Arabidopsis transcriptomic analysis reveals cesium inhibition of root growth involves abscisic acid signaling.

MAIN CONCLUSION: This is the first report on the involvement of abscisic acid signaling in regulating post-germination growth under Cs stress, not related to potassium deficiency. Cesium (Cs) is known to exert toxicity in plants by competition and interference with the transport of potassium (K). However, the precise mechanism of how Cs mediates its damaging effect is still unclear. This fact is mainly attributed to the large effects of lower K uptake in the presence of Cs that shadow other crucial effects by Cs that were not related to K. RNA-seq was conducted on Arabidopsis roots grown to identify putative genes that are functionally involved to investigate the difference between Cs stress and low K stress. Our transcriptome data demonstrated Cs-regulated genes only partially overlap to low K-regulated genes. In addition, the divergent expression trend of High-affinity K+ Transporter (HAK5) from D4 to D7 growth stage suggested participation of other molecular events besides low K uptake under Cs stress. Potassium deficiency triggers expression level change of the extracellular matrix, transfer/carrier, cell adhesion, calcium-binding, and DNA metabolism genes. Under Cs stress, genes encoding translational proteins, chromatin regulatory proteins, membrane trafficking proteins and defense immunity proteins were found to be primarily regulated. Pathway enrichment and protein network analyses of transcriptome data exhibit that Cs availability are associated with alteration of abscisic acid (ABA) signaling, photosynthesis activities and nitrogen metabolism. The phenotype response of ABA signaling mutants supported the observation and revealed Cs inhibition of root growth involved in ABA signaling pathway. The rather contrary response of loss-of-function mutant of Late Embryogenesis Abundant 7 (LEA7) and Translocator Protein (TSPO) further suggested low K stress and Cs stress may activate different salt tolerance responses. Further investigation on the crosstalk between K transport, signaling, and salt stress-responsive signal transduction will provide a deeper understanding of the mechanisms and molecular regulation underlying Cs toxicity.

Annexin 1 Is a Component of eATP-Induced Cytosolic Calcium Elevation in Arabidopsis thaliana Roots.

Extracellular ATP (eATP) has long been established in animals as an important signalling molecule but this is less understood in plants. The identification of Arabidopsis thaliana DORN1 (Does Not Respond to Nucleotides) as the first plant eATP receptor has shown that it is fundamental to the elevation of cytosolic free Ca2+ ([Ca2+]cyt) as a possible second messenger. eATP causes other downstream responses such as increase in reactive oxygen species (ROS) and nitric oxide, plus changes in gene expression. The plasma membrane Ca2+ influx channels involved in eATP-induced [Ca2+]cyt increase remain unknown at the genetic level. Arabidopsis thaliana Annexin 1 has been found to mediate ROS-activated Ca2+ influx in root epidermis, consistent with its operating as a transport pathway. In this study, the loss of function Annexin 1 mutant was found to have impaired [Ca2+]cyt elevation in roots in response to eATP or eADP. Additionally, this annexin was implicated in modulating eATP-induced intracellular ROS accumulation in roots as well as expression of eATP-responsive genes.

Syringic Acid Alleviates Cesium-Induced Growth Defect in Arabidopsis.

Syringic acid, a phenolic compound, serves a variety of beneficial functions in cells. Syringic acid increases in plants in response to cesium, and exogenous application of syringic acid resulted in a significant attenuation of cesium-induced growth defects in Arabidopsis. In addition, cesium or syringic acid application to plants also resulted in increased lignin deposition in interfascicular fibers. To better understand the role of lignin and syringic acid in attenuating cesium-induced growth defects, two mutants for Arabidopsis REDUCED EPIDERMAL FLUORESCENE 4 (REF4) and fourteen laccase mutants, some of which have lower levels of lignin, were evaluated for their response to cesium. These mutants responded differently to cesium stress, compared to control plants, and the application of syringic acid alleviated cesium-induced growth defects in the laccase mutants but not in the ref4 mutants. These findings imply that lignin plays a role in cesium signaling but the attenuation of cesium stress defects by syringic acid is mediated by regulatory components of lignin biosynthesis and not lignin biosynthesis itself. In contrast, syringic acid did not alleviate any low potassium-induced growth defects. Collectively, our findings provide the first established link between lignin and cesium stress via syringic acid in plants.

Cesium Inhibits Plant Growth Primarily Through Reduction of Potassium Influx and Accumulation in Arabidopsis.

Cesium (Cs+) is known to compete with the macronutrient potassium (K+) inside and outside of plants and to inhibit plant growth at high concentrations. However, the detailed molecular mechanisms of how Cs+ exerts its deleterious effects on K+ accumulation in plants are not fully elucidated. Here, we show that mutation in a member of the major K+ channel AKT1-KC1 complex renders Arabidopsis thaliana hypersensitive to Cs+. Higher severity of the phenotype and K+ loss were observed for these mutants in response to Cs+ than to K+ deficiency. Electrophysiological analysis demonstrated that Cs+, but not sodium, rubidium or ammonium, specifically inhibited K+ influx through the AKT1-KC1 complex. In contrast, Cs+ did not inhibit K+ efflux through the homomeric AKT1 channel that occurs in the absence of KC1, leading to a vast loss of K+. Our observation suggests that reduced K+ accumulation due to blockage/competition in AKT1 and other K+ transporters/channels by Cs+ plays a major role in plant growth retardation. This report describes the mechanical role of Cs+ in K+ accumulation, and in turn in plant performance, providing actual evidence at the plant level for what has long been believed, i.e. K+ channels are, therefore AKT1 is, 'blocked' by Cs+.

Arabidopsis CNGC Family Members Contribute to Heavy Metal Ion Uptake in Plants.

Heavy metal ions, including toxic concentrations of essential ions, negatively affect diverse metabolic and cellular processes. Heavy metal ions are known to enter cells in a non-selective manner; however, few studies have examined the regulation of heavy metal ion transport. Plant cyclic nucleotide-gated channels (CNGCs), a type of Ca2+-permeable-channel, have been suggested to be involved in the uptake of both essential and toxic cations. To determine the candidates responsible for heavy metal ion transport, a series of Arabidopsis CNGC mutants were examined for their response to Pb2+ and Cd2+ ions. The primary focus was on root growth and the analysis of the concentration of heavy metals in plants. Results, based on the analysis of primary root length, indicated that AtCNGC1, AtCNGC10, AtCNGC13 and AtCNGC19 play roles in Pb2+ toxicity, while AtCNGC11, AtCNGC13, AtCNGC16 and AtCNGC20 function in Cd2+ toxicity in Arabidopsis. Ion content analysis verified that the mutations of AtCNGC1 and AtCNGC13 resulted in reduced Pb2+ accumulation, while the mutations of AtCNGC11, AtCNGC15 and AtCNGC19 resulted in less Pb2+ and Cd2+ accumulation in plants. These findings provide functional evidence which support the roles of these AtCNGCs in the uptake and transport of Pb2+ or Cd2+ ion in plants.

AtSKIP18 and AtSKIP31, F-box subunits of the SCF E3 ubiquitin ligase complex, mediate the degradation of 14-3-3 proteins in Arabidopsis.

14-3-3 proteins regulate numerous cellular processes through interaction with their target proteins in a phosphorylation dependent manner. Although proteins that are regulated by 14-3-3s have been studied, the regulatory mechanism of 14-3-3s is poorly understood. In the present study, F-box proteins, a component of Skp1-Cullin-F-box E3 ubiquitin ligase, were identified as 14-3-3 targets using yeast two-hybrid screening. Among them, AtSKIP18 and AtSKIP31, were shown to mediate the degradation of Arabidopsis 14-3-3s. Mutational analyses of AtSKIP18 and AtSKIP31 indicated that the phosphorylation of AtSKIPs is critical for interaction and degradation of 14-3-3s. The loss-of-function mutation in AtSKIP31 resulted in enhanced primary root growth under nitrogen deficient conditions. These findings suggest that AtSKIP31 regulates the primary root growth in nitrogen deficiency via degrading 14-3-3s.

14-3-3 proteins participate in light signaling through association with PHYTOCHROME INTERACTING FACTORs.

14-3-3 proteins are regulatory proteins found in all eukaryotes and are known to selectively interact with phosphorylated proteins to regulate physiological processes. Through an affinity purification screening, many light-related proteins were recovered as 14-3-3 candidate binding partners. Yeast two-hybrid analysis revealed that the 14-3-3 kappa isoform (14-3-3κ) could bind to PHYTOCHROME INTERACTING FACTOR3 (PIF3) and CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). Further analysis by in vitro pull-down assay confirmed the interaction between 14-3-3κ and PIF3. Interruption of putative phosphorylation sites on the 14-3-3 binding motifs of PIF3 was not sufficient to inhibit 14-3-3κ from binding or to disturb nuclear localization of PIF3. It was also indicated that 14-3-3κ could bind to other members of the PIF family, such as PIF1 and PIF6, but not to LONG HYPOCOTYL IN FAR-RED1 (HFR1). 14-3-3 mutants, as well as the PIF3 overexpressor, displayed longer hypocotyls, and a pif3 mutant displayed shorter hypocotyls than the wild-type in red light, suggesting that 14-3-3 proteins are positive regulators of photomorphogenesis and function antagonistically with PIF3. Consequently, our results indicate that 14-3-3 proteins bind to PIFs and initiate photomorphogenesis in response to a light signal.

Transport, signaling, and homeostasis of potassium and sodium in plants.

Potassium (K⁺) is an essential macronutrient in plants and a lack of K⁺ significantly reduces the potential for plant growth and development. By contrast, sodium (Na⁺), while beneficial to some extent, at high concentrations it disturbs and inhibits various physiological processes and plant growth. Due to their chemical similarities, some functions of K⁺ can be undertaken by Na⁺ but K⁺ homeostasis is severely affected by salt stress, on the other hand. Recent advances have highlighted the fascinating regulatory mechanisms of K⁺ and Na⁺ transport and signaling in plants. This review summarizes three major topics: (i) the transport mechanisms of K⁺ and Na⁺ from the soil to the shoot and to the cellular compartments; (ii) the mechanisms through which plants sense and respond to K⁺ and Na⁺ availability; and (iii) the components involved in maintenance of K⁺/Na⁺ homeostasis in plants under salt stress.

Identification and characterization of transcription factors regulating Arabidopsis HAK5.

Potassium (K) is an essential macronutrient for plant growth and reproduction. HAK5, an Arabidopsis high-affinity K transporter gene, plays an important role in K uptake. Its expression is up-regulated in response to K deprivation and is rapidly down-regulated when sufficient K levels have been re-established. To identify transcription factors regulating HAK5, an Arabidopsis TF FOX (Transcription Factor Full-length cDNA Over-eXpressor) library containing approximately 800 transcription factors was used to transform lines previously transformed with a luciferase reporter gene whose expression was driven by the HAK5 promoter. When grown under sufficient K levels, 87 lines with high luciferase activity were identified, and endogenous HAK5 expression was confirmed in 27 lines. Four lines overexpressing DDF2 (Dwarf and Delayed Flowering 2), JLO (Jagged Lateral Organs), TFII_A (Transcription initiation Factor II_A gamma chain) and bHLH121 (basic Helix-Loop-Helix 121) were chosen for further characterization by luciferase activity, endogenous HAK5 level and root growth in K-deficient conditions. Further analysis showed that the expression of these transcription factors increased in response to low K and salt stress. In comparison with controls, root growth under low K conditions was better in each of these four TF FOX lines. Activation of HAK5 expression by these four transcription factors required at least 310 bp of upstream sequence of the HAK5 promoter. These results indicate that at least these four transcription factors can bind to the HAK5 promoter in response to K limitation and activate HAK5 expression, thus allowing plants to adapt to nutrient stress.

Cesium Inhibits Plant Growth through Jasmonate Signaling in Arabidopsis thaliana.

It has been suggested that cesium is absorbed from the soil through potassium uptake machineries in plants; however, not much is known about perception mechanism and downstream response. Here, we report that the jasmonate pathway is required in plant response to cesium. Jasmonate biosynthesis mutant aos and jasmonate-insensitive mutant coi1-16 show clear resistance to root growth inhibition caused by cesium. However, the potassium and cesium contents in these mutants are comparable to wild-type plants, indicating that jasmonate biosynthesis and signaling are not involved in cesium uptake, but involved in cesium perception. Cesium induces expression of a high-affinity potassium transporter gene HAK5 and reduces potassium content in the plant body, suggesting a competitive nature of potassium and cesium uptake in plants. It has also been found that cesium-induced HAK5 expression is antagonized by exogenous application of methyl-jasmonate. Taken together, it has been indicated that cesium inhibits plant growth via induction of the jasmonate pathway and likely modifies potassium uptake machineries.

Isolation of novel chemical components and their plant target proteins under selenium stress.

Selenium is recognized as a beneficial nutrient in living organisms. Excessive amounts of selenium, however, can have a significant negative impact on organisms. Screening of novel chemical compounds that regulate and/or moderate selenium in plants was conducted. The present chapter discusses (1) the design of a chemical screening strategy, (2) methods used to identify and select candidate chemicals, and (3) the identification of chemical-binding target proteins. We identified a novel chemical compound, C9H8N2OS2, in our screening program that enhances selenate accumulation and stress tolerance. The target protein, beta-glucosidase 23, in Arabidopsis was found to regulate selenium accumulation, as well as plant response to selenate stress.

Receptor-like activity evoked by extracellular ADP in Arabidopsis root epidermal plasma membrane.

Extracellular purine nucleotides are implicated in the control of plant development and stress responses. While extracellular ATP is known to activate transcriptional pathways via plasma membrane (PM) NADPH oxidase and calcium channel activation, very little is known about signal transduction by extracellular ADP. Here, extracellular ADP was found to activate net Ca(2+) influx in roots of Arabidopsis (Arabidopsis thaliana) and transiently elevate cytosolic free Ca(2+) in root epidermal protoplasts. An inward Ca(2+)-permeable conductance in root epidermal PM was activated within 1 s of ADP application and repeated application evoked a smaller current. Such response speed and densitization are consistent with operation of equivalents to animal ionotropic purine receptors, although to date no equivalent genes for such receptors have been identified in higher plants. In contrast to ATP, extracellular ADP did not evoke accumulation of intracellular reactive oxygen species. While high concentrations of ATP caused net Ca(2+) efflux from roots, equivalent concentrations of ADP caused net influx. Overall the results point to a discrete ADP signaling pathway, reliant on receptor-like activity at the PM.

Nutrient sensing and signaling: NPKS.

Plants often grow in soils that contain very low concentrations of the macronutrients nitrogen, phosphorus, potassium, and sulfur. To adapt and grow in nutrient-deprived environments plants must sense changes in external and internal mineral nutrient concentrations and adjust growth to match resource availability. The sensing and signal transduction networks that control plant responses to nutrient deprivation are not well characterized for nitrogen, potassium, and sulfur deprivation. One branch of the signal transduction cascade related to phosphorus-deprivation response has been defined through the identification of a transcription factor that is regulated by sumoylation. Two different microRNAs play roles in regulating gene expression under phosphorus and sulfur deprivation. Reactive oxygen species increase rapidly after mineral nutrient deprivation and may be one upstream mediator of nutrient signaling. A number of molecular analyses suggest that both short-term and longer-term responses will be important in understanding the progression of signaling events when the external, then internal, supplies of nutrients become depleted.

Calcium channels and transporters: Roles in response to biotic and abiotic stresses.

Calcium (Ca2+) serves as a ubiquitous second messenger by mediating various signaling pathways and responding to numerous environmental conditions in eukaryotes. Therefore, plant cells have developed complex mechanisms of Ca2+ communication across the membrane, receiving the message from their surroundings and transducing the information into cells and organelles. A wide range of biotic and abiotic stresses cause the increase in [Ca2+]cyt as a result of the Ca2+ influx permitted by membrane-localized Ca2+ permeable cation channels such as CYCLIC NUCLEOTIDE-GATE CHANNELs (CNGCs), and voltage-dependent HYPERPOLARIZATION-ACTIVATED CALCIUM2+ PERMEABLE CHANNELs (HACCs), as well as GLUTAMATE RECEPTOR-LIKE RECEPTORs (GLRs) and TWO-PORE CHANNELs (TPCs). Recently, resistosomes formed by some NUCLEOTIDE-BINDING LEUCINE-RICH REPEAT RECEPTORs (NLRs) are also proposed as a new type of Ca2+ permeable cation channels. On the contrary, some Ca2+ transporting membrane proteins, mainly Ca2+-ATPase and Ca2+/H+ exchangers, are involved in Ca2+ efflux for removal of the excessive [Ca2+]cyt in order to maintain the Ca2+ homeostasis in cells. The Ca2+ efflux mechanisms mediate the wide ranges of cellular activities responding to external and internal stimuli. In this review, we will summarize and discuss the recent discoveries of various membrane proteins involved in Ca2+ influx and efflux which play an essential role in fine-tuning the processing of information for plant responses to abiotic and biotic stresses.

Cesium tolerance is enhanced by a chemical which binds to BETA-GLUCOSIDASE 23 in Arabidopsis thaliana.

Cesium (Cs) is found at low levels in nature but does not confer any known benefit to plants. Cs and K compete in cells due to the chemical similarity of Cs to potassium (K), and can induce K deficiency in cells. In previous studies, we identified chemicals that increase Cs tolerance in plants. Among them, a small chemical compound (C17H19F3N2O2), named CsToAcE1, was confirmed to enhance Cs tolerance while increasing Cs accumulation in plants. Treatment of plants with CsToAcE1 resulted in greater Cs and K accumulation and also alleviated Cs-induced growth retardation in Arabidopsis. In the present study, potential target proteins of CsToAcE1 were isolated from Arabidopsis to determine the mechanism by which CsToAcE1 alleviates Cs stress, while enhancing Cs accumulation. Our analysis identified one of the interacting target proteins of CsToAcE1 to be BETA-GLUCOSIDASE 23 (AtßGLU23). Interestingly, Arabidopsis atßglu23 mutants exhibited enhanced tolerance to Cs stress but did not respond to the application of CsToAcE1. Notably, application of CsToAcE1 resulted in a reduction of Cs-induced AtßGLU23 expression in wild-type plants, while this was not observed in a high affinity transporter mutant, athak5. Our data indicate that AtßGLU23 regulates plant response to Cs stress and that CsToAcE1 enhances Cs tolerance by repressing AtßGLU23. In addition, AtHAK5 also appears to be involved in this response.

Plant extracellular ATP signalling by plasma membrane NADPH oxidase and Ca2+ channels.

Extracellular ATP regulates higher plant growth and adaptation. The signalling events may be unique to higher plants, as they lack animal purinoceptor homologues. Although it is known that plant cytosolic free Ca2+ can be elevated by extracellular ATP, the mechanism is unknown. Here, we have studied roots of Arabidopsis thaliana to determine the events that lead to the transcriptional stress response evoked by extracellular ATP. Root cell protoplasts were used to demonstrate that signalling to elevate cytosolic free Ca2+ is determined by ATP perception at the plasma membrane, and not at the cell wall. Imaging revealed that extracellular ATP causes the production of reactive oxygen species in intact roots, with the plasma membrane NADPH oxidase AtRBOHC being the major contributor. This resulted in the stimulation of plasma membrane Ca2+-permeable channels (determined using patch-clamp electrophysiology), which contribute to the elevation of cytosolic free Ca2+. Disruption of this pathway in the AtrbohC mutant impaired the extracellular ATP-induced increase in reactive oxygen species (ROS), the activation of Ca2+ channels, and the transcription of the MAP kinase3 gene that is known to be involved in stress responses. This study shows that higher plants, although bereft of purinoceptor homologues, could have evolved a distinct mechanism to transduce the ATP signal at the plasma membrane.

A nuclear factor regulates abscisic acid responses in Arabidopsis.

Abscisic acid (ABA) is a plant hormone that regulates plant growth as well as stress responses. In this study, we identified and characterized a new Arabidopsis (Arabidopsis thaliana) protein, Nuclear Protein X1 (NPX1), which was up-regulated by stress and treatment with exogenous ABA. Stomatal closure, seed germination, and primary root growth are well-known ABA responses that were less sensitive to ABA in NPX1-overexpressing plants. NPX1-overexpressing plants were more drought sensitive, and the changes in response to drought were due to the altered guard cell sensitivity to ABA in transgenic plants and not to a lack of ABA production. The nuclear localization of NPX1 correlated with changes in the expression of genes involved in ABA biosynthesis and ABA signal transduction. To understand the function of NPX1, we searched for interacting proteins and found that an ABA-inducible NAC transcription factor, TIP, interacted with NPX1. Based on the whole plant phenotypes, we hypothesized that NPX1 acts as a transcriptional repressor, and this was demonstrated in yeast, where we showed that TIP was repressed by NPX1. Our results indicate that the previously unknown protein NPX1 acts as a negative regulator in plant response to changes in environmental conditions through the control of ABA-regulated gene expression. The characterization of this factor enhances our understanding of guard cell function and the mechanisms that plants use to modulate water loss from leaves under drought conditions.

Contribution of KUPs to potassium and cesium accumulation appears complementary in Arabidopsis.

Cesium has no known beneficial effects on plants and while plants have the ability to absorb it through the root system, plant growth is retarded at high concentrations. Recently, we have shown that potassium influx through a potassium channel complex AKT1-KC1 is inhibited by cesium in Arabidopsis thaliana and the resultant reduction in potassium accumulation in the plant is the primary cause of retarded growth. By contrast, a major potassium transporter, HAK5 whose function is crucial under potassium deficiency, was found to be either not affected or complementary under cesium stress in the low affinity potassium range. Here we show the effects of insertional mutation on other members of KUP/HAK/KT gene family in response to cesium stress. Potassium and cesium concentrations in each mutant line demonstrated that disruption of a single KUP/HAK/KT gene was not sufficient to significantly reduce potassium/cesium accumulation, suggesting a complementary effect among these KUP (K+ UPTAKE PERMEASE) transporters.

Glutathione and Its Biosynthetic Intermediates Alleviate Cesium Stress in Arabidopsis.

Phytoremediation is optimized when plants grow vigorously while accumulating the contaminant of interest. Here we show that sulphur supply alleviates aerial chlorosis and growth retardation caused by cesium stress without reducing cesium accumulation in Arabidopsis thaliana. This alleviation was not due to recovery of cesium-induced potassium decrease in plant tissues. Sulphur supply also alleviated sodium stress but not potassium deficiency stress. Cesium-induced root growth inhibition has previously been demonstrated as being mediated through jasmonate biosynthesis and signalling but it was found that sulphur supply did not decrease the levels of jasmonate accumulation or jasmonate-responsive transcripts. Instead, induction of a glutathione synthetase gene GSH2 and reduction of a phytochelatin synthase gene PCS1 as well as increased accumulation of glutathione and cysteine were observed in response to cesium. Exogenous application of glutathione or concomitant treatments of its biosynthetic intermediates indeed alleviated cesium stress. Interestingly, concomitant treatments of glutathione biosynthetic intermediates together with a glutathione biosynthesis inhibitor did not cancel the alleviatory effects against cesium suggesting the existence of a glutathione-independent pathway. Taken together, our findings demonstrate that plants exposed to cesium increase glutathione accumulation to alleviate the deleterious effects of cesium and that exogenous application of sulphur-containing compounds promotes this innate process.

The high affinity K+ transporter AtHAK5 plays a physiological role in planta at very low K+ concentrations and provides a caesium uptake pathway in Arabidopsis.

Caesium (Cs(+)) is a potentially toxic mineral element that is released into the environment and taken up by plants. Although Cs(+) is chemically similar to potassium (K(+)), and much is known about K(+) transport mechanisms, it is not clear through which K(+) transport mechanisms Cs(+) is taken up by plant roots. In this study, the role of AtHAK5 in high affinity K(+) and Cs(+) uptake was characterized. It is demonstrated that AtHAK5 is localized to the plasma membrane under conditions of K(+) deprivation, when it is expressed. Growth analysis showed that AtHAK5 plays a role during severe K(+) deprivation. Under K(+)-deficient conditions in the presence of Cs(+), Arabidopsis seedlings lacking AtHAK5 had increased inhibition of root growth and lower Cs(+) accumulation, and significantly higher leaf chlorophyll concentrations than wild type. These data indicate that, in addition to transporting K(+) in planta, AtHAK5 also transports Cs(+). Further experiments showed that AtHAK5 mediated Cs(+) uptake into yeast cells and that, although the K(+) deficiency-induced expression of AtHAK5 was inhibited by low concentrations of NH(4)(+) in planta, Cs(+) uptake by yeast was stimulated by low concentrations of NH(4)(+). Interestingly, the growth of the Arabidopsis atakt1-1 mutant was more sensitive to Cs(+) than the wild type. This may be explained, in part, by increased expression of AtHAK5 in the atakt1-1 mutant. It is concluded that AtHAK5 is a root plasma membrane uptake mechanism for K(+) and Cs(+) under conditions of low K(+) availability.