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RESEARCHOpenAccessEffectsofdifferentFesuppliesonmineralpartitioningandremobilizationduringthereproductivedevelopmentofrice(OryzasativaL.
)RaulAntonioSperotto1,2,3,MartaWiltonVasconcelos4,MichaelAndrewGrusak5andJanettePalmaFett1,2*AbstractBackground:Minimalinformationexistsonwhole-plantdynamicsofmineralflowthroughriceplantsandonthesourcetissuesresponsibleformineralexporttodevelopingseeds.
Understandingthesephenomenainamodelplantcouldhelpinthedevelopmentofnutritionallyenhancedcropcultivars.
Awhole-plantaccumulationstudy,usingharvestsduringreproductivedevelopmentunderdifferentFesupplies,wasconductedtocharacterizemineralaccumulationinroots,non-flagleaves,flagleaves,stems/sheaths,andpaniclesofKitaakericeplants.
Results:LowFesupplypromotedhigheraccumulationofZn,CuandNiinroots,Mn,Ca,MgandKinleavesandZninstems/sheathsandasmalleraccumulationofFe,MnandCainrootsandZnandNiinleaves.
HighFesupplypromotedhigheraccumulationofFeinrootsandZninleavesandasmalleraccumulationofFeinleavesandstems/sheathsandZn,CuandKinroots.
CorrelationanalyzesindicatedthatfluctuationsinMn-Ca,Zn-Cu,Zn-Ni,Cu-Ni,Mo-S,Ca-Mg,Cu-MnandCu-MgconcentrationsinresponsetodifferentFesupplieswerepositivelycorrelatedinatleastfourofthefiveorgansanalyzed.
Conclusions:Mineralcontentlossanalysisindicatedthatmineralremobilizationfromvegetativeorganscanoccurinriceplants;however,forseedstoacquireminerals,vegetativeremobilizationisnotabsolutelyrequired.
Also,mineralremobilizationfromvegetativetissuesinricewasgreatlydependentofplantFenutrition.
Remobilizationwasobservedforseveralmineralsfromflagleavesandstems/sheaths,buttheamountsweregenerallyfarbelowthetotalmineralaccretionobservedinpanicles,suggestingthatcontinueduptakeandtranslocationofmineralsfromtherootsduringseedfillareprobablymoreimportantthanmineralremobilization.
Keywords:Biofortification,Elementalanalysis,Ionomics,Iron(Fe),Kitaake,Mineralpartitioning,ReproductivedevelopmentofriceBackgroundPlantsaretheprimarysourceofnutrientsforhumannutritiononaglobalbasis.
Stapleseedcrops,suchasrice,supplythemajorityofdailydietarynutrientsforbillionsofpeople.
However,ricehasalowdensityofmineralnutri-ents,andforthosewhosedietsarehighinstaplefoods,micronutrientmalnutritioniswidespread(GrusakandDellaPenna1999).
Biofortification,whichconsistsoftheuseofplantbreedingand/ortransgenicapproachestodevelopnewcultivarswiththepotentialtoincreasethenutrientconcentrationofedibleportionsofcropplants(WhiteandBroadley2005),hasemergedasonepossiblesolutiontoalleviatemalnutrition.
Despitetheincreasingnumberofstudiesaboutthephysiologyandregulationofuptakeofseveralmineralsfromtherhizosphere,thelackofknowledgeabouthowmineralsaremovedintooroutofvasculartissues,translocatedtovegetativetissuesandloadedintoseedsisoneofthebarrierstoseedbiofortifica-tion(ColangeloandGuerinot2006).
InitialseedbiofortificationeffortsforFeandZninricehavefocusedonincreasingtheironstorageproteinfer-ritin(Gotoetal.
1999;Vasconcelosetal.
2003)orrootferricreductaseactivity(Vasconcelosetal.
2004).
Thetransgenicplantsshowedoveraccumulationofminerals*Correspondence:jpfett@cbiot.
ufrgs.
br1CentrodeBiotecnologia,UniversidadeFederaldoRioGrandedoSul,91501-970,PortoAlegre,RS,Brazil2DepartamentodeBotnica,UniversidadeFederaldoRioGrandedoSul,91501-970,PortoAlegre,RS,BrazilFulllistofauthorinformationisavailableattheendofthearticle2012Sperottoetal.
;licenseeSpringer.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Sperottoetal.
Rice2012,5:27http://www.
thericejournal.
com/content/5/1/27inleaves,butonlyasmallincreaseinseeds.
GrainZnandFeconcentrationswereincreasedinbarley(Hordeumvulgare)expressingtheZntransporterZIP1fromArabidopsisthaliana(Rameshetal.
2004),andweredecreasedinwheat(Triticumaestivum)expressingRNAiconstructsthatloweredNAMfamilygeneexpres-sion(Uauyetal.
2006).
Vasconcelosetal.
(2006)consti-tutivelyexpressedtherootferricreductaseFRO2fromA.
thalianainsoybeanandobservedthat,incertainhydroponicgrowthconditions,thetransgenicplantsshowedathreefoldincreaseinleafFeconcentration,butonlya10%increaseinseedFe.
SimilarresultswerefoundwiththebrzmutantofPisumsativum,whichoveraccumulatesFeinleavesbuthasnormalseedFeconcentrations(Grusak1994).
Analysisofmineralover-accumulationmutantsindicatesthattranslocationofmineralstoseedsistightlyregulated,andthatsimplyin-creasingnetmineraluptakeintotheplantwillprobablynotresultinseedswithhighermineralconcentrations.
Additionalregulatorymechanismsortransportcapabil-itiesmustbemanipulatedtoimproveremobilizationandpassthroughofmineralsfromvegetativeorgansintoseeds.
Recently,over-expressionofbarleygenesrelatedtophytosiderophoresynthesisresultedinenhancedFeandZnconcentrationsinriceunpolishedandpolishedseeds(Masudaetal.
2008;Masudaetal.
2009).
Enhancedexpressionofthethreegenesfromthericenicotianaminesynthasefamily(OsNASgenes)alsofacilitatedincreasesinFeandZnconcentrationsofricegrains(Leeetal.
2009;Zhengetal.
2010;Johnsonetal.
2011;Leeetal.
2011).
Alloftheseresultssuggestthatthemostsuccess-fulbreedingortransgenicapproacheswilllikelyneedtotargetmultiplegenessimultaneously(WatersandGrusak2008,Sperottoetal.
2012).
Itisacceptedthatmineralsmayberemobilizedfromvegetativesources(HockingandPate1977;HimelblauandAmasino2001),althoughamajorportionofminer-alsinseedsarelikelysuppliedthroughcontinuousup-takeandtranslocationduringreproductivegrowthtodevelopingseeds.
Inwheat,Zn(Hocking1994)andFe(GarnettandGraham2005)remobilizationfromleaveswasobserved.
AccordingtoJiangetal.
(2007b),when65Znisappliedtoriceleaves(eithertheflagleaforthelowestsenescentleaf),45-50%istransportedoutofthetreatedleaf.
FromthatZn,morethan90%istranslo-catedtoothervegetativeorgans;littleispartitionedtothepaniclepartsandevenlesstothegrains.
Theseresultssuggestthat,inriceplantsgrownundersufficientorsurplusZnsupply,mostoftheZnaccumulatedinthegrainoriginatesfromuptakebyrootsafterfloweringandnotfromZnremobilizationfromleaves.
Inanotherstudy,Znwasappliedtoriceplantsinvariousamountsandatvariousstagesofplantdevelopment.
AnalysisofthetotalplantZnandZncontentinindividualorgansatfloweringandatmaturityindicatedthatthepresenceofZningrainscouldbefullyattributedtoadditionalZnuptakeafterflowering,exceptwhenapplicationrateswereverylow(Jiangetal.
2008).
Underthelattercondi-tions,roots,stemsandsheathscontributedmostlytoZnallocation.
NoneofthericeZn-applicationtreatmentsshowedthatthemainportionofZnloadedingrainwasremobilizedfromleaves(Jiangetal.
2008;Stomphetal.
2009).
Contrastingly,Wuetal.
(2010)showedthatlargeamountsoftheZndepositedinthericegrainsatmatur-ityhadbeenretranslocatedfromotherplantpartsandnotbeentransporteddirectlyafteruptaketothegrainsinthegrain-fillingstage.
Recently,Yoneyamaetal.
(2010)reportedthatZninthericegrainsandpartlyinthehusksmaybeactivelysuppliedviathephloemaftermobilizationfromthebladesoftheflagandupperleavesandalsobyxylem-to-phloemtransferinthenodes.
Festoredintheflagandupperleavesmaybetransportedtothegrainsviathephloem.
Fangetal.
(2008)showedthatfoliarapplicationofZnandSecaninfluencetheZn,Se,andFecontentofricegrains;however,itwasdifficulttoimprovetheFenutritionofricegrainbyFespray,prob-ablyduetoitslimitedmobilityinthephloem.
WatersandGrusak(2008)suggestedthat,inArabidopsis,continuousuptakeandtranslocationofmineralstosourcetissuesdur-ingseedfillareasimportant,ifnotmoreimportant,thanremobilizationofpreviouslystoredminerals.
Uncoveringthegeneticarchitectureunderlyingmin-eralionhomeostasisinplantsisacriticalfirststepto-wardsunderstandingthebiochemicalandphysiologicalprocessesthatregulateaplant'selementalcomposition.
Inthispaper,weassessgrowthdynamicsofthewholeplant(panicles,non-flagleaves,flagleaves,stems/sheathsandroots)overthereproductivedevelopmentofOryzasativa.
Wealsodescribetheconcentrationsandcontentsoftenmineralnutrients(Fe,Zn,Cu,Mn,Mo,Ni,Ca,Mg,KandS)intheseorgansovertime.
Additionally,westud-iedthenetlossofmineralcontentstoinvestigatethepo-tentialofvegetativetissuesassourcesofstoredmineralstoberemobilizedtopanicles.
ToaddressthequestionofwhetherdifferentFesuppliescouldinfluencemineralpar-titioningandmovementofmineralstopanicles,wecom-paredtheseparametersinplantstreatedwithlow,normalandhighFeconcentrations.
ResultsTotestifdifferentFesupplieswouldinfluencemineralaccumulationinriceorgans,wecultivatedKitaakeplantswith5,20or200μMofFe(III)-HEDTAfromthepanicleexertion(PE)untilthefullmaturity(FM)stage.
DifferentFesuppliescausedslightgrowthchangesinpaniclescol-lectedatthegrainfilling(GF)stage,whilenochangeswereseenatthefullmaturitystage(Figure1).
HighFeconcentration(200μM)causedadecreaseofabout50%Sperottoetal.
Rice2012,5:27Page2of11http://www.
thericejournal.
com/content/5/1/27inpanicledryweightcomparedtothecontrolconcen-tration(20μM).
However,noimpactondryweightwasseeninotherorgans,possiblybecausethehighFetreatmentusedinthisworkdidnotleadtoenoughFeaccumulationtoreachtoxiclevels(300μgg-1DW–Sahrawat2004)inallaerialplantorgans(Additionalfile1).
Apanicleweightre-ductionofabout22%(notstatisticallysignificant)wasseenwhenplantswerecultivatedwithlimitedFesupply(5μM),relativeto20μMFecontrols.
Therefore,thedifferentFesuppliesusedinthisworkduringreproductivedevelopmentdidnotresultindifferentdryweightinmostorgans.
Changesinmineralcontents(Additionalfile2)weremainlydependentonchangesinmineralconcentrations.
MostofthechangesinmineralconcentrationsresultingfromdifferentFesupplieswerefoundduringfullmaturitystage.
Panicle(alongwithflagleaf)wastheorganwiththeleastvariationinmineralconcentrationscausedbydiffer-entFesupplies(Additionalfile1,Additionalfile3andAdditionalfile4).
AtGFstage,onlyFe(Additionalfile1a),Ni(Additionalfile3k)andMg(Additionalfile4f)concen-trationsshoweddifferencesduetotheFesupply.
However,thedecreaseinpanicledryweightdetectedinplantsculti-vatedwithhighFeconcentrationduringGFstage(<50%;Figure1)resultedindecreasedcontentinmostofthemineralsanalyzed,exceptFe,MoandK(Additionalfile2).
AtFMstage,noneofthemineralsanalyzedshoweddiffer-enceinthecontentbetweencontrol(20μM)andhighFe(200μM)condition(Additionalfile2).
Innon-flagleaves,noobviousmineralconcentrationdy-namiccouldbedetected.
HighFesupplywasresponsibleforhigherFeconcentration(Additionalfile1b)andforlowerCu(Additionalfile1L),Mo(Additionalfile3g),Ca(Additionalfile4b)andS(Additionalfile4q)concentrations.
LowFesupplywasresponsibleforhigherMn(Additionalfile3b)andNi(Additionalfile3L)concentrationsduringGFandFM;Ca(Additionalfile4b)andMg(Additionalfile4g)duringFMandK(Additionalfile4L)duringGFstage.
Inflagleaves,mostofthemineralconcentrations(includingFeandZn)werenotaffectedbydifferentFesupplies.
DuringGFstage,onlyMo(Additionalfile3h),Mg(Additionalfile4h)andS(Additionalfile4r)concen-trationswerereducedbylowand/orhighFeconcentra-tions.
DuringFMstage,alongwithMo,MgandS,theconcentrationsofCu(Additionalfile1m),Mn(Additionalfile3c)andCa(Additionalfile4c)alsopresentedvaria-tionsaccordingtotheFesupply.
Instems/sheaths,mostofthemodificationsinmineralconcentrationsfoundinplantscollectedattheGFstagewereunderhighFesupply.
OnlyFe(Additionalfile1d),Cu(Additionalfile1n)andNi(Additionalfile3n)main-tainedsimilarconcentrationsindependentoftheFesupply.
DuringFMstage,Fewastheonlymineralwhoseconcen-trationincreasedat200μMFe(Additionalfile1d).
Alltheothermineralsreachedhighermineralconcentrationsat5μMFe.
Inroots,allthemineralsanalyzed,exceptK(Additionalfile4o)andS(Additionalfile4t),presenteddifferentconcentrationswhenplantswerecultivatedwithdifferentFesupplies.
Asexpected,rootFeconcentrationwashigherunder200μMFe(2.
2XinGFand4.
2XinFM;Additionalfile1e).
TheoppositewasfoundforZn(Additionalfile1j),Cu(Additionalfile1o),Mo(Additionalfile3j)andNi(Additionalfile3o),withhigherconcentrationsunderlowerFesupply.
K(Additionalfile4o)andS(Additionalfile4t)concentrationswerenotaffectedbydifferentFesupplies.
Pearson'scorrelationanalysiswasperformedinordertofindrelationshipsamongthetenmineralconcentrationsFigure1Dryweightofriceorgans.
Dryweightofpanicles,non-flagleaves,flagleaves,stems/sheathsandrootscollectedduringgrainfilling(GF)andfullmaturity(FM)stagesofriceplantssuppliedwith5,20or200μMofFe(III)-HEDTAfrompanicleexertion(PE)onwards.
Valuesaretheaveragesofatleastthreesamples±SE.
Differentlettersindicatethatthemeans(betweendifferentFesupplies)aredifferentbytheTukeyHSDtest(P≤0.
05).
Errorbarsmaybetoosmalltobevisibleinthefigure.
Sperottoetal.
Rice2012,5:27Page3of11http://www.
thericejournal.
com/content/5/1/27inresponsetothedifferentFesupplies.
AsseeninFigure2,mostofthecorrelationswerefoundduringfullmaturity(FM)stage,afteralongerperiodunderdifferentFesup-plies.
Negativecorrelationswerefoundmainlyinroots.
Duringgrainfilling(GF)stage,theonlypairofmineralspositivelycorrelatedineverytestedorganwasMn-Ca.
ThesewerealsopositivelycorrelatedduringFMstageinalltestedorgans,alongwithZn-Cu.
PositivecorrelationsatFMbetweenthepairsZn-NiandCu-Niwerefoundinfourofthefivetestedorgans,exceptinflagleaves.
SimilardynamicwasfoundforMo-SandCa-Mg,whichwerenotcorrelatedonlyinpanicles.
Cu-MnandCu-Mgwerealsopositivelycorrelatedinfourofthefivetestedorgans,ex-ceptinroots,showinganegativecorrelationinthisorgan(Figure2).
Differencesinplantsizewereresponsibleforthehighstandarderrorinmineralcontents(Additionalfile2).
Therefore,partitionquotient(PQ)valueswerecalculatedtoallowcomparisonofthedynamicsofpartitioningofmineralsbetweenthereproductivedevelopmentofriceplantscultivatedwithdifferentFesupplies(Figure3),sincethisapproachisabletocheckifonemineralcontentishigherinonespecificorgan,regardlessofdifferencesinplantsize.
Ingeneral,non-flagleaves,flagleavesandrootsweretheorganswhichshowedthehighermineralPQvariationaccordingtoFesupply,whereasstems/sheathswastheorganwithlowervariation(Figure3).
ForFe,PQvaluesabove100werefoundonlyinroots(Figure3a),withhighervaluesinplantscultivatedwith200μMFe.
Inalltheotherorgans,highFesupplywasresponsibleforthelowestFePQvalue,especiallyduringFM(Additionalfile5).
Also,inthisstage,controlFesupply(20μMFe)resultedinhigherPQvalues(Additionalfile5).
ForZn,lowFesupplyresultedinthelowestZnPQvaluesinpani-cles,non-flagleavesandflagleaves,andhighFesupplyresultedinthehighestZnPQvalues(Figure3b).
Instems/sheaths(duringFM)androots(duringbothstages),theoppositewasfound,withhigherZnPQvaluesunderlowFesupply(Figure3b).
SimilarpatternwasfoundforCu(Figure3c)andNi(Figure3f)PQinroots.
ForMn,noobviouspatterncouldbeidentified,exceptthehigherPQvaluesinnon-flagandflagleavesunderFedeprivationandinpaniclesandrootsunderFeexcess,bothduringFM(Figure3d).
Mo(Figure3e)andS(Figure3j)werethemineralswiththemoststablePQvalues.
ForCa(Figure3g)andMg(Figure3h)highPQvalueswereobtainedinnon-flagandflagleavesduringFMstageunderFestarvation.
LowFesupplyalsoresultedinex-tremelyreducedCaPQvaluesinroots(Figure3g),inrela-tiontovaluesseenintheothertreatments.
Toinvestigatethepotentialofvegetativetissuesasasourceofstoredmineralstoberemobilizedtopanicles,weestimatedthenetlossofmineralcontentfromnon-flagleaves,flagleavesandstems/sheaths,bysubtractingfinalmineralcontentfromthepriortimepointwhichhadthehighestmineralcontent.
AsseeninFigure4,netlossofmineralcontentcouldbediscernedunderdifferentFesup-plies.
UndercontrolFeconcentration(20μM),allthemineralsshowedcontentlossfromGFtoFMstage(Figure4b),mostlyfromflagleavesbutalsofromstems/sheaths.
However,astheflagleaves'mineralcontentismuchlowerthanthestems/sheaths'content,themaximumpossiblecontributiontopaniclemineralcontent,assumingthatthetotalnetlossofeachmineralwastranslocatedtopaniclesbeforethefinalcollectionpoint,isingeneralhigherfromstems/sheathsthanfromflagleaves.
DifferentFigure2Mineralcorrelationanalyses.
Pearson'scorrelationanalysisoftenmineralconcentrationsinpanicles(aandf),non-flagleaves(bandg),flagleaves(candh),stems/sheaths(dandi)androots(eandj)ofriceplantscultivatedwith5,20and200μMofFe(III)-HEDTAduringgrainfilling(GF)andfullmaturity(FM)stages.
Solidlinesrepresentasignificantpositivecorrelationanddashedlinesrepresentasignificantnegativecorrelation.
Thinnerlinesindicatesignificanceatthe0.
05levelandthickerlinesindicatesignificanceatthe0.
01level.
Sperottoetal.
Rice2012,5:27Page4of11http://www.
thericejournal.
com/content/5/1/27Fesuppliesinfluencedthecontentlossofmostoftheminerals.
UnderlimitedFecondition(5μM),onlyFe,MnandMgshowedanetremobilizationfromstems/sheaths;andKfromflagleaves.
UnderhighFecondition(200μM),onlyKandSshowedanetremobilizationfromflagleaves;andNifromstems/sheaths.
Surprisingly,noremobilizationwasfoundfromnon-flagleaves,exceptaminimumKremobilizationunder20μMFe,whichcouldhavecontrib-utedatmostonlywith5%oftotalpanicleKcontent(Figure4e).
Formostofthemineralsanalyzed,exceptNiandCa,themaximumpossiblecontributionofremobiliza-tionfromflagleavesandstems/sheathstothefinalpanicle'smineralcontentislessthan40%,beinglessthan20%forFe,Zn,Cu,MnandS.
NiandCaremobilizationfromflagleavesandmostlyfromstems/sheathscouldberesponsibleformorethan100%ofthefinalpaniclecontent(Figure4e),showingthatremobilizationdoesnotnecessarilycorres-pondtomineraltranslocationtothepanicles.
Figure3Partitionquotients(PQ)analyses.
Partitionquotientsofmineralelementsinpanicles,non-flagleaves,flagleaves,stems/sheathsandrootsduringgrainfilling(GF)andfullmaturity(FM)stagesofriceplantssuppliedwith5,20or200μMofFe(III)-HEDTAfrompanicleexertiononwards.
DashedhorizontallinerepresentsPQof100(thepercentagecontributionoftheorgantotheplant'sdryweightisthesameasthepercentagecontributiontotheplant'stotalcontentofthemineralbeingevaluated).
Sperottoetal.
Rice2012,5:27Page5of11http://www.
thericejournal.
com/content/5/1/27TodeterminewhetherdifferentFesuppliescouldresultindifferentmineralexporttodevelopingseeds,wesubmit-tedriceplantstothesameFeconditionsasthepreviousexperiment(although0μMFewasincludedinthisana-lysis).
Ingeneral,thedynamicsofmineralconcentrationsofseeds(Additionalfile6)weresimilartothoseofpanicles;thatis,higherFeandlowerZnandMoconcentrationsunder200μMFe.
Fe,Cu,Mn,MgandSshowedthelow-estandZn,MoandNishowedthehighestconcentrationsunderFedeprivation(Additionalfile6).
DiscussionPlantsarecommonlyfacedwithfluctuationsinnutrientsupplytotheroots.
Tocopewiththesefluctuations,plantsinvokearangeofmechanisms,whichincludedif-ferentuptakerates,changesinrootmorphologyandphysiologyorstorageandremobilizationofmineralnutrients(Marschner1995).
Inthisstudy,wecharacter-izedorgan-specificchangesindrymatterandmineral(Fe,Zn,Cu,Mn,Mo,Ni,Ca,Mg,KandS)contenttomonitorthenetflowofmineralsintoandthroughthericeplantoverthereproductivedevelopmentandunderdifferentFesupplies.
Anyincreaseinmineralcontentinoneorganmusthaveresultedfromuptakeandtrans-locationfromthesoil,orfromremobilizationfromoneorgantoanother.
Byharvestingalltheorgansandtrack-ingthemineralpartitioningintheseorgansovertime,remobilizationcanbeestimatedforeachmineral.
InthisworkweusedthesameremobilizationdefinitionusedbyWatersandGrusak(2008),whichisdefinedasthenetlossofstoredorrecycledmineralcontentfromoneorganovertime,withtheminerallossrepresentingmovementintoanotherorgan.
EffectsoflowandhighFesuppliesonmineraldynamicsAsshowninAdditionalfile2andFigure3,lowFesupply(5μM)allowshigheraccumulationofZn,CuandNiinroots;Mn,Ca,MgandKinleavesandZninstems/sheaths.
Ontheotherhand,lowFesupplyallowsasmalleraccumulationofFe,MnandCainrootsandZnandNiinleaves.
EnhancementofZnuptakeunderlowFesupply,ei-therasadivalentcationorcomplexedtophytosidero-phores(Zhangetal.
1998),couldexplainitshigheraccumulationinroots.
SeveralFetransportersareabletotransportalsoZnandMn(Korshunovaetal.
1999;Eckhardtetal.
2001;Grossetal.
2003;López-Millánetal.
2004).
InductionofFetransportersunderlowFeconcen-trationcouldresultinincreasedZnuptakeandtransloca-tiontostems/sheathsandincreasedMnuptakeandsubsequenttranslocationtoleaves,sinceMnmoveseasilyfromroottoshootinthexylem-saptranspirationstream(RamaniandKannan1987).
Possibly,uptakeofMnandCumayincreaseinriceplantsunderlowFesupply,be-causethepresenceofphytosiderophoresintherhizospheremayincreasetheavailabilityoftheseionsbothintherhizo-sphereitselfandintheapoplast(Zhangetal.
1991).
ThecomplexCu-phytosiderophorecanbetransportedintorootcellsbyFe-phytosiderophoretransporters,althoughwithloweraffinity(Briatetal.
1995).
Fertilizingacalcar-eoussoilwithanFe-deficientsolutionincreasedCuFigure4Mineralremobilizationanalyses.
Percentagedecreaseinnon-flagleaves,flagleavesandstems/sheathsmineralcontentsfromgrainfillingtofullmaturitystages(a)andpotentialcontributionofremobilizedmineraltopaniclemineralcontentsatthefinaltimepoint(b).
Sperottoetal.
Rice2012,5:27Page6of11http://www.
thericejournal.
com/content/5/1/27accumulationbyrootsandshootsintwowheatcultivars(Chaignonetal.
2002).
IthasbeensuggestedthatCu,ZnandNishareacommonuptakesystem(Kochian1991),whichcouldexplainthehigheraccumulationofNiinrootsunderlowFesupply.
Infact,Eckhardtetal.
(2001)pro-posedthattheFetransportersIRT1andIRT2fromtomatocouldalsotransportNi2+.
Differentlyfromourresults,Silveiraetal.
(2007)detectedincreasedCaandKconcen-trationsunderFe-deficiencyinrootsofrice,insteadofinleaves.
PossibleexplanationsforsuchdifferenceinCady-namicunderlowFesupplyarethatwehaveusedadifferentricecultivar,acompletelydifferenttechniqueformineralde-tection(ICP-OESversusPIXE),differentlevelsofFesupply(5μMFeversusFedeprivation)andweanalyzedriceorgansthroughthecourseofreproductivedevelopmentinsteadofduringvegetativedevelopment.
Inrice,Fetoxicityseemstooccurabove500μgFeg-1leafdryweight(Audebert2006)andleadstoadrasticreductionofrootgrowth(BeckerandAsch2005).
AsshowninFigure1andAdditionalfile1,ourplantsculti-vatedwithhighFesupplydidnotpresentreductionofrootgrowthanddidnotreachtoxiclevels.
However,thetreatmentresultedindecreaseddryweightofpaniclescollectedduringgrainfilling(Figure1).
Infact,depend-ingonthesiteandthecultivarsused,reportedcriticalconcentrationscanrangefrom20to2,500μgg–1,indi-catingthatfactorsotherthanFeconcentrationinfluencetheoccurrenceofFetoxicitysymptoms(BeckerandAsch2005).
Inthisway,thefluctuationsinmineralaccumulationsfoundinthepresentworkdonotreflectthechangespromotedbyFetoxicity.
HighFesupply(200μM)promotedahigheraccumulationofFeinrootsandZninleaves.
Ontheotherhand,itpromotedasmalleraccumulationofFeinleavesandstems/sheathsandZn,CuandKinroots(Additionalfile2,Figure3andAdditionalfile5).
Asexpected,highFesupplyresultedinhigherFeaccumulationinroots.
However,rootFemeasurementscouldbeover-estimated.
Theoxi-dationabilityofthericerootprecipitatesinactiveFeattherootapoplastandroot-environmentinterface.
ThesamplingtechniqueuseddidnotseparatetheexistingoxidizedFeattherootsurface.
Thus,therootFecon-tentmeasuredcomprisedtherootFeuptakeandtheoxidizedFeattherootsurface.
MostofthisFeseemstonotbetranslocatedtoshoots,sincesmallerFePQvaluesweredetectedinleavesandstems/sheaths(Figure3),evenwiththeincreasedFecontentinnon-flagleaves(Additionalfile2).
UnderFe-excess(500mgL-1),Silveiraetal.
(2007)reportedthatricerootsandalsoshootsac-cumulatehigherFeconcentration.
Inthatwork,higherconcentrationsofFeweredetectedinI409plants,moresusceptibletoFetoxicity.
Shootconcentrationsdetectedwereupto2.
5XhigherthaninE108plants(toleranttoFetoxicity)after10daysoftreatment.
Theauthorssuggested,therefore,thatE108plantsweremoreresist-anttoexcessFeduetothepossibleinductionofavoid-ancemechanisms,allowingtheplanttodecreaseFetranslocationtoshoots.
ThisavoidancemechanismwasrecentlyreviewedbySperottoetal.
(2010).
Inourwork,Kitaakeplantsseemtousetheavoidancemechanism,butadditionalstudiesareneededtoconfirmthehypoth-esisthatKitaakeisanFe-excess-tolerantricecultivar.
Inaccordancewithourresults,Silveiraetal.
(2007)alsodetectedhigherZnconcentrationinshootsofplantsunderFe-excess.
Alsoinaccordancetoourresults,smallerK(Neueetal.
1998;Ramirezetal.
2002;Mehrabanetal.
2008)andCu(Shaoetal.
2007;Silveiraetal.
2007)ab-sorptionratesunderFe-excesshavealreadybeenreportedinrice.
Itseemsthat,underFe-excess,therelativelyhighferrousironconcentrationinthesoilsolutionanduptakebytheplantmayresultinKdeficiency(Audebert2006).
Ontheotherhand,itappearsthatproperKsupplymayincreaseFeexclusionfromrootsandreducetranslocationofFetoaerialplantorgans,especiallytoupperleaves(Sahrawat2004).
Significantcorrelationswerefoundamongthetenmin-eralelementconcentrationsinriceorgansofplantsfacingdifferentFesupplies.
Inparticular,Mn-CaandZn-Cuwerepositivelycorrelatedinallanalyzedorgans.
Zn-Ni,Cu-Ni,Mo-S,Ca-Mg,Cu-MnandCu-Mgwerepositivelycorre-latedinfourofthefiveorgans(Figure2).
Mostoftheavail-abledataaboutmineralcorrelationinplantswereobtainedingrains.
Asfarasweknow,thisisthefirstworkwhichtriestoidentifysimilarpatternsofmineralfluctuationsindifferentplantorgansduetodifferentFesupplies.
Inthisway,significantpositivecorrelationsbetweenmineralssug-gestthatdifferentFesuppliespromotesimilarchangesintheirconcentrations.
Feconcentrationwaspositivelycorre-latedwithonlytwominerals(MnandMg)inroots(Figure2eandj).
ItisalreadyknownthatFetransporterscanalsotransportMn(Korshunovaetal.
1999;Eckhardtetal.
2001;Grossetal.
2003;López-Millánetal.
2004)andhighpositivecorrelationbetweenFeandMnwasfoundinricegrains,alongwithMn-Ca,Zn-Cu,Ca-Mg,andCu-Mn(Jiangetal.
2007a),inaccordancewithourfindings.
How-ever,noFe-Mncorrelationswerefoundinotherriceorgans.
OnepossibleexplanationisthatMnmoveseasilyfromtheroottotheshootinthexylem-saptranspirationalstream(RamaniandKannan1987),andthemobilityofFeismoreregulated(Marschner1995).
Thephysiologicalsig-nificanceofthecorrelationbetweenFe-Mgisunknown,butJiangetal.
(2007a)havealreadydetectedsuchrelationinmilledrice.
Theseauthorssuggestthatthehighnumberofpositivecorrelationsinriceisprobablyduetotheinter-actionbetweenionswhosechemicalpropertiesaresuffi-cientlysimilar,andsuchsimilarityallowsthecompetitionforsiteofabsorption,transportandfunctioninplanttis-sues.
OneexamplewasshownbyKupperandKupperSperottoetal.
Rice2012,5:27Page7of11http://www.
thericejournal.
com/content/5/1/27(1998),whichreportedthattheheavymetals(Hg,Cu,Cd,Ni,andPb)mightsubstituteMg,thecentralatomofchlorophyll.
Thiscouldhelptoexplainthepositivecorrela-tionswefoundbetweenCuandMginalltheorgans,ex-ceptinroots(Figure2).
Zengetal.
(2005)alsofoundthatMnispositivelyassociatedwithCainricegrainsandMajumderetal.
(1990)reportedthatuptakeofMnandCaarepositivelycorrelatedinricecultivatedinP-deficientsoil,alongwithuptakeofCaandMg.
Variousmoleculesareknowntobindmineralsinriceorgansandcouldexplainsomeofthecorrelationsdetected,suchasCa-Kinpanicles,becausebothmineralsbindtophyticacidinthealeuronelayerofricegrain(Linetal.
2005).
Similarly,manyofthemicronutrientcorrelationsfoundinvariousorganscouldbeattributedtonicotiana-mineordeoxymugineicacid,astheseareknownchelatorsofseveralmetalcationsinplanttissues(Curieetal.
2009).
However,furtherstudiesareneededtoconfirmthepossi-bilityofthesamechelatingmoleculesbeinginvolvedinthemineralcorrelationsdetected.
MineralremobilizationversuscontinuedsupplyfromrootsThedynamicsofmineralconcentrationsinseedsofplantscultivatedwithdifferentFesuppliesshowedthathigherFesupplyresultsinhigherFeconcentrationintheseeds(Additionalfile6),implyingthattheuptakeandtransportsystemswereoperatingbelowcapacitywhenplantsweregrownwithlowerFeconcentrations.
AspreviouslyshownbyFangetal.
(2008),itisdifficulttoimprovetheFenutritionofricegrain,evenbyFespray,possiblyduetoitslimitedmobilityinthephloem.
Probably,enhancedrootuptakecombinedwithleaf/stem/sheatheffluxtransportcanbeaneffectivewaytoincreasetheFeconcentrationinricegrains.
Remobilizationofreservestosupplyriceseedswithmineralshasbeenemphasizedinpreviousstudies(Jiangetal.
2007b;Fangetal.
2008;Jiangetal.
2008;Wuetal.
2010;Yoneyamaetal.
2010),butthecontributionofstoredmineralstototalseedmineralcontentisunclear.
AccordingtoWatersandGrusak(2008),itisexpectedthataminimalamountofeachmineralisincorporatedintostructuralorproteinmoleculesandthusunavailableformobilization,andthatsourcetissueswouldhavetoaccumulatemineralsinexcessofthisminimalamounttoallowmobilizationtogrowingtissuessuchasseeds.
ThiswasthecaseforSinsoy-beanleaves(SunarpiandAnderson1996),whereasolubleSpoolwasavailableforremobilizationandaninsolubleSpoolcouldnotbemobilized.
ThesizeofthesolublepoolwasdependentonSnutrition.
Inthepresentstudy,plantsferti-lizedwithahighFeconcentration(200μM)showedonlyKandSremobilizationfromflagleavesandNifromstems/sheaths(Figure4).
Inthiscase,vegetativetissuesshouldhavebeenabletostoreFequantitiesabovethestructuralminimum,whichwouldhaveprovidedexcessFeforremo-bilization.
However,withtheabundantFesupplyattherootlevel,continueduptakeduringseedfillmayhavereducedorprecludedtheneedforremobilizationtoserveasasourceofFeforseeds.
Ontheotherhand,plantsfertilizedwithlowFeconcentration(5μM)showedthehighestnetFeremobi-lization(Figure4),probablyduetoreduceduptakeduringseedfill.
UnderlimitedFesupply,stems/sheathsarethemajorFesourceforremobilization.
AsimilarpatternwasobservedinotherstudieswhenZnsupplywasverylow.
Underthiscondition,stemsandsheathscontributedmosttoZnallocation(Jiangetal.
2008).
Allthemineralsanalyzedweresubjecttoremobilization,mostlyfromflagleaves,butalsofromstems/sheaths(Figure4),whenplantswereferti-lizedwiththecontrolFeconcentration(20μM).
Theseresultssuggestthatmineralremobilizationfromvegetativetissuescanoccurinriceplants;however,forseedstoacquiremineralsthisremobilizationisnotabsolutelyrequired.
RemobilizationresultsfoundforArabidopsisplantsbyWatersandGrusak(2008)werenotexactlyconsistentbe-tweenexperiments;differenceswerefoundinthemineralsthatwereremobilizedandtheamountsremobilized.
Crafts-Brandner(1992)alsofoundinconsistentresultsinleavesofsoybean,whichremobilizedPinoneexperiment,whileinasecondexperimentnoremobilizationoccurred,yetseedsofbothexperimentshadcomparableseedPconcentrations.
Asshownbyourresults,mineralremobilizationfromvege-tativetissuesinriceismodifiedbyplantFenutrition,be-causedifferentFesuppliesaltertheremobilizationofseveralminerals.
Also,amajorproportionofmineralcontentinpaniclesprobablycomesfromnon-storagesources,thatis,continuedrootuptakeandtranslocationduringtheseedfillperiod.
Wedidobservemineralremobilizationmostlyfromflagleavesbutalsofromstems/sheathswith20μMFe,andatleastaportionofthesemineralswaslikelyincorporatedintopaniclesandseeds.
Intheunlikelyeventthat100%ofthecontentofeachminerallostfromvegetativetissueswenttopanicles,thiscouldaccountfor,atmost,20%ofpanicleFe,Zn,Cu,Mn,Mg,KandS.
However,atleastforZn,itisalreadyknownthatmostofthemineraltransportedoutoftheleaves(eithertheflagleaforthelowestsenescentleaf)istranslocatedtoothervegetativeorgansinsteadofbeingpar-titionedtothepanicles,andevenlesstothegrains(Jiangetal.
2007b).
Wecanalsodiscardthepossibilityof100%NiorCaremobilizationtopanicles,sinceitwouldberespon-sibleformorethan100%ofthefinalpaniclecontent(Figure4).
However,ifremobilized,asignificantpartofthisNipoolwouldbeexpectedtobetransportedtotheseeds,sinceitisalreadyknownthatNirapidlyre-translocatesfromleavestoyoungtissuesinthephloem,particularlyduringre-productivegrowth.
Indeed,upto70%ofNiintheshootswastransportedtotheseedsofsoybean(Tiffin1971).
InArabidopsis,continuousuptakeandtranslocationofminer-alstosourcetissuesduringseedfillareasimportant,ifnotSperottoetal.
Rice2012,5:27Page8of11http://www.
thericejournal.
com/content/5/1/27moreimportant,thanremobilizationofpreviouslystoredminerals(WatersandGrusak2008).
Asshownbyourresults,theflagleafistheorganwithhighestpercentageofmineralcontentloss.
However,consideringthatflagleaveshaveextremelylowmineralcontent,themaximumpossiblecontributiontopaniclemineralcontentwouldbefromstems/sheaths,ratherthanfromflagleaves.
ConclusionInsummary,thisstudysuggeststhatmineralaccumula-tionthroughoutthereproductivedevelopmentofriceorganscanbeaffectedbydifferentFesupplies.
Withre-specttothetranslocationofmineralstopanicles,remo-bilizationfromvegetativeorganscanoccurinriceplants(andnon-optimalFesuppliescanaffectmineralremobi-lization),butapparently,forseedstoacquireminerals,vegetativeremobilizationisnotabsolutelyrequired.
Inthisway,continueduptakeandtranslocationofmineralsduringseedfillareprobablymoreimportantthanremo-bilizationofpreviouslystoredshootminerals.
Thus,inadditiontotargetingsourcetissuesforincreasedmineralremobilization,researchersshouldalsotargetrootup-takeandleaf/stem/sheatheffluxtransporterstoincreasemineralaccumulationinthepaniclesandconsequentlyinthericeseeds.
MethodsPlantmaterialsandgrowthconditionsRice(OryzasativaL.
)seedsfromthefast-growingcultivarKitaakeweregerminatedinPetridisheswithfilterpaperfor8dbeforebeingtransferredtohydroponicsolution.
Plantsweregrowninacontrolledenvironmentchamberwith16-h,20°Cdayand8-h,15°CnightattheUSDA-ARSChildren'sNutritionResearchCenter,Houston,TX.
Rela-tivehumiditywasmaintainedat50%andphotonfluxdensityduringthedaywas350μmolm–2s–1,suppliedbyamixtureofincandescentbulbsandfluorescentlamps.
Thestandardsolutionforhydroponicallygrownplantscontained1mMCa(NO3)2,3mMKNO3,0.
5mMMgSO4,0.
75mMK2SO4,0.
5mMKH2PO4,25μMCaCl2,25μMMnSO4,0.
5μMZnSO4,0.
5μMCuSO4,0.
5μMH2MoO4,0.
1μMNiSO4,0.
1mMK2SiO3,and20μMFe(III)-HEDTA(N-hydroxyethyl-ethylenediaminetriaceticacid).
Allnutri-entswerebufferedwith2mMMES(2,4-morpholino-ethanesulfonicacid),pH5.
5andgrowthsolutionswerereplacedevery3days.
FortreatmentsinvolvingdifferentFeconcentrations,Fe(III)-HEDTAconcentrationwasadjustedto5,20or200μM.
Riceorgans(panicles,non-flagleaves,flagleaves,stems/sheathsandroots)werecollectedatpan-icleexertion,grainfillingandfullmaturity(R3,R5andR9stages,respectively,accordingtoCounceetal.
2000).
De-huskedunpolishedseedswereanalyzedatfullmaturity.
ElementalanalysisbyICPAlltissueswereharvestedanddriedina60°Covenfor48h.
Driedtissueswerepredigestedovernightinboro-silicateglasstubeswith4mlofredistilled98.
8%HNO3.
OnemilliliterofconcentratedtracemetalgradeHClO4wasaddedtothepredigestedtissuesandheatedat100°Cfor1h,150°Cfor1h,180°Cfor1handthenat210°Ctodryness(1–2h).
Digestionswereperformedusingaheat-ingblock(Model1016,Tecator,Hoganas,Sweden)withanexhaust-collectingmanifold.
Digestswereresuspendedin15mlofredistilled2%HNO3.
ConcentrationsofFe,Zn,Cu,Mn,Mo,Ni,Ca,Mg,KandSweredeterminedbyinductivelycoupledplasma-opticalemissionspectroscopy(CIROSICPModelFCE12;Spectro,Kleve,Germany).
Tomatoleavesandriceflourstandards(SRM1573Aand1568A,respectively;NationalInstituteofStandardsandTechnology,Gaithersburg,MD)weredigestedandanalyzedalongwiththericesamplestoensureaccur-acyoftheinstrumentcalibration(Narayananetal.
2007).
Mineralcontentwasdeterminedbymultiplyingeachsample'sconcentrationbydryweight.
Foresti-mationofnetmineralcontentlossfromnon-flagleaves,flagleavesandstems/sheaths,thefinalmineralcontentofeachorganwassubtractedfromthepriortimepointwhichhadthehighestmineralcontent.
Formineralsthathadadecreaseincontent,thenetlosswascomparedwithfinaltotalpaniclemineralcontenttodeterminethecontributionofremobilizedmineralstopaniclemineralcontent.
PartitionquotientcalculationToevaluatethepartitioningofmineralswithinariceplantduringitsreproductivedevelopmentandunderdifferentFesupplies,changesineachtissue'scontentwerenormalizedtochangesineachtissue'sweight,rela-tivetothewholeplant.
TheDWofeachorganwascal-culatedasapercentageoftotalplantweightateachtimepoint(orateachFesupply),andmineralcontentofeachorganwascalculatedasapercentageoftotalplantmineralcontentateachtimepoint(orateachFesup-ply).
Usingthesevalues,thenormalizedpartitioningofeachmineralwithintheplantwascalculatedbydividingeachorgan'spercentagemineralcontentbyitspercent-ageDW,andmultiplyingby100,whichwerefertoasthepartitionquotient(PQ),asdescribedbyWatersandGrusak(2008).
StatisticalanalysesWhenappropriate,dataweresubjectedtoanalysesofvariance(ANOVA)andmeanswerecomparedbytheTukeyHSD(HonestlySignificantDifferences)(P≤0.
05).
TheLevene'stest(forhomogeneityofvariance)wasusedpriortoANOVA.
DatawithunequalvariancesweresubjectedtoWelchanalysisandmeanswerecomparedSperottoetal.
Rice2012,5:27Page9of11http://www.
thericejournal.
com/content/5/1/27bytheDunnett-Ctest.
Pearson'scorrelationanalyseswerecarriedoutusingtwosignificancelevels(P≤0.
05and0.
01).
AllthestatisticalanalyzeswereperformedusingtheSPSSBase19.
0forWindows(SPSSInc.
,USA).
AdditionalfilesAdditionalfile1:Fe,ZnandCuconcentrations.
Fe,ZnandCuconcentrationsinpanicles,non-flagleaves,flagleaves,stems/sheathsandrootscollectedduringgrainfilling(GF)andfullmaturity(FM)stagesofriceplantscultivatedwith5,20or200μMofFe(III)-HEDTA.
Valuesaretheaveragesofatleastthreesamples±SE.
Differentlettersindicatethatthemeans(betweendifferentFesupplies)aredifferentbytheTukeyHSDtest(P≤0.
05).
Errorbarsmaybetoosmalltobevisibleinthefigure.
Additionalfile2:Mineralcontentsinriceorgans.
ContentsofFe,Zn,Cu,Mn,Mo,Ni,Ca,Mg,KandSinpanicles,non-flagleaves,flagleaves,stems/sheathsandrootscollectedfromriceplantssuppliedwithdifferentFeconcentrationsattworeproductivegrowthstages.
Additionalfile3:Mn,MoandNiconcentrations.
Mn,MoandNiconcentrationsinpanicles,non-flagleaves,flagleaves,stems/sheathsandrootscollectedduringgrainfilling(GF)andfullmaturity(FM)stagesofriceplantscultivatedwith5,20or200μMofFe(III)-HEDTA.
Valuesaretheaveragesofatleastthreesamples±SE.
Differentlettersindicatethatthemeans(betweendifferentFesupplies)aredifferentbytheTukeyHSDtest(P≤0.
05).
Errorbarsmaybetoosmalltobevisibleinthefigure.
Additionalfile4:Ca,Mg,KandSconcentrations.
Ca,Mg,KandSconcentrationsinpanicles,non-flagleaves,flagleaves,stems/sheathsandrootscollectedduringgrainfilling(GF)andfullmaturity(FM)stagesofriceplantscultivatedwith5,20or200μMofFe(III)-HEDTA.
Valuesaretheaveragesofatleastthreesamples±SE.
Differentlettersindicatethatthemeans(betweendifferentFesupplies)aredifferentbytheTukeyHSDtest(P≤0.
05).
Errorbarsmaybetoosmalltobevisibleinthefigure.
Additionalfile5:Ironpartitionquotient(PQ)inriceorgans.
Ironpartitionquotientinpanicles,non-flagleaves,flagleavesandstems/sheathsduringgrainfilling(GF)andfullmaturity(FM)stagesofriceplantscultivatedwith5,20or200μMofFe(III)-HEDTA.
Additionalfile6:Mineralconcentrationsinriceseeds.
Fe,Zn,Cu,Mn,Mo,Ni,Ca,Mg,KandSconcentrationsinde-huskedunpolishedseedscollectedduringfullmaturitystageofriceplantscultivatedwith0,5,20or200μMofFe(III)-HEDTA.
Valuesaretheaveragesofthreesamples±SE.
Differentlettersindicatethatthemeans(betweendifferentFesupplies)aredifferentbytheTukeyHSDtest(P≤0.
05).
Errorbarsmaybetoosmalltobevisibleinthefigure.
AbbreviationsFM:Fullmaturity;GF:Grainfilling;ICP-OES:Inductivelycoupledplasmaopticalemissionspectroscopy;PE:Panicleexertion;PQ:Partitionquotient.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsMAGandJPFconceivedandcoordinatedthestudy.
RAS,MWVandMAGdesignedtheexperiments.
RASandMWVobtainedtheICPdata.
RASperformedthestatisticalanalyzesanddraftedthemanuscript.
RAS,MAGandJPFpreparedthefinalmanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsThisresearchwassupportedbyHarvestPlus(Agreementnumber6005–05),ConselhoNacionaldeDesenvolvimentoCientíficoeTecnológico(CNPq)andCoordenaodeAperfeioamentodePessoaldeNívelSuperior(CAPES)toJPF,andbytheUSDA-ARSunderAgreementnumber58-6250-0-008toMAG.
ThecontentsofthispublicationdonotnecessarilyreflecttheviewsorpoliciesoftheUSDepartmentofAgriculture,nordoesmentionoftradenames,commercialproducts,ororganizationsimplyendorsementbytheUSGovernment.
Authordetails1CentrodeBiotecnologia,UniversidadeFederaldoRioGrandedoSul,91501-970,PortoAlegre,RS,Brazil.
2DepartamentodeBotnica,UniversidadeFederaldoRioGrandedoSul,91501-970,PortoAlegre,RS,Brazil.
3CentrodeCiênciasBiológicasedaSaúde,CentroUniversitárioUNIVATES,95900-000,Lajeado,RS,Brazil.
4CBQF/EscolaSuperiordeBiotecnologia,UniversidadeCatólicaPortuguesa,RuaDr.
AntónioBernardinodeAlmeida,4200-072,Porto,Portugal.
5USDA/ARSChildren'sNutritionResearchCenter,DepartmentofPediatrics,BaylorCollegeofMedicine,1100BatesStreet,Houston,TX77030,USA.
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