Graduate School of Agricultural Science, Kobe University, Nada-ku, Kobe 657-8501, Japan.
Plant Physiol Biochem. 2010 Oct-Nov;48(10-11):808-12. doi: 10.1016/j.plaphy.2010.08.002. Epub 2010 Aug 19.
Protease activities and its relation to the contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase were investigated in detached leaves of rice (Oryza sativa L.) floated on the solutions containing abscisic acid (ABA) or benzyladenine (BA). Rubisco and Rubisco activase contents were decreased during the time course and the decreases were enhanced by ABA and suppressed by BA. The decrease in Rubisco activase was faster than that in Rubisco. SDS-dependent protease activities at 50-70 kDa (rice SDS-dependent protease: RSP) analyzed by the gelatin containing PAGE were significantly enhanced by ABA. RSPs were also increased in attached leaves during senescence. RSPs had the pH optimum of 5.5, suggesting that RSPs are vacuolar protease. Both decrease in Rubisco and Rubisco activase contents and increase in RSPs activities were suppressed by cycloheximide. These findings indicate that the activities of RSPs are well correlated with the decrease in these protein contents. Immunoblotting analysis showed that Rubisco in the leaf extracts was completely degraded by 5h at pH 5.5 with SDS where it was optimal condition for RSPs. However, the degradation of Rubisco did not proceed at pH 7.5 without SDS where it is near physiological condition for stromal proteins. Rubisco activase was degraded at similar rate under both conditions. These results suggest that RSPs can functions in a senescence related degradation system of chloroplast protein in rice leaves. Rubisco activase would be more susceptible to proteolysis than Rubisco under physiological condition and this could affect the contents of these proteins in leaves.
在离体漂浮于含有脱落酸（ABA）或苄腺嘌呤（BA）溶液中的水稻（Oryza sativa L.）叶片中，研究了蛋白酶活性及其与核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）和 Rubisco 激活酶含量的关系。在时间过程中，Rubisco 和 Rubisco 激活酶含量下降，ABA 增强，BA 抑制这种下降。Rubisco 激活酶的下降速度快于 Rubisco。通过含有明胶的 PAGE 分析 SDS 依赖性蛋白酶在 50-70 kDa（水稻 SDS 依赖性蛋白酶：RSP）的活性显著增强 ABA。在衰老过程中，附着叶片中的 RSP 也增加。RSP 的最适 pH 为 5.5，表明 RSP 是液泡蛋白酶。Rubisco 和 Rubisco 激活酶含量的下降以及 RSPs 活性的增加均被环己酰亚胺抑制。这些发现表明 RSPs 的活性与这些蛋白质含量的下降密切相关。免疫印迹分析表明，在 pH5.5 下用 SDS 处理 5 小时，叶片提取物中的 Rubisco 完全降解，这是 RSPs 的最佳条件。然而，在没有 SDS 的情况下，即在接近基质蛋白生理条件的 pH7.5 下，Rubisco 的降解不会进行。在这两种条件下，Rubisco 激活酶的降解速率相似。这些结果表明，RSPs 可以在水稻叶片叶绿体蛋白衰老相关降解系统中发挥作用。在生理条件下，Rubisco 激活酶比 Rubisco 更容易受到蛋白水解的影响，这可能会影响叶片中这些蛋白质的含量。
