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.     

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.     

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.     

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.     

Enhanced salt tolerance mediated by AtHKT1 transporter-induced Na unloading from xylem vessels to xylem parenchyma cells

AtHKT1 is a sodium (Na+) transporter that functions in mediating tolerance to salt stress. To investigate the membrane targeting of AtHKT1 and its expression at the translational level, antibodies were generated against peptides corresponding to the first pore of AtHKT1. Immunoelectron microscopy studies using anti-AtHKT1 antibodies demonstrate that AtHKT1 is targeted to the plasma membrane in xylem parenchyma cells in leaves. AtHKT1 expression in xylem parenchyma cells was also confirmed by AtHKT1 promoter-GUS reporter gene analyses. Interestingly, AtHKT1 disruption alleles caused large increases in the Na+ content of the xylem sap and conversely reduced the Na+ content of the phloem sap. The athkt1 mutant alleles had a smaller and inverse influence on the potassium (K+) content compared with the Na+ content of the xylem, suggesting that K+ transport may be indirectly affected. The expression of AtHKT1 was modulated not only by the concentrations of Na+ and K+ but also by the osmolality of non-ionic compounds. These findings show that AtHKT1 selectively unloads sodium directly from xylem vessels to xylem parenchyma cells. AtHKT1 mediates osmolality balance between xylem vessels and xylem parenchyma cells under saline conditions. Thus AtHKT1 reduces the sodium content in xylem vessels and leaves, thereby playing a central role in protecting plant leaves from salinity stress.     

Expression levels of the Na + /K + transporter OsHKT2;1 and vacuolar Na + /H + exchanger OsNHX1 , Na enrichment,maintaining the photosynthetic abilities and growth performances of indica rice seedlings under salt stress

Salt affected soil inhibits plant growth, development and productivity, especially in case of rice crop. Ion homeostasis is a candidate defense mechanism in the salt tolerant plants or halophyte species, where the salt toxic ions are stored in the vacuoles. The aim of this investigation was to determine the OsNHX1 (a vacuolar Na+/H+ exchanger) and OsHKT2;1 (Na+/K+ transporter) regulation by salt stress (200 mM NaCl) in two rice cultivars, i.e. Pokkali (salt tolerant) and IR29 (salt susceptible), the accumulation of Na+ in the root and leaf tissues using CoroNa Green® staining dye and the associated physiological changes in test plants. Na+ content was largely increased in the root tissues of rice seedlings cv. Pokkali (15 min after salt stress) due to the higher expression of OsHKT2;1 gene (by 2.5 folds) in the root tissues. The expression of OsNHX1 gene in the leaf tissues was evidently increased in salt stressed seedlings of Pokkali, whereas it was unchanged in salt stressed seedlings of IR29. Na+ in the root tissues of both Pokkali and IR29 was enriched, when subjected to 200 mM NaCl for 12 h and easily detected in the leaf tissues of salt stressed plants exposed for 24 h, especially in cv. Pokkali. Moreover, the overexpression of OsNHX1 gene regulated the translocation of Na+ from root to leaf tissues, and compartmentation of Na+ into vacuoles, thereby maintaining the photosynthetic abilities in cv. Pokkali. Overall growth performance, maximum quantum yield (Fv/Fm), photon yield of PSII (ΦPSII) and net photosynthetic rate (Pn) was improved in salt stressed leaves of Pokkali than those in salt stressed IR29.     

AtHKT1;1 mediates nernstian sodium channel transport properties in Arabidopsis root stelar cells

The Arabidopsis AtHKT1;1 protein was identified as a sodium (Na⁺) transporter by heterologous expression in Xenopus laevis oocytes and Saccharomyces cerevisiae. However, direct comparative in vivo electrophysiological analyses of a plant HKT transporter in wild-type and hkt loss-of-function mutants has not yet been reported and it has been recently argued that heterologous expression systems may alter properties of plant transporters, including HKT transporters. In this report, we analyze several key functions of AtHKT1;1-mediated ion currents in their native root stelar cells, including Na⁺ and K⁺ conductances, AtHKT1;1-mediated outward currents, and shifts in reversal potentials in the presence of defined intracellular and extracellular salt concentrations. Enhancer trap Arabidopsis plants with GFP-labeled root stelar cells were used to investigate AtHKT1;1-dependent ion transport properties using patch clamp electrophysiology in wild-type and athkt1;1 mutant plants. AtHKT1;1-dependent currents were carried by sodium ions and these currents were not observed in athkt1;1 mutant stelar cells. However, K⁺ currents in wild-type and athkt1;1 root stelar cell protoplasts were indistinguishable correlating with the Na⁺ over K⁺ selectivity of AtHKT1;1-mediated transport. Moreover, AtHKT1;1-mediated currents did not show a strong voltage dependence in vivo. Unexpectedly, removal of extracellular Na⁺ caused a reduction in AtHKT1;1-mediated outward currents in Columbia root stelar cells and Xenopus oocytes, indicating a role for external Na⁺ in regulation of AtHKT1;1 activity. Shifting the NaCl gradient in root stelar cells showed a Nernstian shift in the reversal potential providing biophysical evidence for the model that AtHKT1;1 mediates passive Na⁺ channel transport properties.     

The Arabidopsis phosphatase PP2C49 negatively regulates salt tolerance through inhibition of AtHKT1;1

Type 2C protein phosphatases (PP2Cs) are the largest protein phosphatase family. PP2Cs dephosphorylate substrates for signaling in Arabidopsis, but the functions of most PP2Cs remain unknown. Here, we characterized PP2C49 (AT3G62260, a Group G PP2C), which regulates Na⁺ distribution under salt stress and is localized to the cytoplasm and nucleus. PP2C49 was highly expressed in root vascular tissues and its disruption enhanced plant tolerance to salt stress. Compared with wild type, the pp2c49 mutant contained more Na⁺ in roots but less Na⁺ in shoots and xylem sap, suggesting that PP2C49 regulates shoot Na⁺ extrusion. Reciprocal grafting revealed a root‐based mechanism underlying the salt tolerance of pp2c49. Systemic Na⁺ distribution largely depends on AtHKT1;1 and loss of function of AtHKT1;1 in the pp2c49 background overrode the salt tolerance of pp2c49, resulting in salt sensitivity. Furthermore, compared with plants overexpressing PP2C49 in the wild‐type background, plants overexpressing PP2C49 in the athtk1;1 mutant background were sensitive to salt, like the athtk1;1 mutants. Moreover, protein–protein interaction and two‐voltage clamping assays demonstrated that PP2C49 physically interacts with AtHKT1;1 and inhibits the Na⁺ permeability of AtHKT1;1. This study reveals that PP2C49 negatively regulates AtHKT1;1 activity and thus determines systemic Na⁺ allocation during salt stress.     

转HAL1基因番茄的耐盐性

利用农杆菌介导的叶盘法,把HAL1 基因转入番茄,Southern杂交检测得到转基因植株.耐盐实验表明, T1代转基因番茄在150 mmol/L的NaCl胁迫下仍有43%的发芽率,200 mmol/L的NaCl胁迫下发芽率为6%,而对照种子在100和150 mmol/L的NaCl胁迫下发芽率分别为11.0%和0.转基因番茄的电解质相对外渗率小于对照,而根冠比和叶绿素含量大于对照,转HAL1基因显著提高了番茄的耐盐性.盐胁迫下Na 、K 的累积状况表明,转基因番茄根、茎、叶的K /Na 均有所提高,根系的SK/Na增大,茎、叶的RSK/Na和RLK/Na减小,说明根系对K /Na 离子的选择吸收和运输能力加强.不但选择吸收K /Na ,而且表现出整株水平上的有利于耐盐的K /Na 区域化分配.     

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.     

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.     

整株水平上Na+ 转运体与植物的抗盐性

植物可以利用不同的机制来维持Na+稳态, 从而增强植物的抗盐性。这些机制包括: 限制Na+的内流; 增大Na+的外排; 减少Na+向地上部分的运输; 把进入地上部分的Na+分散到特殊部分(如老叶)或通过泌盐结构排出体外或通过韧皮部的再循环回到根部。本文简要介绍整株水平上Na+转运体与植物抗盐性的研究进展。     

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.     

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.     

Salt stress in Thellungiella halophila activates Na+ transport mechanisms required for salinity tolerance

Salinity is considered one of the major limiting factors for plant growth and agricultural productivity. We are using salt cress (Thellungiella halophila) to identify biochemical mechanisms that enable plants to grow in saline conditions. Under salt stress, the major site of Na+ accumulation occurred in old leaves, followed by young leaves and taproots, with the least accumulation occurring in lateral roots. Salt treatment increased both the H+ transport and hydrolytic activity of salt cress tonoplast (TP) and plasma membrane (PM) H(+)-ATPases from leaves and roots. TP Na(+)/H+ exchange was greatly stimulated by growth of the plants in NaCl, both in leaves and roots. Expression of the PM H(+)-ATPase isoform AHA3, the Na+ transporter HKT1, and the Na(+)/H+ exchanger SOS1 were examined in PMs isolated from control and salt-treated salt cress roots and leaves. An increased expression of SOS1, but no changes in levels of AHA3 and HKT1, was observed. NHX1 was only detected in PM fractions of roots, and a salt-induced increase in protein expression was observed. Analysis of the levels of expression of vacuolar H(+)-translocating ATPase subunits showed no major changes in protein expression of subunits VHA-A or VHA-B with salt treatment; however, VHA-E showed an increased expression in leaf tissue, but not in roots, when the plants were treated with NaCl. Salt cress plants were able to distribute and store Na+ by a very strict control of ion movement across both the TP and PM.