James A. Lomax

Methylation analysis of mesophyll, epidermis, and fibre cell-walls isolated from the leaves of perennial and Italian ryegrass

Abstract Intact, finely milled mesophyll, epidermis, and fibre cell-walls prepared from the leaves of perennial and Italian ryegrass have been subjected to methylation analysis. Methylation of the cell-walls led to a consistently higher recovery of glucose residues than that obtained by analysis of monosaccharide residues as their alditol acetates. Values for other sugars were in close agreement. The partially methylated sugars formed were consistent with the presence, in order of decreasing concentration, of cellulose, (glucurono)arabinoxylan, xyloglucan, rhamnogalacturonan, (1→3),(1→4)-linked glucan, (1→4)-linked galactan, and (1→3),(1→6)-linked galactan. The relative proportions of these polysaccharides differed between the various types of cell. Arabinoxylan comprised 21.6%, 26.7%, and 36.5% of the total sugars recovered from mesophyll, epidermis, and fibre cell-walls, respectively. Mixed-linked glucan and rhamnogalacturonan were found in epidermis walls in amounts 2- to 3-fold higher than in other cell-walls. The xylan backbone of arabinoxylan was more heavily substituted in primary than in secondary-thickened (fibre) cell-walls. Arabinose, found largely as terminal residues in the cell-walls, carried various amounts of alkali-labile substituents, particularly at position 5. The extent of 5-substitution reflected the phenolic content and was substantially higher in fibre cell-walls. The methylation data, coupled with the analytical data for uronic acids and non-carbohydrate components, accounted for ∼98% of the cell-wall dry matter.

Methylation of unfractionated, primary and secondary cell-walls of plants, and the location of alkali-labile substituents

Abstract A procedure has been developed for the methylation of intact (mature) plant cell-walls in good yield. The method depended on the reduction of particle size by dry milling in liquid nitrogen to a level that allowed plant fragments to swell and disperse in the methylation solvent. Quantification of the results was achieved by using methyl β- d -allopyranoside as internal standard. Perennial ryegrass cell-walls, whole barley straw, and beechwood, with degrees of lignification of 1.93, 12.66, and 19.33%, respectively, were used to determine the efficiency of methylation after milling. The recoveries of parent sugars, calculated by summing values for the appropriate partially methylated sugars, were 116, 102, and 109%, respectively, of those determined on the basis of alditol acetates. Acetalation of the free hydroxyl groups of milled, whole cell-walls, using methyl vinyl ether, enabled selective methylation to be performed at sites of alkali-labile substitution. The resulting recovery of pentoses was nearly quantitative, but that of (cellulosic) glucose was very low. These procedures allow linkage analysis of the structural polysaccharides and determination of the location of sites of alkali-labile substitution in the matrix polysaccharides of the intact cell-wall.

Extraction of phenolic-carbohydrate complexes from graminaceous cell walls

Abstract Barley straw and a preparation of perennial ryegrass were sequentially extracted with oxalic acid, dimethyl sulphoxide (DMSO) and a “cellulolytic” enzyme preparation Driselase and the fractions studied by methylation analysis, 13 C NMR and other methods. Oxalic acid, as expected, solubilised the bulk of the arabinose and ferulic acid in both samples, although appreciable amounts of xylose were also solubilised. DMSO yielded polymeric lignin-carbohydrate complexes (LCC), both of which consisted predominantly of a β (1 → 4) xylan. From the results of methylation analysis, sensitivity to oxalic acid hydrolysis and size-exclusion chromatography after alkaline hydrolysis, it was evident that lignin polymers were attached to arabinosyl and xylosyl residues by both ester and aryl-ether linkages. After cellulolytic hydrolysis of the residues, thioacidolysis analysis of the lignin component in the DMSO-soluble and Driselase-insoluble fractions from ryegrass revealed differences in every measurable aspect. The DMSO-soluble lignin was found to be more highly condensed, have a higher S/G ratio and have a higher terminal G/internal G ratio.

Soluble lignin-carbohydrate complexes from sheep rumen fluid: their composition and structural features.

Lignin-carbohydrate complexes, isolated from cell-free rumen fluid of sheep, consisted of polyphenolic material in association with carbohydrate (5.5%) and protein (1.8%). No separation of carbohydrate from the other components could be effected. The complexes showed a bimodal distribution of molecular size with the higher-molecular-weight fraction richer in carbohydrate. Methylation analysis indicated a wide range of linkages and a high proportion of terminal sugars. Reducing sugars were found in the complexes, particularly in the low-molecular-weight fraction (approximately 25%), suggesting that ether linkages to the phenolics were also present.

Lignin-carbohydrate complexes in graminaceous cell walls in relation to digestibility

Abstract Lignin is directly or indirectly covalently bound to polysaccharides in the intact plant cell wall. Relatively little is known about the structures of these lignin-carbohydrate complexes in graminaceous cell walls, despite their evident importance in limiting cell wall degradability. The present state of knowledge on this subject is briefly reviewed with emphasis on results recently obtained by the authors in which two different lignin structures were isolated from a single plant species. It is concluded that much remains to be learnt about lignin-carbohydrate structures especially in terms of their heterogeneity and distribution throughout the cell wall.

Apple-fruit xyloglucans: a comparative study of enzyme digests of whole cell walls and of alkali-extracted xyloglucans

Apple cell walls or alkali-extracted xyloglucans were digested with an endo-glucanase from Trichoderma viride and the resulting oligosaccharides were isolated by chromatography on Bio-Gel P-4. Three main oligosaccharides were present in similar proportions, and their structures were shown to be [Xyl(Glc)]3-Glc, [Xyl(Glc)]2-(FucGalXyl)Glc-Glc, and XylGlc-(GalXyP)Glc-(FucGalXyl)Glc-Glc. Each non-reducing-end Glc was 6-linked, each reducing-end Glc was 4-substituted, and each other Glc was 4,6-disubstituted. The Xyl was either terminal or 2-substituted, the Fuc was terminal, and the Gal was either terminal or 2-substituted. The 1H-NMR spectra of the oligosaccharides extracted directly from the cell wall showed that they are not acetylated. Other oligosaccharides, notably GalXyl3Glc4, Xyl2Glc4, and Xyl2Glc3, were present in smaller proportions in the digest of the cell walls.

A multiple-column approach to the methylation analysis of plant cell-walls

Abstract A procedure has been developed for the improved analysis of the neutral-sugar glycosidic linkages in plant cell-walls, utilising capillary g.l.c. and columns of three different phases for the separation of the products of methylation analysis. Retention coefficients are reported for a wide range of partially methylated alditol acetates on columns of CP-Sil88 and a bonded phase (BP-1) equivalent to OV-1. Using these phases and SP-1000, all cases of co-chromatography can be resolved. Computers were used to process the large amounts of data produced, to identify peaks and to assist in merging the results obtained using the three phases.

Structure of O-(2-hydroxyethyl)celluloses with high degrees of molar substitution : a critical evaluation

Abstract Methylation analysis complete with GLC-MS of three O -(2-dhydroxyethyl)celluloses with degrees of molar substitution (ms) of 1.73, 2.44, and 3.56, determined by hydrolysis with hydriodic acid, revealed at least 35 new monomeric compounds. Except for S 33333 , all of the isomers up to the monomers with ms 5 have been identified and quantified by GLC. The ms, degree of substitution (ds), positional, ds and the ms and ds distribution of the monomers have been derived. The resulting ms values were 1.89,2.30, and 3.39, and the ds values were 1.21, 1.34. and 2.06, respectively. The relative reaction constants for HO-2,3,6 have been computed and that for HO-2 appears not to vary. These calculated constants have been used to determine the homogeneity of the substituent distribution.

Characterisation of polysaccharides by in-source pyrolysis positive- and negative-ion direct chemical ionisation-mass spectrometry

Series of oligosaccharide ions have been generated from a range of polysaccharides by the application of in-source pyrolysis mass spectrometry, using both ammonia positive-ion chemical ionisation and negative-ion chlorine-nucleophilic-addition ionisation. Glucans with alpha-(1----6), beta-(1----6), alpha-(1----4), beta-(1----4), beta-(1----3), and beta-(1----2) linkages were studied, together with pentosans, xyloglucans, and an arabinogalactan. The series of ions correspond to intact, desorbed oligosaccharides with a terminal anhydro-sugar unit, and to similar oligosaccharides with attached sugar ring-cleavage fragments. The ions generated are dependent on the position of the linkage and ring size, and retain significant information on the structure of the original polysaccharide.

Interference from elemental sulphur in the determination of organotins by gas chromatography with flame photometric detection

Elemental sulphur, a common constituent of marine sediments, has been shown to give dialkyl sulphides with the Grignard reagents commonly used to derivatize alkyltin species before their determination by gas chromatography with flame photometric detection (GC-FPD). Further, it has been demonstrated that even with the red filter for 610 nm (normally used for organotin compounds) fitted to the detector, sulphur compounds do give rise to an emission signal, which may be mistaken for tin emission from a pentylated or propylated alkyltin compound, as the respective retention times are in some cases quite close.