strigolactones: host recognition signals for arbuscular ... ppt/hideo... · strigolactones: host...
TRANSCRIPT
Strigolactones: host recognition signalsStrigolactones: host recognition signalsStrigolactones: host recognition signalsStrigolactones: host recognition signals
for arbuscular mycorrhizal fungifor arbuscular mycorrhizal fungifor arbuscular mycorrhizal fungifor arbuscular mycorrhizal fungi
Hideo Hayashi and Kohki Akiyama
Graduate School of Life and EnvironmentalSciences
Osaka Prefecture University
ISC 2008, Padjadjaran University, 31 October
Insecticidal compounds from Indonesian plants
O O
HO
HOH
OHO
O
O
Kalanchoe pinnata Kalanchoe daigremontiana x tubiflora
Bryophyllin C Methyl daigremonate
1) Unang Supratman, T. Fujita, K. Akiyama, H. Hayashi, Biosci. Biotechnol. Biochem., 64646464, 1310-1312 (2000).2) Unang Supratman, T. Fujita, K. Akiyama, H. Hayashi, Phytochemicstry, 58585858, 311-314 (2001).
New anti-brine shrimp compounds from Indonesian mushroom
HN
H
O
H
OCH3
OH
H
OO
OH
Microporellus subsessilis
10-phenyl-[11]-cytochalasans
Dikdik Kurnia, K. Akiyama, H. Hayashi, Phytochemistry, 68686868, 697-702 (2007).
OP053 by Dr. Dikdik
Host recognition signalsHost recognition signalsHost recognition signalsHost recognition signals
for arbuscular mycorrhizal for arbuscular mycorrhizal for arbuscular mycorrhizal for arbuscular mycorrhizal for arbuscular mycorrhizal for arbuscular mycorrhizal for arbuscular mycorrhizal for arbuscular mycorrhizal
fungifungifungifungi
Arbuscular mycorrhizal (AM) fungi
・belong to the phylum Glomeromycota.
・ colonize the roots of 80% of terrestrial plants.
・ existed 400 million years ago (early Denovian)1)・ existed 400 million years ago (early Denovian)1)
460 million years ago (Ordovician)2)
when plants first colonized land.
・ obligate symbionts; the fungi have not been cultured in the absence of a plant host.
1) W. Remy, T.N. Taylor, H. Hass, and H. Kerp, Proc. Natl. Acad. Sci. USA 91919191, 11841-11843 (1994).2) D. Redecker, R. Kodner, and L.E. Graham, Science 289289289289, 1920-1921 (2000).
Life cycle of arbuscular mycorrhizal fungi
SugarSugarSugarSugarHyphaHyphaHyphaHypha
ArbusculeArbusculeArbusculeArbuscule
PiPiPiPi
HHHH2222OOOO
SugarSugarSugarSugar
SporeSporeSporeSpore
External hyphaExternal hyphaExternal hyphaExternal hyphaPromotion of growthEnhancement of resistance against pathogens
Enhancement of drought tolerance
Increase of yield
Mycorrhizal structures in Lotus japonicus roots
Mycorrhizal rootNon-mycorrhizal root
stained with trypan blue
Hyphal branching: host recognition response of AM fungi
host plant roothost plant roothost plant roothost plant root
branchingbranchingbranchingbranchingfactorfactorfactorfactor(BF)(BF)(BF)(BF)
host plant roothost plant roothost plant roothost plant root
Germ tubeGerm tubeGerm tubeGerm tube
nonnonnonnon----host plant roothost plant roothost plant roothost plant rootChenopodiaceaeChenopodiaceaeChenopodiaceaeChenopodiaceaeBrassicaceaeBrassicaceaeBrassicaceaeBrassicaceae
Plant root exudates induce hyphal branching in vitro
Microinjected test samplesMicroinjected test samplesMicroinjected test samplesMicroinjected test samples
wellwellwellwell
2% CO2% CO2% CO2% CO2222, 32 ºC, 32 ºC, 32 ºC, 32 ºC
Spore ofSpore ofSpore ofSpore ofG. giganteaG. giganteaG. giganteaG. giganteagerminated on germinated on germinated on germinated on gelled M medium gelled M medium gelled M medium gelled M medium
---- exudatesexudatesexudatesexudates + exudates+ exudates+ exudates+ exudates
“Hyphal branching” bioassay developed by Nagahashi and Douds “Hyphal branching” bioassay developed by Nagahashi and Douds “Hyphal branching” bioassay developed by Nagahashi and Douds “Hyphal branching” bioassay developed by Nagahashi and Douds
2% CO2% CO2% CO2% CO2222, 32 ºC, 32 ºC, 32 ºC, 32 ºC
24 hours24 hours24 hours24 hours
G. Nagahashi and D.D. Douds, Jr, Mycol. Res. 104104104104, 1453-1464 (2000).
0.1% NaClO0.1% NaClO0.1% NaClO0.1% NaClO10 min10 min10 min10 min
SporesSporesSporesSpores
Gigaspora margarita Gigaspora margarita Gigaspora margarita Gigaspora margarita CGC1411CGC1411CGC1411CGC1411
(Central Glass Co., Ltd. Japan)(Central Glass Co., Ltd. Japan)(Central Glass Co., Ltd. Japan)(Central Glass Co., Ltd. Japan)
32323232℃℃℃℃, 2% CO, 2% CO, 2% CO, 2% CO2 2 2 2
5555----7 days7 days7 days7 days
Paper disc bioassay for hyphal branching in Gigaspora margarita
0.2% Phytagel plate0.2% Phytagel plate0.2% Phytagel plate0.2% Phytagel plate
(containing 3 mM MgSO(containing 3 mM MgSO(containing 3 mM MgSO(containing 3 mM MgSO4444) ) ) )
32 32 32 32 ℃℃℃℃, 2% CO, 2% CO, 2% CO, 2% CO2222
24 hrs24 hrs24 hrs24 hrs
Test samples Test samples Test samples Test samples in 70% EtOHin 70% EtOHin 70% EtOHin 70% EtOH----HHHH2222OOOO
paper discpaper discpaper discpaper discf 6 mmf 6 mmf 6 mmf 6 mm
Hyphal branching
Branching factor from root exudates ofLotus japonicus
- grown in liquid M medium (pH 6.8)under low Pi condition (35 µM)(24 ℃ 16h light / 22 ℃ 8h dark)
Lotus japonicus B-129 Gifu
Hydroponic solutionHydroponic solution- adjusted to pH 2 with HCl- extracted with EtOAc
EtOAc phase Aqueous phase- partitioned with 0.2M K2HPO4 (pH 9)
EtOAc-soluble Aqueous phaseneutral extract
2222°°°°
2°°°°
3333°°°°
Hyphal branching induced by ethyl acetate extracts from root exudates of L. japonicus
Control Control Control Control (70% EtOH(70% EtOH(70% EtOH(70% EtOH----HHHH2222O)O)O)O)
1111°°°° (germ tube)(germ tube)(germ tube)(germ tube)
EtOAc EtOAc EtOAc EtOAc extract extract extract extract
15 15 15 15 µµµµg/discg/discg/discg/disc
2°°°°4444°°°°
5555°°°°
EtOAc EtOAc EtOAc EtOAc ----solublesolublesolublesolubleneutral fraction neutral fraction neutral fraction neutral fraction
16 ng/disc16 ng/disc16 ng/disc16 ng/disc(activated charcoal (activated charcoal (activated charcoal (activated charcoal
method)method)method)method)
Solid phase extraction of BF from hydroponic solution of L. japonicus
Activated charcoalActivated charcoalActivated charcoalActivated charcoalL. japonicusL. japonicusL. japonicusL. japonicus BBBB----129 Gifu129 Gifu129 Gifu129 Gifugrown hydroponicallygrown hydroponicallygrown hydroponicallygrown hydroponicallyin M medium (Pi 35 mM)in M medium (Pi 35 mM)in M medium (Pi 35 mM)in M medium (Pi 35 mM)
Isolation procedure for BFfrom root exudates of L. japonicus
Charcoal cartridge (hydroponic solution 20 L)
- placed in aquarium water pump for 3 days- eluted with acetone
Neutral EtOAc extract (4 mg)Neutral EtOAc extract (4 mg)
- chromatographed on Wakogel C-200(hexane- EtOAc)
40% EtOAc eluate
- chromatographed on Inertsil ODS-3(40% MeCN in water to 100% MeCN)
Active fraction with Rt 22.3 min (ca. 18 µg)
Purification of BF by HPLCAbsorbance at 236 nm (mV)
1000100010001000
500500500500
Rt 22.3 min
Column: Inertsil ODS-3 (φ 10 x 250 mm)Eluent: 40-100% MeCN-H2O, 30min gradientFlow rate: 3.8 ml/min
Absorbance at 236 nm (mV)
500500500500
0000
Retention time (min)
0 5 10 15 20 25 300 5 10 15 20 25 300 5 10 15 20 25 300 5 10 15 20 25 30
97979797
UVUVUVUV (MeCN) (MeCN) (MeCN) (MeCN) λλλλmaxmaxmaxmax 234 nm234 nm234 nm234 nmIRIRIRIR (KBr) (KBr) (KBr) (KBr) ννννmaxmaxmaxmax 1784, 1744, 1683, 1340, 1024 cm1784, 1744, 1683, 1340, 1024 cm1784, 1744, 1683, 1340, 1024 cm1784, 1744, 1683, 1340, 1024 cm----1111
1111HHHH----NMRNMRNMRNMR (400 MHz, CDCl(400 MHz, CDCl(400 MHz, CDCl(400 MHz, CDCl3333) ) ) ) δδδδ 1.08 (3H,s, H1.08 (3H,s, H1.08 (3H,s, H1.08 (3H,s, H----9 or H9 or H9 or H9 or H----10), 10), 10), 10), 1.10 (3H, s, H1.10 (3H, s, H1.10 (3H, s, H1.10 (3H, s, H----9 or H9 or H9 or H9 or H----10), 2.01 (3H, t, J=1.5 Hz, H10), 2.01 (3H, t, J=1.5 Hz, H10), 2.01 (3H, t, J=1.5 Hz, H10), 2.01 (3H, t, J=1.5 Hz, H----7'), 7'), 7'), 7'), 5.49 (1H, br.d. J=8.0 Hz, H5.49 (1H, br.d. J=8.0 Hz, H5.49 (1H, br.d. J=8.0 Hz, H5.49 (1H, br.d. J=8.0 Hz, H----8b), 6.12 (1H, m, H8b), 6.12 (1H, m, H8b), 6.12 (1H, m, H8b), 6.12 (1H, m, H----2'), 2'), 2'), 2'), 6.90 (1H, m, H6.90 (1H, m, H6.90 (1H, m, H6.90 (1H, m, H----3'), 7.39 (1H, d, J=2.7 Hz, H3'), 7.39 (1H, d, J=2.7 Hz, H3'), 7.39 (1H, d, J=2.7 Hz, H3'), 7.39 (1H, d, J=2.7 Hz, H----6')6')6')6')CDCDCDCD (MeCN) (MeCN) (MeCN) (MeCN) λλλλmaxmaxmaxmax ((((∆ε∆ε∆ε∆ε) 262 () 262 () 262 () 262 (----2.86), 230 (21.2) nm 2.86), 230 (21.2) nm 2.86), 230 (21.2) nm 2.86), 230 (21.2) nm
OO
O OO
H
97979797
EIEIEIEI----MSMSMSMS
Spectroscopic analysis of BF
187187187187 215215215215233233233233
330 [M]330 [M]330 [M]330 [M]++++
StrigolactonesStrigolactonesStrigolactonesStrigolactonesseed germination stimulants for the parasiticseed germination stimulants for the parasiticseed germination stimulants for the parasiticseed germination stimulants for the parasiticweeds weeds weeds weeds StrigaStrigaStrigaStriga and and and and OrobancheOrobancheOrobancheOrobanche
5555----DeoxystrigolDeoxystrigolDeoxystrigolDeoxystrigol
Synthesis of 5-deoxystrigol
O O CNCl
O
O O
COOCH3
1) NaNH2, THF
2) CH3I
1) TMSCN, ZnI2
2) POCl3, Py
1) 85% H3PO4
2) SOCl2
SnCl , CH Cl
Si(CH3)31) (CH3O)3CO, NaH, DMF
2) BrCH2COOCH3 6N HCl
O
COOCH3
COOCH3
3SnCl4, CH2Cl2 2) BrCH2COOCH3
K. Frischmuth, E. Samson, A. Kranz, P. Welzel, H. Meuer, and W.S. Sheldrick, Tetrahedron, 47474747, 9793-9806 (1991).
6N HClAcOH
NaBH4, CeCl3, MeOH
OO O
O
OHHCOOEt, KOtBuEt2O/DMF
O O
OO
O
O OBr
K2CO3
N-methylpyrrolidone
Racemic 5-deoxystirigol
Separation by HPLC
Natural (ca. 80 µµµµg)
6' 3' 2'8b 3a
9,10
4a 4b
7'
OO
O OO
H3a
8b
2'
45
9 10
7'3'
6'3 pg/disc
1H-NMR spectra of natural and synthetic 5-deoxystrigol
PPM
7 6 5 4 3 2 1
PPM
7 6 5 4 3 2 1
400 MHz, CDCl3
Synthetic (ca. 3 mg)
17 6 5 4 3 2
6' 3' 2'8b 3a
9,10
4a 4b
7'
ppm
<30 pg/disc
a: natural 5-deoxystrigol(30 pg/disc)
b: synthetic (±±±±)-5-deoxystrigol (3 ng/disc)
Branching activity of strigolactones
c: synthetic (±±±±)-sorgolactone(10 ng/disc)
d: (+)-strigol(3 ng/disc)
Branching factor : 5-Deoxystrigol
O O
O O OH
O O O
K. Akiyama, K. Matsuzaki, & H. Hayashi, Nature, 435, 824-827 (2005).
Exudated from roots of Lotus japonicus
Hyphal branching-inducing activity of known natural strigolactones and
a synthetic analogue GR24
O O O O O O
O
Strigol Sorgolactone GR24Orobanchol(natural) (synthetic, racemic) (synthetic, racemic) (4 isomers)
O O
OH
O O O O O O OOH
<1 ng/disc <30 pg/disc <300 pg/disc 1 ng/disc
Synthetic strigolactone analogues GR24 and GR7 also induce
hyphal branching in Gigaspora rosea
GR24(4 diastereomers)
1 ng/disc
GR70.1-10 ng/disc*
2222'-epi-GR71 mg/disc*
*weak induction of 3º hyphae
G. Bécard et al., WO 2005/077177 A2 (Aug. 25, 2005).
Life cycle of Striga spp.
Germination
ConditioningDormancy
(After-ripening)
Germinationsignal
Initiation of
development Attachment andpenetration
Host
Germination
Haustoriaformation
Parasitism
ParasiteHaustorial
inducing factor
Kindly provided by Prof. Sugimoto
Natural strigolactonesO O
O O
OH
O
O O
O O
OAc
O
O O O O O O
5-Deoxystrigol Strigol Strigyl acetate
O O O O O OOH
O O OOAc
Solgolactone Orobanchol Alectrol
2’-Epiorobanchol Solanacol
O O
O O O
HO
Sorgomol
Distribution of strigolactones in the plant kingdom
Monocots
Gramineaerice plantsorghumcornmilletpearl millet
FabaceaeLotus japonicusred cloverpeanutChinese milk vetchchick peasoybean
Solanaceaeeggplanttomatotobacco
CompositaeCompositaeCompositaeCompositaeburdocksafflower
BrassicaceaeArabidopsis thaliana
Chenopodiaceaspinach
pearl millet
Dicots
Malvaceaecotton
MenispermaceaeMenispermumdauricum
Umbelliferaecarrot
soybeanalfalfacommon beangarden peawinged beancrimson clover broad beanadzuki beancowpeawhite lupin
Cucurbitaceaecucumber?
safflowerChrysanthemum coronarium
Cosmos bipinnatus
sunflowerlettusmarigold
Linaceaeflax
Gymnospermae
PinaceaJapanese red pine
Non-host plants in red letters
Branching factors = Strigolactones
O O
O O OH
AM fungi
(460 million years ago)
Obligate symbiontsRoot parasitic weeds
Obligate parasites
Pi
K. Akiyama & H. Hayashi, Annals of Botany, 97, 925-931 (2006).
Strigolactones Strigolactones
Acknowledgments
Mr. Ken-ichi Matsuzaki (Osaka Prefecture University)Professor Masayoshi Kawaguchi (Tokyo University)Professor Yukihiro Sugimoto (Kobe University)Professor Koichi Yoneyama (Utsunomiya University)
Financial support
Grand-in-Aid for Scientific Research from MEXTCore Research for Evolutional Science and
Technology (CREST) Sumitomo FoundationOsaka Prefecture University