Cytokinins are plant hormones mediating cell division and differentiation. The most important naturally-occurring cytokinin is zeatin, a compound with high biological activity. Various zeatin metabolites are found in plant tissues, some of which are active while others are not, and therefore, it is crucial to understand how zeatin metabolism is regulated. The long-term goal of this project is to identify and characterize elements exerting such controls. Previous research has led to isolation of zeatin-specific enzymes, including O-glycosyltransferases, which transfer a sugar group to zeatin, and cloning of a family of genes corresponding to these enzymes from beans, Arabidopsis, and maize. O-glucosylated zeatin occurs widely in plant tissues where it functions as an inactive, reversible (via hydrolysis) storage form of cytokinin. In contrast to zeatin, O-glucosylated zeatin is resistant to enzymatic breakdown. Consequently, the activities of the glucosyltransferase genes are likely to have significant impact on the level of active cytokinins and, as a consequence, also on plant development. Another important enzyme is zeatin reductase, which converts zeatin to dihydrozeatin, an active cytokinin that is much more stable than zeatin. Although dihydrozeatin occurs in all plants and the reductase has been isolated from plant tissue, genes corresponding to this type of enzyme have not yet been identified. The proposed research focuses on zeatin O-glucosyltransferases and reductases and includes a range of molecular, enzymatic, physiological, and developmental investigations. The specific objectives are to: (1) examine the role of zeatin O-glucosylation in seed germination, transport, and senescence; 2) determine the expression and cytolocalization of zeatin O-glucosyltransferases in Arabidopsis; 3) identify amino acid sequences on the enzymes critical to substrate specificity; and 4) isolate genes encoding zeatin reductases. As to broader impacts, the project involves international collaboration, with Dr. R. Vankova of the Czech Academy of Sciences. The project integrates research with education, with an emphasis on undergraduate training that includes an international experience for students in the laboratory of R. Vankova in Prague. The proposed research also includes collaboration with private industry, which further broadens the impact of the research and enhances the flow of new findings.
