The OitaAG and OitaSTK genes of the orchid Orchis italica: a comparative analysis with other C- and D-class MADS-box genes

According to the ABCDE model of flower development, the C- and D- class MADS box genes are involved in the formation of male and female reproductive organs (fused to form the column in orchids) and in ovule maturation (triggered by fertilization in orchids). In the present study, we report the isolation of the Orchis italica genes OitaAG and OitaSTK, homologs of the C-class AGAMOUS and the D-class SEEDSTICK genes of Arabidopsis, respectively. Analysis of their expression profiles reveals high levels of mRNA in columns and ovaries, particularly after pollination. However, weak expression is also detectable in the inner tepals (OitaAG) and the lip and root (OitaSTK). This expression profile is only partially overlapping with those reported in other orchid species and may be the consequence of a different evolutionary history of these functional gene classes in orchids. The genomic characterization of the OitaAG and OitaSTK genes shows that a high number of traces of mobile elements are present in introns and could have contributed to the size expansion of some of them (e.g., intron 2 and 3 of OitaAG and intron 3, 4 and 5 of OitaSTK). Nucleotide sequences of intron 1 of the OitaSTK gene and other STK-like genes do not share regulatory motifs, whereas sequence comparison of intron 2 of the OitaAG gene with that of intron 2 of other AG-like genes reveals, for the first time in an orchid species, the presence of conserved cis-regulatory boxes and binding sites for transcription factors that positively (e.g., LEAFY and WUSCHEL) or negatively (e.g., BELLRINGER) regulate the expression of the AG homologs in dicots and monocots.