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GDP-L-fucose is the activated nucleotide sugar form of L-fucose, which is a constituent of many structural polysaccharides and glycoproteins in various organisms. The de novo synthesis of GDP-L-fucose from GDP-D-mannose encompasses three catalytic steps, a 4,6-dehydration, a 3,5-epimerization, and a 4-reduction. The mur1 mutant of Arabidopsis is deficient in L-fucose in the shoot and is rescued by growth in the presence of exogenously supplied L-fucose. Biochemical assays of the de novo pathway for the synthesis of GDP-L-fucose indicated that mur1 was blocked in the first nucleotide sugar interconversion step, a GDP-Dmannose-4,6-dehydratase. An expressed sequence tag was identified that showed significant sequence similarity to proposed bacterial GDP-D-mannose-4,6-dehydratases and was tightly linked to the mur1 locus. A full-length clone was isolated from a cDNA library, and its coding region was expressed in Escherichia coli. The recombinant protein exhibited GDP-D-mannose-4,6-dehydratase activity in vitro and was able to complement mur1 extracts in vitro to complete the pathway for the synthesis of GDP-L-fucose. All seven mur1 alleles investigated showed single point mutations in the coding region for the 4,6-dehydratase, confirming that it represents the MUR1 gene.L-Fucose (6-deoxy-L-galactose) is a monosaccharide found in a diverse array of organisms. The sugar is a known component of bacterial lipopolysaccharides, mammalian and plant glycoproteins, and polysaccharides of plant cell walls such as xyloglucan and rhamnogalacturonans I and II. The precise function of L-fucose within these polysaccharides is not clear, but it may stabilize conformations of xyloglucan, which can efficiently bind to cellulose microfibrils (1), possibly aiding in cell wall integrity. Furthermore, xyloglucan fucosylation is essential for the biological activity of some xyloglucan-derived oligosaccharides (2). The pathway for the synthesis of L-fucose has been studied biochemically, but genes for the corresponding enzymes have not been cloned from any eukaryote.GDP-L-fucose (guanosine-diphospho-L-fucose) is the activated form of this sugar, synthesized de novo from GDP-Dmannose via a three-step mechanism or through a salvage pathway involving phosphorylation of free L-fucose and subsequent nucleoside-diphosphate attachment (3-5). The de novo pathway for GDP-L-fucose production is shown in Fig. 1. The first step is catalyzed by GDP-D-mannose-4,6-dehydratase and involves the formation of the intermediate GDP-4-keto-6-deoxy-D-mannose, which is then used in the second and third steps of the pathway by 3,5-epimerase and 4-reductase activities to yield GDP-L-fucose. The pathway was initially elucidated in bacteria but has since been characterized in mammalian and plant systems (6)(7)(8)(9)(10)(11).Recently an L-fucose-deficient cell wall mutant, mur1, was isolated from Arabidopsis thaliana and characterized phenotypically (12). Eight recessive mur1 alleles were obtained from this screen, most of which exhibit 50-to 200-fold reducti...

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With the increasing number of eukaryotic genomes available, high-throughput automated tools for identification of regulatory DNA sequences are becoming increasingly feasible. Several computational approaches for the prediction of regulatory elements were recently developed. Here we combine the prediction of clusters of binding sites for transcription factors with context information taken from genome annotations. Target Explorer automates the entire process from the creation of a customized library of binding sites for known transcription factors through the prediction and annotation of putative target genes that are potentially regulated by these factors. It was specifically designed for the well-annotated Drosophila melanogaster genome, but most options can be used for sequences from other genomes as well. Target Explorer is available at http://trantor.bioc.columbia.edu/Target_Explorer/

SummaryBecause of the limited lysine content in corn grain, synthetic lysine supplements are added to corn meal-based rations for animal feed. The development of biotechnology, combined with the understanding of plant lysine metabolism, provides an alternative solution for increasing corn lysine content through genetic engineering. Here, we report that by suppressing lysine catabolism, transgenic maize kernels accumulated a significant amount of lysine. This was achieved by RNA interference (RNAi) through the endosperm-specific expression of an inverted-repeat (IR) sequence targeting the maize bifunctional lysine degradation enzyme, lysine-ketoglutarate reductase/saccharopine dehydrogenase (ZLKR/SDH). Although plant-short interfering RNA (siRNA) were reported to lack tissue specificity due to systemic spreading, we confirmed that the suppression of ZLKR/SDH in developing transgenic kernels was restricted to endosperm tissue. Furthermore, results from our cloning and sequencing of siRNA suggested the absence of transitive RNAi.These results support the practical use of RNAi for plant genetic engineering to specifically target gene suppression in desired tissues without eliciting systemic spreading and the transitive nature of plant RNAi silencing.

ABSTRACT

GDP-D-mannose 4,6-dehydratase catalyzes the first step in the de novo synthesis of GDP-Lfucose, the activated form of L-fucose, which is a component of glycoconjugates in plants known to be important to the development and strength of stem tissues. We have determined the three-dimensional structure of the MUR1 dehydratase isoform from Arabidopsis thaliana complexed with its NADPH cofactor as well as with the ligands GDP and GDP-D-rhamnose. MUR1 is a member of the nucleosidediphosphosugar modifying subclass of the short-chain dehydrogenase/reductase enzyme family, having homologous structures and a conserved catalytic triad of Lys, Tyr, and Ser/Thr residues. MUR1 is the first member of this subfamily to be observed as a tetramer, the interface of which reveals a close and intimate overlap of neighboring NADP + -binding sites. The GDP moiety of the substrate also binds in an unusual syn conformation. The protein-ligand interactions around the hexose moiety of the substrate support the importance of the conserved triad residues and an additional Glu side chain serving as a general base for catalysis. Phe and Arg side chains close to the hexose ring may serve to confer substrate specificity at the O2 position. In the MUR1/GDP-D-rhamnose complex, a single unique monomer within the protein tetramer that has an unoccupied substrate site highlights the conformational changes that accompany substrate binding and may suggest the existence of negative cooperativity in MUR1 function.The 6-deoxy monosaccharide L-fucose is found as a component of glycoconjugates in organisms from bacteria to mammals and has a diverse range of functions. In humans, L-fucose is most notably an important constituent of glycoproteins such as the blood group antigens as well as cell surface carbohydrate ligands of the cell adhesion family of selectins involved in functions such as inflammation and the immune response (1). Among other organisms, L-fucosecontaining glycoconjugates are involved in developmental signaling in Drosophila (2), are critical components of bacterial cell walls where they may play a role in pathogenicity, and among rhizobial organisms, are components of Nod factors, influencing nodulation efficiency and host specificity (3, 4). In plants, L-fucose has important structural functions as a component of glycoproteins and cell wall polysaccharides, such as xyloglucan and rhamnogalacturonans I and II. Xyloglucan molecules cross-link cellulose microfibrils, one of the major load-bearing elements of the cell wall, and may be involved in the regulation of extension growth. It has been proposed that L-fucose may stabilize conformations that effectively bind cellulose (5, 6); however, this hypothesis has recently been challenged on the basis of the normal growth habit and wall strength of an Arabidopsis mutant specifically deficient in xyloglucan fucosylation (7). Although the function of rhamnogalacturonan II is unknown, the presence of fucose appears to be important for formation of the normal borate di-ester cross-linked f...

The monoclonal antibody, CCRC-M1, which recognizes a fucose (Fuc)-containing epitope found principally in the cell wall polysaccharide xyloglucan, was used to determine the distribution of this epitope throughout the mur1 mutant of Arabidopsis. Immunofluorescent labeling of whole seedlings revealed that mur1 root hairs are stained heavily by CCRC-M1, whereas the body of the root remains unstained or only lightly stained. Immunogold labeling showed that CCRC-M1 labeling within themur1 root is specific to particular cell walls and cell types. CCRC-M1 labels all cell walls at the apex of primary roots 2 d and older and the apices of mature lateral roots, but does not bind to cell walls in lateral root initials. Labeling with CCRC-M1 decreases in mur1 root cells that are undergoing rapid elongation growth such that, in the mature portions of primary and lateral roots, only the walls of pericycle cells and the outer walls of epidermal cells are labeled. Growth of the mutant on Fuc-containing media restores wild-type labeling, where all cell walls are labeled by the CCRC-M1 antibody. No labeling was observed in mur1hypocotyls, shoots, or leaves; stipules are labeled. CCRC-M1 does label pollen grains within anthers and pollen tube walls. These results suggest the Fuc destined for incorporation into xyloglucan is synthesized using one or the other or both isoforms of GDP-d-mannose 4,6-dehydratase, depending on the cell type and/or developmental state of the cell.