Orchid fruit traits scoring.

Fruits play a crucial role in seed dispersal. They open along dehiscence zones. Fruit dehiscence zone formation has been intensively studied in Arabidopsis thaliana. However, little is known about the mechanisms and genes involved in the formation of fruit dehiscence zones in species outside the Brassicaceae. The dehiscence zone of A. thaliana contains a lignified layer, while dehiscence zone tissues of the emerging orchid model Erycina pusilla include a lipid layer. Here we present an analysis of evolution and development of fruit dehiscence zones in orchids. We performed ancestral state reconstructions across the five orchid subfamilies to study the evolution of selected fruit traits and explored dehiscence zone developmental genes using RNA-seq and qPCR. We found that erect dehiscent fruits with non-lignified dehiscence zones and a short ripening period are ancestral characters in orchids. Lignified dehiscence zones in orchid fruits evolved multiple times from non-lignified zones. Furthermore, we carried out gene expression analysis of tissues from different developmental stages of E. pusilla fruits. We found that fruit dehiscence genes from the MADS-box gene family and other important regulators in E. pusilla differed in their expression pattern from their homologs in A. thaliana. This suggests that the current A. thaliana fruit dehiscence model requires adjustment for orchids. Additionally, we discovered that homologs of A. thaliana genes involved in the development of carpel, gynoecium and ovules, and genes involved in lipid biosynthesis were expressed in the fruit valves of E. pusilla, implying that these genes may play a novel role in formation of dehiscence zone tissues in orchids. Future functional analysis of developmental regulators, lipid identification and quantification can shed more light on lipid-layer based dehiscence of orchid fruits.