Ferns the missing link in shoot evolution and development

Shoot development in land plants is a remarkably complex process that gives rise to an extreme diversity of forms. Our current understanding of shoot developmental mechanisms comes almost entirely from studies of angiosperms (flowering plants), the most recently diverged plant lineage. Shoot development in angiosperms is based around a layered multicellular apical meristem that produces lateral organs and/or secondary meristems from populations of founder cells at its periphery. In contrast, nonseed plant shoots develop from either single apical initials or from a small population of morphologically distinct apical cells. Although developmental and molecular information is becoming available for non-flowering plants, such as the model moss Physcomitrella patens, making valid comparisons between highly divergent lineages is extremely challenging. As sister group to the seed plants, the monilophytes (ferns and relatives) represent an excellent phylogenetic midpoint of comparison for unlocking the evolution of shoot developmental mechanisms, and recent technical advances have finally made transgenic analysis possible in the emerging model fern Ceratopteris richardii. This review compares and contrasts our current understanding of shoot development in different land plant lineages with the aim of highlighting the potential role that the fern C. richardii could play in shedding light on the evolution of underlying genetic regulatory mechanisms

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 inferred. 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

Protocol: genetic transformation of the fern Ceratopteris richardii through microparticle bombardment

BACKGROUND: The inability to genetically transform any fern species has been a major technical barrier to unlocking fern biology. Initial attempts to overcome this limitation were based on transient transformation approaches or achieved very low efficiencies. A highly efficient method of stable transformation was recently reported using the fern Ceratopteris richardii, in which particle bombardment of callus tissue achieved transformation efficiencies of up to 72%. As such, this transformation method represents a highly desirable research tool for groups wishing to undertake fern genetic analysis. RESULTS: We detail an updated and optimized protocol for transformation of C. richardii by particle bombardment, including all necessary ancillary protocols for successful growth and propagation of this species in a laboratory environment. The C. richardii lifecycle comprises separate, free-living gametophyte and sporophyte stages. Callus is induced from the sporophyte apex through growth on cytokinin-containing tissue culture medium and can be maintained indefinitely by sub-culturing. Transgene DNA is introduced into callus cells through particle bombardment, and stable genomic integration events are selected by regeneration and growth of T(0) sporophytes for a period of 8 weeks on medium containing antibiotics. Selection of T(1) transgenic progeny is accomplished through screening T(1) gametophytes for antibiotic resistance. In many cases sexual reproduction and development of transgenic embryos requires growth and fertilization of gametophytes in the absence of antibiotics, followed by a separate screen for antibiotic resistance in the resultant sporophyte generation. CONCLUSIONS: Genetic transformation of C. richardii using this protocol was found to be robust under a broad range of bombardment and recovery conditions. The successful expansion of the selection toolkit to include a second antibiotic for resistance screening (G-418) and different resistance marker promoters increases the scope of transformations possible using this technique and offers the prospect of more complex analysis, for example the creation of lines carrying more than one transgene. The introduction of a robust and practicable transformation technique is a very important milestone in the field of fern biology, and its successful implementation in C. richardii paves the way for adoption of this species as the first fern genetic model. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13007-015-0080-8) contains supplementary material, which is available to authorized users