radial oxygen loss

Plant,CellandEnvironment(2014)37,2406–2420doi:10.1111/pce.12294

OriginalArticle

Biochemicalandmolecularcharacterizationofrice(OryzasativaL.)rootsformingabarriertoradialoxygenloss

KonstantinKulichikhin,TakakiYamauchi,KohtaroWatanabe&MikioNakazono

GraduateSchoolofBioagriculturalSciences,NagoyaUniversity,Furo-cho,Chikusa,Nagoya464-8601,Japan

ABSTRACT

Theformationofabarriertoradialoxygen(O2)loss(ROL)intherootisanimportantadaptationofplantstoroot?ood-ing,butthebiochemicalchangesinplantrootswherethebarrierisformedareunclear.Inthisstudy,weanalysedmeta-bolicpro?lesandgeneexpressionpro?lesinrootsofrice(OryzasativaL.)plantsgrownunderstagnantdeoxygenatedconditions,whichinducesuberizationintheoutercelllayersoftherootsandformationofbarriertoROL.Undertheseconditions,twodistinctivebiochemicalfeaturesoftherootsweretheaccumulationsofmalicacidandverylongchainfattyacids(VLCFAs).Wealsoshowedthattheexpressionsofsomegenesencodingplastid-localizedenzymes,whichconvertmalicacidtoacetylcoenzymeA(AcCoA),weresimultaneouslyup-regulatedunderstagnantconditions.Theexpressionlevelsofthesegenesinspeci?croottissuesiso-latedbylasermicrodissectionsuggestedthatmalicacidisconvertedtoAcCoApredominantlyintheplastidsintheoutercelllayersofriceroots.Weproposethatthephysio-logicalroleofmalicacidaccumulationinricerootsgrownunderstagnantconditionsistoprovideasubstrateforthebiosynthesisoffattyacids,which,inturn,areusedinthebiosynthesisofsuberin.

Key-words:lasermicrodissection;malicacid;metabolomics;microarray;rice;ROLbarrier;VLCFA.

INTRODUCTION

Rice(OryzasativaL.)isusuallycultivatedin?ooded,anaerobicenvironments.Themainadaptationsofriceplantstothisextremeconditionare(1)formationofaerenchymatoprovideinternalaerationof?oodedroots(Barberetal.1962;Armstrong1979)and(2)inductionofastrongapoplasticbarrierintheperipheralcelllayersoftheroots(reviewedbyWatanabeetal.2013).Thebarrierreducesradialoxygen(O2)loss(ROL)fromtherootaerenchymatoanaerobicsoilormedium(Armstrong1971;Colmer2003b),aswellaspre-ventstheentryofphytotoxiccompounds(reducedformsofmetalsandorganicacids)totherootsfromthesoil(Armstrong1979).PotentialcomponentsofthebarriertoROLaresuberinand/orlignin,whichareaccumulatedatthe

Correspondence:K.Kulichikhin.Fax:+81527894017;e-mail:konstantin_kulichikhin@http://wendang.chazidian.com;andM.Nakazono.Fax:+81527894017;e-mail:nakazono@agr.nagoya-u.ac.jp2406

cellwallintheperipheralcelllayersoftheroot(DeSimoneetal.2003;Soukupetal.2007;Kotulaetal.2009;Shionoetal.2011).ThechemicalcompositionoftheapoplasticbarrierinricerootssuggeststhatROLisrestrictedbytheformationofasuberizedhypodermis(exodermis)and/orligni?edscleren-chymaintheouterpartoftheroot(Kotulaetal.2009).Suberinisabiopolymerconsistingofthreedistinctgroupsofmonomers:(1)analiphaticdomainrepresentedbyfattyacids,includingverylongchainfattyacids(VLCFAs)andtheirderivatives;(2)anaromaticdomainrepresentedbyferulicandcoumaricacidsandtheirderivatives;and(3)glycerol(Bernards2002;Franke&Schreiber2007).Suberinmonomersaretheproductsofthreedistinctmetabolicpathwaysposses-singdifferentsubcellularlocalizations.Suberizationleadstomassiverearrangementofprimarycarbonmetabolism,increasedenergydemandandredirectionofcarbonandenergy?uxestothebiosynthesisofphenolicacidsandfattyacids.

Thecomponentsofthealiphaticdomainofsuberin–fattyacidsandVLCFAs–areproducedexclusivelyfromacetylcoenzymeA(AcCoA).Apartfromfattyacidbiosynthesis,AcCoAisinvolvedinmanyimportantaspectsofplantmetabolismsuchasthetricarboxylicacid(TCA)cycle(Siedow&Day2000).Inplantcells,AcCoAcanbesynthe-sizedindifferentcompartments,suchasthecytosol,mitochondria,plastidsandglyoxysomes.Inplastidsandmito-chondria,AcCoAisformedfrompyruvicacidthroughoxi-dativedecarboxylationbypyruvatedehydrogenase(PDH).Additionally,AcCoAcanbeformedbyacetyl-CoAsynthetaseusingaceticacidasasubstrate,butthisreactionrequiresadditionalATP.Inthecytosol,ATP-citratelyaseproducesAcCoAfromcitricacidtransportedfromthemito-chondria(Somervilleetal.2000;Rawsthorne2002).CytosolicAcCoAisthemainsourceofAcCoAforVLCFAelongation(Harwood1988).BiosynthesisofVLCFAconsistsoftwosteps:(1)biosynthesisofC16–C18fattyacidsintheplastidsand(2)elongationofthesefattyacidstoC30andlongerintheendoplasmicreticulum(ER)(Beissonetal.2012).Thus,theplastidicandERpoolsofAcCoAarederivedfromdifferentmetabolicpathways.Whenplantsaregrownunder?oodedconditions,theabove-describedprocessesshouldbeabletocopewithpossibleenergyde?citsintheroots;suchde?citsoriginatefromO2restriction,whichcandecreaseATPyieldfrom24–36molpermolofglucosemetabolizedunderaeratedconditionsto2–3molundercompleteanoxia(Gibbs&Greenway2003).

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Metabolicpro?lesofricerootunderwaterlogging

Apoplasticbarriersisolatedfromvariousplantspeciesdifferdramaticallyinsuberinandlignincontents,suberin-to-ligninratio,aliphatic-to-aromaticsuberinmonomerratioandaliphaticsuberincomposition(DeSimoneetal.2003;Soukupetal.2007;Kotulaetal.2009).ItisunclearwhichofthesecomponentsarerequiredtopreventROL(reviewedbyWatanabeetal.2013).Moreover,littleisknownaboutthesolublemetabolitesinroottissuesundergoingsuberizationorligni?cation.Toourknowledge,onlyonestudy(ofpotatowoundperiderm)hasexaminedtheeffectofsuberizationonthepro?leofextractablemetabolites(Yang&Bernards2007).Here,weattemptedtocharacterizethereorganizationofprimarycarbonmetabolisminricerootsduringROLbarrierformation.Tothisend,weobtainedthepro?lesofpolarmetabolitesandoffattyacidsindifferentrootzonesofriceplantsgrowninastagnantdeoxygenatedmediumandawell-aeratedmedium.Tobetterinterpretthedifferencesinmetabolicpro?les,wecombinedthebiochemicaldatawiththeresultsofamicroarrayanalysis,andweusedquantitativeRT-PCR(qRT-PCR)tostudytheexpressionsofseveralgenesthatstoodoutinthemicroarrayanalysis.

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Histochemicalstainingofsuberin

Freshrootswereharvestedasdescribedearlierandwholerootswereembeddedin5%agar.Further,50μmsectionsweremadeatthedistancesof10,20,30,40,50and60mmfromtherootapexusingavibratingmicrotome(VT1200S;LeicaBiosystemsNusslochGmbH,Nussloch,Germany).Rootcrosssectionswereclearedbyincubationat70°Cfor1hinlacticacidsaturatedwithchloralhydrate(Luxetal.2005);thiswasfollowedbystainingwiththelipophilic?uorochromeFluorolYellow088atroomtemperaturefor1htovisualizesuberinlamellae(Brundrettetal.1991).Thealiphaticcomponentofsuberininthecellwallswasdetectedwithacharge-coupleddevice(CCD)camera(DP70;OlympusOpticalCo.Ltd.,Tokyo,Japan)asyellow?uores-cenceuponexcitationbyUVlightundera?uorescencemicroscopeOlympusBX60equippedwithU-MWU?ltrecube(OlympusOpticalCo.Ltd.).

ROLmeasurement

RatesofROLfromtheadventitiousrootsofintactplantsweremeasuredusingroot-sleevingplatinumO2electrodesasdescribedbyKotulaetal.(2009),inaccordancewiththemethodsofArmstrong(1967)andArmstrong&Wright(1975).Therootsystemsofplantsfromeitheraeratedorstagnantculturewereimmersedinachambercontainingadeoxygenatedsolutionof5mmKCl,0.5mmCaSO4and0.1%(w/v)agar(Colmer2003a;Kotula&Steudle2009).Theshootbasewas?xedtothetopofthechambersothattheshootwasinair(21%O2,v/v),therootswereintheO2-freemedium,andtheroot–shootjunctionwas1–2cmbelowthesurfaceofthemedium.The?rstmeasurementsweretaken2haftertransferoftheplanttothechamber.Oneadventitiousroot(100–150mmlong)ofeachplantwasinsertedthroughthecylindricalplatinumelectrode(innerdiameterof2.25mm,heightof5mm),whichwas?ttedwithguidestokeeptherootatthecentreoftheelectrode.ROLwasmeasuredalongtherootwiththecentreoftheelectrodeatpositions5,10,20,30,40,50and60mmfromtheapex.Measurementsweretakenat28°Cinthegrowthchamberwheretheplantshadprevi-ouslybeengrown.

MATERIALSANDMETHODS

Plantmaterials,treatmentsandharvesting

Seedsofrice(O.sativaL.,cv.Nipponbare)weresterilizedwith70%ethanolfor1min;thiswasfollowedbysterilizationwithNaClOsolution(2.5%availablechlorine)for30minandthenwashingseveraltimeswithde-ionizedwater.Thesterilizedseedsweregerminatedinthedarkat28°Cfor2d.Seedlingsweretransferredtoahydroponiccontainerwithquarter-strengthaeratednutrientsolution[28°C,lightcon-ditions,photosyntheticallyactiveradiation200–250μmolm?2s?1]for4d.ThecompositionofthenutrientsolutionwasasdescribedbyColmeretal.(2006).After4d,theseedlingsweretransferredtoaeratedfull-strengthnutrientsolution.After3d(atage9d),halfoftheseedlingsweretransferredtostagnantdeoxygenatednutrientsolution.Thestagnantsolutioncontained0.1%(w/v)dissolvedagarandwasdeoxygenated(dissolvedO2,<0.5mgL?1)beforeusebybeing?ushedwithN2gas.Theaeratedandstagnantnutrientsolutionswererenewedweekly.Topreventironde?ciencyintheseedlingsgrownunderaeratedconditions,FeSO4(uptoa?nalconcentrationof5μm)wasaddedondays9,13,16and20aftergermination.At14dafterthestartofstagnanttreat-ment(23daftergermination),theplantswereusedforROLmeasurementorwereharvestedforbiochemicalanalysis,RNAextractionorenzymeactivitymeasurement.Adventi-tiousroots(100–150mmlong)wereharvestedfromriceplantsgrownineithertheaeratedorthestagnantsolution,and10mmsegmentswerecutwithasterilerazorbladefromregions0to10(0–10)mm,10to20(10–20)mm,20to30(20–30)mmand30to40(30–40)mmfromtherootapex,andcollectedandprocessedseparately.Forenzymeactivitymeasurement,thematerialswereprocessedimmediately.ForbiochemicalanalysisandRNAextraction,therootsegmentswereplunge-cooledinliquidnitrogenandstoredat?80°C.

Metaboliteextractionandanalysis

Metaboliteextraction

Rootsegmentsstoredat?80°Cwerefreeze-driedfor36hbeforetheextractionprocedure.Metaboliteswereextractedfrom3to20mgoflyophilizedtissuegroundinaglasshomogenizerwith1.2mLofbiphasicsolventsystemCHCl3–MeOH–H2O[50:25:25(v:v:v)].MeOHandhalfofCHCl3wereadded?rsttoinhibitenzymeactivity;thiswasfollowedbytheadditionofH2OandtheremainderofCHCl3.Heneicosanoicacid(C21:0,5μgmg?1ofdryweight)wasaddedasanexternalstandardforgaschromatography(GC)analy-sis.Aftercentrifugationoftheextractat800gfor10min,theCHCl3andMeOH–H2Ophaseswerecollectedseparatelyintoglassvials.Extractionwasperformedthreetimes;the

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2408K.Kulichikhinetal.

phased,andthebaselinewasmanuallycorrectedusingthemultipointbaselinecorrectionfeature.Peakswereintegratedmanually,andmetaboliteswerequanti?edbycomparingthepeakareaofthecompoundofinterestwiththepeakareaofTMSP-d4.Metaboliteswereidenti?edbyacquiringtwo-dimensionalspectra[totalcorrelationspectroscopy(TOCSY)andJ-resolvedspectroscopy]torevealthecorrelationbetweenthesignalsandthepatternofsignalmultiplicity,respectively,andthroughcomparisonoftheexperimentaldatawiththedatafrompubliclyavailabledatabases(Fan1996;Cuietal.2008)andwiththespectraofpurecompounds.

non-polarandpolarphaseswerecombinedseparatelyandthesolventwasevaporatedusingastreamofpureN2gasinthecaseoftheCHCl3fractionorbycentrifugationundervacuuminthecaseoftheMeOH–H2Ofraction.Driedsampleswerestoredat?80°C,andvialscontainingtheCHCl3fractionresidueswere?lledwithpureN2gasbeforebeingplacedintotherefrigerator.CHCl3andMeOHandroottissueextractsweredispensedviaglassmicrosyringe(Hamilton,Reno,NV,USA);contactbetweentheorganicsolventsandanyplasticmaterialswasthusavoidedatallstagesofsampleextraction.

Samplepreparationfornuclearmagneticresonance(NMR)analysis

ImmediatelybeforeNMRanalysis,theMeOH/H2Ofractionsofthesampleswerereconstitutedinto220μLofdeuteriumoxide(D2O;99.9%D,CambridgeIsotopeLaboratories,Andover,MA,USA)containing100mmKD2PO4.ThesolventwasevaporatedbyvacuumcentrifugationtoremovethetracesofH2O,theresiduewasreconstitutedin220μLofD2O(99.96%D,CambridgeIsotopeLaboratories)and0.5–5μLof50mmsodium3-(trimethylsilyl)propionate-2,2,3,3-d4(TMSP-d4)wasaddedasachemicalshiftandquanti?cationreference.Sodiumazidewasaddedtoa?nalconcentrationof0.5mmtopreventmicrobialgrowth.ThepDofthesampleswasadjustedto7.40±0.02withKODandDClsolutionsinD2Ousingaglassmicroelectrode.pDvaluewascalculatedfromtheapparentpHvalue(pH*)ofthesampleusingtheequation,pD=pH*+0.4(Glasoe&Long1960).

GC-MSanalysisoffattyacids

Thefattyacidcompositionofthenon-polarfractionofplantextractswasanalysedbytheShimadzuAnalyticalandMeasuringCenter,Kyoto,Japan.Brie?y,theresiduewasreconstitutedin500μLofCHCl3andanaliquotof50–100μLwastransferredintoa10mLscrew-cappedvialwith1mLofMeOH.Thirtymicrolitresof0.3%(w/v)butylatedhydroxytolueneinMeOHwasaddedtopreventsampleoxi-dation,and5μLof0.2%(w/v)nonadecylicacid(C19:0;10μgintotal)wasaddedasaninternalstandard.Finally,1mLof10%HClinMeOHwasaddedtothemixture,thecapwasclosedandthevialwasplacedinadrybathat80°Cfor1hformethanolysis.Afterthemixturehadbeencooled,fattyacidmethylesters(FAMEs)wereextractedbyvortexingwithtwoportionsofn-hexane(1.5and1mL).Thetwopor-tionsofhexanewerecombinedinaglassvial;thesolventwasremovedbyastreamofN2gasat40°C,andtheresiduewasreconstitutedin200μLofn-hexaneandsubjectedtoGCanalysis.TheFAMEswereseparatedinanRtx2330column(ShimadzuCo.Ltd,Kyoto,Japan).

NMRanalysis

ThesampleswereanalysedwithJEOLECA-600orJEOLECA-800NMRspectrometers(JEOLLtd.,Tokyo,Japan).All1

H-NMRspectrawerecollectedwitha3mmbroadbandprobeusingaJEOL-de?nedpulseprogram(single_pulse.ex2).Theprobewastunedmanually(ECA-800)orautomatically(ECA-600).SampleswerelockedbytheD2Osignal,andmagnetic?eldhomogeneitywasoptimizedmanuallyusingupto10shimsfollowedbytheautomaticgradientshimmingoption.Theprobetemperaturewasmaintainedat298K,andspinningat20Hzwasappliedduringacquisition.Freeinduc-tiondecays(FIDs)werecollectedinto65536datapoints.Arelaxationdelayof5swasused,andonedummytransientwasfollowedbytheco-additionof1024scansforatotalexperimenttimeof3.00h.SeveralFIDs(upto?ve,depend-ingupontheamountofmaterialusedintheanalysis)werecollectedforeachsample,andadditionalshimmingwasper-formedinbetweentheacquisitionstoimprovethemagnetic?eldhomogeneity.

RNAextraction

TotalRNAwasextractedfromfrozen-?xedtissuesfromfoursequentialregionsoftheadventitiousrootsusinganRNeasyPlantMiniKit(Qiagen,Valencia,CA,USA)inaccordancewiththemanufacturer’sinstructions.ThequalityoftotalRNAwasassessedwithanRNA6000PicokitonanAgilent2100Bioanalyzer(AgilentTechnologies,PaloAlto,CA,USA).

Microarrayanalysis

TotalRNAs(400ng)werelabelledwithaQuickAmpLabel-ingKit(AgilentTechnologies)inaccordancewiththeman-ufacturer’sinstructions.AliquotsofCy5-labelledcRNAandCy3-labelledcRNA(825ngeach)wereusedforhybridiza-tioninarice44Koligo-DNAmicroarray(AgilentTechnol-ogies).Threebiologicalreplicateswereanalysed.ThehybridizedslideswerescannedwithaDNAmicroarrayscannerG2505C(AgilentTechnologies),andsignalinten-sitieswereextractedusingFeatureExtractionsoftware(version10.5.1.1;AgilentTechnologies).AcompletesetofmicroarraydatawasdepositedintheGeneExpressionOmnibus(GEO;http://www.ncbi.nlm.nih.gov/geo/)reposi-toryunderaccessionnumberGSE52128.

NMRdataprocessing

NMRspectrawereprocessedusingMestReNovaversion8.1(MestrelabResearchS.L.,SantiagodeCompostela,Spain).FIDscollectedforthesamesampleweresummedupbeforeFouriertransformwasapplied.Allspectraweremanually

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Metabolicpro?lesofricerootunderwaterlogging

Formicroarraydataanalysis,theBenjamini–Hochbergfalsediscoveryrate(FDR)methodwasusedtoobtainP-valuescorrectedformultipletesting.Thefoldchangeofeachprobebetweenonesetofconditionsandtheotherwascalculatedusinganaverageofthreebiologicalreplicates.Weidenti?edthegenesforwhichtherewasatleastatwofoldchangeinexpressionbetweenthetwoconditionsonaverageandforwhichtheFDRP-valuewas<0.05.

Forgeneontology(GO)analysis,weanalysedthefre-quencyofGOtermsofup-regulatedanddown-regulatedgenesusingGOSlimAssignments(http://rice.plantbiology.msu.edu/downloads_gad.shtml).

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Enzymeactivitymeasurement

TheactivitiesofNAD-malatedehydrogenase(NAD-MDH;E.C.1.1.1.37),phosphoenolpyruvatecarboxylase(PEPC;E.C.4.1.1.23),NADP-malicenzyme(NADP-ME;E.C.1.1.1.40)andpyruvatephosphatedikinase(PPDK;E.C.2.7.9.1)weremeasuredspectrophotometricallyatawave-lengthof340nmwithaJASCOV-570spectrophotometer(JASCOCorp.,Tokyo,Japan),asdescribedbyMoonsetal.(1998),withsomemodi?cationsinthecaseofPPDK,andbyKulichikhinetal.(2009)inthecaseoftheotherenzymes.Measurementsweretakenat25°CforNAD-MDHandNADP-MEandat30°CforPEPCandPPDK.Theassaymixturewasincubatedatthecorrespondingtemperaturefor3minbeforethereactionwasstartedbytheadditionofthereactionsubstrate.Enzymeactivitywasexpressedinenzymeunits(EU)permilligramofprotein.ProteinintheextractswasassayedwithCoomassieBrilliantBlueG250inaccord-ancewiththemethodofBradford(1976)usingaBio-RadProteinAssayreagent(Bio-Rad,Hercules,CA,USA),withbovineserumalbumin(BSA)asareference.Thedetailedcompositionsoftheassaymixturesforeachenzymearepre-sentedinSupportingInformationFileS1.

Lasermicrodissection(LM)

Segmentsofadventitiousrootsintheregion10–20mmfromtherootapexwere?xedin100%acetone.After?xation,thesampleswereembeddedinparaf?nandsectionedatathick-nessof20μm.SerialsectionswereplacedontoPENmem-braneglassslides(LifeTechnologies,Carlsbad,CA,USA)forLM,asdescribedbyTakahashietal.(2010).Toremoveparaf-?n,slideswereimmersedinHisto-ClearIIsolution(NationalDiagnostics,Atlanta,GA,USA)for10min;thiswasfollowedbyairdryingatroomtemperature.Thecentralcylinder,thecortexandtheoutercelllayerswerecollectedfromtheroottissuesectionsusingaVeritasLaserMicrodissectionSystemLCC1704(MolecularDevices,Toronto,ON,Canada).

RESULTS

ROLfromadventitiousrootsinriceunderaeratedandstagnantconditions

Weplottedthepro?lesofROLalongadventitiousrootsofplants(23dold)grownunderaeratedorstagnantconditionsfor14d(Fig.1).AdventitiousrootsofplantsgrownunderaeratedconditionsshowedthehighestrateofROLatthemostbasalpart;therewasagradualdecreaseinROLtowardstherootapex(Fig.1).Growthunderstagnantcon-ditionsresultedinadramaticalterationintheROL

pro?le

QuantitativeRT-PCR(qRT-PCR)

ForqRT-PCR,5ngoftotalRNAextractedfromtheadven-titiousrootsorthelaser-microdissectedtissueswasusedasatemplate.TranscriptlevelsweremeasuredusingaStepOnePlusReal-TimePCRSystem(AppliedBiosystems,FosterCity,CA,USA)andOneStepSYBRPrimeScriptRT-PCRKitII(TakaraBioInc.,Otsu,Japan),asdescribedbyYamauchietal.(2014).Thequanti?edmRNAlevelsofeachgenewerenormalizedagainstthemRNAlevelsofthetran-scriptionfactorTFIIEgeneasacontrol.qRT-PCRwasper-formedwithtotalRNAfromthreebiologicalreplicates.TheprimersequencesusedfortheexperimentsareshowninSupportingInformationTableS1.

Enzymeactivitymeasurements

Extractionprocedure

ExtractsofplantmaterialwereobtainedasdescribedbyKulichikhinetal.(2009).Freshlyharvestedrootsegments(15–20segments)wereweighedandgroundinaglasshomogenizerwith1mLofextractionmediumcontaining0.1mHEPES-KOH(pH7.5),12.5%(v/v)glycerol,0.5%(w/v)l-ascorbicacid,5mmdithiotreitoland5%(w/v)polyvinylpolypyrrolidone(PVPP,http://wendang.chazidian.completeMiniEDTA-free(ProteaseInhibitorCocktailTablets;RocheDiagnostics,Mannheim,Germany)wasaddedtopreventproteolyticcleav-ageoftheproteins.Thehomogenatewascentrifugedat15000gfor10min.Thepelletwasthendiscardedandthesupernatantwasusedfortheenzymeactivitymeasurements.

Figure1.Pro?lesofradialO2loss(ROL)alongadventitious

rootsofintactriceplantsgrownineitheraeratedorstagnantnutrientsolution.Dataaremeans±SE(n=3).

©2014JohnWiley&SonsLtd,Plant,CellandEnvironment,37,2406–2420

2410K.Kulichikhinetal.

lamellaeweredetectedat40mmorfartherfromtherootapex(Fig.2).OnthebasisoftheresultsoftheROLmeasurementsandsuberinhistochemicalstaining,wechosetheregionfrom0to40mmforfurtherbiochemicalandmolecularstudies.

Metabolicpro?lesinadventitiousrootsunderaeratedandstagnantconditions

1

H-NMRpro?lesofpolarmetabolites

Figure2.Crosssectionsofadventitiousricerootsstainedfor

suberinwithFluorolYellow088.Plantsweregrownundereitheraeratedorstagnantconditions.Yellow?uorescenceindicatesthepresenceofsuberin.Bar=50μm.Thedistancefromtherootapexisindicated.Theappearanceofsuberinat30mmfromtherootapexunderstagnantconditionsisindicatedbyarrows.ep,epidermis;hy,

hypodermis.

(Fig.1).Theadventitiousrootsofplantsgrownunderstag-nantconditionsshowedthehighestratesofROLat5–10mmfromtheapex.Towardstherootbase,theROLdeclinedsubstantially:at20mmfromtheapex,itwasonly7.3%ofthevalueat10mmfromtheapex;andat30mm,itwas6.1%ofthevalue.LittleornoROLwasfoundat40and50mmfromtherootapex,andonlyatinyamountofO2wasreleasedat60mm(Fig.1),probablybecauseofthepresenceoflateralroots(datanotshown).Thus,growthunderstagnantcondi-tionsinducedtheformationofastrongbarriertoROLinriceadventitiousroots.

Thesignalsfrommorethan20compoundshavebeeniden-ti?edonthe1H-NMRspectraofpolarextractsfromriceroots(SupportingInformationTableS2),andmostofthemhavebeenquanti?able.Theconcentrationsofallpolarmetabolites,withtheexceptionofmalicacid,inbothaeratedandstagnantrootsdecreasedinthedirectionfromtherootapextotherootbase(SupportingInformationTableS3).Incontrast,thecontentofmalicacidwasminimalintheapicalregion(0–10mm)andat10–20mminrootsunderstagnantconditions,graduallyincreasingtowardstherootbase(Fig.3).Rootsofplantsgrownunderstagnantconditionswerecharacterizedbyelevatedlevelsofsolublesugars(sucrose,glucoseandfructose;Fig.3),someaminoacids(isoleucine,leucine)andorganicacids(quinicacid,shikimicacidandespeciallymalicacid)(Fig.3;SupportingInforma-tionTableS3).Aeratedrootsweremoreabundantinglutamine,asparagineandalanine,butonlyintheregions0–10mm(alanine)or0–20mm(glutamine,asparagine)fromtherootapex(SupportingInformationTableS3).

Themostabundantpolarmetabolitesinbothaeratedandstagnantrootsweresolublesugars(sucrose,glucoseandfruc-tose)andmalicacid(Fig.3).Thesucrosecontentoftheapicalregionofstagnantrootswas8.1μmolg?1freshweight;thiswas1.7timeshigherthanthecontentintheapicalregionoftherootsofplantsgrownunderaeratedconditions.Intheregion20–30mm,thesucrosecontentdecreasedto3.1and1.9μmolg?1freshweightinthestagnantandaerated

roots,

Histochemicalstainingforsuberin

Weperformedhistochemicalstainingforsuberinincrosssectionsofricerootsfromplantsgrowninaeratedorstagnantsolutions(Fig.2).Underaeratedconditions,nosuberinstain-ingofhypodermalcellwallswasdetectedintheregions10,20and30mmfromtherootapex.Inthemorebasalregions,suberinstainingwasdetectedinsomeindividualcells,butthecellwallsofthemajorityofhypodermalcellswerenotstained(Fig.2).Growthunderstagnantconditionsledtosuberizationofhypodermalcellwalls.Suberinstainingwasdetectedat30mmfromtherootapex,andfullydevelopedsuberin

Figure3.Solublesugarandmalicacidcontentsindifferent

regionsofrootsfromriceplantsgrownundereitheraeratedorstagnantconditions.Signi?cantdifferencesbetweentheaeratedandstagnantconditionsatP<0.05,P<0.01orP<0.001(two-samplet-test)aredenotedby*,**or***,respectively.Dataaremeans±SE(n=3).

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