Arabidopsis R2R3-MYB transcription factor AtMYB60

GENETICTRANSFORMATIONANDHYBRIDIZATIONArabidopsisR2R3-MYBtranscriptionfactorAtMYB60functionsasatranscriptionalrepressorofanthocyaninbiosynthesisinlettuce(Lactucasativa)Jong-SugParkÆJung-BongKimÆKang-JinChoÆChoong-IllCheonÆMi-KyungSungÆMyoung-GunChoungÆKyung-HeeRohReceived:18November2007/Revised:30January2008/Accepted:17February2008/Publishedonline:4March2008
TheAuthor(s)2008AbstractTheMYBtranscriptionfactorsplayimportantrolesintheregulationofmanysecondarymetabolitesatthetranscriptionallevel.WeevaluatedthepossiblerolesoftheArabidopsisR2R3-MYBtranscriptionfactorsinflavonoidbiosynthesisbecausetheyareinducedbyUV-Birradiationbuttheirassociatedphenotypesarelargelyunexplored.WeisolatedtheirgenesbyRACE-PCR,andperformedtrans-genicapproachandmetaboliteanalysesinlettuce(Lactucasativa).Wefoundthatonememberofthisproteinfamily,AtMYB60,inhibitsanthocyaninbiosynthesisinthelettuceplant.Wild-typelettucenormallyaccumulatesanthocya-nin,predominantlycyanidinandtracesofdelphinidin,anddevelopsaredpigmentation.However,theproductionandaccumulationofanthocyaninpigmentsinAtMYB60-over-expressinglettucewasinhibited.UsingRT-PCRanalysis,wealsoidentifiedthecompleteabsenceorreductionofdihydroflavonol4-reductase(DFR)transcriptsinAt-MYB60-overexpressinglettuce(AtMYB60-117andAtMYB60-112lines).Thecorrelationbetweentheover-expressionofAtMYB60andtheinhibitionofanthocyaninaccumulationsuggeststhatthetranscriptionfactor-AtMYB60controlsanthocyaninbiosynthesisinthelettuceleaf.ClarificationoftherolesoftheAtMYB60transcriptionfactorwillfacilitatefurtherstudiesandprovidegenetictoolstobetterunderstandtheregulationinplantsofthegenescontrolledbytheMYB-typetranscriptionfactors.Furthermore,thecharacterizationofAtMYB60hasimpli-cationsforthedevelopmentofnewvarietiesoflettuceandothercommerciallyimportantplantswithmetabolicengi-neeringapproaches.KeywordsR2R3-MYBtranscriptionfactor
Repressor
Arabidopsis
LactucasativaAbbreviationsRT-PCRReversetranscription-polymerasechainreactionRACE-PCRRapidamplificationofcDNAends-polymerasechainreactionCaMVCauliflowermosaicvirusCHSChalconesynthaseDFRDihydroflavonol4-reductaseCHIChalconeisomeraseF3HFlavanone3-hydroxylaseANSAnthocyanidinsynthaseUFGTUDP-glucose:flavonoid3-O-glucosyltransferaseCommunicatedbyJ.R.Liu.J.-S.Park
J.-B.Kim
K.-J.Cho
K.-H.Roh(&)MolecularPhysiologyandBiochemistryDivision,NationalInstituteofAgriculturalBiotechnology,Suwon441-707,SouthKoreae-mail:[e-mail]-mail:[e-mail]ence,SookmyungWomen’sUniversity,Youngsan-gu,Seoul140-742,SouthKoreaM.-K.SungDepartmentofFoodandNutrition,SookmyungWomen’sUniversity,Youngsan-gu,Seoul140-742,SouthKoreaM.-G.ChoungDepartmentofPharmacognosyMaterialDevelopment,KangwonNationalUniversity,Samcheok245-711,SouthKorea123PlantCellRep(2008)27:985–994DOI10.1007/s00299-008-0521-1IntroductionFlavonoidmetabolismisuniquetoplantsandproducesagreatnumberandvarietyofcompoundsincludingflavones,flavonols,anthocyanins,andproanthocyanins(Winkel-Shirley2001;Parketal.2004).Thesemoleculesplayimportantrolesinmanyfundamentalprocessesinplants,suchasthepigmentationofflowersandfruits,UVresis-tance,pollenfertility,responsestowoundingandfungalattacks,andenvironmentalstressresponses(Shirleyetal.1995;WeisshaarandJenkins1998;HarborneandWilliams2000;DixonandPiava1995;Moyanoetal.1996;Pietta2000;Cominellietal.2005).Theregulationoftheantho-cyaninpigmentsinplantsisparticularlyimportantinmetabolicengineeringapplicationsbecausethesecom-poundsactasvisualsignalsthatattracttheinsectsandanimalsrequiredforpollinationandseeddispersal(HoltonandCornish1995).Themetabolicpathwaysinvolvedinanthocyaninbio-synthesisarewellestablished,andthecentralpathwaysinthisprocessarehighlyconservedinplants(Shihetal.2006).Moreover,studiesinmodelplants,suchasArabidopsisandmaize,havefacilitatedasoundunder-standingofthegenesinvolvedandtheassemblyoftherelevantenzymecomplexes(Winkel-Shirley2001).Twogroupsofgenesarerequiredforanthocyaninbiosynthesis:structuralgenesthatencodetheenzymesthatdirectlyparticipateinthebiosyntheticreactions,andgenesfortranscriptionfactorsthatregulatetheexpressionofthesestructuralgenesandtheaccumulationofflavonoidmetabolites.Transcriptionfactorscanactasactivatorsorrepressorsofgeneexpression,andmediateeitherincrea-sesordecreasesintranscriptionthroughsequence-specificDNAbindingandprotein-proteininteractions(Broun2004).MYBproteinshavebeenidentifiedinalargenumberofeukaryotes,includingfungi,plantsandvertebrates(Ohietal.1994;MartinandPaz-Ares1997;Slamonetal.1986),andhaverolesinawidearrayofcellularprocesses.Theseincludetheregulationofsecondarymetabolism,signaltransduction,celldivision,andresponsestoplantdiseasesandvariousformsofstress(UV-Blight,coldanddrought)(Larkinetal.1994;Borevitzetal.2000;Strackeetal.2001;Vailleauetal.2002;Cominellietal.2005).TheR2R3-MYB-relatedproteinsactivatethetranscriptionofstructuralgenesthatfunctionindifferentbranchesofphenylpropanoidmetabolism(MartinandPaz-Ares1997).ThepromotersofthesestructuralgeneseachcontainpotentialMYBproteinrecognitionmotifsandalsobindwiththebasichel