Functional Analysis of Arabidopsis CDKA during plant development

蛋白激酶细胞分裂周期2 （cdc2）在真核生物中是保守的，是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）是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CDKA中的一个功能缺失突变；1，编码唯一的拟南芥CDKA，导致雄性配子体致死。小孢子通常经历不对称的细胞分裂，即花粉有丝分裂I (PMI)，产生双细胞花粉粒。较小的生殖细胞随后进行第二次有丝分裂，即PMII，形成两个精子细胞，从而产生三细胞花粉粒。然而，由于PMII的失败，cdka-1突变体产生成熟的双细胞花粉粒，由单个精子样细胞和一个营养细胞组成。因此,CDKA;在雄性配子体发生中，1对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的更多关联激酶所必需的。Western blot分析显示，当细胞传代培养时，CDKA T161的磷酸化水平显著升高，与p13^suct相关激酶活性的激活平行。虽然诱导过表达与绿色荧光蛋白（GFP）融合的显性阴性CDKA突变体（D146N）在烟草BY-2细胞中导致细胞伸长，但过表达这种具有不可磷酸化丙氨酸的CDKA突变体代替T161 （T161A）对细胞生长没有影响。然而，两种gfp融合的cdka的免疫沉淀均未表现出组蛋白H1激酶活性，这表明两种突变体均形成了激酶无活性复合物。为了进一步阐明T161磷酸化的作用，我们使用了拟南芥CDKA的功能缺失突变；1. CDKA简介；1(T161E)-GFP，模拟磷酸化的T161，导致CDKA -1突变的成功互补，而当CDKA；引入了1(T161A)-GFP。因此，拟南芥CDKA中T161的磷酸化；1在雄性配子体发生过程中对细胞分裂至关重要。少

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