The ancestral duplicated DL/CRC orthologs, PeDL1 and PeDL2, function in orchid reproductive organ innovation.

Orchid gynostemium, the fused organ of the androecium and gynoecium, and ovule development are unique developmental processes. Two DROOPING LEAF/CRABS CLAW (DL/CRC) genes were identified from the Phalaenopsis orchid genome and functionally characterized, including PeDL1 and PeDL2. Phylogenetic analysis indicated that the most recent common ancestor of orchids contained the duplicated DL/CRC-like genes. The temporal and spatial expression analysis indicated PeDL genes are specifically expressed in the gynostemium and at the early stages of ovule development. Both PeDLs could partially complement an Arabidopsis crc-1 mutant. Virus-induced gene silencing (VIGS) of PeDL1 and PeDL2 affects the number of protuberant ovule initials differentiated from the placenta. Transient overexpression of PeDL1 in Phalaenopsis orchids caused abnormal development of ovule and stigmatic cavity of gynostemium. PeDL1, instead of PeDL2, could form a heterodimer with PeCIN8. Paralogous retention and subsequent divergence of the gene sequence of PeDL1 and PeDL2 in P. equestris might result in the differentiation of function and protein behaviors. These results reveal the ancestral duplicated DL/CRC-like genes in orchids play important roles in orchid reproductive organ innovation.