Calcium Regulation of Cytokinin-Stimulated Morphogenesis in Plant Cells

了解细胞分裂素(N6取代的腺嘌呤)对植物细胞分裂的控制，作为植物细胞调控的模型正变得越来越重要。以前关于细胞分裂素诱导苔藓真菌目标茎突细胞不对称分裂的研究表明，作为激活钙调蛋白的第二信使，细胞内钙离子浓度的空间控制上升。提出了一个细胞分裂素刺激细胞分裂的模型，认为细胞分裂素与靶细胞上不对称定位的质膜受体结合，增加了细胞内和细胞外的钙，导致钙依赖的蛋白激酶激活和蛋白磷酸化/去磷酸化。细胞内钙离子和蛋白质磷酸化的空间和时间变化似乎都与激素处理后的细胞质深刻重排有关。越来越清楚的是，通过磷酸化对先前存在的蛋白质进行共价修饰是调节细胞生长和分化的主要机制。钙和蛋白质的磷酸化可能在细胞分裂素对细胞功能的调控中发挥协同作用。本项目的目标是：1.通过使用8-叠氮苯基腺嘌呤的光亲和标记来表征细胞分裂素结合部位，2.使用针对细胞分裂素的抗体分析细胞分裂素结合的定位，3.使用金属显色指示剂分析钙摄取，以及4.监测细胞骨架重排，激素处理后离子变化和磷酸化模式。这项研究将增加我们对细胞分裂素信号的理解，钙的第二信使作用，以及通过蛋白质磷酸化放大细胞反应导致植物发育变化。

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.