| Research Abstract |
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
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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
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