UDP-glucuronic acid pyrophosphorylase

Plant cell walls consist of large amounts of matrix polysaccharides beside the cellulose fibrils. These include hemicelluloses and pectins. The polymers are predominantly synthesized from a common precursor (UDP- glucuronic acid), which accounts for roughly 50% of the cell wall biomass of the model plant Arabidopsis thaliana. Two biosynthetic routes to UDP-glucuronic acid are known for Arabidopsis. Both pathways start from the cellular sugar metabolism with an energitically irreversible entry reaction. This results in a control of metabolites used for storage compounds on one hand and cell wall biosynthesis on the other hand. We will analyze an enzyme of the myo-inositol oxygenation pathway (widely absent in biochemistry books), which catalyzes the terminal conversion of UDP-glucuronic acid-1-phosphate into UDP-glucuronic acid. An alternative function for the pyrophosphorylase might be the recycling of sugar-1-phosphates. Both hypotheses will be tested in several genetic complementation approaches to identify the function of the enzyme for plants. An insertion mutant in the gene for the pyrophosphorylase is lethal for normal pollen development in Arabidopsis. This phenotype can be hardly explained by our current knowledge about the nucleotide conversion pathways in plants. Using a combination of cell biological, biochemical and genetic approaches we will analyze the function of this enzyme for the normal development of Arabidopsis. Heterozygous mutants in the gene of the pyrophosphorylase will be complemented with different DNA-constructs. Cell walls of mutants will be analyzed in depth using a combination of electron microscopy techniques and immuno-light microscopy with monoclonal antibodies against carbohydrate epitopes. This analysis will be complemented by a biochemical analysis of cell walls (and polymers thereof). We will also investigate why no compensation of the defect in the pyrophosphorylase gene occurs by the (major) pathway to UDP-glucuronic acid catalyzed by the enzyme UDP-glucose dehydrogenase. The project will likely contribute novel aspects to the biosynthesis of ascorbic acid in plants. We expect more precise arguments whether an animal like pathway to ascorbate is functional in Arabidopsis.

We are analyzing the question, how solar energy in products from photosynthesis can be long-term stored in plants and used later as food or raw material for humans and within the ecosystem.Sugars are building blocks for cell walls in plants but also needed for connective tissue in animals and humans. We investigate the production of chemically activated energy-rich sugars (nucleotide sugars) by cells. These compounds can be further used for polymer biosynthesis. In leafy plants, roughly 50 % of the cell wall biomass is synthesized from UDP-glucuronic acid. Two pathways for the synthesis of it exist in parallel. The terminal step of the less well known pathway via myo-inositol was investigated in this project using a combination of biochemical, cell biological and molecular methods. The enzyme is called UDP sugar pyrophosphorylase (USP). A plant mutant with a defect in USP does not develop fertile pollen and is thus not viable. Using genetic methods we tried to work around the possible defect. The only successful approach was to use a system in which a residual activity (3-5 %) of USP is retained. Using this genetic approach we found out that the initially favored role of USP, a by pathway to synthesize UDP-glucuronic acid via myo-inositol, is not the major role of USP. Rather it turned out, that a role in recycling of nucleotide sugars seems to be very important. The recycling of arabinose and xylose is an important role for USP in plants. The experiments also allowed us to draw the conclusion that cellular compartimentalization of nucleotide sugar biosynthesis is critical. According to our current knowledge the recycling pathway for nucleotide sugars are located in the cytoplasm whereas the de novo pathways are mainly limited to the Golgi apparatus. Changes in the capacity of both routes have therefore different outcomes for the cell. This unexpected finding has to be worked on in the future to solve the interplay between de novo and recycling pathways.