Inverse regulation of SOS1 and HKT1 protein localization and stability by SOS3/CBL4 in Arabidopsis thaliana.

To control net sodium (Na+) uptake, Arabidopsis plants utilize the plasma membrane (PM) Na+/H+ antiporter SOS1 to achieve Na+ efflux at the root and Na+ loading into the xylem, and the channel-like HKT1;1 protein that mediates the reverse flux of Na+ unloading off the xylem. Together, these opposing transport systems govern the partition of Na+ within the plant yet they must be finely co-regulated to prevent a futile cycle of xylem loading and unloading. Here, we show that the Arabidopsis SOS3 protein acts as the molecular switch governing these Na+ fluxes by favoring the recruitment of SOS1 to the PM and its subsequent activation by the SOS2/SOS3 kinase complex under salt stress, while commanding HKT1;1 protein degradation upon acute sodic stress. SOS3 achieves this role by direct and SOS2-independent binding to previously unrecognized functional domains of SOS1 and HKT1;1. These results indicate that roots first retain moderate amounts of salts to facilitate osmoregulation, yet when sodicity exceeds a set point, SOS3-dependent HKT1;1 degradation switches the balance toward Na+ export out of the root. Thus, SOS3 functionally links and co-regulates the two major Na+ transport systems operating in vascular plants controlling plant tolerance to salinity.

Structure-Guided Identification of Critical Residues in the Vacuolar Cation/Proton Antiporter NHX1 from Arabidopsis thaliana.

Cation/Proton Antiporters (CPA) acting in all biological membranes regulate the volume and pH of cells and of intracellular organelles. A key issue with these proteins is their structure-function relationships since they present intrinsic regulatory features that rely on structural determinants, including pH sensitivity and the stoichiometry of ion exchange. Crystal structures are only available for prokaryotic CPA, whereas the eukaryotic ones have been modeled using the former as templates. Here, we present an updated and improved structural model of the tonoplast-localized K+, Na+/H+ antiporter NHX1 of Arabidopsis as a representative of the vacuolar NHX family that is essential for the accumulation of K+ into plant vacuoles. Conserved residues that were judged as functionally important were mutated, and the resulting protein variants were tested for activity in the yeast Saccharomyces cerevisiae. The results indicate that residue N184 in the ND-motif characteristic of CPA1 could be replaced by the DD-motif of CPA2 family members with minimal consequences for their activity. Attempts to alter the electroneutrality of AtNHX1 by different combinations of amino acid replacements at N184, R353 and R390 residues resulted in inactive or partly active proteins with a differential ability to control the vacuolar pH of the yeast.

Salinity-Induced Cytosolic Alkaline Shifts in Arabidopsis Roots Require the SOS Pathway.

Plants have evolved elaborate mechanisms to sense, respond to and overcome the detrimental effects of high soil salinity. The role of calcium transients in salinity stress signaling is well established, but the physiological significance of concurrent salinity-induced changes in cytosolic pH remains largely undefined. Here, we analyzed the response of Arabidopsis roots expressing the genetically encoded ratiometric pH-sensor pHGFP fused to marker proteins for the recruitment of the sensor to the cytosolic side of the tonoplast (pHGFP-VTI11) and the plasma membrane (pHGFP-LTI6b). Salinity elicited a rapid alkalinization of cytosolic pH (pHcyt) in the meristematic and elongation zone of wild-type roots. The pH-shift near the plasma membrane preceded that at the tonoplast. In pH-maps transversal to the root axis, the epidermis and cortex had cells with a more alkaline pHcyt relative to cells in the stele in control conditions. Conversely, seedlings treated with 100 mM NaCl exhibited an increased pHcyt in cells of the vasculature relative to the external layers of the root, and this response occurred in both reporter lines. These pHcyt changes were substantially reduced in mutant roots lacking a functional SOS3/CBL4 protein, suggesting that the operation of the SOS pathway mediated the dynamics of pHcyt in response to salinity.

S-acylated and nucleus-localized SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 stabilizes GIGANTEA to regulate Arabidopsis flowering time under salt stress.

The precise timing of flowering in adverse environments is critical for plants to secure reproductive success. We report a mechanism in Arabidopsis (Arabidopsis thaliana) controlling the time of flowering by which the S-acylation-dependent nuclear import of the protein SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 (SOS3/CBL4), a Ca2+-signaling intermediary in the plant response to salinity, results in the selective stabilization of the flowering time regulator GIGANTEA inside the nucleus under salt stress, while degradation of GIGANTEA in the cytosol releases the protein kinase SOS2 to achieve salt tolerance. S-acylation of SOS3 was critical for its nuclear localization and the promotion of flowering, but partly dispensable for salt tolerance. SOS3 interacted with the photoperiodic flowering components GIGANTEA and FLAVIN-BINDING, KELCH REPEAT, F-BOX1 and participated in the transcriptional complex that regulates CONSTANS to sustain the transcription of CO and FLOWERING LOCUS T under salinity. Thus, the SOS3 protein acts as a Ca2+- and S-acylation-dependent versatile regulator that fine-tunes flowering time in a saline environment through the shared spatial separation and selective stabilization of GIGANTEA, thereby connecting two signaling networks to co-regulate the stress response and the time of flowering.

A Ca2+-sensor switch for tolerance to elevated salt stress in Arabidopsis.

Excessive Na+ in soils inhibits plant growth. Here, we report that Na+ stress triggers primary calcium signals specifically in a cell group within the root differentiation zone, thus forming a "sodium-sensing niche" in Arabidopsis. The amplitude of this primary calcium signal and the speed of the resulting Ca2+ wave dose-dependently increase with rising Na+ concentrations, thus providing quantitative information about the stress intensity encountered. We also delineate a Ca2+-sensing mechanism that measures the stress intensity in order to mount appropriate salt detoxification responses. This is mediated by a Ca2+-sensor-switch mechanism, in which the sensors SOS3/CBL4 and CBL8 are activated by distinct Ca2+-signal amplitudes. Although the SOS3/CBL4-SOS2/CIPK24-SOS1 axis confers basal salt tolerance, the CBL8-SOS2/CIPK24-SOS1 module becomes additionally activated only in response to severe salt stress. Thus, Ca2+-mediated translation of Na+ stress intensity into SOS1 Na+/H+ antiporter activity facilitates fine tuning of the sodium extrusion capacity for optimized salt-stress tolerance.

Amino Acids 785, 787 of the Na+/H+ Exchanger Cytoplasmic Tail Modulate Protein Activity and Tail Conformation.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a plasma membrane protein ubiquitously present in humans. It regulates intracellular pH by removing an intracellular proton in exchange for an extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, regulatory cytosolic tail. Here, we investigated the effect of mutation of two amino acids of the regulatory tail, Ser785 and Ser787, that were similar in location and context to two amino acids of the Arabidopsis Na+/H+ exchanger SOS1. Mutation of these two amino acids to either Ala or phosphomimetic Glu did not affect surface targeting but led to a slight reduction in the level of protein expressed. The activity of the NHE1 protein was reduced in the phosphomimetic mutations and the effect was due to a decrease in Vmax activity. The Ser to Glu mutations also caused a change in the apparent molecular weight of both the full-length protein and of the cytosolic tail of NHE1. A conformational change in this region was indicated by differential trypsin sensitivity. We also found that a peptide containing amino acids 783-790 bound to several more proximal regions of the NHE1 tail in in vitro protein interaction experiments. The results are the first characterization of these two amino acids and show that they have significant effects on enzyme kinetics and the structure of the NHE1 protein.

The Arabidopsis protein NPF6.2/NRT1.4 is a plasma membrane nitrate transporter and a target of protein kinase CIPK23.

Nitrate and potassium nutrition is tightly coordinated in vascular plants. Physiological and molecular genetics studies have demonstrated that several NPF/NRT1 nitrate transporters have a significant impact on both uptake and the root-shoot partition of these nutrients. However, how these traits are biochemically connected remain controversial since some NPF proteins, e.g. NPF7.3/NRT1.5, have been suggested to mediate K+/H+ exchange instead of nitrate fluxes. Here we show that NPF6.2/NRT1.4, a protein that gates nitrate accumulation at the leaf petiole of Arabidopsis thaliana, also affects the root/shoot distribution of potassium. We demonstrate that NPF6.2/NRT1.4 is a plasma membrane nitrate transporter phosphorylated at threonine-98 by the CIPK23 protein kinase that is a regulatory hub for nitrogen and potassium nutrition. Heterologous expression of NPF6.2/NRT1.4 and NPF7.3/NRT1.5 in yeast mutants with altered potassium uptake and efflux systems showed no evidence of nitrate-dependent potassium transport by these proteins.

Distinct Roles of N-Terminal Fatty Acid Acylation of the Salinity-Sensor Protein SOS3.

The Salt-Overly-Sensitive (SOS) pathway controls the net uptake of sodium by roots and the xylematic transfer to shoots in vascular plants. SOS3/CBL4 is a core component of the SOS pathway that senses calcium signaling of salinity stress to activate and recruit the protein kinase SOS2/CIPK24 to the plasma membrane to trigger sodium efflux by the Na/H exchanger SOS1/NHX7. However, despite the well-established function of SOS3 at the plasma membrane, SOS3 displays a nucleo-cytoplasmic distribution whose physiological meaning is not understood. Here, we show that the N-terminal part of SOS3 encodes structural information for dual acylation with myristic and palmitic fatty acids, each of which commands a different location and function of SOS3. N-myristoylation at glycine-2 is essential for plasma membrane association and recruiting SOS2 to activate SOS1, whereas S-acylation at cysteine-3 redirects SOS3 toward the nucleus. Moreover, a poly-lysine track in positions 7-11 that is unique to SOS3 among other Arabidopsis CBLs appears to be essential for the correct positioning of the SOS2-SOS3 complex at the plasma membrane for the activation of SOS1. The nuclear-localized SOS3 protein had limited bearing on the salt tolerance of Arabidopsis. These results are evidence of a novel S-acylation dependent nuclear trafficking mechanism that contrasts with alternative subcellular targeting of other CBLs by S-acylation.

Insights into the mechanisms of transport and regulation of the arabidopsis high-affinity K+ transporter HAK51.

The high-affinity K+ transporter HAK5 from Arabidopsis (Arabidopsis thaliana) is essential for K+ acquisition and plant growth at low micromolar K+ concentrations. Despite its functional relevance in plant nutrition, information about functional domains of HAK5 is scarce. Its activity is enhanced by phosphorylation via the AtCIPK23/AtCBL1-9 complex. Based on the recently published three-dimensionalstructure of the bacterial ortholog KimA from Bacillus subtilis, we have modeled AtHAK5 and, by a mutational approach, identified residues G67, Y70, G71, D72, D201, and E312 as essential for transporter function. According to the structural model, residues D72, D201, and E312 may bind K+, whereas residues G67, Y70, and G71 may shape the selective filter for K+, which resembles that of K+shaker-like channels. In addition, we show that phosphorylation of residue S35 by AtCIPK23 is required for reaching maximal transport activity. Serial deletions of the AtHAK5 C-terminus disclosed the presence of an autoinhibitory domain located between residues 571 and 633 together with an AtCIPK23-dependent activation domain downstream of position 633. Presumably, autoinhibition of AtHAK5 is counteracted by phosphorylation of S35 by AtCIPK23. Our results provide a molecular model for K+ transport and describe CIPK-CBL-mediated regulation of plant HAK transporters.

Arabidopsis K+ transporter HAK5-mediated high-affinity root K+ uptake is regulated by protein kinases CIPK1 and CIPK9.

The high-affinity K+ transporter HAK5 is the major contributor to root K+ uptake from dilute solutions in K+-starved Arabidopsis plants. Its functionality is tightly regulated and its activity is enhanced under K+ starvation by the transcriptional induction of the AtHAK5 gene, and by the activation of the transporter via the AtCBL1-AtCIPK23 complex. In the present study, the 26 members of the Arabidopsis CIPK protein kinase family were screened in yeast for their capacity to activate HAK5-mediated K+ uptake. Among them, AtCIPK1 was the most efficient activator of AtHAK5. In addition, AtCIPK9, previously reported to participate in K+ homeostasis, also activated the transporter. In roots, the genes encoding AtCIPK1 and AtCIPK9 were induced by K+ deprivation and atcipk1 and atcipk9 Arabidopsis KO mutants showed a reduced AtHAK5-mediated Rb+ uptake. Activation of AtHAK5 by AtCIPK1 did not occur under hyperosmotic stress conditions, where AtCIPK1 function has been shown to be required to maintain plant growth. Taken together, our data contribute to the identification of the complex regulatory networks that control the high-affinity K+ transporter AtHAK5 and root K+ uptake.

Coordinated Transport of Nitrate, Potassium, and Sodium.

Potassium (K+) and nitrogen (N) are essential nutrients, and their absorption and distribution within the plant must be coordinated for optimal growth and development. Potassium is involved in charge balance of inorganic and organic anions and macromolecules, control of membrane electrical potential, pH homeostasis and the regulation of cell osmotic pressure, whereas nitrogen is an essential component of amino acids, proteins, and nucleic acids. Nitrate (NO3 -) is often the primary nitrogen source, but it also serves as a signaling molecule to the plant. Nitrate regulates root architecture, stimulates shoot growth, delays flowering, regulates abscisic acid-independent stomata opening, and relieves seed dormancy. Plants can sense K+/NO3 - levels in soils and adjust accordingly the uptake and root-to-shoot transport to balance the distribution of these ions between organs. On the other hand, in small amounts sodium (Na+) is categorized as a "beneficial element" for plants, mainly as a "cheap" osmolyte. However, at high concentrations in the soil, Na+ can inhibit various physiological processes impairing plant growth. Hence, plants have developed specific mechanisms to transport, sense, and respond to a variety of Na+ conditions. Sodium is taken up by many K+ transporters, and a large proportion of Na+ ions accumulated in shoots appear to be loaded into the xylem by systems that show nitrate dependence. Thus, an adequate supply of mineral nutrients is paramount to reduce the noxious effects of salts and to sustain crop productivity under salt stress. In this review, we will focus on recent research unraveling the mechanisms that coordinate the K+-NO3 -; Na+-NO3 -, and K+-Na+ transports, and the regulators controlling their uptake and allocation.

Recognition and Activation of the Plant AKT1 Potassium Channel by the Kinase CIPK23.

Plant growth largely depends on the maintenance of adequate intracellular levels of potassium (K+). The families of 10 Calcineurin B-Like (CBL) calcium sensors and 26 CBL-Interacting Protein Kinases (CIPKs) of Arabidopsis (Arabidopsis thaliana) decode the calcium signals elicited by environmental inputs to regulate different ion channels and transporters involved in the control of K+ fluxes by phosphorylation-dependent and -independent events. However, the detailed molecular mechanisms governing target specificity require investigation. Here, we show that the physical interaction between CIPK23 and the noncanonical ankyrin domain in the cytosolic side of the inward-rectifier K+ channel AKT1 regulates kinase docking and channel activation. Point mutations on this domain specifically alter binding to CIPK23, enhancing or impairing the ability of CIPK23 to regulate channel activity. Our data demonstrate the relevance of this protein-protein interaction that contributes to the formation of a complex between CIPK23/CBL1 and AKT1 in the membrane for the proper regulation of K+ transport.

Regulation of K+ Nutrition in Plants.

Modern agriculture relies on mineral fertilization. Unlike other major macronutrients, potassium (K+) is not incorporated into organic matter but remains as soluble ion in the cell sap contributing up to 10% of the dry organic matter. Consequently, K+ constitutes a chief osmoticum to drive cellular expansion and organ movements, such as stomata aperture. Moreover, K+ transport is critical for the control of cytoplasmic and luminal pH in endosomes, regulation of membrane potential, and enzyme activity. Not surprisingly, plants have evolved a large ensemble of K+ transporters with defined functions in nutrient uptake by roots, storage in vacuoles, and ion translocation between tissues and organs. This review describes critical transport proteins governing K+ nutrition, their regulation, and coordinated activity, and summarizes our current understanding of signaling pathways activated by K+ starvation.

A Critical Role of Sodium Flux via the Plasma Membrane Na+/H+ Exchanger SOS1 in the Salt Tolerance of Rice.

Rice (Oryza sativa) stands among the world's most important crop species. Rice is salt sensitive, and the undue accumulation of sodium ions (Na+) in shoots has the strongest negative correlation with rice productivity under long-term salinity. The plasma membrane Na+/H+ exchanger protein Salt Overly Sensitive 1 (SOS1) is the sole Na+ efflux transporter that has been genetically characterized to date. Here, the importance of SOS1-facilitated Na+ flux in the salt tolerance of rice was analyzed in a reverse-genetics approach. A sos1 loss-of-function mutant displayed exceptional salt sensitivity that was correlated with excessive Na+ intake and impaired Na+ loading into the xylem, thus indicating that SOS1 controls net root Na+ uptake and long-distance Na+ transport to shoots. The acute Na+ sensitivity of sos1 plants at low NaCl concentrations allowed analysis of the transcriptional response to sodicity stress without effects of the osmotic stress intrinsic to high-salinity treatments. In contrast with that in the wild type, sos1 mutant roots displayed preferential down-regulation of stress-related genes in response to salt treatment, despite the greater intensity of stress experienced by the mutant. These results suggest there is impaired stress detection or an inability to mount a comprehensive response to salinity in sos1 In summary, the plasma membrane Na+/H+ exchanger SOS1 plays a major role in the salt tolerance of rice by controlling Na+ homeostasis and possibly contributing to the sensing of sodicity stress.

K+ Efflux Antiporters 4, 5, and 6 Mediate pH and K+ Homeostasis in Endomembrane Compartments.

KEA4, KEA5, and KEA6 are members of the Arabidopsis (Arabidopsis thaliana) K+ efflux antiporter (KEA) family that share high sequence similarity but whose function remains unknown. Here, we show their gene expression pattern, subcellular localization, and physiological function in Arabidopsis. KEA4, KEA5, and KEA6 had similar tissue expression patterns, and the three KEA proteins localized to the Golgi, the trans-Golgi network, and the prevacuolar compartment/multivesicular bodies, suggesting overlapping roles of these proteins in the endomembrane system. Phenotypic analyses of single, double, and triple mutants confirmed functional redundancy. The triple mutant kea4 kea5 kea6 had small rosettes, short seedlings, and was sensitive to low K+ availability and to the sodicity imposed by high salinity. Also, the kea4 kea5 kea6 mutant plants had a reduced luminal pH in the Golgi, trans-Golgi network, prevacuolar compartment, and vacuole, in accordance with the K/H exchange activity of KEA proteins. Genetic analysis indicated that KEA4, KEA5, and KEA6 as well as endosomal Na+/H+exchanger5 (NHX5) and NHX6 acted coordinately to facilitate endosomal pH homeostasis and salt tolerance. Neither cancelling nor overexpressing the vacuolar antiporters NHX1 and NHX2 in the kea4 kea5 kea6 mutant background altered the salt-sensitive phenotype. The NHX1 and NHX2 proteins in the kea4 kea5 kea6 mutant background could not suppress the acidity of the endomembrane system but brought the vacuolar pH close to wild-type values. Together, these data signify that KEA4, KEA5, and KEA6 are endosomal K+ transporters functioning in maintaining pH and ion homeostasis in the endomembrane network.

Upstream kinases of plant SnRKs are involved in salt stress tolerance.

Sucrose non-fermenting 1-related protein kinases (SnRKs) are important for plant growth and stress responses. This family has three clades: SnRK1, SnRK2 and SnRK3. Although plant SnRKs are thought to be activated by upstream kinases, the overall mechanism remains obscure. Geminivirus Rep-Interacting Kinase (GRIK)1 and GRIK2 phosphorylate SnRK1s, which are involved in sugar/energy sensing, and the grik1-1 grik2-1 double mutant shows growth retardation under regular growth conditions. In this study, we established another Arabidopsis mutant line harbouring a different allele of gene GRIK1 (grik1-2 grik2-1) that grows similarly to the wild-type, enabling us to evaluate the function of GRIKs under stress conditions. In the grik1-2 grik2-1 double mutant, phosphorylation of SnRK1.1 was reduced, but not eliminated, suggesting that the grik1-2 mutation is a weak allele. In addition to high sensitivity to glucose, the grik1-2 grik2-1 mutant was sensitive to high salt, indicating that GRIKs are also involved in salinity signalling pathways. Salt Overly Sensitive (SOS)2, a member of the SnRK3 subfamily, is a critical mediator of the response to salinity. GRIK1 phosphorylated SOS2 in vitro, resulting in elevated kinase activity of SOS2. The salt tolerance of sos2 was restored to normal levels by wild-type SOS2, but not by a mutated form of SOS2 lacking the T168 residue phosphorylated by GRIK1. Activation of SOS2 by GRIK1 was also demonstrated in a reconstituted system in yeast. Our results indicate that GRIKs phosphorylate and activate SnRK1 and other members of the SnRK3 family, and that they play important roles in multiple signalling pathways in vivo.

NRT1.5/NPF7.3 Functions as a Proton-Coupled H+/K+ Antiporter for K+ Loading into the Xylem in Arabidopsis.

Potassium and nitrogen are essential macronutrients for plant growth and have a positive impact on crop yield. Previous studies have indicated that the absorption and translocation of K+ and NO3- are correlated with each other in plants; however, the molecular mechanism that coordinates K+ and NO3- transport remains unknown. In this study, using a forward genetic approach, we isolated a low-K+-sensitive Arabidopsis thaliana mutant, lks2, that showed a leaf chlorosis phenotype under low-K+ conditions. LKS2 encodes the transporter NRT1.5/NPF7.3, a member of the NRT1/PTR (Nitrate Transporter 1/Peptide Transporter) family. The lks2/nrt1.5 mutants exhibit a remarkable defect in both K+ and NO3- translocation from root to shoot, especially under low-K+ conditions. This study demonstrates that LKS2 (NRT1.5) functions as a proton-coupled H+/K+ antiporter. Proton gradient can promote NRT1.5-mediated K+ release out of root parenchyma cells and facilitate K+ loading into the xylem. This study reveals that NRT1.5 plays a crucial role in K+ translocation from root to shoot and is also involved in the coordination of K+/NO3- distribution in plants.

The CBL-Interacting Protein Kinase CIPK23 Regulates HAK5-Mediated High-Affinity K+ Uptake in Arabidopsis Roots.

Plant growth and development requires efficient acquisition of essential elements. Potassium (K(+)) is an important macronutrient present in the soil solution at a wide range of concentrations. Regulation of the K(+) uptake systems in the roots is essential to secure K(+) supply. It has been shown in Arabidopsis (Arabidopsis thaliana) that when the external K(+) concentration is very low (<10 µm), K(+) nutrition depends exclusively on the high-affinity K(+) transporter5 (HAK5). Low-K(+)-induced transcriptional activation of the gene encoding HAK5 has been previously reported. Here, we show the posttranscriptional regulation of HAK5 transport activity by phosphorylation. Expression in a heterologous system showed that the Ca(2+) sensors calcineurin B-like (CBL1), CBL8, CBL9, and CBL10, together with CBL-interacting protein kinase23 (CIPK23), activated HAK5 in vivo. This activation produced an increase in the affinity and the Vmax of K(+) transport. In vitro experiments show that the N terminus of HAK5 is phosphorylated by CIPK23. This supports the idea that phosphorylation of HAK5 induces a conformational change that increases its affinity for K(+). Experiments of K(+) (Rb(+)) uptake and growth measurements in low-K(+) medium with Arabidopsis single mutants hak5, akt1, and cipk23, double mutants hak5 akt1, hak5 cipk23, and akt1 cipk23, and the triple mutant hak5 akt1 cipk23 confirmed the regulatory role of CIPK23 in planta.

Structural basis of the regulatory mechanism of the plant CIPK family of protein kinases controlling ion homeostasis and abiotic stress.

Plant cells have developed specific protective molecular machinery against environmental stresses. The family of CBL-interacting protein kinases (CIPK) and their interacting activators, the calcium sensors calcineurin B-like (CBLs), work together to decode calcium signals elicited by stress situations. The molecular basis of biological activation of CIPKs relies on the calcium-dependent interaction of a self-inhibitory NAF motif with a particular CBL, the phosphorylation of the activation loop by upstream kinases, and the subsequent phosphorylation of the CBL by the CIPK. We present the crystal structures of the NAF-truncated and pseudophosphorylated kinase domains of CIPK23 and CIPK24/SOS2. In addition, we provide biochemical data showing that although CIPK23 is intrinsically inactive and requires an external stimulation, CIPK24/SOS2 displays basal activity. This data correlates well with the observed conformation of the respective activation loops: Although the loop of CIPK23 is folded into a well-ordered structure that blocks the active site access to substrates, the loop of CIPK24/SOS2 protrudes out of the active site and allows catalysis. These structures together with biochemical and biophysical data show that CIPK kinase activity necessarily requires the coordinated releases of the activation loop from the active site and of the NAF motif from the nucleotide-binding site. Taken all together, we postulate the basis for a conserved calcium-dependent NAF-mediated regulation of CIPKs and a variable regulation by upstream kinases.

A constitutively active form of a durum wheat Na⁺/H⁺ antiporter SOS1 confers high salt tolerance to transgenic Arabidopsis.

The SOS signaling pathway has emerged as a key mechanism in preserving the homeostasis of Na⁺ and K⁺ under saline conditions. We have recently identified and functionally characterized, by complementation studies in yeast, the gene encoding the durum wheat plasma membrane Na⁺/H⁺ antiporter (TdSOS1). To extend these functional studies to the whole plant level, we complemented Arabidopsis sos1-1 mutant with wild-type TdSOS1 or with the hyperactive form TdSOS1∆972 and compared them to the Arabidopsis AtSOS1 protein. The Arabidopsis sos1-1 mutant is hypersensitive to both Na⁺ and Li⁺ ions. Compared with sos1-1 mutant transformed with the empty binary vector, seeds from TdSOS1 or TdSOS1∆972 transgenic plants had better germination under salt stress and more robust seedling growth in agar plates as well as in nutritive solution containing Na⁺ or Li⁺ salts. The root elongation of TdSOS1∆972 transgenic lines was higher than that of Arabidopsis sos1-1 mutant transformed with TdSOS1 or with the endogenous AtSOS1 gene. Under salt stress, TdSOS1∆972 transgenic lines showed greater water retention capacity and retained low Na⁺ and high K⁺ in their shoots and roots. Our data showed that the hyperactive form TdSOS1∆972 conferred a significant ionic stress tolerance to Arabidopsis plants and suggest that selection of hyperactive alleles of the SOS1 transport protein may pave the way for obtaining salt-tolerant crops.