Meristem construction and control of CDK activities in plants

本研究旨在揭示植物细胞周期蛋白激酶活性在细胞增殖中的调控机制。分生组织为了使CDK完全活化，不仅需要细胞周期蛋白结合，还需要磷酸化。这种磷酸化由CDK活化激酶（CAK）催化。在拟南芥中，存在四种CAK，CAK 1至CAK 4。其中，CAK 2和CAK 4在酶活性方面可被归类为脊椎动物型CAK，但各自显示出不同的底物特异性。即，CAK 2和CAK 4分别优先磷酸化CDK和RNA聚合酶II的最大亚基的羧基末端结构域（TD）。此外，我们发现CAK 1磷酸化CAK 2和CAK 4，并激活CAK 4的CTD激酶活性。我们利用糖皮质激素诱导系统在水稻中过量表达了B2型细胞周期蛋白CycB 2;2的cDNA。我们的分析表明，过量表达导致根生长加速，而细胞伸长没有任何变化。这表明CYCB 2;2过表达激活了根分生组织中的细胞增殖。通过体外pull-down实验，我们发现CYCB 2;2与CDKB 2特异性相互作用，这种相互作用增强了CDKB 2的激酶活性。基于这些数据，我们得出结论，CYCB 2;2过表达上调CDKB 2活性，这反过来又促进了从G2期到M期的转换，加快了细胞周期的持续时间在根分生组织。

In this project, we aimed to reveal the regulatory :mechanisms underlying CDK activities that control cell proliferation in plant. meristems. For the full activation of CDKs, not only cyclin binding but also phosphorylation is required. This phosphorylation is catalyzed by CDK-activating kinase (CAK). In Arabidopsis, there exist four CAKs, CAKI to CAK4. Among them, CAK2 and CAK4 can be classified into the vertebrate-type CAK in terms of enzyme activity, whereas each shows different substrate specificies. Namely, CAK2 and CAK4 preferentially phosphorylated CDK and the carboxy-terminal domain (TD) of the largest subunit of RNA polymerase II, respectively. Moreover, we found that CAK 1 phosphorylates CAK2 and CAK4, and activate the CTD-kinase activity of CAK4. These results suggested that Arabidopsis has a CDK-and CTD-phosphorylation cascade mediated by three CAKs.We overexpressed the cDNA of a B2-type cyclin, CycB2;2, in rice plants by using a glucocorticoid induction system. Our analyses revealed that the overexpression resulted in acceleration of root growth without any change in cell elongation. This indicated that cell proliferation in the root meristem had been activated by CYCB2;2 overexpression. By in vitro pull-down experiments, we showed that CYCB2;2 specifically interacted with CDKB2, and this interaction enhanced the kinase activity of CDKB2. Based on these data, we concluded that CYCB2;2 overexpression up-regulated the CDKB2 activity, which in turn promoted the transition from G2-to-M phase to accelerate the cell cycle duration in the root meristem.