Proteomics analysis of rice lesion mimic mutant (spl1) reveals tightly localized Probenazole-Induced protein (PBZ1) in cells undergoing programmed cell death
Proteomics analysis of rice lesion mimic mutant (spl1) reveals tightly localized Probenazole-Induced protein (PBZ1) in cells undergoing programmed cell death
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DOI：
10.1021/pr700878t
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发表时间：
2008-04-01
影响因子：
4.4
通讯作者：
Kang, Kyu Young
中科院分区：
文献类型：
--
作者：
Kim, Sun Tae;Kim, Sang Gon;Kang, Kyu Young
许多报告预测/假设丙苯唑诱导蛋白（PBZ1）作为水稻自卫机制中的分子标记的作用。然而，PBZ1的确切功能仍不清楚。在本研究中，我们检查了 PBZ1 作为水稻中假定的细胞死亡标记物。为此，我们将注意力集中在水稻病斑模拟突变体 (LMM)、斑点叶 1 (sp/1) 上，该突变体已被用于研究叶子病斑发展过程中的程序性细胞死亡 (PCD) 现象。利用二维凝胶电泳(2-DGE)，发现sp/1突变体叶片中存在18个胶体考马斯亮蓝染色蛋白点的差异表达。通过 MALDI-TOF-MS 对这些点进行分析后，我们确定 PBZ1 蛋白在 sp/1 中高度诱导。基于这些结果，我们进一步验证了PBZ1在水稻PCD组织中是否高表达。为此，我们进行了免疫印迹分析和免疫定位，并使用了携带与 GFP 融合的 PBZ1 启动子的转基因系。结果表明，PBZ1 的表达水平和定位与经历 PCD 的组织显着一致，即在叶衰老、根通气组织形成、胚芽鞘衰老、根冠和种子糊粉层期间。此外，PBZ1 蛋白的定位也与种子糊粉层中的 TUNEL 信号密切相关。由于 DNA 断裂是 PCD 的一个标志，这一结果清楚地表明了 PBZ1 在经历 PCD 的水稻组织中的作用。总之，我们的结果为以下假设提供了强有力的支持：PBZ1 是水稻防御反应中的分子标记，并且可以作为水稻细胞死亡/PCD 的新型潜在标记。
Numerous reports have predicted/hypothesized a role for probenazole-induced protein (PBZ1) as a molecular marker in rice self-defense mechanism. However, the precise function of PBZ1 remains unknown. In the present study, we examined PBZ1 as a putative cell death marker in rice. For this, we focused our attention on a rice lesion mimic mutant (LMM), spotted leaf 1 (sp/1), which has been used to study the programmed cell death (PCD) phenomenon during lesion development in leaf. Using two-dimensional gel electrophoresis (2-DGE), 18 colloidal Coomassie brilliant blue stained protein spots were found to be differentially expressed in the leaves of sp/1 mutant. After analysis of these spots by MALDI-TOF-MS, we identified the PBZ1 protein to be highly inducible in sp/1. On the basis of these results, we proceeded to verify whether PBZ1 is highly expressed in the tissues undergoing PCD in rice. To do so, we performed immunoblot analysis and immunolocalization and used transgenic lines carrying the PBZ1 promoter fused with GFP. Results demonstrated that the expression levels and localizations of PBZ1 dramatically coincided with tissues undergoing PCD, namely, during leaf senescence, root aerenchyma formation, coleoptiles senescence, root cap, and seed aleurone layer. Furthermore, localization of the PBZ1 protein was also tightly correlated with TUNEL signal in the seed aleurone layer. As DNA fragmentation is a hallmark of PCD, this result clearly indicates a role for PBZ1 in rice tissues undergoing PCD. In conclusion, our results provide strong support for the hypothesis that PBZ1 is a molecular marker in rice defense response, and can serve as a novel potential marker for cell death/PCD in rice.