Golgi apparatus-localized CATION CALCIUM EXCHANGER4 promotes osmotolerance of Arabidopsis.

Calcium (Ca2+) is a major ion in living organisms, where it acts as a second messenger for various biological phenomena. The Golgi apparatus retains a higher Ca2+ concentration than the cytosol and returns cytosolic Ca2+ to basal levels after transient elevation in response to environmental stimuli such as osmotic stress. However, the Ca2+ transporters localized in the Golgi apparatus of plants have not been clarified. We previously found that a wild-type (WT) salt-tolerant Arabidopsis (Arabidopsis thaliana) accession, Bu-5, showed osmotic tolerance after salt acclimatization, whereas the Col-0 WT did not. Here, we isolated a Bu-5 background mutant gene, acquired osmotolerance-defective 6 (aod6), which reduces tolerance to osmotic, salt, and oxidative stresses, with a smaller plant size than the WT. The causal gene of the aod6 mutant encodes CATION CALCIUM EXCHANGER4 (CCX4). The aod6 mutant was more sensitive than the WT to both deficient and excessive Ca2+. In addition, aod6 accumulated higher Ca2+ than the WT in the shoots, suggesting that Ca2+ homeostasis is disturbed in aod6. CCX4 expression suppressed the Ca2+ hypersensitivity of the csg2 (calcium sensitive growth 2) yeast (Saccharomyces cerevisiae) mutant under excess CaCl2 conditions. We also found that aod6 enhanced MAP kinase 3/6 (MPK3/6)-mediated immune responses under osmotic stress. Subcellular localization analysis of mGFP-CCX4 showed GFP signals adjacent to the trans-Golgi apparatus network and co-localization with Golgi apparatus-localized markers, suggesting that CCX4 localizes in the Golgi apparatus. These results suggest that CCX4 is a Golgi apparatus-localized transporter involved in the Ca2+ response and plays important roles in osmotic tolerance, shoot Ca2+ content, and normal growth of Arabidopsis.

Distinct Functions of the Atypical Terminal Hydrophilic Domain of the HKT Transporter in the Liverwort Marchantia polymorpha.

K+/Na+ homeostasis is important for land plants, particularly under salt stress. In this study, the structure and ion transport properties of the high-affinity K+ transporter (HKT) of the liverwort Marchantia polymorpha were investigated. Only one HKT gene, MpHKT1, was identified in the genome of M. polymorpha. Phylogenetic analysis of HKT proteins revealed that non-seed plants possess HKTs grouped into a clade independent of the other two clades including HKTs of angiosperms. A distinct long hydrophilic domain was found in the C-terminus of MpHKT1. Complementary DNA (cDNA) of truncated MpHKT1 (t-MpHKT1) encoding the MpHKT_Δ596-812 protein was used to examine the functions of the C-terminal domain. Both MpHKT1 transporters fused with enhanced green fluorescent protein at the N-terminus were localized to the plasma membrane when expressed in rice protoplasts. Two-electrode voltage clamp experiments using Xenopus laevis oocytes indicated that MpHKT1 mediated the transport of monovalent alkali cations with higher selectivity for Na+ and K+, but truncation of the C-terminal domain significantly reduced the transport activity with a decrease in the Na+ permeability. Overexpression of MpHKT1 or t-MpHKT1 in M. polymorpha conferred accumulation of higher Na+ levels and showed higher Na+ uptake rates, compared to those of wild-type plants; however, phenotypes with t-MpHKT1 were consistently weaker than those with MpHKT1. Together, these findings suggest that the hydrophilic C-terminal domain plays a unique role in the regulation of transport activity and ion selectivity of MpHKT1.

Mechanisms Activating Latent Functions of PIP Aquaporin Water Channels via the Interaction between PIP1 and PIP2 Proteins.

Plant plasma membrane-type plasma membrane intrinsic protein (PIP) aquaporins are classified into two groups, PIP1s and PIP2s. In this study, we focused on HvPIP1;2, a PIP1 in barley (Hordeum vulgare), to dissect the molecular mechanisms that evoke HvPIP1-mediated water transport. No HvPIP1;2 protein was localized to the plasma membrane when expressed alone in Xenopus laevis oocytes. By contrast, a chimeric HvPIP1;2 protein (HvPIP1;2_24NC), in which the N- and C-terminal regions were replaced with the corresponding regions from HvPIP2;4, was found to localize to the plasma membrane of oocytes. However, HvPIP1;2_24NC showed no water transport activity in swelling assays. These results suggested that the terminal regions of PIP2 proteins direct PIP proteins to the plasma membrane, but the relocalization of PIP1 proteins was not sufficient to PIP1s functionality as a water channel in a membrane. A single amino acid replacement of threonine by methionine in HvPIP2;4 (HvPIP2;4T229M) abolished water transport activity. Co-expression of HvPIP1;2_24NC either with HvPIP2;4_12NC or with HvPIP2;4TM_12NC, in which the N- and C-terminal regions were replaced with the corresponding regions of HvPIP1;2, increased the water transport activity in oocytes. These data provided evidence that the HvPIP1;2 molecule has own water transport activity and an interaction with the middle part of the HvPIP2;4 protein (except for the N- and C-termini) is required for HvPIP1;2 functionality as a water channel. This molecular mechanism could be applied to other PIP1s and PIP2s in addition to the known mechanism that the terminal regions of some PIP2s lead some PIP1s to the plasma membrane.

Functions and structure of roots and their contributions to salinity tolerance in plants.

Soil salinity is an increasing threat to the productivity of glycophytic crops worldwide. The root plays vital roles under various stress conditions, including salinity, as well as has diverse functions in non-stress soil environments. In this review, we focus on the essential functions of roots such as in ion homeostasis mediated by several different membrane transporters and signaling molecules under salinity stress and describe recent advances in the impacts of quantitative trait loci (QTLs) or genetic loci (and their causal genes, if applicable) on salinity tolerance. Furthermore, we introduce important literature for the development of barriers against the apoplastic flow of ions, including Na+, as well as for understanding the functions and components of the barrier structure under salinity stress.

Doing 'business as usual' comes with a cost: evaluating energy cost of maintaining plant intracellular K+ homeostasis under saline conditions.

Salinization of agricultural lands is a major threat to agriculture. Many different factors affect and determine plant salt tolerance. Nonetheless, there is a consensus on the relevance of maintaining an optimal cytosolic potassium : sodium ion (K+  : Na+ ) ratio for salinity tolerance in plants. This ratio depends on the operation of plasma membrane and tonoplast transporters. In the present review we focus on some aspects related to the energetic cost of maintaining that K+  : Na+ ratio. One of the factors that affect the cost of the first step of K+ acquisition - root K+ uptake through High Affinity K+ transporter and Arabidopsis K+ transport system 1 transport systems - is the value of the plasma membrane potential of root cells, a parameter that may differ amongst plant species. In addition to its role in nutrition, cytosolic K+ also is important for signalling, and K+ efflux through gated outward-rectifying K+ and nonselective cation channels can be regarded as a switch to redirect energy towards defence reactions. In maintaining cytosolic K+ , the great buffer capacity of the vacuole should be considered. The possible role of high-affinity K+ transporters (HKT)2s in mediating K+ uptake under saline conditions and the importance of cycling of K+ throughout the plant also are discussed.

A Survey of Barley PIP Aquaporin Ionic Conductance Reveals Ca2+-Sensitive HvPIP2;8 Na+ and K+ Conductance.

Some plasma membrane intrinsic protein (PIP) aquaporins can facilitate ion transport. Here we report that one of the 12 barley PIPs (PIP1 and PIP2) tested, HvPIP2;8, facilitated cation transport when expressed in Xenopus laevis oocytes. HvPIP2;8-associated ion currents were detected with Na+ and K+, but not Cs+, Rb+, or Li+, and was inhibited by Ba2+, Ca2+, and Cd2+ and to a lesser extent Mg2+, which also interacted with Ca2+. Currents were reduced in the presence of K+, Cs+, Rb+, or Li+ relative to Na+ alone. Five HvPIP1 isoforms co-expressed with HvPIP2;8 inhibited the ion conductance relative to HvPIP2;8 alone but HvPIP1;3 and HvPIP1;4 with HvPIP2;8 maintained the ion conductance at a lower level. HvPIP2;8 water permeability was similar to that of a C-terminal phosphorylation mimic mutant HvPIP2;8 S285D, but HvPIP2;8 S285D showed a negative linear correlation between water permeability and ion conductance that was modified by a kinase inhibitor treatment. HvPIP2;8 transcript abundance increased in barley shoot tissues following salt treatments in a salt-tolerant cultivar Haruna-Nijo, but not in salt-sensitive I743. There is potential for HvPIP2;8 to be involved in barley salt-stress responses, and HvPIP2;8 could facilitate both water and Na+/K+ transport activity, depending on the phosphorylation status.

Changes in Expression Level of OsHKT1;5 Alters Activity of Membrane Transporters Involved in K+ and Ca2+ Acquisition and Homeostasis in Salinized Rice Roots.

In rice, the OsHKT1;5 gene has been reported to be a critical determinant of salt tolerance. This gene is harbored by the SKC1 locus, and its role was attributed to Na+ unloading from the xylem. No direct evidence, however, was provided in previous studies. Also, the reported function of SKC1 on the loading and delivery of K+ to the shoot remains to be explained. In this work, we used an electrophysiological approach to compare the kinetics of Na+ uptake by root xylem parenchyma cells using wild type (WT) and NIL(SKC1) plants. Our data showed that Na+ reabsorption was observed in WT, but not NIL(SKC1) plants, thus questioning the functional role of HKT1;5 as a transporter operating in the direct Na+ removal from the xylem. Instead, changes in the expression level of HKT1;5 altered the activity of membrane transporters involved in K+ and Ca2+ acquisition and homeostasis in the rice epidermis and stele, explaining the observed phenotype. We conclude that the role of HKT1;5 in plant salinity tolerance cannot be attributed to merely reducing Na+ concentration in the xylem sap but triggers a complex feedback regulation of activities of other transporters involved in the maintenance of plant ionic homeostasis and signaling under stress conditions.

High-Affinity K+ Transporters from a Halophyte, Sporobolus virginicus, Mediate Both K+ and Na+ Transport in Transgenic Arabidopsis, X. laevis Oocytes and Yeast.

Class II high-affinity potassium transporters (HKTs) have been proposed to mediate Na+-K+ co-transport in plants, as well as Na+ and K+ homeostasis under K+-starved and saline environments. We identified class II HKTs, namely SvHKT2;1 and SvHKT2;2 (SvHKTs), from the halophytic turf grass, Sporobolus virginicus. SvHKT2;2 expression in S. virginicus was up-regulated by NaCl treatment, while SvHKT2;1 expression was assumed to be up-regulated by K+ starvation and down-regulated by NaCl treatment. Localization analysis revealed SvHKTs predominantly targeted the plasma membrane. SvHKTs complemented K+ uptake deficiency in mutant yeast, and showed both inward and outward K+ and Na+ transport activity in Xenopus laevis oocytes. When constitutively expressed in Arabidopsis, SvHKTs mediated K+ and Na+ accumulation in shoots under K+-starved conditions, and the K+ concentration in xylem saps of transformants was also higher than in those of wild-type plants. These results suggest transporter-enhanced K+ and Na+ uploading to the xylem from xylem parenchyma cells. Together, our data demonstrate that SvHKTs mediate both outward and inward K+ and Na+ transport in X. laevis oocytes, and possibly in plant and yeast cells, depending on the ionic conditions.

Using membrane transporters to improve crops for sustainable food production.

With the global population predicted to grow by at least 25 per cent by 2050, the need for sustainable production of nutritious foods is critical for human and environmental health. Recent advances show that specialized plant membrane transporters can be used to enhance yields of staple crops, increase nutrient content and increase resistance to key stresses, including salinity, pathogens and aluminium toxicity, which in turn could expand available arable land.

OsHKT1;5 mediates Na+ exclusion in the vasculature to protect leaf blades and reproductive tissues from salt toxicity in rice.

Salt tolerance quantitative trait loci analysis of rice has revealed that the SKC1 locus, which is involved in a higher K+ /Na+ ratio in shoots, corresponds to the OsHKT1;5 gene encoding a Na+ -selective transporter. However, physiological roles of OsHKT1;5 in rice exposed to salt stress remain elusive, and no OsHKT1;5 gene disruption mutants have been characterized to date. In this study, we dissected two independent T-DNA insertional OsHKT1;5 mutants. Measurements of ion contents in tissues and 22 Na+ tracer imaging experiments showed that loss-of-function of OsHKT1;5 in salt-stressed rice roots triggers massive Na+ accumulation in shoots. Salt stress-induced increases in the OsHKT1;5 transcript were observed in roots and basal stems, including basal nodes. Immuno-staining using an anti-OsHKT1;5 peptide antibody indicated that OsHKT1;5 is localized in cells adjacent to the xylem in roots. Additionally, direct introduction of 22 Na+ tracer to leaf sheaths also demonstrated the involvement of OsHKT1;5 in xylem Na+ unloading in leaf sheaths. Furthermore, OsHKT1;5 was indicated to be present in the plasma membrane and found to localize also in the phloem of diffuse vascular bundles in basal nodes. Together with the characteristic 22 Na+ allocation in the blade of the developing immature leaf in the mutants, these results suggest a novel function of OsHKT1;5 in mediating Na+ exclusion in the phloem to prevent Na+ transfer to young leaf blades. Our findings further demonstrate that the function of OsHKT1;5 is crucial over growth stages of rice, including the protection of the next generation seeds as well as of vital leaf blades under salt stress.

A Magnesium Transporter OsMGT1 Plays a Critical Role in Salt Tolerance in Rice.

Salt stress is one of the major factors limiting rice (Oryza sativa) production globally. Although several transporters involved in salt tolerance have been identified in rice, the mechanisms regulating their transport activity are still poorly understood. Here, we show evidence that a rice Mg transporter OsMGT1 is required for salt tolerance probably by regulating transport activity of OsHKT1;5, a key transporter for the removal of Na+ from the xylem sap at the root mature zone. Knockout of OsMGT1 did not affect total Na uptake, but increased Na concentration in the shoots and xylem sap, resulting in a significant increase in salt sensitivity at low external Mg2+ concentration (20-200 µm). However, such differences were abolished at a higher Mg2+ concentration (2 mm), although the total Na uptake was not altered. OsMGT1 was expressed in both the roots and shoots, but only that in the roots was moderately up-regulated by salt stress. Spatial expression analysis revealed that OsMGT1 was expressed in all root cells of the root tips but was highly expressed in the pericycle of root mature zone. OsMGT1 was also expressed in the phloem region of basal node, leaf blade, and sheath. When expressed in Xenopus laevis oocytes, the transport activity of OsHKT1;5 was enhanced by elevating external Mg2+ concentration. Furthermore, knockout of OsHKT1;5 in osmgt1 mutant background did not further increase its salt sensitivity. Taken together, our results suggest that Mg2+ transported by OsMGT1 in the root mature zone is required for enhancing OsHKT1;5 activity, thereby restricting Na accumulation to the shoots.

T-DNA Tagging-Based Gain-of-Function of OsHKT1;4 Reinforces Na Exclusion from Leaves and Stems but Triggers Na Toxicity in Roots of Rice Under Salt Stress.

The high affinity K⁺ transporter 1;4 (HKT1;4) in rice (Oryza sativa), which shows Na⁺ selective transport with little K⁺ transport activity, has been suggested to be involved in reducing Na in leaves and stems under salt stress. However, detailed physiological roles of OsHKT1;4 remain unknown. Here, we have characterized a transfer DNA (T-DNA) insertion mutant line of rice, which overexpresses OsHKT1;4, owing to enhancer elements in the T-DNA, to gain an insight into the impact of OsHKT1;4 on salt tolerance of rice. The homozygous mutant (the O/E line) accumulated significantly lower concentrations of Na in young leaves, stems, and seeds than the sibling WT line under salt stress. Interestingly, however, the mutation rendered the O/E plants more salt sensitive than WT plants. Together with the evaluation of biomass of rice lines, rhizosphere acidification assays using a pH indicator bromocresol purple and 22NaCl tracer experiments have led to an assumption that roots of O/E plants suffered heavier damages from Na which excessively accumulated in the root due to increased activity of Na⁺ uptake and Na⁺ exclusion in the vasculature. Implications toward the application of the HKT1-mediated Na⁺ exclusion system to the breeding of salt tolerant crop cultivars will be discussed.

Identification of an H2 O2 permeable PIP aquaporin in barley and a serine residue promoting H2 O2 transport.

A barley (Hordeum vulgare) plasma membrane type aquaporin, HvPIP2;5, was identified as an H2 O2 permeable aquaporin among 21 barley and rice PIPs examined in the heterologous expression system using Saccharomyces cerevisiae. Four TIPs were also detected as H2 O2 -transporting aquaporins among 15 barley and rice TIPs. Influx of H2 O2 into yeast cells expressing HvPIP2;5 was determined with a florescent-dye-based assay. Indirect immunofluorescence indicated that the expression of HvPIP2;5 protein was ubiquitous in root tissues, and was also weakly observed in leaf epidermal cells and cells in the vascular bundle. Point mutated variants of HvPIP2;5 were generated by the site-directed mutagenesis. Growth assays of yeast cells expressing these mutated HvPIP2;5 proteins suggested that Ser-126 in HvPIP2;5 has a large impact on H2 O2 transport with a minor influence on the HvPIP2;5-mediated water transport.

OsHKT1;4-mediated Na(+) transport in stems contributes to Na(+) exclusion from leaf blades of rice at the reproductive growth stage upon salt stress.

BACKGROUND: Na(+) exclusion from leaf blades is one of the key mechanisms for glycophytes to cope with salinity stress. Certain class I transporters of the high-affinity K(+) transporter (HKT) family have been demonstrated to mediate leaf blade-Na(+) exclusion upon salinity stress via Na(+)-selective transport. Multiple HKT1 transporters are known to function in rice (Oryza sativa). However, the ion transport function of OsHKT1;4 and its contribution to the Na(+) exclusion mechanism in rice remain to be elucidated. RESULTS: Here, we report results of the functional characterization of the OsHKT1;4 transporter in rice. OsHKT1;4 mediated robust Na(+) transport in Saccharomyces cerevisiae and Xenopus laevis oocytes. Electrophysiological experiments demonstrated that OsHKT1;4 shows strong Na(+) selectivity among cations tested, including Li(+), Na(+), K(+), Rb(+), Cs(+), and NH4 (+), in oocytes. A chimeric protein, EGFP-OsHKT1;4, was found to be functional in oocytes and targeted to the plasma membrane of rice protoplasts. The level of OsHKT1;4 transcripts was prominent in leaf sheaths throughout the growth stages. Unexpectedly however, we demonstrate here accumulation of OsHKT1;4 transcripts in the stem including internode II and peduncle in the reproductive growth stage. Moreover, phenotypic analysis of OsHKT1;4 RNAi plants in the vegetative growth stage revealed no profound influence on the growth and ion accumulation in comparison with WT plants upon salinity stress. However, imposition of salinity stress on the RNAi plants in the reproductive growth stage caused significant Na(+) overaccumulation in aerial organs, in particular, leaf blades and sheaths. In addition, (22)Na(+) tracer experiments using peduncles of RNAi and WT plants suggested xylem Na(+) unloading by OsHKT1;4. CONCLUSIONS: Taken together, our results indicate a newly recognized function of OsHKT1;4 in Na(+) exclusion in stems together with leaf sheaths, thus excluding Na(+) from leaf blades of a japonica rice cultivar in the reproductive growth stage, but the contribution is low when the plants are in the vegetative growth stage.

OsHKT2;2/1-mediated Na(+) influx over K(+) uptake in roots potentially increases toxic Na(+) accumulation in a salt-tolerant landrace of rice Nona Bokra upon salinity stress.

HKT transporters are Na(+)-permeable membrane proteins, which mediate Na(+) and K(+) homeostasis in K(+)-depleted and saline environments in plants. Class II HKT transporters, a distinct subgroup found predominantly in monocots, are known to mediate Na(+)-K(+) co-transport in principle. Here we report features of ion transport functions of No-OsHKT2;2/1, a class II transporter identified in a salt tolerant landrace of indica rice, Nona Bokra. We profiled No-OsHKT2;2/1 expression in organs of Nona Bokra plants with or without salinity stress. Dominant accumulation of the No-OsHKT2;2/1 transcript in K(+)-starved roots of Nona Bokra plants largely disappeared in response to 50 mM NaCl. We found that No-OsHKT2;2/1 expressed in the high-affinity K(+) uptake deficient mutant of Saccharomyces cerevisiae and Xenopus laevis oocytes shows robust K(+) selectivity even in the presence of a large amount of NaCl as reported previously. However, No-OsHKT2;2/1-expressing yeast cells exhibited Na(+) hypersensitive growth under various concentrations of K(+) and Na(+) as the cells expressing Po-OsHKT2;2, a similar class II transporter from another salt tolerant indica rice Pokkali, when compared with the growth of cells harboring empty vector or cells expressing OsHKT2;4. The OsHKT2;4 protein expressed in Xenopus oocytes showed strong K(+) selectivity in the presence of 50 mM NaCl in comparison with No-OsHKT2;2/1 and Po-OsHKT2;2. Together with apparent plasma membrane-localization of No-OsHKT2;2/1, these results point to possibilities that No-OsHKT2;2/1 could mediate destructive Na(+) influx over K(+) uptake in Nona Bokra plants upon salinity stress, and that a predominant physiological function of No-OsHKT2;2/1 might be the acquisition of Na(+) and K(+) in K(+)-limited environments.

Dynamic regulation of the root hydraulic conductivity of barley plants in response to salinity/osmotic stress.

Salinity stress significantly reduces the root hydraulic conductivity (Lpr) of several plant species including barley (Hordeum vulgare). Here we characterized changes in the Lpr of barley plants in response to salinity/osmotic stress in detail using a pressure chamber. Salt-tolerant and intermediate barley cultivars, K305 and Haruna-nijyo, but not a salt-sensitive cultivar, I743, exhibited characteristic time-dependent Lpr changes induced by 100 mM NaCl. An identical response was evoked by isotonic sorbitol, indicating that this phenomenon was triggered by osmotic imbalances. Further examination of this mechanism using barley cv. Haruna-nijyo plants in combination with the use of various inhibitors suggested that various cellular processes such as protein phosphorylation/dephosphorylation and membrane internalization appear to be involved. Interestingly, the three above-mentioned barley cultivars did not exhibit a remarkable difference in root cell sap osmolality under hypertonic conditions, in contrast to the case of Lpr. The possible biological significance of the regulation of Lpr in barley plants upon salinity/osmotic stress is discussed.

CO2 transport by PIP2 aquaporins of barley.

CO2 permeability of plasma membrane intrinsic protein 2 (PIP2) aquaporins of Hordeum vulgare L. was investigated. Five PIP2 members were heterologously expressed in Xenopus laevis oocytes. CO2 permeability was determined by decrease of cytosolic pH in CO2-enriched buffer using a hydrogen ion-selective microelectrode. HvPIP2;1, HvPIP2;2, HvPIP2;3 and HvPIP2;5 facilitated CO2 transport across the oocyte cell membrane. However, HvPIP2;4 that is highly homologous to HvPIP2;3 did not. The isoleucine residue at position 254 of HvPIP2;3 was conserved in PIP2 aquaporins of barley, except HvPIP2;4, which possesses methionine instead. CO2 permeability was lost by the substitution of the Ile254 of HvPIP2;3 by methionine, while water permeability was not affected. These results suggest that PIP2 aquaporins are permeable to CO2. and the conserved isoleucine at the end of the E-loop is crucial for CO2 selectivity.

The HKT1 Na+ transporter protects plant fertility by decreasing Na+ content in stamen filaments.

Salinity stress can greatly reduce seed production because plants are especially sensitive to salt during their reproductive stage. Here, we show that the sodium ion transporter AtHKT1;1 is specifically expressed around the phloem and xylem of the stamen in Arabidopsis thaliana to prevent a marked decrease in seed production caused by salt stress. The stamens of AtHKT1;1 mutant under salt stress overaccumulate Na+, limiting their elongation and resulting in male sterility. Specifically restricting AtHKT1;1 expression to the phloem leads to a 1.5-fold increase in the seed yield upon sodium ion stress. Expanding phloem expression of AtHKT1;1 throughout the entire plant is a promising strategy for increasing plant productivity under salinity stress.

K+ transport by the OsHKT2;4 transporter from rice with atypical Na+ transport properties and competition in permeation of K+ over Mg2+ and Ca2+ ions.

Members of class II of the HKT transporters, which have thus far only been isolated from grasses, were found to mediate Na(+)-K(+) cotransport and at high Na(+) concentrations preferred Na(+)-selective transport, depending on the ionic conditions. But the physiological functions of this K(+)-transporting class II of HKT transporters remain unknown in plants, with the exception of the unique class II Na(+) transporter OsHKT2;1. The genetically tractable rice (Oryza sativa; background Nipponbare) possesses two predicted K(+)-transporting class II HKT transporter genes, OsHKT2;3 and OsHKT2;4. In this study, we have characterized the ion selectivity of the class II rice HKT transporter OsHKT2;4 in yeast and Xenopus laevis oocytes. OsHKT2;4 rescued the growth defect of a K(+) uptake-deficient yeast mutant. Green fluorescent protein-OsHKT2;4 is targeted to the plasma membrane in transgenic plant cells. OsHKT2;4-expressing oocytes exhibited strong K(+) permeability. Interestingly, however, K(+) influx in OsHKT2;4-expressing oocytes did not require stimulation by extracellular Na(+), in contrast to other class II HKT transporters. Furthermore, OsHKT2;4-mediated currents exhibited permeabilities to both Mg(2+) and Ca(2+) in the absence of competing K(+) ions. Comparative analyses of Ca(2+) and Mg(2+) permeabilities in several HKT transporters, including Arabidopsis thaliana HKT1;1 (AtHKT1;1), Triticum aestivum HKT2;1 (TaHKT2;1), OsHKT2;1, OsHKT2;2, and OsHKT2;4, revealed that only OsHKT2;4 and to a lesser degree TaHKT2;1 mediate Mg(2+) transport. Interestingly, cation competition analyses demonstrate that the selectivity of both of these class II HKT transporters for K(+) is dominant over divalent cations, suggesting that Mg(2+) and Ca(2+) transport via OsHKT2;4 may be small and would depend on competing K(+) concentrations in plants.