Duplicated C-class MADS-box genes reveal distinct roles in gynostemium development in Cymbidium ensifolium (Orchidaceae).

The orchid floral organs represent novel and effective structures for attracting pollination vectors. In addition, to avoid inbreeding, the androecium and gynoecium are united in a single structure termed the gynostemium. Identification of C-class MADS-box genes regulating reproductive organ development could help determine the level of homology with the current ABC model of floral organ identity in orchids. In this study, we isolated and characterized two C-class AGAMOUS-like genes, denoted CeMADS1 and CeMADS2, from Cymbidium ensifolium. These two genes showed distinct spatial and temporal expression profiles, which suggests their functional diversification during gynostemium development. Furthermore, the expression of CeMADS1 but not CeMADS2 was eliminated in the multitepal mutant whose gynostemium is replaced by a newly emerged flower, and this ecotopic flower continues to produce sepals and petals centripetally. Protein interaction relationships among CeMADS1, CeMADS2 and E-class PeMADS8 proteins were assessed by yeast two-hybrid analysis. Both CeMADS1 and CeMADS2 formed homodimers and heterodimers with each other and the E-class PeMADS protein. Furthermore, transgenic Arabidopsis plants overexpressing CeMADS1 or CeMADS2 showed limited growth of primary inflorescence. Thus, CeMADS1 may have a pivotal C function in reproductive organ development in C. ensifolium.