Plant Omics Division, CSIR-Central Salt and Marine Chemicals Research Institute (CSIR-CSMCRI), Council of Scientific & Industrial Research (CSIR), Gijubhai Badheka Marg, Bhavnagar, 364 002, Gujarat, India.
Plant Omics Division, CSIR-Central Salt and Marine Chemicals Research Institute (CSIR-CSMCRI), Council of Scientific & Industrial Research (CSIR), Gijubhai Badheka Marg, Bhavnagar, 364 002, Gujarat, India.
Plant Physiol Biochem. 2020 Jun;151:689-704. doi: 10.1016/j.plaphy.2020.03.030. Epub 2020 Apr 21.
The maintenance of ROS homeostasis, membrane biogenesis and recycling of molecules are common stress responses involving specific and complex regulatory network. Ubiquitination is an important and common mechanism which facilitates environmental adaptation in eukaryotes. In the present study we have cloned the AlRabring7, an E3-Ub-ligase, previously identified as AlRab7 interacting partner. The role of AlRabring7 for ubiquitinating AlRab7 and facilitating stress tolerance is analysed. The AlRabring7, with an open-reading frame of 702 bp encodes a protein of 233 amino acids, with RING-HC domain of 40 amino acids. In silico analysis shows that AlRabring7 is a C3HC4-type RING E3 Ub ligase. The protein - protein docking show interaction dynamics between AlRab7-AlRabring7-Ubiquitin proteins. The AlRab7 and AlRabring7 transcript showed up-regulation in response to different salts i.e: NaCl, KCl, CaCl, NaCl + KCl, NaCl + CaCl, imposing ionic as well as hyperosmotic stress, and also with oxidative stress by HO treatment. Interestingly, the AlRabring7 showed early transcript expression with maximum expression in shoots on combinatorial stresses. The AlRab7 showed delayed and maximum expression with NaCl + CaCl stress treatment. The AlRab7 complements yeast ypt7Δ mutants and restored the fragmented vacuole. The in vitro ubiquitination assay revealed that AlRabring7 function as E3 ubiquitin ligase and mediates AlRab7 ubiquitination. Overexpression of AlRab7 and AlRabring7 independently and when co-transformed enhanced the growth of yeast cells during stress conditions. Further, the bimolecular fluorescence complementation assay shows the in planta interaction of the two proteins. Our results suggest that AlRab7 and AlRabring7 confers enhanced stress tolerance in yeast.
ROS 平衡、膜生物发生和分子循环的维持是涉及特定和复杂调控网络的常见应激反应。泛素化是一种重要且常见的机制，可促进真核生物适应环境。在本研究中，我们克隆了 AlRabring7，一种先前被鉴定为 AlRab7 相互作用伙伴的 E3-Ub 连接酶。分析了 AlRabring7 对泛素化 AlRab7 和促进应激耐受的作用。AlRabring7 的开放阅读框为 702bp，编码一个 233 个氨基酸的蛋白质，具有 40 个氨基酸的 RING-HC 结构域。计算机分析表明，AlRabring7 是一种 C3HC4 型 RING E3 Ub 连接酶。蛋白-蛋白对接显示了 AlRab7-AlRabring7-泛素蛋白之间的相互作用动力学。AlRab7 和 AlRabring7 转录本在不同盐胁迫下，即 NaCl、KCl、CaCl、NaCl+KCl、NaCl+CaCl 胁迫下，表现出上调，既施加离子胁迫又施加高渗胁迫，用 HO 处理还表现出氧化胁迫。有趣的是，AlRabring7 表现出早期转录表达，在组合胁迫下，在芽中表达量最大。AlRabring7 在 NaCl+CaCl 胁迫处理时表现出延迟和最大表达。AlRab7 补充酵母 ypt7Δ 突变体并恢复了碎片化的液泡。体外泛素化试验表明，AlRabring7 作为 E3 泛素连接酶发挥作用，并介导 AlRab7 泛素化。AlRab7 和 AlRabring7 独立过表达和共转化时，增强了酵母细胞在应激条件下的生长。此外，双分子荧光互补试验显示了两种蛋白质在植物体内的相互作用。我们的结果表明，AlRab7 和 AlRabring7 赋予酵母增强的应激耐受能力。
