The role of phytochelatins in constitutive and adaptive heav

植物修复 重金属污染 植物络合素

DOI:10.1093/jxb/erf107

Theroleofphytochelatinsinconstitutiveandadaptiveheavymetaltolerancesinhyperaccumulatorandnon-hyperaccumulatormetallophytes

ÁLlugany1,4,RietVooijs1,JeanetteHartley-Whitaker2andPetraM.Bleeker1HenkSchat1,3,Merce

DepartmentofEcologyandPhysiologyofPlants,FacultyofEarthandLifeSciences,VrijeUniversiteit,

DeBoelelaan1085,1081HVAmsterdam,TheNetherlands

21

CentreforEcologyandHydrology,Merlewood,Grange-over-Sands,CumbriaLA116JU,UK

Received17May2002;Accepted2August2002

Abstract

Usingtheg-glutamylcysteinesynthetaseinhibitor,L-buthionine-[S,R]-sulphoximine(BSO),theroleforphytochelatins(PCs)wasevaluatedinCu,Cd,Zn,As,Ni,andCotoleranceinnon-metallicolousandmetallicolous,hypertolerantpopulationsofSilenevulgaris(Moench)Garcke,ThlaspicaerulescensJ.&C.Presl.,HolcuslanatusL.,andAgrostiscastel-lanaBoiss.etReuter.Basedonplant-internalPC-thioltometalmolarratios,themetals'tendencytoinducePCaccumulationdecreasedintheorderAs/Cd/Cu>Zn>Ni/Co,andwasconsistentlyhigherinnon-metallicolousplantsthaninhypertolerantones,exceptforthecaseofAs.ThesensitivitiestoCu,Zn,Ni,andCowereconsistentlyunaffectedbyBSOtreatment,bothinnon-metallicolousandhyper-tolerantplants,suggestingthatPC-basedsequestra-tionisnotessentialforconstitutivetoleranceorhypertolerancetothesemetals.CdsensitivitywasconsiderablyincreasedbyBSO,thoughexclusivelyinplantslackingCdhypertolerance,suggestingthatadaptivecadmiumhypertoleranceisnotdependentonPC-mediatedsequestration.BSOdramaticallyincreasedAssensitivity,bothinnon-adaptedandAs-hypertolerantplants,showingthatPC-basedsequestrationisessentialforbothnormalconstitu-tivetoleranceandadaptivehypertolerancetothismetalloid.TheprimaryfunctionofPCsynthaseinplantsandalgaeremainselusive.

34

Keywords:Buthioninesulphoximine,heavymetaltolerance,Holcuslanatus,hyperaccumulator,metallophyte,phyto-chelatins,Silenevulgaris,Thlaspicaerulescens.

Introduction

Phytochelatins(PCs)aresmallmetal-bindingpeptideswiththestructure(g-glu-cys)n-gly,(g-glu-cys)n-b-ala,(g-glu-cys)n-ser,(g-glu-cys)n-glu,(g-glu-cys)n-glnor(g-glu-cys)n,inwhichnvariesfrom2to11(Grilletal.,1985,1986a;MehraandWinge,1988;Meuwlyetal.,1993;Klaphecketal.,1994).Theirsynthesisfromglutathione(Grilletal.,1989),homo-glutathione,hydroxymethyl-glutathione(Klaphecketal.,1995)org-glutamylcysteine(Hayashietal.,1991)iscatalysedbyatranspeptidase,namedphytochelatinsynthase,whichisaconstitutiveenzymerequiringpost-translationalactivitionbyheavymetals(Grilletal.,1989;DeKnechtetal.,1995;Klaphecketal.,1995;Chenetal.,1997).Phytochelatinsynthase(PCS)hasbeenshowntobeactivatedbyabroadrangeofmetalsandmetalloids,inparticularCd,Ag,Pb,Cu,Hg,Zn,Sn,Au,andAs,bothinvivoandinvitro(Grilletal.,1987;Maitanietal.,1996;Chenetal.,1997).ThecapacitytosynthesizePCsissupposedtobepresentinallhigherplants(Gekeleretal.,1989)andthemajorityofalgae(Ahneretal.,1995).Theyalsohavebeendetectedinseveralfungi,includingSchizosaccharomycespombe,Candidaglabrata,andMucorracemosus(Grilletal.,1986b;Mehraetal.,1988;Mierschetal.,2001).Inaddition,thenematodewurm,Caenorhabditeselegans,

Towhomcorrespondenceshouldbeaddressed.Fax:+31204447123.E-mail:hschat@bio.vu.nl

ÂnomadeBarcelona,E-08193Bellaterra,Spain.ÂaVegetal,FaculdaddeCiencias,UniversidadAutoPresentaddress:LaboratoriodeFisiologõ

Abbreviations:BSO:L-buthionine-[S,R]-sulphoximine;PC:phytochelatin;GSH:glutathione;g-ECS:g-glutamylcysteinesynthetase;GS:glutathionesynthase;PCS:phytochelatinsynthase.ãSocietyforExperimentalBiology

2002

植物修复 重金属污染 植物络合素

2382Schatetal.

appearedsynthesistopossessaPCSgene,whichrestoredPCstrain,presentsuggestingandCdtolerancethatfunctionalinanS.pombePCSPCSknock-outVatamaniukincertainetanimalstoo(Clemensgenesetalmay.,2001;befunctionTheretheofhasPCs.beenal.,2001).

Theyconsiderablehavedebateconcerningthemetalcellularbeenassumedtofunctionin1991).nutrients,homeostasisortraf®ckingofessentialheavyminimallyHowever,particularlytheCuandCuZnandexposureZn(Thumannetal.,trationsnutritionalinrequiredplantcellstoinduceareoftenPCsatfarconsiderablelevelsabovetheconcen-thatarenormalthresholdsrequirements,orevenclosetothetoxicitymutants,havesuch(SchatasArabidopsisetal.,2000).cad1Moreover,(HowdenetPC-de®cientforablyessentialneverbeenmetalreportedal.,1995),nutrients,toexhibitincreasedrequirementsCobbett,increasedsensitivitiestoandCudidornotZnshow(Howdenconsider-andecotypic1992).thalianadifferencesConformingwiththeseobservations,metallothioneinwereshowninCutoleranceinArabidopsisratesdisrupted(Murphyandexpression,tobecorrelatedwithtype-2Taiz,ratherthanPCaccumulation(ClemensintheirPCSgene1995).wereHowever,hypersensitiveS.pombetocellsCurequiredforetalCu.,1999),detoxi®cationindicatinginthatPCsynthesismaybeareOntheotherhand,thereisconvincingsomeorganisms,evidencethatatleast.PCsnon-essentialessentialforthemetals,normalparticularlyconstitutiveCd.toleranceFirst,disruptiontoseveralof(ClemensPCSgeneofetalin.,S.pomberesultedinhypersensitivitytoCddramaticallyPCScDNAs1999;fromHawheat,etal.,Arabidopsis1999).Second,,expressioncerevisiaeor,evenincreasedinmutantsCdde®cienttoleranceinSaccharomycesandS.pombenumbervacuolarpeared1995;toofacidi®cation(Clemensetinalvacuole.,1999).formationThird,abeCd-hypersensitiveimpairedinPCsynthesisArabidopsis(Howdenmutantsetap-al.,selectedCobbettsynthesisforhypertoleranceetal.,1998).toInaddition,tomatocelllinescysteineunderGoldsbrough,synthetaseCdexposure,(g-ECS)dueCdtoexhibitedactivityincreasedenhanced(Cheng-glutamylPCandterialjunceag-ECS1994).Furthermore,overexpressionofbac-1999enhancedorglutathionePCsynthesissynthetaseandCdtolerance(GS)in(ZhuBrassicaetessentiala,b).PCsmightalsoberequiredfortolerancetoal.,mutantsCobbett,wereHgandalsoAs.hypersensitivePC-de®cienttoArabidopsisnon-Hg(Howdencad1withenhancedtheg1992).-ECSinhibitor,InhibitionbuthionineofPCsynthesisbytreatmentandVallisneriaHgtreatedspiralissensitivity(GuptainetalHydrillasulphoximine(BSO),.,verticillataandwere2000;foundcellculturesSchmotoÈgerbeoftobaccoand1998).Rauvol®aLikewise,serpentinaBSO-ethypersensitiveal.,2000).toAs(Nakazawaetal.,

andNormalchelationAs,isconstitutiveapparentlynottoleranceentirelytoCdand,possibly,Hg

hypersensitiveofCdbyPCsintheexplainedcytosol.SeveralbythemereCd-synthesisinunderS.Cdpombeexposure,mutantsbutappearedshowedtonormalbede®cientPCthefunctionalettransportHMT1,ofCd±PCanABC-typecomplexestransporterintovacuolesmediating(Ortizvacuolaral.,1992,acid-labileCd-PC1995),orimpairedinafurtherstabilizationof1993).sulphidecomplexes(Speiserthroughetal.,1992theincorporationb;JuangoflardemonstratedaccumulationMgATP-dependentofCd±PCtonoplastcomplexestransportetal.,haveandvacuo-Langeacid-labileandWagner,inoatalso1989;andtobacco,respectively(VoÈbeen

geli-has(SpeiserbeenthatetdemonstratedsulphideincorporationSaltandinRauser,Cd±PC1995).complexesAlso,al.,1992a;DeinKnechtB.junceaandSilenevulgaristionplantsofvacuolaretal.,1994),suggestingCd±PCcompartmentalizationmaybeessentialfornormalandfurtherCdtolerancestabiliza-incommonlyNaturallytoo.

selectedheavymetalhypertolerance,whichenrichedenhancedsoils,founddoesinplantnotpopulationsseemtofromstronglymetal-isequalPCcapacitiesPCsynthesis.DeKnechtetbeal.associated(1995)obtainedwithofsynthesisincrudeandactivationproteinextractsconstantspreparedforCd-inducedfromrootsTheCd/Zn-hypertolerantwererootPCconcentrationsandnon-metallicolousmeasuredinvivoS.,however,paredmuch1994).PCTheatlowerinequalinvivoacid-labileratestheofhypertolerantCdplants,evenwhensulphideuptakecontents(DeKnechtofetal.,identical,complexesstabilitiessuggestingandPCthatchaintheCd±possiblelengthdifferencesdistributionswerewereglutathioneabsentof(DetheKnechtcomplexesformedinbothplantintypesthesimilarly(GSH)concentrationsetal.,1994).intheAlso,rootstherespondedreducedandidentical,GSHtorecoveryCdexposure,afterandarrestingtheratesofPCdegradationhypertolerantsuggestingavailability,etnorplantsthatfromresultedthelowerPCtheaccumulationexposureinweretheahigherPCneitherturnoverfromratealower(DeKnechtGSHextractableal.,1995).hypertolerantrootFinally,Cdwasthefractionofexclusivelyacid-sameplants(DeKnechtconsistentlyetal.,higher1994).inInthethePCsmetallicolousinspecies,rootsCu-inducedalsoappearedandtoZn-inducedbemuchaccumulationhigherofhypertolerantequalplantsplantsrespectively,thaninCu-hypertolerantinnon-bothwhencomparedandZn-accumulationmetalexposureetshownal.,1993).Moreover,intherootslevelsandatequalratesofmetalatdecreased(DeVosetPCalaccumulation.,1992;Harmensbetweentoco-segregatewithCuhypertolerancewas(Schatnon-metallicolousandCu-hypertolerantincrossesplantsexpressionandofKalff,enzymes1992).andThus,transportersalthoughinvolvedarti®cialinover-the

植物修复 重金属污染 植物络合素

PC-basedGS,tolerancePCSmetalorClemensorHMT1,sequestrationCdtolerance,hasbeenmachinery,suchasg-ECS,atleastshown(Ortiztoincreaseetal.,metal1995;severaletal.,1999;Zhuetal.,1999a,b),andarti®ciallyexamplesreportedselectedofenhancedforCdhypertolerancePCsynthesisincellalthoughlinesevidence(ChenhypertolerantofnaturallyandGoldsbrough,selected1994),therehaveisbeennoenvironments.planttheMoreover,populationsenhancedPCsynthesisinBSOfromCd-,Zn-,orCu-toxicecotyperesponsetoa40mMCdtreatmentdidnotdetectablyinaenhancenon-metallicolousofS.vulgarissuggestingecotype,althoughhypertolerantunderitdramaticallyidenticalsensitizedanaturallyKnechtselectedthatPCCdsynthesishypertolerancemightnotberequiredconditions,forratesetal.,1992).Ingeneral,theinstronglythisspeciesdecreased(DetolerantofinS.PCvulgarisaccumulationminepopulationsinCu-,Cd-,(seeandabove)Zn-hyper-PC-independentfact,resultfromincreasedactivitiesmight,decreasedsequestrationmechanismsofleadingalternativetovationnaturally(DecytoplasmicKnechtetmetalal.,1995).availabilitiesOnforPCSacti-foundaccumulationtoselectedbeassociatedAshypertoleranceinHolcustheotherlanatushand,wasinhypertoleranceroots,suggestingandincreasedwithPC-thiolenhancedtoAsratesmolarofratiosPC2001).

toAs,thatatPCleastsynthesis(Hartley-Whitakermightbeessentialetalfor.,PCsThepresentevidencewithregardtothepreciserolefortolerancesinconstitutivetheisoftenandfragmentorynaturallyandselectedambiguous.high-levelMuchmetalofthisevidencecomparedstudy,isthebasedcompleteonsingle-concentrationdose±responseexposures.curveswereInimposedandbyforCu,rootCd,growthZn,Ni,inhibitionCo,andPCaccumulationphytesnon-metallicolouslanaSilenevulgaris,ecotypesandofAstheinpseudometallo-metallicolousTo,andtheZnhyperaccumulator,HolcuslanatusThlaspi,Agrostiscaerulescenscastel-.exposuresassessthesolution.

werepossibledonewithroleandforwithoutPCsinmetalBSOintolerance,thenutrienttheMaterialsandmethods

Plantmaterials

S.(Germany),vulgarisseedswereandazincsmeltercollectedfromacoppermineÁnearres(Belgium),Marsberg

(Amsterdam,anon-metalliferouswastedepositatPlombie

Cu-hypertolerant,ThesiteattheFreeUniversityCampusandandNetherlands).showslowThedegreespopulationofhypertolerancefromMarsbergtoZn

isCd,Cd.Vooijs,andThepopulationfromPlombie

ÁresishypertoleranttoZnandbeencollectedgiven1997).showsinMorepleiotropicSchatdetailedhypertoleranceetal.(1996).siteandtoNiandCo(SchatandSeedspopulationofT.characteristicscaerulescenshavenon-metalliferousfromaZnserpentinehill(MontesiteorewasteatdepositnearLaCalamine(Belgium),wereaPrinzera,WillerwiltzItaly).(Luxemburg),Frompreviousandstudiesfromit

aPhytochelatinsandheavymetaltolerance2383

appearedfromdecreasingthresholdthattheexposureZntolerancelevelsofthesepopulations,asestimatedWillerwiltzin>>(W).theorderLikewise,LaCalamineforleaf(LC)chlorosis,>MontePrinzeravariedstrongly,(MP)>andLC(AssuncCd>accumulationW,andCdtoleranceNiintolerancetheorderdecreasedLC>>WintheorderMPresults).ËÄaoetweremuchhigherinMPand>WMP.thanZn,inNi,LC

As-enrichedSeedsalof.,2001;A.AGLAssuncËÄa

oandHSchat,natusminewastedumpnearJales(Portugal).Non-Campuswas(Amsterdam,collectedfromThetheNetherlands).botanicalgardenofTolerancetesting

Seedstransferredweregerminatedonaerated(1996),MES-bufferedto1.0lpolyethylenemoistpeatand8-d-oldseedlingswerenutrientsolutionpots(threeplantsperpot)withconcentrations.or,incasecomposedasinSchatetal.replacedwerebyafreshAfteroftheone5dgrasses,withhalf-strengthmacronutrientofofthehydroponicsamecomposition.culture,theHalfsolutionofthepotswasconcentrationssuppliedasdidwithnotLproduce-BSOatafurthera250decreasemMconcentrationof(highersolutionsdemonstratedappropriatewereinreplacedpilotexperiments).again,andAfterthemetalsanotherroot5dGSHperiod,levels,thesalts,solutionsexceptconcentrations.As,whichwasAllsuppliedthemetalsassodiumwereaddedwereasaddedsulphateatspikedEDTAsolutions,werethefromsamewhichasduringFe-EDTApreculture,petitivewasAlso,reducedinthetoaAs-spiked10mMlevel,solutions,omittedtopreventthetopreventNHCu-excessive4H2PO4maintainedCu,duringinhibitionofarsenateuptake.TheBSOtreatmentwasBSObycontrastwithmetaltheexposure,othermetals,exceptappearedinthetocasebeofcomplexedCu,becausespeci®cinthetionelectrodesnutrient(Hsolution,Schat,unpublishedasshownbyresults).measurementsbyTherootwithion-methodwasT.(SchatmeasuredandafterTenBookum,4dofexposure,1992)inusingthecharcoalstainingelonga-PCcaerulescensoffand.PartoftheplantswerelefttheunstainedcaseofS.andvulgarisusedandfornewatthemetalstartanalysis.ofthemetalInthetreatment,caseofthegrasses,therootswerecutGrowthrootwaschambermeasuredandthelengthofthelongestconditionsafter4wered.

exactlyasinSchatetal.(1996).PCandmetalanalysis

PriorPb(NOtoharvest,therootsystemsweredesorbedinanice-cold5mMimmediately3)2solutionvacuumfrozenforinliquid30min.nitrogen,Rootslyophilized,andshootswereseparated,materialuntilHCl140andwereanalysis.65%digested(v/v)HNOinTwenty2mloftoa1001:4mg(v/v)aliquotsandmixtureofstoredofgroundunder37%(v/v)dry3,inclosedTe¯oncylindersforabsorption°C.Metalswithspectrophotometerinthedigests6hat(PerkinweremeasuredElmerusinga¯ameatomicextractedacoupledwithandmeasuredMHS-102100),inthecaseofAsbyHPLC,hydrideusingsystem.post-columnPhytochelatinswere(1994),Ellman'sanalysedexceptreagent,forsamplesexactlyofAs-treatedasdescribedplants.inDederivatizationKnechtetal.exactlyderivatizationasafterinpre-columnSnelleretalderivatization.(1999).Correctionswithmonobromobimane,Thelatterwerefordifferential(2000).

ef®ciencesweremadeaccordingtoSnelleretal.Statistics

Thelog-transformationdatawerestatisticallyanalysedusingtwo-wayANOVAconcentrationofthedata.Signi®canceoftheBSOQafterhypersensitivity.

interactionwasusedasacriterionforBSO-imposedmetal

植物修复 重金属污染 植物络合素

2384Schatetal.

Results

Copper

PC-mediatedCutolerancewastestedinnon-metalli-colousandcupricolousS.vulgarisecotypes(populationsAmsterdamandMarsberg,respectively).CopperinducedtheaccumulationofPCsinrootsofbothecotypes.However,thethresholdCuexposurelevelrequiredtoinducesigni®cantPCaccumulationwasmuchhigherinthecupricolousecotypethaninthenon-metallicolousecotype.Moreover,thecupricolousecotypeexhibitedlowerroot-internalPC-thiol(PC-SH)toCumolarratios(Table1).ShootPCconcentrationswerenegligibleinbothecotypes(datanotshown).Intheroots,however,PCswerestronglydecreasedbyBSOinbothecotypes(Table1).Usually,therootcopperconcentrationswerealsodecreased,thoughtoamuchlowerdegree,resultinginlowerPC-SHtoCumolarratiosintheBSO-treatedplants(Table1).TherootgrowthresponsetoCu,however,wascompletelyun-affectedbyBSO,bothinthenon-metallicolousandthecupricolousecotype(Fig.1).

Cadmium

Fig.1.Meanrootelongation(n=15)throughout4dofexposuretoCuinBSO-treated(opensymbols)anduntreated(closedsymbols)non-metallicous(circles)andcupricolous(squares)S.vulgaris.Standarderrorsvariedbetween2%and8%ofthemeans.

PC-mediatedCdtolerancewasestablishedinnon-metallicolousandCd/Zn-hypertolerantSilenevulgaris

Áres,respectively).(populationsAmsterdamandPlombie

Cd,likeCu,stronglyinducedtheaccumulationofPCsinroots(Table2),butbarelyornotinshoots(datanotshown).Again,thePCconcentrationsandtheroot-internalPC-SHtoCdratioswerehigherinthenon-metallicolousecotypethantheywereinthehypertolerantcalamineecotype(Table2).BSOstronglydecreasedrootGSHandPCconcentrationsand,thoughtoamuchlowerdegree,rootCdconcentrations,resultinginconsiderablyde-creasedPC-SHtoCdmolarratios(Table2).BSOsigni®cantlyenhancedCd-imposedrootgrowthinhibitioninthenon-metallicolousecotype(P<0.001),butdidnot

detectablyeffecttherootgrowthresponseofthehypertolerantecotype(Fig.2).

Usingasimilarexperimentaldesign,threeT.caerules-censecotypeswithvaryingdegreesofCdtoleranceandaccumulation,originatingfromserpentine,calamine,andnon-metalliferoussoil(populationsMontePrinzera,LaCalamine,andWillerwiltz,respectively)werecompared.CdinducedconsiderablePCaccumulationintherootsandshootsofalltheseecotypes,thoughtodifferentdegrees.Whencomparedatsimilarroot-internalCdconcentrations,therootPCconcentrationsdecreasedintheorderMontePrinzera>Willerwiltz>LaCalamine(Fig.3),whichisalsotheorderofdecreasingsensitivitytoCd(seeMaterialsandmethods).Thesamepatternwasalsofoundinshoots(datanotshown).Cd-imposedrootgrowthinhibitionwasnotenhancedbyBSOineitheroftheecotypes(Fig.4).Attheendoftheexperiment,i.e.4dafterCdsupply,there

each;SEinparentheses)indesorbedrootsofunpretreatedandBSO-pretreatednon-metallicolous(Amsterdam)andcupricolous(Marsberg)S.vulgaris,aftera4dexposuretoincreasingCuconcentrationsinthenutrientsolution(nd=notdetermined)

Population

Exposure(mMCu)0.10.72.16.318.90.11.07.021.063.0189.0

PC-SH

(mmolg±1DW)+BSO<0.1

0.2(0.07)3.2(0.63)4.6(0.41)1.8(0.16)<0.1

0.2(0.08)0.5(0.04)1.3(0.24)2.7(0.19)1.9(0.45)

±BSO<0.1

0.4(0.11)9.7(2.35)12.8(1.98)2.8(0.61)<0.1

0.3(0.08)1.1(0.23)2.4(0.37)7.9(1.46)2.6(0.09)

PC-SH:Cu(molmol±1)+BSOnd0.81.41.5ndnd0.40.80.30.3nd

(0.23)(0.21)(0.47)(0.04)(0.23)(0.06)(0.10)

±BSOnd1.23.73.6ndnd0.31.41.41.3nd

(0.40)(0.65)(0.15)(0.07)(0.12)(0.16)(0.08)

Table1.Totalphytochelatinthiol(PC-SH)concentrationsandPC-SHtoCumolarratios(meansofthreesamplesofthreeplants

Amsterdam

Marsberg

植物修复 重金属污染 植物络合素

Phytochelatinsandheavymetaltolerance2385

Áres)S.vulgarisafter4dofeach;SEinparentheses)indesorbedrootsofnon-metallicolous(Amsterdam)andcalamine(Plombie

exposure,withandwithoutBSO,toincreasingCdconcentrationsinthenutrientsolution(nd=notdetermined)

Population

Exposure(mMCd)7.515.030.060.0120.0240.07.515.030.060.0120.0240.0

PC-SH

(mmolg±1DW)+BSO5.04.16.79.24.6nd2.83.03.56.15.49.3

(1.07)(0.43)(1.24)(0.89)(0.39)(0.20)(0.08)(0.53)(0.36)(1.14)(2.79)

±BSO21.2(2.07)24.7(4.72)35.3(2.91)40.6(5.33)42.7(2.71)nd

7.3(0.83)9.3(2.07)11.2(0.99)16.(1.74)17.(3.01)26.(2.61)

PC-SH:Cd(molmol±1)+BSO0.90.60.60.70.4nd0.70.50.60.60.30.3

(0.08)(0.12)(0.03)(0.15)(0.10)(0.02)(0.09)(0.05)(0.11)(0.07)(0.10)

±BSO3.42.72.52.61.8nd1.81.71.61.71.10.7

(0.40)(0.12)(0.31)

(0.45)(0.06)(0.25)(0.29)(0.08)(0.31)(0.16)(0.09)

Table2.Totalphytochelatinthiol(PC-SH)concentrationsandPC-SHtoCdmolarratios(meansofthreesamplesofthreeplants

Amsterdam

ÁresPlombie

Fig.2.Meanrootelongation(n=15)throughout4dofexposuretoCdinBSO-treated(opensymbols)anduntreated(closedsymbols)non-metallicolous(circles)andCd-hypertolerant(squares)S.vulgaris.Standarderrorsvariedbetween3%and14%ofthemeans.

Fig.3.MeanPC-thioltoCdmolarratio(n=12)inroots,asafunctionoftheroot-internalCdconcentration,inT.caerulescensfromcalamine(closedcircles),serpentine(opencircles),andnon-metalliferoussoil(opensquares),after4dofexposuretoaseriesofCdconcentrationsinthenutrientsolution.Standarderrorsvariedbetween5%and19%ofthemeans.

werenovisibleeffectsofthetreatmentsonshootperformance.However,themajorsinkforCdinT.caerulescensistheshoot,andpreviousexperiments

Äoetal.,2001)clearlyshowedthatafterlonger(AssuncËa

periodsofexposure(>1week)shootperformancerespondedmuchmoresensitivelytometalexposurethandidrootgrowth.Therefore,anadditionallonger-termexperimentwasperformedwiththeserpentineandthecalamineecotype.After2weeksofexposure,thesensi-tizingeffectofBSOwasclearlyapparentfromtheshootperformanceoftheserpentineecotype.At1mMexternalCd,theshootsoftheBSO-treatedplantswerealmostentirelybrightyelloworwhitewithnecroticparts,whereasthoseoftheuntreatedplantswerestillmoreorlessgreen.Inaddition,asindicatedbythesigni®canceoftheBSOQCdinteraction(P<0.01),BSOincreasedtheshootfreshweightresponsetotheCdtreatment(Fig.5).Inthe

Cd-hypertolerantcalamineecotype,shootfreshweightwasonlysigni®cantlydecreasedatthehighesttreatmentlevel,i.e.125mMCd,andtheBSOQCdinteractionwasnotsigni®cant.Chlorosiswasonlyapparentatthe25mMandthe125mMtreatments,bothwithandwithoutBSO.Inbothecotypes,therootelongationresponsewasunaffectedbyBSO(datanotshown).

Arsenic

BSO-imposedeffectsonAstolerancewereinvestigatedinnon-metallicolousS.vulgaris(Amsterdam)natus(Amsterdam),natusandA.castellanafromastronglyAs-enrichedgoldminewastedeposit(Jales)(seeMaterialsandmethods).ArsenatecausedastrongaccumulationofPCsinS.vulgaris,albeitexclu-sivelyinroots,therootPC-SHtoAsmolarratiosbeing

植物修复 重金属污染 植物络合素

2386Schatetal.

Fig.in4.Meanrootelongation(n=12)throughout4dofexposuretoCdmetallicolousBSO-treated(opensymbols)anduntreated(closedsymbols)non-T.means.

caerulescens(diamonds),.Standardserpentineerrorsvaried(circles),betweenand3%calamineand16%(squares)ofthe

above(Tablerates,3).3,TheexceptgrassesforthemosttoxicexposurelevelsowingbuttotoathevariableresultsalsorecoverywereshowedconsideredhighPCaccumulationofthetobeunreliable,However,thesamples,probablythePC-SHN-acetyl-internalstandardaddedtoAsLmolar-cysteineratios(dataobtained,notgiven).didunderestimated,werebetween1.9and2.7,thoughandtreatmentnotvarytionsdramaticallywiththedegreesofAstolerance.BSOPCg±1concentrations(Table3),particularlydecreasedconsistentlyinthetherootPCconcentra-remainedgrasses,belowwhere0.1therootBSODWbothstronglyintheincreasedBSOtreatment.therootInmmolgrowthalltheresponsespeciestested,theAsgrasses,intolerantBSOandcompletelynon-tolerantarrestedecotypes.rootgrowth,InthecasetoAs,evenofinhibitionexposurerootinthelevelsabsencethatofdidBSOnot(Figscause6,7).rootInfact,growthatcompletely,sytemsofnecrotic,andmosttheiroftheleavesplantswereseemedwiltedtohavetheordiedlargelyoffsomechlorosis,exceptbutforremainedtheyoungerturgid.

ones,whichshowedZinc,Nickel,Cobalt

PCPC-dependentaccumulationinducedbyZn,invulgarisnon-metallicoloustoleranceandtoZn-hypertolerantthesemetalsNi,orwereCoandpossibleecotypesinvestigatepely)Áres,ofS.

Calamine,and(populationsT.caerulescensrespectively),caerulescensAmsterdam(populationsandPlombie

respect-aswellasinWillerwiltzaandLaMaterialsconcentration-dependentandecotypemethods).(populationZnMontePrinzera)serpentine(seeT.non-metallicolousaccumulationexposurecausedofPCsapronouncedinmolarratioswerefarS.vulgarisbelowunity,although(Tablethe4).ThePS-SHrootscalamine

toZnofFig.inserpentineBSO-treated5.Medianshoot(openbiomasssymbols)(n=12)andafteruntreated2weeksof(closedexposuretoCderrorsvaried(circles)betweenand7%calamineand24%(squares)ofthemeans.

T.caerulescens.symbols)StandardecotypeaexhibitedlowerrootdramaticallymuchhighercompareddecreasedrootZnPC-SHaccumulationPCconcentrations,inspiteoftoZnmolarrate,leadingratios,toasbothBSOecotypes,withdecreased(Fig.8),thethenon-metallicolousecotype(Table4).InalthoughrootgrowththeBSOresponsewasunaffectedby(TablethePC-SHtoZnmolarratiostreatmentinbotheffectivelytheZn-induced4).PCaccumulationwasnotapparentinshoots.ecotypesinhowevershoots.T.caerulescensPCaccumulationwasalsofoundineachofThePCecotypes,concentrationsparticularlywereinroots,butalsohigherthan(lessinthancontrol2mmolplantsg±1(aboutdryweight),inconsiderable,0.04mmolalbeitg±1muchweight),theanddidnotincreasewithexposurelevelswithindryinterecotypicrangetested(25±1250mMZn).TherewerenoobviousBSOthenotPC-SHdidnottodetectablydifferencesinPC-SHtoZnmolarratio's.ZnmolarratiosaffectintheeithergrowthoftheresponseecotypestoZn(dataorS.Nishown).

concentrations,vulgarisandCo,inducedalbeitPCaccumulationinbothecotypesof(aboutg±10.06mmolthoughexclusivelyg±1drybeingweight),higherinthanroots.inThecontrolrootplantsPC(5±80dryhypertolerantmweight,MNi;3±243andtendedneverexceeded1mmolmMtodecreasewithexposurelevelhypertoleranceecotype,whichCo)inexhibitsbothecotypes.someTheZn-consistentlytoNiandCo(SchatandVooijs,pleiotropic1997),lowermetallicolousPC-SHdisplayedmetalecotype.tometalmuchBSOmolarlowerPCconcentrationsandneitherratiosaffectedthandidthePC-SHthenon-ecotypemolarratios,northerootgrowthresponseineithertoallPCaccumulation(datanotshown).

underNiexposurewasalsofoundPCofconcentrationsthethreeT.caerulescenswereusuallylowerecotypesthantested.1mmolTheg±1

rootindry

植物修复 重金属污染 植物络合素

Phytochelatinsandheavymetaltolerance2387

each;SEinparentheses)indesorbedrootsofnon-metallicousS.vulgaris(populationAmsterdam),after4dofexposure,withandwithoutBSO,toincreasingAsconcentrationsinthenutrientsolution(nd=notdetermined)

Species(population)

ExposurePC-SH

(mmolg±1DW)PC-SH:As(molmol±1)Table3.Totalphytochelatinthiol(PC-SH)concentrationsandPC-SHtoAsmolarratios(meansofthreesamplesofthreeplants

(mMAs)+BSOS.(Amsterdam)

vulgaris11.71683.7(0.32)327.5(0.21)6.8(0.51)128

64<0.1

3.4(0.79)(0.64)weighttypes,ecotype,andinthebetweennon-metallicolous1and2mmolandg±1theincalaminetheserpentineeco-(15±450moreorlessirrespectiveofthelevelofexposurewereNilowermMNi).ThePS-SHtoNimolarratios,however,0.15inducedintheserpentineecotypethanintheotherones.mmolsomeg±1PCaccumulationintheshoot,uptoserpentineresponseCobalt-inducedtoecotype.dryNiineitherBSOweight,ofthedidalbeitexclusivelyintheecotypesnotaffectthegrowththePCaccumulationwasonly(datainvestigatednotshown).inbelowcalamine1mmolecotype.g±1TherootPCconcentrationswerelevelbelow(3±243responsethetodetectionmMCo).anddecreasedwithincreasingexposurethismetallimit.The(dataBSOshootnotshown).didPCnotconcentrationsaffectthegrowthwereDiscussion

ThedemonstratedaccumulationHowever,inaofPCsundercopperstresshasbeenwithmoreorlesslargeprecisenumberofalgaeandplants.phenomenonregardPCistothedose±effectquantitativerelationshipsinformationofthisthanaccumulationscarce.Wijnholds,forCu(Ahnerappeared,TheandinthresholdMorel,general,exposurelevelsfor1995;tobelowerforCdaccumulation1996).exposuresuggestinglevelwasInseveralstudiesCu-inducedRijstenbilandPCforacutenotapparenttoxicityhaduntilbeenthethresholdsequestrationthatPCsarenormallynotinvolvedexceeded,inCuVosGerringa,etal.,1992;underRijstenbilconditionsofsubtoxicexposure(Deexposuremulationlevels2002).forInrootthepresentetal.,1998;Rijstenbilandgrowthstudy,inhibitiontheandthresholdPCCutolerantseemedtocoincide,bothinnon-tolerantandaccu-Cu-notnormalinduceS.vulgaris(Fig.1,Table1),suggestingthatCudidincreasedcellularPCaccumulationhomeostasishaduntilthecapacityofthereliescapacitymosthomeostaticcapacityinbeenthetolerantexhausted.ecotypeTheconstitutivetoprobablyef¯uxoverexpressionCuonfromacombinationofanenhancedofthea2b-typerootcellsmetallothionein,

andastrong±BSO+BSO±BSO3.42.7(0.40)17.38.6(0.19)(1.26)1.9(0.08)4.13.1(0.27)27(2.50)2.7(0.19)31.741.1(0.12)3.8(0.27)(0.35)<0.1

(6.07)(4.68)0.6(0.32)2.7nd

(0.01)

3.0nd

(0.58)

Fig.arsenate6.Meanrootelongation(n=15)throughout4dofexposuretosymbols)inBSO-treated(opensymbols)anduntreated(closed(squares).Standardnon-metallicolouserrorsvariedS.betweenvulgaris2%(circles)and15%andoftheH.means.

lanatusSvMT2bproperties(VanHoofetal.,2001a,b).Also,thelattercytosolicwouldbeexpectedtoreducetheactivityofthentolerantexplainCutheavailablelowerforPC-SHPCStoactivation,Cumolarwhichratioswouldinaccumulationecotype(Table1).TheabsenceofconsiderablethethelevelslackundersubtoxicexposureobviouslyexplainsPCclearforofrooteffectgrowthofBSOinhibitiononthe(Fig.threshold1).TheCuexposurecurveseffecttoismoreofdif®cultBSOontheslopeofthedose±responseabsenceofaLeopoldbehighlytoexplain.Cu±PCcomplexesseemhaveandGunther,stable1997),(MehraandMulchandani,1995;1989),beensomesuggestingisolatedthatfromS.vulgarisandintactrootsCu±PC(Verkleijcomplexesetal.,Onesynthesismightextent,PCsshouldcontribute,atleasttoarguetoCudetoxi®cationinCu-stressedplants.transportwasthattheBSO-imposedinhibitionofPCPC-SHofGSHnotcomplete,(Depossiblythroughshoot-to-rootdecreasedtoCumolarratiosKnechtintheettreatedal.,1995).plantsHowever,weretheunderCu-toxic(Tableconditions1),showingwasthatnotthelimitedrootperformancemuchbythePC

植物修复 重金属污染 植物络合素

2388Schatetal.

Áres)S.vulgaris,aftera4dexposure,withandeach)indesorbedrootsofnon-metallicolous(Amsterdam)andcalamine(Plombie

withoutBSO,toincreasingZnconcentrationsinthenutrientsolution(nd=notdetermined)

Population

Exposure(mMZn)22550100200400220040080016003200

PC-SH(mmolg±1DW)+BSO<0.1

2.1(0.12)3.4(0.23)3.7(0.42)6.0(1.41)5.8(1.01)<0.1

0.3(0.02)0.5(0.12)2.1(0.03)3.4(0.38)2.1(0.19)

±BSO<0.1

6.1(0.29)8.4(0.13)8.6(0.99)11.9(1.15)14.9(0.98)<0.1

0.5(0.01)1.3(0.11)3.4(0.40)5.0(1.02)2.7(0.34)

PC-SH:Zn(molmol±1)+BSOnd

0.21(0.05)0.12(0.03)0.08(0.01)0.09(0.02)0.11(0.03)nd

0.008(0.002)0.009(0.003)0.004(0.001)0.008(0.001)0.005(0.002)

±BSOnd

0.34(0.01)0.23(0.03)0.22(0.04)0.17(0.02)0.20(0.02)nd

0.014(0.003)0.019(0.002)0.017(0.004)0.014(0.001)0.008(0.002)

Table4.Totalphytochelatinthiol(PC-SH)concentrationsandPC-SHtoZnmolarratios(meansofthreesamplesofthreeplants

Amsterdam

ÁresPlombie

syntheticcapacity.Moreover,thePCS-de®cientcad1mutantofArabidopsisthalianadidnotexhibitconsider-ablyincreasedsensitivitytoCu(HowdenandCobbett,1992),althoughCuhasbeenshowntoinducetheaccumulationofPCsinthisspecies(MurphyandTaiz,1995).Thus,mostoftheevidenceavailablethusfarsuggeststhatPCsmaynoteffectivelycontributetoCudetoxi®cationinmostalgaeandhigherplants,althoughtheyappeartodosoin®ssionyeast(Clemensetal.,1999).Thereasonforthismightlieinthepresenceorabsenceofmoreeffectiveef¯ux-orMT-basedalternativedetoxi®ca-tionsystems.

CdhasbeenshowntobeastronginducerofPCaccumulationinabroadvarietyofalgaeandhigherplants,aswellasinseveralfungi,andPC-basedCdsequestrationisgenerallyconsideredtobeessentialfornormalCdtoleranceinorganismswithfunctionalPCSgenes(HowdenandCobbett,1992;Ortizetal.,1992;Speiseretal.,1992b;Cobbettetal.,1998;Clemensetal.,1999;Vatamaniuketal.,2001).Inagreementwiththisview-point,astrongCd-inducedPCaccumulationandBSO-imposedhypersensitivitytoCdinnon-metallicolousS.vulgariswasobserved(Fig.2).IntheCd-hypertolerantecotype,however,thePC-SHtoCdmolarratiosweremuchlower(Table2),andBSO-imposedhypersensitivitytoCdwasnotapparent,irrespectiveofthelevelofexposure(Fig.2),suggestingthatCdhypertoleranceisachievedthroughenhancedactivityofanasyetunknownPC-independentCdsequestrationmechanismdecreasingtheactivityofcytoplasmicCdavailableforPCSactivation(DeKnechtetal.,1992,1995).TheresultsobtainedwithT.caerulescensarebasicallyinlinewiththis.ThedegreesofCd-imposedPCaccumulation(Fig.3)inthedifferentecotypeswereinverselyrelatedtothelevelsofCdtolerance.Apparently,theCd-hypertolerantecotypedidnotpossessPC-dependentCdtolerance,asshownbytheabsenceofanyBSO-imposedhypersensitivity(Fig.4).Inaccordancewiththis,Ebbsetal.(2002)concludedthatPCsynthesiswasnotresponsibleforCdtoleranceinT.caerulescensfromPrayon(Belgium),whichexhibitsasimilardegreeofCdhypertolerance.However,BSOdidsigni®cantlyincreaseCdsensitivityintheCd-sensitiveserpentineecotype(Fig.4),suggestingthatPC-dependentconstitutiveCdtolerancedoesoccurinnon-metallicolousecotypesofthisspecies.ThemajordifferenceswithS.vulgariswerethatT.caerulescensshowedconsiderableaccumulationofPCsintheshoots,thoughlessthanintheroots,andthattheresponsestotoxicCdexposureandBSOwereprimarilyapparentfromshootperformance,ratherthanfromrootelongation,whichismostprobablyduetothemuchhigherrateofCdtranslocationtotheshoot.ArsenichasbeenshowntoinducehighlevelsofPCaccumulationinavarietyofplantspecies(Grilletal.,1987;Maitanietal.,1996;Nakazawaetal.,2000;

Ègeretal.,2000).IntactAs±PCcomplexeshaveSchmo

beenisolatedfromS.vulgaris(Snelleretal.,1999),andPCsynthesisissupposedtobeessentialforAsdetoxi®cation

Ègeretal.,2000;Hartley-Whitakeretal.,inplants(Schmo

2001).Inagreementwiththis,astrongAs-inducedPCaccumulationwasfoundinallthespeciestested.Moreover,BSOconsistentlyproducedhypersensitivitytoAs(Figs6,7).TherelativelysmallBSOeffectinS.vulgaris,ascomparedwiththegrasses(Fig.6),mightbeduetothefactthatBSOdecreasedrootGSHtoamuchlowerdegreeinS.vulgaris(toabout30%inunexposedcontrols)thanitdidinthegrasses(tolessthan5%).Arsenate,beingaphosphateanalogue,istakenupbyphosphatetransporters,andarsenatehypertolerancehasbeenshowntobeachievedthroughconstitutivesuppres-sionofthehigh-af®nityphosphateuptakesysteminanumberofgrassspecies,natus(MehargandMacnair,1990,1991a,b,1992).Thissuppressionwouldreducethein¯uxofAstoalevelthatcanbecopedwithbytheconstitutivePC-baseddetoxi®cationmachinery

植物修复 重金属污染 植物络合素

Fig.arsenate7.Meansymbols)inBSO-treatedrootelongation(open(n=15)symbols)throughoutand4untreateddofexposure(closedto(squares).StandardmetallicolouserrorsvariedA.castellanabetween4%(circles)and18%andofthe

H.means.

lanatus(Hartley-Whitakerhypothesis.etalobservedlargelyinEventheJalesextreme.,2001).populationAsThesehypertolerance,resultssupportthisofA.castellanasuch,asPClostunderBSOexposure(Fig.7),suggestingwasthatconstitutivesynthesisagreementtoleranceisequallyandhypertoleranceessentialforbothnormalcorrelatedobserveddecreaseswithHartley-WhitakerofPC-SHtoAsetalmolar.(2001),toAs.Alsoinratiostolerance-werenotPC-independentintheminesigni®cantsequestrationecotypes,mechanismssuggestingthatdonotalternative,playanyrelativelyZnand,roleinAshypertolerance.

(Grill1995).etalweakparticularly,.,1988;activatorsNiAhnerandofandPCS,Co,Morel,bothare1995;inconsideredvivoKlapheckandintoetvitrobeal.,tively1997).lowThe(MaitanistabilityoftheZn±PCcomplexiscompara-unknownThelower,atstabilitiesetpresent,ofal.,1996;LeopoldandGunther,butNi±PCmightandbeCo±PCcomplexesareandDaviesCoastosuggestedothercysteine-basedbytherelativelyexpectedligandslowaf®nitiestobeeven(Perrin,1979).ofNiimposedetPC-de®cientrootal.(1991)growthreportedinhibitionthatinBSOFestucadidnotrubraincrease.Also,Zn-theconsiderablyArabidopsisCobbett,enhancedZn-sensitivitycad1mutantdid(HowdennotexhibitPC1992).Inagreementwiththis,low,butdetectableandratiosaccumulationZn,wasNi,wereorCoobservedratesinallandthelowspeciesPC-SHandtoecotypesmetalmolarundercaerulescensconsistentlyexposure.absent,BSO-mediatedbothinS.hypersensensitivityvulgarisandnotHypertoleranceessential,forsuggestingthatPC-basedsequestrationT.isinPC-SHT.caerulescenstotheZn,detoxi®cationNi,andCoofineitherS.vulgarisofthese,andmetals.toNiindicatetothatmetalthesemolarwasagainhypertolerancesratios,associatedwhichwithdecreasedaremayachievedbetakenthroughtoPhytochelatinsandheavymetaltolerance2389

Fig.in8.Meanrootelongation(n=15)throughout4dofexposuretoZnmetallicolousBSO-treatederrorsvariedbetween(circles)(opensymbols)6%andanduntreated(closedsymbols)non-andcalamine17%ofthe(squares)means.

S.vulgaris.Standardsequestrationabilitymechanismsthatdecreasethemetals'avail-increasedforancevacuolarPCSactivationtransportininthecytoplasm,suchastolerance-relatedinS.vulgaris(ChardonnenstheetcaseofZnhypertoler-founddifferencesinPCalaccumulation.,1999).Comparablewerenotecotypes,amonginducedcomparedveryhowever.thethreelittlePCIndistinctlyaccumulationparticular,Zn-tolerantZn,T.caerulescensinincontrasttoCd,mightberelatedtoS.vulgaristotheZn.ThehyperaccumulationreasonforT.thiscaerulescensiselusive,,butasfavourTosummarize,theseresultsdonotprovidetrait.

evidenceinessentialofalthoughmetalaroleforPCsinthedetoxi®cationoftheconsiderableCu,whenmicronutrientspresentZn,Ni,andCuinplants,stablePCscomplexesPCaccumulationattoxicandconcentrations,apparentlyinducedformed(Grillaregeneral,etessentialwithPCs.Also,itishighlyunlikelythatal.,1989;inBrunethedetoxi®cationetal.,1995),ofsuggestingFe,Mo,andthat,MninexcessivelyPCsotheraccumulatedmightnotbemicronutrientsinvolvedinthedetoxi®cationofthehand,thisstudycon®rmsthatPCsinplants.Onthefurtherdetoxi®cationbeevidenceofcertainnon-essentialaremetals.requiredTakingforprimaryhypothesizedfromnon-essentialfunctionofthat,thePCSinliteratureintoaccount,itmightwouldplantslieandalgaeatleast,theaf®nitiesmetalsandmetalloidsinwiththedetoxi®cationrelativelyhighof1992;metals,Guptatosulphur,suchasCd,Hg(HowdenandCobbett,tous,environment,arehowever,etalmostlypresentalthough.,1998)and,particularly,As.Suchatnegligiblebeinghighlyconcentrationstoxicandubiqui-conservationwhichmakesitdif®culttobelievethatinthethekingdomtowouldofbefunctionalultimatelyPCSexplainedthroughouttheplantresultseitherclearlyoftheseshowmetals.adecreasedMoreover,dependencyinthebycasetoxicexposureon

ofPC-based

Cd,these

植物修复 重金属污染 植物络合素

2390Schatetal.

sequestrationcensinhypertolerantS.vulgarisandT.caerules-betothe,suggestingmosteffectivethatPC-mediatedstrategytodetoxi®cationmightnotgenesInthiscopewithtoxicexposureviewmetal,oftheatleast.

(Clemensoverthespreadanimal,ofsigni®cantlyplant,andfungalhomologouskingdomsPCSseemsancestrallikelyetal.,1999,2001;Vatamaniuketal.,2001),itnumbergene.thatPCStheygenesmusthaveseemevolvedtofromanancientconsequenceofanimalandfungallineages,havebeenpossiblylostasinaspeci®cargueMT-basedofthemetalevolutionsequestrationofmoreeffectiveandmoreaaspresentitstillthatseemsPCStooriginallydoinS.pombefunctioned(Clemensinsystems.Cuetdetoxi®cation,Onemightal.,1999).ItsthroughoutfunctionAlthoughtheplantinplantskingdomandareitsstillubiquitousoccurrenceintheprimaryfunctionofPCSenigmatic,doesnotseemhowever.toliemicronutrients,thedetoxi®cationfunctionsitcannotofexcessivelybeexcludedaccumulatedthatitmetaltoxicexposedphysiologicalinmetalmicronutrientconditions,particularlyhomeostasisbecauseundersomehowplantsnon-levelsconcentrations.appeartonormaltocontainnutritionalPCsatmicronutrientlow,butdetectableexposureAcknowledgements

This(CIRIT,workwassupportedbyagrantfromtheCatalonianGeneralityAlessandraprojecttheseedsofLombini,BEAItheMonteUniversity300151).TheauthorsareindeptedtoDrPrinzerapopulationofBologna,ofwhoT.caerulescenskindlyprovided.

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