Expression of floral MADS-box genes in Sinofranchetia chinensis (Lardizabalaceae): implications for the nature of the nectar leaves

The perianths of the Lardizabalaceae are diverse. The second-whorl floral organs of Sinofranchetia chinensis (Lardizabalaceae) are nectar leaves. The aim of this study was to explore the nature of this type of floral organ, and to determine its relationship to nectar leaves in other Ranunculales species, and to other floral organs in Sinofranchetia chinensis. Approaches of evolutionary developmental biology were used, including 3 RACE (rapid amplification of cDNA ends) for isolating floral MADS-box genes, phylogenetic analysis for reconstructing gene evolutionary history, in situ hybridization and tissue-specific RT-PCR for identifying gene expression patterns and SEM (scanning electron microscopy) for observing the epidermal cell morphology of floral organs. Fourteen new floral MADS-box genes were isolated from Sinofranchetia chinensis and from two other species of Lardizabalaceae, Holboellia grandiflora and Decaisnea insignis. The phylogenetic analysis of AP3-like genes in Ranunculales showed that three AP3 paralogues from Sinofranchetia chinensis belong to the AP3-I, -II and -III lineages. In situ hybridization results showed that SIchAP3-3 is significantly expressed only in nectar leaves at the late stages of floral development, and SIchAG, a C-class MADS-box gene, is expressed not only in stamens and carpels, but also in nectar leaves. SEM observation revealed that the adaxial surface of nectar leaves is covered with conical epidermal cells, a hallmark of petaloidy. The gene expression data imply that the nectar leaves in S. chinensis might share a similar genetic regulatory code with other nectar leaves in Ranunculales species. Based on gene expression and morphological evidence, it is considered that the nectar leaves in S. chinensis could be referred to as petals. Furthermore, the study supports the hypothesis that the nectar leaves in some Ranunculales species might be derived from stamens.