Male to hermaphrodite sex change in gametophytes of the fern Ceratopteris richardii is affected by hermaphrodite density, male age, and male size

Gametophytes of the fern Ceratopteris richardii are male or hermaphrodite. The presence of hermaphrodite-secreted antheridiogen influences gametophytes to develop as male (induction), while its absence leads to hermaphrodite development. Males require the continued presence of antheridiogen to remain male. Removal of males from antheridiogen leads to sex change (conversion) of males to hermaphrodites. Density of hermaphrodites that males were exposed to and amount of time males were exposed to antheridiogen was experimentally manipulated to determine if conversion time was affected. Analysis of covariance was employed with male size serving as a covariate. Results suggest that males continuously sample the neighborhood for information regarding the density of hermaphrodites by perceiving hermaphrodite-secreted antheridiogen and use this information to determine whether to convert to hermaphrodite. Larger males converted faster than smaller males suggesting a potential for smaller males to remain male once larger males convert. Conversion time was longer in males grown in higher densities of hermaphrodites and shorter in males exposed to antheridiogen for a longer time, suggesting that males increase sensitivity to antheridiogen with higher doses but decrease sensitivity as they age. It is thought that such decision making is important for C. richardii populations to maintain beneficial sex ratios.

Conditional Stomatal Closure in a Fern Shares Molecular Features with Flowering Plant Active Stomatal Responses

Stomata evolved as plants transitioned from water to land, enabling carbon dioxide uptake and water loss to be controlled. In flowering plants, the most recently divergent land plant lineage, stomatal pores actively close in response to drought. In this response, the phytohormone abscisic acid (ABA) triggers signalling cascades that lead to ion and water loss in the guard cells of the stomatal complex, causing a reduction in turgor and pore closure. Whether this stimulus-response coupling pathway acts in other major land plant lineages is unclear, with some investigations reporting that stomatal closure involves ABA but others concluding that closure is passive. Here we show that in the model fern Ceratopteris richardii active stomatal closure is conditional on sensitisation by pre-exposure to either low humidity or exogenous ABA and is promoted by ABA. RNA-seq analysis and de novo transcriptome assembly reconstructed the protein coding complement of the C. richardii genome with coverage comparable to other plant models, enabling transcriptional signatures of stomatal sensitisation and closure to be identified. In both cases, changes in abundance of homologs of ABA, Ca2+ and ROS-related signalling components were observed, suggesting that the closure response pathway is conserved in ferns and flowering plants. These signatures further suggested that sensitisation is achieved by lowering the threshold required for a subsequent closure-inducing signal to trigger a response. We conclude that the canonical signalling network for active stomatal closure functioned in at least a rudimentary form in the stomata of the last common ancestor of ferns and flowering plants.Significance StatementStomata are valve-like pores that control the uptake of CO2 and the loss of water vapour in almost all land plants. In flowering plants, stomatal opening and closure is actively regulated by a stimulus-response coupling network. Whether active stomatal responses are present in other land plant lineages such as ferns has been hotly debated. Here we show that stomatal responses in the fern Ceratopteris richardii are active but depend on their past growth environment, and demonstrate that fern stomatal closure and sensitisation are associated with the altered expression of genes whose homologs function in the canonical stomatal regulatory network of flowering plants. Genetic pathways for active stomatal regulation therefore most likely evolved before the divergence of ferns and flowering plants.

Development and Cell Cycle Activity of the Root Apical Meristem in the Fern Ceratopteris richardii

Ferns are a representative clade in plant evolution although underestimated in the genomic era. Ceratopteris richardii is an emergent model for developmental processes in ferns, yet a complete scheme of the different growth stages is necessary. Here, we present a developmental analysis, at the tissue and cellular levels, of the first shoot-borne root of Ceratopteris. We followed early stages and emergence of the root meristem in sporelings. While assessing root growth, the first shoot-borne root ceases its elongation between the emergence of the fifth and sixth roots, suggesting Ceratopteris roots follow a determinate developmental program. We report cell division frequencies in the stem cell niche after detecting labeled nuclei in the root apical cell (RAC) and derivatives after 8 h of exposure. These results demonstrate the RAC has a continuous mitotic activity during root development. Detection of cell cycle activity in the RAC at early times suggests this cell acts as a non-quiescent organizing center. Overall, our results provide a framework to study root function and development in ferns and to better understand the evolutionary history of this organ.

Development and cell cycle dynamics of the root apical meristem in the fern Ceratopteris richardii

Ferns are a representative clade in plant evolution although underestimated in the genomic era. Ceratopteris richardii is an emergent model for developmental processes in ferns, yet a complete scheme of the different growth stages is necessary. Here, we present a developmental analysis, at the tissue and cellular levels, of the first shoot-borne root of Ceratopteris. We followed early stages and emergence of the root meristem in sporelings. While assessing root growth, the first shoot-borne root ceases its elongation between the emergence of the fifth and sixth roots, suggesting Ceratopteris roots follow a determinate developmental program. We report cell division frequencies in the stem cell niche after detecting labeled nuclei in the root apical cell (RAC) and derivatives after 8 hours of exposure. These results demonstrate the RAC has a continuous mitotic activity during root development. Detection of cell cycle activity in the RAC at early times suggests this cell acts as a non-quiescent organizing center. Overall, our results provide a framework to study root function and development in ferns and to better understand the evolutionary history of this organ.Summary StatementIn the Ceratopteris root, the apical cell and its derivatives have a high division frequency, suggesting the apical cell acts as a non-quiescent organizing center in the stem cell niche.

Molecular Evolution of Auxin-Mediated Root Initiation in Plants

The root originated independently in euphyllophytes (ferns and seed plants) and lycophytes; however, the molecular evolutionary route of root initiation remains elusive. By analyses of the fern Ceratopteris richardii and seed plants, here we show that the molecular pathway involving auxin, intermediate-clade WUSCHEL-RELATED HOMEOBOX (IC-WOX) genes, and WUSCHEL-clade WOX (WC-WOX) genes could be conserved in root initiation. We propose that the “auxin>IC-WOX>WC-WOX” module in root initiation might have arisen in the common ancestor of euphyllophytes during the second origin of roots, and that this module has further developed during the evolution of different root types in ferns and seed plants.

The C-Fern (Ceratopteris richardii) genome: insights into plant genome evolution with the first partial homosporous fern genome assembly

Ferns are notorious for possessing large genomes and numerous chromosomes. Despite decades of speculation, the processes underlying the expansive genomes of ferns are unclear, largely due to the absence of a sequenced homosporous fern genome. The lack of this crucial resource has not only hindered investigations of evolutionary processes responsible for the unusual genome characteristics of homosporous ferns, but also impeded synthesis of genome evolution across land plants. Here, we used the model fern species Ceratopteris richardii to address the processes (e.g., polyploidy, spread of repeat elements) by which the large genomes and high chromosome numbers typical of homosporous ferns may have evolved and have been maintained. We directly compared repeat compositions in species spanning the green plant tree of life and a diversity of genome sizes, as well as both short- and long-read-based assemblies of Ceratopteris. We found evidence consistent with a single ancient polyploidy event in the evolutionary history of Ceratopteris based on both genomic and cytogenetic data, and on repeat proportions similar to those found in large flowering plant genomes. This study provides a major stepping-stone in the understanding of land plant evolutionary genomics by providing the first homosporous fern reference genome, as well as insights into the processes underlying the formation of these massive genomes.