The Biologically Active Form of Cytokinin Is the Free Base
Classic experiments to determine the active forms of cytokinin generally measured the response of tissues to exogenously supplied cytokinin species. These experiments are complicated by the fact that applied cytokinins are readily converted to other forms, such as the interconversion of cis- and trans-zeatin by zeatin isomerase and the removal of O-linked sugar moieties by glycosidase enzymes. The recent identification of cytokinin receptors has allowed this question to be addressed directly. To test if a cytokinin species is active, the compound is added to a heterologous system in which a plant cytokinin receptor is expressed and in which its activation can be measured, such as in a modified strain of the bacterium E. coli. The host E. coli cell likely lacks the ability to modify or interconvert cytokinin species. If the compound can activate a cytokinin receptor in E. coli, and if binding to a cytokinin receptor can be confirmed in a direct binding assay, then the cytokinin derivative is considered active. These experiments have shown that the most active forms of cytokinins are the free bases; the various riboside derivatives are also active, but display reduced activity, and in some cases the ribotide forms also display some activity (Romanov et al. 2006). The sugar conjugates are inactive. The synthetic diphenylurea-type cytokinins such as thidiazuron, which are structurally distinct from the aminopurine cytokinins, also bind to and activate the same cytokinin receptors. Interestingly, the different receptors show distinct ligand preferences. For example, trans-zeatin ribotide is able to activate some, but not all cytokinin receptors from Arabidopsis. There are also differences between species in the forms of cytokinins that are active. For example, cis-zeatin is capable of activating cytokinin receptors from maize, but not from Arabidopsis.