Abstract
Cytokinins primarily promote cell division to regulate cell growth and differentiation, and also affect apical dominance, axillary bud growth, and leaf senescence. Cytokinin is perceived on the cytokinin receptors CRE1/AHK4 and AHK3 that function as the members of a hybrid sensor histidine kinases and regulates the subset of cytokinin-responsive gene expression. These receptors have a putative ligand binding domain called cyclases/histidine kinases associated sensor extracellular (CHASE) domain and this domain has been thought to be implicated in hormone binding. To identify the cytokinin binding site on CHASE domain of AHK4 receptor, we developed photoaffinity cytokinin probe. The cytokinin probes were designed to show the dual-function on photo-labeling and molecular-tagging for post-labeling. The cytokinin probe has aryl-azide and alkyl-azide for photo-reaction and click-reaction tag with biotin/fluorescent marker, respectively. The cytokinin activity of probe was evaluated by cytokinin-responsive ARR5-GUS reporter line and E. coil bacterial reporter assay expressing AHK4 receptor. The probes were found to be active cytokinin and would be promising tools to dissect cytokinin recognition.
