Bilateral symmetry of flowers is a relevant novelty that has occurred many times throughout the evolution of flowering plants. In Antirrhinum majus, establishment of flower dorso-ventral asymmetry is mainly due to interaction of TCP (CYC and DICH) and MYB (DIV, RAD, and DRIF) transcription factors. In the present study, we characterized 8 DIV-, 4 RAD-, and 2 DRIF-like genes from the transcriptome of Orchis italica, an orchid species with bilaterally symmetric and resupinate flowers. We found a similar number of DIV- and RAD-like genes within the genomes of Phalaenopsis equestris and Dendrobium catenatum orchids. Orchid DIV- and RAD-like proteins share conserved motifs whose distribution reflects their phylogeny and analysis of the genomic organization revealed a single intron containing many traces of transposable elements. Evolutionary analysis has shown that purifying selection acts on the DIV- and RAD-like coding regions in orchids, with relaxation of selective constraints in a branch of the DIV-like genes. Analysis of the expression patterns of DIV- and RAD-like genes in O. italica revealed possible redundant functions for some of them. In the perianth of O. italica, the ortholog of DIV and DRIF of A. majus are expressed in all tissues, whereas RAD is mainly expressed in the outer tepals and lip. These data allow for proposal of an evolutionary conserved model in which the expression of the orthologs of the DIV, RAD, and DRIF genes might be related to establishment of flower bilateral symmetry in the nonmodel orchid species O. italica.

DNA methylation is an epigenetic modification of the genome involved in the regulation of gene expression and modulation of chromatin structure. Plant genomes are widely methylated, and the methylation generally occurs on the cytosine bases through the activity of specific enzymes called DNA methyltransferases. On the other hand, methylated DNA can also undergo demethylation through the action of demethylases. The methylation landscape is finely tuned and assumes a pivotal role in plant development and evolution. This review illustrates different molecular aspects of DNA methylation and some plant physiological processes influenced by this epigenetic modification in model species, crops, and ornamental plants such as orchids. In addition, this review aims to describe the relationship between the changes in plant DNA methylation levels and the response to biotic and abiotic stress. Finally, we discuss the possible evolutionary implications and biotechnological applications of DNA methylation.

The MYB transcription factors DIVARICATA (DIV), DIV-and-RAD-Interacting-Factor (DRIF), and the small interfering peptide RADIALIS (RAD) can interact, forming a regulatory module that controls different plant developmental processes. In the snapdragon Antirrhinum majus, this module, together with the TCP transcription factor CYCLOIDEA (CYC), is responsible for the establishment of floral dorsoventral asymmetry. The spatial gene expression pattern of the OitDIV, OitDRIF, and OitRAD homologs of Orchis italica, an orchid with zygomorphic flowers, has suggested a possible conserved role of these genes in bilateral symmetry of the orchid flower. Here, we have identified four DRIF genes of orchids and have reconstructed their genomic organization and evolution. In addition, we found snapdragon transcriptional cis-regulatory elements of DIV and RAD loci generally conserved within the corresponding orchid orthologues. We have tested the biochemical interactions among OitDIV, OitDRIF1, and OitRAD of O. italica, showing that OitDRIF1 can interact both with OitDIV and OitRAD, whereas OitDIV and OitRAD do not directly interact, as in A. majus. The analysis of the quantitative expression profile of these MYB genes revealed that in zygomorphic orchid flowers, the DIV, DRIF1, and RAD transcripts are present at higher levels in the lip than in lateral inner tepals, whereas in peloric orchid flowers they show similar expression levels. These results indicate that MYB transcription factors could have a role in shaping zygomorphy of the orchid flower, potentially enriching the underlying orchid developmental code.

BackgroundAnalyzing close species with diverse developmental modes is instrumental for investigating the evolutionary significance of physiological, anatomical and behavioral features at a molecular level. Many examples of trait loss are known in metazoan populations living in dark environments. Tunicates are the closest living relatives of vertebrates and typically present a lifecycle with distinct motile larval and sessile adult stages. The nervous system of the motile larva contains melanized cells associated with geotactic and light-sensing organs. It has been suggested that these are homologous to vertebrate neural crest-derived melanocytes. Probably due to ecological adaptation to distinct habitats, several species of tunicates in the Molgulidae family have tailless (anural) larvae that fail to develop sensory organ-associated melanocytes. Here we studied the evolution of Tyrosinase family genes, indispensible for melanogenesis, in the anural, unpigmented Molgula occulta and in the tailed, pigmented Molgula oculata by using phylogenetic, developmental and molecular approaches.ResultsWe performed an evolutionary reconstruction of the tunicate Tyrosinase gene family: in particular, we found that M. oculata possesses genes predicted to encode one Tyrosinase (Tyr) and three Tyrosinase-related proteins (Tyrps) while M. occulta has only Tyr and Tyrp.a pseudogenes that are not likely to encode functional proteins. Analysis of Tyr sequences from various M. occulta individuals indicates that different alleles independently acquired frameshifting short indels and/or larger mobile genetic element insertions, resulting in pseudogenization of the Tyr locus. In M. oculata, Tyr is expressed in presumptive pigment cell precursors as in the model tunicate Ciona robusta. Furthermore, a M. oculata Tyr reporter gene construct was active in the pigment cell precursors of C. robusta embryos, hinting at conservation of the regulatory network underlying Tyr expression in tunicates. In contrast, we did not observe any expression of the Tyr pseudogene in M. occulta embryos. Similarly, M. occulta Tyr allele expression was not rescued in pigmented interspecific M. occulta × M. oculata hybrid embryos, suggesting deleterious mutations also to its cis-regulatory sequences. However, in situ hybridization for transcripts from the M. occulta Tyrp.a pseudogene revealed its expression in vestigial pigment cell precursors in this species.ConclusionsWe reveal a complex evolutionary history of the melanogenesis pathway in tunicates, characterized by distinct gene duplication and loss events. Our expression and molecular data support a tight correlation between pseudogenization of Tyrosinase family members and the absence of pigmentation in the immotile larvae of M. occulta. These results suggest that relaxation of purifying selection has resulted in the loss of sensory organ-associated melanocytes and core genes in the melanogenesis biosynthetic pathway in M. occulta. Electronic supplementary materialThe online version of this article (doi:10.11...