Cell wall biology of the moss Physcomitrium patens.

The moss Physcomitrium (previously Physcomitrella) patens is a non-vascular plant belonging to the bryophytes that has been used as a model species to study the evolution of plant cell wall structure and biosynthesis. Here, we present an updated review of the cell wall biology of P. patens. Immunocytochemical and structural studies have shown that the cell walls of P. patens mainly contain cellulose, hemicelluloses (xyloglucan, xylan, glucomannan, and arabinoglucan), pectin, and glycoproteins, and their abundance varies among different cell types and at different plant developmental stages. Genetic and biochemical analyses have revealed that a number of genes involved in cell wall biosynthesis are functionally conserved between P. patens and vascular plants, indicating that the common ancestor of mosses and vascular plants had already acquired most of the biosynthetic machinery to make various cell wall polymers. Although P. patens does not synthesize lignin, homologs of the phenylpropanoid biosynthetic pathway genes exist in P. patens and they play an essential role in the production of caffeate derivatives for cuticle formation. Further genetic and biochemical dissection of cell wall biosynthetic genes in P. patens promises to provide additional insights into the evolutionary history of plant cell wall structure and biosynthesis.

Genome-Wide Identification and Characterization of Actin-Depolymerizing Factor (ADF) Family Genes and Expression Analysis of Responses to Various Stresses in Zea Mays L.

Actin-depolymerizing factor (ADF) is a small class of actin-binding proteins that regulates the dynamics of actin in cells. Moreover, it is well known that the plant ADF family plays key roles in growth, development and defense-related functions. Results: Thirteen maize (Zea mays L., ZmADFs) ADF genes were identified using Hidden Markov Model. Phylogenetic analysis indicated that the 36 identified ADF genes in Physcomitrella patens, Arabidopsis thaliana, Oryza sativa japonica, and Zea mays were clustered into five groups. Four pairs of segmental genes were found in the maize ADF gene family. The tissue-specific expression of ZmADFs and OsADFs was analyzed using microarray data obtained from the Maize and Rice eFP Browsers. Five ZmADFs (ZmADF1/2/7/12/13) from group V exhibited specifically high expression in tassel, pollen, and anther. The expression patterns of 13 ZmADFs in seedlings under five abiotic stresses were analyzed using qRT-PCR, and we found that the ADFs mainly responded to heat, salt, drought, and ABA. Conclusions: In our study, we identified ADF genes in maize and analyzed the gene structure and phylogenetic relationships. The results of expression analysis demonstrated that the expression level of ADF genes was diverse in various tissues and different stimuli, including abiotic and phytohormone stresses, indicating their different roles in plant growth, development, and response to external stimulus. This report extends our knowledge to understand the function of ADF genes in maize.

Expanding the CRISPR Toolbox in P. patens Using SpCas9-NG Variant and Application for Gene and Base Editing in Solanaceae Crops.

Genome editing has become a major tool for both functional studies and plant breeding in several species. Besides generating knockouts through the classical CRISPR-Cas9 system, recent development of CRISPR base editing holds great and exciting opportunities for the production of gain-of-function mutants. The PAM requirement is a strong limitation for CRISPR technologies such as base editing, because the base substitution mainly occurs in a small edition window. As precise single amino-acid substitution can be responsible for functions associated to some domains or agronomic traits, development of Cas9 variants with relaxed PAM recognition is of upmost importance for gene function analysis and plant breeding. Recently, the SpCas9-NG variant that recognizes the NGN PAM has been successfully tested in plants, mainly in monocotyledon species. In this work, we studied the efficiency of SpCas9-NG in the model moss Physcomitrella patens and two Solanaceae crops (Solanum lycopersicum and Solanum tuberosum) for both classical CRISPR-generated gene knock-out and cytosine base editing. We showed that the SpCas9-NG greatly expands the scope of genome editing by allowing the targeting of non-canonical NGT and NGA PAMs. The CRISPR toolbox developed in our study opens up new gene function analysis and plant breeding perspectives for model and crop plants.

Acyl-CoA synthetases from Physcomitrella, rice and Arabidopsis: different substrate preferences but common regulation by MS188 in sporopollenin synthesis.

MAIN CONCLUSION: ACOS5, OsACOS12 and PpACOS6 are all capable of fatty acyl-CoA synthetase activity but exhibit different substrate preferences. The transcriptional regulation of ACOS for sporopollenin synthesis appears to have been conserved in Physcomitrella, rice and Arabidopsis during evolution. Sporopollenin is the major constituent of spore and pollen exines. In Arabidopsis, acyl-CoA synthetase 5 (ACOS5) is an essential enzyme for sporopollenin synthesis, and its orthologues are PpACOS6 from the moss Physcomitrella and OsACOS12 from monocot rice. However, knowledge regarding the evolutionary conservation and divergence of the ACOS gene in sporopollenin synthesis remains limited. In this study, we analysed the function and regulation of PpACOS6 and OsACOS12. A complementation test showed that OsACOS12 driven by the ACOS5 promoter could partially restore the male fertility of the acos5 mutant in Arabidopsis, while PpACOS6 did not rescue the acos5 phenotype. ACOS5, PpACOS6 and OsACOS12 all complemented the acyl-CoA synthetase-deficient yeast strain (YB525) phenotype, although they exhibited different substrate preferences. To understand the conservation of sporopollenin synthesis regulation, we constructed two constructs with ACOS5 driven by the OsACOS12 or PpACOS6 promoter. Both constructs could restore the fertility of acos5 plants. The MYB transcription factor MS188 from Arabidopsis directly regulates ACOS5. We found that MS188 could also bind the promoters of OsACOS12 and PpACOS6 and activate the genes driven by the promoters, suggesting that the transcriptional regulation of these genes was similar to that of ACOS5. These results show that the ACOS gene promoter region from Physcomitrella, rice and Arabidopsis has been functionally conserved during evolution, while the chain lengths of fatty acid-derived monomers of sporopollenin vary in different plant species.

An event of alternative splicing affects the expression of the NTRC gene, encoding NADPH-thioredoxin reductase C, in seed plants.

The NTRC gene encodes a NADPH-dependent thioredoxin reductase with a joint thioredoxin domain, exclusive of photosynthetic organisms. An updated search shows that although most species harbor a single copy of the NTRC gene, two copies were identified in different species of the genus Solanum, Glycine max and the moss Physcomitrella patens. The phylogenetic analysis of NTRCs from different sources produced a tree with the major groups of photosynthetic organisms: cyanobacteria, algae and land plants, indicating the evolutionary success of the NTRC gene among photosynthetic eukaryotes. An event of alternative splicing affecting the expression of the NTRC gene was identified, which is conserved in seed plants but not in algae, bryophytes and lycophytes. The alternative splicing event results in a transcript with premature stop codon, which would produce a truncated form of the enzyme. The standard splicing/alternative splicing (SS/AS) transcripts ratio was higher in photosynthetic tissues from Arabidopsis, Brachypodium and tomato, in line with the higher content of the NTRC polypeptide in these tissues. Moreover, environmental stresses such as cold or high salt affected the SS/AS ratio of the NTRC gene transcripts in Brachypodium seedlings. These results suggest that the alternative splicing of the NTRC gene might be an additional mechanism for modulating the content of NTRC in photosynthetic and non-photosynthetic tissues of seed plants.

ATG5-knockout mutants of Physcomitrella provide a platform for analyzing the involvement of autophagy in senescence processes in plant cells.

Autophagy is a pathway in which a cell degrades part of its cytoplasm in vacuoles or lysosomes. To identify the physiological functions of autophagy in plants, we disrupted ATG5, an autophagy-related gene, in Physcomitrella, and confirmed that atg5 mutants are deficient in the process of autophagy. On carbon or nitrogen starvation medium, atg5 colonies turned yellow earlier than the wild-type (WT) colonies, showing that Physcomitrella atg5 mutants, like yeast and Arabidopsis, are sensitive to nutrient starvation. In the dark, even under nutrient-sufficient conditions, colonies turned yellow and the net degradation of chlorophyll and Rubisco protein occurred together with the upregulation of several senescence-associated genes. Yellowing reactions were inhibited by the protein synthesis inhibitor cycloheximide, suggesting that protonemal colonies undergo dark-induced senescence like the green leaves of higher plants. Such senescence responses in the dark occurred earlier in atg5 colonies than WT colonies. The sugar content was almost the same between WT and atg5 colonies, indicating that the early-senescence phenotype of atg5 is not explained by sugar deficiency. However, the levels of 7 amino acids showed significantly different alteration between atg5 and WT in the dark: 6 amino acids, particularly arginine and alanine, were much more deficient in the atg5 mutants, irrespective of the early degradation of Rubisco protein. On nutrient-sufficient medium supplemented with casamino acids, the early-senescence phenotype was slightly moderated. We propose that the early-senescence phenotype in atg5 mutants is partly explained by amino acid imbalance because of the lack of cytoplasmic degradation by autophagy in Physcomitrella.

Evolutionary relationships among barley and Arabidopsis core circadian clock and clock-associated genes.

The circadian clock regulates a multitude of plant developmental and metabolic processes. In crop species, it contributes significantly to plant performance and productivity and to the adaptation and geographical range over which crops can be grown. To understand the clock in barley and how it relates to the components in the Arabidopsis thaliana clock, we have performed a systematic analysis of core circadian clock and clock-associated genes in barley, Arabidopsis and another eight species including tomato, potato, a range of monocotyledonous species and the moss, Physcomitrella patens. We have identified orthologues and paralogues of Arabidopsis genes which are conserved in all species, monocot/dicot differences, species-specific differences and variation in gene copy number (e.g. gene duplications among the various species). We propose that the common ancestor of barley and Arabidopsis had two-thirds of the key clock components identified in Arabidopsis prior to the separation of the monocot/dicot groups. After this separation, multiple independent gene duplication events took place in both monocot and dicot ancestors.

Conservation of Male Sterility 2 function during spore and pollen wall development supports an evolutionarily early recruitment of a core component in the sporopollenin biosynthetic pathway.

The early evolution of plants required the acquisition of a number of key adaptations to overcome physiological difficulties associated with survival on land. One of these was a tough sporopollenin wall that enclosed reproductive propagules and provided protection from desiccation and UV-B radiation. All land plants possess such walled spores (or their derived homologue, pollen). We took a reverse genetics approach, consisting of knock-out and complementation experiments to test the functional conservation of the sporopollenin-associated gene MALE STERILTY 2 (which is essential for pollen wall development in Arabidopsis thaliana) in the bryophyte Physcomitrella patens. Knock-outs of a putative moss homologue of the A. thaliana MS2 gene, which is highly expressed in the moss sporophyte, led to spores with highly defective walls comparable to that observed in the A. thaliana ms2 mutant, and extremely compromised germination. Conversely, the moss MS2 gene could not rescue the A. thaliana ms2 phenotype. The results presented here suggest that a core component of the biochemical and developmental pathway required for angiosperm pollen wall development was recruited early in land plant evolution but the continued increase in pollen wall complexity observed in angiosperms has been accompanied by divergence in MS2 gene function.

Phylogenetic analysis of pectin-related gene families in Physcomitrella patens and nine other plant species yields evolutionary insights into cell walls.

BACKGROUND: Pectins are acidic sugar-containing polysaccharides that are universally conserved components of the primary cell walls of plants and modulate both tip and diffuse cell growth. However, many of their specific functions and the evolution of the genes responsible for producing and modifying them are incompletely understood. The moss Physcomitrella patens is emerging as a powerful model system for the study of plant cell walls. To identify deeply conserved pectin-related genes in Physcomitrella, we generated phylogenetic trees for 16 pectin-related gene families using sequences from ten plant genomes and analyzed the evolutionary relationships within these families. RESULTS: Contrary to our initial hypothesis that a single ancestral gene was present for each pectin-related gene family in the common ancestor of land plants, five of the 16 gene families, including homogalacturonan galacturonosyltransferases, polygalacturonases, pectin methylesterases, homogalacturonan methyltransferases, and pectate lyase-like proteins, show evidence of multiple members in the early land plant that gave rise to the mosses and vascular plants. Seven of the gene families, the UDP-rhamnose synthases, UDP-glucuronic acid epimerases, homogalacturonan galacturonosyltransferase-like proteins, ß-1,4-galactan ß-1,4-galactosyltransferases, rhamnogalacturonan II xylosyltransferases, and pectin acetylesterases appear to have had a single member in the common ancestor of land plants. We detected no Physcomitrella members in the xylogalacturonan xylosyltransferase, rhamnogalacturonan I arabinosyltransferase, pectin methylesterase inhibitor, or polygalacturonase inhibitor protein families. CONCLUSIONS: Several gene families related to the production and modification of pectins in plants appear to have multiple members that are conserved as far back as the common ancestor of mosses and vascular plants. The presence of multiple members of these families even before the divergence of other important cell wall-related genes, such as cellulose synthases, suggests a more complex role than previously suspected for pectins in the evolution of land plants. The presence of relatively small pectin-related gene families in Physcomitrella as compared to Arabidopsis makes it an attractive target for analysis of the functions of pectins in cell walls. In contrast, the absence of genes in Physcomitrella for some families suggests that certain pectin modifications, such as homogalacturonan xylosylation, arose later during land plant evolution.

HAK transporters from Physcomitrella patens and Yarrowia lipolytica mediate sodium uptake.

The widespread presence of Na(+)-specific uptake systems across plants and fungi is a controversial topic. In this study, we identify two HAK genes, one in the moss Physcomitrella patens and the other in the yeast Yarrowia lipolytica, that encode Na(+)-specific transporters. Because HAK genes are numerous in plants and are duplicated in many fungi, our findings suggest that some HAK genes encode Na(+) transporters and that Na(+) might play physiological roles in plants and fungi more extensively than is currently thought.

Rice plants expressing the moss sodium pumping ATPase PpENA1 maintain greater biomass production under salt stress.

High cytosolic concentrations of Na+ inhibit plant growth and development. To maintain low cytosolic concentrations of Na+ , higher plants use membrane-bound transporters that drive the efflux of Na+ or partition Na+ ions from the cytosol, either to the extracellular compartment or into the vacuole. Bryophytes also use an energy-dependent Na+ pumping ATPase, not found in higher plants, to efflux Na+ . To investigate whether this transporter can increase the salt tolerance of crop plants, Oryza sativa has been transformed with the Physcomitrella patens Na+ pumping ATPase (PpENA1). When grown in solutions containing 50 mm NaCl, plants constitutively expressing the PpENA1 gene are more salt tolerant and produce greater biomass than controls. Transgenics and controls accumulate similar amounts of Na+ in leaf and root tissues under stress, which indicates that the observed tolerance is not because of Na+ exclusion. Moreover, inductively coupled plasma analysis reveals that the concentration of other ions in the transformants and the controls is similar. The transgenic lines are developmentally normal and fertile, and the transgene expression levels remain stable in subsequent generations. GFP reporter fusions, which do not alter the ability of PpENA1 to complement a salt-sensitive yeast mutant, indicate that when it is expressed in plant tissues, the PpENA1 protein is located in the plasma membrane. PpENA1 peptides are found in plasma membrane fractions supporting the plasma membrane targeting. The results of this study demonstrate the utility of PpENA1 as a potential tool for engineering salinity tolerance in important crop species.

The moss Physcomitrella patens contains cyclopentenones but no jasmonates: mutations in allene oxide cyclase lead to reduced fertility and altered sporophyte morphology.

• Two cDNAs encoding allene oxide cyclases (PpAOC1, PpAOC2), key enzymes in the formation of jasmonic acid (JA) and its precursor (9S,13S)-12-oxo-phytodienoic acid (cis-(+)-OPDA), were isolated from the moss Physcomitrella patens. • Recombinant PpAOC1 and PpAOC2 show substrate specificity against the allene oxide derived from 13-hydroperoxy linolenic acid (13-HPOTE); PpAOC2 also shows substrate specificity against the allene oxide derived from 12-hydroperoxy arachidonic acid (12-HPETE). • In protonema and gametophores the occurrence of cis-(+)-OPDA, but neither JA nor the isoleucine conjugate of JA nor that of cis-(+)-OPDA was detected. • Targeted knockout mutants for PpAOC1 and for PpAOC2 were generated, while double mutants could not be obtained. The ΔPpAOC1 and ΔPpAOC2 mutants showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis.

The SOS1 transporter of Physcomitrella patens mediates sodium efflux in planta.

• SOS1 is an Na(+)/H(+) antiporter that plays a central role in Na(+) tolerance in land plants. SOS1 mediation of Na(+) efflux has been studied in plasma-membrane vesicles and deduced from the SOS1 suppression of the Na(+) sensitivity of yeast mutants defective in Na(+) -efflux. However, SOS1-mediated Na(+) efflux has not been characterized in either plant or yeast cells. Here, we use Physcomitrella patens to investigate the function of SOS1 in planta. • In P. patens, a nonvascular plant in which the study of ion cellular fluxes is technically simple, the existence of two SOS1 genes suggests that the Na(+) efflux remaining after the deletion of the ENA1 ATPase is mediated by a SOS1 system. Therefore, we cloned the P. patens SOS1 and SOS1B genes (PpSOS1 and PpSOS1B, respectively) and complementary DNAs, and constructed the PpΔsos1 and PpΔena1/PpΔsos1 deletion lines by gene targeting. • Comparison of wild-type, and PpΔsos1 and PpΔena1/PpΔsos1 mutant lines revealed that PpSOS1 is crucial for Na(+) efflux and that the PpΔsos1 line, and especially the PpΔena1/PpΔsos1 lines, showed excessive Na(+) accumulation and Na(+)-triggered cell death. The PpΔsos1 and PpΔena1/PpΔsos1 lines showed impaired high-affinity K(+) uptake. • Our data support the hypothesis that PpSOS1 mediates cellular Na(+) efflux and that PpSOS1 enhances K(+) uptake by an indirect effect.

Comparison of the chloroplast peroxidase system in the chlorophyte Chlamydomonas reinhardtii, the bryophyte Physcomitrella patens, the lycophyte Selaginella moellendorffii and the seed plant Arabidopsis thaliana.

BACKGROUND: Oxygenic photosynthesis is accompanied by the formation of reactive oxygen species (ROS), which damage proteins, lipids, DNA and finally limit plant yield. The enzymes of the chloroplast antioxidant system are exclusively nuclear encoded. During evolution, plastid and mitochondrial genes were post-endosymbiotically transferred to the nucleus, adapted for eukaryotic gene expression and post-translational protein targeting and supplemented with genes of eukaryotic origin. RESULTS: Here, the genomes of the green alga Chlamydomonas reinhardtii, the moss Physcomitrella patens, the lycophyte Selaginella moellendorffii and the seed plant Arabidopsis thaliana were screened for ORFs encoding chloroplast peroxidases. The identified genes were compared for their amino acid sequence similarities and gene structures. Stromal and thylakoid-bound ascorbate peroxidases (APx) share common splice sites demonstrating that they evolved from a common ancestral gene. In contrast to most cormophytes, our results predict that chloroplast APx activity is restricted to the stroma in Chlamydomonas and to thylakoids in Physcomitrella. The moss gene is of retrotransposonal origin.The exon-intron-structures of 2CP genes differ between chlorophytes and streptophytes indicating an independent evolution. According to amino acid sequence characteristics only the A-isoform of Chlamydomonas 2CP may be functionally equivalent to streptophyte 2CP, while the weakly expressed B- and C-isoforms show chlorophyte specific surfaces and amino acid sequence characteristics. The amino acid sequences of chloroplast PrxII are widely conserved between the investigated species. In the analyzed streptophytes, the genes are unspliced, but accumulated four introns in Chlamydomonas. A conserved splice site indicates also a common origin of chlorobiont PrxQ.The similarity of splice sites also demonstrates that streptophyte glutathione peroxidases (GPx) are of common origin. Besides a less related cysteine-type GPx, Chlamydomonas encodes two selenocysteine-type GPx. The latter were lost prior or during streptophyte evolution. CONCLUSION: Throughout plant evolution, there was a strong selective pressure on maintaining the activity of all three investigated types of peroxidases in chloroplasts. APx evolved from a gene, which dates back to times before differentiation of chlorobionts into chlorophytes and streptophytes, while Prx and presumably also GPx gene patterns may have evolved independently in the streptophyte and chlorophyte branches.

Characterization of genes encoding poly(A) polymerases in plants: evidence for duplication and functional specialization.

BACKGROUND: Poly(A) polymerase is a key enzyme in the machinery that mediates mRNA 3' end formation in eukaryotes. In plants, poly(A) polymerases are encoded by modest gene families. To better understand this multiplicity of genes, poly(A) polymerase-encoding genes from several other plants, as well as from Selaginella, Physcomitrella, and Chlamydomonas, were studied. METHODOLOGY/PRINCIPAL FINDINGS: Using bioinformatics tools, poly(A) polymerase-encoding genes were identified in the genomes of eight species in the plant lineage. Whereas Chlamydomonas reinhardtii was found to possess a single poly(A) polymerase gene, other species possessed between two and six possible poly(A) polymerase genes. With the exception of four intron-lacking genes, all of the plant poly(A) polymerase genes (but not the C. reinhardtii gene) possessed almost identical intron positions within the poly(A) polymerase coding sequences, suggesting that all plant poly(A) polymerase genes derive from a single ancestral gene. The four Arabidopsis poly(A) polymerase genes were found to be essential, based on genetic analysis of T-DNA insertion mutants. GFP fusion proteins containing three of the four Arabidopsis poly(A) polymerases localized to the nucleus, while one such fusion protein was localized in the cytoplasm. The fact that this latter protein is largely pollen-specific suggests that it has important roles in male gametogenesis. CONCLUSIONS/SIGNIFICANCE: Our results indicate that poly(A) polymerase genes have expanded from a single ancestral gene by a series of duplication events during the evolution of higher plants, and that individual members have undergone sorts of functional specialization so as to render them essential for plant growth and development. Perhaps the most interesting of the plant poly(A) polymerases is a novel cytoplasmic poly(A) polymerase that is expressed in pollen in Arabidopsis; this is reminiscent of spermatocyte-specific cytoplasmic poly(A) polymerases in mammals.

Auxin-binding proteins without KDEL sequence in the moss Funaria hygrometrica.

Whereas the important plant growth regulator auxin has multiple effects in flowering plants, it induces a specific cell differentiation step in the filamentous moss protonema. Here, we analyse the presence of classical auxin-binding protein (ABP1) homologues in the moss Funaria hygrometrica. Microsomal membranes isolated from protonemata of F. hygrometrica have specific indole acetic acid-binding sites, estimated to be about 3-5 pmol/mg protein with an apparent dissociation constant (K (d)) between 3 and 5 microM. Western analyses with anti-ABP1 antiserum detected the canonical endoplasmic reticulum (ER)-localised 22-24 kDa ABP1 in Zea mays, but not in F. hygrometrica. Instead, polypeptides of 31-33 and 46 kDa were labelled in the moss as well as in maize. In F. hygrometrica these proteins were found exclusively in microsomal membrane fractions and were confirmed as ABPs by photo-affinity labelling with 5-azido-[7-(3)H]-indole-3-acetic acid. Unlike the classical corn ABP1, these moss ABPs did not contain the KDEL ER retention sequence. Consistently, the fully sequenced genome of the moss Physcomitrella patens, a close relative of F. hygrometrica, encodes an ABP1-homologue without KDEL sequence. Our study suggests the presence of putative ABPs in F. hygrometrica that share immunological epitopes with ABP1 and bind auxin but are different from the classical corn ABP1.

Modified suppression subtractive hybridization identifies an AP2-containing protein involved in metal responses in Physcomitrella patens.

The moss Physcomitrella patens has two life cycles, filamentous protonema and leafy gametophore. A modified from of suppression subtractive hybridization (SSH), mirror orientation selection (MOS), was applied to screen genes differentially expressed in the P. patens protonema. Using reverse Northern blot analysis, differentially expressed clones were identified. The identified genes were involved mainly in metal binding and detoxification. One of these genes was an AP2 (APETALA2) domain-containing protein (PpACP1), which was highly up-regulated in the protonema. Alignment with other AP2/EREBPs (Ethylene Responsive Element Binding Proteins) revealed significant sequence homology of the deduced amino acid sequence in the AP2/EREBP DNA binding domain. Northern analysis under various stress conditions showed that PpACP1 was induced by ethephon, cadmium, copper, ABA, IAA, and cold. In addition, it was highly expressed in suspension-cultured protonema. We suggest that PpACP1 is involved in responses to metals, and that suspension culture enhance the expression of genes responding to metals.

Heterologous expression of cDNAs encoding monodehydroascorbate reductases from the moss, Physcomitrella patens and characterization of the expressed enzymes.

Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) catalyses the reduction of the monodehydroascorbate (MDHA) radical to ascorbate, using NADH or NADPH as an electron donor, and is believed to be involved in maintaining the reactive oxygen scavenging capability of plant cells. This key enzyme in the ascorbate-glutathione cycle has been studied here in the moss Physcomitrella patens, which is tolerant to a range of abiotic stresses and is increasingly used as a model plant. In the present study, three cDNAs encoding different MDHAR isoforms of 47 kDa were identified in P. patens, and found to exhibit enzymic characteristics similar to MDHARs in vascular plants despite low-sequence identity and a distant evolutionary relationship between the species. The three cDNAs for the P. patens MDHAR enzymes were expressed in Escherichia coli and the active enzymes were purified and characterized. Each recombinant protein displayed an absorbance spectrum typical of flavoenzymes and contained a single non-covalently bound FAD coenzyme molecule. The Km and kcat values for the heterologously expressed PpMDHAR enzymes ranged from 8 to 18 microM and 120-130 s(-1), respectively, using NADH as the electron donor. The Km values were at least an order of magnitude higher for NADPH. The Km values for the MDHA radical were approximately 0.5-1.0 microM for each of the purified enzymes, and further kinetic analyses indicated that PpMDHARs follow a 'ping-pong' kinetic mechanism. In contrast to previously published data, site-directed mutagenesis indicated that the conserved cysteine residue is not directly involved in the reduction of MDHA.

Sulphate assimilation under Cd2+ stress in Physcomitrella patens--combined transcript, enzyme and metabolite profiling.

Cd(2+) causes disturbance of metabolic pathways through severe damage on several levels. Here we present a comprehensive study of Cd(2+)-mediated effects on transcript, enzyme and metabolite levels in a plant without phytochelatin (PC). The moss Physcomitrella patens (Hedw.) B.S.G. was stressed with up to 10 microm Cd(2+) to investigate the regulation of gene transcription and activities of enzymes involved in the assimilatory sulphate reduction pathway and in glutathione biosynthesis. Real-time PCR, specific enzyme assays as well as thiol peptide profiling techniques were applied. Upon supplementation of 10 microm Cd(2+), the moss showed a more than fourfold increase in expression of genes encoding ATP sulphurylase (ATPS), adenosylphosphosulphate reductase, phosphoradenosylphosphorsulphate reductase, sulphite reductase (SiR) and gamma-glutamyl cysteine synthetase (gamma-ECS). Likewise, elevated enzyme activities of gamma-ECS and glutathione synthetase were observed. Contrarily, activity of O-acetylserine (thiol) lyase (OAS-TL), responsible for biosynthesis of cysteine, was diminished. At the metabolite level, nearly doubling of intracellular cysteine and glutathione content was noted, while the moss did not produce any detectable amounts of PCs. These results suggest a Cd(2+)-induced activation of the assimilatory sulphate reduction pathway as well as of glutathione biosynthesis on different levels of regulation.

Identification and characterisation of a bryophyte polyphenol oxidase encoding gene from Physcomitrella patens.

Polyphenol oxidases (PPO) are enzymes of secondary plant metabolism that catalyse the oxidation of polyphenols to quinones. Because of their ubiquitous appearance in the plant kingdom, an important role is assumed; however, the exact physiological function of PPOs remains unclear. In this work, the identification, cloning, and characterisation of a bryophyte PPO from the moss Physcomitrella patens is presented. PPO activity from protein extracts was determined polarographically after activation by SDS. Four Physcomitrella ESTs with homologies to known plant PPOs were selected from publicly accessible databases, and PCR experiments demonstrated that they belong to the same gene, named Pp_ppo1. The identified cDNA was found to be 2402 bp long, containing a single open reading frame of 1611 bp encoding for a 536 amino acid protein with a molecular mass of 60.1 kDa. Cloning and sequencing of a genomic part of Pp_ppo1 revealed the presence of a 94-bp intron. The time course of Pp_ppo1 gene expression in liquid culture was monitored by real time RT-PCR, revealing increasing transcription levels until the 4th day, a maximum between the 4th and the 8th day, and decreasing transcription until the 12th day. A comparison of the deduced amino acid sequence of Pp_ppo1 with seed plant PPOs revealed similarities such as the presence of two highly conserved copper-binding domains and a similar pattern of hydrophobic regions, but also differences such as a stronger membrane association and a shorter signal sequence, thus reflecting the phylogenetic distance of Physcomitrella from seed plants.