Insights into the evo-devo of plant reproduction using next-generation sequencing approaches.

The development of plant model organisms has traditionally been analyzed using resource-heavy, tailored applications that are not easily transferable to distantly related non-model taxa. Thus, our understanding of plant development has been limited to a subset of traits, and evolutionary studies conducted most effectively either across very wide [e.g. Arabidopsis thaliana and Oryza sativa (rice)] or narrow (i.e. population level) phylogenetic distances. As plant biologists seek to capitalize on natural diversity for crop improvement, enhance ecosystem functioning, and better understand plant responses to climate change, high-throughput and broadly applicable forms of existing molecular biology assays are becoming an invaluable resource. Next-generation sequencing (NGS) is increasingly becoming a powerful tool in evolutionary developmental biology (evo-devo) studies, particularly through its application to understanding trait evolution at different levels of gene regulation. Here, I review some of the most common and emerging NGS-based methods, using exemplar studies in reproductive plant evo-devo to illustrate their potential.

Implications of region-specific gene expression for development of the partially fused petunia corolla.

Angiosperm petal fusion (sympetaly) has evolved multiple times independently and is associated with increased specificity between plants and their pollinators. To uncover developmental genetic changes that might have led to the evolution of sympetaly in the asterid core eudicot genus Petunia (Solanaceae), we carried out global and fine-scale gene expression analyses in different regions of the corolla. We found that, despite several similarities with the choripetalous model species Arabidopsis thaliana in the proximal-distal transcriptome, the Petunia axillaris fused and proximal corolla tube expresses several genes that in A. thaliana are associated with the distal petal region. This difference aligns with variation in petal shape and fusion across ontogeny of the two species. Moreover, differential gene expression between the unfused lobes and fused tube of P. axillaris petals revealed three strong candidate genes for sympetaly based on functional annotation in organ boundary specification. Partial silencing of one of these, the HANABA TARANU (HAN)-like gene PhGATA19, resulted in reduced fusion of Petunia hybrida petals, with silencing of both PhGATA19 and its close paralog causing premature plant senescence. Finally, detailed expression analyses for the previously characterized organ boundary gene candidate NO APICAL MERISTEM (NAM) supports the hypothesis that it establishes boundaries between most P. axillaris floral organs, with the exception of boundaries between petals.

Revisiting floral fusion: the evolution and molecular basis of a developmental innovation.

Throughout the evolution of the angiosperm flower, developmental innovations have enabled the modification or elaboration of novel floral organs enabling subsequent diversification and expansion into new niches, for example the formation of novel pollinator relationships. One such developmental innovation is the fusion of various floral organs to form complex structures. Multiple types of floral fusion exist; each type may be the result of different developmental processes and is likely to have evolved multiple times independently across the angiosperm tree of life. The development of fused organs is thought to be mediated by the NAM/CUC3 subfamily of NAC transcription factors, which mediate boundary formation during meristematic development. The goal of this review is to (i) introduce the development of fused floral organs as a key 'developmental innovation', facilitated by a change in the expression of NAM/CUC3 transcription factors; (ii) provide a comprehensive overview of floral fusion phenotypes amongst the angiosperms, defining well-known fusion phenotypes and applying them to a systematic context; and (iii) summarize the current molecular knowledge of this phenomenon, highlighting the evolution of the NAM/CUC3 subfamily of transcription factors implicated in the development of fused organs. The need for a network-based analysis of fusion is discussed, and a gene regulatory network responsible for directing fusion is proposed to guide future research in this area.

Bridging the gaps: evolution and development of perianth fusion.

One of the most striking innovations in flower development is the congenital or postgenital union of petals (sympetaly) which has enabled dramatic specialization in flower structure and possibly accelerated speciation rates. Sympetalous flowers exhibit extraordinary variation in development, including the degree and timing of fusion, and fusion with other floral organs. Different axes of corolla tube complexity can be disentangled at the developmental level, with most variation being explained by differences in coordinated growth between interconnected and lobed regions of neighboring petal primordia, and between lower and upper portions of the corolla tube, defined by the stamen insertion boundary. Genetically, inter- and intra-specific variation in the degree of petal fusion is controlled by various inputs from genes that affect organ boundary and lateral growth, signaling between different cell types, and production of the cuticle. It is thus hypothesized that the evolution and diversification of fused petals, at least within the megadiverse Asteridae clade of core eudicots, have occurred through the modification of a conserved genetic pathway previously involved in free petal development.

Formation and Shaping of the Antirrhinum Flower through Modulation of the CUP Boundary Gene.

Boundary domain genes, expressed within or around organ primordia, play a key role in the formation, shaping, and subdivision of planar plant organs, such as leaves. However, the role of boundary genes in formation of more elaborate 3D structures, which also derive from organ primordia, remains unclear. Here we analyze the role of the boundary domain gene CUPULIFORMIS (CUP) in formation of the ornate Antirrhinum flower shape. We show that CUP expression becomes cleared from boundary subdomains between petal primordia, most likely contributing to formation of congenitally fused petals (sympetally) and modulation of growth at sinuses. At later stages, CUP is activated by dorsoventral genes in an intermediary region of the corolla. In contrast to its role at organ boundaries, intermediary CUP activity leads to growth promotion rather than repression and formation of the palate, lip, and characteristic folds of the closed Antirrhinum flower. Intermediary expression of CUP homologs is also observed in related sympetalous species, Linaria and Mimulus, suggesting that changes in boundary gene activity have played a key role in the development and evolution of diverse 3D plant shapes.

Organ boundary NAC-domain transcription factors are implicated in the evolution of petal fusion.

Research rationale: Evolution of fused petals (sympetaly) is considered to be an important innovation that has repeatedly led to increased pollination efficiency, resulting in accelerated rates of plant diversification. Although little is known about the underlying regulation of sympetaly, genetic pathways ancestrally involved in organ boundary establishment (e.g. CUP SHAPED COTYLEDON [CUC] 1-3 genes) are strong candidates. In sympetalous petunia, mutations in the CUC1/2-like orthologue NO APICAL MERISTEM (NAM) inhibit shoot apical meristem formation. Despite this, occasional 'escape shoots' develop flowers with extra petals and fused inter-floral whorl organs. Central methods: To To determine if petunia CUC-like genes regulate additional floral patterning, we used virus-induced silencing (VIGS) following establishment of healthy shoot apices to re-examine the role of NAM in petunia petal development, and uniquely characterise the CUC3 orthologue NH16. KEY RESULTS: Confirming previous results, we found that reduced floral NAM/NH16 expression caused increased petal-stamen and stamen-carpel fusion, and often produced extra petals. However, further to previous results, all VIGS plants infected with NAM or NH16 constructs exhibited reduced fusion in the petal whorl compared to control plants. MAIN CONCLUSIONS: Together with previous data, our results demonstrate conservation of petunia CUC-like genes in establishing inter-floral whorl organ boundaries, as well as functional evolution to affect the fusion of petunia petals.