Hyphal tip growth in Achlya bisexualis. II. Distribution of cellulose in elongating and non-elongating regions of the wall

Cellulose has been localized in the hyphal wall of elongating and non-elongating hyphae of Achlya bisexualis using a direct enzyme-colloidal-gold method. A number of controls, including several different types of fixation, support the idea that this labeling is specific for cellulose. Both TEM and SEM were used and they gave similar results. The apical area of an elongating hypha lacks cellulose, but the same area of a non-elongating hypha contains cellulose. We have used specific culture media and light microscopic measurements to ensure that we could distinguish between elongating and non-elongating hyphae. The lack of cellulose at the apex of elongating hyphae seems to require a reevaluation of the current concepts of hyphal tip growth in Achlya and related genera. A major question now is to determine whether or not the lack of a microfibrillar component is a universal pattern among all organisms having tip growth.     

Retention of Saccharomyces cerevisiae cell wall proteins through a phosphodiester-linked {beta}-1,3-/{beta}-l,6-glucan heteropolymer

Yeast cell wall proteins, including Cwp1p and      

A beta4 integrin-like protein co-localises with a phosphotyrosine containing protein in the oomycete Achlya bisexualis: inhibition of tyrosine phosphorylation slows tip growth

We present immunocytochemical data that indicate the presence of, and a close association between beta4 integrin-like proteins and proteins containing phosphorylated tyrosine residues in the oomycete Achlya bisexualis. When hyphae were plasmolysed, these proteins were present in wall-membrane attachment sites where there was also F-actin. A combination of immunoblots, ELISA, and a coupled enzyme assay suggest that phosphorylation may occur by both autophosphorylation and through the action of a tyrosine kinase. Tyrphostins, which are inhibitors of tyrosine kinases, abolished the anti-phosphotyrosine staining, inhibited the kinase activity, slowed tip growth and affected the organisation of the actin cytoskeleton, in a dose-dependent manner. By analogy with the integrins and associated kinases of the metazoa we suggest that these proteins could contribute to the process of tip growth by providing a means of bidirectional signaling between the cell wall and the cytoplasm.     

Isolation and characterization of an endo-(1,4)-{beta}-glucanase secreted by Achlya ambisexualis

Models of wall loosening in fungi and other walled eukaryotes require the action of proteins able to reduce the degree of linkage between components of the wall. In the oomycete Achlya ambisexualis, such a role has been proposed for a suite of endoglucanases that are secreted during branching and during the measurable wall softening associated with osmotic stress. We report here the isolation and characterization of one of these isoenzymes. The enzyme has a molecular weight of 32 kDa, a pH optimum of 6.75, a pI of 4.5, and a temperature optimum of 35 C. It is partially inhibited by sulfhydryl-binding reagents and completely inhibited by the tryptophan-binding reagent NBS. The enzyme has an endohydrolytic mode of action with substrate specificity towards glucans that contain β-(1,4) linkages, either alone (carboxymethyl cellulose) or as mixed linkage (1,4-1,3)-β-glucans (e.g., Avena glucan). It does not, however, degrade amorphous insoluble (phosphoric acid swollen) cellulose. Most significantly, the enzyme can also hydrolyze linkages in an Achlya cell wall fraction previously shown to consist of a mixed-linkage (1,4-1,3)-β-glucan. This property is consistent with the long-standing hypothesis that the branching-related endoglucanases of oomycetes play a role in cell wall loosening.     

Autolysis of the cell wall {beta}-D-glucan in corn coleoptiles

Corn coleoptile cell walls prepared and incubated in bufferautolyzed as much as 100 µg per mg dry weight over a 36hr period. This activity was attributed to the release of ß-D-glucanwhich constitutes as much as 110 µg per mg of the cellwall on a dry weight basis. Gel exclusion chromatography (Bio-gelP-2) of the autolytically solubilized products revealed thepresence of a polymeric component and a monosaccharide, andtime course studies showed that the polymeric component wasprogressively converted to monosaccharide. Glucose was the onlymonosaccharide detected. Treatment of the polymeric componentwith a bacterial glucanase specific for ß(13):ß(14)mixed-linkage glucans yielded distinctive tetra- and trisaccharideswhich is consistent with the hypothesis that it was derivedfrom wall ß-D-glucan. At least 90% of the autolysisproducts were derived from this wall component. The tolerance of autolytic activity to detergents and high saltconcentrations provided evidence that the enzymes responsibleare strongly associated with the wall. (Received October 3, 1978; )     

Modulation of Elicitor-Induced Chitinase and {beta}-1,3-Glucanase Activity by Hormones in Phaseolus vulgaris

Chitinase and ß-1 ,3-glucanase induction in Phaseolusvulgaris by cell wall elicitor from Col-letotrichum lindemuthianumhas been studied together with the effects of the hormones IAAand ethylene. Chitinase and ß-1, 3-glucanase increasedin response to the elicitor in the resistant cultivar, Kievit,but not in the susceptible cultivar, Pinto. However, both activitiesincreased in both cultivars in response to hormones in the absenceof elicitor; elicitor did not augment this response in cv. Kievit.Aminoethoxyvinyl glycine (AVG) abolished all responses exceptthose obtained by the application of ethylene. Of other hydrolasestested, only ß -galactosidase was induced by elicitor;this was similar for both cultivars but hormones were withouteffect. Evidence suggests that both chitinase and ß-l,3-glucanase are located within the cell rather than in theintercellular space. It is concluded that chitinase and ß;-l,3-glucanaseare coordinately synthesized as a defence response since theyhydrolyse complementary linkages in pathogen derived polysac-charides.Regulation of the induction of the two enzymes is primarilydue to ethylene and the lack of response in the compatible reactionappears to arise from an inability to synthesize ‘ stress’ ethylene. ¹Present address: School of Chemistry, Molecular and Biological Sciences, University of Sussex, Brighton BN1 9QJ, U.K. (Received March 15, 1991; Accepted June 13, 1991)     

Physiological and Biochemical Changes Associated with Cotton Fibre Development. IV. Glycosidases and {beta}-1,3-Glucanase Activities

Developing cotton fibre was analysed from 12 days post anthesis(DPA) till maturity for the activity of wall degrading enzymes,ß-galactosidase, ß-glucosidase, -mannosidaseand ß-1,3-glucanase. Each enzyme was estimated inthree different fractions namely cytoplasmic, ionically wall-boundand covalently wall-bound. There was a significant correlationbetween ß-galactosidase and ß-glucosidaseactivities in the covalently bound fraction, and the rate offibre elongation. Similarly, covalently bound ß-1,3-glucanaseactivity showed an increasing trend up to 18 DPA, i.e. aboutthe time when maximum rate of fibre elongation was achieved. The results presented here suggest that covalently wall-boundglycosidases may have an importafit role in cell wall loosening.Earlier reports providing evidence against the involvement ofthese enzymes in elongation growth in intact system, may perhapsbe due to scant attention paid to the subcellular distributionof these enzymes. Gossypium hirsutum, cotton fibre, glucanase, glycosidase, wall-loosening     

{alpha}-Galactosyl (Gal{alpha}1-3Gal{beta}1-4GlcNAc-R) epitopes on human cells: synthesis of the epitope on human red cells by recombinant primate {alpha}1,3galactosyltransferase expressed in E.coli

Developing methods for in vitro synthesis of the carbohydratestructure Gal     

The Occurrence of 1,3-{beta}-Glucanase in Healthy and Verticillium-infected, Resistant and Susceptible Tomato Plants

Leaf, stem, and root extracts of near-isogenic tomato plantscv. Craigella, resistant and susceptible to Verticillium albo-atrum,showed constitutive 1,3-ß-glucanase activity whichincreased following inoculation with the pathogen. Partiallypurified enzyme extracts were obtained by dialysing a 30–80%ammonium sulphate fraction of the tissue brei. The enzyme hadpH and temperature optima of 5?5 and 44 ?C respectively, withhigh activity between 50 and 60 ?C. The response to laminarinconcentration was linear between 1?2 and 7?5 mg ml^–1.Root inoculation of susceptible plants with 10⁶ propagules ml^–1V. albo-atrum led to a umform 300 per cent increase in all steminternodes except the terminal one, which was 500 per cent ofthe controls. No spatial relationship of enzyme activity tothe localization of fungus within the stem was apparent. Petioles,leaves, and roots of susceptible infected plants similarly showedan increase in activity but less than that in stems. Changedlevels of stern enzyme activity at different times after inoculationwere associated with reductions in the number of vessels containinghyphae. Extracts of plants of the resistant isoline showed increasedglucanase activity over controls, but this was substantiallylower than that in susceptible plants and was associated withthe greatly reduced mycelial colonization in resistant plants. It is concluded that single gene resistance in tomato to Verticilliumis not associated with innately higher levels of 1,3-ß-glucanasein healthy plants. The increased activity in infected plantsis proportional to the overall quantity of pathogen in the plantor of pathogenic metabolites.     

Hyphal tip growth in Phytophthora. Gradient distribution and ultrahistochemistry of enzymes.

Germinating cysts and isolated walls from germinating cysts incorporated 14C-UDPG into wall material of which 22.5 and 15% respectively were insoluble in boiling 1 N HCl, indicating that part of the synthetase activity is located in the wall itself. A combination of Urografin and Ficoll density gradients was used to separate various intracellular fractions. A consistent separation of beta-glucanase and UDPG-transferase enriched fractions was achieved. The beta-glucanase fraction contained dictyosome vesicles and fragments along with some plasma membranes. The UDPG-transferase fraction was relatively rich in membranes resembling rough and smooth ER. The results suggest the two enzymes are transported to the wall by different intracellular routes, and two types of vesicle may be involved. Alkaline phosphatase, beta-glucosidase and acid phosphatase were found extracellularly and their distribution in density gradients determined. The results of histochemical staining for acid phosphatase, alkaline phosphatase and polysaccharide are described and compared with the biochemical data. beta-1,3-glucanase, found intra- and extracellularly, induced distorted growth of germ tubes and also removed most of the apical wall when added to the incubation medium. None of these responses were observed with cellulase. Determinations of the osmotic pressure of germinating cysts and incubation medium revealed that the turgor of germinating cysts amounts to about 1.8 at under the conditions used.     

Estimation of ^{3}J_{HN\hbox{-}H\alpha} and ^{3}J_{H\alpha\hbox{-}H\beta} coupling constants from heteronuclear TOCSY spectra

(3)J proton-proton coupling constants bear information on the intervening dihedral angles. Methods have been developed to derive this information from NMR spectra of proteins. Using series expansion of the time dependent density matrix, and exploiting the simple topology of amino acid spin-systems, formulae for estimation of (3)J(HN-Halpha) and (3)J(Halpha-Hbeta) from HSQC-TOCSY spectra are derived. The results obtained on a protein entailing both alpha-helix and beta-sheet secondary structure elements agree very well with J-coupling constants computed from the X-ray structure. The method compares well with existing methods and requires only 2D spectra which would be typically otherwise recorded for structural studies.     

Hyphal wall growth in Neurospora crassa and Geotrichum candidum.

Growth of the walls of hyphae of Neurospora crassa and Geotrichum candidum was studied using longitudinal and serial transverse sectioning methods. Rigidification of the hyphal wall below the extension zone did not appear to involve the gross formation of a secondary wall since the transition from extensible to non-extensible wall was not associated with an increase in thickness. However, behind the extension zone the walls leading hyphae of N. crassa increased in thickness until eventually they attained a thickness which was up to five times that of the tip wall. A hypothesis of hyphal wall growth is proposed.     

Tapetum-Specific Expression of the Gene for an Endo-{beta}-1,3-glucanase Causes Male Sterility in Transgenic Tobacco

A cDNA for a pathogenesis-related endo-ß-1,3-glucanaseisolated from soybean, was fused to an anther tapetum-specificpromoter (Osg6B promoter) isolated from rice and the resultingchimeric gene was introduced into tobacco. The Osg6B promoterbecame active in the anther tapetum during formation of tetradsand the tapetal glucanase activity in the transgenic plantscaused in a significant reduction in the number of fertile pollengrains. Most of the pollen grains were aberrant in shape, lackedgerminal apertures and aggregate of the pollen grains. Granulesof ß-1,3-glucan, which have not previously been reported,were often observed to adhere to the surface of the pollen grains.Further observations revealed that the callose wall was almostabsent in the pollen tetrads of transgenic plants. In wild-typeplants, by contrast, the tetrads were surrounded by callosethat was degraded soon after the tetrad stage to release freemicrospores. Thus, the introduced gene for endo-ß-1,3-endoglucanaseunder the control of the Osg6B promoter caused digestion ofthe callose wall at the beginning of the tetrad stage, a timethat was just a little earlier than the time at which endogenousglucanase activity normal appears. These results demonstratethat premature dissolution of the callose wall in pollen tetradscauses male sterility and suggest that the time at which tapetallyproduced glucanase is activate is critical for the normal developmentof microspores. (Received September 29, 1994; Accepted January 30, 1995)     

The Distribution of {beta}-Glycerophosphatase in Young Roots of Pisum sativum L.

The distribution of ß-glycerophosphatase activityin young roots of Pisum sativum, cultivar Alaska, has been examinedby biochemical and histochemical methods. Results obtained bythe two approaches are broadly similar, and indicate that highenzyme activity is associated with cells of the root cap, outerlayers of the cortex, differentiating xylem elements and phloemfibres, and cortical cells surrounding emerging lateral roots.The significance of this distribution in relation to a possiblefunction of ß-glycerophosphatase is discussed.     

Effect of light treatment and endogenous growth hormones on {alpha}-and {beta}-amylase activities in cotyledons of Phaseolus vulgaris L.

The influence of light and endogenous plant-growth regulatorson the evolution of - and ß-amylases in cotyledonsof Phaseolus vulgaris L. was investigated. Both enzymes, whichare not present in ungerminated seeds, appear during germinationof intact seedlings or incubation of excised cotyledons. -Amylaseactivity decreases upon exposure to light. This inhibition iscorrelated with a drastic increase in chlorophyll content anda change in the endogenous gibberellin-inhibitor balance. ß-Amylaseactivity was not affected by light treatment but was by thepresence of endogenous cytokinins. (Received February 3, 1977; )