Protection of plasma membrane K+ transport by the salt overly sensitive1 Na+-H+ antiporter during salinity stress

Physicochemical similarities between K(+) and Na(+) result in interactions between their homeostatic mechanisms. The physiological interactions between these two ions was investigated by examining aspects of K(+) nutrition in the Arabidopsis salt overly sensitive (sos) mutants, and salt sensitivity in the K(+) transport mutants akt1 (Arabidopsis K(+) transporter) and skor (shaker-like K(+) outward-rectifying channel). The K(+)-uptake ability (membrane permeability) of the sos mutant root cells measured electrophysiologically was normal in control conditions. Also, growth rates of these mutants in Na(+)-free media displayed wild-type K(+) dependence. However, mild salt stress (50 mm NaCl) strongly inhibited root-cell K(+) permeability and growth rate in K(+)-limiting conditions of sos1 but not wild-type plants. Increasing K(+) availability partially rescued the sos1 growth phenotype. Therefore, it appears that in the presence of Na(+), the SOS1 Na(+)-H(+) antiporter is necessary for protecting the K(+) permeability on which growth depends. The hypothesis that the elevated cytoplasmic Na(+) levels predicted to result from loss of SOS1 function impaired the K(+) permeability was tested by introducing 10 mm NaCl into the cytoplasm of a patch-clamped wild-type root cell. Complete loss of AKT1 K(+) channel activity ensued. AKT1 is apparently a target of salt stress in sos1 plants, resulting in poor growth due to impaired K(+) uptake. Complementary studies showed that akt1 seedlings were salt sensitive during early seedling development, but skor seedlings were normal. Thus, the effect of Na(+) on K(+) transport is probably more important at the uptake stage than at the xylem loading stage.     

Genetic analysis of salt tolerance in arabidopsis. Evidence for a critical role of potassium nutrition.

A large genetic screen for sos (for salt overly sensitive) mutants was performed in an attempt to isolate mutations in any gene with an sos phenotype. Our search yielded 28 new alleles of sos1, nine mutant alleles of a newly identified locus, SOS2, and one allele of a third salt tolerance locus, SOS3. The sos2 mutations, which are recessive, were mapped to the lower arm of chromosome V, approximately 2.3 centimorgans away from the marker PHYC. Growth measurements demonstrated that sos2 mutants are specifically hypersensitive to inhibition by Na+ or Li+ and not hypersensitive to general osmotic stresses. Interestingly, the SOS2 locus is also necessary for K+ nutrition because sos2 mutants were unable to grow on a culture medium with a low level of K+. The expression of several salt-inducible genes was superinduced in sos2 plants. The salt tolerance of sos1, sos2, and sos3 mutants correlated with their K+ tissue content but not their Na+ tissue content. Double mutant analysis indicated that the SOS genes function in the same pathway. Based on these results, a genetic model for salt tolerance mechanisms in Arabidopsis is presented in which SOS1, SOS2, and SOS3 are postulated to encode regulatory components controlling plant K+ nutrition that in turn is essential for salt tolerance.     

Transgenic evaluation of activated mutant alleles of SOS2 reveals a critical requirement for its kinase activity and C-terminal regulatory domain for salt tolerance in Arabidopsis thaliana

In Arabidopsis thaliana, the calcium binding protein Salt Overly Sensitive3 (SOS3) interacts with and activates the protein kinase SOS2, which in turn activates the plasma membrane Na(+)/H(+) antiporter SOS1 to bring about sodium ion homeostasis and salt tolerance. Constitutively active alleles of SOS2 can be constructed in vitro by changing Thr(168) to Asp in the activation loop of the kinase catalytic domain and/or by removing the autoinhibitory FISL motif from the C-terminal regulatory domain. We expressed various activated forms of SOS2 in Saccharomyces cerevisiae (yeast) and in A. thaliana and evaluated the salt tolerance of the transgenic organisms. Experiments in which the activated SOS2 alleles were coexpressed with SOS1 in S. cerevisiae showed that the kinase activity of SOS2 is partially sufficient for SOS1 activation in vivo, and higher kinase activity leads to greater SOS1 activation. Coexpression of SOS3 with SOS2 forms that retained the FISL motif resulted in more dramatic increases in salt tolerance. In planta assays showed that the Thr(168)-to-Asp-activated mutant SOS2 partially rescued the salt hypersensitivity in sos2 and sos3 mutant plants. By contrast, SOS2 lacking only the FISL domain suppressed the sos2 but not the sos3 mutation, whereas truncated forms in which the C terminus had been removed could not restore the growth of either sos2 or sos3 plants. Expression of some of the activated SOS2 proteins in wild-type A. thaliana conferred increased salt tolerance. These studies demonstrate that the protein kinase activity of SOS2 is partially sufficient for activation of SOS1 and for salt tolerance in vivo and in planta and that the kinase activity of SOS2 is limiting for plant salt tolerance. The results also reveal an essential in planta role for the SOS2 C-terminal regulatory domain in salt tolerance.     

Overexpression of SlSOS2 (SlCIPK24) confers salt tolerance to transgenic tomato

The Ca(2+)-dependent SOS pathway has emerged as a key mechanism in the homeostasis of Na(+) and K(+) under saline conditions. We have identified and functionally characterized the gene encoding the calcineurin-interacting protein kinase of the SOS pathway in tomato, SlSOS2. On the basis of protein sequence similarity and complementation studies in yeast and Arabidopsis, it can be concluded that SlSOS2 is the functional tomato homolog of Arabidopsis AtSOS2 and that SlSOS2 operates in a tomato SOS signal transduction pathway. The biotechnological potential of SlSOS2 to provide salt tolerance was evaluated by gene overexpression in tomato (Solanum lycopersicum L. cv. MicroTom). The better salt tolerance of transgenic plants relative to non-transformed tomato was shown by their faster relative growth rate, earlier flowering and higher fruit production when grown with NaCl. The increased salinity tolerance of SlSOS2-overexpressing plants was associated with higher sodium content in stems and leaves and with the induction and up-regulation of the plasma membrane Na(+)/H(+) (SlSOS1) and endosomal-vacuolar K(+), Na(+)/H(+) (LeNHX2 and LeNHX4) antiporters, responsible for Na(+) extrusion out of the root, active loading of Na(+) into the xylem, and Na(+) and K(+) compartmentalization.     

Conservation of the salt overly sensitive pathway in rice

The salt tolerance of rice (Oryza sativa) correlates with the ability to exclude Na+ from the shoot and to maintain a low cellular Na+/K+ ratio. We have identified a rice plasma membrane Na+/H+ exchanger that, on the basis of genetic and biochemical criteria, is the functional homolog of the Arabidopsis (Arabidopsis thaliana) salt overly sensitive 1 (SOS1) protein. The rice transporter, denoted by OsSOS1, demonstrated a capacity for Na+/H+ exchange in plasma membrane vesicles of yeast (Saccharomyces cerevisiae) cells and reduced their net cellular Na+ content. The Arabidopsis protein kinase complex SOS2/SOS3, which positively controls the activity of AtSOS1, phosphorylated OsSOS1 and stimulated its activity in vivo and in vitro. Moreover, OsSOS1 suppressed the salt sensitivity of a sos1-1 mutant of Arabidopsis. These results represent the first molecular and biochemical characterization of a Na+ efflux protein from monocots. Putative rice homologs of the Arabidopsis protein kinase SOS2 and its Ca2+-dependent activator SOS3 were identified also. OsCIPK24 and OsCBL4 acted coordinately to activate OsSOS1 in yeast cells and they could be exchanged with their Arabidopsis counterpart to form heterologous protein kinase modules that activated both OsSOS1 and AtSOS1 and suppressed the salt sensitivity of sos2 and sos3 mutants of Arabidopsis. These results demonstrate that the SOS salt tolerance pathway operates in cereals and evidences a high degree of structural conservation among the SOS proteins from dicots and monocots.     

The putative plasma membrane Na(+)/H(+) antiporter SOS1 controls long-distance Na(+) transport in plants

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(+) 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-beta-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 wild-type 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.     

SOS4, a pyridoxal kinase gene,is required for root hair development in Arabidopsis

Root hair development in plants is controlled by many genetic, hormonal, and environmental factors. A number of genes have been shown to be important for root hair formation. Arabidopsis salt overly sensitive 4 mutants were originally identified by screening for NaCl-hypersensitive growth. The SOS4 (Salt Overly Sensitive 4) gene was recently isolated by map-based cloning and shown to encode a pyridoxal (PL) kinase involved in the production of PL-5-phosphate, which is an important cofactor for various enzymes and a ligand for certain ion transporters. The root growth of sos4 mutants is slower than that of the wild type. Microscopic observations revealed that sos4 mutants do not have root hairs in the maturation zone. The sos4 mutations block the initiation of most root hairs, and impair the tip growth of those that are initiated. The root hairless phenotype of sos4 mutants was complemented by the wild-type SOS4 gene. SOS4 promoter-beta-glucuronidase analysis showed that SOS4 is expressed in the root hair and other hair-like structures. Consistent with SOS4 function as a PL kinase, in vitro application of pyridoxine and pyridoxamine, but not PL, partially rescued the root hair defect in sos4 mutants. 1-Aminocyclopropane-1-carboxylic acid and 2,4-dichlorophenoxyacetic acid treatments promoted root hair formation in both wild-type and sos4 plants, indicating that genetically SOS4 functions upstream of ethylene and auxin in root hair development. The possible role of SOS4 in ethylene and auxin biosynthesis is discussed.     

Soil bacteria confer plant salt tolerance by tissue-specific regulation of the sodium transporter HKT1

Elevated sodium (Na(+)) decreases plant growth and, thereby, agricultural productivity. The ion transporter high-affinity K(+) transporter (HKT)1 controls Na(+) import in roots, yet dysfunction or overexpression of HKT1 fails to increase salt tolerance, raising questions as to HKT1's role in regulating Na(+) homeostasis. Here, we report that tissue-specific regulation of HKT1 by the soil bacterium Bacillus subtilis GB03 confers salt tolerance in Arabidopsis thaliana. Under salt stress (100 mM NaCl), GB03 concurrently down- and upregulates HKT1 expression in roots and shoots, respectively, resulting in lower Na(+) accumulation throughout the plant compared with controls. Consistent with HKT1 participation in GB03-induced salt tolerance, GB03 fails to rescue salt-stressed athkt1 mutants from stunted foliar growth and elevated total Na(+) whereas salt-stressed Na(+) export mutants sos3 show GB03-induced salt tolerance with enhanced shoot and root growth as well as reduced total Na(+). These results demonstrate that tissue-specific regulation of HKT1 is critical for managing Na(+) homeostasis in salt-stressed plants, as well as underscore the breadth and sophistication of plant-microbe interactions.     

An enhancer mutant of Arabidopsis salt overly sensitive 3 mediates both ion homeostasis and the oxidative stress response

The myristoylated calcium sensor SOS3 and its interacting protein kinase, SOS2, play critical regulatory roles in salt tolerance. Mutations in either of these proteins render Arabidopsis thaliana plants hypersensitive to salt stress. We report here the isolation and characterization of a mutant called enh1-1 that enhances the salt sensitivity of sos3-1 and also causes increased salt sensitivity by itself. ENH1 encodes a chloroplast-localized protein with a PDZ domain at the N-terminal region and a rubredoxin domain in the C-terminal part. Rubredoxins are known to be involved in the reduction of superoxide in some anaerobic bacteria. The enh1-1 mutation causes enhanced accumulation of reactive oxygen species (ROS), particularly under salt stress. ROS also accumulate to higher levels in sos2-1 but not in sos3-1 mutants. The enh1-1 mutation does not enhance sos2-1 phenotypes. Also, enh1-1 and sos2-1 mutants, but not sos3-1 mutants, show increased sensitivity to oxidative stress. These results indicate that ENH1 functions in the detoxification of reactive oxygen species resulting from salt stress by participating in a new salt tolerance pathway that may involve SOS2 but not SOS3.     

Calcium- and salt-stress signaling in plants: shedding light on SOS pathway

As salt stress imposes a major environmental threat to agriculture, understanding the basic physiology and genetics of cell under salt stress is crucial for developing any transgenic strategy. Salt Overly Sensitive (SOS) genes (SOS1-SOS3) were isolated through positional cloning. Since sos mutants are hypersensitive to salt, their characterization resulted in the discovery of a novel pathway, which has helped in our understanding the mechanism of salt-stress tolerance in plants. Genetic analysis confirmed that SOS1-SOS3 function in a common pathway of salt tolerance. This pathway also emphasizes the significance of Ca²⁺ signal in reinstating cellular ion homeostasis. SOS3, a Ca²⁺ sensor, transduces the signal downstream after activating and interacting with SOS2 protein kinase. This SOS3-SOS2 complex activates the Na⁺/H⁺ antiporter activity of SOS1 thereby reestablish cellular ion homeostasis. Recently, SOS4 and SOS5 have also been characterized. SOS4 encodes a pyridoxal (PL) kinase that is involved in the biosynthesis of pyridoxal-5-phosphate (PLP), an active form of vitamin B6. SOS5 has been shown to be a putative cell surface adhesion protein that is required for normal cell expansion. Under salt stress, the normal growth and expansion of a plant cell becomes even more important and SOS5 helps in the maintenance of cell wall integrity and architecture. In this review we focus on the recent advances in salt stress and SOS signaling pathway. A broad coverage of the discovery of SOS mutants, structural aspect of these genes and the latest developments in the field of SOS1-SOS5 has been described.     

SOS3 function in plant salt tolerance requires N-myristoylation and calcium binding

The salt tolerance gene SOS3 (for salt overly sensitive3) of Arabidopsis is predicted to encode a calcium binding protein with an N-myristoylation signature sequence. Here, we examine the myristoylation and calcium binding properties of SOS3 and their functional significance in plant tolerance to salt. Treatment of young Arabidopsis seedlings with the myristoylation inhibitor 2-hydroxymyristic acid caused the swelling of root tips, mimicking the phenotype of the salt-hypersensitive mutant sos3-1. In vitro translation assays with reticulocyte showed that the SOS3 protein was myristoylated. Targeted mutagenesis of the N-terminal glycine-2 to alanine prevented the myristoylation of SOS3. The functional significance of SOS3 myristoylation was examined by expressing the wild-type myristoylated SOS3 and the mutated nonmyristoylated SOS3 in the sos3-1 mutant. Expression of the myristoylated but not the nonmyristoylated SOS3 complemented the salt-hypersensitive phenotype of sos3-1 plants. No significant difference in membrane association was observed between the myristoylated and nonmyristoylated SOS3. Gel mobility shift and (45)Ca(2)+ overlay assays demonstrated that SOS3 is a unique calcium binding protein and that the sos3-1 mutation substantially reduced the capacity of SOS3 to bind calcium. The resulting mutant SOS3 protein was not able to interact with the SOS2 protein kinase and was less capable of activating it. Together, these results strongly suggest that both N-myristoylation and calcium binding are required for SOS3 function in plant salt tolerance.     

Critical role of divalent cations and Na+ efflux in Arabidopsis thaliana salt tolerance

Detrimental effects of salinity on plants are known to be partially alleviated by external Ca²⁺. Previous work demonstrated that the Arabidopsis SOS3 locus encodes a Ca²⁺‐binding protein with similarities to CnB, the regulatory subunit of protein phosphatase 2B (calcineurin). In this study, we further characterized the role of SOS3 in salt tolerance. We found that reduced root elongation of sos3 mutants in the presence of high concentrations of either NaCl or LiCl is specifically rescued by Ca²⁺ and not Mg²⁺, whereas root growth is rescued by both Ca²⁺ and Mg²⁺ in the presence of high concentrations of KCl. Phenocopies of sos3 mutants were obtained in wild‐type plants by the application of calmodulin and calcineurin inhibitors. These data provide further evidence that SOS3 is a calcineurin‐like protein and that calmodulin plays an important role in the signalling pathways involved in plant salt tolerance. The origin of the elevated Na : K ratio in sos3 mutants was investigated by comparing Na⁺ efflux and influx in both mutant and wild type. No difference in Na⁺ influx was recorded between wild type and sos3; however, sos3 plants showed a markedly lower Na⁺ efflux, a property that would contribute to the salt‐oversensitive phenotype of sos3 plants.     

The calcium sensor CBL10 mediates salt tolerance by regulating ion homeostasis in Arabidopsis

Calcium serves as a critical messenger in many adaptation and developmental processes. Cellular calcium signals are detected and transmitted by sensor molecules such as calcium-binding proteins. In plants, the calcineurin B-like protein (CBL) family represents a unique group of calcium sensors and plays a key role in decoding calcium transients by specifically interacting with and regulating a family of protein kinases (CIPKs). We report here that the CBL protein CBL10 functions as a crucial regulator of salt tolerance in Arabidopsis. Cbl10 mutant plants exhibited significant growth defects and showed hypersensitive cell death in leaf tissues under high-salt conditions. Interestingly, the Na(+) content of the cbl10 mutant, unlike other salt-sensitive mutants identified thus far, was significantly lower than in the wild type under either normal or high-salt conditions, suggesting that CBL10 mediates a novel Ca(2+)-signaling pathway for salt tolerance. Indeed, the CBL10 protein physically interacts with the salt-tolerance factor CIPK24 (SOS2), and the CBL10-CIPK24 (SOS2) complex is associated with the vacuolar compartments that are responsible for salt storage and detoxification in plant cells. These findings suggest that CBL10 and CIPK24 (SOS2) constitute a novel salt-tolerance pathway that regulates the sequestration/compartmentalization of Na(+) in plant cells. Because CIPK24 (SOS2) also interacts with CBL4 (SOS3) and regulates salt export across the plasma membrane, our study identifies CIPK24 (SOS2) as a multi-functional protein kinase that regulates different aspects of salt tolerance by interacting with distinct CBL calcium sensors.     

Yeast plasma membrane Ena1p ATPase alters alkali-cation homeostasis and confers increased salt tolerance in tobacco cultured cells

In plants, the plasma membrane Na(+)/H(+) antiporter is the only key enzyme that extrudes cytosolic Na(+) and contributes to salt tolerance. But in fungi, the plasma membrane Na(+)/H(+) antiporter and Na(+)-ATPase are known to be key enzymes for salt tolerance. Saccharomyces cerevisiae Ena1p ATPase encoded by the ENA1/PMR2A gene is primarily responsible for Na(+) and Li(+) efflux across the plasma membrane during salt stress and for K(+) efflux at high pH and high K(+). To test if the yeast ATPase would improve salt tolerance in plants, we expressed a triple hemagglutinin (HA)-tagged Ena1p (Ena1p-3HA) in cultured tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY2) cells. The Ena1p-3HA proteins were correctly localized to the plasma membrane of transgenic BY2 cells and conferred increased NaCl and LiCl tolerance to the cells. Under moderate salt stress conditions, the Ena1p-3HA-expressing BY2 clones accumulated lower levels of Na(+) and Li(+) than nonexpressing BY2 clones. Moreover, the Ena1p-3HA expressing BY2 clones accumulated lower levels of K(+) than nonexpressing cells under no-stress conditions. These results suggest that the yeast Ena1p can also function as an alkali-cation (Na(+), Li(+), and K(+)) ATPase and alter alkali-cation homeostasis in plant cells. We conclude that, even with K(+)-ATPase activity, Na(+)-ATPase activity of the yeast Ena1p confers increased salt tolerance to plant cells during salt stress.     

Vitamer levels, stress response, enzyme activity, and gene regulation of Arabidopsis lines mutant in the pyridoxine/pyridoxamine 5'-phosphate oxidase (PDX3) and the pyridoxal kinase (SOS4) genes involved in the vitamin B6 salvage pathway

PDX3 and SALT OVERLY SENSITIVE4 (SOS4), encoding pyridoxine/pyridoxamine 5'-phosphate oxidase and pyridoxal kinase, respectively, are the only known genes involved in the salvage pathway of pyridoxal 5'-phosphate in plants. In this study, we determined the phenotype, stress responses, vitamer levels, and regulation of the vitamin B(6) pathway genes in Arabidopsis (Arabidopsis thaliana) plants mutant in PDX3 and SOS4. sos4 mutant plants showed a distinct phenotype characterized by chlorosis and reduced plant size, as well as hypersensitivity to sucrose in addition to the previously noted NaCl sensitivity. This mutant had higher levels of pyridoxine, pyridoxamine, and pyridoxal 5'-phosphate than the wild type, reflected in an increase in total vitamin B(6) observed through HPLC analysis and yeast bioassay. The sos4 mutant showed increased activity of PDX3 as well as of the B(6) de novo pathway enzyme PDX1, correlating with increased total B(6) levels. Two independent lines with T-DNA insertions in the promoter region of PDX3 (pdx3-1 and pdx3-2) had decreased PDX3 activity. Both also had decreased activity of PDX1, which correlated with lower levels of total vitamin B(6) observed using the yeast bioassay; however, no differences were noted in levels of individual vitamers by HPLC analysis. Both pdx3 mutants showed growth reduction in vitro and in vivo as well as an inability to increase growth under high light conditions. Increased expression of salvage and some of the de novo pathway genes was observed in both the pdx3 and sos4 mutants. In all mutants, increased expression was more dramatic for the salvage pathway genes.     

Ion exchangers NHX1 and NHX2 mediate active potassium uptake into vacuoles to regulate cell turgor and stomatal function in Arabidopsis

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.     

AtNHX8, a member of the monovalent cation: proton antiporter-1 family in Arabidopsis thaliana, encodes a putative Li/H antiporter

The Arabidopsis monovalent cation:proton antiporter-1 (CPA1) family includes eight members, AtNHX1-8. AtNHX1 and AtNHX7/SOS1 have been well characterized as tonoplast and plasma membrane Na+/H+ antiporters, respectively. The proteins AtNHX2-6 have been phylogenetically linked to AtNHX1, while AtNHX8 appears to be related to AtNHX7/SOS1. Here we report functional characterization of AtNHX8. AtNHX8 T-DNA insertion mutants are hypersensitive to lithium ions (Li+) relative to wild-type plants, but not to the other metal ions such as sodium (Na+), potassium (K+) and caesium (Cs+). AtNHX8 overexpression in a triple-deletion yeast mutant AXT3 that exhibits defective Na+/Li+ transport specifically suppresses sensitivity to Li+, but does not affect Na+ sensitivity. Likewise, AtNHX8 overexpression complemented sensitivity to Li+, but not Na+, in sos1-1 mutant seedlings, and increased Li+ tolerance of both the sos1-1 mutant and wild-type seedlings. Results of Li+ and K+ measurement of loss-of-function and gain-of-function mutants indicate that AtNHX8 may be responsible for Li+ extrusion, and may be able to maintain K+ acquisition and intracellular ion homeostasis. Subcellular localization of the AtNHX8-enhanced green fluorescent protein (EGFP) fusion protein suggested that AtNHX8 protein is targeted to the plasma membrane. Taken together, our findings suggest that AtNHX8 encodes a putative plasma membrane Li+/H+ antiporter that functions in Li detoxification and ion homeostasis in Arabidopsis.