Conserved differential expression of paralogous DEFICIENS- and GLOBOSA-like MADS-box genes in the flowers of Orchidaceae: refining the ‘orchid code

In flowering plants, class-B floral homeotic genes encode MADS-domain transcription factors, which are key in the specification of petal and stamen identity, and have two ancient clades: DEF-like and GLO-like genes. Many species have one gene of each clade, but orchids have typically four DEF-like genes, representing ancient gene clades 1, 2, 3 and 4. We tested the ‘orchid code', a combinatorial genetic model suggesting that differences between the organs of the orchid perianth (outer tepals, inner lateral tepals and labellum) are generated by the combinatorial differential expression of four DEF-like genes. Our experimental test involves highly sensitive and specific measurements, with qRT-PCR of the expression of DEF- and GLO-like genes from the distantly related Vanilla planifolia and Phragmipedium longifolium, as well as from wild-type and peloric Phalaenopsis hybrid flowers. Our findings support the first ‘orchid code' hypothesis, in that absence of clade-3 and -4 gene expression distinguishes the outer tepals from the inner tepals. In contrast to the original hypothesis, however, mRNA of both clade-3 and -4 genes accumulates in wild-type inner lateral tepals and the labellum, and in labellum-like inner lateral tepals of peloric flowers, albeit in different quantities. Our data suggest a revised hypothesis where high levels of clade-1 and -2, and low levels of clade-3 and -4, gene expression specify inner lateral tepals, whereas labellum development requires low levels of clade-1 and -2 expression and high levels of clade-3 and -4 expression.