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.

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.

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.

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.

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.

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.

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.

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.

A galactosyltransferase acting on arabinogalactan protein glycans is essential for embryo development in Arabidopsis.

Arabinogalactan proteins (AGPs) are a complex family of cell-wall proteoglycans that are thought to play major roles in plant growth and development. Genetic approaches to studying AGP function have met limited success so far, presumably due to redundancy within the large gene families encoding AGP backbones. Here we used an alternative approach for genetic dissection of the role of AGPs in development by modifying their glycan side chains. We have identified an Arabidopsis glycosyltransferase of CAZY family GT31 (AtGALT31A) that galactosylates AGP side chains. A mutation in the AtGALT31A gene caused the arrest of embryo development at the globular stage. The presence of the transcript in the suspensor of globular-stage embryos is consistent with a role for AtGALT31A in progression of embryo development beyond the globular stage. The first observable defect in the mutant is perturbation of the formative asymmetric division of the hypophysis, indicating an essential role for AGP proteoglycans in either specification of the hypophysis or orientation of the asymmetric division plane.

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.

ß-galactosyl Yariv reagent binds to the ß-1,3-galactan of arabinogalactan proteins.

Yariv phenylglycosides [1,3,5-tri(p-glycosyloxyphenylazo)-2,4,6-trihydroxybenzene] are a group of chemical compounds that selectively bind to arabinogalactan proteins (AGPs), a type of plant proteoglycan. Yariv phenylglycosides are widely used as cytochemical reagents to perturb the molecular functions of AGPs as well as for the detection, quantification, purification, and staining of AGPs. However, the target structure in AGPs to which Yariv phenylglycosides bind has not been determined. Here, we identify the structural element of AGPs required for the interaction with Yariv phenylglycosides by stepwise trimming of the arabinogalactan moieties using combinations of specific glycoside hydrolases. Whereas the precipitation with Yariv phenylglycosides (Yariv reactivity) of radish (Raphanus sativus) root AGP was not reduced after enzyme treatment to remove α-l-arabinofuranosyl and ß-glucuronosyl residues and ß-1,6-galactan side chains, it was completely lost after degradation of the ß-1,3-galactan main chains. In addition, Yariv reactivity of gum arabic, a commercial product of acacia (Acacia senegal) AGPs, increased rather than decreased during the repeated degradation of ß-1,6-galactan side chains by Smith degradation. Among various oligosaccharides corresponding to partial structures of AGPs, ß-1,3-galactooligosaccharides longer than ß-1,3-galactoheptaose exhibited significant precipitation with Yariv in a radial diffusion assay on agar. A pull-down assay using oligosaccharides cross linked to hydrazine beads detected an interaction of ß-1,3-galactooligosaccharides longer than ß-1,3-galactopentaose with Yariv phenylglycoside. To the contrary, no interaction with Yariv was detected for ß-1,6-galactooligosaccharides of any length. Therefore, we conclude that Yariv phenylglycosides should be considered specific binding reagents for ß-1,3-galactan chains longer than five residues, and seven residues are sufficient for cross linking, leading to precipitation of the Yariv phenylglycosides.

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.

Structural and biochemical characterization of glycoside hydrolase family 79 ß-glucuronidase from Acidobacterium capsulatum.

We present the first structure of a glycoside hydrolase family 79 ß-glucuronidase from Acidobacterium capsulatum, both as a product complex with ß-D-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (ß/α)(8)-barrel domain and a ß-domain with irregular Greek key motifs that is of unknown function. The enzyme showed ß-glucuronidase activity and trace levels of ß-glucosidase and ß-xylosidase activities. In conjunction with mutagenesis studies, these structures identify the catalytic residues as Glu(173) (acid base) and Glu(287) (nucleophile), consistent with the retaining mechanism demonstrated by (1)H NMR analysis. Glu(45), Tyr(243), Tyr(292)-Gly(294), and Tyr(334) form the catalytic pocket and provide substrate discrimination. Consistent with this, the Y292A mutation, which affects the interaction between the main chains of Gln(293) and Gly(294) and the GlcA carboxyl group, resulted in significant loss of ß-glucuronidase activity while retaining the side activities at wild-type levels. Likewise, although the ß-glucuronidase activity of the Y334F mutant is ~200-fold lower (k(cat)/K(m)) than that of the wild-type enzyme, the ß-glucosidase activity is actually 3 times higher and the ß-xylosidase activity is only 2.5-fold lower than the equivalent parameters for wild type, consistent with a role for Tyr(334) in recognition of the C6 position of GlcA. The involvement of Glu(45) in discriminating against binding of the O-methyl group at the C4 position of GlcA is revealed in the fact that the E45D mutant hydrolyzes PNP-ß-GlcA approximately 300-fold slower (k(cat)/K(m)) than does the wild-type enzyme, whereas 4-O-methyl-GlcA-containing oligosaccharides are hydrolyzed only 7-fold slower.

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

A membrane fraction from etiolated 6-day-old primary radish roots (Raphanus sativus L. var hortensis) contained ß-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 ß-(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 ß-Gal chains as well as single branched ß-(1 → 6)-Gal residues through ß-(1 → 6) linkages, producing branched acidic side chains. The enzymes also transferred [(14)C]GlcA residues on to several oligosaccharides, such as ß-(1 → 6)- and ß-(1 → 3)-galactotrioses. A trisaccharide, α-L-Araf-(1 → 3)-ß-Gal-(1 → 6)-Gal, was a good acceptor, yielding a branched tetrasaccharide, α-L-Araf-(1 → 3)[ß-GlcA-(1 → 6)]-ß-Gal-(1 → 6)-Gal. We report the first in vitro assay system for ß-GlcATs involved in the AG synthesis as a step toward full characterization and cloning.

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 ß-(1→3)-galactan backbone with ß-(1→6)-d-galactan side chains. The ß-(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 ß-(1→6)-galactan side chain oligosaccharides are substituted at the nonreducing termini with single 4-O-methyl-glucuronosyl residues via ß-(1→6)-linkages. The ß-(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.

Endo-beta-1,3-galactanase from winter mushroom Flammulina velutipes.

Arabinogalactan proteins are proteoglycans found on the cell surface and in the cell walls of higher plants. The carbohydrate moieties of most arabinogalactan proteins are composed of ß-1,3-galactan main chains and ß-1,6-galactan side chains, to which other auxiliary sugars are attached. For the present study, an endo-ß-1,3-galactanase, designated FvEn3GAL, was first purified and cloned from winter mushroom Flammulina velutipes. The enzyme specifically hydrolyzed ß-1,3-galactan, but did not act on ß-1,3-glucan, ß-1,3:1,4-glucan, xyloglucan, and agarose. It released various ß-1,3-galactooligosaccharides together with Gal from ß-1,3-galactohexaose in the early phase of the reaction, demonstrating that it acts on ß-1,3-galactan in an endo-fashion. Phylogenetic analysis revealed that FvEn3GAL is member of a novel subgroup distinct from known glycoside hydrolases such as endo-ß-1,3-glucanase and endo-ß-1,3:1,4-glucanase in glycoside hydrolase family 16. Point mutations replacing the putative catalytic Glu residues conserved for enzymes in this family with Asp abolished activity. These results indicate that FvEn3GAL is a highly specific glycoside hydrolase 16 endo-ß-1,3-galactanase.

Functional characterization of barley betaglucanless mutants demonstrates a unique role for CslF6 in (1,3;1,4)-ß-D-glucan biosynthesis.

(1,3;1,4)-ß-D-glucans (mixed-linkage glucans) are found in tissues of members of the Poaceae (grasses), and are particularly high in barley (Hordeum vulgare) grains. The present study describes the isolation of three independent (1,3;1,4)-ß-D-glucanless (betaglucanless; bgl) mutants of barley which completely lack (1,3;1,4)-ß-D-glucan in all the tissues tested. The bgl phenotype cosegregates with the cellulose synthase like HvCslF6 gene on chromosome arm 7HL. Each of the bgl mutants has a single nucleotide substitution in the coding region of the HvCslF6 gene resulting in a change of a highly conserved amino acid residue of the HvCslF6 protein. Microsomal membranes isolated from developing endosperm of the bgl mutants lack detectable (1,3;1,4)-ß-D-glucan synthase activity indicating that the HvCslF6 protein is inactive. This was confirmed by transient expression of the HvCslF6 cDNAs in Nicotiana benthamiana leaves. The wild-type HvCslF6 gene directed the synthesis of high levels of (1,3;1,4)-ß-D-glucans, whereas the mutant HvCslF6 proteins completely lack the ability to synthesize (1,3;1,4)-ß-D-glucans. The fine structure of the (1,3;1,4)-ß-D-glucan produced in the tobacco leaf was also very different from that found in cereals having an extremely low DP3/DP4 ratio. These results demonstrate that, among the seven CslF and one CslH genes present in the barley genome, HvCslF6 has a unique role and is the key determinant controlling the biosynthesis of (1,3;1,4)-ß-D-glucans. Natural allelic variation in the HvCslF6 gene was found predominantly within introns among 29 barley accessions studied. Genetic manipulation of the HvCslF6 gene could enable control of (1,3;1,4)-ß-D-glucans in accordance with the purposes of use.

Rice Brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls.

The brittle culm (bc) mutants of Gramineae plants having brittle skeletal structures are valuable materials for studying secondary cell walls. In contrast to other recessive bc mutants, rice Bc6 is a semi-dominant bc mutant with easily breakable plant bodies. In this study, the Bc6 gene was cloned by positional cloning. Bc6 encodes a cellulose synthase catalytic subunit, OsCesA9, and has a missense mutation in its highly conserved region. In culms of the Bc6 mutant, the proportion of cellulose was reduced by 38%, while that of hemicellulose was increased by 34%. Introduction of the semi-dominant Bc6 mutant gene into wild-type rice significantly reduced the percentage of cellulose, causing brittle phenotypes. Transmission electron microscopy analysis revealed that Bc6 mutation reduced the cell wall thickness of sclerenchymal cells in culms. In rice expressing a reporter construct, BC6 promoter activity was detected in the culms, nodes, and flowers, and was localized primarily in xylem tissues. This expression pattern was highly similar to that of BC1, which encodes a COBRA-like protein involved in cellulose synthesis in secondary cell walls in rice. These results indicate that BC6 is a secondary cell wall-specific CesA that plays an important role in proper deposition of cellulose in the secondary cell walls.