N-terminal region is required for functions of the HAM family member.

Shoot meristems contain stem cells, and they sustain growth and development of the above-ground tissues in land plants. The HAIRY MERISTEM (HAM) family genes, encoding GRAS-domain transcriptional regulators, play essential roles in the control of shoot meristem development and stem cell homeostasis in several flowering plants. Similar to other GRAS proteins, the C-terminal regions of HAM family proteins across land plants are conserved, containing signature motifs that define the GRAS domain. In contrast, the N-terminal regions of HAM family proteins display substantial divergence in sequence and length. Whether the variable and divergent N-termini are required for the conserved functions of HAM proteins is unknown. Our recent work showed that CrHAM - the HAM homolog in the fern Ceratopteris richardii was able to replace the role of type-II HAM genes in Arabidopsis, maintaining established shoot apical meristems and promoting the initiation of new stem cell niches. Here, we provide additional information and show that CrHAM contains a much longer N-terminal region compared to Arabidopsis HAM proteins, which is conserved among different fern HAM homologs. The deletion of this region largely compromises the ability of CrHAM to replace the function of Arabidopsis HAM proteins in shoot meristems. These new data together with previous results suggest that, although lacking the sequence conservation among HAM homologs from different plant lineages, the N-termini are important for the conserved functions of HAM family proteins.

Positive root pressure is critical for whole-plant desiccation recovery in two species of terrestrial resurrection ferns.

Desiccation-tolerant (DT) organisms can lose nearly all their water without dying. Desiccation tolerance allows organisms to survive in a nearly completely dehydrated, dormant state. At the cellular level, sugars and proteins stabilize cellular components and protect them from oxidative damage. However, there are few studies of the dynamics and drivers of whole-plant recovery in vascular DT plants. In vascular DT plants, whole-plant desiccation recovery (resurrection) depends not only on cellular rehydration, but also on the recovery of organs with unequal access to water. In this study, in situ natural and artificial irrigation experiments revealed the dynamics of desiccation recovery in two DT fern species. Organ-specific irrigation experiments revealed that the entire plant resurrected when water was supplied to roots, but leaf hydration alone (foliar water uptake) was insufficient to rehydrate the stele and roots. In both species, pressure applied to petioles of excised desiccated fronds resurrected distal leaf tissue, while capillarity alone was insufficient to resurrect distal pinnules. Upon rehydration, sucrose levels in the rhizome and stele dropped dramatically as starch levels rose, consistent with the role of accumulated sucrose as a desiccation protectant. These findings provide insight into traits that facilitate desiccation recovery in dryland ferns associated with chaparral vegetation of southern California.

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.

Field Survey and Comparative Study of Pteris Vittata and Pityrogramma Calomelanos Grown on Arsenic Contaminated Lands with Different Soil pH.

In field survey, Pteris vittata and Pityrogramma calomelanos were only found in arsenic (As) contaminated areas with soil pH 7.2-8.8 and 2.3-4.2, respectively. In the first pot experiment, two fern species were grown on the soil amended with 300 mg kg-1 As at soil pH of 5.1, 7.2 and 9. P. calomelanos survived all pH treatments, and had the highest frond As concentration and soil As removal efficiency at soil pH 5.1. All P. vittata plants were dead at soil pH 5.1. P. vittata had higher frond As concentration, biomass and the amount of As removed from the soil than those of P. calomelanos at soil pH of 7.2 and 9. In the second pot experiment, P. vittata was demonstrated to have greater life time, biomass, As tolerance and accumulation than those of P. calomelanos as planted on alkaline soil (pH 7.8) spiked with various concentrations of As.

Differences in dehydration tolerance among populations of a gametophyte-only fern.

PREMISE OF THE STUDY: For many plant species, historical climatic conditions may have left lasting imprints that are detectable in contemporary populations. Additionally, if these historical conditions also prevented gene flow among populations, these populations may be differentiated with respect to one another and their contemporary environmental conditions. For the fern, Vittaria appalachiana, one theory is that historical conditions during the Pleistocene largely shaped both the distribution and lack of sporophyte production. Our goals-based on this theory-were to examine physiological differences among and within populations spanning the species' geographic range, and the contribution of historical climatic conditions to this differentiation. METHODS: We exposed explants from five populations to four drying treatments and examined differences in physiological response. Additionally, we examined the role of historical and current climatic conditions in driving the observed population differentiation. KEY RESULTS: Populations differ in their ability to tolerate varying levels of dehydration, displaying a pattern of countergradient selection. Exposure to historical and contemporary climatic conditions, specifically variation in temperature and precipitation regimes, resulted in population divergence observed among contemporary populations. CONCLUSIONS: Historical conditions have shaped not only the distribution of V. appalachiana, but also its current physiological limitations. Results from this study support the hypothesis that climatic conditions during the Pleistocene are responsible for the distribution of this species, and may be responsible for the observed differences in dehydration tolerance. Additionally, dehydration tolerance may be the driving factor for previously reported patterns of countergradient selection in this species.

Plasma membrane-anchored chloroplasts are necessary for the gravisensing system of Ceratopteris richardii prothalli.

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.

Not only in the temperate zone: independent gametophytes of two vittarioid ferns (Pteridaceae, Polypodiales) in East Asian subtropics.

Independent gametophyte ferns are unique among vascular plants because they are sporophyteless and reproduce asexually to maintain their populations in the gametophyte generation. Such ferns had been primarily discovered in temperate zone, and usually hypothesized with (sub)tropical origins and subsequent extinction of sporophyte due to climate change during glaciations. Presumably, independent fern gametophytes are unlikely to be distributed in tropics and subtropics because of relatively stable climates which are less affected by glaciations. Nonetheless, the current study presents cases of two independent gametophyte fern species in subtropic East Asia. In this study, we applied plastid DNA sequences (trnL-L-F and matK + ndhF + chlL datasets) and comprehensive sampling (~80%) of congeneric species for molecular identification and divergence time estimation of these independent fern gametophytes. The two independent gametophyte ferns were found belonging to genus Haplopteris (vittarioids, Pteridaceae) and no genetic identical sporophyte species in East Asia. For one species, divergence times between its populations imply recent oversea dispersal(s) by spores occurred during Pleistocene. By examining their ex situ and in situ fertility, prezygotic sterility was found in these two Haplopteris, in which gametangia were not or very seldom observed, and this prezygotic sterility might attribute to their lacks of functional sporophytes. Our field observation and survey on their habitats suggest microhabitat conditions might attribute to this prezygotic sterility. These findings point to consideration of whether recent climate change during the Pleistocene glaciation resulted in ecophysiological maladaptation of non-temperate independent gametophyte ferns. In addition, we provided a new definition to classify fern gametophyte independences at the population level. We expect that continued investigations into tropical and subtropical fern gametophyte floras will further illustrate the biogeographic significance of non-temperate fern gametophyte independence.

Arsenic hyperaccumulation induces metabolic reprogramming in Pityrogramma calomelanos to reduce oxidative stress.

Arsenic (As) pollution is a major environmental concern due to its worldwide distribution and high toxicity to organisms. The fern Pityrogramma calomelanos is one of the few plant species known to be able to hyperaccumulate As, although the mechanisms involved are largely unknown. This study aimed to investigate the metabolic adjustments involved in the As-tolerance of P. calomelanos. For this purpose, ferns with five to seven fronds were exposed to a series of As concentrations. Young fronds were used for biochemical analysis and metabolite profiling using gas chromatography-mass spectrometry. As treatment increased the total concentration of proteins and soluble phenols, enhanced peroxidase activities, and promoted disturbances in nitrogen and carbon metabolism. The reduction of the glucose pool was one of the striking responses to As. Remarkable changes in amino acids levels were observed in As-treated plants, including those related to biosynthesis of glutathione and phenols, osmoregulation and two photorespiratory intermediates. In addition, increases in polyamines levels and antioxidant enzyme activities were observed. In summary, this study indicates that P. calomelanos tolerates high concentration of As due to its capacity to upregulate biosynthesis of amino acids and antioxidants, without greatly disturbing central carbon metabolism. At extremely high As concentrations, however, this protective mechanism fails to block reactive oxygen species production, leading to lipid peroxidation and leaf necrosis.

Sporangium Exposure and Spore Release in the Peruvian Maidenhair Fern (Adiantum peruvianum, Pteridaceae).

We investigated the different processes involved in spore liberation in the polypod fern Adiantum peruvianum (Pteridaceae). Sporangia are being produced on the undersides of so-called false indusia, which are situated at the abaxial surface of the pinnule margins, and become exposed by a desiccation-induced movement of these pinnule flaps. The complex folding kinematics and functional morphology of false indusia are being described, and we discuss scenarios of movement initiation and passive hydraulic actuation of these structures. High-speed cinematography allowed for analyses of fast sporangium motion and for tracking ejected spores. Separation and liberation of spores from the sporangia are induced by relaxation of the annulus (the 'throwing arm' of the sporangium catapult) and conservation of momentum generated during this process, which leads to sporangium bouncing. The ultra-lightweight spores travel through air with a maximum velocity of ~5 m s(-1), and a launch acceleration of ~6300 g is measured. In some cases, the whole sporangium, or parts of it, together with contained spores break away from the false indusium and are shed as a whole. Also, spores can stick together and form spore clumps. Both findings are discussed in the context of wind dispersal.

Adaptation and convergent evolution within the Jamesonia-Eriosorus complex in high-elevation biodiverse Andean hotspots.

The recent uplift of the tropical Andes (since the late Pliocene or early Pleistocene) provided extensive ecological opportunity for evolutionary radiations. We test for phylogenetic and morphological evidence of adaptive radiation and convergent evolution to novel habitats (exposed, high-altitude páramo habitats) in the Andean fern genera Jamesonia and Eriosorus. We construct time-calibrated phylogenies for the Jamesonia-Eriosorus clade. We then use recent phylogenetic comparative methods to test for evolutionary transitions among habitats, associations between habitat and leaf morphology, and ecologically driven variation in the rate of morphological evolution. Páramo species (Jamesonia) display morphological adaptations consistent with convergent evolution in response to the demands of a highly exposed environment but these adaptations are associated with microhabitat use rather than the páramo per se. Species that are associated with exposed microhabitats (including Jamesonia and Eriorsorus) are characterized by many but short pinnae per frond whereas species occupying sheltered microhabitats (primarily Eriosorus) have few but long pinnae per frond. Pinnae length declines more rapidly with altitude in sheltered species. Rates of speciation are significantly higher among páramo than non-páramo lineages supporting the hypothesis of adaptation and divergence in the unique Páramo biodiversity hotspot.

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.

Do asexual polyploid lineages lead short evolutionary lives? A case study from the fern genus Astrolepis.

A life-history transition to asexuality is typically viewed as leading to a heightened extinction risk, and a number of studies have evaluated this claim by examining the relative ages of asexual versus closely related sexual lineages. Surprisingly, a rigorous assessment of the age of an asexual plant lineage has never been published, although asexuality is extraordinarily common among plants. Here, we estimate the ages of sexual diploids and asexual polyploids in the fern genus Astrolepis using a well-supported plastid phylogeny and a relaxed-clock dating approach. The 50 asexual polyploid samples we included were conservatively estimated to comprise 19 distinct lineages, including a variety of auto- and allopolyploid genomic combinations. All were either the same age or younger than the crown group comprising their maternal sexual-diploid parents based simply on their phylogenetic position. Node ages estimated with the relaxed-clock approach indicated that the average maximum age of asexual lineages was 0.4 My, and individual lineages were on average 7 to 47 times younger than the crown- and total-ages of their sexual parents. Although the confounding association between asexuality and polyploidy precludes definite conclusions regarding the effect of asexuality, our results suggest that asexuality limits evolutionary potential in Astrolepis.

Genome-scale cluster analysis of replicated microarrays using shrinkage correlation coefficient.

BACKGROUND: Currently, clustering with some form of correlation coefficient as the gene similarity metric has become a popular method for profiling genomic data. The Pearson correlation coefficient and the standard deviation (SD)-weighted correlation coefficient are the two most widely-used correlations as the similarity metrics in clustering microarray data. However, these two correlations are not optimal for analyzing replicated microarray data generated by most laboratories. An effective correlation coefficient is needed to provide statistically sufficient analysis of replicated microarray data. RESULTS: In this study, we describe a novel correlation coefficient, shrinkage correlation coefficient (SCC), that fully exploits the similarity between the replicated microarray experimental samples. The methodology considers both the number of replicates and the variance within each experimental group in clustering expression data, and provides a robust statistical estimation of the error of replicated microarray data. The value of SCC is revealed by its comparison with two other correlation coefficients that are currently the most widely-used (Pearson correlation coefficient and SD-weighted correlation coefficient) using statistical measures on both synthetic expression data as well as real gene expression data from Saccharomyces cerevisiae. Two leading clustering methods, hierarchical and k-means clustering were applied for the comparison. The comparison indicated that using SCC achieves better clustering performance. Applying SCC-based hierarchical clustering to the replicated microarray data obtained from germinating spores of the fern Ceratopteris richardii, we discovered two clusters of genes with shared expression patterns during spore germination. Functional analysis suggested that some of the genetic mechanisms that control germination in such diverse plant lineages as mosses and angiosperms are also conserved among ferns. CONCLUSION: This study shows that SCC is an alternative to the Pearson correlation coefficient and the SD-weighted correlation coefficient, and is particularly useful for clustering replicated microarray data. This computational approach should be generally useful for proteomic data or other high-throughput analysis methodology.

Profile and analysis of gene expression changes during early development in germinating spores of Ceratopteris richardii.

Analysis of an expressed sequence tag library with more than 5,000 sequences from spores of the fern Ceratopteris richardii reveals that more than 3,900 of them represent distinct genes, and almost 70% of these have significant similarity to Arabidopsis (Arabidopsis thaliana) genes. Eight genes are common between three very different dormant plant systems, Ceratopteris spores, Arabidopsis seeds, and Arabidopsis pollen. We evaluated the pattern of mRNA abundance over the first 48 h of spore development using a microarray of cDNAs representing 3,207 distinct genes of C. richardii and determined the relative levels of RNA abundance for 3,143 of these genes using a Bayesian method of statistical analysis. More than 900 of them (29%) show a significant change between any of the five time points analyzed, and these have been annotated based on their sequence similarity with the Arabidopsis proteome. Novel data arising from these analyses identify genes likely to be critical for the germination and subsequent early development of diverse cells and tissues emerging from dormancy.

Mycothallic/mycorrhizal symbiosis of chlorophyllous gametophytes and sporophytes of a fern, Pellaea viridis (Forsk.) Prantl (Pellaeaceae, Pteridales).

Gametophytes of Pellaea viridis that appeared spontaneously on the surface of substratum originating from an ultramafic area were found to form mycothallic symbiosis with arbuscular mycorrhizal fungi (AMF) under laboratory conditions. In gametophytes and sporophytes grown with Glomus tenue, abundant arbuscule formation was observed at both stages. In gametophytes, the fungus was found in the region where the rhizoids are initiated. If G. intraradices was added to the soil, the gametophytes were colonised mostly by G. tenue, and roots of sporophytes were colonised by G. intraradices. The presence of AM fungi in both gametophytes and sporophytes of P. viridis resulted in the development of larger leaf area and root length of the sporophyte. The analysis of gametophytes from the Botanical Garden in Krakow (Poland) showed that cordate gametophytes of Pteridales, namely Pellaea viridis (Pellaeaceae), Adiantum raddianum and A. formosum (Adiantaceae), were also mycothallic.