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Title: BIOSYNTHESIS OF UDP-GLUCURONIC ACID IN DEVELOPING SOYBEAN EMBRYOS: POSSIBLE ROLE OF UDP-SUGAR PYROPHOSPHORYLASE

Author
item LITTERER, LYNN - UNIVERSITY OF MINNESOTA
item PLAISANCE, KATHRYN - UNIVERSITY OF MINNESOTA
item Schnurr, Judy
item STOREY, KATHLEEN - UNIVERSITY OF MINNESOTA
item Jung, Hans Joachim
item Gronwald, John
item SOMERS, DAVID - UNIVERSITY OF MINNESOTA

Submitted to: Physiologia Plantarum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/27/2006
Publication Date: 10/1/2006
Citation: Litterer, L.A., Plaisance, K.L., Schnurr, J.A., Storey, K.K., Jung, H.G., Gronwald, J.W., Somers, D.A. 2006. Biosynthesis of UDP-glucuronic acid in developing soybean embryos: possible role of UDP-sugar pyrophosphorylase. Physiologia Plantarum. 128:200-211.

Interpretive Summary: There is a need for the U.S. to develop renewable energy sources to reduce dependence on foreign oil. Soybeans hold considerable promise for the production of biodiesel, a renewable fuel. Increasing the oil content of soybean seeds would increase the value of this crop for biodiesel production. By harvesting energy from the sun, soybeans produce carbon compounds that are used to make oil, protein, and cell walls in seeds. A strategy to increase oil content of soybean seeds without decreasing protein is needed. One approach involves altering metabolic pathways in soybean seeds so that more of the carbon compounds produced by the plant are used to make oil and less are used to make cell walls. However, our knowledge about the composition of cell walls in developing soybean seeds and how they are made is limited. We conducted research to identify cell wall components in developing soybean seed and evaluated the relative importance of two metabolic pathways used to make these components. We found that matrix polysaccharides (MPS) make up a major component of cell walls in developing soybean seeds. Two metabolic pathways, the myo-inositol oxidation (MIO) and nucleotide sugar oxidation (NSO) pathways are known to make MPS in plants, but the relative importance of the two pathways in developing soybean seeds is unknown. To determine which was the major pathway, we compared the activities of marker proteins for each pathway. We found that the activity for the marker protein for the MIO pathway was about 30-fold higher than the activity of the marker protein for the NSO pathway. These results suggest that the MIO pathway is probably the major pathway making MPS in developing soybean seeds. We also identified a new gene, UDP-sugar pyrophosphorylase (USP), in soybean. We characterized the protein that the gene makes and the results suggest that it plays a key role in the MIO pathway. Our research has generated new knowledge about metabolic pathways that regulate the synthesis of cell walls in soybean seeds. This knowledge can be used to plan future research to develop soybean seeds with higher oil content and less cell wall material. Achieving this goal will increase the value of soybean as a bioenergy crop and reduce our dependence on foreign oil imports.

Technical Abstract: During soybean (Glycine max L.) embryo development, cell wall polysaccharides (CWP) derived from UDP-GlcA (uronic acids, arabinose, xylose) exhibited a linear increase during the period of 25 to 45 days after flowering (daf). At embryo maturity, CWP derived from UDP-GlcA accounted for 39% of total CWP. To ascertain the relative importance of the nucleotide sugar oxidation (NSO) and the myo-inositol oxidation (MIO) pathways to UDP-GlcA biosynthesis, UDP-Glc dehydrogenase (UDP-Glc DH, EC 1.1.1.22) and UDP-GlcA pyrophosphorylase (UDP-GlcA PPase, EC 2.7.7.44) activities, respectively, were measured in desalted extracts of developing embryos. UDP-Glc DH and UDP-GlcA PPase activities increased 3.5- and 3.9-fold, respectively, during the period of 25 to 45 daf. However, UDP-GlcA PPase activity was 35- to 50-fold greater than UDP-Glc DH activity. Soybean UDP-sugar pyrophosphorylase (USP1), a homolog of pea USP, and a candidate gene for UDP-GlcA PPase, was cloned and the recombinant enzyme characterized. Recombinant soybean USP1 (71 kDa) exhibited high activity with GlcA 1-P, Glc 1-P, and Gal 1-P, but low activity with Man 1-P, GlcNAc 1-P, and Glc 1-P. Determination of kinetic constants indicated that USP1 has a higher affinity for GlcA 1-P (Km = 0.14 ± 0.02) compared to Glc 1-P (Km = 0.23 ± 0.02). Semi-quantitative RT-PCR was used to measure transcript levels of the UDP-glucose DH (UGD) and USP gene families in developing soybean embryos. Transcript levels, normalized to the 18S rRNA controls, were greater for UGD than USP throughout embryo development. The possibility that USP serves as the UDP-GlcA PPase, the terminal enzyme of the MIO pathway, is discussed.