Chang M, Wang B, Chen X, Wu R
Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA.
Plant Mol Biol. 1999 Jan;39(1):105-15. doi: 10.1023/a:1006152223183.
To understand the molecular mechanism of gibberellin-dependent gene regulation, the effect of three phosphatase inhibitors on the germination of rice seeds and the expression of a target gene, the alpha-amylase gene, Osamy-c, were measured. We found that okadaic acid, microcystin-LR, and calyculin A, which are known to specifically inhibit Ser/Thr phosphatases 1 and 2A, strongly inhibit the expression of the Osamy-c and may be involved in the germination of rice seeds. The protein phosphatase enzyme activity assays showed that there is no obvious effect of GA3 on total PP1/PP2A activities. To further understand the possible role of protein phosphatases 1 and 2A in the GA-dependent expression of Osamy-c, we isolated cDNA clones encoding protein phosphatase 1 and protein phosphatase 2A from a rice aleurone cDNA library. These were designated OsPP1c and OsPP2Ac, respectively. Comparison of the deduced amino acid sequences of OsPP1c and OsPP2Ac with the catalytic subunits of PP1 or PP2A of rabbit skeletal muscle, Arabidopsis thaliana, maize and Brassica napus showed that the catalytic subunit sequences of PP1 or PP2A among these organisms are highly conserved (73% to 90% similarity). Genomic Southern blot analysis indicated that there are only one or two copies of OsPP1c genes and more than two copies of OsPP2Ac genes in the rice genome. Northern blot analysis showed that OsPP1c and OsPP2Ac genes are expressed in several organs of rice, including seed, shoot and root. We also showed by using 3' gene-specific probes of OsPP1c and OsPP2Ac cDNA, that the expression of neither gene is regulated by GA. Taken together, our results suggest that protein phosphatases PP1 or PP2A are involved in the GA-dependent expression of the rice Osamy-c gene, though the PP1 or/and PP2A enzymatic activities as well as mRNA levels do not increase upon GA3 treatment.
为了解赤霉素依赖性基因调控的分子机制，测定了三种磷酸酶抑制剂对水稻种子萌发及靶基因α-淀粉酶基因Osamy-c表达的影响。我们发现，已知能特异性抑制丝氨酸/苏氨酸磷酸酶1和2A的冈田酸、微囊藻毒素-LR和花萼海绵诱癌素A，强烈抑制Osamy-c的表达，且可能参与水稻种子的萌发。蛋白质磷酸酶活性测定表明，GA3对总PP1/PP2A活性没有明显影响。为进一步了解蛋白质磷酸酶1和2A在GA依赖的Osamy-c表达中的可能作用，我们从水稻糊粉层cDNA文库中分离出编码蛋白质磷酸酶1和蛋白质磷酸酶2A的cDNA克隆。它们分别被命名为OsPP1c和OsPP2Ac。将OsPP1c和OsPP2Ac推导的氨基酸序列与兔骨骼肌、拟南芥、玉米和甘蓝型油菜的PP1或PP2A催化亚基进行比较，结果表明这些生物体中PP1或PP2A的催化亚基序列高度保守（相似性为73%至90%）。基因组Southern杂交分析表明，水稻基因组中OsPP1c基因只有一两个拷贝，OsPP2Ac基因有两个以上拷贝。Northern杂交分析表明，OsPP1c和OsPP2Ac基因在水稻的几个器官中表达，包括种子、茎和根。我们还使用OsPP1c和OsPP2Ac cDNA的3'基因特异性探针表明，这两个基因的表达均不受GA调控。综上所述，我们的结果表明，蛋白质磷酸酶PP1或PP2A参与了水稻Osamy-c基因的GA依赖表达，尽管GA3处理后PP1或/和PP2A的酶活性以及mRNA水平并未增加。
