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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. [ABSTRACT FROM AUTHOR]

Salt stress reduces land and water productivity and contributes to poverty and food insecurity. Increased salinization caused by human practices and climate change is progressively reducing agriculture productivity despite escalating calls for more food. Plant responses to salt stress are well understood, involving numerous critical processes that are each controlled by multiple genes. Knowledge of the critical mechanisms controlling salt uptake and exclusion from functioning tissues, signaling of salt stress, and the arsenal of protective metabolites is advancing. However, little progress has been made in developing salt-tolerant varieties of crop species using standard (but slow) breeding approaches. The genetic diversity available within cultivated crops and their wild relatives provides rich sources for trait and gene discovery that has yet to be sufficiently utilized. Transforming this knowledge into modern approaches using genomics and molecular tools for precision breeding will accelerate the development of tolerant cultivars and help sustain food production. [ABSTRACT FROM AUTHOR]

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. CATION CALCIUM EXCHANGER4 is a Golgi-localized transporter involved in Ca2+ responses and plays important roles in osmotic tolerance, Ca2+ content in shoots, and Arabidopsis growth. [ABSTRACT FROM AUTHOR]

Plant cells are sensitive to salinity stress and do not require sodium as an essential element for their growth and development. Saline soils reduce crop yields and limit available land. Research shows that HKT transporters provide a potent mechanism for mediating salt tolerance in plants. Knowledge of the molecular ion transport and regulation mechanisms and the control of HKT gene expression are crucial for understanding the mechanisms by which HKT transporters enhance crop performance under salinity stress. This review focuses on HKT transporters in monocot plants and in Arabidopsis as a dicot plant, as a guide to efforts toward improving salt tolerance of plants for increasing the production of crops and bioenergy feedstocks. [ABSTRACT FROM AUTHOR]

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;l. 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 Mg2+ and Ca2+ in the absence of competing K+ ions. Comparative analyses of Ca2+ and Mg2+ permeabilities in several HKT transporters, including Arabidopsis thaliana HKT1;1 (AtHKTl;1), Triticum aestivum HKT2;1 (TaHKT2;l), OsHKT2;l, OsHKT2;2, and OsHKT2;4, revealed that only OsHKT2;4 and to a lesser degree TaHKT2;l mediate Mg2+ 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 Mg2+ and Ca2+ transport via OsHKT2;4 may be small and would depend on competing K+ concentrations in plants. [ABSTRACT FROM AUTHOR]

Water homeostasis is crucial to the growth and survival of plants under water-related stress. Plasma membrane intrinsic proteins (PIPs) have been shown to be primary channels mediating water uptake in plant cells. Here we report the water transport activity and mechanisms for the regulation of barley (Hordeum vulgare) PIP aquaporins. HvPIP2 but not HvPIP1 channels were found to show robust water transport activity when expressed alone in Xenopus laevis oocytes. However, the co-expression of HvPIP1 with HvPIP2 in oocytes resulted in significant increases in activity compared with the expression of HvPIP2 alone, suggesting the participation of HvPIP1 in water transport together with HvPIP2 presumably through heteromerization. Severe salinity stress (200 mM NaCl) significantly reduced root hydraulic conductivity (Lpr) and the accumulation of six of 10 HvPIP mRNAs. However, under relatively mild stress (100 mM NaCl), only a moderate reduction in Lpr with no significant difference in HvPIP mRNA levels was observed. Sorbitol-mediated osmotic stress equivalent to 100 and 200 mM NaCl induced nearly identical Lpr reductions in barley roots. Furthermore, the water transport activity in intact barley roots was suggested to require phosphorylation that is sensitive to a kinase inhibitor, staurosporine. HvPIP2s also showed water efflux activity in Xenopus oocytes, suggesting a potential ability to mediate water loss from cells under hypertonic conditions. Water transport via HvPIP aquaporins and the significance of reductions of Lpr in barley plants during salinity stress are discussed. [ABSTRACT FROM AUTHOR]

Abstract: Potassium ion (K+) plays vital roles in many aspects of cellular homeostasis including competing with sodium ion (Na+) during potassium starvation and salt stress. Therefore, one way to engineer plant cells with improved salt tolerance is to enhance K+ uptake activity of the cells, while keeping Na+ out during salt stress. Here, in search for Na+-insensitive K+ transporter for this purpose, bacterial expression system was used to characterize two K+ transporters, OsHAK2 and OsHAK5, isolated from rice (Oryza sativa cv. Nipponbare). The two OsHAK transporters are members of a KT/HAK/KUP transporter family, which is one of the major K+ transporter families in bacteria, fungi and plants. When expressed in an Escherichia coli K+ transport mutant strain LB2003, both OsHAK transporters rescued the growth defect in K+-limiting conditions by significantly increasing the K+ content of the cells. Under the condition with a large amount of extracellular Na+, we found that OsHAK5 functions as a Na+-insensitive K+ transporter, while OsHAK2 is sensitive to extracellular Na+ and exhibits higher Na+ over K+ transport activities. Moreover, constitutive expression of OsHAK5 in cultured-tobacco BY2 (Nicotiana tabacum cv. Bright Yellow 2) cells enhanced the accumulation of K+ but not Na+ in the cells during salt stress and conferred increased salt tolerance to the cells. Transient expression experiment indicated that OsHAK5 is localized to the plant plasma membrane. These results suggest that the plasma-membrane localized Na+ insensitive K+ transporters, similar to OsHAK5 identified here, could be used as a tool to enhance salt tolerance in plant cells. [Copyright &y& Elsevier]

Increasing soil salinity is a serious threat to agricultural productions worldwide in the 21st century. Several essential Na+ transporters such as AtNHX1 and AtSOS1 function in Na+ tolerance under salinity stress in plants. Recently, evidence for a new primary salt tolerance mechanism has been reported, which is mediated by a class of HKT transporters both in dicots such as Arabidopsis and monocot crops such as rice and wheat. Here we present a review on vital physiological functions of HKT transporters including AtHKT1;1 and OsHKT1;5 in preventing shoot Na+ over-accumulation by mediating Na+ exclusion from xylem vessels in the presence of a large amount of Na+ thereby protecting leaves from salinity stress. Findings of the HKT2 transporter sub-family are also updated in this review. Subjects regarding function and regulation of HKT transporters, which need to be elucidated in future research, are discussed. [ABSTRACT FROM AUTHOR]

The salinization of irrigated lands is increasingly detrimental to plant biomass production and agricultural productivity, as most plant species are sensitive to high concentrations of sodium (Na+), which causes combined Na+ toxicity and osmotic stress. Plants have multiple Na+-transport systems to circumvent Na+ toxicity. Essential physiological functions of major Na+ transporters and their mechanisms mediating salinity resistance have been identified in Arabidopsis , including the AtSOS1, AtNHX and AtHKT1;1 transporters. As we discuss here, recent studies have demonstrated that a class of xylem–parenchyma-expressed Na+-permeable plant HKT transporters represent a primary mechanism mediating salt tolerance and Na+ exclusion from leaves in Arabidopsis, and that major salt-tolerance quantitative trait loci in monocot crop plants are also based on this HKT-mediated mechanism. [Copyright &y& Elsevier]

Excessive accumulation of sodium in plants causes toxicity. No mutation that greatly diminishes sodium (Na+) influx into plant roots has been isolated. The OsHKT2;1 (previously named OsHKT1) transporter from rice functions as a relatively Na+-selective transporter in heterologous expression systems, but the in vivo function of OsHKT2;1 remains unknown. Here, we analyzed transposon-insertion rice lines disrupted in OsHKT2;1. Interestingly, three independent oshkt2;1-null alleles exhibited significantly reduced growth compared with wild-type plants under low Na+ and K+ starvation conditions. The mutant alleles accumulated less Na+, but not less K+, in roots and shoots. OsHKT2;1 was mainly expressed in the cortex and endodermis of roots. 22Na+ tracer influx experiments revealed that Na+ influx into oshkt2;1-null roots was dramatically reduced compared with wild-type plants. A rapid repression of OsHKT2;1-mediated Na+ influx and mRNA reduction were found when wild-type plants were exposed to 30 mM NaCl. These analyses demonstrate that Na+ can enhance growth of rice under K+ starvation conditions, and that OsHKT2;1 is the central transporter for nutritional Na+ uptake into K+-starved rice roots. [ABSTRACT FROM AUTHOR]

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 athkt1-1sos3 and athkt1-2sos3 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary It is thought that Na+ and K+ homeostasis is crucial for salt-tolerance in plants. To better understand the Na+ and K+ homeostasis in important crop rice (Oryza sativa L.), a cDNA homologous to the wheat HKT1 encoding K+-Na+ symporter was isolated from japonica rice, cv Nipponbare (Ni-OsHKT1). We also isolated two cDNAs homologous to Ni-OsHKT1 from salt-tolerant indica rice, cv Pokkali (Po-OsHKT1, Po-OsHKT2). The predicted amino acid sequence of Ni-OsHKT1 shares 100% identity with Po-OsHKT1 and 91% identity with Po-OsHKT2, and they are 66–67% identical to wheat HKT1. Low-K+ conditions (less than 3 mm) induced the expression of all three OsHKT genes in roots, but mRNA accumulation was inhibited by the presence of 30 mm Na+. We further characterized the ion-transport properties of OsHKT1 and OsHKT2 using an expression system in the heterologous cells, yeast and Xenopus oocytes. OsHKT2 was capable of completely rescuing a K+-uptake deficiency mutation in yeast, whereas OsHKT1 was not under K+-limiting conditions. When OsHKTs were expressed in Na+-sensitive yeast, OsHKT1 rendered the cells more Na+-sensitive than did OsHKT2 in high NaCl conditions. The electrophysiological experiments for OsHKT1 expressed in Xenopus oocytes revealed that external Na+, but not K+, shifted the reversal potential toward depolarization. In contrast, for OsHKT2 either Na+ or K+ in the external solution shifted the reversal potential toward depolarization under the mixed Na+ and K+ containing solutions. These results suggest that two isoforms of HKT transporters, a Na+ transporter (OsHKT1) and a Na+- and K+-coupled transporter (OsHKT2), may act harmoniously in the salt tolerant indica rice. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: 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. See also the Commentary on this article by Sanders, 225: 1047–1048. [ABSTRACT FROM AUTHOR]

Highlights: [•] Recent major advances have been made in identifying plant salt-tolerance mechanisms. [•] Potential targets for improvement are uptake, sensing, signaling, and detoxification. [•] Uptake and detoxification are mediated by membrane-bound transporters and channels. [•] Tolerance can be engineered by combining this knowledge with novel genetic techniques. [Copyright &y& Elsevier]

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, Mp HKT1 , 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 Mp HKT1. Complementary DNA (cDNA) of truncated Mp HKT1 (t- Mp HKT1) encoding the MpHKT_Δ596-812 protein was used to examine the functions of the C-terminal domain. Both Mp HKT1 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 Mp HKT1 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 Mp HKT1 or t-Mp HKT1 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-Mp HKT1 were consistently weaker than those with Mp HKT1. Together, these findings suggest that the hydrophilic C-terminal domain plays a unique role in the regulation of transport activity and ion selectivity of Mp HKT1. [ABSTRACT FROM AUTHOR]

Here we describe the development of a microarray-based mapping strategy to rapidly isolate deletion mutant genes. The presented approach is particularly useful for mapping mutant genes that are difficult to phenotype. This strategy uses masking bulk segregant analysis to mask unrelated deletions, thus allowing identification of target deletions by microarray hybridization of pooled genomic DNA from both WT and mutant F2 populations. Elemental profiling has proven to be a powerful tool for isolation of nutrient and toxic metal accumulation mutants in Arabidopsis. Using microarray mapping, a sodium overaccumulation mutant FN1148 was identified as having a 523-bp genomic deletion within the second exon and intron of the AtHKT1 gene. Further cosegregation, complementation, and comparative analyses among different salt-sensitive mutants confirmed that the deletion within the AtHKT1 gene is responsible for the sodium overaccumulation in shoots and leaf sodium sensitivity of the P41148 mutant. These results demonstrate that microarray-based cloning is an efficient and powerful tool to rapidly clone ion accumulation or other genetic deletion mutants that are otherwise difficult to phenotype for mapping, such as metabolic or cell signaling mutants. [ABSTRACT FROM AUTHOR]

The article offers information on 22nd New Phytologist Workshop on "Energy costs of salinity tolerance in crop plants," that was held in Adelaide, Australia, from 10–12 April 2018. It mentions about maintaining energy budget for salinity tolerance in crop plants; and examination of salinity tolerance within leaf sodium (Na+) concentration.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 Os HKT1;5 gene encoding a Na+-selective transporter. However, physiological roles of Os HKT1;5 in rice exposed to salt stress remain elusive, and no Os HKT1;5 gene disruption mutants have been characterized to date. In this study, we dissected two independent T- DNA insertional Os HKT1;5 mutants. Measurements of ion contents in tissues and 22Na+ tracer imaging experiments showed that loss-of-function of Os HKT1;5 in salt-stressed rice roots triggers massive Na+ accumulation in shoots. Salt stress-induced increases in the Os HKT1;5 transcript were observed in roots and basal stems, including basal nodes. Immuno-staining using an anti-Os HKT1;5 peptide antibody indicated that Os HKT1;5 is localized in cells adjacent to the xylem in roots. Additionally, direct introduction of 22Na+ tracer to leaf sheaths also demonstrated the involvement of Os HKT1;5 in xylem Na+ unloading in leaf sheaths. Furthermore, Os HKT1;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 22Na+ allocation in the blade of the developing immature leaf in the mutants, these results suggest a novel function of Os HKT1;5 in mediating Na+ exclusion in the phloem to prevent Na+ transfer to young leaf blades. Our findings further demonstrate that the function of Os HKT1;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. [ABSTRACT FROM AUTHOR]

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, 22Na+ 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Although aquaporins have been known to transport hydrogen peroxide (HO) across cell membranes, the HO-regulated expression patterns and the permeability of every family member of the plasma membrane intrinsic protein (PIP) toward HO have not been determined. This study investigates the HO-regulated expression levels of all plasma membrane aquaporins of Arabidopsis thaliana ( AtPIPs), and determines the permeability of every AtPIP for HO in yeast. Hydrogen peroxide treatment of Arabidopsis down-regulated the expression of AtPIP2 subfamily in roots but not in leaves, whereas the expression of AtPIP1 subfamily was not affected by HO treatment. The growth and survival of yeast cells that expressed AtPIP2;2, AtPIP2;4, AtPIP2;5, or AtPIP2;7 was reduced in the presence of HO, while the growth of yeast cells expressing any other AtPIP family member was not affected by HO. These results show that only certain isoforms of AtPIPs whose expression is regulated by HO treatment are permeable for HO in yeast cells, and suggest that the integrated regulation of aquaporin expression by HO and the capacity of individual aquaporin to transport HO are important for plant response to HO. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Examines the glycine residues in potassium channel-like selectivity filters. Structural basis of the transporters; Origin of glycine to serine mutations; Reduction of salt stress.

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. [ABSTRACT FROM AUTHOR]