The ABC Transporter ABCG1 Is Required for Suberin Formation in Potato Tuber PeridermW

ThePlantCell,Vol.26:3403–3415,August2014,http://wendang.chazidian.comã2014AmericanSocietyofPlantBiologists.Allrightsreserved.

TheABCTransporterABCG1IsRequiredforSuberinFormationinPotatoTuberPeriderm

RamonaLandgraf,aUlrikeSmolka,aSimoneAltmann,a,1LennartEschen-Lippold,aMelanieSenning,b,2

SophiaSonnewald,bBenjaminWeigel,cNadezhdaFrolova,aNadineStrehmel,aGerdHause,dDierkScheel,aChristophBöttcher,a,3andSabineRosahla,4

aLeibniz

InstituteofPlantBiochemistry,DepartmentofStressandDevelopmentalBiology,D-06120Halle(Saale),GermanyAlexanderUniversityErlangen-Nürnberg,DepartmentofBiology,D-91058Erlangen,Germany

cLeibnizInstituteofPlantBiochemistry,DepartmentofBioorganicChemistry,D-06120Halle(Saale),GermanydMartinLutherUniversityHalle-Wittenberg,Biocenter,D-06120Halle(Saale),Germany

bFriedrich

Thelipidbiopolymersuberinplaysamajorroleasabarrierbothatplant-environmentinterfacesandininternaltissues,restrictingwaterandnutrienttransport.Inpotato(Solanumtuberosum),http://wendang.chazidian.comingmicroarrayanalyses,weidenti?edABCG1,encodinganABCtransporter,asageneresponsivetothepathogen-associatedmolecularpatternPep-13.FurtheranalysesrevealedthatABCG1isexpressedinrootsandtuberperiderm,aswellasinwoundedleaves.TransgenicABCG1-RNAipotatoplantswithdownregulatedexpressionofABCG1displaymajoralterationsinbothrootandtubermorphology,whereastheaerialpartoftheABCG1-RNAiplantsappearnormal.Thetuberperidermandrootexodermisshowreducedsuberinstaininganddisorganizedcelllayers.Metaboliteanalysesrevealedreductionofesteri?edsuberincomponentsandhyperaccumulationofputativesuberinprecursorsinthetuberperidermofRNAinterferenceplants,suggestingthatABCG1isrequiredfortheexportofsuberincomponents.

INTRODUCTION

Suberinisabiopolymerdepositedinthecellwalltoformahy-drophobicsurfacethatprotectsagainstwaterlossandregulatesdiffusionofsolutesandgases(Ranathungeetal.,2011).Typicalsuberin-containingtissuesincludetheexo-andendodermisofrootsaswellasoakcork(Quercussuber)andpotatotuber(Solanumtuberosum)periderm.

Thesuberinpolymerconsistsofester-linkedv-hydroxyacids,a,v-dicarboxylicacids(DCAs),fattyacids,andprimaryalcoholswithchainlengthsrangingfromC16uptoC32,aswellasglycerol.Inaddition,suberincontainshydroxycinnamicacids,predominantlyferulicacid,thatarecross-linkedbybothesterandnonesterbonds(FrankeandSchreiber,2007;Pollardetal.,2008).Enzymesinvolvedinthebiosynthesisofsuberinprecursorshavebeenidenti?edprimarilyinArabidopsisthaliana.Theformationofv-hydroxyfattyacidsand,possibly,DCAsiscatalyzedbyCYP86Ahydroxylases(Beissonetal.,2012).AcyltransferasesoftheBAHDsuperfamilytransfertheacylmoietyofferuloyl-CoAtov-hydroxyfattyacids

1Current

address:UniversityofPotsdam,InstituteofBiochemistryand

Biology,Karl-Liebknecht-Str.24-25,14476Potsdam,Germany.

2Currentaddress:ETHZurich,DepartmentofBiology,PlantBiotech-nology,Universitaetstrasse2,CH-8092Zurich,Switzerland.

3Currentaddress:JuliusKühn-Institut,BundesforschungsinstitutfürKulturp?anzen,InstitutfürökologischeChemie,P?anzenanalytik,undVorratsschutz,Königin-Luise-Str.19,14195Berlin,Germany.4Addresscorrespondencetosrosahl@ipb-halle.de.

Theauthorresponsiblefordistributionofmaterialsintegraltothe?ndingspresentedinthisarticleinaccordancewiththepolicydescribedintheInstructionsforAuthors(http://wendang.chazidian.com)is:SabineRosahl(srosahl@ipb-halle.de).OnlineversioncontainsWeb-onlydata.

http://wendang.chazidian.com/cgi/doi/10.1105/tpc.114.124776

andfattyalcohols(Molinaetal.,2009),whereasglycerol-3-phosphateacyltransferaseslinkfattyacylmoietiestoglycerol(Beissonetal.,2007).Inaddition,long-chainacyl-CoAsynthe-tasesarepostulatedtoactivatefreefattyacidsbyconjugationtoCoA.Fattyacyl-CoAreductasesandb-ketoacyl-CoAsynthasescatalyzethebiosynthesisoffattyalcoholsandvery-long-chainfattyacids,respectively(Frankeetal.,2009;Domergueetal.,2010).

Howsuberinmonomersaretransportedthroughtheplasmamembraneintotheapoplastisnotknown.Theformationofcutin,thelipidpolymerofplantcuticles,requiresplasmamembrane-localizedATPbindingcassette(ABC)transporters(Birdetal.,2007;Panikashvilietal.,2011;Bessireetal.,2011);however,noABCtransporterinvolvedinsuberindepositionhasbeenidenti?edsofar.

Potatotuberperidermcellshavehighlysuberizedwalls.Thefunctionoftubersasstorageorgansnecessitatesmechanismstoreducewaterloss.Theimportanceofsuberinasatranspirationbarrierinpotatotuberswasillustratedintransgenicplantswithdefectiveperidermasaconsequenceofthedownregulatedex-pressionofgenesencodingthesuberinbiosyntheticenzymesb-ketoacyl-CoAsynthase(KCS6;Serraetal.,2009a),CYP86A33(Serraetal.,2009b),andv-hydroxyfattyacid/fattyalcoholhy-droxycinnamoyltransferase(FHT;Serraetal.,2010).Reducedlevelsofsuberinintheseplantswerecorrelatedwithcompromisedtranspirationbarrierfunction.

Potatoisthefourthmostimportantcropplantworldwide.Oneofthemajorcausesofyieldlossinpotatoislateblightdisease,causedbytheoomycetePhytophthorainfestans(Fry,2008).ThesusceptiblepotatocultivarDésiréecanbeinducedtomountef-?cientdefenseresponsesagainstP.infestansbytreatmentwiththepathogen-associatedmolecularpattern(PAMP)Pep-13(Halim

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etal.,2004),ultimatelyconferringenhancedresistancetothewholeplant.Uponin?ltrationintopotatoleaves,Pep-13,a13-aminoacidmotiffromanextracellulartransglutaminasefromPhytophthoraspecies(Brunneretal.,2002;Halimetal.,2004),inducesdefenseresponsesinasalicylicacid-andjasmonicacid-dependentmanner(Halimetal.,2009).

AspartofourfunctionalanalysisofgenesthatareactivatedinpotatoinresponsetoPep-13in?ltration,weidenti?edagenepredictedtoencodetheABChalftransporterABCG1.AnalysisofplantswithreducedABCG1expressionindicatesaroleforABCG1insuberinformationofpotatotuberperidermand,thus,inwaterbarrierformation.RESULTS

Identi?cationofthePep-13-ActivatedABCG1GeneSusceptiblepotatoplants(S.tuberosumcvDésirée)displayenhancedresistanceagainstP.infestansafterin?ltrationofthePAMPPep-13(Halimetal.,2009).ToidentifygenesthatareactivatedinresponsetoPep-13treatment,microarrayexperi-mentswereperformedusingPOCIoligoarrays(Kloostermanetal.,2008).RNAwasisolatedfromleavesofpotatoplantsafterin?ltrationwithPep-13oritsinactiveanalogW2A(Brunneretal.,2002).Morethan700geneswereidenti?edasatleast2-foldupregulatedbyPep-13inwild-typeplantscomparedwithW2Atreatment.AnESTencodingaputativeABChalftransporter(ABCG1)waschosenforfurthercharacterization(SupplementalFigure1A).Thecorrespondinggenewasidenti?edfromthepo-tatogenomedatabase(Xuetal.,2011;XM_006345853.1;http://www.ncbi.nlm.nih.gov/).OnlyoneABCG1geneispresentinthereferencegenomeofS.tuberosumgroupphureja.Thecodingregionconsistsoftwoexonsseparatedbyanintronof97bp(SupplementalFigures1Cand1D).Theencodedproteinof750aminoacidsispredictedtobeanABChalftransporterwitha

P-loop-containingATPhydrolaseregionandsixtransmembranedomains(SupplementalFigure1E).TheaminoacidsequenceofABCG1wascomparedwiththatofthe43membersoftheArab-idopsisAtABCGfamily(http://wendang.chazidian.com).Phylogeneticanal-ysesrevealedhighesthomologyofSt-ABCG1toAt-ABCG1andAt-ABCG16(SupplementalFigure2andSupplementalDataSet1).ExpressionofABCG1inPotato

ToverifythePep-13-inducibleexpressionofABCG1observedinthemicroarrays(SupplementalFigure1A),RNAfromleavesofPep-13-treatedwildtypeandemptyvector-containingpotatoplantswassubjectedtoquantitativeRT-PCRanalysis.Inwild-typeandemptyvector-carryingplants,Pep-13,butnotW2A,inducedtheaccumulationofABCG1transcripts(SupplementalFigure1A).Moreover,ABCG1expressionwasinducedbywound-ing(SupplementalFigure1B).

Thetissue-speci?cexpressionofABCG1wasdeterminedusingRNAfromuntreatedsoil-grownplants.Expressionwashardlydetectableinleavesandstems,whereashigherlevelsofABCG1transcriptswerefoundinrootsandintubers(Figure1A).SeparationoftuberskinandtubercortexrevealedhigherlevelsofABCG1transcriptsintuberskin(Figure1B).ABCG1IsLocalizedtothePlasmaMembrane

Thefull-lengthcodingregionofABCG1wasampli?edfromcDNAreversetranscribedfromRNAofPep-13-treatedpotatoleavesandclonedunderthecontroloftheubiquitin10promoterinfrontofthecodingregionofred?uorescentprotein(RFP)inthebinaryvectorpUBC-DEST-RFP(Grefenetal.,2010).TransientexpressionoftheconstructinArabidopsismesophyllprotoplastsrevealedRFP?uorescenceatthecellperiphery(Figure2).Co-localizationwiththeestablishedplasmamembranemarkerFLS2-YFP(Robatzeketal.,2006)suggestedthatSt-ABCG1isplasmamembrane

localized.

Figure1.ABCG1IsExpressedinRootsandTubers.

(A)Tissue-speci?cexpressionofABCG1.RNAwasisolatedfromleaves,stems,roots,andtubersof5-week-oldpotatoplantsgrowninaphy-tochamber,reversetranscribed,andanalyzedforABCG1transcriptlevelsbyquantitativePCR.ExpressionofEF1awasusedasareference.(B)ExpressionofABCG1intubers.RNAwasisolatedfromtuberskinandtubercortexof3-month-oldgreenhouse-grownpotatoplants.

Dataarederivedfromthree([A];n=6)andtwo([B];n=8)independentexperiments.ErrorbarsrepresentSE.Lettersindicatestatisticallydifferentvalues(one-wayANOVA,P#0.05[A]andttest,P#0.01[B]).

Figure2.St-ABCG1ColocalizeswithaPlasmaMembraneProtein.ArabidopsisprotoplastswerecotransfectedwithSt-ABCG1-RFPandAt-FLS2-YFP.Expressionoftheproteinswasdetectedbyconfocallaserscanningmicroscopy.ThechannelsforRFP,YFP,andchlorophyll?uorescenceareshown,aswellasthebright-?eldandmergedimages.Theexperimentwasperformedtwicewithsimilarresults.Bars=5mm.

PotatoPlantswithReducedExpressionofABCG1

ToaddressthefunctionofABCG1,a413-bpfragment,covering210bpoftheendofthe?rstexonand203bpofthesecondexon(SupplementalFigure1D)wasampli?edfrompotatocDNAandclonedintopHellsgate8(Wesleyetal.,2001).Potatoplantsex-pressingtheRNAinterference(RNAi)constructweregeneratedbyAgrobacteriumtumefaciens–mediatedleafdisktransformation.Twentyindependenttransformantswereregeneratedintwoin-dependenttransformationexperiments.Sixteenoftheselinesshowedareductioninwound-inducedABCG1expressioninleaves(SupplementalFigure3A).Moreover,ABCG1expressionwasre-ducedinroots(Figure3A).Thefourrepresentativelines(B4,F2,N,andP)thatweretesteddemonstratedreducedexpressionintuberskin(Figure3B;forindividuallines,seeSupplementalFigure3B).BothrootsandtubersoftheRNAiplants,butnottheaerialparts,displayedalteredmorphology(Figure4).Notably,thetuberskinwasrough,corky,andbrownish,incontrasttothesmoothandreddishtuberskinofwild-typeplants(Figure4A).MicroscopyanalysesofthetubersectionsrevealedmorphologicaldefectsofthetuberskinofABCG1-RNAiplants,possiblycausedbythecollapseofperidermcells.Thiswasvisualizedintoluidineblue-stainedtuberskinsections,revealinganalteredperidermwithout

SuberinABCTransporter3405

thetypicalorganizationofstackedphellemcells(Figure4A;forindividuallines,seeSupplementalFigure4A).Duetothecol-lapsedcellsandrupturedtissue,determinationofcellsizeandnumberwasnotpossible.

Therootsystemofsoil-grownABCG1-RNAiplantswaslessdevelopedthanthatofwild-typeplantsandsectionsofrootsintheroothairzoneshowedmajordefectsintheouterlayers(Figure4B).WithintheindividualABCG1-RNAilines,thesedefectsrangedfromcollapsedindividualexodermalcellstocompletelyrupturedoutercelllayers(SupplementalFigure4B).Incontrasttotheendodermisofwild-typeroots,multiplecelllayerswerepresentaroundthecentralcylinderintheABCG1-RNAilines(SupplementalFigure4B).Asthetuberperidermandtherootexodermisaretissueswithsuberizedcellwalls,?http://wendang.chazidian.comparedwithwild-typeplants,are-ductionin?uorolyellowstainingofcellwallswasobservedforbothtuberperiderm(Figure4C)andtherootexodermisofABCG1-RNAiplants(Figure4D).Incontrasttotheone-layeredwild-typeendodermis,ABCG1-RNAiplantshadmultiplelayersofcellsaroundthecentralcylinderwhosewallswerestainedwith?uorolyellow.Inthesametissues,auto?uorescencewasstrongerthaninwild-typetissue(Figures4Cand4D),suggestinganincreaseinphenoliccompounds.Thus,thereductioninABCG1expressionwasaccompaniedbyreducedsuberindep-ositionsintubersandroots.

Inaccordancewiththereducedsuberizationoftheperiderm,ABCG1-RNAituberssufferedaseverewaterlossduring20dofstorage,resultingina2-foldweightreduction,whereascontroltubersdidnotloseweighttoasigni?cantextent(Figure4E).ABCG1DownregulationReducestheMajorandAltersthePro?leofMinorMonomersofAliphaticTuberSuberinToelucidatetheeffectofABCG1downregulationonthemonomercompositionoftheesteri?edpartofsuberin,tuberperidermofABCG1-RNAiandcontrol(wild-typeandemptyvector)plantswasdigestedwithcellwall-degradingenzymes,delipidated,andde-polymerizedbyacid-catalyzedmethanolysis.Aftertrimethylsily-lation,releasedmonomerswererelativelyquanti?edbygaschromatography-massspectrometry(GC-MS)(SupplementalTables1to4).ABCG1-RNAiperidermhadstronglyreducedlevelsofthemajoraliphaticmonomersv-hydroxy-octadec-9-enoicacid(v-OH-C18:1)andoctadec-9-enedioicacid(a,v-DCA-C18:1),whichaccumulatedonaverageto9and18%ofthecontrolperi-dermlevel,respectively(Figures5Aand5B).Withinthefourin-vestigatedRNAilines,therangeofmonomeraccumulationwas2to16%(v-OH-C18:1)and3to33%(a,v-DCA-C18:1)ofcontrolperidermlevels(SupplementalFigures5Aand5B).Amongtheminormonomers,v-hydroxyfattyacids,fattyacids,andfattyal-coholsofchain-lengthC24,C26,C28,andC30showedasimilarstrongreductioninABCG1-RNAiperidermcomparedwithcontrolperidermasthatobservedforthetwomajormonomers(Figures5A,5C,and5D).Bycontrast,ABCG1downregulationwascorre-latedwithsigni?cantlyhigheramountsofsaturatedv-hydroxyfattyacids,a,v-dicarboxylicacids,andfattyacidsofchainlengthsofC16toC22(Figures5Ato5C).Inaddition,lessferulicacidwasreleasedaftermethanolysisoftuberperidermfromABCG1-RNAiplantscomparedwithcontrolperiderm(Figure

5E).

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Figure3.ABCG1ExpressionIsReducedinABCG1-RNAiLines.

(A)ReducedABCG1expressioninrootsofABCG1-RNAilines.QuantitativeRT-PCRanalysisofRNAisolatedfromuntreatedrootsof3-week-oldwild-typeandemptyvectorplants(“control”)andABCG1-RNAiplants(linesA,B4,F2,andK)growninaphytochamber.ExpressionofEF1awasusedasareference.Dataarederivedfromthreeindependentexperiments(n=18).ErrorbarsrepresentSE.Differentlettersindicatestatisticallysigni?cantdifferences(ttest,P#0.002).

(B)ReducedABCG1expressionintuberskinofABCG1-RNAilines.RNAwasisolatedfromtuberskinof3-month-oldcontrolandABCG1-RNAiplants(linesB4,F2,N,andP)growninagreenhouse.Dataarederivedfromtwoindependentexperiments(control,n=12;ABCG1-RNAi,n=27).Differentlettersrepresentstatisticallysigni?cantdifferences(ttest,P#0.001).

ABCG1DownregulationIsAccompaniedbyAccumulationofMonomericSuberinPrecursorsinTuberPeriderm

Totestwhetherthestrongreductionofthemajorityofesteri?edsuberinmonomersuponABCG1downregulationiscorrelatedwiththeaccumulationofmonomericsuberinprecursors,apolarex-tractsoftuberperidermweresubjectedtoultraperformanceliquidchromatography(UPLC)/photodiodearray(PDA)/electrosprayionization(ESI)-quantitativetime-of-?ightmassspectrometry(http://wendang.chazidian.comparativeanalysesofUVchromatogramsrevealednumerouscompoundswithanabsorptionmaximumat324nmanddramaticallyincreasedlevelsinABCG1-RNAiperidermincomparisontocontrolperiderm(SupplementalFigure6).Amongthem,ahomologousseriesofferuloyloxyfattyacidswithchainlengthsofC16toC28wasidenti?edbycombinedanalysisofaccuratemass,isotopepattern,andcollision-induceddissociation(CID)massspectra(SupplementalFigure7andSupplementalTable5).AlthoughtheacquiredCIDmassspectradonotallowdeductionofthepositionoftheferuloyloxymoietynorthebranchingpatternofthealkylchain,itislikelythatthesecompo-nentsrepresentstraight-chainv-feruloyloxyfattyacids.Inaddition,aderivedhomologousseriesofC16-C28feruloyloxyfattyacidsesteri?edwithglycerolwasidenti?edfromABCG1-RNAiperidermextracts(SupplementalTable6).Incontrasttoferuloyloxyfattyacids,whichgiverisetotwochromatographicallyseparablepeaksduetothepresenceof(E)-and(Z)-con?guredferuloyloxymoieties,fourchromatographicpeakscouldberesolvedforindividualfer-uloyloxyfattyacidglycerolesters(SupplementalFigure8).Thissuggeststhatboth1-/3-O-and2-O-(feruloyloxyalkanoyl)glycerolsarepresentintheextract.Assumingequalresponsefactors,thehomologpro?lesofferuloyloxyfattyacidsandderivedglycerolestersshowamaximumabundanceatachainlengthofC22.Besidesferuloyloxyfattyacidsandtheirglycerolesters,numerousotherferulicacidconjugatescouldbedetectedonthebasisoftheirUVabsorptionandcharacterizedbyESI-QTOFMSasalkylferulates(chainlengthC16-C28),hydroxyalkylferulates(chainlengthC18-C24),andferuloyloxyalkylferulates(chainlengthC16-C24;

SupplementalTables7to9).Relativequanti?cationofthesecomponentsrevealedincreasedlevelsofhydroxyalkylferulates,exceptforchainlengthC21,andferuloyloxyalkylferulatesinABCG1-RNAiperiderminrelationtocontrolperiderm(SupplementalTable10;Figure6forcompoundswitheven-numberedchainlength).Bycontrast,hyperaccumulationofalkylferulatesinABCG1-RNAiperidermincomparisontocontrolperidermwasonlyob-servedforspecieswithchainlengthsofC18,C19,C20,andC22.TheapplicationofUPLC/PDA/ESI(-)-QTOFMSadditionallyfa-cilitatedrelativequanti?cationofotherputativesuberinprecursorsfromapolarperidermextractssuchasv-hydroxyfattyacids,a,v-DCAs,andfattyacids.Here,homologousseriesofv-hydroxyfattyacids(chainlengthC20-C26)anda,v-DCAs(chainlengthC20-C26)werefoundtoaccumulatetosigni?cantlyhigheramountsinABCG1-RNAiperidermincomparisontocontrolperi-derm(Figures6Fand6G).Forfattyacids,homologswithchainlengthsofC16toC24,exceptC18:2,hadincreasedlevelsinABCG1-RNAiperidermincomparisontocontrolperiderm(Figure6H).Inparticular,thelevelofC18:1wasmorethan20-foldhigherinABCG1-RNAiperidermthanincontrolperiderm.StABCG1DownregulationInducesAccumulationofHydroxycinnamicAcidConjugatesinTuberPeridermTostudytheeffectofABCG1downregulationonthesemipolarmetabolitepro?leoftuberperiderm,methanolicperidermextractsofABCG1-RNAiandcontrolperidermweresubjectedtoUPLC/PDA/http://wendang.chazidian.comparativeanalysesoftheresultingdatare-vealeddramaticallyincreasedlevelsof(hydroxy)cinnamoyl/benzoylputrescineconjugates,dihydrohydroxycinnamoylputrescinecon-jugates,andcross-linkeddehydrodimersofferuloyltyramineandferuloyloctopamineinABCG1-RNAiincomparisontocontrolperi-derm(Table1).Bycontrast,caffeicandchlorogenicacid,thelatterbeingthemajorphenoliccompounddetectableincontrolperiderm,werefoundtobereducedinABCG1-RNAiperidermto13and37%ofcontrolperidermlevels,respectively.InagreementwiththebrownishperidermofABCG1-RNAitubers,reducedlevels

of

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Figure4.MorphologicalAlterationsinABCG1-RNAiPlants.

(A)Tuberphenotypeofwild-type(toppanel)andABCG1-RNAi(bottompanel)plants.Tuberswereharvestedfromgreenhouse-grownpotatoplantsandanalyzedafter1weekofstorageat4°C(left).Microscopyofcrosssectionsofwild-typeandABCG1-RNAitubersisshownontheright.pl,phellem;pg,phellogen;pd,phelloderm.Bars=80mm.

(B)Rootphenotypeofwild-type(toppanel)andABCG1-RNAi(bottompanel)plants.MicroscopyofcrosssectionsofemptyvectorandABCG1-RNAirootsisshownontheright.ex,exodermis;co,cortex;en,endodermis;vb,vascularbundle;pe,pericycle.

(C)Reducedsuberinstainingoftuberperidermofwild-type(toppanel)andABCG1-RNAi(bottompanel)plants.Crosssectionsofpotatotuberperidermwerestainedwith?uorolyellowandsubjectedto?uorescencemicroscopy(left,auto?uorescence;middle,?uorolyellow;right,merged).Bars=50mm.

(D)Reducedsuberinstainingoftherootexodermisofwild-type(toppanel)andABCG1-RNAi(bottompanel)plants.Crosssectionsofrootswerestainedwith?uorolyellowandsubjectedto?uorescencemicroscopy(left,auto?uorescence;middle,?uorolyellow;right,merged).Bars=50mm.(E)TuberweightlossofABCG1-RNAiplants.Tuberswereharvestedfromgreenhouse-growncontrol(graybars)andABCG1-RNAiplants(blackbars)andstoredatroomtemperature.Tuberweightwasdeterminedatthetimepointsindicated(n=41,threeindependentexperiments,twoindependentRNAilines,dayspostharvest[dph]).ErrorbarsrepresentSE.Lettersindicatestatisticallydifferentvalues(two-wayANOVA,bP#0.05,c,dP#0.01).