Galactoglucomannan structure of Arabidopsis seed-coat mucilage in GDP-mannose synthesis impaired mutants.

Cell-wall polysaccharides are synthesized from nucleotide sugars by glycosyltransferases. However, in what way the level of nucleotide sugars affects the structure of the polysaccharides is not entirely clear. guanosine diphosphate (GDP)-mannose (GDP-Man) is one of the major nucleotide sugars in plants and serves as a substrate in the synthesis of mannan polysaccharides. GDP-Man is synthesized from mannose 1-phosphate and GTP by a GDP-Man pyrophosphorylase, VITAMIN C DEFECTIVE1 (VTC1), which is positively regulated by the interacting protein KONJAC1 (KJC1) in Arabidopsis. Since seed-coat mucilage can serve as a model of the plant cell wall, we examined the influence of vtc1 and kjc1 mutations on the synthesis of mucilage galactoglucomannan. Sugar composition analysis showed that mannose content in adherent mucilage of kjc1 and vtc1 mutants was only 42% and 11% of the wild-type, respectively, indicating a drastic decrease of galactoglucomannan. On the other hand, structural analysis based on specific oligosaccharides released by endo-ß-1,4-mannanase indicated that galactoglucomannan had a patterned glucomannan backbone consisting of alternating residues of glucose and mannose and the frequency of α-galactosyl branches was also similar to the wild type structure. These results suggest that the structure of mucilage galactoglucomannan is mainly determined by properties of glycosyltransferases rather than the availability of nucleotide sugars.

Root-knot nematode chemotaxis is positively regulated by l-galactose sidechains of mucilage carbohydrate rhamnogalacturonan-I.

Root-knot nematodes (RKNs) are plant parasites and major agricultural pests. RKNs are thought to locate hosts through chemotaxis by sensing host-secreted chemoattractants; however, the structures and properties of these attractants are not well understood. Here, we describe a previously unknown RKN attractant from flaxseed mucilage that enhances infection of Arabidopsis and tomato, which resembles the pectic polysaccharide rhamnogalacturonan-I (RG-I). Fucose and galactose sidechains of the purified attractant were found to be required for attractant activity. Furthermore, the disaccharide α-l-galactosyl-1,3-l-rhamnose, which forms the linkage between the RG-I backbone and galactose sidechains of the purified attractant, was sufficient to attract RKN. These results show that the α-l-galactosyl-1,3-l-rhamnose linkage in the purified attractant from flaxseed mucilage is essential for RKN attraction. The present work also suggests that nematodes can detect environmental chemicals with high specificity, such as the presence of chiral centers and hydroxyl groups.

Unique active-site and subsite features in the arabinogalactan-degrading GH43 exo-ß-1,3-galactanase from Phanerochaete chrysosporium.

Arabinogalactan proteins (AGPs) are plant proteoglycans with functions in growth and development. However, these functions are largely unexplored, mainly because of the complexity of the sugar moieties. These carbohydrate sequences are generally analyzed with the aid of glycoside hydrolases. The exo-ß-1,3-galactanase is a glycoside hydrolase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. However, its structure is not known in relation to its mechanism bypassing side chains. In this study, we solved the apo and liganded structures of Pc1,3Gal43A, which reveal a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family 35 (CBM35) binding domain. GH43_sub24 is known to lack the catalytic base Asp conserved among other GH43 subfamilies. Our structure in combination with kinetic analyses reveals that the tautomerized imidic acid group of Gln263 serves as the catalytic base residue instead. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite -1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the ß-1,6-linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s, including those that bind galactomannan. Specifically, we noted a Gly → Trp substitution, which affects pyranose stacking, and an Asp → Asn substitution in the binding pocket, which recognizes ß-linked rather than α-linked Gal residues. These findings should facilitate further structural analysis of AGPs and may also be helpful in engineering designer enzymes for efficient biomass utilization.

Expression of a fungal exo-ß-1,3-galactanase in Arabidopsis reveals a role of type II arabinogalactans in the regulation of cell shape.

Arabinogalactan-proteins (AGPs) are a family of plant extracellular proteoglycans implicated in many physiological events. AGP is decorated with type II arabinogalactans (AGs) consisting of a ß-1,3-galactan backbone and ß-1,6-galactan side chains, to which other sugars are attached. Based on the fact that a type II AG-specific inhibitor, ß-Yariv reagent, perturbs growth and development, it has been proposed that type II AGs participate in the regulation of cell shape and tissue organization. However, the mechanisms by which type II AGs participate have not yet been established. Here, we describe a novel system that causes specific degradation of type II AGs in Arabidopsis, by which a gene encoding a fungal exo-ß-1,3-galactanase that specifically hydrolyzes ß-1,3-galactan backbones of type II AGs is expressed under the control of a dexamethasone-inducible promoter. Dexamethasone treatment increased the galactanase activity, leading to a decrease in Yariv reagent-reactive AGPs in transgenic Arabidopsis. We detected the typical oligosaccharides released from type II AGs by Il3GAL in the soluble fraction, demonstrating that Il3GAL acted on type II AG in the transgenic plants. Additionally, this resulted in severe tissue disorganization in the hypocotyl and cotyledons, suggesting that the degradation of type II AGs affected the regulation of cell shape.

Structural features conserved in subclass of type II arabinogalactan.

Arabinogalactan-proteins (AGPs) are extracellular proteoglycans, which are presumed to participate in the regulation of cell shape, thus contributing to the excellent mechanical properties of plants. AGPs consist of a hydroxyproline-rich core-protein and large arabinogalactan (AG) sugar chains, called type II AGs. These AGs have a ß-1,3-galactan backbone and ß-1,6-galactan side chains, to which other sugars are attached. The structure of type II AG differs depending on source plant, tissue, and age. Type II AGs obtained from woody plants in large quantity as represented by gum arabic and larch AG, here designated gum arabic-subclass, have a ß-1,3;1,6-galactan structure in which the ß-1,3-galactan backbone is highly substituted with short ß-1,6-galactan side chains. On the other hand, it is unclear whether type II AGs found as the glycan part of AGPs from herbaceous plants, here designated AGP-subclass, also have conserved ß-1,3:1,6-galactan structural features. In the present study we explore similarities of type II AG structures in the AGP-subclass. Type II AGs in fractions obtained from spinach, broccoli, bok choy, komatsuna, and cucumber were hydrolyzed into galactose and ß-1,6-galactooligosaccharides by specific enzymes. Based on the proportion of these sugars, the substitution ratio of the ß-1,3-galactan backbone was calculated as 46-58% in the five vegetables, which is consistently lower than what is seen in gum arabic and larch AG. Although most side chains were short, long chains such as ß-1,6-galactohexaose chains were also observed in these vegetables. The results suggest a conserved ß-1,3;1,6-galactan structure in the AGP-subclass that distinguishes it from the gum arabic-subclass.

Properties of arabinogalactan-proteins in European pear (Pyrus communis L.) fruits.

Arabinogalactans (AGs) and arabinogalactan-proteins (AGPs) were partially purified from an extract of fruits of the European pear (Pyrus communis L.) by DEAE-cellulose ion-exchange and Sepharose 6B gel-filtration chromatography. Among 7 AG(P)-containing fractions, a neutral AGP (SE-1) was confirmed to be highly purified (Mr 67,000) and rich in L-Ara and Gal; this fraction included a small amount (2.6%, w/w) of protein and showed the highest reactivity forming precipitate with ß-Glc Yariv reagent among the 7 fractions, the intensity of which was comparable to that of gum arabic, a standard AGP. Another accompanying minor low-Mr neutral AGP (SE-2; Mr approx. 7200) still contained other polysaccharide (starch fragments) and did not show Yariv reactivity. The carbohydrate moieties of SE-1 consisted of consecutive (1 → 3)-linked ß-galactosyl backbone chains substituted with side chains of (1 → 6)-linked ß-galactosyl residues at O-6, to which mainly single α-l-arabinofuranosyl residues were attached through O-3. This structural feature was also observed for SE-2. Successive digestion of SE-1 with α-l-arabinofuranosidase and exo-ß-(1 → 3)-galactanase with the aid of endo-ß-(1 → 3)-galactanase released most (more than 98%, w/w) of the carbohydrate moieties as low-Mr fragments. These consisted of free L-Ara and Gal, and a series of ß-(1 → 6)-galactooligosaccharides with degree of polymerization (dp) up to at least 17, indicative of attachment of (1 → 6)-linked ß-galactosyl side chains of varying length along the (1 → 3)-linked ß-galactosyl backbone chains.

Degradative enzymes for type II arabinogalactan side chains in Bifidobacterium longum subsp. longum.

Type II arabinogalactan (AG) is a soluble prebiotic fiber stimulating the proliferation of bifidobacteria in the human gut. Larch AG, which is comprised of type II AG, is known to be utilized as an energy source for Bifidobacterium longum subsp. longum (B. longum). We have previously characterized GH43_24 exo-ß-1,3-galactanase (Bl1,3Gal) for the degradation of type II AG main chains in B. longum JCM1217. In this study, we characterized GH30_5 exo-ß-1,6-galactobiohydrolase (Bl1,6Gal) and GH43_22 α-L-arabinofuranosidase (BlArafA), which are degradative enzymes for type II AG side chains in cooperation with exo-ß-1,3-galactanase. The recombinant exo-ß-1,6-galactobiohydrolase specifically released ß-1,6-galactobiose (ß-1,6-Gal2) from the nonreducing terminal of ß-1,6-galactooligosaccharides, and the recombinant α-L-arabinofuranosidase released arabinofuranose (Araf) from α-1,3-Araf-substituted ß-1,6-galactooligosaccharides. ß-1,6-Gal2 was additively released from larch AG by the combined use of type II AG degradative enzymes, including Bl1,3Gal, Bl1,6Gal, and BlArafA. The gene cluster encoding the type II AG degradative enzymes is conserved in all B. longum strains, but not in other bifidobacterial species. The degradative enzymes for type II AG side chains are thought to be important for the acquisition of type II AG in B. longum.

Yariv reactivity of type II arabinogalactan from larch wood.

Larch arabinogalactan (AG) is classified as a plant type II AG. Its basic structure is constituted by a ß-1,3-galactan main chain with ß-1,6-galactan side chains. But its properties are distinct from other type II AGs. Whereas most type II AGs are found as glycan moieties of arabinogalactan-protein (AGP), larch AG lacks a protein moiety. Larch AG itself is also unlike other type II AGs as it lacks Yariv reactivity, the capability of AG to form insoluble precipitate with ß-Yariv reagents, 1,3,5-tri-(p-glycosyloxyphenylazo)-2,4,6-trihydroxybenzene with ß-glucosyl or ß-galactosyl residues at the termini. For the present study, we prepared ß-galactan I, II, and III from larch AG by performing single, double, and triple Smith degradation, which breaks ß-1,6-galactan side chains, and examined Yariv reactivity of the products. Methylation analysis revealed that ß-galactans II and III had lost more than 90% of their ß-1,6-galactan branches. In the radial gel diffusion assay, ß-galactans II and III showed Yariv reactivity, indicating the presence of a Yariv-reactive structure in larch AG, although native larch AG does not have Yariv reactivity. The Yariv reactivity of the ß-galactans was completely lost after treatment with endo-ß-1,3-galactanase. These results confirm that ß-1,3-galactan is necessary for Yariv reactivity of type II AG. The present results also suggest that high substitution of ß-1,3-galactan with ß-1,6-galactan side chains affects Yariv reactivity in larch AG.

Correction to: Metabolism of L-arabinose in plants.

The article "Metabolism of L-arabinose in plants", written by "Toshihisa Kotake, Yukiko Yamanashi, Chiemi Imaizumi, Yoichi Tsumuraya", was originally published Online First without open access. After publication in volume129, issue 5, page 781-792 the Botanical Society of Japan decided to opt for Open Choice and to make the article an open access publication.

Properties of two fungal endo-ß-1,3-galactanases and their synergistic action with an exo-ß-1,3-galactanase in degrading arabinogalactan-proteins.

Arabinogalactan-proteins (AGPs) are plant proteoglycans, which are widely encountered in the plant kingdom, usually localized on the cell surface. The carbohydrate moieties of AGPs consist of ß-1,3-galactan main chains and ß-1,6-galactan side chains, to which other auxiliary sugars are attached. To date, FvEn3GAL isolated from Flammulina velutipes is the sole ß-1,3-galactanase acting on ß-1,3-galactan in an endo-manner. Here we cloned two homologous genes, designated Af3G and NcEn3GAL, possibly encoding endo-ß-1,3-galactanase from Aspergillus flavus and Neurospora crassa, respectively. The recombinant Af3G (rAf3G) and rNcEn3GAL expressed in Pichia pastoris specifically hydrolyzed ß-1,3-galactan in an endo-manner, as did the rFvEn3GAL. Among galactooligosaccharides, ß-1,3-galactotriose was identified as the smallest substrate for these enzymes. These results suggest that enzymatic characteristics are conserved in many endo-ß-1,3-galactanases belonging to the glycoside hydrolase 16 family. On the other hand, rAf3G and rNcEn3GAL generated more ß-1,3-galactobiose from ß-1,3-galactotetraose than did rFvEn3GAL, suggesting that rAf3G and rNcEn3GAL prefer hydrolyzing the central ß-1,3-glycosidic linkage of three in ß-1,3-galactotetraose. Although rAf3G and rNcEn3GAL alone hardly hydrolyze native AGP, they acted synergistically with a fungal exo-ß-1,3-galactanase on the AGP. These endo-ß-1,3-galactanases presumably aid hydrolysis by internally breaking up AGPs, which creates more sites of attack for exo-ß-1,3-galactanase.

Heterologous expression and characterization of an Arabidopsis ß-l-arabinopyranosidase and α-d-galactosidases acting on ß-l-arabinopyranosyl residues.

The major plant sugar l-arabinose (l-Ara) has two different ring forms, l-arabinofuranose (l-Araf) and l-arabinopyranose (l-Arap). Although l-Ara mainly appears in the form of α-l-Araf residues in cell wall components, such as pectic α-1,3:1,5-arabinan, arabinoxylan, and arabinogalactan-proteins (AGPs), lesser amounts of it can also be found as ß-l-Arap residues of AGPs. Even though AGPs are known to be rapidly metabolized, the enzymes acting on the ß-l-Arap residues remain to be identified. In the present study, four enzymes, which we call ß-l-ARAPASE (APSE) and α-GALACTOSIDASE 1 (AGAL1), AGAL2, and AGAL3, are identified as those enzymes that are likely to be responsible for the hydrolysis of the ß-l-Arap residues in Arabidopsis thaliana. An Arabidopsis apse-1 mutant showed significant reduction in ß-l-arabinopyranosidase activity, and an apse-1 agal3-1 double-mutant exhibited even less activity. The apse-1 and the double-mutants both had more ß-l-Arap residues in the cell walls than wild-type plants. Recombinant APSE expressed in the yeast Pichia pastoris specifically hydrolyzed ß-l-Arap residues and released l-Ara from gum arabic and larch arabinogalactan. The recombinant AGAL3 also showed weak ß-l-arabinopyranosidase activity beside its strong α-galactosidase activity. It appears that the ß-l-Arap residues of AGPs are hydrolysed mainly by APSE and partially by AGALs in Arabidopsis.

A protease/peptidase from culture medium of Flammulina velutipes that acts on arabinogalactan-protein.

Arabinogalactan-proteins (AGPs) are highly diverse plant proteoglycans found on the plant cell surface. AGPs have large arabinogalactan (AG) moieties attached to a core-protein rich in hydroxyproline (Hyp). The AG undergoes hydrolysis by various glycoside hydrolases, most of which have been identified, whereas the core-proteins is presumably degraded by unknown proteases/peptidases secreted from fungi and bacteria in nature. Although several enzymes hydrolyzing other Hyp-rich proteins are known, the enzymes acting on the core-proteins of AGPs remain to be identified. The present study describes the detection of protease/peptidase activity toward AGP core-proteins in the culture medium of winter mushroom (Flammulina velutipes) and partial purification of the enzyme by several conventional chromatography steps. The enzyme showed higher activity toward Hyp residues than toward proline and alanine residues and acted on core-proteins prepared from gum arabic. Since the activity was inhibited in the presence of Pefabloc SC, the enzyme is probably a serine protease.

Metabolism of L-arabinose in plants.

L-Arabinose (L-Ara) is a plant-specific sugar accounting for 5-10 % of cell wall saccharides in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). L-Ara occurs in pectic arabinan, rhamnogalacturonan II, arabinoxylan, arabinogalactan-protein (AGP), and extensin in the cell walls, as well as in glycosylated signaling peptides like CLAVATA3 and small glycoconjugates such as quercetin 3-O-arabinoside. This review focuses on recent advances towards understanding the generation of L-Ara and the metabolism of L-Ara-containing molecules in plants.

The AMOR Arabinogalactan Sugar Chain Induces Pollen-Tube Competency to Respond to Ovular Guidance.

Precise directional control of pollen-tube growth by pistil tissue is critical for successful fertilization of flowering plants [1-3]. Ovular attractant peptides, which are secreted from two synergid cells on the side of the egg cell, have been identified [4-6]. Emerging evidence suggests that the ovular directional cue is not sufficient for successful guidance but that competency control by the pistil is critical for the response of pollen tubes to the attraction signal [1, 3, 7]. However, the female molecule for this competency induction has not been reported. Here we report that ovular methyl-glucuronosyl arabinogalactan (AMOR) induces competency of the pollen tube to respond to ovular attractant LURE peptides in Torenia fournieri. We developed a method for assaying the response capability of a pollen tube by micromanipulating an ovule. Using this method, we showed that pollen tubes growing through a cut style acquired a response capability in the medium by receiving a sufficient amount of a factor derived from mature ovules of Torenia. This factor, named AMOR, was identified as an arabinogalactan polysaccharide, the terminal 4-O-methyl-glucuronosyl residue of which was necessary for its activity. Moreover, a chemically synthesized disaccharide, the ß isomer of methyl-glucuronosyl galactose (4-Me-GlcA-ß-(1→6)-Gal), showed AMOR activity. No specific sugar-chain structure of plant extracellular matrix has been identified as a bioactive molecule involved in intercellular communication. We suggest that the AMOR sugar chain in the ovary renders the pollen tube competent to the chemotropic response prior to final guidance by LURE peptides.

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis.

Humans are unable to synthesize l-ascorbic acid (AsA), yet it is required as a cofactor in many critical biochemical reactions. The majority of human dietary AsA is obtained from plants. In Arabidopsis thaliana, a GDP-mannose pyrophosphorylase (GMPP), VITAMIN C DEFECTIVE1 (VTC1), catalyzes a rate-limiting step in AsA synthesis: the formation of GDP-Man. In this study, we identified two nucleotide sugar pyrophosphorylase-like proteins, KONJAC1 (KJC1) and KJC2, which stimulate the activity of VTC1. The kjc1kjc2 double mutant exhibited severe dwarfism, indicating that KJC proteins are important for growth and development. The kjc1 mutation reduced GMPP activity to 10% of wild-type levels, leading to a 60% reduction in AsA levels. On the contrary, overexpression of KJC1 significantly increased GMPP activity. The kjc1 and kjc1kjc2 mutants also exhibited significantly reduced levels of glucomannan, which is also synthesized from GDP-Man. Recombinant KJC1 and KJC2 enhanced the GMPP activity of recombinant VTC1 in vitro, while KJCs did not show GMPP activity. Yeast two-hybrid assays suggested that the stimulation of GMPP activity occurs via interaction of KJCs with VTC1. These results suggest that KJCs are key factors for the generation of GDP-Man and affect AsA level and glucomannan accumulation through the stimulation of VTC1 GMPP activity.

L-Fucose-containing arabinogalactan-protein in radish leaves.

The carbohydrate moieties of arabinogalactan-proteins (AGPs) have ß-(1 → 3)-galactan backbones to which side chains of (1 → 6)-linked ß-Gal residues are attached through O-6. Some of these side chains are further substituted with other sugars. We investigated the structure of L-Fuc-containing oligosaccharides released from the carbohydrate moieties of a radish leaf AGP by digestion with α-L-arabinofuranosidase, followed by exo-ß-(1 → 3)-galactanase. We detected a series of neutral ß-(1 → 6)-galactooligosaccharides branching variously at O-3 of the Gal residues, together with corresponding acidic derivatives terminating in 4-O-methyl-GlcA (4-Me-GlcA) or GlcA at the non-reducing terminals. In neutral oligosaccharides with degree of polymerization (dp) mainly higher than 10, L-Fuc groups were attached through L-Ara residues as the sequence, α-L-Fucp-(1 → 2)-α-L-Araf-(1 →. This sequence was verified by isolation of the pentasaccharide α-L-Fuc-(1 → 2)-α-L-Araf-(1 → 3)-ß-Gal-(1 → 6)-ß-Gal-(1 → 6)-Gal upon digestion of the higher oligosaccharides with endo-ß-(1 → 6)-galactanase. By contrast, in lower polymerized (predominantly dp 4) acidic oligosaccharides, L-Fuc groups were attached directly at the non-reducing terminals through α-(1 → 2)-linkages, resulting in the release of the tetrasaccharides, α-L-Fucp-(1 → 2)-ß-GlcA-(1 → 6)-ß-Gal-(1 → 6)-Gal and α-L-Fucp-(1 → 2)-ß-4-Me-GlcA-(1 → 6)-ß-Gal-(1 → 6)-Gal. In long acidic oligosaccharides with dp mainly higher than 13, L-Fuc groups localized on branches were attached to the uronic acids directly and/or L-Ara residues as in the neutral oligosaccharides.

Action of an endo-ß-1,3(4)-glucanase on cellobiosyl unit structure in barley ß-1,3:1,4-glucan.

ß-1,3:1,4-Glucan is a major cell wall component accumulating in endosperm and young tissues in grasses. The mixed linkage glucan is a linear polysaccharide mainly consisting of cellotriosyl and cellotetraosyl units linked through single ß-1,3-glucosidic linkages, but it also contains minor structures such as cellobiosyl units. In this study, we examined the action of an endo-ß-1,3(4)-glucanase from Trichoderma sp. on a minor structure in barley ß-1,3:1,4-glucan. To find the minor structure on which the endo-ß-1,3(4)-glucanase acts, we prepared oligosaccharides from barley ß-1,3:1,4-glucan by endo-ß-1,4-glucanase digestion followed by purification by gel permeation and paper chromatography. The endo-ß-1,3(4)-glucanase appeared to hydrolyze an oligosaccharide with degree of polymerization 5, designated C5-b. Based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF)/ToF-mass spectrometry (MS)/MS analysis, C5-b was identified as ß-Glc-1,3-ß-Glc-1,4-ß-Glc-1,3-ß-Glc-1,4-Glc including a cellobiosyl unit. The results indicate that a type of endo-ß-1,3(4)-glucanase acts on the cellobiosyl units of barley ß-1,3:1,4-glucan in an endo-manner.

Enzymatic activity and substrate specificity of the recombinant tomato ß-galactosidase 1.

The open reading frame of tomato ß-galactosidase 1 was expressed in yeast, and the enzymatic properties and substrate specificity were investigated. The enzyme had peak activity at pH 5.0 and 40-50°C. TBG1 was active on ß-(1,3)- and ß-(1,6)-galactobiose and lactose. TBG1 released galactose from lupin galactan, tomato fruit alkali soluble pectin, arabinogalactan, gum arabic and methyl ß-(1,6)-galactohexaoside, but not from labeled ß-(1,4)-galactoheptaose. TBG1 was assessed for its ability to degrade three galactosyl-containing cell wall fractions purified from different development and ripening stages of tomato fruit. TBG1 released galactose from all of the fractions from all of the stages tested. TBG1 activity was highest on the hemicellulose fraction at the 10 and 20d after pollination stage. This result is not correlated the with TBG1 expression pattern. TBG1 might act on a small but specific set of polysaccharide containing galactose.

Enzymatic fragmentation of carbohydrate moieties of radish arabinogalactan-protein and elucidation of the structures.

We investigated the structures of L-arabino-galactooligosaccharides released from the sugar moieties of a radish arabinogalactan-protein (AGP) by the action of exo-ß-(1→3)-galactanase. We detected a series of neutral ß-(1 → 6)-linked galactooligosaccharides forming branches of one to up to at least 19 consecutive Gal groups, together with corresponding acidic derivatives terminating in 4-O-methyl-glucuronic acid (4-Me-GlcA) at the non-reducing end. Some oligosaccharide chains of degree of polymerization (dp) higher than 3 for neutral, and 4 for acidic oligomers were modified with L-Araf residues. The acidic tetrasaccharide 4-Me-ß-GlcA-(1 → 6)[α-L-Araf-(1 → 3)]-ß-Gal-(1 → 6)-Gal was detected as an abundant L-Araf-containing oligosaccharide among these neutral and acidic oligomers. A pentasaccharide containing an additional L-Araf group attached to the L-Ara in the tetrasaccharide through an α-(1 → 5)-linkage was also found. We observed L-arabino-galactooligosaccharides substituted with single or disaccharide L-Araf units at different Gal residues along these neutral and acidic ß-(1 → 6)-galactooligosaccharide chains, indicating that these side chains are highly variable in length and substituted variously with L-Araf residues.

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

We have characterized a ß-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 ß-1,6-galactooligosaccharides with degrees of polymerization (DP) ranging from 3-11, and to ß-1,3-galactooligosaccharides of DP5 and 7, indicating that the enzyme is a glucuronosyltransferase that modifies both the ß-1,6- and ß-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-ß-1,6-Gal and ß-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 ß-1,3- and ß-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.