Lowaffinity cation transporter OsLCT1 regulates cadmium transport into rice grains.docx

Lowaffinity cation transporter OsLCT1 regulates cadmium transport into rice grains.docx

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LowaffinitycationtransporterOsLCT1regulatescadmiumtransportintoricegrains

Low-affinitycationtransporter（OsLCT1）regulatescadmiumtransportintoricegrains

1.ShimpeiUraguchia,b,

2.TakehiroKamiyaa,b,

3.TakuyaSakamotob,

4.KojiKasaia,b,

5.YutakaSatoc,

6.YoshiakiNagamurac,

7.AkikoYoshidab,

8.JunkoKyozukab,

9.SatoruIshikawad,and

10.ToruFujiwaraa,b,1

+AuthorAffiliations

1.aBiotechnologyResearchCenterand

2.bGraduateSchoolofAgriculturalandLifeSciences,UniversityofTokyo,Tokyo113-8657,Japan;

3.cGenomeResourceUnit,NationalInstituteofAgrobiologicalSciences,Tsukuba,Ibaraki305-8602,Japan;and

4.dSoilEnvironmentDivision,NationalInstituteforAgro-EnvironmentalSciences,Tsukuba,Ibaraki305-8604,Japan

1.EditedbyEmanuelEpstein,UniversityofCalifornia,Davis,CA,andapprovedNovember15,2011（receivedforreviewOctober7,2011）

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Abstract

Accumulationofcadmium（Cd）inrice（OryzasativaL.）grainsposesapotentialhealthproblem,especiallyinAsia.MostCdinricegrainsaccumulatesthroughphloemtransport,butthemolecularmechanismofthistransporthasnotbeenrevealed.Inthisstudy,weidentifiedariceCdtransporter,OsLCT1,involvedinCdtransporttothegrains.OsLCT1-GFPwaslocalizedattheplasmamembraneinplantcells,andOsLCT1showedCdeffluxactivityinyeast.Inriceplants,strongOsLCT1expressionwasobservedinleafbladesandnodesduringthereproductivestage.Intheuppermostnode,OsLCT1transcriptsweredetectedaroundlargevascularbundlesandindiffusevascularbundles.RNAi-mediatedknockdownofOsLCT1didnotaffectxylem-mediatedCdtransportbutreducedphloem-mediatedCdtransport.TheknockdownplantsofOsLCT1accumulatedapproximatelyhalfasmuchCdinthegrainsasdidthecontrolplants.ThecontentofothermetalsinricegrainsandplantgrowthwerenotnegativelyaffectedbyOsLCT1suppression.TheseresultssuggestthatOsLCT1functionsatthenodesinCdtransportintograinsandthatinastandardjaponicacultivar,theregulationofOsLCT1enablesthegenerationof“low-Cdrice”withoutnegativeeffectsonagronomicaltraits.ThesefindingsidentifyatransportergeneforphloemCdtransportinplants.

heavymetals

foodsafety

Cadmium（Cd）isaheavymetalharmfultohumanhealth.Thebiologicalhalf-lifeofCdinthebodyisestimatedtobenearly30y

（1）,whichleadstochronictoxicity.TheadverseeffectofCdhasbeenaworldwideconcernsincetheoutbreakof“Itai-Itaidisease”inthemid-20thcenturyinJapanthatwascausedbythedailyconsumptionofCd-contaminatedrice（2,3）.Recently,theaveragedietaryintakeofCdinJapanwasestimatedtobe3.0μgCd/kgbodyweightperweek.Thisvalueisnearly50%ofaprovisionaltolerablemonthlyintakeestablishedbytheJointFoodandAgricultureOrganization/WorldHealthOrganization（FAO/WHO）ExpertCommitteeonFoodAdditivesandContaminants（JEFCA）andhigherthanthetolerableweeklyintake（2.5μgCd/kgbodyweight）setbytheEuropeanFoodSafetyAuthority（EFSA）.ReflectingthisgreaterCdintake,theinternalCdlevelamongJapanesepeopleishighamongAsians（4）.RecentsurveysinSweden（5）,theUnitedKingdom（6）,andtheUnitedStates（7,8）alsoshowedmeasurableinternalCdlevelswithinthegeneralpopulation,indicatingtheimportanceofreducingCdexposureinthegeneralpopulation（9）.InAsiancountries,includingJapan,upto50%oftheingestedCdcomesfromriceanditsproducts（4,10）.TheFoodandDrugAdministration's（FDA）TotalDietStudyreportedthatthedietaryintakeofCdamongAmericansincreasedby26%from1990through2003andthatthisrisewascorrelatedwithanincreasedconsumptionofriceandothergrains（11）.

ThesourceofCdinricegrainsissoil.Cdisabsorbedbyricerootsandtransportedtothegrains,resultinginconsiderableCdaccumulationevenwhengrownonslightlyormoderatelyCd-pollutedsoil（12,13）.Transportersformineralnutrients,suchasZnorFe,arepartlyresponsibleforCdtransportinplants（14）.Toimprovethehealthofpeoplewhodependonriceasastaple,theestablishmentofalow-Cdricecultivarisdesirable.AfirststepinthisdirectionistoclarifythemechanismofCdtransportinrice,includingidentificationoftheresponsiblemolecule（s）.

RecentphysiologicalstudieshaveadvancedourunderstandingofhowCdistransportedandaccumulatedinrice.ThemaindeterminantoftheCdconcentrationinshoottissuesistheabilitytotranslocateCdfromroottoshootthroughthexylem,ratherthanCduptakebytheroots（12）.Quantitativetraitloci（QTL）analyseshaveindicatedseveralchromosomalregionscontrollingCdaccumulationinriceshoots;oneoftheselociregulatestheloadingofCdintothexylem（15–19）.Recently,OsHMA3,ariceP-typeATPase,wasidentifiedasaregulatorofthexylemloadingofCdinroots（20,21）.

Despiteitsimportance,littleisknownaboutthemolecularmechanismofphloemCdtransportinplants.Phloem-mediatedCdtransporttograinsfollowingxylem-mediatedroot-to-shoottranslocationiscriticalfortheaccumulationofCdinricegrains.Nearly100%oftheCdinricegrainsisattributabletophloemtransport（22,23）.Fujimakietal.（22）usedanoninvasiveliveimagingtechniquetofollowthetransportof107Cdinintactriceplants.TheydemonstratedtheimportanceofshootnodesforthetransferofCdfromthexylemtothephloem.Forsomeminerals,thecontributionoftransportersiscrucialforthiskindofredirectionofsolutetransportatnodalregions（24,25）.TheCdtranslocationabilityintograinsvariesamongricegenotypes（12）.Katoetal.（26）alsoreportedvariableCdconcentrationsinphloemsapamongcultivarsexhibitingdifferentgrainCdlevelsandshowedthecorrelationbetweengrainandphloemsapCdconcentrations.ThesestudiesindicatedtheexistenceoftransportersmediatingCdtranslocationintograinsandsuggestedthatregulationofthetransporterscanalterthelevelofCddepositioningrains.

Inthisstudy,wefocusedonLOC_Os06g38120asapossibleCdtransportergeneexpressedinriceshootsduringgrainripening.Thisgene,OsLCT1,ispredictedtoencodetheonlyricehomologofthelow-affinitycationtransporter1（TaLCT1）foundinthewheatcDNAlibrary.TaLCT1enhancedtheintakeofvariouscations,includingCd2+inyeast（27）.Here,wepresentevidencethatOsLCT1isaplasmamembrane-localizedCdexporterinvolvedinphloemCdtransport.Bydown-regulatingOsLCT1expression,wegeneratedriceplantswithreducedgrainCdlevels,demonstratingtheimportanceofOsLCT1inCdtransportintograins.

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Results

OsLCT1IsaPlasmaMembrane-LocalizedEffluxTransporterofCd.

OsLCT1cDNAclonedfromajaponicaricecultivar,Nipponbare,wasdeducedtoencode511aawith11transmembranedomains（Fig.S1）.TheprimarysequenceofOsLCT1is23%identicaltothatofTaLCT1.ABasicLocalAlignmentSearchTool（BLAST）searchindicatedthatOsLCT1isasingle-copygeneinthericegenome,andnohomologofOsLCT1wasfoundintheArabidopsisthalianagenome.

ThesubcellularlocalizationofOsLCT1wastheninvestigatedintobaccoBY-2cells.OsLCT1fusedwithGFPwasprimarilycolocalizedwiththeplasmamembranemarkerFM4-64,whereasincellsexpressingGFPalone,greenfluorescencewasobservedmainlyinthecytosolandnucleus（Fig.1A）.ThisstronglysuggeststhatOsLCT1isaplasmamembraneprotein.

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Fig.1.

（A）SubcellularlocalizationofOsLCT1-sGFP.35S-OsLCT1-sGFP（OsLCT1-sGFP）and35S-sGFP（sGFP）wereintroducedseparatelyintoculturedtobaccoBY2cells.FM4-64（25μM）wasusedasaplasmamembranemarker.（Scalebars=10μm.）（B）CdtransportactivityofOsLCT1.Yeast（strainWΔycf1）expressingOsLCT1orharboringtheemptyvectorwereincubatedfor75minwitharginine-phosphatemediumcontaining20μMCdCl2.TheconcentrationsofCdintheharvestedcellsweredeterminedbyICP-MSafternitricaciddigestion.Asterisksrepresentasignificantdifferencefromtheemptyvectorcells（P<0.001,ttest）.Thedataarepresentedasmeans±SD（n=3）.

TaLCT1enhancedthetransportofvariouscations,includingCd2+,inyeast（27）.WetestedwhetherOsLCT1hasCdtransportactivityinayeastheterologousexpressionsystemusingWΔycf1.Thisyeaststrainhasadefectinthegeneencodingavacuolartransporter,Ycf1p,whichmediatesthetransportofaCd–glutathioneconjugateintovacuoles（28）.YeastcellsweretransformedwithavectorcontainingMyc-taggedOsLCT1cDNA,andtheexpressionofOsLCT1wasconfirmedbyWesternblotanalysisusinganti-Mycantibodies（Fig.S2A）.OsLCT1expressionintheyeastdidnotaffecttheCdsensitivityofthecells（Fig.S2B）.However,OsLCT1expressionsignificantlyreducedCdaccumulationinthecellscomparedwithvectorcontrolcellsin10and20μMCdtreatments（Fig.1BandFig.S2C）,suggestingthatOsLCT1isanefflux-typeCdtransporter.Wealsoanalyzedtheconcentrationsofseveralmineralsinthecells.YeastcellsexpressingOsLCT1showedsignificantlyreducedMg,Ca,K,andMnaccumulationcomparedwiththevectorcontrolcells,withnosignificantdifferenceintheconcentrationsofNa,Fe,Zn,orCu（Fig.S2D）.Treatmentwith1mMCaCl2or10mMMgSO4with10μMCdCl2reducedtheCdtransportactivityofOsLCT1-expressingcells（Fig.S2E）comparedwiththatunderthenormalCa（0.1mM）andMg（1mM）conditions（Fig.1BandFig.S2C）.

OsLCT1IsStronglyExpressedinNodesandLeafBladesDuringGrainRipening.

TodeterminetheexpressionprofileofOsLCT1inriceplants,wefirstanalyzedmicroarraydatafr