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

Della Pina, S.

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THE EVOLUTIONARY DIVERGENCE OF THE GENETIC NETWORKS THAT CONTROL FLOWERING IN DISTINCT SPECIES

Cover design: Daniela Lazzini, after an idea of Serena Della Pina.

THE EVOLUTIONARY DIVERGENCE OF THE GENETIC NETWORKS THAT CONTROL FLOWERING IN DISTINCT SPECIES

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus

prof. dr. ir. K.I.J. Maex

ten overstaan van een door het College voor Promoties ingestelde

commissie, in het openbaar te verdedigen in de Agnietenkapel

op donderdag 22 september 2016, te 10:00 uur

door

Serena Della Pina

geboren te Massa, Italië

Promotiecommissie:

Promotor: prof. dr. R.E. Koes Universiteit van Amsterdam

Copromotor: dr. E. Souer Dümmen Orange

Overige leden: prof. dr. G.C. Angenent Wageningen UR dr. A.B. Rebocho John Innes Centre Norwich prof. dr. M.A. Haring Universiteit van Amsterdam dr. M.E. Stam Universiteit van Amsterdam prof. dr. ir. M.W. Prins Universiteit van Amsterdam

Faculteit der Natuurwetenschappen, Wiskunde en Informatie (FNWI)

This work was supported by the Netherlands Organization for Scientific Research (NWO) [grant 819.02.018]

Chapter 1 7 Arguments in the evo-devo debate: Say it with flowers!

Chapter 2 33 Changes in cis-regulatory elements of a key floral regulator are associated with divergence of inflorescence architectures

Chapter 3 71 Rewiring the flowering network in Petunia hybrida

Chapter 4 123 Role of DOUBLE TOP in the activation of ABERRANT LEAF AND FLOWER during flowering time

Chapter 5 145 General Discussion

Summary/Samenvatting 157

Acknowledgments 164

Author contribution list 167

! ! 1!

Arguments!in!the!evo0devo!debate:!Say!it!with!flowers!!

! Serena!Della!Pina,!Erik!Souer,!Ronald!Koes! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Published!in!Journal(of(Experimental(Botany!(2014),!65!(9):!2231A2242.! S.D.P!wrote!the!article!! ! ! Chapter!2!

Abstract!

A! key! question! in! evolutionary! developmental! biology! is! how! DNA! sequence! changes! have! the! evolution! of! morphological! diversity.! The! widelyAaccepted! view! was! that! morphological! changes! resulted!from!differences!in!number!and/or!type!of!transcription!factors,!or!even!from!small!changes! in!the!amino!acid!sequence!of!similar!proteins.!Research!over!the!last!two!decades!indicated!that! most!of!the!developmental!and!genetic!mechanisms!that!produce!new!structures!involve!proteins! that!are!deeply!conserved.!These!proteins!are!encoded!by!a!type!of!genes!known!as!“toolkit”!genes! that! control! a! plethora! of! processes! essential! for! the! correct! development! of! the! organism.! Mutations!in!these!toolkit!genes!produce!deleterious!pleiotropic!effects.!In!contrast,!alterations!in! regulatory! regions! affect! their! expression! only! at! specific! sites! in! the! organism! by! facilitating! morphological!change!at!tissue!and!organ!level.!However,!some!examples!from!the!animal!and!! fields!indicate!that!coding!mutations!also!contributed!to!phenotypic!evolution.!Therefore,!the!main! question! to! ask! at! this! point! is! to! what! extent! these! mechanisms! contributed! to! the! evolution! of! morphological!diversity.!Today,!an!increasing!amount!of!data,!especially!from!the!plant!field,!implies! that!changes!in!cisAregulatory!sequences!in!fact!played!a!major!role!in!evolution.! ! !

8! ! Evolution!of!morphological!diversity!

! !Introduction!

Plant! and! animal! species! both! display! wide! divergent! morphologies! regarding! for! example! the! arrangement!of!different!organs!on!their!bodies!or!the!shape!and!size!of!various!body!parts!A!that!are! thought! to! result! from! evolution! via! mutation! and! selection.! The! Cambrian! explosion! (550! mya)! marks!the!appearance!of!most!animal!phyla!that!are!known!today (Wray!et!al.,!1996),!while!land! ! began! their! first! diversification! 450! mya! (Kenrick! and! Crane,! 1997).! Flowering! plants! (Angiosperms)! appeared! only! around! 90A130! mya! (Crane! et! al.,! 1995).! Compared! to! animals,! the! evolution! of! flowering! plants! on! land! was! relatively! fast.! Plants! “invented”! and! diversified! new! structures,! like! flowers,! over! a! ”small”! lapse! of! time,! making! these! relatively! young! events! more! tractable! for! genetic! dissection.! Since! an! everAgrowing! number! of! plant! species! is! amenable! to! genetic! analyses! and! transgenesis,! plants! offer! excellent! opportunities! for! research! inEvolutionary! Developmental!Biology!(EvoADevo).! Here,!we!summarize!our!current!knowledge!of!the!molecular!basis!of!morphological!changes! during!the!course!of!evolution.!We!present!general!ideas!and!give!examples!of!experimental!data! that!support!these!theories.!While!some!examples!are!taken!from!the!animal!field,!our!emphasis!is! on!plant!development!as!this!represents!our!main!research!subject.!

Lessons!from!the!animal!field!

Although!it!is!widely!accepted!that!morphological!variation!between!organisms!arose!from!genetic! alterations,! the! molecular! details! remain! poorly! understood.! Initially! it! was! assumed! that! species! specific! characters! resulted! from! speciesAspecific! proteins.! However,! as! the! number! of! sequenced! genes,!proteins!and!eventually!entire!genomes!grew,!it!became!clear!that!the!genomes!of!organisms! with! very! different! morphologies! are! composed! mostly! of! conserved! genes! (Martin! et! al.,! 2010;! Miyamoto!et!al.,!1987),!and!genes!controlling!development!were!no!exception.!! Among! the! first! examples! were! the! homeotic! HOX! genes! that! specify! the! identity! of! (para)segments!along!the!anteriorAposterior!axis!of!embryos!ranging!from!insects!to!mammals!(Mallo! et!al.,!2010).!They!encode!conserved!homeodomain!transcription!factors!and!are!arranged!in!clusters! with! a! conserved! gene! order! that! correlates! with! their! expression! patterns! along! the! anteroA posterior!axis!in!early!embryos!(colinearity).!Despite!700!MY!of!evolutionary!separation,!mammalian! HOX! proteins! can! still! functionally! replace! their! Drosophila! homologs.! For! example,! ectopic! expression!of!the!mouse!HOXa5!protein!or!the!Drosophila!homolog!Sex!combs!reduced!(Scr)!caused! similar!defects!in!transgenic!flies,!including!the!transformation!of!antennae!into!T1!legs!(Zhao!et!al.,! 1993),! although! HOXa5! plays! a! different! role! in! mice! (Aubin! et! al.,! 1999).! Similarly,! expression! of! chicken! HOXb1! could! fully! rescue! the! defects! of! Drosophila! labial! mutants!(Lutz! et! al.,! 1996).!

!! 9! Chapter!2!

Comparable! findings!were!made!with!nonAHOX! genes.!For!instance,!EYELESS! of!Drosophila!and!its! mouse! homolog! PAX6/SMALL! EYE! are! both! required! for! the! development! of! eyes.!Even! though! mouse! and! insect! eyes! have! no! obvious! morphological! similarity,! expression! of! mouse! PAX6! can! drive!the!development!of!ectopic!eyes!in!transgenic!flies!(Halder!et!al.,!1995).!A!similar!conservation! was!observed!for!extracellular!proteins,!such!as!HEDGEHOG!and!WNT,!and!their!signalling!pathways,! which!pattern!(regions!of)!embryos!and!various!organs!(De!Robertis,!2008).! These! findings! lead! to! the! conclusion! that! the! development! of! morphologically! disparate! animals!is!governed!by!a!“toolkit”!of!deeply!conserved!genes.!This!triggered!the!hypothesis,!which! was! already! put! forward! early! on! (King! and! Wilson,! 1975),! that! morphological! divergence! results! primarily! from! alterations! in! gene! expression! patterns! (Carroll,! 2000,! 2008;! Doebley! and! Lukens,! 1998).!This!idea!was!supported!by!comparative!analyses!showing,!for!example,!that!changes!in!the! body! plan! of! vertebrate! and! invertebrate! species! correlated! with! shifts! in! HOX! gene! expression! patterns!(Angelini!and!Kaufman,!2005;!Burke!et!al.,!1995;!Heffer!and!Pick,!2013).! Gene! expression! patterns! are! governed! by! complex! gene! regulatory! networks! (GRNs)! consisting! of! transAacting! (transcription)! factors! that! bind! to! specific! cisAacting! DNA! sequences! in! downstream! genes! to! promote! or! inhibit! their! transcription.! Binding! sites! usually! cluster! in! small! regions,! known! as! enhancers,! which! promote! transcription! in! specific! cells.! Genes! often! contain! multiple!enhancers!A!for!expression!on!different!sites!A!that!can!lie!many!kilobases!away!from!the! coding!sequence.!Genes!that!control!body!architecture,!like!HOX!genes,!are!highly!connected!nodes.! They!are!expressed!throughout!development!in!complex!patterns!via!an!array!of!distinct!enhancers! and!transcription!factors!and!in!turn!they!regulate!a!vast!number!of!subordinate!genes!involved!in!a! plethora! of! developmental! processes.! By! contrast,! transcription! factors! involved! in! terminal! differentiation!processes,!or!“physiology”,!(”inputAoutput!genes”)!tend!to!form!less!connected!nodes.!! The! modification! of! a! gene! expression! pattern! requires! (some)! rewiring! of! GRNs! through! changes! in! CREs! or! upstream!transcription!factor! proteins! (Fig.! 1).! Alterations! in! highly! connected! transcription! factors! will! simultaneously! affect! many! other! genes! and! developmental! processes! resulting! in! mostly! deleterious! pleiotropic! effects.! This! may! be! one! reason! for! the! amazing! conservation!of!insect!and!vertebrate!toolkit!proteins!after!>!700!MY.!of!separation.!Changes!in!less! connected!“inputAoutput”!transcription!factors,!are!more!likely!to!be!fixed,!because!they!cause!less! pleiotropic! effects,! and! are! thought! to! account! for! the! subtle! phenotypic! variation! at! the! species! level!(Bhardwaj!et!al.,!2010;!Carrera!et!al.,!2009).!The!complex!expression!patterns!of!toolkit!genes! governing!development!are!often!controlled!by!multiple!enhancers!that!are!active!on!different!sites:! alterations!in!an!individual!enhancer!may!alter!the!expression!of!the!toolkit!gene!on!one!site,!without! affecting! expression! on! other! sites,! and! thus! have! less! pleiotropic! defects! than! alterations! in! the! encoded! protein.! Based! on! such! theoretical! considerations! Carroll! argued! that! the! evolution! of!

10! ! Evolution!of!morphological!diversity!

! “anatomy”!and!“physiology”!is!essentially!different!and!that!alterations!in!CREs,!rather!than!protein! coding!sequences!must!be!the!major!contributor!to!the!evolution!of!form!(Carroll,!2005,!2008).!! Although!the!idea!of!morphological!diversification!via!evolution!of!CREs!is!attractive,!others! criticized! it! for! being! premature! and! based! on! insufficient! evidence!(Hoekstra! and! Coyne,! 2007).! Moreover,!there!is!also!evidence!for!morphological!changes!via!evolution!of!transcription!factors.!In! Arthropods,!for!example,!the!appearance!of!insects!with!only!six!(thoracic)!legs!but!no!legs!on!the! abdomen! is! associated! with! a! change! in! the! HOX! protein! ULTRABITHORAX! (UBX).!This! difference! enables! UBX! to! repress! the! expression! of! DISTALLESS! in! the! abdomen,!which!is!necessary!for!the! development! of! appendages! (Galant! and! Carroll,! 2002;! Ronshaugen! et! al.,! 2002).! Similarly,! the! mammalian!HOX11a!protein!acquired!the!ability!to!activate!certain!target!genes,!possibly!in!relation! to!the!evolution!of!extraAembryonic!membrane!required!for!embryogenesis!in!utero!(Lynch! et!al.,! 2008).! Thus,! studies! with! animals! support! the! evolution! of! body! architecture! both! via! changes! in! CREs!and!changes!in!the!protein!sequence.!However,!a!very!limited!number!of!studies!exist!which! give! solid! support! to! either! one! or! the! other! hypothesis.!As! illustrated! in! the! following! sections,! plants!offer!excellent!tools!in!order!to!study!the!molecular!processes!that!drive!the!evolution!of!body! architecture!and!shape.!

Lessons!from!the!plant!field!

During! evolution! Angiosperms! developed! specialized! organs! and! tissues! that! have! contributed! to! their! diversity! and! success.! According! to! fossil! records,! flowerAlike! structures! originated! 160–147! MYA!(Frohlich,!2006).!Already!in!1970,!Ohno!and!colleagues!hypothesized!a!significant!role!for!gene! duplication! in! the! evolution! of! biological! complexity!(Ohno,!1970).! Land! plants! underwent! several! Whole!Genome!Duplications!(WGD)!(Jiao! et! al.,! 2011)! that! facilitated! their! evolution! by! providing! with! multiple! copies! of! all! the! preAexisting! genes.! Duplicated! genes! were! released! from! the! constraint! of! maintaining! their! original! function! and! could! therefore! evolve! in! different! ways,! through!mutations!in!either!regulatory!or!coding!regions.!In!the!following!paragraphs!we!will!review! data! on! the! importance! of! CRE! and! protein! changes! in! the! evolution! of! flowering! plants,! taking! examples!from!a!variety!of!stages!of!the!plant!lifeAcycle.!! !

!! 11! Chapter!2!

! ! Fig.!1.!Schematic!representation!of!a!gene!with!its!CREs!(CisARegulatory!Elements)!and!the!potential! mutations!that!can!affect!transcriptional!processes.! (A)! Functional! CREs! (AABAC)! together! with! their! respective! Transcription! Factors! (TFs)! allow! expression!of!a!gene!in!a!specific!organ!(or!tissue).! (B)!Mutation!in!one!CRE!(binding!site!of!A!in!this!case!became!of!D)!leads!to!loss!of!expression!in! sepal!but!the!gene!acquires!expression!in!leaf.!! (C)!Mutation!in!a!TF!(A!in!this!example)!leads!to!lack!of!activation!of!the!gene!in!a!specific!organ! (sepal!in!this!case).!

12! ! Evolution!of!morphological!diversity!

! Before!flowering:!Architecture!of!the!vegetative!plant!body!

Angiosperms!show!relatively!little!diversity!during!embryogenesis!(Weijers!and!Jurgens,!2005),!apart! from!the!typical!differences!between!monocots!and!dicots;!most!architectural!differences!arise!later! in!development.!After!germination!the!shoot!apical!meristem!(SAM)!grows!indeterminately!and!leaf! primordia! initiate! at! its! periphery.! Leaves! can! be! simple,! consisting! of! a! single! blade! (Fig.! 2A),! or! compound,!consisting!of!several!leaflets!on!the!same!petiole!(Fig.!2B).!SAM!cells!that!are!located!at! the! periphery!of!the!meristem! downAregulate! KNOX!transcription!factors! to! enable! differentiation! (Long!et!al.,!1996;!Vollbrecht!et!al.,!1991).!In!Arabidopsis,!maize!and!Antirrhinum!KNOX!genes!are! repressed!in!the!developing!simple!leaves!by!orthologous!MYB!proteins!encoded!by!ASYMMETRIC! LEAVES1,! ROUGH! SHEATH2! and! PHANTASTICA!(ARP! genes),! respectively.! In! species! with! lobed! or! compound! leaves,! such! as! Arabidopsis! lyrata! and! Cardamine! hirsuta,! KNOX! genes! are! reactivated! during!leaf!development!(Hay!and!Tsiantis,!2006;!Piazza!et!al.,!2010).!Ectopic!ZmKN1!expression!in! tobacco!causes!severely!lobed!leaves!(Sinha!et!al.,!1993),!while!in!tomato!it!causes!iterations!of!the! compound! pattern,! resulting! in! the! formation! of! superAcompound! leaves! bearing! thousands! of! leaflets! (Hareven! et! al.,! 1996).! In! Arabidopsis! ectopic! expression! of! distinct! class! 1! KNOX! genes! resulted!in!lobed!leaves!in!all!cases!(Hay!and!Tsiantis,!2010),!while!silencing!of!the!KNOX!gene!SHOOT! MERISTEMLESS!(STM)! inhibited! the! lobing! of! leaves! of! Arabidopsis! suecica!(Piazza! et! al.,! 2010).! Swapping!KNOX!genes!or!promoters!between!Arabidopsis!thaliana!and!relatives!with!lobed!leaves! revealed!that!the!divergent!KNOX!expression!patterns!result!from!differences!in!CREs!(Piazza!et!al.,! 2010).! In! addition,! promoter! swaps! between! Arabidopsis! and! Cardamine! hirsuta! suggested! that! a! change!in!the!promoter!of!STM!may!be!sufficient!to!determine!the!leaf!complexity!in!the!latter!(Hay! and!Tsiantis,!2006).!Taken!together,!these!data!show!that!class!1!KNOX!proteins!of!different!species! are! functionally! similar! and! that! changes! in! the! CREs! of! KNOX! genes! were! a! key! factor! in! the! divergence!of!leaf!shape.!

!! 13! Chapter!2!

! Fig.2.!Morphological!variation!between!Angiosperms.!! (A)! Variation! in! leaf! margin! of! Arabidopsis! thaliana! (top)! and! Arabidopsis! lyrata! (bottom)! and,! (B)! from!left!to!right,!compounds!leaves!of!Cardamine!hirsuta,!Pisum!sativus!and!Solanum!lycopersicum.! (C)! Variation! in! inflorescence! architecture:! from! left! to! right,! solitary! flower,! cyme,! raceme! and! compounds!inflorescences.! (DAK):! Variation! in! pigmentation! and! flower! shape/size:! (D)! Fritillaria! Persica,! (E)! Leucospermum! cordifolium,!(F)!Tulipa,!fringed!hybrids,!(G)!Agapanthus,!(H)!Phalaenopsis,!(I)!Passiflora,!!(J)!Cosmos! bipinnatus!and!(K)!Heliconia!Caribaea.! ! A!subclade!of!species!within!the!Fabaceae!forms!compound!leaves!that!do!not!express!KNOX1! genes! (Champagne! et! al.,! 2007).! Leaf! complexity! is! established! in! these! species! by! distinct! genes,! such!as!UNIFOLIATA!(UNI)!and!STAMINA!PETALOIDA!(STP)!from!pea!(Hofer!et!al.,!1997;!Taylor!et!al.,!

14! ! Evolution!of!morphological!diversity!

! 2001)!and!SINGLE!LEAFLET1!(SGL1)!from!alfalfa!(Wang!et!al.,!2008).!UNI!and!SGL1!are!orthologs!of! LEAFY!(LFY)!of!Arabidopsis,!which!is!a!transcription!factor!that!determines!floral!meristem!identity!in! Angiosperms!(Krizek!and!Fletcher,!2005).!STP!is!the!ortholog!of!UNUSUAL!FLORAL!ORGANS,!which!is! an!FABox!protein!that!binds!to!and!activates!LFY!(Chae!et!al.,!2008;!Souer!et!al.,!2008).!Hence,!STP! presumably!controls!leaf!through!activation!of!UNI.! Several! findings! suggest! that! the! role! of! LFY! as! a! promoter! of! meristem! proliferation/! outgrowth! in! the! development! of! compound! leaves! is! more! ancient! than! its! role! in! flower! development.!First,!in!primitive!plants!lacking!flowers,!like!the!moss!Physcomitrella,!LFY!promotes! cell!divisions!at!different!stages!of!development!(Tanahashi!et!al.,!2005).!Second,!tomato!(falsiflora)! and! Lotus! (proliferating! floral! meristem)! mutations! in! LFY! orthologs! also! reduce! leaf! complexity! (MolineroARosales! et! al.,! 1999;! Wang! et! al.,! 2013),! albeit! mildly! because! these! leafs! also! express! KNOX1!genes.!Third,!mutation!of!the!rice!homolog!reduces!outgrowth!of!tillers!(Rao!et!al.,!2008).! Fourth,! recent! work! revealed! that! the! auxin! response! factor! MONOPTEROUS! promotes! LFY! expression! in! floral! primordia! and! that! LFY! induces! auxinAsensing! and! growth/proliferation! of! the! meristem,! which! is! evident! in! certain! double! mutant! combinations! but! not! in! single! lfy! mutants! (Chahtane!et!al.,!2013;!Li!et!al.,!2013;!Yamaguchi!et!al.,!2013).!This!suggests!that!in!early!land!plants! LFY! regulated! cell! division! and! with! the! appearance! of! flowers! acquired! a! new! role! in! the! specification!of!floral!meristem!identity.!Functional!and!structural!comparisons!of!LFY!proteins!from! algae!to!angiosperms!indicated!that!the!acquisition!of!floral!function(s)!was!associated!with!changes! in! its! DNA! binding! domain,! possibly! in! conjunction! with! alterations! in! CREs! of! subordinate! genes.! Interestingly,!LFY!evolution!apparently!involved!a!promiscuous!intermediate!form,!found!today!in!a! hornworts,! with! both! the! old! and! new! DNA! binding! specificities,! which! may! have! helped! to! circumvent!(initial)!deleterious!pleiotropic!effects!(Maizel!et!al.,!2005;!Sayou!et!al.,!2014).! Another!trait!that!diversifies!the!vegetative!plant!body!is!the!formation!of!sidebranches!from! meristems! in! leaf! axils.! The! outgrowth! (or! dormancy)! of! these! axillary! meristems! is! regulated! by! signals!originating!from!the!apex!(auxin)!and!the!basal!part!(strigolactones).!Auxin!produced!in!the! apex!moves!downward!via!the!stem!and!inhibits!the!activity!of!axillary!buds,!a!process!that!is!known! as! apical! dominance.! The! absence! of! lateral! shoots! (tillers)! in! modern! maize,! for! example,! results! from! a! strong! apical! dominance.! This! architecture! evolved! during! its! domestication! from! its! progenitor!Teosinte,!which!displays!less!apical!dominance!and!is!highly!branched!and!could!be!traced! to!the!altered!expression!of!TEOSINTE!BRANCHED!(TB1),!which!encodes!a!transcription!factor!that! supresses!meristem!outgrowth!(Doebley!et!al.,!1997).!In!maize!TB1!is!highly!expressed!and!branching! is! repressed,! whereas! in! teosinte! TB1! expression! is! relatively! low,! resulting! in! more! extensive! branching.!The!elevated!expression!of!TB1!in!maize!correlates!with!the!insertion!of!a!HOPSCOTCH!

!! 15! Chapter!2! retrotransposon! at! approximately! 60! kb! upstream! of! the! TB1! coding! sequence,! thereby! changing! gene!regulation!and!architectural!morphology!(Studer!et!al.,!2011).!

Evolution!of!flowering!time!response!(or!mechanism)!!

Angiosperms!display!an!amazing!variation!regarding!the!moment!when!they!switch!from!vegetative! growth!to!flowering!(flowering!time).!Environmental!and!endogenous!signals!determine!the!moment! that!a!plant!undergoes!this!transition.!Temperature!and!day!length!are!the!principal!cues!that!trigger! the!onset!of! flowering! in! a! particular! season.! Because!these! requirements!are!speciesAdependent,! some! plants! need! a! particular! day! length! (photoperiod)! in! order! to! flower,! while! others! are! photoperiod! insensitive.! In! addition,! some! species! require! exposure! to! low! winter! temperatures! (vernalization),! while! summerAannual! plants! do! not.! External! and! internal! stimuli! converge! in! the! regulation!of!the! main! components! of!the! longAdistance!florigen!signal,!FLOWERING!LOCUS!T!(FT)! and!its!paralog!TWIN!SISTER!OF!FT!(TSF)!(Andres!and!Coupland,!2012;!Song!et!al.,!2013).!After!their! induction!in!leaves,!FT!and!TSF!proteins!are!loaded!into!the!phloem!and!translocated!to!the!apex! where!they!interact!with!a!bZIP!transcription!factor,!FLOWERING!LOCUS!D!(FD)!via!a!14A3A3!protein! (Abe! et! al.,! 2005;! Taoka! et! al.,! 2011;! Wigge! et! al.,! 2005).! These! FT–FD! and! TSFAFD! transcriptional! complexes!activate!the!expression!of!several!floral!pathway!integrators:!SUPPRESSOR!OF!CONSTANS1! (SOC1),! SQUAMOSA! PROMOTER! BINDING! PROTEIN! LIKE! (SPL)! and! Flower! Meristem! Identity! genes! (FMI)! to! promote! flowering!(Pose! et! al.,! 2012).! Once! FMI! genes! are! transcribed,! the! plants! is! irreversibly! committed! to! floral! initiation,! and! FT! and! TSF! become! unnecessary!(Corbesier! et! al.,! 2007).! In! the! long! day! (LD)! plant!Arabidopsis,! FT! is! activated! by! the! zincAfinger! transcription! factor! CONSTANS!(CO).!CO!activity!is!regulated!transcriptionally!by!light!and!the!circadian!clock,!and!postA translationally!by!light,!in!such!a!way!that!during!short!days!(SD)!CO!protein!is!degraded!whereas!in! LD,!CO!is!stabilized!and!FT!transcription!activated!(Turck!et!al.,!2008).!CO!has!an!ancient!origin!as! homologs! that! can! functionally! replace! CO! in! Arabidopsis! can! be! found! even! in! the! alga! Chlamydomonas! reinhardtii.! Interestingly,! CrCO! is! also! regulated! by! dayAlength! and! the! clock! in! Chlamydomonas!and!involved!in!rhythmic!output!processes!(Serrano!et!al.,!2009).! Clearly!FTAlike!proteins!represent!a!universal!flowering!signal!conserved!during!plant!evolution.! The!diversification!of!seasonal!control!of!flowering!appears!to!be!due!to!rewiring!of!the!upstream! network!that!controls!FT!expression!and/or!the!downstream!network!for!the!output!of!FTAsignal.!In! rice,!a!SD!plant,!the!COAhomolog!HEADING!DATE!1!(HD1)!activates!the!FT‑like!genes!HD3A!and!RICE! FT‑LIKE1!but!only!in!SD!conditions.!The!difference!with!Arabidopsis!is!that!HD1!is!in!LDs!converted! from!an!activator!into!a!repressor!of!HD3A!by!a!pathway!that!is!controlled!by!phytochrome!B,!which! confers! a! robust! regulation! by! day! length! and! light! quality!(Ishikawa! et! al.,! 2009;! Ishikawa! et! al.,!

16! ! Evolution!of!morphological!diversity!

! 2005).!In!addition,!EARLY!HEADING!DATE!1!(EHD1),!a!BAtype!response!regulator,!induces!HD3A!in!SDs! independently!of!HD1.!(Doi!et!al.,!2004).!In!another!SD!plant,!Fragaria!vesca!(strawberry),!LDs!induce! expression! of! FvFT! and! (subsequently)! FvSOC1,! as! in! Arabidopsis.! However,! in! strawberry! plants! FvSOC1! activates! a! repressor! of! flowering! that! is! homologous! to! TERMINAL! FLOWER1! from! Arabidopsis!(Mouhu!et!al.,!2013).!! Sugar!beet,!a!biennial!that!requires!vernalization!and!long!days!for!flowering,!contains!two!FT! paralogs.!One!of!these!(BvFT2)!has!a!similar!function!as!FT!in!Arabidopsis,!whereas,!the!other!(BvFT1)! is!a!repressor!of! flowering! that! is!thought!to!be!involved!in!the!vernalization!response!(Pin! et!al.,! 2010).!The!divergence!of!BvFT1!and!BvFT2!function!involved!changes!in!their!CREs,!because!they!are! differentially!expressed!in!beet,!and!changes!in!the!encoded!proteins,!because!BvFT2!induces!and! BvFT1!represses!flowering!when!expressed!in!Arabidopsis!(Pin!et!al.,!2010).!! In! other! species! the! CO/FT! system! acquired! new! roles! in! entirely! different! developmental! processes.! In! Populus! trees,! which! are! perennials,! FT! promotes! the! onset! of! flowering,! as! in! Arabidopsis,!and!in!addition!supresses!the!growth!cessation!and!bud!set!in!the!fall!when!days!are! shortening!(Bohlenius!et!al.,!2006).!Interestingly,!the!critical!day!length!for!downAregulation!of!FT,! growth! cessation! and! bud! set! is! shorter! for! varieties! growing! at! moderate! latitudes! compared! to! those! from! northern! latitudes.! This! adaptive! change,! which! enables! the! timely! preparation! for! winter,!is!associated!with!an!altered!CO!regulation,!because!of!which!CO!mRNA!peaks!several!hours! earlier!after!dawn!in!trees!from!southern!populations!compared!to!northern!populations!(Bohlenius! et!al.,!2006).!The!genetic!changes!that!caused!the!divergent!CO!expression!remain,!however,!to!be! established.!! In!potato!SDs!activate!two!FT!paralogs;!one!(StSP3A)!is!involved!in!the!transition!to!flowering,! and! the! other! (StSP6A)! in! the! transition! to! tuberization!(Navarro! et! al.,! 2011).! Since! StSP6A! can! functionally!replace!FT!in!Arabidopsis!and!rice!Hd3A!promotes!both!flowering!and!tuberization!when! expressed!in!potato,!the!functional!divergence!of!StSP3A!and!StSP6A!seems!mostly!due!to!changes!in! their!transcriptional!regulation!and!CREs,!rather!than!the!encoded!proteins!(Navarro!et!al.,!2011).!

Say!it!with!flowers:!the!evolution!of!inflorescences.!

After!transition!to!flowering,!flowers!emerge!in!specific!positions!on!the!plant!body.!Some!species! form!a!single!(solitary)!flower,!while!others!generate!inflorescence!branches!that!bear!many!flowers! in! a! variety! of! arrays! (Fig.! 2C).! In! cymes! flowers! develop! from! apical! meristems! and! inflorescence! growth! continues! from! a! secondary! lateral! (sympodial)! meristem! formed! at! its! flank,! whereas! in! racemes,! the! apical! meristem! maintains! its! indeterminacy! and! flowers! develop! from! lateral! meristems!(Krizek!and!Fletcher,!2005).!!

!! 17! Chapter!2!

In!Arabidopsis!the!expression!of!AP1!and!LFY!is!largely!restricted!to!floral!meristems,!though! LFY!is!also!expressed!at!low!level!during!vegetative!growths.!These!genes!are!strongly!expressed!in! lateral!(floral)!meristems,!while!in!the!apical!(inflorescence)!meristem!their!expression!is!prevented! by!TERMINAL!FLOWER!1!(TFL1),!in!Arabidopsis,!and!CENTRORADIALIS!in!Antirrhinum!(Bradley!et!al.,! 1996;!Bradley!et!al.,!1997).!Constitutive!expression!of!either!LFY!or!AP1,!which!positively!regulate! each!other!in!a!regulatory!loop,!causes!precocious!flowering!and!converts!the!apical!meristem!into!a! flower,!and,!consequently,!the!racemose!inflorescence!into!a!solitary!flower!(Mandel!and!Yanofsky,! 1995;! Weigel! and! Nilsson,! 1995).! Hence,! the! spatioAtemporal! regulation! of! LFY! and/or! AP1! transcription!is!the!major!factor!determining!when!and!where!flowers!form!in!Arabidopsis.!Similar! results!were!obtained!with!other!species,!including!trees!such!as!aspen!and!citrus!(Pena!et!al.,!2001;! Weigel!and!Nilsson,!1995).!However!in!Petunia,!a!cyme,!constitutive!expression!of!LFY!or!the!Petunia! homolog! ABERRANT! LEAF! AND! FLOWER! (ALF)! does! not! affect! flowering! time! or! inflorescence! architecture.!This!indicates!that!the!transcription!of!ALF,!though!required!for!floral!identity,!is!not!the! limiting!factor!that!restricts!flower!formation!in!time!or!space.!Instead,!the!limiting!factor!is!another! FMI!gene:!DOUBLE!TOP!(DOT)!(Souer!et!al.,!2008).!DOT!is!the!homolog!of!UNUSUAL!FLORAL!ORGANS! (UFO)!from!Arabidopsis.!DOT!and!UFO!are!functionally!interchangeable!FAbox!protein!components!of! an! SCFAtype! ubiquitin! ligase! complex! that! binds! to! ALF/LFY! to! promote! its! transcription! activation! potential!(Chae!et!al.,!2008;!Souer!et!al.,!2008).!While!expression!of!DOT!is!restricted!to!(incipient)! flowers,!UFO!is!expressed!in!many!meristems,!including!the!SAM!in!embryos!and!seedlings.!Indeed,! constitutive! expression! of! DOT! or! UFO! in! Petunia! causes! precocious! flowering! and! changes! the! cymose!inflorescence!into!a!solitary!flower,!while!in!Arabidopsis!no!such!effects!are!seen.!Transgenic! experiments! in! which! promoterAreporter! genes! were! swapped! between! Petunia! and! Arabidopsis! indicated! that! the! diversification! of! DOT! and! UFO! expression! was! caused! at! least! in! part! through! differences!in!their!CREs!(Kusters,!2011).! Interestingly! LFY! expression! patterns! vary! even! between! species! with! similar! inflorescence! architectures.!Brassicaceae!like!Ionopsidium!acaule!(violet!cress,!Iac)!and!Laevenworthia!crassa!(Lcr)! bear! flowers! in! the! axils! of! rosette! leaves! (“rosette! flowering”)! and! can! be! seen! as! racemes! with! reduced! internodes! (Shu! et! al.,! 2000).! When! introduced! in! Arabidopsis! a! LcrLFY! transgene,! or! a! fusion!of!the!LcrLFY!promoter!(LcrLFYp)!to!the!AtLFY!coding!sequence,!rectifies!the!lfy!phenotype,!and! in! addition! converts! the! apical! meristem! into! a! terminal! flower,! which! fits! with! the! finding! that!

LcrLFYp! is! active! in! the! IM! (Sliwinski! et! al.,! 2006;! Yoon! and! Baum,! 2004).! Even! though! IacLFY! is! expressed!in!floral!mersitems!and!the!apical!meristem!in!I.!acaule,!IacLFYp!is!not!active!in!the!apical! meristem! of! Arabidopsis,! and! a! IacLFY! transgene! does! not! affect! the! development! the! apical! meristem! in! Arabidopsis!(Shu! et! al.,! 2000;! Yoon! and! Baum,! 2004).! These! findings! imply! that! LFY! expression!patterns!varied!even!between!closely!related!species,!through!alterations!in!CREs!within!

18! ! Evolution!of!morphological!diversity!

! their!promoters!or!in!the!upstream!network,!without!any!obvious!alterations!in!architecture.!It!may! be!that!the!apical!IM!of!L.crassa!and!I.acaule!does!not!acquire!floral!identity,!even!when!it!expresses! LFY,! because! it! lacks/lost! expression! of! an! essential! partner,! such! as! the! UFO/DOT! homolog.! The! picture!that!emerges!is!that!expression!patterns!of!distinct!FMI!genes!diverged!substantially!between! angiosperms! and! thus! created! new! patterns! where! essential! partners! are! coAexpressed! and! floral! identity!is!established.!!

Flowers!of!all!hues:!variations!in!flower!architecture!

In! Angiosperms! LFY! activates! several! genes! involved! in! floral! morphogenesis,! including! homeotic! floral!organ!identity!genes.!Many!of!these!encode!MADS!box!transcription!factors!which!specify!the! fate! of! emerging! organ! primordia.! According! to! the! ABC! model,! which! is! based! on! phenotypes! of! homeotic!Arabidopsis!thaliana!and!Antirrhinum!majus!mutants,!three!classes!of!genes!(A,!B!and!C)! specify!the!identity!of!floral!organ!primordia!(Coen!and!Meyerowitz,!1991).!A!function!specifies!sepal! identity!in!the!outermost!whorl!(whorl!1),!coAexpression!of!A!and!B!function!specifies!petal!identity!in! whorl! 2,! B! and! C! genes! together! control! stamen! identity! in! whorl! 3,! while! the! CAfunction! alone! specifies! carpel! identity! in! whorl! 4.! In! addition,! the! A! and! C! functions! antagonize! each! other.! E! function!genes!are!a!later!addition!to!the!model!and!act!as!coAregulators!in!controlling!the!four!floral! organ!identities!(Theissen,!2001).!Nowadays!a!significant!amount!of!data!is!available!relating!to!the! conservation/evolution!of!these!TFs.!! Flowers! present! an! enormous! variation! in! organ! number,! size! and! shape! (Fig.! 2DAK).! In! gymnosperm,!male!and!female!sex!organs!develop!on!separate!structures!(cones)!that!lack!most!of! the!features!seen!in!flowers.!Putative!BA!and!CAfunction!orthologs!can!be!found!in!gymnosperms!as! well:!BAclass!genes!are!maleAspecific,!while!C!genes!are!expressed!in!both!male!and!female!organs! (Tandre!et!al.,!1998).!These!genes!still!show!a!significant!level!of!functional!conservation!between! gymnosperm! and! angiosperm! despite! millions! of! years! of! separation! (Rutledge! et! al.,! 1998).! This! finding!has!been!used!as!starting!point!to!explain!flower!origin.!According!to!the!outAofAmale!(or!outA ofAfemale)!theory!(Theissen!and!Becker,!2004),!class!B!genes!worked!as!a!“switch”!to!control!female! (or!male)!organ!development!in!male!(or!female)!cones.!The!formation!of!an!apicalAbasal!dissimilarity! in!B!gene!expression!within!the!cone!could!have!led!to!the!hermaphroditic!precursors!of!flowers.!It!is! conceivable! that! both! trans! and! CREs! alterations! are! responsible! for! alterations! in! the! expression! pattern!of!these!B!genes.!Further!experiments!are!necessary!to!elucidate!their!evolution,!however,! these! studies! are! hampered! by! the! fact! that! basal! angiosperms! are! not! amenable! to! genetic! dissection! and! ancestral! plants! bearing! hermaphroditic! precursors! of! flowers! are! unfortunately! extinct.!!

!! 19! Chapter!2!

APETALA3!(AP3)! together! with! PISTILLATA! belong! to! the! BAtype! MADS! box! genes! that! are! involved! in! the! regulation! of! petal! identity! specification! in! Arabidopsis!(Jenik! and! Irish,! 2001).! In! Ranunculaceae! the! AP3! lineage! has! undergone! many! duplication! events,! giving! rise! to! three! paralogous!AP3!lineages!(AP3Z1,!AP3Z2!and!AP3Z3),!which!are!found!throughout!the!family!(Kramer! et! al.,! 2003).!Species! bearing! petals!express! AP3Z3! orthologs! specifically! in! petal! primordia! and! developing! petals,! while! species! lacking! petals! lack! AP3Z3! expression! (Zhang! et! al.,! 2013).! AP3A3! expression!seems!to!have!been!lost!several!times!independently!by!either!deletions!or!insertions!in! the!coding,!promoter,!or!intronic!regions.!The!independent!occurrence!of!apetalous!species!in!the! Ranunculaceae! reflects! a! possible! advantage:! formation! of! petals! and! nectaries! requires! energy,! which! explains! why! many! taxa! have! evolved! general! pollination! systems! with! sepals,! stamens,! or! filaments!which!are!attractive!to!insects.! Another!gene!duplication,!followed!by!a!frameshift!mutation!in!the!CAterminal!region!of!the! AP3! gene,!formed!two!new!lineages!in!the!core!:!euAP3!and!TM6!(Vandenbussche! et!al.,! 2003).! While! Arabidopsis! and! Antirrhinum! have! lost! their! TM6! copy! and! maintained! only! euAP3! (AtAP3!and!AmDEFICIENS),!Petunia!hybrida!maintained!both!AP3!(PhDEF)!and!TM6!genes.!Mutation! in!PhDEF!shows!a!conversion!of!petals!to!sepals,!but!the!stamens!are!still!made!due!to!the!fact!that! TM6!works!redundantly!in!their!specification!indeed,!the!double!mutant!transforms!petals!to!sepals! and! stamens! to! carpels! (Rijpkema! et! al.,! 2006).! TM6! shows! a! third! and! fourth! whorl! expression! pattern,!while!PhDEF!is!expressed!in!the!second!and!third!whorl.!Interestingly,!the!divergence!of!the! DEF/TM6!expression!patterns!correlates!with!the!loss!of!a!conserved!promoter!element!in!the!TM6! lineage!that!might!be!responsible!for!the!subfunctionalization!of!these!genes.!Ectopic!expression!of! TM6!is!capable!of!inducing!petal!development!in!a!def!mutant!background,!which!shows!that!protein! function!is!identical!and!that!subfunctionalization!results!from!changes!in!TM6!regulation!(Rijpkema! et!al.,!2006;!Vandenbussche!et!al.,!2004).!! Heterotopic! expression! of! BAfunction! genes! in! whorl! one! of! Tulipa! explains! the! presence! of! petals!in!the!outer!whorl,!also!known!as!tepals!(Kanno!et!al.,!2003).!Several!species!of!the!! family!also!show!morphological!alterations!of!whorl!1!too:!the!inflated!calyx!syndrome!(ICS)!is!an! amazing! floral! morphological! novelty! where,! after! pollination,! the! growth! of! sepals! restarts! thus! giving!rise!to!a!balloonAlike!structure!that!encapsulates!the!mature!berry.!Research!in!!has! suggested! that! a! change! in! the! promoter! region! of! MPF2! –! a! member! of! the! StMADS16! clade!A! accounts! for! the! alteration! in! its! expression,! which! is! responsible! for! ICS! in! this! species! (He! and! Saedler,!2005).! An!interesting!exception!to!the!conserved!floral!ground!plan!of!eudicots!is!found!in!Lacandonia! schismatica!where! stamens!occur!in!the!centre! of!the!flower!and!are!surrounded!by!carpels.!This! different!order!in!flower!organs!appears!to!be!due!to!different!spatioAtemporal!expression!patterns!

20! ! Evolution!of!morphological!diversity!

! in! BA! and! CAfunction! genes! (AlvarezABuylla! et! al.,! 2010),! but! further! experiments! are! necessary! in! order!to!elucidate!whether!the!new!expression!patterns!arose!via!mutations!in!CREs!of!B!and!C!genes! or!in!the!upstream!regulatory!network,!or!in!both.! Flowers! can! display! radial! symmetry! (Fig.! 2IAJ),! if! all! petals! develop! and! fuse! equally,! or! bilateral!symmetry!(zygomorphy)!(Fig.!2H),!if!their!petals!develop!in!an!unequal!way.!This!difference! in! flower! symmetry! evolved! multiples! times! during! angiosperm! evolution! and! it! involves! the! expression! of! TEOSINTE! BRANCHED1/CYCLOIDEA/PCF!(TCP)! genes! along! the! dorsoAventral! axis.! In! radial!asymmetric!or!zygomorphic!flowers!these!genes!are!strongly!expressed!in!the!dorsal!petals,! while! in! symmetric! or! actinomorphic! flowers! they! are! present! at! too! low! level! in! order! to! effect! flower!symmetry.!Yang!and!colleagues!(Yang!et!al.,!2012)!found!that!the!promoters!of!two!genes!of! the! CYCLOIDEA2!(CYC)! clade,! CYC1C! and! CYC1D! of! Primulina! heterotricha,! have! consensus! CYC! binding!sites,!resulting!in!positive!autoregulation!and!crossAregulation!of!each!others!expression.!This! feedback!loop!seems!to!operate!in!many!species!which!bear!zygomorphic!flowers!since!consensus! CYC!binding!sites!were!identified!in!the!promoters!of!their!CYC2!genes.!In!contrast,!the!promoters!of! CYC2! genes! in! the! actinomorphic! lineage! lack! these! CYC! binding! sites,! and! consequently! the! autoregulatory! loop! necessary! to! maintain! their! expression! during! flower! development.! It! thus! seems!that!a!similar!change!in!the!CREs!of!CYC2!genes!arose!multiple!times!in!angiosperms,!which! could!explain!the!independent!origin!of!floral!zygomorphy.!!

Terminal!differentiation!of!epidermal!cells.!

Changes! in! the! expression! pattern! also! drove! the! evolution! of! metabolic! pathways,! such! as! plant! pigmentation.! Anthocyanins! are! responsible! for! most! of! the! orange,! red! and! purple! coloration! in! Angiosperms! (Fig.! 2DAK)! and! serve! a! variety! of! (pleiotropic)! functions:! in! vegetative! tissues! they! protect!from!high!light!intensities,!while!in!flowers!and!fruits!they!attract!animals!that!aid!pollination! and!seed!dispersal!(Buer!et!al.,!2010).! Structural! anthocyanin! genes! encoding! the! enzymes! of! the! pathway! are! activated! by! a! conserved!complex!(MBW),!consisting!of!a!MYB,!a!basicAhelixAloopAhelix!(HLH)!and!a!WD40!protein! (Grotewold,!2006;!Koes! et!al.,!2005).! Although!the!MBW!complex!is!conserved!between!monocot! and! dicot! species,! its! function! has! diverged! substantially,! in! terms! of! downstream! genes! and! processes!that!are!activated.!The!MBW!activates!all!structural!genes!from!the!anthocyanin!pathway! in! maize,! but! only! a! subset! of! structural! genes! in! most! dicots.! Exchanging! either! MYB! and! HLH! regulators! or! gene! promoters! between! species! showed! that! this! is! due! to! differences! in! the! structural!gene!promoters,!and!not!in!the!MYB!and!HLH!proteins!(Koes!et!al.,!1994;!Quattrocchio!et! al.,!1998).!

!! 21! Chapter!2!

Phenotypes! of! Arabidopsis! and! Petunia! mutants! revealed! that! the! divergence! of! MBW! function!extends!to!several!other!processes!involved!in!the!differentiation!of!epidermal!cells!(Ramsay! and!Glover,!2005).!In!Petunia!these!include!the!division!and!morphogenesis!of!seed!coat!cells!and! several!vacuolar!processes!that!affect!petal!coloration,!such!as!vacuolar!acidification!and!stabilization! of!vacuolar!anthocyanins.!In!Arabidopsis!MBW!complexes!specify!the!identity!of!incipient!trichomes! in!stems!and!leaves,!nonAhair!cells!in!the!root!epidermis!and!the!production!of!mucilage!by!seeds.! Interestingly,!in!Petunia!or!maize!no!trichome!defects!are!seen!in!the!gain!or!loss!of!function!mutants! for!MBW!components,!indicating!that!in!these!species!the!MBW!complex!has!no!role!in!trichome! development.!Nevertheless,!the!maize!WD40!and!HLH!proteins!PALE!ALEURONE!COLOR!(PAC)!and! RED!(R)!can!drive!trichome!development!when!expressed!in!Arabidopsis!(Carey!et!al.,!2004;!Lloyd!et! al.,!1992).!This!indicates!that!Arabidopsis!MBW!genes!acquired!their!role!in!trichome!development! (and!possibly!noAhair!cells!in!the!root!epidermis)!via!changes!in!the!CREs!of!downstream!trichome! genes!that!brought!them!under!MBW!control.!Genome!wide!analysis!recently!identified!some!20A40! target! genes! that! are! bound! and! regulated! by! GLABRA1! (GL1)! and/or! GL3,! the! MYB! and! HLH! components! of! the! Arabidopsis! MBW! complex!(Morohashi! and! Grotewold,! 2009).! Comparative! analysis!with!other!species,!like!Petunia!or!maize!may!shed!light!on!how!these!“old!genes!learned! new!tricks”.! Because!pigmentation!is!an!easy!readAout!of!gene!expression,!it!is!a!convenient!system!to!use! to!study!(adaptive)!changes!in!gene!expression!patterns.!The!pigmentation!of!floral!tissues!together! with!other!features,!such!as!scent!and!flower!morphology,!attracts!visits!of!specific!animals!(bees,! moths,! birds,! bats)! for! pollination.! Changes! in! any! of! these! floral! features! (pollination! syndrome),! might!lead!to!attraction!of!different!pollinators!and!may,!in!theory,!result!in!genetic!isolation!and! ultimately!speciation!(Wessinger!and!Rausher,!2012).!Petunia!integrifolia,!for!example,!has!colored! flowers!with!a!short!wide!tube!that!are!visited!by!bumblebees,!whereas!P.!axilaris!has!white!scented! flowers!with!a!long!narrow!tube!that!are!visited!by!nocturnal!hawkmoths.!Inactivation!in!P.!axillaris! of!the!ANTHOCYANIN2!gene,!a!MYB!regulator,!caused!different!petal!pigmentation!(Hoballah!et!al.,! 2007;!Quattrocchio!et!al.,!1999).!Interestingly!an2!mutations!were!fixed!in!P.!axillaris!at!least!5!times! independently,!but!mutations!in!other!anthocyanin!genes!were!not.!Presumably!that!is!because!AN2! expression!is!confined!to!the!petal!limb,!while!other!anthocyanin!genes!are!also!expressed!on!many! other!sites,!which!is!the!reason!why!mutations!in!AN2!are!the!least!pleiotropic!(Quattrocchio!et!al.,! 1999).!A!shift!from!bee!to!hummingbird!pollination!is!associated!with!transition!from!blueApurple!to! red! colored! flowers,! together! with! other! morphological! alterations.! The! most! frequent! cause! of! these! color! changes! is! a! reduced! expression! of! Flavonoid! 3’ZHydroxylase!(F3’H)! and/or! F3’5’H! (Wessinger! and! Rausher,! 2012).! Expression! of! F3’5’H,! which! is! primarily! required! for! flower! pigmentation,! was!abolished!by!mutations!in!either!the!coding!sequence!or!a!CRE.! By! contrast! in!

22! ! Evolution!of!morphological!diversity!

! F3’H,! which! is! involved! in! other! processes! besides! flower! pigmentation,! mutations! occurred! predominantly!in!its!CREs,!thus!reducing!F3’H!expression!mainly!in!flowers!(Wessinger!and!Rausher,! 2012).!! The! cases! summarized! above! concern! loss! of! function! mutations! that! reduce! or! eliminate! pigmentation! in! certain! tissues.! They! all! have! an! impact! on! elements! that! cause! relatively! few! pleiotropic! effects,! either! a! CRE! or! the! coding! sequence! of! a! paralog! with! a! restricted! expression! pattern.!Cases!in!which!novel!pigmentation!patterns!were!acquired!are!more!scarcely!documented.! That!may!be!because!such!mutations!are!not!favored!and/or!they!are!more!difficult!to!prove!without! knowing! the! ancestral! state.! A! major! part! of! the! color! difference! between! pale! red! bumblebeeA pollinated! flowers! of! Mimulus! lewisii! and! the! bright! red! hummingbirdApollinated! flowers! of! M.! cardinalis! is! due! to! the! difference! in! expression! level! of! a! MYB! protein,! ROSE! INTENSITY! 1,! that! inhibits!the!activity!of!the!MBW!complex!(Yuan!et!al.,!2013).!The!different!expression!levels!appear! to!be!due!to!an!alteration!in!RO1!CREs,!but!since!the!ancestral!state!is!unknown!it!is!unclear!whether! this!involved!the!loss!of!an!activating!CRE!in!M.!cardinalis,!or!a!gain!in!M.!lewisii.!! The! creation! of! new! pigmentation! patterns! by! gain! of! function! mutations! has! been! documented! in! a! few! domesticated! crops.! In! all! species! analyzed! the! WD40! partner! of! the! MBW! complex!is!more!or!less!ubiquitously!expressed!and!the!pigmentation!pattern!is!largely!dictated!by! the!expression!patterns!of!the!MYB!and!bHLH!partners!(Koes!et!al.,!2005).!Ectopic!expression!of!the! MYB!protein!is!sufficient!to!establish!the!pigmentation!of!new!tissues!(Nesi!et!al.,!2001;!Spelt!et!al.,! 2000).! The! appearance! of! varieties! with! new! pigmentation! patterns! was! indeed! associated! with! alterations!in!the!regulatory!region!of!the!MYB!anthocyanin!regulators.!For!instance!the!appearance! of!blood!oranges!with!anthocyanins!in!the!flesh!of!the!fruit!happened!independently!on!at!least!two! occasion;!in!both!cases!new!CREs!originated!from!a!retrotransposon!insertion!into!the!RUBY!gene,! which! encode! a! MYB! component! of! the! MBW! complex!(Butelli! et! al.,! 2012).! The! color! difference! between!red!and!green!apples!can!also!be!traced!to!a!CRE!in!the!MYB10!allele!of!red!apples!that! results! in! enhanced! MYB10! expression!(Espley! et! al.,! 2007).! This! novel! CRE! originates! from! a! microsatellite!and!creates!a!new!binding!site!for!MYB10!itself!resulting!in!an!autoAregulatory!loop! (Espley!et!al.,!2009).!

Conclusion!

In!this!review!we!have!tried!to!elucidate!some!of!the!general!mechanisms!that!have!been!at!work! throughout! plant! and! animal! evolution.! The! appearance! of! novel! structures! and! their! subsequent! diversification! relied! primarily! on! alterations! in! spatioAtemporal! gene! expression! patterns,! rather! than!the!sudden!appearance!of!completely!new!genes.!Indeed,!in!most!cases!morphological!novelty! originates!from!changes!in!the!timing,!rates!or!pattern!of!expression!of!preAexisting!genes.!To!our!

!! 23! Chapter!2! knowledge!very!few!cases!have!been!documented!where!a!new!gene!expression!pattern!could!be! attributed!to!an!alteration!in!a!transcription!factor!protein.!That!might!be!because!such!events!are! rarely!fixed!during!evolution,!due!to!their!pleiotropic!defects,!and/or!because!such!events!are!more! difficult!to!identify!and!prove!experimentally.!However,!in!a!substantial!number!of!cases!the!cause!of! an!altered!gene!expression!pattern!was!shown!to!result!from!changes!in!gene!promoters,!although! the!specific!CREs!and!DNA!changes!that!were!involved!were!traced!down!in!only!a!few!cases.!This! may!be!due!to!the!fact!that!CREs!can!lie!many!kilobases!away!from!the!coding!sequence!and!are! difficult! to! recognize! since! single! nucleotide! changes! may! be! sufficient! to! create! or! remove! transcription! factor! binding! sites.! The! general! picture! is! that! most! changes,! including! the! few! in! transcription!factor!proteins,!seem!to!have!been!selected!for!minimum!pleiotropic!effects.!! One!could!envisage!several!other!mechanisms,!besides!the!modification!of!tissueAspecific!CREs! involved! in! transcription! control,! to! rewire! GRNs! with! minimal! pleiotropic! effects.! For! example,! alternative!splicing!(Syed!et!al.,!2012)!or!polyadenylation!may!lead!to!the!expression!of!novel!forms! of! a! protein,! with! new! functions,! while! leaving! the! old! function! unharmed.! Alterations! in! (transA acting)!miRNAs,!(cisAacting)!complementary!sequences!within!mRNAs!(Kosik,!2009),!DNA!methylation! (Cortijo! et! al.,! 2014)! and! histone! modifications! (Turck! and! Coupland,! 2013)! could,! theoretically,! modify! gene! expression! patterns! in! a! similar! subtle! ways! as! changes! CREs.! To! what! extent! such! mechanisms!contribute!to!morphological!diversification!remains!to!be!determined.!

Acknowledgements!

This! work! was! supported! by! a! grant! from! the! Netherlands! Organization! for! Scientific! Research! (NWO).! We! apologize! for! not! including! many! of! the! exciting! studies! and! references! due! to! space! limitations! ! !

24! ! Evolution!of!morphological!diversity!

! References

Abe!M,!Kobayashi!Y,!Yamamoto!S,!Daimon!Y,!Yamaguchi!A,!Ikeda!Y,!Ichinoki!H,!Notaguchi!M,!Goto!K,! Araki!T.!2005.!FD,!a!bZIP!protein!mediating!signals!from!the!floral!pathway!integrator!FT!at!the!shoot!apex.! Science!309,!1052A1056.!

Alvarez0Buylla!ER,!Ambrose!BA,!Flores0Sandoval!E,!Vergara0Silva!F,!Englund!M,!Garay0Arroyo!A,!Garcia0 Ponce! B,! de! la! Torre0Barcena! E,! Espinosa0Matias! S,! Martinez! E,! Pineyro0Nelson! A,! Engstrom! P,! Meyerowitz! EM.!2010.!BAFunction!expression!in!the!flower!center!underlies!the!homeotic!phenotype!of! Lacandonia!schismatica!(Triuridaceae).!The!Plant!Cell!22,!3543A3559.!

Andres! F,! Coupland! G.!2012.!The!genetic!basis!of!flowering!responses!to!seasonal!cues.!Nature!Reviews! Genetics!13,!627A639.!

Angelini!DR,!Kaufman!TC.!2005.!Comparative!developmental!genetics!and!the!evolution!of!arthropod!body! plans.!Annual!Review!of!Genetics!39,!95A119.!

Aubin!J,!Chailler!P,!Menard!D,!Jeannotte!L.!1999.!Loss!of!Hoxa5!gene!function!in!mice!perturbs!intestinal! maturation.!American!Journal!of!Physiology!Z!Cell!Physiology!277,!C965AC973.!

Bhardwaj!N,!Yan!KK,!Gerstein!MB.!2010.!Analysis!of!diverse!regulatory!networks!in!a!hierarchical!context! shows!consistent!tendencies!for!collaboration!in!the!middle!levels.!Proceedings!of!the!National!Academy!of! Sciences,!USA!107,!6841A6846.!

Bohlenius!H,!Huang!T,!Charbonnel0Campaa!L,!Brunner!AM,!Jansson!S,!Strauss!SH,!Nilsson!O.!2006.!CO/FT! regulatory!module!controls!timing!of!flowering!and!seasonal!growth!cessation!in!trees.!Science!312,!1040A 1043.!

Bradley! D,! Carpenter! R,! Copsey! L,! Vincent! C,! Rothstein! S,! Coen! E.! 1996.! Control! of! inflorescence! architecture!in!Antirrhinum.!Nature!379,!791A797.!

Bradley!D,!Ratcliffe!O,!Vincent!C,!Carpenter!R,!Coen!E.!1997.!Inflorescence!commitment!and!architecture! in!Arabidopsis.!Science!275,!80A83.!

Buer!CS,!Imin!N,!Djordjevic!MA.!2010.!Flavonoids:!new!roles!for!old!molecules.!Journal!of!Integrative!Plant! Biology!52,!98A111.!

Burke! AC,! Nelson! CE,! Morgan! BA,! Tabin! C.! 1995.! Hox! genes! and! the! evolution! of! vertebrate! axial! morphology.!Development!121,!333A346.!

Butelli! E,! Licciardello! C,! Zhang! Y,! Liu! J,! Mackay! S,! Bailey! P,! Reforgiato0Recupero! G,! Martin! C.! 2012.! Retrotransposons! control! fruitAspecific,! coldAdependent! accumulation! of! anthocyanins! in! blood! oranges.! The!Plant!Cell!24,!1242A1255.!

Carey! CC,! Strahle! JT,! Selinger! DA,! Chandler! VL.! 2004.! Mutations! in! the! pale! aleurone! color1! regulatory! gene!of!the!Zea!mays!anthocyanin!pathway!have!distinct!phenotypes!relative!to!the!functionally!similar! TRANSPARENT!TESTA!GLABRA1!gene!in!Arabidopsis!thaliana.!The!Plant!Cell!16,!450A464.!

Carrera! J,! Rodrigo! G,! Jaramillo! A,! Elena! SF.! 2009.! ReverseAengineering! the! Arabidopsis! thaliana! transcriptional!network!under!changing!environmental!conditions.!Genome!Biology!10.!

!! 25! Chapter!2!

Carroll!SB.!2000.!Endless!forms:!the!evolution!of!gene!regulation!and!morphological!diversity.!Cell!101,!577A 580.!

Carroll!SB.!2005.!Evolution!at!two!levels:!on!genes!and!form.!Plos!Biology!3,!1159A1166.!

Carroll! SB.! 2008.! EvoAdevo! and! an! expanding! evolutionary! synthesis:! a! genetic! theory! of! morphological! evolution.!Cell!134,!25A36.!

Chae!E,!Tan!QKG,!Hill!TA,!Irish!VF.!2008.!An!Arabidopsis!FAbox!protein!acts!as!a!transcriptional!coAfactor!to! regulate!floral!development.!Development!135,!1235A1245.!

Chahtane! H,! Vachon! G,! Le! Masson! M,! Thevenon! E,! Perigon! S,! Mihajlovic! N,! Kalinina! A,! Michard! R,! Moyroud! E,! Monniaux! M,! Sayou! C,! Grbic! V,! Parcy! F,! Tichtinsky! G.!2013.!A!variant!of!LEAFY!reveals!its! capacity!to!stimulate!meristem!development!by!inducing!RAX1.!The!Plant!Journal!74,!678A689.!

Champagne!CEM,!Goliber!TE,!Wojciechowski!MF,!Mei!RW,!Townsley!BT,!Wang!K,!Paz!MM,!Geeta!R,!Sinha! NR.!2007.!Compound!Leaf!Development!and!Evolution!in!the!Legumes.!The!Plant!Cell!19,!3369A3378.!

Coen! ES,! Meyerowitz! EM.! 1991.! The! war! of! the! whorls:! genetic! interactions! controlling! flower! development.!Nature!353,!31A37.!

Corbesier!L,!Vincent!C,!Jang!SH,!Fornara!F,!Fan!QZ,!Searle!I,!Giakountis!A,!Farrona!S,!Gissot!L,!Turnbull!C,! Coupland! G.! 2007.! FT! protein! movement! contributes! to! longAdistance! signaling! in! floral! induction! of! Arabidopsis.!Science!316,!1030A1033.!

Cortijo!S,!Wardenaar!R,!Colome0Tatche!M,!Gilly!A,!Etcheverry!M,!Labadie!K,!Caillieux!E,!Hospital!F,!Aury! JM,!Wincker!P,!Roudier!F,!Jansen!RC,!Colot!V,!Johannes!F.!2014.!Mapping!the!epigenetic!basis!of!complex! traits.!Science,!doi:!10.1126/science.1248127.!

Crane!PR,!Friis!EM,!Pedersen!KR.!1995.!The!origin!and!early!diversification!of!angiosperms.!Nature!374,!27A 33.!

De!Robertis!EM.!2008.!EvoAdevo:!variations!on!ancestral!themes.!Cell!132,!185A195.!

Doebley!J,!Lukens!L.!1998.!Transcriptional!Regulators!and!the!Evolution!of!Plant!Form.!The!Plant!Cell!10,! 1075A1082.!

Doebley!J,!Stec!A,!Hubbard!L.!1997.!The!evolution!of!apical!dominance!in!maize.!Nature!386,!485A488.!

Doi!K,!Izawa!T,!Fuse!T,!Yamanouchi!U,!Kubo!T,!Shimatani!Z,!Yano!M,!Yoshimura!A.!2004.!Ehd1,!a!BAtype! response!regulator!in!rice,!confers!shortAday!promotion!of!flowering!and!controls!FTZlike!gene!expression! independently!of!Hd1.!Genes!&!Development!18,!926A936.!

Espley!RV,!Brendolise!C,!Chagne!D,!Kutty0Amma!S,!Green!S,!Volz!R,!Putterill!J,!Schouten!HJ,!Gardiner!SE,! Hellens! RP,! Allan! AC.! 2009.! Multiple! Repeats! of! a! Promoter! Segment! Causes! Transcription! Factor! Autoregulation!in!Red!Apples.!The!Plant!Cell!21,!168A183.!

Espley!RV,!Hellens!RP,!Putterill!J,!Stevenson!DE,!Kutty0Amma!S,!Allan!AC.!2007.!Red!colouration!in!apple! fruit!is!due!to!the!activity!of!the!MYB!transcription!factor,!MdMYB10.!The!Plant!Journal!49,!414A427.!

Frohlich! MW.! 2006.! Recent! developments! regarding! the! evolutionary! origin! of! flowers.! Advances! in! Botanical!Research:!Incorporating!Advances!in!Plant!Pathology,!Vol!44!44,!63A127.!

26! ! Evolution!of!morphological!diversity!

! Galant! R,! Carroll! SB.! 2002.! Evolution! of! a! transcriptional! repression! domain! in! an! insect! Hox! protein.! Nature!415,!910A913.!

Grotewold!E.!2006.!The!genetics!and!biochemistry!of!floral!pigments.!Annual!Review!of!Plant!Biology!57,! 761A780.!

Halder!G,!Callaerts!P,!Gehring!WJ.!1995.!Induction!of!ectopic!eyes!by!targeted!expression!of!the!eyeless! gene!in!Drosophila.!Science!267,!1788A1792.!

Hareven! D,! Gutfinger! T,! Parnis! A,! Eshed! Y,! Lifschitz! E.! 1996.! The! making! of! a! compound! leaf:! genetic! manipulation!of!leaf!architecture!in!tomato.!Cell!84,!735A744.!

Hay!A,!Tsiantis!M.!2006.!The!genetic!basis!for!differences!in!leaf!form!between!Arabidopsis!thaliana!and!its! wild!relative!Cardamine!hirsuta.!Nature!Genetics!38,!942A947.!

Hay!A,!Tsiantis!M.!2010.!KNOX!genes:!versatile!regulators!of!plant!development!and!diversity.!Development! 137,!3153A3165.!

He!CY,!Saedler!H.!2005.!Heterotopic!expression!of!MPF2!is!the!key!to!the!evolution!of!the!Chinese!lantern! of!Physalis,!a!morphological!novelty!in!Solanaceae.!Proceedings!of!the!National!Academy!of!Sciences,!USA! 102,!5779A5784.!

Heffer!A,!Pick!L.!2013.!Conservation!and!variation!in!Hox!genes:!how!insect!models!pioneered!the!evoAdevo! field.!Annual!review!of!entomology!58,!161A179.!

Hoballah!ME,!Gubitz!T,!Stuurman!J,!Broger!L,!Barone!M,!Mandel!T,!Dell'Olivo!A,!Arnold!M,!Kuhlemeier!C.! 2007.!Single!geneAmediated!shift!in!pollinator!attraction!in!Petunia.!The!Plant!Cell!19,!779A790.!

Hoekstra!HE,!Coyne!JA.!2007.!The!locus!of!evolution:!evo!devo!and!the!genetics!of!adaptation.!Evolution! 61,!995A1016.!

Hofer!J,!Turner!L,!Hellens!R,!Ambrose!M,!Matthews!P,!Michael!A,!Ellis!N.!1997.!UNIFOLIATA!regulates!leaf! and!flower!morphogenesis!in!pea.!Current!Biology!7,!581A587.!

Ishikawa!R,!Shinomura!T,!Takano!M,!Shimamoto!K.!2009.!Phytochrome!dependent!quantitative!control!of! HD3a!transcription!is!the!basis!of!the!night!break!effect!in!rice!flowering.!Genes!&!Genetic!Systems!84,!179A 184.!

Ishikawa!R,!Tamaki!S,!Yokoi!S,!Inagaki!N,!Shinomura!T,!Takano!M,!Shimamoto!K.!2005.!Suppression!of!the! floral!activator!HD3a!is!the!principal!cause!of!the!night!break!effect!in!rice.!The!Plant!Cell!17,!3326A3336.!

Jenik! PD,! Irish! VF.! 2001.! The! Arabidopsis! floral! homeotic! gene! APETALA3! differentially! regulates! intercellular!signaling!required!for!petal!and!stamen!development.!Development!128,!13A23.!

Jiao!YN,!Wickett!NJ,!Ayyampalayam!S,!Chanderbali!AS,!Landherr!L,!Ralph!PE,!Tomsho!LP,!Hu!Y,!Liang!HY,! Soltis! PS,! Soltis! DE,! Clifton! SW,! Schlarbaum! SE,! Schuster! SC,! Ma! H,! Leebens0Mack! J,! dePamphilis! CW.! 2011.!Ancestral!polyploidy!in!seed!plants!and!angiosperms.!Nature!473,!97AU113.!

Kanno! A,! Saeki! H,! Kameya! T,! Saedler! H,! Theissen! G.! 2003.! Heterotopic! expression! of! class! B! floral! homeotic!genes!supports!a!modified!ABC!model!for!tulip!(Tulipa!gesneriana).!Plant!Molecular!Biology!52,! 831A841.!

Kenrick!P,!Crane!PR.!1997.!The!origin!and!early!evolution!of!plants!on!land.!Nature!389,!33A39.!

!! 27! Chapter!2!

King!MC,!Wilson!AC.!1975.!Evolution!at!two!levels!in!humans!and!chimpanzees.!Science!188,!107A116.!

Koes!R,!Verweij!CW,!Quattrocchio!F.!2005.!Flavonoids:!a!colorful!model!for!the!regulation!and!evolution!of! biochemical!pathways.!Trends!Plant!Science!5,!236A242.!

Koes! RE,! Quattrocchio! F,! Mol! JNM.! 1994.! The! flavonoid! biosynthetic! pathway! in! plants:! function! and! evolution.!Bioessays!16,!123A132.!

Kosik!KS.!2009.!MicroRNAs!tell!an!evoAdevo!story.!Nature!Reviews!Neuroscience!10,!754A759.!

Kramer!EM,!Di!Stilio!VS,!Schluter!PM.!2003.!Complex!patterns!of!gene!duplication!in!the!APETALA3!and! PISTILLATA!lineages!of!the!Ranunculaceae.!International!Journal!of!Plant!Sciences!164,!1A11.!

Krizek! BA,! Fletcher! JC.! 2005.! Molecular! mechanisms! of! flower! development:! An! armchair! guide.!Nature! Reviews!Genetics!6,!688A698.!

Kusters!E.!2011.!Genetic!control!of!meristem!identity!in!Petunia.!Vrije!Universiteit!Amsterdam.!

Li!W,!Zhou!Y,!Liu!X,!Yu!P,!Cohen!JD,!Meyerowitz!EM.!2013.!LEAFY!controls!auxin!response!pathways!in! floral!primordium!formation.!Sci!Signal!6,!ra23cr.!

Lloyd! AM,! Walbot! V,! Davis! RW.! 1992.! Arabidopsis! and! Nicotiana! anthocyanin! production! activated! by! maize!regulators!R!and!C1.!Science!258,!1773A1775.!

Long! JA,! Moan! EI,! Medford! JI,! Barton! MK.! 1996.! A! member! of! the! KNOTTED! class! of! homeodomain! proteins!encoded!by!the!STM!gene!of!Arabidopsis.!Nature!379,!66A69.!

Lutz! B,! Lu! HC,! Eichele! G,! Miller! D,! Kaufman! TC.! 1996.! Rescue! of! Drosophila! labial! null! mutant! by! the! chicken!ortholog!HoxbZ1!demonstrates!that!the!function!of!Hox!genes!is!phylogenetically!conserved.!Genes! &!Development!10,!176A184.!

Lynch!VJ,!Tanzer!A,!Wang!Y,!Leung!FC,!Gellersen!B,!Emera!D,!Wagner!GP.!2008.!Adaptive!changes!in!the! transcription!factor!HoxAA11!are!essential!for!the!evolution!of!pregnancy!in!mammals.!Proceedings!of!the! National!Academy!of!Sciences,!USA!105,!14928A14933.!

Maizel! A,! Busch! MA,! Tanahashi! T,! Perkovic! J,! Kato! M,! Hasebe! M,! Weigel! D.!2005.!The!floral!regulator! LEAFY!evolves!by!substitutions!in!the!DNA!binding!domain.!Science!308,!260A263.!

Mallo!M,!Wellik!DM,!Deschamps!J.!2010.!Hox!genes!and!regional!patterning!of!the!vertebrate!body!plan.! Developmental!Biology!344,!7A15.!

Mandel!MA,!Yanofsky!MF.!1995.!A!Gene!Triggering!Flower!Formation!in!Arabidopsis.!Nature!377,!522A524.!

Martin! C,! Ellis! N,! Rook! F.! 2010.! Do! transcription! factors! play! special! roles! in! adaptive! variation?! Plant! Physiology!154,!506A511.!

Miyamoto!MM,!Slightom!JL,!Goodman!M.!1987.!Phylogenetic!relations!of!humans!and!arican!apes!from! DNAAsequences!in!the!PsiAEtaAGlobin!region.!Science!238,!369A373.!

Molinero0Rosales! N,! Jamilena! M,! Zurita! S,! Gomez! P,! Capel! J,! Lozano! R.!1999.!FALSIFLORA,!the!tomato! orthologue! of! FLORICAULA! and! LEAFY,! controls! flowering! time! and! floral! meristem! identity.! The! Plant! Journal!20,!685A693.!

Morohashi!K,!Grotewold!E.!2009.!A!systems!approach!reveals!regulatory!circuitry!for!Arabidopsis!trichome! initiation!by!the!GL3!and!GL1!selectors.!PLoS!genetics!5,!e1000396.!

28! ! Evolution!of!morphological!diversity!

! Mouhu!K,!Kurokura!T,!Koskela!EA,!Albert!VA,!Elomaa!P,!Hytonen!T.!2013.!The!Fragaria!vesca!homolog!of! SUPPRESSOR!OF!OVEREXPRESSION!OF!CONSTANS1!represses!flowering!and!promotes!vegetative!growth.! The!Plant!Cell!25,!3296A3310.!

Navarro!C,!Abelenda!JA,!Cruz0Oro!E,!Cuellar!CA,!Tamaki!S,!Silva!J,!Shimamoto!K,!Prat!S.!2011.!Control!of! flowering!and!storage!organ!formation!in!potato!by!FLOWERING!LOCUS!T.!Nature!478,!119A122.!

Nesi!N,!Jond!C,!Debeaujon!I,!Caboche!M,!Lepiniec!L.!2001.!The!Arabidopsis!TT2!gene!encodes!an!R2R3!MYB! domain!protein!that!acts!as!a!key!determinant!for!proanthocyanidin!accumulation!in!developing!seed.!The! Plant!Cell!13,!2099A2114.!

Ohno!S.!1970.!Enormous!diversity!in!genome!sizes!of!fish!as!a!reflection!of!natures!extensive!experiments! with!gene!duplication.!Transactions!of!the!American!Fisheries!Society!99,!120A&.!

Pena!L,!Martin0Trillo!M,!Juarez!J,!Pina!JA,!Navarro!L,!Martinez0Zapater!JM.!2001.!Constitutive!expression! of!Arabidopsis!LEAFY!or!APETALA1!genes!in!citrus!reduces!their!generation!time.!Nature!Biotechnology!19,! 263A267.!

Piazza!P,!Bailey!C,!Cartolano!M,!Krieger!J,!Cao!J,!Ossowski!S,!Schneeberger!K,!He!F,!de!Meaux!J,!Hall!N,! Macleod!N,!Filatov!D,!Hay!A,!Tsiantis!M.!2010.!Arabidopsis!thaliana!leaf!form!evolved!via!loss!of!KNOX! expression!in!leaves!in!association!with!a!selective!sweep.!Curr!Biol!20,!2223A2228.!

Pin!PA,!Benlloch!R,!Bonnet!D,!Wremerth0Weich!E,!Kraft!T,!Gielen!JJ,!Nilsson!O.!2010.!An!antagonistic!pair! of!FT!homologs!mediates!the!control!of!flowering!time!in!sugar!beet.!Science!330,!1397A1400.!

Pose!D,!Yant!L,!Schmid!M.!2012.!The!end!of!innocence:!flowering!networks!explode!in!complexity.!Current! Opinion!in!Plant!Biology!15,!45A50.!

Quattrocchio!F,!Wing!J,!van!der!Woude!K,!Souer!E,!de!Vetten!N,!Mol!J,!Koes!R.!1999.!Molecular!analysis!of! the!anthocyanin2!gene!of!Petunia!and!its!role!in!the!evolution!of!flower!color.!The!Plant!Cell!11,!1433A1444.!

Quattrocchio!F,!Wing!JF,!van!der!Woude!K,!Mol!JNM,!Koes!R.!1998.!Analysis!of!bHLH!and!MYBAdomain! proteins:!speciesAspecific!regulatory!differences!are!caused!by!divergent!evolution!of!target!anthocyanin! genes.!The!Plant!Journal!13,!475A488.!

Ramsay! NA,! Glover! BJ.! 2005.! MYBAbHLHAWD40! protein! complex! and! the! evolution! of! cellular! diversity.! Trends!Plant!Science!10,!63A70.!

Rao!NN,!Prasad!K,!Kumar!PR,!Vijayraghavan!U.!2008.!Distinct!regulatory!role!for!RFL,!the!rice!LFY!homolog,! in! determining! flowering! time! and! plant! architecture.! Proceedings! of! the! National! Academy! of! Sciences,! USA!105,!3646A3651.!

Rijpkema!AS,!Royaert!S,!Zethof!J,!van!der!Weerden!G,!Gerats!T,!Vandenbussche!M.!2006.!Analysis!of!the! Petunia!TM6!MADS!box!gene!reveals!functional!divergence!within!the!DEF/AP3!lineage.!The!Plant!Cell!18,! 1819A1832.!

Ronshaugen!M,!McGinnis!N,!McGinnis!W.!2002.!Hox!protein!mutation!and!macroevolution!of!the!insect! body!plan.!Nature!415,!914A917.!

Rutledge!R,!Regan!S,!Nicolas!O,!Fobert!P,!Cote!C,!Bosnich!W,!Kauffeldt!C,!Sunohara!G,!Seguin!A,!Stewart! D.!1998.!Characterization!of!an!AGAMOUS!homologue!from!the!conifer!black!spruce!(Picea!mariana)!that! produces!floral!homeotic!conversions!when!expressed!in!Arabidopsis.!The!Plant!Journal!15,!625A634.!

!! 29! Chapter!2!

Sayou!C,!Monniaux!M,!Nanao!MH,!Moyroud!E,!Brockington!SF,!Thevenon!E,!Chahtane!H,!Warthmann!N,! Melkonian! M,! Zhang! Y,! Wong! GK,! Weigel! D,! Parcy! F,! Dumas! R.! 2014.! A! promiscuous! intermediate! underlies!the!evolution!of!LEAFY!DNA!binding!specificity.!Science!343,!645A648.!

Serrano! G,! Herrera0Palau! R,! Romero! JM,! Serrano! A,! Coupland! G,! Valverde! F.! 2009.! Chlamydomonas! CONSTANS!and!the!evolution!of!plant!photoperiodic!signaling.!Current!Biology!19,!359A368.!

Shu! GP,! Amaral! W,! Hileman! LC,! Baum! DA.!2000.!LEAFY!and!the!evolution!of!rosette!flowering!in!violet! cress!(Jonopsidium!acaule,!Brassicaceae).!American!Journal!of!Botany!87,!634A641.!

Sinha!NR,!Williams!RE,!Hake!S.!1993.!Overexpression!of!the!Maize!Homeo!Box!Gene,!KnottedA1,!Causes!a! Switch!from!Determinate!to!Indeterminate!Cell!Fates.!Genes!&!Development!7,!787A795.!

Sliwinski!MK,!White!MA,!Maizel!A,!Weigel!D,!Baum!DA.!2006.!Evolutionary!divergence!of!LFY!function!in! the!mustards!Arabidopsis!thaliana!and!Leavenworthia!crassa.!Plant!Molecular!Biology!62,!279A289.!

Song! YH,! Ito! S,! Imaizumi! T.! 2013.! Flowering! time! regulation:! photoperiodA! and! temperatureAsensing! in! leaves.!Trends!in!Plant!Science!18,!575A583.!

Souer!E,!Rebocho!AB,!Bliek!M,!Kusters!E,!de!Bruin!RAM,!Koes!R.!2008.!Patterning!of!inflorescences!and! flowers! by! the! FAbox! protein! DOUBLE! TOP! and! the! LEAFY! homolog! ABERRANT! LEAF! AND! FLOWER! of! Petunia.!The!Plant!Cell!20,!2033A2048.!

Spelt! C,! Quattrocchio! F,! Mol! J,! Koes! R.!2000.!anthocyanin1!of!Petunia!encodes!a!basicAHelix!Loop!Helix! protein!that!directly!activates!structural!anthocyanin!genes.!The!Plant!Cell!12,!1619A1631.!

Studer!A,!Zhao!Q,!Ross0Ibarra!J,!Doebley!J.!2011.!Identification!of!a!functional!transposon!insertion!in!the! maize!domestication!gene!tb1.!Nature!Genetics!43,!1160AU1164.!

Syed!NH,!Kalyna!M,!Marquez!Y,!Barta!A,!Brown!JWS.!2012.!Alternative!splicing!in!plants!A!coming!of!age.! Trends!in!Plant!Science!17,!616A623.!

Tanahashi! T,! Sumikawa! N,! Kato! M,! Hasebe! M.!2005.!Diversification!of!genie!function:!homologs!of!the! floral! regulator! FLO/LFY! control! the! first! zygotic! cell! division! in! the! moss! Physcomitrella! patens.! Development!132,!1727A1736.!

Tandre!K,!Svenson!M,!Svensson!ME,!Engstrom!P.!1998.!Conservation!of!gene!structure!and!activity!in!the! regulation!of!reproductive!organ!development!of!conifers!and!angiosperms.!The!Plant!Journal!15,!615A623.!

Taoka!K,!Ohki!I,!Tsuji!H,!Furuita!K,!Hayashi!K,!Yanase!T,!Yamaguchi!M,!Nakashima!C,!Purwestri!YA,!Tamaki! S,! Ogaki! Y,! Shimada! C,! Nakagawa! A,! Kojima! C,! Shimamoto! K.!2011.!14A3A3!proteins!act!as!intracellular! receptors!for!rice!Hd3a!florigen.!Nature!476,!332AU397.!

Taylor!S,!Hofer!J,!Murfet!I.!2001.!STAMINA!PISTILLOIDA,!the!pea!ortholog!of!FIM!and!UFO,!is!required!for! normal!development!of!flowers,!inflorescences,!and!leaves.!The!Plant!Cell!13,!31A46.!

Theissen!G.!2001.!Development!of!floral!organ!identity:!stories!from!the!MADS!house.!Current!Opinion!in! Plant!Biology!4,!75A85.!

Theissen!G,!Becker!A.!2004.!Gymnosperm!orthologues!of!class!B!floral!homeotic!genes!and!their!impact!on! understanding!flower!origin.!Critical!Reviews!in!Plant!Sciences!23,!129A148.!

Turck! F,! Coupland! G.! 2013.! Natural! variation! in! epigenetic! gene! regulation! and! its! effect! on! plant! developmental!traits!Evolution,!n/aAn/a.!

30! ! Evolution!of!morphological!diversity!

! Turck! F,! Fornara! F,! Coupland! G.! 2008.! Regulation! and! identity! of! florigen:! FLOWERING! LOCUS! T! moves! center!stage.!Annual!Review!of!Plant!Biology!59,!573A594.!

Vandenbussche! M,! Theissen! G,! Van! de! Peer! Y,! Gerats! T.! 2003.! Structural! diversification! and! neoA functionalization!during!floral!MADSAbox!gene!evolution!by!CAterminal!frameshift!mutations.!Nucleic!Acids! Res!31,!4401A4409.!

Vandenbussche!M,!Zethof!J,!Royaert!S,!Weterings!K,!Gerats!T.!2004.!The!duplicated!BAclass!heterodimer! model:!whorlAspecific!effects!and!complex!genetic!interactions!in!Petunia!hybrida!flower!development.!The! The!16,!741A754.!

Vollbrecht!E,!Veit!B,!Sinha!N,!Hake!S.!1991.!The!developmental!gene!KnottedZ1!Is!a!member!of!a!maize! homeobox!gene!family.!Nature!350,!241A243.!

Wang!H,!Chen!J,!Wen!J,!Tadege!M,!Li!G,!Liu!Y,!Mysore!KS,!Ratet!P,!Chen!R.!2008.!Control!of!compound!leaf! development!by!FLORICAULA/LEAFY!ortholog!SINGLE!LEAFLET1!in!Medicago!truncatula.!Plant!Physiol!146,! 1759A1772.!

Wang!Z,!Chen!J,!Weng!L,!Li!X,!Cao!X,!Hu!X,!Luo!D,!Yang!J.!2013.!Multiple!components!are!integrated!to! determine!leaf!complexity!in!Lotus!japonicus.!Journal!of!Integrative!Plant!Biology!55,!419A433.!

Weigel!D,!Nilsson!O.!1995.!A!developmental!switch!sufficient!for!flower!initiation!in!diverse!plants.!Nature! 377,!495A500.!

Weijers!D,!Jurgens!G.!2005.!Auxin!and!embryo!axis!formation:!the!ends!in!sight?!Curr!Opin!Genet!Dev!8,!32A 37.!

Wessinger!CA,!Rausher!MD.!2012.!Lessons!from!flower!colour!evolution!on!targets!of!selection.!Journal!of! Experimental!Botany!63,!5741A5749.!

Wigge!PA,!Kim!MC,!Jaeger!KE,!Busch!W,!Schmid!M,!Lohmann!JU,!Weigel!D.!2005.!Integration!of!spatial!and! temporal!information!during!floral!induction!in!Arabidopsis.!Science!309,!1056A1059.!

Wray! GA,! Levinton! JS,! Shapiro! LH.! 1996.! Molecular! evidence! for! deep! precambrian! divergences! among! metazoan!phyla.!Science!274,!568A573.!

Yamaguchi!N,!Wu!M0F,!Winter!C,!Berns!M,!Nole0Wilson!S,!Yamaguchi!A,!Coupland!G,!Krizek!B,!Wagner!D.! 2013.! A! molecular! framework! for! auxinAmediated! initiation! of! flower! primordia.! Developmental! Cell! 24,! 271A282.!

Yang! X,! Pang! HB,! Liu! BL,! Qiu! ZJ,! Gao! Q,! Wei! L,! Dong! Y,! Wang! YZ.! 2012.! Evolution! of! double! positive! autoregulatory! feedback! loops! in! CYCLOIDEA2! clade! genes! is! associated! with! the! origin! of! floral! zygomorphy.!The!Plant!Cell!24,!1834A1847.!

Yoon!HS,!Baum!DA.!2004.!Transgenic!study!of!parallelism!in!plant!morphological!evolution.!Proceedings!of! the!National!Academy!of!Sciences,!USA!101,!6524A6529.!

Yuan! YW,! Sagawa! JM,! Young! RC,! Christensen! BJ,! Bradshaw! HD,! Jr.!2013.!Genetic!dissection!of!a!major! anthocyanin! QTL! contributing! to! pollinatorAmediated! reproductive! isolation! between! sister! species! of! Mimulus.!Genetics!194,!255A263.!

Zhang!R,!Guo!CC,!Zhang!WG,!Wang!PP,!Li!L,!Duan!XS,!Du!QG,!Zhao!L,!Shan!HY,!Hodges!SA,!Kramer!EM,! Ren!Y,!Kong!HZ.!2013.!Disruption!of!the!petal!identity!gene!APETALA3Z3!is!highly!correlated!with!loss!of!

!! 31! Chapter!2! petals!within!the!buttercup!family!(Ranunculaceae).!Proceedings!of!the!National!Academy!of!Sciences,!USA! 110,!5074A5079.!

Zhao!JJ,!Lazzarini!RA,!Pick!L.!1993.!The!mouse!HoxA1.3!gene!is!functionally!equivalent!to!the!Drosophila!Sex! combs!reduced!gene.!Genes!&!Development!7,!343A354.!

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Changes(in(cis"regulatory*elements*of*a*key*floral*regulator*are* associated)with)divergence)of)inflorescence)architectures!

Serena&Della&Pina,&Elske&Kusters,&Rob&Castel,&Erik&Souer&and&Ronald&Koes& & & & & & & & & & & & & & & Published&in&Development&(2015),&142(16):2822B31& R.K.,&E.K.,&S.D.P.&and&E.S.&designed&experiments.&E.K.&and&S.D.P.&carried&out&the&majority&of& the& experiments.& E.K.,& S.D.P.,& R.C.& E.S.& and& R.K.& carried& out& experiments,& analyzed& and& interpreted&data.&S.D.P.&wrote&the&paper& & & DOT$cisBelements&and&inflorescence&architecture&

Abstract! Higher&plant&species&diverged&extensively&with®ard&to&the&moment&(flowering&time)&and& the& position& (inflorescence& architecture)& where& flowers& are& formed.& This& seems& largely& caused& by& variation& in& the& expression& patterns& of& conserved& genes& that& specify& floral& meristem& identity& (FMI),& rather& than& changes& in& the& encoded& proteins.& Here& we& report& a& functional& comparison& of& the& promoters& of& homologous& FMI& genes& from& Arabidopsis,& petunia,&tomato&and&Antirrhinum.&Analysis&of&promoterBreporter&constructs&in&petunia&and& Arabidopsis& and& complementation& experiments& showed& that& the& divergent& expression& of& LEAFY&(LFY)& and& the& petunia& homolog& ABERRANT$ LEAF$ AND$ FLOWER$ (ALF)& results& from& alterations& in& the& upstream& regulatory& network& rather& than& cisBregulatory& changes.& The& divergent&expression&of&UNUSUAL$FLORAL$ORGANS&(UFO)&from&Arabidopsis&and&the&petunia& homolog& DOUBLE$ TOP$ $ (DOT),& on& the& other& hand,& is& caused& by& the& loss& or& gain& of& cisB regulatory&promoter&elements,&which&respond&to&transBacting&factors&that&are&expressed&in& similar& patterns& in& both& species.& Introduction& of& pUFO:UFO$ causes& no& obvious& defects& in& Arabidopsis,&but&in&petunia&it&causes&the&precocious&and&ectopic&formation&of&flowers.&This& provides&an&example&of&how&a&change&in&a&cisBregulatory®ion&can&account&for&a&change&in& the&plant&body&plan.& & &

34& Chapter&2&& &

Introduction! Flowering&plants&(Angiosperms)&display&an&enormous&morphological&diversity&and,&because& many& species& are& amenable& to& genetic& analysis& and& transgenesis,& they& offer& excellent& possibilities&to&study&the&evolution&of&developmental&mechanisms&and&morphological&change& (Benlloch& et& al.,& 2007;& Castel& et& al.,& 2010;& Moyroud& et& al.,& 2010;& Della& Pina& et& al.,& 2014).& Angiosperms&differ&widely&with®ard&to&the&moment&(i.e.&the&season&and&age&of&the&plant)& they&switch&from&vegetative&growth&to&flowering,&as&well&as&the&number&of&flowers&that&are& formed&and&their&position&on&the&plant&body&(Weberling,&1989;&Castel&et&al.,&2010).&Distinct& species&may&form&solitary&flowers&or&inflorescences&that&bear&many&flowers&in&a&variety&of& different& patterns& (Rickett,& 1954).& Compound& inflorescences& are& divided& in& three& major& classes&depending&on&the&position&where&flowers&and&shoots&are&formed.&In&(open)&racemes,& the&apical&meristem&remains&undifferentiated&and&flowers&derive&from&lateral&meristems&that& form&at&its&periphery.&In&cymes,&flowers&are&formed&from&apical&meristems&and&inflorescence& growth&continues&from&lateral&meristems,&called&sympodial&inflorescence&meristems&(SIMs),& which& ultimately& will& also& acquire& floral& identity& after& having& formed& a& subsequent& lateral& SIM.& Panicles& go& through& several& rounds& of& producing& lateral& meristems& before& each& meristem&ends&in&a&flower&(Rickett,&1954;&Prusinkiewicz&et&al.,&2007;&Castel&et&al.,&2010).& Distinct& inflorescence& architectures& are& associated& with& differences& in& the& expression& patterns& of& floral& meristem& identity& (FMI)& genes& that& specify& floral& meristem& (FM)& fate& (Benlloch&et&al.,&2007;&Moyroud&et&al.,&2010).&In&racemose&inflorescences,&FMI&genes&such&as& LEAFY$(LFY)&and&APETALA$1$(AP1)&of&Arabidopsis$and&FLORICAULA$(FLO)$of&Antirrhinum&majus$ (snapdragon),&are&expressed&in&lateral&meristems,&which&consequently&develop&into&flowers,& but¬&in&the&apical&meristem,&which&therefore&remains&meristematic&(indeterminate)&(Coen& et&al.,&1990;&Huijser&et&al.,&1992;&Mandel&et&al.,&1992;&Weigel&et&al.,&1992).&Mutations&in&LFY& and/or&AP1&(partially)&convert&lateral&flowers&into&shoots&(Mandel&et&al.,&1992;&Weigel&et&al.,& 1992),& whereas& constitutive& expression& results& in& precocious& flowering& and& conversion& of& apical& meristems& into& flowers& (Mandel& and& Yanofsky,& 1995;& Weigel& and& Nilsson,& 1995).& Hence,&in&Arabidopsis,&the&time&and&place&where&flowers&form&are&primarily®ulated&via&the& transcription& of& LFY& and& its& direct& target& AP1& (Wagner& et& al.,& 1999;& Benlloch& et& al.,& 2007;& Moyroud&et&al.,&2010).&

35& DOT$cisBelements&and&inflorescence&architecture&

Also&in&species&with&cymose&inflorescences,&like&the&nightshades&(Solanaceae)&Nicotiana$ spp.&(tobacco),&Solanum$lycopersicum$(tomato)&and&Petunia$hybrida&(petunia),&LFY&homologs& specify&floral&identity&(Souer&et&al.,&1998;&MolineroBRosales&et&al.,&1999;&Ahearn&et&al.,&2001).& The&encoded&proteins&are&highly&similar&to&LFY&with®ard&to&their&sequences&and&functional& properties,&but&are&expressed&in&very&different&patterns&(Souer&et&al.,&1998;&MolineroBRosales& et& al.,& 1999;& Ahearn& et& al.,& 2001;& Maizel& et& al.,& 2005;& Souer& et& al.,& 2008).& In& tomato,& for& instance,&FALSIFLORA$(FA)&is&already&expressed&during&the&vegetative&phase&in&(incipient)&leaf& primordia& that& emerge& at& the& flanks& of& the& shoot& apical& meristem& (SAM)& and& during& reproductive&development&in&both&(apical)&FMs&and&the&SIMs&that&emerge&in&lateral&positions& (MolineroBRosales&et&al.,&1999).&In&petunia&inflorescences&the&LFYBhomolog&ABERRANT$LEAF$ AND$FLOWER&(ALF)&is&first&activated&in&the&apical&FM&and&with&a&slight&delay&in&the&lateral&SIM& (Souer& et& al.,& 1998).& However,& the& transcription& of& ALF& is& not& the& limiting& factor& that& determines& when& and& where& flowers& form& in& petunia,& because& (i)& ectopic& ALF& expression& does¬&trigger&the&formation&of&precocious&or&ectopic&flowers&and&because&(ii)&ALF,$like$FA& in&tomato,&is&expressed&during&the&vegetative&phase&in&leaf&primordia&(Souer&et&al.,&1998).& The&limiting&factor&that&controls&the&formation&of&flowers&in&petunia&is&DOUBLE$TOP&(DOT),& the&ortholog&of&Arabidopsis&UNUSUAL$FLORAL$ORGANS&(UFO)&(Souer&et&al.,&2008).&UFO&and& DOT&are&interchangeable&FBBox&protein&components&of&an&SCF&ubiquitinBligase&complex&that& binds&to&LFY&and&ALF&in&order&to&promote&the&transcription&of&downstream&genes&(Wang&et& al.,& 2003;& Chae& et& al.,& 2008;& Souer& et& al.,& 2008).& Mutation& of& DOT,& or& its& tomato& ortholog& ANANTHA&(AN),&leads&to&complete&loss&of&floral&identity&and&constitutive&expression&of&DOT& (or&UFO)&in&petunia&causes&the&precocious&formation&of&flowers&in&ectopic&positions.&In&these& species,&the&expression&of&DOT&and&AN&coincides&with&the&development&of&flowers,&both&in& time&and&in&place.&That&is,&during&the&vegetative&growth&both&genes&are&inactive&and&become& first&expressed&upon&the&onset&of&flowering.&Within&developing&flowers,&DOT$and&AN$mRNA& are&initially&seen&at&the&adaxial&side&of&the&first&emerging&sepal&primordium&as&a&stripe&that& expands&into&a&ring&as&the&other&sepals&primordia&initiate&(Souer&et&al.,&2008;&Castel,&2009)& (supplementary&material&Fig.&S1).&&& In& Arabidopsis,& however,& UFO& is& not& limiting& for& flower& formation& because& it& is& widely& expressed& from& the& embryonic& phase& onwards& in& virtually& all& aerial& meristems& (Lee& et& al.,& 1997;&Long&and&Barton,&1998)&and&its&ectopic&expression&does¬&alter&the&time&and&place& where&flowers&appear&(Lee&et&al.,&1997).&Souer&et$al.&postulated&that&the&time&and&place&of&

36& Chapter&2&& & simultaneous& expression& of& LFY& and& UFO& or& their& homologs& determine& the& flowering& time& and&position&of&flowers.&They&expected&that&alterations&in&the&expression&patterns&of&LFY&and& UFO& homologs& therefore& were& a& key& factor& for& the& divergence& of& flowering& time& and& inflorescence&architecture&(Souer&et&al.,&2008).&Consistent&with&this&idea,&the&simultaneous& constitutive&expression&of&LFY&and&UFO&or&ALF&and&DOT&results&in&a&similar&phenotype&in&both& Arabidopsis&and&petunia:&meristems&terminate&at&an&early&seedling&stage&in&structures&with& floral&characteristics&(Parcy&et&al.,&1998;&Souer&et&al.,&2008).& Curiously&the&expression&pattern&of&UFO&and&DOT&within&the&flowers&also&diverged,&albeit& both&genes&are&thought&to&have&similar&functions&in&the&development&of&floral&organs&and&in& the&activation&of&organBidentity&genes.&In&young&FMs&UFO&mRNA&is&expressed&throughout&the& meristem&dome,&overlapping&with&the&expression&of&organ&identity&genes,&and&in&later&stages& it&becomes&confined&to&the&petal/sepal&boundary.&DOT&mRNA,&on&the&other&hand,&is&never& expressed&in&the&FM¢er&(Lee&et&al.,&1997)&(supplementary&material&Fig.&S1).&& What& caused& the& changes& in& the& expression& patterns& of& these& FMI& genes,& however,& remains&unknown.&To&address&whether&the&FMI&gene&expression&patterns&were&altered&by& mutations&in&their&cisBregulatory&elements&(CREs)&and/or&by&changes&in&the&upstream&transB regulatory& network,& we& compared& the& activity& of& homologous& FMI& gene& promoters& from& Arabidopsis,&Antirrhinum,&tomato&and&petunia&by&swapping&them&between&Arabidopsis&and& petunia.&It&appeared&that&the&divergent&expression&of&LFY&homologs&is&caused&by&alterations& in& the& upstream& transBregulatory& network.& Conversely,& the& divergent& expression& of& UFO& homologs& is& due& to& alterations& in& cisBregulatory& elements,& which& make& these& genes& responsive& to& distinct& sets& of& transcription& factors& that& appear& largely& conserved& between& species.& &

37& DOT$cisBelements&and&inflorescence&architecture&

& &

Fig.!1.!Complementation!of!dot!by!pDOT3.1:DOT:tNOS,!pDOT4.6:DOT:t35S!and!pDOT4.6:DOT:tDOT.& (A)&Cymose&inflorescence&of&wild&type&petunia&showing&four&consecutive&flowers&(f1,&f2,&f3,&f4)&with& diagram& showing& the& reiteration& of& modular& sympodial& units& with& flowers& (red& dots)& and& (B)& dot& mutant&in&hybrid&W138/W115&background&with&diagram&showing&the&conversion&of&flowerBtoBshoot& (green&arrows).&(CBE)&Complementation&of&dot&by&pDOT$ 3.1:DOT:tNOS$(C)&producing&“green&flowers”& (green&dots&in&the&diagram),&pDOT4.6:DOT:t35S$(D)&with&partial&developed&flowers&(white&arrow&in&the& picture& and& green& dots& with& red& perimeter& in& the& diagram)& and& fully& complementation& of&& pDOT4.6:DOT:tDOT&(E)&respectively.&& &

Results!

The!promoter!regions!sufficient!for!correct!spatio"temporal!expression! To&study&the&genetic&basis&of&the&different&expression&patterns&of&homologous&FMI&genes&in& petunia$and$Arabidopsis,&we&compared&the&activity&of&their&promoters.&We&isolated&5’&nonB coding& regions& of& ALF& and& DOT& by& PCRBbased& methods,& and& tested& their& activity& by& complementation& experiments.& Therefore& we& fused& the& 2.8kb& ALF& promoter& (pALF)& and& 3.1kb&DOT&promoter&(pDOT3.1)&to&the&ALF&and&DOT&cDNAs,&and&introduced&the&pALF:ALF&and&

38& Chapter&2&& & pDOT3.1:DOT& genes& (supplementary& material& Fig.& S2)& in& petunia& alfW2167& and& dotA2232& null& mutants.& alf&and&dot&mutants&both&have&a&strong&flowerBtoBshoot&phenotype&(Fig.&1ABB&and&S3ABB),& and& their& apical& FMs& develop& as& a& SIM,& which& forms& another& sympodial& unit& instead& of& a& flower.& The& reiteration& of& this& process& results& in& a& green& bushy& structure& lacking& flowers& (Souer&et&al.,&1998;&Souer&et&al.,&2008).&In&two&out&of&nineteen&independent&transgenic&lines,& the&pALF:ALF&transgene&fully&complemented&the&alf&phenotype&(supplementary&material&Fig.& S3B).& In& other& alf& pALF:ALF& plants& the& mutant& phenotype& was& complemented& to& various& degrees.& In& eight& lines,& the& apical& meristem& terminated& in& an& imperfect& flower& having& sepaloid& organs& in& place& of& petals& and& stamens& (supplementary& material& Fig.& S3C).& The& remaining&nine&pALF:ALF$alf&lines&displayed&no&clear&rescue&of&the&mutant&phenotype&at&all.& Nevertheless,& the& two& fully& complementing& lines& showed& that& the& transgene& was& able& to& complement& the& mutant,& indicating& that& the& 2.8& kb& pALF& fragment& contains& sufficient& regulatory&information&for&wild&type&function&of&ALF.& In&pDOT3.1:DOT&dot&transformants&cymose&branching&was&restored&in&seven&out&of&fifteen& independent&lines:&apical&meristems&often&terminated&in&a&‘green&flower’&that&lacked&petals& and& stamens,& but& usually& had& a& wild& type& carpel,& and& occasionally& mosaic& organs& in& the& second&whorl&containing&sepaloid&and&petaloid&tissue&(Fig.&1C).&The&remaining&eight&plants& showed&no&complementation&of&the&dot&phenotype.&& By&extending&the&promoter®ion&with&another&1.5kb&(pDOT4.6:DOT)&the&rescue&of&the&dot& organ& identity& defect& clearly& improved& (Fig.& 1D).& In& eight& of& fifteen& independent& transformants& the& cymose& branching& was& restored& similar& to& dot$ pDOT3.1:DOT,& but,& in& addition,&partially&complete&flowers&with&sepaloid&and&petaloid&tissues&and&fertile&stamens& and&carpel&were&produced.&The&remaining&seven&plants&showed&no&complementation.&This& enhanced&but&still&incomplete&complementation&suggested&that&some®ulatory&elements& were&still&missing.&We&then&replaced&the&t35S&terminator,&which&is&routinely&used&in&many& constructs& (Karimi& et& al.,& 2002),& with& 1& kb& of& the& 3’& flanking& region& of& DOT&(tDOT)& and& repeated& the& complementation.& In& eight& of& fifteen& independent& dot& pDOT4.6:DOT:tDOT$ transformants&both&the&cymose&branching&and&floral&organ&development&were&fully&restored& (Fig.&1E).&Four&lines&showed&partial&complementation,&that&is&complete&restoration&of&cymose& branching& but& formation& of& imperfect& flowers& similar& to& those& described& above& in& transformants& containing& pDOT4.6:DOT:t35S.$ The& remaining& 3& plants& were& not&

39& DOT$cisBelements&and&inflorescence&architecture& complemented.& These& results& indicated& that,& although& the& 3.1& kb& pDOT3.1& fragment& was& sufficient&to&restore&the&floral&meristem&identity&and&the&cymose&inflorescence&architecture,& it&was&unable&to&restore&all&organ&identity&defects&of&dot&mutants.&The&flower&phenotype&of& the&mutant&plants&that&were&complemented&best&is&similar&to&that&of&dot$35S:DOT&(Souer&et& al.,&2008).&The&addition&of&1.5&kb&of&5’&promoter®ion&improved&the&dot&complementation& compared&to&pDOT3.1,&but&the&full&restoration&of&all&dot&defects&was&reached&only&when&we& added&1&kb&of&3’®ion&as&well.& The&2.3&kb®ion&upstream&of&the&translation&start&codon&of&LFY&(pLFY)&used&in&this&study,& when&fused&to&the&LFY&cDNA,&was&able&to&rescue&the&strong&lfyB26&mutant&(Blazquez&et&al.,& 1997),&and&the&3.8&kb&UFO&promoter&(pUFO)&drives&GUS&expression&in&a&pattern&identical&to& that& of& endogenous& UFO& (Lee& et& al.,& 1997).& This& indicates& that& these& promoter& regions& contain&all®ulatory&sequences&necessary&for&promoter&swap&studies.&&

Functional!comparison!of6pALF!and!pLFY!! Divergent& expression& of& two& homologous& genes& can& be& due& to& changes& in& cisBregulatory& regions.&If&so,&those&promoters&will&maintain&their&specific&expression&pattern®ardless&of& the&host.&Alternatively,&the&divergence&in&expression&pattern&can&be&due&to&changes&in&transB regulatory& factors,& in& which& case& the& expression& pattern& of& a& given& promoter& will& be& dependent&on&the&host.& To&determine&which&is&the&case&for&LFY&and&ALF&we&fused&the&pALF&and&pLFY&fragments& described& above& to& the$ coding& sequence& of& the& reporter& gene& βBglucuronidase$ (GUS,& supplementary& material& Fig.& S2)& (Jefferson& et& al.,& 1987)& and& stably& transformed& the& constructs& to& both$ Arabidopsis& and& petunia.& For& each& of& the& construct& and& species& ten& independent&transformants&were&examined&using&histochemical$GUS&staining.&Although&the& expression& level& varied& between& distinct& transformants,& the& expression& pattern& was& highly& similar&& We&observed&that&pALF:GUS&and&pLFY:GUS&are&expressed&in&seemingly&identical&patterns& during& vegetative& growth& both& in& a& petunia& and& in& an& Arabidopsis& background.& That& is,& emerging& petunia& and& Arabidopsis& leaves& expressed$ GUS,& which& quickly& faded& when& the& leaves&grew&older&(Fig.&2ABD).&We&could&observe&pLFY:GUS&expression&from&the&third&leaf&on& in& Arabidopsis,& but& could& not& discern& the& gradual& increase& of& pLFY:GUS$ expression& during& vegetative& development& (Blazquez& et& al.,& 1997).& However,& the& rather& small& quantitative&

40& Chapter&2&& & changes&involved&are&difficult&to&distinguish&by&histochemical&staining,&in&particular&because& the&vegetative&phase&lasted&rather&short&under&the&long&day&conditions&that&we&used.& & & Fig.! 2.! pALF:GUS! and! pLFY:GUS! expression! patterns!in!petunia!and!Arabidopsis.& (A,& B)& In& young& petunia& (Ph)& and& (C,& D)& Arabidopsis&(At)& seedlings& pALF:GUS& and& pLFY:GUS&are&active&in&emerging&leaves,&but¬& in& the& vegetative& meristem.& (EBH)& In& the& reproductive&stage,&pALF:GUS&(E)&and&pLFY:GUS$ (F)$ stained& both& sympodial& inflorescence& and& young& flowers& of& petunia,& while& in& Arabidopsis$ (G,& H)$ they& were& both& expressed& in& FM,& but& excluded& from& the& apical& inflorescence& meristem.&Dashed&lines&indicate&the&outlines&of& organs& that& are& poorly& visible,& subsequent& flowers&are&indicated&from&young&to&old&(f1,&f2).& C,&cotyledon;&*,&sympodial&(E,&F)&or&apical&(G,&H)& meristem;&fm,&floral&meristem;&b,&bract;&s,&sepal.$ & & & & & & & Analysis& of& pALF:GUS& and& pLFY:GUS& plants& after& the& switch& to& flowering& (Fig.& 2EBH)& showed&that&&in&a&petunia&inflorescence&both&transgenes&were&expressed&in&a&similar&pattern& as&the&endogenous&ALF&gene,&while&in&an&Arabidopsis&inflorescence&their&expression&patterns& were&similar&to&that&of&LFY.&In&petunia,&both&promoters&are&highly&active&in&the&apical&FMs,& and&also&in&the&SIMs&(Fig.&2EBF).&The&slightly&delayed&expression&of&endogenous&ALF&in&SIMs&as& compared&to&the&apical&FM&&(Souer&et&al.,&1998)&was¬&observed&with&the&limited&resolution&

41& DOT$cisBelements&and&inflorescence&architecture& of& GUS& assays.& This& is& not& surprising& because& (i)& the& SIM& emerges& as& a& very& small& region& between& the& bract& and& the& apical& FM,& which& both& express& ALF,& and& because& (ii)& ALF& expression&in&the&SIM&is&only&briefly&delayed&(Souer&et&al.,&1998;&Castel&et&al.,&2010).&& In& Arabidopsis& inflorescences,& however,& expression& of& pLFY:GUS& and& pALF:GUS& was& restricted& to& lateral& FMs& (Fig.& 2GBH),& the& only& difference& being& that& pALF:GUS& expression& faded& more& quickly& than& pLFY:GUS$ activity& does& during& development& of& the& flower.& More& importantly,&pALF:GUS&and&pLFY:GUS&are&never&active&in&the&apical&meristems&of&Arabidopsis,& similar&to&endogenous&LFY.&& These&data&indicate&that&pALF&and&pLFY&are&functionally&very&similar,&indicating&that&the& ALF&and&LFY&expression&patterns&in&their&cognate&host&diverged&through&changes&in&upstream& transBregulatory&factors.&&

Functional!heterologous!complementation!! To&obtain&further&evidence&that&pALF&and&pLFY&are&also&functionally&similar,&we&performed& functional& assays& in& transgenic& plants.& Previous& results& revealed& that& the& ALF& and& LFY& proteins&are&functionally&similar&and&interchangeable&(Maizel&et&al.,&2005;&Souer&et&al.,&2008).& In& Arabidopsis& ectopic& expression& of& either& LFY& or& ALF& triggers& precocious& flowering& and& transforms&the&apical&IM&into&a&FM,&which&converts&the&open&raceme&into&a&solitary&flower& (or&a&closed&raceme)&(Weigel&and&Nilsson,&1995;&Souer&et&al.,&2008).&This&predicts&that&if&ALF& (trans)gene&would&introduced&in&Arabidopsis&and&pALF&is&active&in&a&(slightly)&wider&spatioB temporal&expression&pattern&than&pLFY,&this&should&result&in&an&alteration&of&flowering&time& and/or&inflorescence&architecture.&Hence,&we&introduced&promoter:cDNA&fusions&(pLFY:LFY$ and& pALF:ALF)& into& wild& type$ Arabidopsis$ Columbia.& We& found& that& none& of& the& twenty& primary& transformants& analyzed& for& each& construct& displayed& aberrations& in& the& inflorescence& architecture& or& flowering& time& (supplementary& material& Fig.& S4ABC).& This& underlines& that& in& Arabidopsis& pALF& is& not& expressed& ectopically& (e.g.& in& apical& IM& or& the& vegetative& SAM)& when& compared& to& pLFY.& The& same& reasoning& predicts& that& if& pLFY& were& expressed&in&a&more&restricted&pattern&than&pALF,&when&introduced&in&a&petunia,&a&pLFY:LFY& transgene& would& not& be& able& to& fully& rescue& alf& mutants.& Therefore& we& transformed& alf& mutants&with&a&pLFY:LFY&transgene&(supplementary&material&Fig.&S5).&We&found&that&in&six& out& of& twelve& independent& pLFY:LFY& alf& lines& floral& identity& of& the& apical& meristem& was& restored,& resulting& in& a& normal& cymose& architecture.& Two& of& these& pLFY:LFY$ lines& had&

42& Chapter&2&& & abberant&&“green&flowers”&with&supernumerary&whorls&containg&only&sepals&(supplementary& material&Fig.&S5C&and&F);&two&other&lines&had&flowers&with&a&correct&pattern&of&floral&pattern,& but&the&third&whorl&consisted&of&petaloid&stamens&(supplementary&material&Fig.&S5D&and&G);& while& two& transformants& had& perfect& wild& type& flowers& except& for& some& small& sections& of& petal& tissue& on& the& stamens& (supplementary& material& Fig.& S5H).& These& findings& provide& further& support& that& in& petunia& pLFY& is& indeed& active& in& the& appropriate& regions& to& compensate&for&the&loss&of&ALF&activity.&

!Functional!comparison!of6pDOT6and6pUFO66 A& GUS& reporter& gene& driven& by& pDOT3.1& showed& no& expression& during& the& seedling& stage,& neither& in& petunia& nor& in& Arabidopsis& (Fig.& 3ATB),& showing& that& pDOT3.1& reproduces& the& expression& of& the& parental& gene,& independently& of& the& host& plant& species.& The& same& was& observed&for&pDOT4.6:GUS&(Fig.&3CBD).& In&the&petunia&inflorescence,&pDOT3.1:GUS&was&expressed&only&within&the&apical&FMs&on& the&adaxial&side&of&the&sepal&primordia,&but&never&in&the¢er&of&FMs,&nor&in&the&emerging& SIM&(Fig.&4A).&In&pDOT4.6:GUS&transformants,$GUS&expression&was&much&stronger&and&stained& the&entire&FM,&whereas&no&expression&at&all&was&seen&in&the&emerging&SIM&(Fig.&4B).&Even& when&stained&briefly&(30&minutes),&a&strong&GUS&signal&was&seen&in&the&entire&flower&dome.& However,&when&we&analyzed&pDOT4.6:GUS&expression&by&in&situ&hybridization,&we&observed& GUS& mRNA& only& at& the& sepal& petal& boundary& (Fig.& 4C).& This& suggests& that& the& strong& GUS& activity&seen&in&the¢er&of&the&FM&does¬&reflect&the&GUS&mRNA&expression&pattern,&but& results&from&intercellular&movement&of&the&GUS&enzyme&or&XBgluc&reaction&product,&which& becomes& more& evident& at& high& GUS& expression& levels.& Evidently& the& extra& promoter& sequences& included& in& the& pDOT4.6$ construct& contain& some& enhancers& that& drastically& increase&its&activity&compared&to&pDOT3.1,&without&altering&its&expression&pattern.&

& & & &

43& DOT$cisBelements&and&inflorescence&architecture&

& Fig.! 3.! Expression! of! pDOT3.1:GUS,! DOT4.6:GUS! and!pUFO:GUS!in!vegetative!meristems.!! (ABD)& Transgenic& petunia& (Ph)& and& Arabidopsis$ (At)& seedlings& do& not& express& pDOT3.1:GUS& or& pDOT4.6:GUS.& (E,& F)& pUFO:GUS& is& expressed& in& the& SAM& of& young& petunia& and& Arabidopsis& seedlings&respectively.&Dashed&lines&outline&the& youngest&visible&leaves.&C,&cotyledon.& & & & & & & & & & & In& an$ Arabidopsis$ background,& pDOT3.1:GUS& was& expressed& in& lateral& FMs& and& excluded& from& the& apical& meristem& (Fig.& 4EBF).& In& weak& pDOT4.6:GUS$ expressors,& GUS& activity& was& restricted&to&the&FM&exactly&at&the&sepals/petal&boundary&and&was&never&seen&in&the&IM&(Fig.& 4G).& In& strong& expressors& GUS& signal& was& present& in& both& apical& inflorescence& and& lateral& flower&meristem,&in&the&whole&dome&rather&then&in&a&ring&shape&(supplementary&material&Fig.& S7A).& The& different& results& between& weak& versus& strong& expressors& probably& reflect& a& quantitative& difference& in& GUS& staining& rather& than& qualitative.& Indeed,& detection& of& GUS& mRNA& by& in& situ& hybridization& of& strong& expressors& showed& that& expression& was& largely& restricted&to&the&sepal/petal&primordia&boundary&(supplementary&material&Fig.&S7B).& We&tried&to&perform&the&same&analysis&in&petunia&and&Arabidopsis&with&pDOT4.6:GUS:tDOT,& as&the&DOT&cDNA&fused&to&the&same®ulatory&element&completely&complemented&the&dot& phenotype.$However,&among&90&stable&petunia&transformants,&which&were&generated&in&4& independent& transformation& experiments,& none& showed& any& GUS& expression,& while& 32&

44& Chapter&2&& & transformants&had&the&typical&dot&loss&of&function&phenotype&indicating&that&the&transgene(s)& silenced& the& endogenous& DOT& gene& and& itself.& When& we& transformed& the& same& construct& into& Arabidopsis,& none& of& the& 30& independent& transformants& showed& any& GUS& expression,& but&mutant&(ufo)&phenotypes&were¬&seen.&This&suggests&that,&for&unknown&reasons,&the& pDOT4.6:GUS:tDOT$constructs&triggers&RNA&interference&at&high&frequency& &

& Fig.!4.!Expression!of!pDOT3.1:GUS,!pDOT4.6:GUS!and!pUFO:GUS!in!the!inflorescences.! (A)&pDOT3.1:GUS&in&petunia&(Ph)&inflorescence.&GUS&expression&is&first&seen&as&a&stripe&at&the&base&of& the&first&emerging&sepal&primordium&and&at&a&later&stage,&when&all&sepal&primordia&are&visible,&as&a& pentagon.&(B)&pDOT4.6:GUS&petunia&inflorescence.&GUS&activity&is&seen&in&the&entire&FM,&but¬&in&the& SIM&(*).&(C)&In&situ&hybridization&of&a&pDOT4.6:GUS& petunia&inflorescence§ion&showing&that&GUS& mRNA& is& restricted& to& the& sepal/petal& boundary.& (D)& pUFO:GUS& in& petunia& inflorescence& showing& expression&in&both&SIM&(*)&and&young&flower&primordia&(f1,&f2).&(E)&Side&view&and&(F)&top&view&of&an& Arabidopsis$(At)&pDOT3.1:GUS&inflorescence:&GUS&signal&was&observed&only&in&FM.&(G)&In&Arabidopsis& the&expression&of&pDOT4.6:GUS&is&expressed&between&sepals&end&petals&of&FMs&but&is&excluded&from& the& apical& IM.& (H)& In& flowering& pUFO:GUS& Arabidopsis,& both& the& apical& IM& (*)& and& young& flowers& primordia& strongly& expressed& GUS.& In& older& flowers& blue& staining& was& confined& to& the& base& of& the& flower.&Dashed&lines&indicate&the&outlines&of&organs&that&are&poorly&visible,&subsequent&flowers&are& indicated& from& young& to& old& (f1,& f2).& *,& sympodial& (A,& B,& D)$ or& apical& (EBH)$ meristem;& fm,& floral& meristem;&b,&bract;&s,&sepal.& &

45& DOT$cisBelements&and&inflorescence&architecture&

In&Arabidopsis,&UFO&mRNA&is&already&expressed&in&heart&stage&embryos&(Long&and&Barton,& 1998)&and&persists&in&seedlings&in&a&cupBshaped&domain&surrounding&the¢ral&part&of&the& SAM&(Lee&et&al.,&1997).&When&fused&to&GUS,&pUFO&was&already&active&during&embryogenesis& of&petunia&in&a&ring&around&the&root&meristem&and&in&the&apical&meristem&(supplementary& material& Fig.& S6)& and& in& the& seedling& stage& pUFO& remained& active& in& the& vegetative& SAMs& both&in&an&Arabidopsis&and&petunia&background&(Fig.&3EBF).&This&means&that&pUFO&reproduces& during&the&vegetative&phase&the&expression&of&the&parental&gene&from&which&it&is&derived,& irrespective& of& the& host& plant& species. Within& the& petunia& inflorescence& pUFO:GUS& was& strongly&expressed&in&both&the&IM&and&FM&(Fig.&4D)&and&in&Arabidopsis&in&both&the&apical&IM& and&lateral&FMs,&similar&to&UFO&(Fig.&4H).&Moreover,&we&observed&that&in&all&Arabidopsis&and& petunia& pUFO:GUS& transformants& (i.e.& in& both& weak& and& strong& expressors)& GUS& activity& localized&in&young&FMs&throughout&the&entire&meristem&dome,&while&in&expanding&flowers& the&signal&faded&from&the¢er&(Fig.&4D,&H).&These&data&imply&that&the&divergent&expression& of&DOT&and&UFO&within&FMs&also&results&from&differences&in&their&promoters.& Summarizing,&pDOT3.1:GUS,$pDOT4.6:GUS&and&pUFO:GUS&largely&recapitulate&the&divergent& expression&patterns&of&the&corresponding&endogenous&DOT&and&UFO$genes®ardless&of&the& host&species.&This&implies&that&their&different&expression&patterns&in&vegetative&meristems,& IMs&and&FMs&are&caused&by&alterations&in&their&CREs&after&their&divergence&from&their&last& common&ancestor.&

Functional!heterologous!complementation!! To&obtain&direct&evidence&that&the&changes&in&the&CREs&of&pUFO&and&pDOT&are&important&for& the& divergent& racemose& and& cymose& inflorescence& architectures,& we& performed& functional& assays& and& introduced& promoter:cDNA& constructs& (pUFO:UFO$ and& pDOT3.1:DOT)& into& Arabidopsis&and&petunia.&& Based& on& the& above& results& and& because& UFO& and& DOT& encode& functionally& interchangeable&proteins&(Souer&et&al.,&2008)&we&expected&that&pDOT3.1:DOT,&that&is&sufficient& to&restore&inflorescence&architecture&in&petunia,&would¬&alter&flowering&time&in&wild&type& Arabidopsis,& while& pUFO:UFO& would& cause& precocious& flowering& and& inflorescence& architecture&defects&in&wild&type&petunia.& & &

46& Chapter&2&& &

& & Fig.!5.!pUFO:UFO!converts!the!cymose!petunia!inflorescence!to!a!single!flower.! (A)& Wild& type& petunia& plant& during& the& vegetative& phase,& with& diagram& showing& the& production& of& leaves&and&(B)&pUFO:UFO&transformant&of&the&same&age,&which&flowers&early&and&produces&a&terminal& flower& (red& dot& in& the& diagram).& (C)& Cymose& inflorescence& of& wild& type& petunia,& showing& three& consecutive&flowers&(f1,&f2,&f3)&with&diagram&showing&the&reiteration&of&modular&sympodial&units&with& flowers& (red& dots)& .& (D)& In& pUFO:UFO& petunia,& the& cymose& inflorescence& was& reduced& to& a& solitary& flower&with&extra&organs&(red&dot&in&the&diagram).&(E)&Double&transgenic&35S:LFY$pUFO:UFO&petunia& flowers& extremely& early,& after& forming& two& true& leaves.& The& first& whorl& contains& petaloid& sepals& (arrowhead).& & About&twenty&primary&Arabidopsis&transformants&of&each&construct&were&investigated&and& none&of&them&showed&any&aberrant&phenotypical&features&(supplementary&material&Fig.&S4AB B)&or&altered&flowering&time&as&determined&by&counting&of&the&leaves&compared&to&empty& vector&transgene&controls&(supplementary&material&Fig.&S4C).&By&contrast,&the&introduction& pUFO:UFO&in&wild&type&petunia&always&resulted&in&plants&with&an&early&flowering&phenotype& (Fig.&5ABB).&Moreover,&the&cymose&inflorescence&(Fig.&5C)&was&converted&into&a&single&flower& with&supernumerary&petals&and&stamens&subtended&by&extra&leafBlike&organs&(bracts)&directly& under& the& sepal& whorl& (Fig.& 5D& and& S8ABB).& When& pUFO:UFO& was& transformed& into& a& dot& background,& a& single& “green& flower”& was& formed& as& early& as& in& a& wild& type& background& (supplementary&material&Fig.&S8CBD).&However,&in&a&dot&background&this&flower&consisted&of&

47& DOT$cisBelements&and&inflorescence&architecture& whorls&of&sepals&around&a¢ral&carpel&lacking&petals&and&stamens&(supplementary&material& Fig.&S8D&inset);&this&indicates&that&pUFO$could¬&drive&transgene&expression&at&sufficiently& high& levels& during& late& stages& of& FM& development,& when& floral& organs& are& formed.& When& pUFO:UFO& transformants& were& crossed& to& plants& constitutively& expressing& LFY,& the& precocious&flowering&was&enhanced&(Fig.&5E).&&

ANANTHA6and!FIMBRIATA6activity!in!petunia!and!Arabidopsis! Arabidopsis& and& petunia& are& quite& distantly& related& species& that& belong& to& the& Rosids& and& & respectively.& To& study& when& the& alterations& in& pUFO& and& pDOT& occurred,& we& analyzed&the&promoters&from&DOT&homologs&of&Antirrhinum&(FIMBRIATA,$FIM)&and&tomato& (ANANTHA,$ AN),& which& are& both& Asterids.& Antirrhinum& is& member& of& the& Plantaginaceae& (order&Lamiales)&and&has&a&racemose&inflorescence,&while&tomato&belongs,&like&petunia,&to& the&Solanaceae&(order&).& AN& is& in& tomato& essential& to& establish& FM& identity& and& is& expressed& in& a& pattern& very& similar&to&DOT&in&petunia&(Lippman&et&al.,&2008).&During&the&vegetative&stage&pAN:GUS&did& not& show& any& activity& in& a& petunia& or& Arabidopsis& background& (Fig.& 6ABB),& identical& to& the& native&expression&pattern&of&AN$in&tomato&(Lippman&et&al.,&2008).&In&petunia&inflorescences,& the&expression&of&pAN:GUS$was&similar&to&that&of&pDOT4.6.&That&is,&in&low&pAN:GUS$expressors& GUS& activity& was& seen& in& a& pentagonal& domain& on& the& sepals/petals& boundary& (supplementary&material&Fig.&S9A),&in&strong&expressors&it&was&seen&in&the&entire&FM,&and&in& both&cases&it&was&never&seen&in&the&SIM&(Fig.&6E).&In&weak&Arabidopsis$expressors&pAN:GUS$ expression&was&visible&in&the&FM&in&the&sepals/petals&boundary&and¬&in&the&apical&IM&(Fig.& 6F).&In&strong&expressors&GUS&activity&stained&both&FM&and&IM&(supplementary&material&Fig.& S9B).$ In& Antirrhinum& we& found& that& FIM& is& already& expressed& during& the& vegetative& phase& (supplementary&material&Fig.&S10)&and&during&reproductive&growth&its&expression&is&restrict&to& the&(lateral)&FM&and&excluded&from&the&apical&IM&(Simon&et&al.,&1994).&In&petunia&seedlings& we& never& observed& expression& of& pFIM:GUS& (Fig.& 6C),& whereas& in& Arabidopsis& seedlings& pFIM:GUS& was& expressed& at& the& base& of& newly& formed& leaves& (Fig.& 6D).& The& expression& pattern& of& pFIM:GUS& in& inflorescences& was& highly& similar& to& that& of& pDOT4.6:GUS.& In& weak& petunia&pFIM:GUS&expressors&we&observed&GUS&activity&in&emerging&flower&primordia&first&as& stripe& at& the& base& of& incipient& sepals& (supplementary& material& Fig.& S9C)& and& slightly& later,&

48& Chapter&2&& & when&all&the&sepal&primordia&were&visible,&as&a&ring&in&the&flower&dome&(Fig.&6G).&In&strong& expressors& GUS& activity& stained& the& whole& flower& dome,& including& the& FM& center& (supplementary& material& Fig.& S9D).& However,& we& never& observed& GUS& activity& in& the& emerging& inflorescence& meristem.& Also& in& the& Arabidopsis& inflorescence& pFIM:GUS& expression& was& confined& to& the& typical& ring& pattern& at& the& sepal/petal& boundary,& and& was& never&observed&in&the&FM¢er&or&in&the&apical&meristem&(Fig.&6H),&similar&to&DOT4.6:GUS.& In&summary,&the®ulatory&sequences&in&pAN&are&(functionally)&undistinguishable&from&to& those&in&pDOT,&whereas&those&in&pFIM&are&very&similar&but¬&fully&identical&because&pFIM& responds& to& transcription& activators& in& the& base& of& young& Arabidopsis& leaves,& while& pDOT& does¬.& &

& & Fig.!6.!Expression!of!pAN:GUS!and!pFIM:GUS!in!petunia!and!Arabidopsis.!! (ABD)& Expression& in& the& vegative& phase.& (A)& pAN:GUS$ is& not& expressed& in& petunia& (Ph)& or& (B)& Arabidopsis&(At)&seedlings.&(C)&pFIM:GUS&is&inactive&in&petunia&seedlings,&(D)&while&in&Arabidopsis&is& expressed&at&the&base&of&young&leaves&(arrow).&(EBH)&Expression&in&reproductive&phase.&(E)&pAN:GUS$ expression&was&detected&in&the&entire&FM&of&petunia&and&(F)&in&Arabidopsis$in&a&pentagon&pattern&in& sepals/petals& boundary.& (G)& Petunia& expresses& pFIM:GUS$ in& floral& meristems& at& the& sepal/petal& boundary,& but& not& in& the& SIM& (*).& (H)& Arabidopsis& expresses& pFIM:GUS& in& floral& meristems,& at& the& sepal&boundary,&but¬&in&the&apical&IM&(*).&C,&cotyledons;&*,&sympodial&(E,&G)&or&apical&(F,&H)&&

49& DOT$cisBelements&and&inflorescence&architecture&

Discussion! Differences& in& the& spatioBtemporal& regulation& of& meristem& identity& genes& caused& the& divergence& of& inflorescences& with& regard& to& the& positions& where& flowers& and& shoots& are& formed&(Benlloch&et&al.,&2007;&Lippman&et&al.,&2008;&Souer&et&al.,&2008).&Here&we&show&that& the& modification& of& FMI& gene& expression& patterns& results& from& variation& in& their& transcriptional®ulation&due&to&alterations&in&CREs&of&FMI&genes&as&well&as&in&the&upstream& regulatory&genetic&network.&& Our&data&suggest&that&CREs&involved&in&the&transcriptional&activation&of&DOT&reside&in&both& the&3’&and&5’&flanking®ions&of&the&gene.&The&3.1&kb&promoter&(pDOT3.1)&fragment&contains& major&CREs&that&are&sufficient&to&reproduce&the&DOT&expression&pattern&and&to&rescue&FM& identity&when&fused&to&the&DOT&coding&sequence,&but¬&the&identity&of&petals&and&stamens& within& the& developing& flower.& The& phenotype& of& weak& dot& mutants& indicates& that& petal& development& is,& of& all& DOTBregulated& processes,& the& most& dependent& on& full& DOT& activity,& and& is& associated& with& an& extremely& high& abundance& of& DOT& mRNA& in& the& cells& at& the& sepal/petal&boundary&(Souer&et&al.,&2008).&Given&that&pDOT3.1&is&active&in&the&correct&pattern,& its&inability&to&drive&petal&development&is&most&likely&due&to&an&insufficiency&to&drive&the&very& strong&expression&needed&for&petal&development,&rather&than&a&shortcoming&in&the&pattern& of& expression.& Indeed,& expression& of& DOT& in& a& wider& pattern,& either& from& pUFO$ (supplementary&material&Fig.&S8D)&or&p35S&(Souer&et&al.,&2008),&also&results&in&“green&flowers”& lacking&petal&and&stamen&identity.&By&contrast&expression&from&pDOT4.6,&which&is&expressed&in& the&same&pattern&as&pDOT3.1&but&at&much&higher&levels,&leads&to&partial&rescue&of&petal&and& stamen& identity& and,& if& combined& with& the& 3’& flanking& sequence& of& DOT& instead& of& t35S$ fragment,&to&full&rescue.&These&findings&suggest&that&the&CREs&in&the&distal&promoter®ion&(B 3000&to&B4600)&are&largely&redundant&with&those&in&the&proximal®ion&B1&to&B3000.&This&is&in& line&with&recent&functional&data&obtained&with&a&larger&set&of&pDOT&constructs&(S.&Della&Pina,& E.&Souer&and&R.&Koes,&unpublished&data).&The&same&may&hold&true&for&the&3’&flanking®ion&of& DOT,&although&we&cannot&exclude&that&(part&of)&the&effect&of&tDOT&results&from&enhanced& mRNA&processing&and/or&stability.& The&most&obvious&difference&between&DOT&and&UFO&is&that&the&latter&is&expressed&in&all& meristems& during& embryogenesis,& vegetative,& and& reproductive& growth,& whereas& DOT& expression&is&restricted&to&a&defined®ion&in&FMs.&That&pDOT:GUS&and&pUFO:GUS&reproduce& these& different& expression& patterns,& regardless& of& the& host& plant& used,& indicates& that& the&

50& Chapter&2&& & divergent&expression&of&DOT&in&petunia&and&UFO&in&Arabidopsis&is&caused&by&differences&in& the& CREs& within& their& 5’& flanking& sequences.& Furthermore,& it& indicates& that& the& CREs& that& activate& pUFO& in& the& apical& meristem& (SAM)& of& embryos,& vegetative& seedlings& and& inflorescences& (IM)& respond& to& conserved& transcription& factors& that& are& expressed& in& the& same&tissues&in&petunia.&& The& finding& that& in& petunia,& but& not& in& Arabidopsis,& the& pUFO:UFO$ transgene& causes& precocious& flowering& and& the& formation& of& solitary& flowers& provides& direct& evidence& that& alterations&in&the&CREs&of&a&single&gene&may&impinge&major&architectural&differences.&It&is,& however,& difficult& to& directly& link& morphological& changes& to& the& regulatory& divergence& of& pUFO&and&pDOT,&as&it&is&unclear&when&during&evolution&this&occurred.&Many&plants&families& contain&species&with&inflorescences&described&as&racemes&and&cymes&(Watson&and&Dalwitz,& 2007),& suggesting& that& these& structures& may& have& evolved& multiple& times& independently.& However& the& details& are& hard& to& reconstruct& with& certainty& because& inflorescence& architecture& is& (often)& misclassified& for& a& variety& of& reasons& (Castel& et& al.,& 2010).& Second,& assessing&when&the®ulatory&differences&in&pDOT&and&pUFO&arose&during&evolution&requires& data&on&the®ulation&of&DOT/UFO&homologs&in&many&more&(related)&species&than¤tly& available.&Moreover,&as&floral&identity&is&specified&by&the&combined&action&of&several&genes,& not& all& changes& in& the& expression& of& a& single& gene& will& necessarily& alter& development& immediately,&as&outlined&below&& The& large& variation& in& expression& patterns& of& DOT,& UFO,$ FIM$ and& the& Impatiens& UFO& homolog& within& the& FM& are& intriguing& because& they& seem& to& have& very& similar& functions& within& the& flower.& In& Arabidopsis& FMs& UFO& is& initially& expressed& throughout& the& FM,& overlapping&with&the&expression&of&subordinate&organBidentity&genes&that&specify&petal&and& stamen&fate&(Lee&et&al.,&1997),&while&in&petunia&FMs&the&mRNA&expression&patterns&of&DOT& and&the&downstream&B&and&CBtype&genes&have&little&or&no&overlap&(Souer&et&al.,&2008).&Hence,& we&hypothesized&that&DOT&protein&may&move&between&cells&in&the&FM&(Souer&et&al.,&2008).& The&finding&that&pDOT4.6:GUS&mRNA&is&confined&to&the&sepal/petal&boundary,&like&DOT&mRNA,& while& GUS& activity& was& detected& throughout& the& FM& shows& that& GUS& protein& (or& a& XBgluc& reaction&product)&can&indeed&move&from&cells&at&periphery&of&the&FM&toward&the¢er.&If& DOT&(and&UFO)&would&move&in&a&similar&way,&this&might&explain&why&(small)&changes&in&their& expression& pattern& in& the& flower& have& limited& consequences& for& development.& It& is& conceivable&that&the&CREs&and&transcription&factors&that&drive&UFO&expression&in&the¢er&

51& DOT$cisBelements&and&inflorescence&architecture& of&the&emerging&FM&are&the&same&as&those&that&drive&UFO&expression&in&the&other&meristems,& where&it&has&no&known&function,&whereas&UFO&expression&at&the&sepal/petal&boundary&relies& on& distinct& CREs& and& transcription& factors& that& are& probably& similar& to& those& driving& the& expression& of& DOT,$ FIM& and& AN& in& the& same& domain& in& their& hosts.& In& young& FMs& of& Antirrhinum&FIM&is&a&expressed&in&a&rather&thick&8Bcell&wide&ring&with&only&a&small&2Bcells&wide& hole&in&the¢er&(Simon&et&al.,&1994),&which&is&more&similar&to&UFO&than&DOT$expression.& However,& in& petunia& and& Arabidopsis& flowers& pFIM:GUS& is& expressed& in& a& thin& ring& at& the& sepal&boundary,&similar&to&DOT&and&pDOT:GUS,&suggesting&that&this&difference&between&FIM& and& DOT& expression& is& mostly& due& to& alterations& in& the& transBregulatory& network& that& operates&within&the&FM.&Nevertheless,&there&are&clear&functional&differences&between&pFIM& and&pDOT,&as&pFIM&is&active&in&leafs&of&Arabidopsis&seedlings,&in&contrast&to&pDOT&that&is¬.& Whether&expression&of&the&UFOBhomolog&of&Impatiens&B&which&is&expressed&in&leaf&primordia,& like& pFIM& in& Arabidopsis& and& within& the& petal& primordia& rather& than& at& their& boundary& (Pouteau& et& al.,& 1998)& –& diverged& from& FIM& and& DOT& by& cisB& or& transBregulatory& changes& remains&to&be&established.&& Variations& in& the& expression& patterns& of& ALF/LFY& homologs& are& important& for& morphological& divergence& as& those& of& DOT/UFO& homologs.& However,& again,& not& all& the& variation&that&is&seen&relates&necessarily&to&developmental&changes.&Many&species,&with&few& exceptions& (Coen& et& al.,& 1990),& express& their& LFY& homologs& in& vegetative& tissues& with& different&spatioBtemporal&patterns,&where&they&have&no&apparent&(architectural)&role&that&is& obvious&from&mutant&phenotypes&(Weigel&et&al.,&1992;&Kelly&et&al.,&1995;&Souer&et&al.,&1998;& MolineroBRosales&et&al.,&1999),&except&for&a&clade&of&legumes&where&LFY&is&involved&in&the& development& of& compound& leaves& (Champagne& et& al.,& 2007).& Several& Brassicaceae& with& (rosette&flowering)&indeterminate&racemose&inflorescences&express&their&LFY&homologs&in&the& lateral& meristems& that& develop& into& flowers,& but& also& in& the& apical& IM,& which& nevertheless& remains& indeterminate& (Shu& et& al.,& 2000;& Sliwinski& et& al.,& 2007).& Transgenic& experiments& showed& that& the& different& expression& of& IacLFY& in& Ionopsidum$ acuale& compared& to& LFY& in& Arabidopsis,&which&is&repressed&in&the&IM&by&a&pathway&that&involves&TERMINAL$FLOWER$1& (TFL1)& (Bradley& et& al.,& 1997),& is& due& to& a& difference& in& the& upstream& regulatory& network,& whereas&in&Idahao$scapigera&and&Leavenworthia$crassa&it&traced&to&a&difference&in&their&LFY& promoters,& that& causes& lack& of& repression& of& LscLFY& and& LcrLFY& by& TFL1& (Yoon& and& Baum,& 2004;&Sliwinski&et&al.,&2007).&

52& Chapter&2&& & In&this&light&it&is&remarkable&that&the&CREs&in&pALF&and&pLFY&are&so&conserved,&given&that& Arabidopsis& and& petunia& are& distantly& related& dicots,& with& different& inflorescence& architectures& and& different& ALF/LFY& expression& patterns.& In& side& by& side& comparison& the& pLFY:GUS& and& pALF:GUS& expression& patterns& in& petunia& or& in& Arabidopsis& seemed& nearly& identical.&This&indicates&that&(i)&the&divergent&expression&of&ALF&and&LFY&in&their&hosts&is&due& to&differences&in&the&upstream®ulatory&network&that&remain&to&be&identified,&and&(ii)&that& pALF&and&pLFY&are&functionally&similar.&The&latter&was&unexpected&as&it&suggests&that&pALF& still&contains&the&CRE(s)&for&TFL1Bmediated&repression.&In&Arabidopsis&this&repression&persists& from& the& vegetative& to& the& reproductive& phase& (Bradley& et& al.,& 1997)& and& is& conserved& in& Antirrhinum&(Bradley&et&al.,&1996;&Bradley&et&al.,&1997),&but&apparently¬&in&nightshades.& The&TFL1&homolog&from&petunia&was&never&investigated,&but&the&homologs&from&tobacco&and& tomato,&CENTRORADIALIS$(CET4)&and&SELF$PRUNING&(SP)&respectively,&are&expressed&only&in& vegetative&axillary&meristems&and¬&in&the&FM&or&SIMs&(Amaya&et&al.,&1999;&Thouet&et&al.,& 2008).& Moreover,& inactivation& of& SP& only& affects& the& development& of& the& vegetative& sympodial&meristems&(Pnueli&et&al.,&1998),&which&are&lacking&in&petunia&(Castel&et&al.,&2010),& but¬&the&cymose&flower&truss.& Since& Arabidopsis& and& petunia& are& distantly& related& species& pALF& and& pLFY& most& likely& represent&the&ancestral&state&in&dicots,&while&variants&like&pLcrLFY$and&pIscLFY&are&probably& derived.&Because&the&latter&variants&do¬&affect&the&spatial&FMI®ulation,&they&are&most& likely&accompanied&by&compensatory&alterations&in&the&expression&of&the&LcrUFO&and&IscUFO& or&other&FMI&genes&that&remains&to&be&established.

53& DOT$cisBelements&and&inflorescence&architecture&

Materials!and!Methods!

Isolation!of!pALF!and!pDOT! The& 5’& flanking& regions& of& ALF& and& DOT& were& isolated& using& somatic& transposon& insertionB mediated&PCR&(SOTIBPCR)&(Rebocho&et&al.,&2008).&The&sequences&were&submitted&to&GenBank& (see&below).&

Plant!material! The& alfW2167& and& dotA2232& dTPH1& transposon& insertion& alleles& were& in& nonBtransformable& petunia&line&W138,&and&were&described&in&detail&previously&(Souer&et&al.,&1998;&Souer&et&al.,& 2008).&ALFW2167/+&and&DOTA2232/+&were&crossed&to&the&transformable&line&W115.&alf&and&dot& mutants&were&selected&by&phenotype&from&F2&progenies,&their&genotype&confirmed&by&PCR& and& used& for& transformation.& The& phenotypes& of& alf& and& dot& mutants& in& the& hybrid& W115/W138&background&are&comparable&to&those&in&line&W138.&

Construction!of!transgenes!and!plant!transformation! The&coding&sequences&of&ALF,&DOT,&LFY&and&UFO&were&lified&from&the&vectors&described& in& (Souer& et& al.,& 2008);& the& GUS$ sequence& was& amplified& from& pGreenK& vector& (44);& the& upstream/downstream& nonBcoding& regions& were& amplified& from& petunia& W138& line& and& Arabidopsis& thaliana& Columbia& genomic& DNA.& Phusion& HighBFidelity& DNA& Polymerase& (Finnzymes)&was&used&for&all&lification&steps.& All& the& pALF,& pLFY,& pUFO& and& the$ pDOT3.1:GUS& constructs& were& made& by& digestion& (restriction& sites& listed& in& supplementary& material& table& S1& and& Fig.& S2)& and& ligation& into& pRD400tNOS&a&version&of&pRD400$(Datla&et&al.,&1992)&in&which&we&had&ligated&tNOS&as&a&SalI& fragment.& pALF:ALF:tNOS$ was& made& by& digesting& of& pALF& with& KpnI/BamHI& and& ALF& with& BamHI& and& ligation& into& pRD400tNOS$ digested& with& KpnI/BamHI.& pLFY:LFY:tNOS& and& pUFO:UFO:tNOS$were&made&by&ligating&the&corresponding&genomic®ion&of&LFY&or&UFO&as& an&EcoRI/BamHI&or&EcoRI/XbaI$fragment&respectively$into&pRD400tNOS.$ With& the& same& procedure& we& did& the& promoter:GUS& constructs.& For& the& pALF:GUS,& we& digested& pALF& with& BamHI/HinDIII& and& GUS& with& HinDIII& at& both& sites.& For& pDOT3.1:GUS,& pLFY:GUS&and&pUFO:GUS$constructs&we&used&EcoRI/BamHI$for&all&the&promoter&fragments& and& BamHI& at& both& sites& for& GUS.$ After& ligation,& the& promoter:GUS& fragments& were&

54& Chapter&2&& & introduced& into& pRD400tNOS$ digested& with& BamHI/HinDIII& and& EcoRI/BamHI$ respectively.& pDOT3.1:DOT:tNOS& was& made& by& introducing& the& genomic& pDOT:DOT& fragment& into& the& Gateway™& TOPO& Entry& vector& according& to& the& instructions& of& the& manufacturer& and& subsequent&recombination&into&the&binary&vector&pK2GW7.&& The&pFIM&and&pAN&PCR&fragments&were&introduced&into&entry&vector&via&BP&reaction&with& attB1TB2& site.& The& entry& clones& were& subcloned& according& to& Gateway& Technology& with& Clonase&II&(Invitrogen)&into&the&destination&vectors&(Karimi&et&al.,&2002):&we&used&the&pKGW,0& for& promoter:cDNA& constructs& and& pKGWFS7.0& with& GUS& for& promoter& analysis& (supplementary&material&Fig.&S2).& pDOT4.6:DOT:t35S,& pDOT4.6:DOT:tDOT,& pDOT4.6:GUS:t35S$ and& pDOT4.6:GUS:tDOT& were& constructed&with&Gateway&Multisite&Recombination.&pDOT4.6&was&introduced&in&pDONR221& P1BP4& by& a& BP& reaction& using& primer& with& attB1TB4& sites;& GUS$ cDNA& was& inserted& into& pDONR221&P4rBP3r&by&a&BP&reaction&using&primer&with&attB4rTB3r;&DOT&cDNA&sequence&was& also& cloned& into& pDONR221& P4rBP3r& clone& by& a& BP& reaction& using& primer& with& attB4rTB3r;& tDOT&was&inserted&by&a&BP&reaction&into&pDONR221&P3BP2&using&primer&with&attB3TB2;&t35S& was&also&inserted&by&a&BP&reaction&into&pDONR221&P3BP2&using&primer&with&attB3TB2.&By&LR& Clonase& reaction& the& fragments& were& subBcloned& into& pKGW,0& destination& vectors& in& the& different&combination&to&obtain&the&desired&construct.$All&the&primers&used&for&cloning&are& listed&in&Table&S1.& All& transgenes& were& (re)sequenced& before& introduction& into& the& transformable& petunia& line& W115& or& homozygous& alf& and& dot& mutants& using& Agrobacterium$ tumefaciens& (strain& AGL0)&mediated&leaf&disc&transformation&(Horsch&et&al.,&1985).$Arabidopsis$thaliana&Columbia& was&transformed&with&Agrobacterium$tumefaciens&strain&C58C1&(MP90)&using&the&floral&dip& method&(Clough&and&Bent,&1998),&and&transformants&were&selected&on&Murashige&and&Skoog& medium&(Duchefa)&containing&50&mg/l&kanamycin&monoBsulfate.& All& plants& were& grown& in& a& greenhouse.& For& comparisons& of& phenotypes& plants& were& grown&side&by&side&to&exclude&that&any&phenotypic&differences&resulted&from&variations&in& greenhouse&conditions.&

RNA!Extraction!and!Quantitative!Real"Time!PCR! Total&RNA&was&isolated&from&the&SAM&of&2&weeks&old&seedlings&and&fully&developed&IM&of& Arabidopsis&and&Antirrhinum$using&RNAeasy&extraction&kit&(Qiagen)&and&treated&with&DNAB

55& DOT$cisBelements&and&inflorescence&architecture& free&DNase&(Roche)&to&remove&residual&genomic&DNA.&Transcript&levels&were&quantified&with& Eco& Real& time& PCR& system& (Illumina)& using& Power& SYBER& Green& (Applied& Biosystems;& http://appliedbiosystems.com).&The&primers&used&are&shown&in&Table&S2.&Normalization&was& done&based&on&the&expression&of&ACTIN.&

Whole!mount!GUS!staining! We&accurately&followed&the&wholeBmount&GUS&staining&protocol&as&described&in&(Weigel&and& Glazebrook,&2002).&Untransformed&W115&was&always&taken&along&as&negative&control.&The& stained& tissue& was& examined& under& binoculars.& The& brightness& of& the& digital& images& as& a& whole& was& adjusted& for& optimal& visibility& of& the& organs& and& blue& staining& using& Adobe®& Photoshop®&software,&when&necessary.&

Plant!photography! Pictures& of& plants& were& taken& with& a& FujiFilm& FinePix& S2& Pro& digital& camera.& In& figures& the& background&was&blacked&out&using&Adobe®&Photoshop®&software.&

Statistical!analysis!of!flowering!time! We&measured&the&flowering×&of&primary&Arabidopsis$(Columbia)$transformants&by&the& number&of&rosette&and&cauline&leaves&at&bolting.&The&plants&were&grown&under&a&longBday& regime& (16& hours& light/8& hours& darkness).& The& counted& leaf& numbers& were& statistically& analyzed&using&OneBWay&ANOVA&in&SPSS&(SPSS&for&Windows,&Rel.&16.0.1.&2007.&Chicago:&SPSS& Inc.).&

GenBank!accession!numbers! Sequences& of& the& genes& used& in& this& study& can& be& found& in& the& EMBL/GenBank& database& under& the& following& accession& numbers:& ALF& promoter& (JF274656),& ALF& (AF030171),& DOT& promoter&(JF274657),&DOT$(EU352681),&LFY&(NP200993)&and&UFO&(NM102834).&

Acknowledgements! We&thank&Pieter&Hoogeveen,&Daisy&Kloos,&Maartje&Kuijpers&and&Martina&Meesters&for&great& plant&care,&Alexandra&Rebocho&for&the&initial&studies&on&the&DOT&promoter&and&Dr&Raju&Datla& for&kindly&providing&the&pRD400&plasmid.&This&work&was&supported&by&a&grant&to&R.K.&from& the&Netherlands&Organisation&for&Scientific&Research&(NWO).&

56& Chapter&2&& & Supplementary!Information!!

& & Fig.! S1.! Schematic! representation! of! ALF,6 DOT! expression! and! their! homologs! in! tomato! Arabidopsis! and! Antirrhinum.& (ABD)& Expression& in& the& vegetative& SAM& (left),& in& the& flowering& meristems& and& young& FMs& (middle)& and& in& a& stage& 3& flower& (right).& ALF$ (yellow)& and& DOT& (blue)& expression& in& cymose& inflorescence& is& represented& in& (A)& and& (B),& LFY& (yellow)& and& UFO& (blue)& expression&in&raceme&in&(C)&and&(D).&In&green&areas&expression&of&the&two&genes&overlaps.&Numbers& indicate&developmental&stage&of&FMs&(Smyth&et&al.,&1990);&br,&bract;&fm,&floral&meristem;&ilp,&incipient& leaf& primordium;& lp,& leaf& primordium;& sam,& shoot& apical& meristem;& se,& sepal;& sm,& sympodial& meristem.& &

57& DOT$cisBelements&and&inflorescence&architecture&

& & Fig.!S2.!Schematic!representation!of!the!transgenes!used!in!this!study.!Constructs&are&in&the&pRD400& (Datla& et& al.,& 1992)& and& Gateway™& vectors& (see& Materials& and& Methods).& The& primers& used& for& construction&are&indicated&with&arrows,&sequences&are&listed&in&Table&S1&and&numbers&indicate&the& order&in&which&digested&PCR&products&were&cloned&into&the&vector.&Black&bars,&5’&nonBcoding®ions;& blue&bars,&GUS&coding&sequence;&red&bars,&NOS&or&t35S&terminator;&green&bars,&ALF,&DOT,&LFY&or&UFO& coding&sequence,&pink&bar&tDOT;&open&circle,&ATG&start&codon;&closed&circle,&stop&codon.! & &

58& Chapter&2&& &

! & Fig.!S3.!Complementation!of!alf6by!pALF:ALF.!(A)&alf&mutant&in&hybrid&W138/W115&background.&(B)& alf& fully& complemented& by& the& pALF:ALF& transgene;& both& inflorescence& structure& and& flowers& are& indistinguishable&from&wild&type.&Note&that&the&outgrowth&of&axillary&shoots&(ax)&is¬&repressed&like& in&(A).&(C)&alf&partially&complemented&by&pALF:ALF.&The&cymose&inflorescence&structure&is&restored,& but& the& second& and& third& whorl& of& the& flower& consist& of& sepals& instead& of& petals& and& stamens.& Consecutive&flowers&are&numbered&from&old&to&young&(f1,&f2);&b,&bract.&

59& DOT$cisBelements&and&inflorescence&architecture&

& & Fig.! S4.! The! promoter:cDNA! transgenes! have! no! effect! on! Arabidopsis! development.! (A)& The& inflorescence&of&Arabidopsis&containing&the&promoter:cDNA&transgene&appeared&wild&type,&like&those& of&the&empty&vector&control&(left).&(B)&Wild&type&flowers&were&observed&in&Arabidopsis&inflorescences& transformed&with&the&promoter:cDNA&constructs.&(C)&Bar&plot&showing&the&mean&number&of&rosette& plus& cauline& leaves& of& primary& transformants:& empty& vector& control.& The& difference& between& the& means&is¬&significant&in&a&twoBtailed&test&with&α&=&0.05,&P&≥&0,783&for&each&of&the&comparisons&in& OneBWay&ANOVA.! &

60& Chapter&2&& &

! & Fig.!S5.!Complementation!of!alf!by!pLFY:LFY.!(A)&alf&inflorescence&in&W115/W138&hybrid&background& with& schematic& representation& of& the& flowerBtoBshoot& phenotype.& Outgrowth& of& axillaries& is& suppressed.& (B)& Inflorescence& of& wild& type& W115/W138& hybrid.& (C)& alf& partially& complemented& by& pLFY:LFY;& note& that& the& cymose& inflorescence& structure& was& restored,& but& that& flowers& consist& of& whorls&of&sepals&(green&dots&in&the&diagram).&See&also&(F).&(D)&Almost&full&complementation&of&alf,& only&small&flower&defects&remained.&See&(GBH).&(E)&Wild&type&W115/W138&hybrid&flower.&(F)&Partially& complemented& flower& as& seen& on& inflorescences& such& as& in& (C).& (GBH)& Nearly& completely& complemented&flower&of&pLFY:LFY&alf.&In&(H)&the&flower&was&opened&to&show&presence&of&all&floral& organs.& Some& stamens& contained& ‘flags’& of& petal& tissue& (arrow),& or& appeared& filamentous& (arrowhead).&Consecutive&flowers&are&numbered&from&old&to&young&(f1,&f2);&b,&bract;&ax,&axillary;&c,& carpel,&diagrams&as&in&Fig.&1.! & &

61& DOT$cisBelements&and&inflorescence&architecture&

& & Fig.! S6.! pUFO:GUS! is! expressed! in! mature! petunia! embryos.! (A)& Wild& type& W115& and& (B)& pDOT3.1:GUS& embryos& stained& for& GUS.& No& blue& signal& was& observed.& (C)& pUFO:GUS$ is& expressed&in&the&SAM&(arrow)&and&in&the&root&of&mature&petunia&embryos.&C,&cotyledons.! &

& & Fig.!S7.!Strong!pDOT4.6:GUS!Arabidopsis!expressors.!(A)&In&flowering&pDOT4.6:GUS&Arabidopsis$(At),& both&the&SAM&and&young&flower&primordia&strongly&expressed&GUS.&In&older&flowers&blue&staining&was& confined&to&the&base&of&the&flower.&(B)&Longitudinal§ion&showing&GUS&expression&in&Arabidopsis& inflorescence.&Expression&is&absent&in&apical&IM&but&limited&to&sepal&and&petal&primordial.&*,&apical& meristem.&! &

62& Chapter&2&& &

& & Figure!S8.!The!effects!of!pUFO:UFO!in!petunia.!(ABB)&pUFO:UFO&flowers&have&supernumerary&organs.& Wild&type&petunia&flowers&(left)&have&five&petals&(A)&and&five&stamens&(B),&whereas&pUFO:UFO&flowers& have&six&or&more.&Carpels&(c)&appeared&wild&type.&(C)&Repetitively&branching&inflorescence&of&dot&in& W115/W138&background.&(D)&pUFO:UFO&converted&the&dot&inflorescence&to&a&single,&aberrant&flower,& which&terminated&in&a&wild&type&carpel&(inset).! & & & & !

63& DOT$cisBelements&and&inflorescence&architecture&

! ! ! ! ! ! & & & & & Fig.!S9.!Expression!of!pAN:GUS6in!petunia!and!Arabidopsis!inflorescence,!and!pFIM:GUS6in!petunia.! (A)& In& low& petunia& (Ph)& expressors& pAN:GUS& expression& is& first& seen& as& a& stripe& (arrow)& in& the& sepals/petals&boundary.&(B)&In&high&Arabidopsis$(At)&expressors,&pAN:GUS&is&active&in&both&IM&and&FM.& (C)&In&low&petunia&(Ph)&expressors&pFIM:GUS&is&also&first&seen&as&a&stripe&(arrow)&in&the&sepals/petals& boundary.&(D)&In&high&petunia&expressors,&pFIM:GUS$was&observed&in&whole&FM&dome.&*,&sympodial& meristem.& & & Fig.!S10:!LFY,6UFO6and!FIM6are!expressed!in!the! vegetative!and!reproductive!phase,!while!FLO6is! only! expressed! in! the! reproductive! phase.& (A)& Relative& expression& level& of& LFY& and& UFO$ in& vegetative& and& reproductive& phase& of& Arabidopsis.&(B)&Relative&expression&level&of&FLO& and&FIM$in&vegetative&and&reproductive&phase&of& Antirrhinum.& & &

64& Chapter&2&& & Table!S1:!Primers!used!for!the!constructs! ! Name! Num! Description! Sequence! ALF$STOP$R$ flo9R& 530& CGGGATCCTTAGAATGACAACCTAA& BamHI$ flo22F& 1287& ALF$dTPH1$F$ CAGATGGGAACTGCTTGTTGGAG& ALF$T2800F$ flo48F& 2610& CGCGGATCCGTGACTTGGAAGTTGGAACAAACG& BamHI$ flo52R& 2614& ALF$ATG$R$HinDIII$ CCCAAGCTTACTTGCTGAGAAAGCCTCTGGGTCC& flo53R& 3107& ALF$intron$R$ TGGCCTTCCAAAAGTTATGCATGTC& flo59F& 3756& ALF$T2800F$KpnI$ GGGGTACCGTGACTTGGAAGTTGGAACAAACG& flo60R& 3757& ALF$ATG$R$BamHI$ CTTGCTGAGAAAGCCTCTGGATCCATGTTG& flo61F& 3758& ALF$ATG$F$BamHI$ CAACATGGATCCAGAGGCTTTCTCAGCAAG& flo65R& 4229& ALF$dTPH1$R$ CTCATTCTGCCACCGCCTGGC& GUS$ATG$F$ GUS&F4& 2615& CCCAAGCTTGTCCGTCCTGTAGAAACCCCAACC& HinDIII$ GUS$ATG$F$ GUS&F6& 2691& CGCGGATCCGGTCCGTCCTGTAGAAACCCCAAC& BamHI$ GUS$STOP$R$ GUS&R5& 2664& CGCGGATCCTCATTGTTTGCCTCCCTGCTGC& BamHI$ lfy4R& 2694& LFY$ATG$R$BamHI$ CACCGGAATAAGCCACTCGTGAAACCTTCAGGATCC& lfy5F& 2695& LFY$T2300F$EcoRI$ GGAATTCGGCCTATACGACGTCGTTTGAAAGAGATCC& LFY$STOP$R$ lfy10R& 3571& CGGGATCCCTAGAAACGCAAGTCGTCG& BamHI$ lfy11F& 3759& LFY$ATG$F$BamHI$ CGGGATCCTGAAGGTTTCACGAGTGGC& NOS&F1& 2720& tNOS$F$SalI$ ACGCGTCGACCCCGATCGTTCAAACATTTGGCAATA& NOS&F2& 3576& tNOS$F$BamHI$ CGGGATCCCCCGATCGTTCAAACATTTGGCAATA& NOS&F3& 3577& tNOS$F$XbaI$ GCTCTAGACCCGATCGTTCAAACATTTGGCAATA& NOS&R1& 2721& tNOS$R$SalI$ ACGCGTCGACCCGATCTAGTAACATAGATGACACCG& NOS&R2& 2722& tNOS$R$HinDIII$ CCCAAGCTTCCGATCTAGTAACATAGATGACACCG& NOS&R4& 3578& tNOS$R$BamHI$ CGGGATCCCCGATCTAGTAACATAGATGACACCG& NOS&R5& 3579& tNOS$R$XbaI$ GCTCTAGACCGATCTAGTAACATAGATGACACCG& puf3R& 827& DOT$dTPH1$R$ TGGACAAGGAGGAATCCAAAC& puf5R& 836& DOT$dTPH1$R$ CCGCATGGGCGCTTGAATTTTAG& puf14F& 1511& DOT$dTPH1$F$ CTATTGACTTAGCTGTGGCTGG& puf46F& 2661& DOT$T3100F$EcoRI$ CGGAATTCGATTTCATTGCGGTTGGTATTTACGCC& DOT$ATG$R$ puf47R& 2662& CGGGATCCCTAATAGGTGCATGATGAAAAGCTTCC& BamHI$ puf56R& 3962& DOT$STOP$R$SalI$ ACGCGTCGACGTATCAGTTGAAAGATTGAAAGGGTAATGTCAAC& puf57F& 3988& DOT$T3100$TOPO$ CACCGATTTCATTGCGGTTGGTATTTACGCC& ufo4F& 2696& UFO$T3800F$EcoRI$ CCGGAATTCTCTGTTTTAATTGCCCCACTTC& UFO$ATG$R$ ufo5R& 2697& CGCGGATCCTTATTGATGAACACAGTTGAATCC& BamHI$ ufo8R& 3572& UFO$STOP$R$XbaI$ GCTCTAGACTAACAGACTCCAGGAAATGG& PhBOX4attB1& 5636& pDOT4.6$attB1$ GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAAACATTAAGATAAACTCA& PhpDOTE/SattB2& 5936& pDOT4.6$attB2$ GGGGACCACTTTGTACAAGAAAGCTGGGTGTCTAGGATAATTGAAGGTAT&

65& DOT$cisBelements&and&inflorescence&architecture&

DOTstopattB2& 4192& DOTTattB2$ GGGGACCACTTTGTACAAGAAAGCTGGGTAGTTGAAAGATTGAAAGGGTAATGTC& DOTattB2B 5551& tDOTTattB2$ GGGGACCACTTTGTACAAGAAAGCTGGGT&AATGAAAACGTAATGCAATAC& 5510Rev& SlANpromattB1& 5767& pANTattB1$ GGGGACAAGTTTGTACAAAAAAGCAGGCTCAAAGGTGGTAAGATTGATTG& SlANpromattB2& 5768& pANTattB2$ GGGGACCACTTTGTACAAGAAAGCTGGGTTTGAGTTTGAAGCTAGGAGAG& AmFIMpBattB1& 5907& pFIMTattB1$ GGGGACAAGTTTGTACAAAAAAGCAGGCTCACCAATCTCATGATTCCACT& AmFIMpBattB2& 5927& pFIMTattB2$ GGGGACCACTTTGTACAAGAAAGCTGGGTAGTTTTGGATTTGCTAAAGAA& pDOTBattB1& 5636& pDOTBattB1$ GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAAACATTAAGATAAACTCA& pDOTBattB4& 3890& pDOTTattB4$ GGGGACAACTTTGTATAGAAAAGTTGGGTCTTGTCTAGGATAATTGAAGGTATAC& DOTBattB4r& 3895& DOTTattB4r$ GGGGACAACTTTTCTATACAAAGTTGATGGAAGCTTTTCATCATGCA& DOTBattB3r& 3896& DOTTattB3r$ GGGGACAACTTTATTATACAAAGTTGTCAGTTGAAAGATTGAAAGGGTAA& GUSBattB4r& 5024& GUSTattB4r$ GGGGACAACTTTTCTATACAAAGTTGCCATGGTCCGTCCTGTAGAAACC& GUSBattB3r& 3914& GUSTattB3r$ GGGGACAACTTTATTATACAAAGTTGGGTAGCAATTCCCGAGGCT& t35SBattB3& 3887& t35STattB3$ GGGGACAACTTTGTATAATAAAGTTGCGGCCATGCTAGAGTCCGC& t35SBattB2& 3888& t35STattB2$ GGGGACCACTTTGTACAAGAAAGCTGGGTAGGTCACTGGATTTTGGTTTTAG& tDOTBattB3& 5566& tDOTTattB3$ GGGGACAACTTTGTATAATAAAGTTGTACTTTTGAACTGTTTCAAGTGG& tDOTBattB2& 5551& tDOTTattB2$ GGGGACCACTTTGTACAAGAAAGCTGGGTAATGAAAACGTAATGCAATAC&

Table!S2:!Primers!used!for!qPCR!

&

Primer! Primer!ID! Sequence! 6231& AtACTIN"FW& AGTGGTCGTACAACCGGTATTGT&

AtACTIN"RV& 6232& GATGGCATGAGGAAGAGAGAAAC&

AmACTIN"FW& 6951& GCCAAGACAAGCTCCTCTGT&

AmACTIN"RV& 6952& ATTCCGATCATTGATGGCTGGA&

LFY"FW& 5606& CCTCGTCTCTCTATTTGGTATG&

LFY"RV& 6955& CATCCACCACGTCCAGACGT&

UFO"FW& 6956& CTACACACAGTTTGCAGCAGAAG&

UFO"RV& 6957& CTCACTATATCAAACAGCAACTGC&

FLO"FW& 6947& AACCAAAAATGCGCCACTAC&

FLO"RV& 6948& TGTAGCATGCCTGACGCCAT&

FIM"FW& 5780& GATGGACTGCAGAATCTGGAGC&

FIM"RV& 6945& GTAGCCTTCGTAGTTTGTCGGTC& & & &

66& Chapter&2&& & References!! Ahearn,! K.! P.,! Johnson,! H.! A.,! Weigel,! D.! and! Wagner,! D.! R.& (2001)& NFL1,& a& Nicotiana$ tabacum$ LEAFYBlike& gene,& controls& meristem& initiation& and& floral& structure.& Plant$ Cell$ Physiol.&42(10):&1130B1139.& Amaya,!I.,!Ratcliffe,!O.!J.!and!Bradley,!D.!J.&(1999)&Expression&of&CENTRORADIALIS&(CEN)&and& CENBlike& genes& in& tobacco& reveals& a& conserved& mechanism& controlling& phase& change& in& diverse&species.&Plant$Cell&11(8):&1405B18.& Benlloch,! R.,! Berbel,! A.,! Serrano"Mislata,! A.! and! Madueno,! F.& (2007)& Floral& initiation& and& inflorescence&architecture:&a&comparative&view.&Ann.$Bot.$(Lond.)&100(3):&659B76.& Blazquez,!M.!A.,!Soowal,!L.!N.,!Lee,!I.!and!Weigel,!D.&(1997)&LEAFY&expression&and&flower& initiation&in&Arabidopsis.&Development&124(19):&3835B44.& Bradley,!D.,!Carpenter,!R.,!Copsey,!L.,!Vincent,!C.,!Rothstein,!S.!and!Coen,!E.&(1996)&Control& of&inflorescence&architecture&in&Antirrhinum.&Nature&379(6568):&791B797.& Bradley,! D.,! Ratcliffe,! O.,! Vincent,! C.,! Carpenter,! R.! and! Coen,! E.& (1997)& Inflorescence& commitment&and&architecture&in&Arabidopsis.&Science&275(5296):&80B83.& Castel,! R.& (2009)& Molecular$ analysis$ of$ the$ developmental$ mechanisms$ that$ establish$ the$ bodyplan$ of$ petunia& Molecular$ Cell$ Biology,& vol.& PhD.& Amsterdam:& VU& University& Amsterdam.& Castel,!R.,!Kusters,!E.!and!Koes,!R.&(2010)&Inflorescence&development&in&petunia:&through&the& maze&of&botanical&terminology.&J$Exp$Bot&61(9):&2235B46.& Chae,!E.,!Tan,!Q.!K.,!Hill,!T.!A.!and!Irish,!V.!F.&(2008)&An&Arabidopsis$FBbox&protein&acts&as&a& transcriptional&coBfactor&to®ulate&floral&development.&Development&135(7):&1235B45.& Champagne,!C.!E.,!Goliber,!T.!E.,!Wojciechowski,!M.!F.,!Mei,!R.!W.,!Townsley,!B.!T.,!Wang,! K.,! Paz,! M.! M.,! Geeta,! R.! and! Sinha,! N.! R.& (2007)& Compound& leaf& development& and& evolution&in&the&legumes.&Plant$Cell&19(11):&3369B78.& Clough,! S.! J.! and! Bent,! A.! F.& (1998)& Floral& dip:& a& simplified& method& for& AgrobacteriumB mediated&transformation&of&Arabidopsis$thaliana.&Plant$J.&16(6):&735B43.& Coen,! E.! S.,! Romero,! J.! M.,! Doyle,! S.,! Elliott,! R.,! Murphy,! G.! and! Carpenter,! R.& (1990)& floricaula:&a&homeotic&gene&required&for&flower&development&in&Antirrhinum$majus.&Cell& 63(6):&1311B22.& Datla,!R.!S.,!Hammerlindl,!J.!K.,!Panchuk,!B.,!Pelcher,!L.!E.!and!Keller,!W.&(1992)&Modified& binary&plant&transformation&vectors&with&the&wildBtype&gene&encoding&NPTII.&Gene&122(2):& 383B4.& Della!Pina,!S.,!Souer,!E.!and!Koes,!R.&(2014)&Arguments&in&the&evoBdevo&debate:&say&it&with& flowers!&J$Exp$Bot&65(9):&2231B42.& Horsch,!R.!B.,!Fry,!J.!E.,!Hoffmann,!N.!L.,!Eichholtz,!D.,!Rogers,!S.!G.!and!Fraley,!R.!T.&(1985)& A&simple&and&general&method&for&transferring&genes&into&plants.&Science&227(4691):&1229B 1231.& Huijser,! P.,! Klein,! J.,! Lonnig,! W.! E.,! Meijer,! H.,! Saedler,! H.! and! Sommer,! H.& (1992)& Bracteomania,&an&inflorescence&anomaly,&is&caused&by&the&loss&of&function&of&the&MADSB box&gene&squamosa$in&Antirrhinum$majus.&EMBO$J.&11(4):&1239B49.&

67& DOT$cisBelements&and&inflorescence&architecture&

Jefferson,!R.!A.,!Kavanagh,!T.!A.!and!Bevan,!M.!W.&(1987)&GUS&fusions:&betaBglucuronidase& as&a&sensitive&and&versatile&gene&fusion&marker&in&higher&plants.&EMBO$J.&6(13):&3901B7.& Karimi,!M.,!Inze,!D.!and!Depicker,!A.&(2002)&GATEWAY&vectors&for&AgrobacteriumBmediated& plant&transformation.&Trends$in$Plant$Science&7(5):&193B5.& Kelly,!A.!J.,!Bonnlander,!M.!B.!and!Meeks"Wagner,!D.!R.&(1995)&NFL,&the&tobacco&homolog& of& FLORICAULA& and& LEAFY,& is& transcriptionally& expressed& in& both& vegetative& and& floral& meristems.&Plant$Cell&7(2):&225B34.& Lee,! I.,! Wolfe,! D.! S.,! Nilsson,! O.! and! Weigel,! D.& (1997)& A& LEAFY& coBregulator& encoded& by& UNUSUAL&FLORAL&ORGANS.&Curr$Biol&7(2):&95B104.& Lippman,!Z.!B.,!Cohen,!O.,!Alvarez,!J.!P.,!Abu"Abied,!M.,!Pekker,!I.,!Paran,!I.,!Eshed,!Y.!and! Zamir,! D.& (2008)& The& making& of& a& compound& inflorescence& in& tomato& and& related& nightshades.&PLoS$Bio&6(11):&e288.& Long,! J.! A.! and! Barton,! M.! K.& (1998)& The& development& of& apical& embryonic& pattern& in& Arabidopsis.&Development&125(16):&3027B35.& Maizel,!A.,!Busch,!M.!A.,!Tanahashi,!T.,!Perkovic,!J.,!Kato,!M.,!Hasebe,!M.!and!Weigel,!D.& (2005)& The& floral& regulator& LEAFY& evolves& by& substitutions& in& the& DNA& binding& domain.& Science&308(5719):&260B263.& Mandel,! M.! A.,! Gustafson"Brown,! C.,! Savidge,! B.! and! Yanofsky,! M.! F.& (1992)& Molecular& characterization& of& the& Arabidopsis& floral& homeotic& gene& APETALA1.& Nature& 360(6401):& 273B7.& Mandel,! M.! A.! and! Yanofsky,! M.! F.& (1995)& A& Gene& Triggering& Flower& Formation& in& Arabidopsis.&Nature&377(6549):&522B524.& Molinero"Rosales,!N.,!Jamilena,!M.,!Zurita,!S.,!Gomez,!P.,!Capel,!J.!and!Lozano,!R.&(1999)& FALSIFLORA,&the&tomato&orthologue&of&FLORICAULA&and&LEAFY,&controls&flowering&time& and&floral&meristem&identity.&Plant$Journal&20(6):&685B93.& Moyroud,! E.,! Kusters,! E.,! Monniaux,! M.,! Koes,! R.! and! Parcy,! F.& (2010)& LEAFY& blossoms.& Trends$in$Plant$Science&15(6):&346B52.& Parcy,!F.,!Nilsson,!O.,!Busch,!M.!A.,!Lee,!I.!and!Weigel,!D.&(1998)&A&genetic&framework&for& floral&patterning.&Nature&395(6702):&561B566.& Pnueli,!L.,!Carmel"Goren,!L.,!Hareven,!D.,!Gutfinger,!T.,!Alvarez,!J.,!Ganal,!M.,!Zamir,!D.!and! Lifschitz,! E.& (1998)& The& SELFTPRUNING$ gene& of& tomato& regulates& vegetative& to& reproductive& switching& of& sympodial& meristems& and& is& the& ortholog& of& CEN$ and& TFL1.& Development&125(11):&1979&B&1989.& Pouteau,!S.,!Nicholls,!D.,!Tooke,!F.,!Coen,!E.!and!Battey,!N.&(1998)&Transcription&pattern&of&a& FIM& homologue& in& Impatiens& during& floral& development& and& reversion.& Plant$ Journal& 14(2):&235B46.& Prusinkiewicz,! P.,! Erasmus,! Y.,! Lane,! B.,! Harder,! L.! D.! and! Coen,! E.& (2007)& Evolution& and& development&of&inflorescence&architectures.&Science&316(5830):&1452B1456.& Rebocho,! A.! B.,! Bliek,! M.,! Kusters,! E.,! Castel,! R.,! Procissi,! A.,! Roobeek,! I.,! Souer,! E.! and! Koes,! R.& (2008)& Role& of& EVERGREEN$ in& the& development& of& the& cymose& petunia& inflorescence.&Dev.$Cell&15(3):&437B47.&

68& Chapter&2&& & Rickett,!H.!W.&(1954)&Materials&for&a&dictionary&of&botanical&termsBI.&Bulletin$of$the$Torrey$ Botanical$Club&81(1):&1B15.& Shu,!G.!P.,!Amaral,!W.,!Hileman,!L.!C.!and!Baum,!D.!A.&(2000)&LEAFY&and&the&evolution&of& rosette&flowering&in&violet&cress&(Jonopsidium$acaule,&Brassicaceae).&American$Journal$of$ Botany&87(5):&634B641.& Simon,! R.,! Carpenter,! R.,! Doyle,! S.! and! Coen,! E.& (1994)& Fimbriata& controls& flower& development&by&mediating&between&meristem&and&organ&identity&genes.&Cell&78(1):&99B 107.& Sliwinski,!M.!K.,!Bosch,!J.!A.,!Yoon,!H.!S.,!Balthazar,!M.!and!Baum,!D.!A.&(2007)&The&role&of& two& LEAFY& paralogs& from& Idahoa$ scapigera& (Brassicaceae)& in& the& evolution& of& a& derived& plant&architecture.&Plant$Journal&51(2):&211B9.& Smyth,! D.! R.,! Bowman,! J.! L.! and! Meyerowitz,! E.! M.& (1990)& Early& flower& development& in& Arabidopsis.&Plant$Cell&2(8):&755B67.& Souer,! E.,! Rebocho,! A.! B.,! Bliek,! M.,! Kusters,! E.,! de! Bruin,! R.! A.! M.! and! Koes,! R.& (2008)& Patterning&of&inflorescences&and&flowers&by&the&FBbox&protein&DOUBLE&TOP&and&the&LEAFY& homolog&ABERRANT&LEAF&AND&FLOWER&of&Petunia.&Plant$Cell&20(8):&2033B2048.& Souer,!E.,!van!der!Krol,!A.,!Kloos,!D.,!Spelt,!C.,!Bliek,!M.,!Mol,!J.!and!Koes,!R.&(1998)&Genetic& control& of& branching& pattern& and& floral& identity& during& Petunia& inflorescence& development.&Development&125(4):&733B742.& Thouet,! J.,! Quinet,! M.,! Ormenese,! S.,! Kinet,! J.! M.! and! Perilleux,! C.& (2008)& Revisiting& the& involvement&of&SELFTPRUNING$in&the&sympodial&growth&of&tomato.&Plant$Physiol.&148(1):& 61B4.& Wagner,! D.,! Sablowski,! R.! W.! and! Meyerowitz,! E.! M.& (1999)& Transcriptional& activation& of& APETALA1&by&LEAFY.&Science&285(5427):&582B4.& Wang,!X.,!Feng,!S.,!Nakayama,!N.,!Crosby,!W.!L.,!Irish,!V.,!Deng,!X.!W.!and!Wei,!N.&(2003)& The& COP9& signalosome& interacts& with& SCF& UFO& and& participates& in& Arabidopsis& flower& development.&Plant$Cell&15(5):&1071B82.& Watson,! L.! and! Dalwitz,! M.! J.& (2007)& The& families& of& flowering& plants:& descriptions,& illustrations,& identification,& and& information& retrieval.& Version:& 1st& June& 2007.& & (1992& onwards)&http://deltaBintkey.com.& Weberling,!F.&(1989)&Morphology$of$flowers$and$inflorescences.&Cambridge:&Univ.&Press.& Weigel,!D.,!Alvarez,!J.,!Smyth,!D.!R.,!Yanofsky,!M.!F.!and!Meyerowitz,!E.!M.&(1992)&LEAFY& controls&floral&meristem&identity&in&Arabidopsis.&Cell&69(5):&843B59.& Weigel,!D.!and!Glazebrook,!J.&(2002)&Arabidopsis:$a$laboratory$manual.&Cold&Spring&Harbor,& New&York:&Cold&Spring&Harbor&Laboratory&Press&& Weigel,!D.!and!Nilsson,!O.&(1995)&A&developmental&switch&sufficient&for&flower&initiation&in& diverse&plants.&Nature&377(6549):&495B500.& Yoon,!H.!S.!and!Baum,!D.!A.&(2004)&Transgenic&study&of¶llelism&in&plant&morphological& evolution.&Proc$Natl$Acad$Sci$USA&101(17):&6524B6529.& &

69& ! ! 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( (( 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,( (imbricata,(Lycium( barbarum,( Physalis( peruviana,( ( origanifolia,( Lycianthes( biflora,( Brunfelsia( americana,( Anisodus(luridus(,(Normania(triphylla,((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.! ! !

! 108! Chapter!3!

! ! 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!

! 116! Chapter!3!

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!

References!!

Abe!M,!Kobayashi!Y,!Yamamoto!S,!Daimon!Y,!Yamaguchi!A,!Ikeda!Y,!Ichinoki!H,!Notaguchi! M,! Goto! K,! Araki! T.! 2005.! FD,! a! bZIP! protein! mediating! signals! from! the! floral! pathway! integrator!FT!at!the!shoot!apex.!Science!309,!1052Z1056.! Ahearn!KP,!Johnson!HA,!Weigel!D,!Wagner!DR.!2001.!NFL1,!a!Nicotiana(tabacum!LEAFYZlike! gene,! controls! meristen! initiation! and! floral! structure.! Plant( and( Cell( Physiology! 42,! 1130Z 1139.! An! HL,! Roussot! C,! Suarez@Lopez! P,! Corbesler! L,! Vincent! C,! Pineiro! M,! Hepworth! S,! Mouradov! A,! Justin! S,! Turnbull! C,! Coupland! G.! 2004.! CONSTANS! acts! in! the! phloem! to! regulate!a!systemic!signal!that!induces!photoperiodic!flowering!of!Arabidopsis.!Development! 131,!3615Z3626.! Becker! A,! Theissen! G.! 2003.! The! major! clades! of! MADSZbox! genes! and! their! role! in! the! development!and!evolution!of!flowering!plants.!Mol(Phylogenet(Evol!29,!464Z489.! Blazquez!MA,!Soowal!LN,!Lee!I,!Weigel!D.!1997.!LEAFY!expression!and!flower!initiation!in! Arabidopsis.!Development!124,!3835Z3844.! Bomblies! K,! Wang! RL,! Ambrose! BA,! Schmidt! RJ,! Meeley! RB,! Doebley! J.! 2003.! Duplicate! FLORICAULA/LEAFY! homologs! zfl1! and! zfl2! control! inflorescence! architecture! and! flower! patterning!in!maize.!Development!130,!2385Z2395.! Bowman! JL,! Alvarez! J,! Weigel! D,! Meyerowitz! EM,! Smyth! DR.! 1993.! Control! of! Flower! Development!in!ArabidopsisZThaliana!by!Apetala1!and!Interacting!Genes.!Development!119,! 721Z743.! Cardon! G,! Hohmann! S,! Klein! J,! Nettesheim! K,! Saedler! H,! Huijser! P.! 1999.! Molecular! characterisation!of!the!Arabidopsis!SBPZbox!genes.!Gene!237,!91Z104.! Cardon!GH,!Hohmann!S,!Nettesheim!K,!Saedler!H,!Huijser!P.!1997.!Functional!analysis!of! the!Arabidopsis!thaliana!SBPZbox!gene!SPL3:!a!novel!gene!involved!in!the!floral!transition.! Plant(Journal!12,!367Z377.! Castel! R.! 2009.! Molecular! analysis! of! the! developmental! mechanisms! that! establish! the! bodyplan!of!petunia.!Thesis,(VU(University(Amsterdam.( Clough! SJ,! Bent! AF.! 1998.! Floral! dip:! a! simplified! method! for! AgrobacteriumZmediated! transformation!of!Arabidopsis(thaliana.!Plant(Journal!16,!735Z743.! Coen!ES,!Romero!JM,!Doyle!S,!Elliott!R,!Murphy!G,!Carpenter!R.!1990.!floricaula:!a!homeotic! gene!required!for!flower!development!in!Antirrhinum(majus.!Cell!63,!1311Z1322.! Corbesier!L,!Vincent!C,!Jang!SH,!Fornara!F,!Fan!QZ,!Searle!I,!Giakountis!A,!Farrona!S,!Gissot! L,!Turnbull!C,!Coupland!G.!2007.!FT!protein!movement!contributes!to!longZdistance!signaling! in!floral!induction!of!Arabidopsis.!Science!316,!1030Z1033.!

! 118! Chapter!3!

Cousens!DJ,!Greaves!R,!Goding!CR,!O'Hare!P.!1989.!The!CZterminal!79!amino!acids!of!the! herpes! simplex! virus! regulatory! protein,! Vmw65,! efficiently! activate! transcription! in! yeast! and!mammalian!cells!in!chimeric!DNAZbinding!proteins.!Embo(Journal!8,!2337Z2342.! Endrizzi!K,!Moussian!B,!Haecker!A,!Levin!JZ,!Laux!T.!1996.!The!SHOOT!MERISTEMLESS!gene! is! required! for! maintenance! of! undifferentiated! cells! in! Arabidopsis! shoot! and! floral! meristems!and!acts!at!a!different!regulatory!level!than!the!meristem!genes!WUSCHEL!and! ZWILLE.!Plant(Journal!10,!967Z979.! Ferrandiz!C,!Gu!Q,!Martienssen!R,!Yanofsky!MF.!2000.!Redundant!regulation!of!meristem! identity! and! plant! architecture! by! FRUITFULL,! APETALA1! and! CAULIFLOWER.! Development! 127,!725Z734.! Gregis! V,! Sessa! A,! Colombo! L,! Kater! MM.! 2006.! AGL24,! SHORT! VEGETATIVE! PHASE,! and! APETALA1! redundantly! control! AGAMOUS! during! early! stages! of! flower! development! in! Arabidopsis.!Plant(Cell!18,!1373Z1382.! Gregis!V,!Sessa!A,!Colombo!L,!Kater!MM.!2008.!AGAMOUSZLIKE24!and!SHORT!VEGETATIVE! PHASE!determine!floral!meristem!identity!in!Arabidopsis.!Plant(Journal!56,!891Z902.! Hartmann!U,!Hohmann!S,!Nettesheim!K,!Wisman!E,!Saedler!H,!Huijser!P.!2000.!Molecular! cloning!of!SVP:!a!negative!regulator!of!the!floral!transition!in!Arabidopsis.!Plant(Journal!21,! 351Z360.! Jaeger!KE,!Wigge!PA.!2007.!FT!protein!acts!as!a!longZrange!signal!in!Arabidopsis.!Curr(Biol!17,! 1050Z1054.! Jung!JH,!Ju!Y,!Seo!PJ,!Lee!JH,!Park!CM.!2012.!The!SOC1ZSPL!module!integrates!photoperiod! and!gibberellic!acid!signals!to!control!flowering!time!in!Arabidopsis.!Plant(Journal!69,!577Z 588.! Kardailsky!I,!Shukla!VK,!Ahn!JH,!Dagenais!N,!Christensen!SK,!Nguyen!JT,!Chory!J,!Harrison! MJ,!Weigel!D.!1999.!Activation!tagging!of!the!floral!inducer!FT.!Science!286,!1962Z1965.! Kaufmann! K,! Wellmer! F,! Muino! JM,! Ferrier! T,! Wuest! SE,! Kumar! V,! Serrano@Mislata! A,! Madueno!F,!Krajewski!P,!Meyerowitz!EM,!Angenent!GC,!Riechmann!JL.!2010.!Orchestration! of!Floral!Initiation!by!APETALA1.!Science!328,!85Z89.! Kobayashi! Y,! Kaya! H,! Goto! K,! Iwabuchi! M,! Araki! T.! 1999.! A! pair! of! related! genes! with! antagonistic!roles!in!mediating!flowering!signals.!Science!286,!1960Z1962.! Kobayashi!Y,!Weigel!D.!2007.!Move!on!up,!it's!time!for!changeZZmobile!signals!controlling! photoperiodZdependent!flowering.!Genes(Dev!21,!2371Z2384.! Kusters! E.! 2011.! Genetic! control! of! meristem! identity! in! Petunia.! Vrije( Universiteit( Amsterdam.! Kusters!E,!Della!Pina!S,!Castel!R,!Souer!E,!Koes!R.!2015.!Changes!in!cisZregulatory!elements! of! a! key! floral! regulator! are! associated! with! divergence! of! inflorescence! architectures.! Development!142,!2822Z2831.!

119! DOT!regulation!

Lee! J,! Oh! M,! Park! H,! Lee! I.! 2008a.! SOC1! translocated! to! the! nucleus! by! interaction! with! AGL24!directly!regulates!LEAFY.!Plant(Journal!55,!832Z843.! Lee! S,! Choi! SC,! An! G.! 2008b.! Rice! SVPZgroup! MADSZbox! proteins,! OsMADS22! and! OsMADS55,!are!negative!regulators!of!brassinosteroid!responses.!Plant(Journal!54,!93Z105.! Li!D,!Liu!C,!Shen!L,!Wu!Y,!Chen!H,!Robertson!M,!Helliwell!CA,!Ito!T,!Meyerowitz!E,!Yu!H.! 2008.! A! repressor! complex! governs! the! integration! of! flowering! signals! in! Arabidopsis.! Developmental(Cell!15,!110Z120.! Liu!C,!Chen!H,!Er!HL,!Soo!HM,!Kumar!PP,!Han!JH,!Liou!YC,!Yu!H.!2008.!Direct!interaction!of! AGL24!and!SOC1!integrates!flowering!signals!in!Arabidopsis.!Development!135,!1481Z1491.! Liu!C,!Zhou!J,!Bracha@Drori!K,!Yalovsky!S,!Ito!T,!Yu!H.!2007.!Specification!of!Arabidopsis!floral! meristem!identity!by!repression!of!flowering!time!genes.!Development!134,!1901Z1910.! Mandel!MA,!Gustafson@Brown!C,!Savidge!B,!Yanofsky!MF.!1992.!Molecular!characterization! of!the!Arabidopsis!floral!homeotic!gene!APETALA1.!Nature!360,!273Z277.! Mandel!MA,!Yanofsky!MF.!1995.!A!Gene!Triggering!Flower!Formation!in!Arabidopsis.!Nature! 377,!522Z524.! Masiero!S,!Li!MA,!Will!I,!Hartmann!U,!Saedler!H,!Huijser!P,!Schwarz@Sommer!Z,!Sommer!H.! 2004.! INCOMPOSITA:! a! MADSZbox! gene! controlling! prophyll! development! and! floral! meristem!identity!in!Antirrhinum.!Development!131,!5981Z5990.! Mayor!C,!Brudno!M,!Schwartz!JR,!Poliakov!A,!Rubin!EM,!Frazer!KA,!Pachter!LS,!Dubchak!I.! 2000.!VISTA!:!visualizing!global!DNA!sequence!alignments!of!arbitrary!length.!Bioinformatics! 16,!1046Z1047.! Michaels!SD,!Ditta!G,!Gustafson@Brown!C,!Pelaz!S,!Yanofsky!M,!Amasino!RM.!2003.!AGL24! acts!as!a!promoter!of!flowering!in!Arabidopsis!and!is!positively!regulated!by!vernalization.! Plant(Journal!33,!867Z874.! Molinero@Rosales!N,!Jamilena!M,!Zurita!S,!Gomez!P,!Capel!J,!Lozano!R.!1999.!FALSIFLORA,! the! tomato! orthologue! of! FLORICAULA! and! LEAFY,! controls! flowering! time! and! floral! meristem!identity.!Plant(Journal!20,!685Z693.! Mouradov! A,! Glassick! T,! Hamdorf! B,! Murphy! L,! Fowler! B,! Marla! S,! Teasdale! RD.! 1998.! NEEDLY,! a! Pinus! radiata! ortholog! of! FLORICAULA/LEAFY! genes,! expressed! in! both! reproductive!and!vegetative!meristems.!Proc(Natl(Acad(Sci(U(S(A!95,!6537Z6542.! Mutasa@Gottgens!E,!Hedden!P.!2009.!Gibberellin!as!a!factor!in!floral!regulatory!networks.!J( Exp(Bot!60,!1979Z1989.! Parcy!F.!2005.!Flowering:!a!time!for!integration.!Int(J(Dev(Biol!49,!585Z593.! Preston! JC,! Jorgensen! SA,! Jha! SG.! 2014.! Functional! characterization! of! duplicated! Suppressor!of!Overexpression!of!Constans!1Zlike!genes!in!petunia.!PLoS(One!9,!e96108.!

! 120! Chapter!3!

Prusinkiewicz!P,!Erasmus!Y,!Lane!B,!Harder!LD,!Coen!E.!2007.!Evolution!and!development!of! inflorescence!architectures.!Science!316,!1452Z1456.! Quattrocchio!F,!Verweij!W,!Kroon!A,!Spelt!C,!Mol!J,!Koes!R.!2006.!PH4!of!Petunia!is!an!R2R3! MYB!protein!that!activates!vacuolar!acidification!through!interactions!with!basicZhelixZloopZ helix!transcription!factors!of!the!anthocyanin!pathway.!Plant(Cell!18,!1274Z1291.! Rhoades!MW,!Reinhart!BJ,!Lim!LP,!Burge!CB,!Bartel!B,!Bartel!DP.!2002.!Prediction!of!plant! microRNA!targets.!Cell!110,!513Z520.! Samach!A,!Onouchi!H,!Gold!SE,!Ditta!GS,!Schwarz@Sommer!Z,!Yanofsky!MF,!Coupland!G.! 2000.!Distinct!roles!of!CONSTANS!target!genes!in!reproductive!development!of!Arabidopsis.! Science!288,!1613Z1616.! Schwarz!S,!Grande!AV,!Bujdoso!N,!Saedler!H,!Huijser!P.!2008.!The!microRNA!regulated!SBPZ box!genes!SPL9!and!SPL15!control!shoot!maturation!in!Arabidopsis.!Plant(Molecular(Biology! 67,!183Z195.! Souer! E,! Rebocho! AB,! Bliek! M,! Kusters! E,! de! Bruin! RAM,! Koes! R.! 2008.! Patterning! of! inflorescences! and! flowers! by! the! FZbox! protein! DOUBLE! TOP! and! the! LEAFY! homolog! ABERRANT!LEAF!AND!FLOWER!of!Petunia.!Plant(Cell!20,!2033Z2048.! Souer!E,!van!Houwelingen!A,!Kloos!D,!Mol!J,!Koes!R.!1996.!The!no!apical!meristem!gene!of! Petunia! is! required! for! pattern! formation! in! embryos! and! flowers! and! is! expressed! at! meristem!and!primordia!boundaries.!Cell!85,!159Z170.! Spelt! C,! Quattrocchio! F,! Mol! JN,! Koes! R.! 2000.! anthocyanin1! of! petunia! encodes! a! basic! helixZloopZhelix!protein!that!directly!activates!transcription!of!structural!anthocyanin!genes.! Plant(Cell!12,!1619Z1632.! Szymkowiak! EJ,! Irish! EE.! 2006.! JOINTLESS! suppresses! sympodial! identity! in! inflorescence! meristems!of!tomato.!Planta!223,!646Z658.! Taoka! K,! Ohki! I,! Tsuji! H,! Furuita! K,! Hayashi! K,! Yanase! T,! Yamaguchi! M,! Nakashima! C,! Purwestri!YA,!Tamaki!S,!Ogaki!Y,!Shimada!C,!Nakagawa!A,!Kojima!C,!Shimamoto!K.!2011.! 14Z3Z3!proteins!act!as!intracellular!receptors!for!rice!Hd3a!florigen.!Nature!476,!332ZU397.! Triezenberg!SJ,!Kingsbury!RC,!McKnight!SL.!1988.!Functional!dissection!of!VP16,!the!transZ activator!of!herpes!simplex!virus!immediate!early!gene!expression.!Genes(Dev!2,!718Z729.! Unte! US,! Sorensen! AM,! Pesaresi! P,! Gandikota! M,! Leister! D,! Saedler! H,! Huijser! P.! 2003.! SPL8,! an! SBPZbox! gene! that! affects! pollen! sac! development! in! Arabidopsis.! Plant( Cell! 15,! 1009Z1019.! Van!den!Broeck!D,!Maes!T,!Sauer!M,!Zethof!J,!De!Keukeleire!P,!D'Hauw!M,!Van!Montagu! M,! Gerats! T.! 1998.! Transposon! Display! identifies! individual! transposable! elements! in! high! copy!number!lines.!Plant(Journal!13,!121Z129.! Vandenbussche! M,! Zethof! J,! Gerats! T.! 2013.! Transposon! display:! a! versatile! method! for! transposon!tagging.!Methods(Mol(Biol!1057,!239Z250.!

121! DOT!regulation!

Wada! M,! Cao! QF,! Kotoda! N,! Soejima! J,! Masuda! T.! 2002.! Apple! has! two! orthologues! of! FLORICAULA/LEAFY!involved!in!flowering.!Plant(Molecular(Biology!49,!567Z577.! Wagner!D,!Sablowski!RW,!Meyerowitz!EM.!1999.!Transcriptional!activation!of!APETALA1!by! LEAFY.!Science!285,!582Z584.! Wang!H,!Chen!J,!Wen!J,!Tadege!M,!Li!G,!Liu!Y,!Mysore!KS,!Ratet!P,!Chen!R.!2008.!Control!of! compound!leaf!development!by!FLORICAULA/LEAFY!ortholog!SINGLE!LEAFLET1!in!Medicago! truncatula.!Plant(Physiol!146,!1759Z1772.! Wang! JW,! Czech! B,! Weigel! D.!2009.!miR156Zregulated!SPL!transcription!factors!define!an! endogenous!flowering!pathway!in!Arabidopsis!thaliana.!Cell!138,!738Z749.! Weigel!D,!Alvarez!J,!Smyth!DR,!Yanofsky!MF,!Meyerowitz!EM.!1992.!LEAFY!controls!floral! meristem!identity!in!Arabidopsis.!Cell!69,!843Z859.! Weigel!D,!Nilsson!O.!1995.!A!developmental!switch!sufficient!for!flower!initiation!in!diverse! plants.!Nature!377,!495Z500.! Weigel!DaG,!J.!2002.!Arabidopsis:!a!laboratory!manual.!Cold(Spring(Harbor,(New(York:(Cold( Spring(Harbor(Laboratory(Press.! Wigge! PA,! Kim! MC,! Jaeger! KE,! Busch! W,! Schmid! M,! Lohmann! JU,! Weigel! D.! 2005.! Integration! of! spatial! and! temporal! information! during! floral! induction! in! Arabidopsis.! Science!309,!1056Z1059.! Wu!G,!Park!MY,!Conway!SR,!Wang!JW,!Weigel!D,!Poethig!RS.!2009.!The!sequential!action!of! miR156!and!miR172!regulates!developmental!timing!in!Arabidopsis.!Cell!138,!750Z759.! Wu!G,!Poethig!RS.!2006.!Temporal!regulation!of!shoot!development!in!Arabidopsis!thaliana! by!miR156!and!its!target!SPL3.!Development!133,!3539Z3547.! Yamaguchi!A,!Wu!MF,!Yang!L,!Wu!G,!Poethig!RS,!Wagner!D.!2009.!The!microRNAZregulated! SBPZBox! transcription! factor! SPL3! is! a! direct! upstream! activator! of! LEAFY,! FRUITFULL,! and! APETALA1.!Developmental(Cell!17,!268Z278.! Yoo!SD,!Cho!YH,!Sheen!J.!2007.!Arabidopsis!mesophyll!protoplasts:!a!versatile!cell!system!for! transient!gene!expression!analysis.!Nat(Protoc!2,!1565Z1572.! Yu!H,!Xu!Y,!Tan!EL,!Kumar!PP.!2002.!AGAMOUSZLIKE!24,!a!dosageZdependent!mediator!of! the!flowering!signals.!Proc(Natl(Acad(Sci(U(S(A!99,!16336Z16341.!

! 122! ! 4!

Role!of!DOUBLE'TOP!in!the!activation!of!ABERRANT'LEAF'AND' FLOWER!during!flowering!time! Serena&Della&Pina,&Bets&Verbree,&Ronald&Breedveld,&Erik&Manders,&Erik&Souer&and&Ronald&Koes& & & & & & & & & & & & & & & & & & & S.D.P.,& E.S.& and& R.K.& 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& Chapter&4& &

Abstract!

Flowering& plants& display& a& wide& variety& of& inflorescence& architectures& due& to& the& spatioK temporal& expression& patterns& of& two& meristem& identity& genes& that& together& specify& the& “floral& fate”& or& “floral& identity”& of& a& meristem.& In& petunia& DOUBLE' TOP&(DOT)& is& an& FKbox& protein&component&of&SCF&complexes&with&E3&ubiquitin&ligases&function.&DOT&is&essential&to& specify&floral&meristem&identity&and,&trough&interaction&with&ABARRENT'LEAF'AND'FLOWER' (ALF),&activates&floral&organ&identity&genes.&Although&ALF&is&expressed&in&the&whole&flower& meristem& (FM),& DOT& expression& pattern& hardly& overlaps& with& that& of& ABC& genes.& Here& we& show&that&the&DOT&protein,&after&being&synthesized&in&cells&at&the&periphery&of&the&FM&in&the& sepals/petals&boundary,&migrates&into&the¢er&of&floral&meristem.&Moreover&we&showed& that&in&order&to&activate&ABC&genes,&DOT&interacts&with&ALF,&suggesting&that&it&activates&ALF& by&a&posttranslational&mechanism.& & &

124&& DOT&movement&and&function! ! & & Introduction!

The&first&steps&in&the&development&of&a&flower&involve&the&activation&of&a&handful&of&floral& meristem&identity&(FMI)&genes,&which&specify&the&floral&fate&of&meristems.&Hence,&the&spatial& temporal®ulation&of&FMI&gene&expression&is&a&key&factor&that&determines&when&(flowering& time)& and& where& (inflorescence& architecture)& flowers& are& formed,& and& where,& within& the& flower,&organs&like&sepals,&petals,&stamens&and&carpels&are&formed.&LEAFY&from&Arabidopsis& and&homologs&from&a&broad&range&of&Angiosperms,&such&as&ABERRANT'LEAF'AND'FLOWER& (ALF)&from&petunia,&encode&a&unique&transcription&factor&that&is&essential&for&FMI&(Souer'et' al.,&1998;&Weigel&and&Nilsson,&1995).&In&petunia&and&tomato&specification&of&FMI&requires&in& addition& an& FKbox& protein,& encoded& by& DOUBLE' TOP& and& ANANTHA& respectively,& and& mutations& in& DOT& or& AN& cause& a& nearly& complete& transformation& of& flowers& into& inflorescence& shoots& which& lack& floral& characteristics& (Lippman' et' al.,& 2008;& Souer' et' al.,& 2008).& For& unknown& reasons& mutations& in& the& homologous& genes& from& Arabidopsis& (UNUSUAL'FLORAL'ORGANS,'UFO),&Antirrhinum&(FIMBRIATA,'FIM)&and&other&species&cause& only& a& mild& FMI& defect& (Samach' et' al.,& 1999;& Simon' et' al.,& 1994;& Taylor' et' al.,& 2001).& In& Arabidopsis&ufo&mutants,&for&example,&the&very&first&formed&flowers&are&transformed&into&coK inflorescences,&while&later&flowers&do&form&but&present&defects&in&the&development&of&petals& and&stamens&(Hepworth'et'al.,&2006;&Levin&and&Meyerowitz,&1995;&Wilkinson&and&Haughn,& 1995).&& In& Arabidopsis& LFY& binds& to& and& activates& hundreds& of& downstream& genes,& including& different&sets&of&homeotic&floral&organ&identity&genes,&known&as&type&A,&B,&C,&D&and&E&genes,& in& specific& subdomains& of& the& floral& meristem& (Busch' et' al.,& 1999;& Lohmann& and& Weigel,& 2002;&Parcy'et'al.,&1998;&William'et'al.,&2004;&Winter'et'al.,&2011).&Because&LFY&is&expressed& throughout&the&emerging&floral&meristem,&it&is&thought&to&act&in&combination&with&distinct& partners& that& are& expressed& in& more& restricted& patterns,& to&activate& downstream& genes& in& different&subdomains&of&the&flower&(Jack,&2004;&Krizek&and&Fletcher,&2005).&Transcription&of& the&CKtype&organ&identity&gene&AGAMOUS&(AG),&which&specifies&the&identity&of&stamen&and& carpel&primordia,&for&instance,&is&restricted&to&the¢er&of&the&flower&because&that&requires& simultaneous&binding&of&LFY&and&the&homeodomain&transcription&factor&WUSCHEL&(WUS)&to& distinct&sites&in&AG'(Lohmann'et'al.,&2001).&It&was&proposed&early&on&that&UFO&acts&as&a&coK regulator& of& LFY& to& ensure& that& the& B& type& organ& identity& genes& APETELA& 3& (AP3)& and& PISTILATA&(PI)&are&activated&in&whorls&2&and&3&(Chae'et'al.,&2008;&Samach'et'al.,&1999).&This& was&based&on&the&findings&that&(i)&UFO&and&BKgene&expression&patterns&overlap,&(ii)&that&ufo& mutation&reduces&BKgene&expression&and&that&(iii)&ectopic&expression&of&UFO&induces&B&gene& expression&outside&their&normal&domain.&Given&that&UFO&is&the&FKbox&component&of&an&SCFK

& 125& Chapter&4& & type& ubiquitin& ligase,& it& was& assumed& that& UFO& targets,& via& ubiquitinylation,& an& unknown& inhibitory&protein&for°radation.&& A&range&of&subsequent&findings&in&petunia&and&Antirrhinum&were&incompatible&with&this& early&model,&and&suggested&a&slightly&different&function&for&UFO&and&homologs&(Simon'et'al.,& 1994;&Souer'et'al.,&2008).&First,&it&was&found&that&UFO&and&the&petunia&homolog&DOT&directly& interact&with&LFY&and&ALF,&and&that&UFO&can&bind,&via&LFY,&to&the&PI&promoter&(&Souer'et'al.,& 2008;& Chae' et' al.,& 2008).& This& suggested& that& the& role& of& UFO/DOT& is& to& promote& the& transcription& activation& potential& of& ALF/LFY& rather& than& triggering& the& degradation& of& an& unknown& (intermediate)& inhibitor.& Second,& strong& dot& alleles& cause,& unlike& ufo,& a& (near)& complete&transformation&of&flowers&into&shoots,&and&constitutive&coKexpression&of&DOT&(or& UFO)&and&ALF&(or&LFY)&activated&in&petunia&seedlings&a&broad&set&of&organ&identity&genes&of& the&B,&C&and&E&class&(AKtype&genes&were¬&tested),&indicating&that&DOT/UFO&is&involved&in& the&activation&of&many&more&LFYKregulated&genes&than&BKtype&organ&identity&genes.&Third,& within& the& developing& flower& DOT& mRNA& is& restricted& to& narrow& stripe& of& cells& at& the& boundary&of&whorls&1&(sepals)&and&2&(petals)&and,&unlike&UFO,&does&hardly&or¬&overlap&with& the& (future)& expression& domain& of& mRNAs& of& ALF& or& the& downstream& B& and& C& genes.& To& reconcile&these&findings&we&proposed&a&refined&version&of&the&model,&which&stated&that&(i)& the&DOT&protein&may&travel&between&cells&from&the&whorl&1/2&boundary&toward&the¢er&of& the&floral&meristem&into&the®ion&where&B&and&C&genes&are&activated,&potentially&forming&a& gradient,& and& (ii)& that& the& role& of& DOT& and& UFO& is& to& boost& the& transcription& activation& potential& of& ALF/LFY& by& a& postKtranslational& mechanism& that& might& be& similar& to& the& way& some&yeast&transcription&factors&get&activated&by&the&ubiquitin&proteasome&system.&& To& assess& the& validity& of& this& refined& model& we& have& put& the& two& major& underlying& premises&to&a&test.&Here&we&show&that&ALF&can&activate&the&promoters&of&AG,&a&CKtype&organ& identity&gene,&and&AP1,&a&gene&with&FMI&and&AKtype&organ&identity&function&in&yeast,&but&only& if& it& is& coKexpressed& with& DOT& and& that& in& planta& a& genetic& fusion& of& Ubiquitin& to& ALF& can& rescue&the&FMI&and&organ&identity&defects&in&dot&mutants.&Furthermore&we&provide&evidence& that&GFPKtagged&DOT&protein&is&mobile&and&it&can&travel&from&the&sepal/petal&boundary&to& the¢er&of&the&floral&meristem.&& &

Results!

Role!of!DOT!in!posttranslational!activation!of!ALF!!

Although&LFY&can&bind&to&specific&binding&sites&in&subordinate&floral&organ&identity&genes&on& its&own,&additional&factors&are&required&to&activate&transcription.&For&example,&transcriptional& activation& of& AGAMOUS& in& Arabidopsis& or& in& yeast& requires& coKexpression& of& LFY& with& the&

126& DOT&movement&and&function! ! & & transcription&factor&WUS&(Lenhard'et'al.,&2001;&Lohmann'et'al.,&2001).&In&petunia&seedlings,& ALF&can&ectopically&activate&a&wide&array&of&floral&organ&identity&genes,&but&only&when&coK expressed&with&DOT&(Souer'et'al.,&2008).&In&yeast&and&Arabidopsis&cells&the&requirement&of& additional&proteins&for&activation&of&AGAMOUS&promoter&(pAG)&can&be&bypassed&by&fusion&of& an&heterologous&transcription&activation&domain,&such&as&VP16&for&example,&to&LFY&(Lohmann' et'al.,&2001;&Parcy'et'al.,&1998).&We&hypothesized&that&UFO/DOT&may&be&the&coKfactor&that&is& required&in&order&to&activate&B&and&C&genes.&In&order&to&test&this&idea,&we&analyzed&in&a&yeast& oneKhybrid&assay&whether&ALF&and&DOT&can&activate®ulatory&sequences&of&AG,&which&is&a&C& gene&of&Arabidopsis,&using&the&same&pAG:LacZ&&reporter&construct&previously&used&by&Parcy& et&al.&We&found&that&either&ALF&or&DOT&alone&are&incapable&of&promoting&strong&pAG:lacZ& expression& in& yeast,& whereas& coKexpression& of& ALF& and& DOT& strongly& induced& pAG:lacZ& expression&(Figure.&1A).&We&next&quantified&the&activity&of&the&LacZ reporters&fused&to&either& AG& enhancers& or& a& promoter& element& of& the& AKtype& gene& AP1& (Parcy' et' al.,& 1998)& by& measuring& hydrolysis& of& the& chromogenic& substrate& oKnitrophenylKβKDKgalactoside& (ONPG)& (Miller,& 1972)& & (Fig.& 1B).& When& we& expressed& both& ALF& and& DOT& we& observed& a& strong& activation&of&the&pAG:LacZ&reporter,&while&expression&of&βKgalactosidase&in&the&presence&or& absence& of& ALF& alone& the& signal& was& barely& detectable.& Also,& the& activity& of& AP1:LacZ' was& boosted&when&by&ALF&and&DOT,&but&in&this&case&the&basal&activity&of&the&pAP1:LacZ'construct& appeared&slightly&higher.& These& experiments& indicate& that& DOT& is& necessary& and& sufficient& to& induce& the& transcription&activation&potential&of&ALF&and&can&promote&the&expression&the&A&and&C&type& floral&organ&identity&genes&AP1&and&AG.& & & & & & &

& 127& Chapter&4& &

& & Figure!1:!DOT!can!provide!transcriptional!activation!activity!to!ALF.! (A)&Expression&of&pAG:LacZ'in&yeast&cells&expressing&ALF&or&DOT&or&both&(ALF+DOT)&visualized&by&a& XgalKbased& color& assay.& (B)& βKgalactosidase& activity& of& yeast& cells& the& containing& the& reporter& gene& pAG:LacZ&(violet&bars),&pAP1:LacZ&(grey&bars)&or&none&(white&bars)&and&effector&genes&expressing&ALF,& DOT&or&both.& p35S:UB4ALF!partially!rescues!the!dot!mutant!phenotype.!

In&recent&years&it&was&shown&for&several&transcription&factors&that&in&addition&to&DNA&binding& per& se,& the& ubiquitinKproteasome& system& is& required& for& transcription& activation,& either& by& preventing&that&the&transcription&factor&is&stripped&from&the&chromatin,&or&by&enhancing&the& activity&of&the&transcription&activation&domain&(Archer'et'al.,&2008;&Salghetti'et'al.,&2001).&To& examine&whether&DOT&or&the&SCFDOT&complex&acts&to&enhance&the&transcription&activation& domain&of&ALF&by&ubiquitinylation,&we&asked&whether&the&expression&of&a&genetic&fusion&of& Ubiquitin&to&ALF&in&petunia&could&bypass&the&necessity&of&DOT.&To&this&end,&we&generated&a& translational& fusion& between& ALF& and& a& single& UBIQUITIN& and& tested& its& ability& to& complement&the&defects&seen&in&dot&mutants.&Constitutive&expression&of&a&native&ALF&protein& (p35S:ALF)'in&dot&mutant&was&unable&to&rectify&the&inflorescence&architecture&or&the&flower& identity&defects&(Fig.&2C).&This&result&is&consistent&with&earlier&data&showing&that&(i)&ALF&mRNA& remains& normally& expressed& in& dot& meristems& and& that& alf' dot& double& mutants& have& the& same& defects& as& either& single& mutant& and& (ii)& that& ALF& expression& in& vegetative& tissues,& expressed&from&the&endogene&or&a&35S:ALF&transgene,&has&no&effect&on&development,&unless& DOT&is&ectopically&expressed&(Souer'et'al.,&2008).&&

128& DOT&movement&and&function! ! & &

& & Figure! 2:! Expression! of! UB4ALF! restores! the! development! of! flowers! and! floral! organs! in! dot! mutants.! (AKB)&Wild&type&W115&and&dot'mutant&inflorescences.&(C)&Constitutive&expression&of&p35S:ALF&in&a&dot' mutant&does¬&rescue&floral&meristem&or&floral&organ&identity.&(DKG)&Rescue&of&the&dot&phenotype& by&the&p35S:UBOALF&transgene.&(DKF)&Both&inflorescence&structure&and&flowers&are&restored&almost&as& a&wild&type.&(G)&Detail&of&a&flower&on&a&strong&p35S:UBOALF&expressor.&Flowers&appear&identical&to& those& on& wild& type& inflorescences.& (H)& cDNA& and& gDNA& amplification& from& a& p35S:UBOALF' transformant,& dot& mutant& and& a& wildKtype& plant& showing& B& type& gene& expression& in& a& dot& mutant& background&and&the&presence&of&the&transposon&in&native&DOT&gene.& & When&we&ectopically&expressed&the&ubiquitinylated&form&of&ALF&(p35S:UBOALF),&in&six&out& of& twenty& independently& transformed& dot& mutants& the& mutant& phenotype& was& complemented& to& various& degrees.& Two& showed& complementation& of& the& inflorescence& architecture& in& some& branches& with& partially& fertile& flowers& (Fig.& 2D).& Three& transformants& showed&near&complete&complementation&with&a&stronger&restoration&of&the&flower&defects& and&the&inflorescence&architecture&was&restored&in&more&branches&(Fig.&2EKG)&and&one&plant& was&showing&occasional&petal&tissue&along&dot&mutant&branches&(Fig.&2F).&It&was&also&possible& to& spot& petal& tissue& appearing& around& the& main& vein& of& some& bracts,& which& is& a& typical& feature& of& plants& that& ectopically& express& DOT.& We& then& checked& whether& the&

& 129& Chapter&4& & complementation& was& really& due& to& the& presence& of& the& UBOALF& transgene& and& not& to& excision& of& the& transposon& from& the& endogenous& DOT& gene,& although& we& never& saw& any& somatic& reversion& in& the& dotA2232& allele& (used& in& this& experiments);& only& among& progeny& DOT+/A2232&heterozygote,&was&identify&a&partial&revertant&allele&(dotH2082)&where&flowers&and& the&cymose&inflorescence&structure&were&restored,&but&the&petals&showed&stripes&of&sepalK&or& leafKlike&tissue&(Souer'et'al.,&2008).&As&shown&in&Fig.&2H,&the&transposon&was&still&present&in& the&DOT&coding&sequence.&We&also&showed&the&activation&of&the&BKtype&floral&organ&identity& gene& TOMATO' BOX' 6&(TM6)& in& plants& expressing& the& UBOALF& transgene.& These& results& showed&that&when&the&protein&of&ALF&is&artificially&ubiquitined,&and&so&able&to&activate&the& floral&organ&identity&genes,&the&function&of&DOT&is&no&longer&necessary.&&

DOT!protein!migrates!from!the!adaxial!sepal!boundary!to!the!floral!meristem!center!

The& expression& domain& of& DOT& mRNA& hardly& overlaps& with& the& expression& domain& of& the& target&ABC&genes&and&therefore&the&activation&of&these&target&genes&seems&to&occur&in&a&nonK cellKautonomous&manner&(Souer'et'al.,&2008).&To&test&the&possibility&of&cellKtoKcell&movement& of&DOT,&we&generated&a&chimeric&DOT&protein&by&fusing&the&GFP&coding&sequence&at&the&CK terminus& of& the& DOT& ORF.& This& fusion& construct& was& expressed& from& the& native& DOT& promoter,& pDOT:DOTOGFP:tDOT' (Fig.& 3A).& Previous& experiments& (Chapter& 2)& showed& that& these®ulatory®ions&are&sufficient&to&fully&complement&the&dot&defects&when&fused&to&a& DOT&coding&sequence.&& First& we& characterized& the& DOTKGFP& fusion& by& constitutively& expressing& it& from& the& 35S& promoter& in& wild& type& petunia& (Fig.& S1).& We& found& that& the& p35S:DOTOGFP& transformants& flowered& precociously& compared& to& wildKtype& plants,& that& the& cymose& inflorescence& was& reduced& to& a& solitary& flower& (Fig.& S1BKC)& and& that& leaves,& bracts,& and& sepals& contained& patches&of&petalKlike&tissue&(Fig.&S1D),&similar&to&what&was&observed&with&p35S:DOT''(Souer'et' al.,& 2008).& These& results& indicate& that& the& GFP& tag& does& not& affect& the& DOT& function.& Furthermore,& we& confirmed& by& immunoblot& analysis& that& the& DOTKGFP& fusion& protein& remains&intact&and&is¬&cleaved&(Fig.&3B).&These&We&then&expressed&DOTKGFP&using&a&4.6&kb& 5‘Kflanking& sequence& (pDOT4.6)& and& a& 3’& flanking& sequence& of& DOT&(pDOT4.6:DOTOGFP:tDOT' construct)'in&a&dot&mutant&background.&We&previously&demonstrated&that&a&GUS&transgene& driven& by& these& regulatory& (pDOT4.6:GUS:tDOT)& accurately& reproduced& the& DOT& expression& pattern&and,&when&fused&to&DOT&could&fully&rectify&the&phenotype&of&dot&mutants&(Chapter& 2).&We&found&that&a&once&again&that&the&protein&fusion&showed&correct&functional'activity,& since&all&the&defects&caused&by&dot&were&completely&rescued.&The&above&results&showed&that& the&pDOT4.6:DOTOGFP:tDOT&construct&expresses&an&intact&and&functional&DOTKGFP&protein&at& the&correct&time&and&place&in&development.&& &

130& DOT&movement&and&function! ! & & &

& & Figure!3:!The!pDOT:DOT4GFP:tDOT!construct!expresses!an!intact!and!functional!DOTGGFP!protein!at! the!correct!time!and!place!in!development.! (A)&Diagram&of&pDOT:DOTOGFP:tDOT.&The&GFP&protein&is&fused&to&the&CKterminus&of&the&DOT&protein& (green).&(B)&DOTKGFP&fusion&proteins&detected&with&antiKGFP&in&meristems&of&p35S:DOTOGFP&showing& intact&protein&fusion.&A&35S:GFP&transformant&as&well&as&a&untransformed&plant&are&used&as&controls.& (CKD)& A& fully& developed& wild& type& flower& (C)& compared& to& a& flower& from& a& dot& mutant& expressing& pDOT:DOTOGFP:tDOT.&(E)&SEM&analysis&of&reproductive&wild&type&apex&showing&a&terminal&flower&(f3)& and&a&sympodial&meristem&(i).&(F)&Confocal&image&of&an&apex&on&a&pDOT:DOTOGFP:tDOT&transformant& showing& a& reproductive& apex& at& approximately& the& same& stage& as& in& E.& (G)& SEM& analysis& of& reproductive& wild& type& apex& slightly& later& in& development,& showing& a& further& developed& terminal& flower& (f3)& and& a& sympodial& meristem& (i).& (H)& Confocal& image& of& pDOT:DOTOGFP:tDOT& expressors& showing& a& floral& meristem& at& a& similar& stage& as& in& G.& Note& that& the& GFP& signal& seems& to& extend& towards&the¢er&of&the&floral&meristem&Scale&bar,&50&µm& & We&then&analyzed&the&localization&of&the&DOTKGFP&protein&fusion&in&the&apical&meristem& by&laser&scanning&confocalµscopy&&and&observed&a&bright&fluorescence&signal&within&the& emerging&FM&at&the&site&where&DOT&is&normally&expressed&(Fig.&3F&and&3G).&In&the&young&FM,& GFP&signal&was&restricted&to&a&stripe&of&cells&on&the&adaxial&side&of&sepal&primordia&(Fig.&3F).& When&all&sepal&primordia&were&formed,&GFP&expression&had&expanded&into&a&ring&marking& the&boundary&between&whorls&1&and&2.&This&localization&overlaps&with&the&expression&pattern& of&DOT&mRNA.&A&detailed&comparison&of&the&DOT&mRNA&and&the&DOTKGFP&expression&pattern& revealed&that&the&DOT&protein&moves&laterally&from&its&site&of&synthesis&to&the¢er&of&the&

& 131& Chapter&4& & floral&meristem&by&at&least&two&cell&layers&(Fig.&S2).&At&slightly&later&developmental&stages,& GFP&signal&was&visible&in&the&entire&FM&dome&forming&a&gradient&of&intensity:&in&the&source& area,&where&the&DOT&gene&is&transcribed,&the&signal&was&very&strong&and&faded&towards&the& FM& center& (Fig.& 3GKH).& Since& by& in& situ& hybridization& we& never& detected& neither& the& DOT& mRNA&nor&GUS&mRNA&of&pDOT4.6:GUS&in&the&FM¢er&(Fig&4&in&Chapter&2),&we&inferred&that& the& GFP& signal& here& was& due& to& DOT& protein& moving& from& the& cells& of& the& sepal/petal& boundary,&where&it&is&synthesized,&to&adjacent&cells&in&the&FM¢er.& &

& & Figure!4:!DOT!protein!moves!towards!the!center!of!floral!meristems.!! (A–E)&FRAP&in&the&flower&meristem&of&pDOT:DOTOGFP:tDOT&cells.&After&photobleaching&GFP&signal&is& restored&in&the&proximal&and&distal®ion&of&DOT&expression&after&150&seconds.&(A)&Longitudinal&floral& meristem& section& showing& the& situation& before& (A),& immediately& after& (B)& and& 150s& (C)& after& photobleaching.&Scale&bars,&50&µm.&(D)&FRAP&measured&after&photobleaching&of&the&marked&colored& Regions&Of&Interest&(ROIs)&in&three&different&positions&on&the&pDOT:DOTOGFP:tDOT&meristem.&150&s& after&photobleaching&almost&100%&of&the&GFP&signal&was&restored.&(E)&Detail&of&the&quantification&of& green&and&blue®ions.&& &

132& DOT&movement&and&function! ! & & FRAP!reveals!DOT!protein!movement!in!the!FM!center!

To&obtain&further&evidence&that&DOTKGFP&moves&between&cells,&we&performed&fluorescence& recovery&after&photobleaching&(FRAP)&in&several®ions&of&the&FM&dome&(Fig.&4).&FRAP&is&a& conventional& technique& for& studying& GFP& diffusion& in& biological& systems& (Axelrod' et' al.,& 1976).&We&chose&three&different&spots&in&the&flower&meristem&to&quantify&GFP&recovery&(Fig& 4A):&close&to&the&expression&site&(red&square),&just&outside&the&expression&site&(green&square)& and& in& the& FM& center& (blue& square).& Bleaching& with& maximal& laser& intensity& (10s)& was& followed&by&150&seconds&tracking&of&fluorescence&recovery&with&imaging&every&10&seconds& (Fig.&4BKC).&The&FRAP&experiments&showed&that&the&signal&within&the®ions&of&interest&(ROI)& recovered&by&∼45%&after&20&seconds&and&almost&completely&after&30&seconds&or&longer&in&all& the&ROIs&(Fig.&4D).&When&we&focused&on&the&areas&outside&of&DOT&transcription®ion&(green& and&blue&ROIs)&(Fig.&4E),&we&could&clearly&see&a&negative&relationship&between&the&velocity&of& GFP& recovery& and& the& distance& from& the& expression& site.& Moreover,& in& the& center& of& the& meristem& GFP& signal& was& much& lower& compared& to& the& other& ROIs,& which& caused& the& scattering&in&GFP&signal&(“noise”)&that&resulted&in&the&“zigKzag“&quantification&of&the&GFP&(Fig.& 4E).& This& fast& recovery,& especially& in& the& green& and& blue& regions,& could& be& explained& only& assuming&that&the&protein&can&move&between&cellKtoKcell.&& &

Discussion!

Although& the& UFO& gene,& and& its& Antirrhinum& homologs& FIMBRIATA& were& discovered& two& decade& ago,& their& mode& of& action& is& still& poorly& understood.& Here& we& report& data& on& the& petunia& homologs,& DOT,& indicating& that& (i)& DOT& can& boost& the& transcription& activation& potential&of&ALF&and&(ii)&that&DOT&proteins&travels&between&cells&in&a&floral&meristem&and,& hence,&is&involved&in&the&transcriptional&activation&of&a&broad&set&of&ALF®ulated&genes.&& Even& though& LFY& can& bind& functional& cisKregulatory& elements& in& downstream& floral& organ& identity& genes,& it& cannot& activate& transcription& of& reporter& genes& containing& these& sites& in& yeast& cells,& suggesting& that& in' vivo& additional& plant& proteins& are& required.& The& fusion& of& heterologous&transcription&activation&domain&to&LFY&can&byKpass&their&absence&(Parcy'et'al.,& 1998).&Our&finding&that&ALF&can&activate&the&same&reporters&if&coKexpressed&with&DOT&has& multiple&implications.&First,&it&suggests&that&DOT&is&one&of&the&longKsought&proteins&whose& role& can& be& byKpassed& in& yeast,& and& possibly& also& in& planta,& with& the& VP16& fusion.& Interestingly,& the& transcription& activation& capacity& of& VP16& in& yeast& fully& depends& on& an& endogenous& FKbox& protein& (Met30)& that& binds& to& VP16& itself& (Salghetti' et' al.,& 2001).& This& implies&that&VP16&fusion&provides&to&ALF/LFY,&in&the&absence&of&DOT/UFO,&an&alternative&way& to& recruit& an& SCFKcomplex& and& the& related& ubiquitin& proteasome& machinery.& Second,& it&

& 133& Chapter&4& & proves& that& the& binding& of& ALF/LFY& to& UFO/DOT,& seen& in& yeast& two& hybrid& assays& and& bimolecular& fluorescence& complementation& (“split& YFP”),& is& functionally& relevant.& Third,& it& confirms& that& DOT/UFO& can& activate& besides& BKtype& genes,& also& A& and& CKtype& genes& in& a& direct&manner.&& Over& the& past& years& ubiquitinylation& has& arisen& as& an& additional& postKtranslational& regulation& mechanism.& The& consequence& of& polyKubiquitinylation& usually& is& destruction& by& the&26S&proteasome&pathway.&However,&ubiquitinylation&may&have&different&consequences& for& the& substrate& protein& (Haglund& and& Dikic,& 2005;& Kanayama' et' al.,& 2004;& Miranda& and& Sorkin,& 2007;& Weake& and& Workman,& 2008).& Activation& of& a& transcription& factor& appears& difficult& to& reconcile& with& degradation& via& the& ubiquitinylation/proteasome& pathway.& However,&a&range&of&findings&led&to&the&conclusion&that&ubiquitinylation&and&proteolysis&of& transcription& activators& is& essential& to& activate& gene& transcription& (Lipford' et' al.,& 2005;& Muratani'et'al.,&2005;&Muratani&and&Tansey,&2003;&Salghetti'et'al.,&2001),&although&a&general& way&in&which&ubiquitin&acts&has¬&been&described&yet.&Ubiquitin&might&promote&cleavage&of& inhibitory& domains& that& block& nuclear& entry& or& interaction& with& other& proteins& in& a& proteasomeKdependent&manner&(Conaway'et'al.,&2002).&In&our&case,&however,&it&is&unlikely& that& DOT& is& required& for& nuclear& entry& of& ALF& since& it& was& shown& that& ALF& reaches& thKe& nucleus& in& cells& that& lack& DOT& (Souer' et' al.,& 2008).& Recently& it& has& been& proposed& that& monoubiquitylation& protects& an& activator& from& the& “stripping”& activity& of& the& proteasomal& ATPases& (Ferdous' et' al.,& 2007).& In& this& view& the& coupling& between& activator& monoK ubiquitylation&and&the&ability&to&resist&the&destabilization&process&maintain&the&activatorKDNA& binding&allowing&promoter&escape&and&elongation.&Although&ALF&is&already&present&in&both& floral&and&inflorescence&meristems,&there&is&no&expression&of&floral&homeotic&genes&until&also& DOT&is&transcribed.&Thus&monoKubiquitylation&performed&by&DOT&on&ALF&might&be&necessary& to&activate/protect&ALF&function&from&promoter&stripping.&& The&finding&that&a&monoKubiquitinated&form&of&ALF&was&able&to&partially&rescue&the&dot& phenotype,& strongly& suggests& that& ubiquitinylation& of& ALF& performed& by& SCFDOT& complex& is& the&key&process&for&flowers&development&in&petunia.&The&fact&that&the&p35S:UBOALF&was¬& able& to& fully& complement& all& the& dot& defects& was& probably& because& in& the& genetic& fusion& ubiquitin&was&attached&(via&a&normal&peptide&bond)&to&the&NKterminal&methionine,&whereas& ubiquitinylation& normally& occurs& on& internal& lysine& residues.& Although& genetic& fusion& of& ubiquitin& to& ALF& was& sufficient& to& mimic& the& DOT& function& and& so& to& activate& the& B& and& C& genes& (Fig& 2),& it& remains& to& be& established& whether& ALFKUbiquitin& conjugates& also& form& in& vivo.& Previous& reports& suggested& the& presence& of& high& molecular& weight& LFY& isoforms& in& Arabidopsis&plants,&which&were&reduced&or&abolished&in&backgrounds&lacking&UFO&(Chae'et' al.,&2008),&but&we&have&been&unable&to&produce&these&results&(Souer'et'al.,&2008).&In&petunia& seedlings& that& constitutively& expressed& ALF& and& or& DOT& we& detected& a& protein& that& was&

134& DOT&movement&and&function! ! & & slightly&larger&than&native&ALF,&which&may&represent&a&monoKubiquitinated&ALF&isoform&or&a& petalKspecific& protein& that& binds& aKspecifically& to& antiKLFY& antibodies& (coKexpression& of& ALF& and& DOT& triggers& ectopic& petal& identity).& Hence& the& existence& of& ubiquitin& ALF& conjugates,& which& may& be& rare& shortKlived& proteins& formed& only& within& the& chromatin,& remains& to& be& proven.&& Plant&meristems&integrate&several&signals&and&they&are&important&growth&sites&of&plants,& initiating&new&organs&at&specific&developmental&stage.&They&are&composed&of¢ral&cells,& which&are&undifferentiated&and&maintain&the&cell&population,&and&a&peripheral&zone&that&form& the&different&types&of&organ&primordia.&After&cell&division&plant&cells&remain&fixed&at&a&given& position& due& to& the& presence& of& a& rigid& cell& wall,& so& a& precise& communication& between& adjacent&cells&ensures&a&correct&spatial&development&of&primordia&and&what&their&identity&will& be.&This&communication&can&be&achieved&through&transcription&factors&movement&between& cells.& Here& we& have& showed& that& DOT& can& move& between& cells,& as& already& shown& for& LFY& (Sessions' et' al.,& 2000;& Wu' et' al.,& 2003),& resulting& in& a& DOT& gradient& within& the& flower.& As& expected,& the& higher& signal& from& the& DOTKGFP& fusion& was& observed& in& the& sepal/petal& boundary&(Fig&3F),&which&is&the®ion&where&DOT&is&expressed.&Previous&studies&showed&that& the& expression& level& reached& by& pDOT' is& extremely& high,& demonstrated& by& the& fact& that& pDOT4.6:GUS&resulted&in&a&ectopic&coloration&of&the&flower&meristem,&whereas&with&a&shorter& promoter&GUS&signal&was&only&visible&in&the&sepal/petal&boundary&(Chapter&2).&This&would& explain& why& petal& development& is& the& first& processes& affected& by& weak& dot& (Souer' et' al.,& 2008)&and&fim&alleles&(Ingram'et'al.,&1997).&As&the&flower&development&proceeds,&DOTKGFP& signal&is&observed&in&the&whole&flower&dome,&creating&a&gradient&toward&the¢er&of&flower& meristem&(Fig&3H).&This&behavior&resembles&a&special&category&of&signalling&molecules&called& morphogens&that&act&in&a&concentration&depended&matter&diffusing&away&from&their&original& location.&Although&the¤t&data&are&promising,&they&are&insufficient&to&conclude&whether& DOT&act&like&morphogens.&& The&movement&and&nonKcellKautonomous&activity&in&plants&is&predicted&to&occur&for&17– 29%&of&transcription&factors&(Rim'et'al.,&2011).&So&far,&it&is&poorly&understood&what&make&a& transcription& factors& able& to& move,& if& a& specific& sequence& is& sufficient& to& make& mobile& an& immobile&protein.&Generally&transcription&factors&that&localized&in&the&cytoplasm&are&though& to&be&able&to&move,&either&by&nonKtargeted&passive&diffusion&or&active&targeted&movement.& So& far& it& is& still& unclear& whether& DOT& movement& is& passively& by& diffusion& or& facilitated& by& bound&to&a&carrier.&& Taken& together,& our& findings& suggest& that& DOT& protein& moves& from& the& region& were& is& expressed&toward&the¢er&of&the&flower&meristem&dome&forming&a&protein&gradient&(Fig& 5).& Since& the& transcription& factor& ALF& is& the& target& of& DOT,& the& consequence& of& the& DOT&

& 135& Chapter&4& & gradient&is&the&generation&of&an&ubiquitinated&ALF&gradient.&Once&ALF&is&in&the&active&form,&it& can& activate& the& downstream& organ& identity& genes& (Fig& 5).& Whether& this& ALFKDOTKUB& interaction&take&place&before&or&after&DOT&movement&it&still&remain&to&be&proved.&&

& & Figure!5:!Model!for!the!role!of!DOT!in!the!activation!of!distinct!classes!of!organ!identity!genes.!! Scanning&electronµscopy&of&a&mature&flower&meristem&of&petunia&W138,'colors&represent&the& different&organ&tissues:&green&for&sepals,&violet&for&sepals,&yellow&for&stamens&and&red&for&carpels.&The& blocks& at& the& top& indicate& organ& primordia& in& whorls& 1& to& 4& (W1& to& W4).& Bars& indicate& expression& patterns&of&various&mRNAs&and/or&proteins.&Red&color&denotes&a&high&concentration&and&yellow&lower& concentrations&of&proteins.&The&expression&pattern&of&TER/WUS&proteins&is&hypothetical,&as&indicated& by&the&question&marks.&To&date&AKtype&organ&identity&genes&similar&to&AP1&have&still&to&be&identified&in& petunia.& & &

136& DOT&movement&and&function! ! & & Materials!and!methods!

Construction!of!transgenes!and!plant!transformation!

The& p35S:UBOALF& was& created& by& ligating& an& XbaI/BamHI& digested& PCR& fragment& spanning& the&ALF&coding&sequence,&which&we&generated&from&a&petunia&cDNA&library&with&the&primers& listed&in&table&1,&and&of&UB&in&between&the&CaMV35S&promoter&and&the&CaMV&35S&terminator& of& the& binary& vector& pGreen7K& (Hellens' et' al.,& 2000).& The& p35S:DOT:GFP& and& pDOT:DOTO GFP:tDOT'constructs&were&generated&using&the&primers&listed&in&table&1&.&The&p35S&and&t35S& were& amplified& from& the pK2GW7.0& destination& vectors.& The& DOT:GFP& fragment& was& generated& from& a& previous& construct& used& in& Souer& et& al.,& 2008.& The& promoter& and& terminator& region& of& DOT& were& amplified& fro& W115& gDNA.& Each& PCR& fragments& were& recombined&into&the&GATEWAY&pDONR221&vector&(Invitrogen)&and&then&transferred&in&the& desired&destination&vectors&using&the&Gateway&multisite&recombination&system&(Karimi'et'al.,& 2002).& All&the&constructs&were&lified&with&Phusion&HighKFidelity&DNA&Polymerase&(Finzymes)& and& sequenced& with& Big& Dye& terminator& technology& (Perkin& elmer)& before& they& were& transformed& into& petunia& plants& by& Agrobacterium& tumefaciens& mediated& leaf& discs& transformation&(Spelt'et'al.,&2000).&

Yeast!oneGhybrid!assay!

The&yeast&oneKhybrid&experiments&were&performed&with&the&full&length&ALF&and&DOT'coding& sequence&into&the&yeast&expression&vector&pDS848.&AP1&and&AG®ulatory&sequence&were& fused&in&front&at&the&β-GAL&(constructs&provided&by&Detlef&Weigel&(Busch'et'al.,&1999;&Parcy' et'al.,&1998)&).&Yeast&strain&PJ69K4A/α&were&transformed&with&the&ALF&or&DOT&(A)&and&AP1&or& AG&(α)&constructs&using&the&lithium&acetate&method&(Gietz'et'al.,&1992).&Colonies&were&grown& for& a& few& days& on& dropKout& plates& lacking& leucine,& tryptophan& and& histidine& (KLTH).& LacZ' reporter& activation& was& assayed& by& a& semiKquantitative& overlay& assay.& Yeast& spotted& and& grown&on&KLT&selective&plates&was&permeabilized&by&chloroform,&and&subsequently&covered& with&XKGal&containing&topKagar&(1%&low&melting&point&agar&in&0.1M&KPO4&buffer&pH&7.0,&10& mg&XKGal,&at&42ºC).&Pictures&were&taken&after&an&incubation&period&of&1&to&6&hours&at&37ºC.& Positive&colonies&from&the&previous&experiment&were&further&used&for&the&βKgalactosidase& activity& assay& that& was& measured& at& 28°C& and& pH& 7& by& using& oKnitrophenylKβKDK galactopyranoside&(ONPG)&according&to&previously&published&methods&(Miller,&1972).&&

Western!blotting!

Stable& transformed& petunia& leaves& were& used& for& protein& extraction.& The& protein& samples& were& heated& at& 100& °C& for& 2& min& and& sizeKseparated& on& polyacrylamideKSDS& protein& gel.&

& 137& Chapter&4& & Proteins& were& transferred& from& the& gel& to& a& nitrocellulose& membrane& using& the& Wet/Tank& Blotting& Systems& (BioKRad)& according& to& the& manufacturer´s& protocols.& The& primary& antibodies& used& for& western& blotting& was& antiKGFP& (Santa& Cruz& Biotechnology).& Final& concentrations&of&the&antibody&preparations&were&optimized&experimentally.&The&secondary& antibodies&was&goat&antiKmouse&IgG–HRP&(Santa&Cruz&Biotechnology).&

!In!situ!hybridization!

In&situ&hybridization&was&performed&as&described,&(Souer'et'al.,&1996;&Rebocho'et'al.,&2008)& for&double&label&hybridizations.&Antisense&fluorescein&(DOT)&and&digoxigenin&(FBP1)&labeled& RNA&were&synthetized&from&full&coding&sequences&in&vitro&using&the&T7&polymerase&(Roche).& PostKhybridization& washes& included& a& 30& minute& RNaseA& treatment& to& eliminate& nonK specifically& bound& probe& as& well& as& crossKhybridization& to& related& mRNAs.& DigoxigeninK labeled& probes& were& detected& with& an& antidigoxigenin& antibody& conjugated& to& alkaline& phosphatase& (Roche& Applied& Science)& and& Western& Blue& stabilized& alkaline& phosphatase& substrate& (Promega).& This& results& in& a& brownish& signal,& which& turns& dark& blue& after& dehydration&in&anðanol&series.&FluoresceinKlabeled&probes&were&detected&as&a&red&signal& using& an& antiKfluorescein& antibody& conjugated& with& alkaline& phosphatase& and& Fast& Red& tablets&(Roche).&&

Confocal!laser!scanning!microscopy!

Petunia& inflorescences& were& dissected& from& stable& tansformed& W115& with& pDOT:DOTO GFP:tDOT' using& a& scalpel& and& foreceps,& through& a& binocular& microscope.& Samples& were& immerged&in&water&for&confocalµscopy&and&imaged&using&a&Nikon&A1&with&appropriate& filter& sets& for& GFP& detection& (excitation& 488& nm,& emission& 505K550& nm)& and& 40x& water& immersion&objective. For&FRAP&experiments,&a&rectangular®ion&of&interest&(ROI)&of&40x40& pixel&was&applied&interactively&at&the&transversal&FMs§ion.&Bleaching&with&maximal&laser& intensity&was&followed&by&≈170&seconds&tracking&of&fluorescence&recovery&with&imaging&every& 10&seconds.&

Plant!photography!

Pictures& of& plants& were& taken& with& a& Sony& CyberKshot& DSCKRX100.& The& background& was& blacked&out&using&Adobe&Photoshop&Software.&

Acknowledgements!

We& thank& Pieter& Hoogeveen,& Daisy& Kloos,& and& Martina& Meesters& for& their& plant& care.& This& work& was& supported& by& a& grant& to& R.K.& from& the& Netherlands& Organisation& for& Scientific&

Research&(NWO).&

138& DOT&movement&and&function! ! & Supplementary!& Information!

&

& & Figure!S1:!Overexpression!of!p35S:DOT4GFP!cause!the!same!phenotypic!variation!as!p35S:DOT.' (A)& Wild& type& petunia& W115& showing& a& developing& inflorescence.& (B)& Ectopic& expression& of& DOT& causes&the&conversion&of&a&cymose&inflorescence&into&a&solitary&flower.&(C)&p35S:DOTOGFP&also&reduce& the& cymose& inflorescence& into& a& solitary& flower.& (D)& As& p35S:DOT,' strong& p35S:DOTOGFP& overexpressors&show&patches&of&petal&tissues&in&leaves.& & &

& & Figure!S2:!DOT!protein!movement!is!required!for!its!function.!

& 139& Chapter&4& & (A)&Double&localization&in&longitudinal§ion&of&a&flower&meristem&of&DOT&mRNA&(red&arrows)&and& FBP1& mRNA&(black&arrows).&(B)&Side&view&of&flower&meristem&showing&DOTKGFP&protein&extending& from&the&DOT&expression&site&(red&arrow)&toward&the¢er&of&FM.&(C)&GUS&staining&of&pDOT4.6:GUS&in& petunia& inflorescence.& Scale& bars,& 50& µm,& *& inflorescence& meristem,& dashed& line& indicates& the& meristem’s¢er.&

& & Figure!S3:!Quantification!of!GFP!signal!after!photobleaching!GFP!in!the!proximal!and!distal!region! of!DOT!expression.! (A–E)& FRAP& in& the& flower& meristem& pDOT:DOTOGFP:tDOT& cells.& (A)& Longitudinal& floral& meristem& section&showing&the&situation&before&(A),&immediately&after&(B)&and&70s&after&the&photobleaching&(C).& Scale& bars,& 50& µm.& (D)& FRAP& measured& in& three& different& position& of& the& pDOT:DOTOGFP:tDOT& meristem.& 70s& after& photobleaching& 90%& of& the& GFP& signal& was& restored.& (E)& Detailed& of& the& quantification&of&green&and&blue®ions.&& & & & & & & & &

140& DOT&movement&and&function! ! & Primer& & Sequence& Fragment& 5625! AATTCCAGCTGACCACCATG&GGTCGGGATCTGTACGACG& DOT&yeast& 5626! GATCCCCGGGAATTGCCATG&TCAGTTGAAAGATTGAAAGGGTAA& DOT&yeast& 5627! CGGGATCC&ATGCATCATCATCATCATCATAAC& ALF&yeast& 5628! GCGAGCTC&CTAAAAATGAGGCAGATGATCAACAC& ALF&yeast& 1403! GCTCTAGAGACCCAGAGGCTTTCTCAGCA& ALF& 264! GCTCTAGATGAACAATGCAGGGATTTC& ALF& 1404! GCTCTAGAATGCAGATCTTTGTCAAGACC& UB& 1405! GCTCTAGAGGCACCACGGAGACGGAGGAC& UB& 3884! GGGGACAAGTTTGTACAAAAAAGCAGGCTACTAGAGCCAAGCTGATCTCC& p35S& 3886! GGGGACAACTTTGTATAGAAAAGTTGGGTTCGACTAGAATAGTAAATTGTAATG& p35S& 3887! GGGGACAACTTTGTATAATAAAGTTGCGGCCATGCTAGAGTCCGC& t35S& 3888! GGGGACCACTTTGTACAAGAAAGCTGGGTAGGTCACTGGATTTTGGTTTTAG& t35S& 3895! GGGGACAACTTTTCTATACAAAGTTGATGGAAGCTTTTCATCATGCA& DOT:GFP&& 3898! GGGGACAACTTTATTATACAAAGTTGTTACTTGTACAGCTCGTCCATGC& DOT:GFP&& 5636! GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAAACATTAAGATAAACTCA& pDOT& 3891! GGGGACAACTTTTGTATACAAAGTTGCTTGTCTAGGATAATTGAAGGTATAC& pDOT& 5566! GGGGACAACTTTGTATAATAAAGTTG&TACTTTTGAACTGTTTCAAGTGG& tDOT& 5551! GGGGACCACTTTGTACAAGAAAGCTGGGT&AATGAAAACGTAATGCAATAC& tDOT&

& 141& Chapter&4& & References!

Archer! CT,! Delahodde! A,! Gonzalez! F,! Johnston! SA,! Kodadek! T.& 2008.& Activation& domainK dependent& monoubiquitylation& of& Gal4& protein& is& essential& for& promoter& binding& in& vivo.& Journal'of'Biological'Chemistry&283,&12614K12623.& Axelrod!D,!Koppel!DE,!Schlessinger!J,!Elson!E,!Webb!WW.&1976.&Mobility&measurement&by& analysis&of&fluorescence&photobleaching&recovery&kinetics.&Biophys'J&16,&1055K1069.& Busch!MA,!Bomblies!K,!Weigel!D.&1999.&Activation&of&a&floral&homeotic&gene&in&Arabidopsis.& Science&285,&585K587.& Chae! E,! Tan! QKG,! Hill! TA,! Irish! VF.& 2008.& An& Arabidopsis& FKbox& protein& acts& as& a& transcriptional&coKfactor&to®ulate&floral&development.&Development&135,&1235K1245.& Conaway! RC,! Brower! CS,! Conaway! JW.& 2002.& Emerging& roles& of& ubiquitin& in& transcription& regulation.&Science&296,&1254K1258.& Ferdous! A,! Sikder! D,! Gillette! T,! Nalley! K,! Kodadek! T,! Johnston! SA.&2007.&The&role&of&the& proteasomal& ATPases& and& activator& monoubiquitylation& in& regulating& Gal4& binding& to& promoters.&Genes'Dev&21,&112K123.& Gietz! D,! St! Jean! A,! Woods! RA,! Schiestl! RH.& 1992.& Improved& method& for& high& efficiency& transformation&of&intact&yeast&cells.&Nucleic'Acids'Res&20,&1425.& Haglund!K,!Dikic!I.&2005.&Ubiquitylation&and&cell&signaling.&Embo'Journal&24,&3353K3359.& Hellens!RP,!Edwards!EA,!Leyland!NR,!Bean!S,!Mullineaux!PM.&2000.&pGreen:&a&versatile&and& flexible&binary&Ti&vector&for&AgrobacteriumKmediated&plant&transformation.&Plant'Molecular' Biology&42,&819K832.& Hepworth! SR,! Klenz! JE,! Haughn! GW.& 2006.& UFO& in& the& Arabidopsis& inflorescence& apex& is& required&for&floralKmeristem&identity&and&bract&suppression.&Planta&223,&769K778.& Ingram! GC,! Doyle! S,! Carpenter! R,! Schultz! EA,! Simon! R,! Coen! ES.& 1997.& Dual& role& for& fimbriata&in®ulating&floral&homeotic&genes&and&cell&division&in&Antirrhinum.&Embo'Journal& 16,&6521K6534.& Jack!T.&2004.&Molecular&and&genetic&mechanisms&of&floral&control.&Plant'Cell&16!Suppl,&S1K17.& Kanayama!A,!Seth!RB,!Sun!L,!Ea!CK,!Hong!M,!Shaito!A,!Chiu!YH,!Deng!L,!Chen!ZJ.&2004.&TAB2& and&TAB3&activate&the&NFKkappaB&pathway&through&binding&to&polyubiquitin&chains.&Mol'Cell& 15,&535K548.& Karimi! M,! Inze! D,! Depicker! A.&2002.&GATEWAY&vectors&for&AgrobacteriumKmediated&plant& transformation.&Trends'in'Plant'Science&7,&193K195.& Krizek! BA,! Fletcher! JC.& 2005.& Molecular& mechanisms& of& flower& development:& an& armchair& guide.&Nat'Rev'Genet&6,&688K698.&

142& DOT&movement&and&function! ! & & Lenhard!M,!Bohnert!A,!Jurgens!G,!Laux!T.&2001.&Termination&of&stem&cell&maintenance&in& Arabidopsis& floral& meristems& by& interactions& between& WUSCHEL& and& AGAMOUS.& Cell& 105,& 805K814.& Levin!JZ,!Meyerowitz!EM.&1995.&UFO:&an&Arabidopsis&gene&involved&in&both&floral&meristem& and&floral&organ&development.&Plant'Cell&7,&529K548.& Lipford! JR,! Smith! GT,! Chi! Y,! Deshaies! RJ.& 2005.& A& putative& stimulatory& role& for& activator& turnover&in&gene&expression.&Nature&438,&113K116.& Lippman!ZB,!Cohen!O,!Alvarez!JP,!AbuGAbied!M,!Pekker!I,!Paran!I,!Eshed!Y,!Zamir!D.&2008.& The& making& of& a& compound& inflorescence& in& tomato& and& related& nightshades.& PLoS' Bio& 6,& e288.& Lohmann!JU,!Hong!RL,!Hobe!M,!Busch!MA,!Parcy!F,!Simon!R,!Weigel!D.&2001.&A&molecular& link&between&stem&cell®ulation&and&floral&patterning&in&Arabidopsis.&Cell&105,&793K803.& Lohmann! JU,! Weigel! D.& 2002.& Building& beauty:& the& genetic& control& of& floral& patterning.& Developmental'Cell&2,&135K142.& Miller!JH.&1972.&Experiments&in&molecular&genetics.&Cold'Spring'Harbor'Laboratory.& Miranda! M,! Sorkin! A.&2007.&Regulation&of&receptors&and&transporters&by& ubiquitinylation:& new&insights&into&surprisingly&similar&mechanisms.&Mol'Interv&7,&157K167.& Muratani! M,! Kung! C,! Shokat! KM,! Tansey! WP.&2005.&The&F&box&protein&Dsg1/Mdm30&is&a& transcriptional& coactivator& that& stimulates& Gal4& turnover& and& cotranscriptional& mRNA& processing.&Cell&120,&887K899.& Muratani! M,! Tansey! WP.& 2003.& How& the& ubiquitinKproteasome& system& controls& transcription.&Nat'Rev'Mol'Cell'Biol&4,&192K201.& Parcy! F,! Nilsson! O,! Busch! MA,! Lee! I,! Weigel! D.& 1998.& A& genetic& framework& for& floral& patterning.&Nature&395,&561K566.& Rebocho!AB,!Bliek!M,!Kusters!E,!Castel!R,!Procissi!A,!Roobeek!I,!Souer!E,!Koes!R.&2008.&Role& of&EVERGREEN&in&the&development&of&the&cymose&petunia&inflorescence.&Developmental'Cell& 15,&437K447.& Rim!Y,!Huang!L,!Chu!H,!Han!X,!Cho!WK,!Jeon!CO,!Kim!HJ,!Hong!JC,!Lucas!WJ,!Kim!JY.&2011.& Analysis&of&Arabidopsis&transcription&factor&families&revealed&extensive&capacity&for&cellKtoK cell&movement&as&well&as&discrete&trafficking&patterns.&Mol'Cells&32,&519K526.& Salghetti! SE,! Caudy! AA,! Chenoweth! JG,! Tansey! WP.& 2001.& Regulation& of& transcriptional& activation&domain&function&by&ubiquitin.&Science&293,&1651K1653.& Samach!A,!Klenz!JE,!Kohalmi!SE,!Risseeuw!E,!Haughn!GW,!Crosby!WL.&1999.&The&UNUSUAL& FLORAL& ORGANS& gene& of& Arabidopsis& thaliana& is& an& FKbox& protein& required& for& normal& patterning&and&growth&in&the&floral&meristem.&Plant'Journal&20,&433K445.& Sessions! A,! Yanofsky! MF,! Weigel! D.& 2000.& CellKcell& signaling& and& movement& by& the& floral& transcription&factors&LEAFY&and&APETALA1.&Science&289,&779K782.& & 143& Chapter&4& & Simon! R,! Carpenter! R,! Doyle! S,! Coen! E.& 1994.& Fimbriata& controls& flower& development& by& mediating&between&meristem&and&organ&identity&genes.&Cell&78,&99K107.& Souer! E,! Rebocho! AB,! Bliek! M,! Kusters! E,! de! Bruin! RAM,! Koes! R.& 2008.& Patterning& of& inflorescences& and& flowers& by& the& FKbox& protein& DOUBLE& TOP& and& the& LEAFY& homolog& ABERRANT&LEAF&AND&FLOWER&of&Petunia.&Plant'Cell&20,&2033K2048.& Souer!E,!van!der!Krol!A,!Kloos!D,!Spelt!C,!Bliek!M,!Mol!J,!Koes!R.&1998.&Genetic&control&of& branching& pattern& and& floral& identity& during& Petunia& inflorescence& development.& Development&125,&733K742.& Souer!E,!van!Houwelingen!A,!Kloos!D,!Mol!J,!Koes!R.&1996.&The&no&apical&meristem&gene&of& Petunia& is& required& for& pattern& formation& in& embryos& and& flowers& and& is& expressed& at& meristem&and&primordia&boundaries.&Cell&85,&159K170.& Spelt! C,! Quattrocchio! F,! Mol! JN,! Koes! R.& 2000.& anthocyanin1& of& petunia& encodes& a& basic& helixKloopKhelix&protein&that&directly&activates&transcription&of&structural&anthocyanin&genes.& Plant'Cell&12,&1619K1632.& Taylor!S,!Hofer!J,!Murfet!I.&2001.&STAMINA'PISTILLOIDA,&the&pea&ortholog&of&FIM&and&UFO,&is& required&for&normal&development&of&flowers,&inflorescences,&and&leaves.&Plant'Cell&13,&31K46.& Weake! VM,! Workman! JL.&2008.&Histone&ubiquitinylation:&triggering&gene&activity.&Mol'Cell& 29,&653K663.& Weigel!D,!Nilsson!O.&1995.&A&developmental&switch&sufficient&for&flower&initiation&in&diverse& plants.&Nature&377,&495K500.& Wilkinson! MD,! Haughn! GW.& 1995.& Unusual& Floral& Organs& Controls& Meristem& Identity& and& Organ&Primordia&Fate&in&Arabidopsis.&Plant'Cell&7,&1485K1499.& William!DA,!Su!Y,!Smith!MR,!Lu!M,!Baldwin!DA,!Wagner!D.&2004.&Genomic&identification&of& direct&target&genes&of&LEAFY.&Proc'Natl'Acad'Sci'U'S'A&101,&1775K1780.& Winter!CM,!Austin!RS,!BlanvillainGBaufume!S,!Reback!MA,!Monniaux!M,!Wu!MF,!Sang!Y,! Yamaguchi!A,!Yamaguchi!N,!Parker!JE,!Parcy!F,!Jensen!ST,!Li!HZ,!Wagner!D.&2011.&LEAFY& Target& Genes& Reveal& Floral& Regulatory& Logic,& cis& Motifs,& and& a& Link& to& Biotic& Stimulus& Response.&Developmental'Cell&20,&430K443.& Wu!X,!Dinneny!JR,!Crawford!KM,!Rhee!Y,!Citovsky!V,!Zambryski!PC,!Weigel!D.&2003.&Modes& of& intercellular& transcription& factor& movement& in& the& Arabidopsis& apex.& Development& 130,& 3735K3745.& &

144& ! 5"

General"Discussion"

! ! General!Discussion!

Flowering! plants,! also! known! as! Angiosperms,! appeared! relatively! recently! during! evolution! (140! Million!years!ago)!and!nowadays!make!up!the!large!majority!of!extant!plant!species.!Angiosperms! comprise! approximately! 260,000! different! species,! which! display! a! large! variation! with! regard! to! plant!architecture,!shape,!size,!and!colour,!as!well!as!the!time!(different!seasons!and!developmental! age)! that! they! switch! from! vegetative! growth! to! flowering,! and! in! the! position! of! flowers! on! the! flower!bearing!branches!or!inflorescences.!The!variation!when!(flowering!time)!and!where!to!make! flowers!(inflorescence!architecture)!are!mechanistically!connected,!as!that!was!shown!to!relate!to! variation!in!the!spatioJtemporal!expression!pattern!of!the!genes!that!make!flowers,!such!as!floral! meristem!identity!genes.! !The!transition!from!vegetative!to!reproductive!phase!has!been!an!appealing!topic!to!the!plant! science!community!for!many!years,!and!the!importance!of!it!for!plants!itself!and!humanity!has!been! widely!accepted.!However,!although!this!process!has!been!studied!for!a!long!time,!the!mechanisms! behind! this! transition! remain! largely! unknown.! During! their! life,! flowering! plants! go! through! two! developmental!phases.!The!first!one,!the!vegetative!phase,!the!aerial!plant!body!produces!mainly! leaves! and! shoot! tissue,! allowing! the! plant! to! grow,! produce! and! store! energy.! The! second! one,! referred! to! as! the! reproductive! phase,! is! characterized! by! the! formation! of! reproductive! organs,! more!commonly!known!as!flowers.!The!correct!timing!of!the!transition!from!the!vegetative!to!the! reproductive!or!flowering!phase!can!be!regarded!as!the!most!critical!event!in!a!plants!development,! as!it!affects!the!production!of!fruit,!seed!setting!and!germination!of!the!next!generation,!all!which!is! crucial!for!its!reproductive!success!and!can!be!critical!for!species!survival.! Although! the! inflorescence! architectures! vary! enormously! between! and! within! plant! families,! they!represent!the!results!of!a!genetic!network!where!the!major!players!appear!to!be!conserved.!In! order!to!understand!how!similar!genes!can!generate!such!diversity,!it!is!necessary!to!compare!their! function! and! expression! between! different! species.! In! our! work! we! compared! two! distinct! inflorescence! architectures,! cyme! and! raceme,! and! distinct! species,! Arabidopsis,! Antirrhinum,! petunia!and!tomato!in!order!to!gain!such!knowledge.!

Changes"in"the"regulation"of"ALF/LFY"are"due"to"changes"in"the"trans6regulatory"elements""

It!is!generally!accepted!that!the!master!regulators!of!flower!development!in!the!raceme!Arabidopsis! is! represented! by! LEAFY!(LFY)!((Benlloch! et! al.,! 2007;! Blazquez! et! al.,! 2006;! Wagner,! 2009)).! LFY! encodes! a! plantJspecific! transcription! factor,! which! is! expressed! in! leaf! primordia! during! the! vegetative!phase!at!a!basal!level.!However!it!is!progressively!upJregulated!until!a!specific!threshold!is! reached!and!the!floral!transition!is!triggered.!In!developing!floral!meristems,!LFY!protein!is!expressed! homogenously,! whereas! in! the! apical! inflorescence! meristem! TERMINAL8 FLOWER8 1!(TFL1)! antagonizes!its!expression,!thus!maintaining!the!shoot!meristem!indeterminate.!Also!in!cymes!LFY!

146!! Chapter!5!! homologs!specify!floral!identity!and!it!is!known!that,!at!the!protein!level,!these!genes!are!functionally! interchangeable! between! cyme! and! raceme! species! (Souer! et! al.,! 2008).! In! cyme,! however,! LFY! homologs!such!as!ABERRANT8LEAF8AND8FLOWER!(ALF)!in!petunia!and!FALSIFLORA!(FA)!in!tomato!are! expressed!in!a!different!pattern!than!LFY.!In!early!vegetative!phase,!ALF!and!FA!mRNA!is!detected!in! incipient!leaf!primordia,!while!in!inflorescence!meristems!they!are!expressed!in!the!apical!meristem,! which!develops!into!a!flower,!and!with!a!delay!in!the!sympodial!inflorescence!meristem!(SIM),!which! emerges!in!a!lateral!position!to!form!the!next!inflorescence!metamer,!again!consisting!of!an!apical! floral!meristem!and!a!lateral!SIM!(MolineroJRosales!et!al.,!1999;!Souer!et!al.,!1998).!The!different! expression!patterns!of!LFY!homologs!in!spceies!with!cymose!and!racemose!inflorescences!might!be! due! to! differences! in! their! cisJregulatory! elements! or! in! the! upstream! transJregulating! network! (Carroll,!2008).!By!swapping!promoter:GUS!fusion!genes!between!species,!we!have!shown!in"Chapter" 2" (Fig.!1)!that!the!cisJregulatory!regions!of!LFY!and!ALF!are!functionally!similar!and!exchangeable,! despite!the!large!evolutionary!distance!between!petunia,!an!Asterid,!and!Arabidopsis,!a!Rosid.!These! results!suggest!that!alterations!in!the!upstream!network!are,!instead,!responsible!for!the!different! expression!pattern!between!the!two!genes.!! The!promoter!reporter!gene!constructs!(pLFY:GUS!and!pALF:GUS)!showed!that!in!Arabidopsis!and! petunia!as!well!the!transgenes!were!able!to!reproduce!the!same!expression!pattern!of!the!respective! endogenous!genes.!Moreover,!the!pLFY:LFY!could!fully!rescues!all!the!alf!mutant!phenotypes,!further! supporting! the! idea! that! pALF! and! pLFY! are! exchangeable.! Hence,! it! appears! that! pALF! and! pLFY! respond!to!homologous!transcription!factors,!which!have!undergone!changes!in!their!expression.!If! so,! that! has! some! interesting! and! surprising! implications.! For! instance,! it! implies! that! pALF! has! retained! the! capacity! to! be! repressed! by! TFL18 when! expressed! in! Arabidopsis8 inflorescence! meristem,!even!though!the!TFL1!homologs!from!nightshade,!such!as!SELF8PRUNING!(SP)!in!tomato! and!CEN4!in!tobacco,!have!no!role!in!(transiently)!downJregulating!LFY!homologs!in!the!sympodial! inflorescence!meristem!and!are!only!expressed!in!vegetative!axillary!meristem!(Amaya!et!al.,!1999;! Thouet! et! al.,! 2008).! Indeed! the! sp! mutant! does! not! does! not! alter! inflorescence! structure,! but! accelerates!the!termination!of!the!sympodial!meristem!progressively!faster,!leading!to!a!determinate! growth.!This!suggests!that!even!if!pALF!contains!the!responsive!elements!for!TFL1,!this!regulation!is! not!needed!for!the!maintenance!of!the!sympodial!meristem.!

Changes"in"the"regulation"of"DOT/UFO"are"due"to"changes"in"the"cis6regulatory"elements""

In! contrast! to! what! we! observed! with! pALF:GUS! and! pLFY:GUS,! we! discovered! that! reporter! genes!driven!by!pDOT8and!pUFO!maintain!their!distinct!patterns!even!in!a!heterologous!host!plant! (Chapter" 2).! This! indicates! that,! in! this! case,! alterations! in! their! cisJregulatory! elements! are! responsible!for!the!different!expression!patterns!of!UFO!and!DOT,!whereas!the!upstream!network!

! 147! General!Discussion! that!control!their!regulation!is!conserved!between!Arabidopsis!and!petunia.!Indeed,!it!is!remarkable! that!in!a!petunia!background!pUFO:GUS8is8expressed!during!the!vegetative!phase!or!in!the!center!of! young!floral!meristems!in!a!very!similar!pattern!as!pUFO:GUS!and!UFO!in!Arabidopsis,8knowing!that! DOT!is!not!active!there.!This!suggests!that!the!(unknown)!transJregulatory!factors!that!regulate!UFO! expression!in!these!tissues!are!conserved!in!both!species,!but!do!not!activate!DOT8because!it!lacks! the!responsive!cisJelements.! While! the! UFOp! is! expressed! in! all! meristems! from! the! vegetative! to! the! reproductive! stage,! pDOT! expression! is! restricted! to! a! defined! region! in! the! apical! (floral)! meristem! in! petunia.! The! observation! that! introduction! of! a! pUFO:UFO8 in! a! wild! type! petunia! host8 transgene! caused! precocious! flowering! and! the! formation! of! solitary! flowers,! providing! further! evidence! that! alterations!in!the!CREs!of!a!single!gene!may!cause!major!architectural!alterations.!! In!Arabidopsis!floral!meristems!UFO!is!initially!expressed!throughout!the!FM,!overlapping!with! the!expression!of!subordinate!B!and!CJtype!organJidentity!genes!that!specify!petal!and!stamen!fate! (Lee! et! al.,! 1997),! while! in! petunia! floral! meristems! the! expression! of! DOT! mRNA! and! the! downstream!B!and!CJtype!genes!has!little!or!no!overlap!(Souer!et!al.,!2008).!Hence,!it!was!postulated! that!DOT!protein!might!move!between!cells!in!the!FM!(Souer!et!al.,!2008).!

Functional" analysis" of" cis6elements" and" identification" of" upstream" trans6acting" factors" regulating"pDOT1

In!order!to!find!cisJregulatory!elements!in!the!promoter!of!DOT,!several!approaches!were!used!as! described!in!Chapter"3."By!comparing!the!region!upstream!and!downstream!the!DOT!locus!of!several! species,! we! discovered! that! the! overall! synteny! genes! organization! was! still! conserved! within! the! Solanaceae!family,!with!the!exception!of!petunia.!Due!to!the!presence!of!two!MuDR/Mu!transposons! upstream!and!downstream!the!PhDOT!locus,!this!region!underwent!further!rearrangements,!placing! DOT!in!a!different!genomic!environment!(Fig!1,!Chapter"3).!A!wider!analysis!of!the!DOT!loci!outside! of!the!Solanaceae!family!revealed!the!presence!of!an!inversion!in!this!region.!This!could!have!caused! loss/gain!of!transcription!factors!binding!sites,!resulting!in!the!different!expression!pattern!observed! for!UFO!and!DOT.!! When!we!compared!the!5!kB!upstream!upstream!from!the!coding!sequence!of!DOT!homologs! from!different!species,!we!were!able!to!find!four!domains!that!are!highly!conserved!in!nightshade! and! some! of! them! also! in! other! family! (Fig.! S1J5,! Chapter" 3).! This! suggests! that! although! DOT! homologs! underwent! an! inversion,! this! probably! did! not! contribute! to! the! expression! changes! because!the!breakpoint!of!these!rearrangements(s)!was!in!the!far!(>5kb)!upstream!region!and!did! not!affect!the!presence!of!the!conserved!boxes!IJIV!is!the!more!proximal!promoter!region.!

148!! Chapter!5!!

The!four!BOXes!identified!by!sequence!comparison,!and!the!fifth!by!functional!analysis!of!DOT! promoter!deletions!(Fig.!3,!Chapter"3)!might!represent!clusters!of!cisJelements!(enhancer)!involved! in!the!regulation!DOT.!It!was!proposed!that!the!distal!region!of!pDOT,!which!includes!two!of!these! BOXes!(3J4),!is!necessary!to!fully!complemented!dot,!whereas!the!proximal!region,!which!includes! the! first! three! BOXes! (1,2,! and! 5),! represents! the! minimal! promoter! fragment! able! to! mimic! the! correct! spatioJtemporal! expression! pattern! of! DOT! (Kusters! et! al.,! 2015).! We! were! able! to! locate! BOXes!1!and!3!in!most!of!the!DOT!homologs!analyzed,!such!as!FIMBRIATA!(FIM),!VvUFO!and!MgUFO,! but!in!Arabidopsis!we!could!identify!only!BOX1,!suggesting!that!the!UFO!promoter!is!the!different! one.!Indeed,!it!was!shown!that!in!petunia!background!pUFO:GUS!showed!the!a!different!expression! pattern!compared!to!pFIM:GUS!and!pAN:GUS,!that!they!were!expressed!as!pDOT.!! Since!BOX1!lies!close!to!the!transcription!start!site!and!is!indispensable!for!the!activation!of!DOT,! it!could!be!(part)!of!the!core!promoter!and!this!could!explain!why!this!box!is!conserved!in!all!the! species!analyzed.!BOX3!is!able!to!mimic!the!same!expression!pattern!as!pDOT3.18(Fig!2,!Chapter"3),! suggesting!that!this!distal!promoter!region!is!necessary!to!increase!DOT!expression.8BOX5!is!required! for! the! fineJtuning! of! DOT! expression! in! the! sepal/petal! boundary! and! showed! low! sequence! conservation.! A!more!detailed!analysis!of!the!pDOT!sequence!revealed!the!presence!of!CArGJboxes!in!the!distal! and!proximal!promoter!regions.!Interesting!these!binding!sites!for!MADSJbox!proteins!are!conserved! even! in! the! promoters! of! DOT! homologs! from! species! outside! the! Solanaceae! family,! like! in! FIMBRIATA8(FIM)!of!Antirrhinum8majus,!that,!as!mentioned!before,!it!is!functionally!similar!to!DOT.! MADSJbox!proteins!are!known!to!bind!to!this!sequence!and!are!involved!in!flowering!time,!either!as! repressor!or!promoter.!Among!the!flowering!promoters,!AGL24!and!SOC1!are!known!to!work!in!a! positive! feedback! loop! to! activate! the! expression! of! LFY! (Ferrandiz! et! al.,! 2000;! Kaufmann! et! al.,! 2010),!while!SHORT8VEGETATIVE8PHASE8(SVP)!is!known!to!repress!the!switch!to!flowering.!In!in!vivo! experiments! DOT! appears! to! be! under! control! the! same! transcription! factors! as! LFY,! and! more! specifically,!AGL24!and!SPL3!were!involved!in!its!regulation!(Fig!4,!Chapter"3).!In!our!experiments!we! could!not!detect!any!direct!activation!of!pDOT!by!FUL!or!SOC1,!even!it!Although!recent!data!indicate! PhSOC1Jlike!genes!are!also!involved!in!flowering!time!in!petunia!(Preston!et!al.,!2014),!we!could!not! detect! any! direct! activation! of! pDOT! by! FUL! or! SOC1.! That! might! be! because! SOC1Jlike! factors! activate!DOT!indirectly!via!intermediate!regulators,!which!would!be!too!slow!to!be!seen!within!the! 16!hrs!used!in!the!transient!expression!assays,!or!because!SOC1!binding!sites!in!DOT,!if!they!exist!at! all,!lie!outside!pDOT3.1!that!we!used.! The8 petunia8 genes8 EXP,! FBP13,! and! FBP25! encode! MADS! box! proteins! with! high! similarity! to! AGL24.! Phylogenetic! and! micro! synteny! data! suggest! that! the! Arabidopsis! ortholog! of! EXP! is! SVP,! which! is! supported! by! functional! data! showing! that! EXP! acts! as! a! repressor! of! flower! when!

! 149! General!Discussion! constitutively! expressed! in! Arabidopsis! (REF).! We! could,! however,! not! completely! resolve! the! relationship!between!AGL24,!FBP13!and!FBP25.!! When!FBP25!was!overexpressed!in!Arabidopsis!it!caused!a!similar!phenotype!as!35S:AGL24!(Fig.! 7,!Chapter"3),!showing!that!FBP25!is!functionally!very!similar!AGL24.!Interestingly,!35S:FBP258quickly! converted!the!apical!inflorescence!meristem!into!a!terminal!flower!in!Arabidopsis,!which!is!generally! maintained! indeterminate,! probably! due! to! ectopic! expression! of! LFY8 within! the! apical! meristem.! This!conversion!was!never!observed!before!in!35S:AGL24!(Gregis!et!al.,!2008),!suggesting!either!that! FBP25!has!evolved!additional!activity!compare!to!AGL24!–!e.g.!by!interacting!with!different!proteins!J! or!that!the!35S:AGL24!lines!analyzed!in!Arabidopsis!had!a!low!expression!level.!In!petunia!we!did!not! observe!any!alterations!in!the!inflorescence!development!when!we!constitutively!expressed!AGL24,! FBP13! or! FBP25! suggesting! that! in! petunia! these! proteins! alone! are! not! enough! to! specify! floral! identity.! These! results! are! strikingly! similar! to! previous! experiments! showing! that! constitutive! expression!of!LFY!or!ALF!as!well!as!AP1!or!its!putative!petunia!ortholog!PIE7!causes!in!Arabidopsis!the! precocious!formation!of!aberrant!inflorescences!(solitary!flowers),!whereas!in!petunia!no!effect!at!all! is!seen.!! Our! protoplast! experiments! showed! that! indeed! to! activate! pDOT! both! AGL24! and! SPL3! are! necessary,!although!these!proteins!do!not!interact!in!vivo!(fig.!S10,!Chapter"3).!More!telling!is!that! AGL24JVP16! and!SPL3JVP16! can!activate!pDOT! on!their!own,!suggesting!that!AGL24! and!SPL3! can! bind!to!pDOT!independently,!but!that!the!presence!of!both!proteins!is!required!for!transcriptional! activation.! These! results! showed! that! although! the! transcription! factors! involved! in! the! flowering! time! of! Arabidopsis! are! conserved! in! petunia,! the! proteinJDNA! interaction(s)! and! thereby! the! structure! of! the! network! have! been! modify! causing! a! different! spatioJtemporal! regulation! of! the! floral!meristem!identity!genes.! In!the!genome!of!Petunia8axillaris!we!were!able!to!find!several!SBPJbox!proteins!of!varying!size.! According!to!the!alignment!comparison!of!the!SBPJbox!proteins,!the!SBPJbox!appeared!overall!very! conserved! (Fig.! S8,! Chapter" 3)! suggesting! that! the! specific! residues! in! this! domain! have! to! be! preserved! in! order! to! allow! the! SBPJbox! protein! to! bind! DNA! and! perform! its! function! as! a! transcription! factor.! In! this! study! we! further! characterized! PhSPL3,! since! it! showed! an! expression! pattern!that!overlaps!with!the!spatial!expression!of!DOT.!However,!when!we!overexpressed!this!gene! in!petunia,!we!could!not!detect!any!changes!in!the!inflorescence!development!or!in!flowering!time! (Fig.!9,!Chapter"3),!possibly!because!that!re! AgeJdependent!flowering!time!is!known!to!have!central!role!in!Arabidopsis!under!nonJinductive! photoperiods.!As!the!plant!ages,!the!level!of!miR156!declines!allowing!the!activity!of!the!SPLs!and! thus!inducing!flowering!(Wu!and!Poethig,!2006).!Overexpression!of!miR156!in!Arabidopsis,!however,! can! be! overcome! by! inductive! long! day.! The! same! was! observed! in! petunia,! where! the!

150!! Chapter!5!! overexpression!of!the!miR156,!blocked!the!floral!transition!in!short!day!conditions,!did!not!influence! the! floral! transition! in! long! days,! demonstrating! that! also! in! the! annual! petunia,! inductive! photoperiod!can!overcome!the!ageJdependent!flowering!pathway.!! A!peculiarity!of!35S:miR156!in!petunia!was!the!failure!of!this!plant!in!the!development!of!the! sympodial! meristem,! resulting! in! plants! with! a! solitary! flower! instead! of! the! typical! cymose! inflorescence! architecture! in! long! days! condition.! In! SD! condition,! flowers! development! was! completely!abolished!and!plants!grew!only!vegetative!(Fig.!7C,!Chapter"3).!Thus,!overJexpression!of! miRNA156!has!major!effects!in!on!the!switch!from!vegetative!growth!to!flowering!and!on!sympodial! meristem! development.! The! latter! defect! was! already! observed! in! other! two! petunia! mutants:! hermit! and! extrapetals.! In! petunia,! HERMIT!(HER),! the! ortholog! of! the! homeobox! genes8 SHOOTMERISTEMLESS!(STM)!from!Arabidopsis,!is!required!for!the!initiation!and/or!maintenance!of! the!stem!cell!population!in!sympodial!meristems!(Endrizzi!et!al.,!1996).!The!her!mutants!terminates! into!a!flower!and!no!sympodial!shoot!meristem!develops.!A!similar!phenotype!was!also!observed!in! extrapetals!(exp)!mutants,!which!also!lack!sympodial!shoots!(Kusters,!2011).!Although!her!and!exp! mutants! look! similar,! they! are! the! result! of! different! processes:! HER! is! required! for! the! initiation! and/or! maintenance! of! sympodial! meristems,! whereas! EXP! represses! floral! identity! in! sympodial! meristems.! The! fact! that! the! downJregulation! of! the! SPLs,! caused! by! the! ectopic! expression! of! miR156,!also!resulted!in!petunia!plants!with!a!terminal!flower,!leads!to!two!possible!scenarios:!either! the! SPLs! genes! are! involved! in! the! regulation! of! KNOX! gene,! directly! or! indirectly,! so! that! misJ regulation!of!KNOX!results!in!the!failure!of!SYM!initiation/maintenance!or!the!SPLs!genes!“repress! the!repressor”!EXP,!directly!or!indirectly,!causing!ectopic!expression!of!DOT!in!the!apical!meristem.!

DOT"movement"and"function"

It!was!previously!shown!that!when!a!LFY:GFP!protein!fusion!was!express!under!the!control!of!L1!cell! layerJspecific! promoter,! LFY! was! able! to! move! into! inner! layers,!although!it!was!unclear!why!this! transcription! factors! need! to! move! since! it! is! express! in! all! the! three! layers! of! emerging! flower! meristem!(Sessions!et!al.,!2000;!Wu!et!al.,!2003).!In!Chapter" 4!we!report!data!supporting!the!idea! that! also! DOT! can! move! between! cells,! creating! a! protein! gradient! within! the! flower.! By! in! situ! analysis! it! was! shown! that! DOT! mRNA! localize! in! the! sepal/petal! boundary! (Souer! et! al.,! 2008),! leading!to!the!question!on!how!this!gene!could!participate!to!the!activation!of!floral!organ!identity! genes! that! are! expressed! in! the! center! of! flower! meristem.! The! same! is! observed! in! Antirrhinum! majus!where!the!expression!pattern!of!FIM!and!B!and!C!genes!hardly!overlap,!but!in!this!case!was! explained!by!the!cell!nonJautonomus!activity!of!FIM!(Schultz!et!al.,!2001).!In!Chapter"4!we!show!that! the! same! may! hold! in! petunia.! Via! protein! fusion! with! GFP!(pDOT4.6:DOTWGFP),! ,! as! expected,! the! highest!signal!was!observed!in!the!sepal/petal!boundary,!whereas!the!signal!intensity!was!decreasing!

! 151! General!Discussion! toward!the!center!of!the!emerging!flower!meristem,!proving!that!indeed!DOT!protein!was!moving! (Fig.!3F,!Chapter"4).!As!the!flower!development!proceeds,!DOTJGFP!signal!is!observed!in!the!whole! flower! dome,! creating! a! gradient! toward! the! center! of! flower! meristem! (Fig! 3H,! Chapter" 4).! This! behavior! resembles! a! special! category! of! signaling! molecules! called! morphogens! that! act! in! a! concentrationJdependent! matter! diffusing! away! from! their! original! location.! Although! the! current! data!are!promising,!they!are!insufficient!to!conclude!whether!DOT!act!like!morphogens.!! The!movement!and!nonJcellJautonomous!activity!in!plants!is!estimated!to!occur!for!17–29%!of! the!transcription!factors!(Rim!et!al.,!2011).!So!far,!it!is!poorly!understood!what!makes!a!transcription! factor!able!to!move.!Generally!transcription!factors!that!localized!in!the!cytoplasm!are!thought!to!be! able!to!move,!either!by!nonJtargeted!passive!diffusion!or!active!targeted!movement.!Whether!DOT! movement!is!passively!by!diffusion!or!facilitated!by!bound!to!a!carrier!remains!to!be!defined.!! It!was!long!known!that!the!transcription!factor!LFY!and!its!petunia!homolog!ALF!are!essential!for! specification!of!floral!identity!(Souer!et!al.,!1998;!Weigel!and!Nilsson,!1995).!After!floral!evocation! the!inflorescence!meristem!generates!floral!meristems!whose!identity!is!specified!by!ALF/LFY!and!the! FJbox!protein!UNUSUAL8FLORAL8ORGANS8(UFO)!from!Arabidopsis!and!DOUBLE8TOP!(DOT)!in!petunia! (Rebocho! et! al.,! 2008;! Souer! et! al.,! 2008).! It! was! previously! shown! that! although! LFY! can! bind! functional! cisJregulatory! elements! in! downstream! floral! organ! identity! genes,! it! cannot! activate! transcription!of!reporter!genes!containing!these!sites!in!yeast!cells,!suggesting!that!in8vivo!additional! plant!proteins!are!required.!The!fusion!of!heterologous!transcription!activation!domain!to!LFY!can! byJpass!their!absence!(Parcy!et!al.,!1998).!Thus,!during!specification!of!floral!meristem!identity!ALF! and! LFY! have! no! detectable! transcription! activation! activity! of! their! own! and! are! completely! dependent!on!DOT/UFO!for!their!activity.!A!range!of!findings!indicates!that!LFY/ALF!and!UFO/DOT! directly! interact! in8 vivo8 (in! yeast! and! plant! cells)! and! that! this! interaction! takes! places! within! the! chromatin!on!the!promoters!of!their!target!genes!(Chae!et!al.,!2008;!Souer!et!al.,!2008).!In!our!yeast! experiments! we! showed! that! ALF! could! bind,! and! so! activate! transcription,! of! the! regulatory! elements!present!in!the!floral!organ!identity!genes!APETALA81!and!AGAMOUS,!known!to!be!bound!by! LFY! as! well,! but! only! when! coJexpressed! with! DOT! (Fig.! 1J2,! Chapter" 4).! This! finding! has! multiple! implications.! First,! it! suggests! that! DOT! is! one! of! the! longJsought! proteins! whose! role! can! be! byJ passed! in! yeast,! and! possibly! also! in! planta,! with! the! VP16! fusion.! Interestingly,! the! transcription! activation!capacity!of!VP16!in!yeast!fully!depends!on!an!endogenous!FJbox!protein!(Met30)!(Salghetti! et! al.,! 2001).! This! implies! that! VP16! fusion! provides! to! ALF/LFY,! in! the! absence! of! DOT/UFO,! an! alternative!way!to!recruit!an!SCFJcomplex!and!the!related!ubiquitin!proteasome!machinery.!Second,! it!proves!that!the!binding!of!ALF/LFY!to!UFO/DOT,!seen!in!yeast!two!hybrid!assays!and!bimolecular! fluorescence!complementation!(“split!YFP”),!is!functionally!relevant.!Third,!it!confirms!that!DOT/UFO! can!activate!besides!BJtype!genes,!also!A!and!CJtype!genes!in!a!direct!manner.!

152!! Chapter!5!!

Since! ALF! is! the! target! of! DOT,! we! presume! that! the! consequence! of! the! DOT! gradient! is! the! generation!of!an!ubiquitinated!ALF!gradient.!The!finding!that!a!monoJubiquitinated!form!of!ALF!was! able!to!partially!rescue!the!dot!phenotype,!strongly!suggests!that!ubiquitinylation!of!ALF!performed! by! SCFDOT! complex! is! the! key! process! for! flowers! development! in! petunia.! p35S:UBWALF,8 however,! was!not!able!to!fully!complement!all!the!dot!defects!either!because!the!35S!is!not!strong!as!pDOT! promoter! or! because! the! ubiquitin! was! attached! to! the! NJterminal! methionine,! whereas! ubiquitinylation!normally!occurs!on!internal!lysine!residues.! Over! the! past! years! ubiquitination! has! arisen! as! another! postJtranslational! regulation! mechanism,! in! addition! to! other! wellJknown! modifications! like! protein! phosphorylation.! Ubiquitination! occurs! via! a! threeJstep! conjugation! mechanism,! involving! an! UbiquitinJactivating! enzyme! (E1),! an! UbiquitinJconjugating! enzyme! (E2)! and! UbiquitinJligase! (E3)! (Haglund! and! Dikic,! 2005;!Kanayama!et!al.,!2004;!Miranda!and!Sorkin,!2007;!Weake!and!Workman,!2008).!Activation!of!a! transcription! factor! appears! difficult! to! reconcile! with! degradation! via! the! ubiquitinylation/proteasome! pathway.! However,! a! range! of! findings! led! to! the! conclusion! that! ubiquitinylation!and!proteolysis!of!transcription!activators!is!essential!to!activate!gene!transcription! (Lipford!et!al.,!2005;!Muratani!et!al.,!2005;!Muratani!and!Tansey,!2003;!Salghetti!et!al.,!2001).!In!case! of! the! yeast! transcription! factor! GAL4,! ubiquitination! mediated! by! the! FJbox! transcription! factor! Dsg1/MdM30! initiates! transcription! coupled! destruction.! The! removal! and! breakJdown! of! GAL4! is! essential! for! conversion! of! the! RNA! polymerase! II! transcription! preJinitiation! complex! into! an! transcription! elongation! complex! required! for! RNA! processing.! Also,! ubiquitination! of! the! VP16! activation! domain!by!the!FJbox!protein!Met30!is!essential!for!its!transcription!activation!function.! This! soJcalled! “activation! by! destruction”! model! seems! to! regulate! a! large! class! of! transcription! factors.! Influence! on! RNA! processing! is! not! the! only! way! by! which! ubiquitination! influences! transcription.! One! of! the! first! steps! in! transcriptional! activation! is! formation! of! a! stable! DNA/transcription! factor! complex.! Recently! it! was! shown! that! in! case! of! GAL4,! DNA! binding! is! mediated!by!monoJubiquitination!of!the!GAL4!DNAJbinding!domain!(Archer!et!al.,!2008;!Ferdous!et! al.,!2007).!It!thus!becomes!obvious!that!the!regulation!of!transcription!factors!by!ubiquitination!is! complex!and!involves!not!only!proteolysis!but!also!DNA!binding,!transcription!and!RNA!processing.! Although!ALF!is!already!present!in!both!floral!and!inflorescence!meristems,!there!is!no!expression!of! floral!homeotic!genes!until!also!DOT!is!transcribed.!Thus!monoJubiquitylation!performed!by!DOT!on! ALF!might!be!necessary!to!activate/protect!ALF!function!from!promoter!stripping.! Our!findings!suggest!that!DOT!protein!moves!from!the!cells!at!the!sepal/petal!boundary,!where!it! is!synthesized,!towards!the!center!of!the!flower!meristem!dome!forming!a!protein!gradient!(Fig!5,! Chapter! 4).! Since! the! transcription! factor! ALF! is! the! target! of! DOT,! the! consequence! of! the! DOT! gradient!is!the!generation!of!an!ubiquitinated!ALF!gradient.!Once!ALF!is!in!the!active!form,!it!can!

! 153! General!Discussion! activate!the!downstream!organ!identity!genes.!Thus!far,!it!remains!to!establish!if!the!ALFJDOTJUB! interaction!take!place!before!or!after!DOT!movement.! ! !

154!! Chapter!5!!

References"

Amaya,"I.,"Ratcliffe,"O."J."and"Bradley,"D."J."(1999).!Expression!of!CENTRORADIALIS!(CEN)!and!CENJ like! genes! in! tobacco! reveals! a! conserved! mechanism! controlling! phase! change! in! diverse! species.!The8Plant8cell!11,!1405J1418.! Archer," C." T.," Delahodde," A.," Gonzalez," F.," Johnston," S." A." and" Kodadek," T." (2008).! Activation! domainJdependent!monoubiquitylation!of!Gal4!protein!is!essential!for!promoter!binding!in! vivo.!J8Biol8Chem!283,!12614J12623.! Benlloch," R.," Berbel," A.," Serrano6Mislata," A." and" Madueno," F." (2007).! Floral! initiation! and! inflorescence!architecture:!a!comparative!view.!Annals8of8botany!100,!659J676.! Blazquez," M." A.," Ferrandiz," C.," Madueno," F." and" Parcy," F." (2006).!How!floral!meristems!are!built.! Plant8Mol8Biol!60,!855J870.! Carroll," S." B." (2008).! EvoJdevo! and! an! expanding! evolutionary! synthesis:! a! genetic! theory! of! morphological!evolution.!Cell!134,!25J36.! Chae," E.," Tan," Q." K." G.," Hill," T." A." and" Irish," V." F." (2008).! An! Arabidopsis!FJbox! protein! acts! as! a! transcriptional!coJfactor!to!regulate!floral!development.!Development!135,!1235J1245.! Endrizzi,"K.,"Moussian,"B.,"Haecker,"A.,"Levin,"J."Z."and"Laux,"T."(1996).!The!SHOOT!MERISTEMLESS! gene! is! required! for! maintenance! of! undifferentiated! cells! in! Arabidopsis! shoot! and! floral! meristems!and!acts!at!a!different!regulatory!level!than!the!meristem!genes!WUSCHEL!and! ZWILLE.!Plant8J!10,!967J979.! Ferdous,"A.,"Sikder,"D.,"Gillette,"T.,"Nalley,"K.,"Kodadek,"T."and"Johnston,"S."A."(2007).!The!role!of!the! proteasomal! ATPases! and! activator! monoubiquitylation! in! regulating! Gal4! binding! to! promoters.!Genes8Dev!21,!112J123.! Ferrandiz,"C.,"Gu,"Q.,"Martienssen,"R."and"Yanofsky,"M."F."(2000).!Redundant!regulation!of!meristem! identity! and! plant! architecture! by! FRUITFULL,! APETALA1! and! CAULIFLOWER.! Development! 127,!725J734.! Gregis," V.," Sessa," A.," Colombo," L." and" Kater," M." M." (2008).! AGAMOUSJLIKE24! and! SHORT! VEGETATIVE!PHASE!determine!floral!meristem!identity!in!Arabidopsis.!Plant8J!56,!891J902.! Haglund,"K."and"Dikic,"I."(2005).!Ubiquitylation!and!cell!signaling.!Embo8J!24,!3353J3359.! Kanayama,"A.,"Seth,"R."B.,"Sun,"L.,"Ea,"C."K.,"Hong,"M.,"Shaito,"A.,"Chiu,"Y."H.,"Deng,"L."and"Chen,"Z."J." (2004).! TAB2! and! TAB3! activate! the! NFJkappaB! pathway! through! binding! to! polyubiquitin! chains.!Molecular8cell!15,!535J548.! Kaufmann,"K.,"Wellmer,"F.,"Muino,"J."M.,"Ferrier,"T.,"Wuest,"S."E.,"Kumar,"V.,"Serrano6Mislata,"A.," Madueno," F.," Krajewski," P.," Meyerowitz," E." M.," et" al." (2010).! Orchestration! of! Floral! Initiation!by!APETALA1.!Science!328,!85J89.! Kusters,"E."(2011).!Genetic!control!of!meristem!identity!in!Petunia.!Vrije8Universiteit8Amsterdam.! Kusters," E.," Della" Pina," S.," Castel," R.," Souer," E." and" Koes," R." (2015).! Changes! in! cisJregulatory! elements! of! a! key! floral! regulator! are! associated! with! divergence! of! inflorescence! architectures.!Development!142,!2822J2831.! Lee,"I.,"Wolfe,"D."S.,"Nilsson,"O."and"Weigel,"D."(1997).!A!LEAFY!coJregulator!encoded!by!UNUSUAL! FLORAL!ORGANS.!Current8biology8:8CB!7,!95J104.! Lipford,"J."R.,"Smith,"G."T.,"Chi,"Y."and"Deshaies,"R."J."(2005).!A!putative!stimulatory!role!for!activator! turnover!in!gene!expression.!Nature!438,!113J116.! Miranda,"M."and"Sorkin,"A."(2007).!Regulation!of!receptors!and!transporters!by!ubiquitination:!new! insights!into!surprisingly!similar!mechanisms.!Molecular8interventions!7,!157J167.!

! 155! General!Discussion!

Molinero6Rosales," N.," Jamilena," M.," Zurita," S.," Gomez," P.," Capel," J." and" Lozano," R." (1999).! FALSIFLORA,!the!tomato!orthologue!of!FLORICAULA!and!LEAFY,!controls!flowering!time!and! floral!meristem!identity.!Plant8J!20,!685J693.! Muratani,"M.,"Kung,"C.,"Shokat,"K."M."and"Tansey,"W."P."(2005).!The!F!box!protein!Dsg1/Mdm30!is!a! transcriptional! coactivator! that! stimulates! Gal4! turnover! and! cotranscriptional! mRNA! processing.!Cell!120,!887J899.! Muratani," M." and" Tansey," W." P." (2003).! How! the! ubiquitinJproteasome! system! controls! transcription.!Nature8reviews.8Molecular8cell8biology!4,!192J201.! Parcy," F.," Nilsson," O.," Busch," M." A.," Lee," I." and" Weigel," D." (1998).!A!genetic!framework!for!floral! patterning.!Nature!395,!561J566.! Preston," J." C.," Jorgensen," S." A." and" Jha," S." G." (2014).! Functional! characterization! of! duplicated! Suppressor!of!Overexpression!of!Constans!1Jlike!genes!in!petunia.!PloS8one!9,!e96108.! Rebocho," A." B.," Bliek," M.," Kusters," E.," Castel," R.," Procissi," A.," Roobeek," I.," Souer," E." and" Koes," R." (2008).! Role! of! EVERGREEN! in! the! development! of! the! cymose! petunia! inflorescence.! Dev8 Cell!15,!437J447.! Rim,"Y.,"Huang,"L.,"Chu,"H.,"Han,"X.,"Cho,"W."K.,"Jeon,"C."O.,"Kim,"H."J.,"Hong,"J."C.,"Lucas,"W."J."and" Kim," J." Y." (2011).! Analysis! of! Arabidopsis! transcription! factor! families! revealed! extensive! capacity!for!cellJtoJcell!movement!as!well!as!discrete!trafficking!patterns.!Molecules8and8cells! 32,!519J526.! Salghetti," S." E.," Caudy," A." A.," Chenoweth," J." G." and" Tansey," W." P." (2001).! Regulation! of! transcriptional!activation!domain!function!by!ubiquitin.!Science!293,!1651J1653.! Schultz," E.," Carpenter," R.," Doyle," S." and" Coen," E." (2001).! The! gene! fimbriata! interacts! nonJcell! autonomously!with!floral!regulatory!genes.!Plant8J!25,!499J507.! Sessions,"A.,"Yanofsky,"M."F."and"Weigel,"D."(2000).!CellJcell!signaling!and!movement!by!the!floral! transcription!factors!LEAFY!and!APETALA1.!Science!289,!779J782.! Souer,"E.,"Rebocho,"A."B.,"Bliek,"M.,"Kusters,"E.,"de"Bruin,"R."A."M."and"Koes,"R."(2008).!Patterning!of! inflorescences! and! flowers! by! the! FJbox! protein! DOUBLE! TOP! and! the! LEAFY! homolog! ABERRANT!LEAF!AND!FLOWER!of!Petunia.!The8Plant8cell!20,!2033J2048.! Souer,"E.,"van"der"Krol,"A.,"Kloos,"D.,"Spelt,"C.,"Bliek,"M.,"Mol,"J."and"Koes,"R."(1998).!Genetic!control! of! branching! pattern! and! floral! identity! during! Petunia! inflorescence! development.! Development!125,!733J742.! Thouet,"J.,"Quinet,"M.,"Ormenese,"S.,"Kinet,"J."M."and"Perilleux,"C."(2008).!Revisiting!the!involvement! of!SELFJPRUNING!in!the!sympodial!growth!of!tomato.!Plant8physiology!148,!61J64.! Wagner,"D."(2009).!Flower!morphogenesis:!timing!is!key.!Dev8Cell!16,!621J622.! Weake,"V."M."and"Workman,"J."L."(2008).!Histone!ubiquitination:!triggering!gene!activity.!Molecular8 cell!29,!653J663.! Weigel,"D."and"Nilsson,"O."(1995).!A!developmental!switch!sufficient!for!flower!initiation!in!diverse! plants.!Nature!377,!495J500.! Wu,"G."and"Poethig,"R."S."(2006).!Temporal!regulation!of!shoot!development!in!Arabidopsis!thaliana! by!miR156!and!its!target!SPL3.!Development!133,!3539J3547.! Wu," X.," Dinneny," J." R.," Crawford," K." M.," Rhee," Y.," Citovsky," V.," Zambryski," P." C." and" Weigel," D." (2003).! Modes! of! intercellular! transcription! factor! movement! in! the! Arabidopsis! apex.! Development!130,!3735J3745.! !

156!! !

SUMMARY!

!

THE!EVOLUTIONARY!DIVERGENCE!OF!THE!GENETIC!NETWORKS!THAT!CONTROL! FLOWERING!IN!DISTINCT!SPECIES!!

Plant! and! animal! species! evolved! an! astonishing! variation! with! regard! to! the! basic! architecture! of! their! body! and! overall! morphology,! which! is! nowadays! obvious! from! the! variety! of! types,! shape! and! size! of! organs! and! their! arrangement! on! the! plant! body.! Angiosperms,!the!flowering!plants,!comprise!approximately!260,000!different!species!with! an!enormous!variation!in!size,!ranging!in!size!from!a!few!centimeters!(e.g.!Arabidopsis)!to! more! than! 90! meters! (e.g.! Eucalyptus),! and! morphology.! One! of! the! most! peculiar! differences! between! Angiosperm! species! concerns! the! moment! that! they! commence! flowering!and!the!position!on!the!plant!body!where!flowers!are!formed.!Most!plants!start!to! flower!during!spring!or!summer,!while!others!during!do!so!in!autumn!or!winter,!either!in! their! first! year! (annuals),! their! second! year! (biennials)! or! repeatedly! over! many! years! (perenials).! Some! species! form! a! single! flower,! while! others! develop! more! complicated! branched! structures,! called! inflorescences,! bearing! multiple! flowers! in! different! arrangements.!!

This!thesis!aims!to!identify!the!molecular!changes!underlying!the!divergence!of!inflorescence! architectures! by! comparing! genes! that! regulate! the! development! of! the! racemose! inflorescence! in! of! Arabidopsis0 and! the! cymose! inflorescence! of! Petunia0 hybrida.! By! analyzing!and!comparing!the!genetic!mechanisms!that!govern!the!formation!of!flowers!in! both!species!we!attempt!to!define!the!genetic!alterations!that!gave!rise!to!the!variation!of! inflorescence!architectures!found!in!nature.!!

Some! of! the! DNA! changes! that! are! responsible! for! specific! morphological! differences! observed!in!closely!related!species!have!been!already!reported!and!are!reviewed!in!Chapter( 1.!In!the!light!of!the!EvoZDevo!debate,!we!explain!the!reasons!why!most!of!these!changes! happen! in! the! regulatory! regions! rather! than! in! the! protein! coding! sequences! of! the!

! 157! !

regulatory! genes! that! control! several! important! morphological! processes.! From! the! first! stage!of!the!embryo!development!to!the!setting!and!dispersal!of!seeds,!we!describe!how! such!changes!influenced!the!evolution!of!different!shapes!and!the!adaptation!of!plants!to! the!different!environment.!!

In! Chapter( 2( we! describe! the! divergence! of! the! inflorescence! architectures! observed! in! Arabidopsis0thaliana0and!Anthirrhinum0majus,!both!racemes,!compared!to!Petunia0hybrida0 and! Solanum0 lycopersicum,! both! cymes.! Two! genes,! LEAFY0 (LFY)! and! UNUSUAL0 FLORAL0 ORGANS0 (UFO)! are! important! in! order! to! trigger! the! flower! formation! in! Arabidopsis0 and! Anthirrhinum.!Although!orthologous!genes,!named!ABERRANT0LEAF0and0FLOWERS0(ALF)!and! DOUBLE0 TOP0 (DOT)! respectively! in! petunia! and! FALSIFLORA0 (FA)! and! ANANTHA0 (AN)! in! tomato,!have!the!same!function!and!encode!proteins!that!are!functionally!interchangeable! with!LFY!and!UFO,!they!are!expressed!in!different!patterns.!Starting!from!the!idea!that!genes! can!change!their!expression!pattern!either!by!changes!in!the!upstream!regulatory!network! of! transZacting! factors! that! control! their! expression! pattern! or! by! changes! in! the! cisZ regulatory! elements! within! the! gene! itself,! we! swapped! promoterZreporter! gene! fusions! between!the!four!aforementioned!species.!This!revealed!that!in!the!case!of!LFY0and!ALF!the! different! expression! pattern! in! their! cognate! host! species! results! from! changes! in! the! upstream!network,!whereas!the!divergence!of!the!DOT!and!UFO!expression!patterns!results! from! changes! in! their! cisZregulatory! regions.! Moreover,! expression! of! Arabidopsis0 UFO,! driven!by!its!own!promoter!converts!the!cymose!petunia!inflorescence!into!a!solitary!flower,! showing! that! these! differences! in! the! DOT0 and! UFO0 promoter! are! important! for! the! development!of!the!correct!sympodial!inflorescence!architecture.!!

To!unravel!how!the!DOT!and!UFO!promoters!diverged!during!evolution,!we!analyzed,!as!a! first! step,! ! (putative)! cisZregulatory! elements! by! combining! promoter! shadowing! and! functional! analysis,! as! described! in! Chapter( 3.! By! comparing! the! promoters! from! DOT! homologs! of! several! species! belonging! to! the! Solanaceae! we! found! four! highly! conserved! regions!that!we!named!BOX1!to!BOX4,!two!of!which!are!also!conserved!in!the!regulatory! region! of! DOT! homologs! from! species! belonging! to! other! plant! families! (i.e.! grape,! snapdragon!and!monkey!flower).!Given!that!regulatory!elements!generally!cluster!together,! we! predicted! transcription! factors! binding! site! (TFBS)! with! the! TFBS! prediction! software! JASPAR,!but!only!BOX1!and!3!contain!possible!binding!sites!for!MADS!box!!and!SPLs!proteins.!

158!! !

In! Arabidopsis0 is! it! known! that! several! MADS! box! genes! are! involved! in! the! regulation! of! flowering! time! and! flower! development! in! response! to! photoperiod,! whereas! the! microRNA156ZSPLs!module!operate!in!ageZdependent!flowering!time!regulation.!The!finding! that!activation!of!the!a!the!MADS!box!protein,!AGAMOUS0LIKE0240(AGL24),!and!SQUAMOSA0 PROMOTER0 BINDING0 PROTEIN0 LIKE0 30 (SPL3)! can! activate! the! DOT! promoter! in! transient! protoplast!expression!assays,!provides!direct!indication!for!the!regulation!of!pDOT!by!AGL24Z like!and!SPL3Zlike!proteins!in!petunia.!!We!then!show!that!overexpression!of!the!single!gene! (AGL24!or!SPL3)!is!able!to!induce!early!flowering,!but!only!in!short!day!conditions!where!the! photoperiod! pathway! is! not! active.! Moreover,! ectopic! expression! of! the! mir156,! which! downZregulates!expression!of!SPL!genes,!does!not!change!the!flowering!time!but!it!affects! the! flower! development.! Given! that! the! petunia! genome! is! available,! we! identified! the! putative! AtAGL240 Z! FBP250 and! FBP130 Z! and! AtSPL30 (PhSPL30 to! 6)! orthologs! and! further! characterized! their! function.! We! show! that! overexpression! of! FBP25! only! influenced! the! flowering!time!in!Arabidopsis,0whereas!FBP130does!not.!Our!findings!show!that!despite!the! strong! conservation! of! the! proteins! themselves,! their! role! in! the! genetic! networks! controlling!flower!formation!diverged!significantly,!as!indicated!by!the!divergent!phenotypes! seen!in!petunia!and!Arabidopsis.!This!suggests!that!the!genetic!networks!governing!flower! formation! in! Arabidopsis! and! petunia,! have! been! rewired! to! a! much! greater! extent! than! generally!thought.!

The!mechanism!by!which!DOT!and!UFO!promotes!the!identity!of!floral!meristems!and!floral! organs! had! remained! unclear.! Given! that! DOT! and! UFO! are! part! of! an! SCFZtype! ubiquitin! ligase! complex! and! that! ubiquitination! of! a! protein! often! leads! to! its! destruction! by! the! proteasome,!it!seemed!likely!that!DOT!and!UFO!target!an!unknown!inhibitor!of!floral!identity! for! degradation.! Genetic! and! biochemical! evidence,! on! the! other! hand,! revealed! that! the! main!interactors!of!DOT!and!UFO!are!ALF!and!LFY.!Given!that,!like!UFO!and!DOT,!ALF!and!LFY! promote!floral!identity!this!would!invoke!the!paradoxal!conclusion!of!an!FZbox!protein!(DOT! and!UFO)!promoting!the!activity!of!a!transcription!factor!(ALF!or!LFY),!instead!on!inhibiting!it.! Another!problem!a!model!in!which!DOT!and!ALF!together!trigger!the!activation!of!ABC!organ! identity!genes!was!that!the!mRNAs!of!ALF,!DOT!and!the!target!ABC!genes!are!expressed!in! different!patterns!with!little!or!no!overlap.!In!Chapter( 4!we!now!solved!both!issues!by!(i)! providing!direct!evidence!for!the!role!of!DOT!in!the!posttranslational!activation!of!ALF!and!

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(ii)! demonstrations! that! DOT! protein! can! move! between! cells.! First,! we! rebuild! the! SCFDOTcomplex!in!yeast!and!we!show!that!binding!of!DOT!is!necessary!and!sufficient!for!the! capacity! of! ALF! to! promote! expression! of! a! reporter! gene! driven! by! regulatory! elements! from!the!organ!identity!genes!APETALA010(AP1)!and!AGAMOUS0(AG).!Second,!in!a!dot!mutant! background!the!expression!of!a!genetic!fusion!of!ALF!and!UBIQUITIN!(UBI)is!able!to!partially! restore!the!floral!transition,!indicating!that!conjugation!of!UBI!to!ALF!indeed!enhances!the! transcription!activation!potential!of!ALF.!Interestingly,!the!yeast!experiments!show!that!DOT! is!a!direct!activator!of!the!ABC!organ!identity!genes,!although!these!genes!are!express!in! different!position!in!the!meristem.!DOT0mRNA!displays!a!specific!expression!pattern!in!the! sepal/petal! boundary,! suggesting! that! DOT! protein! moves! within! the! floral! meristem! in! order! to! activate! its! target! gene.! By! transforming! pDOT:DOTTGFP:tDOT! in! petunia! we! obtained! direct! evidence! that! DOT! protein! is! able! to! move! between! cells! within! the! meristem!and!by!FRAP!analysis!we!show!that!DOT!movement!is!toward!the!floral!meristem! center!creating!a!gradient!of!protein.!Our!results!show!a!potential!role!for!an!FZ!box!protein! gradient!(DOT)!in!patterning!the!floral!meristem,!and!may!explain!why!UFO!and!DOT!have! similar!functions!within!the!developing!flower,!even!though!their!mRNAs!are!expressed!in! different!patterns.!

! ! !

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!

SAMENVATTING!

!

THE!EVOLUTIONARY!DIVERGENCE!OF!THE!GENETIC!NETWORKS!THAT!CONTROL! FLOWERING!IN!DISTINCT!SPECIES!!

Angiospermae,! de! bloeiende! planten,! omvatten! ongeveer! 260.000! verschillende! soorten! met! een! enorme! variatie! in! grootte,! variërend! van! enkele! centimeters! (bijvoorbeeld! Arabidopsis)! tot! meer! dan! 90! meter! (bijvoorbeeld! Eucalyptus),! en! morfologie.! Een! van! de! meest!opvallende!verschillen!tussen!plantensoorten!betreft!het!moment!dat!ze!gaan!bloeien! en!de!positie!aan!de!plant!waar!de!bloemen!worden!gevormd.!De!meeste!planten!begint!de! bloei!in!het!voorjaar!of!de!zomer,!terwijl!andere!soorten!gaan!bloeien!in!de!herfst!of!winter,! ofwel!in!hun!eerste!jaar!(eenjarigen),!hun!tweede!jaar!(tweejarigen)!of!vele!jaren!achtereen! (vaste! planten).! Sommige! soorten! vormen! een! enkele! bloem,! terwijl! andere! soorten! gecompliceerde! vertakte! structuren! vormen,! bloeiwijzen,! die! meerder! bloemen! dragen! in! verschillende!patronen.!

! Dit!proefschrift!beoogt!moleculaire!veranderingen!te!identificeren!die!ten!grondslag!liggen! aan!de!evolutie!van!bloeiwijzen!met!verschillende!vertakking!patronen!door!het!vergelijken! van!genen!die!de!ontwikkeling!sturen!van!een!tros!of!aar!(raceme),!zoals!de!bloeiwijze!van! Arabidopsis0thaliana,!en!die!van!een!waaier!(cyme),!zoals!de!bloeiwijze!van!Petunia0hybrida.! Door! het! analyseren! en! vergelijken! van! de! genetische! mechanismen! die! de! vorming! van! bloemen!controleert!in!beide!soorten!konden!we!enkele!van!de!genetische!veranderingen! die!leidden!tot!de!evolutie!van!verschillende!bloeiwijze!architecturen!nader!definiëren.!

! Zoals! samengevat! in( hoofdstuk( 1!zijn! slechts! enkele! van! de! genetische! veranderingen! die! verantwoordelijk! zijn! voor! het! ontstaan! van! specifieke! morfologische! verschillen! tussen! nauw! verwante! (planten)soorten! zijn! tot! dusver! beschreven.! In! dit! hoofdstuk! beargumenteren! we! waarom! de! meeste! van! deze! veranderingen! plaatsvonden! in! de! regulatoire! elementen! van! belangrijke! ontwikkelingsgenen! en! waarom! veranderingen! in!

! 161! !

eiwit! coderende! sequenties! veel! zeldzamer! zijn.! We! beschrijven! hoe! dergelijke! mutaties! verschillende!stappen!in!de!!ontwikkeling!van!planten!hebben!veranderd,!van!embryogenese! tot!de!bevruchting!en!verspreiding!van!zaden,!als!aanpassing!aan!de!omgeving.! ! In! hoofdstuk( 2! beschrijven! we! de! divergentie! van! de! architectuur! van! de! bloeiwijzes! van! Arabidopsis0 thaliana! (zandraket)! en! Anthirrhinum0 majus0 (leeuwenbek),! beide! aren! of! trossen,!vergeleken!met!die!van!Petunia0hybrida!en!Solanum0lycopersicum!(tomaat),!beide! waaiers.!In!Arabidopsis!en!Anthirrhinum!controleren!twee!genen,!LEAFY!(LFY)!en!UNUSUAL0 FLORAL0 ORGANS!(UFO),! de! identiteit! van! een! bloemmeristeem,! de! eerste! stap! in! de! ontwikkeling!van!de!bloem.!De!orthologe!genen!van!petunia,!respectievelijk!ABERRANT0LEAF0 AND0FLOWER!(ALF)!en!DOUBLE0TOP!(DOT),!en!tomaat,!FALSIFLORA!(FA)!en!ANANTHA!(AN),! hebben!dezelfde!functie!en!coderen!voor!eiwitten!die!functioneel!uitwisselbaar!zijn!met!LFY! en!UFO,!maar!hebben!een!zeer!verschillend!expressiepatroon.!Uitgaande!van!de!gedachte! dat! genen! hun! expressiepatroon! op! twee! manieren! kunnen! veranderen,! hetzij! door! veranderingen!in!hogere!lagen!van!het!genetische!netwerk!van!transZregulerende!factoren,! hetzij! door! veranderingen! in! de! cisZregulerende! elementen! in! het! gen! zelf,! hebben! we! promotorZ!reporter!genfusies!tussen!bovengenoemde!soorten!uitgewisseld.!Hieruit!bleek!(i)! dat!de!verschillen!in!de!expressiepatronen!in!het!geval!van!LFY!en!ALF!zijn!veroorzaakt!door! veranderingen!in!hogere!regionen!van!het!regulatoire!netwerk!en!(ii)!dat!de!divergentie!van! de! DOT! en! UFO! expressiepatronen! is! ontstaan! door! veranderingen! in! de! cisZregulatoire! gebieden!van!UFO!en!DOT!zelf.!Bovendien!bleek!dat!door!introductie!van!het!Arabidopsis! UFO! gen! inclusief! de! UFO! promotor! in! petunia,! de! complexe! meerbloemige! bloeiwijze! (waaier)!verandert!in!een!enkele!bloem.!Dit!levert!een!direct!bewijs!dat!veranderingen!in!de! cisZregulatoire!elementen!van!genen!een!belangrijke!rol!hebben!gespeeld!in!de!evolutie!van! verschillende!bloeiwijzen.! ! Om! nader! te! bepalen! hoe! de! promotors! van! DOT! en! UFO! zijn! gedivergeerd! tijdens! de! evolutie,!hebben!we,!als!eerste!stap,!de!(vermeende)!cisZregulerende!elementen!in!de!DOT! promotor! geïdentificeerd! door! een! combinatie! van! “promotorZshadowing”! en! functionele! analyse,s! zoals! beschreven! in! hoofdstuk( 3.! Door! de! sequenties! te! vergelijken! van! de! promotors!van!DOT!homologen!van!verschillende!soorten!Solanaceae!vonden!we!vier!zeer! geconserveerde! regio's,! BOX1! tot! en! met! ! BOX4;! twee! van! deze! elementen! bleken! ook!

162!! !

geconserveerd!te!zijn!in!DOT!homologen!uit!minder!verwante!plantenfamilies!(d.w.z.!druif,! Antirrhinum!en!Mimulus).!Aangezien!de!cisZregulerende!elementen!van!genen!(enhancers)! vaak! bestaan! uit! clusters! van! bindingsplaatsen! voor! transcriptiefactoren,! hebben! we! de! sequenties! van! BOX1! tot! BOX4! geanalyseerd! m.b.v.! software! (JASPER)! die! potentiele! bindingsplaatsen!van!bekende!(klassen!van)!transcriptie!factoren!voorspelt!en!vonden!dat! alleen!BOX1!en!3!potentiele!bindingsplaatsen!voor!MADS!box!en!SPL!eiwitten!bevatten.!Het! is!bekend!dat!in!Arabidopsis0verschillende!MADS!box!transcriptiefactoren!betrokken!zijn!bij! de! regulering! van! de! bloeitijd! en! bloemontwikkeling! door! daglengte,! terwijl! de! microRNA156ZSPL! module! een! rol! speelt! in! leeftijdsafhankelijke! regulatie! van! bloei.! De! bevinding!dat!coZexpressie!van!het!MADSZbox!eiwit!AGAMOUS!LIKE!24!(AGL24)!en!het!SPL! eiwit!SQUAMOSA!PROMOTOR!BINDING!PROTEIN3!(SPL3)!voldoende!is!om!de!DOTZpromotor! (pDOT)!te!activeren!in!een!transiente!protoplast!expressie!assays,!is!een!directe!aanwijzing! voor!de!regulering!van!pDOT!door!een!combinatie!van!een!AGL24Z!en!SPL3Zachtige!eiwitten! in!petunia.!! Om! de! rol! van! AGL24! en! SPL3Zachtige! eiwitten! in! de! regulatie! van! DOT! expressie,! en! de! ontwikkeling!van!bloemen!in!petunia!verder!te!karakteriseren!hebben!we!potentiele!AGL24! en!SPL3!homologen!geïdentificeerd!en!hun!expressie!veranderd.!We!vonden!dat!continue! expressie! van! enkel! AGL24! of! SPL3! in! petunia! vervroegde! bloei! induceert,! maar! alleen! in! korte! dag! omstandigheden! wanneer! de! langeZdag! route! inactief! is.! Tevens! bleek! dat! ectopische!expressie!van!het!micro!RNA!mir156,!welke!de!expressie!van!SPL!genen!verlaagd,! geen! effect! heeft! op! de! bloeitijd,! maar! de! ontwikkeling! van! de! bloemen! ontregelt.! Het! petunia! genoom! bevat! twee! potentiele! AGL24! homologen,! FBP25! en! FBP13! genaamd,! en! meerdere! orthologen! van! AtSPL3! (PhSPL3! tot! 6),! voorts! gekenmerkt! hun! functie.! Constitutieve! expressie! van! FBP25! vervroegd,! net! als! AGL24,! de! bloeitijd! in! Arabidopsis,! maar!constitutieve!expressie!van!FBP13!niet.!Deze!bevindingen!laten!zien!dat!ondanks!de! sterke!conservering!van!de!eiwitten!zelf,!hun!rol!in!genetische!netwerken!die!bloemvorming! controleren!sterk!is!gedivergeerd,!zoals!blijkt!uit!de!uiteenlopende!fenotypes!bij!petunia!en! Arabidopsis.!Dit!suggereert!dat!de!genetische!netwerken!die!bloemvorming!controleren!in! Arabidopsis! en! petunia,! in! veel! grotere! mate! zijn! gereorganiseerd! dan! algemeen! werd! gedacht.!

!

! 163! !

Het! mechanisme! waarmee! DOT! en! UFO! de! identiteit! van! bloemmeristemen! en! bloem! organen!bepaald!was!tot!dusver!onduidelijk.!Gezien!het!feit!dat!de!DOT!en!UFO!onderdeel! zijn! van! een! SCFZtype! ubiquitineZligase! complex! is! en! dat! ubiquitinatie! van! een! eiwit! vaak! leidt!tot!de!vernietiging!door!het!proteasoom,!werd!lang!gedacht!dat!het!doelwit!van!DOT! en!UFO!een!onbekend!eiwit!was!dat!de!bloemidentiteit!van!een!meristeem!remt.!Genetische! en! biochemische! gegevens,! aan! de! ander! kant,! suggereerden! dan! de! voornaamste! doelwitten!van!DOT!en!UFO!de!transcriptiefactoren!ALF!en!LFY!zijn.!Aangezien!ALF!en!LFY,! net! als! DOT! en! UFO,! de! bloemidentiteit! van! een! meristeem! bevorderen! leidt! dit! tot! de! paradoxale! conclusie! dat! een! FZboxZeiwit! (DOT! en! UFO)! de! activiteit! van! een! transcriptiefactor!(ALF!of!LFY)!zou!stimuleren,!i.p.v.!remmen.!Een!tweede!probleem!met!het! model! DOT! en! ALF! samen! de! expressie! ABC! orgaanidentiteitsgenen! stimuleren! is! dat! de! mRNA!expressiepatronen!van!ALF,!DOT!en!de!orgaanidentiteitsgenen!weinig!of!geen!overlap! vertonen.! In! hoofdstuk( 4! hebben! we! nu! beide! kwesties! opgelost! door! (i)! direct! bewijs! te! leveren! dat! DOT! de! activiteit! van! ALF! stimuleert! en! (ii)! aan! te! tonen! dat! DOT! eiwit! kan! bewegen!tussen!de!cellen.!We!laten!zien!dat!DOT!functioneel!is!in!gist!cellen!samen!met!ALF! nodig!en!voldoende!is!voor!de!om!transcriptie!te!stimuleren!via!regulerende!elementen!van! de!orgaanidentiteitsgenen!APETALA!1!(AP1)!en!AGAMOUS!(AG).!Bovendien!vonden!we!dat,! in!een!dot!mutant!achtergrond!de!expressie!van!een!genetische!fusie!van!ALF!en!ubiquitine! (UBI)! in! staat! is! de! ontwikkeling! van! bloemen! gedeeltelijk! te! herstellen,! wat! aangeeft! dat! fusie!van!UBI!aan!ALF!de!transcriptieactiverings!capaciteit!van!ALF!stimuleert.!Interessant!is! dat! de! gist! experimenten! tonen! aan! dat! DOT! is! een! directe! activator! is! van! de! ABC! orgaanidentiteitsgenen,!ondanks!de!mRNAs!van!ALF,!DOT!en!de!orgaanidentiteitsgenen!in! verschillende!cellen!van!het!bloemmeristeem!tot!expressie!komen.!Middels!experimenten! met! een! met! een! DOTZGREEN! FLUORESCENT! PROTEIN! (DOTZGFP)! fusie! eiwit! in! transgene! petunia! planten! konden! we! aantonen! dat! het! DOTZGFP! functioneel! is! en! in! het! bloemmeristeem! van! cel! naar! cel! kan! bewegen.! Onze! resultaten! tonen! een! mogelijke! rol! voor! een! FZ! box! eiwit! gradiënt! (DOT)! in! patroonvorming! binnen! bloemmeristemen,! en! kunnen!verklaren!hoe!het!kan!dat!UFO!en!DOT!binnen!de!bloem!een!vergelijkbare!functie! hebben!ondanks!dat!hun!mRNAs!is!zeer!verschillende!patronen!tot!expressie!komen.! !

164!! Acknowledgments

It is finally the end of this thesis and, with it, the end of a chapter of my life. It has been a long journey that gave me the opportunity to know new people and renew the relationship with old “friends“. Hence, I would like to thank…

My promoter Ronald, for giving me the opportunity to carry out my PhD project in his lab, but also for his helpfulness, critical remarks and useful recommendations.

My copromoter Erik for helping me in the lab, for giving me faith when I was losing it and for trying to teach me to be more patient (sorry, it did not work!).

Francesca, for welcoming me in the group at the beginning, without you all this experience would not have been possible. Thanks for taking care of all of us.

All the members of the group, Kees, Tijs, Michel, Valentina, Roeska, Afke, Yanbang, Tarcies, Henk, Peter, Martina, Daisy and the students for the nice atmosphere, for taking care of the lab and the plants and for the biertje time after work. In particular, I would like to thank Bets, my paraninf, that not only helped me with the experiments but she was also my rock in the black days.

My Italian lab mates Sofia, Marianna and Giovanni for bringing a piece of Italy in Amsterdam, for the nice last minute beer and chips meeting and for cheering up each other in the down moments. In particular, thanks to Giovanni for teaching me that not always we have to be harsh, we are human after all. Thanks to Marianna for having always everything under control, so we could relax. And a special thanks to Sofia, my paraninf, for the wonderful time together in the lab and at home, for being such a good friend even if we live in different countries and for not killing yourself at the end (even if you tried really hard!).

My students Colin, for contributing with some experiments of chapter 3, and Hjalmar (together with Lenneke) for being my fist Dutch friends.

My Italian friends in Amsterdam Erika, Roberto, Ilaria, Maurizio, Annacarla and Enrico for the nice evenings together.

All my expat friends for sharing the same homesickness and for having the same problems understanding the Dutcheis.

My new lab mates and friends in Cologne Elisa, Eva, Virginia, Fernando, Diarmuid, Vicky, Ulla, Amaury, Tobias, Youbong, Ana, Maida and Liron for supporting and suffering me at the same time.

My new supervisor George Coupland for giving me the possibility to carry out my post-doc, even without the official PhD.

Sebbene Amsterdam sia diventata la mia nuova casa, l’Italia è sempre nel mio cuore, quindi grazie a…

I miei genitori che mi hanno sempre sostenuto e aiutato, anche quando non comprendevano le mie scelte fino in fondo.

Mia sorella, che mi ha sopportato per cosi lungo tempo e continuerà a farlo! Grazie per ascoltarmi nei momenti di sclero e per infondermi ottimismo.

Le mie amiche massesi perché ci sono sempre ogni volta che torno e per non farmi sentire il peso della distanza. In particolare ringrazio Daniela per aver creato la copertina di questa tesi.

La mia famiglia acquisita per avermi accolto come una figlia e per il costante aiuto in ogni situazione.

Gli amici sardi, che mi hanno accolto a braccia aperte, per non avermi mai fatta sentire fuori luogo e per cercare di convincermi a rimanere in Sardegna ogni volta che scendo.

Ed infine la persona piú importante della mia vita, Carlo. Sinceramente non ci sono parole che riescano ad esprimere quanto sono grata per averti al mio fianco… Grazie per esserti imbarcato come me in questa avventura in Amsterdam, anche senza sapere cosa ci aspettava. Grazie per aver sostenuto la mia scelta di trasferirmi in Germania, anche se la distanza sucks! Grazie per essere sempre paziente e per cercare di farmi ragionare anche quando perdo la testa. Ma soprattutto, grazie per essere cosi cretins a farmi ridere sempre!

Author contribution list

Serena Della Pina designed the experiments, carried out the majority of the experiments, analyzed and interpreted data and wrote all the chapters.

Ronald Koes designed the experiments, analyzed and interpreted data and revised all the chapters.

Erik Souer designed the experiments, analyzed and interpreted data and revised all the chapters.

Elske Kusters designed part of the experiments of chapter 2, carried out part of those experiments, analyzed and interpreted data and contributed to write chapter 2.

Rob Castel carried out part of the experiments of chapter 2.

Colin Rijkenberg carried out part of the experiments of chapter 3, especially the phylogenetic analysis.

Bets Verbree carried out part of the experiments of chapter 3 and 4 especially scored flowering time, performed yeasts experiments and helped with the Solanaceae DOT sequencing.

Ronald Breedveld contributed to define the experiments of the DOT movement of chapter 4 and helped with the settings of the confocal.

Erik Manders contributed to define the experiments of the DOT movement of chapter 4.