Identification of three potent hydroxyproline O-galactosyltransferases in Arabidopsis

Arabinogalactan proteins (AGPs) are plant-specific extracellular glycoproteins implicated in a variety of processes during growth and development. AGP biosynthesis involves O-galactosylation of hydroxyproline (Hyp) residues followed by a stepwise elongation of the complex sugar chains. However, functionally dominant Hyp O-galactosyltransferases, such that their disruption produces phenocopies of AGP-deficient mutants, remain to be identified. Here, we purified and identified three potent Hyp O-galactosyltransferases, HPGT1, HPGT2 and HPGT3, from Arabidopsis microsomal fractions. Loss-of-function analysis indicated that approximately 90% of the endogenous Hyp O-galactosylation activity is attributable to these three enzymes. AGP14 expressed in the triple mutant migrated much faster on SDS-PAGE than when expressed in wild-type, confirming a considerable decrease in levels of glycosylation of AGPs in the mutant. Loss-of-function mutant plants exhibited a pleiotropic phenotype of longer lateral roots, longer root hairs, radial expansion of the cells in the root tip, small leaves, shorter inflorescence stems, reduced fertility and shorter siliques. Our findings provide genetic evidence that Hyp-linked arabinogalactan polysaccharide chains are critical for AGP function and clues to how arabinogalactan moieties of AGPs contribute to cell-to-cell communication during plant growth and development.