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The evolutionary divergence of the genetic networks that control flowering in distinct species

Della Pina, S.

Publication date 2016 Document Version Final published version

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Citation for published version (APA): Della Pina, S. (2016). The evolutionary divergence of the genetic networks that control flowering in distinct species.

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Download date:25 Sep 2021 ! 3!

Rewiring!the!flowering!network!in!Petunia(hybrida(

! Serena!Della!Pina,!Colin!Rijkenberg,!Bets!Verbree,!Erik!Souer!and!Ronal!Koes!

S.D.P.,! R.K.! and! E.S.! designed! experiments.! S.D.P.! carried! out! the! majority! of! the! experiments.!S.D.P.!and!B.V.!carried!out!experiments,!analyzed!and!interpreted!data.!S.D.P.! wrote!the!paper!

DOT!regulation!

Abstract!

It! is! largely! accepted! that! the! set! of! genes! that! gives! floral! identity! to! meristems! are! widely! conserved! in! the! plant! kingdom,! whereas! their! divergent! expression! patterns! correlate! with! distinct! inflorescence! architectures! seen! in! nature.! Here! we! show! that! changes!in!the!regulatory!elements!of!one!of!these!floral!identity!genes,!DOUBLE(TOP!(DOT),! caused!the!rewiring!of!the!genetic!networks!that!control!flowering!time!in!petunia.!Through! phylogenetic! shadowing! and! functional! assays! in! transgenic! plants! we! localized! important! regulatory! elements! in! the! promoter! regions! of! this! gene! and,! using! transient! assays,! we! show! that! known! flowering! time! integrators! in! Arabidopsis! are! also! involved! in! flowering! time!regulation!of!petunia.!! ! !

! 72! Chapter!3!

Introduction!

The!transition!from!vegetative!to!reproduction!phase!represents!one!of!the!most!important! events!during!plant!life!and!the!correct!temporal!regulation!of!this!switch!is!critical!for!the! survival! of! the! species.! This! is! the! reason! why! multiple! pathways! control! this! important! developmental! process.! External! signals,! such! as! day! length,! ambient! temperature,! and! exposure!to!cold,!as!well!as!plant!age!are!perceived!via!distinct!pathways,!which!converge!in! the!regulation!of!several!key!genes,!called!floral!integrators,!which!ultimately!activate!the! floral! meristem! (FM)! identity! genes! (Parcy,! 2005).! The! expression! of! these! FM! identity! genes,!in!turn,!determines!when!and!where!flowers!are!formed!(Prusinkiewicz(et(al.,!2007).! In! Arabidopsis( thaliana!(Arabidopsis)! FM! identity! is! specified! by! two! transcription! factors! that!are!encoded!by!LEAFY!(LFY)!and!APETALA1!(AP1)!(Mandel( et(al.,! 1992;! Weigel(et(al.,! 1992).!The!position!where!flowers!are!formed!is!largely!determined!via!the!transcriptional! regulation!of!LFY,!which!in!turn!directly!activates!AP1!(Wagner(et(al.,!1999;!Weigel(et(al.,! 1992).! Indeed,! in! lfy! and! ap1! mutants! floral! identity! is! (partially)! lost,! whereas! ectopic! expression!of!LFY!or!AP1!is!sufficient!to!trigger!precocious!flowering!and!to!convert!the!apex! into!a!solitary!flower!(Mandel!and!Yanofsky,!1995;!Weigel!and!Nilsson,!1995).!! In!Arabidopsis,!inductive!long!day!conditions!are!perceived!in!leaves!and!transduced!to! CONSTANS!(CO)!(An(et(al.,!2004;!Kobayashi!and!Weigel,!2007;!Samach(et(al.,!2000),!which!in! turn!activates!the!expression!of!the!FLOWERING(LOCUS!T!(FT),!in!the!vascular!tissues!of!the! leaves!(Corbesier(et(al.,!2007).!FT!is!a!mobile!signaling!protein,!largely!identical!to!the!longZ sought! florigen! signal,! which! moves! from! the! leaves! to! the! shoot! apical! meristem! (SAM),! where!it!activates,!in!a!complex!with!FLOWERING!LOCUS!D!(FD)!and!a!14:3:3!protein,!several! MADS! box! genes,! including! AP1! (Abe( et( al.,! 2005;! Jaeger! and! Wigge,! 2007;! Taoka( et( al.,! 2011;!Wigge(et(al.,!2005).!In!Arabidopsis,!two!closely!related!MADSZbox!genes!belonging!to! the!StMADS11!clade,!SHORT(VEGETATIVE(PHASE!(SVP)!and!AGAMOUSILIKE(24!(AGL24),!have! been! shown! to! have! opposite! functions! in! the! regulation! of! flowering! time.! Ectopic! expression! of! SVP! causes! late! flowering,! floral! reversion! and! conversion! of! flowers! into! shootZlike!structures!(Gregis(et(al.,!2006,!2008;!Hartmann(et(al.).!By!contrast,!overexpression! of! AGL24! results! in! early! flowering! plants! bearing! abnormal! flowers! and! ectopic! inflorescences!from!swollen!ovaries!(Gregis(et(al.,!2006;!Yu(et(al.,!2002).!Both!genes!carry! out! their! opposite! function! by! directly! regulating! SUPPRESSOR( OF( OVERIEXPRESSION( OF(

73! ! DOT!regulation!

CONSTANS1!(SOC1),! which! encodes! another! MADS! box! transcription! factor:! SVP! directly! suppresses!SOC1!expression!and!flowering!(Li(et(al.,!2008),!whereas!AGL24!promotes!SOC1! expression!and!flowering!(Liu(et(al.,!2007).!Interestingly,!AGL24!and!SOC1!are!also!able!to! upZregulate! each! other's! expression,! creating! a! positive! feedback! loop,! which! may! be! necessary!to!integrate!flowering!signals!(Liu(et(al.,!2007;!Michaels(et(al.,!2003).!Furthermore,! AGL24!and!SOC1!together!directly!upZregulate!LFY!under!inductive!long!day!conditions.! Under! nonZinductive! shortZday! conditions,! gibberellin! (GA)! (MutasaZGottgens! and! Hedden,!2009)!and!aging!play!critical!roles!in!flowering!(Cardon(et(al.,!1999;!Rhoades(et(al.,! 2002).! In! the! latter! case,! several! reports! suggested! that! a! group! of! transcription! factors! called! SQUAMOSA! PROMOTER! BINDING–LIKE( proteins! (SPLs)! directly! influence! the! transitions! from! juvenile! to! adult! as! well! as! from! vegetative! to! reproductive! phase.! SPL! genes! are! postZtranscriptionally! silenced! by! microRNA156!(miR156)! in! an! ageZdependent! manner!(Wu!and!Poethig,!2006).!During!the!juvenile!phase!miR156(expression!is!high,!but! decreases!as!the!plant!ages,!resulting!in!an!increased!expression!of!SPLs.!The!activation!of! SPLs!promotes!juvenile/adult!shift!and!flowering!by!directly!regulating!microRNA172,!several! MADSZbox! genes! and! LFY! (Wang( et( al.,! 2009;! Wu( et( al.,! 2009;! Yamaguchi( et( al.,! 2009).! Interestingly,!some!of!these!SPLs!are!upstream!activators!of!the!floral!integrators!SOC1,!AP1,! FUL! and! LFY,! but! also! their! downstream! target.! It! has! been! shown! that! SPL3! is! directly! regulated! by! SOC1! establishing! a! connection! between! photoperiod! and! GA! signals! to! promote!flowering!in!Arabidopsis!(Jung(et(al.,!2012),!while!SPL3!directly!activates!LFY,!AP1( and!FRUITFULL!(FUL)!in!Arabidopsis!(Yamaguchi(et(al.,!2009).!Indeed,!overexpression!of!SPL3! accelerates!flowering!under!both!short!and!long!day!conditions,!whereas!reduction!of!SPL! activity,! by! overexpression! of! miR156,! delays! flowering! (Cardon( et( al.,! 1997;! Wu! and! Poethig,!2006).! Although! the! pathways! controlling! flowering! time! are! best! described! in! Arabidopsis,! homologs!of!LFY!can!be!found!in!several!species!(Ahearn(et(al.,!2001;!Bomblies(et(al.,!2003;! Coen(et(al.,!1990;!MolineroZRosales(et(al.,!1999;!Mouradov(et(al.,!1998;!Souer(et(al.,!2008;! Wada( et( al.,! 2002;! Wang( et( al.,! 2008).! In! petunia,! for! instance,! the! specification! of! floral! meristem!identity!requires!ABERRANT(LEAF(AND(FLOWER!(ALF),!which!is!orthologous!to!LFY! and!encodes!a!functionally!exchangeable!protein!(Souer(et(al.,!2008).!In!contrast!to!LFY,!ALF! is! already! expressed! during! the! vegetative! phase! in! leaf! primordia! and,! within! the! inflorescence,!in!the!apical!and!lateral!meristems.!Although!the!expression!pattern!of!ALF!is!

! 74! Chapter!3! remarkably! different! from! that! of! LFY,! this! does! fully! not! account! for! the! divergent! inflorescence!architectures!of!petunia!(cyme)!and!Arabidopsis!(raceme),!as!in!petunia!ALF!is! not!the!limiting!factor!that!determines!when!and!where!flowers!are!formed!(Souer(et(al.,! 2008).!Indeed,!constitutive!expression!of!either!LFY!or!AP1!does!not!alter!flowering!time!or! inflorescence!architecture!in!petunia.!Hence,!petunia!does!not!determine!when!and!where! flowers!are!formed!via!the!transcription!of!ALF/LFY!but!via!a!different!floral!identity!gene! that!was!identify!as!DOUBLE(TOP!(DOT)!(Souer(et(al.,!2008).! DOT!is!essential!to!specify!floral!meristem!identity!in!petunia.!It!encodes!an!FZbox!protein! that! is! functionally! exchangeable! with! UNUSUAL( FLORAL( ORGANS!(UFO)! from! Arabidopsis! and!it!is!the!substrateZbinding!component!of!an!SCFZtype!ubiquitin!ligase!complex!(SCFDOT!).! The!major!target!of!SCFDOT(is!ALF!and!its!role!is!not!to!downZregulate!ALF!by!targeting!it!for! proteasomeZmediated! degradation,! but! to! promote! ALF! activity! via! a! posttranslational! mechanism!(Souer(et(al.,!2008).!Although!DOT!and!UFO!encode!functionally!similar!proteins,! the! genes! have! clearly! distinct! roles! in! development! due! to! the! divergence! of! their! expression! patterns! (Souer( et( al.,! 2008).! In! Arabidopsis! UFO! is! expressed! in! all! meristems! throughout!the!vegetative!phase!and!after!the!onset!of!flowering!in!both!apical!and!lateral! meristems.!Consequently!LFY,!rather!than!UFO,!is!the!limiting!factor!that!determines!where! and!when!flowers!form!in!Arabidopsis.!In!petunia!it!is!the!opposite:!DOT!is!inactive!during! vegetative!phase!and!is!only!transcribed!after!the!onset!of!flowering!in!the!apical!meristem,! where!it!promotes!floral!fate!and!activates!floral!organ!identity!genes.!Furthermore,!ectopic! expression!of!DOT!results!in!precocious!flowering,!converting!the!cymose!inflorescence!into! a! solitary! flower! and! homeotically! transforming! leaves! into! petals! (Souer( et( al.,! 2008)! (Chapter!2).!Thus,!in!petunia,!the!time!and!position!where!flowers!appear!is!controlled!via! transcriptional!regulation!of!DOT!rather!than!ALF/LFY.! The!divergent!function!and!expression!patterns!of!DOT!and!UFO,!which!correlate!with! distinct!inflorescence!architectures,!result!from!differences!in!their!promoters!(Chapter!2).! To!understand!in!more!detail!how!such!changes!rewired!the!genetic!networks!that!control! flowering!time,!we!aimed!to!identify!cisZregulatory!elements!(CREs)!and!transZacting!factors! that! control! DOT! expression.! Through! phylogenetic! shadowing! and! functional! assays! in! transgenic!plants!we!localized!important!cisZregulatory!regions!in!the!5’!flanking!region!of! DOT!(pDOT),!some!of!which!appear!well!conserved!in!other!species.!Using!transient!assays!in! protoplasts,!we!show!that!AGL24!and!SPL3,!known!in!Arabidopsis!as!regulators!of!LFY,!can!

75! ! DOT!regulation! activate! pDOT! in! petunia.! We! then! analyzed! protein! function! in! petunia! and! Arabidopsis,! expression! pattern! and! proteinZprotein! interaction! of! AGL24/FBP25/FBP13! and! SPL3/PhSPL3/4/5/6.!

Results!

Synteny!analysis!of!DOT/UFO@like!genes!

Previous!results!(Souer(et(al.,!2008)!showed!that!the!divergent!inflorescence!architecture!of! racemes! and! cymes! was! mainly! due! to! differences! in! the! expression! pattern! of! floral! meristem!identity!genes.!Through!promoter!swap!experiments!we!showed!that!the!different! spatioZtemporal! expression! of! the! petunia! DOT! and! Arabidopsis( UFO! genes! was! due! to! changes! in! regulatory! elements! present! in! their! promoter! region! (Chapter! 2).! Such! differences! may! have! evolved! after! the! separation! of! the! two! species! through! the! accumulation! of! numerous! small! mutations! over! time! resulting! in! the! gain! and/or! loss! of! transcription!factor!binding!sites,!or!in!one!or!few!big!steps!by!genomic!rearrangements.!In! order!to!gain!insight!in!the!rearrangements!that!might!have!occurred!during!evolution!at!the! DOT/UFO! locus,! we! analyzed! the! gene! organization! surrounding! the! DOT! locus! in! several! species!(Fig.!1).!The!Generic!Genome!Browser!9.1v!on!the!Phytozome!platform!showed!a! remarkable!difference!in!the!location!of!the!ERAIrelated(GTPase!(ERG)!gene!(violet!arrow,! labeled!“a”)!relative!to!the!DOT/UFO!homologs!(black!arrow).!In!eudicots!belonging!to!the! Rosids! clade,( like! Arabidopsis( thaliana!(AtUFO)( and! Vitis! vinifera( (VvUFO),! but! also! in! Antirrhinum(majus((AmFIM)!and!Mimulus(guttatus!(MgUFO)!members!of!the!Asterid!clade,! ERG!is!downstream!of!DOT.(In!species!belonging!to!the!Solanaceae!family!(Asterids)!ERG!is! either! upstream! of( DOTIlike( genes,! like! in! Solanum( tuberosum! and! Solanum( lycopersicum( (AN)(or(absent!at!this!genomic!location!(Petunia).!Strikingly!the!DOT!locus!in!petunia!seems! to!have!underwent!a!further!inversion!since!the!Nuclear(Complex(2!(NCO2)!gene!(red!arrow,! b)! is! upstream! of! DOT,! while! in! Solanum( tuberosum! and! Solanum( lycopersicum( NCO2! is! downstream.(Moreover!the!DOT!locus!is!surrounded!by!MULEItype(transposon!like!elements! (light!grey!arrows,!@)!and!this!could!have!caused!the!excision!of!DOT!locus!from!the!region! where! ERG! is! present.! The! analysis! of! the! current! data! hints! towards! specific! genome! reZ arrangements! in! Solanaceae! species! that! might! be! correlated! with! altered! expression! patterns!of!DOTZlike!genes!and!inflorescence!architecture.!

! 76! Chapter!3!

!

Figure! 1:! Synteny! plot! reports! of! the! local! gene! organization! flanking! DOT! homologs! in! several! families.!! The! diagrams! indicate! the! relative! position! and! orientation! of! DOT! homologs! (black! arrows)! and! immediate! flanking! genes.! Maps! are! not! to! size.! The! petunia! ERG! locus! is! shown! at! the! bottom.! Similarity!between!flanking!genes!is!indicated!by!colorZcoding!and!letters.!White!fillings!and!lack!of! letters!denote!genes!occurring!only!once!in!this!plot.!Violet!arrow!(a)!GTPIBINDING(PROTEIN((ERG),! red!arrow!(b)!NUCLEOLAR(COMPLEX(2((NCO2),!yellow!arrow!(c)!FIBOX(PROTEIN,!orange!arrow!(d)! UDPIGLUCOSYLTRANSFERASE,! pink! arrow! (e)! PHOSPHOGLICERATE( MUTASE( FAMILY( PROTEIN,! blue! arrow!(f)!RNA(POLYMERASE(III(TRANSCRIPTION(INITIATION(FACTOR(B,!light!blue!arrow!(g)!GAGIPOL( POLYMERASE,!green!arrow!(h)!PROTEASOME(SUBUNIT(ALPHA(TYPE(4,(light!grey!arrow!(@)!MuDR(A( (MULE)(TRANSPOSONE(LIKE.!

Identification!of!putative!DOT!regulatory!sequences!by!phylogenetic!shadowing.!

Functional!analyses!of!the!regulatory!regions!of!DOT/UFOIlike!genes!showed!that!most!of! the!elements!required!for!correct!expression!are!located!in!the!5’!flanking!region!of!those!

77! ! DOT!regulation! genes!(Chapter!2).!To!identify!conserved!regions!that!might!contain!cisZregulatory!elements! (enhancers)!we!first!compared!promoter!sequences!of!DOT/UFOIlike!genes!from!different! Solanaceae!for!which!sequence!information!is!available.!! We!aligned!5.0!kb!upstream!region!of!the!DOT!transcription!start!from(Petunia(axillaris! (PaDOT;!Figure!2A),!Solanum(lycopersicum((ANANTHA,(AN),(Capsicum(annuum((CaAN),!and! Solanum( tuberosum( (StAN)( using! mVISTA! (Mayor( et( al.,! 2000).! These! pairwise! alignments! revealed!four!highly!conserved!sequence!blocks!(Fig.!2A,!highlighted!in!light!gray),!which!we! named!BOX1(to(BOX4,!with!BOX1!closest!to!the!DOT!transcription!start.!Next,!we!used!the! sequence!conservation!in!the!identified!boxes!to!design!specific!primers!in!order!to!amplify! fragments!of!DOT!homologs!from!additional!Solanaceous!species.!We!chose!several!species,! more!or!less!closely!related!to!petunia,!and!we!sequenced!the!obtained!promoter!fragments! (Fig.!S1ZS4).!In!each!of!the!species!analyzed!we!could!easily!detect!BOX1,(3!and!4,!while!that! was! not! always! possible! for! BOX2! (Fig! S2A).!The! regions! between! boxes! were! not! always! amplifiable,!suggesting!that!the!distances!between!boxes!can!vary!a!lot!among!species!(Fig! S5A)! and! may! exceed! the! size! limits! for! PCR! amplification,! or! that! the! order! and! relative! orientation!of!the!conserved!boxes!in!variable.!! We!then!used!JASPAR!database!(http://jaspar.genereg.net)!to!predict!transcription!factor! binding! sites! within! the! promoters! and! more! specifically! within! the! identified! BOXes.! This! analysis! revealed! conserved! binding! sites! for! a! number! of! transcription! factors.! BOX1( contains!two!putative!CArGIbox!sites,!the!typical!binding!site!for! MADS!box!proteins,!and! two!SQUAMOSA(PROMOTER(BINDING(PROTEIN!binding!sites!(Fig.!S1).!In!BOX3!two!CArGIbox! sites!can!be!identified!in!a!tandem!repetition!(Fig.!S3).!In!BOX2!(Fig.!S2)!as!well!as!in!BOX4( (Fig.! S4),! the! most! upstream! conserved! region! that! lies! at! 4.2! kb! from! the! DOT! ATG,! no! obvious!hypothetical!TF!binding!sites!could!be!identified.! We!also!analyzed!the!5’!flanking!regions!of!DOT/UFOIlike(genes!from!species!outside!the! Solanaceae!family!(Fig!S5B).!Specifically,!we!analyzed!the!promoter!regions!of!AtUFO((3.8kb)! from! Arabidopsis,! VvUFO( (6kb)! from! grape,( AmFIM( (3.6! kb)! from! Antirrhinum( majus( and( MgUFO( (6.5kb)! from! Mimulus( guttatus.( In! AtUFO! we! could! identify! only! BOX1,! while! in! VvUFO,!AmFIM!and!MgUFO!we!found!BOX1!and!BOX3,!but!none!of!them!contained!BOX2!or! BOX4.!!

! 78! Chapter!3!

! Figure! 2:! Identification! and! functional! characterization! of! conserved! domains! in! the! 5’! flanking! region!of!DOT.!! (A)! Sequence! comparison! of! pDOT! from( Petunia.axillaris!(Pa)! to! 5’! flanking! sequences! of! DOT! homologs!from!Capsicum!annuum((Ca),!Solanum!lycopersicum!(Sl)(and!Solanum!tuberosum!(St).!The! pDOT!sequence!used!for!pairwise!alignment!is!represented!by!as!a!black!line.(Pairwise!alignment!was! done!using!mVISTA.!Graphical!output!shows!percent!base!pair!identity!in!a!sliding!window!of!200!bp! in!a!range!of!50!to!100%.!LightZgrey!areas!highlight!conserved!blocks.!! (B)!Functional!analysis!of!BOX1Z!BOX4.!In!petunia!seedlings!mp35S:GUS!is!expressed!at!the!periphery! of! cotyledons,! but! not! in! the! root! tip! or! the! apical! meristem.! Addition! of! single! pDOT! boxes! to! mp35S:GUS! minimal! promoter! spread! the! activity! to! the! root! tip! and! the! apical! meristem.! In! the! inflorescence!meristem,!35S!minimal!promoter!alone!doesn’t!show!any!activity!while!the!addition!of! BOX1!enhances!the!expression!in!an!ectopic!pattern!in!both!IM!and!FM.!BOX2!drives!GUS!activity!in! young!and!mature!floral!meristem.!BOX3!is!able!to!recapitulate!the!correct!expression!pattern!of!the! pDOT! showing! activity! in! the! typical! ring! pattern! in! the! flower! meristem.! BOX4! shows! the! same! ectopic!pattern!like!BOX1.!

79! ! DOT!regulation!

Functional!relevance!of!the!conserved!sequence!elements!present!in!DOT/UFO@like!genes!

To!understand!the!function!of!the!four!conserved!boxes!in!the!DOT!promoter!we!inserted! each! of! them! upstream! of! a! minimal! 35S! promoter! and! the! GUS! coding! sequence! and! generated!stable!transformants!containing!the!different!BOXImp35S:GUS!fusion!genes!in!the! P.( hybrida! W115! background! (Fig.! 2B).! As! a! control! we! generated! W115! transformants! containing! mp35S:GUS! alone.! These! transformants! showed! weak! GUS! expression! in! seedlings!at! the! periphery! of! cotyledons! but! none,! after! floral! transition! in! the! sympodial! inflorescence!meristems.! In! seedlings,! the! BOX1Imp35S:GUS,! BOX2Imp35S:GUS! and! BOX3Imp35S:GUS! fusions! showed! GUS!activity! in!the!tip!and! the!veins! of!cotyledons,!in! root!tips,! and!in!the!shoot! apical!meristem.!BOX4Imp35S:GUS!was!active!only!in!the!root!tip.!In!inflorescence,!BOX1I mp35S:GUS! and! BOX4Imp35S:GUS! transgenes! caused! GUS! expression! in! a! much! broader! domain! than! the! DOT! endogene! or! a! GUS! gene! driven! by! a! 3.1! kb! DOT! promoter! (pDOT3.1:GUS)!(Fig.!3!Chapter!2),!while!BOX2Zmp35S:GUS!showed!signal!mainly!in!flowers.! Most!interestingly!the!transgenic!plants!containing!BOX3Imp35S:GUS!accurately!reproduced! the!expression!pattern!of!pDOT3.1:GUS!and!the!DOT!endogene!(Souer(et(al.,!2008).!In!floral! meristems! of! BOX3Imp35S:GUS! expressors,! GUS! activity! was! visible! in! a! ring! at! the! sepals/petals! boundary.! Given! that! the! 3.1! kb! pDOT3.1! promoter,! which! lacks! BOX4! and! BOX3,!can!drive!correct!expression!of!the!GUS!reporter,!it!seems!that!the!BOX3!enhancer! elements!are!redundant!with!elements!within!pDOT3.1!(Chapter!2).!

Promoter!deletions!to!identify!the!regulatory!information!for!correct!PhDOT!regulation.!

Previously,!we!showed!that!a!transgene!consisting!of!3.1!kb!sequence!upstream!of!the!DOT! translation!start!site!Z!which!includes!only!BOX1(and!BOX2!Z!fused!to!the!cDNA!of!GUS!was! sufficient! to! reproduce! the! spatioZtemporal! expression! of! DOT! (Chapter! 2,! Fig! 3).! The! addition! of! another! 1.5! kb! –!including!BOX3! and! BOX4! –! only! seems! to! have! quantitative! effects,!causing!GUS!activity!in!both!apical!and!lateral!meristem,!although!the!expression!of! the!transgene!was!restricted!to!the!sepals/petals!primordia!boundary!(Chapter!2,!Fig!4).!! To!fully!value!the!importance!of!the!boxes!present!in!the!DOT!promoter!and!at!the!same! time! delimit! the! upstream! regulatory! region! of! DOT,! we! generated! a! set! of! promoter! deletion!constructs!starting!from!the!pDOT3.1:GUS!(Fig.!3AZB).!When!we!removed!a!spurious! TATA!box,!close!to!what!we!considered!to!be!the!real!TATA!box,!and!a!small!part!of!the!3’!

! 80! Chapter!3! end!of!BOX1,!GUS!expression!was!still!visible!in!a!correct!spatioZtemporal!expression!pattern! (Fig!3B,!deletion!ΔA).!When!BOX1(was!completely!removed,!we!could!not!detect!any!GUS! signal!in!any!of!the!20!independent!transformants!that!we!raised!(Fig!3B,!deletion!ΔB).!The! exclusion!of!BOX2!left!the!pDOT!activity!unchanged,!since!GUS!signal!was!visible!in!a!ring! shape!in!the!floral!meristem!(deletion!ΔC).!When!we!removed!another!750!bp,!GUS!activity! in! emerging! floral! meristems! was! lost,! but! we! could! still! detect! GUS! activity! in! mature! flowers!(Fig!3B,!deletion!ΔD).!A!similar!picture!emerged!upon!deletion!of!another!400!bp:! GUS!expression!was!only!visible!in!mature!flowers,!but!not!in!emerging!floral!meristems!(Fig! 3B,!deletion!ΔE).!Shortening!the!DOT!promoter!to!360!bp!upstream!of!the!translation!start! site,! thereby! also! removing! BOX1! entirely,! disrupted! the! ability! to! drive! GUS! expression! completely!(Fig!3B,!deletion!ΔF).!! In!summary,!the!PhDOT!promoter!sequence!1.8!kb!upstream!of!the!translation!start!site,! as! present! in! deletion! ΔC,! contains! all! regulatory! elements! required! to! mediate! correct! spatial!and!temporal!expression!of!PhDOT.!Further!shortening!abolished!promoter!activity!in! the!emerging!floral!meristem,!where!DOT!is!necessary!to!specify!floral!identity.!Our!analysis! of!DOT!promoter!deletions!and!BOXes!thus!revealed!the!presence!of!redundant!enhancer! elements! for! spatial! and! temporal! regulation! of( DOT! in! BOX3! and! in! the! 1! kb! region! just! upstream! of! BOX1,! while! BOX1! is! fundamental! for! the! transcriptional! activation! of! DOT,! since!its!deletion!caused!complete!loss!of!activity.!Furthermore,!it!reveals!the!presence!of!an! additional!enhancer,!BOX5,!which!is!located!between!BOX1!and!2(and!is(necessary!for!the! expression!of!DOT!in!sepal/petals!boundary,!but!which!does!not!stand!out!as!a!conserved! sequence!in!promoter!comparison.!

81! ! DOT!regulation!

! ! Figure!3:!Correct!induction!of!DOT!requires!1.8!kb!of!5’!promoter!sequence!region.!! (A)!Schematic!representation!of!pDOT!and!the!position!of!conserved!BOXes(1I4.!Sequences!included! in!the!deletion!derivatives!(ΔAZΔF)!are!indicated!with!a!black!line.!Numbers!indicate!the!start!and!the! end!of!each!pDOT!fragment.!(B)!Histochemical!localization!of!GUS!activity!in!flowering!petunia!apices! containing!GUS!transgenes!driven!by!the!pDOT!deletion!derivatives!ΔA!to!ΔF,!a!3.1!kb!(pDOT3.1)!or!4.6! kb!pDOT!fragments!(pDOT4.6).!FM.!*,!sympodial!meristem.!Consecutive!flowers!are!numbered!from! young!to!old!(f1,!f2,!etc.).! !

Identification!of!trans@acting!factors!necessary!for!the!activation!of!pDOT!

Several!studies!in!Arabidopsis!showed!that!a!complex!gene!regulatory!network!that!involves! numerous! transcription! factors! (TFs)! controls! the! onset! of! flowering! by! activating! transcription!of!LFY!and/or!AP1.!As!the!onset!of!flowering!and!the!positioning!of!flowers!in!

! 82! Chapter!3! petunia! are! primarily! determined! by! the! activation! of! DOT,! we! examined! whether! pDOT! responds!to!the!same!TFs!that!control!LFY!expression!in!Arabidopsis.!! To!explore!which!of!these!TFs!are!able!to!activate!DOT!we!fused!the!3.1!kb!fragment!of! DOT! promoter! to! the! GFP! coding! sequence!(pDOT3.1:GFP).! Subsequently,! we! transformed! petunia! leaf! protoplasts! with! this! construct! in! combination! with! constructs! expressing! Arabidopsis!TFs!known!to!be!involved!in!the!switch!from!vegetative!to!reproductive!growth! (flowering)!and/or!the!specification!of!floral!meristem!identity.!Therefore!we!fused!the!full! length!coding!sequences!of!these!TFs!to!the!35S!promoter:!35S:AtFT((Kardailsky(et(al.,!1999;! Kobayashi( et( al.,! 1999),( 35S:AtFD( (Wigge( et( al.,! 2005),( 35S:AtLFY( (Blazquez( et( al.,! 1997),( 35S:AtFUL((Ferrandiz!et!al.,!2000!Science),(35S:AtAP1!(Bowman(et(al.,!1993),(35S:AtSOC1(and( 35S:AtAGL24( (Lee( et( al.,! 2008a;! Liu( et( al.,! 2008),! 35S:SPL3( (Cardon( et( al.,! 1997).! Equal! amounts! of! these! eight! constructs! and! pDOT3.1:GFP! were! mixed! and! used! to! transform! petunia!leaf!protoplasts!(Fig.!4).!If!one!or!more!TF(s)!of!the!mix!can!activate!pDOT3.1:GFP,!we! should! see! free! GFP! signal.! First! we! tested! whether! pDOT3.1:GFP( was! activated! by! TFs! present!in!leaf!protoplasts.!No!GFP!signal!was!visible!when!we!transfected!leaf!protoplasts! with!pDOT3.1:GFP(alone!(Fig!4A).! When!we!coZtransformed!the!protoplasts!with!a!cocktail!of!eight!constructs!expressing! the! aforementioned! Arabidopsis! TFs,! we! observed! GFP( expression! in! nearly! 70%! of! the! protoplasts!(Fig.!4B).!To!find!out!which!of!the!TFs!in!the!8!factor!cocktail!were!responsible! for! the! activation! of! pDOT3.1:GFP( we! repeated! this! experiment! with! different! cocktails! lacking! one! or! more! of! these! TFs.! Cocktails! lacking! FT,! FD( and( FUL! could! still! drive! pDOT3.1:GFP( expression!(Fig.! 4C).! Additional! exclusion! of! AP1! and! LFY!(Fig.! 4D)! and! subsequently!SOC1!left!the!GFP!signal!unchanged!(Fig.!4E).!As!in!this!last!experiment!only! AGL24!and!SPL3!were!present,!either!one!or!both!of!these!TFs!are!responsible!for!pDOT3.1! activation.!Indeed,!when!we!removed!both!(Fig!4F),!AGL24!alone!(Fig.4G),!or!only!SPL3!alone! (Fig.4H)!from!the!complete!TF!cocktail,!the!induction!of!pDOT3.1:GFP!was!no!longer!seen.!! Thus! both! AGL24! and! SPL3! seem! to! be! necessary! and! together! sufficient! for! (ectopic)! 3.1 3.1 pDOT !activation!in!leaf!cells,!whereas!the!other!six!factors!cannot!activate!pDOT .!! The!finding!AGL24!and!SPL3!can!only!activate!pDOT3.1!when!they!coZexpressed,!but!not! when! expressed! alone,! might! be! because! cooperation! of! ALG24! and! SPL3! is! required! for! binding! to! pDOT,! for! example! as! a! complex,! or! because! both! factors! can! bind! pDOT! independently!but!are!needed!together!for!transcription!activation.!To!discriminate!between!

83! ! DOT!regulation! these!possibilities!we!fused!AGL24!and!SPL3(to!the!strong!activation!domain!from!the!viral! transcription!factor!VP16!(Cousens(et(al.,!1989;!Triezenberg(et(al.,!1988),!which!should!make! these!proteins!lessZdependent!on!partners!for!transcription!activation.!When!expressed!in! protoplasts! both! AGL24ZVP16! and! SPL3ZVP16! could! induce! pDOT3.1:GFP! on! their! own,! suggesting! that! AGL24! and! SPL3! have! the! capacity! to! bind! pDOT! independently,! but! are! mutually!dependent!to!activate!transcription!(Fig!4IZJ).! Next,!we!considered!the!possibility!that!the!inability!of(FUL,(AP1,(LFY,(SOC1,(AGL24(and( SPL3(to!activate(pDOT3.1!was!due!to!the!absence!of!a!suitable!partner!protein.!Therefore,!we! tested!whether!corresponding!VP16!fusions!could!activate!pDOT3.1:GFP!in!protoplasts!(Fig!4IZ L).!When!we!expressed!FULZVP16!or!SOCZVP16!in!protoplasts!we!did!not!observe!expression! of!pDOT3.1:GFP!(Fig!4!KZL)!suggesting!that!neither!FUL!nor!SOC1!is!a!direct!regulator!of!pDOT( (Fig.!4K).!! ! ! ! ! ! !

! 84! Chapter!3!

! Figure! 4:! AGL24! and! SPL3! are! necessary! and! sufficient! to! activate! pDOT:GFP( in! petunia! leaf! protoplasts.! (AZH)! Transient! coexpression! of! pDOT:GFP! and! with! a! “cocktail”! of! Transcription! Factors( in! leaf! protoplasts.!(A)!pDOT3.1(alone.!(B)!pDOT3.1!with!the!complete!mix!of!eight!TFs.!(C)!pDOT3.1!with!LFY,( AP1,(SOC1,(AGL24! and!SPL3.((D)!pDOT3.1! with( SOC1,(AGL24! and!SPL3.! (E)!pDOT3.1! with!AGL24! and! SPL3.((F)!pDOT3.1!with!FT,(FD,(FUL,(LFY,(AP1(and(SOC1.!(G)(pDOT3.1!with!FT,(FD,(FUL,(LFY,(AP1,(SOC1( and(AGL24!(H)!pDOT3.1!with!FT,(FD,(FUL,(LFY,(AP1,(SOC1(and!SPL3.!(IZM)!pDOT3.:GFP1(can!be!activated! by! coZtransformation! of! 35S:AGL24IVP16! (I)! 35S:SPL3IVP16( (J)! alone,! but! not! by! cotransformation! with! 35S:FULIVP16( (K)! or( 35S:SOC1IVP16( (L).! (M)! RTZPCR! analysis! of! GFP! mRNA! from! protoplasts! transfected!with!pDOT3.1:GFP(alone!and!in!combination!with!35S:AGL24IVP16(or!35S:SOC1IVP16.( !

85! ! DOT!regulation!

Identification!of!putative!AGL24!and!SPL3!orthologues!in!petunia!

The! findings! that! the! Arabidopsis! TFs! AGL24! and! SPL3! can! activate! of! the! pDOT3.1! promoter! encouraged! us! to! identify! their! homologs! from! petunia.! AGL24! belongs! to! the! MIKC!type!MADS!box!family,!of!which!the!genome!of!Arabidopsis,!rice,!and!poplar!have!been! reported!to!contain!39,!47,!and!64!members,!respectively!(Becker!and!Theissen,!2003).!In! order! to! identify! the! putative! ortholog(s)! of! AGL24! in! petunia,! we! performed! a! tBLASTn! searches! against! the! Petunia! inflata! and! axillaris! genome! using! the! protein! sequence! of! AGL24( as! a! query.! As! expected,( this! identified! a! large! number! of! proteins! with! various! degrees!of!similarity.!The!three!most!similar!genes!were!selected!as!hypothetical!AGL24Ilike! genes:! EXP,! FBP13! and! FBP25.! All! three! genes! belong! to! the! STMADS11! subZclade! of! the! MADSZbox! family! (Becker! and! Theissen,! 2003;! Hartmann( et( al.,! 2000;! Lee( et( al.,! 2008b;! Szymkowiak!and!Irish,!2006),!which!includes!known(floral!meristem!identity!repressors!like! INCOMPOSITA!(AmINCO)! from! Antirrhinum,! SHORT! VEGETATIVE( PHASE!(AtSVP)! from! Arabidopsis,( and! JOINTLESS!(J)! from! tomato( (Gregis( et( al.,! 2008;! Hartmann( et( al.,! 2000;! Masiero( et( al.,! 2004).! In! order! to! establish! which! of! the! three! candidate! petunia! genes! is! orthologous! to! AGL24,( we! performed! phylogenetic! analyses.! The! sequences! of! 52! MIKC! MADS!proteins!belonging!to!11!different!species!were!retrieved!from!the!UNIPROT!database! (http://www.uniprot.org)! and! included! in! the! phylogenetic! analysis.! We! first! aligned! the! amino!acid!sequences!representing!the!MADSZdomain!(60!amino!acids)!(Fig.!S6)!and!KZbox! (95Z110!amino!acids)!(Fig.!S7).!The!alignment!was!restricted!to!these!domains,!because!the! MADSZdomain!and!KZbox!are!the!most!conserved,!fulfilling!important!functions,!while!the! intervening!region!and!CZterminal!domain!are!very!variable!and!difficult!to!align!properly,! thereby! possibly! reducing! the! reliability! of! phylogenetic! analysis.! The! alignment! was! performed!with!the!internal!CLUSTALW!feature!of!the!MEGA5.1!software!and!adjusted!by! hand!afterwards.!The!MADSZdomain!appeared!overall!to!be!very!conserved,!and!even!12!of! the!60!amino!acid!positions!were!found!to!be!identical!in!all!proteins!(Fig.!S6).!The!KZbox,!on! the! other! hand,! displayed! more! variation! in! its! sequence,! especially! in! the! 5’Zregion,! potentially!resulting!in!the!preference!for!different!interaction!partners.!Not!a!single!amino! acid!residue!showed!100!%!conservation!among!the!52!sequences!(Fig.!S7).!Both!the!MADSZ domain!and!KZbox!of!AGL24,(EXP,(FBP13(and(FBP25!showed!a!high!degree!of!similarity!as! expected,!although!the!MADS!domain!sequence!of!FBP25!appeared!to!be!more!dissimilar!to!

! 86! Chapter!3! that!of!AGL24!than!the!MADS!domain!of!EXP!and!FBP13.!We!then!performed!a!phylogenetic! analysis!with!both!the!MADSZdomain!and!KZbox!sequences!(Fig!5).!This!indicated!that!EXP!is! closely!related,!and!presumably!orthologous,!to!the!floral!meristem!identity!repressors!(SVP,! INCO! and! J)! (Gregis( et( al.,! 2008;! Hartmann( et( al.,! 2000;! Masiero( et( al.,! 2004),! which! is! supported! by! the! finding! that! several! genes! that! flank! SVP! also! flanking! J! (microsynteny).! FB13,!on!the!other!hand,!groups!in!the!same!clade!of!the!MPF2Zlike!protein!and!the!potato! StMADS16.!FBP25!grouped!with!the!potato!StSTMDS11,!being!somewhat!less!similar!to!SVP! but! could! not! be! clearly! identified! as! homologous! to! either! SVP! or! AGL24.( Previous! experiments!showed!that!EXP(transiently!represses!floral!identity!in!sympodial!meristems!of! petunia,!while!in!Arabidopsis!it!causes!late!flowering!and!green!petals,!indicating!that!EXP! protein!is!functionally!more!similar!to!SVP,!INCO!and!J!than!to!AGL24!(Kusters,!2011).! To!identify!the!putative!petunia!orthologs!of!SPL3(we!used!a!similar!procedure!as!above.! SPL3!belongs!to!the!SBPZbox!gene!family.!This!family!received!its!name!from!the!first!two! isolated! members! SQUAMOSA( PROMOTER( BINDING( PROTEIN( 1( and( 2!(SBP1! and! SBP2),! which!were!shown!to!interact!with!the!SQUAMOSA!(SQUA)!promoter!of!Antirrhinum(majus.! This!gene!family!encodes!plantZspecific!transcription!factors!that!contain!a!highly!conserved! domain,!known!as!the!SBPZbox,!which!is!generally!encoded!by!the!first!and!second!exon!of! the!gene!(Cardon(et(al.,!1999).!! In!order!to!find!the!putative!PhSPL3(homolog,!we!used!the!protein!sequence!of!SPL3(as!a! query!to!search!the!Petunia!inflata!and!P.!axillaris!genome!with!tBLASTn.!We!found!a!large! number!of!scaffolds!encoding!proteins!with!different!degrees!of!similarity.!Top!hits!from!the! query!were!subsequently!used!for!further!analysis.!Since!none!of!these!SPL!homologs!had! been! annotated,! we! predicted! their! structure! and! exon/intron! sequences! by! using! the! known!sequence!and!structure!of!SPL3.!In!order!to!increase!the!accuracy!of!the!predicted! gene!structures!of!the!putative!SPL3!orthologs,!a!tBLASTn!was!carried!out!on!the!genome!of! Petunia( axillaris( with! other! members! of! the! Arabidopsis(SPL! gene! family! that! displayed! a! similar! gene! structure! (SPL4! and! SPL5).! Information! on! shared! genetic! sequence! and! structure!was!then!used!to!improve!the!prediction!of!the!putative!petunia!orthologs!of!SPL3.( Besides! identifying! and! predicting! the! gene! structure! of! putative! PhSPL3! orthologs,! an! additional! 9! putative! SBPZbox! proteins! of! varying! size! were! identified! in! the! genome! of! Petunia( axillaris.! The! nomenclature! of! the! Arabidopsis! SBPZbox! genes! was! adopted,! and! petunia!genes!were!numbered!according!to!nucleotide!similarity!to!the(SPL!genes.!

87! ! DOT!regulation!

61 TaMPF2-like 32 WsMPF2-like2

11 VbMPF2-like WsMPF2-like

58 49 PpMPF2 95 PpMPF2-like 55 WcMPF2-like 31 StMADS16 98 SmMSM2 60 PhFBP13 20 CaSTMADS11

28 VvSVP AtSVP AmINCO 55 73 72 PhEXP 94 100 SlJ AtAGL24 PhFBP25 100 StMADS11 AtAGL12

81 AtSOC1

97 PhFBP28 AtAGL14 99 AtAGL19 44 AtAG

51 AtAGL7 25 AtAGL3

100 AtAGL8 30 FLC 12 AtAGL6 99 AtAGL13

! Figure!5:!Phylogenetic!analysis!by!Maximum!Likelihood!method!of!fused!MADS!domain!and!K!box! sequences!from!different!plant!species.!! Numbers!on!branches!indicate!percentage!bootstrap!support!(1000!replicates).!The!tree!is!based!on! the!alignment!of!amino!acid!sequences!of!conserved!domains!shown!in!Fig.!S6Z7.!At(=(A.(thaliana,(Ca( =(C.(annuum,(Ph(=(P.(hybrida,(Pp(=(P.(pubescens,(Sl(=(S.(lycopersicum,(Sm(=(S.(macrocarpon,(St(=(S.( tuberosum,(Ta(=(T.(anomalum,(Vb(=(V.(breviflora,(Vv(=(V.(vinifera,(Wc(=(W.(coccoloboides,(Ws(=(W.( somnifera.!

! 88! Chapter!3!

! !

! Figure!6:!Unrooted!phylogenetic!tree!of!SBP@box!genes!from!petunia!and!Arabidopsis.! The! tree! was! generated! using! MEGA! 5.1! with! the! neighborZjoining! method.! Multiple! sequence! alignment! of! the! SBPZbox! proteins! was! performed! using! clustal! X2.0.! Numbers! above! branches! indicate!bootstrap!value.!The!tree!is!based!on!an!alignment!of!amino!acid!sequences!of!conserved! domains!(Fig.!S8).! ! ! To!determine!the!phylogenetic!relationships!between!the!SBPZbox!genes!of!petunia!and! Arabidopsis,!we!constructed!a!phylogenetic!tree!based!on!multiple!sequence!alignments!of! the! SBPZdomain! amino! acid! sequence! (80! AA)! of! the! putative! SBPZbox! proteins.! The!

89! ! DOT!regulation! alignment!was!performed!with!the!internal!CLUSTALW!feature!of!the!MEGA5.1!software!and! adjusted! by! hand! afterwards.! As! expected! the,! SBPZbox! appeared! to! be! overall! very! conserved!(Fig.!S8),!including!the!amino!acid!residues!that!form!the!two!zincZfingers!and!the! Nuclear!Localization!Signal!(NLS)!domain.!The!first!three!cysteine!residues!of!the!first!zincZ finger! were! completely! conserved! in! all! proteins,! whereas! the! fourth! residue,! which! is! histidine!in!most!proteins,!appeared!to!be!changed!into!cysteine!in!SPL7.!The!second!zincZ finger!(CZCZH—C)!however!showed!100%!conservation!at!all!positions.!The!25!SBPZbox!genes! formed! nine! groups! in! the! unrooted! phylogenetic! tree! (Fig.! 6).! PhSPL3a! and! PhSPL3b! clustered!together!with!SPL3.!PhSPL4/5/6!were!also!clustering!together!with!SPL3/4/5,!but!it! was!not!possible!to!have!a!more!defined!one!to!one!correspondence.!PhSPL10!and!PhSPL12! reside! in! a! clade! together! with! SPL1,! SPL12,! SPL14! and! SPL16,! which! are! the! longer! SPL! proteins!of!Arabidopsis.!PhSPL11!was!in!the!same!clade!as!SPL8,!known!to!be!involved!in! pollen! sac! development! (Unte( et( al.,! 2003).! While! PhSPL7! was! close! to! SPL9! and! SPL15,! known! to! be! involved! in! the! regulation! of! the! juvenileZtoZadult! and! floral! transitions! (Schwarz(et(al.,!2008).!As!shown!in!the!tree,!the!evolutionary!closest!petunia!homologue!of! SPL3!appeared!to!be!PhSPL3a!and!PhSPL3b.(Given!that!PhSPL3b!contains!a!premature!stop! codon!in!the!first!exon!we!decided!to!exclude!it!in!further!analysis.!

Analysis!of!expression!profiles!of!FBP13,(FBP25!and!PhSPL3!

The! expression! pattern! of! a! gene! is! often! indicative! of! its! possible! function! in! certain! developmental! processes,! and! suitable! if! no! mutant! plants! are! available.! The! spatial! and! temporal! expression! of! the! identified! putative! petunia! orthologs! of! AGL24! and! SPL3! was! explored! using! qRTZPCR! and! in! situ! hybridization.! RNA! analyses! revealed! that! FBP13! is! primarily! expressed! in! inflorescence! apices! (which! contain! SIMs,! floral! meristems! and! developing! bracts)! and! in! leaves,! but! not! in! petals,! while! the! expression! of( FBP25! was! constant!in!all!the!tissues!analysed!(Fig.!S10A).!The!SBPZbox!genes!PhSPL3/4/6!showed!the! highest!expression!in!the!inflorescence!apices!and!low!expression!in!the!other!tissues,!while! SPL5!mRNA!was!detected!mainly!in!petals!(Fig.!S10B).! To!further!investigate!the!expression!in!the!developing!petunia!inflorescence!apices,!we! used!in!situ!hybridisation!and!specific!probes!to!examine!the!expression!patterns!of!FBP13,! FBP25,! and! PhSPL3,! to! discern! whether! they! are! expressed! in! the! SIM! and/or! the! floral! meristem((Fig.!S10CZF).!If!one!of!these!genes!is!involved!in!the!regulation!of!DOT,!we!expect!

! 90! Chapter!3! it!to!be!expressed!in!regions!that!overlap!with!or!are!adjacent!to!the!sepal/petal!boundaries.! We!observed!FBP13(transcripts!in!the!SIM,!but!not!in!developing!floral!meristems,!making!it! unlikely!that!this!gene!is!(directly)!involved!in!the!activation!of!DOT!(Fig.!S10D).!Transcripts! of! FBP25! were! detected! in! the! developing! apical! FM! and! emerging! SIM! (Fig.!S10E).! The! expression!of!SPL3!was!observed!in!the!young!petunia!flowers!when!the!IM!and!FM!are!not! yet!completely!separated!(Fig.!S10F).!Since!FBP25!and!SPL3!are!expressed!in!the!entire!dome! of!emerging!flower!meristem,!before!the!IMZFM!separation,!they!might!be!involved!in!the! regulation!of!DOT.!

Determination!of!physical!interactions!between!putative!activators!of!pDOT!!

Activation! of! the! pDOT3.1:GFP! construct! in! petunia! protoplasts! requires! the! simultaneous! expression! of! AGL24! and! SPL3,! or! versions! of! these! proteins! with! constitutively! active! transcription!activation!domains!(VP16).!One!scenario!is!that!these!two!proteins!physically! interact!at!the!DOT!promoter.!Therefore,!we!performed!a!yeast!twoZhybrid!interaction!assay! using!AGL24,!SOC1!and!SPL3,!EXP,!FBP13,!FBP25,!PhSPL3a!and!FBP28.!Except!for!FBP28,!all! genes! were! fused! to! the! transcription! activation! domain! (GAL4AD)! and! the! DNAZbinding! domain!(GAL4BD)!of!GAL4.!By!mating,!yeast!strains!expressing!all!combinations!of!GAL4AD! and!GAL4BD!fusions!were!produced!and!tested!for!their!ability!to!activate!the!LacZ!reporter! gene!(Fig.!S10).!All!created!fusion!constructs!contained!the!entire!coding!sequence,!except! for!PhFBP28IGAL4AD,!which!missed!the!first!74!amino!acids!that!represent!the!MADS!domain! and!the!first!four!amino!acids!of!the!KZbox.!The!combinations!EXPZFBP28,!EXPZEXP(and!SOC1Z AGL24! served! as! positive! controls,! since! these! interactions! were! reported! previously! (Kusters,!2011;!Lee(et(al.,!2008a;!Souer(et(al.,!2008).!In!order!to!determine!possible!autoZ activation!of!some!GAL4!fusions,!they!were!also!tested!in!combination!with!empty!GAL4AD! and! GAL4BD.! FBP13ZGAL4BD! appeared! to! activate! the! reporter! gene! irrespectively! of! its! interaction!partner,!even!with!the!empty!GAL4AD!vector!(Fig.!S11),!suggesting!that!FBP13Z GAL4BD! autoactivates! the! reporter! genes,! either! on! its! own,! or! by! interaction! with! an! endogenous!yeast!protein.!The!only!MADS!box!protein!that!showed!ability!to!dimerize!was! EXP,!while!FBP25,!AGL24!and!SOC1!did!not!show!any!interaction.!We!found!interactions!of! AGL24!with!PhFBP28!and!of!SOC1!with!FBP13!and!FBP25,!showing!functional!conservation! between!SOCZlike!and!AGL24Zlike!genes.!Interestingly,!FBP13!and!AGL24!were!able!to!form! heterodimers,!whereas!FBP25!and!AGL24!did!not!show!any!interaction!(Fig.!S11).!The!yeast!

91! ! DOT!regulation! twoZhybrid!analysis!showed!that!both! FBP13!and!FBP25!maintained!the!ability!to!interact! with!SOC1!and!FBP28,!suggesting!that!the!proteinprotein!interaction!between!the!AGL24Z like!and!SOC1ZLike!proteins!is!evolutionarily!conserved!in!Arabidopsis!and!petunia.!PhSPL3a! and!SPL3,!on!the!other!hand,!did!not!show!any!interaction!with!the!other!proteins!tested.!

A C E F

WT 35S:FBP13 35S:FBP25 B D G

WT 35S:FBP13 35S:FBP25 35S:FBP25 ! ! Figure!7:!Overexpression!of!FBP25,(but!not(FBP13,(accelerates!flowering!time!in!Arabidopsis!under! short!day!conditions.(! (AZE)!Comparison!of!flowering!time!of!wild!type!Arabidopsis((AZB),!and!transgenic!siblings!expressing! 35S:FB13!(CZD)!or!35S:FBP25((E)!under!SD!conditions.!(FZG)!In!strong!FBP25!expressors!inflorescences! terminate!with!an!apical!flower!with!an!incomplete!number!of!floral!organs.! !

Function!of!FBP13!and!FBP25!in!Arabidopsis(and!in(petunia(!

To!determine!whether!the!petunia!AGL24!homologs!can!influence!flowering!time(in!petunia! and!Arabidopsis,!we!tested!the!effect!of!their!ectopic!expression!in!both!plants.!Constitutive! expression!of!FBP13!from!the!35S!promoter!did!not!alter!the!flowering!time!of!Arabidopsis( plants,!whereas!constitutive!expression!of!FBP25!caused!early!flowering!compared!to!wild! type! plants! grown! under! the! same! conditions! (Fig.! 7AZE).! Furthermore,! strong! 35S:FBP25( expressors(produced!fewer!flowers!due!to!a!conversion!of!the!apical!inflorescence!meristem! into!a!terminal!flower!(Fig.!7FZG).!This!indicates!that!ectopic!expression!of!FBP25!results!in! ectopic!expression!of!floral!meristem!identity,!both!in!time!and!in!space.!

! 92! Chapter!3!

In! wild! type! petunia,! however,! the! constitutively! expressed! 35S:FBP13! or! 35S:FBP25! transgenes!did!not!cause!precocious!flowering!or!changes!of!the!inflorescence!architecture! under!long!day!(LD)!conditions!(Fig.!S12AZC).!The!only!difference!with!wild!type!plants!was! an!extra!outgrowth!of!petal!tissue!(“spurs”)!on!the!abaxial!side!of!petal!tubes!(Fig.!S12DZF).! This!suggests!that!although!FBP25!protein!is!functionally!similar!to!AGL24!in!Arabidopsis,!its! function!in!petunia!has!been!changed!so!that!its!ectopic!expression!is!not!sufficient!to!trigger! early! flowering.! Furthermore,! a! transposon! insertion! at! the! beginning! of! the! coding! sequence!of!FBP25,!which!results!in!a!premature!stop!codon!and!complete!knock!out!of!the! gene,! did! not! cause! any! obvious! phenotypic! changes! (M.! Vandenbussche! personal! communication).!Often,!single!mutant!of!the!MADS!box!gene!family!in!petunia!are!wild!type! because!of!redundancy!with!other!paralogs.!

AGL24!in!petunia!caused!early!flowering!but!only!in!short!day!

In!Arabidopsis!constitutive!expression!of!AGL24!is!known!to!induce!early!flowering!(Yu(et(al.,! 2002).!Thus!we!asked!whether!the!same!would!happen!in!petunia.!To!our!surprise!we!found! that!constitutive!expression!of!35S:AGL24!in!wild!type!petunia!W115!did!not!affect!flowering! time!or!the!development!of!the!inflorescences!and!flowers!(Fig.!8AZC).!Also!counting!of!the! leaves!before!the!first!flower!did!not!show!a!significant!difference!with!WT!(Fig.!8A).!The! only! alteration! that! we! observed! was! extra! petal! tissue! outgrowth! (“spurs”),! similar! to! 35S:FBP13!and!35S:FBP25!transformants!(Fig.!8DZE).!Because!the!acceleration!of!flowering! time! in! 35S:AGL24( plants! might! be! hidden! under! inductive! long! day! conditions! by! the! photoperiod! pathway,! which! is! controlled! by! FT! and! homologs,( we! grew! progeny! of! the! 35S:AGL24! transformants! under! short! day! (SD)! conditions! and! compared! their! flowering! time!to!wild!type!W115.! We!found!that! under!these!conditions! the!ectopic!expression!of! AGL24!accelerated!the!onset!of!flowering!(Fig.!8A!and!FZG).!On!average,!AGL24!expressors! produced!16!leaves!less!than!wild!type!plants!before!they!switched!to!flowering.!

93! ! DOT!regulation!

These!results!show!that!ectopic!expression!of!AGL24!could!alter!the!flowering!time!of! petunia,!but!only!in!short!day!conditions.!Probably!in!long!day!conditions!other!factors,!like! the!photoperiod!pathway,!accelerate!flowering!making!it!difficult!to!appreciate!difference! between!wild!type!and!35S:AGL24!plants.!!

! Figure!8:!Constitutive!expression!of!AGL24!in!petunia.! (A)!Flowering!times!of!petunia!W115!and!35S:AGL24(independent!transformants!in!short!days!(SD)! and! long! days! (LD)! condition.! (BZC)! Comparison! of! flowering! time! and! inflorescence! architecture! between!wild!type!W115!(B)!and!35S:AGL24!(D)!under!LD!conditions.!(DZF)!Extra!petal!tissue!growth! in!35S:AGL24.!(FZG)!Comparison!of!flowering!time!and!inflorescence!architecture!between!wild!type! W115!(F)!and!35S:AGL24!(F)!and!in!SD!conditions.(! !

! 94! Chapter!3!

SPL3(in!petunia!caused!early!flowering!in!short!days!whereas!35S:PhmiR156(altered!flower! meristem!identity.!

To! determine! whether! SPL3! regulates! flowering! time! in! response! to! ambient! condition! in! petunia,! we! tested! the! effect! of! its! ectopic! expression! in! wild! type! petunia.! Under! LD! condition!we!did!not!observe!any!difference!between!35S:SPL3(transformants!and!wild!type! siblings((Fig.!9AZC),!both!plants!flowered!after!developing!approximately!28!leaves!(Fig.!9A).! However,! in! SDZconditions! the! 35S:SPL3( plants! flowered! much! earlier! than! wild! type! and! also! showed! less! secondary! growth! compared! to! wild! type! (Fig.! 9A! and! EZF).! In! both! conditions,!the!inflorescence!architecture!was!not!altered.!! Also!in!this!case!we!wanted!to!assess!a!loss!of!function!phenotype!but,!since!it!is!known! that! mutation! of! SPL3! in! Arabidopsis! causes! no! visible! phenotype,! most! likely! because! of! functional!redundancy!with!other!related!SPL!proteins!(SPL4/5)!(Wu!and!Poethig,!2006),!we! decided!to!down!regulate!these!genes!through!overexpression!of!microRNA(156!(miR156).! This!microRNA!family!is!known!to!be!involved!in!phase!transition!in!Arabidopsis!and!ectopic! expression! of! miR156! results! in! prolongation! of! the! vegetative! phase,! as! well! as! delayed! flowering!(Wu!and!Poethig,!2006)!.! Using! the! sequence! of! Arabidopsis! miR156b! we! were! able! to! find! the! corresponding! petunia!miR156b!and,!after!confirming!that!PhSPL3/4/5/6!indeed!have!the!specific!binding! site!for!the!miR156!in!their!3’!UTR,!we!overexpressed!miR156!in!wild!type!W115.!We!then! analyzed!its!effect!in!both!LD!and!SD!conditions!since!from!Arabidopsis(studies!it!is!known! that!photoperiod!can!overcome!the!repression!of!35S:miR156.!In!LDs,!ectopic!expression!of! the! PhmiR156! did! not! alter! the! flowering! time! (Fig.! 9A)! but! it! had! a! strong! effect! on! the! inflorescence!architecture.!As!shown!in!Figure!9D,!the!main!branch!ended!in!a!solitary!flower! without! producing! any! further! flowers.! After! termination! of! the! main! branch,! growth! restarted! from! the! dormant! meristems! in! the! axils! of! more! basipetal! leaves,! which! also! ended! into! solitary! flowers! after! producing! some! leaves! first.! This! phenotype! was! the! opposite!of!what!we!expected!and!it!seemed!to!caused!ovrerexpression!of!DOT!in!the!shoot! apical! meristem! rather! then! downregulation,! since! 35S:DOT! plants! show! a! similar! phenotype.!This!phenotype!also!resembles!that!of!hermit!mutants,!which!have!a!defect!in! the! initiation! of! the! sympodial! inflorescence! meristem! due! to! a! defect! in! a! KNOX! gene! (Castel,!2009)!and!exp!mutants,!in!which!the!SIM!precociously!acquires!floral!identity.!!

95! ! DOT!regulation!

!

! ! Figure!9:!Constitutive!expression!of!SPL3!and!PhmiR156!in!petunia! (A)! Flowering! times! of! petunia! W115,! 35S:SPL3! and! 35S:PhmR156( independent! transformants! in! short! days! (SD)! and! long! days! (LD)! condition.! NF! indicates! never! flowered.! (BZD)! Comparison! of! flowering! time! and! inflorescence! architecture! between! wild! type! W115! (B),! 35S:SPL3! (C)! and! 35S:PhmiR156( (D)( under! LD! conditions.! (EZG)! Comparison! of! flowering! time! and! inflorescence! architecture!between!wild!type!W115!(E),!35S:SPL3!(F)!and!35S:PhmR156((G)(in!SD!condition.! ! These!results!were!surprising!since!they!showed!an!opposite!phenotype!than!what!we! would!have!expected.!In!several!species,!the!overexpression!of!miR156!cause!mainly!delay!in! flowering! time,! together! with! several! secondary! effects! as! trichomes! reduction,! simpler! leaves! development! and! extensive! secondary! growth.! In! petunia! it! shows! and! additional! phenotype,!the!failure!of!FMI!repression!in!the!emerging!sympodial!inflorescence!meristem,!

! 96! Chapter!3! causing!the!conversion!of!the!typical!cyme!inflorescence!of!petunia!into!a!solitary!flower.!A! similar! phenotype! was! already! observed! in! petunia! mutants! as! HERMIT!(HER)! and/or! EXTRAPETALS!(EXP)!where!the!initiation!of!sympodial!meristems!and/or!its!maintenance!is! compromised! causing! the! formation! of! a! solitary! flower.! When! we! analyzed! the! overexpressing!plants!in!short!days!condition,!the!phenotype!caused!by!miR156!was!even! stranger:!once!again!these!plants!were!not!able!to!maintain!the!inflorescence!meristem,!but! in!this!case!all!the!flowers!produced!were!aborted!(Fig.9G!and!insert)!and!the!plants!kept!a! vegetative!growth.!In!SD!condition!these!plants!were!never!able!to!generate!fully!developed! flowers!in!the!8!months!we!conducted!the!experiments.!

Simultaneous!ectopic!expression!of!SPL3!and!AGL24(in!petunia!

Since!from!the!protoplast!experiments!we!knew!that!in!order!to!activate!pDOT!both!AGL24! and!SPL3!were!necessary,!we!decided!to!cross!the!single!overexpressors!in!order!to!have! both!transgene!in!the!same!plants.!After!genotyping!around!30!plants!from!four!different! crosses,!none!of!the!plant!we!analyzed!was!carrying!both!genes.!In!a!specific!cross!we!could! noticed!that!a!quarter!of!the!seedlings!were!dying!just!after!germination.!We!then!decided! to!overexpress!the!single!genes!fused!to!the!VP16,!since!from!the!protoplast!experiments!we! knew! that! these! proteins! fusion! were! positively! regulating! pDOT.! We! then! try! to! stably! transformed! into! wild! type! W115! petunia! the! SPL3:VP16! and! AGL24:VP16,! but! we! never! were!able!to!produced!any!transformants!since,!already!during!the!transformation,!the!calli! were!dying.!Most!likely!the!overexpression!of!the!activated!form!of!these!proteins!(AGL24Z VP16!or!SPL3ZVP16)!or!coZexpression!of!the!two!native!proteins!has!lethal!effects!! To! overcome! this! problem! we! expressed! the! VP16! fusion! under! the! control! of! the! Estradiol!inducible!promoter!and!we!stably!transformed!the!constructs!into!wild!type!W115.! Unfortunately!it!seems!that!petunia!produces!hormones!similar!to!estradiol!so!the!transgene! was!already!induced!along!transformation!process!and!we!never!were!able!to!produce!any! expressing!plant.!

Discussion!

Changes! in! the! inflorescence! architecture! are! associated! with! differences! in! the! spatioZ temporal!regulation!of!meristem!identity!genes!ALF/LFY,!but!more!significantly,!of!DOT/UFO! (Kusters(et(al.,!2015).!Here!we!have!shown!that!the!rewiring!of!the!genetic!networks!that!

97! ! DOT!regulation! control!flowering!time!in!petunia!caused!change!in!the!expression!pattern!of!DOT!compared! to!that!of!UFO!and!we!further!identifying!the!cluster!of!cisZregulatory!elements!(CREs)!and! transZacting!factors!that!might!account!for!the!different!expression!pattern!between!these! two!genes.! Through!promoter!shadowing!and!bashing!we!have!shown!that!the!DOT!locus!underwent! several! changes! compared! to! its! homologs.! Genome! and! gene! duplications! followed! by! structural! rearrangements! happen! frequently! during! plant! evolution! and! are! thought! to! strongly! contribute! to! the! amazing! genetic! and! morphological! plant! diversity.! It! was! interesting!to!notice!that,!despite!the!evolutionary!distance!between!the!plants!analyzed,! the!overall!synteny!genes!organization!is!still!conserved!within!the!Solanaceae!family,!with! the! exception! of! petunia! that,! compared! to! Solanum( lycopersicum! and! S.! tuberosum,! underwent!further!rearrangements,!placing!DOT!in!a!different!genomic!environment!(Fig!1).! This! might! be! explained! by! the! presence! of! two! MuDR/Mu! transposons! upstream! and! downstream! the! PhDOT! locus:! it! is! known! that! the! genome! of! petunia! is! rich! of! active! transposons!(Van!den!Broeck(et(al.,!1998),!successfully!used!in!the!generation!of!mutants!via! transposon!tagging!(Vandenbussche(et(al.,!2013).!When!we!widen!the!analysis!to!the!other! families! present! in! the! tree! we! could! immediately! observed! an! inversion! between! the! different! DOT! loci.! Such! rearrangements! might! have! brought! DOT! under! the! control! of! enhancers!originating!from!distinct!genes,!resulting!in!a!new!expression!pattern.!However,! the!finding!that!the!5!kb!region!upstream!of!the!DOT!coding!sequence,!which!contains!most! of!the!cisZregulatory!elements,!contains!four!boxes!that!are!conserved!among!species!within! and! outside! of! the! Solanaceae! family! (Fig.! S1Z5)! suggests! a! different! scenario.! First,! it! indicates!that!the!break!point!of!the!rearrangement!lies!>!5kb!upstream!of!the!DOT!locus! and! did! not! (directly)! contribute! to! changes! in! the! more! proximal! cisZelements! and! expression!changes.!Second,!also!in!the!Brassicaceae!family!we!observed!a!further!genomic! rearrangement,! lineage! specific,! which! could! cause! the! loss/gain! of! transcription! factors! binding!sites!in!UFO.!Together!with!the!observation!that!the!UFO!promoter!contains!only! BOX1,! this! may! suggest! that! UFO! might! be! the! only! one! to! have! a! different! expression! pattern,!whereas!the!others!(DOT,!AN!and!FIM)!show!a!similar!expression!pattern!(Kusters(et( al.,!2015).! Conserved!sequences!identified!by!phylogenetic!shadowing!in!nonZcoding!regions!are!in! most!of!the!cases!a!combination!of!cisZelements!(enhancer)!involved!in!the!regulation!of!a!

! 98! Chapter!3! given! promoter.! The! four! BOXes! identified! by! sequence! comparison,! and! the! fifth! by! promoter!deletion,!apparently!represent!clusters!of!regulatory!elements!necessary!for!the! correct!spatioZtemporal!expression!of!DOT.(Previous!analyses!showed!that!the!distal!region! of!pDOT,!which!includes!BOX3!and!BOX4,!boosts!the!expression!of!DOT!when!added!to!the! proximal! region! (Chapter! 2)! and! is! necessary! to! fully! complement! the! dot! mutant.! The! analysis! of! the! single! BOXes! revealed! that! BOX3( alone! was! able! to! recapitulate! the! same! expression! pattern! as! pDOT3.1( (Fig! 2),! suggesting! that! both! regions! act! in! an! additive,! or! perhaps!even!synergistic!way,!to!increase!DOT!expression!high!levels,!which!is!particularly! important!to!rescue!the!defects!in!petal!and!stamen!development!in!dot.!It!is!known!that! DOT!is!expressed!at!high!level!in!a!restricted!zone!of!the!FM!and!that!of!all!functions!of!DOT! the!one!in!flower!organ!development!is!the!most!sensitive!to!reduction!of!DOT!expression! (Souer( et( al.,! 2008).! It! is! interesting! that! BOX3! is! also! conserved! outside! the! Solanaceae! clade,!especially!in!the!promoter!region!of!FIMBRIATA((FIM),!given!that!pFIM!is!active!in!a! similar! pattern! as! pDOT! when! introduced! in! both! Arabidopsis! and! petunia! even! if! it! originates!from!a!species!(Antirrhinum)!with!a!racemose!inflorescence!architecture!(Kusters( et(al.,!2015);!whereas!we!could!not!find!any!conservation!between!pDOT!and!pUFO,!other! than!BOX1.!BOX1(is!necessary!for!the!activation!of!DOT!and!lies!close!to!the!transcription! start! site,! suggesting! that! this! box! represent! (part)! of! the! core! promoter! and! this! could! explain!why!this!box!is!conserved!in!all!the!species!analyzed.(Although!we!could!not!detect! obvious!conserved!sequences!in!between!nucleotides!Z1800!and!Z1080,!the!functional!data! show!that!this!region!contains!one!or!more!elements!(“BOX5”)!that!drive!DOT!expression! within!the!floral!meristem!at!the!sepal!boundary.( (It!appears!that!the!DOT!locus!underwent!several!alterations!that!caused!the!gain/loss!of! different!transcription!factor!binding!sites!that!made!DOT!to!be!responsive!to!different!TFs! compared! to! UFO! and,! more! importantly,! to! be! under! control! of! the! floral! integrators! pathway.!As!shown!in!Fig!S1!and!S3,!we!were!able!to!find!the!same!CArGIboxes!in!the!distal! and! proximal! promoter! regions.! Interestingly! these! binding! sites! are! conserved! even! in! species!outside!the!Solanaceae!family,!like!in!FIMBRIATA!(FIM)!of!Antirrhinum(majus,(that,!as! mention!before,!it!is!functionally!similar!to!DOT.!CaRGIbox(sequences!are!among!the!most! extensively! characterized! proteinZbinding! sites! and! are! bound! by! MADSZbox! protein.! In! Arabidopsis!these!protein!are!known!to!be!involved!in!several!aspects!of!plant!development! either! as! repressor! or! promoter.! Among! the! flowering! promoters,! AGL24( and( SOC1( are!

99! ! DOT!regulation! known!to!work!in!a!positive!feedback!loop!to!activate!the!expression!of!LFY((Ferrandiz(et(al.,! 2000;!Kaufmann(et(al.,!2010),!while!SHORT(VEGETATIVE(PHASE((SVP)(is!known!to!repress!the! switch! to! flowering! and! transiently! represses! floral! organ! identity! genes! during! the! early! stages!of!flower!development.!In!vivo!experiments!we!were!able!to!show!that!DOT!can!be! activated!by!the!same!TFs!(AGL24!and!SPL3)!that!activate!LFY!in!Arabidopsis!(Fig!4).!Although! it!was!recently!shown!that!PhSOC1Ilike!genes!promote!flowering!in!petunia!(Preston(et(al.,! 2014),!neither!SOC1!nor!FUL!could!activate!pDOT!ectopically!in!leaf!cells.!This!suggests!that! the!pDOT3.1!fragment!lacks!specific!binding!sites!for!these!proteins.!However,!we!cannot!rule! out! that! SOC1! and/or! FUL! (or! any! of! the! other! TFs! that! we! tested)! activate! pDOT! via! sequences! that! are! outside! pDOT3.1,! (e.g.! via! BOX3),! or! indirectly,! via! the! activation! of! intermediate! regulators.! Since! we! analyzed! the! activation! of! pDOT3.1:GFP( 16h! after! the! protoplasts!transfection,!we!were!able!to!detect!only!the!primary!activators.( EXP,! FBP13,! and! FBP25! are,! of! all! known! MADS! box! proteins! from! petunia,! the! most! similar!to!AGL24.!In!our!phylogenetic!analysis!EXP!grouped!with!JOINTLESS!(J)!of!Solanum( lycopersicum(and(SVP,!which!is!involved!in!the!repression!of!floral!transition!of!inflorescence! architecture.!However,!it!was!not!possible!to!distinguish!whether!FBP13!and/or!FBP25!are! true!homologs!of!AGL24!(Fig.!5).!At!the!functional!level!it!appears!that!FBP25!is!more!similar! to!AGL24((Fig.!7),!at!least!when!expressed!in!Arabidopsis,!supporting!the!idea!that!FBP25!is! the!possible!activator!of!DOT.!Moreover,!its!overexpression!in!Arabidopsis!quickly!converted! the! apical! inflorescence! meristem! into! a! terminal! flower,! which! is! generally! maintained! indeterminate,!probably!by!causing!an!ectopic!expression!of!LFY.!This!conversion!was!never! observed!before!in!35S:AGL24((Gregis(et(al.,!2008),!suggesting!either!that,!although!these! two!genes!have!maintained!similar!proteins!properties,!FBP25!has!evolved!additional!activity! compare!to!AGL24(Z!i.e.!by!interacting!with!different!proteins!Z(or!that!the!35S:AGL24!lines! analyzed!in!Arabidopsis!had!a!low!expression!level.!The!finding!that!constitutive!expression! of!AGL24,!FBP13!or!FBP25!does!not!affect!inflorescence!development!in!petunia!indicates! that!AGL24!or!its!homologs!are!alone!not!sufficient!to!specify!floral!identity.!Our!protoplast! experiments! showed! that! in! order! to! activate! pDOT( both! AGL24! and! SPL3! are! necessary,! although! these! proteins! do! not! interact! in( vivo( (fig.! S10).! More! interesting! is! that! AGL24Z VP16!and!SPL3ZVP16!can!activate!pDOT!on!their!own,!suggesting!that!each!of!this!protein! can!bind!to!pDOT!on!its!own!These!results!strengthen!the!idea!that,!even!if!the!transcription! factors!involved!in!the!flowering!of!Arabidopsis!are!conserved!in!petunia,!the!proteinZDNA!

! 100! Chapter!3! interaction(s)! and! thereby! the! structure! of! the! network! have! been! modified! causing! a! divergence!in!the!spatioZtemporal!regulation!of!the!floral!meristem!identity!genes.! We!identified!nine!putative!SBPZbox!proteins!of!varying!size!in!the!genome!of!Petunia( axillaris.!This!study,!however,!was!not!specifically!directed!at!the!identification!of!all!petunia! SBPZbox!genes.!We!therefore!expect!the!petunia!genome!to!contain!more!genes!than!the! nine!identified!here,!especially!as!most!plants,!including!related!Solanaceae!such!as!tomato,! possess! between! 15! and! 30! members.! The! SBPZbox! appeared! overall! very! conserved:! the! first!three!cysteine!residues!of!the!first!zincZfinger!were!completely!conserved!in!all!proteins,! but!the!fourth!histidine!residue!appeared!to!be!changed!into!another!cysteine!in!AtASPL7.! The! second! zincZfinger! (CZCZH—C),! however,! showed! 100! %! conservation! at! all! positions.! Also!the!nuclear!localization!signal!sequence!was!conserved!between!the!different!SPLs!(fig! S8).!This!suggest!that!the!specific!residues!in!this!domain!have!to!be!preserved!in!order!to! allow!the!SBPZbox!protein!to!bind!DNA!and!perform!its!function!as!a!transcription!factor.!The! only!SPL!gene!that!was!further!characterized!in!this!study!(PhSPL3)!showed!an!expression! pattern!that!overlaps!with!the!spatial!expression!of!DOT:!it!was!detected!in!young!bracts!and! emerging! meristems! of! the! inflorescence,! with! the! strongest! expression! in! the! premature! FM,!especially!at!the!boundary!between!the!emerging!FM!and!IMs.!This!pattern!suggests!a! role!of!PhSPL3!in!FMI!or!IMI!initiation.!! Under! inductive! photoperiods! flowering! occurs! in! young! Arabidopsis! plants! before! the! decline!of!the!miR156!levels!showing!that!the!SPL!activity!is!not!needed!for!flowering!(Wu! and! Poethig,! 2006).! The! same! was! observed! in! petunia,! where! the! overexpression! of! the! miR156(blocked!the!floral!transition!in!short!days!but!not!in!long!days,!demonstrating!that! also! in! the! annual! petunia,! inductive! photoperiod! can! overcome! the! ageZdependent! flowering! pathway.! Surprisingly,! constitutive! miR156( expression! caused! the! conversion! of! the! typical! cyme! architecture! into! a! solitary! flower! followed! by! a! breakdown! of! apical! dominance!and!outgrowth!of!axillary!meristem.!This!phenotype!is!reminiscent!of!the!effects! of! misZregulation! of! homeobox! genes.! KNOX! domainZcontaining! transcription! factors! are! known!to!be!involved!in!several!developmental!processes,!including!the!establishment!and! maintenance!of!the!shoot!apical!meristem!(Endrizzi(et(al.,!1996).!In!petunia!HERMIT!(HER),! the!ortholog!of!SHOOTMERISTEMLESS!(STM)!from!Arabidopsis,!is!required!for!the!initiation! and/or!maintenance!of!the!stem!cell!population!in!sympodial!meristems!(Castel,!2009).!The! peculiarity! of! her( mutants! is! that! after! the! main! axis! has! terminated! into! a! flower,! no!

101! DOT!regulation! sympodial!shoot!meristem!develops.!extrapetals!(exp)!mutants!and!35S:DOT!plants!also!lack! sympodial!shoots,!but!in!this!case!is!thought!to!result!from!a!failure!to!transiently!repress! floral!identity!in!the!sympodial!meristem!(Kusters,!2011).!Hence,!the!observation!that!the! downZregulation!of!the!SPLs,!caused!by!the!ectopic!expression!of!miR156,!also!converted!the! cymose!inflorescence!into!solitary!flowers,!can!be!explained!in!at!least!two!ways:!either!the! SPLs!genes!are!involved!in!the!regulation!of!KNOX!gene(s),!directly!or!indirectly,!so!that!misZ regulation! of! KNOX! results! in! the! failure! of! SIM! initiation/maintenance! or! the! SPLs! genes! “repress!the!repressor”!EXP,!directly!or!indirectly,!causing!ectopic!expression!of!DOT(in!the! apical!meristem.! We! previously! showed! that! the! regulation! of! the! floral! meristem! identity! genes! diversified! considerably! between! Arabidopsis! and! petunia! although! the! major! players,! LFY/ALF! and! UFO/DOT,! are! conserved.! Our! findings! indicate! that! the! divergence! in! the! regulation!of!UFO!and!DOT!arose!after!genomic!arrangement!on!the!DOT!locus,!placing!this! gene!under!the!control!of!the!same!floral!integrators!that!regulate!LFY!in!Arabidopsis.!This! suggests! that! although! the! upstream! network! that! regulates! floral! transition! is! conserved! between! these! two! plants,! the! proteinZprotein! and! DNAZprotein! interaction! has! been! rewired!in!petunia!compared!to!Arabidopsis.!Several!questions!immediately!arise!from!these! results,! which! remain! to! be! further! answered:! i)! can! AGL24/FBP13Z25! and! AtZPhSPL3! also! activate!FIM!and!UFO!expression?!ii)!are!AGL24/FBP13Z25!and/or!AtZPhSPL3!activating!the! DOT!homologues!by!binding!to!the!conserved!BOXes!found!in!this!study!and!iii)!are!these! boxes,!or!distinct!AGL24!and!SPL3!binding!sites,!also!present!in!the!regulatory!region!of!LFY! and!homologs!from!other!species?!

! 102! Chapter!3!

Materials!and!Methods!

Plasmid!construction!and!plant!transformation!!

To!generate!binary!destination!vectors!with!deletions!of!the!3.1!kb!the!pDOT3.1!promoter,! we!used!the!construct!previously!described!in!Chapter!2!(pDOT3.1:GUS).!We!digested!with! EcoRI/KpnI,! to! get! deletion! ΔF,! with! EcoRI/SacI! for! deletion! ΔE,! and! with! EcoRI/PacI! for! deletion! ΔD.! To! obtain! deletion! ΔAZC,! we! first! digested! pDOT3.1:GUS( with! EcoRI/KpnI,! we! then! amplified! three! PCR! fragments! from! the! undigested! plasmid! (pDOT3.1:GUS)! with! primers!#2661!and!#5276!(ΔA),!#2661!and!#5277!(ΔB)!and!#2662!and!#5274!(ΔC),!containing! either! EcoRI! or! KpnI! site! (Table! 1),! and! we! ligated! the! different! fragments! back! into! the! pDOTEcoRI/KpnI:GUS.! The! coding! sequences! of( Arabidopsis! floral! integrators! genes! were! amplified! from! Columbia!cDNA!and!their!petunia!homologs!from!W115!cDNA.!The!PhmiR156!and!individual! BOXes!from!pDOT!were!amplified!by!PCR!from!petunia!W115!gDNA!using!primers!listed!in! Table! 1.! PCR! products! were! then! introduced! into! the! GATEWAY! pDONR221! vector! (Invitrogen)!and!sequenced!with!Big!Dye!terminator!technology!(Perkin!Elmer)!before!they! were!transferred!into!the!desired!destination!vectors!(pK7GW7.0,!pB7YWG2.0,!pK2GW7.0).!! All! constructs! were! introduced! into! Agrobacterium( tumefaciens! strain! AGL0! by! electroporation.!A!single!colony!was!used!for!an!overnight!culture!and!used!to!infect!leaf! discs!(Spelt(et(al.,!2000).!Arabidopsis!(Columbia)!was!transformed!with!the!floral!dip!method! (Clough!and!Bent,!1998),!and!transformants!were!selected!on!Murashige!and!Skoog!medium! (Duchefa)!containing!50!mg/l!kanamycin!monoZsulfate.! All! plants! were! grown! in! a! greenhouse.! Care! was! taken! that! for! comparisons! of! phenotypes! plants! were! grown! side! by! side! to! exclude! that! any! phenotypic! differences! resulted!from!(seasonal)!variations!in!greenhouse!conditions.!

Protoplast!Isolation,!transformation!and!imaging!

Protoplasts!were!prepared!from!petunia!leaves!using!an!enzyme!solution!containing!0.2%! Macerozyme!R10!and!0.4%!Cellulase!R10!(Yakult)!as!previously!described!(Yoo(et(al.,!2007)! with! minor! modifications.! Protoplasts! were! coZtransformed! with! 30! μg! of! (supercoiled)! plasmid! DNA! extract! with! Maxi! kits! (QIAGEN).! DNA! was! introduced! into! protoplasts! by! incubating! protoplasts! with! 40%! PEG! 4000! for! 30! min.! After! removal! of! PEG,! protoplasts!

103! DOT!regulation! were!kept!in!the!dark!at!room!temperature!for!16h!to!18h!to!avoid!secondary!activation.! Protoplasts!were!analyzed!with!a!Zeiss!florescence!microscope.!

RNA!preparation!

Total! RNA! was! isolated! using! the! Plant! Total! RNA! kit! (SigmaZAldrich)! according! to! the! manufacturer’s! instructions.! RNA! concentrations! were! determined! using! a! Nanodrop! NDZ 1000! spectrophotometer! (Thermo! Fisher! Scientific).! Total! RNA! preparations! used! for! qRTZ PCR!analysis!were!treated!with!DNase!I!(Ambion)!to!reduce!contaminations!with!genomic! DNA.!Protoplasts!were!lysed!with!cell!culture!lysis!buffer!(QIAGEN)!and!RNA!was!extracted! using! the! RNeasy! Micro! Kit! (QIAGEN)! followed! by! treatment! with! DNase! I! (Ambion)! to! reduce!contaminations!with!genomic!DNA.! qRT@PCR!experiments!

First! strand! cDNA! synthesis! was! performed! using! DNase! IZtreated! total! RNA! preparations! (see!above),!oligoZdT!primers!and!the!Superscript!reverse!transcriptase!(Invitrogen).!Relative! transcript! abundance! of! selected! genes! (see! Table! 1! for! a! list! of! the! primers! used)! was! determined! using! the! RealZtime! PCR! analysis! was! done! with! Eco! Real! time! PCR! system! (Illumina)!using!the!SensiMix!(Bioline!QT650Z05)!following!instructions!of!the!producer.!

In!situ!hybridization!

In! situ! hybridization! was! performed! as! described! (Souer( et( al.,! 1996).! FluoresceinZlabeled! (DOT)!and!digoxigenin!(FBP13,(FBP25(and(PhSPL3)!antisense!RNA!probes!that!spanned!the! full!coding!sequences!were!in!vitro!synthesized!with!T7!polymerase!using!the!fluorescein!and! digoxigenin!labeling!kits!(Roche)!according!to!the!manufacturer’s!instructions.!FluoresceinZ labeled!probes!were!detected!as!a!red!signal!using!an!antiZfluorescein!antibody!conjugated! with!alkaline!phosphatase!and!Fast!Red!tablets!(Roche).!

Phylogenetic!analysis!!

Phylogenetic!analyses!were!performed!using!multiple!alignments!of!amino!acid!sequences! generated! with! CLUSTALW! program! from! the! MEGA5.1! software! package.! The! alignment! was! followed! by! manual! adjustment! if! found! necessary.! The! phylogeny! was! then! reconstructed!using!the!Maximum!Likelihood!method!based!on!the!JonesZTaylorZThornton! (JTT)! matrixZbased! model,! gaps/missing! data! treatment! was! set! at! ‘Use! all! sites’! and! a!

! 104! Chapter!3! discrete! Gamma! (+G,! 5! categories)! distribution! was! used! to! model! evolutionary! rate! differences! among! sites.! The! phylogenetic! reconstructions! were! performed! using! varying! parameters!and!different!protein!sequence!selections.!The!selected!sequences!consisted!of:! the!MADSZbox!(~60!amino!acids),!KZbox!(95Z110!amino!acids),!MADSZbox!+!KZbox!of!all!52! sequences,!the!SBPZbox!(79/80!amino!acids)!of!all!51!sequences.!!

Yeast!two@hybrid!

A!yeast!twoZhybrid!assay!was!performed!with!the!full!length!coding!sequence!of!petunia!and! Arabidopsis(MADS!box!and!SPL!genes!ligated!into!pADZGAL4!and!pBDZGAL4,!and!tested!for! the! activation! of! GAL4Zresponsive! HIS,! ADE,! and! LacZ! reporters! as! described! as! described! before! (Quattrocchio( et( al.,! 2006).! lacZ! reporter! activation! was! assayed! by! a! semiZ quantitative! XZgal! overlay! assay.! Yeasts! spotted! and! grown! on! ZLT! selective! plates! were! permeabilized!by!chloroform,!and!subsequently!covered!with!XZGal!containing!topZagar!(1%! low!melting!point!agar!in!0.1M!KPO4!buffer!pH!7.0,!10!mg!XZGal,!at!42ºC).!Pictures!were! taken!after!an!incubation!period!of!1!to!6!hours!at!37ºC.!

Flowering!Time!Measurement!

Flowering!time!was!measured!by!scoring!the!number!of!leaves!on!the!main!stem!of!at!least! 15!individuals!for!each!stable!transformants!petunia.!LD!condition:!16!hr!light!and!8!hr!dark! at!23ºC!degrees!during!the!day!and!21ºC!during!the!night,!humidity!70%.!SD!condition:!8hr! light!and!16hr!dark!at!20ºC!degrees!during!the!day!and!19ºC!during!the!night,!humidity!70%.! The!counted!leaf!numbers!were!statistically!analyzed!using!OneZWay!ANOVA!in!SPSS.!

Whole!mount!GUS!staining!

Petunia! seedlings! of! four! independent! line! of! each! transgenic! BOXes:GUS! were! harvested! when! they! had! made! one! or! two! leaves.! Inflorescences! were! dissected! from! stable! transformed! W115! plants! using! a! scalpel! and! forceps.! We! accurately! followed! the! wholeZ mount!GUS!staining!protocol!as!described!in!(Weigel,!2002).!

Plant!photography!

Pictures! of! plants! were! taken! with! a! Sony! CyberZshot! DSCZRX100.! The! background! was! blacked!out!using!Adobe!Photoshop!Software.! ! !

105! DOT!regulation!

Supplementary!Information!!

! ! Figure!S1:!Conservation!of!DOTEBOX(1(Promoter!Sequences.! Sequence! alignment! of! BOX( 3( in! genomes! present! in! NCBI!(Solanum( lycopersicum,( Capsicum( annuum,( Solanum( tuberosum,( Nicotiana( tabacum,( Citrus( sinensis,( Citrus( clementina,( Vitis( vinifera,(

! 106! Chapter!3!

Mimulus(guttatus(and(Antirrhinum(majus)!and!fragments!obtained!by!PCR!amplification!from!plants! belonging! to! the! Solanaceae! family( (Calibrachoa( parviflora,( Lycianthes( biflora,( Anisodus( luridus,( Lycium(barbarum,(Normania(triphylla(and(Brunfelsia(americana).!Putative!binding!sites!for!SBP!and! MADSZbox! protein! were! identified! using! the! JASPAR! database! (http://jaspar.genereg.net)! and! are! indicated!with!green!and!orange!boxes!respectively.! !

! ! Figure!S2:!Conservation!of!DOT(BOX(2(Promoter!Sequences.! Sequence! alignment! of! BOX( 2( in! genomes! present! in! NCBI!(Solanum( lycopersicum,( Capsicum( annuum,( Solanum( tuberosum,( Nicotiana( tabacum)! and! fragments! obtained! by! PCR! amplification! from!plants!belonging!to!the!Solanaceae!family!(Acnistus(lorentzii,!Calibrachoa(parviflora,(Brunfelsia( americana,(Lycium(barbarum,(Normania(triphylla,(Solanum(villosum(and!Solanum(lidii)! !

107!! DOT!regulation!

! ! Figure!S3:!Conservation!of!DOTEBOX(3(Promoter!Sequences.! Sequence!alignment!of!BOX(3(in!genome!present!in!NCBI!(Solanum(lycopersicum,(Capsicum(annuum,( Solanum( tuberosum,( Nicotiana( tabacum,( Citrus( sinensis,( Citrus( clementina,( Vitis( vinifera,! Populus( trichocarpa,(Mimulus(guttatus(and(Antirrhinum(majus)!and!fragments!obtained!by!PCR!amplification! from!plants!belonging!to!the!Solanaceae!family!(Calibrachoa(parviflora,( Fabiana(imbricata,(Lycium( barbarum,( Physalis( peruviana,( Salpichroa( origanifolia,( Lycianthes( biflora,( Brunfelsia( americana,( Anisodus(luridus(,(Normania(triphylla,(Withania(coagulans(and!Withania(somnifera).!Putative!binding! sites! for! MADSZbox! protein! were! identified! using! the! JASPAR! database! (http://jaspar.genereg.net)! and!are!indicated!with!orange!boxes.! ! !

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! ! Figure!S4:!Conservation!of!DOTEBOX(4(Promoter!Sequences.! Sequence!alignment!of!BOX(4(in!genome!present!in!NCBI!(Solanum(lycopersicum,(Capsicum(annuum,( Solanum( tuberosum,( Nicotiana( tabacum,( Citrus( sinensis,( Citrus( clementina,( Vitis( vinifera,! Populus( trichocarpa,(Mimulus(guttatus(and(Antirrhinum(majus)!and!fragments!obtained!by!PCR!amplification! from! plants! belonging! to! the! Solanaceae! family! (Acnistus( lorentzii,! Calibrachoa( parviflora,( Brugmansia( suaveolens,( Brunfelsia( Americana,( Fabiana( imbricata,( Lycianthes( biflora,( Lycianthes( rantonnei,( Lycium( barbarum,( Lycium( pallidum,( Normania( triphylla,( Physalis( peruviana,( Salpichroa( origanifolia,(Solanum(lidii,(Tubocapsicum(anomalum,(Withania(coagulans(and!Withania(somnifera)! .!

109! DOT!regulation!

! !

! 110! Chapter!3!

Figure!S5:!The!4!BOXes!are!all!present!within!the!Solanaceae!family,!but!in!other!families!BOX2!and! 4!are!missing.! (A)! Schematic! representation! of! the! 4! BOXes! within! the! Solanaceae! family! obtained! from! the! available!genome!sequences!or!by!PCR!sequencing.!As!indicated!by!numbers,!boxes!size!and!distance! are!usually!different.!Shaded!boxes!with!question!marks!indicate!boxes!that!could!not!be!amplified.! Half!dashed!boxes!with!an!asterisk!indicate!boxes!that!could!not!completely!amplified.!Dashed!lines! indicate!promoter!sequence!between!boxes!that!could!not!be!amplified.!(B)!In!Mimmulus(guttatus,! Antirrhinum(majus,!(both!Asterids)(and(Vitis(vinifera!(Rosid)!we!could!detect!only!BOX1!and!BOX3.!In! Arabidopsis(thaliana((Rosid)!only!BOX1!is!detectable.! !

! ! Figure!S6:!Alignment!of!the!MADS@domain!from!52!MADS@box!proteins!of!different!plant!species.! MADSZdomains! were! identified! and! annotated! by! SMART! (http://smart.embl.de).! Amino! acids! are! shown!in!single!letter!code.!At!=!Arabidopsis(thaliana,(Ca!=!Capsicum(annuum,!Ph!=!Petunia(hybrida,! Pp!=!Physalis(pubescens,!Sl!=!Solanum(lycopersicum,!Sm!=!Solanum(macrocarpon,!Ta!=!Tubocapsicum( anomalum,! Vb! =! Vassobia( breviflora,! Vv! =! Vitis( vinifera,! Wc! =! Witheringia( coccoloboides,! Ws! =! Withania(somnifera.! ! !

111! DOT!regulation!

!

! Figure!S7:!Amino!acid!alignment!of!the!K@box!sequences!from!52!MADS!proteins!of!different!plant! species.!! KZbox!sequences!were!identified!and!annotated!by!SMART!(http://smart.embl.de).!Amino!acids!are! shown!in!single!letter!code.!At!=!Arabidopsis(thaliana,(Ca!=!Capsicum(annuum,!Ph!=!Petunia(hybrida,! Pp!=!Physalis(pubescens,!Sl!=!Solanum(lycopersicum,!Sm!=!Solanum(macrocarpon,!Ta!=!Tubocapsicum( anomalum,! Vb! =! Vassobia( breviflora,! Vv! =! Vitis( vinifera,! Wc! =! Witheringia( coccoloboides,! Ws! =! Withania(somnifera.! ! !

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! ! Figure!S8:!Multiple!sequence!alignment!and!sequence!logo!of!the!petunia!and!Arabidopsis!SBP@box! domain.! Multiple!sequence!alignment!was!performed!using!AliView.!The!two!conserved!zinc!fingers!and!NLS! are!indicated.!The!sequence!logo!was!obtained!from!Weblogo!online!software.!The!overall!height!of! the!stack!indicates!the!sequence!conservation!at!that!position.! ! ! ! ! ! ! ! ! ! !

113! DOT!regulation!

(

! ! Figure!S9:!Expression!of!FBP13,!FBP25!and!PhSPL3!in!petunia.! (A)!RealZtime!PCR!analysis!of!FBP13!and!FBP25!mRNAs!expressed!in!inflorescence!apices,!leaf!and! petal!of!W115.!(B)!RealZtime!PCR!analysis!of!PhSPL3I4I5I6!in!inflorescence!top,,!leaf,!petal!and!root! of!W115.!(CZF)!Expression!of!DOT((C),!FBP13!(D),!FBP25!(E)!and!PhSPL3!(F)!in!6!in!inflorescence!and! flower!meristems!of!W115.!*,!inflorescence!meristem;!br,!bract;!se,!sepal.! ( ( (

! 114! Chapter!3!

! Figure!S10:!Yeast!to!hybrid!screen!between!AGL24!and!SPL3!homologs.! Activation!of!a!GAL4Zresponsive!LacZ!gene!in!yeast!strains!expressing!different!GAL4BD!and!GAL4AD! fusions.!! !

! ! Figure!S11.!Constitutive!expression!of!FBP13!and!FBP25!in!petunia.! (AZC)! Comparison! of! flowering! time! and! inflorescence! architecture! between! wild! type! W115! (A),! 35S:FBP13!(B)!and!35S:FBP25!(C)!under!LD!conditions.!(DZF)!Extra!petal!tissues!growth!in!35S:FBP13! and!35S:FBP25!! ! !

115! DOT!regulation!

Table!1:!Sequences!of!primers!used!!

Primer! Sequence! USE! ID! 5619! GGGGACAAGTTTGTACAAAAAAGCAGGCTGAAGGACTATGTGAGAGAAGA! BOX1mp35S:GUS! 5631! GGGGACAACTTTTGTATACAAAGTTGTGCAAAGAGAAAGGAGAAGTACTG! BOX1mp35S:GUS! 5632! GGGGACAAGTTTGTACAAAAAAGCAGGCTCTGGCTATAAGATCTTAAGAT! BOX2mp35S:GUS! 5633! GGGGACAACTTTTGTATACAAAGTTGTGAATTTGTTTCACCAATATATGT! BOX2mp35S:GUS! 5634! GGGGACAAGTTTGTACAAAAAAGCAGGCTGAGAATATTCAAAGCAAAATG! BOX3mp35S:GUS! 5635! GGGGACAACTTTTGTATACAAAGTTGTTGCAATTTGGCGTAAATACCAAC! BOX3mp35S:GUS! 5636! GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAAACATTAAGATAAACTCA! BOX4mp35S:GUS! 5637! GGGGACAACTTTTGTATACAAAGTTGTTTTTCTTTTATGAATTCATTGAG! BOX4mp35S:GUS! 5638! GGGGACAAGTTTGTACAAAAAAGCAGGCTGTCTTCAAAGCAAGTGGATTG! mp35S:GUS! 5639! GGGGACCACTTTGTACAAGAAAGCTGGGTAGTAAATTGTAATGTTGTTTG! mp35S:GUS! 5640! GGGGACAACTTTTGTATACAAAGTTGGTCTTCAAAGCAAGTGGATTGATG! mp35S:GUS! 2661! CGGAATTCGATTTCATTGCGGTTGGTATTTACGCC! pDOTΔA/ΔB! 5276! GGGGTACCAATCAGCAGAGTACTGCAG! pDOTΔA! 5277! GGGGTACCTTGGTCTCCTTTCTACTTTG! pDOTΔB! 2662! CGGGATCCCTAATAGGTGCATGATGAAAAGCTTCC! pDOTΔC! 5274! GGGAATTCTCTACAGTGCTCCAACATC! pDOTΔC! 5441! GGGGACAAGTTTGTACAAAAAAGCAGGCTATGTTGTCATCAGCTAAGCAT! p35S:FD! 5572! GGGGACCACTTTGTACAAGAAAGCTGGGTAAAATGGAGCTGTGGAAGACC! p35S:FD! 5443! GGGGACAAGTTTGTACAAAAAAGCAGGCTATGTCTATAAATATAAGAGAC! p35S:FT! 5444! GGGGACCACTTTGTACAAGAAAGCTGGGTAATATCAATTGGTTATAAAGG! p35S:FT! 5445! GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGTGAGGGGCAAAACTCAG! p35S:SOC1! 5446! GGGGACCACTTTGTACAAGAAAGCTGGGTTCACTTTCTTGAAGAACAAGG! p35S:SOC1! 5447! GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGGAAGGGGTAGGGTTCAA! p35S:AP1! 5448! GGGGACCACTTTGTACAAGAAAGCTGGGTTCATGCGGCGAAGCAGCCAAG! p35S:AP1! 5449! GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGCGAGAGAGAAGATAAGG! p35S:AGL24! 5450! GGGGACCACTTTGTACAAGAAAGCTGGGTTCATTCCCAAGATGGAAGCCC! p35S:AGL24! 5451! GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAGTATGAGAAGAAGCAAA! p35S:SPL3! 5452! GGGGACCACTTTGTACAAGAAAGCTGGGTTTAGTCAGTTGTGCTTTTCCG! p35S:SPL3! 5453! GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGATCCTGAAGGTTTCACG! p35S:LFY! 5454! GGGGACCACTTTGTACAAGAAAGCTGGGTCTAGAAACGCAAGTCGTCGCC! p35S:LFY! 5455! GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGGAAGAGGTAGGGTTCAG! p35S:FUL!

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5456! GGGGACCACTTTGTACAAGAAAGCTGGGTCTACTCGTTCGTAGTGGTAGG! p35S:FUL! 5462! GGGGACCACTTTGTACAAGAAAGC!TGGGTACTTTCTTGAAGAACAAGGTA! p35S:SOC1ZVP16! 5463! GGGGACCACTTTGTACAAGAAAGCTGGGTATGCGGCGAAGCAGCCAAGGT! p35S:AP1ZVP16! 5464! GGGGACCACTTTGTACAAGAAAGCTGGGTATTCCCAAGATGGAAGCCCAA! p35S:AGL24Z VP16! 5465! GGGGACCACTTTGTACAAGAA!AGCTGGGTAGTCAGTTGTGCTTTTCCGCC! p35S:SPL3ZVP16! 5466! GGGGACCACTTTGTACAAGAAAGCTGGGTAGAAACGCAAGTCGTCGCCGC! p35S:LFYZVP16! 6056! GGGGACAAGTTTGTACAAAAAAGCAGGCTGTATGGCAAGAGAGAAGATCA! p35S:FBP13! 6057! GGGGACAAGTTTGTACAAAAAAGCAGGCTTTATGGTGAGACAAAAGATTC! p35S:FBP25! 5947! GGGGACCACTTTGTACAAGAAAGCTGGGTCTAGTTGAATGGTAGCCCTAA! p35S:FBP13! 5949! GGGGACCACTTTGTACAAGAAAGCTGGGTTCAATAAGGAAAAGGCAACCC! p35S:FBP25! 5937! GGGGACAAGTTTGTACAAAAAAGCAGGCTGGTTTCAGATCTGAGACAGAC! p35S:PhmiR156! 5938! GGGGACCACTTTGTACAAGAAAGCTGGGTCACGTGTGTGTAGCATATAAC! p35S:PhmiR156! 5655! GTACAACTACAACAGCCACAACG! GFP!qPCR! 5656! GATCTTGAAGTTCACCTTGATGC! GFP!qPCR! 5922! TGCACTCCCACATGCTATCCT! ACT!qPCR! 5922! TCAGCCGAAGTGGTGAAAGAG! ACT!qPCR! 6326! CAGTACTGATAGGTGGACACCAA!! PhSPL3!qPCR! 6327! GGAGAATCACATGAACTTTTACGG!! PhSPL3!qPCR! 6328! CCATGCCAAGGCTCCAATTGTTC!! PhSPL4!qPCR! 6329! GGCGGCAACTCCTTTTTGTCC!! PhSPL4!qPCR! 6330! AATGGAGGGTCAAGTTCAGTGAG!! PhSPL5!qPCR! 6331! GGATTCATCAAACTCCCCCAG!! PhSPL5!qPCR! 6332! GCTCAAGCCGTCGTCGTTG!! PhSPL6!qPCR! 6333! TGCAGATGATGATGATGAGCTGA!! PhSPL6!qPCR! 3930! AGGGTACAAGAATTATGGATG! FBP13!qPCR! 3931! CCCTAACTTCAGAAATGTATCTGAG! FBP13!qPCR! 3932! AGGGTGACAAGTTTATGAAAGAG! FBP25!qPCR! 3933! CTAACTTGAGGCTAGTATCTGAG! FBP25!qPCR! ! ! !

117! DOT!regulation!

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