Tuber, or not tuber: Molecular and morphological basis of underground storage organ development.

Underground storage organs occur in phylogenetically diverse plant taxa and arise from multiple tissue types including roots and stems. Thickening growth allows underground storage organs to accommodate carbohydrates and other nutrients and requires proliferation at various lateral meristems followed by cell expansion. The WOX-CLE module regulates thickening growth via the vascular cambium in several eudicot systems, but the molecular mechanisms of proliferation at other lateral meristems are not well understood. In potato, onion, and other systems, members of the phosphatidylethanolamine-binding protein (PEBP) gene family induce underground storage organ development in response to photoperiod cues. While molecular mechanisms of tuber development in potato are well understood, we lack detailed mechanistic knowledge for the extensive morphological and taxonomic diversity of underground storage organs in plants.

Evolution of major flowering pathway integrators in Orchidaceae.

The Orchidaceae is a mega-diverse plant family with ca. 29,000 species with a large variety of life forms that can colonize transitory habitats. Despite this diversity, little is known about their flowering integrators in response to specific environmental factors. During the reproductive transition in flowering plants a vegetative apical meristem (SAM) transforms into an inflorescence meristem (IM) that forms bracts and flowers. In model grasses, like rice, a flowering genetic regulatory network (FGRN) controlling reproductive transitions has been identified, but little is known in the Orchidaceae. In order to analyze the players of the FRGN in orchids, we performed comprehensive phylogenetic analyses of CONSTANS-like/CONSTANS-like 4 (COL/COL4), FLOWERING LOCUS D (FD), FLOWERING LOCUS C/FRUITFULL (FLC/FUL) and SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) gene lineages. In addition to PEBP and AGL24/SVP genes previously analyzed, here we identify an increase of orchid homologs belonging to COL4, and FUL gene lineages in comparison with other monocots, including grasses, due to orchid-specific gene lineage duplications. Contrariwise, local duplications in Orchidaceae are less frequent in the COL, FD and SOC1 gene lineages, which points to a retention of key functions under strong purifying selection in essential signaling factors. We also identified changes in the protein sequences after such duplications, variation in the evolutionary rates of resulting paralogous clades and targeted expression of isolated homologs in different orchids. Interestingly, vernalization-response genes like VERNALIZATION1 (VRN1) and FLOWERING LOCUS C (FLC) are completely lacking in orchids, or alternatively are reduced in number, as is the case of VERNALIZATION2/GHD7 (VRN2). Our findings point to non-canonical factors sensing temperature changes in orchids during reproductive transition. Expression data of key factors gathered from Elleanthus auratiacus, a terrestrial orchid in high Andean mountains allow us to characterize which copies are actually active during flowering. Altogether, our data lays down a comprehensive framework to assess gene function of a restricted number of homologs identified more likely playing key roles during the flowering transition, and the changes of the FGRN in neotropical orchids in comparison with temperate grasses.

Expression and Functional Studies of Leaf, Floral, and Fruit Developmental Genes in Non-model Species.

Researchers working on evolutionary developmental plant biology are inclined to choose non-model taxa to address how specific features have been acquired during ontogeny and fixed during phylogeny. In this chapter we describe methods to extract RNA, to assemble de-novo transcriptomes, to isolate orthologous genes within gene families, and to evaluate expression and function of target genes. We have successfully optimized these protocols for non-model plant species including ferns, gymnosperms, and a large assortment of angiosperms. In the latter, we have ranged a large number of families including Aristolochiaceae, Apodanthaceae, Chloranthaceae, Orchidaceae, Papaveraceae, Rubiaceae, Solanaceae, and Tropaeolaceae.

Evolution and expression of the MADS-box flowering transition genes AGAMOUS-like 24/SHORT VEGETATIVE PHASE with emphasis in selected Neotropical orchids.

In angiosperms the reproductive transition results in the transformation of a vegetative apical meristem (SAM) into an inflorescence meristem (IM), capable of forming floral meristems (FM). Two key players in the flowering transition are AGAMOUS-like 24 (AGL24) and SHORT VEGETATIVE PHASE (SVP). They are eudicot MADS-box paralogs performing opposite roles, as AGL24 positively regulates flowering while SVP represses the reproductive transition in Arabidopsis. We confirm that the Arabidopsis functional reference cannot be readily extrapolated to all eudicots as there are additional duplications of AGL24 in early divergent eudicots and core eudicots with significant sequence variation. In addition, we found that in monocots, two additional independent duplication events have resulted in at least three clades of AGL24/SVP homologs, some only found in Orchidaceae. Protein sequence analyses and comparative evolutionary rates point to higher rates of relaxed negative selection in the Core Eudicot AGL24 B and the Orch SVP-like B clades, in eudicots and monocots respectively. On the other hand, expression data points to plesiomorphic pleiotropic roles of AGL24/SVP genes likely similar to SVP core eudicot genes, and the acquisition of new roles as flowering positive regulators in Core Eudicot AGL24 A genes. Our research presents evidence on the diversification and recruitment of AGL24/SVP homologs in flowering transition in orchids. Although, broad expression of most copies does not allow to determine if they act as flowering repressors or promoters, the restricted expression of some homologs in the SAM suggests putative roles in maintaining the vegetative phase. If so studying in detail the function of AGL24/SVP homologs in orchids is critical to identify putative flowering repressors in a lineage where other canonical repressors remain elusive.

Evolution of Class II TCP genes in perianth bearing Piperales and their contribution to the bilateral calyx in Aristolochia.

Controlled spatiotemporal cell division and expansion are responsible for floral bilateral symmetry. Genetic studies have pointed to class II TCP genes as major regulators of cell division and floral patterning in model core eudicots. Here we study their evolution in perianth-bearing Piperales and their expression in Aristolochia, a rare occurrence of bilateral perianth outside eudicots and monocots. The evolution of class II TCP genes reveals single-copy CYCLOIDEA-like genes and three paralogs of CINCINNATA (CIN) in early diverging angiosperms. All class II TCP genes have independently duplicated in Aristolochia subgenus Siphisia. Also CIN2 genes duplicated before the diversification of Saruma and Asarum. Sequence analysis shows that CIN1 and CIN3 share motifs with Cyclin proteins and CIN2 genes have lost the miRNA319a binding site. Expression analyses of all paralogs of class II TCP genes in Aristolochia fimbriata point to a role of CYC and CIN genes in maintaining differential perianth expansion during mid- and late flower developmental stages by promoting cell division in the distal and ventral portion of the limb. It is likely that class II TCP genes also contribute to cell division in the leaf, the gynoecium and the ovules in A. fimbriata.

Evolution and Expression of Reproductive Transition Regulatory Genes FT/TFL1 With Emphasis in Selected Neotropical Orchids.

Flowering is a rigorously timed and morphologically complex shift in plant development. This change depends on endogenous as well as environmental factors. FLOWERING LOCUS T (FT) integrates several cues from different pathways acting as a flowering promoter. Contrary to the role of FT, its paralog TERMINAL FLOWER 1 (TFL1) delays floral transition. Although FT/TFL1 homologs have been studied in model eudicots and monocots, scarce studies are available in non-model monocots like the Orchidaceae. Orchids are very diverse and their floral complexity is translated into a unique aesthetic display, which appeals the ornamental plant market. Nonetheless, orchid trade faces huge limitations due to their long vegetative phase and intractable indoor flowering seasons. Little is known about the genetic basis that control reproductive transition in orchids and, consequently, manipulating their flowering time remains a challenge. In order to contribute to the understanding of the genetic bases that control flowering in orchids we present here the first broad-scale analysis of FT/TFL1-like genes in monocots with an expanded sampling in Orchidaceae. We also compare expression patterns in three selected species and propose hypotheses on the putative role of these genes in their reproductive transition. Our findings show that FT-like genes are by far more diversified than TFL1-like genes in monocots with six subclades in the former and only one in the latter. Within MonFT1, the comparative protein sequences of MonFT1A and MonFT1B suggest that they could have recruited functional roles in delaying flowering, a role typically assigned to TFL1-like proteins. On the other hand, MonFT2 proteins have retained their canonical motifs and roles in promoting flowering transition. This is also shown by their increased expression levels from the shoot apical meristem (SAM) and leaves to inflorescence meristems (IM) and floral buds (FBs). Finally, TFL1-like genes are retained as single copy and often times are lost. Their loss could be linked to the parallel recruitment of MonFT1A and MonFT1B homologs in delaying flowering and maintaining indeterminacy of the inflorescence meristem. These hypotheses lay the foundation for future functional validation in emerging model orchid species and comparative analyses in orchids with high horticultural potential in the market.

Evolution of RADIALIS and DIVARICATA gene lineages in flowering plants with an expanded sampling in non-core eudicots.

PREMISE OF THE STUDY: Bilateral symmetry in core eudicot flowers is established by the differential expression of CYCLOIDEA (CYC), DICHOTOMA (DICH), and RADIALIS (RAD), which are restricted to the dorsal portion of the flower, and DIVARICATA (DIV), restricted to the ventral and lateral petals. Little is known regarding the evolution of these gene lineages in non-core eudicots, and there are no reports on gene expression that can be used to assess whether the network predates the diversification of core eudicots. METHODS: Homologs of the RAD and DIV lineages were isolated from available genomes and transcriptomes, including those of three selected non-core eudicot species, the magnoliid Aristolochia fimbriata and the monocots Cattleya trianae and Hypoxis decumbens. Phylogenetic analyses for each gene lineage were performed. RT-PCR was used to evaluate the expression and putative contribution to floral symmetry in dissected floral organs of the selected species. KEY RESULTS: RAD-like genes have undergone at least two duplication events before eudicot diversification, three before monocots and at least four in Orchidaceae. DIV-like genes also duplicated twice before eudicot diversification and underwent independent duplications specific to Orchidaceae. RAD-like and DIV-like genes have differential dorsiventral expression only in C. trianae, which contrasts with the homogeneous expression in the perianth of A. fimbriata. CONCLUSIONS: Our results point to a common genetic regulatory network for floral symmetry in monocots and core eudicots, while alternative genetic mechanisms are likely driving the bilateral perianth symmetry in the early-diverging angiosperm Aristolochia.

Evolution and Expression Patterns of TCP Genes in Asparagales.

CYCLOIDEA-like genes are involved in the symmetry gene network, limiting cell proliferation in the dorsal regions of bilateral flowers in core eudicots. CYC-like and closely related TCP genes (acronym for TEOSINTE BRANCHED1, CYCLOIDEA, and PROLIFERATION CELL FACTOR) have been poorly studied in Asparagales, the largest order of monocots that includes both bilateral flowers in Orchidaceae (ca. 25.000 spp) and radially symmetrical flowers in Hypoxidaceae (ca. 200 spp). With the aim of assessing TCP gene evolution in the Asparagales, we isolated TCP-like genes from publicly available databases and our own transcriptomes of Cattleya trianae (Orchidaceae) and Hypoxis decumbens (Hypoxidaceae). Our matrix contains 452 sequences representing the three major clades of TCP genes. Besides the previously identified CYC specific core eudicot duplications, our ML phylogenetic analyses recovered an early CIN-like duplication predating all angiosperms, two CIN-like Asparagales-specific duplications and a duplication prior to the diversification of Orchidoideae and Epidendroideae. In addition, we provide evidence of at least three duplications of PCF-like genes in Asparagales. While CIN-like and PCF-like genes have multiplied in Asparagales, likely enhancing the genetic network for cell proliferation, CYC-like genes remain as single, shorter copies with low expression. Homogeneous expression of CYC-like genes in the labellum as well as the lateral petals suggests little contribution to the bilateral perianth in C. trianae. CIN-like and PCF-like gene expression suggests conserved roles in cell proliferation in leaves, sepals and petals, carpels, ovules and fruits in Asparagales by comparison with previously reported functions in core eudicots and monocots. This is the first large scale analysis of TCP-like genes in Asparagales that will serve as a platform for in-depth functional studies in emerging model monocots.