Structural Characterization of Arabidopsis Leaf Arabinogalactan Polysaccharides

Proteins decorated with arabinogalactan (AG) have important roles in cell wall structure and plant development, yet the structure and biosynthesis of this polysaccharide are poorly understood. To facilitate the analysis of biosynthetic mutants, water-extractable arabinogalactan proteins (AGPs) were isolated from the leaves of Arabidopsis (Arabidopsis thaliana) plants and the structure of the AG carbohydrate component was studied. Enzymes able to hydrolyze specifically AG were utilized to release AG oligosaccharides. The released oligosaccharides were characterized by high-energy matrix-assisted laser desorption ionization-collision-induced dissociation mass spectrometry and polysaccharide analysis by carbohydrate gel electrophoresis. The Arabidopsis AG is composed of a β-(1→3)-galactan backbone with β-(1→6)-d-galactan side chains. The β-(1→6)-galactan side chains vary in length from one to over 20 galactosyl residues, and they are partly substituted with single α-(1→3)-l-arabinofuranosyl residues. Additionally, a substantial proportion of the β-(1→6)-galactan side chain oligosaccharides are substituted at the nonreducing termini with single 4-O-methyl-glucuronosyl residues via β-(1→6)-linkages. The β-(1→6)-galactan side chains are occasionally substituted with α-l-fucosyl. In the fucose-deficient murus1 mutant, AGPs lack these fucose modifications. This work demonstrates that Arabidopsis mutants in AGP structure can be identified and characterized. The detailed structural elucidation of the AG polysaccharides from the leaves of Arabidopsis is essential for insights into the structure-function relationships of these molecules and will assist studies on their biosynthesis.Arabinogalactans (AGs) are structurally complex large-branched polysaccharides attached to Hyp residues of many plant cell wall polypeptides. Most proteins glycosylated with AGs (AGPs) have both AG glycosylated domains (glycomodules) and structural or enzymatic domains. However, typical AGPs commonly contain less than 10% protein, suggesting that the AG is the functional part of the molecule (Clarke et al., 1979; Fincher et al., 1983; Kieliszewski and Lamport, 1994; Borner et al., 2003; Xu et al., 2008). Hyp is the most characteristic amino acid present at the glycosylated domain of the AGP, but other amino acids such as Ser, Ala, and Thr are also very common. Type II AG polysaccharides share common structural features based on a β-(1→3)-galactan backbone with β-(1→6)-linked galactan side chains and can be found both on AGPs and rhamnogalacturonan-I (RG-I) pectin (Renard et al., 1991). The galactopyranosyl (Galp) residues can be further substituted with l-arabinofuranosyl (l-Araf) and occasionally also l-rhamnosyl (l-Rha), l-fucosyl (l-Fuc), and glucuronosyl (GlcA; with or without 4-O-methylation) residues (Tsumuraya et al., 1988; Tan et al., 2004; Tryfona et al., 2010). (Sugars mentioned in this work belong to the D-series unless otherwise stated.)The structure of AGs is poorly characterized, and this is mainly due to the great heterogeneity of glycan structures, not only between different AGPs but also even on the same peptide sequence in the same tissue (Estévez et al., 2006). The glycan structure can also be different depending on the developmental stage and tissue type (Tsumuraya et al., 1988), adding to the great heterogeneity of these molecules and therefore limiting their detailed characterization. Molecular and biochemical evidence has indicated that AGPs have specific functions during root formation, promotion of somatic embryogenesis (van Hengel et al., 2002), and attraction of pollen tubes to the style (Cheung et al., 1995). In addition, enhanced secretion efficiency or stability in the cell wall are properties that the AG may confer on the glycosylated protein (Borner et al., 2003). However, it has been difficult to differentiate one species of AGP from another in plant tissues and to assign specific roles to individual AGPs.l-Fuc is present in AGPs in Arabidopsis (Arabidopsis thaliana; van Hengel et al., 2002), radish (Raphanus sativus; Nakamura et al., 1984; Tsumuraya et al., 1984a, 1984b, 1988), and several other dicot plants such as thyme (Thymus vulgaris; Chun et al., 2001) and celery (Apium graveolens; Lin et al., 2011). Reduction in l-Fuc by 40% in roots of murus1 (mur1) plants resulted in a decrease of 50% in root cell elongation, and eel lectin binding assays suggested that the phenotype was the result of alterations in the composition of root AGPs (van Hengel and Roberts, 2002). An α-(1→2)-fucosyltransferase (FUT) activity for radish primary root AGPs has been described, where an α-l-Araf-(1→3)-β-Galp-(1→6)-Galp trisaccharide was used as exogenous substrate acceptor to mimic an AG polysaccharide in the enzymatic assay (Misawa et al., 1996). Linkage analysis, reactivity with eel lectin, and digestion with α-(1→2)-fucosidase indicated that the l-Fuc residues added are terminal and attached via an α-linkage to the C-2 position of an adjacent l-Araf residue (Nakamura et al., 1984; Tsumuraya et al., 1984a, 1984b, 1988). Recently, Wu et al. (2010) identified AtFUT4 and AtFUT6 genes encoding FUT proteins specific to AGPs, but the structures of the fucosylated AG generated have not been fully characterized.To gain insights into the synthesis and function of plant AGPs, it would be useful to have mutants altered in their carbohydrate moieties. However, no AG-specific biosynthetic mutants have been characterized, and this, among other reasons, is due to the very limited knowledge of the structure of Arabidopsis AGs (Qu et al., 2008). Moreover, characterization of AG in candidate mutants remains challenging. Even though the structures of some AGs have been proposed using NMR and sugar linkage analyses, the complete structural elucidation of a native AG still remains a formidable task, because NMR spectroscopy and methylation analysis have been largely used to provide information regarding the amount and type of linkages between adjacent glycosyl residues, and AG heterogeneity can confound attempts to build complete structural models. Recently, a modular structure was proposed for AGs on heterologously expressed proteins in tobacco (Nicotiana tabacum; Tan et al., 2010). Tan et al. (2010) proposed that approximately 15-residue repeating blocks of decorated β-(1→3)-trigalactosyl subunits connected by β-(1→6)-linkages were the building blocks of type II AG polysaccharides and concluded that these molecules are far less complex than commonly supposed. Most characterized β-(1→6)-galactan side chains in AGs are reported to be short, of one or two residues (Neukom and Markwalder, 1975; Gane et al., 1995; Gaspar et al., 2001). On the contrary, there are reports of long β-(1→6)-galactan side chains in radish root AGPs (Haque et al., 2005). Similarly, we recently found evidence that wheat (Triticum aestivum) flour endosperm AGP extracts contained long β-(1→6)-galactan side chains heavily substituted with l-Araf at C-3 (Tryfona et al., 2010). This partial structure of the carbohydrate component of wheat flour AGP isolated from water extracts of wheat endosperm was elucidated utilizing a combination of analytical approaches, such as the use of enzymes able to release oligosaccharides specifically from AGs, high-energy matrix-assisted laser desorption ionization (MALDI)-collision-induced dissociation (CID) mass spectrometry (MS), and polysaccharide analysis by carbohydrate gel electrophoresis (PACE; Tryfona et al., 2010). In this work, we applied these techniques to study the carbohydrate component of Arabidopsis leaf AGPs. AG-specific enzyme digestion products were analyzed by PACE and MS, allowing a partial structure to be proposed. We show that endogenous Arabidopsis leaf AG is composed of a β-(1→3)-galactan backbone with β-(1→6)-galactan side chains. These side chains are substituted with l-Araf residues via α-(1→3)-linkages and can vary in length from one up to at least 20 Galp residues. We also found that the β-(1→6)-galactan side chains are substituted mainly with 4-O-methyl-glucuronosyl (4-O-Me-GlcA) at their nonreducing termini, while occasional l-Fuc substitutions were also present via α-(1→2)-linkages on l-Araf residues. In addition, AG oligosaccharides from leaves of the mur1 mutant were identified, and their structures were compared with those isolated from wild-type plants.