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.     

Involvement of mitochondrial-targeted RecA in the repair of mitochondrial DNA in the moss, Physcomitrella patens

Homologous recombination is a universal process that contributes to genetic diversity and genomic integrity. Bacterial-type RecA generally exists in all bacteria and plays a crucial role in homologous recombination. Although RecA homologues also exist in plant mitochondria, there have been few reports about the in vivo functions of these homologues. We identified a recA gene orthologue (named PprecA1) in a cDNA library of the moss, Physcomitrella patens. N-terminal fusion of the putative organellar targeting sequence of PpRecA1 to GFP caused a targeting of PpRecA1 to mitochondria. PprecA1 partially complemented the effects of a DNA damaging agent in an Escherichia coli recA deficient strain. Additionally, the expression of PprecA1 was induced by treating the plants with DNA damaging agents. Disruption of PprecA1 by targeted replacement resulted lower rate of the recovery of the mitochondrial DNA from methyl methan sulfonate damage. This is the first report about the characteristics of a null mutant of bacterial-type recA gene in plant. The data suggest that PprecA1 participates in the repair of mitochondrial DNA in P. patens.     

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.     

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.     

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...     

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.     

Expression and Complementation Analyses of a Chloroplast-Localized Homolog of Bacterial RecA in the Moss Physcomitrella patens

RecA protein is widespread in bacteria, and it plays a crucial role in homologous recombination. We have identified two bacterial-type recA gene homologs (PprecA1, PprecA2) in the cDNA library of the moss Physcomitrella patens. N-terminal fusion of the putative organellar targeting sequence of PpRecA2 to the green fluorescent protein (GFP) caused a targeting of PpRecA2 to the chloroplasts. Mutational analysis showed that the first AUG codon acts as initiation codon. Fusion of the full-length PpRecA2 to GFP caused the formation of foci that were colocalized with chloroplast nucleoids. The amounts of PprecA2 mRNA and protein in the cells were increased by treatment with DNA damaging agents. PprecA2 partially complemented the recA mutation in Escherichia coli. These results suggest the involvement of PpRecA2 in the repair of chloroplast DNA.     

Characterization of a PDK1 homologue from the moss Physcomitrella patens

The serine/threonine protein kinase 3-phosphoinositide-dependent protein kinase 1 (PDK1) is a highly conserved eukaryotic kinase that is a central regulator of many AGC kinase subfamily members. Through its regulation of AGC kinases, PDK1 controls many basic cellular processes, from translation to cell survival. While many of these PDK1-regulated processes are conserved across kingdoms, it is not well understood how PDK1 may have evolved within kingdoms. In order to better understand PDK1 evolution within plants, we have isolated and characterized the PDK1 gene from the moss Physcomitrella patens (PpPDK1), a nonvascular representative of early land plants. PpPDK1 is similar to other plant PDK1s in that it can functionally complement a yeast PDK1 knockout line. However, unlike PDK1 from other plants, the P. patens PDK1 protein does not bind phospholipids due to a lack of the lipid-binding pleckstrin homology domain, which is used for lipid-mediated regulation of PDK1 activity. Sequence analysis of several PDK1 proteins suggests that lipid regulation of PDK1 may not commonly occur in algae and nonvascular land plants. PpPDK1 can phosphorylate AGC kinase substrates from tomato (Solanum lycopersicum) and P. patens at the predicted PDK1 phosphorylation site, indicating that the PpPDK1 substrate phosphorylation site is conserved with higher plants. We have also identified residues within the PpPDK1 kinase domain that affect kinase activity and show that a mutant with highly reduced kinase activity can still confer cell viability in both yeast and P. patens. These studies lay the foundation for further analysis of the evolution of PDK1 within plants.     

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.     

Photoperiod-regulated expression of the PpCOL1 gene encoding a homolog of CO/COL proteins in the moss Physcomitrella patens

The CONSTANS (CO) protein is a critical regulator of the photoperiodic control of flowering in Arabidopsis thaliana and Oryza sativa. We isolated a cDNA PpCOL1 encoding a homolog of the CO/CO-LIKE (COL) family proteins from a cryptogam Physcomitrella patens. The predicted PpCOL1 protein has N-terminal zinc finger and C-terminal CCT domains, which are conserved in the angiosperm CO/COL proteins. Structurally, PpCOL1 is the most closely related to the Group Ia or Ic proteins, which include AtCO and AtCOL1/2, among diverged members of the family. A transient expression assay using GFP showed that the CCT domain of PpCOL1 contains a nuclear-localizing signal. Northern blotting analyses revealed that the PpCOL1 expression is controlled by the circadian clock, and moreover, it is photoperiodically regulated at a gametophore stage when the rate of sporophyte formation is affected by day length. These observations indicate a possible involvement of PpCOL1 as a nuclear factor in the photoperiodic regulation of reproduction of Physcomitrella.     

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.     

Molecular cloning and characterization of a sodium-pump ATPase of the moss Physcomitrella patens

Physcomitrella patens grew slowly at 600 mm Na+, pH 6.0, affected by the low water potential but without signs of suffering Na+ toxicity. At pH 8.0, tolerance seemed to be lower but it grew at 200 mm Na+, again without signs of Na+ toxicity. The resistance of Physcomitrella cells to the toxic effects of Na+ can be accounted for by their capacity to keep high K+:Na+ ratios and to extrude Na+ by a system that is not dependent on DeltapH. Physcomitrella expresses two P-type ATPases similar in sequence to fungal ENA-type Na+-ATPases. A functional study in yeast demonstrated that one of these ATPases, PpENA1, is an Na+-pump. We also found that P. patens has a plant-type SOS1 Na+/H+ antiporter. We discuss that Na+-ATPases existed in early land plants but that they were lost during the evolution of bryophytes to flowering plants.     

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.).