Regulation of SOS1, a plasma membrane Na+/H+ exchanger in Arabidopsis thaliana, by SOS2 and SOS3

Abstract

Maintaining low levels of sodium ions in the cell cytosol is critical for plant growth and development. Biochemical studies suggest that Na(+)/H(+) exchangers in the plasma membrane of plant cells contribute to cellular sodium homeostasis by transporting sodium ions out of the cell; however, these exchangers have not been identified at the molecular level. Genetic analysis has linked components of the salt overly sensitive pathway (SOS1-3) to salt tolerance in Arabidopsis thaliana. The predicted SOS1 protein sequence and comparisons of sodium ion accumulation in wild-type and sos1 plants suggest that SOS1 is involved directly in the transport of sodium ions across the plasma membrane. To demonstrate the transport capability of SOS1, we studied Na(+)/H(+)-exchange activity in wild-type and sos plants using highly purified plasma membrane vesicles. The results showed that plasma membrane Na(+)/H(+)-exchange activity was present in wild-type plants treated with 250 mM NaCl, but this transport activity was reduced by 80% in similarly treated sos1 plants. In vitro addition of activated SOS2 protein (a protein kinase) increased Na(+)/H(+)-exchange activity in salt-treated wild-type plants 2-fold relative to transport without added protein. However, the addition of activated SOS2 did not have any stimulatory effect on the exchange activity in sos1 plants. Although vesicles of sos2 and sos3 plants had reduced plasma membrane Na(+)/H(+)-exchange activity, transport activity in both increased with the addition of activated SOS2 protein. These results demonstrate that SOS1 contributes to plasma membrane Na(+)/H(+) exchange and that SOS2 and SOS3 regulate SOS1 transport activity.

Figure 1

Vesicles isolated from wild-type plants are transport-competent and enriched for plasma membrane. Plasma membrane vesicles were isolated by two-phase partitioning from the leaves of wild-type plants treated with 250 mM NaCl for 3 days. Transport assays were performed as described in Materials and Methods. (A) When added at the start of the reaction, the proton channel inhibitor N,N′-dicyclohexylcarbodiimide (DCCD, 20 μM) prevented the formation of ΔpH. When added after the ΔpH formation reached steady state, the protonophore carbonylcyanide p-trifluoro-methoxyphenylhydrazone (FCCP, 5 μM) and the uncoupler NH₄Cl (1 mM) dissipated the existing ΔpH. (B) When added at the start of the reaction, 100 μM vanadate (a plasma membrane H⁺-ATPase inhibitor) reduced ΔpH formation 48%, while control levels of ΔpH formation were measured in the presence of 50 mM nitrate (a vacuolar H⁺-ATPase inhibitor). (C) After the formation of ΔpH, NaCl (50 mM) was added to initiate Na⁺/H⁺-exchange activity (dissipation of ΔpH). The electroneutral Na⁺/H⁺ exchanger, monensin (150 μM), was added at the end of the assay to eliminate any remaining ΔpH. The data shown in A–C are representative at least three experiments.

Figure 2

Vesicles isolated from sos1 plants have reduced plasma membrane Na⁺/H⁺-exchange activity relative to that of vesicles isolated from wild-type (WT) plants. Plasma membrane vesicles were isolated from the leaves of wild-type and sos1 plants treated with 250 mM NaCl for 3 days. Transport assays were performed as described in Materials and Methods. When ΔpH reached steady state, NaCl was added over a range of final concentrations (0–100 mM), and initial rates of dissipation were measured. ● and ■, vesicles isolated from wild-type and sos1 plants, respectively. The units of Na⁺/H⁺-exchange activity are Δ%F/min per mg of protein.

Figure 3

Vesicles isolated from sos2 and sos3 plants have reduced plasma membrane Na⁺/H⁺-exchange activity relative to that of vesicles isolated from wild-type (WT) plants. Plasma membrane vesicles were isolated from the leaves of wild-type, sos2, and sos3 plants treated with 250 mM NaCl for 3 days. Transport assays were performed as described in Materials and Methods. When ΔpH reached steady state, NaCl was added over a range of final concentrations (0–100 mM), and the initial rates of dissipation were measured. ●, ■, and ▴, vesicles isolated from wild-type, sos2, and sos3 plants, respectively. The units of Na⁺/H⁺-exchange activity are Δ%F/min per mg of protein.

Figure 4

T/DSOS2DF is a constitutive, highly active form of the SOS2 serine/threonine protein kinase. The kinase activities (autophosphorylation and phosphorylation of an in vitro substrate) of altered forms of the serine/threonine kinase SOS2 (GST-fusion proteins of SOS2DF, T/DSOS2DF, and T/DSOS2) were evaluated. After the autophosphorylation assays, protein (100 ng per lane) was separated by SDS/PAGE, and the gel was stained with Coomassie blue (A) and exposed to x-ray film (B). The arrows indicate SOS2 and SOS3 proteins. (C) The ability of the same GST-SOS2 fusion proteins to phosphorylate the peptide substrate p3 (400 pmol per assay) was determined. The units of protein phosphorylation are nmol/min per mg of protein.

Figure 5

Constitutively active recombinant SOS2 protein stimulates plasma membrane Na⁺/H⁺-exchange activity in vesicles isolated from wild-type, sos2, and sos3 plants but not in those isolated from sos1 plants. Transport assays were performed as described in Materials and Methods. ΔpH was formed in the absence (●) or presence (○) of active recombinant SOS2 protein (GST-T/DSOS2DF). When ΔpH reached steady state, NaCl was added over a range of final concentrations (0–100 mM), and the initial rates of dissipation were measured. (A) Wild-type. (B) sos1. (C) sos2. (D) sos3. The units of Na⁺/H⁺-exchange activity are Δ%F/min per mg of protein.