Plant primary cell walls are hydrated extracellular complexes composed largely of polysaccharides: cellulose, hemicellulose and pectin. Cell wall constituents and composition vary in cell-, environment-, and species-dependent manners. For example, within land plant hemicelluloses xyloglucan is ubiquitous while mixedlinkage (1→3),(1→4)-β-D-glucan (MLG) is found only in the Poales and Equisetum. Glycosyl hydrolase 16 (GH16) enzyme family members include numerous enzymes with pertinence to the understanding of the ‘lives’ of cell wall hemicelluloses. However, despite this, the details of the interactions between GH16 enzymes and their substrates have often not been elucidated. Likewise, the true preferences of many of these enzymes and the range of substrates which they can utilise remain to be fully explored. By providing a greater wealth of information for the correlation of enzyme structure with reaction catalysed, such an understanding would enable better predictions of the activities of novel enzymes. Crucially, this would also allow better identification of roles performed by these enzymes in planta as well as of the potential applications of these enzymes. This work sought to further our understanding of the interactions between GH16 enzymes and their substrates by the study of five activities exhibited by GH16 enzymes – xyloglucan endotransglucosylase (XET), xyloglucan endoglucanase/hydrolase (XEG/XEH), mixed-linkage glucan : xyloglucan endotransglucosylase (MXE), lichenase and cellulose : xyloglucan endotransglucosylase (CXE). All of the analysed activities act on xyloglucan and/or MLG. Of particular focus is the novel enzyme MXE from the evolutionarily isolated genus Equisetum (horsetail), which acts on both. Notable findings include: identification of MXE/CXE gene; determination of the substrate specificity of MXE; defining of the sites of attack of lichenase, XEG, XET and MXE; discovery of novel xyloglucan structures and discrepancies between the xyloglucan present in different barley organs.

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.