Presence of a basic secretory protein in xylem sap and shoots of poplar in winter and its physicochemical activities against winter environmental conditions.

XSP25, previously shown to be the most abundant hydrophilic protein in xylem sap of Populus nigra in winter, belongs to a secretory protein family in which the arrangement of basic and acidic amino acids is conserved between dicotyledonous and monocotyledonous species. Its gene expression was observed at the same level in roots and shoots under long-day conditions, but highly induced under short-day conditions and at low temperatures in roots, especially in endodermis and xylem parenchyma in the root hair region of Populus trichocarpa, and its protein level was high in dormant buds, but not in roots or branches. Addition of recombinant PtXSP25 protein mitigated the denaturation of lactate dehydrogenase by drying, but showed only a slight effect on that caused by freeze-thaw cycling. Recombinant PtXSP25 protein also showed ice recrystallization inhibition activity to reduce the size of ice crystals, but had no antifreezing activity. We suggest that PtXSP25 protein produced in shoots and/or in roots under short-day conditions and at non-freezing low temperatures followed by translocation via xylem sap to shoot apoplast may protect the integrity of the plasma membrane and cell wall functions from freezing and drying damage in winter environmental conditions.

UDP-arabinopyranose mutase 3 is required for pollen wall morphogenesis in rice (Oryza sativa).

l-Arabinose is one of the main constituents of cell wall polysaccharides such as pectic rhamnogalacturonan I (RG-I), glucuronoarabinoxylans and other glycoproteins. It is found predominantly in the furanose form rather than in the thermodynamically more stable pyranose form. UDP-L-arabinofuranose (UDP-Araf), rather than UDP-L-arabinopyranose (UDP-Arap), is a sugar donor for the biosynthesis of arabinofuranosyl (Araf) residues. UDP-arabinopyranose mutases (UAMs) have been shown to interconvert UDP-Araf and UDP-Arap and are involved in the biosynthesis of polysaccharides including Araf. The UAM gene family has three members in Oryza sativa. Co-expression network in silico analysis showed that OsUAM3 expression was independent from OsUAM1 and OsUAM2 co-expression networks. OsUAM1 and OsUAM2 were expressed ubiquitously throughout plant development, but OsUAM3 was expressed primarily in reproductive tissue, particularly at the pollen cell wall formation developmental stage. OsUAM3 co-expression networks include pectin catabolic enzymes. To determine the function of OsUAMs in reproductive tissues, we analyzed RNA interference (RNAi)-knockdown transformants (OsUAM3-KD) specific for OsUAM3. OsUAM3-KD plants grew normally and showed abnormal phenotypes in reproductive tissues, especially in terms of the pollen cell wall and exine. In addition, we examined modifications of cell wall polysaccharides at the cellular level using antibodies against polysaccharides including Araf. Immunolocalization of arabinan using the LM6 antibody showed low levels of arabinan in OsUAM3-KD pollen grains. Our results suggest that the function of OsUAM3 is important for synthesis of arabinan side chains of RG-I and is required for reproductive developmental processes, especially the formation of the cell wall in pollen.

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.

Rice BRITTLE CULM 3 (BC3) encodes a classical dynamin OsDRP2B essential for proper secondary cell wall synthesis.

"Brittle culm" mutants found in Gramineae crops are suitable materials to study the mechanism of secondary cell wall formation. Through positional cloning, we have identified a gene responsible for the brittle culm phenotype in rice, brittle culm 3 (bc3). BC3 encodes a member of the classical dynamin protein family, a family known to function widely in membrane dynamics. The bc3 mutation resulted in reductions of 28-36% in cellulose contents in culms, leaves, and roots, while other cell wall components remained unaffected. Reductions of cell wall thickness and birefringence were observed in both fiber (sclerenchyma) and parenchymal cells, together with blurring of the wall's layered structures. From promoter-GUS analyses, it was suggested that BC3 expression is directly correlated with active secondary cell wall synthesis. These results suggest that BC3 is tightly involved in the synthesis of cellulose and is essential for proper secondary cell wall construction.

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.

Beta-1,3:1,4-glucan synthase activity in rice seedlings under water.

BACKGROUND AND AIMS: The metabolism of beta-1,3 : 1,4-glucan regulates the mechanical properties of cell walls, and thereby changes the elongation growth of Poaceae plants. A previous study has shown that elongation growth of rice coleoptiles under water is enhanced by increased activity of beta-1,3 : 1,4-glucan hydrolases; however, the involvement of beta-1,3 : 1,4-glucan synthase activity in elongation growth under water has not yet been clarified. METHODS: The beta-1,3 : 1,4-glucan synthase activity in a microsomal fraction prepared from rice seedlings grown under water was compared with that from control seedlings grown in air. The change under water in the relative expression level of CslF6, a major isoform of the beta-1,3 : 1,4-glucan synthase genes, was examined by quantitative reverse-transcriptase PCR. KEY RESULTS: The level of beta-1,3 : 1,4-glucan synthase activity in submerged seedlings decreased to less than 40 % of that of the control seedlings and was accompanied by a significant reduction in the amount of beta-1,3 : 1,4-glucan in the cell walls. Under water, the expression of CslF6 was reduced to less than 20 % of the unsubmerged control. Bubble aeration partially restored both beta-1,3 : 1,4-glucan synthase activity and the expression of CslF6 under water, correlating with suppression of the submergence-induced elongation growth of coleoptiles. CONCLUSIONS: Submergence down-regulates the expression of the CslF6 gene, leading to a decreased level of beta-1,3 : 1,4-glucan synthase activity. Together with the increased activity of beta-1,3 : 1,4-glucan hydrolases, the decreased activity of beta-1,3 : 1,4-glucan synthase contributes to the decrease in the amount of beta-1,3 : 1,4-glucan in the cell walls under water. The suppression of beta-1,3 : 1,4-glucan synthesis under water may be mainly due to oxygen depletion.

Short day length-induced decrease of cesium uptake without altering potassium uptake manner in poplar.

Short day length-induced alteration of potassium (K) localization in perennial trees is believed to be a mechanism for surviving and adapting to severe winters. To investigate the relationship between cesium (Cs) and K localizations, a model tree poplar, hybrid aspen T89, was employed. Under short day length conditions, the amount of 137Cs absorbed through the root and translocated to the root was drastically reduced, but 42K was not. Potassium uptake from the rhizosphere is mediated mainly by KUP/HAK/KT and CNGC transporters. In poplar, however, these genes were constantly expressed under short-day conditions except for a slight increase in the expression a KUP/HAK/KT gene six weeks after the onset of the short-day treatment. These results indicated that the suppression of 137Cs uptake was triggered by short day length but not regulated by competitive Cs+ and K+ transport. We hypothesize that there are separately regulated Cs+ and K+ transport systems in poplar.

Down-regulation of UDP-arabinopyranose mutase reduces the proportion of arabinofuranose present in rice cell walls.

Arabinoxylans may account for up to 25% of the mass of grass cell walls. The interactions of these polysaccharides with themselves and with cellulose and lignin is believed to affect the walls physical properties and increase the walls resistance to biochemical conversion to fermentable sugars. Arabinoxylans have a backbone composed of 1,4-linked ß-D-xylosyl residues, some of which are substituted at O-2 or O-3 with single arabinofuranosyl (Araf) residues. The Araf residues are likely transferred from UDP-Araf to the xylan backbone by arabinofuranosyltransferases. UDP-Araf is itself formed from UDP-arabinopyranose (UDP-Arap) by UDP-arabinopyranose mutase (UAM). In this study, RNA interference (RNAi) was used to suppress UAM expression in rice plants and thereby reduce the amounts of UDP-Araf available for cell wall synthesis. Several of the transgenic plants had reduced proportions of Araf in their walls together with a decrease in the extent of substitution of the xylan backbone, and a reduction of between 25% and 80% in ferulic acid and p-coumaric acid contents of the cell walls. Those transgenic plants with >25% reduction in the amounts of Araf were dwarfed and infertile.

Properties and physiological functions of UDP-sugar pyrophosphorylase in Arabidopsis.

UDP-sugar pyrophosphorylase catalyzes the conversion of various monosaccharide 1-phosphates to the respective UDP-sugars in the salvage pathway. Using the genomic database, we cloned a putative gene for UDP-sugar pyrophosphorylase from Arabidopsis. Although relatively stronger expression was detected in the vascular tissue of leaves and the pollen, AtUSP is expressed in most cell types of Arabidopsis, indicating a housekeeping function in nucleotide sugar metabolism. Recombinant AtUSP expressed in Escherichia coli exhibited broad specificity toward monosaccharide 1-phosphates, resulting in the formation of various UDP-sugars such as UDP-glucose, -galactose, -glucuronic acid, -xylose and -L-arabinose. A loss-of-function mutation in the AtUSP gene caused by T-DNA insertion completely abolished male fertility. These results indicate that AtUSP functions as a UDP-sugar pyrophosphorylase in the salvage pathway, and that the generation of UDP-sugars from monosaccharide 1-phosphates catalyzed by AtUSP is essential for pollen development in Arabidopsis.

An alpha-L-arabinofuranosidase/beta-D-xylosidase from immature seeds of radish (Raphanus sativus L.).

The carbohydrate moieties of arabinogalactan proteins (AGPs) are essential for their physiological functions and undergo rapid turnover in vivo. Degradation of the carbohydrate moieties of AGPs seems to occur by concerted action of several glycosidases, among them alpha-L-arabinofuranosidase, beta-D-galactosidase, and beta-D-glucuronidase. Here, a bifunctional alpha-L-arabinofuranosidase/beta-D-xylosidase from immature seeds of radish (Raphanus sativus L.), which hydrolyses alpha-L-arabinofuranosyl residues of the carbohydrate moieties of AGPs, has been cloned by reverse transcriptase-PCR. The gene, designated RsAraf1, contained an open reading frame of 2343 bp (780 amino acids), including a putative signal sequence (33 amino acids) at the N-terminus. RsAraf1 is highly similar to barley alpha-L-arabinofuranosidase/beta-D-xylosidases and belongs to family 3 of the glycosyl hydrolases based on sequence homology. Southern blot analysis revealed that several related genes exist in the radish genome. RsAraf1 is expressed throughout seed development and weakly expressed in young seedlings. It was found that alpha-L-arabinofuranosidase activity in a cell-wall protein fraction prepared from transgenic Arabidopsis plants with enhanced expression of RsAraf1 was significantly higher than that in a wild-type protein fraction; the crude enzyme preparation released L-arabinose from radish AGPs as well as alpha-(1-->5)-arabinan and arabinoxylan. Accordingly, the amount of L-arabinosyl residues in the cell walls of transgenic plants was significantly decreased. These results indicate that RsAraf1 encodes a bifunctional alpha-L-arabinofuranosidase/beta-D-xylosidase and suggest that RsAraf1 is involved in the hydrolysis of the carbohydrate moieties of AGPs in immature radish seeds.