Investigating the synthesis and regulation of (1,3;1,4)-β-glucan biosynthesis

Cereals such as rice (Oryza sativa (Os)), barley (Hordeum vulgare (Hv)) and sorghum (Sorghum bicolor (Sb)) provide a considerable portion of our daily energy requirements. Their cell wall constituents, such as (1,3;1,4)-β-glucan, survive relatively intact through much of the upper human digestive system to reach the colon, where they are fermented by a range of commensal microorganisms. The products of this fermentation help reduce blood cholesterol levels and ameliorate diseases including coronary heart disease, type II diabetes and colorectal cancer. Efforts have therefore been directed toward understanding the regulation and mechanism of (1,3;1,4)-β-glucan biosynthesis to enhance the human health potential and industrial utility of cereal grain. Numerous reports suggest that the CELLULOSE SYNTHASE-LIKE F6 (CslF6) gene encodes the synthase responsible for producing the majority of (1,3;1,4)-β-glucan in cereals. These synthase genes contain species-specific polymorphisms that have been shown to influence the amount and structure of (1,3;1,4)-β-glucan produced when they are expressed heterologously in Nicotiana benthamiana and barley grain. Here, a chimeric approach exchanged sections of the barley (Hv) and sorghum (Sb) CSLF6 synthases to identify regions influencing (1,3;1,4)-β-glucan production and structure. Using this approach an 80 amino acid stretch, which contains the conserved TED and QxxRW motifs, was shown to be responsible for much of the difference in (1,3;1,4)-β-glucan production and structure between the barley and sorghum synthases. Of the six amino acid polymorphisms contained within this section, one affected polysaccharide structure whilst another dictated the amount of (1,3;1,4)-β-glucan. Co-expression in N. benthamiana was used to investigate CSLF6 modulation and complex formation. Results from a variety of chimeric, truncated and mutated constructs suggest that a highly variable section of unknown function, termed the class-specific region (CSR), and the NH2-terminal region of CSLF6 are separately able to mediate complex formation and increase (1,3;1,4)-β-glucan production. Expression of a construct missing the CSR indicated that the region was not structurally or functionally required for (1,3;1,4)-β-glucan synthesis in N. benthamiana. A PilZ domain responsible for cofactor binding and cellulose synthase activation in bacteria was also identified at the COOH-terminal end of the NH2-terminal region of CSLF6, and was shown to influence (1,3;1,4)-β-glucan production. Overall, the results presented here have furthered our understanding of the action of the CSLF6 isoform of the (1,3;1,4)-β-glucan synthase enzyme. This brings us closer to having the capacity to precisely control the synthase’s function, and allowing the prospect of manipulating cereal tissues to contain the optimal amount of (1,3;1,4)-β-glucan with a defined structure for specific human health and industrial applications.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2016