Reactive oxygen species mediate Na+-induced SOS1 mRNA stability in Arabidopsis.

Salt Overly Sensitive 1 (SOS1), a plasma membrane Na+/H+ antiporter in Arabidopsis, is a salt tolerance determinant crucial for the maintenance of ion homeostasis in saline stress conditions. SOS1 mRNA is unstable at normal growth conditions, but its stability is substantially increased under salt stress and other ionic and dehydration stresses. In addition, H2O2 treatment increases the stability of SOS1 mRNA. SOS1 mRNA is inherently unstable and rapidly degraded with a half-life of approximately 10 min. Rapid decay of SOS1 mRNA requires new protein synthesis. Stress-induced SOS1 mRNA stability is mediated by reactive oxygen species (ROS). NADPH oxidase is also involved in the upregulation of SOS1 mRNA stability, presumably through the control of extracellular ROS production. The cis-element required for SOS1 mRNA instability resides in the 500-bp region within the 2.2 kb at the 3' end of the SOS1 mRNA. Furthermore, mutations in the SOS1 gene render sos1 mutants more tolerant to paraquat, a non-selective herbicide causing oxidative stress, indicating that SOS1 plays negative roles in tolerance of oxidative stress. A hypothetical model for the signaling pathway involving SOS1-mediated pH changes, NADPH oxidase activation, apoplastic ROS production and downstream signaling transduction is proposed, and the biological significance of ROS-mediated induction of SOS1 mRNA stability is discussed.     

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.     

A cellulose synthase‐like protein is required for osmotic stress tolerance in Arabidopsis

Osmotic stress imposed by soil salinity and drought stress significantly affects plant growth and development, but osmotic stress sensing and tolerance mechanisms are not well understood. Forward genetic screens using a root‐bending assay have previously identified salt overly sensitive (sos) mutants of Arabidopsis that fall into five loci, SOS1 to SOS5. These loci are required for the regulation of ion homeostasis or cell expansion under salt stress, but do not play a major role in plant tolerance to the osmotic stress component of soil salinity or drought. Here we report an additional sos mutant, sos6‐1, which defines a locus essential for osmotic stress tolerance. sos6‐1 plants are hypersensitive to salt stress and osmotic stress imposed by mannitol or polyethylene glycol in culture media or by water deficit in the soil. SOS6 encodes a cellulose synthase‐like protein, AtCSLD5. Only modest differences in cell wall chemical composition could be detected, but we found that sos6‐1 mutant plants accumulate high levels of reactive oxygen species (ROS) under osmotic stress and are hypersensitive to the oxidative stress reagent methyl viologen. The results suggest that SOS6/AtCSLD5 is not required for normal plant growth and development but has a critical role in osmotic stress tolerance and this function likely involves its regulation of ROS under stress.     

Reactive oxygen species mediate Na+-induced SOS1 mRNA stability in Arabidopsis

Salt Overly Sensitive 1 (SOS1), a plasma membrane Na⁺/H⁺ antiporter in Arabidopsis, is a salt tolerance determinant crucial for the maintenance of ion homeostasis in saline stress conditions. SOS1 mRNA is unstable at normal growth conditions, but its stability is substantially increased under salt stress and other ionic and dehydration stresses. In addition, H₂O₂ treatment increases the stability of SOS1 mRNA. SOS1 mRNA is inherently unstable and rapidly degraded with a half-life of approximately 10 min. Rapid decay of SOS1 mRNA requires new protein synthesis. Stress-induced SOS1 mRNA stability is mediated by reactive oxygen species (ROS). NADPH oxidase is also involved in the upregulation of SOS1 mRNA stability, presumably through the control of extracellular ROS production. The cis -element required for SOS1 mRNA instability resides in the 500-bp region within the 2.2 kb at the 3' end of the SOS1 mRNA. Furthermore, mutations in the SOS1 gene render sos1 mutants more tolerant to paraquat, a non-selective herbicide causing oxidative stress, indicating that SOS1 plays negative roles in tolerance of oxidative stress. A hypothetical model for the signaling pathway involving SOS1-mediated pH changes, NADPH oxidase activation, apoplastic ROS production and downstream signaling transduction is proposed, and the biological significance of ROS-mediated induction of SOS1 mRNA stability is discussed.     

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.     

SCABP8/CBL10, a putative calcium sensor, interacts with the protein kinase SOS2 to protect Arabidopsis shoots from salt stress

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.     

Interaction of SOS2 with nucleoside diphosphate kinase 2 and catalases reveals a point of connection between salt stress and H2O2 signaling in Arabidopsis thaliana

SOS2, a class 3 sucrose-nonfermenting 1-related kinase, has emerged as an important mediator of salt stress response and stress signaling through its interactions with proteins involved in membrane transport and in regulation of stress responses. We have identified additional SOS2-interacting proteins that suggest a connection between SOS2 and reactive oxygen signaling. SOS2 was found to interact with the H2O2 signaling protein nucleoside diphosphate kinase 2 (NDPK2) and to inhibit its autophosphorylation activity. A sos2-2 ndpk2 double mutant was more salt sensitive than a sos2-2 single mutant, suggesting that NDPK2 and H2O2 are involved in salt resistance. However, the double mutant did not hyperaccumulate H2O2 in response to salt stress, suggesting that it is altered signaling rather than H2O2 toxicity alone that is responsible for the increased salt sensitivity of the sos2-2 ndpk2 double mutant. SOS2 was also found to interact with catalase 2 (CAT2) and CAT3, further connecting SOS2 to H2O2 metabolism and signaling. The interaction of SOS2 with both NDPK2 and CATs reveals a point of cross talk between salt stress response and other signaling factors including H2O2.     

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.     

Calcium regulation of sodium hypersensitivities of sos3 and athkt1 mutants

T-DNA disruption mutations in the AtHKT1 gene have previously been shown to suppress the salt sensitivity of the sos3 mutant. However, both sos3 and athkt1 single mutants show sodium (Na+) hypersensitivity. In the present study we further analyzed the underlying mechanisms for these non-additive and counteracting Na+ sensitivities by characterizing athkt1-1 sos3 and athkt1-2 sos3 double mutant plants. Unexpectedly, mature double mutant plants grown in soil clearly showed an increased Na+ hypersensitivity compared with wild-type plants when plants were subjected to salinity stress. The salt sensitive phenotype of athkt1 sos3 double mutant plants was similar to that of athkt1 plants, which showed chlorosis in leaves and stems. The Na+ content in xylem sap samples of soil-grown athkt1 sos3 double and athkt1 single mutant plants showed dramatic Na+ overaccumulation in response to salinity stress. Salinity stress analyses using basic minimal nutrient medium and Murashige-Skoog (MS) medium revealed that athkt1 sos3 double mutant plants show a more athkt1 single mutant-like phenotype in the presence of 3 mM external Ca2+, but show a more sos3 single mutant-like phenotype in the presence of 1 mM external Ca2+. Taken together multiple analyses demonstrate that the external Ca2+ concentration strongly impacts the Na+ stress response of athkt1 sos3 double mutants. Furthermore, the presented findings show that SOS3 and AtHKT1 are physiologically distinct major determinants of salinity resistance such that sos3 more strongly causes Na+ overaccumulation in roots, whereas athkt1 causes an increase in Na+ levels in the xylem sap and shoots and a concomitant Na+ reduction in roots.     

SOS3 mediates lateral root development under low salt stress through regulation of auxin redistribution and maxima in Arabidopsis

? The SOS signaling pathway plays an important role in plant salt tolerance. However, little is known about how the SOS pathway modulates organ development in response to salt stress. Here, the involvement of SOS signaling in NaCl-induced lateral root (LR) development in Arabidopsis was assessed. ? Wild-type and sos3-1 mutant seedlings on iso-osmotic concentrations of NaCl and mannitol were analyzed. The marker lines for auxin accumulation, auxin transport, cell division activity and stem cells were also examined. ? The results showed that ionic effect alleviates the inhibitory effects of osmotic stress on LR development. LR development of the sos3-1 mutant showed increased sensitivity specifically to low salt. Under low-salt conditions, auxin in cotyledons and LR primordia (LRP) of the sos3-1 mutant was markedly reduced. Decreases in auxin polar transport of mutant roots may cause insufficient auxin supply, resulting in defects not only in LR initiation but also in cell division activity in LRP. ? Our data uncover a novel role of the SOS3 gene in modulation of LR developmental plasticity and adaptation in response to low salt stress, and reveal a new mechanism for plants to sense and adapt to small changes of salt.     

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.     

SOS1, a Genetic Locus Essential for Salt Tolerance and Potassium Acquisition

To begin to determine which genes are essential for salt tolerance in higher plants, we identified four salt-hypersensitive mutants of Arabidopsis by using a root-bending assay on NaCl-containing agar plates. These mutants (sos1-1, sos1-2, sos1-3, and sos1-4) are allelic to each other and were caused by single recessive nuclear mutations. The SOS1 gene was mapped to chromosome 2 at 29.5 [plusmn] 6.1 centimorgans. The mutants showed no phenotypic changes except that their growth was >20 times more sensitive to inhibition by NaCl. Salt hypersensitivity is a basic cellular trait exhibited by the mutants at all developmental stages. The sos1 mutants are specifically hypersensitive to Na+ and Li+. The mutants were unable to grow on media containing low levels (below ~1 mM) of potassium. Uptake experiments using 86Rb showed that sos1 mutants are defective in high-affinity potassium uptake. sos1 plants became deficient in potassium when treated with NaCl. The results demonstrate that potassium acquisition is a critical process for salt tolerance in glycophytic plants.     

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

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.     

Signal transduction during cold, salt, and drought stresses in plants

Abiotic stresses, especially cold, salinity and drought, are the primary causes of crop loss worldwide. Plant adaptation to environmental stresses is dependent upon the activation of cascades of molecular networks involved in stress perception, signal transduction, and the expression of specific stress-related genes and metabolites. Plants have stress-specific adaptive responses as well as responses which protect the plants from more than one environmental stress. There are multiple stress perception and signaling pathways, some of which are specific, but others may cross-talk at various steps. In this review article, we first expound the general stress signal transduction pathways, and then highlight various aspects of biotic stresses signal transduction networks. On the genetic analysis, many cold induced pathways are activated to protect plants from deleterious effects of cold stress, but till date, most studied pathway is ICE-CBF-COR signaling pathway. The Salt-Overly-Sensitive (SOS) pathway, identified through isolation and study of the sos1, sos2, and sos3 mutants, is essential for maintaining favorable ion ratios in the cytoplasm and for tolerance of salt stress. Both ABA-dependent and -independent signaling pathways appear to be involved in osmotic stress tolerance. ROS play a dual role in the response of plants to abiotic stresses functioning as toxic by-products of stress metabolism, as well as important signal transduction molecules and the ROS signaling networks can control growth, development, and stress response. Finally, we talk about the common regulatory system and cross-talk among biotic stresses, with particular emphasis on the MAPK cascades and the cross-talk between ABA signaling and biotic signaling.     

Salt stress affects cortical microtubule organization and helical growth in Arabidopsis

Cortical microtubule arrays are critical in determining the growth axis of diffusely growing plant cells, and various environmental and physiological factors are known to affect the array organization. Microtubule organization is partly disrupted in the spiral1 mutant of Arabidopsis thaliana, which displays a right-handed helical growth phenotype in rapidly elongating epidermal cells. We show here that mutations in the plasma membrane Na(+)/H(+) antiporter SOS1 and its regulatory kinase SOS2 efficiently suppressed both microtubule disruption and helical growth phenotypes of spiral1, and that sos1 and sos2 roots in the absence of salt stress exhibited altered helical growth response to microtubule-interacting drugs at low doses. Salt stress also altered root growth response to the drugs in wild-type roots. Suppression of helical growth appeared to be specific to spiral1 since other helical growth mutants were not rescued. The effects of sos1 in suppressing spiral1 defects and in causing abnormal drug responses were nullified in the presence of the hkt1 Na(+) influx carrier mutation in roots but not in hypocotyls. These results suggest that cytoplasmic salt imbalance caused by insufficient SOS1 activity compromises cortical microtubule functions in which microtubule-localized SPIRAL1 is specifically involved.     

Increased tolerance to salt stress in the phosphate-accumulating <Emphasis Type="Italic">Arabidopsis</Emphasis> mutants <Emphasis Type="Italic">siz1</Emphasis> and <Emphasis Type="Italic">pho2</Emphasis>

High salinity is an environmental factor that inhibits plant growth and development, leading to large losses in crop yields. We report here that mutations in SIZ1 or PHO2, which cause more accumulation of phosphate compared with the wild type, enhance tolerance to salt stress. The siz1 and pho2 mutations reduce the uptake and accumulation of Na⁺. These mutations are also able to suppress the Na⁺ hypersensitivity of the sos3-1 mutant, and genetic analyses suggest that SIZ1 and SOS3 or PHO2 and SOS3 have an additive effect on the response to salt stress. Furthermore, the siz1 mutation cannot suppress the Li⁺ hypersensitivity of the sos3-1 mutant. These results indicate that the phosphate-accumulating mutants siz1 and pho2 reduce the uptake and accumulation of Na⁺, leading to enhanced salt tolerance, and that, genetically, SIZ1 and PHO2 are likely independent of SOS3-dependent salt signaling.