New Generation of Arabidopsis thaliana Cytokinin Oxidase/Dehydrogenase Inhibitors Affect Shoot/Root Growth and Seed Yield

This chapter describes the preparation of a new group of cytokinin oxidase/dehydrogenase (CKX) enzyme inhibitors usable for different agricultural applications. Recently developed CKX inhibitors, 2-chloro-6-(3-methoxyphenylamino)purine (INCYDE) and 2-fluoro-6-(3-methoxyphenylamino)purine (INCYDE-F) were modified by the tetrahydropyran-2-yl (THP) group at the N9-position to obtain 2-chloro-6-(3-methoxyanilino)-9-(tetrahydropyran-2-yl)purine (INCYDE-THP) and 2-fluoro-6-(3-methoxyanilino)-9-(tetrahydropyran-2-yl)purine (INCYDE-F-THP), respectively. These compounds were used to inhibit CKX from Arabidopsis thaliana (AtCKX2). Further, their biological activity was investigated in a receptor binding assay where its ability to activate cytokinin receptors was assessed. Activation of the cytokinin signalling pathway was determined by ARR5/GUS reporter gene assay. All the assays showed that INCYDE-THP is a medium AtCKX2 inhibitor, average cytokinin receptor agonist as well as inducer of ARR5 gene expression (a member of the A-type response regulator family classified as cytokinin primary response genes). INCYDE-F-THP was, however, found to be almost inactive. Their biological activity was also compared in three different bioassays based on the stimulation of tobacco callus growth, the retention of chlorophyll in excised wheat leaves and the dark induction of betacyanin synthesis in Amaranthus cotyledons. The two compounds were, however, only mildly active (<10% of BAP) in tobacco callus and Amaranthus bioassay and almost inactive in the senescence test. Finally, in vitro and in vivo experiments carried out with INCYDE-THP on different plant species showed positive effects on plant growth and development. In summary, new perspective inhibitors of AtCKX2 were found. These positive effects are also reflected in increased yields of some crops, such as wheat, corn and oilseed rape, and some vegetables, especially when applied by watering. In conclusion, INCYDE-THP is a promising candidate for plant irrigation and seed coating that could contribute as an agrochemical approach to the growth of shoot/root biomass than standard molecular biology methods.