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    Structural studies of a fucogalactoxyloglucan from pinus radiata primary cell walls : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
    (Massey University, 1980) Little, John William Lester
    1. The changes in carbohydrate composition of elongating Pinus radiata primary cell walls were investigated. In the hemicellulose B extracts, a large increase in the percentage of non-starch, non-cellulosic, glucose was found to occur on cessation of cell-wall elongation. 2. By fractionation of the hemicellulose B extracts, with a variety of methods involving precipitation from an aqueous solution, a xyloglucan was purified. This xyloglucan was the major hemicellulose of the Pinus radiata hypocotyl cell wall. 3. Characterisation studies on the xyloglucan involved: quantitative analysis of the monosaccharides derived by nitric acid/urea hydrolysis; identification of the partial hydrolysis products derived by trifluoroacetic acid hydrolysis; quantitation of the sugar linkages using methylation by the Hakomori method; and analysis of the anomeric configuration of component sugars using chromium trioxide oxidation. 4. From the results a tentative structure has been suggested for the xyloglucan, consisting of a backbone of B-D-gluco-pyranose residues linked together by 1-4 glycosidic bonds, and with sidechains of single xylose residues linked through C-6 of the glucose units. Galacto and fuco-1,2- galacto sidechains are attached to some of the xylose residues, probably through the C-2 of the xylose.
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    Carbohydrate fractionation and elongation of lupin hypocotyl cell walls : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Botany at Massey University
    (Massey University, 1974) Monro, John Alexander
    The relationship between extensibility, growth rate and carbohydrate composition in different sections of lupin hypocotyl has been investigated. Although significant differences in extensibility were found, the carbohydrate composition of elongating and non-elongating regions were similar when delignified tissue was examined. However, it was subsequently found that the delignification removed all of the wall hydroxyproline, most of the arabinose, and much galactose and that all of these were higher in non-elongating than in elongating hypocotyl. The acid conditions of delignification caused about half of the loss of the sugars but did not cause the loss of hydroxyproline. Extraction of the hypocotyl cell walls with guanidinium thiocyanate and other denaturants, both before and after treatment with dilute acid or sodium methoxide in methanol did not dissolve the hydroxyproline, indicating that compounds containing this amino acids are sprobably covalently linked to insoluble wall constituents other than through acid labile arabinofuranose-hydroxyproline or ester links alone. 10% KOH extracted most of the wall hydroxyproline and hemicellulose largely as non-dialysable material. The hemicellulose thus extracted may be fractionated into hemicelluloses A and B and the latter into linear 1-4 linked polysaccharides and branched polysaccharides. Most of the hydroxyproline containing polymer is co-precipitated with the linear 1-4 linked hemicellulose-B arabinoxylan. When cell walls from elongating and non-elongating hypocotyl sections were compared the hemicellulose-B arabinoxylan fraction from the non-elongating wall had a much higher proportion or arabinose, galactose and hydroxyproline than the same polymer from elongating wall. Extraction of cell walls with 10% KOH at O°C removed about two thirds of the hemicellulose-B but little hydroxyproline. Subsequent treatment with 10% KOH at room temperature removed most of the hydroxyproline and remaining hemicellulose-B. The hemicellulose-B removed at room temperature showed the greatest increases in arabinose and galactose accompanying cessation of elongation. The polysaccharide extracted at 0°C is mainly xylan while that removed at room temperature contains large amounts of galactose and arabinose. The release of galactose at room temperature was accompanied by destruction or serine and appeared to parallel β-elimination of galactosylserine The kinetics of release of arabinose and galactose at room temperature differed. The above and other results are discussed particularly in relation to wall structure and a tentative model for the extensin-polysacchsride complex of lupin hypocotyl cell walls is proposed.
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    Extraction, composition and some of the physical and chemical properties of dietary fibre : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Department of Food Technology, Massey University
    (Massey University, 1985) Holloway, Warren Desmond
    The extraction and some of the chemical and physical properties of components from plant cell walls are described in this thesis. The chemical composition of the extracted polymers and the morphological and physical changes occurring in wheat bran at various stages of an extraction sequence and the metal binding capacities of the extracts were determined. A sequential extraction procedure using water, amylase, oxalate and alkali (before and after delignification) was used to isolate components of plant cell walls. This enabled water soluble and water insoluble fibres from bean, cabbage, lettuce, tomato, peach, pumpkin, kumera, onion, pear, wheat bran, lucerne, clover and ryegrass to be obtained. The water soluble fibres were shown to be composed predominantly of arabinose, galactose and uronic acid, whereas the water insoluble fibres contained mainly arabinose and xylose. The viscosities of the alkali soluble fibres extracted from wheat bran, before and after chlorite delignification, and after solubilisation in N-methyl-morpholine-N-oxide were determined. The arabinoxylan extracted before delignification, yield of 7.9 g/100 g, had a limiting viscosity number of 220.6 ml/g, whereas the arabinoxylan extracted after chlorite delignification, yield of 3.8 g/100 g, had a limiting viscosity number of 74.2 ml/g. when the solvent N-methyl-morpholine-N-oxide had been used to dissolve the nondelignified arabinoxylan, a considerable decrease in viscosity, to 6.3 ml/g, was observed. It was concluded that direct extraction (no delignification) of wheat bran, enables a less degraded arabinoxylan to be extracted in adequate yields. The use of N-methyl-morpholine-N-oxide as a solvent for arabinoxylan resulted in extensive degradation. The structural changes in wheat bran at each stage of the extraction sequence and when dimethylsulphoxide (DMSO) was substituted for alkali were observed using light and scanning electron microscopy. It was shown that the commercially ground sample of wheat bran contained a high proportion of starch, which was removed after the amylase treatment. Alkali removed cell wall material predominantly from the aleurone layer. DMSO was not an efficient extractor of arabinoxylans from cell walls, a yield of only 0.4% being obtained and the aleurone cell walls remaining intact. The arabinoxylan, extracted with DMSO, had a higher ferulic acid and acetyl content than the arabinoxylan extracted with alkali. The interactions of fibres with metal ions (copper, iron, zinc, calcium, potassium, magnesium, manganese and sodium) using concentrations that would be expected in the human small bowel after a 'typical' meal were investigated. It was found that the water soluble fibres bound more copper, iron and zinc than the water insoluble fibres. The copper, iron and zinc binding occurred with a displacement of calcium, magnesium and manganese. The water insoluble fibres (hemicelluloses) contained a higher calcium content than the soluble fibres (pectins). After acid treatment, sodium was bound preferentially rather than calcium to hemicellulose. Possibly divalent calcium ions play a role in stabilising the hemicellulose components of plant cell walls. The binding capacities and mechanisms of zinc binding to wheat bran, its components and to phytate were determined. Zinc binding capacities (µM/g dry weight of plant material) in order of magnitude were; phytate (6582 ± 192), DMSO soluble hemicellulose (5089 ± 921), water soluble fibre (4038 ± 216), cell walls (1012.6 ± 193), lignocellulose (510 ± 41.9), cold water soluble fibre (440.0 ± 15.3), alkali soluble hemicellulose (227.9 ± 61.4), bran (167.7 ± 12.7), bran ex oxalate (148.3 ± 50.0), bran ex ethanol (142.3 ± 4.4) and cellulose (57.4 ± 5.3). The water soluble fibre, fractionated using ammonium sulphate, composed predominantly of arabinose (24.0%), galactose (20.3%), xylose (18.6%), mannose (16.2%), glucose (10.9%) and rhamnose (6.0%), bound zinc more strongly than phytate or the DMSO hemicellulose. The Scatchard plots of zinc binding to phytate and to the fibres, except for the water soluble fibres, were concave and markedly nonlinear, suggesting that the binding mechanism is by negative cooperativity or site heterogeneity. The Scatchard plots of zinc binding to the water soluble fibres showed well pronounced maximum, indicating the binding mechanism is by positive cooperativity. Part I of this thesis describes the studies undertaken to isolate and determine the chemical composition of different types of fibres from bean, cabbage, sweet potato, lettuce, onion, peach, pear, pumpkin, tomato, wheat bran, white clover, lucerne and ryegrass. This study has been published in the Journal of the Science of Food and Agriculture 1983, 34: 1236-1240 (see Appendix F). Part II of this thesis describes studies undertaken to investigate the viscosity of hemicelluloses obtained using the extraction procedure and after solubilisation in N-methyl-morpholine-N-oxide. The work has been published in carbohydrate Research 1985, 143: 271-274 (see Appendix G). Part III describes studies undertaken to observe the morphological structure of wheat bran, changes occurring during the extraction sequence and influence of DMSO when substituted for alkali. Part IV describes studies on binding of metals to water soluble and insoluble fibres from fruits, vegetables, bran and grasses. Part v describes a more detailed study of zinc binding to wheat bran, its fibre components, and to phytate. The thesis concludes with a general discussion of the findings and a summary of the conclusions.
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    Structural studies on cell walls of Pinus radiata : with particular reference to callus cultured cells : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, New Zealand
    (Massey University, 1982) Fenemor, Dallas R.
    A study of the composition of the primary cell wall of Pinus radiata has been undertaken. 1. Preliminary work with hypocotyl tissue showed that hemicelluloses of hypocotyl consisted of a xylan (probably (4-O-methylglucurono)xylan) and a xyloglucan. Acidic sugars examined, showed that galacturonic acid was the main component, and that 4-O-MeGlcA was present. 2. Cell walls were prepared from callus tissue either by wet sieving in 80% ethanol (Batch 1) or by disruption in a French Pressure cell and washing with aqueous potassium phosphate buffer; (Batch 2). Each batch was submitted to a series of extractions with different reagents in order to investigate the mode of bonding of polymers within the walls. The polysaccharide and protein components of each fraction were studied by monosaccharide and amino acid analysis. Fractions of Batch 1 were assayed for lignin and selected fractions from both batches were studied by methylation analysis. 3. The results of investigations led to the following major conclusions. The non-cellulosic components recognised in the wall preparations were: - a) A(1→3)-linked galactan and an arabino-3, 6-galactan which were largely extractable from the cell walls by hot water and may be only loosely bound in the cell wall. b) The pectic components consisting of; i) pectin, a galacturonate polymer containing a linear (1→4)-galacturonan back bone interspersed with branched rhamnose residues, ii) branched (1→5)-arabinan and iii) linear (1→4)-galactan, which occurred together in cell wall fractions and were not all extracted by classical extractants (such as hot aqueous EDTA), some being tightly bound in the cellulosic residue after alkali extraction. c) A fucogalactoxyloglucan some of which was extracted by water or EDTA, but the majority was extracted by subsequent treatment with either alkali or in part by a strong chaotropic reagent (6M GTC). Thus the fucogalactoxyloglucan was probably bound in the cell wall by strong hydrogen bonding. Some other bonding may be involved in the GTC-resistant fraction. d) A branched xylan which was removed by GTC and alkali, the larger level being removed by GTC. e) A galactoglucomannan, identified only by 4-linked mannose residues in hot water extracts and strong alkali fractions. f) Hydroxyproline-containing protein which was extracted from the cell wall by a variety of reagents but hydroxyproline-rich protein remains tighly bound after alkali extraction. g) Lignin which was tentatively identified in the cell wall. It appeared likely that cross-linking with lignin would be responsible for the non-extractability of some polysaccharides and protein from the walls. A mild acid chlorite treatment followed by alkali extraction removed most of the residual pectic components, xylan and protein from the walls. A basis has been laid for the further investigation of the wall structure and isolation of polysaccharides.