IDENTIFICATION OF THE ROLE OF [METHYL]GLUCURONIC ACID ON ARABINOGALACTAN POLYSACCHARIDES IN ARABIDOPSIS THALIANA

Abstract

Arabinogalactan proteins (AGPs) are proteoglycans heavily substituted by arabinogalactan polysaccharides. These are composed of arabinose and galactose, and minor sugars such as glucuronic acid (GlcA), fucose and xylose. The arabinogalactan polysaccharides do not decorate classical AGPs exclusively, but they can also be found decorating a wide range of proteins. Arabinogalactan proteins have been implicated in many processes of plant development. Recently, AGPs were proposed to bind and store calcium at the plasma membrane. They are extracellular, and are localised mainly at the plasma membrane via a GPI-anchor. They can also be soluble in the apoplast. Their low abundance, chemical similarity and high functional redundancy have hindered their study. My strategy to overcome these difficulties was to study knock-out Arabidopsis thaliana plants of glycosyltransferases that transfer sugars specifically onto AG-polysaccharides. Glucuronic acid makes up about 10% of the arabinogalactan polysaccharide structure in Arabidopsis thaliana cell culture AGPs. Previously, the glucuronic acid transferase A TGLCA T14A, a member of the CAZy Glycosyl Transferase 14 family, was shown to transfer GlcA specifically onto AGPs, and knock-out Arabidopsis plants showed a 30% reduction in [Me]GlcA substitution in AGP-enriched preparations. However, no clear growth phenotype was observed. The characterisation of knock-out plants of other GT14 family members and combinations thereof is described here. Based on previous studies (Lamport and Várnai, 2013), I assayed in vitro the calcium binding capacity of AGP extracts from WT and knock-out plants. The results showed that AGP extracts from knock-out plants can hold less calcium than WT plants in vitro. A wide range of plant growth phenotypes were identified. Growth phenotypes can be explained by changes in the cytoskeleton and deficiencies in calcium signaling. Our evidence suggests links between structural deficiencies of extracellular proteoglycans to extracellular calcium and cytoskeleton. This research has the potential to create a new model system for the study of molecular mechanisms dependent on calcium that drive cell expansion, division and differentiation in plants.