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Title: CHARACTERIZATION AND EXPRESSION OF ARABIDOPSIS UDP-SUGAR PYROPHOSPHORYLASE

Author
item LITTERER, LYNN - UNIVERSITY OF MINNESOTA
item Plaisance, Kathryn
item Schnurr, Judy
item STOREY, KATHLEEN - UNIVERSITY OF MINNESOTA
item Gronwald, John
item SOMERS, DAVID - UNIVERSITY OF MINNESOTA

Submitted to: Plant Physiology and Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/9/2006
Publication Date: 6/1/2006
Citation: 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.

Interpretive Summary: A large proportion of plant cell walls consist of matrix sugars (pectin and hemicellulose). In forages, such as alfalfa, the amount and structure of these compounds plays an important role in determining digestibility by dairy cows. Cell wall matrix sugars also influence the efficiency of converting cell wall biomass into bioethanol. Two different pathways are involved in the synthesis of cell wall matrix sugars; the nucleotide sugar oxidation (NSO) pathway and the inositol oxidation (IO) pathway. Greater understanding of factors that regulate these two pathways is needed to improve the sugar content of alfalfa cell walls. The terminal step in the IO pathway involves a protein call UDP-sugar pyrophosphorylase (USP) that activates sugars so that they can be incorporated into the cell wall. Little is known about the role that this protein plays in the regulation of the IO pathway. To learn more about USP, we cloned the gene from the model plant Arabidopsis. We expressed the gene and characterized the protein that it makes. We learned that the USP protein requires magnesium to function and can rapidly activate sugars that are incorporated into cell walls. We also learned that the USP gene is expressed at high levels in stems, flowers, and young leaves. This research generated new knowledge about a key protein that regulates the synthesis of matrix sugars in plant cell walls. The results indicate that the USP gene is a promising candidate for future research to engineer alfalfa cell walls to improve sugar composition. The new knowledge generated in this research can be utilized to improve the digestibility of alfalfa by dairy cows and increase the potential for developing this crop as a bioethanol feedstock.

Technical Abstract: The myo-inositol oxidation pathway is an important source of UDP-glucuronic acid (UDP-GlcUA) for plant cell wall biosynthesis. UDP-sugar pyrophosphorylase (USP) is the terminal enzyme in this pathway. To better understand the contribution of USP to UDP-GlcUA synthesis, we used a bioinformatics approach to identify this gene (atUSP) from Arabidopsis thaliana. Phylogenetic analysis indicated that AtUSP belongs to a pyrophosphorylase family distantly related to the UDP-Glc and UDP-GlcNAc pyrophosphorylase families. The recombinant USP enzyme, purified from E. coli, exhibited high activity with GlcUA-1-P, Glc-1-P, and Gal-1-P, but very low activity with GlcNAc-1-P, Fuc-1-P, Man-1-P, inositol-1-P, or Glc-6-P. Activity in the pyrophosphorylase direction was not significantly inhibited by UDP-Xyl or UDP-Ara. The enzyme was specific in its requirement for Mg2+ and preference for UTP. AtUSP had apparent Km values for GlcUA-1-P, Glc-1-P, and UTP of 0.13 mM, 0.42 mM, and 0.14 mM, respectively. In the reverse direction, the apparent Km values were 0.56 mM for UDP-GlcUA, 0.72 mM for UDP-Glc, and 0.15 mM for pyrophosphate. Semi-quantitative RT-PCR analysis showed high levels of AtUSP transcript accumulation in flowers, stems, and young leaves.