4157 RESEARCH ARTICLE INTRODUCTION Nearly all angiosperm flowers possess a perianth, a series of sterile organs that surround the reproductive organs – the stamens and carpels. In many flowering plant lineages, the perianth is differentiated into distinct outer organs called sepals, and inner organs called petals; this is termed a bipartite perianth. Petals are therefore defined as occupying the spatial position outside of the stamens but internal to the sepals, and are often large, showy and pigmented, and contain specialized reflective papillate epidermal cells (Endress and Matthews, 2006). The evolution of flowers with elaborated petals is likely to have increased fitness through facilitating pollinator interactions (Clegg and Durbin, 2003). Our current understanding of the molecular mechanisms controlling petal-identity specification rests largely on functional analyses carried out in several core eudicot species, including Arabidopsis thaliana and Antirrhinum majus (Weigel and Meyerowitz, 1994). In Arabidopsis, for example, the APETALA3 (AP3) and PISTILLATA (PI) MADS-box genes are required to specify petal and stamen identity, and mutations in these genes result in homeotic transformations of petals into sepals and stamens into carpeloid structures (Bowman et al., 1991; Goto and Meyerowitz, 1994; Jack et al., 1992). The AP3 and PI gene products heterodimerize, and are likely to act in vivo as part of larger MADS- box protein complexes, in order to specify petal as well as stamen identity (Bowman et al., 1991; Goto and Meyerowitz, 1994; Honma and Goto, 2001; Jack et al., 1992; Pelaz et al., 2001; Riechmann et al., 1996). Although a considerable amount is known about the molecular mechanisms specifying petal identity in Arabidopsis and other core eudicot species, there is little functional evidence that homologs of these genes play similar roles in petal-identity specification outside of the core eudicots. It is generally accepted that a bipartite perianth with distinct petals evolved independently multiple times within the flowering plants (Drinnan et al., 1994; Takhtajan, 1991). However, exactly when such events occurred is still unresolved. Phylogenetic analyses have been used to suggest that transitions between a unipartite and bipartite perianth have occurred multiple times within the eudicots (Albert et al., 1998; Soltis et al., 2005; Zanis et al., 2003). These analyses, though, are equivocal in determining the direction of such evolutionary transitions. One possibility is that a bipartite perianth represents independent evolutionary events in core eudicots as compared with the Ranunculales (Fig. 1A). Alternatively, a bipartite perianth might have been ancestral, and was lost in only a few, derived, non-core eudicot lineages (Fig. 1B). Determining whether core eudicots and Ranunculales species possess similar or divergent developmental genetic mechanisms to condition petal identity would be valuable in assessing the merits of these two hypotheses. A variety of studies have already been carried out to assess the roles of AP3 homologs in core eudicot species. A duplication in the AP3 gene lineage at the base of the core eudicots gave rise to the euAP3 and TM6 lineages, which are characterized by having distinct C-terminal sequence motifs (Kramer et al., 1998). The sequence motifs in the TM6 lineage genes are more similar to those of the paleoAP3 genes, which are found in non-core eudicot angiosperms (Kramer et al., 1998). Genetic analyses of euAP3 genes in core eudicot species, such as of the Arabidopsis AP3 gene, support the idea that these genes have a common function in petal-identity specification, as well as in stamen specification (de Martino et al., 2006; Jack et al., 1992; Schwarz-Sommer et al., 1992; Vandenbussche et al., 2004). By contrast, core eudicot TM6 genes appear to have a more restricted role in conditioning stamen identity (de Martino et al., 2006; Rijpkema et al., 2006). Functional analyses of genetic pathways controlling petal specification in poppy Sinéad Drea 1 , Lena C. Hileman 1, *, Gemma de Martino 1 and Vivian F. Irish 1,2,† MADS-box genes are crucial regulators of floral development, yet how their functions have evolved to control different aspects of floral patterning is unclear. To understand the extent to which MADS-box gene functions are conserved or have diversified in different angiosperm lineages, we have exploited the capability for functional analyses in a new model system, Papaver somniferum (opium poppy). P. somniferum is a member of the order Ranunculales, and so represents a clade that is evolutionarily distant from those containing traditional model systems such as Arabidopsis, Petunia, maize or rice. We have identified and characterized the roles of several candidate MADS-box genes in petal specification in poppy. In Arabidopsis, the APETALA3 (AP3) MADS-box gene is required for both petal and stamen identity specification. By contrast, we show that the AP3 lineage has undergone gene duplication and subfunctionalization in poppy, with one gene copy required for petal development and the other responsible for stamen development. These differences in gene function are due to differences both in expression patterns and co- factor interactions. Furthermore, the genetic hierarchy controlling petal development in poppy has diverged as compared with that of Arabidopsis. As these are the first functional analyses of AP3 genes in this evolutionarily divergent clade, our results provide new information on the similarities and differences in petal developmental programs across angiosperms. Based on these observations, we discuss a model for how the petal developmental program has evolved. KEY WORDS: Petal identity, Homeotic genes, MADS-box genes, Poppy, Papaver somniferum Development 134, 4157-4166 (2007) doi:10.1242/dev.013136 1 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA. 2 Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06520, USA. *Present address: Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045, USA † Author for correspondence (e-mail: vivian.irish@yale.edu) Accepted 7 September 2007 DEVELOPMENT