Exploration and identification of Physcomitrella patens moss peptides

In the current study the isolation and identification of Physcomitrella patens (Hedw.) B.S.G. moss peptides are described. Physcomitrella patens moss is actively used in recent years as a model organism to study the biology of plants. Protoplasts, protonemata and gametophores of the moss are demonstrated for the first time to contain diverse small peptides. From gametophores was isolated and identified 58 peptides that are fragments of 14 proteins, and from protonemata - 49 peptides, fragments of 15 proteins. It was found that the protonemata and gametophores Ph. patens, which are the successive stages of development of this plant, significantly different from each other as a peptide composition and the spectrum of the precursor protein of identified peptides. Isolation of protoplasts of the enzymatic destruction of cell wall protonemata accompanied by massive degradation of intracellular proteins, many of whom are proteins of photosynthesis, which is a characteristic response of plants to stress the impact of environmental factors. A total of moss protoplasts were isolated and identified 323 peptides that are fragments of 79 proteins.     

MicroRNAs in the moss Physcomitrella patens

Having diverged from the lineage that lead to flowering plants shortly after plants have established on land, mosses, which share fundamental processes with flowering plants but underwent little morphological changes by comparison with the fossil records, can be considered as an evolutionary informative place. Hence, they are especially useful for the study of developmental evolution and adaption to life on land. The transition to land exposed early plants to harsh physical conditions that resulted in key physiological and developmental changes. MicroRNAs (miRNAs) are an important class of small RNAs (sRNAs) that act as master regulators of development and stress in flowering plants. In recent years several groups have been engaged in the cloning of sRNAs from the model moss Physcomitrella patens. These studies have revealed a wealth of miRNAs, including novel and conserved ones, creating a unique opportunity to broaden our understanding of miRNA functions in land plants and their contribution to the latter??s evolution. Here we review the current knowledge of moss miRNAs and suggest approaches for their functional analysis in P. patens.     

RNA interference in the moss Physcomitrella patens

The moss Physcomitrella patens performs efficient homologous recombination, which allows for the study of individual gene function by generating gene disruptions. Yet, if the gene of study is essential, gene disruptions cannot be isolated in the predominantly haploid P. patens. Additionally, disruption of a gene does not always generate observable phenotypes due to redundant functions from related genes. However, RNA interference (RNAi) can provide mutants for both of these situations. We show that RNAi disrupts gene expression in P. patens, adding a significant tool for the study of plant gene function. To assay for RNAi in moss, we constructed a line (NLS-4) expressing a nuclearly localized green fluorescent protein (GFP):beta-glucuronidase (GUS) fusion reporter protein. We targeted the reporter protein with two RNAi constructs, GUS-RNAi and GFP-RNAi, expressed transiently by particle bombardment. Transformed protonemal cells are marked by cobombardment with dsRed2, which diffuses between the nucleus and cytoplasm. Cells transformed with control constructs have nuclear/cytoplasmic red fluorescence and nuclear green fluorescence. In cells transformed with GUS-RNAi or GFP-RNAi constructs, the nuclear green fluorescence was reduced on average 9-fold as soon as 48 h after transformation. Moreover, isolated lines of NLS-4 stably transformed with GUS-RNAi construct have silenced nuclear GFP, indicating that RNAi is propagated stably. Thus, RNAi adds a powerful tool for functional analysis of plant genes in moss.     

Stable transformation of the moss Physcomitrella patens

Summary We report the stable transformation of Physcomitrella patens to either G418 or hygromycin B resistance following polyethylene glycol-mediated direct DNA uptake by protoplasts. The method described in this paper was used successfully in independent experiments carried out in our two laboratories. Transformation was assessed by the following criteria: selection of antibiotic-resistant plants, mitotic and meiotic stability of phenotypes after removal of selective pressure and stable transmission of the character to the offspring; Southern hybridisation analysis of genomic DNA to show integration of the plasmid DNA; segregation of the resistance gene following crosses with antibiotic-sensitive strains; and finally Southern hybridisation analysis of both resistant and sensitive progeny. In addition to stable transformants, a heterogeneous class of unstable transformants was obtained.     

Isolation of mutant lines with decreased numbers of chloroplasts per cell from a tagged mutant library of the moss Physcomitrella patens

Eleven mutant lines exhibiting decreased numbers of chloroplasts per cell were isolated from 8 800 tagged mutant lines of Physcomitrella patens by microscopic observations. Chloronema subapical cells in wild-type plants had a mean of 48 chloroplasts, whereas chloroplast numbers in subapical cells in mutant lines 215 and 222 decreased to 75 % of that in the wild type. Seven mutant lines - 473, 122, 221, 129, 492, 207, and 138 - had about half as many chloroplasts as the wild type. Mutant line 11 had a few remarkably enlarged chloroplasts, and mutant line 347 had chloroplasts of various sizes. Whereas the cell volume was the same as in the wild type in mutant lines 222, 473, 221, 129, 492, and 207, the cell volume of the other mutants increased. The chloroplast number of leaf cells was the same as that of chloronema cells in each mutant line when gametophores could be formed. Treatment with ampicillin decreased the number of chloroplasts in all mutant lines. Southern hybridization using DNA in tags as probes showed that only one insertion occurred in mutant lines 473 and 221. To determine whether the tagged DNA inserted into the known genes for plastid division, we isolated the PpMinD1, PpMinD2, and PpMinE1 genes. Genomic polymerase chain reaction analysis showed that the PpFtsZ and PpMinD/E genes were not disrupted by the insertion of the tags in mutant lines 11 and 347, respectively.     

Efficient gene targeting in the moss Physcomitrella patens

The moss Physcomitrella patens is used as a genetic model system to study plant development, taking advantage of the fact that the haploid gametophyte dominates in its life cycle. Transformation experiments designed to target three single-copy genomic loci were performed to determine the efficiency of gene targeting in this plant. Mean transformation rates were 10-fold higher with the targeting vectors and molecular evidence for the integration of exogenous DNA into each targeted locus by homologous recombination is provided. The efficiency of gene targeting determined in these experiments is above 90%, which is in the range of that observed in yeast and several orders of magnitude higher than previous reports of gene targeting in plants. Thus, gene knock-out and allele replacement approaches are directly accessible to study plant development in the moss Physcomitrella patens . Moreover, efficient gene targeting has so far only been observed in lower eukaryotes such as protozoa, yeasts and filamentous fungi, and, as shown here the first example from the plant kingdom is a haplobiontic moss. This suggests a possible correlation between efficient gene targeting and haplo-phase in eukaryotes.     

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.     

Somatic mutagenesis of the moss,Physcomitrella patens

Molecular Genetics and Genomics - Spores have been preferred for mutagenic treatment of Physcomitrella patens. Many mutant strains are, however, sexually sterile and so do not produce spores. We...     

Biosynthesis of C9-aldehydes in the moss Physcomitrella patens

After wounding, the moss Physcomitrella patens emits fatty acid derived volatiles like octenal, octenols and (2E)-nonenal. Flowering plants produce nonenal from C18-fatty acids via lipoxygenase and hydroperoxide lyase reactions, but the moss exploits the C20 precursor arachidonic acid for the formation of these oxylipins. We describe the isolation of the first cDNA (PpHPL) encoding a hydroperoxide lyase from a lower eukaryotic organism. The physiological pathway allocation and characterization of a downstream enal-isomerase gives a new picture for the formation of fatty acid derived volatiles from lower plants. Expression of a fusion protein with a yellow fluorescent protein in moss protoplasts showed that PpHPL was found in clusters in membranes of plastids. PpHPL can be classified as an unspecific hydroperoxide lyase having a substrate preference for 9-hydroperoxides of C18-fatty acids but also the predominant substrate 12-hydroperoxy arachidonic acid is accepted. Feeding experiments using arachidonic acid show an increase in the 12-hydroperoxide being metabolized to C8-aldehydes/alcohols and (3Z)-nonenal, which is rapidly isomerized to (2E)-nonenal. PpHPL knock out lines failed to emit (2E)-nonenal while formation of C8-volatiles was not affected indicating that in contrast to flowering plants, PpHPL is only involved in formation of a specific subset of volatiles.     

Establishment of gene-trap and enhancer-trap systems in the moss Physcomitrella patens

Because of its simple body plan and ease of gene knockout and allele replacement, the moss Physcomitrella patens is often used as a model system for studies in plant physiology and developmental biology. Gene-trap and enhancer-trap systems are useful techniques for cloning genes and enhancers that function in specific tissues or cells. Additionally, these systems are convenient for obtaining molecular markers specific for certain developmental processes. Elements for gene-trap and enhancer-trap systems were constructed using the uidA reporter gene with either a splice acceptor or a minimal promoter. Through a high rate of transformation conferred by a method utilizing homologous recombination, 235 gene-trap and 1073 enhancer-trap lines were obtained from 5637 and 3726 transgenic lines, respectively. The expression patterns of these trap lines in the moss gametophyte varied. The candidate gene trapped in a gene-trap line YH209, which shows rhizoid-specific expression, was obtained by 5' and 3' RACE. This gene was named PpGLU, and forms a clade with plant acidic alpha-glucosidase genes. Thus, these gene-trap and enhancer-trap systems should prove useful to identify tissue- and cell-specific genes in Physcomitrella.     

Adaptive basis of codon usage in the haploid moss Physcomitrella patens

Patterns of codon usage bias were studied in the moss model species Physcomitrella patens. A total of 92 nuclear, protein coding genes were employed, and estimated levels of gene expression were tested for association with two measures of codon usage bias and other variables hypothesized to be associated with gene expression. Codon bias was found to be positively associated both with estimated levels of gene expression and GC content in the coding parts of studied genes. However, GC content in noncoding parts, that is, introns and 5' and 3' untranslated regions (UTRs), was not associated with estimated levels of gene expression. It is argued that codon bias is not shaped by mutational bias, but rather by weak natural selection for translational efficiency in P. patens. The possible role of life history characteristics in shaping patterns of codon usage in this species is discussed.     

Calcium-dependent membrane depolarisation activated by phytochrome in the moss Physcomitrella patens

In caulonemal filaments of Physcomitrella patens which had been preincubated in the dark for 24 h, irradiation with red light (640 nm, fluence rate 85 mol · m^–2 · s^–1) evoked (i) the development of side branch initials and (ii) a rapid, but transient, depolarisation of the plasma membrane by 90 ± 13 mV from a resting potential of -178 ± 13 mV. This was followed by a transient hyperpolarisation to a value 21± 8 mV more negative than the original membrane potential. The refractory period for the transient depolarisation was between 12 and 15 min. The fluence rate of red light required to evoke maximal depolarisation was about 80 mol · m^–2 · s^–1 for a 1-min pulse. At this fluence rate, a depolarising response could be recorded for pulse lengths as small as 7 s. The transient depolarisation was insensitive to 3-(3,4dichlorophenyl)-1,1-dimethyl urea (DCMU) and was unchanged in plants bleached by growth on norflurazon (SAN 9789). Furthermore, the electrical response could be blocked by simultaneous application of far-red light. These results suggest the involvement of the photoreceptor phytochrome in the response. Removing Ca²⁺ from the external medium or replacing Ca²⁺ with Mg²⁺ blocked the depolarisation. The depolarisation could also be blocked by the K⁺ channel-blocker tetraethylammonium (10 mM) and the Cl^– channel-blocker niflumic acid (1 M). Conversely, although calcium channel-antagonists such as nifedipine and lanthanides, applied at a concentration of 100 M, also altered the response, they did not block it. A possible ionic mechanism for the membrane potential transient is advanced, and the physiological significance discussed in the context of early events in the phytochrome signalling pathway.Abbreviations [Ca²⁺]_c cytosolic Ca²⁺ concentration - DCMU 3-(3,4-dichlorophenyt)-1,1-dimethylurea - TEA tetraethylammonium We thank Prof. David Cove (Department of Genetics, University of Leeds) for fruitful discussions, providing plants and advice on culturing methods, Dr. Richard Firn (York) for stimulating discussions, Ian Jennings (York) for technical advice on the electrophysiological apparatus, and Anna Bate (York) for looking after the plant cultures. Financial support was received from the Biotechnology and Biological Sciences Research Council (Grant P87/4043 to D.S. and Grant PDF/14 to E.J.) and The New Phytologist Trust (studentship support to E.E.).     

Profilin is essential for tip growth in the moss Physcomitrella patens

The actin cytoskeleton is critical for tip growth in plants. Profilin is the main monomer actin binding protein in plant cells. The moss Physcomitrella patens has three profilin genes, which are monophyletic, suggesting a single ancestor for plant profilins. Here, we used RNA interference (RNAi) to determine the loss-of-function phenotype of profilin. Reduction of profilin leads to a complete loss of tip growth and a partial inhibition of cell division, resulting in plants with small rounded cells and fewer cells. We silenced all profilins by targeting their 3' untranslated region sequences, enabling complementation analyses by expression of profilin coding sequences. We show that any moss or a lily (Lilium longiflorum) profilin support tip growth. Profilin with a mutation in its actin binding site is unable to rescue profilin RNAi, while a mutation in the poly-l-proline binding site weakly rescues. We show that moss tip growing cells contain a prominent subapical cortical F-actin structure composed of parallel actin cables. Cells lacking profilin lose this structure; instead, their F-actin is disorganized and forms polarized cortical patches. Plants expressing the actin and poly-l-proline binding mutants exhibited similar F-actin disorganization. These results demonstrate that profilin and its binding to actin are essential for tip growth. Additionally, profilin is not needed for formation of F-actin, but profilin and its interactions with actin and poly-l-proline ligands are required to properly organize F-actin.     

Autophagy is required for gamete differentiation in the moss Physcomitrella patens

Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abiotic stresses (through the induction or repression of programmed cell death, PCD) as well as to promotion of developmentally regulated PCD, starch degradation or caloric restriction important for life span. Much less is known regarding its role in plant cell differentiation. Here we show that macroautophagy, the autophagy pathway driven by engulfment of cytoplasmic components by autophagosomes and its subsequent degradation in vacuoles, is highly active during germ cell differentiation in the early diverging land plant Physcomitrella patens. Our data provide evidence that suppression of ATG5-mediated autophagy results in reduced density of the egg cell-mediated mucilage that surrounds the mature egg, pointing toward a potential role of autophagy in extracellular mucilage formation. In addition, we found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility. The similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking, strongly pointing toward an ancestral function of autophagy not only as a protector against nutrient stress, but also in gamete differentiation.