ABSTRACT Floral homeotic MADS-box transcription factors ensure the correct development of floral organs with all their mature features, i.e. organ shape, size, colour and cellular identity. Furthermore, all plant organs develop from clonally-independent cell layers, deriving from the meristematic epidermal (L1) and internal (L2 and L3) layers. How cells from these distinct layers acquire their floral identities and coordinate their growth to ensure reproducible organ development is unclear. Here we study the development of the Petunia x hybrida (petunia) corolla, which consists of five fused petals forming a tube and pigmented limbs. We present petunia flowers expressing the B-class MADS-box gene PhDEF in the epidermis or in the mesophyll of the petal only, that we called wico and star respectively. Strikingly, the wico flowers form a very small tube while their limbs are almost normal, and the star flowers form a normal tube but very reduced and unpigmented limbs. Therefore, the star and wico phenotypes indicate that in the petunia petal, the epidermis mainly drives limb growth and pigmentation while the mesophyll mainly drives tube growth. As a first step towards the identification of candidate genes involved in specification of petal layer identities and tube/limb development, we sequenced the star and wico whole petal transcriptome at three developmental stages. Among downregulated genes in star petals, we found the major regulator of anthocyanin biosynthesis ANTHOCYANIN 1 (AN1) , and we showed that, in vitro , PhDEF directly binds to its terminator sequence, suggesting that it might regulate its expression. Altogether this study shows that layer-specific expression of PhDEF drives petunia tube or limb development in a highly modular fashion, which adds an extra layer of complexity to the petal development process.