SOS2 promotes salt tolerance in part by interacting with the vacuolar H+-ATPase and upregulating its transport activity

Abstract

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.

FIG. 1.

Expression of a functional TAP-tagged SOS2 complements the salt sensitivity of a sos2-2 mutant. (A) Western blot analysis with SOS2-specific antibody detected NTAP-SOS2 expressed in the sos2-2 mutant at the molecular mass expected for a fusion of the TAPi tag with SOS2 (molecular mass of SOS2 alone is 50.6 kDa). SOS2 was not detected in the wild type (WT) because of its relatively low level of expression compared to the expression of NTAP-SOS2 driven by the 35S promoter. (B) Complementation of the salt sensitivity phenotype of the sos2-2 mutant by NTAP-SOS2. Photographs were taken 10 days after the transfer of seedlings to control or salt-containing media.

FIG. 2.

Both NTAP-SOS2 and VHA-B are present after TAP. Western blot analysis using SOS2- or VHA-B-specific antibodies detected both proteins in samples before (input) and after TAP.

FIG. 3.

Purification of NTAP-SOS2 protein complexes from control and salt-treated plants. (A) Coomassie-stained SDS-PAGE gel showing protein extracts from control (Cont) and salt-stressed (Salt) NTAP-SOS2 sos2-2 plants. Similar amounts of total protein were present in both extracts. (B) Western blot analysis showing similar amounts of NTAP-SOS2 and VHA-B in control and salt-stressed samples. (C) Coomassie-stained SDS-PAGE gel of protein isolated from control and salt-stressed plants after TAP. Positions of the VHA subunits identified by LC-MS/MS analysis are indicated on the right side of the gel. Note the similar amounts of total protein in the control versus salt-stressed samples. (D) Western blot analysis with anti-SOS2 antibody of TAP-purified samples. While the amounts of N-TAPiSOS2 recovered are similar in both samples, in salt-stressed plants the antibody recognizes additional bands, perhaps indicating posttranslational modifications of SOS2 occurring only under salt stress conditions.

FIG. 4.

Yeast two-hybrid analysis detects interaction of SOS2 with VHA-B1 and -B2. VHA-B2 (A) and VHA-B1 (B) were cloned into the prey plasmid pACT2 and cotransformed with the bait plasmid encoding SOS2, SOS2-K40N, or SOS1 (a negative control) or the empty pACT2 vector into the yeast strain Y190. Yeast grown on synthetic complete (SC) medium and the β-galactosidase filter assay (β- gal) are shown. SOS2-K40N is a catalytically inactive mutant of SOS2. Middle sections of both panels A and B show the interaction of SOS2 or SOS2-K40N with VHA-B2 Δ140 or VHA-B1 Δ140, which are lacking the N-terminal 140 amino acids. Bottom sections of panels A and B show negative controls.

FIG. 5.

The det3 mutant, which is impaired in V-ATPase activity, is salt sensitive. (A, left) Effect of NaCl on root elongation in the wild type (WT; ecotype Columbia) and det3 mutant. Root elongation is presented as a percentage of the elongation on control media for each genotype. Data are means ± standard errors (n = 14 to 16). (A, right) Pictures of representative seedlings 6 days after transfer to either control media (half-strength MS without sugar) or the same media with the addition of 100 mM NaCl. (B) Phenotypes of 6-day-old wild-type and det3 seedlings germinated and grown on control media (half-strength MS, 0.5% sucrose) in either light or dark.

FIG. 6.

Effect of sos2 and sos3 mutants on V-ATPase activity. (A) V-ATPase activity of tonoplast vesicles isolated from the wild type (WT) and sos2-2 and sos3-1 mutants. Tonoplast vesicles were isolated from cell cultures of each genotype by dextran gradient purification, and transport assays were performed as described in Materials and Methods. Initial rates of fluorescence quench were calculated over a range of ATP concentrations from 0 to 3 mM. All data represent means ± standard errors of at least three replicate experiments. Each replicate experiment was performed using independent membrane preparations. (B) V-ATPase activity of vesicles from wild-type cells with or without the addition of recombinant T/DSOS2DF. Assay conditions are as described for panel A, and wild-type data are the same as in panel A and are reproduced in this panel for ease of comparison. (C) V-ATPase activity of vesicles from sos2-2 cells with or with the addition of T/DSOS2DF. sos2-2 cell data are the same as in panel A and are reproduced here for ease of comparison.

FIG. 7.

Diagram of SOS2 regulation of ion transport. In response to salt stress, SOS2 is activated and is recruited to the plasma membrane by binding to SOS3. The activated SOS3-SOS2 regulatory complex activates the Na⁺/H⁺ antiporter SOS1, which reduces Na⁺ concentration in the cytosol by extrusion to the apoplast. On the plasma membrane, the H⁺ gradient needed to drive Na⁺ extrusion is maintained by the plasma membrane H⁺-ATPase (P-ATPase). SOS2 also activates tonoplast-located NHX antiporters to compartmentalize Na⁺ ions to the vacuole and the H⁺/Ca²⁺ transporter CAX1. SOS2 has been shown to regulate these tonoplast transporters independently of SOS3, perhaps by mechanisms other than phosphorylation of the target transporter. Instead, an SCaBP may be responsible for targeting SOS2 to the tonoplast. Posttranslational modification of SOS2 may also be important for these interactions. The H⁺ gradient needed to drive Na⁺ transport across the tonoplast is maintained by the tonoplast H⁺-pyrophosphatase (not shown) and the V-ATPase. The data reported here indicate that SOS2 interacts with and regulates the V-ATPase and that V-ATPase activity is required for salt tolerance. The FISL domain of SOS2 is known to be required for SOS3 binding and could also have a role in the interaction of SOS2 with the V-ATPase either directly or through SCaBPs.