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Abstract
Apiose is a unique, branched-chain monosaccharide. It is a conserved initial residue of side chains A and B in the vascular plant cell wall polysaccharide rhamnogalacturonan II, an abundant side chain in the seagrass and duckweed-specific pectic polysaccharide apiogalacturonan, and a glycoside decorating numerous plant secondary metabolites. Detection of aqueous methanolic-soluble apiosides in mosses, liverworts, hornworts, and green algae revealed that apiosides are not exclusive to vascular plants. Recently, our research uncovered apiosyl-residues in extracts from the marine bacteria Geminicoccus roseus and plant pathogen Xanthomonas pisi. These apiose-containing glycans are synthesized using UDP-apiose as the donor. UDP-apiose together with UDP-xylose is formed from UDP-glucuronic acid by the bifunctional UDP-apiose/UDP-xylose synthase (UAS). UASs from vascular plants, avascular plants, and bacteria (bUAS) were identified and functionally characterized to distinguish them from other short-chain dehydrogenase/reductases. The initial goals of this research were to 1) determine if UDP-apiose synthesis regulates apioside deposition in plant cell walls and 2) identify and characterize an apiosyltransferase (ApiT) capable of utilizing UDP-apiose to synthesize wall apiosides. To address the first aim, we over-expressed duckweed UAS in the moss Physcomitrella patens, which led to an increase in aqueous methanolic-soluble apiose but did not result in discernible amounts of cell wall-associated apiose. Thus, bryophytes and algae likely lack the ApiT required to synthesize apiose-containing cell wall glycans but can synthesize secondary metabolite apiosides. Over-expression of duckweed UAS in Arabidopsis also led to increased methanolic-soluble apiose but not a significant increase in pectic apiose, suggesting the limiting factor for plant wall apiose is the glycosyltransferase machinery. The second aim was addressed when small amounts of apiose were detected in the cell pellet of Xanthomonas pisi. Examination of the bacterial genome revealed putative GTs (termed XpGTA and XpGTB) directly downstream of the bUAS ORF. Heterologous co-expression of UAS and XpGTB in E. coli demonstrated that these cells utilize UDP-apiose to generate an apioside detectable in the cell pellet fraction. Enzyme purification and assay demonstrated XpGTB is an apiosyltransferase that specifically uses UDP-apiose.