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

The carbohydrate moieties of arabinogalactan-proteins (AGPs) have β-(1 → 3)-galactan backbones to which side chains of (1 → 6)-linked β-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-β-(1 → 3)-galactanase. We detected a series of neutral β-(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)-β-Gal-(1 → 6)-β-Gal-(1 → 6)-Gal upon digestion of the higher oligosaccharides with endo-β-(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)-β-GlcA-(1 → 6)-β-Gal-(1 → 6)-Gal and α-L-Fucp-(1 → 2)-β-4-Me-GlcA-(1 → 6)-β-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.The authors would like to thank Prof. M. Hisamatsu, Mie University, Tsu, Japan, for a gift of cyclic β-(1→2)-glucan. This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grant-in-Aid for Scientific Research no. 23570048 to Y.T. and no. 24114006 to Y.T. and T.K.). Support was also provided by BBSRC Sustainable Bioenergy Centre: Cell wall sugars program (Grant No. BB/G016240/1) to P.D.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.carres.2015.07.00

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

β-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 β-1,3-glucosidic linkages, but it also contains minor structures such as cellobiosyl units. In this study, we examined the action of an endo-β-1,3(4)-glucanase from Trichoderma sp. on a minor structure in barley β-1,3:1,4-glucan. To find the minor structure on which the endo-β-1,3(4)-glucanase acts, we prepared oligosaccharides from barley β-1,3:1,4-glucan by endo-β-1,4-glucanase digestion followed by purification by gel permeation and paper chromatography. The endo-β-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 β-Glc-1,3-β-Glc-1,4-β-Glc-1,3-β-Glc-1,4-Glc including a cellobiosyl unit. The results indicate that a type of endo-β-1,3(4)-glucanase acts on the cellobiosyl units of barley β-1,3:1,4-glucan in an endo-manner.This work was supported in part by a grant-in-aid for Scientific Research to T. Kotake [Grant-in-Aid for Scientific Research no. 25514001] from Japan Society of the Promotion of Science; Y. Tsumuraya and T. Kotake [Grant-in-Aid for Scientific Research no. 24114006] from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Supports were also provided by BBSRC Sustainable Bioenergy Centre: Cell wall sugars program to P. Dupree [grant number BB/G016240/1].This is the final version of the article. It first appeared from Taylor & Francis via http://dx.doi.org/10.1080/09168451.2015.104636

Group 3 sigma factor gene, sigJ, a key regulator of desiccation tolerance, regulates the synthesis of extracellular polysaccharide in cyanobacterium Anabaena sp. strain PCC 7120

The changes in the expression of sigma factor genes during dehydration in terrestrial Nostoc HK-01 and aquatic Anabaena PCC 7120 were determined. The expression of the sigJ gene in terrestrial Nostoc HK-01, which is homologous to sigJ (alr0277) in aquatic Anabaena PCC 7120, was significantly induced in the mid-stage of dehydration. We constructed a higher-expressing transformant of the sigJ gene (HE0277) in Anabaena PCC 7120, and the transformant acquired desiccation tolerance. The results of Anabaena oligonucleotide microarray experiments showed that a comparatively large number of genes relating to polysaccharide biosynthesis were upregulated in the HE0277 cells. The extracellular polysaccharide released into the culture medium of the HE0277 cells was as much as 3.2-fold more than that released by the control cells. This strongly suggests that the group 3 sigma factor gene sigJ is fundamental and conducive to desiccation tolerance in these cyanobacteria

Rice BRITTLE CULM 5 (BRITTLE NODE) is Involved in Secondary Cell Wall Formation in the Sclerenchyma Tissue of Nodes

Several brittle culm (bc) mutants known in grasses are considered excellent materials to study the process of secondary cell wall formation. The brittle phenotype of the rice bc5 (brittle node) mutant appears exclusively in the developed nodes, which is distinct from other bc mutants (bc1, 2, 3, 4, 6 and 7) that show the brittle phenotype in culms and leaves. To address the defects of the rice bc5 mutant in node-specific cell wall formation, we analyzed tissue morphology and cell wall composition. The bc5 mutation was found to affect the cell wall deposition of node sclerenchyma tissues at 1 week after heading, the stage at which the cell wall sugar content is reduced, in the bc5 nodes, compared with wild-type nodes. Moreover, decreased accumulation of lignin and thickness of cell walls in the sclerenchyma tissues were also observed in the bc5 nodes. The amounts of cellulose and hemicellulose were reduced to 53 and 65% of those in the wild-type plants, respectively. Sugar composition and glycosidic linkage analyses of the hemicellulose showed that the accumulation of glucuronosyl arabinoxylan in bc5 nodes was perturbed by the mutation. The bc5 locus was narrowed to an approximately 3.1 Mb region of chromosome 2, where none of the other bc genes is located. The bc5 mutation appeared to reduce the expression levels of the OsCesA genes in the nodes after heading. The results indicate that the BC5 gene regulates the development of secondary cell walls of node sclerenchyma tissues

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

(1,3;1,4)-β-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)-β-D-glucanless (betaglucanless; bgl) mutants of barley which completely lack (1,3;1,4)-β-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)-β-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)-β-D-glucans, whereas the mutant HvCslF6 proteins completely lack the ability to synthesize (1,3;1,4)-β-D-glucans. The fine structure of the (1,3;1,4)-β-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)-β-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)-β-D-glucans in accordance with the purposes of use

A 110-kDa WGA-binding Glycoprotein Involved in Cell Adhesion Acts as a Receptor for Aggregation Factor in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus(Developmental Biology)

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Characterization of an Endo-β-1,6-Galactanase from Streptomyces avermitilis NBRC14893▿

The putative endo-β-1,6-galactanase gene from Streptomyces avermitilis was cloned and expressed in Escherichia coli, and the enzymatic properties of the recombinant enzyme were characterized. The gene consisted of a 1,476-bp open reading frame and encoded a 491-amino-acid protein, comprising an N-terminal secretion signal sequence and glycoside hydrolase family 5 catalytic module. The recombinant enzyme, Sa1,6Gal5A, catalyzed the hydrolysis of β-1,6-linked galactosyl linkages of oligosaccharides and polysaccharides. The enzyme produced galactose and a range of β-1,6-linked galacto-oligosaccharides, predominantly β-1,6-galactobiose, from β-1,6-galactan chains. There was a synergistic effect between the enzyme and Sa1,3Gal43A in degrading tomato arabinogalactan proteins. These results suggest that Sa1,6Gal5A is the first identified endo-β-1,6-galactanase from a prokaryote