UDP-arabinopyranose mutase gene expressions are required for the biosynthesis of the arabinose side chain of both pectin and arabinoxyloglucan, and normal leaf expansion in Nicotiana tabacum.

Plant cell walls are composed of polysaccharides such as cellulose, hemicelluloses, and pectins, whose location and function differ depending on plant type. Arabinose is a constituent of many different cell wall components, including pectic rhamnogalacturonan I (RG-I) and II (RG-II), glucuronoarabinoxylans (GAX), and arabinoxyloglucan (AXG). Arabinose is found predominantly in the furanose rather than in the thermodynamically more stable pyranose form. The UDP-arabinopyranose mutases (UAMs) have been demonstrated to convert UDP-arabinopyranose (UDP-Arap) to UDP-arabinofuranose (UDP-Araf) in rice (Oryza sativa L.). The UAMs have been implicated in polysaccharide biosynthesis and developmental processes. Arabinose residues could be a component of many polysaccharides, including branched (1→5)-α-arabinans, arabinogalactans in pectic polysaccharides, and arabinoxyloglucans, which are abundant in the cell walls of solanaceous plants. Therefore, to elucidate the role of UAMs and arabinan side chains, we analyzed the UAM RNA interference transformants in tobacco (Nicotiana tabacum L.). The tobacco UAM gene family consists of four members. We generated RNAi transformants (NtUAM-KD) to down-regulate all four of the UAM members. The NtUAM-KD showed abnormal leaf development in the form of a callus-like structure and many holes in the leaf epidermis. A clear reduction in the pectic arabinan content was observed in the tissue of the NtUAM-KD leaf. The arabinose/xylose ratio in the xyloglucan-rich cell wall fraction was drastically reduced in NtUAM-KD. These results suggest that UAMs are required for Ara side chain biosynthesis in both RG-I and AXG in Solanaceae plants, and that arabinan-mediated cell wall networks might be important for normal leaf expansion.

The gentio-oligosaccharide gentiobiose functions in the modulation of bud dormancy in the herbaceous perennial Gentiana.

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 [ß-D-Glcp-(1→6)-D-Glc] and gentianose [ß-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 ß-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.

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-[(14)C]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.

Hyaluronidase-inhibiting Polysaccharide from Caulerpa lentillifera.

Algal sulfated polysaccharides are known to be effective hyaluronidase inhibitors. We evaluated hyaluronidase inhibitory activity of sulfated polysaccharide (SP) from Caulerpa lentillifera. Results showed that SP with IC50 of 163 µg/mL appears to allosterically inhibit the hyaluronidase activity. Main sugar composition and sulfate content of SP was estimated to be Gal, Glc, Xyl, Man, uronic acids, and sulfate in the weight percent of 27.7: 28.9: 14.6: 22.5: 3.4: 21.7. We modified the SP by desulfation and partial hydrolysis with trifluoroacetic acid (TFA) to investigate the effect of sulfate content and molecular weight on inhibition. Hyaluronidase inhibitory activity of desulfated SP, 0.1 M TFA-hydrolyzed SP and 0.5 M TFA-hydrolyzed SP were significantly lower than that of native SP, revealing that sulfate content or molecular weight is important for hyaluronidase inhibition.

Fractionation and characterization of cell wall polysaccharides from the brown alga Cladosiphon okamuranus.

Brown algae contain a polysaccharide-rich cell wall, mainly composed of alginate and fucoidan which have been extensively studied for their individual structure and bioactivities. Particularly, the cell wall of Cladosiphon okamuranus is rich in fucoidan rather than alginate. However, little is known about its arrangement or interlinking with other polysaccharides such as cellulose in the cell wall. To determine its structure in detail, the cell wall was sequentially fractionated into five fractions: hot water (HW), ammonium oxalate, hemicellulose-I (HC-I), HC-II, and cellulose. Almost 80% of the total cell wall recovered from alcohol insoluble residue in C. okamuranus consisted of HW and HC-I, which mainly contained fucoidan composed of fucose, glucuronic acid, and sulfate in molar ratios of 1.0:0.3:0.9 and 1.0:0.2:0.3, respectively. Methylation analysis revealed that fucoidan in HW and HC-I structurally differed in terms of content of sulfate, and sugar residue which was 1,4-linked xylose and 1,4-linked fucose. Small angle X-ray scattering measurements also showed distinct conformational differences between HW and HC-I. These structural heterogeneities of fucoidan may be related to their localization, and fucoidan in HC-I may be involved in reinforcing cell wall structure by cross-linking to cellulose.

The Cell Wall Characterization of Brown Alga Cladosiphon okamuranus during Growth.

The present study provides new insights into the growth of the brown algal cell wall by showing that cell wall polysaccharides play an important role in the process of growth, considering the physicochemical characteristic of young and old Cladosiphon okamuranus. To determine its structural variation in detail, the cell wall was sequentially fractionated into five fractions: hot water (HW), ammonium oxalate, hemicellulose-I (HC-I), HC-II, and cellulose, and analyzed physicochemically. Results showed that almost 80% of the total recovery cell wall from both young and old thalli was HW, and HC-I contained mainly fucoidan composed of Fucose, Glucuronic acid, and sulfate in molar ratios of 1.0:0.3:0.6~0.7 and 1.0:0.3:0.2~0.3, respectively. Fucoidan in HW was a highly sulfated matrix polysaccharide abundance in young thalli, while fucoidan in HC-I was rich in old thalli and functions as hemicellulose in land plants, crosslinking with cellulose and strengthening the cell wall. We found that HW and HC-I were particularly involved in the growth and strength of old thalli appeared to be due to the deposition of HC-I and the reduction in water content during the growth process.

Biochemical characterization of Magnaporthe oryzae ß-glucosidases for efficient ß-glucan hydrolysis.

ß-Glucosidases designated MoCel3A and MoCel3B were successfully overexpressed in Magnaporthe oryzae. MoCel3A and MoCel3B showed optimal activity at 50 °C and pH 5.0-5.5. MoCel3A exhibited higher activity on higher degree of polymerization (DP) oligosaccharides and on ß-1,3-linked oligosaccharides than on ß-1,4-linked oligosaccharides. Furthermore, MoCel3A could liberate glucose from polysaccharides such as laminarin, 1,3-1,4-ß-glucan, phosphoric acid-swollen cellulose, and pustulan, of which laminarin was the most suitable substrate. Conversely, MoCel3B preferentially hydrolyzed lower DP oligosaccharides such as cellobiose, cellotriose, and laminaribiose. Furthermore, the synergistic effects of combining enzymes including MoCel3A and MoCel3B were investigated. Depolymerization of 1,3-1,4-ß-glucan by M. oryzae cellobiohydrolase (MoCel6A) enhanced the production of glucose by the actions of MoCel3A and MoCel3B. In these reactions, MoCel3A hydrolyzed higher DP oligosaccharides, resulting in the release of glucose and cellobiose, and MoCel3B preferentially hydrolyzed lower DP oligosaccharides including cellobiose. On the other hand, MoCel3A alone produced glucose from laminarin at levels equivalent to 80% of maximal hydrolysis obtained by the combined action of MoCel3A, MoCel3B, and endo-1,3-ß-glucanase. Therefore, MoCel3A and MoCel3B activities yield glucose from not only cellulosic materials but also hemicellulosic polysaccharides.

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.

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

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), ß-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.

Structural characteristics and in vitro macrophage activation of acetyl fucoidan from Cladosiphon okamuranus.

We investigated a structural characteristics of acetyl fucoidan (CAF) isolated from commercially cultured Cladosiphon okamuranus. The CAF-induced macrophage activation and its signaling pathways in murine macrophage cell line, RAW 264.7 were also investigated. From the results of methylation analysis, CAF consisted of alpha-1-->3 linked L: -fucosyl residues and substituted sulfate and acetyl groups at C-4 on the main chain. CAF induced production of nitric oxide (NO), tumor necrosis factor-alpha and interleukin-6 in RAW 264.7 cells. Sulfate and acetyl groups of CAF involved in CAF-induced NO production. Neutralizing anti-Toll-like receptor 4 (TLR4), anti-CD14 and anti-scavenger receptor class A (SRA) but not anti-complement receptor type 3 monoclonal antibodies decreased CAF-induced NO production. The results of immunoblot analysis indicated that CAF activated mitogen-activated protein kinases (MAPKs) such as p38 MAPK, extracellular signal-regulated kinase (ERK)1/2 and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). SB203580 (p38 MAPK inhibitor) and SP600125 (SAPK/JNK inhibitor), but not U0126 (MAPK/ERK kinase 1/2 inhibitor) decreased CAF-induced NO production. The results suggested that CAF induced macrophage activation through membrane receptors TLR4, CD14 and SRA, and MAPK signaling pathways.

Isolation and characterization of pectin from peel of Citrus tankan.

A pectin was extracted from the peel of Citrus tankan with a yield of 2.75%. The uronic acid content was 80.0%, and the degree of methoxylation was 63.2%. The pectin was composed of D-GalA, D-Gal, L-Ara and L-Rha in the molar ratio of 100:11.3:3.6:2.6. The molecular weight was estimated to be approximately 9.2 x 10(4). The pectin formed a gel by conventional procedures.

Anti-proliferative activity of oversulfated fucoidan from commercially cultured Cladosiphon okamuranus TOKIDA in U937 cells.

Fucoidan from Cladosiphon okamuranus and its sulfate derivatives were prepared. Sulfate contents of native and oversulfated fucoidan were estimated to be 13.5% and 32.8%, respectively. The results of (1)H NMR suggest that 2,4-di-O-sulfo-, 2-mono-O-sulfo- and 4-mono-O-sulfo-l-fucopyranose were involved in oversulfated fucoidan and 4-mono-O-sulfo-l-fucopyranose was involved in native fucoidan. The oversulfated fucoidan reduced the proliferation of U937 cells in a dose-dependent manner, but the activity of native fucoidan was weak. The sulfate content and substituting position of sulfate group might be important factors of anti-proliferative activity in U937 cells. To examine whether the anti-proliferative activity of oversulfated fucoidan was caused by induction of apoptosis, apoptosis assay, caspase-3 activity assay and Western blotting analysis were performed. These results indicated that the oversulfated fucoidan induced apoptosis via caspase-3 and -7 activation-dependent pathway.

Cellulose metabolism in plants.

Many bacterial genomes contain a cellulose synthase operon together with a cellulase gene, indicating that cellulase is required for cellulose biosynthesis. In higher plants, there is evidence that cell growth is enhanced by the overexpression of cellulase and prevented by its suppression. Cellulase overexpression could modify cell walls not only by trimming off the paracrystalline sites of cellulose microfibrils, but also by releasing xyloglucan tethers between the microfibrils. Mutants for membrane-anchored cellulase (Korrigan) also show a typical phenotype of prevention of cellulose biosynthesis in tissues. All plant cellulases belong to family 9, which endohydrolyzes cellulose, but are not strong enough to cause the bulk degradation of cellulose microfibrils in a plant body. It is hypothesized that cellulase participates primarily in repairing or arranging cellulose microfibrils during cellulose biosynthesis in plants. A scheme for the roles of plant cellulose and cellulases is proposed.

A draft genome of the brown alga, Cladosiphon okamuranus, S-strain: a platform for future studies of 'mozuku' biology.

The brown alga, Cladosiphon okamuranus (Okinawa mozuku), is economically one of the most important edible seaweeds, and is cultivated for market primarily in Okinawa, Japan. C. okamuranus constitutes a significant source of fucoidan, which has various physiological and biological activities. To facilitate studies of seaweed biology, we decoded the draft genome of C. okamuranus S-strain. The genome size of C. okamuranus was estimated as ∼140 Mbp, smaller than genomes of two other brown algae, Ectocarpus siliculosus and Saccharina japonica Sequencing with ∼100× coverage yielded an assembly of 541 scaffolds with N50 = 416 kbp. Together with transcriptomic data, we estimated that the C. okamuranus genome contains 13,640 protein-coding genes, approximately 94% of which have been confirmed with corresponding mRNAs. Comparisons with the E. siliculosus genome identified a set of C. okamuranus genes that encode enzymes involved in biosynthetic pathways for sulfated fucans and alginate biosynthesis. In addition, we identified C. okamuranus genes for enzymes involved in phlorotannin biosynthesis. The present decoding of the Cladosiphon okamuranus genome provides a platform for future studies of mozuku biology.

A beta-(1-->3)-arabinopyranosyltransferase that transfers a single arabinopyranose onto arabino-oligosaccharides in mung bean (Vigna radiate) hypocotyls.

Arabinopyranosyltransferase (ArapT) activity that results in the transfer of a single arabinopyranose (Arap) residue from UDP-beta-L-arabinopyranose (UDP-Arap) to exogenous (1-->5)-linked alpha-L-arabino-oligosaccharides labeled with 2-aminobenzamide (2-AB) at their reducing ends was identified in a particulate preparation obtained from 3-day-old mung bean (Vigna radiate L. Wilezek) hypocotyls. The transferred Ara residue was shown to be beta-(1-->3)-linked to O-3 of the non-reducing terminal Araf residues of the oligosaccharide using nuclear magnetic resonance spectroscopy together with glycosyl composition and glycosyl linkage composition analyses. The 2AB-labeled arabino-octasaccharide was the most effective acceptor substrate analyzed, although arabino-oligosaccharides with a degree of polymerization between 4 and 7 were also acceptor substrates. Maximum ArapT activity was obtained at pH 6.5-7.0, and 20 degrees C in the presence of 25 mM Mn(2+) and 0.5% Triton X-100.

Structure of a novel α-glucan substitute with the rare 6-deoxy-D-altrose from Lactarius lividatus (mushroom).

A novel α-glucan substituted rare 6-deoxy-D-altropyranose was isolated from edible fruiting bodies of a mushroom (Lactarius lividatus) grown in Okinawa, Japan. The polysaccharide consists of D-glucose, D-galactose and 6-deoxy-D-altrose in a molar ratio of 3.0:1.0:1.0. The specific rotation [α](589) was estimated as +64.3° (0.2% in water) at 25 °C. Based on results of IR, NMR ((1)H, (13)C, 2D-COSY, 2D-HMQC, 2D-ROESY and 2D-HMBC), and methylation analyses, the structure of the polysaccharide was determined as [formula, see text] This work is the first demonstration of rare 6-deoxy-D-altropyranose moiety on polysaccharides.

Identification of rare 6-deoxy-D-altrose from an edible mushroom (Lactarius lividatus).

6-Deoxy-L-altrose is well known as a constituent sugar moiety of lipopolysaccharides in Gram-negative bacteria. However, its isomer, 6-deoxy-D-altrose, is little known. Identification of 6-deoxy-D-altrose isolated from a polysaccharide extracted from an edible mushroom (Lactarius lividatus), its comparison with chemically synthesized 6-deoxy-D-altrose using (1)H and (13)C NMR including COSY, HMQC spectroscopy, and investigation of its specific optical rotation were all conducted in this study. The 6-deoxy-hexose isolated from acid hydrolysate of the polysaccharide extracted from L. lividatus was involved in four anomeric isomers (α-pyranose and ß-pyranose, and α-furanose and ß-furanose), as was chemically synthesized 6-deoxy-d-altrose in an aqueous solution because of mutarotation. Almost all signals of 1D ((1)H NMR and (13)C NMR) and 2D (COSY and HMQC)-NMR spectra agreed with those of the authentic 6-deoxy-D-altrose. The specific optical rotation [α](589) of 6-deoxy-sugar showed a value of +18.2°, which was in agreement with that of authentic 6-deoxy-D-altrose. Consequently, 6-deoxy-hexose was identified as the 6-deoxy-D-altrose. This work is the first complete identification of 6-deoxy-D-altrose in a natural environment.

Gelation and Retrogradation Mechanism of Wheat Amylose.

The flow behavior, dynamic viscoelasticity, and optical rotation of aqueous solutions of wheat amylose were measured using a rheogoniometer and a polarimeter. The amylose solutions, at 25 °C, showed shear-thinning behavior at a concentration of 1.2%, but plastic behavior at 1.4 and 1.6%, the yield values of which were estimated to be 0.6 and 1.0 Pa, respectively. The viscosity of the wheat amylose increased a little with increase in temperature up to 10 or 20 °C at 1.2% or 1.4 and 1.6%, which was estimated to be a transition temperature. The elastic modulus increased with increase in concentration, and increased with increasing temperature up to 20, 25 and 30 °C, which was estimated to be a transition temperature, respectively, then decreased gradually but stayed at a large value even at high temperature (80 °C). A very low elastic modulus of the wheat amylose was observed upon addition of urea (4.0 M) and in alkaline solution (0.05 M NaOH) even at low temperature. The optical rotation of wheat amylose solution increased a little with decreasing temperature down to 25 °C, then increased rapidly with further decrease in the temperature. The mode of gelation mechanism of amylose molecules, which was previously proposed, was confirmed and a retrogradation mechanism of wheat amylose was proposed.

An arginyl residue in rice UDP-arabinopyranose mutase is required for catalytic activity and autoglycosylation.

Plants use UDP-arabinofuranose (UDP-Araf) to donate Araf residues in the biosynthesis of Araf-containing complex carbohydrates. UDP-Araf itself is formed from UDP-arabinopyranose (UDP-Arap) by UDP-arabinopyranose mutase (UAM). However, the mechanism by which this enzyme catalyzes the interconversion of UDP-Arap and UDP-Araf has not been determined. To gain insight into this reaction, functionally recombinant rUAMs were reacted with UDP-Glc or UDP-Araf. The glycosylated recombinant UAMs were fragmented with trypsin, and the glycopeptides formed were then identified and sequenced by LC-MS/MS. The results of these experiments, together with site-directed mutagenesis studies, suggest that in functional UAMs an arginyl residue is reversibly glycosylated with a single glycosyl residue, and that this residue is required for mutase activity. We also provide evidence that a DXD motif is required for catalytic activity.

A plant mutase that interconverts UDP-arabinofuranose and UDP-arabinopyranose.

Plant cell walls constitute the bulk of the earth renewable source of energy and are a component in the diet of humans and herbivores. l-Arabinofuranosyl (Araf) residues are a quantifiably important constituent of these walls. Plants use uridine diphosphate (UDP)-l-arabinofuranose (UDP-Araf) to donate Araf residues in the biosynthesis of Araf-containing polysaccharides, proteoglycans, and glycoproteins. However, little is known about the formation of UDP-Araf. We now describe the purification and partial characterization of a rice UDP-arabinopyranose mutase (UAM) that catalyzes the formation of UDP-Araf from UDP-arabinopyranose (UDP-Arap). The reaction is reversible and at thermodynamic equilibrium the pyranose form is favored over the furanose form (90 : 10). Three related proteins that are encoded by rice gene loci Os03g40270, Os04g56520, and Os07g41360 were identified from partial amino acid sequences of UAM. These proteins have >80% sequence identity with polypeptides that are reversibly glycosylated in the presence of UDP-sugars. The rice mutase and two functionally active recombinant mutases were shown to be reversibly glycosylated in the presence of UDP-Glc. The cofactor, flavin-adenine-dinucleotide (FAD), is required for the catalytic activity of UDP-galactose mutases of prokaryotes, fungi, and protozoa. The plant mutases, which do not require a cofactor, must therefore have a different catalytic mechanism. Putative UAM-encoding genes are present in the green algae Chlamydomonas reinhardtii, the moss Physcomitrella patens, the gymnosperm Pinus taeda (loblolly pine), and in numerous dicots and monocots, indicating that UAMs are widespread in green plants.