Fractionation of cell-wall preparations from grass leaves by centrifuging in non-aqueous density gradients.

1. Dried preparations of cell walls from perennial-ryegrass (Lolium perenne) and Italian-ryegrass (L. multiflorum) leaves were suspended in mixtures of carbon tetrachloride with light-petroleum (b.p. 45--50 degrees C) or alcohols and layered on density gradients formed from the same solvents. 2. On centrifugation, the cell walls become distributed throughout a suitably chosen gradient. Fractions corresponding to various regions of the gradient were separated, examined under the microscope and analysed. 3. Cell-wall preparations made from leaf material ground in liquid N2, or in a triple roll mill, showed considerable heterogeneity in particle size, and their behaviour in the density gradient was variable, although there was a general indication that walls derived from vascular bundles were less dense than those from sclerenchyma. 4 Treatment in a vibratory ball mill decreased the size of the particles and produced a more uniform material, but made it impossible to distinguish the origins of the particles. This material behaved more reproducibly in the density gradient. 5. Some fractionations were also made by successive centrifugation in media of increasing relative density. 6. Analyses of the fractions obtained by each method indicated that the less dense had a greater proportion of xylose in the polysaccharide components, and higher contents of acetyl groups and lignin, confirming the close relationship between these components in plant cell walls. 7. The results show that there are differences in polysaccharide composition between the cell-wall types in the grass leaf, the vascular tissue being richer in hemicellulose relative to cellulose than the sclerenchyma.

The relationship between acetyl content and sugar composition in expanding leaves of beech (Fagus sylvatica L.).

Samples of beech (Fagus sylvatica) leaves were taken from the time of leaf emergence until leaf fall. They were extracted with boiling aqueous ethanol and the residues analysed for their acetyl-group content and their content of sugars released by acid hydrolysis. The accumulation of acetyl groups closely paralled that of xylose, and the two were present in approximately equimolecular proportions, except during the period of leaf expansion, when the acetyl-group/xylose ratio exceeded 3:2. The galactose present in each leaf reached a maximum as leaf expansion ceased and then declined during the period of rapid xylose accumulation. The results are discussed in relation to the known association of acetyl groups with xylans and pectic substancesand to the disappearance of constituents during cell-wall maturation.

Electron microscopy of the microbial populations present and their modes of attack on various cellulosic substrates undergoing digestion in the sheep rumen.

Cotton fibers and various cell wall preparations from grass leaves and from the feces of sheep fed on dried grass were placed in the sheep rumen in bags made from 5-mum-mesh nylon cloth. After periods of from 3 to 48 h, bags were removed, and the contents were fixed, embedded, sectioned, and stained for electron microscopy. Some of the bacteria present were seen to be closely associated with the cell walls, either tunneling within them or making very close contact. Evidence was obtained for differential digestion of cell walls and of the layers within them. Distinct differences were noticed between bacterial populations attacking the more susceptible wall types and those attacking feces cell walls and cotton fibers. Among the latter, the dominant form was a long, thin rod with a typical gramnegative cell wall structure, different from that described for Bacteroides succinogenes S85 or for Butyrivibrio fibrisolvens.

Acetyl groups in cell-wall preparations from higher plants.

O-Acetyl groups were detected by i.r. spectroscopy in cell-wall preparations from grasses and other higher plants and their presence was confirmed chemically. The amounts present are likely to influence both the physical state of the cell-wall polysaccharides and also their digestion by enzymes.

Co-operative action by endo- and exo-beta-(1 leads to 3)-glucanases from parasitic fungi in the degradation of cell-wall glucans of Sclerotinia sclerotiorum (Lib.) de Bary.

1. Two fungi, Coniothyrium minitans Campbell and Trichoderma viride Pers. ex Fr., were grown on autoclaved crushed sclerotia of the species Sclerotinia sclerotiorum, which they parasitize. 2. In vitro the crude culture filtrates would lyse walls isolated from hyphal cells or the inner pseudoparenchymatous cells of the sclerotia, in which a branched beta-(1-->3)-beta-(1-->6)-glucan, sclerotan, is a major constituent. 3. Chromatographic fractionation of the enzymes in each culture filtrate revealed the presence of several laminarinases, the most active being an exo-beta-(1-->3)-glucanase, known from previous studies to attack sclerotan. Acting alone this brought about a limited degradation of the glucan, but the addition of fractions containing an endo-beta-(1-->3)-glucanase led to almost complete breakdown. A similar synergism between the two enzymes was found in their lytic action on cell walls. 4. When acting alone the endo-beta-(1-->3)-glucanase had a restricted action, the products including a trisaccharide, tentatively identified as 6(2)-beta-glucosyl-laminaribiose. 5. These results are discussed in relation to the structure of the cell walls and of their glucan constituents.

Apiose and mono-O-methyl sugars as minor constituents of the leaves of deciduous trees and various other species.

1. Leaves of a number of species were hydrolysed with aqueous sulphuric acid and the resulting mixtures of sugars were fractionated by chromatography on activated charcoal. Paper chromatography of the fractions showed the presence in all the hydrolysates of minor constituents with R(F) values similar to or greater than those of the common hexoses and pentoses. 2. Two of these were identified as 2-O-methylxylose and 2-O-methylfucose. Estimates of the amounts present in whole leaves, and in fractions prepared from them, showed that they were associated with the hemicelluloses. 3. A third constituent was identified, by the formation of its di-isopropylidene derivative, as apiose. It also was associated chiefly with the hemicellulose fraction; none could be found in aqueous extracts from leaves of Tilia vulgaris, nor in aqueous extracts of Zostera marina, in which apiose is a major constituent of the water-insoluble polysaccharide. 4. A further constituent, after further purification by preparative paper chromatography, was tentatively identified, by gas-liquid chromatography of derivatives, as 3-O-methylgalactose, and was probably accompanied by small amounts of 4-O-methylgalactose. 5. These observations confirm the widespread occurrence of 2-O-methylxylose, 2-O-methylfucose and apiose, but 3-O-methylgalactose was hitherto known only in slippery-elm mucilage, and 4-O-methylgalactose in soil polysaccharides. Some experiments on the digestion of leaf hemicellulose fractions by snail crop-juice suggested that the mono-O-methyl sugars might confer resistance to enzymic degradation.

The separation of beta-glucanases produced by Cytophaga johnsonii and their role in the lysis of yeast cell walls.

1. When Cytophaga johnsonii was grown in the presence of suitable inducers the culture fluid was capable of lysing thiol-treated yeast cell walls in vitro. 2. Autoclaved or alkali-extracted cells, isolated cell walls and glucan preparations made from them were effective inducers, but living yeast cells or cells killed by minimal heat treatment were not. 3. Chromatographic fractionation of lytic culture fluids showed the presence of two types of endo-beta-(1-->3)-glucanase and several beta-(1-->6)-glucanases; the latter may be induced separately by growing the myxo-bacterium in the presence of lutean. 4. Extensive solubilization of yeast cell walls was obtained only with preparations of one of these glucanases, an endo-beta-(1-->3)-glucanase producing as end products mainly oligosaccharides having five or more residues. Lysis by the other endo-beta-(1-->3)-glucanase was incomplete. 5. The beta-(1-->6)-glucanases produced a uniform thinning of the cell walls, and mannan-peptide was found in the solution. 6. These results, and the actions of the enzyme preparations on a variety of wall-derived preparations made from baker's yeast, are discussed in the light of present conceptions of yeast cell-wall structure.

The glucan components of the cell wall of baker's yeast (Saccharomyces cerevisiae) considered in relation to its ultrastructure.

1. Commercial pressed baker's yeast, and cell walls prepared from it, were extracted in various ways and the products examined by a number of techniques, including infrared spectroscopy and electron microscopy. 2. The glucan components of the walls cannot be extracted from intact yeast cells by 3% (w/v) sodium hydroxide at 75 degrees , but at least one-third of the glucan of cell wall preparations is dissolved under these conditions, and more will dissolve after ultrasonic treatment. 3. If intact cells are given a preliminary treatment with acid the wall glucans dissolve in dilute aqueous alkali. 4. Acid conditions as mild as sodium acetate buffer, pH5.0, for 3hr. at 75 degrees are sufficient for this preliminary treatment; the glucan then dissolves in 3% sodium hydroxide at 75 degrees leaving a very small residue, which contains chitin and about 1% of the initial glucan of the wall. Dissolution is hindered by exclusion of air, or by a preliminary reduction with sodium borohydride, suggesting that some degradation of the glucan by alkali is taking place. 5. After treatment with 0.5m-acetic acid for 24hr. at 90 degrees the glucan dissolves slowly at room temperature in 3% sodium hydroxide, or in dimethyl sulphoxide. The extraction with acetic acid removes glycogen and a predominantly beta-(1-->6)-linked glucan (not hitherto recognized as a component of baker's yeast), but none of the beta-(1-->3)-glucan, which remains water-insoluble. 6. Without treatment with acid, the glucan is not significantly soluble in dimethyl sulphoxide, but can be induced to dissolve by ultrasonic treatment. 7. These results are interpreted by postulating the presence of an enclosing membrane, composed of chitin and glucan, that when intact acts as a semipermeable membrane preventing the escape of the alkali- and dimethyl sulphoxide-soluble fraction of the glucan. Mild acid treatments damage this membrane, and ultrasonic and ballistic disintegration disrupt it. 8. Some support for this hypothesis is given by the effects of certain enzyme preparations, which have been found to render a substantial part of the glucan extractable by dimethyl sulphoxide.

Cell wall residues in yeast protoplast preparations.

Protoplast preparations made from Saccharomyces cerevisiae by prolonged treatment with snail digestive juice contained fibrils and chitinous bud-scar residues from the original cell wall.

The effect of illumination of the malic acid content and anion/cation balance of mustard leaves (Sinapis alba).

1. Mustard plants have been grown under conditions in which the length of artificial day could be controlled. 2. Leaf samples were analysed for malic acid and citric acid, and for a number of inorganic anions and cations. A simple method is described by which sap was obtained from 0.5g. samples of leaves. 3. In days of 16hr. or more, malic acid was accumulated; the chief cation accumulated was calcium. 4. When the day-length was reduced the malic acid content decreased considerably but the calcium content remained the same. There was little change in the pH value of the sap, the balance of anions and cations having been maintained mainly by increases in citrate and nitrate contents. Analyses of the whole leaf still showed some deficiency in anion after sodium, potassium, calcium, magnesium, nitrate, sulphate, inorganic phosphate, chloride, malate and citrate had been accounted for. 5. Analyses at shorter intervals revealed a large diurnal variation in malic acid content, which increased during the first 5-6hr. of the light period, and fell during darkness. 6. The significance of these findings is discussed, and it is suggested that malic acid accumulation is a by-product of photosynthesis, calcium being taken up irreversibly to maintain anion/cation balance, and hence creating a continuing need for anions to balance it.