role du silicium sur la tolerance au cuivre et la croissance des

AIXMARSEILLEUNIVERSITE

N°bibliothèque:XXX

ROLE DU SILICIUM SUR LA TOLERANCE AU CUIVRE

ET LA CROISSANCE DES BAMBOUS

THESE

pourobtenirlegradede

DOCTEURDEL’UNIVERSITEPAULCEZANNE

FacultédesSciencesetTechniques

Discipline:Géosciencesdel’Environnement

Présentéeetsoutenuepubliquementpar

Blanche COLLIN

Le29novembre2011

JURY

M.Jean‐ClaudeDAVIDIAN Professeur,MontpellierSupAgro RapporteurMmeGéraldineSARRET ChargéedeRecherche,CNRS RapporteurMmeCamilleDUMAT Professeur,INP‐ENSAT ExaminateurMmeCatherineKELLER Professeur,UniversitéAix‐Marseille ExaminateurM.Jean‐DominiqueMEUNIER DirecteurdeRecherche,CNRS DirecteurdethèseM.EmmanuelDOELSCH ChargédeRecherche,CIRAD Co‐directeurdethèseMmeVeroniqueARFI DirectriceGénérale,Phytorem InvitéeM.FrédéricPANFILI ResponsableR&D,Phytorem Invité

ANNEE 2011

iii

TABLE DES MATIERES

TABLEDESMATIERES...................................................................................................................................................iii

LISTEDESTABLES.........................................................................................................................................................vii

LISTEDESFIGURES ........................................................................................................................................................ ix

INTRODUCTION ............................................................................................................................... 1

CHAPITRE1SYNTHESEBIBLIOGRAPHIQUE......................................................................... 9

1. LECUIVREDANSLESPLANTES.........................................................................................................................111.1. Lecuivre,oligoélément ........................................................................................................................................................ 111.2. Mécanismesdeprélèvement................................................................................................................................................ 131.2.1 Adsorptionducuivredansl’apoplasmeracinaire .......................................................................................141.2.2 Absorptionracinaire.................................................................................................................................................15

1.3. Homéostasieauseindelacellule ...................................................................................................................................... 151.4. Transportetcomplexation .................................................................................................................................................. 161.5. Distribution................................................................................................................................................................................. 191.6. Toxicité.......................................................................................................................................................................................... 191.6.1 Rhizotoxicité.................................................................................................................................................................201.6.2 Stressoxydatifetgénotoxicité..............................................................................................................................201.6.3 Phytotoxicitéauniveaudespartiesaériennes..............................................................................................21

1.7. MécanismedetoléranceàlatoxicitédeCu.................................................................................................................. 221.8. Utilisationdelaspectroscopied’absorptiondesrayonsXpourétudierlaspéciationdeCudanslesplantes ........................................................................................................................................................................................................ 24

2. LESILICIUM.............................................................................................................................................................322.1. TeneurenSidanslesplantesetdanslebambou ....................................................................................................... 322.2. Absorptionracinaire............................................................................................................................................................... 332.3. Transportetstockage ............................................................................................................................................................ 342.4. Fonctiondusiliciumdanslebambou .............................................................................................................................. 382.5. Métauxetsilicium .................................................................................................................................................................... 402.5.1 Modificationdesconditionsenvironnementales .........................................................................................402.5.2 Augmentationdelabiomasse...............................................................................................................................412.5.3 Diminutiondel’absorptiondesmétaux ...........................................................................................................412.5.4 Modificationdelarépartitiondesmétaux ......................................................................................................42

iv

2.5.5 Modificationdel’expressiondegènes ..............................................................................................................43

3. BILANDUCHAPITREI .........................................................................................................................................44

4. REFERENCESBIBLIOGRAPHIQUES..................................................................................................................45

CHAPITREIIDISTRIBUTIONANDVARIABILITYOFSILICON,COPPERANDZINCINDIFFERENTBAMBOOSPECIES ..................................................................................................57

1. INTRODUCTION.....................................................................................................................................................65

2. MATERIALSANDMETHODS ..............................................................................................................................672.1. Geographicalareaofthestudy,soildescriptionandsamplingprocedure..................................................... 672.2. Soilmaterialandanalysis .................................................................................................................................................... 682.3. Plantmaterialandanalysis................................................................................................................................................. 692.4. Statisticalanalyses .................................................................................................................................................................. 69

3. RESULTS...................................................................................................................................................................703.1. Soil .................................................................................................................................................................................................. 703.2. Betweenplantparts ................................................................................................................................................................ 703.3. Betweenplantspecies ............................................................................................................................................................ 71

4. DISCUSSION.............................................................................................................................................................754.1. PhytoavailabilityofSi,CuandZn ..................................................................................................................................... 754.2. OriginofSivariationinbamboos ..................................................................................................................................... 754.3. OriginofCuandZnvariabilityinbamboo.................................................................................................................... 784.4. InteractionsbetweenmetalsandSi ................................................................................................................................. 80

5. BILANDUCHAPITREII........................................................................................................................................81


CHAPITREIIIEFFECTSOFSILICONANDCOPPERONBAMBOOGROWNHYDROPONICALLY........................................................................................................................87

1 INTRODUCTION ......................................................................................................................................................95

2 MATERIALANDMETHODS .................................................................................................................................962.1 Plantmaterial,experimentaldesignandpreculture............................................................................................... 962.2 Experimentinnutrientsolution.......................................................................................................................................... 972.2.1 PhaseI:Sitreatment .................................................................................................................................................972.2.2 PhaseII:Cu+Sitreatment .....................................................................................................................................98

2.3 Plantanalysis .............................................................................................................................................................................. 992.3.1 Growthparameters ...................................................................................................................................................992.3.2 Analysisoftissues ...................................................................................................................................................100

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2.4 Statisticalanalyses ................................................................................................................................................................ 100

3 RESULTS................................................................................................................................................................. 1013.1 Evolutionoftheelementconcentrationsinthenutrientsolution.................................................................... 1013.2 Bamboogrowthparametersandelementaldistribution .................................................................................... 105

4 DISCUSSION........................................................................................................................................................... 1094.1 Siliconinbamboo................................................................................................................................................................... 1094.1.1 Mechanismsofsiliconaccumulation ..............................................................................................................1094.1.2 EffectofSiuptakeonthenutrientconcentrationandgrowthparameters ...................................111

4.2 Effectsofcopperonbamboogrowth............................................................................................................................. 1124.3 EffectsofbambooSionCusensitivity ........................................................................................................................... 115

5 BILANDUCHAPITREIII .................................................................................................................................... 117

6 REFERENCESBIBLIOGRAPHIQUES................................................................................................................ 118

CHAPITREIVCOPPERDISTRIBUTIONANDSPECIATIONINBAMBOOS“Phyllostachysfastuosa”EXPOSEDTODIFFERENTCOPPERANDSILICONCONCENTRATIONS..................................................................................................................... 125

1. INTRODUCTION.................................................................................................................................................. 133

2. MATERIALANDMETHODS ............................................................................................................................. 1342.1. Plantmaterial,experimentaldesignandpreculture........................................................................................... 1342.2. Siliconandcoppertreatment .......................................................................................................................................... 1352.3. Plantanalysis .......................................................................................................................................................................... 1362.3.1 Growthparameters ................................................................................................................................................1362.3.2 Analysisofmacroandmicronutrients...........................................................................................................136

2.4. Desorptionprocedure.......................................................................................................................................................... 1362.5. Anionassays ............................................................................................................................................................................ 1372.6. Totalsolubleaminoacid.................................................................................................................................................... 1382.7. SEMEDX ................................................................................................................................................................................... 1382.8. LaboratoryBasedµXRF ..................................................................................................................................................... 1382.9. EXAFSandXANES:dataacquisitionandanalysis.................................................................................................. 1392.10. Statisticalanalyses............................................................................................................................................................. 140

3. RESULTS................................................................................................................................................................ 1403.1. Siliconandcopperinnutrientsolution ....................................................................................................................... 1403.2. Growthparameters .............................................................................................................................................................. 1413.3. CuandSidistributionbetweenplantparts ............................................................................................................... 1423.4. CuandSilocalizationintheroot ................................................................................................................................... 1443.5. Theeffectsofcopperandsilicononsomeplantcomponents ............................................................................ 1453.6. XANESfeatures....................................................................................................................................................................... 149

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3.7. EXAFSfeatures ....................................................................................................................................................................... 151

4. DISCUSSION.......................................................................................................................................................... 156

5. SUPPORTINGINFORMATIONS....................................................................................................................... 162

6. BILANDUCHAPITREIV.................................................................................................................................... 170

7. REFERENCESBIBLIOGRAPHIQUES............................................................................................................... 171

CONCLUSIONSETPERSPECTIVES ......................................................................................... 178

RESUME.......................................................................................................................................................................... 185

ABSTRACT..................................................................................................................................................................... 185

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L ISTE DES TABLES

Table I.1 Synthèse des concentrations en Si publiées dans le bambou..............................33

Table I.2 Relation entre la teneur de SiO2 des feuilles de bambou et la productivité de la bambousaie (Ma et Takahashi, 2002)..........................................................39

Table II.1 Characteristics of the total soil population (N=18)............................................70

Table II.2 SiO2, Cu and Zn concentrations in the different plant parts (N=47). Data are expressed as mg kg-1 of dry matter for Cu and Zn concentrations and mg g-1 of dry matter for SiO2 concentrations. ............................................................71

Table II.3 Mean SiO2, Cu and Zn concentrations in different plant parts in each species. For each species, the sample number is 3. The type of bamboo is indicated: S: Sympodial bamboo species, M: Monopodial bamboo species. .........................74

Table II.4 Inventory of Si data found in the literature......................................................77

Table III.1 Ca, Mg, P, Fe and Zn concentrations in the nutrient solution with Si0 and Si1.5 treatments in the input and output solutions at three different time periods during the experiment.....................................................................102

Table III.2 Concentration of macro and micronutrients in the leaves, stems, and roots of the bamboo plants that were grown with different Si concentr ation in the nutrient solution. .......................................................................................107

Table III.3 Effect of different levels of Si nutrition on the following growth parameters: number of stems and leaves that developed during the experiment in phase I and phase II. ..........................................................................................107

Table IV.1 Cu and Si concentrations in plant parts of bamboos grown in the five treatments (n=4, mean ± standard deviation)...............................................144

Table IV.2 N, P, K, Ca, Mg, Fe, Mn and Zn concentrations in leaves and roots of bamboos from control, Cu100 and Cu100+Si treatments (n=4, mean ± standard deviation)....................................................................................147

Table IV.3 Composition of inorganic acids, carboxylic acids and amino acid in leaves, stems and roots of plant grown in treatments control, Cu100 and Cu100Si (mmoles kg dry weight-1, n=4 for inorganic acids and carboxylic acids, n=3 for amino acids). The standard deviation was not indicated for the lisiblity of the table. .................................................................................................148

Table IV.4 Proportion of Cu species determined by LCF of the Cu K-edge EXAFS spectra. The proportions were adjusted to reach 100 %..............................................155

Table IV.5 Reference compounds database...................................................................162

Table IV.6 Component output parameters determined by principal component analysis of the whole set of XANES spectra ...............................................................164

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Table IV.7 Results of target transform of XANES spectra (Chi square, R value and Spoil values) of the main Cu references................................................................165

Table IV.8 Proportion of Cu species determined by LCF of the Cu K-edge XANES spectra ....166

Table IV.9 Results of target transform of EXAFS spectra (Chi square, R value and Spoil values) of the main Cu references................................................................168

Table IV.10 Proportion of Cu species determined by LCF of the Cu K-edge EXAFS spectra.....168

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L ISTE DES F IGURES

Figure I.1 Courbe de croissance en réponse au statut nutritionnel de la plante, d’après Reuter and Robinson (1997) .........................................................................13

Figure I.2 Transfert radial des minéraux dans une racine, de la solution de sol au xylème (image issue du site internet biologie-forums.com) ...............................14

Figure I.3 Schéma récapitulant les différents transports de Cu identifiés dans une cellule générique (essentiellement dans Arabidopsis). Les protéines membranaires transporteurs de Cu sont représentées en orange, les Cu chaperonnes en violet. Abréviations : CCH, copper chaperone; ATX : antioxydant1 ; COPT, copper transporter; FRO, ferric reductase oxidase; HMA, heavy metal P-type ATPase; MT, metallothioneins; ZIP, IRT-like protein. Schéma modifié d’après (Yruela 2009)..............................................................................................16

Figure I.4 Structure chimique d’acides aminés, d’acides carboxyliques et de la phytochélatine et du glutathion .....................................................................18

Figure I.5 Distribution du cuivre sur une racine de niébé (cowpea) observée par µ-fluorescence X (Kopittke et al. 2011)..............................................................19

Figure I.6 Schéma présentant les caractéristiques du XANES et de l’EXAFS ........................25

Figure I.7 Spectre XANES au seuil K du Cu des feuilles, tiges, racines de créosotier et du sol d’origine des plantes (Polette et al. 2000)..............................................26

Figure I.8 Spectres EXAFS normalisés et transformées de Fourier des échantillons de feuilles de Thalspi caerulescens de l’étude de Mijovilovich et al (2009). Les données sont représentées en traits pleins et les fits en pointillés.......................29

Figure I.9 Spectres EXAFS (A et C) et XANES (B et D) au seuil K du Cu des racines de niébé (cowpea) soumises à 1,5 µM Cu pendant 3h (en haut) ou 24h (en bas). Les spectres des composés de référence les plus importants (i.e. acide polygalacturonique ou cystéine) et les résultats des combinaisons linéaires sont également représentés. .........................................................................31

Figure I.10 Processus de formation de particules de silice par différentes réactions de polymérisation (dimères, oligomères et agrégats) de monomères de silicium (Currie and Perry 2007). ..............................................................................34

Figure I.11 Absorption et localisation de Si dans le bambou. Le mécanisme d’absorption est celui qui est décrit pour le blé et le maïs (Mitani et al. 2009a; Mitani et al. 2009b). La localisation de Si a été schématisée sur les photographies MEB de feuilles (B) et de racines (C) de bambou selon les analyses EDX des études de Motomura et al (2006) (B) et de Lux et al (2003) (C)...................................36

Figure I.12 Teneurs en SiO2 dans des feuilles de bambous pendant trois ans : 1998 (carrés), 1999 (cercles), 2000 (triangles) (Motomura, 2002) .............................38

Figure I.13 Résumé des fonctions du silicium (schéma provenant de Ma and Yamaji 2006) ........................................................................................................40

x

Figure II.0 Types de rhizomes, figure modifiée d’après (Soderstrom and Young 1983) ..........62

Figure II.1 Comparison of SiO2, Cu, Zn concentrations in stem bases, stem tips and leaves of monopodial bamboos (n= 18) and sympodial bamboos (n = 29). DM: dry matter; Boxes represent the median (vertical solid line) and 25–75 % percentile. Whiskers represent the 90th and 10th percentile. Significant differences were determined by a post hoc comparison of means (Tukey test after ANOVA; P < 0.05) and are indicated by different letters. ...........................73

Figure III.1 Concentrations of macronutrients (Ca, Mg and P) and micronutrients (Fe and Zn) in the Si0.4 treatment during phase II from days 196 to 201. ....................102

Figure III.2 Bamboo Si uptake throughout the experiment, which was calculated as the difference between the Si concentrations that were measured in the input and output solutions. “Theo. max” is the theoretical maximum uptake that could be obtained if the entire Si in solution is taken up by the plants...............103

Figure III.3 Bamboo Cu uptake throughout the experiment, which was calculated from the difference in the Cu concentrations in the input and output solutions. “Theo. max” is the theoretical maximum uptake that would be obtained if all of the Cu in the solution was taken up by the plants.......................................104

Figure III.4 Si and Cu concentrations in the nutrient solution during the Si0.4 treatment in phase II from days 196 to 201.................................................................105

Figure III.5 Si concentrations in different bamboo organs as a function of the Si concentration in the nutrient solution at 1.5 µM Cu. The error bars represent the standard deviation of the SiO2 concentrations in the 5 different bamboo plants. .....................................................................................................106

Figure III.6 Repartitioning of the Cu concentration in bamboo plants. ................................108

Figure IV.1 Si and Cu amount (µmol) in the nutrient solution in treatment Cu1.5Si and Cu100Si from day 27 to 34 .........................................................................140

Figure IV.2 Mean number of bamboo leaves in the 5 treatments, throughout the experiment from day 0 to day 70. Values followed by same letter for the same days are statistically not different according to Tukey test at the 95 % confidence level.........................................................................................141

Figure IV.3 Photographs of whole plants harvested after 70 days in treatments: (A) control; (B) Cu100; (C) Cu100 Si.................................................................142

Figure IV.4 Cu and Si concentration in leaves, stems, roots in the different treatments. Values followed by same letter between treatments are statistically not different according to Tukey test at the 95 % confidence level. ........................143

Figure IV.5 cryo-SEM image with two EDX spectra taken in the root epidermis cells (A) and in the endodermis cells (B) ...................................................................145

Figure IV.6 µXRF image of a root cross-section from treatment Cu100Si, tricolour image combining Si (red), K (blue) and Cu (green)..................................................145

Figure IV.7 (a) Normalized Cu K-edge XANES spectra of plant samples and (b) the corresponding first derivatives of Cu K-edge XANES spectra. ...........................150

Figure IV.8 The derivative of XANES spectra showing the evolution of the feature B in different part of the plant and the splitting of the 4p orbitals resulting from the Jahn-Teller distortion, in leaf Cu100, desorbed root Cu100 and root Cu100......................................................................................................150

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Figure IV.9 Proportions of Cu(I) and Cu(II) determined by LCF on XANES spectra. The proportions were adjusted to reach 100 %....................................................151

Figure IV.10 (a) Cu K-edge extended X-ray absorption fine structure spectra; (b) radial distribution function (RDF) for plant samples. k = photoelectron wave number (Å−1), R = distance to the neighboring atom (Å). ..............................152

Figure IV.11 Cu K-edge k3-weighted EXAFS (solid line) and linear combination fits (dotted line) for plant samples................................................................................153

Figure IV.12 Cu K-edge k3-weighted EXAFS spectra of leaf in treatment Cu100Si (solid line) and fits (dotted line) with and without the contribution of Cu2S compound (Cu(I)S-inorganic)......................................................................154

Figure IV.13 Proportions of Cu compounds determined by LCF on EXAFS spectra. The proportions were adjusted to reach 100 %....................................................155

Figure IV.14 XANES spectra and their first derivative of reference compounds .....................166

Figure IV.15 a) Component spectra of the principal component analysis, b) first to eight component output parameters determined by principal component analysis of a whole set of EXAFS spectra ......................................................................167

Figure IV.16 EXAFS spectra of reference compounds ........................................................169

Introductiongénérale

1

INTRODUCTION

Face à la croissance démographique, la gestion des déchets et des eaux usées représente un

enjeutechnologique,économiqueetscientifiquemajeurdesannéesàvenir,aussibiendansles

paysduNordquedansceuxduSud.Ilestindispensabledetravailleràl’élaborationdesolutions

detraitementquipermettentuneépurationoptimaledeseauxuséesavantleurréutilisationou

leurrejetdans lemilieunaturel.Afindepallier les limitesenvironnementales,économiqueset

sociétalesdesméthodesconventionnellesqui sontactuellementemployéespour le traitement

des sols pollués, telles que l’incinération et le confinement, les recherches s’orientent, depuis

unequinzained’années,versdenouvellesméthodesbiologiquesdetraitementdessolsbasées

surl’utilisationdesplantes.Cesméthodes,quiseregroupentsousletermedephytoremédiation,

apparaissent comme des voies alternatives, moins coûteuses, plus extensives et plus

respectueuses des sols et de l’environnement (Jabeen et al. 2009; Keller 2005; Pilon‐Smits

2005).

La société PHYTOREM® a développé une technologie d’épuration utilisant le procédé de

phytoremédiation sur sol en place : le BAMBOU ASSAINISSEMENT®. Cette technologie est

optimiséeparlechoixdubambou,planteligneusedelafamilledespoaceae,quipossèdel’undes

plus forts tauxde croissancedu règnevégétal.Une forteproductiondebiomasse, combinéeà

unefortecapacitéàabsorberdel’eauetdesélémentsminéraux,permetaubamboud’êtreune

plante adaptée aux techniques de phytoépuration (Arfi et al. 2009). Le BAMBOU

ASSAINISSEMENT® est une technologie rustique et simple dans sa mise en œuvre et son

exploitation.PHYTOREM®conçoitetgèredesstationsquiexploitentaumaximumcesprocessus

pourépurerplusieurs typesd'effluents :eauxusées,effluents industrielsetvinicoles, lixiviats,

eauxpluviales,etc.

LesactivitéshumainesliéesauxévolutionsindustriellesdesXIXeetXXesièclesontdéversédes

quantités importantes d'éléments traces métalliques (ETM) dans les compartiments liquides,

solides et gazeux de la terre, augmentant ainsi leur concentration dans lemilieu naturel. Ces

métauxsontnonbiodégradablesetontdonctendanceàs’accumulerdanslesdéchetsetleseaux


2

usées que nous produisons. Mais, jusqu’à présent, le comportement des bambous face à la

présencedemétauxn’apasétéévalué.Commel’undessymptômesdelatoxicitémétalliqueest

la réductionde la croissancedesplantes, il convientdedéterminer si l’efficienceduBAMBOU

ASSAINNISSEMENT®estaffectéeparlaprésencedesETMdansleseffluentsàtraiter.Ainsi,ilest

nécessaire de comprendre le comportement conjoint des ETM et du bambou: comme leur

capacité d’absorption, la localisation des éléments dans la plante et leur toxicité. Nous avons

choisi le cuivre (Cu) comme ETM car c’est l’un des contaminants les plus abondants dans

certains polluants qui peuvent être traités par le BAMBOU ASSAINNISSEMENT®, comme par

exemplele lisierdeporc(Legrosetal.2010).Lecuivreestégalementintéressantpuisqu’il fait

partiedesoligoéléments:ilestnécessairepourledéveloppementdelaplanteàfaibledosemais

induitunefortetoxicitéquandsaconcentrationcellulaireesttropélevée.

Lebamboupossèdelaparticularitéd’absorberdefortesquantitésdesilicium,jusqu’à40%de

matièresèchedeSiO2dans les feuilles.Orderécentesétudesmontrentque lesiliciumpermet

d’améliorer la croissanceet la résistancedesplantes soumisesàdes stress,notammentàune

toxicité métallique (da Cunha and do Nascimento 2009; Gu et al. 2011). Ainsi il est donc

intéressant,danslebutd’améliorerlatechnologiedephytoremédiationdubambou,d’étudierle

rôle que peut avoir sur lui le silicium, en particulier lorsque celui‐ci fait face à une toxicité

métallique.

Lesobjectifsdecetravaildethèsesontlessuivants:

1‐ Déterminerlerôledusiliciumdanslebambou.

– L’apportdesiliciumpermet‐ild’augmenterlabiomassedubambou?

– Les nombreuses espèces de bambous présentent‐elles des différences

d’accumulationsdeSi?

– L’amendement en silicium peut‐il être envisagé pour le BAMBOU

ASSAINNISSEMENT®?

2‐ DéterminerlecomportementdeCudanslebambou

– QuelleestlarépartitiondeCudanslesdifférentespartiesdubambou?

– A quelle concentration Cu devient toxique pour le bambou, quels sont les

symptômes?

– Est‐cequeCuauncomportementdifférentenfonctiondel’espècedebambou?

3‐ DéterminersilaprésencedeSiminimiselatoxicitédeCu.


3

CetteétudeapprofondiedesdifférentsprocessusimpliquésdanslatoxicitédeCuetlerôledeSi

dans le bambou a été réalisée à différentes échelles et en couplant plusieurs techniques

analytiques.

Toutd’abordlarépartitionetlavariabilitédeCuetSiontétéétudiéesdansplusieursespècesde

bambous sedéveloppantdansun contextepédoclimatiquenaturel. Commeaucunedonnéede

concentrationdeCuetZnn’existedanslalittérature,cetteapprocheadoncpermisd’établirdes

concentrationsderéférencesdeCuetZndanslesbambous.

Ensuite,desexpériencesen culturehydroponiqueontété réaliséesafinde contrôler finement

l’apport de Si et Cu aux bambous. Ces expériences ont permis de caractériser de manière

«macroscopique»laréponsedesbambousenfonctiondesapportsdeSietàCu.Parallèlement,

nousavonsétudiélaspéciationdeCuauseindesdifférentsorganesdubambou.Laspéciation

(spectroscopie d’absorption des rayons X) associée à la localisation des éléments

(microfluorescence X et microscopie électronique) sont deux caractérisations indispensables

pourmieuxappréhenderlecomportementdeCudanslebambou.

Cedocuments’articuleenquatrepartiesprincipales:

Danslechapitre1,seraprésentéunétatdel’artsurlecomportementdeCuetdeSidans

lesplantesetlebambouenparticulier.

Le chapitre 2 décrit et discute la variabilité et la localisation de Si, Cu et Zn dans

plusieursespècesdebambousdéveloppéesenconditionspédoclimatiquesnaturelles.

Lechapitre3présentel’effetdeSietCusurdesbambouscultivésenhydroponie.

Le chapitre 4 traite de l’effet d’une toxicité au Cu chez le bambou et le rôle de Si, en

culturehydroponique.Nousprésenteronsdanscechapitrel’étudedelaspéciationdeCu.

Ce projet de recherche a été mené dans le cadre d’une bourse CIFRE entre le CEREGE (Centre

Européen de Recherche et d’Enseignement des Géosciences de l’Environnement) et la société

PHYTOREMetd’uncontratdecollaborationderechercheentreleCEREGEetPHYTOREMsoutenu

parlaDirectionGénéraledesEntreprises.


4

REFERENCESBIBLIOGRAPHIQUES

ArfiV,BagoudouD,KorboulewskyNandBoisG2009Initialefficiencyofabamboogrove‐basedtreatmentsystemforwinerywastewater.Desalination246,69‐77.

daCunhaKPVanddoNascimentoCWA2009SiliconEffectsonMetalToleranceandStructural

ChangesinMaize(ZeamaysL.)GrownonaCadmiumandZincEnrichedSoil.WaterAirandSoilPollution197,323‐330.

GuHH,QiuH,TianT,ZhanSS,DengTHB,ChaneyRL,WangSZ,TangYT,MorelJLandQiuRL

2011Mitigationeffectsofsiliconrichamendmentsonheavymetalaccumulationinrice(Oryza sativa L.) planted on multi‐metal contaminated acidic soil. Chemosphere 83,1234‐1240.

Jabeen R, Ahmad A and Iqbal M 2009 Phytoremediation of Heavy Metals: Physiological and

MolecularMechanisms.TheBotanicalReview75,339‐364.Keller C 2005 Efficiency and Limitations of Phytoextraction byHigh Biomass Plants. In Trace

ElementsintheEnvironment.pp611‐630.CRCPress.LegrosS,ChaurandP,Rose J,MasionA,BrioisV,Ferrasse JH,SaintMacaryH,Bottero JYand

Doelsch E 2010 Investigation of Copper Speciation in Pig Slurry by a MultitechniqueApproach.EnvironmentalScience&Technology44,6926‐6932.

Pilon‐SmitsE2005Phytoremediation.AnnualReviewofPlantBiology56,15‐39.


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6

C H A P I T R E 1

SYNTHESE B IBL IOGRAPHIQUE

ChapitreI:Synthèsebibliographique

9

TABLE DES MATIERES

CHAPITRE1SYNTHESEBIBLIOGRAPHIQUE.......................................................................11

1. LECUIVREDANSLESPLANTES.........................................................................................................................111.1. Lecuivre,oligoélément ........................................................................................................................................................ 111.2. Mécanismesdeprélèvement................................................................................................................................................ 131.2.1 Adsorptionducuivredansl’apoplasmeracinaire .......................................................................................141.2.2 Absorptionracinaire.................................................................................................................................................15

1.3. Homéostasieauseindelacellule ...................................................................................................................................... 151.4. Transportetcomplexation .................................................................................................................................................. 161.5. Distribution................................................................................................................................................................................. 191.6. Toxicité.......................................................................................................................................................................................... 191.6.1 Rhizotoxicité.................................................................................................................................................................201.6.2 Stressoxydatifetgénotoxicité..............................................................................................................................201.6.3 Phytotoxicitéauniveaudespartiesaériennes..............................................................................................21

1.7. MécanismedetoléranceàlatoxicitédeCu.................................................................................................................. 221.8. Utilisationdelaspectroscopied’absorptiondesrayonsXpourétudierlaspéciationdeCudanslesplantes ........................................................................................................................................................................................................ 24

2. LESILICIUM.............................................................................................................................................................322.1. TeneurenSidanslesplantesetdanslebambou ....................................................................................................... 322.2. Absorptionracinaire............................................................................................................................................................... 332.3. Transportetstockage ............................................................................................................................................................ 342.4. Fonctiondusiliciumdanslebambou .............................................................................................................................. 382.5. Métauxetsilicium .................................................................................................................................................................... 402.5.1 Modificationdesconditionsenvironnementales .........................................................................................402.5.2 Augmentationdelabiomasse...............................................................................................................................412.5.3 Diminutiondel’absorptiondesmétaux ...........................................................................................................412.5.4 Modificationdelarépartitiondesmétaux ......................................................................................................422.5.5 Modificationdel’expressiondegènes ..............................................................................................................43

3. BILANDUCHAPITREI .........................................................................................................................................44



10


11

CHAPITRE 1

SYNTHESE B IBL IOGRAPHIQUE

Cette synthèse bibliographique est organisée en deux axes principaux dédiésau cuivre et au

siliciumdanslesplantes.

Commeaucunedonnéen’estencorepubliéesurCudanslebambou,lapremièrepartieprésente

les connaissances actuelles sur la présence de Cu dans les plantes: ses caractéristiques, ses

mécanismesdeprélèvement,detransportetsatoxicité.Lesapprochesetlesoutilsutiliséspour

caractériser le comportement de Cu dans les plantes sont nombreux et couvrent différentes

échelles:de l’observationmacroscopiquedeseffetsdeCuàdesétudesdegénomique.Comme

nousavonsutilisélaspectroscopied’absorptiondesrayonsXpourcaractériserlaspéciationdu

Cudanslesbambous,uneprésentationdesprincipauxrésultatsdespéciationdeCuacquisgrâce

àcetteméthodedanslesplantescomplètecettepremièrepartie.

Lasecondepartiedétaillelecomportementdusiliciumdanslesplantesetdanslebambou:ses

mécanismesdeprélèvement,detransport,sarépartitiondans lesdifférentstissusdubambou,

sesfonctionsdanslesplantesetsonrôlefaceàunetoxicitémétallique.

1. LE CUIVRE DANS LES PLANTES

1.1. Le cuivre, oligo-élément

Lecuivrefaitpartiedesmétauxdetransition,c’estunoligo‐élémentprésentenproportiontrès

faibledans lestissusbiologiquesmais indispensableà lavie.En1930,Cuestreconnuélément

trace essentiel pour les plantes (Alloway 1995). Certains éléments traces cationiques

plurivalentscommeCupeuventseprésentersousdifférentsétatsd'oxydation(Cu2++e‐↔Cu+)

etjouentainsiunrôled'accepteuroudedonneurd'électrons,cequiesttrèsimportantdansles

multiplessystèmesenzymatiquesmettanten jeudesréactionsd'oxydoréduction.Lecuivreest

également nécessaire à de nombreuses enzymes, comme activateur ou comme constituant

spécifique des protéines (Marschner 1995). Dans le cas d'une croissance optimale, les

concentrationsenCudanslesplantesatteignentdesvaleurscomprisesentre5et20mg.kg‐1de


12

matièresèche(MS)danslespartiesaériennes(Marschner1995).Endessousd’unseuilde2‐5

mg.kg‐1MSdanslaplante,sacroissanceestsévèrementréduiteetdessymptômesdedéficience

peuventsemanifester(FigureI.1).Ceseuilpeutvariersuivantlesespècesvégétalesetl’étatde

développementdelaplante.Al’inverse, lateneurenCumesuréedanslaplantepeutatteindre

une concentration critique à partir de laquelle apparaissent des symptômes de phytotoxicité

(Marschner1995).

Les concentrations demétaux libres ou chélates dans les solutions de sol sont généralement

faibles, même si cela dépend des propriétés du sol (Marschner 1995). Que ce soit dans la

solution ou dans la phase solide, Cu est majoritairement présent sous forme complexée. Les

minéraux secondaires, comme lesargileset lesoxyhydroxydesdeFeetdeMnet lesmatières

organiques,sontlesprincipauxconstituantsdusolquicontribuentàl'adsorptiondeséléments

traces(Kabata‐PendiasandPendias2001).Enquantitémoindre,Cupeutégalementêtreprésent

dans les réseaux cristallinsd’autresminéraux secondaires comme les carbonates, les sulfures,

lesphosphateset certainsoxydes (Dasetal.1995).LadistributiondeCuparmicesdifférents

composantsdusolinfluencesamobilitéet,ainsi,laquantitépotentiellementdisponiblepourles

plantes.LarétentiondeCuparlaphasesolidedusolestfortementdépendantedupH.Eneffet,

lorsque le pHdu sol augmente, la charge nette de surface des phases adsorbantes devient de

moinsenmoinspositive(oxyhydroxydesFeetAl)oudeplusenplusnégative(oxyhydroxydes

MnetMOS),cequiaccroîtleuraffinitépourlescationsmétalliques(Alloway1995).

Cependant le Cu biodisponible va également être influencé par les processus physiques,

chimiques et biologiques qui ont lieu à l’interface sol‐racine, dans la rhizosphère, tels qu’une

modificationdupHoudelaquantitédecarboneorganiquedissous(Hinsingeretal.2009).


13

Figure I.1 Courbe de croissance en réponse au statut nutritionnel de la plante, d’après Reuter and Robinson (1997)

1.2. Mécanismes de prélèvement

Le cuivre pénètre dans la plante par son système racinaire. Le transfert radial des ions

métalliques de la surface des racines jusqu’aux vaisseaux du xylème peut suivre deux voies

(FigureI.2):

‐ lavoieapoplasmiqueenpassantdanslesespacesintercellulaires(enrouge).Letransfert

des solutés de faible poids moléculaire (éléments traces complexés ou non, acides

organiques, acides aminés, sucres) de la solution du sol vers l'espace pariétal (parois

cellulaires et apoplasme ou espace intercellulaire) se fait passivement par diffusion

(Marschner,1995)avantd’êtrebloquéparlabandedeCasparydansl’endoderme.

‐ la voie symplasmique (en bleu) en traversant la membrane plasmique d’une cellule,

généralementdelacouchelaplusexterneducortex,puisenpassantdecelluleàcellule

parlesplasmodesmes(lesporesdelaparoicellulaire).Letransportdesmétauxdansle

symplasmeestprisenchargepardifférentesfamillesdetransporteurs.


14

Figure I.2 Transfert radial des minéraux dans une racine, de la solution de sol au xylème (image issue du site internet biologie-forums.com)

Lesparoiscellulairesdesracinessontlepremiercompartimentdelaplanteencontactavecles

métaux,etvont influencer le transfertdeCuauseinde l’apoplasmeetdusymplasme.Ainsi le

prélèvement des métaux intègre deux processus distincts: (i) l’adsorption des métaux dans

l’apoplasmeracinaireetàlasurfacedesmembranesplasmiqueset(ii)l’absorptiondesmétaux

autraversdesmembranesplasmiques.

1.2.1 Adsorption du cuivre dans l’apoplasme racinaire

Lecompartimentapoplasmiquesesubdiviseentroiscouchessuccessives:lalamellemoyenne,

la paroi primaire et la paroi secondaire qui est en contact avec la membrane plasmique

(Sattelmacher2001).Cettematriceestprincipalementconstituéedecellulose,d’hémicellulose,

de pectines et de protéines. L’apoplasme racinaire présente la particularité d’être chargé

négativement,cequiestprincipalementdûàlaprédominancedesfonctionscarboxyliquesdans

la lamelle moyenne et la paroi primaire (Sattelmacher 2001). Sa composition joue un rôle

important dans la complexation desmétaux, les parois cellulaires se comportant comme des

échangeurs d'ions spécifiques (Allan and Jarrell 1989). Cu peut alors être fixé sous forme

cationiqueparleschargesnégativesdesparoiscellulaires.Cupeutaussiêtreliésousformenon

ionique par des réactions de coordination aux groupements contenant de l'azote, aux

phosphatases et peroxydases également présents dans les parois cellulaires. L’apoplasme

racinaireformel’undesprincipauxcompartimentsaccumulantlesmétaux(Krzeslowska2011).


15

1.2.2 Absorption racinaire

Lesmécanismes d’absorption de Cu restent à l’heure actuelle encoremal identifiés et ont été

essentiellement caractérisés chez Arabidopsis thaliana L. (Burkhead et al. 2009; Puig et al.

2007b;Sancenonetal.2004;Sancenonetal.2003;Yruela2009).L’absorptionracinairedeCu

semble principalement dépendre de transporteurs à haute affinité spécifique de Cu(I)

appartenantàlafamilleCTR(CopperTRansporter)etenparticulierCOPT1(COPper Transporter

protein), fortement exprimé dans les racines (Sancenon et al. 2004). L'expression de ce

transporteur est contrôlée négativement par le cuivre (Sancenon et al. 2004). Ces protéines

COPT transportent le cuivre sous une forme réduite Cu(I), mais celui‐ci est majoritairement

présentsous formeoxydéeCu(II)dans lasolutiondesol.Laréductionducuivrepourraitêtre

assuréepardesprotéinesréductasesferreuses(FRO2etFRO3)présentesdans lamembrane

plasmique,quisontimpliquéesdanslaréductionduFe3+chezlesdicotylédones(Mukherjeeet

al.2006;Welchetal.1993).Cependantlaprésencedecesréductaseschezlesmonocotylédones

n’estpasconfirmée.Enplusdel'importdeCu(I)parCOPT1,Cu(II)pourraitêtreimportépardes

membresde la familleZIP(GrotzandGuerinot2006;Wintzetal.2003).Cependant, lerôlede

cesprotéinesdansletransportdecuivreinplantaresteàvérifier.

1.3. Homéostasie au sein de la cellule

Il est primordial pour les cellules de contrôler finement l’homéostasie des métaux afin de

pouvoirfairefaceàunexcèsdecesions,etéviterdesdommagestelsquedesstressoxydatifs.

Unefoistransportésdanslesdifférentstissus,lesmétauxdoiventêtrecorrectementdistribués

dansl'ensembledescompartimentssubcellulairesetàdesconcentrationsnontoxiquespourla

cellule. Ceci peut se faire grâce à la présencede transporteurs spécifiques et demolécules ou

protéinesquivontprendreenchargecesmétaux.Danslacellulevégétale,lecuivreestessentiel

pour de nombreux processus comme la photosynthèse, la respiration, la perception de

l'éthylène,lemétabolismedesdérivésréactifsdel’oxygène....Lecuivredoitdoncêtreacheminé

dans de nombreux compartiments subcellulaires comme le réticulum endoplasmique, la

mitochondrie, lechloroplasteet l’apoplaste(Marschner1995).Dans lecytosol,Cu(I)peutêtre

chélatépardesphytochélatinesetdesmétallothionines(CobbettandGoldsbrough2002)oupris

en charge par des métallochaperonnes. Les métallochaperonnes permettent d'acheminer le

métal à une protéine cible spécifique via des interactions protéine‐protéine. Ces

métallochaperonnespermettraientaussid'augmenter la capacitéde la celluleà chélaterCu(I).

ChezArabidopsis (Figure I.3), deux protéines ont été identifiées: CCH (Copper CHaperone) et

ATX1quiinteragiraientaveclesATPasesHMA7etHMA5(Andrés‐Colásetal.2006).Différents

facteurs entraînent une augmentation de leur expression comme une carence en cuivre, un


16

stress oxydatif, la sénescence ou encore des stress mécaniques (Burkhead et al. 2009).

Récemment,deuxnouvellesmétallochaperonnesquicomplexentCu(II),CUTAetPCaP1,ontété

identifiéesetcaractérisées(Burkeadetal.,2003).CUTAestlocaliséedanslechloroplaste,PCaP1

seraitassociéeàlamembraneplasmiqueavecuneforteaffinitépourCu(II).Ellenepossèdepas

de résidus cystéine ou histidine mais de nombreux résidus glutamate qui pourraient être

impliquésdanslaliaisonducuivre(Nagasaki‐Takeuchietal.2008).

Figure I.3 Schéma récapitulant les différents transports de Cu identifiés dans une cellule générique (essentiellement dans Arabidopsis). Les protéines membranaires transporteurs de Cu sont représentées en orange, les Cu chaperonnes en violet. Abréviations : CCH, copper chaperone; ATX : antioxydant1 ; COPT, copper transporter; FRO, ferric reductase oxidase; HMA, heavy metal P-type ATPase; MT, metallothioneins; ZIP, IRT-like protein. Schéma modifié d’après (Yruela 2009).

1.4. Transport et complexation

Une fois incorporé dans les cellules racinaires, le cuivre est libéré dans les vaisseaux

conducteursdelasèvebrute, lexylème,pourêtretransportéjusqu’auxpartiesaériennesdela

plante. L'efflux de Cu(I) de la racine vers le xylème, s’effectue en partie par le transporteur

HMA5(ATPasedetypeP)(Andrés‐Colásetal.2006).Celaaétéenpartiemontréparlaprésence


17

deHMA5danslesracinesetunblocagedelatranslocationdeCu,quis’accumulealorsdansles

racines,dansunmutantdéficientenHMA5.

Dans la sève, Cu peut modifier sa spéciation et former des complexes avec les substances

organiquesprésentes(Pohlmeier1999).Lesligandspotentielssontlessuivants:

- carboxylates(citrate,oxalate,malate,succinate,tartrate,phtalate,salicylate,acétate),

- acides aminés et acides mercaptiques, ils possèdent au moins un groupement

carboxylique et un groupement amine (glycine, acide glutamique, histidine, cystéine,

asparagine,nicotianamine)(FigureI.4)(Whiteetal.1981a;Whiteetal.1981b),

- polymères (protéines, pectines, ADN, ARN, lignine et polysaccharides). Ils sont formés

d’unechaînedemonomèresquipossèdentdesgroupementsfonctionnelscarboxyliques

ouaminés.

Lecuivreestdistribuédanslesdifférentsorganes,puispeutêtreremobilisé,c’estàdirechanger

d’organeenpassantparlesvaisseauxconducteursdesèveélaborée,lephloème.


18

Figure I.4 Structure chimique d’acides aminés, d’acides carboxyliques et de la phytochélatine et du glutathion


19

1.5. Distribution

Dans laplupartdesplantesétudiées, lesconcentrationsracinairesdeCusontsupérieuresaux

concentrationsdespartiesaériennes,quecesoitpourdesplantescapablesdetolérerdefortes

quantités de Cu comme Haumaniastrum katangense, Nicotiana plumbaginifolia, Elsholtzia

haichowensis,Silenevulgaris(Chipengetal.2009;XiaandShen2007),oupourdesplantesnon

tolérantes,commeleblé, lemaïs, lesroseaux, leniébéetc…(Alietal.2002;Bravinetal.2010;

KopittkeandMenzies2006).Ainsi,lerapportdesconcentrationstotalesdeCuentrelesracines

et les parties aériennes peut considérablement varier et être compris entre 2.5 et 166 en

fonctionduniveaud’expositiondesplantes(Brunetal.2001;Santibanezetal.2008).Cetteforte

accumulation racinaire s’explique en partie par la quantité importante de Cu liée aux parois

cellulaireset/ouàunmécanismedeséquestrationducuivre.Enséparantlesparoiscellulaires

ducytoplasmedecellulesderacinesdeAthyriumyokoscense,Nishizonoetal.(1987)montrent

que 70 à 90 % de Cu de la racine est localisé dans les parois cellulaires. Par une méthode

similaire, Lou et al. (2004)mesurent 60% du cuivre racinaire lié aux parois cellulaires dans

Elsholtziahaichowensis.Ceciestégalementobservéparobservationsinsitu,commeparexemple

danslarécenteétudedeKopittkeetal.(2011)oul’onobserveuneforteaccumulationduCusur

lescellulesdel’épidermeracinaire(FigureI.5).

Figure I.5 Distribution du cuivre sur une racine de niébé (cowpea) observée par µ-fluorescence X (Kopittke et al. 2011)

1.6. Toxicité

L’origine des concentrations en Cu des sols est souvent multipleavec d’une part une origine

naturelleoufondpédogéochimiquenatureletd’autrepartuneorigineanthropiquesuiteàdes

apports atmosphériques ou agricoles (épandages de fumier, de lisier, de boues de station

d’épuration, de compost urbain, et l’utilisation de produits de traitement phytosanitaires


20

(Kabata‐Pendias and Pendias 2001; Pilon‐Smits 2005)). Au‐delà de la concentration en Cu

requise pour une croissance optimale, Cu devient toxique, excepté pour quelques plantes

tolérantesàde fortesconcentrationdeCu(i.e.ArabidopsishalleriL.Silenevulgaris (Moench).,

Elshotziasplendens)(Lietal.2009;Qianetal.2005;Shietal.2008;Shietal.2004;Songetal.

2004;Yangetal.2002).

1.6.1 Rhizotoxicité

Un excès de Cu inhibe la croissance racinaire avant d’inhiber la croissance des tiges. Comme

nous l’avons vu auparavant, Cu a une très forte affinité pour les parois cellulaires, et a donc

tendance à s’accumuler dans ce compartiment (Iwasaki et al. 1990). Une inhibition de

l’élongation racinaire et les dégâts causés à la membrane plasmique sont des symptômes

immédiatsd’unetoxicitéauCu(DeVosetal.1991).L’excèsdeCuentraîneégalementcertaines

modificationsmorphologiques tel qu’un épaississement de la racine, une coloration brunâtre,

unediminutionde l’élongationracinaireetuneaugmentationde la ramification(Kopittkeand

Menzies 2006; Kopittke et al. 2009; Panou‐Filotheou and Bosabalidis 2004). Ces symptômes

sontprincipalementattribuésà la rigidificationdesparoiscellulairespar l’adsorptionmassive

des métaux en remplacement du Ca dans l’apoplasme, en particulier au niveau de la zone

d’élongation. Cette rigidité de l’apoplasme freine l’élongation racinaire et peut provoquer des

ruptures au niveau des cellules du rhizoderme et du cortex externe qui seraient dues à des

vitessesdecroissancedifférentesentrelescellulesdescortexinternesetexternes(Kopittkeet

al.2008).

LaconcentrationenCuracinairepeutêtreuneindicationdelatoléranceauCudesplantesplus

fiable que celle qui est donnée par les parties aériennes (Marschner 1995). En effet, la

translocationdeCuauxpartiesaériennesest limitéepar la forteaccumulationdeCudans les

racines, donc les phénomènes de toxicité et les atteintes physiologiques (i.e. réduction de la

croissance racinaire et de l’absorption des nutriments) se produisent avant que les parties

aériennes n’atteignent des teneurs en Cu anormalement hautes (Michaud et al. 2008). Ainsi,

Michaudetal.(2008)ontdéterminéunseuilde100‐150mgCukg‐1danslesracines,enincluant

lecompartimentapoplasmique,pourunerhizotoxicitémodérée(EC10)etunseuilde250‐300

mgCukg‐1pourunerhizotoxicitéimportante(EC50).

1.6.2 Stress oxydatif et génotoxicité

UneconcentrationtropélevéedeCuinduitlaformationd’espècesréactivesdel’oxygène(ROS)

par la réaction d’Haber‐Weiss et de Fenton. Cette formation d’ions superoxydes (oxygène


21

moléculaire ionisé par addition d’un électron supplémentaire) entraîne des dommages

oxydatifs. Les plantes ont développé des systèmes de défense antioxydants qui, dans des

conditionsnonoupeustressantes,sontsuffisantspourassurerl’homéostasieetainsiempêcher

desdégâtsdusauxstressoxydatifs(Foyeretal.1994).Lesenzymesantioxydantesprésentesen

majorité sontdes enzymesdétoxifiantes : superoxidesdismutases (SOD),Cu/ZnSOD, catalase

(CAT),etdesenzymesperoxydasesquidécomposentlescomposéspéroxydes(H202),commeles

glutathions peroxydases. De nombreux antioxydants non‐enzymatiques sont également

impliqués: le glutathion, l’acide ascorbique, les caroténoïdes, l’acide urique, plusieurs acides

aminésetdesthiols.

Quandlesystèmenesuffitplusàempêcherlestressoxydatif, lesROSparleurnatureinstable,

sont particulièrement réactives et sont capables de provoquer des dégâts cellulaires

importants(BriatandLebrun1999;DietzandBaier1999):

• la peroxydation des membranes lipidiques; les acides gras polyinsaturés des

membranescellulairesoudeslipoprotéinessontlacibleprincipaledesROS(Lunaetal.

1994),

• cassuresetmutationsdel’ADN(Lloydetal.1997),

• inactivationdesprotéinesetdesenzymes,

• oxydationdespigments,dessucres…,

• dégradation de la chlorophylle et inhibition de la photosynthèse (Luna et al. 1994;

Patsikkaetal.2002).

Le cuivre peut également être génotoxique, c’est à dire capable de générer des mutations

génétiques (génique, chromosomiques ou génomiques). Les effets génotoxiques sont

vraisemblablement causés par les radicaux libres et agents oxydants libérés par l’action du

métal(Souguiretal.2008;Yildizetal.2009).

1.6.3 Phytotoxicité au niveau des parties aériennes

Laphytotoxicitéauniveaudespartiesaérienness’observeparuneréductiondebiomasse,des

symptômeschlorotiques,desnécrosesetune inhibitionde lacroissance.Unediminutionde la

quantitédechlorophylle,desaltérationsdelastructuredeschloroplastesetde lacomposition

delamembranedesthylakoïdesontétéobservéesdansleriz,leblé,leharicotetl’origandans

des conditions toxiques (Panou‐Filotheou and Bosabalidis 2004; Patsikka et al. 2002; Yruela

2009). Il a été proposé que Cu interfère avec la biosynthèse de l’appareil photosynthétique,

modifiantlacompositiondespigmentsetdelamembraneinternedeschloroplastes(Maksymiec


22

etal.1994).Patsikkaetal.(2002)attribuentcetteréductiondechlorophylleàunedéficienceen

Fe induitepar la toxicitédeCu.Commeon l’a signalédans leparagrapheprécédent, celapeut

égalementêtredûaustressoxydatifquientraîneladégradationdesmembranesdesthylakoides

(Lunaetal.1994).

La phytotoxicité de Cu semble également se traduire par l’apparition de déficiences en

nutriments.DesdiminutionssignificativesdesconcentrationsenCa,Fe,K,Mg,MnetZndansles

partiesaériennesdeRhodesGrass,duniébé(cowpea),etdumaïsontétéobservées(AitAlietal.

2002; Kopittke and Menzies 2006; Sheldon and Menzies 2005). Des décolorations

internervaires,synonymesdechloroseferrique,ontégalementétédécrites.L’origineexactede

cesphénomènesdedéficiencen’estpasconnueprécisément.Cependant,l’apparitionsimultanée

de déficiences multiples laisse penser qu’il s’agit d’une conséquence de l’altération de la

structuredusystèmeracinaire,commeladégradationde lamembraneplasmique(Kopittkeet

al.2009;Panou‐FilotheouandBosabalidis2004).Danslecasparticulierdesmonocotylédones,

l’induction d’une déficience en Fe lors d’une forte exposition à Cu pourrait être due à un

mécanisme plus spécifique, impliquant la perturbation par Cu2+ du rôle complexant des

phytosidérophoresvis‐à‐visdeFe3+,ouàunantagonismeFe‐Cuauniveaudestransporteursdes

complexesformésparlesphytosidérophoresaveccesdeuxmétaux(Michaudetal.2008).

1.7. Mécanisme de tolérance à la toxicité de Cu

Les mécanismes cellulaires impliqués dans la tolérance au cuivre peuvent avoir les effets

suivants(Clemens2006;Hall2002;Yruela2009):

• réduire l’absorption métallique via l’action des mycorhizes ou des exsudats extra

cellulaires,

• immobiliserl’excèsdeCudanslesracinesetainsiexclurelemétaldespartiesaériennes,

• stimulerlapompeàeffluxdumétaldanslamembraneplasmique,

• chélaterCupardesphytochélatines,desmétallothionines,desacidesorganiquesoudes

protéinesdechocthermique(HSP),

• séquestrerCudanslesvacuoles.

Des acides organiques (citrate, malate, oxalate), carbohydrates, protéines ou peptides sont

excrétésparlesplantesetpeuventfaciliterl’absorptiondesmétaux,maiscesmoléculespeuvent

également inhiber leur acquisition en formant des complexes extérieurs à la racine.

L’importancedecesmécanismessemblevarierselonlaconcentrationdemétaux,lesespèceset

variétés impliquées et le temps d’exposition. Des dépôts foncés extracellulaires riches en Cu,

couplésàuneforteconcentrationdecitrateetdemalateontétéobservéssurdescellulesisolées


23

desoja,enprésencede10µMdeCu(Bernaletal.2006).Desconcentrationssimilairesdecitrate

etdemalateontétéobservéesdansdescellulestolérantesauCudeNicotianaplumbaginifoliaL.

(KishinamiandWidholm1987).Lerôledesacidesorganiquesdanslarhizosphèreestimportant

pourtolérerunecontaminationaluminique(Jones1998),mêmesilesmécanismesquiactivent

etrégulentlasynthèsedesacidesorganiquesnesonttoujourspasbienidentifiés(Walkeretal.

2003).

Une fois internalisés, lesmétauxenexcèsvontêtre stockésdansunendroitmétaboliquement

peuactif.Uneséquestrationdesmétauxpeutavoirlieudanslavacuole,l’apoplasme,oudansdes

cellulesspécialiséescommelescellulesépidermalesoudestrichômes.

Malgrélaprésencedemétallothioneines(MTs)etlaforteexpressiondecertainsgènesdesMTs,

leurs fonctionsdans lesplantesrestentpeuconnues.L’expressiondeplusieursgènesdesMTs

estinduiteparlaprésencedeCu(Guoetal.2003),leniveaud’expressiondugènedutype2de

MTsestassociéàlatoléranceauCuchezlesAthaliana(MurphyandTaiz1995)etlatolérance

desplantesmétallophytesSilenevulgarisetSileneparadoxaestassociéeàuneaugmentationde

l’expression des gènes MTs (van Hoof et al. 2001). Cependant, l’implication des MTs dans la

détoxificationdeCudanslesplantesn’apasétédémontréedemanièreconcluante(Hassinenet

al.2011).LadivergencedesséquencesdesprotéinesMTsetleprofild’expressioncomplexedes

gènesMTslaissesuggérerquelesfonctionsdeMTsnesontpaslimitéesàladétoxificationdeCu.

Récemment,despreuvesontétéapportéesquelesMTscontribuentàl’homéostasiedesmétaux

danslesplantes(Guoetal.2008;Roosensetal.2004).

Lerôledesphytochélatines(PCs)dansladétoxificationdeCun’apasétémisenévidence.Cuest

unactivateurde labiosynthèsedePCs,mais lesplantesmutantesdéficientesenPCsmontrent

peu de sensibilité au cuivre (Lee and Kang 2005; Schat et al. 2002). Comme les PCs peuvent

formerdescomplexesavecCu,ilestpossiblequelescomplexesPC‐Cunesoientpasséquestrés

danslavacuole(CobbettandGoldsbrough2002).RécemmentSinghetal.(2010)montrentaussi

quelaproductiondePCsn’estpasliéeàlatolérancedeCudanslepoischiche(Cicerarietinum).

Lemétabolismedel’azoteaunrôlecentraldanslaréponsedesplantesaustressmétallique(Lea

andAzevedo2007).L’accumulationdecertainsacidesaminés,commelaprolineoul’histidinea

étémesuréedansdesplantesayantétésoumisesàdefortesteneursdeCulaissantsuggérerun

rôleentantquechélateurouantioxydant(SharmaandDietz2006).

LesATPasesdetypesP1BquitransportentCusontimportantes,pasuniquementpourimporter

les quantités de Cu nécessaires au fonctionnement de la cellule, mais aussi pour limiter leur

accumulation.Lecuivreestchélatéàdeschaperonnesqui letransportent jusqu’auxorganelles


24

oudirectementàdesprotéinescytosoliquesdépendantesdeCu.Ilsemblequelachélationetle

transport de Cu ne soit pas seulement requis pour l’absorption de Cu mais aussi pour des

processus de détoxification (Puig et al. 2007). Par exemple, l’inactivation du gène ActP, qui

encodeuneATPasedetypePentraîneunehypersensibilitéduRhizobiumlegominosarumetdu

Sinorhizobiummeliloti (Reeveetal.2002). IlaétémontréquechezA. thaliana le transporteur

ATPaseHMA5amélioreladétoxificationdeCu(Andrés‐Colásetal.2006).

1.8. Utilisation de la spectroscopie d’absorption des rayons X

pour étudier la spéciation de Cu dans les plantes

La spectroscopie d’absorption des rayons X est utilisée pour caractériser la spéciation des

éléments tracesmétalliquesdans lesplantes (Krameretal.2000;Saltetal.2002;Sarretetal.

2009). L’utilisation de la XAS présente de nombreux avantages comme l’absence de

prétraitementdel’échantillon,quiestsusceptibledemodifier laspéciationdel’élémentétudié

(Gardea‐Torresdey et al. 2005). Lesméthodesbasées surdes extractions séquentielles ou sur

une homogénéisation des tissus frais (par exemple la chromatographie) entraînent une

dégradation desmembranes intracellulaires. Lesmétaux faiblement liés initialement localisés

dans les vacuoles, peuvent se retrouver en contact avec divers ligands forts du cytoplasme,

entraînantalorsdeschangementsartificielsdelaspéciation.Parmilesautresavantagesdecette

technique spectroscopique, nous pouvons citer la sélectivité chimique ainsi que les seuils de

détectionbasquipermettentdetravailleravecdesconcentrationsphysiologiques.LaFigureI.6

résume les principales informations que l’on peut obtenir grâce à l’analyse d’un spectre

d’absorptiondesrayonsX.

La spéciation du cuivre (état d’oxydation, nombre et nature des voisins) dans les tissus des

plantes est peu étudiée malgré une bonne connaissance de la structure et des fonctions de

nombreusesenzymesquidépendentducuivreetdesCuchaperonnes(voir lareviewdePilon

parexemple(2006)).


25

Figure I.6 Schéma présentant les caractéristiques du XANES et de l’EXAFS

LapremièreétudedécrivantlaspéciationducuivredansuneplanteaétéréaliséeparPoletteet

al. (2000) dans Larrea tridentata (créosotier). Ces plantes ont accumulé des concentrations

élevéesdeCu:953mg.kg‐1dans lesracines,493mg.kg‐1dans lestigeset370mg.kg‐1dans les

feuilles. Après l’échantillonnage des plantes, les échantillons ont été immédiatement congelés

dans l’azote liquide. L’analyse XAS a ensuite été effectuée avec un cryostat à hélium. La

préparation des échantillons et leur analyse à froid répond à un double enjeu: i) éviter de

modifierlaspéciationinsituquipeutêtreprovoquéeparunealtérationdesstructures,lorsdu

séchage par exemple, ii) limiter au maximum les dommages dus au faisceau de photons sur

l’échantillon.Dansunéchantillonorganique,lecuivrepeutêtreréduitenCu(I)ouCu(0)parles

électrons,radicauxlibres,H2ouautresespècesréactivesdel’oxygènequiontétéformésparla

radiolysede l’eausuiteà l’effet ionisantdesrayonsX(ManceauandMatynia2010;Mesuetal.

2006). Unemanière de retarder l’apparition de ces dégâts est d’analyser l’échantillon à froid

(Manceauetal.2002).

L’observationduspectreXANESdusol,desracines,delatigeetdesfeuilles(FigureI.7)permet

de décrire l’état d’oxydation de Cu. La position de la raie blanche (environ 8998 eV) indique


26

danslestroispartiesdelaplanteetdanslesollaprésencedeCu(II).L’épaulementsituéautour

de 8984 eV est caractéristique des composés Cu(I). Les auteurs observent un épaulement de

plusenplusmarquéde la racineaux feuilles, et suggèrentdoncque la réductionduCu(II)en

Cu(I)alieulorsdutransportdanslaplante.

Figure I.7 Spectre XANES au seuil K du Cu des feuilles, tiges, racines de créosotier et du sol d’origine des plantes (Polette et al. 2000)

L’affinementdesspectresEXAFSmontrelaprésencedeliaisonsdetypeCu‐O(1,83Ået1,90Å)

et Cu‐S (2,24 et 2,27 Å) dans les racines, tiges et feuilles. Ils proposent également l’existence

d’interactionsCu‐Cude2,78Ådans les feuilleset les tigesetune interactionCu‐Cude3,70Å

danstoutelaplante.

Lesauteursfontalorsleshypothèsessuivantes:

- unephasedestockage:Cu‐phytochélatines,

- unephaseprécipitée,soitforméedanslaplante(exchalcopyrite)oud’origineextérieure

etinternaliséeparlebiaisdesstomates.


27

Une autre étudedeGardea‐Torresdey et al. (2001) a également porté sur la spéciation de Cu

danslecréosotier,maisenutilisantdesplantesdéveloppéesenculturehydroponique,exposées

àuneconcentrationde635mg.L‐1deCuSO4.Lesplantesontétémisesaucontactd’unesolution

deCuSO4pendant48h.Lapréparationdesplantesprécédantl’analyseXASn’estpasdétaillée,il

semble que les différentes parties de la plante ont été séchées à 59°C. Contrairement à la

précédente étude, Cu est présent uniquement sous forme oxydée dans les tiges, racines et

feuilles et est complexéàun ligandde typeacideorganique.Un résultat similaireestobservé

chez la plante Sesbania drummondii (Sahi et al. 2007) qui a été cultivée en hydroponie et

exposée à des concentrations de 25 mg.L‐1 et 100 mg.L‐1 de CuSO4 pendant 10 jours.

Préalablementàl’analyseXAS,leséchantillonsontétélyophilisés.Lesauteursneprécisentpas

quellepartiedelaplanteestanalysée,noussupposonsqu’ils’agitdelaplanteentière.Lecuivre

présente un seul degré d’oxydation Cu(II) dans la plante. Des analyses par combinaisons

linéairesmontrent que Cu est complexé à des ligands semblables à des sucres ou des acides

organiques (référence utilisée: Cu(II)‐gluconate) et une forme précipitée (Cu(II)oxide et

Cu(II)nitrate).LamodélisationdesspectresEXAFSindiquelaprésencede4voisinsOà1.95Å,2

voisinsOà2,32Å,et4voisinsCà2,80Å.

Cependantdanscesdeuxétudes,laspéciationdeCupeutavoirétéinfluencéeparleséchagede

l’échantillon. Le séchage peut entraîner une oxydation de Cu présent dans les tissus et

éventuellementlaprécipitationdelaphasedécriteparSahietal(2007)dansleurséchantillons.

Shietal(2008)ontétudiélaspéciationdeCuprésentd’uneplantetoléranteElshotziasplendens

quiaétécultivéeenhydroponieetmiseaucontactd’unesolutionde300µMdeCupendant10

ou 60 jours. Les concentrations en Cu des tissus varient entre 165 et 248 mg.kg‐1 dans les

feuilles, 202 à 263 mg.kg‐1 dans les tiges et 11 755 et 13 796 mg.kg‐1 dans les racines. Les

échantillons frais ont été congelés à l’azote liquide. La spéciation de Cu est similaire dans les

feuilles, tiges et racines et est caractérisée par une forte proportion de Cu(II), même si la

présencedeCu(I)estdétectée.L’analyseparcombinaisonlinéairedesspectresXANESindique

quelesespècesdetype:Cu‐histidine(33‐52%)etCu‐paroiscellulairessontmajoritaires,alors

que les espèces de type Cu‐oxalate (9‐18 %) et Cu‐glutathione (7‐16 %) sont minoritaires.

D’après lamodélisationdes spectresEXAFS, l’interactiondominanteestCu‐N/Oà1,94‐1,97Å

danslesracines,lestigesetlesfeuilles.

Ainsi,lesmécanismesdetolérancedeCudansE.Splendensimpliqueraient:

‐ LacomplexationdeCusurlesparoiscellulaires,notammentsurlesgroupesoxygénés‐

OHou–COOHprésentssurlacellulose,lalignineoulapectine,

‐ Lacomplexationàdesacidesaminés(detypehistidine)ouorganiques,éventuellement

séquestrésdanslavacuole,


28

‐ Uneabsencedecomplexationàdescomposésdetypephytochélatines.

Deuxétudesrécentesontétudiélacomplexationetlatoxicitéducuivredansdeuxplantes:une

plante accumulatrice de Cu Crassula helmsii et une plante non tolérante Thlaspi caerulescens

(Kupperetal.2009;Mijovilovichetal.2009).

DesgrainesdeT.caerulescensontétéplacéesenculturehydroponiqueetsoumisesà10µMde

Cupendant8semaines.Certainesplantesdites«résistantes»sesontmieuxadaptéesàl’apport

deCuetontétécultivéespendant4mois.LesconcentrationsenCudesfeuillesdesplantesdites

«sensibles» sont de 31,4mg.kg‐1MS et celles des plantes «résistantes» de 42,7mg.kg‐1MS.

L’affinement des spectres EXAFS (Figure I.8) indique la présence de ligands O et S dans la

premièresphèredecoordinationetdesinteractionsCu‐Cuàplusgrandedistance.Unepartiede

ligandOayantunecontributionà2,8Åproviendraitdelanicotianamide.LaprésencedeCuaété

proposéecarl’interactionCu‐CuexpliqueraitlepicobservésurlatransforméedeFourierà4,4Å

et à 5,2 Å. L’analyse par combinaison linéaire indique la présence de ligands de type: soufré

(Cu(I/II)‐glutathione), acide organique (CuSO4, Cu(II)‐malate, Cu(II)‐citrate, Cu(II)‐Pro),

nicotianamine,histidine,dontlesproportionsvarientselonlarésistanceàCuetl’âgedesfeuilles

(juvénilesoumatures).

Ainsi,lesauteurssuggèrent:

‐ la présence de Cu lié à des métallothioneines, en quantité plus importante dans les

individusrésistants,

‐ de labiominéralisationdeCu.Celapourrait êtreexpliquéenpartiepar laprésencede

moolooite, une forme hydratée cristalline de Cu oxalate, détectée auparavant dans du

lichen,etuneautreforme,commeunoxydedeCu,ouunehydrocalcite(CaCO3.H2O),

‐ LaprésencedeCuliéàdelanicotianamide.


29

Figure I.8 Spectres EXAFS normalisés et transformées de Fourier des échantillons de feuilles de Thalspi caerulescens de l’étude de Mijovilovich et al (2009). Les données sont représentées en traits pleins et les fits en pointillés.

Lasecondeétuderéaliséeparlamêmeéquipe(Kupperetal.2009)présentelaspéciationdeCu

dansuneplanteamphibienneCrassulahelmsiiquisembletolérerdefortesquantitésdeCu.Les

quantités de Cu accumulées sont de 9200 et 9100 mg.kg‐1 MS dans les parties émergées et

immergées respectivement. L’analyse EXAFS des spectres de ces deux parties des plantes

indiqueuniquementlaprésencedeligandsoxygénés.Ilsproposentdonclaprésencedeligands

detypemalateoucitrate.

D’après les résultats de ces deux études, les auteurs proposent l’existence de deux stratégies

pourfairefaceàunetoxicitédeCu:

‐ Séquestration de Cu dans les vacuoles ou les parois cellulaires, associé à des ligands

acidesorganiquesouacidesaminésdanslesplantesaccumulatrices.

‐ DétoxificationparcomplexationdeCuàdesPCsoudesMTs,minéralisationdeCu,dans

lesplantesnonaccumulatrices.


30

Enfin,Koppitkeetal.(2011)ontétudiélalocalisationetlaspéciationdeCudansdesracinesde

niébé(cowpea)ayantétémisesaucontactde1,5µMdeCupendantunecourtepériode(3het

24h).Lesconcentrationsracinairessontégalesà96mg.kg‐1MSaprès3het190mg.kg‐1MSaprès

24h.L’analyseparcombinaisonslinéairesdesspectresXANESetEXAFSdesplantesaucontact

deCupendant3h,indiqueunmélangededifférentsligandsdeCucomposéde45à60%deCu‐

cysteine,de40à55%deCu‐citrate(EXAFS)oudeCu‐histidine(XANES)pourlesracines.Après

24h, leCu racinaire estmajoritairement associé à60%d’acidepolygalacturonique, et ade la

cystéineoudu citrate (Figure I.9). Les auteurs concluentqueCuestprincipalement accumulé

dans la paroi cellulaire, l’acide polygalacturonique étant le principal composant de la pectine,

maisaussiliéàdescomposésthioltelsquePCsouMTs.

Les auteurs ne s’interrogent pas sur la présence de Cu(I) dans l’échantillon. Pourtant, le

complexe Cu‐cystéine synthétisé peut contenir du Cu(I). Il apparaît cependant difficile de

comparer les spectres XANES car le maximum d’absorption des spectres XANES de leurs

échantillonsestsituéàuneénergieautourde8982eV(FigureI.9),alorsquelatransition1s‐4p

des composés Cu(II) est normalement située autour de 8995eV. On voit bien cependant que

l’épaulement présent dans la cystéine est présent à plus faible énergie que l’épaulement de

l’acidepolygalacturonique.Cestransitionspourraientalorsêtre1s‐4pxyduCu(I)de lacystéine

et1s‐4pzduCu(II)del’acidepolygalacturonique.Ilauraitétéintéressantd’avoirlesdérivéesde

cesspectresafindemieuxvisualiserlestransitions.LeCuprésentdanslaracineaucontactde

Cupendant3hayantunesignatureprochedelacystéine,pourraitdonccontenirCu(I).


31

Figure I.9 Spectres EXAFS (A et C) et XANES (B et D) au seuil K du Cu des racines de niébé (cowpea) soumises à 1,5 µM Cu pendant 3h (en haut) ou 24h (en bas). Les spectres des composés de référence les plus importants (i.e. acide polygalacturonique ou cystéine) et les résultats des combinaisons linéaires sont également représentés.

AuvudeladescriptiondesquelquespublicationsdécrivantlaspéciationdeCudanslesplantes,

ilapparaîtdifficiledecomparerlaspéciationentrelesespèces.Eneffet, lesrésultatsprésentés

varient en fonction du mode de préparation des plantes (congélation ou séchage), de la

concentrationdeCudumilieu,deladuréed’exposition,etpeuventaussiêtreinfluencésparles

différentesméthodesd’analysedesspectresEXAFS(combinaison linéaireoumodélisationdes

différentessphèresdecoordination).


32

2. LE SILICIUM

Lesilicium(Si)représentelesecondélémentleplusabondantdanslesol,aprèsl’oxygène.Ainsi,

la plupart des plantes se développant sur du sol contiennent de la silice dans leurs tissus

(Epstein 1994). La question du rôle de Si dans le développement des plantes a été soulevée

depuisplusd’unsiècle(Epstein1994;JonesandHandreck1965;Sachs1862).LesteneursenSi

danslesplantessontcomparablesauxteneursdesmacronutriments,maislesiliciumn’estpas

considérécommeunélément«essentiel»pourlacroissancedesplantes(Epstein1994;Epstein

1999).Laprincipaleraisonestl’absencedepreuvedel’implicationdeSidanslemétabolismede

la plante, qui est l’un des trois critères requis pour l’essentialité établis par Arnon et Stout

(1939).Cependant,denombreusesétudesmettentenévidencelerôlebénéfiquedecetélément

pour les plantes: amélioration de la croissance, meilleure résistance aux stress biotiques et

abiotiques(MaandTakahashi2002).

2.1. Teneur en Si dans les plantes et dans le bambou

Lesteneursd’acidesiliciquemesuréesdanslesplantesvariententre1et100mg.g‐1dematière

sèche (MS) en fonction de l’espèce (Epstein 1999; Hodson et al. 2005). Hodson et al. (2005)

montrent que la concentration en Si des plantes dépend majoritairement de la position

phylogénétiquedel’espèceplutôtquedesonenvironnement(i.econcentrationenSidanslesol

et la solution de sol, pH, climat…). Les espèces les plus riches font partie de la famille des

Poacées (Poaceae) dans la classe des monocotylédones. La capacité d’accumuler Si dans les

parties aériennes dépend de l’absorption racinaire, qui n’a pas la même efficacité selon les

espèces. Les plantes sont considérées comme «accumulatrices» quand la teneur en Si est

supérieure à 10 mg.g‐1 (Ma and Takahashi 2002). Avec une concentration en Si pouvant

atteindre jusqu'à410mg.g‐1 (Table I.1), lesbambous fontpartiedesplantesquiaccumulent le

plus de silicium (Motomura et al. 2002). Comme le montre la Table I.1, le nombre de

publications présentant des teneurs dans les bambous reste faible et quelques questions

importantesrestentsujettesàcontroverse.Parexemple,Dingetal.,(2008b)mesurentdansles

racines une concentration de 7,9 mg.g‐1, concentration qui se situe entre celle des tiges

(3,9mg.g‐1) et des branches (17,8 mg.g‐1). Alors que Li et al.,(2006) et Lux et al., (2003)

reportent des teneurs dans les racines supérieuresaux feuilles : 73,2 mg.g‐1 et 24 mg.g‐1

respectivement.


33

Table I.1 Synthèse des concentrations en Si publiées dans le bambou

LesconcentrationsenSisontexpriméesenSiO2mgg‐1dematièresèche.1:Dingetal.2008;2:Lietal.2006;3:Luxetal.2003;4:Meunieretal.1999;5:Motomuraetal.2002;6:UedaandUeda.1961(fromMaandTakahashi,2002).

2.2. Absorption racinaire

Danslesol,ilexistetroisprincipauxréservoirsdesilicium:(a)lasiliceetlesminérauxsiliceux

(b), les phytolithes et (c) le silicium dissous dans la solution du sol (Sauer et al. 2006). La

solution du sol est dominée par la présence d’acide silicique monomère, Si(OH)4. La

concentration d’acide siliciquemesurée dans les solutions du sol varie de 0.1 à 0.6mmol.L‐1

(Epstein1994). Il aétésuggérédansdenombreusesétudesque lesplantesabsorbentSi sous

cetteforme.Danslebambou,Dingetal.(2008b)confirmentcelaparuneétudeisotopiquedeSi

danslebambou,lesoletlasolutiondusol.

Lesmécanismesd’absorptiondeSi auniveaude la racine (passagede la solutionexterneaux

cellules corticales) font l’objet de controverses. Elle s’effectuerait par troismécanismes: de la

diffusionpassive,untransportactif,maisaussiunprocessusd’exclusion(Maetal.2004;Mitani

andMa2005).Lesespècesdites«nonaccumulatrices»ontdansleurstissusdesteneursenSi

inférieures aux teneurs de la solution du sol. Elles ont développé un

processusd’exclusionempêchant l’absorption de Si par diffusion passive (Raven 2001). Lors

d’unprocessusd’absorptionpassif,laplanteabsorbelesiliciumcontenudanslasolutiondusol

selon le gradient de concentration. Le flux passif est principalement contrôlé par

l’évapotranspiration.Cettehypothèseestbaséesurlefaitquelessitesdeprécipitationdesilice

coïncidentaveclesprincipauxsitesd’évapotranspiration(Dingetal.2008a;Dingetal.2008b).

Le silicium tend à s’accumuler là où l’eau s’évapore le plus: feuilles les plus hautes, les plus

exposées à l’ensoleillement (Motomura et al. 2002; Raven 2001; 2003). Dans le cas d’un

processus actif, le silicium est absorbé grâce à des protéines membranaires spécifiques qui

permettentàSides’accumulerdanslaplanteendépitdugradientdeconcentration.Cetypede


34

transporteurestplusrapidequeletransportdel’eauetindépendantdelatranspiration.Rains

et al., (2006) ont mis en évidence ce phénomène actif dans le blé (Triticum aestivum) en

montrantquel’absorptiondeSisuitunecinétiquedeMichaelis‐Menten.Al’aidedemutants,de

nombreusesétudesréaliséesparl’équipedeMaetsescollègues(voirreview(Maetal.2011))

ont identifié deux gènes responsables du transport et de l’absorption de Si dans plusieurs

plantes(FigureI.11):

‐ Lsi1:transporteurd’influxquifaitpénétrerleSidanslacellule

‐ Lsi2:transporteurd’effluxquifaitsortirleSidelacellule

LetransporteurLsi1initialementdécouvertdanslerizadeshomologuesprésentsdansleblé,le

maïsetlacitrouille(curcurbitacée)(Chibaetal.2009;Mitanietal.2011;Mitanietal.2009b).Le

transporteurLsi2aétéidentifiédansleriz,lebléetlemaïs(Mitanietal.2009a).Cependant,des

différencesdelocalisationdecestransporteursdanslescellulesracinairessontobservéesentre

lesespècesetpourraientêtreàl’originedescapacitésd’accumulationsdifférentes.

2.3. Transport et stockage

Après l’absorption racinaire, Si est transporté aux parties aériennes via le xylème. Le

transporteurquipermetàSid’entrerdanslexylèmen’apasétéidentifié.Unepartiedusilicium

transportédanslefluxdesèvevapolymériseretformerungeldesilice(SiO2,nH20).Eneffet,

lorsquelaconcentrationdeSiestsupérieureà2mM,leprocessusdepolymérisationconvertit

lesmonomèresd’acidesiliciqueenoligomères,puisenparticulesdesiliceformantdesagrégats

(PerryandKeeling‐Tucker2003)(FigureI.10).

Figure I.10 Processus de formation de particules de silice par différentes réactions de polymérisation (dimères, oligomères et agrégats) de monomères de silicium (Currie and Perry 2007).

Quandlesparticulesdesilicesesontdéveloppéespouratteindreunetaillecompriseentre1et3

nm(taillesobservéesdanslanature),lesparticulesportentalorsunechargedesurfacenégative

et interagissent avec l’environnement local, comme les parois cellulaires (Currie and Perry


35

2007).MalgrécesobservationsetlaforteconcentrationdeSidanslasèveduxylème,quipeut

atteindre18mM,Siestmajoritairementdétectésousuneformemonomérique:H4SiO4(Caseyet

al.2004;Dingetal.2005;Mitanietal.2005).Mais lemécanismeempêchantlapolymérisation

n’estpasconnu.


36

Figure I.11 Absorption et localisation de Si dans le bambou. Le mécanisme d’absorption est celui qui est décrit pour le blé et le maïs (Mitani et al. 2009a; Mitani et al. 2009b). La localisation de Si a été schématisée sur les photographies MEB de feuilles (B) et de racines (C) de bambou selon les analyses EDX des études de Motomura et al (2006) (B) et de Lux et al (2003) (C).


37

Le silicium doit ensuite être exporté du xylème vers les feuilles. Il est observé dans de

nombreuses études que Si précipite majoritairement au niveau des sites d’évaporation. Sa

répartition au sein de la feuille est alors la résultante d’un phénomène passif (Epstein 1999;

Raven2003).Dans lebambou,Dingetal. (2008b)mesurentuneaccumulationplus fortedans

les feuilles, puis les branches, puis les tiges. C’est une tendance générale que l’on retrouve en

comparantlesmoyennesdesconcentrationsenSidonnéesdanslesdifférentesétudes:feuilles

94mg.g‐1>branches17mg.g‐1>tiges8mg.g‐1SiO2(TableI.1).Cetterépartitionpeutindiquerle

rôledelatranspirationetdel’évaporationpourletransportdusiliciumdanslebambou.Dinget

al. (2008b) confirment également cela par une distribution des valeurs de δ30Si qui sont plus

élevéesdanslespartiesdelaplanteoùalieulatranspiration,commecelaaétédécritpourleriz

et la banane (Ding et al. 2005; Ding et al. 2008b; Opfergelt et al. 2005). Plusieurs études de

Motomuraetal(2000;2004;2006;2008;2002)ontétémenéespourlocaliserlesiliciumdans

lesfeuillesdebambouetcomprendresil’accumulationdeSiestliéeàunmécanismepassifou

actif.Auseindelafeuille,lesiliciums’accumulemajoritairementdanslescellulesdel’épiderme,

notammentdescellulesbulliformesetdespoilsbicellulaires (Luxetal.2003;Motomuraetal.

2000).Orsil’accumulationdeSiétaituniquementlerésultatdelatranspiration,laprécipitation

deSidevraitêtreobservéedanslescellulesduchlorenchymecarbeaucoupd’eauestévaporée

via les stomates. Ces auteurs ont également étudié l’accumulation de Si dans les feuilles de

Phyllostachys pubescens et P. Bambusoidesdurant leurduréede vie.Les feuilles accumulent Si

pendant laphasedecroissancemaisaussiaprès leurmaturation(Figure I.12).L’accumulation

deSiestplusforteauprintempsetenété,etdiminueenhiver.Cetteobservationestexpliquée

parlesvariationsdel’activitéphysiologiquedubambou,enfonctiondessaisons.Enété,laperte

d’eauestplusforte,lesstomatesouvertsetlaphotosynthèseplusactive(Motomura,2008).Ce

«schémad’accumulation»est réitérépendant laduréedeviedes feuilles,quiestde troisans

pourl’espèceconsidérée.Cesrésultatsmontrentquel’accumulationdesiliciumprovientsurtout

del’activitéphysiologique(évapo‐transpiration,photosynthèse,etc.)et nonpasdesprocessus

dedéveloppementetdecroissancedelaplante.


38

Figure I.12 Teneurs en SiO2 dans des feuilles de bambous pendant trois ans : 1998 (carrés), 1999 (cercles), 2000 (triangles) (Motomura, 2002)

Ainsi,ilsemblequedanslebambou,onnepuisseexpliquerletransportetl’accumulationdeSi

danslespartiesaériennesuniquementparuntransportpassif.Deplusuntransporteur(Lsi6),

permettantd’exporterSiduxylèmeverscertainescellulesaccumulatricesdeSidelafeuille(ex

cellulebulliforme),aétémisenévidencedansleriz(Yamajietal.2008).Ainsilaprésencedece

transporteur indique que le dépôt de Si dans les cellules spécialisées est dépendant d’un

transportsymplasmiqueplutôtqued’untransportapoplasmique(Yamajietal.2008).

2.4. Fonction du silicium dans le bambou

MaetTakahashi(2002)montrentquelaquantitédesilicedessolsinfluesurlaquantitédesilice

concentrée dans les feuilles et le développement des bambousaies. Ils observent que les

bambousaies ayant une forte productivité se développent sur des sols riches en silice

biodisponibleetsecaractérisentpardesteneursélevéesensiliciumdanslesfeuilles(voirTable

I.2).Al’inverse,lesfeuillesdesbambousaiesatteintesdemaladieoucellesquisontcaractérisées

paruneproductivitéfaible,ontdesteneursplusfaiblesenSi.


39

Table I.2 Relation entre la teneur de SiO2 des feuilles de bambou et la productivité de la bambousaie (Ma et Takahashi, 2002).

Lesexpériencesfaitessurdesbambousaiesmontrentégalementdeseffetsremarquablessurla

croissanceavecuneaugmentationnotabledelabiomassedesfeuilles,dunombredebourgeons

etdelarigiditédeschaumes(MaetTakahashi,2002).Cependant,l’applicationdescoriesriches

en silice dans différentes plantations de bambous en Chine ne s’accompagne pas d’une

augmentation du rendement (Hong, 1994), mais au contraire d’une baisse dans certaines

conditions expérimentales (absence d’engrais N, P et K). De plus, Motomura et al. (2008)

montrent qu’à partir de teneurs en SiO2 supérieures à 250mg.g‐1, la photosynthèse peut être

affectéeparledépôtdeSidanslescellulesduchlorenchyme.

MalgrélestrèsfortesquantitésdeSiaccumuléesdanslebambou,lerôledusiliciumn’aétéque

trèspeuétudié.PourtantdenombreuxeffetsbénéfiquesdeSisontdécritspourd’autrePoacées

telquelerizetleblé.

Leseffetsbénéfiquesdusiliciumpeuventserépartirentroistypesdefonctions:

• une fonction structurale, le silicium conférant une certaine résistance et rigidité des

paroiscellulaires;

• une fonction protectrice, induisant une résistance aux pathogènes, aux insectes, aux

champignons;

• une fonction physiologique, en réduisant l'évapo‐transpiration, en améliorant

l'oxygénationdes racineset les interactionsavecd'autreséléments (dont les éléments

tracesmétalliques).


40

Figure I.13 Résumé des fonctions du silicium (schéma provenant de Ma and Yamaji 2006)

LessynthèsesbibliographiquesdétaillantleseffetspositifsdeSisontnombreuses(Guntzeretal.

2011; Liang et al. 2007; Ma and Yamaji 2006) (Figure I.13). Nous allons présenter dans le

paragraphesuivantuniquementlerôledusiliciumfaceàunetoxicitémétallique.

2.5. Métaux et silicium

Plusieursétudestestentl’efficacitéd’unapportdesiliciumsurlatoxicitédecertainsmétaux:Al,

Mn, Cd, Zn, Cu, As, B. Toutes les publications montrent qu’un apport de silicium diminue la

toxicitédesmétaux, cependant lesmécanismesd’actiondusiliciumdans laplanterestentpeu

connus.Leshypothèsesproposéesdanslalittératuresontprésentéesci‐dessous.Danslaplupart

des études, c’est la combinaison d’une ou de plusieurs hypothèses qui est observée pour

expliquerlerôledeSi.

2.5.1 Modification des conditions environnementales

Dansdesexpérimentationssursol,l’apportdeSi(silicatedecalcium,silicatedepotassium)peut

faire varier les propriétés physicochimiques du sol, en augmentant le pH par exemple,


41

entraînantainsiunediminutiondelaphytodisponibilitédesmétaux(Chenetal.2000;Guetal.

2011;Liangetal.2005;TrederandCieslinski2005;ZhaoandMasaihiko2007).L’apportdeSi

danslasolutiondesolpeutégalementmodifierlaspéciationdesmétauxetnotammentformer

uncomplexesilicaté:Si‐Al(Liangetal.2001;Maetal.1997).Cependantceteffetindirectn’est

passuffisantpourexpliquerlameilleuretoléranceauxmétaux,etestassociéàuneffetduSiin

planta.

2.5.2 Augmentation de la biomasse

L’apport de silicium peut entraîner une augmentation la biomasse, diluant ainsi l’effet du

polluant.Lorsd’uneexpérienceenpot,desajoutsdesilicatedecalcium(de50à200mg.kg‐1)

dansdusolenrichienZn(100mg.kg‐1)etenCd(10mg.kg‐1)ontprovoquéuneaugmentationde

biomassechezlemaïs(daCunhaanddoNascimento2009).Zhangetal(2008)ontobservéune

augmentationsignificativedelabiomassederacinesetdefeuillesderizcultivéenhydroponieà

lasuited’unapportde2mMSiensolution,quecesoitsansajoutdeCdouavecajoutdeCden

solution. De même Si améliore la croissance de plants de maïs (augmentation de la surface

foliaire, du poids frais) (Vaculik et al. 2009), et de pak‐choï, une brassicacée (variété de chou

chinois)sanstoxicitémétallique(Songetal.2009).Cependant,enconditionenvironnementale

non stressante, l’augmentation de la biomasse n’est pas systématique (Hammond et al. 1995;

Olivaetal.2011;RichmondandSussman2003).

2.5.3 Diminution de l’absorption des métaux

Un des principaux effets du Si vis‐à‐vis de la diminution d’une toxicité métallique est une

réduction de la concentration des métaux dans les parties aériennes des plantes. Cela a été

observépourCd,Zn,Mn,Cudansdesmonocotylédonescommeleriz(Shietal.2005b;Zhanget

al.2008), lemaïs(Liangetal.2005),maisaussidesespècesdicotylédonescomme lepak‐choï

(Song et al. 2009), le concombre (Feng et al. 2009), l’arachide (Shi et al. 2010), Erica

Andevalensis (Olivaetal.2011).Cettediminutionpeutêtreexpliquéepar labarrièrephysique

que forment les dépôts de Si sur les parois de l’endoderme entraînant une diminution de la

porosité des parois cellulaires, et réduisant ainsi le passage des métaux dans le xylème (da

CunhaanddoNascimento2009).Shietal.(2005b)ontégalementmontré laréductionduflux

apoplastiqueinduitparSilorsd’unetoxicitéauCddansleriz,enmesurantlafluorescenced’un

traceur apoplastique (PTS) qui renseigne sur l’efficacité du transport apoplastique. Il est

cependant peu probable que le mécanisme réduisant la concentration des métaux dans les

parties aériennes soit communpour toutes les espèces étudiéeset s’expliqueuniquementpar

une réduction du flux de métaux entrant. De plus, dans certaines études, Si entraine une


42

augmentationdes teneurs enmétauxdans la plante tout en réduisant leur toxicité (daCunha

anddoNascimento2009;Vaculiketal.2009).

2.5.4 Modification de la répartition des métaux

Plusieurs études ont observé la localisation desmétaux et du silicium dans les tissus afin de

comprendre leurs interactions. Da Cunha and doNascimento (2009) ont localisé, par analyse

optiquehisto‐chimique,Cd,ZnetSidanslesmêmescellules,auniveaudel’épidermefoliaireet

auniveaude l’endodermed’uneracinedemaïs.Zhangetal. (2008)ontobservéparMEB‐EDX

une répartition similaire de Cd et Si dans une cellule bulliforme de feuille de riz. Ces auteurs

proposent alors une co‐précipitation de Si avec Cd ou Zn. Grâce à une observation par

spectrométrie de perte d'énergie des électrons (EELS) Neumann and zur Nieden (2001) ont

déterminélacompositiondesdépôtsdesilicedansCardaminopsis.Cesauteurssuggèrentquele

silicium intracellulaire forme des complexes Zn‐silicate dans le cytoplasme des feuilles de

Cardaminopsis.Ces silicates seraientensuite transformésenSi02dans le cytoplasmealorsque

lesmétauxseraientséquestrésdanslavacuole.

De nombreuses études analysent Si présent dans les différents compartiments en faisant des

extractions chimiques: fluide apoplastique, fluide symplastique, parois cellulaires. Les

premières études de ce type ont porté sur l’effet de Si face à une toxicité auMn. Si réduit le

développementdetâchesbrunessur les feuilles,quiestunsymptômetypiqued’unetoxicitéà

Mn.Pourtantlesteneursmesuréesdanslesplantesnesontpastoujoursdiminuéesaprèsapport

deSi,maisc’est ladistributionduMndansune feuillequiestmodifiée.AprèsunapportdeSi

dans le haricot et la vigne, Mn est réparti de manière homogène dans la feuille, ce qui

expliquerait la réduction de sa toxicité (Horst and Marschner 1978; Iwasaki and Matsumura

1999). Les mécanismes d’action de Si entraînent donc une meilleure tolérance à Mn dans la

celluleplutôtqu’unmécanismed’exclusion(RogallaandRomheld2002b).Dans leconcombre,

l’apport de Si augmente la quantité de Mn lié aux parois cellulaires (90 %) et diminue la

proportionsymplasmique(10%).IlestdoncproposéparlesauteursqueSifavoriselaliaisonde

Mn aux parois cellulaires ce qui permet de diminuer la quantité deMn du cytosol et donc sa

toxicité(RogallaandRomheld2002a;b;Wangetal.2000).Danslepotiron(Cucurbitamoschata

Duch.),1,44mMSidiminuelatoxicitédeMnparuneaccumulationdeMnetSisousuneforme

métabolique inactive autour de la base des trichomes sur la surface des feuilles (Iwasaki and

Matsumura,1999).DansdesfeuillesdeCowpea,Iwasakietal.(2002)observentunediminution

delaquantitédeMndanslefluideapoplastiquecombinéàunplusforttauxdeMnadsorbésur

lesparoiscellulairesaprèsunapportdeSide50µM.


43

Cette modification de distribution a été observée pour d’autres métaux. Lors d’une étude en

hydroponie, la concentration totale en Al des racines de maïs n’est pas influencée par Si, en

revanchelapartieéchangeabledeAlprésentsurlesparoiscellulairesestmodifiée(Wangetal.

2004). La partie facilement échangeable est réduite avec l’apport de Si et la répartition deAl

dans les tissus des racines est modifiée. Ces auteurs proposent la formation de complexes

biologiquement inactifs d’hydroxyaluminosilicate (HAS) dans l’apex des racines. Dans

Arabidopsis Thaliana, Li et al. (2008) expliquent la meilleure tolérance à Cu par une

séquestration de Cu dans les parois cellulaires. Leur étude montre également que la

concentration enCudesplantesn’est pasmodifiéemais que la plantemodifie sa stratégiede

toléranceàCu.

Cependant,cesrésultatssontendésaccordavecceuxdeShietal.(2010)dansArachishypogaea

(arachide). Ces auteurs montrent que Si affecte la distribution de Cd dans les feuilles en

diminuantlafractionduCdliéeauxparoiscellulaires.Cettedifférencepeutêtreexpliquéeparle

comportement de Si qui diffère entre des espèces accumulatrices de Si comme le riz, et une

espècenonaccumulatrice:l’arachide.

2.5.5 Modification de l’expression de gènes

Un mécanisme qui semble être responsable d’une meilleure tolérance aux métaux est la

stimulationdusystèmeanti‐oxydant.Ceteffetaétémisenévidenceparlamesuredel’activité

d’enzymes anti‐oxydantes (par exemple SOD, POD, CAT, APX) dans Arabidopsis lors d’une

toxicité à Cu (Khandekar and Leisner 2011), dans Brassica chinensis (pak‐choï) et Arachis

hypogaea (Arachide) lorsd’une toxicitéàCd(Shietal.2010;Songetal.2009), dansDucumis

sativusenréponseàunetoxicitéàMn(Shietal.2005a),etdansleblé,latomateetlesépinards

enréponseàunetoxicitéàB(Gunesetal.2007a;Gunesetal.2007b).

Récemment,KhandekarandLeisner(2011)ontmontréqu’unapportde1,5mMdeSi,lorsd’une

toxicité au Cu chez Arabidopsis Thaliana, maintient ou augmente l’expression des gènes

responsables de la production de métallothioneines (MTs) qui peuvent être des molécules

chélatantesdeCu(CobbettandGoldsbrough2002).

IlsembleraitégalementqueSiaitunrôlesurl’activitédesgènesPAL(PhenylalanineAmmonia

Lyase),diminuantleurexpressionlorsd’unstressàCu(Lietal.2008).Cependantleurrôledans

ladiminutiondelatoxicitén’estpasbiencompris(KhandekarandLeisner2011).


44

3. BILAN DU CHAPITRE I

Danslesracines,unepartieimportantedeCuestimmobiliséedanslesparoiscellulaires.Le

cuivre semblemajoritairement absorbé sous forme réduiteCu(I) par le transporteurCOPT1.

Dans la cellule, mais aussi dans les vaisseaux conducteurs, Cu peut être chélaté par de

nombreuxligands,commelesacidesorganiques,lesmétallochaperonnes,lesphytochélatines,

les glutathions, lesmétallothionéines. Dans la plante, le cuivre estmajoritairement accumulé

danslesracines.

PlusieursmécanismesdetolérancesontproposéspourréduireunetoxicitédeCu:

• réductiondel’absorption,

• séquestrationdeCudanslesracines,

• séquestrationduCudanslapartieapoplasmique(paroiscellulaires,vacuoles),

• complexation de Cu sous une forme moins toxique (phytochélatines,

métallothionéines,acidesorganiques).

Lebambouestuneplanteaccumulatricedesilicium.Sis’accumulemajoritairementdansles

feuilles où il peut atteindre une concentration de 410 mg.g‐1 Si02. Son absorption et son

transportsemblentêtrecontrôléspardesmécanismesactifsetpassifs.

PlusieursétudesontmontréquelesiliciumdiminuelatoxicitédesETM.Danslaplante,les

principauxmécanismesd’actionobservéssontlessuivants:

• augmentationdelabiomasse,diluantl’effetdupolluant,

• améliorationdelacapacitédelaplanteàséquestrerlesmétauxdanslesracines,

• modification de la répartition des métaux entre la partie symplastique et

apoplastique,

• modificationdel’expressiondecertainsgènes.


45

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56

C H A P I T R E I I

DISTRIBUTION AND VARIABIL ITY OF

S IL ICON, COPPER AND ZINC IN

DIFFERENT BAMBOO SPECIES

ChapitreII:Distributionandvariabilityofsilicon,copperandzincindifferentbamboosspecies

58


59

TABLE DES MATIERES

CHAPITREIIDISTRIBUTIONANDVARIABILITYOFSILICON,COPPERANDZINCINDIFFERENTBAMBOOSPECIES ..................................................................................................61

1. INTRODUCTION.....................................................................................................................................................65

2. MATERIALSANDMETHODS ..............................................................................................................................672.1. Geographicalareaofthestudy,soildescriptionandsamplingprocedure..................................................... 672.2. Soilmaterialandanalysis .................................................................................................................................................... 682.3. Plantmaterialandanalysis................................................................................................................................................. 692.4. Statisticalanalyses .................................................................................................................................................................. 69

3. RESULTS...................................................................................................................................................................703.1. Soil .................................................................................................................................................................................................. 703.2. Betweenplantparts ................................................................................................................................................................ 703.3. Betweenplantspecies ............................................................................................................................................................ 71

4. DISCUSSION.............................................................................................................................................................754.1. PhytoavailabilityofSi,CuandZn ..................................................................................................................................... 754.2. OriginofSivariationinbamboos ..................................................................................................................................... 754.3. OriginofCuandZnvariabilityinbamboo.................................................................................................................... 784.4. InteractionsbetweenmetalsandSi ................................................................................................................................. 80

5. BILANDUCHAPITREII........................................................................................................................................81



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C H A P I T R E I I

DISTRIBUTION AND VARIABIL ITY OF S IL ICON, COPPER AND ZINC IN

DIFFERENT BAMBOO SPECIES

Nous avons vu dans le premier chapitre que le bambou possède la capacité d’accumuler de

fortesquantitésdesiliciumdanssestissus,maislesquantitésaccumuléesvarienttrèsfortement

d’uneétudeàl’autre.Atitred’exemple,Lietal.(2006)mesurentuneconcentrationmoyennede

31mg.g‐1danslesfeuillesdebambouPhyllostachysheterocyclavar.pubescensalorsqueDinget

al. (2008) reportent une concentration de 90,4 mg.g‐1 en moyenne dans différentes espèces.

Nousnoussommesdoncinterrogéssurl’originedecettevariabilité: tient‐elleauxparamètres

environnementaux contrastés entre les études précitées ou bien à l’impact des différentes

espècesétudiées? Sachantqu’il existeplusde1200espèces, appartenant àplusde70genres

(KleinhenzandMidmore2001),ilapparaîtimportantdedéterminersil’accumulationdeSiest

influencéeparl’espècedebambou.Deuxtypesdebamboussontdistinguésselonlamorphologie

deleursrhizomes(FigureII.0)(McClure1966):

• lesbambousmonopodiaux(outraçants)ayantdesrhizomes«leptomorphes»,

• lesbamboussympodiaux(oucespiteux)ayantdesrhizomes«pachymorphes».

Lesentrenœudsdesrhizomesleptomorphessontlongsetminces,alorsqueceuxdesrhizomes

pachymorphes sont courts et épais. Les différences d’anatomie du rhizome peuvent être

considérées comme une adaptation aux conditions climatiques de la zone d’origine des

bambous. Les bambous monopodiaux sont originaires de régions marquées par un climat

tempéréavecdeshiversfroidsethumides,alorsquelesbamboussympodiauxsontoriginaires

derégionsàclimattropicalavecunesaisonsèchemarquée.Grâceàdesentrenœudspluscourts,

le rhizome pachymorphe est moins sensible à la sècheresse. Contrairement aux bambous


62

monopodiauxquipeuvent coloniserune surface très importante, lesbambous sympodiauxne

s’étendentquetrèslentementetleurschaumesdemeurentserrésenunetouffetrèsdense.

Figure II.0 Types de rhizomes, figure modifiée d’après (Soderstrom and Young 1983)

Ainsi, le nombre important d’espèces de bambous et la distinction entre deux typesmajeurs

nousontamenéeànousinterrogersurl’influencedesespècessurlesconcentrationsenSimais

aussienCuetZn.Lesobjectifsdecechapitresontde:

1) déterminerlavariabilitédesconcentrationsdeCu,ZnetSiencomparant16espècesde

bambous se développant dans les mêmes conditions environnementales, sur un sol

volcaniquedel’îledelaRéunion,

2) obtenirdesconcentrationsderéférencespourSi,CuetZndanslespartiesaériennesdes

bambous,

3) identifier d’éventuelles corrélations entre les concentrations de Si, Cu et Zn dans les

bambous.

L’article présenté dans ce chapitre intitulé “Distribution and variability of silicon, copper and

zinc indifferentbamboospecies”décrit lesvariationsdeconcentrationsdeSi,Cu,Zndans les

différents tissusdesbambouset entre lesespèces, endiscutantdesmécanismes impliquésau

regarddesconnaissancesissuesdelabibliographie.

CetarticleaétéacceptédanslarevuePlantandSoil.

Rhizomeleptomorphe

Rhizomepachymorphe


63

Distribution and variability of silicon, copper and zinc in

different bamboo species

BlancheCollin*a,b,EmmanuelDoelschc,CatherineKellerb,FredericPanfilia,Jean‐Dominique

Meunierb

a Département de recherche développement de la société PHYTOREM S.A., 13140 Miramas,

France

bCEREGE,CNRS,Aix‐MarseilleUniversité,Europôleméditerranéendel'ArboisBP80,13545Aix

enProvence,France

cCIRAD,UPRRecyclageetrisque,34398Montpellier,France

*Correspondingauthor.

DOI:10.1007/s11104‐011‐0974‐9

ABSTRACT

With a high growth rate and biomass production, bamboos are frequently used for industrial

applications and recently have proven to be useful for wastewater treatment. Bamboos are

consideredasSiaccumulatorsandthereisincreasingevidencethatsiliconmayalleviateabiotic

stresses such as metal toxicity. The aim of this study was to investigate the extent of metal

concentrationsandpossiblecorrelationswithSi concentrations inplants.Thisstudypresents,

for the first time,referencevalues forsilicon(Si), copper(Cu)andzinc(Zn)concentrations in

stemsandleavesofvariousbamboospeciesgrownunderthenaturalpedo‐climaticconditions

oftheislandofRéunion(IndianOcean).Abroadrangeofsiliconconcentrations,from0(inferior

todetectionlimit)to183mgg‐1drymatter(DM),werefoundinstemsandleaves.MeanleafCu

andZnconcentrationswerelow,i.e.5.1mgkg‐1DMand15.7mgkg‐1DM,respectively.Silicon,

CuandZnconcentrationsincreasedoverthefollowinggradient:stembase<stemtip<leaves.

Significant differences in Si, Cu and Zn contents (except Zn in the stem)were noted between

bamboospecies,particularlybetweenmonopodialandsympodialbamboospecies,whichdiffer


64

intheirrhizomemorphology.SympodialbamboosaccumulatedmoreSiandCuthanmonopodial

bamboos,inbothstemsandleaves,whereassympodialbamboosaccumulatedlessZninleaves

thanmonopodialbamboos.Thefindingsofthisstudysuggestthatagenotypiccharactermaybe

responsibleforSi,CuandZnaccumulationinbamboo.

Keywords:traceelement,silica,Poaceae,genotypicvariability,islandofRéunion


65

1. INTRODUCTION

Bamboos arewidespread plants belonging to the grass family (Poaceae). They are commonly

foundintemperate,tropicalandsubtropicalregionsandwidelyusedforindustrialpurposes,as

freshedibleshoots,makingpaper,buildingmaterialandeveninmedicines.Bamboosareknown

for their resistance to awide range of stress factors and their high growth rate and biomass

production, with potential uses in phytoremediation. The PHYTOREM company (France) has

developedBAMBOO‐ASSAINISSEMENT®technologyforwastewatertreatment(Arfietal.2009).

Thecompanyiscurrentlyoptimizingthephytoremediationcapacityofbamboosundertropical

climaticconditions.

TheuptakeandaccumulationofessentialnutrientssuchasN,PandKarewelldocumentedin

bamboos (Embaye et al. 2005; Shanmughavel and Francis 2001), butmetal accumulation has

been poorly documented so far. Metals are naturally present in the pedo‐geochemical

backgroundofsoilsatvarious levelsandmanymetalsareessential toplants,buttheymaybe

toxic at higher concentrations.Metals accumulate in soil due to anthropogenic contamination

throughfertilizerandorganicmanureapplications, industrialandmunicipalwastes, irrigation,

and wet and/or dry deposits (Doelsch et al. 2010; Novak J et al. 2004). Phytoremediation

techniques have been put forward as alternatives to remediate metal contaminated soils,

especially agricultural soils (McCutcheon and Schnoor 2003). One of the limitations of such

technologiesistheavailabilityofplantspeciesadaptedtospecificenvironmentalconditionsand,

accumulatingand/ortolerantto largemetalormetalloidconcentrationsinsoils(Keller2005).

Bamboos are potentially good candidates for phytoremediation because of their widespread

distribution,theireasyandwell‐knownpropagationmode,thebroadrangeofspeciesandtheir

possibleadditionaluseasrawmaterial.Toourknowledge, there isnodataavailableonmetal

concentrationsinbamboos,especiallyundernaturalconditions.Inthisstudy,wefocusedonthe

case of copper (Cu) and zinc (Zn), twometalswhichmaybepresent at high concentration in

wastewater.

Like many Poaceae species (sugarcane, rice, wheat, etc.), bamboos are considered as Si

accumulators,withSiconcentrationsrangingfrom3to410mgg‐1SiO2(DM)(Dingetal.2008;Li

etal.2006).ThevariabilityinSicontentinbamboomaybeexplainedby:(i)theavailablepoolof

Si insoil (Henrietetal.2008; Jonesetal.1967); (ii) the increase inSicontentduringbamboo

ageing (Motomura et al. 2002) and (iii) genetic variability among species. This last point has


66

never been studied in bamboos,whereas itmay be important because of the high number of

bamboo species: a total of about 1030 bamboo species (77 genera) are grouped in the sub‐

familyBambusoideae,withinthefamilyPoaceae.Inordertoclarifytheroleofthespeciesinthe

Siuptakecapacity,dataarethusneededonSiconcentrations inbamboosof thesameageand

growninsimilarsoils.

Inriceorwheat,thereisincreasingevidencethatsiliconmayalleviatemetaltoxicity(Liangetal.

2007).SeveralmechanismshavebeenproposedtoexplaintheroleofSiinmetaltolerance,such

as limitation of metal uptake, reduction of root‐to‐shoot translocation or changes in metal

allocation within the plant. In bamboos, the role of Si on metal tolerance has not been

investigated. The first step before any attempt to test bamboos in contaminated soils for

phytoremediation technologies is to investigate, under natural conditions, the extent ofmetal

concentrationsandpossiblecorrelationswithSiconcentrationsinplants.

Theobjectivesofthepresentstudywereto:(1)determinethevariabilityinCuandZn(forthe

firsttime)andSiconcentrationsbetweenspeciesbycomparing16bamboospeciesgrownunder

similar environmental conditions, i.e. a volcanic soil on the island of Réunion (Indian Ocean,

France),(2)obtainreferencevaluesforSi,Cu,Znconcentrationsinthedifferentabove‐ground

partsofbamboosand,(3)highlightthepossiblerelationshipbetweenSi,ZnandCuinbamboos

growinginanaturalsoilundertropicalclimaticconditions.


67

2. MATERIALS AND METHODS

2.1. Geographical area of the study, soil description and

sampling procedure

Bamboo samples were collected from a 3.5‐ha bamboo nursery in Réunion (Indian Ocean

island), France (Mr Perrussot, Le Guillaume, Saint Paul). 130 different species have been

maintained in thisnursery since1987.The climate is both tropical andoceanic,with easterly

prevailingwinds.Thestudiedsite(21°03’51S,55°19’28E)isonthewestsideofthePitondes

Neigesshieldvolcano,1035mabovesealevel.Themeanannualprecipitationis1700mm,and

the temperature ranges from 10 to 28°C, with hot and humid summers and warm and wet

winters. Soil in this area is classified as a chromic Andosol developed from Piton des Neiges

volcanicmaterial(Raunet1991).

SixteenbamboospeciesbelongingtosixgeneraweresampledinthesamefieldinOctober2008.

Thesebambooswereselectedbecausetheymaybegoodcandidatesforphytoremediationunder

tropical conditions, due to their high biomass production and good adaptations to tropical

climates.Bamboospeciesdiffer the rhizome form, i.e. theundergroundpart fromwhich roots

andshootsgrowfromnodes.Accordingtotherhizomemorphology,bamboosaredividedinto

monopodial bamboos with leptomorph rhizome systems, and sympodial bamboos with

pachymorph rhizome systems (McClure 1966). These differences in rhizome systems can be

regarded as adaptations to the climatic conditions from which the bamboos originated:

monopodial bamboos are native to temperate climates and sympodial bamboos are native to

tropical climates (Kleinhenz andMidmore 2001). Ten out of the 16 speciesweremonopodial

bamboos, i.e. Dendrocalamus giganteus, Dendrocalamus strictus, Bambusa bambos, Bambusa

oldhamii, Bambusa vulgaris 'Vittata', Bambusa multiplex ‘Golden Goddess’, Bambusa multiplex

'AlphonseKarr',Bambusatuldoides,Gigantochloasp.'MalayDwarf'andThyrsostachyssiamensis;

andsixweresympodialbamboos,i.e.Phyllostachysaurea,Phyllostachysbambusoïdes‘Castillon’,

Phyllostachysbissetii,Phyllostachysflexuosa,PhyllostachyshumilisandPseudosasajaponica.

Foreachspecies, threedifferent1‐year‐oldbamboospecimenswereselected tosamplestems

andleaves.Twosamplesweretakenfromeachstem:oneatthethirdinternodeabovethesoil

surface, further referred to as the “stem base”, and the second at the tip of the stem, further

referredtoasthe“stemtip”.Foreachstem,onebulkleafsamplewastaken.Atotalof94stem

samplesand47leafsampleswerecollected.


68

2.2. Soil material and analysis

Soilwas sampled under at least one bamboo of each species. A total of 18 soil sampleswere

obtainedbycollectingtopsoils(0‐25cm)withagaugeaugerandmixingfivereplicatesforeach

sample.Onlysteelorplastictools(knife,spadeandshovel)wereusedforsamplinginorderto

avoidheavymetalcontamination.

Soilsampleswereair‐dried,crushedandpassedthrougha2‐mmsievebeforeanalysis.pHwater

(soil/waterratio=1:5)wasmeasuredaccordingtoISO10390.

For Si and trace element analyses, a representative soil subsample was ground to 100 µm

particlesizebeforedigestion.ForCuandZnanalysis,calcinationat450°Cwasfollowedbytotal

dissolutionperformedusingamixtureofHF,HNO3andHClO4(ISO14869‐1).ForSiO2analysis,

complete dissolution was obtained by alkaline fusion of the soil sample in the presence of

sodiumperoxide(AFNORstandardBPX30‐428).

Phytoavailable fractions of Cu and Znwere estimated using an extractionmethod (Collin and

Doelsch2010).Aftershaking50mlof1molL‐1NH4NO3solutionand20gdrysoilsamplefor2h

at 30 rpm in a room at 20 ± 2°C, the extracts were centrifuged at 1000 g for 15 min. The

supernatantwasfilteredthroughamembraneunitfilter(0.22µm).

AllextractswereacidifiedwithHNO3andstoredinpolyethylenebottlesat4°Cbeforeanalysis.

All reagentswere analytical grade andonlyultrapurewater (PurelabPrimaplusClassic from

Elga Labwater)was used. All glass and plasticware used for the experimentswas previously

soaked overnight in nitric acid and rinsed with ultrapure water. Three replicates were

performed for each sample. Blank tubes (containing reagent but no soil) were also taken

throughouteachprocedure.

Silicon and trace element concentrations were then determined with an inductively coupled

plasma‐opticalemissionspectrometer(ICP–OESVista‐PRO,Varian,Inc.)withanaxiallyviewed

plasma system and a charge coupled device detector. For quality control, in‐house reference

samplesandcertifiedsamples(CRM7001LightSandySoilandCRM7004Loam,Analytica)were

usedevery10samplesandeachmeasurementwasconductedinduplicate.Thedetectionlimits

were0.025mg.kg‐1forCuandZn.Themeasurementuncertaintywaslessthan10%.


69

2.3. Plant material and analysis

Stems and leaves were washed with distilled water and dried at 60°C until constant weight.

Theyweresubsequentlymixed,groundandhomogenised.Sub‐samplesweredriedat80°Cuntil

constantweighttodeterminethedryweight.

TheplantsamplesunderwentdrymineralisationforZnandCutraceelementanalyses.During

mineralisation, the Si contentwas determined by gravimetric quantification: 500mg of dried

plant material was placed in a platinum dish and gradually heated to 500°C. Silica was

eliminated in the ash with HF. The Si weight was determined after cooling. The ash was

dissolved in HCl and the Cu and Zn contents in the solutionswere analysed by ICP‐OES. For

qualitycontrol,in‐housereferencesamplesandcertifiedsamples(Astrasol‐Mix,Analytika)were

used every 20 samples and each analysis was conducted in duplicate. The measurement

uncertaintywas±15%.ThequantificationlimitforSiwas5mgg‐1.

Copper and Zn concentrations are expressed as mg kg‐1 of dry matter (DM) and Si

concentrations are expressed inmg g‐1 DM SiO2 in order to have results directly comparable

withtheliterature.

2.4. Statistical analyses

The Minitab 15.1 software package (Minitab, Inc.) was used for statistical analyses. Mean

concentrationsatthestembase,thestemtipandleaveswerecomparedusingapairedt‐testat

the95%confidencelevel.

Foreachplantpart,concentrationsinthedifferentspecieswereanalysedbyANOVA.Weused

one‐wayANOVA at the 95% confidence levelwith bamboo “species” (16 levels) as themain

factor,followedbyTukey’sposthoctestatthe95%confidenceleveltoevaluatedifferencesin

Cu,SiandZnconcentrationatthestembases,thestemtipsandtheleaves.Differencesbetween

monopodialandsympodialbambooswereanalysedusingagenerallinearmodelwiththetype

ofbambooasfactor(2levels).Meanconcentrationsfrombothtypes(sympodialormonopodial)

werethencomparedusingTukey’sposthoctestatthe95%confidencelevel.


70

3. RESULTS

3.1. Soil

TotalSi,CuandZnsoilconcentrationsarepresentedinTableII.1.Themeantotalconcentrations

in the soil samples were 213 ± 23.3 mg g‐1 for SiO2, 26.5 ± 5.8 mg kg‐1 for Cu and

113±18.2mgkg‐1forZn,andtheaveragesoilpHwas6.1±0.3.Withinthestudyframework,soil

variations in Si, Cu, Zn concentration were reduced, while the coefficient of variation of

concentrationswithinthesampledareawasof11%forSiand21.8%forCuand16.2%(Table

II.1).TheCuNH4NO3‐extractable fractions (CuNH4NO3)werebelow thedetection limit inall soil

samplesandthemeanZnNH4NO3‐extractablefractions(ZnNH4NO3)were0.3±0.23mgkg‐1(Table

II.1).

Table II.1 Characteristics of the total soil population (N=18)

aDatafromDoelschetal.(2006)andCollinandDoelsch(2010)bDatafromKabata‐PendiasandMukherjee,(2007)

3.2. Between plant parts

TableII.2showstheaverageSi,Cu,Znconcentrationsatthestembase,atthestemtipandinthe

leaves.Withinthestem,Si,CuandZnconcentrationssignificantlyincreasedfromthestembase

tothetip.ThemeanconcentrationsatthestembasewereunderquantificationlimitforSi,3.5

mgCukg‐1and7.0mgZnkg‐1,whereasatthestemtip,themeanconcentrationswere21mgSig‐

1,4.5mgCukg‐1and14.8mgZnkg‐1.TheSicontentatthestemtipwasthusmorethan4.1‐fold

higherthanatthestembase,andthisdifferencewasgreaterthanthatnotedforCuandZn:1.3‐

and 2.1‐fold, respectively. The leaf Si and Cu concentrations, i.e. 109 mg g‐1 and 5.1 mg kg‐1

respectively,weresignificantlyhigherthaninthestem.


71

NocorrelationsbetweenCu,ZnandSicontentswerefoundinstemsandleaves(R2<0.18).There

wasnolongeranycorrelationbetweenthetotalSi,CuandZnsoilconcentrationandtheSi,Cu

and Zn plant concentration (R2<0.2), or between the ZnNH4NO3 fraction and the Zn plant

concentration(R2<0.3)(forCu,CuNH4NO3fractionsarebelowthedetectionlimit).

Table II.2 SiO2, Cu and Zn concentrations in the different plant parts (N=47). Data are expressed as mg kg-1 of dry matter for Cu and Zn concentrations and mg g-1 of dry matter for SiO2 concentrations.

Stembases Stemtips Leaves

Numberofsamples 47 45 47

Mean±standarddeviation <QLτ 21aττ±16 109b±30

Coefficientofvariation <QL 785 279

Range 5‐12 5‐102 23‐183

SiO2

ANOVAFSPECIES ‐ 2.12* 2.23*

Mean±standarddeviation 3.5a±1.0 4.5b±2.0 5.1c±1.0

Coefficientofvariation 28.1 43.9 18.8

Range 1.8‐6.4 1.7‐10.9 3.4‐7

Cu

ANOVAFSPECIES 4.99*** 5.53*** 6.8***

Mean±standarddeviation 7.0a±6.3 14.8b±16 15.7b±5.6

Coefficientofvariation 89.5 110 35.9

Range 1.5‐36.7 1.7‐87.6 9.8‐37.8

Zn

ANOVAFSPECIES 4.73*** 1.56NS 2.54*

τ<QL:underthequantificationlimitof5mgg‐1ττValues followedbysame letterwithin thesame lineare statisticallynotdifferentaccording toTukeytestatthe95%confidencelevel.

0ne‐wayanalysisofvarianceandF‐testforSiO2,Cu,Zn,concentrationsinstembases,stemtipsandleavesrelativetothespeciesfactor.NS:notsignificantp>0.05Sig.:*=P<0.05,***P<0.001

3.3. Between plant species

MeanSi concentrationswere significantlydifferentbetween species in leaves and in the stem

tips(TableII.2).Inourstudy,therewassubstantialvariationintheSicontentrangebetweenthe

16 species: from 5.7mg g‐1 inPhyllostachys aurea to 56mg g‐1 inBambusamultiplex ‘Golden

Goddess’ at the stem tip, and from 82 mg g‐1 in Phyllostachys bissetii to 159 mg g‐1 in

Dendrocalamus strictus in the leaves (Table II.3).We assumed that thiswide Si content range

could mainly be explained by the genotypic variation since both soil and climatic conditions

weresimilarforallthesampledspecies.


72

In order to explain the differences in Si content between species, we first compared Si

concentrationsbetweenthetwogeneraPhyllostachysandBambusa(datanotshown),andtheSi

concentrationwasnotsignificantlydifferent.However,wefoundsignificantdifferencesbetween

sympodialandmonopodialbamboos(FigureII.1),withtheSiconcentrationsbeingsignificantly

higherinsympodialthaninmonopodialbamboos,bothinstemsandleaves.

For Cu, differences between species were significant both in stems and leaves (Table II.2).

Bambusamultiplex“AlphonseKar”hadthehighestCuconcentration,with:7.6mgkg‐1 instem

tips,whilePhyllostachysbambusoides“Castillon”hadthelowestconcentration,with2.0mgkg‐1

instemtips,and3.5mgkg‐1intheleaves(TableII.3).GroupingspeciesbytypesshowedthatCu

concentrationsweresignificantlyhigherinsympodialthaninmonopodialbamboospecies,both

instemsandleaves(FigureII.1).TherangeofZnconcentrationsintheleavesandatthestem

base was broad and significantly different between species (Table II.2). For example, the Zn

concentration in the stem bases ranged from 2.3 to 21.7 mg kg‐1 in Gigantochloa sp. “Malay

Dwarf” andThyrsostachys siamensis, respectively (Table II.3)but the influenceof specieswith

respect to concentrations in the stem tipswasnot significant.UnlikeCu,Zn concentrations in

leavesweresignificantlyhigherinmonopodialthaninsympodialbamboospecies.Instems,Zn

concentrationswerenotinfluencedbythetypeofbamboo(FigureII.1).


73

Figure II.1 Comparison of SiO2, Cu, Zn concentrations in stem bases, stem tips and leaves of

monopodial bamboos (n= 18) and sympodial bamboos (n = 29). DM: dry matter; Boxes represent the median (vertical solid line) and 25–75 % percentile. Whiskers represent the 90th and 10th percentile. Significant differences were determined by a post hoc comparison of means (Tukey test after ANOVA; P < 0.05) and are indicated by different letters.


74

Tab

le II

.3

Mea

n Si

O2,

Cu

and

Zn

conc

entr

atio

ns i

n di

ffere

nt p

lant

par

ts i

n ea

ch s

peci

es.

For

each

spe

cies

, th

e sa

mpl

e nu

mbe

r is

3.

The

ty

pe o

f bam

boo

is in

dica

ted:

S: S

ympo

dial

bam

boo

spec

ies,

M: M

onop

odia

l bam

boo

spec

ies.


75

4. DISCUSSION

4.1. Phytoavailability of Si, Cu and Zn

Themean total SiO2 concentration of the soil samples (213 ± 23.3mg g‐1)was similar to the

meanSiconcentrationpreviouslymeasuredinRéunionsoils,i.e.216mgg‐1(TableII.1)(Doelsch

etal.2006).Thisvolcanicsoilcontainseasilyweatherablesilicatemineralsduetothepresence

ofpoorlycrystallinemineralsandparticularlyverylowpolymerizedaluminosilicatesthatmay

contributethephytoavailableSipool(Basile‐Doelschetal.2005).

ThemeantotalCuconcentration(TableII.1)wasclosetothemeanconcentrationinworldsoil

(20mgkg‐1),butlowerthanthemeanconcentrationmeasuredinasetofRéunionsoils(Doelsch

etal.2006).ThemeanZnconcentrationwasalsomuchhigherthanthemeansoilconcentration

givenbyKabata‐PendiasandMukherjee(2007),andslightlylowerthanthemeanconcentration

calculated for Réunion soils (Doelsch et al. 2006). These larger Cu and Zn concentrations as

comparedtoworldsoilconcentrationscouldbeexplainedbytheoriginoftheparentmaterial:

soilsformedfromthePitondesNeigesvolcanicmaterialarecharacterisedbylowCr,CuandNi

concentrationsandrelativelyhighZnconcentrations(Doelschetal.2006). Indeed,theselatter

authorsdemonstratedthatthenaturalpedogeochemicalbackgroundcouldaccountforthehigh

Cr, Cu, Ni and Zn concentrations in Réunion soils. In spite of these higher average total

concentrations,theCuandZnNH4NO3‐extractablefractionswerelow,whichisconsistentwith

thefindingsofCollinandDoelsch(2010),whodemonstratedthelowphytoavailabilityofCu,Cr,

NiandZninRéunionsoils.TheabsenceofcorrelationsbetweenCuandZntotalconcentrationin

soilandCu,Znconcentrationinplants(R2<0.2)wasthusnotsurprisingandconfirmedthelow

phytoavailabilityoftheseelements.

4.2. Origin of Si variation in bamboos

Silica concentrations in leaveswerewithin the 20 to 410mg g‐1 Si range reported in several

previous studies (Table II.4). Silicon concentrations in stems ranged from <5 to 102 mg g‐1,

whichisabroaderrangethanreportedintheliteraturepresentedinTableII.4(3–44mgg‐1).

The mean Si concentration measured at the stem tip in the 16 species (21 mg g‐1) was

significantlyhigherthantheconcentrationatthestembase(underthedetectionlimit),whichis

out of linewith the findings of Li et al. (2006). Indeed, at the stembase in theMosobamboo

stand(Phyllostachysheterocyclavar.pubescens),theseauthorsmeasuredaconcentrationof44

mgg‐1Si,whereastheymeasured1.5mgg‐1Siinthestem.However,theexactlocationsofthe

analysedsamplescorrespondingto“baseofthestem”andthe“stem”werenotgivenbyLietal.


76

(2006),thuslimitingthepossibilityofcomparisonwithourresults.Siliconconcentrationsthus

variedbetweenplantparts,withtheaccumulationofSi in leavesandaconcentrationgradient

along thestem.Toexplain thedistributionwith theplant, transpirationhasbeenproposedas

the main mechanism for Si transportation and precipitation in Chinese bamboos (Ding et al.

2008).Theevidenceisbasedonthetotalsiliconcontentandδ30Sivalues,whichbothincrease

fromthestem,throughthebranchestotheleaves.Theresultsofthisstudyareconsistentwith

the hypothesis of silica being carried passively through the transpiration stream and being

depositedwherewaterislostinlargestquantities,asproposedbyDingetal.(2008).However,

inanSiaccumulator,itislikelythatactivesilicondistributionmechanismsinbambooshootsare

alsorequired(MaandYamaji2008).

In leaves and at the stem tips, therewasmarked variation in the Si content between the 16

sampledspecies(TableII.2).ThisresultisingoodagreementwiththefindingsofHodsonetal.,

(2005)whoshowedthatvariabilityinSicontentover735plantspeciesismainlyexplainedby

genotypic variation rather than environment. However, although differences in Si content

between plant families are well known (Hodson et al. 2005), genotypic variation in Si

concentration within species is less documented. The effect of genotypic variation has been

studiedinafewotherplants,particularlyinotherPoaceaespecies.Forexample,in52sugarcane

genotypesgrowninthesamesoil,theshootSiconcentrationrangedfrom6to8mgg‐1(Derenet

al. 1993). In rice, the sameauthors compared theSi concentration inplants from18cultivars

growningreenhouseexperimentsandinfieldsamendedwithvariousamountsofSi.Thesilica

concentrationinplanttissuesrangedfrom3to60mgg‐1dependingontheSisupply,butwithin

eachSitreatmentthecoefficientofvariationduetogenotypicdifferenceswas9to17%(Deren

etal.2001;Derenetal.1993).Infieldtrials,Winslowetal.(1997)observeddifferencesbetween

japonica and indica rice cultivars, with mean Si concentrations in husks of 2 and 1.2 mg g‐1,

respectively.Inasurveyofabout400barleyspecies,theSiconcentrationingrainrangedfrom0

(under the detection limit) to 3.8 mg g‐1 in hulled barley cultivars (Feng Ma et al. 2003).

However, no significant differences in Si absorptionwere observed in three different banana

genotypes grown in hydroponic culture conditions (Henriet et al. 2006). Although genotypic

differencesinSicontenthavebeenfoundinotherPoaceaespeciessuchasrice,sugarcaneand

wheat,thisisnotsystematicinallspeciesandmayquestionthegeneticmechanismsthatcontrol

Siaccumulationindifferentspecies.


77

Table II.4 Inventory of Si data found in the literature.

DataareexpressesasSiO2mgg‐1ofdrymatterforSiconcentrations

1:Dingetal.2008;2:Lietal.2006;3:Luxetal.2003;4:Meunieretal.1999;5:Motomuraetal.2002;6:UedaandUeda.1961(fromMaandTakahashi,2002).

Siconcentrationsweresignificantlyhigherinsympodialbamboosthaninmonopodialbamboos,

inbothstemsand leaves(Figure II.1 ).This isconsistentwith findings in the literature(Table

II.4).Forexample,higherSiconcentrationswerereportedinsympodialbambooleaves(114mg

g‐1)thaninmonopodialbamboosleaves(82mgg‐1)(TableII.4).

There aremarkedmorphological differences betweenpachymorph rhizomes (sympodial) and

leptomorph rhizomes (monopodial), but no differences in root development and nutrient

absorption capacity have been noted between the two types (Kleinhenz andMidmore 2001).

Since theseauthorsalsoreportedthatat least80%of the totalrootbiomass is located in the

topsoil (0‐30 cm) regardless of the species (Kleinhenz and Midmore 2001), we assume that

rootsofbothbambootypeswerewithinthesamesoillayer,sothecharacteristicsofthesoilin

contactwithrootsweresimilar.

DifferencesinSiaccumulationmaybeattributedtodifferencesinthesiliconuptakecapacityof

roots.Recently,twogenes(Lowsiliconrice1:Lsi1andLsi2)encodingsilicontransporterswere

identified in japonica rice (MaandYamaji2008).Maetal., (2007)have shown thatgenotypic

differences between two rice species, japonica and indica, were due to the difference in

abundanceofSitransportersinriceroots.Therefore,differencesbetweenbamboosspecies,and


78

toafurtherextentbetweenthetwotypesofbamboocouldreflectadifferenceinexpressionof

genesresponsibleforsiliconuptake.

Afterrootuptake,Siistranslocatedtotheshootviathexylem.Itislikelythattransportersare

also required for xylem Si loading and unloading and for distributing Si to the above‐ground

plant parts (Ma and Yamaji 2008). Genetic differencesmay also be expressed in the relative

distribution of Si in stems and leaves. For example, Keeping et al., (2009) found that cultivar

differences in sugarcane stalks could be explained by differing propensities of cultivars to

deposit Si within the stalk epidermis. Active processes could partly explain differences in Si

concentrationsbetweenspecies,buttheunderstandingofthesilicadepositionprocessandthe

identificationoftransportersinplantshootsstillneedtobestudied.

In the above‐ground parts of bamboos, Kleinhenz and Midmore (2001) highlighted that the

lifespanofleaveswassubstantiallydifferentbetweenmonopodialandsympodialbamboos.The

canopyofmonopodialspeciesisrejuvenatedeveryyearwhen2‐year‐oldleavesarereplacedby

newones.Thoseofsympodialspeciesremainonculmslonger,i.e.uptoabout6years.Therefore

culms of sympodial bamboos of over 2‐years‐old contain relatively older and less productive

leavesthanmonopodialbamboos.Motomuraetal.(2002)reportedthatinbambooleavessilica

iscontinuouslyaccumulated inthetissuesthroughouttheir life.Leavesofsympodialbamboos

may thereforehave a higher Si content than leaves ofmonopodial bamboos.However, in this

study,weonly sampled1‐year‐old stems, so the leaf ages shouldall havebeen the same.The

differencesobservedinourstudybetweenmonopodialandsympodialbambooswerethusnot

relatedtothischaracter.

4.3. Origin of Cu and Zn variability in bamboo

DuetothelackofpublishedZnandCuconcentrationsinbamboos,wefirstcomparedourresults

withanotherPoaceaespecies,i.e.sugarcane(stems),growinginRéuniononsimilarsoils(Collin

andDoelsch2010).Copper concentrations inbamboo sampleswerehigher than in sugarcane

samples,withanaverageof3.5±1.0and4.5±2.0mgkg‐1atthestembaseandtip,respectively

(Table II.2), whereas in sugarcane the average was 2.1 ± 0.6 mg kg‐1. Zinc concentrations in

bamboostemsweresimilartoconcentrationsinsugarcanestems,withanaverageof7.0±6.3

and14.8±16mgkg‐1atthebamboostembaseandthetip,respectively(TableII.2),and10±5.2

mgk‐1 insugarcane.Wethencomparedleafconcentrationswithdatacompiledformatureleaf

tissue from various plant species (Kabata‐Pendias and Mukherjee 2007). The concentration

rangesthattheseauthorsconsiderednormalwere5‐30mgkg−1forCuand27‐150mgkg−1for

Zn. The concentration measured in bamboo leaves were within this range for Cu, with an


79

averageof5.1±1.0mgCukg‐1,andlowerforZn,withanaverageof15.7±5.6mgZnkg‐1(Table

II.2).CuandZnconcentrationsinabove‐groundpartsofRéunionbambooswerethusrelatively

low, which may confirm the low phytoavailability of these elements measured by NH4NO3‐

extraction,aswellasthenonspecificabilityofbamboostoaccumulateCuandZn.

Within thestem,CuandZnconcentrations,similar toSi, significantly increased fromthestem

base to the tip (Table II.2), suggesting that part of this element translocation was driven by

transpiration. However, metals do not move freely in the plant. Interactions of cations with

negativelychargedsites(mainlywithpectins)inxylemorphloemcellwallsleadtodecoupling

ofiontransportandwaterflow(Francoetal.2002).Inaddition,mostmetalsarecomplexedby

organic acids, amino acids, peptides, metallothionine or phytochelatin (Broadley et al. 2007;

CobbettandGoldsbrough2002;Liaoetal.2000).ThegreaterZnaccumulationatthestemtipas

comparedtothatofCumaythusbeexplainedbydifferencesinthemobilityofmetalcomplexes

formedwithintheplant.

SignificantdifferencesbetweenspeciesweremeasuredforCuinstemsandleavesandforZnin

leaves (Table II.2). This finding is in good agreementwith the studyofBroadley et al. (2007)

who reported that, in a dataset of 365 species, therewere substantial differences in shoot Zn

contentbetweenandwithingeneraand species.Metal absorption inplants isbothactiveand

passive,andmetabolicmechanisms,suchastheexpressionofspecifictransportersinrootcells,

is genetically controlledandmay thusvarybetweenspecies. Forexample, theexpressionand

gene organization of proton pumping ATPase genes in the plant plasmamembrane seems to

differbetweenspecies(MorsommeandBoutry2000).Moreover,theavailabilityofelementsin

thesoilandrootuptakecanbeaffectedbyplantfactorssuchasrootexudates,rootsurfacearea,

rootabsorptionabilityandmycorrhization(Kelleretal.2003;Langeretal.2010;Whitingetal.

2000). For example in rice, Zn uptake efficiency is correlated with exudation rates of low

molecularweightorganicanionsandasubstantialproportionofthephenotypicvariationinZn

uptakeefficiencyisundergeneticcontrol(Wissuwaetal.2006).Inwheat,genotypicvariations

inZnuptakemaybe related to the releaseofphytosiderophores (mugineic andavenic acids),

whichwereshowntosignificantlyenhanceZnbioavailability(Cakmaketal.1996;Rengeletal.

1998; Tolay et al. 2001). Thus, these mechanisms and inherent differences in uptake,

translocationandaccumulationmaypartlyexplainthesignificantdifferencesnotedinCuandZn

concentrationsamongthe16bamboospecies.

DifferencesbetweenmonopodialandsympodialbamboosweresignificantforCuandZn,butno

information is available in the literature on any possible specific behaviour regardingmetals


80

between the two typesofbamboo.Analysesof thedifferenceswithSi contentwerediscussed

above,andmayalsoapplyforCuandZn.

4.4. Interactions between metals and Si

NocorrelationsbetweenCu,ZnandSicontentswerefoundinstemsandleaves.Inthenatural

pedo‐climaticenvironmentofRéunion,withasoilhavinglowNH4NO3‐extractableCuandZn,the

Si and trace metal behaviours in bamboos seem to be independent. This could be easily

explained since thebeneficial effects of Si areusually expressedwhenplants are subjected to

stressconditions(Liangetal.2007).Ithasbeenshown,forexample,thatSialleviatesZntoxicity

inheavymetal‐tolerantCardaminopsishalleri(NeumannandzurNieden2001).Theseauthors

suggestedthattheformationofZn‐silicatecompoundsinthecytoplasmmayberesponsiblefor

the alleviation of the Zn toxicity. InArabidopsis thaliana, Li et al. (2008) have shown that Si

improvestheresistancetoCustress.ArecentstudyhasshownthatSimodulatestheexpression

of various genes involved in Cu tolerance in A. thaliana (Khandekar and Leisner 2011). Our

studywasnotdesignedtoassesstheinteractionbetweenSiandmetalsatthecellularlevel,but

wecanassumethat,withsuchahighSiabsorptioncapacityinitstissues,bamboomaybeableto

toleratehighermetalconcentrationsthanthosepresent inRéunionsoils.However, thiswould

needfurthertestingwithincreasingmetalconcentrations.

In conclusion, we report the first data on Cu and Zn concentrations in stems and leaves of

variousbamboospeciesandnewdataonSiinbamboos.Ourresultshighlighttheimportanceof

thevariabilitybetweenspecies,particularlybetweenmonopodialandsympodialspeciesforZn,

Cu, andSi contents inbamboos, suggesting that a genotypic charactermaybe responsible for

theiraccumulation.


81

ACKNOWLEDGMENTS

This work was financially supported by the Direction Générale des Entreprises, Direction

GénéraledelaCompétitivité,del’IndustrieetdesServices,régionRéunion,régionPACAinthe

frame of the research program RUN INNOVATION II and by the Association Nationale de la

RechercheetdelaTechnologie(CIFREgrant).

TheauthorsthankMrAlexandrePerrussot,MrGregoryBoisandMmeVeroniqueArfifortheir

assistanceandfruitfuldiscussions.

5. BILAN DU CHAPITRE II

Le silicium est majoritairement accumulé dans les feuilles, au sein desquelles sa

concentration varie entre 82±2,5 et 159±2,3 mg.g‐1 de MS. Les concentrations en Cu et Zn

mesurées dans les bambous sont relativement faibles, elles sont comprises entre 2,0±0,3 et

7,6±1,8mgCukg‐1deMSetentre2,3±0,5et41±40mgZnkg‐1dansleschaumesetlesfeuilles.

Unepartiede la variabilitéobservéeest expliquéepardesdifférencesd’accumulation entre

les16espècesdebambous.Nousavonsmontréquelesbamboussympodiaux,présentsdans

lesrégionsàclimattropical,accumulentplusdeSietCudansleschaumesetlesfeuilles,que

les bambous monopodiaux. A l’inverse, les bambous sympodiaux accumulent moins de Zn

danslesfeuillesquelesbambousmonopodiaux.

Danscesconditionspédoclimatiquesnaturelles,lescomportementsdeSietCudanslebambou

semblentindépendants.


82



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86

C H A P I T R E I I I

EFFECTS OF S IL ICON AND COPPER

ON BAMBOO GROWN

HYDROPONICALLY

ChapitreIII:Effectsofsiliconandcopperonbamboogrownhydroponically

88


89

TABLE DES MATIERES

CHAPITREIIIEFFECTSOFSILICONANDCOPPERONBAMBOOGROWNHYDROPONICALLY........................................................................................................................91

1 INTRODUCTION ......................................................................................................................................................95

2 MATERIALANDMETHODS .................................................................................................................................962.1 Plantmaterial,experimentaldesignandpreculture............................................................................................... 962.2 Experimentinnutrientsolution.......................................................................................................................................... 972.2.1 PhaseI:Sitreatment .................................................................................................................................................972.2.2 PhaseII:Cu+Sitreatment .....................................................................................................................................98

2.3 Plantanalysis .............................................................................................................................................................................. 992.3.1 Growthparameters ...................................................................................................................................................992.3.2 Analysisoftissues ...................................................................................................................................................100

2.4 Statisticalanalyses ................................................................................................................................................................ 100

3 RESULTS................................................................................................................................................................. 1013.1 Evolutionoftheelementconcentrationsinthenutrientsolution.................................................................... 1013.2 Bamboogrowthparametersandelementaldistribution .................................................................................... 105

4 DISCUSSION........................................................................................................................................................... 1094.1 Siliconinbamboo ................................................................................................................................................................... 1094.1.1 Mechanismsofsiliconaccumulation ..............................................................................................................1094.1.2 EffectofSiuptakeonthenutrientconcentrationandgrowthparameters ...................................111

4.2 Effectsofcopperonbamboogrowth............................................................................................................................. 1124.3 EffectsofbambooSionCusensitivity ........................................................................................................................... 115

5 BILANDUCHAPITREIII .................................................................................................................................... 117

6 REFERENCES ......................................................................................................................................................... 118


90


91

C H A P I T R E I I I

EFFECTS OF S IL ICON AND COPPER ON BAMBOO GROWN

HYDROPONICALLY

L’un des objectifs principaux de ce travail de thèse est d’évaluer le rôle du silicium sur les

bambous, en vue d’améliorer, si possible, leur croissance et donc leur productivité dans un

systèmedephytoremédiation.Nousavonsvudanslechapitreprécédentquelesconcentrations

deSiaccumuléesdanslesbamboussonttrèsfortes,109mg.g‐1enmoyennedanslesfeuilles.On

peut donc se demander si la quantité de Si biodisponible dans le milieu va influencer leur

croissance.UndéficitdeSiaffecte‐t‐illeurdéveloppement?Lacroissancevat’elleêtrecorrélée

àlaquantitédeSidisponible?

Surunsolprésentantdesconcentrationsdecuivrephytodisponiblesfaibles,nousavonsmontré,

danslechapitreprécédent,quelesbambousaccumulentdesconcentrationsrelativementbasses

deCudansleurstissus:de3,5à6,3mg.g‐1danslesfeuilles.Danscecontexte,lescomportements

deSietdeCudans lebambousemblent indépendants.Maisonpeutsedemandersi les fortes

concentrationsdeSiaccumuléesdans lesbambousvontpermettred’améliorer latolérancedu

bambouàdeplusfortesconcentrationsdeCu.Jusqu’àprésentlesétudesmontrantuneffetdeSi

surlesmétaux,ontétéeffectuéesdansdesconditionsdefortetoxicitémétallique.Parexemple,

Olivaetal. (2011)ontmis leursplantesaucontactd’unesolutionde300µMdeCuenculture

hydroponique. Des concentrations de Cu libre si élevées ne sont pasmesurées en conditions

environnementales,enraisondelaforteaffinitédeCupourlamatièreorganiqueprésentedans

le sol (Sauvé et al. 1997). Pour cette étude, nous avons choisi d’étudier le rôle de Si sur des

bambousquisontexposésàuneconcentrationdeCuélevéemaiscohérenteavecdesconditions

environnementales.

Afindecontrôler les concentrationsdeCuetSiapportéesauxbambous,nousavonscultivé le

bambouenhydroponie.Cette techniquenousapermisde fairevarieruniquementSietCu,de


92

suivreleursconcentrationsdanslessolutionsnutritivesetd’observerleseffetsdeceséléments

surlesbambous.

Lesobjectifsdecetteexpériencesontlessuivants:

• Localiser et quantifier Si dans les tissus du bambou exposés à une large gamme de

concentrationdeSidanslasolutionnutritive;

• Evaluerl’effetdeSisurlacroissanceetledéveloppementdubambou;

• Evaluerl’effetdeSisurlaconcentrationetlasensibilitéaucuivredubambou;

L’article ci‐après décrit l’expérience d’hydroponie qui a été réalisée pendant 11 mois. Nous

avons choisi une espèce de bambou sympodial: Gigantochloa sp «Malay Dwarf» pour cette

expérience.LesbambousontétéexposésàunelargegammedeSiensolution(de0à1,5mM)

pendant6mois,puisàuneconcentrationpotentiellementtoxiquedeCu(1.5µMCu2+)pendant2

mois.

CetarticleestsoumisàlarevuePlantandSoil.


93

Effects of silicon and copper on bamboo grown

hydroponically

BlancheCollin*a,b,EmmanuelDoelschc,CatherineKellerb,FredericPanfilia,Jean‐Dominique

Meunierb


France

bCEREGE,CNRS,Aix‐MarseilleUniv.,Europôleméditerranéende l'ArboisBP80,13545Aixen

Provence,France

cCIRAD,UPRRecyclageetrisque,34398Montpellier,France


ABSTRACT

Due to its high growth rate and biomass production, bamboo is frequently used in industrial

applicationsandhasrecentlyproventobeusefulinwastewatertreatment.Bambooaccumulates

highsilicon(Si)levelsinitstissues,whichmayimproveitsdevelopmentandtolerancetoabiotic

stresses, such as metal toxicity. This study investigates the beneficial effect of Si on bamboo

growth and copper (Cu) sensitivity. A hydroponic culture of bamboo Gigantocloa sp. “Malay

Dwarf”wasperformedfor8months.Thebambooplantswere firstsubmittedtoarangeofSi

supplementation(0to1.5mM)thatwasaddedtoapotentiallytoxicCuconcentrationforplants

(1.5µMCu2+)after6months.

Silicon affected neither the development (number of stems and leaves, height and total dry

mass) nor the nutrient content of the bamboo plants. The Si concentration in the plants

increasedmarkedlywithanincreasingSiconcentrationinthenutrientsolution.Thedistribution


94

ofSiamongthedifferentplantpartsinalloftheSitreatmentswasasfollows:root<stem<leaf.

In the leaves, the Si concentration ranged from below the detection limit to 218 ± 19mg g‐1,

whichisoneofthehighestSivaluesofSifoundthathavebeenobservedinbambooplantsthat

weregrowninnature.Aftereachrenewalofthenutrientsolution,theSicontentinthesolution

decreased progressively, whereas the Cu concentration decreased abruptly during the first

hours and then remained constant in the solution.Thesedifferences inCuandSi uptakemay

indicatethatbambootakesupandallocatesthesetwoelementsdifferently.TheCuadditiondid

not induce any toxicity symptoms in the bambooplants. The absorption of Cu in the bamboo

plantswasnotalteredbytheSisupplementation;Cuaccumulatedmainly inroots(131±39.3

mgkg‐1),whichwasfollowedbyitsaccumulationinleaves(16.6±3.6mgkg‐1)andstems(9.8±

3.8mgkg‐1).

Hydroponic culture has proven to be an efficient technique to grow the sympodial bamboo

Gigantocloa sp. “MalayDwarf”. Bamboo growthdoes not dependon Si levels even though the

bambooGigantocloasp.“MalayDwarf”didabsorbSiinlargeamounts.Thedifferentabsorption

mechanismsforCuandSimaypartiallyexplainwhySididnotinfluencetheCurepartitionand

concentration in bamboo. Given the high biomass and its adsorption capacity, the bamboo

tolerateshighCuconcentrationsinnutrientsolutions.

Keywords:hydroponics,metal,siliconaccumulator,Poaceae


95

1 INTRODUCTION

Extensive agronomic evidence supports the beneficial effects of silicon (Si) on the growth,

developmentandyieldofplantsandtheprotectionofplantsagainstbioticandabioticstresses

(Cooke and Leishman 2011; Epstein 1994; Epstein 1999). Bamboo species that contain large

deposits of silica in the leaves (Motomura et al. 2004) are classified among the silicon‐

accumulating plants (Ma and Takahashi 2002). Although Si distribution in bamboo has been

describedinseveralstudies(Collinetal.2011;Dingetal.2008b;Lietal.2006;Luxetal.2003),

the effects of Si on bamboo growth are poorly described (Ma and Takahashi 2002). Bamboo

speciesarecommonlyfoundintemperate,tropicalandsubtropicalregions,andtheyarewidely

usedforindustrialpurposes,asfreshedibleshoots,formakingpaper,asbuildingmaterialand

even inmedicine. They are known for their resistance to awide range of stress factors, their

high growth rate and their high biomass production, and they have potential uses in

phytoremediation. The PHYTOREM company (France) has developed BAMBOO‐

ASSAINISSEMENT® technology for wastewater treatment (Arfi et al. 2009). Therefore, the

potential beneficial effects of Si on bamboo may be used to optimise the phytoremediation

capacityofbamboo.

For various reasons, there is increasing concern regarding copper (Cu) toxicity in agriculture.

TheseconcernsinvolvethehighsoilCucontentsthatarecausedbythelong‐termuseofcopper–

containing fungicides (e.g., in vineyards) (Michaudet al. 2007), industrial andurbanactivities

(airpollution,citywasteandsewagesludge),andtheapplicationofpigandpoultryslurriesthat

arerichincopper(Legrosetal.2010;Marschner1995).

Silicon is known to play a significant role in minimising the toxic effects of metals. Several

mechanisms that are responsible formetal‐stress alleviationhavebeenproposed, such as the

modificationofthemetaldistributioninsidetheplant,anincreaseofmetalbindingtocellwalls,

andtheco‐precipitationofSi‐metal(RogallaandRomheld2002).Thesestudieshaveprimarily

focussed on Cd, Zn, Mn and Al, but Cu has been poorly studied to date. Two recent studies

regardingtheCu‐tolerantspeciesEricaandevalensisandArabidopsisthalianahavedescribedthe

beneficial effects of Si onCu toxicity (Khandekar andLeisner2011; Li et al. 2008;Oliva et al.

2011).Olivaetal.(2011)reportedthatadditionofSireducedtheleafmetalconcentration,while

Li et al. (2008) suggested that Si affects metal distribution or bioavailability. The Cu

concentration in bamboo and its interaction with Si has been recently assessed (Collin et al.


96

2011). Collin et al. (2011) provided the first data regarding Cu concentrations in various

bamboospecies thatweregrownundersimilarnaturalpedo‐climaticconditions; theyshowed

that theCuconcentrationswererelatively low,whereasSiwasaccumulated in the leavesata

levelof82to159mgg‐1dryweightdependingonthespecies.Withsuchahighvariability,itwill

be interesting to assess the extent of Si accumulation under increasing available Si

concentrations and the effects that this supplementationmayhaveonCuuptake and toxicity.

We chose to grow bamboo using hydroponics. Themain advantage of this procedure is that

plantscanbegrownunderoptimalnutrientconditions,andthenutrientsupplycanbeadapted

over time as necessary. To our knowledge, this is the first example of the application of this

techniquetobamboo.Thequestionsthatareaddressedinthisstudyareasfollows.1)Howare

bamboogrowthanddevelopmentaffectedbyvariousSisupplementations?2)HowdoestheSi

quantityandlocalisationindifferentbambooorganschangewithSisupplementation?3)DoesSi

supplementationmodifythecoppersensitivityofbamboo?

2 MATERIAL AND METHODS

2.1 Plant material, experimental design and pre-culture

Fiftyone‐year‐oldsympodialbambooplantsGigantocloasp.“MalayDwarf”thatweregrownon

the same substratewere provided by a bamboo nursery (A. PERRUSSOT, Le Guillaume, Saint

Paul,Réunion Island). Inmost of thehydroponic experiments that havebeenperformedwith

other species, seeds were directly used; however bamboo seeds are not typically used to

propagate bamboo plants. Indeed, bamboo seeds are difficult to obtain and have a low

germination rate. Most of the time, the propagation of bamboo is performed by vegetative

reproduction, which involves culm cutting for sympodial bamboo and rhizome cutting for

monopodialspecies.However,thegrowthfromcuttingisslowandproducesnon‐homogeneous

plants; therefore, we decided to use soil‐grown bamboo andmade a posteriori selections for

theirsimilarsizeanddevelopmentsothatwecouldworkwithahomogeneouspopulation.The

adaptation of the bamboo plants to hydroponic conditions was delicate because the bamboo

rootswerefirstadaptedtosoil.Afteracarefulwashingoftherootsincludingremovalofallof

thesoilparticles,thebambooplantsweretransferredintoaeratednutrientsolutiontanks.

Theexperimentwasperformedfor11months.Athree‐monthpre‐culturephasewasconducted;

during this period, the bamboo plants lost their leaves and began to develop new ones. The

bamboogrowthwasconducted for6months (phase I).During thisperiod, thebambooplants


97

weresupplementedwitha largerangeofSiconcentrationstoobservetheSieffectonbamboo

growth. After the bamboo was well‐developed with several news stems and leaves, Cu was

addedtotheSitreatmentsfor2months(phaseII).Thissecondphasewasperformedtoobserve

theeffectofSionCuconcentrationsinbamboo.

The composition of the pre‐culture nutrient solution consisted of a 25 %‐strength complete

Hoaglandsolution.Thecompositionofthemacroelements(mM)wasasfollows:1Ca(NO3)2,1.5

KNO3, 0.24MgSO4, and 0.22 (NH4)2HPO4; the composition of themicroelements (µM)was as

follows:11.6H3BO3,0.08CuSO4,0.2ZnSO4,0.03MoO3,2.3MnCl2,and100FeNaEDTA.ThepHof

thissolutionwasadjustedtoapHof6withNaOHorHNO3.Thenutrientsolutionwasrenewed

every7days.Analytical‐gradechemicalreagentsandultrapurewaterwereusedtopreparethe

nutrient solutions. No glassware was used, which minimised Si contamination. All of the

plasticwarethatwasusedintheexperimentswaspreviouslysoakedovernightinnitricacidand

rinsed with ultrapure water. The growth chamber parameters were set to the following

(day/night):28/24°C,90/70%relativehumidityand450μmolphotonsm−2s−1lightintensity.

2.2 Experiment in nutrient solution

2.2.1 Phase I: Si treatment

At the end of the pre‐culture stage, 25 bamboo plants of similar sizes were selected and

transferred to individual pots. The plants were then grown in cylindrical PVC pots that

contained 2.5 L of nutrient solution. For 6months, fromday 0 to day 191, five Si treatments

wereappliedasfollows:0.0(control),0.38,0.75,1.13and1.5mM,whicharereferredtoasSi0,

Si04, Si08, Si1.1, and Si1.5, respectively. These concentrations were chosen to cover a large

range of environmentally relevant Si concentrations. Indeed, in natural soil solutions,

orthosilicicacid(H4SiO4)concentrationsrange from0.1 to1.7mM(Knightetal.2001).Siwas

provided as amonosilicic acid from potassiummetasilicate Si(KOH)2,which has amolar K:Si

ratio of 2 (Metso 400 ‐ YARA) (Voogt et al. 2001). The potassium and hydroxide levelswere

adjusted to compensate for the additional input of K and OH from the addition of Si. The

compositionof themacroelements(mM) in thismodifiednutrientsolutionwas therefore0.35

Ca(NO3)2, 0.3 CaCl2, 0.24MgSO4, and 0.22 (NH4)2HPO4, and the composition ofmicroelements

(µM)was11.6H3BO3,0.08CuSO4,0.2ZnSO4,0.03MoO3,and6.5MnCl2.Fewasprovidedas15

µM Fe‐N, N9‐di (2‐hydroxybenzyl) ethylenediamine‐N, N9‐diacetic acid monohydrochloride

hydrate (HBED; Strem chemical, USA). Fe(III)‐HBEDwas prepared according to Chaney et al.

(1998) in a manner such that all of the HBED was saturated with Fe. The KNO3 and HNO3


98

concentrations were adjusted in each treatment such that the concentrations of K and NO3

reached 3 mM. The solution was set to a pH of 6.0 (±0.2) and was buffered with 1 mM 2‐

morpholinoethanesulphonicacid(MES).

Five replicates per treatment were conducted. The solution was continuously supplied by a

peristalticpumpfromthe15‐L“reservetank”tothebaseofthefive2.5‐Lpots,andthesolution

thatexceeded2.5Lwasrecoveredviaanoverflowpipeandreturnedtothereservetank.The

nutrientsolutionswerecontinuouslyaeratedwithanairpumpineachpot.Allofthesolutions

fromeachpotandthereservetankwerecompletelyrenewedevery7days.

The nutrient solution was sampled throughout the experiment at the beginning of the week

(beforecontactwithbamboos),referredtoas“inputsolution”.Thesolutionwasalsosampledat

theendoftheweek(justbeforetherenewal),whenitwasreferredtoasthe“outputsolution”.

ThepHwasmeasuredinallofthesolutionswithacombinedglasselectrode.Thesolutionswere

thenfiltered(0.2µm,Sartorius)andacidifiedtopH<2usingconcentratedHNO3,andtheywere

refrigerated(4°C)before theanalysis.Thesolutionswereanalysed for thepresenceofcations

(Si,Ca,Fe,Mg,Mn,P,andZn)byinductivelycoupledplasma‐atomicemissionspectrometer(ICP‐

AESJobinYvonJ38).

2.2.2 Phase II: Cu + Si treatment

Fromday191untilday226(2months),1.5µMCuwasaddedtoeachSitreatmentusingCuSO4.

TheSiandnutrientconcentrationsweremaintainedatlevelsthatwereidenticaltophaseI.The

CuconcentrationwaschosenbecauseithadbeenpreviouslyshowntoinducetoxicityinPoaceae

plants(Bravinetal.2010),anditwasconsistentwithCuconcentrationsthatweremeasuredin

contaminated soil (Sauvé et al. 1997). Due to the high bamboo biomass at this stage, which

consequentlyrequiredhigherwateruptake,thenutrientsolutionswererenewedevery5days.

GeochemicalcalculationswereperformedusingPHREEQC(version2‐17)(Parkhurst1995)and

theMINTEQdatabaseofthermodynamicconstantstodeterminetheCuspeciationintheinput

solution.Thecalculationsindicatedthatfreecoppershouldaccountfor97.6%ofthetotalCuin

thesolutionatpH6.ThequalityofthespeciationmodellingwasconfirmedbymeasuringCu2+

withacupricionselectiveelectrode(Cu‐ISE)(Orion9629BNWP).TheISEmeasurementswere

calibratedbetweenpCu2+9.0and5.2ina10‐4MCusolutionthatwasbufferedwithiminodiacetic

acidandpotassiumphthalateaccordingtotheprocedurepreviouslydescribedbyRachouetal.

(2007).

In phase II, 40‐mL nutrient solution samples were taken from each of the input and output

nutrient solutions. In addition, to more precisely evaluate the changes in the nutrient


99

concentrationsduringthe5days(betweentheinputandoutputsolutions),thenutrientsolution

wasalsosampledfromday196today201at1,2,3,4,5,20,23,27,43,72and96hafterthe

solutionwas renewed. Additional sampleswere taken at day 216 1 h, 3 h and 44 h after the

renewalofthesolution.

Allofthesolutionswerefiltered(0.2µm,Sartorius)andkeptata4°Ctemperature.Asubsample

wasacidified,andthesolutionswereanalysedforthepresenceofcations(Si,Ca,Fe,Mg,Mn,P

and Zn) by ICP‐AES; the total Cu concentrationwasmeasured by inductively coupled plasma

mass spectrometer (ICP‐MS, Agilent 7500 CE). Free Cu2+ was analysed by Cu‐ISE in another

subsample immediately following the solution sampling. The Cu‐ISE measurement was

conducted on the following selected samples: in the input solution on day 196 and in the

solutionsthatweresampledafter1,2,4,27,43,and96h;intheinputsolutiononday216and

at1hand3h;andintheoutputsolutionthatwascollectedonday221.

The Si and Cu uptake levels from the nutrient solution were calculated throughout the

experiment.TheuptakeofSiorCucorrespondstothequantityofSiorCuthatwasmeasuredin

the input solution (mmol) minus the quantity of Si or Cu that was measured in the output

solution(mmol).Theelementalquantity(SiorCu)inthesolutionistheelementconcentration

thatwasmeasuredbyICP‐AESorICP‐MSmultipliedbythevolumeofthesolution.Thevolume

oftheinputsolutionwas15L,andthevolumeoftheoutputsolutionswasmeasuredthroughout

theexperimentandvariedbetween13and8L. Inthe figures thatrepresentCuandSiuptake

(Figure III.2 and Figure III.3 ), the errors are estimated to be 20%, and these errors were

calculatedbasedontheerrorsintheoutputvolumeofthesolutionsandtheanalyticalerrors.In

theother figures (Figure III.1andFigure III.4 ), errorbars represent theuncertainty thatwas

estimatedfromtheanalyticalerrors(ICPAESorICP‐MS).

2.3 Plant analysis

2.3.1 Growth parameters

Thenumberandheightofthestemsandthenumberofleaveswereassayedatthebeginningof

theexperiment(day0),attheendofphaseI(day191)andattheendofthephaseII(day226).


100

2.3.2 Analysis of tissues

AttheendofphaseII,all25plantswereharvested.Theplantleaves,stems,rhizomesandroots

wereseparated.Thesampleswerecarefullywashedwithultrapurewater,andthefreshmasses

weredetermined.

Beforetheanalysis,allofthepartsweredriedat60°Cuntiltheyreachedaconstantweight.The

sampleswere subsequentlymixed, ground and homogenised. The sub‐sampleswere dried at

80%untiltheyreachedaconstantweighttodeterminetheirdryweight.

Theplant samples (leaves, stems and roots) underwent drymineralisation for the analysis of

trace elements. During the mineralisation, the Si content was determined by gravimetry as

follows:500mgofdriedplantmaterialwasplaced inaplatinumdishandgraduallyheatedto

500°C.ThesilicawaseliminatedintheashwithHF.Aftercooling,theSiweightwasdetermined

based on the difference from the 500°C weight. The ashes were dissolved into HCl, and the

contentof traceelements in thesolutionswasanalysedby inductivelycoupledplasma‐optical

spectrometer (ICP‐OES Vista‐Pro, Varian, CIRAD,Montpellier, France). For quality control, in‐

house reference samples and certified samples (Astrasol‐Mix, Analytika) were used every 20

samples,andeachanalysiswasconductedinduplicate.Themeasurementuncertaintywasless

than15%.ThequantificationlimitforSiwas5mgg‐1ofdryweight(DW).

Theconcentrationsofmacro‐andmicronutrientsareexpressedasgkg‐1ormgkg‐1DW,andthe

Siconcentrationsareexpressedinmgg‐1DWSiO2suchthattheresultsaredirectlycomparable

withtheliterature.

2.4 Statistical analyses

TheMinitab15.1softwarepackage(Minitab,Inc.)wasusedforthestatisticalanalyses.Foreach

portionoftheplants,theconcentrationsofmicro‐andmacronutrientswereanalysedbyANOVA.

Weusedaone‐wayANOVAatthe95%confidencelevelwiththeSitreatments(5levels)asthe

main factor, which was followed by a Tukey’s post hoc test at the 95% confidence level to

evaluate thedifferences in the treatments.ThemeanconcentrationsofSi andCu in the roots,

stemsandleaveswerecomparedusingapairedt‐Testatthe95%confidencelevel.


101

3 RESULTS

3.1 Evolution of the element concentrations in the nutrient

solution

The concentrationsof thenutrients thatweremeasured in theoutput solutionsdiffered from

their concentrations in the input solutions. Table III.1 provides the Ca, Mg, P and Fe

concentrations that weremeasured in the input and output solutions at three different time

periods during the experiment. These time periods are used as an example because the

variations throughout the experiment were reproducible. The Ca, Mg and Fe concentrations

increased in the nutrient solution. The P concentration varied throughout the experiment; it

increasedslightly in thesolutionat thebeginningofphase I from0.22mMto0.24mM in the

input andoutput solutions, respectively (Table III.1), but after theday135duringphase I, its

concentration decreased in the output solution. Throughout the experiment, the Zn

concentration remainedconstant (datanot shown).The resultsof theextreme treatmentsSi0

andSi15 (Table III.1) illustrate thatnoSi treatment effectwasobservedon theuptakeof the

measurednutrientsineitherphaseIorII.Thevariationsofthemacro‐andmicronutrients(Mg,

Ca,P,FeandZn) throughout the tworenewals(day196to201)duringphase IIareshown in

FigureIII.1.TheincreaseintheMg,CaandFeconcentrationsandthedecreaseofPandZnwere

constantovertime.

Despite the use of a pH buffer (MES), the pH of the solution changed over time (Table III.1).

During the first90daysof theexperiment, thepHdecreasedandreachedvaluesbetween5.5

and5.9intheoutputsolution.Fromday91today191,thepHofthesolutionincreasedweekly

and ranged from 6.3 to 7.3. During phase II, the pH consistently decreased following contact

withtheplants,andthefinalpHvariedbetween5.4and6.


102

Table III.1 Ca, Mg, P, Fe and Zn concentrations in the nutrient solution with Si0 and Si1.5 treatments in the input and output solutions at three different time periods during the experiment

Figure III.1 Concentrations of macronutrients (Ca, Mg and P) and micronutrients (Fe and Zn) in the Si0.4 treatment during phase II from days 196 to 201.


103

Figure III.2 Bamboo Si uptake throughout the experiment, which was calculated as the difference between the Si concentrations that were measured in the input and output solutions. “Theo. max” is the theoretical maximum uptake that could be obtained if the entire Si in solution is taken up by the plants.

Thesiliconconcentrationsinthesolutiondecreasedduringthecontactwithbamboo.Siuptake

from theSi concentrations in the input andoutput solutionswas calculated for each renewal,

andtheevolutionthroughouttheexperimentisshowninFigureIII.2.Forallofthetreatments,

atthebeginningoftheexperiment,theuptakeincreaseduntilitreachedaplateau.Thisplateau

indicates that the solution was entirely depleted during its contact with the bamboo. The Si

content in the solutionwas initially depleted in the less‐concentrated solutions, starting from

day 65, 73 and 106 for the Si0.4, Si0.8 and Si1.1 treatments, respectively. The uptake then

decreased starting on days 176, 157 and 164 for the Si0.4, Si0.8 and Si1.1 treatments,

respectively.TheSiuptakeinthehighertreatment(1.5mM)increaseduntilday114,anditthen

decreaseduntilday176.TheSiuptakewassimilar in theSi1.1andSi1.5 treatments,with the

exception of the period between days 114 and 126. This uptake decrease was observed,

althoughnothingwaschangedintheexperiment.Cuwasaddedtothesolutionbeginningonday


104

191.DuringphaseII,theCuthatwasaddedtothesolutiondidnotmodifythedownwardtrend

ofSiuptakeintheSi0.8,Si1.1andSi1.5treatments.However,inthesetreatments,theSiuptake

was variable throughout phase II. In the Si0.4 and Si0.8 treatments, Cu seemed to slightly

decreasetheSiuptake.

The Cu uptake differences between the Si treatments during phase II were not significant

(Figure III.3 ), and the Cu concentration in the output solution throughout phase II varied

between0.72and0.91µM.Duringthecontactwiththebambooplants,i.e.,for5days,theSiand

Cuconcentrationsbothdecreased(FigureIII.4),unliketheCa,MgandFeconcentrations(Figure

III.1).TherateofdecreasewasdifferentforSiandCu;theSiconcentrationgraduallydeclinedin

thesolutionovertheperiodof5days,whereastheCuconcentrationdeclinedsharplyinthefirst

hoursafterthenutrientsolutionwasrenewed(FigureIII.4).ThetotalCuconcentrationthatwas

measured insolutionby ICP‐MScorrelatedwellwith thepCuvalue thatwasmeasuredby ISE

(R2=0.7)(datanotshown).

Figure III.3 Bamboo Cu uptake throughout the experiment, which was calculated from the difference in the Cu concentrations in the input and output solutions. “Theo. max” is the theoretical maximum uptake that would be obtained if all of the Cu in the solution was taken up by the plants.


105

Figure III.4 Si and Cu concentrations in the nutrient solution during the Si0.4 treatment in phase II from days 196 to 201.

3.2 Bamboo growth parameters and elemental distribution

At the end of phase I (day 191), the number of stems and leaves was similar between the

differentSitreatments(TableIII.3).Thenumberofleavesvariedbetween170±117intheSi1.5

treatmentand377±243intheSi0.8treatment.AttheendofphaseII(day224),nosignificant

differences in the plant growth parameters, including the final fresh weight content (g), the

numberofstemsandtheirheights(cm),andthenumberofleaves,wereobservedbetweenthe

Si treatments (Table III.3). The numbers of leaves and stems that developed during phase II

were also similar between the treatments. At the harvest, each bamboo plant contained an

averageof20±14stemsthatwere20±4.5cmhighandhadatotalof241±168leaves(Table

III.3).ThefreshmatterfromthefinalharvestwasnotsignificantlyaffectedbytheSitreatments.

TheSiconcentrationsinthebambooplantsarepresentedinFigureIII.5 .TheSicontentinthe

leavesvariedfrombelowthedetectionlimit(5mgg‐1)fortheSi0treatmentto218±19mgg‐1

fortheSi1.5treatment.TheSicontentinthestemsvariedfrombelowthedetectionlimitinthe

Si0 treatment to 65 ± 14mg g‐1 for the Si1.5 treatment, and the root Si content ranged from

below the detection limit for the Si0 treatment to 11 ± 3 for the Si1.5 treatment. Our results

demonstratethattheSiconcentrationinbambooGigantocloasp.“MalayDwarf”decreasedinthe

following order: leaf > stem > root. The larger Si content that wasmeasured in the nutrient

solutionresulted inhighlysignificant linear increases in theSiconcentrations from0.4mMto

1.5mM(R2>0.99fortheleaves).TheslopeoftheregressionlinebetweentheSicontentinthe

solutionandtheSiconcentrationinthebamboopartswasdifferentbetween0and0.4mMand


106

between0.4mMto1.5mMSiinsolution.DuetotheuncertaintyinthebambooSiconcentration

in the Si0 treatment (which was below the detection limit), we have drawn a dotted line

between Si0 and Si0.4. The nitrogen, P, K, Ca andMg concentrationswere affected by the Si

treatment(TableIII.2).TheseconcentrationsdeclinedsignificantlyinresponsetoincreasingSi

concentrations.TheFe,MnandZnconcentrationswerenotaffectedbytheSitreatments.

At the end of phase II, no Cu toxicity symptoms (chlorosis, major root damage or growth

inhibition)wereobserved regardlessof theSi treatment.TheSi treatmentsdidnot causeany

significant effect on the Cu concentrations in the plants. Because Si did not influence the Cu

concentrationinanyoftheplantparts, theCuconcentrations inthedifferenttreatmentswere

averagedinFigureIII.6 .Copperaccumulatedintheplantsinthefollowingorder:root>leaf>

stem;theaverageswere131±39.3mgkg‐1intheroots,16.6±3.6mgkg‐1intheleavesand9.8±

3.8mgkg‐1inthestems.

Figure III.5 Si concentrations in different bamboo organs as a function of the Si concentration in the nutrient solution at 1.5 µM Cu. The error bars represent the standard deviation of the SiO2 concentrations in the 5 different bamboo plants.


107

Tab

le II

I.2

Con

cent

rati

on o

f m

acro

and

mic

ronu

trie

nts

in t

he l

eave

s, s

tem

s, a

nd r

oots

of

the

bam

boo

plan

ts t

hat

wer

e gr

own

wit

h di

ffere

nt S

i con

cent

rati

on i

n th

e nu

trie

nt s

olut

ion

*DW:dryweight;**Foreachpart,differentlettersindicatesignificantmeansdifferencesbyTukeytest

Tab

le II

I.3

Effe

ct o

f diff

eren

t le

vels

of S

i nut

riti

on o

n th

e fo

llow

ing

grow

th p

aram

eter

s: n

umbe

r of

ste

ms

and

leav

es t

hat

deve

lope

d du

ring

th

e ex

peri

men

t in

pha

se I

and

phas

e II

. .*Foreachpart,differentlettersindicatesignificantmeansdifferencesbyTukeytest


108

Figure III.6 Repartitioning of the Cu concentration in bamboo plants.


109

4 DISCUSSION

4.1 Silicon in bamboo

4.1.1 Mechanisms of silicon accumulation

TheSiuptakevariedgreatlythroughouttheexperiment.The increaseduptakeduringthe first

106 days that is shown in Figure III.2 is likely associated with biomass production. At the

beginning of the experiment (day 0), the bamboo plants contained an average of 18 leaves

(Table III.3), and they developed until they each contained between 124 and 324 leaves at

harvest. Si uptake decreased beginning on day 114 in the Si1.5 treatment and day 164 in the

Si0.4, Si0.8 and Si1.1 treatments. One hypothesis thatwould explain this decrease is that the

plants developed at a slower rate. Indeed, the development of the bamboo plants and,

particularly,therootsmayhavebeeninhibitedbythepotsize.

During phase I, the pH of the output solution ranged from 5.5 to 7.3 (Table III.1). The pH

decreasedat thebeginningof theexperimentduringplantcontact,whereasafter90days, the

pHincreased.ThepHinthesolutionseemstocorrelatewiththebamboodevelopment.ThepH

is an important property in the regulation of solubility and the speciation of elements in the

nutrientsolution.However,atpH8,Sispeciationisnotaffected,andSiisprimarilypresentasan

orthosilicicacidinthenutrientsolution,whichshouldnotmodifySiuptake.

Silica concentrations in the leaves and stemsofbambooGigantocloa sp. “MalayDwarf” that is

grown in hydroponics can be comparedwith data that have been obtained from field‐grown

plants (Collin et al. 2011). The same species that were grown under natural conditions

contained a leaf Si content of 135mg g‐1 and a stem Si content thatwas between 5.9mg g‐1

(base) and 30 mg g‐1 (tip of the stem). These concentrations are similar to those that were

measured in the Si0.4 and Si0.8 treatments; the leaves contained 94 and 142 mg g‐1 Si,

respectively, and the stemscontained37and49mgg‐1, respectively.TheSi concentrations in

the leaves and stems of the Si1.1 and Si1.5 treatments were higher than those that were

measuredinallofthestudiedbamboospeciesasreportedbyCollinetal.(2011),whoreporteda

Sirangeof55to159mgg‐1Si02intheleavesof16temperateandtropicalbamboospecies.The

Si concentrations in the leaves reached an average of 218mg g‐1 in the highest Si treatment,

whichwashigherthananySicontentthathasbeenpreviouslyreportedintheliterature(Dinget


110

al. 2008b; Li et al. 2006; Lux et al. 2003)with the exception ofMotomura et al. (2002). The

authorsreportedaSicontentof410mgg‐1inthe3‐year‐oldleavesofthebambooSasaveitchii,

whileinourstudy,theSicontentwasmeasuredinbulkfromamaximumof8month‐oldleaves.

Inall of the treatments, the root silica contentwasalways lower than the stemand leaf silica

contents (Figure III.5 ). This pattern is different from the pattern thatwas found in previous

studiesconcerningSiinbamboo,whichshowedthattherootSicontentwashigherthanthatof

thestemsbutlowerthanthoseofthebranchesandleaves(Dingetal.2008b;Luxetal.2003).

Thesepreviousstudieswereperformedonbamboothatwasgrowninsoil,andtheroot‐cleaning

proceduremayhavebeeninefficientatremovingalloftheSi‐containingsoilparticles.However,

thispossibilitycanberuledoutforthestudyofDingetal.(2008b),whoattemptedtoreducethe

potential bias throughout the cleaning procedure. However, this concentration pattern is

consistentwithpreviousstudies thatwereperformed inhydroponicswith twootherPoaceae,

rice(Dingetal.2008a)andbanana(Henrietetal.2006;Opfergeltetal.2006).Inthesespecies,

theconcentrationsalsoincreasedfromtherootstothestemsandtheleaves.

AfterSiwassuppliedinsolution,asignificantlinearrelationshipbetweenthetissue(leaf,stem

and root) Si content and the nutritive Si concentration was observed (Figure III.5 ). This Si

distribution in thedifferentpartsof theplant suggestspassiveuptake. Si translocationwithin

theplantislargelydependentonthetranspirationstream,whichcarriessilicicacidanddeposits

itintheformofamorphoussilica(Jonesetal.1967);thisprimarilyoccursintheleaves.Inthe

leavesandroots, theSicontentdidnotreachaplateau,which indicatesthattheSiuptakecan

increase even more; this is contrary to rice, which reached maximum Si uptake at a Si

concentrationgreaterthan1.28mM(MaandTakahashi2002).However,asSimaypolymerise

insolutionatconcentrationsabove2mM,itisnotpossibletotestlargerSiconcentrationsand

thuspossiblyobservealargeruptakebybamboo.Theonlypossibilitywouldbetoincreasethe

lengthoftheexperimentunderthehypothesisthatbamboocantakeupSimorequicklythanit

cangrow.

Recent studies have demonstrated the existence of active Si transporters in roots (Lsi1 and

Lsi2),which are responsible for the high Si uptake capacities of rice andmaize (Mitani et al.

2009; Yamaji and Ma 2011). Therefore, Si uptake in Si accumulators is explained by the

coexistenceofactiveandpassiveuptakemechanisms(Dingetal.2008b;Maetal.2007;Mitani

et al. 2009). In our study, the depletion of Si in the nutrient solution of the Si0.4 and Si0.8

treatments indicates that silicon, which is transported together with water, is not the only

absorption mechanism; this further suggests that an active process is involved in Si uptake

(Henriet et al. 2006; Rafi and Epstein 1999). Therefore, the coexistence of passive and active


111

uptakeandtransportationmechanismswouldbeconsistentwithpreviousstudiesonbamboo,

riceandwheat(Dingetal.2008b;MaandYamaji2008;Motomuraetal.2004).

4.1.2 Effect of Si uptake on the nutrient concentration and growth

parameters

Thevariationsinthemacro‐andmicronutrient(Mg,Ca,P,FeandZn)concentrationsthroughout

the two renewals (Figure III.1 )were attributed to bothplant uptake and thedecrease in the

solutionvolumeduetoevaporationandwateruptake(waterdecreaseof13%atthebeginning

and 47% at the end of the experiment). The variation of the P concentration in the output

solutionthroughouttheexperiment(i.e.,anincreasethatwasfollowedbyadecreasefromday

135 in theoutputsolution) indicates that thenutritionalneedof thebambooplants increased

afterday135.TheSisupplydidnotaffect theuptakeofanyof themeasurednutrients(Table

III.1).However,theadditionofSisignificantlyreducedtheleafconcentrationsofmacronutrients

(N, P, K, Ca and Mg) (Table III.2). The decrease in these concentrations is correlated to the

increaseinSi intheleaves.Infact,thehighamountofsilicathatprecipitatedintheleavescan

explain thedecrease in thenutrientconcentrations. Indeed, ifwesubtract thequantityofSiO2

from the dry matter, the nutrient concentrations were identical between the treatments.

Therefore,thequantityofthenutrientsinthebamboopartsisthesamebetweentreatments,but

theconcentrationdecreasedbecauseofthecontributionoftheprecipitatedSitothedrymatter.

Thisresult isconsistentwiththesimilaruptakeofnutrientsthatwasobservedbetweentheSi

treatments.

IncreasingtheSiconcentrationinthenutrientsolutiondidnotsignificantlymodifythegrowth

parameters:ourresultsdidnotrevealanypositiveeffectonthenumbersof leavesandstems,

the height of the stems or the total biomass. Si has often been described in the literature to

promoteplantgrowth.Indeed,Hattorietal.(2003)showedthatsupplementationwith1.67mM

Siinhydroponiccultureenhancedsorghumrootelongation,andHossainetal.(2002)reported

thatSipromotedtheseedlinggrowthofseveralPoaceaespecies,namelyrice,oatandwheat,by

increasing cell‐wall extensibility. Dakora et al. (2003) reported that cowpea root growth

increased significantly in hydroponic culture when Si was added. In our study, neither stem

growthnor total rootweightwasaffectedbySi,androotgrowthwasnotmeasured; this is in

contrasttosomehorticulturalcrops,suchascucumber,courgetteandrose,forwhichthetotal

weightincreasedfollowingtheapplicationofpotassiumsilicate(Hwangetal.2005;Voogtetal.

2001).Inmostcases,however,itisuncertainwhetherthegrowthstimulationwasattributable

to a nutritional effect or to the alleviation of biotic and abiotic stresses. Indeed, the

misinterpretationof theresultsof someexperimentshavebeendescribedbyMarschneretal.


112

(1990). For example, Miyake and Takahashi (1978) (tomato), Miyake and Takahashi (1983)

(cucumber), Miyake and Takahashi (1985) (soybean) and Miyake and Takahashi (1986)

(strawberry)arguedthatSiwasessentialforplantgrowthbecausetheyobservedthatthelack

of Si in the nutrient solution caused severe leaf chlorosis, depressions in flower and fruit

formation,malformation andwilting. However,Marschner et al. (1990) demonstrated that in

these experiments, the toxicity symptoms were caused by a nutrient imbalance, which is

characterisedbyahighPconcentrationandalowZnconcentration;theyalsodemonstratedthat

theSieffectwasindirectbecauseitincreasedthephysiologicalavailabilityofZn,whichthereby

reduceditsdeficiency.

SeveralstudieshavealsoreportedthatSifertilisationinfieldtrialsmayincreaseandsustainthe

productivity of rice, sugarcane and non‐accumulator species, such as tomato and strawberry

(Korndorferetal.2001).Tavakkolietal.(2011)foundasignificantincreaseinriceyieldinared

Ferrosolfollowingcalciumsilicatefertilisation.However,infieldstudies,itislikelythatSimay

increaseyieldbyalleviatingbioticandabioticstresses,suchasinsectdamageandlodging(Ma

andYamaji2006). Inhydroponicculture,Fauteuxetal. (2006)demonstratedthatSialonedid

notaffectArabidopsis thalianametabolism. In someother Poaceae plants,high levelsofSidid

notaffectwheatgrowth(RafiandEpstein1999),bananagrowth,therateofwaterandnutrient

uptake(Henrietetal.2006)orBrachiariabrizanthaforageproduction(deMeloetal.2010).One

couldthereforespeculatethatSiindirectlybenefitsplantgrowthbyalleviatingstress.

Inthisstudy,itisnoteworthythatthebamboointheSi0treatmentdevelopednormallyanddid

notdisplayanydeficiencysymptoms.BecausetheSicontentintheplantsthatweretreatedwith

Si0werebelowthedetectionlimit(<5mgg‐1),itseemsthattherequirementofbambooforSiis

low. This should be investigated in further studies that can assess the requirement of Si as a

mineralelementforbamboo;forexample,thecontentofSi inthesolutioncouldbedecreased,

andtheSicontentinbamboocouldbequantifiedwithamoresensitivetechnique(Guntzeretal.

2010). Indeed, despite the results of several studies that have described the effect of Si, its

requirementforplantgrowthisstilldebated(Fauteuxetal.2006).

4.2 Effects of copper on bamboo growth

On average, the leaves and stems contained 16.6 ± 3.6mg kg‐1 and 9.8 ± 3.8mg Cu kg‐1 DW.

Theseconcentrationswerehigherthanthoseofseveralbamboospeciesthatweregrowninnon‐

contaminatedsoil; theseplantscontained3.5and4.5mgCukg‐1DWinthestembaseandtip,

respectively,and5.1mgCukg‐1DWintheleaf(Collinetal.2011),whichindicatesthatbamboo


113

respondedtoCuinsolution.However,theleafconcentrationswerestillwithintherangethatis

considered to be normal for mature leaf tissues (5‐30 mg kg‐1 Cu) (Kabata‐Pendias and

Mukherjee 2007). With an average of 131 ± 39.3 mg kg‐1, more than 80 % of the Cu was

concentrated in the roots of the bamboo plants. This pattern is consistent with a number of

studiesthatindicatethatCuaccumulatesmoreinplantrootsthanintheshootsorleaves(Aliet

al. 2002; Ouzounidou et al. 1995). Because we did not desorb Cu from the roots, we cannot

distinguishbetweenadsorptionandabsorptionbytheroots.However,ahighproportionofCu

intherootsmaybeboundtothecellwall(Nishizonoetal.1987).Indeed,cellwallsarerichin

compounds that can bind Cu, such as pectin polysaccharide and glycoprotein constituents

(Krzeslowska2011).It is likelythattherapiddecreaseinthenutrientsolutionduringthefirst

hoursmaybeexplainedbyCuadsorptionontotherootsurface.Weobservedthisrapiddecline

throughout phase II; it occurred following each contact with the bamboo plants, which may

indicate that the Cu‐binding capacity of the roots was not saturated even at the end of the

experiment.Bravinetal.(2010)suggestedthattheCu‐bindingcapacityoftherootapoplasmcan

reach approximately 1 g Cu kg‐1 root DW. In the present study, the quantity of Cu that was

delivered to theplantmayrepresentapproximately0.4gCukg‐1 rootDWperplant (datanot

shown). Thus, the binding capacity of the bamboo roots was certainly not entirely saturated

withCu.DuetoitsabilitytobindCu2+ontoitsroots,theuseofbamboohasbeenproposedfor

theadsorbentremovalofCuinwastewater(Babatundeetal.2009).

RegardlessoftheSitreatments,notoxicitysymptoms(i.e.,leafchlorosis,visiblerootchangesor

biomassdecrease)weredetectedwithasupplyof1.5µMCu2+duringphaseII,althoughsimilar

concentrationshavebeenfoundtobetoxicinPoaceae.Severalauthorshaveobservedchlorosis

indurumwheatseedlingsatCu2+solution levels thatwereaboveeither0.55µM(Bravinetal.

2010)or1µM(Michaudetal.,2008);a50%reductionoftheSabigrassfreshshootbiomassat

1.2µMCu2+andthefreshrootweightat1.0µMCu2+(Kopittkeetal.2009);anda50%reduction

inrootelongationindurumwheatataCu2+solutionconcentrationofeither0.06µM(Bravinet

al. 2010) or 0.6 µM (Michaud et al. 2008).Root damagewas observed in the roots ofRhodes

grassthatwasgrownwithasolutionconcentrationof0.22µMCu(SheldonandMenzies2005).

Moreover,arecentreviewoftheliteratureoverthepast34years(Kopittkeetal.2010)reported

thatthemediantoxicCuconcentrationwas2µMinhydroponicstudies.

Severalhypothesesmayexplaintheabsenceoftoxicitysymptomsinourexperiments.First,this

resultmaybeduetoamodificationoftheCuconcentrationandspeciationinthesolution.The

initialconcentrationwaseffectively1.5µMCu2+;thiswasconfirmedbybothchemicalmodelling

and Cu‐ISE. Once it was in contact with the plant, the Cu content in the solution decreased

abruptlyinthefirsthoursandstabilisedtoavaluebetween0.72and0.91µM.After24h,theCu


114

concentration increased slightly and homogeneously until the next renewal, but this increase

maybe explainedby thedecrease in volumeof thenutrient solutiondue toplantuptake and

evaporation.TheISEresultsthatwerecombinedwiththecalculationswithPHREEQCusingthe

compositionandpHoftheoutputsolutionindicatethattheremainingCuisprimarilypresentas

freecopper.Therefore, theCu2+content inthesolutionwassmallerthaninthe inputsolution;

however,whencomparedtotheworkofBravinetal.(2010)andSheldonandMenzies(2005),

theCu2+inthesolutionshouldhavebeensufficienttoinducetoxicitysymptomsindurumwheat.

Second,theabsenceofanyobservedtoxicitymaybeaconsequenceofthehighbiomassthatwas

produced during phase I. Typically, and in the previous cited studies, toxicity measurements

wereperformedon2‐or3‐week‐oldplants;theseyoungplantshavealowerbiomassthanthe

plantsthatwereusedinthecurrentstudy(Kopittkeetal.2010). Inourcase,oncetheCuwas

absorbedand translocated, itmayhavebeendiluted in the largenumberof stemsand leaves,

whichmayhaveinducedanimprovedCutolerancethroughalowerinternalCuconcentration.

This hypothesis can be ruled out because in our experiment, the Cu concentrations in the

bambooleaveswere16.6mgCukg‐1,whiletheyreached21mgCukg‐1intheexperimentsthat

were performed by Michaud et al. (2008) and Kopittke et al. (2009) when the plants were

exposedtoasimilarCucontent(1.1‐1.2µMCu2+).

Finally, the main differences between the previous studies and our study are the exposure

length (2months vs. 8 to 10 days) and the plantmaturity (mature plants vs. seedlings). The

largerootbiomassmightmayhavecontributedtotolerancethroughaCudilution in/ontothe

roots.Incontrasttotheshoots,theCucontentinthebambooroots(anaverageof131±39.3mg

Cu kg‐1 DW) was lower than the concentrations that were measured in the roots of the

aforementioned studies (approximately 350 mg Cu kg‐1 DW in durum wheat (Michaud et al.

2008)and890mgCukg‐1DWinSabiGrass(Kopittkeetal.2009)).ThebindingofCuontothe

cellwallreducestheamountofCuthatisinternalised;ifthebindingsitesarenotsaturated,the

roots may be better able to prevent translocation to the stems and leaves, which therefore

protectsthemostsensitivesitesfromCutoxicity.Ithasbeensuggestedthatthesequestrationof

tracemetalsincellwallsisoneofthemainstrategiesthatplantsusetocopewithmetalstress

(Krzeslowska2011).Indurumwheat,Michaudetal.(2008)suggestedthatarootCucontentof

approximately100‐150mgkg‐1 is thecritical level thatcorrespondstoearlyandmoderateCu

phytotoxicity. Incomparison, theCucontent in thebambooroots iswithin the lowerrangeof

theCuconcentrationthatmayinducetoxicity.

Based on its high biomass and its adsorption capacity, the bamboo Gigantocloa sp. “Malay

Dwarf”tolerateshighCuconcentrationsinnutrientsolution.


115

4.3 Effects of bamboo Si on Cu sensitivity

Despite the addition of Cu into the nutrient solution, no differences regarding the biomass

measurements or the Cu content in plants were observed between the Si treatments. These

resultscontrastwiththoseofthestudyofNowakowskiandNowakowska(1997),whichshowed

that the additionof 0.4mmolL‐1 Si significantlydecreased theCu content in the roots and in

shootsof7‐day‐oldwheatseedlings.TwootherstudiesalsodemonstratedtheSialleviationof

CutoxicityinArabidopsisthaliana(Brassicaceaea)(KhandekarandLeisner2011;Lietal.2008)

andEricaandevalensis(Ericaceaea)(Olivaetal.2011). Inbothstudies, theSisupplementation

inducedanincreaseintheshootbiomassandareductionofchlorosissymptoms.However,the

mechanisms by which Si alleviates Cu toxicity are still poorly understood. Li et al. (2008)

demonstratedthatsupplementalSididnotchangethetotalCuconcentrationsintheleaftissue,

suggestingthatSiinfluencedthedistributionorbioavailabilityofCuwithintheleaves(Rogalla

and Romheld 2002). Conversely, Oliva et al. (2011) observed that Si induced a significant

reduction of the Cu concentration in the leaves, which likely occurred by inhibiting the

translocation of Cu from the roots to the shoots. Interestingly, Li (2008) and Khandekar and

Leisner(2011)observedthatSialteredgeneexpression.InresponsetohighCu,Sisignificantly

decreased the expression of two Arabidopsis copper transport genes, copper transporter 1

(COPT1)andheavymetalATPasesubunit5(HMA5).Sihelps toreducestress throughamore

efficient response toCu toxicitybymaintainingorup‐regulatingCu‐bindingmoleculesandby

increasingtheexpressionoffree‐radical‐metabolisingenzymes(KhandekarandLeisner2011).

ThequantitiesofCu thatwereused in thesestudieswerehigher than theconcentrations that

werechoseninthepresentstudy;Olivaetal.used500µM(2011),andKhandekarandLeisner

(2011) and Li et al. (2008) used 30 µM. However, in Erica plants, even at the lowest Cu

treatment(1µM),SisignificantlydecreasedtheCucontent,whereasinthepresentstudy,theCu

contentwasnotaffectedbySisupplementation.

Inourstudy,weidentifiedtwodifferentmechanismsthatregulatetheuptakeofSiandCu.Siis

absorbedhomogeneouslybetweentworenewals,whereasCuisprimarilyadsorbedinthefirst

hoursaftereachsolutionrenewal.Asaresult,Cuismainlyaccumulatedintheroots,andithasa

low concentration in aerial parts,whereas Siwas accumulated in high amounts in the leaves.

Thesedifferences in theuptakeandrepartitionofCuandSi in theplantmay indicate that the

bamboo plant utilises two different strategies to govern the allocation of these elements.

Therefore,differencesintheresponseofbambooplantswhencomparedtoArabidopsisorErica

maybeduetothedifferencesinCuandSiacquisition,transportorinteractioninplants.


116

Copper uptake mechanisms in monocotyledonous species are poorly understood when

compared to those in dicotyledonous species; the mechanisms in monocotyledons have

primarily been studied in Arabidopsis (Yruela 2009). However, it is known that Si uptake

mechanisms and their ability to accumulate Si differ between phylogenetic groups and plant

species(Hodsonetal.2005;MaandYamaji2006).CuandSiinEricaarebothaccumulatedinthe

roots (Olivaetal.2011),whereas inbamboo,Si isprimarilyaccumulated in the leaves.TheSi

concentration variedwidely between different plant families; for example, the leaf Si content

rangedfrom0.565to1.2mgg‐1inErica,rangedfrom0.084to0.566mgg‐1inArabidopsis,and

rangedfrombelowthedetectionlimitto218mgg‐1inbamboo(ourstudy).Therefore,theplant

responsetotheSisupply iscertainlydifferentbetweenSi‐accumulatingandnon‐accumulating

plants.

Our data lead us to conclude that Si displayed no significant effects when it was supplied to

plants that were growing under controlled conditions, although the bamboo Gigantocloa sp.

“MalayDwarf”didabsorbSiinlargeamounts.ThedifferentabsorptionmechanismsforCuand

Si may partially explain why Si did not influence the Cu repartition and concentration in

bamboo. No toxicity symptoms were observed for the applied Cu concentration, which were

relevanttoCuconcentrationsthathavebeenmeasuredincontaminatedsoil;thisindicatesthat

bambooplantsaretoleranttoCu.Therefore, itseemsthatbamboocancopewith largeCusoil

concentrations and could thus be used in phytoremediation schemes. However, this result

shouldbefurther investigatedbytestinghigherCuconcentrations,anditshouldbeconfirmed

withfieldexperiments.

Acknowledgments

This work was financially supported by the Direction Générale des Entreprises, Direction

GénéraledelaCompétitivité,del’IndustrieetdesServices,régionRéunion,régionPACAinthe

frame of the research program RUN INNOVATION II and by the Association Nationale de la

RechercheetdelaTechnologie(CIFREgrant).

Theauthors thankDr.WimVoogt forproviding thepotassiummetasilicateand forhishelpful

advice,BernardAngeletti forperforming the ICP‐MSmeasurements andDr.GregoryBois and

VéroniqueArfifromPHYTOREMfortheirassistanceandfruitfuldiscussions.


117

5 BILAN DU CHAPITRE III

Malgré des temps de culture longs, la culture hydroponique s’est révélée adaptée à la

croissancedesbamboussympodiaux,etnousapermisdesuivreavecprécisionl’évolutiondes

nutrimentsdanslasolutionnutritive.

L’accumulationdeSidanslebambouGigantochloasp«MalayDwarf»estproportionnelleàla

concentrationdeSiensolution.DansletraitementdeSileplusélevé,laconcentrationenSides

feuilles est de 218 ± 19mg g‐1 enmoyenne. En revanche, Si n’a pas eu d’influence sur le

développementetlacroissancedesbambous.

LesuividesconcentrationsdeSietCudanslasolutionnutritivenousapermisd’identifierdeux

mécanismesd’absorptiondifférentspourcesdeuxélémentsdanslaplante:uneabsorption

progressivedeSi,etuneadsorptionrapideetimportantedeCusurlesracines.

L’accumulationdeSidanslesdifférentespartiesdelaplantesuitlatendancesuivante:feuilles

>tiges>racines,alorsqueladistributiondeCuest lasuivante :racines>tiges≥feuilles.Les

concentrationsdeCunesontpasmodifiéesparlesdifférentstraitementsdeSi.

Grâceàsafortebiomasseracinaire,etàsacapacitéàséquestrerCudanslesracines,lebambou

atolérélaforteconcentrationdeCuàlaquelleilaétéexposé.


118

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124

C H A P I T R E I V

COPPER DISTRIBUTION AND

SPECIATION IN BAMBOOS

“Phy l l o s ta chy s f a s tuo sa” EXPOSED TO

DIFFERENT COPPER AND S IL ICON

CONCENTRATIONS

ChapitreIV:Copperdistributionandspeciationinbamboos“Phyllostachysfastuosa”

126


127

TABLE DES MATIERES

CHAPITREIVCOPPERDISTRIBUTIONANDSPECIATIONINBAMBOOS“Phyllostachysfastuosa”EXPOSEDTODIFFERENTCOPPERANDSILICONCONCENTRATIONS..................................................................................................................... 128

1. INTRODUCTION.................................................................................................................................................. 133

2. MATERIALANDMETHODS ............................................................................................................................. 1342.1. Plantmaterial,experimentaldesignandpreculture........................................................................................... 1342.2. Siliconandcoppertreatment .......................................................................................................................................... 1352.3. Plantanalysis .......................................................................................................................................................................... 1362.3.1 Growthparameters ................................................................................................................................................1362.3.2 Analysisofmacroandmicronutrients...........................................................................................................136

2.4. Desorptionprocedure.......................................................................................................................................................... 1362.5. Anionassays ............................................................................................................................................................................ 1372.6. Totalsolubleaminoacid.................................................................................................................................................... 1382.7. SEMEDX ................................................................................................................................................................................... 1382.8. LaboratoryBasedµXRF ..................................................................................................................................................... 1382.9. EXAFSandXANES:dataacquisitionandanalysis.................................................................................................. 1392.10. Statisticalanalyses............................................................................................................................................................. 140

3. RESULTS................................................................................................................................................................ 1403.1. Siliconandcopperinnutrientsolution ....................................................................................................................... 1403.2. Growthparameters .............................................................................................................................................................. 1413.3. CuandSidistributionbetweenplantparts ............................................................................................................... 1423.4. CuandSilocalizationintheroot ................................................................................................................................... 1443.5. Theeffectsofcopperandsilicononsomeplantcomponents ............................................................................ 1453.6. XANESfeatures....................................................................................................................................................................... 1493.7. EXAFSfeatures ....................................................................................................................................................................... 151

4. DISCUSSION.......................................................................................................................................................... 156

5. SUPPORTINGINFORMATIONS....................................................................................................................... 162

6. BILANDUCHAPITREIV.................................................................................................................................... 170

7. REFERENCESBIBLIOGRAPHIQUES............................................................................................................... 171


128


129

CHAPITRE IV

COPPER DISTRIBUTION AND SPECIATION IN BAMBOO

“Phy l l o s ta chy s f a s tuo sa” EXPOSED TO DIFFERENT COPPER AND S IL ICON

CONCENTRATIONS

Dansdesconditionsnonoupeustressantes,lesiliciumn’apaseud’effetssurlacroissanceetle

développement du bambou sympodial Gigantocloa sp. «Malay Dwarf» malgré la forte

accumulation de Si dans ses tissus. Cela est cohérent avec des observations effectuées pour

d’autresplantes,commelebananier(Henrietetal.2006)oudesplantulesdeconifères(Cornelis

et al. 2010): le silicium jouerait un rôle lorsque laplante est soumise àun stressbiotiqueou

abiotique (Liang et al. 2007). Or, il s’avère que la concentration de Cu choisie dans l’étude

précédenten’apasinduitdetoxicitéchezlebambou,contrairementàcequiaétéobservépour

d’autres Poaceae telles que le blé et la Sabi grass (Bravin et al. 2010; Kopittke et al. 2009;

Michaudetal.2008).Nousn’avonsdoncpaspu,danscetteétude,statuersurl’effetdeSifaceà

une toxicité de Cu. En revanche, nous avons identifié deux comportements différents de

l’absorptionausenslarge,deCuetSi:alorsqueSiestprélevédemanièrecontinueaucoursdu

temps,Cuestprélevédemanièrerapidelespremièresheuresaprèschaquerenouvellementde

lasolutionnutritive.CelasembleindiqueruneadsorptionrapidedeCusurlesparoisracinaires

qu’ilseraitintéressantdevérifierparuneobservationprécisedelalocalisationducuivredansla

racineententantd’estimerlaproportiondeCuadsorbée.

La toxicité du cuivre dépend non seulement de sa localisation dans les différents tissusmais

aussidesaspéciation.Laplanteadoptedifférentesstratégiespourgérerunexcèsdecuivredans

sestissus,commeuneséquestrationdanslesparoiscellulaires(Krzeslowska2011),oubienune

complexationpardifférents ligands(CfchapitreI).Undesmécanismespermettantd’expliquer

laréductiond’unetoxicitégrâceàSiestlaformationdecomplexesmétaux‐Si,commecelaaété

proposé pour Zn dans Cardaminopsis halleri (Neumann and zur Nieden 2001). L’étude de la

spéciationdeCuparspectroscopied’absorptiondesrayonsXdoitnouspermettred’identifierla


130

nature des complexes formés avec Cu, d’éventuelles interactions entre Cu et Si et/ou une

modificationdelaspéciationdeCuinduiteparSitellequ’unemodificationdesligands.

NousavonsvudanslechapitreIIque,danslesconditionspédoclimatiquesdel’îledelaRéunion,

les bambous sympodiaux accumulent significativement plus de Si et de Cu que les bambous

monopodiaux.Lesbambousmonopodiaux,qui sont lesplus répandusdans lespays tempérés,

présentent‐ilségalement lacapacitéde toléreruneconcentration fortedeCu(1.5µMCu2+)en

solution?C’est cequenousavons testédanscetteexpérienceconduiteenhydroponieavec le

bambouPhyllostachys fastuosa et deux niveaux de concentrations en Cu: 1.5 et 100 µMCu2+.

Cettedernièreconcentrationaétéchoisieafind’induireunefortetoxicitéetd’observerl’effetde

Sisurcettetoxicité.

Lesobjectifsdecechapitresontlessuivants:

• Evaluer la distribution et la localisation de Cu et Si dans les différentes parties du

bambou,

• Evaluerl’effetdeSisurlaconcentrationetlatoxicitédeCu,

• Evaluer l’effet de Si et Cu sur la nutrition (acides organiques et inorganiques) du

bambou,

• CaractériserlaspéciationdeCudanslebambou.

Pour répondre à ces objectifs, plusieurs techniques ont été utilisées: en premier lieu des

analyses chimiques desmacro etmicronutriments, puis l’identification et la quantification de

composésorganiquesetinorganiquesparchromatographieionique,ensuitelalocalisationdeSi

etCudanslesracinesparmicroscopeélectroniqueetmicro‐fluorescenceX,etenfinlaspéciation

de Cu par spectroscopie d’absorption X en couplant le XANES (X‐ray Absorption Near Edge

Structure)et l’EXAFS(ExtendedX‐rayAbsorptionFineStructure).Lesrésultatssontprésentés

dansl’articleci‐après,quiaétérédigéafind’êtresoumisàlarevueNewPhytologist.


131

Copper distribution and speciation in bamboo

“Phyllostachys fastuosa” exposed to different copper

and silicon concentrations. First evidence of the

presence of reduced copper bound to sulfur compound

in a Poaceae species.

BlancheCollin*a,b,EmmanuelDoelschc,CatherineKellerb,PatrickCazevieillec,FredericPanfilia,

DenisTestemaled,Jean‐LouisHazemannd,Jean‐DominiqueMeunierb


France

bCEREGE,CNRS,Aix‐MarseilleUniv.,Europôleméditerranéende l'ArboisBP80,13545Aixen

Provence,France

cCIRAD,UPRRecyclageetrisque,F‐34398Montpellier,France

dInstitutNéel,CNRSetUniversitéJosephFourier,BP166,F‐38042GrenobleCedex9,France.


SUMMARY

• The purpose of this study was to examine copper (Cu) absorption, repartition and

toxicityanddeterminetheroleofaSisupplementationinapoaceaespecies:thebamboo

Phyllostachysfastuosa.

• Anhydroponicculturewascarriedoutfor3months,bamboosweresubmittedtothree

contrastedcopperconcentrations,andtwosilicon(Si)concentrations.Copperspeciation

and Cu and Si distributionwere investigated by chemical analyses, spectroscopic and

microscopictechniques.


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• Copper ismainlyaccumulated inbambooroots,wherea largepartofCu isretained in

epidermis.Inroots,siliconismainlyaccumulatedinendodermalcells.Inroots,stemand

leaves, Cu was present under two oxidation states Cu(I) and Cu(II) with different

proportions.

• Silicon supplementation modified the Cu speciation but did not induced significant

improve of bamboo Cu tolerance. Themain strategy of bamboo to copewith high Cu

concentrationinitstissuesis(i)animportantsequestrationintherootsapoplast,mainly

in epidermis, and (ii) a Cu(I) complexation with sulfur ligands both organic and

inorganic.

Keywords:toxicity,alleviation,ExtendedX‐rayabsorptionfinestructurespectroscopy(EXAFS),

X‐rayAbsorptionNear‐EdgeSpectroscopy(XANES),microfluorescenceX(µ‐XRF)


133

1. INTRODUCTION

Copperplaysacentralroleinplantmetabolismbutisdetrimentalwhencriticalthresholdsare

exceeded. Excess copper is cytotoxic, induces stress and causes injury to plants. Cu

contamination is widespread as a result of its use in fungicides, application of pig slurries,

atmospheric deposition from industrial and urban activities and metalliferous mining.

Phytoremediation of tracemetals has been proposed forwastewater treatment. Bamboos are

knownfortheirresistancetoawiderangeofstressfactorsaswellastheirhighgrowthrateand

biomassproduction.Bamboosarealsousedinphytoremediationtechnologies(Arfietal.2009).

ThereforeinordertooptimizethistechnologyitisessentialtotesttheextentofCutolerancein

bamboos and thereafter to identifymechanisms used by bamboos to copewith an excess Cu

content.SpeciationofCuassociatedtoitsdistributionwithintheplantultimatelydeterminesthe

various mechanisms of availability, detoxification and toxicity (Lombi et al. 2011). X‐ray

absorptionspectroscopy(XAS)isapowerfultooltoassessthechemicalspeciationinplants(Salt

etal.2002)andcanbeusedtodeterminetheoxidationsateandthephysicalstructureofsites

surroundingCuinplanttissues.Cuspeciationhadbeenreportedinthefollowingplants;Larrea

tridentate(Gardea‐Torresdeyetal.2001;Poletteetal.2000),Vignaunguiculata(Kopittkeetal.

2011), Sesbania drummondii (Sahi et al. 2007), Elsholtzia splendens (Shi et al. 2008), Thlaspi

caerulescens (Mijovilovich et al. 2009), Crassula helmsii (Kupper et al. 2009). However, no

studies have been performed on monocotyledonous species, and particularly poaceae family.

Poaceaeplants,andbamboosinparticular,areabletoaccumulatehighamountsofsilicon(Si)in

theirtissues(Collinetal.2011).Recently,ithasbeenshownthatsiliconalleviatesCutoxicityin

Arabidopsis thaliana (Khandekar and Leisner 2011; Li et al. 2008) and in Erica andevalensis

(Olivaetal.2011).However,weshowedinapreviousstudy,thatawiderangeofSiinsolution

didnotinfluencethegrowthanddevelopmentofbamboosGigantocloasp“Malaydwarf”which

weresubmittedtoaCuconcentrationof1.5µMCu2+inhydroponics(Collinetal,chapIII).This

Cuconcentrationdidnotinducetoxicitysymptomsinbambooscontrarytoeffectsdescribedin

otherPoaceae plants such aswheat (Bravin et al. 2010) and Sabi grass (Kopittke et al. 2009)

exposedat similarCu content. Silicon isknown tobebeneficialmostlyunderabioticorbiotic

stress(Liangetal.2007).ThefactthatCollinetal.(chapIII)didnotobservetheeffectofSion

bamboomayberelated to theabsenceof stressconditions.Toconfirm that, it isnecessary to

testSiwithahigherCuconcentration,thatmayinducetoxicityinbamboos.


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Theprevioushydroponiccultureofbamboos(Collinetal,chapIII)weredoneonamonopodial

type of bamboo, which has been shown to accumulate significantly more Cu and Si than

sympodialbamboosinnaturalpedo‐climaticenvironment(Collinetal.2011).Wethereforecan

wonderiftheabilitytotoleratehighCucontentdependsonthebamboospecies.Thuswealso

chosetotestCutoleranceonasympodialbamboo:Phyllostachysfastuosa.Bamboosweregrown

in a 4‐month hydroponic experiment and submitted to the same Cu content (1.5 µM) as in

(Collinetal,chapIII)whichis largebutenvironmentallyrelevant(Sauvéetal.1997),andtoa

higherCuconcentrationinordertoreachacutetoxicityandtestthepossibleCualleviatingeffect

ofSi.InordertoidentifythepossibleCutolerancemechanismsinbamboossupplementedornot

with Si, we investigated the in situ distribution and speciation of Cu using several combined

approaches:mineralandorganicanalyses,CuandSilocalizationintherootsinvestigatedbyX‐

ray fluorescence micro‐spectroscopy (µXRF) and scanning electron microscopy coupled to

energydispersiveX‐rayanalysis(SEM‐EDX),CuspeciationdeterminedbyCuK‐edgeExtended

X‐ray absorption fine structure spectroscopy (EXAFS) and X‐ray Absorption Near‐Edge

Spectroscopy(XANES).

2. MATERIAL AND METHODS

2.1. Plant material, experimental design and pre-culture

Thirty‐five one‐year old monopodial bamboos Phyllostachys Fastuosa grown on the same

substrate were provided by PHYTOREM (Miramas, France). Bamboos were transferred in

hydroponiccultureafteracarefulwashingoftherootsinordertoremoveallthesoilparticles.A

preliminaryexperimentwas carriedout toadapthydroponic culture tobamboos (Collinet al,

chapIII).Theexperimentwasperformedduring4months.Apre‐culturephasewasconducted

over2months.Plantsweregrown in cylindricalPVCpots containing3Lofnutrient solution.

The compositionof thepre‐culturenutrient solutionwasa25%strength completeHoagland

solution.Thecompositioninmacroelements(mM)was:1Ca(NO3)2,1.5KNO3,0.24MgSO4,0.22

(NH4)2HPO4, and in microelements (µM): 11.6 H3BO3, 0.08 CuSO4, 0.2 ZnSO4, 0.03 MoO3, 2.3

MnCl2, 100 FeNaEDTA. The pH of this solution was adjusted to 6 with NaOH or HNO3. The

nutrient solutions were continuously aerated with an air pump in each pot. The nutrient

solutionwas renewed every 7 days. Analytical‐grade chemical reagents and ultra purewater

were used in the preparation of nutrients solutions. No glassware was used to minimize Si

contamination.Allplasticwareused for theexperimentswerepreviously soakedovernight in

nitric acid and rinsed with ultrapure water. The growth chamber parameters were set at

(day/night):28/25°C,90/75%ofrelativehumidityand450μmolphotonsm−2s−1lightintensity.


135

2.2. Silicon and copper treatment

At the end of the pre‐culture phase, 25 bamboo plants of uniform size were selected. Five

treatmentswereappliedduring70days,2concentrationsofSiand3concentrationsofCuwith

thefollowingcombinations:0mMSi+0.1µMCu,0mMSi+1,5µMCu,1.1mMSi+1,5µMCu,0

mMSi+100µMCu,1.1µMSi+100µMCuthereafterreferredtorespectivelyascontrol,Cu1.5,

Cu1.5Si, Cu100, Cu100Si. Si was provided as monosilic acid from potassium‐metasilicate

Si(KOH)2,whichhasaK:Simoleratioof2(Metso400‐YARA)(Voogtetal.2001).Cuwasadded

using CuSO4. Potassium and hydroxide levels were adjusted in the nutrient solution to

compensate for the additional input of K and OH from the silicon addition. KNO3 and HNO3

concentrationswereadjustedineachtreatmenttoreachK=NO3=2.25mM.Thecompositionof

macroelements(mM)inthisnutrientsolutionwas1CaCl2,0.24MgSO4,0.22(NH4)2HPO4andthe

compositionofmicroelements (µM)was:11.6H3BO3,0.2ZnSO4,0.03MoO3,6.5MnCl2.Fewas

provided as 20 µM Fe‐N, N9‐di (2‐hydroxybenzyl) ethylenediamine‐N, N9‐diacetic acid

monohydrochloride hydrate (HBED; Strem chemical, USA). Fe(III)‐HBED was prepared

followingthedescriptionofChaneyetal.(1998)insuchawaythatallHBEDwassaturatedwith

Fe. SolutionpHwas set at6.0 (±0.2)bufferedwith1mMMES (2‐morpholinoethanesulphonic

acid).Five replicatesper treatmentwereconducted.Foreach treatment, thenutrient solution

circulated continuously between the five pots and the 15L “reserve tank”. The solution was

continuouslysuppliedbyaperistalticpumpfromthereservetanktothebaseofthepotandthe

solution inexcessof2.5 lwasreleasedandcollectedviaanoverflowpipetocomeback inthe

reservetank.Thenutrientsolutionswerecontinuouslyaeratedwithanairpumpineachpot.All

solutionscontainedineachpotandinthereservetankweretotallyrenewedevery7days.

GeochemicalcalculationswereperformedwithPHREEQC(version2‐17)(Parkhurst1995)and

the MINTEQ database of thermodynamic constant. The quality of speciation modelling was

checked by measuring Cu2+ with a Cupric Ion Selective Electrode (Orion 9629BNWP). To

measure Cu in treatment Cu1.5 and Cu1.5Si, Cu‐ISE measurements were calibrated between

pCu2+ 12.0‐4.2 in 10‐4M Cu solution buffered with iminodiacetic acid and potassium phtalate

accordingtotheproceduredescribedbyRachouetal.(2007).DuetothehighCuconcentration

oftreatmentCu100andCu100Si,ISEmeasurementswerecalibratedbetween30‐500µMusing

adirectmeasurementinCustandards.

Throughouttheexperiment,nutritivesolutionwassampledatthebeginningoftheweek(before

contact with bamboos) further referred to as “input solution” and just before the renewal,

further referred as “output solution”. All the solutions were filtered (0.2 µm membrane,

Sartorius)andkeptatatemperatureof4°Ctemperature.ThepHineachsolutionwasmeasured


136

withacombinedglass‐electrode.AsubsamplewasacidifiedandsolutionswereanalysedforSi

byICP‐AES(JobinYvonJ38),andtotalCuconcentrationwasmeasuredbyICP‐MS(Agilent7500

CE, Montpellier). Free Cu2+ was analysed by Cu‐ISE in another subsample, immediately after

solutionsampling.TheCu‐ISEmeasurementwasconductedonthefollowingselectedsamples:

fortreatmentCu100andCu100Si,inalltheinputandoutputsolutionsandfortreatmentCu1.5,

Cu1.5Si:intheinputsolutiononday196andinthesolutionsthatweresampledafter1,2,4,27,

43,and96h; in the input solutiononday27andatday28and29 (afteroneand twodayof

solutioncontactwithplant);andintheoutputsolutionthatwascollectedonday34.

The total volumesof each inputandoutput solutionweremeasured.Thevolumeof the input

solutionwas14,5Lonaverage,andoutputvolumevariedbetween9,2and5L.Theamountof

element(SiorCu)inthesolutionistheelementconcentrationmeasuredbyICP‐AESorICP‐MS

multipliedbythevolumeofthesolution.Errorscalculatedfromtheerrorsonoutputvolumeof

solutionsandanalyticalerrorsareestimatedtobe15%.

2.3. Plant analysis

2.3.1 Growth parameters

Thenumberandheightofthestems,thenumberofleavesandthefreshweightwereassayedat

thebeginningoftheexperiment(day0),duringtheexperiment(day21andday49),andatthe

endoftheexperiment(day226).

2.3.2 Analysis of macro and micronutrients

At theendof theexperiment, the25plantswereharvested.Plant leaves,stems,rhizomesand

rootswere separated.The sampleswere carefullywashedwithultrapurewaterand the fresh

massesweredetermined.

Beforetheanalysis,allofthepartsweredriedat60°Cuntiltheyreachedaconstantweight.The

sampleswere subsequentlymixed, ground and homogenised. The sub‐sampleswere dried at

80% until they reached a constantweight to determine their dryweight. The plant samples

(leaves, stems and roots) underwent dry mineralisation for the analysis of trace elements.

Duringthemineralisation, theSicontentwasdeterminedbygravimetryas follows:500mgof

driedplantmaterialwasplacedinaplatinumdishandgraduallyheatedto500°C.Thesilicawas

eliminated in the ash with HF. After cooling, the Si weight was determined based on the

difference fromthe500°Cweight.Theashesweredissolved intoHCl,and thecontentof trace


137

elements in the solutions was analysed by inductively coupled plasma‐optical spectrometer

(ICP‐OESVista‐Pro,Varian,CIRAD,Montpellier,France).Forqualitycontrol,in‐housereference

samplesandcertifiedsamples(Astrasol‐Mix,Analytika)wereusedevery20samples,andeach

analysis was conducted in duplicate. Themeasurement uncertainty was less than 15%. The

quantificationlimitforSiwas5mgg‐1ofdryweight(DW).

Theconcentrationsofmacro‐andmicronutrientsareexpressedasgkg‐1ormgkg‐1DW,andthe

Siconcentrationsareexpressedinmgg‐1DWSiO2suchthattheresultsaredirectlycomparable

withtheliterature.

2.4. Desorption procedure

Asetof frozenrootsub‐sampleswasused tomeasure theadsorbedCuafter theextractionof

apoplasmicCuwithHClonthawnroots.Theextractionprocedurewaspreviouslydetailedand

testedbyChaignonetal.(2002).Briefly,asubsampleof0.4goffresh(thawed)rootswasshaken

end‐over‐endwith20mlof1mMHClfor3min,then180μlof1MHClwereaddedtoyieldafinal

concentration of 10mMHCl. After shaking for a further 5min, the suspensionswere filtered

throughanashlessfilterpaper(Whatman40).CopperinthesuspensionswereanalysedbyICP‐

MS(MSE,Montpellier).Rootsampleswerethenrinsed3timeswithultrapurewater,apartof

thesampleweredryat80°Cinordertomeasurethedryweightandtheotherpartwasfrozenin

liquidnitrogen tostudycopperspeciation: these roots sampleswere further referredas “root

desorbed”.Copperadsorbedontherootswerecalculatedandexpressedinµg.kg‐1DWfromthe

quantityofCuinthesuspensions.TherootdesorbedconcentrationswerecalculatedastotalCu

concentrationinrootsminusCuadsorbedconcentration.

2.5. Anion assays

Plantmaterialwerefreeze‐driedandgroundinablender(Retsh,MM301).Approximately10mg

of resulting powder were immersed in 1 ml of 50 % (v/v) methanol at 4°C, then 300 µL

chloroformwasadded.Thesampleswereshakenfor20min,40rpmat4°Candcentrifugedat

13200 rpm, 4°C, for 10min. 800 µL of upper phasewas collected and evaporated to dryness

usingacentrifugalvacuumdryeratambienttemperaturefor4handredissolvedin1600µLof

ultra‐pure water. Anions concentrations were determined by High Performance Ionic

Chromatography(DionexDX600)usingtheIonPaqAS11HCanionexchangecolumnandaNaOH

gradient.NaOHconcentrationwasraised linearlyfrom1.25to25.0mMover8min, thenfrom

25.0to65.0mMover45min.Identificationandquantificationofeachanionwereperformedby


138

comparison of the retention times and peak areas –integrated using Chromeleon software

(Dionex)–withthestandards.

2.6. Total soluble amino acid

Frozenplantmaterialswerehomogenisedwithapestleandmortarinliquidnitrogen.Analiquot

of1mgofthehomogenatewasmixedin10mlof0.1Mandcentrifugedat12500rpm,4°Cfor10

min.Theupperphasewerecollected, filteredat0.22µmand immediatelyanalyzed foramino

acid determination. Amino acids were detected by High Performance Ionic Chromatography

(DionexICS3000)usingatrapcolumn(CRC)andanaminoacidcolumn(AminoPacPA10)witha

NaOHgradient.Weusedavolumeinjectionof20µMandaflowrateof0.25mL/min.Detection

wasperformedwithAuworkingelectrodes(060082Dionex). Identificationandquantification

of asparagine, threonine, glycine, histidine, cysteine were performed by comparison to

standardsAAADirectmethod(Dionex).

2.7. SEM-EDX

TwosamplesofrootsoftreatmentCu100Siwerefreeze‐fracturedinacryospecimenchamber,

sputter‐coatedwithamixtureofgoldandpalladiuminargonatmosphere,andexaminedusinga

PhilipsXL30SFEGscanningelectronmicroscope (SEM)atnitrogen liquid temperature (North

Billerica, MA) coupled to an Oxford Instruments (Oxfordshire, UK) energy dispersive X‐ray

spectrometer(EDX).TheSEMwasoperatedat15kVwithacountingtimeof60sperpoint.With

SEM‐EDS,chemicalmicroanalysisresultscanbeobtainedwithaspatialresolutionofabout2to

5μmandapenetrationdepthofaboutthesamerange.

2.8. Laboratory-Based µXRF

Root samples were dried at 40°C, embedded in an epoxy resin and cut transversely with

diamondwire saw.The thicknessof theobtainedsectionswasof200µm.Themeasurements

werecarriedoutonaHORIBAXGT7000microscopeequippedwithanX‐rayguidetubeproducing

a finely focused and high‐intensity beamwith a 10‐μm spot size (Rh X‐ray tube, accelerating

voltageof30kVandacurrentof1mA).X‐raysemittedfromtheirradiatedsampleweredetected

with an energy‐dispersive X‐ray (EDX) spectrometer equipped with a liquid nitrogen cooled

high‐puritySidetector.Elementalmaps(256px2,pixelsizeof8mm),showinginparticularCu,

Si and K distribution in root cross sections, were recorded with a total counting time of

20*1000sundertotalvacuummode(toenhanceSidetection).Thebackgroundcontributionwas


139

removedforSiandCu.ImageswereobtainedfromtheintensityoftheSi(Kα),K(Kα)andCu(Kα)

emissionlines.

2.9. EXAFS and XANES: data acquisition and analysis

Copper K‐edge bulk X ray absorption spectra for the plant leaves, stems, roots and reference

compoundswererecordedonaFAMEBeamlineattheEuropeanSynchrotronRadiationFacility

(ESRF,Grenoble,France).Inordertoavoidartifactualchangesofspeciationduetothedryingof

samples, thesampleswereplunged in liquidnitrogen immediatelyafter theharvestandplant

washing, and stored frozen. Frozen leaves, stems and rootswere ground and compacted into

pressedpellets in liquidnitrogen (77K),with special care tokeep thepellets frozen in liquid

nitrogen until the XAS analysis. Pellets of frozen‐hydrated plant samples and referencecompounds were transferred to a He cryostat and cooled to 15 K. Spectra were recorded in

fluorescencemodeusingaSi(220)doublecrystalmonochromatoranda30‐elementsolid‐state

Gedetector(Canberra,France).Foreachsample,1to14scansof45mineachwereaveraged.To

reduce the riskofbeamdamageand toobtain representative spectra, each scanwasdoneon

differentpositionineachspecimen.TheenergywascalibratedusingaCufoil(thresholdenergy

takenatthezero‐crossingpointofthesecondderivativespectrum).Thedatawerenormalized

usingtheAthenasoftware(RavelandNewville2005).EXAFSspectrawereFouriertransformed

from the k to R space using Kaiser–Bessel apodization Windows. This procedure results in

pseudo‐radialdistributionfunctions(RDF)uncorrectedfromphaseshiftfunctions.

In this study, we used a combination of principal component analysis (PCA), target

transformation(TT),andlinearcombinationfitting(LCF)tofitCuKedgeXANESspectra(‐30‐

50keV) andk3‐weightedEXAFS (2.6‐10.5Å‐1) recordedon theplants.Thegoodnessof fitwas

assessed with the normalized sum‐square (NSS) equation

€

NSS =100 × k 3χ(ki)measured − k3[

i=1

N

∑ χ(ki) fitted ]2

/ k 3χ(ki)measured[ ]

2

i=1

N

∑

, N is the number of

points, k3χ(ki)measured is theEXAFS spectrumof the sample in thek‐space andk3χ(ki)fitted is the

EXAFSfit inthek‐space).PCAandTTweredonewiththeSixPacksoftwareandLCFwasdone

withAthenasoftware.Thisprocedurehasalreadybeenusedto fitXANESspectrathatcontain

multiple chemical species (Beaucheminet al. 2002;Legroset al. 2010).Reference compounds

arechosenfromalargepanelofpotentialchemicalspeciesthatcouldcontributetoexplaining

thecoordination,oxidationstate,andatomicenvironmentofCuwithinplantsamples.CuK‐edge

XANESspectraandk3‐weightedEXAFSspectraforCumodelcompounds,contained10inorganic

and 9 organic compounds with various Cu oxidation states (Cu(0), Cu(I), and Cu(II)) were


140

recorded(listedinSII).ForfurtherdetailsaboutthisXANESandEXAFSanalysisSeeSIIIandSI

III.

2.10. Statistical analyses

TheMinitab15.1softwarepackage(Minitab,Inc.)wasusedforstatisticalanalyses.Foreachpart

oftheplants,concentrationsofmicroandmacronutrientsstudiedwereanalysedbyANOVA.We

usedaone‐wayANOVAat the95%confidence levelwithSi treatments(5 levels)as themain

factor, followedbyLSD’sposthoc test at the95%confidence level to evaluatedifferences in

treatments. Average concentrations of Si and Cu in root, stem and leafwere compared using

pairedt‐Testatthe95%confidencelevel.

3. RESULTS

3.1. Silicon and copper in nutrient solution

During the contact with bamboos, i.e. during 7 days, amounts of Si and Cu in the nutrient

solutiondecreased(FigureIV.1).CuamountdecreasedsharplyduringthefirstdaysinbothCu

treatmentsandthendecreasedslowlyduringthelast6days.TotalCumeasuredinsolutionby

ICP‐MSwaswellcorrelatedtopCumeasuredbyISE(R2=0.88)(datanotshown).Theadditionof

Si in nutrient solution did not change Cu concentration in solution (data not shown). The

decreaseofSiamountinthenutrientsolutionwassmallerinCu100SitreatmentthaninCu1.5Si

(FigureIV.1).Throughouttheexperimentand inall treatments,pHslightly increasedafter the

contactwithplants,withafinalpHvaryingbetween6and6.8.

Figure IV.1 Si and Cu amount (µmol) in the nutrient solution in treatment Cu1.5Si and Cu100Si from day 27 to 34

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141

3.2. Growth parameters

Duringthefirst22days,thenumbersofleavesandstemsdevelopedinalltreatmentswerenot

significantlydifferent,rangingfrom86±31to169±103leavesperbamboos(FigureIV.2).After

that period, the average number of leaves in treatment Cu100 and Cu100Si decreased and

reached an average number of 31±23 and 48±52 leaves per bamboo at the end of the

experiment,whichissignificantlydifferentfromthe230±52leavesinthecontrol.Throughout

theexperiment,allthegrowthmeasurementswerestatisticallysimilarfromtreatmentsControl,

Cu1.5 and Cu1.5Si (data not shown). Significant differences betweenmean numbers of stems

and freshmasseswere alsomeasured between treatments Cu100, Cu100Si on one hand and

treatmentsCu1.5,Cu1.5Si,controlontheotherhand.Thereforethe100µMCuconcentrationin

solution induceda significant growth reduction contrary to the1.5µMCu concentration.This

toxicity was confirmed by visual symptoms: chlorotic leaves and brown coloration of roots,

whichwas lesspronouncedinCu100Si treatmentthan inCu100treatment.(FigureIV.3).The

presence of Si in nutritive solution increased the leaves number for Cu100Si treatment as

comparedtoCu100treatmentbutthisincreasewasnotsignificant(FigureIV.2).

Figure IV.2 Mean number of bamboo leaves in the 5 treatments, throughout the experiment from day 0 to day 70. Values followed by same letter for the same days are statistically not different according to Tukey test at the 95 % confidence level.


142

Figure IV.3 Photographs of whole plants harvested after 70 days in treatments: (A) control;

(B) Cu100; (C) Cu100 Si

3.3. Cu and Si distribution between plant parts

TableIV.1showstheaverageCuandSiconcentrationsandtheirstatisticalsignificancebetween

differentpartsofbamboos,whereasFigureIV.4comparesconcentrationsbetweentreatments.

Cu concentrations in all bamboo parts in treatments Cu1.5, Cu1.5Si and control were similar

(Figure IV.4). The Cu concentrations in plant tissues, both leaves, stems and roots, are

significantly higher in treatments Cu100 and Cu100Si than in other treatments. Copper

concentrations in aerial parts of bamboo were not affected by Si in both Cu treatments.

DifferencesbetweenroottotalCucontentintreatmentCu1.5andCu1.5Siwerenotsignificant

duetohighindividualvariabilityofCucontent(147±145forCu1.5treatment)(FigureIV.4).The

adsorbed Cu concentrationwas significantly higher in treatment Cu100Si (2409±1263mg g‐1

DW)than in treatmentCu100(1333±327mgg‐1DW)(Figure IV.4),butconcentrations inroot

desorbed were not different. The adsorbed copper concentration represented 41.5 % and

61.5% of total root Cu concentration in treatment Cu100 and Cu100Si. In all treatments, Cu

accumulatio followed the order root>stem≥leaf, with 70 to 96% of Cu retained in the roots

(TableIV.1).Rootsconcentration(totalCu)rangedfrom14.7to3915mgg‐1DWwhereasleaves


143

concentrationrangedfrom2.4±0.1to35.9±8mgg‐1DW.Siliconaccumulationfollowedtheorder

leaf>stem>root. Copper supply decreased significantly Si concentration in leaves with an

averageof92.8±31.3mgSig‐1intreatmentCu1.5Si,and42±11.8mg.g‐1intreatmentCu100Si.

Figure IV.4 Cu and Si concentration in leaves, stems, roots in the different treatments. Values

followed by same letter between treatments are statistically not different according to Tukey test at the 95 % confidence level.


144

Table IV.1 Cu and Si concentrations in plant parts of bamboos grown in the five treatments (n=4, mean ± standard deviation)

ValuesfollowedbysameletterwithineachtreatmentarestatisticallynotdifferentaccordingtoTukeytestatthe95%confidencelevel.<QL:underthequantificationlimitof5mgg−1

3.4. Cu and Si localization in the root

TheexaminationofarootcrosssectionbelongingtothetreatmentCu100Siwasperformedwith

SEMwhich is a powerful imaging technique because of its nanometer‐scale spatial resolution

andallowstheindividual‘spot’analysis.Thistechniquewaschosentocompareconcentrations

in different tissues of the roots (i.e epidermis, cortex, endodermis, stele)(Figure IV.5). EDX

spectrawere thenrecorded todetermine theirelementalcomposition.Siliconwasdetected in

differentcellsbutmostlyintheendodermiscells.DuetothelowdetectionlimitofCuwithSEM‐

EDX, Cuwas onlyweakly detected in epidermal cells (Figure IV.6). In order to better localize

these elements, another root cross section was examined with μXRF, which has a better

detectionlimitforCuandallowsfor2Dimagingofelementaldistributions.TheµXRFmapping

confirmedtheMEBobservations:Siwasdetectedmostly inendodermalcells,whereasCuwas

detectedmainlyinepidermalcells.


145

Figure IV.5 cryo-SEM image with two EDX spectra taken in the root epidermis cells (A) and

in the endodermis cells (B)

Figure IV.6 µXRF image of a root cross-section from treatment Cu100Si, tricolour image combining Si (red), K (blue) and Cu (green).

3.5. The effects of copper and silicon on some plant

components

The 100 µM Cu treatment decreased significantly the concentrations of the macro and

micronutrients N, Ca, Mg, Mn in leaves and K, Mg in roots (Table IV.2). The addition of Si

increased Ca content in roots and decreased Zn content in stems. The content of P and Fe in

leaves and roots were not significantly modified by Cu and Si treatments. The content of

inorganic acids, carboxylic acids and amino acids are presented in Table IV.3. Among the

inorganicacid,nitrateandphosphateconcentrationsinrootsweresignificantlydecreasedbyCu

both in treatment Cu1.5 and Cu100, with or without Si. Sulfate concentration in roots were

significantly smaller in Cu100 and Cu100Si treatment than in other treatments. Sulfate

concentration in leaves were decreased in all the Cu treatments as compared to the control,

500µm


146

sulfate concentration in stemswas also reducedbut itwasnot significantly. Chlorure content

wasnotmodifiedwhateverthetreatments.

Fewchangeswereobservedforthecarboxilicandaminoacids:measuredbetweentreatments.

CuintreatmentCu100andCu100Siinducedasignificantdecreaseincitrateandmalatecontents

inrootsandinisocitratecontentinleaves.Theonlysignificantmodificationofaminoacidwas

observed forhistidineconcentration inrootsand,contrarytootherelements,an increasewas

observedintreatmentCu100(0.78mmol.kg‐1)andCu100Si(0.88mmol.kg‐1)ascomparedtothe

control(0.34mmol.kg‐1)(TableIV.3).


147

Tab

le IV

.2

N, P

, K, C

a, M

g, F

e, M

n an

d Z

n co

ncen

trat

ions

in le

aves

and

roo

ts o

f bam

boos

from

con

trol

, Cu1

00 a

nd C

u100

Si

trea

tmen

ts (

n=4,

mea

n ±

stan

dard

dev

iati

on)

Valuesfollowedbysameletterwithinplantpartsforeachelem

entarestatisticallynotdifferentaccordingtoTukeytestatthe95%

confidencelevel


148

Tab

le IV

.3

Com

posi

tion

of

inor

gani

c ac

ids,

car

boxy

lic a

cids

and

am

ino

acid

in le

aves

, ste

ms

and

root

s of

pla

nt g

row

n in

tre

atm

ents

co

ntro

l, C

u100

and

Cu1

00Si

(m

mol

es k

g dr

y w

eigh

t-1, n

=4 f

or in

orga

nic

acid

s an

d ca

rbox

ylic

aci

ds, n

=3 f

or a

min

o ac

ids)

. T

he s

tand

ard

devi

atio

n w

as n

ot in

dica

ted

for

the

lisib

lity

of t

he t

able

.

Valuesfollowedbysameletterwithinplantpartsforeachelem

entarestatisticallynotdifferentaccordingtoTukeytestatthe95%

confidence

level


149

3.6. XANES features

XANESspectraanalysiswasusedtoinvestigatethelocalatomiccoordinationsurroundingCuin

different parts of bambooplants. Several inflections (A, B, C,D), defined in XANES references

spectraandpeaksinthefirstderivative(FigureIV.7),provideinformationontheCuoxidation

state and the Cu symmetry compound (Legros et al. 2010). The Cu K‐edge XANES spectra of

plantsamplesdifferedbetweenplantparts(FigureIV.7)suggestingthatCuspeciationinroots,

stemsandleaveswasdifferent.WhenCuismonovalentawelldefinedabsorptionfeature(B)is

locatedat8982eVwhichcorrespondstothetransitions1s‐4pxyfortheCu(I)compounds(Kauet

al.1987).ThisfeatureiswellexhibitedinleafandstemsamplesoftreatmentCu1.5andCu100

inbothSitreatments,indicatingthepresenceofCu(I).Indesorbedrootssamples,thepeakBin

thefirstderivativeallowsustoidentifyalsoCu(I)butataweakerintensitythaninleaforstem

samples (Figure IV.8). The pre‐edge feature (peak A) corresponding to dipole‐forbidden

electronic transitions1s to 3d(quadrupole) is visible only in root and root‐desorbed samples

around8977.8 eV. FeatureC (8988eV) andD (8995eV) are the1s to4pmain electronedge

transitionsofCu(II)compound(FigureIV.8).Thesefeaturescorrespondrespectivelytothe1sto

4pzelectrontransitionand1sto4pxy(Furnareetal.2005).Thesplittingofthemainedgepeak

results from the anisotropic symmetry of Cu(II) compounds (Jahn‐Taller distortion). The

featureD is present in all plant samples, indicating that a fraction of Cu is divalent Cu. Roots

sampleswithSiandwithoutSishowthesameXANESspectrum,whichisclosetoanorganicacid

spectrasuchasmalate(FigureIV.7).Thesplittingofthe4porbitalis5.3and5.5eVinrootand

desorbed root samples, corresponding to an axial Cu‐O distance of approximately 2.35 Å as

comparedtoabout1.95Åfortheequatorialdistance(FigureIV.7)(Dupontetal.2002;Manceau

andMatynia2010).

Inordertoevaluatethenumberofindividualspecies,whichreconstructthewholesetofXANES

spectra (n=10), principal component analysis (PCA)was applied (See SI Table IV.6 andTable

IV.7).Acombinationof twocomponentswassufficient toprovidesatisfactory fits.TheXANES

spectra were best fitted with the reference compounds Cu(I)cysteine and Cu(II)malate or

Cu(II)acetate,indifferentproportionsdependingonplantparts.Themaindifferencesbetween

aerialpartsandrootswastheproportionofCu(I)andCu(II)(FigureIV.9andSITableIV.8).We

observethattheproportionofCu(I)inleaves,stems,androotswashigherintreatmentCu100Si

thanintreatmentCu100.


150

Figure IV.7 (a) Normalized Cu K-edge XANES spectra of plant samples and (b) the

corresponding first derivatives of Cu K-edge XANES spectra.

Figure IV.8 The derivative of XANES spectra showing the evolution of the feature B in

different part of the plant and the splitting of the 4p orbitals resulting from the Jahn-Teller distortion, in leaf Cu100, desorbed root Cu100 and root Cu100.


151

Figure IV.9 Proportions of Cu(I) and Cu(II) determined by LCF on XANES spectra. The

proportions were adjusted to reach 100 %.

3.7. EXAFS features

To gain more insight into the molecular structure of Cu in plant, the EXAFS Spectra were

compared (Figure IV.10)with two reference compounds that have coordination shells: Cu(I)‐

cysteine and Cu(II)‐malate. For Cu(I)‐cysteine, Cuwas surrounded by S atoms located 2.34 Å

fromthecentralatom(Cu‐Sbinding),whereasforCu(II)‐malate,CuwasboundtofourOatoms

at a distance ranging from 1.95 to 1.97 Å (Cu‐O binding). This was reflected on the EXAFS

spectra of these reference compounds by out‐of‐phase oscillations and by RDF peaks, for the

firstcoordinationshell,whichwasshiftedto1.9ÅforCu(I)‐cysteine(featureD)andto1.4Åfor

Cu(II)‐malate (feature C) (distances uncorrected for phase shifts). The leaf and stem EXAFS

spectra(FigureIV.10)showedoscillationssimilartoCu(I)‐cysteineat6.2and7.7Å‐1(featureA),

whereasrootsspectrashowedoscillationssimilartoCu(II)‐malateat6.5and8.5Å‐1(featureB).

ComparingtheEXAFSspectra,wedetectedashiftfromadominantCu‐Obindingpattern(root)

to a dominant Cu‐S binding pattern (leaves). This pattern was clearly expressed by the RDF

pattern. The peak at 1.4 Å (uncorrected for phase shifts) was dominant in roots and its

amplitudegraduallydecreasedintheaerialpartofbamboos,whereastheintensityofthepeak

at1.9Å(uncorrectedforphaseshifts)increased.ThereforeEXAFSspectraconfirmthatdifferent

typesofligandsareinvolvedinmetalcomplexationinvariouspartsoftheplant.


152

Figure IV.10 (a) Cu K-edge extended X-ray absorption fine structure spectra; (b) radial

distribution function (RDF) for plant samples. k = photoelectron wave number (Å−1), R = distance to the neighboring atom (Å).


153

Figure IV.11 Cu K-edge k3-weighted EXAFS (solid line) and linear combination fits (dotted

line) for plant samples.

TheproportionsoftheCuspeciesintheeightsamplesweredeterminedbyLCFuponthemodel

compounds suggested. In roots, best component fits were obtainedwith 50% Cu(II)‐malate,

50%Cu(II)‐histidine for the Cu100 treatment andwith 100%Cu(II)‐malate for the Cu100Si

treatment(FigureIV.11andTableIV.10).MalatewasusedasarepresentativeofCu(II)binding

to alcohol groups and carboxyl groups, and histidine as of a Cu(II) binding to amino N and

carboxylCOOHgroups.ManceauetMatynia (2010)showedthatCu in these twocomplexes is

bound to two 5‐(O/N)‐rings. It is worth noting that oxygen can not be distinguished from

nitrogenbyEXAFS.Thisisduetotheirsimilaratomicnumberandtothefactthatthedistance

betweenOandCuinCu‐OboundingisusuallyverysimilartothedistancebetweenNandCuin

Cu‐Nbounding.However, theLCFofCu100was significantly improvedby the introductionof

Cu‐histidinereference,thismayindicateacontributionofaminegroupsinCucomplexationfor

theCu100treatmentandnotfortheCu100Sitreatment.


154

IndesorbedrootsoftheCu100andCu100Sitreatments,approximately20%ofCu(I)‐cysteine

was detected, and the remaining proportion shows the same ligands than roots of the Cu100

treatment (Cu(II)‐histidine and Cu(II)‐malate) and Cu100Si (Cu(II)‐malate) (Table IV.4). LCF

analysisrevealedthatCuinleavesandstemswaspredominantlyassociatedwithsulfurligands

(Cu2S and Cu(I)cysteine) and the remaining fraction associated with an oxygen or nitrogen

containing organic acid (Cu(II)‐malate) (Table IV.4 Figure IV.13). The presence of both Cu(I)‐

sulphur ligands, i.e. Cu2S : Cu(I)S‐inorganic and Cu(I)‐cysteine: Cu(I)S organic, could be

questioned. But attempts to model leaf and stem samples with Cu2S contributions always

decreasedNSSsignificantly (>30%).Forexample, in leafof treatmentCu100Si, addingCu(I)S

inorganicasthirdcomponent improvedthespectralmatchanddecreasetheNSSby33%,the

beststatisticalagreementbeingobtainedwithamixtureof33%Cu(I)‐cysteine,43%Cu2S,24%

Cu(II)‐malate(FigureIV.12).InstemsamplesCu(II)‐malateimprovedtheLCFqualitycompared

toCu(II)‐histidine(NSSdecreasefrom12.1%to9.8%),butinleavestheLCFwassimilarwith

Cu(II)‐histidineandCu(II)‐malate.

Figure IV.12 Cu K-edge k3-weighted EXAFS spectra of leaf in treatment Cu100Si (solid line)

and fits (dotted line) with and without the contribution of Cu2S compound (Cu(I)S-inorganic).


155

Table IV.4 Proportion of Cu species determined by LCF of the Cu K-edge EXAFS spectra. The proportions were adjusted to reach 100 %.

Figure IV.13 Proportions of Cu compounds determined by LCF on EXAFS spectra. The

proportions were adjusted to reach 100 %.

TheproportionofCu(I)andCu(II)measuredbyXANESandEXAFSareclosed,andtheyshowthe

same tendency between samples, i.e, an increase of Cu(I) proportions in Cu100Si treatment

comparedCu100.Moreover,thebestXANESandEXAFSLCFweredonewiththesamereference

compounds. The accordance between XANES and EXAFS results shows the robustness of the

analysis.

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156

4. DISCUSSION

ThebambooPhyllostachysfastuosaexposedtoaCuconcentrationof1.5µMCu2+inhydroponics

did not express any toxicity symptoms. This result is in accordancewith the tolerance of the

bambooGigantocloasp.“Malaydwarf”exposedtothesameCucontentinaprevioushydroponic

study (Collin et al., Chap III). Therefore, monopodial bamboos Phyllostachys fastuosa and

sympodial bamboos Gigantocloa sp. “Malay dwarf” seem to have the same ability to tolerate

copper.Thisnon‐toxicitydiffers fromobservedtoxicity inhydroponiccultureofdurumwheat

(Bravin et al. 2010) and Sabi Grass (Kopittke et al. 2009) for similar Cu contents. Cu

concentrationinleaves,stemsandrootsincontrolandtreatmentCu1.5andCu1.5Siwerenot

significantlydifferentandthemeansrangedfrom2.4mgkg‐1 in leavesto147mgkg‐1 inroots

(TableIV.1),whichisclosertotheCuconcentrationmeasuredinGigantocloasp.“Malaydwarf”

exposed to1.5µM: 16.6mgkg‐1 in leavesand131mgkg‐1 in roots (Collinet al.Chap III).Cu

contentinleavesandstemsareclosedtothoseofseveralbamboospeciesinanon‐contaminated

soil:3.5and4.5instembaseandtipand5.1mgkg‐1inleaf(Collinetal.2011).Withtheaddition

of100µMCu2+ insolution,Cucontent in leaveshadanaverageof35.9and35.5mgkg‐1anda

rootaverageof3171and3915mgkg‐1inthetreatmentCu100andCu100Sirespectively(Table

IV.1),whichisthehighestCucontentreportedsofarinbambooplants(Collinetal.2011,Collin

et al, chap III). These concentrations are above the toxic thresholds of 20‐30 mg kg‐1 DW

proposed by Marschner (1995) and above the critical root concentration of 250‐300mg.kg‐1

measuredindurumwheat(Michaudetal.2008).WhatevertheSisupplementation,weobserved

strongphytotoxicitysymptomsasevidencedbythebiomassdecrease(FigureIV.2).InbothCu

treatments, Si supplementation did not significantly modify the absorption of Cu whereas Si

concentrations in bamboos parts were increased. However, visual symptoms (chlorosis and

brown coloration of roots) were reduced by the Si supplementation. The measured Si

concentrationinleavesofPhyllostachysfastuosa(averageof92.8mg.g‐1,TableIV.1)wassmaller

than Si concentration measured in a previous hydroponic study with the same Si content in

solution for the bamboo Gigantocloa sp “Malay Dwarf” (179mg.g‐1, Collin et al, chap III).

Bamboos are known to accumulate Si throughout their live (Motomura et al. 2004), thus this

differencemaybeexplainedbythesmallerdurationofthisstudy(3months)ascomparedtothe

previous8monthsstudyofCollinetal.(ChapIII).

Toxic metal may interfere with essential nutrient uptake and transport, thereby disturbing

mineralnutritioncomposition. Inourstudy,CureducedtheuptakeofN,Ca,Mg,K,MnandZn

(Table IV.2) in treatment Cu100 and Cu100Si. It is generally accepted that damage to the


157

plasmalemmaofrootcellsconstitutesthefirsteffectofCutoxicitybycausingalossofions,such

asKorothersolutes(DeVosetal.1991).ThestrongdecreaseofNconcentrationinleavesand

NO3‐inroots(TableIV.2andTableIV.3)showsthatexcessiveCuaffectednitrogenandprotein

metabolisminplants.TheseobservationsareconsistentwiththedecreaseofNO32‐,aminoacids

andproteincontentinducedbyexcessCuinE.haichowensis(Lietal.2007)andinSilenevulgaris

(Weberetal.1991).

ThemajorityofCuwasstoredor immobilized inbambooroots,andonlya little fraction(4 to

30%)of total Cuwas translocated from the root to aerial parts (Figure IV.4). Thedesorption

procedurehas shown that the fractionadsorbed, i.e. stored in theapoplast, represented42%

and 61.5 % of total root Cu concentration in respectively treatments Cu100 and Cu100Si.

Moreover, the examination of a root cross‐section by SEM‐EDX and µXRF, revealed that

substantialdepositionofCuoccurredintheepidermalregion.Thisresultis inagreementwith

severalstudiesusingdifferentapproaches:Cuanalysesafterisolatingthecellwalls(Konnoetal.

2010;Louetal.2004;Nishizonoetal.1987),observationandanalysesbySEM‐EDX(Brunneret

al. 2008; MacFarlane and Burchett 2000), localization using synchrotron‐based X‐ray

fluorescencemicroscopy(Kopittkeetal.2011).Inaprevioushydroponicstudywiththebamboo

Gigantocloasp“MalayDwarf”,weobservedahighandrapiddecreaseofCucontentinsolution

duringthefirsthoursafternutrientsolutionrenewal.Inthisstudyweconfirmthebehaviourof

Cu insolution,whichdecreasedsharplyduring the firstday inbothCu treatments.Therefore,

the desorption procedure, the localization, and the evolution of Cu in the nutrient solution

indicate the important role of Cu adsorption in bamboo roots. The ability of bamboo roots to

bind Cu2+ has been suggested for a use as an adsorbent for the removal of Cu inwastewater

(Babatundeet al. 2009).The cellwallsofplantshave theability tobind copperonnegatively

charged sites, mainly on polysaccharide compounds rich in carboxyl groups (Krzeslowska

2011). The binding to plant cell walls may be a mechanism of heavy metal tolerance and a

defensivestrategyofplantstometalstress(Krzeslowska2011;Nishizonoetal.1987).

The localisation of Si within root tissue, mainly in the region of endodermal cells, is in

accordancewithSEMobservationsinthebambooPhyllostachysheterocycla(Luxetal.2003)and

Sidepositioninotherpoaceaespecies(Hattorietal.2003;Sparksetal.2011).Endodermalcells

are directly connectedwith the Casparian stripswhich provide selectivity againstmetalwith

reduced concentration entering the stele of root (MacFarlane and Burchett 2000). It is

hypothesized that silica deposits in the endodermis cellwall alongwith the thickening of the

Casparianstripsisoneofthemechanismsthatreducethemetalsapoplastictransport(daCunha

anddoNascimento2009).Thiswouldbeexplainedbyaco‐precipitationofSiandmetalinthe

endodermandbyareductioninthetranspirationby‐passflowinducedbytheSideposition(Shi


158

etal.2005).Inthepresentstudy,CuwasnotdetectedintheendodermisandCuconcentration

in aerial partwas not affected by Si supplementation. Despite the important Si deposition in

endodermis,thereductionofapoplasticby‐passofCuandaco‐precipitationdoesnotseemtobe

arelevantmechanismhere.

ThequantityofadsorbedCuissignificantlyhigherintreatmentCu100SithantreatmentCu100.

Another hypothesis to explain the alleviation of excess metal is the mediation of the metal

binding to the cell wall, thereby lowering the symplasmic concentration while the total

concentrationwas the same. Thismechanism has been proposed for the Si alleviation ofMn

toxicityinCucumissativus(RogallaandRomheld2002).MorerecentlyLietal.(2008)suggested

thatSidecreasesCutoxicitybyitssequestrationincellwalls inArabidopsisthaliana.Asimilar

mechanism may occur in bamboo roots since Cu speciation in roots is influenced by the

presenceofSiinnutrientsolution:Cuisboundto50%Cu(II)malate‐likecompoundsand50%

Cu(II)histidine‐like compounds in the treatment Cu100, whereas Cu is entirely bound to

Cu(II)malate‐like compounds in treatment Cu100Si (Table IV.3). In rice, Wang et al. (2004)

suggested the formationofAl‐Si complexes as amechanism for the sequestrationofAl into a

non‐phytotoxic form. But if Si seems to affect Cu speciation, Si was not detected in the local

environmentofCu,contrarytowhatwassuggestedforAl‐Siinriceroots(Wangetal.2004).

XAS results on desorbed roots confirmed that a significant part of Cu in roots (≈40%Figure

IV.9)ispresentasCu(I).WeexplainedthefactthatCu(I)washigherindesorbedrootcompared

toroot,becauseitssignaturewasdilutedbythehighamountofCu(II)presentintheadsorbed

pool.AsCu is known tobe a redox activemetal in plants, thepresenceof the twooxidations

stateinplanttissueisinlinewithphysiologicalstudies(Yruela2009).Coppermaybereduced

before its acquisition by root cells, or Cumay be reduced in the cytoplasm after acquisition,

eitherchemically,metabolisedenzymaticallyorbycellmacro‐molecules(Meharg2005).Ithas

beenshown,inArabidospisthaliana,thatCuismostlikelytoenterthecytosolofrootcellsviaa

cellsurfaceCOPT‐familytransporter(Sancenonetal.2003).TheCOPTproteinstransportCuin

a reduced form, so a reduction step may be necessary at the cell surface. The prevaling

hypothesis is that the Cu(II) would be reduced before by root cell surface ferric reductase

(Mukherjeeetal.2006;Welchetal.1993).Besides,noevidenceofaCureductionbyaplasma

membranereductaseinpoaceaehasbeenshownyet.TheresultofthisstudyshowsthatCu(I)is

foundinrootsbutasCuspeciationwasanalyzedonabulkofroots,wecannotassesswhether

reduction occured at the root surface, in the apoplast or in the symplast. Cu(I) is also

translocatedtostemsandleaveswhereitrepresentsfrom63to85%basedonXANESresults.

InpreviousXASstudy,Cu(I)wasdetectedinCreosotebush(Zygophyllaceae)(Poletteetal.2000)

andThalspi caerulescens (Brassicaceae) (Mijovilovich et al. 2009), and in small proportions in


159

Elsholtziasplendens(Laminaceae)(Shietal.2008)butourresultsarethefirstevidenceforthe

presenceofCu(I)inthetissuesofamonocotyledonspecies.

In treatments Cu100 and Cu100Si, the EXAFS LCF results indicate that Cu was bound to S‐

containing ligands, in two different types of complexes: Cu(I)S‐organic and Cu(I)S‐inorganic

(FigureIV.13).CopperfromCu(I)S‐organiccomplexcanoriginatedfromthiolgroupspresenton

cysteineormethionineligand.TheconsiderablecontentofS‐containgligandstobindCumaybe

correlated to the decrease of sulfate content in bamboo leaves, stems and roots (Table IV.3),

indeed inorganic S is converted to nutritionally and functionally important S‐containing

compounds and their synthesis might be a major factor in plant stress‐defense (Sirko et al.

2007). Several cysteine‐rich binding ligands are involved in the homeostasie, transport and

tolerance mechanisms of Cu, namely opper chaperones, Glutathione (GSH), Phytochelatines

(PCs) and Metallothioneins (MTs). Copper chaperones are involved in the intracellular

trafficking ofmetal ions and insert the Cu into the active sites of specific proteins (Gonzalez‐

GuerreroandArguello2008).GSHandPCsmaybeinvolvedinhomeostasisandinCutransport

both in the phloem (Mendoza‐Cozatl et al. 2008) and xylem (Wei et al. 2007). PCs play an

important role in Cu toxicity by forming stable Cu complexes (Clemens 2006; Cobbett and

Goldsbrough2002;Qianetal.2005).MetallothioneinsarealsoinvolvedinCuhomeostasisand

Cu tolerance of Arabidopsis thaliana (Guo et al. 2008; Roosens et al. 2004). Therefore the

strategy of bamboo to cope with Cu is the internal Cu sequestration by S‐rich ligands and

probablymanyof them.ThehigherproportionofCu(I)S‐compoundinaerialpart isconsistent

withthefindingofQianetal.(2005)whichshowsthatPCshaveamoreactiveroleintheleaves

thanintheroots.

ThepresenceofaCu(I)Sinorganicligandismoresurprising,andrevealthepresenceofanother

Cu S‐rich complex. To our knowledge, this is the first time that a Cu(I)S inorganic phase is

described.Mineralizationcouldbeadefenseagainsttoxiccopper,butreportsofCubiominerals

arerare:coppersulfidemineralizationinyeast(Yuetal.1996),copperoxalateinlichens(Purvis

etal.2008),infungus(Fominaetal.2005)andinThalspicaerulescens(Mijovilovichetal.2009).

It has been shown that in plants cadmium may be stored in a stable high‐molecular weight

complex that contains Cd, PCs and sulfides (Pickering et al. 1999; Reese et al. 1992). Some

complexeswithhighCdratiosconsistofaggregatedparticleswhichthemselvesconsistofCdS

crystallite core coatedwith PCs (Bae andMehra 1998; Dameron andWinge 1990).We could

makethehypothesisthatasimilarprocessoccurswithCuandPCsandinorganicsulfides,and

participatetothestorageofCuinordertoreduceavailableCu.


160

PartofCu instemsand leavesofbamboo ispresentasaCu(II)‐malatecomplex. In leaves,we

cannotdiscriminatetheCu(II)asCu(II)‐malatecomplexfromCu(II)‐histidinecomplex.Inplants,

S‐free ligands include free aminoacids (histidine, asparagine, andalanine), organic carboxylic

acids(citric,malicetc.)andphytosiderophores(nicotinamine(NA),mugineicacid,avenicacid.

Simulation models (Mullins et al. 1986; White et al. 1981a; White et al. 1981b) and in vivo

studiesof xylemsap (Irtelli et al.2009;Liaoetal.2000;PichandScholz1996) show that the

most important long‐distance Cu‐chelating compounds are amino acids mostly histidine,

asparagineandnicotianamine.Inbamboos,thehistidineconcentrationincreasedsignificantlyin

treatment Cu100 and Cu100Si in roots, but not in stems (Table IV.3). Therefore, the role of

aminoacidscouldbeinvolvedintheCucomplexes,althoughtheconcentrationofsolubleamino

acidscontentissmallerthanthoseofcarboxylicacids.ForexampleinstemsintreatmentCu100,

the average histidine concentration was 0.64 mmol kg‐1, whereas malate concentration was

20mmol kg‐1 (Table IV.3). Carboxilic acidsmay also bind to Cu and thus contribute to in its

sequestration or transport (Callahan et al. 2006). EXAFS data demonstrated that malonate,

citrateappearedunlikelytocoordinateCu(II),whilemalatewasfoundtobethemostlikelytodo

so. Although data resulting from malate or histidine complexes were difficult to separate

becauseoftheirchemicalsimilarity,theirevaluationinLCFandtheirdifferentconcentrationin

bamboos,somewhatfavourmalateoverhistidineasthemorelikelymainligand.

ThespeciationofCuinleavesofCu1.5treatmentandCu100treatmentissimilar(FigureIV.9),

thereforeitseemsthatthebambooappliesthesamestrategytocopewithCuconcentrationsin

itstissuesthatareeithertoxicornon‐toxic.

Inleavesandstems,thesupplementationofSiinnutrientsolutionintreatmentCu100Siinduced

an increase of the Cu(I) proportion compared to treatment Cu100. Recently, Khandekar and

Leisner(2011)examinedtheexpressionofthreeMTgenesinArabidopsis,subsequenttoaCu

toxicity and Si supplementationwhich alleviatesmetal toxicity. Cu induced the expression of

these genes, as previously reported (Guo et al. 2003), but the levels of expression either

remainedelevatedorwereboostedtoevenhigherlevelsinplantsprovidedwithextraSi.Inthis

study,wecanassessthatanincreaseofMTsproductioninpresenceofSimayinduceanincrease

in complexes Cu(I)‐MT concentration. Therefore, the formation of these complexes will be a

strategytocopewiththehighCutoxicitylevelincells.However,silicon‐mediatedalleviationof

CutoxicityhasnotbeenconfirmedbythisstudywithbambooPhyllostachysfastuosa.


161

Insummary,thisstudyprovidesimportantevidencesforthepresenceofCu(I)boundtosulfur

compound in a Poaceae species. The main strategy of bamboo to cope with high Cu

concentration in its tissues is (i) an important sequestration in the roots apoplast, mainly in

epidermis,and(ii)acomplexationwithCu(I)Scompoundorganic,thatcanbephytochelatines,

metallothioneins,metalochaperonnes, etc (iii) formationof aCu(I) sulfur compound inorganic

thatmay be involved in Cu storage. Si supplementation has induced low beneficial effects on

bambooCutolerance,butmodifiedtheCuspeciation.


162

5. SUPPORTING INFORMATIONS

SI I References compounds

Table IV.5 Reference compounds database

AnextensivelibraryofCumodelcompoundswasusedandcontained10inorganicand9organic

compounds with various Cu oxidation states (Cu(0), Cu(I), and Cu(II)). Inorganic reference

compoundweredescribed in thestudyofLegrosetal. (2010).Thecrystallineorganometallic

complexes Cu(II)‐acetate dihydrate; Cu(II)‐formate, Cu(I)‐thiophenolate, Cu(I)‐acetate were

purchased from Sigma aldrich. In addition, 4 crystalline organometallic complexes of known

structureweresynthesized.Thecomplexeswerechosenfortheirdiversityinfunctionalgroups

(‐COOOH,‐OH,‐SH,‐NH2)representativeofnaturalorganicmattermoieties.

• Copper(II)di(hydrogenmalonate)dehydrate(LenstraandKataeva,2001);

• Diaquabis(malato‐κ²O1O2)copper(II)(Zhang,2007);

• Malonic andmalic acid were selected among several hydroxy‐carboxylic acids for thisdatabase due to their capacity to model copper(II) binding with NOM (Manceau andMatynia,2010)


163

• Bis‐L‐Histidinecopper(II)DinitrateDihydrate(Evertsson,1969).

• Copper(I)cysteine(Rigoetal,2004).

Synthesisof copper(II) complexeswith thio‐organicacidswereattempted:Cu(II)with cystein

(Dokken et al., 2001) and Cu(II) with thioglycolic acid (synthesis adapted from Ogawa et al.,

1981).Unfortunately,copperreductionwasobservedresultinginpresenceofCu(I).Itwasthus

conductedthatCu(II)isunlikelytobestableinthepresenceofligandcontainingreducedsulfur;

thesedatawerenotconsideredinfurtheranalysis.


164

SI II XANES spectra analysis

Theminimumnumberofreferencespectraneededtofittheunknownsamplewasdetermined

byprincipalcomponentanalysis(PCA)(Beaucheminetal.2002).Principalcomponentanalysis

was performed using SixPack software (Stanford, CA). The indicator function IND provides a

meanstodeterminethenumberofrelevantcomponentsandshouldbeminimalforthenumber

of relevant components. For the relevant component, we also verified that their progressive

introductionduringthespectralreconstructiondecreasesNSSatleast20%.Relevantreference

compoundswereidentifiedbytargettransformationandtheSPOILfunction(Malinowski1978)

wasusedtoevaluateifagivenmodelcompoundwasanacceptabletarget.Thisisanon‐negative

dimensionless number forwhich values <1.5 are considered excellent, 1.5–3 good, 3–4.5 fair,

4.5–6poor,and>6unacceptable.TheuncertaintyintheproportionofCuspeciesisestimatedto

beapproximatively10%ofthetotalamountofCu(Manceauetal.2002a).

TableIV.6givesthePCAoutputparameters,includingtheeigenvalue,indicatorvalues(IND)and

variability explained by the component (Var) for the nine components. IND is assumed to be

minimumwhenthenumberofprincipalcomponentsisreached(Manceauetal.2002b;Sarretet

al.2004a).Inthisstudy,INDwasminimumwith4components(0.037).But,thethirdandfourth

components contained essentially noise. Moreover, the whole set of spectra (n = 10) were

reconstructedcorrectlywith2components.

Table IV.6 Component output parameters determined by principal component analysis of the whole set of XANES spectra

aEigenvalue:valuesofthediagonalmatrixinthePCAafterconsecutiveeliminationofthecomponents.EigenvaluesrankPCsaccordingtotheirimportancetoreproducedata.bMalinowski(1978)indicatorvalue.


165

The nature of Cu species was determined by target transformation and the SPOIL function

(Manceauetal.2002b)wasusedtoassesswhetheragivenmodelcompoundwasanacceptable

target. This is a non‐negative dimensionless number for which values < 1.5 are considered

excellent, 1.5‐3 good, 3‐4.5 fair, 4.5‐6 poor, and> 6 unacceptable. Table IV.7 shows results of

targettransformbyselectingthetwofirstcomponents.

Table IV.7 Results of target transform of XANES spectra (Chi square, R value and Spoil values) of the main Cu references


166

Table IV.8 Proportion of Cu species determined by LCF of the Cu K-edge XANES spectra

Figure IV.14 XANES spectra and their first derivative of reference compounds

treatment Cu(I)-cysteine Cu(II)-malate Cu(II)-acetate Somme NSS (%)

Cu1.5 0.79 0.25 1.04 0.146

Cu1.5Si 0.72 0.32 1.04 0.144

Cu100 0.64 0.37 1.01 0.026

Cu100Si 0.85 0.15 1.00 0.024

Cu100 0.72 0.29 1.02 0.018

Cu100Si 0.82 0.17 0.99 0.022

Cu100 0.40 0.56 0.95 0.031

Cu100Si 0.39 0.56 0.95 0.033

Cu100 .010 0.84 0.94 0.058

Cu100Si 0.17 0.78 0.95 0.045

leaf

stem

root

desorbed

root


167

SI III EXAFS spectra analysis

ThenumbersofcomplexesidentifiablebyEXAFSspectroscopyinthesampleswereevaluatedby

PCAonthewholesetofbamboospectra(n=8).BasedontheINDlocalminimumcriterion,the

dataset contains 2 relevant components (Figure IV.15). However, only 66 % of the total

experimentalvariancecouldbedescribedbythe first twocomponentsandtheexaminationof

thefirstfiveprincipalcomponentssuggestthatthethirdcomponentcontainsignal.Itisshown

inpreviousstudiesthattheINDdeterminationmayunderestimateoroverestimatedthenumber

of independent component required (Manceau et al. 2002a; Panfili et al. 2005; Sarret et al.

2004b).Moreover,thenumberofcomponentwasestimatedherebycalculatinghowmuchthe

fit is degraded upon varying the proportions of the reference spectra in the LCF. Using this

procedure,thequalityofthereconstructionimprovessignificantlywith3components.Then,the

nature of Cu species was determined by target transformation, and the SPOIL function

(Malinowski.E.R.1978;Manceauetal.2002b)wasusedtoevaluateifagivenmodelcompound

wasanacceptabletarget(TableIV.9).

Figure IV.15 a) Component spectra of the principal component analysis, b) first to eight component output parameters determined by principal component analysis of a whole set of EXAFS spectra


168

Table IV.9 Results of target transform of EXAFS spectra (Chi square, R value and Spoil values) of the main Cu references

Table IV.10 Proportion of Cu species determined by LCF of the Cu K-edge EXAFS spectra

Reference Chi Sq Rvalue SPOIL

Cu(II)-malate 41 0.03 1.44

Cu(II)-histidine 58 0.06 2.03

Brochantite (Cu4(SO4)(OH)6) 215 0.13 2.85

Chalcocite (Cu2S) 158 0.16 3.00

Cu(I)-cysteine 244 0.15 3.03

Cu(II)-formate 230 0.17 3.39

Cornetite (Cu3(PO4)(OH)3) 1172 0.36 3.69

Dioptase (CuSiO2(OH)2) 373 0.18 4.43

Cu(II)-acetate 246 0.22 4.47

Cu(I)-thiophenolate 193 0.19 4.49

Cu(II)-malonate 531 0.21 4.66

Cu(OH)2 1667 0.49 5.21

Cuprite (Cu2O) 4911 0.91 6.07

Cu(I)-thioglycolique 731 0.36 6.31

Malachite (Cu2(CO3)(OH2)) 386 0.37 6.42

Elemental Cu 29771 0.75 6.82

Tenorite (CuO) 1830 0.57 7.26

Cu(I)-acetate 371 0.71 7.27

Covellite (CuS) 1213 0.46 8.42

Cu2S Cu(I)-cysteine Cu(II)-malate Cu(II)-histidine somme NSS (%)

Cu100 0.22 0.19 0.32 0.73 14.2

Cu100Si 0.38 0.29 0.21 0.88 12.3

Cu100 0.31 0.26 0.29 0.86 9.8

Cu100Si 0.31 0.33 0.24 0.88 9.3

Cu100 0.19 0.32 0.33 0.84 4.1

Cu100Si 0.19 0.54 0.74 6.9

Cu100 0.43 0.42 0.85 3.2

Cu100Si 0.72 0.72 3.9

stem

root

desorbed

root

leaf


169

Figure IV.16 EXAFS spectra of reference compounds


170

6. BILAN DU CHAPITRE IV

LesbambousmonopodiauxPhyllostachysfastuosaonttoléréuneconcentrationde1.5µMCu2+

en solution, en accumulant des concentrations de Cu similaires à celle de l’expérience

d’hydroponie précédente effectuée avec des bambous sympodiaux Gigantocloa sp. «Malay

Dwarf».Enrevanche,uneconcentrationde100µMCu2+ensolutionainduitunefortetoxicité

chezlesbambous:diminutiondelabiomasse,apparitiondechlorose,concentrationenCudans

les tissus élevée, diminution de certains nutriments. L’ajout de silicium n’a pas eu d’effets

significatifs sur ledéveloppementdesbambousquelquesoit le traitement,maisa induitune

diminutiondessymptômesvisuelsdelatoxicitédeCu.

Lecuivreestessentiellementaccumulédanslesracines,cecompartimentretenant70à96%

deCudubambou.Enraisondesaforteaffinitépourlesparoisracinaires,unepartimportante

deCuestretenuedanslecompartimentapoplasmiqueetenparticulierauniveaudescellules

de l’épiderme. Cela a été observé par microscopie et est confirmé par la spéciation de Cu

racinaire: il est complexé majoritairement sous forme Cu(II) par des composés

carboxyliques,detypemalate,oudescomposésaminésdetypehistidine.Laproportionde

cescomposésdiminuelorsquel’ondésorbeleCuracinaireauprofitdecomposésCu(I).L’ajout

de silicium à entraîné une augmentation de la proportion de Cu apoplasmique etmodifié la

spéciation de Cu (forme Cu(II)‐malate uniquement), sans toutefois entraîner la formation de

liaisonSi‐Cu.

Lecuivreprésentedeuxétatsd’oxydationdanslestissusdubambou:Cu(I)etCu(II),etcedès

laracine.Lesplantesexposéesà1.5µMet100µMCu2+semblentavoirdesstratégiessimilaires

pour faire face à la toxicité du Cu dans la plante. Dans les parties aériennes, Cu(I) est

majoritairement lié à des composés organosulfurés de type cystéine, mais aussi à des

composés inorganiques soufrés. Une proportion mineure de Cu est présente sous forme

oxydée Cu(II) liée à des composés organiques non soufrés: des acides carboxyliques ou

aminésdetypemalateouhistidine.L’ajoutdesiliciumaugmentesensiblementlaproportionde

composés Cu(I) soufrés dans les bambous exposés à 100 µM Cu2+. Le silicium, en induisant

l’augmentationdelaproportiondeCucomplexéeàdesligandsorgano‐sulfurés,pourraitalors

diminuerlatoxicitédeCu.


171



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Conclusionetperspectives

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CONCLUSIONS ET PERSPECTIVES

Rôlebénéfiquedusiliciumchezlebambou

Cestravauxderecherchetrouventleuroriginedanslepostulatsuivant:l’apportdeSiaméliore

la croissance et la tolérance des bambous aux métaux, permettant une amélioration de la

technologiedephytoremédiation.Onassiste,depuisunevingtained’années,àuneaugmentation

du nombre d’études concernant le Si dans les plantes. Certaines abordent la question de

l’essentialité,d’autresdel’identificationdestransporteurs,d’autresencoredesonrôlefaceàde

nombreuxstressbiotiquesouabiotiques.SicompréhensiondutransportdeSidanslesplantesa

progressé,onremarque toutefoisque lesmécanismesd’actionduSipermettantà laplantede

réduirel’impactd’unstresssonttrèsdiversetqu’ilconvientdelesétudier.L’actiondusilicium

semble être différente selon les types de stress, les espèces étudiées, les conditions

environnementales,etc.

Lesbambouspossèdentune fortecapacitéàaccumulerSi,principalementdans les feuilles.Ce

travail a permis de montrer que cette capacité varie significativement entre les espèces, et

particulièrement entre les deux principaux types de bambous: les bambous monopodiaux, à

rhizomes traçants et les bambous sympodiaux, à rhizomes cespiteux. Grâce aux

expérimentationsenhydroponie,nousavonsmesuréuneplusfaibleconcentrationdeSidansles

racinesquedanslestigesetlesfeuilles.Cerésultat,quiestendésaccordaveclesconcentrations

deSimesuréesdans lesracinesdebambous lorsd’étudespubliéesprécédemment(Dingetal.

2008;Luxetal.2003),estnéanmoinsimportantpourcomprendrelemécanismed’accumulation

deSidanslebambou.LarépartitiondeSidanslesbambous,etlesuividesaconcentrationdans

les solutions nutritives (notamment un appauvrissement total de Si en solution) confirme la

présencedemécanismesd’absorptionàlafoisactifsetpassifs.

Aucoursdesexpériencesd’hydroponie,Sin’apaseud’effetbénéfiquesurledéveloppementdes

bambouslorsqu’ilsnesontpassoumisàunstressetce,malgréleurfortecapacitéàaccumuler

Si, que ce soit dans le cas d’un bambou sympodial Gigantocloa sp «Malay Dwarf» ou d’un

bamboumonopodial:Phyllostachys fastuosa. FaceauxdeuxconcentrationsdeCu testéesdans

lescultureshydroponiques(1.5µMet100µM),l’apportdeSinemodifienilesmacronutriments


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absorbés,nilesteneursenacidesinorganiques,acidesorganiquesouacidesaminés.Cependant,

face à une forte toxicité de Cu (100 µM), la présence de Si dans le milieu induit une légère

diminutiondessymptômesvisuelsdelatoxicitédeCuainsiqu’unemodificationdelaspéciation

deCuauniveaudesracinesetdespartiesaériennes.L’apportdeSipourraitdoncdiminuer la

toxicité de Cu par une plus forte immobilisation de Cu dans l’apoplasme racinaire, et une

augmentationdanslespartiesaériennesdeligandscomplexantstelsquelesmétallotionéinesou

lesphytochélatines.

Variabilitéducuivredanslesdifférentesespècesdebambou

Ce travail est le premier à étudier le cuivre dans le bambou : à savoir les concentrations

absorbées en conditions naturelles d’une part et sa toxicité d’autre part. Dans les conditions

naturelles pédoclimatiques de l’île de la Réunion, les concentrations en Cu du bambou sont

faiblesmaissimilairesàcellesdelacanneàsucre,uneautrePoacée(Poaceae)développéesur

dessolssemblables(CollinandDoelsch2010).Malgrécela, lesconcentrationsmesuréesentre

les différentes espèces sont significativement différentes, et il apparaît que les bambous

sympodiauxaccumulentplusdeCuque lesbambousmonopodiaux.Nousavonsalors testé,au

cours de nos expériences en hydroponie, la réponse à un apport de Cu dans deux espèces de

bambous appartenant aux deux différents types. Les bambous sympodiaux Gigantocloa sp

«MalayDwarf»commelesbambousmonopodiauxPhyllostachysfastuosaontprésentélamême

réponsefaceàlaconcentrationdeCuappliquéeensolution(1,5µMCu2+):concentrationdeCu

similairedans lesdifférents tissus, absencede symptômesde toxicité.Ainsi, nousn’avonspas

mis en évidence de différence de réaction entre les deux types de bambous lorsqu’ils sont

exposés à une forte concentration de Cu contrairement à ce qui avait été observé dans les

conditionsenvironnementalesdessolsdelaRéunion.

Stratégiedetoléranceaucuivrechezlebambou

LapremièreetprincipalestratégiedubamboupourtolérerCuest laséquestrationdeCudans

ses racines, diminuant ainsi la quantité de Cu internalisée et potentiellement toxique. Cela

s’expliqueaisémentparsaforteaffinitéauxparoiscellulaires.UnegrandepartiedeCuracinaire

est en effetprésentedans le compartiment apoplastique et localisée, enparticulier, auniveau

des cellules épidermiques des racines. Ce mécanisme semble être suffisant pour tolérer une

concentrationde1,5µMCu2+ensolution.Eneffet,aucunsignedetoxicitén’aétédétectéchezles

bambousexposésàcetteconcentration,quecesoitsurlesracinesousurlespartiesaériennes.

LasecondestratégiepourgérerCuestunecomplexation inplantaquipermetdediminuersa

toxicité.Malgré les nombreux travaux dédiés à Cu dans les plantes, sa spéciation au sein des


180

tissusestpeuconnue.Unrésultatimportantapportéparcetteétudeestlamiseenévidencede

Cu réduit dans les racines. Cela avait été observé chez deux dicotylédones (Mijovilovich et al.

2009;Poletteetal.2000),maiscetteétudeapportelapremièreévidencedelaprésencedeCu(I)

dansunePoaceae.CommeCuestuncofacteurdenombreusesenzymes,laprésencedeCu(I)et

deCu(II)dans laplanteestenaccordavec les fonctionsphysiologiquesqui luisontconférées.

UneformeminoritairedeCuestprésentedanslespartiesaériennessousformedeCu(II)O/N‐

organique, forme qui peut être impliquée dans le transport, en formant des complexes à des

acidescarboxyliquesouacidesaminés.Nousmontronsdanscetteétudequelaphasemajoritaire

deCudanslespartiesaériennesdubambouestsousformedeCu(I)complexéàdescomposés

soufrésdedeuxtypes: uncomposéCu(I)S‐organiqueetCu(I)S‐inorganique.Cetteétudeest la

premièreàdétecter laprésenced’uncomposésoufré inorganiquedans laplante.Uncomplexe

de type Cu(I)S‐organique indique une complexation à des acides aminés soufrés tels que la

cystéineet laméthionine,quisontdesconstituantsdenombreusesmoléculesimpliquéesdans

l’homéostasie,letransportetlestockage.

PERSPECTIVES

SpéciationdeCudanslaplante

Danslestigesetlesfeuilles,nousavonsidentifiéuncomplexeCu(I)inorganiquesoufré.Ilserait

intéressant de confirmer la présence de cette phase. Plusieurs techniques sont utilisées pour

identifier la localisation des éléments traces dans les plantes, comme par exemple la

spectroscopie par perte d'énergie d'électrons (EELS) (Liu and Kottke 2004), l’analyse par

faisceaux d’ions (proton‐induced X‐ray émission: PIXE)(Kramer et al. 1997), la micro‐

fluorescence X induite par rayonnement synchrotron (Sarret et al. 2009; Shi et al. 2011). Ces

techniquessontleplussouventutiliséespouruneétudeàl’échelledutissuoudescellules,avec

une résolution spatiale de l’ordre de 1 µm. Le développement récent de l’utilisation du

NanoSIMS(Nano‐scaleSecondaryIonMassspectrometry)enbiologiesembleparticulièrement

intéressant. En effet, cette technique permet d’analyser des échantillons avec une très bonne

résolutionspatiale(<50nm),unetrèsbonnesensibilité,toutendiscriminantclairementtousles

élémentsetisotopesprésents(Smartetal.2010).Desétudesrécentesontutilisécettetechnique

pourétudierladistributiondunickeldanslescellulesd’uneplanteaccumulatrice(Smartetal.

2010), localiser l’arsenicet leséléniumdansdesgrainesdecéréales(Mooreetal.2010),ou la

distribution subcellulaire du silicium et de l’arsenic (As) dans les racines de riz (Moore et al.

2011).Danscettedernièreétude,lesauteurssontcapablesdelocaliserlesiliciumsurlesparois


181

cellulairesdel’endodermeetdeco‐localiserl’arsenicetlesoufredanslesvacuolesdecertaines

cellules, confirmant une forme de stockage de type As‐phytochelatin (Moore et al. 2011). Les

analysesnanoSIMSs’effectuant surdeséchantillons secs, la contraintemajeurede l’utilisation

du nanoSIMS est la préparation des échantillons qui doit préserver les structures

morphologiquesetconserverladistributionchimique(Grovenoretal.2006).

Ce travail de thèse a apporté des résultats importants concernant la spéciation de Cu et la

localisation de Cu et Si dans la racine.Mais l’utilisation d’une technique d’imagerie ayant une

meilleurerésolutionspatialeetunemeilleuresensibilitépourraitêtreutiliséepourlocaliserles

élémentsdanslescellulesdestigesetdesfeuilles.Celapourraitpermettredemieuxcomprendre

lecomportementdeCudans laplante,commeparexemple la localisationdescomplexesCu‐S

danslescellules:sont‐ilsséquestrédanslavacuole,danslecytoplasme?LescomplexesCu‐O/N

organiques sont‐ils des formes de transport, ou bien des formes de stockage? Au niveau

racinaire,celapermettraitégalementdemieuxcomprendrelerôledusiliciumobservélorsdela

secondeexpérienced’hydroponie.NousavonsvuquelaprésencedeSiaugmentelaproportion

deCuapoplasmiqueetmodifiesaspéciation.Ilestpossiblequedesobservationscommecelles

qui sont décrites dans l’étude de Moore et al (2011) pourraient révéler des distributions

différentesdes éléments enprésencedeSi, etdoncunemeilleure compréhensionde son rôle

danslaracine.

La famille des Poacées, dont fait partie le bambou, représente plus de 20%de la couverture

végétaleterrestreetrassembledenombreusesespècesessentiellespourl’alimentationhumaine

(Arber 2010). Il est probable que le comportement de Cu présente des similarités entre les

espèces de Poacées. Ainsi, unemeilleure connaissance de la spéciation de Cu représente une

avancéeimportantepourestimersatoxicitéaucoursdelachaînealimentaire,sondevenirdans

lessols,etc.

Ilseraitdoncintéressant,pourpoursuivrecetravail,d’étudierlaspéciationetlalocalisationde

Cu dans d’autres Poacées. De plus, même si le bambou à un fort taux de croissance, son

développementenhydroponieest longetsaréponse faceàune forte toxicitén’estobservable

qu’aprèsunmoisdeculture.Afind’avoirdesrésultatsquantifiables,nousavonsdûmenernos

deux expériences de 11mois et 6 mois respectivement, en incluant le temps de pré‐culture.

Ainsi, l’utilisation d’une Poacée plus facile à cultiver (croissance à partir de graines,

développementplusrapide)tellequeleblé,seraitintéressantepourcompléterlesrésultatsde

spéciation et de localisation de Cu. En outre, nos résultats nous renseignent sur l’effet d’une

toxicitéàlongtermedeCuchezlebambou,cequiestadaptéaucontextedephytoremédiation.

Cependantcommelesmétauxinduisentunetoxicitédèslespremièresheuresd’expositiondela


182

plante (ex,Blameyetal.2011;Kopittkeetal.2011) il seraitégalement intéressantd’effectuer

desétudesàcourttermeafindecomprendrelaréponsedesplantesàunetoxicitédeCu.

Améliorationdelatechnologiedephytoremédiation

Le cuivre est un contaminant abondant des effluents qui peuvent être traités par le BAMBOU

ASSAINNISSEMENT®;lacompréhensiondesoncomportement,desmécanismesdeprélèvement

etdetoxicité,estunenjeufortpouraméliorerlatechnologie.

Ilestdélicatd’extrapolerlesrésultatsobtenusenculturehydroponiqueàceuxquel’onpourrait

obtenirsurunsolenconditionsnaturelles.Toutefois, ilsembleque lebambousoitcapablede

tolérerdefortesconcentrationsdeCu.NousavonsvuquelafortecapacitéàtolérerCuestliéeà

saséquestrationdanslesracines,etnotammentàsaforteadsorptionsurlesparoiscellulaires.

Ainsi,uneaugmentationdebiomasseaugmentelaquantitédesitesd’adsorptiondisponibles.Il

semble donc que, grâce à son fort taux de croissance, le bambou présente des capacités de

toléranceàCusupérieuresàd’autresespèces, lebléparexemple(Bravinetal.2010).Dansun

sol, la forteproductiondebiomasse racinairedubamboupourraitpermettredesmécanismes

similairesd’adsorptiondeCusurlesparoiscellulairesdesracines,etainsiunebonnetolérance

àCu.

CettetoléranceàCupermetaubamboudeconserversonefficacitépourl’épurationdesautres

polluants. Lamajeure partie de Cu prélevée par le bambou est difficilement exportable de la

parcellepuisqueelleest localiséedans lesracines.Enrevanche,cettecapacitéàséquestrerCu

dans ses tissus racinaires semble être intéressante pour une utilisation du bambou dans un

objectif de phytostabilisation. Cela permettrait de réduire la mobilité de Cu dans le sol en

l’immobilisantinsitu,etderéduireainsilesrisquesdedispersiondanslemilieuenvironnant.

Nous avons montré que le silicium n’influence pas directement la croissance et le

développement du bambou en conditions contrôlées. Cependant, en conditions

environnementales,lesbamboussontexposésàdenombreuxstressnonétudiésici(desstress

salins, stress hydriques, stress biotiques…), par conséquent Si pourrait avoir un rôle indirect

maisbénéfiquesurlebambou.Enfin,iln’estpasexclunonplusqueSipuisseavoirunrôleplus

importantdans lebamboufaceàunetoxicité induitepard’autresmétaux,commelecadmium

parexemple,oufaceàunepollutionmultimétalliquecommecelaaétédécritrécemmentpour

leriz(Guetal.2011).


183

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RESUME

Cette étude vise à évaluer le rôle du silicium (Si) sur l’amélioration de la croissance et de latoléranceaucuivre(Cu)dubambou,planteutiliséeenphytoremédiation.Plusieursapprochesont été choisies. Dans un premier temps, la répartition et la variabilité de Cu et Si ont étéétudiéesdansplusieursespècesdebamboussedéveloppantdansuncontextepédoclimatiquenaturel afin d’établir des concentrations de référence pour ces éléments. Des cultureshydroponiques ont ensuite permis de caractériser demanièremacroscopique la réponse desbambous à des apports de Si et Cu, et, parallèlement, d’étudier la spéciation de Cu et lalocalisationdeSietCuauseindesdifférentsorganesdubambou(racines,tiges,feuilles).Les concentrations en Si et Cu présentent des différences significatives entre les principauxtypes de bambous, suggérant l’importance d’un caractère génotypique responsable del’absorptiondeceséléments.Faceàunetoxicitéaucuivre,unapportdeSimodifielaspéciationde Cu dans les tissus du bambou, sans toutefois améliorer significativement sa tolérance. Ilapparaît que la stratégie principale dubamboupour gérer de fortes concentrationsdeCu esttoutd’abordune importanteséquestrationdans lesracines,puisunecomplexationdeCuavecdes composés soufrés organiques et inorganiques. Les résultats de cette étude permettrontd’optimiser les technologies liées aux capacités épuratives des bambous face à une pollutionmétallique.Mots clefs: silicium, cuivre, bambou, phytoremédiation, culture hydroponique, spectroscopied’absorptionX(EXAFS,XANES)

ABSTRACT

Thisstudyaimsatassessingtheroleofsilicon(Si)ontheplantgrowthandalleviationofcopper(Cu)toxicityinbamboos.Severalapproacheshavebeenperformed.Firstly,thedistributionandvariabilityofSiandCuwereinvestigatedinseveralbamboospeciesgrownundernaturalpedo‐climatic conditions in order to obtain reference values for Cu and Si in bamboos. Secondly,hydroponicexperimentswerecarriedouttocharacterizethemacroscopicresponseofbambooplants exposed to Si and Cu and, investigated in parallel the Cu speciation and Si and Culocalisationindifferentpartofbamboos(roots,stems,leaves).Significant differences were measured between bamboo species, suggesting that a genotypiccharactermayberesponsibleforSiandCuaccumulation.SiliconsupplementationmodifiedtheCuspeciationbutdidnotinducesignificantimprovementofCutolerance.ThemainstrategyofbambootocopewithhighCuconcentrationsinitstissuesisinitiallyanimportantsequestrationin the roots apoplast, mainly in epidermis, and then a Cu complexation with organic andinorganic sulphur compounds. These results will allow the optimisation of phytoremediationprocessesusingbambooplants.

Keywords: silicon, copper, bamboo, phytoremediation, hydroponic, X‐ray absorptionspectroscopy(EXAFS,XANES)

role du silicium sur la tolerance au cuivre et la croissance des

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