A β–glucuronosyltransferase from Arabidopsis thaliana involved in biosynthesis of type II arabinogalactan has a role in cell elongation during seedling growth

Abstract

These authors contributed equally to this work. SUMMARYWe have characterized a b-glucuronosyltransferase (AtGlcAT14A) from Arabidopsis thaliana that is involved in the biosynthesis of type II arabinogalactan (AG). This enzyme belongs to the Carbohydrate Active Enzyme database glycosyltransferase family 14 (GT14). The protein was localized to the Golgi apparatus when transiently expressed in Nicotiana benthamiana. The soluble catalytic domain expressed in Pichia pastoris transferred glucuronic acid (GlcA) to b-1,6-galactooligosaccharides with degrees of polymerization (DP) ranging from 3-11, and to b-1,3-galactooligosaccharides of DP5 and 7, indicating that the enzyme is a glucuronosyltransferase that modifies both the b-1,6-and b-1,3-galactan present in type II AG. Two allelic T-DNA insertion mutant lines showed 20-35% enhanced cell elongation during seedling growth compared to wild-type. Analyses of AG isolated from the mutants revealed a reduction of GlcA substitution on Gal-b-1,6-Gal and b-1,3-Gal, indicating an in vivo role of AtGlcAT14A in synthesis of those structures in type II AG. Moreover, a relative increase in the levels of 3-, 6-and 3,6-linked galactose (Gal) and reduced levels of 3-, 2-and 2,5-linked arabinose (Ara) were seen, suggesting that the mutation in AtGlcAT14A results in a relative increase of the longer and branched b-1,3-and b-1,6-galactans. This increase of galactosylation in the mutants is most likely caused by increased availability of the O6 position of Gal, which is a shared acceptor site for AtGlcAT14A and galactosyltransferases in synthesis of type II AG, and thus addition of GlcA may terminate Gal chain extension. We discuss a role for the glucuronosyltransferase in the biosynthesis of type II AG, with a biological role during seedling growth.

The Effects of High Steady State Auxin Levels on Root Cell Elongation in Brachypodium

“…Error bars indicate SE of the mean. Differences were not statistically significant (Student's t test) unless indicated as follows: *P < 0.05, **P < 0.01, and ***P < 0.001. transgenic lines with mildly reduced arabinogalactan complexity, root elongation is substantially stimulated (van Hengel and Roberts, 2002;Eudes et al, 2008;Knoch et al, 2013). However, whether this is mainly due to enhanced cell elongation has not been reported.…”

Recombinant therapeutic proteins produced in plants: towards engineering of human-type O-and N-glycosylation

“…Much of the recent work on AGPs has focused on their biosynthesis and several of the biosynthetic glycosyltransferase (GT) enzymes responsible for AG polysaccharide production have been identified (103,104) and have been grouped into families based on primary structure (http://www.cazy.org/). Wu et al modifies both the b-1,6-and b-1,3-galactan present in type II AGP with a biological role during seedling growth (111). Moreover, Dilokpimol and Geshi (2014) reported the enzyme activities for other two of the Arabidopsis GT14 isoforms, AtGlcAT14B and AtGlcAT14C, which were also found to possess the glucuronosyltransferase activity adding glucuronic acid residues to b-1,3-and b-1,6-linked galactans (112).…”

Glycobiology in yeast: production of bio-ative biopolymers and small molecules

“…For the gene discovery part of the gum Arabic (AGP) glycosylation, we identified four novel glycosyltransferases. We have biochemically characterized the enzymes expressed heterologously (deliverable 16), which resulted in the several international peerreviewed articles (Geshi et al, 2013) (Knoch et al, 2013) ) . Also, we found that the arabinogalactan glycosyltransferases target to an unknown subcellular compartment partially overlapped with unconventional protein secretion pathway when expressed heterologously .…”

“…Further, we identified that AtGlcAT14A (Knoch et al, 2013) adds extra glucuronic acids to gum Arabic and improves the performance of the gum (deliverable 20). We have tested the emulsifier property using the method described in (Heidebach et al, 2013) and found that the enzyme-modified gum Arabic is superior than the unmodified gum in the formation of oil-in-water emulsions (Dilokpimol and Geshi, manuscript submitted).…”