Understanding mercury accumulation in mosses of two subalpine forests in China.

The role of forest ecosystems in the global mercury (Hg) biogeochemical cycle is widely recognized; however, using litterfall as a surrogate to assess the Hg sink function of forests encounters limitations. We investigated the accumulation characteristics and influencing factors of Hg in mosses from two remote subalpine forests in southwestern China. The results indicated that there was high Hg accumulation in subalpine forest mosses, with average concentrations of 82 ± 49 ng g-1 for total mercury (THg) and 1.3 ± 0.8 ng g-1 for methylmercury (MeHg). We demonstrated that the accumulation capacity of Hg in mosses was significantly dependent on species and substrates (micro-habitats), the mosses on tree trunks exhibited significantly elevated Hg accumulation levels (THg 132 ± 56 ng g-1, MeHg 1.6 ± 0.2 ng g-1) compared to mosses in other substrates. The surface morphologies and biochemical components of leaf (phyllidia), such as cation exchange capacity (CEC), pectin, uronic acid, and metallothionein, play a crucial role in the accumulation of Hg by mosses. These findings provide valuable insights into Hg accumulation in forest mosses. Suggesting that the contribution of mosses Hg accumulation should be considered when assessing atmospheric Hg sinks of forests.

Divergent evolution of the alcohol-forming pathway of wax biosynthesis among bryophytes.

The plant cuticle is a hydrophobic barrier, which seals the epidermal surface of most aboveground organs. While the cuticle biosynthesis of angiosperms has been intensively studied, knowledge about its existence and composition in nonvascular plants is scarce. Here, we identified and characterized homologs of Arabidopsis thaliana fatty acyl-CoA reductase (FAR) ECERIFERUM 4 (AtCER4) and bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase 1 (AtWSD1) in the liverwort Marchantia polymorpha (MpFAR2 and MpWSD1) and the moss Physcomitrium patens (PpFAR2A, PpFAR2B, and PpWSD1). Although bryophyte harbor similar compound classes as described for angiosperm cuticles, their biosynthesis may not be fully conserved between the bryophytes M. polymorpha and P. patens or between these bryophytes and angiosperms. While PpFAR2A and PpFAR2B contribute to the production of primary alcohols in P. patens, loss of MpFAR2 function does not affect the wax profile of M. polymorpha. By contrast, MpWSD1 acts as the major wax ester-producing enzyme in M. polymorpha, whereas mutations of PpWSD1 do not affect the wax ester levels of P. patens. Our results suggest that the biosynthetic enzymes involved in primary alcohol and wax ester formation in land plants have either evolved multiple times independently or undergone pronounced radiation followed by the formation of lineage-specific toolkits.

Morphological Innovation Drives Sperm Release in Bryophytes.

Plant movements for survival are nontrivial. Antheridia in the moss Physcomitrium patens (P. patens) use motion to eject sperm in the presence of water. However, the biological and mechanical mechanisms that actuate the process are unknown. Here, the burst of the antheridium of P. patens, triggered by water, results from elastic instability and is determined by an asymmetric change in cell geometry. The tension generated in jacket cell walls of antheridium arises from turgor pressure, and is further promoted when the inner walls of apex burst in hydration, causing water and cellular contents of apex quickly influx into sperm chamber. The outer walls of the jacket cells are strengthened by NAC transcription factor VNS4 and serve as key morphomechanical innovations to store hydrostatic energy in a confined space in P. patens. However, the antheridium in liverwort Marchantia polymorpha (M. polymorpha) adopts a different strategy for sperm release; like jacket cell outer walls of P. patens, the cells surrounding the antheridium of M. polymorpha appear to play a similar role in the storage of energy. Collectively, the work shows that plants have evolved different ingenious devices for sperm discharge and that morphological innovations can differ.

Multilevel analysis between Physcomitrium patens and Mortierellaceae endophytes explores potential long-standing interaction among land plants and fungi.

The model moss species Physcomitrium patens has long been used for studying divergence of land plants spanning from bryophytes to angiosperms. In addition to its phylogenetic relationships, the limited number of differential tissues, and comparable morphology to the earliest embryophytes provide a system to represent basic plant architecture. Based on plant-fungal interactions today, it is hypothesized these kingdoms have a long-standing relationship, predating plant terrestrialization. Mortierellaceae have origins diverging from other land fungi paralleling bryophyte divergence, are related to arbuscular mycorrhizal fungi but are free-living, observed to interact with plants, and can be found in moss microbiomes globally. Due to their parallel origins, we assess here how two Mortierellaceae species, Linnemannia elongata and Benniella erionia, interact with P. patens in coculture. We also assess how Mollicute-related or Burkholderia-related endobacterial symbionts (MRE or BRE) of these fungi impact plant response. Coculture interactions are investigated through high-throughput phenomics, microscopy, RNA-sequencing, differential expression profiling, gene ontology enrichment, and comparisons among 99 other P. patens transcriptomic studies. Here we present new high-throughput approaches for measuring P. patens growth, identify novel expression of over 800 genes that are not expressed on traditional agar media, identify subtle interactions between P. patens and Mortierellaceae, and observe changes to plant-fungal interactions dependent on whether MRE or BRE are present. Our study provides insights into how plants and fungal partners may have interacted based on their communications observed today as well as identifying L. elongata and B. erionia as modern fungal endophytes with P. patens.

Conservation of the moss RNA editing factor PPR78 despite the loss of its known cytidine-to-uridine editing sites is explained by a hidden extra target.

Cytidine (C)-to-uridine (U) RNA editing in plant organelles relies on specific RNA-binding pentatricopeptide repeat (PPR) proteins. In the moss Physcomitrium patens, all such RNA editing factors feature a C-terminal DYW domain that acts as the cytidine deaminase for C-to-U conversion. PPR78 of Physcomitrium targets 2 mitochondrial editing sites, cox1eU755SL and rps14eU137SL. Remarkably, the latter is edited to highly variable degrees in different mosses. Here, we aimed to unravel the coevolution of PPR78 and its 2 target sites in mosses. Heterologous complementation in a Physcomitrium knockout line revealed that the variable editing of rps14eU137SL depends on the PPR arrays of different PPR78 orthologues but not their C-terminal domains. Intriguingly, PPR78 has remained conserved despite the simultaneous loss of editing at both known targets among Hypnales (feather mosses), suggesting it serves an additional function. Using a recently established RNA editing assay in Escherichia coli, we confirmed site-specific RNA editing by PPR78 in the bacterium and identified 4 additional off-targets in the bacterial transcriptome. Based on conservation profiles, we predicted ccmFNeU1465RC as a candidate editing target of PPR78 in moss mitochondrial transcriptomes. We confirmed editing at this site in several mosses and verified that PPR78 targets ccmFNeU1465RC in the bacterial editing system, explaining the conservation and functional adaptation of PPR78 during moss evolution.

Molecular characterization of a moss isoprene synthase provides insight into its evolution.

This is the first report on the molecular characterization of isoprene synthase (ISPS) from the moss Calohypnum plumiforme. After isoprene emission from C. plumiforme was confirmed, the cDNA encoding C. plumiforme ISPS (CpISPS) was narrowed down using a genome database associated with protein structure prediction, and a CpISPS gene was identified. The recombinant CpISPS, produced in Escherichia coli, converted dimethylallyl diphosphate to isoprene. Phylogenetic analysis indicated similarity between the amino acid sequences of CpISPS and moss diterpene cyclases (DTCs) but not ISPSs of higher plants, implying that CpISPS is derived from moss DTCs and is evolutionarily unrelated to canonical ISPSs of higher plants. CpISPS is a novel class I cyclase of the terpene synthase-c subfamily harboring αß domains. This study will help further study of isoprene biosynthesis and the physiological functions of isoprene in mosses.

A comprehensive view of metabolic responses to CYP98 perturbation in ancestral plants.

Cytochrome P450 monooxygenase 98 (CYP98) is a critical rate-limiting enzyme of the phenylpropanoid pathway. One of the end-product of the phenylpropanoid pathway is a lignin monomer, although the occurrence of lignin in bryophytes is controversial. Here we investigated the functions of PpCYP98 in Physcomitrium patens by transcriptome and metabolome analyses. We identified 5266 differentially expressed genes (DEGs) and 68 differentially abundant secondary metabolites between wild-type and ΔPpCYP98 gametophores. Of the identified metabolites, 23 phenolic acids were identified, with only one showing upregulation. Among the phenolic acids, 4-coumaroyl tartaric acid and chlorogenic acid showed significant decreases. Declines were also observed in coniferylaldehyde and coniferin, precursor substances and downstream products of the lignin monomer coniferyl alcohol, respectively. Thus, the pre-lignin synthesis pathway already exists in bryophytes, and PpCYP98 plays vital roles in this pathway. Besides, most flavonoids show significant reductions, including eriodyctiol, dihydroquecetin, and dihydromyricetin, whereas naringenin chalone and dihydrokaempferol were increased after PpCYP98 knockout. Therefore, the synthesis of flavonoids shares the core pathway with phenylpropanoids and mainly starts from caffeoyl-CoA, that is the compound of divergence between the two pathways in moss. PpCYP98 showed systemic effects on metabolisms, including carbohydrate, fatty acid, and hormonal signaling transductions, suggesting that PpCYP98 might indirectly regulate carbon influx allocation. Our results demonstrated roles of PpCYP98 were essential for the development of the early landing plant.

Genome-Wide Analysis of Long Non-Coding RNAs Related to UV-B Radiation in the Antarctic Moss Pohlia nutans.

Antarctic organisms are consistently suffering from multiple environmental pressures, especially the strong UV radiation caused by the loss of the ozone layer. The mosses and lichens dominate the vegetation of the Antarctic continent, which grow and propagate in these harsh environments. However, the molecular mechanisms and related regulatory networks of these Antarctic plants against UV-B radiation are largely unknown. Here, we used an integrated multi-omics approach to study the regulatory mechanism of long non-coding RNAs (lncRNAs) of an Antarctic moss (Pohlia nutans) in response to UV-B radiation. We identified a total of 5729 lncRNA sequences by transcriptome sequencing, including 1459 differentially expressed lncRNAs (DELs). Through functional annotation, we found that the target genes of DELs were significantly enriched in plant-pathogen interaction and the flavonoid synthesis pathway. In addition, a total of 451 metabolites were detected by metabonomic analysis, and 97 differentially change metabolites (DCMs) were found. Flavonoids account for 20% of the total significantly up-regulated metabolites. In addition, the comprehensive transcriptome and metabolome analyses revealed the co-expression pattern of DELs and DCMs of flavonoids. Our results provide insights into the regulatory network of lncRNA under UV-B radiation and the adaptation of Antarctic moss to the polar environments.

How the pH Controls Photoprotection in the Light-Harvesting Complex of Mosses.

In response to varying light conditions, light-harvesting complexes (LHCs) switch from a light-harvesting state to a quenched state to protect the photosynthetic organism from excessive light irradiation in a strategy known as nonphotochemical quenching (NPQ). NPQ is activated by an acidification of the thylakoid lumen, which is sensed directly or indirectly by the LHC, resulting in a conformational change of the complex that leads to the quenched state. The conformational changes responsible for NPQ activation and their connection to specific quenching mechanisms are still unknown. Here, we investigate the pH-triggered conformational changes in the light-harvesting complex stress-related (LHCSR) of mosses. By combining constant-pH molecular dynamics and enhanced sampling techniques, we find that the pH sensitivity of the complex is driven by the coupled protonation of three residues modulating the conformation of the short amphipathic helix placed at the lumen side of the embedding membrane. Combining these results with quantum mechanics/molecular mechanics calculations, we show that the quenching mechanism sensitive to the pH goes through a charge-transfer between a carotenoid and an excited chlorophyll, which is controlled by the protein conformation.

A Physcomitrella PIN protein acts in spermatogenesis and sporophyte retention.

The auxin efflux PIN-FORMED (PIN) proteins are conserved in all land plants and important players in plant development. In the moss Physcomitrella (Physcomitrium patens), three canonical PINs (PpPINA-C) are expressed in the leafy shoot (gametophore). PpPINA and PpPINB show functional activity in vegetative growth and sporophyte development. Here, we examined the role of PpPINC in the life cycle of Physcomitrella. We established reporter and knockout lines for PpPINC and analysed vegetative and reproductive tissues using microscopy and transcriptomic sequencing of moss gametangia. PpPINC is expressed in immature leaves, mature gametangia and during sporophyte development. The sperm cells (spermatozoids) of pinC knockout mutants exhibit increased motility and an altered flagella phenotype. Furthermore, the pinC mutants have a higher portion of differentially expressed genes related to spermatogenesis, increased fertility and an increased abortion rate of premeiotic sporophytes. Here, we show that PpPINC is important for spermatogenesis and sporophyte retention. We propose an evolutionary conserved way of polar growth during early moss embryo development and sporophyte attachment to the gametophore while suggesting the mechanical function in sporophyte retention of a ring structure, the Lorch ring.

Horizontally acquired fungal killer protein genes affect cell development in mosses.

The moss Physcomitrium patens is crucial for studying plant development and evolution. Although the P. patens genome includes genes acquired from bacteria, fungi and viruses, the functions and evolutionary significance of these acquired genes remain largely unclear. Killer protein 4 (KP4) is a toxin secreted by the phytopathogenic fungus Ustilago maydis that inhibits the growth of sensitive target strains by blocking their calcium uptake. Here, we show that KP4 genes in mosses were acquired from fungi through at least three independent events of horizontal gene transfer. Two paralogous copies of KP4 (PpKP4-1 and PpKP4-2) exist in P. patens. Knockout mutants ppkp4-1 and ppkp4-2 showed cell death at the protonemal stage, and ppkp4-2 also exhibited defects in tip growth. We provide experimental evidence indicating that PpKP4-1/2 affects P. patens protonemal cell development by mediating cytoplasmic calcium and that KP4 genes are functionally conserved between P. patens and fungi. The present study provides additional insights into the role of horizontal gene transfer in land plant development and evolution.

The ScAPD1-like gene from the desert moss Syntrichia caninervis enhances resistance to Verticillium dahliae via phenylpropanoid gene regulation.

Soloist is a member of a distinct and small subfamily within the AP2/ERF transcriptional factor family that play important roles in plant biotic and abiotic stress responses. There are limited studies of Soloist genes and their functions are poorly understood. We characterized the abiotic and biotic stress tolerance function of the ScSoloist gene (designated as ScAPD1-like) from the desert moss Syntrichia caninervis. ScAPD1-like responded to multiple abiotic, biotic stresses and plant hormone treatments. ScAPD1-like protein located to the nucleus and bound to several DNA elements. Overexpression of ScAPD1-like in Arabidopsis did not alter abiotic stress resistance or inhibit Pseudomonas syringae pv. tomato (Pst) DC3000 infection. However, overexpression of ScAPD1-like significantly increased the resistance of transgenic Arabidopsis and S. caninervis to Verticillium dahliae infection, decreased reactive oxygen species accumulation and improved reactive oxygen species scavenging activity. ScAPD1-like overexpression plants altered the abundance of transcripts for lignin synthesis and promoted lignin accumulation in Arabidopsis. ScAPD1-like directly bind to RAV1, AC elements, and TATA-box in the promoters of AtPAL1 and AtC4H genes, respectively, in vitro. Chromatin immunoprecipitation-quantitative polymerase chain reaction assays demonstrated ScAPD1-like directly bound to PAL and C4H genes promoters in Arabidopsis and their homologs in S. caninervis. In S. caninervis, ScAPD1-like overexpression and RNAi directly regulated the abundance of ScPAL and ScC4H transcripts and modified the metabolites of phenylpropanoid pathway. We provide insight into the function of Soloist in plant defense mechanisms that likely occurs through activation of the phenylpropanoid biosynthesis pathway. ScAPD1-like is a promising candidate gene for breeding strategies to improve resistance to Verticillium wilt.

BaZFP1, a C2H2 Subfamily Gene in Desiccation-Tolerant Moss Bryum argenteum, Positively Regulates Growth and Development in Arabidopsis and Mosses.

C2H2 zinc finger protein (C2H2-ZFP) plays an important role in regulating plant growth, development, and response to abiotic stress. To date, there have been no analyses of the C2H2-ZFP family in desiccation-tolerant moss. In this study, we identified 57 BaZFP transcripts across the Bryum argenteum (B. argenteum) transcriptome. The BaZFP proteins were phylogenetically divided into four groups (I-IV). Additionally, we studied the BaZFP1 gene, which is a nuclear C2H2-ZFP and acts as a positive regulator of growth and development in both moss and Arabidopsis thaliana. The complete coding sequence of the BaZFP1 gene was isolated from B. argenteum cDNA, which showed a high expression level in a dehydration-rehydration treatment process. The overexpression of the BaZFP1 gene in the Physcomitrium patens and B. argenteum promoted differentiation and growth of gametophytes. Heterologous expression in Arabidopsis regulated the whole growth and development cycle. In addition, we quantitatively analyzed the genes related to growth and development in transgenic moss and Arabidopsis, including HLS1, HY5, ANT, LFY, FT, EIN3, MUS, APB4, SEC6, and STM1, and found that their expression levels changed significantly. This study may pave the way for substantial insights into the role of C2H2-ZFPs in plants as well as suggest appropriate candidate genes for crop breeding.

Syntrichia caninervis adapt to mercury stress by altering submicrostructure and physiological properties in the Gurbantünggüt Desert.

Sewage and industrial waste discharges have been found to have a deleterious effect on plant growth and environmental safety through the accumulation of trace metal mercury (Hg) in soils. Although the effects of Hg on vascular plants have been reported in terms of enzyme activity, oxidative damage and physiology, few studies have been done on non-vascular plants. A simulation experiment including 7 Hg concentrations (0, 10, 20, 30, 40, 50, 75 µM) was conducted to investigate the influence of Hg stress on ultrastructure and physiological properties of biocrust moss Syntrichia. caninervis across 7 consecutive days. The results showed that the lowest lethal concentration of S. caninervis was 30 µM Hg. The mortality rate of the plants increased significantly with Hg concentrations. The ultrastructure did not change significantly at Hg concentration ≤ 20 µM, while exceeding which, cell walls began to separate, nuclei began to blur, and chloroplasts began to expand. The soluble sugars (SS), peroxidase (POD), and superoxide dismutase (SOD) activities increased initially and then decreased with the increase of concentration in the time gradient, with the largest values at 20 µM. The contents of malondialdehyde (MDA) and proline (Pro) increased with the increase of Hg concentration, both reached peak value at 50 µM. However, chlorophyll (Chl) contents continued to decrease along both the concentration and time gradients. Pearson correlation and principal component analysis showed that two principal components (PC1 and PC2) explained 73.9% of the variance in plant adaptation to Hg stress. SOD, POD, Chl, SS, and Pro all responded well to Hg in S. caninervis. Our study showed that Hg stress caused changes in ultrastructure and physiological metabolism of S. caninervis. 20 µM was the maximum concentration of Hg that biocrust moss S. caninervis can tolerate. S. caninervis mainly adopted two adaptation strategies related to exclusion and accumulation to reduce Hg stress.

Limitations to photosynthesis in bryophytes: certainties and uncertainties regarding methodology.

Bryophytes are the group of land plants with the lowest photosynthetic rates, which was considered to be a consequence of their higher anatomical CO2 diffusional limitation compared with tracheophytes. However, the most recent studies assessing limitations due to biochemistry and mesophyll conductance in bryophytes reveal discrepancies based on the methodology used. In this study, we compared data calculated from two different methodologies for estimating mesophyll conductance: variable J and the curve-fitting method. Although correlated, mesophyll conductance estimated by the curve-fitting method was on average 4-fold higher than the conductance obtained by the variable J method; a large enough difference to account for the scale of differences previously shown between the biochemical and diffusional limitations to photosynthesis. Biochemical limitations were predominant when the curve-fitting method was used. We also demonstrated that variations in bryophyte relative water content during measurements can also introduce errors in the estimation of mesophyll conductance, especially for samples which are overly desiccated. Furthermore, total chlorophyll concentration and soluble proteins were significantly lower in bryophytes than in tracheophytes, and the percentage of proteins quantified as Rubisco was also significantly lower in bryophytes (<6.3% in all studied species) than in angiosperms (>16% in all non-stressed cases). Photosynthetic rates normalized by Rubisco were not significantly different between bryophytes and angiosperms. Our data suggest that the biochemical limitation to photosynthesis in bryophytes is more relevant than so far assumed.

When time is not of the essence: constraints to the carbon balance of bryophytes.

The data available so far indicate that the photosynthetic and relative growth rates of bryophytes are 10% of those reported for tracheophytes. By examining the existing literature and reanalysing data published in over 100 studies, this review examines the ecophysiological, biochemical, and structural reasons behind this phenomenon. The limiting Rubisco content and surface for gas exchange are the internal factors that can explain the low photosynthetic and growth rates of bryophytes. The role of the thicker cell walls of bryophytes in limiting CO2 diffusion is unclear, due to the current uncertainties regarding their porosity and permeability to CO2. From this review, it is also evident that, despite bryophytes having low photosynthetic rates, their positive carbon balance is tightly related to their capacity to deal with extreme conditions. Contributing factors include their capacity to deal with large daily temperature oscillations, and their capacity to delay the cessation of photosynthesis under water deficit (or to tolerate desiccation in extreme situations). Although further studies on bryophytes are needed before more solid conclusions can be drawn, it seems that their success relies on their remarkable tolerance to a highly variable environment, possibly at the expense of their maximum photosynthetic rate.

CLAVATA modulates auxin homeostasis and transport to regulate stem cell identity and plant shape in a moss.

The CLAVATA pathway is a key regulator of stem cell function in the multicellular shoot tips of Arabidopsis, where it acts via the WUSCHEL transcription factor to modulate hormone homeostasis. Broad-scale evolutionary comparisons have shown that CLAVATA is a conserved regulator of land plant stem cell function, but CLAVATA acts independently of WUSCHEL-like (WOX) proteins in bryophytes. The relationship between CLAVATA, hormone homeostasis and the evolution of land plant stem cell functions is unknown. Here we show that in the moss, Physcomitrella (Physcomitrium patens), CLAVATA affects stem cell activity by modulating hormone homeostasis. CLAVATA pathway genes are expressed in the tip cells of filamentous tissues, regulating cell identity, filament branching, plant spread and auxin synthesis. The receptor-like kinase PpRPK2 plays the major role, and Pprpk2 mutants have abnormal responses to cytokinin, auxin and auxin transport inhibition, and show reduced expression of PIN auxin transporters. We propose a model whereby PpRPK2 modulates auxin gradients in filaments to determine stem cell identity and overall plant form. Our data indicate that CLAVATA-mediated auxin homeostasis is a fundamental property of plant stem cell function, probably exhibited by the last shared common ancestor of land plants.

Chemotaxonomic and evolutionary perspectives of Bryophyta based on multivariate analysis of fatty acid fingerprints of Eastern Himalayan mosses.

Bryophyta comprises one of the earliest lineages of land plants that had implemented remarkable innovations to their lipid metabolic systems for successful adaptation to terrestrial habitat. This study presents a comprehensive investigation of fatty acid profiles of mosses from Eastern Himalayas with an aim to trace their chemotaxonomic and evolutionary implications. Fatty acid compositions of 40 random mosses belonging to major families of Bryophyta were explored by gas chromatographic analysis. A diverse array of saturated, monounsaturated and polyunsaturated fatty acids including rare acetylenic fatty acids were detected. Hexadecanoic acid (C16:0), 9,12 (Z,Z)-octadecadienoic acid (C18:2n6) and 9,12,15 (Z,Z,Z)-octadecatrienoic acid (C18:3n3) were the predominant fatty acids in all the mosses. However, quantitative variation of C20 polyunsaturated fatty acids (PUFAs), specifically 5,8,11,14 (Z,Z,Z,Z)-eicosatetraenoic acid (C20:4n6), among the investigated mosses was the most prominent outcome. The diplolepidous members of Bryidae, especially the mosses of Hypnales, Bryales and Bartramiales contained higher amount of C20 PUFAs compared with the haplolepidous orders. Principal component analyses based on individual fatty acids and other related parameters validated C20:4n6 content and the ratio of C20:4n6/C18:2n6 as the apparent chemotaxonomic discriminants. The prevalent notion of considering 9,12,15-octadecatrien-6-ynoic acid (C18:4a) as the chemomarker of Dicranaceae has also been challenged, since the compound was detected not only in different families of Dicranales, but also in a Pottiales member, Leptodontium viticulosoides. Therefore, an ensemble of fatty acids instead of a single one can be considered as the chemical signature for taxonomic interpretation which may also be vital from an evolutionary standpoint.

A conserved loop structure of GH19 chitinases assists the enzyme function from behind the core-functional region.

Plant GH19 chitinases have several loop structures, which may define their enzymatic properties. Among these loops, the longest loop, Loop-III, is most frequently conserved in GH19 enzymes. A GH19 chitinase from the moss Bryum coronatum (BcChi-A) has only one loop structure, Loop-III, which is connected to the catalytically important ß-sheet region. Here, we produced and characterized a Loop-III-deleted mutant of BcChi-A (BcChi-A-ΔIII) and found that its stability and chitinase activity were strongly reduced. The deletion of Loop-III also moderately affected the chitooligosaccharide binding ability as well as the binding mode to the substrate-binding groove. The crystal structure of an inactive mutant of BcChi-A-ΔIII was successfully solved, revealing that the remaining polypeptide chain has an almost identical fold to that of the original protein. Loop-III is not necessarily essential for the folding of the enzyme protein. However, closer examination of the crystal structure revealed that the deletion of Loop-III altered the arrangement of the catalytic triad, Glu61, Glu70 and Ser102, and the orientation of the Trp103 side chain, which is important for sugar residue binding. We concluded that Loop-III is not directly involved in the enzymatic activity but assists the enzyme function by stabilizing the conformation of the ß-sheet region and the adjacent substrate-binding platform from behind the core-functional regions.

Cytotoxicity, antimicrobial and antioxidant activities of mosses obtained from open habitats.

Mosses are mainly the object of ecological and taxonomic research. This group of plants are still underestimated by scientists in other aspects of research. Recent research has shown that these plants contain remarkable and unique substances with high biological activity. Five species of mosses from a large urban ecosystem were identified for present study. In order to determine their biological potential, multifaceted studies were carried out, including: total phenolics content, antioxidant activity, antimicrobial and antifungal study, cytotoxicity evaluation, and scratch assay to assess pro-regenerative effect in the context of their possible use as the ingredients of biologically active cosmetics. Additionally, determination of individual phenolic compounds in selected extracts of the tested mosses was made. Research showed that Ceratodon purpureus and Dryptodon pulvinatus extracts had the greatest potential as antioxidants and antimicrobial activity. The cytotoxicity assessment indicated that the extracts from Dryptodon pulvinatus and Rhytidiadelphus squarossus exerted the strongest negative effect on mouse fibroblast line L929 viability at higher concentrations. While, the extract from Tortulla muralis best stimulated human foreskin fibroblast line HFF-1 proliferation and wound healing. The research on individual phenolic compounds content in the extracts tested indicated over 20 peaks on UPLC chromatograms. The conducted study has shown that mosses, especially so far unexplored species of open ecosystems, and e.g. epilytic habitats, may be a valuable source of biologically active substances and thus may constitute important medical and cosmetic possibilities.