Batelli Giorgia, Verslues Paul E, Agius Fernanda, Qiu Quansheng, Fujii Hiroaki, Pan Songqin, Schumaker Karen S, Grillo Stefania, Zhu Jian-Kang
Institute for Integrative Genome Biology and Department of Botany and Plant Sciences, University of California, Riverside, California 92521, USA.
Mol Cell Biol. 2007 Nov;27(22):7781-90. doi: 10.1128/MCB.00430-07. Epub 2007 Sep 17.
The salt overly sensitive (SOS) pathway is critical for plant salt stress tolerance and has a key role in regulating ion transport under salt stress. To further investigate salt tolerance factors regulated by the SOS pathway, we expressed an N-terminal fusion of the improved tandem affinity purification tag to SOS2 (NTAP-SOS2) in sos2-2 mutant plants. Expression of NTAP-SOS2 rescued the salt tolerance defect of sos2-2 plants, indicating that the fusion protein was functional in vivo. Tandem affinity purification of NTAP-SOS2-containing protein complexes and subsequent liquid chromatography-tandem mass spectrometry analysis indicated that subunits A, B, C, E, and G of the peripheral cytoplasmic domain of the vacuolar H+-ATPase (V-ATPase) were present in a SOS2-containing protein complex. Parallel purification of samples from control and salt-stressed NTAP-SOS2/sos2-2 plants demonstrated that each of these V-ATPase subunits was more abundant in NTAP-SOS2 complexes isolated from salt-stressed plants, suggesting that the interaction may be enhanced by salt stress. Yeast two-hybrid analysis showed that SOS2 interacted directly with V-ATPase regulatory subunits B1 and B2. The importance of the SOS2 interaction with the V-ATPase was shown at the cellular level by reduced H+ transport activity of tonoplast vesicles isolated from sos2-2 cells relative to vesicles from wild-type cells. In addition, seedlings of the det3 mutant, which has reduced V-ATPase activity, were found to be severely salt sensitive. Our results suggest that regulation of V-ATPase activity is an additional key function of SOS2 in coordinating changes in ion transport during salt stress and in promoting salt tolerance.
盐过度敏感（SOS）途径对于植物耐盐性至关重要，并且在盐胁迫下调节离子运输中起关键作用。为了进一步研究由SOS途径调控的耐盐因子，我们在sos2-2突变体植物中表达了改良的串联亲和纯化标签与SOS2的N端融合蛋白（NTAP-SOS2）。NTAP-SOS2的表达挽救了sos2-2植物的耐盐缺陷，表明该融合蛋白在体内具有功能。对含有NTAP-SOS2的蛋白质复合物进行串联亲和纯化，随后进行液相色谱-串联质谱分析，结果表明液泡H + -ATP酶（V-ATPase）外周胞质结构域的亚基A、B、C、E和G存在于含有SOS2的蛋白质复合物中。从对照和盐胁迫的NTAP-SOS2/sos2-2植物中平行纯化样品表明，这些V-ATPase亚基中的每一个在从盐胁迫植物中分离的NTAP-SOS2复合物中含量更高，这表明盐胁迫可能增强了这种相互作用。酵母双杂交分析表明，SOS2与V-ATPase调节亚基B1和B2直接相互作用。在细胞水平上，相对于野生型细胞的液泡，从sos2-2细胞分离的液泡膜囊泡的H + 转运活性降低，这表明SOS2与V-ATPase相互作用的重要性。此外，发现具有降低的V-ATPase活性的det3突变体幼苗对盐极度敏感。我们的结果表明，V-ATPase活性的调节是SOS2在协调盐胁迫期间离子运输变化和促进耐盐性方面的另一个关键功能。
