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Mediator Subunit18 Controls Flowering Time and Floral Organ Identity in Arabidopsis
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Title: Mediator Subunit18 Controls Flowering Time and Floral Organ Identity in Arabidopsis
Series Title: Zheng Z, Guan H, Leal F, Grey PH, Oppenheimer DG (2013) Mediator Subunit18 Controls Flowering Time and Floral Organ Identity in Arabidopsis. PLoS ONE 8(1): e53924. doi:10.1371/journal.pone.0053924
Physical Description: Journal Article
Creator: Oppenheimer, David
Publisher: Public Library of Science
Place of Publication: San Francisco, CA 94111, USA
Publication Date: January 11, 2013
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Abstract: Mediator is a conserved multi-protein complex that plays an important role in regulating transcription by mediating interactions between transcriptional activator proteins and RNA polymerase II. Much evidence exists that Mediator plays a constitutive role in the transcription of all genes transcribed by RNA polymerase II. However, evidence is mounting that specific Mediator subunits may control the developmental regulation of specific subsets of RNA polymerase II-dependent genes. Although the Mediator complex has been extensively studied in yeast and mammals, only a few reports on Mediator function in flowering time control of plants, little is known about Mediator function in floral organ identity. Here we show that in Arabidopsis thaliana, MEDIATOR SUBUNIT 18 (MED18) affects flowering time and floral organ formation through FLOWERING LOCUS C (FLC) and AGAMOUS (AG). A MED18 loss-of-function mutant showed a remarkable syndrome of later flowering and altered floral organ number. We show that FLC and AG mRNA levels and AG expression patterns are altered in the mutant. Our results support parallels between the regulation of FLC and AG and demonstrate a developmental role for Mediator in plants.
Acquisition: Collected for University of Florida's Institutional Repository by the UFIR Self-Submittal tool. Submitted by David Oppenheimer.
Publication Status: Published
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Holding Location: University of Florida
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System ID: IR00001385:00001

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MediatorSubunit18ControlsFloweringTimeandFloral OrganIdentityinArabidopsisZhenguiZheng1,2* ,HexinGuan1¤,FranciscaLeal1,ParisH.Grey1,DavidG.Oppenheimer1*1 DepartmentofBiology,UniversityofFlorida,Gainesville,Florida,UnitedStatesofAmerica, 2 HowardHughesMedicalInstitute,DepartmentofMolecularGeneticsand Microbiology,UniversityofFlorida,Gainesville,Florida,UnitedStatesofAmericaAbstractMediatorisaconservedmulti-proteincomplexthatplaysanimportantroleinregulatingtranscriptionbymediating interactionsbetweentranscriptionalactivatorproteinsandRNApolymeraseII.MuchevidenceexiststhatMediatorplaysa constitutiveroleinthetranscriptionofallgenestranscribedbyRNApolymeraseII.However,evidenceismountingthat specificMediatorsubunitsmaycontrolthedevelopmentalregulationofspecificsubsetsofRNApolymeraseII-dependent genes.AlthoughtheMediatorcomplexhasbeenextensivelystudiedinyeastandmammals,onlyafewreportsonMediator functioninfloweringtimecontrolofplants,littleisknownaboutMediatorfunctioninfloralorganidentity.Hereweshow thatin Arabidopsisthaliana ,MEDIATORSUBUNIT18(MED18)affectsfloweringtimeandfloralorganformationthrough FLOWERINGLOCUSC ( FLC )and AGAMOUS ( AG ).A MED18 loss-of-functionmutantshowedaremarkablesyndromeoflater floweringandalteredfloralorgannumber.Weshowthat FLC and AG mRNAlevelsand AG expressionpatternsarealteredin themutant.Ourresultssupportparallelsbetweentheregulationof FLC and AG anddemonstrateadevelopmentalrolefor Mediatorinplants.Citation: ZhengZ,GuanH,LealF,GreyPH,OppenheimerDG(2013) MediatorSubunit18 ControlsFloweringTimeandFloralOrganIdentityin Arabidopsis .PLoS ONE8(1):e53924.doi:10.1371/journal.pone.0053924 Editor: XiaoyuZhang,UniversityofGeorgia,UnitedStatesofAmerica Received September10,2012; Accepted December4,2012; Published January11,2013 Copyright: 2013Zhengetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding: ThisworkwassupportedbyNationalScienceFoundationgrantDBI-0115684toDGO.PublicationofthisarticlewasfundedinpartbytheUniversityof FloridaOpen-AccessPublishingFund.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofth emanuscript. CompetingInterests: Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:patrickz@ufl.edu(ZZ);oppenhe@ufl.edu(DGO) ¤Currentaddress:AgriculturalCollege,GuangxiUniversity,Nannin,People’sRepublicofChinaIntroductionMediatorplaysanimportantroleinregulatingRNApolymeraseII(PolII)transcription.TheMediatorcomplexcontains22–28 subunits,andmediatesinteractionsbetweentranscriptionalcoactivatorsandPolII[1,2].Mediatorisevolutionarilyconserved andhasanancienteukaryoticorigin[3];itisfoundinorganisms fromfungitomammalsandplants,althoughtheevolutionary conservationofindividualsubunitsismoderate[4–6].Morethan 30differentsubunitshavebeendescribedthatarepartofthe Mediatorcomplexindifferentorganisms,butonlyabout20 subunitsarefoundinalleukaryotes[6–8].Theothersconsistof eitherspecies-specificsubunitsorotherancillarysubunitsassociatedwithactivationofspecificgenes.Med18isonesubunitofthe Mediatorcomplexandacomponentoftheheadmodulethatis involvedinstimulatingbasalRNAPolIItranscriptioninyeast [4,9]. Arabidopsis Mediatorwasfirstfoundtocontain27subunits,and mostofthemareconservedineukaryotes[5].Untilnow,allthe knownyeast/metazoanMediatorcomponentshavebeenidentifiedinplants[10].PHYTOCHROMEandFLOWERING TIME1(PFT1),nowknownasMED25,wasidentifiedasafactor ofaPhytochromeB(phyB)signalingpathwaythatpromotes floweringandcontrolsfinalorgansize[5,11–14].STRUWWELPETER(SWP)/MED14wasreportedtobeanuclearproteinthat playsaroleindefiningthedurationofcellproliferation[15].Some MediatorsubunitslikeMED25,MED8,MED16,andMED21act asintegratorsinresponsetoenvironmentalcuesin Arabidopsis [16– 21].MED18isasubunitoftheheadsubmoduleoftheplant Mediatorcomplex[10].Recently,Kimetal.(2011)reportedthat severalMediatorsubunitsincludingMediator18(MED18)are requiredformicroRNAbiogenesis[22].Littleisknownabout Mediatorfunctionduringfloralorganformationoritsroleinthe regulationoffloweringtime. Thetransitionfromvegetativegrowthtoreproductivedevelopmentin Arabidopsis isregulatedbymultiplefloralinduction pathways.Geneticstudiesofthetimingoffloweringin Arabidopsis haverevealed5majorpathways[23].Thephotoperiodand vernalizationpathwaysintegrateexternalsignalsintothefloral decision,theautonomousandgibberellin(GA)pathwaysact independentlyofenvironmentalcues,whereastheendogenous pathwayaddsplantagetothecontroloffloweringtime.The floweringpathwaysareinterconnectedandconvergeonafew floralintegrators,suchas FLOWERINGLOCUST ( FT )and SUPPRESSOROFOVEREXPRESSIONOFCONSTANS1 ( SOC1 or AGL20 )[24–26].Oneimportantregulatoroffloralinitiationisthe MADS-boxtranscriptionfactorFLOWERINGLOCUSC(FLC), whichactsasanegativeregulatoroffloweringinresponsetoboth endogenousandenvironmentalsignals;itisalsoanintegratorof theautonomousandvernalizationpathways[23,27,28].The autonomousandvernalizationpathwaysbothsuppressthe expressionof FLC [29],resultinginadecreasedexpressionof FLC andconsequentlyincreasedexpressionof FT and/or SOC1 in alaterdevelopmentalstageorafteraprolongedexposureofplants PLOSONE|www.plosone.org1January2013|Volume8|Issue1|e53924

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tolowtemperature[23,30].Theflorigen,FT,directlyregulates floralmeristemidentitygenessuchas APETALA1 ( AP1 )and initiatesfloralmorphogenesis[31,32]. Followingthevegetativetofloraltransition, Arabidopsis flowers developfourdifferentorgantypesthatarearrangedinconcentric whorls:thefirstwhorlcontainsfoursepals,thesecondwhorl containsfourpetals,sixstamensdevelopinthethirdwhorl,and twofusedcarpelsforminthefourthwhorl.Thecontroloffloral organidentityhasbeenintensivelystudiedinthepast25years. Analysisoffloralhomeoticmutantsledtotheproposalofasimple geneticmodel,explaininghowthreegroupsofregulatorygenes (classA,BandCgenes)aloneorbyinteractions,controltheorgan identityofthefourfloralwhorls[33,34].Theterminationofstem cellsinthefloralmeristemrequires AGAMOUS ( AG ),aMADSdomaintranscriptionfactor[35];AsaclassCfloralhomeotic gene, AG specifiesstamenidentitytogetherwiththeBclassand SEPALLATA ( SEP )genesandcarpelidentitytogetherwiththe SEP genes[34,36]. AG activates SPOROCYTELESS ( SPL ),which controlssporogenesisinbothstamensandcarpels[37].Clearly, AGisoneofthemostimportantregulatorsforthefloraltransition, floralorganidentity,andsporeformation. Inthisstudy,wedescribethefunctionof Arabidopsis Mediator subunit18(MED18),andshowthatitcontrolsfloweringtimeand floralorganidentitybytranscriptionalregulationof FLC and AG .MaterialsandMethods PlantMaterialsandGrowthConditionsAllthetransgenicandmutant Arabidopsis linesusedinthisstudy wereofecotypeColumbia(Col)exceptforthe ag-1 and pi-1 mutants,whichwereintheLandsberg erecta (L er )background. Plantsweregrowninatemperaturecontrolledgreenhouseat22– 24 u C,witharelativehumidityof60%–70%,anda16hourslight and8hoursdarkphotoperiod. Forshortdayexperiments,Plantsweregrowninagrowth chamberequippedwith40Wfluorescentlighttubeswitha9 hourslightand15hoursdarkphotoperiod.AnalysisofTranscriptsLevelsSemi-quantitativeRT-PCRwasusedtomeasurethetranscript levelsof MED18 andthefloralhomeoticgenesusing ACTIN4 (At5g59370)and GAPC (AT3G04120)asloadingcontrols.Total RNAsamplesweretreatedextensivelywithRNase-freeDNaseIto removeanycontaminatinggenomicDNA.First-strandcDNAwas synthesizedusing1mgoftotalRNAina20mlreactionvolume usingHighCapacitycDNAArchiveKitfromAppliedBiosystems (FosterCity,CA)accordingtothemanufacturer’sinstructions, followedbyphenol/chloroformpurification,andethanolprecipitation.ThecDNAweredissolvedin30mlTEbufferand1mlwas subjectedtoPCRina20mlreactionvolume.TheRT-PCRruns were20to30cycles,dependingonthelinearrangeofPCR amplificationforeachgene,withcycleparametersof94 u Cfor 0.5min,58 u Cfor0.5min,and72 u Cfor1minforeachcycle, withafinalincubationof72 u Cfor10min. AP1 AP2 AP3 PI ,and AG ,primersweredesignedaccordingtopublishedsequences[38]. AllotherprimersdesignedinthisstudywereinTableS3. QuantitativeRT-PCRwasmodifiedfromapreviously publishedmethod[39].Mutantandwildtypeseedlingorflowers weredissectedandpooled.TotalRNAwasextractedusing RNeasyplusmicrokit(Qiagen)andRNAquantity( 100ng/ml) andpurity(260/280 2.0,260/230 1.65)weredeterminedusing aNanodrop.RNAintegrity(RIN 8.5)and28S/18Sratio( 1.5) wasassessedusingaBioanalyzer2100(AgilentTechnologies).A quantityof500ngofhigh-qualityRNAforeachpooledsample wasconvertedintocDNAusingtheiScriptcDNASynthesisKit (Bio-Rad).GeneexpressionwasdeterminedusingtheCFX96TM RealTimesystem(Bio-Rad)with Act2/8 [40]asacontrol. FT SOC1 and FLC PrimersweredesignedaccordingtoZhouandNi [41].Theresultsweredeterminedusing DD Ctmethod[42],4 replicatesofpooledsampleswereusedforbothwildtypeand mutantseedlingsandflowers.InsituHybridizationWeusedpreviouslyestablishedmethodsfor insitu hybridization [43]withthefollowingmodifications.Togeneratetemplatesfor insitu probesynthesis,acDNAwasPCRamplifiedusingprimers thatcontainedthephageT7RNApolymeraseinitiationsequence. ThePCRproductwasusedfor invitro transcriptionofdigoxigenin-labeledprobesusingaDIG-RNAlabelingkit(Roche AppliedScience).DIG-labeledRNAprobeswerenothydrolyzed, andusedatafinalconcentrationof400ng/mlinthehybridizationsolution.Slideswerephotographedunderbrightfield illumination.StatisticalAnalysisAllgroupdifferencesinourdependentvariableswererevealed usingtwo-tailedStudent’sT-tests,and a -Levelsweresetat0.05.Results MED18 ControlsFloweringTimeandFloralOrgan IdentityThe MED18 (NP_565534;At2g22370)genewasoriginally identifiedthroughaphylogenomiccomparisonofsingle-copy genesconservedinangiosperms[44].Additionaldatabase searchesrevealedthatMED18wasaplanthomologofMediator subunit18(Fig.S1,ref[3,5]).Togainfurtherinsightintothe functionofthisgenein Arabidopsis ,weexaminedthephenotypesof plantshomozygousforT-DNAinsertionsinthe MED18 coding sequence.WeidentifiedT-DNAinsertionalleles(SAIL_889_C08, med18-1 ;SALK_027178, med18-2 )fromtheSALKT-DNA insertiondatabase(http://signal.salk.edu/cgi-bin/tdnaexpress) [45,46],andconfirmedthelocationoftheT-DNAinsertsby PCRusing MED18 andT-DNAspecificprimers(Fig.1A).We alsoexamined MED18 mRNAlevelsinplantshomozygousfor eachoftheallelesusingRT-PCR. MED18 mRNAwas undetectablein med18-1 plantscomparedtowildtype.Thisresult suggeststhat med18-1 isanullallele.Kimetal.[22]reportedweak expressionof MED18 mRNAin med18-2 plants,butwefailedto detectany MED18 mRNAusingprimerslocatedoneithersideof theT-DNAinsertion(Fig.S2). Wheneither med18-1 or med18-2 wascrossedwithwildtype plants,theF1showedawildtypephenotype,whichdemonstrates thatboth med18-1 and med18-2 arerecessive.Acomplementation testwasalsoperformedusingthe med18-1 and med18-2 alleles.The F1plantsfromacrossof med18-1andmed18-2 plantsshoweda med18 phenotype(TableS1),whichisdescribedbelow.Thisresult demonstratesthat med18-1 and med18-2 areallelic. Inadditiontothepreviouslydescribedphenotype[22], med18 mutationscauseasyndromeofrelatedphenotypesaffecting floweringtime,inflorescencestructure,andflowermorphology. Underlongdayconditions(16hourlight,8hourdark),both med18-1 and med18-2 mutantsdidnotflowerevenafter35days aftergermination(DAG)(Fig.1C).Incontrast,wildtypeplants floweredapproximately14DAG(12leaves)(Fig.1B).The med18-1 mutationalsoaffectsflowermorphology.Inwildtypeflowers,the sepalsfullyenclosethedevelopingflowersuntilshortlyafterthe beginningofanthesis(Fig.1D).Thesepalsof med18-1 flowersdidMediatorSubunit18 ControlsFlowerDevelopment PLOSONE|www.plosone.org2January2013|Volume8|Issue1|e53924

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notfullyenclosethedevelopingflowerssuchthatthebuds appearedprematurelyopen(Fig.1E). Moststrikingofallwasthat med18-1 mutantsshowed dramaticallyalteredfloralorgannumbersascomparedto wildtype.Allfloralorganswereaffected(TableS1,Fig.1F–H), morethan40and80percentoftheflowerson med18-1 plants hadabnormalsepalsandpetalsnumberrespectively,while 80%oftheflowershadfewerthan6stamens.Approximately Figure1.PhenotypesofMediatorsubunit18(med18)mutants. (A)SchematicdiagramoftheMED18geneshowingthelocationsofT-DNA insertionsinthe med18 mutants.Blackrectanglesrepresentexons,linesrepresentintrons,andtrianglesrepresentT-DNAinsertions;the med18-1 mutationcorrespondstoinsertionlineSAIL_889_C08,whereasthe med18-2 mutationcorrespondstoinsertionline,SALK_027178.(B,C)35dayold wildtype(B), med18-1 (C)plants.(D,E)Inflorescenceofwildtype(D),and med18-1 (E)plants.(F–H) Arabidopsis wildtype(F), med18-1 (G),and dissected med18-1 (H)flower.(I,J)Transversesectionofwildtype(I),and med18-1 (J)carpels.(K,L)Anthersofwildtype(K)and med18-1 (L)(stained withKI/I2)attimeofflowering.Scalebars:1mminD,E,F,GandH;100mminI,J,KandL. doi:10.1371/journal.pone.0053924.g001 MediatorSubunit18 ControlsFlowerDevelopment PLOSONE|www.plosone.org3January2013|Volume8|Issue1|e53924

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25%of med18-1 mutantflowershadmorethantwocarpels (TableS1,Fig.1I,J).Inadditiontothealteredfloralorgan number, med18-1 mutantsalsoshoweddelayedstamendevelopmentandlatermaturationofpollen(Fig.1K,L),whichled toreduceseedset. Overexpressionof MED18 causedincreasedcarpelandstamen numbers(Fig.S3A),andreducedpetalnumbers(Fig.S3B), Carpel-likesepalswerealsoobservedin MED18 overexpressing plants(Fig.S3C),andthese MED18 overexpressingplants floweredearlierthanwildtype(datanotshown).MED18 Expressionduring Arabidopsis DevelopmentThefloweringtimeandfloralorganpatterningdefectsinthe med18 mutantssuggeststhat MED18 playsaroleinregulating genesimportantforfloweringandfloralorgandevelopment.To determineif MED18 expressioncoincideswiththephenotype,we examinedtheRNAexpressionpatternof MED18 inwildtype plantsusing insitu hybridization. MED18 transcriptscouldbe detectedindevelopingseeds(Fig.2A),theinflorescencemeristem, thefloralmeristem,andfloralorganprimordia(Fig.2BandC). Transcriptabundanceappearedhighestinthedevelopingstamens andpistils(Fig.2D).Inlaterstages, MED18 expressionwas abundantindevelopingovulesandpollen(Fig.2E)andin addition, MED18 mRNAwasweaklyexpressedinpetals,sepals, andthewallsofcarpelsuptostage12(Fig.2E).Thestrong expressionof MED18 intheinflorescencemeristemandfloral organsisconsistentwiththefloraldefectsobservedin med18-1 mutants.med18 MutantsAffectFloweringTimeinBothLongand ShortDaysInlongdaycondition(16hlight,8hdark),themutantplants didnotfloweruntilmorethan40leaves;andinshortday(9h light,15hdark),after95days(morethan60leaves), med18 mutantsstilldidnotflower,andmanyrosetteandcaulineleaves showedsenescence(Fig.3A).Comparedtowildtypeplants (12leavesunderlongdaysandabout40leavesundershortdays), med18 islatefloweringunderbothlongandshortdays(Fig.3A).med18 MutantsAffectFloweringTimethroughupregulationof FLC ExpressionTounderstandhowMED18regulatesfloweringtime,we comparedtheexpressionlevelsofkeygenesofdifferentpathways inmutantandwildtypeseedlingsusingRT-PCR. FT and FLC showexpressiondifferencesinthe med18 mutantsascomparedto wildtypeplants(Fig.S4).WeperformedQPCRandshowedthat FT mRNAwasdownregulatedapproximately4.7fold(Fig.3B, p # 0.004),whilethe FLC transcriptlevelwasmuchhigher (9.5fold)in med18-1 thaninthewildtypeplants(Fig.3B, Figure2.ExpressionpatternsofMED18. ( A–D )Longitudinalsectionsofwildtypeshow MED18 mRNAdetectedin(A)developingseed,(B) inflorescencemeristem,floralmeristemandsepals,(C,D)petal,stamen,andcarpelprimordia.( E )Transversesectionofawildtypeflowershows MED18 mRNAexpressioninallfloralorgans.( F ) MED18 mRNAsensestrandcontrolshowingnonon-specifichybridization.NumbersinB–DandF indicateflowerstages;IMinBandFindicatesinflorescencemeristem.Scalebars:100mminB–Fand25mminA. doi:10.1371/journal.pone.0053924.g002 MediatorSubunit18 ControlsFlowerDevelopment PLOSONE|www.plosone.org4January2013|Volume8|Issue1|e53924

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p # 0.001).For FT and SOC1 ,whicharedownstreamof FLC ,only FT mRNAwassignificantlysuppressedbythelossof MED18 function,whereas SOC1 mRNAlevelsshownosignificant difference(Fig.3B,p=0.106).med18 MutantsareResponsivetoVernalizationbutnot GATreatmentToexaminetheresponsivenessof med18 tovernalization,the med18-1 plantswereplantedandkeptat4 u Cfor4weeksinthe darkandthentransferredtonormalgrowthtemperature(23 u C). Thevernalization-treated med18 plantsfloweredmuchearlierthan theuntreatedplants,afterproducingabout14leaves,whichis comparabletothatofwildtypeplants(Fig.3C.Theuntreated plantsfloweredafterproducingcloseto40leaves,whichsuggests thatthefloweringofthemutantplantswasdecreasedby vernalization(Fig.3C).ToexaminetheeffectsofGAtreatment, aGAsolutionof20mMwassprayedtwiceaweekafter germinationuntilflowering.TheresultsshowthatGAhasno obviouseffectonthefloweringof med18 mutantsascomparedto wildtypeplants.TheGA-treated med18-1 plantsinitiatedflowering afterproducingmorethan35leaves,whichisnotsignificantly differentthanuntreatedmutantplants(p=0.115,Fig.3D).Our resultsareconsistentwithpreviousresultsthatshowthat vernalizationpromotesfloweringbyrepressing FLC andreleasing FT fromrepression[23,47].Theresponsivenessof med18-1 to vernalizationsuggeststhat MED18 regulatesfloweringtime throughthevernalizationpathway.MED18 AffectsFloralOrganFormationinallFourWhorlsBothmutationandoverexpressionof MED18 alteredthe numberoffloralorgans(Fig.1G–I,TableS1,Fig.S3).The increasednumberofpetalsandfewerthannormalstamensseenin the med18 mutantswasreminiscentofthefloralphenotypeof agamous ( ag )mutants[48,49].Tofurtherexplorethefloral developmentalpathwayinwhich MED18 isinvolved,we constructeddoublemutantswiththewell-studiedfloraldevelopmentalregulators, ag pistillata ( pi )and apetala2 ( ap2 ).Flowersonthe med18-1ag-1 doublemutantshowedthestrikingreiterationof sepalsandpetalscharacteristicof ag mutants(Fig.4A–C,Table S2).The med18-1pi-1 doublemutantflowershadabnormal carpels,butfewersepalsthanthe pi-1 singlemutant(Fig.4D,E, TableS2).Theflowersonthe med18-1ap2-5 doublemutant displayedamuchmorecomplexphenotype.Carpelsinboththe firstandfourthwhorlswerepresentasseeninthe ap2-5 single mutant.Inaddition,doublemutantflowersexhibitedpetaloid stamensandotherpetaloidstructuresinwhorls2and3(Fig.4F, G,TableS2).MED18 AffectsFloralOrganFormationthrough RegulationofFloralHomeoticGeneExpressionTheclearepistasisobservedinthe med18-1ag-1 doublemutants suggestedthat MED18 and AG functioninthesamepathwayto controlfloraldevelopment.Therefore,weexamined AG mRNA levelsaswellasthelevelsofmRNAforotherkeyfloralregulators in med18 mutantsusingsemi-quantitativeRT-PCR.Ourresults showedthatthemRNAexpressionlevelsof AG AP1 and PI were Figure3.MED18controlsfloweringtime. ( A ) MED18 controlsfloweringtimeunderbothlongdayandshortdayconditions.Arrowindicatesthat floweringwasnotachievedwhentheexperimentwasterminated.( B )Transcriptslevelsof FT and FLC arealteredin med18 mutants.( C,D )Flowering timeinvernalization(C)andgibberellin(D)treatment.Vm:vernalization,GA:gibberellin.Scalebarsindicatemean 6 s.e.;**p # 0.01. doi:10.1371/journal.pone.0053924.g003 MediatorSubunit18 ControlsFlowerDevelopment PLOSONE|www.plosone.org5January2013|Volume8|Issue1|e53924

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downregulatedin med18-1 mutantswhile AP2 and AP3 mRNA showednoobviouschange(Fig.S5).QPCRresultsrevealed AG mRNAwasdownregulatedupto4.8fold(Fig.5A,p # 0.007), AP1 and PI mRNAexpressionlevelswerealsoreducedinthe med18-1 mutant1.9(p # 0.014)and2.4fold(p # 0.011)respectively,while AP2 and AP3 transcriptlevelsshowednosignificantdifferences between med18-1 andthewildtype(Fig.5A,p=0.068and0.082). Thedecreased AG expressionislikelytocauseastamentopetal transitionaswellasabnormalcarpeldevelopment[49],as observedin med18-1andMED18 overexpressionflowers(Fig.1F– H,Fig.S3).Theseresultssupportthehypothesisthat MED18 controlsfloralorganidentitythroughitsregulationoffloral homeoticgeneexpression.Altered AG ExpressionPatternsareObservedin med18 MutantsTofurtherexaminetheexpressionof AG in med18-1 mutants, in situ hybridizationusingan AG antisenseRNAprobewas performedontissuesectionsfromwildtype(Fig.5B,D,F,H) and med18-1 plants(Fig.5C,E,G,I).Asdescribedpreviously [50,51], AG isnotexpressedintheinflorescencemeristem,norin stage1orstage2floralmeristemsinwildtypeplants.Strong AG expressionisfirstfoundinthecenterofstage3andstage4 wildtypeflowers,butnotintheemergingsepalprimordia(Fig.5B). Duringlaterstagesofwildtypeflowerdevelopment, AG expression ispresentinstamensandcarpels(Fig.5D,F,H).In med18-1 mutants,apatternof AG expressionsimilartothatseeninwildtype wasobservedinboththeinflorescencemeristemandinstage1 andstage2floralmeristems(Fig.5C).Inflowersfromstage4to stage7,weak AG expressionwasdetectedinthecenterofthe carpelprimordia,butstrong AG expressionwasobservedin stamenprimordia,althoughnoexpressionwasobservedinpetal andsepalprimordia(Fig.5C,E,G).Inlaterdevelopmentstages (stage11,Fig.5H,I), AG expressionwasobservedindeveloping pollensimilartothatobservedinwildtypeflowers(Fig.5H),butit wasdifficulttodetectanysignalinvascularbundlesofstamens andcarpels(Fig.5I).Thisresult,togetherwiththeresultsofthe QPCRanalysis,stronglysuggeststhat MED18 isrequiredto maintainthenormal AG expressionlevelandpatternduringearly stamenandgynoeciumdevelopment.DiscussionUndermanyconditionsMediatorappearstofunctionasa generaltranscriptionfactor[52].Nonetheless,expressionprofiling ofyeastMediatorsubunitmutantshasrevealedthedirect regulationofspecificsetsofgenesbyMediator[53],andanalysis ofMediatorin Arabidopsis hasshownthattheMediatorsubunits areimportantinregulatingspecificdevelopmentalprocesseslike earlyembryopatterning[54,55],cellnumberandorgansize [12,15],floweringtimecontrol[5,14,56]environmentalregulation anddefensegeneregulation[16,18–21,57]. MED18wasfirstcharacterizedasageneraltranscriptionfactor thatpromotesPolIItranscriptionthroughpromotionofthe transcriptionofmiRNA,andknockingdown MED18 expression causedabnormalcotyledonandsiliquedevelopmentaswellasa laterfloweringphenotype[22].However,themechanismsby whichMED18regulatesfloweringarepoorlyunderstood,and nothingisknownabouthowMED18regulatesfloralorgan identity.Inthisstudy,weidentifiedMED18asaregulatorofboth floweringtimeandfloralorganidentity.Ourfindingsshowthat MED18controlsfloweringtimebyup-regulating FLC expression, whichalsoaffectstheexpressionofthedownstreamgene, FT Figure4.Doublemutantofmed18-1andfloralhomeoticgenes. ( A ) ag-1 flower.( B ) med18-1ag-1 doublemutantflower.( C )dissectionof med18-1ag-1 doublemutantflower.( D ) pi-1 flower.( E ) med18-1pi-1 doublemutantflower.( F ) ap2-5 flower.( G ) med18-1ap2-5 doublemutant flower.Scalebars:1mm. doi:10.1371/journal.pone.0053924.g004 MediatorSubunit18 ControlsFlowerDevelopment PLOSONE|www.plosone.org6January2013|Volume8|Issue1|e53924

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Afterthefloweringtransition,MED18playsaroleinfloralorgan identitybyregulatingthe AG expressionlevelandpatternaswell as AP1 expressionlevels(Fig.5,6).Theseresultssuggestthat MED18isimportantintheintegrationofkeysignalingpathways inplantsbycontrollingtargetgenestranscription(Fig.6). The med18 mutantsdisplayvariousdevelopmentaldefects, suggestingthatMED18regulatesmultiplepathways.Thisisin agreementwiththefunctionofanimalandyeastMediatorproteins, whichhavebeensuggestedtoregulatebothbasicandspecific transcription[8,58,59].WehavefocusedonastudyofMED18 functionduringfloweringandfloralorgandevelopment.Wefound that MED18 mRNAislocalizedmainlytotheprecursorcellsof inflorescencemeristem,thefourfloralwhorlsandstronglyinthe pollenandovuleprimordia.ThissuggestsaroleforMED18inthe controloffloralandreproductiveorganinitiationanddevelopment. Floralprimordiaarisefromtheinflorescencemeristem,andfloral organsarethenformedinthefloralmeristem.TheABCmodelof flowerdevelopmentexplainshowthreeclassesofgenescontrol sepal,petal,stamen,andcarpelidentity[33].Furthermore,the modelindicatesthatclassAandCgenesaremutuallyantagonistic [60].Thepreviouslyisolatedfloralhomeoticgenesallseemtocode forpotentialtranscriptionfactors.AP1,AP3,PI,AG,andtheSEP proteinscontaintheMADSdomainknowntobindtoDNA[36]. AP2containsanotherDNAbindingdomain,theAP2domain[61]. GeneralregulatorslikeMediatorarelikelytoregulatethesegenes. Ourstudieshaveshownthatmutationsin MED18 causedown regulationofseveralhomeoticgenes,suchas AP1 PI and AG ,but notallofthem;thisfindingsuggeststhattheeffectofMED18on homeoticgeneexpressionisgenespecific. BecauseMED18affectstheexpressionofmultiplegenes,the flowersof med18 mutantsshowvariablephenotypes,suchas4–14 petals,2–6stamens,0–6sepalsand1–3carpels.Inoneofthe flowersthathad14petals,fourstamensareclearlyvisible(Fig.S6). Therefore,theeffectofthe med18 mutationcannotbeasimple homeotictransformationofthefloralorgansintopetals.Itis interestingtonotethatthe med18 mutationcausesadown Figure5.MED18regulatesfloralhomeoticgeneexpression. (A) Relativetranscriptlevelsofsixfloralhomeoticgenesdeterminedby real-timeRT-PCRinwildtypeand med18-1 mutants(**p # 0.01, *p # 0.05).(B,D,F,H) AG expressionpatternasdeterminedby insitu hybridizationinwildtypeflowers.(C,E,G,I)The AG expressionpattern in med18-1 mutantflowers.NumbersinB–Ishowflowerstages,IMinB andCindicatesinflorescencemeristem.Arrowsin(H)and(I)indicate expressioninvascularbundlesofstamensandcarpels,in(F)and(G) showingdevelopingpedals.ScalebarsinB–I:50mm. doi:10.1371/journal.pone.0053924.g005 Figure6.ModelofMED18controlfloweringtimeandfloral organidentity. GrowthsignalsaretransmittedtotheMediator complexbydirectactionorthroughHEN3onMED18toregulatethe transcriptionoftargetgenes.BothfloweringtimeintegratorFLCand floralorganidentityorganizerAGtranscriptslevelsaredeterminedby MED18.Inaddition,MED18affectsthetranscriptionofothertarget genes,whicharefloweringtimeandfloralorganidentityregulators, suchas FT AP1 and PI .Theseregulatorsworktogetherandwithother factorstocontrolfloweringandfloralorganformation. doi:10.1371/journal.pone.0053924.g006 MediatorSubunit18 ControlsFlowerDevelopment PLOSONE|www.plosone.org7January2013|Volume8|Issue1|e53924

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regulationof AG expression,yetsomeflowershavethreecarpels. AccordingtotheABCmodel,downregulationofanAclassgene (suchasAP1)willcauseectopiccarpelformation. Thelaterfloweringphenotypeobservedin med18 mutantsis causedbyup-regulationof FLC mRNAexpression.FLCalso containsaMADSdomain,andinterestingly,both FLC and AG expressionlevelsarealteredin med18 mutants,butinopposite directions.TherearesimilaritiesbetweentheDNAstructureof FLC and AG .Mostofthegenesinthe Arabidopsisthaliana genome haveshort( 1kb)introns[62],butintron1in FLC andintron2 in AG arebothgreaterthan3.0kbinlength,andbothofthese intronshavecis-elementsthatareimportantfortranscriptional control[27,63,64]. HUA1 HUA2 HUAENHANCER2 [ HEN2 ] and HEN4 wereshowntopositivelyregulate AG expression,either byinhibitingprematurepolyadenylationwithin AG intron2orby promotingthesplicingofthisintron,and hua1hua2 double mutantshavereducedlevelsof FLC mRNA[65].Alltheseresults andourdatathat MED18 regulates FLC and AG mRNA expressionlevels,stronglysupporttheideaofparallelregulation of FLC and AG [66]. Theresultofup-regulationof FLC anddown-regulationof AG suggestthat MED18 isnotageneraltranscriptionrepressor,but ratheritplaysdifferentrolesdependingontheidentityofitstarget, and/orthedevelopmentalstage.Themechanismbywhich MED18affects FLC and AG expressioniscurrentlyunknown. Highlyspecificgeneregulationisthoughttobedeterminedby activatorsandcombinatorialuseofcofactors.Inyeast,Med18acts downstreamofCDK8,andmayactasadirectprocessorof signalingpathwaysfordeterminingspecificgeneexpression[53]. Med18wasalsoreportedtoberequiredforproperterminationof transcriptionofasubsetofgenesduringyeastbudding[67].In Arabidopsis ,theCDK8homologis HUAENHANCER3 [ HEN3 ], whichalsocontrolsorganidentityandshowsimilarloss-of-Cfunctionphenotypes(WangandChen,2004),suggestingthat HEN3 mayregulateorganidentitythrough MED18in Arabidopsis (Fig.6).Ourresultssuggestaconservedmechanismmayexistin yeastandplants.SupportingInformationFigureS1Phylogenetictreeofeukaryotic MEDIATOR SUBUNIT18 ( MED18 ). TheBayesianinferenceanalysiswas derivedfrom406aminoacidpositionsofMED18indifferent speciesofEukaryotes.Thebestmodelaminoacidreplacementfor MED18sequenceswasJTTandgammamodelforsubstitution rateheterogeneitybetweensites.Bayesianphylogeneticinference wasperformedwithMrBayesVersion3.0usingfourchainsand 2,000,000generations.Numbersatnodeindicateposterior probabilities;scalebarshows0.2aminoacidsubstitutionspersite. (TIF)FigureS2RT-PCRresultof MED18 showingthat MED18 mRNAwasnotdetectedin med18-1 med18-2 and med18-1/med18-2 T1plantsusingprimersdesigned forthePCRproductincludingbothinsertionsites. The GAPC genewasusedasacontrol. (TIF)FigureS3Phenotypeofoverexpressionof MED18 in transgenicplantstransformedwitha 35S::MED18 construct. (TIF)FigureS4RT-PCRresultsofselectedfloweringtime regulatorsinwildtypeand med18-1 seedlings. In med18-1 plants, FLC isup-regulated, FT isdown-regulated,butothersshow noobviousdifference. (TIF)FigureS5RT-PCRresultsofselectedfloralorgan identitygenesinwildtypeand med18-1 flowers. In med18-1 plants, AP2 PI and AG aredown-regulated,butothers shownoobviousdifference. (TIF)FigureS6A med18-1 mutantflowerthatshows14petals and4stamens. (TIF)TableS1Numberofdifferentorgantypesin med18 flowers.aa200flowerswerecounted.bmeanof200lowers.c Organnumberis6ormore.dOrgannumberis4orless.eOrgan numberis1or1.5(somecarpelsonlyhavehalforonethirdof normalsizelocatedatthetipofpistil).fOrgannumberisbetween 2and3(samease).Mutantplantsweregrowninthegreenhouse (16hlight,23 6 2 u C),wildtypeplantsweregrownunderthesame conditions.Thefloralorganson200wildtypeplantswerealso counted,andallflowersshowed4sepals,4petals,6stamensand2 fusedcarpels,except2flowersshowed5petals.The med18-1 allele isastrongalleleandallfourfloralorgansshowsignificant differencesfromwildtype(**p 0.01), med18-2 isaweakerallele, andonlypetalsandstamensshowsignificantdifferencesfrom wildtype.TheF1plantsfromacrossof med18-1 with med18-2 ( med18-1 6 med18-2 )alsoshowobviousfloralorgannumber changes. (DOCX)TableS2Numberofdifferentorgantypesinfloral homeoticmutantsanddoublemutantswith med18-1 flowers.aa,atleast15flowerswerecountedforeachmutant.b, thefirstwhorlofeachorganshowedthenormalorgannumberfor thatwhorl.c,manyflowersshowed1connectedcarpel-likesepal. (DOCX)TableS3Primersdesignedinthisstudy. (DOCX)AcknowledgmentsTheauthorsgratefullyacknowledgetheSALKInstituteGenomicAnalysis LaboratoryandSyngentaforsupplyingtheT-DNAinsertionalleles.AuthorContributionsConceivedanddesignedtheexperiments:ZZDGO.Performedthe experiments:ZZHGPG.Analyzedthedata:ZZFLDGO.Wrotethe paper:ZZDGO.References1.BlazekE,MittlerG,MeisterernstM(2005)ThemediatorofRNApolymerase II.Chromosoma113:399–408. 2.CasamassimiA,NapoliC(2007)Mediatorcomplexesandeukaryotic transcriptionregulation:anoverview.Biochimie89:1439–1446. 3.BourbonHM(2008)Comparativegenomicssupportsadeepevolutionaryorigin forthelarge,four-moduletranscriptionalmediatorcomplex.NucleicAcidsRes 36:3993–4008. 4.KornbergRD(2005)Mediatorandthemechanismoftranscriptionalactivation. 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64.HongRL,HamaguchiL,BuschMA,WeigelD(2003)Regulatoryelementsof thefloralhomeoticgeneAGAMOUSidentifiedbyphylogeneticfootprinting andshadowing.PlantCell15:1296–1309. 65.ChengY,KatoN,WangW,LiJ,ChenX(2003)TwoRNAbindingproteins, HEN4andHUA1,actintheprocessingofAGAMOUSpre-mRNAin Arabidopsisthaliana.DevCell4:53–66. 66.SimpsonGG(2004)Theautonomouspathway:epigeneticandpost-transcriptionalgeneregulationinthecontrolofArabidopsisfloweringtime.CurrOpin PlantBiol7:570–574. 67.MukundanB,AnsariA(2011)NovelroleformediatorcomplexsubunitSrb5/ Med18interminationoftranscription.TheJournalofbiologicalchemistry286: 37053–37057.MediatorSubunit18 ControlsFlowerDevelopment PLOSONE|www.plosone.org10January2013|Volume8|Issue1|e53924