Golgi-localized exo-β1,3-galactosidases involved in AGP modification and root cell expansion in Arabidopsis

Abstract

13 14 15 16 *corresponding author: Totte Niittylä, totte.niittyla@slu.se 17 18 19 20 48Plant arabinogalactan proteins (AGPs) are a diverse group of cell surface-and wall-associated 49 glycoproteins. Functionally important AGP glycans are synthesized in the Golgi apparatus, 50 but the relationships between their glycosylation, processing, and functionality are poorly 51 understood. Here we report the identification and functional characterization of two Golgi-52 localized exo-β-1,3-galactosidases from the glycosyl hydrolase 43 (GH43) family in 53 Arabidopsis thaliana. GH43 loss of function mutants exhibit root cell expansion defects in 54 sugar-containing growth media. This root phenotype is associated with an increase in the 55 extent of AGP cell wall association, as demonstrated by Yariv phenylglycoside dye 56 quantification and comprehensive microarray polymer profiling of sequentially extracted cell 57 walls. Recombinant GH43 characterization showed that the exo-β-1,3-galactosidase activity 58 of GH43s is hindered by β-1,6 branches on β-1,3-galactans. In line with this steric hindrance, 59 the recombinant GH43s did not release galactose from cell wall extracted glycoproteins or 60 AGP rich gum arabic. These results show that Arabidopsis GH43s are involved in AGP 61 glycan biosynthesis in the Golgi, and suggest their exo-β-1,3-galactosidase activity influences 62 AGP and cell wall matrix interactions, thereby adjusting cell wall extensibility. 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 et al. , 2010; Rose and Lee, 2010; Scheller and Ulvskov, 2010). The cell wall-associated 87 glycoproteins include arabinogalactan proteins (AGPs), which are proposed to have diverse 88 roles during cell expansion, including as structural components, modifiers of the extracellular 89 matrix, and ligands for cell surface receptors (Showalter et al., 2010; Xue et al., 2017). 90 91Glycans account for 90-98% of the molecular weight of AGPs, and are thought to be critical 92 for their functionality (Showalter, 1993). Characterisation of the enzymes responsible for 93 AGP glycosylation can elucidate AGP functions and functional redundancies (Showalter et 94 al., 2010; Showalter and Basu, 2016). AGP glycosylation is initiated by proline hydroxylation 95 in the ER and is then advanced in the Golgi apparatus by specialized glycosyl transferases 96 (GTs) (Rose and Lee, 2010; Hijazi et al., 2014). The glycosyl transferase family 31 (GT31) is 97 responsible for the synthesis of the hydroxyproline galactose and the β-1,3-galactan backbone 98