recent

Review

Recentfindingsonthephytoremediationofsoilscontaminated

withenvironmentallytoxicheavymetalsandmetalloids

suchaszinc,cadmium,lead,andarnic

a1,dez-Allica2,il3,a3,2&u2,*

1UnidaddeBiofı´sica,CentroMixtoUPV/EHU,Apdo.644,E-48080Bilbao,Spain;2NEIKER,Basque

InstituteofAgriculturalRearchandDevelopment,Berreaga1,E-48160Derio,Spain;3DepartmentofPlant

BiologyandEcology,UniversityoftheBasqueCountry,Apdo.644,E-48080Bilbao,Spain(*authorfor

correspondence:phone:+34-94-403-43-00;fax:+34-94-403-43-10;e-mail:cgarbisu@)

Keywords:metalloids,metals,phytochelatins,phytoextraction,phytoremediation,transgenicplants

Abstract

Duetotheirimmutablenature,ultofhuman

activitiessuchasminingandsmeltingofmetalliferousores,electroplating,gaxhaust,energyandfuel

production,fertilizerandpesticideapplication,etc.,metalpollutionhasbecomeoneofthemostrious

emediation,anemergingcost-effective,non-intrusive,andaesthet-

icallypleasingtechnology,thatustheremarkableabilityofplantstoconcentrateelementsandcom-

poundsfromtheenvironmentandtometabolizevariousmoleculesintheirtissues,appearsverypromising

thisfieldofphytoremediation,theutilizationof

plantstotransportandconcentratemetalsfromthesoilintotheharvestablepartsofrootsandabove-

groundshoots,i.e.,phytoextraction,maybe,atprent,ementof

thecapacityofplantstotolerateandaccumulatemetalsbygeneticengineeringshouldopenupnew

kofunderstandingpertainingtometaluptakeandtranslocation

mechanisms,enhancementamendments,andexternaleffectsofphytoremediationishinderingitsfullscale

tsgreatpotentialasaviablealternativetotraditionalcontaminatedlandremediation

methods,phytoremediationiscurrentlyanexcitingareaofactiverearch.

nmentalmetalpollution

andphytoremediation

Soilpollutionhasrecentlybeenattractingconsid-

erablepublicattentionsincethemagnitudeofthe

probleminoursoilscallsforimmediateaction

(Garbisu&Alkorta2003).Asaresultofhuman

activitiessuchasminingandsmeltingofmetallif-

erous,electroplating,gaxhaust,energyandfuel

production,fertilizerandpesticideapplication,

etc.,metalpollutionhasbecomeoneofthemost

theirimmutablenature,metalsareagroupof

,although

veralmetalsareesntialforbiologicalsystems

andmustbeprentwithinacertainconcentration

range(Garbisu&Alkorta2003),athighconcen-

trations,metalscanactinadeleteriousmannerby

blockingesntialfunctionalgroups,displacing

othermetalions,ormodifyingtheactiveconfor-

mationofbiologicalmolecules(Collins&Stotzky

1989).Metaltoxicityforlivingorganismsinvolves

oxidativeand/orgenotoxicmechanisms(Briat&

Lebrun1999).

Badontheirchemicalandphysicalproper-

ties,threedifferentmolecularmechanismsof

heavymetaltoxicitycanbedistinguished:(i)pro-

ductionofreactivespeciesbyautooxidationand

Fentonreaction(Fe,Cu),(ii)blockingofesntial

functionalgroupsinbiomolecules(Cd,Hg),and

(iii)displacementofesntialmetalionsfrom

biomolecules(Schutzendubel&Polle2002).

ReviewsinEnvironmentalScienceandBio/Technology3:71–90,2004.

ÓdintheNetherlands.

71

Metal-contaminatedsoilsarenotoriouslyhard

ttechnologiesresorttosoil

excavationandeitherlandfillingorsoilwashing

followedbyphysicalorchemicalparationofthe

ghhighlyvariableand

dependentonthecontaminantsofconcern,soil

properties,siteconditions,andsoon,theusually

enormouscostsassociatedwiththeremovalof

metalsfromsoilsbymeansoftraditionalphysi-

cochemicalmethodxplainwhymostcompanies

hefactthat

veryoftenlargeareasareaffectedbyheavymetal

contamination,aremovaliscertainlydifficult.

Therefore,somemethodsaredevelopedtokeep

themetalsinthesoilbutreducetherisksrelatedto

thisprence(e.g.,bydecreasingbioavailabilityby

insituimmobilisationprocess)(Dieltal.

2002).Onewaytofacilitatesuchimmobilisationis

byalteringthephysicochemicalpropertiesofthe

metal-soilcomplexbyintroducingamultipurpo

anion,suchasphosphate,thatenhancesmetal

adsorptionviaanion-inducednegativechargeand

metalprecipitation(Bolanetal.2003a).

Heavymetalscannotbedestroyedbiologically

(no‘‘degradation’’,changeinthenuclearstruc-

tureoftheelement,occurs)butareonlytrans-

formedfromoneoxidationstateororganic

complextoanother(Garbisu&Alkorta2001).

Althoughmicroorganismsthatumetalsaster-

minalelectronacceptorsorreducethemaspart

ofadetoxificationmechanismcanbeudfor

metalremediation(Garbisu&Alkorta1997),

whenconsideringtheremediationofmetal-pol-

lutedsoil,metal-accumulatingplantsoffer

numerousadvantagesovermicrobialprocess

sinceplantscanactuallyextractmetalsfromthe

pollutedsoils,theoreticallyrenderingthemclean

(metal-free)(Garbisu&Alkorta2001;Garbisu

etal.2002).

Phytoremediation,theuofplantstoextract,

quester,and/ordetoxifypollutants,hasbeen

reportedtobeaneffective,non-intrusive,inex-

pensive,aestheticallypleasing,sociallyaccepted

technologytoremediatepollutedsoils(Alkorta&

Garbisu2001;Weberetal.2001;Garbisuetal.

2002).Phytoremediationiswidelyviewedasthe

ecologicallyresponsiblealternativetotheenvi-

ronmentallydestructivephysicalremediation

methodscurrentlypracticed(Meagher2000).The

USphytoremediationmarketixpectedtoex-

pandmorethanten-foldbetween1998and2005,

toover$214million(Evans2002).

Inthelastfewyears,someexcellentreviewshave

beenpublishedfocusingondifferentaspectsof

phytoremediation(Saltetal.1995a,1998;Chaney

etal.1997;Raskinetal.1997;Chaudhryetal.

1998;Wenzeletal.1999;Meagher2000;Navari-

Izzo&Quartacci2001;Lasat2002;McGrathetal.

2002;McGrath&Zhao2003;McIntyre2003;Singh

etal.2003).Inanyca,andincontrasttoitsmany

positiveaspects,phytoremediationdoeshavecer-

taindisadvantagesandlimitations(Table1).

agesandlimitationsofthephytoremediationtechnology

AdvantagesLimitations

Applicabletoawidevarietyofinorganicandorganic

contaminants.

Limitedbydepth(roots)andsolubilityandavailabilityofthe

contaminant.

Reducestheamountofwastegoingtolandfighfasterthannaturalattenuation,itrequireslongtime

periods(veralyears).

Doesnotrequireexpensiveequipmentorhighly

specializedpersonnel.

Restrictedtositeswithlowcontaminantconcentration.

ssoildisturbanceandthe

spreadofcontaminants.

Plantbiomassfromphytoextractionrequiresproperdisposalas

hazardouswaste.

Earlyestimatesofthecostsindicatethatphytoremediation

ischeaperthanconventionalremediationmethods.

lsoloitffectiveness

whendamageoccurstothevegetationfromdiaorpests.

areacheap

andrenewableresource,easilyavailable.

Introductionofinappropriateorinvasiveplantspeciesshouldbe

avoided(non-nativespeciesmayaffectbiodiversity).

Environmentallyfriendly,aestheticallypleasing,socially

accepted,low-techalternative.

Contaminantsmaybetransferredtoanothermedium,the

environment,and/orthefoodchain.

ly,

treesmayreducenoifromindustrialactivities.

Amendmentsandcultivationpracticesmayhavenegative

conquencesoncontaminantmobility.

72

Withinthefieldofphytoremediation,different

categorieshavebeendefinedsuchas,amongothers,

phytoextraction,phytofiltration(rhizofiltration,

blastofiltration),phytostabilization,phytovolatil-

ization,phytodegradation(phytotransformation),

plant-assistedbioremediation(plant-assisteddeg-

radation,plant-aidedinsitubiodegradation,phyt-

ostimulation,enhancedrhizospheredegradation,

rhizodegradation),etc.(Table2).

Plantsforphytoextraction,i.e.,metalremoval

fromsoil,shouldhavethefollowingcharacteris-

tics:(i)toleranttohighlevelsofthemetal,(ii)

accumulatereasonablyhighlevelsofthemetal,

(iii)rapidgrowthrate,(iv)producereasonably

highbiomassinthefield,and(v)profuroot

system(Garbisuetal.2002).

Theideaofusingplantstoremediatemetal

pollutedsoilscamefromthediscoveryof‘‘hyper-

accumulators’’(Table3),definedasplants,often

endemictonaturallymineralizedsoils,thataccu-

mulatehighconcentrationsofmetalsintheirfo-

liage(Baker&Brooks1989;Raskinetal.1997;

Brooks1998).Infact,plantsgrowingonmetal-

liferoussoilscanbegroupedintothreecategories

accordingtoBaker(1981):(i)excluders,where

metalconcentrationsintheshootaremaintained,

uptoacriticalvalue,atalowlevelacrossawide

rangeofsoilconcentration;(ii)accumulators,

wheremetalsareconcentratedinabove-ground

plantpartsfromlowtohighsoilconcentrations;

and(iii)indicators,whereinternalconcentration

reflectxternallevels(McGrathetal.2002).The

criterionfordefiningNihyperaccumulationis

1000lgNig

)1

onadryleafbasis(Brooktal.

1977),whereasforZnandMnthethresholdis

10,000lgg

)1

andforCd100lgCdg

)1

.Finally,

thecriterionforCo,Cu,PbandSehyperaccu-

mulationisalso1,000lgg

)1

inshootdrymatter

(Brooks1998;Bakeretal.2000;McGrathetal.

2002).Ingeneralterms,metalconcentrationsin

hyperaccumulatorsareabout100–1000-foldhigh-

erthanthofoundinnormalplantsgrowingon

soilswithbackgroundmetalconcentrations,and

about10–100-foldhigherthanmostotherplants

growingonmetal-comtaminatedsoils(McGrath

etal.2002).Hyperaccumulatorsarealsocharac-

terizedbyashoot-to-rootmetalconcentration

ratioof>1(i.e.,hyperaccumulatorplantsshowa

highlyefficienttransportofmetalsfromrootsto

shoots),whereasnon-hyperaccumulatorsusually

havehighermetalconcentrationsinrootsthanin

shoots(Baker1981;Gabbriellietal.1990;Homer

riesofphytoremediation

TermDefinition

PhytoextractionTheuofplantstoremovepollutants(mostly,metals)fromsoils.

PhytofiltrationTheuofplantsroots(rhizofiltration)oredlings(blastofiltration)toabsorb

oradsorbpollutants(mostly,metals)fromwater.

PhytostabilizationTheuofplantstoreducethebioavailabilityofpollutantsintheenvironment.

PhytovolatilizationTheuofplantstovolatilizepollutants.

PhytodegradationTheuofplantstodegradeorganicpollutants

Phytotransformation

PhytostimulationTheuofplantrootsinconjunctionwiththeirrhizosphericmicroorganismsto

edrhizospheredegradation

Rhizodegradation

Plant-assistedbioremediation

Plant-asssisteddegradation

Plant-aidedinsitubiodegradation

esofhyperaccumulators

MetalSpecies

Zinc(Zn)escens

Cadmium(Cd)escens

Nickel(Ni)Berkheyacoddii

Selenium(Se)Astragalusracemosa

Thallium(Tl)Iberisintermedia

Copper(Cu)Ipomoeaalpina

Cobalt(Co)Haumaniastrumrobertii

Arnic(As)a

73

etal.1991;Bakeretal.1994a,b;Brownetal.

1995;Krameretal.1996;Shenetal.1997;Zhao

etal.2000;McGrathetal.2002).

Hyperaccumulationofheavymetalionsisin-

deedastrikingphenomenonexhibitedby<0.2%

ofangiosperms(Baker&Whiting2002).Most

recently,aplethoraofpapersarebeingpublished

inanattempttodisctthemechanismsofmetal

uptake,transportandaccumulation,bothatthe

physiologicalandmolecularlevel(Baker&

Whiting2002).The‘‘model’’hyperaccumulator

Thlaspicaerulescenshasbeenmuchscreenedinthe

archfornewandmoreextremeecotypes

(McGrathetal.2001;Lombietal.2002).Our

understandingoftheinternalprocessthatconfer

thehyperaccumulationphenotypeisadvancingin

leapsandbounds,andthemechanismsoftrans-

port,toleranceandquestrationinsomespecies,

atleastinthegeneraThlaspiandAlyssum,are

partiallyelucidated(Lasat2002).

Treeshavealsobeenconsideredforphyto-

remediationofheavymetal-contaminatedland,

withwillowandpoplarbeingpromisingcandi-

dates,amongothers,inthisrespect(Pulford&

Watson2003).Accordingtosomeauthors,trees

potentiallyarethelowest-costplanttypetoufor

phytoremediation(Stompetal.1994).Manytrees

cangrowonlandofmarginalquality,havemas-

siverootsystems,andtheirabove-groundbiomass

canbeharvestedwithsubquentresprouting

withoutdisturbanceofthesite(Stompetal.1994).

Followingtheharvestofmetal-enrichedplants,

theweightandvolumeofthecontaminated

materialcanbefurtherreducedbyashingor

composting(Garbisu&Alkorta2001;Garbisu

etal.2002).Metal-enrichedplantscanbedispod

ofashazardousmaterialor,ifeconomicallyfea-

sible,udformetalrecovery(Saltetal.1998).

Recently,somestudieshavereportedontheutili-

zationofpyrolysistoparateheavymetalsfrom

hyperaccumulators(Koppolu&Clements2003).

Althoughplantsacquireesntialmineralssuch

asFe,Cu,Ni,ZnandSefromthesoil,forreasons

thatarenotyetclear,theyalsohavetheabilityto

acquireanddetoxifynon-esntialelementssuch

asAs,Cd,CrandPb(Saltetal.2002).Certain

themesinthephysiologyandbiochemistryoftrace

elementaccumulationbyplantsappearcommon

(Saltetal.2002).

Mostphytoremediationstudieshaveconsid-

eredmetalextractionefficiencyinrelationtometal

concentrationofbulksoilsamplesormetalcon-

centrationofthesoilsolution,butlittleisknown

abouttheeffectofvariousmetal-bearingsolidson

,it

hasbeenshownthatitisntialtoconsiderthe

natureofthemetal-bearingsolidstobetterpredict

theefficiencyofplantextraction(Dahmani-Muller

etal.2001).Besides,itisalsoimportanttocon-

siderthatmetalbioavailabilitychangesbetween

thebulksoilandtherhizosphere,thelatterbeinga

microbiospherewhichhasquitedifferentchemical,

physicalandbiologicalpropertiesfrombulksoils

(Wangetal.2002b).Inthisrespect,recently,ithas

beenreportedthatrootgrowthisamorensitive

endpointofmetalavailabilitythanchlorophyll

assays(Morganetal.2002).Inordertoimprove

phytoremediationofheavymetalpollutedsites,

thespeciationandbioavailabilityofthemetalsin

thesoil,theroleofplant-associatedsoilmicroor-

ganismsandfungiinphytoremediation,andthat

ofplantshavetobeelucidated(Kamnev&vander

Lelie2000).

Phytoremediationhasbeenudinminedsoil

restoration,sincethesoilsaresourcesofairand

waterpollution,bymeansofphytostabilization

andphytoextractiontechniquestostabilizetoxic

minespoilsandremovetoxicmetalsfromthe

spoils,respectively(Wong2003).

Somehigherplantspecieshavedeveloped

heavymetaltolerancestrategieswhichenablethem

tosurviveandreproduceinhighly-metalcontam-

i-Mulleretal.(2000)inves-

tigatedmetaluptakeandaccumulationstrategies

oftwoabsolutemetallophytespeciesandone

ormertwospecies,

realhyperaccumulationintheleavesaswellas

metalimmobilisationinrootsand/oradetoxifi-

cationmechanismbyleaffallwerefoundas

possiblestrategiestodealwiththehighmetal

rast,thestrategyofthe

pudometallophyte,i.e.,Agrostistenuis,pre-

ntedasignificantmetalimmobilisationbythe

roots.

Mostplantshavemycorrhizalfungiassociated

withthem,providingtheirhostswithanincread

capacitytoabsorbwaterandnutrientsfromthe

mationandfunctionofmycorrhizal

relationshipsareaffectedbyanthropogenic

stressorsincludingmetals(Entryetal.2002).

Arbuscularmycorrhizalfungiareofinterest

fortheirreportedrolesinalleviationofdiver

74

soil-associatedplantstressors,includingthoin-

ducedbymetals,soithasbeenclaimedthatthe

evaluationoftheefficacyofplant-mycorrhizal

associationstoremediatemetal-pollutedsoilsde-

rvesincreadattention(Entryetal.2002).In

addition,phytoextractionpractices,e.g.,the

choiceofplantspeciesandsoilamendments,may

haveagreatinfluenceonthequantityandspecies

compositionofglomaleanpropagulesaswellason

arbuscularmycorrhizalfungifunctioningduring

long-termmetal-remediationtreatments(Paw-

lowskaetal.2000).

Auniquetestingsystem,thetarget-neighbour

method,hasbeendescribedtoallowevaluationof

howplantingdensityinfluencesmetaluptake,so

thattheinformationneededtomanipulateplant

densityforoptimizationofmetalremovalcouldbe

obtained(Shann1995).

Finally,mostrecently,phytoremediationhas

beencombinedwithelectrokineticremediation,

applyingaconstantvoltageof30Vacrossthesoil,

concludingthatthecombinationofbothtech-

niquesreprentsaverypromisingapproachto

thedecontaminationofmetalpollutedsoils

(O0Connoretal.2003).

findingsonthephytoextractionofsome

ofthemostrelevantenvironmentallytoxicheavy

metals(zinc,cadmium,lead)andmetalloids

(arnic)

Thisctionisdividedintofourdifferentsub-

firstthreecorrespondtothreeofthe

mostenvironmentallyrelevantheavymetals,i.e.,

Zn,Cd,rthsub-headingdealswith

As,portant

toemphasizeherethatveryofteninformation

regardingonemetalappearsunderadifferent,

apparentlywrong,anyofthe

reviewedpublicationsdealwithmorethanone

metalatthesametime,ithasbeenpreferredto

prentthemaspartofthesamerearch,despite

thefactthatctionstructurecouldnotbemain-

tainedasdesired.

ZincandCdareubiquitouspollutantsthattendto

Znisoftenphytotoxic,Cdrarelyinhibitsplant

escens,anintegratedmolecular

andphysiologicalinvestigationofthefundamental

mechanismsofheavymetalaccumulationwas

conducted(Penceetal.2000).Ametaltransporter

cDNA,znt1(expresdatveryhighlevelsinroots

andshootsofthisplant),wasclonedfrom

escensthroughfunctionalcomplementa-

tioninyeastandwasshowntomediatehigh-

affinityZnuptakeaswellaslowaffinityCd

tionintheregulationofznt1gene

expressionbyplantZnstatusresultsintheover-

expressionofthistransporterandinincreadZn

influxinroots,evenwhenintracellularZnlevels

,specificalterationsinZn-respon-

siveelements(e.g.,transcriptionalactivators)

possiblyplayanimportantroleinZnhyperaccu-

escens(Penceetal.2000).In

thisrespect,Lasatetal.(1998)foundthattheen-

es-

censwas,atleastpartly,achievedthroughan

alteredZncompartmentationintherootsym-

plasm,whichreducesZnquestrationinroot

erstepatelucidatingthemech-

anismsunderlyingZnhyperaccumulationwasgi-

venthankstothecloningofmetaltransporter

genencodingputativevacuolariontransport

escens(Assuncaoetal.2001).

Cd

Ni

Zn

Pb

As

Cu

CoTl

Rootuptake

Translocation

Accumulation

Harvesting

Disposal/Recovery

xtractionofmetals.

75

Infact,ZTP1(atransporterbelongingtothecat-

ion-effluxfamily)hasbeenfoundtobehighlyex-

escens,predominantlyinleaves

(Assuncaoetal.2001).

Longetal.(2002)haverecentlydescribeda

largebiomassZnhyperaccumulatingplant,i.e.,

rly,Wangetal.(2003)have

reportedonthediscoveryoftwonewplantswith

potentialforphytoremediationofZn-polluted

soils,i.e.,PolygonumhydropiperandRumexace-

amestudy,theauthorsindicatethat

theconsumptionofricegrowninpaddysoils

contaminatedwithCd,CrorZnmaypoa

riousrisktohumanhealth,becaufrom22to

24%ofthetotalmetalcontentinthericebiomass

stingly,

Platanusacerifoliagrowingonheavilycontami-

natedsoilaccumulatedonlyverylowlevelsof

heavymetals,andthismechanismforexcluding

metaluptakemayhavevalueincropimprovement

(Wangetal.2003).

pestoleranttoZn

toxicityemtogrowbetterthanZn-nsitive

genotypes,eveninZn-deficientsoil,becauof

theirgreatercapacityfortakingupZnfromZn-

deficientsoil,revealingthecoexistenceoftraitsfor

tolerancetoZntoxicityandZndeficiencyina

singleplantgenotype(Rengel2000).

MetalresponsinthemetallophyteArabidop-

sishalleri,aclorelativetothemodelplant

na,thatisCdhypertolerantandZnhy-

peraccumulating,havebeenstudied,andmetal-

regulatedgenesisolatedandmolecularlyanalyzed

asinterestingcandidategenesforphytoremedia-

tion(Bertetal.2000;Dahmani-Mulleretal.2000;

Clemens2001;Macnair2002).UnlikeThlaspi,

iemstobelargelyallogamous,eds

profulyoveralongergrowingasonandcan

alsospreadvegetativelybystolons(Baker&

Whiting2002).Macnair(2002)demonstratedthat

theheritabilityofZnaccumulationwasbetween

25and50%,thehighestyetrecordedforanyhy-

peraccumulator,probablybecauoftheout-

uingly,the

i

wasmanifestedmorewhengrowninlow-Znmedia

thaninhigh(Baker&Whiting2002).

beendeveloped,andthismighthelpinthelection

andcharacterizationofheavymetaltolerancein

plantsforbreedingprogrammes(Routetal.1999).

InapaperfocusingonsoilsolutionZnandpH

-

rulescensJ.&,dataindicatethatthepo-

tentialofthishyperaccumulatortoremoveZn

fromcontaminatedsoilmaynotberelatedeither

toacidificationoftherhizosphere(McGrathetal.

1997;Luoetal.2000)ortoexudationofspecific

metal-mobilizingcompounds(Zhaoetal.2001).

Intriguinglyenough,somestudieshaveprovided

escens

areabletonandactivelyforageintheZnrich

patchesinsoil(Schwartzetal.1999;Whitingetal.

2000).

Amodifiedglassbeadcompartmentcultiva-

tionsystemforstudiesonnutrientandtrace

metaluptakebyarbuscularmycorrhizausingtwo

hostplantspecies,maize(ZeamaysL.)andred

clover(TrifoliumpratenL.),andtwoarbuscu-

larmycorrhizalfungi,GlomusmossaeandG.

versiforme,foundastriking,veryhighaffinityof

thefungalmyceliumforZn,suggestingthepo-

tentialuofarbuscularmycorrhizainthephy-

toremediationofZn-pollutedsoils(Chenetal.

2001).

m

Cadmiumisoneofthemoremobileheavymetals

inthesoil-plantsystem,easilytakenupbyplants

andwithnoesntialfunctionknowntodate

(Lehoczkyetal.2000).Thilementcanaccumu-

lateinplantswithoutcausingtoxicitysymptoms

(Lehoczkyetal.1998).

Insoybeanplants,resultsrevealthatthecon-

tentofCdindifferentpartsoftheplantswas

roots>>stems>eds,indicatingthattheaccu-

mulationofCdbyrootsismuchlargerthanthat

ofanyotherpartofthesoybeanplant,andmight

caudeleteriouffectstorootsystems(i.e.,de-

creadnodulation,changesintheultrastructure

ofrootnodule,etc.)(Chenetal.2003).

Ithasbeensuggestedthatvetivergrasscouldbe

udtoremediateCd-pollutedsoil,sinceitaccu-

mulated218gCdha

)1

atasoilCdconcentration

of0.33mgCdkg

)1

(Chenetal.2000).Although

questrationofCdbyrhizospheremicroorgan-

ismsmayhaveanimportantinfluenceonplantCd

uptake,furtherrearchisstillrequiredtoestab-

lishwhethertheaccumulationofCdby

rhizobacteriainhibits,oraccelerates,Cduptakeby

thehostplant(Robinsonetal.2001).

76

Recentworkshaveaimedtoidentifytheroleof

antioxidativemetabolisminheavymetaltolerance

escens(Boominathan&Doran2003a,

b).Hairyrootswereudtotesttheeffectsofhigh

Cdenvironments,demonstratingthatmetal-in-

ducedoxidativestressoccursinhyperaccumulator

tissueventhoughgrowthisunaffectedbythe

orantioxidant

defens,particularlycatalaactivity,mayplay

animportantroleinthehyperaccumulatorphe-

escens.

Phragmitesaustralisplantswereexpodtoa

highconcentrationofCd,findingoutthatmostof

thilementaccumulatedinroots,followedby

leaves(Iannellietal.2002).Inrootsfrom

Cd-treatedplants,boththehighamountof

glutathioneandtheparallelincreaofglutathione-

S-transferaactivityemedtobeassociatedwith

aninductionofthedetoxificationprocessin

xide

dismuta,ascorbateperoxida,glutathione

reductaandcatalaactivitiesaswellasreduced

andoxidizedglutathionecontentsinallsamplesof

leaves,rootsandstolonswereincreadinthe

ethefactthatCdhasaredox

characteristicnotcompatiblewiththeFenton-type

chemistrythatproducesactiveoxygenspecies,Cd

toleranceinPhragmitesplantsmightbeassociated

totheefficiencyofthemechanisms.

TheeffectofFestatusontheuptakeofCdand

escens,i.e.,Gan-

gesandPrayon(theformerbeingfarsuperiorin

Cduptake)wasstudied(Lombietal.2002).

Moreover,escenszip(Zn-regulated

transporter/Fe-regulatedtransporter-likeprotein)

genes,TcZNT1-GandTcIRT1-G,werecloned

fromGangesandtheirexpressionunderFe-suf-

ficientand-defi-

miumuptakewassignificantlyenhancedbyFe

deficiencyinGanges,whileZnuptakewasnot

influencedbytheFestatusoftheplantsineither

esultsareinagreement

withthegeneexpressionstudy,sincetheabun-

danceofZNT1-GmRNAwasalwayssimilar,

independentlyoftheFestatusorecotype,while

thatofTcIRT1-GmRNAwasgreatlyincread

onlyinGangesroottissueunderFe-deficient

conditions,suggestingapossiblerelationshipwith

anup-regulationintheexpressionofgenes

encodingFeuptake,possiblyTcIRT1-G(Lombi

etal.2002).

Recently,Songetal.(2003)reportedonthe

utilityoftheyeastproteinYCF1,aproteinwhich

detoxifiesCdbytransportingitintovacuoles,for

theremediationofCdandPbcontamination,

findingoutthattransgenicArabidopsisthaliana

plantsoverexpressingYCF1showedenhanced

toleranceandaccumulatedgreateramountsofCd

stinglyenough,Lahneretal.(2003)

analyzedveralesntialandnonesntialele-

na

plants,demonstratingtheutilityofgenomicscale

profilingofnutrientsandtraceelementsasa

runderstanding

ofhowplantshandlemineralelementshasthe

potentialofyieldingnewphytoremediationcapa-

bilities(Rea,2003).

WhileCddetoxificationiscertainlyacomplex

phenomenon,probablyunderpolygeniccontrol,

Cdrealtolerancefoundinmineplantsemstobe

asimplerphenomenon,possiblyinvolvingonly

monogenic/oligogeniccontrol(SanitadiToppi&

Gabbrielli1999).Theauthorsconcludedthat

adaptivetoleranceissupportedbyconstitutive

detoxificationmechanisms,whichinturnrelyon

constitutivehomeostaticprocess.

Symmetricandasymmetricsomatichybridiza-

tionshavebeenudtointroducetoxicmetal-

escensintoBrassica

juncea(Dushenkovetal.2002).hypo-

escens

mesophyllprotoplasts,andallputativehybrids

.

Hybridplants,producedbyasymmetricsomatic

hybridizationbetweenthetwospecies,demon-

stratedhighmetalaccumulationpotential,toler-

ancetotoxicmetals,andgoodbiomass

a,isbyitlfa

goodcandidateforefficientphytoextractionof

heavymetals-suchasCd-frompollutedsoils

(Schneideretal.1999).

Twoyearsafterthetoxicspillcaudbythe

failureofatailingponddamattheAznalcollar

pyritemine(SWSpain)in1998,noneofthetrace

elementsmeasured-As,Cd,Cu,Pb,Tl-reached

levelitherphytotoxicortoxicforhumansor

animalsinedsandtheabove-groundpartof

spill-affectedsunflowerplants(Madejonetal.

2003).However,thepotentialforphytoextraction

oftheplantsisverylow,thoughtheymightbe

ductionofoil

(usableforindustrialpurpos,whoproduction

77

wasgreaterinthespill-affectedplantsascompared

withunaffectedsunflowerplantsgrowinginadja-

centsoil)mayaddsomevaluetothiscrop

(Madejonetal.2003).

Fieldandglasshouinvestigationswerecon-

ductedoftheresponsontwolegumes(fieldpea

andfoddervetch)andthreenon-leguminouscrops

(maize,wheatandrapeed)totheheavymetals

Cd,Cr,Zn,Pb,CuandMninsoilwithmultiple

metalcontamination(Wangetal.2002a).Among

thecrops,maizehadthehighestconcentrationsof

Mn,ZnandCd,whilewheatwasthehighest

wasaccumulatedin

thegrainofwheatthanofmaize,suggestingthat

growingwheatwouldreprentahigherriskof

foodcontaminationthangrowingmaizein

oncludedthatmaize

couldperhapsbeudforphytoremediationof

lightlycontaminatedsoils(Wangetal.2002a).

y

metalsmakesitagoodprospectforreclamation

projects(Jauertetal.2002).Intheplants,there

emstobeanegativecorrelationbetweenrhizo-

spherepHandCduptake.

Mejare&Bulow(2001)reviewedthemetal-

bindingproteinsandpeptidesinbioremediation

bymicroorganismsandphytoremediationofhea-

vymetals,withspecialemphasisonCd,indicating

thattheexpressionofthemoleculestoenhance

heavymetalaccumulationand/ortolerancehas

greatpotential.

Toaugmenthigherplantmetalquestration,

theyeastmetallothioneinCUP1(metallothioneins

aremetal-bindingproteinsthatconferheavymetal

toleranceandaccumulation)wasintroducedinto

tobaccoplants,andthecup1geneexpressionand

CuandCdphytoextractionweredetermined

(Thomatal.2003).Althoughpooledleavesof

transgenicplantscontainedtwotothreetimesthe

Cucontentasthatofthecontrolplants,CUP1

edlingdidnotsignificantlyquesterordemon-

stratetolerancetoCd.

Phytosiderophores,Fechelatorxcretedby

graminaceousplantsunderconditionsofFelimi-

tation,mhasbeen

showntoincreatherateofphytosiderophore2¢-

deoxymugineicacidreleainmaizeunderboth

Fe-sufficientandFe-limitingconditions(Hilletal.

2002).Collectively,resultsindicatedthatCdstress

causFedeficiencysymptomsthatresultin

greater2¢-deoxymugineicacidproductionby

maizeroots,andthenthe2¢-deoxymugineicacid

appearstoreduceCdaccumulation.

HairyrootswereudtoinvestigateCduptake

escens,findingoutthattheaccumula-

tionofthilementincreadaftertreatingthe

rootswithH(+)-ATPainhibitor(Nedelkoska&

Doran2000).MeasurementofCdlevelsrevealed

significantdiff-

rulescensandNicotianatabacum:mostmetalwas

-

bacumrootswithin3daysofexposure,while,in

contrast,escensrootsstoredvirtuallyall

oftheirCdinthecellwallfractionforthefirst7to

layintransmembraneuptakemay

reprentanimportantdefensivestrategyagainst

Cdpoisoning,allowingtimeforactivationof

intracellularmechanismsforheavymetaldetoxi-

fication(Nedelkoska&Doran2000).Inthis

respect,itshouldbenotedthat,duetotheirfast

growthratesandbiochemicalstability,‘hairyroot’

culturesremainunsurpasdasthechoicefor

modelrootsystemswithawiderangeofapplica-

tions,includingasamodelforphytoremediation

(Shanks&Morgan1999).

HairyrootsoftheCdhyperaccumulator

escenshavebeenshowntocontainhigh

constitutivelevelsofcitric,malicandmalonic

acids(Boominathan&Doran2003a,b).-

rulescenshairyrootsremainedhealthyandgrew

wellathighCdconcentrations,withmostofthe

of

escenshairyrootswasverelyreducedin

theprenceofdiethylstilbestrol,aninhibitorof

plasmamembraneH+-entwith

diethylstilbestrolincreadtheconcentrationof

escensabout6-fold

esultssug-

gestthatthemechanismsofCdtoleranceand

escenshairyroots

arecapableofwithstandingtheeffectsofplasma

membranedepolarization(Boominathan&Doran

2003a,b).

Cadmiumcausatransientdepletionofglu-

thathioneandaninhibitionofantioxidativeen-

zymes,especiallyofglutathionereducta

(Schutzendubel&Polle2002).Thedepletionof

glutathioneisapparentlyacriticalstepinCd

nsitivity,andthereareindicationsthatCd,when

notdetoxifiedrapidlyenough,maytriggera

quenceofreactionsleadingtogrowthinhibition,

stimulationofcondarymetabolism,lignification

78

and,finally,celldeath,incontrasttotheideathat

Cdresultsinunspecificnecrosis(Schutzendubel&

Polle2002).Mycorrhizationstimulatesthephe-

nolicdefencesysteminthePaxillus-Pinusmycor-

rhizalsymbiosis,butitisstillnotknownwhether

mycorrhizationprotectsrootsfromCd-induced

injurybypreventingaccessofCdtonsitiveex-

tra-orintracellularsites,orbyexcretedorintrinsic

metal-chelators,orbyanyotherdefencesystem

(Schutzendubel&Polle2002).Thus,thedevelop-

mentofstress-tolerantplant-mycorrhizalassocia-

tionsmaybeapromisingnewstrategyfor

phytoremediation.

Tobetterexaminethephytoremediationof

transgenicIndianmustard()plants,

overproducingtheenzymesgamma-glutamylcy-

steinesynthetaorglutathionesyntheta(which

haveincreadlevelsofthemetal-bindingthiol

peptidesphytochelatinsandglutathione,anden-

hancedCdtoleranceandaccumulation),and

overexpressingadenosinetriphosphatesulfuryla

(showntohavehigherlevelsofglutathioneand

totalthiols),agreenhouexperimentwascon-

ductedinwhichthetransgenicsweregrownon

metal-contaminatedsoilcollectedfromaUSEPA

Superfundsite(Bennettetal.2003).Alltrans-

genicsremovesignificantlymoremetalfromthe

soilcomparedwithwild-typeIndianmustard,

confirmingtheimportanceofmetal-bindingpep-

udyis

thefirsttodemonstrateenhancedphytoextraction

potentialoftransgenicplantsusingpollutedenvi-

ronmentalsoil.

Previously,Indianmustardhadbeengeneti-

callyengineeredtooverexpresstheEscherichiacoli

gshIgeneencodinggamma-glutamylcysteinesyn-

theta,targetedtotheplastids,obtaininged-

lingswithincreadtolerancetoCdandhigher

concentrationsofphytochelatins,gamma-GluCys,

glutathione,andtotalnon-proteinthiolscom-

paredwithwild-typeedlings(Zhuetal.1999b).

IntheprenceofCd,glutathionesynthetais

ratelimitingforthebiosynthesisofphytochelatins

andglutathione(theirprecursor)(Zhuetal.

1999a).

InordertostudythenatureofCdbindingin

phytochelatinsandrelatedcysteine-richpoly-

peptidesinmaize(),Pickeringetal.

(1999)udX-rayabsorptionspectroscopyto

revealapredominantlytetrahedralcoordination

ofCdbysulfurinthomolecules,suggesting

theprenceofapolynuclearCdclusterinmaize

phytochelatin.

TheeffectofCa(OH)

2

additiononimmobili-

zationandphytoavailabilityofCdfromsoilswas

plants(Bolanetal.

2003b).TheadditionofCa(OH)

2

increassoil

pH,therebyincreasingtheadsorptionofCd,and

effhere

isnodirectevidenceforCdCO

3

orCd(OH)

2

pre-

cipitationinthevariablechargesoiludforthe

plantgrowthexperiment,alleviationofphytotox-

icityisattributedprimarilytoimmobilizationof

CdbyenhancedpH-inducedincreasinnegative

charge.

nageneencodingphytochelatin

syntha(AtPCS),

obrvingamarkedaccumulationofphytochela-

tinswithaconcomitantdecreainglutathione

cellularcontent(Sauge-Merleetal.2003).When

thebacterialcellxpressingAtPCSareplacedin

theprenceofCdorAs,cellularmetalcontents

aresignifiensupthe

possibilityofusinggenesfromthephytochelatin

biosyntheticpathwaytodesignbacteria(bio-

remediation)orhigherplants(phytoremediation)

withincreadabilitiestoaccumulatetoxicmetals.

Bymakinguofnuclearmicroscopytechniques,

suchasmicro-PIXE,ithasbeenobrvedthatCd

isquesteredwithinthetrichomesontheleaf

na(Ageretal.2002).

Usingthec-glutamylcysteinesynthetainhib-

itor,L-buthionine-[S,R]-sulphoximine(BSO),the

roleforphytochelatinswavaluatedinCu,Cd,

Zn,As,Ni,andCotoleranceinnon-metallicolous

andmetallicoloushypertolerantpopulationsof

Silenevulgaris,escens,sand

Agrostiscastelana(Schatetal.2002).Phytochela-

tin-badquestrationemstobeesntialfor

constitutivetolerancebutnotforhypertolerance

mnsitivityisconsider-

ablyincreadbyBSO(aninhibitorofphyto-

chelatinsynthesis),althoughexclusivelyinplants

lackingCdhypertolerance,suggestingthatadap-

tativeCdhypertoleranceisnotdependenton

phytochelatin-mediatedquestration(Schatetal.

2002).

Phytochelatin-badCdquestrationisgen-

erallyconsideredesntialforconstitutiveCdtol-

eranceinorganismswithfunctionalPCSgenes

(Cobbett&Goldsbrough2002;Schatetal.2002).

TheroleofPCsinCddetoxificationwas

79

supportedbytheisolationoftwomutantsof

Arabidopsis,cad1andcad2(deficientinPCand

GSHbiosynthesis,respectively),whichweremore

nsitivetoCd(Cobbetetal.1998).Similarly,a

strongCd-inducedPCaccumulationhasbeen

obrvedinbothnon-tolerantThlaspispecies

(e)(Ebbtal.2002)andnon-tolerant

escens(Schatetal.2002).

Furthermore,Saltetal.(1995b)foundenough

PCstochelatealltheCdintherootsofCd-treated

uthorsconsistentlyfound

themajorityofCdboundtoSligands,witha

probableCd-S

4

coordination,andabondlength

coincidentwiththatofthepurifiedCd-PCcom-

gCd-inducedPCaccumulationhas

escenco-

types(i.e.,Prayon)(Ebbtal.2002)aswellasin

aCd-tolerantecotypeisolatedfromcalaminesoils

(Schatetal.2002).However,thecomparisonbe-

tweennon-tolerantandCd-tolerantecotypesled

totheconclusionthatPCscanonlyplayacrucial

roleforCddetoxificationinnon-tolerantecotypes

(Ebbtal.2002;Schatetal.2002).Twomain

argumentssustainthisconclusion:(i)atequalor

higherrootorshootCdinternalconcentration,

PCsaccumulationislowerinthemoretolerant

hyperacumulatorecotype(themoretolerantthe

ecotype,thelowerPC-SHtoCdmolarratio);and

(ii)theBSOimpodhypernsitivitytoCdin

non-tolerantecotypewasnotapparentinthetol-

erantecotype,irrespectivetothelevelofCd

findings,regardingthelackofa

signifiescensCdhy-

peraccumulatorecotypes,areconsistentwiththo

is

(DeKnechtetal.1994).

TranslocationofCdinthexylemsapappears

tobedrivenbytranspirationfromtheleavesand

tobeindependentofPCproduction(Saltetal.

1995b).Complexationwithlowmolecularweight

organicligands,suchascitrateandhistidine,

m

K-edgeEXAFSofxylemsapisolatedfromB.

junceaplantxpodto0.6mgCdml

)1

for

7daysshowedCdinteractionswithNorO,with

bondlengthsdifferenttothatofCd-PC(Saltetal.

1999).However,veryrecently,Gongetal.(2003)

havefoundthefirstevidenceoflong-distanceroot-

obrvedthatthetransgenicexpressionofwheat

TaPCS1inrootsofcad1-3mutantsofArabidopsis

supresdCdnsitivity,reducedCdaccumulation

inroots,and,mostinterestingly,ledtoPCaccu-

rmore,

Gongetal.(2003)foundaBSOimpodroot-to-

shoottransportinhibitioninTaPCS1transgenic

ementioned,

shootcellularquestrationthroughvacuolar

compartimentationappearstobeakeycompo-

nentinZndetoxifiescens,and

thesamemayholdtrueforCddetoxification

(Ebbtal.2002;Schatetal.2002).Tolerant

escensmayquesterCdin

leafcellsmoreefficiently,eitherviatonoplast

transportersforPCand/oraCd-PCcomplex

(Ebbtal.2002).

AType1metallothioneingenemcMT1was

clonedfromtheCuinducedcDNAlibraryof

andbymeansoffunc-

tionalcomplementationstudiesusingSaccharo-

mycescerevisiaecup1mutantABDE-1(metal

nsitive),thefunctionalnatureofthismcMT1

geneinquesteringbothesntial(Cu,Zn)and

non-esntialmetals(Cd,Pb,Cr)wasconfirmed

(Maetal.2003).

Itisinterestingtonotethatphytochelatin

synthaisnotonlyrestrictedtoplantsandsome

fungi,aswasoncethought,butalsohasan

esntialroleinheavy-metaldetoxificationinthe

modelnematodeCaenorhabditilegans,sonow

phytochelatins,thopost-translationallysynthe-

sizedpeptides,willalsobeinvestigatedfroma

clinicalparasitologicalstandpoint(Vatamaniuk

etal.2002).

Earlytranscriptionalresponsofacellwall-

deficientmutantofChlamydomonasreinhardtiito

heavymetalstresshavebeeninvestigated,identi-

fying,quencingandquantifyingtheinductionof

anumberoftranscriptsthatareup-regulatedby

exposuretoCd(Rubinellietal.2002).The

Chlamydomonasstrainscouldbeufulforfunc-

-

tion,themagnitudeofinductionandfunctional

analyssuggestpossibleutilityforthegenesin

thestudyofmetalstressnsingingreenplants

anddevelopmentofphytoremediationstrategies.

Hemp(CannabissativaL.)hasbeenudto

examineitscapabilityasarenewableresourceto

decontaminateheavymetalpollutedsoils(Linger

etal.2002).Metalaccumulationindifferentparts

oftheplantwasstudied(i.e.,eds,leaves,fibres

andhurds),andthehighestconcentrationsofall

80

examinedmetals(i.e.,Ni,Pb,Cd)arefoundinthe

owsaphytoremediationpotential

of126gCdha

)1

vegetationperperiod.

IntheirstudiesontheeffectofmixedCd,Cu,

Ni,andZnatdifferentpHsuponalfalfagrowth

andheavymetaluptake,Peralta-Videaetal.

(2002)foundthatthemaximumrelativeuptakes

(elementinplant/elementinsoil-water-solution)

are26timesforNi,23timesforCd,12timesfor

Zn,and6timesforCu,indicatingtheabilityof

alfalfaplantstotakeupelementsfromasoilma-

trixcontaminatedwithamixtureofthemetals.

Onceintroducedintothesoilmatrix,Pbisvery

diffiacityofthesoilto

adsorbPbincreaswithincreasingpH,cation

exchangecapacity,organiccarboncontent,soil/

waterEh(redoxpotential)andphosphatelevels

(UnitedStatesEnvironmentalProtectionAgency

1992).

Amodelfortheuptake,translocation,and

accumulationofPbbymaizeforthepurpoof

phytoextractionhasbeenpropod,suggesting

thatprecipitationofPbasaPb-phoshateisoneof

themostimportantmechanismsinthissystem,

withmaximumsaturableuptakerateofPband

effectiverootsmassbeingalsopossiblekeyplant

parameters(Brennan&Shelley1999).

Aftercollectingsoilmaterialandplanttissue

alongtranctsintwoheavilycontaminatedsoil

facilitiesandanalyzingthemformetalcontent,it

wasobrvedthattissuePbcorrelatedslightlywith

exchageableandsolublesoilPb,buttissueCdwas

poorlycorrelatedwithsoilCdspecies(Pichtel

etal.2000).LeadandCduptakewasmaximalin

AgrostemmagithagorootandinTaraxacumoffi-

cinale,lantsthatremoved

mostPbandCdwerepredominantlyherbaceous

species,someofwhichproducesufficientbiomass

abilityofT.officinaleandAmbrosiaartemisiifolia

tosuccessfullyremovesoilPbandCdduringre-

peatedcroppingswasdemonstratedingrowth

chamberstudies(Pichteletal.2000).

Certainplants(mostly,belongingtothe

Brassicaceae,Euphorbiaceae,Asteraceae,Lamia-

ceae,andScrophulariaceaefamilies)havebeen

identifiedwhichhavethepotentialtouptakePb

(UnitedStatesEnvironmentalProtectionAgency

2000a,b).hasbeenfoundtohaveagood

abilitytotransportPbfromrootstotheshoots.A

studyontheeffectsofdifferentconcentrationsof

Pbontheuptakeandaccumulationofthilement

bytheroots,

hizashowedthat,

hasconsiderableabilitytoremovePbfromsolu-

tionsandaccumulateit,theplantstransportedand

concentratedonlyasmallamountofPbinhy-

pocotylsandshoots(Liuetal.2000).

ifoliumhasbeenfoundtogrowinsoils

a,onemajor

factorlimitingthepotentialforPbphytoextrac-

tionislowmetalbioavailabilityforplantuptake.

Toovercomethislimitation,syntheticchelators

havebeenpropodtobeaddedtothesoiltoin-

creatheamountofavailablePb(UnitedStates

EnvironmentalProtectionAgency2000a,b).Sahi

etal.(2002)reportedonaleguminousfastgrow-

ingshrub(Sesbaniadrummondii)thataccumulates

upto10,000mgPbkg

)1

inshootafterexposureto

on

ofEDTA(100lM)toamediumcontaining

1gPbl

)1

increaduptakeby21%.

AgeneticallymodifiedNicotianaglauca

(shrubtobacco)hasproventoaccu-

mulatePb,makingitespeciallypromisingfor

phytoremediation(Gisbertetal.2003).Thisplant

hasawidegeographicdistribution,isfast-growing

withahighbiomass,andisrepulsivetohervibores.

Theinductionandoverexpressionofawheatgene

encodingphytochelatingsynthainthisplant

greatlyincreaditstolerancetoPbandCd,and

edlingsofthetransformedplantsaccumulated

doubleconcentrationofPbcomparedtothewild

type.

Usingradiolabeledrecombinantcalmodulinas

aprobetoscreenatobaccocDNAlibrary,Arazi

etal.(1999)discoveredaprotein,NtCBP4,that

canmodulateplanttolerancetoheavymetals.

Severalindependenttransgeniclinexpressing

NtCBP4hadhigherthannormallevelsof

NtCBP4,exhibitingimprovedtolerancetoNiand

hyperntitivitytoPb,whichareassociatedwith

reducedNiaccumulationandenhancedPbaccu-

mulation,sthefirstreportof

aplantprotein(probablyinvolvedinmetaluptake

acrosstheplasmamembrane)thatmodulates

ne

couldbeufulforimprovingphytoremediation

strategies.

81

c

AspointedoutinthereviewbyOremlandand

Stolz(2003),Aspollutionisaproblemofcritical

importancecurrentlyaffectingthehealthofmil-

bovementioned

paper,theauthorsreviewedwhatisknownabout

As-metabolizingbacteriaandindicatedthatar-

niteoxidationisbeingstudiedasthebasisfor

bioremediationofsystemswherearnite,As(III),

isapollutant.

Inthiscontext,phytoremediationhasrecently

beenpropodasaneff

fact,theChinebrakefern(Pterisvittata)hasbeen

reportedtohyperaccumulateAstoextremelyhigh

concentrations,upto23,000lgarnicg

)1

,inits

shoots(fronds)(Maetal.2001).Thisprimitive

plantactuallythrivesonAs,doublingitsbiomassin

strikingdiffaandAsnon-

accumulatorsistheremarkabletransportofthis

a,accu-

mulatingupto95%oftheAsintheabove-ground

tissue(Doucleff&Terry2002).Zhangetal.(2002)

havehypothesizedthatthebrakeferntakesupAsas

arnate,subquentlyconvertingittoarnite

ahasbeenshowntoreacha

bioconcentrationfactor(ratioofplantAsconcen-

trationtowater-solubleAsinsoil)of1450(Tuetal.

2002).Oncethemolecularmechanismsinvolvedin

aareknown,

genesresponsiblefortheremarkableabilitiesofthis

plantcouldbeudtotransformfast-growing,high-

biomassphytoremediators(Doucleff&Terry2002).

OtherplantssuchasPityrogrammacalomelanos,

Mimosapudica,andMelastomamalabrathricum

mightbesuitableforAsphytoremediation(Vi-

soottivithetal.2002).lanosaccumu-

latesmostofthismetalloidinthefrondswhilethe

rhizoidscontainthelowestconcentrationsofAs

(Francesconietal.2002).

AtransgenicsystemforremovingAsfromsoil,

inrtingtwogenes,arsC(arnatereducta)and

ECS(c-glutamylcysteinesyntheta)

naplantshasbeendeveloped

(Dhankheretal.2002).WhengrownonAs,the

transgenicplantsaccumulated4-to17-foldgreater

freshshootweightandaccumulated2-to3-fold

moreAspergramoftissuethanwildtypeor

plantxpressingc-ECSorArsCalone(Dhankher

etal.2002).Inanyca,thetoleranceandaccu-

mulationvaluesreachedbythetransgenicplants

a.

Sauge-Merleetal.(2003)havedescribedadif-

ferentapproach,nagene

encodingphytochelatinsyntheta(AtPCS)in

cterialcellxpressingAtPCS

wereplacedintheprenceofAs,cellularmetal

archis

stillneededonthepossibilityofusinggenesofthe

phytochelatinbiosyntheticpathwaytodesign

bacterialstrainsorhigherplantswithincread

abilitiestoacumulateAsforremediationpro-

cess.

,onceintheshoot,Asisstoredas

anAs(III)tris-thiolatecomplex,beingthemost

probablethiolatedonoritherglutathioneor

phytochelatins(Pickeringetal.2000).Inthisre-

spect,theadditionofthedithiolAschelatordi-

mercaptosuccinatetoAs-contaminatedsoilshas

beensuggestedtopromoteAsbioaccumulationin

plantshoots.

Astudyoftheinfluenceofroottemperatureon

phytoaccumulationofAs,amongotherelements,

inpotatoplants(.

Spunta),inducedbytheapplicationofmulches,

indicatedthatdifferentroottemperatureswere

accompaniedbysignificantlydifferentresponsin

Asphytoaccumulation(Baghouretal.2001).In

termsoftherelativedistributionofthephytoac-

cumulatedmetals(withrespecttothetotalofthe

plant),Asaccumulatedmainlyintherootsand

leaflgardtophytoremediationusing

tomatoplants,itisimportanttoconsiderthe

thermalregimeofthesoiltooptimizephytoex-

traction(Baghouretal.2001).

Thec-glutamylcysteinesynthetainhibitor,

L-buthionine-[S,R]-sulphoximine(BSO),dra-

maticallyincreasAsnsitivity,bothinnon-

adaptedandAs-hypertolerantplants,showing

thatphytochelatin-badquestrationisntial

forbothnormalconstitutivetoleranceand

adaptativehypertolerancetothismetalloid(Schat

etal.2002).

lnewfindingsandoutlook

Improvementofplantsbygeneticengineering

opensupnewpossibilitiesforphytoremediation

(Karenlampietal.2000).Also,genesinvolvedin

theregulationofthepartitioningofmetals

82

betweenrootandshootwillbeofhighinterestin

thefuture(Delhaize1996).

Sofar,onlyafewcashavebeenreported

wheremetaluptake,transportandaccumulation

aswellasmetaltolerancehavebeensuccesfully

ncludeHgionreductioncausing

improvedresistanceandphytoextraction(Heaton

etal.1998;Rughetal.1996,1998a,b),andme-

tallothioneincausingenhancedCdtolerance

(Misra&Gedamu1989;Evantal.1992;

Elmayan&Tepfer1994;Hattorietal.1994;

Hagawaetal.1997;Karenlampietal.2000;

Kramer&Chardonnens2001).

Veryexcitingworksontheapplicationof

moleculargeneticapproachestoimprovemetal

phytoremediationwerepublishedbyMeagher¢s

teaminrelationtoHgpollution(Rughetal.1996,

1998a,b).TheyudexpressionofmerAgene

(mercuricreducta,MerA,convertstoxicHg2+

tothelesstoxic,relativelyinertHg0inbacteria)in

transgenicplantsfortheremediationofHgpol-

enizedmerAquence,merApe9,

naed-

lingxpressingthisquencegerminatedandgrew

onHg-containingmedium,evolvingconsiderable

abilityofyellowpoplar(Liriodendrontulipifera)

tissueculturesandplantletstoexpressmodified

mercuryreductageneconstructshasalsobeen

reported(Rughetal.1998a).Thesignificanceof

thestudyofMeagher¢srearchgroupisthatit

canleadtothemoreefficientandaffordable

cleanupofenvironmentalHgpollution,andina

broadercontext,itprovesthepowerofgenetic

engineeringforphytoremediation(Pilon-Smits&

Pilon2000).Mostrecently,Bizilyetal.(2003)

haveengineeredplantsthatexpressthebacterial

Hgresistanceenzymesorganomercuriallya

merBgenewangineeredtotargetMerBfor

accumulationintheendoplasmicreticulumand

oteinin

endoplasmicreticulum-targetedplantsappearedto

accumulateinlargevesicularstructuresand

moderateincreasintargetedMerBexpression

ledtosignificantgainsindetoxification.

escensor

shouldbegoodsourcesforgenes

olerance

andaccumulationarelargelyindependentprop-

erties,theyshouldbebothengineeredtogeta

suitableplantformetalphytoextraction(Mac-

nairetal.1999;Karemlampietal.2000).With

respecttometalphytoextraction,twostrategies

couldbeconsidered:(i)convertslow-growing,

low-biomasshyperaccumulatorsintofast-grow-

ing,high-biomassvarieties,or,alternatively,(ii)

introducemetal-hyperaccumulationtraitsinto

fast-growing,high-biomassplants(Cunningham

&Ow1996).

Raskin(1996)suggestedthattransgenicplants

couldbedevelopedtocretemetal-lectiveli-

gandsintotherhizospherewhichcouldspecifically

solubilizeelementsofphytoremediationinterest.

Sincelittleisknownabouttherhizosphereof

hyperaccumulators,Delormeetal.(2001)com-

paredtheinfl-

rulescenswiththeeffonsoil

microbes,findingoutthatmicrobialpopulations

but

higherratiosofmetal-resistantbacteriawere

-

escensacidifiesits

rhizosphere,increasingavailablemetalsaround

therootsand,conquently,increaslectionfor

metal-resistantbacteria.

Sincemetaltransportfromthecytosoltothe

vacuoleisthoughttobeanimportantcomponent

ofiontoleranceandofaplantspotentialoruin

phytoremediation,somestudieshavebeenper-

formedtounderstandthisprocess(Hirschietal.

2000).Infact,intobacco(m)plants,it

hasbeenfoundthattheArabidopsisantiporter

CAX2(calciumexchanger2)maybeakeymedi-

atorofthemetaltransportfromthecytosoltothe

vacuole(Hirschietal.2000).Tobaccoplants

expressingCAX2accumulatedmoreCa,Cd,and

MnandweremoretoleranttoelevatedMnlevels.

ExpressionofCAX2alsoincreadCdandMn

transportinisolatedroottonoplastvesicles.

Modulationofthistransportercouldbeufulfor

phytoremediation.

Someauthors(Kramer&Chardonnens2001)

considerthatfieldtrialshavesuggestedthatthe

rateofcontaminantremovalusingconventional

traitsandgrowthconditionsisinsufficient,andso

theintroductionofnoveltraitsintohighbiomass

plantsinatransgenicapproachisneededfor

effa,the

engineeringofaphytoremediatorplantrequires

theoptimizationofprocesssuchastraceele-

mentmobilizationinthesoil,uptakeintothe

83

root,anddetoxificationandallocationwithinthe

plant(Kramer&Chardonnens2001;Singhetal.

2003).

Moleculartechniquessuchastheanalysisof

molecularvarianceoftherandomamplifiedpoly-

morphicDNAmarkersareufultoinvestigate

thegeneticdiversityandheavymetaltolerancein

plantpopulations,providingtheopportunityto

investigatethefirststepsinthedifferentiationof

plantpopulationsunderverelectionpressure

andtolectplantsforphytoremediation

(Mengonietal.2000).

Onestrategyforincreasingtheefficiencyof

phytoextractionistoincreametaltranslocation

determinewhethergeneticallyincreadtranspi-

rationwouldincreatheefficiencyofphytoex-

traction,weremutagenizedwith

ethylmethanesulfonate,andmatureplantswere

thenlf-pollinated(Glebaetal.1999).Insub-

quentsteps,alineinwhichthetranspirationrate

exceededthatofthewild-typeplantsby130%in

soil,phytoextractionofPbwastestedinPb-con-

h-

transpirationlinephytoextracted104%morePb

.

Amuchbetterunderstandingofthegenetic,

biochemicalandphysiologicalbasisofmetalhy-

peraccumulationinplantsisofkeyimportancefor

hanisms

deployedintheacquisitionofesntialheavyme-

talmicronutrientshavenotbeenclearlydefined

althoughanumberofgeneshavenowbeeniden-

tifi

classofmembranetransportershavebeen

implicatedinthetransportofheavymetalsina

varietyoforganismsandcouldrvesucharolein

plants:(i)theheavymetal(CPx-type)ATPas,(ii)

thenaturalresistance-associatedmacrophage

protein(Nramp)family,and(iii)membersofthe

cationdiffusionfacilitator(CDF)family(Williams

etal.2000),recentlyrenamedascationefflux

family(CEF)(Maretal.2001).

MembersoftheZIPgenefamilyarealso

capableoftransportingavarietyofcations,

includingCd,Fe,MnandZn(Guerinot2000).

InformationonwhereintheplanteachoftheZIP

transportersfunctionsandaboutitsregulation,

wouldopenthepossibilityofmanipulatingplant

mineralstatuswithaneyetodevelopingcropsthat

bioaccumulateorexcludetoxicmetals.

AccordingtoRughetal.(1998a),theexcep-

tionalphysiologicalabilitiesofplantscouldbe

augmentedwithgenesfromthe‘‘moleculartool-

boxes’’ofmicrobialmetabolismtoallowthe

developmentofpowerfulweaponsforpollution

control.

Inrecentyears,veralkeystepshavebeen

identifiedatthemolecularlevel,enablingusto

initiatetransgenicapproachestoengineerthe

transitionmetalcontentofplants(Clemental.

2002).Inthisrespect,transgenictomatoplants

(902)express-

ingthebacterialgene1-aminocyclopropane-1-

carboxylicacid(ACC)deaminawerecompared

tonon-transgenictomatoplantsintheirabilityto

growintheprenceofandaccumulateCd,Co,

Cu,Mg,Ni,PborZn(Grichkoetal.2000).In

general,transgenictomatoplantxpressingACC

deaminaacquireagreateramountofmetal

withintheplanttissues,andarelesssubjecttothe

deleteriouffectsofthemetalsonplantgrowth

thanthenon-transgenicplants.

Finally,andsinceconsiderableexpectationsare

placedongeneticmodificationtoproduceideal-

izedplantsforphytoextraction,itisimportantto

rememberthattheever-increasingpublicconcern

overthereleaofgeneticallymodifiedorganisms

-

podbyBaker&Whiting(2002),itwouldbe

prudenttocomplementgeneticalapproacheswith

archesfornaturalhyperaccumulatorsandfur-

therexploreconventionalplantbreedingpractices

(Chaneyetal.2000),withtheadditionalbenefitof

catalogingandconrvingtheuniqueglobalbio-

r-

more,Kramer(2001)suggestedthattheGanges

escencotype(Lombietal.2000)should

beconsideredamodelpopulationoftobecen-

trallypropagated,crosdandmadeavailablefor

allscientists,na

uldeventuallyre-

ducethegeneticvariabilitywithintheedpopu-

lation,amajordrawbackintheworkwithmetal

hyperaccumulators.

Althoughphytoremediationisstillanewtech-

nology,inthelastfewyearsalotofbasicrearch

hasbeencarriedoutinanattempttounderstand

howplantstakeuplargequantitiesofmetals,to-

getherwiththemechanismsofmetaltranslocation

fromrootstoshoots,storageanddetoxification.

Nevertheless,itisnotyetclearhowthisinforma-

84

tionshouldbeudtoefficientlyremovemetals

frompollutedfi,apartfromdeeperbasic

rearch,moreappliedprojectsinthefieldare

neededtoclarifytherealpotentialofthis

technology.

Indeed,beforeattemptingfieldimplementation

ofthistechnology,itisntialtoconductbasic

laboratoryworkfirst,ifwearetoavoidinflated

expectationscomingfromexaggeratedclaimsof

success(aswasthecawithbacterialremediation

regardingitscontaminantremovalefficiency)

(Boyajian&Carreira,1997).

Inthisrespect,twostrategieswilldefinitelybe

consideredinthefuture:(i)optimizationofagro-

nomicpractices,includingfertilization,croppro-

tectionchemicals,methodsofharvestingand

sowing,etc.,especiallyforhyperaccumulating

plants,sincetheknowledgeneededforproper

cultivationofmanyofthereportedhyperaccu-

mulatorsisstilllacking;and(ii)geneticmanipu-

lationofplants,although,asabove-mentioned,

theutilizationoftransgenicplantsprentlydoes

notenjoyhighpublicacceptance,atleastregard-

ingtheutilizationofGMcropsforhumancon-

r,althoughcertainlyextremely

difficulttopredict,publicopinionmightbedif-

ferentwhentransgenicplantsareaimedtowards

environmentalcleanup,suchastransgenicphyto-

a,beforeGMtech-

niquescanbeappliedsuccessfully,adetailed

knowledgeofthemechanismsofuptake,translo-

cation,questration,lsinplantsis

required,andthatinformationisatprentcer-

ll,thefunctionsofmany

planttransportersthatarecentraltophytoreme-

diationremainstilluncharacterized.

Itislikelythatconventionalbreedingtech-

niques,whichundoubtedlyenjoyhighpublic

acceptance,mightprovideasuitablealternative

although,sofar,crossingbetweenhyperaccumu-

latorsandcropsplantshasnotbeensuccessful

a

species,theuofthelectionofindividuals

withgreatermetalremovalefficiencyhasappar-

entlynotbeenachievedyet(McGrathetal.

2002).Breweretal.(1999)udsomatichybrid-

izationtocombinehyperaccumulatingtraitswith

thoofhigherbiomasscropspecies,butcom-

bininggenomesandlectingaprogenyusing

non-GMmethodsrequiresrelativelylongperiods

oftime.

Onefrequentlyignoredaspectwhendiscussing

futuredirectionswithinthephytoextractionfieldis

theneedtodevelopeconomicallyfeasibletech-

niquesforthedisposalofmetal-enrichedplantsor,

whenpractical,omical

methodofreclaimingmetalsfromplantresiduesis

requiredtoeliminatetheneedforcostlyoff-site

portantly,rearchmustalso

inevitablybefocudtowardsfindingwaysto

avoidtransferofmetalstoothermedia,theenvi-

ronmentingeneral,andparticularlytothefood

chain.

Althoughphytoextractionisnotamagicsolu-

tion,commercially,itisgainingappealbecauit

ischeaperthanconventionalclean-upmethods.

Butitisnotaneasytechnologyjustconsistingof

pickingupsomehyperaccumulatingplantsand

contrary,itishighlytechnical,requiringexpert

projectdesignerswithplentyoffieldexperience

thatcarefullychootheproperspeciesandculti-

varsforparticularmetals(andcombinationsof

them)andregions,andmanagetheentiresystem

tomaximizepollutantremovalefficiency.

Forthetimebeing,phytoextractionisnotthe

appropriatechoiceforallmetalpollutedsites

since,forinstance,inmanycas,thehighmetal

concentrationsand/ortheprenceofharshcon-

ditions(hostileclimate,soilproperties)donot

uently,phytoextrac-

tionwillmostlikelybeudinareaswithmedium

tolowlevelsofmetals,orasafinalpolishingstep

(toreducethecostandimpactofothermethods)

afterotherclean-uptechniqueshavebeenudto

ably,phytoextraction

(limitedtotherootingzone)willfinditswidest

applicationintheremediationofsurface-polluted

soils.

Inanyevent,phytoextractionofmetalsdefi-

nitelyholdsgreatpotentialfortheremovalof

xtractionisa

morecost-effectivealternativethanconventional

remediationmethodsandsincemanydecisionsare

badonthebasisofstrictcost-benefitanalysis,

economicpressurestogetherwithpublicaccep-

tancewillprobablycontinuetoinfluencedecisions

s,al-

thoughremediationdoesnotreprentprofitto

thepollutingcompanies,itreducestheprobability

ormagnitudeoflegalliability,oftenmakingit

worththeinvestment.

85

Phytoextractionislittlebylittlecarvingoutits

ownnicheasasureandaestheticallypleasing

-

thoughstudiedextensivelyinrearchandin

small-scaledemonstrations,yetfull-scaleapplica-

tionsofphytoextractionarecurrentlylimitedtoa

xtractionneedsa

transdisciplinary(notsimplymultidisciplinary)

approachwithinputsfrommanyfieldssuchas

botany,plantphysiology,biochemistry,geochem-

istry,agriculturalengineering,agronomy,soilsci-

ence,geneticengineeringandsoon.

Althoughonlythefuturewilltelluswhether

phytoremediationwillbecomeawidelyud

technology,thestudyoftheutilizationofthe

remarkableabilityofplantstoremovepollutants

fromtheenvironmentisatprentafascinating

fieldofrearch.

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