The salt tolerance locus SOS1 from Arabidopsis has been shown to encode a putative plasma membrane Na ؉ /H ؉ antiporter. In this study, we examined the tissue-specific pattern of gene expression as well as the Na ؉ transport activity and subcellular localization of SOS1. When expressed in a yeast mutant deficient in endogenous Na ϩ transporters, SOS1 was able to reduce Na ؉ accumulation and improve salt tolerance of the mutant cells. Confocal imaging of a SOS1-green fluorescent protein fusion protein in transgenic Arabidopsis plants indicated that SOS1 is localized in the plasma membrane. Analysis of SOS1 promoter-␤ -glucuronidase transgenic Arabidopsis plants revealed preferential expression of SOS1 in epidermal cells at the root tip and in parenchyma cells at the xylem/symplast boundary of roots, stems, and leaves. Under mild salt stress (25 mM NaCl), sos1 mutant shoot accumulated less Na ؉ than did the wildtype shoot. However, under severe salt stress (100 mM NaCl), sos1 mutant plants accumulated more Na ؉ than did the wild type. There also was greater Na ؉ content in the xylem sap of sos1 mutant plants exposed to 100 mM NaCl. These results suggest that SOS1 is critical for controlling long-distance Na ؉ transport from root to shoot. We present a model in which SOS1 functions in retrieving Na ؉ from the xylem stream under severe salt stress, whereas under mild salt stress it may function in loading Na ؉ into the xylem. INTRODUCTIONPlant growth depends on mineral nutrients absorbed from the soil by roots. Although Na ϩ is a major cation present in soil solutions, Na ϩ is not considered an essential mineral for most plants. In saline soils, high concentrations of Na ϩ disrupt K ϩ and other mineral nutrition, create hyperosmotic stress, and cause secondary problems such as oxidative stress (Zhu, 2001). These adverse effects contribute to plant growth inhibition and even plant death.Many cytosolic enzymes are activated by K ϩ and inhibited by Na ϩ (Flowers et al., 1977). Three mechanisms are available to plant cells to prevent excessive accumulation of Na ϩ in the cytosol (Niu et al., 1995;Blumwald et al., 2000; Zhu, 2001). First, Na ϩ entry to plant cells may be restricted by selective ion uptake. Nonselective cation channels have been proposed to mediate substantial Na ϩ entry into plant roots, but genes encoding these channels have yet to be identified (Amtmann and Sanders, 1999; Tyerman and Skerrett, 1999). The cloned transporters HKT1 and LCT1 have Na ϩ permeability when expressed in yeast or oocytes, suggesting that they also may be candidate Na ϩ influx transporters (Rubio et al., 1995;Schachtman et al., 1997). Recently, studies in yeast demonstrated that the magnitude of the membrane potential affects net Na ϩ influx into cells. Mutations in the yeast PMP3 gene lead to membrane hyperpolarization, increased Na ϩ influx, and salt sensitivity (Navarre and Goffeau, 2000).Second, internalized Na ϩ can be stored in vacuoles. Vacuolar compartmentation is an efficient strategy for plant cells to deal with salt s...

The SOS (for Salt Overly Sensitive) pathway plays essential roles in conferring salt tolerance in Arabidopsis thaliana. Under salt stress, the calcium sensor SOS3 activates the kinase SOS2 that positively regulates SOS1, a plasma membrane sodium/ proton antiporter. We show that SOS3 acts primarily in roots under salt stress. By contrast, the SOS3 homolog SOS3-LIKE CALCIUM BINDING PROTEIN8 (SCABP8)/CALCINEURIN B-LIKE10 functions mainly in the shoot response to salt toxicity. While root growth is reduced in sos3 mutants in the presence of NaCl, the salt sensitivity of scabp8 is more prominent in shoot tissues. SCABP8 is further shown to bind calcium, interact with SOS2 both in vitro and in vivo, recruit SOS2 to the plasma membrane, enhance SOS2 activity in a calcium-dependent manner, and activate SOS1 in yeast. In addition, sos3 scabp8 and sos2 scabp8 display a phenotype similar to sos2, which is more sensitive to salt than either sos3 or scabp8 alone. Overexpression of SCABP8 in sos3 partially rescues the sos3 salt-sensitive phenotype. However, overexpression of SOS3 fails to complement scabp8. These results suggest that SCABP8 and SOS3 are only partially redundant in their function, and each plays additional and unique roles in the plant salt stress response.

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...

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Intracellular NHX proteins are Na + ,K + /H + antiporters involved in K + homeostasis, endosomal pH regulation, and salt tolerance. Proteins NHX1 and NHX2 are the two major tonoplast-localized NHX isoforms. Here, we show that NHX1 and NHX2 have similar expression patterns and identical biochemical activity, and together they account for a significant amount of the Na + ,K + /H + antiport activity in tonoplast vesicles. Reverse genetics showed functional redundancy of NHX1 and NHX2 genes. Growth of the double mutant nhx1 nhx2 was severely impaired, and plants were extremely sensitive to external K + . By contrast, nhx1 nhx2 mutants showed similar sensitivity to salinity stress and even greater rates of Na + sequestration than the wild type. Double mutants had reduced ability to create the vacuolar K + pool, which in turn provoked greater K + retention in the cytosol, impaired osmoregulation, and compromised turgor generation for cell expansion. Genes NHX1 and NHX2 were highly expressed in guard cells, and stomatal function was defective in mutant plants, further compromising their ability to regulate water relations. Together, these results show that tonoplast-localized NHX proteins are essential for active K + uptake at the tonoplast, for turgor regulation, and for stomatal function.

SummaryThe Arabidopsis thaliana vacuolar Na + /H + antiporter AtNHX1 is a salt tolerance determinant. Predicted amino acid sequence similarity, protein topology and the presence of functional domains conserved in AtNHX1 and prototypical mammalian NHE Na + /H + exchangers led to the identi®cation of ®ve additional AtNHX genes (AtNHX2±6). The AtNHX1 and AtNHX2 mRNAs are the most prevalent transcripts among this family of genes in seedling shoots and roots. A lower-abundance AtNHX5 mRNA is present in both shoots and roots, whereas AtNHX3 transcript is expressed predominantly in roots. AtNHX4 and AtNHX6 mRNAs were detected only by RT±PCR. AtNHX1, 2 or 5 suppress, with differential ef®cacy, the Na + /Li + -sensitive phenotype of a yeast mutant that is de®cient in the endosomal/vacuolar Na + /H + antiporter ScNHX1. Ion accumulation data indicate that these AtNHX proteins function to facilitate Na + ion compartmentalization and maintain intracellular K + status. Seedling steady-state mRNA levels of AtNHX1 and AtNHX2 increase similarly after treatment with NaCl, an equi-osmolar concentration of sorbitol, or ABA, whereas AtNHX5 transcript abundance increases only in response to salt treatment. Hyper-osmotic up-regulation of AtNHX1, 2 or 5 expression is not dependent on the SOS pathway that controls ion homeostasis. However, steady-state AtNHX1, 2 and 5 transcript abundance is greater in sos1, sos2 and sos3 plants growing in medium that is not supplemented with sorbitol or NaCl, providing evidence that transcription of these genes is negatively affected by the SOS pathway in the absence of stress. AtNHX1 and AtNHX2 transcripts accumulate in response to ABA but not to NaCl in the aba2-1, mutant indicating that the osmotic responsiveness of these genes is ABA-dependent. An as yet unde®ned stress signal pathway that is ABA-and SOS-independent apparently controls transcriptional up-regulation of AtNHX5 expression by hyper-saline shock. Similar to AtNHX1, AtNHX2 is localized to the tonoplast of plant cells. Together, these results implicate AtNHX2 and 5, together with AtNHX1, as salt tolerance determinants, and indicate that AtNHX2 has a major function in vacuolar compartmentalization of Na + .

Soil salinity is a growing problem around the world with special relevance in farmlands. The ability to sense and respond to environmental stimuli is among the most fundamental processes that enable plants to survive. At the cellular level, the Salt Overly Sensitive (SOS) signaling pathway that comprises SOS3, SOS2, and SOS1 has been proposed to mediate cellular signaling under salt stress, to maintain ion homeostasis. Less well known is how cellularly heterogenous organs couple the salt signals to homeostasis maintenance of different types of cells and to appropriate growth of the entire organ and plant. Recent evidence strongly indicates that different regulatory mechanisms are adopted by roots and shoots in response to salt stress. Several reports have stated that, in roots, the SOS proteins may have novel roles in addition to their functions in sodium homeostasis. SOS3 plays a critical role in plastic development of lateral roots through modulation of auxin gradients and maxima in roots under mild salt conditions. The SOS proteins also play a role in the dynamics of cytoskeleton under stress. These results imply a high complexity of the regulatory networks involved in plant response to salinity. This review focuses on the emerging complexity of the SOS signaling and SOS protein functions, and highlights recent understanding on how the SOS proteins contribute to different responses to salt stress besides ion homeostasis.

Uptake and translocation of cations play essential roles in plant nutrition, signal transduction, growth, and development. Among them, potassium (K+) and sodium (Na+) have been the focus of numerous physiological studies because K+ is an essential macronutrient and the most abundant inorganic cation in plant cells, whereas Na+ toxicity is a principal component of the deleterious effects associated with salinity stress. Although the homeostasis of these two ions was long surmised to be fine tuned and under complex regulation, the myriad of candidate membrane transporters mediating their uptake, intracellular distribution, and long-distance transport is nevertheless perplexing. Recent advances have shown that, in addition to their function in vacuolar accumulation of Na+, proteins of the NHX family are endosomal transporters that also play critical roles in K+ homeostasis, luminal pH control, and vesicle trafficking. The plasma membrane SOS1 protein from Arabidopsis thaliana, a highly specific Na+/H+ exchanger that catalyses Na+ efflux and that regulates its root/shoot distribution, has also revealed surprising interactions with K+ uptake mechanisms by roots. Finally, the function of individual members of the large CHX family remains largely unknown but two CHX isoforms, AtCHX17 and AtCH23, have been shown to affect K+ homeostasis and the control of chloroplast pH, respectively. Recent advances on the understanding of the physiological processes that are governed by these three families of cation exchangers are reviewed and discussed.