The search for and identification of peptides from the moss Physcomitrella patens

The isolation and identification of peptides from the moss Physcomitrella patens (Hedw.) B.S.G., which has been widely used in recent years as a model for studying plant biology, has been described. It was shown for the first time that protoplasts, the protonemata, and gametophores of Ph. patens contain a variety of peptides. From gametophores, 58 peptides, which are the fragments of 14 proteins, and from the protonemata, 49 peptides, the fragments of 15 proteins, were isolated and identified. It was found that the protonemata and gametophores of Ph. patens, which are the successive stages of the development of this plant, significantly differ from each other in both the peptide composition and the spectrum of precursor proteins of the identified peptides. The isolation of protoplasts during the enzymatic destruction of the protonema cell wall is accompanied by massive degradation of intracellular proteins, many of which are the proteins of the protosynthetic system, which is a characteristic response of higher plants to environmental stress factors. In all, 323 peptides, which are the fragments of 79 proteins, were isolated and identified from moss protoplasts.     

Desiccation tolerance in Physcomitrella patens: Rate of dehydration and the involvement of endogenous abscisic acid (ABA)

The moss Physcomitrella patens , a model system for basal land plants, tolerates several abiotic stresses, including dehydration. We previously reported that Physcomitrella patens survives equilibrium dehydration to ?13 MPa in a closed system at 91% RH. Tolerance of desiccation to water potentials below ?100 MPa was only achieved by pretreatment with exogenous abscisic acid (ABA). We report here that gametophores, but not protonemata, can survive desiccation below ?100 MPa after a gradual drying regime in an open system, without exogenous ABA. In contrast, faster equilibrium drying at 90% RH for 3–5 days did not induce desiccation tolerance in either tissue. Endogenous ABA accumulated in protonemata and gametophores under both drying regimes, so did not correlate directly with desiccation tolerance. Gametophores of a Ppabi3a/b/c triple knock out transgenic line also survived the gradual dehydration regime, despite impaired ABA signaling. Our results suggest that the initial drying rate, and not the amount of endogenous ABA, may be critical in the acquisition of desiccation tolerance. Results from this work will provide insight into ongoing studies to uncover the role of ABA in the dehydration response and the underlying mechanisms of desiccation tolerance in this bryophyte.     

Genetic analysis of a mutant class of Physcomitrella patens in which the polarity of gravitropism is reversed.

Summary In the moss Physcomitrella patens, single-cell protonemata and multicellular gametophores respond to reorientation relative to the gravity vector by growing negatively gravitropically. A mutant class in which the protonemata, but not the gametophores, respond by growing towards gravity has been identified. In this paper, we describe the isolation of additional mutants of this class. Complementation and segregation ratio analyses were carried out on these mutants, which indicate that a single gene may mutate to switch the polarity of gravitropism.     

A CELLULOSE SYNTHASE (CESA) gene essential for gametophore morphogenesis in the moss Physcomitrella patens

In seed plants, different groups of orthologous genes encode the CELLULOSE SYNTHASE (CESA) proteins that are responsible for cellulose biosynthesis in primary and secondary cell walls. The seven CESA sequences of the moss Physcomitrella patens (Hedw.) B. S. G. form a monophyletic sister group to seed plant CESAs, consistent with independent CESA diversification and specialization in moss and seed plant lines. The role of PpCESA5 in the development of P. patens was investigated by targeted mutagenesis. The cesa5 knockout lines were tested for cellulose deficiency using carbohydrate-binding module affinity cytochemistry and the morphology of the leafy gametophores was analyzed by 3D reconstruction of confocal images. No defects were identified in the development of the filamentous protonema or in production of bud initials that normally give rise to the leafy gametophores. However, the gametophore buds were cellulose deficient and defects in subsequent cell expansion, cytokinesis, and leaf initiation resulted in the formation of irregular cell clumps instead of leafy shoots. Analysis of the cesa5 knockout phenotype indicates that a biophysical model of organogenesis can be extended to the moss gametophore shoot apical meristem.     

Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes

Abscisic acid (ABA)-induced genes are implicated in the development of freezing tolerance during cold acclimation in higher plants, but their roles in lower land plants have not been determined. We examined ABA- and cold-induced changes in freezing tolerance and gene expression in the moss Physcomitrella patens. Slow equilibrium freezing to -4 degrees C of P. patens protonemata grown under normal growth conditions killed more than 90% of the cells, indicating that the protonema cells are freezing-sensitive. ABA treatment for 24 h dramatically increased the freezing tolerance of the protonemata, while cold treatment only slightly increased the freezing tolerance within the same period. We examined the expressions of fourteen Physcomitrella patens ABA-responsive genes (PPARs), isolated from ABA-treated protonemata. ABA treatment resulted in a remarkable increase in the expression of all the PPAR genes within 24 h. Several of the PPAR genes (PPAR 1 to 8, and 14) were also responsive to cold, but the response was much slower than that to ABA. Treatment with hyperosmotic concentrations of NaCl and mannitol increased freezing tolerance of protonemata and also increased the expression levels of eleven PPAR genes (PPAR2, 3, 5 to 8, and 10 to 14). These results suggest that ABA and environmental stresses positively affect the expression of common genes that participate in protection of protonema cells leading to the development of freezing tolerance.     

Water transport by aquaporins in the extant plant Physcomitrella patens

Although aquaporins (AQPs) have been shown to increase membrane water permeability in many cell types, the physiological role of this increase was not always obvious. In this report, we provide evidence that in the leafy stage of development (gametophore) of the moss Physcomitrella patens, AQPs help to replenish more rapidly the cell water that is lost by transpiration, at least if some water is in the direct vicinity of the moss plant. Three AQP genes were cloned in P. patens: PIP2;1, PIP2;2, and PIP2;3. The water permeability of the membrane was measured in protoplasts from leaves and protonema. A significant decrease was measured in protoplasts from leaves and protonema of PIP2;1 or PIP2;2 knockouts but not the PIP2;3 knockout. No phenotype was observed when knockout plants were grown in closed petri dishes with ample water supply. Gametophores isolated from the wild type and the pip2;3 mutant were not sensitive to moderate water stress, but pip2;1 or pip2;2 gametophores expressed a water stress phenotype. The knockout mutant leaves were more bent and twisted, apparently suffering from an important loss of cellular water. We propose a model to explain how the AQPs PIP2;1 and PIP2;2 delay leaf dessication in a drying atmosphere. We suggest that in ancestral land plants, some 400 million years ago, APQs were already used to facilitate the absorption of water.     

Sequence analysis of expressed sequence tags from an ABA-treated cDNA library identifies stress response genes in the moss Physcomitrella patens.

Partial cDNA sequencing was used to obtain 169 expressed sequence tags (ESTs) in the moss, Physcomitrella patens. The source of ESTs was a random cDNA library constructed from 7 day-old protonemata following treatment with 10(-4) M abscisic acid (ABA). Analysis of the ESTs identified 69% with homology to known sequences, 61% of which had significant homology to sequences of plant origin. More importantly, at least 11 ESTs had significant similarities to genes which are implicated in plant stress-responses, including responses which may involve ABA. These included a cDNA associated with desiccation tolerance, two heat shock protein genes, one cold acclimation protein cDNA and five others that may be involved in either oxidative or chemical stress or both, i.e., Zn/Cu-superoxide dismutase, NADPH protochlorophyllide oxidoreductase (PorB), selenium binding protein, glutathione peroxidase and glutathione S transferase. Analysis of codon usage between P. patens and seed plants indicated that although mosses and higher plants are to a large extent similar, minor variations also exists that may represent the distinctiveness of each group.     

Actin-related protein2/3 complex component ARPC1 is required for proper cell morphogenesis and polarized cell growth in Physcomitrella patens

The actin-related protein2/3 (Arp2/3) complex functions as a regulator of actin filament dynamics in a wide array of eukaryotic cells. Here, we focus on the role of the Arp2/3 complex subunit ARPC1 in elongating tip cells of protonemal filaments of the moss Physcomitrella patens. Using RNA interference (RNAi) to generate loss-of-function mutants, we show dramatic defects in cell morphology manifested as short, irregularly shaped cells with abnormal division patterns. The arpc1 RNAi plants lack the rapidly elongating caulonemal cell type found in wild-type protonemal tissue. The absence of this cell type prevents normal bud formation even in response to cytokinin treatment and results in filamentous colonies lacking leafy gametophores. In addition, arpc1 protoplasts show an increased sensitivity to osmotic shock and are defective in their ability to properly establish a polarized outgrowth during regeneration from a single cell. This failure of arpc1 protoplasts to undergo proper tip growth is rescued by ARPC1 overexpression and is phenocopied in wild-type protoplasts treated with Latrunculin B, a potent inhibitor of actin polymerization. We show in moss that ARPC1, and by inference the Arp2/3 complex, plays a critical role in controlling polarized growth and cell division patterning through its regulation of actin dynamics at the cell apex.     

Identification and characterization of cDNAs encoding pentatricopeptide repeat proteins in the basal land plant, the moss Physcomitrella patens

A large gene family encoding proteins with a pentatricopeptide repeat (PPR) motif exists in flowering plants but not in algae, fungi, or animals. This suggests that PPR protein genes expanded vastly during the evolution of the land plants. To investigate this possibility, we analysed PPR protein genes in the basal land plant, the moss Physcomitrella patens. An extensive survey of the Physcomitrella expressed sequence tag (EST) databases revealed 36 ESTs encoding PPR proteins. This indicates that a large gene family of PPR proteins originated before the divergence of the vascular plant and moss lineages. We also characterized five full-length cDNAs encoding PPR proteins, designated PPR513-10, PPR566-6, PPR868-14, PPR986-12, and PPR423-6. Intracellular localization analysis demonstrated two PPR proteins in chloroplasts (cp), whereas the cellular localization of the other three PPR proteins is unclear. The genes of the cp-localized PPR513-10 and PPR566-6 were expressed differentially in protonemata grown under different light-dark conditions, suggesting they have distinctive functions in cp. This is the first report and analysis of genes encoding PPR proteins in bryophytes.     

Toc64 is not required for import of proteins into chloroplasts in the moss Physcomitrella patens

Toc64 has been suggested to be part of the chloroplast import machinery in Pisum sativum. A role for Toc64 in protein transport has not been established, however. To address this, we generated knockout mutants in the moss Physcomitrella patens using the moss's ability to perform homologous recombination with nuclear DNA. Physcomitrella patens contains two genes that encode Toc64-like proteins. Both of those proteins appear to be localized in the chloroplast. The double-mutant plants were lacking Toc64 protein in the chloroplasts but showed no growth phenotype. In addition, these plants accumulated other plastid proteins at wild-type levels and showed no difference from wild type in in vitro protein import assays. These plants did have a slightly altered chloroplast shape in some tissues, however. The evidence therefore indicates that Toc64 proteins are not required for import of proteins in Physcomitrella, but may point to involvement in the determination of plastid shape.     

Acclimation and endogenous abscisic acid in the moss Physcomitrella patens during acquisition of desiccation tolerance

The moss Physcomitrella patens has been used as a model organism to study the induction of desiccation tolerance (DT), but links between dehydration rate, the accumulation of endogenous abscisic acid (ABA) and DT remain unclear. In this study, we show that prolonged acclimation of P. patens at 89% relative humidity (RH) [?16 MPa] can induce tolerance of desiccation at 33% RH (?153 MPa) in both protonema and gametophore stages. During acclimation, significant endogenous ABA accumulation occurred after 1 day in gametophores and after 2 days in protonemata. Physcomitrella patens expressing the ABA‐inducible EARLY METHIONINE promoter fused to a cyan fluorescent protein (CFP) reporter gene revealed a mostly uniform distribution of the CFP increasing throughout the tissues during acclimation. DT was measured by day 6 of acclimation in gametophores, but not until 9 days of acclimation for protonemata. These results suggest that endogenous ABA accumulating when moss cells experience moderate water loss requires sufficient time to induce the changes that permit cells to survive more severe desiccation. These results provide insight for ongoing studies of how acclimation induces metabolic changes to enable DT in P. patens.     

Proteome analysis of chloroplasts from the moss Physcomitrella patens (Hedw.) B.S.G.

Intact chloroplasts were prepared from protoplasts of the moss Physcomitrella patens according to an especially developed method. They were additionally separated into stroma and thylakoid fractions. The proteomes of intact plastids, stroma, and thylakoids were analyzed by 1D-electrophoresis under denaturing conditions followed by protein digestion and nano-LC-ESI-MS/MS of tryptic peptides from gel bands. A total of 624 unique proteins were identified, 434 of which were annotated as chloroplast resident proteins. The majority of proteins belonged to a photosynthetic group (21.3%) and to the group of proteins implicated in protein degradation, posttranslational modification, folding, and import (20.6%). Among proteins assigned to chloroplasts, the following groups are prominent combining proteins implicated in metabolism of: amino acids (6.9%), nucleotides (2.5%), lipids (2.2%), carbohydrates (2.4%), hormones (1.5%), isoprenoids (1.25%), vitamins and cofactors (1%), sulfur (1.25%), and nitrogen (1%); as well as proteins involved in the pentose-phosphate cycle (1.75%), tetrapyrrole synthesis (3.7%), and redox processes (3.6%). The data can be used in physiological and photobiological studies as well as in further studies of P. patens chloroplast proteome including structural and functional specifics of plant protein localization in organelles.     

Large-scale analysis of 73 329 physcomitrella plants transformed with different gene disruption libraries: production parameters and mutant phenotypes

Gene targeting in the moss Physcomitrella patens has created a new platform for plant functional genomics. We produced a mutant collection of 73 329 Physcomitrella plants and evaluated the phenotype of each transformant in comparison to wild type Physcomitrella. Production parameters and morphological changes in 16 categories, such as plant structure, colour, coverage with gametophores, cell shape, etc., were listed and all data were compiled in a database (mossDB). Our mutant collection consists of at least 1804 auxotrophic mutants which showed growth defects on minimal Knop medium but were rescued on supplemented medium. 8129 haploid and 11 068 polyploid transformants had morphological alterations. 9 % of the haploid transformants had deviations in the leaf shape, 7 % developed less gametophores or had a different leaf cell shape. Other morphological deviations in plant structure, colour, and uniformity of leaves on a moss colony were less frequently observed. Preculture conditions of the plant material and the cDNA library (representing genes from either protonema, gametophore or sporophyte tissue) used to transform Physcomitrella had an effect on the number of transformants per transformation. We found correlations between ploidy level and plant morphology and growth rate on Knop medium. In haploid transformants correlations between the percentage of plants with specific phenotypes and the cDNA library used for transformation were detected. The number of different cDNAs present during transformation had no effect on the number of transformants per transformation, but it had an effect on the overall percentage of plants with phenotypic deviations. We conclude that by linking incoming molecular, proteome, and metabolome data of the transformants in the future, the database mossDB will be a valuable biological resource for systems biology.     

Light requirements for regeneration of protoplasts of the moss Physcomitrella patens

Protoplasts prepared by enzymic treatment of protonemata of the moss Physcomitrella patens regenerate rapidly in white light (15 W m^?2). The great majority of protoplasts follow a simple regenerative sequence, namely: cell wall synthesis; formation of an asymmetric cell; division of the asymmetric cell, and further extension and division to produce a new chloronemal filament. Only cell wall formation occurs independently of light. The production of an asymmetric cell requires relatively high photon fluence rates of blue or red light and ceases upon transfer to darkness. The subsequent stages of regeneration require much lower photon fluence rates, and red light is considerably more effective than blue or far-red light in permitting cell division. This system is of interest in the study of the induction of cell polarity in plants.     

Gravitropic responses of wild-type and mutant strains of the moss Physcomitrella patens

The gravitropic responses of dark-grown caulonemata and gametophores of wild-type and mutant strains of the moss Physcomitrella patens have been investigated. In the wild-type both caulonemata and gametophores show negative orthogravitropism. No gravitropic response is observed when plants are rotated slowly on a clinostat and the inductive effect of gravity can be replaced by centrifugal force. The gravitropic response of caulonemanta is biphasic, consisting of an initial phase producing a bend of about 20 degrees within 12 h of 90 degrees reorientation and a subsequent slower phase leading to completion of the 90 degrees curvature. No obvious sedimentation of statoliths accompanies this response. Several mutants have been isolated that are either partially or completely impaired in caulonemal gravitropism and one mutant shows a positive gravitropic response. Complementation analysis using somatic hybrids obtained following protoplast fusion indicates that at least three genes can mutate to give an altered gravitropic phenotype. None of these mutants is altered in gametophore gravitropism, suggesting that the gravitropic response of caulonemal filaments may require at least some gene products that are not required for the response of the multicellular gametophores. One class of mutant with impaired caulonemal gravitropism shows a pleiotropic alteration in leaf shape.