Characterization and Expression Analysis of a Glutathione Reductase Gene from Antarctic Moss Pohlia nutans

Glutathione reductase (GR) is a flavoprotein oxidoreductase and plays an important role in response to oxidative stresses in plants. A cDNA-encoding cytosolic GR [GenBank accession number GACA01029426, designated as Pohlia nutans glutathione reductase gene (PnGR)] was successfully cloned from Antarctic moss P. nutans. The full-length PnGR cDNA has 1,654 bp nucleotides with an open reading frame of 1,494 bp, encoding 497 amino acid residues. The deduced amino acid sequence of PnGR had 87.0 % identity with GR in Physcomitrella patens subsp. patens. The phylogenetic analysis showed that PnGR is clustered together with known cytosolic GR in other plants. In addition, the subcellular localization analysis by observing the transient expression of PnGR–green fluorescent protein fusion protein in Arabidopsis thaliana mesophyll protoplasts also revealed PnGR targeting to cytosol in plant cells. The expression patterns of PnGR under different abiotic stresses were determined by real-time PCR. Compared to the normal condition, the maximal mRNA accumulation of PnGR increased 3.82-fold at 4 °C, 2.92-fold at 10 °C, 4.14-fold with 200 mM NaCl, and 3.17-fold with drought stress, respectively. Together, our results suggested that the inducible PnGR might play an important role in Antarctic moss P. nutans acclimatizing to polar environment.     

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 μM. 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-[7-³H]-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.     

Mosses in High-Arctic lakes: in situ measurements of annual primary production and decomposition

Aquatic mosses are important primary producers in High-Arctic lakes, but little information is available on their contribution to the overall production in these lakes. In order to predict effects of climate change on whole-lake ecosystem characteristics, more knowledge is needed on the role of moss in primary production, the extent of nutrient limitation of moss primary production and whether moss serves as food resource for secondary producers. In this study, we conducted an in situ growth experiment of an aquatic moss in a High-Arctic lake in NE Greenland and used these data to determine annual net production of this moss in the whole lake. We also measured tissue-N and tissue-P in order to assess nutrient limitation of moss production, measured in situ decomposition rates by litter bag experiments over 1 year and assessed the role of moss as food source by analysing stable isotope ¹⁵N and ¹³C of relevant organism groups in the lake. Net primary production of moss was 1.3 gC m^?2 year^?1 and constituted 23 % of the total benthic primary production and 18 % of the total lake primary production. Stoichiometric assessments suggested N and P limitation of moss growth. On average, 15 % of the standing biomass was decomposed per year. Our results also indicate that moss is not directly used as food resource by herbivores, but the most abundant herbivore, Lepidurus arcticus, is feeding on the epiphytic biofilm on the moss. Moss biomass is instead incorporated into the microbial decomposer pathway. All together, the study shows that moss plays an important ecological role as primary producer in High-Arctic lakes and functions as substrate for periphytic biofilm that serves as food resource for important herbivore invertebrates.     

Novel localization of callose in the spores of Physcomitrella patens and phylogenomics of the callose synthase gene family

Background and Aims

Callose involvement in spore development is a plesiomorphic feature of land plants. Correlated light, fluorescence and immuno-electron microscopy was conducted on the developing spores of Physcomitrella patens to probe for callose. Using a bioinformatic approach, the callose synthase (PpCalS) genes were annotated and PpCalS and AtCalS gene families compared, testing the hypothesis that an exine development orthologue is present in P. patens based on deduced polypeptide similarity with AtCalS5, a known exine development gene.

Methods

Spores were stained with aniline blue fluorescent dye. Capsules were prepared for immuno-light and immuno-electron microscopy by gold labelling callose epitopes with monoclonal antibody. BLAST searches were conducted using the AtCalS5 sequence as a query against the P. patens genome. Phylogenomic analysis of the CalS gene family was conducted using PAUP (v.4·1b10).

Key Results

Callose is briefly present in the aperture of developing P. patens spores. The PpCalS gene family consists of 12 copies that fall into three distinct clades with AtCalS genes. PpCalS5 is an orthologue to AtCalS5 with highly conserved domains and 64 % similarity of their deduced polypeptides.

Conclusions

This is the first study to identify the presence of callose in moss spores. AtCalS5 was previously shown to be involved in pollen exine development, thus making PpCalS5 a suspect gene involved in moss spore exine development.Key words: Bryophyte, callose, callose synthase, exine development, moss, Physcomitrella patens, spores, sporogenesis     

Light- and dark-induced action potentials in Physcomitrella patens

Glass microelectrodes were inserted into Physcomitrella patens gametophyte leaves and action potentials (APs) were recorded in response to sudden illumination as well as after darkening, i.e., when the dark-induced membrane depolarization crossed a threshold. Application of 5 mM La³⁺ (a calcium channel inhibitor), 10 mM TEA⁺ (a potassium channel inhibitor) and increased free Ca²⁺ resulted in a loss of excitability. Lack of Ca²⁺ in the external medium did not prevent APs from occurring. It was concluded that during light- dark-induced excitation of Physcomitrella patens, APs might rely upon calcium influxes from the intracellular compartments. APs were not blocked by the proton pump inhibitors (DES, DCCD), although the resting potential (RP) diminished significantly.Key words: action potential, calcium, moss, Physcomitrella patens, plant     

Targeted knock-out of a gene encoding sulfite reductase in the moss Physcomitrella patens affects gametophytic and sporophytic development

A key step in sulfate assimilation into cysteine is the reduction of sulfite to sulfide by sulfite reductase (SiR). This enzyme is encoded by three genes in the moss Physcomitrella patens. To obtain a first insight into the roles of the individual isoforms, we deleted the gene encoding the SiR1 isoform in P. patens by homologous recombination and subsequently analysed the ΔSiR1 mutants. While ΔSiR1 mutants showed no obvious alteration in sulfur metabolism, their regeneration from protoplasts and their ability to produce mature spores was significantly affected, highlighting an unexpected link between moss sulfate assimilation and development, that is yet to be characterized.     

Effect of the metals iron, copper and silver on fluorobenzene biodegradation by Labrys portucalensis

Organic and metallic pollutants are ubiquitous in the environment. Many metals are reported to be toxic to microorganisms and to inhibit biodegradation. The effect of the metals iron, copper and silver on the metabolism of Labrys portucalensis F11 and on fluorobenzene (FB) biodegradation was examined. The results indicate that the addition of 1 mM of Fe²⁺ to the culture medium has a positive effect on bacterial growth and has no impact in the biodegradation of 1 and 2 mM of FB. The presence of 1 mM of Cu²⁺ was found to strongly inhibit the growth of F11 cultures and to reduce the biodegradation of 1 and 2 mM of FB to ca. 50 %, with 80 % of stoichiometrically expected fluoride released. In the experiments with resting cells, the FB degraded (from 2 mM supplied) was reduced ca. 20 % whereas the fluoride released was reduced to 45 % of that stoichiometrically expected. Ag⁺ was the most potent inhibitor of FB degradation. In experiments with growing cells, the addition of 1 mM of Ag⁺ to the culture medium containing 1 and 2 mM of FB resulted in no fluoride release, whereas FB degradation was only one third of that observed in control cultures. In the experiments with resting cells, the addition of Ag⁺ resulted in 25 % reduction in substrate degradation and fluoride release was only 20 % of that stoichiometrically expected. The accumulation of catechol and 4-fluorocatechol in cultures supplemented with Cu²⁺ or Ag⁺ suggest inhibition of the key enzyme of FB metabolism—catechol 1,2-dioxygenase.     

<Emphasis Type="Italic">Pythium</Emphasis> infection activates conserved plant defense responses in mosses

The moss Physcomitrella patens (P. patens) is a useful model to study abiotic stress responses since it is highly tolerant to drought, salt and osmotic stress. However, very little is known about the defense mechanisms activated in this moss after pathogen assault. In this study, we show that P. patens activated multiple and similar responses against Pythium irregulare and Pythium debaryanum, including the reinforcement of the cell wall, induction of the defense genes CHS, LOX and PAL, and accumulation of the signaling molecules jasmonic acid (JA) and its precursor 12-oxo-phytodienoic acid (OPDA). However, theses responses were not sufficient and infection could not be prevented leading to hyphae colonization of moss tissues and plant decay. Pythium infection induced reactive oxygen species production and caused cell death of moss tissues. Taken together, these data indicate that Pythium infection activates in P. patens common responses to those previously characterized in flowering plants. Microscopic analysis also revealed intracellular relocation of chloroplasts in Pythium-infected tissues toward the infection site. In addition, OPDA, JA and its methyl ester methyl jasmonate induced the expression of PAL. Our results show for the first time JA and OPDA accumulation in a moss and suggest that this defense pathway is functional and has been maintained during the evolution of plants. Authors Juan Pablo Oliver and Alexandra Castro contributed equally to this work.     

Genome analysis of the moss Physcomitrella patens (Hedw.) B.S.G.

A wild-type (WT) strain of the moss Physcomitrella patens (Hedw.) B.S.G., two mutants derived from it (PC22 and P24), and a somatic hybrid, PC22(+)P24, were analysed. Staining of metaphases revealed 54±2 chromosomes in the somatic hybrid and 27 chromosomes in the wild type and the two mutants. Using flow cytometry (FCM), DNA contents were calculated to be 0.6 pg (WT, PC22), 1.2 pg (P24), and 1.6 pg (PC22(+)P24) per nucleus, respectively. Southern hybridization provided evidence for at least one family of highly repetitive DNA and, furthermore, revealed different amounts of repetitive DNA in the four genotypes. However, these sequences cannot account for the 100% increase in the nuclear DNA amount in mutant P24, relative to wild type. In FCM analyses every moss geno-type generated just one single peak of fluorescence, indicating an arrest in the cell cycle during the daytime. Thermal denaturation of wild-type DNA revealed a G+C content of 34.6% for total DNA and 38.6% for plastid DNA. A cDNA library of 1.2 × 10⁶ independent clones was established, from which sequences homologous to cab and rbcS, respectively, were isolated. These genes show significant homologies to those of higher plants, and, likewise, comprise multigene families. No restriction fragment length polymorphisms could be detected between the four moss genotypes using these cDNA probes.     

2-Ketogluconic Acid Secretion by Incorporation of Pseudomonas putida KT 2440 Gluconate Dehydrogenase (gad) Operon in Enterobacter asburiae PSI3 Improves Mineral Phosphate Solubilization

Enterobacter asburiae PSI3 is known to efficiently solubilize rock phosphate by secretion of approximately 50 mM gluconic acid in Tris-buffered medium in the presence of 75 mM glucose and in a mixture of seven aldosugars each at 15 mM concentration, mimicking alkaline vertisol soils. Efficacy of this bacterium in the rhizosphere requires P release in the presence of low amount of sugars. To achieve this, E. asburiae PSI3 has been manipulated to express gluconate dehydrogenase (gad) operon of Pseudomonas putida KT 2440 to produce 2-ketogluconic acid. E. asburiae PSI3 harboring gad operon had 438 U of GAD activity, secreted 11.63 mM 2-ketogluconic and 21.65 mM gluconic acids in Tris-rock phosphate-buffered medium containing 45 mM glucose. E. asburiae PSI3 gad transformant solubilized 0.84 mM P from rock phosphate in TRP-buffered liquid medium. In the presence of a mixture of seven sugars each at 12 mM, the transformant brought about a drop in pH to 4.1 and released 0.53 mM P.     

Cation-permeable vacuolar ion channels in the moss Physcomitrella patens: a patch-clamp study

Patch-clamp studies carried out on the tonoplast of the moss Physcomitrella patens point to existence of two types of cation-selective ion channels: slowly activated (SV channels), and fast-activated potassium-selective channels. Slowly and instantaneously saturating currents were observed in the whole-vacuole recordings made in the symmetrical KCl concentration and in the presence of Ca²⁺ on both sides of the tonoplast. The reversal potential obtained at the KCl gradient (10 mM on the cytoplasmic side and 100 mM in the vacuole lumen) was close to the reversal potential for K⁺ (E _K), indicating K⁺ selectivity. Recordings in cytoplasm-out patches revealed two distinct channel populations differing in conductance: 91.6 ± 0.9 pS (n = 14) at ?80 mV and 44.7 ± 0.7 pS (n = 14) at +80 mV. When NaCl was used instead of KCl, clear slow vacuolar SV channel activity was observed both in whole-vacuole and cytoplasm-out membrane patches. There were no instantaneously saturating currents, which points to impermeability of fast-activated potassium channels to Na⁺ and K⁺ selectivity. In the symmetrical concentration of NaCl on both sides of the tonoplast, currents have been measured exclusively at positive voltages indicating Na⁺ influx to the vacuole. Recordings with different concentrations of cytoplasmic and vacuolar Ca²⁺ revealed that SV channel activity was regulated by both cytoplasmic and vacuolar calcium. While cytoplasmic Ca²⁺ activated SV channels, vacuolar Ca²⁺ inhibited their activity. Dependence of fast-activated potassium channels on the cytoplasmic Ca²⁺ was also determined. These channels were active even without Ca²⁺ (2 mM EGTA in the cytosol and the vacuole lumen), although their open probability significantly increased at 0.1 μM Ca²⁺ on the cytoplasmic side. Apart from monovalent cations (K⁺ and Na⁺), SV channels were permeable to divalent cations (Ca²⁺ and Mg²⁺). Both monovalent and divalent cations passed through the channels in the same direction—from the cytoplasm to the vacuole. The identity of the vacuolar ion channels in Physcomitrella and ion channels already characterised in different plants is discussed.     

Production and characterization of a solvent-tolerant protease from a novel marine isolate Bacillus tequilensis P15

Thirty-six proteolytic bacteria were isolated from the Jakhau coast, Kutch, India, amongst which isolate P15 identified as Bacillus tequilensis (JQ904626) was found to produce an extracellular solvent-- and detergent-tolerant protease (116.69?±?0.48 U/ml) and was selected for further investigation. Deoiled Jatropha seedcake (JSC) was found to be a suitable substrate for protease production under submerged condition. Upon optimization of process parameters following one-factor-at-a-time approach, an overall 6.4-fold (860.27?±?18.48 U/ml) increase in protease production was achieved. The maximum protease yield was obtained using a medium containing 2 % (w/v) deoiled JSC as substrate (pH of 8.0) upon 36 h of fermentation at 30 °C. The optimum temperature and pH for activity of B. tequilensis P15 protease was found to be 50 °C and 8.0, respectively. The enzyme exhibited a half-life of 190 min at 50 °C, which was enhanced to 270 min in presence of 5 mM Ca²⁺. The enzyme exhibited significant stability in almost all the solvents tested in the range of log P _ow varying from 8.8 to ?0.76. The enzyme activity was strongly inhibited by PMSF at 5 mM concentration, whereas the presence of EDTA (5 mM) and pCMB (5 mM) enhanced enzyme activity by 20.9 and 13.7 %, respectively. The enzyme was also found to be stable in the presence of surfactants, commercial detergents and bleach-oxidant (H₂O₂). This protease was demonstrated to be effective in removal of blood stains from fabrics, dehairing of hide, and stripping off the gelatin from used photographic films.     

The role of antioxidants enzymes of E. coli ASU3, a tolerant strain to heavy metals toxicity, in combating oxidative stress of copper

The current study describes the isolation and characterization of E. coli from wastewater that collected from El-Malah canal in Assiut, Egypt. Twelve isolates were investigated for heavy metal resistance by which one of them showed multiple metal resistances. Furthermore, the bacterium was identified as E. coli ASU3 according to biochemical tests and then, preserved at Assuit University Mycological Centre with accession number AUMC B83. It exhibited high minimal inhibitory concentrations for metals and antibiotic resistance. The order of metals toxicity to the bacterium was Cr⁶⁺ > Cu²⁺ > Co²⁺ > Pb²⁺ > Ni²⁺ > Cr³⁺ > Cd²⁺ > Zn²⁺. Total protein content of E. coli ASU3 decreased with the increase of copper concentration. Under exposure of different concentrations of copper, the induction of antioxidant enzymes such as catalase, peroxidase and ascorbate peroxidase was increased and these antioxidant enzymes can contribute to combating oxidative stresses.     

Characteristics of ammonia oxidation potentials and ammonia oxidizers in mineral soil under <Emphasis Type="Italic">Salix polaris</Emphasis>–moss vegetation in Ny-Ålesund,Svalbard

Although nitrification is a unique and important process in the nitrogen cycle with respect to ammonium consumption and nitrate production, limited information on this process is available for high-Arctic soils. We elucidated the ammonia oxidation potentials (AOPs) and characteristics of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in mineral soils under climax vegetation, i.e., Salix polaris (polar willow)–moss vegetation, on a coastal hill in Ny-Ålesund, Svalbard. AOPs at 10 °C were determined by incubation with sufficient substrate (2 mM ammonium). The ammonia monooxygenase subunit A (amoA) genes of AOB and AOA were analyzed by using quantitative polymerase chain reaction and pyrosequencing. AOPs ranged from 1.1 to 14.1 ng N g^?1 dry soil h^?1—relatively low but of a similar order to the gross nitrification rates reported in another Svalbard study. AOP was positively correlated with thickness of the moss layer (P < 0.01), soil water content, and ammonium nitrogen content (P < 0.05). The population sizes of both AOB and AOA were not significantly related to AOP or edaphic factors. For AOB-amoA, six major operational taxonomic units (OTUs) were identified, all of which were classified into the Nitrosospira Mount Everest cluster. For AOA-amoA, six major OTUs were also identified, five of which were grouped with sequences from cold environments within clade A of the Nitrososphaera cluster, i.e., species known to have low, or no, AOP. It is, therefore, possible that the AOPs measured at the study site were driven mainly by psychrotolerant AOB.     

Rhizosphere colonization and arsenic translocation in sunflower (Helianthus annuus L.) by arsenate reducing Alcaligenes sp. strain Dhal-L

In the present study, six arsenic-resistant strains previously isolated were tested for their plant growth promoting characteristics and heavy metal resistance, in order to choose one model strain as an inoculum for sunflower plants in pot experiments. The aim was to investigate the effect of arsenic-resistant strain on sunflower growth and on arsenic uptake from arsenic contaminated soil. Based on plant growth promoting characteristics and heavy metal resistance, Alcaligenes sp. strain Dhal-L was chosen as an inoculum. Beside the ability to reduce arsenate to arsenite via an Ars operon, the strain exhibited 1-amino-cyclopropane-1-carboxylic acid deaminase activity and it was also able to produce siderophore and indole acetic acid. Pot experiments were conducted with an agricultural soil contaminated with arsenic (214 mg kg^?1). A real time PCR method was set up based on the quantification of ACR3(2) type of arsenite efflux pump carried by Alcaligenes sp. strain Dhal-L, in order to monitor presence and colonisation of the strain in the bulk and rhizospheric soil. As a result of strain inoculation, arsenic uptake by plants was increased by 53 %, whereas ACR3(2) gene copy number in rhizospheric soil was 100 times higher in inoculated than in control pots, indicating the colonisation of strain. The results indicated that the presence of arsenate reducing strains in the rhizosphere of sunflower influences arsenic mobilization and promotes arsenic uptake by plant.