Reconstitution in yeast of the Arabidopsis SOS signaling pathway for Na ⁺ homeostasis

Abstract

The Arabidopsis thaliana SOS1 protein is a putative Na ؉ ͞H ؉ antiporter that functions in Na ؉ extrusion and is essential for the NaCl tolerance of plants. sos1 mutant plants share phenotypic similarities with mutants lacking the protein kinase SOS2 and the Ca 2؉ sensor SOS3. To investigate whether the three SOS proteins function in the same response pathway, we have reconstituted the SOS system in yeast cells. Expression of SOS1 improved the Na ؉ tolerance of yeast mutants lacking endogenous Na ؉ transporters. Coexpression of SOS2 and SOS3 dramatically increased SOS1-dependent Na ؉ tolerance, whereas SOS2 or SOS3 individually had no effect. The SOS2͞SOS3 kinase complex promoted the phosphorylation of SOS1. A constitutively active form of SOS2 phosphorylated SOS1 in vitro independently of SOS3, but could not fully substitute for the SOS2͞SOS3 kinase complex for activation of SOS1 in vivo. Further, we show that SOS3 recruits SOS2 to the plasma membrane. Although sos1 mutant plants display defective K ؉ uptake at low external concentrations, neither the unmodified nor the SOS2͞SOS3-activated SOS1 protein showed K ؉ transport capacity in vivo, suggesting that the role of SOS1 on K ؉ uptake is indirect. Our results provide an example of functional reconstitution of a plant response pathway in a heterologous system and demonstrate that the SOS1 ion transporter, the SOS2 protein kinase, and its associated Ca 2؉ sensor SOS3 constitute a functional module. We propose a model in which SOS3 activates and directs SOS2 to the plasma membrane for the stimulatory phosphorylation of the Na ؉ transporter SOS1.S oil salinity is a prevalent abiotic stress for crop plants. Excess salts in the soil solution interfere with mineral nutrition and water uptake, and lead to the undue accumulation of toxic ions (1). Maladies associated to salt stress are membrane disorganization, impaired nutrient and water acquisition, metabolic toxicity, inhibition of photosynthesis, and production of reactive oxygen species. In most instances, ion toxicity results from immoderate Na ϩ uptake caused by its steep inward electrochemical gradient. Plant growth under salt stress depends, among other concomitant processes, on the re-establishment of proper cellular ion homeostasis. Low cytosolic Na ϩ content is preserved by the concerted interplay of regulated ion uptake, vacuolar compartmentation, and active extrusion to the extracellular milieu (2). Vacuolar partitioning of Na ϩ and other ions also contributes to the maintenance of cellular water relations in a hypertonic medium. Energy-dependent exclusion of Na ϩ from the cytosol is coupled to downhill reverse transport of H ϩ by Na ϩ ͞H ϩ antiporters located in both the plasma membrane and tonoplast.The Arabidopsis thaliana SOS1 protein is the first putative plasma membrane Na ϩ ͞H ϩ antiporter to be described in plants (3,4). Arabidopsis sos1 mutants were isolated in a genetic screen for plants hypersensitive to NaCl, together with sos2 and sos3 mutants (5). SOS2 is a Ser͞Thr protein kinase in which t...

Structural Insights on the Plant Salt-Overly-Sensitive 1 (SOS1) Na+/H+ Antiporter

“…1 SOS1 is a plasma membrane Na + /H + antiporter that is regulated by the SOS2 Ser/Thr protein kinase and two alternative interacting activators, the calcium sensors SOS3/CBL4 and SCaBP8/CBL10. [2][3][4] An active SOS2-SOS3 complex is responsible for the phosphorylation and subsequent transport activation of SOS1 in response to salt stress. The transporter, in turn, extrudes the toxic Na + from the cytoplasm.…”

“…The transporter, in turn, extrudes the toxic Na + from the cytoplasm. 2,3,5 The SOS1 protein is predicted to have an N-terminal transmembrane (TM) region composed of 13 -helices and a long cytoplasmic tail of around 700 amino acids at the C-terminal end. 6 The TM portion has sequence similarities with plasma membrane Na + /H + exchangers from animal, bacterial, and fungal cells.…”

Salt Stress Signaling and Mechanisms of Plant Salt Tolerance

“…Consistent with this finding, these mutants also accumulate higher levels of Na + , similar to those accumulated by the sos1 mutant. However, the addition of activated SOS2 is sufficient to rescue the Na + /H + exchange activity of plasma membrane vesicles from sos3 and sos2 mutants (64,67). The SOS3-SOS2 kinase complex phosphorylates the SOS1 protein and activates SOS1 Na + /H + antiporter activity (67).…”

“…However, the addition of activated SOS2 is sufficient to rescue the Na + /H + exchange activity of plasma membrane vesicles from sos3 and sos2 mutants (64,67). The SOS3-SOS2 kinase complex phosphorylates the SOS1 protein and activates SOS1 Na + /H + antiporter activity (67). SOS1 upregulation during salt stress is also under the regulatory control of the SOS pathway, as shown by the impaired expression of SOS1 in salt-stressed sos2 and sos3 mutants (60) significantly increased SOS1-dependent Na + tolerance in the yeast mutant (67).…”

“…The SOS3-SOS2 kinase complex phosphorylates the SOS1 protein and activates SOS1 Na + /H + antiporter activity (67). SOS1 upregulation during salt stress is also under the regulatory control of the SOS pathway, as shown by the impaired expression of SOS1 in salt-stressed sos2 and sos3 mutants (60) significantly increased SOS1-dependent Na + tolerance in the yeast mutant (67). This evidence demonstrates that SOS3 senses the salt-stress induced Ca 2+ signals and activates SOS2 kinase, which in turn regulates the Na + /H + exchange activity and expression of SOS1 ( Fig.…”