Progress 10/01/04 to 09/30/05
Outputs
In the previous funding period we discovered the UDP-glucuronic acid decarboxylase genes (UXS) involved in the synthesis of UDP-xylose in plants. We further characterized and biochemically determined the function and kinetic properties of each distinct isoforms. This established unambiguously that all the isoforms of the UXS gene family are involved in the synthesis of UDP-xylose in plants. During the previous granting period we published two research papers and we anticipate publishing one more describing polysaccharide alterations in an uxs2 mutant.
Impacts
Wall polysaccharides play major roles in plant development. Our long-term goal is to identify the molecular components required for the synthesis of xylose-containing wall polysaccharides such as xylan and xyloglucan. Their synthesis, which takes place in different Golgi cisternae, requires xylosyltransferases and UDP-xylose. It remains unclear how the different Golgi-localized xylosyltransferases receive their supply of UDP-xylose since the synthesis of UDP-xylose itself occurs both in the cytosol and in subcellular compartments. The synthesis of UDP-xylose is catalyzed by a total of 6 different UDP-glucuronic acid decarboxylase (Uxs) isoforms. UDP-xylose also feedback inhibits upstream enzymes involved in UDP-sugar synthesis and is a precursor for the formation of other UDP-sugars. How the metabolic flux and balance of these UDP-sugars are regulated and affect the biosynthesis of glycans is far from clear. Therefore, the goals of this project are to (1) biochemically
characterize UXS-specific mutants; (2) define the subcellular localization of each UXS type by immuno-EM; and (3) identify the domain(s) required for targeting each distinct Uxs to its final sub-cellular location. We expect that these studies will lead to the isolation of molecular components involved in the synthesis of xylan and xyloglucan in plants. These polysaccharides are important components of dietary fibers and these studies should have an impact on the USDA's strategic goal of improving the nation's nutrition and health.
Publications
- Gu, X. and M. Bar-Peled. 2004. The biosynthesis of UDP-galacturonic acid in plants. Functional cloning and characterization of Arabidopsis UDP-D-glucuronic acid 4-epimerase. Plant Physiol. 136: 4256-4264.
- Pattathil, S., A. Harper, and M. Bar-Peled. 2005. Biosynthesis of UDP-xylose: Characterization of a membrane bound UXS2. Planta 221: 538-548.
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Progress 10/01/03 to 09/30/04
Outputs
The wall matrix plays major roles in plant development and structure. Our goal is to identify the molecular components required for the synthesis of xylose-containing wall polysaccharides. Their synthesis requires UDP-xylose. The synthesis of UDP-xylose is catalyzed by several distinct UDP-glucuronic acid decarboxylase isoforms (Uxs), whose function is still unclear. The proposed research will elucidate the roles of UDP-xylose related to polysaccharide synthesis by characterizing the isoforms encoded by the UXS gene family. a) Biochemistry. The enzymatic properties of Uxs1,2,3, the soluble and membrane isoform was determine (Harper and Bar-Peled, 2002, Pattathil et al., 2005). Each member of the Uxs generates UDP-Xyl. Unlike fungal Uxs, the plant Uxs is not inhibited by NADH. Nucleosides enhance Uxs2 activity by 30%, but strong inhibition observed with TDP, UDP, UTP, and TTP suggest that small metabolites can up- or down-regulate the activity at the protein level. b)
Cell biology. For subcellular studies, antibodies were raised to specific Uxs2 peptide region; and translation fusion between each Uxs and GFP were made. Uxs3-GFP is enzymatically active and localized to the cytosol, and fractionate with soluble proteins (Pattahil et al., 2005). Both Uxs2-GFP and Uxs1-GFP are membrane proteins and appear to be localized in the Golgi. (Pattahil et al., 2005). Current studies are underway to define their exact subcellular location by immuno-EM. c) Gene expression. Uxs members are transcribed in all tissues (Watt et al. 2004). Microarray gene expression studies indicate that the three distinct Uxs isoforms are expressed in all root cell types and in each of the 3 zones of the root. This suggest that each cell transcribes all three distinct UXSs. d) Genetics. We are characterizing mutant plants with T-DNA inserts in their exons. Further work is underway to chemically characterize the wall sugar composition of the mutants at their phenotype. Future double
cross will be used to deplete xylose from the cell.
Impacts
UDP-xylose is an important building block for synthesis of numerous sugar-containing polymers [carbohydrates and proteins] especially in wheat, rice and woody plants. It also appears to regulate, in-vitro, the synthesis of other nucleotide-sugars. The ability to manipulate UDP-Xyl synthesis could generate plants with different wall properties, a potential valuable trait for wood, fiber and paper industries.
Publications
- 4)Publication Harper AD, Bar-Peled M (2002) Biosynthesis of UDP-Xylose. Cloning and characterization of a novel Arabidopsis gene family, UXS, encoding soluble and putative membrane-bound UDP-Glucuronic acid decarboxylase isoforms, Plant Physiol 130:2188-2198
- Watt G, Leoff C, Harper AD, Bar-Peled M. (2004). A bifunctional 3,5-epimerase/4-keto reductase for nucleotide-rhamnose synthesis in Arabidopsis. Plant Physiol. 134:1337-1346
- Gu X, Bar-Peled, M (2004) The biosynthesis of UDP-galacturonic acid in plants: Functional cloning and characterization of Arabidopsis UDP-D-galacturonic acid 4-epimerase. Plant Physiol 36:4256-4264
- Pattathil S, Harper AD, Bar-Peled M (2005) Biosynthesis of UDP Xylose: characterization of membrane-bound AtUxs2. Planta in press & online
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Progress 10/01/02 to 09/30/03
Outputs
The wall matrix plays major roles in plant development and structure. Our goal is to identify the molecular components required for the synthesis of xylose-containing wall polysaccharides. Their synthesis requires XylT and a supply of UDP-xylose. The synthesis of UDP-xylose is catalyzed by several distinct UDP-glucuronic acid decarboxylase isoforms, whose function is still unclear. The proposed research will elucidate the roles of UDP-xylose related to polysaccharide synthesis by characterizing the isoforms encoded by the UXS gene family. a) Biochemistry. The cloning and enzymatic properties of Uxs3, the soluble isoform was determine (Harper and Bar-Peled, 2002), and current studies are in progress for the membrane-bound Uxs2 and Uxs1. Each Uxs generates UDP-Xyl and its activity does not require exogenous NAD. Uxs2 and Uxs1 are active under similar pH and temperature conditions that differ from Uxs3 (manuscript in preparation). Unlike fungal Uxs, the plant Uxs is not
inhibited by NADH. Nucleosides enhance Uxs2 activity by 30%, but strong inhibition observed with TDP, UDP, UTP, and TTP suggest that small metabolites can up- or down-regulate the activity at the protein level. b) Cell biology. For subcellular studies, antibodies were raised to recombinant GST-fused to specific Uxs peptide regions (40 aa). In addition, translation fusion between each Uxs and GFP were made. Initial studies indicate that recombinant Uxs3-GFP is enzymatically active, localized to the cytosol, and fractionate with soluble proteins. Both recombinant Uxs2-GFP and Uxs1-GFP were found to fractionate with membranes and appear to be localized in the Golgi. Current studies are underway to define their exact subcellular location by immuno-EM. c) To understand roles of isoforms in plants, the expression pattern of Uxs isoforms was compared to isoforms involved in synthesis of a other nucleotide-sugars. Uxs members are transcribed in all tissues (Watt et al. 2004), and the 3
distinct UXSs (type 1, 2, and 3) are expressed in the same root cell type. Microarray gene expression studies indicate that the three distinct Uxs isoforms are expressed in all root cell types (stele, endodermis, epidermis, cortex, lateral root cap) and in each of the 3 zones of the root. More interestingly, within each unique cell type, unique expression patterns are observed for each of the distinct Uxs. This suggests that distinct isoforms of Uxs may have unique functions in a given cell. If our hypothesis is correct, each distinct member of the Uxs family provides a pool of UDP-Xyl for the synthesis of a specific polymer. Results obtained from EM analysis will be valuable to re-examine this hypothesis. Results of this study will also be useful to isolate molecular components involved in the synthesis of xylan and xyloglucan. d) Genetics. Mutant collection and analysis. We obtained several lines with T-DNA inserts in the exon and 5 region of Uxs2 and Uxs1. Unfortunately valuable
time was wasted on the wrong seed lots. Further progress is underway to characterize the mutants at the phenotype and wall. Recently, inserts in Uxs3 were generated, and RNAi transgenic plants are currently being screen.
Impacts
UDP-xylose is an important building block for synthesis of numerous sugar-containing polymers [carbohydrates and proteins]. It also appears to regulate, in-vitro, the synthesis of other nucleotide-sugars. The ability to manipulate UDP-Xyl synthesis could generate plants with different wall properties, a potential valuable trait for wood and paper industries.
Publications
- -Harper, A.D., and Bar-Peled, M. 2002. Biosynthesis of UDP-xylose. Cloning and characterization of a novel Arabidopsis gene family, UXS, encoding soluble and putative membrane-bound UDP-glucuronic acid decarboxylase isoforms. Plant Physiol. 130:2188-2198.
- -Watt, G., Leoff, C., Harper, A.D., and Bar-Peled, M. 2004. A bifunctional 3,5-epimerase/4-keto reductase for nucleotide-rhamnose synthesis in Arabidopsis. Plant Physiol. In press.
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