OBJECTIVES: Investigate the importance of the myo-inositol and nucleotide sugar oxidation pathways in the biosynthesis of cell wall matrix polysaccharides in alfalfa. Characterize the myo-inositol and nucleotide sugar oxidation pathways in transgenic alfalfa modified with genes that alter cell wall matrix polysaccharide biosynthesis.

APPROACH: Characterize the expression of UDP-glucose dehydrogenase and glucuronic acid 1-P pyrophosphorylase in alfalfa. Compare expression of UDP-glucose dehydrogenase and glucuronic acid 1-P pyrophosphorylase with changes in cell wall structure and composition in developing alfalfa stems.

KEYWORDS: alfalfa; cell wall; ethanol; fuel; fuel sources; molecular biology; energy sources; biochemistry; plant physiology; engineering; metabolic pathways; inositol; transgenic plants; carbohydrate metabolism; gene action; gene expression; glucuronic acid; dehydrogenases; glucose; pyrophosphorylases; renewable resources; bioconversion; pectin; hemicellulose

PROGRESS: 2006/01 TO 2006/12
A large proportion of plant cell walls consists of matrix polysaccharides (pectin and hemicellulose). In alfalfa, the amount and structure of these polysaccharides influence the efficiency of converting cell wall biomass into ethanol. The myo-inositol oxidation pathway plays a key role in the synthesis of cell wall matrix polysaccharides in plants and UDP-sugar pyrophosphorylase (USP) is an important protein in this pathway. In earlier research conducted with the model plant Arabidopsis, we identified the gene that makes the USP protein. Our research also provided the first report that USP plays a critical role in cell wall synthesis in pollen. In current research to better define the role of USP in cell wall development, we prepared an antibody to the purified Arabidopsis USP protein. Research conducted with the antibody indicated that there are two isoforms of USP in Arabidopsis tissues. Immunoprecipitation assays conducted with the antibody indicated that USP is responsible for UDP-glucuronic acid pyrophosphorylase activity measured in Arabidopsis tissue extracts. These results provide evidence that USP serves as the terminal enzyme of the myo-inositol pathway and therefore plays a key role in cell wall synthesis. We have also examined the role of USP in cell wall formation in developing soybean embryos. Both gene expression and enzyme activity of USP increased during the growth of soybean embryos suggesting that USP plays an important role in cell wall synthesis. In future research, we plan to use gene silencing to block USP in developing soybean embryos and determine whether this extra carbon will be redirected toward the synthesis of more seed oil and protein. The results of this research have advanced understanding of an important metabolic pathway that regulates the formation and composition of plant cell walls. This new knowledge will be used to plan future research to modify alfalfa cell walls and increase the potential of alfalfa as a bioenergy crop. This knowledge will also be used to develop molecular approaches to redirect carbon into metabolic pathways that increase soybean seed oil and protein content.

IMPACT: 2006/01 TO 2006/12
There is a need to develop renewable energy sources to decrease dependence on imported fossil fuels and reduce the negative impact of fossil fuel use on the environment. In Minnesota, alfalfa offers considerable potential for development as a cellulosic feedstock for ethanol production. Basic knowledge generated by this research project will be used to develop transgenic alfalfa varieties that exhibit enhanced potential for cellulosic ethanol production. Knowledge regarding the regulation of cell wall synthesis in developing soybean seeds will be used to increase oil and protein content.

PUBLICATIONS (not previously reported): 2006/01 TO 2006/12
1. Litterer, L.A., Schnurr, J.A, Plaisance, K.L., Storey, K.K., Gronwald, J.W., Somers, D.A. 2006. Characterization and expression of Arabidopsis UDP-sugar pyrophosphorylase. Plant Physiology and Biochemistry 44:171-180.
2. Schnurr, J.A., Storey, K.K., Jung, H.G., Somers, D.A., Gronwald, J.W. 2006. UDP-sugar pyrophosphorylase is essential for pollen development in Arabidopsis. Planta 224:520-532.
3. 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.

PROJECT CONTACT:
Name: Gronwald, J. W.
Phone: 612-625-8186
Fax: 651-649-5058
Email: gronw001@umn.edu