Functional Analyses of AGAMOUS Family Members In

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Accepted Article View metadata,citationandsimilarpapersatcore.ac.uk GeneBank accessions: VIGS,dehiscence, Nicotiana benthamiana, development fruit words: Key title: Running Additional e-mailaddresses Tel. +34 963877892; Fax +34 963877859; e-mail: [email protected] la Universidad deValencia,Campus Valencia, Politécnica de 46022 Spain Molecular deBiología Instituto yUPV-CSIC CelulardePlantas, FerrándizCristina For* correspondence : Naranjosde los Av. Valencia, 46022 s/n Spain Molecular deBiología Instituto dePlantas yCelular (IBMCP), UPV-CSIC. Chloe Fourquin and Cristina Ferrándiz* earlyof evolutionof the C-functionroles eudicots andlate genesin Functional analyses of AGAMOUS in family members OriginalArticle : type Article DateAccepted :03-May-2012 Revised Date : 30-Apr-2012 Received Date : 29-Feb-2012 © 2012 The 2012The© Authors. Plant Journal © 2012 Blackwell Ltd Publishing this cite article as an 'AcceptedArticle', doi:10.1111/j.1365-313X.2012.05046.x between may versiondifferences towhich this Versionlead the andPlease ofRecord. been not paginationthrough typesetting, the copyediting, proofreading and process has This article been accepted for publication andundergone peer full but review has

C-function, AGAMOUS, SHATTERPROOF,development, flower AG-like genes in Nicotiana benthamiana JQ699177, JQ699178, JQ699179, JQ699180 : ChloeFourquin: [email protected]:

Nicotiana benthamiana

clarify brought toyouby provided byDigital.CSIC CORE Accepted Article processes in dry (dehiscence) and fleshy (ripening) fruits. a common andsuggests evolutionary developmentaldevelopment late origin of whichdehiscence, strongly theancestral supports PLE-like roleof genes in late fruit weMoreover, the PLE-like aconserved roleof genesin haveshown controlling fruit and (rosidsasterids). major the groupswithin eudicots the of divergence following the gene lineages these of likely evolution on the providing insights species, inthis function downregulation.specific show bothparalogs We redundantly that C- to the contribute their causedby the defects of phenotypic characterization analyses and expression genesinPLE-like euAG- and the characterization of to evolutionaryspecies validate are hypotheses scarce.Here, we reportthe functional role of analyses thesethe genesinadditionalrepresentative of detailed functional euAGthe evolutionary but the andPLEreconstruct eudicots, lineages in history of respective Different scenarioshavebeenthe C-function. the paralogs to proposedto thaliana euAG and PLE, extensively characterized in coretwo eudicots, In sublineages coexist, Angiosperms. in conserved broadlymanner ina the C-function the genefamilyspecify largeMADS-box AG lineage of the of provide determinacy.andcarpel andto development meristem Members floral stamen according ABCofto The organidentity, floral the C-function, is required model for SUMMARY © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 , where, strong subfunctionalization highly hasledto contributions of divergent Antirrhinum majus Nicotiana benthamiana and Arabidopsis

through Accepted Article the C-function is mainly carried out by a single AG-like gene, gene, by is carried out a singleAG-like mainly C-function the Antirrhinum majus C-class genes were in coreeudicots, the identified In al. et eudicots (Bowmanand core or mutant of including thecharacterization andinbasallines in knockdown monocots, asbyrevealed studies functional inabroadly C-function many conserved manner, the the AG the of largeMADS-box Members genefamilydeterminacy. lineage of specify withcombination B-functiongenes inwhorl 3andalsocontrol floral meristem stamenin centralwhorl the specifies identity carpelidentity the providing flower, of in model of organidentityfloral (Coenand Meyerowitz C-function,1991). in additionto is byby ABCas defined genes,specified identityC-function the inAngiosperms Carpel a thereforestudies. question inEvo-Devofundamental Understanding the evolution and diversifications offunctional carpels isand fruits which inturnprotectfruits, developing the seedsandensure seed dispersal. includingpollenization, and, incompatibility after developinto mechanisms, fertilization, extant species,300,000 isthecarpel. Carpels protect the provideovules, support for A themajor evolutionaryfactor for oftheAngiosperms, consisting of success of more INTRODUCTION © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 core the place of early history duplication genes.tookA event in the major orthologous AtAG petals (Bowmanpetals are totallyinthe flowerorgans areabsent and replacedby and whorls sepals of several their throughout development. Inloss-of-function of mutants genesthese thesexual sexual organprimordiaandearly carpel floral inthe remain stamensthe to specific and Antirrhinum refer to as 2011, Mena 2011, and . AmPLE AtAG AtAG et al. et et al. et , where , they have thoroughly characterized. Inbothspecies,been and for in for clarity) belong to the belongto MADSthe box AG-subfamily, althoughthey arenot 1989; Bradley 1989; 1996,Pan AmPLE et al. et encodecloselyrelated genes whose startexpression etal. 1989,Bradley Arabidopsis et al. 2010, Pnueli 1993). 1993). and et al. et etal. 1993,Davies PLENA 1994,Yellina Arabidopsis thaliana AGAMOUS ( AmPLE etal. et al. et (which wewill hereafter) in 1999, 1999, Dreni 2010).

andin

Accepted Article (Airoldi asa toexplain keyidentified the of the element functional differences two proteins been has in AmFAR) (Q173insertion andAmFAR betweenAmPLE difference acid (Moyroud participation intheC-function minor in activation been retained by a different member of thegene pairin of member different bybeen retained a (Busch meristem flower the ( regulatorgeneThe floral andproteinactivity. bothingeneregulation changes and maybe more generallyand maybe in rosidsand theand enlighten asterids, plasticitymore of (Causier rosids asterids after dehiscence the of theto control occurred andthe divergence fruit genes specify euAG andPLElineages subfunctionalization to of C-function andthe the suggestedThese studies anevolutionary wherescenario switch a betweenfunctional (Davies pollen development ( al. double in observed mutant the AtSHP withvery recentduplication Incontrast within theBrassicaceae. paralog their Arabidopsis gene C-function the AtAG PLE (Kramer euAG and the AG two subfamily: lineagesgene within theorigin of ledto that eudicots © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 gene duplication(Causier evolution functional following LFY AmFAR 2000). Likewise, in ) is a key activator of of activator key a is ) corresponds tocorrespondswhile euAG the lineage in etal. genes do significantlynot contribute since the onlyC-function, to the phenotype hereafter), which appears to provide only a minor contribution to C-function in C-function contributionwhich to provideonlyaminor hereafter), appearsto are the two arethe AtSHP et al. et 2010). All together, these results indicate that primaryAll 2010). theseresults together, indicatehas that C-function et al. et 2004). In In 2004). and AmPLE 2005). switch This hasbeensubsequently to functional attributed Antirrhinum AmFAR SHATTERPROOF AtAG et al. corresponds to the PLE lineage. The PLE lineagegenes in et al. Arabidopsis shp1shp2 and promotesits veryinthe early expression of center has then been suggested as an explanation for been asanexplanationtheir suggested hasthen for 1999,Parcy 1999). , the is a is infruitdehiscence latedefect (Liljegren , a , the of member rosids, C-function genethe euAG genes ( genes et al. et al. et lineage is represented by Antirrhinum 1998). A reduced response LFYA reduced to 1998). 2011). In addition, a 2011).In single amino AtSHP et al. Arabidopsis , a asterids, of , the member 2005). hereafter), products of hereafter), a and Antirrhinum FARINELLI FARINELLI

LEAFY AtAG SHP et , ,

Accepted Article role of Arabidopsis. Likewise, expression studies in different peach varieties (rosids) suggest a development, similar to what hasbeendescribed for the PLE-like Therefore, Therefore, reportedthe onAG-lineage studies eudicots genes incore leaveopen PLE-lineage in hamper mutants validity genes, speculations. the these of and dry ( dehiscent fruits However, theprofound differencesstructural between fleshy berries peach)(tomato, the to specific organ specificationin floral prominent and development, role amore notlate in fruit the that These indicate may studies 2007). Vrebalov (Giménez specification in organ cause defects floral to not PLE-like genedownregulationof the PLE lineage genes outside are obviousPLE genes of Little information isavailable the functioncandidates. onthe suggesting otherthat genes participate in the inC-function these species, whichfor whereexperiments euAG orthologshavebeen downregulated strongly inSolanaceae, loss-of-C-functioncomplete C-function couldberestrictedtothe al. et of inthecontroldeterminacyas meristem (Kapoor appearand carpelas well besignificantly to specification, stamen orthologs involvedin regardingevolutionary timing the least of euAG Inpetunia,and events. tobacco, tomato results conflicting provide undermine that hypothesisthis on evolution, C-function at However, several functional analyses inconducted theasterid family Solanaceae of © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 in C-function otherthan species contribute to questions. First,relevant itremains be studiedto PLE-like whether genes significantly 2010, Pnueli 2010, PLE etal. orthologs in fruit ripening and lignification of pericarp cells(Tani pericarp of ripeningandlignification infruit orthologs 2009). Thus, theroleof Arabidopsis et al. et 1994). Thus, 1994). Thus, the reduced contributionthe euAG of ortholog to Arabidopsis lineage but more generallylineage more but inothereudicots. conserved

phenotype hasnotbeen inobserved reportedthe Arabidopsis TAGL1 ), together with thelack of additional of examples AmFAR TAGL1 PLE has affect been shown to fruit ripening, but and in the in Antirrhinum genes could have a reduced early geneshave areduced role could appears to be late restrictedappears to fruit to etal. Antirrhinum Antirrhinum 2002, Kempin 2002, . In this sense, itiscurrently sense, Inthis . et al. et . Intomato (asterid),. 2010, Pan 2010, However,lineage. a et al. SHP et al. et 1993, Pan 1993, genesin

et al. et 2010,

Accepted Article The large second intron ofeuAG/PLEThehas largesecond intron genes beenshown beimportanttheir to for andeuAGclade, NbSHP inthe PLE clade. TheAmPLE. phylogenetic analysis shown in Figure 1a clearly groups NbAG in the shows 67% and 64% identity to AtSHP1 and AtSHP2, respectively, and76% to identity) or to similar and ismore NAG1, with identity shows aa 96% tabaccum Nicotiana usingavailable of flowers sequenceinformation AG-like genes closethe relativefrom cloned corresponding the likelyfull-length by orthologs cDNA young RT-PCR of from To functionally characterize the RESULTS PLE-like the ofgenesanddevelopment the function in into evolution. possibly fruit fruit Nicotiana divergencegene lineagesfollowing the of rosidsandasterids.the as In addition, toC-functioncontributions betterto and therefore understandtheevolutionthese of Solanaceae species, we a Here, detailedreport analysisfunctional oftheeuAG- and PLE-like genes in a and dehiscence ripeningindryfruit and respectively. fleshy fruits originof developmental common indicate a development maytherefore fruit late rosidsandof divergence This asterids. ancestral role PLEputative of lineages genes in iswhetherlate question PLE-likethe role of genes controlling dehiscencepredatesthe generalmore Finally, a evolutionary formulate hypotheses. to used andcan be function relative ofC- AG-like the contribution genes to valueinassessing good predictive expression clearwhether coupledtoregulatoryhavea sequenceanalyses not studies © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 toestablish as reported earlycritical in flower C-functionexpression development of andtocontain regulation severalconserved motifs, including a LFY binding site dryforms and dehiscent fruits, ithas allowed ustogather deeper insights Antirrhinum from public databases ( databases public from AmFAR (79% of identity) than to AtSHP or AmPLE. NbSHP NbSHP AmPLE. or AtSHP to than identity) of (79% AmFAR Nicotiana benthamiana euAG and NAG1 PLE , aiming , unravel theirto respective genes in and NtPLE36 Nicotiana benthamiana Arabidopsis , see methods). NbAG, see methods). AtAG (67%

, we Accepted Article could share C-function specification, although share C-function specification, could summary,In sequence analysesof expected. NbAG andNbSHPproducedby be changeshouldaminoacid proteins not this this glutaminelacked residue, thus suggesting differences that functional between NbAGboth and NbSHP amino acid sequences showed both that deduced proteins organidentity. inpossible of subfunctionalization male Examination specifying mainly insertion in glutamine the AmFAR sequenceof been has protein correlated to its addition to In the role of consensus of (Moyroud bindingsearch sites regionsgenomic (termed “Predicted occupancy” orPOcc), outperforming classical the quantitatively that basedtool onabiophysical specific predicts LFY affinity for model in role C-functionreduced S1). (Figure However, we a useof bioinformatic also made NbSHP et 2005)and Causier al., in was found LFY 2003 al., bindingin HongA consensusfor et (CCANTGG, site asdefined element. NbAG of thesecond of sequences intron (Moyroudspecies C-function indifferent genes to AG-like relative contributionof on the been suggestedhave some predictive value has to motifs CCAATCA repeat (Causier andtwo other anaAGAATgenes, motifs, 70bp element characterized boxanda by a © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 onearly rolebased predictedLFY occupancyof intron.the second for for the later, similar to what hasbeenreported for the pair LFY POcclevel of inboth Antirrhinum and might havelosttheearlyandtherefore expressionof have onset might might a NbSHP and opposite to second introns to have introns second the conservedto aAGAAT andthe70bp box cis NbAG et al. et AtAG -elements in-elements the evolution genes,C-function of a single and / AtSHP 2009). Because the presence Because 2009). of the these conserved NbAG NbAG NbSHP NbAG in (3897 kb) and and and Arabidopsis et al. second introns, beingsignificantly higher introns, second intron but not in butnot intron NbSHP 2011). 2011). Interestingly, a we high found NbSHP (Figure 1bandS1). may have a more prominent have a more may NbSHP suggestedthat both factors et al. NbSHP 2011), we analyzedthe 2011), (2742 bp). found (2742bp). We AmFAR , suggesting that / AmPLE

from Accepted Article by Mandel NbSHP 81% Both(64% MADS-box codingregion). inthe two genes versus the 500 bp of each coding sequence, a region presenting low sequence similarity between situ detailedToexpression elucidate the patterns of genes NbAG © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 At stage 4, when 4, At stage were petalprimordia visible, appearedexpression to externally accumulate more inthan the floral meristem both stage, this showedexpressiontranscripts although similar domains, andcarpel2ab).At (Figure primordia tostamen give rise whichlater will flower, cells that would develop into stamens and carpels (Figure 2c and d). At At 2c andcarpels andd). (Figurestage 6, wouldinto stamens that develop cells expression expression patterns, similar to those described Arabidopsisfor development. flowerand throughout early stages sexual organs from the to specific remained theseexpression of two genes the that theseexperiments,weconclude From can (Figure 2i and j). ovules inthe developing observed transcripts wasalso both of expression stages, later In inthestrong 2gandh). placenta(Figure but instamens weak geneswasboth thepistilcentral zoneof (Figure2eand apical f). In the theflower,mature expressionof and AmPLE hybridization on young buds of youngbuds of hybridization on NbSHP and transcripts began accumulate transcripts stage at to 2of flower development asdefined genes. genes. et al. et transcripts transcripts were uniformly in present developing the stamens and inthe NbSHP NbAG (1992), when the sepal primordia arise, in the central domain of thewhen (1992), primordia of thesepal domain arise,inthecentral are expressed during areexpressed floral like development typical C-function and NbSHP Nicotiana benthamiana showed nearly identical temporal andspatial NbAG NbAG1 and and NbSHP . We used as probesthe last used as . We NbSHP were detected in the the in detected were AtAG , we performed or Antirrhinum or NbAG

NbSHP NbAG NbAG and in .

Accepted Article fused to the secondfused whorl; and the whorlfourth is occupied by a two-carpellate the whorlcorolla; third stamens iscomposed of whosefive long adnatelyfilaments are length; of developtubular their most insecond whorl,thefor fused petals ina five fused thewildIn type flower of that proving the TRV2-NbAG construct was gene specific. the to whereas WT, ofthe expression observed that in all expressionthe cases, of different treated bymeasured RT-PCR plants. onflowers five quantitative from We level the of theVIGS treatment, of and specificity wereinoculatedTwelve by plants the TRV2-NbAG Toefficiencyconstruct. evaluate the NbAG-VIGS 20 identical nucleotides. presentcontiguous stretchesthan sequencesimilarity not showed more of low anddid the used for codingfragments sequence same carrying the (Constantin degradation ofthe endogenous in mRNAs Virus Tobacco (TRV), Rattle case the hasbeenshown and efficiently direct in our it to plantviruses, genevia modified aspecific of theexpression downregulates transiently order to specifically to silence order reduce to contribution of each gene to these roles, we used Virus InducedGene Silencing (VIGS) development in roles infloral early andlate both that Expression analyses suggested andflower affect development fruit Silencing of © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 et al. et NbAG NbAG 2004, Hileman 2004, and and and NbSHP NbSHP N. benthamiana NbAG in in transcript levelsin transcript et al. N. benthamiana N. and 2005, Ratcliff 2005, NbSHP NbSHP NbSHP NbAG , the whorl , first byis composed sepals five N. benthamiana NbAG was practically S2),(Figure unaffected wegenes, TRV generated constructs Nicotiana and was significantly reduced compared NbAG using VIGS technology differentially et al. et NbSHP N. benthamiana N. as well as other species and and 2001, Wege . To investigate the specific . To investigate genes could have similar have could genes NbSHP NbSHP in situ analyses, which transcripts were transcripts et al. et . This method This method . 2007).In

Accepted Article required to both toboth andrequired specify carpel stamen identity anddeterminacy. control to floral that indicated results Our flowerof the 4i). (Figure NbAG-VIGSthe plants occasionally producedanectopic floral inmeristem the center (Figure Moreover,i). 4c,handflower the prolonged in the activity floral the of meristem 3l).(Figure These supernumerary organswere evidentin histologicalthe sections of developed petalsand additionalwhorls of sepals insidethesepalloid gynoecium theindeterminacy NbAG-VIGS caused floral the treatment meristem. of flowers addition toIn homeotic transformation stamens of and carpels, the NbAG-VIGS and 4h). VIGS flowers also retained carpelloid some like tissuefeatures stigmatic (Figure 3m abaxialsurface 5c). sepal (Figure However,the the in NbAG- organs fourth whorlthe of whichstomata,and aretypical andnumerous puzzletrichomes of found shapedcells, of the Instead small and rectangular cells typical theovary of (Figure epidermis we5e), structures.these identity of thesepal Acloser inspection byconfirmed SEMk). and organdeveloped, green havewhich appearedto sepal-carpel identitymixed (Figure 3f tubularof NbAG-VIGS centerashort the flowers, In 10) (Figure4c). 6 and (between atubularstructure in into 74% and of flowers the there werethan petals more five 3band (Figure Thef). petalsdeveloping in the third whorl instead were of free fused all of flowers whorl, plants.third the In treated the stamens were replacedby petals the inapproximately wasof Thisobserved flower. the phenotype 92% organsof sexual NbAG-VIGS plants showed transform homeotic 3and 4).(Figure placentation,central andaveryby ends round and longstyle, which a stigmaflat aTheofwith short ovary composed two locules mature gynoecium. pistil is dividedinto © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 analyzedflowers that alsosuggested additional likelyfactors, most carpel all NbAG-VIGS whorlfourth tissueinthe presence the of However, of the NbAG had a role major in providing C-function, being ations specifically the restricted to NbSHP

, could Accepted Article In addition, most of the NbSHP-VIGS flowers displayed mild to strong defects in floral strongdefects the to displayedNbSHP-VIGS of addition, mild flowers most In the whorlfourth of the NbSHP-VIGS flowers. inconversion occurred partial homeotic a that indicate These 5f). observations (Figure onwere alsotrichomes style the observed theNbSHP-VIGS of epidermis gynoecium and stomata characteristics trichomes, of sepal of(Figure tissue 5e). andStomata morphologywas between typical intermediate with ovary development andsepalcells, theepidermal of NbSHP-VIGScellsthe of ovarymodified revealed thatcell and shorter Closein the wildinspection 4f). g, n 3c, (Figure than was type plants much Moreover, style and stigma. style acomplete of carpelthe split led to Incomplete fusion development gynoecium was severely affected NbSHP-VIGSin 94% of flowers. oftransformations anthers into petaloidtissue were observed (Figure S4). contrast, In developmentStamen was although occasionallymostly unaffected, mild NbSHP-VIGS plants showed phenotypic alterations restrictedto flower development. significantly altered or even increased (Figure S2). expression of the whereas NbSHP-VIGS flowers stronglyreduced inthe NbSHP TRV-NbSHPexperiment with aspecific construct. The quantification of we development, C-function fruit and to equivalent ancontribution to performed To functionally characterize NbSHP-VIGS development. activity in VIGS-treatedthe plants could topartly be sufficient carpel direct in participate carpel identity or,alternatively, specification, the that residual NbAG © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 two internal carpelsandwhorls developed determinacy.meristem In43% of the flowers, thegynoecium was formed by than more transcripts transcripts in treated plants revealed thattheexpression of NbSHP andto possible specifically evaluateits of carpeloidof organs(Figure 3n,4band j). NbAG NbSHP NbAG

was not not was was was and Accepted Article maturity, a phenotype similar to that caused by loss of function of of function of caused byloss to that similar maturity,a phenotype NbSHP-VIGSthe that fruits were remainedformed indehiscent even weeks after carpelcentral carpel (Figuretwo the andto margins fused 6a). all In contrast, midveins dispersalalong dehiscence seed zones top of four at the correspond that thefruit tothe Arabidopsis dehiscencegreatlyshattering role infruit andthat is reduced incrucial lignification zone dehiscencezones playsArabidopsis (Figure is 6b).that lignification of a known It The wild-type of fruit ovary of close inspection cell ovarymorphology andof wall S5). (Figure organization anddidnotwild-typehomeotic fruits show any transformations, signof asrevealedby identicalto development. were almost NbSHP Thesein fruits anatomically fruit of role onlyflowers after weeks inoculationand several allowed possible evaluatethe usto proportion NbSHP-VIGS of sterile, on asmall develop fruits therefore ableto were vast Although theNbSHP-VIGSthe majority of gynoeciawere andaffected severely 4b). stamensto 10ofwild thetypical up developedinstead 5 in formed (Figure type flowers Supernumerary inthewere also found organs © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 determinacy, while also participating in the specification of carpel identity and, to a to carpel identity and, of specification inthe participating also while determinacy, control meristem role inthe of play this to amajor indicated that geneseemed plants addition toitsrole In in fruitdehiscence, describedthe phenotypes NbSHP-VIGSof NbAG1-NbSHP-VIGS NbSHP-VIGSindehiscent (Figure 6d).fruits the carpels in the of the midveins or carpel margins fused alongthe was detected By tothe nolignificationzones 6c). (Figure which corresponded contrast, dehiscence four stronglytype fruit, narrowlignified stripes were observedatthetopthe of fruit, pattern in lignification NbSHP-VIGS mature comparedfruits towild In type. wildthe shp1shp2 N. benthamiana mutants (Liljegren mutants is a dry is capsule, whichopens at maturityto allow et al. third whorl of 36% of theflowers, where 36%third whorl of of 2000). For reason,this we studied the AtSHP genes in

Accepted Article able to assess the specific contribution of a contribution of thePLE-lineage of specific assessmember the inasterids to able to questions regarding theAG-like evolution of genes in First, eudicots. we have been benthamianaNicotiana Solanaceaedevelopmentin euAGand PLE inflower the andfruit species lineages we thiswork, theroleofAG-like presentadetailedcharacterization In of genes from DISCUSSION specification.meristem (Figure 3o),(Figure suggesting that observed was also whorl athird organ axilof the flower from emerging ectopic an been described for similar sepalsandpetals(Figure3i), of new towhat has composed developed flower the 5%around flowers of showed phenotype: adifferent whorlthe of fourth instead a reiterating pattern petals inthe second and third whorls (Figure3hand However, 4d). corollanew petals developed inthe of tubular whorl,fourth which enclosednumerous whorl,third were stamens converted into petals (Figure 3d). 90% of In the aflowers, supernumeraryalthough occasionally some organswere (Figure In the observed S4). andpetalsdeveloped normally, Sepals theindividual genes. downregulation of specific the to indeterminacy compared enhanced floral greatly showed a theNbAG-NbSHP-VIGS95% of flowers and stamens completely and lacked carpels, stronglydownregulated (Figure S2). PCR inthe treated of Quantitative plants confirmed flowers five that geneswereboth with TRV2-NbAG-NbSHPsimultaneously 12plants the genes inoculating vector. downregulation of theinNbAG-VIGS residual presumptive see if inorder Thus, flowers. to the C-function stamenof degree,lesser identity. this sense, In © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 Arabidopsis NbSHP . Our . studies provide additional datatoaddress several open modified theNbAG-VIGS phenotype,modified silenced both we NbAG

ag mutants (BowmanIn etal.,1989). very flowers, few and NbSHP NbSHP could alsohavea roleminor in floral appearedto be responsible for

Accepted Article series areseries available,ithas beenshown the that componentsofdifferent the C-function In similarly floral meristem determination. downregulation of NbAG NbAG contribution specific of The the floral of meristem. center the arisingfrom new flowers petals or of whorls of carpels, and determinacy, loss lack floral numerousof of with concentric formation stamensintopetals, of phenotypes,transformation homeotic withcomplete of-function NbAG both wework, that thishypothesis.demonstrate Inthis to confirm fully insufficient thereforewere PLE-like genes, were especiallyonly partialand data, for functional (Kapoor function the PLE andeuAG C- from lineages couldbesignificantly requiredtocontributethe to in suggested contrastthat, in situation to the performedPrevious works inotherSolanaceaepetuniaor tomato like species The C-functionin distantly related species. in regulationconserved PLE-like role dehiscencefruit of in genes the shown the of have we Finally, C-function. of aspects different the to factors AG-like of requirements Second, C-function. the ourresults help tounderstand temporal the and quantitative © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 (Causier identity, carpel(stamen identity anddeterminacy) canbegenetically separated Simultaneous dowregulation ofboth in the Solanaceae species Arabidopsis and butnot and lineage) (euAG et al. NbSHP 2009, Davies and NbSHP Nicotiana benthamiana NbAG et al. et were requiredcarpel to confer identity,although specific Antirrhinum appeared to contribute significantly to stamen identity;both significantly to stamen contribute appeared to NbAG 2002, Pan 2002, hadastronger effect; and both Nicotiana benthamiana NbSHP et al. et and , where functional studiesare extensive andallelic 1999,Mizukami and Ma 1992). The phenotypes of (PLE lineage) genes jointly carry out the C-function jointly outthe carry genes (PLElineage) NbSHP NbAG et al.

to these phenotypes was also assessed: phenotypeswas also to these and 2010, Vrebalov 2010, Arabidopsis NbSHP . genescausedtypical C-loss- or NbAG Antirrhinum et al. et and 2009). However, 2009). NbSHP , both paralogs , both controlled

Accepted Article need both need both of absence identity,whileenough C-activityspecify to provide stamen Thus, inprotein function. differences probably reflecting activity, NbSHP biochemical different potential. Our results indicate that in overexpression did not (Kater et al, 1998), therefore indicating that the two proteins had FBP6 andpetals, sepals of transformations inducedhomeotic overexpression ( hybrida supported bysupported Petunia thephenotypes overexpressing of plants indirectly hypothesis is this fact, In withmodel. the threshold activity, compatible protein differences in on rely C-function the genes may to both contributionof quantitative different the beproposedthat carpelidentity,can of it loss onlycauses mild Since 2011). both, although both, quantitatively higher for expression early both of predict onsetof genes that for inthesecond intron elements inpartialagreement previouslyexpression, with regulatory presenceof described the we show that (Causier development onlyfloral laterstagesof is attained at still C-function termination and activationcarpel canbeachievedif formation meristem identity,whilecorrect to specify is needed stamen expression an early of onset that be deduced the phenotypesfrom heterochronic of alleles of levelsC-function increasing activity. temporal In of requirementsdifferent addition, can stamen identity, identitycarpel and floral determinacymeristem wouldeach require of alleles mutant weak © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 Petunia hybrida euAG-like gene, NbAG)sequence euAG-like or identical 91%in deducedaato would beredundantly C-function, required for but NbAG NbAG NbSHP NbAG and PLE-like gene, 90% identical toNbSHP): while pMADS3 ; the higher requirements of C-function for carpelspecificationhigherrequirements C-functionof would ; the for and downregulation shows little or no effect on stamen no effect downregulation on formation littleor and shows NbSHP AtAG NbSHP , but the absence of the absence NbSHP but lessactive of the , would factor or or exhibit highly similar temporal and temporal exhibithighly spatial of similar patterns AmPLE NbSHP genes support genes a thresholdsupport model inwhich (Causier et al. et NbAG N. benthamiana N. NbSHP et al. et AmPLE 2009, Moyroud 2009, pMADS3 would providehigher NbAG 2009). In work 2009).this In would not inthewould not , which indicate which , alone would alone (the (the

NbAG

Petunia FBP6 et al. and and

Accepted Article treated plants frequently produces a concommitant increase in increase a concommitant produces plants frequently treated thereduction of wethat have observed Interestingly, andpatterns protein activity. in expression changes both inevolutioninvolving times multiple would haveoccurred subfunctionalization AG-like of genes to carry out different the aspects C-functionof Yellina independently but also diversified redundantly the provide (Dreni C-funcion or basal wheregenes evidence have dicots withTogether AG-likesimilar in monocots An ancestral role of PLEAn ancestralroleof genes inlatecarpel development C-function duplicated genes. wouldthat compensation themechanism subfunctionalization independent facilitate of C-function of genes could theautoregulation Thus, providea following duplication. regulation would andthatthis autoregulated would maintained been function be have al.2010,Kapoor etal. Vrebalovet 2002, al.2009) et C- suggesting that ancestral the effects wereSimilar previously otheral. et 1999,Giménezfor reported species (Davies evolutionary hypotheses. evolutionary hypotheses. analyses based regulatory on bioinformatic to predictions sequences of formulate addressofimportance evolutionary to data functional validatethe andto questions Antirrhinum contributions relative theC-function to euAG/PLEthe for paralogs in Expression and ineudicots. proteinC-function of patterns, activities the evolution clarify to valuableinformation adds inNicotiana genes and PLE euAG redundancy of The The C-functionevolution of These expression from notanticipated conclusions, or NbSHP only and of defects; cause bothfinally,mild only expression simultaneous © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 et al. et would provide sufficient C-activity topromote floral meristem termination. and 2010, Zahn Nicotiana areeachin different species, et al. 2006), itstrongly an supports scenario where the NbSHP in silico levels in VIGS- mRNA analyses, highlight the analyses, NbAG mRNA mRNA levels. Arabidopsis et al. et NbAG

2011, and , Accepted Article downregulation of downregulationthe of addition In to different the phenotypesfloral discussed above, we show the that PLEAn ancestralroleof lineagein development fruit genes three thegenes couldwhere this function. redundantly regulate fruits phenocopy fruits the presumptivestrongly dehiscence atthe zone. reducedlignification these Remarkably, evident style in defects inexpressed apicalthe regionsof the developing togetherpistil, with the absence of arepistil development bound to go unnoticed. The fact that backgrounds (Colombo only observed incertain is mutant roleof late stylethe regions. andstigma In in unfused development, carpels with pistil inlate NbSHP-VIGS showdefects flowers maintenance, phenotypefloral meristem organ identity insexual its andin additionto In © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 isin well process shattering The documented fruit ag develop.tissue Likewise, stigmatic only of and patches NbAG-VIGSaffected isstrongly in Carpel genes development participate euAG-like whether function. inthis could also However, rosids. it isdifficult assess ifto this role is specific to the PLE lineage, or was already inthe present PLE lineage before the separation the asteridsof and the Arabidopsis genesappearlike role toshareacommon development in bothin style and stigma Ferrándiz 2002,Ferrandiz mutants do not form any carpels in Arabidopsis, and therefore, late roles of roles late of therefore, andArabidopsis, anyin not carpels form do mutants AtSHP and Nicotiana genes in the promotion of the apical regions of the pistil, although pistil,it apical genes the promotionof regions the of inthe shp1shp2 NbSHP shp1shp2 , it is possible to speculate that this function is ancestral and isancestral this function that to speculate possible itis , etal. gene leads to the development of indehiscentwith of development to the geneleads fruits double mutants in double mutants 2010). The 2010). Arabidopsis mutants, may also support an alternative scenarioan also support alternative may mutants, AtSHP , a recent study hasuncoveredasimilar Arabidopsis valve in genes are expressed the Arabidopsis et al. et AtAG (Liljegren (Liljegren 2010). asPLE- Thus, and (for see review AtSHP et al. et genes are genes

AtAG 2000). in Accepted Article formation in distantlyformation speciessuch as related this gene directing dehiscence zone same network roleof withtogether theconserved in fr FUL-SHP fleshy the network of role (cited Angiosperms taxa of main inPabon-Morawithin 2011). and most The Litt likely phylogenetic into treessuggesttransformations these thathave occurred times multiple (Tanilignification fruits) expression analyses expression of (Giménez in sylvestris Nicotiana beenpreviouslystudied. has When adjacent cells. the of differentiation dehiscencethe zone inthe andlatermargins in the dehiscence zones, wherefruit they required are for © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 Arabidopsis ligninreduced (Ferrándiz AP1/FULthe from clade expressed inthevalves.In by exerted regulation species with fleshy fruits, have shown that haveshown that fleshy fruits, with species therefore, it can be proposed that this network was already present in the common in the present was network already this that be proposed can it therefore, dehiscence zone formation hasbeen established twice eudicotevolution,andduring isverydirectingthis regulatory andlignification. It unlikely that dehiscence network ( overexpression phenocopiesthe Recent studiesRecent of the ofancestor asterids and rosids. AtFUL Arabidopsis

in suggest a conserved function of these genesin controlling peach fruit Arabidopsis et al. et , NbSHP and in 2010, Itkin 2010, et al. et al. AtSHP , indehiscent fruits are also formed (Smykal indehiscent arealsoformed , fruits in in FRUITFULL , Nicotiana 2007). A wealth studiesof mapping fleshy-to-dry dry-to-fleshy or FUL NtFUL N. benthamiana N. PLE/SHP et al. et expression isrestricted bythe valve to negativemargins the etal. and in 2000). The , the downregulation, thePLE of orthologs ( PLE/SHP 2009,Pan shp1shp2 N. sylvestris ortholog ( AtFUL NtFUL ), orthe the of overexpression FUL orthologs uit across development rosids and asterids, ), another of MADS the member box family orthologs in peach (rosid speciesorthologs inpeach (rosid with fleshy FUL mutant, resulting in indehiscent fruits with resulting inindehiscent fruits mutant, is overexpressed in the relatedspecies isoverexpressed inthe Arabidopsis TAGL1 et al. et ) produce nearly effects in identical fruit Arabidopsis ortholog of of ortholog TAGL1 2010,Vrebalov in tomato, another Solanaceae is required for fruit ripening requiredfor is silique and the lignification silique of andthe and Nicotiana tabacum et al. et Nicotiana Arabidopsis 2007). Thus, both 2007). Thus, et al. 2009).Also, allows us to to allows us AtSHP ,

, NtFUL AtFUL in , Accepted Article For the phylogeneticFor the acid thededucedamino tree, usingthe sequenceswere aligned JQ699178. number NbSHP with accessionretrotranscription). toGenbank hasbeendeposited used oligodT primersfor NbSHPFor2 inthe primer and RT(sequence present the partial sequence of oligos NbSHPFor5'ATGthe amplifywere designedandused a to andNbSHPRev partial sequenceof of sequence gene the isolate used to anddesigned accession L23925)ortholog, number oligos the NbAGForwere andNbAGRev ( tomato distant of information by RT-PCR youngon cDNA of flowers of Full-length coding sequence of of codingsequenceFull-length sequence analysesCloning and with supplementedoligosolution (Hewitt, elements 1966). dark with in photoperiod, a 16-hlight/8-h 1(night) soilirrigatedwith Hoagland no. benthamianaNicotiana Plant material and growth condition PROCEDURES EXPERIMENTAL versa downstreamofvice theFUL-SHP genetic network. allowsdrytransformationthe genetic switch that and of place intofleshyfruits capsules dehiscenceevolutionary of origin in dry and ripening fruits in fleshythatwould fruits andita common pointsto Second, asterids. rosids divergence of the predate subfunctionalizationPLE-like of genestodirect development late fruit thatwould anancestral ideaof strongly the it supports First, conclusions: main draw two © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 TAGL1 AG-like Solanum lycopersicum NbSHP (Tomato (Tomato NtPLE36 genes from the close relative relative close the genes from plantsweregrown 22°C in greenhouse, (day) at the and18°C . The 3'end of The. of 3'end PLE ( N. tabacum PLE tabacum N. N. benthamiana orthologous, accession number AY098735)orthologous,accessionthe number and ) NbAG . From thesequenceof NbSHP N. benthamiana (accession number JQ699177). Fromthe(accession

AG ortholog, accession numberortholog, U63163) was then isolated by RT-PCR using thenisolatedby was and Nicotiana tabacum PLE using available sequence genes have been isolatedgenes havebeen NAG1 ( N. tabacum and the more more the and

Accepted Article RNA antisense andsenseRNAwere antisense probes a generated 504-bp the from of fragment sectionsof paraffin The same regions of The same Virus-Induced GeneSilencing (VIGS)in polymerases. and antisenseprobes were synthesized using thecorresponding SP6 or T7 Both 754). fragments cloned were the pGemT-Easy into (Promega), and vector sense (positions244cDNA to747) and RNA In situ sequences. primer fragment. SeetableS1 the different binding for presentonthe integrates sites biophysical thata using model DNA a fragment of molecules factor transcription in DNAfragments. The occupancy calculates program expected the number of bound transcription binding program The tolocalize best permits sites score factor the matrix. Morpheus webpage facility (http://biodev.cea.fr/morpheus/Default.aspx)LFY withthe probabilitythe presence of LFY of binding in intron sites the sequences we usedthe and NbSHPintronR in the ofcase intheNbAGintronRev, case of were obtainedby NbAGintronFor The PCR primers usingthe secondintrons and OsMADS13 10,000bootstrap replicates to androoted from distance matrices estimated from weredistances alignmentthe from estimated and a treewasneighbour joining andgenetic refined further byPairwisehttp://www.macvector.com/) hand. Poisson toolinCLUSTALW MACVECTOR software 12.0 (MacVector, Inc., © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 were the VIGS experiments. In used for casethe the single of agene constructions, in situ in hybridization , an hybridization with performed digoxigenin-labeled probeswas on8- hybridization Oryza sativa N. benthamiana NbAG and ortholog of the NbAG NbSHP NbSHP NbSHP NbSHP NbSHP buds as described by Ferrándiz et al. (2000).by The budsasdescribed Ferrándiz etal. (accession number JQ699179), NbSHPintronF(accession number coding sequence used for used coding sequence for N. benthamiana (accession number JQ699180). To predict from a 510-bp fragment (positions 244to 510-bp fragment a from Arabidopsis class-Dgene

in situ in situ SEEDSTICK hybridization

NbAG . μ m

Accepted Article restriction site wasrestriction the to added was amplicon 3' The end. digested by Xba1 © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 sequencing, before being introduced into introduced being before sequencing, NbAG, pTRV2-NbSHPand pTRV2-NbAG-NbSHP bydigestion and wereconfirmed using NbSHPthe vector the construction fragment of BamH1 prepared usingtheprepared iQTM SYBR GreenSupermix (Bio-rad). The used primers to First-Strand (Ithe kit cDNA Synthesis Fourwere RNA (Qiagen). of usedtotal withmicrograms cDNAsynthesis performed for Plant Minikit with theRNeasy anthesisflowersfrom at was extracted Total RNA RT-PCRQuantitative Kumar The Agroinoculationof Results wereResults totheexpressionof the normalized qNbSHPRev) generated 81bpand of products di amplify VIGS-treated plants were analyzed by CryoSEM on fresh tissue under the underthe tissue microscopeonfresh plants wereVIGS-treatedby CryoSEM analyzed Scanning electron microscopy(SEM)histology and sequences. Sequence detectionsystem (Applied Biosystems). SeeTableS1 primers for ThereactionsPCR similar. wereand analyzed run ABI usingthe PRISM 7700 of amplification in the efficiency Theand qNbEF1rev. numberamplified byqNbEF1for AY206004), (accession Young buds were collected inYoung buds were histologicalanalyses, for FAAcollected underfixed andvacuum JSM JEOL, CT Instrument 5410,CRIOSEM model Instruments). 15000-C(Oxford restriction site was added to the 5' end of the PCR fragment andPCR a the fragment 5' endof to the added was restriction site et al and cloned intoasimilarly digestedpTRV2 For the vector. double gene NbAG . 2003).. (qNbAGFor and qNbAGRev) and (qNbAGForandqNbAGRev) N. bentahamiana EcoRI NbAG restriction site. The restriction site. resulting three plasmids, pTRV2- NbAG coding sequence was into thepTRV2- introduced , nvitrogen) and qPCRthe master wasmix leaves was as performed described (Dinesh- NbSHP Agrobacterium tumefaciens andthe d not show any cross-amplification. d notshowany cross-amplification. Elongation Factor1 NbSHP EF1 reference gene was gene reference (qNbSHPFor and strain GV3101. ( EF1 Xba1

) mRNA BamH1 and

Accepted Article Table S1.Primers usedthis work in MorphologyFigure S5. indehiscentNbSHP-VIGS of fruits Figure S4. Additional phenotypes of NbSHP-VIGS flowers. flowers TRV2-NbAG-NbSHP or TRV2-NbSHP NbAG, levelof Expression Figure S3. Figure S2.Sensecontrols i for Figure S1. Analyses of SUPPORTING INFORMATION Cienciae Ministerio de C.Ferrándiz.Innovación to of the reading was bymanuscript. BIO2009-09920 funded grant Work Spanish the from and Francisco tool Madueño, Monica Colombo and Barbara Ambrose critical for andMorpheuswith LFY help matrix the EugenioMinguet for plasmids, VIGSproviding Martínez-Pardo Rafael thank We andEugeni Acknowledgements HCl. in2.5% in50% inFAA, phloroglucinolandsoaked 5 30 sec stained were fixed min for and observed underamicroscope Nikon Eclipse For E-600. lignin observation, fruits paraffin. into embedded 10- © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 NbAG μ and m ections were bluesolution stained ina0.2%m toluidine n situ NbAG NbSHP hybridization . and second intron. NbSHP o Grau for technical support, Amy Litt Amy support, technical for Litt Grau for o by real time PCRTRV2- analysis in by time real

Accepted Article development in Arabidopsis. in development Bowman, J.L., Smyth, and Meyerowitz, D.R. E.M. 18902. or organidentity. specifyability male female to B. Davies, and C.A.,Bergonzi, S. Airoldi, REFERENCES © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 in Arabidopsis. Busch,and M.A.,Bomblies, Weigel, K. D. at theplenalocus Antirrhinum. oftransposon Complementary homeotic phenotypesfloral opposite result a of orientations from E. Coen, N.and Hartley, Sommer, H., R., D.,Carpenter, Bradley, elements were evolution of elements for the critical floralthe C-function. B. andDavies, Cook, H. D., B.,Bradley, Causier, in a legume species. in and Lund, O.S. MacFarlane, Krath, B.N., Johansen, M., S.A.,Constantin, I.E. G.D., Nicolaisen, development. L. Brambilla,Marcheselli,Colombo, Caporali, M., V., E., R., and M. Kater, Colombo, development.controlling flower Coen, E.S. and Meyerowitz, E.M. genes. genes. B. Davies, R., Causier, B.,Castillo, Zhou, J., Ingram, R.,Xue, Schwarz-Sommer, Y., Z.and (2010) A (2010) new SHATTERPROOF the rolefor genes during Arabidopsis gynoecium Curr Biol (2005) Evolution inaction: function induplicated following homeotic floral Dev Biol Science , (2004) Virus-induced gene silencing as a tool for (2004)functional genesilencing Virus-induced genomics atoolfor as 15 , 1508-1512. Plant J . 337 , 285 : 294-302. , , 585-587., Plant Cell 40 Nature , 622-631. (1991) The warof the whorls: genetic interactions , , 353 1 , 37-52. , 31-37., (2010) Single amino acid change alters the (1999) Activation of a floral homeotic gene afloral Activation (1999) of Cell Proc NatlProc Acad Sci USA , 72 , 85-95., (1989) Genesdirecting flower (2009) intragenic Conserved Plant J , 58 :41-52. , 107

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, 4023- , (2003) , Accepted Article Network. FruitTOMATO Ripeningthe of 1 isaComponent AGAMOUS-LIKE Regulatory Seybold, M.,Itkin, H., D.,Rogachev, Breitel, I., Meir,Aharoni, S.and A. shadowing. the homeotic floral of gene AGAMOUS and identified by footprinting phylogenetic Hong, Hamaguchi,R.L., L., Busch, M.A. and Weigel, D. Papaverspecies somniferum poppy). (opium toolgene silencing isaneffective assaying for in gene the basalfunction eudicot Drea,A.Martino,Hileman,L.C.,V.F.and G., S., Litt, Irish, © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 box genesinangiosperms.box evolution theMADSand functional diversificationduring the AGAMOUS of subfamily of Kramer, Jaramillo, E.M., M.A.V.S.Stilio, andDi Plant Physiol homeotic of organsby tobacco ectopic expression the floral genereproductive NAG1. Kempin, S.A., Mandel, M.A. and Yanofsky, M.F. 10 and cucumber petunia indiffer their ability toinduce reproductive organ fate. meristem identity, of as revealedmeristem by its loss function. H. Takatsuji, and S. Tsuchimoto, Y., Okuyama, T., Mayama, Y., Tanaka, S., Tsuda, M., Kapoor, Nature M.F. Liljegren, S.J., Ditta, Eshed, G.S., Y., Savidge, Bowman,B., J.L. and Yanofsky, Campagne, M.M.,Angenent, G.C. M.M.,Kater, Colombo, Franken,L., J., Busscher, M., Masiero, S., Van Lookeren , 171-182. , (2000) SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis. , 404 Plant J , 766-770. Plant Cell Plant , 103 (2002) Roleof petunia pMADS3(2002) organand floral indetermination of , 60 , 1041-1046. , :1081-95. :1081-95. , 15 , 1296–1309. , Genetics , 166 (1998) , 1011-1023., Plant J

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, Accepted Article Yanofsky, M.F. M.F. Yanofsky, and E.M. Meyerowitz, H., Ma, S.A., Kempin, J.L., Bowman, M.A., Mandel, © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 analysis of dry and fleshy fruits of Solanaceae. of dry fruits of andfleshy analysis Pabon-Mora, N.A. and Litt, Arabidopsis LEAFYFactor. Transcription the ModelGenomefor SequencesUsing aBiophysical from RegulatoryInteractions of Bastien, O.,Thévenon, Weigel, E., D.,Schmid, M. andParcy, F. Moyroud, Minguet, E., F.,E.G., Ott,Yant, L., Posé, D.,Monniaux, M., S.,Blanchet, 131. AGAMOUS transgenic Arabidopsis alters organidentity. in plants floral and Y. Mizukami, Ma, H. 274 R.J. Ambrose, M., Mena, B.A., Briggs, R.B., Meeley, S.P., Yanofsky, M.F. and Schmidt, 133-143. Plant J TobaccoAdvance. rattle as virus for analysisa vector gene function by of silencing. Ratcliff, F., Martin-Hernandez,A.M. and Baulcombe, D.C. plants.transgenic homeotic roleinits AGAMOUStomato the of Isolation andanalysis gene TAG1 of Rounsley, Yanofsky, L.,Hareven,Pnueli, S.D., D., Lifschitz, andM.F. E. patterning. floral for framework M.A.,Nilsson,D. Busch, O., Lee,I.andWeigel,F., Parcy, development. flowerand fruit regulatingtomato AGAMOUSof genes in lineage diversification V.F. Irish, and J.J. Giovannoni, R., McQuinn, I.L., Pan, 1436. , 1537-1540., (1996) Diversification of C-function activity in maize flower development. development. flower maize activityin C-function (1996) of Diversification , 25 , 237-245. J Exp Bot J Exp (1992) Manipulation inflower of structure transgenictobacco. Plant Cell ., 61 , :1795-806. 6 (1992) Ectopic expression of genefloral homeotic the , 163-173., Nature (2011) Comparative anatomical anddevelopmental , 395 Plant Cell , 561-566. American Botany Journalof . 23 :1293-306. (2010) Functional (1998) A genetic (2001) Technical (2011) Prediction Cell , , 98

Science 71 Cell , 1415- , (1994) , 119- , 71 , , Accepted Article neofunctionalization andsub- events. the AGAMOUS in divergence MADS-boxgenes: subfamily of evidenceof independent dePamphilis, C.W., Becker,A., Theissen, G. and H. Ma, Arrington, L.M.,Leebens-Mack,Zahn, J.H., Landherr, Y., J.M.,Hu, L.L., Evodevo californica). (Eschscholzia poppy California eudicot thewhorl basal the carpel of in A.Becker, A.L.,Yellina, Orashakova, S., Lange, S., Erdmann, Leebens-Mack, R., and J. BotanyAnnals of VIGSof evaluation as a strategy obtain functional non-model to from data plants. induced gene silencing (VIGS) in California poppy (Eschscholzia an californica): Wege, Scholz,A., S., Gleissberg, S. and Becker,A. Plant Cell SHATTERPROOFare regulatedby Tomato expansion andripening the gene TAGL1. Seymour,J., G., Grandillo, S., Giovannoni, J. and Irish, V.F. Vrebalov, J., Pan, I.L., Arroyo,A.J.M., McQuinn, R.,Chung, M., Poole, M., Rose, andpersica) in their role split-pitformation. FRUITFULL-analysis and of SHATTERPROOF-like genes peachfrom (Prunus Tani, E., Polidoros, A.N. and Tsaftaris,A.S. approaches. conditions innon-inductive strictphotoperiodicby Nicotianavarieties of transgenic and Ranganath, S. Gennen, Melzer, V. S., Bodt, Smykal, J., P., De © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012

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8 , 30-45. , , 27 (2007) Highly efficient virus-(2007) Highlyefficient , 649-659. (2006) Conservation and (2009) Fleshy fruit (2007) Flowering

Accepted Article indicate bootstrapindicate values10000 for replicates. Thegene AtSHP2 TAG1 (M55553), TAGL1 (L26295), (AY098735) Numbers onbranches PLE pMADS3 (S53900), PPERSHP(X72912), (DQ777635), AtSHP1 (M55550), (X68675), OsMADS3 (L37528),OsMADS13 (AF151693), OsMADS58 (AB232157), EScaAG1(X53579), (DQ088996),EScaAG2 AmFAR(AJ239057), (DQ088997), FBP6 AG AG and PLEsequences homologues, protein of from the Phylogenetic tree (a) Figure 1 LEGENDS FIGURE © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 AG ofgreydark greylargestintronPrediction(b) LFY and respectively. the of occupancy of analysis. Sequences belonging euAGto and euPLE lineages are shadowed inlight the of ortholog expression isdetected placenta, carpel expression in ovules, wallIn weakly and(i-j) instamens. developing the (g-h)In organs. expressionis specific sexual to mature flowers, flowers, the of infloral stage4 meristem flowers, when begin to petals (e-f) form. stage In 6 when 2 are clearly Expression sepalprimordia (c-d) center stage remains visible. in the (a-b) wereprobedright with Sectionsbuds. were ontheleft withprobed In situ Figure 2 presentbinding sites onthe fragment. ofnumber boundLFY of eachDNAmolecules sequenceintegrating different the benthamiana benthamiana AtSHP2 and NbAG expression analyses of expressionanalyses of PLE At2g42830), and homologues from from homologues ( NbAG Arabidopsis NbSHP NbSHP and Antirrhinum NbSHP expression restricted in the center of the floral meristem at meristem the floral of center in the restricted expression NbSHP class-Dgene (b, d, f, h (b,d,and f, j). Arabidopsis NbAG ( ). The). occupancy relative the calculates expected AmPLE and AY935269 and SEEDSTICK ( NbSHP NbAG AtAG At4g18960, (a, c, e, gand(a, c,e, onthe i).All sections in wild type inwild , was usedasoutgroup in this AmFAR OsMADS13 AtSHP1 AtSHP1 N. benthamiana AJ239057 )and At3g58780 and , Oryza sativa

floral N. N. Accepted Article presence of inner petals.of presence (m) view theCloser apical of the partof a whorlfourth of NbAG-VIGSa NbAG-VIGSwhorl(l) the flower.the of Opened of flower revealing fourth whorl ovary fourth inthehighly (k)Absenceof differentiation flower. modified and style a Centralwhorl to(m) (k) stigma. NbAG-VIGSovary, of shaped" alongstyle andaflat whorlfourth of sepals develops anew enclosing (o) (j) flower whorl petals. Floralto of organsatthe byreplaced The petals.(h) whorl fourth iscomposed of petals. (i) the In of center the are stamens both flowers In NbAG-NbSHP-VIGS style.(h) and(i) flowers. reduced andwith unfused whorl. NbSHP-VIGSa pistil an flower fourth (g) presenting abnormal flower showing homeotic conversions of stamens intopetals and the presence of an are adnatelywhose filaments petals, the fused andthe to central pistil. (f) NbAG-VIGS stamens a the then whorltubular sepals, petals, of of consecutively awhorl presenting Longitudinalsection of (i) petals(e) toorgans. VIGSwhorls presenting andnovisiblesexual flower additional of Note absence flower. the center in stigma of of the the anthers. NbAG-NbSHP-five (d) (c) NbSHP-VIGS stigma. and novisible intopetals stamens of the conversion showing anthers petals and surroundingfive presenting five stigma. (b) the NbAG-VIGS flower (a) (d)to Top view of pTRV2-NbAG-NbSHP. Phenotypes of Figure 3 with sense probes are shown in Figure S2. stages ovule. Mandel Developmental from o: al.(1992).pistil, Control et hybridizations stages, expression later is inovules. p:mainly detected s: sepal, petal,st: stamen, pi: © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 benthamiana NbAG-VIGS indicatingtheflower presence ofstigmatic tissue. (n)NbSHP-VIGS pistils showing fusion pistils showing reductiondefects, fusion styleof length and increased N. benthamiana N. benthamiana N. benthamiana N. benthamiana plants plants treated with pTRV2-NbAG, pTRV2-NbSHP or flowers. (j) Wild type pistil composed of an"egg- of (j) composed flowers. type pistil Wild flowers. (a) typeWild flower in anthesis flowers. (e) Wild type anthesis flower flowers. type (e) Wild anthesis

N. Accepted Article free petal, s: sepal,stigma, petal, s: sg: free sy:style. andnumerousp*: styles. center, carpel wall, ov:ovary, petal, meristem, floral cw: p: fm: a NbSHP-VIGS gynoecium presenting additional whorls carpels of developing in its Longitudinal of (j) section the flower. of center inthe meristem a new of floral presence whorl fourth are shown.(i) Longitudinal NbAG-VIGS section of showing flower the Conversion stamens of into petalsandthe presence some of stigmatic tissue inthe benthamiana NbSHP-VIGS unfused a styleof with (g) wild Longitudinal section andstigma. type stigma and thepistil. upperstyle. theapicalWT of (e) Longitudinal section of (f) part and(f) petals. (e) whorls of andnumerous whorl anouter sepals of of composed aNbAG-NbSHP-VIGS of section sexualTransverse flower lacking organsand (d) ovulesandplacenta. whorl carpeltissuedifferentiation, lacking andafourth petals intofree converted stamens surrounding eight petals whorl of enlarged second an Transverse section aNbAG-VIGS(c) of whorlflower showing first anormal sepals, of filaments stamen thepetals with fused and at least carpels developingfive in the ovary. secondwhorls, eight and a NbSHP-VIGSfirst wild of type presenting flower section stamens andthebi-carpellateadnately filaments ovary. central fused Transverse(b) petal whorlwith sepalwhorl,five tubular wildan external flower a showing (a) type to (d) Transverse(a) section of or pTRV2-NbAG-NbSHP.NbSHP Sections of Figure 4 whorl NbAG-NbSHP-VIGS fourth the ina cm.= 0.5 Scaleflower. bar styles of Presence number anof andstigma. the baseof (o) ectopic floral at meristem © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 Figure 5 N. benthamiana flower. (h) and (i) Longitudinal section of NbAG-VIGS (h)section of flower. (h) and(i)Longitudinal flower. plants flowers from treated with pTRV2-NbAG, pTRV2- N. benthamiana flower at 0.5 cm up flower at from thepedicel.

N. N. Accepted Article Cellular types observed in a VIGS NbSHP fourth whorl, in the ovary (e) and in the style in (e) and ovarywhorl,VIGS fourth inthe NbSHP a in Cellular types observed Cellsareirregularwhorl. andtrichomes and canbe stomata and observed. (e) (f) stomata of a wild-type sepal.(d)Cellular types the at surface of VIGS NbAG fourth Elongatedovary. (b) acells of wild type (c)Puzzle style. shaped trichomes andcells, wild-type fruit. (d) Absence of lignin aof mature capsule ofaNbSHP-VIGS plant. (c)Lignification patternofa of a NbSHP. (a)Open capsule mature altered in dehiscence andlignification Fruit Figure 6 (f). Note the presence of trichomes and stomata in both cases. Scale bar = 60 VIGS epidermalScanning electron analyses cell of inflowersmicroscope of morphology © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd Blackwell Publishing 2012 © Journal The Plant Authors. The © 2012 N.benthamiana treated plants. (a)Smalltreatedandrectangular cells awild of type N. benthamiana N. benthamiana N. benthamianaN. NbSHP-VIGS fruit. NbSHP-VIGS fruit. wild-type Closed plant. (b) plants treated with pTRV2- treated with plants N. benthaniana μ

Accepted Article