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.

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.

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.

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.