Functional Analysis of Arabidopsis CDKA during plant development

拟南芥CDKA在植物发育过程中的功能分析

基本信息

批准号：
18570046
负责人：
SEKINE Masami
金额：
$ 2.57万
依托单位：
Ishikawa Prefectural University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18570046/
关键词：
cell cycle Arabidopsis cyclin gametogenesis plant development 分子生物学

项目摘要

The protein kinase cell division cycle 2 (cdc2) is conserved throughout eukaryotes, and acts as a key regulator of the cell cycle. In plants, A-type cyclin-dependent kinase (CDKA), a homologue of cdc2, has a role throughout the cell cycle. In this study, we show that a loss-of-function mutation in CDKA;1, encoding the only Arabidopsis CDKA, results in male gametophyte lethality. Microspores normally undergo an asymmetric cell division, pollen mitosis I (PMI), to produce bicellular pollen grains. The smaller generative cell undergoes subsequently second mitosis, PMII, to form the two sperm cells, thereby generating tricellular pollen grains. The cdka-1 mutant, however, produces mature bicellular pollen grains, consisting of a single sperm-like cell and a vegetative cell, due to failure of PMII. Thus, CDKA; 1 is essential for cell division of the generative cell in male gametogenesis.In addition, phosphorylation of threonine 161 (T161) in CDKA is required for the activation of its associ … More ated kinase. Western blot analysis revealed that phosphorylation of CDKA T161 increased greatly, in parallel with activation of p13^suct-associated kinase activity when cells were subcultured. Although induced overexpression of a dominant-negative CDKA mutant (D146N) fused with green fluorescent protein (GFP) in tobacco BY-2 cells resulted in elongated cells, overexpression of this CDKA mutant possessing a non-phosphorylatable alanine in place of T161 (T161A) had no effect on cellular growth. However, immunoprecipitates of both GFP-fused CDKAs exhibited no histone H1 kinase activity, suggesting that both mutants formed kinase-inactive complexes. To further elucidate the role of T161 phosphorylation, we used a loss-of-function mutation of Arabidopsis CDKA; 1. Introduction of CDKA; 1(T161E)-GFP, which mimics phosphorylated T161, resulted in successful complementation of the cdka-1 mutation, whereas no recovery was observed when CDKA; 1(T161A)-GFP was introduced. Thus, phosphorylation of T161 in Arabidopsis CDKA;1 is essential for cell division during male gametogenesis. Less

蛋白激酶细胞分裂周期 2 (cdc2) 在整个真核生物中是保守的，并作为细胞周期的关键调节因子，在植物中，A 型细胞周期蛋白依赖性激酶 (CDKA)（cdc2 的同源物）在整个细胞周期中发挥作用。在这项研究中，我们发现编码唯一拟南芥 CDKA 的 CDKA;1 的功能丧失突变通常会导致小孢子死亡。不对称细胞分裂，花粉有丝分裂 I (PMI)，产生双细胞花粉粒。较小的生殖细胞经历随后的第二次有丝分裂，PMII，形成两个精子细胞，从而产生三细胞花粉粒。然而，cdka-1 突变体产生。由于 PMII 的失败，成熟的双细胞花粉粒由单个精子样细胞和营养细胞组成，因此，CDKA; 1 对于雄性生殖细胞的细胞分裂至关重要。此外，CDKA 中苏氨酸 161 (T161) 的磷酸化是激活其相关激酶所必需的。蛋白质印迹分析显示，CDKA T161 的磷酸化大大增加，与 p13^suct 相关激酶的激活同时发生。尽管在烟草中诱导了与绿色荧光蛋白（GFP）融合的显性失活CDKA突变体（D146N）的过度表达。 BY-2 细胞导致细胞伸长，这种具有不可磷酸化丙氨酸代替 T161 (T161A) 的 CDKA 突变体的过度表达对细胞生长没有影响，然而，两种 GFP 融合的 CDKA 的免疫沉淀物均不表现出组蛋白 H1 激酶活性。这表明两种突变体都形成了激酶失活复合物。为了进一步阐明 T161 磷酸化的作用，我们使用了功能丧失突变。拟南芥CDKA；1.引入模仿磷酸化T161的CDKA；1(T161E)-GFP，导致cdka-1突变的成功互补，而引入CDKA；1(T161A)-GFP则没有观察到恢复。，拟南芥 CDKA 中 T161 的磷酸化；1 对于雄性配子发生过程中的细胞分裂至关重要。