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.     

A novel protein kinase involved in Na+ exclusion revealed from positional cloning

Salinity is a major abiotic stress which affects crop plants around the world, resulting in substantial loss of yield and millions of dollars of lost revenue. High levels of Na⁺ in shoot tissue have many adverse effects and, crucially, yield in cereals is commonly inversely proportional to the extent of shoot Na⁺ accumulation. We therefore need to identify genes, resistant plant cultivars and cellular processes that are involved in salinity tolerance, with the goal of introducing these factors into commercially available crops. Through the use of an Arabidopsis thaliana mapping population, we have identified a highly significant quantitative trait locus (QTL) linked to Na⁺ exclusion. Fine mapping of this QTL identified a protein kinase (AtCIPK16), related to AtSOS2, that was significantly up‐regulated under salt stress. Greater Na⁺ exclusion was associated with significantly higher root expression of AtCIPK16, which is due to differences in the gene's promoter. Constitutive overexpression of the gene in Arabidopsis leads to plants with significant reduction in shoot Na⁺ and greater salinity tolerance. amiRNA knock‐downs of AtCIPK16 in Arabidopsis show a negative correlation between the expression levels of the gene and the amount of shoot Na⁺. Transgenic barley lines overexpressing AtCIPK16 show increased salinity tolerance.     

A teosinte-derived allele of an HKT1 family sodium transporter improves salt tolerance in maize

The sodium cation (Na⁺) is the predominant cation with deleterious effects on crops in salt-affected agricultural areas. Salt tolerance of crop can be improved by increasing shoot Na⁺ exclusion. Therefore, it is crucial to identify and use genetic variants of various crops that promote shoot Na⁺ exclusion. Here, we show that a HKT1 family gene ZmNC3 (Zea mays L. Na⁺ Content 3; designated ZmHKT1;2) confers natural variability in shoot-Na⁺ accumulation and salt tolerance in maize. ZmHKT1;2 encodes a Na⁺-preferential transporter localized in the plasma membrane, which mediates shoot Na⁺ exclusion, likely by withdrawing Na⁺ from the root xylem flow. A naturally occurring nonsynonymous SNP (SNP947-G) increases the Na⁺ transport activity of ZmHKT1;2, promoting shoot Na⁺ exclusion and salt tolerance in maize. SNP947-G first occurred in the wild grass teosinte (at a allele frequency of 43%) and has become a minor allele in the maize population (allele frequency 6.1%), suggesting that SNP947-G is derived from teosinte and that the genomic region flanking SNP947 likely has undergone selection during domestication or post-domestication dispersal of maize. Moreover, we demonstrate that introgression of the SNP947-G ZmHKT1;2 allele into elite maize germplasms reduces shoot Na⁺ content by up to 80% and promotes salt tolerance. Taken together, ZmNC3/ZmHKT1;2 was identified as an important QTL promoting shoot Na⁺ exclusion, and its favourable allele provides an effective tool for developing salt-tolerant maize varieties.     

The sodium transporter encoded by the HKT1;2 gene modulates sodium/potassium homeostasis in tomato shoots under salinity

Excessive soil salinity diminishes crop yield and quality. In a previous study in tomato, we identified two closely linked genes encoding HKT1‐like transporters, HKT1;1 and HKT1;2, as candidate genes for a major quantitative trait locus (kc7.1) related to shoot Na⁺/K⁺ homeostasis – a major salt tolerance trait – using two populations of recombinant inbred lines (RILs). Here, we determine the effectiveness of these genes in conferring improved salt tolerance by using two near‐isogenic lines (NILs) that were homozygous for either the Solanum lycopersicum allele (NIL17) or for the Solanum cheesmaniae allele (NIL14) at both HKT1 loci; transgenic lines derived from these NILs in which each HKT1;1 and HKT1;2 had been silenced by stable transformation were also used. Silencing of ScHKT1;2 and SlHKT1;2 altered the leaf Na⁺/K⁺ ratio and caused hypersensitivity to salinity in plants cultivated under transpiring conditions, whereas silencing SlHKT1;1/ScHKT1;1 had a lesser effect. These results indicate that HKT1;2 has the more significant role in Na⁺ homeostasis and salinity tolerance in tomato.     

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.     

Differential Responses of <i>NHX1</i> and <i>SOS1</i> Gene Expressions to Salinity in two <i>Miscanthus sinensis</i> Anderss. Accessions with Different Salt Tolerance

The lignocellulosic crop Miscanthus spp. has been identified as a good candidate for biomass production. The responses of Miscanthus sinensis Anderss. to salinity were studied to satisfy the needs for high yields in marginal areas and to avoid competition with food production. The results indicated that the relative advantages of the tolerant accession over the sensitive one under saline conditions were associated with restricted Na⁺ accumulation in shoots. Seedlings of two accessions (salt-tolerant ‘JM0119’ and salt-sensitive ‘JM0099’) were subjected to 0 (control), 100, 200, and 300 mM NaCl stress to better understand the salt-induced biochemical responses of genes involved in Na⁺ accumulation in M. sinensis. The adaptation responses of genes encoding for Na⁺ /H⁺ antiporters, NHX1 and SOS1 to NaCl stress were examined in JM0119 and JM0099.The cDNA sequences of genes examined were highly conserved among the relatives of M. sinensis based on the sequencing on approximate 600 bp-long cDNA fragments obtained from degenerate PCR. These salt-induced variations of gene expression investigated by quantitative real-time PCR provided evidences for insights of the molecular mechanisms of salt tolerance in M. sinensis. The expression of NHX1 was up-regulated by salt stress in JM0119 shoot and root tissues. However, it was hardly affected in JM0099 shoot tissue except for a significant increase at the 100 mM salt treatment, and it was salt-suppressed in the JM0099 root tissue. In the root tissue, the expression of SOS1 was induced by the high salt treatment in JM0119 but repressed by all salt treatments in JM0099. Thus, the remarkably higher expression of NHX1 and SOS1 were associated with the resistance to Na⁺ toxicity by regulation of the Na⁺ influx, efflux, and sequestration under different salt conditions.     

Control of xylem Na+ loading and transport to the shoot in rice and barley as a determinant of differential salinity stress tolerance

Control of xylem Na⁺ loading has often been named as the essential component of salinity tolerance mechanism. However, it is less clear to what extent the difference in this trait may determine differential salinity tolerance between species. In this study, barley (Hordeum vulgare L. cv. CM72) and rice (Oryza sativa L. cv. Dongjin) plants were grown under two levels of salinity. Na⁺ and K⁺ concentrations in the xylem sap, and shoot and root tissues were measured at different time points after stress onset. Salt‐exposed rice plants prevented xylem Na⁺ loading for several days, but failed to control this process in the longer term, ultimately resulting in a massive Na⁺ shoot loading. Barley plants quickly increased xylem Na⁺ concentration and its delivery to the shoot (most likely for the purpose of osmotic adjustment) but were able to reduce this process later on, keeping most of accumulated Na⁺ in the root, thus maintaining non‐toxic shoot Na⁺ level. Rice plants increased shoot K⁺ concentration, while barley plants maintained higher root K⁺ concentration. Control of xylem Na⁺ loading is remarkably different between rice and barley; this difference may differentiate the extent of the salinity tolerance between species. This trait should be investigated in more detail to be used in the breeding programs aimed to improve salinity tolerance in crops.     

Cloning of two SOS1 transporters from the seagrass <Emphasis Type="Italic">Cymodocea nodosa</Emphasis>. SOS1 transporters from <Emphasis Type="Italic">Cymodocea</Emphasis> and <Emphasis Type="Italic">Arabidopsis</Emphasis> mediate potassium uptake in bacteria

Two cDNAs isolated from Cymodocea nodosa, CnSOS1A, and CnSOS1B encode proteins with high-sequence similarities to SOS1 plant transporters. CnSOS1A expressed in a yeast Na⁺-efflux mutant under the control of a constitutive expression promoter mimicked AtSOS1 from Arabidopsis; the wild type cDNA did not improve the growth of the recipient strain in the presence of Na⁺, but a cDNA mutant that expresses a truncated protein suppressed the defect of the yeast mutant. In similar experiments, CnSOS1B was not effective. Conditional expression, under the control of an arabinose responsive promoter, of the CnSOS1A and CnSOS1B cDNAs in an Escherichia coli mutant defective in Na⁺ efflux was toxic, and functional analyses were inconclusive. The same constructs transformed into an E. coli K⁺-uptake mutant revealed that CnSOS1A was also toxic, but that it slightly suppressed defective growth at low K⁺. Truncation in the C-terminal hydrophilic tail of CnSOS1A relieved the toxicity and proved that CnSOS1A was an excellent low-affinity K⁺ and Rb⁺ transporter. CnSOS1B mediated a transient, extremely rapid K⁺ or Rb⁺ influx. Similar tests with AtSOS1 revealed that it was not toxic and that the whole protein exhibited excellent K⁺ and Rb⁺ uptake characteristics in bacteria.     

Salt tolerance of barley: Relations between expression of isoforms of vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript>-antiporter and <Superscript>22</Superscript>Na<Superscript>+</Superscript> accumulation

Two barley cultivars (Hordeum vulgare L., cvs. Elo and Belogorskii) differing in salt tolerance were used to study ²²Na⁺ uptake, expression of three isoforms of the Na⁺/H⁺ antiporter HvNHX1-3, and the cellular localization of these isoforms in the elongation zone of seedling roots. During short (1 h) incubation, seedling roots of both cultivars accumulated approximately equal quantities of ²²Na⁺. However, after 24-h incubation the content of ²²Na⁺ in roots of a salt-tolerant variety Elo was 40% lower than in roots of the susceptible variety Belogorskii. The content of ²²Na⁺ accumulated in shoots of cv. Elo after 24-h incubation was 6.5 times lower than in shoots of cv. Belogorskii and it was 4 times lower after the salt stress treatment. The cytochemical examination revealed that three proteins HvNHX1-3 are co-localized in the same cells of almost all root tissues; these proteins were present in the tonoplast and prevacuolar vesicles. Western blot analysis of HvNHX1-3 has shown that the content of isoforms in vacuolar membranes increased in response to salt stress in seedling roots and shoots of both cultivars, although the increase was more pronounced in the tolerant cultivar. The content of HvNHX1 in the seedlings increased in parallel with the enhanced expression of HvNHX1, whereas the increase in HvNHX2 and HvNHX3 protein content was accompanied by only slight changes in expression of respective genes. The results provide evidence that salt tolerance of barley depends on plant ability to restrict Na⁺ transport from the root to the shoot and relies on regulatory pathways of HvNHX1-3 expression in roots and shoots during salt stress.     

Molecular mechanisms of potassium and sodium uptake in plants

Potassium (K⁺) is an essential nutrient and the most abundant cation in plants, whereas the closely related ion sodium (Na⁺) is toxic to most plants at high millimolar concentrations. K⁺ deficiency and Na⁺ toxicity are both major constraints to crop production worldwide. K⁺ counteracts Na⁺ stress, while Na⁺, in turn, can to a certain degree alleviate K⁺ deficiency. Elucidation of the molecular mechanisms of K⁺ and Na⁺ transport is pivotal to the understanding – and eventually engineering – of plant K⁺ nutrition and Na⁺ sensitivity. Here we provide an overview on plant K⁺ transporters with particular emphasis on root K⁺ and Na⁺ uptake. Plant K⁺-permeable cation transporters comprise seven families: Shaker-type K⁺ channels, `two-pore' K⁺ channels, cyclic-nucleotide-gated channels, putative K⁺/H⁺ antiporters, KUP/HAK/KT transporters, HKT transporters, and LCT1. Candidate genes for Na⁺ transport are the KUP/HAK/KTs, HKTs, CNGCs, and LCT1. Expression in heterologous systems, localization in plants, and genetic disruption in plants will provide insight into the roles of transporter genes in K⁺ nutrition and Na⁺ toxicity.     

Increased salt tolerance with overexpression of cation/proton antiporter 1 genes: a meta‐analysis

Cation/proton antiporter 1 (CPA1) genes encode cellular Na⁺/H⁺ exchanger proteins, which act to adjust ionic balance. Overexpression of CPA1s can improve plant performance under salt stress. However, the diversified roles of the CPA1 family and the various parameters used in evaluating transgenic plants over‐expressing CPA1s make it challenging to assess the complex functions of CPA1s and their physiological mechanisms in salt tolerance. Using meta‐analysis, we determined how overexpression of CPA1s has influenced several plant characteristics involved in response and resilience to NaCl stress. We also evaluated experimental variables that favour or reduce CPA1 effects in transgenic plants. Viewed across studies, overexpression of CPA1s has increased the magnitude of 10 of the 19 plant characteristics examined, by 25% or more. Among the ten moderating variables, several had substantial impacts on the extent of CPA1 influence: type of culture media, donor and recipient type and genus, and gene family. Genes from monocotyledonous plants stimulated root K⁺, root K⁺/Na⁺, total chlorophyll, total dry weight and root length much more than genes from dicotyledonous species. Genes transformed to or from Arabidopsis have led to smaller CPA1‐induced increases in plant characteristics than genes transferred to or from other genera. Heterogeneous expression of CPA1s led to greater increases in leaf chlorophyll and root length than homologous expression. These findings should help guide future investigations into the function of CPA1s in plant salt tolerance and the use of genetic engineering for breeding of resistance.     

Disorder of trafficking system of plasma membrane and vacuole antiporter proteins causes hypersensitive response to salinity stress in <Emphasis Type="Italic">Arabidopsis Thaliana</Emphasis>

Rab GTPases play an important role in regulating intracellular vesicular trafficking in eukaryotic cells. Previously, we found that Oryza sativa rice Rab11 (OsRab11) is required for the regulation of vesicular trafficking from the trans- Golgi network (TGN) to the plasma membrane (PM) and/or vacuoles. To further elucidate the relationship between vesicular trafficking and abiotic and biotic stresses, we determined OsRab11 expression levels under several environmental stress conditions. OsRab11 expression was induced by pathogens, jasmonic acid (JA), and high salt treatment. Under high salt conditions, dominant negative OsRab11(S28N) mutant plants exhibited a hypersensitive phenotype similar to that of sos1-1, whereas overexpressed-OsRab11 plants showed resistance to high salt stress. When the expression of vacuolar and PM Na⁺/H⁺ antiporter genes such as AtNHX1, AtNHX2, and AtSOS1 was examined, there was no significant difference between the wild-type and OsRab11(S28N) mutant plants. However, PM trafficking of AtSOS1-green fluorescent protein (GFP) in 35S::AtSOS1-GFP sos1-1 plants was severely impaired by T7-OsRab11(S28N) expression. Similarly, vacuolar trafficking of AtNHX2-GFP was inhibited by T7-OsRab11 (S28N) expression. These results indicate that trafficking of PM and vacuolar antiporter proteins by OsRab11 is important for high salt stress resistance.     

Salt tolerance of maize (Zea mays L.): the role of sodium exclusion

The influence of NaCl and Na₂SO₄ on growth of two maize cultivars (Zea mays cv. Pioneer 3906 and cv. Across 8023) differing in Na⁺ uptake was investigated in two green-house experiments. Na⁺ treatment with different accompanying anions (Cl^?/SO₄^2?) showed that ion toxicity was caused by Na⁺. While shoot growth of the two cultivars was markedly affected by salt in comparison to the control during the first 2–3 weeks, there were only slight differences between the cultivars. The shoot Ca²⁺ concentration was reduced in both cultivars, and the youngest leaves contained an even lower concentration compared with the rest of the shoot. During this first phase, Across 8023 tended to have higher concentrations of Ca²⁺ than Pioneer 3906. The Na⁺-excluding cultivar Pioneer 3906 showed continuous, although reduced, growth compared with the control, while the Na⁺ concentration in the shoot decreased until flowering. Cultivar Across 8023 accumulated Na⁺ until flowering: the reduction in the growth of stressed plants was greater than that for Pioneer 3906. Leaves of cultivar Across 8023 showed clear toxic symptoms, while those of the more salt-tolerant cultivar Pioneer 3906 did not. It is concluded that Na⁺ exclusion contributes to the salt tolerance of maize.     

Arbuscular mycorrhizal fungi native from a Mediterranean saline area enhance maize tolerance to salinity through improved ion homeostasis

Soil salinity restricts plant growth and productivity. Na⁺ represents the major ion causing toxicity because it competes with K⁺ for binding sites at the plasma membrane. Inoculation with arbuscular mycorrhizal fungi (AMF) can alleviate salt stress in the host plant through several mechanisms. These may include ion selection during the fungal uptake of nutrients from the soil or during transfer to the host plant. AM benefits could be enhanced when native AMF isolates are used. Thus, we investigated whether native AMF isolated from an area with problems of salinity and desertification can help maize plants to overcome the negative effects of salinity stress better than non‐AM plants or plants inoculated with non‐native AMF. Results showed that plants inoculated with two out the three native AMF had the highest shoot dry biomass at all salinity levels. Plants inoculated with the three native AMF showed significant increase of K⁺ and reduced Na⁺ accumulation as compared to non‐mycorrhizal plants, concomitantly with higher K⁺/Na⁺ ratios in their tissues. For the first time, these effects have been correlated with regulation of ZmAKT2, ZmSOS1 and ZmSKOR genes expression in the roots of maize, contributing to K⁺ and Na⁺ homeostasis in plants colonized by native AMF.     

Expression of the AKT1-type K+ channel gene from Puccinellia tenuiflora, PutAKT1, enhances salt tolerance in Arabidopsis

Potassium channels are important for many physiological functions in plants, one of which is to regulate plant adaptation to stress conditions. In this study, a K⁺ channel PutAKT1 cDNA was isolated from the salt-tolerant plant Puccinellia tenuiflora. A phylogenetic analysis showed that PutAKT1 belongs to the AKT1-subfamily in the Shaker K⁺ channel family. PutAKT1 was localized in the plasma membrane and it was preferentially expressed in the roots. The expression of PutAKT1 was induced by K⁺-starvation stress in the roots and was not down-regulated by the presence of excess Na⁺. Arabidopsis plants over-expressing PutAKT1 showed enhanced salt tolerance compared to wild-type plants as shown by their shoot phenotype and dry weight. Expression of PutAKT1 increased the K⁺ content of Arabidopsis under normal, K⁺-starvation, and NaCl-stress conditions. Arabidopsis expressing PutAKT1 also showed a decrease in Na⁺ accumulation both in the shoot and in the root. These results suggest that PutAKT1 is involved in mediating K⁺ uptake (i) both in low- and in high-affinity K⁺ uptake range, and (ii) unlike its homologs in rice, even under salt-stress condition.