Xyloglucan endotransglucosylase activity (XET), one of the two main activities of wall xyloglucan endotransglucosylase/hydrolase proteins (XTHs), is of interest because it is responsible for cutting and re-joining xyloglucan of the hemicellulose-cellulose microfibril network in the plant cell wall. XET activity causes transient matrix cleavage without hydrolysis, thus providing a molecular mechanism for controlled, turgor-driven wall expansion. XET activity can be involved in both wall-loosening, thus facilitating cell expansion, and wall-tightening, thus suppressing cell expansion depending on the molecular size, location and age of the participating xyloglucan chains. I have studied the existence of an ‘XET activating factor’ (XAF) in the cold-water-extractable polymers of cauliflower florets. Remaining water-soluble on boiling but losing activity upon proteinase K- and trypsin-digestions implied a heavily glycosylated glycoprotein. XAF was extracted from a wide range of plants and organs. XAF solubilised Arabidopsis cell-wall XTHs, increasing their XET activity on soluble xyloglucan up to 120-fold, tested by a novel method developed in my project. XAF had effects similar to those of 15 mM Ca2+ and 100 mM Na+ in this respect, although it was only weakly ionic. Interestingly, XAF had the unique ability to solubilise XET activity but no other tested wall enzymes from Arabidopsis cell walls, suggesting a specific interaction of XAF to XTH proteins. XAF was successfully purified by the use of several methods, developed in this project. These included cation-exchange column chromatography followed by anion-exchange column chromatography, resulting in two main XAF-activity fractions; or a native- PAGE electro-elution, resulting in three main fractions. Purified XAF contained a major amount of glucose, arabinose, galactose and uronic acid residues. Both boiled cauliflower preparation (BCP) and partially purified XAF were positive with AGP antibodies but the purification of AGP from BCP by the use of Yariv reagent did not enrich XAF activity. Mass-spectrometry analyses of the purified XAF fractions showed some candidates for XAF, including fasciclin-like arabinogalactan-protein 7 (FLA7), stress-responsive protein (LTI65, LTI140) and early nodulin-like protein 14 (ENODL14). Homozygous Arabidopsis mutants (confirmed by genotyping) defective in these genes were used to determine XAF as well as its biological role on plant cell growth. Although there was no phenotype observed, several organs of the mutant plants had significant increases or decreases in XAF activity compared to that of wild type plants. This is the first work that suggests a role of fla7, enodl14 and lti65 in the solubilisation, and thus activation, of Arabidopsis XET.

Acacia senegal and Acacia seyal are important agroforestry cash crops indigenous to several countries of sub-Saharan Africa, including Nigeria. The gum exudate produced by these species is termed gum Arabic which is an approved food additive (E414), primarily used as an emulsifier. In the current study, the molecular structure, composition and biophysical properties of gum samples harvested from mature trees of Acacia senegal at two specific ecolocations in Nigeria (NG1 and NG2), have been investigated together with two previously characterised gum samples harvested from A. senegal and A. seyal originating from Sudan. The monosaccharide sugar composition analyses have shown that the A. seyal gum had a lower rhamnose and glucuronic acid content than the A. senegal gum, but had higher arabinose content. No significant difference was observed between the sugar composition of the A. senegal gums from Sudan and Nigeria. The total protein content of the Nigerian gum samples were significantly higher than recorded for the Sudanese samples. The principal amino acids present in all the gum samples are hydroxyproline, serine, aspartame, threonine and proline which is in agreement with literature values. The hydrodynamic size of the molecules present in the gums was studied using dynamic light scattering and it was found that molecular association occurred in solution over time which was inhibited in the presence of an electrolyte. The comparison of droplet size distribution for emulsions prepared with A. senegal (NG1) and A. seyal gum samples showed that A. senegal sample was a better emulsifier than the A. seyal. Multilayer adsorption of the samples onto polystyrene latex particles was observed, which resulted in an increase in thickness of the adsorbed layer as a consequence of the interaction between the protein and carbohydrate within the molecules adsorbed on the emulsion surface. Preliminary analyses of the gums using transmission electron microscopy showed the presence of varied macromolecules, ranging in size from ~12 - ~60 nm. Immuno-gold negative staining (using JIM8 monoclonal antibody) indicated clear labelling of arabinogalactan-proteins present in the gums harvested from A. senegal, the labelling of the A. seyal sample was inconclusive. In summary, the data presented represents the first detailed comparison of the structure, composition and physicochemical characteristics of Nigerian Acacia gum exudates versus Sudanese samples (main global supplier) which have shown that gum obtained from Nigerian sources is a viable alternative to ensure future supply of this valuable natural resource.

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