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.

Summary

The class B MADS-box genes belong to two distinct functional groups: the AP3/DEF-like and the PI/GLO-like sub-families. In orchids, AP3/DEF-like genes are present in four copies, each with a different role in floral organ formation, which is described in the "orchid code" model. Interestingly, the orchid PI/GLO-like genes are present in two copies in Orchidinae, whereas they are described as single copy in the other orchid lineages. The two PI/GLO-like paralogs have site-specific different selective constraints; in addition, they show relaxation of purifying selection when compared to the single-copy lineages. In this study, we present a comparative analysis of the expression patterns of the two PI/GLO-like paralogs, OrcPI and OrcPI2, in floral tissues of Orchis italica in different developmental stages using real-time PCR. The two genes show similar expression profiles in the tissue examined, with differences detectable between immature and mature inflorescence. In all cases, OrcPI2 is expressed at a higher level than OrcPI. Real-time PCR results reveal that the co-expression of the two duplicated loci could have a fully or partially redundant function. The possible evolutionary fate of OrcPI and OrcPI2 is discussed as well as their involvement in ovary development.

The AP2/ERF proteins are plant-specific transcription factors involved in multiple regulatory pathways, from plant organ development to response to various environmental stresses. One of the mechanisms that regulates the AP2-like genes involves the microRNA miR172, which controls their activity at the post-transcriptional level. Extensive studies on AP2-like genes are available in many different species; however, in orchids, one of the largest plant families, studies are restricted to a few species, all belonging to the Epidendroideae subfamily. In the present study, we report the isolation of an AP2-like gene in the Mediterranean orchid Orchis italica (Orchidoideae). The OitaAP2 locus includes 10 exons and 9 introns, and its transcript is alternatively spliced, resulting in the long OitaAP2 and the short OitaAP2_ISO isoforms, with the latter skipping exon 9. Both isoforms contain the conserved target site for miR172, whose action is demonstrated by the presence of cleaved OitaAP2 mRNA. The OitaAP2 and OitaAP2_ISO mRNAs are present in the tepals and lip before and after anthesis at different expression levels. In addition, the OitaAP2_ISO isoform is expressed in the ovary before pollination and in the root and stem. The isoform-specific expression pattern suggests a functional differentiation of the OitaAP2 alternatively spliced transcripts. The expression profile of miR172 is complementary to that of the OitaAP2 isoforms in inflorescence tissues before anthesis, whereas after anthesis and in ovary tissue before and after pollination, this relationship disappears, suggesting the existence of OitaAP2 inhibitory mechanisms in these tissues that differ from that involving miR172.

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