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.

Fitness of an allopolyploid rupicolous fern compared with its diploid progenitors: from sporogenesis to sporophyte formation.

PREMISE: When two populations of related cytotypes grow in sympatry, the rarer cytotype tends to be excluded due to a frequency-dependent mating disadvantage. Evolutionary models predict that polyploids, which are typically the rarer cytotype upon first formation, should have higher relative fitness and/or higher selfing rates to establish and then coexist with diploid parents. METHODS: Performance during early recruitment was compared among three co-occurring rupicolous fern species: the allotetraploid Cheilanthes tinaei and its diploid ancestors, C. hispanica and C. maderensis. In culture experiments, fresh spores and samples of soil spore banks were tested for variation among cytotypes in germination, survival, fecundity, and mating system of gametophytes. RESULTS: Compared with its diploid parents, C. tinaei fresh spores had higher abortion percentages, lower dispersal ability as a result of its larger spores, and similar vigor at germination. For gametophytes from soil spore banks, C. tinaei had high survival similar to C. maderensis, but its sex expression resembled that of C. hispanica, with a high proportion of males. Patterns of sporophyte formation by females and bisexuals indicate that the polyploid does not have an increased gametophytic selfing rate. Gametophytes were larger in C. tinaei, but its reproductive success (sporophyte formation) was intermediate relative to diploids. CONCLUSIONS: Our results show no evidence of higher selfing or fitness advantage of the allopolyploid over both diploid parents at any stage of early recruitment. These two unexpected findings suggest that further factors, such as niche differentiation, play a more important role in cytotype coexistence.

Comparative glycan profiling of Ceratopteris richardii 'C-Fern' gametophytes and sporophytes links cell-wall composition to functional specialization.

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.

RNA-seq analysis identifies potential modulators of gravity response in spores of Ceratopteris (Parkeriaceae): evidence for modulation by calcium pumps and apyrase activity.

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 nifedipine 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 identify 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 Ca(2+)-ATPase pumps and apyrases in the gravity response were further 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 Ca(2+)-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 number of calcium-responsive and light-receptive transcripts. Pharmacologic assays indicate endomembrane Ca(2+)-ATPases and extracellular nucleotides may play regulatory roles in the gravity response of Ceratopteris spores.

Gene expression changes induced by space flight in single-cells of the fern Ceratopteris richardii.

This work describes a rare high-throughput evaluation of gene expression changes induced by space flight in a single plant cell. The cell evaluated is the spore of the fern Ceratopteris richardii, which exhibits both perception and response to gravity. cDNA microarray and Q RT-PCR analysis of spores germinating in microgravity onboard NASA space shuttle flight STS-93 revealed changes in the mRNA expression of roughly 5% of genes analyzed. These gene expression changes were compared with gene expression changes that occur during gravity perception and response in animal cells and multicellular plants. Our data contribute to a better understanding of the impact of space flight conditions, including microgravity, on cellular growth and development, and provide insights into the adaptive strategies of individual cells in response to these conditions.

Nitric oxide and cGMP signaling in calcium-dependent development of cell polarity in Ceratopteris richardii.

Single-celled spores of the fern Ceratopteris richardii undergo gravity-directed cell polarity development that is driven by polar calcium currents. Here we present results that establish a role for nitric oxide (NO)/cGMP signaling in transducing the stimulus of gravity to directed polarization of the spores. Application of specific NO donors and scavengers inhibited the calcium-dependent gravity response in a dose-dependent manner. The effects of NO donor exposure were antagonized by application of NO scavenger compounds. Similarly, the guanylate cyclase inhibitors 6-anilino-5,8-quinolinedione and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin, and the phosphodiesterase inhibitor Viagra, which modulate NO-dependent cGMP levels in the cells, disrupted gravity-directed cell polarity in a dose-dependent manner. Viagra effects were antagonized by application of NO scavengers, consistent with the postulate that NO and cGMP are linked in the signaling pathway. To identify other components of the signaling system we analyzed gene expression changes induced by Viagra treatment using microarrays and quantitative real-time reverse transcription-polymerase chain reaction. Preliminary microarray analysis revealed several genes whose expression was significantly altered by Viagra treatment. Three of these genes had strong sequence similarity to key signal transduction or stress response genes and quantitative real-time reverse transcription-polymerase chain reaction was used to more rigorously quantify the effects of Viagra on their expression in spores and to test how closely these effects could be mimicked by treatment with dibutyryl cGMP. Taken together our results implicate NO and cGMP as downstream effectors that help link the gravity stimulus to polarized growth in C. richardii spores. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers BE 640669 to BE 643506, BQ 086920 to BQ 087668, and CV 734654 to CV 736151.

Developmental anatomy and auxin response of lateral root formation in Ceratopteris richardii.

The homosporous fern Ceratopteris richardii exhibits a homorhizic root system where roots originate from the shoot system. These shoot-borne roots form lateral roots (LRs) that arise from the endodermis adjacent to the xylem poles, which is in contrast to flowering plants where LR formation arises from cell division in the pericycle. A detailed study of the fifth shoot-borne root showed that one lateral root mother cell (LRMC) develops in each two out of three successive merophytes. As a result, LRs emerge alternately in two ranks from opposite positions on a parent root. From LRMC initiation to LR emergence, three developmental stages were identified based on anatomical criteria. The addition of auxins (either indole-3-acetic acid or indole-3-butyric acid) to the growth media did not induce additional LR formation, but exogenous applications of both auxins inhibited parent root growth rate. Application of the polar auxin-transport inhibitor N-(1-naphthyl)phthalamic acid (NPA) also inhibited parent root growth without changing the LR initiation pattern. The results suggest that LR formation does not depend on root growth rate per se. The result that exogenous auxins do not promote LR formation in C. richardii is similar to reports for certain species of flowering plants, in which there is an acropetal LR population and the formation of the LRs is insensitive to the application of auxins. It also may indicate that different mechanisms control LR development in non-seed vascular plants compared with angiosperms, taking into consideration the long and independent evolutionary history of the two groups.

External Ca(2+) is essential for chloroplast movement induced by mechanical stimulation but not by light stimulation.

In the fern Adiantum capillus-veneris, chloroplast movement is induced by mechanical stimulation as well as by light stimulation. Directional movement of both types depends on an actin-based motile system. To investigate the physiological relationship between mechanical and light signaling in the regulation of chloroplast movement, we examined the mechano-response of chloroplasts whose motility had been already restricted after photo-relocation. Chloroplast mechano-avoidance movement was induced under all of the photo-relocation conditions tested, indicating that mechano-specific signals generated by mechanical stimulation dominate over the light signals and reactivate the motility of chloroplasts. When the effects of external Ca(2+) on the induction of mechano- and light responses were examined, strikingly different requirements of external Ca(2+) were found for each. In medium without Ca(2+), the mechano-response was suppressed but no effects were observed on photo-response. Mechano-relocation movement of chloroplasts was inhibited by 100 microM lanthanum (La(3+)), a plasma membrane calcium channel blocker, and by 10 microM gadolinium (Gd(3+)), a stretch-activated channel blocker. However, the same concentrations of these drugs did not affect the photo-relocation movement at all. These results suggest that the influx of external Ca(2+) is crucial for the early signaling step of chloroplast mechano-relocation but not for that of photo-relocation. This is the first report showing the separation of signaling pathways in mechano- and photo-relocation of chloroplasts.