Proteins from the lichenXanthoria parietina which bind to phycobiont cell walls. Correlation between binding patterns and cell wall cytochemistry

Summary A protein fraction was isolated from the lichenXanthoria parietina which bound to the appropriate cultured phycobiont, but not to the freshly isolated symbiotic alga. The protein also appeared to discriminate between five other strains of cultured phycobionts from different lichens; phycobionts isolated from lichens in the familyTeloschistaceae bound the protein whereas phycobionts isolated from lichens in other families did not. Using cytochemical techniques, it was shown that protein binding ability was correlated with high levels of acidic polysaccharide in the cell wall, and the presence of a protein coat on the cell wall surface of the phycobiont. The possible role of this protein in recognition between lichen symbionts is briefly discussed.     

Oxidative stress induces distinct physiological responses in the two Trebouxia phycobionts of the lichen Ramalina farinacea

Background and Aims

Most lichens form associations with Trebouxia phycobionts and some of them simultaneously include genetically different algal lineages. In other symbiotic systems involving algae (e.g. reef corals), the relative abundances of different endosymbiotic algal clades may change over time. This process seems to provide a mechanism allowing the organism to respond to environmental stress. A similar mechanism may operate in lichens with more than one algal lineage, likewise protecting them against environmental stresses. Here, the physiological responses to oxidative stress of two distinct Trebouxia phycobionts (provisionally named TR1 and TR9) that coexist within the lichen Ramalina farinacea were analysed.

Methods

Isolated phycobionts were exposed to oxidative stress through the reactive oxygen species propagator cumene hydroperoxide (CuHP). Photosynthetic pigments and proteins, photosynthesis (through modulated chlorophyll fluorescence), the antioxidant enzymes superoxide dismutase (SOD) and glutathione reductase (GR), and the stress-related protein HSP70 were analysed.

Key Results

Photosynthetic performance was severely impaired by CuHP in phycobionts, as indicated by decreases in the maximal PSII photochemical efficiency (F_v/F_m), the quantum efficiency of PSII (Φ_PSII) and the non-photochemical dissipation of energy (NPQ). However, the CuHP-dependent decay in photosynthesis was significantly more severe in TR1, which also showed a lower NPQ and a reduced ability to preserve chlorophyll a, carotenoids and D1 protein. Additionally, differences were observed in the capacities of the two phycobionts to modulate antioxidant activities and HPS70 levels when exposed to oxidative stress. In TR1, CuHP significantly diminished HSP70 and GR but did not change SOD activities. In contrast, in TR9 the levels of both antioxidant enzymes and those of HSP70 increased in response to CuHP.

Conclusions

The better physiological performance of TR9 under oxidative conditions may reflect its greater capacity to undertake key metabolic adjustments, including increased non-photochemical quenching, higher antioxidant protection and the induction of repair mechanisms.     

Phycobionts of some Japanese species ofPertusaria (Pertusariaceae)

12 species ofPertusaria DC. were collected from various areas in Japan. Their phycobionts were isolated and studied taxonomically. The phycobionts of these species were indentified asTrebouxia potteri Ahmadjian exGärtner.Dedicated to Prof. DrLothar Geitler on the occasion of the 90th anniversary of his birthday.     

Isolation and characterization of a cyanobacterium-binding protein and its cell wall receptor in the lichen Peltigera canina

Peltigera canina, a cyanolichen containing Nostoc as cyanobiont, produces and secretes arginase to a medium containing arginine. Secreted arginase acts as a lectin by binding to the surface of Nostoc cells through a specific receptor which develops urease activity. The enzyme urease has been located in the cell wall of recently isolated cyanobionts. Cytochemical detection of urease is achieved by producing a black, electron-dense precipitate of cobalt sulfide proceeding from CO₂ evolved from urea hydrolysis in the presence of cobalt chloride. This urease has been pre-purified by affinity chromatography on a bead of active agarose to which arginase was attached. Urease was eluted from the beads by 50 mM α-D-galactose. The experimentally probed fact that a fungal lectin developing subsidiary arginase activity acts as a recognition factor of compatible algal cells in chlorolichens can now been expanded to cyanolichens.Key words: arginase, lectin, Peltigera canina, recognition, urease     

Response surface analysis of nano-ureases from Canavalia ensiformis and Cajanus cajan

Ureases isolated from leguminous sources, Canavalia ensiformis and Cajanus cajan were immobilized onto gold nanoparticles (nano-ureases). Optimization of the urease immobilization was carried using response surface methodology based on Central Composite Design. Immobilization efficiency of nano-urease from C. ensiformis and C. cajan were found to be 215.10% and 255.92%, respectively. The methodology adopted has deviation of 2.56% and 3.01% with respect to experimental values in case of C. ensiformis and C. cajan, respectively. Nano-urease from C. cajan has broad physico-chemical parameters with pH optimum from 7.1 to 7.3 and temperature optimum from 50 to 70 °C. Nano-urease from C. ensiformis has sharp pH and temperature optima at 7.3 and 70 °C, respectively. Fourier transform infra-red spectroscopy has revealed involvement of groups viz. amino, glycosyl moiety, etc. in urease immobilization onto gold nano-particles. Transmission and scanning electron micrographs revealed that arrangement of urease onto gold nano-particles from C. ensiformis was uniform while it was localized in case of C. cajan. Nano-urease from C. ensiformis has higher specificity and catalysis toward urea as compared to nano-urease from C. cajan. Nano-ureases from both sources are equally stable for 6 months under dried conditions and can be used for 10 washes.     

Maize stem tissues: ferulate deposition in developing internode cell walls

It has been hypothesized that ferulates are only deposited in the primary cell wall of grasses. To test this hypothesis, the fourth elongating, above-ground internode of maize (Zea mays l.) was sampled from three maize hybrids throughout development. Cell wall composition was determined by the Uppsala Dietary Fibre method. Ester- and ether-linked ferulates were determined by HPLC analysis of ferulic acid released from the internodes by low and high temperature alkaline treatments. Internode length increased from 9 to 152 mm over 96 days of growth, with elongation being complete in the first 12 days. More than half of the cell wall material in the maize internodes accumulated after elongation had ended. Deposition of cell wall material appeared to reach its maximum extent 40 days after sampling began, well before physiological maturity of the maize plants. Galactose and arabinose began to accumulate early in cell wall development which was presumed to be associated with primary wall growth during internode elongation. The major secondary wall constituents (analyzed as glucose, xylose, and Klason lignin) did not begin to accumulate rapidly until shortly before internode elongation ended. Ferulate ester deposition began before ferulate ethers were observed in the cell wall, but both forms of ferulate continued to accumulate in secondary cell walls, long after internode elongation had ceased. These data clearly show that contrary to the hypothesis, ferulate deposition was not restricted to the primary wall and that active lignin/polysaccharide cross-linking mediated by ferulates occurs in the secondary wall.     

Absorption of mulberry root urease to the hemolymph of the silkworm, Bombyx mori

Mulberry leaves are the sole diet of the silkworm, Bombyx mori. The host urease is incorporated into the larval hemolymph and involved in nitrogen metabolism in the insect. To investigate the selective absorption of the host urease to the larvae, crude urease was prepared from mulberry leaves and roots. Root urease was identical to leaf urease on the basis of electrophoretic analyses: (1) the urease activity appeared in the same migration position in a native gel; (2) There was no difference in molecular mass of the subunit. The root urease was orally injected to the fifth instar larvae of the silkworm. Just before spinning, the larvae absorbed intact urease from the midgut lumen to the hemolymph without the loss of activity. The capacity to absorb urease occurred only at the specific stage. Localization of host urease in midgut tissue was observed using confocal laser scanning microscopy and transmission electron microscopy. Based on spatial distribution of immunofluorescent signals and immunogold particles, host urease specifically attached to the surfaces of microvilli existing in the apical side of columnar cells and appeared in the cytoplasm of the cells for transport to the hemolymph. The incorporation efficiency of root urease into the hemolymph was significantly higher than for ureases from jack bean seeds and Bacillus pasteurii. The urease that was transported to the hemolymph was electrophoretically altered, compared with the host urease extracted.     

Construction of two ureolytic model organisms for the study of microbially induced calcium carbonate precipitation

Two bacterial strains, Pseudomonas aeruginosa MJK1 and Escherichia coli MJK2, were constructed that both express green fluorescent protein (GFP) and carry out ureolysis. These two novel model organisms are useful for studying bacterial carbonate mineral precipitation processes and specifically ureolysis-driven microbially induced calcium carbonate precipitation (MICP). The strains were constructed by adding plasmid-borne urease genes (ureABC, ureD and ureFG) to the strains P. aeruginosa AH298 and E. coli AF504gfp, both of which already carried unstable GFP derivatives. The ureolytic activities of the two new strains were compared to the common, non-GFP expressing, model organism Sporosarcina pasteurii in planktonic culture under standard laboratory growth conditions. It was found that the engineered strains exhibited a lower ureolysis rate per cell but were able to grow faster and to a higher population density under the conditions of this study. Both engineered strains were successfully grown as biofilms in capillary flow cell reactors and ureolysis-induced calcium carbonate mineral precipitation was observed microscopically. The undisturbed spatiotemporal distribution of biomass and calcium carbonate minerals were successfully resolved in 3D using confocal laser scanning microscopy. Observations of this nature were not possible previously because no obligate urease producer that expresses GFP had been available. Future observations using these organisms will allow researchers to further improve engineered application of MICP as well as study natural mineralization processes in model systems.     

Effects of selenium, iron and cobalt addition to growth and yessotoxin production of the toxic marine dinoflagellate Protoceratium reticulatum in culture

The marine dinoflagellate Protoceratium reticulatum has been recently identified as a source for the disulfated polyether toxin, yessotoxin (YTX), and may pose a risk to human health, aquaculture development and coastal environments. The requirements of P. reticulatum for selenium, iron and cobalt were assessed in culture. P. reticulatum was grown in nutrient enriched seawater (1/10 GP medium) without selenium or with 0.003 and 0.0003 μM selenium added; without iron or with 0.076 and 0.0076 μM iron added; and without cobalt or with 0.008 μM cobalt added. Test flasks were monitored for growth rate, cell yield and YTX production. P. reticulatum was found to exhibit a strong requirement for both selenium and iron. Growth rate and cell yield in treatments without added selenium were significantly (P<0.05) reduced to 60.2% (μ=0.15 day⁻¹) and 20.2% (4942 cell ml⁻¹), respectively, of those with selenium added (μ=0.23 day⁻¹ and 24, 387 cell ml⁻¹). YTX production was significantly increased by addition of selenium in two of three transfers tested. Cells of P. reticulatum subjected to medium without selenium added showed morphological changes observable at the light microscope level which included enlarged cell size. The diameter of cells in medium without selenium added were significantly (P<0.05) enlarged to 36.7±0.90 μm compared to cells in the medium with selenium added, 27.5±1.25 μm. Growth rate and cell yield in treatments without added iron were also significantly reduced to 70.1% (μ=0.16 day⁻¹) and 34.2% (8003 cells ml⁻¹), respectively, of those with iron added (μ=0.23 day⁻¹ and 23,416 cells ml⁻¹). No significant effect on YTX production was measured. In contrast to selenium and iron, no limitation of growth or cell yield or differences in YTX production were observed for flasks without cobalt as compared to those with cobalt added. The possibility that harmful algal events of P. reticulatum may be influenced by selenium or iron in neritic waters is discussed.     

Increase of free cysteine and citric acid in plant cells exposed to cobalt ions

Cobalt complexation was investigated in a suspension cell culture of the cobalt hyperaccumulator Crotalaria cobalticola. C. cobalticola cells were more tolerant towards cobalt ions than the suspension cells of the non-accumulators Rauvolfia serpentina and Silene cucubalus. While the concentration of various compounds increased in cells of C. cobalticola challenged with cobalt ions, phytochelatin biosynthesis was not induced. Instead, the exposure to cobalt ions resulted in the increase of citrate and cysteine in cells. Size exclusion chromatography demonstrated the co-elution of cobalt and cysteine in C. cobalticola cell extracts. A significant increase in cysteine was observed also in cells of R. serpentina and S. cucubalus when they were exposed to cobalt ions. These results suggest that free cysteine is involved in cobalt ion complexation in plant cells.     

Evaluation of different yeast cell wall mutants and microalgae strains as feed for gnotobiotically grown brine shrimp Artemia franciscana

The nutritional value of isogenic yeast strains and two microalgal species for gnotobiotically grown Artemia was examined. Yeast cell wall mutants were always better feed for Artemia than their respective wild type. Yeast cells harbouring null mutants for enzymes involved early in the biochemical pathway for cell wall mannoproteins synthesis performed best as feed for Artemia. Yeast cells defective in chitin or β-glucan production were scored in second order. The mnn6 isogenic yeast mutant, harbouring a null mutation for mannoprotein phosphorylation, performed poorly as feed for Artemia, although with good growth. These results suggest that any mutation affecting the yeast cell wall scaffolding by reducing the amount of covalent links between the major components of yeast cell wall, namely mannoproteins, β-glucans and chitin, is sufficient to improve the digestibility for Artemia. The results with microalgae indicated that within one species, strains can have different nutritional value under gnotobiotic conditions. The growth phase was another parameter influencing feed quality, although here it was not possible to reveal the exact cause. It is anticipated that the standard Artemia gnotobiotic growth test is an excellent tool to study the mode of action of bacteria, with a probiotic as well as with a pathogenic character.     

Non-lignified helical cell wall thickenings in root cortical cells of Aspleniaceae (Polypodiales): histology and taxonomical significance

Background and Aims

Extraxylary helical cell wall thickenings in vascular plants are not well documented, except for those in orchid velamen tissues which have been studied extensively. Reports on their occurrence in ferns exist, but detailed information is missing. The aim of this study is to focus on the broad patterns of structure and composition and to study the taxonomic occurrence of helical cell wall thickenings in the fern family Aspleniaceae.

Methods

Structural and compositional aspects of roots have been examined by means of light, electron, epifluorescence and laser scanning confocal microscopy. To assess the taxonomical distribution of helical cell wall thickenings a molecular phylogenetic analysis based on rbcL sequences of 64 taxa was performed.

Key Results

The helical cell wall thickenings of all examined species showed considerable uniformity of design. The pattern consists of helical, regularly bifurcating and anastomosing strands. Compositionally, the cell wall thickenings were found to be rich in homogalacturonan, cellulose, mannan and xyloglucan. Thioacidolysis confirmed our negative phloroglucinol staining tests, demonstrating the absence of lignins in the root cortex. All taxa with helical cell wall thickenings formed a monophyletic group supported by a 100 % bootstrap value and composed of mainly epiphytic species.

Conclusions

This is the first report of non-lignified pectin-rich secondary cell walls in ferns. Based on our molecular analysis, we reject the hypothesis of parallel evolution of helical cell wall thickenings in Aspleniaceae. Helical cell wall thickenings can mechanically stabilize the cortex tissue, allowing maximal uptake of water and nutrients during rainfall events. In addition, it can also act as a boundary layer increasing the diffusive pathway towards the atmosphere, preventing desiccation of the stele of epiphytic growing species.     

Effect of salinity and pH on cobalt biosorption by the estuarine microalgaChlorella saliva

Summary Accumulation of cobalt (⁶⁰Co) by the estuarine microalgaChlorella salina has been characterized. At cobalt concentrations ranging over 3.125–100 M, a significant amount of cobalt was bound within 1 min. This was metabolism-independent and unaffected by incubation in light or dark conditions. This initial rapid phase of biosorption was followed by a slower phase of uptake which was apparently active and inhibited by incubation in the dark, or by the uncoupler dinitrophenol and the respiratory and photosynthetic inhibitor potassium cyanide in the light. For cells suspended in 10 mM Taps pH 8, cobalt biosorption followed a Freundlich adsorption isotherm. However, in the presence of 0.5 M NaCl, biosorption deviated from the Freundlich model because of competition by Na⁺. Cobalt biosorption was decreased by increasing concentrations of Na⁺, decreasing pH and the presence of Cs⁺, Li⁺, Rb⁺, Zn²⁺. Mn²⁺ and Sr²⁺ (added as chlorides). This was a result of competition between Co²⁺ and the other cations, including H⁺, for available binding sites on the cell wall and was confirmed by increased desorption of cobalt by solutions of low pH or high salinity. Increasing cell density resulted in increased removal of cobalt from solution but decreased the specific amount of cobalt taken up by the cells.     

The role of nickel in urea assimilation by algae

Nickel is required for urease synthesis by Phaeodactylum tricornutum and Tetraselmis subcordiformis and for growth on urea by Phaeodactylum. There is no requirement for nickel for urea amidolyase synthesis by Chlorella fusca var. vacuolata. Neither copper nor palladium can substitute for nickel but cobalt partially restored urease activity in Phaeodactylum. The addition of nickel to nickel-deficient cultures of Phaeodactylum or Tetraselmis resulted in a rapid increase of urease activity to 7–30 times the normal level; this increase was not inhibited by cycloheximide. It is concluded that nickel-deficient cells over-produce a non-functional urease protein and that either nickel or the functional urease enzyme participates in the regulation of the production of urease protein.Abbreviation UALase ATP; urea amidolyase     

Chemoprevention by thyme oils of Aspergillus parasiticus growth and aflatoxin production

The essential oils from Thymus eriocalyx and Thymus X-porlock obtained by hydrodistillation were analyzed by GC/MS. The major components of T. eriocalyx and T. X-porlock oils were thymol (63.8, 31.7%), beta-phellandrene (13.30, 38.7%), cis-sabinene hydroxide (8.1, 9.6%), 1,8-cineole (2, 1.7%), and beta-pinene (1.31, 2%), respectively. Antifungal activities of the oils were studied with special reference to the inhibition of Aspergillus parasiticus growth and aflatoxin production. Minimal inhibitory (MIC) and minimal fungicidal (MFC) concentrations of the oils were determined. Static effects of the above oils against A. parasiticus were at 250 ppm and lethal effects of T. eriocalyx and T. X-porlock were 500 and 1000 ppm of the oils, respectively. Aflatoxin production was inhibited at 250 ppm of both oils with that of T. eriocalyx being stronger inhibitor. Transmission electron microscopy (TEM) of A. parasiticus exposed to MIC level (250 ppm) of the oils showed irreversible damage to cell wall, cell membrane, and cellular organelles. It is concluded that the essential oils could be safely used as preservative materials on some kinds of foods at low concentrations to protect them from fungal infections.     

A novel ATP/ADP hydrolysis activity of hyperthermostable group II chaperonin in the presence of cobalt or manganese ion

A novel ATPase activity that was strongly activated in the presence of either cobalt or manganese ion was discovered in the chaperonin from hyperthermophilic Pyrococcus furiosus (Pfu-cpn). Surprisingly, a significant ADPase activity was also detected under the same conditions. A more extensive search revealed similar nucleotide hydrolysis activities in other thermostable chaperonins. Chaperonin activity, i.e., thermal stabilization and refolding of malate dehydrogenase from the guanidine-hydrochloride unfolded state were also detected for Pfu-cpn under the same conditions. We propose that the novel cobalt/manganese-dependent ATP/ADPase activity may be a common trait of various thermostable chaperonins.     

Purification and characterization of urease from dehusked pigeonpea (Cajanus cajan L) seeds

Urease has been purified from the dehusked seeds of pigeonpea (Cajanus cajan L.) to apparent electrophoretic homogeneity with approximately 200 fold purification, with a specific activity of 6.24 x10(3) U mg(-1) protein. The enzyme was purified by the sequence of steps, namely, first acetone fractionation, acid step, a second acetone fractionation followed by gel filtration and anion-exchange chromatographies. Single band was observed in both native- and SDS-PAGE. The molecular mass estimated for the native enzyme was 540 kDa whereas subunit values of 90 kDa were determined. Hence, urease is a hexamer of identical subunits. Nickel was observed in the purified enzyme from atomic absorption spectroscopy with approximately 2 nickel ions per enzyme subunit. Both jack bean and soybean ureases are serologically related to pigeonpea urease. The amino acid composition of pigeonpea urease shows high acidic amino acid content. The N-terminal sequence of pigeonpea urease, determined up to the 20th residue, was homologous to that of jack bean and soybean seed ureases. The optimum pH was 7.3 in the pH range 5.0-8.5. Pigeonpea urease shows K(m) for urea of 3.0+/-0.2 mM in 0.05 M Tris-acetate buffer, pH 7.3, at 37 degrees C. The turnover number, k(cat), was observed to be 6.2 x 10(4) s(-1) and k(cat)/K(m) was 2.1 x 10(7) M(-1) s(-1). Pigeonpea urease shows high specificity for its primary substrate urea.     

Alterations of O-glycosylation, cell wall, and mitochondrial metabolism in Kluyveromyces lactis cells defective in KlPmr1p, the Golgi Ca(2+)-ATPase

In yeast the P-type Ca(2+)-ATPase of the Golgi apparatus, Pmr1p, is the most important player in calcium homeostasis. In Kluyveromyces lactis KlPMR1 inactivation leads to pleiotropic phenotypes, including reduced N-glycosylation and altered cell wall morphogenesis. To study the physiology of K. lactis when KlPMR1 was inactivated microarrays containing all Saccharomyces cerevisiae coding sequences were utilized. Alterations in O-glycosylation, consistent with the repression of KlPMT2, were found and a terminal N-acetylglucosamine in the O-glycans was identified. Klpmr1Delta cells showed increased expression of PIRs, proteins involved in cell wall maintenance, suggesting that responses to cell wall weakening take place in K. lactis. We found over-expression of KlPDA1 and KlACS2 genes involved in the Acetyl-CoA synthesis and down-regulation of KlIDP1, KlACO1, and KlSDH2 genes involved in respiratory metabolism. Increases in oxygen consumption and succinate dehydrogenase activity were also observed in mutant cells. The described approach highlighted the unexpected involvement of KlPMR1 in energy-yielding processes.     

Dynamic changes in cell wall polysaccharides during wheat seedling development

Changes in arabinoxylan content and composition during development of wheat seedlings were investigated. The cell walls isolated from the seedlings showed an increasing content of arabinoxylan during development, which could be correlated to increased activity of xylan synthase and arabinoxylan arabinosyltransferase. Arabinoxylan changed from initially having a high degree of arabinose substitution to a much lower degree of substitution. beta-Glucan was present in the walls at the early stages of development, but was actively degraded after day 4. Increased deposition of arabinoxylan did not take place until beta-glucan had been fully degraded. Ferulic and p-coumaric acid esters were present at all points but increased significantly from day 3 to 6, where lignification began. Ferulic acid dimers did not appear in the cell wall until day three and the different ferulic acid dimers varied in the course of accumulation. The ratio of ferulic acid dimers to free ferulic acid was maximal at the time when the wall had been depleted for beta-glucan, which had not yet been fully replaced by arabinoxylan. This pattern suggests a role for ferulic acid dimers in stabilizing the wall during the transition from a flexible to a more rigid structure. To investigate if the same changes could be observed within a single seedling, 7 day old seedlings were divided into four sections and the walls were analyzed. Some of the changes observed during the seedling development could also be observed within a single seedling, when analyzing the segments from the elongation zone at the base to the top of the leaf. However, the expanding region of older seedlings was much richer in hydroxycinnamates than the expanding region of younger seedlings. Diferulic acids are stabilizing the wall in the transition phase from an expanding to a mature wall. This transition can take place in different manners depending on the cell and tissue type.     

Glucogalactan: A polysaccharide isolated from the cell-wall of Verticillium Lecanii

A water-soluble polysaccharide was extracted with alkali from the cell wall of Verticillium lecanii (also called Lecanicillium lecanii). After freezing and thawing, the water-soluble fraction was purified by gel filtration chromatography on Sepharose CL-6B and eluted as one peak by HPSEC/RID. Monosaccharide analysis showed galactose and glucose (1.1:1), with traces of mannose (<1%). The structural characteristics were determined by spectroscopic analysis, FT-IR and 1D and 2D ¹H and ¹³C NMR, and methylation results. On the basis of the data obtained, the following structure of the polysaccharide (E₃S_IV fraction) was established: