Lucibelli, Francesca (2023) Comparative genomics and transcriptomics to identify key regulators of orchid flower symmetry and organ identity. [Tesi di dottorato]

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Abstract

The Orchidaceae are a monocot angiosperm family with an extraordinary diversification of flower architecture and unique ecological characteristics. In addition to the scientific interest, orchids have a considerable economic value, representing the most commercialized ornamental plants. For these reasons, a complete understanding of the complex molecular network that regulates orchid flower development is an interesting challenge. The class B MADS-box AP3/DEF genes lead to orchid perianth morphogenesis together with other MADS-box genes (AGL6 and SEP-like). This work aims to identify novel candidates that may be part of the genetic program at the base of orchid flower symmetry and organ identity determination. The in silico differential expression analysis on RNA-seq data of wild-type and peloric Phalaenopsis orchids suggested that the YABBY DROOPING LEAF-like gene PeDL2 could be a novel regulator of the orchid lip specification. In wild-type Phalaenopsis, PeDL2 is differentially expressed in lip and lateral inner tepals, while in the perianth organs of the peloric mutant it is expressed at similar levels. This is the first evidence of the expansion of a DL-like gene expression domain to the perianth, since it is generally involved in reproductive organs and leaf development. The regulatory relationship between DL-like and class B MADS-box genes of other angiosperms, together with the central role of the MADS-box genes in orchid perianth development, supports the hypothesis that DL2 can be involved in the regulatory network underlying the orchid flower development. These premises prompted me to focus my Ph.D. research project on the orchid DL-like genes. Genomics and transcriptomics revealed that the orchids with zygomorphic flowers have two DL genes, whereas the ancestral orchids with actinomorphic flowers have only one. These observations and the expression pattern of the Phalaenopsis DL genes suggest that the DL paralogs conserved their function in the development of the reproductive organs; however, after a duplication event, the neofunctionalization of the DL2 gene could explain its acquisition of a role in orchid lip development and bilateral symmetry determination. The PeDL2 expression pattern is similar to that of the AP3/DEF genes belonging to clades 3 and 4 and opposite to that of the AP3/DEF genes belonging to clades 1 and 2, suggesting a possible regulatory link. To support the hypothesis of the orchid DL2 neofunctionalization, I evaluated the expression of the DL- and DEF-like genes outside the Phalaenopsis genus (Vanilla planifolia, Phragmipedium logifolium, and Rhyncholaeliocattleya). The obtained results led to a reformulation of the initial hypothesis, suggesting that DL2 could have acquired a specific role in lip development only in evolutionarily more recent orchids. In the basal zygomorphic orchids, DL2 is expressed at similar levels in all the perianth organs. Possibly, after gene duplication, DL2 acquired a new function in the perianth specification, and later assumed a specific role in the lip. To characterize PeDL2, I performed intracellular protein localization experiments, confirming its nuclear localization. In addition, I predicted the DLs structure and interactions by computational approach and confirmed the results by yeast two-hybrid analysis. Finally, I evaluated the DL2 transcriptional regulation by in silico screening of possible promoter interactors. I validated these results through yeast one-hybrid assay and protoplasts dsRNAi experiments. According to these analyses, DL2 transcription could be modulated by different transcription factors; in particular, the Phalaenopsis clade 1 DEF-like protein MADS2 could repress DL2 expression.

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