Cell layer-specific expression of the B-class MADS-box genePhDEFdrives petal tube or limb development in petunia flowers

ABSTRACT Floral homeotic MADS-box transcription factors ensure the correct morphogenesis of floral organs, which are organized in different cell layers deriving from the meristematic L1, L2 and L3 layers. How cells from these distinct layers acquire their respective identity and coordinate their growth to ensure normal floral organ morphogenesis is unresolved. Here, we study petunia petals that form a limb and tube through congenital fusion, a complex morphology that coevolved with pollinators. We have identified petunia mutants expressing the B-class MADS-box gene PhDEF in the epidermis or in the mesophyll of the petal only, called wico and star respectively. Strikingly, wico flowers form a strongly reduced tube while their limbs are almost normal, while star flowers form a normal tube but very reduced and unpigmented limbs, showing that petunia petal morphogenesis is highly modular. Comparative transcriptome analysis of star, wico and wild-type petals revealed a strong down-regulation of the anthocyanin production pathway in star petals including its major regulator ANTHOCYANIN2 (AN2). We found that PhDEF directly binds to AN2 regulatory sequence in vitro by gel shift assay, and in vivo by chromatin immunoprecipitation, suggesting that PhDEF directly activates the petal pigmentation pathway by activating AN2 . Altogether, we show that cell-layer specific homeotic activity in petunia petals differently impacts tube and limb development, revealing the relative importance of the different cell layers in the modular architecture of petunia petals.