Bruce A. Stone

A simple and rapid preparation of alditol acetates for monosaccharide analysis

Abstract A simple and rapid method is described for the preparation of alditol acetates from monosaccharides. It can be performed in a single tube without transfers or evaporations. Monosaccharides are reduced with sodium borohydride in dimethyl sulphoxide and the resulting alditols acetylated using 1-methylimidazole as the catalyst. Removal of borate is unnecessary and acetylation is complete in 10 min at room temperature. Monosaccharides are quantitatively reduced and acetylated by this procedure. The alditol acetates are completely separated by glass-capillary, gas-liquid chromatography on Silar 10C. The method has been applied to the analysis of monosaccharides in acid hydrolysates of a plant cell-wall.

An improved procedure for the methylation analysis of oligosaccharides and polysaccharides.

An improved procedure for the methylation analysis of oligosaccharides and polysaccharides is described. Steps in the procedure were examined and optimised for quantitative recovery and speed. Methylation was shown to be complete by using [14C]methyl iodide. All operations were performed in the same tube and the need to concentrate solutions containing acetylated alditols of methylated sugars was eliminated, thus minimising losses due to volatilization. The method is convenient, gives high recoveries of acetylated alditols of methylated sugars, and allows analysis of the glycosyl linkages of oligo- or poly-saccharides to be completed within a working day. A wide range of oligo- and poly-saccharides were methylated by this procedure.

Form and function of arabinogalactans and arabinogalactan-proteins

Abstract The occurrence, isolation, chemistry and physico-chemistry of plant arabino-3,6-galactans and arabino-3,6-galactan-proteins is reviewed. The structural relationships between arabino-3,6-galactans from gymnosperm wood, gum exudates of Acacia and other trees, and from plant callus cells and whole tissues are discussed. The nature of these proteoglycans is compared with the arabinose and galactose containing cell wall glycoproteins. Interactions of the arabino-3,6-galactan proteoglycans with carbohydrate binding proteins and with Yariv antigens are described. The utility of these reactions for both cellular and subcellular localization of the proteoglycans is discussed. The possible biological roles of the arabinogalactans and the arabinogalactan-proteins are reviewed.

Cellulose Synthase-Like CslF Genes Mediate the Synthesis of Cell Wall (1,3;1,4)-ß-d-Glucans

A characteristic feature of grasses and commercially important cereals is the presence of (1,3;1,4)-β-d-glucans in their cell walls. We have used comparative genomics to link a major quantitative trait locus for (1,3;1,4)-β-d-glucan content in barley grain to a cluster of cellulose synthase–like CslF genes in rice. After insertion of rice CslF genes into Arabidopsis, we detected (1,3;1,4)-β-d-glucan in walls of transgenic plants using specific monoclonal antibodies and enzymatic analysis. Because wild-type Arabidopsis does not contain CslF genes or have (1,3;1,4)-β-d-glucans in its walls, these experiments provide direct, gain-of-function evidence for the participation of rice CslF genes in (1,3;1,4)-β-d-glucan biosynthesis.

Water-soluble (1→3), (1→4)-β-D-glucans from barley (Hordeum vulgare) endosperm. II. Fine structure

Abstract Water-soluble (1→3),(1→4)-β- d -glucans isolated from barleys grown in Australia and the UK were depolymerised using a purified (1→3),(1→4)-β- d -glucan 4-glucanohydrolase (EC 3.2.1.73). Oligomeric products were quantitatively separated by high resolution gel filtration chromatography and their structures defined by methylation analysis. Approximately 90% (w/w) of each polysaccharide consists of cellotriosyl and cellotetraosyl residues separated by single (1→3)-linkages but blocks of 5–11 (1→4)-linked glucosyl residues are also present in significant proportions. Periodate oxidation followed by Smith degradation suggested that contiguous (1→3)-linked β-glucosyl residues are either absent, or present in very low frequency. The potential for misinterpretation of data due to incomplete Smith degradation was noted. The irregularly-spaced (1→3)-linkages interrupt the relatively rigid, ribbon-like (1→4)-β-glucan conformation and confer a flexibility and ‘irregular’ shape on the barley (1→3),(1→4)-β- d -glucan, consistent with its solubility in water. Molecular models incorporating the major structural features confirm that the polysaccharide is likely to assume a worm-like conformation in solution. Non-covalent interactions between long blocks of (1→4)-linkages in (1→3),(1→4)-β- d -glucans, or between these blocks and other polysaccharides, offer a possible explanation for the organisation of polysaccharides in the framework of the cell wall.

Cinnamic acid bridges between cell wall polymers in wheat and Phalaris internodes

Abstract A method has been devised for the quantitative determination of cinnamic acids participating in ester—ether bridges between cell wall polymers based on the different reactivities of free carboxylic acids and their esters towards borohydride reductants and the different susceptibilities of cinnamic acid ester and benzyl ether linkages to alkaline treatments. Lignin—polysaccharide containing fractions extracted with dioxane—H 2 O from cell walls of wheat ( Triticum aestivum ) and phalaris ( Phalaris aquatica ) internodes are hydrogenated using a Pd/C catalyst at room temperature to convert cinnamic acids to their corresponding dihydrocinnamic acids. The sample is subsequently reduced with LiBH 4 in ether—toluene to convert ester-linked dihydrocinnamates to their corresponding alcohols, hydrolysed with 4 M NaOH at 170°, and the dihydrocinnamic acid derivatives released from their etherified forms determined by GC. Using model compounds it was shown that these reactions proceeded quantitatively. The results indicate that all of the etherified ferulic acid in the dioxane—H 2 O-soluble fractions of walls of wheat and phalaris internodes is also ester-linked. It has been calculated that there are nine to 10 ferulic acid ester—ether bridges for every 100 C 6 —C 6 lignin monomers. p -Coumaric acid is not involved in ester-ether bridges.

The location of (1→3)-β-glucans in the walls of pollen tubes of Nicotiana alata using a (1→3)-β-glucan-specific monoclonal antibody

The location of the (1→3)-β-glucan, callose, in the walls of pollen tubes in the style of Nicotiana alata Link et Otto was studied using specific monoclonal antibodies. The antibodies were raised against a laminarinhaemocyanin conjugate. One antibody selected for further characterization was specific for (1→3)-β-glucans and showed no binding activity against either a cellopentaose-bovine serum albumin (BSA) conjugate or a (1→3, 1→4)-β-glucan-BSA conjugate. Binding was inhibited by (1→3)-β-oligoglucosides (DP, 3–6) with maximum competition being shown by laminaripentaose and laminarihexaose, indicating that the epitope included at least five (1→3)-β-linked glucopyranose residues. The monoclonal antibody was determined to have an affinity constant for laminarihexaose of 2.7. 104M−1. When used with a second-stage gold-labelled, rabbit anti-mouse antibody, the monoclonal antibody probe specifically located the (1→3)-β-glucan in the inner wall layer of thin sections of the N. alata pollen tubes.

Solution properties of wheat-flour arabinoxylans and enzymically modified arabinoxylans

Abstract A series of water-soluble arabinoxylans has been prepared from purified wheat-flour arabinoxylan by partial removal of arabinosyl side-branches using an α- L -arabinofuranosidase. Their solubilities, molecular weights, intrinsic viscosities, and degrees of hydration were measured. The results suggest that the arabinoxylan assumes an extended rod-like conformation in solution. The solubilizing effect of arabinosyl substituents was not the result of increased hydration, but due to their ability to prevent intermolecular aggregation of unsubstituted xylose residues. The extended conformation of arabinoxylan is consistent with its known organization and function in plant cell-walls.

A new substrate for investigating the specificity of β-glucan hydrolases

The tine distinctions between the substrate speciflcities of the /3-glucan endo-hydrolases [ 1 ] can only be defined precisely by the use of substrates with known, and preferably regular, structures. A new 1,3; 1,4-/Iglucan with these properties has been prepared and its use in studies on the specificity of /3-glucan endohydrolases is described. the proportions of 1,3and 1,4linkages in the mixedlinked substrates and in the hydrolysis products, that for the Streptomyces enzyme, x can only be a 1,4&kage. However, unequivocal proof was not provided by these experiments; An Aspergillus niger fi-glucan hydrolase [5] and an enzyme from Trichoderma uiride [6] appear to have the same specificity as the Streptomyces enzyme.

The application of sirofluor, a chemically defined fluorochrome from aniline blue for the histochemical detection of callose

SummarySirofluor, a chemically defined fluorochrome from aniline blue in aqueous unbuffered solutions, complexes with isolated (1 → 3)-β-glucans, but not (1 → 4)-β-glucans, after embedding in JB-4 resin and sectioning. Under these conditions, callose deposits in plant tissues give a brilliant yellow fluorescence with essentially no background fluorescence.