Ancestral duplicated DL/CRC orthologs display function on orchid reproductive organ innovation

Abstract

The orchid flower is renowned for complexity of flower organ morphogenesis and has attracted great interest from scientists. The YABBY genes encode plant-specific transcription factors with important roles in vegetative and reproductive development in seed plants. DROOPING LEAF/CRABS CLAW (DL/CRC) orthologs are involved in reproductive organ development (especially carpels) of angiosperms. Orchid gynostemium (the fused organ of the androecium and gynoecium) and ovule development are unique developmental processes. Understanding the DL/CRC-like genes controlling the developmental program of the gynostemium and ovule could provide accessible information for reproductive organ molecular regulation in orchids. Two DL/CRC-like genes, named PeDL1 and PeDL2, were cloned from Phalaenopsis equestris. The orchid DL/CRC forms a monophyletic clade with two subclades including AshDL, PeDL1 and DcaDL1 in subclade I, and PeDL2 and DcaDL2 in subclade II. 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. 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. Paralogue 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 important roles of PeDLs involved in orchid gynostemium and ovule development and provide new insights for further understanding the molecular mechanisms underlying orchid reproductive organ development.