In Vivo pH Regulation by a Na/H Antiporter in the Halotolerant Alga Dunaliella salina

Na⁺/H⁺ exchange activity in whole cells of the halotolerant alga Dunaliella salina can be elicited by intracellular acidification due to addition of weak acids at appropriate external pH. The changes in both intracellular pH and Na⁺ were followed. Following a mild intracellular acidification, intracellular Na⁺ content increased dramatically and then decreased. We interpret the phase of Na⁺ influx as due to the activation of the plasma membrane Na⁺/H⁺ antiporter and the phase of Na⁺ efflux as due to an active Na⁺ extrusion process. The following observations are in agreement with this interpretation: (a) the Na⁺ influx phase was sensitive to Li⁺, which is an inhibitor of the Na⁺/H⁺ antiporter, did not require energy, and was insensitive to vanadate; (b) the Na⁺ efflux phase is energy-dependent and sensitive to the plasma membrane ATPase inhibitor, vanadate. Following intracellular acidification, a drastic decrease in the intracellular ATP content is observed that is reversed when the cells regain their neutral pH value. We suggest that the intracellular acidification-induced change in the internal Na⁺ concentration is due to a combination of Na⁺ uptake via the Na⁺/H⁺ antiporter and an active, ATPase-dependent, Na⁺ extrusion. The Na⁺/H⁺ antiporter seems, therefore, to play a principal role in internal pH regulation in Dunaliella.     

Auxin and Fusicoccin Enhancement of beta-Glucan Synthase in Peas : An Intracellular Enzyme Activity Apparently Modulated by Proton Extrusion

Fusicoccin (FC), like indoleacetic acid (IAA), causes Golgi-localized β-1,4-glucan synthase (GS) activity to increase when applied to pea third internode segments whose GS activity has declined after isolation from the plant. This suggests that GS activity is modulated by H⁺ extrusion; in agreement, vanadate and nigericin inhibit the GS response. The GS response is not due to acidification of the cell wall. Treatment of tissue with heavy water, which in effect raises intracellular pH, mimics the IAA/FC GS response. However, various treatments that tend to raise cytoplasmic pH directly, other than IAA- or FC-induced H⁺ extrusion, failed to increase GS activity, suggesting that cytoplasmic pH is not the link between H⁺ extrusion and increased GS activity. Although FC stimulates H⁺ extrusion more strongly than IAA does, FC enhances GS activity at most only as much as, and often somewhat less than, IAA does. This and other observations indicate that GS enhancement is probably not due to membrane hyperpolarization, stimulated sugar uptake, or changes in ATP level, but leave open the possibility that GS is controlled by H⁺ transport-driven changes in intracellular concentrations of ions other than H⁺.     

Ethanol-Induced Activation of ATP-Dependent Proton Extrusion in Elodea densa Leaves

In Elodea densa leaves, ethanol up to 0.17 m stimulates H⁺ extrusion activity. This effect is strictly dependent on the presence of K⁺ in the medium and is suppressed by the presence of the plasmalemma H⁺-ATPase inhibitor vanadate. Stimulation of H⁺ extrusion is associated with (a) a decrease in cellular ATP level, (b) a marked hyperpolarization of transmembrane electrical potential, and (c) an increase in net K⁺ influx. These results suggest that ethanol-induced H⁺ extrusion is mediated by an activation of the plasma membrane ATP-dependent, electrogenic proton pump. This stimulating effect is associated with an increase of cell sap pH and of the capacity to take up the weak acid 5,5-dimethyloxazolidine-2,4-dione, which is interpretable as due to an increase of cytosolic pH. This indicates that the stimulation of H⁺ extrusion by ethanol does not depend on a cytosolic acidification by products of ethanol metabolism. The similarity of the effects of ethanol and those of photosynthesis on proton pump activity in E. densa leaves suggests that a common metabolic situation is responsible for the activation of the ATP-dependent H⁺-extruding mechanism.     

Plasmalemma redox activity and h extrusion: I. Activation of the h-pump by ferricyanide-induced potential depolarization and cytoplasm acidification

Ferricyanide reduction by Elodea densa leaves, in the dark, is associated with: (a) acidification of the medium; (b) decrease (about 0.2-0.3 units) of intracellular pH (measured in cell sap, cytoplasm, and vacuole); (c) depolarization of the transmembrane potential; (d) net efflux of K⁺ to the medium. Ferricyanide-induced acid secretion is markedly increased by the presence of fusicoccin (FC), and this effect is severely inhibited by the proton pump inhibitors erythrosine B and vanadate. In the presence of ferricyanide FC-induced H⁺ extrusion no longer requires the presence of K⁺ in the medium. The (ferricyanide reduced)/(H⁺ extruded) ratio varies from about 2, in the absence of FC, to about 1 when the toxin is present, and to more than 4, when ATP-driven H⁺ extrusion is inhibited by erythrosine B or by vanadate. Fusicoccin markedly reduces K⁺ release to the medium. The ratio (ferricyanide reduced)/(H⁺ extruded + K⁺ released) approaches unity under all of the three conditions considered. These results indicate that ferricyanide reduction depends on a plasmalemma system transporting only electrons to the extracellular acceptor, with consequent potential depolarization and cytoplasm acidification. Most of the protons released in the cytoplasm would be secondarily extruded by the ATP-driven pump, stimulated by both intracellular acidification and depolarization. K⁺ efflux would depend on potential depolarization.     

Effects of salt-adaptation and salt-stress on extracellular acidification and microsome phosphohydrolase activities in tomato cell suspensions

The effects of NaCl-adaptation and NaCl-stress on in vivo H⁺ extrusion and microsomal vanadate- and bafilomycin-sensitive ATPase and PPase activities were studied in tomato cell suspensions. Acidification of the external medium by 50 mM NaCl-adapted and non-adapted (control) tomato cells was similar. Extracellular acidification by both types of cells during the first hour of incubation with 2 μM fusicoccin (FC) in the presence of 100 mM NaCl was lightly increased while in the presence of 100 mM KCl it was increased by 3 (control)- and 6.5 (adapted)-fold. Extracellular alkalinization after 2 h of cell incubation in 100 mM NaCl indicated the possibility that a Na⁺/H⁺ exchange activity could be operating in both types of cells. Moreover, acidification induced by adding 100 mM NaCl + FC to non-adapted cells was relatively less affected by vanadate than that induced by 5 mM KCl + FC, which suggested that salt stress could induce some component other than H⁺ extrusion by H⁺-ATPase. In addition, no differences were observed in microsomal vanadate-sensitive ATPase activity among control, NaCl-adapted and NaCl-stressed cells, while K⁺-stimulated H⁺-PPase and bafilomycin-sensitive H⁺-ATPase activities were higher in microsomes from NaCl-adapted than in those from control cells. Likewise, the stimulation of in vivo H⁺ extrusion in NaCl adapted cells under NaCl or KCl stress in the presence of FC occurred with an inhibition of H⁺-PPase and bafilomycin-sensitive H⁺-ATPase activities and without changes in the vanadate-sensitive H⁺-ATPase activity. These results suggest that the stimulation of tonoplast proton pumps in NaCl-adapted cells, without changes in plasmalemma H⁺-ATPase, could serve to energize Na⁺ efflux across the plasmalemma and Na⁺ fluxes into vacuoles catalyzed by the Na⁺/H⁺ antiports. This revised version was published online in June 2006 with corrections to the Cover Date.     

Early changes of Cl− efflux and H+ extrusion induced by osmotic stress in Arabidopsis thaliana cells

In various plant materials changes in turgor pressure, following hyper- or hypo-osmotic stress, were associated with the activation or inactivation of the plasma membrane H⁺-ATPase, respectively. To see if the turgor changes might indirectly influence H⁺-ATPase activity by regulating ion fluxes through plasma membrane, we investigated, in cultured cells of Arabidopsis thaliana (L.) Heynh., the early effects of hyper- and hypo-osmotic stress on Cl^? fluxes in comparison, in the case of hyper-osmotic treatment, with its effect on net H⁺ extrusion. The results obtained showed that hyper-osmotic stress (200 mM mannitol) quickly reduced Cl^? efflux (?70%) from cells preloaded with ³⁶C1^? for 18 h. This inhibiting effect was independent of the simultaneous mannitol-induced stimulation of Cl^? influx and rapidly reversible after removal of the hyper-osmotic treatment. The inhibition of Cl^? efflux was associated with a stimulation of net H⁺ extrusion, and these two effects showed the same dependence on the external mannitol concentration. Fusicoccin (FC, 20 µM), which stimulated H⁺ extrusion to about the same extent as 200 mM mannitol, did not affect Cl^? efflux. When cells preloaded with ³⁶C1^? for 18 h in the presence of mannitol (from 25 up to 200 mM) were eluted in a mannitol-free medium an early and strong increase in Cl^? efflux was found. The increase of Cl- efflux was already detectable for a small hypo-osmotic jump (25 mM), and was reduced (?50%) by the anion channel inhibitor A9C (300 µM). These results lead to exclude a direct causal relationship mediated by E_m changes between the effects of osmoticum on Cl^? efflux and net H⁺ extrusion, and favour the view that the changes in turgor pressure induced by hyper/hypo-osmotic stress may respectively induce an early inactivation/activation of stretch-sensitive anion channels.     

Regulation of Apoplastic pH in Source Leaves of Vicia faba by Gibberellic Acid

Leaf discs of broad bean (Vicia faba L.), peeled on the spongy mesophyll side, rapidly altered the pH of the surrounding medium (apoplast). Using pH indicator paper appressed against the leaf, immediately after peeling, initial apoplastic pH was estimated to be 4.5. Changes in the apoplastic pH were measured with a microelectrode placed into a 100-microliter drop of an unbuffered solution (2 millimolar KCl, 0.5 millimolar CaCl₂, and 200 millimolar mannitol) on the peeled surface. Discs acidified the medium until the pH stabilized at about 5.0 (about 10 minutes). Acidification was inhibited by 50 micromolar sodium vanadate, an inhibitor of the plasmalemma H⁺-ATPase and attenuated by omitting the osmoticum or potassium ions from the medium. Fusicoccin (10 micromolar) greatly enhanced the rate of acidification. The presence of 0.1 to 1 micromolar gibberellic acid resulted in a slower rate of medium acidification. Gibberellic acid appeared to modulate the activity of the H⁺-translocating ATPase located at the plasma membrane of the mesophyll cells.     

Effects of Two Plasmalemma ATPase Inhibitors on H+ Extrusion and Intracellular pH in Elodea densa Leaves

Beffagna, N. and Romani, G. 1988. Effects of two plasmalemmaATPase inhibitors on H⁺ extrusion and intracellular pH in Elodeadensa leaves.—J. exp. Bot. 39: 1033–1043. Elodea leaves in the dark show very little exchange of H⁺ withthe medium in the external pH range between 5.0 and 6.0. Thepresence of fusicoccin and potassium in the medium markedlystimulates H⁺ extrusion. Fusicoccin- and K⁺ -induced H⁺ extrusionis inhibited by either erythrosin B (EB) or Na-orthovanadate,two inhibitors of H⁺ transporting plasma membrane ATPase. EBcompletely inhibits it from the first 30 min of treatment, whensupplied at pH 5.5 at a concentration of 30 mmol m^–3.Vanadate also inhibits H⁺ extrusion, this effect becoming evidentonly after 45 min of treatment. After this time inhibition iscomplete with 250 mmol m^–3 vanadate but only partial forlower concentrations. In the presence of either inhibitor the intracellular pH, measuredas cell sap pH, is significantly lowered. When the intracellularpH changes are determined on vacuole and, separately, on cytoplasmby the weak acid and base distribution method, acidificationof both compartments is found to accompany the blocking of H⁺extrusion by either of the inhibitors. Key words: Intracellular pH, vanadate, erythrosin B, H⁺pumping     

Elucidation of antimicrobial activity and mechanism of action by N-substituted carbazole derivatives

Compounds belonging to a carbazole series have been identified as potent fungal plasma membrane proton adenosine triphophatase (H⁺-ATPase) inhibitors with a broad spectrum of antifungal activity. The carbazole compounds inhibit the adenosine triphosphate (ATP) hydrolysis activity of the essential fungal H⁺-ATPase, thereby functionally inhibiting the extrusion of protons and extracellular acidification, processes that are responsible for maintaining high plasma membrane potential. The compound class binds to and inhibits the H⁺-ATPase within minutes, leading to fungal death after 1–3 h of compound exposure in vitro. The tested compounds are not selective for the fungal H⁺-ATPase, exhibiting an overlap of inhibitory activity with the mammalian protein family of P-type ATPases; the sarco(endo)plasmic reticulum calcium ATPase (Ca²⁺-ATPase) and the sodium potassium ATPase (Na⁺,K⁺-ATPase). The ion transport in the P-type ATPases is energized by the conversion of ATP to adenosine diphosphate (ADP) and phosphate and a general inhibitory mechanism mediated by the carbazole derivative could therefore be blocking of the active site. However, biochemical studies show that increased concentrations of ATP do not change the inhibitory activity of the carbazoles suggesting they act as allosteric inhibitors. Furthermore decreased levels of intracellular ATP would suggest that the compounds inhibit the H⁺-ATPase indirectly, but Candida albicans cells exposed to potent H⁺-ATPase-inhibitory carbazoles result in increased levels of intracellular ATP, indicating direct inhibition of H⁺-ATPase.     

Changes in Chloride Fluxes and Cytosolic pH Induced by Abscisic Acid in Elodea densa Leaves

The effects of ABA on intracellular pH, net H⁺ extrusion, Cl^? fluxes and E_m values were studied in Elodea densa leaves, and the possible relationships between the ABA-induced changes of cytosolic pH and of Cl^? and H⁺ fluxes were investigated. Cytosolic and vacuolar pH were calculated by the weak acid and weak base distribution method. The data show that, also in this material (a water plant without stomata), ABA induces a decrease in both net H⁺ extrusion and intracellular pH, and strongly inhibits Cl^? efflux. No significant effect of ABA is detectable on E_m values, either at short or long intervals in the presence or absence of K⁺. Cl^? efflux is apparently independent of the activity of the plasmalemma H⁺ pump and of the E_m values. Conversely, it strongly depends on the value of cytosolic pH, a larger efflux occurring for the lower pH values both in the presence and in the absence of ABA. These results indicate that the ABA-induced cytosolic acidification cannot be the cause but, possibly, a consequence of the decrease in Cl^? efflux, and are consistent with the hypothesis of a primary role of ABA in regulating Cl^? efflux, presumably by directly affecting a class of Cl^?-permeable channels.     

Effects of vanadate on the ATP content,ATPase activity and phosphate absorption capacity of maize roots

Although the sensitivity of the plasma membrane H⁺-ATPase to vanadate is well known, the metabolic response of plant cells to vanadate is less well characterised in vivo and its use as an inhibitor in whole plant experiments has had mixed success. Experiments with maize (Zea mays, L.) roots and with purified plasma membrane fractions from the same tissues showed that exposure to vanadate caused: (i) a reduction in the capacity for phosphate uptake; (ii) a reduction in the extractable ATPase activity from the tissue; and (iii) a significant increase in the ATP level. The measurements on the extractable ATPase activity and the ATP level showed that the effect of vanadate developed slowly, apparently reflecting the slow accumulation of intracellular vanadate. The marked effect of vanadate on the ATP level-exposure to 500 M vanadate for 5 h doubled the ATP content of the roots tips-indicates that there is no stringent control over the ATP level in the roots and that the plasma membrane H⁺-ATPase activity is likely to have a significant role in determining the ATP level under normal conditions.     

Plasma-membrane H+-ATPases are expressed in pitchers of the carnivorous plant Nepenthes alata Blanco

Nepenthes is a unique genus of carnivorous plants that can capture insects in trapping organs called pitchers and digest them in pitcher fluid. The pitcher fluid includes digestive enzymes and is strongly acidic. We found that the fluid pH decreased when prey accumulates in the pitcher fluid of Nepenthes alata. The pH decrease may be important for prey digestion and the absorption of prey-derived nutrients. To identify the proton pump involved in the acidification of pitcher fluid, plant proton-pump homologs were cloned and their expressions were examined. In the lower part of pitchers with natural prey, expression of one putative plasma-membrane (PM) H⁺-ATPase gene, NaPHA3, was considerably higher than that of the putative vacuolar H⁺-ATPase (subunit A) gene, NaVHA1, or the putative vacuolar H⁺-pyrophosphatase gene, NaVHP1. Expression of one PM H⁺-ATPase gene, NaPHA1, was detected in the head cells of digestive glands in the lower part of pitchers, where proton extrusion may occur. Involvement of the PM H⁺-ATPase in the acidification of pitcher fluid was also supported by experiments with proton-pump modulators; vanadate inhibited proton extrusion from the inner surface of pitchers, whereas bafilomycin A₁ did not, and fusicoccin induced proton extrusion. These results strongly suggest that the PM H⁺-ATPase is responsible for acidification of the pitcher fluid of Nepenthes. Received: 8 June 2000 / Accepted: 8 August 2000     

The effect of divalent cations on Na+ tolerance in Charophytes. I: Char a buckellii

Abstract The comparative Na⁺ tolerance of Chora buckellii cultured in freshwater (FW) or artificial Waldsea water (AWW, which contains about 110 mol m^?3 each Na ⁺, Mg²⁺, Cl^? and SO^2-₄ was tested with respect to the external Na⁺ to Ca²⁺ ratio (Na: Ca). Fifty per cent of FW cells subjected to 70 mol m^?3 NaCl, which raised Na:Ca from 10: 1 to 700: 1 and the external osmotic pressure from 0.024 to 0.402 MPa, died within 6 d. Death was associated with the loss of Na/K selectivity, H⁺ -pump activity and turgor. Restoration of Na:Ca to 10:1 in high Na⁺ medium with CaCl₂ ensured 100% survival and maintained H⁺-pump activity and Na/K selectivity of FW cells. Turgor was regulated within 3 d with net uptake of Na ⁺, K⁺ and Cl^? in the vacuolc. Mg²⁺ was not as effective as Ca²⁺ in enhancing survival or maintaining H⁺ -pump activity and Na/K selectivity of FW cells in the presence of elevated Na⁺. However, turgor was regulated within 3 d by accumulation of Cl^? and an unknown cation in the vacuole. All AWW cells subjected to an increase of 70 mol m ^?3 NaCl, which raised Na: Ca from 16:1 to 25: 1 and the external osmotic pressure from 0.915 to 1.22 MPa, survived and maintained H ⁺ -pump activity. Turgor was regulated within 6d by accumulating Na ⁺, K⁺ and Cl^? in the vacuole. All AWW cells subjected to 70molm^?3 NaCl in a medium in which Na:Ca was equal to 700:1 survived and maintained H ⁺ -pump activity, but showed loss of Na/K selectivity. Turgor was regulated with an unknown osmoticum(a) within 6 d.     

New Drugs for the Na+/H+ Exchanger. Influence of Na+ Concentration and Determination of Inhibition Constants with a Microphysiometer

The NHE-1 isoform of the Na⁺/H⁺ exchanger is excessively activated in cardiac cells during ischemia. Hence NHE-1 specific inhibitors are being developed since they could be of beneficial influence under conditions of cardiac ischemia and reperfusion. In this study, the Cytosensor™ microphysiometer was used to measure the potency of four new drug molecules, i.e., EMD 84021, EMD 94309, EMD 96785 and HOE 642 which are inhibitors of the isoform 1 of the Na⁺/H⁺ exchanger. The experiments were performed with Chinese hamster ovary cells (CHO K1) which are enriched in the NHE-1 isoform of the Na⁺/H⁺ antiporter. The Na⁺/H⁺ exchanger was stimulated with NaCl and the rate of extracellular acidification was quantified with the Cytosensor. The proton exchange rate was measured as a function of the NaCl concentration in the range of 10–138 mm NaCl stimulation. The proton exchange rate followed Michaelis-Menten kinetics with a K _M = 30 ± 4 mm for Na⁺. Addition of either one of the four inhibitors decreased the acidification rate. The IC₅₀ values of the four compounds could be determined as 23 ± 7 nm for EMD 84021, 5 ± 1 nm for EMD 94309, 9 ± 2 nm for EMD 96785 and 8 ± 2 nm for HOE 642 at 138 mm NaCl, in good agreement with more elaborate biological assays. The IC₅₀ values increased with the NaCl concentration indicating competitive binding of the inhibitor. The microphysiometer approach is a fast and simple method to measure the activity of the Na⁺/H⁺ antiporter and allows a quantitative kinetic analysis of the proton excretion rate. Received: 3 September 1998/Revised: 20 November 1998     

Boric acid stimulates the plasma membrane H+-ATPase of ungerminated lily pollen grains

The stimulation of the plasma membrane (PM) H⁺-ATPase by boric acid was studied on a microsomal fraction (MF) obtained from ungerminated, boron-dependent pollen grains of Lilium longiflorum Thunb. which usually need boron for germination and tube growth. ATP hydrolysis and H+ transport activity increased by 14 and 18%, respectively, after addition of 2-4 mM boric acid. The optimum of boron stimulation was at pH 6.5-8.5 for ATP hydrolysis and at pH 6.5-7.5 for H⁺ transport. No boron stimulation was detected when vanadate was added to the MF, whereas an increase of 10-20% in ATP hydrolysis and H⁺ transport was still measured in the presence of inhibitors specific for V -type ATPase (nitrate and bafilomycin) and F-type ATPase (azide), respectively. A vanadate-sensitive increase in ATP hydrolysis activity was also observed in partially permeabilized vesicles (0.001%[w/v] Triton X-100) suggesting a direct interaction between borate and the PM H⁺-ATPase rather than a weak acid-induced stimulation. Additionally, we measured the effect of boron on membrane voltage (V_m) of ungerminated pollen grains and observed small hyperpolarizations in 48% of all experiments. Exposing pollen grains to a more acidic pH of 4 caused a depolarization, followed in some experiments by a repolarization (21%). In the presence of 2 mM boron such hyperpolarizations, perhaps caused by an enhanced activity of the H⁺-ATPase, were measured in 58% of all tested pollen grains. The effects of boron on V_m may be reduced by additional stimulation of a K⁺ inward current of opposite direction to the H⁺-ATPase. All experiments indicate that boron stimulates an electrogenic transport system in the plasma membrane which is sensitive to vanadate and has a pH optimum around 7, i.e. the plasma membrane H⁺-ATPase. A boron-increased PM H⁺-ATPase activity in turn may stimulate germination and growth of pollen tubes.     

Osmotic adaptation in halotolerant yeast, Debaryomyces nepalensis NCYC 3413: role of osmolytes and cation transport

Debaryomyces nepalensis NCYC 3413, a food spoiling yeast isolated from rotten apple, has been previously demonstrated as halotolerant yeast. In the present study, we assessed its growth, change in cell size, and measured the intracellular polyol and cations (Na⁺ or K⁺) accumulated during growth in the absence and presence of different concentrations of salts (NaCl and KCl). Cells could tolerate 2 M NaCl and KCl in defined medium. Scanning electron microscopic results showed linear decrease in mean cell diameter with increase in medium salinity. Cells accumulated high amounts of K⁺ during growth at high concentrations of KCl. However, it accumulated low amounts of Na⁺ and high amounts of K⁺ when grown in the presence of NaCl. Cells grown in the absence of salt showed rapid influx of Na⁺/K⁺ on incubation with high salt. On incubation with 2 M KCl, cells grown at 2 M NaCl showed an immediate efflux of Na⁺ and rapid uptake of K⁺ and vice versa. To withstand the salt stress, osmotic adjustment of intracellular cation was accompanied by intracellular accumulation of polyol (glycerol, arabitol, and sorbitol). Based on our result, we hypothesize that there exists a balanced efflux and synthesis of osmolytes when D. nepalensis was exposed to hypoosmotic and hyperosmotic stress conditions, respectively. Our findings suggest that D. nepalensis is an Na⁺ excluder yeast and it has an efficient transport system for sodium extrusion.     

Scanning ion‐selective electrode technique and X‐ray microanalysis provide direct evidence of contrasting Na+ transport ability from root to shoot in salt‐sensitive cucumber and salt‐tolerant pumpkin under NaCl stress

Grafting onto salt‐tolerant pumpkin rootstock can increase cucumber salt tolerance. Previous studies have suggested that this can be attributed to pumpkin roots with higher capacity to limit the transport of Na⁺ to the shoot than cucumber roots. However, the mechanism remains unclear. This study investigated the transport of Na⁺ in salt‐tolerant pumpkin and salt‐sensitive cucumber plants under high (200 mM) or moderate (90 mM) NaCl stress. Scanning ion‐selective electrode technique showed that pumpkin roots exhibited a higher capacity to extrude Na⁺, and a correspondingly increased H⁺ influx under 200 or 90 mM NaCl stress. The 200 mM NaCl induced Na⁺/H⁺ exchange in the root was inhibited by amiloride (a Na⁺/H⁺ antiporter inhibitor) or vanadate [a plasma membrane (PM) H⁺‐ATPase inhibitor], indicating that Na⁺ exclusion in salt stressed pumpkin and cucumber roots was the result of an active Na⁺/H⁺ antiporter across the PM, and the Na⁺/H⁺ antiporter system in salt stressed pumpkin roots was sufficient to exclude Na⁺. X‐ray microanalysis showed higher Na⁺ in the cortex, but lower Na⁺ in the stele of pumpkin roots than that in cucumber roots under 90 mM NaCl stress, suggesting that the highly vacuolated root cortical cells of pumpkin roots could sequester more Na⁺, limit the radial transport of Na⁺ to the stele and thus restrict the transport of Na⁺ to the shoot. These results provide direct evidence for pumpkin roots with higher capacity to limit the transport of Na⁺ to the shoot than cucumber roots.     

H-pumping driven by the vanadate-sensitive ATPase in membrane vesicles from corn roots

The initial rate of quenching of quinacrine fluorescence was used to monitor Mg:ATP-dependent H⁺-pumping in membrane vesicles from corn (Zea mays L. cv WF9 × MO17) roots and obtain a preparation in which vanadate-sensitive H⁺-pumping could be observed. Separation of membranes on a linear sucrose density gradient resulted in two distinct peaks of H⁺-pumping activity: a major one, at density 1.11 grams per cubic centimeter, was sensitive to NO₃⁻ and resistant to vanadate, while a minor one, at density 1.17 grams per cubic centimeter, was substantially resistant to NO₃⁻ and sensitive to vanadate. A membrane fraction enriched in the vanadate-sensitive H⁺-pump could be obtained by washing microsomes prepared in the presence of 10% glycerol with 0.25 molar KI. The kinetics of inhibition of H⁺-pumping by vanadate in this membrane preparation indicated that most of the H⁺-pumping activity in this fraction is sensitive to inhibition by vanadate, 50% inhibition being reached at about 60 micromolar vanadate. This value is fairly close to that observed for inhibition by vanadate of the ATPase activity in similar experimental conditions (40 micromolar). The inhibitor sensitivity, divalent cation dependence, pH optimum (6.5), and K_m for ATP (0.7 millimolar) of the H⁺-pumping activity match quite closely those reported for the plasma membrane ATPase of corn roots and other plant materials.     

Glucose- and K+-induced acidification in different yeast species

The process of acidification of the external medium after addition of glucose and subsequently of KCl to a suspension of yeast cells varies substantially from species to species. After glucose it is most pronounced inSaccharomyces cerevisiae andSchizosaccharomyces pombe but is very much lower inLodderomyces elongisporus, Dipodascus magnusii andRhodotorula gracilis. Both the buffering capacity and the varied effects of vanadate, suloctidil and erythrosin B indicate that the acidification is by about one-half due to the activity of plasma membrane H⁺-ATPase and by about one-half to the extrusion of acidic metabolites from cells. This is supported by the finding that a respiratory quotient greater than one (in various strains ofS. cerevisiae and inS. pombe) is indicative of a greater buffering capacity and overall acidification of the medium. Taking into account the virtually negligible buffering capacity of the medium in the pH range where the effect of K⁺ is observed, the effect of K⁺ is generally of a similar magnitude as that of adding glucose. It is clearly dependent on (anaerobic) production of metabolic energy, quite distinct from the dependence of the H⁺-ATPase-caused acidification.     

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.