An Arabidopsis callose synthase

-1,3-glucan polymers are major structural components of fungal cell walls, while cellulosic -1,4-glucan is the predominant polysaccharide in plant cell walls. Plant -1,3-glucan, called callose, is produced in pollen and in response to pathogen attack and wounding, but it has been unclear whether callose synthases can also produce cellulose and whether plant cellulose synthases may also produce -1,3-glucans. We describe here an Arabidopsis gene, AtGsl5, encoding a plasma membrane-localized protein homologous to yeast -1,3-glucan synthase whose expression partially complements a yeast -1,3-glucan synthase mutant. AtGsl5 is developmentally expressed at highest levels in flowers, consistent with flowers having high -1,3-glucan synthase activities for deposition of callose in pollen. A role for AtGsl5 in callose synthesis is also indicated by AtGsl5expression in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated -1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5mRNA accumulation is induced by SA in wild-type plants, while expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant.     

Several phenotypic changes in the cell envelope of Agrobacterium tumefaciens chvB mutants are prevented by calcium limitation

The chvB gene of Agrobacterium tumefaciens encodes a 235 kDa proteinaceous intermediate involved in the synthesis of -1,2-glucan. chvB mutants show a pleiotropic phenotype. Besides not to produce cyclic -1,2-glucan, chvB mutants have been reported to be avirulent, attachment-deficient, and nonmotile. In this study we report additional differences from the parent strain, probably all linked to changes in the cell envelope. This pleiotropic phenotype — except for attachment and virulence — could largely be prevented by growing chvB cells with low levels of calcium. Although a role for -1,2-glucan in osmoadaptation has been proposed, the mode of action of -1,2-glucan is not known. We speculate that in A. tumefaciens -1,2-glucan stabilizes membranes, which would be important especially in hypotonic media containing calcium.Abbreviations Cb carbenicillin - Km kanamycin - TCA trichloroacetic acid - K_av fraction of the stationary gel volume available for diffusion - LPS lipopolysaccharide - SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Infectivity of Chlorella species for the ciliate Paramecium bursaria is not based on sugar residues of their cell wall components, but on their ability to localize beneath the host cell membrane after escaping from the host digestive vacuole in the early infection process

Summary. Paramecium bursaria cells harbor several hundred symbiotic algae in their cytoplasm. Algae-free cells can be reinfected with algae isolated from algae-bearing cells or cultivated Chlorella species through the digestive vacuoles. To determine the relationship between the infectivity of various Chlorella species and the nature of their cell wall components, algae-free P. bursaria cells were mixed with 15 strains of cultivated Chlorella species and observed for the establishment of endosymbiosis at 1 h and 3 weeks after mixing. Only 2 free-living algal strains, C. sorokiniana C-212 and C. kessleri C-531, were maintained in the host cells, whereas free-living C. sorokiniana C-43, C. kessleri C-208, C. vulgaris C-27, C. ellipsoidea C-87 and C-542, C. saccharophila C-183 and C-169, C. fusca var. vacuolata C-104 and C-28, C. zofingiensis C-111, and C. protothecoides C-150 and C-206 and the cultivated symbiotic Chlorella sp. strain C-201 derived from Spongilla fluviatilis could not be maintained. These infection-incapable strains could escape from the host digestive vacuole but failed to localize beneath the host cell membrane and were eventually digested. Labeling of their cell walls with Alexa Fluor 488-conjugated wheat germ agglutinin, GS-II, or concanavalin A, with or without pretreatment with 0.4 N NaOH, showed no relationship between their infectivity and the stainability with these lectins. Our results indicate that the infectivity of Chlorella species for P. bursaria is not based on the sugar residues on their cell wall and on the alkali-insoluble part of the cell wall components, but on their ability to localize just beneath the host cell membrane after escaping from the host digestive vacuole. Correspondence and reprints: Environmental Science and Engineering, Graduate School of Science and Engineering, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan.     

Heterologous expression of cDNAs encoding Chlorella sorokiniana NADP-specific glutamate dehydrogenase wild-type and mutant subunits in Escherichia coli cells and comparison of kinetic and thermal stability properties of their homohexamers

Full-length cDNAs encoding the - and -subunits and a truncated mutant subunit of the Chlorella sorokiniana NADP-GDH isozymes were constructed and expressed in Escherichia coli cells. The kinetic and thermal stability properties of the resultant homohexamers were examined. The electrophoretic mobility of the recombinant - and -subunits was identical to that of the native subunits as determined by immunoblotting. The homohexamers were purified by anion-exchange and gel-filtration chromatography. The - and -homohexamers that were synthesized in the bacterial cells were shown to have similar Michaelis constants for their substrates as previously shown after synthesis in C. sorokiniana cells (Bascomb and Schmidt, 1987). The homohexamer synthesized in the bacterium was allosteric with respect to NADPH but to a lesser degree than when isolated from the alga. The mutant homohexamer was composed of subunits that were truncated by 40 amino acids at their N-termini. This mutant isozyme was kinetically similar to the larger, anabolic -homohexamer, but it did not display the allosteric response to NADPH shown by the -homohexamer. The three isozymes had significant thermal tolerance and were stable at 50 °C. The temperature optimum for catalytic activity for the - and -homohexamers was 60 °C, and 65 °C for the 40N homohexamer. This study demonstrated that most of the kinetic properties of the Chlorella sorokiniana NADP-GDH isozymes were retained after their synthesis in a heterologous system, and that the distinctive N-terminal domains of these isozymes have dramatic effects on their biochemical characteristics.     

Observations on the cell-wall compositions of the bracket fungi Laetiporus sulphureus and Piptoporus betulinus

Homogenized tissues and their alkali-soluble and alkali-insoluble fractions of fruiting bodies of the basidiomycetes Laetiporus sulphureus and Piptoporus betulinus were investigated using X-ray diffraction, infrared spectrometry and chemical methods. The presence of (13)--d-glucan, (13)--d-glucan and chitin was established. The relative amounts of these polysaccharides were different in the two species and differences were also found between context and trama. The proportion of (13)--d-glucan was exceptionally high in the context of L. sulphureus (about 78%). In addition, the trama of both species contained a substance resembling a cyclic wax by its X-ray pattern and solubility properties. The substances identified are considered to belong to the hyphal wall     

Cell wall-lytic activity in Chlorella fusca

The soluble fraction of homogenates of synchronous Chlorella fusca was tested for carbohydrate-lyzing activities. With isolated cell walls and -1,4-mannan or carboxymethyl cellulose as substrates, a sharp increase in activity occurred shortly before release of the daughter cells followed by a decline during release. The lytic activities were partially purified by ammonium sulphate precipitation and analyzed by gel filtration on a calibrated column. Apparent molecular weights were 27,000 for cell wall autolysin(s) and -1,4-mannanase, 36,000 for carboxymethyl cellulase and 70,000 for another -1,4-mannanase. Incubation of isolated cell walls with an enzyme preparation purified by ammonium sulphate precipitation resulted in release of up to 70% of the cell wall carbohydrate as monosaccharide, predominantly mannose and glucose. The carbohydrate released in vivo into the culture medium shortly before and during liberation of the daughter cells consisted largely of polymeric material with rhamnose, fucose and mannose as main constitutents. Upon poisoning the cells with NaN₃ or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, however, a monosaccharide fraction consisting of mannose and glucose was predominant in the medium. It is suggested that the major products of cell wall lysis in vivo are monosaccharides which are rapidly taken up and metabolized by the developing daughter cells in an energy-dependent manner.     

β-D-glucan-hydrolase activities in pure cell-wall-enriched fractions from Valerianella olitoria cells

An effective method for the preparation of purified cell walls from mesophyll cells of Valerianella olitoria has been developed. Cells were isolated by a mechanical procedure only and crude cell walls were prepared from cell homogenates. Crude wall suspensions were fractionated in a discontinuous sucrose gradient and the wall fragments recovered were examined by scanning electron microscopy. An evaluation of the degree of purity and physiological integrity of the wall fragments showed that the material found at the 50–60% (w/w) interface consisted mostly of wall particles of high purity. Some characteristics of the purified walls are reported, especially the following enzyme activities: -d-glucosidase (EC 3.2.1.21) and the -d-glucanases, 1,4--glucan glucanohydrolase (EC 3.2.1.4), 1,4--glucan cellobiohydrolase (EC 3.2.1.91), 1,3--glucan glucanohydrolase (EC 3.2.1.39), 1,3--glucan glucohydrolase (EC 3.2.1.58). The results provided evidence for the microlocalization of some hydrolases and indicated that enzymes extracted only with a high-salt-concentration buffer were confined to walls whereas the 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris)-solubilized enzymes could have multiple sites, e.g. walls and membranes of the endoplasmic reticulum.Abbreviations CMC carboxymethylcellulose - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - PM plasma membrane(s) - ER endoplasmic reticulum     

Cell wall polysaccharides from Talaromyces species

The neutral sugars and amino sugars, released by acid hydrolysis of walls and polysaccharidic fractions, of six species of Talaromyces and the infrared spectra have been used to study their interspecific relationships. In whole cell walls neutral sugars ranged from 23 to 39.6% dry weight and were identified as glucose, galactose and mannose. Glucosamine varied from 8 to 19.8% in the samples. Galactosamine (2% or less) was found in T. emersonii and T. rotundus and no galactosamine in the other species. Sequential fractionation of the cell walls with alkali and acid gave several polysaccharidic fractions. The main differences among species were found in the alkali-soluble fraction at 20° (F1). This fraction represented 8 to 33.2% of the whole cell wall and was characterized as an -glucan in T. bacillisporus, T. emersonii, T. luteus and T. rotundus (Group A) and as a -galactofuranosyl containing glucan in T. ohiensis and T. stipitatus (Group B). The alkali-insoluble residue (F4) represented the bulk of the cell wall in all species tested (33.2% to 57.3%) and was characterized as a -glucan/chitin complex. The results may indicate degrees of interspecific relationship in the genus Talaromyces.Abbreviations CWM cell wall material - GLC gas-liquid chromatography - IR infrared - wt weight - CBS Centraal Bureau voor Schimmelcultures (Baarn. The Netherlands) - Ara arabinose - Xyl xylose - Man mannose - Gal galactose - Glc glucose - GlcNH₂ glucosamine - GalNH₂ galactosamine     

Yeast Kre1p is a cell surface O-glycoprotein

The Saccharomyces cerevisiae KRE1 gene encodes a secretory protein required for the production of the cell wall polymer (1 6)--glucan. Here we report further characterization of the KRE1 gene product, Krelp. A functional, epitope-tagged Krelp is shown to be highly modified in a SEC53-dependent manner. Krelp is O-glycosylated, but the basis for the majority of its post-translational modification is unknown. Fractionation of Kre1p reveals a cell wall-associated form and a less abundant membrane-associated species. Indirect immunoflurorescence demonstrates that Kre1p localizes to the cell surface, where it becomes concentrated at the surface of mother cells. Such a localization of Kre1p seems to parallel the CAL1/CSD2-dependent cell wall deposition of chitin found in S. cerevisiae, and is consistent with evidence from Schizophyllum commune that (1 6)--glucan accumulates during maturation of the subapical region of the wall distal to the hyphal tip.     

Cell wall assembly ofNeurospora crassa: Isolation and analysis of cell wall-less mutants

Cell-wall-less mutants ofNeurospora crassa were isolated by mutagenizing cells of the temperature-sensitive, protoplast-formingosmotic-1 mutant and screening for cultures that did not regenerate cell wall. Of the 24 strains isolated, 22 were found to have significantly reduced levels of (1–3)-glucan synthase activity; none was found to be defective in chitin synthase activity. Genetic analysis by standard transmission genetics revealed that the glucan synthase defect was ascospore lethal. Complementation analysis by forced heterokaryosis revealed four complementation groups. In each case in which complementation of cell wall formation was observed, there was concomitant restoration of (1–3)-glucan synthase activity. Our results indicate that glucan synthase activity is necessary, but not sufficient, for cell wall assembly and resulting morphogenesis.     

Natural vitamin E enrichment of Artemia salina fed freshwater and marine microalgae

Three species of microalga, the freshwater Euglena gracilis and the marine Dunaliella salina and Tetraselmis suecica, were compared in terms of vitamin E enrichment and survival and growth of the brine shrimp Artemia salina. The tocopherol content was investigated using HPLC for the post-larval and pre-adult stages of Artemia fed the microalgae and the results compared to the initial content of unfed newly hatched nauplii. There was a markedly higher content of tocopherols (about two-fold) in Artemia fed Euglena. Since this microalga contains other antioxidants such as -carotene, vitamin C and glutathione, bioactive molecules such as PUFA, and the immunostimulant polysaccharide -glucan, it represents a valuable alternative for enriching the diets of Artemia and increase its nutritional value as a food item.     

Antioxidants from carbon dioxide fixing Chlorella sorokiniana

Chlorella sorokiniana H-84, which has toleranceto high temperatures and high concentrations ofCO₂, has been isolated from a hot spring inJapan. Large-scale culturing of C. sorokinianawas carried out in air containing 10% CO₂.Analysis of the biomass shows that protein, carbohydrate and lipids comprised 68.5, 11.9 and10.0% of dry matter, respectively. The totalcarotenoids comprised 0.69% dry matter. The luteinand -carotene contents were 4300 and 600 gg^-1 dry weight, respectively. The-tocopherol content was 112 g g^-1 dry weight. These carotenoids and -tocopherolare known to possess radical scavenging activity.Two fractions with radical scavenging activity wereisolated from the aqueous extract of C.sorokiniana H-84. The extract showed several singlepeaks by reversed phase HPLC analysis and two of themhad molecular weights of 710 and 1286, respectively.     

Surface carbohydrates and cell wall structure of in vitro-induced uredospore infection structures ofUromyces viciae-fabae before and after treatment with enzymes and alkali

Summary Uredospores ofUromyces viciae-fabae differentiate to form germ tubes, appressoria, infection hyphae and haustorial mother cells on oil-containing collodion membranes. The cell walls of these infection structures were studied with the electron microscope and with FITC-labeled lectins before and after treatment with enzymes and inorganic solvents. Binding of the FITC-labeled lectins was measured with a microscope photometer. The enzymes pronase E, laminarinase, chitinase and lipase had different effects on each infection structure. Pronase treatment uncovered the chitin of germ tubes, appressoria and haustorial mother cells, but not of substomatal vesicles and infection hyphae. A mixture of - and -1,3-glucanase which also contained chitinase activity dissolved germ tubes and appressoria completely, but not infection pegs, substomatal vesicles, infection hyphae and haustorial mother cells. After treatment with laminarinase or lipase, an additional layer, which is especially obvious over the substomatal vesicle, infection hypha and haustorial mother cell, bound to LCA-FITC. In the wall of the haustorial mother cell, a ring, which surrounds the presumed infection peg, had strong affinity for WGA after protease and sodium hydroxide treatment. The infection structures have a fibrillar skeleton. The main constituent seems to be chitin. This skeleton is more dense or has a higher chitin content in the walls of appressoria and haustorial mother cells. The fibrils of the skeleton extend throughout the cell wall of the germ tube and appressorium. They are embedded within amorphous material of complex chemical composition (-1,3-glucan, -1,3-glucan, glycoprotein). The chitin of the infection peg, substomatal vesicle, infection hypha and haustorial mother cell is covered completely with this amorphous material. These results show, that each infection structure has distinct surface and wall characteristics. They may reflect the different tasks of the infection structures during host recognition and leaf penetration.Abbreviations AP appressorium - FITC fluorescein isothiocyanate - GT germ tube - HC haustorial mother cell - IH infection hypha - IP infection peg - LCA Lens culinaris agglutinin - n nucleus - neu neuramic acid - p pyranoside - R ring - s septum - SV substomatal vesicle - WGA wheat germ agglutinin     

Effect of <Emphasis Type="Italic">Agave tequilana</Emphasis> juice on cell wall polysaccharides of three <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains from different origins

In this study, a characterization of cell wall polysaccharide composition of three yeasts involved in the production of agave distilled beverages was performed. The three yeast strains were isolated from different media (tequila, mezcal and bakery) and were evaluated for the β(1,3)-glucanase lytic activity and the β-glucan/mannan ratio during the fermentation of Agave tequilana juice and in YPD media (control). Fermentations were performed in shake flasks with 30 g l⁻¹ sugar concentration of A. tequilana juice and with the control YPD using 30 g l⁻¹ of glucose. The three yeasts strains showed different levels of β-glucan and mannan when they were grown in A. tequilana juice in comparison to the YPD media. The maximum rate of cell wall lyses was 50% lower in fermentations with A. tequilana juice for yeasts isolated from tequila and mezcal than compared to the bakery yeast.     

Genetical investigations into β-glucan content in barley

Summary Random inbred lines produced by doubled haploidy (DH) and single seed descent (SSD) have been used to investigate the genetics of -glucan (gum) content in barley (Hordeum vulgare). Genetical analyses indicated that gum content is controlled by a simple additive genetic system. Significant negative genetic correlations were observed between -glucan content, thousand grain weight and height in the DH samples. These correlations were much reduced in the SSD samples and would suggest linkage of the genes controlling these characters. The presence of repulsion linkages could be exploited in a barley breeding programme by producing F1 derived DH to generate recombinants with high thousand grain weight and low -glucan content. Genetical parameters estimated from DH and F3 samples have successfully been used to predict the number of inbred lines transgressing the parental range for -glucan content and bivariate combinations involving -glucan.     

Mapping of β-glucan content and β-glucanase activity loci in barley grain and malt

Genetic study of -glucan content and -glucanase activity has been facilitated by recent developments in quantitative trait loci (QTL) analysis. QTL for barley and malt -glucan content and for green and finished malt -glucanase activity were mapped using a 123-point molecular marker linkage map from the cross of Steptoe/Morex. Three QTL for barley -glucan, 6 QTL for malt -glucan, 3 QTL for -glucanase in green malt and 5 QTL for -glucanase in finished malt were detected by interval mapping procedures. The QTL with the largest effects on barley -glucan, malt glucan, green malt -glucanase and finished malt glucanase were identified on chromosomes 2,1,4 and 7, respectively. A genome map-based approach allows for dissection of relationships among barley and malt glucan content, green and finished malt -glucanase activity, and other malting quality parameters.     

Cell wall composition of the yeast and mycelial forms of Yarrowia lipolytica

The cell walls of the yeast and mycelial forms of Yarrowia lipolytica were isolated and purified. Electron microscopy studies showed no differences between both types of cell walls. Chemical analysis revealed that the yeast cell wall contained 70% neutral carbohydrate, 7% amino sugars, 15% protein, 5% lipids and 0.8% phosphorus. Mycelial cell walls contained 70% carbohydrate, 14% aminosugars, 6% protein, 5% lipids and 0.6% phosphorus. Three polysaccharides: -glucan, mannan and chitin were detected. Proteins were solubilized from both cell wall fractions and separated by polyacrylamide gel electrophoresis. About 50 protein bands were detected, four of them corresponding to glycoproteins. The cell walls of the yeast and mycelial forms of Y. lipolytica were qualitatively similar and only quantitative differences were found.Abbreviations GlcNAc N-acetylglucosamine - FITC-WGA fluorescein isothiocyanate-wheat germ agglutinin - PAS periodic acid Schiff     

Characterization of theGeosiphon pyriforme symbiosome by affinity techniques: confocal laser scanning microscopy (CLSM) and electron microscopy

Summary Geosiphon pyriforme represents a photoautotrophic endosymbiosis of aGlomus-like fungus with the cyanobacteriumNostoc punctiforme. The fungus forms unicellular bladders of up to 2 mm in length and 0.5 mm in diameter growing on the soil surface and harboring the endosymbioticNostoc filaments. The cyanobacteria are located in a compartment (the symbiosome) bordered by a host membrane. The space between this symbiosome membrane (SM) and theNostoc cell wall is filled with an about 30–40 nm thick layer of amorphous material, which is present also in the regions of the symbiosome where noNostoc filaments are located. At these sites the amorphous material consists of a 20–30 nm thick layer separating the SM. The region between the SM and the cyanobacterium is defined as symbiosome space (SS). Fungal bladders, hyphae and free livingNostoc were analyzed by affinity techniques as well as the material occurring in the SS. FITC-coupled lectins with sugar specificity to -D-mannosyl/-D-glucosyl (Con A), N-acetyl--D-glucosamine oligomers (WGA), -L-fucosyl (UEA-I), -D-galactosyl (RCA-120), -D-galactosyl (BS-I-B₄), N-acetyl--D-galactosamine (HPA), and sialic acid (EBL) residues were tested. WGA binding and calcofluor white staining demonstrated that the bladder wall as well as the SS contain fibrillar chitin. Of the other lectins only Con A clearly labeled the symbiosome. On the contrary, the lectin binding properties of the slime produced by free livingNostoc-colonies indicate the presence of mannose, fucose, GalNAc, sialic acid, and galactose, while chitin or GlucNAc-oligomers could not be detected. The symbiosome was also investigated electron microscopically. WGA-gold binding confirmed the presence of chitin, while a slight PATAg reaction indicated some polysaccharidic molecules within the SS. Our results show that the amorphous material within the SS contains molecules typical of the fungal cell wall and suggest that the SM is related to the fungal plasma membrane. The applied lectins all bind to the hyphal surface, indicating a high molecular complexity. Mannosyl, -galactosyl, and sialic acid residues are strongly exposed at the outer cell wall layer, whereas GlucNAc, GalNAc, and -galactosyl residues seem to be present in smaller amounts. The symbiotic interface established between the fungus andNostoc inGeosiphon shows many similarities to that occurring between fungi and root cells in arbuscular mycorrhizas.Abbreviations AM arbuscular mycorrhiza - BS-I-B₄ Bandeiraea simplicifolia lectin I isolectin B₄ - CLSM confocal laser scanning microscopy - Con A Concanavalin A - EBL elderberry bark lectin I - FITC fluorescein isothiocyanate - HPA Helix pomatia agglutinin - PATAg periodic acid-thiocarbohydrazide-Ag proteinate - SM symbiosome membrane - SS symbiosome space - RCA-120 Ricinus communis agglutinin 120 - UEA-I Ulex europaeus agglutinin I - WGA wheat germ agglutinin Dedicated to Professor Dr. Peter Sitte at the occasion of his 65th birthday     

Stimulation of membrane-associated polysaccharide synthetases by a membrane potential in developing cotton fibers

Conditions which induce a transmembrane electrical potential, positive with respect to the inside of membrane vesicles, result in a substantial (4–12-fold) stimulation of the activity of membrane-associated -glucan synthetases in a membrane preparation derived from the developing cotton (Gossypium hirsutum L.) fiber. Induction of electrical potentials which are negative with respect to the inside of the membrane vesicle results in little or no stimulation of -glucan synthesis. Those products whose synthesis is stimulated are mainly -1,3-glucan, but there is also a considerable increase in -1,4-glucan. No -1,4-glucan (starch) was detected in the reaction products. A transmembrane pH gradient was found to have no effect on -glucan synthesis. The results indicate that a transmembrane electrical potential can influence, either directly or indirectly, the activity of membrane-associated polysaccharide synthetases.Abbreviations UDP-glucose uridine-5-diphosphoglucose - PEG polyethylene glycol - BTP bistrispropane (1,3-bis[tris(hydroxymethyl)methylamino]propane) - MES 2(N-morpholino)ethanesulfonic acid - VAL valinomycin