Search

Intracellular potassium (K + ) homeostasis is fundamental to cell viability. In addition to channels, K + levels are maintained by various ion transporters. One major family is the proton-driven K + efflux transporters, which in gram-negative bacteria is important for detoxification and in plants is critical for efficient photosynthesis and growth. Despite their importance, the structure and molecular basis for K + -selectivity is poorly understood. Here, we report ~3.1 Å resolution cryo-EM structures of the Escherichia coli glutathione (GSH)-gated K + efflux transporter KefC in complex with AMP, AMP/GSH and an ion-binding variant. KefC forms a homodimer similar to the inward-facing conformation of Na + /H + antiporter NapA. By structural assignment of a coordinated K + ion, MD simulations, and SSM-based electrophysiology, we demonstrate how ion-binding in KefC is adapted for binding a dehydrated K + ion. KefC harbors C-terminal regulator of K + conductance (RCK) domains, as present in some bacterial K + -ion channels. The domain-swapped helices in the RCK domains bind AMP and GSH and they inhibit transport by directly interacting with the ion-transporter module. Taken together, we propose that KefC is activated by detachment of the RCK domains and that ion selectivity exploits the biophysical properties likewise adapted by K + -ion-channels., (© 2024. The Author(s).)

Variations in light intensity induce cytosol pH changes in photosynthetic tissues, providing a possible signal to adjust a variety of biochemical, physiological and developmental processes to the energy status of the cells. It was shown that these pH changes are partially due to the transport of protons in or out of the thylakoid lumen. However, the ion transporters in the chloroplast that transmit these pH changes to the cytosol are not known. KEA1 and KEA2 are K + /H + antiporters in the chloroplast inner envelope that adjust stromal pH in light-to-dark transitions. We previously determined that stromal pH is higher in kea1kea2 mutant cells. In this study, we now show that KEA1 and KEA2 are required to attenuate cytosol pH variations upon sudden light intensity changes in leaf mesophyll cells, showing they are important components of the light-modulated pH signalling module. The kea1kea2 mutant mesophyll cells also have a considerably less negative membrane potential. Membrane potential is dependent on the activity of the plasma membrane proton ATPase and is regulated by secondary ion transporters, mainly potassium channels in the plasma membrane. We did not find significant differences in the activity of the plasma membrane proton pump but found a strongly increased membrane permeability to protons, especially potassium, of the double mutant plasma membranes. Our results indicate that chloroplast envelope K + /H + antiporters not only affect chloroplast pH but also have a strong impact on cellular ion homeostasis and energization of the plasma membrane., (© 2024 The Author(s). Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Arabidopsis K + -efflux antiporter (KEA)1 and KEA2 are chloroplast inner envelope membrane K + /H + antiporters that play an important role in plastid development and seedling growth. However, the function of KEA1 and KEA2 during early seedling development is poorly understood. In this work, we found that in Arabidopsis, KEA1 and KEA2 mediated primary root growth by regulating photosynthesis and the ABA signaling pathway. Phenotypic analyses revealed that in the absence of sucrose, the primary root length of the kea1kea2 mutant was significantly shorter than that of the wild-type Columbia-0 (Col-0) plant. However, this phenotype could be remedied by the external application of sucrose. Meanwhile, HPLC-MS/MS results showed that in sucrose-free medium, ABA accumulation in the kea1kea2 mutant was considerably lower than that in Col-0. Transcriptome analysis revealed that many key genes involved in ABA signals were repressed in the kea1kea2 mutant. We concluded that KEA1 and KEA2 deficiency not only affected photosynthesis but was also involved in primary root growth likely through an ABA-dependent manner. This study confirmed the new function of KEA1 and KEA2 in affecting primary root growth., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Potassium (K) is important in the regulation of plant growth and development. It is the most abundant mineral element in kiwifruit, and its content increases during fruit ripening. However, how K + transporter works in kiwifruit postharvest maturation is not yet clear., Results: Here, 12 K + transporter KT/HAK/KUP genes, AcKUP1 ~ AcKUP12, were isolated from kiwifruit, and their phylogeny, genomic structure, chromosomal location, protein properties, conserved motifs and cis-acting elements were analysed. Transcription analysis revealed that AcKUP2 expression increased rapidly and was maintained at a high level during postharvest maturation, consistent with the trend of K content; AcKUP2 expression was induced by ethylene, suggesting that AcKUP2 might play a role in ripening. Fluorescence microscopy showed that AcKUP2 is localised in the plasma membrane. Cis-elements, including DER or ethylene response element (ERE) responsive to ethylene, were found in the AcKUP2 promoter sequence, and ethylene significantly enhanced the AcKUP2 promoter activity. Furthermore, we verified that AcERF15, an ethylene response factor, directly binds to the AcKUP2 promoter to promote its expression. Thus, AcKUP2 may be an important potassium transporter gene which involved in ethylene-regulated kiwifruit postharvest ripening., Conclusions: Therefore, our study establishes the first genome-wide analysis of the kiwifruit KT/HAK/KUP gene family and provides valuable information for understanding the function of the KT/HAK/KUP genes in kiwifruit postharvest ripening., (© 2022. The Author(s).)

The high-affinity K+ transporter HAK5 from Arabidopsis (Arabidopsis thaliana) is essential for K+ acquisition and plant growth at low micromolar K+ concentrations. Despite its functional relevance in plant nutrition, information about functional domains of HAK5 is scarce. Its activity is enhanced by phosphorylation via the AtCIPK23/AtCBL1-9 complex. Based on the recently published three-dimensionalstructure of the bacterial ortholog KimA from Bacillus subtilis, we have modeled AtHAK5 and, by a mutational approach, identified residues G67, Y70, G71, D72, D201, and E312 as essential for transporter function. According to the structural model, residues D72, D201, and E312 may bind K+, whereas residues G67, Y70, and G71 may shape the selective filter for K+, which resembles that of K+shaker-like channels. In addition, we show that phosphorylation of residue S35 by AtCIPK23 is required for reaching maximal transport activity. Serial deletions of the AtHAK5 C-terminus disclosed the presence of an autoinhibitory domain located between residues 571 and 633 together with an AtCIPK23-dependent activation domain downstream of position 633. Presumably, autoinhibition of AtHAK5 is counteracted by phosphorylation of S35 by AtCIPK23. Our results provide a molecular model for K+ transport and describe CIPK-CBL-mediated regulation of plant HAK transporters., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Potassium (K + ) exerts key physiological functions such as osmoregulation, stomatal movement, membrane transport, protein synthesis and photosynthesis among others. Previously, it was demonstrated in Arabidopsis thaliana that the loss of function of the chloroplast K + Efflux Antiporters KEA1 and KEA2, located in the inner envelope membrane, provokes inefficient photosynthesis. Therefore, the main goal of this study was to evaluate the potential impact of the loss of function of those cation transport systems in the metabolism of reactive oxygen and nitrogen species (ROS and RNS). Using 14-day-old seedlings from Arabidopsis double knock-out kea1kea2 mutants, ROS metabolism and NO content in roots and green cotyledons were studied at the biochemical level. The loss of function of AtKEA1 and AtKEA2 did not cause oxidative stress but it provoked an alteration of the ROS homeostasis affecting some ROS-generating enzymes. These included glycolate oxidase (GOX) and NADPH-dependent superoxide generation activity, enzymatic and non-enzymatic antioxidants and both NADP-isocitrate dehydrogenase and NADP-malic enzyme activities. NO content, analyzed by confocal laser scanning microscopy (CLSM), was negatively affected in both photosynthetic and non-photosynthetic organs in kea1kea2 mutant seedlings. Furthermore, the S-nitrosoglutathione reductase (GSNOR) protein expression and activity were downregulated in kea1kea2 mutants, whereas the tyrosine nitrated protein profile, analyzed by immunoblot, was unaffected but the relative expression of each immunoreactive band changed. Moreover, kea1kea2 mutants showed an increased photorespiratory pathway and stomata closure, thus promoting a higher resilience to drought stress. Data suggest that the chloroplast osmotic balance and integrity maintained by AtKEA1 and AtKEA2 are necessary to keep the balance of ROS/RNS metabolism. Moreover, these data open new questions about how endogenous NO generation might be affected by the K + /H + transport located in the chloroplasts., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Photosynthesis and carbon fixation depend critically on the regulation of pH in chloroplast compartments in the daylight and at night. While it is established that an alkaline stroma is required for carbon fixation, it is not known how alkaline stromal pH is formed, maintained or regulated. We tested whether two envelope transporters, AtKEA1 and AtKEA2, directly affected stromal pH in isolated Arabidopsis chloroplasts using the fluorescent probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). External K + -induced alkalinization of the stroma was observed in chloroplasts from wild-type (WT) plants but not from kea1kea2 mutants, suggesting that KEA1 and KEA2 mediate K + uptake/H + loss to modulate stromal pH. While light-stimulated alkalinization of the stroma was independent of KEA1 and KEA2, the rate of decay to neutral pH in the dark is delayed in kea1kea2 mutants. However, the dark-induced loss of a pH gradient across the thylakoid membrane was similar in WT and mutant chloroplasts. This indicates that proton influx from the cytosol mediated by envelope K + /H + antiporters contributes to adjustment of stromal pH upon light to dark transitions., (© 2020 The Authors New Phytologist © 2020 New Phytologist Foundation.)

In angiosperms, the NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport around PSI (CET). K + Efflux Antiporter3 (KEA3) is a putative thylakoid H + /K + antiporter and allows an increase in membrane potential at the expense of the ∆pH component of the proton motive force. In this study, we discovered that the chlororespiratory reduction2-1 ( crr2-1 ) mutation, which abolished NDH-dependent CET, enhanced the kea3-1 mutant phenotypes in Arabidopsis ( Arabidopsis thaliana ). The NDH complex pumps protons during CET, further enhancing ∆pH, but its physiological function has not been fully clarified. The observed effect only took place upon exposure to light of 110 µmol photons m -2 s -1 after overnight dark adaptation. We propose two distinct modes of NDH action. In the initial phase, within 1 min after the onset of actinic light, the NDH-dependent CET engages with KEA3 to enhance electron transport efficiency. In the subsequent phase, in which the ∆pH-dependent down-regulation of the electron transport is relaxed, the NDH complex engages with KEA3 to relax the large ∆pH formed during the initial phase. We observed a similar impact of the crr2-1 mutation in the genetic background of the PROTON GRADIENT REGULATION5 overexpression line, in which the size of ∆pH was enhanced. When photosynthesis was induced at 300 µmol photons m -2 s -1 , the contribution of KEA3 was negligible in the initial phase and the ∆pH-dependent down-regulation was not relaxed in the second phase. In the crr2-1 kea3-1 double mutant, the induction of CO 2 fixation was delayed after overnight dark adaptation., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Improving salinity tolerance in the most widely cultivated cereal, bread wheat (Triticum aestivum L.), is essential to increase grain yields on saline agricultural lands. A Portuguese landrace, Mocho de Espiga Branca accumulates up to sixfold greater leaf and sheath sodium (Na + ) than two Australian cultivars, Gladius and Scout, under salt stress in hydroponics. Despite high leaf and sheath Na + concentrations, Mocho de Espiga Branca maintained similar salinity tolerance compared to Gladius and Scout. A naturally occurring single nucleotide substitution was identified in the gene encoding a major Na + transporter TaHKT1;5-D in Mocho de Espiga Branca, which resulted in a L190P amino acid residue variation. This variant prevents Mocho de Espiga Branca from retrieving Na + from the root xylem leading to a high shoot Na + concentration. The identification of the tissue-tolerant Mocho de Espiga Branca will accelerate the development of more elite salt-tolerant bread wheat cultivars., (© 2020 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

The high-affinity K+ transporter HAK5 is the major contributor to root K+ uptake from dilute solutions in K+-starved Arabidopsis plants. Its functionality is tightly regulated and its activity is enhanced under K+ starvation by the transcriptional induction of the AtHAK5 gene, and by the activation of the transporter via the AtCBL1-AtCIPK23 complex. In the present study, the 26 members of the Arabidopsis CIPK protein kinase family were screened in yeast for their capacity to activate HAK5-mediated K+ uptake. Among them, AtCIPK1 was the most efficient activator of AtHAK5. In addition, AtCIPK9, previously reported to participate in K+ homeostasis, also activated the transporter. In roots, the genes encoding AtCIPK1 and AtCIPK9 were induced by K+ deprivation and atcipk1 and atcipk9 Arabidopsis KO mutants showed a reduced AtHAK5-mediated Rb+ uptake. Activation of AtHAK5 by AtCIPK1 did not occur under hyperosmotic stress conditions, where AtCIPK1 function has been shown to be required to maintain plant growth. Taken together, our data contribute to the identification of the complex regulatory networks that control the high-affinity K+ transporter AtHAK5 and root K+ uptake., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The plastid potassium cation efflux antiporters (KEAs) are important for chloroplast function, development, and photosynthesis. To understand their regulation at the protein level is therefore of fundamental importance. Prior studies have focused on the regulatory K + transport and NAD-binding (KTN) domain in the C-terminus of the thylakoid carrier KEA3 but the localization of this domain remains unclear. While all three plastid KEA members are highly conserved in their transmembrane region and the C-terminal KTN domain, only the inner envelope KEA family members KEA1 and KEA2 carry a long soluble N-terminus. Interestingly, this region is acetylated at lysine 168 by the stromal acetyltransferase enzyme NSI. If an odd number of transmembrane domains existed for inner envelope KEAs, as it was suggested for all three plastid KEA carriers, regulatory domains and consequently protein regulation would occur on opposing sides of the inner envelope. In this study we therefore set out to investigate the topology of inner envelope KEA proteins. Using a newly designed antibody specific to the envelope KEA1 N-terminus and transgenic Arabidopsis plants expressing a C-terminal KEA1-YFP fusion protein, we show that both, the N-terminal and C-terminal, regulatory domains of KEA1 reside in the chloroplast stroma and not in the intermembrane space. Considering the high homology between KEA1 and KEA2, we therefore reason that envelope KEAs must consist of an even number of transmembrane domains.

The composition of the thylakoid proton motive force (pmf) is regulated by thylakoid ion transport. Passive ion channels in the thylakoid membrane dissipate the membrane potential (Δψ) component to allow for a higher fraction of pmf stored as a proton concentration gradient (ΔpH). K + /H + antiport across the thylakoid membrane via K+ EXCHANGE ANTIPORTER3 (KEA3) instead reduces the ΔpH fraction of the pmf. Thereby, KEA3 decreases nonphotochemical quenching (NPQ), thus allowing for higher light use efficiency, which is particularly important during transitions from high to low light. Here, we show that in the background of the Arabidopsis ( Arabidopsis thaliana ) chloroplast (cp)ATP synthase assembly mutant cgl160 , with decreased cpATP synthase activity and increased pmf amplitude, KEA3 plays an important role for photosynthesis and plant growth under steady-state conditions. By comparing cgl160 single with cgl160 kea3 double mutants, we demonstrate that in the cgl160 background loss of KEA3 causes a strong growth penalty. This is due to a reduced photosynthetic capacity of cgl160 kea3 mutants, as these plants have a lower lumenal pH than cgl160 mutants, and thus show substantially increased pH-dependent NPQ and decreased electron transport through the cytochrome b 6 f complex. Overexpression of KEA3 in the cgl160 background reduces pH-dependent NPQ and increases photosystem II efficiency. Taken together, our data provide evidence that under conditions where cpATP synthase activity is low, a KEA3-dependent reduction of ΔpH benefits photosynthesis and growth., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Main Conclusion: There is a need to integrate conceptual framework based on the current understanding of salt stress responses with different approaches for manipulating and improving salt tolerance in crop plants. Soil salinity exerts significant constraints on global crop production, posing a serious challenge for plant breeders and biotechnologists. The classical transgenic approach for enhancing salinity tolerance in plants revolves by boosting endogenous defence mechanisms, often via a single-gene approach, and usually involves the enhanced synthesis of compatible osmolytes, antioxidants, polyamines, maintenance of hormone homeostasis, modification of transporters and/or regulatory proteins, including transcription factors and alternative splicing events. Occasionally, genetic manipulation of regulatory proteins or phytohormone levels confers salinity tolerance, but all these may cause undesired reduction in plant growth and/or yields. In this review, we present and evaluate novel and cutting-edge approaches for engineering salt tolerance in crop plants. First, we cover recent findings regarding the importance of regulatory proteins and transporters, and how they can be used to enhance salt tolerance in crop plants. We also evaluate the importance of halobiomes as a reservoir of genes that can be used for engineering salt tolerance in glycophytic crops. Additionally, the role of microRNAs as critical post-transcriptional regulators in plant adaptive responses to salt stress is reviewed and their use for engineering salt-tolerant crop plants is critically assessed. The potentials of alternative splicing mechanisms and targeted gene-editing technologies in understanding plant salt stress responses and developing salt-tolerant crop plants are also discussed.

Root cells take up K + from the soil solution, and a fraction of the absorbed K + is translocated to the shoot after being loaded into xylem vessels. K + uptake and translocation are spatially separated processes. K + uptake occurs in the cortex and epidermis whereas K + translocation starts at the stele. Both uptake and translocation processes are expected to be linked, but the connection between them is not well characterized. Here, we studied K + uptake and translocation using Rb + as a tracer in wild-type Arabidopsis thaliana and in T-DNA insertion mutants in the K + uptake or translocation systems. The relative amount of translocated Rb + to the shoot was positively correlated with net Rb + uptake rates, and the akt1 athak5 T-DNA mutant plants were more efficient in their allocation of Rb + to shoots. Moreover, a mutation of SKOR and a reduced plant transpiration prevented the full upregulation of AtHAK5 gene expression and Rb + uptake in K + -starved plants. Lastly, Rb + was found to be retrieved from root xylem vessels, with AKT1 playing a significant role in K + -sufficient plants. Overall, our results suggest that K + uptake and translocation are tightly coordinated via signals that regulate the expression of K + transport systems., (© 2019 John Wiley & Sons Ltd.)

Arabidopsis thaliana contains the putative K + efflux transporters KEA1-KEA6, similar to KefB and KefC of Escherichia coli. KEA1-KEA3 are involved in the regulation of photosynthetic electron transport and chloroplast development. KEA4-KEA6 mediate pH regulation of the endomembrane network during salinity stress. However, the ion transport activities of KEA1-KEA6 have not been directly characterized. In this study, we used an E. coli expression system to examine KEA activity. KEA1-KEA3 and KEA5 showed bi-directional K + transport activity, whereas KEA4 and KEA6 functioned as a K + uptake system. The thylakoid membrane-localized Na + /H + antiporter NhaS3 from the model cyanobacterium Synechocystis is the closest homolog of KEA3. Changing the putative Na + /H + selective site of KEA3 (Gln-Asp) to that of NhaS3 (Asp-Asp) did not alter the ion selectivity without loss of K + transport activity. The first residue in the conserved motif was not a determinant for K + or Na + selectivity. Deletion of the possible nucleotide-binding KTN domain from KEA3 lowered K + transport activity, indicating that the KTN domain was important for this function. The KEA3-G422R mutation discovered in the Arabidopsis dpgr mutant increased K + transport activity, consistent with the mutant phenotype. These results indicate that Arabidopsis KEA1-KEA6 act as K + transport systems, and support the interpretation that KEA3 promotes dissipation of ΔpH in the thylakoid membrane.

The absence of high-affinity potassium uptake in Candida glabrata, the consequence of the deletion of the TRK1 gene encoding the sole potassium-specific transporter, has a pleiotropic effect. Here, we show that in addition to changes in basic physiological parameters (e.g., membrane potential and intracellular pH) and decreased tolerance to various cell stresses, the loss of high affinity potassium uptake also alters cell-surface properties, such as an increased hydrophobicity and adherence capacity. The loss of an efficient potassium uptake system results in diminished virulence as assessed by two insect host models, Drosophila melanogaster and Galleria mellonella, and experiments with macrophages. Macrophages kill trk1Δ cells more effectively than wild type cells. Consistently, macrophages accrue less damage when co-cultured with trk1Δ mutant cells compared to wild-type cells. We further show that low levels of potassium in the environment increase the adherence of C. glabrata cells to polystyrene and the propensity of C. glabrata cells to form biofilms.

In most halophiles, K + generally acts as a major osmotic solute for osmotic adjustment and pH homeostasis. However, strains also need to extrude excessive intracellular K + to avoid its toxicity. In the halotolerant and alkaliphilic Halomonas sp. Y2, an Na + -induced K + extrusion process was observed when the cells were confronted with high extracellular K + pressure and supplementation by millimolar Na + ions. Among three mechanosensitive channels (KefA) and two K + /H + antiporters founded in the genome of the strain, ke1 displayed around 3-5-fold upregulation to ion stress at pH 8.0, while much higher upregulation of Ha-mrp was observed at pH 10.0. Compared to the growth of wild-type Halomonas sp. Y2, deletion of these genes from the strain resulted in different growth phenotypes in response to the osmotic pressure of potassium. In combination with the transcriptional response of these genes, we proposed that the KefA channel of Ke1 is the main contributor to the K + -extrusion process under weak alkalinity, while the Mrp system plays critical roles in alleviating K + contents at high pH. The combination of these strategies allows Halomonas sp. Y2 to grow over a range of extracellular pH and ion concentrations, and thus protect cells under high osmotic stress conditions.

Since their discovery, twenty years ago, KT-HAK-KUP transporters have become a keystone to understand how alkali cation fluxes are controlled in major land-dwelling photosynthetic organisms. In this review we focus on their discovery, phylogeny, and functions, as well as the regulation of its canonical member, AtHAK5. We also address issues related to structure-function studies, and the technological possibilities opened up by recent findings. Available evidence suggests that this family of transporters underwent an early divergence into major groups following the conquest of land by embryophytes. KT-HAK-KUPs are necessary to accomplish several major developmental and growth processes, as well as to ensure plant responses to environmental injuries. Although the primary function of these transporters is to mediate potassium (K + ) fluxes, some of them can also mediate sodium (Na + ) and cesium (Cs + ) transport, and contribute to maintenance of K + (and Na + ) homeostasis in different plant tissues. In addition, there is evidence for a role of some members of this family in auxin movement and in adenylate cyclase activity. Recent research, focusing on the regulation of the canonical member of this family, AtHAK5, revealed the existence of a complex network that involves transcriptional and post-transcriptional phenomena which control the enhancement of AtHAK5-mediated K + uptake when Arabidopsis thaliana plants are faced with low K + supply. In spite of the formidable advances made since their discovery, important subjects remain to be elucidated to gain a more complete knowledge of the roles and regulation of KT-HAK-KUPs, as well as to improve their use for innovative procedures in crop breeding., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Plants remodel their cells through the dynamic endomembrane system. Intracellular pH is important for membrane trafficking, but the determinants of pH homeostasis are poorly defined in plants. Electrogenic proton (H + ) pumps depend on counter-ion fluxes to establish transmembrane pH gradients at the plasma membrane and endomembranes. Vacuolar-type H + -ATPase-mediated acidification of the trans-Golgi network is crucial for secretion and membrane recycling. Pump and counter-ion fluxes are unlikely to fine-tune pH; rather, alkali cation/H + antiporters, which can alter pH and/or cation homeostasis locally and transiently, are prime candidates. Plants have a large family of predicted cation/H + exchangers (CHX) of obscure function, in addition to the well-studied K + (Na + )/H + exchangers (NHX). Here, we review the regulation of cytosolic and vacuolar pH, highlighting the similarities and distinctions of NHX and CHX members. In planta, alkalinization of the trans-Golgi network or vacuole by NHXs promotes membrane trafficking, endocytosis, cell expansion, and growth. CHXs localize to endomembranes and/or the plasma membrane and contribute to male fertility, pollen tube guidance, pollen wall construction, stomatal opening, and, in soybean ( Glycine max ), tolerance to salt stress. Three-dimensional structural models and mutagenesis of Arabidopsis ( Arabidopsis thaliana ) genes have allowed us to infer that AtCHX17 and AtNHX1 share a global architecture and a translocation core like bacterial Na + /H + antiporters. Yet, the presence of distinct residues suggests that some CHXs differ from NHXs in pH sensing and electrogenicity. How H + pumps, counter-ion fluxes, and cation/H + antiporters are linked with signaling and membrane trafficking to remodel membranes and cell walls awaits further investigation., (© 2018 American Society of Plant Biologists. All rights reserved.)

Cation antiporters of the NHX family are widely regarded as determinants of salt tolerance due to their capacity to drive sodium (Na) and sequester it into vacuoles. Recent work shows, however, that NHX transporters are primarily involved in vacuolar potassium (K) storage. Over-expression of the K/H antiporter AtNHX1 in tomato increases K accumulation into vacuoles and plant sensitivity to K deprivation. Here we show that the appearance of early leaf symptoms of K deficiency was associated with higher concentration of polyamines. Transgenic roots exhibited a greater sensitivity than shoots to K deprivation with changes in the composition of the free amino acids pool, total sugars and organic acids. Concentrations of amides (glutamine), amino acids (arginine) and sugars significantly increased in root, together with a reduction in malate and succinate concentrations. The concentration of pyruvate and the activity of pyruvate kinase were greater in the transgenic roots before K withdrawal although both parameters were depressed by K deprivation and approached wild-type levels. In the longer term, the over-expression of the NHX1 antiporter affected root growth and biomass partitioning (shoot/root ratio). Greater ethylene release produced longer stem internodes and leaf curling in the transgenic line. Our data show that enhanced sequestration of K by the NHX antiporter in the vacuoles altered cellular K homeostasis and had deeper physiological consequences than expected. Early metabolic changes lead later on to profound morphological and physiological adjustments resulting eventually in the loss of nutrient use efficiency., (© 2017 Scandinavian Plant Physiology Society.)

Halomonas alkalicola CICC 11012s is an alkaliphilic and halotolerant bacterium isolated from a soap-making tank (pH > 10) from a household-product plant. This strain can propagate at pH 12.5, which is fatal to most bacteria. Genomic analysis revealed that the genome size was 3,511,738 bp and contained 3295 protein-coding genes, including a complete cell wall and plasma membrane lipid biosynthesis pathway. Furthermore, four putative Na + /H + and K + /H + antiporter genes, or gene clusters, designated as HaNhaD, HaNhaP, HaMrp and HaPha, were identified within the genome. Heterologous expression of these genes in antiporter-deficient Escherichia coli indicated that HaNhaD, an Na + /H + antiporter, played a dominant role in Na + tolerance and pH homeostasis in acidic, neutral and alkaline environments. In addition, HaMrp exhibited Na + tolerance; however, it functioned mainly in alkaline conditions. Both HaNhaP and HaPha were identified as K + /H + antiporters that played an important role in high alkalinity and salinity. In summary, genome analysis and heterologous expression experiments demonstrated that a complete set of adaptive strategies have been developed by the double extremophilic strain CICC 11012s in response to alkalinity and salinity. Specifically, four antiporters exhibiting different physiological roles for different situations worked together to support the strain in harsh surroundings.

Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding.

KEA3 is a thylakoid membrane localized K + /H + antiporter that regulates photosynthesis by modulating two components of proton motive force (pmf), the proton gradient (∆pH) and the electric potential (∆ψ). We identified a mutant allele of KEA3, disturbed proton gradient regulation (dpgr) based on its reduced non-photochemical quenching (NPQ) in artificial (CO 2 -free with low O 2 ) air. This phenotype was enhanced in the mutant backgrounds of PSI cyclic electron transport (pgr5 and crr2-1). In ambient air, reduced NPQ was observed during induction of photosynthesis in dpgr, the phenotype that was enhanced after overnight dark adaptation. In contrast, the knockout allele of kea3-1 exhibited a high-NPQ phenotype during steady state in ambient air. Consistent with this kea3-1 phenotype in ambient air, the membrane topology of KEA3 indicated a proton efflux from the thylakoid lumen to the stroma. The dpgr heterozygotes showed a semidominant and dominant phenotype in artificial and ambient air, respectively. In dpgr, the protein level of KEA3 was unaffected but the downregulation of its activity was probably disturbed. Our findings suggest that fine regulation of KEA3 activity is necessary for optimizing photosynthesis., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

Halomonas sp. Y2 is a halotolerant alkaliphilic strain from Na + -rich pulp mill wastewater with high alkalinity (pH >11.0). Transcriptome analysis of this isolate revealed this strain may use various transport systems for pH homeostasis. In particular, the genes encoding four putative Na + /H + antiporters were differentially expressed upon acidic or alkaline conditions. Further evidence, from heterologous expression and mutant studies, suggested that Halomonas sp. Y2 employs its Na + /H + antiporters in a labor division way to deal with saline and alkaline environments. Ha-NhaD2 displayed robust Na + (Li + ) resistance and high transport activities in Escherichia coli; a ΔHa-nhaD2 mutant exhibited growth inhibition at high Na + (Li + ) concentrations at pH values of 6.2, 8.0, and 10.0, suggesting its physiological role in osmotic homeostasis. In contrast, Ha-NhaD1 showed much weaker activities in ion exporting and pH homeostasis. Ha-Mrp displayed a combination of properties similar to those of Mrp transporters from some Bacillus alkaliphiles and neutrophiles. This conferred obvious Na + (Li + , K + ) resistance in E. coli-deficient strains, as those ion transport spectra of some neutrophil Mrp antiporters. Conversely, similar to the Bacillus alkaliphiles, Ha-Mrp showed central roles in the pH homeostasis of Halomonas sp. Y2. An Ha-mrp-disrupted mutant was seriously inhibited by high concentrations of Na + (Li + , K + ) but only under alkaline conditions. Ha-NhaP was determined to be a K + /H + antiporter and shown to confer strong K + resistance both at acidic and alkaline stresses., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Potassium (K + ) plays crucial roles in plant growth and development. In natural environments, K + availability in soils is relatively low and fluctuating. Transcriptional regulation of K + transporter genes is one of the most important mechanisms in the plant's response to K + deficiency. In this study, we demonstrated that the transcription factor ARF2 (Auxin Response Factor 2) modulates the expression of the K + transporter gene HAK5 (High Affinity K + transporter 5) in Arabidopsis thaliana The arf2 mutant plants showed a tolerant phenotype similar to the HAK5-overexpressing lines on low-K + medium, whose primary root lengths were longer than those of wild-type plants. High-affinity K + uptake was significantly increased in these plants. ARF2-overexpressing lines and the hak5 mutant were both sensitive to low-K + stress. Disruption of HAK5 in the arf2 mutant abolished the low-K + -tolerant phenotype of arf2 As a transcriptional repressor, ARF2 directly bound to the HAK5 promoter and repressed HAK5 expression under K + sufficient conditions. ARF2 can be phosphorylated after low-K + treatment, which abolished its DNA binding activity to the HAK5 promoter and relieved the inhibition on HAK5 transcription. Therefore, HAK5 transcript could be induced, and HAK5-mediated high-affinity K + uptake was enhanced under K + deficient conditions. The presented results demonstrate that ARF2 plays important roles in the response to external K + supply in Arabidopsis and regulates HAK5 transcription accordingly., (© 2016 American Society of Plant Biologists. All rights reserved.)

It is well established that thylakoid membranes of chloroplasts convert light energy into chemical energy, yet the development of chloroplast and thylakoid membranes is poorly understood. Loss of function of the two envelope K(+)/H(+) antiporters AtKEA1 and AtKEA2 was shown previously to have negative effects on the efficiency of photosynthesis and plant growth; however, the molecular basis remained unclear. Here, we tested whether the previously described phenotypes of double mutant kea1kea2 plants are due in part to defects during early chloroplast development in Arabidopsis (Arabidopsis thaliana). We show that impaired growth and pigmentation is particularly evident in young expanding leaves of kea1kea2 mutants. In proliferating leaf zones, chloroplasts contain much lower amounts of photosynthetic complexes and chlorophyll. Strikingly, AtKEA1 and AtKEA2 proteins accumulate to high amounts in small and dividing plastids, where they are specifically localized to the two caps of the organelle separated by the fission plane. The unusually long amino-terminal domain of 550 residues that precedes the antiport domain appears to tether the full-length AtKEA2 protein to the two caps. Finally, we show that the double mutant contains 30% fewer chloroplasts per cell. Together, these results show that AtKEA1 and AtKEA2 transporters in specific microdomains of the inner envelope link local osmotic, ionic, and pH homeostasis to plastid division and thylakoid membrane formation., (© 2016 American Society of Plant Biologists. All rights reserved.)

Plants experience hyperosmotic stress when faced with saline soils and possibly with drought stress, but it is currently unclear how plant roots perceive this stress in an environment of dynamic water availabilities. Hyperosmotic stress induces a rapid rise in intracellular Ca(2+) concentrations ([Ca(2+)]i) in plants, and this Ca(2+) response may reflect the activities of osmo-sensory components. Here, we find in the reference plant Arabidopsis thaliana that the rapid hyperosmotic-induced Ca(2+) response exhibited enhanced response magnitudes after preexposure to an intermediate hyperosmotic stress. We term this phenomenon "osmo-sensory potentiation." The initial sensing and potentiation occurred in intact plants as well as in roots. Having established a quantitative understanding of wild-type responses, we investigated effects of pharmacological inhibitors and candidate channel/transporter mutants. Quintuple mechano-sensitive channels of small conductance-like (MSL) plasma membrane-targeted channel mutants as well as double mid1-complementing activity (MCA) channel mutants did not affect the response. Interestingly, however, double mutations in the plastid K(+) exchange antiporter (KEA) transporters kea1kea2 and a single mutation that does not visibly affect chloroplast structure, kea3, impaired the rapid hyperosmotic-induced Ca(2+) responses. These mutations did not significantly affect sensory potentiation of the response. These findings suggest that plastids may play an important role in early steps mediating the response to hyperosmotic stimuli. Together, these findings demonstrate that the plant osmo-sensory components necessary to generate rapid osmotic-induced Ca(2+) responses remain responsive under varying osmolarities, endowing plants with the ability to perceive the dynamic intensities of water limitation imposed by osmotic stress., Competing Interests: The authors declare no conflict of interest.

Calcium is important for chloroplast, not only in its photosynthetic but also nonphotosynthetic functions. Multiple Ca(2+)/H(+) transporters and channels have been described and studied in the plasma membrane and organelle membranes of plant cells; however, the molecular identity and physiological roles of chloroplast Ca(2+)/H(+) antiporters have remained unknown. Here we report the identification and characterization of a member of the UPF0016 family, CCHA1 (a chloroplast-localized potential Ca(2+)/H(+) antiporter), in Arabidopsis thaliana. We observed that the ccha1 mutant plants developed pale green leaves and showed severely stunted growth along with impaired photosystem II (PSII) function. CCHA1 localizes to the chloroplasts, and the levels of the PSII core subunits and the oxygen-evolving complex were significantly decreased in the ccha1 mutants compared with the wild type. In high Ca(2+) concentrations, Arabidopsis CCHA1 partially rescued the growth defect of yeast gdt1Δ null mutant, which is defective in a Ca(2+)/H(+) antiporter. The ccha1 mutant plants also showed significant sensitivity to high concentrations of CaCl2 and MnCl2, as well as variation in pH. Taken these results together, we propose that CCHA1 might encode a putative chloroplast-localized Ca(2+)/H(+) antiporter with critical functions in the regulation of PSII and in chloroplast Ca(2+) and pH homeostasis in Arabidopsis., (Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.)

During sexual reproduction of flowering plants, the pollen tube grows fast and over a long distance within the pistil to deliver two sperms for double fertilization. Growing plant cells need to communicate constantly with external stimuli as well as monitor changes in surface tension of the cell wall and plasma membrane to coordinate these signals and internal growth machinery; however, the underlying mechanisms remain largely unknown. Here we show that the rice member of plant-specific receptor-like kinase CrRLK1Ls subfamily, Ruptured Pollen tube (RUPO), is specifically expressed in rice pollen. RUPO localizes to the apical plasma membrane and vesicle of pollen tubes and is required for male gamete transmission. K+ levels were greater in pollen of homozygous CRISPR-knockout lines than wild-type plants, and pollen tubes burst shortly after germination. We reveal the interaction of RUPO with high-affinity potassium transporters. Phosphorylation of RUPO established and dephosphorylation abolished the interaction. These results have revealed the receptor-like kinase as a regulator of high-affinity potassium transporters via phosphorylation-dependent interaction, and demonstrated a novel receptor-like kinase signaling pathway that mediates K+ homeostasis required for pollen tube growth and integrity.

Molecular evolution of large protein families in closely related species can provide useful insights on structural functional relationships. Phylogenetic analysis of the grass-specific group II HKT genes identified two distinct subfamilies, I and II. Subfamily II was represented in all species, whereas subfamily I was identified only in the small grain cereals and possibly originated from an ancestral gene duplication post divergence from the coarse grain cereal lineage. The core protein structures were highly analogous despite there being no more than 58% amino acid identity between members of the two subfamilies. Distinctly variable regions in known functional domains, however, indicated functional divergence of the two subfamilies. The subsets of codons residing external to known functional domains predicted signatures of positive Darwinian selection potentially identifying new domains of functional divergence and providing new insights on the structural function and relationships between protein members of the two subfamilies.

Crop canopies create environments of highly fluctuating light intensities. In such environments, photoprotective mechanisms and their relaxation kinetics have been hypothesized to limit photosynthetic efficiency and therefore crop yield potential. Here, we show that overexpression of the Arabidopsis thylakoid K + /H + antiporter KEA3 accelerates the relaxation of photoprotective energy-dependent quenching after transitions from high to low light in Arabidopsis and tobacco. This, in turn, enhances PSII quantum efficiency in both organisms, supporting that in wild-type plants, residual light energy quenching following a high to low light transition represents a limitation to photosynthetic efficiency in fluctuating light. This finding underscores the potential of accelerating quenching relaxation as a building block for improving photosynthetic efficiency in the field. Additionally, by overexpressing natural KEA3 variants with modification to the C-terminus, we show that KEA3 activity is regulated by a mechanism involving its lumen-localized C-terminus, which lowers KEA3 activity in high light. This regulatory mechanism fine-tunes the balance between photoprotective energy dissipation in high light and maximum quantum yield in low light, likely to be critical for efficient photosynthesis in fluctuating light conditions., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Acquisition and distribution of metal ions support a number of biological processes. Here we show that respiratory growth of and iron acquisition by the yeast Saccharomyces cerevisiae relies on potassium (K(+)) compartmentalization to the trans-Golgi network via Kha1p, a K(+)/H(+) exchanger. K(+) in the trans-Golgi network facilitates binding of copper to the Fet3p multi-copper ferroxidase. The effect of K(+) is not dependent on stable binding with Fet3p or alteration of the characteristics of the secretory pathway. The data suggest that K(+) acts as a chemical factor in Fet3p maturation, a role similar to that of cations in folding of nucleic acids. Up-regulation of KHA1 gene in response to iron limitation via iron-specific transcription factors indicates that K(+) compartmentalization is linked to cellular iron homeostasis. Our study reveals a novel functional role of K(+) in the binding of copper to apoFet3p and identifies a K(+)/H(+) exchanger at the secretory pathway as a new molecular factor associated with iron uptake in yeast., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Although the HKT transporter genes ascertain some of the key determinants of crop salt tolerance mechanisms, the diversity and functional role of group II HKT genes are not clearly understood in bread wheat. The advanced knowledge on rice HKT and whole genome sequence was, therefore, used in comparative gene analysis to identify orthologous wheat group II HKT genes and their role in trait variation under different saline environments., Results: The four group II HKTs in rice identified two orthologous gene families from bread wheat, including the known TaHKT2;1 gene family and a new distinctly different gene family designated as TaHKT2;2. A single copy of TaHKT2;2 was found on each homeologous chromosome arm 7AL, 7BL and 7DL and each gene was expressed in leaf blade, sheath and root tissues under non-stressed and at 200 mM salt stressed conditions. The proteins encoded by genes of the TaHKT2;2 family revealed more than 93% amino acid sequence identity but ≤52% amino acid identity compared to the proteins encoded by TaHKT2;1 family. Specifically, variations in known critical domains predicted functional differences between the two protein families. Similar to orthologous rice genes on chromosome 6L, TaHKT2;1 and TaHKT2;2 genes were located approximately 3 kb apart on wheat chromosomes 7AL, 7BL and 7DL, forming a static syntenic block in the two species. The chromosomal region on 7AL containing TaHKT2;1 7AL-1 co-located with QTL for shoot Na(+) concentration and yield in some saline environments., Conclusion: The differences in copy number, genes sequences and encoded proteins between TaHKT2;2 homeologous genes and other group II HKT gene families within and across species likely reflect functional diversity for ion selectivity and transport in plants. Evidence indicated that neither TaHKT2;2 nor TaHKT2;1 were associated with primary root Na(+) uptake but TaHKT2;1 may be associated with trait variation for Na(+) exclusion and yield in some but not all saline environments.

Plant growth and development requires efficient acquisition of essential elements. Potassium (K(+)) is an important macronutrient present in the soil solution at a wide range of concentrations. Regulation of the K(+) uptake systems in the roots is essential to secure K(+) supply. It has been shown in Arabidopsis (Arabidopsis thaliana) that when the external K(+) concentration is very low (<10 µm), K(+) nutrition depends exclusively on the high-affinity K(+) transporter5 (HAK5). Low-K(+)-induced transcriptional activation of the gene encoding HAK5 has been previously reported. Here, we show the posttranscriptional regulation of HAK5 transport activity by phosphorylation. Expression in a heterologous system showed that the Ca(2+) sensors calcineurin B-like (CBL1), CBL8, CBL9, and CBL10, together with CBL-interacting protein kinase23 (CIPK23), activated HAK5 in vivo. This activation produced an increase in the affinity and the Vmax of K(+) transport. In vitro experiments show that the N terminus of HAK5 is phosphorylated by CIPK23. This supports the idea that phosphorylation of HAK5 induces a conformational change that increases its affinity for K(+). Experiments of K(+) (Rb(+)) uptake and growth measurements in low-K(+) medium with Arabidopsis single mutants hak5, akt1, and cipk23, double mutants hak5 akt1, hak5 cipk23, and akt1 cipk23, and the triple mutant hak5 akt1 cipk23 confirmed the regulatory role of CIPK23 in planta., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Plants are exposed to various environmental stresses during their life cycle such as salt, drought and cold. Natural variation mediated plant growth adaptation has been employed as an effective approach in response to the diverse environmental cues such as salt stress. However, the molecular mechanism underlying this process is not well understood. In the present study, a collection of 82 Arabidopsis thaliana accessions (ecotypes) was screened with a view to identify variation for salinity tolerance. Seven accessions showed a higher level of tolerance than Col-0. The young seedlings of the tolerant accessions demonstrated a higher K(+) content and a lower Na(+)/K(+) ratio when exposed to salinity stress, but its Na(+) content was the same as that of Col-0. The K(+) transporter genes AtHAK5, AtCHX17 and AtKUP1 were up-regulated significantly in almost all the tolerant accessions, even in the absence of salinity stress. There was little genetic variation or positive transcriptional variation between the selections and Col-0 with respect to Na+-related transporter genes, as AtSOS genes, AtNHX1 and AtHKT1;1. In addition, under salinity stress, these selections accumulated higher compatible solutes and lower reactive oxygen species than did Col-0. Taken together, our results showed that natural variation in salinity tolerance of Arabidopsis seems to have been achieved by the strong capacity of K(+) retention.

Iron (Fe) is an essential microelement but is highly toxic when in excess. The response of plant roots to Fe toxicity and the nature of the regulatory pathways engaged are poorly understood. Here, we examined the response to excess Fe exposure in Arabidopsis wild type and ethylene mutants with a focus on primary root growth and the role of ethylene. We showed that excess Fe arrested primary root growth by decreasing both cell elongation and division, and principally resulteds from direct external Fe contact at the root tip. Pronounced ethylene, but not abscisic acid, evolution was associated with excess Fe exposure. Ethylene antagonists intensified root growth inhibition in the wild type, while the inhibition was significantly reduced in ethylene-overproduction mutants. We showed that ethylene plays a positive role in tissue Fe homeostasis, even in the absence of iron-plaque formation. Ethylene reduced Fe concentrations in the stele, xylem, and shoot. Furthermore, ethylene increased the expression of genes encoding Fe-sequestering ferritins. Additionally, ethylene significantly enhanced root K(+) status and upregulated K(+)-transporter (HAK5) expression. Our findings highlight the important role of ethylene in tissue Fe and K homeostasis and primary root growth under Fe stress in Arabidopsis., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Trichoblasts of trh1 plants form root-hair initiation sites that fail to undergo tip growth resulting in a tiny root-hair phenotype. TRH1 belongs to Arabidopsis KT/KUP/HAK potassium transporter family controlling root-hair growth and gravitropism. Double mutant combinations between trh1 and root-hair mutants affecting cell fate or root-hair initiation exhibited additive phenotypes, suggesting that TRH1 acts independently and developmentally downstream of root-hair initiation. Bimolecular Fluorescence Complementation (BiFC), upon TRH1-YFP(C) and TRH1-YFP(N) co-transformation into tobacco epidermal cells, led to fluorescence emission indicative of TRH1 subunit homodimerization. Yeast two-hybrid analysis revealed two types of interactions. The hydrophilic segment between the second and the third transmembrane domain extending from residues Q105 to T141 is competent for a relatively weak interaction, whereas the region at the C-terminal beyond the last transmembrane domain, extending from amino acids R565 to A729, strongly self-interacts. These domains likely facilitate the co-assembly of TRH1 subunits forming an active K(+) transport system within cellular membrane structures. The results support the role of TRH1 acting as a convergence point between the developmental root-hair pathway and the environmental/hormonal signaling pathway to preserve auxin homeostasis ensuring plant adaptation in changing environments., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The KT/HAK/KUP family members encoding high-affinity potassium (K(+)) transporters mediate K(+) transport across the plasma membranes of plant cells to maintain plant normal growth and metabolic activities. In this paper, we identified 16 potassium transporter genes in the peach (Prunus persica) using the Hidden Markov model scanning strategy and searching the peach genome database. Utilizing the Arabidopsis KT/HAK/KUP family as a reference, phylogenetic analysis indicates that the KT/HAK/KUP family in the peach can be classified into 3 groups. Genomic localization indicated that 16 KT/HAK/KUP family genes were well distributed on 7 scaffolds. Gene structure analysis showed that the KT/HAK/KUP family genes have 6-9 introns. In addition, all of the KT/HAK/KUP family members were hydrophobic proteins; they exhibited similar secondary structure patterns and homologous tertiary structures. Putative cis-elements involved in abiotic stress adaption, Ca(2+) response, light and circadian rhythm regulation, and seed development were observed in the promoters of the KT/HAK/KUP family genes. Subcellular localization prediction indicated that the KT/HAK/KUP members were mainly located in the plasma membrane. Expression levels of the KT/HAK/ KUP family genes were much higher in the fruit and flower than those in the other 7 tissues examined, indicating that the KT/HAK/KUP family genes may have important roles in K(+) uptake and transport, which mainly contribute to flower formation and fruit development in the peach.

The high-affinity K(+) transporter HAK5 is a key system for root K(+) uptake and, under very low external K(+), the only one capable of supplying K(+) to the plant. Functional HAK5-mediated K(+) uptake should be tightly regulated for plant adaptation to different environmental conditions. Thus, it has been described that the gene encoding the transporter is transcriptionally regulated, being highly induced under K(+) limitation. Here we show that environmental conditions, such as the lack of K(+), NO(3)(-) or P, that induced a hyperpolarization of the plasma membrane of root cells, induce HAK5 transcription. However, only the deprivation of K(+) produces functional HAK5-mediated K(+) uptake in the root. These results suggest on the one hand the existence of a posttranscriptional regulation of HAK5 elicited by the low K(+) signal and on the other that HAK5 may be involved in yet-unknown functions related to NO(3)(-) and P deficiencies. These results have been obtained here with Solanum lycopersicum (cv. Micro-Tom) as well as Arabidopsis thaliana plants, suggesting that the posttranscriptional regulation of high-affinity HAK transporters take place in all plant species., (© 2014 Scandinavian Plant Physiology Society.)

kefB is annotated as a potassium/proton antiporter in M. tuberculosis. There have been divergent reports on the involvement of KefB in phagosomal maturation in M. bovis BCG and no investigation has been carried out on its role in M. tuberculosis, the pathogenic species responsible for causing tuberculosis. This study was taken up to ascertain the involvement of KefB in the growth of M. tuberculosis and its role in phagosomal maturation and survival of the pathogen in guinea pigs. Our findings show that kefB mutant of M. tuberculosis (MtbΔkefB) was impaired i) for growth in high concentrations of potassium and ii) in arresting phagosomal acidification. However, the disruption of kefB had no adverse effect on the survival of M. tuberculosis in macrophages as well as in guinea pigs suggesting that the role of KefB in phagosomal acidification is unrelated to the survival of the pathogen in the host.

AtKEAs, homologs of bacterial KefB/KefC, are predicted to encode K(+)/H(+) antiporters in Arabidopsis. The AtKEA family contains six genes forming two subgroups in the cladogram: AtKEA1-3 and AtKEA4-6. AtKEA1 and AtKEA2 have a long N-terminal domain; the full-length AtKEA1 was inactive in yeast. The transport activity was analyzed by expressing the AtKEA genes in yeast mutants lacking multiple ion carriers. AtKEAs conferred resistance to high K(+) and hygromycin B but not to salt and Li(+) stress. AtKEAs expressed in both the shoot and root of Arabidopsis. The expression of AtKEA1, -3 and -4 was enhanced under low K(+) stress, whereas AtKEA2 and AtKEA5 were induced by sorbitol and ABA treatments. However, osmotic induction of AtKEA2 and AtKEA5 was not observed in aba2-3 mutants, suggesting an ABA regulated mechanism for their osmotic response. AtKEAs' expression may not be regulated by the SOS pathway since their expression was not affected in sos mutants. The GFP tagging analysis showed that AtKEAs distributed diversely in yeast. The Golgi localization of AtKEA3 was demonstrated by both the stably transformed seedlings and the transient expression in protoplasts. Overall, AtKEAs expressed and localized diversely, and may play roles in K(+) homeostasis and osmotic adjustment in Arabidopsis.

The subcellular localization of a wheat NHX antiporter, TaNHX2, was studied in Arabidopsis protoplasts, and its function was evaluated using Saccharomyces cerevisiae as a heterologous expression system. Fluorescence patterns of TaNHX2-GFP fusion protein in Arabidopsis cells indicated that TaNHX2 localized at endomembranes. TaNHX2 has significant sequence homology to NHX sodium exchangers from Arabidopsis, is abundant in roots and leaves and is induced by salt or dehydration treatments. Western blot analysis showed that TaNHX2 could be expressed in transgenic yeast cells. Expressed TaNHX2 protein suppressed the salt sensitivity of a yeast mutant strain by increasing its K(+) content when exposed to salt stress. TaNHX2 also increased the tolerance of the strain to potassium stress. However, the expression of TaNHX2 did not affect the sodium concentration in transgenic cells. Western blot analysis for tonoplast proteins indicated that the TaNHX2 protein localized at the tonoplast of transgenic yeast cells. The tonoplast vesicles from transgenic yeast cells displayed enhanced K(+)/H(+) exchange activity but very little Na(+/)H(+) exchange compared with controls transformed with the empty vector; Na(+)/H(+) exchange was not detected with concentrations of less than 37.5 mM Na(+) in the reaction medium. Our data suggest that TaNHX2 is a endomembrane-bound protein and may primarily function as a K(+)/H(+) antiporter, which is involved in cellular pH regulation and potassium nutrition under normal conditions. Under saline conditions, the protein mediates resistance to salt stress through the intracellular compartmentalization of potassium to regulate cellular pH and K(+) homeostasis.

High soil Na concentrations damage plants by increasing cellular Na accumulation and K loss. Excess soil Na stimulates ethylene-induced soil-salinity tolerance, the mechanism of which we here define via characterization of an Arabidopsis thaliana mutant displaying transpiration-dependent soil-salinity tolerance. This phenotype is conferred by a loss-of-function allele of ethylene overproducer1 (ETO1; mutant alleles of which cause increased production of ethylene). We show that lack of ETO1 function confers soil-salinity tolerance through improved shoot Na/K homeostasis, effected via the ethylene resistant1-constitutive triple response1 ethylene signaling pathway. Under transpiring conditions, lack of ETO1 function reduces root Na influx and both stelar and xylem sap Na concentrations, thereby restricting root-to-shoot delivery of Na. These effects are associated with increased accumulation of respiratory burst oxidase homolog F (RBOHF)-dependent reactive oxygen species in the root stele. Additionally, lack of ETO1 function leads to significant enhancement of tissue K status by an RBOHF-independent mechanism associated with elevated high-affinity K(+) TRANSPORTER5 transcript levels. We conclude that ethylene promotes soil-salinity tolerance via improved Na/K homeostasis mediated by RBOHF-dependent regulation of Na accumulation and RBOHF-independent regulation of K accumulation.

This study aims to increase our understanding of the functions of CHX transporters in plant cells using the model plant Physcomitrella patens, in which four CHX genes have been identified, PpCHX1-PpCHX4. Two of these genes, PpCHX1 and PpCHX2, are expressed at approximately the same level as the PpACT5 gene, but the other two genes show an extremely low expression. PpCHX1 and PpCHX2 restored growth of Escherichia coli mutants on low K(+)-containing media, suggesting that they mediated K+ uptake that may be energized by symport with H+. In contrast, these genes suppressed the defect associated with the kha1 mutation in Saccharomyces cerevisiae, which suggests that they might mediate K+/H+ antiport. PpCHX1-green fluorescent protein (GFP) fusion protein transiently expressed in P. patens protoplasts co-localized with a Golgi marker. In similar experiments, the PpCHX2-GFP protein appeared to localize to tonoplast and plasma membrane. We constructed the ΔPpchx1 and ΔPpchx2 single mutant lines, and the ΔPpchx2 ΔPphak1 double mutant. Single mutant plants grew normally under all the conditions tested and exhibited normal K+ and Rb+ influxes; the ΔPpchx2 mutation did not increase the defect of ΔPphak1 plants. In long-term experiments, ΔPpchx2 plants showed slightly higher Rb+ retention than wild-type plants, which suggests that PpCHX2 mediates the transfer of Rb+ either from the vacuole to the cytosol or from the cytosol to the external medium in parallel with other transporters. The distinction between these two possibilities is technically difficult. We suggest that K+ transporters of several families are involved in the pH homeostasis of organelles by mediating either K+/H+ antiport or K(+)-H(+) symport.

The function of HAK transporters in high-affinity K+ uptake in plants is well established; this study aims to demonstrate that some transporters of the same family play important roles in endomembranes. The PpHAK2-PpHAK4 genes of Physcomitrella patens encode three transporters of high sequence similarity. Quantitative PCR showed that PpHAK2 and PpHAK3 transcripts are expressed at approximately the same level as the PpACT5 gene, while the expression of PpHAK4 seems to be restricted to specific conditions that have not been determined. KHA1 is an endomembrane K+/H+ antiporter of Saccharomyces cerevisiae, and the expression of the PpHAK2 cDNA, but not that of PpHAK3, suppressed the defect of a kha1 mutant. Transient expression of the PpHAK2-green fluorescent protein (GFP) and PpHAK3-GFP fusion proteins in P. patens protoplasts localized to the endoplasmic reticulum and Golgi complex, respectively. To determine the function of PpHAK2 and PpHAK3 in planta, we constructed ΔPphak2 and ΔPphak2 ΔPphak3 plants. ΔPphak2 plants were normal under all of the conditions tested except under K+ starvation or at acidic pH in the presence of acetic acid, whereupon they die. The defect observed under K+ starvation was suppressed by the presence of Na+. We propose that PpHAK2 may encode either a K(+)-H(+) symporter or a K+/H+ antiporter that mediates the transfer of H+ from the endoplasmic reticulum lumen to the cytosol. PpHAK2 may be a model of the second function of HAK transporters in plant cells. The disruption of the PpHAK3 gene in ΔPphak2 plants showed no effect.

Heterologous expression of a putative K+/H+ antiporter of Streptomyces coelicolor A3(2) (designated as sha4) in E. coli and Streptomyces hygroscopicus JCM4427 showed enhanced tolerance to K+ stress, acidic-pH shock, and/or geldanamycin production under K+ stress. In a series of K+ extrusion experiments with sha4-carrying E. coli deficient in the K+/H+ antiporter, a restoration of impaired K+ extrusion activity was observed. Based on this, it was concluded that sha4 was a true K+/H+ antiporter. In different sets of experiments, the sha4-carrying E. coli showed significantly improved tolerances to K+ stresses and acidic-pH shock, whereas sha4-carrying S. hygroscopicus showed an improvement in K+ stress tolerance only. The sha4-carrying S. hygroscopicus showed much higher geldanamycin productivity than the control under K+ stress condition. In another set of experiments with a production medium, the secretion of geldanamycin was also significantly enhanced by the expression of sha4.

Vibrio cholerae has adapted to a wide range of salinity, pH and osmotic conditions, enabling it to survive passage through the host and persist in the environment. Among the many proteins responsible for bacterial survival under these diverse conditions, we have identified Vc-NhaP1 as a K(+)(Na(+))/H(+) antiporter essential for V. cholerae growth at low environmental pH. Deletion of the V. cholerae nhaP1 gene caused growth inhibition when external potassium was either limited (100 mM and below) or in excess (400 mM and above). This growth defect was most apparent at mid-exponential phase, after 4-6 h of culture. Using a pH-sensitive GFP, cytosolic pH was shown to be dependent on K(+) in acidic external conditions in a Vc-NhaP1-dependent manner. When functionally expressed in an antiporterless Escherichia coli strain and assayed in everted membrane vesicles, Vc-NhaP1 operated as an electroneutral alkali cation/proton antiporter, exchanging K(+) or Na(+) ions for H(+) within a broad pH range (7.25-9.0). These data establish the putative V. cholerae NhaP1 protein as a functional K(+)(Na(+))/H(+) antiporter of the CPA1 family that is required for bacterial pH homeostasis and growth in an acidic environment.

The first chloride transporter identified in the superfamily of ClC chloride channels was from Escherichia coli (EClC) (Accardi, A., and Miller, C. (2004) Nature 427, 803-807). Pathways, energetics, and mechanism of proton and chloride translocation and their coupling are up to now unclear. To bridge the hydrophobic gap of proton transport, we modeled four stable buried waters into both subunits of the WT EClC structure. Together they form a "water wire" connecting Glu-203 with the chloride at the central site, which in turn connects to Glu-148, the hypothetical proton exit site. Assuming the transient production of hydrochloride in the central chloride binding site of EClC, the water wire could establish a transmembrane proton transport pathway starting from Glu-203 all the way downstream onto Glu-148. We demonstrated by electrostatic and quantum chemical computations that protonation of the central chloride is energetically feasible. We characterized all chloride occupancies and protonation states possibly relevant for the proton-chloride transport cycle in EClC and constructed a working model. Accordingly, EClC evolves through states involving up to two excess protons and between one and three chlorides, which was required to fulfill the experimentally observed 2:1 stoichiometry. We show that the Y445F and E203H mutants of EClC can operate similarly, thus explaining why they exhibit almost WT activity levels. The proposed mechanism of coupled chloride-proton transport in EClC is consistent with available experimental data and allows predictions on the importance of specific amino acids, which may be probed by mutation experiments.

Survival of exposure to methylglyoxal (MG) in Gram-negative pathogens is largely dependent upon the operation of the glutathione-dependent glyoxalase system, consisting of two enzymes, GlxI (gloA) and GlxII (gloB). In addition, the activation of the KefGB potassium efflux system is maintained closed by glutathione (GSH) and is activated by S-lactoylGSH (SLG), the intermediate formed by GlxI and destroyed by GlxII. Escherichia coli mutants lacking GlxI are known to be extremely sensitive to MG. In this study we demonstrate that a ΔgloB mutant is as tolerant of MG as the parent, despite having the same degree of inhibition of MG detoxification as a ΔgloA strain. Increased expression of GlxII from a multicopy plasmid sensitizes E. coli to MG. Measurement of SLG pools, KefGB activity and cytoplasmic pH shows these parameters to be linked and to be very sensitive to changes in the activity of GlxI and GlxII. The SLG pool determines the activity of KefGB and the degree of acidification of the cytoplasm, which is a major determinant of the sensitivity to electrophiles. The data are discussed in terms of how cell fate is determined by the relative abundance of the enzymes and KefGB., (© 2010 Blackwell Publishing Ltd.)

Three vacuolar cation/H+ antiporters, AtNHX1 (At5g27150), 2 (At3g05030) and 5 (At1g54370), have been characterized as functional Na+/H+ antiporters in Arabidopsis. However, the physiological functions of AtNHX3 (At5g55470) still remain unclear. In this study, the physiological functions of AtNHX3 were studied using T-DNA insertion mutant and 35S-driven AtNHX3 over-expression Arabidopsis plants. RT-PCR analyses revealed that AtNHX3 is highly expressed in germinating seeds, flowers and siliques. Experiments with AtNHX3::YFP fusion protein in tobacco protoplasts indicated that AtNHX3 is mainly localized to vacuolar membrane, with a minor localization to pre-vacuolar compartments (PVCs) and endoplasmic reticulum (ER). Seedlings of null nhx3 mutants were hypersensitive to K+-deficient conditions. Expression of AtNHX3 complemented the sensitivity to K+ deficiency in nhx3 seedlings. Tonoplast vesicles isolated from transgenic plants over-expressing AtNHX3 displayed significantly higher K+/H+ exchange rates than those isolated from wild-type plants. Furthermore, nhx3 seeds accumulated less K+ and more Na+ when both wild-type and nhx3 were grown under normal growth condition. The overall results indicate that AtNHX3 encodes a K+/H+ antiporter required for low-potassium tolerance during germination and early seedling development, and may function in K+ utilization and ion homeostasis in Arabidopsis., (© 2010 Blackwell Publishing Ltd.)