Takumi Takeda

Suppression and acceleration of cell elongation by integration of xyloglucans in pea stem segments.

Xyloglucan is a key polymer in the walls of growing plant cells. Using split pea stem segments and stem segments from which the epidermis had been peeled off, we demonstrate that the integration of xyloglucan mediated by the action of wall-bound xyloglucan endotransglycosylase suppressed cell elongation, whereas that of its fragment oligosaccharide accelerated it. Whole xyloglucan was incorporated into the cell wall and induced the rearrangement of cortical microtubules from transverse to longitudinal; in contrast, the oligosaccharide solubilized xyloglucan from the cell wall and maintained the microtubules in a transverse orientation. This paper proposes that xyloglucan metabolism controls the elongation of plant cells.

Characterization of a cellobiohydrolase (MoCel6A) produced by Magnaporthe oryzae.

ABSTRACT Three GH-6 family cellobiohydrolases are expected in the genome of Magnaporthe grisea based on the complete genome sequence. Here, we demonstrate the properties, kinetics, and substrate specificities of a Magnaporthe oryzae GH-6 family cellobiohydrolase (MoCel6A). In addition, the effect of cellobiose on MoCel6A activity was also investigated. MoCel6A contiguously fused to a histidine tag was overexpressed in M. oryzae and purified by affinity chromatography. MoCel6A showed higher hydrolytic activities on phosphoric acid-swollen cellulose (PSC), β-glucan, and cellooligosaccharide derivatives than on cellulose, of which the best substrates were cellooligosaccharides. A tandemly aligned cellulose binding domain (CBD) at the N terminus caused increased activity on cellulose and PSC, whereas deletion of the CBD (catalytic domain only) showed decreased activity on cellulose. MoCel6A hydrolysis of cellooligosaccharides and sulforhodamine-conjugated cellooligosaccharides was not inhibited by exogenously adding cellobiose up to 438 mM, which, rather, enhanced activity, whereas a GH-7 family cellobiohydrolase from M. oryzae (MoCel7A) was severely inhibited by more than 29 mM cellobiose. Furthermore, we assessed the effects of cellobiose on hydrolytic activities using MoCel6A and Trichoderma reesei cellobiohydrolase (TrCel6A), which were prepared in Aspergillus oryzae. MoCel6A showed increased hydrolysis of cellopentaose used as a substrate in the presence of 292 mM cellobiose at pH 4.5 and pH 6.0, and enhanced activity disappeared at pH 9.0. In contrast, TrCel6A exhibited slightly increased hydrolysis at pH 4.5, and hydrolysis was severely inhibited at pH 9.0. These results suggest that enhancement or inhibition of hydrolytic activities by cellobiose is dependent on the reaction mixture pH.

The Gentio-Oligosaccharide Gentiobiose Functions in the Modulation of Bud Dormancy in the Herbaceous Perennial Gentiana

This study examines the mechanisms regulating dormancy in gentians, a group of alpine perennials that produce overwintering buds. Multivariate analysis of metabolite profiles revealed that gentiobiose, a rare disaccharide, acts as a signal for dormancy release of overwintering buds through the ascorbate-glutathione cycle, a pathway that detoxifies reactive oxygen species. Bud dormancy is an adaptive strategy that perennials use to survive unfavorable conditions. Gentians (Gentiana), popular alpine flowers and ornamentals, produce overwintering buds (OWBs) that can persist through the winter, but the mechanisms regulating dormancy are currently unclear. In this study, we conducted targeted metabolome analysis to obtain clues about the metabolic mechanisms involved in regulating OWB dormancy. Multivariate analysis of metabolite profiles revealed metabolite patterns characteristic of dormant states. The concentrations of gentiobiose [β-d-Glcp-(1→6)-d-Glc] and gentianose [β-d-Glcp-(1→6)-d-Glc-(1→2)-d-Fru] significantly varied depending on the stage of OWB dormancy, and the gentiobiose concentration increased prior to budbreak. Both activation of invertase and inactivation of β-glucosidase resulted in gentiobiose accumulation in ecodormant OWBs, suggesting that gentiobiose is seldom used as an energy source but is involved in signaling pathways. Furthermore, treatment with exogenous gentiobiose induced budbreak in OWBs cultured in vitro, with increased concentrations of sulfur-containing amino acids, GSH, and ascorbate (AsA), as well as increased expression levels of the corresponding genes. Inhibition of GSH synthesis suppressed gentiobiose-induced budbreak accompanied by decreases in GSH and AsA concentrations and redox status. These results indicate that gentiobiose, a rare disaccharide, acts as a signal for dormancy release of gentian OWBs through the AsA-GSH cycle.

Characterization of β-N-acetylhexosaminidase (LeHex20A), a member of glycoside hydrolase family 20, from Lentinula edodes (shiitake mushroom)

We purified and cloned a β-N-acetylhexosaminidase, LeHex20A, with a molecular mass of 79 kDa from the fruiting body of Lentinula edodes (shiitake mushroom). The gene lehex20a gene had 1,659 nucleotides, encoding 553 amino acid residues. Sequence analysis indicated that LeHex20A belongs to glycoside hydrolase (GH) family 20, and homologues of lehex20a are broadly represented in the genomes of basidiomycetes. Purified LeHex20A hydrolyzed the terminal monosaccharide residues of β-N-acetylgalactosaminides and β-N-acetylglucosaminides, indicating that LeHex20A is a β-N-acetylhexosaminidase classified into EC 3.2.1.52. The maximum LeHex20A activity was observed at pH 4.0 and 50°C. The kinetic constants were estimated using chitooligosaccharides with degree of polymerization 2-6. GH20 β-N-acetylhexosaminidases generally prefer chitobiose among natural substrates. However, LeHex20A had the highest catalytic efficiency (kcat/Km) for chitotetraose, and the Km values for GlcNAc6 were 3.9-fold lower than for chitobiose. Furthermore, the enzyme partially hydrolyzed amorphous chitin polymers. These results indicate that LeHex20A can produce N-acetylglucosamine from long-chain chitomaterials.

A novel glycosylphosphatidylinositol-anchored glycoside hydrolase from Ustilago esculenta functions in β-1,3-glucan degradation.

ABSTRACT A glycoside hydrolase responsible for laminarin degradation was partially purified to homogeneity from a Ustilago esculenta culture filtrate by weak-cation-exchange, strong-cation-exchange, and size-exclusion chromatography. Three proteins in enzymatically active fractions were digested with chymotrypsin followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis, resulting in the identification of three peptide sequences that shared significant similarity to a putative β-1,3-glucanase, a member of glucoside hydrolase family 16 (GH16) from Sporisorium reilianum SRZ2. A gene encoding a laminarin-degrading enzyme from U. esculenta, lam16A, was isolated by PCR using degenerate primers designed based on the S. reilianum SRZ2 β-1,3-glucanase gene. Lam16A possesses a GH16 catalytic domain with an N-terminal signal peptide and a C-terminal glycosylphosphatidylinositol (GPI) anchor peptide. Recombinant Lam16A fused to an N-terminal FLAG peptide (Lam16A-FLAG) overexpressed in Aspergillus oryzae exhibited hydrolytic activity toward β-1,3-glucan specifically and was localized both in the extracellular and in the membrane fractions but not in the cell wall fraction. Lam16A without a GPI anchor signal peptide was secreted extracellularly and was not detected in the membrane fraction. Membrane-anchored Lam16A-FLAG was released completely by treatment with phosphatidylinositol-specific phospholipase C. These results suggest that Lam16A is anchored in the plasma membrane in order to modify β-1,3-glucan associated with the inner cell wall and that Lam16A is also used for the catabolism of β-1,3-glucan after its release in the extracellular medium.

Purification and Characterization of UDP-Arabinopyranose Mutase from Chlamydomonas reinhardtii

Chlamydomonas reinhardtii cells are surrounded by a mixture of hydroxyprolin-rich glycoproteins consisting of L-arabinose, D-galactose, D-glucose, and D-mannose residues. The L-arabinose residue is thought to be attached by a transfer of UDP-L-arabinofuranose (UDP-Araf), which is produced from UDP-L-arabinopyranose (UDP-Arap) by UDP-arabinopyranose mutase (UAM). UAM was purified from the cytosol to determine the involvement of C. reinhardtii UAM (CrUAM) in glycoprotein synthesis. CrUAM was purified 94-fold to electrophoretic homogeneity by hydrophobic and size-exclusion chromatography. CrUAM catalyzed the reversible conversion between UDP-Arap and UDP-Araf and exhibited autoglycosylation activity when UDP-D-[14C]glucose was added as substrate. Compared to the properties of native and recombinant CrUAM overexpressed in Escherichia coli, native CrUAM showed a higher affinity for UDP-Arap than recombinant CrUAM did. This increased affinity for UDP-Arap might have been caused by post-translational modifications that occur in eukaryotes but not in prokaryotes.

Fungal hemicellulose-degrading enzymes cause physical property changes concomitant with solubilization of cell wall polysaccharides

Main conclusionPhysical properties of wheat coleoptile segments decreased after treatment with hemicellulose-degrading enzymes, indicating that hemicellulosic polysaccharides function to control the strength of primary cell walls.AbstractChanges in the physical properties of plant cell walls, a viscoelastic structure, are thought to be one of the growth-limiting factors for plants and one of the infection-affecting factors for fungi. To study the significance of hemicellulosic polysaccharides that form cross-bridges between cellulose microfibrils in controlling cell wall strength in monocot plants, the effects of hemicellulose degradation by recombinant Magnaporthe oryzae xylanase and 1,3-1,4-β-glucanase, and recombinant Aspergillus oryzae xyloglucanase on the physical properties and polysaccharide solubilization were investigated using wheat (Triticum aestivum L.) coleoptiles. Treatments with xylanase or 1,3-1,4-β-glucanase significantly decreased the viscosity and elasticity of wheat coleoptile segments. In addition, xyloglucanase treatment slightly decreased the viscoelasticity. Furthermore, 1,3-1,4-β-glucan polymer was solubilized during hydrolysis with xylanase and xyloglucanase, even though neither enzyme had hydrolytic activity towards 1,3-1,4-β-glucan. These results suggest that xylan and xyloglucan interact with 1,3-1,4-β-glucan and that the composites and hemicellulosic polysaccharides form inter-molecular bridges. Degradation of these bridges causes decreases in the physical properties, resulting in increased extensibility of the cell walls. These findings provide a testable model in which wheat coleoptile cell walls are loosened by the degradation of hemicellulosic polysaccharides and hemicellulose-degrading enzymes play a significant role in loosening the walls during fungal infection.

Polysaccharide-Inducible Endoglucanases from Lentinula edodes Exhibit a Preferential Hydrolysis of 1,3–1,4-β-Glucan and Xyloglucan

Three genes encoding glycoside hydrolase family 12 (GH12) enzymes from Lentinula edodes, namely Lecel12A, Lecel12B, and Lecel12C, were newly cloned by PCR using highly conserved sequence primers. To investigate enzymatic properties, recombinant enzymes encoded by L. edodes DNAs and GH12 genes from Postia placenta (PpCel12A and PpCel12B) and Schizophyllum commune (ScCel12A) were prepared in Brevibacillus choshinensis. Recombinant LeCel12A, PpCel12A, and PpCel12B, which were grouped in GH12 subfamily 1, preferentially hydrolyzed 1,3-1,4-β-glucan. By contrast, LeCel12B, LeCel12C, and ScCel12A, members of the subfamily 2, exhibited specific hydrolysis of xyloglucan. These results suggest that two subfamilies of GH12 are separated based on the substrate specificity. Transcript levels of L. edodes genes increased 72 h after growth of L. edodes mycelia cells in the presence of plant cell wall polymers such as xyloglucan, 1,3-1,4-β-glucan, and cellulose. These results suggest that L. edodes GH12 enzymes have evolved to hydrolyze 1,3-1,4-β-glucan and xyloglucan, which might enhance hyphal extension and nutrient acquisition.

Transferase activity of GhCesA2 (putative cotton cellulose 4-β-glucosyltransferase) expressed in Pichia pastoris

GhCesA2 is a cotton (Gossypium hirsutum) homologue of the bacterial cellulose synthase gene that encodes cellulose 4-β-glucosyltransferase. The central catalytic region of GhCesA2 was expressed as a soluble protein in the methylotrophic yeastPichia pastoris. The molecular size of the recombinant protein was 100kDa, which decreased to 85 kDa after treatment with endoglycosidase H. The recombinant GhCesA2 catalyzed transfer of glucose from UDP-glucose into unknown products in the presence of an extract of cotton hypocotyls, but the products were notβ-1,4-glucan.

Expression of Gene Encoding Endo-1,4-β-glucanase in Suspension-cultured Poplar (Poplus alba L.) Cells†

The level of mRNA for endo-1,4-β-glucanase was increased before the exponential phase of growth and decreased rapidly during the exponential phase in suspension-cultured poplar cells. The level of mRNA was increased for a short period after the addition of either 2,4-D or sucrose to the culture medium at the stationary phase. The level was increased to the maximal level when both 2,4-D and sucrose were provided together, but one did not increase the effect of the other. These findings suggest that expression of gene encoding poplar endo-1,4-β-glucanase is controlled during cell growth by independent systems activated by auxin and sucrose.