Patrice Morel

Pierre Chambrier

Véronique Boltz

Sophy Chamot

Frédérique Rozier

Suzanne Rodrigues Bento

Christophe Trehin

Marie Monniaux

Jan Zethof

Michiel Vandenbussche
email: michiel.vandenbussche@ens-lyon.fr

Divergent Functional Diversification Patterns in the SEP/AGL6/AP1 MADS-box Transcription Factor Superclade

Keywords: SEPALLATA, APETALA1, AP1/SQUA, AGL6, MADS-box, floral meristem identity, inflorescence meristem identity, plant evolution, ABC model, Petunia, Arabidopsis, inflorescence architecture

établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Over the last two decades, the ABC model of floral organ identity has served as a genetic framework for the understanding of flower development in other species, and across evolution [START_REF] Bowman | The ABC model of flower development: then and now[END_REF]. Members of the MADS-box transcription factor family play a central role in this model, and especially the MADS-BOX proteins encoding the floral B-and Cfunctions have been studied in a wide range of species [START_REF] Krizek | Molecular mechanisms of flower development: an armchair guide[END_REF], providing a better understanding of the evolution and diversification of floral development at the molecular level. By contrast, much less comparative data is available for members of the AP1/SQUA and the SEPALLATA MADS-box transcription factor subfamilies. Compared to the B-and C-class MADS-box subfamilies, the SEP and AP1/SQUA subfamilies have substantially expanded via several gene duplication events during angiosperm evolution [START_REF] Litt | Duplication and diversification in the APETALA1/FRUITFULL floral homeotic gene lineage: implications for the evolution of floral development[END_REF][START_REF] Zahn | The evolution of the SEPALLATA subfamily of MADS-box genes: a preangiosperm origin with multiple duplications throughout angiosperm history[END_REF]. Together with reported extensive redundancy among individual SEP and among AP1/SQUA genes (see below), this makes comparative functional studies challenging, and probably underlies the relative lack of functional data in a broad range of species.

Moreover, the extensive sequence similarity observed among members within both subfamilies may render the interpretation of phenotypes obtained by gene-silencing approaches (such as RNAi/co-suppression/VIGS) difficult. In addition, in several species members of the closely related AGL6 MADS-box subfamily also perform SEP-like functions [START_REF] Ohmori | MOSAIC FLORAL ORGANS1, an AGL6-like MADS box gene, regulates floral organ identity and meristem fate in rice[END_REF][START_REF] Rijpkema | The petunia AGL6 gene has a SEPALLATA-like function in floral patterning[END_REF][START_REF] Thompson | bearded-ear encodes a MADS box transcription factor critical for maize floral development[END_REF][START_REF] Dreni | Flower development: the evolutionary history and functions of the AGL6 subfamily MADS-box genes[END_REF], adding further genetic complexity to a comparative analysis of the SEP function across species borders.

The SEP and AP1/SQUA MADS-box transcription factor families are unique to angiosperms, while AGL6 genes are present both in gymnosperms and angiosperms [START_REF] Becker | The major clades of MADS-box genes and their role in the development and evolution of flowering plants[END_REF][START_REF] Litt | Duplication and diversification in the APETALA1/FRUITFULL floral homeotic gene lineage: implications for the evolution of floral development[END_REF][START_REF] Zahn | The evolution of the SEPALLATA subfamily of MADS-box genes: a preangiosperm origin with multiple duplications throughout angiosperm history[END_REF]. Interestingly, the AGL6, SEP and AP1/SQUA subfamilies together compose a monophyletic superclade within the MADS-box family (further referred to as the AP1/SEP/AGL6 superclade), suggesting a common ancestral origin predating the angiosperm/gymnosperm divergence, although the evolutionary relationship between the different subfamilies had not been completely resolved [START_REF] Purugganan | Molecular evolution of flower development: diversification of the plant MADS-box regulatory gene family[END_REF][START_REF] Purugganan | The MADS-box floral homeotic gene lineages predate the origin of seed plants: phylogenetic and molecular clock estimates[END_REF][START_REF] Becker | The major clades of MADS-box genes and their role in the development and evolution of flowering plants[END_REF]. A more recent phylogenetic analysis based on exon/intron structural changes suggests that AGL6 genes are sister to both SEP and AP1 subfamilies [START_REF] Yu | Prevalent Exon-Intron Structural Changes in the APETALA1/FRUITFULL, SEPALLATA, AGAMOUS-LIKE6, and FLOWERING LOCUS C MADS-Box Gene Subfamilies Provide New Insights into Their Evolution[END_REF].

Thus far, Arabidopsis is the only core eudicot species for which a functional characterization of all its AP1/SEP/AGL6 superclade genes has been achieved in sufficient detail, including the identification of redundant functions through higher order mutant analysis, but a wealth of functional data has been accumulated also in tomato and rice in recent years (see further). The Arabidopsis SEP subfamily consists of four members, named SEP1, SEP2, SEP3 and SEP4, and petals, stamens and carpels in the sep1 sep2 sep3 triple mutant are transformed into sepals [START_REF] Pelaz | B and C floral organ identity functions require SEPALLATA MADS-box genes[END_REF], while all floral organs in a sep1 sep2 sep3 sep4 mutant develop as leaf-like organs [START_REF] Ditta | The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity[END_REF]. This led to the conclusion that SEP genes are required for the identity of all floral organs, and function in a largely, but not completely redundant fashion. In addition, SEP genes were shown to be involved in floral meristem identity and determinacy [START_REF] Pelaz | B and C floral organ identity functions require SEPALLATA MADS-box genes[END_REF][START_REF] Ditta | The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity[END_REF]. SEP proteins are proposed to act as 'bridge proteins' enabling higher order complex formation (floral quartets) with the products of the homeotic B and C function organ identity genes, and to provide transcriptional activation capacity to these complexes [START_REF] Honma | Complexes of MADS-box proteins are sufficient to convert leaves into floral organs[END_REF][START_REF] Theissen | Plant biology. Floral quartets[END_REF][START_REF] Immink | SEPALLATA3: the 'glue' for MADS box transcription factor complex formation[END_REF][START_REF] Melzer | The class E floral homeotic protein SEPALLATA3 is sufficient to loop DNA in 'floral quartet'-like complexes in vitro[END_REF]. These findings have inspired the addition of the SEP (or E-) function to the classic ABC model of floral development [START_REF] Bowman | Genetic interactions among floral homeotic genes of Arabidopsis[END_REF][START_REF] Coen | The war of the whorls: genetic interactions controlling flower development[END_REF], summarized in a floral quartet model [START_REF] Theissen | Plant biology. Floral quartets[END_REF]. In contrast to the function of AGL6 genes in other species, the two Arabidopsis AGL6 subfamily members AGL6 and AGL13 do not seem to perform a SEP-like function in floral organ identity determination [START_REF] Koo | Control of lateral organ development and flowering time by the Arabidopsis thaliana MADS-box Gene AGAMOUS-LIKE6[END_REF][START_REF] Huang | Epistatic natural allelic variation reveals a function of AGAMOUS-LIKE6 in axillary bud formation in Arabidopsis[END_REF][START_REF] Hsu | AGAMOUS-LIKE13, a putative ancestor for the E functional genes, specifies male and female gametophyte morphogenesis[END_REF]. The Arabidopsis AP1/SQUA subfamily is composed of 4 members, of which the roles of AP1, CAL (CAULIFLOWER) and FUL (FRUITFULL) in floral development have been particularly well studied. Arabidopsis ap1 mutants lack petals and have sepals displaying bract like features [START_REF] Irish | Function of the apetala-1 gene during Arabidopsis floral development[END_REF][START_REF] Mandel | Molecular characterization of the Arabidopsis floral homeotic gene APETALA1[END_REF]. For these reasons, AP1 has been classified as an A-function gene in the ABC model, required for the identity specification of sepals and petals.

Furthermore, AP1 plays also a major role in specifying floral meristem identity, in a largely redundant fashion with CAL [START_REF] Bowman | Control of flower development in Arabidopsis thaliana by APETALA1 and interacting genes[END_REF][START_REF] Kempin | Molecular basis of the cauliflower phenotype in Arabidopsis[END_REF]. FUL was initially identified for its unique role in Arabidopsis carpel and fruit development [START_REF] Gu | The FRUITFULL MADSbox gene mediates cell differentiation during Arabidopsis fruit development[END_REF], but in addition was later shown to function redundantly with AP1 and CAL to control inflorescence architecture [START_REF] Ferrandiz | Redundant regulation of meristem identity and plant architecture by FRUITFULL, APETALA1 and CAULIFLOWER[END_REF].

To provide more insight in floral development and in the evolution of the floral gene regulatory network in higher eudicot species in general, we have been systematically analyzing the genetics underlying floral development in the Asterid species Petunia x hybrida. While the genes encoding the floral A, B and C-functions in petunia have been well characterized [START_REF] Angenent | Petal and stamen formation in petunia is regulated by the homeotic gene fbp1[END_REF][START_REF] Van Der Krol | Functional analysis of petunia floral homeotic MADS box gene pMADS1[END_REF][START_REF] Kater | Multiple AGAMOUS Homologs from Cucumber and Petunia Differ in Their Ability to Induce Reproductive Organ Fate[END_REF][START_REF] Kapoor | Role of petunia pMADS3 in determination of floral organ and meristem identity, as revealed by its loss of function[END_REF][START_REF] Vandenbussche | The duplicated B-class heterodimer model: whorl-specific effects and complex genetic interactions in Petunia hybrida flower development[END_REF][START_REF] Rijpkema | Analysis of the Petunia TM6 MADS box gene reveals functional divergence within the DEF/AP3 lineage[END_REF][START_REF] Cartolano | A conserved microRNA module exerts homeotic control over Petunia hybrida and Antirrhinum majus floral organ identity[END_REF][START_REF] Heijmans | Redefining C and D in the Petunia ABC[END_REF][START_REF] Morel | Divergence of the Floral A-Function between an Asterid and a Rosid Species[END_REF][START_REF] Morel | The Floral C-Lineage Genes Trigger Nectary Development in Petunia and Arabidopsis[END_REF], only a few of the 10 previously described genes belonging to the large petunia AP1/SEP/AGL6 superclade [START_REF] Immink | A petunia MADS box gene involved in the transition from vegetative to reproductive development[END_REF][START_REF] Ferrario | The MADS box gene FBP2 is required for SEPALLATA function in petunia[END_REF][START_REF] Immink | Analysis of the petunia MADS-box transcription factor family[END_REF]Vandenbussche et al., 2003a;[START_REF] Vandenbussche | Toward the analysis of the petunia MADS box gene family by reverse and forward transposon insertion mutagenesis approaches: B, C, and D floral organ identity functions require SEPALLATA-like MADS box genes in petunia[END_REF][START_REF] Rijpkema | The petunia AGL6 gene has a SEPALLATA-like function in floral patterning[END_REF] have been functionally analyzed thus far.

Research on petunia SEPALLATA genes dates back a long time and provided, together with a study in tomato, the first indication of the existence of SEP-function in floral development: transgenic lines in which the SEP3-like petunia FBP2 or tomato TM5 genes were silenced by co-suppression both exhibited simultaneous homeotic conversion of whorls 2, 3, and 4 into sepal-like organs and loss of determinacy in the center of the flower [START_REF] Angenent | Cosuppression of the petunia homeotic gene fbp2 affects the identity of the generative meristem[END_REF][START_REF] Pnueli | The TM5 MADS Box Gene Mediates Organ Differentiation in the Three Inner Whorls of Tomato Flowers[END_REF], a phenotype similar to that later found in Arabidopsis sep1 sep2 sep3 mutants. However, at that time, multimeric complex formation of MADS-box proteins still remained to be discovered [START_REF] Egea-Cortines | Ternary complex formation between the MADS-box proteins SQUAMOSA, DEFICIENS and GLOBOSA is involved in the control of floral architecture in Antirrhinum majus[END_REF], and it was not clear how many genes where co-suppressed in these lines. Therefore, the molecular basis of these phenotypes in petunia and tomato was not immediately understood. Later, it was shown in yeast that petunia SEP proteins also bind to B-class heterodimers and to C-class proteins, mediate quaternary complex formation with B-and C-class proteins and display transcriptional activation capacity [START_REF] Ferrario | The MADS box gene FBP2 is required for SEPALLATA function in petunia[END_REF], compatible with the proposed quartet model in Arabidopsis.

Interestingly, among the six petunia SEP-like proteins, also clear differences in protein-protein interactions were revealed in a yeast 2-hybrid assay, suggesting functional diversification [START_REF] Ferrario | The MADS box gene FBP2 is required for SEPALLATA function in petunia[END_REF][START_REF] Immink | Analysis of the petunia MADS-box transcription factor family[END_REF]. Especially FBP2 and FBP5 showed a much broader range of interaction partners compared to the other petunia SEP proteins. Furthermore, it was shown in planta that petunia SEP proteins may be crucial to import at least some other MADSbox transcription factors into the nucleus [START_REF] Immink | Analysis of MADS box protein-protein interactions in living plant cells[END_REF].

Using a gene-specific approach, we showed that the fbp2 co-suppression phenotype was indeed not gene specific, since single fbp2 mutants showed only a very incomplete sep-like phenotype, with primarily the margins of the petals exhibiting a petal-to sepal homeotic conversion [START_REF] Vandenbussche | Toward the analysis of the petunia MADS box gene family by reverse and forward transposon insertion mutagenesis approaches: B, C, and D floral organ identity functions require SEPALLATA-like MADS box genes in petunia[END_REF]. We also reported fbp5 mutants that as single mutants develop as wild type. Flowers of fbp2 fbp5 mutants, however, showed an enhanced phenotype compared to fbp2 mutants: the sepaloid regions at the petal edges extended slightly further towards the center; sepal-like structures appeared on top of the anthers, and a sudden dramatic phenotype appeared in the ovary, which continued to grow long after development has arrested in wild-type (WT) flowers of comparable stages, resulting in a giant ovary. While the general architecture of the ovary was maintained (carpels containing an interior placenta), inside all ovules were homeotically converted to sepal-like organs [START_REF] Vandenbussche | Toward the analysis of the petunia MADS box gene family by reverse and forward transposon insertion mutagenesis approaches: B, C, and D floral organ identity functions require SEPALLATA-like MADS box genes in petunia[END_REF]. This directly demonstrated that not only the identity of petals, stamens and carpels depends on SEP activity in petunia, but also ovule identity, as was also reported in Arabidopsis in the same journal issue [START_REF] Favaro | MADS-box protein complexes control carpel and ovule development in Arabidopsis[END_REF]. More recently, we demonstrated that petunia AGL6 also exhibits SEP-like functions [START_REF] Rijpkema | The petunia AGL6 gene has a SEPALLATA-like function in floral patterning[END_REF], and performs a major role in petal identity, redundantly with FBP2. In addition, a function in stamen development was revealed by fbp2 fbp5 agl6 triple mutant analysis. In line with the proposed SEP-function for AGL6, we found that AGL6 and FBP2 in yeast overall interact with the same the partners [START_REF] Rijpkema | The petunia AGL6 gene has a SEPALLATA-like function in floral patterning[END_REF].

Thus far, three petunia AP1/SQUA genes have been described, called PFG, FBP26 and FBP29 [START_REF] Immink | Analysis of the petunia MADS-box transcription factor family[END_REF], and only the function of PFG was analyzed, using a cosuppression approach, resulting in a dramatic nonflowering phenotype, although the occasional development of single solitary flowers in these lines was also reported [START_REF] Immink | A petunia MADS box gene involved in the transition from vegetative to reproductive development[END_REF].

However, as for the FBP2 co-suppression line, the full-length coding sequence including the highly conserved MADS-domain was used to generate the co-suppression construct, questioning the specificity of the obtained phenotype.

To provide more insight in the functions of the AP1/SEP/AGL6 superclade members in petunia, and more broadly in the evolutionary trajectory of the AP1/SEP/AGL6 superclade in the core eudicots, we aimed to uncover unique and redundant functions of the complete SEP/AGL6 and AP1/SQUA subfamilies during petunia flower development.

First, we present a genetic fine-dissection of the petunia SEP-function obtained from the analysis of a series of single and multiple knock-out mutants, combining putative null mutations in the six petunia SEP genes and AGL6. Most remarkably, we found that the FBP9 subclade members FBP9 and FBP23, for which no clear ortholog is present in Arabidopsis [START_REF] Zahn | The evolution of the SEPALLATA subfamily of MADS-box genes: a preangiosperm origin with multiple duplications throughout angiosperm history[END_REF], play an essential role in determining floral meristem identity together with FBP4, with only moderate contributions to the classic SEP floral organ identity function.

Furthermore, we show that the petunia genetic equivalent of the Arabidopsis sep1 sep2 sep3 mutant still displays residual B-and C-function activity, while a full sepallata phenotype was obtained in a sextuple fbp2 fbp4 fbp5 fbp9 pm12 agl6 mutant. The analysis further suggests that the petunia SEP3 ortholog FBP2 performs a master floral organ identity SEP-function as in Arabidopsis. In addition, we have analyzed the dependence of homeotic gene expression on the SEP function, by comparing the dynamics of expression between wild-type and the sextuple fbp2 fbp4 fbp5 fbp9 pm12 agl6 mutant.

Finally, we show that the petunia AP1/SQUA subfamily is composed of four members (PFG, FBP26, FBP26 and the here described euAP1 gene) that function in a largely redundant way. We found that they are required for inflorescence meristem identity, but surprisingly, pfg fbp26 fbp29 euap1 mutants developed fully functional terminal flowers. In addition, we show that they act as B-function repressors in the first floral whorl, together with BEN/ROB genes [START_REF] Morel | Divergence of the Floral A-Function between an Asterid and a Rosid Species[END_REF]. Overall, comparison of these data with previous studies in mainly Arabidopsis, tomato and rice reveal major differences in the functional diversification of the SEP/AGL6 and AP1/SQUA MADS-box subfamilies during evolution of the angiosperms.

RESULTS

Petunia Floral Development

To facilitate the comparison of the phenotypes presented in this study with the equivalent Arabidopsis mutants, we summarize first the relevant differences in WT floral architecture between petunia and Arabidopsis (Figure 1). Petunia flowers consist, from the outside towards the center, of five sepals partly fused at their basis, five large congenitally fused petals, five stamens of which the filaments are partly fused with the petal tube, and a central pistil composed of two congenitally fused carpels (Figure 1A). Some important differences in flower development between petunia and Arabidopsis, and relevant for this study, concern sepal identity, placentation topology and inflorescence architecture. Indeed in Arabidopsis, epidermal cell types and trichome architecture found on sepals can clearly be distinguished from those of leaves [START_REF] Ditta | The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity[END_REF]. By contrast, petunia sepals display a similar kind of epidermal cell types as found in bracts and leaves, and are covered with the same type of multicellular trichomes (Figure 1C). While Arabidopsis sepals dehisce rapidly after fertilization of the flower and subsequently fall off together with petals and stamens, petunia sepals physiologically behave more as leaf-like organs: they stay firmly attached to the pedicel and may remain green, even long after the fruit has fully matured (Figure 1B). Note that the same occurs in flowers that were not fertilized (see further Figure 4F). The parietal placenta and ovules in Arabidopsis develop from the inner ovary wall, after termination of the floral meristem. In petunia, the central placenta arises directly from the center of the floral meristem in between the two emerging carpel primordia (Figure 1D), suggesting that the floral meristem is terminated later compared to Arabidopsis [START_REF] Colombo | Arabidopsis ovule development and its evolutionary conservation[END_REF]. Finally, Petunia species develop a cymose inflorescence (Figure 1E, inset) as opposed to the raceme in Arabidopsis (reviewed in [START_REF] Castel | Inflorescence development in petunia: through the maze of botanical terminology[END_REF]). During petunia cymose inflorescence development, the apical meristem terminates by forming a flower, while an inflorescence meristem (IM) emerges laterally, repeating the same pattern [START_REF] Souer | Genetic control of branching pattern and floral identity during Petunia inflorescence development[END_REF]. This results in the typical zigzag-shaped petunia inflorescence with alternating flowers on each node subtended by bracts.

Petunia SEP/AGL6 Expression Analysis and Mutant Identification

Six SEP genes and one AGL6 gene [START_REF] Ferrario | The MADS box gene FBP2 is required for SEPALLATA function in petunia[END_REF][START_REF] Vandenbussche | Toward the analysis of the petunia MADS box gene family by reverse and forward transposon insertion mutagenesis approaches: B, C, and D floral organ identity functions require SEPALLATA-like MADS box genes in petunia[END_REF][START_REF] Rijpkema | The petunia AGL6 gene has a SEPALLATA-like function in floral patterning[END_REF] were described in petunia compared to 4 SEP genes and 2 AGL6-like genes in Arabidopsis. A survey of the recently released Petunia axillaris and Petunia inflata genome sequences [START_REF] Bombarely | Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida[END_REF] indicated that these sequences represent the total number of SEP/AGL6 genes in petunia (Supplemental Table 1). Several detailed and robust phylogenetic studies of the SEP family [START_REF] Zahn | The evolution of the SEPALLATA subfamily of MADS-box genes: a preangiosperm origin with multiple duplications throughout angiosperm history[END_REF][START_REF] Yu | Prevalent Exon-Intron Structural Changes in the APETALA1/FRUITFULL, SEPALLATA, AGAMOUS-LIKE6, and FLOWERING LOCUS C MADS-Box Gene Subfamilies Provide New Insights into Their Evolution[END_REF] as well as the more limited phylogenetic analysis presented here (Figure 1F), identified FBP2 as the sole SEP3 ortholog in petunia, meaning that the petunia SEP3 clade contains only one member as in Arabidopsis. Petunia FBP5 and PMADS12 (PM12) were shown to be the closest relatives of SEP1 and SEP2, with the FBP5/PM12 and SEP1/SEP2 paralogous pairs originating from independent gene duplications in the lineages leading to petunia and Arabidopsis respectively.

Finally, petunia FBP4 grouped in the SEP4 subclade, while FBP9 and FBP23 genes were members of the FBP9 subclade, a subclass of SEP genes that is absent from the Arabidopsis genome and potentially may have been lost in the lineage leading to Arabidopsis [START_REF] Malcomber | SEPALLATA gene diversification: brave new whorls[END_REF][START_REF] Zahn | The evolution of the SEPALLATA subfamily of MADS-box genes: a preangiosperm origin with multiple duplications throughout angiosperm history[END_REF]. The larger number of SEP genes in petunia compared to Arabidopsis is therefore entirely due to the presence of the two petunia FBP9 subclade genes.

As expected based on their close taxonomic relationship, the petunia proteins overall showed the closest relationship with SEP/AGL6 members from tomato [START_REF] Tomato | The tomato genome sequence provides insights into fleshy fruit evolution[END_REF][START_REF] Soyk | Bypassing Negative Epistasis on Yield in Tomato Imposed by a Domestication Gene[END_REF] compared to Arabidopsis and rice (Figure 1F). Like petunia, tomato contained one AGL6 gene, one SEP3 copy and two FBP9 members, but slight differences in the number of genes belonging to the SEP1/SEP2 and SEP4 subclades could be observed between the two species. Notably, tomato contained only one SEP1/SEP2 copy, while having two SEP4-like genes. Among the members of the tomato SEP/AGL6 family, the SEP4like RIN gene initially received most of the attention, because the classical rin mutation has been widely used in tomato breeding as it improves shelf-life of tomato fruits when present in a heterozygous state, while the homozygous rin mutation prevents initiation of ripening [START_REF] Vrebalov | A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus[END_REF]. Interestingly, more recent studies in tomato have shed a first light on the function of the enigmatic FBP9 subclade genes. First of all, it was found that SLMBP21/J2 (JOINTLESS 2) is required for the development of the pedicel abscission zone [START_REF] Liu | The SEPALLATA MADS-box protein SLMBP21 forms protein complexes with JOINTLESS and MACROCALYX as a transcription activator for development of the tomato flower abscission zone[END_REF][START_REF] Roldan | Natural and induced loss of function mutations in SlMBP21 MADS-box gene led to jointless-2 phenotype in tomato[END_REF][START_REF] Soyk | Bypassing Negative Epistasis on Yield in Tomato Imposed by a Domestication Gene[END_REF]. Furthermore, a breakthrough functional study [START_REF] Soyk | Bypassing Negative Epistasis on Yield in Tomato Imposed by a Domestication Gene[END_REF] based on both natural and CRISPR induced mutant alleles showed that the two tomato FBP9 clade genes SLMBP21/J2 and SlMADS1/EJ2 (ENHANCER OF JOINTLESS 2) have overlapping functions in meristem maturation and the control of inflorescence branching together with LIN (LONG INFLORESCENCE), the second tomato SEP4-like gene.

Remarkably, triple j2 ej2 lin knockout mutants exhibit a dramatic phenotype consisting of massively overproliferated sympodial inflorescence meristems (SIMs) without the formation of flowers, indicating that the transition towards FM identity is not made.

As a first step in the characterization of the complete SEP/AGL6 clade in petunia, we performed RT-qPCR expression analysis (Figure 1G) in three floral bud developmental stages (Figure 1E) with the stage 1 floral bud sample also including very early flower primordia, bracts and the inflorescence meristem, and in various other tissues. This allows for a more quantitative analysis than a previous study by RNA gel blot and in situ hybridization [START_REF] Ferrario | The MADS box gene FBP2 is required for SEPALLATA function in petunia[END_REF].

We detected important differences in expression levels among the SEP/AGL6 genes and clear differences in expression patterns, both correlated with their phylogenetic position, suggesting functional divergence: FBP2, FBP5, and AGL6 were the most abundantly expressed genes, reaching expression levels roughly tenfold higher than the SEP4 homolog FBP4, and the FBP9 subclade members FBP9 and FBP23. Furthermore, FBP2, FBP5 and AGL6 expression was restricted to floral tissues, with expression levels strongly increasing during floral bud development, while FBP4, FBP9 and FBP23 were more broadly expressed, and expression levels did not show a strong upregulation during later stages of floral bud development.

Expression outside the floral domain was most marked in bracts for FBP4, and in the inflorescence stem tissue for both FBP4 and FBP9. One exception to these general differences observed between SEP1/SEP2/SEP3/AGL6 and SEP4/FBP9 genes was PM12, which was expressed ~100 fold lower than its close paralog FBP5, and for which expression was detected also in bracts and stems. For all genes analyzed, expression levels varied considerably between the different floral organs: Expression may be much lower in one particular organ type compared to the three other floral organs (e.g. very low FBP2 and FBP5 expression in sepals; low levels of PMADS12, AGL6 and FBP23 expression in stamens), or may peak in one specific floral organ (FBP4 in sepals; FBP9 in petals). Our results are in agreement with the in situ data previously obtained for FBP2 and FBP5, showing mainly expression in the three inner floral whorls during early flower development, while some minor differences with the PMADS12 in situ data suggest that the PM12 expression pattern is not constant as floral buds further develop [START_REF] Ferrario | The MADS box gene FBP2 is required for SEPALLATA function in petunia[END_REF].

To perform a functional analysis, we used a reverse genetics strategy [START_REF] Koes | Targeted gene inactivation in petunia by PCR-based selection of transposon insertion mutants[END_REF][START_REF] Vandenbussche | Toward the analysis of the petunia MADS box gene family by reverse and forward transposon insertion mutagenesis approaches: B, C, and D floral organ identity functions require SEPALLATA-like MADS box genes in petunia[END_REF][START_REF] Vandenbussche | Generation of a 3D indexed Petunia insertion database for reverse genetics[END_REF] to identify dTph1 transposon insertions in the coding sequences of the petunia SEP and AGL6 genes. In total, we identified and confirmed 16 independent transposon insertions in planta, including some earlier reported alleles [START_REF] Vandenbussche | Toward the analysis of the petunia MADS box gene family by reverse and forward transposon insertion mutagenesis approaches: B, C, and D floral organ identity functions require SEPALLATA-like MADS box genes in petunia[END_REF][START_REF] Rijpkema | The petunia AGL6 gene has a SEPALLATA-like function in floral patterning[END_REF] in all of the 7 different members of the SEP/AGL6 clade (Figure 1H). Because the 284 bp dTph1 sequence encodes stop codons in all six possible reading frames, and based on their insert position (either disrupting the first exon encoding the MADS DNA binding domain, or the K-region required for protein-protein interactions in the case of the fbp2 insertions, all of the selected insertion alleles most likely represent null alleles. We obtained and analyzed homozygous mutants for all insertion alleles, but remarkably, only homozygous mutants for fbp2 insertions displayed floral homeotic defects (Figure 1H), suggesting extensive functional redundancy among the petunia SEP/AGL6 genes, and that FBP2 function is more essential than that of any other SEP/AGL6 gene. These results clearly indicated the need for multiple mutant analyses to further uncover putative redundant functions.

The Petunia fbp2 fbp5 pmads12 Mutant, Genetic Equivalent of the Arabidopsis sep1 sep2 sep3 Mutant, Displays Floral Characteristics Indicating Residual B-and C-Function

Activity

In Arabidopsis, the simultaneous loss of SEP1, SEP2 and SEP3 results in flowers consisting only of sepals [START_REF] Pelaz | B and C floral organ identity functions require SEPALLATA MADS-box genes[END_REF]. To compare the petunia genetic equivalent, we aimed to analyze fbp2 fbp5 pm12 triple mutants (Figure 2). As mentioned earlier, of the three single mutants, only fbp2 mutants displayed a phenotype different from WT (Figures 2A to 2D). Moreover, fbp2/+ fbp5 pm12 flowers (Figure 2E) developed morphologically as WT, demonstrating that FBP2 even in a heterozygous state can fully compensate for the loss of FBP5 and PM12 functions. In addition, fbp2 pm12 double mutants were not markedly different from fbp2 single mutants (Figure 2F), in contrast to the earlier reported fbp2 fbp5 mutants (Figures 2G to 2K) and [START_REF] Vandenbussche | Toward the analysis of the petunia MADS box gene family by reverse and forward transposon insertion mutagenesis approaches: B, C, and D floral organ identity functions require SEPALLATA-like MADS box genes in petunia[END_REF]. However, fbp2 fbp5 pm12 flowers could be easily distinguished from fbp2 fbp5 flowers: a clear enhancement of stamen to sepal identity could be observed in the third whorl, although still some antheroid tissue remained, as in fbp2 fbp5 mutants (Figure 2M). Furthermore, while the extremely enlarged fbp2 fbp5 mutant pistil still exhibited partial carpel identity, the carpels of fbp2 fbp5 pm12 mutants acquired clear sepal/leaf-like epidermal characteristics (Figures 2N to 2Q), and were densely covered with trichomes. The latter are never observed on WT pistils, and only at very low frequency on fbp2 fbp5 pistils (Figure 2N). Furthermore, no stigma and style structures remained in the triple mutant, but the overall internal organization of the ovary was maintained, with a placenta structure covered by a few hundred leaf-like organs that represented homeotically converted ovules, as observed in fbp2 fbp5 mutants (Figures 2K and2L). In the second whorl of fbp2 fbp5 pm12 flowers, the partial petal to sepal conversion at the corolla border was only subtly enhanced compared to fbp2 fbp5 mutants (Figures 2G and 2H). Given that the effect of the pm12 mutation only became apparent in an fbp2 fbp5 mutant background, we conclude that PM12 plays a less essential role than its close paralog FBP5. Overall, the remnant petal and stamen tissues and the maintenance of a placenta structure in fbp2 fbp5 pm12 flowers show that unlike in Arabidopsis, genes outside the SEP3 and SEP1/SEP2 subfamilies are able to rescue part of the B-and C-functions in a petunia sep1 sep2 sep3 mutant background.

The FBP9 Subclade Genes FBP9 and FBP23 Function as Floral Meristem Identity Genes together with FBP4

We showed earlier that petunia AGL6 is one of the genes outside the classical SEP1/SEP2/SEP3 group that plays a prominent role in performing a SEP-like floral organ identity function, especially in the determination of petal identity, redundantly with FBP2 [START_REF] Rijpkema | The petunia AGL6 gene has a SEPALLATA-like function in floral patterning[END_REF].

However, FBP4 as a SEP4-like gene may also participate, as found in Arabidopsis [START_REF] Ditta | The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity[END_REF] and potentially also FBP9 and FBP23, the petunia representatives of the FBP9 subclade.

Earlier we found that the fbp9, fbp23 and fbp4 single mutants displayed a WT phenotype (Figure 1H), and that expression levels of all three genes peak early during floral developmental stages compared to the other petunia SEP genes and AGL6 (Figure 1G), potentially indicating a redundant (common) function for FBP4, FBP9 and FBP23. Indeed, a functional overlap was recently demonstrated among corresponding SEP subclade members in tomato [START_REF] Soyk | Bypassing Negative Epistasis on Yield in Tomato Imposed by a Domestication Gene[END_REF].

To test such a putative functional redundancy among the petunia FBP9, FBP23 and FBP4 genes, we first created and analyzed fbp9 fbp23 double mutants, since FBP9 and FBP23 are close paralogs belonging to the same FBP9 SEP-subclade. Interestingly, we found that fbp9 fbp23 mutants were dramatically affected in their inflorescence architecture, with new inflorescence shoots developing instead of flowers, resulting in a highly branched inflorescence structure. However, flower development was not completely abolished in these mutants, because after several weeks of a highly branched inflorescence development, frequently a flower appeared on one or more branches of the same plant, after which these branches switched again to the initial phenotype (Figures 3B and 3F). This indicated that the capacity to form floral meristems was not completely abolished in fbp9 fbp23 mutants and that (an)other factor(s) can partly rescue floral meristem determinacy in the absence of FBP9/FBP23 function.

Because we assumed FBP4 being a likely candidate, we next analyzed fbp4 fbp9 fbp23 triple mutants. Indeed, we found that the fbp9 fbp23 phenotype was further enhanced in these triple mutants, resulting in a highly branched flowerless inflorescence architecture (Figures 3C,3G and 3H), phenotypically very similar to that reported earlier for the petunia floral meristem identity mutant alf [START_REF] Souer | Genetic control of branching pattern and floral identity during Petunia inflorescence development[END_REF], with ALF being orthologous to Arabidopsis LEAFY (LFY) [START_REF] Weigel | LEAFY controls floral meristem identity in Arabidopsis[END_REF] and snapdragon FLORICAULA (FLO) [START_REF] Coen | floricaula: a homeotic gene required for flower development in antirrhinum majus[END_REF] genes.

Note that over a long period (> 6 months) of highly branched inflorescence development, some triple mutants produced 1-2 isolated flowers, while other individuals never flowered at all.

To study the fbp4 fbp9 fbp23 phenotype in more detail, we analyzed fbp4 fbp9 fbp23 inflorescence apices by scanning electron microscopy in comparison with WT (Figures 3I to 3L). At very early developmental stages, the fbp4 fbp9 fbp23 plants exhibited a phenotype very comparable to alf mutants [START_REF] Souer | Genetic control of branching pattern and floral identity during Petunia inflorescence development[END_REF]: as in alf mutants, the bifurcation pattern of fbp4 fbp9 fbp23 inflorescence apices was similar to WT, but the two resulting meristems both behaved as inflorescence meristems, as indicated by the continuous bifurcation of each newly formed meristem and the repetitive formation of bracts flanking these meristems. Together, this indicates that floral meristems in fbp4 fbp9 fbp23 mutants are homeotically transformed into inflorescence meristems.

Finally, floral meristem identity was not visibly affected in fbp4 fbp9 fbp23/+ and fbp4 fbp23 fbp9/+ plants, as judged by the presence of a normal cymose inflorescence architecture in these mutant combinations (Figures 3M to O). This shows that the presence of either FBP9 or FBP23 in heterozygote state is sufficient to rescue floral meristem identity. Together with the already strong phenotype observed in fbp9 fbp23 plants compared to fbp4 fbp9 fbp23 plants (Figures 3B to 3C,3F to 3G,3P to 3Q), we conclude that the FBP9 clade members FBP23 and FBP9 play a major role in floral meristem identity determination in a largely redundant fashion, while FBP4 is involved in the same function, but plays a less essential role compared to the FBP9/FBP23 gene pair.

Although the phenotypes of tomato j2 ej2 lin and petunia fbp9 fbp23 fbp4 mutants at first sight do not look very similar (see discussion), overall, this shows that in both species, FBP9 clade genes together with a SEP4 gene play an essential role in floral meristem identity, different from the classical SEP organ identity functions.

To test whether FBP4, FBP9 and FBP23 also function later in conferring floral organ identity, we introduced the corresponding mutations into the fbp2 mutant background, the only petunia sep mutation with a visible phenotype as a single mutant. However, we found that flowers of fbp2 fbp4, fbp2 fbp9 and fbp2 fbp23 mutants were not markedly different from fbp2 mutants (Figures 3R to 3U), while fbp2 fbp4 fbp23 and fbp2 fbp4 fbp9 flowers only showed a moderate enhancement of the fbp2 petal-to-sepal conversion phenotype (Figure Figures 3V to 3W). In fbp2 fbp4 fbp23 flowers, the green margin appeared to be broader in all five petals while in fbp2 fbp4 fbp9 flowers this was most visible in the two basal petals. In comparison with the earlier described floral phenotypes of fbp2 fbp5 [START_REF] Vandenbussche | Toward the analysis of the petunia MADS box gene family by reverse and forward transposon insertion mutagenesis approaches: B, C, and D floral organ identity functions require SEPALLATA-like MADS box genes in petunia[END_REF], fbp2 agl6, fbp2 fbp5 agl6 [START_REF] Rijpkema | The petunia AGL6 gene has a SEPALLATA-like function in floral patterning[END_REF] and fbp2 fbp5 pmads12 mutants, this suggests that FBP4, FBP9 and FBP23 do play a role in floral organ identity, but contribute only moderately to this function compared to the petunia SEP1/SEP2/SEP3 homologs and AGL6. Note that we also obtained fbp2 fbp4 fbp9 fbp23 quadruple mutants, but as expected, these developed a highly branched flowerless inflorescence structure as in fbp4 fbp9 fbp23 mutants.

The Sextuple fbp2 fbp4 fbp5 fbp9 pm12 agl6 Mutant Displays a Classic sepallata Phenotype

By analyzing AGL6, FBP2 and FBP5 functions and the fbp2 fbp5 pmads12 and fbp4 fbp9 fbp23 triple and fbp2 fbp4 fbp9 fbp23 mutants, we could reveal specific/specialized SEP functions for certain members of the petunia SEP/AGL6 clade and surprisingly, the requirement of FBP9/FBP23 function (and to a lesser extend FBP4) for floral meristem identity, as was also recently shown in tomato. However, a classic floral sepallata phenotype as described for Arabidopsis was not obtained, indicating further redundancy, possibly shared between the majority of the petunia SEP/AGL6 genes. To test this further, we embarked on a long-term crossing scheme aimed to combine all of the sep/agl6 mutant alleles in a single plant. However, we chose to exclude the fbp23 mutation in this scheme since this would completely abolish flower formation when combined with the fbp9 and fbp4 mutations and thus prevent visualization of additive floral phenotypes. After years of crossing, we finally obtained homozygous sextuple fbp2-332 fbp4-44 fbp5-129 fbp9-90 pm12-37 agl6-118 mutant plants, hereafter referred to as sextuple sep/agl6 mutants. In contrast to the earlier described lower order mutants, all organs in the flowers of sextuple sep/agl6 mutants were green and densely covered by trichomes (Figure 4), exhibiting sepal/leaf-like characteristics (Figures 4A to 4E).

Note that as mentioned earlier, it is not possible to discriminate between sepal, bract and leaf identity in petunia based on epidermal cell characteristics (Figure 1C). As expected, scanning electron microscopy of these organs revealed the conversion of the typical petal, stamen and carpel epidermal cell types into epidermal cells characteristic for sepals, bracts and leaves, including stomata and multicellular trichomes (Figure 4E). The second whorl, which in WT consists of five large brightly colored fused petals, was occupied by five green organs that remained fused at their bases (Figure 1C). Although dramatically smaller than WT petals, they remained larger than first whorl sepals. Similarly, in the third whorl, the five stamens were replaced by sepal/leaf like organs. The overall shape of these organs did retain some of the stamen architecture, since the region corresponding to the WT stamen filament remained smaller compared to the more leaf blade-like upperparts. Stamen filaments in WT are fused along half of their length with the inside of the petal tube. By contrast, third whorl organs in the sextuple mutant completely lost this partial fusion. In the fourth whorl, normally occupied by two carpels that are entirely fused and enclose the placenta and ovules, two (sometimes three) unfused sepal/leaf-like organs were found. Internally, the placenta was entirely replaced by a new emerging flower reiterating the same floral phenotype (Figure 1D). Thus in contrast to lower order sep mutants, the sextuple mutant was fully indeterminate. In the majority of the flowers (Figure 4F), this secondary flower further developed and emerged from the primary flower supported by a pedicel, while containing on itself another flower in its center. This third flower usually did not further grow out, although occasionally we observed up to three consecutive fully developed flowers (Figure 4F). Note that the sextuple sep/agl6 mutant displayed a normal cymose inflorescence architecture as in WT (Figure 4F), in sharp contrast to fbp4 fbp9 fbp23 mutants. This demonstrates that FBP23 alone can fully rescue floral meristem identity in a sextuple mutant background, but not floral organ identity.

Homeotic Gene Expression in Sextuple sep/agl6 Mutant Flowers

To further characterize the sextuple sep/agl6 mutant at the molecular level, we quantified and compared the dynamics of homeotic gene expression levels between WT and the sextuple mutant (Figure 4G) at three different stages of floral bud development, as described earlier (Figure 1E). Encoding of the B-function in petunia is more complex compared to Arabidopsis and Antirrhinum, and involves the two PI/GLO-like MADS-box transcription factors Petunia hybrida GLO1 and GLO2, the DEF/AP3 ortholog PhDEF, and PhTM6, the petunia representative of the ancestral B-class TM6 lineage that has been lost in Arabidopsis, but which is present in many species [START_REF] Angenent | Petal and stamen formation in petunia is regulated by the homeotic gene fbp1[END_REF][START_REF] Van Der Krol | Functional analysis of petunia floral homeotic MADS box gene pMADS1[END_REF]Vandenbussche et al., 2004;[START_REF] Rijpkema | Analysis of the Petunia TM6 MADS box gene reveals functional divergence within the DEF/AP3 lineage[END_REF]. In WT, we found that all four B-class genes were progressively upregulated as floral buds developed, with an upregulation from stage 1 to stage 3 varying roughly from three to six times, depending on the gene. In the sextuple mutant, we observed expression levels of PhGLO1, PhGLO2 and PhDEF initially similar to WT in the youngest stage analyzed. However, upregulation in older stages was strongly affected, especially for PhGLO1 and PhGLO2, which remained expressed at initial levels. PhDEF remained progressively upregulated in the different sextuple mutant samples, but reached only one third of the expression compared to WT in the final stage. By contrast, PhTM6 expression levels were strongly downregulated from stage 1 floral buds onwards, remaining at similarly low levels in the two older stages.

The C-function in petunia is redundantly encoded by PMADS3 and FBP6, orthologs of Arabidopsis AG and SHP1/2 respectively [START_REF] Heijmans | Redefining C and D in the Petunia ABC[END_REF][START_REF] Morel | The Floral C-Lineage Genes Trigger Nectary Development in Petunia and Arabidopsis[END_REF]. As for the B-function genes, FBP6 and PMADS3 expression in WT is progressively upregulated in developing floral buds (6,5 and 11 times respectively), and initial expression levels in stage 1 buds were very similar between WT and sextuple mutants for both C-class genes. Both C-genes still displayed a clear upregulation in the older sextuple mutant flower buds, and seemed in general less affected by the sextuple loss of SEP/AGL6 function than the B-function genes, especially in stage 2. In stage 3 buds, FBP6 expression was not different between WT and sextuple mutants, while PMADS3 in the sextuple mutant was expressed at around 50% of its WT levels. FBP11 is a petunia D-lineage MADS-box gene orthologous to STK (Angenent et al., 1995;[START_REF] Colombo | The Petunia MADS box gene FBP11 determines ovule identity[END_REF], and that with FBP7 (another D-lineage member) and the Cgenes PMADS3 and FBP6 redundantly is required to confer ovule identity, and to arrest the floral meristem [START_REF] Heijmans | Redefining C and D in the Petunia ABC[END_REF]. Consistent with its later function in floral development, the FBP11 expression profile showed a very strong upregulation in the WT developmental series (~30 fold). By contrast, in the sextuple mutant samples, FBP11 expression was barely detectable in all stages tested. Finally, we choose to monitor the expression of petunia UNS (UNSHAVEN), a member of the SOC1 subfamily, because of its particular expression pattern reported to be mainly restricted to green tissues including stems, leaves, bracts and the first whorl (sepals) in the flower [START_REF] Immink | Analysis of the petunia MADS-box transcription factor family[END_REF][START_REF] Ferrario | Ectopic expression of the petunia MADS box gene UNSHAVEN accelerates flowering and confers leaf-like characteristics to floral organs in a dominant-negative manner[END_REF]. Moreover, UNS ectopic expression was shown to confer leaflike characteristics to floral organs. We first used the cDNA series from Figure 1F to analyze its expression pattern in a more quantitative manner compared to earlier gel blot data, confirming highest expression levels in bracts, inflorescence stems, and in the sepals within the flower (Supplemental Figure 1). In the WT developmental series, we found UNS to be progressively downregulated as flower buds further developed, corresponding to a ~4 fold drop in expression levels compared to the youngest stage (Fig 4G). Interestingly, UNS was expressed at higher levels in all sextuple mutant floral bud stages compared to WT, with the largest difference found in the oldest bud stage (~ 5-fold upregulation compared to WT). Moreover, a linear downregulation as in WT was not observed.

The Petunia AP1/SQUA Subfamily: Phylogeny, Expression Analysis and Mutant

Identification

We found that the petunia SEP genes FBP9, FBP23 and FBP4 function primarily as floral meristem identity genes, a function which is in Arabidopsis mainly associated with members of the AP1/SQUA MADS-box subfamily [START_REF] Irish | Function of the apetala-1 gene during Arabidopsis floral development[END_REF][START_REF] Mandel | Molecular characterization of the Arabidopsis floral homeotic gene APETALA1[END_REF][START_REF] Kempin | Molecular basis of the cauliflower phenotype in Arabidopsis[END_REF][START_REF] Ferrandiz | Redundant regulation of meristem identity and plant architecture by FRUITFULL, APETALA1 and CAULIFLOWER[END_REF]. This raised the obvious question to what extent the petunia AP1/SQUA members are implicated in floral meristem identity determination. For these reasons, we aimed to functionally analyze the members of the petunia AP1/SQUA subfamily.

Thus far, three petunia AP1/SQUA genes have been described, called PFG, FBP26 and FBP29 [START_REF] Immink | A petunia MADS box gene involved in the transition from vegetative to reproductive development[END_REF][START_REF] Immink | Analysis of the petunia MADS-box transcription factor family[END_REF]. In addition, based on sequence similarity, we identified a fourth AP1/SQUA member by the presence of an insertion mutant and associated transposon flanking sequence encountered in our transposon flanking sequence database, which we have called Ph-euAP1 (Petunia x hybrida euAP1), based on the presence of the highly conserved euAP1 motif [START_REF] Litt | Duplication and diversification in the APETALA1/FRUITFULL floral homeotic gene lineage: implications for the evolution of floral development[END_REF]Vandenbussche et al., 2003a) in its C-terminus, as also found in the Arabidopsis AP1 and CAL genes (Supplemental Figure 2A). To provide further proof for the euAP1 classification of the new Petunia AP1/SQUA member, we conducted a phylogenetic analysis (Figure 5) including all AP1/SQUA subfamily members from Arabidopsis, tomato (Hileman et al., 2006; The Tomato Genome Consortium, 2012 and rice [START_REF] Lee | Systematic reverse genetic screening of T-DNA tagged genes in rice for functional genomic analyses: MADS-box genes as a test case[END_REF][START_REF] Yu | Prevalent Exon-Intron Structural Changes in the APETALA1/FRUITFULL, SEPALLATA, AGAMOUS-LIKE6, and FLOWERING LOCUS C MADS-Box Gene Subfamilies Provide New Insights into Their Evolution[END_REF]. Similar to the SEP/AGL6 phylogenetic analysis, overall the petunia proteins showed the closest relationship with AP1/SQUA members from tomato (Figure 5A), while all four rice AP1 members grouped apart from the eudicot proteins as shown previously [START_REF] Yu | Prevalent Exon-Intron Structural Changes in the APETALA1/FRUITFULL, SEPALLATA, AGAMOUS-LIKE6, and FLOWERING LOCUS C MADS-Box Gene Subfamilies Provide New Insights into Their Evolution[END_REF]. The analysis further showed that petunia euAP1 indeed is orthologous to the tomato MACROCALYX (MC) gene [START_REF] Vrebalov | A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus[END_REF] and the Arabidopsis AP1 and CAL genes, all previously demonstrated as belonging to the euAP1 clade [START_REF] Litt | Duplication and diversification in the APETALA1/FRUITFULL floral homeotic gene lineage: implications for the evolution of floral development[END_REF][START_REF] Yu | Prevalent Exon-Intron Structural Changes in the APETALA1/FRUITFULL, SEPALLATA, AGAMOUS-LIKE6, and FLOWERING LOCUS C MADS-Box Gene Subfamilies Provide New Insights into Their Evolution[END_REF]. Petunia therefore is similar to tomato in having only one euAP1 clade member compared to two members in Arabidopsis. MC, the tomato euAP1 representative, was shown to regulate sepal size, fruit abscission and maintenance of inflorescence meristem identity. Indeed, mc mutants develop flowers with enlarged sepals, have an incomplete pedicel abscission zone, and develop inflorescences that revert to vegetative growth after forming two to three flowers [START_REF] Vrebalov | A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus[END_REF][START_REF] Nakano | MACROCALYX and JOINTLESS interact in the transcriptional regulation of tomato fruit abscission zone development[END_REF][START_REF] Yuste-Lisbona | Characterization of vegetative inflorescence (mc-vin) mutant provides new insight into the role of MACROCALYX in regulating inflorescence development of tomato[END_REF].

The previously described FBP29 gene fell into the AGL79 subclade to which the tomato genes MBP10 and MBP20 also belonged, while the PFG and FBP26 genes grouped into the euFUL subclade together with the tomato FUL1 (TDR4/TM4) and FUL2 (MBP7) genes as previously shown [START_REF] Yu | Prevalent Exon-Intron Structural Changes in the APETALA1/FRUITFULL, SEPALLATA, AGAMOUS-LIKE6, and FLOWERING LOCUS C MADS-Box Gene Subfamilies Provide New Insights into Their Evolution[END_REF]. While stable mutants remain to be described for these four tomato genes, RNAi mediated downregulation of FUL1 and FUL2 indicate a role for these genes in fleshy fruit ripening [START_REF] Bemer | The tomato FRUITFULL homologs TDR4/FUL1 and MBP7/FUL2 regulate ethyleneindependent aspects of fruit ripening[END_REF][START_REF] Shima | Tomato FRUITFULL homologues act in fruit ripening via forming MADS-box transcription factor complexes with RIN[END_REF][START_REF] Wang | Members of the tomato FRUITFULL MADS-box family regulate style abscission and fruit ripening[END_REF]. Furthermore, a role for MBP20 and FUL1 was proposed in the regulation of compound leaf development [START_REF] Burko | A role for APETALA1/fruitfull transcription factors in tomato leaf development[END_REF]. Finally, to date no function has been proposed for tomato MBP10 but an evolutionary study of the FUL genes in the Solanaceous family suggest that the MBP10 lineage, which is absent in petunia, may be undergoing pseudogenization [START_REF] Maheepala | Evolution and Diversification of FRUITFULL Genes in Solanaceae[END_REF]. A sequence analysis of the Petunia axillaris and Petunia inflata genome sequences [START_REF] Bombarely | Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida[END_REF] further indicated that euAP1, PFG, FBP26 and FBP29 represent the total number of AP1/SQUA family members in petunia (Supplemental Table 1), similar to the size of the AP1/SQUA subfamily in Arabidopsis and rice, and one less compared to tomato (due to the absence of a MBP10 lineage member in petunia).

A quantitative expression analysis in different tissues and three floral bud developmental stages in WT (Figure 5B) showed that the expression patterns of the four genes were quite similar, although some minor differences did exist. Interestingly, expression levels of all four genes gradually decreased during floral bud development, suggesting an early developmental function, similar as what we observed for e.g. FBP9 and FBP4. Furthermore, during later flower development, moderate expression levels were detected in sepals, petals (except for FBP29) and carpels, while expression in stamens was considerably lower compared to the other floral organs. The four genes were also well expressed in inflorescence stem tissues as well as in bracts (with the exception of Ph-euAP1). Finally, PFG showed the broadest expression pattern, since moderate expression levels were also observed in vegetative apices and leaves. In addition, the peak values of PFG expression levels were around tenfold higher compared to those of Ph-euAP1, FBP26 and FBP29. The PFG expression data were in line with the broad expression pattern previously observed by RNA gel blot analysis and in situ hybridization [START_REF] Immink | A petunia MADS box gene involved in the transition from vegetative to reproductive development[END_REF], which revealed PFG expression in vegetative, inflorescence and floral meristems, in newly formed leaves, the vascular tissues, during early flower organ development and in carpel walls and ovules during later phases of pistil development.

To determine the function of the four petunia AP1/SQUA genes, we screened for dTph1 transposon insertions in their coding sequences, similarly as for the SEP genes. In total, we identified and confirmed 6 independent transposon insertions in planta (Figure 5C), including two earlier reported alleles [START_REF] Vandenbussche | Toward the analysis of the petunia MADS box gene family by reverse and forward transposon insertion mutagenesis approaches: B, C, and D floral organ identity functions require SEPALLATA-like MADS box genes in petunia[END_REF], potentially yielding putative null mutants for all four genes based on the insertion position of the dTph1 transposon, either disrupting the first exon encoding the MADS DNA binding domain or the K-region required for protein-protein interactions in the case of the euap1 allele. We obtained and analyzed homozygous mutants for all insertion alleles, but all these homozygous mutants developed normally (Figure 5C). Moreover, when we analyzed some double mutants to overcome putative genetic redundancy, flowers in these double mutants developed normally, and inflorescence architecture was not affected (Supplemental Figure 2B).

Petunia AP1/SQUA Family Members are Required for Inflorescence Meristem Identity

Because of the absence of clear phenotypes in the above-described mutants, we decided to create and analyze pfg fbp26 fbp29 euap1 quadruple mutants (Figure 6). Remarkably, the flowers that developed on these quadruple mutants were fertile, and organ identity of the carpels, stamens and petals was not visibly affected (Figures 6A to 6C). However, sepals were considerably enlarged and contained sectors that exhibited homeotic conversion towards petal identity, as indicated by the red pigmentation and the presence of petal conical cells in these regions (Figure 6D). Overall, the general mildness of the pfg fbp26 fbp29 euap1 flower phenotype was very surprising, compared to the already dramatic phenotypes found in Arabidopsis ap1 single and ap1 cal double mutants, and compared to the complete absence of flowers in ap1 cal ful triple mutants [START_REF] Irish | Function of the apetala-1 gene during Arabidopsis floral development[END_REF][START_REF] Mandel | Molecular characterization of the Arabidopsis floral homeotic gene APETALA1[END_REF][START_REF] Kempin | Molecular basis of the cauliflower phenotype in Arabidopsis[END_REF][START_REF] Ferrandiz | Redundant regulation of meristem identity and plant architecture by FRUITFULL, APETALA1 and CAULIFLOWER[END_REF].

Quadruple pfg fbp26 fbp29 euap1 mutants did display a severe phenotype in inflorescence development. In fact, the normal cyme inflorescence architecture was completely abolished, and instead a large number of leaves were produced from the main apical meristem before terminating into a solitary flower (Figures 6E and6G). In addition, branches that developed from the base of the plant followed exactly the same developmental pattern (Figure 6L). The leaves produced on the main stem and side branches were generated in a spiral phyllotaxy (Figures 6F and6Q), characteristic of vegetative development (Figure 6P), in contrast to the opposite positioning of bracts in a WT inflorescence meristem. Finally, after the production of usually >25 leaves, this vegetative meristem was fully converted into a floral meristem resulting in a solitary flower (Figures 6H to 6I) as opposed to the normal cyme inflorescence structure in WT (Figure 6M). Note that quadruple mutant flowers consistently displayed an increase in floral organ number (e.g. the flower shown in Figure 6A has 10 petals), possibly because the full conversion of the vegetative meristem into a floral meristem resulted in a larger floral meristem size. In addition, the corolla of these flowers was not always properly organized, as the petal tube was often disrupted on one side.

Once this terminal flower was fully developed, new branches started to grow from vegetative meristems that were present in the axils of the leaves further down on the stem (Figure 6I). These branches produced again a large number of leaves before terminating with a solitary flower (Figure 6X), after which the same process was repeated. Together these results indicate that petunia AP1/SQUA genes are required to establish inflorescence meristem identity and associated cymose branching of the petunia inflorescence.

Interestingly, intermediate phenotypes could be observed in different triple mutants in which the fourth AP1 subfamily member was still in a heterozygous state (Figures 6S to 6Q The partial sepal-to-petal homeotic conversion in flowers of pfg fbp26 fbp29 euap1 mutants (Figure 6D) suggests that petunia AP1/SQUA genes negatively regulate the B-function in the first floral whorl. Recently we demonstrated that the AP2-type REPRESSOR OF B (ROB1), ROB2 and ROB3 genes repress the B-function in the first whorl, together with BEN, a TOEtype AP2 gene [START_REF] Morel | Divergence of the Floral A-Function between an Asterid and a Rosid Species[END_REF]. To further explore the implication of the petunia AP1/SQUA genes in patterning the B-function, we tested their genetic interaction with ROB genes. We crossed pfg fbp26 fbp29 euap1 and rob1 rob2 rob3 mutants and screened progenies for an enhanced sepal-to-petal homeotic conversion phenotype compared to pfg fbp26 fbp29 euap1 and rob1 rob2 rob3 mutants. Among a large progeny, we found individuals displaying the pfg fbp26 fbp29 euap1 inflorescence phenotype while bearing terminal flowers of which the first whorl organs showed a much more pronounced sepal-to-petal conversion compared to pfg fbp26 fbp29 euap1 mutants. We genotyped several of these plants for the seven insertions, and found that plants with the strongest phenotype were rob1 rob2/+ rob3 pfg fbp26 fbp29 euap1 (Figures 6J and6K). Flowers of these mutants had first whorl organs that clearly formed the beginning of a petal tube (Figure 6J), although not fused along its entire length, and with strongly expanded petaloid regions compared to the first whorl organs of pfg fbp26 fbp29 euap1 flowers (Figures 6D and6K). The presence of pale pigmentation at the basal end of the organs and bright red at the distal end (Figure 6K) was also characteristic for the modular tube/corolla architecture of a WT petunia petal (Figures 1A and2I). For comparison, first whorl sepals of rob1 rob2/+ rob3 plants had a phenotype similar to WT (Figures 6N to 6O), while rob1 rob2 rob3 flowers exhibit a very subtle sepal-to-petal conversion at the margins of their sepals, and which is only clearly visible in the first 2-3 flowers that develop [START_REF] Morel | Divergence of the Floral A-Function between an Asterid and a Rosid Species[END_REF]. Although we did not obtain plants homozygous for all seven mutations, the synergistic interaction observed between rob1 rob2/+ rob3 and pfg fbp26 fbp29 euap1 mutations strongly supports a role for petunia AP1/SQUA genes in repressing the B-function in the first whorl, together with the ROB/BEN genes. In this study, we exploited the natural dTph1 transposon mutagenesis system in petunia to identify mutants for all 11 members of the petunia AP1/SEP/AGL6 superclade, and created a series of higher order mutants to uncover putative redundant functions. Here we discuss and compare our findings with the available functional data from mainly Arabidopsis, tomato and rice (see Figures 1F and5A for the composition of their SEP/AGL6 and AP1/SQUA subfamilies). Petunia and tomato on the one hand, and Arabidopsis on the other hand are representatives of the Asterids and Rosids respectively, which constitute the two major groups in the core eudicots, and are thought to have diverged >100 million years ago [START_REF] Moore | Phylogenetic analysis of 83 plastid genes further resolves the early diversification of eudicots[END_REF]. Comparison of the molecular mechanisms controlling flower development in these species therefore helps to assess conservation and divergence of the floral regulatory gene network in the core eudicots [START_REF] Vandenbussche | Petunia, Your Next Supermodel?[END_REF]. Petunia and tomato both belong to the Solanaceous family, and the lineages leading to petunia and tomato are estimated to have diverged around 30 MYA [START_REF] Bombarely | Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida[END_REF]. Their close relationship is indeed reflected in a high degree of sequence similarity between tomato/petunia orthologous pairs in the AP1/SEP/AGL6 superclade (see also Supplemental Data Files 1, 2, 3 and 4), which makes the petunia/tomato comparison particularly well suited to evaluate functional diversification patterns on a shorter evolutionary time-scale, as opposed to the comparison with the distant monocot model species rice.

DISCUSSION

Implication of SEP and AGL6 Gene Functions in Floral Organ Identity

Our genetic analysis in petunia indicates that its SEP3 ortholog FBP2 encodes the major SEP organ identity function: FBP2 is capable of fully rescuing flower development in a fbp2/+ fbp5 pm12 mutant background, and fbp2 is the only sep single mutant with a clearly visible phenotype. In Arabidopsis, all available genetic data indicate that SEP3 is also the most important SEP gene. Indeed, it was reported that single sep3 mutants display a phenotype on their own, showing a mild petal to sepal conversion, while sep1, sep2 and sep4 single mutants showed no developmental abnormalities [START_REF] Pelaz | APETALA1 and SEPALLATA3 interact to promote flower development[END_REF]. Secondly, sep1 sep2 sep4 mutants show no significant perturbation of floral organ development, indicating that SEP3 can fully rescue WT development in a triple mutant background [START_REF] Ditta | The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity[END_REF]. Thus SEP3 seems to perform a master SEP floral organ identity function in both species.

While the gene-silencing approaches used to analyze SEP3 function in tomato and rice

do not yet allow such detailed conclusions, these experiments suggest that their SEP3 orthologs play also a major role in floral organ identity: Tomato TM5 co-suppression lines genes exhibited homeotic conversion of whorls 2, 3, and 4 into sepal-like organs and loss of determinacy in the center of the flower [START_REF] Pnueli | The TM5 MADS Box Gene Mediates Organ Differentiation in the Three Inner Whorls of Tomato Flowers[END_REF] and a Y2H study found that TM5 was the preferred bridge protein of the 5 SEP tomato proteins tested [START_REF] Leseberg | Interaction study of MADS-domain proteins in tomato[END_REF]. Transgenic lines carrying a construct aimed at simultaneously downregulating the two SEP3-like rice OsMADS7 and OsMADS8 genes were late flowering, and carried flowers exhibiting partial homeotic conversions of the floral organs in the three inner whorls into palea/lemma-like organs, and a partial loss of floral determinacy [START_REF] Cui | Functional conservation and diversification of class E floral homeotic genes in rice (Oryza sativa)[END_REF].

Arabidopsis sep1 sep2 sep3 mutants display a full conversion of petals, stamens and carpels into sepals, and flowers are fully indeterminate [START_REF] Pelaz | B and C floral organ identity functions require SEPALLATA MADS-box genes[END_REF]. By contrast, the genetically equivalent fbp2 fbp5 pmads12 mutant in petunia retains -albeit reduced-petal and stamen tissues, and the basic organization of the placenta structure in the flower center is maintained. Thus unlike in Arabidopsis, genes outside the SEP3 and SEP1/SEP2 clades are able to rescue part of the B-and C-functions in a petunia sep1/sep2/sep3 mutant background.

We identified petunia AGL6 as one of these genes [START_REF] Rijpkema | The petunia AGL6 gene has a SEPALLATA-like function in floral patterning[END_REF]. Similarly, the two rice AGL6 genes OsMADS6/MFO1 and OsMADS17 perform SEP-like functions, partly in a redundant fashion with the SEP gene OsMADS1/LHS1 [START_REF] Ohmori | MOSAIC FLORAL ORGANS1, an AGL6-like MADS box gene, regulates floral organ identity and meristem fate in rice[END_REF][START_REF] Dreni | Flower development: the evolutionary history and functions of the AGL6 subfamily MADS-box genes[END_REF]. More recently, a floral organ identity function was also proposed for the tomato AGL6 gene, based on RNAi [START_REF] Yu | Silencing SlAGL6, a tomato AGAMOUS-LIKE6 lineage gene, generates fused sepal and green petal[END_REF]. Despite that the Arabidopsis genome encodes two AGL6 homologs (AGL6 and AGL13), the phenotype of the sep1 sep2 sep3 mutant demonstrates that Arabidopsis AGL6 genes may have lost most of their SEP-like activity compared to petunia, rice and tomato AGL6 genes, in agreement with the diverse proposed functions for Arabidopsis AGL6 and AGL13 [START_REF] Koo | Control of lateral organ development and flowering time by the Arabidopsis thaliana MADS-box Gene AGAMOUS-LIKE6[END_REF][START_REF] Huang | Epistatic natural allelic variation reveals a function of AGAMOUS-LIKE6 in axillary bud formation in Arabidopsis[END_REF][START_REF] Hsu | AGAMOUS-LIKE13, a putative ancestor for the E functional genes, specifies male and female gametophyte morphogenesis[END_REF]. Furthermore, we showed that in a petunia sextuple sep/agl6 mutant a full sepallata phenotype was obtained, including complete loss of floral meristem termination. Remarkably, the obtained phenotype was similar to that of the earlier described FBP2 co-suppression line [START_REF] Ferrario | The MADS box gene FBP2 is required for SEPALLATA function in petunia[END_REF], demonstrating the efficiency of co-suppression to silence multiple genes simultaneously. The expression levels of all six petunia SEP genes (but not of AGL6) were monitored in the co-suppression line, but only FBP2 and FBP5 were found to be downregulated. This strongly suggests that other genes were silenced at the post-transcriptional level as was reported to frequently occur in gene silencing experiments [START_REF] Stam | The silence of genes in transgenic plants[END_REF].

Measuring mRNA levels of paralogous genes thus appears to be a limited method to assess the specificity of a silencing construct.

The addition of the sep4 mutation to the Arabidopsis triple sep1 sep2 sep3 mutant resulted in the conversion of sepal-like organs into leaf-like organs, indicating that SEP genes are required to specify sepal identity [START_REF] Ditta | The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity[END_REF]. The fact that we could not observe a transition from sepal towards leaf-identity in the sextuple sep/agl6 mutant is most likely directly related to the 'leaf'-like identity of petunia WT sepals. Such basic differences in sepal identity between Arabidopsis and other species such as petunia may be contributing to the difficulties to formulate a broadly applicable A-function [START_REF] Litt | An Evaluation of A-Function: Evidence from the APETALA1 and APETALA2 Gene Lineages[END_REF][START_REF] Causier | Floral organ identity: 20 years of ABCs[END_REF].

Transgenic lines in which at least four of the rice SEP-like genes (OsMADS1/LHS1 (LEAFY HULL STERILE1)), OsMADS5, OsMADS7 and OsMADS8) were downregulated, showed homeotic transformation of all floral organs except for the lemma into leaf-like organs [START_REF] Cui | Functional conservation and diversification of class E floral homeotic genes in rice (Oryza sativa)[END_REF], reminiscent of the Arabidopsis sep1 sep2 sep3 sep4 quadruple mutant flower phenotype. Remarkably however, severe loss-of-function mutations in the LOFSEP gene OsMADS1/LHS1 alone can cause complete homeotic conversion of organs of the three inner whorls into lemma/palea-like structures, and loss of floral meristem determinacy [START_REF] Agrawal | Conservation of the E-function for floral organ identity in rice revealed by the analysis of tissue culture-induced loss-of-function mutants of the OsMADS1 gene[END_REF], while also dominant-negative and milder phenotypes were reported for other OsMADS1/LHS1 alleles [START_REF] Jeon | leafy hull sterile1 is a homeotic mutation in a rice MADS box gene affecting rice flower development[END_REF][START_REF] Chen | Morphogenesis and molecular basis on naked seed rice, a novel homeotic mutation of OsMADS1 regulating transcript level of AP3 homologue in rice[END_REF]. More recently, Wu and colleagues specifically investigated unique and redundant functions of the three LOFSEP genes using mutant alleles and found that OsMADS1/LHS, OsMADS5, and OsMADS34/PAP2 (PANICLE PHYTOMER2) together regulate determinacy of the floral meristem and specify the identities of spikelet organs by positively regulating the other MADS-box floral homeotic genes including B-, C-, SEP3 and AGL6 genes (Wu et al., 2017a).

In petunia sextuple sep/agl6 mutant flowers, we found that the initial expression levels of the B-class genes PhGLO1, PhGLO2 and PhDEF and of the C-class genes PMADS3 and FBP6 were comparable to WT, indicating that initial activation and expression of these genes does not depend on the SEP/AGL6 floral organ identity function. In Arabidopsis, a similar observation has been made, showing normal patterning and accumulation of AP3, PI and AG expression in young sep1 sep2 sep3 floral buds [START_REF] Pelaz | B and C floral organ identity functions require SEPALLATA MADS-box genes[END_REF]. With perhaps the exception of FBP6 (SHP1/2), we found that further upregulation during later stages of development was impaired, especially for the PI homologs PhGLO1 and PhGLO2, while PhDEF (AP3) and PMADS3 (AG) still showed upregulation, but with a smaller incremental rate. These results are in agreement with the idea that in Arabidopsis, complex formation of SEP proteins with B-and C-class MADS-box proteins is required for their positive autoregulation [START_REF] Gomez-Mena | Transcriptional program controlled by the floral homeotic gene AGAMOUS during early organogenesis[END_REF][START_REF] Kaufmann | Target genes of the MADS transcription factor SEPALLATA3: integration of developmental and hormonal pathways in the Arabidopsis flower[END_REF].

In sharp contrast with the other B-class genes, PhTM6 expression levels in the sextuple mutant were almost completely abolished during all stages tested, indicating a full dependence on SEP/AGL6 activity for all stages of its expression. Earlier, we showed that regulation of PhTM6 expression is atypical for a B-class gene, since its expression largely depends on the activity of the C-genes PMADS3 and FBP6 [START_REF] Heijmans | Redefining C and D in the Petunia ABC[END_REF], resulting in a WT expression pattern mainly in stamens and carpels from early developmental stages onwards, and in all floral whorls when the C-genes are ectopically expressed [START_REF] Vandenbussche | The duplicated B-class heterodimer model: whorl-specific effects and complex genetic interactions in Petunia hybrida flower development[END_REF][START_REF] Rijpkema | Analysis of the Petunia TM6 MADS box gene reveals functional divergence within the DEF/AP3 lineage[END_REF]. Together, this indicates that both SEP and C-class genes are absolutely required for PhTM6 expression, most likely as interaction partners in a MADS-box protein complex [START_REF] Ferrario | The MADS box gene FBP2 is required for SEPALLATA function in petunia[END_REF]. For FBP11 (STK) expression, we found the same SEP dependence, but since FBP11 is expressed relatively late during flower development in the developing placenta and ovules [START_REF] Angenent | A Novel Class of MADS Box Genes Is Involved in Ovule Development in Petunia[END_REF], this may also be an indirect effect, since these tissues are completely absent in the sextuple mutant. Thus, B-and C-class MADSbox proteins may have an absolute requirement of SEP function to activate their downstream developmental programs, but depend only partly on it for upregulation of their own expression. This suggests differences in the molecular mechanisms involved in autoregulation versus downstream target gene activation/repression. Finally, we found that UNS, a petunia member of the SOC1 family, was strongly upregulated in the sextuple sep/agl6 mutant from early stages onwards, suggesting that SEP genes repress UNS during WT flower development. SOC1 was identified as a direct target of SEP3 in a genome wide study in Arabidopsis, with the expression of SOC1 being already reduced after only 8h of SEP3 induction in seedlings [START_REF] Kaufmann | Target genes of the MADS transcription factor SEPALLATA3: integration of developmental and hormonal pathways in the Arabidopsis flower[END_REF]. Interestingly, it was shown that constitutive UNS expression in petunia and Arabidopsis flowers lead to the unshaven floral phenotype, which is characterized by ectopic trichome formation on floral organs and conversion of petals into organs with leaf-like features [START_REF] Ferrario | Ectopic expression of the petunia MADS box gene UNSHAVEN accelerates flowering and confers leaf-like characteristics to floral organs in a dominant-negative manner[END_REF]. All these observations are consistent with the finding of Ó'Maoiléidigh and colleagues, who demonstrated that the floral homeotic organ identity gene AG not only functions by positively conferring floral identity to organ primordia in the flower, but also by actively repressing components of the leaf developmental program [START_REF] Omaoileidigh | Control of reproductive floral organ identity specification in Arabidopsis by the C function regulator AGAMOUS[END_REF].

The FBP9 Subclade Genes together with a SEP4-like Gene are Required to Confer Floral

Meristem Identity in petunia and tomato.

We found that the FBP9 subclade members FBP9 and FBP23 together with FBP4 play a crucial role in floral meristem identity, as illustrated by the homeotic transformation of flower meristems into inflorescence meristems in fbp9 fbp23 fbp4 triple mutants. In contrast, genetic interactions with the fbp2 mutant revealed only mild contributions to the classical SEP organ identity function. The phenotype of the fbp9 fbp23 fbp4 triple mutant is strikingly similar to that of the floral meristem identity mutants alf and dot [START_REF] Souer | Genetic control of branching pattern and floral identity during Petunia inflorescence development[END_REF][START_REF] Souer | Patterning of inflorescences and flowers by the F-Box protein DOUBLE TOP and the LEAFY homolog ABERRANT LEAF AND FLOWER of petunia[END_REF], but it remains to be investigated how these genes are hierarchically positioned. However, it was found that simultaneous overexpression of ALF and DOT in young seedlings led to strong activation of FBP9 and FBP23 expression [START_REF] Souer | Patterning of inflorescences and flowers by the F-Box protein DOUBLE TOP and the LEAFY homolog ABERRANT LEAF AND FLOWER of petunia[END_REF], suggesting that ALF/DOT specify floral meristem identity at least in part by activating FBP9 and FBP23 expression. An expression analysis of ALF, DOT, FBP9, FBP23 and FBP4 in the different mutant backgrounds may provide further support for this hypothesis.

Importantly, our analysis of the fbp4 fbp9 fbp23 mutant combined with a recent study of tomato FBP9 and SEP4 subclade members [START_REF] Soyk | Bypassing Negative Epistasis on Yield in Tomato Imposed by a Domestication Gene[END_REF] demonstrates that the requirement of FBP9 and SEP4 clade genes for floral meristem identity is conserved between tomato and petunia, and therefore likely also in other Solanaceous species. Note that although in both cases floral meristem identity is compromised, the phenotypes of tomato j2 ej2 lin and petunia fbp9 fbp23 fbp4 mutants superficially do look quite different. We believe that this may be explained for an important part by basic differences in the inflorescence architecture between the two species. First of all, in petunia, every flower arises from a node that bears two bracts, while the tomato inflorescence is bractless. As a consequence, loss of FM identity in petunia leads to a highly branched structure composed of a lot of bracts, while in tomato this leads to a more naked structure consisting of proliferating SIMs. Also, the compound tomato inflorescence architecture is more complex compared to petunia and involves the transition of a vegetative meristem into a transition meristem (TM) that terminates in a floral meristem (FM) resulting in the first flower of the inflorescence. Additional flowers then develop from the axillary SIM, resulting in an inflorescence bearing multiple flowers [START_REF] Park | Meristem maturation and inflorescence architecture-lessons from the Solanaceae[END_REF].

While the strongest phenotype was obtained in the tomato triple mutants, analysis of single and double mutants revealed also individual contributions to tomato development: LIN limits internode length and the number of flowers that develop per inflorescence, EJ2 negatively regulates sepal size, while both J2 and EJ2 are involved in the control of branching of the tomato inflorescence [START_REF] Soyk | Bypassing Negative Epistasis on Yield in Tomato Imposed by a Domestication Gene[END_REF]. In addition, J2 is required for the development of the pedicel abscission zone [START_REF] Liu | The SEPALLATA MADS-box protein SLMBP21 forms protein complexes with JOINTLESS and MACROCALYX as a transcription activator for development of the tomato flower abscission zone[END_REF][START_REF] Roldan | Natural and induced loss of function mutations in SlMBP21 MADS-box gene led to jointless-2 phenotype in tomato[END_REF][START_REF] Soyk | Bypassing Negative Epistasis on Yield in Tomato Imposed by a Domestication Gene[END_REF].

Finally, remark that our phylogenetic analysis indicates that within the SEP4 clade, RIN in fact is more closely related to petunia FBP4 compared to LIN. However, RIN shows a much more restricted expression pattern limited to the developing fruit [START_REF] Vrebalov | A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus[END_REF], indicating that RIN has evolved a specialized role compared to FBP4 and LIN. Because of the rin phenotype, RIN has long time been considered to function as a major regulator that is essential for the induction of ripening, but a recent study using a CRISPR/Cas9-mediated RINknockout mutation shows that inactivation of RIN does not repress initiation of ripening and that the original rin mutation is rather a gain-of-function mutation resulting in an aberrant protein that actively represses ripening [START_REF] Ito | Re-evaluation of the rin mutation and the role of RIN in the induction of tomato ripening[END_REF].

While Arabidopsis doesn't have FBP9 subclade members [START_REF] Zahn | The evolution of the SEPALLATA subfamily of MADS-box genes: a preangiosperm origin with multiple duplications throughout angiosperm history[END_REF], it was found that Arabidopsis SEP proteins, in addition to their role in floral organ identity, are also involved in maintaining floral meristem identity, as evidenced by the frequent appearance of secondary flowers in the axils of first-whorl organs in sep1 sep2 sep3 sep4 quadruple mutants and much less frequently in sep1 sep2 sep 3 mutants [START_REF] Ditta | The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity[END_REF]. Moreover, an ap1 sep1 sep2 sep4 quadruple mutant was shown to produce a cauliflower phenotype similar to ap1 cal mutants, while an ap1 sep4 mutant had a meristem identity defect intermediate between that of ap1 and ap1 cal mutants. Although these data clearly demonstrate the implication of Arabidopsis SEP genes in floral meristem identity, the very severe floral meristem defects observed in ap1 cal or ap1 cal ful mutants, indicate that in Arabidopsis, floral meristem is mainly determined by members of the AP1/SQUA subfamily.

In rice, the three LOFSEP genes OsMADS1/LHS, OsMADS5, and OsMADS34/PAP2 were proposed to be involved in the transition of the spikelet meristem into a floral meristem (Wu et al., 2017a). However, floral meristem formation in the triple osmads1 osmads5 osmads34 mutants was not completely abolished, only strongly delayed, possibly because the insertion alleles are not complete null mutants as suggested by the authors (Wu et al., 2017a).

The Petunia AP1 ortholog euAP1 is not required for petal development, and acts redundantly with the other AP1 clade members as a B-function Repressor in the First Floral Whorl.

Because Arabidopsis ap1 mutants lack petals and have sepals displaying bract like features [START_REF] Irish | Function of the apetala-1 gene during Arabidopsis floral development[END_REF][START_REF] Mandel | Molecular characterization of the Arabidopsis floral homeotic gene APETALA1[END_REF] and AP1 is negatively regulated by AG in whorls three and four [START_REF] Gustafson-Brown | Regulation of the arabidopsis floral homeotic gene APETALA1[END_REF], AP1 has been classified as an Afunction gene in the ABC model, required for the identity specification of sepals and petals. In sharp contrast with the phenotype of Arabidopsis ap1 mutants, we found that petal development does not at all require euAP1 activity in petunia. This may not come as a complete surprise since it was shown before that also in Arabidopsis, AP1 is not essential for petal development, as evidenced by the nearly complete restauration of petal development in ap1 ag mutants [START_REF] Bowman | Control of flower development in Arabidopsis thaliana by APETALA1 and interacting genes[END_REF] and in 35S: SEP3 ap1 flowers [START_REF] Castillejo | A new role of the Arabidopsis SEPALLATA3 gene revealed by its constitutive expression[END_REF], and a partial restauration in ap1 agl24 double mutants [START_REF] Yu | Repression of AGAMOUS-LIKE 24 is a crucial step in promoting flower development[END_REF]. In addition, single euap1 mutants that still develop petals have previously been described in other species such as e.g. the squa mutant in snapdragon [START_REF] Huijser | Bracteomania, an inflorescence anomaly, is caused by the loss of function of the MADS-box gene squamosa in Antirrhinum majus[END_REF], the pim mutant in pea [START_REF] Berbel | Analysis of PEAM4, the pea AP1 functional homologue, supports a model for AP1-like genes controlling both floral meristem and floral organ identity in different plant species[END_REF][START_REF] Taylor | PROLIFERATING INFLORESCENCE MERISTEM, a MADS-box gene that regulates floral meristem identity in pea[END_REF], mtap1 in Medicago [START_REF] Benlloch | Isolation of mtpim proves Tnt1 a useful reverse genetics tool in Medicago truncatula and uncovers new aspects of AP1-like functions in legumes[END_REF][START_REF] Cheng | Dissection of genetic regulation of compound inflorescence development in Medicago truncatula[END_REF], and mc in tomato [START_REF] Vrebalov | A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus[END_REF][START_REF] Nakano | MACROCALYX and JOINTLESS interact in the transcriptional regulation of tomato fruit abscission zone development[END_REF][START_REF] Yuste-Lisbona | Characterization of vegetative inflorescence (mc-vin) mutant provides new insight into the role of MACROCALYX in regulating inflorescence development of tomato[END_REF].

Restricting the activity of the floral homeotic B-and C-functions to their proper domains is crucial for the correct development of the flower structure, and it appears that the molecular mechanisms underlying these cadastral functions are much more diverse compared to the floral organ identity functions (reviewed in [START_REF] Monniaux | How to Evolve a Perianth: A Review of Cadastral Mechanisms for Perianth Identity[END_REF]). Here we identified the petunia AP1/SQUA genes as repressors of the B-function in the first whorl, as evidenced by the partial conversion of sepals into petaloid tissue in pfg fbp26 fbp29 euap1 mutants, and the strong enhancement of this phenotype in combination with mutations in the ROB genes, which were previously identified as B-function repressors in the first whorl [START_REF] Morel | Divergence of the Floral A-Function between an Asterid and a Rosid Species[END_REF]. Such a phenotype has so far never been reported in flowers of Arabidopsis ap1, cal or ful mutants, or any combination of these mutations [START_REF] Ferrandiz | Redundant regulation of meristem identity and plant architecture by FRUITFULL, APETALA1 and CAULIFLOWER[END_REF]. Nevertheless, it was proposed that AP1 in combination with AGL24 (AGAMOUS LIKE 24) and SVP (SHORT VEGETATIVE PHASE) represses both the B-and C-function genes during early phases of floral development [START_REF] Gregis | AGL24, SHORT VEGETATIVE PHASE, and APETALA1 Redundantly Control AGAMOUS during Early Stages of Flower Development in Arabidopsis[END_REF][START_REF] Gregis | The Arabidopsis floral meristem identity genes AP1, AGL24 and SVP directly repress class B and C floral homeotic genes[END_REF], but it is not clear if other Arabidopsis AP1/SQUA genes would be also involved in this process and whether this is specific to the first floral whorl. Finally in rice, deregulation of B-and C-expression patterns was observed in osmads14 osmads15/+ and osmads14/+ osmads15 flowers [START_REF] Wu | The ABCs of flower development: mutational analysis of AP1/FUL-like genes in rice provides evidence for a homeotic (A)-function in grasses[END_REF], suggesting that these rice AP1/SQUA transcription factors are also involved in patterning the homeotic B-and C-functions.

In summary, the observation that the petunia AP1/SQUA genes repress the B-function in the first floral whorl but do not seem to be required for 2 nd whorl petal development demonstrates that petunia AP1/SQUA genes cannot be easily classified as "A-function" genes according to the original definition of the A-function in the ABC model. Earlier, we encountered the same difficulties when trying to integrate the function of the petunia AP2-like transcription factors AP2 and ROB1-3 into a simple ABC model [START_REF] Morel | Divergence of the Floral A-Function between an Asterid and a Rosid Species[END_REF]. This led us to propose a modified model for petunia floral organ identity in which the original A-function is replaced by a combinatorial function describing the cadastral (boundary setting) mechanisms that pattern the floral B-and C-functions [START_REF] Morel | Divergence of the Floral A-Function between an Asterid and a Rosid Species[END_REF]. The above described cadastral function of the petunia AP1/SQUA genes during flower development perfectly fits into this alternative model, and is also compatible with the proposed modified (A)BC model [START_REF] Causier | Floral organ identity: 20 years of ABCs[END_REF], in which a more broadly defined (A)-function provides the genetic context in which the B-and C-functions are active and regulates both their spatial and temporal expression domains. Our findings for both the AP1/SQUA and AP2-like gene functions in petunia entirely explain the struggles to translate the Arabidopsis definition of the A-function to distant flowering species [START_REF] Litt | An Evaluation of A-Function: Evidence from the APETALA1 and APETALA2 Gene Lineages[END_REF].

Petunia AP1/SQUA Family Members Function in a Largely Redundant Fashion and are

Required for Inflorescence Meristem Identity.

Different members of the AP1/SQUA subfamily in Arabidopsis have evolved unique roles during development as exemplified by the distinct phenotypes of the single ap1 and ful mutants. Swapping experiments suggest that functional divergence between AP1 and FUL is due to changes in both expression pattern and coding sequence [START_REF] Mccarthy | Functional Divergence of APETALA1 and FRUITFULL is due to Changes in both Regulation and Coding Sequence[END_REF]. At the same time, AP1, CAL and FUL have retained a redundant function in inflorescence architecture [START_REF] Ferrandiz | Redundant regulation of meristem identity and plant architecture by FRUITFULL, APETALA1 and CAULIFLOWER[END_REF], whereas CAL shares a cryptic role in petal development redundantly with AP1 [START_REF] Castillejo | A new role of the Arabidopsis SEPALLATA3 gene revealed by its constitutive expression[END_REF]. While the function of AGL79 (a euFUL-like gene) has remained elusive for a long time, a recent study suggests a role for AGL79 in lateral root development and control of lateral shoot branching [START_REF] Gao | miR156/SPL10 Modulates Lateral Root Development, Branching and Leaf Morphology in Arabidopsis by Silencing AGAMOUS-LIKE 79[END_REF]. It remains to be established if AGL79 overlaps in function with AP1, CAL and FUL.

Although we cannot exclude to have overlooked some very subtle defects, the absence of clear floral developmental defects in mutants for any of the four petunia AP1/SQUA genes suggests that individual members of the petunia AP1/SQUA subfamily did not functionally diverge, independent from their euAP1 or euFUL/paleoAP1 clade identity. In line with that, we found that all four genes show overlapping expression patterns in most tissues tested. It remains to be tested whether this broad functional redundancy is also observed during other developmental processes, such root or fruit development, which were not analyzed in this study.

One of the striking aspects of the phenotype of quadruple pfg fbp26 fbp29 euap1 mutants is that these plants develop fully functional flowers, suggesting that floral meristem identity does not require AP1/SQUA activity in petunia. Our finding that this function is apparently taken care off by a specific subset of SEP genes fully fits this hypothesis. However, we can currently not fully exclude that some residual AP1/SQUA activity remains in the pfg fbp26 fbp29 euap1 mutants, possibly explaining the formation of terminal flowers. Especially the pfg-12 insertion allele potentially could be a hypomorphic allele, since an alternative startcodon is present in the first exon (AA nr 8, Supplemental Data File 2) shortly after the transposon insertion site. This could in theory lead to the production of a protein with an N-terminal truncation of the MADS-domain, perhaps displaying some residual functionality. Other alleles will have to be identified in the future to fully proof the hypothesis that floral meristem identity in petunia does not require AP1/SQUA activity.

On the other hand, the phenotype of the quadruple pfg fbp26 fbp29 euap1 mutants indicate that the petunia AP1/SQUA genes appear to be essential for the development of the cymose inflorescence, indicating a role in inflorescence meristem identity. Such a role also has been proposed for AP1/SQUA genes in other core eudicot species: VEG1 and its ortholog MtFUL are essential for the specification of the secondary inflorescence meristem in the compound inflorescences of pea and Medicago respectively, but are not required for floral meristem identity [START_REF] Berbel | VEGETATIVE1 is essential for development of the compound inflorescence in pea[END_REF][START_REF] Cheng | Dissection of genetic regulation of compound inflorescence development in Medicago truncatula[END_REF].

Interestingly, it was earlier found that the tomato mc mutants also play a role in inflorescence meristem development, since mc inflorescences revert to vegetative growth after forming two to three flowers. In addition, these flowers developed enlarged sepals and have an incomplete pedicel abscission zone [START_REF] Vrebalov | A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus[END_REF][START_REF] Nakano | MACROCALYX and JOINTLESS interact in the transcriptional regulation of tomato fruit abscission zone development[END_REF][START_REF] Yuste-Lisbona | Characterization of vegetative inflorescence (mc-vin) mutant provides new insight into the role of MACROCALYX in regulating inflorescence development of tomato[END_REF]. Moreover, the implication of MC in the development of the pedicel abscission zone is proposed to occur via a higher order MADS-box complex including the SVP-like protein JOINTLESS (J), and J2/SLMBP21 a SEP FBP9 clade member . Except for the pedicel abscission zone which does not exist in petunia, the mc phenotypes are reminiscent of what we observed in petunia quadruple ap1 mutants, suggesting a conserved role in inflorescence meristem identity and first whorl development. Because mc single mutants have a clear phenotype on their own, it also shows that MC exhibits less functional overlap with the other AP1 family members compared to petunia. However, as suggested by the relative mildness of the inflorescence meristem defect in mc mutants compared to the petunia quadruple mutants, this does not exclude possible partial redundancy with one or more of the other tomato AP1 family members, something that still remains to be tested. RNAi mediated downregulation of tomato FUL1 and FUL2 suggested a role for these genes in fleshy fruit ripening [START_REF] Bemer | The tomato FRUITFULL homologs TDR4/FUL1 and MBP7/FUL2 regulate ethyleneindependent aspects of fruit ripening[END_REF][START_REF] Shima | Tomato FRUITFULL homologues act in fruit ripening via forming MADS-box transcription factor complexes with RIN[END_REF][START_REF] Wang | Members of the tomato FRUITFULL MADS-box family regulate style abscission and fruit ripening[END_REF], indicating that the implication of FUL genes in fruit development is conserved between tomato and Arabidopsis, despite that these two species have very different fruit types (fleshy versus dry). Petunia on the other hand develops a dry fruit capsule, but the implication of AP1/FUL members in its development remains to be investigated.

Finally, of the four identified rice AP1 subfamily members called OsMADS14, OsMADS15, OsMADS18 and OsMADS20 [START_REF] Lee | Systematic reverse genetic screening of T-DNA tagged genes in rice for functional genomic analyses: MADS-box genes as a test case[END_REF][START_REF] Yu | Prevalent Exon-Intron Structural Changes in the APETALA1/FRUITFULL, SEPALLATA, AGAMOUS-LIKE6, and FLOWERING LOCUS C MADS-Box Gene Subfamilies Provide New Insights into Their Evolution[END_REF], it was found that OsMADS14, OsMADS15 and OsMADS18 are specifically activated in the meristem at phase transition together with the LOFSEP gene PAP2/OsMADS34 [START_REF] Kobayashi | PANICLE PHYTOMER2 (PAP2), encoding a SEPALLATA subfamily MADS-box protein, positively controls spikelet meristem identity in rice[END_REF][START_REF] Kobayashi | Inflorescence meristem identity in rice is specified by overlapping functions of three AP1/FUL-like MADS box genes and PAP2, a SEPALLATA MADS box gene[END_REF]. While downregulation of these three AP1/FUL-like genes by RNAi caused only a slight delay in reproductive transition, further depletion of PAP2 function from these triple knockdown plants inhibited the transition of the meristem to the IM [START_REF] Kobayashi | Inflorescence meristem identity in rice is specified by overlapping functions of three AP1/FUL-like MADS box genes and PAP2, a SEPALLATA MADS box gene[END_REF], indicating that the AP1/FUL-like OsMADS14, OsMADS15, OsMADS18 and the LOFSEP gene PAP2/OsMADS34 coordinately act in the meristem to specify inflorescence meristem identity. In addition, it was shown that OsMADS14 and OsMADS15, besides to their function of specifying meristem identity, are also involved in the specification of palea and lodicule identities, using stable mutant alleles [START_REF] Wu | The ABCs of flower development: mutational analysis of AP1/FUL-like genes in rice provides evidence for a homeotic (A)-function in grasses[END_REF].

Functional Diversification Patterns in the AP1/SEP/AGL6 Superclade during Angiosperm evolution.

Above, we compared AP1/SEP/AGL6 functions between different species, mainly focusing on Arabidopsis, petunia, tomato and rice, revealing important differences in the functions performed by their respective members. Perhaps the most striking observation is that a subclass of SEP genes (all belonging to the LOFSEP group) in petunia, tomato and possibly also rice are required to confer floral meristem identity, while in Arabidopsis the floral meristem identity function is mainly associated with members of the AP1/SQUA subfamily. It thus seems that during angiosperm evolution, members of different subfamilies within the AP1/SEP/AGL6 superclade have evolved specialized/subfunctionalized roles either in floral organ identity or inflorescence and/or floral meristem determination, providing further genetic support for the monophyletic origin of the AP1/SEP/AGL6 superclade. Within MADS-box subfamilies, it is not unusual that functions have been distributed differently between paralogs in different species. One of the first well documented cases concerns the C-function MADS-box subfamily, showing that the canonical C-function is encoded by nonorthologous genes in Arabidopsis and Antirrhinum [START_REF] Causier | Evolution in Action: Following Function in Duplicated Floral Homeotic Genes[END_REF]. However, careful comparison of gene functions in the AP1/SEP/AGL6 superclade suggest that this random distribution of functions after gene duplication has occurred also during the earlier phases of the evolution of the MADS-box gene family, resulting in functions that are differently distributed beyond the subfamily level. In addition, comparison between tomato and petunia indicates major functional differences that have arisen on a relatively short evolutionary time-scale. Of note is the involvement of several tomato AP1/SEP/AGL6 members in the development of the pedicel abscission zone and in compound leaf development, all processes that do not occur in petunia.

Together, these observations illustrate that gene function cannot accurately be predicted solely based on sequence homology and phylogenetic analysis, and that final gene function may be strongly dependent on species-specific developmental contexts. Furthermore, it also illustrates that demonstration of gene function conservation between only two species, even if they are very distantly related (e.g. a monocot versus a dicot species), cannot safely be used to extrapolate a more general conservation of a particular gene function. Together with other studies, this further enforces the argument that plant biology in general, and plant evo-devo in particular would strongly benefit from a broader range of available model systems [START_REF] Vandenbussche | Petunia, Your Next Supermodel?[END_REF]. 5D) was described previously (current allele naming based on exact insert position; old allele names in between brackets): fbp2-332 (fbp2-1); fbp2-440 (fbp2-2); fbp4-44 (fbp4-2); fbp4-55 (fbp4-3); fbp5-129 (fbp5-1); fbp9-110 (fbp9-1); pfg-12 (pfg-1); fbp26-76 (fbp26-1) [START_REF] Vandenbussche | Toward the analysis of the petunia MADS box gene family by reverse and forward transposon insertion mutagenesis approaches: B, C, and D floral organ identity functions require SEPALLATA-like MADS box genes in petunia[END_REF], and agl6-118 (agl6-1) [START_REF] Rijpkema | The petunia AGL6 gene has a SEPALLATA-like function in floral patterning[END_REF]. Note that the previously determined insert positions for some of these alleles differ by a few nucleotides compared to the data presented here, due to the imperfect manual sequencing method used at that time combined with the characterization of only the right border of the transposon flanking sequences, not taking the dTph1 8bp target site duplication into account. Also, it was mentioned that homozygous fbp9-1 (fbp9-110) mutants exhibited aberrations in plant architecture during the reproductive phase [START_REF] Vandenbussche | Toward the analysis of the petunia MADS box gene family by reverse and forward transposon insertion mutagenesis approaches: B, C, and D floral organ identity functions require SEPALLATA-like MADS box genes in petunia[END_REF]. However, later outcrossing analysis of the fbp9-1 allele showed that this defect was closely linked to fbp9-1, but not due to the fbp9-1 insertion, as confirmed by the absence of this phenotype in the new fbp9-7 and fbp9-90 alleles.

MATERIALS AND METHODS

Plant

The following alleles fbp2-209; fbp4-23; fbp5-51; pm12-37; pm12-118; pm12-325; euap1-317; fbp29-31; fbp29-123 and fbp29-153 were identified by BLAST-searching our sequenceindexed dTph1 transposon flanking sequence database [START_REF] Vandenbussche | Generation of a 3D indexed Petunia insertion database for reverse genetics[END_REF]) that was enlarged with the addition of extra populations. Exact insert positions were determined by aligning the transposon flanking sequences with the corresponding genomic and coding sequences. The insertion alleles were named after their exact insert position, expressed in bp downstream of the ATG in the coding sequence (Figures 1G and5D). Offspring of candidate insertion lines were grown and genotyped by PCR using gene specific primer pairs flanking the insertion site (Supplemental Table 2). The following thermal profile was used for segregation analysis PCR: 11 cycles (94°C for 15s, 71°C for 20s minus 1°C/cycle, 72°C for 30s), followed by 40 cycles (94°C for 15s, 60°C for 20s, 72°C for 30s). For all alleles, homozygous mutants were obtained in offspring of the originally heterozygous insertion mutants, either containing the original transposon insertion allele, or a stably inherited out-of-frame derived footprint allele that was confirmed by sequencing, fully maintaining the mutation. Insertion alleles that were used in crosses for higher order mutant analysis are indicated in red in Figures 1G and5D. The different insertion alleles were further systematically genotyped in subsequent crosses and segregation analyses. To test genetic interactions with the rob mutations (Figure 6), a pfg fbp26 fbp29 euap1 mutant was crossed with the earlier described rob1 rob2 rob3 mutant [START_REF] Morel | Divergence of the Floral A-Function between an Asterid and a Rosid Species[END_REF]. Phenotypic analysis of all single and higher order mutants was focused and limited to the screening for defects in floral organ development and inflorescence architecture.

Phylogenetic Analysis

The phylogenetic analyses shown in Figures 1F and5A were conducted using the advanced PhyML/oneClick workflow at ngphylogeny.fr [START_REF] Lemoine | NGPhylogeny.fr: new generation phylogenetic services for non-specialists[END_REF]. Full-length protein sequences of either SEP/AGL6 (Figure 1F) or AP1/SQUA (Figure 5A) subfamily members from petunia, tomato, arabidopsis and rice (Supplemental Table 1) were first aligned using MAFFT [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: improvements in performance and usability[END_REF] applying the following options: Data type: Autodetection; MAFFT flavor: auto; Gap extend penalty: 0.123; Gap opening penalty: 1.53; Matrix selection: no matrix; Reorder output? true. Output format: FASTA (Supplemental Data files 1 and 2).

Next, alignment curation was done using BMGE [START_REF] Criscuolo | BMGE (Block Mapping and Gathering with Entropy): a new software for selection of phylogenetic informative regions from multiple sequence alignments[END_REF] with the following options: Sequence coding: AA; matrix: BLOSUM; Estimated matrix BLOSUM: 62; Sliding windows size: 3; Maximum entropy threshold: 0.5; Gap Rate cut-off: 0.5; Minimum block size: 3 and 5 for Figures 1F and5A respectively. Using the resulting BMGE files, Maximum Likelyhood trees were calculated using PhyML [START_REF] Lemoine | Renewing Felsenstein's phylogenetic bootstrap in the era of big data[END_REF] initiate the random number generator: 123456. The tree was rendered using Newick Display [START_REF] Junier | The Newick utilities: high-throughput phylogenetic tree processing in the UNIX shell[END_REF]. For the visual representation of the SEP/AGL6 analysis (Figure 1F), mid-point rooting was applied on the node separating SEP and AGL6 subfamilies, while for the AP1/SQUA analysis (Figure 5A), mid-point rooting was applied on the node separating rice from eudicot AP1/SQUA proteins.

Imaging and Microscopy

Electron microscopy images were obtained as previously described [START_REF] Vandenbussche | Differential recruitment of WOX transcription factors for lateral development and organ fusion in Petunia and Arabidopsis[END_REF] or by using a HIROX SH-1500 benchtop environmental electron microscope equipped with a cooled stage. Macroscopic floral phenotypes were imaged by conventional digital photography using a glass plate as a support and black velvet tissue around 10 cm below the glass plate in order to generate a clean black background. When needed, backgrounds were further equalized by removing dust particles and light reflections with Photoshop. Images in Figures 6H, 6I and 6M were photographed using a Zeiss Imager M2 microscope equipped with an AxioCam MRc camera (Zeiss).

RT-qPCR Expression Analysis.

Total RNA was extracted using the Spectrum Plant Total RNA kit (Sigma Aldrich) and treated with Turbo DNA-free DNase I (Ambion). RNA was reverse transcribed using RevertAid M-MuLV reverse transcriptase (Fermentas) according to the manufacturer's protocol. PCR reactions were performed in an optical 384-well plate in the QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems), using FastStart Universal SYBR Green Master (Roche), in a final volume of 10µl, according to the manufacturer's instructions. Primers (Supplemental Table 2) were designed using the online Universal ProbeLibrary Assay Design Center (Roche).

Data were analyzed using the QuantStudio™ 6 and 7 Flex Real-Time PCR System Software v1.0 (Applied Biosystems). Petunia ACTIN, GAPDH, and RAN were used as reference genes.

PCR efficiency (E) was estimated from the data obtained from standard curve amplification using the equation E=10 -1/slope . Relative expression (R.E.) values on the y-axes are the average of nine data points resulting from the technical triplicates of three biological replicates ± sd and normalized to the geometrical average of three E -ΔCt , where ΔCt = CtGOI -CtACTIN, GAPDH and RAN.

The floral bud series (marked floral buds 1-3 in Figures 1F,4G, 5C and Supplemental Figure 1) are successive developmental stages of complete floral buds harvested from the same inflorescences (Figure 1E). Young bracts were harvested from the node bearing stage 3 flowers, while inflorescence stem tissue was collected from the internode connecting node stage 4 and stage 5 bearing flowers. For each biological replicate, corresponding stages harvested from three inflorescences were pooled. Stage 3 corresponds to flower buds with a diameter of ∼5 mm and from which individual floral organs can be easily dissected by hand. All analyses showing expression in separate floral organ types are from this stage. Biological replicates of the different floral organ types were composed of pooled stage 3 organs harvested from three different flowers each time. Floral buds marked "2" (diameter ∼2.5 mm) and "1" (diameter ∼1.5 mm) are younger stages and were harvested from the next two nodes produced after bud stage 3. In addition to 1.5-mm buds, stage 1 also includes the inflorescence meristem and very young developing floral primordia subtended by bracts, which are attached to the base of the pedicel of the 1.5-mm bud. For the sextuple mutant flower buds analyzed in Figure 4G, developmental stages in relation to wild-type development were deduced based on their position on the inflorescence. Vegetative apices (including very small leaf primordia) were harvested from 3week-old seedlings by manually removing cotyledons, roots, and developed leaves. Young leaf primordia were isolated from the same 3-week-old seedlings. Each biological replicate of the vegetative apices and young leaf primordia consisted of pooled material harvested from each time 10 seedlings. The root samples were obtained by pooling 10-15 actively growing 2 cm root tips per biological replicate.

Accession Numbers

Sequence data for the genes that were functionally analyzed in this article can be found in the GenBank/EMBL libraries under accession numbers FBP2 (M91666.1); FBP5 (AF335235.1); PMADS12 (AY370527.1); FBP9 (AF335236.1); FBP23 (AF335241.1); FBP4 (AF335234.1); Ph-AGL6 (AB031035.1); PFG (AF176782.1); FBP29 (AF335245.1); FBP26 (AF176783.1); Ph-euAP1 (MK598839) (see also Supplemental Table 1). 1. Naming of subfamilies and subfamily clades is based on previously described phylogenies for the SEP subfamily [START_REF] Malcomber | SEPALLATA gene diversification: brave new whorls[END_REF][START_REF] Zahn | The evolution of the SEPALLATA subfamily of MADS-box genes: a preangiosperm origin with multiple duplications throughout angiosperm history[END_REF][START_REF] Yu | Prevalent Exon-Intron Structural Changes in the APETALA1/FRUITFULL, SEPALLATA, AGAMOUS-LIKE6, and FLOWERING LOCUS C MADS-Box Gene Subfamilies Provide New Insights into Their Evolution[END_REF]. (G) RT-qPCR expression analysis of the petunia SEP/AGL6 genes. Relative expression (R.E.) levels are plotted as the mean value of three biological and three technical replicates ±SE, normalized against three Table 1. Naming of subfamilies and subfamily clades is based on previously described phylogenies for the AP1/SQUA subfamily [START_REF] Litt | Duplication and diversification in the APETALA1/FRUITFULL floral homeotic gene lineage: implications for the evolution of floral development[END_REF][START_REF] Yu | Prevalent Exon-Intron Structural Changes in the APETALA1/FRUITFULL, SEPALLATA, AGAMOUS-LIKE6, and FLOWERING LOCUS C MADS-Box Gene Subfamilies Provide New Insights into Their Evolution[END_REF][START_REF] Maheepala | Evolution and Diversification of FRUITFULL Genes in Solanaceae[END_REF].

  ), resulting in inflorescences in which each time several leaves developed before the next flowerbearing node was produced. Together this indicates that all four genes contribute to cymose inflorescence development in petunia. Petunia AP1/SQUA Family Members Repress the B-Function in the First Whorl in Concert with the ROB/BEN Genes.

A

  Comparison of SEP/AGL6 and AP1/SQUA Functions in Petunia, Arabidopsis, Tomato and Rice

  Material, Genotyping and Phenotyping Petunia plants were grown in soil (FAVORIT-argile 10) either in a greenhouse (16 h day/8 h night: natural light supplemented with Philips Sodium HPS 400W SON-T AGRO light bulbs; 55.000 lumens) or outside protected by an agricultural tunnel (from April to October), both under conditions that depend on local seasonal changes (45.72°N 4.82°E), or in growth chambers (settings: 16 h day 22°C /8 h night 18°C, 75W Valoya NS12 LED bars, light intensity 130 µE). The identification of the following dTph1 transposon insertion alleles (Figures 1G and

  with the following settings: Data type: amino acids; Evolutionary model: LG; Equilibrium frequencies: ML/Model. Proportion of invariant sites: estimated; Number of categories for the discrete gamma model: 4; Parameter of the gamma model: estimated; Tree topology search: Best of NNI and SPR. Optimize parameter: Tree topology, Branch length, Model parameter; Statistical test for branch support: Bootstrap; Number of bootstrap replicates: 1000. Seed value used to

FIGURE LEGENDS Figure 1 .

 1 FIGURE LEGENDS Figure 1. Characterization of the Petunia SEP/AGL6 MADS-box Genes. (A) Section through a WT petunia W138 flower showing inner whorls. (B) Petunia seedpod ~4 weeks post-pollination surrounded by green sepals. (C) SEM (scanning electron microscopy) images of sepal, bract and leaf adaxial and abaxial epidermal surfaces. Bars = 50 µm. (D) Longitudinal sections of developing petunia floral buds showing the placenta developing from the center of the floral meristem s = sepal; p = petal; st = stamen; c = carpel; pl = placenta. Bars = 200 µm. (E) W138 floral bud developmental stages for RT-qPCR analysis shown in (G), dissected from the top of an inflorescence (inset), of which the large floral bud at the right is just prior to opening. Numbers indicate sampled stages. 1 to 3 correspond to floral bud diameters of ~1.5, 2.5 and 5 mm respectively. Stage 1 also includes very early flower primordia, bracts and the inflorescence meristem. Bar = 1 cm. (F) Maximum Likelihood phylogenetic analysis of the SEP and AGL6 subfamily members of Petunia hybrida (Ph), Solanum lycopersicum (Sl), Arabidopsis thaliana (At) and Oryza sativa (Os). Bootstrap values marked in red (expressed in %, based on 1000 replicates) supporting tree branching are indicated near

  reference genes (see Material and Methods). Expression levels were measured in vegetative tissues (green bars; infl. stem = inflorescence stem); entire floral buds (orange bars) from 3 developmental stages shown in (E) and dissected floral organs (red bars) obtained from flower buds corresponding to stage 3. (H) Schematic representations of the gene structures and insertion alleles of the petunia SEP and AGL6 genes and floral phenotypes of the corresponding insertion mutants used in further crosses. Black boxes and lines represent exons and introns respectively. All gene models start at the start codon and end at the stop codon. Scale bars = 500 bp. Red triangles indicate positions of dTph1 transposon insertions. Alleles are named after the exact insert position of the dTph1 element in number of base pairs downstream of the ATG in the coding sequence. The names of the insertion alleles that have been selected for the creation of double and higher order mutants are marked in red.

Figure 2 .

 2 Figure 2. The Petunia fbp2 fbp5 pm12 Mutant, Genetic Equivalent of the Arabidopsis sep1 sep2 sep3 Mutant, Still Displays B-and C-function Floral Characteristics. (A) to (H) Top view of flowers from WT, single, double and triple mutants of petunia SEP1/SEP2/SEP3 homologs. All images are at the same magnification. (I) to (L) Side view of WT and mutant flowers sectioned through the middle. All images are at the same magnification. (M) Close-up of dissected third whorl organs (stamens). (N) Close-up of dissected fourth whorl organs (carpels). (O) to (Q) SEM images of the outer ovary surface. Scale bars = 100 µm.

Figure 3 .

 3 Figure 3. Petunia Floral Meristem Identity Depends on FBP9/FBP23/FBP4 Activity. (A) to (C) and (E) to (G) Top and side view of WT, fbp9 fbp23 and fbp9 fbp23 fbp4 plants 13 weeks after sowing. (D) and (H) Schematic representation of inflorescence phenotypes. (I) to (L) SEM images of inflorescence apices in WT and fbp4 fbp9 fbp23 mutants. Br: bracts; Se: sepals; F: flower; Fm: Flower meristem; Im: Inflorescence meristem. Scale bars = 100 µm. (M) to (Q) Inflorescence architecture of lower order mutants compared to WT and fbp9 fbp23 fbp4 mutants. (R) to (W) Flower phenotypes of fbp4, fbp23 and fbp9 mutations in combination with fbp2. All flowers are at the same magnification.

Figure 4 .

 4 Figure 4. Characterization of the Sextuple fbp2 fbp4 fbp5 fbp9 pm12 agl6 Mutant Compared to WT. Genotypes in each panel are indicated as follows: sext: sextuple fbp2 fbp4 fbp5 fbp9 pm12 agl6 mutant. WT: wild-type. (A) and (B) Top view of young (A) and mature flower (B). (C) Dissected floral organs of a flower similar to the stage as indicated by the asterisk in (F). W# indicate whorl numbers. (D) Longitudinal section through an older flower similar to the stage as indicated by the double asterisk in (F). (E) SEM images of the epidermis of the four different floral whorls (indicated by W#) in WT (left panels) and the sextuple mutant (right panels). (F) Inflorescences showing flowers at various stages of development and aging. The arrows indicate an example where three consecutive fully developed flowers arose from a single floral meristem. Scale bars: 0.25 cm in (A); 0.5 cm in (B, D); 1 cm in (C, F); 50 µm in (E). (G) RT-qPCR expression analysis of the petunia floral homeotic genes in WT versus sextuple fbp2 fbp4 fbp5 fbp9 pm12 agl6 mutants. Petunia genes are indicated and names of corresponding Arabidopsis orthologs are shown in between brackets. *No TM6 ortholog exists in the Arabidopsis genome. **Petunia FBP6 is orthologous to SHP1/SHP2, but is functionally homologous to AG. Relative expression (R.E.) levels are plotted as the mean value of three biological and three technical replicates ±SE, normalized against three reference genes (see Material and Methods). Expression levels were measured in entire floral buds from three developmental stages as shown in Figure 1E.

Figure 5 .

 5 Figure 5. Characterization of the Petunia AP1/SQUA MADS-box Subfamily. (A) Maximum likelihood phylogenetic analysis of the AP1/SQUA subfamily members of Petunia hybrida (Ph), Solanum lycopersicum (Sl), Arabidopsis thaliana (At) and Oryza sativa (Os). Bootstrap values marked in red (expressed in %, based on 1000 replicates) supporting branching are indicated near the branch points. The scale bar represents number of substitutions/site. Accession codes for the corresponding sequences are shown in Supplemental

  (B) RT-qPCR expression analysis of the petunia AP1/SQUA genes. Relative expression (R.E.) levels are plotted as the mean value of three biological and three technical replicates ±SE, normalized against three reference genes (see Material and Methods). See legend of Figure 1G for sample description. (C) Schematic representations of the gene structures and insertion alleles of the petunia AP1/SQUA genes and corresponding floral

Acknowledgments

M.V. was supported by a CNRS ATIP-AVENIR award and the French National Research Agency program DODO (ANR-16CE20-0024-03). We thank A. Lacroix, J. Berger and P.

Bolland for plant care assistance, and V. Bayle for electron microscopy technical support performed at the PLATIM platform, IFR BioScience Lyon (UMS3444/US8). We thank A. Rijpkema for help with preliminary studies.

Supplemental Data

Supplemental Figure 1. RT-qPCR 1. Naming of subfamilies and subfamily clades are based on previously described phylogenies for the AP1/SQUA subfamily [START_REF] Litt | Duplication and diversification in the APETALA1/FRUITFULL floral homeotic gene lineage: implications for the evolution of floral development[END_REF][START_REF] Yu | Prevalent Exon-Intron Structural Changes in the APETALA1/FRUITFULL, SEPALLATA, AGAMOUS-LIKE6, and FLOWERING LOCUS C MADS-Box Gene Subfamilies Provide New Insights into Their Evolution[END_REF][START_REF] Maheepala | Evolution and Diversification of FRUITFULL Genes in Solanaceae[END_REF]. (B) RT-qPCR expression analysis of the petunia AP1/SQUA genes. Relative expression (R.E.) levels are plotted as the mean value of three biological and three technical replicates ±SE, normalized against three reference genes (see Material and Methods). See legend of Figure 1G