Barry J.H. Stevens

Structural features of cell-wall polymers of the apple

Abstract Cell-wall material was isolated from ripe-apple cortical tissues by sequential extraction with aqueous 1.5% sodium dodecyl sulphate and aqueous 90% methyl sulphoxide. The wall material, which contained ∼1% of protein, with proline and hydroxyproline as the preponderant amino acids, was sequentially extracted with water at 80°, oxalate at 80°, m KOH at 1°, and m and 4 m KOH at 20°, to leave a residue of α-cellulose, which was associated with an appreciable amount of arabinose-rich pectic material. The depectinated material was also extracted with 6 m guanidinium thiocyanate at 20° to solubilise preferentially polysaccharides rich in mannose. The hot-water-soluble pectic substances were richer in arabinose compared with the oxalate-soluble ones and were resolved into five fractions by anion-exchange chromatography. The bulk of the hemicelluloses, which were xyloglucans, were solubilised by 4 m KOH. The alkali-soluble hemicellulose polymers were resolved by anion-exchange chromatography into polysaccharides, mainly xyloglucans, arabinoxylan-pectic-xyloglucan, and arabinoxylan-pectic complexes. Small amounts of polysaccharide-protein-polyphenol complexes (where the polysaccharide moieties were arabinoxylans), pectic substances, and xyloglucans were also present. The glycosidic linkages of the above polymers were determined by methylation analysis. The general structural features of the cell-wall polymers are discussed.

Structural features of cell-wall polysaccharides of the carrot Daucus carota

Abstract Cell-wall material was isolated from the alcohol-insoluble residue of carrot by treatment with Pronase, phenol—acetic acid—water, and aqueous 90% methyl sulphoxide. Some pectic material was solubilised, but the major component was a highly esterified, acidic arabinogalactan. The purified cell-wall material, which contained ∼1% of protein, was sequentially extracted with water at 80°, ammonium oxalate at 80°, and m and 4 m KOH at 20°, to leave a residue of α-cellulose, which contained some pectic material. From the hot-water-soluble fraction, a major pectic polymer was isolated by anion-exchange chromatography. Methylation analysis showed that it was a rhamnogalacturonan, probably having highly branched arabinan and slightly branched galactan side-chains linked to O-4 of rhamnopyranosyl residues. An unusual feature of this pectic polymer is that it contained a small but significant proportion of 1,4-linked xylopyranosyl residues. From the alkali-soluble fractions, a range of pectic polymers associated with various amounts of xylans and possibly xyloglucans was isolated. The main linkages present in these complexes were 1,4-linked galactopyranosyluronic acid, 1,4-linked galactopyranosyl, and 1,5-linked arabinofuranosyl residues, terminal arabinofuranosyl and galactopyranosyl groups, and, in some fractions, 1,4-linked xylopyranosyl residues. The possible association of some of these polymers with proteins and phenolics is discussed.

Hemicellulosic polymers of cabbage leaves

Abstract The hemicellulosic polymers of depectinated cell-wall material of immature cabbage leaves have been extracted by alkali, fractionated by ion-exchange chromatography and their structural features studied. In the 1 M potassium hydroxide-soluble fraction the main polymers are arabinoxylan-xyloglucan-pectic, arabinoxylan-pectic-protein and arabinoxylan-xyloglucan-pectic-protein complexes. Small amounts of polysaccharide-protein-polyphenol complexes are also present. In the 4 M potassium hydroxide-soluble fraction the predominant polymers are two xyloglucans, the major one of which appears to have ca 10% of(1 → 4)-linked and 3% of(1 → 4,6)-linked mannose residues associated with it, and both have terminal galactose, fucose and possibly arabinose residues in the side chains. Methylation analysis of the oligosaccharides, formed by degradation with cellulase, and partial hydrolysis of the methylated xyloglucan, followed by re-methylation with CD 3 I, have enabled the formulation of a tentative structure which is similar to other plant galactoxyloglucans. It was not possible to establish whether the mannose residues are part of an associated glucomannan or whether they are an integral part of the glucan backbone. The general structural features of the hemicellulosic polymers are discussed in the light of these results.

Changes in composition and structure of wheat bran resulting from the action of human faecal bacteria in vitro

Cell-wall material of wheat bran was incubated with human faecal bacteria for 24-72 h and the resulting structural changes were studied by methylation analysis. Of the carbohydrate content, approximately 39% was degraded after 24 h, increasing to only 44% after 72 h. Arabinoxylans and mixed-linkage beta-D-glucans from the aleurone layer were degraded preferentially. After treatment of the bran with alkali, the extent of degradation was increased three-fold as a result of saponification of ester cross-links which facilitated increased degradation of the polymers from both the aleurone and outer, lignified, layers. There was evidence that ester linkages between the glucuronosyl residues, attached to O-2 of the (1----4)-linked xylosyl residues, and phenolic groups of lignin were also saponified. The treatment with alkali also rendered the cellulose more susceptible to bacterial attack. The alkali-soluble acidic arabinoxylan fractions of the bran were degraded readily by bacterial action, but the xyloglucans cross-linked to arabinoxylans by phenolics were relatively resistant.

Pectic polysaccharides of cabbage (Brassica oleracea)

Abstract Pectic substances extracted from cabbage cell walls with water, at 80°, and (NH 4 ) 2 C 2 O 4 , at 80°, accounted for 45%(w/w) of the purified cell wall material. Only a small amount of neutral arabinan was isolated. Partial acid hydrolysis and methylation analysis revealed that the major pectic polysaccharide had a rhamnogalacturonan backbone to which a highly branched arabinan was linked, at C-4 of the rhamnose units, mainly through short chains of (1→4)-linked galactopyranose residues. The bulk of the soluble pectic substances had only small amounts of proteins associated with them. After further extraction of the depectinated material with 1M and 4M KOH, to remove the hemicelluloses, the cellulose residue was found to contain a pectic polysaccharide which was solubilized by treatment with cellulase. The general structural features of the pectic polymers are discussed in the light of these results.

Structural investigation of an arabinan from cabbage (Brassica oleracea var. capitata)

Abstract An arabinan has been isolated from the hot water-soluble pectic substances of cabbage cell wall material. Methylation analysis involving GC-MS of methylated alditol acetates formed from the methylated arabinan has shown that the parent polysaccharide is highly branched and of the same structural type as other arabinans associated with seed pectins.

Use of oligonucleotide probes to identify members of two-component regulatory systems in Xanthomonas campestris pathovar campestris.

SummaryTwo-component regulatory systems comprising a sensor and a regulator protein, both with highly conserved amino acid domains, and commonly genetically linked, have been described in a range of bacterial species and are involved in sensing environmental stimuli. We used two oligonucleotide probes matching the postulated coding regions for domains of sensor and regulator proteins respectively in Xanthomonas campestris pathovar campestris (Xcc) to identify possible two-component regulatory systems in Xcc. Two different fragments of Xcc DNA with homology to both of these probes were cloned. The DNA sequence of part of one of these fragments encompassed a potential open reading frame (ORF), the predicted amino acid sequence of which had extensive homology with regulator proteins of two-component regulatory systems. Analysis of the predicted amino acid sequence for the 3′ end of an adjacent ORF revealed a very high level of homology with the C-terminal end of sensor proteins. Strains of Xcc with Tn5-induced mutations in the regulator gene were affected in extracellular polysaccharide production, and also in resistance to salt and chloramphenicol. No effects of mutation in the second clone were observed.

Investigation of the heterogeneity of xyloglucans from the cell walls of apple

Abstract Cell-wall material from parenchymatous tissues of apple was sequentially extracted with 50m m NaOH at 1°, m KOH at 1° and 20°, and 4 m KOH at 20°, to leave a residue of α-cellulose. From the 4 m KOH-soluble fraction, a crude xyloglucan was isolated by anion-exchange chromatography, and further resolved into seven xyloglucans by borate anion-exchange chromatography. The relative amounts of the xyloglucans, in order of elution, were 2.7:1.3:29.7:1.0:3.2:1.2:10.3. The structural features of five of the xyloglucans were determined by methylation analysis. These results show that apple xyloglucans exhibit heterogeneity.

Cation-dependent gelation of the acidic extracellular polysaccharides of Rhizobium leguminosarum: A non-specific mechanism for the attachment of bacteria to plant roots

Abstract The extracellular acidic heteropolysaccharides produced by various species of Rhizobium leguminosarum possess the same backbone but have different sidechains. Aqueous solutions of these polysaccharides form gels in the presence of excess of salt and divalent cations are more effective at inducing gelation than are monovalent cations, although the moduli of the gels are similar at comparable ionic strength. Gelation of the extracellular polysaccharide is proposed as a mechanism for attaching the bacteria to the tips of plant roots.

Characterisation of cyclosophorans produced by an acidic polysaccharide-negative mutant of Rhizobium leguminosaram

Abstract The cyclosophorans produced by the EPS− mutant Rhizobium leguminosarum 8401 pRL1JI pssl :: Tn5 have been isolated and characterised. The cyclic glucans were recovered from the clarified culture broth by absorption and subsequent elution from a charcoal column. Individual ring sizes were identified by fast atom bombardment mass spectrometry and the crude mixture fractionated by HPLC. The spectrum of ring sizes produced is typical of that produced by wild-type R. leguminosarum.