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ARS Home » Pacific West Area » Corvallis, Oregon » Forage Seed and Cereal Research Unit » Research » Publications at this Location » Publication #232450
Title: Isolation of legume glycosyltransferases and active site mapping of the Phaseolus lunatus zeatin O-glucosyltransferase ZOG1

Author
item MEEK, LAURA - OSU
item Martin, Ruth
item SHAN, XUEYAN - OSU
item KARPLUS, P - OSU
item MOK, DAVID - OSU
item MOK, MACHTELD - OSU

Submitted to: Journal of Plant Growth Regulation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/25/2008
Publication Date: 4/30/2008
Citation: Meek, L., Martin, R.C., Shan, X., Karplus, P.A., Mok, D.W., Mok, M.C. 2008. Isolation of legume glycosyltransferases and active site mapping of the Phaseolus lunatus zeatin O-glucosyltransferase ZOG1. Journal of Plant Growth Regulation. 27: 192-201.

Interpretive Summary: Cytokinins are essential for normal plant growth and development. Zeatin is a natural occurring cytokinin found in many plant tissues. Appropriate levels of zeatin are maintained in the plant through degradation, biosynthesis and transport. The O-glycosylated forms of zeatin are protected against degradative enzymes and may serve as cytokinin reserves. Two zeatin glycosyltransferase (GT) genes, an O-glucosyltransferase (ZOG1) from Phaseolus lunatus and an O-xylosyltransferase (ZOX1) from P. vulgaris, were previously isolated. Several genes from bean, soybean, rice and tomato similar to ZOG1 were isolated and analyzed. The proteins encoded for by these genes were expressed but were not able to glycosylate zeatin. By analyzing the 3-D model of another glucosyltransferase, Medicago truncatula UGT71G1, and comparing it to the ZOG1 sequence, four regions possibly important to zeatin binding were identified. Recombinant proteins with mutations in these regions were analyzed for enzyme activity and one amino acid within each region (R59, D87, L127, and F149) whose mutation strongly impaired enzyme activity was identified. The new bean and soybean GTs differ from ZOG1 in one (PlGT2 and GmGT2) to three (GmGT1) of these residues. Mutation of one such GT (PlGT2) to render it identical to ZOG1 at the four implicated residues conferred low enzyme activity, providing further support for the importance of these amino acids in recognizing zeatin as substrate.

Technical Abstract: O-Glycosides of the cytokinin zeatin are found in many plant tissues. They provide protection against degradative enzymes and may serve as cytokinin reserves. Two zeatin glycosyltransferase (GT) genes, an O-glucosyltransferase (ZOG1) from Phaseolus lunatus and an O-xylosyltransferase (ZOX1) from P. vulgaris, were previously isolated. Five novel bean and soybean GT genes with high sequence identity to ZOG1 were isolated, sequenced, and expressed, along with two such genes from rice and one from tomato. None of the recombinant proteins showed GT activity with zeatin. By comparing the ZOG1 sequence to the 3-D model of Medicago truncatula UGT71G1, four regions possibly important to zeatin binding were identified, and mutation studies identified one amino acid within each region (R59, D87, L127, and F149) whose mutation strongly impaired enzyme activity. The new bean and soybean GTs differ from ZOG1 in one (PlGT2 and GmGT2) to three (GmGT1) of these residues. Mutation of one such GT (PlGT2) to render it identical to ZOG1 at the four implicated residues conferred low enzyme activity, providing further support for the importance of these amino acids in recognizing zeatin as substrate.