Understanding the Genetic Basis of Floral Diversity

The major radiations that punctuate angiosperm evolution are often associated with innovations in floral architecture. The field of plant developmental evolution seeks to understand the generation of this diversity using comparative genetic approaches that build on work done in model species such as Arabidopsis. This strategy has been particularly successful when applied to the genetic program controlling floral organ identity, but is also making advances in the area of floral symmetry. As useful as the "candidate gene" approach has been, alternatives are required to investigate morphological phenomena that are not represented in model species. Other options include methods based on quantitative trait loci and DNA sequencing, which have the additional benefit of facilitating the development of new model species. The combination of all of these approaches across a wide range of species has the potential to elucidate the genetic underpinnings of both recent and ancient radiations of flowering plants.a wide range of species has the potential to elucidate the genetic underpinnings of both reeent and ancient radiations of flowering plants.Organismic and Evolutionary Biolog

Virus-induced Gene Silencing as a Tool for Functional Analyses in the Emerging Model Plant Aquilegia (Coumbine, Ranunculaceae)

Background: The lower eudicot genus Aquilegia, commonly known as columbine, is currently the subject of extensive genetic and genomic research aimed at developing this taxon as a new model for the study of ecology and evolution. The ability to perform functional genetic analyses is a critical component of this development process and ultimately has the potential to provide insight into the genetic basis for the evolution of a wide array of traits that differentiate flowering plants. Aquilegia is of particular interest due to both its recent evolutionary history, which involves a rapid adaptive radiation, and its intermediate phylogenetic position between core eudicot (e. g., Arabidopsis) and grass (e. g., Oryza) model species. Results: Here we demonstrate the effective use of a reverse genetic technique, virus-induced gene silencing (VIGS), to study gene function in this emerging model plant. Using Agrobacterium mediated transfer of tobacco rattle virus (TRV) based vectors, we induce silencing of PHYTOENE DESATURASE (AqPDS) in Aquilegia vulgaris seedlings, and ANTHOCYANIDIN SYNTHASE (AqANS) and the B-class floral organ identity gene PISTILLATA in A. vulgaris flowers. For all of these genes, silencing phenotypes are associated with consistent reduction in endogenous transcript levels. In addition, we show that silencing of AqANS has no effect on overall floral morphology and is therefore a suitable marker for the identification of silenced flowers in dual-locus silencing experiments. Conclusion: Our results show that TRV-VIGS in Aquilegia vulgaris allows data to be rapidly obtained and can be reproduced with effective survival and silencing rates. Furthermore, this method can successfully be used to evaluate the function of early-acting developmental genes. In the future, data derived from VIGS analyses will be combined with large-scale sequencing and microarray experiments already underway in order to address both recent and ancient evolutionary questions.Organismic and Evolutionary Biolog

Evolution of the Petal and Stamen Developmental Programs: Evidence from Comparative Studies of the Basal Angiosperms

Our recently acquired understanding of the ABC program, which controls floral organ identity in model plant species such as Arabidopsis thaliana and Antirrhinum majus, has provided a new set of characters with which to evaluate floral evolution. What is still lacking, however, is a clear assessment of the actual degree of conservation of this genetic program across the angiosperms. To this end, we have begun to investigate the evolution of members of the B class gene lineages, which are known to control petal and stamen identity in the higher eudicots, and to analyze their expression patterns in selected species from the lower eudicots and basal angiosperms. The B class genes comprise the homologues of the A. thaliana genes APETALA3 (AP3) and PISTILLATA (PI), which are closely related paralogues encoding MADS box-containing DNA-binding proteins. This study has uncovered many examples of gene duplication and divergence in both the AP3 and PI lineages as well as complex and variable patterns of gene expression. These findings indicate that although some aspects of the ABC program are conserved, others display a high degree of plasticity and may not have become fixed until later in angiosperm evolution.Organismic and Evolutionary Biolog

The Role of Developmental Genetics in Understanding Homology and Morphological Evolution in Plants

Homology assessments are critical to comparative biological studies. Although gene expression data have been proposed as instrumental for defining homologous relationships, several lines of evidence suggest that this type of data can be misleading if used in isolation. The correspondence between the homology of genes and that of structures is not simple, and conclusions can be derived only after careful examination of all available data. For instance, the MADS-box gene family is one of the best-studied families of transcription factors, and it provides several examples of dissociation between genetic and morphological homology. In this regard, we examine the role of APETALA3 and PISTILLATA homologs in the development of petaloid organs, a feature thought to have originated multiple times. We also consider the role of members of the AGAMOUS subfamily in the development of the pistil, a character that originated only once. Additionally, we discuss how serial homology makes gene co-option a very common phenomenon in plants. In spite of the multiple cases of this type of dissociation, comparative developmental genetics can yield other types of information that help assess homologies. Furthermore, comparative gene expression studies provide useful data for dissecting the origin of morphological innovations and are, therefore, key to understanding character evolution. Finally, we provide some guidelines for the critical examination of comparative gene expression data in the context of studying morphological innovations.Organismic and Evolutionary Biolog

Aquilegia as a Model System for the Evolution and Ecology of Petals

The ranunculid genus Aquilegia holds extraordinary promise as a model system for investigating a wide range of questions relating to the evolution and ecology of petals. New genetic and genomic resources, including an extensive EST database, BAC libraries and physical maps, as well as virus-induced gene silencing are facilitating this research on multiple fronts. At the developmental genetic level, Aquilegia has been important for elucidating the developmental programme for specifying petals and petaloid characteristics. Data suggest that duplication events among the petal and stamen identity genes have resulted in sub- and neofunctionalization. This expansion of gene function does not include the petaloidy of Aquilegia sepals, however, which does not depend on the same loci that control identity of the second whorl petals. Of special interest is the elaboration of the petal into a nectar spur, a major innovation for the genus. Intra- and interspecific variation in the shape and colour of petals, especially the spurs, has been shown to be adaptative for different pollinators. Thus, understanding the genetic basis of these traits will help us connect the ecological interactions driving speciation with the genetic changes responsible for remodelling morphology. Progress in this area has focused on the multiple, parallel transitions in flower colour and nectar spur length across the genus. For flower colour, upstream transcription factors appear to be primarily targets of natural selection. Thus research in Aquilegia spans the initial evolution of petals and petaloidy to the diversification of petal morphology to the ecological basis of petal form, thereby providing a comprehensive picture of the evolutionary biology of this critical angiosperm feature.Organismic and Evolutionary Biolog

The evolution of reproductive structures in seed plants: a re-examination based on insights from developmental genetics

The study of developmental genetics is providing insights into how plant morphology can and does evolve, and into the fundamental nature of specific organs. This new understanding has the potential to revise significantly the way we think about seed plant evolution, especially with regard to reproductive structures. Here, we have sought to take a step in bridging the divide between genetic data and critical fields such as paleobotany and systematics. We discuss the evidence for several evolutionarily important interpretations, including the possibility that ovules represent meristematic axes with their own type of lateral determinate organs (integuments) and a model that considers carpels as analogs of complex leaves. In addition, we highlight the aspects of reproductive development that are likely to be highly labile and homoplastic, factors that have major implications for the understanding of seed plant relationships. Although these hypotheses may suggest that some long-standing interpretations are misleading, they also open up whole new avenues for comparative study and suggest concrete best practices for evolutionary analyses of development.Organismic and Evolutionary Biolog

Identification of Conserved Aquilegia Coerulea MicroRNAs and Their Targets

Aquilegia is an emerging model organism that is phylogenetically intermediate between the core eudicot and monocot models, Arabidopsis and Oryza. In this study, we have used a comparative genomics approach to identify 45 Aquilegia microRNAs that comprise 20 separate plant microRNA families. We have predicted 84 targets of these newly identified Aquilegia microRNAs including transcription factors and loci involved in metabolism, stress responses, transport, and auxin signaling. microRNA families from 16 plant species and the newly identified microRNAs from Aquilegia were analyzed in a phylogenetic context revealing 40 distantly conserved microRNA families. In addition to these highly conserved plant microRNA families, several families with disjointed phylogenetic distribution were identified. This study provides a phylogenetically important dataset for plant microRNA evolution studies. The current study is the first to identify miRNAs in a lower eudicot in which comprehensive genomic resources are becoming available.Organismic and Evolutionary Biolog

Conserved Roles for Polycomb Repressive Complex 2 in the Regulation of Lateral Organ Development in Aquilegia X Coerulea 'Origami'

Background: Epigenetic regulation is necessary for maintaining gene expression patterns in multicellular organisms. The Polycomb Group (PcG) proteins form several complexes with important and deeply conserved epigenetic functions in both the plant and animal kingdoms. One such complex, the Polycomb Repressive Complex 2 (PRC2), is critical to many developmental processes in plants including the regulation of major developmental transitions. In addition, PRC2 restricts the expression domain of various transcription factor families in Arabidopsis, including the class I KNOX genes and several of the ABCE class MADS box genes. While the functions of these transcription factors are known to be deeply conserved, whether or not their regulation by PRC2 is similarly conserved remains an open question. Results: Here we use virus-induced gene silencing (VIGS) to characterize the function of the PRC2 complex in lateral organ development of Aquilegia x coerulea 'Origami', a member of the lower eudicot order Ranunculales. Leaves with PRC2 down-regulation displayed a range of phenotypes including ruffled or curled laminae, additional lobing, and an increased frequency of higher order branching. Sepals and petals were also affected, being narrowed, distorted, or, in the case of the sepals, exhibiting partial homeotic transformation. Many of the petal limbs also had a particularly intense yellow coloration due to an accumulation of carotenoid pigments. We show that the A. x coerulea floral MADS box genes AGAMOUS1 (AqAG1), APETALA3-3 (AqAP3-3) and SEPALLATA3 (AqSEP3) are up-regulated in many tissues, while expression of the class I KNOX genes and several candidate genes involved in carotenoid production or degradation are largely unaffected. Conclusions: PRC2 targeting of several floral MADS box genes may be conserved in dicots, but other known targets do not appear to be. In the case of the type I KNOX genes, this may reflect a regulatory shift associated with the evolution of compound leaves.Organismic and Evolutionary Biolog

Characterization of Aquilegia Polycomb Repressive Complex 2 homologs reveals absence of imprinting

Epigenetic regulation is important for maintaining gene expression patterns in multicellular organisms. The Polycomb Group (PcG) proteins form several complexes with important and deeply conserved epigenetic functions in both the plant and animal kingdoms. The plant Polycomb Repressive Complex 2 (PRC2) contains four core proteins, Enhancer of Zeste (E(z)), Suppressor of Zeste 12 (Su(z)12), Extra Sex Combs (ESC), and Multicopy Suppressor of IRA 1 (MSI1), and functions in many developmental transitions. In some plant species, including rice and Arabidopsis, duplications in the core PRC2 proteins allow the formation of PRC2s with distinct developmental functions. In addition, members of the plant specific VEL PHD family have been shown to associate with the PRC2 complex in Arabidopsis and may play a role in targeting the PRC2 to specific loci. Here we examine the evolution and expression of the PRC2 and VEL PHD families in Aquilegia, a member of the lower eudicot order anunculales and an emerging model for the investigation of plant ecology, evolution and developmental genetics. We find that Aquilegia has a relatively simple PRC2 with only one homolog of Su(z)12, ESC and MSI1 and two ancient copies of E(z), AqSWN and AqCLF. Aquilegia has four members of the VEL PHD family, three of which appear to be closely related to Arabidopsis proteins known to associate with the PRC2. The PRC2 and VEL PHD family proteins are expressed at a relatively constant level throughout Aquilegia vulgaris development, with the VEL PHD family and MSI1 expressed at higher levels during and after vernalization and in the inflorescence. Both AqSWN and AqCLF are expressed in Aquilegia endosperm but neither copy is imprinted.Organismic and Evolutionary Biolog