mir-ifica cell suspension cultures Plant Cell Tissue Organ Cult 103 (3) 333ndash342 httpsdoiorg101007s11240-010-9785-6
Lalitha K Kulothungan SR 2006 Selective determination of mimosine and its dihy-droxypyridinyl derivative in plant systems Amino Acids 31 (3) 279ndash287 httpsdoiorg101007s00726-005-0226-5
Lalitha K Kulothungan SR 2007 Mimosine mitigates oxidative stress in seleniumdeficient seedlings of Vigna radiata - Part I restoration of mitochondrial functionBiol Trace Elem Res 118 (1) 84ndash96 httpsdoiorg101007s12011-007-0013-0
Liu J Li Y Wang Y Zhang Z-H Zu Y-G Efferth T Tang Z-H 2016 Thecombined effects of ethylene and MeJA on metabolic profiling of phenolic com-pounds in Catharanthus roseus revealed by metabolomics analysis Front Physiol 71ndash11 httpsdoiorg103389fphys201600217 Article 217
Malekzadeh P Khara J Heydari R 2014 Alleviating effects of exogenous Gamma-aminobutiric acid on tomato seedling under chilling stress Physiol Mol Biol Plants20 (1) 133ndash137 httpsdoiorg101007s12298-013-0203-5
Martiacutenez-Luumlscher J Torres N Hilbert G Richard T Saacutenchez-Diacuteaz M Delrot SAguirreolea J Pascual I Gomegraves E 2014 Ultraviolet-B radiation modifies thequantitative and qualitative profile of flavonoids and amino acids in grape berriesPhytochemistry 102 106ndash114 httpsdoiorg101016jphytochem201403014
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation ofbioactive secondary metabolites by ultraviolet radiation mechanisms strategies andapplications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology forMedicinal Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash1902012
Matsuura HN Fragoso V Paranhos JT Rau MR Fett-Neto AG 2016 Thebioactive monoterpene indole alkaloid N szlig-D-glucopyranosylvincosamide is regu-lated by irradiance quality and development in Psychotria leiocarpa Ind Crop Prod86 210ndash218 httpsdoiorg101016jindcrop201603050
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo RBhambra AS Strnad M Bonfill M Fett-Neto AG 2018 Specialized plant me-tabolism characteristics and impact on target molecule biotechnological productionMol Biotechnol 60 (2) 169ndash183 httpsdoiorg101007s12033-017-0056-1
Murakoshi S Ohmiya S Haginiwa J 1972 Enzymic synthesis of mimoside a meta-bolite of mimosine in Mimosa pudica and Leucaena leucocephala Chem Pharm Bull20 (4) 855ndash857
Nakagawa T Kawaguchi M 2006 Shoot-applied MeJA suppresses root nodulation inLotus japonicus Plant Cell Physiol 47 (1) 176ndash180 httpsdoiorg101093pcppci222
Nascimento NC Menguer PK Henriques AT Fett-Neto AG 2013 Accumulation ofbrachycerine an antioxidant glucosidic indole alkaloid is induced by abscisic acidheavy metal and osmotic stress in leaves of Psychotria brachyceras Plant PhysiolBiochem 73 33ndash40 httpsdoiorg101016jplaphy201308007
Neelamegam R Sutha T 2015 UV-C irradiation effect on seed germination seedling
growth and productivity of groundnut (Arachis hypogaea L) Int J Curr MicrobiolApp Sci 4 (8) 430ndash443
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase fromLeucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol164 (2) 922ndash934 httpsdoiorg101104pp113230870
Negi VS Borthakur D 2016 Heterologous expression and characterization of mimo-sinase from Leucaena leucocephala In Fett-Neto Arthur Germano (Ed)Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods inMolecular Biology vol 1405 copySpringer Science+Business Media New York httpsdoiorg101007978-1-4939-3393-8_7 2016
Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636
Parmar F Kushawaha N Highland H George L-B 2015 In vitro antioxidant andanticancer activity of Mimosa pudica Linn extract and L-mimosine on lymphomaDaudi cells Int J Pharm Sci 12 100ndash104
Porto DD Matsuura HN Vargas LRB Henriques AT Fett-Neto AG 2014 Shootaccumulation kinetics and effects on herbivores of the wound-induced antioxidantindole alkaloid brachycerine of Psychotria brachyceras Nat Prod Commun 9 (5)629ndash632
Rai R Meena RP Smita SS Shukla A Rai SK Pandey-Rai S 2011 UV-B and UV-C pre-treatments induce physiological changes and artemisinin biosynthesis inArtemisia annua L ndash an antimalarial plant J Photochem Photobiol B Biol 105 (3)216ndash225 httpsdoiorg101016jjphotobiol201109004
Shabani L Ehsanpour AA Asghari G Emami J 2009 Glycyrrhizin production by invitro cultured Glycyrrhiza glabra elicited by methyl jasmonate and salicylic acid RussJ Plant Physiol 56 (5) 621ndash626 httpsdoiorg101134S1021443709050069
Shah J 2003 The salicylic acid loop in plant defense Curr Opin Plant Biol 6 (4)365ndash371
Shi J Fu XZ Peng T Huang XS Fan QJ Liu JH 2010 Spermine pretreatmentconfers dehydration tolerance of citrus in vitro plants via modulation of antioxidativecapacity and stomatal response Tree Physiol 30 (7) 914ndash922 httpsdoiorg101093treephystpq030
Smith IK Fowden L 1966 A study of mimosine toxicity in plants J Exp Bot 17750ndash761 httpsdoiorg101093jxb174750
Soedarjo M Borthakur D 1996 Simple procedures to remove mimosine from youngleaves pods and seeds of Leucaena leucocephala used as food Int J Food SciTechnol 31 (1) 97ndash103
Soedarjo M Borthakur D 1998 Mimosine a toxin produced by the tree-legumeLeucaena provides a nodulation competition advantage to mimosine-degradingRhizobium strains Soil Biol Biochem 30 1605ndash1613
Suda S 1960 On the physiological properties of mimosine Bot Mag Tokyo 73 (862)142ndash147 httpsdoiorg1015281jplantres188773142
Tangendjaja B Lowry JB Wills RBH 1986 Isolation of a mimosine degrading en-zyme from leucaena leaf J Sci Food Agric 37 523ndash526 httpsdoiorg101002jsfa2740370603
Tiburcio F Pintildeol MT Serrano M 1985 Effect of UV-C on growth soluble protein andalkaloids in Nicotiana rustica plants Environ Exp Bot 25 (3) 203ndash210 httpsdoiorg1010160098-8472(85)90004-8
Vestena S Fett-Neto AG Duarte RC Ferreira A 2001 Regulation of mimosineaccumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 httpsdoiorg101016S0168-9452(01)00448-4
Vlot AC Dempsey DMA Klessig DF 2009 Salicylic acid a multifaceted hormone tocombat disease Annu Rev Phytopathol 47 177ndash206 httpsdoiorg101146annurevphyto050908135202 2009
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene-induced vinblastine accumulation is related to activated expression of downstreamTIA pathway genes in Catharanthus roseus BioMed Res Int 2016 Article ID 3708187httpsdoiorg10115520163708187
Wasternack C Strnad M 2016 Jasmonate signaling in plant stress responses and de-velopment ndash active and inactive compounds N Biotech 33 (5B) 604ndash613 httpsdoiorg101016jnbt201511001
Wencomo HB Ortiz R Caacuteceres J 2017 Afr J Agric Res 12 (4) 279ndash285 httpsdoiorg105897AJAR201510604 26
Wong CC Devendra C 1983 Research on leucaena forage production in Malaysia InLeucaena Research in the Asian Pacific Region pp 55ndash60 Ottawa Ontario Canada
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX TsimKWK 2018 Jasmonate-elicited stress induces metabolic change in the leaves ofLeucaena leucocephala Molecules 23 (2) httpsdoiorg103390molecules23020188 E188
Yafuso JT Negi VS Bingham J-P Borthakur D 2014 An O-acetylserine (thiol)lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthaseAppl Biochem Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z
Zhao J Zheng S-H Fujita K Sakai K 2004 Jasmonate and ethylene signalling andtheir interaction are integral parts of the elicitor signalling pathway leading to b-thujaplicin biosynthesis in Cupressus lusitanica cell cultures J Exp Bot 55 (399)1003ndash1012 httpsdoiorg101093jxberh127
Zhishen J Mengcheng T Jianming W 1999 The determination of flavonoid contentsin mulberry and their scavenging effects on superoxide radicals Food Chem 64 (4)555ndash559 httpsdoiorg101016S0308-8146(98)00102-2
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
440
61
Supplementary Fig 1 Basal mimosine concentration in adult trees of common leucaena (L leucocephala
var leucocephala) Samples were collected from 10 field grown trees at Manoa Valley Honolulu Hawairsquoi
on June 25th 2017 Bars sharing a letter do not differ by Tukey test (P le 005) The error bars represent the
standard error
Supplementary Fig 2 Bar diagram showing mimosine concentration in shoots of 12-week-old common
leucaena seedlings treated with different elicitors CTRL = Milli-Q water SA = Salicylic Acid MeJA =
Methyl Jasmonate CEPA = 2-Chloroethylphosphonic acid (an ethylene releasing compound) Bars sharing a
letter of same case do not differ by Tukey test (P le 005) Capital letters (A B) compare treatments on day
two and lower-case letters (a b) compare treatments on day four Indicates significant statistical difference
ABB
A A
0
200
400
600
800
1000
1200
LEAVES GREEN FLOWERBUDS
POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
con
cen
trat
ion
(micro
gg
-1o
f FW
)
B AB AB AB B A
b
a
ab b
ab
0
2
4
6
8
10
12
14
16
18
20
CTRL SA 10 ppm SA 100 ppm CEPA 10 ppm CEPA 100 ppm MeJA 90 ppm
Mim
osi
ne
co
nce
ntr
atio
n (
gg
-1o
f FW
)
DAY 02 DAY 04
62
between day two and day four in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
Supplementary Fig 3 Bar diagram showing the effects of UV-C radiation exposure for 5 10 and 15 min on
mimosine accumulation in shoots of 12-week-old seedlings of common leucaena Bars sharing a letter of
same case do not differ by Tukey test (P le 005) Capital letters (A B C) compare treatments on day three
and lower-case letters (a b) compare treatments on day six Indicates significant statistical difference
between day three and day six in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
C BC AB A
bb
a
a
0
10
20
30
40
50
60
CTRL UV-C 5 UV-C 10 UV-C 15
Mim
osi
ne
co
nce
ntr
atio
n (
gg-1
of
FW)
DAY 03 DAY 06
63
Supplementary Fig 4 Model depicting induction of mimosine synthesis in leucaena following application of
stress elicitors such as CEPA and jasmonic acid or exposure to UV-C radiation The additional mimosine
synthesized may serve to alleviate oxidative stress induced by UV-C radiation
64
Supplementary Table 1 Mimosine contents in leaves of common and giant leucaena
Leucaena
type
Mimosine content
( FW)
Mimosine
content ( DW)
Dry matter
content ( FW)
Water content
( FW)
Common (1) 050 plusmn 009 245 plusmn 051 2011 plusmn 054 7989 plusmn 054
Common (2) 043 plusmn 006 214 plusmn 037 1998 plusmn 050 8002 plusmn 050
K636 (1) 070 plusmn 014 356 plusmn 077 1908 plusmn 052 8092 plusmn 052
K636 (2) 042 005 205 plusmn 033 2008plusmn 093 7992plusmn 093
KX2 (1) 122 plusmn 011 608 plusmn 082 1939 plusmn 123 8061 plusmn 123
KX2 (2) 134 plusmn 010 623 plusmn 056 2029 plusmn 114 7971 plusmn 114
KX3 (1) 044 plusmn 006 221 plusmn 030 1945 plusmn 073 8055 plusmn 073
KX3 (2) 054 plusmn 005 273 plusmn 023 1930 plusmn 038 8070 plusmn 038
KX4 (1) 086 plusmn 011 471 plusmn 065 1753 plusmn 084 8247 plusmn 084
KX4 (2) 089 plusmn 011 476 plusmn 065 180 plusmn 072 820 plusmn 072
KX5 (1) 099 plusmn 012 489 plusmn 048 1907 plusmn060 8093 plusmn 060
KX5 (2) 115 plusmn 015 548 plusmn080 1992 plusmn 053 8008 plusmn 053
Common leucaena variety koa haole grows widely on the island of Orsquoahu K636 is widely
grown variety of giant leucaena KX2 KX3 KX4 and KX5 are giant leucaena varieties
developed through interspecies hybridization (Brewbaker 2016) (1) and (2) indicate plants
from two separate locations within the University of Hawaii Waimanalo Research Center The
values are shown as mean plusmn standard error obtained from at least three biological replicates
65
Supplementary Table 2 GenBank accession numbers of the tested cysteine pathway genes isoforms
Gene name GenBank accession
OAS-TL (o-acetylserine-thiol-lyase) GDRZ01032940
GDRZ01061620
GDRZ01153117
GDSA01187555
GDSA01196891
GDSA01214467
Cys syn (cysteine synthase) GDRZ01015860
GDRZ01050898
GDRZ01086813
GDRZ01193515
GDRZ01202579
GDSA01180863
GDSA01215622
SAT (serine acetyltransferase) GDRZ01187456
GDRZ01189631
CAS (β-cyanoalanine synthase) GDRZ01054066
GDRZ01175418
GDSA01118400
66
SHORT COMMUNICATION 1
Mimosine occurrence and accumulation in Mimosa bimucronata var bimucronata (DC) 2
Kuntze 3
Kelly Cristine da Silva Rodrigues-Correcirca1 Lana Dorneles Pedroso2 Fernanda de Costa1 4
Arthur Germano Fett-Neto1 5
1Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal 6
University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970 7
Porto Alegre Rio Grande do Sul Brazil 2Department of Biological Sciences Unipampa ndash 8
Campus Satildeo Gabriel 9
Corresponding author 10
E-mail addresses krodriguescbiotufrgsbr (KCdaS Rodrigues-Correcirca) 11
lanalima2012gmailcom (LD Pedroso) fernandadecostayahoocombr (F de Costa) 12
fettnetocbiotufrgsbr (AG Fett-Neto) 13
14
15
16
17
18
19
20
21
22
67
ABSTRACT 23
Mimosine is a non-protein aromatic amino acid present in plants of Leucaena spp 24
and Mimosa spp Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) is a native 25
tree from Brazil which occurs as a pioneer species on plant succession processes In the 26
current study the presence of mimosine in M bimucronata was verified by HPLC analyses 27
Moreover mimosine accumulation upon exposure to UV-C and chemical elicitors of 28
specialized metabolism (salicylic acid - SA methyl jasmonate - MeJA sodium nitroprusside 29
- SNP and ethephon - ETH) most of which also known as promoters of the amino acid 30
production in leucaena plants was evaluated The results showed a lower concentration of 31
constitutive mimosine present in both maricaacute seedlings and mature trees when compared to 32
leucaena plants In spite of a trend towards increased mimosine accumulation observed in 33
MeJA and ETH treatments no statistical differences were found with the various stressors 34
used to induce its biosynthesis in maricaacute seedlings Data suggest that mimosine in M 35
bimucronata is probably a phytoanticipin-like metabolite or its accumulation is driven by 36
other types of stresses 37
38
39
Keywords Mimosine Mimosa bimucronata stress 40
41
42
43
44
45
46
68
Introduction 47
Mimosa bimucronata commonly known as maricaacute is a native tree from Brazil 48
(REFLORA 2019) ecologically important in plant succession and in processes of degraded 49
land recovery (Bitencourt et al 2007 Silva et al 2011) occurring as a pioneer species 50
(Pilatti et al 2019) Maricaacute is a deciduous or semi-deciduous plant which reaches up to 15 51
m in height and 40 cm of diameter at breast height (DBH) displays shrub or tree habit and 52
bears typical sharp thorns (Carvalho 2004) This species belongs to Fabaceae one of the 53
most economically important families of flowering plants due to its high diversity and 54
occurrence in different types of habitats (Gomes et al 2018) As well as several others 55
Mimosa spp maricaacute is usually referred to as a multipurpose tree (Olkoski and Wittmann 56
2011) employed for alternative medicinal uses (Champanerkar et al 2010 Silva et al 57
2011) honey production constructions and remodeling of landscape architecture (living 58
fences) for instance (Marchiori 1993 Lorenzi 1998) 59
In southern Brazil maricaacute is widely distributed and typically found either in wetland 60
areas close to river banks (Patreze and Cordeiro 2004) or composing large and almost pure 61
landscape formations on hillsides (Jacobi and Ferreira 1991) In dense populations this 62
species like several Mimosa spp (Simon and Proenccedila 2000) is considered an important and 63
highly invasive weed by preventing cattle to reach pasturesand water bodies as a result of its 64
thorny branches (Lorenzi 2008 Kestring et al 2009) Its dominant and nearly exclusive 65
pattern of distribution in those areas has led Jacobi and Ferreira (1991) to test its allelopathic 66
potential on cultivated species Indeed extracts of leaves and ripe fruits (but not the green 67
ones) of maricaacute showed phytotoxic effects on germination and initial radical growth of most 68
of the target species tested 69
69
Several investigations have been performed on maricaacute floristics (Silva et al 2011) 70
distribution (Simon and Proenccedila 2000) wood anatomy (Marchiori 1993) cytogenetic 71
parameters (Olkoski and Wittmann 2011) and allelopathic potential (Jacobi and Ferreira 72
1991 Ferreira et al 1992) However excluding two recent publications on maricaacute 73
constitutive chemical composition (Schlickmann et al 2017 Pilatti et al 2019) which 74
identified phenolic compounds (methyl gallate and water-soluble tannins) as its major 75
compounds little is known regarding this subject In other Mimosa species (eg M pudica 76
and M pigra) mimosine has been identified (Soedarjo and Borthakur 1998) as one of the 77
major specialized metabolites present in the different organs of the plant (Champanerkar et 78
al 2010) The presence of this molecule was also reported for M bimucronata in a thin layer 79
chromatography-based preliminary study performed by Ferreira et al (1992) showing co-80
chromatography of a leaf extract component with authentic mimosine The authors attributed 81
the allelopathic effect of maricaacute to the accumulation of this metabolite in its leaves 82
Mimosine is an aromatic non-protein amino acid initially found in plants of Mimosa 83
pudica and later in Leucaena leucocephala (Lam) de Wit (Soedarjo and Borthakur 1998) a 84
leguminous tree which biosynthesizes large amounts of this nitrogen-containing compound 85
(Rodrigues-Correcirca et al 2019) It is believed that the accumulation of high contents of 86
mimosine in L leucocephala tissues confers among other traits defense against herbivores 87
and pathogens (Vestena et al 2001) tolerance to drought (Negi et al 2014) as well as 88
general oxidative stress protection (Rodrigues-Correcirca et al 2019) Interestingly drought is 89
the opposite environmental and physiological condition to that observed in the wet habitats 90
occupied by native populations of M bimucronata in Brazil (Patreze and Cordeiro 2004 91
Kestring et al 2009) and Mimosa pudica Linn in India (Champanerkar et al 2010) 92
70
Nonetheless flooding is also associated with oxidative stress particularly as water levels 93
change (Fukao et al 2019) 94
In Leucaena leucocephala var leucocephala (common leucaena) and Leucaena 95
leucocephala var glabrata (giant leucaena) mimosine accumulation has been shown to be 96
both constitutive and inducible by stress-related phytohormones such as jasmonic acid (JA) 97
Ethephon (ETH an ethylene- releasing compound) salicylic acid (SA - only common 98
leucaena) (Vestena et al 2001) as well as by UV-C radiation (Xu et al 2018 Rodrigues-99
Correcirca et al 2019) On the other hand there is a lack of information regarding mimosine 100
content and elicitation effects in Mimosa spp plants 101
The aim of this study was to examine the presence of mimosine in Mimosa 102
bimucronata and examine the effects of stresses and stress-signaling molecules on its 103
accumulation in leaves 104
Material and Methods 105
Plant material 106
For all experiments the plant material was collected at Morro Santana campus do 107
Vale of UFRGS (Federal University of Rio Grande do Sul) Porto Alegre RS Brazil 108
(3004rsquoS 5108rsquoW) Authorization for access to genetic material was obtained from 109
SISGEN-Brazil (license number A845493) Constitutive mimosine content in adult plants of 110
M bimucronata var bimucronata (DC) Kuntze was determined in plant material (leaves 111
green flower buds post-anthesis flowers and green pods) harvested in January 2017 112
(summer) A voucher herbarium specimen (ICN 187953) was deposited in the ICN ndash UFRGS 113
herbarium (Herbaacuterio do Instituto de Biociecircncias of UFRGS) 114
71
For mimosine elicitation experiments legumes (fruits) of maricaacute were collected in 115
the end of June 2017 (winter) Seeds were then removed from the dry fruits and kept in the 116
dark until sowing and seedling development for use in the assays 117
Seed germination 118
To break the coat-imposed seed dormancy after surface sterilization dry seeds of 119
maricaacute were acid scarified by immersion in H2SO4 (95 ndash 98 ) for 2 min (see Correcirca et al 120
2008) and repeatedly washed in distilled water to remove any residue of the acid Then seeds 121
were distributed in 50 mL individual plastic tubes (dibble-tubes) (30 cm diameter x 120 cm 122
depth) filled up with 11 (vv) of commercial top soil and vermiculite Tubes were watered 123
every 2 days to avoid substrate dryness and were kept in a growth room under controlled 124
conditions of light (circa 75 μmol mminus2s minus1 photosynthetically active radiation photoperiod 125
of 16 h light and 8 h dark) and temperature (24plusmn2C) 126
127
Treatments 128
In order to verify inducibility of mimosine accumulation in M bimucronata fifty 12-129
week-old maricaacute seedlings (per treatment) exhibiting similar features were selected and 130
sprayed (saturated) with solutions of different chemical stressors (plant specialized 131
metabolism elicitors) as follows (for further details see Rodrigues-Correcirca et al 2019) 10 132
and 50 mM SA (pathogen-signaling molecule Shah 2003) 007 and 035 mM 2-133
chloroethylphosphonic acid (ETH ethylene releasing-compound Kim et al 2016 Wang et 134
al 2016) 100 and 200 mM MeJA (Dar et al 2015) 10 and 50 mM SNP (a nitric oxide 135
donor Perotti et al 2015) Alternatively maricaacute seedlings were also supplemented with UV-136
C radiation (13 minutes 105 kJ cm2) (elicitor of plant specialized metabolism Kara 2013) 137
72
After 2 and 4 days of exposure to the chemical treatments and 3 and 6 days of UV-138
C supplementation maricaacute shoots were harvested immediately frozen in liquid nitrogen and 139
stored at ndash 80 C until mimosine extraction and HPLC analyses 140
Mimosine extraction and detection 141
Mimosine extraction was conducted according to the modified protocol described by 142
Rodrigues-Correcirca et al (2019) for L leucocephala HPLC (Thermo Scientific Surveyor) 143
analyses (mimosine detection and quantification) were performed following previously 144
published procedures (Negi et al 2014) A C18 column (ACE C18 5 μm 46times250 mm) and 145
isocratic solvent system of 002M o-phosphoric acid with a linear flow rate of 1 mL min minus1 146
were used to separate and quantify the amino acid Mimosine detection was performed at 280 147
nm by photodiode array detection (200ndash400 nm) and retention time (229plusmn0024 min) 148
Mimosine quantification was done by means of the method of external standard curve 149
Additional confirmation of mimosine identity was performed by co-chromatography with 150
standard (Acros Organics authentic mimosine 99 used as reference) and peak purity check 151
The analyses of the chromatograms were done with the ChromQuest software 152
153
154
Results and Discussion 155
Constitutive accumulation of mimosine in M bimucronata 156
Mimosine was detected in all analyzed samples positively meeting all identification 157
criteria In agreement with what has been found for other Mimosa spp (Soedarjo and 158
Borthakur 1998) compared to L leucocephala adult plants (Rodrigues-Correcirca 2019) 159
mimosine content was lower in M bimucronata Of the adult plant tissues analyzed the 160
73
highest content of mimosine in maricaacute (per gram of fresh weight - FW) was found in post-161
anthesis flowers (36644 microg versus 89448 microg in common leucaena followed by leaves 162
(28838 microg x 67358 microg) green flower buds (28094 microg x 51247 microg) and green pods (19002 163
microg x 82687 microg) (Fig 1)The same pattern is observed for seedlings when both species are 164
compared In this study untreated 12-week-old maricaacute seedlings (control at day 2) showed a 165
shoot content of mimosine of 23029plusmn007 microg g-1 of (FW) Five-week-old untreated giant 166
leucaena seedlings cultivated in similar conditions exhibited between 83640 and 178736 167
microg g-1 of FW (Rodrigues-Correcirca et al 2019) In the same way mimosine concentration 168
percentage in dry matter of Mimosa pigra was found to be rather low (002 in nodules and 169
roots and 007 in leaves) (Soedarjo and Borthakur 1998) 170
In this investigation the lowest constitutive mimosine content was found in green 171
pods (Fig 1) This result may partly explain the absence of phytotoxic effect observed for 172
green pods on germination and growth of crop target plants tested by Jacobi and Ferreira 173
(1991) compared to the other maricaacute parts analyzed 174
Elicitation of mimosine biosynthesis in M bimucronata 175
Chemical stressors 176
Secondary metabolites (or natural products) are structural- and chemically 177
specialized compounds derived from primary metabolism These molecules are mainly 178
biosynthesized as part of a complex defense mechanism in response to biotic and abiotic 179
stresses such as pathogens herbivores water status metal toxicity and UV radiation for 180
example (Matsuura et al 2018) Ethephon SA SNP MeJA have been extensively used as 181
chemical elicitors of specialized metabolism (Wang et al 2016 Vestena et al 2001 Perotti 182
74
et al 2015 Zhang and Memelink 2009 Xu et al 2018) These phytohormonal signals can 183
simulate environmental challenges and modulate plant homeostasis often leading to 184
alterations in gene expression (Shinozaki et al 2015) Except SNP all treatments tested in 185
the present study showed positive effect on mimosine accumulation in common or giant 186
leucaena (Vestena et al 2001 Rodrigues-Correcirca 2019 Rodrigues-Correcirca unpublished 187
data) However in spite of the trend of increasing the mimosine content observed in seedlings 188
treated with 007 mM Ethephon (at day 2) and 100 mM MeJA (at day 4) no statistical 189
difference was confirmed for these treatments when compared to the control 190
On the other hand a within treatment difference on mimosine induction was seen 191
between day 2 and 4 in seedlings treated with 100 mM MeJA (Fig 2) In a lower 192
concentration (04 mM) jasmonic acid (JA)promoted a near threefold increase in mimosine 193
accumulation of giant leucaena seedlings after 2 days of application 194
UV-C radiation 195
Albeit UV-C radiation is not biologically active in natural environments it has been 196
widely used under controlled experimental conditions to generate acute responses of plant 197
specialized metabolism within a shorter period of time compared to that required to with UV-198
B radiation (Kara 2013 Cetin 2014) This fast response is due to the higher energy of UV-199
C photons that act as potent reactive oxygen species (ROS) generators causing extensive 200
damage to the cells either at the physiological level or on DNA structure (Gregianini et al 201
2003 Matsuura et al 2013) 202
Although divergent responses can be observed in plants exposed to UV-C radiation 203
the deleterious processes are usually reported on primary metabolism (decreasing of 204
chlorophyll content and plant height eg) (Kara 2013) In the present study no statistical 205
75
differences were observed in the mimosine concentration in maricaacute seedlings supplemented 206
with UV-C radiation However a decreasing in its content was found for both control and 207
treatment at day 6 post-treatment (Fig 03) Taking into account the lower constitutive 208
concentration of mimosine observed in maricaacute compared to the leucaena plants besides its 209
relative thermolability (Nguyen and Tawata 2016) it seems to be plausible to consider the 210
effect of the temperature inside the UV-C and the white light (control) chambers as an 211
additional abiotic factor contributing to the decrease of mimosine accumulation in both group 212
of plants 213
Besides mimosine identification the presence of 34-dihydroxypyridine (34-DHP or 214
3-hydroxy-4-pyridone - 3H4P) a mimosine degradation product (Negi et al 2014 Nguyen 215
and Tawata 2016) was also reported for maricaacute leaf extracts analyzed by TLC by Ferreira 216
et al (1992) In our chromatograms we detected a second large peak after that of mimosine 217
(229plusmn0024) and similar to that identified by Negi et al (2014) as 3H4P (data not shown) 218
Comparing the chromatogram profiles obtained from seedlings elicited with chemical 219
stressors and those supplemented with UV-C the largest area for this peak was found (in all 220
samples) in the latter treatment at day 6 It might indicate that the constitutive andor the 221
initially UV-C-induced mimosine was degraded into 3H4P to cope with the cellular damage 222
caused by this treatment associated with an increased temperature inside the chambers 223
Nevertheless it was not possible to determine 3H4P concentration (or confirm its identity) 224
in maricaacute plants since there is no commercial standard (pure 3H4P) available for purchase 225
to be used as a reference in calculations Establishment of improved protocols for obtaining 226
in house 3H4P reference substance by acid hydrolysis is ongoing 227
228
229
76
Conclusion 230
On the basis of the overall absence of effect of the treatments tested here on mimosine 231
concentration it is possible to suggest that its accumulation profile is similar to that of 232
phytoanticipins unlike what is observed for the same amino acid production in leucaena 233
which shows features of inducibility resembling phytoalexin-like metabolites Alternatively 234
a putative inducible pool of mimosine in maricaacute might be involved in other types of stress 235
such as extended drought periods If involved in protection against oxidative stress as 236
described for leucaena mimosine in maricaacute may act predominantly by physical quenching 237
of ROS as indicated by the lack of overt chemical degradation Nevertheless further 238
investigations are needed to assess these hypotheses 239
To sum up mimosine biosynthesis was not modulated by the treatments evaluated as 240
in L leucocephala (Lam) de Wit To the best of our knowledge this is the first work that 241
analytically identifies and quantifies mimosine accumulation in M bimucronata 242
243
REFERENCES 244
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Mimosa bimucronata (DC) O Kuntze em aacutereas degradadas pela mineraccedilatildeo de carvatildeo R 246
Bras Bioci 5 750-752 247
Carvalho PER 2004 Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 248
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Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitis 250
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Champanerkar PA Vaidya VV Shailajan S Menon SN 2010 A sensitive rapid and 253
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determination of Mimosine in Mimosa pudica Linn Nat Sci 2 713-717 255
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Gomes GS Silva GS Silva DLS Oliveira RR Conceiccedilatildeo GM 2018 Botanical 257
Composition of Fabaceae Family in the Brazilian Northeast Maranhatildeo Brazil Asian J 258
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Correcirca LR Soares GLG Fett-Neto AG 2008 Allelopathic potential of Psychotria 260
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Ferreira AG Aquila MEA Jacobi US Rizvi V 1992 Allelopathy in Brazil In Allelopathy 263
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Fukao T Barrera-Figueroa BE Juntawong P Pentildea-Castro JM 2019 Submergence 265
and waterlogging stress in plants a review highlighting research opportunities and 266
understudied aspects Front Plant Sci 10 340 httpsdoiorg103389fpls201900340 267
Gregianini TS Silveira VC Porto DD Kerber VA Henriques AT Fett-Neto AG 268
2003 The alkaloid brachycerine is induced by ultraviolet radiation and is a singlet oxygen 269
quencher Photochem Photobiol 78(5) 470ndash474 httpsdoiorg1015620031-270
8655(2003)0784070TABIIB20CO2 271
Jacobi US Ferreira AG 1991 Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK 272
sobre espeacutecies cultivadas Pesq Agropec Bras 26(7) 935-943 273
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant 274
(Phaseolus vulgaris) Biosci Res 10(1) 29ndash32 275
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Kestring D Klein J Menezes LCCR Rossi MN 2009 Imbibition phases and 276
germination response of Mimosa bimucronata (Fabaceae Mimosoideae) to water 277
submersion Aquat Bot 91 105ndash109 httpsdoiorg101016jaquabot200903004 278
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect of 279
Ethephon as an ethylene-releasing compound on the metabolic profile of Chlorella vulgaris 280
J Agric Food Chem 64(23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541 281
Lorenzi H 1998 Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 282
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 283
Lorenzi H 2008 Plantas daninhas do Brasil terrestres aquaacuteticas parasitas e toacutexicas 4 ed 284
Nova Odessa Instituto Plantarum 640 p 285
Marchiori JNC 1993 Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 286
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Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation of 288
bioactive secondary metabolites by ultraviolet radiation mechanisms strategies and 289
applications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology for Medicinal 290
Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash190= 291
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo R Bhambra AS 292
Strnad M Bonfill M Fett-Neto AG 2018 Specializedplant 293
metabolismcharacteristicsandimpactontargetmoleculebiotechnologicalproduction 294
Molecular Biotechnology 60(2) 169ndash183httpsdoiorg101007s12033-017-0056-1 295
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase from 296
Leucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 297
922ndash934 httpsdoiorg101104pp113230870 298
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Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine 299
areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636 300
Olkoski D Wittmann MTS 2011 Cytogenetics of Mimosa bimucronata (DC) O Kuntze 301
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop Breed 302
Appl Biotechnol 11 27-35 httpdxdoiorg101590S1984-70332011000100004 303
Patreze CM Cordeiro L 2004 Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 304
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecol Manag 196 275ndash305
285 httpdxdoiorg101016jforeco200403034 306
Perotti JC Rodrigues-Correcirca KCS Fett-Neto AG 2015 Control of resin production in 307
Araucaria angustifolia an ancient South American conifer Plant Biology 17 852ndash859 308
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG 2019 Mimosine 309
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UV exposure Plant Physiology and Biochemistry 135 432ndash440 311
Pilatti DM Fortes AMT Jorge TCM Boiago NP 2019 Comparison of the phytochemical 312
profiles of five native plant species in two different forest formations Brazilian Journal of 313
Biology 79(2) 233-242 314
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS 2011 Biologia da reproduccedilatildeo 315
deMimosa bimucronatandash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 316
Simon MF and Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 317
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 318
endemism Biological Conservation 96 279-296 319
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 320
LM Andrade SF Cechinel-Filho V 2017 Chemical composition and diuretic natriuretic and 321
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kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its majority 322
constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash1624 323
Shah J 2003 The salicylic acid loop in plant defense Current Opinion Plant Biology6 (4) 324
365ndash371 325
Shinozaki K Uemura M Serres JB Bray EA Weretilnyk E 2015 Responses to Abiotic 326
Stress In Buchanan BB Gruissem W Jones RL (Eds) Biochemistry and Molecular 327
Biology of Plants Second Edition John Wiley and Sons Ltd 328
Soedarjo M and Borthakur D 1998 Mimosine a toxin produced by the tree-legume 329
Leucaena provides a nodulation competition advantage to mimosine-degrading Rhizobium 330
strains Soil Biology and Biochemistry 30(12)1605-1613 331
Vestena S Fett-Neto AG Duarte RC Ferreira AG 2001 Regulation of mimosine 332
accumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 333
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene induced 334
vinblastine accumulation is related to activated expression of downstream TIA pathway 335
genes in Catharanthus roseus BioMed Research International Article ID 3708187 336
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX Tsim KWK 2018 337
Jasmonate-elicited stress induces metabolic change in the leaves of Leucaena leucocephala 338
Molecules 23 (2) 339
Zhang H Memelink J 2009 Regulation of Secondary Metabolism by Jasmonate Hormones 340
In AE Osbourn and V Lanzotti (eds) Plant-derived Natural Products 3 DOI 101007978-341
0-387-85498-4_1 copy Springer Science + Business Media LLC 342
343
344
345
81
346
Figure 1 Constitutive concentration of mimosine in different plant organs of Mimosa 347
bimucronata Bars sharing the same letter do not differ statistically by Tukey test (Ple005) 348
The error bars denote standard error of 10 replicates 349
350
351
352
353
354
355
356
357
B B A C0
5
10
15
20
25
30
35
40
LEAVES GREEN FLOWER BUDS POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
co
nce
ntr
atio
n u
gg-1
Mimosine concentration in adult plants of Mimosa bimucronata (DC) Kuntze
82
C T R L S A
1 0 m M
S A
5 0 m M
E T H
0 0 7 m M
E T H
0 3 5 m M
M e J A
1 0 0 m M
M e J A
2 0 0 m M
S N P
1 0 m M
S N P
5 0 m M
0
1 0
2 0
3 0
T re a tm e n ts
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1) D A Y 2
D A Y 4
A B C C B C A B C C A B C A B C A
a b b b a a b a a b b a b
358
Figure 2 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 359
bimucronata treated with different signaling molecules SA = Salicylic Acid ETH = 360
Ethephon MeJA = Methyl Jasmonate SNP = Sodium Nitroprusside Uppercase and 361
lowercase letters indicate statistical differences among treatments in days 2 and 4 362
respectively Bars sharing a letter of the same case do not differ statistically by Tukey test 363
(Ple005) Indicates statistical difference in the same treatment between day 2 and 4 by t-364
test (Ple005) The error bars denote standard error of 5 replicates (25 individual seedlings 365
arranged in 5 groups of 5) 366
367
368
83
D AY 3 D AY 6
0
5
1 0
1 5
2 0
2 5
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1)
C O N TR O L
U V -C
369
Figure 3 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 370
bimucronata supplemented with UV-C radiation Indicates statistical difference in the same 371
treatment between day 3 and 6 by t-test (Ple005) The error bars denote standard error of 5 372
replicates (25 individual seedlings arranged in 5 groups of 5) 373
374
375
376
377
378
379
380
381
382
383
384
385
84
Consideraccedilotildees finais 386
- Experimentos que avaliam os efeitos da aplicaccedilatildeo exoacutegena de ANPs em diferentes espeacutecies 387
vegetais tecircm sido realizados principalmente com GABA Dentre os principais efeitos 388
conferidos pela aplicaccedilatildeo dessa moleacutecula em espeacutecies de mono e eudicotiledocircneas satildeo 389
relatados a toleracircncia agrave seca agrave salinidade e agraves temperaturas extremas 390
- Como metaboacutelitos especializados claacutessicos os ANPs podem ter sua concentraccedilatildeo basal 391
endoacutegena aumentada em resposta agrave induccedilatildeo mediada por uma vasta gama de tratamentos com 392
moleacuteculas sinalizadoras de estresse e fontes alternativas de estressores De um modo geral 393
observa-se o acuacutemulo das diferentes classes de ANPs em resposta agrave radiaccedilatildeo UV elicitores 394
quiacutemicos que mimetizam ataques por patoacutegenos dano mecacircnico agentes osmoacuteticos metais 395
pesados entre outros 396
- Especificamente em leucena a resposta observada em relaccedilatildeo aos diferentes tratamentos 397
testados indica que apesar do seu alto teor constitutivo nessa espeacutecie a biossiacutentese e o 398
acuacutemulo de mimosina podem ser modulados por fatores causadores de estresses exibindo -399
nessa espeacutecie - um padratildeo de acumulaccedilatildeo similar agrave fitoalexinas Em maricaacute por outro lado 400
aumento de acuacutemulo dessa moleacutecula natildeo foi observado para os mesmos tratamentos testados 401
para leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas 402
- O padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno 403
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado pela 404
sua degradaccedilatildeo 405
- As respostas observadas nos testes que avaliaram a atividade de mitigaccedilatildeo de espeacutecies 406
reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode agir como um agente 407
antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de estresse 408
85
Perspectivas 409
- Confirmaccedilatildeo em espectrocircmetro de massas eou ressonacircncia nuclear magneacutetica da natureza 410
quiacutemica da lsquomimosinarsquo presente em maricaacute 411
- Avaliaccedilatildeo do efeito de concentraccedilotildees mais elevadas e em diferentes periacuteodos de aplicaccedilatildeo 412
das moleacuteculas sinalizadoras testadas sobre o acuacutemulo de mimosina em leucena e maricaacute 413
- Ampliar a investigaccedilatildeo dos padrotildees de expressatildeo gecircnica dos genes que codificam para 414
mimosinase (em maricaacute) mimosina sintase (em ambas as espeacutecies testadas) bem como o 415
perfil de precursores e cataboacutelitos de mimosina em resposta aos tratamentos mencionados 416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
86
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Ahmed AMM Saacutenchez FJS Bavileacutes LRY Mahdy REZ Camaal JBC (2016) Tannins and 443
mimosine in Leucaena genotypes and their relations to Leucaena resistance against 444
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model systems Food Sci Technol Int 19(4)365-76 448
httpsdoiorg1011771082013212455186 449
Bitencourt F Zocche JJ Costa S Souza PZ Mendes AR (2007) Nucleaccedilatildeo de Mimosa 450
bimucronata (DC) O Kuntze em aacutereas degradadas pela mineraccedilatildeo de carvatildeo Revista 451
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A Montes-Perez R Muntildeoz-Rodriguez D Ku-Vera J (2015) Ovarian activity and estrus 454
behavior in early postpartum cows grazing Leucaena leucocephala in the tropics Trop Anim 455
Health Prod 47(8)1481-6 456
Carvalho PER (2004) Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 457
Teacutecnica 941-10 458
Chowtivannakul P Srichaikul B Talubmook C (2016) Antidiabetic and antioxidant activities 459
of seed extract from Leucaena leucocephala (Lam) de Wit Agriculture and Natural 460
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Chung H-H Chen M-K Chang Y-C Yang S-F Lin C-C Lin C-W (2017) Inhibitory effects 462
of Leucaena leucocephala on the metastasis and invasion of human oral cancer cells 463
Environmental Toxicology 321765ndash1774 httpsdoiorg101002tox22399 464
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Crowe B Poynter JA Manukyan MC Wang Y Brewster BD Herrmann JL Abarbanell 465
AM Weil BR Meldrum DR (2001) Pretreatment with intracoronary mimosine improves 466
postischemic myocardial functional recovery Surgery 150(2) 191-106 467
Fallon (2015) Effects of mimosine on Wolbachia in mosquito cells cell cycle suppression 468
reduces bacterial abundance In Vitro Cell Dev Biol Anim 51(9)958-63 469
httpsdoiorg101007s11626-015-9918-7 Epub 2015 May 28 470
Fernaacutendez-Salas A Alonso-Diacuteaza MA Acosta-Rodriacuteguez A Torres-Acosta JFJ Sandoval-471
Castro CA Rodriacuteguez-Vivas RI (2011) In vitro acaricidal effect of tannin-rich plants against 472
the cattle tick Rhipicephalus (Boophilus) microplus (Acari Ixodidae) Veterinary 473
Parasitology 175113ndash118 2010 httpsdoiorg101016jvetpar201009016 474
Ferreira AG Aquila MEA Jacobi US Rizvi V (1992) Allelopathy in Brazil In Allelopathy 475
basic and applied aspects Rizvi V and Jacobi US (Eds) Chapman and Hall PP 243-250 476
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad Hossain Md Anai 477
T Oku H (2018) Cytosolic cysteine synthase switch cysteine and mimosine production in 478
Leucaena leucocephala Appl Biochem Biotechnol 186 (3) 613ndash632 479
httpsdoiorg101007s12010-018-2745-z 480
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of the thyrotoxic 481
amino acid mimosine by cysteine synthase Phytochemistry 29 (11) 3461ndash3465 482
httpsdoiorg1010160031-9422(90)85258-H 483
Jacobi US Ferreira AG (1991) Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK Sobre 484
espeacutecies cultivadas Pesquisa Agropecuaacuteria Brasileira 26(7) 935-943 485
Jamous RM Ali-Shtayeh MS Abu-Zaitoun SY Markovics A Azaizeh H (2017) Effects of 486
selected Palestinian plants on the in vitro exsheathment of the third stage larvae of 487
gastrointestinal nematodes BMC Veterinary Research 13308 488
httpdxdoiorg101186s12917-017-1237-7 489
Jiao CJ Jiang J-L Ke L-M Cheng W Li F-M Li Z-X Wang C-Y (2011) Factors affecting 490
β-ODAP content in Lathyrus sativus and their possible physiological mechanisms Food 491
Chem Toxicol 49 543ndash549 httpsdoiorg101016jfct201004050 492
Kubota S Fukumoto Y Ishibashi K Soeda S Kubota SS Yuki R Nakayama Y Aoyama K 493
Yamaguchi N (2014) Activation of the prereplication complex is blocked by mimosine 494
88
through reactive oxygen species-activated ataxia telangiectasia mutated (ATM) protein 495
without DNA damage J Biol Chem 28 289(9)5730-46 496
Kuppusamy UR Arumugam B Azaman N Wai CJ (2014) Leucaena leucocephala Fruit 497
Aqueous Extract Stimulates Adipogenesis Lipolysis and Glucose Uptake in Primary Rat 498
Adipocytes Hindawi Publishing Corporation e Scientific World Journal Article ID 737263 499
8 pages httpdxdoiorg1011552014737263 500
Kusama-Eguchi K (2019) Research in motor neuron diseases caused by natural substances 501
focus on pathological mechanisms of neurolathyrism Yakugaku Zasshi 139 (4) 609-502
615 httpsdoiorg101248yakushi18-00202 503
Kutchan TM Gershenzon J Moslashller BL Gang DR (2015) Natural Products In Buchanan 504
BB Gruissem W and Jones RL (eds) Biochemistry amp Molecular Biology of Plants 2nd edn 505
Wiley Blackwell Chichester pp 1135-1205 506
Lalande M (1990) A reversible arrest point in the late G1 phase of the mammalian cell cycle 507
Exp Cell Res 186 332ndash339 508
Li X-W Hu C-P Li Y-J Gao Y-X Wang XM Yang J-R (2015) Inhibitory effect of L-509
mimosine on bleomycin-induced pulmonary fibrosis in rats Role of eIF3a and p27 Int 510
Immunopharmacol 27(1) 53ndash64 511
Little Jr EL Skolmen RG (1989) Koa haole Agriculture Handbook 679 USDA 512
Lorenzi H (1998) Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 513
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 514
Marchiori JNC (1993) Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 515
O Kuntze Ciecircncia Florestal 3 85-106 516
Mohammed RS El Souda SS Taie HAA Moharam ME Shaker KH (2015) Antioxidant 517
antimicrobial activities of flavonoids glycoside from Leucaena leucocephala leaves Journal 518
of Applied Pharmaceutical Science 5(06)138-147 519
httpdxdoiorg107324JAPS201550623 520
Negi VS Bingham J-P Li QX Borthakur D (2014) A carbon-nitrogen lyase from Leucaena 521
leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 (2) 922ndash522
934 httpsdoiorg101104pp113230870 523
89
Olkoski D Wittmann MTS (2011) Cytogenetics of Mimosa bimucronata (DC) O Kuntze 524
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop 525
Breeding and Applied Biotechnology 11 27-35 526
Patreze CM Cordeiro L (2004) Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 527
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecology and Management 528
196275ndash285 529
Pilatti DM Fortes AMT Jorge TCM Boiago NP (2019) Comparison of the phytochemical 530
profiles of five native plant species in two different forest formations Brazilian Journal of 531
Biology 79(2) 233-242 532
Ramos-Ruiz R Poirot E Flores-Mosquera M (2018) GABA a non-protein amino acid 533
ubiquitous in food matrices Cogent Food Agric 41534323 534
httpsdoiorg1010802331193220181534323 535
REFLORA (2019) httpfloradobrasiljbrjgovbrreflora Acesso em agosto de 2019 536
Rodgers KJ Samardzic K Main BJ (2015) Toxic Nonprotein Amino Acids Plant Toxins 537
httpsdoiorg 101007978-94-007-6728-7_9-1 538
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG (2019) Mimosine 539
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 540
UV exposure Plant Physiology and Biochemistry 135 432ndash440 541
httpsdoiorg101016jplaphy201811018 542
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 543
LM Andrade SF Cechinel-Filho V (2017) Chemical composition and diuretic natriuretic 544
and kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its 545
majority constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash546
1624 547
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS (2011) Biologia da reproduccedilatildeo 548
de Mimosa bimucronata ndash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 549
Simon MF Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 550
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 551
endemism Biological Conservation 96 279-296 552
90
Soares AMS Arauacutejo SA Lopes SG Costa Junior LM (2015) Anthelmintic activity of 553
Leucaena leucocephala protein extracts on Haemonchus contortus Braz J Vet Parasitol 554
Jaboticabal 24(4) 396-401 httpdxdoiorg101590S1984-29612015072 555
Soerdajo M Borthakur D (1998) Mimosine a toxin produced by the tree-legume Leucaena 556
provides a nodulation competition advantage to mimosine-degrading Rhizobium strains Soil 557
Biol Biochem 30(12) 16051613 558
Souza-Lima ES Sinani TR Pott A Sartori ALB (2017) Mimosoideae (Leguminosae) in the 559
Brazilian Chaco of Porto Murtinho Mato Grosso do Sul Rodrigueacutesia 68(1) 263-290 2017 560
httpdxdoiorg1015902175-7860201768131 561
Taiz L amp Zeiger E (2010) Plant Physiology 5th edition Sinauer Associates Inc Sunderland 562
Verma VK Rani KV Kumara SR Prakash O (2018) Leucaena leucocephala pod seed 563
protein as an alternate to animal protein in fish feed and evaluation of its role to fight against 564
infection caused by Vibrio harveyi and Pseudomonas aeruginosa Fish and Shellfish 565
Immunology 76 (2018) 324ndash332 httpsdoiorg101016jfsi201803011 566
Yafuso JT Negi VS Bingham J-P Borthakur D (2014) An O-acetylserine (thiol) lyase from 567
Leucaena leucocephala is a cysteine synthase but not a mimosine synthase Appl Biochem 568
Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z 569
Zarin RMA Wan HY Isha A Armani N (2016) Antioxidant antimicrobial and cytotoxic 570
potential of condensed tannins from Leucaena leucocephala hybrid Food Science and 571
Human Wellness 5 65ndash75 httpdxdoiorg101016jfshw201602001 572
573
574
Contents lists available at ScienceDirect
Industrial Crops amp Productsjournal homepage wwwelseviercomlocateindcrop
Resin tapping transcriptome in adult slash pine (Pinus elliottii var elliottii)Camila Fernanda de Oliveira Junkes1 Artur Teixeira de Arauacutejo Juacutenior1 Juacutelio Ceacutesar de LimaFernanda de Costa Thanise Fuumlller Maacutercia Rodrigues de Almeida Franciele Antocircnia NeisKelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-NetoCenter for Biotechnology and Department of Botany Federal University of Rio Grande do Sul Porto Alegre PO Box 15005 91501-970 Brazil
A R T I C L E I N F O
KeywordsPinus elliottiResinResinosisTranscriptomeAdjuvant paste
A B S T R A C T
To better understand the bases of resin production a major source of terpenes for industry the transcriptome ofadult Pinus elliottii var elliottii (slash pine) trees under field commercial resinosis was obtained Samples werecollected from cambium after 5 and 15 days of treatment application which included tapping followed byapplication of commercial resin stimulant paste or control wounding without paste Overall mean number ofreads of all 16 libraries (2 treatments x 2 times x 4 replicated trees) was 34582048 Of these 89 were mappedagainst the reference sequence with a mismatch of 058 Using the Blast2Go 570 candidate genes were de-tected based on sequence annotation By comparing the expression profile between paste and control 310differentially expressed genes (DEGs) were identified at 5 days and 190 at 15 days with a significant fold changeof log2gt 12 Regarding changes in time comparisons within each treatment 210 and 105 DEGs were identifiedwithin control and paste treatment respectively Genes with different expression patterns in the times andtreatments examined included ethylene responsive transcription factors geranylgeranyl diphosphate synthasediterpene synthase cytochrome P450 and ABC transporters all of which may play important roles in resinproduction RT-qPCR analysis correlated well with the data obtained by RNAseq Resin composition changedover time This is the first transcriptomic investigation of resinosis of the main species used in the bioresinindustry and of molecular analyses of resinosis under field operations with implications for stand managementstimulant paste development genotype selection and breeding for high resinosis
1 Introduction
The adaptive success of conifers is largely due to the development ofa defense system based on the synthesis and secretion of terpenes in allmajor organs and different tissues (Miller et al 2005 Hall et al 2013Warren et al 2015) Conifer resin is a viscous fluid composed of acomplex mixture of terpenoids such as monoterpenes sesquiterpenesand diterpenes (Zulak and Bohlmann 2010) These terpenoids are se-creted from severed resin ducts when the tree is under biotic attack(Ralph et al 2006 Lange 2015 Geisler et al 2016) acting as pro-tectants (Schiebe et al 2012 Liu et al 2015)Biosynthesis of terpenes in conifers starts from isomerization of two
isoprenoid (C5) units dimethylallyl diphosphate (DMAPP) and iso-pentenyl diphosphate (IPP) These molecules can be biosynthesized viatwo separate routes in plants the methyl-erythritol 4-phosphate andmevalonate pathways IPP is synthesized and isomerized to DMAPP byisopentenyl diphosphate isomerase then prenyl transferases catalyze
the condensation of these two C5-units to geranyl diphosphate (Pazoukiand Niinemets 2016) Their elongation to prenyl diphosphates withaddition of IPP molecules leads to monoterpenes (C10) sesquiterpenes(C15) and diterpenes (C20) which are the substrates for terpene syn-thases (TPS) (Keeling and Bohlmann 2006b)TPSs are part of a large family of mechanistically related enzymes
involved in both primary and secondary metabolism (Keeling andBohlmann 2006b) The events of evolutionary diversification and ex-pansion of plant TPSs appear to have originated from gene duplicationsdomain losses and sub- or neofunctionalizations with subsequent di-vergence of an ancestral TPS gene of primary metabolism (Hall et al2013) Modification of TPS products changes their physical propertiesand may alter their biological activities (Chen et al 2011) TPSs of highsequence identity may have different functions even in closely relatedspecies Low sequence identity of TPSs in phylogenetically distantspecies does not preclude the possibility of independent evolution of thesame or related function of these enzymes (Zerbe and Bohlmann 2015)
httpsdoiorg101016jindcrop2019111545Received 4 January 2019 Received in revised form 10 June 2019 Accepted 4 July 2019
Corresponding authorE-mail address fettnetocbiotufrgsbr (AG Fett-Neto)1 These authors have equally contributed to this work
doi 1015900102-33062019abb0114
Acta Botanica Brasilica
Sustainable production of bioactive alkaloids in Psychotria L of
southern Brazil propagation and elicitation strategies
Yve Verocircnica da Silva Magedans1 Kelly Cristine da Silva Rodrigues-Correcirca1 Cibele Tesser da Costa1
Heacutelio Nitta Matsuura1 and Arthur Germano Fett-Neto1
Received April 1 2019Accepted June 28 2019
ABSTRACTPsychotria is the largest genus in Rubiaceae South American species of the genus are promising sources of natural
products mostly due to bioactive monoterpene indole alkaloids they accumulate ese alkaloids can have analgesic
antimutagenic and antioxidant activities in dierent experimental models among other pharmacological properties
of interest Propagation of genotypes with relevant pharmaceutical interest is important for obtaining natural
products in a sustainable and standardized fashion Besides the clonal propagation of elite individuals the alkaloid
content of Psychotria spp can also be increased by applying moderate stressors or stress-signaling molecules is
review explores advances in research on methods for plant propagation and elicitation techniques for obtaining
bioactive alkaloids from Psychotria spp of the South Region of Brazil
Keywords abiotic stress alkaloids elicitation monoterpenes plant propagation Psychotria southern Brazil
sustainability
Introduction
Psychotria belongs to Rubiaceae one of the major families
of $owering plants having economic interest e family
includes coee a few signicant poisonous plants to livestock
besides several important ornamental and medicinal species
(Souza amp Lorenzi 2012) Psychotria has captured researchersrsquo
attention mostly because of its medicinal properties
Psychotria colorata is an Amazonian species that produces
polyindolinic alkaloids with analgesic activity (Matsuura et
al 2013) e promising results obtained with P colorata
motivated the investigation of southern Brazilian Psychotria
species and the discovery of new bioactive alkaloids (Porto
et al 2009) Moreover leads on in planta alkaloid functions
were also topic of experimental evaluation
One of the key elements that needs to be addressed early
on during the process of developing new bioactive molecules
from plants is the capacity to generate catalytically active
biomass to support extraction and steady supply ere are a
number of ways through which these goals may be reached
including greenhouse rooting of cuttings (mini-cutting
1 Laboratoacuterio de Fisiologia Vegetal Departamento de Botacircnica Instituto de Biociecircncias e Centro de Biotecnologia Universidade Federal do Rio
Grande do Sul 91501-970 Porto Alegre RS Brazil
Corresponding author fettnetocbiotufrgsbr
Review
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Biomass yield of resin in adult Pinus elliottii Engelm trees is differentially
regulated by environmental factors and biochemical effectors
Franciele Antocircnia Neis Fernanda de Costa Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima
Kelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-Neto
Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) CP 15005 CEP 91501-970 Porto Alegre RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Biomass
Terpene resin
Seasonal
Benzoic acid
Regenerated forest
A B S T R A C T
Biomass of pine resin finds several applications in the chemical pharmaceutical biofuel and food industries
Resin exudation after injury is a key defense response in Pinaceae since this complex mixture of terpenes has
insecticidal antimicrobial and wound repair properties Resin yield is increased by effectors applied on the
wound area including phytohormones and metal cofactors of terpene synthases The interaction of resinosis
mechanism effectors is not fully understood particularly in adult forest setups under natural environmental
variations The aim of this work was to determine how resin exudation by wounded trunks of adult P elliottii
responded to combined chemical effectors involved in different regulatory pathways of resinosis (metal cofactors
of terpene synthases benzoic acid and plant growth regulators) and whether seasonal and tree distribution
variations affected these responses Symmetrically planted and scattered trees regenerated from the seed bank
had similar resin biomass yields suggesting that the homogeneity in development and spatial arrangement were
not significant factors in resin yield This new finding is of practical importance with the used tapping system
since costs of implanting forests by regeneration can be advantageous compared to planting In addition it was
shown for the first time that the salicylic acid precursor benzoic acid and the auxin naphthalene acetic acid
promoted resin exudation when individually applied to wound sites Both these adjuvants are two orders of
magnitude less costly compared to the conventionally used ethylene precursors besides facing less environ-
mental and health restrictions for use Most adjuvant-treated trees showed higher resin flow in the second year
indicating mechanisms of response build up Overall temperature was more important than rainfall as en-
vironmental parameter affecting resin biosynthesis which was higher in the warmer months of spring and
summer The combination of resinosis stimulant effectors from different signaling pathways showed no sig-
nificant synergistic or additive effect suggesting possible converging signaling pathways andor limitation of
common intermediate transducing molecules
1 Introduction
Pines occupy highly diverse environments over a range of tem-
peratures water and nutrient availabilities irradiance levels and pho-
toperiods being able to effectively face attacks from diverse herbivore
and pathogen guilds The success of conifers is linked to their complex
terpene biochemistry hosted by specialized secretory cells The terpe-
noid resin synthesized by Pinus spp is one of the main mechanisms of
defense of these trees particularly against bark beetles and the fungi
they carry (Fett-Neto and Rodrigues-Correcirca 2012) Pine resin biomass
is essentially composed of a monoterpene and sesquiterpene-rich tur-
pentine and diterpenoid-rich rosin fraction both finding numerous in-
dustrial applications as non-wood forest products (Rodrigues-Correcirca
et al 2012)
Molecules capable of modulating different signaling pathways have
been identified as resin yield stimulators including sulfuric acid (ex-
tends wound damage) 2-chloroethylphosphonic acid (CEPA a syn-
thetic ethylene precursor) paraquat (free radical generator) yeast ex-
tract (mimics attack by pathogens) salicylic acid (pathogen signaling
molecule) auxin (promotes ethylene biosynthesis and resin canal dif-
ferentiation) jasmonic acid (signals mechanical damage and promotes
secondary metabolism) and metal ions such as potassium iron and
manganese (cofactors of terpene synthases in conifers) and copper (a
component of ethylene receptors) (Clements 1970 Conrath et al
2002 Fett-Neto and Rodrigues-Correcirca 2012 Hudgins and Franceschi
2004 Lewinsohn et al 1994 Martin et al 2002 Popp et al 1995
httpsdoiorg101016jindcrop201803027
Received 12 December 2017 Received in revised form 9 March 2018 Accepted 13 March 2018
Corresponding author
E-mail addresses franci_neisyahoocombr (FA Neis) fernandadecostayahoocombr (F de Costa) thanisenfyahoocombr (TN Fuumlller)
jjuliocesarlimagmailcom (JC de Lima) krodriguescbiotufrgsbr (KC da Silva Rodrigues-Correcirca) jpfettcbiotufrgsbr (JP Fett) fettnetocbiotufrgsbr (AG Fett-Neto)
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Research Paper
Dual allelopathic effects of subtropical slash pine (Pinus elliottii Engelm)
needles Leads for using a large biomass reservoir
Kelly Cristine da Silva Rodrigues-Correcircaa Gelson Halmenschlagera Joseacuteli Schwambachb
Fernanda de Costaa Emili Mezzomo-Trevizana Arthur Germano Fett-Netoa
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 Brazilb University of Caxias do Sul Institute of Biotechnology Caxias do Sul RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Seasonality
Growth
Germination
Litter
Substrate
A B S T R A C T
Pinus elliottii Engelm (slash pine) is distributed along the maritime coast of Southern Brazil where it shows
invasive pattern and typical allelopathic features Large quantities of needle litter are produced by pine trees a
biomass that is little explored in areas where this species is alien Little is known about the dynamics of needle
and litter phytochemical interactions particularly in subtropical environments To elucidate the full range of
needle and litter allelopathic potential the effects of litter (superficial and deep) and seasonally harvested fresh
slash pine needles stored for different times were evaluated against lettuce tomato and cucumber seeds and
seedlings Increasing concentrations (0 1 2 4 and 8 wv) of hot and cold aqueous extracts of needles
and litter affected in different ways target plant development Growth and germination inhibition were directly
related to the highest extract concentrations (regardless of the season and mainly in hot water extracts) of
needles On the other hand stimulatory effects of litter extracts on lettuce growth were observed Growth and
germination of cucumber and tomato were not affected by pine litter as substrate when compared to rice husk
The presumable high polarity and thermal stability of slash pine leaf biomass allelochemicals and their transient
toxic effect or growth promoting impact suggest potential applications of this largely available biomass both as a
biological herbicide and growth substrate in plant propagation
1 Introduction
Native from the Northern Hemisphere Pinus is one of the most
widely distributed genera throughout different climate regions of the
globe growing either as native or alien species even in extreme habi-
tats (Rodrigues-Correcirca and Fett-Neto 2012) Despite the high economic
value currently attributed to pine wood and oleoresin (Rodrigues-
Correcirca et al 2012) there is increasing concern about the aggressive
potential of invasiveness displayed by Pinus species especially those
cultivated out of their native range of distribution (Richardson et al
2008 Rolon et al 2011) These species are dispersed by wind and there
is notably low plant diversity observed in most understories of pine
plantations (Kato-Noguchi et al 2009) This latter feature has been
considered an important trait of allelopathic interference
The term ldquoallelopathyrdquo was coined by Molisch in 1937 as a chemical
reciprocal interaction established among plants (including micro-
organisms) sharing the same site by means of the release of secondary
metabolites named allelochemicals (Rice 1984) For the most part
these metabolites are derived from the shikimic acid or isoprenoid
pathway and their biosynthesis can be modulated by biotic and abiotic
stresses (Nascimento and Fett-Neto 2010) including seasonal-related
changes (Sartor et al 2013) Allelopathy studies may range from sterile
assays (Aryakia et al 2015) to soil (Correcirca et al 2008 Sharma et al
2016) and field tests being a complex biological phenomenon to as-
certain in several circumstances due to issues of solubility release
mechanisms and stability of bioactive compounds (Scognamiglio et al
2013) Often the use of complementary methods provides more in-
formative data
The allelopathic effects of soil leachates green needles and litter
extracts of Pinus spp on germination and seedling growth aspects of
wild and crop species have been evaluated in natural and cultivated
pine stands and have proven to be stimulatory or inhibitory (Lodhi and
Killingbeck 1982 Kil and Yim 1983 Nektarios et al 2005 Akkaya
et al 2006 Machado 2007 Alrababah et al 2009 Sartor et al 2009
Kato-Noguchi et al 2011 Rolon et al 2011 Valera-Burgos et al
2012) exhibiting in some cases autotoxicity (Garnett et al 2004
Fernandez et al 2008 Zhu et al 2009 Monnier et al 2011) Studies
on potential dual allelopathic effects of Pinus elliottii Engelm (slash
httpdxdoiorg101016jindcrop201706019
Received 23 March 2017 Received in revised form 15 May 2017 Accepted 7 June 2017
Corresponding author
E-mail address fettnetocbiotufrgsbr (AG Fett-Neto)
ORIGINAL RESEARCHpublished 16 June 2016
doi 103389fpls201600849
Frontiers in Plant Science | wwwfrontiersinorg 1 June 2016 | Volume 7 | Article 849
Edited by
Juan Francisco Jimenez Bremont
Instituto Potosino de Investigacioacuten
Cientiacutefica y Tecnoloacutegica Mexico
Reviewed by
Mariacutea De La Luz Guerrero Gonzaacutelez
Universidad Autoacutenoma de San Luis
Potosiacute Mexico
Rosalia Cristina Paz
CIGEOBIO (CONICETFCEFN UNSJ)
Argentina
Correspondence
Arthur G Fett-Neto
fettnetocbiotufrgsbr
daggerThese authors have contributed
equally to this work
Specialty section
This article was submitted to
Plant Physiology
a section of the journal
Frontiers in Plant Science
Received 08 December 2015
Accepted 30 May 2016
Published 16 June 2016
Citation
de Lima JC de Costa F Fuumlller TN
Rodrigues-Correcirca KCdS Kerber MR
Lima MS Fett JP and Fett-Neto AG
(2016) Reference Genes for qPCR
Analysis in Resin-Tapped Adult Slash
Pine As a Tool to Address the
Molecular Basis of Commercial
Resinosis Front Plant Sci 7849
doi 103389fpls201600849
Reference Genes for qPCR Analysisin Resin-Tapped Adult Slash Pine Asa Tool to Address the MolecularBasis of Commercial Resinosis
Juacutelio C de Lima 1dagger Fernanda de Costa 1 dagger Thanise N Fuumlller 1
Kelly C da Silva Rodrigues-Correcirca 2 Magnus R Kerber 1 Mariano S Lima 1
Janette P Fett 1 and Arthur G Fett-Neto 1
1 Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul
Porto Alegre Brazil 2 Biological Sciences Department Regional Integrated University of Alto Uruguai and Missotildees (URI-FW)
Frederico Westphalen Brazil
Pine oleoresin is a major source of terpenes consisting of turpentine (mono- and
sesquiterpenes) and rosin (diterpenes) fractions Higher oleoresin yields are of economic
interest since oleoresin derivatives make up a valuable source of materials for chemical
industries Oleoresin can be extracted from living trees often by the bark streak method
in which bark removal is done periodically followed by application of stimulant paste
containing sulfuric acid and other chemicals on the freshly wounded exposed surface
To better understand the molecular basis of chemically-stimulated and wound induced
oleoresin production we evaluated the stability of 11 putative reference genes for the
purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis
Samples for RNA extraction were collected from field-grown adult trees under tapping
operations using stimulant pastes with different compositions and at various time points
after paste application Statistical methods established by geNorm NormFinder and
BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3
and UBI To confirm expression stability of the candidate reference genes expression
profiles of putative P elliottii orthologs of resin biosynthesis-related genes encoding Pinus
contorta β-pinene synthase [PcTPS-(minus)β-pin1] P contorta levopimaradieneabietadiene
synthase (PcLAS1) Pinus taeda α-pinene synthase [PtTPS-(+)αpin] and P taeda
α-farnesene synthase (PtαFS) were examined following stimulant paste application
Increased oleoresin yields observed in stimulated treatments using phytohormone-based
pastes were consistent with higher expression of pinene synthases Overall the
expression of all genes examined matched the expected profiles of oleoresin-related
transcript changes reported for previously examined conifers
Keywords resin Pinus gene expression normalizer genes terpene synthase
19
Chapter 2
Stimulant Paste Preparation and Bark Streak Tapping Technique for Pine Oleoresin Extraction
Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
Tapping technique comprises the extraction of pine oleoresin a non-wood forest product consisting of a
complex mixture of mono sesqui and diterpenes biosynthesized and exuded as a defense response to
wounding Oleoresin is used to produce gum rosin turpentine and their multiple derivatives Oleoresin
yield and quality are objects of interest in pine tree biotechnology both in terms of environmental and
genetic control Monitoring these parameters in individual trees grown in the fi eld provides a means to
examine the control of terpene production in resin canals as well as the identifi cation of genetic-based
differences in resinosis A typical method of tapping involves the removal of bark and application of a
chemical stimulant on the wounded area Here we describe the methods for preparing the resin-stimulant
paste with different adjuvants as well as the bark streaking process in adult pine trees
Key words Oleoresin Pine Tapping Chemical stimulant Wounding
1 Introduction
Several conifer species produce oleoresin a complex mixture of isoprenoid compounds relevant for defense against herbivores and pathogens Two major fractions can be recognized in oleoresin (a) turpentine the volatile fraction containing mono- and sesquiter-penes and (b) rosin the nonvolatile diterpene fraction Oleoresin is a forest commodity of global interest fi nding applications in diverse industry sectors Rosin is used in adhesives printing ink manufacture and paper sizing Turpentine can be used either as a solvent for paints and varnishes or as a raw material for fraction-ation of high-value chemicals used in the pharmaceutical agro-chemical and food industry [ 1 ndash 3 ]
During the extraction activity resin is obtained from the tree in a similar way as rubber tree tapping which generally involves the
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_2 copy Springer Science+Business Media New York 2016
These authors have equally contributed to this work
fettnetocbiotufrgsbr
27
Chapter 3
A Modifi ed Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines
Juacutelio Ceacutesar de Lima Thanise Nogueira Fuumlller Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes
through high-throughput sequencing technologies microarray and also northern blots RT-PCR and
RTqPCR Even though many specifi c protocols designed for plants with high content of secondary metab-
olites have been developed these are often expensive time consuming and not suitable for a wide range
of tissues Here we present a modifi cation of the method previously described using the commercially
available Concerttrade Plant RNA Reagent (Invitrogen) buffer for fi eld-grown adult pine trees with high
oleoresin content
Key words RNA Pines Concert plant RNA reagent Stem RNA extraction Oleoresin Conifers
1 Introduction
Several conifer species especially within the Pinaceae have tissues with high concentrations of phenolics terpenes and polysaccha-rides [ 1 ] Many specifi c protocols that are appropriate for plants rich in secondary metabolite s have been developed but the extrac-tion of high-quality RNA from these tissues using commercial kits is often diffi cult and usually not applicable to woody tissues [ 2 ndash 6 ] One of the major issues during RNA extraction concerns the pres-ence of phenolic compounds which oxidize and form quinones Aromatic compounds bind RNA which interferes in downstream steps and applications [ 3 7 ] Another point of concern is the har-vest of plant samples in the experimental fi eld which constitutes another obstacle in the efforts to avoid degradation of RNA [ 8 ] These problems often result in RNAs of low quality and insuffi -cient amounts especially for methodologies that normally require
These authors have equally contributed to this work
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_3 copy Springer Science+Business Media New York 2016
fettnetocbiotufrgsbr
RESEARCH PAPER
Control of resin production in Araucaria angustifolia an ancientSouth American coniferJ C Perotti1 K C da Silva Rodrigues-Correa123 amp A G Fett-Neto12
1 Plant Physiology Laboratory Department of Botany Federal University of Rio Grande do Sul (UFRGS) Porto Alegre RS Brazil
2 Center for Biotechnology UFRGS Porto Alegre RS Brazil
3 Present address Regional Integrated University of Alto Uruguai and Miss~oes (URI-FW) Frederico Westphalen RS Brazil
Keywords
Araucaria ethylene jasmonic acid nitric
oxide resin salicylic acid terpenes
Correspondence
A G Fett-Neto Plant Physiology Laboratory
Center for Biotechnology Federal University
of Rio Grande do Sul (UFRGS) PO Box 15005
Av Bento Goncalves 9500 91501-970 Porto
Alegre Brazil
E-mail fettnetocbiotufrgsbr
Editor
K Leiss
Received 22 July 2014 Accepted 11
December 2014
doi101111plb12298
ABSTRACT
Araucaria angustifolia is an ancient slow-growing conifer that characterises parts ofthe Southern Atlantic Forest biome currently listed as a critically endangered speciesThe species also produces bark resin although the factors controlling its resinosis arelargely unknown To better understand this defence-related process we examined theresin exudation response of A angustifolia upon treatment with well-known chemicalstimulators used in fast-growing conifers producing both bark and wood resin suchas Pinus elliottii The initial hypothesis was that A angustifolia would display signifi-cant differences in the regulation of resinosis The effect of Ethrel (ET ndash ethylene pre-cursor) salicylic acid (SA) jasmonic acid (JA) sulphuric acid (SuA) and sodiumnitroprusside (SNP ndash nitric oxide donor) on resin yield and composition in youngplants of A angustifolia was examined In at least one of the concentrations testedand frequently in more than one an aqueous glycerol solution applied on fresh woundsites of the stem with one or more of the adjuvants examined promoted an increase inresin yield as well as monoterpene concentration (a-pinene b-pinene camphene andlimonene) Higher yields and longer exudation periods were observed with JA and ETanother feature shared with Pinus resinosis The results suggest that resinosis controlis similar in Araucaria and Pinus In addition A angustifolia resin may be a relevantsource of valuable terpene chemicals whose production may be increased by usingstimulating pastes containing the identified adjuvants
INTRODUCTION
Many conifer species produce terpenoid-based resins that havelong been studied for their industrial importance and role indefence against attack by herbivores and pathogens The twomost important resin-producing families of conifers are Pina-ceae and Araucariaceae (Langenheim 1996) The viscous resinsecretion is generally composed of a complex mixture ofterpenoids consisting of roughly equal parts of volatile mono-(C10) and sesquiterpene (C15 turpentine) fractions and non-volatile diterpenic (C20 rosin) components (Rodrigues-Correaet al 2013) Terpenes act in a complex and multilayereddefence response providing toxicity against bark beetles andfungi bark wound sealing disruption of insect developmentand attraction of herbivore predators (Phillips amp Croteau1999)Most conifers rely on some combination of preformed and
inducible resin defences (Trapp amp Croteau 2001 Zulak amp Bohl-mann 2010) Resin defences are controlled by environmentaland genetic factors to various extents depending on species(Roberds et al 2003 Sampedro et al 2010 Moreira et al2013) Resin traits have been reported as highly variable havingmoderate heritability indicating that breeding efforts towardssuper-resinous forests are promising (Tadasse et al 2001Roberds et al 2003) Several chemicals are known as stimulantsof resin production Commercial extraction of resin from pine
trees uses periodic bark streaking and application of resin stim-ulant pastes to the wound
Resin-stimulant paste based on sulphuric acid (SuA) iswidely used for the commercial production of pine resin Cur-rent stimulant pastes usually have two chemically active com-ponents SuA to magnify the wounding and an ethyleneprecursor (2-chloroethylphosphonic acid CEPA or Ethrel ndash
ET) to stimulate resin flow (Rodrigues et al 2011 Rodrigues-Correa amp Fett-Neto 2013) Jasmonic acid (JA) and its methylester methyl jasmonate (MeJa) are among the most widelyused chemical elicitors of plant secondary metabolism It hasbeen shown that the exogenous application of MeJa or herbi-vore attack induce chemical and anatomical defence responsesin conifers such as the formation of traumatic resin ducts andresin accumulation in stems along with increased biosynthesisof terpenes and phenolics (Franceschi et al 2002 Martin et al2002 Heijari et al 2005 Zeneli et al 2006 Moreira et al 2008Gould et al 2009) JA commercial use however is limited byits high cost
The effects of exogenous salicylic acid (SA) on conifer ter-pene production have also been studied In Pinus elliottiiapplication of 10 molm3 of SA induced resin productionin wound panels but in Pseudotsuga menziesii and Sequoia-dendron giganteum it had no apparent effect on resinaccumulation (Hudgins amp Franceschi 2004 Rodrigues ampFett-Neto 2009) Nitric oxide (NO) has also emerged as an
Plant Biology 17 (2015) 852ndash859 copy 2014 German Botanical Society and The Royal Botanical Society of the Netherlands852
Plant Biology ISSN 1435-8603
ix
RESUMO
Ao longo de sua evoluccedilatildeo as plantas desenvolveram estrateacutegias estruturais e quiacutemicas de
defesa em resposta aos estresses bioacuteticos e abioacuteticos impostos pelo ambiente Dentre
essas satildeo reconhecidas moleacuteculas quimicamente especializadas denominadas
metaboacutelitos secundaacuterios produtos naturais ou metaboacutelitos especializados Aminoaacutecidos
natildeo proteicos (ANPs) satildeo compostos nitrogenados que constituem aleacutem de componentes
do arsenal de defesa quiacutemica vegetal uma importante fonte de reserva de carbono e
nitrogecircnio para diversos taxa especialmente aqueles pertencentes agrave famiacutelia Fabaceae de
Angiospermas Esse grupo de moleacuteculas quimicamente heterogecircneo eacute assim definido por
natildeo participar da formaccedilatildeo de estruturas proteicas funcionais sendo frequentemente
toacutexicos quando erroneamente incorporados nas cadeias polipeptiacutedicas em formaccedilatildeo em
funccedilatildeo da similaridade estrutural que apresentam com os aminoaacutecidos proteicos Sob o
ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados ANPs satildeo
em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou abioacutetica como
o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de elicitores
quiacutemicos por exemplo O objetivo da presente tese foi investigar o papel bioloacutegico da
mimosina endoacutegena em Leucaena leucocephala (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute) a partir da avaliaccedilatildeo do efeito de tratamentos
relacionados ao estresse abioacutetico (UV-C aacutecido saliciacutelico metil jasmonato e etileno)
Mimosina eacute um ANP aromaacutetico anaacutelogo da L-tirosina com atividade toacutexica para ceacutelulas
de procariotos e eucariotos Dentre as atividades descritas para esse ANP destacam-se a
atividade anti-mitoacutetica ou bloqueadora do ciclo celular atividade alelopaacutetica e
antioxidante Os resultados indicaram que em leucena a biossiacutentese e o acuacutemulo de
mimosina podem ser modulados por fatores causadores de estresses exibindo um padratildeo
de acumulaccedilatildeo similar ao das fitoalexinas Em maricaacute por outro lado a induccedilatildeo do
acuacutemulo dessa moleacutecula natildeo foi observada para os mesmos tratamentos testados para
leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas Aleacutem disso
o padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado
pela sua degradaccedilatildeo As respostas observadas nos testes que avaliaram a atividade de
mitigaccedilatildeo de espeacutecies reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode
agir como um agente antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de
estresse
1
Introduccedilatildeo
Na condiccedilatildeo de organismos seacutesseis ao longo de sua evoluccedilatildeo as plantas
desenvolveram estrateacutegias estruturais e quiacutemicas de defesa em resposta aos estresses bioacuteticos
e abioacuteticos impostos pelo ambiente Dentre essas satildeo reconhecidas moleacuteculas quimicamente
especializadas denominadas metaboacutelitos secundaacuterios produtos naturais (Kutchan et al 2015)
ou mais recentemente metaboacutelitos especializados
Entre as trecircs classes mais gerais de metaboacutelitos secundaacuterios (terpenos compostos
fenoacutelicos e compostos nitrogenados) aminoaacutecidos natildeo-proteicos (ANPs) satildeo incluiacutedos no
terceiro grupo e constituem aleacutem de componentes do arsenal de defesa quiacutemica uma
importante fonte de reserva de carbono e nitrogecircnio para diversos taxa especialmente aqueles
pertencentes agrave famiacutelia Fabaceae de Angiospermas (leguminosas sensu lato)
Aleacutem dos 20 aminoaacutecidos proteicos estima-se que existam entre 600 e 1000 ANPs
(Acamovic amp Brooker 2005 Rodgers et al 2015) Esse grupo de moleacuteculas quimicamente
heterogecircneo eacute assim definido por natildeo participar da formaccedilatildeo de estruturas proteicas
funcionais sendo frequentemente toacutexicos quando erroneamente incorporados nas cadeias
polipeptiacutedicas em formaccedilatildeo em funccedilatildeo da similaridade estrutural que apresentam com os
aminoaacutecidos proteicos (Taiz amp Zeiger 2010)
Conforme mencionado a ocorrecircncia de ANPs eacute comum em espeacutecies de leguminosas
e sua distribuiccedilatildeo pode ser restrita a alguns gecircneros de plantas circunscritos nessa famiacutelia
botacircnica (eg mimosina e canavanina) Por outro lado alguns ANPs como GABA por
exemplo podem apresentar distribuiccedilatildeo ubiacutequa no Reino Plantae assim como ocorrer em
outros tipos de organismos como animais por exemplo (Ramos-Ruiz et al 2018)
2
Apesar de representarem uma fonte nutricional importante sem tratamento preacutevio o
consumo de plantas que acumulam ANPs por animais eacute limitado Isso ocorre pois em longo
prazo a ingestatildeo prolongada de plantas (especialmente sementes) que acumulam ANPs pode
representar risco agrave sauacutede uma vez que estes comprometem o funcionamento de mecanismos
basais de manutenccedilatildeo da homeostase celular e podem tambeacutem em um quadro mais severo
desencadear doenccedilas neurotoacutexicas degenerativas como por exemplo o latirismo causado
por aacutecido β-N-oxalil-l-αβ-diaminopropiocircnico (β-ODAP) (Jiao et al 2011 Kusama-Eguchi
2019)
Sob o ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados
ANPs satildeo em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou
abioacutetica como o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de
elicitores quiacutemicos por exemplo No que concerne ao estudo dos efeitos da induccedilatildeo abioacutetica
sobre o acuacutemulo de ANPs em diferentes espeacutecies vegetais (Monocotiledocircneas e
Eudicotiledocircneas) as moleacuteculas mais amplamente investigadas ateacute o momento satildeo GABA
L-DOPA e mais recentemente mimosina (vide Tabela 1 do capiacutetulo primeiro) Em termos
de efeitos causados a partir da aplicaccedilatildeo exoacutegena de ANPs GABA tambeacutem figura como o
principal aminoaacutecido investigado seguido de L-DOPA e canavanina (vide Tabela 2 do
capiacutetulo primeiro)
No primeiro capiacutetulo da presente tese estatildeo descritas as caracteriacutesticas gerais dos
principais ANPs estudados seus possiacuteveis papeacuteis bioloacutegicos in planta e seus efeitos quando
aplicados exogenamente bem como os estresses abioacuteticos capazes de induzir seu(s)
acuacutemulo(s) nos diferentes tecidos vegetais Nos segundo e terceiro capiacutetulos
respectivamente satildeo elucidados os efeitos dos tratamentos de UV-C aacutecido saliciacutelico etileno
e jasmonato (claacutessicos elicitores do metabolismo secundaacuterio vegetal) sobre o acuacutemulo de
3
mimosina em Leucaena leucocephala var glabrata (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute)
Mimosina eacute um aminoaacutecido aromaacutetico natildeo-proteico anaacutelogo da L-tirosina com
atividade toacutexica para ceacutelulas de procariotos e eucariotos Embora em menor concentraccedilatildeo
mimosina foi primeiramente identificada em Mimosa pudica sendo posteriormente detectada
em outras espeacutecies do gecircnero como Mimosa pigra por exemplo (Soedarjo amp Borthakur
1998) Seu efeito toacutexico eacute atribuiacutedo agrave capacidade de quelar metais o que impede o
funcionamento adequado das metalo-proteiacutenas que dependem dos mesmos como co-fatores
(Negi et al 2014)
A concentraccedilatildeo basal de mimosina em espeacutecies de leucaena pode variar entre 1 e 12
do peso seco do oacutergatildeo (Soedarjo amp Borthakur 1998) Como eacute comum para outros ANPs
que ocorrem em espeacutecies de leguminosas em sementes de Leucaena spp eacute observada uma
maior concentraccedilatildeo de mimosina quando comparada aos demais oacutergatildeos da planta
(Rodrigues-Correcirca et al 2019) sendo esta a fonte de extraccedilatildeo comercial do padratildeo quiacutemico
de mimosina vendido por empresas de reagentes de pesquisa
Diversas atividades foram descritas para mimosina em outros organismos eou tipos
celulares Dentre essas destacam-se a atividade anti-mitoacutetica ou bloqueadora do ciclo
celular em ceacutelulas de eucariotos e procariotos Isto ocorre porque a mimosina impede a
formaccedilatildeo da forquilha de replicaccedilatildeo (e portanto a siacutentese de DNA) interrompendo assim o
avanccedilo do ciclo de divisatildeo celular na fase tardia G1 (Lalande 1990) Foram tambeacutem descritas
para mimosina atividade alelopaacutetica observada sobre o desenvolvimento de outras espeacutecies
de leguminosas e atividade antioxidante entre outras (Tabela 1)
A rota de biossiacutentese de mimosina eacute compartilhada em grande parte com a de cisteiacutena
um aminoaacutecido proteico sulfurado (Figura 1) A siacutentese da cisteiacutena se daacute a partir da conversatildeo
4
de serina e acetil-CoA em o-acetilserina pela enzima SAT (serina acetiltransferase) seguida
da conversatildeo de o-acetilserina e aacutecido sulfiacutedrico em cisteiacutena em uma reaccedilatildeo catalisada pela
OAS-TL (o-acetilserina tiol-liase) A siacutentese de mimosina por sua vez eacute compartilhada com
a da cisteiacutena ateacute esse ponto e acredita-se que pelo menos uma das isoformas de OAS-TL
catalise a conversatildeo de o-acetilserina e 3-hidroxi-4-piridona em mimosina
Tabela 1 Atividades descritas para mimosina de Leucaena leucocephala (Lam) de Wit
ATIVIDADE
ALVO AVALIADO
(organismo eou tecido tipo
celular)
REFEREcircNCIA
Bloqueio do complexo de ativaccedilatildeo
da preacute-replicaccedilatildeo do DNA
Ceacutelulas de mamiacuteferos
KUBOTA et al
(2014)
Alteraccedilatildeo no ciclo ovariano e
extensatildeo da duraccedilatildeo do corpo luacuteteo
bovino no periacuteodo poacutes-parto
Bovinos
(Bos taurus x
Bos indicus)
BOTTINI-
LUZARDO et al
(2015)
Supressatildeo do ciclo celular e reduccedilatildeo
da abundacircncia bacteriana em
mosquitos
Wolbachia pipientis
Aedes albopictus
FALLON
(2015)
Accedilatildeo inibitoacuteria da fibrose
pulmonar induzida
Ratos SD
LI et al
(2015)
Recuperaccedilatildeo da funccedilatildeo do
miocaacuterdio poacutes-isquemia
Miocaacuterdio de ratos (SD)
machos
CROWE et al
(2001)
Inseticida
Heteropsylla cubana
Crawford 1914 e Thrips tabaci
Lindemann 1889
AHMED et al
(2016)
Alelopaacutetica
Albizia procera Vigna
unguiculata Cicer arietinum
Cajanus cajan
AHMED et al
(2008)
Antioxidante
Sistemas modelo de oxidaccedilatildeo
lipiacutedica (β-caroteno - aacutecido
linolecircico e lecitina)
BENJAKUL et al
(2013)
Ateacute momento versotildees divergentes sobre a enzima responsaacutevel pela biossiacutentese de
mimosina (mimosina sintase) tecircm sido publicadas Em 1990 Ikegami e colaboradores
5
identificaram uma OAS-TL responsaacutevel pela formaccedilatildeo de cisteiacutena como sendo tambeacutem uma
mimosina sintase Mais tarde Yafuso et al (2014) realizaram a expressatildeo heteroacuteloga do gene
que codifica para OAS-TL em Escherichia coli e natildeo foi observada a formaccedilatildeo de mimosina
mesmo quando dadas as condiccedilotildees oacutetimas para tanto Mais recentemente Harun-Ur-Rashid
et al (2018) elucidaram a mimosina sintase como sendo uma isoforma da OAS-TL
corroborando o postulado por Ikegami e colaboradores em 1990
Figura 1 Rota de biossiacutentese da mimosina Fonte Ikegami et al (1990)
Espeacutecies estudadas
Leucaena leucocephala (Lam) de Wit (leucaena koa haole ou ldquoacaacutecia exoacuteticardquo na
liacutengua Hawairsquoiana) eacute uma espeacutecie de haacutebito arboacutereo ou arbustivo pertencente agrave famiacutelia
Fabaceae de Angiospermas e caracterizada pelo acuacutemulo de mimosina em todos os seus
oacutergatildeos Eacute nativa da Ameacuterica Central (especificamente da regiatildeo sudeste do Meacutexico) mas
irradiou-se atraveacutes de praticamente todas as zonas tropicais e subtropicais da Terra No
Brasil leucena eacute amplamente distribuiacuteda e classificada como naturalizada pelo REFLORA
(2019) ocorrendo em todo territoacuterio Nacional Satildeo reconhecidas no miacutenimo duas
6
subespeacutecies de leucena ocorrentes no Brasil L leucocephala var leucocephala e L
leucocephala var glabrata sendo a primeira a mais abundante
Leucaena apresenta atributos morfoloacutegicos caracteriacutesticos das leguminosas como o
fruto do tipo vagem deiscente no periacuteodo poacutes-maturaccedilatildeo folhas compostas e bipinadas As
flores satildeo seacutesseis actinomorfas e polistecircmones apresentam caacutelice sinseacutepala e corola
gamopeacutetala e satildeo dispostas em inflorescecircncias do tipo glomeacuterulo (Figura 2)
Figura 2 Oacutergatildeos vegetativos e reprodutivos de L leucocephala (Lam) de Wit Fonte Little Jr amp Skolmen
(1989)
Com base no conhecimento etnobotacircnico disponiacutevel acerca dessa espeacutecie em
diversas regiotildees tropicais e subtropicais leucena eacute utilizada para vaacuterios fins Extratos de
diferentes oacutergatildeos de leucena apresentam atividade anti-diabeacutetica (Kuppusamy et al 2014
Chowtivannakul et al 2016) antioxidante (Mohammed et al 2015 Chowtivannakul et al
2016 Zarin et al 2016) antimicrobiana (Zarin et al 2016) anti-helmiacutentica (Soares et al
2015 Jamous et al 2017) bactericida (Mohammed et al 2015) acaricida (Fernaacutendez-Salas
et al 2011) anti-tumoral (Chung et al 2017) e potencializadora da resposta imune em
peixes (Verma et al 2018) entre outras
7
Leucaena apresenta alta toleracircncia agrave seca sendo capaz de enfrentar estaccedilotildees sazonais
inteiras com deacuteficit hiacutedrico sem prejuiacutezo permanente de seus oacutergatildeos e de recuperar
vigorosamente sua biomassa vegetativa tatildeo logo o regime de precipitaccedilatildeo retome a
regularidade em frequecircncia Acredita-se que a toleracircncia agrave seca apresentada por essa espeacutecie
ocorra em funccedilatildeo do acuacutemulo de mimosina nos diferentes tecidos da planta a qual
funcionaria como um agente osmoregulador responsaacutevel pela preservaccedilatildeo da integridade das
membranas a das macromoleacuteculas intracelulares em periacuteodos de escassez de aacutegua no
ambiente
Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) eacute uma leguminosa
nativa natildeo endecircmica do Brasil amplamente distribuiacuteda nos domiacutenios fitogeograacuteficos da
Caatinga do Cerrado e da Mata Atlacircntica (Simon amp Proenccedila 2000 REFLORA 2019) Como
espeacutecie pioneira (Pilatti et al 2019) exerce importante papel ecoloacutegico na recuperaccedilatildeo de
aacutereas degradadas (Bitencourt et al 2007 Silva et al 2011) no estabelecimento de processos
de sucessatildeo vegetacional
Maricaacute eacute uma espeacutecie semi-deciacutedua a deciacutedua a qual atinge ateacute 15 m em altura (e
diacircmetro agrave altura do peito de ateacute 40 cm) na idade adulta com haacutebito arboacutereo ou arbustivo
(REFLORA 2019) e espinhos caracteriacutesticos desde os estaacutegios iniciais de desenvolvimento
(Carvalho 2004) Apresenta folhas compostas alternas e bipinadas (Figura 2) amplas
inflorescecircncias brancas com flores reunidas em glomeacuterulos esfeacutericos dispostos em grandes
paniacuteculas As flores satildeo diplostecircmones actinomorfas hipoacuteginas e unicarpelares (Silva et al
2011)
Assim como descrito para leucena maricaacute eacute considerado uma espeacutecie multifuncional
sendo comumente empregada para produccedilatildeo de mel como combustiacutevel (Olkoski amp
8
Wittmann 2011) em edificaccedilotildees na carpintaria e como lsquocerca-vivarsquo (Marchiori 1993
Lorenzi 1998) entre outras aplicaccedilotildees
Figura 2 Folhas e fruto de Mimosa bimucronata (DC) Kuntze Fonte Souza-Lima et al (2017)
Em contraste com a amplitude de habitats explorados por leucena (especialmente os
aacuteridos) no Sul do Brasil maricaacute ocorre preferencialmente em ambientes uacutemidos a alagadiccedilos
em aacutereas proacuteximas agraves margens de rios (Patreze amp Cordeiro 2004) embora possa tambeacutem
ocorrer em formaccedilotildees quase exclusivas dessa espeacutecie nas encostas de morros (Jacobi amp
Ferreira 1991)
Em relaccedilatildeo agraves atividades elucidadas para os extratos de maricaacute foram relatados os
efeitos alelopaacutetico (Jacobi amp Ferreira 1991 Ferreira et al 1992) diureacutetico natriureacutetico e
caliureacutetico (Schlickmann et al 2017)
9
Hipoacutetese
Mimosina apresenta perfil dinacircmico de acuacutemulo em Leucaena leucocephala e
Mimosa bimucronata frente a estresses associado a alteraccedilotildees significativas na expressatildeo de
genes relacionados ao metabolismo deste ANP o qual contribui para mitigar o desequiliacutebrio
oxidativo inerente a vaacuterios tipos de estresse
Objetivo geral
O objetivo da presente tese foi investigar o papel bioloacutegico da mimosina endoacutegena
em leucena e maricaacute a partir da avaliaccedilatildeo do efeito de tratamentos relacionados a estresses
ou sinalizadores de estresse
Objetivos especiacuteficos
- Analisar a concentraccedilatildeo constitutiva de mimosina nos diferentes oacutergatildeos de L leucocephala
(Lam) de Wit (leucena) e M bimucronata (DC) Kuntze (maricaacute)
- Verificar se apesar do seu alto teor constitutivo em plantas de leucena o acuacutemulo de
mimosina pode ser induzido com tratamentos que mimetizam diferentes estresses a partir da
avaliaccedilatildeo do efeito de moleacuteculas sinalizadoras (aacutecido saliciacutelico jasmonato etileno) e da
exposiccedilatildeo agrave radiaccedilatildeo UV-C na modulaccedilatildeo do acuacutemulo de mimosina em leucena bem como
em maricaacute
- Determinar se a expressatildeo de genes relacionados ao metabolismo de mimosina estaacute
associada agrave induccedilatildeo por estresses fisioloacutegicos
- Avaliar o potencial antioxidante da mimosina em experimentos realizados in situ
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry
journal homepage wwwelseviercomlocateplaphy
Research article
Mimosine accumulation in Leucaena leucocephala in response to stresssignaling molecules and acute UV exposure
Kelly Cristine da Silva Rodrigues-Correcircaab Michael DH Hondab Dulal BorthakurbArthur Germano Fett-Netoalowast
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970Porto Alegre Rio Grande do Sul BrazilbDepartment of Molecular Biosciences and Bioengineering University of Hawaii at Manoa Honolulu HI 96822 USA
A R T I C L E I N F O
KeywordsLeucaena leucocephalaMimosineMimosine amidohydrolaseJasmonic acidEthyleneSalicylic acidUV-C radiation
A B S T R A C T
Mimosine is a non-protein amino acid of Fabaceae such as Leucaena spp and Mimosa spp Several relevantbiological activities have been described for this molecule including cell cycle blocker anticancer antifungalantimicrobial herbivore deterrent and allelopathic activities raising increased economic interest in its pro-duction In addition information on mimosine dynamics in planta remains limited In order to address this topicand propose strategies to increase mimosine production aiming at economic uses the effects of several stress-related elicitors of secondary metabolism and UV acute exposure were examined on mimosine accumulation ingrowth room-cultivated seedlings of Leucaena leucocephala spp glabrata Mimosine concentration was not sig-nificantly affected by 10 ppm salicylic acid (SA) treatment but increased in roots and shoots of seedlings treatedwith 84 ppm jasmonic acid (JA) and 10 ppm Ethephon (an ethylene-releasing compound) and in shoots treatedwith UV-C radiation Quantification of mimosine amidohydrolase (mimosinase) gene expression showed thatethephon yielded variable effect over time whereas JA and UV-C did not show significant impact Consideringthe strong induction of mimosine accumulation by acute UV-C exposure additional in situ ROS localization aswell as in vitro antioxidant assays were performed suggesting that akin to several secondary metabolitesmimosine may be involved in general oxidative stress modulation acting as a hydrogen peroxide and superoxideanion quencher
1 Introduction
Different plant groups synthesize a large diversity of secondary orspecialized metabolites These molecules are generally produced inresponse to biotic and abiotic environmental stresses Indeed inductionof secondary metabolism usually involves stress-generating factorswhich have also been explored in biotechnological processes aiming atthe production of target metabolites of economic interest (Matsuuraet al 2018) Metabolic control of nitrogen-containing secondarycompounds (eg alkaloids and non-protein amino acids) has beenshown to be complex and influenced by phytohormones environmentalstresses (seasonality herbivory pathogen attack drought) UV radia-tion (Holloacutesy 2002) methyl jasmonate (MeJA) salicylic acid (SA)yeast extract (Cho et al 2008) abscisic acid (ABA) heavy metals os-motic stress (Nascimento et al 2013) and mechanical wounding (Portoet al 2014)
Due to their particular trait of associating with N-fixing micro-organisms Fabaceae species (leguminous sensu lato) are often proteinrich hence the relevance of several of these species as forage Fabaceaespecies are also known for accumulating nitrogen containing secondarymetabolites which play important roles as ecochemical molecules andat least for the case of non-protein amino acids potential cell reservoirsof nitrogen (Huang et al 2011)
High contents of mimosine a toxic aromatic non-protein aminoacid are found in species of two leguminous genera Leucaena spp andMimosa spp Leucaena leucocephala (Lam) de Wit (leucaena koa haole)is a fast-growing leguminous tree native from Central America (south-eastern Mexico) widely distributed in tropical and subtropical zonesThis species is also characterized by its high tolerance to droughtamong other environmental stresses (Honda et al 2018) Leucaena canbe divided into two subspecies (i) L leucocephala subsp leucocephala(common leucaena a bushy shrub) and (ii) L leucocephala subsp
httpsdoiorg101016jplaphy201811018Received 1 August 2018 Received in revised form 9 November 2018 Accepted 14 November 2018
lowast Corresponding authorE-mail addresses krodriguescbiotufrgsbr (KCdS Rodrigues-Correcirca) mhonda2hawaiiedu (MDH Honda) dulalhawaiiedu (D Borthakur)
fettnetocbiotufrgsbr (AG Fett-Neto)
Plant Physiology and Biochemistry 135 (2019) 432ndash440
Available online 19 November 20180981-9428 copy 2018 Elsevier Masson SAS All rights reserved
T
glabrata (giant leucaena a tree) The latter has been used as a fastgrowing tree for production of wood and paper pulp The foliage ofboth common and giant leucaena is used as a fodder because of its highprotein content and palatability to farm animals The foliage containsup to 18 protein 142 crude fiber and 64 ether extractcrude fat(Soedarjo and Borthakur 1996)
Production of nitrogen-containing secondary metabolites such asmimosine requires large amounts of carbon and nitrogen resourcesNegi et al (2014) estimated that up to 21 of the carbon-nitrogenresources may be used for production of mimosine in leucaenaBrewbaker et al (1972) determined the mimosine content of 96 Lleucocephala cultivars and 8 other Leucaena species collected from 38different countries by growing them in an observational nursery inHawaii and found that basal mimosine content varied from 189 to477 of the dry weight
Mimosine is biosynthesized from OAS (o-acetylserine) and 3H4P (3-hydroxy-4-pyridone or its tautoisomer 3-hydroxy-4-pyridine) A pre-vious analysis suggested that mimosine synthase is an OAS-TL (o-acetylserine-thiol-lyase) of the cysteine biosynthesis pathway (Ikegamiet al 1990) Later however recombinant enzyme tests did not supportan OAS-TL identity of mimosine synthase (Yafuso et al 2014) Recentfindings on mimosine biosynthesis revealed that a cytosolic cysteine-OAS-TL isoform can also catalyze the formation of mimosine underspecific conditions (Harun-Ur-Rashid et al 2018)
Mimosine toxicity is related to its ability of reducing the availabilityof divalent metal ions such as Fe(II) Zn(II) Cu(II) Co(II) and Mn(II)by chelating co-factors and preventing their association with metal-dependent enzymes Furthermore this non-protein amino acid is cap-able of forming a stable complex with pyridoxal-5prime-phosphate (PLP)leading to the inactivation of PLP-dependent enzymes (eg Asp-Glutransaminase and cystathionine synthetase) (Negi et al 2014)
Mimosine features several useful biological activities such as alle-lopathic antimicrobial insecticide cell cycle inhibitor agent antic-ancer phytoremediator (Nguyen and Tawata 2016) as well as anti-oxidant (Benjakul et al 2013) Despite the relatively well establishedbiological activities of purified mimosine on other organisms or celltypes little is known about its biological role in leguminous speciesHowever it has been suggested that at least in part its activity ismainly related to defense mechanisms against some biotic and abioticstresses and as nitrogen source during fast growth (Vestena et al2001)
Suda (1960) and Smith and Fowden (1966) identified enzymes in-volved in mimosine degradation in seedling extracts of L leucocephalaand Mimosa pudica A mimosine-degrading enzyme named mimosinase(mimosine amidohydrolase EC 35161 CAS registry number 104118-49-2) (IUBMB 2018) a carbon-nitrogen lyase which degrades mimo-sine into 3H4P was later purified by Tangendjaja et al (1986) Itsbiochemical characterization was described and the cDNA was isolatedby Negi et al (2014)
Although mimosinase has been described and isolated only fewstudies on the role played by biotic and abiotic factors on the dynamicmodulation of mimosine metabolism in leguminous species have beenconducted (Vestena et al 2001 Xu et al 2018) In aseptic cultures ofleucaena mechanical injury of shoots promoted local mimosine accu-mulation (Vestena et al 2001) In the same study cultivation in pre-sence of auxin or SA in culture medium also had a positive effect on
mimosine accumulation More recently the effect of drought treatmenton gene expression of leucaena was also evaluated by Honda et al(2018) However several potential factors regulating mimosine meta-bolism need to be further examined
To date there is a lack of information on the biological role ofmimosine in planta as well as on details of its metabolic dynamicsMoreover its overt potential for pharmaceutical applications and de-velopment of new drugs as well as the possible use as tool to addressheavy metal soil contamination or plant mineral nutrition improve-ment justify additional research The objective of this study was toinvestigate the effect of stress signaling molecules and acute UV ex-posure on modulation of mimosine accumulation and metabolism in Lleucocephala spp glabrata in order to better understand its biologicalrole and to identify strategies for yield improvement aiming at ex-ploring its useful bioactivities
2 Methods
21 Plant material
For the experiments carried out to evaluate the effects of elicitors onmimosine accumulation seeds of leucaena were kindly provided by DrJames Brewbaker and harvested at CTAHRs (College of TropicalAgriculture and Human Resources of the University of Hawaii atManoa) Waimanalo Research Station at Oahu Hawaii This plantmaterial was originated from the accession K636 of Leucaena leucoce-phala ssp glabrata (Brewbaker 2008)
22 Induced mimosine content in 5-week-old giant leucaena
221 Seed germinationIn order to overcome seed coat dormancy seeds were submitted to a
chemical scarification with sulfuric acid 95ndash98 for 20min and re-peatedly rinsed in distilled water to remove any residual trace of thisreagent Then seeds were distributed in 254 cmtimes508 cm plastictrays containing 11 vv of vermiculite and commercial soil watereduntil reaching substrate field capacity Three weeks after seed imbibi-tion seedlings displaying similar size and shape (eg number of com-pound leaves and leaflets) were transplanted to individual pots(250mL) in number of three plants per container
During the experimental period (except in the UV-C radiationtreatment) all tested seedlings were kept in a growth chamber andsubmitted to controlled conditions of temperature (circa 25 degC) and ir-radiance (approximately 100 μmol photons mminus2sdot s minus1) with a photo-period of 16 h light and 8 h dark
222 Treatments2221 JA Ethephon and SA Five-week-old giant leucaena seedlingswere treated with different solutions as described in Table 1 Idealconcentrations were defined in preliminary experiments under the sameconditions indicated above At the beginning of the experiments 30plants were sprayed with 84 ppm JA 10 ppm SA 10 or 100 ppmEthephon or Milli-Qreg water (control) until the point of imminent runoffPlant pots were kept closed inside transparent plastic bags for 24 h toavoid solution volatilization Fifteen plants arranged in 5 sets of 3 (5biological replicates) were harvested 48 h and 96 h after being treated
Table 1Treatments used to modulate mimosine biosynthesis in giant leucaena
ELICITOR CONCENTRATION UV FLUENCE EXPOSURE TIME RATIONALE FOR USE
Salicylic acid (SA) 10 ppm 24 h Pathogen signaling molecule (Shah 2003)Jasmonic acid (JA) 84 ppm 24 h Chemical elicitor of plant secondary metabolism (Dar et al 2015)Ethephon 10 ppm 24 h Ethylene releasing-compound (Kim et al 2016) elicitor of plant secondary metabolism (Wang
et al 2016)UV-C radiation 3 Jcmminus2 10min or 15min Elicitor of plant secondary metabolism (Kara 2013 Neelamegam and Sutha 2015)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
433
After collection shoots were separated from roots immediately frozenin liquid nitrogen and stored at ndash 80 degC prior to HPLC analyses
2222 UV-C Thirty seedlings of giant leucaena were exposed to UV-Cradiation (3 Jcmminus2) for 10 or 15min and kept in a growth chamberunder controlled conditions as described above until the end of theexperiments Fifteen plants arranged in groups of 3 were harvested at96 h and 120 h after UV-C exposure and processed as previouslydescribed
223 Mimosine extractionMimosine extraction was based on a modified version of the pro-
tocol published by Lalitha and Kulothungan (2006) as follows a knownweight of fresh tissue (shoots or roots) of giant leucaena was first addedto Milli-Qreg boiling water in a proportion of 110 (g of plant per mL ofsolvent) in test tubes Tubes were covered with foil to avoid solutionevaporation and placed on a hot stirrer at 100 degC for 10min A pro-portional volume of 01M HCl was added to the cooled suspensions andhomogenized using mortar and pestle The plant extracts were filteredthrough cotton and centrifuged twice for 7min in a bench top re-frigerated centrifuge at 4 degC and 13200 rpm Before being analyzed theextracts were diluted 13 with ondashphosphoric acid (OPA)
224 Mimosine detectionHPLC analyses were carried out as described by Negi and Borthakur
(2016) Pure mimosine (L-mimosine from koa haole seeds Sigma-Al-drich CAS number 500-44-7) was used as standard Separation andquantification of mimosine was done with a C18 column (PhenomenexC18 5 μm 46times250mm) under an isocratic solvent system of 002MOPA with a linear flow rate of 1mLsdotminminus1 Mimosine detection wasdone at 280 nm by photodiode array detection (200ndash400 nm) showingretention time of 277 plusmn 0042min Quantification was done using themethod of external standard curve Further confirmation of mimosineidentity was performed by co-chromatography with standard and peakpurity check Chromatograms were analyzed using the Waters Em-power 3 software
23 Quantitative real-time PCR analysis of mimosinase gene expression
Fifteen 8-week-old giant leucaena plants arranged in 4 sets of 3 (4biological replicates) were treated with either water (control) or10 ppm Ethephon 84 ppm JA acid or 15min of UV-C radiation ex-posure following the methods described above Following treatmentleucaena plants were harvested at 48 and 96 h or 72 and 144 h (UV-Ctreated plants only) after treatments Total RNA of samples was ex-tracted and purified from roots and shoots of giant leucaena by meansof a modified method using Qiagen RNeasy Plant Kit (Valencia CAUSA) and Fruit-mate (Takara Japan) according to the protocol de-scribed by Ishihara et al (2016a) The assessment of RNA quality andquantity was carried out at 230 260 and 280 nm by using a NanoDropSpectrophotometer ND-1000 (NanoDrop Technologies DE USA) Inorder to avoid genomic DNA contamination RNA samples were treatedwith TURBO DNAfree Kit (Invitrogen Carlsbad CA) Two microgramsof DNase-treated RNA were used to synthesize the first-strand cDNAusing M-MLV Reverse Transcriptase (Promega WI USA)
Quantitative real-time (qPCR) analysis was carried out to examinepossible differential expression of the mimosinase gene (GenBank ac-cession number AB2985971) in seedlings treated with 84 ppm JA10mM Ethephon or 15min of UV-C exposure Shoots and roots wereharvested 24 h before the time of mimosine concentration peak for eachtreatment previously observed as assessed by HPLC assays The 10 μLqPCR reaction consisted of 5 μL of PowerUpTM SYBRreg Green MasterMix (Applied Biosystems Foster City CA) 1 μL MgCl2 (50mM) 03 μLforward primer (10 μM) 03 μL reverse primer (10 μM) and 1 μL cDNAfirst-strand In the experimental validation through qPCR reactionconditions and melting curve analysis of the amplicon were performed
following the protocol published by Ishihara et al (2016b) for the sameleucaena variety qPCR analysis was conducted using StepOnetrade Real-Time PCR System (Applied Biosystems) Measurements were performedusing 4 biological and 3 technical replicates Relative expression wascalculated with the 2-ΔΔct method using OAS-TL as reference gene sinceits expression showed a consistently stable profile comparable to that ofUBQ-5 and ELF1α expressions Mimosinase primer sequences used forthese analyses were (FWD) 5prime- GAA AGG CAG GAA TCA CAG TGA AGAG ndash 3rsquo (REV) 5prime GGA GAC TCT AGC CAC ACC AAC TTA ndash 3rsquo
24 Antioxidant assays
241 Mimosine effect on hydrogen peroxide (H2O2) accumulationAs a follow up to the induction of mimosine accumulation profiles
under stress signals and conditions tests were conducted to verify mi-mosine antioxidant capacity In situ histological localization of hy-drogen peroxide (H2O2) accumulation was evaluated on foliar disks ofPhaseolus vulgaris L according to the protocol described by Shi et al(2010) Briefly the plant foliar tissue was exposed to 1 mgmiddotmLminus1 dia-minobenzidine (DAB) solution in 10 mM KH2PO4 (control) in presenceor absence of 10mM mimosine (equivalent to the average mimosineconcentration induced by UV-C radiation in giant leucaena) or 10mMascorbic acid (positive antioxidant control) Oxidative response wasidentified by the formation of a brown polymer on the injured leafareas indicating the presence of H2O2 and registered in a Leica M165FC stereomicroscope (Leica Microsystems)
242 Mimosine quenching of superoxide radicalsGeneration of superoxide radical and subsequent analysis was per-
formed by a modified protocol based on Zhishen et al (1999) Nitroblue tetrazolium (NBT) reduction was used to measure superoxide an-ions quenching activity Shortly a 50mM KH2PO4 pH 78 solutioncontaining 6 μM riboflavin 100mM methionine 1 mM NBT in pre-sence or absence of 5mM mimosine was exposed to white light(22 Jsdotcmminus2) for 25min on a white light transilluminator Five micro-molar rutin was used as positive control (Matsuura et al 2016) Theabsorbance was read at 560 nm before and after light exposure in aSpectraMaxreg M2 Microplate Reader (Molecular Devices LLC)
25 Statistical analyses
For HPLC and superoxide anions data simple analyses of variance(ANOVA) followed by Tukey or Welch ANOVA followed by Dunnetts Ctest were used as appropriate for data distribution characteristics InqPCR analysis results were analyzed by t-test In all cases at least fourbiological triplicates were used and experiments were repeated twiceindependently All data were analyzed using the statistical packageSPSS 200 for Windows (SPSS Inc USA) In all cases a ple 005 wasused
3 Results and discussion
31 Increased mimosine concentrations in giant leucaena treated withchemical elicitors
Leucaena produces high amounts of mimosine that accumulate in allparts of the plants including leaves stem flowers pods seeds rootsand root nodules (Soedarjo and Borthakur 1998) The highest con-centrations of mimosine can be found in the growing shoot tips andseeds (Wong and Devendra 1983) It is not known why leucaena pro-duces such high amounts of mimosine Negi et al (2014) estimated thatleucaena plants would be able to grow 21 larger if the nutrient re-sources spent on mimosine production were diverted for biomass in-crease In a previous analysis performed to quantify the basal con-centration of mimosine present in adult plants of common leucaena thehighest constitutive amount of mimosine per gram of fresh weight in
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
434
the analyzed organs was found in post-anthesis flowers (89448 μg)followed by green pods (82687 μg) leaves (67358 μg) and greenflower buds (51247 μg) which showed significantly less mimosineconcentration compared to the other reproductive structures(Supplementary Fig 1) Since mature seeds have very low moisturecontent (Wencomo et al 2017) its mimosine concentration was esti-mated as 338253 μgsdotgminus1 of dry weight Additionally it was also ob-served that the basal mimosine distribution in shoots of field-grownadult plants of leucaena is dependent on the variety type(Supplementary Table 1)
Phytohormones such as salicylic acid and jasmonic acid are knownto be produced by plants in response to various abiotic and bioticstresses These phytohormones trigger adaptive responses to stress byregulating major plant metabolic processes such as photosynthesisnitrogen metabolism defense systems and plant-water relationsthereby providing protection (for review see Khan et al 2015)
Secondary or specialized metabolite production and accumulationare also known to be controlled by biotic and abiotic stresses (Matsuuraet al 2018) In this study exposure of 5-week-old giant leucaenaseedlings to JA or Ethephon treatments significantly enhanced mimo-sine accumulation in shoots and roots in at least one of the two timepoints tested (48 and 96 h) albeit in a different way (Fig 1) Thehighest concentrations of mimosine in shoots were found in seedlingstreated with JA 84 ppm (43441 μgsdotgminus1) and Ethephon 100 ppm(38412 μgsdotgminus1) two days after application of the respective phyto-hormones Nevertheless after four days shoots yielded the highestconcentration of mimosine (approximately 460 μgsdotgminus1) upon treatmentwith 10 or 100 ppm Ethephon (Fig 1A) In roots after two and four
days JA 84 ppm and Ethephon 10 ppm resulted in highest mimosineaccumulation 18488 μgsdotgminus1 and 15801 μgsdotgminus1 respectively (Fig 1B)These observations show that mimosine accumulation response tospecific elicitors may vary over time after exposure
Although all treatments were applied exclusively on shoots of giantleucaena seedlings roots of some of them were also able to respond tothe different elicitors Overall shoots displayed higher basal and in-duced mimosine concentration compared to roots (Fig 1) which agreeswith previous observations in 1 to 3-week-old aseptic seedlings ofcommon leucaena (Vestena et al 2001) However as previouslymentioned significant post-induction increase of mimosine concentra-tion in roots and shoots simultaneously was only observed for JA andEthephon 10 ppm on day 02 and 04 respectively (Fig 1)
It is well established that perceived regulatory signals or elicitorsgenerate a transduction network mediated by secondary messengersresulting in changes in gene expression profiles that afford adaptiveresponses to environmental stimuli These modulation events are oftenmediated by transcription factors (TFs) which directly bind to specificgene promoters or act by forming complexes with repressor proteinslabeling them to degradation subsequently releasing other TFs toproceed with the gene expression program This is the case of the actionmechanism of JA and its active form jasmonoyl isoleucine for example(Kazan 2015 Wasternack and Strnad 2016)
JA ethylene and SA are known as important stress regulatory sig-nals in plants JA however is thought to be the most effective signal forinduction of plant secondary metabolism (Wasternack and Strnad2016) thereby contributing to mitigation of damage caused by severalstresses (Dar et al 2015) JA is mainly derived from linolenic acid
Fig 1 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings treated with differentelicitors CTRL=Milli-Q water SA = Salicylic AcidJA= Jasmonic Acid ETH=Ethephon Bars sharing a letterof same case do not differ by Tukey test (P le 005) Capitalletters (A B) compare treatments on day two and lowercaseletters (a b) compare treatments on day four Indicatessignificant statistical difference between day two and dayfour in the same treatment by t-test (Ple 005) The errorbars represent standard error of five replicates (each meanwas calculated with 15 individual seedlings organized in 5groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
435
(Wasternack and Strnad 2016) playing important roles in differentprocesses of plant growth and development such as plant defensemechanisms against herbivory pathogen attack fungal elicitation andsome abiotic factors such as osmotic temperature and salt stresses (Daret al 2015)
JA and its methyl ester MeJA have several different effects on le-guminous species MeJA exogenous application has increased iso-flavonoid content in cell suspension cultures of Pueraria candollei varcandollei and P candollei var mirifica (Korsangruang et al 2010) aswell as the production of the triterpenoid glycyrrhizin in Glycyrrhizaglabra roots Enhanced production of the triterpenoid however waspartly at the expense of root growth (Shabani et al 2009) MeJA ap-plication on shoots was observed to suppress root nodulation and lat-eral root formation in Lotus japonicus (Nakagawa and Kawaguchi2006) In grapevine a non-leguminous species proteinogenic aminoacids did not show an expressive increase under MeJA treatment(Gutieacuterrez-Gamboa et al 2017)
The effects of the application of four different jasmonate forms (JAMeJA jasmonoyl-L-isoleucine (JA-Ile) and 6-ethyl indanoyl glycineconjugate (2-[(6-ethyl-1-oxo-indane-4-carbonyl)-amino]-acetic acidmethyl ester - CGM) on leucaena metabolite profile has recently beenreported by Xu et al (2018) JA-Ile form was most effective althoughno major alteration was observed on monitored metabolite abundancesAlanine threonine and 34-dihydroxypyridine (34 DHP a metabolitederived from mimosine degradation) (Nguyen and Tawata 2016)among others were the major metabolites elicited by JA-Ile In contrastto the results described here mimosine concentration did not changesignificantly These divergent results on mimosine accumulation maybe due to a number of factors including mode of application jasmonateform used (JA-Ile x JA) and L leucocephala subspecies (common x giantleucaena)
Ethylene is also a phytohormone involved in plant response me-chanisms to different types of challenges such as mechanical damageand insect attack among others The integration mechanism betweenJA and ethylene signaling pathways is not completely understoodhowever it has been shown that they may work cooperatively in abioticstress tolerance (Kazan 2015) MeJA can induce ethylene production(Zhao et al 2004) and when applied simultaneously these moleculesseem to work in a synergic way by enhancing the magnitude of theplant response to external stimuli (Liu et al 2016)
Treatment with SA was able to significantly increase mimosine ac-cumulation in 12-week-old plants of common leucaena (SupplementaryFig 2) However no significant effect of SA treatment on mimosineconcentration was seen in 5-week-old seedlings of giant leucaena(Fig 1) suggesting some degree of genotype andor age dependency inelicitation by this phytohormone On the other hand several treat-ments including 90 ppm MeJA 10 and 100 ppm 2-chloroethylpho-sphonic acid (CEPA an ethylene-releasing compound) significantlyincreased mimosine accumulation (Supplementary Fig 2) in agree-ment with the data obtained for giant leucaena The lack of systemiceffects of externally applied SA on mimosine accumulation was alsoobserved when the phytohormone was supplied in the culture mediumof aseptically-grown seedlings in which case only roots had highercontent of mimosine (Vestena et al 2001) This could be due totransport limitations or to low methyl salicylate production from ap-plied SA since the former is recognized as the main systemic signalingform (Vlot et al 2009)
32 Increased mimosine concentrations in giant leucaena exposed to UV-Cradiation
UV-C treatment promoted increased concentration of the aminoacid in shoots but not in roots of giant leucaena (Fig 2) Increasedaccumulation of mimosine in shoots was also observed in 12-week-oldseedlings of common leucaena exposed to UV-C radiation for 10 and15min (Supplementary Fig 3) Similar to the SA treatment in giant
leucaena UV-C radiation did not induce mimosine biosynthesis in rootsregardless of time after exposure The absence of mimosine induction inroots by SA and UV indicates that these effectors do not cause a sys-temic response Moreover roots are shielded from irradiance by thepresence of substrate
UV radiation effects on different aspects of plant metabolism anddevelopment have been described However compared to UV-B (en-vironmentally relevant type of UV radiation) assays there are less re-ports related to the UV-C effects on secondary metabolites biosynthesisand accumulation (Cetin 2014) especially in leguminous (Fabaceae)plants They generally concern primary metabolism aspects such asgrowth and development For instance seedlings of Phaseolus vulgaris L(Fabaceae) exposed to low intensity UV-C radiation have displayeddecreased chlorophyll content and reduced height after 14 days of ex-posure (Kara 2013) Negative effects on growth parameters and ni-trogen metabolism were also observed in Vigna radiata L (Fabaceae)after UV-B radiation treatment in addition to adverse effects on JA SAand antioxidant compounds accumulation (Choudhary and Agrawal2014a) The same authors reported increased accumulation of flavo-noids SA and JA besides negative effects on growth biomass yieldnitrogen fixation and accumulation in 2 cultivars of Pisum sativum L(Fabaceae) under elevated UV-B treatment (Choudhary and Agrawal2014b) Despite the negative UV influence on growth reported for thepreviously mentioned leguminous UV-C radiation on groundnut plants(Arachis hypogaea L Fabaceae) increased seedling vigor and biomassand had no adverse effect on germination or other development para-meters (Neelamegam and Sutha 2015)
Besides its impact on growth and primary metabolism UV exposurecan cause important changes in secondary metabolism depending onintensity and time of exposure (Matsuura et al 2013) UV-B and UV-Cpre-treatments of Artemisia annua (Asteraceae) seedlings yielded in-creased biosynthesis of artemisinin a drug which displays anti-malarialproperties and activity against some others infectious diseases (egschistosomiasis leishmaniasis and hepatitis B) and several kinds oftumors (Rai et al 2011) The accumulation of nicotine in Nicotianarustica plants (Solanaceae) was also increased by UV-C treatment(Tiburcio et al 1985) Similar inducing effects on production of severalsecondary metabolites were observed in callus cultures of Vitis viniferaL Oumlkuumlzgoumlzuuml (grapevine Vitaceae) treated with a UV-C source for 5 or10min (Cetin 2014)
Regarding amino acid biosynthesis in response to UV radiationMartiacutenez-Luumlscher et al (2014) have found that in spite of not causingchanges in total amino acid content UV-B radiation exposure can affecttheir profile in grape berries Proteinogenic amino acids have beenknown to be important targets of the deleterious effects of UV radiation(Holloacutesy 2002) On the other hand in the present study acute UV-Ctreatment was found to increase mimosine accumulation in shoots byover twofold (Fig 2) which may suggest a possible participation of thismolecule as part of the antioxidant defense system in L leucocephalaThis possibility is further supported by the induction of the amino acidaccumulation by JA and Ethephon involved in abiotic and biotic stressresponses which are generally associated with oxidative imbalance andare signaling components in high UV stress (Matsuura et al 2013)
33 Mimosinase gene expression
In order to determine if increases in mimosine content upon ex-posure to JA CEPA or UV-C radiation were related to changes intranscription of mimosine metabolism-related genes RT-qPCR analysiswas carried out The complete pathway for mimosine biosynthesis hasnot yet been determined although the final step has been character-ized Based on transcription analysis (Ishihara et al 2016a) leucaenaappears to encode for multiple cysteine synthases one or more of whichmay be able to catalyze mimosine synthesis In addition a leucaenagene encoding a mimosinase (an enzyme responsible for mimosinedegradation) has been identified and characterized (Negi et al 2014)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
436
In addition to mimosinase gene expression several gene isoformsbelonging to the cysteine pathway [cysteine synthase (CYS SYN) serineacetyltransferase (SAT) and β-cyanoalanine synthase (CAS) Table 2 -supplementary material] were also tested in this study (data notshown) However expressions of these genes did not vary in giantleucaena throughout the experiments suggesting that the increasedcontent of mimosine observed in the treated plants might not be relatedto the expression of these genes but presumably to increased enzymeactivity andor release from conjugates such as mimoside a mimosineβ-D-glucoside (Murakoshi et al 1972)
Considering the time variation of mimosine accumulation observedin this work mimosinase gene expression in shoots and roots wasevaluated 24 h before the increase of mimosine concentration in giantleucaena seedlings (ie 24 h and 72 h after the chemical elicitorstreatments and 48 h and 120 h after UV-C exposure)
Ethylene signaling has been shown to up-regulate expression ofseveral genes related to secondary metabolism pathways as is the caseof phenolic compounds (Liu et al 2016) and terpenoid indole alkaloids(Wang et al 2016) Among all elicitors tested in the present workEthephon was the only one able to significantly change mimosinasegene expression Leucaena plants treated with Ethephon showed sig-nificant increases in mimosine concentration at both day 2 and 4 fol-lowing treatment which coincided with low-level expression of mi-mosinase Up-regulation of mimosinase gene expression was detected24 h before the increase of mimosine concentration in shoots treatedwith 10 ppm of Ethephon (Fig 3A) but not after JA or UV-C treatments(Fig 3C-D and 3E-F respectively) Nevertheless 72 h after treatment
application (24 h before the highest mimosine content measured inshoots) down regulation of mimosinase gene was seen in both shootsand roots treated with 10 ppm of Ethephon (Fig 3B) These data in-dicate that mimosine content in leucaena plants is at least partlyregulated by mimosinase expression in Ethephon exposed plants Onthe other hand the fact that mimosinase mRNA was not significantlyaffected by JA and UV-C treatments despite their stimulating effects onmimosine biosynthesis in giant leucaena may indicate that other levelsof regulation are at play or that the chosen harvesting time window wasunable to detect relevant changes
34 In situ and in vitro antioxidant assays
Considering the stimulation of mimosine accumulation byEthephon JA and UV all of which are often associated or known tocause oxidative imbalance the antioxidant capacity of mimosine wasevaluated Mimosine has been shown to have antioxidant activities oncultured cancer cells (Parmar et al 2015) In the present study it washypothesized that mimosine could confer radical scavenging propertieswhich would contribute to plant protection from possible damagecaused by reactive oxygen species generated during stress(Supplementary Fig 4)
Foliar disks of P vulgaris L were treated with 10mM mimosine for15min Treated disks showed less hydrogen peroxide accumulationinduced by wounding in contrast to untreated ones being comparableto those treated with ascorbic acid (a known hydrogen peroxide neu-tralizer) (Fig 4A) These observations support a possible antioxidant
Fig 2 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings exposed to UV-C lightCTRL= visible light (100 μmol photons mminus2 s minus1) UV-C 10primeand UV-C 15rsquo=UV-C exposure time (10 and 15min re-spectively) Bars sharing a letter of same case do not differ byTukey test (P le 005) Capital letters (A B) compare treat-ments on day three and lowercase letters (a b) comparetreatments on day six Indicates significant statistical dif-ference between day three and day six in the same treatmentby t-test (Ple 005) The error bars represent standard errorof five replicates (each mean was calculated with 15 in-dividual seedlings organized in 5 groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
437
role of mimosine as an in situ hydrogen peroxide scavengerMimosine was also able to quench superoxide anions generated by
light exposure Mimosine exhibited equivalent antioxidant effect com-pared to rutin (Fig 4B) a well-established effective superoxide anionquencher (Matsuura et al 2016) The radical scavenging activity ofmimosine may be due to the 3-OH group of the pyridine ring of mi-mosine (Fig 5) The pKa of the 3-OH of mimosine has been estimated tobe 88 (M Honda unpublished results) At physiological pH this OHgroup is expected to remain in a protonated state and therefore mayscavenge a radical by donating a proton and an electron In this processmimosine itself is converted to a stable radical form which is perhapsless toxic and less reactive than the reactive oxygen species generatedduring oxidative stress It is likely that the less toxic radical mimosineproduced may react with another radical or molecule and becomeconverted to a non-reactive indole molecule
In vivo antioxidant activity of mimosine has been previously eval-uated by means of its exogenous application on selenium-deficientseedlings of Vigna radiata In spite of its allelopathic properties (Ahmedet al 2008) the results showed mitigation of mitochondrial oxidativestress by treatment with 01mM mimosine (Lalitha and Kulothungan2007) DPPH radical scavenging activity was also reported for aqueous
seed extracts of leucaena rich in mimosine and phenolic compounds inin vitro assays (Benjakul et al 2014) Mimosine antioxidant activityshown in the present work is in good agreement with data reported forother non-protein amino acids such as L-DOPA (Dhanani et al 2015)and GABA (Malekzadeh et al 2014) for instance
4 Conclusion
Taken together results show that mimosine biosynthesis and ac-cumulation can be modulated by stress-related factors despite its re-latively high constitutive content in leucaena plants The pattern ofgene expression in stressed plants suggests mimosine steady-state con-trol may be regulated by its degradation in possible connection withdynamic changes in carbon and nitrogen metabolism of stressed plantsMimosine quenching activity against hydrogen peroxide and super-oxide anions in the in situ staining and in vitro assays respectivelyshowed that this non-protein amino acid can act as non-enzymaticantioxidant agent Increase in mimosine content in response to elicitorsmimicking environmental challenges in addition to its antiherbivoreand antimicrobial properties may be related to its activity as protectivemolecule against oxidative damage in line with other classes of plant
Fig 3 Relative expression of the mimosinase gene in shoots (A E and F) and shoots and roots (B C and D) of giant leucaena 24 h (A and C) 48 h (E) 72 h (B and D)and 120 h (F) after treatment with stress signaling molecules or UV-C exposure ETH = Ethephon JA = Jasmonic Acid Indicates significant statistical differencebetween control and treatment by t-test (Ple 005) The error bars represent standard error of four replicates
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
438
secondary metabolites
Funding
This work was funded by the National Council for Scientific andTechnological Development (CNPq-Brazil) grant 3060792013-5Coordenaccedilatildeo de Aperfeiccediloamento de Pessoal de Niacutevel Superior - Brazil(CAPES) - Finance Code 001 and the USDA NIFA Hatch projectHA05029-H managed by CTAHR
CRediT authorship contribution statement
Kelly Cristine da Silva Rodrigues-Correcirca InvestigationValidation Writing ndash original draft Michael DH HondaInvestigation Validation Dulal Borthakur Supervision Writing ndashreview amp editing Funding acquisition Arthur Germano Fett-NetoSupervision Funding acquisition Writing ndash review amp editing
Acknowledgements
The authors would like to thank Dr Jorge Ernesto Mariath fromLaVeg-UFRGS for kindly lending the Leica M165 FC stereomicroscopefor in situ analysis
Appendix A Supplementary data
Supplementary data to this article can be found online at httpsdoiorg101016jplaphy201811018
References
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leucocephala leaf litter on some forest and agricultural crops grown in nursery J ForRes 19 298 httpsdoi 101007s11676-008-0053-0
Benjakul S Kittiphattanabawon P Shahidi F Maqsood S 2013 Antioxidant activityand inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipidoxidation in model systems Food Sci Technol Int 19 (4) 365ndash376 httpsdoiorg1011771082013212455186
Benjakul S Kittiphattanabawon P Sumpavapol P Maqsood S 2014 Antioxidantactivities of lead (Leucaena leucocephala) seed as affected by extraction solvent priordechlorophyllisation and drying methods extracts against lipid oxidation in modelsystems Food Sci Technol 51 (11) 3026ndash3037 httpsdoiorg101007s13197-012-0846-1
Brewbaker JL Pluckett D Gonzalez V 1972 Varietal variation and yield trials ofLeucaena leucocephala (koa haole) in Hawaii Hawaii Agric Exp Stn Bull 166 26
Brewbaker JL 2008 Registration of KX2 ndash Hawaii interspecific-hybrid leucaena JPlant Registrations 1 (3) 190ndash193 httpsdoiorg103198jpr2007050298crc
Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitisvinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-6287-47-37
Cho H-Y Son SY Rhee HS Yoon S-YH Lee-Parsons CWT Park JM 2008Synergistic effects of sequential treatment with methyl jasmonate salicylic acid andyeast extract on benzophenanthridine alkaloid accumulation and protein expressionin Eschscholtzia californica suspension cultures J Biotechnol 135 117ndash122 httpsdoiorg101016jjbiotec200802020
Choudhary KK Agrawal SB 2014a Cultivar specificity of tropical mung bean (Vignaradiata L) to elevated ultraviolet-B changes in antioxidative defense system ni-trogen metabolism and accumulation of jasmonic and salicylic acids Environ ExpBot 99 122ndash132 httpsdoiorg101016jenvexpbot201311006
Choudhary KK Agrawal SB 2014b Ultraviolet-B induced changes in morphologicalphysiological and biochemical parameters of two cultivars of pea (Pisum sativum L)Ecotoxicol Environ Saf 100 178ndash187 httpsdoiorg101016jecoenv201310032
Dar TA Uddin M Khan MMA Hakeem KR Jaleel H 2015 Jasmonates counterplant stress a Review Environ Exp Bot 115 49ndash57 httpsdoiorg101016jenvexpbot201502010
Dhanani T Singh R Shah S Kumari P Kumar S 2015 Comparison of green ex-traction methods with conventional extraction method for extract yield L-DOPAconcentration and antioxidant activity of Mucuna pruriens seed Green Chem LettRev 8 (2) 43ndash48 httpsdoiorg1010801751825320151075070
Gutieacuterrez-Gamboa G Portu J Santamariacutea P Loacutepez R Garde-Cerdaacuten T 2017Effects on grape amino acid concentration through foliar application of three dif-ferent elicitors Food Res Int 99 688ndash692 httpsdoiorg101016jfoodres201706022
Fig 4 A In situ antioxidant assay Foliar disksof Phaseolus vulgaris L treated with (a) No an-tioxidant added (negative control) (b) 10 mMMimosine (c) 10mM ascorbic acid (positivecontrol) The oxidative damage can be seen bythe formation of a brown polymer in leaf veinsand injured areas B In vitro superoxidescavenging assay carried out with mimosineDifferent letters indicate significant differenceby Tukey test (Ple 005) The error bars re-present standard error of four replicates (Forinterpretation of the references to colour in thisfigure legend the reader is referred to the Webversion of this article)
Fig 5 Predicted mimosine radical formed followingquenching of hydroxyl radical Mimosine is first converted toa stable mimosine radical which may be then converted to anontoxic indole form
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
439
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad HossainMd Anai T Oku H 2018 Cytosolic cysteine synthase switch cysteine and mi-mosine production in Leucaena leucocephala Appl Biochem Biotechnol 186 (3)613ndash632 httpsdoiorg101007s12010-018-2745-z
Holloacutesy F 2002 Effects of ultraviolet radiation on plant cells Micron 33 (2) 179ndash197Honda MDH Ishihara KL Pham DT Borthakur D 2018 Identification of drought-
induced genes in giant leucaena (Leucaena leucocephala subsp glabrata) Trees 32571ndash585 httpsdoiorg101007s00468-018-1657-4
Huang T Jander G de Vos M 2011 Non-protein amino acids in plant defense againstinsect herbivores representative cases and opportunities for further functional ana-lysis Phytochemistry 72 1531ndash1537 httpsdoiorg101016jphytochem201103019
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of thethyrotoxic amino acid mimosine by cysteine synthase Phytochemistry 29 (11)3461ndash3465 httpsdoiorg1010160031-9422(90)85258-H
Ishihara K Lee EKW Borthakur D 2016a An improved method for RNA extractionfrom woody legume species Acacia koa A Gray and Leucaena leucocephala (Lam) deWit Int J For Wood Sci 3 (1) 031ndash035
Ishihara KL Honda MDH Pham DT Borthakur D 2016b Transcriptome analysisof Leucaena leucocephala and identification of highly expressed genes in roots andshoots Transcriptomics 4 135 httpsdoiorg1041722329-89361000135
IUBMB 2018 Enzyme Nomenclature EC 35161 httpwwwsbcsqmulacukiubmbenzymeEC35161html Accessed date 8 February 2018
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant(Phaseolus vulgaris) Biosci Res 10 (1) 29ndash32
Kazan K 2015 Diverse roles of jasmonates and ethylene in abiotic stress toleranceTrends Plant Sci 20 (4) 219ndash229 httpsdoiorg101016jtplants201502001
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect ofEthephon as an ethylene-releasing compound on the metabolic profile of Chlorellavulgaris J Agric Food Chem 64 (23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541
Khan MIR Fatma M Per TS Anjum NA Khan NA 2015 Salicylic acid-inducedabiotic stress tolerance and underlying mechanisms in plants Front Plant Sci 6 462httpsdoiorg103389fpls201500462
Korsangruang S Soonthornchareonnon N Chintapakorn Y Saralamp PPrathanturarug S 2010 Effects of abiotic and biotic elicitors on growth and iso-flavonoid accumulation in Pueraria candollei var candollei and P candollei var mir-ifica cell suspension cultures Plant Cell Tissue Organ Cult 103 (3) 333ndash342 httpsdoiorg101007s11240-010-9785-6
Lalitha K Kulothungan SR 2006 Selective determination of mimosine and its dihy-droxypyridinyl derivative in plant systems Amino Acids 31 (3) 279ndash287 httpsdoiorg101007s00726-005-0226-5
Lalitha K Kulothungan SR 2007 Mimosine mitigates oxidative stress in seleniumdeficient seedlings of Vigna radiata - Part I restoration of mitochondrial functionBiol Trace Elem Res 118 (1) 84ndash96 httpsdoiorg101007s12011-007-0013-0
Liu J Li Y Wang Y Zhang Z-H Zu Y-G Efferth T Tang Z-H 2016 Thecombined effects of ethylene and MeJA on metabolic profiling of phenolic com-pounds in Catharanthus roseus revealed by metabolomics analysis Front Physiol 71ndash11 httpsdoiorg103389fphys201600217 Article 217
Malekzadeh P Khara J Heydari R 2014 Alleviating effects of exogenous Gamma-aminobutiric acid on tomato seedling under chilling stress Physiol Mol Biol Plants20 (1) 133ndash137 httpsdoiorg101007s12298-013-0203-5
Martiacutenez-Luumlscher J Torres N Hilbert G Richard T Saacutenchez-Diacuteaz M Delrot SAguirreolea J Pascual I Gomegraves E 2014 Ultraviolet-B radiation modifies thequantitative and qualitative profile of flavonoids and amino acids in grape berriesPhytochemistry 102 106ndash114 httpsdoiorg101016jphytochem201403014
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation ofbioactive secondary metabolites by ultraviolet radiation mechanisms strategies andapplications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology forMedicinal Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash1902012
Matsuura HN Fragoso V Paranhos JT Rau MR Fett-Neto AG 2016 Thebioactive monoterpene indole alkaloid N szlig-D-glucopyranosylvincosamide is regu-lated by irradiance quality and development in Psychotria leiocarpa Ind Crop Prod86 210ndash218 httpsdoiorg101016jindcrop201603050
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo RBhambra AS Strnad M Bonfill M Fett-Neto AG 2018 Specialized plant me-tabolism characteristics and impact on target molecule biotechnological productionMol Biotechnol 60 (2) 169ndash183 httpsdoiorg101007s12033-017-0056-1
Murakoshi S Ohmiya S Haginiwa J 1972 Enzymic synthesis of mimoside a meta-bolite of mimosine in Mimosa pudica and Leucaena leucocephala Chem Pharm Bull20 (4) 855ndash857
Nakagawa T Kawaguchi M 2006 Shoot-applied MeJA suppresses root nodulation inLotus japonicus Plant Cell Physiol 47 (1) 176ndash180 httpsdoiorg101093pcppci222
Nascimento NC Menguer PK Henriques AT Fett-Neto AG 2013 Accumulation ofbrachycerine an antioxidant glucosidic indole alkaloid is induced by abscisic acidheavy metal and osmotic stress in leaves of Psychotria brachyceras Plant PhysiolBiochem 73 33ndash40 httpsdoiorg101016jplaphy201308007
Neelamegam R Sutha T 2015 UV-C irradiation effect on seed germination seedling
growth and productivity of groundnut (Arachis hypogaea L) Int J Curr MicrobiolApp Sci 4 (8) 430ndash443
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase fromLeucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol164 (2) 922ndash934 httpsdoiorg101104pp113230870
Negi VS Borthakur D 2016 Heterologous expression and characterization of mimo-sinase from Leucaena leucocephala In Fett-Neto Arthur Germano (Ed)Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods inMolecular Biology vol 1405 copySpringer Science+Business Media New York httpsdoiorg101007978-1-4939-3393-8_7 2016
Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636
Parmar F Kushawaha N Highland H George L-B 2015 In vitro antioxidant andanticancer activity of Mimosa pudica Linn extract and L-mimosine on lymphomaDaudi cells Int J Pharm Sci 12 100ndash104
Porto DD Matsuura HN Vargas LRB Henriques AT Fett-Neto AG 2014 Shootaccumulation kinetics and effects on herbivores of the wound-induced antioxidantindole alkaloid brachycerine of Psychotria brachyceras Nat Prod Commun 9 (5)629ndash632
Rai R Meena RP Smita SS Shukla A Rai SK Pandey-Rai S 2011 UV-B and UV-C pre-treatments induce physiological changes and artemisinin biosynthesis inArtemisia annua L ndash an antimalarial plant J Photochem Photobiol B Biol 105 (3)216ndash225 httpsdoiorg101016jjphotobiol201109004
Shabani L Ehsanpour AA Asghari G Emami J 2009 Glycyrrhizin production by invitro cultured Glycyrrhiza glabra elicited by methyl jasmonate and salicylic acid RussJ Plant Physiol 56 (5) 621ndash626 httpsdoiorg101134S1021443709050069
Shah J 2003 The salicylic acid loop in plant defense Curr Opin Plant Biol 6 (4)365ndash371
Shi J Fu XZ Peng T Huang XS Fan QJ Liu JH 2010 Spermine pretreatmentconfers dehydration tolerance of citrus in vitro plants via modulation of antioxidativecapacity and stomatal response Tree Physiol 30 (7) 914ndash922 httpsdoiorg101093treephystpq030
Smith IK Fowden L 1966 A study of mimosine toxicity in plants J Exp Bot 17750ndash761 httpsdoiorg101093jxb174750
Soedarjo M Borthakur D 1996 Simple procedures to remove mimosine from youngleaves pods and seeds of Leucaena leucocephala used as food Int J Food SciTechnol 31 (1) 97ndash103
Soedarjo M Borthakur D 1998 Mimosine a toxin produced by the tree-legumeLeucaena provides a nodulation competition advantage to mimosine-degradingRhizobium strains Soil Biol Biochem 30 1605ndash1613
Suda S 1960 On the physiological properties of mimosine Bot Mag Tokyo 73 (862)142ndash147 httpsdoiorg1015281jplantres188773142
Tangendjaja B Lowry JB Wills RBH 1986 Isolation of a mimosine degrading en-zyme from leucaena leaf J Sci Food Agric 37 523ndash526 httpsdoiorg101002jsfa2740370603
Tiburcio F Pintildeol MT Serrano M 1985 Effect of UV-C on growth soluble protein andalkaloids in Nicotiana rustica plants Environ Exp Bot 25 (3) 203ndash210 httpsdoiorg1010160098-8472(85)90004-8
Vestena S Fett-Neto AG Duarte RC Ferreira A 2001 Regulation of mimosineaccumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 httpsdoiorg101016S0168-9452(01)00448-4
Vlot AC Dempsey DMA Klessig DF 2009 Salicylic acid a multifaceted hormone tocombat disease Annu Rev Phytopathol 47 177ndash206 httpsdoiorg101146annurevphyto050908135202 2009
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene-induced vinblastine accumulation is related to activated expression of downstreamTIA pathway genes in Catharanthus roseus BioMed Res Int 2016 Article ID 3708187httpsdoiorg10115520163708187
Wasternack C Strnad M 2016 Jasmonate signaling in plant stress responses and de-velopment ndash active and inactive compounds N Biotech 33 (5B) 604ndash613 httpsdoiorg101016jnbt201511001
Wencomo HB Ortiz R Caacuteceres J 2017 Afr J Agric Res 12 (4) 279ndash285 httpsdoiorg105897AJAR201510604 26
Wong CC Devendra C 1983 Research on leucaena forage production in Malaysia InLeucaena Research in the Asian Pacific Region pp 55ndash60 Ottawa Ontario Canada
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX TsimKWK 2018 Jasmonate-elicited stress induces metabolic change in the leaves ofLeucaena leucocephala Molecules 23 (2) httpsdoiorg103390molecules23020188 E188
Yafuso JT Negi VS Bingham J-P Borthakur D 2014 An O-acetylserine (thiol)lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthaseAppl Biochem Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z
Zhao J Zheng S-H Fujita K Sakai K 2004 Jasmonate and ethylene signalling andtheir interaction are integral parts of the elicitor signalling pathway leading to b-thujaplicin biosynthesis in Cupressus lusitanica cell cultures J Exp Bot 55 (399)1003ndash1012 httpsdoiorg101093jxberh127
Zhishen J Mengcheng T Jianming W 1999 The determination of flavonoid contentsin mulberry and their scavenging effects on superoxide radicals Food Chem 64 (4)555ndash559 httpsdoiorg101016S0308-8146(98)00102-2
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
440
61
Supplementary Fig 1 Basal mimosine concentration in adult trees of common leucaena (L leucocephala
var leucocephala) Samples were collected from 10 field grown trees at Manoa Valley Honolulu Hawairsquoi
on June 25th 2017 Bars sharing a letter do not differ by Tukey test (P le 005) The error bars represent the
standard error
Supplementary Fig 2 Bar diagram showing mimosine concentration in shoots of 12-week-old common
leucaena seedlings treated with different elicitors CTRL = Milli-Q water SA = Salicylic Acid MeJA =
Methyl Jasmonate CEPA = 2-Chloroethylphosphonic acid (an ethylene releasing compound) Bars sharing a
letter of same case do not differ by Tukey test (P le 005) Capital letters (A B) compare treatments on day
two and lower-case letters (a b) compare treatments on day four Indicates significant statistical difference
ABB
A A
0
200
400
600
800
1000
1200
LEAVES GREEN FLOWERBUDS
POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
con
cen
trat
ion
(micro
gg
-1o
f FW
)
B AB AB AB B A
b
a
ab b
ab
0
2
4
6
8
10
12
14
16
18
20
CTRL SA 10 ppm SA 100 ppm CEPA 10 ppm CEPA 100 ppm MeJA 90 ppm
Mim
osi
ne
co
nce
ntr
atio
n (
gg
-1o
f FW
)
DAY 02 DAY 04
62
between day two and day four in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
Supplementary Fig 3 Bar diagram showing the effects of UV-C radiation exposure for 5 10 and 15 min on
mimosine accumulation in shoots of 12-week-old seedlings of common leucaena Bars sharing a letter of
same case do not differ by Tukey test (P le 005) Capital letters (A B C) compare treatments on day three
and lower-case letters (a b) compare treatments on day six Indicates significant statistical difference
between day three and day six in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
C BC AB A
bb
a
a
0
10
20
30
40
50
60
CTRL UV-C 5 UV-C 10 UV-C 15
Mim
osi
ne
co
nce
ntr
atio
n (
gg-1
of
FW)
DAY 03 DAY 06
63
Supplementary Fig 4 Model depicting induction of mimosine synthesis in leucaena following application of
stress elicitors such as CEPA and jasmonic acid or exposure to UV-C radiation The additional mimosine
synthesized may serve to alleviate oxidative stress induced by UV-C radiation
64
Supplementary Table 1 Mimosine contents in leaves of common and giant leucaena
Leucaena
type
Mimosine content
( FW)
Mimosine
content ( DW)
Dry matter
content ( FW)
Water content
( FW)
Common (1) 050 plusmn 009 245 plusmn 051 2011 plusmn 054 7989 plusmn 054
Common (2) 043 plusmn 006 214 plusmn 037 1998 plusmn 050 8002 plusmn 050
K636 (1) 070 plusmn 014 356 plusmn 077 1908 plusmn 052 8092 plusmn 052
K636 (2) 042 005 205 plusmn 033 2008plusmn 093 7992plusmn 093
KX2 (1) 122 plusmn 011 608 plusmn 082 1939 plusmn 123 8061 plusmn 123
KX2 (2) 134 plusmn 010 623 plusmn 056 2029 plusmn 114 7971 plusmn 114
KX3 (1) 044 plusmn 006 221 plusmn 030 1945 plusmn 073 8055 plusmn 073
KX3 (2) 054 plusmn 005 273 plusmn 023 1930 plusmn 038 8070 plusmn 038
KX4 (1) 086 plusmn 011 471 plusmn 065 1753 plusmn 084 8247 plusmn 084
KX4 (2) 089 plusmn 011 476 plusmn 065 180 plusmn 072 820 plusmn 072
KX5 (1) 099 plusmn 012 489 plusmn 048 1907 plusmn060 8093 plusmn 060
KX5 (2) 115 plusmn 015 548 plusmn080 1992 plusmn 053 8008 plusmn 053
Common leucaena variety koa haole grows widely on the island of Orsquoahu K636 is widely
grown variety of giant leucaena KX2 KX3 KX4 and KX5 are giant leucaena varieties
developed through interspecies hybridization (Brewbaker 2016) (1) and (2) indicate plants
from two separate locations within the University of Hawaii Waimanalo Research Center The
values are shown as mean plusmn standard error obtained from at least three biological replicates
65
Supplementary Table 2 GenBank accession numbers of the tested cysteine pathway genes isoforms
Gene name GenBank accession
OAS-TL (o-acetylserine-thiol-lyase) GDRZ01032940
GDRZ01061620
GDRZ01153117
GDSA01187555
GDSA01196891
GDSA01214467
Cys syn (cysteine synthase) GDRZ01015860
GDRZ01050898
GDRZ01086813
GDRZ01193515
GDRZ01202579
GDSA01180863
GDSA01215622
SAT (serine acetyltransferase) GDRZ01187456
GDRZ01189631
CAS (β-cyanoalanine synthase) GDRZ01054066
GDRZ01175418
GDSA01118400
66
SHORT COMMUNICATION 1
Mimosine occurrence and accumulation in Mimosa bimucronata var bimucronata (DC) 2
Kuntze 3
Kelly Cristine da Silva Rodrigues-Correcirca1 Lana Dorneles Pedroso2 Fernanda de Costa1 4
Arthur Germano Fett-Neto1 5
1Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal 6
University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970 7
Porto Alegre Rio Grande do Sul Brazil 2Department of Biological Sciences Unipampa ndash 8
Campus Satildeo Gabriel 9
Corresponding author 10
E-mail addresses krodriguescbiotufrgsbr (KCdaS Rodrigues-Correcirca) 11
lanalima2012gmailcom (LD Pedroso) fernandadecostayahoocombr (F de Costa) 12
fettnetocbiotufrgsbr (AG Fett-Neto) 13
14
15
16
17
18
19
20
21
22
67
ABSTRACT 23
Mimosine is a non-protein aromatic amino acid present in plants of Leucaena spp 24
and Mimosa spp Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) is a native 25
tree from Brazil which occurs as a pioneer species on plant succession processes In the 26
current study the presence of mimosine in M bimucronata was verified by HPLC analyses 27
Moreover mimosine accumulation upon exposure to UV-C and chemical elicitors of 28
specialized metabolism (salicylic acid - SA methyl jasmonate - MeJA sodium nitroprusside 29
- SNP and ethephon - ETH) most of which also known as promoters of the amino acid 30
production in leucaena plants was evaluated The results showed a lower concentration of 31
constitutive mimosine present in both maricaacute seedlings and mature trees when compared to 32
leucaena plants In spite of a trend towards increased mimosine accumulation observed in 33
MeJA and ETH treatments no statistical differences were found with the various stressors 34
used to induce its biosynthesis in maricaacute seedlings Data suggest that mimosine in M 35
bimucronata is probably a phytoanticipin-like metabolite or its accumulation is driven by 36
other types of stresses 37
38
39
Keywords Mimosine Mimosa bimucronata stress 40
41
42
43
44
45
46
68
Introduction 47
Mimosa bimucronata commonly known as maricaacute is a native tree from Brazil 48
(REFLORA 2019) ecologically important in plant succession and in processes of degraded 49
land recovery (Bitencourt et al 2007 Silva et al 2011) occurring as a pioneer species 50
(Pilatti et al 2019) Maricaacute is a deciduous or semi-deciduous plant which reaches up to 15 51
m in height and 40 cm of diameter at breast height (DBH) displays shrub or tree habit and 52
bears typical sharp thorns (Carvalho 2004) This species belongs to Fabaceae one of the 53
most economically important families of flowering plants due to its high diversity and 54
occurrence in different types of habitats (Gomes et al 2018) As well as several others 55
Mimosa spp maricaacute is usually referred to as a multipurpose tree (Olkoski and Wittmann 56
2011) employed for alternative medicinal uses (Champanerkar et al 2010 Silva et al 57
2011) honey production constructions and remodeling of landscape architecture (living 58
fences) for instance (Marchiori 1993 Lorenzi 1998) 59
In southern Brazil maricaacute is widely distributed and typically found either in wetland 60
areas close to river banks (Patreze and Cordeiro 2004) or composing large and almost pure 61
landscape formations on hillsides (Jacobi and Ferreira 1991) In dense populations this 62
species like several Mimosa spp (Simon and Proenccedila 2000) is considered an important and 63
highly invasive weed by preventing cattle to reach pasturesand water bodies as a result of its 64
thorny branches (Lorenzi 2008 Kestring et al 2009) Its dominant and nearly exclusive 65
pattern of distribution in those areas has led Jacobi and Ferreira (1991) to test its allelopathic 66
potential on cultivated species Indeed extracts of leaves and ripe fruits (but not the green 67
ones) of maricaacute showed phytotoxic effects on germination and initial radical growth of most 68
of the target species tested 69
69
Several investigations have been performed on maricaacute floristics (Silva et al 2011) 70
distribution (Simon and Proenccedila 2000) wood anatomy (Marchiori 1993) cytogenetic 71
parameters (Olkoski and Wittmann 2011) and allelopathic potential (Jacobi and Ferreira 72
1991 Ferreira et al 1992) However excluding two recent publications on maricaacute 73
constitutive chemical composition (Schlickmann et al 2017 Pilatti et al 2019) which 74
identified phenolic compounds (methyl gallate and water-soluble tannins) as its major 75
compounds little is known regarding this subject In other Mimosa species (eg M pudica 76
and M pigra) mimosine has been identified (Soedarjo and Borthakur 1998) as one of the 77
major specialized metabolites present in the different organs of the plant (Champanerkar et 78
al 2010) The presence of this molecule was also reported for M bimucronata in a thin layer 79
chromatography-based preliminary study performed by Ferreira et al (1992) showing co-80
chromatography of a leaf extract component with authentic mimosine The authors attributed 81
the allelopathic effect of maricaacute to the accumulation of this metabolite in its leaves 82
Mimosine is an aromatic non-protein amino acid initially found in plants of Mimosa 83
pudica and later in Leucaena leucocephala (Lam) de Wit (Soedarjo and Borthakur 1998) a 84
leguminous tree which biosynthesizes large amounts of this nitrogen-containing compound 85
(Rodrigues-Correcirca et al 2019) It is believed that the accumulation of high contents of 86
mimosine in L leucocephala tissues confers among other traits defense against herbivores 87
and pathogens (Vestena et al 2001) tolerance to drought (Negi et al 2014) as well as 88
general oxidative stress protection (Rodrigues-Correcirca et al 2019) Interestingly drought is 89
the opposite environmental and physiological condition to that observed in the wet habitats 90
occupied by native populations of M bimucronata in Brazil (Patreze and Cordeiro 2004 91
Kestring et al 2009) and Mimosa pudica Linn in India (Champanerkar et al 2010) 92
70
Nonetheless flooding is also associated with oxidative stress particularly as water levels 93
change (Fukao et al 2019) 94
In Leucaena leucocephala var leucocephala (common leucaena) and Leucaena 95
leucocephala var glabrata (giant leucaena) mimosine accumulation has been shown to be 96
both constitutive and inducible by stress-related phytohormones such as jasmonic acid (JA) 97
Ethephon (ETH an ethylene- releasing compound) salicylic acid (SA - only common 98
leucaena) (Vestena et al 2001) as well as by UV-C radiation (Xu et al 2018 Rodrigues-99
Correcirca et al 2019) On the other hand there is a lack of information regarding mimosine 100
content and elicitation effects in Mimosa spp plants 101
The aim of this study was to examine the presence of mimosine in Mimosa 102
bimucronata and examine the effects of stresses and stress-signaling molecules on its 103
accumulation in leaves 104
Material and Methods 105
Plant material 106
For all experiments the plant material was collected at Morro Santana campus do 107
Vale of UFRGS (Federal University of Rio Grande do Sul) Porto Alegre RS Brazil 108
(3004rsquoS 5108rsquoW) Authorization for access to genetic material was obtained from 109
SISGEN-Brazil (license number A845493) Constitutive mimosine content in adult plants of 110
M bimucronata var bimucronata (DC) Kuntze was determined in plant material (leaves 111
green flower buds post-anthesis flowers and green pods) harvested in January 2017 112
(summer) A voucher herbarium specimen (ICN 187953) was deposited in the ICN ndash UFRGS 113
herbarium (Herbaacuterio do Instituto de Biociecircncias of UFRGS) 114
71
For mimosine elicitation experiments legumes (fruits) of maricaacute were collected in 115
the end of June 2017 (winter) Seeds were then removed from the dry fruits and kept in the 116
dark until sowing and seedling development for use in the assays 117
Seed germination 118
To break the coat-imposed seed dormancy after surface sterilization dry seeds of 119
maricaacute were acid scarified by immersion in H2SO4 (95 ndash 98 ) for 2 min (see Correcirca et al 120
2008) and repeatedly washed in distilled water to remove any residue of the acid Then seeds 121
were distributed in 50 mL individual plastic tubes (dibble-tubes) (30 cm diameter x 120 cm 122
depth) filled up with 11 (vv) of commercial top soil and vermiculite Tubes were watered 123
every 2 days to avoid substrate dryness and were kept in a growth room under controlled 124
conditions of light (circa 75 μmol mminus2s minus1 photosynthetically active radiation photoperiod 125
of 16 h light and 8 h dark) and temperature (24plusmn2C) 126
127
Treatments 128
In order to verify inducibility of mimosine accumulation in M bimucronata fifty 12-129
week-old maricaacute seedlings (per treatment) exhibiting similar features were selected and 130
sprayed (saturated) with solutions of different chemical stressors (plant specialized 131
metabolism elicitors) as follows (for further details see Rodrigues-Correcirca et al 2019) 10 132
and 50 mM SA (pathogen-signaling molecule Shah 2003) 007 and 035 mM 2-133
chloroethylphosphonic acid (ETH ethylene releasing-compound Kim et al 2016 Wang et 134
al 2016) 100 and 200 mM MeJA (Dar et al 2015) 10 and 50 mM SNP (a nitric oxide 135
donor Perotti et al 2015) Alternatively maricaacute seedlings were also supplemented with UV-136
C radiation (13 minutes 105 kJ cm2) (elicitor of plant specialized metabolism Kara 2013) 137
72
After 2 and 4 days of exposure to the chemical treatments and 3 and 6 days of UV-138
C supplementation maricaacute shoots were harvested immediately frozen in liquid nitrogen and 139
stored at ndash 80 C until mimosine extraction and HPLC analyses 140
Mimosine extraction and detection 141
Mimosine extraction was conducted according to the modified protocol described by 142
Rodrigues-Correcirca et al (2019) for L leucocephala HPLC (Thermo Scientific Surveyor) 143
analyses (mimosine detection and quantification) were performed following previously 144
published procedures (Negi et al 2014) A C18 column (ACE C18 5 μm 46times250 mm) and 145
isocratic solvent system of 002M o-phosphoric acid with a linear flow rate of 1 mL min minus1 146
were used to separate and quantify the amino acid Mimosine detection was performed at 280 147
nm by photodiode array detection (200ndash400 nm) and retention time (229plusmn0024 min) 148
Mimosine quantification was done by means of the method of external standard curve 149
Additional confirmation of mimosine identity was performed by co-chromatography with 150
standard (Acros Organics authentic mimosine 99 used as reference) and peak purity check 151
The analyses of the chromatograms were done with the ChromQuest software 152
153
154
Results and Discussion 155
Constitutive accumulation of mimosine in M bimucronata 156
Mimosine was detected in all analyzed samples positively meeting all identification 157
criteria In agreement with what has been found for other Mimosa spp (Soedarjo and 158
Borthakur 1998) compared to L leucocephala adult plants (Rodrigues-Correcirca 2019) 159
mimosine content was lower in M bimucronata Of the adult plant tissues analyzed the 160
73
highest content of mimosine in maricaacute (per gram of fresh weight - FW) was found in post-161
anthesis flowers (36644 microg versus 89448 microg in common leucaena followed by leaves 162
(28838 microg x 67358 microg) green flower buds (28094 microg x 51247 microg) and green pods (19002 163
microg x 82687 microg) (Fig 1)The same pattern is observed for seedlings when both species are 164
compared In this study untreated 12-week-old maricaacute seedlings (control at day 2) showed a 165
shoot content of mimosine of 23029plusmn007 microg g-1 of (FW) Five-week-old untreated giant 166
leucaena seedlings cultivated in similar conditions exhibited between 83640 and 178736 167
microg g-1 of FW (Rodrigues-Correcirca et al 2019) In the same way mimosine concentration 168
percentage in dry matter of Mimosa pigra was found to be rather low (002 in nodules and 169
roots and 007 in leaves) (Soedarjo and Borthakur 1998) 170
In this investigation the lowest constitutive mimosine content was found in green 171
pods (Fig 1) This result may partly explain the absence of phytotoxic effect observed for 172
green pods on germination and growth of crop target plants tested by Jacobi and Ferreira 173
(1991) compared to the other maricaacute parts analyzed 174
Elicitation of mimosine biosynthesis in M bimucronata 175
Chemical stressors 176
Secondary metabolites (or natural products) are structural- and chemically 177
specialized compounds derived from primary metabolism These molecules are mainly 178
biosynthesized as part of a complex defense mechanism in response to biotic and abiotic 179
stresses such as pathogens herbivores water status metal toxicity and UV radiation for 180
example (Matsuura et al 2018) Ethephon SA SNP MeJA have been extensively used as 181
chemical elicitors of specialized metabolism (Wang et al 2016 Vestena et al 2001 Perotti 182
74
et al 2015 Zhang and Memelink 2009 Xu et al 2018) These phytohormonal signals can 183
simulate environmental challenges and modulate plant homeostasis often leading to 184
alterations in gene expression (Shinozaki et al 2015) Except SNP all treatments tested in 185
the present study showed positive effect on mimosine accumulation in common or giant 186
leucaena (Vestena et al 2001 Rodrigues-Correcirca 2019 Rodrigues-Correcirca unpublished 187
data) However in spite of the trend of increasing the mimosine content observed in seedlings 188
treated with 007 mM Ethephon (at day 2) and 100 mM MeJA (at day 4) no statistical 189
difference was confirmed for these treatments when compared to the control 190
On the other hand a within treatment difference on mimosine induction was seen 191
between day 2 and 4 in seedlings treated with 100 mM MeJA (Fig 2) In a lower 192
concentration (04 mM) jasmonic acid (JA)promoted a near threefold increase in mimosine 193
accumulation of giant leucaena seedlings after 2 days of application 194
UV-C radiation 195
Albeit UV-C radiation is not biologically active in natural environments it has been 196
widely used under controlled experimental conditions to generate acute responses of plant 197
specialized metabolism within a shorter period of time compared to that required to with UV-198
B radiation (Kara 2013 Cetin 2014) This fast response is due to the higher energy of UV-199
C photons that act as potent reactive oxygen species (ROS) generators causing extensive 200
damage to the cells either at the physiological level or on DNA structure (Gregianini et al 201
2003 Matsuura et al 2013) 202
Although divergent responses can be observed in plants exposed to UV-C radiation 203
the deleterious processes are usually reported on primary metabolism (decreasing of 204
chlorophyll content and plant height eg) (Kara 2013) In the present study no statistical 205
75
differences were observed in the mimosine concentration in maricaacute seedlings supplemented 206
with UV-C radiation However a decreasing in its content was found for both control and 207
treatment at day 6 post-treatment (Fig 03) Taking into account the lower constitutive 208
concentration of mimosine observed in maricaacute compared to the leucaena plants besides its 209
relative thermolability (Nguyen and Tawata 2016) it seems to be plausible to consider the 210
effect of the temperature inside the UV-C and the white light (control) chambers as an 211
additional abiotic factor contributing to the decrease of mimosine accumulation in both group 212
of plants 213
Besides mimosine identification the presence of 34-dihydroxypyridine (34-DHP or 214
3-hydroxy-4-pyridone - 3H4P) a mimosine degradation product (Negi et al 2014 Nguyen 215
and Tawata 2016) was also reported for maricaacute leaf extracts analyzed by TLC by Ferreira 216
et al (1992) In our chromatograms we detected a second large peak after that of mimosine 217
(229plusmn0024) and similar to that identified by Negi et al (2014) as 3H4P (data not shown) 218
Comparing the chromatogram profiles obtained from seedlings elicited with chemical 219
stressors and those supplemented with UV-C the largest area for this peak was found (in all 220
samples) in the latter treatment at day 6 It might indicate that the constitutive andor the 221
initially UV-C-induced mimosine was degraded into 3H4P to cope with the cellular damage 222
caused by this treatment associated with an increased temperature inside the chambers 223
Nevertheless it was not possible to determine 3H4P concentration (or confirm its identity) 224
in maricaacute plants since there is no commercial standard (pure 3H4P) available for purchase 225
to be used as a reference in calculations Establishment of improved protocols for obtaining 226
in house 3H4P reference substance by acid hydrolysis is ongoing 227
228
229
76
Conclusion 230
On the basis of the overall absence of effect of the treatments tested here on mimosine 231
concentration it is possible to suggest that its accumulation profile is similar to that of 232
phytoanticipins unlike what is observed for the same amino acid production in leucaena 233
which shows features of inducibility resembling phytoalexin-like metabolites Alternatively 234
a putative inducible pool of mimosine in maricaacute might be involved in other types of stress 235
such as extended drought periods If involved in protection against oxidative stress as 236
described for leucaena mimosine in maricaacute may act predominantly by physical quenching 237
of ROS as indicated by the lack of overt chemical degradation Nevertheless further 238
investigations are needed to assess these hypotheses 239
To sum up mimosine biosynthesis was not modulated by the treatments evaluated as 240
in L leucocephala (Lam) de Wit To the best of our knowledge this is the first work that 241
analytically identifies and quantifies mimosine accumulation in M bimucronata 242
243
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J Agric Food Chem 64(23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541 281
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Molecular Biotechnology 60(2) 169ndash183httpsdoiorg101007s12033-017-0056-1 295
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase from 296
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Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine 299
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Olkoski D Wittmann MTS 2011 Cytogenetics of Mimosa bimucronata (DC) O Kuntze 301
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Appl Biotechnol 11 27-35 httpdxdoiorg101590S1984-70332011000100004 303
Patreze CM Cordeiro L 2004 Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 304
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Perotti JC Rodrigues-Correcirca KCS Fett-Neto AG 2015 Control of resin production in 307
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UV exposure Plant Physiology and Biochemistry 135 432ndash440 311
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Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS 2011 Biologia da reproduccedilatildeo 315
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Simon MF and Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 317
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 318
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Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 320
LM Andrade SF Cechinel-Filho V 2017 Chemical composition and diuretic natriuretic and 321
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kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its majority 322
constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash1624 323
Shah J 2003 The salicylic acid loop in plant defense Current Opinion Plant Biology6 (4) 324
365ndash371 325
Shinozaki K Uemura M Serres JB Bray EA Weretilnyk E 2015 Responses to Abiotic 326
Stress In Buchanan BB Gruissem W Jones RL (Eds) Biochemistry and Molecular 327
Biology of Plants Second Edition John Wiley and Sons Ltd 328
Soedarjo M and Borthakur D 1998 Mimosine a toxin produced by the tree-legume 329
Leucaena provides a nodulation competition advantage to mimosine-degrading Rhizobium 330
strains Soil Biology and Biochemistry 30(12)1605-1613 331
Vestena S Fett-Neto AG Duarte RC Ferreira AG 2001 Regulation of mimosine 332
accumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 333
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene induced 334
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Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX Tsim KWK 2018 337
Jasmonate-elicited stress induces metabolic change in the leaves of Leucaena leucocephala 338
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Zhang H Memelink J 2009 Regulation of Secondary Metabolism by Jasmonate Hormones 340
In AE Osbourn and V Lanzotti (eds) Plant-derived Natural Products 3 DOI 101007978-341
0-387-85498-4_1 copy Springer Science + Business Media LLC 342
343
344
345
81
346
Figure 1 Constitutive concentration of mimosine in different plant organs of Mimosa 347
bimucronata Bars sharing the same letter do not differ statistically by Tukey test (Ple005) 348
The error bars denote standard error of 10 replicates 349
350
351
352
353
354
355
356
357
B B A C0
5
10
15
20
25
30
35
40
LEAVES GREEN FLOWER BUDS POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
co
nce
ntr
atio
n u
gg-1
Mimosine concentration in adult plants of Mimosa bimucronata (DC) Kuntze
82
C T R L S A
1 0 m M
S A
5 0 m M
E T H
0 0 7 m M
E T H
0 3 5 m M
M e J A
1 0 0 m M
M e J A
2 0 0 m M
S N P
1 0 m M
S N P
5 0 m M
0
1 0
2 0
3 0
T re a tm e n ts
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1) D A Y 2
D A Y 4
A B C C B C A B C C A B C A B C A
a b b b a a b a a b b a b
358
Figure 2 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 359
bimucronata treated with different signaling molecules SA = Salicylic Acid ETH = 360
Ethephon MeJA = Methyl Jasmonate SNP = Sodium Nitroprusside Uppercase and 361
lowercase letters indicate statistical differences among treatments in days 2 and 4 362
respectively Bars sharing a letter of the same case do not differ statistically by Tukey test 363
(Ple005) Indicates statistical difference in the same treatment between day 2 and 4 by t-364
test (Ple005) The error bars denote standard error of 5 replicates (25 individual seedlings 365
arranged in 5 groups of 5) 366
367
368
83
D AY 3 D AY 6
0
5
1 0
1 5
2 0
2 5
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1)
C O N TR O L
U V -C
369
Figure 3 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 370
bimucronata supplemented with UV-C radiation Indicates statistical difference in the same 371
treatment between day 3 and 6 by t-test (Ple005) The error bars denote standard error of 5 372
replicates (25 individual seedlings arranged in 5 groups of 5) 373
374
375
376
377
378
379
380
381
382
383
384
385
84
Consideraccedilotildees finais 386
- Experimentos que avaliam os efeitos da aplicaccedilatildeo exoacutegena de ANPs em diferentes espeacutecies 387
vegetais tecircm sido realizados principalmente com GABA Dentre os principais efeitos 388
conferidos pela aplicaccedilatildeo dessa moleacutecula em espeacutecies de mono e eudicotiledocircneas satildeo 389
relatados a toleracircncia agrave seca agrave salinidade e agraves temperaturas extremas 390
- Como metaboacutelitos especializados claacutessicos os ANPs podem ter sua concentraccedilatildeo basal 391
endoacutegena aumentada em resposta agrave induccedilatildeo mediada por uma vasta gama de tratamentos com 392
moleacuteculas sinalizadoras de estresse e fontes alternativas de estressores De um modo geral 393
observa-se o acuacutemulo das diferentes classes de ANPs em resposta agrave radiaccedilatildeo UV elicitores 394
quiacutemicos que mimetizam ataques por patoacutegenos dano mecacircnico agentes osmoacuteticos metais 395
pesados entre outros 396
- Especificamente em leucena a resposta observada em relaccedilatildeo aos diferentes tratamentos 397
testados indica que apesar do seu alto teor constitutivo nessa espeacutecie a biossiacutentese e o 398
acuacutemulo de mimosina podem ser modulados por fatores causadores de estresses exibindo -399
nessa espeacutecie - um padratildeo de acumulaccedilatildeo similar agrave fitoalexinas Em maricaacute por outro lado 400
aumento de acuacutemulo dessa moleacutecula natildeo foi observado para os mesmos tratamentos testados 401
para leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas 402
- O padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno 403
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado pela 404
sua degradaccedilatildeo 405
- As respostas observadas nos testes que avaliaram a atividade de mitigaccedilatildeo de espeacutecies 406
reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode agir como um agente 407
antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de estresse 408
85
Perspectivas 409
- Confirmaccedilatildeo em espectrocircmetro de massas eou ressonacircncia nuclear magneacutetica da natureza 410
quiacutemica da lsquomimosinarsquo presente em maricaacute 411
- Avaliaccedilatildeo do efeito de concentraccedilotildees mais elevadas e em diferentes periacuteodos de aplicaccedilatildeo 412
das moleacuteculas sinalizadoras testadas sobre o acuacutemulo de mimosina em leucena e maricaacute 413
- Ampliar a investigaccedilatildeo dos padrotildees de expressatildeo gecircnica dos genes que codificam para 414
mimosinase (em maricaacute) mimosina sintase (em ambas as espeacutecies testadas) bem como o 415
perfil de precursores e cataboacutelitos de mimosina em resposta aos tratamentos mencionados 416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
86
Referecircncias Bibliograacuteficas 436
437
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effects in animals P Nutr Soc 64 403ndash412 httpsdoiorg101079PNS2005449 439
Ahmed R Hoque ATMR Hossain MK (2008) Allelopathic effects of Leucaena 440
leucocephala leaf litter on some forest and agricultural crops grown in nursery J Forestry 441
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Ahmed AMM Saacutenchez FJS Bavileacutes LRY Mahdy REZ Camaal JBC (2016) Tannins and 443
mimosine in Leucaena genotypes and their relations to Leucaena resistance against 444
Leucaena Psyllid and Onion thrips Agroforestry Systems 1-8 445
Benjakul S Kittiphattanabawon P Shahidi F Maqsood S (2013) Antioxidant activity and 446
inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipid oxidation in 447
model systems Food Sci Technol Int 19(4)365-76 448
httpsdoiorg1011771082013212455186 449
Bitencourt F Zocche JJ Costa S Souza PZ Mendes AR (2007) Nucleaccedilatildeo de Mimosa 450
bimucronata (DC) O Kuntze em aacutereas degradadas pela mineraccedilatildeo de carvatildeo Revista 451
Brasileira de Biociecircncias 5 750-752 452
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A Montes-Perez R Muntildeoz-Rodriguez D Ku-Vera J (2015) Ovarian activity and estrus 454
behavior in early postpartum cows grazing Leucaena leucocephala in the tropics Trop Anim 455
Health Prod 47(8)1481-6 456
Carvalho PER (2004) Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 457
Teacutecnica 941-10 458
Chowtivannakul P Srichaikul B Talubmook C (2016) Antidiabetic and antioxidant activities 459
of seed extract from Leucaena leucocephala (Lam) de Wit Agriculture and Natural 460
Resources 50 (2016) 357e361 httpdxdoiorg101016janres201606007 461
Chung H-H Chen M-K Chang Y-C Yang S-F Lin C-C Lin C-W (2017) Inhibitory effects 462
of Leucaena leucocephala on the metastasis and invasion of human oral cancer cells 463
Environmental Toxicology 321765ndash1774 httpsdoiorg101002tox22399 464
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Crowe B Poynter JA Manukyan MC Wang Y Brewster BD Herrmann JL Abarbanell 465
AM Weil BR Meldrum DR (2001) Pretreatment with intracoronary mimosine improves 466
postischemic myocardial functional recovery Surgery 150(2) 191-106 467
Fallon (2015) Effects of mimosine on Wolbachia in mosquito cells cell cycle suppression 468
reduces bacterial abundance In Vitro Cell Dev Biol Anim 51(9)958-63 469
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Fernaacutendez-Salas A Alonso-Diacuteaza MA Acosta-Rodriacuteguez A Torres-Acosta JFJ Sandoval-471
Castro CA Rodriacuteguez-Vivas RI (2011) In vitro acaricidal effect of tannin-rich plants against 472
the cattle tick Rhipicephalus (Boophilus) microplus (Acari Ixodidae) Veterinary 473
Parasitology 175113ndash118 2010 httpsdoiorg101016jvetpar201009016 474
Ferreira AG Aquila MEA Jacobi US Rizvi V (1992) Allelopathy in Brazil In Allelopathy 475
basic and applied aspects Rizvi V and Jacobi US (Eds) Chapman and Hall PP 243-250 476
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad Hossain Md Anai 477
T Oku H (2018) Cytosolic cysteine synthase switch cysteine and mimosine production in 478
Leucaena leucocephala Appl Biochem Biotechnol 186 (3) 613ndash632 479
httpsdoiorg101007s12010-018-2745-z 480
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of the thyrotoxic 481
amino acid mimosine by cysteine synthase Phytochemistry 29 (11) 3461ndash3465 482
httpsdoiorg1010160031-9422(90)85258-H 483
Jacobi US Ferreira AG (1991) Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK Sobre 484
espeacutecies cultivadas Pesquisa Agropecuaacuteria Brasileira 26(7) 935-943 485
Jamous RM Ali-Shtayeh MS Abu-Zaitoun SY Markovics A Azaizeh H (2017) Effects of 486
selected Palestinian plants on the in vitro exsheathment of the third stage larvae of 487
gastrointestinal nematodes BMC Veterinary Research 13308 488
httpdxdoiorg101186s12917-017-1237-7 489
Jiao CJ Jiang J-L Ke L-M Cheng W Li F-M Li Z-X Wang C-Y (2011) Factors affecting 490
β-ODAP content in Lathyrus sativus and their possible physiological mechanisms Food 491
Chem Toxicol 49 543ndash549 httpsdoiorg101016jfct201004050 492
Kubota S Fukumoto Y Ishibashi K Soeda S Kubota SS Yuki R Nakayama Y Aoyama K 493
Yamaguchi N (2014) Activation of the prereplication complex is blocked by mimosine 494
88
through reactive oxygen species-activated ataxia telangiectasia mutated (ATM) protein 495
without DNA damage J Biol Chem 28 289(9)5730-46 496
Kuppusamy UR Arumugam B Azaman N Wai CJ (2014) Leucaena leucocephala Fruit 497
Aqueous Extract Stimulates Adipogenesis Lipolysis and Glucose Uptake in Primary Rat 498
Adipocytes Hindawi Publishing Corporation e Scientific World Journal Article ID 737263 499
8 pages httpdxdoiorg1011552014737263 500
Kusama-Eguchi K (2019) Research in motor neuron diseases caused by natural substances 501
focus on pathological mechanisms of neurolathyrism Yakugaku Zasshi 139 (4) 609-502
615 httpsdoiorg101248yakushi18-00202 503
Kutchan TM Gershenzon J Moslashller BL Gang DR (2015) Natural Products In Buchanan 504
BB Gruissem W and Jones RL (eds) Biochemistry amp Molecular Biology of Plants 2nd edn 505
Wiley Blackwell Chichester pp 1135-1205 506
Lalande M (1990) A reversible arrest point in the late G1 phase of the mammalian cell cycle 507
Exp Cell Res 186 332ndash339 508
Li X-W Hu C-P Li Y-J Gao Y-X Wang XM Yang J-R (2015) Inhibitory effect of L-509
mimosine on bleomycin-induced pulmonary fibrosis in rats Role of eIF3a and p27 Int 510
Immunopharmacol 27(1) 53ndash64 511
Little Jr EL Skolmen RG (1989) Koa haole Agriculture Handbook 679 USDA 512
Lorenzi H (1998) Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 513
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 514
Marchiori JNC (1993) Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 515
O Kuntze Ciecircncia Florestal 3 85-106 516
Mohammed RS El Souda SS Taie HAA Moharam ME Shaker KH (2015) Antioxidant 517
antimicrobial activities of flavonoids glycoside from Leucaena leucocephala leaves Journal 518
of Applied Pharmaceutical Science 5(06)138-147 519
httpdxdoiorg107324JAPS201550623 520
Negi VS Bingham J-P Li QX Borthakur D (2014) A carbon-nitrogen lyase from Leucaena 521
leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 (2) 922ndash522
934 httpsdoiorg101104pp113230870 523
89
Olkoski D Wittmann MTS (2011) Cytogenetics of Mimosa bimucronata (DC) O Kuntze 524
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop 525
Breeding and Applied Biotechnology 11 27-35 526
Patreze CM Cordeiro L (2004) Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 527
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecology and Management 528
196275ndash285 529
Pilatti DM Fortes AMT Jorge TCM Boiago NP (2019) Comparison of the phytochemical 530
profiles of five native plant species in two different forest formations Brazilian Journal of 531
Biology 79(2) 233-242 532
Ramos-Ruiz R Poirot E Flores-Mosquera M (2018) GABA a non-protein amino acid 533
ubiquitous in food matrices Cogent Food Agric 41534323 534
httpsdoiorg1010802331193220181534323 535
REFLORA (2019) httpfloradobrasiljbrjgovbrreflora Acesso em agosto de 2019 536
Rodgers KJ Samardzic K Main BJ (2015) Toxic Nonprotein Amino Acids Plant Toxins 537
httpsdoiorg 101007978-94-007-6728-7_9-1 538
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG (2019) Mimosine 539
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 540
UV exposure Plant Physiology and Biochemistry 135 432ndash440 541
httpsdoiorg101016jplaphy201811018 542
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 543
LM Andrade SF Cechinel-Filho V (2017) Chemical composition and diuretic natriuretic 544
and kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its 545
majority constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash546
1624 547
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS (2011) Biologia da reproduccedilatildeo 548
de Mimosa bimucronata ndash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 549
Simon MF Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 550
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 551
endemism Biological Conservation 96 279-296 552
90
Soares AMS Arauacutejo SA Lopes SG Costa Junior LM (2015) Anthelmintic activity of 553
Leucaena leucocephala protein extracts on Haemonchus contortus Braz J Vet Parasitol 554
Jaboticabal 24(4) 396-401 httpdxdoiorg101590S1984-29612015072 555
Soerdajo M Borthakur D (1998) Mimosine a toxin produced by the tree-legume Leucaena 556
provides a nodulation competition advantage to mimosine-degrading Rhizobium strains Soil 557
Biol Biochem 30(12) 16051613 558
Souza-Lima ES Sinani TR Pott A Sartori ALB (2017) Mimosoideae (Leguminosae) in the 559
Brazilian Chaco of Porto Murtinho Mato Grosso do Sul Rodrigueacutesia 68(1) 263-290 2017 560
httpdxdoiorg1015902175-7860201768131 561
Taiz L amp Zeiger E (2010) Plant Physiology 5th edition Sinauer Associates Inc Sunderland 562
Verma VK Rani KV Kumara SR Prakash O (2018) Leucaena leucocephala pod seed 563
protein as an alternate to animal protein in fish feed and evaluation of its role to fight against 564
infection caused by Vibrio harveyi and Pseudomonas aeruginosa Fish and Shellfish 565
Immunology 76 (2018) 324ndash332 httpsdoiorg101016jfsi201803011 566
Yafuso JT Negi VS Bingham J-P Borthakur D (2014) An O-acetylserine (thiol) lyase from 567
Leucaena leucocephala is a cysteine synthase but not a mimosine synthase Appl Biochem 568
Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z 569
Zarin RMA Wan HY Isha A Armani N (2016) Antioxidant antimicrobial and cytotoxic 570
potential of condensed tannins from Leucaena leucocephala hybrid Food Science and 571
Human Wellness 5 65ndash75 httpdxdoiorg101016jfshw201602001 572
573
574
Contents lists available at ScienceDirect
Industrial Crops amp Productsjournal homepage wwwelseviercomlocateindcrop
Resin tapping transcriptome in adult slash pine (Pinus elliottii var elliottii)Camila Fernanda de Oliveira Junkes1 Artur Teixeira de Arauacutejo Juacutenior1 Juacutelio Ceacutesar de LimaFernanda de Costa Thanise Fuumlller Maacutercia Rodrigues de Almeida Franciele Antocircnia NeisKelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-NetoCenter for Biotechnology and Department of Botany Federal University of Rio Grande do Sul Porto Alegre PO Box 15005 91501-970 Brazil
A R T I C L E I N F O
KeywordsPinus elliottiResinResinosisTranscriptomeAdjuvant paste
A B S T R A C T
To better understand the bases of resin production a major source of terpenes for industry the transcriptome ofadult Pinus elliottii var elliottii (slash pine) trees under field commercial resinosis was obtained Samples werecollected from cambium after 5 and 15 days of treatment application which included tapping followed byapplication of commercial resin stimulant paste or control wounding without paste Overall mean number ofreads of all 16 libraries (2 treatments x 2 times x 4 replicated trees) was 34582048 Of these 89 were mappedagainst the reference sequence with a mismatch of 058 Using the Blast2Go 570 candidate genes were de-tected based on sequence annotation By comparing the expression profile between paste and control 310differentially expressed genes (DEGs) were identified at 5 days and 190 at 15 days with a significant fold changeof log2gt 12 Regarding changes in time comparisons within each treatment 210 and 105 DEGs were identifiedwithin control and paste treatment respectively Genes with different expression patterns in the times andtreatments examined included ethylene responsive transcription factors geranylgeranyl diphosphate synthasediterpene synthase cytochrome P450 and ABC transporters all of which may play important roles in resinproduction RT-qPCR analysis correlated well with the data obtained by RNAseq Resin composition changedover time This is the first transcriptomic investigation of resinosis of the main species used in the bioresinindustry and of molecular analyses of resinosis under field operations with implications for stand managementstimulant paste development genotype selection and breeding for high resinosis
1 Introduction
The adaptive success of conifers is largely due to the development ofa defense system based on the synthesis and secretion of terpenes in allmajor organs and different tissues (Miller et al 2005 Hall et al 2013Warren et al 2015) Conifer resin is a viscous fluid composed of acomplex mixture of terpenoids such as monoterpenes sesquiterpenesand diterpenes (Zulak and Bohlmann 2010) These terpenoids are se-creted from severed resin ducts when the tree is under biotic attack(Ralph et al 2006 Lange 2015 Geisler et al 2016) acting as pro-tectants (Schiebe et al 2012 Liu et al 2015)Biosynthesis of terpenes in conifers starts from isomerization of two
isoprenoid (C5) units dimethylallyl diphosphate (DMAPP) and iso-pentenyl diphosphate (IPP) These molecules can be biosynthesized viatwo separate routes in plants the methyl-erythritol 4-phosphate andmevalonate pathways IPP is synthesized and isomerized to DMAPP byisopentenyl diphosphate isomerase then prenyl transferases catalyze
the condensation of these two C5-units to geranyl diphosphate (Pazoukiand Niinemets 2016) Their elongation to prenyl diphosphates withaddition of IPP molecules leads to monoterpenes (C10) sesquiterpenes(C15) and diterpenes (C20) which are the substrates for terpene syn-thases (TPS) (Keeling and Bohlmann 2006b)TPSs are part of a large family of mechanistically related enzymes
involved in both primary and secondary metabolism (Keeling andBohlmann 2006b) The events of evolutionary diversification and ex-pansion of plant TPSs appear to have originated from gene duplicationsdomain losses and sub- or neofunctionalizations with subsequent di-vergence of an ancestral TPS gene of primary metabolism (Hall et al2013) Modification of TPS products changes their physical propertiesand may alter their biological activities (Chen et al 2011) TPSs of highsequence identity may have different functions even in closely relatedspecies Low sequence identity of TPSs in phylogenetically distantspecies does not preclude the possibility of independent evolution of thesame or related function of these enzymes (Zerbe and Bohlmann 2015)
httpsdoiorg101016jindcrop2019111545Received 4 January 2019 Received in revised form 10 June 2019 Accepted 4 July 2019
Corresponding authorE-mail address fettnetocbiotufrgsbr (AG Fett-Neto)1 These authors have equally contributed to this work
doi 1015900102-33062019abb0114
Acta Botanica Brasilica
Sustainable production of bioactive alkaloids in Psychotria L of
southern Brazil propagation and elicitation strategies
Yve Verocircnica da Silva Magedans1 Kelly Cristine da Silva Rodrigues-Correcirca1 Cibele Tesser da Costa1
Heacutelio Nitta Matsuura1 and Arthur Germano Fett-Neto1
Received April 1 2019Accepted June 28 2019
ABSTRACTPsychotria is the largest genus in Rubiaceae South American species of the genus are promising sources of natural
products mostly due to bioactive monoterpene indole alkaloids they accumulate ese alkaloids can have analgesic
antimutagenic and antioxidant activities in dierent experimental models among other pharmacological properties
of interest Propagation of genotypes with relevant pharmaceutical interest is important for obtaining natural
products in a sustainable and standardized fashion Besides the clonal propagation of elite individuals the alkaloid
content of Psychotria spp can also be increased by applying moderate stressors or stress-signaling molecules is
review explores advances in research on methods for plant propagation and elicitation techniques for obtaining
bioactive alkaloids from Psychotria spp of the South Region of Brazil
Keywords abiotic stress alkaloids elicitation monoterpenes plant propagation Psychotria southern Brazil
sustainability
Introduction
Psychotria belongs to Rubiaceae one of the major families
of $owering plants having economic interest e family
includes coee a few signicant poisonous plants to livestock
besides several important ornamental and medicinal species
(Souza amp Lorenzi 2012) Psychotria has captured researchersrsquo
attention mostly because of its medicinal properties
Psychotria colorata is an Amazonian species that produces
polyindolinic alkaloids with analgesic activity (Matsuura et
al 2013) e promising results obtained with P colorata
motivated the investigation of southern Brazilian Psychotria
species and the discovery of new bioactive alkaloids (Porto
et al 2009) Moreover leads on in planta alkaloid functions
were also topic of experimental evaluation
One of the key elements that needs to be addressed early
on during the process of developing new bioactive molecules
from plants is the capacity to generate catalytically active
biomass to support extraction and steady supply ere are a
number of ways through which these goals may be reached
including greenhouse rooting of cuttings (mini-cutting
1 Laboratoacuterio de Fisiologia Vegetal Departamento de Botacircnica Instituto de Biociecircncias e Centro de Biotecnologia Universidade Federal do Rio
Grande do Sul 91501-970 Porto Alegre RS Brazil
Corresponding author fettnetocbiotufrgsbr
Review
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Biomass yield of resin in adult Pinus elliottii Engelm trees is differentially
regulated by environmental factors and biochemical effectors
Franciele Antocircnia Neis Fernanda de Costa Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima
Kelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-Neto
Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) CP 15005 CEP 91501-970 Porto Alegre RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Biomass
Terpene resin
Seasonal
Benzoic acid
Regenerated forest
A B S T R A C T
Biomass of pine resin finds several applications in the chemical pharmaceutical biofuel and food industries
Resin exudation after injury is a key defense response in Pinaceae since this complex mixture of terpenes has
insecticidal antimicrobial and wound repair properties Resin yield is increased by effectors applied on the
wound area including phytohormones and metal cofactors of terpene synthases The interaction of resinosis
mechanism effectors is not fully understood particularly in adult forest setups under natural environmental
variations The aim of this work was to determine how resin exudation by wounded trunks of adult P elliottii
responded to combined chemical effectors involved in different regulatory pathways of resinosis (metal cofactors
of terpene synthases benzoic acid and plant growth regulators) and whether seasonal and tree distribution
variations affected these responses Symmetrically planted and scattered trees regenerated from the seed bank
had similar resin biomass yields suggesting that the homogeneity in development and spatial arrangement were
not significant factors in resin yield This new finding is of practical importance with the used tapping system
since costs of implanting forests by regeneration can be advantageous compared to planting In addition it was
shown for the first time that the salicylic acid precursor benzoic acid and the auxin naphthalene acetic acid
promoted resin exudation when individually applied to wound sites Both these adjuvants are two orders of
magnitude less costly compared to the conventionally used ethylene precursors besides facing less environ-
mental and health restrictions for use Most adjuvant-treated trees showed higher resin flow in the second year
indicating mechanisms of response build up Overall temperature was more important than rainfall as en-
vironmental parameter affecting resin biosynthesis which was higher in the warmer months of spring and
summer The combination of resinosis stimulant effectors from different signaling pathways showed no sig-
nificant synergistic or additive effect suggesting possible converging signaling pathways andor limitation of
common intermediate transducing molecules
1 Introduction
Pines occupy highly diverse environments over a range of tem-
peratures water and nutrient availabilities irradiance levels and pho-
toperiods being able to effectively face attacks from diverse herbivore
and pathogen guilds The success of conifers is linked to their complex
terpene biochemistry hosted by specialized secretory cells The terpe-
noid resin synthesized by Pinus spp is one of the main mechanisms of
defense of these trees particularly against bark beetles and the fungi
they carry (Fett-Neto and Rodrigues-Correcirca 2012) Pine resin biomass
is essentially composed of a monoterpene and sesquiterpene-rich tur-
pentine and diterpenoid-rich rosin fraction both finding numerous in-
dustrial applications as non-wood forest products (Rodrigues-Correcirca
et al 2012)
Molecules capable of modulating different signaling pathways have
been identified as resin yield stimulators including sulfuric acid (ex-
tends wound damage) 2-chloroethylphosphonic acid (CEPA a syn-
thetic ethylene precursor) paraquat (free radical generator) yeast ex-
tract (mimics attack by pathogens) salicylic acid (pathogen signaling
molecule) auxin (promotes ethylene biosynthesis and resin canal dif-
ferentiation) jasmonic acid (signals mechanical damage and promotes
secondary metabolism) and metal ions such as potassium iron and
manganese (cofactors of terpene synthases in conifers) and copper (a
component of ethylene receptors) (Clements 1970 Conrath et al
2002 Fett-Neto and Rodrigues-Correcirca 2012 Hudgins and Franceschi
2004 Lewinsohn et al 1994 Martin et al 2002 Popp et al 1995
httpsdoiorg101016jindcrop201803027
Received 12 December 2017 Received in revised form 9 March 2018 Accepted 13 March 2018
Corresponding author
E-mail addresses franci_neisyahoocombr (FA Neis) fernandadecostayahoocombr (F de Costa) thanisenfyahoocombr (TN Fuumlller)
jjuliocesarlimagmailcom (JC de Lima) krodriguescbiotufrgsbr (KC da Silva Rodrigues-Correcirca) jpfettcbiotufrgsbr (JP Fett) fettnetocbiotufrgsbr (AG Fett-Neto)
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Research Paper
Dual allelopathic effects of subtropical slash pine (Pinus elliottii Engelm)
needles Leads for using a large biomass reservoir
Kelly Cristine da Silva Rodrigues-Correcircaa Gelson Halmenschlagera Joseacuteli Schwambachb
Fernanda de Costaa Emili Mezzomo-Trevizana Arthur Germano Fett-Netoa
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 Brazilb University of Caxias do Sul Institute of Biotechnology Caxias do Sul RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Seasonality
Growth
Germination
Litter
Substrate
A B S T R A C T
Pinus elliottii Engelm (slash pine) is distributed along the maritime coast of Southern Brazil where it shows
invasive pattern and typical allelopathic features Large quantities of needle litter are produced by pine trees a
biomass that is little explored in areas where this species is alien Little is known about the dynamics of needle
and litter phytochemical interactions particularly in subtropical environments To elucidate the full range of
needle and litter allelopathic potential the effects of litter (superficial and deep) and seasonally harvested fresh
slash pine needles stored for different times were evaluated against lettuce tomato and cucumber seeds and
seedlings Increasing concentrations (0 1 2 4 and 8 wv) of hot and cold aqueous extracts of needles
and litter affected in different ways target plant development Growth and germination inhibition were directly
related to the highest extract concentrations (regardless of the season and mainly in hot water extracts) of
needles On the other hand stimulatory effects of litter extracts on lettuce growth were observed Growth and
germination of cucumber and tomato were not affected by pine litter as substrate when compared to rice husk
The presumable high polarity and thermal stability of slash pine leaf biomass allelochemicals and their transient
toxic effect or growth promoting impact suggest potential applications of this largely available biomass both as a
biological herbicide and growth substrate in plant propagation
1 Introduction
Native from the Northern Hemisphere Pinus is one of the most
widely distributed genera throughout different climate regions of the
globe growing either as native or alien species even in extreme habi-
tats (Rodrigues-Correcirca and Fett-Neto 2012) Despite the high economic
value currently attributed to pine wood and oleoresin (Rodrigues-
Correcirca et al 2012) there is increasing concern about the aggressive
potential of invasiveness displayed by Pinus species especially those
cultivated out of their native range of distribution (Richardson et al
2008 Rolon et al 2011) These species are dispersed by wind and there
is notably low plant diversity observed in most understories of pine
plantations (Kato-Noguchi et al 2009) This latter feature has been
considered an important trait of allelopathic interference
The term ldquoallelopathyrdquo was coined by Molisch in 1937 as a chemical
reciprocal interaction established among plants (including micro-
organisms) sharing the same site by means of the release of secondary
metabolites named allelochemicals (Rice 1984) For the most part
these metabolites are derived from the shikimic acid or isoprenoid
pathway and their biosynthesis can be modulated by biotic and abiotic
stresses (Nascimento and Fett-Neto 2010) including seasonal-related
changes (Sartor et al 2013) Allelopathy studies may range from sterile
assays (Aryakia et al 2015) to soil (Correcirca et al 2008 Sharma et al
2016) and field tests being a complex biological phenomenon to as-
certain in several circumstances due to issues of solubility release
mechanisms and stability of bioactive compounds (Scognamiglio et al
2013) Often the use of complementary methods provides more in-
formative data
The allelopathic effects of soil leachates green needles and litter
extracts of Pinus spp on germination and seedling growth aspects of
wild and crop species have been evaluated in natural and cultivated
pine stands and have proven to be stimulatory or inhibitory (Lodhi and
Killingbeck 1982 Kil and Yim 1983 Nektarios et al 2005 Akkaya
et al 2006 Machado 2007 Alrababah et al 2009 Sartor et al 2009
Kato-Noguchi et al 2011 Rolon et al 2011 Valera-Burgos et al
2012) exhibiting in some cases autotoxicity (Garnett et al 2004
Fernandez et al 2008 Zhu et al 2009 Monnier et al 2011) Studies
on potential dual allelopathic effects of Pinus elliottii Engelm (slash
httpdxdoiorg101016jindcrop201706019
Received 23 March 2017 Received in revised form 15 May 2017 Accepted 7 June 2017
Corresponding author
E-mail address fettnetocbiotufrgsbr (AG Fett-Neto)
ORIGINAL RESEARCHpublished 16 June 2016
doi 103389fpls201600849
Frontiers in Plant Science | wwwfrontiersinorg 1 June 2016 | Volume 7 | Article 849
Edited by
Juan Francisco Jimenez Bremont
Instituto Potosino de Investigacioacuten
Cientiacutefica y Tecnoloacutegica Mexico
Reviewed by
Mariacutea De La Luz Guerrero Gonzaacutelez
Universidad Autoacutenoma de San Luis
Potosiacute Mexico
Rosalia Cristina Paz
CIGEOBIO (CONICETFCEFN UNSJ)
Argentina
Correspondence
Arthur G Fett-Neto
fettnetocbiotufrgsbr
daggerThese authors have contributed
equally to this work
Specialty section
This article was submitted to
Plant Physiology
a section of the journal
Frontiers in Plant Science
Received 08 December 2015
Accepted 30 May 2016
Published 16 June 2016
Citation
de Lima JC de Costa F Fuumlller TN
Rodrigues-Correcirca KCdS Kerber MR
Lima MS Fett JP and Fett-Neto AG
(2016) Reference Genes for qPCR
Analysis in Resin-Tapped Adult Slash
Pine As a Tool to Address the
Molecular Basis of Commercial
Resinosis Front Plant Sci 7849
doi 103389fpls201600849
Reference Genes for qPCR Analysisin Resin-Tapped Adult Slash Pine Asa Tool to Address the MolecularBasis of Commercial Resinosis
Juacutelio C de Lima 1dagger Fernanda de Costa 1 dagger Thanise N Fuumlller 1
Kelly C da Silva Rodrigues-Correcirca 2 Magnus R Kerber 1 Mariano S Lima 1
Janette P Fett 1 and Arthur G Fett-Neto 1
1 Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul
Porto Alegre Brazil 2 Biological Sciences Department Regional Integrated University of Alto Uruguai and Missotildees (URI-FW)
Frederico Westphalen Brazil
Pine oleoresin is a major source of terpenes consisting of turpentine (mono- and
sesquiterpenes) and rosin (diterpenes) fractions Higher oleoresin yields are of economic
interest since oleoresin derivatives make up a valuable source of materials for chemical
industries Oleoresin can be extracted from living trees often by the bark streak method
in which bark removal is done periodically followed by application of stimulant paste
containing sulfuric acid and other chemicals on the freshly wounded exposed surface
To better understand the molecular basis of chemically-stimulated and wound induced
oleoresin production we evaluated the stability of 11 putative reference genes for the
purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis
Samples for RNA extraction were collected from field-grown adult trees under tapping
operations using stimulant pastes with different compositions and at various time points
after paste application Statistical methods established by geNorm NormFinder and
BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3
and UBI To confirm expression stability of the candidate reference genes expression
profiles of putative P elliottii orthologs of resin biosynthesis-related genes encoding Pinus
contorta β-pinene synthase [PcTPS-(minus)β-pin1] P contorta levopimaradieneabietadiene
synthase (PcLAS1) Pinus taeda α-pinene synthase [PtTPS-(+)αpin] and P taeda
α-farnesene synthase (PtαFS) were examined following stimulant paste application
Increased oleoresin yields observed in stimulated treatments using phytohormone-based
pastes were consistent with higher expression of pinene synthases Overall the
expression of all genes examined matched the expected profiles of oleoresin-related
transcript changes reported for previously examined conifers
Keywords resin Pinus gene expression normalizer genes terpene synthase
19
Chapter 2
Stimulant Paste Preparation and Bark Streak Tapping Technique for Pine Oleoresin Extraction
Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
Tapping technique comprises the extraction of pine oleoresin a non-wood forest product consisting of a
complex mixture of mono sesqui and diterpenes biosynthesized and exuded as a defense response to
wounding Oleoresin is used to produce gum rosin turpentine and their multiple derivatives Oleoresin
yield and quality are objects of interest in pine tree biotechnology both in terms of environmental and
genetic control Monitoring these parameters in individual trees grown in the fi eld provides a means to
examine the control of terpene production in resin canals as well as the identifi cation of genetic-based
differences in resinosis A typical method of tapping involves the removal of bark and application of a
chemical stimulant on the wounded area Here we describe the methods for preparing the resin-stimulant
paste with different adjuvants as well as the bark streaking process in adult pine trees
Key words Oleoresin Pine Tapping Chemical stimulant Wounding
1 Introduction
Several conifer species produce oleoresin a complex mixture of isoprenoid compounds relevant for defense against herbivores and pathogens Two major fractions can be recognized in oleoresin (a) turpentine the volatile fraction containing mono- and sesquiter-penes and (b) rosin the nonvolatile diterpene fraction Oleoresin is a forest commodity of global interest fi nding applications in diverse industry sectors Rosin is used in adhesives printing ink manufacture and paper sizing Turpentine can be used either as a solvent for paints and varnishes or as a raw material for fraction-ation of high-value chemicals used in the pharmaceutical agro-chemical and food industry [ 1 ndash 3 ]
During the extraction activity resin is obtained from the tree in a similar way as rubber tree tapping which generally involves the
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_2 copy Springer Science+Business Media New York 2016
These authors have equally contributed to this work
fettnetocbiotufrgsbr
27
Chapter 3
A Modifi ed Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines
Juacutelio Ceacutesar de Lima Thanise Nogueira Fuumlller Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes
through high-throughput sequencing technologies microarray and also northern blots RT-PCR and
RTqPCR Even though many specifi c protocols designed for plants with high content of secondary metab-
olites have been developed these are often expensive time consuming and not suitable for a wide range
of tissues Here we present a modifi cation of the method previously described using the commercially
available Concerttrade Plant RNA Reagent (Invitrogen) buffer for fi eld-grown adult pine trees with high
oleoresin content
Key words RNA Pines Concert plant RNA reagent Stem RNA extraction Oleoresin Conifers
1 Introduction
Several conifer species especially within the Pinaceae have tissues with high concentrations of phenolics terpenes and polysaccha-rides [ 1 ] Many specifi c protocols that are appropriate for plants rich in secondary metabolite s have been developed but the extrac-tion of high-quality RNA from these tissues using commercial kits is often diffi cult and usually not applicable to woody tissues [ 2 ndash 6 ] One of the major issues during RNA extraction concerns the pres-ence of phenolic compounds which oxidize and form quinones Aromatic compounds bind RNA which interferes in downstream steps and applications [ 3 7 ] Another point of concern is the har-vest of plant samples in the experimental fi eld which constitutes another obstacle in the efforts to avoid degradation of RNA [ 8 ] These problems often result in RNAs of low quality and insuffi -cient amounts especially for methodologies that normally require
These authors have equally contributed to this work
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_3 copy Springer Science+Business Media New York 2016
fettnetocbiotufrgsbr
RESEARCH PAPER
Control of resin production in Araucaria angustifolia an ancientSouth American coniferJ C Perotti1 K C da Silva Rodrigues-Correa123 amp A G Fett-Neto12
1 Plant Physiology Laboratory Department of Botany Federal University of Rio Grande do Sul (UFRGS) Porto Alegre RS Brazil
2 Center for Biotechnology UFRGS Porto Alegre RS Brazil
3 Present address Regional Integrated University of Alto Uruguai and Miss~oes (URI-FW) Frederico Westphalen RS Brazil
Keywords
Araucaria ethylene jasmonic acid nitric
oxide resin salicylic acid terpenes
Correspondence
A G Fett-Neto Plant Physiology Laboratory
Center for Biotechnology Federal University
of Rio Grande do Sul (UFRGS) PO Box 15005
Av Bento Goncalves 9500 91501-970 Porto
Alegre Brazil
E-mail fettnetocbiotufrgsbr
Editor
K Leiss
Received 22 July 2014 Accepted 11
December 2014
doi101111plb12298
ABSTRACT
Araucaria angustifolia is an ancient slow-growing conifer that characterises parts ofthe Southern Atlantic Forest biome currently listed as a critically endangered speciesThe species also produces bark resin although the factors controlling its resinosis arelargely unknown To better understand this defence-related process we examined theresin exudation response of A angustifolia upon treatment with well-known chemicalstimulators used in fast-growing conifers producing both bark and wood resin suchas Pinus elliottii The initial hypothesis was that A angustifolia would display signifi-cant differences in the regulation of resinosis The effect of Ethrel (ET ndash ethylene pre-cursor) salicylic acid (SA) jasmonic acid (JA) sulphuric acid (SuA) and sodiumnitroprusside (SNP ndash nitric oxide donor) on resin yield and composition in youngplants of A angustifolia was examined In at least one of the concentrations testedand frequently in more than one an aqueous glycerol solution applied on fresh woundsites of the stem with one or more of the adjuvants examined promoted an increase inresin yield as well as monoterpene concentration (a-pinene b-pinene camphene andlimonene) Higher yields and longer exudation periods were observed with JA and ETanother feature shared with Pinus resinosis The results suggest that resinosis controlis similar in Araucaria and Pinus In addition A angustifolia resin may be a relevantsource of valuable terpene chemicals whose production may be increased by usingstimulating pastes containing the identified adjuvants
INTRODUCTION
Many conifer species produce terpenoid-based resins that havelong been studied for their industrial importance and role indefence against attack by herbivores and pathogens The twomost important resin-producing families of conifers are Pina-ceae and Araucariaceae (Langenheim 1996) The viscous resinsecretion is generally composed of a complex mixture ofterpenoids consisting of roughly equal parts of volatile mono-(C10) and sesquiterpene (C15 turpentine) fractions and non-volatile diterpenic (C20 rosin) components (Rodrigues-Correaet al 2013) Terpenes act in a complex and multilayereddefence response providing toxicity against bark beetles andfungi bark wound sealing disruption of insect developmentand attraction of herbivore predators (Phillips amp Croteau1999)Most conifers rely on some combination of preformed and
inducible resin defences (Trapp amp Croteau 2001 Zulak amp Bohl-mann 2010) Resin defences are controlled by environmentaland genetic factors to various extents depending on species(Roberds et al 2003 Sampedro et al 2010 Moreira et al2013) Resin traits have been reported as highly variable havingmoderate heritability indicating that breeding efforts towardssuper-resinous forests are promising (Tadasse et al 2001Roberds et al 2003) Several chemicals are known as stimulantsof resin production Commercial extraction of resin from pine
trees uses periodic bark streaking and application of resin stim-ulant pastes to the wound
Resin-stimulant paste based on sulphuric acid (SuA) iswidely used for the commercial production of pine resin Cur-rent stimulant pastes usually have two chemically active com-ponents SuA to magnify the wounding and an ethyleneprecursor (2-chloroethylphosphonic acid CEPA or Ethrel ndash
ET) to stimulate resin flow (Rodrigues et al 2011 Rodrigues-Correa amp Fett-Neto 2013) Jasmonic acid (JA) and its methylester methyl jasmonate (MeJa) are among the most widelyused chemical elicitors of plant secondary metabolism It hasbeen shown that the exogenous application of MeJa or herbi-vore attack induce chemical and anatomical defence responsesin conifers such as the formation of traumatic resin ducts andresin accumulation in stems along with increased biosynthesisof terpenes and phenolics (Franceschi et al 2002 Martin et al2002 Heijari et al 2005 Zeneli et al 2006 Moreira et al 2008Gould et al 2009) JA commercial use however is limited byits high cost
The effects of exogenous salicylic acid (SA) on conifer ter-pene production have also been studied In Pinus elliottiiapplication of 10 molm3 of SA induced resin productionin wound panels but in Pseudotsuga menziesii and Sequoia-dendron giganteum it had no apparent effect on resinaccumulation (Hudgins amp Franceschi 2004 Rodrigues ampFett-Neto 2009) Nitric oxide (NO) has also emerged as an
Plant Biology 17 (2015) 852ndash859 copy 2014 German Botanical Society and The Royal Botanical Society of the Netherlands852
Plant Biology ISSN 1435-8603
1
Introduccedilatildeo
Na condiccedilatildeo de organismos seacutesseis ao longo de sua evoluccedilatildeo as plantas
desenvolveram estrateacutegias estruturais e quiacutemicas de defesa em resposta aos estresses bioacuteticos
e abioacuteticos impostos pelo ambiente Dentre essas satildeo reconhecidas moleacuteculas quimicamente
especializadas denominadas metaboacutelitos secundaacuterios produtos naturais (Kutchan et al 2015)
ou mais recentemente metaboacutelitos especializados
Entre as trecircs classes mais gerais de metaboacutelitos secundaacuterios (terpenos compostos
fenoacutelicos e compostos nitrogenados) aminoaacutecidos natildeo-proteicos (ANPs) satildeo incluiacutedos no
terceiro grupo e constituem aleacutem de componentes do arsenal de defesa quiacutemica uma
importante fonte de reserva de carbono e nitrogecircnio para diversos taxa especialmente aqueles
pertencentes agrave famiacutelia Fabaceae de Angiospermas (leguminosas sensu lato)
Aleacutem dos 20 aminoaacutecidos proteicos estima-se que existam entre 600 e 1000 ANPs
(Acamovic amp Brooker 2005 Rodgers et al 2015) Esse grupo de moleacuteculas quimicamente
heterogecircneo eacute assim definido por natildeo participar da formaccedilatildeo de estruturas proteicas
funcionais sendo frequentemente toacutexicos quando erroneamente incorporados nas cadeias
polipeptiacutedicas em formaccedilatildeo em funccedilatildeo da similaridade estrutural que apresentam com os
aminoaacutecidos proteicos (Taiz amp Zeiger 2010)
Conforme mencionado a ocorrecircncia de ANPs eacute comum em espeacutecies de leguminosas
e sua distribuiccedilatildeo pode ser restrita a alguns gecircneros de plantas circunscritos nessa famiacutelia
botacircnica (eg mimosina e canavanina) Por outro lado alguns ANPs como GABA por
exemplo podem apresentar distribuiccedilatildeo ubiacutequa no Reino Plantae assim como ocorrer em
outros tipos de organismos como animais por exemplo (Ramos-Ruiz et al 2018)
2
Apesar de representarem uma fonte nutricional importante sem tratamento preacutevio o
consumo de plantas que acumulam ANPs por animais eacute limitado Isso ocorre pois em longo
prazo a ingestatildeo prolongada de plantas (especialmente sementes) que acumulam ANPs pode
representar risco agrave sauacutede uma vez que estes comprometem o funcionamento de mecanismos
basais de manutenccedilatildeo da homeostase celular e podem tambeacutem em um quadro mais severo
desencadear doenccedilas neurotoacutexicas degenerativas como por exemplo o latirismo causado
por aacutecido β-N-oxalil-l-αβ-diaminopropiocircnico (β-ODAP) (Jiao et al 2011 Kusama-Eguchi
2019)
Sob o ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados
ANPs satildeo em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou
abioacutetica como o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de
elicitores quiacutemicos por exemplo No que concerne ao estudo dos efeitos da induccedilatildeo abioacutetica
sobre o acuacutemulo de ANPs em diferentes espeacutecies vegetais (Monocotiledocircneas e
Eudicotiledocircneas) as moleacuteculas mais amplamente investigadas ateacute o momento satildeo GABA
L-DOPA e mais recentemente mimosina (vide Tabela 1 do capiacutetulo primeiro) Em termos
de efeitos causados a partir da aplicaccedilatildeo exoacutegena de ANPs GABA tambeacutem figura como o
principal aminoaacutecido investigado seguido de L-DOPA e canavanina (vide Tabela 2 do
capiacutetulo primeiro)
No primeiro capiacutetulo da presente tese estatildeo descritas as caracteriacutesticas gerais dos
principais ANPs estudados seus possiacuteveis papeacuteis bioloacutegicos in planta e seus efeitos quando
aplicados exogenamente bem como os estresses abioacuteticos capazes de induzir seu(s)
acuacutemulo(s) nos diferentes tecidos vegetais Nos segundo e terceiro capiacutetulos
respectivamente satildeo elucidados os efeitos dos tratamentos de UV-C aacutecido saliciacutelico etileno
e jasmonato (claacutessicos elicitores do metabolismo secundaacuterio vegetal) sobre o acuacutemulo de
3
mimosina em Leucaena leucocephala var glabrata (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute)
Mimosina eacute um aminoaacutecido aromaacutetico natildeo-proteico anaacutelogo da L-tirosina com
atividade toacutexica para ceacutelulas de procariotos e eucariotos Embora em menor concentraccedilatildeo
mimosina foi primeiramente identificada em Mimosa pudica sendo posteriormente detectada
em outras espeacutecies do gecircnero como Mimosa pigra por exemplo (Soedarjo amp Borthakur
1998) Seu efeito toacutexico eacute atribuiacutedo agrave capacidade de quelar metais o que impede o
funcionamento adequado das metalo-proteiacutenas que dependem dos mesmos como co-fatores
(Negi et al 2014)
A concentraccedilatildeo basal de mimosina em espeacutecies de leucaena pode variar entre 1 e 12
do peso seco do oacutergatildeo (Soedarjo amp Borthakur 1998) Como eacute comum para outros ANPs
que ocorrem em espeacutecies de leguminosas em sementes de Leucaena spp eacute observada uma
maior concentraccedilatildeo de mimosina quando comparada aos demais oacutergatildeos da planta
(Rodrigues-Correcirca et al 2019) sendo esta a fonte de extraccedilatildeo comercial do padratildeo quiacutemico
de mimosina vendido por empresas de reagentes de pesquisa
Diversas atividades foram descritas para mimosina em outros organismos eou tipos
celulares Dentre essas destacam-se a atividade anti-mitoacutetica ou bloqueadora do ciclo
celular em ceacutelulas de eucariotos e procariotos Isto ocorre porque a mimosina impede a
formaccedilatildeo da forquilha de replicaccedilatildeo (e portanto a siacutentese de DNA) interrompendo assim o
avanccedilo do ciclo de divisatildeo celular na fase tardia G1 (Lalande 1990) Foram tambeacutem descritas
para mimosina atividade alelopaacutetica observada sobre o desenvolvimento de outras espeacutecies
de leguminosas e atividade antioxidante entre outras (Tabela 1)
A rota de biossiacutentese de mimosina eacute compartilhada em grande parte com a de cisteiacutena
um aminoaacutecido proteico sulfurado (Figura 1) A siacutentese da cisteiacutena se daacute a partir da conversatildeo
4
de serina e acetil-CoA em o-acetilserina pela enzima SAT (serina acetiltransferase) seguida
da conversatildeo de o-acetilserina e aacutecido sulfiacutedrico em cisteiacutena em uma reaccedilatildeo catalisada pela
OAS-TL (o-acetilserina tiol-liase) A siacutentese de mimosina por sua vez eacute compartilhada com
a da cisteiacutena ateacute esse ponto e acredita-se que pelo menos uma das isoformas de OAS-TL
catalise a conversatildeo de o-acetilserina e 3-hidroxi-4-piridona em mimosina
Tabela 1 Atividades descritas para mimosina de Leucaena leucocephala (Lam) de Wit
ATIVIDADE
ALVO AVALIADO
(organismo eou tecido tipo
celular)
REFEREcircNCIA
Bloqueio do complexo de ativaccedilatildeo
da preacute-replicaccedilatildeo do DNA
Ceacutelulas de mamiacuteferos
KUBOTA et al
(2014)
Alteraccedilatildeo no ciclo ovariano e
extensatildeo da duraccedilatildeo do corpo luacuteteo
bovino no periacuteodo poacutes-parto
Bovinos
(Bos taurus x
Bos indicus)
BOTTINI-
LUZARDO et al
(2015)
Supressatildeo do ciclo celular e reduccedilatildeo
da abundacircncia bacteriana em
mosquitos
Wolbachia pipientis
Aedes albopictus
FALLON
(2015)
Accedilatildeo inibitoacuteria da fibrose
pulmonar induzida
Ratos SD
LI et al
(2015)
Recuperaccedilatildeo da funccedilatildeo do
miocaacuterdio poacutes-isquemia
Miocaacuterdio de ratos (SD)
machos
CROWE et al
(2001)
Inseticida
Heteropsylla cubana
Crawford 1914 e Thrips tabaci
Lindemann 1889
AHMED et al
(2016)
Alelopaacutetica
Albizia procera Vigna
unguiculata Cicer arietinum
Cajanus cajan
AHMED et al
(2008)
Antioxidante
Sistemas modelo de oxidaccedilatildeo
lipiacutedica (β-caroteno - aacutecido
linolecircico e lecitina)
BENJAKUL et al
(2013)
Ateacute momento versotildees divergentes sobre a enzima responsaacutevel pela biossiacutentese de
mimosina (mimosina sintase) tecircm sido publicadas Em 1990 Ikegami e colaboradores
5
identificaram uma OAS-TL responsaacutevel pela formaccedilatildeo de cisteiacutena como sendo tambeacutem uma
mimosina sintase Mais tarde Yafuso et al (2014) realizaram a expressatildeo heteroacuteloga do gene
que codifica para OAS-TL em Escherichia coli e natildeo foi observada a formaccedilatildeo de mimosina
mesmo quando dadas as condiccedilotildees oacutetimas para tanto Mais recentemente Harun-Ur-Rashid
et al (2018) elucidaram a mimosina sintase como sendo uma isoforma da OAS-TL
corroborando o postulado por Ikegami e colaboradores em 1990
Figura 1 Rota de biossiacutentese da mimosina Fonte Ikegami et al (1990)
Espeacutecies estudadas
Leucaena leucocephala (Lam) de Wit (leucaena koa haole ou ldquoacaacutecia exoacuteticardquo na
liacutengua Hawairsquoiana) eacute uma espeacutecie de haacutebito arboacutereo ou arbustivo pertencente agrave famiacutelia
Fabaceae de Angiospermas e caracterizada pelo acuacutemulo de mimosina em todos os seus
oacutergatildeos Eacute nativa da Ameacuterica Central (especificamente da regiatildeo sudeste do Meacutexico) mas
irradiou-se atraveacutes de praticamente todas as zonas tropicais e subtropicais da Terra No
Brasil leucena eacute amplamente distribuiacuteda e classificada como naturalizada pelo REFLORA
(2019) ocorrendo em todo territoacuterio Nacional Satildeo reconhecidas no miacutenimo duas
6
subespeacutecies de leucena ocorrentes no Brasil L leucocephala var leucocephala e L
leucocephala var glabrata sendo a primeira a mais abundante
Leucaena apresenta atributos morfoloacutegicos caracteriacutesticos das leguminosas como o
fruto do tipo vagem deiscente no periacuteodo poacutes-maturaccedilatildeo folhas compostas e bipinadas As
flores satildeo seacutesseis actinomorfas e polistecircmones apresentam caacutelice sinseacutepala e corola
gamopeacutetala e satildeo dispostas em inflorescecircncias do tipo glomeacuterulo (Figura 2)
Figura 2 Oacutergatildeos vegetativos e reprodutivos de L leucocephala (Lam) de Wit Fonte Little Jr amp Skolmen
(1989)
Com base no conhecimento etnobotacircnico disponiacutevel acerca dessa espeacutecie em
diversas regiotildees tropicais e subtropicais leucena eacute utilizada para vaacuterios fins Extratos de
diferentes oacutergatildeos de leucena apresentam atividade anti-diabeacutetica (Kuppusamy et al 2014
Chowtivannakul et al 2016) antioxidante (Mohammed et al 2015 Chowtivannakul et al
2016 Zarin et al 2016) antimicrobiana (Zarin et al 2016) anti-helmiacutentica (Soares et al
2015 Jamous et al 2017) bactericida (Mohammed et al 2015) acaricida (Fernaacutendez-Salas
et al 2011) anti-tumoral (Chung et al 2017) e potencializadora da resposta imune em
peixes (Verma et al 2018) entre outras
7
Leucaena apresenta alta toleracircncia agrave seca sendo capaz de enfrentar estaccedilotildees sazonais
inteiras com deacuteficit hiacutedrico sem prejuiacutezo permanente de seus oacutergatildeos e de recuperar
vigorosamente sua biomassa vegetativa tatildeo logo o regime de precipitaccedilatildeo retome a
regularidade em frequecircncia Acredita-se que a toleracircncia agrave seca apresentada por essa espeacutecie
ocorra em funccedilatildeo do acuacutemulo de mimosina nos diferentes tecidos da planta a qual
funcionaria como um agente osmoregulador responsaacutevel pela preservaccedilatildeo da integridade das
membranas a das macromoleacuteculas intracelulares em periacuteodos de escassez de aacutegua no
ambiente
Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) eacute uma leguminosa
nativa natildeo endecircmica do Brasil amplamente distribuiacuteda nos domiacutenios fitogeograacuteficos da
Caatinga do Cerrado e da Mata Atlacircntica (Simon amp Proenccedila 2000 REFLORA 2019) Como
espeacutecie pioneira (Pilatti et al 2019) exerce importante papel ecoloacutegico na recuperaccedilatildeo de
aacutereas degradadas (Bitencourt et al 2007 Silva et al 2011) no estabelecimento de processos
de sucessatildeo vegetacional
Maricaacute eacute uma espeacutecie semi-deciacutedua a deciacutedua a qual atinge ateacute 15 m em altura (e
diacircmetro agrave altura do peito de ateacute 40 cm) na idade adulta com haacutebito arboacutereo ou arbustivo
(REFLORA 2019) e espinhos caracteriacutesticos desde os estaacutegios iniciais de desenvolvimento
(Carvalho 2004) Apresenta folhas compostas alternas e bipinadas (Figura 2) amplas
inflorescecircncias brancas com flores reunidas em glomeacuterulos esfeacutericos dispostos em grandes
paniacuteculas As flores satildeo diplostecircmones actinomorfas hipoacuteginas e unicarpelares (Silva et al
2011)
Assim como descrito para leucena maricaacute eacute considerado uma espeacutecie multifuncional
sendo comumente empregada para produccedilatildeo de mel como combustiacutevel (Olkoski amp
8
Wittmann 2011) em edificaccedilotildees na carpintaria e como lsquocerca-vivarsquo (Marchiori 1993
Lorenzi 1998) entre outras aplicaccedilotildees
Figura 2 Folhas e fruto de Mimosa bimucronata (DC) Kuntze Fonte Souza-Lima et al (2017)
Em contraste com a amplitude de habitats explorados por leucena (especialmente os
aacuteridos) no Sul do Brasil maricaacute ocorre preferencialmente em ambientes uacutemidos a alagadiccedilos
em aacutereas proacuteximas agraves margens de rios (Patreze amp Cordeiro 2004) embora possa tambeacutem
ocorrer em formaccedilotildees quase exclusivas dessa espeacutecie nas encostas de morros (Jacobi amp
Ferreira 1991)
Em relaccedilatildeo agraves atividades elucidadas para os extratos de maricaacute foram relatados os
efeitos alelopaacutetico (Jacobi amp Ferreira 1991 Ferreira et al 1992) diureacutetico natriureacutetico e
caliureacutetico (Schlickmann et al 2017)
9
Hipoacutetese
Mimosina apresenta perfil dinacircmico de acuacutemulo em Leucaena leucocephala e
Mimosa bimucronata frente a estresses associado a alteraccedilotildees significativas na expressatildeo de
genes relacionados ao metabolismo deste ANP o qual contribui para mitigar o desequiliacutebrio
oxidativo inerente a vaacuterios tipos de estresse
Objetivo geral
O objetivo da presente tese foi investigar o papel bioloacutegico da mimosina endoacutegena
em leucena e maricaacute a partir da avaliaccedilatildeo do efeito de tratamentos relacionados a estresses
ou sinalizadores de estresse
Objetivos especiacuteficos
- Analisar a concentraccedilatildeo constitutiva de mimosina nos diferentes oacutergatildeos de L leucocephala
(Lam) de Wit (leucena) e M bimucronata (DC) Kuntze (maricaacute)
- Verificar se apesar do seu alto teor constitutivo em plantas de leucena o acuacutemulo de
mimosina pode ser induzido com tratamentos que mimetizam diferentes estresses a partir da
avaliaccedilatildeo do efeito de moleacuteculas sinalizadoras (aacutecido saliciacutelico jasmonato etileno) e da
exposiccedilatildeo agrave radiaccedilatildeo UV-C na modulaccedilatildeo do acuacutemulo de mimosina em leucena bem como
em maricaacute
- Determinar se a expressatildeo de genes relacionados ao metabolismo de mimosina estaacute
associada agrave induccedilatildeo por estresses fisioloacutegicos
- Avaliar o potencial antioxidante da mimosina em experimentos realizados in situ
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry
journal homepage wwwelseviercomlocateplaphy
Research article
Mimosine accumulation in Leucaena leucocephala in response to stresssignaling molecules and acute UV exposure
Kelly Cristine da Silva Rodrigues-Correcircaab Michael DH Hondab Dulal BorthakurbArthur Germano Fett-Netoalowast
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970Porto Alegre Rio Grande do Sul BrazilbDepartment of Molecular Biosciences and Bioengineering University of Hawaii at Manoa Honolulu HI 96822 USA
A R T I C L E I N F O
KeywordsLeucaena leucocephalaMimosineMimosine amidohydrolaseJasmonic acidEthyleneSalicylic acidUV-C radiation
A B S T R A C T
Mimosine is a non-protein amino acid of Fabaceae such as Leucaena spp and Mimosa spp Several relevantbiological activities have been described for this molecule including cell cycle blocker anticancer antifungalantimicrobial herbivore deterrent and allelopathic activities raising increased economic interest in its pro-duction In addition information on mimosine dynamics in planta remains limited In order to address this topicand propose strategies to increase mimosine production aiming at economic uses the effects of several stress-related elicitors of secondary metabolism and UV acute exposure were examined on mimosine accumulation ingrowth room-cultivated seedlings of Leucaena leucocephala spp glabrata Mimosine concentration was not sig-nificantly affected by 10 ppm salicylic acid (SA) treatment but increased in roots and shoots of seedlings treatedwith 84 ppm jasmonic acid (JA) and 10 ppm Ethephon (an ethylene-releasing compound) and in shoots treatedwith UV-C radiation Quantification of mimosine amidohydrolase (mimosinase) gene expression showed thatethephon yielded variable effect over time whereas JA and UV-C did not show significant impact Consideringthe strong induction of mimosine accumulation by acute UV-C exposure additional in situ ROS localization aswell as in vitro antioxidant assays were performed suggesting that akin to several secondary metabolitesmimosine may be involved in general oxidative stress modulation acting as a hydrogen peroxide and superoxideanion quencher
1 Introduction
Different plant groups synthesize a large diversity of secondary orspecialized metabolites These molecules are generally produced inresponse to biotic and abiotic environmental stresses Indeed inductionof secondary metabolism usually involves stress-generating factorswhich have also been explored in biotechnological processes aiming atthe production of target metabolites of economic interest (Matsuuraet al 2018) Metabolic control of nitrogen-containing secondarycompounds (eg alkaloids and non-protein amino acids) has beenshown to be complex and influenced by phytohormones environmentalstresses (seasonality herbivory pathogen attack drought) UV radia-tion (Holloacutesy 2002) methyl jasmonate (MeJA) salicylic acid (SA)yeast extract (Cho et al 2008) abscisic acid (ABA) heavy metals os-motic stress (Nascimento et al 2013) and mechanical wounding (Portoet al 2014)
Due to their particular trait of associating with N-fixing micro-organisms Fabaceae species (leguminous sensu lato) are often proteinrich hence the relevance of several of these species as forage Fabaceaespecies are also known for accumulating nitrogen containing secondarymetabolites which play important roles as ecochemical molecules andat least for the case of non-protein amino acids potential cell reservoirsof nitrogen (Huang et al 2011)
High contents of mimosine a toxic aromatic non-protein aminoacid are found in species of two leguminous genera Leucaena spp andMimosa spp Leucaena leucocephala (Lam) de Wit (leucaena koa haole)is a fast-growing leguminous tree native from Central America (south-eastern Mexico) widely distributed in tropical and subtropical zonesThis species is also characterized by its high tolerance to droughtamong other environmental stresses (Honda et al 2018) Leucaena canbe divided into two subspecies (i) L leucocephala subsp leucocephala(common leucaena a bushy shrub) and (ii) L leucocephala subsp
httpsdoiorg101016jplaphy201811018Received 1 August 2018 Received in revised form 9 November 2018 Accepted 14 November 2018
lowast Corresponding authorE-mail addresses krodriguescbiotufrgsbr (KCdS Rodrigues-Correcirca) mhonda2hawaiiedu (MDH Honda) dulalhawaiiedu (D Borthakur)
fettnetocbiotufrgsbr (AG Fett-Neto)
Plant Physiology and Biochemistry 135 (2019) 432ndash440
Available online 19 November 20180981-9428 copy 2018 Elsevier Masson SAS All rights reserved
T
glabrata (giant leucaena a tree) The latter has been used as a fastgrowing tree for production of wood and paper pulp The foliage ofboth common and giant leucaena is used as a fodder because of its highprotein content and palatability to farm animals The foliage containsup to 18 protein 142 crude fiber and 64 ether extractcrude fat(Soedarjo and Borthakur 1996)
Production of nitrogen-containing secondary metabolites such asmimosine requires large amounts of carbon and nitrogen resourcesNegi et al (2014) estimated that up to 21 of the carbon-nitrogenresources may be used for production of mimosine in leucaenaBrewbaker et al (1972) determined the mimosine content of 96 Lleucocephala cultivars and 8 other Leucaena species collected from 38different countries by growing them in an observational nursery inHawaii and found that basal mimosine content varied from 189 to477 of the dry weight
Mimosine is biosynthesized from OAS (o-acetylserine) and 3H4P (3-hydroxy-4-pyridone or its tautoisomer 3-hydroxy-4-pyridine) A pre-vious analysis suggested that mimosine synthase is an OAS-TL (o-acetylserine-thiol-lyase) of the cysteine biosynthesis pathway (Ikegamiet al 1990) Later however recombinant enzyme tests did not supportan OAS-TL identity of mimosine synthase (Yafuso et al 2014) Recentfindings on mimosine biosynthesis revealed that a cytosolic cysteine-OAS-TL isoform can also catalyze the formation of mimosine underspecific conditions (Harun-Ur-Rashid et al 2018)
Mimosine toxicity is related to its ability of reducing the availabilityof divalent metal ions such as Fe(II) Zn(II) Cu(II) Co(II) and Mn(II)by chelating co-factors and preventing their association with metal-dependent enzymes Furthermore this non-protein amino acid is cap-able of forming a stable complex with pyridoxal-5prime-phosphate (PLP)leading to the inactivation of PLP-dependent enzymes (eg Asp-Glutransaminase and cystathionine synthetase) (Negi et al 2014)
Mimosine features several useful biological activities such as alle-lopathic antimicrobial insecticide cell cycle inhibitor agent antic-ancer phytoremediator (Nguyen and Tawata 2016) as well as anti-oxidant (Benjakul et al 2013) Despite the relatively well establishedbiological activities of purified mimosine on other organisms or celltypes little is known about its biological role in leguminous speciesHowever it has been suggested that at least in part its activity ismainly related to defense mechanisms against some biotic and abioticstresses and as nitrogen source during fast growth (Vestena et al2001)
Suda (1960) and Smith and Fowden (1966) identified enzymes in-volved in mimosine degradation in seedling extracts of L leucocephalaand Mimosa pudica A mimosine-degrading enzyme named mimosinase(mimosine amidohydrolase EC 35161 CAS registry number 104118-49-2) (IUBMB 2018) a carbon-nitrogen lyase which degrades mimo-sine into 3H4P was later purified by Tangendjaja et al (1986) Itsbiochemical characterization was described and the cDNA was isolatedby Negi et al (2014)
Although mimosinase has been described and isolated only fewstudies on the role played by biotic and abiotic factors on the dynamicmodulation of mimosine metabolism in leguminous species have beenconducted (Vestena et al 2001 Xu et al 2018) In aseptic cultures ofleucaena mechanical injury of shoots promoted local mimosine accu-mulation (Vestena et al 2001) In the same study cultivation in pre-sence of auxin or SA in culture medium also had a positive effect on
mimosine accumulation More recently the effect of drought treatmenton gene expression of leucaena was also evaluated by Honda et al(2018) However several potential factors regulating mimosine meta-bolism need to be further examined
To date there is a lack of information on the biological role ofmimosine in planta as well as on details of its metabolic dynamicsMoreover its overt potential for pharmaceutical applications and de-velopment of new drugs as well as the possible use as tool to addressheavy metal soil contamination or plant mineral nutrition improve-ment justify additional research The objective of this study was toinvestigate the effect of stress signaling molecules and acute UV ex-posure on modulation of mimosine accumulation and metabolism in Lleucocephala spp glabrata in order to better understand its biologicalrole and to identify strategies for yield improvement aiming at ex-ploring its useful bioactivities
2 Methods
21 Plant material
For the experiments carried out to evaluate the effects of elicitors onmimosine accumulation seeds of leucaena were kindly provided by DrJames Brewbaker and harvested at CTAHRs (College of TropicalAgriculture and Human Resources of the University of Hawaii atManoa) Waimanalo Research Station at Oahu Hawaii This plantmaterial was originated from the accession K636 of Leucaena leucoce-phala ssp glabrata (Brewbaker 2008)
22 Induced mimosine content in 5-week-old giant leucaena
221 Seed germinationIn order to overcome seed coat dormancy seeds were submitted to a
chemical scarification with sulfuric acid 95ndash98 for 20min and re-peatedly rinsed in distilled water to remove any residual trace of thisreagent Then seeds were distributed in 254 cmtimes508 cm plastictrays containing 11 vv of vermiculite and commercial soil watereduntil reaching substrate field capacity Three weeks after seed imbibi-tion seedlings displaying similar size and shape (eg number of com-pound leaves and leaflets) were transplanted to individual pots(250mL) in number of three plants per container
During the experimental period (except in the UV-C radiationtreatment) all tested seedlings were kept in a growth chamber andsubmitted to controlled conditions of temperature (circa 25 degC) and ir-radiance (approximately 100 μmol photons mminus2sdot s minus1) with a photo-period of 16 h light and 8 h dark
222 Treatments2221 JA Ethephon and SA Five-week-old giant leucaena seedlingswere treated with different solutions as described in Table 1 Idealconcentrations were defined in preliminary experiments under the sameconditions indicated above At the beginning of the experiments 30plants were sprayed with 84 ppm JA 10 ppm SA 10 or 100 ppmEthephon or Milli-Qreg water (control) until the point of imminent runoffPlant pots were kept closed inside transparent plastic bags for 24 h toavoid solution volatilization Fifteen plants arranged in 5 sets of 3 (5biological replicates) were harvested 48 h and 96 h after being treated
Table 1Treatments used to modulate mimosine biosynthesis in giant leucaena
ELICITOR CONCENTRATION UV FLUENCE EXPOSURE TIME RATIONALE FOR USE
Salicylic acid (SA) 10 ppm 24 h Pathogen signaling molecule (Shah 2003)Jasmonic acid (JA) 84 ppm 24 h Chemical elicitor of plant secondary metabolism (Dar et al 2015)Ethephon 10 ppm 24 h Ethylene releasing-compound (Kim et al 2016) elicitor of plant secondary metabolism (Wang
et al 2016)UV-C radiation 3 Jcmminus2 10min or 15min Elicitor of plant secondary metabolism (Kara 2013 Neelamegam and Sutha 2015)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
433
After collection shoots were separated from roots immediately frozenin liquid nitrogen and stored at ndash 80 degC prior to HPLC analyses
2222 UV-C Thirty seedlings of giant leucaena were exposed to UV-Cradiation (3 Jcmminus2) for 10 or 15min and kept in a growth chamberunder controlled conditions as described above until the end of theexperiments Fifteen plants arranged in groups of 3 were harvested at96 h and 120 h after UV-C exposure and processed as previouslydescribed
223 Mimosine extractionMimosine extraction was based on a modified version of the pro-
tocol published by Lalitha and Kulothungan (2006) as follows a knownweight of fresh tissue (shoots or roots) of giant leucaena was first addedto Milli-Qreg boiling water in a proportion of 110 (g of plant per mL ofsolvent) in test tubes Tubes were covered with foil to avoid solutionevaporation and placed on a hot stirrer at 100 degC for 10min A pro-portional volume of 01M HCl was added to the cooled suspensions andhomogenized using mortar and pestle The plant extracts were filteredthrough cotton and centrifuged twice for 7min in a bench top re-frigerated centrifuge at 4 degC and 13200 rpm Before being analyzed theextracts were diluted 13 with ondashphosphoric acid (OPA)
224 Mimosine detectionHPLC analyses were carried out as described by Negi and Borthakur
(2016) Pure mimosine (L-mimosine from koa haole seeds Sigma-Al-drich CAS number 500-44-7) was used as standard Separation andquantification of mimosine was done with a C18 column (PhenomenexC18 5 μm 46times250mm) under an isocratic solvent system of 002MOPA with a linear flow rate of 1mLsdotminminus1 Mimosine detection wasdone at 280 nm by photodiode array detection (200ndash400 nm) showingretention time of 277 plusmn 0042min Quantification was done using themethod of external standard curve Further confirmation of mimosineidentity was performed by co-chromatography with standard and peakpurity check Chromatograms were analyzed using the Waters Em-power 3 software
23 Quantitative real-time PCR analysis of mimosinase gene expression
Fifteen 8-week-old giant leucaena plants arranged in 4 sets of 3 (4biological replicates) were treated with either water (control) or10 ppm Ethephon 84 ppm JA acid or 15min of UV-C radiation ex-posure following the methods described above Following treatmentleucaena plants were harvested at 48 and 96 h or 72 and 144 h (UV-Ctreated plants only) after treatments Total RNA of samples was ex-tracted and purified from roots and shoots of giant leucaena by meansof a modified method using Qiagen RNeasy Plant Kit (Valencia CAUSA) and Fruit-mate (Takara Japan) according to the protocol de-scribed by Ishihara et al (2016a) The assessment of RNA quality andquantity was carried out at 230 260 and 280 nm by using a NanoDropSpectrophotometer ND-1000 (NanoDrop Technologies DE USA) Inorder to avoid genomic DNA contamination RNA samples were treatedwith TURBO DNAfree Kit (Invitrogen Carlsbad CA) Two microgramsof DNase-treated RNA were used to synthesize the first-strand cDNAusing M-MLV Reverse Transcriptase (Promega WI USA)
Quantitative real-time (qPCR) analysis was carried out to examinepossible differential expression of the mimosinase gene (GenBank ac-cession number AB2985971) in seedlings treated with 84 ppm JA10mM Ethephon or 15min of UV-C exposure Shoots and roots wereharvested 24 h before the time of mimosine concentration peak for eachtreatment previously observed as assessed by HPLC assays The 10 μLqPCR reaction consisted of 5 μL of PowerUpTM SYBRreg Green MasterMix (Applied Biosystems Foster City CA) 1 μL MgCl2 (50mM) 03 μLforward primer (10 μM) 03 μL reverse primer (10 μM) and 1 μL cDNAfirst-strand In the experimental validation through qPCR reactionconditions and melting curve analysis of the amplicon were performed
following the protocol published by Ishihara et al (2016b) for the sameleucaena variety qPCR analysis was conducted using StepOnetrade Real-Time PCR System (Applied Biosystems) Measurements were performedusing 4 biological and 3 technical replicates Relative expression wascalculated with the 2-ΔΔct method using OAS-TL as reference gene sinceits expression showed a consistently stable profile comparable to that ofUBQ-5 and ELF1α expressions Mimosinase primer sequences used forthese analyses were (FWD) 5prime- GAA AGG CAG GAA TCA CAG TGA AGAG ndash 3rsquo (REV) 5prime GGA GAC TCT AGC CAC ACC AAC TTA ndash 3rsquo
24 Antioxidant assays
241 Mimosine effect on hydrogen peroxide (H2O2) accumulationAs a follow up to the induction of mimosine accumulation profiles
under stress signals and conditions tests were conducted to verify mi-mosine antioxidant capacity In situ histological localization of hy-drogen peroxide (H2O2) accumulation was evaluated on foliar disks ofPhaseolus vulgaris L according to the protocol described by Shi et al(2010) Briefly the plant foliar tissue was exposed to 1 mgmiddotmLminus1 dia-minobenzidine (DAB) solution in 10 mM KH2PO4 (control) in presenceor absence of 10mM mimosine (equivalent to the average mimosineconcentration induced by UV-C radiation in giant leucaena) or 10mMascorbic acid (positive antioxidant control) Oxidative response wasidentified by the formation of a brown polymer on the injured leafareas indicating the presence of H2O2 and registered in a Leica M165FC stereomicroscope (Leica Microsystems)
242 Mimosine quenching of superoxide radicalsGeneration of superoxide radical and subsequent analysis was per-
formed by a modified protocol based on Zhishen et al (1999) Nitroblue tetrazolium (NBT) reduction was used to measure superoxide an-ions quenching activity Shortly a 50mM KH2PO4 pH 78 solutioncontaining 6 μM riboflavin 100mM methionine 1 mM NBT in pre-sence or absence of 5mM mimosine was exposed to white light(22 Jsdotcmminus2) for 25min on a white light transilluminator Five micro-molar rutin was used as positive control (Matsuura et al 2016) Theabsorbance was read at 560 nm before and after light exposure in aSpectraMaxreg M2 Microplate Reader (Molecular Devices LLC)
25 Statistical analyses
For HPLC and superoxide anions data simple analyses of variance(ANOVA) followed by Tukey or Welch ANOVA followed by Dunnetts Ctest were used as appropriate for data distribution characteristics InqPCR analysis results were analyzed by t-test In all cases at least fourbiological triplicates were used and experiments were repeated twiceindependently All data were analyzed using the statistical packageSPSS 200 for Windows (SPSS Inc USA) In all cases a ple 005 wasused
3 Results and discussion
31 Increased mimosine concentrations in giant leucaena treated withchemical elicitors
Leucaena produces high amounts of mimosine that accumulate in allparts of the plants including leaves stem flowers pods seeds rootsand root nodules (Soedarjo and Borthakur 1998) The highest con-centrations of mimosine can be found in the growing shoot tips andseeds (Wong and Devendra 1983) It is not known why leucaena pro-duces such high amounts of mimosine Negi et al (2014) estimated thatleucaena plants would be able to grow 21 larger if the nutrient re-sources spent on mimosine production were diverted for biomass in-crease In a previous analysis performed to quantify the basal con-centration of mimosine present in adult plants of common leucaena thehighest constitutive amount of mimosine per gram of fresh weight in
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
434
the analyzed organs was found in post-anthesis flowers (89448 μg)followed by green pods (82687 μg) leaves (67358 μg) and greenflower buds (51247 μg) which showed significantly less mimosineconcentration compared to the other reproductive structures(Supplementary Fig 1) Since mature seeds have very low moisturecontent (Wencomo et al 2017) its mimosine concentration was esti-mated as 338253 μgsdotgminus1 of dry weight Additionally it was also ob-served that the basal mimosine distribution in shoots of field-grownadult plants of leucaena is dependent on the variety type(Supplementary Table 1)
Phytohormones such as salicylic acid and jasmonic acid are knownto be produced by plants in response to various abiotic and bioticstresses These phytohormones trigger adaptive responses to stress byregulating major plant metabolic processes such as photosynthesisnitrogen metabolism defense systems and plant-water relationsthereby providing protection (for review see Khan et al 2015)
Secondary or specialized metabolite production and accumulationare also known to be controlled by biotic and abiotic stresses (Matsuuraet al 2018) In this study exposure of 5-week-old giant leucaenaseedlings to JA or Ethephon treatments significantly enhanced mimo-sine accumulation in shoots and roots in at least one of the two timepoints tested (48 and 96 h) albeit in a different way (Fig 1) Thehighest concentrations of mimosine in shoots were found in seedlingstreated with JA 84 ppm (43441 μgsdotgminus1) and Ethephon 100 ppm(38412 μgsdotgminus1) two days after application of the respective phyto-hormones Nevertheless after four days shoots yielded the highestconcentration of mimosine (approximately 460 μgsdotgminus1) upon treatmentwith 10 or 100 ppm Ethephon (Fig 1A) In roots after two and four
days JA 84 ppm and Ethephon 10 ppm resulted in highest mimosineaccumulation 18488 μgsdotgminus1 and 15801 μgsdotgminus1 respectively (Fig 1B)These observations show that mimosine accumulation response tospecific elicitors may vary over time after exposure
Although all treatments were applied exclusively on shoots of giantleucaena seedlings roots of some of them were also able to respond tothe different elicitors Overall shoots displayed higher basal and in-duced mimosine concentration compared to roots (Fig 1) which agreeswith previous observations in 1 to 3-week-old aseptic seedlings ofcommon leucaena (Vestena et al 2001) However as previouslymentioned significant post-induction increase of mimosine concentra-tion in roots and shoots simultaneously was only observed for JA andEthephon 10 ppm on day 02 and 04 respectively (Fig 1)
It is well established that perceived regulatory signals or elicitorsgenerate a transduction network mediated by secondary messengersresulting in changes in gene expression profiles that afford adaptiveresponses to environmental stimuli These modulation events are oftenmediated by transcription factors (TFs) which directly bind to specificgene promoters or act by forming complexes with repressor proteinslabeling them to degradation subsequently releasing other TFs toproceed with the gene expression program This is the case of the actionmechanism of JA and its active form jasmonoyl isoleucine for example(Kazan 2015 Wasternack and Strnad 2016)
JA ethylene and SA are known as important stress regulatory sig-nals in plants JA however is thought to be the most effective signal forinduction of plant secondary metabolism (Wasternack and Strnad2016) thereby contributing to mitigation of damage caused by severalstresses (Dar et al 2015) JA is mainly derived from linolenic acid
Fig 1 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings treated with differentelicitors CTRL=Milli-Q water SA = Salicylic AcidJA= Jasmonic Acid ETH=Ethephon Bars sharing a letterof same case do not differ by Tukey test (P le 005) Capitalletters (A B) compare treatments on day two and lowercaseletters (a b) compare treatments on day four Indicatessignificant statistical difference between day two and dayfour in the same treatment by t-test (Ple 005) The errorbars represent standard error of five replicates (each meanwas calculated with 15 individual seedlings organized in 5groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
435
(Wasternack and Strnad 2016) playing important roles in differentprocesses of plant growth and development such as plant defensemechanisms against herbivory pathogen attack fungal elicitation andsome abiotic factors such as osmotic temperature and salt stresses (Daret al 2015)
JA and its methyl ester MeJA have several different effects on le-guminous species MeJA exogenous application has increased iso-flavonoid content in cell suspension cultures of Pueraria candollei varcandollei and P candollei var mirifica (Korsangruang et al 2010) aswell as the production of the triterpenoid glycyrrhizin in Glycyrrhizaglabra roots Enhanced production of the triterpenoid however waspartly at the expense of root growth (Shabani et al 2009) MeJA ap-plication on shoots was observed to suppress root nodulation and lat-eral root formation in Lotus japonicus (Nakagawa and Kawaguchi2006) In grapevine a non-leguminous species proteinogenic aminoacids did not show an expressive increase under MeJA treatment(Gutieacuterrez-Gamboa et al 2017)
The effects of the application of four different jasmonate forms (JAMeJA jasmonoyl-L-isoleucine (JA-Ile) and 6-ethyl indanoyl glycineconjugate (2-[(6-ethyl-1-oxo-indane-4-carbonyl)-amino]-acetic acidmethyl ester - CGM) on leucaena metabolite profile has recently beenreported by Xu et al (2018) JA-Ile form was most effective althoughno major alteration was observed on monitored metabolite abundancesAlanine threonine and 34-dihydroxypyridine (34 DHP a metabolitederived from mimosine degradation) (Nguyen and Tawata 2016)among others were the major metabolites elicited by JA-Ile In contrastto the results described here mimosine concentration did not changesignificantly These divergent results on mimosine accumulation maybe due to a number of factors including mode of application jasmonateform used (JA-Ile x JA) and L leucocephala subspecies (common x giantleucaena)
Ethylene is also a phytohormone involved in plant response me-chanisms to different types of challenges such as mechanical damageand insect attack among others The integration mechanism betweenJA and ethylene signaling pathways is not completely understoodhowever it has been shown that they may work cooperatively in abioticstress tolerance (Kazan 2015) MeJA can induce ethylene production(Zhao et al 2004) and when applied simultaneously these moleculesseem to work in a synergic way by enhancing the magnitude of theplant response to external stimuli (Liu et al 2016)
Treatment with SA was able to significantly increase mimosine ac-cumulation in 12-week-old plants of common leucaena (SupplementaryFig 2) However no significant effect of SA treatment on mimosineconcentration was seen in 5-week-old seedlings of giant leucaena(Fig 1) suggesting some degree of genotype andor age dependency inelicitation by this phytohormone On the other hand several treat-ments including 90 ppm MeJA 10 and 100 ppm 2-chloroethylpho-sphonic acid (CEPA an ethylene-releasing compound) significantlyincreased mimosine accumulation (Supplementary Fig 2) in agree-ment with the data obtained for giant leucaena The lack of systemiceffects of externally applied SA on mimosine accumulation was alsoobserved when the phytohormone was supplied in the culture mediumof aseptically-grown seedlings in which case only roots had highercontent of mimosine (Vestena et al 2001) This could be due totransport limitations or to low methyl salicylate production from ap-plied SA since the former is recognized as the main systemic signalingform (Vlot et al 2009)
32 Increased mimosine concentrations in giant leucaena exposed to UV-Cradiation
UV-C treatment promoted increased concentration of the aminoacid in shoots but not in roots of giant leucaena (Fig 2) Increasedaccumulation of mimosine in shoots was also observed in 12-week-oldseedlings of common leucaena exposed to UV-C radiation for 10 and15min (Supplementary Fig 3) Similar to the SA treatment in giant
leucaena UV-C radiation did not induce mimosine biosynthesis in rootsregardless of time after exposure The absence of mimosine induction inroots by SA and UV indicates that these effectors do not cause a sys-temic response Moreover roots are shielded from irradiance by thepresence of substrate
UV radiation effects on different aspects of plant metabolism anddevelopment have been described However compared to UV-B (en-vironmentally relevant type of UV radiation) assays there are less re-ports related to the UV-C effects on secondary metabolites biosynthesisand accumulation (Cetin 2014) especially in leguminous (Fabaceae)plants They generally concern primary metabolism aspects such asgrowth and development For instance seedlings of Phaseolus vulgaris L(Fabaceae) exposed to low intensity UV-C radiation have displayeddecreased chlorophyll content and reduced height after 14 days of ex-posure (Kara 2013) Negative effects on growth parameters and ni-trogen metabolism were also observed in Vigna radiata L (Fabaceae)after UV-B radiation treatment in addition to adverse effects on JA SAand antioxidant compounds accumulation (Choudhary and Agrawal2014a) The same authors reported increased accumulation of flavo-noids SA and JA besides negative effects on growth biomass yieldnitrogen fixation and accumulation in 2 cultivars of Pisum sativum L(Fabaceae) under elevated UV-B treatment (Choudhary and Agrawal2014b) Despite the negative UV influence on growth reported for thepreviously mentioned leguminous UV-C radiation on groundnut plants(Arachis hypogaea L Fabaceae) increased seedling vigor and biomassand had no adverse effect on germination or other development para-meters (Neelamegam and Sutha 2015)
Besides its impact on growth and primary metabolism UV exposurecan cause important changes in secondary metabolism depending onintensity and time of exposure (Matsuura et al 2013) UV-B and UV-Cpre-treatments of Artemisia annua (Asteraceae) seedlings yielded in-creased biosynthesis of artemisinin a drug which displays anti-malarialproperties and activity against some others infectious diseases (egschistosomiasis leishmaniasis and hepatitis B) and several kinds oftumors (Rai et al 2011) The accumulation of nicotine in Nicotianarustica plants (Solanaceae) was also increased by UV-C treatment(Tiburcio et al 1985) Similar inducing effects on production of severalsecondary metabolites were observed in callus cultures of Vitis viniferaL Oumlkuumlzgoumlzuuml (grapevine Vitaceae) treated with a UV-C source for 5 or10min (Cetin 2014)
Regarding amino acid biosynthesis in response to UV radiationMartiacutenez-Luumlscher et al (2014) have found that in spite of not causingchanges in total amino acid content UV-B radiation exposure can affecttheir profile in grape berries Proteinogenic amino acids have beenknown to be important targets of the deleterious effects of UV radiation(Holloacutesy 2002) On the other hand in the present study acute UV-Ctreatment was found to increase mimosine accumulation in shoots byover twofold (Fig 2) which may suggest a possible participation of thismolecule as part of the antioxidant defense system in L leucocephalaThis possibility is further supported by the induction of the amino acidaccumulation by JA and Ethephon involved in abiotic and biotic stressresponses which are generally associated with oxidative imbalance andare signaling components in high UV stress (Matsuura et al 2013)
33 Mimosinase gene expression
In order to determine if increases in mimosine content upon ex-posure to JA CEPA or UV-C radiation were related to changes intranscription of mimosine metabolism-related genes RT-qPCR analysiswas carried out The complete pathway for mimosine biosynthesis hasnot yet been determined although the final step has been character-ized Based on transcription analysis (Ishihara et al 2016a) leucaenaappears to encode for multiple cysteine synthases one or more of whichmay be able to catalyze mimosine synthesis In addition a leucaenagene encoding a mimosinase (an enzyme responsible for mimosinedegradation) has been identified and characterized (Negi et al 2014)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
436
In addition to mimosinase gene expression several gene isoformsbelonging to the cysteine pathway [cysteine synthase (CYS SYN) serineacetyltransferase (SAT) and β-cyanoalanine synthase (CAS) Table 2 -supplementary material] were also tested in this study (data notshown) However expressions of these genes did not vary in giantleucaena throughout the experiments suggesting that the increasedcontent of mimosine observed in the treated plants might not be relatedto the expression of these genes but presumably to increased enzymeactivity andor release from conjugates such as mimoside a mimosineβ-D-glucoside (Murakoshi et al 1972)
Considering the time variation of mimosine accumulation observedin this work mimosinase gene expression in shoots and roots wasevaluated 24 h before the increase of mimosine concentration in giantleucaena seedlings (ie 24 h and 72 h after the chemical elicitorstreatments and 48 h and 120 h after UV-C exposure)
Ethylene signaling has been shown to up-regulate expression ofseveral genes related to secondary metabolism pathways as is the caseof phenolic compounds (Liu et al 2016) and terpenoid indole alkaloids(Wang et al 2016) Among all elicitors tested in the present workEthephon was the only one able to significantly change mimosinasegene expression Leucaena plants treated with Ethephon showed sig-nificant increases in mimosine concentration at both day 2 and 4 fol-lowing treatment which coincided with low-level expression of mi-mosinase Up-regulation of mimosinase gene expression was detected24 h before the increase of mimosine concentration in shoots treatedwith 10 ppm of Ethephon (Fig 3A) but not after JA or UV-C treatments(Fig 3C-D and 3E-F respectively) Nevertheless 72 h after treatment
application (24 h before the highest mimosine content measured inshoots) down regulation of mimosinase gene was seen in both shootsand roots treated with 10 ppm of Ethephon (Fig 3B) These data in-dicate that mimosine content in leucaena plants is at least partlyregulated by mimosinase expression in Ethephon exposed plants Onthe other hand the fact that mimosinase mRNA was not significantlyaffected by JA and UV-C treatments despite their stimulating effects onmimosine biosynthesis in giant leucaena may indicate that other levelsof regulation are at play or that the chosen harvesting time window wasunable to detect relevant changes
34 In situ and in vitro antioxidant assays
Considering the stimulation of mimosine accumulation byEthephon JA and UV all of which are often associated or known tocause oxidative imbalance the antioxidant capacity of mimosine wasevaluated Mimosine has been shown to have antioxidant activities oncultured cancer cells (Parmar et al 2015) In the present study it washypothesized that mimosine could confer radical scavenging propertieswhich would contribute to plant protection from possible damagecaused by reactive oxygen species generated during stress(Supplementary Fig 4)
Foliar disks of P vulgaris L were treated with 10mM mimosine for15min Treated disks showed less hydrogen peroxide accumulationinduced by wounding in contrast to untreated ones being comparableto those treated with ascorbic acid (a known hydrogen peroxide neu-tralizer) (Fig 4A) These observations support a possible antioxidant
Fig 2 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings exposed to UV-C lightCTRL= visible light (100 μmol photons mminus2 s minus1) UV-C 10primeand UV-C 15rsquo=UV-C exposure time (10 and 15min re-spectively) Bars sharing a letter of same case do not differ byTukey test (P le 005) Capital letters (A B) compare treat-ments on day three and lowercase letters (a b) comparetreatments on day six Indicates significant statistical dif-ference between day three and day six in the same treatmentby t-test (Ple 005) The error bars represent standard errorof five replicates (each mean was calculated with 15 in-dividual seedlings organized in 5 groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
437
role of mimosine as an in situ hydrogen peroxide scavengerMimosine was also able to quench superoxide anions generated by
light exposure Mimosine exhibited equivalent antioxidant effect com-pared to rutin (Fig 4B) a well-established effective superoxide anionquencher (Matsuura et al 2016) The radical scavenging activity ofmimosine may be due to the 3-OH group of the pyridine ring of mi-mosine (Fig 5) The pKa of the 3-OH of mimosine has been estimated tobe 88 (M Honda unpublished results) At physiological pH this OHgroup is expected to remain in a protonated state and therefore mayscavenge a radical by donating a proton and an electron In this processmimosine itself is converted to a stable radical form which is perhapsless toxic and less reactive than the reactive oxygen species generatedduring oxidative stress It is likely that the less toxic radical mimosineproduced may react with another radical or molecule and becomeconverted to a non-reactive indole molecule
In vivo antioxidant activity of mimosine has been previously eval-uated by means of its exogenous application on selenium-deficientseedlings of Vigna radiata In spite of its allelopathic properties (Ahmedet al 2008) the results showed mitigation of mitochondrial oxidativestress by treatment with 01mM mimosine (Lalitha and Kulothungan2007) DPPH radical scavenging activity was also reported for aqueous
seed extracts of leucaena rich in mimosine and phenolic compounds inin vitro assays (Benjakul et al 2014) Mimosine antioxidant activityshown in the present work is in good agreement with data reported forother non-protein amino acids such as L-DOPA (Dhanani et al 2015)and GABA (Malekzadeh et al 2014) for instance
4 Conclusion
Taken together results show that mimosine biosynthesis and ac-cumulation can be modulated by stress-related factors despite its re-latively high constitutive content in leucaena plants The pattern ofgene expression in stressed plants suggests mimosine steady-state con-trol may be regulated by its degradation in possible connection withdynamic changes in carbon and nitrogen metabolism of stressed plantsMimosine quenching activity against hydrogen peroxide and super-oxide anions in the in situ staining and in vitro assays respectivelyshowed that this non-protein amino acid can act as non-enzymaticantioxidant agent Increase in mimosine content in response to elicitorsmimicking environmental challenges in addition to its antiherbivoreand antimicrobial properties may be related to its activity as protectivemolecule against oxidative damage in line with other classes of plant
Fig 3 Relative expression of the mimosinase gene in shoots (A E and F) and shoots and roots (B C and D) of giant leucaena 24 h (A and C) 48 h (E) 72 h (B and D)and 120 h (F) after treatment with stress signaling molecules or UV-C exposure ETH = Ethephon JA = Jasmonic Acid Indicates significant statistical differencebetween control and treatment by t-test (Ple 005) The error bars represent standard error of four replicates
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
438
secondary metabolites
Funding
This work was funded by the National Council for Scientific andTechnological Development (CNPq-Brazil) grant 3060792013-5Coordenaccedilatildeo de Aperfeiccediloamento de Pessoal de Niacutevel Superior - Brazil(CAPES) - Finance Code 001 and the USDA NIFA Hatch projectHA05029-H managed by CTAHR
CRediT authorship contribution statement
Kelly Cristine da Silva Rodrigues-Correcirca InvestigationValidation Writing ndash original draft Michael DH HondaInvestigation Validation Dulal Borthakur Supervision Writing ndashreview amp editing Funding acquisition Arthur Germano Fett-NetoSupervision Funding acquisition Writing ndash review amp editing
Acknowledgements
The authors would like to thank Dr Jorge Ernesto Mariath fromLaVeg-UFRGS for kindly lending the Leica M165 FC stereomicroscopefor in situ analysis
Appendix A Supplementary data
Supplementary data to this article can be found online at httpsdoiorg101016jplaphy201811018
References
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leucocephala leaf litter on some forest and agricultural crops grown in nursery J ForRes 19 298 httpsdoi 101007s11676-008-0053-0
Benjakul S Kittiphattanabawon P Shahidi F Maqsood S 2013 Antioxidant activityand inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipidoxidation in model systems Food Sci Technol Int 19 (4) 365ndash376 httpsdoiorg1011771082013212455186
Benjakul S Kittiphattanabawon P Sumpavapol P Maqsood S 2014 Antioxidantactivities of lead (Leucaena leucocephala) seed as affected by extraction solvent priordechlorophyllisation and drying methods extracts against lipid oxidation in modelsystems Food Sci Technol 51 (11) 3026ndash3037 httpsdoiorg101007s13197-012-0846-1
Brewbaker JL Pluckett D Gonzalez V 1972 Varietal variation and yield trials ofLeucaena leucocephala (koa haole) in Hawaii Hawaii Agric Exp Stn Bull 166 26
Brewbaker JL 2008 Registration of KX2 ndash Hawaii interspecific-hybrid leucaena JPlant Registrations 1 (3) 190ndash193 httpsdoiorg103198jpr2007050298crc
Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitisvinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-6287-47-37
Cho H-Y Son SY Rhee HS Yoon S-YH Lee-Parsons CWT Park JM 2008Synergistic effects of sequential treatment with methyl jasmonate salicylic acid andyeast extract on benzophenanthridine alkaloid accumulation and protein expressionin Eschscholtzia californica suspension cultures J Biotechnol 135 117ndash122 httpsdoiorg101016jjbiotec200802020
Choudhary KK Agrawal SB 2014a Cultivar specificity of tropical mung bean (Vignaradiata L) to elevated ultraviolet-B changes in antioxidative defense system ni-trogen metabolism and accumulation of jasmonic and salicylic acids Environ ExpBot 99 122ndash132 httpsdoiorg101016jenvexpbot201311006
Choudhary KK Agrawal SB 2014b Ultraviolet-B induced changes in morphologicalphysiological and biochemical parameters of two cultivars of pea (Pisum sativum L)Ecotoxicol Environ Saf 100 178ndash187 httpsdoiorg101016jecoenv201310032
Dar TA Uddin M Khan MMA Hakeem KR Jaleel H 2015 Jasmonates counterplant stress a Review Environ Exp Bot 115 49ndash57 httpsdoiorg101016jenvexpbot201502010
Dhanani T Singh R Shah S Kumari P Kumar S 2015 Comparison of green ex-traction methods with conventional extraction method for extract yield L-DOPAconcentration and antioxidant activity of Mucuna pruriens seed Green Chem LettRev 8 (2) 43ndash48 httpsdoiorg1010801751825320151075070
Gutieacuterrez-Gamboa G Portu J Santamariacutea P Loacutepez R Garde-Cerdaacuten T 2017Effects on grape amino acid concentration through foliar application of three dif-ferent elicitors Food Res Int 99 688ndash692 httpsdoiorg101016jfoodres201706022
Fig 4 A In situ antioxidant assay Foliar disksof Phaseolus vulgaris L treated with (a) No an-tioxidant added (negative control) (b) 10 mMMimosine (c) 10mM ascorbic acid (positivecontrol) The oxidative damage can be seen bythe formation of a brown polymer in leaf veinsand injured areas B In vitro superoxidescavenging assay carried out with mimosineDifferent letters indicate significant differenceby Tukey test (Ple 005) The error bars re-present standard error of four replicates (Forinterpretation of the references to colour in thisfigure legend the reader is referred to the Webversion of this article)
Fig 5 Predicted mimosine radical formed followingquenching of hydroxyl radical Mimosine is first converted toa stable mimosine radical which may be then converted to anontoxic indole form
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
439
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad HossainMd Anai T Oku H 2018 Cytosolic cysteine synthase switch cysteine and mi-mosine production in Leucaena leucocephala Appl Biochem Biotechnol 186 (3)613ndash632 httpsdoiorg101007s12010-018-2745-z
Holloacutesy F 2002 Effects of ultraviolet radiation on plant cells Micron 33 (2) 179ndash197Honda MDH Ishihara KL Pham DT Borthakur D 2018 Identification of drought-
induced genes in giant leucaena (Leucaena leucocephala subsp glabrata) Trees 32571ndash585 httpsdoiorg101007s00468-018-1657-4
Huang T Jander G de Vos M 2011 Non-protein amino acids in plant defense againstinsect herbivores representative cases and opportunities for further functional ana-lysis Phytochemistry 72 1531ndash1537 httpsdoiorg101016jphytochem201103019
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of thethyrotoxic amino acid mimosine by cysteine synthase Phytochemistry 29 (11)3461ndash3465 httpsdoiorg1010160031-9422(90)85258-H
Ishihara K Lee EKW Borthakur D 2016a An improved method for RNA extractionfrom woody legume species Acacia koa A Gray and Leucaena leucocephala (Lam) deWit Int J For Wood Sci 3 (1) 031ndash035
Ishihara KL Honda MDH Pham DT Borthakur D 2016b Transcriptome analysisof Leucaena leucocephala and identification of highly expressed genes in roots andshoots Transcriptomics 4 135 httpsdoiorg1041722329-89361000135
IUBMB 2018 Enzyme Nomenclature EC 35161 httpwwwsbcsqmulacukiubmbenzymeEC35161html Accessed date 8 February 2018
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant(Phaseolus vulgaris) Biosci Res 10 (1) 29ndash32
Kazan K 2015 Diverse roles of jasmonates and ethylene in abiotic stress toleranceTrends Plant Sci 20 (4) 219ndash229 httpsdoiorg101016jtplants201502001
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect ofEthephon as an ethylene-releasing compound on the metabolic profile of Chlorellavulgaris J Agric Food Chem 64 (23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541
Khan MIR Fatma M Per TS Anjum NA Khan NA 2015 Salicylic acid-inducedabiotic stress tolerance and underlying mechanisms in plants Front Plant Sci 6 462httpsdoiorg103389fpls201500462
Korsangruang S Soonthornchareonnon N Chintapakorn Y Saralamp PPrathanturarug S 2010 Effects of abiotic and biotic elicitors on growth and iso-flavonoid accumulation in Pueraria candollei var candollei and P candollei var mir-ifica cell suspension cultures Plant Cell Tissue Organ Cult 103 (3) 333ndash342 httpsdoiorg101007s11240-010-9785-6
Lalitha K Kulothungan SR 2006 Selective determination of mimosine and its dihy-droxypyridinyl derivative in plant systems Amino Acids 31 (3) 279ndash287 httpsdoiorg101007s00726-005-0226-5
Lalitha K Kulothungan SR 2007 Mimosine mitigates oxidative stress in seleniumdeficient seedlings of Vigna radiata - Part I restoration of mitochondrial functionBiol Trace Elem Res 118 (1) 84ndash96 httpsdoiorg101007s12011-007-0013-0
Liu J Li Y Wang Y Zhang Z-H Zu Y-G Efferth T Tang Z-H 2016 Thecombined effects of ethylene and MeJA on metabolic profiling of phenolic com-pounds in Catharanthus roseus revealed by metabolomics analysis Front Physiol 71ndash11 httpsdoiorg103389fphys201600217 Article 217
Malekzadeh P Khara J Heydari R 2014 Alleviating effects of exogenous Gamma-aminobutiric acid on tomato seedling under chilling stress Physiol Mol Biol Plants20 (1) 133ndash137 httpsdoiorg101007s12298-013-0203-5
Martiacutenez-Luumlscher J Torres N Hilbert G Richard T Saacutenchez-Diacuteaz M Delrot SAguirreolea J Pascual I Gomegraves E 2014 Ultraviolet-B radiation modifies thequantitative and qualitative profile of flavonoids and amino acids in grape berriesPhytochemistry 102 106ndash114 httpsdoiorg101016jphytochem201403014
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation ofbioactive secondary metabolites by ultraviolet radiation mechanisms strategies andapplications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology forMedicinal Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash1902012
Matsuura HN Fragoso V Paranhos JT Rau MR Fett-Neto AG 2016 Thebioactive monoterpene indole alkaloid N szlig-D-glucopyranosylvincosamide is regu-lated by irradiance quality and development in Psychotria leiocarpa Ind Crop Prod86 210ndash218 httpsdoiorg101016jindcrop201603050
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo RBhambra AS Strnad M Bonfill M Fett-Neto AG 2018 Specialized plant me-tabolism characteristics and impact on target molecule biotechnological productionMol Biotechnol 60 (2) 169ndash183 httpsdoiorg101007s12033-017-0056-1
Murakoshi S Ohmiya S Haginiwa J 1972 Enzymic synthesis of mimoside a meta-bolite of mimosine in Mimosa pudica and Leucaena leucocephala Chem Pharm Bull20 (4) 855ndash857
Nakagawa T Kawaguchi M 2006 Shoot-applied MeJA suppresses root nodulation inLotus japonicus Plant Cell Physiol 47 (1) 176ndash180 httpsdoiorg101093pcppci222
Nascimento NC Menguer PK Henriques AT Fett-Neto AG 2013 Accumulation ofbrachycerine an antioxidant glucosidic indole alkaloid is induced by abscisic acidheavy metal and osmotic stress in leaves of Psychotria brachyceras Plant PhysiolBiochem 73 33ndash40 httpsdoiorg101016jplaphy201308007
Neelamegam R Sutha T 2015 UV-C irradiation effect on seed germination seedling
growth and productivity of groundnut (Arachis hypogaea L) Int J Curr MicrobiolApp Sci 4 (8) 430ndash443
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase fromLeucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol164 (2) 922ndash934 httpsdoiorg101104pp113230870
Negi VS Borthakur D 2016 Heterologous expression and characterization of mimo-sinase from Leucaena leucocephala In Fett-Neto Arthur Germano (Ed)Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods inMolecular Biology vol 1405 copySpringer Science+Business Media New York httpsdoiorg101007978-1-4939-3393-8_7 2016
Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636
Parmar F Kushawaha N Highland H George L-B 2015 In vitro antioxidant andanticancer activity of Mimosa pudica Linn extract and L-mimosine on lymphomaDaudi cells Int J Pharm Sci 12 100ndash104
Porto DD Matsuura HN Vargas LRB Henriques AT Fett-Neto AG 2014 Shootaccumulation kinetics and effects on herbivores of the wound-induced antioxidantindole alkaloid brachycerine of Psychotria brachyceras Nat Prod Commun 9 (5)629ndash632
Rai R Meena RP Smita SS Shukla A Rai SK Pandey-Rai S 2011 UV-B and UV-C pre-treatments induce physiological changes and artemisinin biosynthesis inArtemisia annua L ndash an antimalarial plant J Photochem Photobiol B Biol 105 (3)216ndash225 httpsdoiorg101016jjphotobiol201109004
Shabani L Ehsanpour AA Asghari G Emami J 2009 Glycyrrhizin production by invitro cultured Glycyrrhiza glabra elicited by methyl jasmonate and salicylic acid RussJ Plant Physiol 56 (5) 621ndash626 httpsdoiorg101134S1021443709050069
Shah J 2003 The salicylic acid loop in plant defense Curr Opin Plant Biol 6 (4)365ndash371
Shi J Fu XZ Peng T Huang XS Fan QJ Liu JH 2010 Spermine pretreatmentconfers dehydration tolerance of citrus in vitro plants via modulation of antioxidativecapacity and stomatal response Tree Physiol 30 (7) 914ndash922 httpsdoiorg101093treephystpq030
Smith IK Fowden L 1966 A study of mimosine toxicity in plants J Exp Bot 17750ndash761 httpsdoiorg101093jxb174750
Soedarjo M Borthakur D 1996 Simple procedures to remove mimosine from youngleaves pods and seeds of Leucaena leucocephala used as food Int J Food SciTechnol 31 (1) 97ndash103
Soedarjo M Borthakur D 1998 Mimosine a toxin produced by the tree-legumeLeucaena provides a nodulation competition advantage to mimosine-degradingRhizobium strains Soil Biol Biochem 30 1605ndash1613
Suda S 1960 On the physiological properties of mimosine Bot Mag Tokyo 73 (862)142ndash147 httpsdoiorg1015281jplantres188773142
Tangendjaja B Lowry JB Wills RBH 1986 Isolation of a mimosine degrading en-zyme from leucaena leaf J Sci Food Agric 37 523ndash526 httpsdoiorg101002jsfa2740370603
Tiburcio F Pintildeol MT Serrano M 1985 Effect of UV-C on growth soluble protein andalkaloids in Nicotiana rustica plants Environ Exp Bot 25 (3) 203ndash210 httpsdoiorg1010160098-8472(85)90004-8
Vestena S Fett-Neto AG Duarte RC Ferreira A 2001 Regulation of mimosineaccumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 httpsdoiorg101016S0168-9452(01)00448-4
Vlot AC Dempsey DMA Klessig DF 2009 Salicylic acid a multifaceted hormone tocombat disease Annu Rev Phytopathol 47 177ndash206 httpsdoiorg101146annurevphyto050908135202 2009
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene-induced vinblastine accumulation is related to activated expression of downstreamTIA pathway genes in Catharanthus roseus BioMed Res Int 2016 Article ID 3708187httpsdoiorg10115520163708187
Wasternack C Strnad M 2016 Jasmonate signaling in plant stress responses and de-velopment ndash active and inactive compounds N Biotech 33 (5B) 604ndash613 httpsdoiorg101016jnbt201511001
Wencomo HB Ortiz R Caacuteceres J 2017 Afr J Agric Res 12 (4) 279ndash285 httpsdoiorg105897AJAR201510604 26
Wong CC Devendra C 1983 Research on leucaena forage production in Malaysia InLeucaena Research in the Asian Pacific Region pp 55ndash60 Ottawa Ontario Canada
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX TsimKWK 2018 Jasmonate-elicited stress induces metabolic change in the leaves ofLeucaena leucocephala Molecules 23 (2) httpsdoiorg103390molecules23020188 E188
Yafuso JT Negi VS Bingham J-P Borthakur D 2014 An O-acetylserine (thiol)lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthaseAppl Biochem Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z
Zhao J Zheng S-H Fujita K Sakai K 2004 Jasmonate and ethylene signalling andtheir interaction are integral parts of the elicitor signalling pathway leading to b-thujaplicin biosynthesis in Cupressus lusitanica cell cultures J Exp Bot 55 (399)1003ndash1012 httpsdoiorg101093jxberh127
Zhishen J Mengcheng T Jianming W 1999 The determination of flavonoid contentsin mulberry and their scavenging effects on superoxide radicals Food Chem 64 (4)555ndash559 httpsdoiorg101016S0308-8146(98)00102-2
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
440
61
Supplementary Fig 1 Basal mimosine concentration in adult trees of common leucaena (L leucocephala
var leucocephala) Samples were collected from 10 field grown trees at Manoa Valley Honolulu Hawairsquoi
on June 25th 2017 Bars sharing a letter do not differ by Tukey test (P le 005) The error bars represent the
standard error
Supplementary Fig 2 Bar diagram showing mimosine concentration in shoots of 12-week-old common
leucaena seedlings treated with different elicitors CTRL = Milli-Q water SA = Salicylic Acid MeJA =
Methyl Jasmonate CEPA = 2-Chloroethylphosphonic acid (an ethylene releasing compound) Bars sharing a
letter of same case do not differ by Tukey test (P le 005) Capital letters (A B) compare treatments on day
two and lower-case letters (a b) compare treatments on day four Indicates significant statistical difference
ABB
A A
0
200
400
600
800
1000
1200
LEAVES GREEN FLOWERBUDS
POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
con
cen
trat
ion
(micro
gg
-1o
f FW
)
B AB AB AB B A
b
a
ab b
ab
0
2
4
6
8
10
12
14
16
18
20
CTRL SA 10 ppm SA 100 ppm CEPA 10 ppm CEPA 100 ppm MeJA 90 ppm
Mim
osi
ne
co
nce
ntr
atio
n (
gg
-1o
f FW
)
DAY 02 DAY 04
62
between day two and day four in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
Supplementary Fig 3 Bar diagram showing the effects of UV-C radiation exposure for 5 10 and 15 min on
mimosine accumulation in shoots of 12-week-old seedlings of common leucaena Bars sharing a letter of
same case do not differ by Tukey test (P le 005) Capital letters (A B C) compare treatments on day three
and lower-case letters (a b) compare treatments on day six Indicates significant statistical difference
between day three and day six in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
C BC AB A
bb
a
a
0
10
20
30
40
50
60
CTRL UV-C 5 UV-C 10 UV-C 15
Mim
osi
ne
co
nce
ntr
atio
n (
gg-1
of
FW)
DAY 03 DAY 06
63
Supplementary Fig 4 Model depicting induction of mimosine synthesis in leucaena following application of
stress elicitors such as CEPA and jasmonic acid or exposure to UV-C radiation The additional mimosine
synthesized may serve to alleviate oxidative stress induced by UV-C radiation
64
Supplementary Table 1 Mimosine contents in leaves of common and giant leucaena
Leucaena
type
Mimosine content
( FW)
Mimosine
content ( DW)
Dry matter
content ( FW)
Water content
( FW)
Common (1) 050 plusmn 009 245 plusmn 051 2011 plusmn 054 7989 plusmn 054
Common (2) 043 plusmn 006 214 plusmn 037 1998 plusmn 050 8002 plusmn 050
K636 (1) 070 plusmn 014 356 plusmn 077 1908 plusmn 052 8092 plusmn 052
K636 (2) 042 005 205 plusmn 033 2008plusmn 093 7992plusmn 093
KX2 (1) 122 plusmn 011 608 plusmn 082 1939 plusmn 123 8061 plusmn 123
KX2 (2) 134 plusmn 010 623 plusmn 056 2029 plusmn 114 7971 plusmn 114
KX3 (1) 044 plusmn 006 221 plusmn 030 1945 plusmn 073 8055 plusmn 073
KX3 (2) 054 plusmn 005 273 plusmn 023 1930 plusmn 038 8070 plusmn 038
KX4 (1) 086 plusmn 011 471 plusmn 065 1753 plusmn 084 8247 plusmn 084
KX4 (2) 089 plusmn 011 476 plusmn 065 180 plusmn 072 820 plusmn 072
KX5 (1) 099 plusmn 012 489 plusmn 048 1907 plusmn060 8093 plusmn 060
KX5 (2) 115 plusmn 015 548 plusmn080 1992 plusmn 053 8008 plusmn 053
Common leucaena variety koa haole grows widely on the island of Orsquoahu K636 is widely
grown variety of giant leucaena KX2 KX3 KX4 and KX5 are giant leucaena varieties
developed through interspecies hybridization (Brewbaker 2016) (1) and (2) indicate plants
from two separate locations within the University of Hawaii Waimanalo Research Center The
values are shown as mean plusmn standard error obtained from at least three biological replicates
65
Supplementary Table 2 GenBank accession numbers of the tested cysteine pathway genes isoforms
Gene name GenBank accession
OAS-TL (o-acetylserine-thiol-lyase) GDRZ01032940
GDRZ01061620
GDRZ01153117
GDSA01187555
GDSA01196891
GDSA01214467
Cys syn (cysteine synthase) GDRZ01015860
GDRZ01050898
GDRZ01086813
GDRZ01193515
GDRZ01202579
GDSA01180863
GDSA01215622
SAT (serine acetyltransferase) GDRZ01187456
GDRZ01189631
CAS (β-cyanoalanine synthase) GDRZ01054066
GDRZ01175418
GDSA01118400
66
SHORT COMMUNICATION 1
Mimosine occurrence and accumulation in Mimosa bimucronata var bimucronata (DC) 2
Kuntze 3
Kelly Cristine da Silva Rodrigues-Correcirca1 Lana Dorneles Pedroso2 Fernanda de Costa1 4
Arthur Germano Fett-Neto1 5
1Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal 6
University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970 7
Porto Alegre Rio Grande do Sul Brazil 2Department of Biological Sciences Unipampa ndash 8
Campus Satildeo Gabriel 9
Corresponding author 10
E-mail addresses krodriguescbiotufrgsbr (KCdaS Rodrigues-Correcirca) 11
lanalima2012gmailcom (LD Pedroso) fernandadecostayahoocombr (F de Costa) 12
fettnetocbiotufrgsbr (AG Fett-Neto) 13
14
15
16
17
18
19
20
21
22
67
ABSTRACT 23
Mimosine is a non-protein aromatic amino acid present in plants of Leucaena spp 24
and Mimosa spp Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) is a native 25
tree from Brazil which occurs as a pioneer species on plant succession processes In the 26
current study the presence of mimosine in M bimucronata was verified by HPLC analyses 27
Moreover mimosine accumulation upon exposure to UV-C and chemical elicitors of 28
specialized metabolism (salicylic acid - SA methyl jasmonate - MeJA sodium nitroprusside 29
- SNP and ethephon - ETH) most of which also known as promoters of the amino acid 30
production in leucaena plants was evaluated The results showed a lower concentration of 31
constitutive mimosine present in both maricaacute seedlings and mature trees when compared to 32
leucaena plants In spite of a trend towards increased mimosine accumulation observed in 33
MeJA and ETH treatments no statistical differences were found with the various stressors 34
used to induce its biosynthesis in maricaacute seedlings Data suggest that mimosine in M 35
bimucronata is probably a phytoanticipin-like metabolite or its accumulation is driven by 36
other types of stresses 37
38
39
Keywords Mimosine Mimosa bimucronata stress 40
41
42
43
44
45
46
68
Introduction 47
Mimosa bimucronata commonly known as maricaacute is a native tree from Brazil 48
(REFLORA 2019) ecologically important in plant succession and in processes of degraded 49
land recovery (Bitencourt et al 2007 Silva et al 2011) occurring as a pioneer species 50
(Pilatti et al 2019) Maricaacute is a deciduous or semi-deciduous plant which reaches up to 15 51
m in height and 40 cm of diameter at breast height (DBH) displays shrub or tree habit and 52
bears typical sharp thorns (Carvalho 2004) This species belongs to Fabaceae one of the 53
most economically important families of flowering plants due to its high diversity and 54
occurrence in different types of habitats (Gomes et al 2018) As well as several others 55
Mimosa spp maricaacute is usually referred to as a multipurpose tree (Olkoski and Wittmann 56
2011) employed for alternative medicinal uses (Champanerkar et al 2010 Silva et al 57
2011) honey production constructions and remodeling of landscape architecture (living 58
fences) for instance (Marchiori 1993 Lorenzi 1998) 59
In southern Brazil maricaacute is widely distributed and typically found either in wetland 60
areas close to river banks (Patreze and Cordeiro 2004) or composing large and almost pure 61
landscape formations on hillsides (Jacobi and Ferreira 1991) In dense populations this 62
species like several Mimosa spp (Simon and Proenccedila 2000) is considered an important and 63
highly invasive weed by preventing cattle to reach pasturesand water bodies as a result of its 64
thorny branches (Lorenzi 2008 Kestring et al 2009) Its dominant and nearly exclusive 65
pattern of distribution in those areas has led Jacobi and Ferreira (1991) to test its allelopathic 66
potential on cultivated species Indeed extracts of leaves and ripe fruits (but not the green 67
ones) of maricaacute showed phytotoxic effects on germination and initial radical growth of most 68
of the target species tested 69
69
Several investigations have been performed on maricaacute floristics (Silva et al 2011) 70
distribution (Simon and Proenccedila 2000) wood anatomy (Marchiori 1993) cytogenetic 71
parameters (Olkoski and Wittmann 2011) and allelopathic potential (Jacobi and Ferreira 72
1991 Ferreira et al 1992) However excluding two recent publications on maricaacute 73
constitutive chemical composition (Schlickmann et al 2017 Pilatti et al 2019) which 74
identified phenolic compounds (methyl gallate and water-soluble tannins) as its major 75
compounds little is known regarding this subject In other Mimosa species (eg M pudica 76
and M pigra) mimosine has been identified (Soedarjo and Borthakur 1998) as one of the 77
major specialized metabolites present in the different organs of the plant (Champanerkar et 78
al 2010) The presence of this molecule was also reported for M bimucronata in a thin layer 79
chromatography-based preliminary study performed by Ferreira et al (1992) showing co-80
chromatography of a leaf extract component with authentic mimosine The authors attributed 81
the allelopathic effect of maricaacute to the accumulation of this metabolite in its leaves 82
Mimosine is an aromatic non-protein amino acid initially found in plants of Mimosa 83
pudica and later in Leucaena leucocephala (Lam) de Wit (Soedarjo and Borthakur 1998) a 84
leguminous tree which biosynthesizes large amounts of this nitrogen-containing compound 85
(Rodrigues-Correcirca et al 2019) It is believed that the accumulation of high contents of 86
mimosine in L leucocephala tissues confers among other traits defense against herbivores 87
and pathogens (Vestena et al 2001) tolerance to drought (Negi et al 2014) as well as 88
general oxidative stress protection (Rodrigues-Correcirca et al 2019) Interestingly drought is 89
the opposite environmental and physiological condition to that observed in the wet habitats 90
occupied by native populations of M bimucronata in Brazil (Patreze and Cordeiro 2004 91
Kestring et al 2009) and Mimosa pudica Linn in India (Champanerkar et al 2010) 92
70
Nonetheless flooding is also associated with oxidative stress particularly as water levels 93
change (Fukao et al 2019) 94
In Leucaena leucocephala var leucocephala (common leucaena) and Leucaena 95
leucocephala var glabrata (giant leucaena) mimosine accumulation has been shown to be 96
both constitutive and inducible by stress-related phytohormones such as jasmonic acid (JA) 97
Ethephon (ETH an ethylene- releasing compound) salicylic acid (SA - only common 98
leucaena) (Vestena et al 2001) as well as by UV-C radiation (Xu et al 2018 Rodrigues-99
Correcirca et al 2019) On the other hand there is a lack of information regarding mimosine 100
content and elicitation effects in Mimosa spp plants 101
The aim of this study was to examine the presence of mimosine in Mimosa 102
bimucronata and examine the effects of stresses and stress-signaling molecules on its 103
accumulation in leaves 104
Material and Methods 105
Plant material 106
For all experiments the plant material was collected at Morro Santana campus do 107
Vale of UFRGS (Federal University of Rio Grande do Sul) Porto Alegre RS Brazil 108
(3004rsquoS 5108rsquoW) Authorization for access to genetic material was obtained from 109
SISGEN-Brazil (license number A845493) Constitutive mimosine content in adult plants of 110
M bimucronata var bimucronata (DC) Kuntze was determined in plant material (leaves 111
green flower buds post-anthesis flowers and green pods) harvested in January 2017 112
(summer) A voucher herbarium specimen (ICN 187953) was deposited in the ICN ndash UFRGS 113
herbarium (Herbaacuterio do Instituto de Biociecircncias of UFRGS) 114
71
For mimosine elicitation experiments legumes (fruits) of maricaacute were collected in 115
the end of June 2017 (winter) Seeds were then removed from the dry fruits and kept in the 116
dark until sowing and seedling development for use in the assays 117
Seed germination 118
To break the coat-imposed seed dormancy after surface sterilization dry seeds of 119
maricaacute were acid scarified by immersion in H2SO4 (95 ndash 98 ) for 2 min (see Correcirca et al 120
2008) and repeatedly washed in distilled water to remove any residue of the acid Then seeds 121
were distributed in 50 mL individual plastic tubes (dibble-tubes) (30 cm diameter x 120 cm 122
depth) filled up with 11 (vv) of commercial top soil and vermiculite Tubes were watered 123
every 2 days to avoid substrate dryness and were kept in a growth room under controlled 124
conditions of light (circa 75 μmol mminus2s minus1 photosynthetically active radiation photoperiod 125
of 16 h light and 8 h dark) and temperature (24plusmn2C) 126
127
Treatments 128
In order to verify inducibility of mimosine accumulation in M bimucronata fifty 12-129
week-old maricaacute seedlings (per treatment) exhibiting similar features were selected and 130
sprayed (saturated) with solutions of different chemical stressors (plant specialized 131
metabolism elicitors) as follows (for further details see Rodrigues-Correcirca et al 2019) 10 132
and 50 mM SA (pathogen-signaling molecule Shah 2003) 007 and 035 mM 2-133
chloroethylphosphonic acid (ETH ethylene releasing-compound Kim et al 2016 Wang et 134
al 2016) 100 and 200 mM MeJA (Dar et al 2015) 10 and 50 mM SNP (a nitric oxide 135
donor Perotti et al 2015) Alternatively maricaacute seedlings were also supplemented with UV-136
C radiation (13 minutes 105 kJ cm2) (elicitor of plant specialized metabolism Kara 2013) 137
72
After 2 and 4 days of exposure to the chemical treatments and 3 and 6 days of UV-138
C supplementation maricaacute shoots were harvested immediately frozen in liquid nitrogen and 139
stored at ndash 80 C until mimosine extraction and HPLC analyses 140
Mimosine extraction and detection 141
Mimosine extraction was conducted according to the modified protocol described by 142
Rodrigues-Correcirca et al (2019) for L leucocephala HPLC (Thermo Scientific Surveyor) 143
analyses (mimosine detection and quantification) were performed following previously 144
published procedures (Negi et al 2014) A C18 column (ACE C18 5 μm 46times250 mm) and 145
isocratic solvent system of 002M o-phosphoric acid with a linear flow rate of 1 mL min minus1 146
were used to separate and quantify the amino acid Mimosine detection was performed at 280 147
nm by photodiode array detection (200ndash400 nm) and retention time (229plusmn0024 min) 148
Mimosine quantification was done by means of the method of external standard curve 149
Additional confirmation of mimosine identity was performed by co-chromatography with 150
standard (Acros Organics authentic mimosine 99 used as reference) and peak purity check 151
The analyses of the chromatograms were done with the ChromQuest software 152
153
154
Results and Discussion 155
Constitutive accumulation of mimosine in M bimucronata 156
Mimosine was detected in all analyzed samples positively meeting all identification 157
criteria In agreement with what has been found for other Mimosa spp (Soedarjo and 158
Borthakur 1998) compared to L leucocephala adult plants (Rodrigues-Correcirca 2019) 159
mimosine content was lower in M bimucronata Of the adult plant tissues analyzed the 160
73
highest content of mimosine in maricaacute (per gram of fresh weight - FW) was found in post-161
anthesis flowers (36644 microg versus 89448 microg in common leucaena followed by leaves 162
(28838 microg x 67358 microg) green flower buds (28094 microg x 51247 microg) and green pods (19002 163
microg x 82687 microg) (Fig 1)The same pattern is observed for seedlings when both species are 164
compared In this study untreated 12-week-old maricaacute seedlings (control at day 2) showed a 165
shoot content of mimosine of 23029plusmn007 microg g-1 of (FW) Five-week-old untreated giant 166
leucaena seedlings cultivated in similar conditions exhibited between 83640 and 178736 167
microg g-1 of FW (Rodrigues-Correcirca et al 2019) In the same way mimosine concentration 168
percentage in dry matter of Mimosa pigra was found to be rather low (002 in nodules and 169
roots and 007 in leaves) (Soedarjo and Borthakur 1998) 170
In this investigation the lowest constitutive mimosine content was found in green 171
pods (Fig 1) This result may partly explain the absence of phytotoxic effect observed for 172
green pods on germination and growth of crop target plants tested by Jacobi and Ferreira 173
(1991) compared to the other maricaacute parts analyzed 174
Elicitation of mimosine biosynthesis in M bimucronata 175
Chemical stressors 176
Secondary metabolites (or natural products) are structural- and chemically 177
specialized compounds derived from primary metabolism These molecules are mainly 178
biosynthesized as part of a complex defense mechanism in response to biotic and abiotic 179
stresses such as pathogens herbivores water status metal toxicity and UV radiation for 180
example (Matsuura et al 2018) Ethephon SA SNP MeJA have been extensively used as 181
chemical elicitors of specialized metabolism (Wang et al 2016 Vestena et al 2001 Perotti 182
74
et al 2015 Zhang and Memelink 2009 Xu et al 2018) These phytohormonal signals can 183
simulate environmental challenges and modulate plant homeostasis often leading to 184
alterations in gene expression (Shinozaki et al 2015) Except SNP all treatments tested in 185
the present study showed positive effect on mimosine accumulation in common or giant 186
leucaena (Vestena et al 2001 Rodrigues-Correcirca 2019 Rodrigues-Correcirca unpublished 187
data) However in spite of the trend of increasing the mimosine content observed in seedlings 188
treated with 007 mM Ethephon (at day 2) and 100 mM MeJA (at day 4) no statistical 189
difference was confirmed for these treatments when compared to the control 190
On the other hand a within treatment difference on mimosine induction was seen 191
between day 2 and 4 in seedlings treated with 100 mM MeJA (Fig 2) In a lower 192
concentration (04 mM) jasmonic acid (JA)promoted a near threefold increase in mimosine 193
accumulation of giant leucaena seedlings after 2 days of application 194
UV-C radiation 195
Albeit UV-C radiation is not biologically active in natural environments it has been 196
widely used under controlled experimental conditions to generate acute responses of plant 197
specialized metabolism within a shorter period of time compared to that required to with UV-198
B radiation (Kara 2013 Cetin 2014) This fast response is due to the higher energy of UV-199
C photons that act as potent reactive oxygen species (ROS) generators causing extensive 200
damage to the cells either at the physiological level or on DNA structure (Gregianini et al 201
2003 Matsuura et al 2013) 202
Although divergent responses can be observed in plants exposed to UV-C radiation 203
the deleterious processes are usually reported on primary metabolism (decreasing of 204
chlorophyll content and plant height eg) (Kara 2013) In the present study no statistical 205
75
differences were observed in the mimosine concentration in maricaacute seedlings supplemented 206
with UV-C radiation However a decreasing in its content was found for both control and 207
treatment at day 6 post-treatment (Fig 03) Taking into account the lower constitutive 208
concentration of mimosine observed in maricaacute compared to the leucaena plants besides its 209
relative thermolability (Nguyen and Tawata 2016) it seems to be plausible to consider the 210
effect of the temperature inside the UV-C and the white light (control) chambers as an 211
additional abiotic factor contributing to the decrease of mimosine accumulation in both group 212
of plants 213
Besides mimosine identification the presence of 34-dihydroxypyridine (34-DHP or 214
3-hydroxy-4-pyridone - 3H4P) a mimosine degradation product (Negi et al 2014 Nguyen 215
and Tawata 2016) was also reported for maricaacute leaf extracts analyzed by TLC by Ferreira 216
et al (1992) In our chromatograms we detected a second large peak after that of mimosine 217
(229plusmn0024) and similar to that identified by Negi et al (2014) as 3H4P (data not shown) 218
Comparing the chromatogram profiles obtained from seedlings elicited with chemical 219
stressors and those supplemented with UV-C the largest area for this peak was found (in all 220
samples) in the latter treatment at day 6 It might indicate that the constitutive andor the 221
initially UV-C-induced mimosine was degraded into 3H4P to cope with the cellular damage 222
caused by this treatment associated with an increased temperature inside the chambers 223
Nevertheless it was not possible to determine 3H4P concentration (or confirm its identity) 224
in maricaacute plants since there is no commercial standard (pure 3H4P) available for purchase 225
to be used as a reference in calculations Establishment of improved protocols for obtaining 226
in house 3H4P reference substance by acid hydrolysis is ongoing 227
228
229
76
Conclusion 230
On the basis of the overall absence of effect of the treatments tested here on mimosine 231
concentration it is possible to suggest that its accumulation profile is similar to that of 232
phytoanticipins unlike what is observed for the same amino acid production in leucaena 233
which shows features of inducibility resembling phytoalexin-like metabolites Alternatively 234
a putative inducible pool of mimosine in maricaacute might be involved in other types of stress 235
such as extended drought periods If involved in protection against oxidative stress as 236
described for leucaena mimosine in maricaacute may act predominantly by physical quenching 237
of ROS as indicated by the lack of overt chemical degradation Nevertheless further 238
investigations are needed to assess these hypotheses 239
To sum up mimosine biosynthesis was not modulated by the treatments evaluated as 240
in L leucocephala (Lam) de Wit To the best of our knowledge this is the first work that 241
analytically identifies and quantifies mimosine accumulation in M bimucronata 242
243
REFERENCES 244
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Carvalho PER 2004 Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 248
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Jacobi US Ferreira AG 1991 Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK 272
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Kestring D Klein J Menezes LCCR Rossi MN 2009 Imbibition phases and 276
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Ethephon as an ethylene-releasing compound on the metabolic profile of Chlorella vulgaris 280
J Agric Food Chem 64(23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541 281
Lorenzi H 1998 Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 282
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 283
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metabolismcharacteristicsandimpactontargetmoleculebiotechnologicalproduction 294
Molecular Biotechnology 60(2) 169ndash183httpsdoiorg101007s12033-017-0056-1 295
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase from 296
Leucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 297
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Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine 299
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Olkoski D Wittmann MTS 2011 Cytogenetics of Mimosa bimucronata (DC) O Kuntze 301
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Appl Biotechnol 11 27-35 httpdxdoiorg101590S1984-70332011000100004 303
Patreze CM Cordeiro L 2004 Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 304
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285 httpdxdoiorg101016jforeco200403034 306
Perotti JC Rodrigues-Correcirca KCS Fett-Neto AG 2015 Control of resin production in 307
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Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG 2019 Mimosine 309
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UV exposure Plant Physiology and Biochemistry 135 432ndash440 311
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Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS 2011 Biologia da reproduccedilatildeo 315
deMimosa bimucronatandash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 316
Simon MF and Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 317
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 318
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Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 320
LM Andrade SF Cechinel-Filho V 2017 Chemical composition and diuretic natriuretic and 321
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kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its majority 322
constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash1624 323
Shah J 2003 The salicylic acid loop in plant defense Current Opinion Plant Biology6 (4) 324
365ndash371 325
Shinozaki K Uemura M Serres JB Bray EA Weretilnyk E 2015 Responses to Abiotic 326
Stress In Buchanan BB Gruissem W Jones RL (Eds) Biochemistry and Molecular 327
Biology of Plants Second Edition John Wiley and Sons Ltd 328
Soedarjo M and Borthakur D 1998 Mimosine a toxin produced by the tree-legume 329
Leucaena provides a nodulation competition advantage to mimosine-degrading Rhizobium 330
strains Soil Biology and Biochemistry 30(12)1605-1613 331
Vestena S Fett-Neto AG Duarte RC Ferreira AG 2001 Regulation of mimosine 332
accumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 333
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene induced 334
vinblastine accumulation is related to activated expression of downstream TIA pathway 335
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Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX Tsim KWK 2018 337
Jasmonate-elicited stress induces metabolic change in the leaves of Leucaena leucocephala 338
Molecules 23 (2) 339
Zhang H Memelink J 2009 Regulation of Secondary Metabolism by Jasmonate Hormones 340
In AE Osbourn and V Lanzotti (eds) Plant-derived Natural Products 3 DOI 101007978-341
0-387-85498-4_1 copy Springer Science + Business Media LLC 342
343
344
345
81
346
Figure 1 Constitutive concentration of mimosine in different plant organs of Mimosa 347
bimucronata Bars sharing the same letter do not differ statistically by Tukey test (Ple005) 348
The error bars denote standard error of 10 replicates 349
350
351
352
353
354
355
356
357
B B A C0
5
10
15
20
25
30
35
40
LEAVES GREEN FLOWER BUDS POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
co
nce
ntr
atio
n u
gg-1
Mimosine concentration in adult plants of Mimosa bimucronata (DC) Kuntze
82
C T R L S A
1 0 m M
S A
5 0 m M
E T H
0 0 7 m M
E T H
0 3 5 m M
M e J A
1 0 0 m M
M e J A
2 0 0 m M
S N P
1 0 m M
S N P
5 0 m M
0
1 0
2 0
3 0
T re a tm e n ts
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1) D A Y 2
D A Y 4
A B C C B C A B C C A B C A B C A
a b b b a a b a a b b a b
358
Figure 2 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 359
bimucronata treated with different signaling molecules SA = Salicylic Acid ETH = 360
Ethephon MeJA = Methyl Jasmonate SNP = Sodium Nitroprusside Uppercase and 361
lowercase letters indicate statistical differences among treatments in days 2 and 4 362
respectively Bars sharing a letter of the same case do not differ statistically by Tukey test 363
(Ple005) Indicates statistical difference in the same treatment between day 2 and 4 by t-364
test (Ple005) The error bars denote standard error of 5 replicates (25 individual seedlings 365
arranged in 5 groups of 5) 366
367
368
83
D AY 3 D AY 6
0
5
1 0
1 5
2 0
2 5
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1)
C O N TR O L
U V -C
369
Figure 3 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 370
bimucronata supplemented with UV-C radiation Indicates statistical difference in the same 371
treatment between day 3 and 6 by t-test (Ple005) The error bars denote standard error of 5 372
replicates (25 individual seedlings arranged in 5 groups of 5) 373
374
375
376
377
378
379
380
381
382
383
384
385
84
Consideraccedilotildees finais 386
- Experimentos que avaliam os efeitos da aplicaccedilatildeo exoacutegena de ANPs em diferentes espeacutecies 387
vegetais tecircm sido realizados principalmente com GABA Dentre os principais efeitos 388
conferidos pela aplicaccedilatildeo dessa moleacutecula em espeacutecies de mono e eudicotiledocircneas satildeo 389
relatados a toleracircncia agrave seca agrave salinidade e agraves temperaturas extremas 390
- Como metaboacutelitos especializados claacutessicos os ANPs podem ter sua concentraccedilatildeo basal 391
endoacutegena aumentada em resposta agrave induccedilatildeo mediada por uma vasta gama de tratamentos com 392
moleacuteculas sinalizadoras de estresse e fontes alternativas de estressores De um modo geral 393
observa-se o acuacutemulo das diferentes classes de ANPs em resposta agrave radiaccedilatildeo UV elicitores 394
quiacutemicos que mimetizam ataques por patoacutegenos dano mecacircnico agentes osmoacuteticos metais 395
pesados entre outros 396
- Especificamente em leucena a resposta observada em relaccedilatildeo aos diferentes tratamentos 397
testados indica que apesar do seu alto teor constitutivo nessa espeacutecie a biossiacutentese e o 398
acuacutemulo de mimosina podem ser modulados por fatores causadores de estresses exibindo -399
nessa espeacutecie - um padratildeo de acumulaccedilatildeo similar agrave fitoalexinas Em maricaacute por outro lado 400
aumento de acuacutemulo dessa moleacutecula natildeo foi observado para os mesmos tratamentos testados 401
para leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas 402
- O padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno 403
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado pela 404
sua degradaccedilatildeo 405
- As respostas observadas nos testes que avaliaram a atividade de mitigaccedilatildeo de espeacutecies 406
reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode agir como um agente 407
antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de estresse 408
85
Perspectivas 409
- Confirmaccedilatildeo em espectrocircmetro de massas eou ressonacircncia nuclear magneacutetica da natureza 410
quiacutemica da lsquomimosinarsquo presente em maricaacute 411
- Avaliaccedilatildeo do efeito de concentraccedilotildees mais elevadas e em diferentes periacuteodos de aplicaccedilatildeo 412
das moleacuteculas sinalizadoras testadas sobre o acuacutemulo de mimosina em leucena e maricaacute 413
- Ampliar a investigaccedilatildeo dos padrotildees de expressatildeo gecircnica dos genes que codificam para 414
mimosinase (em maricaacute) mimosina sintase (em ambas as espeacutecies testadas) bem como o 415
perfil de precursores e cataboacutelitos de mimosina em resposta aos tratamentos mencionados 416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
86
Referecircncias Bibliograacuteficas 436
437
Acamovic T Brooker JD (2005) Biochemistry of plant secondary metabolites and their 438
effects in animals P Nutr Soc 64 403ndash412 httpsdoiorg101079PNS2005449 439
Ahmed R Hoque ATMR Hossain MK (2008) Allelopathic effects of Leucaena 440
leucocephala leaf litter on some forest and agricultural crops grown in nursery J Forestry 441
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Ahmed AMM Saacutenchez FJS Bavileacutes LRY Mahdy REZ Camaal JBC (2016) Tannins and 443
mimosine in Leucaena genotypes and their relations to Leucaena resistance against 444
Leucaena Psyllid and Onion thrips Agroforestry Systems 1-8 445
Benjakul S Kittiphattanabawon P Shahidi F Maqsood S (2013) Antioxidant activity and 446
inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipid oxidation in 447
model systems Food Sci Technol Int 19(4)365-76 448
httpsdoiorg1011771082013212455186 449
Bitencourt F Zocche JJ Costa S Souza PZ Mendes AR (2007) Nucleaccedilatildeo de Mimosa 450
bimucronata (DC) O Kuntze em aacutereas degradadas pela mineraccedilatildeo de carvatildeo Revista 451
Brasileira de Biociecircncias 5 750-752 452
Bottini-Luzardo M Aguilar-Perez C Centurion-Castro F Solorio-Sanchez F Ayala-Burgos 453
A Montes-Perez R Muntildeoz-Rodriguez D Ku-Vera J (2015) Ovarian activity and estrus 454
behavior in early postpartum cows grazing Leucaena leucocephala in the tropics Trop Anim 455
Health Prod 47(8)1481-6 456
Carvalho PER (2004) Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 457
Teacutecnica 941-10 458
Chowtivannakul P Srichaikul B Talubmook C (2016) Antidiabetic and antioxidant activities 459
of seed extract from Leucaena leucocephala (Lam) de Wit Agriculture and Natural 460
Resources 50 (2016) 357e361 httpdxdoiorg101016janres201606007 461
Chung H-H Chen M-K Chang Y-C Yang S-F Lin C-C Lin C-W (2017) Inhibitory effects 462
of Leucaena leucocephala on the metastasis and invasion of human oral cancer cells 463
Environmental Toxicology 321765ndash1774 httpsdoiorg101002tox22399 464
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Crowe B Poynter JA Manukyan MC Wang Y Brewster BD Herrmann JL Abarbanell 465
AM Weil BR Meldrum DR (2001) Pretreatment with intracoronary mimosine improves 466
postischemic myocardial functional recovery Surgery 150(2) 191-106 467
Fallon (2015) Effects of mimosine on Wolbachia in mosquito cells cell cycle suppression 468
reduces bacterial abundance In Vitro Cell Dev Biol Anim 51(9)958-63 469
httpsdoiorg101007s11626-015-9918-7 Epub 2015 May 28 470
Fernaacutendez-Salas A Alonso-Diacuteaza MA Acosta-Rodriacuteguez A Torres-Acosta JFJ Sandoval-471
Castro CA Rodriacuteguez-Vivas RI (2011) In vitro acaricidal effect of tannin-rich plants against 472
the cattle tick Rhipicephalus (Boophilus) microplus (Acari Ixodidae) Veterinary 473
Parasitology 175113ndash118 2010 httpsdoiorg101016jvetpar201009016 474
Ferreira AG Aquila MEA Jacobi US Rizvi V (1992) Allelopathy in Brazil In Allelopathy 475
basic and applied aspects Rizvi V and Jacobi US (Eds) Chapman and Hall PP 243-250 476
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad Hossain Md Anai 477
T Oku H (2018) Cytosolic cysteine synthase switch cysteine and mimosine production in 478
Leucaena leucocephala Appl Biochem Biotechnol 186 (3) 613ndash632 479
httpsdoiorg101007s12010-018-2745-z 480
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of the thyrotoxic 481
amino acid mimosine by cysteine synthase Phytochemistry 29 (11) 3461ndash3465 482
httpsdoiorg1010160031-9422(90)85258-H 483
Jacobi US Ferreira AG (1991) Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK Sobre 484
espeacutecies cultivadas Pesquisa Agropecuaacuteria Brasileira 26(7) 935-943 485
Jamous RM Ali-Shtayeh MS Abu-Zaitoun SY Markovics A Azaizeh H (2017) Effects of 486
selected Palestinian plants on the in vitro exsheathment of the third stage larvae of 487
gastrointestinal nematodes BMC Veterinary Research 13308 488
httpdxdoiorg101186s12917-017-1237-7 489
Jiao CJ Jiang J-L Ke L-M Cheng W Li F-M Li Z-X Wang C-Y (2011) Factors affecting 490
β-ODAP content in Lathyrus sativus and their possible physiological mechanisms Food 491
Chem Toxicol 49 543ndash549 httpsdoiorg101016jfct201004050 492
Kubota S Fukumoto Y Ishibashi K Soeda S Kubota SS Yuki R Nakayama Y Aoyama K 493
Yamaguchi N (2014) Activation of the prereplication complex is blocked by mimosine 494
88
through reactive oxygen species-activated ataxia telangiectasia mutated (ATM) protein 495
without DNA damage J Biol Chem 28 289(9)5730-46 496
Kuppusamy UR Arumugam B Azaman N Wai CJ (2014) Leucaena leucocephala Fruit 497
Aqueous Extract Stimulates Adipogenesis Lipolysis and Glucose Uptake in Primary Rat 498
Adipocytes Hindawi Publishing Corporation e Scientific World Journal Article ID 737263 499
8 pages httpdxdoiorg1011552014737263 500
Kusama-Eguchi K (2019) Research in motor neuron diseases caused by natural substances 501
focus on pathological mechanisms of neurolathyrism Yakugaku Zasshi 139 (4) 609-502
615 httpsdoiorg101248yakushi18-00202 503
Kutchan TM Gershenzon J Moslashller BL Gang DR (2015) Natural Products In Buchanan 504
BB Gruissem W and Jones RL (eds) Biochemistry amp Molecular Biology of Plants 2nd edn 505
Wiley Blackwell Chichester pp 1135-1205 506
Lalande M (1990) A reversible arrest point in the late G1 phase of the mammalian cell cycle 507
Exp Cell Res 186 332ndash339 508
Li X-W Hu C-P Li Y-J Gao Y-X Wang XM Yang J-R (2015) Inhibitory effect of L-509
mimosine on bleomycin-induced pulmonary fibrosis in rats Role of eIF3a and p27 Int 510
Immunopharmacol 27(1) 53ndash64 511
Little Jr EL Skolmen RG (1989) Koa haole Agriculture Handbook 679 USDA 512
Lorenzi H (1998) Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 513
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 514
Marchiori JNC (1993) Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 515
O Kuntze Ciecircncia Florestal 3 85-106 516
Mohammed RS El Souda SS Taie HAA Moharam ME Shaker KH (2015) Antioxidant 517
antimicrobial activities of flavonoids glycoside from Leucaena leucocephala leaves Journal 518
of Applied Pharmaceutical Science 5(06)138-147 519
httpdxdoiorg107324JAPS201550623 520
Negi VS Bingham J-P Li QX Borthakur D (2014) A carbon-nitrogen lyase from Leucaena 521
leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 (2) 922ndash522
934 httpsdoiorg101104pp113230870 523
89
Olkoski D Wittmann MTS (2011) Cytogenetics of Mimosa bimucronata (DC) O Kuntze 524
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop 525
Breeding and Applied Biotechnology 11 27-35 526
Patreze CM Cordeiro L (2004) Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 527
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecology and Management 528
196275ndash285 529
Pilatti DM Fortes AMT Jorge TCM Boiago NP (2019) Comparison of the phytochemical 530
profiles of five native plant species in two different forest formations Brazilian Journal of 531
Biology 79(2) 233-242 532
Ramos-Ruiz R Poirot E Flores-Mosquera M (2018) GABA a non-protein amino acid 533
ubiquitous in food matrices Cogent Food Agric 41534323 534
httpsdoiorg1010802331193220181534323 535
REFLORA (2019) httpfloradobrasiljbrjgovbrreflora Acesso em agosto de 2019 536
Rodgers KJ Samardzic K Main BJ (2015) Toxic Nonprotein Amino Acids Plant Toxins 537
httpsdoiorg 101007978-94-007-6728-7_9-1 538
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG (2019) Mimosine 539
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 540
UV exposure Plant Physiology and Biochemistry 135 432ndash440 541
httpsdoiorg101016jplaphy201811018 542
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 543
LM Andrade SF Cechinel-Filho V (2017) Chemical composition and diuretic natriuretic 544
and kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its 545
majority constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash546
1624 547
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS (2011) Biologia da reproduccedilatildeo 548
de Mimosa bimucronata ndash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 549
Simon MF Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 550
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 551
endemism Biological Conservation 96 279-296 552
90
Soares AMS Arauacutejo SA Lopes SG Costa Junior LM (2015) Anthelmintic activity of 553
Leucaena leucocephala protein extracts on Haemonchus contortus Braz J Vet Parasitol 554
Jaboticabal 24(4) 396-401 httpdxdoiorg101590S1984-29612015072 555
Soerdajo M Borthakur D (1998) Mimosine a toxin produced by the tree-legume Leucaena 556
provides a nodulation competition advantage to mimosine-degrading Rhizobium strains Soil 557
Biol Biochem 30(12) 16051613 558
Souza-Lima ES Sinani TR Pott A Sartori ALB (2017) Mimosoideae (Leguminosae) in the 559
Brazilian Chaco of Porto Murtinho Mato Grosso do Sul Rodrigueacutesia 68(1) 263-290 2017 560
httpdxdoiorg1015902175-7860201768131 561
Taiz L amp Zeiger E (2010) Plant Physiology 5th edition Sinauer Associates Inc Sunderland 562
Verma VK Rani KV Kumara SR Prakash O (2018) Leucaena leucocephala pod seed 563
protein as an alternate to animal protein in fish feed and evaluation of its role to fight against 564
infection caused by Vibrio harveyi and Pseudomonas aeruginosa Fish and Shellfish 565
Immunology 76 (2018) 324ndash332 httpsdoiorg101016jfsi201803011 566
Yafuso JT Negi VS Bingham J-P Borthakur D (2014) An O-acetylserine (thiol) lyase from 567
Leucaena leucocephala is a cysteine synthase but not a mimosine synthase Appl Biochem 568
Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z 569
Zarin RMA Wan HY Isha A Armani N (2016) Antioxidant antimicrobial and cytotoxic 570
potential of condensed tannins from Leucaena leucocephala hybrid Food Science and 571
Human Wellness 5 65ndash75 httpdxdoiorg101016jfshw201602001 572
573
574
Contents lists available at ScienceDirect
Industrial Crops amp Productsjournal homepage wwwelseviercomlocateindcrop
Resin tapping transcriptome in adult slash pine (Pinus elliottii var elliottii)Camila Fernanda de Oliveira Junkes1 Artur Teixeira de Arauacutejo Juacutenior1 Juacutelio Ceacutesar de LimaFernanda de Costa Thanise Fuumlller Maacutercia Rodrigues de Almeida Franciele Antocircnia NeisKelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-NetoCenter for Biotechnology and Department of Botany Federal University of Rio Grande do Sul Porto Alegre PO Box 15005 91501-970 Brazil
A R T I C L E I N F O
KeywordsPinus elliottiResinResinosisTranscriptomeAdjuvant paste
A B S T R A C T
To better understand the bases of resin production a major source of terpenes for industry the transcriptome ofadult Pinus elliottii var elliottii (slash pine) trees under field commercial resinosis was obtained Samples werecollected from cambium after 5 and 15 days of treatment application which included tapping followed byapplication of commercial resin stimulant paste or control wounding without paste Overall mean number ofreads of all 16 libraries (2 treatments x 2 times x 4 replicated trees) was 34582048 Of these 89 were mappedagainst the reference sequence with a mismatch of 058 Using the Blast2Go 570 candidate genes were de-tected based on sequence annotation By comparing the expression profile between paste and control 310differentially expressed genes (DEGs) were identified at 5 days and 190 at 15 days with a significant fold changeof log2gt 12 Regarding changes in time comparisons within each treatment 210 and 105 DEGs were identifiedwithin control and paste treatment respectively Genes with different expression patterns in the times andtreatments examined included ethylene responsive transcription factors geranylgeranyl diphosphate synthasediterpene synthase cytochrome P450 and ABC transporters all of which may play important roles in resinproduction RT-qPCR analysis correlated well with the data obtained by RNAseq Resin composition changedover time This is the first transcriptomic investigation of resinosis of the main species used in the bioresinindustry and of molecular analyses of resinosis under field operations with implications for stand managementstimulant paste development genotype selection and breeding for high resinosis
1 Introduction
The adaptive success of conifers is largely due to the development ofa defense system based on the synthesis and secretion of terpenes in allmajor organs and different tissues (Miller et al 2005 Hall et al 2013Warren et al 2015) Conifer resin is a viscous fluid composed of acomplex mixture of terpenoids such as monoterpenes sesquiterpenesand diterpenes (Zulak and Bohlmann 2010) These terpenoids are se-creted from severed resin ducts when the tree is under biotic attack(Ralph et al 2006 Lange 2015 Geisler et al 2016) acting as pro-tectants (Schiebe et al 2012 Liu et al 2015)Biosynthesis of terpenes in conifers starts from isomerization of two
isoprenoid (C5) units dimethylallyl diphosphate (DMAPP) and iso-pentenyl diphosphate (IPP) These molecules can be biosynthesized viatwo separate routes in plants the methyl-erythritol 4-phosphate andmevalonate pathways IPP is synthesized and isomerized to DMAPP byisopentenyl diphosphate isomerase then prenyl transferases catalyze
the condensation of these two C5-units to geranyl diphosphate (Pazoukiand Niinemets 2016) Their elongation to prenyl diphosphates withaddition of IPP molecules leads to monoterpenes (C10) sesquiterpenes(C15) and diterpenes (C20) which are the substrates for terpene syn-thases (TPS) (Keeling and Bohlmann 2006b)TPSs are part of a large family of mechanistically related enzymes
involved in both primary and secondary metabolism (Keeling andBohlmann 2006b) The events of evolutionary diversification and ex-pansion of plant TPSs appear to have originated from gene duplicationsdomain losses and sub- or neofunctionalizations with subsequent di-vergence of an ancestral TPS gene of primary metabolism (Hall et al2013) Modification of TPS products changes their physical propertiesand may alter their biological activities (Chen et al 2011) TPSs of highsequence identity may have different functions even in closely relatedspecies Low sequence identity of TPSs in phylogenetically distantspecies does not preclude the possibility of independent evolution of thesame or related function of these enzymes (Zerbe and Bohlmann 2015)
httpsdoiorg101016jindcrop2019111545Received 4 January 2019 Received in revised form 10 June 2019 Accepted 4 July 2019
Corresponding authorE-mail address fettnetocbiotufrgsbr (AG Fett-Neto)1 These authors have equally contributed to this work
doi 1015900102-33062019abb0114
Acta Botanica Brasilica
Sustainable production of bioactive alkaloids in Psychotria L of
southern Brazil propagation and elicitation strategies
Yve Verocircnica da Silva Magedans1 Kelly Cristine da Silva Rodrigues-Correcirca1 Cibele Tesser da Costa1
Heacutelio Nitta Matsuura1 and Arthur Germano Fett-Neto1
Received April 1 2019Accepted June 28 2019
ABSTRACTPsychotria is the largest genus in Rubiaceae South American species of the genus are promising sources of natural
products mostly due to bioactive monoterpene indole alkaloids they accumulate ese alkaloids can have analgesic
antimutagenic and antioxidant activities in dierent experimental models among other pharmacological properties
of interest Propagation of genotypes with relevant pharmaceutical interest is important for obtaining natural
products in a sustainable and standardized fashion Besides the clonal propagation of elite individuals the alkaloid
content of Psychotria spp can also be increased by applying moderate stressors or stress-signaling molecules is
review explores advances in research on methods for plant propagation and elicitation techniques for obtaining
bioactive alkaloids from Psychotria spp of the South Region of Brazil
Keywords abiotic stress alkaloids elicitation monoterpenes plant propagation Psychotria southern Brazil
sustainability
Introduction
Psychotria belongs to Rubiaceae one of the major families
of $owering plants having economic interest e family
includes coee a few signicant poisonous plants to livestock
besides several important ornamental and medicinal species
(Souza amp Lorenzi 2012) Psychotria has captured researchersrsquo
attention mostly because of its medicinal properties
Psychotria colorata is an Amazonian species that produces
polyindolinic alkaloids with analgesic activity (Matsuura et
al 2013) e promising results obtained with P colorata
motivated the investigation of southern Brazilian Psychotria
species and the discovery of new bioactive alkaloids (Porto
et al 2009) Moreover leads on in planta alkaloid functions
were also topic of experimental evaluation
One of the key elements that needs to be addressed early
on during the process of developing new bioactive molecules
from plants is the capacity to generate catalytically active
biomass to support extraction and steady supply ere are a
number of ways through which these goals may be reached
including greenhouse rooting of cuttings (mini-cutting
1 Laboratoacuterio de Fisiologia Vegetal Departamento de Botacircnica Instituto de Biociecircncias e Centro de Biotecnologia Universidade Federal do Rio
Grande do Sul 91501-970 Porto Alegre RS Brazil
Corresponding author fettnetocbiotufrgsbr
Review
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Biomass yield of resin in adult Pinus elliottii Engelm trees is differentially
regulated by environmental factors and biochemical effectors
Franciele Antocircnia Neis Fernanda de Costa Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima
Kelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-Neto
Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) CP 15005 CEP 91501-970 Porto Alegre RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Biomass
Terpene resin
Seasonal
Benzoic acid
Regenerated forest
A B S T R A C T
Biomass of pine resin finds several applications in the chemical pharmaceutical biofuel and food industries
Resin exudation after injury is a key defense response in Pinaceae since this complex mixture of terpenes has
insecticidal antimicrobial and wound repair properties Resin yield is increased by effectors applied on the
wound area including phytohormones and metal cofactors of terpene synthases The interaction of resinosis
mechanism effectors is not fully understood particularly in adult forest setups under natural environmental
variations The aim of this work was to determine how resin exudation by wounded trunks of adult P elliottii
responded to combined chemical effectors involved in different regulatory pathways of resinosis (metal cofactors
of terpene synthases benzoic acid and plant growth regulators) and whether seasonal and tree distribution
variations affected these responses Symmetrically planted and scattered trees regenerated from the seed bank
had similar resin biomass yields suggesting that the homogeneity in development and spatial arrangement were
not significant factors in resin yield This new finding is of practical importance with the used tapping system
since costs of implanting forests by regeneration can be advantageous compared to planting In addition it was
shown for the first time that the salicylic acid precursor benzoic acid and the auxin naphthalene acetic acid
promoted resin exudation when individually applied to wound sites Both these adjuvants are two orders of
magnitude less costly compared to the conventionally used ethylene precursors besides facing less environ-
mental and health restrictions for use Most adjuvant-treated trees showed higher resin flow in the second year
indicating mechanisms of response build up Overall temperature was more important than rainfall as en-
vironmental parameter affecting resin biosynthesis which was higher in the warmer months of spring and
summer The combination of resinosis stimulant effectors from different signaling pathways showed no sig-
nificant synergistic or additive effect suggesting possible converging signaling pathways andor limitation of
common intermediate transducing molecules
1 Introduction
Pines occupy highly diverse environments over a range of tem-
peratures water and nutrient availabilities irradiance levels and pho-
toperiods being able to effectively face attacks from diverse herbivore
and pathogen guilds The success of conifers is linked to their complex
terpene biochemistry hosted by specialized secretory cells The terpe-
noid resin synthesized by Pinus spp is one of the main mechanisms of
defense of these trees particularly against bark beetles and the fungi
they carry (Fett-Neto and Rodrigues-Correcirca 2012) Pine resin biomass
is essentially composed of a monoterpene and sesquiterpene-rich tur-
pentine and diterpenoid-rich rosin fraction both finding numerous in-
dustrial applications as non-wood forest products (Rodrigues-Correcirca
et al 2012)
Molecules capable of modulating different signaling pathways have
been identified as resin yield stimulators including sulfuric acid (ex-
tends wound damage) 2-chloroethylphosphonic acid (CEPA a syn-
thetic ethylene precursor) paraquat (free radical generator) yeast ex-
tract (mimics attack by pathogens) salicylic acid (pathogen signaling
molecule) auxin (promotes ethylene biosynthesis and resin canal dif-
ferentiation) jasmonic acid (signals mechanical damage and promotes
secondary metabolism) and metal ions such as potassium iron and
manganese (cofactors of terpene synthases in conifers) and copper (a
component of ethylene receptors) (Clements 1970 Conrath et al
2002 Fett-Neto and Rodrigues-Correcirca 2012 Hudgins and Franceschi
2004 Lewinsohn et al 1994 Martin et al 2002 Popp et al 1995
httpsdoiorg101016jindcrop201803027
Received 12 December 2017 Received in revised form 9 March 2018 Accepted 13 March 2018
Corresponding author
E-mail addresses franci_neisyahoocombr (FA Neis) fernandadecostayahoocombr (F de Costa) thanisenfyahoocombr (TN Fuumlller)
jjuliocesarlimagmailcom (JC de Lima) krodriguescbiotufrgsbr (KC da Silva Rodrigues-Correcirca) jpfettcbiotufrgsbr (JP Fett) fettnetocbiotufrgsbr (AG Fett-Neto)
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Research Paper
Dual allelopathic effects of subtropical slash pine (Pinus elliottii Engelm)
needles Leads for using a large biomass reservoir
Kelly Cristine da Silva Rodrigues-Correcircaa Gelson Halmenschlagera Joseacuteli Schwambachb
Fernanda de Costaa Emili Mezzomo-Trevizana Arthur Germano Fett-Netoa
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 Brazilb University of Caxias do Sul Institute of Biotechnology Caxias do Sul RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Seasonality
Growth
Germination
Litter
Substrate
A B S T R A C T
Pinus elliottii Engelm (slash pine) is distributed along the maritime coast of Southern Brazil where it shows
invasive pattern and typical allelopathic features Large quantities of needle litter are produced by pine trees a
biomass that is little explored in areas where this species is alien Little is known about the dynamics of needle
and litter phytochemical interactions particularly in subtropical environments To elucidate the full range of
needle and litter allelopathic potential the effects of litter (superficial and deep) and seasonally harvested fresh
slash pine needles stored for different times were evaluated against lettuce tomato and cucumber seeds and
seedlings Increasing concentrations (0 1 2 4 and 8 wv) of hot and cold aqueous extracts of needles
and litter affected in different ways target plant development Growth and germination inhibition were directly
related to the highest extract concentrations (regardless of the season and mainly in hot water extracts) of
needles On the other hand stimulatory effects of litter extracts on lettuce growth were observed Growth and
germination of cucumber and tomato were not affected by pine litter as substrate when compared to rice husk
The presumable high polarity and thermal stability of slash pine leaf biomass allelochemicals and their transient
toxic effect or growth promoting impact suggest potential applications of this largely available biomass both as a
biological herbicide and growth substrate in plant propagation
1 Introduction
Native from the Northern Hemisphere Pinus is one of the most
widely distributed genera throughout different climate regions of the
globe growing either as native or alien species even in extreme habi-
tats (Rodrigues-Correcirca and Fett-Neto 2012) Despite the high economic
value currently attributed to pine wood and oleoresin (Rodrigues-
Correcirca et al 2012) there is increasing concern about the aggressive
potential of invasiveness displayed by Pinus species especially those
cultivated out of their native range of distribution (Richardson et al
2008 Rolon et al 2011) These species are dispersed by wind and there
is notably low plant diversity observed in most understories of pine
plantations (Kato-Noguchi et al 2009) This latter feature has been
considered an important trait of allelopathic interference
The term ldquoallelopathyrdquo was coined by Molisch in 1937 as a chemical
reciprocal interaction established among plants (including micro-
organisms) sharing the same site by means of the release of secondary
metabolites named allelochemicals (Rice 1984) For the most part
these metabolites are derived from the shikimic acid or isoprenoid
pathway and their biosynthesis can be modulated by biotic and abiotic
stresses (Nascimento and Fett-Neto 2010) including seasonal-related
changes (Sartor et al 2013) Allelopathy studies may range from sterile
assays (Aryakia et al 2015) to soil (Correcirca et al 2008 Sharma et al
2016) and field tests being a complex biological phenomenon to as-
certain in several circumstances due to issues of solubility release
mechanisms and stability of bioactive compounds (Scognamiglio et al
2013) Often the use of complementary methods provides more in-
formative data
The allelopathic effects of soil leachates green needles and litter
extracts of Pinus spp on germination and seedling growth aspects of
wild and crop species have been evaluated in natural and cultivated
pine stands and have proven to be stimulatory or inhibitory (Lodhi and
Killingbeck 1982 Kil and Yim 1983 Nektarios et al 2005 Akkaya
et al 2006 Machado 2007 Alrababah et al 2009 Sartor et al 2009
Kato-Noguchi et al 2011 Rolon et al 2011 Valera-Burgos et al
2012) exhibiting in some cases autotoxicity (Garnett et al 2004
Fernandez et al 2008 Zhu et al 2009 Monnier et al 2011) Studies
on potential dual allelopathic effects of Pinus elliottii Engelm (slash
httpdxdoiorg101016jindcrop201706019
Received 23 March 2017 Received in revised form 15 May 2017 Accepted 7 June 2017
Corresponding author
E-mail address fettnetocbiotufrgsbr (AG Fett-Neto)
ORIGINAL RESEARCHpublished 16 June 2016
doi 103389fpls201600849
Frontiers in Plant Science | wwwfrontiersinorg 1 June 2016 | Volume 7 | Article 849
Edited by
Juan Francisco Jimenez Bremont
Instituto Potosino de Investigacioacuten
Cientiacutefica y Tecnoloacutegica Mexico
Reviewed by
Mariacutea De La Luz Guerrero Gonzaacutelez
Universidad Autoacutenoma de San Luis
Potosiacute Mexico
Rosalia Cristina Paz
CIGEOBIO (CONICETFCEFN UNSJ)
Argentina
Correspondence
Arthur G Fett-Neto
fettnetocbiotufrgsbr
daggerThese authors have contributed
equally to this work
Specialty section
This article was submitted to
Plant Physiology
a section of the journal
Frontiers in Plant Science
Received 08 December 2015
Accepted 30 May 2016
Published 16 June 2016
Citation
de Lima JC de Costa F Fuumlller TN
Rodrigues-Correcirca KCdS Kerber MR
Lima MS Fett JP and Fett-Neto AG
(2016) Reference Genes for qPCR
Analysis in Resin-Tapped Adult Slash
Pine As a Tool to Address the
Molecular Basis of Commercial
Resinosis Front Plant Sci 7849
doi 103389fpls201600849
Reference Genes for qPCR Analysisin Resin-Tapped Adult Slash Pine Asa Tool to Address the MolecularBasis of Commercial Resinosis
Juacutelio C de Lima 1dagger Fernanda de Costa 1 dagger Thanise N Fuumlller 1
Kelly C da Silva Rodrigues-Correcirca 2 Magnus R Kerber 1 Mariano S Lima 1
Janette P Fett 1 and Arthur G Fett-Neto 1
1 Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul
Porto Alegre Brazil 2 Biological Sciences Department Regional Integrated University of Alto Uruguai and Missotildees (URI-FW)
Frederico Westphalen Brazil
Pine oleoresin is a major source of terpenes consisting of turpentine (mono- and
sesquiterpenes) and rosin (diterpenes) fractions Higher oleoresin yields are of economic
interest since oleoresin derivatives make up a valuable source of materials for chemical
industries Oleoresin can be extracted from living trees often by the bark streak method
in which bark removal is done periodically followed by application of stimulant paste
containing sulfuric acid and other chemicals on the freshly wounded exposed surface
To better understand the molecular basis of chemically-stimulated and wound induced
oleoresin production we evaluated the stability of 11 putative reference genes for the
purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis
Samples for RNA extraction were collected from field-grown adult trees under tapping
operations using stimulant pastes with different compositions and at various time points
after paste application Statistical methods established by geNorm NormFinder and
BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3
and UBI To confirm expression stability of the candidate reference genes expression
profiles of putative P elliottii orthologs of resin biosynthesis-related genes encoding Pinus
contorta β-pinene synthase [PcTPS-(minus)β-pin1] P contorta levopimaradieneabietadiene
synthase (PcLAS1) Pinus taeda α-pinene synthase [PtTPS-(+)αpin] and P taeda
α-farnesene synthase (PtαFS) were examined following stimulant paste application
Increased oleoresin yields observed in stimulated treatments using phytohormone-based
pastes were consistent with higher expression of pinene synthases Overall the
expression of all genes examined matched the expected profiles of oleoresin-related
transcript changes reported for previously examined conifers
Keywords resin Pinus gene expression normalizer genes terpene synthase
19
Chapter 2
Stimulant Paste Preparation and Bark Streak Tapping Technique for Pine Oleoresin Extraction
Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
Tapping technique comprises the extraction of pine oleoresin a non-wood forest product consisting of a
complex mixture of mono sesqui and diterpenes biosynthesized and exuded as a defense response to
wounding Oleoresin is used to produce gum rosin turpentine and their multiple derivatives Oleoresin
yield and quality are objects of interest in pine tree biotechnology both in terms of environmental and
genetic control Monitoring these parameters in individual trees grown in the fi eld provides a means to
examine the control of terpene production in resin canals as well as the identifi cation of genetic-based
differences in resinosis A typical method of tapping involves the removal of bark and application of a
chemical stimulant on the wounded area Here we describe the methods for preparing the resin-stimulant
paste with different adjuvants as well as the bark streaking process in adult pine trees
Key words Oleoresin Pine Tapping Chemical stimulant Wounding
1 Introduction
Several conifer species produce oleoresin a complex mixture of isoprenoid compounds relevant for defense against herbivores and pathogens Two major fractions can be recognized in oleoresin (a) turpentine the volatile fraction containing mono- and sesquiter-penes and (b) rosin the nonvolatile diterpene fraction Oleoresin is a forest commodity of global interest fi nding applications in diverse industry sectors Rosin is used in adhesives printing ink manufacture and paper sizing Turpentine can be used either as a solvent for paints and varnishes or as a raw material for fraction-ation of high-value chemicals used in the pharmaceutical agro-chemical and food industry [ 1 ndash 3 ]
During the extraction activity resin is obtained from the tree in a similar way as rubber tree tapping which generally involves the
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_2 copy Springer Science+Business Media New York 2016
These authors have equally contributed to this work
fettnetocbiotufrgsbr
27
Chapter 3
A Modifi ed Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines
Juacutelio Ceacutesar de Lima Thanise Nogueira Fuumlller Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes
through high-throughput sequencing technologies microarray and also northern blots RT-PCR and
RTqPCR Even though many specifi c protocols designed for plants with high content of secondary metab-
olites have been developed these are often expensive time consuming and not suitable for a wide range
of tissues Here we present a modifi cation of the method previously described using the commercially
available Concerttrade Plant RNA Reagent (Invitrogen) buffer for fi eld-grown adult pine trees with high
oleoresin content
Key words RNA Pines Concert plant RNA reagent Stem RNA extraction Oleoresin Conifers
1 Introduction
Several conifer species especially within the Pinaceae have tissues with high concentrations of phenolics terpenes and polysaccha-rides [ 1 ] Many specifi c protocols that are appropriate for plants rich in secondary metabolite s have been developed but the extrac-tion of high-quality RNA from these tissues using commercial kits is often diffi cult and usually not applicable to woody tissues [ 2 ndash 6 ] One of the major issues during RNA extraction concerns the pres-ence of phenolic compounds which oxidize and form quinones Aromatic compounds bind RNA which interferes in downstream steps and applications [ 3 7 ] Another point of concern is the har-vest of plant samples in the experimental fi eld which constitutes another obstacle in the efforts to avoid degradation of RNA [ 8 ] These problems often result in RNAs of low quality and insuffi -cient amounts especially for methodologies that normally require
These authors have equally contributed to this work
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_3 copy Springer Science+Business Media New York 2016
fettnetocbiotufrgsbr
RESEARCH PAPER
Control of resin production in Araucaria angustifolia an ancientSouth American coniferJ C Perotti1 K C da Silva Rodrigues-Correa123 amp A G Fett-Neto12
1 Plant Physiology Laboratory Department of Botany Federal University of Rio Grande do Sul (UFRGS) Porto Alegre RS Brazil
2 Center for Biotechnology UFRGS Porto Alegre RS Brazil
3 Present address Regional Integrated University of Alto Uruguai and Miss~oes (URI-FW) Frederico Westphalen RS Brazil
Keywords
Araucaria ethylene jasmonic acid nitric
oxide resin salicylic acid terpenes
Correspondence
A G Fett-Neto Plant Physiology Laboratory
Center for Biotechnology Federal University
of Rio Grande do Sul (UFRGS) PO Box 15005
Av Bento Goncalves 9500 91501-970 Porto
Alegre Brazil
E-mail fettnetocbiotufrgsbr
Editor
K Leiss
Received 22 July 2014 Accepted 11
December 2014
doi101111plb12298
ABSTRACT
Araucaria angustifolia is an ancient slow-growing conifer that characterises parts ofthe Southern Atlantic Forest biome currently listed as a critically endangered speciesThe species also produces bark resin although the factors controlling its resinosis arelargely unknown To better understand this defence-related process we examined theresin exudation response of A angustifolia upon treatment with well-known chemicalstimulators used in fast-growing conifers producing both bark and wood resin suchas Pinus elliottii The initial hypothesis was that A angustifolia would display signifi-cant differences in the regulation of resinosis The effect of Ethrel (ET ndash ethylene pre-cursor) salicylic acid (SA) jasmonic acid (JA) sulphuric acid (SuA) and sodiumnitroprusside (SNP ndash nitric oxide donor) on resin yield and composition in youngplants of A angustifolia was examined In at least one of the concentrations testedand frequently in more than one an aqueous glycerol solution applied on fresh woundsites of the stem with one or more of the adjuvants examined promoted an increase inresin yield as well as monoterpene concentration (a-pinene b-pinene camphene andlimonene) Higher yields and longer exudation periods were observed with JA and ETanother feature shared with Pinus resinosis The results suggest that resinosis controlis similar in Araucaria and Pinus In addition A angustifolia resin may be a relevantsource of valuable terpene chemicals whose production may be increased by usingstimulating pastes containing the identified adjuvants
INTRODUCTION
Many conifer species produce terpenoid-based resins that havelong been studied for their industrial importance and role indefence against attack by herbivores and pathogens The twomost important resin-producing families of conifers are Pina-ceae and Araucariaceae (Langenheim 1996) The viscous resinsecretion is generally composed of a complex mixture ofterpenoids consisting of roughly equal parts of volatile mono-(C10) and sesquiterpene (C15 turpentine) fractions and non-volatile diterpenic (C20 rosin) components (Rodrigues-Correaet al 2013) Terpenes act in a complex and multilayereddefence response providing toxicity against bark beetles andfungi bark wound sealing disruption of insect developmentand attraction of herbivore predators (Phillips amp Croteau1999)Most conifers rely on some combination of preformed and
inducible resin defences (Trapp amp Croteau 2001 Zulak amp Bohl-mann 2010) Resin defences are controlled by environmentaland genetic factors to various extents depending on species(Roberds et al 2003 Sampedro et al 2010 Moreira et al2013) Resin traits have been reported as highly variable havingmoderate heritability indicating that breeding efforts towardssuper-resinous forests are promising (Tadasse et al 2001Roberds et al 2003) Several chemicals are known as stimulantsof resin production Commercial extraction of resin from pine
trees uses periodic bark streaking and application of resin stim-ulant pastes to the wound
Resin-stimulant paste based on sulphuric acid (SuA) iswidely used for the commercial production of pine resin Cur-rent stimulant pastes usually have two chemically active com-ponents SuA to magnify the wounding and an ethyleneprecursor (2-chloroethylphosphonic acid CEPA or Ethrel ndash
ET) to stimulate resin flow (Rodrigues et al 2011 Rodrigues-Correa amp Fett-Neto 2013) Jasmonic acid (JA) and its methylester methyl jasmonate (MeJa) are among the most widelyused chemical elicitors of plant secondary metabolism It hasbeen shown that the exogenous application of MeJa or herbi-vore attack induce chemical and anatomical defence responsesin conifers such as the formation of traumatic resin ducts andresin accumulation in stems along with increased biosynthesisof terpenes and phenolics (Franceschi et al 2002 Martin et al2002 Heijari et al 2005 Zeneli et al 2006 Moreira et al 2008Gould et al 2009) JA commercial use however is limited byits high cost
The effects of exogenous salicylic acid (SA) on conifer ter-pene production have also been studied In Pinus elliottiiapplication of 10 molm3 of SA induced resin productionin wound panels but in Pseudotsuga menziesii and Sequoia-dendron giganteum it had no apparent effect on resinaccumulation (Hudgins amp Franceschi 2004 Rodrigues ampFett-Neto 2009) Nitric oxide (NO) has also emerged as an
Plant Biology 17 (2015) 852ndash859 copy 2014 German Botanical Society and The Royal Botanical Society of the Netherlands852
Plant Biology ISSN 1435-8603
2
Apesar de representarem uma fonte nutricional importante sem tratamento preacutevio o
consumo de plantas que acumulam ANPs por animais eacute limitado Isso ocorre pois em longo
prazo a ingestatildeo prolongada de plantas (especialmente sementes) que acumulam ANPs pode
representar risco agrave sauacutede uma vez que estes comprometem o funcionamento de mecanismos
basais de manutenccedilatildeo da homeostase celular e podem tambeacutem em um quadro mais severo
desencadear doenccedilas neurotoacutexicas degenerativas como por exemplo o latirismo causado
por aacutecido β-N-oxalil-l-αβ-diaminopropiocircnico (β-ODAP) (Jiao et al 2011 Kusama-Eguchi
2019)
Sob o ponto de vista de defesa vegetal como claacutessicos metaboacutelitos especializados
ANPs satildeo em sua maioria passiacuteveis de induccedilatildeo por estresses de natureza bioacutetica eou
abioacutetica como o ataque de herbiacutevoros exposiccedilatildeo agrave radiaccedilatildeo UV e aplicaccedilatildeo exoacutegena de
elicitores quiacutemicos por exemplo No que concerne ao estudo dos efeitos da induccedilatildeo abioacutetica
sobre o acuacutemulo de ANPs em diferentes espeacutecies vegetais (Monocotiledocircneas e
Eudicotiledocircneas) as moleacuteculas mais amplamente investigadas ateacute o momento satildeo GABA
L-DOPA e mais recentemente mimosina (vide Tabela 1 do capiacutetulo primeiro) Em termos
de efeitos causados a partir da aplicaccedilatildeo exoacutegena de ANPs GABA tambeacutem figura como o
principal aminoaacutecido investigado seguido de L-DOPA e canavanina (vide Tabela 2 do
capiacutetulo primeiro)
No primeiro capiacutetulo da presente tese estatildeo descritas as caracteriacutesticas gerais dos
principais ANPs estudados seus possiacuteveis papeacuteis bioloacutegicos in planta e seus efeitos quando
aplicados exogenamente bem como os estresses abioacuteticos capazes de induzir seu(s)
acuacutemulo(s) nos diferentes tecidos vegetais Nos segundo e terceiro capiacutetulos
respectivamente satildeo elucidados os efeitos dos tratamentos de UV-C aacutecido saliciacutelico etileno
e jasmonato (claacutessicos elicitores do metabolismo secundaacuterio vegetal) sobre o acuacutemulo de
3
mimosina em Leucaena leucocephala var glabrata (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute)
Mimosina eacute um aminoaacutecido aromaacutetico natildeo-proteico anaacutelogo da L-tirosina com
atividade toacutexica para ceacutelulas de procariotos e eucariotos Embora em menor concentraccedilatildeo
mimosina foi primeiramente identificada em Mimosa pudica sendo posteriormente detectada
em outras espeacutecies do gecircnero como Mimosa pigra por exemplo (Soedarjo amp Borthakur
1998) Seu efeito toacutexico eacute atribuiacutedo agrave capacidade de quelar metais o que impede o
funcionamento adequado das metalo-proteiacutenas que dependem dos mesmos como co-fatores
(Negi et al 2014)
A concentraccedilatildeo basal de mimosina em espeacutecies de leucaena pode variar entre 1 e 12
do peso seco do oacutergatildeo (Soedarjo amp Borthakur 1998) Como eacute comum para outros ANPs
que ocorrem em espeacutecies de leguminosas em sementes de Leucaena spp eacute observada uma
maior concentraccedilatildeo de mimosina quando comparada aos demais oacutergatildeos da planta
(Rodrigues-Correcirca et al 2019) sendo esta a fonte de extraccedilatildeo comercial do padratildeo quiacutemico
de mimosina vendido por empresas de reagentes de pesquisa
Diversas atividades foram descritas para mimosina em outros organismos eou tipos
celulares Dentre essas destacam-se a atividade anti-mitoacutetica ou bloqueadora do ciclo
celular em ceacutelulas de eucariotos e procariotos Isto ocorre porque a mimosina impede a
formaccedilatildeo da forquilha de replicaccedilatildeo (e portanto a siacutentese de DNA) interrompendo assim o
avanccedilo do ciclo de divisatildeo celular na fase tardia G1 (Lalande 1990) Foram tambeacutem descritas
para mimosina atividade alelopaacutetica observada sobre o desenvolvimento de outras espeacutecies
de leguminosas e atividade antioxidante entre outras (Tabela 1)
A rota de biossiacutentese de mimosina eacute compartilhada em grande parte com a de cisteiacutena
um aminoaacutecido proteico sulfurado (Figura 1) A siacutentese da cisteiacutena se daacute a partir da conversatildeo
4
de serina e acetil-CoA em o-acetilserina pela enzima SAT (serina acetiltransferase) seguida
da conversatildeo de o-acetilserina e aacutecido sulfiacutedrico em cisteiacutena em uma reaccedilatildeo catalisada pela
OAS-TL (o-acetilserina tiol-liase) A siacutentese de mimosina por sua vez eacute compartilhada com
a da cisteiacutena ateacute esse ponto e acredita-se que pelo menos uma das isoformas de OAS-TL
catalise a conversatildeo de o-acetilserina e 3-hidroxi-4-piridona em mimosina
Tabela 1 Atividades descritas para mimosina de Leucaena leucocephala (Lam) de Wit
ATIVIDADE
ALVO AVALIADO
(organismo eou tecido tipo
celular)
REFEREcircNCIA
Bloqueio do complexo de ativaccedilatildeo
da preacute-replicaccedilatildeo do DNA
Ceacutelulas de mamiacuteferos
KUBOTA et al
(2014)
Alteraccedilatildeo no ciclo ovariano e
extensatildeo da duraccedilatildeo do corpo luacuteteo
bovino no periacuteodo poacutes-parto
Bovinos
(Bos taurus x
Bos indicus)
BOTTINI-
LUZARDO et al
(2015)
Supressatildeo do ciclo celular e reduccedilatildeo
da abundacircncia bacteriana em
mosquitos
Wolbachia pipientis
Aedes albopictus
FALLON
(2015)
Accedilatildeo inibitoacuteria da fibrose
pulmonar induzida
Ratos SD
LI et al
(2015)
Recuperaccedilatildeo da funccedilatildeo do
miocaacuterdio poacutes-isquemia
Miocaacuterdio de ratos (SD)
machos
CROWE et al
(2001)
Inseticida
Heteropsylla cubana
Crawford 1914 e Thrips tabaci
Lindemann 1889
AHMED et al
(2016)
Alelopaacutetica
Albizia procera Vigna
unguiculata Cicer arietinum
Cajanus cajan
AHMED et al
(2008)
Antioxidante
Sistemas modelo de oxidaccedilatildeo
lipiacutedica (β-caroteno - aacutecido
linolecircico e lecitina)
BENJAKUL et al
(2013)
Ateacute momento versotildees divergentes sobre a enzima responsaacutevel pela biossiacutentese de
mimosina (mimosina sintase) tecircm sido publicadas Em 1990 Ikegami e colaboradores
5
identificaram uma OAS-TL responsaacutevel pela formaccedilatildeo de cisteiacutena como sendo tambeacutem uma
mimosina sintase Mais tarde Yafuso et al (2014) realizaram a expressatildeo heteroacuteloga do gene
que codifica para OAS-TL em Escherichia coli e natildeo foi observada a formaccedilatildeo de mimosina
mesmo quando dadas as condiccedilotildees oacutetimas para tanto Mais recentemente Harun-Ur-Rashid
et al (2018) elucidaram a mimosina sintase como sendo uma isoforma da OAS-TL
corroborando o postulado por Ikegami e colaboradores em 1990
Figura 1 Rota de biossiacutentese da mimosina Fonte Ikegami et al (1990)
Espeacutecies estudadas
Leucaena leucocephala (Lam) de Wit (leucaena koa haole ou ldquoacaacutecia exoacuteticardquo na
liacutengua Hawairsquoiana) eacute uma espeacutecie de haacutebito arboacutereo ou arbustivo pertencente agrave famiacutelia
Fabaceae de Angiospermas e caracterizada pelo acuacutemulo de mimosina em todos os seus
oacutergatildeos Eacute nativa da Ameacuterica Central (especificamente da regiatildeo sudeste do Meacutexico) mas
irradiou-se atraveacutes de praticamente todas as zonas tropicais e subtropicais da Terra No
Brasil leucena eacute amplamente distribuiacuteda e classificada como naturalizada pelo REFLORA
(2019) ocorrendo em todo territoacuterio Nacional Satildeo reconhecidas no miacutenimo duas
6
subespeacutecies de leucena ocorrentes no Brasil L leucocephala var leucocephala e L
leucocephala var glabrata sendo a primeira a mais abundante
Leucaena apresenta atributos morfoloacutegicos caracteriacutesticos das leguminosas como o
fruto do tipo vagem deiscente no periacuteodo poacutes-maturaccedilatildeo folhas compostas e bipinadas As
flores satildeo seacutesseis actinomorfas e polistecircmones apresentam caacutelice sinseacutepala e corola
gamopeacutetala e satildeo dispostas em inflorescecircncias do tipo glomeacuterulo (Figura 2)
Figura 2 Oacutergatildeos vegetativos e reprodutivos de L leucocephala (Lam) de Wit Fonte Little Jr amp Skolmen
(1989)
Com base no conhecimento etnobotacircnico disponiacutevel acerca dessa espeacutecie em
diversas regiotildees tropicais e subtropicais leucena eacute utilizada para vaacuterios fins Extratos de
diferentes oacutergatildeos de leucena apresentam atividade anti-diabeacutetica (Kuppusamy et al 2014
Chowtivannakul et al 2016) antioxidante (Mohammed et al 2015 Chowtivannakul et al
2016 Zarin et al 2016) antimicrobiana (Zarin et al 2016) anti-helmiacutentica (Soares et al
2015 Jamous et al 2017) bactericida (Mohammed et al 2015) acaricida (Fernaacutendez-Salas
et al 2011) anti-tumoral (Chung et al 2017) e potencializadora da resposta imune em
peixes (Verma et al 2018) entre outras
7
Leucaena apresenta alta toleracircncia agrave seca sendo capaz de enfrentar estaccedilotildees sazonais
inteiras com deacuteficit hiacutedrico sem prejuiacutezo permanente de seus oacutergatildeos e de recuperar
vigorosamente sua biomassa vegetativa tatildeo logo o regime de precipitaccedilatildeo retome a
regularidade em frequecircncia Acredita-se que a toleracircncia agrave seca apresentada por essa espeacutecie
ocorra em funccedilatildeo do acuacutemulo de mimosina nos diferentes tecidos da planta a qual
funcionaria como um agente osmoregulador responsaacutevel pela preservaccedilatildeo da integridade das
membranas a das macromoleacuteculas intracelulares em periacuteodos de escassez de aacutegua no
ambiente
Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) eacute uma leguminosa
nativa natildeo endecircmica do Brasil amplamente distribuiacuteda nos domiacutenios fitogeograacuteficos da
Caatinga do Cerrado e da Mata Atlacircntica (Simon amp Proenccedila 2000 REFLORA 2019) Como
espeacutecie pioneira (Pilatti et al 2019) exerce importante papel ecoloacutegico na recuperaccedilatildeo de
aacutereas degradadas (Bitencourt et al 2007 Silva et al 2011) no estabelecimento de processos
de sucessatildeo vegetacional
Maricaacute eacute uma espeacutecie semi-deciacutedua a deciacutedua a qual atinge ateacute 15 m em altura (e
diacircmetro agrave altura do peito de ateacute 40 cm) na idade adulta com haacutebito arboacutereo ou arbustivo
(REFLORA 2019) e espinhos caracteriacutesticos desde os estaacutegios iniciais de desenvolvimento
(Carvalho 2004) Apresenta folhas compostas alternas e bipinadas (Figura 2) amplas
inflorescecircncias brancas com flores reunidas em glomeacuterulos esfeacutericos dispostos em grandes
paniacuteculas As flores satildeo diplostecircmones actinomorfas hipoacuteginas e unicarpelares (Silva et al
2011)
Assim como descrito para leucena maricaacute eacute considerado uma espeacutecie multifuncional
sendo comumente empregada para produccedilatildeo de mel como combustiacutevel (Olkoski amp
8
Wittmann 2011) em edificaccedilotildees na carpintaria e como lsquocerca-vivarsquo (Marchiori 1993
Lorenzi 1998) entre outras aplicaccedilotildees
Figura 2 Folhas e fruto de Mimosa bimucronata (DC) Kuntze Fonte Souza-Lima et al (2017)
Em contraste com a amplitude de habitats explorados por leucena (especialmente os
aacuteridos) no Sul do Brasil maricaacute ocorre preferencialmente em ambientes uacutemidos a alagadiccedilos
em aacutereas proacuteximas agraves margens de rios (Patreze amp Cordeiro 2004) embora possa tambeacutem
ocorrer em formaccedilotildees quase exclusivas dessa espeacutecie nas encostas de morros (Jacobi amp
Ferreira 1991)
Em relaccedilatildeo agraves atividades elucidadas para os extratos de maricaacute foram relatados os
efeitos alelopaacutetico (Jacobi amp Ferreira 1991 Ferreira et al 1992) diureacutetico natriureacutetico e
caliureacutetico (Schlickmann et al 2017)
9
Hipoacutetese
Mimosina apresenta perfil dinacircmico de acuacutemulo em Leucaena leucocephala e
Mimosa bimucronata frente a estresses associado a alteraccedilotildees significativas na expressatildeo de
genes relacionados ao metabolismo deste ANP o qual contribui para mitigar o desequiliacutebrio
oxidativo inerente a vaacuterios tipos de estresse
Objetivo geral
O objetivo da presente tese foi investigar o papel bioloacutegico da mimosina endoacutegena
em leucena e maricaacute a partir da avaliaccedilatildeo do efeito de tratamentos relacionados a estresses
ou sinalizadores de estresse
Objetivos especiacuteficos
- Analisar a concentraccedilatildeo constitutiva de mimosina nos diferentes oacutergatildeos de L leucocephala
(Lam) de Wit (leucena) e M bimucronata (DC) Kuntze (maricaacute)
- Verificar se apesar do seu alto teor constitutivo em plantas de leucena o acuacutemulo de
mimosina pode ser induzido com tratamentos que mimetizam diferentes estresses a partir da
avaliaccedilatildeo do efeito de moleacuteculas sinalizadoras (aacutecido saliciacutelico jasmonato etileno) e da
exposiccedilatildeo agrave radiaccedilatildeo UV-C na modulaccedilatildeo do acuacutemulo de mimosina em leucena bem como
em maricaacute
- Determinar se a expressatildeo de genes relacionados ao metabolismo de mimosina estaacute
associada agrave induccedilatildeo por estresses fisioloacutegicos
- Avaliar o potencial antioxidante da mimosina em experimentos realizados in situ
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry
journal homepage wwwelseviercomlocateplaphy
Research article
Mimosine accumulation in Leucaena leucocephala in response to stresssignaling molecules and acute UV exposure
Kelly Cristine da Silva Rodrigues-Correcircaab Michael DH Hondab Dulal BorthakurbArthur Germano Fett-Netoalowast
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970Porto Alegre Rio Grande do Sul BrazilbDepartment of Molecular Biosciences and Bioengineering University of Hawaii at Manoa Honolulu HI 96822 USA
A R T I C L E I N F O
KeywordsLeucaena leucocephalaMimosineMimosine amidohydrolaseJasmonic acidEthyleneSalicylic acidUV-C radiation
A B S T R A C T
Mimosine is a non-protein amino acid of Fabaceae such as Leucaena spp and Mimosa spp Several relevantbiological activities have been described for this molecule including cell cycle blocker anticancer antifungalantimicrobial herbivore deterrent and allelopathic activities raising increased economic interest in its pro-duction In addition information on mimosine dynamics in planta remains limited In order to address this topicand propose strategies to increase mimosine production aiming at economic uses the effects of several stress-related elicitors of secondary metabolism and UV acute exposure were examined on mimosine accumulation ingrowth room-cultivated seedlings of Leucaena leucocephala spp glabrata Mimosine concentration was not sig-nificantly affected by 10 ppm salicylic acid (SA) treatment but increased in roots and shoots of seedlings treatedwith 84 ppm jasmonic acid (JA) and 10 ppm Ethephon (an ethylene-releasing compound) and in shoots treatedwith UV-C radiation Quantification of mimosine amidohydrolase (mimosinase) gene expression showed thatethephon yielded variable effect over time whereas JA and UV-C did not show significant impact Consideringthe strong induction of mimosine accumulation by acute UV-C exposure additional in situ ROS localization aswell as in vitro antioxidant assays were performed suggesting that akin to several secondary metabolitesmimosine may be involved in general oxidative stress modulation acting as a hydrogen peroxide and superoxideanion quencher
1 Introduction
Different plant groups synthesize a large diversity of secondary orspecialized metabolites These molecules are generally produced inresponse to biotic and abiotic environmental stresses Indeed inductionof secondary metabolism usually involves stress-generating factorswhich have also been explored in biotechnological processes aiming atthe production of target metabolites of economic interest (Matsuuraet al 2018) Metabolic control of nitrogen-containing secondarycompounds (eg alkaloids and non-protein amino acids) has beenshown to be complex and influenced by phytohormones environmentalstresses (seasonality herbivory pathogen attack drought) UV radia-tion (Holloacutesy 2002) methyl jasmonate (MeJA) salicylic acid (SA)yeast extract (Cho et al 2008) abscisic acid (ABA) heavy metals os-motic stress (Nascimento et al 2013) and mechanical wounding (Portoet al 2014)
Due to their particular trait of associating with N-fixing micro-organisms Fabaceae species (leguminous sensu lato) are often proteinrich hence the relevance of several of these species as forage Fabaceaespecies are also known for accumulating nitrogen containing secondarymetabolites which play important roles as ecochemical molecules andat least for the case of non-protein amino acids potential cell reservoirsof nitrogen (Huang et al 2011)
High contents of mimosine a toxic aromatic non-protein aminoacid are found in species of two leguminous genera Leucaena spp andMimosa spp Leucaena leucocephala (Lam) de Wit (leucaena koa haole)is a fast-growing leguminous tree native from Central America (south-eastern Mexico) widely distributed in tropical and subtropical zonesThis species is also characterized by its high tolerance to droughtamong other environmental stresses (Honda et al 2018) Leucaena canbe divided into two subspecies (i) L leucocephala subsp leucocephala(common leucaena a bushy shrub) and (ii) L leucocephala subsp
httpsdoiorg101016jplaphy201811018Received 1 August 2018 Received in revised form 9 November 2018 Accepted 14 November 2018
lowast Corresponding authorE-mail addresses krodriguescbiotufrgsbr (KCdS Rodrigues-Correcirca) mhonda2hawaiiedu (MDH Honda) dulalhawaiiedu (D Borthakur)
fettnetocbiotufrgsbr (AG Fett-Neto)
Plant Physiology and Biochemistry 135 (2019) 432ndash440
Available online 19 November 20180981-9428 copy 2018 Elsevier Masson SAS All rights reserved
T
glabrata (giant leucaena a tree) The latter has been used as a fastgrowing tree for production of wood and paper pulp The foliage ofboth common and giant leucaena is used as a fodder because of its highprotein content and palatability to farm animals The foliage containsup to 18 protein 142 crude fiber and 64 ether extractcrude fat(Soedarjo and Borthakur 1996)
Production of nitrogen-containing secondary metabolites such asmimosine requires large amounts of carbon and nitrogen resourcesNegi et al (2014) estimated that up to 21 of the carbon-nitrogenresources may be used for production of mimosine in leucaenaBrewbaker et al (1972) determined the mimosine content of 96 Lleucocephala cultivars and 8 other Leucaena species collected from 38different countries by growing them in an observational nursery inHawaii and found that basal mimosine content varied from 189 to477 of the dry weight
Mimosine is biosynthesized from OAS (o-acetylserine) and 3H4P (3-hydroxy-4-pyridone or its tautoisomer 3-hydroxy-4-pyridine) A pre-vious analysis suggested that mimosine synthase is an OAS-TL (o-acetylserine-thiol-lyase) of the cysteine biosynthesis pathway (Ikegamiet al 1990) Later however recombinant enzyme tests did not supportan OAS-TL identity of mimosine synthase (Yafuso et al 2014) Recentfindings on mimosine biosynthesis revealed that a cytosolic cysteine-OAS-TL isoform can also catalyze the formation of mimosine underspecific conditions (Harun-Ur-Rashid et al 2018)
Mimosine toxicity is related to its ability of reducing the availabilityof divalent metal ions such as Fe(II) Zn(II) Cu(II) Co(II) and Mn(II)by chelating co-factors and preventing their association with metal-dependent enzymes Furthermore this non-protein amino acid is cap-able of forming a stable complex with pyridoxal-5prime-phosphate (PLP)leading to the inactivation of PLP-dependent enzymes (eg Asp-Glutransaminase and cystathionine synthetase) (Negi et al 2014)
Mimosine features several useful biological activities such as alle-lopathic antimicrobial insecticide cell cycle inhibitor agent antic-ancer phytoremediator (Nguyen and Tawata 2016) as well as anti-oxidant (Benjakul et al 2013) Despite the relatively well establishedbiological activities of purified mimosine on other organisms or celltypes little is known about its biological role in leguminous speciesHowever it has been suggested that at least in part its activity ismainly related to defense mechanisms against some biotic and abioticstresses and as nitrogen source during fast growth (Vestena et al2001)
Suda (1960) and Smith and Fowden (1966) identified enzymes in-volved in mimosine degradation in seedling extracts of L leucocephalaand Mimosa pudica A mimosine-degrading enzyme named mimosinase(mimosine amidohydrolase EC 35161 CAS registry number 104118-49-2) (IUBMB 2018) a carbon-nitrogen lyase which degrades mimo-sine into 3H4P was later purified by Tangendjaja et al (1986) Itsbiochemical characterization was described and the cDNA was isolatedby Negi et al (2014)
Although mimosinase has been described and isolated only fewstudies on the role played by biotic and abiotic factors on the dynamicmodulation of mimosine metabolism in leguminous species have beenconducted (Vestena et al 2001 Xu et al 2018) In aseptic cultures ofleucaena mechanical injury of shoots promoted local mimosine accu-mulation (Vestena et al 2001) In the same study cultivation in pre-sence of auxin or SA in culture medium also had a positive effect on
mimosine accumulation More recently the effect of drought treatmenton gene expression of leucaena was also evaluated by Honda et al(2018) However several potential factors regulating mimosine meta-bolism need to be further examined
To date there is a lack of information on the biological role ofmimosine in planta as well as on details of its metabolic dynamicsMoreover its overt potential for pharmaceutical applications and de-velopment of new drugs as well as the possible use as tool to addressheavy metal soil contamination or plant mineral nutrition improve-ment justify additional research The objective of this study was toinvestigate the effect of stress signaling molecules and acute UV ex-posure on modulation of mimosine accumulation and metabolism in Lleucocephala spp glabrata in order to better understand its biologicalrole and to identify strategies for yield improvement aiming at ex-ploring its useful bioactivities
2 Methods
21 Plant material
For the experiments carried out to evaluate the effects of elicitors onmimosine accumulation seeds of leucaena were kindly provided by DrJames Brewbaker and harvested at CTAHRs (College of TropicalAgriculture and Human Resources of the University of Hawaii atManoa) Waimanalo Research Station at Oahu Hawaii This plantmaterial was originated from the accession K636 of Leucaena leucoce-phala ssp glabrata (Brewbaker 2008)
22 Induced mimosine content in 5-week-old giant leucaena
221 Seed germinationIn order to overcome seed coat dormancy seeds were submitted to a
chemical scarification with sulfuric acid 95ndash98 for 20min and re-peatedly rinsed in distilled water to remove any residual trace of thisreagent Then seeds were distributed in 254 cmtimes508 cm plastictrays containing 11 vv of vermiculite and commercial soil watereduntil reaching substrate field capacity Three weeks after seed imbibi-tion seedlings displaying similar size and shape (eg number of com-pound leaves and leaflets) were transplanted to individual pots(250mL) in number of three plants per container
During the experimental period (except in the UV-C radiationtreatment) all tested seedlings were kept in a growth chamber andsubmitted to controlled conditions of temperature (circa 25 degC) and ir-radiance (approximately 100 μmol photons mminus2sdot s minus1) with a photo-period of 16 h light and 8 h dark
222 Treatments2221 JA Ethephon and SA Five-week-old giant leucaena seedlingswere treated with different solutions as described in Table 1 Idealconcentrations were defined in preliminary experiments under the sameconditions indicated above At the beginning of the experiments 30plants were sprayed with 84 ppm JA 10 ppm SA 10 or 100 ppmEthephon or Milli-Qreg water (control) until the point of imminent runoffPlant pots were kept closed inside transparent plastic bags for 24 h toavoid solution volatilization Fifteen plants arranged in 5 sets of 3 (5biological replicates) were harvested 48 h and 96 h after being treated
Table 1Treatments used to modulate mimosine biosynthesis in giant leucaena
ELICITOR CONCENTRATION UV FLUENCE EXPOSURE TIME RATIONALE FOR USE
Salicylic acid (SA) 10 ppm 24 h Pathogen signaling molecule (Shah 2003)Jasmonic acid (JA) 84 ppm 24 h Chemical elicitor of plant secondary metabolism (Dar et al 2015)Ethephon 10 ppm 24 h Ethylene releasing-compound (Kim et al 2016) elicitor of plant secondary metabolism (Wang
et al 2016)UV-C radiation 3 Jcmminus2 10min or 15min Elicitor of plant secondary metabolism (Kara 2013 Neelamegam and Sutha 2015)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
433
After collection shoots were separated from roots immediately frozenin liquid nitrogen and stored at ndash 80 degC prior to HPLC analyses
2222 UV-C Thirty seedlings of giant leucaena were exposed to UV-Cradiation (3 Jcmminus2) for 10 or 15min and kept in a growth chamberunder controlled conditions as described above until the end of theexperiments Fifteen plants arranged in groups of 3 were harvested at96 h and 120 h after UV-C exposure and processed as previouslydescribed
223 Mimosine extractionMimosine extraction was based on a modified version of the pro-
tocol published by Lalitha and Kulothungan (2006) as follows a knownweight of fresh tissue (shoots or roots) of giant leucaena was first addedto Milli-Qreg boiling water in a proportion of 110 (g of plant per mL ofsolvent) in test tubes Tubes were covered with foil to avoid solutionevaporation and placed on a hot stirrer at 100 degC for 10min A pro-portional volume of 01M HCl was added to the cooled suspensions andhomogenized using mortar and pestle The plant extracts were filteredthrough cotton and centrifuged twice for 7min in a bench top re-frigerated centrifuge at 4 degC and 13200 rpm Before being analyzed theextracts were diluted 13 with ondashphosphoric acid (OPA)
224 Mimosine detectionHPLC analyses were carried out as described by Negi and Borthakur
(2016) Pure mimosine (L-mimosine from koa haole seeds Sigma-Al-drich CAS number 500-44-7) was used as standard Separation andquantification of mimosine was done with a C18 column (PhenomenexC18 5 μm 46times250mm) under an isocratic solvent system of 002MOPA with a linear flow rate of 1mLsdotminminus1 Mimosine detection wasdone at 280 nm by photodiode array detection (200ndash400 nm) showingretention time of 277 plusmn 0042min Quantification was done using themethod of external standard curve Further confirmation of mimosineidentity was performed by co-chromatography with standard and peakpurity check Chromatograms were analyzed using the Waters Em-power 3 software
23 Quantitative real-time PCR analysis of mimosinase gene expression
Fifteen 8-week-old giant leucaena plants arranged in 4 sets of 3 (4biological replicates) were treated with either water (control) or10 ppm Ethephon 84 ppm JA acid or 15min of UV-C radiation ex-posure following the methods described above Following treatmentleucaena plants were harvested at 48 and 96 h or 72 and 144 h (UV-Ctreated plants only) after treatments Total RNA of samples was ex-tracted and purified from roots and shoots of giant leucaena by meansof a modified method using Qiagen RNeasy Plant Kit (Valencia CAUSA) and Fruit-mate (Takara Japan) according to the protocol de-scribed by Ishihara et al (2016a) The assessment of RNA quality andquantity was carried out at 230 260 and 280 nm by using a NanoDropSpectrophotometer ND-1000 (NanoDrop Technologies DE USA) Inorder to avoid genomic DNA contamination RNA samples were treatedwith TURBO DNAfree Kit (Invitrogen Carlsbad CA) Two microgramsof DNase-treated RNA were used to synthesize the first-strand cDNAusing M-MLV Reverse Transcriptase (Promega WI USA)
Quantitative real-time (qPCR) analysis was carried out to examinepossible differential expression of the mimosinase gene (GenBank ac-cession number AB2985971) in seedlings treated with 84 ppm JA10mM Ethephon or 15min of UV-C exposure Shoots and roots wereharvested 24 h before the time of mimosine concentration peak for eachtreatment previously observed as assessed by HPLC assays The 10 μLqPCR reaction consisted of 5 μL of PowerUpTM SYBRreg Green MasterMix (Applied Biosystems Foster City CA) 1 μL MgCl2 (50mM) 03 μLforward primer (10 μM) 03 μL reverse primer (10 μM) and 1 μL cDNAfirst-strand In the experimental validation through qPCR reactionconditions and melting curve analysis of the amplicon were performed
following the protocol published by Ishihara et al (2016b) for the sameleucaena variety qPCR analysis was conducted using StepOnetrade Real-Time PCR System (Applied Biosystems) Measurements were performedusing 4 biological and 3 technical replicates Relative expression wascalculated with the 2-ΔΔct method using OAS-TL as reference gene sinceits expression showed a consistently stable profile comparable to that ofUBQ-5 and ELF1α expressions Mimosinase primer sequences used forthese analyses were (FWD) 5prime- GAA AGG CAG GAA TCA CAG TGA AGAG ndash 3rsquo (REV) 5prime GGA GAC TCT AGC CAC ACC AAC TTA ndash 3rsquo
24 Antioxidant assays
241 Mimosine effect on hydrogen peroxide (H2O2) accumulationAs a follow up to the induction of mimosine accumulation profiles
under stress signals and conditions tests were conducted to verify mi-mosine antioxidant capacity In situ histological localization of hy-drogen peroxide (H2O2) accumulation was evaluated on foliar disks ofPhaseolus vulgaris L according to the protocol described by Shi et al(2010) Briefly the plant foliar tissue was exposed to 1 mgmiddotmLminus1 dia-minobenzidine (DAB) solution in 10 mM KH2PO4 (control) in presenceor absence of 10mM mimosine (equivalent to the average mimosineconcentration induced by UV-C radiation in giant leucaena) or 10mMascorbic acid (positive antioxidant control) Oxidative response wasidentified by the formation of a brown polymer on the injured leafareas indicating the presence of H2O2 and registered in a Leica M165FC stereomicroscope (Leica Microsystems)
242 Mimosine quenching of superoxide radicalsGeneration of superoxide radical and subsequent analysis was per-
formed by a modified protocol based on Zhishen et al (1999) Nitroblue tetrazolium (NBT) reduction was used to measure superoxide an-ions quenching activity Shortly a 50mM KH2PO4 pH 78 solutioncontaining 6 μM riboflavin 100mM methionine 1 mM NBT in pre-sence or absence of 5mM mimosine was exposed to white light(22 Jsdotcmminus2) for 25min on a white light transilluminator Five micro-molar rutin was used as positive control (Matsuura et al 2016) Theabsorbance was read at 560 nm before and after light exposure in aSpectraMaxreg M2 Microplate Reader (Molecular Devices LLC)
25 Statistical analyses
For HPLC and superoxide anions data simple analyses of variance(ANOVA) followed by Tukey or Welch ANOVA followed by Dunnetts Ctest were used as appropriate for data distribution characteristics InqPCR analysis results were analyzed by t-test In all cases at least fourbiological triplicates were used and experiments were repeated twiceindependently All data were analyzed using the statistical packageSPSS 200 for Windows (SPSS Inc USA) In all cases a ple 005 wasused
3 Results and discussion
31 Increased mimosine concentrations in giant leucaena treated withchemical elicitors
Leucaena produces high amounts of mimosine that accumulate in allparts of the plants including leaves stem flowers pods seeds rootsand root nodules (Soedarjo and Borthakur 1998) The highest con-centrations of mimosine can be found in the growing shoot tips andseeds (Wong and Devendra 1983) It is not known why leucaena pro-duces such high amounts of mimosine Negi et al (2014) estimated thatleucaena plants would be able to grow 21 larger if the nutrient re-sources spent on mimosine production were diverted for biomass in-crease In a previous analysis performed to quantify the basal con-centration of mimosine present in adult plants of common leucaena thehighest constitutive amount of mimosine per gram of fresh weight in
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
434
the analyzed organs was found in post-anthesis flowers (89448 μg)followed by green pods (82687 μg) leaves (67358 μg) and greenflower buds (51247 μg) which showed significantly less mimosineconcentration compared to the other reproductive structures(Supplementary Fig 1) Since mature seeds have very low moisturecontent (Wencomo et al 2017) its mimosine concentration was esti-mated as 338253 μgsdotgminus1 of dry weight Additionally it was also ob-served that the basal mimosine distribution in shoots of field-grownadult plants of leucaena is dependent on the variety type(Supplementary Table 1)
Phytohormones such as salicylic acid and jasmonic acid are knownto be produced by plants in response to various abiotic and bioticstresses These phytohormones trigger adaptive responses to stress byregulating major plant metabolic processes such as photosynthesisnitrogen metabolism defense systems and plant-water relationsthereby providing protection (for review see Khan et al 2015)
Secondary or specialized metabolite production and accumulationare also known to be controlled by biotic and abiotic stresses (Matsuuraet al 2018) In this study exposure of 5-week-old giant leucaenaseedlings to JA or Ethephon treatments significantly enhanced mimo-sine accumulation in shoots and roots in at least one of the two timepoints tested (48 and 96 h) albeit in a different way (Fig 1) Thehighest concentrations of mimosine in shoots were found in seedlingstreated with JA 84 ppm (43441 μgsdotgminus1) and Ethephon 100 ppm(38412 μgsdotgminus1) two days after application of the respective phyto-hormones Nevertheless after four days shoots yielded the highestconcentration of mimosine (approximately 460 μgsdotgminus1) upon treatmentwith 10 or 100 ppm Ethephon (Fig 1A) In roots after two and four
days JA 84 ppm and Ethephon 10 ppm resulted in highest mimosineaccumulation 18488 μgsdotgminus1 and 15801 μgsdotgminus1 respectively (Fig 1B)These observations show that mimosine accumulation response tospecific elicitors may vary over time after exposure
Although all treatments were applied exclusively on shoots of giantleucaena seedlings roots of some of them were also able to respond tothe different elicitors Overall shoots displayed higher basal and in-duced mimosine concentration compared to roots (Fig 1) which agreeswith previous observations in 1 to 3-week-old aseptic seedlings ofcommon leucaena (Vestena et al 2001) However as previouslymentioned significant post-induction increase of mimosine concentra-tion in roots and shoots simultaneously was only observed for JA andEthephon 10 ppm on day 02 and 04 respectively (Fig 1)
It is well established that perceived regulatory signals or elicitorsgenerate a transduction network mediated by secondary messengersresulting in changes in gene expression profiles that afford adaptiveresponses to environmental stimuli These modulation events are oftenmediated by transcription factors (TFs) which directly bind to specificgene promoters or act by forming complexes with repressor proteinslabeling them to degradation subsequently releasing other TFs toproceed with the gene expression program This is the case of the actionmechanism of JA and its active form jasmonoyl isoleucine for example(Kazan 2015 Wasternack and Strnad 2016)
JA ethylene and SA are known as important stress regulatory sig-nals in plants JA however is thought to be the most effective signal forinduction of plant secondary metabolism (Wasternack and Strnad2016) thereby contributing to mitigation of damage caused by severalstresses (Dar et al 2015) JA is mainly derived from linolenic acid
Fig 1 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings treated with differentelicitors CTRL=Milli-Q water SA = Salicylic AcidJA= Jasmonic Acid ETH=Ethephon Bars sharing a letterof same case do not differ by Tukey test (P le 005) Capitalletters (A B) compare treatments on day two and lowercaseletters (a b) compare treatments on day four Indicatessignificant statistical difference between day two and dayfour in the same treatment by t-test (Ple 005) The errorbars represent standard error of five replicates (each meanwas calculated with 15 individual seedlings organized in 5groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
435
(Wasternack and Strnad 2016) playing important roles in differentprocesses of plant growth and development such as plant defensemechanisms against herbivory pathogen attack fungal elicitation andsome abiotic factors such as osmotic temperature and salt stresses (Daret al 2015)
JA and its methyl ester MeJA have several different effects on le-guminous species MeJA exogenous application has increased iso-flavonoid content in cell suspension cultures of Pueraria candollei varcandollei and P candollei var mirifica (Korsangruang et al 2010) aswell as the production of the triterpenoid glycyrrhizin in Glycyrrhizaglabra roots Enhanced production of the triterpenoid however waspartly at the expense of root growth (Shabani et al 2009) MeJA ap-plication on shoots was observed to suppress root nodulation and lat-eral root formation in Lotus japonicus (Nakagawa and Kawaguchi2006) In grapevine a non-leguminous species proteinogenic aminoacids did not show an expressive increase under MeJA treatment(Gutieacuterrez-Gamboa et al 2017)
The effects of the application of four different jasmonate forms (JAMeJA jasmonoyl-L-isoleucine (JA-Ile) and 6-ethyl indanoyl glycineconjugate (2-[(6-ethyl-1-oxo-indane-4-carbonyl)-amino]-acetic acidmethyl ester - CGM) on leucaena metabolite profile has recently beenreported by Xu et al (2018) JA-Ile form was most effective althoughno major alteration was observed on monitored metabolite abundancesAlanine threonine and 34-dihydroxypyridine (34 DHP a metabolitederived from mimosine degradation) (Nguyen and Tawata 2016)among others were the major metabolites elicited by JA-Ile In contrastto the results described here mimosine concentration did not changesignificantly These divergent results on mimosine accumulation maybe due to a number of factors including mode of application jasmonateform used (JA-Ile x JA) and L leucocephala subspecies (common x giantleucaena)
Ethylene is also a phytohormone involved in plant response me-chanisms to different types of challenges such as mechanical damageand insect attack among others The integration mechanism betweenJA and ethylene signaling pathways is not completely understoodhowever it has been shown that they may work cooperatively in abioticstress tolerance (Kazan 2015) MeJA can induce ethylene production(Zhao et al 2004) and when applied simultaneously these moleculesseem to work in a synergic way by enhancing the magnitude of theplant response to external stimuli (Liu et al 2016)
Treatment with SA was able to significantly increase mimosine ac-cumulation in 12-week-old plants of common leucaena (SupplementaryFig 2) However no significant effect of SA treatment on mimosineconcentration was seen in 5-week-old seedlings of giant leucaena(Fig 1) suggesting some degree of genotype andor age dependency inelicitation by this phytohormone On the other hand several treat-ments including 90 ppm MeJA 10 and 100 ppm 2-chloroethylpho-sphonic acid (CEPA an ethylene-releasing compound) significantlyincreased mimosine accumulation (Supplementary Fig 2) in agree-ment with the data obtained for giant leucaena The lack of systemiceffects of externally applied SA on mimosine accumulation was alsoobserved when the phytohormone was supplied in the culture mediumof aseptically-grown seedlings in which case only roots had highercontent of mimosine (Vestena et al 2001) This could be due totransport limitations or to low methyl salicylate production from ap-plied SA since the former is recognized as the main systemic signalingform (Vlot et al 2009)
32 Increased mimosine concentrations in giant leucaena exposed to UV-Cradiation
UV-C treatment promoted increased concentration of the aminoacid in shoots but not in roots of giant leucaena (Fig 2) Increasedaccumulation of mimosine in shoots was also observed in 12-week-oldseedlings of common leucaena exposed to UV-C radiation for 10 and15min (Supplementary Fig 3) Similar to the SA treatment in giant
leucaena UV-C radiation did not induce mimosine biosynthesis in rootsregardless of time after exposure The absence of mimosine induction inroots by SA and UV indicates that these effectors do not cause a sys-temic response Moreover roots are shielded from irradiance by thepresence of substrate
UV radiation effects on different aspects of plant metabolism anddevelopment have been described However compared to UV-B (en-vironmentally relevant type of UV radiation) assays there are less re-ports related to the UV-C effects on secondary metabolites biosynthesisand accumulation (Cetin 2014) especially in leguminous (Fabaceae)plants They generally concern primary metabolism aspects such asgrowth and development For instance seedlings of Phaseolus vulgaris L(Fabaceae) exposed to low intensity UV-C radiation have displayeddecreased chlorophyll content and reduced height after 14 days of ex-posure (Kara 2013) Negative effects on growth parameters and ni-trogen metabolism were also observed in Vigna radiata L (Fabaceae)after UV-B radiation treatment in addition to adverse effects on JA SAand antioxidant compounds accumulation (Choudhary and Agrawal2014a) The same authors reported increased accumulation of flavo-noids SA and JA besides negative effects on growth biomass yieldnitrogen fixation and accumulation in 2 cultivars of Pisum sativum L(Fabaceae) under elevated UV-B treatment (Choudhary and Agrawal2014b) Despite the negative UV influence on growth reported for thepreviously mentioned leguminous UV-C radiation on groundnut plants(Arachis hypogaea L Fabaceae) increased seedling vigor and biomassand had no adverse effect on germination or other development para-meters (Neelamegam and Sutha 2015)
Besides its impact on growth and primary metabolism UV exposurecan cause important changes in secondary metabolism depending onintensity and time of exposure (Matsuura et al 2013) UV-B and UV-Cpre-treatments of Artemisia annua (Asteraceae) seedlings yielded in-creased biosynthesis of artemisinin a drug which displays anti-malarialproperties and activity against some others infectious diseases (egschistosomiasis leishmaniasis and hepatitis B) and several kinds oftumors (Rai et al 2011) The accumulation of nicotine in Nicotianarustica plants (Solanaceae) was also increased by UV-C treatment(Tiburcio et al 1985) Similar inducing effects on production of severalsecondary metabolites were observed in callus cultures of Vitis viniferaL Oumlkuumlzgoumlzuuml (grapevine Vitaceae) treated with a UV-C source for 5 or10min (Cetin 2014)
Regarding amino acid biosynthesis in response to UV radiationMartiacutenez-Luumlscher et al (2014) have found that in spite of not causingchanges in total amino acid content UV-B radiation exposure can affecttheir profile in grape berries Proteinogenic amino acids have beenknown to be important targets of the deleterious effects of UV radiation(Holloacutesy 2002) On the other hand in the present study acute UV-Ctreatment was found to increase mimosine accumulation in shoots byover twofold (Fig 2) which may suggest a possible participation of thismolecule as part of the antioxidant defense system in L leucocephalaThis possibility is further supported by the induction of the amino acidaccumulation by JA and Ethephon involved in abiotic and biotic stressresponses which are generally associated with oxidative imbalance andare signaling components in high UV stress (Matsuura et al 2013)
33 Mimosinase gene expression
In order to determine if increases in mimosine content upon ex-posure to JA CEPA or UV-C radiation were related to changes intranscription of mimosine metabolism-related genes RT-qPCR analysiswas carried out The complete pathway for mimosine biosynthesis hasnot yet been determined although the final step has been character-ized Based on transcription analysis (Ishihara et al 2016a) leucaenaappears to encode for multiple cysteine synthases one or more of whichmay be able to catalyze mimosine synthesis In addition a leucaenagene encoding a mimosinase (an enzyme responsible for mimosinedegradation) has been identified and characterized (Negi et al 2014)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
436
In addition to mimosinase gene expression several gene isoformsbelonging to the cysteine pathway [cysteine synthase (CYS SYN) serineacetyltransferase (SAT) and β-cyanoalanine synthase (CAS) Table 2 -supplementary material] were also tested in this study (data notshown) However expressions of these genes did not vary in giantleucaena throughout the experiments suggesting that the increasedcontent of mimosine observed in the treated plants might not be relatedto the expression of these genes but presumably to increased enzymeactivity andor release from conjugates such as mimoside a mimosineβ-D-glucoside (Murakoshi et al 1972)
Considering the time variation of mimosine accumulation observedin this work mimosinase gene expression in shoots and roots wasevaluated 24 h before the increase of mimosine concentration in giantleucaena seedlings (ie 24 h and 72 h after the chemical elicitorstreatments and 48 h and 120 h after UV-C exposure)
Ethylene signaling has been shown to up-regulate expression ofseveral genes related to secondary metabolism pathways as is the caseof phenolic compounds (Liu et al 2016) and terpenoid indole alkaloids(Wang et al 2016) Among all elicitors tested in the present workEthephon was the only one able to significantly change mimosinasegene expression Leucaena plants treated with Ethephon showed sig-nificant increases in mimosine concentration at both day 2 and 4 fol-lowing treatment which coincided with low-level expression of mi-mosinase Up-regulation of mimosinase gene expression was detected24 h before the increase of mimosine concentration in shoots treatedwith 10 ppm of Ethephon (Fig 3A) but not after JA or UV-C treatments(Fig 3C-D and 3E-F respectively) Nevertheless 72 h after treatment
application (24 h before the highest mimosine content measured inshoots) down regulation of mimosinase gene was seen in both shootsand roots treated with 10 ppm of Ethephon (Fig 3B) These data in-dicate that mimosine content in leucaena plants is at least partlyregulated by mimosinase expression in Ethephon exposed plants Onthe other hand the fact that mimosinase mRNA was not significantlyaffected by JA and UV-C treatments despite their stimulating effects onmimosine biosynthesis in giant leucaena may indicate that other levelsof regulation are at play or that the chosen harvesting time window wasunable to detect relevant changes
34 In situ and in vitro antioxidant assays
Considering the stimulation of mimosine accumulation byEthephon JA and UV all of which are often associated or known tocause oxidative imbalance the antioxidant capacity of mimosine wasevaluated Mimosine has been shown to have antioxidant activities oncultured cancer cells (Parmar et al 2015) In the present study it washypothesized that mimosine could confer radical scavenging propertieswhich would contribute to plant protection from possible damagecaused by reactive oxygen species generated during stress(Supplementary Fig 4)
Foliar disks of P vulgaris L were treated with 10mM mimosine for15min Treated disks showed less hydrogen peroxide accumulationinduced by wounding in contrast to untreated ones being comparableto those treated with ascorbic acid (a known hydrogen peroxide neu-tralizer) (Fig 4A) These observations support a possible antioxidant
Fig 2 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings exposed to UV-C lightCTRL= visible light (100 μmol photons mminus2 s minus1) UV-C 10primeand UV-C 15rsquo=UV-C exposure time (10 and 15min re-spectively) Bars sharing a letter of same case do not differ byTukey test (P le 005) Capital letters (A B) compare treat-ments on day three and lowercase letters (a b) comparetreatments on day six Indicates significant statistical dif-ference between day three and day six in the same treatmentby t-test (Ple 005) The error bars represent standard errorof five replicates (each mean was calculated with 15 in-dividual seedlings organized in 5 groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
437
role of mimosine as an in situ hydrogen peroxide scavengerMimosine was also able to quench superoxide anions generated by
light exposure Mimosine exhibited equivalent antioxidant effect com-pared to rutin (Fig 4B) a well-established effective superoxide anionquencher (Matsuura et al 2016) The radical scavenging activity ofmimosine may be due to the 3-OH group of the pyridine ring of mi-mosine (Fig 5) The pKa of the 3-OH of mimosine has been estimated tobe 88 (M Honda unpublished results) At physiological pH this OHgroup is expected to remain in a protonated state and therefore mayscavenge a radical by donating a proton and an electron In this processmimosine itself is converted to a stable radical form which is perhapsless toxic and less reactive than the reactive oxygen species generatedduring oxidative stress It is likely that the less toxic radical mimosineproduced may react with another radical or molecule and becomeconverted to a non-reactive indole molecule
In vivo antioxidant activity of mimosine has been previously eval-uated by means of its exogenous application on selenium-deficientseedlings of Vigna radiata In spite of its allelopathic properties (Ahmedet al 2008) the results showed mitigation of mitochondrial oxidativestress by treatment with 01mM mimosine (Lalitha and Kulothungan2007) DPPH radical scavenging activity was also reported for aqueous
seed extracts of leucaena rich in mimosine and phenolic compounds inin vitro assays (Benjakul et al 2014) Mimosine antioxidant activityshown in the present work is in good agreement with data reported forother non-protein amino acids such as L-DOPA (Dhanani et al 2015)and GABA (Malekzadeh et al 2014) for instance
4 Conclusion
Taken together results show that mimosine biosynthesis and ac-cumulation can be modulated by stress-related factors despite its re-latively high constitutive content in leucaena plants The pattern ofgene expression in stressed plants suggests mimosine steady-state con-trol may be regulated by its degradation in possible connection withdynamic changes in carbon and nitrogen metabolism of stressed plantsMimosine quenching activity against hydrogen peroxide and super-oxide anions in the in situ staining and in vitro assays respectivelyshowed that this non-protein amino acid can act as non-enzymaticantioxidant agent Increase in mimosine content in response to elicitorsmimicking environmental challenges in addition to its antiherbivoreand antimicrobial properties may be related to its activity as protectivemolecule against oxidative damage in line with other classes of plant
Fig 3 Relative expression of the mimosinase gene in shoots (A E and F) and shoots and roots (B C and D) of giant leucaena 24 h (A and C) 48 h (E) 72 h (B and D)and 120 h (F) after treatment with stress signaling molecules or UV-C exposure ETH = Ethephon JA = Jasmonic Acid Indicates significant statistical differencebetween control and treatment by t-test (Ple 005) The error bars represent standard error of four replicates
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
438
secondary metabolites
Funding
This work was funded by the National Council for Scientific andTechnological Development (CNPq-Brazil) grant 3060792013-5Coordenaccedilatildeo de Aperfeiccediloamento de Pessoal de Niacutevel Superior - Brazil(CAPES) - Finance Code 001 and the USDA NIFA Hatch projectHA05029-H managed by CTAHR
CRediT authorship contribution statement
Kelly Cristine da Silva Rodrigues-Correcirca InvestigationValidation Writing ndash original draft Michael DH HondaInvestigation Validation Dulal Borthakur Supervision Writing ndashreview amp editing Funding acquisition Arthur Germano Fett-NetoSupervision Funding acquisition Writing ndash review amp editing
Acknowledgements
The authors would like to thank Dr Jorge Ernesto Mariath fromLaVeg-UFRGS for kindly lending the Leica M165 FC stereomicroscopefor in situ analysis
Appendix A Supplementary data
Supplementary data to this article can be found online at httpsdoiorg101016jplaphy201811018
References
Ahmed R Hoque ATMR Hossain MK 2008 Allelopathic effects of Leucaena
leucocephala leaf litter on some forest and agricultural crops grown in nursery J ForRes 19 298 httpsdoi 101007s11676-008-0053-0
Benjakul S Kittiphattanabawon P Shahidi F Maqsood S 2013 Antioxidant activityand inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipidoxidation in model systems Food Sci Technol Int 19 (4) 365ndash376 httpsdoiorg1011771082013212455186
Benjakul S Kittiphattanabawon P Sumpavapol P Maqsood S 2014 Antioxidantactivities of lead (Leucaena leucocephala) seed as affected by extraction solvent priordechlorophyllisation and drying methods extracts against lipid oxidation in modelsystems Food Sci Technol 51 (11) 3026ndash3037 httpsdoiorg101007s13197-012-0846-1
Brewbaker JL Pluckett D Gonzalez V 1972 Varietal variation and yield trials ofLeucaena leucocephala (koa haole) in Hawaii Hawaii Agric Exp Stn Bull 166 26
Brewbaker JL 2008 Registration of KX2 ndash Hawaii interspecific-hybrid leucaena JPlant Registrations 1 (3) 190ndash193 httpsdoiorg103198jpr2007050298crc
Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitisvinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-6287-47-37
Cho H-Y Son SY Rhee HS Yoon S-YH Lee-Parsons CWT Park JM 2008Synergistic effects of sequential treatment with methyl jasmonate salicylic acid andyeast extract on benzophenanthridine alkaloid accumulation and protein expressionin Eschscholtzia californica suspension cultures J Biotechnol 135 117ndash122 httpsdoiorg101016jjbiotec200802020
Choudhary KK Agrawal SB 2014a Cultivar specificity of tropical mung bean (Vignaradiata L) to elevated ultraviolet-B changes in antioxidative defense system ni-trogen metabolism and accumulation of jasmonic and salicylic acids Environ ExpBot 99 122ndash132 httpsdoiorg101016jenvexpbot201311006
Choudhary KK Agrawal SB 2014b Ultraviolet-B induced changes in morphologicalphysiological and biochemical parameters of two cultivars of pea (Pisum sativum L)Ecotoxicol Environ Saf 100 178ndash187 httpsdoiorg101016jecoenv201310032
Dar TA Uddin M Khan MMA Hakeem KR Jaleel H 2015 Jasmonates counterplant stress a Review Environ Exp Bot 115 49ndash57 httpsdoiorg101016jenvexpbot201502010
Dhanani T Singh R Shah S Kumari P Kumar S 2015 Comparison of green ex-traction methods with conventional extraction method for extract yield L-DOPAconcentration and antioxidant activity of Mucuna pruriens seed Green Chem LettRev 8 (2) 43ndash48 httpsdoiorg1010801751825320151075070
Gutieacuterrez-Gamboa G Portu J Santamariacutea P Loacutepez R Garde-Cerdaacuten T 2017Effects on grape amino acid concentration through foliar application of three dif-ferent elicitors Food Res Int 99 688ndash692 httpsdoiorg101016jfoodres201706022
Fig 4 A In situ antioxidant assay Foliar disksof Phaseolus vulgaris L treated with (a) No an-tioxidant added (negative control) (b) 10 mMMimosine (c) 10mM ascorbic acid (positivecontrol) The oxidative damage can be seen bythe formation of a brown polymer in leaf veinsand injured areas B In vitro superoxidescavenging assay carried out with mimosineDifferent letters indicate significant differenceby Tukey test (Ple 005) The error bars re-present standard error of four replicates (Forinterpretation of the references to colour in thisfigure legend the reader is referred to the Webversion of this article)
Fig 5 Predicted mimosine radical formed followingquenching of hydroxyl radical Mimosine is first converted toa stable mimosine radical which may be then converted to anontoxic indole form
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
439
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad HossainMd Anai T Oku H 2018 Cytosolic cysteine synthase switch cysteine and mi-mosine production in Leucaena leucocephala Appl Biochem Biotechnol 186 (3)613ndash632 httpsdoiorg101007s12010-018-2745-z
Holloacutesy F 2002 Effects of ultraviolet radiation on plant cells Micron 33 (2) 179ndash197Honda MDH Ishihara KL Pham DT Borthakur D 2018 Identification of drought-
induced genes in giant leucaena (Leucaena leucocephala subsp glabrata) Trees 32571ndash585 httpsdoiorg101007s00468-018-1657-4
Huang T Jander G de Vos M 2011 Non-protein amino acids in plant defense againstinsect herbivores representative cases and opportunities for further functional ana-lysis Phytochemistry 72 1531ndash1537 httpsdoiorg101016jphytochem201103019
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of thethyrotoxic amino acid mimosine by cysteine synthase Phytochemistry 29 (11)3461ndash3465 httpsdoiorg1010160031-9422(90)85258-H
Ishihara K Lee EKW Borthakur D 2016a An improved method for RNA extractionfrom woody legume species Acacia koa A Gray and Leucaena leucocephala (Lam) deWit Int J For Wood Sci 3 (1) 031ndash035
Ishihara KL Honda MDH Pham DT Borthakur D 2016b Transcriptome analysisof Leucaena leucocephala and identification of highly expressed genes in roots andshoots Transcriptomics 4 135 httpsdoiorg1041722329-89361000135
IUBMB 2018 Enzyme Nomenclature EC 35161 httpwwwsbcsqmulacukiubmbenzymeEC35161html Accessed date 8 February 2018
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant(Phaseolus vulgaris) Biosci Res 10 (1) 29ndash32
Kazan K 2015 Diverse roles of jasmonates and ethylene in abiotic stress toleranceTrends Plant Sci 20 (4) 219ndash229 httpsdoiorg101016jtplants201502001
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect ofEthephon as an ethylene-releasing compound on the metabolic profile of Chlorellavulgaris J Agric Food Chem 64 (23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541
Khan MIR Fatma M Per TS Anjum NA Khan NA 2015 Salicylic acid-inducedabiotic stress tolerance and underlying mechanisms in plants Front Plant Sci 6 462httpsdoiorg103389fpls201500462
Korsangruang S Soonthornchareonnon N Chintapakorn Y Saralamp PPrathanturarug S 2010 Effects of abiotic and biotic elicitors on growth and iso-flavonoid accumulation in Pueraria candollei var candollei and P candollei var mir-ifica cell suspension cultures Plant Cell Tissue Organ Cult 103 (3) 333ndash342 httpsdoiorg101007s11240-010-9785-6
Lalitha K Kulothungan SR 2006 Selective determination of mimosine and its dihy-droxypyridinyl derivative in plant systems Amino Acids 31 (3) 279ndash287 httpsdoiorg101007s00726-005-0226-5
Lalitha K Kulothungan SR 2007 Mimosine mitigates oxidative stress in seleniumdeficient seedlings of Vigna radiata - Part I restoration of mitochondrial functionBiol Trace Elem Res 118 (1) 84ndash96 httpsdoiorg101007s12011-007-0013-0
Liu J Li Y Wang Y Zhang Z-H Zu Y-G Efferth T Tang Z-H 2016 Thecombined effects of ethylene and MeJA on metabolic profiling of phenolic com-pounds in Catharanthus roseus revealed by metabolomics analysis Front Physiol 71ndash11 httpsdoiorg103389fphys201600217 Article 217
Malekzadeh P Khara J Heydari R 2014 Alleviating effects of exogenous Gamma-aminobutiric acid on tomato seedling under chilling stress Physiol Mol Biol Plants20 (1) 133ndash137 httpsdoiorg101007s12298-013-0203-5
Martiacutenez-Luumlscher J Torres N Hilbert G Richard T Saacutenchez-Diacuteaz M Delrot SAguirreolea J Pascual I Gomegraves E 2014 Ultraviolet-B radiation modifies thequantitative and qualitative profile of flavonoids and amino acids in grape berriesPhytochemistry 102 106ndash114 httpsdoiorg101016jphytochem201403014
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation ofbioactive secondary metabolites by ultraviolet radiation mechanisms strategies andapplications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology forMedicinal Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash1902012
Matsuura HN Fragoso V Paranhos JT Rau MR Fett-Neto AG 2016 Thebioactive monoterpene indole alkaloid N szlig-D-glucopyranosylvincosamide is regu-lated by irradiance quality and development in Psychotria leiocarpa Ind Crop Prod86 210ndash218 httpsdoiorg101016jindcrop201603050
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo RBhambra AS Strnad M Bonfill M Fett-Neto AG 2018 Specialized plant me-tabolism characteristics and impact on target molecule biotechnological productionMol Biotechnol 60 (2) 169ndash183 httpsdoiorg101007s12033-017-0056-1
Murakoshi S Ohmiya S Haginiwa J 1972 Enzymic synthesis of mimoside a meta-bolite of mimosine in Mimosa pudica and Leucaena leucocephala Chem Pharm Bull20 (4) 855ndash857
Nakagawa T Kawaguchi M 2006 Shoot-applied MeJA suppresses root nodulation inLotus japonicus Plant Cell Physiol 47 (1) 176ndash180 httpsdoiorg101093pcppci222
Nascimento NC Menguer PK Henriques AT Fett-Neto AG 2013 Accumulation ofbrachycerine an antioxidant glucosidic indole alkaloid is induced by abscisic acidheavy metal and osmotic stress in leaves of Psychotria brachyceras Plant PhysiolBiochem 73 33ndash40 httpsdoiorg101016jplaphy201308007
Neelamegam R Sutha T 2015 UV-C irradiation effect on seed germination seedling
growth and productivity of groundnut (Arachis hypogaea L) Int J Curr MicrobiolApp Sci 4 (8) 430ndash443
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase fromLeucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol164 (2) 922ndash934 httpsdoiorg101104pp113230870
Negi VS Borthakur D 2016 Heterologous expression and characterization of mimo-sinase from Leucaena leucocephala In Fett-Neto Arthur Germano (Ed)Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods inMolecular Biology vol 1405 copySpringer Science+Business Media New York httpsdoiorg101007978-1-4939-3393-8_7 2016
Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636
Parmar F Kushawaha N Highland H George L-B 2015 In vitro antioxidant andanticancer activity of Mimosa pudica Linn extract and L-mimosine on lymphomaDaudi cells Int J Pharm Sci 12 100ndash104
Porto DD Matsuura HN Vargas LRB Henriques AT Fett-Neto AG 2014 Shootaccumulation kinetics and effects on herbivores of the wound-induced antioxidantindole alkaloid brachycerine of Psychotria brachyceras Nat Prod Commun 9 (5)629ndash632
Rai R Meena RP Smita SS Shukla A Rai SK Pandey-Rai S 2011 UV-B and UV-C pre-treatments induce physiological changes and artemisinin biosynthesis inArtemisia annua L ndash an antimalarial plant J Photochem Photobiol B Biol 105 (3)216ndash225 httpsdoiorg101016jjphotobiol201109004
Shabani L Ehsanpour AA Asghari G Emami J 2009 Glycyrrhizin production by invitro cultured Glycyrrhiza glabra elicited by methyl jasmonate and salicylic acid RussJ Plant Physiol 56 (5) 621ndash626 httpsdoiorg101134S1021443709050069
Shah J 2003 The salicylic acid loop in plant defense Curr Opin Plant Biol 6 (4)365ndash371
Shi J Fu XZ Peng T Huang XS Fan QJ Liu JH 2010 Spermine pretreatmentconfers dehydration tolerance of citrus in vitro plants via modulation of antioxidativecapacity and stomatal response Tree Physiol 30 (7) 914ndash922 httpsdoiorg101093treephystpq030
Smith IK Fowden L 1966 A study of mimosine toxicity in plants J Exp Bot 17750ndash761 httpsdoiorg101093jxb174750
Soedarjo M Borthakur D 1996 Simple procedures to remove mimosine from youngleaves pods and seeds of Leucaena leucocephala used as food Int J Food SciTechnol 31 (1) 97ndash103
Soedarjo M Borthakur D 1998 Mimosine a toxin produced by the tree-legumeLeucaena provides a nodulation competition advantage to mimosine-degradingRhizobium strains Soil Biol Biochem 30 1605ndash1613
Suda S 1960 On the physiological properties of mimosine Bot Mag Tokyo 73 (862)142ndash147 httpsdoiorg1015281jplantres188773142
Tangendjaja B Lowry JB Wills RBH 1986 Isolation of a mimosine degrading en-zyme from leucaena leaf J Sci Food Agric 37 523ndash526 httpsdoiorg101002jsfa2740370603
Tiburcio F Pintildeol MT Serrano M 1985 Effect of UV-C on growth soluble protein andalkaloids in Nicotiana rustica plants Environ Exp Bot 25 (3) 203ndash210 httpsdoiorg1010160098-8472(85)90004-8
Vestena S Fett-Neto AG Duarte RC Ferreira A 2001 Regulation of mimosineaccumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 httpsdoiorg101016S0168-9452(01)00448-4
Vlot AC Dempsey DMA Klessig DF 2009 Salicylic acid a multifaceted hormone tocombat disease Annu Rev Phytopathol 47 177ndash206 httpsdoiorg101146annurevphyto050908135202 2009
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene-induced vinblastine accumulation is related to activated expression of downstreamTIA pathway genes in Catharanthus roseus BioMed Res Int 2016 Article ID 3708187httpsdoiorg10115520163708187
Wasternack C Strnad M 2016 Jasmonate signaling in plant stress responses and de-velopment ndash active and inactive compounds N Biotech 33 (5B) 604ndash613 httpsdoiorg101016jnbt201511001
Wencomo HB Ortiz R Caacuteceres J 2017 Afr J Agric Res 12 (4) 279ndash285 httpsdoiorg105897AJAR201510604 26
Wong CC Devendra C 1983 Research on leucaena forage production in Malaysia InLeucaena Research in the Asian Pacific Region pp 55ndash60 Ottawa Ontario Canada
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX TsimKWK 2018 Jasmonate-elicited stress induces metabolic change in the leaves ofLeucaena leucocephala Molecules 23 (2) httpsdoiorg103390molecules23020188 E188
Yafuso JT Negi VS Bingham J-P Borthakur D 2014 An O-acetylserine (thiol)lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthaseAppl Biochem Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z
Zhao J Zheng S-H Fujita K Sakai K 2004 Jasmonate and ethylene signalling andtheir interaction are integral parts of the elicitor signalling pathway leading to b-thujaplicin biosynthesis in Cupressus lusitanica cell cultures J Exp Bot 55 (399)1003ndash1012 httpsdoiorg101093jxberh127
Zhishen J Mengcheng T Jianming W 1999 The determination of flavonoid contentsin mulberry and their scavenging effects on superoxide radicals Food Chem 64 (4)555ndash559 httpsdoiorg101016S0308-8146(98)00102-2
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
440
61
Supplementary Fig 1 Basal mimosine concentration in adult trees of common leucaena (L leucocephala
var leucocephala) Samples were collected from 10 field grown trees at Manoa Valley Honolulu Hawairsquoi
on June 25th 2017 Bars sharing a letter do not differ by Tukey test (P le 005) The error bars represent the
standard error
Supplementary Fig 2 Bar diagram showing mimosine concentration in shoots of 12-week-old common
leucaena seedlings treated with different elicitors CTRL = Milli-Q water SA = Salicylic Acid MeJA =
Methyl Jasmonate CEPA = 2-Chloroethylphosphonic acid (an ethylene releasing compound) Bars sharing a
letter of same case do not differ by Tukey test (P le 005) Capital letters (A B) compare treatments on day
two and lower-case letters (a b) compare treatments on day four Indicates significant statistical difference
ABB
A A
0
200
400
600
800
1000
1200
LEAVES GREEN FLOWERBUDS
POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
con
cen
trat
ion
(micro
gg
-1o
f FW
)
B AB AB AB B A
b
a
ab b
ab
0
2
4
6
8
10
12
14
16
18
20
CTRL SA 10 ppm SA 100 ppm CEPA 10 ppm CEPA 100 ppm MeJA 90 ppm
Mim
osi
ne
co
nce
ntr
atio
n (
gg
-1o
f FW
)
DAY 02 DAY 04
62
between day two and day four in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
Supplementary Fig 3 Bar diagram showing the effects of UV-C radiation exposure for 5 10 and 15 min on
mimosine accumulation in shoots of 12-week-old seedlings of common leucaena Bars sharing a letter of
same case do not differ by Tukey test (P le 005) Capital letters (A B C) compare treatments on day three
and lower-case letters (a b) compare treatments on day six Indicates significant statistical difference
between day three and day six in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
C BC AB A
bb
a
a
0
10
20
30
40
50
60
CTRL UV-C 5 UV-C 10 UV-C 15
Mim
osi
ne
co
nce
ntr
atio
n (
gg-1
of
FW)
DAY 03 DAY 06
63
Supplementary Fig 4 Model depicting induction of mimosine synthesis in leucaena following application of
stress elicitors such as CEPA and jasmonic acid or exposure to UV-C radiation The additional mimosine
synthesized may serve to alleviate oxidative stress induced by UV-C radiation
64
Supplementary Table 1 Mimosine contents in leaves of common and giant leucaena
Leucaena
type
Mimosine content
( FW)
Mimosine
content ( DW)
Dry matter
content ( FW)
Water content
( FW)
Common (1) 050 plusmn 009 245 plusmn 051 2011 plusmn 054 7989 plusmn 054
Common (2) 043 plusmn 006 214 plusmn 037 1998 plusmn 050 8002 plusmn 050
K636 (1) 070 plusmn 014 356 plusmn 077 1908 plusmn 052 8092 plusmn 052
K636 (2) 042 005 205 plusmn 033 2008plusmn 093 7992plusmn 093
KX2 (1) 122 plusmn 011 608 plusmn 082 1939 plusmn 123 8061 plusmn 123
KX2 (2) 134 plusmn 010 623 plusmn 056 2029 plusmn 114 7971 plusmn 114
KX3 (1) 044 plusmn 006 221 plusmn 030 1945 plusmn 073 8055 plusmn 073
KX3 (2) 054 plusmn 005 273 plusmn 023 1930 plusmn 038 8070 plusmn 038
KX4 (1) 086 plusmn 011 471 plusmn 065 1753 plusmn 084 8247 plusmn 084
KX4 (2) 089 plusmn 011 476 plusmn 065 180 plusmn 072 820 plusmn 072
KX5 (1) 099 plusmn 012 489 plusmn 048 1907 plusmn060 8093 plusmn 060
KX5 (2) 115 plusmn 015 548 plusmn080 1992 plusmn 053 8008 plusmn 053
Common leucaena variety koa haole grows widely on the island of Orsquoahu K636 is widely
grown variety of giant leucaena KX2 KX3 KX4 and KX5 are giant leucaena varieties
developed through interspecies hybridization (Brewbaker 2016) (1) and (2) indicate plants
from two separate locations within the University of Hawaii Waimanalo Research Center The
values are shown as mean plusmn standard error obtained from at least three biological replicates
65
Supplementary Table 2 GenBank accession numbers of the tested cysteine pathway genes isoforms
Gene name GenBank accession
OAS-TL (o-acetylserine-thiol-lyase) GDRZ01032940
GDRZ01061620
GDRZ01153117
GDSA01187555
GDSA01196891
GDSA01214467
Cys syn (cysteine synthase) GDRZ01015860
GDRZ01050898
GDRZ01086813
GDRZ01193515
GDRZ01202579
GDSA01180863
GDSA01215622
SAT (serine acetyltransferase) GDRZ01187456
GDRZ01189631
CAS (β-cyanoalanine synthase) GDRZ01054066
GDRZ01175418
GDSA01118400
66
SHORT COMMUNICATION 1
Mimosine occurrence and accumulation in Mimosa bimucronata var bimucronata (DC) 2
Kuntze 3
Kelly Cristine da Silva Rodrigues-Correcirca1 Lana Dorneles Pedroso2 Fernanda de Costa1 4
Arthur Germano Fett-Neto1 5
1Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal 6
University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970 7
Porto Alegre Rio Grande do Sul Brazil 2Department of Biological Sciences Unipampa ndash 8
Campus Satildeo Gabriel 9
Corresponding author 10
E-mail addresses krodriguescbiotufrgsbr (KCdaS Rodrigues-Correcirca) 11
lanalima2012gmailcom (LD Pedroso) fernandadecostayahoocombr (F de Costa) 12
fettnetocbiotufrgsbr (AG Fett-Neto) 13
14
15
16
17
18
19
20
21
22
67
ABSTRACT 23
Mimosine is a non-protein aromatic amino acid present in plants of Leucaena spp 24
and Mimosa spp Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) is a native 25
tree from Brazil which occurs as a pioneer species on plant succession processes In the 26
current study the presence of mimosine in M bimucronata was verified by HPLC analyses 27
Moreover mimosine accumulation upon exposure to UV-C and chemical elicitors of 28
specialized metabolism (salicylic acid - SA methyl jasmonate - MeJA sodium nitroprusside 29
- SNP and ethephon - ETH) most of which also known as promoters of the amino acid 30
production in leucaena plants was evaluated The results showed a lower concentration of 31
constitutive mimosine present in both maricaacute seedlings and mature trees when compared to 32
leucaena plants In spite of a trend towards increased mimosine accumulation observed in 33
MeJA and ETH treatments no statistical differences were found with the various stressors 34
used to induce its biosynthesis in maricaacute seedlings Data suggest that mimosine in M 35
bimucronata is probably a phytoanticipin-like metabolite or its accumulation is driven by 36
other types of stresses 37
38
39
Keywords Mimosine Mimosa bimucronata stress 40
41
42
43
44
45
46
68
Introduction 47
Mimosa bimucronata commonly known as maricaacute is a native tree from Brazil 48
(REFLORA 2019) ecologically important in plant succession and in processes of degraded 49
land recovery (Bitencourt et al 2007 Silva et al 2011) occurring as a pioneer species 50
(Pilatti et al 2019) Maricaacute is a deciduous or semi-deciduous plant which reaches up to 15 51
m in height and 40 cm of diameter at breast height (DBH) displays shrub or tree habit and 52
bears typical sharp thorns (Carvalho 2004) This species belongs to Fabaceae one of the 53
most economically important families of flowering plants due to its high diversity and 54
occurrence in different types of habitats (Gomes et al 2018) As well as several others 55
Mimosa spp maricaacute is usually referred to as a multipurpose tree (Olkoski and Wittmann 56
2011) employed for alternative medicinal uses (Champanerkar et al 2010 Silva et al 57
2011) honey production constructions and remodeling of landscape architecture (living 58
fences) for instance (Marchiori 1993 Lorenzi 1998) 59
In southern Brazil maricaacute is widely distributed and typically found either in wetland 60
areas close to river banks (Patreze and Cordeiro 2004) or composing large and almost pure 61
landscape formations on hillsides (Jacobi and Ferreira 1991) In dense populations this 62
species like several Mimosa spp (Simon and Proenccedila 2000) is considered an important and 63
highly invasive weed by preventing cattle to reach pasturesand water bodies as a result of its 64
thorny branches (Lorenzi 2008 Kestring et al 2009) Its dominant and nearly exclusive 65
pattern of distribution in those areas has led Jacobi and Ferreira (1991) to test its allelopathic 66
potential on cultivated species Indeed extracts of leaves and ripe fruits (but not the green 67
ones) of maricaacute showed phytotoxic effects on germination and initial radical growth of most 68
of the target species tested 69
69
Several investigations have been performed on maricaacute floristics (Silva et al 2011) 70
distribution (Simon and Proenccedila 2000) wood anatomy (Marchiori 1993) cytogenetic 71
parameters (Olkoski and Wittmann 2011) and allelopathic potential (Jacobi and Ferreira 72
1991 Ferreira et al 1992) However excluding two recent publications on maricaacute 73
constitutive chemical composition (Schlickmann et al 2017 Pilatti et al 2019) which 74
identified phenolic compounds (methyl gallate and water-soluble tannins) as its major 75
compounds little is known regarding this subject In other Mimosa species (eg M pudica 76
and M pigra) mimosine has been identified (Soedarjo and Borthakur 1998) as one of the 77
major specialized metabolites present in the different organs of the plant (Champanerkar et 78
al 2010) The presence of this molecule was also reported for M bimucronata in a thin layer 79
chromatography-based preliminary study performed by Ferreira et al (1992) showing co-80
chromatography of a leaf extract component with authentic mimosine The authors attributed 81
the allelopathic effect of maricaacute to the accumulation of this metabolite in its leaves 82
Mimosine is an aromatic non-protein amino acid initially found in plants of Mimosa 83
pudica and later in Leucaena leucocephala (Lam) de Wit (Soedarjo and Borthakur 1998) a 84
leguminous tree which biosynthesizes large amounts of this nitrogen-containing compound 85
(Rodrigues-Correcirca et al 2019) It is believed that the accumulation of high contents of 86
mimosine in L leucocephala tissues confers among other traits defense against herbivores 87
and pathogens (Vestena et al 2001) tolerance to drought (Negi et al 2014) as well as 88
general oxidative stress protection (Rodrigues-Correcirca et al 2019) Interestingly drought is 89
the opposite environmental and physiological condition to that observed in the wet habitats 90
occupied by native populations of M bimucronata in Brazil (Patreze and Cordeiro 2004 91
Kestring et al 2009) and Mimosa pudica Linn in India (Champanerkar et al 2010) 92
70
Nonetheless flooding is also associated with oxidative stress particularly as water levels 93
change (Fukao et al 2019) 94
In Leucaena leucocephala var leucocephala (common leucaena) and Leucaena 95
leucocephala var glabrata (giant leucaena) mimosine accumulation has been shown to be 96
both constitutive and inducible by stress-related phytohormones such as jasmonic acid (JA) 97
Ethephon (ETH an ethylene- releasing compound) salicylic acid (SA - only common 98
leucaena) (Vestena et al 2001) as well as by UV-C radiation (Xu et al 2018 Rodrigues-99
Correcirca et al 2019) On the other hand there is a lack of information regarding mimosine 100
content and elicitation effects in Mimosa spp plants 101
The aim of this study was to examine the presence of mimosine in Mimosa 102
bimucronata and examine the effects of stresses and stress-signaling molecules on its 103
accumulation in leaves 104
Material and Methods 105
Plant material 106
For all experiments the plant material was collected at Morro Santana campus do 107
Vale of UFRGS (Federal University of Rio Grande do Sul) Porto Alegre RS Brazil 108
(3004rsquoS 5108rsquoW) Authorization for access to genetic material was obtained from 109
SISGEN-Brazil (license number A845493) Constitutive mimosine content in adult plants of 110
M bimucronata var bimucronata (DC) Kuntze was determined in plant material (leaves 111
green flower buds post-anthesis flowers and green pods) harvested in January 2017 112
(summer) A voucher herbarium specimen (ICN 187953) was deposited in the ICN ndash UFRGS 113
herbarium (Herbaacuterio do Instituto de Biociecircncias of UFRGS) 114
71
For mimosine elicitation experiments legumes (fruits) of maricaacute were collected in 115
the end of June 2017 (winter) Seeds were then removed from the dry fruits and kept in the 116
dark until sowing and seedling development for use in the assays 117
Seed germination 118
To break the coat-imposed seed dormancy after surface sterilization dry seeds of 119
maricaacute were acid scarified by immersion in H2SO4 (95 ndash 98 ) for 2 min (see Correcirca et al 120
2008) and repeatedly washed in distilled water to remove any residue of the acid Then seeds 121
were distributed in 50 mL individual plastic tubes (dibble-tubes) (30 cm diameter x 120 cm 122
depth) filled up with 11 (vv) of commercial top soil and vermiculite Tubes were watered 123
every 2 days to avoid substrate dryness and were kept in a growth room under controlled 124
conditions of light (circa 75 μmol mminus2s minus1 photosynthetically active radiation photoperiod 125
of 16 h light and 8 h dark) and temperature (24plusmn2C) 126
127
Treatments 128
In order to verify inducibility of mimosine accumulation in M bimucronata fifty 12-129
week-old maricaacute seedlings (per treatment) exhibiting similar features were selected and 130
sprayed (saturated) with solutions of different chemical stressors (plant specialized 131
metabolism elicitors) as follows (for further details see Rodrigues-Correcirca et al 2019) 10 132
and 50 mM SA (pathogen-signaling molecule Shah 2003) 007 and 035 mM 2-133
chloroethylphosphonic acid (ETH ethylene releasing-compound Kim et al 2016 Wang et 134
al 2016) 100 and 200 mM MeJA (Dar et al 2015) 10 and 50 mM SNP (a nitric oxide 135
donor Perotti et al 2015) Alternatively maricaacute seedlings were also supplemented with UV-136
C radiation (13 minutes 105 kJ cm2) (elicitor of plant specialized metabolism Kara 2013) 137
72
After 2 and 4 days of exposure to the chemical treatments and 3 and 6 days of UV-138
C supplementation maricaacute shoots were harvested immediately frozen in liquid nitrogen and 139
stored at ndash 80 C until mimosine extraction and HPLC analyses 140
Mimosine extraction and detection 141
Mimosine extraction was conducted according to the modified protocol described by 142
Rodrigues-Correcirca et al (2019) for L leucocephala HPLC (Thermo Scientific Surveyor) 143
analyses (mimosine detection and quantification) were performed following previously 144
published procedures (Negi et al 2014) A C18 column (ACE C18 5 μm 46times250 mm) and 145
isocratic solvent system of 002M o-phosphoric acid with a linear flow rate of 1 mL min minus1 146
were used to separate and quantify the amino acid Mimosine detection was performed at 280 147
nm by photodiode array detection (200ndash400 nm) and retention time (229plusmn0024 min) 148
Mimosine quantification was done by means of the method of external standard curve 149
Additional confirmation of mimosine identity was performed by co-chromatography with 150
standard (Acros Organics authentic mimosine 99 used as reference) and peak purity check 151
The analyses of the chromatograms were done with the ChromQuest software 152
153
154
Results and Discussion 155
Constitutive accumulation of mimosine in M bimucronata 156
Mimosine was detected in all analyzed samples positively meeting all identification 157
criteria In agreement with what has been found for other Mimosa spp (Soedarjo and 158
Borthakur 1998) compared to L leucocephala adult plants (Rodrigues-Correcirca 2019) 159
mimosine content was lower in M bimucronata Of the adult plant tissues analyzed the 160
73
highest content of mimosine in maricaacute (per gram of fresh weight - FW) was found in post-161
anthesis flowers (36644 microg versus 89448 microg in common leucaena followed by leaves 162
(28838 microg x 67358 microg) green flower buds (28094 microg x 51247 microg) and green pods (19002 163
microg x 82687 microg) (Fig 1)The same pattern is observed for seedlings when both species are 164
compared In this study untreated 12-week-old maricaacute seedlings (control at day 2) showed a 165
shoot content of mimosine of 23029plusmn007 microg g-1 of (FW) Five-week-old untreated giant 166
leucaena seedlings cultivated in similar conditions exhibited between 83640 and 178736 167
microg g-1 of FW (Rodrigues-Correcirca et al 2019) In the same way mimosine concentration 168
percentage in dry matter of Mimosa pigra was found to be rather low (002 in nodules and 169
roots and 007 in leaves) (Soedarjo and Borthakur 1998) 170
In this investigation the lowest constitutive mimosine content was found in green 171
pods (Fig 1) This result may partly explain the absence of phytotoxic effect observed for 172
green pods on germination and growth of crop target plants tested by Jacobi and Ferreira 173
(1991) compared to the other maricaacute parts analyzed 174
Elicitation of mimosine biosynthesis in M bimucronata 175
Chemical stressors 176
Secondary metabolites (or natural products) are structural- and chemically 177
specialized compounds derived from primary metabolism These molecules are mainly 178
biosynthesized as part of a complex defense mechanism in response to biotic and abiotic 179
stresses such as pathogens herbivores water status metal toxicity and UV radiation for 180
example (Matsuura et al 2018) Ethephon SA SNP MeJA have been extensively used as 181
chemical elicitors of specialized metabolism (Wang et al 2016 Vestena et al 2001 Perotti 182
74
et al 2015 Zhang and Memelink 2009 Xu et al 2018) These phytohormonal signals can 183
simulate environmental challenges and modulate plant homeostasis often leading to 184
alterations in gene expression (Shinozaki et al 2015) Except SNP all treatments tested in 185
the present study showed positive effect on mimosine accumulation in common or giant 186
leucaena (Vestena et al 2001 Rodrigues-Correcirca 2019 Rodrigues-Correcirca unpublished 187
data) However in spite of the trend of increasing the mimosine content observed in seedlings 188
treated with 007 mM Ethephon (at day 2) and 100 mM MeJA (at day 4) no statistical 189
difference was confirmed for these treatments when compared to the control 190
On the other hand a within treatment difference on mimosine induction was seen 191
between day 2 and 4 in seedlings treated with 100 mM MeJA (Fig 2) In a lower 192
concentration (04 mM) jasmonic acid (JA)promoted a near threefold increase in mimosine 193
accumulation of giant leucaena seedlings after 2 days of application 194
UV-C radiation 195
Albeit UV-C radiation is not biologically active in natural environments it has been 196
widely used under controlled experimental conditions to generate acute responses of plant 197
specialized metabolism within a shorter period of time compared to that required to with UV-198
B radiation (Kara 2013 Cetin 2014) This fast response is due to the higher energy of UV-199
C photons that act as potent reactive oxygen species (ROS) generators causing extensive 200
damage to the cells either at the physiological level or on DNA structure (Gregianini et al 201
2003 Matsuura et al 2013) 202
Although divergent responses can be observed in plants exposed to UV-C radiation 203
the deleterious processes are usually reported on primary metabolism (decreasing of 204
chlorophyll content and plant height eg) (Kara 2013) In the present study no statistical 205
75
differences were observed in the mimosine concentration in maricaacute seedlings supplemented 206
with UV-C radiation However a decreasing in its content was found for both control and 207
treatment at day 6 post-treatment (Fig 03) Taking into account the lower constitutive 208
concentration of mimosine observed in maricaacute compared to the leucaena plants besides its 209
relative thermolability (Nguyen and Tawata 2016) it seems to be plausible to consider the 210
effect of the temperature inside the UV-C and the white light (control) chambers as an 211
additional abiotic factor contributing to the decrease of mimosine accumulation in both group 212
of plants 213
Besides mimosine identification the presence of 34-dihydroxypyridine (34-DHP or 214
3-hydroxy-4-pyridone - 3H4P) a mimosine degradation product (Negi et al 2014 Nguyen 215
and Tawata 2016) was also reported for maricaacute leaf extracts analyzed by TLC by Ferreira 216
et al (1992) In our chromatograms we detected a second large peak after that of mimosine 217
(229plusmn0024) and similar to that identified by Negi et al (2014) as 3H4P (data not shown) 218
Comparing the chromatogram profiles obtained from seedlings elicited with chemical 219
stressors and those supplemented with UV-C the largest area for this peak was found (in all 220
samples) in the latter treatment at day 6 It might indicate that the constitutive andor the 221
initially UV-C-induced mimosine was degraded into 3H4P to cope with the cellular damage 222
caused by this treatment associated with an increased temperature inside the chambers 223
Nevertheless it was not possible to determine 3H4P concentration (or confirm its identity) 224
in maricaacute plants since there is no commercial standard (pure 3H4P) available for purchase 225
to be used as a reference in calculations Establishment of improved protocols for obtaining 226
in house 3H4P reference substance by acid hydrolysis is ongoing 227
228
229
76
Conclusion 230
On the basis of the overall absence of effect of the treatments tested here on mimosine 231
concentration it is possible to suggest that its accumulation profile is similar to that of 232
phytoanticipins unlike what is observed for the same amino acid production in leucaena 233
which shows features of inducibility resembling phytoalexin-like metabolites Alternatively 234
a putative inducible pool of mimosine in maricaacute might be involved in other types of stress 235
such as extended drought periods If involved in protection against oxidative stress as 236
described for leucaena mimosine in maricaacute may act predominantly by physical quenching 237
of ROS as indicated by the lack of overt chemical degradation Nevertheless further 238
investigations are needed to assess these hypotheses 239
To sum up mimosine biosynthesis was not modulated by the treatments evaluated as 240
in L leucocephala (Lam) de Wit To the best of our knowledge this is the first work that 241
analytically identifies and quantifies mimosine accumulation in M bimucronata 242
243
REFERENCES 244
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Mimosa bimucronata (DC) O Kuntze em aacutereas degradadas pela mineraccedilatildeo de carvatildeo R 246
Bras Bioci 5 750-752 247
Carvalho PER 2004 Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 248
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Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitis 250
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Champanerkar PA Vaidya VV Shailajan S Menon SN 2010 A sensitive rapid and 253
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determination of Mimosine in Mimosa pudica Linn Nat Sci 2 713-717 255
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Gomes GS Silva GS Silva DLS Oliveira RR Conceiccedilatildeo GM 2018 Botanical 257
Composition of Fabaceae Family in the Brazilian Northeast Maranhatildeo Brazil Asian J 258
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Correcirca LR Soares GLG Fett-Neto AG 2008 Allelopathic potential of Psychotria 260
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Ferreira AG Aquila MEA Jacobi US Rizvi V 1992 Allelopathy in Brazil In Allelopathy 263
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Fukao T Barrera-Figueroa BE Juntawong P Pentildea-Castro JM 2019 Submergence 265
and waterlogging stress in plants a review highlighting research opportunities and 266
understudied aspects Front Plant Sci 10 340 httpsdoiorg103389fpls201900340 267
Gregianini TS Silveira VC Porto DD Kerber VA Henriques AT Fett-Neto AG 268
2003 The alkaloid brachycerine is induced by ultraviolet radiation and is a singlet oxygen 269
quencher Photochem Photobiol 78(5) 470ndash474 httpsdoiorg1015620031-270
8655(2003)0784070TABIIB20CO2 271
Jacobi US Ferreira AG 1991 Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK 272
sobre espeacutecies cultivadas Pesq Agropec Bras 26(7) 935-943 273
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant 274
(Phaseolus vulgaris) Biosci Res 10(1) 29ndash32 275
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Kestring D Klein J Menezes LCCR Rossi MN 2009 Imbibition phases and 276
germination response of Mimosa bimucronata (Fabaceae Mimosoideae) to water 277
submersion Aquat Bot 91 105ndash109 httpsdoiorg101016jaquabot200903004 278
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect of 279
Ethephon as an ethylene-releasing compound on the metabolic profile of Chlorella vulgaris 280
J Agric Food Chem 64(23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541 281
Lorenzi H 1998 Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 282
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 283
Lorenzi H 2008 Plantas daninhas do Brasil terrestres aquaacuteticas parasitas e toacutexicas 4 ed 284
Nova Odessa Instituto Plantarum 640 p 285
Marchiori JNC 1993 Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 286
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Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation of 288
bioactive secondary metabolites by ultraviolet radiation mechanisms strategies and 289
applications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology for Medicinal 290
Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash190= 291
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo R Bhambra AS 292
Strnad M Bonfill M Fett-Neto AG 2018 Specializedplant 293
metabolismcharacteristicsandimpactontargetmoleculebiotechnologicalproduction 294
Molecular Biotechnology 60(2) 169ndash183httpsdoiorg101007s12033-017-0056-1 295
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase from 296
Leucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 297
922ndash934 httpsdoiorg101104pp113230870 298
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Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine 299
areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636 300
Olkoski D Wittmann MTS 2011 Cytogenetics of Mimosa bimucronata (DC) O Kuntze 301
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop Breed 302
Appl Biotechnol 11 27-35 httpdxdoiorg101590S1984-70332011000100004 303
Patreze CM Cordeiro L 2004 Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 304
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecol Manag 196 275ndash305
285 httpdxdoiorg101016jforeco200403034 306
Perotti JC Rodrigues-Correcirca KCS Fett-Neto AG 2015 Control of resin production in 307
Araucaria angustifolia an ancient South American conifer Plant Biology 17 852ndash859 308
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG 2019 Mimosine 309
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UV exposure Plant Physiology and Biochemistry 135 432ndash440 311
Pilatti DM Fortes AMT Jorge TCM Boiago NP 2019 Comparison of the phytochemical 312
profiles of five native plant species in two different forest formations Brazilian Journal of 313
Biology 79(2) 233-242 314
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS 2011 Biologia da reproduccedilatildeo 315
deMimosa bimucronatandash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 316
Simon MF and Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 317
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 318
endemism Biological Conservation 96 279-296 319
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 320
LM Andrade SF Cechinel-Filho V 2017 Chemical composition and diuretic natriuretic and 321
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kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its majority 322
constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash1624 323
Shah J 2003 The salicylic acid loop in plant defense Current Opinion Plant Biology6 (4) 324
365ndash371 325
Shinozaki K Uemura M Serres JB Bray EA Weretilnyk E 2015 Responses to Abiotic 326
Stress In Buchanan BB Gruissem W Jones RL (Eds) Biochemistry and Molecular 327
Biology of Plants Second Edition John Wiley and Sons Ltd 328
Soedarjo M and Borthakur D 1998 Mimosine a toxin produced by the tree-legume 329
Leucaena provides a nodulation competition advantage to mimosine-degrading Rhizobium 330
strains Soil Biology and Biochemistry 30(12)1605-1613 331
Vestena S Fett-Neto AG Duarte RC Ferreira AG 2001 Regulation of mimosine 332
accumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 333
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene induced 334
vinblastine accumulation is related to activated expression of downstream TIA pathway 335
genes in Catharanthus roseus BioMed Research International Article ID 3708187 336
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX Tsim KWK 2018 337
Jasmonate-elicited stress induces metabolic change in the leaves of Leucaena leucocephala 338
Molecules 23 (2) 339
Zhang H Memelink J 2009 Regulation of Secondary Metabolism by Jasmonate Hormones 340
In AE Osbourn and V Lanzotti (eds) Plant-derived Natural Products 3 DOI 101007978-341
0-387-85498-4_1 copy Springer Science + Business Media LLC 342
343
344
345
81
346
Figure 1 Constitutive concentration of mimosine in different plant organs of Mimosa 347
bimucronata Bars sharing the same letter do not differ statistically by Tukey test (Ple005) 348
The error bars denote standard error of 10 replicates 349
350
351
352
353
354
355
356
357
B B A C0
5
10
15
20
25
30
35
40
LEAVES GREEN FLOWER BUDS POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
co
nce
ntr
atio
n u
gg-1
Mimosine concentration in adult plants of Mimosa bimucronata (DC) Kuntze
82
C T R L S A
1 0 m M
S A
5 0 m M
E T H
0 0 7 m M
E T H
0 3 5 m M
M e J A
1 0 0 m M
M e J A
2 0 0 m M
S N P
1 0 m M
S N P
5 0 m M
0
1 0
2 0
3 0
T re a tm e n ts
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1) D A Y 2
D A Y 4
A B C C B C A B C C A B C A B C A
a b b b a a b a a b b a b
358
Figure 2 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 359
bimucronata treated with different signaling molecules SA = Salicylic Acid ETH = 360
Ethephon MeJA = Methyl Jasmonate SNP = Sodium Nitroprusside Uppercase and 361
lowercase letters indicate statistical differences among treatments in days 2 and 4 362
respectively Bars sharing a letter of the same case do not differ statistically by Tukey test 363
(Ple005) Indicates statistical difference in the same treatment between day 2 and 4 by t-364
test (Ple005) The error bars denote standard error of 5 replicates (25 individual seedlings 365
arranged in 5 groups of 5) 366
367
368
83
D AY 3 D AY 6
0
5
1 0
1 5
2 0
2 5
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1)
C O N TR O L
U V -C
369
Figure 3 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 370
bimucronata supplemented with UV-C radiation Indicates statistical difference in the same 371
treatment between day 3 and 6 by t-test (Ple005) The error bars denote standard error of 5 372
replicates (25 individual seedlings arranged in 5 groups of 5) 373
374
375
376
377
378
379
380
381
382
383
384
385
84
Consideraccedilotildees finais 386
- Experimentos que avaliam os efeitos da aplicaccedilatildeo exoacutegena de ANPs em diferentes espeacutecies 387
vegetais tecircm sido realizados principalmente com GABA Dentre os principais efeitos 388
conferidos pela aplicaccedilatildeo dessa moleacutecula em espeacutecies de mono e eudicotiledocircneas satildeo 389
relatados a toleracircncia agrave seca agrave salinidade e agraves temperaturas extremas 390
- Como metaboacutelitos especializados claacutessicos os ANPs podem ter sua concentraccedilatildeo basal 391
endoacutegena aumentada em resposta agrave induccedilatildeo mediada por uma vasta gama de tratamentos com 392
moleacuteculas sinalizadoras de estresse e fontes alternativas de estressores De um modo geral 393
observa-se o acuacutemulo das diferentes classes de ANPs em resposta agrave radiaccedilatildeo UV elicitores 394
quiacutemicos que mimetizam ataques por patoacutegenos dano mecacircnico agentes osmoacuteticos metais 395
pesados entre outros 396
- Especificamente em leucena a resposta observada em relaccedilatildeo aos diferentes tratamentos 397
testados indica que apesar do seu alto teor constitutivo nessa espeacutecie a biossiacutentese e o 398
acuacutemulo de mimosina podem ser modulados por fatores causadores de estresses exibindo -399
nessa espeacutecie - um padratildeo de acumulaccedilatildeo similar agrave fitoalexinas Em maricaacute por outro lado 400
aumento de acuacutemulo dessa moleacutecula natildeo foi observado para os mesmos tratamentos testados 401
para leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas 402
- O padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno 403
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado pela 404
sua degradaccedilatildeo 405
- As respostas observadas nos testes que avaliaram a atividade de mitigaccedilatildeo de espeacutecies 406
reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode agir como um agente 407
antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de estresse 408
85
Perspectivas 409
- Confirmaccedilatildeo em espectrocircmetro de massas eou ressonacircncia nuclear magneacutetica da natureza 410
quiacutemica da lsquomimosinarsquo presente em maricaacute 411
- Avaliaccedilatildeo do efeito de concentraccedilotildees mais elevadas e em diferentes periacuteodos de aplicaccedilatildeo 412
das moleacuteculas sinalizadoras testadas sobre o acuacutemulo de mimosina em leucena e maricaacute 413
- Ampliar a investigaccedilatildeo dos padrotildees de expressatildeo gecircnica dos genes que codificam para 414
mimosinase (em maricaacute) mimosina sintase (em ambas as espeacutecies testadas) bem como o 415
perfil de precursores e cataboacutelitos de mimosina em resposta aos tratamentos mencionados 416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
86
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Ahmed AMM Saacutenchez FJS Bavileacutes LRY Mahdy REZ Camaal JBC (2016) Tannins and 443
mimosine in Leucaena genotypes and their relations to Leucaena resistance against 444
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model systems Food Sci Technol Int 19(4)365-76 448
httpsdoiorg1011771082013212455186 449
Bitencourt F Zocche JJ Costa S Souza PZ Mendes AR (2007) Nucleaccedilatildeo de Mimosa 450
bimucronata (DC) O Kuntze em aacutereas degradadas pela mineraccedilatildeo de carvatildeo Revista 451
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A Montes-Perez R Muntildeoz-Rodriguez D Ku-Vera J (2015) Ovarian activity and estrus 454
behavior in early postpartum cows grazing Leucaena leucocephala in the tropics Trop Anim 455
Health Prod 47(8)1481-6 456
Carvalho PER (2004) Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 457
Teacutecnica 941-10 458
Chowtivannakul P Srichaikul B Talubmook C (2016) Antidiabetic and antioxidant activities 459
of seed extract from Leucaena leucocephala (Lam) de Wit Agriculture and Natural 460
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Chung H-H Chen M-K Chang Y-C Yang S-F Lin C-C Lin C-W (2017) Inhibitory effects 462
of Leucaena leucocephala on the metastasis and invasion of human oral cancer cells 463
Environmental Toxicology 321765ndash1774 httpsdoiorg101002tox22399 464
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Crowe B Poynter JA Manukyan MC Wang Y Brewster BD Herrmann JL Abarbanell 465
AM Weil BR Meldrum DR (2001) Pretreatment with intracoronary mimosine improves 466
postischemic myocardial functional recovery Surgery 150(2) 191-106 467
Fallon (2015) Effects of mimosine on Wolbachia in mosquito cells cell cycle suppression 468
reduces bacterial abundance In Vitro Cell Dev Biol Anim 51(9)958-63 469
httpsdoiorg101007s11626-015-9918-7 Epub 2015 May 28 470
Fernaacutendez-Salas A Alonso-Diacuteaza MA Acosta-Rodriacuteguez A Torres-Acosta JFJ Sandoval-471
Castro CA Rodriacuteguez-Vivas RI (2011) In vitro acaricidal effect of tannin-rich plants against 472
the cattle tick Rhipicephalus (Boophilus) microplus (Acari Ixodidae) Veterinary 473
Parasitology 175113ndash118 2010 httpsdoiorg101016jvetpar201009016 474
Ferreira AG Aquila MEA Jacobi US Rizvi V (1992) Allelopathy in Brazil In Allelopathy 475
basic and applied aspects Rizvi V and Jacobi US (Eds) Chapman and Hall PP 243-250 476
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad Hossain Md Anai 477
T Oku H (2018) Cytosolic cysteine synthase switch cysteine and mimosine production in 478
Leucaena leucocephala Appl Biochem Biotechnol 186 (3) 613ndash632 479
httpsdoiorg101007s12010-018-2745-z 480
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of the thyrotoxic 481
amino acid mimosine by cysteine synthase Phytochemistry 29 (11) 3461ndash3465 482
httpsdoiorg1010160031-9422(90)85258-H 483
Jacobi US Ferreira AG (1991) Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK Sobre 484
espeacutecies cultivadas Pesquisa Agropecuaacuteria Brasileira 26(7) 935-943 485
Jamous RM Ali-Shtayeh MS Abu-Zaitoun SY Markovics A Azaizeh H (2017) Effects of 486
selected Palestinian plants on the in vitro exsheathment of the third stage larvae of 487
gastrointestinal nematodes BMC Veterinary Research 13308 488
httpdxdoiorg101186s12917-017-1237-7 489
Jiao CJ Jiang J-L Ke L-M Cheng W Li F-M Li Z-X Wang C-Y (2011) Factors affecting 490
β-ODAP content in Lathyrus sativus and their possible physiological mechanisms Food 491
Chem Toxicol 49 543ndash549 httpsdoiorg101016jfct201004050 492
Kubota S Fukumoto Y Ishibashi K Soeda S Kubota SS Yuki R Nakayama Y Aoyama K 493
Yamaguchi N (2014) Activation of the prereplication complex is blocked by mimosine 494
88
through reactive oxygen species-activated ataxia telangiectasia mutated (ATM) protein 495
without DNA damage J Biol Chem 28 289(9)5730-46 496
Kuppusamy UR Arumugam B Azaman N Wai CJ (2014) Leucaena leucocephala Fruit 497
Aqueous Extract Stimulates Adipogenesis Lipolysis and Glucose Uptake in Primary Rat 498
Adipocytes Hindawi Publishing Corporation e Scientific World Journal Article ID 737263 499
8 pages httpdxdoiorg1011552014737263 500
Kusama-Eguchi K (2019) Research in motor neuron diseases caused by natural substances 501
focus on pathological mechanisms of neurolathyrism Yakugaku Zasshi 139 (4) 609-502
615 httpsdoiorg101248yakushi18-00202 503
Kutchan TM Gershenzon J Moslashller BL Gang DR (2015) Natural Products In Buchanan 504
BB Gruissem W and Jones RL (eds) Biochemistry amp Molecular Biology of Plants 2nd edn 505
Wiley Blackwell Chichester pp 1135-1205 506
Lalande M (1990) A reversible arrest point in the late G1 phase of the mammalian cell cycle 507
Exp Cell Res 186 332ndash339 508
Li X-W Hu C-P Li Y-J Gao Y-X Wang XM Yang J-R (2015) Inhibitory effect of L-509
mimosine on bleomycin-induced pulmonary fibrosis in rats Role of eIF3a and p27 Int 510
Immunopharmacol 27(1) 53ndash64 511
Little Jr EL Skolmen RG (1989) Koa haole Agriculture Handbook 679 USDA 512
Lorenzi H (1998) Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 513
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 514
Marchiori JNC (1993) Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 515
O Kuntze Ciecircncia Florestal 3 85-106 516
Mohammed RS El Souda SS Taie HAA Moharam ME Shaker KH (2015) Antioxidant 517
antimicrobial activities of flavonoids glycoside from Leucaena leucocephala leaves Journal 518
of Applied Pharmaceutical Science 5(06)138-147 519
httpdxdoiorg107324JAPS201550623 520
Negi VS Bingham J-P Li QX Borthakur D (2014) A carbon-nitrogen lyase from Leucaena 521
leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 (2) 922ndash522
934 httpsdoiorg101104pp113230870 523
89
Olkoski D Wittmann MTS (2011) Cytogenetics of Mimosa bimucronata (DC) O Kuntze 524
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop 525
Breeding and Applied Biotechnology 11 27-35 526
Patreze CM Cordeiro L (2004) Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 527
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecology and Management 528
196275ndash285 529
Pilatti DM Fortes AMT Jorge TCM Boiago NP (2019) Comparison of the phytochemical 530
profiles of five native plant species in two different forest formations Brazilian Journal of 531
Biology 79(2) 233-242 532
Ramos-Ruiz R Poirot E Flores-Mosquera M (2018) GABA a non-protein amino acid 533
ubiquitous in food matrices Cogent Food Agric 41534323 534
httpsdoiorg1010802331193220181534323 535
REFLORA (2019) httpfloradobrasiljbrjgovbrreflora Acesso em agosto de 2019 536
Rodgers KJ Samardzic K Main BJ (2015) Toxic Nonprotein Amino Acids Plant Toxins 537
httpsdoiorg 101007978-94-007-6728-7_9-1 538
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG (2019) Mimosine 539
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 540
UV exposure Plant Physiology and Biochemistry 135 432ndash440 541
httpsdoiorg101016jplaphy201811018 542
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 543
LM Andrade SF Cechinel-Filho V (2017) Chemical composition and diuretic natriuretic 544
and kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its 545
majority constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash546
1624 547
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS (2011) Biologia da reproduccedilatildeo 548
de Mimosa bimucronata ndash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 549
Simon MF Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 550
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 551
endemism Biological Conservation 96 279-296 552
90
Soares AMS Arauacutejo SA Lopes SG Costa Junior LM (2015) Anthelmintic activity of 553
Leucaena leucocephala protein extracts on Haemonchus contortus Braz J Vet Parasitol 554
Jaboticabal 24(4) 396-401 httpdxdoiorg101590S1984-29612015072 555
Soerdajo M Borthakur D (1998) Mimosine a toxin produced by the tree-legume Leucaena 556
provides a nodulation competition advantage to mimosine-degrading Rhizobium strains Soil 557
Biol Biochem 30(12) 16051613 558
Souza-Lima ES Sinani TR Pott A Sartori ALB (2017) Mimosoideae (Leguminosae) in the 559
Brazilian Chaco of Porto Murtinho Mato Grosso do Sul Rodrigueacutesia 68(1) 263-290 2017 560
httpdxdoiorg1015902175-7860201768131 561
Taiz L amp Zeiger E (2010) Plant Physiology 5th edition Sinauer Associates Inc Sunderland 562
Verma VK Rani KV Kumara SR Prakash O (2018) Leucaena leucocephala pod seed 563
protein as an alternate to animal protein in fish feed and evaluation of its role to fight against 564
infection caused by Vibrio harveyi and Pseudomonas aeruginosa Fish and Shellfish 565
Immunology 76 (2018) 324ndash332 httpsdoiorg101016jfsi201803011 566
Yafuso JT Negi VS Bingham J-P Borthakur D (2014) An O-acetylserine (thiol) lyase from 567
Leucaena leucocephala is a cysteine synthase but not a mimosine synthase Appl Biochem 568
Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z 569
Zarin RMA Wan HY Isha A Armani N (2016) Antioxidant antimicrobial and cytotoxic 570
potential of condensed tannins from Leucaena leucocephala hybrid Food Science and 571
Human Wellness 5 65ndash75 httpdxdoiorg101016jfshw201602001 572
573
574
Contents lists available at ScienceDirect
Industrial Crops amp Productsjournal homepage wwwelseviercomlocateindcrop
Resin tapping transcriptome in adult slash pine (Pinus elliottii var elliottii)Camila Fernanda de Oliveira Junkes1 Artur Teixeira de Arauacutejo Juacutenior1 Juacutelio Ceacutesar de LimaFernanda de Costa Thanise Fuumlller Maacutercia Rodrigues de Almeida Franciele Antocircnia NeisKelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-NetoCenter for Biotechnology and Department of Botany Federal University of Rio Grande do Sul Porto Alegre PO Box 15005 91501-970 Brazil
A R T I C L E I N F O
KeywordsPinus elliottiResinResinosisTranscriptomeAdjuvant paste
A B S T R A C T
To better understand the bases of resin production a major source of terpenes for industry the transcriptome ofadult Pinus elliottii var elliottii (slash pine) trees under field commercial resinosis was obtained Samples werecollected from cambium after 5 and 15 days of treatment application which included tapping followed byapplication of commercial resin stimulant paste or control wounding without paste Overall mean number ofreads of all 16 libraries (2 treatments x 2 times x 4 replicated trees) was 34582048 Of these 89 were mappedagainst the reference sequence with a mismatch of 058 Using the Blast2Go 570 candidate genes were de-tected based on sequence annotation By comparing the expression profile between paste and control 310differentially expressed genes (DEGs) were identified at 5 days and 190 at 15 days with a significant fold changeof log2gt 12 Regarding changes in time comparisons within each treatment 210 and 105 DEGs were identifiedwithin control and paste treatment respectively Genes with different expression patterns in the times andtreatments examined included ethylene responsive transcription factors geranylgeranyl diphosphate synthasediterpene synthase cytochrome P450 and ABC transporters all of which may play important roles in resinproduction RT-qPCR analysis correlated well with the data obtained by RNAseq Resin composition changedover time This is the first transcriptomic investigation of resinosis of the main species used in the bioresinindustry and of molecular analyses of resinosis under field operations with implications for stand managementstimulant paste development genotype selection and breeding for high resinosis
1 Introduction
The adaptive success of conifers is largely due to the development ofa defense system based on the synthesis and secretion of terpenes in allmajor organs and different tissues (Miller et al 2005 Hall et al 2013Warren et al 2015) Conifer resin is a viscous fluid composed of acomplex mixture of terpenoids such as monoterpenes sesquiterpenesand diterpenes (Zulak and Bohlmann 2010) These terpenoids are se-creted from severed resin ducts when the tree is under biotic attack(Ralph et al 2006 Lange 2015 Geisler et al 2016) acting as pro-tectants (Schiebe et al 2012 Liu et al 2015)Biosynthesis of terpenes in conifers starts from isomerization of two
isoprenoid (C5) units dimethylallyl diphosphate (DMAPP) and iso-pentenyl diphosphate (IPP) These molecules can be biosynthesized viatwo separate routes in plants the methyl-erythritol 4-phosphate andmevalonate pathways IPP is synthesized and isomerized to DMAPP byisopentenyl diphosphate isomerase then prenyl transferases catalyze
the condensation of these two C5-units to geranyl diphosphate (Pazoukiand Niinemets 2016) Their elongation to prenyl diphosphates withaddition of IPP molecules leads to monoterpenes (C10) sesquiterpenes(C15) and diterpenes (C20) which are the substrates for terpene syn-thases (TPS) (Keeling and Bohlmann 2006b)TPSs are part of a large family of mechanistically related enzymes
involved in both primary and secondary metabolism (Keeling andBohlmann 2006b) The events of evolutionary diversification and ex-pansion of plant TPSs appear to have originated from gene duplicationsdomain losses and sub- or neofunctionalizations with subsequent di-vergence of an ancestral TPS gene of primary metabolism (Hall et al2013) Modification of TPS products changes their physical propertiesand may alter their biological activities (Chen et al 2011) TPSs of highsequence identity may have different functions even in closely relatedspecies Low sequence identity of TPSs in phylogenetically distantspecies does not preclude the possibility of independent evolution of thesame or related function of these enzymes (Zerbe and Bohlmann 2015)
httpsdoiorg101016jindcrop2019111545Received 4 January 2019 Received in revised form 10 June 2019 Accepted 4 July 2019
Corresponding authorE-mail address fettnetocbiotufrgsbr (AG Fett-Neto)1 These authors have equally contributed to this work
doi 1015900102-33062019abb0114
Acta Botanica Brasilica
Sustainable production of bioactive alkaloids in Psychotria L of
southern Brazil propagation and elicitation strategies
Yve Verocircnica da Silva Magedans1 Kelly Cristine da Silva Rodrigues-Correcirca1 Cibele Tesser da Costa1
Heacutelio Nitta Matsuura1 and Arthur Germano Fett-Neto1
Received April 1 2019Accepted June 28 2019
ABSTRACTPsychotria is the largest genus in Rubiaceae South American species of the genus are promising sources of natural
products mostly due to bioactive monoterpene indole alkaloids they accumulate ese alkaloids can have analgesic
antimutagenic and antioxidant activities in dierent experimental models among other pharmacological properties
of interest Propagation of genotypes with relevant pharmaceutical interest is important for obtaining natural
products in a sustainable and standardized fashion Besides the clonal propagation of elite individuals the alkaloid
content of Psychotria spp can also be increased by applying moderate stressors or stress-signaling molecules is
review explores advances in research on methods for plant propagation and elicitation techniques for obtaining
bioactive alkaloids from Psychotria spp of the South Region of Brazil
Keywords abiotic stress alkaloids elicitation monoterpenes plant propagation Psychotria southern Brazil
sustainability
Introduction
Psychotria belongs to Rubiaceae one of the major families
of $owering plants having economic interest e family
includes coee a few signicant poisonous plants to livestock
besides several important ornamental and medicinal species
(Souza amp Lorenzi 2012) Psychotria has captured researchersrsquo
attention mostly because of its medicinal properties
Psychotria colorata is an Amazonian species that produces
polyindolinic alkaloids with analgesic activity (Matsuura et
al 2013) e promising results obtained with P colorata
motivated the investigation of southern Brazilian Psychotria
species and the discovery of new bioactive alkaloids (Porto
et al 2009) Moreover leads on in planta alkaloid functions
were also topic of experimental evaluation
One of the key elements that needs to be addressed early
on during the process of developing new bioactive molecules
from plants is the capacity to generate catalytically active
biomass to support extraction and steady supply ere are a
number of ways through which these goals may be reached
including greenhouse rooting of cuttings (mini-cutting
1 Laboratoacuterio de Fisiologia Vegetal Departamento de Botacircnica Instituto de Biociecircncias e Centro de Biotecnologia Universidade Federal do Rio
Grande do Sul 91501-970 Porto Alegre RS Brazil
Corresponding author fettnetocbiotufrgsbr
Review
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Biomass yield of resin in adult Pinus elliottii Engelm trees is differentially
regulated by environmental factors and biochemical effectors
Franciele Antocircnia Neis Fernanda de Costa Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima
Kelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-Neto
Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) CP 15005 CEP 91501-970 Porto Alegre RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Biomass
Terpene resin
Seasonal
Benzoic acid
Regenerated forest
A B S T R A C T
Biomass of pine resin finds several applications in the chemical pharmaceutical biofuel and food industries
Resin exudation after injury is a key defense response in Pinaceae since this complex mixture of terpenes has
insecticidal antimicrobial and wound repair properties Resin yield is increased by effectors applied on the
wound area including phytohormones and metal cofactors of terpene synthases The interaction of resinosis
mechanism effectors is not fully understood particularly in adult forest setups under natural environmental
variations The aim of this work was to determine how resin exudation by wounded trunks of adult P elliottii
responded to combined chemical effectors involved in different regulatory pathways of resinosis (metal cofactors
of terpene synthases benzoic acid and plant growth regulators) and whether seasonal and tree distribution
variations affected these responses Symmetrically planted and scattered trees regenerated from the seed bank
had similar resin biomass yields suggesting that the homogeneity in development and spatial arrangement were
not significant factors in resin yield This new finding is of practical importance with the used tapping system
since costs of implanting forests by regeneration can be advantageous compared to planting In addition it was
shown for the first time that the salicylic acid precursor benzoic acid and the auxin naphthalene acetic acid
promoted resin exudation when individually applied to wound sites Both these adjuvants are two orders of
magnitude less costly compared to the conventionally used ethylene precursors besides facing less environ-
mental and health restrictions for use Most adjuvant-treated trees showed higher resin flow in the second year
indicating mechanisms of response build up Overall temperature was more important than rainfall as en-
vironmental parameter affecting resin biosynthesis which was higher in the warmer months of spring and
summer The combination of resinosis stimulant effectors from different signaling pathways showed no sig-
nificant synergistic or additive effect suggesting possible converging signaling pathways andor limitation of
common intermediate transducing molecules
1 Introduction
Pines occupy highly diverse environments over a range of tem-
peratures water and nutrient availabilities irradiance levels and pho-
toperiods being able to effectively face attacks from diverse herbivore
and pathogen guilds The success of conifers is linked to their complex
terpene biochemistry hosted by specialized secretory cells The terpe-
noid resin synthesized by Pinus spp is one of the main mechanisms of
defense of these trees particularly against bark beetles and the fungi
they carry (Fett-Neto and Rodrigues-Correcirca 2012) Pine resin biomass
is essentially composed of a monoterpene and sesquiterpene-rich tur-
pentine and diterpenoid-rich rosin fraction both finding numerous in-
dustrial applications as non-wood forest products (Rodrigues-Correcirca
et al 2012)
Molecules capable of modulating different signaling pathways have
been identified as resin yield stimulators including sulfuric acid (ex-
tends wound damage) 2-chloroethylphosphonic acid (CEPA a syn-
thetic ethylene precursor) paraquat (free radical generator) yeast ex-
tract (mimics attack by pathogens) salicylic acid (pathogen signaling
molecule) auxin (promotes ethylene biosynthesis and resin canal dif-
ferentiation) jasmonic acid (signals mechanical damage and promotes
secondary metabolism) and metal ions such as potassium iron and
manganese (cofactors of terpene synthases in conifers) and copper (a
component of ethylene receptors) (Clements 1970 Conrath et al
2002 Fett-Neto and Rodrigues-Correcirca 2012 Hudgins and Franceschi
2004 Lewinsohn et al 1994 Martin et al 2002 Popp et al 1995
httpsdoiorg101016jindcrop201803027
Received 12 December 2017 Received in revised form 9 March 2018 Accepted 13 March 2018
Corresponding author
E-mail addresses franci_neisyahoocombr (FA Neis) fernandadecostayahoocombr (F de Costa) thanisenfyahoocombr (TN Fuumlller)
jjuliocesarlimagmailcom (JC de Lima) krodriguescbiotufrgsbr (KC da Silva Rodrigues-Correcirca) jpfettcbiotufrgsbr (JP Fett) fettnetocbiotufrgsbr (AG Fett-Neto)
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Research Paper
Dual allelopathic effects of subtropical slash pine (Pinus elliottii Engelm)
needles Leads for using a large biomass reservoir
Kelly Cristine da Silva Rodrigues-Correcircaa Gelson Halmenschlagera Joseacuteli Schwambachb
Fernanda de Costaa Emili Mezzomo-Trevizana Arthur Germano Fett-Netoa
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 Brazilb University of Caxias do Sul Institute of Biotechnology Caxias do Sul RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Seasonality
Growth
Germination
Litter
Substrate
A B S T R A C T
Pinus elliottii Engelm (slash pine) is distributed along the maritime coast of Southern Brazil where it shows
invasive pattern and typical allelopathic features Large quantities of needle litter are produced by pine trees a
biomass that is little explored in areas where this species is alien Little is known about the dynamics of needle
and litter phytochemical interactions particularly in subtropical environments To elucidate the full range of
needle and litter allelopathic potential the effects of litter (superficial and deep) and seasonally harvested fresh
slash pine needles stored for different times were evaluated against lettuce tomato and cucumber seeds and
seedlings Increasing concentrations (0 1 2 4 and 8 wv) of hot and cold aqueous extracts of needles
and litter affected in different ways target plant development Growth and germination inhibition were directly
related to the highest extract concentrations (regardless of the season and mainly in hot water extracts) of
needles On the other hand stimulatory effects of litter extracts on lettuce growth were observed Growth and
germination of cucumber and tomato were not affected by pine litter as substrate when compared to rice husk
The presumable high polarity and thermal stability of slash pine leaf biomass allelochemicals and their transient
toxic effect or growth promoting impact suggest potential applications of this largely available biomass both as a
biological herbicide and growth substrate in plant propagation
1 Introduction
Native from the Northern Hemisphere Pinus is one of the most
widely distributed genera throughout different climate regions of the
globe growing either as native or alien species even in extreme habi-
tats (Rodrigues-Correcirca and Fett-Neto 2012) Despite the high economic
value currently attributed to pine wood and oleoresin (Rodrigues-
Correcirca et al 2012) there is increasing concern about the aggressive
potential of invasiveness displayed by Pinus species especially those
cultivated out of their native range of distribution (Richardson et al
2008 Rolon et al 2011) These species are dispersed by wind and there
is notably low plant diversity observed in most understories of pine
plantations (Kato-Noguchi et al 2009) This latter feature has been
considered an important trait of allelopathic interference
The term ldquoallelopathyrdquo was coined by Molisch in 1937 as a chemical
reciprocal interaction established among plants (including micro-
organisms) sharing the same site by means of the release of secondary
metabolites named allelochemicals (Rice 1984) For the most part
these metabolites are derived from the shikimic acid or isoprenoid
pathway and their biosynthesis can be modulated by biotic and abiotic
stresses (Nascimento and Fett-Neto 2010) including seasonal-related
changes (Sartor et al 2013) Allelopathy studies may range from sterile
assays (Aryakia et al 2015) to soil (Correcirca et al 2008 Sharma et al
2016) and field tests being a complex biological phenomenon to as-
certain in several circumstances due to issues of solubility release
mechanisms and stability of bioactive compounds (Scognamiglio et al
2013) Often the use of complementary methods provides more in-
formative data
The allelopathic effects of soil leachates green needles and litter
extracts of Pinus spp on germination and seedling growth aspects of
wild and crop species have been evaluated in natural and cultivated
pine stands and have proven to be stimulatory or inhibitory (Lodhi and
Killingbeck 1982 Kil and Yim 1983 Nektarios et al 2005 Akkaya
et al 2006 Machado 2007 Alrababah et al 2009 Sartor et al 2009
Kato-Noguchi et al 2011 Rolon et al 2011 Valera-Burgos et al
2012) exhibiting in some cases autotoxicity (Garnett et al 2004
Fernandez et al 2008 Zhu et al 2009 Monnier et al 2011) Studies
on potential dual allelopathic effects of Pinus elliottii Engelm (slash
httpdxdoiorg101016jindcrop201706019
Received 23 March 2017 Received in revised form 15 May 2017 Accepted 7 June 2017
Corresponding author
E-mail address fettnetocbiotufrgsbr (AG Fett-Neto)
ORIGINAL RESEARCHpublished 16 June 2016
doi 103389fpls201600849
Frontiers in Plant Science | wwwfrontiersinorg 1 June 2016 | Volume 7 | Article 849
Edited by
Juan Francisco Jimenez Bremont
Instituto Potosino de Investigacioacuten
Cientiacutefica y Tecnoloacutegica Mexico
Reviewed by
Mariacutea De La Luz Guerrero Gonzaacutelez
Universidad Autoacutenoma de San Luis
Potosiacute Mexico
Rosalia Cristina Paz
CIGEOBIO (CONICETFCEFN UNSJ)
Argentina
Correspondence
Arthur G Fett-Neto
fettnetocbiotufrgsbr
daggerThese authors have contributed
equally to this work
Specialty section
This article was submitted to
Plant Physiology
a section of the journal
Frontiers in Plant Science
Received 08 December 2015
Accepted 30 May 2016
Published 16 June 2016
Citation
de Lima JC de Costa F Fuumlller TN
Rodrigues-Correcirca KCdS Kerber MR
Lima MS Fett JP and Fett-Neto AG
(2016) Reference Genes for qPCR
Analysis in Resin-Tapped Adult Slash
Pine As a Tool to Address the
Molecular Basis of Commercial
Resinosis Front Plant Sci 7849
doi 103389fpls201600849
Reference Genes for qPCR Analysisin Resin-Tapped Adult Slash Pine Asa Tool to Address the MolecularBasis of Commercial Resinosis
Juacutelio C de Lima 1dagger Fernanda de Costa 1 dagger Thanise N Fuumlller 1
Kelly C da Silva Rodrigues-Correcirca 2 Magnus R Kerber 1 Mariano S Lima 1
Janette P Fett 1 and Arthur G Fett-Neto 1
1 Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul
Porto Alegre Brazil 2 Biological Sciences Department Regional Integrated University of Alto Uruguai and Missotildees (URI-FW)
Frederico Westphalen Brazil
Pine oleoresin is a major source of terpenes consisting of turpentine (mono- and
sesquiterpenes) and rosin (diterpenes) fractions Higher oleoresin yields are of economic
interest since oleoresin derivatives make up a valuable source of materials for chemical
industries Oleoresin can be extracted from living trees often by the bark streak method
in which bark removal is done periodically followed by application of stimulant paste
containing sulfuric acid and other chemicals on the freshly wounded exposed surface
To better understand the molecular basis of chemically-stimulated and wound induced
oleoresin production we evaluated the stability of 11 putative reference genes for the
purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis
Samples for RNA extraction were collected from field-grown adult trees under tapping
operations using stimulant pastes with different compositions and at various time points
after paste application Statistical methods established by geNorm NormFinder and
BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3
and UBI To confirm expression stability of the candidate reference genes expression
profiles of putative P elliottii orthologs of resin biosynthesis-related genes encoding Pinus
contorta β-pinene synthase [PcTPS-(minus)β-pin1] P contorta levopimaradieneabietadiene
synthase (PcLAS1) Pinus taeda α-pinene synthase [PtTPS-(+)αpin] and P taeda
α-farnesene synthase (PtαFS) were examined following stimulant paste application
Increased oleoresin yields observed in stimulated treatments using phytohormone-based
pastes were consistent with higher expression of pinene synthases Overall the
expression of all genes examined matched the expected profiles of oleoresin-related
transcript changes reported for previously examined conifers
Keywords resin Pinus gene expression normalizer genes terpene synthase
19
Chapter 2
Stimulant Paste Preparation and Bark Streak Tapping Technique for Pine Oleoresin Extraction
Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
Tapping technique comprises the extraction of pine oleoresin a non-wood forest product consisting of a
complex mixture of mono sesqui and diterpenes biosynthesized and exuded as a defense response to
wounding Oleoresin is used to produce gum rosin turpentine and their multiple derivatives Oleoresin
yield and quality are objects of interest in pine tree biotechnology both in terms of environmental and
genetic control Monitoring these parameters in individual trees grown in the fi eld provides a means to
examine the control of terpene production in resin canals as well as the identifi cation of genetic-based
differences in resinosis A typical method of tapping involves the removal of bark and application of a
chemical stimulant on the wounded area Here we describe the methods for preparing the resin-stimulant
paste with different adjuvants as well as the bark streaking process in adult pine trees
Key words Oleoresin Pine Tapping Chemical stimulant Wounding
1 Introduction
Several conifer species produce oleoresin a complex mixture of isoprenoid compounds relevant for defense against herbivores and pathogens Two major fractions can be recognized in oleoresin (a) turpentine the volatile fraction containing mono- and sesquiter-penes and (b) rosin the nonvolatile diterpene fraction Oleoresin is a forest commodity of global interest fi nding applications in diverse industry sectors Rosin is used in adhesives printing ink manufacture and paper sizing Turpentine can be used either as a solvent for paints and varnishes or as a raw material for fraction-ation of high-value chemicals used in the pharmaceutical agro-chemical and food industry [ 1 ndash 3 ]
During the extraction activity resin is obtained from the tree in a similar way as rubber tree tapping which generally involves the
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_2 copy Springer Science+Business Media New York 2016
These authors have equally contributed to this work
fettnetocbiotufrgsbr
27
Chapter 3
A Modifi ed Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines
Juacutelio Ceacutesar de Lima Thanise Nogueira Fuumlller Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes
through high-throughput sequencing technologies microarray and also northern blots RT-PCR and
RTqPCR Even though many specifi c protocols designed for plants with high content of secondary metab-
olites have been developed these are often expensive time consuming and not suitable for a wide range
of tissues Here we present a modifi cation of the method previously described using the commercially
available Concerttrade Plant RNA Reagent (Invitrogen) buffer for fi eld-grown adult pine trees with high
oleoresin content
Key words RNA Pines Concert plant RNA reagent Stem RNA extraction Oleoresin Conifers
1 Introduction
Several conifer species especially within the Pinaceae have tissues with high concentrations of phenolics terpenes and polysaccha-rides [ 1 ] Many specifi c protocols that are appropriate for plants rich in secondary metabolite s have been developed but the extrac-tion of high-quality RNA from these tissues using commercial kits is often diffi cult and usually not applicable to woody tissues [ 2 ndash 6 ] One of the major issues during RNA extraction concerns the pres-ence of phenolic compounds which oxidize and form quinones Aromatic compounds bind RNA which interferes in downstream steps and applications [ 3 7 ] Another point of concern is the har-vest of plant samples in the experimental fi eld which constitutes another obstacle in the efforts to avoid degradation of RNA [ 8 ] These problems often result in RNAs of low quality and insuffi -cient amounts especially for methodologies that normally require
These authors have equally contributed to this work
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_3 copy Springer Science+Business Media New York 2016
fettnetocbiotufrgsbr
RESEARCH PAPER
Control of resin production in Araucaria angustifolia an ancientSouth American coniferJ C Perotti1 K C da Silva Rodrigues-Correa123 amp A G Fett-Neto12
1 Plant Physiology Laboratory Department of Botany Federal University of Rio Grande do Sul (UFRGS) Porto Alegre RS Brazil
2 Center for Biotechnology UFRGS Porto Alegre RS Brazil
3 Present address Regional Integrated University of Alto Uruguai and Miss~oes (URI-FW) Frederico Westphalen RS Brazil
Keywords
Araucaria ethylene jasmonic acid nitric
oxide resin salicylic acid terpenes
Correspondence
A G Fett-Neto Plant Physiology Laboratory
Center for Biotechnology Federal University
of Rio Grande do Sul (UFRGS) PO Box 15005
Av Bento Goncalves 9500 91501-970 Porto
Alegre Brazil
E-mail fettnetocbiotufrgsbr
Editor
K Leiss
Received 22 July 2014 Accepted 11
December 2014
doi101111plb12298
ABSTRACT
Araucaria angustifolia is an ancient slow-growing conifer that characterises parts ofthe Southern Atlantic Forest biome currently listed as a critically endangered speciesThe species also produces bark resin although the factors controlling its resinosis arelargely unknown To better understand this defence-related process we examined theresin exudation response of A angustifolia upon treatment with well-known chemicalstimulators used in fast-growing conifers producing both bark and wood resin suchas Pinus elliottii The initial hypothesis was that A angustifolia would display signifi-cant differences in the regulation of resinosis The effect of Ethrel (ET ndash ethylene pre-cursor) salicylic acid (SA) jasmonic acid (JA) sulphuric acid (SuA) and sodiumnitroprusside (SNP ndash nitric oxide donor) on resin yield and composition in youngplants of A angustifolia was examined In at least one of the concentrations testedand frequently in more than one an aqueous glycerol solution applied on fresh woundsites of the stem with one or more of the adjuvants examined promoted an increase inresin yield as well as monoterpene concentration (a-pinene b-pinene camphene andlimonene) Higher yields and longer exudation periods were observed with JA and ETanother feature shared with Pinus resinosis The results suggest that resinosis controlis similar in Araucaria and Pinus In addition A angustifolia resin may be a relevantsource of valuable terpene chemicals whose production may be increased by usingstimulating pastes containing the identified adjuvants
INTRODUCTION
Many conifer species produce terpenoid-based resins that havelong been studied for their industrial importance and role indefence against attack by herbivores and pathogens The twomost important resin-producing families of conifers are Pina-ceae and Araucariaceae (Langenheim 1996) The viscous resinsecretion is generally composed of a complex mixture ofterpenoids consisting of roughly equal parts of volatile mono-(C10) and sesquiterpene (C15 turpentine) fractions and non-volatile diterpenic (C20 rosin) components (Rodrigues-Correaet al 2013) Terpenes act in a complex and multilayereddefence response providing toxicity against bark beetles andfungi bark wound sealing disruption of insect developmentand attraction of herbivore predators (Phillips amp Croteau1999)Most conifers rely on some combination of preformed and
inducible resin defences (Trapp amp Croteau 2001 Zulak amp Bohl-mann 2010) Resin defences are controlled by environmentaland genetic factors to various extents depending on species(Roberds et al 2003 Sampedro et al 2010 Moreira et al2013) Resin traits have been reported as highly variable havingmoderate heritability indicating that breeding efforts towardssuper-resinous forests are promising (Tadasse et al 2001Roberds et al 2003) Several chemicals are known as stimulantsof resin production Commercial extraction of resin from pine
trees uses periodic bark streaking and application of resin stim-ulant pastes to the wound
Resin-stimulant paste based on sulphuric acid (SuA) iswidely used for the commercial production of pine resin Cur-rent stimulant pastes usually have two chemically active com-ponents SuA to magnify the wounding and an ethyleneprecursor (2-chloroethylphosphonic acid CEPA or Ethrel ndash
ET) to stimulate resin flow (Rodrigues et al 2011 Rodrigues-Correa amp Fett-Neto 2013) Jasmonic acid (JA) and its methylester methyl jasmonate (MeJa) are among the most widelyused chemical elicitors of plant secondary metabolism It hasbeen shown that the exogenous application of MeJa or herbi-vore attack induce chemical and anatomical defence responsesin conifers such as the formation of traumatic resin ducts andresin accumulation in stems along with increased biosynthesisof terpenes and phenolics (Franceschi et al 2002 Martin et al2002 Heijari et al 2005 Zeneli et al 2006 Moreira et al 2008Gould et al 2009) JA commercial use however is limited byits high cost
The effects of exogenous salicylic acid (SA) on conifer ter-pene production have also been studied In Pinus elliottiiapplication of 10 molm3 of SA induced resin productionin wound panels but in Pseudotsuga menziesii and Sequoia-dendron giganteum it had no apparent effect on resinaccumulation (Hudgins amp Franceschi 2004 Rodrigues ampFett-Neto 2009) Nitric oxide (NO) has also emerged as an
Plant Biology 17 (2015) 852ndash859 copy 2014 German Botanical Society and The Royal Botanical Society of the Netherlands852
Plant Biology ISSN 1435-8603
3
mimosina em Leucaena leucocephala var glabrata (Lam) de Wit (leucena) e Mimosa
bimucronata (DC) Kuntze (maricaacute)
Mimosina eacute um aminoaacutecido aromaacutetico natildeo-proteico anaacutelogo da L-tirosina com
atividade toacutexica para ceacutelulas de procariotos e eucariotos Embora em menor concentraccedilatildeo
mimosina foi primeiramente identificada em Mimosa pudica sendo posteriormente detectada
em outras espeacutecies do gecircnero como Mimosa pigra por exemplo (Soedarjo amp Borthakur
1998) Seu efeito toacutexico eacute atribuiacutedo agrave capacidade de quelar metais o que impede o
funcionamento adequado das metalo-proteiacutenas que dependem dos mesmos como co-fatores
(Negi et al 2014)
A concentraccedilatildeo basal de mimosina em espeacutecies de leucaena pode variar entre 1 e 12
do peso seco do oacutergatildeo (Soedarjo amp Borthakur 1998) Como eacute comum para outros ANPs
que ocorrem em espeacutecies de leguminosas em sementes de Leucaena spp eacute observada uma
maior concentraccedilatildeo de mimosina quando comparada aos demais oacutergatildeos da planta
(Rodrigues-Correcirca et al 2019) sendo esta a fonte de extraccedilatildeo comercial do padratildeo quiacutemico
de mimosina vendido por empresas de reagentes de pesquisa
Diversas atividades foram descritas para mimosina em outros organismos eou tipos
celulares Dentre essas destacam-se a atividade anti-mitoacutetica ou bloqueadora do ciclo
celular em ceacutelulas de eucariotos e procariotos Isto ocorre porque a mimosina impede a
formaccedilatildeo da forquilha de replicaccedilatildeo (e portanto a siacutentese de DNA) interrompendo assim o
avanccedilo do ciclo de divisatildeo celular na fase tardia G1 (Lalande 1990) Foram tambeacutem descritas
para mimosina atividade alelopaacutetica observada sobre o desenvolvimento de outras espeacutecies
de leguminosas e atividade antioxidante entre outras (Tabela 1)
A rota de biossiacutentese de mimosina eacute compartilhada em grande parte com a de cisteiacutena
um aminoaacutecido proteico sulfurado (Figura 1) A siacutentese da cisteiacutena se daacute a partir da conversatildeo
4
de serina e acetil-CoA em o-acetilserina pela enzima SAT (serina acetiltransferase) seguida
da conversatildeo de o-acetilserina e aacutecido sulfiacutedrico em cisteiacutena em uma reaccedilatildeo catalisada pela
OAS-TL (o-acetilserina tiol-liase) A siacutentese de mimosina por sua vez eacute compartilhada com
a da cisteiacutena ateacute esse ponto e acredita-se que pelo menos uma das isoformas de OAS-TL
catalise a conversatildeo de o-acetilserina e 3-hidroxi-4-piridona em mimosina
Tabela 1 Atividades descritas para mimosina de Leucaena leucocephala (Lam) de Wit
ATIVIDADE
ALVO AVALIADO
(organismo eou tecido tipo
celular)
REFEREcircNCIA
Bloqueio do complexo de ativaccedilatildeo
da preacute-replicaccedilatildeo do DNA
Ceacutelulas de mamiacuteferos
KUBOTA et al
(2014)
Alteraccedilatildeo no ciclo ovariano e
extensatildeo da duraccedilatildeo do corpo luacuteteo
bovino no periacuteodo poacutes-parto
Bovinos
(Bos taurus x
Bos indicus)
BOTTINI-
LUZARDO et al
(2015)
Supressatildeo do ciclo celular e reduccedilatildeo
da abundacircncia bacteriana em
mosquitos
Wolbachia pipientis
Aedes albopictus
FALLON
(2015)
Accedilatildeo inibitoacuteria da fibrose
pulmonar induzida
Ratos SD
LI et al
(2015)
Recuperaccedilatildeo da funccedilatildeo do
miocaacuterdio poacutes-isquemia
Miocaacuterdio de ratos (SD)
machos
CROWE et al
(2001)
Inseticida
Heteropsylla cubana
Crawford 1914 e Thrips tabaci
Lindemann 1889
AHMED et al
(2016)
Alelopaacutetica
Albizia procera Vigna
unguiculata Cicer arietinum
Cajanus cajan
AHMED et al
(2008)
Antioxidante
Sistemas modelo de oxidaccedilatildeo
lipiacutedica (β-caroteno - aacutecido
linolecircico e lecitina)
BENJAKUL et al
(2013)
Ateacute momento versotildees divergentes sobre a enzima responsaacutevel pela biossiacutentese de
mimosina (mimosina sintase) tecircm sido publicadas Em 1990 Ikegami e colaboradores
5
identificaram uma OAS-TL responsaacutevel pela formaccedilatildeo de cisteiacutena como sendo tambeacutem uma
mimosina sintase Mais tarde Yafuso et al (2014) realizaram a expressatildeo heteroacuteloga do gene
que codifica para OAS-TL em Escherichia coli e natildeo foi observada a formaccedilatildeo de mimosina
mesmo quando dadas as condiccedilotildees oacutetimas para tanto Mais recentemente Harun-Ur-Rashid
et al (2018) elucidaram a mimosina sintase como sendo uma isoforma da OAS-TL
corroborando o postulado por Ikegami e colaboradores em 1990
Figura 1 Rota de biossiacutentese da mimosina Fonte Ikegami et al (1990)
Espeacutecies estudadas
Leucaena leucocephala (Lam) de Wit (leucaena koa haole ou ldquoacaacutecia exoacuteticardquo na
liacutengua Hawairsquoiana) eacute uma espeacutecie de haacutebito arboacutereo ou arbustivo pertencente agrave famiacutelia
Fabaceae de Angiospermas e caracterizada pelo acuacutemulo de mimosina em todos os seus
oacutergatildeos Eacute nativa da Ameacuterica Central (especificamente da regiatildeo sudeste do Meacutexico) mas
irradiou-se atraveacutes de praticamente todas as zonas tropicais e subtropicais da Terra No
Brasil leucena eacute amplamente distribuiacuteda e classificada como naturalizada pelo REFLORA
(2019) ocorrendo em todo territoacuterio Nacional Satildeo reconhecidas no miacutenimo duas
6
subespeacutecies de leucena ocorrentes no Brasil L leucocephala var leucocephala e L
leucocephala var glabrata sendo a primeira a mais abundante
Leucaena apresenta atributos morfoloacutegicos caracteriacutesticos das leguminosas como o
fruto do tipo vagem deiscente no periacuteodo poacutes-maturaccedilatildeo folhas compostas e bipinadas As
flores satildeo seacutesseis actinomorfas e polistecircmones apresentam caacutelice sinseacutepala e corola
gamopeacutetala e satildeo dispostas em inflorescecircncias do tipo glomeacuterulo (Figura 2)
Figura 2 Oacutergatildeos vegetativos e reprodutivos de L leucocephala (Lam) de Wit Fonte Little Jr amp Skolmen
(1989)
Com base no conhecimento etnobotacircnico disponiacutevel acerca dessa espeacutecie em
diversas regiotildees tropicais e subtropicais leucena eacute utilizada para vaacuterios fins Extratos de
diferentes oacutergatildeos de leucena apresentam atividade anti-diabeacutetica (Kuppusamy et al 2014
Chowtivannakul et al 2016) antioxidante (Mohammed et al 2015 Chowtivannakul et al
2016 Zarin et al 2016) antimicrobiana (Zarin et al 2016) anti-helmiacutentica (Soares et al
2015 Jamous et al 2017) bactericida (Mohammed et al 2015) acaricida (Fernaacutendez-Salas
et al 2011) anti-tumoral (Chung et al 2017) e potencializadora da resposta imune em
peixes (Verma et al 2018) entre outras
7
Leucaena apresenta alta toleracircncia agrave seca sendo capaz de enfrentar estaccedilotildees sazonais
inteiras com deacuteficit hiacutedrico sem prejuiacutezo permanente de seus oacutergatildeos e de recuperar
vigorosamente sua biomassa vegetativa tatildeo logo o regime de precipitaccedilatildeo retome a
regularidade em frequecircncia Acredita-se que a toleracircncia agrave seca apresentada por essa espeacutecie
ocorra em funccedilatildeo do acuacutemulo de mimosina nos diferentes tecidos da planta a qual
funcionaria como um agente osmoregulador responsaacutevel pela preservaccedilatildeo da integridade das
membranas a das macromoleacuteculas intracelulares em periacuteodos de escassez de aacutegua no
ambiente
Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) eacute uma leguminosa
nativa natildeo endecircmica do Brasil amplamente distribuiacuteda nos domiacutenios fitogeograacuteficos da
Caatinga do Cerrado e da Mata Atlacircntica (Simon amp Proenccedila 2000 REFLORA 2019) Como
espeacutecie pioneira (Pilatti et al 2019) exerce importante papel ecoloacutegico na recuperaccedilatildeo de
aacutereas degradadas (Bitencourt et al 2007 Silva et al 2011) no estabelecimento de processos
de sucessatildeo vegetacional
Maricaacute eacute uma espeacutecie semi-deciacutedua a deciacutedua a qual atinge ateacute 15 m em altura (e
diacircmetro agrave altura do peito de ateacute 40 cm) na idade adulta com haacutebito arboacutereo ou arbustivo
(REFLORA 2019) e espinhos caracteriacutesticos desde os estaacutegios iniciais de desenvolvimento
(Carvalho 2004) Apresenta folhas compostas alternas e bipinadas (Figura 2) amplas
inflorescecircncias brancas com flores reunidas em glomeacuterulos esfeacutericos dispostos em grandes
paniacuteculas As flores satildeo diplostecircmones actinomorfas hipoacuteginas e unicarpelares (Silva et al
2011)
Assim como descrito para leucena maricaacute eacute considerado uma espeacutecie multifuncional
sendo comumente empregada para produccedilatildeo de mel como combustiacutevel (Olkoski amp
8
Wittmann 2011) em edificaccedilotildees na carpintaria e como lsquocerca-vivarsquo (Marchiori 1993
Lorenzi 1998) entre outras aplicaccedilotildees
Figura 2 Folhas e fruto de Mimosa bimucronata (DC) Kuntze Fonte Souza-Lima et al (2017)
Em contraste com a amplitude de habitats explorados por leucena (especialmente os
aacuteridos) no Sul do Brasil maricaacute ocorre preferencialmente em ambientes uacutemidos a alagadiccedilos
em aacutereas proacuteximas agraves margens de rios (Patreze amp Cordeiro 2004) embora possa tambeacutem
ocorrer em formaccedilotildees quase exclusivas dessa espeacutecie nas encostas de morros (Jacobi amp
Ferreira 1991)
Em relaccedilatildeo agraves atividades elucidadas para os extratos de maricaacute foram relatados os
efeitos alelopaacutetico (Jacobi amp Ferreira 1991 Ferreira et al 1992) diureacutetico natriureacutetico e
caliureacutetico (Schlickmann et al 2017)
9
Hipoacutetese
Mimosina apresenta perfil dinacircmico de acuacutemulo em Leucaena leucocephala e
Mimosa bimucronata frente a estresses associado a alteraccedilotildees significativas na expressatildeo de
genes relacionados ao metabolismo deste ANP o qual contribui para mitigar o desequiliacutebrio
oxidativo inerente a vaacuterios tipos de estresse
Objetivo geral
O objetivo da presente tese foi investigar o papel bioloacutegico da mimosina endoacutegena
em leucena e maricaacute a partir da avaliaccedilatildeo do efeito de tratamentos relacionados a estresses
ou sinalizadores de estresse
Objetivos especiacuteficos
- Analisar a concentraccedilatildeo constitutiva de mimosina nos diferentes oacutergatildeos de L leucocephala
(Lam) de Wit (leucena) e M bimucronata (DC) Kuntze (maricaacute)
- Verificar se apesar do seu alto teor constitutivo em plantas de leucena o acuacutemulo de
mimosina pode ser induzido com tratamentos que mimetizam diferentes estresses a partir da
avaliaccedilatildeo do efeito de moleacuteculas sinalizadoras (aacutecido saliciacutelico jasmonato etileno) e da
exposiccedilatildeo agrave radiaccedilatildeo UV-C na modulaccedilatildeo do acuacutemulo de mimosina em leucena bem como
em maricaacute
- Determinar se a expressatildeo de genes relacionados ao metabolismo de mimosina estaacute
associada agrave induccedilatildeo por estresses fisioloacutegicos
- Avaliar o potencial antioxidante da mimosina em experimentos realizados in situ
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry
journal homepage wwwelseviercomlocateplaphy
Research article
Mimosine accumulation in Leucaena leucocephala in response to stresssignaling molecules and acute UV exposure
Kelly Cristine da Silva Rodrigues-Correcircaab Michael DH Hondab Dulal BorthakurbArthur Germano Fett-Netoalowast
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970Porto Alegre Rio Grande do Sul BrazilbDepartment of Molecular Biosciences and Bioengineering University of Hawaii at Manoa Honolulu HI 96822 USA
A R T I C L E I N F O
KeywordsLeucaena leucocephalaMimosineMimosine amidohydrolaseJasmonic acidEthyleneSalicylic acidUV-C radiation
A B S T R A C T
Mimosine is a non-protein amino acid of Fabaceae such as Leucaena spp and Mimosa spp Several relevantbiological activities have been described for this molecule including cell cycle blocker anticancer antifungalantimicrobial herbivore deterrent and allelopathic activities raising increased economic interest in its pro-duction In addition information on mimosine dynamics in planta remains limited In order to address this topicand propose strategies to increase mimosine production aiming at economic uses the effects of several stress-related elicitors of secondary metabolism and UV acute exposure were examined on mimosine accumulation ingrowth room-cultivated seedlings of Leucaena leucocephala spp glabrata Mimosine concentration was not sig-nificantly affected by 10 ppm salicylic acid (SA) treatment but increased in roots and shoots of seedlings treatedwith 84 ppm jasmonic acid (JA) and 10 ppm Ethephon (an ethylene-releasing compound) and in shoots treatedwith UV-C radiation Quantification of mimosine amidohydrolase (mimosinase) gene expression showed thatethephon yielded variable effect over time whereas JA and UV-C did not show significant impact Consideringthe strong induction of mimosine accumulation by acute UV-C exposure additional in situ ROS localization aswell as in vitro antioxidant assays were performed suggesting that akin to several secondary metabolitesmimosine may be involved in general oxidative stress modulation acting as a hydrogen peroxide and superoxideanion quencher
1 Introduction
Different plant groups synthesize a large diversity of secondary orspecialized metabolites These molecules are generally produced inresponse to biotic and abiotic environmental stresses Indeed inductionof secondary metabolism usually involves stress-generating factorswhich have also been explored in biotechnological processes aiming atthe production of target metabolites of economic interest (Matsuuraet al 2018) Metabolic control of nitrogen-containing secondarycompounds (eg alkaloids and non-protein amino acids) has beenshown to be complex and influenced by phytohormones environmentalstresses (seasonality herbivory pathogen attack drought) UV radia-tion (Holloacutesy 2002) methyl jasmonate (MeJA) salicylic acid (SA)yeast extract (Cho et al 2008) abscisic acid (ABA) heavy metals os-motic stress (Nascimento et al 2013) and mechanical wounding (Portoet al 2014)
Due to their particular trait of associating with N-fixing micro-organisms Fabaceae species (leguminous sensu lato) are often proteinrich hence the relevance of several of these species as forage Fabaceaespecies are also known for accumulating nitrogen containing secondarymetabolites which play important roles as ecochemical molecules andat least for the case of non-protein amino acids potential cell reservoirsof nitrogen (Huang et al 2011)
High contents of mimosine a toxic aromatic non-protein aminoacid are found in species of two leguminous genera Leucaena spp andMimosa spp Leucaena leucocephala (Lam) de Wit (leucaena koa haole)is a fast-growing leguminous tree native from Central America (south-eastern Mexico) widely distributed in tropical and subtropical zonesThis species is also characterized by its high tolerance to droughtamong other environmental stresses (Honda et al 2018) Leucaena canbe divided into two subspecies (i) L leucocephala subsp leucocephala(common leucaena a bushy shrub) and (ii) L leucocephala subsp
httpsdoiorg101016jplaphy201811018Received 1 August 2018 Received in revised form 9 November 2018 Accepted 14 November 2018
lowast Corresponding authorE-mail addresses krodriguescbiotufrgsbr (KCdS Rodrigues-Correcirca) mhonda2hawaiiedu (MDH Honda) dulalhawaiiedu (D Borthakur)
fettnetocbiotufrgsbr (AG Fett-Neto)
Plant Physiology and Biochemistry 135 (2019) 432ndash440
Available online 19 November 20180981-9428 copy 2018 Elsevier Masson SAS All rights reserved
T
glabrata (giant leucaena a tree) The latter has been used as a fastgrowing tree for production of wood and paper pulp The foliage ofboth common and giant leucaena is used as a fodder because of its highprotein content and palatability to farm animals The foliage containsup to 18 protein 142 crude fiber and 64 ether extractcrude fat(Soedarjo and Borthakur 1996)
Production of nitrogen-containing secondary metabolites such asmimosine requires large amounts of carbon and nitrogen resourcesNegi et al (2014) estimated that up to 21 of the carbon-nitrogenresources may be used for production of mimosine in leucaenaBrewbaker et al (1972) determined the mimosine content of 96 Lleucocephala cultivars and 8 other Leucaena species collected from 38different countries by growing them in an observational nursery inHawaii and found that basal mimosine content varied from 189 to477 of the dry weight
Mimosine is biosynthesized from OAS (o-acetylserine) and 3H4P (3-hydroxy-4-pyridone or its tautoisomer 3-hydroxy-4-pyridine) A pre-vious analysis suggested that mimosine synthase is an OAS-TL (o-acetylserine-thiol-lyase) of the cysteine biosynthesis pathway (Ikegamiet al 1990) Later however recombinant enzyme tests did not supportan OAS-TL identity of mimosine synthase (Yafuso et al 2014) Recentfindings on mimosine biosynthesis revealed that a cytosolic cysteine-OAS-TL isoform can also catalyze the formation of mimosine underspecific conditions (Harun-Ur-Rashid et al 2018)
Mimosine toxicity is related to its ability of reducing the availabilityof divalent metal ions such as Fe(II) Zn(II) Cu(II) Co(II) and Mn(II)by chelating co-factors and preventing their association with metal-dependent enzymes Furthermore this non-protein amino acid is cap-able of forming a stable complex with pyridoxal-5prime-phosphate (PLP)leading to the inactivation of PLP-dependent enzymes (eg Asp-Glutransaminase and cystathionine synthetase) (Negi et al 2014)
Mimosine features several useful biological activities such as alle-lopathic antimicrobial insecticide cell cycle inhibitor agent antic-ancer phytoremediator (Nguyen and Tawata 2016) as well as anti-oxidant (Benjakul et al 2013) Despite the relatively well establishedbiological activities of purified mimosine on other organisms or celltypes little is known about its biological role in leguminous speciesHowever it has been suggested that at least in part its activity ismainly related to defense mechanisms against some biotic and abioticstresses and as nitrogen source during fast growth (Vestena et al2001)
Suda (1960) and Smith and Fowden (1966) identified enzymes in-volved in mimosine degradation in seedling extracts of L leucocephalaand Mimosa pudica A mimosine-degrading enzyme named mimosinase(mimosine amidohydrolase EC 35161 CAS registry number 104118-49-2) (IUBMB 2018) a carbon-nitrogen lyase which degrades mimo-sine into 3H4P was later purified by Tangendjaja et al (1986) Itsbiochemical characterization was described and the cDNA was isolatedby Negi et al (2014)
Although mimosinase has been described and isolated only fewstudies on the role played by biotic and abiotic factors on the dynamicmodulation of mimosine metabolism in leguminous species have beenconducted (Vestena et al 2001 Xu et al 2018) In aseptic cultures ofleucaena mechanical injury of shoots promoted local mimosine accu-mulation (Vestena et al 2001) In the same study cultivation in pre-sence of auxin or SA in culture medium also had a positive effect on
mimosine accumulation More recently the effect of drought treatmenton gene expression of leucaena was also evaluated by Honda et al(2018) However several potential factors regulating mimosine meta-bolism need to be further examined
To date there is a lack of information on the biological role ofmimosine in planta as well as on details of its metabolic dynamicsMoreover its overt potential for pharmaceutical applications and de-velopment of new drugs as well as the possible use as tool to addressheavy metal soil contamination or plant mineral nutrition improve-ment justify additional research The objective of this study was toinvestigate the effect of stress signaling molecules and acute UV ex-posure on modulation of mimosine accumulation and metabolism in Lleucocephala spp glabrata in order to better understand its biologicalrole and to identify strategies for yield improvement aiming at ex-ploring its useful bioactivities
2 Methods
21 Plant material
For the experiments carried out to evaluate the effects of elicitors onmimosine accumulation seeds of leucaena were kindly provided by DrJames Brewbaker and harvested at CTAHRs (College of TropicalAgriculture and Human Resources of the University of Hawaii atManoa) Waimanalo Research Station at Oahu Hawaii This plantmaterial was originated from the accession K636 of Leucaena leucoce-phala ssp glabrata (Brewbaker 2008)
22 Induced mimosine content in 5-week-old giant leucaena
221 Seed germinationIn order to overcome seed coat dormancy seeds were submitted to a
chemical scarification with sulfuric acid 95ndash98 for 20min and re-peatedly rinsed in distilled water to remove any residual trace of thisreagent Then seeds were distributed in 254 cmtimes508 cm plastictrays containing 11 vv of vermiculite and commercial soil watereduntil reaching substrate field capacity Three weeks after seed imbibi-tion seedlings displaying similar size and shape (eg number of com-pound leaves and leaflets) were transplanted to individual pots(250mL) in number of three plants per container
During the experimental period (except in the UV-C radiationtreatment) all tested seedlings were kept in a growth chamber andsubmitted to controlled conditions of temperature (circa 25 degC) and ir-radiance (approximately 100 μmol photons mminus2sdot s minus1) with a photo-period of 16 h light and 8 h dark
222 Treatments2221 JA Ethephon and SA Five-week-old giant leucaena seedlingswere treated with different solutions as described in Table 1 Idealconcentrations were defined in preliminary experiments under the sameconditions indicated above At the beginning of the experiments 30plants were sprayed with 84 ppm JA 10 ppm SA 10 or 100 ppmEthephon or Milli-Qreg water (control) until the point of imminent runoffPlant pots were kept closed inside transparent plastic bags for 24 h toavoid solution volatilization Fifteen plants arranged in 5 sets of 3 (5biological replicates) were harvested 48 h and 96 h after being treated
Table 1Treatments used to modulate mimosine biosynthesis in giant leucaena
ELICITOR CONCENTRATION UV FLUENCE EXPOSURE TIME RATIONALE FOR USE
Salicylic acid (SA) 10 ppm 24 h Pathogen signaling molecule (Shah 2003)Jasmonic acid (JA) 84 ppm 24 h Chemical elicitor of plant secondary metabolism (Dar et al 2015)Ethephon 10 ppm 24 h Ethylene releasing-compound (Kim et al 2016) elicitor of plant secondary metabolism (Wang
et al 2016)UV-C radiation 3 Jcmminus2 10min or 15min Elicitor of plant secondary metabolism (Kara 2013 Neelamegam and Sutha 2015)
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After collection shoots were separated from roots immediately frozenin liquid nitrogen and stored at ndash 80 degC prior to HPLC analyses
2222 UV-C Thirty seedlings of giant leucaena were exposed to UV-Cradiation (3 Jcmminus2) for 10 or 15min and kept in a growth chamberunder controlled conditions as described above until the end of theexperiments Fifteen plants arranged in groups of 3 were harvested at96 h and 120 h after UV-C exposure and processed as previouslydescribed
223 Mimosine extractionMimosine extraction was based on a modified version of the pro-
tocol published by Lalitha and Kulothungan (2006) as follows a knownweight of fresh tissue (shoots or roots) of giant leucaena was first addedto Milli-Qreg boiling water in a proportion of 110 (g of plant per mL ofsolvent) in test tubes Tubes were covered with foil to avoid solutionevaporation and placed on a hot stirrer at 100 degC for 10min A pro-portional volume of 01M HCl was added to the cooled suspensions andhomogenized using mortar and pestle The plant extracts were filteredthrough cotton and centrifuged twice for 7min in a bench top re-frigerated centrifuge at 4 degC and 13200 rpm Before being analyzed theextracts were diluted 13 with ondashphosphoric acid (OPA)
224 Mimosine detectionHPLC analyses were carried out as described by Negi and Borthakur
(2016) Pure mimosine (L-mimosine from koa haole seeds Sigma-Al-drich CAS number 500-44-7) was used as standard Separation andquantification of mimosine was done with a C18 column (PhenomenexC18 5 μm 46times250mm) under an isocratic solvent system of 002MOPA with a linear flow rate of 1mLsdotminminus1 Mimosine detection wasdone at 280 nm by photodiode array detection (200ndash400 nm) showingretention time of 277 plusmn 0042min Quantification was done using themethod of external standard curve Further confirmation of mimosineidentity was performed by co-chromatography with standard and peakpurity check Chromatograms were analyzed using the Waters Em-power 3 software
23 Quantitative real-time PCR analysis of mimosinase gene expression
Fifteen 8-week-old giant leucaena plants arranged in 4 sets of 3 (4biological replicates) were treated with either water (control) or10 ppm Ethephon 84 ppm JA acid or 15min of UV-C radiation ex-posure following the methods described above Following treatmentleucaena plants were harvested at 48 and 96 h or 72 and 144 h (UV-Ctreated plants only) after treatments Total RNA of samples was ex-tracted and purified from roots and shoots of giant leucaena by meansof a modified method using Qiagen RNeasy Plant Kit (Valencia CAUSA) and Fruit-mate (Takara Japan) according to the protocol de-scribed by Ishihara et al (2016a) The assessment of RNA quality andquantity was carried out at 230 260 and 280 nm by using a NanoDropSpectrophotometer ND-1000 (NanoDrop Technologies DE USA) Inorder to avoid genomic DNA contamination RNA samples were treatedwith TURBO DNAfree Kit (Invitrogen Carlsbad CA) Two microgramsof DNase-treated RNA were used to synthesize the first-strand cDNAusing M-MLV Reverse Transcriptase (Promega WI USA)
Quantitative real-time (qPCR) analysis was carried out to examinepossible differential expression of the mimosinase gene (GenBank ac-cession number AB2985971) in seedlings treated with 84 ppm JA10mM Ethephon or 15min of UV-C exposure Shoots and roots wereharvested 24 h before the time of mimosine concentration peak for eachtreatment previously observed as assessed by HPLC assays The 10 μLqPCR reaction consisted of 5 μL of PowerUpTM SYBRreg Green MasterMix (Applied Biosystems Foster City CA) 1 μL MgCl2 (50mM) 03 μLforward primer (10 μM) 03 μL reverse primer (10 μM) and 1 μL cDNAfirst-strand In the experimental validation through qPCR reactionconditions and melting curve analysis of the amplicon were performed
following the protocol published by Ishihara et al (2016b) for the sameleucaena variety qPCR analysis was conducted using StepOnetrade Real-Time PCR System (Applied Biosystems) Measurements were performedusing 4 biological and 3 technical replicates Relative expression wascalculated with the 2-ΔΔct method using OAS-TL as reference gene sinceits expression showed a consistently stable profile comparable to that ofUBQ-5 and ELF1α expressions Mimosinase primer sequences used forthese analyses were (FWD) 5prime- GAA AGG CAG GAA TCA CAG TGA AGAG ndash 3rsquo (REV) 5prime GGA GAC TCT AGC CAC ACC AAC TTA ndash 3rsquo
24 Antioxidant assays
241 Mimosine effect on hydrogen peroxide (H2O2) accumulationAs a follow up to the induction of mimosine accumulation profiles
under stress signals and conditions tests were conducted to verify mi-mosine antioxidant capacity In situ histological localization of hy-drogen peroxide (H2O2) accumulation was evaluated on foliar disks ofPhaseolus vulgaris L according to the protocol described by Shi et al(2010) Briefly the plant foliar tissue was exposed to 1 mgmiddotmLminus1 dia-minobenzidine (DAB) solution in 10 mM KH2PO4 (control) in presenceor absence of 10mM mimosine (equivalent to the average mimosineconcentration induced by UV-C radiation in giant leucaena) or 10mMascorbic acid (positive antioxidant control) Oxidative response wasidentified by the formation of a brown polymer on the injured leafareas indicating the presence of H2O2 and registered in a Leica M165FC stereomicroscope (Leica Microsystems)
242 Mimosine quenching of superoxide radicalsGeneration of superoxide radical and subsequent analysis was per-
formed by a modified protocol based on Zhishen et al (1999) Nitroblue tetrazolium (NBT) reduction was used to measure superoxide an-ions quenching activity Shortly a 50mM KH2PO4 pH 78 solutioncontaining 6 μM riboflavin 100mM methionine 1 mM NBT in pre-sence or absence of 5mM mimosine was exposed to white light(22 Jsdotcmminus2) for 25min on a white light transilluminator Five micro-molar rutin was used as positive control (Matsuura et al 2016) Theabsorbance was read at 560 nm before and after light exposure in aSpectraMaxreg M2 Microplate Reader (Molecular Devices LLC)
25 Statistical analyses
For HPLC and superoxide anions data simple analyses of variance(ANOVA) followed by Tukey or Welch ANOVA followed by Dunnetts Ctest were used as appropriate for data distribution characteristics InqPCR analysis results were analyzed by t-test In all cases at least fourbiological triplicates were used and experiments were repeated twiceindependently All data were analyzed using the statistical packageSPSS 200 for Windows (SPSS Inc USA) In all cases a ple 005 wasused
3 Results and discussion
31 Increased mimosine concentrations in giant leucaena treated withchemical elicitors
Leucaena produces high amounts of mimosine that accumulate in allparts of the plants including leaves stem flowers pods seeds rootsand root nodules (Soedarjo and Borthakur 1998) The highest con-centrations of mimosine can be found in the growing shoot tips andseeds (Wong and Devendra 1983) It is not known why leucaena pro-duces such high amounts of mimosine Negi et al (2014) estimated thatleucaena plants would be able to grow 21 larger if the nutrient re-sources spent on mimosine production were diverted for biomass in-crease In a previous analysis performed to quantify the basal con-centration of mimosine present in adult plants of common leucaena thehighest constitutive amount of mimosine per gram of fresh weight in
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the analyzed organs was found in post-anthesis flowers (89448 μg)followed by green pods (82687 μg) leaves (67358 μg) and greenflower buds (51247 μg) which showed significantly less mimosineconcentration compared to the other reproductive structures(Supplementary Fig 1) Since mature seeds have very low moisturecontent (Wencomo et al 2017) its mimosine concentration was esti-mated as 338253 μgsdotgminus1 of dry weight Additionally it was also ob-served that the basal mimosine distribution in shoots of field-grownadult plants of leucaena is dependent on the variety type(Supplementary Table 1)
Phytohormones such as salicylic acid and jasmonic acid are knownto be produced by plants in response to various abiotic and bioticstresses These phytohormones trigger adaptive responses to stress byregulating major plant metabolic processes such as photosynthesisnitrogen metabolism defense systems and plant-water relationsthereby providing protection (for review see Khan et al 2015)
Secondary or specialized metabolite production and accumulationare also known to be controlled by biotic and abiotic stresses (Matsuuraet al 2018) In this study exposure of 5-week-old giant leucaenaseedlings to JA or Ethephon treatments significantly enhanced mimo-sine accumulation in shoots and roots in at least one of the two timepoints tested (48 and 96 h) albeit in a different way (Fig 1) Thehighest concentrations of mimosine in shoots were found in seedlingstreated with JA 84 ppm (43441 μgsdotgminus1) and Ethephon 100 ppm(38412 μgsdotgminus1) two days after application of the respective phyto-hormones Nevertheless after four days shoots yielded the highestconcentration of mimosine (approximately 460 μgsdotgminus1) upon treatmentwith 10 or 100 ppm Ethephon (Fig 1A) In roots after two and four
days JA 84 ppm and Ethephon 10 ppm resulted in highest mimosineaccumulation 18488 μgsdotgminus1 and 15801 μgsdotgminus1 respectively (Fig 1B)These observations show that mimosine accumulation response tospecific elicitors may vary over time after exposure
Although all treatments were applied exclusively on shoots of giantleucaena seedlings roots of some of them were also able to respond tothe different elicitors Overall shoots displayed higher basal and in-duced mimosine concentration compared to roots (Fig 1) which agreeswith previous observations in 1 to 3-week-old aseptic seedlings ofcommon leucaena (Vestena et al 2001) However as previouslymentioned significant post-induction increase of mimosine concentra-tion in roots and shoots simultaneously was only observed for JA andEthephon 10 ppm on day 02 and 04 respectively (Fig 1)
It is well established that perceived regulatory signals or elicitorsgenerate a transduction network mediated by secondary messengersresulting in changes in gene expression profiles that afford adaptiveresponses to environmental stimuli These modulation events are oftenmediated by transcription factors (TFs) which directly bind to specificgene promoters or act by forming complexes with repressor proteinslabeling them to degradation subsequently releasing other TFs toproceed with the gene expression program This is the case of the actionmechanism of JA and its active form jasmonoyl isoleucine for example(Kazan 2015 Wasternack and Strnad 2016)
JA ethylene and SA are known as important stress regulatory sig-nals in plants JA however is thought to be the most effective signal forinduction of plant secondary metabolism (Wasternack and Strnad2016) thereby contributing to mitigation of damage caused by severalstresses (Dar et al 2015) JA is mainly derived from linolenic acid
Fig 1 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings treated with differentelicitors CTRL=Milli-Q water SA = Salicylic AcidJA= Jasmonic Acid ETH=Ethephon Bars sharing a letterof same case do not differ by Tukey test (P le 005) Capitalletters (A B) compare treatments on day two and lowercaseletters (a b) compare treatments on day four Indicatessignificant statistical difference between day two and dayfour in the same treatment by t-test (Ple 005) The errorbars represent standard error of five replicates (each meanwas calculated with 15 individual seedlings organized in 5groups of three)
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(Wasternack and Strnad 2016) playing important roles in differentprocesses of plant growth and development such as plant defensemechanisms against herbivory pathogen attack fungal elicitation andsome abiotic factors such as osmotic temperature and salt stresses (Daret al 2015)
JA and its methyl ester MeJA have several different effects on le-guminous species MeJA exogenous application has increased iso-flavonoid content in cell suspension cultures of Pueraria candollei varcandollei and P candollei var mirifica (Korsangruang et al 2010) aswell as the production of the triterpenoid glycyrrhizin in Glycyrrhizaglabra roots Enhanced production of the triterpenoid however waspartly at the expense of root growth (Shabani et al 2009) MeJA ap-plication on shoots was observed to suppress root nodulation and lat-eral root formation in Lotus japonicus (Nakagawa and Kawaguchi2006) In grapevine a non-leguminous species proteinogenic aminoacids did not show an expressive increase under MeJA treatment(Gutieacuterrez-Gamboa et al 2017)
The effects of the application of four different jasmonate forms (JAMeJA jasmonoyl-L-isoleucine (JA-Ile) and 6-ethyl indanoyl glycineconjugate (2-[(6-ethyl-1-oxo-indane-4-carbonyl)-amino]-acetic acidmethyl ester - CGM) on leucaena metabolite profile has recently beenreported by Xu et al (2018) JA-Ile form was most effective althoughno major alteration was observed on monitored metabolite abundancesAlanine threonine and 34-dihydroxypyridine (34 DHP a metabolitederived from mimosine degradation) (Nguyen and Tawata 2016)among others were the major metabolites elicited by JA-Ile In contrastto the results described here mimosine concentration did not changesignificantly These divergent results on mimosine accumulation maybe due to a number of factors including mode of application jasmonateform used (JA-Ile x JA) and L leucocephala subspecies (common x giantleucaena)
Ethylene is also a phytohormone involved in plant response me-chanisms to different types of challenges such as mechanical damageand insect attack among others The integration mechanism betweenJA and ethylene signaling pathways is not completely understoodhowever it has been shown that they may work cooperatively in abioticstress tolerance (Kazan 2015) MeJA can induce ethylene production(Zhao et al 2004) and when applied simultaneously these moleculesseem to work in a synergic way by enhancing the magnitude of theplant response to external stimuli (Liu et al 2016)
Treatment with SA was able to significantly increase mimosine ac-cumulation in 12-week-old plants of common leucaena (SupplementaryFig 2) However no significant effect of SA treatment on mimosineconcentration was seen in 5-week-old seedlings of giant leucaena(Fig 1) suggesting some degree of genotype andor age dependency inelicitation by this phytohormone On the other hand several treat-ments including 90 ppm MeJA 10 and 100 ppm 2-chloroethylpho-sphonic acid (CEPA an ethylene-releasing compound) significantlyincreased mimosine accumulation (Supplementary Fig 2) in agree-ment with the data obtained for giant leucaena The lack of systemiceffects of externally applied SA on mimosine accumulation was alsoobserved when the phytohormone was supplied in the culture mediumof aseptically-grown seedlings in which case only roots had highercontent of mimosine (Vestena et al 2001) This could be due totransport limitations or to low methyl salicylate production from ap-plied SA since the former is recognized as the main systemic signalingform (Vlot et al 2009)
32 Increased mimosine concentrations in giant leucaena exposed to UV-Cradiation
UV-C treatment promoted increased concentration of the aminoacid in shoots but not in roots of giant leucaena (Fig 2) Increasedaccumulation of mimosine in shoots was also observed in 12-week-oldseedlings of common leucaena exposed to UV-C radiation for 10 and15min (Supplementary Fig 3) Similar to the SA treatment in giant
leucaena UV-C radiation did not induce mimosine biosynthesis in rootsregardless of time after exposure The absence of mimosine induction inroots by SA and UV indicates that these effectors do not cause a sys-temic response Moreover roots are shielded from irradiance by thepresence of substrate
UV radiation effects on different aspects of plant metabolism anddevelopment have been described However compared to UV-B (en-vironmentally relevant type of UV radiation) assays there are less re-ports related to the UV-C effects on secondary metabolites biosynthesisand accumulation (Cetin 2014) especially in leguminous (Fabaceae)plants They generally concern primary metabolism aspects such asgrowth and development For instance seedlings of Phaseolus vulgaris L(Fabaceae) exposed to low intensity UV-C radiation have displayeddecreased chlorophyll content and reduced height after 14 days of ex-posure (Kara 2013) Negative effects on growth parameters and ni-trogen metabolism were also observed in Vigna radiata L (Fabaceae)after UV-B radiation treatment in addition to adverse effects on JA SAand antioxidant compounds accumulation (Choudhary and Agrawal2014a) The same authors reported increased accumulation of flavo-noids SA and JA besides negative effects on growth biomass yieldnitrogen fixation and accumulation in 2 cultivars of Pisum sativum L(Fabaceae) under elevated UV-B treatment (Choudhary and Agrawal2014b) Despite the negative UV influence on growth reported for thepreviously mentioned leguminous UV-C radiation on groundnut plants(Arachis hypogaea L Fabaceae) increased seedling vigor and biomassand had no adverse effect on germination or other development para-meters (Neelamegam and Sutha 2015)
Besides its impact on growth and primary metabolism UV exposurecan cause important changes in secondary metabolism depending onintensity and time of exposure (Matsuura et al 2013) UV-B and UV-Cpre-treatments of Artemisia annua (Asteraceae) seedlings yielded in-creased biosynthesis of artemisinin a drug which displays anti-malarialproperties and activity against some others infectious diseases (egschistosomiasis leishmaniasis and hepatitis B) and several kinds oftumors (Rai et al 2011) The accumulation of nicotine in Nicotianarustica plants (Solanaceae) was also increased by UV-C treatment(Tiburcio et al 1985) Similar inducing effects on production of severalsecondary metabolites were observed in callus cultures of Vitis viniferaL Oumlkuumlzgoumlzuuml (grapevine Vitaceae) treated with a UV-C source for 5 or10min (Cetin 2014)
Regarding amino acid biosynthesis in response to UV radiationMartiacutenez-Luumlscher et al (2014) have found that in spite of not causingchanges in total amino acid content UV-B radiation exposure can affecttheir profile in grape berries Proteinogenic amino acids have beenknown to be important targets of the deleterious effects of UV radiation(Holloacutesy 2002) On the other hand in the present study acute UV-Ctreatment was found to increase mimosine accumulation in shoots byover twofold (Fig 2) which may suggest a possible participation of thismolecule as part of the antioxidant defense system in L leucocephalaThis possibility is further supported by the induction of the amino acidaccumulation by JA and Ethephon involved in abiotic and biotic stressresponses which are generally associated with oxidative imbalance andare signaling components in high UV stress (Matsuura et al 2013)
33 Mimosinase gene expression
In order to determine if increases in mimosine content upon ex-posure to JA CEPA or UV-C radiation were related to changes intranscription of mimosine metabolism-related genes RT-qPCR analysiswas carried out The complete pathway for mimosine biosynthesis hasnot yet been determined although the final step has been character-ized Based on transcription analysis (Ishihara et al 2016a) leucaenaappears to encode for multiple cysteine synthases one or more of whichmay be able to catalyze mimosine synthesis In addition a leucaenagene encoding a mimosinase (an enzyme responsible for mimosinedegradation) has been identified and characterized (Negi et al 2014)
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In addition to mimosinase gene expression several gene isoformsbelonging to the cysteine pathway [cysteine synthase (CYS SYN) serineacetyltransferase (SAT) and β-cyanoalanine synthase (CAS) Table 2 -supplementary material] were also tested in this study (data notshown) However expressions of these genes did not vary in giantleucaena throughout the experiments suggesting that the increasedcontent of mimosine observed in the treated plants might not be relatedto the expression of these genes but presumably to increased enzymeactivity andor release from conjugates such as mimoside a mimosineβ-D-glucoside (Murakoshi et al 1972)
Considering the time variation of mimosine accumulation observedin this work mimosinase gene expression in shoots and roots wasevaluated 24 h before the increase of mimosine concentration in giantleucaena seedlings (ie 24 h and 72 h after the chemical elicitorstreatments and 48 h and 120 h after UV-C exposure)
Ethylene signaling has been shown to up-regulate expression ofseveral genes related to secondary metabolism pathways as is the caseof phenolic compounds (Liu et al 2016) and terpenoid indole alkaloids(Wang et al 2016) Among all elicitors tested in the present workEthephon was the only one able to significantly change mimosinasegene expression Leucaena plants treated with Ethephon showed sig-nificant increases in mimosine concentration at both day 2 and 4 fol-lowing treatment which coincided with low-level expression of mi-mosinase Up-regulation of mimosinase gene expression was detected24 h before the increase of mimosine concentration in shoots treatedwith 10 ppm of Ethephon (Fig 3A) but not after JA or UV-C treatments(Fig 3C-D and 3E-F respectively) Nevertheless 72 h after treatment
application (24 h before the highest mimosine content measured inshoots) down regulation of mimosinase gene was seen in both shootsand roots treated with 10 ppm of Ethephon (Fig 3B) These data in-dicate that mimosine content in leucaena plants is at least partlyregulated by mimosinase expression in Ethephon exposed plants Onthe other hand the fact that mimosinase mRNA was not significantlyaffected by JA and UV-C treatments despite their stimulating effects onmimosine biosynthesis in giant leucaena may indicate that other levelsof regulation are at play or that the chosen harvesting time window wasunable to detect relevant changes
34 In situ and in vitro antioxidant assays
Considering the stimulation of mimosine accumulation byEthephon JA and UV all of which are often associated or known tocause oxidative imbalance the antioxidant capacity of mimosine wasevaluated Mimosine has been shown to have antioxidant activities oncultured cancer cells (Parmar et al 2015) In the present study it washypothesized that mimosine could confer radical scavenging propertieswhich would contribute to plant protection from possible damagecaused by reactive oxygen species generated during stress(Supplementary Fig 4)
Foliar disks of P vulgaris L were treated with 10mM mimosine for15min Treated disks showed less hydrogen peroxide accumulationinduced by wounding in contrast to untreated ones being comparableto those treated with ascorbic acid (a known hydrogen peroxide neu-tralizer) (Fig 4A) These observations support a possible antioxidant
Fig 2 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings exposed to UV-C lightCTRL= visible light (100 μmol photons mminus2 s minus1) UV-C 10primeand UV-C 15rsquo=UV-C exposure time (10 and 15min re-spectively) Bars sharing a letter of same case do not differ byTukey test (P le 005) Capital letters (A B) compare treat-ments on day three and lowercase letters (a b) comparetreatments on day six Indicates significant statistical dif-ference between day three and day six in the same treatmentby t-test (Ple 005) The error bars represent standard errorof five replicates (each mean was calculated with 15 in-dividual seedlings organized in 5 groups of three)
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role of mimosine as an in situ hydrogen peroxide scavengerMimosine was also able to quench superoxide anions generated by
light exposure Mimosine exhibited equivalent antioxidant effect com-pared to rutin (Fig 4B) a well-established effective superoxide anionquencher (Matsuura et al 2016) The radical scavenging activity ofmimosine may be due to the 3-OH group of the pyridine ring of mi-mosine (Fig 5) The pKa of the 3-OH of mimosine has been estimated tobe 88 (M Honda unpublished results) At physiological pH this OHgroup is expected to remain in a protonated state and therefore mayscavenge a radical by donating a proton and an electron In this processmimosine itself is converted to a stable radical form which is perhapsless toxic and less reactive than the reactive oxygen species generatedduring oxidative stress It is likely that the less toxic radical mimosineproduced may react with another radical or molecule and becomeconverted to a non-reactive indole molecule
In vivo antioxidant activity of mimosine has been previously eval-uated by means of its exogenous application on selenium-deficientseedlings of Vigna radiata In spite of its allelopathic properties (Ahmedet al 2008) the results showed mitigation of mitochondrial oxidativestress by treatment with 01mM mimosine (Lalitha and Kulothungan2007) DPPH radical scavenging activity was also reported for aqueous
seed extracts of leucaena rich in mimosine and phenolic compounds inin vitro assays (Benjakul et al 2014) Mimosine antioxidant activityshown in the present work is in good agreement with data reported forother non-protein amino acids such as L-DOPA (Dhanani et al 2015)and GABA (Malekzadeh et al 2014) for instance
4 Conclusion
Taken together results show that mimosine biosynthesis and ac-cumulation can be modulated by stress-related factors despite its re-latively high constitutive content in leucaena plants The pattern ofgene expression in stressed plants suggests mimosine steady-state con-trol may be regulated by its degradation in possible connection withdynamic changes in carbon and nitrogen metabolism of stressed plantsMimosine quenching activity against hydrogen peroxide and super-oxide anions in the in situ staining and in vitro assays respectivelyshowed that this non-protein amino acid can act as non-enzymaticantioxidant agent Increase in mimosine content in response to elicitorsmimicking environmental challenges in addition to its antiherbivoreand antimicrobial properties may be related to its activity as protectivemolecule against oxidative damage in line with other classes of plant
Fig 3 Relative expression of the mimosinase gene in shoots (A E and F) and shoots and roots (B C and D) of giant leucaena 24 h (A and C) 48 h (E) 72 h (B and D)and 120 h (F) after treatment with stress signaling molecules or UV-C exposure ETH = Ethephon JA = Jasmonic Acid Indicates significant statistical differencebetween control and treatment by t-test (Ple 005) The error bars represent standard error of four replicates
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secondary metabolites
Funding
This work was funded by the National Council for Scientific andTechnological Development (CNPq-Brazil) grant 3060792013-5Coordenaccedilatildeo de Aperfeiccediloamento de Pessoal de Niacutevel Superior - Brazil(CAPES) - Finance Code 001 and the USDA NIFA Hatch projectHA05029-H managed by CTAHR
CRediT authorship contribution statement
Kelly Cristine da Silva Rodrigues-Correcirca InvestigationValidation Writing ndash original draft Michael DH HondaInvestigation Validation Dulal Borthakur Supervision Writing ndashreview amp editing Funding acquisition Arthur Germano Fett-NetoSupervision Funding acquisition Writing ndash review amp editing
Acknowledgements
The authors would like to thank Dr Jorge Ernesto Mariath fromLaVeg-UFRGS for kindly lending the Leica M165 FC stereomicroscopefor in situ analysis
Appendix A Supplementary data
Supplementary data to this article can be found online at httpsdoiorg101016jplaphy201811018
References
Ahmed R Hoque ATMR Hossain MK 2008 Allelopathic effects of Leucaena
leucocephala leaf litter on some forest and agricultural crops grown in nursery J ForRes 19 298 httpsdoi 101007s11676-008-0053-0
Benjakul S Kittiphattanabawon P Shahidi F Maqsood S 2013 Antioxidant activityand inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipidoxidation in model systems Food Sci Technol Int 19 (4) 365ndash376 httpsdoiorg1011771082013212455186
Benjakul S Kittiphattanabawon P Sumpavapol P Maqsood S 2014 Antioxidantactivities of lead (Leucaena leucocephala) seed as affected by extraction solvent priordechlorophyllisation and drying methods extracts against lipid oxidation in modelsystems Food Sci Technol 51 (11) 3026ndash3037 httpsdoiorg101007s13197-012-0846-1
Brewbaker JL Pluckett D Gonzalez V 1972 Varietal variation and yield trials ofLeucaena leucocephala (koa haole) in Hawaii Hawaii Agric Exp Stn Bull 166 26
Brewbaker JL 2008 Registration of KX2 ndash Hawaii interspecific-hybrid leucaena JPlant Registrations 1 (3) 190ndash193 httpsdoiorg103198jpr2007050298crc
Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitisvinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-6287-47-37
Cho H-Y Son SY Rhee HS Yoon S-YH Lee-Parsons CWT Park JM 2008Synergistic effects of sequential treatment with methyl jasmonate salicylic acid andyeast extract on benzophenanthridine alkaloid accumulation and protein expressionin Eschscholtzia californica suspension cultures J Biotechnol 135 117ndash122 httpsdoiorg101016jjbiotec200802020
Choudhary KK Agrawal SB 2014a Cultivar specificity of tropical mung bean (Vignaradiata L) to elevated ultraviolet-B changes in antioxidative defense system ni-trogen metabolism and accumulation of jasmonic and salicylic acids Environ ExpBot 99 122ndash132 httpsdoiorg101016jenvexpbot201311006
Choudhary KK Agrawal SB 2014b Ultraviolet-B induced changes in morphologicalphysiological and biochemical parameters of two cultivars of pea (Pisum sativum L)Ecotoxicol Environ Saf 100 178ndash187 httpsdoiorg101016jecoenv201310032
Dar TA Uddin M Khan MMA Hakeem KR Jaleel H 2015 Jasmonates counterplant stress a Review Environ Exp Bot 115 49ndash57 httpsdoiorg101016jenvexpbot201502010
Dhanani T Singh R Shah S Kumari P Kumar S 2015 Comparison of green ex-traction methods with conventional extraction method for extract yield L-DOPAconcentration and antioxidant activity of Mucuna pruriens seed Green Chem LettRev 8 (2) 43ndash48 httpsdoiorg1010801751825320151075070
Gutieacuterrez-Gamboa G Portu J Santamariacutea P Loacutepez R Garde-Cerdaacuten T 2017Effects on grape amino acid concentration through foliar application of three dif-ferent elicitors Food Res Int 99 688ndash692 httpsdoiorg101016jfoodres201706022
Fig 4 A In situ antioxidant assay Foliar disksof Phaseolus vulgaris L treated with (a) No an-tioxidant added (negative control) (b) 10 mMMimosine (c) 10mM ascorbic acid (positivecontrol) The oxidative damage can be seen bythe formation of a brown polymer in leaf veinsand injured areas B In vitro superoxidescavenging assay carried out with mimosineDifferent letters indicate significant differenceby Tukey test (Ple 005) The error bars re-present standard error of four replicates (Forinterpretation of the references to colour in thisfigure legend the reader is referred to the Webversion of this article)
Fig 5 Predicted mimosine radical formed followingquenching of hydroxyl radical Mimosine is first converted toa stable mimosine radical which may be then converted to anontoxic indole form
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
439
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad HossainMd Anai T Oku H 2018 Cytosolic cysteine synthase switch cysteine and mi-mosine production in Leucaena leucocephala Appl Biochem Biotechnol 186 (3)613ndash632 httpsdoiorg101007s12010-018-2745-z
Holloacutesy F 2002 Effects of ultraviolet radiation on plant cells Micron 33 (2) 179ndash197Honda MDH Ishihara KL Pham DT Borthakur D 2018 Identification of drought-
induced genes in giant leucaena (Leucaena leucocephala subsp glabrata) Trees 32571ndash585 httpsdoiorg101007s00468-018-1657-4
Huang T Jander G de Vos M 2011 Non-protein amino acids in plant defense againstinsect herbivores representative cases and opportunities for further functional ana-lysis Phytochemistry 72 1531ndash1537 httpsdoiorg101016jphytochem201103019
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of thethyrotoxic amino acid mimosine by cysteine synthase Phytochemistry 29 (11)3461ndash3465 httpsdoiorg1010160031-9422(90)85258-H
Ishihara K Lee EKW Borthakur D 2016a An improved method for RNA extractionfrom woody legume species Acacia koa A Gray and Leucaena leucocephala (Lam) deWit Int J For Wood Sci 3 (1) 031ndash035
Ishihara KL Honda MDH Pham DT Borthakur D 2016b Transcriptome analysisof Leucaena leucocephala and identification of highly expressed genes in roots andshoots Transcriptomics 4 135 httpsdoiorg1041722329-89361000135
IUBMB 2018 Enzyme Nomenclature EC 35161 httpwwwsbcsqmulacukiubmbenzymeEC35161html Accessed date 8 February 2018
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant(Phaseolus vulgaris) Biosci Res 10 (1) 29ndash32
Kazan K 2015 Diverse roles of jasmonates and ethylene in abiotic stress toleranceTrends Plant Sci 20 (4) 219ndash229 httpsdoiorg101016jtplants201502001
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect ofEthephon as an ethylene-releasing compound on the metabolic profile of Chlorellavulgaris J Agric Food Chem 64 (23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541
Khan MIR Fatma M Per TS Anjum NA Khan NA 2015 Salicylic acid-inducedabiotic stress tolerance and underlying mechanisms in plants Front Plant Sci 6 462httpsdoiorg103389fpls201500462
Korsangruang S Soonthornchareonnon N Chintapakorn Y Saralamp PPrathanturarug S 2010 Effects of abiotic and biotic elicitors on growth and iso-flavonoid accumulation in Pueraria candollei var candollei and P candollei var mir-ifica cell suspension cultures Plant Cell Tissue Organ Cult 103 (3) 333ndash342 httpsdoiorg101007s11240-010-9785-6
Lalitha K Kulothungan SR 2006 Selective determination of mimosine and its dihy-droxypyridinyl derivative in plant systems Amino Acids 31 (3) 279ndash287 httpsdoiorg101007s00726-005-0226-5
Lalitha K Kulothungan SR 2007 Mimosine mitigates oxidative stress in seleniumdeficient seedlings of Vigna radiata - Part I restoration of mitochondrial functionBiol Trace Elem Res 118 (1) 84ndash96 httpsdoiorg101007s12011-007-0013-0
Liu J Li Y Wang Y Zhang Z-H Zu Y-G Efferth T Tang Z-H 2016 Thecombined effects of ethylene and MeJA on metabolic profiling of phenolic com-pounds in Catharanthus roseus revealed by metabolomics analysis Front Physiol 71ndash11 httpsdoiorg103389fphys201600217 Article 217
Malekzadeh P Khara J Heydari R 2014 Alleviating effects of exogenous Gamma-aminobutiric acid on tomato seedling under chilling stress Physiol Mol Biol Plants20 (1) 133ndash137 httpsdoiorg101007s12298-013-0203-5
Martiacutenez-Luumlscher J Torres N Hilbert G Richard T Saacutenchez-Diacuteaz M Delrot SAguirreolea J Pascual I Gomegraves E 2014 Ultraviolet-B radiation modifies thequantitative and qualitative profile of flavonoids and amino acids in grape berriesPhytochemistry 102 106ndash114 httpsdoiorg101016jphytochem201403014
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation ofbioactive secondary metabolites by ultraviolet radiation mechanisms strategies andapplications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology forMedicinal Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash1902012
Matsuura HN Fragoso V Paranhos JT Rau MR Fett-Neto AG 2016 Thebioactive monoterpene indole alkaloid N szlig-D-glucopyranosylvincosamide is regu-lated by irradiance quality and development in Psychotria leiocarpa Ind Crop Prod86 210ndash218 httpsdoiorg101016jindcrop201603050
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo RBhambra AS Strnad M Bonfill M Fett-Neto AG 2018 Specialized plant me-tabolism characteristics and impact on target molecule biotechnological productionMol Biotechnol 60 (2) 169ndash183 httpsdoiorg101007s12033-017-0056-1
Murakoshi S Ohmiya S Haginiwa J 1972 Enzymic synthesis of mimoside a meta-bolite of mimosine in Mimosa pudica and Leucaena leucocephala Chem Pharm Bull20 (4) 855ndash857
Nakagawa T Kawaguchi M 2006 Shoot-applied MeJA suppresses root nodulation inLotus japonicus Plant Cell Physiol 47 (1) 176ndash180 httpsdoiorg101093pcppci222
Nascimento NC Menguer PK Henriques AT Fett-Neto AG 2013 Accumulation ofbrachycerine an antioxidant glucosidic indole alkaloid is induced by abscisic acidheavy metal and osmotic stress in leaves of Psychotria brachyceras Plant PhysiolBiochem 73 33ndash40 httpsdoiorg101016jplaphy201308007
Neelamegam R Sutha T 2015 UV-C irradiation effect on seed germination seedling
growth and productivity of groundnut (Arachis hypogaea L) Int J Curr MicrobiolApp Sci 4 (8) 430ndash443
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase fromLeucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol164 (2) 922ndash934 httpsdoiorg101104pp113230870
Negi VS Borthakur D 2016 Heterologous expression and characterization of mimo-sinase from Leucaena leucocephala In Fett-Neto Arthur Germano (Ed)Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods inMolecular Biology vol 1405 copySpringer Science+Business Media New York httpsdoiorg101007978-1-4939-3393-8_7 2016
Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636
Parmar F Kushawaha N Highland H George L-B 2015 In vitro antioxidant andanticancer activity of Mimosa pudica Linn extract and L-mimosine on lymphomaDaudi cells Int J Pharm Sci 12 100ndash104
Porto DD Matsuura HN Vargas LRB Henriques AT Fett-Neto AG 2014 Shootaccumulation kinetics and effects on herbivores of the wound-induced antioxidantindole alkaloid brachycerine of Psychotria brachyceras Nat Prod Commun 9 (5)629ndash632
Rai R Meena RP Smita SS Shukla A Rai SK Pandey-Rai S 2011 UV-B and UV-C pre-treatments induce physiological changes and artemisinin biosynthesis inArtemisia annua L ndash an antimalarial plant J Photochem Photobiol B Biol 105 (3)216ndash225 httpsdoiorg101016jjphotobiol201109004
Shabani L Ehsanpour AA Asghari G Emami J 2009 Glycyrrhizin production by invitro cultured Glycyrrhiza glabra elicited by methyl jasmonate and salicylic acid RussJ Plant Physiol 56 (5) 621ndash626 httpsdoiorg101134S1021443709050069
Shah J 2003 The salicylic acid loop in plant defense Curr Opin Plant Biol 6 (4)365ndash371
Shi J Fu XZ Peng T Huang XS Fan QJ Liu JH 2010 Spermine pretreatmentconfers dehydration tolerance of citrus in vitro plants via modulation of antioxidativecapacity and stomatal response Tree Physiol 30 (7) 914ndash922 httpsdoiorg101093treephystpq030
Smith IK Fowden L 1966 A study of mimosine toxicity in plants J Exp Bot 17750ndash761 httpsdoiorg101093jxb174750
Soedarjo M Borthakur D 1996 Simple procedures to remove mimosine from youngleaves pods and seeds of Leucaena leucocephala used as food Int J Food SciTechnol 31 (1) 97ndash103
Soedarjo M Borthakur D 1998 Mimosine a toxin produced by the tree-legumeLeucaena provides a nodulation competition advantage to mimosine-degradingRhizobium strains Soil Biol Biochem 30 1605ndash1613
Suda S 1960 On the physiological properties of mimosine Bot Mag Tokyo 73 (862)142ndash147 httpsdoiorg1015281jplantres188773142
Tangendjaja B Lowry JB Wills RBH 1986 Isolation of a mimosine degrading en-zyme from leucaena leaf J Sci Food Agric 37 523ndash526 httpsdoiorg101002jsfa2740370603
Tiburcio F Pintildeol MT Serrano M 1985 Effect of UV-C on growth soluble protein andalkaloids in Nicotiana rustica plants Environ Exp Bot 25 (3) 203ndash210 httpsdoiorg1010160098-8472(85)90004-8
Vestena S Fett-Neto AG Duarte RC Ferreira A 2001 Regulation of mimosineaccumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 httpsdoiorg101016S0168-9452(01)00448-4
Vlot AC Dempsey DMA Klessig DF 2009 Salicylic acid a multifaceted hormone tocombat disease Annu Rev Phytopathol 47 177ndash206 httpsdoiorg101146annurevphyto050908135202 2009
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene-induced vinblastine accumulation is related to activated expression of downstreamTIA pathway genes in Catharanthus roseus BioMed Res Int 2016 Article ID 3708187httpsdoiorg10115520163708187
Wasternack C Strnad M 2016 Jasmonate signaling in plant stress responses and de-velopment ndash active and inactive compounds N Biotech 33 (5B) 604ndash613 httpsdoiorg101016jnbt201511001
Wencomo HB Ortiz R Caacuteceres J 2017 Afr J Agric Res 12 (4) 279ndash285 httpsdoiorg105897AJAR201510604 26
Wong CC Devendra C 1983 Research on leucaena forage production in Malaysia InLeucaena Research in the Asian Pacific Region pp 55ndash60 Ottawa Ontario Canada
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX TsimKWK 2018 Jasmonate-elicited stress induces metabolic change in the leaves ofLeucaena leucocephala Molecules 23 (2) httpsdoiorg103390molecules23020188 E188
Yafuso JT Negi VS Bingham J-P Borthakur D 2014 An O-acetylserine (thiol)lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthaseAppl Biochem Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z
Zhao J Zheng S-H Fujita K Sakai K 2004 Jasmonate and ethylene signalling andtheir interaction are integral parts of the elicitor signalling pathway leading to b-thujaplicin biosynthesis in Cupressus lusitanica cell cultures J Exp Bot 55 (399)1003ndash1012 httpsdoiorg101093jxberh127
Zhishen J Mengcheng T Jianming W 1999 The determination of flavonoid contentsin mulberry and their scavenging effects on superoxide radicals Food Chem 64 (4)555ndash559 httpsdoiorg101016S0308-8146(98)00102-2
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
440
61
Supplementary Fig 1 Basal mimosine concentration in adult trees of common leucaena (L leucocephala
var leucocephala) Samples were collected from 10 field grown trees at Manoa Valley Honolulu Hawairsquoi
on June 25th 2017 Bars sharing a letter do not differ by Tukey test (P le 005) The error bars represent the
standard error
Supplementary Fig 2 Bar diagram showing mimosine concentration in shoots of 12-week-old common
leucaena seedlings treated with different elicitors CTRL = Milli-Q water SA = Salicylic Acid MeJA =
Methyl Jasmonate CEPA = 2-Chloroethylphosphonic acid (an ethylene releasing compound) Bars sharing a
letter of same case do not differ by Tukey test (P le 005) Capital letters (A B) compare treatments on day
two and lower-case letters (a b) compare treatments on day four Indicates significant statistical difference
ABB
A A
0
200
400
600
800
1000
1200
LEAVES GREEN FLOWERBUDS
POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
con
cen
trat
ion
(micro
gg
-1o
f FW
)
B AB AB AB B A
b
a
ab b
ab
0
2
4
6
8
10
12
14
16
18
20
CTRL SA 10 ppm SA 100 ppm CEPA 10 ppm CEPA 100 ppm MeJA 90 ppm
Mim
osi
ne
co
nce
ntr
atio
n (
gg
-1o
f FW
)
DAY 02 DAY 04
62
between day two and day four in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
Supplementary Fig 3 Bar diagram showing the effects of UV-C radiation exposure for 5 10 and 15 min on
mimosine accumulation in shoots of 12-week-old seedlings of common leucaena Bars sharing a letter of
same case do not differ by Tukey test (P le 005) Capital letters (A B C) compare treatments on day three
and lower-case letters (a b) compare treatments on day six Indicates significant statistical difference
between day three and day six in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
C BC AB A
bb
a
a
0
10
20
30
40
50
60
CTRL UV-C 5 UV-C 10 UV-C 15
Mim
osi
ne
co
nce
ntr
atio
n (
gg-1
of
FW)
DAY 03 DAY 06
63
Supplementary Fig 4 Model depicting induction of mimosine synthesis in leucaena following application of
stress elicitors such as CEPA and jasmonic acid or exposure to UV-C radiation The additional mimosine
synthesized may serve to alleviate oxidative stress induced by UV-C radiation
64
Supplementary Table 1 Mimosine contents in leaves of common and giant leucaena
Leucaena
type
Mimosine content
( FW)
Mimosine
content ( DW)
Dry matter
content ( FW)
Water content
( FW)
Common (1) 050 plusmn 009 245 plusmn 051 2011 plusmn 054 7989 plusmn 054
Common (2) 043 plusmn 006 214 plusmn 037 1998 plusmn 050 8002 plusmn 050
K636 (1) 070 plusmn 014 356 plusmn 077 1908 plusmn 052 8092 plusmn 052
K636 (2) 042 005 205 plusmn 033 2008plusmn 093 7992plusmn 093
KX2 (1) 122 plusmn 011 608 plusmn 082 1939 plusmn 123 8061 plusmn 123
KX2 (2) 134 plusmn 010 623 plusmn 056 2029 plusmn 114 7971 plusmn 114
KX3 (1) 044 plusmn 006 221 plusmn 030 1945 plusmn 073 8055 plusmn 073
KX3 (2) 054 plusmn 005 273 plusmn 023 1930 plusmn 038 8070 plusmn 038
KX4 (1) 086 plusmn 011 471 plusmn 065 1753 plusmn 084 8247 plusmn 084
KX4 (2) 089 plusmn 011 476 plusmn 065 180 plusmn 072 820 plusmn 072
KX5 (1) 099 plusmn 012 489 plusmn 048 1907 plusmn060 8093 plusmn 060
KX5 (2) 115 plusmn 015 548 plusmn080 1992 plusmn 053 8008 plusmn 053
Common leucaena variety koa haole grows widely on the island of Orsquoahu K636 is widely
grown variety of giant leucaena KX2 KX3 KX4 and KX5 are giant leucaena varieties
developed through interspecies hybridization (Brewbaker 2016) (1) and (2) indicate plants
from two separate locations within the University of Hawaii Waimanalo Research Center The
values are shown as mean plusmn standard error obtained from at least three biological replicates
65
Supplementary Table 2 GenBank accession numbers of the tested cysteine pathway genes isoforms
Gene name GenBank accession
OAS-TL (o-acetylserine-thiol-lyase) GDRZ01032940
GDRZ01061620
GDRZ01153117
GDSA01187555
GDSA01196891
GDSA01214467
Cys syn (cysteine synthase) GDRZ01015860
GDRZ01050898
GDRZ01086813
GDRZ01193515
GDRZ01202579
GDSA01180863
GDSA01215622
SAT (serine acetyltransferase) GDRZ01187456
GDRZ01189631
CAS (β-cyanoalanine synthase) GDRZ01054066
GDRZ01175418
GDSA01118400
66
SHORT COMMUNICATION 1
Mimosine occurrence and accumulation in Mimosa bimucronata var bimucronata (DC) 2
Kuntze 3
Kelly Cristine da Silva Rodrigues-Correcirca1 Lana Dorneles Pedroso2 Fernanda de Costa1 4
Arthur Germano Fett-Neto1 5
1Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal 6
University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970 7
Porto Alegre Rio Grande do Sul Brazil 2Department of Biological Sciences Unipampa ndash 8
Campus Satildeo Gabriel 9
Corresponding author 10
E-mail addresses krodriguescbiotufrgsbr (KCdaS Rodrigues-Correcirca) 11
lanalima2012gmailcom (LD Pedroso) fernandadecostayahoocombr (F de Costa) 12
fettnetocbiotufrgsbr (AG Fett-Neto) 13
14
15
16
17
18
19
20
21
22
67
ABSTRACT 23
Mimosine is a non-protein aromatic amino acid present in plants of Leucaena spp 24
and Mimosa spp Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) is a native 25
tree from Brazil which occurs as a pioneer species on plant succession processes In the 26
current study the presence of mimosine in M bimucronata was verified by HPLC analyses 27
Moreover mimosine accumulation upon exposure to UV-C and chemical elicitors of 28
specialized metabolism (salicylic acid - SA methyl jasmonate - MeJA sodium nitroprusside 29
- SNP and ethephon - ETH) most of which also known as promoters of the amino acid 30
production in leucaena plants was evaluated The results showed a lower concentration of 31
constitutive mimosine present in both maricaacute seedlings and mature trees when compared to 32
leucaena plants In spite of a trend towards increased mimosine accumulation observed in 33
MeJA and ETH treatments no statistical differences were found with the various stressors 34
used to induce its biosynthesis in maricaacute seedlings Data suggest that mimosine in M 35
bimucronata is probably a phytoanticipin-like metabolite or its accumulation is driven by 36
other types of stresses 37
38
39
Keywords Mimosine Mimosa bimucronata stress 40
41
42
43
44
45
46
68
Introduction 47
Mimosa bimucronata commonly known as maricaacute is a native tree from Brazil 48
(REFLORA 2019) ecologically important in plant succession and in processes of degraded 49
land recovery (Bitencourt et al 2007 Silva et al 2011) occurring as a pioneer species 50
(Pilatti et al 2019) Maricaacute is a deciduous or semi-deciduous plant which reaches up to 15 51
m in height and 40 cm of diameter at breast height (DBH) displays shrub or tree habit and 52
bears typical sharp thorns (Carvalho 2004) This species belongs to Fabaceae one of the 53
most economically important families of flowering plants due to its high diversity and 54
occurrence in different types of habitats (Gomes et al 2018) As well as several others 55
Mimosa spp maricaacute is usually referred to as a multipurpose tree (Olkoski and Wittmann 56
2011) employed for alternative medicinal uses (Champanerkar et al 2010 Silva et al 57
2011) honey production constructions and remodeling of landscape architecture (living 58
fences) for instance (Marchiori 1993 Lorenzi 1998) 59
In southern Brazil maricaacute is widely distributed and typically found either in wetland 60
areas close to river banks (Patreze and Cordeiro 2004) or composing large and almost pure 61
landscape formations on hillsides (Jacobi and Ferreira 1991) In dense populations this 62
species like several Mimosa spp (Simon and Proenccedila 2000) is considered an important and 63
highly invasive weed by preventing cattle to reach pasturesand water bodies as a result of its 64
thorny branches (Lorenzi 2008 Kestring et al 2009) Its dominant and nearly exclusive 65
pattern of distribution in those areas has led Jacobi and Ferreira (1991) to test its allelopathic 66
potential on cultivated species Indeed extracts of leaves and ripe fruits (but not the green 67
ones) of maricaacute showed phytotoxic effects on germination and initial radical growth of most 68
of the target species tested 69
69
Several investigations have been performed on maricaacute floristics (Silva et al 2011) 70
distribution (Simon and Proenccedila 2000) wood anatomy (Marchiori 1993) cytogenetic 71
parameters (Olkoski and Wittmann 2011) and allelopathic potential (Jacobi and Ferreira 72
1991 Ferreira et al 1992) However excluding two recent publications on maricaacute 73
constitutive chemical composition (Schlickmann et al 2017 Pilatti et al 2019) which 74
identified phenolic compounds (methyl gallate and water-soluble tannins) as its major 75
compounds little is known regarding this subject In other Mimosa species (eg M pudica 76
and M pigra) mimosine has been identified (Soedarjo and Borthakur 1998) as one of the 77
major specialized metabolites present in the different organs of the plant (Champanerkar et 78
al 2010) The presence of this molecule was also reported for M bimucronata in a thin layer 79
chromatography-based preliminary study performed by Ferreira et al (1992) showing co-80
chromatography of a leaf extract component with authentic mimosine The authors attributed 81
the allelopathic effect of maricaacute to the accumulation of this metabolite in its leaves 82
Mimosine is an aromatic non-protein amino acid initially found in plants of Mimosa 83
pudica and later in Leucaena leucocephala (Lam) de Wit (Soedarjo and Borthakur 1998) a 84
leguminous tree which biosynthesizes large amounts of this nitrogen-containing compound 85
(Rodrigues-Correcirca et al 2019) It is believed that the accumulation of high contents of 86
mimosine in L leucocephala tissues confers among other traits defense against herbivores 87
and pathogens (Vestena et al 2001) tolerance to drought (Negi et al 2014) as well as 88
general oxidative stress protection (Rodrigues-Correcirca et al 2019) Interestingly drought is 89
the opposite environmental and physiological condition to that observed in the wet habitats 90
occupied by native populations of M bimucronata in Brazil (Patreze and Cordeiro 2004 91
Kestring et al 2009) and Mimosa pudica Linn in India (Champanerkar et al 2010) 92
70
Nonetheless flooding is also associated with oxidative stress particularly as water levels 93
change (Fukao et al 2019) 94
In Leucaena leucocephala var leucocephala (common leucaena) and Leucaena 95
leucocephala var glabrata (giant leucaena) mimosine accumulation has been shown to be 96
both constitutive and inducible by stress-related phytohormones such as jasmonic acid (JA) 97
Ethephon (ETH an ethylene- releasing compound) salicylic acid (SA - only common 98
leucaena) (Vestena et al 2001) as well as by UV-C radiation (Xu et al 2018 Rodrigues-99
Correcirca et al 2019) On the other hand there is a lack of information regarding mimosine 100
content and elicitation effects in Mimosa spp plants 101
The aim of this study was to examine the presence of mimosine in Mimosa 102
bimucronata and examine the effects of stresses and stress-signaling molecules on its 103
accumulation in leaves 104
Material and Methods 105
Plant material 106
For all experiments the plant material was collected at Morro Santana campus do 107
Vale of UFRGS (Federal University of Rio Grande do Sul) Porto Alegre RS Brazil 108
(3004rsquoS 5108rsquoW) Authorization for access to genetic material was obtained from 109
SISGEN-Brazil (license number A845493) Constitutive mimosine content in adult plants of 110
M bimucronata var bimucronata (DC) Kuntze was determined in plant material (leaves 111
green flower buds post-anthesis flowers and green pods) harvested in January 2017 112
(summer) A voucher herbarium specimen (ICN 187953) was deposited in the ICN ndash UFRGS 113
herbarium (Herbaacuterio do Instituto de Biociecircncias of UFRGS) 114
71
For mimosine elicitation experiments legumes (fruits) of maricaacute were collected in 115
the end of June 2017 (winter) Seeds were then removed from the dry fruits and kept in the 116
dark until sowing and seedling development for use in the assays 117
Seed germination 118
To break the coat-imposed seed dormancy after surface sterilization dry seeds of 119
maricaacute were acid scarified by immersion in H2SO4 (95 ndash 98 ) for 2 min (see Correcirca et al 120
2008) and repeatedly washed in distilled water to remove any residue of the acid Then seeds 121
were distributed in 50 mL individual plastic tubes (dibble-tubes) (30 cm diameter x 120 cm 122
depth) filled up with 11 (vv) of commercial top soil and vermiculite Tubes were watered 123
every 2 days to avoid substrate dryness and were kept in a growth room under controlled 124
conditions of light (circa 75 μmol mminus2s minus1 photosynthetically active radiation photoperiod 125
of 16 h light and 8 h dark) and temperature (24plusmn2C) 126
127
Treatments 128
In order to verify inducibility of mimosine accumulation in M bimucronata fifty 12-129
week-old maricaacute seedlings (per treatment) exhibiting similar features were selected and 130
sprayed (saturated) with solutions of different chemical stressors (plant specialized 131
metabolism elicitors) as follows (for further details see Rodrigues-Correcirca et al 2019) 10 132
and 50 mM SA (pathogen-signaling molecule Shah 2003) 007 and 035 mM 2-133
chloroethylphosphonic acid (ETH ethylene releasing-compound Kim et al 2016 Wang et 134
al 2016) 100 and 200 mM MeJA (Dar et al 2015) 10 and 50 mM SNP (a nitric oxide 135
donor Perotti et al 2015) Alternatively maricaacute seedlings were also supplemented with UV-136
C radiation (13 minutes 105 kJ cm2) (elicitor of plant specialized metabolism Kara 2013) 137
72
After 2 and 4 days of exposure to the chemical treatments and 3 and 6 days of UV-138
C supplementation maricaacute shoots were harvested immediately frozen in liquid nitrogen and 139
stored at ndash 80 C until mimosine extraction and HPLC analyses 140
Mimosine extraction and detection 141
Mimosine extraction was conducted according to the modified protocol described by 142
Rodrigues-Correcirca et al (2019) for L leucocephala HPLC (Thermo Scientific Surveyor) 143
analyses (mimosine detection and quantification) were performed following previously 144
published procedures (Negi et al 2014) A C18 column (ACE C18 5 μm 46times250 mm) and 145
isocratic solvent system of 002M o-phosphoric acid with a linear flow rate of 1 mL min minus1 146
were used to separate and quantify the amino acid Mimosine detection was performed at 280 147
nm by photodiode array detection (200ndash400 nm) and retention time (229plusmn0024 min) 148
Mimosine quantification was done by means of the method of external standard curve 149
Additional confirmation of mimosine identity was performed by co-chromatography with 150
standard (Acros Organics authentic mimosine 99 used as reference) and peak purity check 151
The analyses of the chromatograms were done with the ChromQuest software 152
153
154
Results and Discussion 155
Constitutive accumulation of mimosine in M bimucronata 156
Mimosine was detected in all analyzed samples positively meeting all identification 157
criteria In agreement with what has been found for other Mimosa spp (Soedarjo and 158
Borthakur 1998) compared to L leucocephala adult plants (Rodrigues-Correcirca 2019) 159
mimosine content was lower in M bimucronata Of the adult plant tissues analyzed the 160
73
highest content of mimosine in maricaacute (per gram of fresh weight - FW) was found in post-161
anthesis flowers (36644 microg versus 89448 microg in common leucaena followed by leaves 162
(28838 microg x 67358 microg) green flower buds (28094 microg x 51247 microg) and green pods (19002 163
microg x 82687 microg) (Fig 1)The same pattern is observed for seedlings when both species are 164
compared In this study untreated 12-week-old maricaacute seedlings (control at day 2) showed a 165
shoot content of mimosine of 23029plusmn007 microg g-1 of (FW) Five-week-old untreated giant 166
leucaena seedlings cultivated in similar conditions exhibited between 83640 and 178736 167
microg g-1 of FW (Rodrigues-Correcirca et al 2019) In the same way mimosine concentration 168
percentage in dry matter of Mimosa pigra was found to be rather low (002 in nodules and 169
roots and 007 in leaves) (Soedarjo and Borthakur 1998) 170
In this investigation the lowest constitutive mimosine content was found in green 171
pods (Fig 1) This result may partly explain the absence of phytotoxic effect observed for 172
green pods on germination and growth of crop target plants tested by Jacobi and Ferreira 173
(1991) compared to the other maricaacute parts analyzed 174
Elicitation of mimosine biosynthesis in M bimucronata 175
Chemical stressors 176
Secondary metabolites (or natural products) are structural- and chemically 177
specialized compounds derived from primary metabolism These molecules are mainly 178
biosynthesized as part of a complex defense mechanism in response to biotic and abiotic 179
stresses such as pathogens herbivores water status metal toxicity and UV radiation for 180
example (Matsuura et al 2018) Ethephon SA SNP MeJA have been extensively used as 181
chemical elicitors of specialized metabolism (Wang et al 2016 Vestena et al 2001 Perotti 182
74
et al 2015 Zhang and Memelink 2009 Xu et al 2018) These phytohormonal signals can 183
simulate environmental challenges and modulate plant homeostasis often leading to 184
alterations in gene expression (Shinozaki et al 2015) Except SNP all treatments tested in 185
the present study showed positive effect on mimosine accumulation in common or giant 186
leucaena (Vestena et al 2001 Rodrigues-Correcirca 2019 Rodrigues-Correcirca unpublished 187
data) However in spite of the trend of increasing the mimosine content observed in seedlings 188
treated with 007 mM Ethephon (at day 2) and 100 mM MeJA (at day 4) no statistical 189
difference was confirmed for these treatments when compared to the control 190
On the other hand a within treatment difference on mimosine induction was seen 191
between day 2 and 4 in seedlings treated with 100 mM MeJA (Fig 2) In a lower 192
concentration (04 mM) jasmonic acid (JA)promoted a near threefold increase in mimosine 193
accumulation of giant leucaena seedlings after 2 days of application 194
UV-C radiation 195
Albeit UV-C radiation is not biologically active in natural environments it has been 196
widely used under controlled experimental conditions to generate acute responses of plant 197
specialized metabolism within a shorter period of time compared to that required to with UV-198
B radiation (Kara 2013 Cetin 2014) This fast response is due to the higher energy of UV-199
C photons that act as potent reactive oxygen species (ROS) generators causing extensive 200
damage to the cells either at the physiological level or on DNA structure (Gregianini et al 201
2003 Matsuura et al 2013) 202
Although divergent responses can be observed in plants exposed to UV-C radiation 203
the deleterious processes are usually reported on primary metabolism (decreasing of 204
chlorophyll content and plant height eg) (Kara 2013) In the present study no statistical 205
75
differences were observed in the mimosine concentration in maricaacute seedlings supplemented 206
with UV-C radiation However a decreasing in its content was found for both control and 207
treatment at day 6 post-treatment (Fig 03) Taking into account the lower constitutive 208
concentration of mimosine observed in maricaacute compared to the leucaena plants besides its 209
relative thermolability (Nguyen and Tawata 2016) it seems to be plausible to consider the 210
effect of the temperature inside the UV-C and the white light (control) chambers as an 211
additional abiotic factor contributing to the decrease of mimosine accumulation in both group 212
of plants 213
Besides mimosine identification the presence of 34-dihydroxypyridine (34-DHP or 214
3-hydroxy-4-pyridone - 3H4P) a mimosine degradation product (Negi et al 2014 Nguyen 215
and Tawata 2016) was also reported for maricaacute leaf extracts analyzed by TLC by Ferreira 216
et al (1992) In our chromatograms we detected a second large peak after that of mimosine 217
(229plusmn0024) and similar to that identified by Negi et al (2014) as 3H4P (data not shown) 218
Comparing the chromatogram profiles obtained from seedlings elicited with chemical 219
stressors and those supplemented with UV-C the largest area for this peak was found (in all 220
samples) in the latter treatment at day 6 It might indicate that the constitutive andor the 221
initially UV-C-induced mimosine was degraded into 3H4P to cope with the cellular damage 222
caused by this treatment associated with an increased temperature inside the chambers 223
Nevertheless it was not possible to determine 3H4P concentration (or confirm its identity) 224
in maricaacute plants since there is no commercial standard (pure 3H4P) available for purchase 225
to be used as a reference in calculations Establishment of improved protocols for obtaining 226
in house 3H4P reference substance by acid hydrolysis is ongoing 227
228
229
76
Conclusion 230
On the basis of the overall absence of effect of the treatments tested here on mimosine 231
concentration it is possible to suggest that its accumulation profile is similar to that of 232
phytoanticipins unlike what is observed for the same amino acid production in leucaena 233
which shows features of inducibility resembling phytoalexin-like metabolites Alternatively 234
a putative inducible pool of mimosine in maricaacute might be involved in other types of stress 235
such as extended drought periods If involved in protection against oxidative stress as 236
described for leucaena mimosine in maricaacute may act predominantly by physical quenching 237
of ROS as indicated by the lack of overt chemical degradation Nevertheless further 238
investigations are needed to assess these hypotheses 239
To sum up mimosine biosynthesis was not modulated by the treatments evaluated as 240
in L leucocephala (Lam) de Wit To the best of our knowledge this is the first work that 241
analytically identifies and quantifies mimosine accumulation in M bimucronata 242
243
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Bitencourt F Zocche JJ Costa S Souza PZ Mendes AR 2007 Nucleaccedilatildeo de 245
Mimosa bimucronata (DC) O Kuntze em aacutereas degradadas pela mineraccedilatildeo de carvatildeo R 246
Bras Bioci 5 750-752 247
Carvalho PER 2004 Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 248
Teacutecnica 94 1-10 249
Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitis 250
vinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-251
6287-47-37 252
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Champanerkar PA Vaidya VV Shailajan S Menon SN 2010 A sensitive rapid and 253
validated liquid chromatography ndash tandem mass spectrometry (LC-MS-MS) method for 254
determination of Mimosine in Mimosa pudica Linn Nat Sci 2 713-717 255
httpsdoiorg104236ns201027088 256
Gomes GS Silva GS Silva DLS Oliveira RR Conceiccedilatildeo GM 2018 Botanical 257
Composition of Fabaceae Family in the Brazilian Northeast Maranhatildeo Brazil Asian J 258
Environ Ecol 6(4) 1-10 httpsdoiorg109734AJEE201841207 259
Correcirca LR Soares GLG Fett-Neto AG 2008 Allelopathic potential of Psychotria 260
leiocarpa a dominant understorey species of subtropical forests S Afri J Bot 74 583ndash261
590 httpsdoiorg101016jsajb200802006 262
Ferreira AG Aquila MEA Jacobi US Rizvi V 1992 Allelopathy in Brazil In Allelopathy 263
basic and applied aspects Rizvi V and Jacobi US (Eds) Chapman and Hall pp 243-250 264
Fukao T Barrera-Figueroa BE Juntawong P Pentildea-Castro JM 2019 Submergence 265
and waterlogging stress in plants a review highlighting research opportunities and 266
understudied aspects Front Plant Sci 10 340 httpsdoiorg103389fpls201900340 267
Gregianini TS Silveira VC Porto DD Kerber VA Henriques AT Fett-Neto AG 268
2003 The alkaloid brachycerine is induced by ultraviolet radiation and is a singlet oxygen 269
quencher Photochem Photobiol 78(5) 470ndash474 httpsdoiorg1015620031-270
8655(2003)0784070TABIIB20CO2 271
Jacobi US Ferreira AG 1991 Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK 272
sobre espeacutecies cultivadas Pesq Agropec Bras 26(7) 935-943 273
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant 274
(Phaseolus vulgaris) Biosci Res 10(1) 29ndash32 275
78
Kestring D Klein J Menezes LCCR Rossi MN 2009 Imbibition phases and 276
germination response of Mimosa bimucronata (Fabaceae Mimosoideae) to water 277
submersion Aquat Bot 91 105ndash109 httpsdoiorg101016jaquabot200903004 278
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect of 279
Ethephon as an ethylene-releasing compound on the metabolic profile of Chlorella vulgaris 280
J Agric Food Chem 64(23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541 281
Lorenzi H 1998 Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 282
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 283
Lorenzi H 2008 Plantas daninhas do Brasil terrestres aquaacuteticas parasitas e toacutexicas 4 ed 284
Nova Odessa Instituto Plantarum 640 p 285
Marchiori JNC 1993 Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 286
O Kuntze Ciecircncia Florestal 3 85-106 287
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation of 288
bioactive secondary metabolites by ultraviolet radiation mechanisms strategies and 289
applications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology for Medicinal 290
Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash190= 291
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo R Bhambra AS 292
Strnad M Bonfill M Fett-Neto AG 2018 Specializedplant 293
metabolismcharacteristicsandimpactontargetmoleculebiotechnologicalproduction 294
Molecular Biotechnology 60(2) 169ndash183httpsdoiorg101007s12033-017-0056-1 295
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase from 296
Leucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 297
922ndash934 httpsdoiorg101104pp113230870 298
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Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine 299
areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636 300
Olkoski D Wittmann MTS 2011 Cytogenetics of Mimosa bimucronata (DC) O Kuntze 301
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop Breed 302
Appl Biotechnol 11 27-35 httpdxdoiorg101590S1984-70332011000100004 303
Patreze CM Cordeiro L 2004 Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 304
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecol Manag 196 275ndash305
285 httpdxdoiorg101016jforeco200403034 306
Perotti JC Rodrigues-Correcirca KCS Fett-Neto AG 2015 Control of resin production in 307
Araucaria angustifolia an ancient South American conifer Plant Biology 17 852ndash859 308
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG 2019 Mimosine 309
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 310
UV exposure Plant Physiology and Biochemistry 135 432ndash440 311
Pilatti DM Fortes AMT Jorge TCM Boiago NP 2019 Comparison of the phytochemical 312
profiles of five native plant species in two different forest formations Brazilian Journal of 313
Biology 79(2) 233-242 314
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS 2011 Biologia da reproduccedilatildeo 315
deMimosa bimucronatandash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 316
Simon MF and Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 317
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 318
endemism Biological Conservation 96 279-296 319
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 320
LM Andrade SF Cechinel-Filho V 2017 Chemical composition and diuretic natriuretic and 321
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kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its majority 322
constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash1624 323
Shah J 2003 The salicylic acid loop in plant defense Current Opinion Plant Biology6 (4) 324
365ndash371 325
Shinozaki K Uemura M Serres JB Bray EA Weretilnyk E 2015 Responses to Abiotic 326
Stress In Buchanan BB Gruissem W Jones RL (Eds) Biochemistry and Molecular 327
Biology of Plants Second Edition John Wiley and Sons Ltd 328
Soedarjo M and Borthakur D 1998 Mimosine a toxin produced by the tree-legume 329
Leucaena provides a nodulation competition advantage to mimosine-degrading Rhizobium 330
strains Soil Biology and Biochemistry 30(12)1605-1613 331
Vestena S Fett-Neto AG Duarte RC Ferreira AG 2001 Regulation of mimosine 332
accumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 333
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene induced 334
vinblastine accumulation is related to activated expression of downstream TIA pathway 335
genes in Catharanthus roseus BioMed Research International Article ID 3708187 336
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX Tsim KWK 2018 337
Jasmonate-elicited stress induces metabolic change in the leaves of Leucaena leucocephala 338
Molecules 23 (2) 339
Zhang H Memelink J 2009 Regulation of Secondary Metabolism by Jasmonate Hormones 340
In AE Osbourn and V Lanzotti (eds) Plant-derived Natural Products 3 DOI 101007978-341
0-387-85498-4_1 copy Springer Science + Business Media LLC 342
343
344
345
81
346
Figure 1 Constitutive concentration of mimosine in different plant organs of Mimosa 347
bimucronata Bars sharing the same letter do not differ statistically by Tukey test (Ple005) 348
The error bars denote standard error of 10 replicates 349
350
351
352
353
354
355
356
357
B B A C0
5
10
15
20
25
30
35
40
LEAVES GREEN FLOWER BUDS POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
co
nce
ntr
atio
n u
gg-1
Mimosine concentration in adult plants of Mimosa bimucronata (DC) Kuntze
82
C T R L S A
1 0 m M
S A
5 0 m M
E T H
0 0 7 m M
E T H
0 3 5 m M
M e J A
1 0 0 m M
M e J A
2 0 0 m M
S N P
1 0 m M
S N P
5 0 m M
0
1 0
2 0
3 0
T re a tm e n ts
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1) D A Y 2
D A Y 4
A B C C B C A B C C A B C A B C A
a b b b a a b a a b b a b
358
Figure 2 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 359
bimucronata treated with different signaling molecules SA = Salicylic Acid ETH = 360
Ethephon MeJA = Methyl Jasmonate SNP = Sodium Nitroprusside Uppercase and 361
lowercase letters indicate statistical differences among treatments in days 2 and 4 362
respectively Bars sharing a letter of the same case do not differ statistically by Tukey test 363
(Ple005) Indicates statistical difference in the same treatment between day 2 and 4 by t-364
test (Ple005) The error bars denote standard error of 5 replicates (25 individual seedlings 365
arranged in 5 groups of 5) 366
367
368
83
D AY 3 D AY 6
0
5
1 0
1 5
2 0
2 5
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1)
C O N TR O L
U V -C
369
Figure 3 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 370
bimucronata supplemented with UV-C radiation Indicates statistical difference in the same 371
treatment between day 3 and 6 by t-test (Ple005) The error bars denote standard error of 5 372
replicates (25 individual seedlings arranged in 5 groups of 5) 373
374
375
376
377
378
379
380
381
382
383
384
385
84
Consideraccedilotildees finais 386
- Experimentos que avaliam os efeitos da aplicaccedilatildeo exoacutegena de ANPs em diferentes espeacutecies 387
vegetais tecircm sido realizados principalmente com GABA Dentre os principais efeitos 388
conferidos pela aplicaccedilatildeo dessa moleacutecula em espeacutecies de mono e eudicotiledocircneas satildeo 389
relatados a toleracircncia agrave seca agrave salinidade e agraves temperaturas extremas 390
- Como metaboacutelitos especializados claacutessicos os ANPs podem ter sua concentraccedilatildeo basal 391
endoacutegena aumentada em resposta agrave induccedilatildeo mediada por uma vasta gama de tratamentos com 392
moleacuteculas sinalizadoras de estresse e fontes alternativas de estressores De um modo geral 393
observa-se o acuacutemulo das diferentes classes de ANPs em resposta agrave radiaccedilatildeo UV elicitores 394
quiacutemicos que mimetizam ataques por patoacutegenos dano mecacircnico agentes osmoacuteticos metais 395
pesados entre outros 396
- Especificamente em leucena a resposta observada em relaccedilatildeo aos diferentes tratamentos 397
testados indica que apesar do seu alto teor constitutivo nessa espeacutecie a biossiacutentese e o 398
acuacutemulo de mimosina podem ser modulados por fatores causadores de estresses exibindo -399
nessa espeacutecie - um padratildeo de acumulaccedilatildeo similar agrave fitoalexinas Em maricaacute por outro lado 400
aumento de acuacutemulo dessa moleacutecula natildeo foi observado para os mesmos tratamentos testados 401
para leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas 402
- O padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno 403
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado pela 404
sua degradaccedilatildeo 405
- As respostas observadas nos testes que avaliaram a atividade de mitigaccedilatildeo de espeacutecies 406
reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode agir como um agente 407
antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de estresse 408
85
Perspectivas 409
- Confirmaccedilatildeo em espectrocircmetro de massas eou ressonacircncia nuclear magneacutetica da natureza 410
quiacutemica da lsquomimosinarsquo presente em maricaacute 411
- Avaliaccedilatildeo do efeito de concentraccedilotildees mais elevadas e em diferentes periacuteodos de aplicaccedilatildeo 412
das moleacuteculas sinalizadoras testadas sobre o acuacutemulo de mimosina em leucena e maricaacute 413
- Ampliar a investigaccedilatildeo dos padrotildees de expressatildeo gecircnica dos genes que codificam para 414
mimosinase (em maricaacute) mimosina sintase (em ambas as espeacutecies testadas) bem como o 415
perfil de precursores e cataboacutelitos de mimosina em resposta aos tratamentos mencionados 416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
86
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Benjakul S Kittiphattanabawon P Shahidi F Maqsood S (2013) Antioxidant activity and 446
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model systems Food Sci Technol Int 19(4)365-76 448
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Bitencourt F Zocche JJ Costa S Souza PZ Mendes AR (2007) Nucleaccedilatildeo de Mimosa 450
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Crowe B Poynter JA Manukyan MC Wang Y Brewster BD Herrmann JL Abarbanell 465
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β-ODAP content in Lathyrus sativus and their possible physiological mechanisms Food 491
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Yamaguchi N (2014) Activation of the prereplication complex is blocked by mimosine 494
88
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8 pages httpdxdoiorg1011552014737263 500
Kusama-Eguchi K (2019) Research in motor neuron diseases caused by natural substances 501
focus on pathological mechanisms of neurolathyrism Yakugaku Zasshi 139 (4) 609-502
615 httpsdoiorg101248yakushi18-00202 503
Kutchan TM Gershenzon J Moslashller BL Gang DR (2015) Natural Products In Buchanan 504
BB Gruissem W and Jones RL (eds) Biochemistry amp Molecular Biology of Plants 2nd edn 505
Wiley Blackwell Chichester pp 1135-1205 506
Lalande M (1990) A reversible arrest point in the late G1 phase of the mammalian cell cycle 507
Exp Cell Res 186 332ndash339 508
Li X-W Hu C-P Li Y-J Gao Y-X Wang XM Yang J-R (2015) Inhibitory effect of L-509
mimosine on bleomycin-induced pulmonary fibrosis in rats Role of eIF3a and p27 Int 510
Immunopharmacol 27(1) 53ndash64 511
Little Jr EL Skolmen RG (1989) Koa haole Agriculture Handbook 679 USDA 512
Lorenzi H (1998) Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 513
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 514
Marchiori JNC (1993) Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 515
O Kuntze Ciecircncia Florestal 3 85-106 516
Mohammed RS El Souda SS Taie HAA Moharam ME Shaker KH (2015) Antioxidant 517
antimicrobial activities of flavonoids glycoside from Leucaena leucocephala leaves Journal 518
of Applied Pharmaceutical Science 5(06)138-147 519
httpdxdoiorg107324JAPS201550623 520
Negi VS Bingham J-P Li QX Borthakur D (2014) A carbon-nitrogen lyase from Leucaena 521
leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 (2) 922ndash522
934 httpsdoiorg101104pp113230870 523
89
Olkoski D Wittmann MTS (2011) Cytogenetics of Mimosa bimucronata (DC) O Kuntze 524
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop 525
Breeding and Applied Biotechnology 11 27-35 526
Patreze CM Cordeiro L (2004) Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 527
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecology and Management 528
196275ndash285 529
Pilatti DM Fortes AMT Jorge TCM Boiago NP (2019) Comparison of the phytochemical 530
profiles of five native plant species in two different forest formations Brazilian Journal of 531
Biology 79(2) 233-242 532
Ramos-Ruiz R Poirot E Flores-Mosquera M (2018) GABA a non-protein amino acid 533
ubiquitous in food matrices Cogent Food Agric 41534323 534
httpsdoiorg1010802331193220181534323 535
REFLORA (2019) httpfloradobrasiljbrjgovbrreflora Acesso em agosto de 2019 536
Rodgers KJ Samardzic K Main BJ (2015) Toxic Nonprotein Amino Acids Plant Toxins 537
httpsdoiorg 101007978-94-007-6728-7_9-1 538
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG (2019) Mimosine 539
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 540
UV exposure Plant Physiology and Biochemistry 135 432ndash440 541
httpsdoiorg101016jplaphy201811018 542
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 543
LM Andrade SF Cechinel-Filho V (2017) Chemical composition and diuretic natriuretic 544
and kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its 545
majority constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash546
1624 547
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS (2011) Biologia da reproduccedilatildeo 548
de Mimosa bimucronata ndash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 549
Simon MF Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 550
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 551
endemism Biological Conservation 96 279-296 552
90
Soares AMS Arauacutejo SA Lopes SG Costa Junior LM (2015) Anthelmintic activity of 553
Leucaena leucocephala protein extracts on Haemonchus contortus Braz J Vet Parasitol 554
Jaboticabal 24(4) 396-401 httpdxdoiorg101590S1984-29612015072 555
Soerdajo M Borthakur D (1998) Mimosine a toxin produced by the tree-legume Leucaena 556
provides a nodulation competition advantage to mimosine-degrading Rhizobium strains Soil 557
Biol Biochem 30(12) 16051613 558
Souza-Lima ES Sinani TR Pott A Sartori ALB (2017) Mimosoideae (Leguminosae) in the 559
Brazilian Chaco of Porto Murtinho Mato Grosso do Sul Rodrigueacutesia 68(1) 263-290 2017 560
httpdxdoiorg1015902175-7860201768131 561
Taiz L amp Zeiger E (2010) Plant Physiology 5th edition Sinauer Associates Inc Sunderland 562
Verma VK Rani KV Kumara SR Prakash O (2018) Leucaena leucocephala pod seed 563
protein as an alternate to animal protein in fish feed and evaluation of its role to fight against 564
infection caused by Vibrio harveyi and Pseudomonas aeruginosa Fish and Shellfish 565
Immunology 76 (2018) 324ndash332 httpsdoiorg101016jfsi201803011 566
Yafuso JT Negi VS Bingham J-P Borthakur D (2014) An O-acetylserine (thiol) lyase from 567
Leucaena leucocephala is a cysteine synthase but not a mimosine synthase Appl Biochem 568
Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z 569
Zarin RMA Wan HY Isha A Armani N (2016) Antioxidant antimicrobial and cytotoxic 570
potential of condensed tannins from Leucaena leucocephala hybrid Food Science and 571
Human Wellness 5 65ndash75 httpdxdoiorg101016jfshw201602001 572
573
574
Contents lists available at ScienceDirect
Industrial Crops amp Productsjournal homepage wwwelseviercomlocateindcrop
Resin tapping transcriptome in adult slash pine (Pinus elliottii var elliottii)Camila Fernanda de Oliveira Junkes1 Artur Teixeira de Arauacutejo Juacutenior1 Juacutelio Ceacutesar de LimaFernanda de Costa Thanise Fuumlller Maacutercia Rodrigues de Almeida Franciele Antocircnia NeisKelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-NetoCenter for Biotechnology and Department of Botany Federal University of Rio Grande do Sul Porto Alegre PO Box 15005 91501-970 Brazil
A R T I C L E I N F O
KeywordsPinus elliottiResinResinosisTranscriptomeAdjuvant paste
A B S T R A C T
To better understand the bases of resin production a major source of terpenes for industry the transcriptome ofadult Pinus elliottii var elliottii (slash pine) trees under field commercial resinosis was obtained Samples werecollected from cambium after 5 and 15 days of treatment application which included tapping followed byapplication of commercial resin stimulant paste or control wounding without paste Overall mean number ofreads of all 16 libraries (2 treatments x 2 times x 4 replicated trees) was 34582048 Of these 89 were mappedagainst the reference sequence with a mismatch of 058 Using the Blast2Go 570 candidate genes were de-tected based on sequence annotation By comparing the expression profile between paste and control 310differentially expressed genes (DEGs) were identified at 5 days and 190 at 15 days with a significant fold changeof log2gt 12 Regarding changes in time comparisons within each treatment 210 and 105 DEGs were identifiedwithin control and paste treatment respectively Genes with different expression patterns in the times andtreatments examined included ethylene responsive transcription factors geranylgeranyl diphosphate synthasediterpene synthase cytochrome P450 and ABC transporters all of which may play important roles in resinproduction RT-qPCR analysis correlated well with the data obtained by RNAseq Resin composition changedover time This is the first transcriptomic investigation of resinosis of the main species used in the bioresinindustry and of molecular analyses of resinosis under field operations with implications for stand managementstimulant paste development genotype selection and breeding for high resinosis
1 Introduction
The adaptive success of conifers is largely due to the development ofa defense system based on the synthesis and secretion of terpenes in allmajor organs and different tissues (Miller et al 2005 Hall et al 2013Warren et al 2015) Conifer resin is a viscous fluid composed of acomplex mixture of terpenoids such as monoterpenes sesquiterpenesand diterpenes (Zulak and Bohlmann 2010) These terpenoids are se-creted from severed resin ducts when the tree is under biotic attack(Ralph et al 2006 Lange 2015 Geisler et al 2016) acting as pro-tectants (Schiebe et al 2012 Liu et al 2015)Biosynthesis of terpenes in conifers starts from isomerization of two
isoprenoid (C5) units dimethylallyl diphosphate (DMAPP) and iso-pentenyl diphosphate (IPP) These molecules can be biosynthesized viatwo separate routes in plants the methyl-erythritol 4-phosphate andmevalonate pathways IPP is synthesized and isomerized to DMAPP byisopentenyl diphosphate isomerase then prenyl transferases catalyze
the condensation of these two C5-units to geranyl diphosphate (Pazoukiand Niinemets 2016) Their elongation to prenyl diphosphates withaddition of IPP molecules leads to monoterpenes (C10) sesquiterpenes(C15) and diterpenes (C20) which are the substrates for terpene syn-thases (TPS) (Keeling and Bohlmann 2006b)TPSs are part of a large family of mechanistically related enzymes
involved in both primary and secondary metabolism (Keeling andBohlmann 2006b) The events of evolutionary diversification and ex-pansion of plant TPSs appear to have originated from gene duplicationsdomain losses and sub- or neofunctionalizations with subsequent di-vergence of an ancestral TPS gene of primary metabolism (Hall et al2013) Modification of TPS products changes their physical propertiesand may alter their biological activities (Chen et al 2011) TPSs of highsequence identity may have different functions even in closely relatedspecies Low sequence identity of TPSs in phylogenetically distantspecies does not preclude the possibility of independent evolution of thesame or related function of these enzymes (Zerbe and Bohlmann 2015)
httpsdoiorg101016jindcrop2019111545Received 4 January 2019 Received in revised form 10 June 2019 Accepted 4 July 2019
Corresponding authorE-mail address fettnetocbiotufrgsbr (AG Fett-Neto)1 These authors have equally contributed to this work
doi 1015900102-33062019abb0114
Acta Botanica Brasilica
Sustainable production of bioactive alkaloids in Psychotria L of
southern Brazil propagation and elicitation strategies
Yve Verocircnica da Silva Magedans1 Kelly Cristine da Silva Rodrigues-Correcirca1 Cibele Tesser da Costa1
Heacutelio Nitta Matsuura1 and Arthur Germano Fett-Neto1
Received April 1 2019Accepted June 28 2019
ABSTRACTPsychotria is the largest genus in Rubiaceae South American species of the genus are promising sources of natural
products mostly due to bioactive monoterpene indole alkaloids they accumulate ese alkaloids can have analgesic
antimutagenic and antioxidant activities in dierent experimental models among other pharmacological properties
of interest Propagation of genotypes with relevant pharmaceutical interest is important for obtaining natural
products in a sustainable and standardized fashion Besides the clonal propagation of elite individuals the alkaloid
content of Psychotria spp can also be increased by applying moderate stressors or stress-signaling molecules is
review explores advances in research on methods for plant propagation and elicitation techniques for obtaining
bioactive alkaloids from Psychotria spp of the South Region of Brazil
Keywords abiotic stress alkaloids elicitation monoterpenes plant propagation Psychotria southern Brazil
sustainability
Introduction
Psychotria belongs to Rubiaceae one of the major families
of $owering plants having economic interest e family
includes coee a few signicant poisonous plants to livestock
besides several important ornamental and medicinal species
(Souza amp Lorenzi 2012) Psychotria has captured researchersrsquo
attention mostly because of its medicinal properties
Psychotria colorata is an Amazonian species that produces
polyindolinic alkaloids with analgesic activity (Matsuura et
al 2013) e promising results obtained with P colorata
motivated the investigation of southern Brazilian Psychotria
species and the discovery of new bioactive alkaloids (Porto
et al 2009) Moreover leads on in planta alkaloid functions
were also topic of experimental evaluation
One of the key elements that needs to be addressed early
on during the process of developing new bioactive molecules
from plants is the capacity to generate catalytically active
biomass to support extraction and steady supply ere are a
number of ways through which these goals may be reached
including greenhouse rooting of cuttings (mini-cutting
1 Laboratoacuterio de Fisiologia Vegetal Departamento de Botacircnica Instituto de Biociecircncias e Centro de Biotecnologia Universidade Federal do Rio
Grande do Sul 91501-970 Porto Alegre RS Brazil
Corresponding author fettnetocbiotufrgsbr
Review
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Biomass yield of resin in adult Pinus elliottii Engelm trees is differentially
regulated by environmental factors and biochemical effectors
Franciele Antocircnia Neis Fernanda de Costa Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima
Kelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-Neto
Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) CP 15005 CEP 91501-970 Porto Alegre RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Biomass
Terpene resin
Seasonal
Benzoic acid
Regenerated forest
A B S T R A C T
Biomass of pine resin finds several applications in the chemical pharmaceutical biofuel and food industries
Resin exudation after injury is a key defense response in Pinaceae since this complex mixture of terpenes has
insecticidal antimicrobial and wound repair properties Resin yield is increased by effectors applied on the
wound area including phytohormones and metal cofactors of terpene synthases The interaction of resinosis
mechanism effectors is not fully understood particularly in adult forest setups under natural environmental
variations The aim of this work was to determine how resin exudation by wounded trunks of adult P elliottii
responded to combined chemical effectors involved in different regulatory pathways of resinosis (metal cofactors
of terpene synthases benzoic acid and plant growth regulators) and whether seasonal and tree distribution
variations affected these responses Symmetrically planted and scattered trees regenerated from the seed bank
had similar resin biomass yields suggesting that the homogeneity in development and spatial arrangement were
not significant factors in resin yield This new finding is of practical importance with the used tapping system
since costs of implanting forests by regeneration can be advantageous compared to planting In addition it was
shown for the first time that the salicylic acid precursor benzoic acid and the auxin naphthalene acetic acid
promoted resin exudation when individually applied to wound sites Both these adjuvants are two orders of
magnitude less costly compared to the conventionally used ethylene precursors besides facing less environ-
mental and health restrictions for use Most adjuvant-treated trees showed higher resin flow in the second year
indicating mechanisms of response build up Overall temperature was more important than rainfall as en-
vironmental parameter affecting resin biosynthesis which was higher in the warmer months of spring and
summer The combination of resinosis stimulant effectors from different signaling pathways showed no sig-
nificant synergistic or additive effect suggesting possible converging signaling pathways andor limitation of
common intermediate transducing molecules
1 Introduction
Pines occupy highly diverse environments over a range of tem-
peratures water and nutrient availabilities irradiance levels and pho-
toperiods being able to effectively face attacks from diverse herbivore
and pathogen guilds The success of conifers is linked to their complex
terpene biochemistry hosted by specialized secretory cells The terpe-
noid resin synthesized by Pinus spp is one of the main mechanisms of
defense of these trees particularly against bark beetles and the fungi
they carry (Fett-Neto and Rodrigues-Correcirca 2012) Pine resin biomass
is essentially composed of a monoterpene and sesquiterpene-rich tur-
pentine and diterpenoid-rich rosin fraction both finding numerous in-
dustrial applications as non-wood forest products (Rodrigues-Correcirca
et al 2012)
Molecules capable of modulating different signaling pathways have
been identified as resin yield stimulators including sulfuric acid (ex-
tends wound damage) 2-chloroethylphosphonic acid (CEPA a syn-
thetic ethylene precursor) paraquat (free radical generator) yeast ex-
tract (mimics attack by pathogens) salicylic acid (pathogen signaling
molecule) auxin (promotes ethylene biosynthesis and resin canal dif-
ferentiation) jasmonic acid (signals mechanical damage and promotes
secondary metabolism) and metal ions such as potassium iron and
manganese (cofactors of terpene synthases in conifers) and copper (a
component of ethylene receptors) (Clements 1970 Conrath et al
2002 Fett-Neto and Rodrigues-Correcirca 2012 Hudgins and Franceschi
2004 Lewinsohn et al 1994 Martin et al 2002 Popp et al 1995
httpsdoiorg101016jindcrop201803027
Received 12 December 2017 Received in revised form 9 March 2018 Accepted 13 March 2018
Corresponding author
E-mail addresses franci_neisyahoocombr (FA Neis) fernandadecostayahoocombr (F de Costa) thanisenfyahoocombr (TN Fuumlller)
jjuliocesarlimagmailcom (JC de Lima) krodriguescbiotufrgsbr (KC da Silva Rodrigues-Correcirca) jpfettcbiotufrgsbr (JP Fett) fettnetocbiotufrgsbr (AG Fett-Neto)
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Research Paper
Dual allelopathic effects of subtropical slash pine (Pinus elliottii Engelm)
needles Leads for using a large biomass reservoir
Kelly Cristine da Silva Rodrigues-Correcircaa Gelson Halmenschlagera Joseacuteli Schwambachb
Fernanda de Costaa Emili Mezzomo-Trevizana Arthur Germano Fett-Netoa
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 Brazilb University of Caxias do Sul Institute of Biotechnology Caxias do Sul RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Seasonality
Growth
Germination
Litter
Substrate
A B S T R A C T
Pinus elliottii Engelm (slash pine) is distributed along the maritime coast of Southern Brazil where it shows
invasive pattern and typical allelopathic features Large quantities of needle litter are produced by pine trees a
biomass that is little explored in areas where this species is alien Little is known about the dynamics of needle
and litter phytochemical interactions particularly in subtropical environments To elucidate the full range of
needle and litter allelopathic potential the effects of litter (superficial and deep) and seasonally harvested fresh
slash pine needles stored for different times were evaluated against lettuce tomato and cucumber seeds and
seedlings Increasing concentrations (0 1 2 4 and 8 wv) of hot and cold aqueous extracts of needles
and litter affected in different ways target plant development Growth and germination inhibition were directly
related to the highest extract concentrations (regardless of the season and mainly in hot water extracts) of
needles On the other hand stimulatory effects of litter extracts on lettuce growth were observed Growth and
germination of cucumber and tomato were not affected by pine litter as substrate when compared to rice husk
The presumable high polarity and thermal stability of slash pine leaf biomass allelochemicals and their transient
toxic effect or growth promoting impact suggest potential applications of this largely available biomass both as a
biological herbicide and growth substrate in plant propagation
1 Introduction
Native from the Northern Hemisphere Pinus is one of the most
widely distributed genera throughout different climate regions of the
globe growing either as native or alien species even in extreme habi-
tats (Rodrigues-Correcirca and Fett-Neto 2012) Despite the high economic
value currently attributed to pine wood and oleoresin (Rodrigues-
Correcirca et al 2012) there is increasing concern about the aggressive
potential of invasiveness displayed by Pinus species especially those
cultivated out of their native range of distribution (Richardson et al
2008 Rolon et al 2011) These species are dispersed by wind and there
is notably low plant diversity observed in most understories of pine
plantations (Kato-Noguchi et al 2009) This latter feature has been
considered an important trait of allelopathic interference
The term ldquoallelopathyrdquo was coined by Molisch in 1937 as a chemical
reciprocal interaction established among plants (including micro-
organisms) sharing the same site by means of the release of secondary
metabolites named allelochemicals (Rice 1984) For the most part
these metabolites are derived from the shikimic acid or isoprenoid
pathway and their biosynthesis can be modulated by biotic and abiotic
stresses (Nascimento and Fett-Neto 2010) including seasonal-related
changes (Sartor et al 2013) Allelopathy studies may range from sterile
assays (Aryakia et al 2015) to soil (Correcirca et al 2008 Sharma et al
2016) and field tests being a complex biological phenomenon to as-
certain in several circumstances due to issues of solubility release
mechanisms and stability of bioactive compounds (Scognamiglio et al
2013) Often the use of complementary methods provides more in-
formative data
The allelopathic effects of soil leachates green needles and litter
extracts of Pinus spp on germination and seedling growth aspects of
wild and crop species have been evaluated in natural and cultivated
pine stands and have proven to be stimulatory or inhibitory (Lodhi and
Killingbeck 1982 Kil and Yim 1983 Nektarios et al 2005 Akkaya
et al 2006 Machado 2007 Alrababah et al 2009 Sartor et al 2009
Kato-Noguchi et al 2011 Rolon et al 2011 Valera-Burgos et al
2012) exhibiting in some cases autotoxicity (Garnett et al 2004
Fernandez et al 2008 Zhu et al 2009 Monnier et al 2011) Studies
on potential dual allelopathic effects of Pinus elliottii Engelm (slash
httpdxdoiorg101016jindcrop201706019
Received 23 March 2017 Received in revised form 15 May 2017 Accepted 7 June 2017
Corresponding author
E-mail address fettnetocbiotufrgsbr (AG Fett-Neto)
ORIGINAL RESEARCHpublished 16 June 2016
doi 103389fpls201600849
Frontiers in Plant Science | wwwfrontiersinorg 1 June 2016 | Volume 7 | Article 849
Edited by
Juan Francisco Jimenez Bremont
Instituto Potosino de Investigacioacuten
Cientiacutefica y Tecnoloacutegica Mexico
Reviewed by
Mariacutea De La Luz Guerrero Gonzaacutelez
Universidad Autoacutenoma de San Luis
Potosiacute Mexico
Rosalia Cristina Paz
CIGEOBIO (CONICETFCEFN UNSJ)
Argentina
Correspondence
Arthur G Fett-Neto
fettnetocbiotufrgsbr
daggerThese authors have contributed
equally to this work
Specialty section
This article was submitted to
Plant Physiology
a section of the journal
Frontiers in Plant Science
Received 08 December 2015
Accepted 30 May 2016
Published 16 June 2016
Citation
de Lima JC de Costa F Fuumlller TN
Rodrigues-Correcirca KCdS Kerber MR
Lima MS Fett JP and Fett-Neto AG
(2016) Reference Genes for qPCR
Analysis in Resin-Tapped Adult Slash
Pine As a Tool to Address the
Molecular Basis of Commercial
Resinosis Front Plant Sci 7849
doi 103389fpls201600849
Reference Genes for qPCR Analysisin Resin-Tapped Adult Slash Pine Asa Tool to Address the MolecularBasis of Commercial Resinosis
Juacutelio C de Lima 1dagger Fernanda de Costa 1 dagger Thanise N Fuumlller 1
Kelly C da Silva Rodrigues-Correcirca 2 Magnus R Kerber 1 Mariano S Lima 1
Janette P Fett 1 and Arthur G Fett-Neto 1
1 Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul
Porto Alegre Brazil 2 Biological Sciences Department Regional Integrated University of Alto Uruguai and Missotildees (URI-FW)
Frederico Westphalen Brazil
Pine oleoresin is a major source of terpenes consisting of turpentine (mono- and
sesquiterpenes) and rosin (diterpenes) fractions Higher oleoresin yields are of economic
interest since oleoresin derivatives make up a valuable source of materials for chemical
industries Oleoresin can be extracted from living trees often by the bark streak method
in which bark removal is done periodically followed by application of stimulant paste
containing sulfuric acid and other chemicals on the freshly wounded exposed surface
To better understand the molecular basis of chemically-stimulated and wound induced
oleoresin production we evaluated the stability of 11 putative reference genes for the
purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis
Samples for RNA extraction were collected from field-grown adult trees under tapping
operations using stimulant pastes with different compositions and at various time points
after paste application Statistical methods established by geNorm NormFinder and
BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3
and UBI To confirm expression stability of the candidate reference genes expression
profiles of putative P elliottii orthologs of resin biosynthesis-related genes encoding Pinus
contorta β-pinene synthase [PcTPS-(minus)β-pin1] P contorta levopimaradieneabietadiene
synthase (PcLAS1) Pinus taeda α-pinene synthase [PtTPS-(+)αpin] and P taeda
α-farnesene synthase (PtαFS) were examined following stimulant paste application
Increased oleoresin yields observed in stimulated treatments using phytohormone-based
pastes were consistent with higher expression of pinene synthases Overall the
expression of all genes examined matched the expected profiles of oleoresin-related
transcript changes reported for previously examined conifers
Keywords resin Pinus gene expression normalizer genes terpene synthase
19
Chapter 2
Stimulant Paste Preparation and Bark Streak Tapping Technique for Pine Oleoresin Extraction
Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
Tapping technique comprises the extraction of pine oleoresin a non-wood forest product consisting of a
complex mixture of mono sesqui and diterpenes biosynthesized and exuded as a defense response to
wounding Oleoresin is used to produce gum rosin turpentine and their multiple derivatives Oleoresin
yield and quality are objects of interest in pine tree biotechnology both in terms of environmental and
genetic control Monitoring these parameters in individual trees grown in the fi eld provides a means to
examine the control of terpene production in resin canals as well as the identifi cation of genetic-based
differences in resinosis A typical method of tapping involves the removal of bark and application of a
chemical stimulant on the wounded area Here we describe the methods for preparing the resin-stimulant
paste with different adjuvants as well as the bark streaking process in adult pine trees
Key words Oleoresin Pine Tapping Chemical stimulant Wounding
1 Introduction
Several conifer species produce oleoresin a complex mixture of isoprenoid compounds relevant for defense against herbivores and pathogens Two major fractions can be recognized in oleoresin (a) turpentine the volatile fraction containing mono- and sesquiter-penes and (b) rosin the nonvolatile diterpene fraction Oleoresin is a forest commodity of global interest fi nding applications in diverse industry sectors Rosin is used in adhesives printing ink manufacture and paper sizing Turpentine can be used either as a solvent for paints and varnishes or as a raw material for fraction-ation of high-value chemicals used in the pharmaceutical agro-chemical and food industry [ 1 ndash 3 ]
During the extraction activity resin is obtained from the tree in a similar way as rubber tree tapping which generally involves the
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_2 copy Springer Science+Business Media New York 2016
These authors have equally contributed to this work
fettnetocbiotufrgsbr
27
Chapter 3
A Modifi ed Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines
Juacutelio Ceacutesar de Lima Thanise Nogueira Fuumlller Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes
through high-throughput sequencing technologies microarray and also northern blots RT-PCR and
RTqPCR Even though many specifi c protocols designed for plants with high content of secondary metab-
olites have been developed these are often expensive time consuming and not suitable for a wide range
of tissues Here we present a modifi cation of the method previously described using the commercially
available Concerttrade Plant RNA Reagent (Invitrogen) buffer for fi eld-grown adult pine trees with high
oleoresin content
Key words RNA Pines Concert plant RNA reagent Stem RNA extraction Oleoresin Conifers
1 Introduction
Several conifer species especially within the Pinaceae have tissues with high concentrations of phenolics terpenes and polysaccha-rides [ 1 ] Many specifi c protocols that are appropriate for plants rich in secondary metabolite s have been developed but the extrac-tion of high-quality RNA from these tissues using commercial kits is often diffi cult and usually not applicable to woody tissues [ 2 ndash 6 ] One of the major issues during RNA extraction concerns the pres-ence of phenolic compounds which oxidize and form quinones Aromatic compounds bind RNA which interferes in downstream steps and applications [ 3 7 ] Another point of concern is the har-vest of plant samples in the experimental fi eld which constitutes another obstacle in the efforts to avoid degradation of RNA [ 8 ] These problems often result in RNAs of low quality and insuffi -cient amounts especially for methodologies that normally require
These authors have equally contributed to this work
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_3 copy Springer Science+Business Media New York 2016
fettnetocbiotufrgsbr
RESEARCH PAPER
Control of resin production in Araucaria angustifolia an ancientSouth American coniferJ C Perotti1 K C da Silva Rodrigues-Correa123 amp A G Fett-Neto12
1 Plant Physiology Laboratory Department of Botany Federal University of Rio Grande do Sul (UFRGS) Porto Alegre RS Brazil
2 Center for Biotechnology UFRGS Porto Alegre RS Brazil
3 Present address Regional Integrated University of Alto Uruguai and Miss~oes (URI-FW) Frederico Westphalen RS Brazil
Keywords
Araucaria ethylene jasmonic acid nitric
oxide resin salicylic acid terpenes
Correspondence
A G Fett-Neto Plant Physiology Laboratory
Center for Biotechnology Federal University
of Rio Grande do Sul (UFRGS) PO Box 15005
Av Bento Goncalves 9500 91501-970 Porto
Alegre Brazil
E-mail fettnetocbiotufrgsbr
Editor
K Leiss
Received 22 July 2014 Accepted 11
December 2014
doi101111plb12298
ABSTRACT
Araucaria angustifolia is an ancient slow-growing conifer that characterises parts ofthe Southern Atlantic Forest biome currently listed as a critically endangered speciesThe species also produces bark resin although the factors controlling its resinosis arelargely unknown To better understand this defence-related process we examined theresin exudation response of A angustifolia upon treatment with well-known chemicalstimulators used in fast-growing conifers producing both bark and wood resin suchas Pinus elliottii The initial hypothesis was that A angustifolia would display signifi-cant differences in the regulation of resinosis The effect of Ethrel (ET ndash ethylene pre-cursor) salicylic acid (SA) jasmonic acid (JA) sulphuric acid (SuA) and sodiumnitroprusside (SNP ndash nitric oxide donor) on resin yield and composition in youngplants of A angustifolia was examined In at least one of the concentrations testedand frequently in more than one an aqueous glycerol solution applied on fresh woundsites of the stem with one or more of the adjuvants examined promoted an increase inresin yield as well as monoterpene concentration (a-pinene b-pinene camphene andlimonene) Higher yields and longer exudation periods were observed with JA and ETanother feature shared with Pinus resinosis The results suggest that resinosis controlis similar in Araucaria and Pinus In addition A angustifolia resin may be a relevantsource of valuable terpene chemicals whose production may be increased by usingstimulating pastes containing the identified adjuvants
INTRODUCTION
Many conifer species produce terpenoid-based resins that havelong been studied for their industrial importance and role indefence against attack by herbivores and pathogens The twomost important resin-producing families of conifers are Pina-ceae and Araucariaceae (Langenheim 1996) The viscous resinsecretion is generally composed of a complex mixture ofterpenoids consisting of roughly equal parts of volatile mono-(C10) and sesquiterpene (C15 turpentine) fractions and non-volatile diterpenic (C20 rosin) components (Rodrigues-Correaet al 2013) Terpenes act in a complex and multilayereddefence response providing toxicity against bark beetles andfungi bark wound sealing disruption of insect developmentand attraction of herbivore predators (Phillips amp Croteau1999)Most conifers rely on some combination of preformed and
inducible resin defences (Trapp amp Croteau 2001 Zulak amp Bohl-mann 2010) Resin defences are controlled by environmentaland genetic factors to various extents depending on species(Roberds et al 2003 Sampedro et al 2010 Moreira et al2013) Resin traits have been reported as highly variable havingmoderate heritability indicating that breeding efforts towardssuper-resinous forests are promising (Tadasse et al 2001Roberds et al 2003) Several chemicals are known as stimulantsof resin production Commercial extraction of resin from pine
trees uses periodic bark streaking and application of resin stim-ulant pastes to the wound
Resin-stimulant paste based on sulphuric acid (SuA) iswidely used for the commercial production of pine resin Cur-rent stimulant pastes usually have two chemically active com-ponents SuA to magnify the wounding and an ethyleneprecursor (2-chloroethylphosphonic acid CEPA or Ethrel ndash
ET) to stimulate resin flow (Rodrigues et al 2011 Rodrigues-Correa amp Fett-Neto 2013) Jasmonic acid (JA) and its methylester methyl jasmonate (MeJa) are among the most widelyused chemical elicitors of plant secondary metabolism It hasbeen shown that the exogenous application of MeJa or herbi-vore attack induce chemical and anatomical defence responsesin conifers such as the formation of traumatic resin ducts andresin accumulation in stems along with increased biosynthesisof terpenes and phenolics (Franceschi et al 2002 Martin et al2002 Heijari et al 2005 Zeneli et al 2006 Moreira et al 2008Gould et al 2009) JA commercial use however is limited byits high cost
The effects of exogenous salicylic acid (SA) on conifer ter-pene production have also been studied In Pinus elliottiiapplication of 10 molm3 of SA induced resin productionin wound panels but in Pseudotsuga menziesii and Sequoia-dendron giganteum it had no apparent effect on resinaccumulation (Hudgins amp Franceschi 2004 Rodrigues ampFett-Neto 2009) Nitric oxide (NO) has also emerged as an
Plant Biology 17 (2015) 852ndash859 copy 2014 German Botanical Society and The Royal Botanical Society of the Netherlands852
Plant Biology ISSN 1435-8603
4
de serina e acetil-CoA em o-acetilserina pela enzima SAT (serina acetiltransferase) seguida
da conversatildeo de o-acetilserina e aacutecido sulfiacutedrico em cisteiacutena em uma reaccedilatildeo catalisada pela
OAS-TL (o-acetilserina tiol-liase) A siacutentese de mimosina por sua vez eacute compartilhada com
a da cisteiacutena ateacute esse ponto e acredita-se que pelo menos uma das isoformas de OAS-TL
catalise a conversatildeo de o-acetilserina e 3-hidroxi-4-piridona em mimosina
Tabela 1 Atividades descritas para mimosina de Leucaena leucocephala (Lam) de Wit
ATIVIDADE
ALVO AVALIADO
(organismo eou tecido tipo
celular)
REFEREcircNCIA
Bloqueio do complexo de ativaccedilatildeo
da preacute-replicaccedilatildeo do DNA
Ceacutelulas de mamiacuteferos
KUBOTA et al
(2014)
Alteraccedilatildeo no ciclo ovariano e
extensatildeo da duraccedilatildeo do corpo luacuteteo
bovino no periacuteodo poacutes-parto
Bovinos
(Bos taurus x
Bos indicus)
BOTTINI-
LUZARDO et al
(2015)
Supressatildeo do ciclo celular e reduccedilatildeo
da abundacircncia bacteriana em
mosquitos
Wolbachia pipientis
Aedes albopictus
FALLON
(2015)
Accedilatildeo inibitoacuteria da fibrose
pulmonar induzida
Ratos SD
LI et al
(2015)
Recuperaccedilatildeo da funccedilatildeo do
miocaacuterdio poacutes-isquemia
Miocaacuterdio de ratos (SD)
machos
CROWE et al
(2001)
Inseticida
Heteropsylla cubana
Crawford 1914 e Thrips tabaci
Lindemann 1889
AHMED et al
(2016)
Alelopaacutetica
Albizia procera Vigna
unguiculata Cicer arietinum
Cajanus cajan
AHMED et al
(2008)
Antioxidante
Sistemas modelo de oxidaccedilatildeo
lipiacutedica (β-caroteno - aacutecido
linolecircico e lecitina)
BENJAKUL et al
(2013)
Ateacute momento versotildees divergentes sobre a enzima responsaacutevel pela biossiacutentese de
mimosina (mimosina sintase) tecircm sido publicadas Em 1990 Ikegami e colaboradores
5
identificaram uma OAS-TL responsaacutevel pela formaccedilatildeo de cisteiacutena como sendo tambeacutem uma
mimosina sintase Mais tarde Yafuso et al (2014) realizaram a expressatildeo heteroacuteloga do gene
que codifica para OAS-TL em Escherichia coli e natildeo foi observada a formaccedilatildeo de mimosina
mesmo quando dadas as condiccedilotildees oacutetimas para tanto Mais recentemente Harun-Ur-Rashid
et al (2018) elucidaram a mimosina sintase como sendo uma isoforma da OAS-TL
corroborando o postulado por Ikegami e colaboradores em 1990
Figura 1 Rota de biossiacutentese da mimosina Fonte Ikegami et al (1990)
Espeacutecies estudadas
Leucaena leucocephala (Lam) de Wit (leucaena koa haole ou ldquoacaacutecia exoacuteticardquo na
liacutengua Hawairsquoiana) eacute uma espeacutecie de haacutebito arboacutereo ou arbustivo pertencente agrave famiacutelia
Fabaceae de Angiospermas e caracterizada pelo acuacutemulo de mimosina em todos os seus
oacutergatildeos Eacute nativa da Ameacuterica Central (especificamente da regiatildeo sudeste do Meacutexico) mas
irradiou-se atraveacutes de praticamente todas as zonas tropicais e subtropicais da Terra No
Brasil leucena eacute amplamente distribuiacuteda e classificada como naturalizada pelo REFLORA
(2019) ocorrendo em todo territoacuterio Nacional Satildeo reconhecidas no miacutenimo duas
6
subespeacutecies de leucena ocorrentes no Brasil L leucocephala var leucocephala e L
leucocephala var glabrata sendo a primeira a mais abundante
Leucaena apresenta atributos morfoloacutegicos caracteriacutesticos das leguminosas como o
fruto do tipo vagem deiscente no periacuteodo poacutes-maturaccedilatildeo folhas compostas e bipinadas As
flores satildeo seacutesseis actinomorfas e polistecircmones apresentam caacutelice sinseacutepala e corola
gamopeacutetala e satildeo dispostas em inflorescecircncias do tipo glomeacuterulo (Figura 2)
Figura 2 Oacutergatildeos vegetativos e reprodutivos de L leucocephala (Lam) de Wit Fonte Little Jr amp Skolmen
(1989)
Com base no conhecimento etnobotacircnico disponiacutevel acerca dessa espeacutecie em
diversas regiotildees tropicais e subtropicais leucena eacute utilizada para vaacuterios fins Extratos de
diferentes oacutergatildeos de leucena apresentam atividade anti-diabeacutetica (Kuppusamy et al 2014
Chowtivannakul et al 2016) antioxidante (Mohammed et al 2015 Chowtivannakul et al
2016 Zarin et al 2016) antimicrobiana (Zarin et al 2016) anti-helmiacutentica (Soares et al
2015 Jamous et al 2017) bactericida (Mohammed et al 2015) acaricida (Fernaacutendez-Salas
et al 2011) anti-tumoral (Chung et al 2017) e potencializadora da resposta imune em
peixes (Verma et al 2018) entre outras
7
Leucaena apresenta alta toleracircncia agrave seca sendo capaz de enfrentar estaccedilotildees sazonais
inteiras com deacuteficit hiacutedrico sem prejuiacutezo permanente de seus oacutergatildeos e de recuperar
vigorosamente sua biomassa vegetativa tatildeo logo o regime de precipitaccedilatildeo retome a
regularidade em frequecircncia Acredita-se que a toleracircncia agrave seca apresentada por essa espeacutecie
ocorra em funccedilatildeo do acuacutemulo de mimosina nos diferentes tecidos da planta a qual
funcionaria como um agente osmoregulador responsaacutevel pela preservaccedilatildeo da integridade das
membranas a das macromoleacuteculas intracelulares em periacuteodos de escassez de aacutegua no
ambiente
Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) eacute uma leguminosa
nativa natildeo endecircmica do Brasil amplamente distribuiacuteda nos domiacutenios fitogeograacuteficos da
Caatinga do Cerrado e da Mata Atlacircntica (Simon amp Proenccedila 2000 REFLORA 2019) Como
espeacutecie pioneira (Pilatti et al 2019) exerce importante papel ecoloacutegico na recuperaccedilatildeo de
aacutereas degradadas (Bitencourt et al 2007 Silva et al 2011) no estabelecimento de processos
de sucessatildeo vegetacional
Maricaacute eacute uma espeacutecie semi-deciacutedua a deciacutedua a qual atinge ateacute 15 m em altura (e
diacircmetro agrave altura do peito de ateacute 40 cm) na idade adulta com haacutebito arboacutereo ou arbustivo
(REFLORA 2019) e espinhos caracteriacutesticos desde os estaacutegios iniciais de desenvolvimento
(Carvalho 2004) Apresenta folhas compostas alternas e bipinadas (Figura 2) amplas
inflorescecircncias brancas com flores reunidas em glomeacuterulos esfeacutericos dispostos em grandes
paniacuteculas As flores satildeo diplostecircmones actinomorfas hipoacuteginas e unicarpelares (Silva et al
2011)
Assim como descrito para leucena maricaacute eacute considerado uma espeacutecie multifuncional
sendo comumente empregada para produccedilatildeo de mel como combustiacutevel (Olkoski amp
8
Wittmann 2011) em edificaccedilotildees na carpintaria e como lsquocerca-vivarsquo (Marchiori 1993
Lorenzi 1998) entre outras aplicaccedilotildees
Figura 2 Folhas e fruto de Mimosa bimucronata (DC) Kuntze Fonte Souza-Lima et al (2017)
Em contraste com a amplitude de habitats explorados por leucena (especialmente os
aacuteridos) no Sul do Brasil maricaacute ocorre preferencialmente em ambientes uacutemidos a alagadiccedilos
em aacutereas proacuteximas agraves margens de rios (Patreze amp Cordeiro 2004) embora possa tambeacutem
ocorrer em formaccedilotildees quase exclusivas dessa espeacutecie nas encostas de morros (Jacobi amp
Ferreira 1991)
Em relaccedilatildeo agraves atividades elucidadas para os extratos de maricaacute foram relatados os
efeitos alelopaacutetico (Jacobi amp Ferreira 1991 Ferreira et al 1992) diureacutetico natriureacutetico e
caliureacutetico (Schlickmann et al 2017)
9
Hipoacutetese
Mimosina apresenta perfil dinacircmico de acuacutemulo em Leucaena leucocephala e
Mimosa bimucronata frente a estresses associado a alteraccedilotildees significativas na expressatildeo de
genes relacionados ao metabolismo deste ANP o qual contribui para mitigar o desequiliacutebrio
oxidativo inerente a vaacuterios tipos de estresse
Objetivo geral
O objetivo da presente tese foi investigar o papel bioloacutegico da mimosina endoacutegena
em leucena e maricaacute a partir da avaliaccedilatildeo do efeito de tratamentos relacionados a estresses
ou sinalizadores de estresse
Objetivos especiacuteficos
- Analisar a concentraccedilatildeo constitutiva de mimosina nos diferentes oacutergatildeos de L leucocephala
(Lam) de Wit (leucena) e M bimucronata (DC) Kuntze (maricaacute)
- Verificar se apesar do seu alto teor constitutivo em plantas de leucena o acuacutemulo de
mimosina pode ser induzido com tratamentos que mimetizam diferentes estresses a partir da
avaliaccedilatildeo do efeito de moleacuteculas sinalizadoras (aacutecido saliciacutelico jasmonato etileno) e da
exposiccedilatildeo agrave radiaccedilatildeo UV-C na modulaccedilatildeo do acuacutemulo de mimosina em leucena bem como
em maricaacute
- Determinar se a expressatildeo de genes relacionados ao metabolismo de mimosina estaacute
associada agrave induccedilatildeo por estresses fisioloacutegicos
- Avaliar o potencial antioxidante da mimosina em experimentos realizados in situ
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry
journal homepage wwwelseviercomlocateplaphy
Research article
Mimosine accumulation in Leucaena leucocephala in response to stresssignaling molecules and acute UV exposure
Kelly Cristine da Silva Rodrigues-Correcircaab Michael DH Hondab Dulal BorthakurbArthur Germano Fett-Netoalowast
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970Porto Alegre Rio Grande do Sul BrazilbDepartment of Molecular Biosciences and Bioengineering University of Hawaii at Manoa Honolulu HI 96822 USA
A R T I C L E I N F O
KeywordsLeucaena leucocephalaMimosineMimosine amidohydrolaseJasmonic acidEthyleneSalicylic acidUV-C radiation
A B S T R A C T
Mimosine is a non-protein amino acid of Fabaceae such as Leucaena spp and Mimosa spp Several relevantbiological activities have been described for this molecule including cell cycle blocker anticancer antifungalantimicrobial herbivore deterrent and allelopathic activities raising increased economic interest in its pro-duction In addition information on mimosine dynamics in planta remains limited In order to address this topicand propose strategies to increase mimosine production aiming at economic uses the effects of several stress-related elicitors of secondary metabolism and UV acute exposure were examined on mimosine accumulation ingrowth room-cultivated seedlings of Leucaena leucocephala spp glabrata Mimosine concentration was not sig-nificantly affected by 10 ppm salicylic acid (SA) treatment but increased in roots and shoots of seedlings treatedwith 84 ppm jasmonic acid (JA) and 10 ppm Ethephon (an ethylene-releasing compound) and in shoots treatedwith UV-C radiation Quantification of mimosine amidohydrolase (mimosinase) gene expression showed thatethephon yielded variable effect over time whereas JA and UV-C did not show significant impact Consideringthe strong induction of mimosine accumulation by acute UV-C exposure additional in situ ROS localization aswell as in vitro antioxidant assays were performed suggesting that akin to several secondary metabolitesmimosine may be involved in general oxidative stress modulation acting as a hydrogen peroxide and superoxideanion quencher
1 Introduction
Different plant groups synthesize a large diversity of secondary orspecialized metabolites These molecules are generally produced inresponse to biotic and abiotic environmental stresses Indeed inductionof secondary metabolism usually involves stress-generating factorswhich have also been explored in biotechnological processes aiming atthe production of target metabolites of economic interest (Matsuuraet al 2018) Metabolic control of nitrogen-containing secondarycompounds (eg alkaloids and non-protein amino acids) has beenshown to be complex and influenced by phytohormones environmentalstresses (seasonality herbivory pathogen attack drought) UV radia-tion (Holloacutesy 2002) methyl jasmonate (MeJA) salicylic acid (SA)yeast extract (Cho et al 2008) abscisic acid (ABA) heavy metals os-motic stress (Nascimento et al 2013) and mechanical wounding (Portoet al 2014)
Due to their particular trait of associating with N-fixing micro-organisms Fabaceae species (leguminous sensu lato) are often proteinrich hence the relevance of several of these species as forage Fabaceaespecies are also known for accumulating nitrogen containing secondarymetabolites which play important roles as ecochemical molecules andat least for the case of non-protein amino acids potential cell reservoirsof nitrogen (Huang et al 2011)
High contents of mimosine a toxic aromatic non-protein aminoacid are found in species of two leguminous genera Leucaena spp andMimosa spp Leucaena leucocephala (Lam) de Wit (leucaena koa haole)is a fast-growing leguminous tree native from Central America (south-eastern Mexico) widely distributed in tropical and subtropical zonesThis species is also characterized by its high tolerance to droughtamong other environmental stresses (Honda et al 2018) Leucaena canbe divided into two subspecies (i) L leucocephala subsp leucocephala(common leucaena a bushy shrub) and (ii) L leucocephala subsp
httpsdoiorg101016jplaphy201811018Received 1 August 2018 Received in revised form 9 November 2018 Accepted 14 November 2018
lowast Corresponding authorE-mail addresses krodriguescbiotufrgsbr (KCdS Rodrigues-Correcirca) mhonda2hawaiiedu (MDH Honda) dulalhawaiiedu (D Borthakur)
fettnetocbiotufrgsbr (AG Fett-Neto)
Plant Physiology and Biochemistry 135 (2019) 432ndash440
Available online 19 November 20180981-9428 copy 2018 Elsevier Masson SAS All rights reserved
T
glabrata (giant leucaena a tree) The latter has been used as a fastgrowing tree for production of wood and paper pulp The foliage ofboth common and giant leucaena is used as a fodder because of its highprotein content and palatability to farm animals The foliage containsup to 18 protein 142 crude fiber and 64 ether extractcrude fat(Soedarjo and Borthakur 1996)
Production of nitrogen-containing secondary metabolites such asmimosine requires large amounts of carbon and nitrogen resourcesNegi et al (2014) estimated that up to 21 of the carbon-nitrogenresources may be used for production of mimosine in leucaenaBrewbaker et al (1972) determined the mimosine content of 96 Lleucocephala cultivars and 8 other Leucaena species collected from 38different countries by growing them in an observational nursery inHawaii and found that basal mimosine content varied from 189 to477 of the dry weight
Mimosine is biosynthesized from OAS (o-acetylserine) and 3H4P (3-hydroxy-4-pyridone or its tautoisomer 3-hydroxy-4-pyridine) A pre-vious analysis suggested that mimosine synthase is an OAS-TL (o-acetylserine-thiol-lyase) of the cysteine biosynthesis pathway (Ikegamiet al 1990) Later however recombinant enzyme tests did not supportan OAS-TL identity of mimosine synthase (Yafuso et al 2014) Recentfindings on mimosine biosynthesis revealed that a cytosolic cysteine-OAS-TL isoform can also catalyze the formation of mimosine underspecific conditions (Harun-Ur-Rashid et al 2018)
Mimosine toxicity is related to its ability of reducing the availabilityof divalent metal ions such as Fe(II) Zn(II) Cu(II) Co(II) and Mn(II)by chelating co-factors and preventing their association with metal-dependent enzymes Furthermore this non-protein amino acid is cap-able of forming a stable complex with pyridoxal-5prime-phosphate (PLP)leading to the inactivation of PLP-dependent enzymes (eg Asp-Glutransaminase and cystathionine synthetase) (Negi et al 2014)
Mimosine features several useful biological activities such as alle-lopathic antimicrobial insecticide cell cycle inhibitor agent antic-ancer phytoremediator (Nguyen and Tawata 2016) as well as anti-oxidant (Benjakul et al 2013) Despite the relatively well establishedbiological activities of purified mimosine on other organisms or celltypes little is known about its biological role in leguminous speciesHowever it has been suggested that at least in part its activity ismainly related to defense mechanisms against some biotic and abioticstresses and as nitrogen source during fast growth (Vestena et al2001)
Suda (1960) and Smith and Fowden (1966) identified enzymes in-volved in mimosine degradation in seedling extracts of L leucocephalaand Mimosa pudica A mimosine-degrading enzyme named mimosinase(mimosine amidohydrolase EC 35161 CAS registry number 104118-49-2) (IUBMB 2018) a carbon-nitrogen lyase which degrades mimo-sine into 3H4P was later purified by Tangendjaja et al (1986) Itsbiochemical characterization was described and the cDNA was isolatedby Negi et al (2014)
Although mimosinase has been described and isolated only fewstudies on the role played by biotic and abiotic factors on the dynamicmodulation of mimosine metabolism in leguminous species have beenconducted (Vestena et al 2001 Xu et al 2018) In aseptic cultures ofleucaena mechanical injury of shoots promoted local mimosine accu-mulation (Vestena et al 2001) In the same study cultivation in pre-sence of auxin or SA in culture medium also had a positive effect on
mimosine accumulation More recently the effect of drought treatmenton gene expression of leucaena was also evaluated by Honda et al(2018) However several potential factors regulating mimosine meta-bolism need to be further examined
To date there is a lack of information on the biological role ofmimosine in planta as well as on details of its metabolic dynamicsMoreover its overt potential for pharmaceutical applications and de-velopment of new drugs as well as the possible use as tool to addressheavy metal soil contamination or plant mineral nutrition improve-ment justify additional research The objective of this study was toinvestigate the effect of stress signaling molecules and acute UV ex-posure on modulation of mimosine accumulation and metabolism in Lleucocephala spp glabrata in order to better understand its biologicalrole and to identify strategies for yield improvement aiming at ex-ploring its useful bioactivities
2 Methods
21 Plant material
For the experiments carried out to evaluate the effects of elicitors onmimosine accumulation seeds of leucaena were kindly provided by DrJames Brewbaker and harvested at CTAHRs (College of TropicalAgriculture and Human Resources of the University of Hawaii atManoa) Waimanalo Research Station at Oahu Hawaii This plantmaterial was originated from the accession K636 of Leucaena leucoce-phala ssp glabrata (Brewbaker 2008)
22 Induced mimosine content in 5-week-old giant leucaena
221 Seed germinationIn order to overcome seed coat dormancy seeds were submitted to a
chemical scarification with sulfuric acid 95ndash98 for 20min and re-peatedly rinsed in distilled water to remove any residual trace of thisreagent Then seeds were distributed in 254 cmtimes508 cm plastictrays containing 11 vv of vermiculite and commercial soil watereduntil reaching substrate field capacity Three weeks after seed imbibi-tion seedlings displaying similar size and shape (eg number of com-pound leaves and leaflets) were transplanted to individual pots(250mL) in number of three plants per container
During the experimental period (except in the UV-C radiationtreatment) all tested seedlings were kept in a growth chamber andsubmitted to controlled conditions of temperature (circa 25 degC) and ir-radiance (approximately 100 μmol photons mminus2sdot s minus1) with a photo-period of 16 h light and 8 h dark
222 Treatments2221 JA Ethephon and SA Five-week-old giant leucaena seedlingswere treated with different solutions as described in Table 1 Idealconcentrations were defined in preliminary experiments under the sameconditions indicated above At the beginning of the experiments 30plants were sprayed with 84 ppm JA 10 ppm SA 10 or 100 ppmEthephon or Milli-Qreg water (control) until the point of imminent runoffPlant pots were kept closed inside transparent plastic bags for 24 h toavoid solution volatilization Fifteen plants arranged in 5 sets of 3 (5biological replicates) were harvested 48 h and 96 h after being treated
Table 1Treatments used to modulate mimosine biosynthesis in giant leucaena
ELICITOR CONCENTRATION UV FLUENCE EXPOSURE TIME RATIONALE FOR USE
Salicylic acid (SA) 10 ppm 24 h Pathogen signaling molecule (Shah 2003)Jasmonic acid (JA) 84 ppm 24 h Chemical elicitor of plant secondary metabolism (Dar et al 2015)Ethephon 10 ppm 24 h Ethylene releasing-compound (Kim et al 2016) elicitor of plant secondary metabolism (Wang
et al 2016)UV-C radiation 3 Jcmminus2 10min or 15min Elicitor of plant secondary metabolism (Kara 2013 Neelamegam and Sutha 2015)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
433
After collection shoots were separated from roots immediately frozenin liquid nitrogen and stored at ndash 80 degC prior to HPLC analyses
2222 UV-C Thirty seedlings of giant leucaena were exposed to UV-Cradiation (3 Jcmminus2) for 10 or 15min and kept in a growth chamberunder controlled conditions as described above until the end of theexperiments Fifteen plants arranged in groups of 3 were harvested at96 h and 120 h after UV-C exposure and processed as previouslydescribed
223 Mimosine extractionMimosine extraction was based on a modified version of the pro-
tocol published by Lalitha and Kulothungan (2006) as follows a knownweight of fresh tissue (shoots or roots) of giant leucaena was first addedto Milli-Qreg boiling water in a proportion of 110 (g of plant per mL ofsolvent) in test tubes Tubes were covered with foil to avoid solutionevaporation and placed on a hot stirrer at 100 degC for 10min A pro-portional volume of 01M HCl was added to the cooled suspensions andhomogenized using mortar and pestle The plant extracts were filteredthrough cotton and centrifuged twice for 7min in a bench top re-frigerated centrifuge at 4 degC and 13200 rpm Before being analyzed theextracts were diluted 13 with ondashphosphoric acid (OPA)
224 Mimosine detectionHPLC analyses were carried out as described by Negi and Borthakur
(2016) Pure mimosine (L-mimosine from koa haole seeds Sigma-Al-drich CAS number 500-44-7) was used as standard Separation andquantification of mimosine was done with a C18 column (PhenomenexC18 5 μm 46times250mm) under an isocratic solvent system of 002MOPA with a linear flow rate of 1mLsdotminminus1 Mimosine detection wasdone at 280 nm by photodiode array detection (200ndash400 nm) showingretention time of 277 plusmn 0042min Quantification was done using themethod of external standard curve Further confirmation of mimosineidentity was performed by co-chromatography with standard and peakpurity check Chromatograms were analyzed using the Waters Em-power 3 software
23 Quantitative real-time PCR analysis of mimosinase gene expression
Fifteen 8-week-old giant leucaena plants arranged in 4 sets of 3 (4biological replicates) were treated with either water (control) or10 ppm Ethephon 84 ppm JA acid or 15min of UV-C radiation ex-posure following the methods described above Following treatmentleucaena plants were harvested at 48 and 96 h or 72 and 144 h (UV-Ctreated plants only) after treatments Total RNA of samples was ex-tracted and purified from roots and shoots of giant leucaena by meansof a modified method using Qiagen RNeasy Plant Kit (Valencia CAUSA) and Fruit-mate (Takara Japan) according to the protocol de-scribed by Ishihara et al (2016a) The assessment of RNA quality andquantity was carried out at 230 260 and 280 nm by using a NanoDropSpectrophotometer ND-1000 (NanoDrop Technologies DE USA) Inorder to avoid genomic DNA contamination RNA samples were treatedwith TURBO DNAfree Kit (Invitrogen Carlsbad CA) Two microgramsof DNase-treated RNA were used to synthesize the first-strand cDNAusing M-MLV Reverse Transcriptase (Promega WI USA)
Quantitative real-time (qPCR) analysis was carried out to examinepossible differential expression of the mimosinase gene (GenBank ac-cession number AB2985971) in seedlings treated with 84 ppm JA10mM Ethephon or 15min of UV-C exposure Shoots and roots wereharvested 24 h before the time of mimosine concentration peak for eachtreatment previously observed as assessed by HPLC assays The 10 μLqPCR reaction consisted of 5 μL of PowerUpTM SYBRreg Green MasterMix (Applied Biosystems Foster City CA) 1 μL MgCl2 (50mM) 03 μLforward primer (10 μM) 03 μL reverse primer (10 μM) and 1 μL cDNAfirst-strand In the experimental validation through qPCR reactionconditions and melting curve analysis of the amplicon were performed
following the protocol published by Ishihara et al (2016b) for the sameleucaena variety qPCR analysis was conducted using StepOnetrade Real-Time PCR System (Applied Biosystems) Measurements were performedusing 4 biological and 3 technical replicates Relative expression wascalculated with the 2-ΔΔct method using OAS-TL as reference gene sinceits expression showed a consistently stable profile comparable to that ofUBQ-5 and ELF1α expressions Mimosinase primer sequences used forthese analyses were (FWD) 5prime- GAA AGG CAG GAA TCA CAG TGA AGAG ndash 3rsquo (REV) 5prime GGA GAC TCT AGC CAC ACC AAC TTA ndash 3rsquo
24 Antioxidant assays
241 Mimosine effect on hydrogen peroxide (H2O2) accumulationAs a follow up to the induction of mimosine accumulation profiles
under stress signals and conditions tests were conducted to verify mi-mosine antioxidant capacity In situ histological localization of hy-drogen peroxide (H2O2) accumulation was evaluated on foliar disks ofPhaseolus vulgaris L according to the protocol described by Shi et al(2010) Briefly the plant foliar tissue was exposed to 1 mgmiddotmLminus1 dia-minobenzidine (DAB) solution in 10 mM KH2PO4 (control) in presenceor absence of 10mM mimosine (equivalent to the average mimosineconcentration induced by UV-C radiation in giant leucaena) or 10mMascorbic acid (positive antioxidant control) Oxidative response wasidentified by the formation of a brown polymer on the injured leafareas indicating the presence of H2O2 and registered in a Leica M165FC stereomicroscope (Leica Microsystems)
242 Mimosine quenching of superoxide radicalsGeneration of superoxide radical and subsequent analysis was per-
formed by a modified protocol based on Zhishen et al (1999) Nitroblue tetrazolium (NBT) reduction was used to measure superoxide an-ions quenching activity Shortly a 50mM KH2PO4 pH 78 solutioncontaining 6 μM riboflavin 100mM methionine 1 mM NBT in pre-sence or absence of 5mM mimosine was exposed to white light(22 Jsdotcmminus2) for 25min on a white light transilluminator Five micro-molar rutin was used as positive control (Matsuura et al 2016) Theabsorbance was read at 560 nm before and after light exposure in aSpectraMaxreg M2 Microplate Reader (Molecular Devices LLC)
25 Statistical analyses
For HPLC and superoxide anions data simple analyses of variance(ANOVA) followed by Tukey or Welch ANOVA followed by Dunnetts Ctest were used as appropriate for data distribution characteristics InqPCR analysis results were analyzed by t-test In all cases at least fourbiological triplicates were used and experiments were repeated twiceindependently All data were analyzed using the statistical packageSPSS 200 for Windows (SPSS Inc USA) In all cases a ple 005 wasused
3 Results and discussion
31 Increased mimosine concentrations in giant leucaena treated withchemical elicitors
Leucaena produces high amounts of mimosine that accumulate in allparts of the plants including leaves stem flowers pods seeds rootsand root nodules (Soedarjo and Borthakur 1998) The highest con-centrations of mimosine can be found in the growing shoot tips andseeds (Wong and Devendra 1983) It is not known why leucaena pro-duces such high amounts of mimosine Negi et al (2014) estimated thatleucaena plants would be able to grow 21 larger if the nutrient re-sources spent on mimosine production were diverted for biomass in-crease In a previous analysis performed to quantify the basal con-centration of mimosine present in adult plants of common leucaena thehighest constitutive amount of mimosine per gram of fresh weight in
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
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the analyzed organs was found in post-anthesis flowers (89448 μg)followed by green pods (82687 μg) leaves (67358 μg) and greenflower buds (51247 μg) which showed significantly less mimosineconcentration compared to the other reproductive structures(Supplementary Fig 1) Since mature seeds have very low moisturecontent (Wencomo et al 2017) its mimosine concentration was esti-mated as 338253 μgsdotgminus1 of dry weight Additionally it was also ob-served that the basal mimosine distribution in shoots of field-grownadult plants of leucaena is dependent on the variety type(Supplementary Table 1)
Phytohormones such as salicylic acid and jasmonic acid are knownto be produced by plants in response to various abiotic and bioticstresses These phytohormones trigger adaptive responses to stress byregulating major plant metabolic processes such as photosynthesisnitrogen metabolism defense systems and plant-water relationsthereby providing protection (for review see Khan et al 2015)
Secondary or specialized metabolite production and accumulationare also known to be controlled by biotic and abiotic stresses (Matsuuraet al 2018) In this study exposure of 5-week-old giant leucaenaseedlings to JA or Ethephon treatments significantly enhanced mimo-sine accumulation in shoots and roots in at least one of the two timepoints tested (48 and 96 h) albeit in a different way (Fig 1) Thehighest concentrations of mimosine in shoots were found in seedlingstreated with JA 84 ppm (43441 μgsdotgminus1) and Ethephon 100 ppm(38412 μgsdotgminus1) two days after application of the respective phyto-hormones Nevertheless after four days shoots yielded the highestconcentration of mimosine (approximately 460 μgsdotgminus1) upon treatmentwith 10 or 100 ppm Ethephon (Fig 1A) In roots after two and four
days JA 84 ppm and Ethephon 10 ppm resulted in highest mimosineaccumulation 18488 μgsdotgminus1 and 15801 μgsdotgminus1 respectively (Fig 1B)These observations show that mimosine accumulation response tospecific elicitors may vary over time after exposure
Although all treatments were applied exclusively on shoots of giantleucaena seedlings roots of some of them were also able to respond tothe different elicitors Overall shoots displayed higher basal and in-duced mimosine concentration compared to roots (Fig 1) which agreeswith previous observations in 1 to 3-week-old aseptic seedlings ofcommon leucaena (Vestena et al 2001) However as previouslymentioned significant post-induction increase of mimosine concentra-tion in roots and shoots simultaneously was only observed for JA andEthephon 10 ppm on day 02 and 04 respectively (Fig 1)
It is well established that perceived regulatory signals or elicitorsgenerate a transduction network mediated by secondary messengersresulting in changes in gene expression profiles that afford adaptiveresponses to environmental stimuli These modulation events are oftenmediated by transcription factors (TFs) which directly bind to specificgene promoters or act by forming complexes with repressor proteinslabeling them to degradation subsequently releasing other TFs toproceed with the gene expression program This is the case of the actionmechanism of JA and its active form jasmonoyl isoleucine for example(Kazan 2015 Wasternack and Strnad 2016)
JA ethylene and SA are known as important stress regulatory sig-nals in plants JA however is thought to be the most effective signal forinduction of plant secondary metabolism (Wasternack and Strnad2016) thereby contributing to mitigation of damage caused by severalstresses (Dar et al 2015) JA is mainly derived from linolenic acid
Fig 1 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings treated with differentelicitors CTRL=Milli-Q water SA = Salicylic AcidJA= Jasmonic Acid ETH=Ethephon Bars sharing a letterof same case do not differ by Tukey test (P le 005) Capitalletters (A B) compare treatments on day two and lowercaseletters (a b) compare treatments on day four Indicatessignificant statistical difference between day two and dayfour in the same treatment by t-test (Ple 005) The errorbars represent standard error of five replicates (each meanwas calculated with 15 individual seedlings organized in 5groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
435
(Wasternack and Strnad 2016) playing important roles in differentprocesses of plant growth and development such as plant defensemechanisms against herbivory pathogen attack fungal elicitation andsome abiotic factors such as osmotic temperature and salt stresses (Daret al 2015)
JA and its methyl ester MeJA have several different effects on le-guminous species MeJA exogenous application has increased iso-flavonoid content in cell suspension cultures of Pueraria candollei varcandollei and P candollei var mirifica (Korsangruang et al 2010) aswell as the production of the triterpenoid glycyrrhizin in Glycyrrhizaglabra roots Enhanced production of the triterpenoid however waspartly at the expense of root growth (Shabani et al 2009) MeJA ap-plication on shoots was observed to suppress root nodulation and lat-eral root formation in Lotus japonicus (Nakagawa and Kawaguchi2006) In grapevine a non-leguminous species proteinogenic aminoacids did not show an expressive increase under MeJA treatment(Gutieacuterrez-Gamboa et al 2017)
The effects of the application of four different jasmonate forms (JAMeJA jasmonoyl-L-isoleucine (JA-Ile) and 6-ethyl indanoyl glycineconjugate (2-[(6-ethyl-1-oxo-indane-4-carbonyl)-amino]-acetic acidmethyl ester - CGM) on leucaena metabolite profile has recently beenreported by Xu et al (2018) JA-Ile form was most effective althoughno major alteration was observed on monitored metabolite abundancesAlanine threonine and 34-dihydroxypyridine (34 DHP a metabolitederived from mimosine degradation) (Nguyen and Tawata 2016)among others were the major metabolites elicited by JA-Ile In contrastto the results described here mimosine concentration did not changesignificantly These divergent results on mimosine accumulation maybe due to a number of factors including mode of application jasmonateform used (JA-Ile x JA) and L leucocephala subspecies (common x giantleucaena)
Ethylene is also a phytohormone involved in plant response me-chanisms to different types of challenges such as mechanical damageand insect attack among others The integration mechanism betweenJA and ethylene signaling pathways is not completely understoodhowever it has been shown that they may work cooperatively in abioticstress tolerance (Kazan 2015) MeJA can induce ethylene production(Zhao et al 2004) and when applied simultaneously these moleculesseem to work in a synergic way by enhancing the magnitude of theplant response to external stimuli (Liu et al 2016)
Treatment with SA was able to significantly increase mimosine ac-cumulation in 12-week-old plants of common leucaena (SupplementaryFig 2) However no significant effect of SA treatment on mimosineconcentration was seen in 5-week-old seedlings of giant leucaena(Fig 1) suggesting some degree of genotype andor age dependency inelicitation by this phytohormone On the other hand several treat-ments including 90 ppm MeJA 10 and 100 ppm 2-chloroethylpho-sphonic acid (CEPA an ethylene-releasing compound) significantlyincreased mimosine accumulation (Supplementary Fig 2) in agree-ment with the data obtained for giant leucaena The lack of systemiceffects of externally applied SA on mimosine accumulation was alsoobserved when the phytohormone was supplied in the culture mediumof aseptically-grown seedlings in which case only roots had highercontent of mimosine (Vestena et al 2001) This could be due totransport limitations or to low methyl salicylate production from ap-plied SA since the former is recognized as the main systemic signalingform (Vlot et al 2009)
32 Increased mimosine concentrations in giant leucaena exposed to UV-Cradiation
UV-C treatment promoted increased concentration of the aminoacid in shoots but not in roots of giant leucaena (Fig 2) Increasedaccumulation of mimosine in shoots was also observed in 12-week-oldseedlings of common leucaena exposed to UV-C radiation for 10 and15min (Supplementary Fig 3) Similar to the SA treatment in giant
leucaena UV-C radiation did not induce mimosine biosynthesis in rootsregardless of time after exposure The absence of mimosine induction inroots by SA and UV indicates that these effectors do not cause a sys-temic response Moreover roots are shielded from irradiance by thepresence of substrate
UV radiation effects on different aspects of plant metabolism anddevelopment have been described However compared to UV-B (en-vironmentally relevant type of UV radiation) assays there are less re-ports related to the UV-C effects on secondary metabolites biosynthesisand accumulation (Cetin 2014) especially in leguminous (Fabaceae)plants They generally concern primary metabolism aspects such asgrowth and development For instance seedlings of Phaseolus vulgaris L(Fabaceae) exposed to low intensity UV-C radiation have displayeddecreased chlorophyll content and reduced height after 14 days of ex-posure (Kara 2013) Negative effects on growth parameters and ni-trogen metabolism were also observed in Vigna radiata L (Fabaceae)after UV-B radiation treatment in addition to adverse effects on JA SAand antioxidant compounds accumulation (Choudhary and Agrawal2014a) The same authors reported increased accumulation of flavo-noids SA and JA besides negative effects on growth biomass yieldnitrogen fixation and accumulation in 2 cultivars of Pisum sativum L(Fabaceae) under elevated UV-B treatment (Choudhary and Agrawal2014b) Despite the negative UV influence on growth reported for thepreviously mentioned leguminous UV-C radiation on groundnut plants(Arachis hypogaea L Fabaceae) increased seedling vigor and biomassand had no adverse effect on germination or other development para-meters (Neelamegam and Sutha 2015)
Besides its impact on growth and primary metabolism UV exposurecan cause important changes in secondary metabolism depending onintensity and time of exposure (Matsuura et al 2013) UV-B and UV-Cpre-treatments of Artemisia annua (Asteraceae) seedlings yielded in-creased biosynthesis of artemisinin a drug which displays anti-malarialproperties and activity against some others infectious diseases (egschistosomiasis leishmaniasis and hepatitis B) and several kinds oftumors (Rai et al 2011) The accumulation of nicotine in Nicotianarustica plants (Solanaceae) was also increased by UV-C treatment(Tiburcio et al 1985) Similar inducing effects on production of severalsecondary metabolites were observed in callus cultures of Vitis viniferaL Oumlkuumlzgoumlzuuml (grapevine Vitaceae) treated with a UV-C source for 5 or10min (Cetin 2014)
Regarding amino acid biosynthesis in response to UV radiationMartiacutenez-Luumlscher et al (2014) have found that in spite of not causingchanges in total amino acid content UV-B radiation exposure can affecttheir profile in grape berries Proteinogenic amino acids have beenknown to be important targets of the deleterious effects of UV radiation(Holloacutesy 2002) On the other hand in the present study acute UV-Ctreatment was found to increase mimosine accumulation in shoots byover twofold (Fig 2) which may suggest a possible participation of thismolecule as part of the antioxidant defense system in L leucocephalaThis possibility is further supported by the induction of the amino acidaccumulation by JA and Ethephon involved in abiotic and biotic stressresponses which are generally associated with oxidative imbalance andare signaling components in high UV stress (Matsuura et al 2013)
33 Mimosinase gene expression
In order to determine if increases in mimosine content upon ex-posure to JA CEPA or UV-C radiation were related to changes intranscription of mimosine metabolism-related genes RT-qPCR analysiswas carried out The complete pathway for mimosine biosynthesis hasnot yet been determined although the final step has been character-ized Based on transcription analysis (Ishihara et al 2016a) leucaenaappears to encode for multiple cysteine synthases one or more of whichmay be able to catalyze mimosine synthesis In addition a leucaenagene encoding a mimosinase (an enzyme responsible for mimosinedegradation) has been identified and characterized (Negi et al 2014)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
436
In addition to mimosinase gene expression several gene isoformsbelonging to the cysteine pathway [cysteine synthase (CYS SYN) serineacetyltransferase (SAT) and β-cyanoalanine synthase (CAS) Table 2 -supplementary material] were also tested in this study (data notshown) However expressions of these genes did not vary in giantleucaena throughout the experiments suggesting that the increasedcontent of mimosine observed in the treated plants might not be relatedto the expression of these genes but presumably to increased enzymeactivity andor release from conjugates such as mimoside a mimosineβ-D-glucoside (Murakoshi et al 1972)
Considering the time variation of mimosine accumulation observedin this work mimosinase gene expression in shoots and roots wasevaluated 24 h before the increase of mimosine concentration in giantleucaena seedlings (ie 24 h and 72 h after the chemical elicitorstreatments and 48 h and 120 h after UV-C exposure)
Ethylene signaling has been shown to up-regulate expression ofseveral genes related to secondary metabolism pathways as is the caseof phenolic compounds (Liu et al 2016) and terpenoid indole alkaloids(Wang et al 2016) Among all elicitors tested in the present workEthephon was the only one able to significantly change mimosinasegene expression Leucaena plants treated with Ethephon showed sig-nificant increases in mimosine concentration at both day 2 and 4 fol-lowing treatment which coincided with low-level expression of mi-mosinase Up-regulation of mimosinase gene expression was detected24 h before the increase of mimosine concentration in shoots treatedwith 10 ppm of Ethephon (Fig 3A) but not after JA or UV-C treatments(Fig 3C-D and 3E-F respectively) Nevertheless 72 h after treatment
application (24 h before the highest mimosine content measured inshoots) down regulation of mimosinase gene was seen in both shootsand roots treated with 10 ppm of Ethephon (Fig 3B) These data in-dicate that mimosine content in leucaena plants is at least partlyregulated by mimosinase expression in Ethephon exposed plants Onthe other hand the fact that mimosinase mRNA was not significantlyaffected by JA and UV-C treatments despite their stimulating effects onmimosine biosynthesis in giant leucaena may indicate that other levelsof regulation are at play or that the chosen harvesting time window wasunable to detect relevant changes
34 In situ and in vitro antioxidant assays
Considering the stimulation of mimosine accumulation byEthephon JA and UV all of which are often associated or known tocause oxidative imbalance the antioxidant capacity of mimosine wasevaluated Mimosine has been shown to have antioxidant activities oncultured cancer cells (Parmar et al 2015) In the present study it washypothesized that mimosine could confer radical scavenging propertieswhich would contribute to plant protection from possible damagecaused by reactive oxygen species generated during stress(Supplementary Fig 4)
Foliar disks of P vulgaris L were treated with 10mM mimosine for15min Treated disks showed less hydrogen peroxide accumulationinduced by wounding in contrast to untreated ones being comparableto those treated with ascorbic acid (a known hydrogen peroxide neu-tralizer) (Fig 4A) These observations support a possible antioxidant
Fig 2 Mimosine concentration in shoots (A) and roots (B) of5-week-old giant leucaena seedlings exposed to UV-C lightCTRL= visible light (100 μmol photons mminus2 s minus1) UV-C 10primeand UV-C 15rsquo=UV-C exposure time (10 and 15min re-spectively) Bars sharing a letter of same case do not differ byTukey test (P le 005) Capital letters (A B) compare treat-ments on day three and lowercase letters (a b) comparetreatments on day six Indicates significant statistical dif-ference between day three and day six in the same treatmentby t-test (Ple 005) The error bars represent standard errorof five replicates (each mean was calculated with 15 in-dividual seedlings organized in 5 groups of three)
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
437
role of mimosine as an in situ hydrogen peroxide scavengerMimosine was also able to quench superoxide anions generated by
light exposure Mimosine exhibited equivalent antioxidant effect com-pared to rutin (Fig 4B) a well-established effective superoxide anionquencher (Matsuura et al 2016) The radical scavenging activity ofmimosine may be due to the 3-OH group of the pyridine ring of mi-mosine (Fig 5) The pKa of the 3-OH of mimosine has been estimated tobe 88 (M Honda unpublished results) At physiological pH this OHgroup is expected to remain in a protonated state and therefore mayscavenge a radical by donating a proton and an electron In this processmimosine itself is converted to a stable radical form which is perhapsless toxic and less reactive than the reactive oxygen species generatedduring oxidative stress It is likely that the less toxic radical mimosineproduced may react with another radical or molecule and becomeconverted to a non-reactive indole molecule
In vivo antioxidant activity of mimosine has been previously eval-uated by means of its exogenous application on selenium-deficientseedlings of Vigna radiata In spite of its allelopathic properties (Ahmedet al 2008) the results showed mitigation of mitochondrial oxidativestress by treatment with 01mM mimosine (Lalitha and Kulothungan2007) DPPH radical scavenging activity was also reported for aqueous
seed extracts of leucaena rich in mimosine and phenolic compounds inin vitro assays (Benjakul et al 2014) Mimosine antioxidant activityshown in the present work is in good agreement with data reported forother non-protein amino acids such as L-DOPA (Dhanani et al 2015)and GABA (Malekzadeh et al 2014) for instance
4 Conclusion
Taken together results show that mimosine biosynthesis and ac-cumulation can be modulated by stress-related factors despite its re-latively high constitutive content in leucaena plants The pattern ofgene expression in stressed plants suggests mimosine steady-state con-trol may be regulated by its degradation in possible connection withdynamic changes in carbon and nitrogen metabolism of stressed plantsMimosine quenching activity against hydrogen peroxide and super-oxide anions in the in situ staining and in vitro assays respectivelyshowed that this non-protein amino acid can act as non-enzymaticantioxidant agent Increase in mimosine content in response to elicitorsmimicking environmental challenges in addition to its antiherbivoreand antimicrobial properties may be related to its activity as protectivemolecule against oxidative damage in line with other classes of plant
Fig 3 Relative expression of the mimosinase gene in shoots (A E and F) and shoots and roots (B C and D) of giant leucaena 24 h (A and C) 48 h (E) 72 h (B and D)and 120 h (F) after treatment with stress signaling molecules or UV-C exposure ETH = Ethephon JA = Jasmonic Acid Indicates significant statistical differencebetween control and treatment by t-test (Ple 005) The error bars represent standard error of four replicates
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
438
secondary metabolites
Funding
This work was funded by the National Council for Scientific andTechnological Development (CNPq-Brazil) grant 3060792013-5Coordenaccedilatildeo de Aperfeiccediloamento de Pessoal de Niacutevel Superior - Brazil(CAPES) - Finance Code 001 and the USDA NIFA Hatch projectHA05029-H managed by CTAHR
CRediT authorship contribution statement
Kelly Cristine da Silva Rodrigues-Correcirca InvestigationValidation Writing ndash original draft Michael DH HondaInvestigation Validation Dulal Borthakur Supervision Writing ndashreview amp editing Funding acquisition Arthur Germano Fett-NetoSupervision Funding acquisition Writing ndash review amp editing
Acknowledgements
The authors would like to thank Dr Jorge Ernesto Mariath fromLaVeg-UFRGS for kindly lending the Leica M165 FC stereomicroscopefor in situ analysis
Appendix A Supplementary data
Supplementary data to this article can be found online at httpsdoiorg101016jplaphy201811018
References
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Benjakul S Kittiphattanabawon P Shahidi F Maqsood S 2013 Antioxidant activityand inhibitory effects of lead (Leucaena leucocephala) seed extracts against lipidoxidation in model systems Food Sci Technol Int 19 (4) 365ndash376 httpsdoiorg1011771082013212455186
Benjakul S Kittiphattanabawon P Sumpavapol P Maqsood S 2014 Antioxidantactivities of lead (Leucaena leucocephala) seed as affected by extraction solvent priordechlorophyllisation and drying methods extracts against lipid oxidation in modelsystems Food Sci Technol 51 (11) 3026ndash3037 httpsdoiorg101007s13197-012-0846-1
Brewbaker JL Pluckett D Gonzalez V 1972 Varietal variation and yield trials ofLeucaena leucocephala (koa haole) in Hawaii Hawaii Agric Exp Stn Bull 166 26
Brewbaker JL 2008 Registration of KX2 ndash Hawaii interspecific-hybrid leucaena JPlant Registrations 1 (3) 190ndash193 httpsdoiorg103198jpr2007050298crc
Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitisvinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-6287-47-37
Cho H-Y Son SY Rhee HS Yoon S-YH Lee-Parsons CWT Park JM 2008Synergistic effects of sequential treatment with methyl jasmonate salicylic acid andyeast extract on benzophenanthridine alkaloid accumulation and protein expressionin Eschscholtzia californica suspension cultures J Biotechnol 135 117ndash122 httpsdoiorg101016jjbiotec200802020
Choudhary KK Agrawal SB 2014a Cultivar specificity of tropical mung bean (Vignaradiata L) to elevated ultraviolet-B changes in antioxidative defense system ni-trogen metabolism and accumulation of jasmonic and salicylic acids Environ ExpBot 99 122ndash132 httpsdoiorg101016jenvexpbot201311006
Choudhary KK Agrawal SB 2014b Ultraviolet-B induced changes in morphologicalphysiological and biochemical parameters of two cultivars of pea (Pisum sativum L)Ecotoxicol Environ Saf 100 178ndash187 httpsdoiorg101016jecoenv201310032
Dar TA Uddin M Khan MMA Hakeem KR Jaleel H 2015 Jasmonates counterplant stress a Review Environ Exp Bot 115 49ndash57 httpsdoiorg101016jenvexpbot201502010
Dhanani T Singh R Shah S Kumari P Kumar S 2015 Comparison of green ex-traction methods with conventional extraction method for extract yield L-DOPAconcentration and antioxidant activity of Mucuna pruriens seed Green Chem LettRev 8 (2) 43ndash48 httpsdoiorg1010801751825320151075070
Gutieacuterrez-Gamboa G Portu J Santamariacutea P Loacutepez R Garde-Cerdaacuten T 2017Effects on grape amino acid concentration through foliar application of three dif-ferent elicitors Food Res Int 99 688ndash692 httpsdoiorg101016jfoodres201706022
Fig 4 A In situ antioxidant assay Foliar disksof Phaseolus vulgaris L treated with (a) No an-tioxidant added (negative control) (b) 10 mMMimosine (c) 10mM ascorbic acid (positivecontrol) The oxidative damage can be seen bythe formation of a brown polymer in leaf veinsand injured areas B In vitro superoxidescavenging assay carried out with mimosineDifferent letters indicate significant differenceby Tukey test (Ple 005) The error bars re-present standard error of four replicates (Forinterpretation of the references to colour in thisfigure legend the reader is referred to the Webversion of this article)
Fig 5 Predicted mimosine radical formed followingquenching of hydroxyl radical Mimosine is first converted toa stable mimosine radical which may be then converted to anontoxic indole form
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
439
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad HossainMd Anai T Oku H 2018 Cytosolic cysteine synthase switch cysteine and mi-mosine production in Leucaena leucocephala Appl Biochem Biotechnol 186 (3)613ndash632 httpsdoiorg101007s12010-018-2745-z
Holloacutesy F 2002 Effects of ultraviolet radiation on plant cells Micron 33 (2) 179ndash197Honda MDH Ishihara KL Pham DT Borthakur D 2018 Identification of drought-
induced genes in giant leucaena (Leucaena leucocephala subsp glabrata) Trees 32571ndash585 httpsdoiorg101007s00468-018-1657-4
Huang T Jander G de Vos M 2011 Non-protein amino acids in plant defense againstinsect herbivores representative cases and opportunities for further functional ana-lysis Phytochemistry 72 1531ndash1537 httpsdoiorg101016jphytochem201103019
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of thethyrotoxic amino acid mimosine by cysteine synthase Phytochemistry 29 (11)3461ndash3465 httpsdoiorg1010160031-9422(90)85258-H
Ishihara K Lee EKW Borthakur D 2016a An improved method for RNA extractionfrom woody legume species Acacia koa A Gray and Leucaena leucocephala (Lam) deWit Int J For Wood Sci 3 (1) 031ndash035
Ishihara KL Honda MDH Pham DT Borthakur D 2016b Transcriptome analysisof Leucaena leucocephala and identification of highly expressed genes in roots andshoots Transcriptomics 4 135 httpsdoiorg1041722329-89361000135
IUBMB 2018 Enzyme Nomenclature EC 35161 httpwwwsbcsqmulacukiubmbenzymeEC35161html Accessed date 8 February 2018
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant(Phaseolus vulgaris) Biosci Res 10 (1) 29ndash32
Kazan K 2015 Diverse roles of jasmonates and ethylene in abiotic stress toleranceTrends Plant Sci 20 (4) 219ndash229 httpsdoiorg101016jtplants201502001
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect ofEthephon as an ethylene-releasing compound on the metabolic profile of Chlorellavulgaris J Agric Food Chem 64 (23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541
Khan MIR Fatma M Per TS Anjum NA Khan NA 2015 Salicylic acid-inducedabiotic stress tolerance and underlying mechanisms in plants Front Plant Sci 6 462httpsdoiorg103389fpls201500462
Korsangruang S Soonthornchareonnon N Chintapakorn Y Saralamp PPrathanturarug S 2010 Effects of abiotic and biotic elicitors on growth and iso-flavonoid accumulation in Pueraria candollei var candollei and P candollei var mir-ifica cell suspension cultures Plant Cell Tissue Organ Cult 103 (3) 333ndash342 httpsdoiorg101007s11240-010-9785-6
Lalitha K Kulothungan SR 2006 Selective determination of mimosine and its dihy-droxypyridinyl derivative in plant systems Amino Acids 31 (3) 279ndash287 httpsdoiorg101007s00726-005-0226-5
Lalitha K Kulothungan SR 2007 Mimosine mitigates oxidative stress in seleniumdeficient seedlings of Vigna radiata - Part I restoration of mitochondrial functionBiol Trace Elem Res 118 (1) 84ndash96 httpsdoiorg101007s12011-007-0013-0
Liu J Li Y Wang Y Zhang Z-H Zu Y-G Efferth T Tang Z-H 2016 Thecombined effects of ethylene and MeJA on metabolic profiling of phenolic com-pounds in Catharanthus roseus revealed by metabolomics analysis Front Physiol 71ndash11 httpsdoiorg103389fphys201600217 Article 217
Malekzadeh P Khara J Heydari R 2014 Alleviating effects of exogenous Gamma-aminobutiric acid on tomato seedling under chilling stress Physiol Mol Biol Plants20 (1) 133ndash137 httpsdoiorg101007s12298-013-0203-5
Martiacutenez-Luumlscher J Torres N Hilbert G Richard T Saacutenchez-Diacuteaz M Delrot SAguirreolea J Pascual I Gomegraves E 2014 Ultraviolet-B radiation modifies thequantitative and qualitative profile of flavonoids and amino acids in grape berriesPhytochemistry 102 106ndash114 httpsdoiorg101016jphytochem201403014
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation ofbioactive secondary metabolites by ultraviolet radiation mechanisms strategies andapplications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology forMedicinal Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash1902012
Matsuura HN Fragoso V Paranhos JT Rau MR Fett-Neto AG 2016 Thebioactive monoterpene indole alkaloid N szlig-D-glucopyranosylvincosamide is regu-lated by irradiance quality and development in Psychotria leiocarpa Ind Crop Prod86 210ndash218 httpsdoiorg101016jindcrop201603050
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo RBhambra AS Strnad M Bonfill M Fett-Neto AG 2018 Specialized plant me-tabolism characteristics and impact on target molecule biotechnological productionMol Biotechnol 60 (2) 169ndash183 httpsdoiorg101007s12033-017-0056-1
Murakoshi S Ohmiya S Haginiwa J 1972 Enzymic synthesis of mimoside a meta-bolite of mimosine in Mimosa pudica and Leucaena leucocephala Chem Pharm Bull20 (4) 855ndash857
Nakagawa T Kawaguchi M 2006 Shoot-applied MeJA suppresses root nodulation inLotus japonicus Plant Cell Physiol 47 (1) 176ndash180 httpsdoiorg101093pcppci222
Nascimento NC Menguer PK Henriques AT Fett-Neto AG 2013 Accumulation ofbrachycerine an antioxidant glucosidic indole alkaloid is induced by abscisic acidheavy metal and osmotic stress in leaves of Psychotria brachyceras Plant PhysiolBiochem 73 33ndash40 httpsdoiorg101016jplaphy201308007
Neelamegam R Sutha T 2015 UV-C irradiation effect on seed germination seedling
growth and productivity of groundnut (Arachis hypogaea L) Int J Curr MicrobiolApp Sci 4 (8) 430ndash443
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase fromLeucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol164 (2) 922ndash934 httpsdoiorg101104pp113230870
Negi VS Borthakur D 2016 Heterologous expression and characterization of mimo-sinase from Leucaena leucocephala In Fett-Neto Arthur Germano (Ed)Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods inMolecular Biology vol 1405 copySpringer Science+Business Media New York httpsdoiorg101007978-1-4939-3393-8_7 2016
Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636
Parmar F Kushawaha N Highland H George L-B 2015 In vitro antioxidant andanticancer activity of Mimosa pudica Linn extract and L-mimosine on lymphomaDaudi cells Int J Pharm Sci 12 100ndash104
Porto DD Matsuura HN Vargas LRB Henriques AT Fett-Neto AG 2014 Shootaccumulation kinetics and effects on herbivores of the wound-induced antioxidantindole alkaloid brachycerine of Psychotria brachyceras Nat Prod Commun 9 (5)629ndash632
Rai R Meena RP Smita SS Shukla A Rai SK Pandey-Rai S 2011 UV-B and UV-C pre-treatments induce physiological changes and artemisinin biosynthesis inArtemisia annua L ndash an antimalarial plant J Photochem Photobiol B Biol 105 (3)216ndash225 httpsdoiorg101016jjphotobiol201109004
Shabani L Ehsanpour AA Asghari G Emami J 2009 Glycyrrhizin production by invitro cultured Glycyrrhiza glabra elicited by methyl jasmonate and salicylic acid RussJ Plant Physiol 56 (5) 621ndash626 httpsdoiorg101134S1021443709050069
Shah J 2003 The salicylic acid loop in plant defense Curr Opin Plant Biol 6 (4)365ndash371
Shi J Fu XZ Peng T Huang XS Fan QJ Liu JH 2010 Spermine pretreatmentconfers dehydration tolerance of citrus in vitro plants via modulation of antioxidativecapacity and stomatal response Tree Physiol 30 (7) 914ndash922 httpsdoiorg101093treephystpq030
Smith IK Fowden L 1966 A study of mimosine toxicity in plants J Exp Bot 17750ndash761 httpsdoiorg101093jxb174750
Soedarjo M Borthakur D 1996 Simple procedures to remove mimosine from youngleaves pods and seeds of Leucaena leucocephala used as food Int J Food SciTechnol 31 (1) 97ndash103
Soedarjo M Borthakur D 1998 Mimosine a toxin produced by the tree-legumeLeucaena provides a nodulation competition advantage to mimosine-degradingRhizobium strains Soil Biol Biochem 30 1605ndash1613
Suda S 1960 On the physiological properties of mimosine Bot Mag Tokyo 73 (862)142ndash147 httpsdoiorg1015281jplantres188773142
Tangendjaja B Lowry JB Wills RBH 1986 Isolation of a mimosine degrading en-zyme from leucaena leaf J Sci Food Agric 37 523ndash526 httpsdoiorg101002jsfa2740370603
Tiburcio F Pintildeol MT Serrano M 1985 Effect of UV-C on growth soluble protein andalkaloids in Nicotiana rustica plants Environ Exp Bot 25 (3) 203ndash210 httpsdoiorg1010160098-8472(85)90004-8
Vestena S Fett-Neto AG Duarte RC Ferreira A 2001 Regulation of mimosineaccumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 httpsdoiorg101016S0168-9452(01)00448-4
Vlot AC Dempsey DMA Klessig DF 2009 Salicylic acid a multifaceted hormone tocombat disease Annu Rev Phytopathol 47 177ndash206 httpsdoiorg101146annurevphyto050908135202 2009
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene-induced vinblastine accumulation is related to activated expression of downstreamTIA pathway genes in Catharanthus roseus BioMed Res Int 2016 Article ID 3708187httpsdoiorg10115520163708187
Wasternack C Strnad M 2016 Jasmonate signaling in plant stress responses and de-velopment ndash active and inactive compounds N Biotech 33 (5B) 604ndash613 httpsdoiorg101016jnbt201511001
Wencomo HB Ortiz R Caacuteceres J 2017 Afr J Agric Res 12 (4) 279ndash285 httpsdoiorg105897AJAR201510604 26
Wong CC Devendra C 1983 Research on leucaena forage production in Malaysia InLeucaena Research in the Asian Pacific Region pp 55ndash60 Ottawa Ontario Canada
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX TsimKWK 2018 Jasmonate-elicited stress induces metabolic change in the leaves ofLeucaena leucocephala Molecules 23 (2) httpsdoiorg103390molecules23020188 E188
Yafuso JT Negi VS Bingham J-P Borthakur D 2014 An O-acetylserine (thiol)lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthaseAppl Biochem Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z
Zhao J Zheng S-H Fujita K Sakai K 2004 Jasmonate and ethylene signalling andtheir interaction are integral parts of the elicitor signalling pathway leading to b-thujaplicin biosynthesis in Cupressus lusitanica cell cultures J Exp Bot 55 (399)1003ndash1012 httpsdoiorg101093jxberh127
Zhishen J Mengcheng T Jianming W 1999 The determination of flavonoid contentsin mulberry and their scavenging effects on superoxide radicals Food Chem 64 (4)555ndash559 httpsdoiorg101016S0308-8146(98)00102-2
KCdS Rodrigues-Correcirca et al Plant Physiology and Biochemistry 135 (2019) 432ndash440
440
61
Supplementary Fig 1 Basal mimosine concentration in adult trees of common leucaena (L leucocephala
var leucocephala) Samples were collected from 10 field grown trees at Manoa Valley Honolulu Hawairsquoi
on June 25th 2017 Bars sharing a letter do not differ by Tukey test (P le 005) The error bars represent the
standard error
Supplementary Fig 2 Bar diagram showing mimosine concentration in shoots of 12-week-old common
leucaena seedlings treated with different elicitors CTRL = Milli-Q water SA = Salicylic Acid MeJA =
Methyl Jasmonate CEPA = 2-Chloroethylphosphonic acid (an ethylene releasing compound) Bars sharing a
letter of same case do not differ by Tukey test (P le 005) Capital letters (A B) compare treatments on day
two and lower-case letters (a b) compare treatments on day four Indicates significant statistical difference
ABB
A A
0
200
400
600
800
1000
1200
LEAVES GREEN FLOWERBUDS
POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
con
cen
trat
ion
(micro
gg
-1o
f FW
)
B AB AB AB B A
b
a
ab b
ab
0
2
4
6
8
10
12
14
16
18
20
CTRL SA 10 ppm SA 100 ppm CEPA 10 ppm CEPA 100 ppm MeJA 90 ppm
Mim
osi
ne
co
nce
ntr
atio
n (
gg
-1o
f FW
)
DAY 02 DAY 04
62
between day two and day four in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
Supplementary Fig 3 Bar diagram showing the effects of UV-C radiation exposure for 5 10 and 15 min on
mimosine accumulation in shoots of 12-week-old seedlings of common leucaena Bars sharing a letter of
same case do not differ by Tukey test (P le 005) Capital letters (A B C) compare treatments on day three
and lower-case letters (a b) compare treatments on day six Indicates significant statistical difference
between day three and day six in the same treatment by t-test (P le 005) The error bars represent standard error
of five replicates (each mean was calculated with 15 individual seedlings organized in 5 groups of three)
C BC AB A
bb
a
a
0
10
20
30
40
50
60
CTRL UV-C 5 UV-C 10 UV-C 15
Mim
osi
ne
co
nce
ntr
atio
n (
gg-1
of
FW)
DAY 03 DAY 06
63
Supplementary Fig 4 Model depicting induction of mimosine synthesis in leucaena following application of
stress elicitors such as CEPA and jasmonic acid or exposure to UV-C radiation The additional mimosine
synthesized may serve to alleviate oxidative stress induced by UV-C radiation
64
Supplementary Table 1 Mimosine contents in leaves of common and giant leucaena
Leucaena
type
Mimosine content
( FW)
Mimosine
content ( DW)
Dry matter
content ( FW)
Water content
( FW)
Common (1) 050 plusmn 009 245 plusmn 051 2011 plusmn 054 7989 plusmn 054
Common (2) 043 plusmn 006 214 plusmn 037 1998 plusmn 050 8002 plusmn 050
K636 (1) 070 plusmn 014 356 plusmn 077 1908 plusmn 052 8092 plusmn 052
K636 (2) 042 005 205 plusmn 033 2008plusmn 093 7992plusmn 093
KX2 (1) 122 plusmn 011 608 plusmn 082 1939 plusmn 123 8061 plusmn 123
KX2 (2) 134 plusmn 010 623 plusmn 056 2029 plusmn 114 7971 plusmn 114
KX3 (1) 044 plusmn 006 221 plusmn 030 1945 plusmn 073 8055 plusmn 073
KX3 (2) 054 plusmn 005 273 plusmn 023 1930 plusmn 038 8070 plusmn 038
KX4 (1) 086 plusmn 011 471 plusmn 065 1753 plusmn 084 8247 plusmn 084
KX4 (2) 089 plusmn 011 476 plusmn 065 180 plusmn 072 820 plusmn 072
KX5 (1) 099 plusmn 012 489 plusmn 048 1907 plusmn060 8093 plusmn 060
KX5 (2) 115 plusmn 015 548 plusmn080 1992 plusmn 053 8008 plusmn 053
Common leucaena variety koa haole grows widely on the island of Orsquoahu K636 is widely
grown variety of giant leucaena KX2 KX3 KX4 and KX5 are giant leucaena varieties
developed through interspecies hybridization (Brewbaker 2016) (1) and (2) indicate plants
from two separate locations within the University of Hawaii Waimanalo Research Center The
values are shown as mean plusmn standard error obtained from at least three biological replicates
65
Supplementary Table 2 GenBank accession numbers of the tested cysteine pathway genes isoforms
Gene name GenBank accession
OAS-TL (o-acetylserine-thiol-lyase) GDRZ01032940
GDRZ01061620
GDRZ01153117
GDSA01187555
GDSA01196891
GDSA01214467
Cys syn (cysteine synthase) GDRZ01015860
GDRZ01050898
GDRZ01086813
GDRZ01193515
GDRZ01202579
GDSA01180863
GDSA01215622
SAT (serine acetyltransferase) GDRZ01187456
GDRZ01189631
CAS (β-cyanoalanine synthase) GDRZ01054066
GDRZ01175418
GDSA01118400
66
SHORT COMMUNICATION 1
Mimosine occurrence and accumulation in Mimosa bimucronata var bimucronata (DC) 2
Kuntze 3
Kelly Cristine da Silva Rodrigues-Correcirca1 Lana Dorneles Pedroso2 Fernanda de Costa1 4
Arthur Germano Fett-Neto1 5
1Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal 6
University of Rio Grande do Sul (UFRGS) PO Box CP 15005 91501-970 7
Porto Alegre Rio Grande do Sul Brazil 2Department of Biological Sciences Unipampa ndash 8
Campus Satildeo Gabriel 9
Corresponding author 10
E-mail addresses krodriguescbiotufrgsbr (KCdaS Rodrigues-Correcirca) 11
lanalima2012gmailcom (LD Pedroso) fernandadecostayahoocombr (F de Costa) 12
fettnetocbiotufrgsbr (AG Fett-Neto) 13
14
15
16
17
18
19
20
21
22
67
ABSTRACT 23
Mimosine is a non-protein aromatic amino acid present in plants of Leucaena spp 24
and Mimosa spp Mimosa bimucronata var bimucronata (DC) Kuntze (maricaacute) is a native 25
tree from Brazil which occurs as a pioneer species on plant succession processes In the 26
current study the presence of mimosine in M bimucronata was verified by HPLC analyses 27
Moreover mimosine accumulation upon exposure to UV-C and chemical elicitors of 28
specialized metabolism (salicylic acid - SA methyl jasmonate - MeJA sodium nitroprusside 29
- SNP and ethephon - ETH) most of which also known as promoters of the amino acid 30
production in leucaena plants was evaluated The results showed a lower concentration of 31
constitutive mimosine present in both maricaacute seedlings and mature trees when compared to 32
leucaena plants In spite of a trend towards increased mimosine accumulation observed in 33
MeJA and ETH treatments no statistical differences were found with the various stressors 34
used to induce its biosynthesis in maricaacute seedlings Data suggest that mimosine in M 35
bimucronata is probably a phytoanticipin-like metabolite or its accumulation is driven by 36
other types of stresses 37
38
39
Keywords Mimosine Mimosa bimucronata stress 40
41
42
43
44
45
46
68
Introduction 47
Mimosa bimucronata commonly known as maricaacute is a native tree from Brazil 48
(REFLORA 2019) ecologically important in plant succession and in processes of degraded 49
land recovery (Bitencourt et al 2007 Silva et al 2011) occurring as a pioneer species 50
(Pilatti et al 2019) Maricaacute is a deciduous or semi-deciduous plant which reaches up to 15 51
m in height and 40 cm of diameter at breast height (DBH) displays shrub or tree habit and 52
bears typical sharp thorns (Carvalho 2004) This species belongs to Fabaceae one of the 53
most economically important families of flowering plants due to its high diversity and 54
occurrence in different types of habitats (Gomes et al 2018) As well as several others 55
Mimosa spp maricaacute is usually referred to as a multipurpose tree (Olkoski and Wittmann 56
2011) employed for alternative medicinal uses (Champanerkar et al 2010 Silva et al 57
2011) honey production constructions and remodeling of landscape architecture (living 58
fences) for instance (Marchiori 1993 Lorenzi 1998) 59
In southern Brazil maricaacute is widely distributed and typically found either in wetland 60
areas close to river banks (Patreze and Cordeiro 2004) or composing large and almost pure 61
landscape formations on hillsides (Jacobi and Ferreira 1991) In dense populations this 62
species like several Mimosa spp (Simon and Proenccedila 2000) is considered an important and 63
highly invasive weed by preventing cattle to reach pasturesand water bodies as a result of its 64
thorny branches (Lorenzi 2008 Kestring et al 2009) Its dominant and nearly exclusive 65
pattern of distribution in those areas has led Jacobi and Ferreira (1991) to test its allelopathic 66
potential on cultivated species Indeed extracts of leaves and ripe fruits (but not the green 67
ones) of maricaacute showed phytotoxic effects on germination and initial radical growth of most 68
of the target species tested 69
69
Several investigations have been performed on maricaacute floristics (Silva et al 2011) 70
distribution (Simon and Proenccedila 2000) wood anatomy (Marchiori 1993) cytogenetic 71
parameters (Olkoski and Wittmann 2011) and allelopathic potential (Jacobi and Ferreira 72
1991 Ferreira et al 1992) However excluding two recent publications on maricaacute 73
constitutive chemical composition (Schlickmann et al 2017 Pilatti et al 2019) which 74
identified phenolic compounds (methyl gallate and water-soluble tannins) as its major 75
compounds little is known regarding this subject In other Mimosa species (eg M pudica 76
and M pigra) mimosine has been identified (Soedarjo and Borthakur 1998) as one of the 77
major specialized metabolites present in the different organs of the plant (Champanerkar et 78
al 2010) The presence of this molecule was also reported for M bimucronata in a thin layer 79
chromatography-based preliminary study performed by Ferreira et al (1992) showing co-80
chromatography of a leaf extract component with authentic mimosine The authors attributed 81
the allelopathic effect of maricaacute to the accumulation of this metabolite in its leaves 82
Mimosine is an aromatic non-protein amino acid initially found in plants of Mimosa 83
pudica and later in Leucaena leucocephala (Lam) de Wit (Soedarjo and Borthakur 1998) a 84
leguminous tree which biosynthesizes large amounts of this nitrogen-containing compound 85
(Rodrigues-Correcirca et al 2019) It is believed that the accumulation of high contents of 86
mimosine in L leucocephala tissues confers among other traits defense against herbivores 87
and pathogens (Vestena et al 2001) tolerance to drought (Negi et al 2014) as well as 88
general oxidative stress protection (Rodrigues-Correcirca et al 2019) Interestingly drought is 89
the opposite environmental and physiological condition to that observed in the wet habitats 90
occupied by native populations of M bimucronata in Brazil (Patreze and Cordeiro 2004 91
Kestring et al 2009) and Mimosa pudica Linn in India (Champanerkar et al 2010) 92
70
Nonetheless flooding is also associated with oxidative stress particularly as water levels 93
change (Fukao et al 2019) 94
In Leucaena leucocephala var leucocephala (common leucaena) and Leucaena 95
leucocephala var glabrata (giant leucaena) mimosine accumulation has been shown to be 96
both constitutive and inducible by stress-related phytohormones such as jasmonic acid (JA) 97
Ethephon (ETH an ethylene- releasing compound) salicylic acid (SA - only common 98
leucaena) (Vestena et al 2001) as well as by UV-C radiation (Xu et al 2018 Rodrigues-99
Correcirca et al 2019) On the other hand there is a lack of information regarding mimosine 100
content and elicitation effects in Mimosa spp plants 101
The aim of this study was to examine the presence of mimosine in Mimosa 102
bimucronata and examine the effects of stresses and stress-signaling molecules on its 103
accumulation in leaves 104
Material and Methods 105
Plant material 106
For all experiments the plant material was collected at Morro Santana campus do 107
Vale of UFRGS (Federal University of Rio Grande do Sul) Porto Alegre RS Brazil 108
(3004rsquoS 5108rsquoW) Authorization for access to genetic material was obtained from 109
SISGEN-Brazil (license number A845493) Constitutive mimosine content in adult plants of 110
M bimucronata var bimucronata (DC) Kuntze was determined in plant material (leaves 111
green flower buds post-anthesis flowers and green pods) harvested in January 2017 112
(summer) A voucher herbarium specimen (ICN 187953) was deposited in the ICN ndash UFRGS 113
herbarium (Herbaacuterio do Instituto de Biociecircncias of UFRGS) 114
71
For mimosine elicitation experiments legumes (fruits) of maricaacute were collected in 115
the end of June 2017 (winter) Seeds were then removed from the dry fruits and kept in the 116
dark until sowing and seedling development for use in the assays 117
Seed germination 118
To break the coat-imposed seed dormancy after surface sterilization dry seeds of 119
maricaacute were acid scarified by immersion in H2SO4 (95 ndash 98 ) for 2 min (see Correcirca et al 120
2008) and repeatedly washed in distilled water to remove any residue of the acid Then seeds 121
were distributed in 50 mL individual plastic tubes (dibble-tubes) (30 cm diameter x 120 cm 122
depth) filled up with 11 (vv) of commercial top soil and vermiculite Tubes were watered 123
every 2 days to avoid substrate dryness and were kept in a growth room under controlled 124
conditions of light (circa 75 μmol mminus2s minus1 photosynthetically active radiation photoperiod 125
of 16 h light and 8 h dark) and temperature (24plusmn2C) 126
127
Treatments 128
In order to verify inducibility of mimosine accumulation in M bimucronata fifty 12-129
week-old maricaacute seedlings (per treatment) exhibiting similar features were selected and 130
sprayed (saturated) with solutions of different chemical stressors (plant specialized 131
metabolism elicitors) as follows (for further details see Rodrigues-Correcirca et al 2019) 10 132
and 50 mM SA (pathogen-signaling molecule Shah 2003) 007 and 035 mM 2-133
chloroethylphosphonic acid (ETH ethylene releasing-compound Kim et al 2016 Wang et 134
al 2016) 100 and 200 mM MeJA (Dar et al 2015) 10 and 50 mM SNP (a nitric oxide 135
donor Perotti et al 2015) Alternatively maricaacute seedlings were also supplemented with UV-136
C radiation (13 minutes 105 kJ cm2) (elicitor of plant specialized metabolism Kara 2013) 137
72
After 2 and 4 days of exposure to the chemical treatments and 3 and 6 days of UV-138
C supplementation maricaacute shoots were harvested immediately frozen in liquid nitrogen and 139
stored at ndash 80 C until mimosine extraction and HPLC analyses 140
Mimosine extraction and detection 141
Mimosine extraction was conducted according to the modified protocol described by 142
Rodrigues-Correcirca et al (2019) for L leucocephala HPLC (Thermo Scientific Surveyor) 143
analyses (mimosine detection and quantification) were performed following previously 144
published procedures (Negi et al 2014) A C18 column (ACE C18 5 μm 46times250 mm) and 145
isocratic solvent system of 002M o-phosphoric acid with a linear flow rate of 1 mL min minus1 146
were used to separate and quantify the amino acid Mimosine detection was performed at 280 147
nm by photodiode array detection (200ndash400 nm) and retention time (229plusmn0024 min) 148
Mimosine quantification was done by means of the method of external standard curve 149
Additional confirmation of mimosine identity was performed by co-chromatography with 150
standard (Acros Organics authentic mimosine 99 used as reference) and peak purity check 151
The analyses of the chromatograms were done with the ChromQuest software 152
153
154
Results and Discussion 155
Constitutive accumulation of mimosine in M bimucronata 156
Mimosine was detected in all analyzed samples positively meeting all identification 157
criteria In agreement with what has been found for other Mimosa spp (Soedarjo and 158
Borthakur 1998) compared to L leucocephala adult plants (Rodrigues-Correcirca 2019) 159
mimosine content was lower in M bimucronata Of the adult plant tissues analyzed the 160
73
highest content of mimosine in maricaacute (per gram of fresh weight - FW) was found in post-161
anthesis flowers (36644 microg versus 89448 microg in common leucaena followed by leaves 162
(28838 microg x 67358 microg) green flower buds (28094 microg x 51247 microg) and green pods (19002 163
microg x 82687 microg) (Fig 1)The same pattern is observed for seedlings when both species are 164
compared In this study untreated 12-week-old maricaacute seedlings (control at day 2) showed a 165
shoot content of mimosine of 23029plusmn007 microg g-1 of (FW) Five-week-old untreated giant 166
leucaena seedlings cultivated in similar conditions exhibited between 83640 and 178736 167
microg g-1 of FW (Rodrigues-Correcirca et al 2019) In the same way mimosine concentration 168
percentage in dry matter of Mimosa pigra was found to be rather low (002 in nodules and 169
roots and 007 in leaves) (Soedarjo and Borthakur 1998) 170
In this investigation the lowest constitutive mimosine content was found in green 171
pods (Fig 1) This result may partly explain the absence of phytotoxic effect observed for 172
green pods on germination and growth of crop target plants tested by Jacobi and Ferreira 173
(1991) compared to the other maricaacute parts analyzed 174
Elicitation of mimosine biosynthesis in M bimucronata 175
Chemical stressors 176
Secondary metabolites (or natural products) are structural- and chemically 177
specialized compounds derived from primary metabolism These molecules are mainly 178
biosynthesized as part of a complex defense mechanism in response to biotic and abiotic 179
stresses such as pathogens herbivores water status metal toxicity and UV radiation for 180
example (Matsuura et al 2018) Ethephon SA SNP MeJA have been extensively used as 181
chemical elicitors of specialized metabolism (Wang et al 2016 Vestena et al 2001 Perotti 182
74
et al 2015 Zhang and Memelink 2009 Xu et al 2018) These phytohormonal signals can 183
simulate environmental challenges and modulate plant homeostasis often leading to 184
alterations in gene expression (Shinozaki et al 2015) Except SNP all treatments tested in 185
the present study showed positive effect on mimosine accumulation in common or giant 186
leucaena (Vestena et al 2001 Rodrigues-Correcirca 2019 Rodrigues-Correcirca unpublished 187
data) However in spite of the trend of increasing the mimosine content observed in seedlings 188
treated with 007 mM Ethephon (at day 2) and 100 mM MeJA (at day 4) no statistical 189
difference was confirmed for these treatments when compared to the control 190
On the other hand a within treatment difference on mimosine induction was seen 191
between day 2 and 4 in seedlings treated with 100 mM MeJA (Fig 2) In a lower 192
concentration (04 mM) jasmonic acid (JA)promoted a near threefold increase in mimosine 193
accumulation of giant leucaena seedlings after 2 days of application 194
UV-C radiation 195
Albeit UV-C radiation is not biologically active in natural environments it has been 196
widely used under controlled experimental conditions to generate acute responses of plant 197
specialized metabolism within a shorter period of time compared to that required to with UV-198
B radiation (Kara 2013 Cetin 2014) This fast response is due to the higher energy of UV-199
C photons that act as potent reactive oxygen species (ROS) generators causing extensive 200
damage to the cells either at the physiological level or on DNA structure (Gregianini et al 201
2003 Matsuura et al 2013) 202
Although divergent responses can be observed in plants exposed to UV-C radiation 203
the deleterious processes are usually reported on primary metabolism (decreasing of 204
chlorophyll content and plant height eg) (Kara 2013) In the present study no statistical 205
75
differences were observed in the mimosine concentration in maricaacute seedlings supplemented 206
with UV-C radiation However a decreasing in its content was found for both control and 207
treatment at day 6 post-treatment (Fig 03) Taking into account the lower constitutive 208
concentration of mimosine observed in maricaacute compared to the leucaena plants besides its 209
relative thermolability (Nguyen and Tawata 2016) it seems to be plausible to consider the 210
effect of the temperature inside the UV-C and the white light (control) chambers as an 211
additional abiotic factor contributing to the decrease of mimosine accumulation in both group 212
of plants 213
Besides mimosine identification the presence of 34-dihydroxypyridine (34-DHP or 214
3-hydroxy-4-pyridone - 3H4P) a mimosine degradation product (Negi et al 2014 Nguyen 215
and Tawata 2016) was also reported for maricaacute leaf extracts analyzed by TLC by Ferreira 216
et al (1992) In our chromatograms we detected a second large peak after that of mimosine 217
(229plusmn0024) and similar to that identified by Negi et al (2014) as 3H4P (data not shown) 218
Comparing the chromatogram profiles obtained from seedlings elicited with chemical 219
stressors and those supplemented with UV-C the largest area for this peak was found (in all 220
samples) in the latter treatment at day 6 It might indicate that the constitutive andor the 221
initially UV-C-induced mimosine was degraded into 3H4P to cope with the cellular damage 222
caused by this treatment associated with an increased temperature inside the chambers 223
Nevertheless it was not possible to determine 3H4P concentration (or confirm its identity) 224
in maricaacute plants since there is no commercial standard (pure 3H4P) available for purchase 225
to be used as a reference in calculations Establishment of improved protocols for obtaining 226
in house 3H4P reference substance by acid hydrolysis is ongoing 227
228
229
76
Conclusion 230
On the basis of the overall absence of effect of the treatments tested here on mimosine 231
concentration it is possible to suggest that its accumulation profile is similar to that of 232
phytoanticipins unlike what is observed for the same amino acid production in leucaena 233
which shows features of inducibility resembling phytoalexin-like metabolites Alternatively 234
a putative inducible pool of mimosine in maricaacute might be involved in other types of stress 235
such as extended drought periods If involved in protection against oxidative stress as 236
described for leucaena mimosine in maricaacute may act predominantly by physical quenching 237
of ROS as indicated by the lack of overt chemical degradation Nevertheless further 238
investigations are needed to assess these hypotheses 239
To sum up mimosine biosynthesis was not modulated by the treatments evaluated as 240
in L leucocephala (Lam) de Wit To the best of our knowledge this is the first work that 241
analytically identifies and quantifies mimosine accumulation in M bimucronata 242
243
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Bitencourt F Zocche JJ Costa S Souza PZ Mendes AR 2007 Nucleaccedilatildeo de 245
Mimosa bimucronata (DC) O Kuntze em aacutereas degradadas pela mineraccedilatildeo de carvatildeo R 246
Bras Bioci 5 750-752 247
Carvalho PER 2004 Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 248
Teacutecnica 94 1-10 249
Cetin ES 2014 Induction of secondary metabolite production by UV-C radiation in Vitis 250
vinifera L Oumlkuumlzgoumlzuuml callus cultures Biol Res 47 (1) 37 httpsdoiorg1011860717-251
6287-47-37 252
77
Champanerkar PA Vaidya VV Shailajan S Menon SN 2010 A sensitive rapid and 253
validated liquid chromatography ndash tandem mass spectrometry (LC-MS-MS) method for 254
determination of Mimosine in Mimosa pudica Linn Nat Sci 2 713-717 255
httpsdoiorg104236ns201027088 256
Gomes GS Silva GS Silva DLS Oliveira RR Conceiccedilatildeo GM 2018 Botanical 257
Composition of Fabaceae Family in the Brazilian Northeast Maranhatildeo Brazil Asian J 258
Environ Ecol 6(4) 1-10 httpsdoiorg109734AJEE201841207 259
Correcirca LR Soares GLG Fett-Neto AG 2008 Allelopathic potential of Psychotria 260
leiocarpa a dominant understorey species of subtropical forests S Afri J Bot 74 583ndash261
590 httpsdoiorg101016jsajb200802006 262
Ferreira AG Aquila MEA Jacobi US Rizvi V 1992 Allelopathy in Brazil In Allelopathy 263
basic and applied aspects Rizvi V and Jacobi US (Eds) Chapman and Hall pp 243-250 264
Fukao T Barrera-Figueroa BE Juntawong P Pentildea-Castro JM 2019 Submergence 265
and waterlogging stress in plants a review highlighting research opportunities and 266
understudied aspects Front Plant Sci 10 340 httpsdoiorg103389fpls201900340 267
Gregianini TS Silveira VC Porto DD Kerber VA Henriques AT Fett-Neto AG 268
2003 The alkaloid brachycerine is induced by ultraviolet radiation and is a singlet oxygen 269
quencher Photochem Photobiol 78(5) 470ndash474 httpsdoiorg1015620031-270
8655(2003)0784070TABIIB20CO2 271
Jacobi US Ferreira AG 1991 Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK 272
sobre espeacutecies cultivadas Pesq Agropec Bras 26(7) 935-943 273
Kara Y 2013 Morphological and physiological effects of UV-C radiation on bean plant 274
(Phaseolus vulgaris) Biosci Res 10(1) 29ndash32 275
78
Kestring D Klein J Menezes LCCR Rossi MN 2009 Imbibition phases and 276
germination response of Mimosa bimucronata (Fabaceae Mimosoideae) to water 277
submersion Aquat Bot 91 105ndash109 httpsdoiorg101016jaquabot200903004 278
Kim SH Lim SR Hong SJ Cho BK Lee H Lee CG Choi HK 2016 Effect of 279
Ethephon as an ethylene-releasing compound on the metabolic profile of Chlorella vulgaris 280
J Agric Food Chem 64(23) 4807ndash4816 httpsdoiorg101021acsjafc6b00541 281
Lorenzi H 1998 Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 282
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 283
Lorenzi H 2008 Plantas daninhas do Brasil terrestres aquaacuteticas parasitas e toacutexicas 4 ed 284
Nova Odessa Instituto Plantarum 640 p 285
Marchiori JNC 1993 Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 286
O Kuntze Ciecircncia Florestal 3 85-106 287
Matsuura HN De Costa F Yendo ACA Fett-Neto AG 2013 Photoelicitation of 288
bioactive secondary metabolites by ultraviolet radiation mechanisms strategies and 289
applications In Chandra S Lata H Varma A (Eds) (Org) Biotechnology for Medicinal 290
Plants1ed vol 1 Springer Berlin Heidelberg New York pp 171ndash190= 291
Matsuura HN Malik S de Costa F Yousefzadi M Mirjalili MH Arroo R Bhambra AS 292
Strnad M Bonfill M Fett-Neto AG 2018 Specializedplant 293
metabolismcharacteristicsandimpactontargetmoleculebiotechnologicalproduction 294
Molecular Biotechnology 60(2) 169ndash183httpsdoiorg101007s12033-017-0056-1 295
Negi VS Bingham J-P Li QX Borthakur D 2014 A carbon-nitrogen lyase from 296
Leucaena leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 297
922ndash934 httpsdoiorg101104pp113230870 298
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Nguyen BCQ Tawata S 2016 The chemistry and biological activities of mimosine 299
areview Phytother Res 30 1230ndash1242 httpsdoiorg101002ptr5636 300
Olkoski D Wittmann MTS 2011 Cytogenetics of Mimosa bimucronata (DC) O Kuntze 301
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop Breed 302
Appl Biotechnol 11 27-35 httpdxdoiorg101590S1984-70332011000100004 303
Patreze CM Cordeiro L 2004 Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 304
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecol Manag 196 275ndash305
285 httpdxdoiorg101016jforeco200403034 306
Perotti JC Rodrigues-Correcirca KCS Fett-Neto AG 2015 Control of resin production in 307
Araucaria angustifolia an ancient South American conifer Plant Biology 17 852ndash859 308
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG 2019 Mimosine 309
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 310
UV exposure Plant Physiology and Biochemistry 135 432ndash440 311
Pilatti DM Fortes AMT Jorge TCM Boiago NP 2019 Comparison of the phytochemical 312
profiles of five native plant species in two different forest formations Brazilian Journal of 313
Biology 79(2) 233-242 314
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS 2011 Biologia da reproduccedilatildeo 315
deMimosa bimucronatandash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 316
Simon MF and Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 317
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 318
endemism Biological Conservation 96 279-296 319
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 320
LM Andrade SF Cechinel-Filho V 2017 Chemical composition and diuretic natriuretic and 321
80
kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its majority 322
constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash1624 323
Shah J 2003 The salicylic acid loop in plant defense Current Opinion Plant Biology6 (4) 324
365ndash371 325
Shinozaki K Uemura M Serres JB Bray EA Weretilnyk E 2015 Responses to Abiotic 326
Stress In Buchanan BB Gruissem W Jones RL (Eds) Biochemistry and Molecular 327
Biology of Plants Second Edition John Wiley and Sons Ltd 328
Soedarjo M and Borthakur D 1998 Mimosine a toxin produced by the tree-legume 329
Leucaena provides a nodulation competition advantage to mimosine-degrading Rhizobium 330
strains Soil Biology and Biochemistry 30(12)1605-1613 331
Vestena S Fett-Neto AG Duarte RC Ferreira AG 2001 Regulation of mimosine 332
accumulation in Leucaena leucocephala seedlings Plant Sci 161 597ndash604 333
Wang X Pan Y-J Chang B-W Hu Y-B Guo X-R Tang ZH 2016 Ethylene induced 334
vinblastine accumulation is related to activated expression of downstream TIA pathway 335
genes in Catharanthus roseus BioMed Research International Article ID 3708187 336
Xu Y Tao Z Jin Y Chen S Zhou Z Gong AGW Yuan Y Dong TTX Tsim KWK 2018 337
Jasmonate-elicited stress induces metabolic change in the leaves of Leucaena leucocephala 338
Molecules 23 (2) 339
Zhang H Memelink J 2009 Regulation of Secondary Metabolism by Jasmonate Hormones 340
In AE Osbourn and V Lanzotti (eds) Plant-derived Natural Products 3 DOI 101007978-341
0-387-85498-4_1 copy Springer Science + Business Media LLC 342
343
344
345
81
346
Figure 1 Constitutive concentration of mimosine in different plant organs of Mimosa 347
bimucronata Bars sharing the same letter do not differ statistically by Tukey test (Ple005) 348
The error bars denote standard error of 10 replicates 349
350
351
352
353
354
355
356
357
B B A C0
5
10
15
20
25
30
35
40
LEAVES GREEN FLOWER BUDS POST-ANTHESISFLOWERS
GREEN PODS
Mim
osi
ne
co
nce
ntr
atio
n u
gg-1
Mimosine concentration in adult plants of Mimosa bimucronata (DC) Kuntze
82
C T R L S A
1 0 m M
S A
5 0 m M
E T H
0 0 7 m M
E T H
0 3 5 m M
M e J A
1 0 0 m M
M e J A
2 0 0 m M
S N P
1 0 m M
S N P
5 0 m M
0
1 0
2 0
3 0
T re a tm e n ts
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1) D A Y 2
D A Y 4
A B C C B C A B C C A B C A B C A
a b b b a a b a a b b a b
358
Figure 2 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 359
bimucronata treated with different signaling molecules SA = Salicylic Acid ETH = 360
Ethephon MeJA = Methyl Jasmonate SNP = Sodium Nitroprusside Uppercase and 361
lowercase letters indicate statistical differences among treatments in days 2 and 4 362
respectively Bars sharing a letter of the same case do not differ statistically by Tukey test 363
(Ple005) Indicates statistical difference in the same treatment between day 2 and 4 by t-364
test (Ple005) The error bars denote standard error of 5 replicates (25 individual seedlings 365
arranged in 5 groups of 5) 366
367
368
83
D AY 3 D AY 6
0
5
1 0
1 5
2 0
2 5
Mim
os
ine
co
nc
en
tra
tio
n (
gg
-1)
C O N TR O L
U V -C
369
Figure 3 Mimosine concentration in shoots of 12-week-old seedlings of Mimosa 370
bimucronata supplemented with UV-C radiation Indicates statistical difference in the same 371
treatment between day 3 and 6 by t-test (Ple005) The error bars denote standard error of 5 372
replicates (25 individual seedlings arranged in 5 groups of 5) 373
374
375
376
377
378
379
380
381
382
383
384
385
84
Consideraccedilotildees finais 386
- Experimentos que avaliam os efeitos da aplicaccedilatildeo exoacutegena de ANPs em diferentes espeacutecies 387
vegetais tecircm sido realizados principalmente com GABA Dentre os principais efeitos 388
conferidos pela aplicaccedilatildeo dessa moleacutecula em espeacutecies de mono e eudicotiledocircneas satildeo 389
relatados a toleracircncia agrave seca agrave salinidade e agraves temperaturas extremas 390
- Como metaboacutelitos especializados claacutessicos os ANPs podem ter sua concentraccedilatildeo basal 391
endoacutegena aumentada em resposta agrave induccedilatildeo mediada por uma vasta gama de tratamentos com 392
moleacuteculas sinalizadoras de estresse e fontes alternativas de estressores De um modo geral 393
observa-se o acuacutemulo das diferentes classes de ANPs em resposta agrave radiaccedilatildeo UV elicitores 394
quiacutemicos que mimetizam ataques por patoacutegenos dano mecacircnico agentes osmoacuteticos metais 395
pesados entre outros 396
- Especificamente em leucena a resposta observada em relaccedilatildeo aos diferentes tratamentos 397
testados indica que apesar do seu alto teor constitutivo nessa espeacutecie a biossiacutentese e o 398
acuacutemulo de mimosina podem ser modulados por fatores causadores de estresses exibindo -399
nessa espeacutecie - um padratildeo de acumulaccedilatildeo similar agrave fitoalexinas Em maricaacute por outro lado 400
aumento de acuacutemulo dessa moleacutecula natildeo foi observado para os mesmos tratamentos testados 401
para leucena o que sugere um perfil de acumulaccedilatildeo similar ao das fitoanticipinas 402
- O padratildeo de expressatildeo gecircnica observado nas plantas de leucena estressadas com etileno 403
sugere que o controle steady-state da mimosina pode ser pelo menos em parte regulado pela 404
sua degradaccedilatildeo 405
- As respostas observadas nos testes que avaliaram a atividade de mitigaccedilatildeo de espeacutecies 406
reativas de oxigecircnio por mimosina sugerem que essa moleacutecula pode agir como um agente 407
antioxidante natildeo-enzimaacutetico em plantas de leucena em situaccedilatildeo de estresse 408
85
Perspectivas 409
- Confirmaccedilatildeo em espectrocircmetro de massas eou ressonacircncia nuclear magneacutetica da natureza 410
quiacutemica da lsquomimosinarsquo presente em maricaacute 411
- Avaliaccedilatildeo do efeito de concentraccedilotildees mais elevadas e em diferentes periacuteodos de aplicaccedilatildeo 412
das moleacuteculas sinalizadoras testadas sobre o acuacutemulo de mimosina em leucena e maricaacute 413
- Ampliar a investigaccedilatildeo dos padrotildees de expressatildeo gecircnica dos genes que codificam para 414
mimosinase (em maricaacute) mimosina sintase (em ambas as espeacutecies testadas) bem como o 415
perfil de precursores e cataboacutelitos de mimosina em resposta aos tratamentos mencionados 416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
86
Referecircncias Bibliograacuteficas 436
437
Acamovic T Brooker JD (2005) Biochemistry of plant secondary metabolites and their 438
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Ahmed R Hoque ATMR Hossain MK (2008) Allelopathic effects of Leucaena 440
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Ahmed AMM Saacutenchez FJS Bavileacutes LRY Mahdy REZ Camaal JBC (2016) Tannins and 443
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Leucaena Psyllid and Onion thrips Agroforestry Systems 1-8 445
Benjakul S Kittiphattanabawon P Shahidi F Maqsood S (2013) Antioxidant activity and 446
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model systems Food Sci Technol Int 19(4)365-76 448
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Bitencourt F Zocche JJ Costa S Souza PZ Mendes AR (2007) Nucleaccedilatildeo de Mimosa 450
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Health Prod 47(8)1481-6 456
Carvalho PER (2004) Maricaacute ndash Mimosa bimucronata EMBRAPA Colombo ndash PR Circular 457
Teacutecnica 941-10 458
Chowtivannakul P Srichaikul B Talubmook C (2016) Antidiabetic and antioxidant activities 459
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Resources 50 (2016) 357e361 httpdxdoiorg101016janres201606007 461
Chung H-H Chen M-K Chang Y-C Yang S-F Lin C-C Lin C-W (2017) Inhibitory effects 462
of Leucaena leucocephala on the metastasis and invasion of human oral cancer cells 463
Environmental Toxicology 321765ndash1774 httpsdoiorg101002tox22399 464
87
Crowe B Poynter JA Manukyan MC Wang Y Brewster BD Herrmann JL Abarbanell 465
AM Weil BR Meldrum DR (2001) Pretreatment with intracoronary mimosine improves 466
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Fallon (2015) Effects of mimosine on Wolbachia in mosquito cells cell cycle suppression 468
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Fernaacutendez-Salas A Alonso-Diacuteaza MA Acosta-Rodriacuteguez A Torres-Acosta JFJ Sandoval-471
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Parasitology 175113ndash118 2010 httpsdoiorg101016jvetpar201009016 474
Ferreira AG Aquila MEA Jacobi US Rizvi V (1992) Allelopathy in Brazil In Allelopathy 475
basic and applied aspects Rizvi V and Jacobi US (Eds) Chapman and Hall PP 243-250 476
Harun-Ur-Rashid Md Iwasaki H Parveen S Oogai1 S Fukuta M Amzad Hossain Md Anai 477
T Oku H (2018) Cytosolic cysteine synthase switch cysteine and mimosine production in 478
Leucaena leucocephala Appl Biochem Biotechnol 186 (3) 613ndash632 479
httpsdoiorg101007s12010-018-2745-z 480
Ikegami F Mizuno M Kihara M Murakoshi I 1990 Enzymatic synthesis of the thyrotoxic 481
amino acid mimosine by cysteine synthase Phytochemistry 29 (11) 3461ndash3465 482
httpsdoiorg1010160031-9422(90)85258-H 483
Jacobi US Ferreira AG (1991) Efeitos alelopaacuteticos de Mimosa bimucronata (DC) OK Sobre 484
espeacutecies cultivadas Pesquisa Agropecuaacuteria Brasileira 26(7) 935-943 485
Jamous RM Ali-Shtayeh MS Abu-Zaitoun SY Markovics A Azaizeh H (2017) Effects of 486
selected Palestinian plants on the in vitro exsheathment of the third stage larvae of 487
gastrointestinal nematodes BMC Veterinary Research 13308 488
httpdxdoiorg101186s12917-017-1237-7 489
Jiao CJ Jiang J-L Ke L-M Cheng W Li F-M Li Z-X Wang C-Y (2011) Factors affecting 490
β-ODAP content in Lathyrus sativus and their possible physiological mechanisms Food 491
Chem Toxicol 49 543ndash549 httpsdoiorg101016jfct201004050 492
Kubota S Fukumoto Y Ishibashi K Soeda S Kubota SS Yuki R Nakayama Y Aoyama K 493
Yamaguchi N (2014) Activation of the prereplication complex is blocked by mimosine 494
88
through reactive oxygen species-activated ataxia telangiectasia mutated (ATM) protein 495
without DNA damage J Biol Chem 28 289(9)5730-46 496
Kuppusamy UR Arumugam B Azaman N Wai CJ (2014) Leucaena leucocephala Fruit 497
Aqueous Extract Stimulates Adipogenesis Lipolysis and Glucose Uptake in Primary Rat 498
Adipocytes Hindawi Publishing Corporation e Scientific World Journal Article ID 737263 499
8 pages httpdxdoiorg1011552014737263 500
Kusama-Eguchi K (2019) Research in motor neuron diseases caused by natural substances 501
focus on pathological mechanisms of neurolathyrism Yakugaku Zasshi 139 (4) 609-502
615 httpsdoiorg101248yakushi18-00202 503
Kutchan TM Gershenzon J Moslashller BL Gang DR (2015) Natural Products In Buchanan 504
BB Gruissem W and Jones RL (eds) Biochemistry amp Molecular Biology of Plants 2nd edn 505
Wiley Blackwell Chichester pp 1135-1205 506
Lalande M (1990) A reversible arrest point in the late G1 phase of the mammalian cell cycle 507
Exp Cell Res 186 332ndash339 508
Li X-W Hu C-P Li Y-J Gao Y-X Wang XM Yang J-R (2015) Inhibitory effect of L-509
mimosine on bleomycin-induced pulmonary fibrosis in rats Role of eIF3a and p27 Int 510
Immunopharmacol 27(1) 53ndash64 511
Little Jr EL Skolmen RG (1989) Koa haole Agriculture Handbook 679 USDA 512
Lorenzi H (1998) Aacutervores brasileiras manual de identificaccedilatildeo e cultivo de plantas arboacutereas 513
nativas do Brasil Vol II Plantarum Nova Odessa 368 p 514
Marchiori JNC (1993) Anatomia da madeira e casca do maricaacute Mimosa bimucronata (DC) 515
O Kuntze Ciecircncia Florestal 3 85-106 516
Mohammed RS El Souda SS Taie HAA Moharam ME Shaker KH (2015) Antioxidant 517
antimicrobial activities of flavonoids glycoside from Leucaena leucocephala leaves Journal 518
of Applied Pharmaceutical Science 5(06)138-147 519
httpdxdoiorg107324JAPS201550623 520
Negi VS Bingham J-P Li QX Borthakur D (2014) A carbon-nitrogen lyase from Leucaena 521
leucocephala catalyzes the first step of mimosine degradation Plant Physiol 164 (2) 922ndash522
934 httpsdoiorg101104pp113230870 523
89
Olkoski D Wittmann MTS (2011) Cytogenetics of Mimosa bimucronata (DC) O Kuntze 524
(Mimosoideae Leguminosae) chromosome number polysomaty and meiosis Crop 525
Breeding and Applied Biotechnology 11 27-35 526
Patreze CM Cordeiro L (2004) Nitrogen-fixing and vesicularndasharbuscular mycorrhizal 527
symbioses in some tropical legume trees of tribe Mimoseae Forest Ecology and Management 528
196275ndash285 529
Pilatti DM Fortes AMT Jorge TCM Boiago NP (2019) Comparison of the phytochemical 530
profiles of five native plant species in two different forest formations Brazilian Journal of 531
Biology 79(2) 233-242 532
Ramos-Ruiz R Poirot E Flores-Mosquera M (2018) GABA a non-protein amino acid 533
ubiquitous in food matrices Cogent Food Agric 41534323 534
httpsdoiorg1010802331193220181534323 535
REFLORA (2019) httpfloradobrasiljbrjgovbrreflora Acesso em agosto de 2019 536
Rodgers KJ Samardzic K Main BJ (2015) Toxic Nonprotein Amino Acids Plant Toxins 537
httpsdoiorg 101007978-94-007-6728-7_9-1 538
Rodrigues-Correcirca KCS Honda MDH Borthakur D Fett-Neto AG (2019) Mimosine 539
accumulation in Leucaena leucocephala in response to stress signaling molecules and acute 540
UV exposure Plant Physiology and Biochemistry 135 432ndash440 541
httpsdoiorg101016jplaphy201811018 542
Schlickmann F Souza P Boeing T Mariano LNB Steimbach VMB Krueger CMA Silva 543
LM Andrade SF Cechinel-Filho V (2017) Chemical composition and diuretic natriuretic 544
and kaliuretic effects of extracts of Mimosa bimucronata (DC) Kuntze leaves and its 545
majority constituent methyl gallate in rats Journal of Pharmacy and Pharmacology 69 1615ndash546
1624 547
Silva LA Guimaratildees E Rossi MN Maimoni-Rodella RCS (2011) Biologia da reproduccedilatildeo 548
de Mimosa bimucronata ndash uma espeacutecie ruderal Planta Daninha Viccedilosa-MG 29 1011-1021 549
Simon MF Proenccedila C 2000 Phytogeographic patterns of Mimosa (Mimosoideae 550
Leguminosae) in the Cerrado biome of Brazil an indicator genus of high-altitude centers of 551
endemism Biological Conservation 96 279-296 552
90
Soares AMS Arauacutejo SA Lopes SG Costa Junior LM (2015) Anthelmintic activity of 553
Leucaena leucocephala protein extracts on Haemonchus contortus Braz J Vet Parasitol 554
Jaboticabal 24(4) 396-401 httpdxdoiorg101590S1984-29612015072 555
Soerdajo M Borthakur D (1998) Mimosine a toxin produced by the tree-legume Leucaena 556
provides a nodulation competition advantage to mimosine-degrading Rhizobium strains Soil 557
Biol Biochem 30(12) 16051613 558
Souza-Lima ES Sinani TR Pott A Sartori ALB (2017) Mimosoideae (Leguminosae) in the 559
Brazilian Chaco of Porto Murtinho Mato Grosso do Sul Rodrigueacutesia 68(1) 263-290 2017 560
httpdxdoiorg1015902175-7860201768131 561
Taiz L amp Zeiger E (2010) Plant Physiology 5th edition Sinauer Associates Inc Sunderland 562
Verma VK Rani KV Kumara SR Prakash O (2018) Leucaena leucocephala pod seed 563
protein as an alternate to animal protein in fish feed and evaluation of its role to fight against 564
infection caused by Vibrio harveyi and Pseudomonas aeruginosa Fish and Shellfish 565
Immunology 76 (2018) 324ndash332 httpsdoiorg101016jfsi201803011 566
Yafuso JT Negi VS Bingham J-P Borthakur D (2014) An O-acetylserine (thiol) lyase from 567
Leucaena leucocephala is a cysteine synthase but not a mimosine synthase Appl Biochem 568
Biotechnol 173 (5) 1157ndash1168 httpsdoiorg101007s12010-014-0917-z 569
Zarin RMA Wan HY Isha A Armani N (2016) Antioxidant antimicrobial and cytotoxic 570
potential of condensed tannins from Leucaena leucocephala hybrid Food Science and 571
Human Wellness 5 65ndash75 httpdxdoiorg101016jfshw201602001 572
573
574
Contents lists available at ScienceDirect
Industrial Crops amp Productsjournal homepage wwwelseviercomlocateindcrop
Resin tapping transcriptome in adult slash pine (Pinus elliottii var elliottii)Camila Fernanda de Oliveira Junkes1 Artur Teixeira de Arauacutejo Juacutenior1 Juacutelio Ceacutesar de LimaFernanda de Costa Thanise Fuumlller Maacutercia Rodrigues de Almeida Franciele Antocircnia NeisKelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-NetoCenter for Biotechnology and Department of Botany Federal University of Rio Grande do Sul Porto Alegre PO Box 15005 91501-970 Brazil
A R T I C L E I N F O
KeywordsPinus elliottiResinResinosisTranscriptomeAdjuvant paste
A B S T R A C T
To better understand the bases of resin production a major source of terpenes for industry the transcriptome ofadult Pinus elliottii var elliottii (slash pine) trees under field commercial resinosis was obtained Samples werecollected from cambium after 5 and 15 days of treatment application which included tapping followed byapplication of commercial resin stimulant paste or control wounding without paste Overall mean number ofreads of all 16 libraries (2 treatments x 2 times x 4 replicated trees) was 34582048 Of these 89 were mappedagainst the reference sequence with a mismatch of 058 Using the Blast2Go 570 candidate genes were de-tected based on sequence annotation By comparing the expression profile between paste and control 310differentially expressed genes (DEGs) were identified at 5 days and 190 at 15 days with a significant fold changeof log2gt 12 Regarding changes in time comparisons within each treatment 210 and 105 DEGs were identifiedwithin control and paste treatment respectively Genes with different expression patterns in the times andtreatments examined included ethylene responsive transcription factors geranylgeranyl diphosphate synthasediterpene synthase cytochrome P450 and ABC transporters all of which may play important roles in resinproduction RT-qPCR analysis correlated well with the data obtained by RNAseq Resin composition changedover time This is the first transcriptomic investigation of resinosis of the main species used in the bioresinindustry and of molecular analyses of resinosis under field operations with implications for stand managementstimulant paste development genotype selection and breeding for high resinosis
1 Introduction
The adaptive success of conifers is largely due to the development ofa defense system based on the synthesis and secretion of terpenes in allmajor organs and different tissues (Miller et al 2005 Hall et al 2013Warren et al 2015) Conifer resin is a viscous fluid composed of acomplex mixture of terpenoids such as monoterpenes sesquiterpenesand diterpenes (Zulak and Bohlmann 2010) These terpenoids are se-creted from severed resin ducts when the tree is under biotic attack(Ralph et al 2006 Lange 2015 Geisler et al 2016) acting as pro-tectants (Schiebe et al 2012 Liu et al 2015)Biosynthesis of terpenes in conifers starts from isomerization of two
isoprenoid (C5) units dimethylallyl diphosphate (DMAPP) and iso-pentenyl diphosphate (IPP) These molecules can be biosynthesized viatwo separate routes in plants the methyl-erythritol 4-phosphate andmevalonate pathways IPP is synthesized and isomerized to DMAPP byisopentenyl diphosphate isomerase then prenyl transferases catalyze
the condensation of these two C5-units to geranyl diphosphate (Pazoukiand Niinemets 2016) Their elongation to prenyl diphosphates withaddition of IPP molecules leads to monoterpenes (C10) sesquiterpenes(C15) and diterpenes (C20) which are the substrates for terpene syn-thases (TPS) (Keeling and Bohlmann 2006b)TPSs are part of a large family of mechanistically related enzymes
involved in both primary and secondary metabolism (Keeling andBohlmann 2006b) The events of evolutionary diversification and ex-pansion of plant TPSs appear to have originated from gene duplicationsdomain losses and sub- or neofunctionalizations with subsequent di-vergence of an ancestral TPS gene of primary metabolism (Hall et al2013) Modification of TPS products changes their physical propertiesand may alter their biological activities (Chen et al 2011) TPSs of highsequence identity may have different functions even in closely relatedspecies Low sequence identity of TPSs in phylogenetically distantspecies does not preclude the possibility of independent evolution of thesame or related function of these enzymes (Zerbe and Bohlmann 2015)
httpsdoiorg101016jindcrop2019111545Received 4 January 2019 Received in revised form 10 June 2019 Accepted 4 July 2019
Corresponding authorE-mail address fettnetocbiotufrgsbr (AG Fett-Neto)1 These authors have equally contributed to this work
doi 1015900102-33062019abb0114
Acta Botanica Brasilica
Sustainable production of bioactive alkaloids in Psychotria L of
southern Brazil propagation and elicitation strategies
Yve Verocircnica da Silva Magedans1 Kelly Cristine da Silva Rodrigues-Correcirca1 Cibele Tesser da Costa1
Heacutelio Nitta Matsuura1 and Arthur Germano Fett-Neto1
Received April 1 2019Accepted June 28 2019
ABSTRACTPsychotria is the largest genus in Rubiaceae South American species of the genus are promising sources of natural
products mostly due to bioactive monoterpene indole alkaloids they accumulate ese alkaloids can have analgesic
antimutagenic and antioxidant activities in dierent experimental models among other pharmacological properties
of interest Propagation of genotypes with relevant pharmaceutical interest is important for obtaining natural
products in a sustainable and standardized fashion Besides the clonal propagation of elite individuals the alkaloid
content of Psychotria spp can also be increased by applying moderate stressors or stress-signaling molecules is
review explores advances in research on methods for plant propagation and elicitation techniques for obtaining
bioactive alkaloids from Psychotria spp of the South Region of Brazil
Keywords abiotic stress alkaloids elicitation monoterpenes plant propagation Psychotria southern Brazil
sustainability
Introduction
Psychotria belongs to Rubiaceae one of the major families
of $owering plants having economic interest e family
includes coee a few signicant poisonous plants to livestock
besides several important ornamental and medicinal species
(Souza amp Lorenzi 2012) Psychotria has captured researchersrsquo
attention mostly because of its medicinal properties
Psychotria colorata is an Amazonian species that produces
polyindolinic alkaloids with analgesic activity (Matsuura et
al 2013) e promising results obtained with P colorata
motivated the investigation of southern Brazilian Psychotria
species and the discovery of new bioactive alkaloids (Porto
et al 2009) Moreover leads on in planta alkaloid functions
were also topic of experimental evaluation
One of the key elements that needs to be addressed early
on during the process of developing new bioactive molecules
from plants is the capacity to generate catalytically active
biomass to support extraction and steady supply ere are a
number of ways through which these goals may be reached
including greenhouse rooting of cuttings (mini-cutting
1 Laboratoacuterio de Fisiologia Vegetal Departamento de Botacircnica Instituto de Biociecircncias e Centro de Biotecnologia Universidade Federal do Rio
Grande do Sul 91501-970 Porto Alegre RS Brazil
Corresponding author fettnetocbiotufrgsbr
Review
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Biomass yield of resin in adult Pinus elliottii Engelm trees is differentially
regulated by environmental factors and biochemical effectors
Franciele Antocircnia Neis Fernanda de Costa Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima
Kelly Cristine da Silva Rodrigues-Correcirca Janette Palma Fett Arthur Germano Fett-Neto
Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) CP 15005 CEP 91501-970 Porto Alegre RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Biomass
Terpene resin
Seasonal
Benzoic acid
Regenerated forest
A B S T R A C T
Biomass of pine resin finds several applications in the chemical pharmaceutical biofuel and food industries
Resin exudation after injury is a key defense response in Pinaceae since this complex mixture of terpenes has
insecticidal antimicrobial and wound repair properties Resin yield is increased by effectors applied on the
wound area including phytohormones and metal cofactors of terpene synthases The interaction of resinosis
mechanism effectors is not fully understood particularly in adult forest setups under natural environmental
variations The aim of this work was to determine how resin exudation by wounded trunks of adult P elliottii
responded to combined chemical effectors involved in different regulatory pathways of resinosis (metal cofactors
of terpene synthases benzoic acid and plant growth regulators) and whether seasonal and tree distribution
variations affected these responses Symmetrically planted and scattered trees regenerated from the seed bank
had similar resin biomass yields suggesting that the homogeneity in development and spatial arrangement were
not significant factors in resin yield This new finding is of practical importance with the used tapping system
since costs of implanting forests by regeneration can be advantageous compared to planting In addition it was
shown for the first time that the salicylic acid precursor benzoic acid and the auxin naphthalene acetic acid
promoted resin exudation when individually applied to wound sites Both these adjuvants are two orders of
magnitude less costly compared to the conventionally used ethylene precursors besides facing less environ-
mental and health restrictions for use Most adjuvant-treated trees showed higher resin flow in the second year
indicating mechanisms of response build up Overall temperature was more important than rainfall as en-
vironmental parameter affecting resin biosynthesis which was higher in the warmer months of spring and
summer The combination of resinosis stimulant effectors from different signaling pathways showed no sig-
nificant synergistic or additive effect suggesting possible converging signaling pathways andor limitation of
common intermediate transducing molecules
1 Introduction
Pines occupy highly diverse environments over a range of tem-
peratures water and nutrient availabilities irradiance levels and pho-
toperiods being able to effectively face attacks from diverse herbivore
and pathogen guilds The success of conifers is linked to their complex
terpene biochemistry hosted by specialized secretory cells The terpe-
noid resin synthesized by Pinus spp is one of the main mechanisms of
defense of these trees particularly against bark beetles and the fungi
they carry (Fett-Neto and Rodrigues-Correcirca 2012) Pine resin biomass
is essentially composed of a monoterpene and sesquiterpene-rich tur-
pentine and diterpenoid-rich rosin fraction both finding numerous in-
dustrial applications as non-wood forest products (Rodrigues-Correcirca
et al 2012)
Molecules capable of modulating different signaling pathways have
been identified as resin yield stimulators including sulfuric acid (ex-
tends wound damage) 2-chloroethylphosphonic acid (CEPA a syn-
thetic ethylene precursor) paraquat (free radical generator) yeast ex-
tract (mimics attack by pathogens) salicylic acid (pathogen signaling
molecule) auxin (promotes ethylene biosynthesis and resin canal dif-
ferentiation) jasmonic acid (signals mechanical damage and promotes
secondary metabolism) and metal ions such as potassium iron and
manganese (cofactors of terpene synthases in conifers) and copper (a
component of ethylene receptors) (Clements 1970 Conrath et al
2002 Fett-Neto and Rodrigues-Correcirca 2012 Hudgins and Franceschi
2004 Lewinsohn et al 1994 Martin et al 2002 Popp et al 1995
httpsdoiorg101016jindcrop201803027
Received 12 December 2017 Received in revised form 9 March 2018 Accepted 13 March 2018
Corresponding author
E-mail addresses franci_neisyahoocombr (FA Neis) fernandadecostayahoocombr (F de Costa) thanisenfyahoocombr (TN Fuumlller)
jjuliocesarlimagmailcom (JC de Lima) krodriguescbiotufrgsbr (KC da Silva Rodrigues-Correcirca) jpfettcbiotufrgsbr (JP Fett) fettnetocbiotufrgsbr (AG Fett-Neto)
Contents lists available at ScienceDirect
Industrial Crops amp Products
journal homepage wwwelseviercomlocateindcrop
Research Paper
Dual allelopathic effects of subtropical slash pine (Pinus elliottii Engelm)
needles Leads for using a large biomass reservoir
Kelly Cristine da Silva Rodrigues-Correcircaa Gelson Halmenschlagera Joseacuteli Schwambachb
Fernanda de Costaa Emili Mezzomo-Trevizana Arthur Germano Fett-Netoa
a Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul (UFRGS) PO Box CP 15005 Brazilb University of Caxias do Sul Institute of Biotechnology Caxias do Sul RS Brazil
A R T I C L E I N F O
Keywords
Pinus elliottii
Seasonality
Growth
Germination
Litter
Substrate
A B S T R A C T
Pinus elliottii Engelm (slash pine) is distributed along the maritime coast of Southern Brazil where it shows
invasive pattern and typical allelopathic features Large quantities of needle litter are produced by pine trees a
biomass that is little explored in areas where this species is alien Little is known about the dynamics of needle
and litter phytochemical interactions particularly in subtropical environments To elucidate the full range of
needle and litter allelopathic potential the effects of litter (superficial and deep) and seasonally harvested fresh
slash pine needles stored for different times were evaluated against lettuce tomato and cucumber seeds and
seedlings Increasing concentrations (0 1 2 4 and 8 wv) of hot and cold aqueous extracts of needles
and litter affected in different ways target plant development Growth and germination inhibition were directly
related to the highest extract concentrations (regardless of the season and mainly in hot water extracts) of
needles On the other hand stimulatory effects of litter extracts on lettuce growth were observed Growth and
germination of cucumber and tomato were not affected by pine litter as substrate when compared to rice husk
The presumable high polarity and thermal stability of slash pine leaf biomass allelochemicals and their transient
toxic effect or growth promoting impact suggest potential applications of this largely available biomass both as a
biological herbicide and growth substrate in plant propagation
1 Introduction
Native from the Northern Hemisphere Pinus is one of the most
widely distributed genera throughout different climate regions of the
globe growing either as native or alien species even in extreme habi-
tats (Rodrigues-Correcirca and Fett-Neto 2012) Despite the high economic
value currently attributed to pine wood and oleoresin (Rodrigues-
Correcirca et al 2012) there is increasing concern about the aggressive
potential of invasiveness displayed by Pinus species especially those
cultivated out of their native range of distribution (Richardson et al
2008 Rolon et al 2011) These species are dispersed by wind and there
is notably low plant diversity observed in most understories of pine
plantations (Kato-Noguchi et al 2009) This latter feature has been
considered an important trait of allelopathic interference
The term ldquoallelopathyrdquo was coined by Molisch in 1937 as a chemical
reciprocal interaction established among plants (including micro-
organisms) sharing the same site by means of the release of secondary
metabolites named allelochemicals (Rice 1984) For the most part
these metabolites are derived from the shikimic acid or isoprenoid
pathway and their biosynthesis can be modulated by biotic and abiotic
stresses (Nascimento and Fett-Neto 2010) including seasonal-related
changes (Sartor et al 2013) Allelopathy studies may range from sterile
assays (Aryakia et al 2015) to soil (Correcirca et al 2008 Sharma et al
2016) and field tests being a complex biological phenomenon to as-
certain in several circumstances due to issues of solubility release
mechanisms and stability of bioactive compounds (Scognamiglio et al
2013) Often the use of complementary methods provides more in-
formative data
The allelopathic effects of soil leachates green needles and litter
extracts of Pinus spp on germination and seedling growth aspects of
wild and crop species have been evaluated in natural and cultivated
pine stands and have proven to be stimulatory or inhibitory (Lodhi and
Killingbeck 1982 Kil and Yim 1983 Nektarios et al 2005 Akkaya
et al 2006 Machado 2007 Alrababah et al 2009 Sartor et al 2009
Kato-Noguchi et al 2011 Rolon et al 2011 Valera-Burgos et al
2012) exhibiting in some cases autotoxicity (Garnett et al 2004
Fernandez et al 2008 Zhu et al 2009 Monnier et al 2011) Studies
on potential dual allelopathic effects of Pinus elliottii Engelm (slash
httpdxdoiorg101016jindcrop201706019
Received 23 March 2017 Received in revised form 15 May 2017 Accepted 7 June 2017
Corresponding author
E-mail address fettnetocbiotufrgsbr (AG Fett-Neto)
ORIGINAL RESEARCHpublished 16 June 2016
doi 103389fpls201600849
Frontiers in Plant Science | wwwfrontiersinorg 1 June 2016 | Volume 7 | Article 849
Edited by
Juan Francisco Jimenez Bremont
Instituto Potosino de Investigacioacuten
Cientiacutefica y Tecnoloacutegica Mexico
Reviewed by
Mariacutea De La Luz Guerrero Gonzaacutelez
Universidad Autoacutenoma de San Luis
Potosiacute Mexico
Rosalia Cristina Paz
CIGEOBIO (CONICETFCEFN UNSJ)
Argentina
Correspondence
Arthur G Fett-Neto
fettnetocbiotufrgsbr
daggerThese authors have contributed
equally to this work
Specialty section
This article was submitted to
Plant Physiology
a section of the journal
Frontiers in Plant Science
Received 08 December 2015
Accepted 30 May 2016
Published 16 June 2016
Citation
de Lima JC de Costa F Fuumlller TN
Rodrigues-Correcirca KCdS Kerber MR
Lima MS Fett JP and Fett-Neto AG
(2016) Reference Genes for qPCR
Analysis in Resin-Tapped Adult Slash
Pine As a Tool to Address the
Molecular Basis of Commercial
Resinosis Front Plant Sci 7849
doi 103389fpls201600849
Reference Genes for qPCR Analysisin Resin-Tapped Adult Slash Pine Asa Tool to Address the MolecularBasis of Commercial Resinosis
Juacutelio C de Lima 1dagger Fernanda de Costa 1 dagger Thanise N Fuumlller 1
Kelly C da Silva Rodrigues-Correcirca 2 Magnus R Kerber 1 Mariano S Lima 1
Janette P Fett 1 and Arthur G Fett-Neto 1
1 Plant Physiology Laboratory Center for Biotechnology and Department of Botany Federal University of Rio Grande do Sul
Porto Alegre Brazil 2 Biological Sciences Department Regional Integrated University of Alto Uruguai and Missotildees (URI-FW)
Frederico Westphalen Brazil
Pine oleoresin is a major source of terpenes consisting of turpentine (mono- and
sesquiterpenes) and rosin (diterpenes) fractions Higher oleoresin yields are of economic
interest since oleoresin derivatives make up a valuable source of materials for chemical
industries Oleoresin can be extracted from living trees often by the bark streak method
in which bark removal is done periodically followed by application of stimulant paste
containing sulfuric acid and other chemicals on the freshly wounded exposed surface
To better understand the molecular basis of chemically-stimulated and wound induced
oleoresin production we evaluated the stability of 11 putative reference genes for the
purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis
Samples for RNA extraction were collected from field-grown adult trees under tapping
operations using stimulant pastes with different compositions and at various time points
after paste application Statistical methods established by geNorm NormFinder and
BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3
and UBI To confirm expression stability of the candidate reference genes expression
profiles of putative P elliottii orthologs of resin biosynthesis-related genes encoding Pinus
contorta β-pinene synthase [PcTPS-(minus)β-pin1] P contorta levopimaradieneabietadiene
synthase (PcLAS1) Pinus taeda α-pinene synthase [PtTPS-(+)αpin] and P taeda
α-farnesene synthase (PtαFS) were examined following stimulant paste application
Increased oleoresin yields observed in stimulated treatments using phytohormone-based
pastes were consistent with higher expression of pinene synthases Overall the
expression of all genes examined matched the expected profiles of oleoresin-related
transcript changes reported for previously examined conifers
Keywords resin Pinus gene expression normalizer genes terpene synthase
19
Chapter 2
Stimulant Paste Preparation and Bark Streak Tapping Technique for Pine Oleoresin Extraction
Thanise Nogueira Fuumlller Juacutelio Ceacutesar de Lima Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
Tapping technique comprises the extraction of pine oleoresin a non-wood forest product consisting of a
complex mixture of mono sesqui and diterpenes biosynthesized and exuded as a defense response to
wounding Oleoresin is used to produce gum rosin turpentine and their multiple derivatives Oleoresin
yield and quality are objects of interest in pine tree biotechnology both in terms of environmental and
genetic control Monitoring these parameters in individual trees grown in the fi eld provides a means to
examine the control of terpene production in resin canals as well as the identifi cation of genetic-based
differences in resinosis A typical method of tapping involves the removal of bark and application of a
chemical stimulant on the wounded area Here we describe the methods for preparing the resin-stimulant
paste with different adjuvants as well as the bark streaking process in adult pine trees
Key words Oleoresin Pine Tapping Chemical stimulant Wounding
1 Introduction
Several conifer species produce oleoresin a complex mixture of isoprenoid compounds relevant for defense against herbivores and pathogens Two major fractions can be recognized in oleoresin (a) turpentine the volatile fraction containing mono- and sesquiter-penes and (b) rosin the nonvolatile diterpene fraction Oleoresin is a forest commodity of global interest fi nding applications in diverse industry sectors Rosin is used in adhesives printing ink manufacture and paper sizing Turpentine can be used either as a solvent for paints and varnishes or as a raw material for fraction-ation of high-value chemicals used in the pharmaceutical agro-chemical and food industry [ 1 ndash 3 ]
During the extraction activity resin is obtained from the tree in a similar way as rubber tree tapping which generally involves the
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_2 copy Springer Science+Business Media New York 2016
These authors have equally contributed to this work
fettnetocbiotufrgsbr
27
Chapter 3
A Modifi ed Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines
Juacutelio Ceacutesar de Lima Thanise Nogueira Fuumlller Fernanda de Costa Kelly C S Rodrigues-Correcirca and Arthur G Fett-Neto
Abstract
RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes
through high-throughput sequencing technologies microarray and also northern blots RT-PCR and
RTqPCR Even though many specifi c protocols designed for plants with high content of secondary metab-
olites have been developed these are often expensive time consuming and not suitable for a wide range
of tissues Here we present a modifi cation of the method previously described using the commercially
available Concerttrade Plant RNA Reagent (Invitrogen) buffer for fi eld-grown adult pine trees with high
oleoresin content
Key words RNA Pines Concert plant RNA reagent Stem RNA extraction Oleoresin Conifers
1 Introduction
Several conifer species especially within the Pinaceae have tissues with high concentrations of phenolics terpenes and polysaccha-rides [ 1 ] Many specifi c protocols that are appropriate for plants rich in secondary metabolite s have been developed but the extrac-tion of high-quality RNA from these tissues using commercial kits is often diffi cult and usually not applicable to woody tissues [ 2 ndash 6 ] One of the major issues during RNA extraction concerns the pres-ence of phenolic compounds which oxidize and form quinones Aromatic compounds bind RNA which interferes in downstream steps and applications [ 3 7 ] Another point of concern is the har-vest of plant samples in the experimental fi eld which constitutes another obstacle in the efforts to avoid degradation of RNA [ 8 ] These problems often result in RNAs of low quality and insuffi -cient amounts especially for methodologies that normally require
These authors have equally contributed to this work
Arthur Germano Fett-Neto (ed) Biotechnology of Plant Secondary Metabolism Methods and Protocols Methods in Molecular Biology vol 1405 DOI 101007978-1-4939-3393-8_3 copy Springer Science+Business Media New York 2016
fettnetocbiotufrgsbr
RESEARCH PAPER
Control of resin production in Araucaria angustifolia an ancientSouth American coniferJ C Perotti1 K C da Silva Rodrigues-Correa123 amp A G Fett-Neto12
1 Plant Physiology Laboratory Department of Botany Federal University of Rio Grande do Sul (UFRGS) Porto Alegre RS Brazil
2 Center for Biotechnology UFRGS Porto Alegre RS Brazil
3 Present address Regional Integrated University of Alto Uruguai and Miss~oes (URI-FW) Frederico Westphalen RS Brazil
Keywords
Araucaria ethylene jasmonic acid nitric
oxide resin salicylic acid terpenes
Correspondence
A G Fett-Neto Plant Physiology Laboratory
Center for Biotechnology Federal University
of Rio Grande do Sul (UFRGS) PO Box 15005
Av Bento Goncalves 9500 91501-970 Porto
Alegre Brazil
E-mail fettnetocbiotufrgsbr
Editor
K Leiss
Received 22 July 2014 Accepted 11
December 2014
doi101111plb12298
ABSTRACT
Araucaria angustifolia is an ancient slow-growing conifer that characterises parts ofthe Southern Atlantic Forest biome currently listed as a critically endangered speciesThe species also produces bark resin although the factors controlling its resinosis arelargely unknown To better understand this defence-related process we examined theresin exudation response of A angustifolia upon treatment with well-known chemicalstimulators used in fast-growing conifers producing both bark and wood resin suchas Pinus elliottii The initial hypothesis was that A angustifolia would display signifi-cant differences in the regulation of resinosis The effect of Ethrel (ET ndash ethylene pre-cursor) salicylic acid (SA) jasmonic acid (JA) sulphuric acid (SuA) and sodiumnitroprusside (SNP ndash nitric oxide donor) on resin yield and composition in youngplants of A angustifolia was examined In at least one of the concentrations testedand frequently in more than one an aqueous glycerol solution applied on fresh woundsites of the stem with one or more of the adjuvants examined promoted an increase inresin yield as well as monoterpene concentration (a-pinene b-pinene camphene andlimonene) Higher yields and longer exudation periods were observed with JA and ETanother feature shared with Pinus resinosis The results suggest that resinosis controlis similar in Araucaria and Pinus In addition A angustifolia resin may be a relevantsource of valuable terpene chemicals whose production may be increased by usingstimulating pastes containing the identified adjuvants
INTRODUCTION
Many conifer species produce terpenoid-based resins that havelong been studied for their industrial importance and role indefence against attack by herbivores and pathogens The twomost important resin-producing families of conifers are Pina-ceae and Araucariaceae (Langenheim 1996) The viscous resinsecretion is generally composed of a complex mixture ofterpenoids consisting of roughly equal parts of volatile mono-(C10) and sesquiterpene (C15 turpentine) fractions and non-volatile diterpenic (C20 rosin) components (Rodrigues-Correaet al 2013) Terpenes act in a complex and multilayereddefence response providing toxicity against bark beetles andfungi bark wound sealing disruption of insect developmentand attraction of herbivore predators (Phillips amp Croteau1999)Most conifers rely on some combination of preformed and
inducible resin defences (Trapp amp Croteau 2001 Zulak amp Bohl-mann 2010) Resin defences are controlled by environmentaland genetic factors to various extents depending on species(Roberds et al 2003 Sampedro et al 2010 Moreira et al2013) Resin traits have been reported as highly variable havingmoderate heritability indicating that breeding efforts towardssuper-resinous forests are promising (Tadasse et al 2001Roberds et al 2003) Several chemicals are known as stimulantsof resin production Commercial extraction of resin from pine
trees uses periodic bark streaking and application of resin stim-ulant pastes to the wound
Resin-stimulant paste based on sulphuric acid (SuA) iswidely used for the commercial production of pine resin Cur-rent stimulant pastes usually have two chemically active com-ponents SuA to magnify the wounding and an ethyleneprecursor (2-chloroethylphosphonic acid CEPA or Ethrel ndash
ET) to stimulate resin flow (Rodrigues et al 2011 Rodrigues-Correa amp Fett-Neto 2013) Jasmonic acid (JA) and its methylester methyl jasmonate (MeJa) are among the most widelyused chemical elicitors of plant secondary metabolism It hasbeen shown that the exogenous application of MeJa or herbi-vore attack induce chemical and anatomical defence responsesin conifers such as the formation of traumatic resin ducts andresin accumulation in stems along with increased biosynthesisof terpenes and phenolics (Franceschi et al 2002 Martin et al2002 Heijari et al 2005 Zeneli et al 2006 Moreira et al 2008Gould et al 2009) JA commercial use however is limited byits high cost
The effects of exogenous salicylic acid (SA) on conifer ter-pene production have also been studied In Pinus elliottiiapplication of 10 molm3 of SA induced resin productionin wound panels but in Pseudotsuga menziesii and Sequoia-dendron giganteum it had no apparent effect on resinaccumulation (Hudgins amp Franceschi 2004 Rodrigues ampFett-Neto 2009) Nitric oxide (NO) has also emerged as an
Plant Biology 17 (2015) 852ndash859 copy 2014 German Botanical Society and The Royal Botanical Society of the Netherlands852
Plant Biology ISSN 1435-8603
