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.     

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

The salt tolerance locus SOS1 from Arabidopsis has been shown to encode a putative plasma membrane Na(+)/H(+) antiporter. In this study, we examined the tissue-specific pattern of gene expression as well as the Na(+) transport activity and subcellular localization of SOS1. When expressed in a yeast mutant deficient in endogenous Na(+) transporters, SOS1 was able to reduce Na(+) accumulation and improve salt tolerance of the mutant cells. Confocal imaging of a SOS1-green fluorescent protein fusion protein in transgenic Arabidopsis plants indicated that SOS1 is localized in the plasma membrane. Analysis of SOS1 promoter-beta-glucuronidase transgenic Arabidopsis plants revealed preferential expression of SOS1 in epidermal cells at the root tip and in parenchyma cells at the xylem/symplast boundary of roots, stems, and leaves. Under mild salt stress (25 mM NaCl), sos1 mutant shoot accumulated less Na(+) than did the wild-type shoot. However, under severe salt stress (100 mM NaCl), sos1 mutant plants accumulated more Na(+) than did the wild type. There also was greater Na(+) content in the xylem sap of sos1 mutant plants exposed to 100 mM NaCl. These results suggest that SOS1 is critical for controlling long-distance Na(+) transport from root to shoot. We present a model in which SOS1 functions in retrieving Na(+) from the xylem stream under severe salt stress, whereas under mild salt stress it may function in loading Na(+) into the xylem.     

AtHKT1 facilitates Na+ homeostasis and K+ nutrition in planta

Genetic and physiological data establish that Arabidopsis AtHKT1 facilitates Na(+) homeostasis in planta and by this function modulates K(+) nutrient status. Mutations that disrupt AtHKT1 function suppress NaCl sensitivity of sos1-1 and sos2-2, as well as of sos3-1 seedlings grown in vitro and plants grown in controlled environmental conditions. hkt1 suppression of sos3-1 NaCl sensitivity is linked to higher Na(+) content in the shoot and lower content of the ion in the root, reducing the Na(+) imbalance between these organs that is caused by sos3-1. AtHKT1 transgene expression, driven by its innate promoter, increases NaCl but not LiCl or KCl sensitivity of wild-type (Col-0 gl1) or of sos3-1 seedlings. NaCl sensitivity induced by AtHKT1 transgene expression is linked to a lower K(+) to Na(+) ratio in the root. However, hkt1 mutations increase NaCl sensitivity of both seedlings in vitro and plants grown in controlled environmental conditions, which is correlated with a lower K(+) to Na(+) ratio in the shoot. These results establish that AtHKT1 is a focal determinant of Na(+) homeostasis in planta, as either positive or negative modulation of its function disturbs ion status that is manifested as salt sensitivity. K(+)-deficient growth of sos1-1, sos2-2, and sos3-1 seedlings is suppressed completely by hkt1-1. AtHKT1 transgene expression exacerbates K(+) deficiency of sos3-1 or wild-type seedlings. Together, these results indicate that AtHKT1 controls Na(+) homeostasis in planta and through this function regulates K(+) nutrient status.     

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

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

Effects of sodium chloride stress and calcium supply on growth, potassium uptake, and internal chloride and sodium levels of winter wheat seedlings

The effects of saline conditions on the K+ (86Rb), Na+ and Cl- uptake and growth of 6-day-old wheat (Triticum aestivum L. cv. GK Szeged) seedlings were studied in the absence and presence of Ca2+. It was found that on direct NaCl treatment the K+ uptake of the roots in the absence of Ca2+ declined significantly with increasing salinity. The reverse was true, however, in the case of NaCl pretreatment: seedlings grown under highly saline conditions (50 mM NaCl) absorbed more K+ than those pretreated with low levels of NaCl (1 or 10 mM NaCl). The data indicate a definite Na(+)-induced K+ uptake inhibition and/or feed-back regulation in the K+ uptake of roots under the above-mentioned growth conditions. As regards the Ca2+ effect, it was established that supplemental Ca2+ counteracts the unfavourable effect of saline conditions as concerns both the K+ uptake of the roots and the dry matter yield of the seedlings. The internal concentrations of Na+ and Cl- in the seedlings increased in proportion to increasing salinity. Marked differences were experienced, however, in the internal concentrations of Na+ and Cl- in the roots and shoots, respectively. It was concluded that under these experimental conditions the salt tolerance of wheat could be related to its capability of restricting the transport of Na+ at low and moderate levels to the shoots, where it is highly toxic.     

Regulation of vacuolar Na+/H+ exchange in Arabidopsis thaliana by the salt-overly-sensitive (SOS) pathway

For plants growing in highly saline environments, accumulation of sodium in the cell cytoplasm leads to disruption of metabolic processes and reduced growth. Maintaining low levels of cytoplasmic sodium requires the coordinate regulation of transport proteins on numerous cellular membranes. Our previous studies have linked components of the Salt-Overly-Sensitive pathway (SOS1-3) to salt tolerance in Arabidopsis thaliana and demonstrated that the activity of the plasma membrane Na+/H+ exchanger (SOS1) is regulated by SOS2 (a protein kinase) and SOS3 (a calcium-binding protein). Current studies were undertaken to determine if the Na+/H+ exchanger in the vacuolar membrane (tonoplast) of Arabidopsis is also a target for the SOS regulatory pathway. Characterization of tonoplast Na+/H+ exchange demonstrated that it represents activity originating from the AtNHX proteins since it could be inhibited by 5-(N-methyl-N-isobutyl)amiloride and by anti-NHX1 antibodies. Transport activity was selective for sodium (apparent Km=31 mm) and electroneutral (one sodium ion for each proton). When compared with tonoplast Na+/H+-exchange activity in wild type, activity was significantly higher, greatly reduced, and unchanged in sos1, sos2, and sos3, respectively. Activated SOS2 protein added in vitro increased tonoplast Na+/H+-exchange activity in vesicles isolated from sos2 but did not have any effect on activity in vesicles isolated from wild type, sos1, or sos3. These results demonstrate that (i) the tonoplast Na+/H+ exchanger in Arabidopsis is a target of the SOS regulatory pathway, (ii) there are branches to the SOS pathway, and (iii) there may be coordinate regulation of the exchangers in the tonoplast and plasma membrane.     

Sodium influx and accumulation in Arabidopsis

Arabidopsis is frequently used as a genetic model in plant salt tolerance studies, however, its physiological responses to salinity remain poorly characterized. This study presents a characterization of initial Na+ entry and the effects of Ca2+ on plant growth and net Na+ accumulation in saline conditions. Unidirectional Na+ influx was measured carefully using very short influx times in roots of 12-d-old seedlings. Influx showed three components with distinct sensitivities to Ca2+, diethylpyrocarbonate, and osmotic pretreatment. Pharmacological agents and known mutants were used to test the contribution of different transport pathways to Na+ uptake. Influx was stimulated by 4-aminobutyric acid and glutamic acid; was inhibited by flufenamate, quinine, and cGMP; and was insensitive to modulators of K+ and Ca2+ channels. Influx did not differ from wild type in akt1 and hkt1 insertional mutants. These data suggested that influx was mediated by several different types of nonselective cation channels. Na+ accumulation in plants grown in 50 mM NaCl was strongly reduced by increasing Ca2+ activity (from 0.05-3.0 mM), and plant survival was improved. However, plant biomass was not affected by shoot Na+ concentration, suggesting that in Arabidopsis Na+ toxicity is not dependent on shoot Na+ accumulation. These data suggest that Arabidopsis is a good model for investigation of Na+ transport, but may be of limited utility as a model for the study of Na+ toxicity.     

Ammonium inhibition of Arabidopsis root growth can be reversed by potassium and by auxin resistance mutations aux1, axr1, and axr2.

A novel effect of ammonium ions on root growth was investigated to understand how environmental signals affect organ development. Ammonium ions (3-12 mM) were found to dramatically inhibit Arabidopsis thaliana seedling root growth in the absence of potassium even if nitrate was present. This inhibition could be reversed by including in the growth medium low levels (20-100 microM) of potassium or alkali ions Rb+ and Cs+ but not alkali ions Na+ and Li+. The protective effect of low concentrations of potassium is not due to an inhibition of ammonium uptake. Ammonium inhibition is reversible, because root growth was restored in ammonium-treated seedlings if they were subsequently transferred to medium containing potassium. It is known that plant hormones can inhibit root growth. We found that mutants of Arabidopsis resistant to high levels of auxin and other hormones (aux1, axr1, and axr2) are also resistant to the ammonium inhibition and produce roots in the absence of potassium. Thus, the mechanisms that mediate the ammonium inhibition of root development are linked to hormone metabolic or signaling pathways. These findings have important implications for understanding how environmental signals, especially mineral nutrients, affect plant root development.     

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

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

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

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

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

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

Genes that are uniquely stress regulated in salt overly sensitive (sos) mutants

Repetitive rounds of differential subtraction screening, followed by nucleotide sequence determination and northern-blot analysis, identified 84 salt-regulated (160 mM NaCl for 4 h) genes in Arabidopsis wild-type (Col-0 gl1) seedlings. Probes corresponding to these 84 genes and ACP1, RD22BP1, MYB2, STZ, and PAL were included in an analysis of salt responsive gene expression profiles in gl1 and the salt-hypersensitive mutant sos3. Six of 89 genes were expressed differentially in wild-type and sos3 seedlings; steady-state mRNA abundance of five genes (AD06C08/unknown, AD05E05/vegetative storage protein 2 [VSP2], AD05B11/S-adenosyl-L-Met:salicylic acid carboxyl methyltransferase [SAMT], AD03D05/cold regulated 6.6/inducible2 [COR6.6/KIN2], and salt tolerance zinc finger [STZ]) was induced and the abundance of one gene (AD05C10/circadian rhythm-RNA binding1 [CCR1]) was reduced in wild-type plants after salt treatment. The expression of CCR1, SAMT, COR6.6/KIN2, and STZ was higher in sos3 than in wild type, and VSP2 and AD06C08/unknown was lower in the mutant. Salt-induced expression of VSP2 in sos1 was similar to wild type, and AD06C08/unknown, CCR1, SAMT, COR6.6/KIN2, and STZ were similar to sos3. VSP2 is regulated presumably by SOS2/3 independent of SOS1, whereas the expression of the others is SOS1 dependent. AD06C08/unknown and VSP2 are postulated to be effectors of salt tolerance whereas CCR1, SAMT, COR6.6/KIN2, and STZ are determinants that must be negatively regulated during salt adaptation. The pivotal function of the SOS signal pathway to mediate ion homeostasis and salt tolerance implicates AD06C08/unknown, VSP2, SAMT, 6.6/KIN2, STZ, and CCR1 as determinates that are involved in salt adaptation.     

Binding of sulfonylurea by AtMRP5, an Arabidopsis multidrug resistance-related protein that functions in salt tolerance

Recently, a new member of the ABC transporter superfamily of Arabidopsis, AtMRP5, was identified and characterized. In the present work, we found that AtMRP5 can bind specifically to sulfonurea when it is expressed in HEK293 cells. We also present evidence for a new role of AtMRP5 in the salt stress response of Arabidopsis. We used reverse genetics to identify an Arabidopsis mutant (atmrp5-2) in which the AtMRP5 gene was disrupted by transferred DNA insertion. In root-bending assays using Murashige and Skoog medium supplemented with 100 mm NaCl, root growth of atmrp5-2 was substantially inhibited in contrast to the almost normal growth of wild-type seedlings. This hypersensitive response of the atmrp5-2 mutant was not observed during mannitol treatment. The root growth of the wild-type plant grown in Murashige and Skoog medium supplemented with the MRP inhibitor glibenclamide and NaCl was inhibited to a very similar extent as the root growth of atmrp5-2 grown in NaCl alone. The Na(+)-dependent reduction of root growth of the wild-type plant in the presence of glibenclamide was partially restored by diazoxide, a known K+ channel opener that reverses the inhibitory effects of sulfonylureas in animal cells. Moreover, the atmrp5-2 mutant was defective in 86Rb+ uptake. When seedlings were treated with 100 mm NaCl, atmrp5-2 seedlings accumulated less K+ and more Na+ than those of the wild type. These observations suggest that AtMRP5 is a putative sulfonylurea receptor that is involved in K+ homeostasis and, thus, also participates in the NaCl stress response.     

Altered shoot/root Na+ distribution and bifurcating salt sensitivity in Arabidopsis by genetic disruption of the Na+ transporter AtHKT1

Sodium (Na+) is toxic to most plants, but the molecular mechanisms of plant Na+ uptake and distribution remain largely unknown. Here we analyze Arabidopsis lines disrupted in the Na+ transporter AtHKT1. AtHKT1 is expressed in the root stele and leaf vasculature. athkt1 null plants exhibit lower root Na+ levels and are more salt resistant than wild-type in short-term root growth assays. In shoot tissues, however, athkt1 disruption produces higher Na+ levels, and athkt1 and athkt1/sos3 shoots are Na+-hypersensitive in long-term growth assays. Thus wild-type AtHKT1 controls root/shoot Na+ distribution and counteracts salt stress in leaves by reducing leaf Na+ accumulation.     

NKS1, Na(+)- and K(+)-sensitive 1, regulates ion homeostasis in an SOS-independent pathway in Arabidopsis

An Arabidopsis thaliana mutant, nks1-1, exhibiting enhanced sensitivity to NaCl was identified in a screen of a T-DNA insertion population in the genetic background of Col-0 gl1sos3-1. Analysis of the genome sequence in the region flanking the T-DNA left border indicated two closely linked mutations in the gene encoded at locus At4g30996. A second allele, nks1-2, was obtained from the Arabidopsis Biological Resource Center. NKS1 mRNA was detected in all parts of wild-type plants but was not detected in plants of either mutant, indicating inactivation by the mutations. Both mutations in NKS1 were associated with increased sensitivity to NaCl and KCl, but not to LiCl or mannitol. NaCl sensitivity was associated with nks1 mutations in Arabidopsis lines expressing either wild type or null alleles of SOS1, SOS2 or SOS3. The NaCl-sensitive phenotype of the nks1-2 mutant was complemented by expression of a full-length NKS1 allele from the CaMV35S promoter. When grown in medium containing NaCl, nks1 mutants accumulated more Na(+) than wild type and K(+)/Na(+) homeostasis was perturbed. It is proposed NKS1, a plant-specific gene encoding a 19kDa endomembrane-localized protein of unknown function, is part of an ion homeostasis regulation pathway that is independent of the SOS pathway.     

Monosaccharide/proton symporter AtSTP1 plays a major role in uptake and response of Arabidopsis seeds and seedlings to sugars

The aim of this study was to investigate the in vivo properties and function of the high-affinity monosaccharide/proton symporter AtSTP1 of Arabidopsis. We isolated an Atstp1 knock-out mutant and found that this plant grows and develops normally. The AtSTP1 gene is expressed in germinating seeds and seedlings, with AtSTP1 activity found mainly in the seedling root. The rate of uptake of [(14)C]-3-O-methylglucose and [(14)C]-D-glucose is 60% less in Atstp1 seedlings than in the wild type, showing that AtSTP1 is the major monosaccharide transporter in Arabidopsis seedlings. Transport of D-galactose and D-mannose is also up to 60% less in Atstp1 seedlings compared to wild type, but transport of D-fructose, L-arabinose and sucrose is not reduced. Germination of Atstp1 seed shows reduced sensitivity to D-mannose, demonstrating that AtSTP1 is active before germination. Atstp1 seedlings grow effectively on concentrations of D-galactose that inhibit wild-type growth, even at up to 100 mM D-galactose, indicating that active transport by AtSTP1 plays a major role at very high concentrations of exogenous sugar. These findings provide insight into the physiological function of AtSTP1 and clearly establish its importance in the uptake of extracellular sugars by the embryo and in seedlings.     

Mutants of Arabidopsis thaliana Capable of Germination under Saline Conditions

Three mutant strains of Arabidopsis thaliana var Columbia were selected for their ability to germinate in elevated concentrations of NaCl. They were not more tolerant than wild type at subsequent development stages. Wild-type strains could not germinate at concentrations > 125 mM NaCl. Two of mutant strains, RS17 and RS20, could withstand up to 225 mM, whereas RS19 was resistant to 175 mM. The RS mutants could also germinate under even lower osmotic potentials imposed by high concentrations of exogenous mannitol (550 mM), whereas the effects of elevated levels of KCl, K2SO4, and LiCl were similar among the mutants and wild type. Therefore, the mutants are primarily osmotolerant, but they also possess a degree of ionic tolerance for sodium. Sodium and potassium contents of seeds exposed to high salinities indicated that the NaCl-tolerant mutants absorbed more of these respective cations during imbibition. These higher internal concentrations of potassium and sodium could contribute to the osmotic adjustment of the germinating seeds to the low osmotic potential of the external medium. Genetic analysis of F1 and F2 progeny of outcrosses suggest that the salt-tolerant mutations are recessive and that they define three complementation groups.     

Relationship between xylem ion concentration and bean growth responses to short-term salinisation in spring and summer

Bean plants, Phaseolus vulgaris L. cv. Contender, were grown in the spring and summer seasons to study the relationship between xylem Na+/Cl-, transpiration rate, and salt tolerance. Eight-day-old seedlings were transplanted to 50% modified Hoagland solution with 1 mM NaCl. Four days after transfer, one of two treatments was applied: a control of 1 mM NaCl or a treatment of 25 mM NaCl every two days to reach a final treatment concentration of 75 mM NaCl. Plants were sampled on the fourth day after the final salt concentration was reached, eight days after the salinisation treatment began. Relative growth rate was 2.6-fold greater in summer than in spring. However, while no differences were found between treatments in spring, summer salt-treated plants had growth rates that were 31% lower than those of controls. In summer, CO2 assimilation, stomatal conductance, and transpiration rate of salinised plants declined with respect to controls. Leaf Na+ and trifoliolate leaf Cl- were higher in salt-treated plants in summer, although root Na+ was significantly higher in spring. Moreover, in summer salinity inhibited Ca2+ and K+ uptake and changed its distribution. Summer salt-treated plants had an average of 17-fold higher xylem Na+ during the daily cycle, while xylem Cl-, only in the afternoon, showed higher values (1.5-fold) compared to spring-grown plants. Our results suggest that the faster growth response to salt in summer-grown bean was at least partly due to an increase in xylem Na+ independent of the transpiration rate and possibly related to an increase in xylem Na+ influx or/and Na+ recirculation.     

Site density of the sodium-calcium exchange carrier in reconstituted vesicles from bovine cardiac sarcolemma

The site density of the Na2+-Ca2+ exchanger in bovine cardiac sarcolemma was estimated from measurements of the fraction of reconstituted proteoliposomes exhibiting exchange activity. Sarcolemmal vesicles were solubilized with 1% Triton X-100 in the presence of either 100 mM NaCl or 100 mM KCl; after a 20-40-min incubation period on ice, sufficient KCl, NaCl, CaCl2, and soybean phospholipids were added to each extract to give final concentrations of 40 mM NaCl, 120 mM KCl, 0.1 mM CaCl2, and 10 mg/ml phospholipid. These mixtures were then reconstituted into proteoliposomes, and the rate of 45Ca2+ isotopic exchange was measured under equilibrium conditions. Control studies showed that Na+-Ca2+ exchange activity was completely lost if Na+ was not present during solubilization. The difference in 45Ca2+ uptake between vesicles initially solubilized in the presence or absence of NaCl therefore reflected exchange activity and corresponded to 3.1 +/- 0.3% of the total 45Ca2+ uptake by the entire population of vesicles, as measured in the presence of the Ca2+ ionophore A23187. Assuming that each vesicle with exchange activity contained 1 molecule of the Na+-Ca2+ exchange carrier, a site density of 10-20 pmol/mg of protein for the exchanger was calculated. The Vmax for Na+-Ca2+ exchange activity in the proteoliposomes was approximately 20 nmol/mg of protein.s which indicates that the turnover number of the exchange carrier is 1000 s-1 or more. Thus, the Na+-Ca2+ exchanger is a low density, high turnover transport system.