Quantitative live imaging of floral organ initiation and floral meristem termination in Aquilegia.

In-depth investigation of any developmental process in plants requires knowledge of both the underpinning molecular networks and how they directly determine patterns of cell division and expansion over time. Floral meristems (FMs) produce floral organs, after which they undergo floral meristem termination (FMT); precise control of organ initiation and FMT is crucial to the reproductive success of any flowering plant. Using live confocal imaging, we characterized developmental dynamics during floral organ primordia initiation and FMT in Aquilegia coerulea (Ranunculaceae). Our results uncover distinct patterns of primordium initiation between stamens and staminodes compared with carpels, and provide insight into the process of FMT, which is discernable based on cell division dynamics that precede carpel initiation. To our knowledge, this is the first quantitative live imaging of meristem development in a system with numerous whorls of floral organs, as well as an apocarpous gynoecium. This study provides crucial information for our understanding of how the spatial-temporal regulation of floral meristem behavior is achieved in both evolutionary and developmental contexts. This article has an associated 'The people behind the papers' interview.

An ontogenetic framework for functional studies in the model fern Ceratopteris richardii.

As the sister group to seed plants, ferns are a phylogenetically informative lineage. Functional studies in representatives of the fern lineage are helping bridge the knowledge gap in developmental mechanisms between angiosperms and non-vascular plants. The fern life cycle has the advantage of combining a sizable free-living haploid gametophyte, more amenable for developmental studies than the reduced seed plant gametophyte, with an indeterminate and complex diploid sporophyte. Ceratopteris richardii has long been proposed as a model fern and has recently become tractable due to stable transgenesis and increasing genomic resources, allowing researchers to test explicit questions about gene function in a fern for the first time. As with any model system, a detailed understanding of wild-type morphology and a staged ontogeny are indispensable for the characterization of mutant phenotypes resulting from genetic manipulations. Therefore, the goal of this study is to provide a unified reference ontogeny for this emerging model fern as a tool for comparative evolutionary and developmental studies. It complements earlier research by filling gaps in major stages of development of the haploid gametophyte and diploid sporophyte generations, and provides additional descriptions of the shoot apical meristem and early leaf development. This resource is meant to facilitate not only studies of candidate genes within C. richardii, but also broader ontogenetic comparisons to other model plants.

Brassinosteroids regulate petal spur length in Aquilegia by controlling cell elongation.

BACKGROUND AND AIMS: Aquilegia produce elongated, three-dimensional petal spurs that fill with nectar to attract pollinators. Previous studies have shown that the diversity of spur length across the Aquilegia genus is a key innovation that is tightly linked with its recent and rapid diversification into new ranges, and that evolution of increased spur lengths is achieved via anisotropic cell elongation. Previous work identified a brassinosteroid response transcription factor as being enriched in the early developing spur cup. Brassinosteroids are known to be important for cell elongation, suggesting that brassinosteroid-mediated response may be an important regulator of spur elongation and potentially a driver of spur length diversity in Aquilegia. In this study, we investigated the role of brassinosteroids in the development of the Aquilegia coerulea petal spur. METHODS: We exogenously applied the biologically active brassinosteroid brassinolide to developing petal spurs to investigate spur growth under high hormone conditions. We used virus-induced gene silencing and gene expression experiments to understand the function of brassinosteroid-related transcription factors in A. coerulea petal spurs. KEY RESULTS: We identified a total of three Aquilegia homologues of the BES1/BZR1 protein family and found that these genes are ubiquitously expressed in all floral tissues during development, yet, consistent with the previous RNAseq study, we found that two of these paralogues are enriched in early developing petals. Exogenously applied brassinosteroid increased petal spur length due to increased anisotropic cell elongation as well as cell division. We found that targeting of the AqBEH genes with virus-induced gene silencing resulted in shortened petals, a phenotype caused in part by a loss of cell anisotropy. CONCLUSIONS: Collectively, our results support a role for brassinosteroids in anisotropic cell expansion in Aquilegia petal spurs and highlight the brassinosteroid pathway as a potential player in the diversification of petal spur length in Aquilegia.

Evolution of the YABBY gene family in seed plants.

Members of the YABBY gene family of transcription factors in angiosperms have been shown to be involved in the initiation of outgrowth of the lamina, the maintenance of polarity, and establishment of the leaf margin. Although most of the dorsal-ventral polarity genes in seed plants have homologs in non-spermatophyte lineages, the presence of YABBY genes is restricted to seed plants. To gain insight into the origin and diversification of this gene family, we reconstructed the evolutionary history of YABBY gene lineages in seed plants. Our findings suggest that either one or two YABBY genes were present in the last common ancestor of extant seed plants. We also examined the expression of YABBY genes in the gymnosperms Ephedra distachya (Gnetales), Ginkgo biloba (Ginkgoales), and Pseudotsuga menziesii (Coniferales). Our data indicate that some YABBY genes are expressed in a polar (abaxial) manner in leaves and female cones in gymnosperms. We propose that YABBY genes already acted as polarity genes in the last common ancestor of extant seed plants.

Origin of a novel regulatory module by duplication and degeneration of an ancient plant transcription factor.

It is commonly believed that gene duplications provide the raw material for morphological evolution. Both the number of genes and size of gene families have increased during the diversification of land plants. Several small proteins that regulate transcription factors have recently been identified in plants, including the LITTLE ZIPPER (ZPR) proteins. ZPRs are post-translational negative regulators, via heterodimerization, of class III Homeodomain Leucine Zipper (C3HDZ) proteins that play a key role in directing plant form and growth. We show that ZPR genes originated as a duplication of a C3HDZ transcription factor paralog in the common ancestor of euphyllophytes (ferns and seed plants). The ZPRs evolved by degenerative mutations resulting in loss all of the C3HDZ functional domains, except the leucine zipper that modulates dimerization. ZPRs represent a novel regulatory module of the C3HDZ network unique to the euphyllophyte lineage, and their origin correlates to a period of rapid morphological changes and increased complexity in land plants. The origin of the ZPRs illustrates the significance of gene duplications in creating developmental complexity during land plant evolution that likely led to morphological evolution.

Quantitative Live Confocal Imaging in AquilegiaFloral Meristems.

In this study, we present a detailed protocol for live imaging and quantitative analysis of floral meristem development in Aquilegia coerulea, a member of the buttercup family (Ranunculaceae). Using confocal microscopy and the image analysis software MorphoGraphX, we were able to examine the cellular growth dynamics during floral organ primordia initiation, and the transition from floral meristem proliferation to termination. This protocol provides a powerful tool to study the development of the meristem and floral organ primordia, and should be easily adaptable to many plant lineages, including other emerging model systems. It will allow researchers to explore questions outside the scope of common model systems.

LEAFY maintains apical stem cell activity during shoot development in the fern Ceratopteris richardii.

During land plant evolution, determinate spore-bearing axes (retained in extant bryophytes such as mosses) were progressively transformed into indeterminate branching shoots with specialized reproductive axes that form flowers. The LEAFY transcription factor, which is required for the first zygotic cell division in mosses and primarily for floral meristem identity in flowering plants, may have facilitated developmental innovations during these transitions. Mapping the LEAFY evolutionary trajectory has been challenging, however, because there is no functional overlap between mosses and flowering plants, and no functional data from intervening lineages. Here, we report a transgenic analysis in the fern Ceratopteris richardii that reveals a role for LEAFY in maintaining cell divisions in the apical stem cells of both haploid and diploid phases of the lifecycle. These results support an evolutionary trajectory in which an ancestral LEAFY module that promotes cell proliferation was progressively co-opted, adapted and specialized as novel shoot developmental contexts emerged.