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

Significance

Plants have evolved several strategies to cope with salinity, which in most species involve efficient Na⁺ management. We show that the Ca²⁺-sensor protein SOS3, a critical determinant of the salt tolerance of Arabidopsis, is the missing molecular link that co-regulates inversely the two major Na⁺ transport systems operating in vascular plants, the SOS1- and HKT1-like proteins that mediate Na⁺ loading and unloading at the xylem, respectively. Under salt stress, SOS3 promotes the recruitment of SOS1 to the plasma membrane and its activation by the SOS2/SOS3 kinase complex, while simultaneously SOS3 interacts with and commands the degradation of HKT1;1. Thus, SOS3 acts as the molecular switch shifting the balance from Na⁺ retention in the root toward delivery to the shoot.

Abstract

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.

Cultivation of crops in many regions requires irrigation, which often leads to the accumulation of high amounts of salt at the topsoil. The resulting salinization reduces growth and yield through ion (mainly Na⁺) toxicity, dehydration stress, and nutrient deficiency (1). Nearly all crops are salt-sensitive glycophytes easily intoxicated by Na⁺ intake (1, 2). Plants have evolved several strategies to cope with salinity that in most species involve efficient Na⁺ management (2). Primary processes are the restriction of Na⁺ uptake by roots, controlled Na⁺ delivery to shoots via the xylem and return via the phloem, and compartmentalizing cellular Na⁺ into vacuoles to avert ion toxicity while improving osmotic balance (1, 3). All these processes involve dedicated ion transporters acting in specific cell membranes and compartments. Genetic evidence has shown that the SOS (salt overly sensitive) pathway is paramount to the Na⁺ tolerance of plants (4–6). The core SOS pathway consists of three major components, the plasma membrane (PM)-localized Na⁺/H⁺ antiporter SOS1/NHX7, the serine/threonine protein kinase SOS2/CIPK24, and the alternative calcineurin B-like Ca²⁺-binding proteins SOS3/CBL4, SCaBP8/CBL10, and CBL8. Under salt stress, the interacting CBLs bind to and recruit SOS2/CIPK24 to the PM to phosphorylate and activate SOS1 (7–10). Work in Arabidopsis thaliana, its halophytic relative Eutrema salsuginea (a.k.a Thellungiella salsuginea), and the crops tomato and rice, has shown the twofold contribution of the SOS pathway to Na⁺ homeostasis: reducing net Na⁺ uptake by roots and favoring the long-distance transport to shoots via xylem loading (5, 6, 11–14).

Opposing the SOS1-mediated fluxes at the vasculature are the HKT1 transporters. All HKT family members behave as Na⁺ transporters mediating either channel-like Na⁺ uniport (class-I, HKT1 group) or Na⁺-K⁺ symport (class-II, HKT2 group) (15, 16). Generally, dicot species have only a few class-I HKT genes in their genomes, while monocots have larger HKT gene families comprising both classes. Quantitative genetics has shown that HKT1 proteins governing the long-distance transport of Na⁺ underpin a significant proportion of the natural genetic variability of salt tolerance in cereal crops (3, 17, 18). The Arabidopsis genome encodes a single HKT1;1 protein that principally mediates Na⁺-selective retrieval out of the xylem sap, thereby reducing the amount of Na⁺ ions delivered to aerial parts (19, 20). Genetic diversity at the HKT1;1 locus impinges on the salt tolerance of A. thaliana ecotypes (21).

Despite the critical importance that the SOS pathway and HKT1-like proteins have in the salt tolerance of plants, little is known about how these opposing fluxes in and out of the xylem are coordinately regulated to achieve fine-tuned long-distance delivery of Na⁺ and root/shoot ion partition (22). The Nax1 and Nax2 loci encoding alleles of HKT1;4 and HKT1;5, respectively, contribute to the salt tolerance of wheat by enhancing Na⁺ retrieval from the xylem and also reducing the expression of SOS1, presumably to lessen the xylem Na⁺ load (23). The cross talk of these Na⁺ transport systems is essential to avoid wasting energy in a potentially futile cycle of Na⁺ loading into and retrieval off the xylem sap. Here, we show that the Ca²⁺-sensor protein SOS3/CBL4, a critical determinant of the salt tolerance of Arabidopsis roots, constitutes a molecular switch that upon salt stress promotes the recruitment of SOS1 to the PM and its subsequent activation by the SOS2/SOS3 complex. Simultaneously, SOS3 commands the degradation of HKT1;1 to shift the balance from Na⁺ retention in the root toward delivery to the shoot. SOS3 achieves this role by direct and SOS2-independent binding to previously unrecognized functional domains of SOS1 and HKT1;1.

Results

SOS3 Interacts with SOS1.

The established model of SOS pathway operation is that SOS2 and SOS3 make a complex at the PM to activate SOS1 transport (Fig. 1A) (7, 10). Notably, SOS3 and SOS1 were also found to interact in a BiFC (bimolecularfluorescence complementation) assay in Nicotiana benthamiana, albeit with a distinctive punctate fluorescence pattern (Fig. 1A and SI Appendix, Fig. S1A). The fluorescence produced by the SOS2/SOS3 complex and by GFP-tagged SOS1 expressed alone delineated the cell contour with a continuous fluorescent rim characteristic of the PM, whereas the SOS1-SOS3 complex showed a distinctive patchy pattern that might correspond to endomembrane compartments or derived vesicles. The interaction of SOS3 and SOS1 was confirmed by co-immunoprecipitation of tagged SOS3-MYC and SOS1-GFP proteins transiently expressed in tobacco leaves (Fig. 1B).

Fig. 1.

SOS3 interacts with SOS1. (A) SOS1-SOS3 interaction visualized by BiFC. SOS1 and SOS2 fused to the N terminus of the fluorescent protein YFP, and wild-type SOS3 and mutant SOS3-1 proteins fused to the C terminus of YFP, were transiently co-expressed in N. benthamiana leaves in the combinations indicated. Combinations that included an empty vector produced no fluorescence. SOS1 fused to full-length GFP (lower row) was used as a control to label the PM. Fluorescence signals for interaction were detected by confocal microscopy 2 to 3 d after infiltration. (Scale bar is 20 μm.) (B) Co-immunoprecipitation of SOS1 and SOS3. The translational fusion SOS1:GFP was transiently co-expressed with MYC-tagged wild-type SOS3 (SOS3:MYC) or the mutant protein SOS3-1 (SOS3-1:MYC) in N. benthamiana leaves. Total proteins were extracted and SOS1:GFP and associating proteins were pulled down with α-GFP antibody. SDS-PAGE and immunoblots were performed with α-GFP and α-MYC antibodies to detect the proteins expressed (Input) and pulled-down (IP). (C) Schematic representation of SOS1 topology and known functional domains. The N-terminal transmembrane segments (gray) extend up to amino acid Gly-441. The large cytosolic part of SOS1 contains the cyclic-nucleotide binding homologous domain (blue, G765-L841), the auto-inhibitory domain (red, L1005-L1047), and the phosphorylation site by the SOS2/SOS3 kinase complex at Ser-1138 (green). The SOS3 binding domain (K460-L482) is shown in yellow. Amino acid residues flanking each domain are indicated. (D) The SOS3 binding domain of SOS1 is necessary and sufficient for interaction. Full-length SOS1, or with an internal deletion of the SOS3 binding domain (SOS1ΔS3BD), or the SOS3 binding domain alone (S3BD) were fused to the N-terminal part of YFP and co-expressed in N. benthamiana leaves with SOS3 fused to the C-terminal part of YFP. The C-terminally truncated SOS1 protein at Gln-998 that lacks the AID and SOS2 phosphorylation site (SOS1Δ998) was fused to the N terminus of YFP, whereas SOS2 was fused to the C terminus of YFP. Recombinant proteins were co-expressed as indicated in each row. Fluorescence signals were detected by confocal microscopy 3 d after infiltration. (Scale bar is 20 μm.)

In the absence of salt stress, the C-terminal auto-inhibitory domain located between residues L1005 and L1047 keeps SOS1 in a resting state (Fig. 1C). The SOS2/SOS3 complex releases this auto-inhibition by phosphorylating SOS1 at S1138, thereby activating Na⁺ transport (10). Deleting both the auto-inhibitory domain and the phosphorylation site in the truncated protein SOS1Δ998 abolished SOS2 interaction but did not prevent SOS3 binding, suggesting different binding sites (Fig. 1D). The SOS3-binding domain (S3BD) of SOS1 was mapped between amino acids K460 and L482, a protein stretch downstream the last transmembrane predicted in the pore domain of SOS1 (Fig. 1C). Deletion of S3BD while preserving the rest of the SOS1 protein (SOS1ΔS3BD) abrogated the interaction with SOS3 but not with SOS2, whereas expression of the S3BD fragment alone was sufficient to restore BiFC with SOS3 (Fig. 1D and SI Appendix, Fig. S1B).

To provide further evidence of the interaction of SOS3 and the S3BD of SOS1, we analyzed the binding of SOS3 to a synthetic peptide spanning the S3BD, residues K460 to D484. Monitoring the intrinsic fluorescence of the unique tryptophan W177 of SOS3 upon excitation at 285 nm was suitable for this purpose since this residue is located in the central hydrophobic cavity through which SOS3 interacts with the partner protein SOS2 (Fig. 2C) (24). The addition of S3BD to SOS3 decreased the ratio of the fluorescence intensity at 330/350 nm in a dose-dependent manner, indicating that SOS3 binds the S3BD through the central cavity of SOS3 (Fig. 2). Furthermore, we modeled the structure of the SOS1 dimer using residues 1 to 697 that comprise the pore domain of SOS1 and the adjacent alpha-helical domain including S3BD (25, 26). The model showed that the S3BD segment folds as a single amphipathic helix that exposes a patch of hydrophobic residues suitable for SOS3 binding (Fig. 2). The predicted SOS3-S3BD complex buries this solvent-accessible hydrophobic patch of S3BD in the hydrophobic crevice of SOS3 that also binds SOS2, suggesting that SOS3 binding to SOS1 is independent of and alternative to SOS2 binding. Deletion of S3BD did not prevent the formation of the SOS1 dimer (SI Appendix, Fig. S1B). These conclusions are consistent with the recent release of the cryogenic electron microscopy (cryo-EM) structure of SOS1 (27, 28). The bi-partite autoinhibitory domain (AID) we had described (10) interacts with the domain termed HC1-HC2 that lies underneath the HC7-HC8 domain corresponding to the SOS3-binding domain (27, 28). Deletion of S3BD could alter the topological relationship between AID and HC1-HC2 and modify the basal activity of SOS1.

Fig. 2.

SOS3 binding to SOS1. (A) Fluorescence spectroscopy titration of SOS3 with S3BD. The squares represent the average value of three independent series of experiments, whose individual values are shown as dots. The error bars are SD. (B) Predicted structure of the S3BD of SOS1. Residues forming the hydrophobic surface exposed to the solvent are indicated. (C) Complex of SOS3 bound to the S3BD of SOS1. The hydrophobic surface of S3BD faces the crevice of SOS3 that serves to bind interacting proteins. Residue W177 used to analyze protein binding is indicated. (D) SOS3 bound to the SOS1 dimer. The SOS3-S3BD complex (C) was overlaid with the structure of the SOS1 dimer. The dashed line separates the transmembrane domain forming the transporting pore of SOS1 and the helical domain underneath the membrane, which contains the S3BD.

We next analyzed whether SOS1 interacted with other CBLs besides SOS3/CBL4. Of the full complement of 10 CBLs encoded in the Arabidopsis genome, only SOS3/CBL4 and CBL8 showed robust interaction with SOS1 in BiFC assays (SI Appendix, Fig. S2). Notably, CBL10/SCaBP8, a SOS3 counterpart that is known to interact with SOS2/CIPK24 to regulate SOS1 mainly in shoots (9), failed to interact with SOS1.

SOS3 Promotes SOS1 Recruitment to the PM.

Expression of the core SOS elements in Saccharomyces cerevisiae reconstitutes a functional SOS pathway that conveys Na⁺ tolerance to yeast cells (7, 9). Using this system, we assessed whether the Na⁺/H⁺ exchanger activity of SOS1 was affected by its interaction with SOS3 by measuring the Na⁺ tolerance of yeast co-expressing SOS2, SOS3, and either wild-type SOS1, SOS1ΔS3BD or SOS1Δ998. Although deletion of S3BD increased the basal activity of SOS1, the SOS1ΔS3BD protein was still further activated by SOS2/SOS3 (Fig. 3A). These results are in agreement with the notion that the removal of S3BD alters the topology of the SOS1 protein so that the auto-inhibitory domain is less effective to keep SOS1 in the resting state (10, 27) and that the direct binding of SOS3 to SOS1 is not a requisite for the SOS2/SOS3 kinase complex to activate SOS1 in vivo. Removal of the auto-inhibitory domain of SOS1 (SOS1Δ998) resulted in a constitutively active protein imparting robust tolerance of yeast to very high NaCl concentrations (Fig. 3A). Co-expression of SOS3 with SOS1Δ998, which still retains S3BD, had no significant effect on SOS1Δ998 activity (Fig. 3B). Collectively, these results indicate that the direct interaction with SOS3 is not a significant factor in setting the catalytic activity of SOS1, in contrast with the large activation achieved by phosphorylation by the SOS2/SOS3 kinase complex (10).

Fig. 3.

Relevance of SOS3 binding for SOS1 activity. (A) Full-length SOS1 or a recombinant protein in which the SOS3-binding domain had been spliced out (SOS1ΔS3BD) was expressed alone or together with SOS2 and SOS3 in yeast cells to reconstitute a functional SOS module. Four serial decimal dilutions (10^-1 to 10^-4) of overnight liquid cultures of transformed cells were spotted in selective AP medium supplemented with the indicated NaCl concentrations. Plates were pictured after 2 to 3 d at 30 °C. Two independent transformants for each combination are shown. (B) Full-length SOS1 or a constitutively active SOS1 protein lacking the auto-inhibitory and phosphorylation domains (SOS1Δ998) was co-expressed or not with SOS3. Samples were processed as in (A). Note that maximal NaCl concentration used was 800 mM. (C) Mutant sos1-1 seedlings transformed with constructs pSOS1:SOS1:GFP or pSOS1:SOS1ΔS3BD:GFP to express wild-type SOS1 or a recombinant form with the SOS3 binding domain spliced out (SOS1ΔS3BD) at nearly native protein levels, were initially grown on regular ½MS medium and then transferred to fresh media plates with and without 50 mM NaCl to test for phenotypic complementation. The box plots show root growth. Centerlines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum values (n = 14). Asterisks indicate significantly different means relative to WT, based on one-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.001.

We next tested the functionality of the SOS1ΔS3BD protein in planta. To mimic the native expression pattern and protein abundance of SOS1, a genomic fragment comprising the SOS1 gene promoter and the first 13 exons and 13 introns of the SOS1 gene were fused to the cDNA sequences encoding the remainder of SOS1 and SOS1ΔS3BD proteins with C-terminal GFP translational fusions to the SOS1 protein variants. Transformation of sos1-1 plants with the resulting pSOS1:SOS1:GFP and pSOS1:​SOS1ΔS3BD:GFP constructs showed that, in contrast to full-length SOS1, the SOS1ΔS3BD protein failed to complement the sos1-1 mutant, indicating that the interaction with SOS3 is essential for the function of SOS1 in planta (Fig. 3C). To investigate this discrepancy between biological systems, we examined the localization and protein stability of SOS1 in the sos3-1 mutant that expresses a dysfunctional SOS3 protein with a defective EF-hand calcium-binding site (11, 29). First, we confirmed that the SOS3-1 protein was expressed but incompetent to interact with SOS1 in BiFC and co-immunoprecipitation assays (Fig. 1B and SI Appendix, Fig. S1 A and C). Under non-saline control conditions and using spinning disk confocal microscopy, we could image in meristematic root cells the weak SOS1:GFP fluorescence localizing at the PM and in yet uncharacterized cytosolic compartments (Fig. 4). Salt treatment promoted the recruitment of SOS1:GFP to the PM in control SOS3 roots over 1 to 3 d at the expense of the gradually disappearing cytosolic signal (Fig. 4 and SI Appendix, Fig. S3A). However, the opposite was observed in the sos3-1 mutant, in which most of SOS1:GFP re-localized to cytosolic compartments and then to the vacuole after 2 d of treatment (Fig. 4). We confirmed the SOS1 re-localization pattern in a complete sos3/cbl4 knock-out mutant not producing SOS3 protein (30) (SI Appendix, Fig. S3B). In the converse experiment, the expression of the SOS1ΔS3BD-GFP mutant protein (pSOS1:SOS1ΔS3BD:GFP) in the sos1-1 plant resulted in extensive fluorescence in cytosolic compartments and weak signal at the PM (Fig. 4 C and D). Roots were shortly stained with the fluorescent lipophilic dye FM4-64 to visualize the PM. Together, these results indicate that SOS1 requires the binding of SOS3 at the S3BD for the salinity-induced recruitment to the PM and subsequent activation by the SOS2/SOS3 kinase complex.

Fig. 4.

Salinity- and SOS3-dependent recruitment of SOS1 to the PM. (A) Representative spinning disk confocal images of epidermal cells at the root meristem of Col-0 gl1 (wild-type, WT) and sos3-1 transgenic lines expressing pSOS1:SOS1:GFP and treated with 50 mM NaCl over 1 to 3 d. (Scale bar is 10 μm.) (B) Quantification of SOS1:GFP fluorescence partition between PM and intracellular compartments (IC) compartments of samples shown in (A). Violin plots: centerlines show the medians, dotted lines indicate the 25th and 75th percentiles. Data are from n ≥ 70 cells from ≥7 plants. Letters indicate significantly different means, One-way ANOVA followed by Tukey’s HSD, P < 0.05. (C) Representative spinning disk confocal images of epidermal cells at the root meristem of sos1-1 plants transformed with the indicated plasmids. Samples were shortly treated with FM4-64 to stain the PM and imaged in control conditions. (Scale bars, 10 μm.) (D) Quantification of fluorescence partition between PM and IC compartments of samples in (C). Violin plots are as in (B). Data are from 60 cells of 6 plants. Asterisks indicate significantly different means at P < 0.0001, Welch’s unpaired t test.

HKT1;1 Interacts with SOS3.

Genetic interactions between sos3 and hkt1 mutants of Arabidopsis indicated that the SOS and HKT1 systems must be counterbalanced to achieve salt tolerance (31). We hypothesized that SOS1- and HKT1-dependent Na⁺ fluxes at the xylem parenchyma should be co-regulated in opposing ways to prevent a futile cycle of antagonizing loading and unloading fluxes (3, 22). To test this idea, we examined the root growth of wild-type (Col-gl1), single mutants sos1-1, sos2-2, sos3-1, and hkt1-1, and the double mutants sos1-1 hkt1-1, sos2-2 hkt1-1, and sos3-1 hkt1-1 on high-salt media (SI Appendix, Fig. S4). In young seedlings under limited transpiration, the salt sensitivity of all sos mutants was alleviated when combined with the hkt1-1 mutation (Fig. 5A and SI Appendix, Fig. S4) as previously reported for sos3-1 (32).

Fig. 5.

Physical and genetic interactions of SOS3 and HKT1. (A) Mutation hkt1-1 suppresses the salt hypersensitivity of sos3-1. Seedlings of wild-type, mutants sos3-1, hkt1-1, hkt1-1 sos3-1, and the complemented line sos3-1 35S:SOS3 (SOS3ox) were transferred to ½MS medium supplemented with 50 and 100 mM NaCl. Pictures were taken 1 wk after transfer. (Scale bar size is 1 cm.) (B) BiFC assay to map the interaction domain of HKT1;1 with SOS3. SOS3 fused to the C-terminal part of the Venus fluorescent protein (SOS3:VC) was transiently co-expressed in tobacco epidermal cells with the Venus N-terminal part fused to the full-length HKT1;1 (HKT1:VN) or the indicated protein fragments. Fluorescence signals produced by protein interaction were detected by confocal microscopy on the third day after infiltration. (Scale bar size is 20 μm.) (C) Schematic representation of HKT1;1 protein topology consisting of 4 pore-loop domains (P_A to P_D) and the transmembrane fragments (M1_A/M2_A to M1_D/M2_D).

We found that SOS3 also interacted with HKT1;1 in BiFC and co-IP experiments (Figs. 5B and 6A). The mutant protein SOS3-1 failed to interact with HKT1;1 in these assays (Fig. 6A), and neither did SOS1, SOS2, or CBL10 (SI Appendix, Fig. S5). The topology of HKT1;1 consists of four repeated membrane/pore-loop/membrane domain motifs (M1PM2_A, M1PM2_B, M1PM2_C, M1PM2_D) (Fig. 5C) (33). The SOS3 interaction domain of HKT1;1 was located between residues 104 and 152, comprising the intervening cytosolic loop between the transmembranes M2_A and M1_B, a protein fragment to which SOS3 bound strongly when tested separately from the rest of the HKT1;1 protein (Fig. 5B). We have modeled the HKT1;1 protein folding using the known 3D structures of bacterial potassium transporters KtrB (PDB entry 4J7C) and TrkH (PDB entry 3PJZ) as templates. The domain comprising amino acids 110 to 150 is predicted to produce a helical structure protruding at the surface of HKT1;1 protein, near the PM and facing inward to the cytosol (SI Appendix, Fig. S6), which is well suited to serve as the SOS3 docking site.

Fig. 6.

SOS3 controls HKT1 protein stability. (A) The HKT1:GFP fusion protein was transiently co-expressed with SOS3:MYC or SOS3-1:MYC in tobacco leaves. Total proteins were extracted and HKT1:GFP-associating proteins were pulled down with α-GFP antibody. SDS-PAGE and immunoblots were performed to detect expressed proteins (Input) and pulled-down proteins (IP). α-GFP and α-MYC antibodies were used to detect HKT1;1 and SOS3 or SOS3-1, respectively. (B) Total proteins were extracted from 2-wk-old Arabidopsis wild-type seedlings transformed with 35S:HKT1:GFP and treated with 100 mM NaCl for the indicated periods. The HKT1:GFP protein was detected by western blot using α-GFP antibody. Coomassie Brilliant Blue (CBB) staining is shown as a loading reference. (C) Arabidopsis seedlings of genotype hkt1-3 transformed with pHKT1:HKT1:CFP levels were salt-treated 100 mM NaCl, with and without 50 µM MG132, for 12 h. The HKT1:CFP protein was detected with α-GFP antibody, and α-tubulin antibodies were used for protein loading control. (D) Ten-day-old seedlings of wild-type, sos1-1, sos2-2, and sos3-1 transformed with construct 35S:HKT1:GFP were treated with 100 mM NaCl and with or without 100 µM MG132 for 12 h, and total protein was extracted from root tissues. Immunoblots were performed to detect HKT1:GFP protein with α-GFP. CBB staining and western blotting of HSP90 show protein loading.

Salt- and SOS3-Dependent Degradation of HKT1.

The channel-like activity of HKT1;1 was registered in Xenopus laevis oocytes, alone or in combination with SOS3 to determine whether the interaction altered the Na⁺ transport of HKT1;1. In the presence of external Na⁺ (97 mM NaCl plus 3 mM KCl), HKT1;1 generated Na⁺-dependent inward currents that were much higher than those recorded in control oocytes (mock injection) or in the presence of 100 mM KCl. Co-expression of SOS3 produced currents that were no different from HKT1;1 alone (SI Appendix, Fig. S7). Oocytes co-expressing HKT1;1 with tagged SOS3:RFP, whose expression and localization could be confirmed under the confocal microscope, produced similar recordings of Na⁺ fluxes.

Next, we examined HKT1;1 protein stability under salt stress. In transgenic seedlings of genotype 35S:HKT1:GFP salt treatment reduced the amount of the HKT1:GFP protein, which gradually disappeared over a period of 24 h (Fig. 6B). Testing HKT1;1 protein stability in a pHKT1:HKT1:CFP/hkt1-1 complementation line that mimicked the native expression pattern and protein abundance of HKT1;1 confirmed the salt-induced degradation, which was prevented by the 26S proteasome inhibitor MG132 (Fig. 6C). Moreover, a western blot with polyclonal antibodies against the native HKT1;1 protein in Col-0 gl1 plants confirmed the steady decay of HKT1;1 that was again counteracted by MG132 (SI Appendix, Fig. S8A). Treatment with 3 mM CaCl₂ produced similar results to those of salinity stress (SI Appendix, Fig. S8 B and C). Together, these data demonstrate that HKT1;1 stability is substantially decreased by salinity stress or high-calcium treatment reminiscent of the salt-induced raise in cytosolic free calcium.

To test whether SOS3 was involved in the salt-induced degradation of HKT1;1, the 35S:HKT1:GFP reporter line was crossed with sos1-1, sos2-2, and sos3-1 mutant backgrounds. Seedlings were treated with 100 mM NaCl for 12 h with and without 50 µM MG132. Western blotting with α-GFP antibody showed that salt-induced degradation of HKT1;1 occurred in sos1-1 and sos2-2 mutants but not in the sos3-1 mutant (Fig. 6D). Degradation of HKT1;1 protein upon high-calcium treatment was also blocked in sos3-1 35S:HKT1:GFP plants (SI Appendix, Fig. S8D). Notably, salt and calcium treatments had the opposite effect on SOS3 and stabilized SOS3:MYC and SOS3:GFP proteins in tobacco leaves (SI Appendix, Fig. S9). Myristoylation at position Gly2 serves for anchoring SOS3 to the PM and is essential for SOS1 activation (34). Western blotting of native HKT1;1 in sos3-1 plants expressing the non-myristoylated SOS3-G2A protein indicated that SOS3 recruitment to the PM was a requisite for HKT1;1 degradation (SI Appendix, Fig. S10). Together, these data indicate that HKT1;1 activity is detrimental under acute salt stress and that SOS3 is specifically involved in HKT1;1 degradation. Accordingly, HKT1;1 mRNA abundance declined in salt-treated seedlings of wild-type and sos mutants (SI Appendix, Fig. S8E).

The salt-induced degradation of HKT1 predicted that acute sodicity stress should minimize differences in shoot/root partition of Na⁺ between wild-type and hkt1 plants. To test this, plants were treated with 5 mM Na⁺ in the presence of 0.1, 1 or 10 mM K⁺ to modulate the intensity of the Na⁺-K⁺ imbalance while avoiding the osmotic stress imposed by a high-concentration NaCl treatment. The much greater Na⁺/K⁺ ratios at 0.1 mM K⁺ compared to 10 mM K⁺ confirmed the efficacy of the differential sodicity treatment (SI Appendix, Fig. S11A). As expected from SOS1 and HKT1;1 function in organismal Na⁺ fluxes, Na⁺/K⁺ ratios were maximal in sos1 roots and in hkt1 shoots. In the 10 mM K⁺ treatment, the Na⁺ content in hkt1 shoots exceeded WT values (P = 6.38 × 10^-6, Fisher’s LSD test) (SI Appendix, Fig. S11B). However, in 0.1 mM K⁺ the Na⁺ content of WT plants approached that of the hkt1 mutant (P = 0.29, Fisher’s LSD test). The sos1 mutant roots had the greatest Na⁺ contents in all K⁺ conditions owing to reduced Na⁺ efflux (11), but enhanced sodicity stress partly redirected Na⁺ toward sos1 leaves (P = 0.04; 0.1 vs. 10 mM K⁺) and alleviated the root Na⁺ load (P = 0.02, 0.1 vs. 10 mM K⁺). Last, Na⁺ profiles and shoot/root partition were compared for single sos1, sos3 and double sos1 sos3 mutant seedlings under sodicity stress (5 mM Na⁺ in 0.1 mM K⁺). The high root Na⁺ content in sos1 and sos3 plants was increased further in the sos1 sos3 mutant, whereas the shoot content decreased. This doubled the root-to-shoot ratio of sos1 sos3 plants relative to the single mutants (Fig. 7A). Collectively, these results are coherent with the sodicity-induced and SOS3-dependent inactivation of HKT1;1.

Fig. 7.

SOS3 controls long-distance Na⁺ transport from roots to shoots. (A) Na⁺ concentration in roots and leaves. Two-week old plants of genotypes Col-0 gl1 (WT), hkt1-1, sos1-1, sos3-1, and the double mutant sos1-1 sos3-1 were transferred to hydroponic LAK medium supplemented with 5 mM NaCl and 0.1 mM KCl for another 2 wk. Plants were collected, weighted, and had their root and leaves separated and homogenized individually. Shown are the means and SE of the Na⁺ contents (% of dry weight) determined by atomic emission spectrometry. The ratios of root-to-shoot contents are given in the Lower panel. Different letters indicate statistical differences by Fisher’s LSD (P < 0.05). Smaller values are given in numerals for clarity. (B) Mechanistic model of SOS3 operation. Under non-saline or moderately saline conditions, roots take up salts, including Na⁺ ions, to reduce root water potential and draw water from the soil. The net balance of basal activity of SOS1 and the retrieval of Na⁺ ions from the xylem sap by HKT1;1 system allows Na⁺ uptake and the retention of salts in the root. When salinity reaches stress levels, activated SOS3 promotes the delivery of SOS1 to the PM and enhances its Na⁺/H⁺ exchanger activity via phosphorylation by SOS2. Whether binding of SOS3 to SOS1 and SOS1 activation by the SOS2/SOS3 complex (not shown) occurs simultaneously or they proceed stepwise so that SOS1 is first trafficked to the PM and subsequently activated by SOS2/SOS3, remains unknown. In concert, activated SOS3 targets HKT1;1 for degradation to prevent further Na⁺ unloading from the xylem sap. These events lead to increased Na⁺ efflux toward the soil and active loading into the xylem, thereby allowing the evapotranspiration stream to evacuate Na⁺ toward the shoot, where potentially toxic Na⁺ ions will be compartmentalized into vacuoles and diluted by growth.

Discussion

Although Na⁺ toxicity is a major physiological constraint imposed by salinity, controlled Na⁺ uptake and compartmentation may be beneficial to preserve cell turgor (35–37). The long-distance transport through the xylem and subsequent shoot-root partition of the potentially toxic Na⁺ ions have been recognized as critical determinants of salt tolerance in several plant species, including the crops rice and wheat that are the staple foods for most human societies (3, 15). Two major systems, with opposing fluxes, have been identified controlling the Na⁺ xylem content: the SOS1 protein that actively loads Na⁺ into the root xylem for delivery to shoots (5, 11–13), and HKT1-like proteins that mediate Na⁺ reabsorption from the xylem stream to protect the photosynthetic and reproductive structures in the shoot (3, 17, 18, 20, 38). If left unchecked and running autonomously, these two counter-acting systems would result in a futile cycle of Na⁺ loading and unloading (22). Using Arabidopsis as a dicot model, here we report that binding of SOS3 to SOS1 and HKT1;1 proteins constitutes a molecular switch that shifts the balance from Na⁺ reabsorption to evacuation via the xylem under salinity stress. Our data also reveal a regulatory layer controlling in a coordinated manner the localization and abundance of SOS1 and HKT1;1 proteins, representing the two most critical and universal Na⁺ transport systems in vascular plants. SOS3 promotes the recruitment of SOS1 to the PM of root cells through the direct and SOS2-independent binding to a previously unrecognized functional domain of SOS1. Moreover, the results demonstrating that SOS3 controls HKT1;1 protein stability provide a foundation for further research on the regulation of HKT-like proteins.

The SOS3 protein contains four EF-hand motifs for Ca²⁺ binding, which triggers the dimerization of SOS3, and EF3 appears to be critically important for this process (39). The mutant protein SOS3-1 carries a deletion of three amino acids at EF3, which results in a dysfunctional mutant protein in biological assays (39–41). The mutant protein SOS3-1 does not interact with SOS2 (40), and here we demonstrate that it also fails to interact with SOS1 and HKT1;1 (Figs. 1 and 5). Together, these results indicate that Ca²⁺-activated SOS3 protein is what triggers the recruitment of SOS1 to the PM under salt stress, the assembly of the SOS2/SOS3 kinase complex that phosphorylates SOS1 to promote Na⁺ efflux, and the concurrent degradation of HKT1;1. Hence, we propose a model (Fig. 7B) in which roots under regular non-saline or moderately saline conditions allow the uptake of salts, among which are the usually abundant and osmotically active Na⁺ ions, to reduce the water potential of the root symplast and to draw water from the soil (36). In the absence of a threatening salinity stress level, the SOS system operates with basal activity minimizing Na⁺ efflux and xylem loading (5, 10, 11) while HKT1;1 recovers from the xylem sap Na⁺ ions leaked into the xylem (20). However, when Na⁺ ions reach potentially toxic levels in roots, a stress-signaling rise in cytosolic Ca²⁺ activates SOS3, which triggers the delivery of SOS1 to the PM and the activation of its Na⁺/H⁺ exchanger activity via phosphorylation by SOS2. These events lead to increased Na⁺ efflux toward the soil in the undifferentiated root tissues and the epidermis of older roots, and the active loading of Na⁺ into the xylem in the differentiated stele (5, 11). Simultaneously, activated SOS3 targets HKT1;1 for degradation to prevent Na⁺ unloading from the xylem sap, thereby allowing the evapotranspiration stream to evacuate Na⁺ from the root toward the shoot, where eventually the Na⁺ load could be counteracted by sequestration into vacuoles, dilution by growth, and the shedding of senescent leaves (36, 42). Failure of either the loading or unloading system leads to uncontrolled Na⁺ uptake (sos mutants) or untenable delivery to shoots (hkt1 mutant), and to salt sensitivity. The simultaneous loss of SOS3 and HKT1;1 results in a precarious but somehow balanced root/shoot Na⁺ partition that explains the mutually suppressive effects of these mutations (Fig. 5 and SI Appendix, Fig. S4) (31, 43). In this regard, it is noteworthy that SOS1 protein stability is partly controlled by diurnal cycles in Arabidopsis plants submitted to salinity stress, with maximal SOS1 abundance at the end of the light period (ZT8-12) when shoot Na⁺ accumulation driven by evapotranspiration peaks (44). Moreover, SOS1 functions in circadian clock regulation, pointing to a superior regulatory role for SOS1 in addition to its function as a transporter to maintain Na⁺ homeostasis under daily fluctuating salt levels (45).

The PM is dynamically and continuously reorganized in response to environmental inputs. Salinity stress enhances endocytosis and cargo protein shuttling between the PM and endosomes (46). Patellin1 (PATL1), a phosphoinositide-binding protein that physically interacts with SOS1, negatively regulates vesicle trafficking and might function in the attenuation of SOS1 activity by repressing endocytosis and protein turnover (47). By contrast, we show here that SOS3 is required for anterograde trafficking of SOS1 toward the PM during salt stress. This mechanism regulating SOS1 recruitment to the PM appears to be substantially different from the previously described process by which SOS3/CBL4 facilitates the translocation of the K⁺ channel AKT2 from the endoplasmic reticulum to the PM (30). In the latter case, SOS3/CBL4 acts in concert with the partnering kinase CIPK6, and it is the complex between SOS3/CBL4 and the C-terminal non-catalytic domain of CIPK6 what promotes the kinase-dependent but phosphorylation-independent translocation of AKT2 to the PM. However, the function of SOS3 in sorting SOS1 is independent of SOS2 and mediated solely by the binding of SOS3 to a domain identified in SOS1. The low and localized expression of HKT1 in differentiated vascular tissues, made unfeasible the microscopy cell biology characterization of the pHKT1:HKT1:GFP line, as we show for pSOS1:SOS1:GFP.

The SOS3-SOS1 interaction resembles that of mammalian calcineurin-B homologous proteins (CHP) as obligatory binding partners for the PM Na⁺/H⁺ exchangers (NHE). NHE antiporters are physiologically important regulators of ionic and pH homeostasis through Na⁺/H⁺ exchange at the PM and other intracellular (IC) membranes. CHP1 and CHP2 are essential cofactors supporting the physiological activity of family members NHE1 and NHE3. Mutant NHE proteins defective in CHP binding exhibited low exchange activity and altered the IC pH‐dependence, suggesting that CHP binding is a key feature to maintain the physiologically active conformation of NHEs (48, 49). On the other hand, binding of CHP3 appeared to regulate NHE1 maturation and protein half-life at the cell surface, thereby increasing the steady-state abundance of functional NHE1 protein (47). CHPs bind to a cytoplasmic α‐helix of NHEs proximal to the pore region and the PM (48, 49) which is topologically similar to the SOS3-binding site of SOS1 (Fig. 1C). The same holds true for the SOS3 binding domain of HKT1;1, which by protein modeling constitutes a protrusion underneath the cytosolic face of the PM (SI Appendix, Fig. S6). Although SOS3 binding appeared not to affect the catalytic activity of SOS1 and only its trafficking to the PM, the striking similarities between interacting modules SOS1-SOS3 and NHE-CHP are evidence of the evolutionary conservation of regulation of eukaryotic CPA1 exchangers (animal NHE and NHXs of plants and fungi) by calcineurin-B like proteins (CHP and CBL). Vascular plants may have co-opted this intrinsically cellular mechanism to achieve the coordinated regulation of SOS1- and HKT1-like proteins for the long-distance transport of Na⁺ and inter-organ distribution.

Our data unravel a key process in the regulation of HKT1;1. Thus far, our understanding of the regulation of HKT1-like channels was limited to gene transcription. In Arabidopsis, the expression of HKT1 in shoots is negatively regulated by small RNA-mediated promoter methylation (50). Cytokinin and ABA reduce HKT1 expression in roots through ARR1 and ARR12 (type B Arabidopsis Response Regulator 1 and 12) (51) and ABI4 (ABSCISIC ACID INSENSITIVE 4) (52). The calmodulin-binding transcription factor CAMTA6 induces HKT1 expression in response to NaCl and ABA in germinating seedlings (53). Importantly, HKT1 expression is determined by the intensity of the saline stress. Low salt concentrations, up to 30 mM NaCl, induced HKT1 expression, but mRNA levels dropped to very low abundance at higher salt concentrations (20). With smaller amplitudes in mRNA abundance, the same behavior was observed in response to increasing amounts of KCl, sorbitol, and mannitol, suggesting that, as we propose here, the primary response of osmotically challenged plants is to favor salt retention in the root, in part by enhancing HKT1;1 activity. However, in conditions in which further root Na⁺ retention could become damaging, stress-activated SOS3 binds to and promotes the degradation of HKT1;1 (Fig. 6) at the same time that gene transcription is reduced (SI Appendix, Fig. S8). SOS3 must reach the PM to trigger HKT1;1 degradation by the MG132-sensitive proteasome (SI Appendix, Fig. S10), yet clarifying the molecular details of how HKT1;1 degradation proceeds requires additional research. Several ubiquitin E3 ligases have been shown to regulate plant salt tolerance. EST1 (ENHANCED SALT TOLERANCE 1), an F-box ubiquitin E3 ligase of Arabidopsis, negatively regulates MKK4 protein level and the activity of a PM Na⁺/H⁺ antiporter (54). The salt tolerance of the est1 mutant was suppressed in the est1/mpk6 and est1/sos1 double mutants, suggesting that the salt tolerance of ste1 was largely due to enhanced SOS1 activity by the MKK4/MPK6 module (55). STRF1 (SALT TOLERANCE RING FINGER 1), a membrane-associated RING finger E3 ligase, is involved in the response to salt stress and protein trafficking, but the targets of STRF1 are still unknown (56). These reports strongly suggest the involvement of ubiquitin E3 ligases in the regulation of Na⁺ tolerance in Arabidopsis. We are currently characterizing F-box E3 ligases potentially interacting with SOS1 and HKT1 proteins.

In conclusion, we have shown that the opposing Na⁺ fluxes mediated by the antagonistic activity of SOS1 and HKT1;1 transporters are under the inverse control of the Ca²⁺-activated sensor SOS3 through direct protein interactions. SOS3 activation upon salinity stress results in the recruitment of SOS1 to the PM and the degradation of HKT1;1, presumably to switch from Na⁺ retention in the root to controlled delivery to the shoot.

Materials and Methods

Plant Materials.

Wild-type Arabidopsis Col-gl1 was used in this work. Arabidopsis mutants sos1-1, sos2-2, and sos3-1 have been described (57). Double mutants sos1-1 hkt1-1, sos2-2 hkt1-1, and sos3-1 hkt1-1 were kindly provided by Ana Rus (31, 32), and the sos1-1 sos3-1 line was produced by crossing. The sos3-1 SOS3ox transgenic line was obtained from Jian-Kang Zhu (29). Transgenic Arabidopsis plants were produced by Agrobacterium tumefaciens-mediated transformation and floral dipping (58). In all plant transformations made for this study, several primary transgenic lines were tested and representative homozygous lines of the T3 generation were used for further experimentation.

Plant Salt Tolerance Tests.

Surface sterilized Arabidopsis seeds were germinated on ½ Murashige and Skoog (MS) medium for 4 d. Then seedlings were transferred to vertical ½ MS plates (pH 5.7 and 1.2% agar) supplemented with the indicated NaCl concentrations. To measure ion profiles under sodicity stress, plants were grown in hydroponic LAK medium, which has only traces of Na⁺ and K⁺ (59), supplemented with KCl and NaCl as indicated for each experiment. For HKT1;1 protein stability tests, 10- to 12-d-old Arabidopsis HKT1:GFP overexpression plants were treated with 100 mM NaCl or 3 mM CaCl₂ with or without 50 to 100 µM MG132 for the time indicated in each experiment. Tobacco leaves transiently expressing HKT1:GFP were treated with 100 mM salt or 3 mM CaCl₂ for 8 h. HKT1 transcript level was checked by quantitative real-time PCR. Ten-day-old Arabidopsis seedlings of the indicated genotypes were treated with 100 mM NaCl for 8 h. Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen) and treated with DNase (Sigma). First-strand cDNA was synthesized using the ReverTra Ace-α- (Toyobo Co. Ltd.). Amplified products were detected using iQTM SYBR green supermix in a Bio-Rad C1000TM thermal cycler CFX384. Transcript levels of HKT1 were measured and normalized to that of UBQ10. Errors bars represent means ± SEM from two biological replicates with three technical repeats each. Primers used for qRT-PCR were AtHKT1-860F, AtHKT1-960R, UBQ-qRT-For, and UBQ-qRT-Rev (SI Appendix, Table S1).

Yeast Assays.

Plasmid pYPGE15 expressing the wild-type SOS1 or the hyperactive mutant SOS1Δ998 were described elsewhere (10). A cDNA version of SOS1 harboring a deletion of the SOS3 binding domain (S3BD) was generated by overlap extension PCR to produce plasmid pYPGE15-SOS1ΔS3BD (SI Appendix, Supplementary Methods). The SOS3 cDNA was subcloned in the yeast expression vector p425GPD as a 0.7 kb BgIII/XhoI fragment. For simultaneous expression of SOS2 and SOS3 plasmid pFL32T was used (7). Plasmids were transformed in the salt-sensitive yeast strain ANT5 (ena1::KanMX4::ena4, nha1::hisG) (60) using the PEG/LiAc method (61). Salt tolerance drop tests were performed in the alkali cation-free medium AP (62) solidified with 2% (w/v) agar and supplemented with 1 mM KCl and NaCl at the concentrations indicated.

Co-Immunoprecipitation Assays.

Combinations of Agrobacterium cultures carrying HKT1:GFP, SOS3:MYC, and/or SOS3-1:MYC were infiltrated into epidermal cell layers of N. benthamiana leaves. Samples were harvested after 2 or 3 d and ground. Total proteins were extracted and HKT1;1-associated protein complexes were immunoprecipitated with anti-GFP fused Protein Agarose A protein beads (Invitrogen, #A11120). Eluted proteins and aliquot from inputs were subjected to SDS-PAGE and immunoblotting (see detailed protocols in SI Appendix, Supplementary Methods).

Constructs for GFP-Based Localization In Planta.

The 35S:SOS1:GFP construct for transient expression in N. benthamiana has been described elsewhere (25). For the transient expression of SOS3:GFP and SOS3-1:GFP, amplicons produced with primers SOS3-HindIII-For and SOS3-GFP-3, were inserted in pGreenII as a HindIII-NotI fragment. A plasmid construct for the expression of the C-terminal GFP-tagged SOS1 protein under the control of its own gene promoter (pSOS1:SOS1:GFP) was produced for this work (SI Appendix, Supplementary Methods). The construct pSOS1:SOS1:GFP was transformed in Arabidopsis Col0-gl1 and mutant lines sos1-1 and sos3-1, and was confirmed to complement the sos1-1 mutant (Fig. 2B). To express a SOS1 protein unable to interact with SOS3, a fragment spanning the SOS1ΔS3DB internal deletion was used to replace the equivalent fragment in pSOS1:SOS1:GFP to produce the plasmid pSOS1:SOS1ΔS3BD:GFP (SI Appendix, Supplementary Methods), which was used to transform sos1-1. In this case, no complementation of the salt-sensitive phenotype was observed (Fig. 2B).

Arabidopsis Col-gl1 plants and hkt1-1 mutants were transformed with a 35S:HKT1:GFP construct (63) to generate HKT1:GFP and hkt1-1 HKT1:GFP transgenic lines. Lines sos1-1 HKT1:GFP, sos2-2 HKT1:GFP, and sos3-1 HKT1:GFP were obtained by crossing. Plasmid pHKT1:HKT1:CFP was produced to direct HKT1:CFP expression from its own promoter (SI Appendix, Supplementary Methods). Genotypes were verified by immunoblotting and PCR analysis.

BIFC.

The SOS1 fusions to the N-terminal half of YFP in SPYNE173 or the C-terminal part of YFP in SPYCE(M) have been described previously (25). Constructs for the co-expression of protein SOS3-1, the SOS3-binding domain (S3BD) of SOS1 and proteins SOS1Δ998 and SOS1ΔS3BD are described in SI Appendix, Supplementary Methods. The coding sequences of the 10 members of the Arabidopsis CBL family subcloned in pSPYCE(M) were kindly provided by Jörg Kudla (64, 65). Fusion of SOS2(CIPK24) and CIPK9 to the N-terminal half of the YFP in pSPYNE(R)173 were used as positive controls when the interaction of SOS1 with the CBL tested was negative. For BiFC with HKT1;1 and SOS3, the corresponding cDNAs were cloned using Gateway technology (Invitrogen), generating the constructs pDEST-HKT1-VYNE and pDEST-SOS3 VYCE (66). Reciprocal empty plasmids were used as negative controls with pDEST-HKT1-VYNE or pDEST-SOS3-VYCE.

Proteins were transiently expressed in N. benthamiana tobacco leaf epidermal cells through A. tumefaciens infiltration. Two-three days after infiltration, YFP fluorescence signals were detected at an excitation wavelength of 515 nm using a confocal laser scanning microscope (Olympus FluoView FV1000).

Laser Scanning and Spinning Disk Confocal Microscopy.

Laser scanning confocal microscopy of GFP-tagged SOS3 and HKT1;1 proteins was done with an excitation wavelength of 488 nm using the multi-line Argon laser source. For spinning-disk confocal microscopy of SOS1:GFP, pictures were generated using a CSU-W1 spinning disk head (Yokogawa) fitted to a Nikon Eclipse Ti-E-inverted microscope with a CFI PlanApo × 100 N.A. 1.40 oil immersion objective and an EM-CCD ImageEM 1K (c9100-14) camera. Seeds were sown in ½ MS plates with 1% sucrose in a long-day chamber at 21 °C. Five days after germination, seedlings were transferred to ½ MS plates with 1% sucrose with or without NaCl, keeping the same growth conditions. Roots were stained with FM4-64 (2 μM from a stock of 2 mM in water, Molecular Probes) in ½ MS for 10 min before imaging. GFP and FM4-64 were imaged using a 488 nm and 561 nm solid-state diode laser, and 525/50 and 605/52 nm emission filters, respectively. Fluorescence intensity (as mean gray value, arbitrary units) of microscopy images was measured at the PM and IC compartment avoiding nuclei. Image analysis was performed with Fiji software, version 1.57.

Fluorescence Peptide Binding Assay.

Fluorescence assays were carried out using a Tycho NT.6 device (NanoTemper). Samples were prepared using 10 μL of 6 μM purified SOS3 (39) in a buffer containing 5 mM Tris pH 7.5, 5 mM NaCl, 100 mM KCl, and 5 mM CaCl₂ mixed with 10 μL of S3BD resuspended in the same buffer at increasing protein:peptide ratios. The sample was then loaded into Tycho NT.6 capillaries (NanoTemper) and the intrinsic fluorescence of the mix at 35 °C was recorded at 330 and 350 nm after excitation at 285 nm. Three independent series of experiments were carried out.

Structure Modeling.

Homology modeling of HKT1;1 structure was performed using the Phyre2 Protein Fold Recognition Server Intensive mode (67). The crystal structures of the potassium transporters KtrB from Bacillus subtilis, PDB code 4J7C (68) and TrkH from Vibrio parahaemolyticus, PDB code 3PJZ (69) were used as templates. The dimeric structure of SOS1 (residues 1 to 697) and the complex SOS3-S3BD (residues 460 to 484) were modeled using the AlphaFold Advanced Interface (26).

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

All study data are included in the article and/or SI Appendix. Research materials and the underlying data supporting the findings in this study are available on request to the corresponding authors (C.S-R., D-J.Y. and F.J.Q), upon signing a Material Transfer Agreement with disclosure of the intended use of the data or materials requested.