FTproteinisa“florigen”CO–atranscriptionfactorin

leaves,respondtoday-length(photoperiodsensor)FT–atranscriptionalco-activator,travels

inphloemfromleaftoSAM,targetofCOSo……..PhotoperiodicfloweringinplantsFTproteinFTproteinHd3aproteinGI1.我們當(dāng)時(shí)用一個(gè)金屬板來(lái)熱擊葉片，金屬板下焊接的一個(gè)Ｕ型金屬管同恒溫水浴鍋相連。當(dāng)特定溫度的水流經(jīng)金屬管時(shí)會(huì)加熱金屬板，而固定在金屬板上的葉片就會(huì)被熱擊誘導(dǎo)ＦＴ基因的表達(dá)。2.問(wèn)題是當(dāng)水流入金屬管時(shí)的溫度比較高，而當(dāng)繞金屬板一周經(jīng)過(guò)熱交換再流出金屬管時(shí)，其溫度就降低了很多。因此金屬板表面的溫度不均勻，在金屬板溫度較低區(qū)域的一部分植株的熱擊誘導(dǎo)效果并不好。當(dāng)時(shí)對(duì)這部分植株也取了樣并進(jìn)行了檢測(cè)，結(jié)果發(fā)現(xiàn)如同預(yù)料的一樣，不能檢測(cè)到FTmRNA的轉(zhuǎn)運(yùn)。因此，當(dāng)時(shí)我將這部分樣品的結(jié)果明顯標(biāo)出并排除在統(tǒng)計(jì)之外3.還有，我從來(lái)就沒(méi)有向尼爾森承認(rèn)過(guò)作假，一開始我就明明白白的給他解釋為什么將那些數(shù)據(jù)排除在統(tǒng)計(jì)之外，不知尼爾森此說(shuō)有何用意？4.在2006年9月份我回到中國(guó)，然后由一個(gè)新的研究人員來(lái)接手我的工作。她在重復(fù)兩次沒(méi)有得到預(yù)期結(jié)果的情況下，對(duì)我們以前的數(shù)據(jù)處理方法提出質(zhì)疑。如果那些熱擊效果不好的樣品的結(jié)果也加入計(jì)算的話，會(huì)影響到該次實(shí)驗(yàn)的結(jié)果。我提出是金屬板的問(wèn)題，并在中國(guó)重新設(shè)計(jì)和做了一個(gè)新的金屬板郵寄給他們，但不知為何他們并沒(méi)有使用。5.我認(rèn)為在僅有的兩次重復(fù)而沒(méi)有得到預(yù)期結(jié)果的情況下就匆匆撤稿，未免太草率了（我推測(cè)可能跟最近發(fā)表的一個(gè)研究結(jié)果有關(guān)，該研究結(jié)果不支持我們的結(jié)論。但由于試驗(yàn)體系的不同，我認(rèn)為該結(jié)果并不能否定我們的研究.）。HuangTao’sResponsemainpointsFTmRNAcontributestothesystemicflorigensignallinginfloralinductionShortDay(10wpi)LongDayshortDay(6wpi)(6wpi)mFTmRNAaloneisnotadirectinducerorgeneexpressionregulatorthatisessentialforMMplantstoflower.mFT:startcodonATG->TAGDeletionof11thbaseYiguoHongHangzhouNormalUnivAproposedmodelillustratingthebindingofanFTRNA-bindingprotein(s)to“zipcodes”locatedwithin102ntofthe5’endoftheFTmRNAtoformanRNAproteincomplexwhichthenmovesfromtheleafthroughthephloemandintotheSAM.FTproteinmayeitherassociate,andbetransported,withthisRNA–proteincomplex,oritmaymoveindependentlyoftheFTmRNAwithitsmovementthroughtheplasmodesmataandintotheSAM.ProposedmodelforFTRNA/proteinmovementFTproteinsequenceanalysis------aputativetranscriptionalregulatorbutlacksacanonicalDNAbindingdomainQuestion:howdoesFTpromotefloraltransition?35S:FTleadstoearlyflowering,andup-regulatesAP1andSOC1geneexpressionGR-FTexperiment(DEX+CHX)suggeststhatAP1andSOC1aredirecttargetgensofFTChIPassay---------YesEMSAassay---------NoWhatisyourmodel?AP1orSOC1XFTAP1SOC1LFY

Aworkingmodel…..AP1orSOC1XFTHowtofindX?1,biochemicalapproach2,molecularbiologyapproach3,geneticapproach35S:FTisanearly-floweringplantCol(wildtype)35S:FT/ColSuppressorof35S:FT/ColWhatgenescouldbeSOFT(suppressorsof35S:FT)?1,35S:FTtransgene2,XAP1orSOC1XFTFTFTFT3,AP1,SOC1,orLFY4,RNAipathwaygenesAbZIPprotein,FD,isrequiredforFTfunction與野生型相比：

35S:FT

早花

fd-2晚花

35S:FT/fd晚于35S:FTScreenfor35S:FTsuppressor-----fdmutantfd-1isastrongsuppressorof35S:FT,buthasnoeffectonsoc1-101Dor35S:LFY-------FDisdownstreamofFTbutupstreamofSOC1andLFYFDup-regulatesAP1expressionwhichrequiresFTfunctionFTprovidesanactivationdomainforFDTFactivity,whereasGF14(14-3-3protein)actsasalinkerproteinFT&14-3-3&FDformanhetero-hexamericstructureModelledstructureoftheFAC–promoter-DNAcomplexFAC：FlorigenActivationComplexTranscriptionalactivatorvsrepressorTFL1andFThavehighlyconservedaminoacidsequencesbutoppositefunctions.FTpromotes?oweringandTFL1repressesit.ThetransitiontofloweringinvolvesmultiplefactorsandpathwaysMeristemidentitygenes14-3-3Besidesthephotoperiod-dependentregulation,floraltransitionisundercontrolsofmanyothercues.Vernalization(春化)–

cold-promotingfloweringVernalization:Acquisitionofthecompetencetoflowerinthespringbyexposuretotheprolongedcoldofwinter.SomeplantsneedwintertoflowerNovernalizationVernalizationPlantsaregeneticallyidenticalExposedasaseedlingto4oCfor40days.VernalizationblockstheexpressionofFLCincold-requiringwinterannualArabidopsisecotypesWinterannualwithoutcoldFLCmRNAWinterannualafter40dayscoldwinterannualecotypesflcmutant(Winterannualwithoutcold)FLOWERINGLOCUSC(FLC)-encodesa

MADSboxtranscriptionfactor,delayingfloraltransition,i.e.arepressorofflowering-FLCdirectlyrepressestheexpression

offlowering-timeintegratorsincludingFT,FD,andSOC1highlyexpressedinnon-vernalizedSAMitsexpressionisprogressivelyswitchedoffduringthevernalizationperiodFLCrepressionbyvernalizationismitoticallystable

but

meioticallyunstableVIN3VIN3UBQFLCthecold-mediatedFLCrepression

isstablymaintainedaftertheplantreturnstowarmconditions;however,FLCexpressionisre-activatedduring

embryogenesissothatthenextgenerationrequiresvernalization

again.4C40

days+22C20daysV:vernalizationT:normaltemperatureinitiationmaintenanceofrepressionColdFLCFloweringGeneticapproach1,4Ctreatmentfor40days,lookforlatefloweringmutants2,nocoldtreatment,lookforearlyfloweringmutantsFT/FDSOC1-----flc

XY？ColdFLCFloweringFT/FDSOC1XY-----x-----ft,fd,soc1-----y-----otherFLC

activators

VRN2:HomologofSu(z)12VRN1:MybDNAbindingproteinVIN3:PHDfingerproteinVIL1(VIN3-Like1):PHDfingerproteinLHP1:LIKE-HETEROCHROMATINPROTEINVIN5:HistoneargininemethyltransferaseGenesareinvolvedininitiationofrepressedchromatinGenesareinvolvedinmaintenanceofrepressedchromatinGeneticstudyforvernalizationpathway----IsolationofVernalizationinsensitive(vin)mutantsgenesinvolvedincontroloffloweringbyvernalizationVERNALIZATION(VRN)1andVRN2botharerequiredformaintenanceoflowlevelsofFLCmRNAthatare establishedbycoldtreatmentonceplantsareexposedtowarmerconditionsRoleofVRN2istomaintaintherepressionofFLCexpression.Timevernalized（weeks）Timevernalized（weeks）Daysat20oCaftervernalizationDaysat20oCaftervernalizationFLCmRNAFLCmRNAWildtypevrn2VRN2encodesagenerelatedto

Drosophilab-group(PcG)genesInDrosophila,PcGproteinsactinlargeproteincomplexes.b-groupproteinscatalyzethemethylationofH3K9andH3K27,thusinvlovingingenerepression.VRN2ArabidopsisSU(Z)12DrosophilaEnzymeswritingHistonecodebRepressiveComplex(PRC)Trithorax

ActiveComplex(TrxG)HistonemethylationenzymesFLCexpressionisactivatedbyFRI,PAF1c(H3K4Me),theSWR1c-mediateddepositionofthehistonevariantH2A.Z,andtheH2Bmonoubiquitination.ProlongedcoldexposureinducesVIN3expression,andVIN3functionsinthecontextofaPRC2(PHD-PRC2)(H3K27Me3)toinitiaterepression.brepressivecomplex2(PRC2)-mediatedH3K27trimethylation(CLF-PRC2)actstomaintaintheFLCrepression.Inaddition,FLD,FCA,PRMT5,PRMT10andFPAalsorepressFLCexpression.FLCrepressestheexpressionofflowering-timeintegratorsincludingFT,FD,andSOC1.Regulatory

Networkcontrolling

FloweringinArabidopsisPAF1complex(PAF1forRNAPolymeraseIIAssociated

Factor1)recruitsanH3K4methyltransferase(TrxG)totargetgenechromatin,

whichcatalyzesH3K4trimethylationand

leads

toactiveexpressionoftargetgenesActivationofFLCexpressionassociated

with‘a(chǎn)ctive’chromatin

modificationsH2BmonoubiquitinationinFLCchromatinisrequiredfor

FLCexpression

(HUB1,HUB2,UBC1,andUBC2mediateH2B-ub1inArabidopsis)DepositionoftheHistoneVariantH2A.ZinFLCchromatinisrequiredforFLCactivation

(aSWR1c-likecomplexthatdepositsH2A.Zinactively

transcribedgenes)RepressionofFLCexpressionassociated

with‘silencing’chromatin

modificationsFLDisaplanthomologofthehumanLysine-SpecificDemethylase1(LSD1),

ahistoneH3K4demethylaseFVE,ahomologofthehumanRbAp46/48thathasbeen

foundinseveralchromatin-modifyingrepressorcomplexes,

isinvolvedinhistone

deacetylation

inFLCchromatinBothFCAandFPAcontainmultipleRRM-typeRNA-binding

Domains,involvedinsilencingofendogenoustransposons

andotherrepeatedsequencesviaaRNA-mediatedchromatin

silencingmechanism.BothFCAandFPA

partlyactthroughFLDtorepressFLCexpressionbrepressivecomplex2(PRC2)-mediatedH3K27trimethylation(PHD-PRC2,CLF-PRC2)----VIN3inducedbyvernalizationPRMT5:

histoneargininemethylationisinvolvedinFLC

repressionDynamicsofFLCchromatinActiveFLCChromatinHighin

Ac;H3K4Me

H2A.ZHistonevariantRepressedFLCChromatinHighin

H3K9Me;H3K27Me

H4R3Me;HP1VIL1,VIN5VIN3,VIL1,VIN5VRN2,VRN1,LHP1,etc.(heterochromatin

structure)ON;FallWINTER!OFF;SpringHowdoesvernalizatoninitiateFLCrepression?VIN3VIN3UBQFLC--progressiveinductionofVIN3maybeonemechanism,soaVIN3-PRC2complexisformedonFLCgene.20

days

after

40d

vernalization--butVIN3(aPhDfingerprotein)doesnothaveaDNAbindingdomain,sohowdoesitrecruitPRC2tospecificallytargetFLCgene?Twolongnon-codingRNAs,COOLAIRandCOLDAIR,regulateFLCexpressionCOLDASSISTEDINTRONICNONCODINGRNA(COLDAIR)(a)FLCcontrolsthetransitionfromvegetativetoreproductivedevelopmentinArabidopsis.FLCexpressionisregulatedbymanypathways:vernalization,acold-inducedepigeneticsilencingthatoccursduringwinter;FRIGIDA,acoiled-coilproteinthatupregulatesFLCexpression;theautonomouspathway,whichiscomposedofmanyindependentrepressiveactivities;ArabidopsisTrithorax-likepathway,whichstimulatesFLCgeneexpression;RNAinterferencesuppressesFLCindifferentArabidopsisaccessions31

and

81.(b)TheFLClocus(～7

kbinlength)expressesmultipletypesoftranscripts.FLCmRNAencodesaMADSboxtranscriptionfactor.COOLAIRisanon-codingtranscriptthatfullypassesFLCintheantisensedirection.Itisalternativelypolyadenylated,withaproximalpoly(A)siteinsenseintron6andadistalpoly(A)siteinthesensepromoterregion,andisdifferentiallyexpressedinwarmandcoldconditions.COLDAIR,expressedundercoldconditionsfromwithinintron1ofFLCinthesensedirection,isacappedbutnon-polyadenylatedlncRNA.COOLAIR,anantisenseRNAwithalternativepolyAtails,repressesFLCexpressionWild-type(+FCA)fca(-FCA)FCA:RRM-typeRNA-bindingproteinFLD:H3K4demethylaseFY/CstF64/77:polyadenylationenzymesIntheabsenceofendogenousFCA,COOLAIRtranscripts(red)arepolyadenylatedundertheinfluenceofFY,CstF64,andCstF77atadistalpoly(A)site.ThisisassociatedwithhighH3K4me2(me2inblue)levelsinthebodyoftheFLCgene,andwithhighlevelsoffunctionalFLCmRNA(green)andCOOLAIRexpression.FCApromotesalternativepolyadenylationofCOOLAIRthroughtargetingFYandCstFactivitytoaproximalpoly(A)site.ThiseventislikelytotriggerFLD-dependentdemethylationinthebodyofFLC,leadingtoatranscriptionallyrepressedstateandlowlevelsoffunctionalFLCmRNA.TheautonomouspathwayrepressesFLCexpressionthroughantisenseRNA(asRNA)-mediatedchromatinmodifications.Acold-inducedlongnoncodingRNA(COLDAIR)fromthefirstintronofFLCtransientlyappearduringthecoldexposureCOLDAIRassociateswithPRC2duringthecoldexposure(CLF,ahomologofPRC2)(GFP-CLFimmunoprecipitation)(COLDAIRin