A CPD photolyase gene PnPHR1 from Antarctic moss Pohlia nutans is involved in the resistance to UV-B radiation and salinity stress.

In Antarctic continent, the organisms are exposed to high ultraviolet (UV) radiation because of damaged stratospheric ozone. UV causes DNA lesions due to the accumulation of photoproducts. Photolyase can repair UV-damaged DNA in a light-dependent process by electron transfer mechanism. Here, we isolated a CPD photolyase gene PnPHR1 from Antarctic moss Pohlia nutans, which encodes a protein of theoretical molecular weight of 69.1 KDa. The expression level of PnPHR1 was increased by UV-B irradiation. Enzyme activity assay in vitro showed that PnPHR1 exhibited photoreactivation activity, which can repair CPD photoproducts in a light-dependent manner. The complementation assay of repair-deficient E. coli strain SY2 demonstrated that PnPHR1 gene enhanced the survival rate of SY2 strain after UV-B radiation. Additionally, overexpression of PnPHR1 enhanced the Arabidopsis resistance to UV-B radiation and salinity stress, which also conferred plant tolerance to oxidative stress by decreasing ROS production and increasing ROS clearance. Our work shows that PnPHR1 encodes an active CPD photolyase, which may participate in the adaptation of P. nutans to polar environments.

Function of the HYDROXYCINNAMOYL-CoA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE is evolutionarily conserved in embryophytes.

The plant phenylpropanoid pathway generates a major class of specialized metabolites and precursors of essential extracellular polymers that initially appeared upon plant terrestrialization. Despite its evolutionary significance, little is known about the complexity and function of this major metabolic pathway in extant bryophytes, which represent the non-vascular stage of embryophyte evolution. Here, we report that the HYDROXYCINNAMOYL-CoA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE (HCT) gene, which plays a critical function in the phenylpropanoid pathway during seed plant development, is functionally conserved in Physcomitrium patens (Physcomitrella), in the moss lineage of bryophytes. Phylogenetic analysis indicates that bona fide HCT function emerged in the progenitor of embryophytes. In vitro enzyme assays, moss phenolic pathway reconstitution in yeast and in planta gene inactivation coupled to targeted metabolic profiling, collectively indicate that P. patens HCT (PpHCT), similar to tracheophyte HCT orthologs, uses shikimate as a native acyl acceptor to produce a p-coumaroyl-5-O-shikimate intermediate. Phenotypic and metabolic analyses of loss-of-function mutants show that PpHCT is necessary for the production of caffeate derivatives, including previously reported caffeoyl-threonate esters, and for the formation of an intact cuticle. Deep conservation of HCT function in embryophytes is further suggested by the ability of HCT genes from P. patens and the liverwort Marchantia polymorpha to complement an Arabidopsis thaliana CRISPR/Cas9 hct mutant, and by the presence of phenolic esters of shikimate in representative species of the three bryophyte lineages.

An endocannabinoid catabolic enzyme FAAH and its paralogs in an early land plant reveal evolutionary and functional relationship with eukaryotic orthologs.

Endocannabinoids were known to exist only among Animalia but recent report of their occurrence in early land plants prompted us to study its function and metabolism. In mammals, anandamide, as an endocannabinoid ligand, mediates several neurological and physiological processes, which are terminated by fatty acid amide hydrolase (FAAH). We identified nine orthologs of FAAH in the moss Physcomitrella patens (PpFAAH1 to PpFAAH9) with amidase signature and catalytic triad. The optimal amidase activity for PpFAAH1 was at 37 °C and pH 8.0, with higher specificity to anandamide. Further, the phylogeny and predicted structural analyses of the nine paralogs revealed that PpFAAH1 to PpFAAH4 were closely related to plant FAAH while PpFAAH6 to PpFAAH9 were to the rat FAAH, categorized based on the membrane binding cap, membrane access channel and substrate binding pocket. We also identified that a true 'dynamic paddle' that is responsible for tighter regulation of FAAH is recent in vertebrates and absent or not fully emerged in plants and non-vertebrates. These data reveal evolutionary and functional relationship among eukaryotic FAAH orthologs and features that contribute to versatility and tighter regulation of FAAH. Future studies will utilize FAAH mutants of moss to elucidate the role of anandamide in early land plants.

PnSAG1, an E3 ubiquitin ligase of the Antarctic moss Pohlia nutans, enhanced sensitivity to salt stress and ABA.

Plant U-box (PUB) E3 ubiquitin ligases play crucial roles in the plant response to abiotic stress and the phytohormone abscisic acid (ABA) signaling, but little is known about them in bryophytes. Here, a representative U-box armadillo repeat (PUB-ARM) ubiquitin E3 ligase from Antarctic moss Pohlia nutans (PnSAG1), was explored for its role in abiotic stress response in Arabidopsis thaliana and Physcomitrella patens. The expression of PnSAG1 was rapidly induced by exogenous abscisic acid (ABA), salt, cold and drought stresses. PnSAG1 was localized to the cytoplasm and showed E3 ubiquitin ligase activity by in vitro ubiquitination assay. The PnSAG1-overexpressing Arabidopsis enhanced the sensitivity with respect to ABA and salt stress during seed germination and early root growth. Similarly, heterogeneous overexpression of PnSAG1 in P. patens was more sensitive to the salinity and ABA in their gametophyte growth. The analysis by RT-qPCR revealed that the expression of salt stress/ABA-related genes were downregulated in PnSAG1-overexpressing plants after salt treatment. Taken together, our results indicated that PnSAG1 plays a negative role in plant response to ABA and salt stress.

Evolutionary History of the Glycoside Hydrolase 3 (GH3) Family Based on the Sequenced Genomes of 48 Plants and Identification of Jasmonic Acid-Related GH3 Proteins in Solanum tuberosum.

Glycoside Hydrolase 3 (GH3) is a phytohormone-responsive family of proteins found in many plant species. These proteins contribute to the biological activity of indolacetic acid (IAA), jasmonic acid (JA), and salicylic acid (SA). They also affect plant growth and developmental processes as well as some types of stress. In this study, GH3 genes were identified in 48 plant species, including algae, mosses, ferns, gymnosperms, and angiosperms. No GH3 representative protein was found in algae, but we identified 4 genes in mosses, 19 in ferns, 7 in gymnosperms, and several in angiosperms. The results showed that GH3 proteins are mainly present in seed plants. Phylogenetic analysis of all GH3 proteins showed three separate clades. Group I was related to JA adenylation, group II was related to IAA adenylation, and group III was separated from group II, but its function was not clear. The structure of the GH3 proteins indicated highly conserved sequences in the plant kingdom. The analysis of JA adenylation in relation to gene expression of GH3 in potato (Solanum tuberosum) showed that StGH3.12 greatly responded to methyl jasmonate (MeJA) treatment. The expression levels of StGH3.1, StGH3.11, and StGH3.12 were higher in the potato flowers, and StGH3.11 expression was also higher in the stolon. Our research revealed the evolution of the GH3 family, which is useful for studying the precise function of GH3 proteins related to JA adenylation in S. tuberosum when the plants are developing and under biotic stress.

Evolution of intron-poor clades and expression patterns of the glycosyltransferase family 47.

MAIN CONCLUSION: A large-scale bioinformatics analysis revealed the origin and evolution of GT47 gene family, and identified two clades of intron-poor genes with putative functions in drought stress responses and seed development in maize. Glycosyltransferase family 47 (GT47) genes encode ß-galactosyltransferases and ß-glucuronyltransferases that synthesize pectin, xyloglucans and xylan, which are important components of the plant cell wall. In this study, we performed a systematic and large-scale bioinformatics analysis of GT47 gene family using 352 GT47 proteins from 15 species ranging from cyanobacteria to seed plants. The analysis results showed that GT47 family may originate in cyanobacteria and expand along the evolutionary trajectory to moss. Further analysis of 47 GT47 genes in maize revealed that they can divide into five clades with diverse exon-intron structures. Among these five clades, two were mainly composed with intron-poor genes, which may originate in the moss. Gene duplication analysis revealed that the expansion of GT47 gene family in maize was significantly driven from tandem duplication events and segmental duplication events. Significantly, almost all duplicated genes are intron-poor genes. Expression analysis indicated that several intron-poor GT47 genes may be involved in the drought stress response and seed development in maize. This work provides insight into the origin and evolutionary process, expansion mechanisms and expression patterns of GT47 genes, thus facilitating their functional investigations in the future.

Enhanced multiple stress tolerance in Arabidopsis by overexpression of the polar moss peptidyl prolyl isomerase FKBP12 gene.

KEY MESSAGE: PaFKBP12 overexpression in Arabidopsis resulted in stress tolerance to heat, ABA, drought, and salt stress, in addition to growth promotion under normal conditions. Polytrichastrum alpinum (alpine haircap moss) is one of polar organisms that can withstand the severe conditions of the Antarctic. In this study, we report the isolation of a peptidyl prolyl isomerase FKBP12 gene (PaFKBP12) from P. alpinum collected in the Antarctic and its functional implications in development and stress responses in plants. In P. alpinum, PaFKBP12 expression was induced by heat and ABA. Overexpression of PaFKBP12 in Arabidopsis increased the plant size, which appeared to result from increased rates of cell cycle. Under heat stress conditions, PaFKBP12-overexpressing lines (PaFKBP12-OE) showed better growth and survival than the wild type. PaFKBP12-OE also showed higher root elongation rates, better shoot growth and enhanced survival at higher concentrations of ABA in comparison to the wild type. In addition, PaFKBP12-OE were more tolerant to drought and salt stress than the wild type. All these phenotypes were accompanied with higher induction of the stress responsive genes in PaFKBP12-OE than in the wild type. Taken together, our findings revealed important functions of PaFKBP12 in plant development and abiotic stress responses.

The ALDH21 gene found in lower plants and some vascular plants codes for a NADP+ -dependent succinic semialdehyde dehydrogenase.

Lower plant species including some green algae, non-vascular plants (bryophytes) as well as the oldest vascular plants (lycopods) and ferns (monilophytes) possess a unique aldehyde dehydrogenase (ALDH) gene named ALDH21, which is upregulated during dehydration. However, the gene is absent in flowering plants. Here, we show that ALDH21 from the moss Physcomitrella patens codes for a tetrameric NADP+ -dependent succinic semialdehyde dehydrogenase (SSALDH), which converts succinic semialdehyde, an intermediate of the γ-aminobutyric acid (GABA) shunt pathway, into succinate in the cytosol. NAD+ is a very poor coenzyme for ALDH21 unlike for mitochondrial SSALDHs (ALDH5), which are the closest related ALDH members. Structural comparison between the apoform and the coenzyme complex reveal that NADP+ binding induces a conformational change of the loop carrying Arg-228, which seals the NADP+ in the coenzyme cavity via its 2'-phosphate and α-phosphate groups. The crystal structure with the bound product succinate shows that its carboxylate group establishes salt bridges with both Arg-121 and Arg-457, and a hydrogen bond with Tyr-296. While both arginine residues are pre-formed for substrate/product binding, Tyr-296 moves by more than 1 Å. Both R121A and R457A variants are almost inactive, demonstrating a key role of each arginine in catalysis. Our study implies that bryophytes but presumably also some green algae, lycopods and ferns, which carry both ALDH21 and ALDH5 genes, can oxidize SSAL to succinate in both cytosol and mitochondria, indicating a more diverse GABA shunt pathway compared with higher plants carrying only the mitochondrial ALDH5.

Cloning, expression, and characterization of Fe-SOD from Isöetes sinensis.

Although the palynology and sporophyte stage of Isöetes sinensis have been well studied, the biology of its gametophyte and embryo is less well understood. To date, the functions of several genes of I. sinensis and the molecular mechanisms of enzymes encoded by them remain to be studied. In the present study, the Fe-SOD gene of I. sinensis was successfully cloned using RT-PCR and rapid amplification of cDNA ends (RACE), and termed IsFeSOD. IsFeSOD has certain reference value in the classification of system evolution. The study also accumulated data for further research on the SOD gene. Bioinformatic analysis was employed to compare IsFeSOD with gene sequences obtained from other plants present in the GenBank. Furthermore, the recombinant pET32-FeSOD plasmids were transformed into Escherichia coli BL21 for expression. IsFeSOD was observed to have 1469 nucleotides that were predicted to encode 247 amino acids. The bioinformatic analysis revealed that IsFeSOD contained conserved TGGGA sequences, similar to eight other species, in addition to five other conserved sequences. The recombinant protein was about 43 kDa. Recombinant FeSOD was expressed, purified, and confirmed by western blotting. Alignment of complete Fe-SOD mRNA sequences from 9 species revealed several conserved sequences. A phylogenetic tree was constructed using MEGA4.1 and ClustalX multiple-sequence alignment programs. This study could be helpful in further characterization of SOD genes and for classification of system evolution status.

Biomonitoring of air pollution using antioxidative enzyme system in two genera of family Pottiaceae (Bryophyta).

Bryophyte particularly mosses, have been found to serve as reliable indicators of air pollution and can serve as bryometers-biological instruments for measuring air pollution. They are remarkable colonizers, as they have the ability to survive in adverse environments and are also particular in their requirement of environmental conditions, which makes them appropriate ecological indicators. The purpose of this study was to evaluate the activity of antioxidative enzymes in two mosses viz., Hyophila rosea R.S. Williams and Semibarbula orientalis (Web.) Wijk. & Marg. and assess their suitability as biomonitors. Three different locations viz., Lucknow University, Residency (contaminated sites) and Dilkusha Garden (reference site) within Lucknow city with different levels of air pollutants were used for comparison. Our results indicate that air pollution caused marked enhancement in activity of antioxidative enzymes viz., catalase, peroxidase and superoxide dismutase. All the three are capable of scavenging reactive oxygen species. In the genus S. orientalis, catalase, peroxidase and superoxide dismutase activity was minimum at the reference site Dilkusha Garden and was significantly higher at the two contaminated sites for catalase and peroxidase, whereas the difference was non significant for superoxide dismutase. In H. rosea the activity of catalase and peroxidase at the three locations was almost similar, however superoxide dismutase activity showed a significant increase in the two contaminated sites when compared to the reference site, the value being highest for Lucknow University site. It was thus observed that the two genera, from the same location, showed difference in the activity of the antioxidative enzymes. Based on our results, we recommend bryophytes as good monitors of air pollution.

HpDTC1, a Stress-Inducible Bifunctional Diterpene Cyclase Involved in Momilactone Biosynthesis, Functions in Chemical Defence in the Moss Hypnum plumaeforme.

Momilactones, which are diterpenoid phytoalexins with antimicrobial and allelopathic functions, have been found only in rice and the moss Hypnum plumaeforme. Although these two evolutionarily distinct plant species are thought to produce momilactones as a chemical defence, the momilactone biosynthetic pathway in H. plumaeforme has been unclear. Here, we identified a gene encoding syn-pimara-7,15-diene synthase (HpDTC1) responsible for the first step of momilactone biosynthesis in the moss. HpDTC1 is a bifunctional diterpene cyclase that catalyses a two-step cyclization reaction of geranylgeranyl diphosphate to syn-pimara-7,15-diene. HpDTC1 transcription was up-regulated in response to abiotic and biotic stress treatments. HpDTC1 promoter-GUS analysis in transgenic Physcomitrella patens showed similar transcriptional responses as H. plumaeforme to the stresses, suggesting that a common response system to stress exists in mosses. Jasmonic acid (JA), a potent signalling molecule for inducing plant defences, could not activate HpDTC1 expression. In contrast, 12-oxo-phytodienoic acid, an oxylipin precursor of JA in vascular plants, enhanced HpDTC1 expression and momilactone accumulation, implying that as-yet-unknown oxylipins could regulate momilactone biosynthesis in H. plumaeforme. These results demonstrate the existence of an evolutionarily conserved chemical defence system utilizing momilactones and suggest the molecular basis of the regulation for inductive production of momilactones in H. plumaeforme.

PpASCL, the Physcomitrella patens Anther-Specific Chalcone Synthase-Like Enzyme Implicated in Sporopollenin Biosynthesis, Is Needed for Integrity of the Moss Spore Wall and Spore Viability.

Sporopollenin is the main constituent of the exine layer of spore and pollen walls. The anther-specific chalcone synthase-like (ASCL) enzyme of Physcomitrella patens, PpASCL, has previously been implicated in the biosynthesis of sporopollenin, the main constituent of exine and perine, the two outermost layers of the moss spore cell wall. We made targeted knockouts of the corresponding gene, PpASCL, and phenotypically characterized ascl sporophytes and spores at different developmental stages. Ascl plants developed normally until late in sporophytic development, when the spores produced were structurally aberrant and inviable. The development of the ascl spore cell wall appeared to be arrested early in microspore development, resulting in small, collapsed spores with altered surface morphology. The typical stratification of the spore cell wall was absent with only an abnormal perine recognisable above an amorphous layer possibly representing remnants of compromised intine and/or exine. Equivalent resistance of the spore walls of ascl mutants and the control strain to acetolysis suggests the presence of chemically inert, defective sporopollenin in the mutants. Anatomical abnormalities of late-stage ascl sporophytes include a persistent large columella and an air space incompletely filled with spores. Our results indicate that the evolutionarily conserved PpASCL gene is needed for proper construction of the spore wall and for normal maturation and viability of moss spores.

Biochemical characterization of an isoprene synthase from Campylopus introflexus (heath star moss).

Each year, plants emit terragram quantities of the reactive hydrocarbon isoprene (2-methyl-1,3-butadiene) into the earth's atmosphere. In isoprene-emitting plants, the enzyme isoprene synthase (ISPS) catalyzes the production of isoprene from the isoprenoid intermediate dimethylallyl diphosphate (DMADP). While isoprene is emitted from all major classes of land plants, to date ISPSs from angiosperms only have been characterized. Here, we report the identification and initial biochemical characterization of a DMADP-dependent ISPS from the isoprene-emitting bryophyte Campylopus introflexus (heath star moss). The partially-purified C. introflexus ISPS (CiISPS) exhibited a Km for DMADP of 0.37 ± 0.28 mM, a pH optimum of 8.6 ± 0.5, and a temperature optimum of 40 ± 3 °C in vitro. Like ISPSs from angiosperms, the CiISPS required the presence of a divalent cation. However, unlike angiosperm ISPSs, the CiISPS utilized Mn(2+) preferentially over Mg(2+). Efforts are currently underway in our laboratory to further purify the CiISPS and clone the cDNA sequence encoding this novel enzyme. Our discovery of the first bryophyte ISPS paves the way for future studies concerning the evolutionary origins of isoprene emission in land plants and may help generate new bryophyte model systems for physiological and biochemical research on plant isoprene function.

The nitric oxide production in the moss Physcomitrella patens is mediated by nitrate reductase.

During the last 20 years multiple roles of the nitric oxide gas (•NO) have been uncovered in plant growth, development and many physiological processes. In seed plants the enzymatic synthesis of •NO is mediated by a nitric oxide synthase (NOS)-like activity performed by a still unknown enzyme(s) and nitrate reductase (NR). In green algae the •NO production has been linked only to NR activity, although a NOS gene was reported for Ostreococcus tauri and O. lucimarinus, no other Viridiplantae species has such gene. As there is no information about •NO synthesis neither for non-vascular plants nor for non-seed vascular plants, the interesting question regarding the evolution of the enzymatic •NO production systems during land plant natural history remains open. To address this issue the endogenous •NO production by protonema was demonstrated using Electron Paramagnetic Resonance (EPR). The •NO signal was almost eliminated in plants treated with sodium tungstate, which also reduced the NR activity, demonstrating that in P. patens NR activity is the main source for •NO production. The analysis with confocal laser scanning microscopy (CLSM) confirmed endogenous NO production and showed that •NO signal is accumulated in the cytoplasm of protonema cells. The results presented here show for the first time the •NO production in a non-vascular plant and demonstrate that the NR-dependent enzymatic synthesis of •NO is common for embryophytes and green algae.

Identification of the flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase genes from Antarctic moss and their regulation during abiotic stress.

Flavonoids are ubiquitous plant secondary metabolites, and their hydroxylation pattern determines their color, stability, and antioxidant capacity. The hydroxylation pattern of the B-ring of flavonoids is determined by the activity of two members of cytochrome P450 protein (P450) family, the flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3',5'H). However, they are still not well documented in lower plants such as bryophytes. We report the identification of gene encoding F3'H, F3',5'H from Antarctic moss Pohlia nutans and their transcriptional regulation under different stress conditions. Totally, sixteen cDNAs were isolated from P. nutans by RT-PCR and RACE techniques, all of which were predicted to code for F3'Hs or F3',5'Hs based on their annotations of Blast results. Amino acid alignment showed that they possessed the featured conserved domains of flavonoid hydroxylase, including proline-rich "hinge" region, EXXR motif, oxygen binding pocket motif, heme binding domain and substrate recognition sites. Phylogenetic analysis indicated that moss F3'Hs and F3',5'Hs were highly conserved and have independent evolution from the monocots, dicots and ferns. Meanwhile, real-time PCR analysis revealed that the expression profiling of flavonoid hydroxylase genes was influenced by diverse abiotic stresses including cold, salinity, drought or UV-B radiation and plant hormone abscisic acid (ABA) or jasmonic acid (JA) treatment. Since 3',4',5'-hydroxylated flavonoid-derivatives may serve a multitude of functions, including antioxidant activity and UV filters, the evolution and expression profile of flavonoid hydroxylase probably reflect the adaptive value of Antarctic moss in the acclimation of polar environment.

The capability to synthesize phytochelatins and the presence of constitutive and functional phytochelatin synthases are ancestral (plesiomorphic) characters for basal land plants.

Bryophytes, a paraphyletic group which includes liverworts, mosses, and hornworts, have been stated as land plants that under metal stress (particularly cadmium) do not synthesize metal-binding peptides such as phytochelatins. Moreover, very little information is available to date regarding phytochelatin synthesis in charophytes, postulated to be the direct ancestors of land plants, or in lycophytes, namely very basal tracheophytes. In this study, it was hypothesized that basal land plants and charophytes have the capability to produce phytochelatins and possess constitutive and functional phytochelatin synthases. To verify this hypothesis, twelve bryophyte species (six liverworts, four mosses, and two hornworts), three charophytes, and two lycophyte species were exposed to 0-36 µM cadmium for 72 h, and then assayed for: (i) glutathione and phytochelatin quali-quantitative content by HPLC and mass spectrometry; (ii) the presence of putative phytochelatin synthases by western blotting; and (iii) in vitro activity of phytochelatin synthases. Of all the species tested, ten produced phytochelatins in vivo, while the other seven did not. The presence of a constitutively expressed and functional phytochelatin synthase was demonstrated in all the bryophyte lineages and in the lycophyte Selaginella denticulata, but not in the charophytes. Hence, current knowledge according to phytochelatins have been stated as being absent in bryophytes was therefore confuted by this work. It is argued that the capability to synthesize phytochelatins, as well as the presence of active phytochelatin synthases, are ancestral (plesiomorphic) characters for basal land plants.

Evidence for the absence of enzymatic reactions in the glassy state. A case study of xanthophyll cycle pigments in the desiccation-tolerant moss Syntrichia ruralis.

Desiccation-tolerant plants are able to withstand dehydration and resume normal metabolic functions upon rehydration. These plants can be dehydrated until their cytoplasm enters a 'glassy state' in which molecular mobility is severely reduced. In desiccation-tolerant seeds, longevity can be enhanced by drying and lowering storage temperature. In these conditions, they still deteriorate slowly, but it is not known if deteriorative processes include enzyme activity. The storage stability of photosynthetic organisms is less studied, and no reports are available on the glassy state in photosynthetic tissues. Here, the desiccation-tolerant moss Syntrichia ruralis was dehydrated at either 75% or <5% relative humidity, resulting in slow (SD) or rapid desiccation (RD), respectively, and different residual water content of the desiccated tissues. The molecular mobility within dry mosses was assessed through dynamic mechanical thermal analysis, showing that at room temperature only rapidly desiccated samples entered the glassy state, whereas slowly desiccated samples were in a 'rubbery' state. Violaxanthin cycle activity, accumulation of plastoglobules, and reorganization of thylakoids were observed upon SD, but not upon RD. Violaxanthin cycle activity critically depends on the activity of violaxanthin de-epoxidase (VDE). Hence, it is proposed that enzymatic activity occurred in the rubbery state (after SD), and that in the glassy state (after RD) no VDE activity was possible. Furthermore, evidence is provided that zeaxanthin has some role in recovery apparently independent of its role in non-photochemical quenching of chlorophyll fluorescence.

Polyphenol oxidases in Physcomitrella: functional PPO1 knockout modulates cytokinin-dependent development in the moss Physcomitrella patens.

Polyphenol oxidases (PPOs) are copper-binding enzymes of the plant secondary metabolism that oxidize polyphenols to quinones. Although PPOs are nearly ubiquitous in seed plants, knowledge on their evolution and function in other plant groups is missing. This study reports on the PPO gene family in the moss Physcomitrella patens (Hedw.) B.S.G. asan example for an early divergent plant. The P. patens PPO multigene family comprises 13 paralogues. Phylogenetic analyses suggest that plant PPOs evolved with the colonization of land and that PPO duplications within the monophyletic P. patens paralogue clade occurred after the separation of the moss and seed plant lineages. PPO functionality was demonstrated for recombinant PPO6. P. patens was analysed for phenolic compounds and six substances were detected intracellularly by LC-MS analysis: 4-hydroxybenzoic acid, p-cumaric acid, protocatechuic acid, salicylic acid, caffeic acid, and an ester of caffeic acid. Targeted PPO1 knockout (d|ppo1) plants were generated and plants lacking PPO1 exhibited only ~30% of the wild-type PPO activity in the culture medium, thus suggesting extracellular localization of PPO1, which is in contrast to the mostly plastidic PPO localization in seed plants. Further, d|ppo1 lines formed significantly more gametophores with a reduced areal plant size, which could be related to an increase of endogenously produced cytokinins and indicates an impact of PPO1 on plant development. d|ppo1 plants were less tolerant towards applied 4-methylcatechol compared to the wild type, which suggests a role of extracellular PPO1 in establishing appropriate conditions by the removal of inhibitory extracellular phenolic compounds.

Three different mechanisms of energy dissipation of a desiccation-tolerant moss serve one common purpose: to protect reaction centres against photo-oxidation.

Three different types of non-photochemical de-excitation of absorbed light energy protect photosystem II of the sun- and desiccation-tolerant moss Rhytidium rugosum against photo-oxidation. The first mechanism, which is light-induced in hydrated thalli, is sensitive to inhibition by dithiothreitol. It is controlled by the protonation of a thylakoid protein. Other mechanisms are activated by desiccation. One of them permits exciton migration towards a far-red band in the antenna pigments where fast thermal deactivation takes place. This mechanism appears to be similar to a mechanism detected before in desiccated lichens. A third mechanism is based on the reversible photo-accumulation of a radical that acts as a quencher of excitation energy in reaction centres of photosystem II. On the basis of absorption changes around 800 nm, the quencher is suggested to be an oxidized chlorophyll. The data show that desiccated moss is better protected against photo-oxidative damage than hydrated moss. Slow drying of moss thalli in the light increases photo-protection more than slow drying in darkness.

A glycosynthase derived from an inverting GH19 chitinase from the moss Bryum coronatum.

BcChi-A, a GH19 chitinase from the moss Bryum coronatum, is an endo-acting enzyme that hydrolyses the glycosidic bonds of chitin, (GlcNAc)(n) [a ß-1,4-linked polysaccharide of GlcNAc (N-acetylglucosamine) with a polymerization degree of n], through an inverting mechanism. When the wild-type enzyme was incubated with α-(GlcNAc)2-F [α-(GlcNAc)(2) fluoride] in the absence or presence of (GlcNAc)(2), (GlcNAc)(2) and hydrogen fluoride were found to be produced through the Hehre resynthesis-hydrolysis mechanism. To convert BcChi-A into a glycosynthase, we employed the strategy reported by Honda et al. [(2006) J. Biol. Chem. 281, 1426-1431; (2008) Glycobiology 18, 325-330] of mutating Ser(102), which holds a nucleophilic water molecule, and Glu(70), which acts as a catalytic base, producing S102A, S102C, S102D, S102G, S102H, S102T, E70G and E70Q. In all of the mutated enzymes, except S102T, hydrolytic activity towards (GlcNAc)(6) was not detected under the conditions we used. Among the inactive BcChi-A mutants, S102A, S102C, S102G and E70G were found to successfully synthesize (GlcNAc)(4) as a major product from α-(GlcNAc)(2)-F in the presence of (GlcNAc)(2). The S102A mutant showed the greatest glycosynthase activity owing to its enhanced F(-) releasing activity and its suppressed hydrolytic activity. This is the first report on a glycosynthase that employs amino sugar fluoride as a donor substrate.