The Na+ transporter AtHKT1;1 controls retrieval of Na+ from the xylem in Arabidopsis

HKT-type transporters appear to play key roles in Na(+) accumulation and salt sensitivity in plants. In Arabidopsis HKT1;1 has been proposed to influx Na(+) into roots, recirculate Na(+) in the phloem and control root : shoot allocation of Na(+). We tested these hypotheses using (22)Na(+) flux measurements and ion accumulation assays in an hkt1;1 mutant and demonstrated that AtHKT1;1 contributes to the control of both root accumulation of Na(+) and retrieval of Na(+) from the xylem, but is not involved in root influx or recirculation in the phloem. Mathematical modelling indicated that the effects of the hkt1;1 mutation on root accumulation and xylem retrieval were independent. Although AtHKT1;1 has been implicated in regulation of K(+) transport and the hkt1;1 mutant showed altered net K(+) accumulation, (86)Rb(+) uptake was unaffected by the hkt1;1 mutation. The hkt1;1 mutation has been shown previously to rescue growth of the sos1 mutant on low K(+); however, HKT1;1 knockout did not alter K(+) or (86)Rb(+) accumulation in sos1.     

Natural variants of AtHKT1 enhance Na+ accumulation in two wild populations of Arabidopsis

Plants are sessile and therefore have developed mechanisms to adapt to their environment, including the soil mineral nutrient composition. Ionomics is a developing functional genomic strategy designed to rapidly identify the genes and gene networks involved in regulating how plants acquire and accumulate these mineral nutrients from the soil. Here, we report on the coupling of high-throughput elemental profiling of shoot tissue from various Arabidopsis accessions with DNA microarray-based bulk segregant analysis and reverse genetics, for the rapid identification of genes from wild populations of Arabidopsis that are involved in regulating how plants acquire and accumulate Na(+) from the soil. Elemental profiling of shoot tissue from 12 different Arabidopsis accessions revealed that two coastal populations of Arabidopsis collected from Tossa del Mar, Spain, and Tsu, Japan (Ts-1 and Tsu-1, respectively), accumulate higher shoot levels of Na(+) than do Col-0 and other accessions. We identify AtHKT1, known to encode a Na(+) transporter, as being the causal locus driving elevated shoot Na(+) in both Ts-1 and Tsu-1. Furthermore, we establish that a deletion in a tandem repeat sequence approximately 5 kb upstream of AtHKT1 is responsible for the reduced root expression of AtHKT1 observed in these accessions. Reciprocal grafting experiments establish that this loss of AtHKT1 expression in roots is responsible for elevated shoot Na(+). Interestingly, and in contrast to the hkt1-1 null mutant, under NaCl stress conditions, this novel AtHKT1 allele not only does not confer NaCl sensitivity but also cosegregates with elevated NaCl tolerance. We also present all our elemental profiling data in a new open access ionomics database, the Purdue Ionomics Information Management System (PiiMS; http://www.purdue.edu/dp/ionomics). Using DNA microarray-based genotyping has allowed us to rapidly identify AtHKT1 as the casual locus driving the natural variation in shoot Na(+) accumulation we observed in Ts-1 and Tsu-1. Such an approach overcomes the limitations imposed by a lack of established genetic markers in most Arabidopsis accessions and opens up a vast and tractable source of natural variation for the identification of gene function not only in ionomics but also in many other biological processes.     

The Arabidopsis kinase-associated protein phosphatase regulates adaptation to Na+ stress

The kinase-associated protein phosphatase (KAPP) is a regulator of the receptor-like kinase (RLK) signaling pathway. Loss-of-function mutations rag1-1 (root attenuated growth1-1) and rag1-2, in the locus encoding KAPP, cause NaCl hypersensitivity in Arabidopsis thaliana. The NaCl hypersensitive phenotype exhibited by rag1 seedlings includes reduced shoot and primary root growth, root tip swelling, and increased lateral root formation. The phenotype exhibited by rag1-1 seedlings is associated with a specific response to Na(+) toxicity. The sensitivity to Na(+) is Ca(2+) independent and is not due to altered intracellular K(+)/Na(+). Analysis of the genetic interaction between rag1-1 and salt overly sensitive1 (sos1-14) revealed that KAPP is not a component of the SOS signal transduction pathway, the only Na(+) homeostasis signaling pathway identified so far in plants. All together, these results implicate KAPP as a functional component of the RLK signaling pathway, which also mediates adaptation to Na(+) stress. RLK pathway components, known to be modulated by NaCl at the messenger RNA level, are constitutively down-regulated in rag1-1 mutant plants. The effect of NaCl on their expression is not altered by the rag1-1 mutation.     

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.     

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.     

Reduced Na+ uptake in the NaCl-hypersensitive sos1 mutant of Arabidopsis thaliana.

Sos1 is an Arabidopsis thaliana mutant with > 20 times higher sensitivity toward Na+ inhibition due to a defective high-affinity potassium-uptake system. We report here that sos1 accumulates less Na+ than the wild type in response to NaCl stress. The Na+ contents in sos1 seedlings exposed to 25 mM NaCl for 2 or more d are about 43% lower than those in the wild type. When assayed at 20 mM external NaCl, sos1 seedlings pretreated with low potassium have 32% lower Na+ uptake than the wild type. However, little difference in Na+ uptake could be measured when the seedlings were not pretreated with low potassium. Low-potassium treatment was shown to induce high-affinity potassium-uptake activity in Arabidopsis seedlings. No substantial difference in Na+ efflux between sos1 and the wild type was detected. The results show that the reduced Na+ accumulation in sos1 is due to a lower Na+ influx rate. Therefore, the sos1 mutation appears to disrupt low-affinity Na+ uptake in addition to its impairment of high-affinity K+ uptake.     

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.     

Coordination of AtHKT1;1 and AtSOS1 facilitates Na+ and K+ homeostasis in Arabidopsis thaliana under salt stress

Reducing Na⁺ accumulation and maintaining K⁺ stability in plant is one of the key strategies for improving salt tolerance. AtHKT1;1 and AtSOS1 are not only the salt tolerance determinants themselves, but also mediate K⁺ uptake and transport indirectly. To assess the contribution of AtHKT1;1 and AtSOS1 to Na⁺ homeostasis and K⁺ nutrition in plant, net Na⁺ and K⁺ uptake rate, Na⁺ and K⁺ distributions in Arabidopsis thaliana wild type (WT), hkt1;1 mutant (athkt1;1) and sos1 mutant (atsos1) were investigated. Results showed that under 2.5 mM K⁺ plus 25 or 100 mM NaCl, athkt1;1 shoot concurrently accumulated more Na⁺ and less K⁺ than did WT shoot, suggesting that AtHKT1;1 was critical for controlling Na⁺ and K⁺ distribution in plant; while atsos1 root accumulated more Na⁺ and absorbed lower K⁺ than did WT root, implying that AtSOS1 was determiner of Na⁺ excretion and K⁺ acquisition. Under 0.01 mM K⁺, athkt1;1 absorbed lower Na⁺ than did WT with 100 mM NaCl, suggesting that AtHKT1;1 is involved in Na⁺ uptake in roots; while atsos1 shoot accumulated less Na⁺ than did WT shoot no matter with 25 or 100 mM NaCl, implying that AtSOS1 played a key role in controlling long-distance Na⁺ transport from root to shoot. We present a model in which coordination of AtHKT1;1 and AtSOS1 facilitates Na⁺ and K⁺ homeostasis in A. thaliana under salt stress: under the normal K⁺, the major function of AtHKT1;1 is Na⁺ unloading and AtSOS1 is mainly involved in Na⁺ exclusion, whereas under the low K⁺, AtHKT1;1 may play a dominant role in Na+ uptake and AtSOS1 may be mainly involved in Na⁺ loading into the xylem.     

Regulated AtHKT1 gene expression by a distal enhancer element and DNA methylation in the promoter plays an important role in salt tolerance

Through sos3 (salt overly sensitive 3) suppressor screening, two allelic suppressor mutants that are weak alleles of the strong sos3 suppressor sos3hkt1-1 were recovered. Molecular characterization identified T-DNA insertions in the distal promoter region of the Arabidopsis thaliana HKT1 (AtHKT1, At4g10310) in these two weak sos3 suppressors, which results in physical separation of a tandem repeat from the proximal region of the AtHKT1 promoter. The tandem repeat is approximately 3.9 kb upstream of the ATG start codon and functions as an enhancer element to promote reporter gene expression. A putative small RNA target region about 2.6 kb upstream of the ATG start codon is heavily methylated. CHG and CHH methylation but not CG methylation is significantly reduced in the small RNA biogenesis mutant rdr2, indicating that non-CG methylation in this region is mediated by small RNAs. Analysis of AtHKT1 expression in rdr2 suggests that non-CG methylation in the putative small RNA target region represses AtHKT1 expression in shoots. The DNA methylation-deficient mutant met1-3 has nearly complete loss of total cytosine methylation in the putative small RNA target region and is hypersensitive to salt stress. The putative small RNA target region and the tandem repeat are essential for maintaining AtHKT1 expression patterns crucial for salt tolerance.     

Calcium regulation of sodium hypersensitivities of sos3 and athkt1 mutants

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

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.     

Functional analysis of AtHKT1 in Arabidopsis shows that Na(+) recirculation by the phloem is crucial for salt tolerance

Two allelic recessive mutations of Arabidopsis, sas2-1 and sas2-2, were identified as inducing sodium overaccumulation in shoots. The sas2 locus was found (by positional cloning) to correspond to the AtHKT1 gene. Expression in Xenopus oocytes revealed that the sas2-1 mutation did not affect the ionic selectivity of the transporter but strongly reduced the macro scopic (whole oocyte current) transport activity. In Arabidopsis, expression of AtHKT1 was shown to be restricted to the phloem tissues in all organs. The sas2-1 mutation strongly decreased Na(+) concentration in the phloem sap. It led to Na(+) overaccumulation in every aerial organ (except the stem), but to Na(+) underaccumulation in roots. The sas2 plants displayed increased sensitivity to NaCl, with reduced growth and even death under moderate salinity. The whole set of data indicates that AtHKT1 is involved in Na(+) recirculation from shoots to roots, probably by mediating Na(+) loading into the phloem sap in shoots and unloading in roots, this recirculation removing large amounts of Na(+) from the shoot and playing a crucial role in plant tolerance to salt.     

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.     

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.     

The Arabidopsis HKT1 gene homolog mediates inward Na(+) currents in xenopus laevis oocytes and Na(+) uptake in Saccharomyces cerevisiae

The Na(+)-K(+) co-transporter HKT1, first isolated from wheat, mediates high-affinity K(+) uptake. The function of HKT1 in plants, however, remains to be elucidated, and the isolation of HKT1 homologs from Arabidopsis would further studies of the roles of HKT1 genes in plants. We report here the isolation of a cDNA homologous to HKT1 from Arabidopsis (AtHKT1) and the characterization of its mode of ion transport in heterologous systems. The deduced amino acid sequence of AtHKT1 is 41% identical to that of HKT1, and the hydropathy profiles are very similar. AtHKT1 is expressed in roots and, to a lesser extent, in other tissues. Interestingly, we found that the ion transport properties of AtHKT1 are significantly different from the wheat counterpart. As detected by electrophysiological measurements, AtHKT1 functioned as a selective Na(+) uptake transporter in Xenopus laevis oocytes, and the presence of external K(+) did not affect the AtHKT1-mediated ion conductance (unlike that of HKT1). When expressed in Saccharomyces cerevisiae, AtHKT1 inhibited growth of the yeast in a medium containing high levels of Na(+), which correlates to the large inward Na(+) currents found in the oocytes. Furthermore, in contrast to HKT1, AtHKT1 did not complement the growth of yeast cells deficient in K(+) uptake when cultured in K(+)-limiting medium. However, expression of AtHKT1 did rescue Escherichia coli mutants carrying deletions in K(+) transporters. The rescue was associated with a less than 2-fold stimulation of K(+) uptake into K(+)-depleted cells. These data demonstrate that AtHKT1 differs in its transport properties from the wheat HKT1, and that AtHKT1 can mediate Na(+) and, to a small degree, K(+) transport in heterologous expression systems.     

NADPH oxidase AtrbohD and AtrbohF function in ROS-dependent regulation of Na⁺/K⁺homeostasis in Arabidopsis under salt stress

Maintaining cellular Na(+)/K(+) homeostasis is pivotal for plant survival in saline environments. However, knowledge about the molecular regulatory mechanisms of Na(+)/K(+) homeostasis in plants under salt stress is largely lacking. In this report, the Arabidopsis double mutants atrbohD1/F1 and atrbohD2/F2, in which the AtrbohD and AtrbohF genes are disrupted and generation of reactive oxygen species (ROS) is pronouncedly inhibited, were found to be much more sensitive to NaCl treatments than wild-type (WT) and the single null mutant atrbohD1 and atrbohF1 plants. Furthermore, the two double mutant seedlings had significantly higher Na(+) contents, lower K(+) contents, and resultant greater Na(+)/K(+) ratios than the WT, atrbohD1, and atrbohF1 under salt stress. Exogenous H(2)O(2) can partially reverse the increased effects of NaCl on Na(+)/K(+) ratios in the double mutant plants. Pre-treatments with diphenylene iodonium chloride, a widely used inhibitor of NADPH oxidase, clearly enhanced the Na(+)/K(+) ratios in WT seedlings under salt stress. Moreover, NaCl-inhibited inward K(+) currents were arrested, and NaCl-promoted increases in cytosolic Ca(2+) and plasma membrane Ca(2+) influx currents were markedly attenuated in atrbohD1/F1 plants. No significant differences in the sensitivity to osmotic or oxidative stress among the WT, atrbohD1, atrbohF1, atrbohD1/F1, and atrbohD2/F2 were observed. Taken together, these results strongly suggest that ROS produced by both AtrbohD and AtrbohF function as signal molecules to regulate Na(+)/K(+) homeostasis, thus improving the salt tolerance of Arabidopsis.