The role of plasma membrane H+‐ATPase in jasmonate‐induced ion fluxes and stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) elicits stomatal closure in many plant species. Stomatal closure is accompanied by large ion fluxes across the plasma membrane (PM). Here, we recorded the transmembrane ion fluxes of H⁺, Ca²⁺ and K⁺ in guard cells of wild‐type (Col‐0) Arabidopsis, the CORONATINE INSENSITIVE1 (COI1) mutant coi1‐1 and the PM H⁺‐ATPase mutants aha1‐6 and aha1‐7, using a non‐invasive micro‐test technique. We showed that MeJA induced transmembrane H⁺ efflux, Ca²⁺ influx and K⁺ efflux across the PM of Col‐0 guard cells. However, this ion transport was abolished in coi1‐1 guard cells, suggesting that MeJA‐induced transmembrane ion flux requires COI1. Furthermore, the H⁺ efflux and Ca²⁺ influx in Col‐0 guard cells was impaired by vanadate pre‐treatment or PM H⁺‐ATPase mutation, suggesting that the rapid H⁺ efflux mediated by PM H⁺‐ATPases could function upstream of the Ca²⁺ flux. After the rapid H⁺ efflux, the Col‐0 guard cells had a longer oscillation period than before MeJA treatment, indicating that the activity of the PM H⁺‐ATPase was reduced. Finally, the elevation of cytosolic Ca²⁺ concentration and the depolarized PM drive the efflux of K⁺ from the cell, resulting in loss of turgor and closure of the stomata.     

Involvement of plasma membrane potential in the tolerance mechanism of plant roots to aluminium toxicity

H⁺-ATPase activity of a plasma membrane-enriched fraction decreased after the treatment of barley (Hordeum vulgare) seedlings with Al for 5 days. A remarkably high level of Al was found in the membrane fraction of Al-treated roots. A long-term effect of Al was identified as the repression of the H⁺-ATPase of plasma membranes isolated from the roots of barley and wheat (Triticum aestivum) cultivars, Atlas 66 (Al-tolerant) and Scout 66 (Al-sensitive). To monitor short-term effects of Al, the electrical membrane potentials across plasma membranes of both wheat cultivars were compared indirectly by measuring the efflux of K⁺ for 40 min under various conditions. The rate of efflux of K⁺ in Scout was twice that in Atlas at low pH values such as 4.2. Vanadate, an inhibitor of the H⁺-ATPase of the plasma membrane, increased the efflux of K⁺. Al repressed this efflux at low pH, probably through an effect on K⁺ channels, and repression was more pronounced in Scout. Al strongly repressed the efflux of K⁺ irrespective of the presence of vanadate. Ca²⁺ also had a repressive effect on the efflux of K⁺ at low pH. The effect of Ca²⁺, greater in Scout, might be related to the regulation of the net influx of H⁺, since the effect was negated by vanadate. The results suggest that extracellular low pH may cause an increase in the influx of H⁺, which in turn is counteracted by the efflux of K⁺ and H⁺. These results suggest that the ability to maintain the integrity of the plasma membrane and the ability to recover the electrical balance at the plasma membrane through a net influx of H⁺ and the efflux of K⁺ seem to participate in the mechanism of tolerance to Al stress under acidic conditions.     

Mechanisms of Aluminum Tolerance in Wheat : An Investigation of Genotypic Differences in Rhizosphere pH, K, and H Transport, and Root-Cell Membrane Potentials

Control of rhizosphere pH and exclusion of Al by the plasma membrane have been hypothesized as possible mechanisms for Al tolerance. To test primarily the rhizosphere pH hypothesis, wheat cultivars (Triticum aestivum L. `Atlas 66' and `Scout'), which differ in Al tolerance, were grown in either complete nutrient solution, or 0.6 millimolar CaSO₄, with and without Al at pH 4.50. A microelectrode system was used to simultaneously measure rhizosphere pH, K⁺, and H⁺ fluxes, and membrane potentials (E_m) along the root at various distances from the root apex. In complete nutrient solution, the rhizosphere pH associated with mature root cells (measured 10-40 millimeters from the root apex) of Al-tolerant `Atlas 66' was slightly higher than that of the bulk solution, whereas roots of Al-sensitive `Scout' caused a very small decrease in the rhizosphere pH. In CaSO₄ solution, no significant differences in rhizosphere pH were found between wheat cultivars, while differential Al tolerance was still observed, indicating that the rhizosphere pH associated with mature root tissue is not directly involved in the mechanism(s) of differential Al tolerance. In Al-tolerant `Atlas 66', growth in a CaSO<sub>4</sub> solution with 5 micromolar Al (pH 4.50) had little effect on net K<sup>+</sup> influx, H<sup>+</sup> efflux, and root-cell membrane potential measured in cells of mature root tissue (from 10-40 mm back from apex). However, in Al-sensitive `Scout', Al treatment caused a dramatic inhibition of K⁺ influx and both a moderate reduction of H⁺ efflux and depolarization of the membrane potential. These results demonstrate that increased Al tolerance in wheat is associated with the increased ability of the tolerant plant to maintain normal ion fluxes and membrane potentials across the plasmalemma of root cells in the presence of Al.     

H2O2 and cytosolic Ca2+ signals triggered by the PM H+‐coupled transport system mediate K+/Na+ homeostasis in NaCl‐stressed Populus euphratica cells

Using confocal microscopy, X‐ray microanalysis and the scanning ion‐selective electrode technique, we investigated the signalling of H₂O₂, cytosolic Ca²⁺ ([Ca²⁺]_cyt) and the PM H⁺‐coupled transport system in K⁺/Na⁺ homeostasis control in NaCl‐stressed calluses of Populus euphratica. An obvious Na⁺/H⁺ antiport was seen in salinized cells; however, NaCl stress caused a net K⁺ efflux, because of the salt‐induced membrane depolarization. H₂O₂ levels, regulated upwards by salinity, contributed to ionic homeostasis, because H₂O₂ restrictions by DPI or DMTU caused enhanced K⁺ efflux and decreased Na⁺/H⁺ antiport activity. NaCl induced a net Ca²⁺ influx and a subsequent rise of [Ca²⁺]_cyt, which is involved in H₂O₂‐mediated K⁺/Na⁺ homeostasis in salinized P. euphratica cells. When callus cells were pretreated with inhibitors of the Na⁺/H⁺ antiport system, the NaCl‐induced elevation of H₂O₂ and [Ca²⁺]_cyt was correspondingly restricted, leading to a greater K⁺ efflux and a more pronounced reduction in Na⁺/H⁺ antiport activity. Results suggest that the PM H⁺‐coupled transport system mediates H⁺ translocation and triggers the stress signalling of H₂O₂ and Ca²⁺, which results in a K⁺/Na⁺ homeostasis via mediations of K⁺ channels and the Na⁺/H⁺ antiport system in the PM of NaCl‐stressed cells. Accordingly, a salt stress signalling pathway of P. euphratica cells is proposed.     

Effect of potassium deficiency on the plasma membrane H+-ATPase of the wood ray parenchyma in poplar

The effect of K⁺ deficiency on the plasma membrane (PM) H⁺‐ATPase was studied in young stems of poplar plants (Populus tremula × tremuloides) grown with low or full‐strength K⁺ supply. Immunological assays using different antibodies were applied to test if K⁺ deficiency affects the amount of immunodetectable PM H⁺‐ATPases in the stem tissue. The monoclonal antibody clone 46 E5 B11 revealed an increased abundance of PM H⁺‐ATPases under conditions of low K⁺ supply, and immunolabelling experiments showed that this increase was restricted to vessel‐associated cells (VACs) of the wood ray parenchyma. Replacement of the monoclonal antibody by a polyclonal antibody against PM H⁺‐ATPase gave a specific immunoreactivity on blots as well as tissue sections too, but the labelling intensity showed no difference between plants with low or full‐strength K⁺ supply. Measurements of extracellular H⁺ concentrations using non‐invasive, H⁺‐selective microelectrodes revealed a lowering of the pH at the surface of VACs and an enhancement of net efflux of H⁺ in plants grown with low K⁺ supply. The present results indicate an up‐regulation of specific isoforms of the PM H⁺‐ATPase in VACs under K⁺‐deficient conditions and suggest a key role for these PM H⁺‐ATPases in unloading K⁺ from the xylem stream.     

The involvement of wheat U‐box E3 ubiquitin ligase TaPUB1 in salt stress tolerance

U‐box E3 ubiquitin ligases play important roles in the ubiquitin/26S proteasome machinery and in abiotic stress responses. TaPUB1‐overexpressing wheat (Triticum aestivum L.) were generated to evaluate its function in salt tolerance. These plants had more salt stress tolerance during seedling and flowering stages, whereas the TaPUB1‐RNA interference (RNAi)‐mediated knock‐down transgenic wheat showed more salt stress sensitivity than the wild type (WT). TaPUB1 overexpression upregulated the expression of genes related to ion channels and increased the net root Na⁺ efflux, but decreased the net K⁺ efflux and H⁺ influx, thereby maintaining a low cytosolic Na⁺/K⁺ ratio, compared with the WT. However, RNAi‐mediated knock‐down plants showed the opposite response to salt stress. TaPUB1 could induce the expression of some genes that improved the antioxidant capacity of plants under salt stress. TaPUB1 also interacted with TaMP (Triticum aestivum α‐mannosidase protein), a regulator playing an important role in salt response in yeast and in plants. Thus, low cytosolic Na⁺/K⁺ ratios and better antioxidant enzyme activities could be maintained in wheat with overexpression of TaPUB1 under salt stress. Therefore, we conclude that the U‐box E3 ubiquitin ligase TaPUB1 positively regulates salt stress tolerance in wheat.     

High affinity k uptake in maize roots: a lack of coupling with h efflux

We report here on the putative coupling between a high affinity K⁺ uptake system which operates at low external K⁺ concentrations (K_m = 10-20 micromolar), and H⁺ efflux in roots of intact, low-salt-grown maize plants. An experimental approach combining electrophysiological measurements, quantification of unidirectional K⁺(⁸⁶Rb⁺) influx, and the simultaneous measurement of net K⁺ and H⁺ fluxes associated with individual cells at the root surface with K⁺- and H⁺-selective microelectrodes was utilized. A microelectrode system described previously (IA Newman, LV Kochian, MA Grusak, and WJ Lucas [1987] Plant Physiol 84: 1177-1184) was used to quantify net ion fluxes from the measurement of electrochemical potential gradients for K⁺ and H⁺ ions within the unstirred layer at the root surface. No evidence for coupling between K⁺ uptake and H⁺ efflux could be found based on: (a) extremely variable K⁺:H⁺ flux stoichiometries, with K⁺ uptake often well in excess of H⁺ efflux; (b) dramatic time-dependent variability in H⁺ extrusion when both fluxes were measured at a particular location along the root over time; and (c) a lack of pH sensitivity by the high affinity K⁺ uptake system (to changes in external pH) when net K⁺ uptake, unidirectional K⁺(⁸⁶Rb⁺) influx, and K⁺-induced depolarizations of the membrane potential were determined in uptake solutions buffered at pH values from pH 4 to 8. Based on the results presented here, we propose that high affinity active K⁺ absorption into maize root cells is not mediated by a K⁺/H⁺ exchange mechanism. Instead, it is either due to the operation of a K⁺-H⁺ cotransport system, as has been hypothesized for Neurospora, or based on the striking lack of sensitivity to changes in extracellular pH, uptake could be mediated by a K⁺-ATPase as reported for Escherichia coli and Saccharomyces.     

Ionic and osmotic components of salt stress specifically modulate net ion fluxes from bean leaf mesophyll

Ionic mechanisms of salt stress perception were investigated by non‐invasive measurements of net H⁺, K⁺, Ca²⁺, Na⁺, and Cl^? fluxes from leaf mesophyll of broad bean (Vicia faba L.) plants using vibrating ion‐selective microelectrodes (the MIFE technique). Treatment with 90 m M NaCl led to a significant increase in the net K⁺ efflux and enhanced activity of the plasma membrane H⁺‐pump. Both these events were effectively prevented by high (10 m M ) Ca²⁺ concentrations in the bath. At the same time, no significant difference in the net Na⁺ flux has been found between low‐ and high‐calcium treatments. It is likely that plasma membrane K⁺ and H⁺ transporters, but not the VIC channels, play the key role in the amelioration of negative salt effects by Ca²⁺ in the bean mesophyll. Experiments with isotonic mannitol application showed that cell ionic responses to hyperosmotic treatment are highly stress‐specific. The most striking difference in response was shown by K⁺ fluxes, which varied from an increased net K⁺ efflux (NaCl treatment) to a net K⁺ influx (mannitol treatment). It is concluded that different ionic mechanisms are involved in the perception of the ‘ionic’ and ‘osmotic’ components of salt stress.     

Rapid calcium exchange for protons and potassium in cell walls of Chara

Net fluxes of Ca²⁺, H⁺ and K⁺ were measured from intact Chara australis cells and from isolated cell walls, using ion-selective microelectrodes. In both systems, a stimulation in Ca²⁺ efflux (up to 100 nmol m^?2 s^?1, from an influx of ～40 nmol m^?2 s^?1) was detected as the H⁺ or K⁺ concentration was progressively increased in the bathing solution (pH 7.0 to 4.6 or K⁺ 0.2 to 10mol m^?3, respectively). A Ca²⁺ influx of similar size occurred following the reverse changes. These fluxes decayed exponentially with a time constant of about 10 min. The threshold pH for Ca²⁺ efflux (pH 5.2) is similar to a reported pH threshold for acid-induced wall extensibility in a closely related characean species. Application of NH₄⁺ to intact cells caused prolonged H⁺ efflux and also transient Ca²⁺ efflux. We attribute all these net Ca²⁺ fluxes to exchange in the wall with H⁺ or K⁺. A theoretical treatment of the cell wall ion exchanges, using the ‘weak acid Donnan Manning’ (WADM) model, is given and it agrees well with the data. The role of Ca²⁺ in the cell wall and the effect of Ca²⁺ exchanges on the measured fluxes of other ions, including bathing medium acidification by H⁺ efflux, are discussed.     

Glucose-6-phosphate dehydrogenase-dependent hydrogen peroxide production is involved in the regulation of plasma membrane H+-ATPase and Na+/H+ antiporter protein in salt-stressed callus from Carex moorcroftii

Glucose‐6‐phosphate dehydrogenase (G6PDH) is important for the activation of plant resistance to environmental stresses, and ion homeostasis is the physiological foundation for living cells. In this study, we investigated G6PDH roles in modulating ion homeostasis under salt stress in Carex moorcroftii callus. G6PDH activity increased to its maximum in 100 mM NaCl treatment and decreased with further increased NaCl concentrations. K⁺/Na⁺ ratio in 100 mM NaCl treatment did not exhibit significant difference compared with the control; however, in 300 mM NaCl treatment, it decreased. Low‐concentration NaCl (100 mM) stimulated plasma membrane (PM) H⁺‐ATPase and NADPH oxidase activities as well as Na⁺/H⁺ antiporter protein expression, whereas high‐concentration NaCl (300 mM) decreased their activity and expression. When G6PDH activity and expression were reduced by glycerol treatments, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein level and K⁺/Na⁺ ratio dramatically decreased. Simultaneously, NaCl‐induced hydrogen peroxide (H₂O₂) accumulation was abolished. Exogenous application of H₂O₂ increased G6PDH, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein expression and K⁺/Na⁺ ratio in the control and glycerol treatments. Diphenylene iodonium (DPI), the NADPH oxidase inhibitor, which counteracted NaCl‐induced H₂O₂ accumulation, decreased G6PDH, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein level and K⁺/Na⁺ ratio. Western blot result showed that G6PDH expression was stimulated by NaCl and H₂O₂, and blocked by DPI. Taken together, G6PDH is involved in H₂O₂ accumulation under salt stress. H₂O₂, as a signal, upregulated PM H⁺‐ATPase activity and Na⁺/H⁺ antiporter protein level, which subsequently resulted in the enhanced K⁺/Na⁺ ratio. G6PDH played a central role in the process.     

Herbivore exposure alters ion fluxes and improves salt tolerance in a desert shrub

Plants have evolved complex mechanisms that allow them to withstand multiple environmental stresses, including biotic and abiotic stresses. Here, we investigated the interaction between herbivore exposure and salt stress of Ammopiptanthus nanus, a desert shrub. We found that jasmonic acid (JA) was involved in plant responses to both herbivore attack and salt stress, leading to an increased NaCl stress tolerance for herbivore-pretreated plants and increase in K⁺/Na⁺ ratio in roots. Further evidence revealed the mechanism by which herbivore improved plant NaCl tolerance. Herbivore pretreatment reduced K⁺ efflux and increased Na⁺ efflux in plants subjected to long-term, short-term, or transient NaCl stress. Moreover, herbivore pretreatment promoted H⁺ efflux by increasing plasma membrane H⁺-adenosine triphosphate (ATP)ase activity. This H⁺ efflux creates a transmembrane proton motive force that drives the Na⁺/H⁺ antiporter to expel excess Na⁺ into the external medium. In addition, high cytosolic Ca²⁺ was observed in the roots of herbivore-treated plants exposed to NaCl, and this effect may be regulated by H⁺-ATPase. Taken together, herbivore exposure enhance s A. nanus tolerance to salt stress by activating the JA-signalling pathway, increasing plasma membrane H ⁺ - ATPase activity, promoting cytosolic Ca²⁺ accumulation, and then restricting K⁺ leakage and reducing Na⁺ accumulation in the cytosol.     

Reactions of corn root tissue to calcium

Washing corn (Zea mays L.) root tissue in water causes loss of about one-third of the exchangeable Ca²⁺ over the first 10 to 15 minutes. Upon transfer to K⁺-containing solutions, the tissue shows a short period of rapid K⁺ influx which subsequently declines. Addition of 0.1 millimolar Ca²⁺ decreases the initial rapid K⁺ influx, but increases the sustained rate of K⁺ and Cl⁻ uptake. It was confirmed (Elzam and Hodges 1967 Plant Physiol 42: 1483-1488) that 0.1 millimolar Ca²⁺ is more effective than higher concentrations for the initial inhibition, and that Mg²⁺ will substitute.

The inhibition arises from a mild shock affect of restoring Ca²⁺. With 0.1 millimolar Ca²⁺ net H⁺ efflux is blocked for 10 to 15 minutes and the cells are depolarized by about 30 millivolts. However, 1 millimolar Ca²⁺ rapidly produces increased K⁺ influx and blocks net H⁺ efflux for only a few minutes; blockage is preceded by a brief net H⁺ influx which may restore and increase ion transport by reactivating the plasmalemma H⁺-ATPase.

Stimulation of electrogenic H⁺-pumping with fusicoccin eliminates the shock responses and minimizes Ca²⁺ effects on K⁺ influx. Fusicoccin also strongly decreases Ca²⁺ influx, but has no effect on Ca²⁺ efflux. Ice temperatures and high pH decreased Ca²⁺ efflux, but uncoupler and chlorpromazine did not.

It is suggested that the inhibitory and promotive actions of Ca²⁺ are manifested through decreases or increases in the protonmotive force.

     

Sucrose and proton cotransport in Ricinus cotyledons

In Ricinus cotyledons, evidence for proton extrusion came from observation of direct acidification of the medium in the presence of potassium salts. Increasing K⁺ influx with increasing pH suggested a link between K⁺ influx and H⁺ efflux by an H⁺ pump. The kinetics of K⁺ influx and H⁺ efflux were consistent with a 1:1 stoichiometry K⁺:H⁺, which may indicate either electrical coupling or carrier mediated exchange. The results were consistent with an H⁺ pump setting up an electrochemical potential gradient which provides the driving force for an H⁺-sucrose cotransport and the movement of K⁺. With reference to this, a model for phloem loading is suggested.     

Variations of Intracellular pH in Human Erythrocytes via K+(Na+)/H+ Exchange Under Low Ionic Strength Conditions

The change of intracellular pH of erythrocytes under different experimental conditions was investigated using the pH-sensitive fluorescent dye BCECF and correlated with (ouabain + bumetanide + EGTA)-insensitive K⁺ efflux and Cl⁻ loss. When human erythrocytes were suspended in a physiological NaCl solution (pH _o = 7.4), the measured pH _i was 7.19 ± 0.04 and remained constant for 30 min. When erythrocytes were transferred into a low ionic strength (LIS) solution, an immediate alkalinization increased the pH _i to 7.70 ± 0.15, which was followed by a slower cell acidification. The alkalinization of cells in LIS media was ascribed to a band 3 mediated effect since a rapid loss of approximately 80% of intracellular Cl⁻ content was observed, which was sensitive to known anion transport inhibitors. In the case of cellular acidification, a comparison of the calculated H⁺ influx with the measured unidirectional K⁺ efflux at different extracellular ionic strengths showed a correlation with a nearly 1:1 stoichiometry. Both fluxes were enhanced by decreasing the ionic strength of the solution resulting in a H⁺ influx and a K⁺ efflux in LIS solution of 108.2 ± 20.4 mmol (l _cells hr)⁻¹ and 98.7 ± 19.3 mmol (l _cells hr)⁻¹, respectively. For bovine and porcine erythrocytes, in LIS media, H⁺ influx and K⁺ efflux were of comparable magnitude, but only about 10% of the fluxes observed in human erythrocytes under LIS conditions. Quinacrine, a known inhibitor of the mitochondrial K⁺(Na⁺)/H⁺ exchanger, inhibited the K⁺ efflux in LIS solution by about 80%. Our results provide evidence for the existence of a K⁺(Na⁺)/H⁺ exchanger in the human erythrocyte membrane. Received: 22 December 1999/Revised: 10 April 2000     

The cyclic nucleotide‐gated channel AtCNGC10 transports Ca2+ and Mg2+ in Arabidopsis

The suppression of the cyclic nucleotide‐gated channel (CNGC) AtCNGC10 alters K⁺ transport in Arabidopsis plants. Other CNGCs have been shown to transport Ca²⁺, K⁺, Li⁺, Cs⁺ and Rb⁺ across the plasma membrane when expressed in heterologous systems; however, the ability of the AtCNGC10 channel to transport nutrients other than K⁺ in plants has not been previously tested. The ion fluxes along different zones of the seedling roots, as estimated by the non‐invasive ion‐specific microelectrode technique, were significantly different in two AtCNGC10 antisense lines (A2 and A3) in comparison to the wild type (WT). Most notably, the influxes of H⁺, Ca²⁺ and Mg²⁺ in the meristem and distal elongation zones of the antisense A2 and A3 lines were significantly lower than in the WT. The lower Ca²⁺ influx from the external media corresponded to a lower intracellular Ca²⁺ activity, which was estimated by fluorescence lifetime imaging measurements (FLIM). On the other hand, the intracellular pH values in the meristem zone of the roots of A2 and A3 seedlings were significantly lower (more acidic) than that of the WT, which might indicate a feedback block of H⁺ influx into meristematic cells caused by low intracellular pH. Under the control conditions, mature plants from the A2 and A3 lines contained significantly higher K⁺ and lower Ca²⁺ and Mg²⁺ content in the shoots, indicating disturbed long‐distance ion transport of these cations, possibly because of changes in xylem loading/retrieval and/or phloem loading. Exposing the plants in the flowering stage to various K⁺, Ca²⁺ and Mg²⁺ concentrations in the solution led to altered K⁺, Ca²⁺ and Mg²⁺ content in the shoots of A2 and A3 plants in comparison with the WT, suggesting a primary role of AtCNGC10 in Ca²⁺ (and probably Mg²⁺) transport in plants, which in turn regulates K⁺ transporters' activities.     

Na+/H+ antiporter activity of the SOS1 gene: lifetime imaging analysis and electrophysiological studies on Arabidopsis seedlings

Based on sequence analysis, the salt overly sensitive (SOS1) gene has been suggested to function as a Na⁺/H⁺ antiporter located at the plasma membrane of plant cells, being expressed mostly in the meristem zone of the root and in the parenchyma cells surrounding the vascular tissue of the stem. In this study, we compared net H⁺ and Ca²⁺ fluxes and intracellular pH and [Ca²⁺]_cyt in the root meristem zone of Arabidopsis wild‐type (WT) and sos mutants before and after salt stress. In addition, we studied the effect of pretreatment with amiloride (an inhibitor of Na⁺/H⁺ antiporters) on net ion fluxes, intracellular pH and intracellular Ca²⁺ activity ([Ca²⁺]_cyt) in WT plants and sos1 mutants before and after salt stress. Net ion fluxes were measured using microelectrode ion flux estimation (MIFE) and intracellular pH and [Ca²⁺]_cyt using fluorescence lifetime imaging microscopy (FLIM) techniques. During the first 15 min after NaCl application, sos1 mutants showed net H⁺ efflux and intracellular alkalinization in the meristem zone, whereas sos2 and sos3 mutants and WT showed net H⁺ influx and slight intracellular acidification in the meristem zone. Treatment with amiloride led to intracellular acidification and lower net H⁺ flux in WT plants and to a decrease in intracellular Ca²⁺ in WT and sos1 plants. WT plants pretreated with amiloride did not show positive net H⁺ flux and intracellular acidification. After NaCl application, internal pH shifted to higher values in WT and sos1 plants. However, absolute values of H⁺ fluxes were higher and internal pH values were lower in WT plants pretreated with amiloride compared with sos1 mutants. Therefore, the SOS1 transporter is involved in H⁺ influx into the meristem zone of Arabidopsis roots, or it may function as a Na⁺/H⁺ antiporter. Amiloride affects SOS1 and other Na⁺/H⁺ antiporters in plant cells because of its ability to decrease the H⁺ gradient across the plasma membrane.     

Electrogenic pump activity in red beet: Its relation to ATP levels and to cation influx

Summary In storage tissue ofBeta vulgaris L., carbonyl cyanidem-chlorophenylhydrazone or cyanide+salicylhydroxamic acid reduce cell electropotentials from about –200 to below –100 mV. The relationship between potential and cellular ATP level is examined during treatment with different concentrations of inhibitiors. At low ATP levels the potential rises sharply with increases in ATP, but above an ATP level of approximately 50% of the uninhibited level the potential changes very little with ATP concentration. A plot of membrane potentialvs.⁸⁶Pb⁺ influx or of potentialvs. net K⁺ uptake indicates that as the level of inhibition is decreased, the potential tends to reach a limit while cation influx and net uptake continue to increase. Resistance measurements, although subject to difficulties of interpretation, indicate no change in conductance with potential, ion flux, or ATP level. Thus the membrane potential should directly reflect electrogenic pump activity, attributed to active uncoupled H⁺ efflux. K⁺ uptake can occur against its electrochemical gradient and is attributed to a coupled K⁺ influx/H⁺ efflux pump. The results show that the electrogenic pump activity is independent of the K⁺/H⁺ exchange rate. Thus electrogenic H⁺ efflux and K⁺/H⁺ exchange may represent different transport systems, or different modes of operation of a single pump with variable stoichiometry.     

K+/H+ antiporter in alkaliphilic Bacillus sp. no. 66 (JCM 9763)

A K⁺/H⁺ antiport system was detected for the first time in right-side-out membrane vesicles prepared from alkaliphilic Bacillus sp. no. 66 (JCM 9763). An outwardly directed K⁺ gradient (intravesicular K⁺ concentration, K_in, 100 mM; extravesicular K⁺ concentration, K_out, 0.25 mM) stimulated uphill H⁺ influx into right-side-out vesicles and created the inside-acidic pH gradient (ΔpH). This H⁺ influx was pH-dependent and increased as the pH increased from 6.8 to 8.4. Addition of 100 μM quinine inhibited the H⁺ influx by 75%. This exchange process was electroneutral, and the H⁺ influx was not stimulated by the imposition of the membrane potential (interior negative). Addition of K⁺ at the point of maximum ΔpH caused a rapid K⁺-dependent H⁺ eflux consistent with the inward exchange of external K⁺ for internal H⁺ by a K⁺/H⁺ antiporter. Rb⁺ and Cs⁺ could replace K⁺ but Na⁺ and Li⁺ could not. The H⁺ efflux rate was a hyperbolic function of K⁺ and increased with increasing extravesicular pH (pH_out) from 7.5 to 8.5. These findings were consistent with the presence of K⁺/H⁺ antiport activity in these membrane vesicles. Received: March 20, 1997 / Accepted: May 22, 1997     

K+ Uptake by Fermenting Escherichia coli Cells: pH Dependent Mode of the TrkA System Operating

Escherichia coli accumulates K⁺ by means of multiple transportsystems, of which TrkA is the most prominent at neutral and alkalinepH while Kup is major at acidic pH. In the present study, K⁺ uptakewas observed with cells grown under fermentative conditions at an initialpH of 9.0 and 7.3 (the medium pH decreased to 8.4 and 6.8, respectively,during the mid-logarithmic growth phase), washed with distilled water andresuspended in a K⁺ containing medium at pH 7.5 in the presence ofglucose. The kinetics for this K⁺ uptake and the amount of K⁺accumulated by the wild type and mutants having a functional TrkA orKup could confirm that K⁺ uptake by E. coli grown either at pH 9.0or pH 7.3 occurs mainly through TrkA. The following results distinguishpH dependent mode of TrkA operating: (1) K⁺ uptake was inhibited byDCCD in cells grown either at pH 9.0 or pH 7.3, although the stoichiometryof K⁺ influx to DCCD-inhibited H⁺ efflux for bacteria grownat pH 9.0 varied with external K⁺ concentration, but remained constantfor cells grown at pH 7.3; (2) K⁺ uptake was observed with an atpDmutant grown at pH 9.0 but not at pH 7.3; (3) The DCCD-inhibited H⁺efflux was increased 8-fold less by 5 mM K⁺ added into a K⁺ freemedium for bacteria grown at pH 9.0 than that for cells grown at pH 7.3;(4) the DCCD-inhibited ATPase activity of membrane vesicles from bacteriagrown at pH 9.0 was reduced a little in the presence of 100 mM K⁺,but stimulated more than 2.4-fold at pH 7.3.