Characterization of B- and C-class MADS-box genes in medicinal plant Epimedium sagittatum

The basal eudicot Epimedium (Barrenwort) exhibits innovative floral morphology in the form of petal spurs filled with nectar and petaloid sepals. The B-class MADS-box genes APETALAS3 (AP3) and PISTILLA (PI) determine sepal and petal identity while the C-class gene AGAMOUS (AG) determines carpel and stamen identity in Arabidopsis. Complex histories of gene duplication resulted in subsequent subfunctionalization or neofunctionalization of paralogs. Here, a total of four B- and two C-class genes were successfully isolated from E. sagittatum. Phylogenetic analysis showed that EsAP3-1, EsAP3-2, and EsAP3-3 are part of the AP3 clade; EsPI is part of the PI group; and EsAG and EsAG11 clustered into the AG and AG11 groups, respectively. Quantitative real-time PCR was utilized to detect the expression patterns of these genes, and all B-class genes except for EsAP3-3 were found to be universally expressed. The transcribed EsAG and EsAG11 genes were confined to reproductive organs. In addition, yeast three and two hybrid assays were used to explore the status of protein complexes. EsAP3-1 was found to have broadly interactive partners, and EsPI can form a heterodimer with EsAP3-1 and EsAP3-2. Transgenic EsAG overexpression in wild-type Arabidopsis confirmed the conserved function determining carpel development.