Chemical composition of the yeast ascospore wall. The second outer layer consists of chitosan

In a preceding paper (Briza, P., Winkler, G., Kalchhauser, H., and Breitenbach, M. (1986) J. Biol. Chem. 261, 4288-4294), we reported the presence of dityrosine in the outer layers of yeast ascospore walls. Both outer layers seen in electron micrographs of yeast ascospore walls are sporulation-specific. Here we show that the second of these two outer layers consists of chitosan. In intact spores, it is shielded from staining with primulin by the outermost layer. However, in purified spore walls, the second layer is brightly stained by primulin, and hydrolysates of such preparations contain about 10% glucosamine relative to spore wall dry weight. The spore wall material staining with primulin is resistant to chitinase, but readily degraded by treatment with HNO2. Acetylation prior to HNO2 treatment completely prevents its degradation. A partial acid hydrolysate of spore walls contains predominantly soluble poly-beta-(1,4)-glucosamine as determined by 13C NMR spectroscopy. By these criteria, the glucosamine polymer of yeast ascospore walls is chitosan. As spore walls treated with alkali lack the inner layers but contain chitosan and as chitosan is not exposed at the surface of the spore, we conclude that it is localized in the second outer layer of the spore wall.     

Production and isolation of chitosan from Mucor rouxii.

A method for the lab-scale production and isolation of chitosan (polyglucosamine) from hyphal walls of Mucor rouxii was developed. Hyphal wall yields were generally 16 to 22% on a dry cell weight basis, of which 35 to 40% was glucosamine. Chitosan was readily extracted from purified, mycelial walls with acetic, formic, and hydrochloric acids; the last named was the most efficient. The yield of chitosan isolated ranged from 4 to 8% of the dry weight of the cell wall material.     

Location of glycoproteins that contain glucosamine in plant tissues

When radioactive d-glucosamine is provided to Acer pseudoplatanus cells in liquid culture in order to label those glycoproteins that contain amino sugars, it is incorporated predominantly into a crude cell wall fraction. This observation was confirmed histologically by preparing autoradiographs of thin tissue sections from plasmolyzed cells. Highly purified cell wall material from unlabeled cells has also been shown to contain small amounts of glucosamine. Similarly, about one-half of the amino sugar recovered from cultured cells of Nicotiana tabacum is present in their cell walls. In corn roots, however, the labeled glycoproteins that are formed after glucosamine incorporation are predominantly cytoplasmic and not deposited outside the protoplast.     

An Ultrastructural Study on the Developmental Phases and Silicification of the Glumes of Phalaris canariensis L.

In the glume of Phalaris canariensis L. silicon deposition takesplace in the macrohairs, papillae, prickle hairs and silicacells of the abaxial epidermis before panicle emergence. Early in their development the macrohairs have large vacuolesand thin walls. At maturity the walls become thickened and aremajor sites of silica deposition. Dry ashing reveals a helicalpattern within the hair walls. Distinct papillae and prickle hairs were first observed oneweek before panicle emergence. Here silicification was initiallyconfined to outer tangential walls, but by two weeks after emergencetheir cytoplasmic contents had broken down, and the lumina werefilled with siliceous granules. Cork-silica twin cells werealso present in the abaxial epidermis. By panicle emergencethe silica cells were infilled, but the cork cells retainedtheir cytoplasmic contents. The long cells of the abaxial epidermiswere initially thin walled, but thickening occurred in the outertangential wall, this being complete by one week after emergence.These cells remained relatively unsilicified throughout. After panicle emergence the adaxial epidermal cells, and theirassociated parenchyma cell layers began to lose their cellularcontents and collapse. This process was complete two weeks afteremergence when the collapsed walls formed a thin internal layerbetween the two epidermi. Electron opaque granular material,containing several elements, but predominantly calcium, waspresent between the collapsed cell walls. The results are comparedwith those for the lemma, and silica deposition mechanisms arealso discussed. Phalaris canariensis L., canary grass, silicification, trichome, glume, ultrastructure     

Studies on the Cell Wall of Chlorella V. Comparison of the Cell Wall Chemical Compositions in Strains of Chlorella ellipsoidea

Cell walls of four strains of Chlorella ellipsoidea (IAM C-27,C-87, C-102 and C-183) were compared as to their chemical compositions.Many differences were found: (1) The sugar composition of alkali-soluble cell walls differedin quantity as well as quality with glucuronic acid being foundonly in C-27 and C-87. (2) In alkali-insoluble cell walls glucosamine was found onlyin C-27. The other three strains contained mainly glucose. (3) The amino acid compositions of the alkali-insoluble cellwalls markedly differed among the four strains. The cell wallof C-102 contained more amino acids than carbohydrates, butC-27 and C-87 contained extremely little amino acid. In addition to the variation in cell wall composition, the opticalanisotropy findings also differed for these cell walls of Chlorellastrains which had been grouped as the same species. (Received August 16, 1983; Accepted December 27, 1983)     

Studies on the Cell Wall of Chlorella III. Incorporation of Photosynthetically Fixed Carbon into Cell Walls of Synchronously Growing Cells of Chlorella ellipsoidea

The carbon metabolism in cell walls of Chlorella ellipsoideawas studied by following ¹⁴C incorporation into cell wall constituentsin photosynthesizing, synchronously growing cells. The rateof incorporation was higher at an early growth phase of thecell cycle. The ¹⁴C was incorporated into both the major cellwall constituents, hemicellulose and ‘rigid wall’,and the radioactivity in the latter was distributed into itstwo components, glucosamine and amino acids. In pulse-chaseexperiments, the ¹⁴C fixed photosynthetically in the precedingcell cycle was rapidly transferred into the cell wall constituentsat the early growth phase of the ongoing cell cycle, and thereafterwas gradually released from the cell walls, although the totalamount of ¹⁴C in the cells remained constant. It was concludedthat the cell wall constituents are turned over during the growthphase of the algal cell cycle, and that the cell wall metabolismin the ongoing cell cycle is closely connected with the carbonmetabolism in the preceding cell cycle. (Received February 3, 1982; Accepted June 21, 1982)     

Aluminate-Induced Changes in Morphology and Ultrastructure of Thinopyrum Roots

The effects of aluminate [Al(OH)_4$$$] on the morphology andultrastructure of root cells were studied in the salt-tolerantgrasses Thinopyrum bessarabicum (Savul. and Rayss) A. Lve (2) and Thinopyrum junceum (L.) A. Lve (6) by light and transmissionelectron microscopy. Seedlings were grown in nutrient solutioncontaining 1 mol m^–3 [Al] and 5 mol m^–3 Na₂CO₃ atpH 100. Light microscopy revealed that root tips of [Al]-treated plantsdisplayed bending. Many cells of the cortex in the elongatingregion contained a fibrillar/granular material which renderedthem densely staining. Radial (anticlinal) walls of the epidermalcells were either cleft apart of unusually thickened. Amyloplastsof the central root cap cells contained fewer starch grains,while their distribution was disturbed. Electron microscopy showed that the most serious effects of[Al] toxicity occurred at the cell walls of the epidermal androot cap cells, as they lost their fibrillar fine structureand contained an amorphous electron-dense material distributedall over the wall section. Electron-opaque droplets were encounteredat the plasma membrane region of epidermal cells, while theelectron-dense material observed in the vacuoles of cortex cellscould be aluminate which had accumulated there. Thus, despitethe presence of a barrier to aluminate uptake, some [Al] doesenter the symplast. However, the cytoplasm of many epidermalcells displayed a normal fine structure and contained the usualsubcellular components. Dictyosomes, in particular, were abundantand surrounded by many vesicles denoting an active state. Theseobservations stress the role of cell walls as the major [Al]pool and of the plasma membrane as the ultimate barrier thatprotects the cytoplasm. Results are further discussed in relation to the findings inother plant species and it is concluded that, although aluminateis less toxic than Al³⁺, it causes morphological, structuraland, presumably, functional damage-to the roots of the speciesinvestigated. Key words: Thinopyrum, aluminate toxicity, cell walls, root bending     

Amino Acid-Sugar Linkages in Rice Coleoptile Cell Walls

The nature of amino acid-sugar linkages in cell walls was investigatedin a monocotyledonous tissue, rice coleoptiles. The molar ratiosof aspartic acid, threonine, and serine in cell walls were decreasedby hydrazinolysis in coleoptiles grown both on and under water.The molar ratios of threonine and serine were decreased alsoby a NaOHNaBH₄ treatment, while the alanine content was increased,and -aminobutyric acid was not formed. The cell walls were treated with NaOH in the presence of NaB³H₄,hydrolyzed, then divided into amino acid and sugar fractions.Two distinct radioactive peaks were detected in the thin-layerchromatography of the amino acid fractions. One was identifiedas alanine derived from glycosylated serine; the other was confirmedto be an oxidation product of glucosaminitol. There was justone ³H-labeled product in the sugar fractions, galactitol. Theseresults suggest the presence of serine-O-galactose and asparagine-N-N-acetylglucosamine linkages in rice coleoptile cell walls. The existence of glucosamine linked to amino acids was furthersupported by the incorporation of ¹⁴C-glucosamine into cellwalls. These linkages were also detected in the cell walls ofa dicotyledonous tissue, Vicia epicotyls. (Received April 2, 1981; Accepted June 24, 1981)     

Chitosan as a Component of Pea-Fusarium solani Interactions

Chitosan, a polymer of β-1,4-linked glucosamine residues with a strong affinity for DNA, was implicated in the pea pod-Fusarium solani interaction as an elicitor of phytoalexin production, an inhibitor of fungal growth and a chemical which can protect pea tissue from infection by F. solani f. sp. pisi. Purified Fusarium fungal cell walls can elicit phytoalexin production in pea pod tissue. Enzymes from acetone powders of pea tissue release eliciting components from the F. solani f. sp. phaseoli cell walls. Hydrochloric acid-hydrolyzed F. solani cell walls are about 20% glucosamine. The actual chitosan content of F. solani cell walls is about 1%. However, chitosan assays and histochemical observations indicate that chitosan content of F. solani spores and adjacent pea cells increases following inoculation. Dormant F. solani spores also accumulate chitosan. Concentrations of nitrous acid-cleaved chitosan as low as 0.9 microgram per milliliter and 3 micrograms per milliliter elicit phytoalexin induction and inhibit germination of F. solani macroconidia, respectively. When chitosan is applied to pea pod tissue with or prior to F. solani f. sp. pisi, the tissue is protected from infection.     

On the Occurrence of Intracellular Blue-green Algae in Cortical Cells of the Apogeotropic Roots of Macrozamia communis L. Johnson

The occurrence of species of the cyanophytes Nostoc and Anabaenain the cortex near the algal zone is reported for apogeotropicroots of Macrozamia communis L. Johnson. Algae were found tooccur both intercellularly and intracellularly in cells of theinner and outer cortex. This is the first record of intracellularalgae in the cycads and only the second report of this phenomenonin vascular plants. By examination of cells at various stagesof invasion by algae, it is interpreted that algal invasionof cortical cells and intercellular spaces is preceded by mucusapparently secreted by algal zone cells of the host, and depositedin intercellular spaces of cortical parenchyma cells nearby.Also algal penetration of cortical cells is preceded by an algalinvasion front of finely granular mucal material which bypassesmucus already deposited in intercellular spaces and may eitherlyse part of the host cell wall or enter through the plasmo-desmata,filling much of the cell cavity. Subsequently, large numbersof the algal symbionts enter the cell and may be enclosed withinhost wall material. Electron microscopic techniques are nowbeing employed to further clarify these invasion processes.     

The Role of Cellulase in Hyalocyst Pore Formation in the Moss Syrrhopodon texanus

The sheathing leaf bases of Syrrhopodon texanus are primarilycomposed of porose, thin-walled cells called hyalocysts. Largepores develop in several of the hyalocyst walls through thegradual removal of wall material. A cellulase with a pH optimumof 4.5 was detected in extracts of Syrrhopodon gametophoresby viscometric assays and dinitrosalicylic acid assay for reducingsugars. Cytochemical localization of cellulase activity in associationwith thinning hyalocyst cell walls implicate this enzyme inpore formation and is the first direct evidence of cellulaseactivity in a member of the Bryophyta. Syrrhopodon texanus, cellulase, cytochemistry, Bryophyta, hyalocyst     

Occurrence of Glucosamine Residues with Free Amino Groups in Cell Wall Peptidoglycan from Bacilli as a Factor Responsible for Resistance to Lysozyme

Analysis by dinitrophenylation techniques revealed the occurrence of significant amounts of glucosamine residues with free amino groups in the peptidoglycan component of cell walls isolated from Bacillus cereus, Bacillus subtilis, and Bacillus megaterium. A close correlation was demonstrated between the content of N-unacetylated glucosamine residues in the peptidoglycan component and the resistance of the cell walls to lysozyme. These lysozyme-resistant cell walls and peptidoglycan were converted into a lysozyme-sensitive form by means of N-acetylation with acetic anhydride. Thus, the occurrence of the N-unacetylated glucosamine residues in the peptidoglycan component accounts for the resistance of these cell walls to lysozyme. The N-unacetylated glucosamine residues were not found in a significant amount in the cell walls of Micrococcus lysodeikticus, Staphylococcus aureus, Streptococcus faecalis, Lactobacillus casei, or Lactobacillus arabinosus.     

Lysis of cell walls ofMucor ramannianus möller by aStreptomyces sp.

A study has been made of some chemical and ultrastructural changes that occur in the hyphal, arthrospore and sporangiospore walls ofMucor ramannianus during lysis by a soil streptomycete.Arthrospore and hyphal walls, which were shown to contain chitin, chitosan, other polysaccharides and phosphate (principally as polyphosphate), were lysed by culture fluid of the streptomycete after this organism had been grown on the same material. Alcohol-insoluble material found in the supernatants of the incubation mixtures gave on hydrolysis glucosamine, galactose, mannose and fucose. No laminarinase activity was detected in these culture fluids. Culture fluids of the streptomycete after growth on chitin and chitosan were also found to lyse the walls of arthrospores and hyphae.Despite the chemical similarities the walls were very different in thin section.A major component in the sporangiospore walls was glucan and an active laminarinase was shown to be present in the culture fluids of the streptomycete after growth on them. Further, ultrathin sections showed that an inner fibrillar layer of the sporangiospore wall was lysed leaving an outer electron-dense layer.     

The Floral Nectaries of Hibiscus rosa-sinensis: 1. Development of the Secretory Hairs

Floral nectaries of Hibiscus rosa-sinensis occur on the lowerinner side of the fused sepals and each one consists of numerous(50000–55000) secretory hairs, occupying a cylinder-likezone completely lining the inner side of the sepals. Each hairoriginates from a single protodermal mother cell and, at maturity,it is built up of a basal cell, a stalk, 35–40 intermediatecells and a tip secretory cell. Development of protodermal cellsinto secretory hairs is asynchronous, the first cells to initiatedevelopment being those situated in the lowermost part of thecylindrical zone, and development progressing upwards. Volume increase of protodermal mother cells initiating developmentis accompanied by cell polarization manifested by organelledisplacement towards the apical region. Secretory hairs areformed through a sequence of transverse and, later on, anticlinaldivisions. Divisions of apical cells are preceded by well definedpre-prophase microtubule bands, which foreshadow the plane ofthe forthcoming division and predict with accuracy the sitesof parental walls where the new cell plate fuses at cytokinesis. Stalks consist of either one or two cells. Two-celled stalksoccur in 40 per cent of secretory hairs and derive from a transversedivision of one stalk cell; the wall formed is always depositedparallel to the proximal and distal walls, but never to thelateral ones. The significance of this mode of division is discussedin relation to the fact that lateral walls are entirely impregnatedwith a cutin-like material that blocks apoplastic movement ofsolutes. Hibiscus rosa-sinensis, nectaries, development, preprophase microtuble bands, stalk cells     

Immunocytochemical Localization of Phenylalanine Ammonia-Lyase and Cinnamyl Alcohol Dehydrogenase in Differentiating Tracheary Elements Derived from Zinnia Mesophyll Cells

Secondary wall thickening is the most characteristic morphologicalfeature of the differentiation of tracheary elements. Isolatedmesophyll cells of Zinnia elegans L. cv. Canary Bird in differentiationmedium are converted to tracheary elements, which develop lignifiedsecondary wall thickenings. Using this system, we investigatedthe distribution of two enzymes, phenylalanine ammonia-Iyase(PAL) (EC 4.3.1.5 [EC] ) and cinnamyl alcohol dehydrogenase (CAD)(EC 1.1.1.195 [EC] ), by both biochemical and immunological methods.Both PAL and CAD appear to be key enzymes in the biosynthesisof lignin precursors, and they have been shown to be associatedwith the differentiation of tracheary elements. Cultured cellswere collected after various times in culture. The culture mediumwas separated from cells by centrifugation and designated fraction(1), the extracellular fraction. The collected cells were homogenizedand separated into four fractions: (2) cytosol; (3) microsomes;(4) cell walls (loosely bound material); and (5) cell walls(tightly bound material). PAL activity was detected in eachfraction. The extracellular fraction consistently had the greatestPAL activity. Moreover, PAL activity in the cytosolic fractionincreased rapidly prior to lignification, as it did in boththe microsomal and the cell wall (tightly bound) fractions duringlignification. Antisera against PAL and against CAD detectedthe proteins with molecular masses that corresponded to thoseof PAL and CAD in Zinnia. Immuno-electron microscopy revealedthat, in differentiating tracheary elements, PAL was dispersedin the cytoplasmic matrix and was located on Golgi-derived vesiclesand on the secondary wall thickenings. "Cell-free" immuno-lightmicroscopy supported the putative distribution of PAL on lignifyingsecondary walls. The pattern of distribution of CAD was similarto that of PAL. Thus, both PAL and CAD seemed to be localizedin secondary wall thickenings. From the results of both biochemicalassays and immunocytochemical staining, it appeared that atleast two types of PAL and CAD are present in differentiatingcells. One type of each enzyme is distributed in the cytosol,while the other is secreted from the Golgi apparatus and transportedby Golgi-derived vesicles to the secondary wall thickenings. (Received April 19, 1996; Accepted November 18, 1996)     

Cytochemical Localization of Calcium in Cap Cells of Primary Roots of Zea mays L.

The distribution of calcium (Ca) in caps of vertically- andhorizontally-oriented roots of Zea mays was monitored to determineits possible role in root graviresponsiveness. A modificationof the antimonate precipitation procedure was used to localizeCa in situ. In vertically-oriented roots, the presumed graviperceptive(i.e., columella) cells were characterized by minimal and symmetricstaining of the plasmalemma and mitochondria. No precipitatewas present in plasmodesmata or cell walls. Within 5 min afterhorizontal reorientation, staining was associated with the portionof the cell wall adjacent to the distal end of the cell. Thisasymmetric staining persisted throughout the onset of gravicurvature.No staining of lateral cell walls of columella cells was observedat any stage of gravicurvature, suggesting that a lateral flowof Ca through the columella tissue of horizontally-orientedroots does not occur. The outermost peripheral cells of rootsoriented horizontally and vertically secrete Ca through plasmodesmata-likestructures in their cell walls. These results are discussedrelative to proposed roles of root-cap Ca in root gravicurvature. Key words: Antimonate, calcium, columella cell, peripheral cell, root gravitropism, Zea mays L.     

Isolation,characterization and heterologous expression of a novel chitosanase from <Emphasis Type="Italic">Janthinobacterium</Emphasis> sp. strain 4239

Background  

Chitosanases (EC 3.2.1.132) hydrolyze the polysaccharide chitosan, which is composed of partially acetylated β-(1,4)-linked glucosamine residues. In nature, chitosanases are produced by a number of Gram-positive and Gram-negative bacteria, as well as by fungi, probably with the primary role of degrading chitosan from fungal and yeast cell walls for carbon metabolism. Chitosanases may also be utilized in eukaryotic cell manipulation for intracellular delivery of molecules formulated with chitosan as well as for transformation of filamentous fungi by temporal modification of the cell wall structures.     

Oligogalacturonic Acid and Chitosan Reduce Stomatal Aperture by Inducing the Evolution of Reactive Oxygen Species from Guard Cells of Tomato and Commelina communis

Stomatal opening provides access to inner leaf tissues for many plant pathogens, so narrowing stomatal apertures may be advantageous for plant defense. We investigated how guard cells respond to elicitors that can be generated from cell walls of plants or pathogens during pathogen infection. The effect of oligogalacturonic acid (OGA), a degradation product of the plant cell wall, and chitosan (beta-1,4-linked glucosamine), a component of the fungal cell wall, on stomatal movements were examined in leaf epidermis of tomato (Lycopersicon esculentum L.) and Commelina communis L. These elicitors reduced the size of the stomatal aperture. OGA not only inhibited light-induced stomatal opening, but also accelerated stomatal closing in both species; chitosan inhibited light-induced stomatal opening in tomato epidermis. The effects of OGA and chitosan were suppressed when EGTA, catalase, or ascorbic acid was present in the medium, suggesting that Ca(2+) and H(2)O(2) mediate the elicitor-induced decrease of stomatal apertures. We show that the H(2)O(2) that is involved in this process is produced by guard cells in response to elicitors. Our results suggest that guard cells infected by pathogens may close their stomata via a pathway involving H(2)O(2) production, thus interfering with the continuous invasion of pathogens through the stomatal pores.     

Pectolytic Activity by the Haustorium of the Parasitic PlantOrobancheL. (Orobanchaceae) in Host Roots

The possible involvement of enzymes in the penetration of intrusivecells of the parasitic angiospermOrobancheinto host root tissueswas studied using cytochemical and immunocytochemical methods.Pectin methyl esterase (PME) was detected, with specific antibodies,in the cytoplasm and cell walls ofOrobancheintrusive cells andin adjacent host apoplast. Depletion and chemical changes ofpectins in host cell walls were shown by histochemical stainingwith PATAg, which detects carbohydrates that are sensitive toperiodic acid, especially pectins, and with the monoclonal antibodiesJIM 5 and JIM 7 that label pectins with low and high rates ofesterification, respectively. Galacturonic sequences with lowrates of esterification were more abundant in host cell wallsadjacent to the parasite, which is consistent with pectin de-methylationby PME release from the parasite. Pectins were absent in middlelamellae and in host cell walls neighbouring mature intrusivecells of the parasite, consistent with further degradation ofpectins by other enzymes. These results provide the first directevidence for the presence and activity of a pectolytic enzymein the infection zone of the haustorium of a parasitic angiosperminsitu.Copyright 1998 Annals of Botany Company Broomrape;Orobanche; parasitic weed; haustorium; pectin methyl esterase; pectin; cell wall.     

Alkalinity-induced aggregation in Chlorella vulgaris I. Changes in cell volume and cell-wall structure

In Chlorella vulgaris cell aggregation, the clustering of singlecells into groups is induced by an alkaline pH (9.5). The processof alkalinity-induced aggregation may be divided into two stages:the first stage (0–24 hr after exposure to the alkalinepH) is characterized by enhanced precipitation of cells fromthe medium, as well as by a seven fold increase in cell volume.The second stage (24–120 hr) is associated with a furtherincrease in the extent of cell precipitation in the culture,which seems to result from the aggregation of clusters of enlargedcells. Electron micrographs reveal the existence, at this phase,of a number of autospores in the cells within a modified multi-layeredmother cell wall. The pectin content of cells at this stageis twice that of control cells grown at pH 6.3. In addition,the relative content of the different pectin fractions is modifiedas a result of the exposure to alkalinity. It is suggested thatthe aggregates result from the repeated failure of the cellsto detach from their original mother cell walls, thus formingclusters which represent several generations of cells. ¹Present address: Division of Food Storage, ARO Beit Dagan,P.O.B. 6, Israel. (Received September 3, 1979; )