N-glucosylation of cytokinins by glycosyltransferases of Arabidopsis thaliana

Cytokinins are plant hormones that can be glucosylated to form O-glucosides and N-glucosides. The glycoconjugates are inactive and are thought to play a role in homeostasis of the hormones. Although O-glucosyltransferases have been identified that recognize cytokinins, the enzymes involved in N-glucosylation have not been identified even though the process has been recognized for many years. This study utilizes a screening strategy in which 105 recombinant glycosyltransferases (UGTs) of Arabidopsis have been analyzed for catalytic activity toward the classical cytokinins: trans-zeatin, dihydrozeatin, N-6-benzyladenine, N-6-isopentenyladenine, and kinetin. Five UGTs were identified in the screen. UGT76C1 and UGT76C2 recognized all cytokinins and glucosylated the hormones at the N-7 and N-9 positions. UGT85A1, UGT73C5, and UGT73C1 recognized trans-zeatin and dihydrozeatin, which have an available hydroxyl group for glucosylation and formed the O-glucosides. The biochemical characteristics of the N-glucosyltransferases were analyzed, and highly effective inhibitors of their activities were identified. Constitutive overexpression of UGT76C1 in transgenic Arabidopsis confirmed that the recombinant enzyme functioned in vivo to glucosylate cytokinin applied to the plant. The role of the N-glucosyltransferases in cytokinin metabolism is discussed.