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Background and Aims In flowering plants, regeneration can be achieved by a variety of approaches, and different sets of transcriptional factors are involved in these processes. However, regeneration in taxa other than flowering plants remains a mystery. Ceratopteris richardii is a representative fern capable of both direct and indirect organogenesis, and we aimed to investigate the genetics underlying the transition from callus proliferation to differentiation. Methods Morphological and histological analyses were used to determine the type of regeneration involved. RNA sequencing and differential gene expression were used to investigate how the callus switches from proliferation to differentiation. Phylogenetic analysis and RNA in situ hybridization were used to understand whether transcriptional factors are involved in this transition. Key Results The callus formed on nascent leaves and subsequently developed the shoot pro-meristem and shoot meristem, thus completing indirect de novo shoot organogenesis in C. richardii. Genes were differentially expressed during the callus transition from proliferation to differentiation, indicating a role for photosynthesis, stimulus response and transmembrane signalling in this transition and the involvement of almost all cell layers that make up the callus. Transcriptional factors were either downregulated or upregulated, which were generally in many-to-many orthology with genes known to be involved in callus development in flowering plants, suggesting that the genetics of fern callus development are both conserved and divergent. Among them, an STM -like, a PLT -like and an ethylene- and salt-inducible ERF gene3 -like gene were expressed simultaneously in the vasculature but not in the other parts of the callus, indicating that the vasculature played a role in the callus transition from proliferation to differentiation. Conclusions Indirect de novo shoot organogenesis could occur in ferns, and the callus transition from proliferation to differentiation required physiological changes, differential expression of transcriptional factors and involvement of the vasculature. [ABSTRACT FROM AUTHOR]

SUMMARY: Significant changes have occurred in plant cell wall composition during evolution and diversification of tracheophytes. As the sister lineage to seed plants, knowledge on the cell wall of ferns is key to track evolutionary changes across tracheophytes and to understand seed plant‐specific evolutionary innovations. Fern cell wall composition is not fully understood, including limited knowledge of glycoproteins such as the fern arabinogalactan proteins (AGPs). Here, we characterize the AGPs from the leptosporangiate fern genera Azolla, Salvinia, and Ceratopteris. The carbohydrate moiety of seed plant AGPs consists of a galactan backbone including mainly 1,3‐ and 1,3,6‐linked pyranosidic galactose, which is conserved across the investigated fern AGPs. Yet, unlike AGPs of angiosperms, those of ferns contained the unusual sugar 3‐O‐methylrhamnose. Besides terminal furanosidic arabinose, Ara (Araf), the main linkage type of Araf in the ferns was 1,2‐linked Araf, whereas in seed plants 1,5‐linked Araf is often dominating. Antibodies directed against carbohydrate epitopes of AGPs supported the structural differences between AGPs of ferns and seed plants. Comparison of AGP linkage types across the streptophyte lineage showed that angiosperms have rather conserved monosaccharide linkage types; by contrast bryophytes, ferns, and gymnosperms showed more variability. Phylogenetic analyses of glycosyltransferases involved in AGP biosynthesis and bioinformatic search for AGP protein backbones revealed a versatile genetic toolkit for AGP complexity in ferns. Our data reveal important differences across AGP diversity of which the functional significance is unknown. This diversity sheds light on the evolution of the hallmark feature of tracheophytes: their elaborate cell walls. [ABSTRACT FROM AUTHOR]

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) system allows precise and easy editing of genes in many plant species. However, this system has not yet been applied to any fern species through gametophytes due to the complex characteristics of fern genomes, genetics, and physiology. Here, we established a protocol for gametophyte-based screening of single-guide RNAs (sgRNAs) with high efficiency for CRISPR/Cas9-mediated gene knockout in a model fern species, Ceratopteris richardii. We utilized the C. richardii ACTIN promoter to drive sgRNA expression and the enhanced CaMV 35S promoter to drive the expression of Streptococcus pyogenes Cas9 in this CRISPR-mediated editing system, which was employed to successfully edit a few genes, such as Nucleotidase/phosphatase 1 (CrSAL1) and Phytoene Desaturase (CrPDS), which resulted in an albino phenotype in C. richardii. Knockout of CrSAL1 resulted in significantly (P<0.05) reduced stomatal conductance (gs), leaf transpiration rate (E), guard cell length, and abscisic acid (ABA)-induced reactive oxygen species (ROS) accumulation in guard cells. Moreover, CrSAL1 overexpressing plants showed significantly increased net photosynthetic rate (A), gs, and E as well as most of the stomatal traits and ABA-induced ROS production in guard cells compared to the wild-type (WT) plants. Taken together, our optimized CRISPR/Cas9 system provides a useful tool for functional genomics in a model fern species, allowing the exploration of fern gene functions for evolutionary biology, herbal medicine discovery, and agricultural applications., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Gene expression in roots has been assessed in different plant species in studies ranging from complete organs to specific cell layers, and more recently at the single cell level. While certain genes or functional categories are expressed in the root of all or most plant species, lineage-specific genes have also been discovered. An increasing amount of transcriptomic data is available for angiosperms, while a limited amount of data is available for ferns, and few studies have focused on fern roots. Here, we present a de novo transcriptome assembly from three different parts of the Ceratopteris richardii young sporophyte. Differential gene expression analysis of the root tip transcriptional program showed an enrichment of functional categories related to histogenesis and cell division, indicating an active apical meristem. Analysis of a diverse set of orthologous genes revealed conserved expression in the root meristem, suggesting a preserved role for different developmental roles in this tissue, including stem cell maintenance. The reconstruction of evolutionary trajectories for ground tissue specification genes suggests a high degree of conservation in vascular plants, but not for genes involved in root cap development, showing that certain genes are absent in Ceratopteris or have intricate evolutionary paths difficult to track. Overall, our results suggest different processes of conservation and divergence of genes involved in root development. [ABSTRACT FROM AUTHOR]

Phytohormones play an important role in the adaptive evolution of terrestrial plants. Brassinosteroids (BRs) are essential hormones that regulate multiple aspects of plant growth and development in angiosperms, but the presence of BR signaling in non-seed plants such as ferns remains unknown. Here, we found that BR promotes the growth of Ceratopteris richardii, while the synthetic inhibitor PCZ inhibits the growth. Using full-length transcriptome sequencing, we identified four BRI1-like receptors. By constructing chimeric receptors, we found that the kinase domains of these four receptors could trigger BR downstream signaling. Further, the extracellular domains of two receptors were functionally interchangeable with that of BRI1. In addition, we identified a co-receptor, CtSERK1, that could phosphorylate with CtBRL2s in vitro. Together, these proved the presence of a receptor complex in Ceratopteris richardii that might perceive BR and activate downstream hormone signaling. Our results shed light on the biological and molecular mechanisms of BR signaling in ferns and the role of BR hormone signaling in the adaptive evolution of terrestrial plants. [ABSTRACT FROM AUTHOR]

Gene expression in roots has been assessed in different plant species in studies ranging from complete organs to specific cell layers, and more recently at the single cell level. While certain genes or functional categories are expressed in the root of all or most plant species, lineage-specific genes have also been discovered. An increasing amount of transcriptomic data is available for angiosperms, while a limited amount of data is available for ferns, and few studies have focused on fern roots. Here, we present a de novo transcriptome assembly from three different parts of the Ceratopteris richardii young sporophyte. Differential gene expression analysis of the root tip transcriptional program showed an enrichment of functional categories related to histogenesis and cell division, indicating an active apical meristem. Analysis of a diverse set of orthologous genes revealed conserved expression in the root meristem, suggesting a preserved role for different developmental roles in this tissue, including stem cell maintenance. The reconstruction of evolutionary trajectories for ground tissue specification genes suggests a high degree of conservation in vascular plants, but not for genes involved in root cap development, showing that certain genes are absent in Ceratopteris or have intricate evolutionary paths difficult to track. Overall, our results suggest different processes of conservation and divergence of genes involved in root development.

The fern Ceratopteris richardii has been studied as a model organism for over 50 years because it is easy to grow and has a short life cycle. In particular, as the first homosporous vascular plant for which genomic resources were developed, C. richardii has been an important system for studying plant evolution. However, we know relatively little about the natural history of C. richardii. In this article, we summarize what is known about this aspect of C. richardii, and discuss how learning more about its natural history could greatly increase our understanding of the evolution of land plants.

Sequence-specific cytidine to uridine (C-to-U) and adenosine to inosine editing tools can alter RNA and DNA sequences and utilize a hydrolytic deamination mechanism requiring an active site zinc ion and a glutamate residue. In plant organelles, DYW-PG domain containing enzymes catalyze C-to-U edits through the canonical deamination mechanism. Proteins developed from consensus sequences of the related DYW-KP domain family catalyze what initially appeared to be uridine to cytidine (U-to-C) edits leading to this investigation into the U-to-C editing mechanism. The synthetic DYW-KP enzyme KP6 was found sufficient for C-to-U editing activity stimulated by the addition of carboxylic acids in vitro. Despite addition of putative amine/amide donors, U-to-C editing by KP6 could not be observed in vitro. C-to-U editing was found not to be concomitant with U-to-C editing, discounting a pyrimidine transaminase mechanism. RNAs containing base modifications were highly enriched in interphase fractions consistent with covalent crosslinks to KP6, KP2, and KP3 proteins. Mass spectrometry of purified KP2 and KP6 proteins revealed secondary peaks with mass shifts of 319 Da. A U-to-C crosslinking mechanism was projected to explain the link between crosslinking, RNA base changes, and the ∼319 Da mass. In this model, an enzymatic lysine attacks C4 of uridine to form a Schiff base RNA-protein conjugate. Sequenced RT-PCR products from the fern Ceratopteris richardii indicate U-to-C base edits do not preserve proteinaceous crosslinks in planta. Hydrolysis of a protonated Schiff base conjugate releasing cytidine is hypothesized to explain the completed pathway in plants., Competing Interests: Conflict of interests The authors declare no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The alternation of generations in land plants occurs between the sporophyte phase and the gametophyte phase. The sporophytes of seed plants develop self-maintained, multicellular meristems, and these meristems determine plant architecture. The gametophytes of seed plants lack meristems and are heterotrophic. In contrast, the gametophytes of seed-free vascular plants, including ferns, are autotrophic and free-living, developing meristems to sustain their independent growth and proliferation. Compared with meristems in the sporophytes of seed plants, the cellular mechanisms underlying meristem development in fern gametophytes remain largely unknown. Here, using confocal time-lapse live imaging and computational segmentation and quantification, we determined different patterns of cell divisions associated with the initiation and proliferation of two distinct types of meristems in gametophytes of two closely related Pteridaceae ferns, Pteris vittata and Ceratopteris richardii. Our results reveal how the simple timing of a switch between two meristems has considerable consequences for the divergent gametophyte morphologies of the two ferns. They further provide evolutionary insight into the function and regulation of gametophyte meristems in seed-free vascular plants. [ABSTRACT FROM AUTHOR]

Abstract Background Post-embryonic growth of land plants originates from meristems. Genetic networks in meristems maintain the stem cells and direct acquisition of cell fates. WUSCHEL-RELATED HOMEOBOX (WOX) transcription factors involved in meristem networks have only been functionally characterized in two evolutionarily distant taxa, mosses and seed plants. This report characterizes a WOX gene in a fern, which is located phylogenetically between the two taxa. Results CrWOXB transcripts were detected in proliferating tissues, including gametophyte and sporophyte meristems of Ceratopteris richardii. In addition, CrWOXB is expressed in archegonia but not the antheridia of gametophytes. Suppression of CrWOXB expression in wild-type RN3 plants by RNAi produced abnormal morphologies of gametophytes and sporophytes. The gametophytes of RNAi lines produced fewer cells, and fewer female gametes compared to wild-type. In the sporophyte generation, RNAi lines produced fewer leaves, pinnae, roots and lateral roots compared to wild-type sporophytes. Conclusions Our results suggest that CrWOXB functions to promote cell divisions and organ development in the gametophyte and sporophyte generations, respectively. CrWOXB is the first intermediate-clade WOX gene shown to function in both generations in land plants.

Phytohormones play an important role in the adaptive evolution of terrestrial plants. Brassinosteroids (BRs) are essential hormones that regulate multiple aspects of plant growth and development in angiosperms, but the presence of BR signaling in non-seed plants such as ferns remains unknown. Here, we found that BR promotes the growth of Ceratopteris richardii, while the synthetic inhibitor PCZ inhibits the growth. Using full-length transcriptome sequencing, we identified four BRI1-like receptors. By constructing chimeric receptors, we found that the kinase domains of these four receptors could trigger BR downstream signaling. Further, the extracellular domains of two receptors were functionally interchangeable with that of BRI1. In addition, we identified a co-receptor, CtSERK1, that could phosphorylate with CtBRL2s in vitro. Together, these proved the presence of a receptor complex in Ceratopteris richardii that might perceive BR and activate downstream hormone signaling. Our results shed light on the biological and molecular mechanisms of BR signaling in ferns and the role of BR hormone signaling in the adaptive evolution of terrestrial 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. [ABSTRACT FROM AUTHOR]

Abstract Ceratopteris richardii is a model fern species widely used to analyze various developmental processes and their regulation in gametophytes. The form of mature C. richardii gametophytes depends on the activity of the marginal meristem, but knowledge on meristem formation and structure is limited. Therefore, we analyzed cellular events accompanying the development of gametophytes using cell lineage and proliferation analyses to explain the establishment and functioning of the marginal meristem. We show that: i) gametophytes are devoid of the apical initial cell or the apical cell-based meristem in the early developmental stages; ii) the cells that are predestined to form the marginal meristem divide according to a stable pattern; iii) only one transient initial cell is present in the marginal meristem, and the selection of a new functioning initial cell is related to a stable sequence of its divisions. Our results contribute to a better understanding of the developmental events underlying gametophyte growth and marginal meristem functioning in Ceratopteris. The principles, which were established in this study and enabled the identification of functioning initial cells, can be applied to analyze genetic and/or physiological mechanism(s) governing meristem maintenance in vascular plants, both in developmental and evolutionary contexts. Highlights • Ceratopteris gametophyte growth does not relate to the initial cell activity in early development. • The cells which form the marginal meristem divide according to a stable pattern. • Only one transient initial cell is present in the marginal meristem. • Selection of a new initial cell is related to a stable sequence of its divisions. [ABSTRACT FROM AUTHOR]

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.

Abstract: Resource allocation plasticity enables individuals to alter patterns of nutrient use between reproductive and vegetative output to better fit their current environment. In sexually labile plant species, abiotic environmental factors can influence expression of dimorphic gender, resulting in environmental sex determination (ESD), which potentially reduces the need for plasticity of resource allocation by preemptively matching an individual’s future nutrient demands to resource availability in its location. Ceratopteris richardii gametophytes exhibit gender‐dependent differences in relative carbon and nitrogen content, and ESD in certain nutrient environments. This study examined whether prior ESD in C. richardii gametophyte populations reduced subsequent plasticity of reproductive allocation compared to instances where no ESD occurred, by quantifying phenotypic responses to reduced P, N, or CO2 availabilities. All three nutrient‐limited environments resulted in decreased size of egg‐bearing (meristic) gametophytes compared to nonlimited environments, but gametophytes failed to respond to N and CO2 limitation at the time of sex determination, resulting in no ESD. N limitation resulted in a predictable allometric re‐allocation of resources based on small gametophyte size, whereas CO2 limitation caused a change in reproductive output consistent with true plasticity. Withholding exogenous P caused ESD and had no effect on relative reproductive output of resultant meristic gametophytes because the size decrease was minor. Under P limitation, ESD matched the resource demands of gender phenotypes to their environment before the onset of developmental dimorphism, reducing the need for large allocation adjustments after sex determination. [ABSTRACT FROM AUTHOR]

Class I KNOTTED- LIKE HOMEOBOX ( KNOX) proteins regulate development of the multicellular diploid sporophyte in both mosses and flowering plants; however, the morphological context in which they function differs., In order to determine how Class I KNOX function was modified as land plants evolved, phylogenetic analyses and cross-species complementation assays were performed., Our data reveal that a duplication within the charophyte sister group to land plants led to distinct Class I and Class II KNOX gene families. Subsequently, Class I sequences diverged substantially in the nonvascular bryophyte groups (liverworts, mosses and hornworts), with moss sequences being most similar to those in vascular plants. Despite this similarity, moss mutants were not complemented by vascular plant KNOX genes. Conversely, the Arabidopsis brevipedicellus ( bp-9) mutant was complemented by the Pp MKN2 gene from the moss Physcomitrella patens. Lycophyte KNOX genes also complemented bp-9 whereas fern genes only partially complemented the mutant. This lycophyte/fern distinction is mirrored in the phylogeny of KNOX-interacting BELL proteins, in that a gene duplication occurred after divergence of the two groups., Together, our results imply that the moss MKN2 protein can function in a broader developmental context than vascular plant KNOX proteins, the narrower scope having evolved progressively as lycophytes, ferns and flowering plants diverged. [ABSTRACT FROM AUTHOR]

Asexual reproduction is widespread in land plants, including ferns where 10% of all species are obligate asexuals. In these ferns, apogamous sporophytes are generated directly from gametophytes, bypassing fertilization. In the model fern Ceratopteris richardii, a sexual species, apogamy can be induced by culture on high sugar media. BABY BOOM ( BBM) genes in angiosperms are known to promote somatic embryogenesis, which like apogamy produce sporophytes without fertilization. Here, a Brassica napus BBM ( Bn BBM) was used to investigate genetic similarity between apogamy in ferns and somatic embryogenesis in angiosperms. A C. richardii transcriptome was constructed from which one AINTEGUMENTA- LIKE unigene, Cr ANT, was identified. Whole mount in situ hybridization showed that Cr ANT is expressed in sperm and fertilized eggs. Phylogenetic analysis grouped Cr ANT with other non-seed-plant ANT genes to the eu ANT clade but in a branch separate from BBM genes. Overexpression of Cr ANT or Bn BBM promotes apogamy in C. richardii without sugar supplement. Cr ANT knockdown gametophytes responded weakly to sugar for apogamy promotion. Theses results suggest some genetic conservation between apogamy and somatic embryogenesis and that such asexual reproduction may be ancient. [ABSTRACT FROM AUTHOR]

The prothalli of the fern Ceratopteris richardii exhibit negative gravitropism when grown in darkness. However, no sedimentable organelles or substances have been detected in the prothallial cells, suggesting that a non-sedimentable gravisensor exists. We investigated whether chloroplasts are involved in the gravisensing system of C. richardii prothalli. We used a clumped-chloroplast mutant, clumped chloroplast 1 ( cp1), in which the chloroplasts are detached from the plasma membrane and clustered around the nucleus likely because of a partial deletion in the KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 gene. The cp1 mutation resulted in prothalli that had a significantly diminished gravitropic response, while the phototropic response occurred normally. These results suggest that plasma membrane-anchored chloroplasts in prothallial cells function as one of the gravisensors in C. richardii prothalli. [ABSTRACT FROM AUTHOR]

Sexual reproduction in animals and plants shares common elements, including sperm and egg production, but unlike animals, little is known about the regulatory pathways that determine the sex of plants. Here we use mutants and gene silencing in a fern species to identify a core regulatory mechanism in plant sexual differentiation. A key player in fern sex differentiation is the phytohormone abscisic acid (ABA), which regulates the sex ratio of male to hermaphrodite tissues during the reproductive cycle. Our analysis shows that in the fern Ceratopteris richardii, a gene homologous to core ABA transduction genes in flowering plants [SNF1-related kinase2s (SnRK2s)] is primarily responsible for the hormonal control of sex determination. Furthermore, we provide evidence that this ABA-SnRK2 signaling pathway has transitioned from determining the sex of ferns to controlling seed dormancy in the earliest seed plants before being co-opted to control transpiration and CO2 exchange in derived seed plants. By tracing the evolutionary history of this ABA signaling pathway from plant reproduction through to its role in the global regulation of plant-atmosphere gas exchange during the last 450 million years, we highlight the extraordinary effect of the ABA-SnRK2 signaling pathway in plant evolution and vegetation function. [ABSTRACT FROM AUTHOR]

When habitats are heterogeneous regarding key abiotic factors, and individual organisms have no control over the environment in which they develop, labile sex expression can allow individuals to adjust their sex based on local environmental conditions, resulting in increased individual fitness. Sexual lability is found extensively in homosporous ferns, where sex expression is often regulated via the pheromone antheridiogen. Nutrient availability may provide additional signals for sex determination in fern gametophytes, particularly if nutrient demands required for sexual development differ by sex. The model fern Ceratopteris richardii Brongn. has a well-characterized antheridiogen response and short time to sexual maturity. Although tests for nutrient effects on sex determination have been conducted in this fern, tests for differences in nutrient demands by sex have not. Elemental analysis demonstrated that 14-day-old ameristic male and meristic female or hermaphrodite gametophytes of C. richardii differ significantly in their relative carbon and nitrogen masses, resulting in significantly dissimilar C:N ratios between the sexes. Average gametophyte dry mass in ameristic males was approximately half that of meristic plants of the same age, and contained less N than meristic gametophytes in both relative and absolute terms. Those characteristic differences in elemental composition imply that variation in nutrient availability could potentially influence sex expression in C. richardii gametophyte populations, rather than regulation of sex determination by the antheridiogen system alone. [ABSTRACT FROM AUTHOR]

Carbon (C), nitrogen (N), and phosphorous (P) are needed by all organisms for basic biological processes. When an individual macronutrient is not accessible, nutrient limitation occurs. The stochiometric balance between multiple nutrients and individual concentrations are both vital for normal growth and development. Labile sex expression in plants is a phenotypic trait predicted to be sensitive to local nutrient conditions because males and females differ in their nutritional demands. We applied concepts from ecological stoichiometry to assess the effects of variation in individual nutrient concentration and multiple macronutrient stoichiometry on sexual development in the fern Ceratopteris richardii Brongn. Manipulation of N, P, and organic and inorganic C was expected to yield variation in the ratio of males to females, consistent with environmental sex-determination theory. Our results suggest nutrient stoichiometry, not strictly concentration, influences sex determination at ambient CO2. However, an early response to population density preempted nutrient effects in elevated CO2 environments with exogenous glucose, in which C. richardii gametophytes presumably grow naturally. Although sex determination is not nutrient-dependent in the latter environment, C:N in the dry mass of meristic gametophytes is influenced by the external nutrient context, suggesting sex determination takes place before abiotic environmental factors subsequently influence plant nutrient uptake. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 T0 sporophytes for a period of 8 weeks on medium containing antibiotics. Selection of T1 transgenic progeny is accomplished through screening T1 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. [ABSTRACT FROM AUTHOR]

The development of male Ceratopteris richardii Brongn. gametophytes (induction) is well studied. Males develop in response to hermaphrodite-produced antheridiogen, which coincides with induced expression of ANI1 (antheridiogen-induced 1). Induction is blocked by exogenous abscisic acid (ABA). Conversion of male gametophytes to hermaphrodites occurs when males are removed from antheridiogen and is a less understood process. In this study, males were exposed to ABA at 0 μmol·L−1, 1 μmol·L−1, 10 μmol·L−1, 100 μmol·L−1, and 300 μmol·L−1 and monitored for conversion. RT-qPCR methodology was developed to examine ANI1 expression levels in gametophytes outside of induction. Conversion of male gametophytes occurred more often and more quickly in media lacking ABA, and at lower ABA concentrations than at higher concentrations. ANI1 expression dropped significantly in males transferred to media lacking antheridiogen, but remained high in males remaining in the presence of antheridiogen and in those transferred to ABA. ANI1 expression was higher in 24 day old hermaphrodites relative to 24 day old males and at comparable expression levels to 6 day old gametophytes. These results suggest ABA serves a distinct role in conversion compared with induction, and ANI1 is expressed throughout the male's lifespan and in older hermaphrodites. [ABSTRACT FROM AUTHOR]

Premise of the study: According to Cope's 'law of the unspecialized' highly dependent species interactions are 'evolutionary dead ends,' prone to extinction because reversion to more generalist interactions is thought to be unlikely. Cases of extreme specialization, such as those seen between obligate mutualists, are cast as evolutionarily inescapable, inevitably leading to extinction rather than diversification of participating species. The pollination mutualism between Yucca plants and yucca moths (Tegeticula and Parategeticula) would seem to be locked into such an obligate mutualism. Yucca aloifolia populations, however, can produce large numbers of fruit lacking moth oviposition scars. Here, we investigate the pollination ecology of Y. aloifolia, in search of the non-moth pollination of a Yucca species. • Methods: We perform pollinator exclusion studies on Yucca aloifolia and a sympatric yucca species, Y. filamentosa. We then perform postvisit exclusion treatments, an analysis of dissected fruits, and a fluorescent dye transfer experiment. • Key results: As expected, Yucca filamentosa plants set fruit only when inflorescences were exposed to crepuscular and nocturnal pollinating yucca moths. In contrast, good fruit set was observed when pollinators were excluded from Y. aloifolia inflorescences from dusk to dawn, and nofruit set was observed when pollinators were excluded during the day. Follow up experiments indicated that European honeybees (Apis mellifera) were passively yet effectively pollinating Y. aloifolia flowers. • Conclusions: These results indicate that even highly specialized mutualisms may not be entirely obligate interactions or evolutionary dead ends. [ABSTRACT FROM AUTHOR]

Background and Aims Innovations in vegetative and reproductive characters were key factors in the evolutionary history of land plants and most of these transformations, including dramatic changes in life cycle structure and strategy, necessarily involved cell-wall modifications. To provide more insight into the role of cell walls in effecting changes in plant structure and function, and in particular their role in the generation of vascularization, an antibody-based approach was implemented to compare the presence and distribution of cell-wall glycan epitopes between (free-living) gametophytes and sporophytes of Ceratopteris richardii ‘C-Fern’, a widely used model system for ferns. Methods Microarrays of sequential diamino-cyclohexane-tetraacetic acid (CDTA) and NaOH extractions of gametophytes, spores and different organs of ‘C-Fern’ sporophytes were probed with glycan-directed monoclonal antibodies. The same probes were employed to investigate the tissue- and cell-specific distribution of glycan epitopes. Key Results While monoclonal antibodies against pectic homogalacturonan, mannan and xyloglucan widely labelled gametophytic and sporophytic tissues, xylans were only detected in secondary cell walls of the sporophyte. The LM5 pectic galactan epitope was restricted to sporophytic phloem tissue. Rhizoids and root hairs showed similarities in arabinogalactan protein (AGP) and xyloglucan epitope distribution patterns. Conclusions The differences and similarities in glycan cell-wall composition between ‘C-Fern’ gametophytes and sporophytes indicate that the molecular design of cell walls reflects functional specialization rather than genetic origin. Glycan epitopes that were not detected in gametophytes were associated with cell walls of specialized tissues in the sporophyte. [ABSTRACT FROM PUBLISHER]

Background Although plants and many algae (e.g. the Phaeophyceae, brown, and Rhodophyceae, red) are only very distantly related they are united in their possession of carbohydrate-rich cell walls, which are of integral importance being involved in many physiological processes. Furthermore, wall components have applications within food, fuel, pharmaceuticals, fibres (e.g. for textiles and paper) and building materials and have long been an active topic of research. As shown in the 27 papers in this Special Issue, as the major deposit of photosynthetically fixed carbon, and therefore energy investment, cell walls are of undisputed importance to the organisms that possess them, the photosynthetic eukaryotes (plants and algae). The complexities of cell wall components along with their interactions with the biotic and abiotic environment are becoming increasingly revealed. Scope The importance of plant and algal cell walls and their individual components to the function and survival of the organism, and for a number of industrial applications, are illustrated by the breadth of topics covered in this issue, which includes papers concentrating on various plants and algae, developmental stages, organs, cell wall components, and techniques. Although we acknowledge that there are many alternative ways in which the papers could be categorized (and many would fit within several topics), we have organized them as follows: (1) cell wall biosynthesis and remodelling, (2) cell wall diversity, and (3) application of new technologies to cell walls. Finally, we will consider future directions within plant cell wall research. Expansion of the industrial uses of cell walls and potentially novel uses of cell wall components are both avenues likely to direct future research activities. Fundamentally, it is the continued progression from characterization (structure, metabolism, properties and localization) of individual cell wall components through to defining their roles in almost every aspect of plant and algal physiology that will present many of the major challenges in future cell wall research. [ABSTRACT FROM PUBLISHER]

Ferns represent the most closely related extant lineage to seed plants. The aquatic fern Ceratopteris richardii has been subject to research for a considerable period of time, but analyses of the genetic programs underpinning developmental processes have been hampered by a large genome size, a lack of available mutants, and an inability to create stable transgenic lines. In this paper, we report a protocol for efficient stable genetic transformation of C. richardii and a closely related species Ceratopteris thalictroides using microparticle bombardment. Indeterminate callus was generated and maintained from the sporophytes of both species using cytokinin treatment. In proof-of-principle experiments, a 35S::β-glucuronidase (GUS) expression cassette was introduced into callus cells via tungsten microparticles, and stable transformants were selected via a linked hygromycin B resistance marker. The presence of the transgene in regenerated plants and in subsequent generations was validated using DNA-blot analysis, reverse transcription-polymerase chain reaction, and GUS staining. GUS staining patterns in most vegetative tissues corresponded with constitutive gene expression. The protocol described in this paper yields transformation efficiencies far greater than those previously published and represents a significant step toward the establishment of a tractable fern genetic model. [ABSTRACT FROM AUTHOR]

Plant cell walls are essential for most aspects of plant growth, development, and survival, including cell division, expansive cell growth, cell-cell communication, biomechanical properties, and stress responses. Therefore, characterizing cell wall diversity contributes to our overall understanding of plant evolution and development. Recent biochemical analyses, concomitantly with whole genome sequencing of plants located at pivotal points in plant phylogeny, have helped distinguish between homologous characters and those which might be more derived. Most plant lineages now have at least one fully sequenced representative and although genome sequences for fern species areinprogress they are not yet available for this group. Ferns offer key advantages for the study of developmental processes leading to vascularisation and complex organs as well as the specific differences between diploid sporophyte tissues and haploid gametophyte tissues and the interplay between them. Ceratopteris richardii has been well investigated building a body of knowledge which combined with the genomic and biochemical information available for other plants will progress our understanding of wall diversity and its impact on evolution and development. [ABSTRACT FROM AUTHOR]

* Premise of the study: Gravity regulates the magnitude and direction of a trans-cell calcium current in germinating spores of Ceratopteris richardii. Blocking this current with nitedipine blocks the spore's downward polarity alignment, a polarization that is fixed by gravity -10 h after light induces the spores to germinate. RNA-seq analysis at 10 h was used to identity genes potentially important for the gravity response. The data set will be valuable for other developmental and phylogenetic studies. * Methods: De novo Newbler assembly of 958 527 reads from Roche 454 sequencing was executed. The sequences were identified and analyzed using in silico methods. The roles of endomembrane Ca2+-ATPase pumps and apyrases in the gravity response were fnrther tested using pharmacological agents. * Key results: Transcripts related to calcium signaling and ethylene biosynthesis were identified as notable constituents of the transcriptome. Inhibiting the activity of endomembrane Ca2+-ATPase pumps with 2.5-di-(t-butyl)-1,4-hydroquinone diminished the trans-cell current, but increased the orientation of the polar axis to gravity. The effects of applied nucleotides and purinoceptor antagonists gave novel evidence implicating extracellular nucleotides as regulators of the gravity response in these fern spores. * Conclusions: In addition to revealing general features of the transcriptome of germinating spores, the results highlight a num- ber of calcium-responsive and light-receptive transcripts. Pharmacologic assays indicate endomembrane Ca2+-ATPases and extracellular nucleotides may play regulatory roles in the gravity response of Ceratopteris spores. [ABSTRACT FROM AUTHOR]

Apogamy is a phenomenon in which a sporophyte develops asexually, directly from a cell or cells of a gametophyte. It is a phenomenon described mainly in lower plants, but shares certain aspects with apomixis in angiosperms. The genes involved in apogamy commitment in ferns are unknown. We hypothesize that the mechanism of asexual reproduction is controlled in lower and higher plants by overlapping sets of genes. To this end, we created a normalized subtracted cDNA library that represents genes with increased expression during apogamy commitment in the fern Ceratopteris richardii. The cDNA library consists of 170 unique sequences. Compared to the mature gametophyte transcriptome of the fern Pteridium aquilinum, the apogamy library is enriched in plant GO-Slim terms that are associated with stress and metabolism. In silico expression analyses of the closest Arabidopsis homologs of the apogamy library revealed many genes that display preferential expression in seed and flower tissues, structures that are absent in ferns. This apogamy library provides a rich resource for investigations into the genetic control of apogamy in ferns and comparisons with the asexual processes of higher plants. [ABSTRACT FROM AUTHOR]

This study examined whether gravity influences the growth direction of dark-grown gametophytes of the fern Ceratopteris richardii. Analyses of directional growth of gametophytes in response to gravitropic stimulation demonstrated that gametophytes showed negative gravitropism. Dark-grown gametophytes of dkgl herl mutants, which germinate in complete darkness, displayed a more distinct negative gravitropism. Unlike herl spores, dkgl herl spores do riot require light irradiation to induce spore germination. Therefore, light irradiation on herl spores was possibly inhibiting the negative gravitropism of herl gametophytes. In the present study, prolonged white-light irradiation on herl spores inhibited negative gravitropism in the gametophytes. Light irradiation on spores therefore affects the later negative gravitropism of dark-grown gametophytes. [ABSTRACT FROM AUTHOR]

In many plants females invest more in reproduction than males. In organisms that exhibit environmental sex determination, individuals in low-quality environments or who are slow growing are expected to develop into males. The gametophytes of Brongn., a homosporous fern, may develop as males or hermaphrodites. Hermaphrodites secrete a pheromone called antheridiogen that induces undifferentiated spores to develop as males. Given that induction is not 100% in the presence of antheridiogen, it is hypothesized that resources may alter C. richardii gender decisions. An experiment was undertaken to determine ( i) whether spore size predicts gender, ( ii) whether spore size predicts gametophyte size, ( iii) whether antheridiogen negatively affects the growth of C. richardii, and ( iv) whether wild-type C. richardii and him1 mutants (genetic mutants disposed to male development regardless of antheridiogen presence) behave similarly in their response to antheridiogen. Spore size was not predictive of gender but was positively related to both male and hermaphrodite gametophyte size. Antheridiogen was found to slow the growth of male and hermaphrodite gametophytes of the wild type and male gametophytes of the him1 mutant. These results are supportive of the idea that gender may be determined indirectly through antheridiogen's effect on gametophyte growth. [ABSTRACT FROM AUTHOR]

Understanding how green sink strength is regulated in planta poses a difficult problem because non-structural carbohydrate (NSC) levels can have integrated, simultaneous feedback effects on photosynthesis, sugar uptake, and respiration that depend on specific NSC moieties. Photosynthetic gametophytes of the fern Ceratopteris richardii provide a simple land plant model to assess how different NSCs imported from the apoplast of intact plants affect green sink strength. Sink strength was quantified as the amount of exogenous sugar that plants grown in low light depleted from their liquid media, and the relative contributions of carbon assimilation by photosynthesis and sugar uptake was estimated from stable isotope analysis of plant dry mass. Gametophytes absorbed fructose and glucose with equal affinity when cultured on either hexose alone, or in the presence of an equimolar blend of both sugars. Plants also depleted sucrose from the surrounding media, although a portion of this disaccharide that was hydrolysed into fructose and glucose by putative cell wall invertase activity remained in the media. The δ13C in plant dry masses harvested from sugar treatments were all close to –18‰, indicating that 25–39% of total plant carbon was from C3 photosynthesis (δ13C=–29‰) and 61–75% was from uptake of exogenous sugars (δ13C=–11‰). Carbon-use efficiency (i.e. carbon accumulated/carbon depleted) was significantly improved when plants had a blend of exogenous sugars available compared with plants grown in a single hexose alone. Plants avoided complete down-regulation of photosynthesis even though a large excess of exogenous carbon fluxed through their cells. [ABSTRACT FROM PUBLISHER]

The article presents a study which determines the effects of nutrient level to the induction of male gametophyte through antheridiogen in Ceratopteris richardii. The Analysis of Covariance (ANCOVA) was performed to determine the relationship between the density of gametophytes to the hermaphroditic gametophyte and the effect of varying nutrient level to this relationship. The study reveals that the nutrient levels have no significant effect on the male induction rate by means of antheridiogen.

Fern gametophytes are extremely shade-tolerant, potentially existing for long periods under conditions of extreme light limitation. Many previous studies have demonstrated an increase in gametophyte growth and incidence of spontaneous transition to sporophyte morphology (apogamy) under culture on media containing exogenous sugar. However, these studies did not verify sugar uptake or quantify relative growth on media containing different sugar types. Here, we examine the extent of heterotrophy and underlying mechanisms of sugar transport in photosynthetic gametophytes of the fern Ceratopteris richardii Brongn. Exogenous sugar uptake, growth, and sugar transport were evaluated with assays of exogenous glucose depletion, experimental culture of gametophytes under different sugar and light conditions, and bioinformatic approaches. The glucose from the growth media was significantly depleted by gametophytes growing under all conditions, especially those in the dark compared with those exposed to higher light. Gametophyte area increased similarly when cultured on equimolar concentrations of either glucose or the disaccharide sucrose, likely due to preferential uptake of one of the monomers of sucrose. Although at least one gene with similarity to sucrose transporters is expressed in germinating spores, our results suggest a reliance on monosaccharide transport for exogenous sugar uptake. Glucose assimilation in both light and dark conditions constitutes nutritional opportunism and may enhance gametophyte survival in very low light. [ABSTRACT FROM AUTHOR]