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Zeatin and its derivatives are major consituents of higher plant cytokinins. Metabolic steps modifying the isoprenoid side chain, such as O-glycosylation, are expected to have a direct bearing on cytokinin-mediated processes. To examine this possibility, transgenic tobacco plants were generated harboring a gene (ZOG1) encoding a zeatin O-glucosyltransferase from Phaseolus lunatus under the control of a constitutive (35S) and an inducible (Tet) promoter. The presence of the transgene resulted in elevated enzyme production and conversion of exogenous zeatin to its O-glucoside, confirming the expression of the ZOG1 gene in transgenic plants. Endogenous O-glucosylzeatin was increased from less than 1 pmol per g fresh weight in leaves and roots of controls to 26 and 68 pmol per g fresh weight in leaves and roots of 35S-ZOG1 transformants, respectively. In cytokinin/auxin interaction experiments, Tet-ZOG1 leaf discs, in the presence of tetracycline, required 10-fold higher zeatin concentrations for the formation of shoots and callus than the controls. In 35S-ZOG1 plants, developmental changes included adventitious root formation on the lower stems, shorter stature, and axillary shoot growth. Thus, increased zeatin O-glucosylation in detached, cytokinin-dependent tissues leads to a shift in the response to exogenous zeatin indicative of cytokinin sequestering. In whole plants the effect can simulate a reduction or a rise in cytokinin activity depending on the tissue and stage of development. The use of tissue- and stagespecific promoters in the future will allow more precise analyses and targeted growth alterations.