CHITIN AND CELLULOSE IN THE CELL WALLS OF RHIZIDIOMYCES SP

Fuller , Melvin S. (Brown U. Providence, R. I.), and Isaac Barshad . Chitin and cellulose in the cell walls of Rhizidiomyces sp. Amer. Jour. Bot. 47(2): 105-109. Illus. 1960.–Chemically isolated cell wall preparations of the aquatic Phycomycete, Rhizidiomyces sp., were analyzed by means of X-rays. The resulting diffraction patterns had maxima corresponding with known values for chitin and mercerized cellulose. The findings in this study are discussed with respect to Von Wettstein's hypothesis that the aquatic Phycomycetes can be separated into groups on the basis of whether their cell walls contain chitin or cellulose.     

Metal loading and enzymatic degradation of fungal cell walls and chitin

The capacity of chitin (from crab shells) and of fungal cell walls from Trichoderma harzianum to accumulate zinc, cadmium and mercury was studied as well as the effects of adsorbed metals on the enzymatic hydrolysis by Novozym 234 of the two substrates. The total adsorbing capacity with respect to these metals was estimated to be at least 10 mmol kg^–1 chitin (dry weight) and 50 mmol kg^–1 fungal cell walls (dry weight), respectively, at pH 6.1. Enzymatic digestion of fungal cell walls preloaded with mercury and cadmium was significantly reduced, while zinc did not cause any significant inhibition. The effect of metal complexation by chitin on the enzymatic digestion was not as pronounced as for fungal cell walls. This could reflect the fact that chitin sorbed a lower total amount of metals. The inhibitory effect of metals on the enzymatic hydrolysis was caused by the association of the metals with the two substrates and not by the presence of free metals in solution.     

On the cell wall composition of the apiculate yeasts

Summary Sonic oscillation was used for the purpose of obtaining clean, chemically intact cell walls. The rate of disruption was determined for cells ofHanseniaspora uvarum andSaccharomyces cerevisiae. The carbohydrate fractions of cell walls ofHanseniaspora uvarum, H. valbyensis, Kloeckera apiculata, Saccharomycodes ludwigii andSaccharmyces cerevisiae were shown to be similar. Chromatography of cell wall hydrolysates of all these species demonstrated that glucose and mannose were the only sugars present (in about equal amounts) besides traces of glucosamine. The cell walls ofH. uvarum contained 78.1 per cent carbohydrates, 7 per cent protein and approximately 0.05 per cent of chitin. Fractionation of the polysaccharides lead to a recovery of 83.3 per cent of the carbohydrates present (30.4 per cent glucan and 34.9 per cent mannan). Saccharomyces cerevisiae cell walls were found to have a carbohydrate content of 82.8 per cent, 6.5 per cent protein and a trace of chitin (0.04 per cent). Nadsonia elongata contained a relatively large amount of chitin (ca. 5 per cent) and lacked mannan in its cell walls. It was concluded thatHanseniaspora andSaccharomycodes are closely related to theSaccharomyceteae but they have little in common with species ofNadsonia.     

THE ULTRASTRUCTURE OF PHYCOPELTIS (CHROOLEPIDACEAE: CHLOROPHYTA). I. SPOROPOLLENIN IN THE CELL WALLS

Electron microscope observations on Phycopeltis epiphyton, a subaerial green alga found growing on the leaves of vascular plants and bryophytes, revealed the presence of a densely staining material within the inner and outer zones of the cell walls. This material resists acetolysis, is degraded by chromic acid, is unaffected by ethanolamine and exhibits secondary fluorescence when stained with the fluorochrome Primuline. These characteristics, together with infrared absorption spectra indicate that, on the basis of currently accepted criteria, the densely staining material is a sporopollenin and that it is a major component of the cell wall. Tests for cellulose, chitin, and lignin were negative, and little if any silica is present. It is suggested that negative results in tests for cellulose may be due to a masking effect by the sporopollenin. Comparison of the fine structure of the cell walls of P. epiphyton, pollen grains, and algal cells (known to contain sporopollenin) supports the suggestion that sporopollenin deposition on “unit membranes” is universal. Morphological similarity among sporopollenin lamellae in P. epiphyton, pollen grains, spores of land plants, and the trilaminar sporopollenin sheath in Chlorella, Scenedesmus, and Pediastrum indicates that the structures may be analogous. As in pollen grains, sporopollenin may provide protection against desiccation and parasitism. It may also be involved in the adhesion of Phycopeltis to host plants and in the adhesion between adjacent filaments of the thallus.     

Studies of the cell walls of Schizophyllum commune

Mechanically isolated cell wall materials of eight strains of Schizophyllum commune were studied by chemical and enzymatic procedures. Isolated wall material of each strain was separated by chemical methods into three fractions: A (cold alkali-soluble, , amorphous), B (warm alkali-soluble, amorphous), and C (alkali-insoluble, retaining appearance of hyphal fragments). Chemical tests indicated the presence of chitin in Fraction C and the absence of cellulose, lignin and pectic substances from all fractions. Analyses of acid hydrolysates indicated the presence of glucose in Fractions A, B and C, of hexosamine in Fraction C and the absence of galactose, mannose, 6-deoxyhexoses, xylose and other pentoses from all fractions. Unfractionated material, Fraction A and Fraction B were slightly attacked by commercial cellulase whereas Fraction C was heavily attacked. Commercial chitinase by itself did not attack Fraction C or unfractionated material to a significant extent. In the presence of cellulase, it was active upon Fraction C. Qualitative differences in cell wall composition between strains were not detected; however, quantitative differences were observed in the proportion of Fraction A and Fraction C as well as in the amount of the various breakdown products in certain strains. It is visualized that the cell wall of this fungus consists of a core of chitin covered by or intermeshed with glucose-containing polymers.     

Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active

Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA⁻ cell lines are shown.     

Chitin and cellulose in the cell walls of the oomycete,Apodachlya sp.

Summary Hyphal walls of Apodachlya sp. (Leptomitales) gave a positive reaction when tested cytochemically for chitin. The color reaction indicative of the presence of chitin developed uniformly throughout the walls, but did not appear in the numerous cellulin granules found in this fungus. Chitin and cellulose fractions were prepared from chemically isolated walls and identified by X-ray diffraction.     

Colonial growth and ramihyphin A — Induced changes in cell walls ofNeurospora sitophila

Colonial growth ofNeurospora sitophila phenotypically induced by ramihyphin A is accompanied by marked changes in the contents of DNA, RNA and proteins in the mycelium, and in the relative proportion of hexoses in cell wall hydrolysates. The glucosamine/glucose ratio is also characteristic for colonial growth. X-ray analysis of cell walls showed that ramihyphin A suppresses the crystalline arrangement of chitin in cell walls. A combination of microbiological, biochemical and physico-chemical methods yielded a general picture of the changes accompanying the colonial growth ofNeurospora sitophila.     

High‐level hemicellulosic arabinose predominately affects lignocellulose crystallinity for genetically enhancing both plant lodging resistance and biomass enzymatic digestibility in rice mutants

Rice is a major food crop with enormous biomass residue for biofuels. As plant cell wall recalcitrance basically decides a costly biomass process, genetic modification of plant cell walls has been regarded as a promising solution. However, due to structural complexity and functional diversity of plant cell walls, it becomes essential to identify the key factors of cell wall modifications that could not much alter plant growth, but cause an enhancement in biomass enzymatic digestibility. To address this issue, we performed systems biology analyses of a total of 36 distinct cell wall mutants of rice. As a result, cellulose crystallinity (CrI) was examined to be the key factor that negatively determines either the biomass enzymatic saccharification upon various chemical pretreatments or the plant lodging resistance, an integrated agronomic trait in plant growth and grain production. Notably, hemicellulosic arabinose (Ara) was detected to be the major factor that negatively affects cellulose CrI probably through its interlinking with β‐1,4‐glucans. In addition, lignin and G monomer also exhibited the positive impact on biomass digestion and lodging resistance. Further characterization of two elite mutants, Osfc17 and Osfc30, showing normal plant growth and high biomass enzymatic digestion in situ and in vitro, revealed the multiple GH9B candidate genes for reducing cellulose CrI and XAT genes for increasing hemicellulosic Ara level. Hence, the results have suggested the potential cell wall modifications for enhancing both biomass enzymatic digestibility and plant lodging resistance by synchronically overexpressing GH9B and XAT genes in rice.     

Comparative Study of the Cell Walls of the Yeastlike and Mycelial Phases of Histoplasma capsulatum

Cell walls were prepared from the yeastlike and mycelial phases (YP and MP) of Histoplasma capsulatum and from Saccharomyces cerevisiae by mechanical disruption and washing. Lipids were extracted with methanol-ether, chloroform, and acidified methanol:ether; a final extraction was made with ethylenediamine. The lipid contents of H. capsulatum YP and MP walls were about the same. Qualitative and quantitative analyses were made of the products obtained from treatment of the cell walls, or fractions from them, with weak acid or with enzymatic preparations containing glucanase and chitinase activities. YP walls contained much larger quantities of chitin and smaller quantities of mannose and amino acids than the MP walls. H. capsulatum MP was shown to resemble S. cerevisiae by low chitin content and by the presence of a mannose polymer, soluble in ethylenediamine and water. H. capsulatum MP chitin appeared to be intimately associated with glucose in the wall, since enzymatic hydrolysis of the residue after mild acid hydrolysis of cell walls or fractions from them resulted in the release of glucose and acetylglucosamine; only acetylglucosamine was released from YP walls with such treatment. By electron microscopic observations, the unextracted MP cell walls were much thinner than the YP, and neither wall appeared laminated.     

哈氏木霉分生孢子发育的超微结构和细胞化学

通过电子显微镜和细胞化学标记研究了哈氏木霉分生孢子发育的超微结构和细胞化学。分生孢子发育的超微结构研究表明,分生孢子壁的发育是有个由薄而光滑到厚而有疣的过程;期间脂肪体在分生孢子和产孢细胞中不断累积,最后脂肪体沿着内壁排列成一层。免疫金标记结果显示,幼嫩的分生孢子壁中缺乏几丁质和纤维素,只有在成熟的分生孢子壁中含有几丁质;出乎意料的是在成熟分生孢子中发现有少量纤维素的存在。     

CELL WALL CHEMISTRY AND ULTRASTRUCTURE OF CHLOROCOCCUM OLEOFACIENS (CHLOROPHYCEAE)1,2

Cell walls of Chlorococcum oleofadens Trainor & Bold were examined ultrastructurally and chemically. The wall of zoospores has a uniform 30 nm width and a regular lamellar pattern. Zoospores and young vegetative cell walk exhibit periodicities, consisting of 20 nm ridges on the outer layer. Vegetative cell walls have a variable thickness of Up to 800 nm and are composed of multiple layers of electron dense material. Further, vegetative walk contain a microfibrillar material composed predominantly of glucose and presumed to be cellulose. Except for this cellulose, vegetative cell wall chemistry is very similar to that of Chlamydomemas being composed of glycoprotein rich in hydroxyproline. The hydroxyproline in Chlorococcum walls is linked glycosidically to a mixture of hetrooligosaccharides composed of arabinose and galactose, and in one instance, an unknown 6-deoxyhexose. Altogether, the glycoprotein complex accounts for at least 52% of the wall. The amino acid composition of the walls is stikingly similar to those of widely different plant species. Indirect evidence indicates zoospore cell walls are also chemically similar to those of Chlamydomonas, and like them, are cellulose free. Thus a major chemical difference between zoospore and vegetative cell walk of Chlorococcum is the presence of cellulose in the latter. The contribution of this microfibrillar cellulose to the physical properties of the vegetative wall is discussed.     

Characterization of chitin and chitin synthase from the cellulosic cell wall fungusSaprolegnia monoi¨ca

The presence of chitin in hyphal cell walls and regenerating protoplast walls ofSaprolegnia monoi¨ca was demonstrated by biochemical and biophysical analyses. α-Chitin was characterized by X-ray diffraction, electron diffraction, and infrared spectroscopy. In hyphal cell walls, chitin appeared as small globular particles while cellulose, the other crystalline cell wall component, had a microfibrillar structure. Chitin synthesis was demonstrated in regenerating protoplasts by the incorporation of radioactiveN-acetylglucosamine into a KOH-insoluble product. Chitin synthase activity of cell-free extracts was particulate. This activity was stimulated by trypsin and inhibited by the competitive inhibitor polyoxin D (K_i 20 μM). The reaction product was insoluble in 1M KOH or 1M acetic acid and was hydrolyzed by chitinase into diacetylchitobiose. Fungal growth and cell wall chitin content were reduced when mycelia were grown in the presence of polyoxin D. However, hyphal morphology was not altered by the presence of the antibiotic indicating that chitin does not seem to play an important role in the morphogenesis ofSaprolegnia.     

Composition of cell wall phenolics and polysaccharides of the potential bioenergy crop –Miscanthus

The composition and concentrations of cell wall polysaccharides and phenolic compounds were analyzed in mature stems of several Miscanthus genotypes, in comparison with switchgrass and reed (Arundo donax), and biomass characteristics were correlated with cell wall saccharification efficiency. The highest cellulose content was found in cell walls of M. sinensis‘Grosse Fontaine’ (55%) and in A. donax (47%) and lowest (about 32%) in M. sinensis‘Adagio’. There was little variation in lignin contents across M. sinensis samples (all about 22–24% of cell wall), however, Miscanthus×giganteus (M × g) cell walls contained about 28% lignin, reed – 23% and switchgrass – 26%. The highest ratios of cellulose/lignin and cellulose/xylan were in M. sinensis‘Grosse Fontaine’ across all samples tested. About the same total content of ester‐bound phenolics was found in different Miscanthus genotypes (23–27 μg/mg cell wall), while reed cell walls contained 17 μg/mg cell wall and switchgrass contained a lower amount of ester‐bound phenolics, about 15 μg/mg cell wall. Coumaric acid was a major phenolic compound ester‐bound to cell walls in plants analyzed and the ratio of coumaric acid/ferulic acid varied from 2.1 to 4.3, with the highest ratio being in M × g samples. Concentration of ether‐bound hydroxycinnamic acids varied greatly (about two‐three‐fold) within Miscanthus genotypes and was also the highest in M × g cell walls, but at a concentration lower than ester‐bound hydroxycinnamic acids. We identified four different forms of diferulic acid esters bound to Miscanthus cell walls and their concentration and proportion varied in genotypes analyzed with the 5‐5‐coupled dimer being the predominant type of diferulate in most samples tested. The contents of lignin and ether‐bound phenolics in the cell wall were the major determinants of the biomass degradation caused by enzymatic hydrolysis.     

OsCESA9 conserved‐site mutation leads to largely enhanced plant lodging resistance and biomass enzymatic saccharification by reducing cellulose DP and crystallinity in rice

Genetic modification of plant cell walls has been posed to reduce lignocellulose recalcitrance for enhancing biomass saccharification. Since cellulose synthase (CESA) gene was first identified, several dozen CESA mutants have been reported, but almost all mutants exhibit the defective phenotypes in plant growth and development. In this study, the rice (Oryza sativa) Osfc16 mutant with substitutions (W481C, P482S) at P‐CR conserved site in CESA9 shows a slightly affected plant growth and higher biomass yield by 25%–41% compared with wild type (Nipponbare, a japonica variety). Chemical and ultrastructural analyses indicate that Osfc16 has a significantly reduced cellulose crystallinity (CrI) and thinner secondary cell walls compared with wild type. CESA co‐IP detection, together with implementations of a proteasome inhibitor (MG132) and two distinct cellulose inhibitors (Calcofluor, CGA), shows that CESA9 mutation could affect integrity of CESA4/7/9 complexes, which may lead to rapid CESA proteasome degradation for low‐DP cellulose biosynthesis. These may reduce cellulose CrI, which improves plant lodging resistance, a major and integrated agronomic trait on plant growth and grain production, and enhances biomass enzymatic saccharification by up to 2.3‐fold and ethanol productivity by 34%–42%. This study has for the first time reported a direct modification for the low‐DP cellulose production that has broad applications in biomass industries.     

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae

Chitin deacetylation results in the formation of chitosan, a polymer of β1,4‐linked glucosamine. Chitosan is known to have important functions in the cell walls of a number of fungal species, but its role during hyphal growth has not yet been investigated. In this study, we have characterized the role of chitin deacetylation during vegetative hyphal growth in the filamentous phytopathogen Magnaporthe oryzae. We found that chitosan localizes to the septa and lateral cell walls of vegetative hyphae and identified 2 chitin deacetylases expressed during vegetative growth—CDA1 and CDA4. Deletion strains and fluorescent protein fusions demonstrated that CDA1 is necessary for chitin deacetylation in the septa and lateral cell walls of mature hyphae in colony interiors, whereas CDA4 deacetylates chitin in the hyphae at colony margins. However, although the Δcda1 strain was more resistant to cell wall hydrolysis, growth and pathogenic development were otherwise unaffected in the deletion strains. The role of chitosan hydrolysis was also investigated. A single gene encoding a putative chitosanase (CSN) was discovered in M. oryzae and found to be expressed during vegetative growth. However, chitosan localization, vegetative growth, and pathogenic development were unaffected in a CSN deletion strain, rendering the role of this enzyme unclear.     

THE CELL WALL OF PYROCYSTIS SPP. (DINOCOCCALES)1,2

The cell of Pyrocystis spp. is covered by an outer layer of material resistant to strong acids and bases. Internal to this layer much of the cell wall is composed of cellulose fibrils. The presence of cellulose fibrils was established by staining raw and ultra-violet–peroxide-cleaned cell walls and by combining X-ray diffraction spectroscopy with electron microscope observation. Carbon replicas of freeze-etched preparations and thin sections of P. lunula walls show outer layers, inside them ca. 24 layers of crossed parallel cellulose fibrils (4–5 nm thick, ca. 12 nm wide), then a region of smaller (ca. 6–12 nm diameter) fibrils in a disperse texture, and then the plasma membrane. Cellulose fibrils in the parallel texture are constructed of 3–5 elementary fibrils ca. 3 nm in diameter. Walls of P. fusiformis and P. pseudonctiluca also have cellulose fibrils in a crossed parallel texture similar to those of P. lunula. The Gymnodinium-type swarmer from lunate P. lunula appears to have a cell wall ultrastructure typical of other “naked” dinoflagellates.     

GROWTH AND DEVELOPMENT OF THE WATER MOLD RHIZIDIOMYCES IN PURE CULTURE

Fuller , Melvin S. (Brown U., Providence, R. I.) Growth and development of the water mold Rhizidiomyces in pure culture. Amer. Jour. Bot. 49(1): 64–71. Illus. 1962.—A method for growing pure cultures of many of the single or few-celled phycomycetous fungi such as Rhizidiomyces is described. This method facilitates both qualitative and quantitative study of such fungi during their growth and development. Zoospores of Rhizidiomyces start to grow within 3 hr after being placed in a liquid medium, and are ready to discharge new zoospores after about 40 hr growth at 25 C. The single nucleus of the zoospore goes through many synchronous mitotic divisions during the development of the mature multinucleate cell. Under carefully controlled environmental conditions, sporangium discharge occurs in a definite and predictable manner. Observations suggesting a possible mechanism of sporangium discharge are presented and discussed.     

HYPHAL WALL COMPOSITION OF MINDENIELLA SPINOSPORA AND ARAIOSPORA SP.

Mechanically isolated hyphal walls of the rhipidiacean fungi Mindeniella spinospora Kanouse and Araiospora sp. (Oomycetes) were examined by biochemical, cytochemical and x-ray diffraction analyses. In both fungi, the most abundant wall constituents were 1 → 3- and 1 → 6-linked β-glucans accounting for 91% of wall dry weight in M. spinospora and 87% in Araiospora sp. In addition, hyphal walls of M. spinospora contained 1.7% mannose, 4.3% protein, 2.0% ash and 1.0% lipid. The quantities of these components in Araiospora sp. were 1.9%, 1.8%, 1.5%, and 1.3%, respectively. Both species had cellulose contents ranging from one-fifth to one-fourth of wall dry weight and chitin was apparently absent. In general, wall composition of both fungi is quite similar to that of the related species Sapromyces elongatus, lending support to the assertion that a biochemical dichotomy exists with respect to hyphal wall composition between Rhipidiaceae and Leptomitaceae, the two families comprising the order Leptomitales.     

Unexpected Effects of Chitin,Cellulose, and Lignin Addition on Soil Dynamics in a Wet Tropical Forest

Decades of studies on the role of decomposition in carbon (C) and nitrogen (N) cycling have focused on organic matter (OM) of plant origin. Despite potentially large inputs of belowground OM from fungal cell walls and invertebrate exoskeletons, studies of the decomposability of their major constituent, chitin, are scarce. To explore effects on soil C dynamics of chitin, in comparison with two plant-derived chemicals, cellulose and lignin, I conducted a field-based chemical-addition experiment. The design contained three chemical treatments plus a control, with four replicates in each of two species of tropical trees grown in plantations. The chemicals were added in reagent-grade form at a rate that doubled the natural detrital C inputs of 1000 g C m^?2 y^?1. Despite its purported recalcitrance, chitin was metabolized quickly, with soil respiration (R _soil) increasing by 64% above the control within days, coupled with a 32% increase in soil extractable ammonium. Cellulose, which was expected to be labile, was not readily decomposed, whereas lignin was rapidly metabolized at least partially in one of the forest types. I examined effects of stoichiometry by adding to all treatments ammonium nitrate in a quantity that adjusted the C:N of cellulose (166) to that of chitin (10), using both field and in vitro experiments. For cellulose, CO₂ release increased more than five- to eightfold after N addition in root-free soil incubated in vitro, but only 0–20% in situ where roots were intact. By the end of the 2-year-long field experiment, fine-root biomass tended to be higher in the chitin treatment, where R _soil was significantly higher. Together these findings suggest that soil N availability limited cellulose decomposition, even in this Neotropical forest with high soil N stocks, and also that trees successfully competed for N that became available as chitin decomposed. These results indicate that the major constituent of cell walls of soil fungi, chitin, can decompose rapidly and release substantial N that is available for plant and microbial growth. As a consequence, soil fungi can stimulate soil OM decomposition and N cycling, and thereby play a disproportionate role in ecosystem C and N dynamics.