Moss Cells Research Articles

Physcomitrella PpORS, Basal to Plant Type III Polyketide Synthases in Phylogenetic Trees, Is a Very Long Chain 2′-Oxoalkylresorcinol Synthase

The plant type III polyketide synthases (PKSs), which produce diverse secondary metabolites with different biological activities, have successfully co-evolved with land plants. To gain insight into the roles that ancestral type III PKSs played during the early evolution of land plants, we cloned and characterized PpORS from the moss Physcomitrella. PpORS has been proposed to closely resemble the most recent common ancestor of the plant type III PKSs. PpORS condenses a very long chain fatty acyl-CoA with four molecules of malonyl-CoA and catalyzes decarboxylative aldol cyclization to yield the pentaketide 2'-oxoalkylresorcinol. Therefore, PpORS is a 2'-oxoalkylresorcinol synthase. Structure modeling and sequence alignments identified a unique set of amino acid residues (Gln(218), Val(277), and Ala(286)) at the putative PpORS active site. Substitution of the Ala(286) to Phe apparently constricted the active site cavity, and the A286F mutant instead produced triketide alkylpyrones from fatty acyl-CoA substrates with shorter chain lengths. Phylogenetic analysis and comparison of the active sites of PpORS and alkylresorcinol synthases from sorghum and rice suggested that the gramineous enzymes evolved independently from PpORS to have similar functions but with distinct active site architecture. Microarray analysis revealed that PpORS is exclusively expressed in nonprotonemal moss cells. The in planta function of PpORS, therefore, is probably related to a nonprotonemal structure, such as the cuticle.

A STUDY OF SOLUTIONS TO PREVENT THE EROSION OF THE MOSS GROWING ON ARCHITECTURES OF HUE IMPERIAL CITADEL

Background: The erosion of mosses on the surface of Hue imperial citadel has caused a lot of harm and is currently an urgent problem to be solved. Objectives:1. Identifying some moss species growing on Hue relics. 2. Determining the cause of slipperiness and the living environment mosses. 3. Finding a solution to limit the intrusion of the mosses. Methods: The methods are: identifying scientific name by comparing patterns, identify mucus, pH measurement and evaluation of the effects of certain chemicals on the growth of mosses in the laboratory. Result: The moss species that develop popularly in Hue relics is Funaria hygrometrica, Bryum apiculatum, Trichostomum orthodontum, Fissidens lycopodioides. Funaria hygrometrica is the most popular moss. The cause of the slipperiness is mucus in moss cells released outside the moss after death. Mosses grow well in moist environments, clear and slightly acidic surface. After two days assessing the effect of citric acid 18%, 15%, 10% and 7% NaHCO3, 5%, 3% on the growth of moss, the mosses all died completely. Since then identified two types of chemicals have been found to completely dead moss, inexpensive, environmentally friendly and does not affect the surface of architectures are citric acid 10% solution and NaHCO3 3% solution. Keywords: The moss erosion, Funaria hygrometrica, mucus, citric acid, NaHCO3 Key words: The moss erosion, Funaria hygrometrica, mucus, citric acid, NaHCO 3

Bioacumulation and ultrastructural effects of Cd, Cu, Pb and Zn in the moss Scorpiurum circinatum (Brid.) Fleisch. & Loeske

This paper tested if culturing the moss Scorpiurum circinatum (Brid.) Fleisch. & Loeske with metal solutions (Cd, Cu, Pb and Zn) for 30 days causes metal bioaccumulation and ultrastructural changes. The results showed that despite the high heavy metal concentrations in treatment solutions, treated samples did not show severe ultrastructural changes and cells were still alive and generally well preserved. Bioaccumulation highlighted that moss cells survived to heavy metal toxicity by immobilizing most toxic ions extracellularly, likely in binding sites of the cell wall, which is the main site of metal detoxification.

Fungi infecting cultivated moss can also cause diseases in crop plants

AbstractBryophytes (mosses) are non‐vascular plants inhabited by a large number of fungal species, but whether mosses can act as reservoirs of fungal pathogens of crop plants has gained little attention. A few moss species including the Sunagoke moss (Racomitrium japonicum; family Grimmiaceae) are found to have modern economical applications in uses such as greening of urban environments. In a previous study, we identified fungi causing symptoms of varying severity in the commercially grown Sunagoke moss. The aim of this study was to test whether the same fungal isolates are pathogenic to vascular plants. An isolate of Fusarium avenaceum lethal to the Sunagoke moss caused root and crown rot in barley (Hordeum vulgare) and reduced germination of tomato (Solanum lycopersicum) and carrot (Daucus carota) grown in the infested soil. An isolate of Cladosporium oxysporum causing mild symptoms in moss reduced growth and caused reddening and premature death of carrot seedlings. On the other hand, isolates of Alternaria alternata and Fusarium oxysporum lethal to the Sunagoke moss caused no detectable symptoms in any tested vascular plant, suggesting specialisation of these isolates to moss. Chloroplast repositioning was observed in the neighbouring cells towards the initially infected cell following infection with F. avenaceum and A. alternata in Physcomitrella patens (family Funariaceae), a model moss used to study microscopic symptoms. Infection of P. patens with a non‐virulent Apiospora montagnei isolate induced formation of papillae in the moss cells, indicating activation of host defence as described in vascular plants. Results suggest that mosses and vascular plants may be linked by a common microbial interface constituted by pathogenic fungi. The findings have epidemiological implications that have gained little previous attention.

Coarse-Grained Model of Cooperative Chloroplast Transport in Moss

Organelle motility is essential for the functioning of the eukaryotic cell. Actively modifying intracellular structures allows cells to change and adapt to different conditions. One of these cellular structures is the microtubule cytoskeleton, which is comprised of polarized filaments that function as tracks to transport cargo via molecular motors. Recent studies have revealed the importance of cooperative transport in living cells. In the moss Physcomitrella patens, reorganization of the chloroplasts is critical to adapt to changes in light quality and intensity. In this work, we performed detailed analysis of the transport of chloroplasts in protonemal moss cells, and showed that their transport is facilitated by molecular motors and the microtubule cytoskeleton. Our findings were recapitulated using coarse-grained modeling of this cargo transport over different microtubule network topologies. Our simulations include a detailed description of motor and microtubule dynamics, in the presence of thermal fluctuations. Our modeling sheds light into the molecular mechanisms at play in the photo-relocation response of chloroplasts in moss.

Anomalous concentrations of zinc and copper in highmoor peat bog, southeast coast of Lake Baikal

When examining the peat deposit discovered in Vydrinaya bog, South Baikal region, the authors encountered anomalous Zn and Cu concentrations for highmoors being up to 600–500 ppm on a dry matter basis in the Early Holocene beds (360–440 cm) formed 11 000–8500 years ago. It has been demonstrated that Zn and Cu are present inside the plant cells of peat moss in the form of authigenic sulfide minerals of micron size. Apart from Zn and Cu, native Ag particles (5–7 um) have been encountered in the peat of the Vydrinaya bog at a depth of 390–410 cm; these particles formed inside the organic matter of the plasma membrane of peat moss containing Ca, Al, S, and Cu. This study suggests probable patterns of the formation of zinc sulfides, copper sulfides, and native silver in peat moss. The results obtained indicate that biogenic mineral formation plays a significant role in this system, which is a very important argument in the discussion on the ore genesis, in which physicochemical processes are normally favored, while the role of living matter is quite frequently disregarded.

Enhancing the growth of Physcomitrella patens by combination of monochromatic red and blue light – a kinetic study

In the current work we demonstrate the relevance of monochromatic light conditions in moss plant cell culture. Light intensity and illumination wavelength are important cultivation parameters due to their impact on growth and chlorophyll formation kinetics of the moss Physcomitrella patens. This moss was chosen as a model organism due to its capability to produce complex recombinant pharmaceutical proteins. Filamentous moss cells were cultivated in mineral medium in shaking flasks. The flasks were illuminated by light emitting diodes (LED) providing nearly monochromatic red and blue light as well as white light as a reference. A maximum growth rate of 0.78 day((1) was achieved under additional CO(2) aeration and no growth inhibition was observed under high light illumination. The application of dual red and blue light is the most effective way to reach high growth and chlorophyll formation rates while minimizing energy consumption of the LEDs. These observations are discussed as effects of photo sensory pigments in the moss. The combination of monochromatic red and blue light should be considered when a large scale process is set up.

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss <em>Physcomitrella patens</em>

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1. Due to its capacity to undergo efficient mitotic homologous recombination, Physcomitrella patens has emerged as an important model system in recent years2. This capacity allows high frequencies of gene targeting3-9, which is not seen in other model plants such as Arabidopsis. To take full advantage of this system, we need an effective and easy method to deliver DNA into moss cells. The most common ways to transform this moss are particle bombardment10 and PEG-mediated DNA uptake11. Although particle bombardment can produce a high transformation efficiency12, gene guns are not readily available to many laboratories and the protocol is difficult to standardize. On the other hand, PEG mediated transformation does not require specialized equipments, and can be performed in any laboratory with a sterile hood. Here, we show a simple and highly efficient method for transformation of moss protoplasts. This method can generate more than 120 transient transformants per microgram of DNA, which is an improvement from the most efficient protocol previously reported13. Because of its simplicity, efficiency, and reproducibility, this method can be applied to projects requiring large number of transformants as well as for routine transformation.

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss <em>Physcomitrella patens</em>

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1. Due to its capacity to undergo efficient mitotic homologous recombination, Physcomitrella patens has emerged as an important model system in recent years2. This capacity allows high frequencies of gene targeting3-9, which is not seen in other model plants such as Arabidopsis. To take full advantage of this system, we need an effective and easy method to deliver DNA into moss cells. The most common ways to transform this moss are particle bombardment10 and PEG-mediated DNA uptake11. Although particle bombardment can produce a high transformation efficiency12, gene guns are not readily available to many laboratories and the protocol is difficult to standardize. On the other hand, PEG mediated transformation does not require specialized equipments, and can be performed in any laboratory with a sterile hood. Here, we show a simple and highly efficient method for transformation of moss protoplasts. This method can generate more than 120 transient transformants per microgram of DNA, which is an improvement from the most efficient protocol previously reported13. Because of its simplicity, efficiency, and reproducibility, this method can be applied to projects requiring large number of transformants as well as for routine transformation.

Desiccation sensitivity and tolerance in the moss Physcomitrella patens: assessing limits and damage

The moss Physcomitrella patens is becoming the model of choice for functional genomic studies at the cellular level. Studies report that Physcomitrella survives moderate osmotic and salt stress, and that desiccation tolerance can be induced by exogenous ABA. Our goal was to quantify the extent of dehydration tolerance in wild type moss and to examine the nature of cellular damage caused by desiccation. We exposed Physcomitrella to humidities that generate water potentials from −4 (97% RH) to −273 MPa (13% RH) and monitored water loss until equilibrium. Water contents were measured on a dry matter basis to determine the extent of dehydration because fresh weights (FW) were found to be variable and, therefore, unreliable. We measured electrolyte leakage from rehydrating moss, assessed overall regrowth, and imaged cells to evaluate their response to drying and rehydration. Physcomitrella did not routinely survive water potentials <−13 MPa. Upon rehydration, moss dried to water contents >0.4 g g dm−1 maintained levels of leakage similar to those of hydrated controls. Moss dried to lower water contents leaked extensively, suggesting that plasma membranes were damaged. Moss protonemal cells were shrunken and their walls twisted, even at −13 MPa. Moss cells rehydrated after drying to −273 MPa failed to re-expand completely, again indicating membrane damage. ABA treatment elicited tolerance of desiccation to at least −273 MPa and limited membrane damage. Results of this work will form the basis for ongoing studies on the functional genomics of desiccation tolerance at the cellular level.

The CaMV 35S promoter has a weak expression activity in dark grown tissues of moss Physcomitrella patens

The constitutive Cauliflower Mosaic Virus 35S promoter (CaMV 35S) is widely used as a tool to express recombinant proteins in plants, but with different success. We previously showed that the expression of an F-actin marker, GFP-talin, in Physcomitrella patens using the CaMV 35S promoter failed to homogenously label moss tissues. Here, we show a significant diminution of the GFP fluorescence in dark grown old moss cells and complete lack of labelling in newly differentiated cells. Furthermore, we demonstrate that stable moss lines harbouring a resistance cassette driven by the CaMV 35S are unable to grow in darkness in the presence of the antibiotic. In contrast to the CaMV 35S, the heat inducible promoter, hsp17.3B showed uniform expression pattern in all cells and tissues following a mild heat shock.

C2 Domain Protein MIN1 Promotes Eyespot Organization inChlamydomonas reinhardtii

Assembly and asymmetric localization of the photosensory eyespot in the biflagellate, unicellular green alga Chlamydomonas reinhardtii requires coordinated organization of photoreceptors in the plasma membrane and pigment granule/thylakoid membrane layers in the chloroplast. min1 (mini-eyed) mutant cells contain abnormally small, disorganized eyespots in which the chloroplast envelope and plasma membrane are no longer apposed. The MIN1 gene, identified here by phenotypic rescue, encodes a protein with an N-terminal C2 domain and a C-terminal LysM domain separated by a transmembrane sequence. This novel domain architecture led to the hypothesis that MIN1 is in the plasma membrane or the chloroplast envelope, where membrane association of the C2 domain promotes proper eyespot organization. Mutation of conserved C2 domain loop residues disrupted association of the MIN1 C2 domain with the chloroplast envelope in moss cells but did not abolish eyespot assembly in Chlamydomonas. In min1 null cells, channelrhodopsin-1 (ChR1) photoreceptor levels were reduced, indicating a role for MIN1 in ChR1 expression and/or stability. However, ChR1 localization was only minimally disturbed during photoautotrophic growth of min1 cells, conditions under which the pigment granule layers are disorganized. The data are consistent with the hypothesis that neither MIN1 nor proper organization of the plastidic components of the eyespot is essential for localization of ChR1.

In vivo visualization of F‐actin structures during the development of the moss Physcomitrella patens

* The 'in planta' visualization of F-actin in all cells and in all developmental stages of a plant is a challenging problem. By using the soybean heat inducible Gmhsp17.3B promoter instead of a constitutive promoter, we have been able to label all cells in various developmental stages of the moss Physcomitrella patens, through a precise temperature tuning of the expression of green fluorescent protein (GFP)-talin. * A short moderate heat treatment was sufficient to induce proper labeling of the actin cytoskeleton and to allow the visualization of time-dependent organization of F-actin structures without impairment of cell viability. * In growing moss cells, dense converging arrays of F-actin structures were present at the growing tips of protonema cell, and at the localization of branching. Protonema and leaf cells contained a network of thick actin cables; during de-differentiation of leaf cells into new protonema filaments, the thick bundled actin network disappeared, and a new highly polarized F-actin network formed. * The controlled expression of GFP-talin through an inducible promoter improves significantly the 'in planta' imaging of actin.

Accumulation of theanderose in association with development of freezing tolerance in the moss Physcomitrella patens

Mosses are known to have the ability to develop high degrees of resistance to desiccation and freezing stress at cellular levels. However, underlying cellular mechanisms leading to the development of stress resistance in mosses are not understood. We previously showed that freezing tolerance in protonema cells of the moss Physcomitrella patens was rapidly increased by exogenous application of the stress hormone abscisic acid (ABA) [Minami, A., Nagao, M., Arakawa, K., Fujikawa, S., Takezawa, D., 2003a. Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes. J. Plant Physiol. 160, 475–483]. Herein it is shown that protonema cells with acquired freezing tolerance specifically accumulate low-molecular-weight soluble sugars. Analysis of the most abundant trisaccharide revealed that the cells accumulated theanderose (G 6-α-glucosyl sucrose) in close association with enhancement of freezing tolerance by ABA treatment. The accumulation of theanderose was inhibited by cycloheximide, an inhibitor of nuclear-encoded protein synthesis, coinciding with a remarkable decrease in freezing tolerance. Furthermore, theanderose accumulation was promoted by cold acclimation and treatment with hyperosmotic solutes, both of which had been shown to enhance cellular freezing tolerance. These results reveal a novel role for theanderose, whose biological function has been obscure, in high freezing tolerance in moss cells.

Use of endogenous signal sequences for transient production and efficient secretion by moss (Physcomitrella patens) cells

BackgroundEfficient targeting to appropriate cell organelles is one of the bottlenecks for the production of recombinant proteins in plant systems. A common practice is to use the native secretory signal peptide of the heterologous protein to be produced. Though general features of secretion signals are conserved between plants and animals, the broad sequence variability among signal peptides suggests differing efficiency of signal peptide recognition.ResultsAiming to improve secretion in moss bioreactors, we quantitatively compared the efficiency of two human signal peptides and six signals from recently isolated moss (Physcomitrella patens) proteins. We therefore used fusions of the different signals to heterologous reporter sequences for transient transfection of moss cells and measured the extra- and intracellular accumulation of the recombinant proteins rhVEGF and GST, respectively. Our data demonstrates an up to fivefold higher secretion efficiency with endogenous moss signals compared to the two utilised human signal peptides.ConclusionFrom the distribution of extra- and intracellular recombinant proteins, we suggest translational inhibition during the signal recognition particle-cycle (SRP-cycle) as the most probable of several possible explanations for the decreased extracellular accumulation with the human signals. In this work, we report on the supremacy of moss secretion signals over the utilised heterologous ones within the moss-bioreactor system. Though the molecular details of this effect remain to be elucidated, our results will contribute to the improvement of molecular farming systems.

The Evolution of the Abscisic Acid-response in Land Plants: Comparative Analysis of Group 1 LEA Gene Expression in Moss and Cereals

The moss Physcomitrella patens possesses a single copy of a Group 1 LEA gene, designated PpLEA-1. Sequence analysis of the PpLEA-1 gene reveals the gene to contain a single intron in a position conserved in all members of the Group 1 LEA gene family, but also to contain a premature termination codon within the first exon. Nevertheless, a PpLEA-1 transcript accumulates in moss tissues in response both to the imposition of osmotic stress, and to the plant growth regulator abscisic acid (ABA). This response appears to be mediated at the transcriptional level, and observation of the pattern of gene expression, reported by histochemical staining of plants expressing a PpLea-1::GUS transgene suggests that the promoter responds preferentially to ABA in protonemal filaments, whereas osmotic stress induces gene expression primarily in the gametophores. Quantitative analysis of promoter activity by transient expression in Physcomitrella protoplasts shows the PpLEA-1 promoter to be highly active in response to ABA and osmotic stress. ABA-mediated transgene expression from the PpLea-1 promoter occurs at a level similar to that driven by the highly active promoter of the wheat Group 1 LEA gene, E(m). Site-directed mutagenesis of the PpLEA-1 promoter indicates that ABA-inducibility is mediated via an ACGT-core motif similar to that seen in the ABA response elements of higher plant LEA genes. However, whereas the wheat E(m )promoter is active in moss tissues, the moss promoter is not reciprocally active in cereal cells: no activity, ABA-inducible or otherwise was detected in barley aleurone protoplasts transfected with the PpLEA-1::GUS construct. We propose that ABA activation of gene expression in moss cells represents an ancestral state, with only minimal requirements for promoter recognition, whereas cereal cells require the interaction of additional factors with ABA-responsive promoters.

Toxic Effects, Oxidative Stress and Ultrastructural Changes in Moss Taxithelium Nepalense (Schwaegr.) Broth. Under Chromium and Lead Phytotoxicity

The effect of short-term Pb and Cr (0, 100 and 1000μM) stress in moss Taxithelium nepalense (Schwaegr.) Broth., the possible generation of oxidative stress, antioxidant metabolism and changes in the chloroplast and cell membrane ultrastructure were investigated. In moss cells, treatment of Pb and Cr for 12 and 24 h decreased the dry mass and total chlorophyll content with marked inhibition under Pb. Both Pb and Cr accumulated after 24 h of their treatment where highest accumulation of Pb was visible than that of Cr. The ultrastructural studies at 1000 μM of Pb and Cr showed distortion of the thylakoid, distortion of chloroplast membrane and changes in the chloroplast structure. Chloroplast distortion was highly visible under Pb than that of Cr. The distortion in the cell membrane was evident at high concentration of Pb, while under Cr, minor changes were visible as compared to controls. Both Pb and Cr significantly increased the production of ROS like H2O2 and O2− radical with marked production after 24 h under Pb than that of Cr. The alteration in metabolism of activated oxygen in moss cells was evidenced by the increase in the lipid peroxidation in moss cells, with pronounced effect after 24 h than that of 12 h after Pb and Cr treatment. The SOD activity showed an increasing trend followed by decrease in CAT, POX and GR activity after 12 and 24 h. Both ascorbate and glutathione showed higher accumulation under Pb followed by Cr. The results showed that at high concentration of metals, oxidative stress could be induced in moss cells characterized by the generation of ROS and initiation lipid peroxidation that inhibited the major antioxidant metabolism. Both physiological and ultrastructural studies suggested the possible induction of oxidative stress in Taxithelium nepalense (Schwaegr.) Broth. under Pb and Cr toxicity.

NEW WRINGKLE IN CARBON CYCLING

A BACTERIUM LIVING ON and inside the cells of Sphagnum moss in peat bogs has been discovered to oxidize methane to carbon dioxide, which the moss then uses as a carbon source for photosynthesis. This symbiotic relationship helps to better explain the fate of CH 4 produced by decaying plants in wetland ecosystems, animportant part of Earth's carbon cycle that affects the concentration of greenhouse gases in the atmosphere. Jaap S. Sinninghe Damste of the Royal Netherlands Institute for Sea Research, Alfons J. P. Smolders and Ashna A. Raghoebarsing of Radboud University, in the Netherlands, and colleagues identified the bacterium as a relative of a Methylocetta species ( Nature 2005, 436, 1153). In lab studies, the researchers incubated moss plants with 13 C-labeled methane to show rapid in situ bacterial oxidation of 13 CH 4 to 13 CO 2 . Over several days, the moss fixed the 13 CO 2 into carbohydrates, which was monitored by measuring the formation of a 13 C-containing sitosterol. The team confirmed the fi...

Gravitropic moss cells default to spiral growth on the clinostat and in microgravity during spaceflight

In addition to shoots and roots, the gravity (g)-vector orients the growth of specialized cells such as the apical cell of dark-grown moss protonemata. Each apical cell of the moss Ceratodon purpureus senses the g-vector and adjusts polar growth accordingly producing entire cultures of upright protonemata (negative gravitropism). The effect of withdrawing a constant gravity stimulus on moss growth was studied on two NASA Space Shuttle (STS) missions as well as during clinostat rotation on earth. Cultures grown in microgravity (spaceflight) on the STS-87 mission exhibited two successive phases of non-random growth and patterning, a radial outgrowth followed by the formation of net clockwise spiral growth. Also, cultures pre-aligned by unilateral light developed clockwise hooks during the subsequent dark period. The second spaceflight experiment flew on STS-107 which disintegrated during its descent on 1 February 2003. However, most of the moss experimental hardware was recovered on the ground, and most cultures, which had been chemically fixed during spaceflight, were retrieved. Almost all intact STS-107 cultures displayed strong spiral growth. Non-random culture growth including clockwise spiral growth was also observed after clinostat rotation. Together these data demonstrate the existence of default non-random growth patterns that develop at a population level in microgravity, a response that must normally be overridden and masked by a constant g-vector on earth.

The influence of high temperature on cell damage and shoot survival rates of Plagiomnium acutum

Cell damage and shoot survival rates of Plagiomnium acutum under high temperatures were studied by immersing the moss in hot water in the light (wet) and in dark incubators in air (dry) at various constant temperatures for up to 8 h. At 35–40°C no moss cells were damaged (indicated by expansion, contraction, or breach of chloroplasts, total loss of green colour in cells, or protoplast of cell disintegrating), and all moss shoots survived. At 45°C, damage to both wet and dry moss cells, and death of moss shoots increased with exposure time. No samples survived prolonged exposure at temperatures of 50°C and higher, indicating that the lethal high temperature of Plagiomnium acutum is about 45°C. Mosses in air in a dark constanttemperature chamber required a longer time to exhibit cell damage and death, reaching 100% only at 50°C and above. Age of tissue, exemplified by position on stem, had no apparent effect on these responses.