SOS2 Promotes Salt Tolerance in Part by Interacting with the Vacuolar H⁺-ATPase and Upregulating Its Transport Activity

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 saltstressed 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.