Control of leaf development in the water fern Ceratopteris richardii by the auxin efflux transporter CrPINMa in the CRISPR/Cas9 analysis.

BACKGROUND: PIN-FORMED genes (PINs) are crucial in plant development as they determine the directionality of auxin flow. They are present in almost all land plants and even in green algae. However, their role in fern development has not yet been determined. This study aims to investigate the function of CrPINMa in the quasi-model water fern Ceratopteris richardii. RESULTS: CrPINMa possessed a long central hydrophilic loop and characteristic motifs within it, which indicated that it belonged to the canonical rather than the non-canonical PINs. CrPINMa was positioned in the lineage leading to Arabidopsis PIN6 but not that to its PIN1, and it had undergone numerous gene duplications. CRISPR/Cas9 genome editing had been performed in ferns for the first time, producing diverse mutations including local frameshifts for CrPINMa. Plants possessing disrupted CrPINMa exhibited retarded leaf emergence and reduced leaf size though they could survive and reproduce at the same time. CrPINMa transcripts were distributed in the shoot apical meristem, leaf primordia and their vasculature. Finally, CrPINMa proteins were localized to the plasma membrane rather than other cell parts. CONCLUSIONS: CRISPR/Cas9 genome editing is feasible in ferns, and that PINs can play a role in fern leaf development.

A De Novo Transcriptome Assembly of Ceratopteris richardii Provides Insights into the Evolutionary Dynamics of Complex Gene Families in Land Plants.

As the closest extant sister group to seed plants, ferns are an important reference point to study the origin and evolution of plant genes and traits. One bottleneck to the use of ferns in phylogenetic and genetic studies is the fact that genome-level sequence information of this group is limited, due to the extreme genome sizes of most ferns. Ceratopteris richardii (hereafter Ceratopteris) has been widely used as a model system for ferns. In this study, we generated a transcriptome of Ceratopteris, through the de novo assembly of the RNA-seq data from 17 sequencing libraries that are derived from two sexual types of gametophytes and five different sporophyte tissues. The Ceratopteris transcriptome, together with 38 genomes and transcriptomes from other species across the Viridiplantae, were used to uncover the evolutionary dynamics of orthogroups (predicted gene families using OrthoFinder) within the euphyllophytes and identify proteins associated with the major shifts in plant morphology and physiology that occurred in the last common ancestors of euphyllophytes, ferns, and seed plants. Furthermore, this resource was used to identify and classify the GRAS domain transcriptional regulators of many developmental processes in plants. Through the phylogenetic analysis within each of the 15 GRAS orthogroups, we uncovered which GRAS family members are conserved or have diversified in ferns and seed plants. Taken together, the transcriptome database and analyses reported here provide an important platform for exploring the evolution of gene families in land plants and for studying gene function in seed-free vascular 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.

Tolerance mechanism of cadmium in Ceratopteris pteridoides: Translocation and subcellular distribution.

Hydroponic experiment was conducted to investigate the biochemical responses and accumulation behaviour of cadmium (Cd) in aquatic fern, Ceratopteris pteridoides, under four different levels of exposure. Plants were grown in 10 µM (CdT1), 20 µM (CdT2), 40 µM (CdT3) and 60 µM (CdT4) concentrations of Cd for 12 consecutive days and Cd accumulation in different plant parts, cell levels and growth medium was estimated. In C. pteridoides, Cd removal kinetics was best described by pseudo-second-order kinetic model. Increased accumulation of Cd in the plants was detected in a concentration dependent manner with maximum under 60 µM of Cd (CdT4) exposure (191.38 mg kg-1, 186.19 mg kg-1 and 1316.34 mg kg-1 in leaves, stems and roots, respectively). Cell wall of C. pteridoides is identified as crucial Cd storage site with the highest (28-69%) accumulation followed by organelles (14-44%) and soluble fraction (6-46%). Increased leaf proline, malondialdehyde (MDA) and protein content with significant reduction (P < 0.05) in chlorophyll concentration and upregulation of antioxidant enzymes catalase (CAT), guaiacol peroxidase (POD) and superoxide dismutase (SOD) reveals the presence of Cd resistance mechanism in C. pteridoides. Calculated higher (>1) bioconcentration factor (BCF) and lower (<1) translocation factor (TF) values evinced the suitability of C. pteridoides in Cd phytostabilization rather than phytoextraction.

Proteomic analysis of gametophytic sex expression in the fern Ceratopteris thalictroides.

Ceratopteris thalictroides, a model fern, has two kinds of gametophytes with different sex expression: male and hermaphrodite. Hermaphroditic gametophytes have one or several archegonia beneath the growing point and a few antheridia at the base or margin. Male gametophytes show a spoon-like shape with much longer than the width and produce many antheridia at the margin and surface. The results of chlorophyll fluorescence detection showed that the photochemical efficiency of hermaphrodites was higher than that of males. By using two-dimensional electrophoresis and mass spectrometry, the differentially abundant proteins in hermaphroditic and male gametophytes were identified. A total of 1136 ± 55 protein spots were detected in Coomassie-stained gels of proteins from hermaphroditic gametophytes, and 1130 ± 65 spots were detected in gels of proteins from male gametophytes. After annotation, 33 spots representing differentially abundant proteins were identified. Among these, proteins involved in photosynthesis and chaperone proteins were over-represented in hermaphrodites, whereas several proteins involved in metabolism were increased in male gametophytes in order to maintain their development under relatively nutritionally deficient conditions. Furthermore, the differentially abundant cytoskeletal proteins detected in this study, such as centrin and actin, may be involved in the formation of sexual organs and are directly related to sex expression. These differentially abundant proteins are important for maintaining the development of gametophytes of different sexes in C. thalictroides.

Detection of chlorophylls in spores of seven ferns.

Fern spores were traditionally classified into chlorophyllous (green) and nonchlorophyllous (nongreen) types based on the color visible to the naked eye. Recently, a third type, "cryptochlorophyllous spores", is recognized, and these spores are nongreen under white light but contain chlorophylls. Epifluorescence microscopy was previously used to detect chlorophylls in cryptochlorophyllous spores. In addition to epifluorescence microscopy, current study performed some other approaches, including spore-squash epifluorescence, absorption spectra, laser-induced fluorescence emission spectra, thin layer chromatography (TLC), and ultra-high performance liquid chromatography with ultraviolet and mass spectrometric detection (UHPLC-UV-MS) in order to detect chlorophylls of spores of seven ferns (Sphaeropteris lepifera, Ceratopteris thalictroides, Leptochilus wrightii, Leptochilus pothifolius, Lepidomicrosorum buergerianum, Osmunda banksiifolia, and Platycerium grande). Destructive methods, such as TLC and UHPLC-UV-MS, successfully detected chlorophylls inside the spores when their signals of red fluorescence under epifluorescence microscope were masked by spore wall. Although UHPLC-UV-MS analysis was the most sensitive and reliable for determining the chlorophylls of spores, spore-squash epifluorescence is not only reliable but also cost- and time-effective one among our study methods. In addition, we first confirmed that Lepidomicrosorium buergerianum, Leptochilus pothifolius, Leptochilus wrightii, and Platycerium grande, produce cryptochlorophyllous spores.

Arabinogalactan proteins and arabinan pectins abound in the specialized matrices surrounding female gametes of the fern Ceratopteris richardii.

MAIN CONCLUSION: Both male and female gametes of archegoniates are highly specialized cells surrounded by an extraprotoplasmic matrix rich in AGPs, which are speculated to facilitate development and gamete fusion through Ca 2+) oscillations. An additional layer, the egg envelope, forms around the egg periphery, except at the fertilization pore, and contains arabinose-rich polymers that presumably impart flexibility for the rapidly growing zygote and embryo. The abundant AGPs and arabinan pectins associated with the eggs of C. richardii not only are integral to development, fertilization, and early embryogenesis, but also may be involved in desiccation tolerance important to the survival of the reproductive gametophyte. A defining feature of gametogenesis in archegoniates is the deposition of a special matrix outside of the plasmalemma of both egg and sperm cells that displaces the primary cell wall away from the protoplasm. It is within this matrix that gamete differentiation occurs. In leptosporangiate ferns, maturation of the egg cell involves the deposition of a second specialized wall, the so-called egg envelope that surrounds the cell except at the fertilization pore, a narrow site where gamete fusion takes place. We provide the first conclusive evidence of the macromolecular constituents in the unique structures surrounding fern egg cells before and after fertilization. To test the hypotheses that the egg extracellular matrix contains arabinogalactan proteins (AGPs) as does the sperm cell matrix, and that cell wall polysaccharides, especially pectins, are components of the egg envelope, we examined the expression patterns of AGPs and cell wall constituents during oogenesis in Ceratopteris richardii. Utilizing histochemical stains for callose, cellulose and AGPs coupled with immunogold localizations employing a suite of monoclonal antibodies to cell wall components (JIM13, JIM8, LM2, LM5, LM6, LM19, LM20 and anticallose), we demonstrate that AGPs, but not pectins, are abundant in the matrix around egg cells and degrading neck canal and ventral canal cells during archegonial development. A striking finding is that both AGPs and (1,5)-α-L-arabinan pectin epitopes are principle components of the egg envelope before and after fertilization, suggesting that they are important in both egg maturation and gamete fusion.

Arsenic-induced responses in Pityrogramma calomelanos (L.) Link: Arsenic speciation, mineral nutrition and antioxidant defenses.

Arsenic (As) hyperaccumulation trait has been described in a limited number of fern species. The physiological basis of hyperaccumulation remains unclear, especially in non-Pteris species such as Pityrogramma calomelanos. Aiming at a better understanding of As-induced responses, P. calomelanos plants were exposed to 1 mM As for 21 days and compared with control plants. Chemical analyses revealed that As accumulation was ten times higher in pinnae then in roots and stipes. In pinnae, As was present mainly as arsenite, whereas arsenate was the dominant form in stipes and roots. Arsenic promoted an increase in antioxidant enzyme activities in both fern parts and several alterations in mineral nutrition, especially with regard to P and K. A higher content of non-protein thiols was observed in pinnae of plants exposed to As, whereas As induced the increase in lipid peroxidation in roots. The results showed that Pityrogramma calomelanos shares with Pteris vittata several aspects of As metabolism. High root-shoot As translocation showed to be essential to avoid toxic effects in roots, since the root is more sensitive to the metalloid. The higher capacity of P. calomelanos to sequester arsenite in the pinna and its efficient antioxidant system maintain the reactive oxygen species at a low level, thus enhancing the continuous accumulation of As. Molecular investigations are needed to elucidate the evolution of As-tolerance mechanisms in Pteridaceae species, especially with regard to membrane transporters and ROS signaling.

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.

A preliminary study on the phytoremediation of antibiotic contaminated sediment.

In Vietnam's coastal wetlands, fluoroquinolones, a widely used class of antibiotics in shrimp farming, are frequently detected in sediments of former shrimp farms. This phenomenon could lead to negative impacts on the aquatic ecosystem, since the antibiotic residues could induce changes in the microorganism communities of the water body. The potential of native wetland plants (Acrostichum aureum L. and Rhizophora apiculata Blume Fl. Javae) for phytoremediation of fluoroquinolones (ciprofloxacin and norfloxacin) was investigated. The half-life for each antibiotic was estimated at approximately 10 days in the planted sediment. With respect to the accumulation of ciprofloxacin and norfloxacin in plants, these antibiotics were found mainly in roots. Antibiotic translocation from root to stem and leaves occurred at a low rate. The results showed that A. aureum and R. apiculata can be valuable for the phytoremediation of antibiotic-contaminated sediments. Additionally, the initialfindings of the presence of resistant bacteria indicated that bacteria could play a role in facilitating the phytodegradation.

Natural revegetation of alkaline tailing heaps at Taxco, Guerrero, Mexico.

The aim of this research was to identify adapted native plant species with potential for use in phytoremediation of a metalliferous mine tailings heap in Guerrero, Mexico. Physicochemical characterization, total, DTPA-extractable and fractionation of metals in rhizospheric and non-rhizopheric samples were carried out to gain information about their potential risks. Metal concentrations in plant and bioconcentration factors (BCF) were also determined. Organic matter (OM) and total N contents were higher in the rhizospheric samples, which could improve the conditions for plant establishment. Total Cu, Zn, and Pb concentration were above those for normal soils. The highest metals concentration was found in the residual and organic fractions. Eleven plant species were recorded at the site; three behaved as metal accumulator plants: Gnaphalium chartaceum (accumulator of Cu, Mn, Zn, and Ph), Wigandia urens and Senecio salignus (1027 and 2477 mg kg(-1) of Zn). These species and Brickellia sp. presented high Pb-BCF; they may be suitable for metals phytoextraction. Seven species behaved as excluder plants; Guardiola tulocarpus, Juniperus flaccida, and Ficus goldmanii, presented low BCFs. These species are well suited to cope with the toxic conditions, and they could be propagated for revegetation and stabilization of these residues and to decrease metal bioavailability.

Changes in gravity rapidly alter the magnitude and direction of a cellular calcium current.

In single-celled spores of the fern Ceratopteris richardii, gravity directs polarity of development and induces a directional, trans-cellular calcium (Ca(2+)) current. To clarify how gravity polarizes this electrophysiological process, we measured the kinetics of the cellular response to changes in the gravity vector, which we initially estimated using the self-referencing calcium microsensor. In order to generate more precise and detailed data, we developed a silicon microfabricated sensor array which facilitated a lab-on-a-chip approach to simultaneously measure calcium currents from multiple cells in real time. These experiments revealed that the direction of the gravity-dependent polar calcium current is reversed in less than 25 s when the cells are inverted, and that changes in the magnitude of the calcium current parallel rapidly changing g-forces during parabolic flight on the NASA C-9 aircraft. The data also revealed a hysteresis in the response of cells in the transition from 2g to micro-g in comparison to cells in the micro-g to 2-g transition, a result consistent with a role for mechanosensitive ion channels in the gravity response. The calcium current is suppressed by either nifedipine (calcium-channel blocker) or eosin yellow (plasma membrane calcium pump inhibitor). Nifedipine disrupts gravity-directed cell polarity, but not spore germination. These results indicate that gravity perception in single plant cells may be mediated by mechanosensitive calcium channels, an idea consistent with some previously proposed models of plant gravity perception.

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.

Exposure to the fern Onychium contiguum causes increase in lipid peroxidation and alters antioxidant status in urinary bladder.

The status of lipid peroxidation, glutathione, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, superoxide dismutase, catalase, ascorbic acid, and alpha-tocopherol was studied in the urinary bladder of guinea pigs exposed to the carcinogenic fern Onychium contiguum. There was significant increase in the preformed lipid peroxides in the urinary bladders from fern exposed animals. The amount of lipid peroxides produced on incubation of urinary bladder homogenates with or without catalyst was significantly higher in the fern exposed animals. The concentrations of glutathione and alpha-tocopherol and the activities of glutathione reductase and catalase were elevated in the urinary bladders of the animals exposed to the fern. No effect was observed on the concentration of ascorbic acid and the activities of glutathione peroxidase, glutathione-S-transferase, and superoxide dismutase. It is summarized that the fern toxins increased oxidative stress in the urinary bladder and antioxidant status was altered. However, the altered antioxidant status did not provide protection from the toxin induced injury. Histopathology of the urinary bladder in the fern exposed animals revealed oedema, haemorrhages, and congestion. This is the first study to show increase in lipid peroxidation along with altered antioxidant status in the urinary bladder of fern exposed animals.

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.