Quantitative cell biology of tip growth in moss.

KEY MESSAGE: Here we review, from a quantitative point of view, the cell biology of protonemal tip growth in the model moss Physcomitrium patens. We focus on the role of the cytoskeleton, vesicle trafficking, and cell wall mechanics, including reviewing some of the existing mathematical models of tip growth. We provide a primer for existing cell biological tools that can be applied to the future study of tip growth in moss. Polarized cell growth is a ubiquitous process throughout the plant kingdom in which the cell elongates in a self-similar manner. This process is important for nutrient uptake by root hairs, fertilization by pollen, and gametophyte development by the protonemata of bryophytes and ferns. In this review, we will focus on the tip growth of moss cells, emphasizing the role of cytoskeletal organization, cytoplasmic zonation, vesicle trafficking, cell wall composition, and dynamics. We compare some of the existing knowledge on tip growth in protonemata against what is known in pollen tubes and root hairs, which are better-studied tip growing cells. To fully understand how plant cells grow requires that we deepen our knowledge in a variety of forms of plant cell growth. We focus this review on the model plant Physcomitrium patens, which uses tip growth as the dominant form of growth at its protonemal stage. Because mosses and vascular plants shared a common ancestor more than 450 million years ago, we anticipate that both similarities and differences between tip growing plant cells will provide mechanistic information of tip growth as well as of plant cell growth in general. Towards this mechanistic understanding, we will also review some of the existing mathematical models of plant tip growth and their applicability to investigate protonemal morphogenesis. We attempt to integrate the conclusions and data across cell biology and physical modeling to our current state of knowledge of polarized cell growth in P. patens and highlight future directions in the field.

3D Dissection of Structural Membrane-Wall Contacts in Filamentous Moss Protonemata.

In conventional light microscopy, the adjacent cell walls of filamentous moss protonemata are seen from its narrow side thereby obscuring the major area of cell-cell connection. Optical sectioning, segmentation and 3D reconstructions allow the tilting and rotation of intracellular structures thereby greatly improving our understanding of interaction between organelles, membranes and the cell wall. Often, the findings also allow for conclusions on the respective functions. The moss Physcomitrium (Physcomitrella) patens is a model organism for growth, development and morphogenesis. Its filamentous protonemata are ideal objects for microscopy. Here, we investigated the cell wall between two neighboring cells and the connection of membranes towards this wall after plasmolysis in 0.8 M mannitol. An m-green fluorescent protein (GFP)-HDEL cell line was used to visualize the endoplasmatic reticulum (ER), the plasma membrane (PM) was stained with FM4-64. Our studies clearly show the importance of cell-cell contacts in P. patens protonemata. In 86% of the investigated cell pairs, at least one of the protoplasts remained fully attached to the adjacent cell wall. By tilting of z-stacks, volume renderings and 3D reconstructions, we visualized the amount of attached/detached PM and ER components after plasmolysis and membrane piercings through the wall of cell neighbors.

Photoprotection enhanced by red cell wall pigments in three East Antarctic mosses.

BACKGROUND: Antarctic bryophytes (mosses and liverworts) are resilient to physiologically extreme environmental conditions including elevated levels of ultraviolet (UV) radiation due to depletion of stratospheric ozone. Many Antarctic bryophytes synthesise UV-B-absorbing compounds (UVAC) that are localised in their cells and cell walls, a location that is rarely investigated for UVAC in plants. This study compares the concentrations and localisation of intracellular and cell wall UVAC in Antarctic Ceratodon purpureus, Bryum pseudotriquetrum and Schistidium antarctici from the Windmill Islands, East Antarctica. RESULTS: Multiple stresses, including desiccation and naturally high UV and visible light, seemed to enhance the incorporation of total UVAC including red pigments in the cell walls of all three Antarctic species analysed. The red growth form of C. purpureus had significantly higher levels of cell wall bound and lower intracellular UVAC concentrations than its nearby green form. Microscopic and spectroscopic analyses showed that the red colouration in this species was associated with the cell wall and that these red cell walls contained less pectin and phenolic esters than the green form. All three moss species showed a natural increase in cell wall UVAC content during the growing season and a decline in these compounds in new tissue grown under less stressful conditions in the laboratory. CONCLUSIONS: UVAC and red pigments are tightly bound to the cell wall and likely have a long-term protective role in Antarctic bryophytes. Although the identity of these red pigments remains unknown, our study demonstrates the importance of investigating cell wall UVAC in plants and contributes to our current understanding of UV-protective strategies employed by particular Antarctic bryophytes. Studies such as these provide clues to how these plants survive in such extreme habitats and are helpful in predicting future survival of the species studied.

Quantitative moss cell biology.

Research on mosses has provided answers to many fundamental questions in the life sciences, with the model moss Physcomitrella patens spearheading the field. Recent breakthroughs in cell biology were obtained in the quantification of chlorophyll fluorescence, signalling via calcium waves, the creation of designer organelles, gene identification in cellular reprogramming, reproduction via motile sperm and egg cells, asymmetric cell division, visualization of the actin cytoskeleton, identification of genes responsible for the shift from 2D to 3D growth, the structure and importance of the cell wall, and in the live imaging and modelling of protein networks in general. Highly standardized growth conditions, simplicity of most moss tissues, and an outstandingly efficient gene editing facilitate quantitative moss cell biology.

Photoprotection enhanced by red cell wall pigments in three East Antarctic mosses

BACKGROUND: Antarctic bryophytes (mosses and liverworts) are resilient to physiologically extreme environmental conditions including elevated levels of ultraviolet (UV) radiation due to depletion of stratospheric ozone. Many Antarctic bryophytes synthesise UV-B-absorbing compounds (UVAC) that are localised in their cells and cell walls, a location that is rarely investigated for UVAC in plants. This study compares the concentrations and localisation of intracellular and cell wall UVAC in Antarctic Ceratodon purpureus, Bryum pseudotriquetrum and Schistidium antarctici from the Windmill Islands, East Antarctica. RESULTS: Multiple stresses, including desiccation and naturally high UV and visible light, seemed to enhance the incorporation of total UVAC including red pigments in the cell walls of all three Antarctic species analysed. The red growth form of C. purpureus had significantly higher levels of cell wall bound and lower intracellular UVAC concentrations than its nearby green form. Microscopic and spectroscopic analyses showed that the red colouration in this species was associated with the cell wall and that these red cell walls contained less pectin and phenolic esters than the green form. All three moss species showed a natural increase in cell wall UVAC content during the growing season and a decline in these compounds in new tissue grown under less stressful conditions in the laboratory. CONCLUSIONS: UVAC and red pigments are tightly bound to the cell wall and likely have a long-term protective role in Antarctic bryophytes. Although the identity of these red pigments remains unknown, our study demonstrates the importance of investigating cell wall UVAC in plants and contributes to our current understanding of UV-protective strategies employed by particular Antarctic bryophytes. Studies such as these provide clues to how these plants survive in such extreme habitats and are helpful in predicting future survival of the species studied.

Stomatal development in time: the past and the future.

Stomata have significantly diversified in nature since their first appearance around 400 million years ago. The diversification suggests the active reprogramming of molecular machineries of stomatal development during evolution. This review focuses on recent progress that sheds light on how this rewiring occurred in different organisms. Three specific aspects are discussed in this review: (i) the evolution of the transcriptional complex that governs stomatal state transitions; (ii) the evolution of receptor-ligand pairs that mediate extrinsic signaling; and (iii) the loss of stomatal development genes in an astomatous angiosperm.

The enigmatic mossy cell of the dentate gyrus.

Mossy cells comprise a large fraction of the cells in the hippocampal dentate gyrus, suggesting that their function in this region is important. They are vulnerable to ischaemia, traumatic brain injury and seizures, and their loss could contribute to dentate gyrus dysfunction in such conditions. Mossy cell function has been unclear because these cells innervate both glutamatergic and GABAergic neurons within the dentate gyrus, contributing to a complex circuitry. It has also been difficult to directly and selectively manipulate mossy cells to study their function. In light of the new data generated using methods to preferentially eliminate or activate mossy cells in mice, it is timely to ask whether mossy cells have become any less enigmatic than they were in the past.

Dehydration-responsive features of Atrichum undulatum.

Drought is an increasingly important limitation on plant productivity worldwide. Understanding the mechanisms of drought tolerance in plants can lead to new strategies for developing drought-tolerant crops. Many moss species are able to survive desiccation-a more severe state of dehydration than drought. Research into the mechanisms and evolution of desiccation tolerance in basal land plants is of particular significance to both biology and agriculture. In this study, we conducted morphological, cytological, and physiological analyses of gametophytes of the highly desiccation-tolerant bryophyte Atrichum undulatum (Hedw.) P. Beauv during dehydration and rehydration. Our results suggested that the mechanisms underlying the dehydration-recovery cycle in A. undulatum gametophytes include maintenance of membrane stability, cellular structure protection, prevention of reactive oxygen species (ROS) generation, elimination of ROS, protection against ROS-induced damage, and repair of ROS-induced damage. Our data also indicate that this dehydration-recovery cycle consists not only of the physical removal and addition of water, but also involves a highly organized series of cytological, physiological, and biochemical changes. These attributes are similar to those reported for other drought- and desiccation-tolerant plant species. Our findings provide major insights into the mechanisms of dehydration-tolerance in the moss A. undulatum.

Infraspecific variation within and across complete organellar genomes and nuclear ribosomal repeats in a moss.

Bryophytes (mosses, liverworts, and hornworts) are diverse and ecologically and evolutionarily significant yet genome scale data sets and analyses remain extremely sparse relative to other groups of plants, and are completely lacking at the infraspecific level. By sequencing the complete organellar genomes and nuclear ribosomal repeat from seven patches of a South American sub-Antarctic neo-endemic non-model moss, we present the first characterization of infraspecific polymorphism within and across the three genomic compartments for a bryophyte. Diversity within patches is accounted for by both intraindividual and interindividual variation for the nuclear ribosomal repeat and plastid genome, respectively. This represents the most extensive infraspecific genomic dataset generated for an early land plant lineage thus far and provides insight into relative rates of substitution between organellar genomes, including high rates of nonsynonymous to synonymous substitutions.

Exploring the fossil history of pleurocarpous mosses: Tricostaceae fam. nov. from the Cretaceous of Vancouver Island, Canada.

PREMISE OF THE STUDY: Mosses, very diverse in modern ecosystems, are currently underrepresented in the fossil record. For the pre-Cenozoic, fossil mosses are known almost exclusively from compression fossils, while anatomical preservation, which is much more taxonomically informative, is rare. The Lower Cretaceous of Vancouver Island (British Columbia, Canada) hosts a diverse anatomically preserved flora at Apple Bay. While the vascular plant component of the Apple Bay flora has received much attention, the numerous bryophytes identified at the locality have yet to be characterized. METHODS: Fossil moss gametophytes in more than 20 carbonate concretions collected from the Apple Bay locality on Vancouver Island were studied in serial sections prepared using the cellulose acetate peel technique. KEY RESULTS: We describe Tricosta plicata gen. et sp. nov., a pleurocarpous moss with much-branched gametophytes, tricostate plicate leaves, rhizoid-bearing bases, and delicate gametangia (antheridia and archegonia) borne on specialized branches. A new family of hypnanaean mosses, Tricostaceae fam. nov., is recognized based on the novel combination of characters of T. plicata. CONCLUSIONS: Tricosta plicata reveals pleurocarpous moss diversity unaccounted for in extant floras. This new moss adds the first bryophyte component to an already diverse assemblage of vascular plants described from the Early Cretaceous at Apple Bay and, as the oldest representative of the Hypnanae, provides a hard minimum age for the group (136 Ma).

All green, but equal? Morphological traits and ecological implications on spores of three species of mosses in the Brazilian Atlantic forest.

Spores of the tropical mosses Pyrrhobryum spiniforme, Neckeropsis undulata and N. disticha were characterized regarding size, number per capsule and viability. Chemical substances were analyzed for P. spiniforme and N. undulata spores. Length of sporophyte seta (spore dispersal ability) was analyzed for P. spiniforme. Four to six colonies per species in each site (lowland and highland areas of an Atlantic Forest; Serra do Mar State Park, Brazil) were visited for the collection of capsules (2008 - 2009). Neckeropsis undulata in the highland area produced the largest spores (ca. 19 µm) with the highest viability. The smallest spores were found in N. disticha in the lowland (ca. 13 µm). Pyrrhobryum spiniforme produced more spores per capsule in the highland (ca. 150,000) than in lowland (ca. 40,000); longer sporophytic setae in the lowland (ca. 64 mm) than in the highland (ca. 43 mm); and similar sized spores in both areas (ca. 16 µm). Spores of N. undulata and P. spiniforme contained lipids and proteins in the cytoplasm, and acid/neutral lipids and pectins in the wall. Lipid bodies were larger in N. undulata than in P. spiniforme. No starch was recorded for spores. Pyrrhobryum spiniforme in the highland area, different from lowland, was characterized by low reproductive effort, but presented many spores per capsule.

350 my of mitochondrial genome stasis in mosses, an early land plant lineage.

Among land plants, angiosperms have the structurally most labile mitochondrial (mt) genomes. In contrast, the so-called early land plants (e.g., mosses) seem to have completely static mt chromosomes. We assembled the complete mt genomes from 12 mosses spanning the moss tree of life, to assess 1) the phylogenetic depth of the conserved mt gene content and order and 2) the correlation between scattered sequence repeats and gene order lability in land plants. The mt genome of most mosses is approximately 100 kb in size, and thereby the smallest among land plants. Based on divergence time estimates, moss mt genome structure has remained virtually frozen for 350 My, with only two independent gene losses and a single gene relocation detected across the macroevolutionary tree. This is the longest period of mt genome stasis demonstrated to date in a plant lineage. The complete lack of intergenic repeat sequences, considered to be essential for intragenomic recombinations, likely accounts for the evolutionary stability of moss mt genomes.

Desiccation tolerance of Hymenophyllacea filmy ferns is mediated by constitutive and non-inducible cellular mechanisms.

The Hymenophyllaceae is a primitive family within the Filicopsidae. One of the most exceptional features of this family of ferns is the presence of fronds with one or just a few cell layers (hence their name of filmy ferns), and the absence of stomata. Hymenophyllum caudiculatum and Hymenophyllum dentatum are able to lose more than 82% of their fully hydrated water content, to remain dry for extended periods of time (days or weeks), and to survive and remain viable following rehydration. The aim of this work was to understand whether the adaptive strategy of the Hymenophyllaceae for desiccation tolerance is constitutive or inducible. A proteomic approach was adopted in combination with physiological parameters to assess whether there were changes in the protein content during dehydration and following rehydration. Detached fronds were used to monitor the rates of photosynthesis in desiccation experiments, sugar accumulation, and high-resolution 2-DE to analyze proteome variation during a desiccation-rehydration cycle. The analyzed proteome exhibited little variation (3-4%) between hydrated and desiccated states, while variation was greater between the desiccated and rehydrated states (8.7-10%). Eighty-two discrete proteins were analyzed by MS/MS, and 65 were identified. About 21% of the analyzed proteins (17) were mixtures of two or more different polypeptides. Of the identified proteins, more than a half (33 spots, 55%) had functions related to energy-photosynthesis. The second largest category with known function (five spots, 8%) was related to cell rescue, defense, and virulence. More than one in every four proteins analyzed belonged to a group of hypothetical proteins (18 spots, 28%). The results suggest that the Hymenophyllaceae represent an example of a change in adaptive strategy from a typical vascular to the poikilohydric homoiochlorophyllous adaptation, which they share with the bryophytes that grow in profusion in the same habitats. The speed at which desiccation takes place therefore precludes the induction of protective systems, suggesting a constitutive mechanism of cellular protection.

Regeneration of Little Ice Age bryophytes emerging from a polar glacier with implications of totipotency in extreme environments.

Across the Canadian Arctic Archipelago, widespread ice retreat during the 20th century has sharply accelerated since 2004. In Sverdrup Pass, central Ellesmere Island, rapid glacier retreat is exposing intact plant communities whose radiocarbon dates demonstrate entombment during the Little Ice Age (1550-1850 AD). The exhumed bryophyte assemblages have exceptional structural integrity (i.e., setae, stem structures, leaf hair points) and have remarkable species richness (60 of 144 extant taxa in Sverdrup Pass). Although the populations are often discolored (blackened), some have developed green stem apices or lateral branches suggesting in vivo regrowth. To test their biological viability, Little Ice Age populations emerging from the ice margin were collected for in vitro growth experiments. Our results include a unique successful regeneration of subglacial bryophytes following 400 y of ice entombment. This finding demonstrates the totipotent capacity of bryophytes, the ability of a cell to dedifferentiate into a meristematic state (analogous to stem cells) and develop a new plant. In polar ecosystems, regrowth of bryophyte tissue buried by ice for 400 y significantly expands our understanding of their role in recolonization of polar landscapes (past or present). Regeneration of subglacial bryophytes broadens the concept of Ice Age refugia, traditionally confined to survival of land plants to sites above and beyond glacier margins. Our results emphasize the unrecognized resilience of bryophytes, which are commonly overlooked vis-a-vis their contribution to the establishment, colonization, and maintenance of polar terrestrial ecosystems.

Spores before sporophytes: hypothesizing the origin of sporogenesis at the algal-plant transition.

Fossil spores from mid-Ordovician deposits (475 million yr old) are the first indication of plants on land and predate megafossils of plants by 30-50 million yr. Sporopollenin-walled spores distinguish land plants from algae, which typically have heavy-walled zygotes that germinate via meiosis into motile or protonemal cells. All land plants are embryophytes with spores produced by the sporophyte generation. It is generally assumed that retention of the zygote and delay in meiosis led to matrotrophic embryo development and intercalation of the diploid sporophyte before spore production. However, new data on the cell biology of sporogenesis in extant bryophytes suggest that spores were produced directly from zygotes in protoembryophytes. The mechanism of wall transfer from zygote to meiospores was a three-phase heterochrony involving precocious initiation of cytokinesis, acceleration of meiosis, and concomitant delay in wall deposition. In bryophyte sporogenesis, cytokinesis is typically initiated in advance of meiosis, and quadrilobing of the cytoplasm is followed by development of a bizarre quadripolar spindle that assures coordination of nuclear distribution with predetermined spore domains. This concept of the innovation of sporogenesis at the onset of terrestrialization provides a new perspective for interpreting fossil evidence and understanding the evolution of land plants.

Cloning, expression and characterization of the putative nuclear transport factor 2 (NTF2) gene from moss Conocephalum conicum(L.) Dum.

Biomacromolecules import into the nucleus is a complex progress which requires the participation of several cytosolic factors, and nuclear transport factor 2 (NTF2) is one of essential components in nuclear trafficking. Its main role is to transport RanGDP from cytoplasm to nucleus by interacting with FxFG nucleoporin repeats. In the study a putative new gene, designated as CcNTF2, was obtained from the moss (Conocephalum conicum) cDNA library we have constructed. The full-length cDNA sequence is 913 bp in size contains a 372 bp open reading frame (ORF) flanked by a 195 bp 5'-untranslated sequence and a long 346 bp 3'-non-coding region, encoding 123 amino acids of 13,575.3 Da. Part of the genomic sequence was also cloned and sequenced, which is 1,602 bp long and possesses two exons and one intron. Alignment analysis showed that the CcNTF2 protein is high conserved among plant NTF2 and shares 81% similarity with the ones from Arabidopsis thaliana and Brassica rapa. The expression of wild-type CcNTF2 was detected by immunoblotting of extraction of C. conicum and it indicated the putative protein is integral. Through functional expression of CcNTF2-green fluorescent protein (GFP) in tobacco, it was demonstrated that CcNTF2 can accumulate at the nuclear rim. Site-directed mutagenesis analysis confirmed CcNTF2 P71K has influence on the protein import into nucleus. In addition, overexpression of CcNTF2 P71K was observed to be deleterious for the plant cell. It is the first illumination of NTF2 in moss, and our study established the primary foundation for further research on moss NTF2.

Complex mutation and weak selection together determined the codon usage bias in bryophyte mitochondrial genomes.

Mutation and selection are two major forces causing codon usage biases. How these two forces influence the codon usages in green plant mitochondrial genomes has not been well investigated. In the present study, we surveyed five bryophyte mitochondrial genomes to reveal their codon usage patterns as well as the determining forces. Three interesting findings were made. First, comparing to Chara vulgaris, an algal species sister to all extant land plants, bryophytes have more G, C-ending codon usages in their mitochondrial genes. This is consistent with the generally higher genomic GC content in bryophyte mitochondria, suggesting an increased mutational pressure toward GC. Second, as indicated by Wright's Nc-GC3s plot, mutation, not selection, is the major force affecting codon usages of bryophyte mitochondrial genes. However, the real mutational dynamics seem very complex. Context-dependent analysis indicated that nucleotide at the 2nd codon position would slightly affect synonymous codon choices. Finally, in bryophyte mitochondria, tRNA genes would apply a weak selection force to fine-tune the synonymous codon frequencies, as revealed by data of Ser4-Pro-Thr-Val families. In summary, complex mutation and weak selection together determined the codon usages in bryophyte mitochondrial genomes.

Comparative development of the sporophyte-gametophyte junction in six moss species.

Developmental anatomy of the sporophyte-gametophyte junction in six moss species is described with special reference to sporophyte penetration into the gametophytic tissue. The sporophyte-gametophyte junction in mosses is classified into two types based on vaginula morphology: in the "true vaginula" type, the junction involves only an epigonium derived from the archegonium, and in the other "shoot vaginula" type, it involves a shoot and an epigonium. In both of the types, the sporophyte penetrates into an epigonial tissue accompanied by degeneration of epigonium cells under the developing sporophyte. In the "shoot vaginula" type, the sporophyte further penetrates into the conducting strand or similar cells that seem to be induced by stimulation of fertilization. It is likely that the difference in growth rate between the epigonium and the capped sporophyte is a mechanical force for sporophyte penetration.

Vitrification of mosses: a useful method for the cryopreservation of Splachnum ampullaceum Hedw.

The source of germplasm as well as the technique used for storage of mosses can enhance survival after cryopreservation. Samples of gametophores, protonemata and protonemal brood cells from in vitro cultures of Splachnum ampullaceum were cryopreserved following exposure to a plant vitrification solution (PVS2) for two different times (5 and 10 min) at 0 degree C. Half of the samples were pretreated with a loading solution containing 2 M glycerol and 0.4 M sucrose before exposure to PVS2. After one week storage in liquid nitrogen, S. ampullaceum samples were regenerated on Gamborg's B5 mineral medium with B5 vitamins. Exposure to a loading solution was a prerequisite for high survival in all samples. Four weeks after cryopreservation, 92.3 percent brood cells, 60.0 percent gametophores and 46.0 percent protonemata pretreated with a loading solution had regenerated, displaying normal growth and development, thus demonstrating that vitrification is a useful method for moss cryopreservation.

Cytological insights into the desiccation biology of a model system: moss protonemata.

*Set out here is the first generic account of the cytological effects of dehydration and rehydration and exogenous abscisic acid on moss protonemata. *Protonemal cells were subjected to slow and fast drying regimes, with and without prior exposure to abscisic acid. The cytological changes associated with de- and rehydration were analysed by light, fluorescence and transmission electron microscopy, together with pharmacological studies. *Protonemata survive slow but not fast drying, unless pretreated with abscisic acid. Dehydration elicits profound cytological changes, namely vacuolar fragmentation, reorganization of the endomembrane domains, changes in the thickness of the cell wall and in the morphology of plastids and mitochondria, and the controlled dismantling of the cytoskeleton; these dynamic events are prevented by fast drying. In control cells, abscisic acid elicits changes that partially mimic those associated with slow drying, including controlled disassembly of cytoskeletal elements, thus enabling protonemal cells to survive normally lethal rates of water loss. *Our demonstration that moss protonemata are an ideal system for visualizing and manipulating the cytological events associated with vegetative desiccation tolerance in land plants now opens up the way for genomic dissection of the underlying mechanisms.