Analytical Techniques for Determining the Role of Domain of Unknown Function 579 Proteins in the Synthesis of O-Methylated Plant Polysaccharides

Matrix polysaccharides are a diverse group of structurally complex carbohydrates and account for a large portion of the biomass consumed as food or used to produce fuels and materials. Glucuronoxylan and arabinogalactan protein are matrix glycans that have sidechains decorated with 4-O-methyl glucuronosyl residues. Methylation is a key determinant of the physical properties of these wall glycopolymers and consequently affects both their biological function and ability to interact with other wall polymers. Indeed, there is increasing interest in determining the distribution and abundance of methyl-etherified polysaccharides in different plant species, tissues, and developmental stages. There is also a need to understand the mechanisms involved in their biosynthesis. Members of the Domain of Unknown Function (DUF) 579 family have been demonstrated to have a role in the biosynthesis of methyl-etherified glycans. Here we describe methods for the analysis of the 4-O-methyl glucuronic acid moieties that are present in sidechains of arabinogalactan proteins. These methods are then applied toward the analysis of loss-of-function mutants of two DUF579 family members that lack this modification in muro. We also present a procedure to assay DUF579 family members for enzymatic activity in vitro using acceptor oligosaccharides prepared from xylan of loss-of-function mutants. Our approach facilitates the characterization of enzymes that modify glycosyl residues during cell wall synthesis and the structures that they generate.