UC Berkeley

Xyloglucan (XyG) represents the most abundant hemicellulose in the primary cell wall of many dicot and non-graminaceous monocot plant species. Found throughout the land plant lineage, the structure of this polysaccharide varies by species, tissue, and developmental stage. Some proteins responsible for the biosynthesis and metabolism of XyG have been previously identified but additional genes remain to be uncovered. In the work presented here, several genetic and genomic approaches were used to identify previously unknown genes involved in XyG biosynthesis and metabolism. A forward genetic screen of mutagenized Arabidopsis thaliana resulted in the identification of the Altered Xyloglucan 9 (AXY9) gene, coding for a protein of unknown function, as part of an apparently plant-specific pathway for the acetylation of polysaccharides. The AXY9 protein may be part of a system for shuttling activated acetyl groups across the Golgi membrane for use by polysaccharide-specific acetyltransferases. A transcriptional profiling approach used on developing XyG-rich Tropaeolum majus seeds revealed a galactosyltransferase that acts on XyG, as demonstrated by mutant analysis in A. thaliana. By utilizing comparative genomics, two putative XyG arabinofuranosyltransferase genes were discovered from tomato, which are able to act in vivo to arabinosylate XyG. Expression of these genes rescued mechanical and morphological phenotypes of an A. thaliana mutant deficiency for XyG galactosylation, providing insight into the function of XyG substitution. To provide a testing platform for candidate genes involved in XyG biosynthesis and to investigate the activities of these genes independently from endogenous plant proteins, an attempt was made to reconstruct the XyG biosynthetic pathway in a heterologous host. This effort resulted in the production of a glucan backbone and the UDP-xylose donor substrate required for the xylosyltransferases, but a XyG polysaccharide was not detected.