Understanding the control of cell division in plants by cytokinins (N6-substituted adenines) is becoming increasingly important as a model for cellular regulation in plants. Previous research on cytokinin-induced asymmetrical division in target caulonema cells of the moss Funaria has implicated a spatially controlled rise in intracellular calcium ion concentration as a second messenger that activates calmodulin. A model for cytokinin stimulation of cell division is proposed that suggests that the binding of cytokinin to asymmetrically localized plasma membrane receptors on target cells increases intracellular calcium from both intracellular and extracellular stores leading to calcium-dependent protein kinase activation and protein phosphorylation/dephosphorylation. Both the spatial and temporal changes in intracellular calcium and the protein phosphorylation appear to be involved in the profound cytoplasmic rearrangements seen after hormone treatment. It is becoming increasingly clear that covalent modification of preexisting proteins by phosphorylation is a major mechanism in the regulation of cell growth and differentiation. Calcium and protein phosphorylation may play a synergistic role in cytokinin control of cellular function. The goals of the current project are to: 1. characterize cytokinin binding sites by photoaffinity labeling using 8-azido benzyladenine, 2. analyze the localization of cytokinin binding using antibodies directed against cytokinins, 3. analyze calcium uptake using metallochromic indicators, and 4. monitor the cytoskeletal rearrangements, ion changes and phosphorylation patterns after hormone treatment. This research will increase our understanding of cytokinin signalling, the second messenger role of calcium, and the amplification of cellular response by protein phosphorylation leading to developmental changes in plants.
