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Biosynthesis of the carbohydrate moieties of arabinogalactan proteins by membrane-bound β-glucuronosyltransferases from radish primary roots.

ABSTRACT: A membrane fraction from etiolated 6-day-old primary radish roots (Raphanus sativus L. var hortensis) contained β-glucuronosyltransferases (GlcATs) involved in the synthesis of the carbohydrate moieties of arabinogalactan proteins (AGPs). The GlcATs transferred [(14)C]GlcA from UDP-[(14)C]GlcA on to β-(1 → 3)-galactan as an exogenous acceptor substrate, giving a specific activity of 50-150 pmol min(-1) (mg protein)(-1). The enzyme specimen also catalyzed the transfer of [(14)C]GlcA on to an enzymatically modified AGP from mature radish root. Analysis of the transfer products revealed that the transfer of [(14)C]GlcA occurred preferentially on to consecutive (1 → 3)-linked β-Gal chains as well as single branched β-(1 → 6)-Gal residues through β-(1 → 6) linkages, producing branched acidic side chains. The enzymes also transferred [(14)C]GlcA residues on to several oligosaccharides, such as β-(1 → 6)- and β-(1 → 3)-galactotrioses. A trisaccharide, α-L-Araf-(1 → 3)-β-Gal-(1 → 6)-Gal, was a good acceptor, yielding a branched tetrasaccharide, α-L-Araf-(1 → 3)[β-GlcA-(1 → 6)]-β-Gal-(1 → 6)-Gal. We report the first in vitro assay system for β-GlcATs involved in the AG synthesis as a step toward full characterization and cloning.

SUBMITTER: Endo M

PROVIDER: S-EPMC3898515 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundArabinogalactan-proteins (AGPs) are heavily glycosylated with type II arabinogalactan (AG) polysaccharides attached to hydroxyproline residues in their protein backbone. Type II AGs are necessary for plant growth and critically important for the establishment of normal cellular functions. Despite the importance of type II AGs in plant development, our understanding of the underlying role of these glycans/sugar residues in mucilage formation and seed coat epidermal cell development is poorly understood and far from complete. One such sugar residue is the glucuronic acid residues of AGPs that are transferred onto AGP glycans by the action of β-glucuronosyltransferase genes/enzymes.ResultsHere, we have characterized two β-glucuronosyltransferase genes, GLCAT14A and GLCAT14C, that are involved in the transfer of β-glucuronic acid (GlcA) to type II AGs. Using a reverse genetics approach, we observed that glcat14a-1 mutants displayed subtle alterations in mucilage pectin homogalacturonan (HG) compared to wild type (WT), while glcat14a-1glcat14c-1 mutants displayed much more severe mucilage phenotypes, including loss of adherent mucilage and significant alterations in cellulose ray formation and seed coat morphology. Monosaccharide composition analysis showed significant alterations in the sugar amounts of glcat14a-1glcat14c-1 mutants relative to WT in the adherent and non-adherent seed mucilage. Also, a reduction in total mucilage content was observed in glcat14a-1glcat14c-1 mutants relative to WT. In addition, glcat14a-1glcat14c-1 mutants showed defects in pectin formation, calcium content and the degree of pectin methyl-esterification (DM) as well as reductions in crystalline cellulose content and seed size.ConclusionsThese results raise important questions regarding cell wall polymer interactions and organization during mucilage formation. We propose that the enzymatic activities of GLCAT14A and GLCAT14C play partially redundant roles and are required for the organization of the mucilage matrix and seed size in Arabidopsis thaliana. This work brings us a step closer towards identifying potential gene targets for engineering plant cell walls for industrial applications.

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Biosynthesis of the carbohydrate moieties of arabinogalactan proteins by membrane-bound β-glucuronosyltransferases from radish primary roots.

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