UBC Social Ecological Economic Development Studies (SEEDS) Student Report

Qualitative study of the effects of Cadmium ion on the morphology, physiology and

protein profile of Lolium perenne (Perennial Ryegrass) and Trifolium hybridum (Alsike

Clover)

Yinuo (Yvette) Guo

University of British Columbia

BIOL 448

June 1, 2012

Disclaimer: “UBC SEEDS provides students with the opportunity to share the findings of their studies, as well as their opinions,

conclusions and recommendations with the UBC community. The reader should bear in mind that this is a student project/report and

is not an official document of UBC. Furthermore readers should bear in mind that these reports may not reflect the current status of

activities at UBC. We urge you to contact the research persons mentioned in a report or the SEEDS Coordinator about the current

status of the subject matter of a project/report”.

[Academic work for this project was completed on 2010/April 30]

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UBC Social Ecological Economic Development Studies (SEEDS) Student Report

Qualitative study of the effects of Cadmium ion on the morphology,

physiology and protein profile of Lolium perenne (Perennial Ryegrass)

and Trifolium hybridum (Alsike Clover)

Yinuo (Yvette) Guo

University of British Columbia UBC SEEDS Project

June 1, 2012

Disclaimer: UBC SEEDS provides students with the opportunity to share the findings of their studies, as well as their opinions,

conclusions and recommendations with the UBC community. The reader should bear in mind that this is a student project/report and

is not an official document of UBC. Furthermore readers should bear in mind that these reports may not reflect the current status of

activities at UBC. We urge you to contact the research persons mentioned in a report or the SEEDS Coordinator about the current

status of the subject matter of a project/report.

2

Qualitative study of the effects of Cadmium ion on the morphology, physiology and

protein profile of Lolium perenne (Perennial Ryegrass) and Trifolium hybridum (Alsike

Clover)

UBC SEEDS RESEARCH PROJECT

YINUO (YVETTE) GUO

June 1, 2012

Research Supervisor: DR. SANTOKH SINGH Department of Botany Faculty of Science University of British Columbia

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Abstract RoadsideplantsareexposedtoheavymetalionseveryyearintheLowerMainland.Onanaveragerainyday,roadsidegroundwatermaycontainpotentiallytoxiccadmiumconcentrationsaswellasotherheavymetals.Doesthisposesevereproblemsforplantgrowthandsurvival?ThecurrentstudyinvestigatedthephysiologicalsymptomsofRyegrass(L.perenne)andClover(T.hybridum)inresponsetocadmiumstress.Usingplateculturingandhydroponicgrowthmedium,wefoundthatbothplantspeciesinhighcadmiumconcentration(1000µM)experienced1)reductioninthemorphologicaldevelopmentofseedlingandyoungplants,and2)reductioninmetabolism(e.g.photosynthesisrate).Thesefindingsshedlightonthetolerancelimitsofroadsideplantsandtheirsustainablegrowthinheavy‐metalproneenvironments.

Introduction

Oneoftherecoursesofindustrializationistheeffectofchemicalleachingonthe

environment.Miningprocessescanbeamajorsourceofmineralleaching,whichnotonly

cansignificantlyaffectthechemicalandstructuralcompositionoftheenvironment

impactedbutalsotheanimalsandplantsthatresidethere(Laws,2000).Miningcanvery

oftenproduceenvironmentaltoxinssuchascadmium(Cd2+),iron(Fe),zinc(Zn2+),lead

(Pb2+)andcopper(Cu2+orCu4+)ataconcentrationsuspectedtobeintolerabletomany

species(Laws,2000;EPA2000).Inhumans,Cd2+isnoteasilydegraded,long‐term

exposuretowhichcancausekidneytomalfunction,adverselyaffectcalciummetabolism

andboneformation,e.g.osteoporosis(Laws,2000).Forthepresentstudy,thefocuswasto

determineatdifferentconcentrationsofCd2+anddifferentlengthsofexposurewhether

certainplantspeciescommonlygrownontheBCroadsideexhibit1)tolerancereflectedin

morphology,transpirationandphotosynthesisrates,andtoidentify2)proteinsthatmay

behighlyexpressedasafunctionoftoxintolerance.Itwasthoughtthatduetotherarityof

cadmiuminthenaturalenvironment,organismswouldnotbegenerallyadaptedtohigh

Cd2+concentrations,e.g.nomorethan1‐10μM(EPA,2000).However,astudyon

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tumbleweedCd2+tolerancebydelaRosaetal(2005)ledtothehypothesisthatsome

plantsmaybefairlyresistantevenathigherconcentrations.Thehopeisthatiftolerant

speciesarefound,theycanbefavouredtogrowatindustrialsites.Moreover,speciesthat

tolerateinternallyhighamountsofcadmiumhavethepotentialtoactasabufferingzoneto

preventchemicalleachingtonearbywatersheds(Laws,2000).Therefore,thepotential

existsforsomeplantstoplayimportantrolesinbioremediationsespeciallyconsideringthe

growingdemandforzinc,whichwhenminedyieldsreleasescadmiumasabyproductthat

accountsformostofthecadmiumemittedtodate(Laws,2000;EPA2000).

Methods and material I. Plantspeciesselection(seeFigure1)

Seedsofthirteen(13)commonroadsidegrownplantspeciesnearBChighways

wereincubatedinseparatepetridishes(n=15)onfilterpapersoakedin1.5mLof

waterorvariousdilutedconcentrationsofcadmiumintheformofcadmium

chloride.Aftersealinginthemoisturewithparafilm,onesetoftheseedswere

incubatedinthedarkandanotheridenticalsetinthelightatconstantroom

temperaturefor2‐3days,thenormaltimefoundforgerminationoftheseseeds.The

morphology(colour,texture)oftheseedlingswasnoted,inadditiontotherootsand

shootlengthsofeachseedlingbeingmeasuredusingacomputertracingprogram.

Theresultsforeachpetridishwereaveragedfromthespecimenthatgerminated.

Withthewatertreatmentasacontrol,theseedlingsthatshowedlittle/nochangeat

highconcentrationsofcadmiumwerethenselectedforthenextstep.

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II. FromthestepI,itwasdeterminedthatCloverandRyegrasswerethemosttolerant

amongtheelevenothercommonlygrownplantspeciesalongBChighways.Itwas

nowtodetermineatwhatconcentrationandthelengthofexposuretocadmiumdo

seedlingsbecomeintolerantalongwiththeaccompanyingmorphologicaland

physiologicalcharacteristicstheyexhibit.

Plantseedlings(n>3)thatgerminatedsuccessfullyathighCd2+concentrationwere

transferredfromthepetridishestosmall,shortboxesfilledwitheitherdiluted

nutrientsolutions(seelabbookforafullrecipe)ornutrientsolutionsmixedwith

variousconcentrationsofCd2+.SincehydroponicgrowthofRyegrass(startedbefore

Clover)revealednovisibleeffectfromexposuretoCd2+concentrationbelow1M,it

wasassumedthat10MCd2+wouldbeareasonableminimumconcentrationfor

Clover.PlasticnettingwithStyrofoamedgeswasusedtosupporttheseedlings

uprightwhiletheirrootswereexposedtothemediumsolution.Allmediawere

aeratedusingahandmadebubblingdevicewithavalvecontrolledairsource(see

Figure2).Theincubationcontinuedaslongastheseedlingssustainedahealthy

appearance.Leafsampleswerefrozenforproteinanalysiswhenchangeswere

observedandwhenpossible,photosynthesisandtranspirationratesweretaken

withtheLiCORmachineasmeasuresoftheoverallphysiologicalperformanceof

eachreplicate.Technicallyspeaking,LiCORmeasuresandrecordstheCO2

concentrationandwaterloss,whichwerelaterconvertedtophotosynthesisrate

andtranspirationratewithacomputerprogram.

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III. Proteinanalysis

WiththefrozenleafsamplefromstepII,standardSDSgelelectrophoresiswere

performedtodetectifmajorproteingroups(e.g.RUBISCO/ATPSynthaseandLight

Harvestingcomplexes)increaseordecreasewithhighercadmiumconcentration.

Thesegroupscangiveusanindicationofwhethercadmiuminhibitsorstimulates

themajorbiochemicalfunctionsoftheplants.WithproteinsmigratedontotheSDS

gel,WesternblottingusingtheheatshockproteingroupDehydrin(AgriSara™

enzyme)ataratioof1:5000(vol/volproteinversus5‐10mLofmilkprotein

blottingsolution)wasperformedasabiochemicalindicatorofstress.Itisimportant

tonotethatthebiochemicalcuesdonotusuallyexplainallmacroscopicchanges

becausetherearemany,interactingmicroscopicprocesses.

Limitationstothemethods

Itwasdifficulttokeeptheconditionsforhydroponicgrowthconsistentthroughout

theexperiment,especiallyconsideringtheunwantedgrowthofalgaeinthenutrient

solutionandtheperiodicevaporationofmediumthroughtheatmosphereviaaeration.

Thesedifficultiescouldhaveledtonon‐negligiblenutrientdeficiencies,confoundingthe

resultsfromboththecontrolsandthetreatments.

FortheWesternBlot,thesampleswereonlyassayedwithDehydrinthoughthere

couldbeotherproteinsup‐ordown‐regulatedasaresultofcadmiumstress.Therationale

fortestingwithDehydrinwasthatthisfamilyofproteinshasbeenlinkedtostress

symptomsandheavymetaldetoxificationinplants(Zhangetal,2006).Jonaketal(2004)

havedemonstratedtheupregulationofcertainmitogen‐activatedproteinkinasesaidingin

stressresponseinalfalfaexposedtohigh[Cd2+].However,anotherreasonforstudying

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Dehydrinisthatitisgenerallylesscharacterizedthanotherproteingroupsknownfortheir

rolesinplantmetabolism.

Duetotimeconstraints,thehydroponicsexperimentwasnotabletobereplicated.

Roots,shootsandmetabolicratessampledateachsamplingtimewerebasedonone

replicatefromeachtreatment.Theresultsfromthecurrentstudyweretherefore

qualitativeonlyandnotstatisticallyrobust.

Results & Discussion PerennialRyegrass(Loliumperenne)

Morphology

After2daysofhydroponicgrowthwithvariousconcentrationsofcadmium,the

Ryegrassseemedtoexhibitnochangesinrootorshootlengthrelativetothecontrol(Fig.

3).Onthe8thday,however,plantstreatedat1000uMbegantoshowstuntedgrowth(Fig.

3),yellowingoftheshootsandlackofturgorascomparedtothecontrolandtoother

treatmentsatlowerCd2+concentrations.Thesesymptomsaretypicalofplantsunderheavy

metalstress,whichindicatesaninterferencewiththenormalphysiologicalfunctions(Zhu

etal,2005)

Physiology

Photosynthesisrates–onday2,therewasaten‐folddecreaseinphotosynthesis

ratefromthecontrolforthetreatmentat1000uMCd2+(Fig.4).Asimilardecreasing

patternwasexpectedonday7butthiswasnotreflectedinthegraph.Wesuspectedthat

theaerationmechanisminthehydroponicswasnotconsistentthroughouttheexperiment

andthusoccasionallackofO2inthemediumcouldhaveinducedstressonsomeofthe

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plants,includingthecontrol,resultinginphotosynthesisratesgenerallybeinglowonday7

comparedtoday2.Decreaseinphotosynthesiswasexpectedbecauseplantsunderionic

stresstendtoproducefewerstomataduetoinhibitionofguardcellprecursors(Zhuetal,

2005),leadingtodecreasedintakeofCO2,thesubstrateforphotosynthesis.

Transpirationrates–onday2,therewasatwo‐threefoldincreaseintranspiration

inthe1‐100uMCd2+treatmentscomparedtothecontrol(Fig.5).Similarlyonday7,there

wasatwo‐foldincreaseintranspirationrateat0.1uMandanevenlargerincrease(fivefold)

at1000uM.ThisfindingiscontrarytothatofLeitaetal(1995),whocitedattributedto

lowertranspirationinsoybeanplantstostomatalclosure.However,thismaybedueto

differentplantusedsinceZhuetal(2005)foundlargerstomatalapertureinBrassica

juncea,whichiffoundtobethecaseinthisexperiment,wouldexplaininpartthe

heightenedtranspirationinRyegrass.

Proteinanalysis

Forallbutonetreatment(1000μM),itwaspossibletodetectdistinct,near‐uniform

bandsforRubiscoandATPsynthaseandlight‐harvestingcomplexes(LHC)I&IIonthe

stainedgel(Fig.6).Thiscouldexplainthelackofmorphologicalchangesintreatmentsat

Cd2+concentrationsunder1000μM,butgenerallydonotreflectthechangesinphysiology,

i.e.decreaseinphotosynthesisandriseintranspirationwithincreasingcadmium

concentrations,theexceptionbeingthe“smeared”ordegradedgelpatternofplantsthat

becameincreasinglystressedbyhighCd2+asthetreatmentscontinued(i.e.undergoing

proteolysis).However,thelackofbiochemicalsignalsmayinsteadsuggestthatcadmium

doesnotdirectlyantagonizephysiologicalfunctionsdirectly,butrathermoredirectly

throughtheclosureofstomata,whichleadindirectlytotheobservedphysiologicalchanges

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(Zhuetal,2005).Dehydrinwaspresentinvariousamountsinthecontrolsandtreatments

(Fig.7),whichindicatedthatexperimentalconditionsingeneralwerenotproperly

controlled(e.g.throughunequallysustainedairpressureandalgalgrowthonnutrient

solutionpastday2)andcouldhaveresultedinplantsfunctioningoutoftheirnormalrange.

AlsikeClover(Trifoliumhybridum)Morphology

Itwaspossibletovisualizetoxiceffectsofcadmiuminthe1000uMCd2+treated

cloverseedlingsonday2.Notably,theplantsshowedstuntedgrowth,yellowingandcrispy

textureonmostofitsleavesaswellaslackofturgorthroughouttheplant(Figs.8and12).

Bythe7thday,the100uMtreatmenthadalsobeguntoexhibitthesameeffects(Fig.8).

ComparedtoRyegrass,Cloverappearedtobemorevulnerabletotheheavymetal.This

couldbepartiallyduetothelargesurfaceareaofcloverleaveswhichmakestheplantmore

susceptibletofunctionaldeclinewhichleadstomorevisiblesignsofstress.

Physiology

Photosynthesisrates–a100%dropinphotosynthesisfromthecontroltreatmentin

1000uMCd2+treatedcloveronday2wasfound(Fig.9).Thoughthedataarenotshown

here,thedecreasingtrendshowedsignsofcontinuationonday7.

Transpirationrates‐therewasaslighttonegligibledeclineintranspirationofall

treatmentsonday2(Fig.10)andcadmiumseemtohaveresultedinmoretoxiceffectson

Cloverasmoretimepassed(day7dataincomplete,notshown).

Altogether,theratesforCloverweremuchlowerthanthoseoftheRyegrass,which

iscounter‐intuitivesincethelargesurfaceareashouldfavourhighermetabolicrates.Aside

frompotentiallystressfulconditionscausedbythealgaeintheCloverhydroponics,we

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suspectedtheremayhavebeenerrorsincalibrationoftheLiCOR,whichtendedto

underestimateratesastheCO2filtersbecamecongestedovertime.Thiswouldhavecaused

anoveralllackofphysiologicalstresssignalexpressed.Thisisanotherimportantreason

thattheexperimentneedstobereplicatedinthefuturetoconfirmthecurrentfindings.

Proteinanalysis

UnliketheRyegrass,eventhoughtherewasnoappreciabledifferenceinprotein

patternsbetweenthecontrolandthetreatmentsfrom0.1‐100μMCd2+inbothdays2and,

the1000uMtreatmentappearedtobeaffectedsincethegellanehadasmearpatternthat

isusuallyaccompaniedbyproteindegradationofadyingplant,whichcorrespondsto

morphologicalsymptomsofstressdescribedearlier.Whilethegeneralproteinpatterndid

notindicateadifferencebetweenthecontrolandastressedcadmiumtreatment,Dehydrin

wasfoundtobehighlyexpressedinthe1000μMtreatment,suggestingthatpartofthe

plant’sstressresponsecanbetracedtotheupregulationofDehydrin.Onday7,however,

onlyasmearofDehydrinwasfoundinthe100μMsample,possiblyresultingfrom

proteolysis.

Synthesis

ItappearsthatRyegrass(L.perenne)isgenerallyverytolerantofhighCd2+

concentrations,onlyshowingmajorsignsofstressat1000μM,aconcentrationthatistwo

tofourmagnitudeshigherthanwhatisgenerallyfoundatindustrialsites(Peralta‐Videaet

al,2009;EPA,2000).Clover(T.hybridum)exhibitedfairlyhightolerancetocadmium,

thoughnotasmuchasL.perenneintermsofmorphologyandphotosynthesis.The

differencesinstressresponses,especiallyinmorphologybetweenthetwoplantsmaybe

attributedtodifferentialuptakebyleaves(delaRosa,2005),possiblymorecontrolledin

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RyegrassthanitisinClover.Or,aswasmentionedearlier,themorphologyoftheplants

couldalsobeafactorbyrelatingourresultstoHaghiri(1973)wheretherelative

sensitivityofsoybean(atrifoliate)toCd2+wasfoundtobehigherthaninwheat(agrass

species).T.hybridumappearstoberesistantoverextendedperiodsoftimeat10uM

Cd2+butnotathigherconcentrations(Fig.12).T.hybridum’sresponsetocadmiumisalso

timeandconcentrationdependent,whichwasreflectedinthepresenceofDehydrin.Where

Dehydrinwasnotdetected(duetoabsenceofexpressionortoproteolysis)inRyegrass,it

wasinClover,suggestingthatDehydrincanplayasignificantinmediatingthestress

responseoftheseplantstocadmium.Amongtheveryfewstudiesavailableexaminingthe

relationshipbetweenDehydrinandheavy‐metalstress,Zhangetal(2005)andXuetal

(2008)haveproposedthatthefoundDehydrin‐likeproteins(twospecificproteinscloned

bytheauthors)seekoutoxidativederivativesharmfultotheplantthatisunderthestress

ofheavymetals.Krugeretal(2002,citedinXuetal,2008),however,foundheavy‐metal

bindingDehydrins.ThereappearstobediversebutimportantrolesofDehydrininheavy‐

metaltoleranceofplants.

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Figures

Figure1.PlateculturingexperimentwithRyegrass(above)andClover(below)seedlingsincubatedinvariousconcentrationsofCd2+inlightanddarkconditions.

Figure2.HydroponicssetupofCloverseedlingsgrownatvariousconcentrationsofCd2+.

Figure3.Ryegrassmorphology(rootsandshoots)subjectto2days(left)and8days(right)ofcadmiumtreatments(concentrationsindicatedabove).

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-0.2

0

0.2

0.4

0.6

0.8

1

Control 0.1µM 1µM 10µM 100µM 1000µM

Treatment concentration (µM of Cd)

Ph

oto

syn

thes

is r

ate

(µm

ol/m

2/s)

Day 2 Day 7

Figure4.PhotosynthesisratesofRyegrasstreatedatvariousconcentrationsondays2and7.

0

0.5

1

1.5

2

2.5

3

Control 0.1µM 1µM 10µM 100µM 1000µM

Treatment concentration (µM of Cd)

Tra

nsp

irat

ion

rat

e (g

H20

/m2/

min

)

Day 2 Day 7

Figure5.TranspirationratesofRyegrasstreatedatvariousconcentrationsondays2and7.

Figure6.Day8RyegrassSDSelectrophoresisgelshowingdegradativeproteinprofileat1000μMCd2+

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Figure7.Day2RyegrassDehydrinprofile(Westernblotting)atvariousCd2+concentrationsandaccompanyingmorphology.

Figure8.Clovermorphology(rootsandshoots)subjectto2days(left)and7days(right)ofcadmiumtreatments(concentrationsindicatedabove).

-0.005

0

0.005

0.01

0.015

0.02

0.025

0.03

Control 10µM 100µM 1000µM

Treatment concentration (µM Cd)

Ph

oto

syn

thes

is r

ate

(µm

ol/m

2/s)

Day 2

Figure9.PhotosynthesisrateofClovertreatedatvariousconcentrationsonday2;day7datanotavailable.

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0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

Control 10µM 100µM 1000µM

Treatment concentration (µM of Cd)

Tra

nsp

irat

ion

rat

e (g

H2O

/m2/

min

)Day 2

Figure10.TranspirationrateofClovertreatedatvariousconcentrationsonday2;day7datanotavailable.

Figure11.Top:Day2and7SDSelectrophoresisgel(lefttoright)ofClovertreatedatvariousconcentrationsofCd2+.ArrowsindicateproteingroupsspecifiedinFigure#.Bottom:accompanyingDehydrinprofile.

Figure12.CloverhydroponicsrevealingmorphologicaldifferencesbetweendifferenttreatmentsofCd2+(controlattopleft,[Cd2+]increaseisseencounterclockwise.

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Conclusions and future direction

ThehighestconcentrationsdetectedinbothCloverandRyegrassinthisstudyare

moreelevatedthanthetypicalCd2+levelsreportedatindustrialsites,usuallynotmore

thanthe10μMrange(Peralta‐Videa,2009;EPA,2000),andnearhighways(usually0.04‐

0.1µM).Consideringtheresultsofthisstudy,itisreasonablylikelythatbothCloverand

Ryegrasshavetheabilitytogerminateandsurviveforsometimeathigherthanusual

concentration.Itisworthnotingthatthehighestconcentrationsofheavymetalusually

occurinstormyseasonswhenthemineralleachatesarethemosteasilyflushedoutintothe

surroundingbiologicalenvironmentbutthisisexpectedtolastafewdays(Laws,2000).In

thecaseofClover,tolerancetohighbutshort‐livedflushesofCd2+seemsquitepossible.All

inall,heavymetalresistancedemonstratesinbothCloverandRyegrasswouldmakethem

competitiveinCd2+‐proneenvironmentinadditiontotheirbeingfastgerminatingspecies.

Itisstilldifficulttoextrapolateourresulttothenaturalsoilenvironmentwhere

manyfactorscanimpactcadmiumtoxicity.Forinstance,itispossiblethatcadmiumions

(positivelycharged)areadsorbedbythenegativelychargedsoilparticles,whichmakes

Cd2+morebioavailabletoplants.Inaddition,somestudieshavefoundcadmiumtobeco‐

transportedwithironandzincundercertaincircumstances,whichleadstoincreasedCd2+

uptake(Peralta‐Videaetal,2009;delaRosaetal,2005).Thisstudydidnotinvestigate

whethercadmiumabsorptionbyCloverandRyegrassrootswasindeedhappeningandifso,

whethereither/bothplantshaveathresholdforitsintake.Thestrongtoleranceof

RyegrasstohighCd2+concentrationsuggestthatcadmiummaybetakenuponlypasta

certainconcentrationoritmayhaveaveryhighthresholdofaccumulation.Bycontrast,

Cloverexhibitslimitedtolerancetocadmium,suggestingaCd2+isreadilymetabolized.The

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possibilitiesofCd2+bindingandcompartmentalizationcannotberuledouteither.These

considerationscanbehelpfulforfuturestudies,whichmaybeinterestedinestablishing

whethercertainplantspeciesasparticularlystrongabsorbantsforcadmiumwhilebeing

resistanttoit,thuscapableofactingasbufferzonestopreventheavymetalrunoffsto

watersheds.Fromtheviewpointofphysiologicalstudies,itwouldbeinsightfulto

determinethefateofthecadmiumiononceintheplant,i.e.whetheritistransportedto

differentorgans,storedormetabolized.Arelevantimplicationofthisisthatstudieshave

shownCd2+tobeselectivelyaccumulatedindifferentplantorgans,whichwhilenot

phytotoxicatthislevel,canbebioaccumulatedorposesignificanthealthriskstohumans

andmammals(Peralta‐Videaetal,2009,EPA2000;Haghiri,1973).

Acknowledgments

IwouldliketothankDr.SantokhSinghforadviceandsupportthroughoutthisproject,andMr.JarnailMahrokeandfortechnicalhelp.ThankyoutoBrendaSawadafromUBCSEEDSforcoordinatingthisproject,andtoKellyCoulsonforsupplyingseedsofvariousgrasses.

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