Impact of salinity stress on soil-borne fungi of sugarbeet

The sugarbeet cultivar Kaumera was found to be highly susceptible to infection by the root-rot pathogens Rhizoctonia solani and Sclerotium rolfsii in the absence of salinity stress. Under this environmental condition, R. solani was more efficient than S. rolfsii in producing cell wall-degrading enzymes in infected hypocotyls. Xylanase and galactanase were most effective. The rate of cell wall degradation by R. solani was nearly 2.5 times that of S. rolfsii when cells walls of healthy hypocotyls were used as sole carbon substrate for the in vitro produced crude enzymes.Under salinity stress the pathogenicity and the performance of cell wall-degrading enzymes of R. solani and S. rolfsii varied profoundly. Pathogenicity studies showed that R. solani appeared to be more tolerant than S. rolfsii of the salinity stresses applied, and relatively more virulent to cv Kaumera. The activities of cell wall enzymes of R. solani decreased and those of S. rolfsii increased with increased salt concentration when cell wall material was used as a sole carbon source. The metabolic products produced under salinity stress by R. solani and R. solani in the cell wall amended culture media shifted the initial pH towards neutrality or slight alkalinity for R. solani and to high acidity for S. rolfsii.When model substrates were used, xyland and galactan were the most responsive substrates for degradation by the cell wall enzymes of the two fungi studied. The rate of degradation was higher for S. rolfsii than for R. solani. The excessive acidity in salt stressed S. rolfsii culture media suggested reduced activities of the enzymes involved in cell wall degradation in vivo. This may explain the decreased virulence potentialities.     

A major stress-inducible Mr-42000 wall glycoprotein of French bean (Phaseolus vulgaris L.)

A major wall protein of suspension-cultured cells of French bean has been isolated and characterised. It can be prepared from walls or the culture filtrate and in composition it is particularly rich in proline, valine and glutamic acid/glutamine and contains appreciable amounts of hydroxyproline. The N-terminus shows some glycosylation, while following chemical deglycosylation the first 38 residues were found to be identical to those of proline-rich proteins from soybean. However, the composition of the highly purified M_r-42000 bean protein differs considerably from the soybean proteins and must contain its own specific domains. An antibody was raised and used to demonstrate the inducibility of the M_r-42000 bean protein in response to elicitor action. The protein was found to be mainly localised in the intercellular spaces of the cortical cells of bean hypocotyls and at the wall-plasmalemma interface of xylem vessels, another potentially accessible compartment for pathogens. Following wounding, the protein was found to be generally distributed in the wall of epidermal and cortical cells of the hypocotyls. The M_r-42000 protein is cross reactive with antibodies raised to glycoproteins of the Rhizobium infection thread and the chitin-binding hydroxyproline-rich glycoprotein, potato lectin. These common epitopes together with the previously demonstrated chitin-binding properties of the bean protein indicate a role in host-microbial interactions. Furthermore, the M_r-42000 protein itself bound to the growing hyphal tips of the bean pathogen, Colletotrichum lindemuthianum.Abbreviations FITC fluorescein isothiocyanate - IgG immunoglobulin G - PAL phenylalanine ammonia-lyase We thank Dr Nick Brewin for advice on interpretation of immunolocalisations and for the gift of MCA 265. We thank Dudley Fernandino for carrying out the confocal microscopy. GPB thanks the Science and Engineering Research Council for funding.     

Auxin-induced H+-pump stimulation does not depend on the presence of epidermal cells in corn coleoptiles

Cell-wall acidification and electrical reactions (depolarization and hyperpolarization) are typical auxin responses in maize (Zea mays L.) coleoptiles. In an attempt to test the role of the outer epidermis in these responses, they have been measured and compared in intact and peeled coleoptile fragments. To exclude interactions between parenchymal and epidermal cells, the coleoptile pieces were completely stripped of their outer epidermis. This preparation was monitored by means of a scanning electron microscope. When externally applied indole-3-acetic acid was tested, we found that neither cell-wall acidification nor the electrical membrane responses depended on the presence of intact epidermal cells.Abbreviations IAA Indole-3-acetic acid - MES 2-[N morpholino-ethane-sulfonic acid - TRIS 2-Amino-2-hydroxymethyl-1,3-propanediol We thank Kuki Kaethner for her excellent technical assistance. This work was supported by the Hessische Graduiertenförderung and the Deutsche Forschungsgemeinschaft.     

Purification and properties of a lytic enzyme from the cell wall of Chlorella ellipsoidea C-87

Cell wall lytic activity was detected in the culture medium and cell wall of 1AM Chlorella ellipsoidea C-87. The enzymes of both fractions had their highest activity at pH 5. The lytic activity bound to the cell wall consisted of a polysaccharide releasing enzyme, an exo-type enzyme releasing disaccharide, and glucosidase; but only the polysaccharide releasing enzyme was solubilized by lithium chloride. A polysaccharide releasing enzyme with a molecular weight around 40 kDa was isolated from the culture medium. Hemicellulose is degraded by the polysaccharide releasing enzyme, and the rigid wall by the exo-type enzyme.     

Preinfection cell wall formation in roots and developing nodules ofAlnus rubra Bong

Summary The establishment of actinorhizal root nodules involves penetration of host cell walls and intracellular colonization by the nitrogen-fixing endosymbiont,Frankia (Actinomycetales). In the early stages of the infection process inAlnus, unusual cell walls with undulate profiles were observed in root tip meristematic derivatives, and in early (preinfection) derivatives of the nodule lobe meristem, inFrankia-inoculated plants. The irregular cell walls attached obliquely to preexisting walls, but were not discontinuous. Serial sections revealed that the unusual walls divided two daughter cells. Microtubules in bundled arrays were abundant near the undulate walls, and radiated in several planes. In the root tips, the anomalous cell walls were observed within one day of inoculation withFrankia.     

Relationship of actin organization to growth in the two forms of the dimorphic yeastCandida tropicalis

Summary Candida tropicalis is a dimorphic yeast capable of growing both as a budding yeast and as filamentous hyphae depending upon the source of the carbon used in the culture medium. The organization of F-actin during growth of the yeast form (Y-form) and the hyphal form (H-form) was visualized by rhodamine-conjugated phalloidin by using a conventional fluorescence microscope as well as a laser scanning confocal fluorescence microscope. In single cells without a bud or non-growing hyphae, actin dots were evenly distributed throughout the cytoplasm. Before the growth of the bud or hypha, the actin dots were concentrated at one site. During bud growth, actin dots were located solely in the bud. They filled the small bud and then filled the apical two-thirds of the cytoplasm of the middlesized bud. During growth of the large bud, actin dots which had filled the apical half of the cytoplasm gradually moved to the tip of the bud. In the formation of the septum, actin dots were arranged in two lines at the conjunction of the bud and the mother cell. During hyphal growth, the majority of actin dots were concentrated at the hyphal apex. A line of clustered spots or a band of actin was observed only at the site where the formation of a new septum was imminent. This spatial and temporal organization of actin in both categories of cells was demonstrated to be closely related to the growth and local deposition of new cell wall material by monitoring the mode of growth with Calcofluor staining. Treatment of both forms of cells with cytochalasin A (CA) confirmed the close relationship between actin and new cell wall deposition. CA treatment revealed lightly stained unlocalized actin which was associated with abnormal cell wall deposition as well as changes in morphology. These results suggest that actin is required for proper growth and proper deposition of cell wall material and also for maintaining the morphology of both forms of cells.Abbrevations FM fluorescence microscopy - EM electron microscopy - rh rhodamine - CA cytochalasin A - CD cytochalasin D - PBS phosphate-buffered saline - DMSO dimethylsulfoxide - GA glutaraldehyde     

Rapid calcium exchange for protons and potassium in cell walls of Chara

Net fluxes of Ca²⁺, H⁺ and K⁺ were measured from intact Chara australis cells and from isolated cell walls, using ion-selective microelectrodes. In both systems, a stimulation in Ca²⁺ efflux (up to 100 nmol m^?2 s^?1, from an influx of ～40 nmol m^?2 s^?1) was detected as the H⁺ or K⁺ concentration was progressively increased in the bathing solution (pH 7.0 to 4.6 or K⁺ 0.2 to 10mol m^?3, respectively). A Ca²⁺ influx of similar size occurred following the reverse changes. These fluxes decayed exponentially with a time constant of about 10 min. The threshold pH for Ca²⁺ efflux (pH 5.2) is similar to a reported pH threshold for acid-induced wall extensibility in a closely related characean species. Application of NH₄⁺ to intact cells caused prolonged H⁺ efflux and also transient Ca²⁺ efflux. We attribute all these net Ca²⁺ fluxes to exchange in the wall with H⁺ or K⁺. A theoretical treatment of the cell wall ion exchanges, using the ‘weak acid Donnan Manning’ (WADM) model, is given and it agrees well with the data. The role of Ca²⁺ in the cell wall and the effect of Ca²⁺ exchanges on the measured fluxes of other ions, including bathing medium acidification by H⁺ efflux, are discussed.     

Ultrastructural and biochemical aspects of cell wall reconstitution in recalcitrant (grapevine) and regenerating (tobacco) leaf protoplasts

Summary To identify possible reasons that may contribute to recalcitrance in plant protoplasts, the time course of new cell wall deposition was studied by scanning electron microscopy in protoplasts of a recalcitrant species, the grapevine. Results showed that microfibrils were developed after 2 days of culture, that complete cell wall formation occurred on Day 6 to 7 of protoplast culture, and its ultrastructural appearance was identical to that of grapevine leaf-derived callus cells. In addition, a comparative study was undertaken on [U-¹⁴C]glucose uptake and incorporation in ethanol-soluble, cellulosic, and noncellulosic polysaccharide fractions in protoplasts of grapevine and of a readily regenerating species, tobacco, during culture. There was a significantly higher [U-¹⁴C]glucose uptake by tobacco than by grapevine protoplasts. The label distribution in the ethanol-soluble, cellulosic, and noncellulosic fractions of newly synthesized cell walls differed quantitatively between the two species. In particular, the labeled glucose incorporated in the noncellulosic cell wall fraction was threefold greater in tobacco than in grapevine protoplasts. Differences were also revealed in the monosaccharide composition of this fraction between the two species. Addition of dimethyl sulfoxide to the culture medium resulted in a dramatic increase in [U-¹⁴C]glucose uptake by grapevine protoplasts, whereas it exhibited a limited effect in tobacco protoplasts. It showed no effect on the ultrastructural characteristics of new cell wall nor on the incorporation rate of labeled glucose in the cellulosic and noncellulosic cell wall fractions.     

Cell wall turnover in phosphate and potassium limited chemostat cultures of Bacillus subtilis W23

Turnover in phosphate and potassium limited chemostat cultures of Bacillus subtilis W23 results in the release of over 80% of the wall material present at the time of chasing equilibrium-labelled cultures. The rate at which turnover proceeds is faster in potassium limited cultures than in phosphate limited cultures but in both cases a fraction of the wall material appears to be conserved, or to undergo turnover at a lower rate. Previously we have shown that the polar wall is less active metabolically than the cylindrical wall and it is possible that the apparently conserved wall is that present in the pole.