Chlorophyll is not the primary photoreceptor for the stimulation of P-type H+ pump and growth in variegated leaves of Coleus×hybridus

There has been persisting controversy over the role of photosynthesis in the stimulation of the plasma membrane H⁺-ATPase and growth of dicotyledonous leaves by light. To investigate this, we compared the effects of light on growth, H⁺ net efflux and membrane potential (V_m) of strips which contained either only chlorophyll-free (white) mesophyll cells or chlorophyll-containing (green) cells cut from variegated Coleus leaves. White mesophyll cells responded to white, blue and red light with a hyperpolarization of V_m, an acidification of the apoplast and a promotion of growth, all of which began after a lag of 2–7 min. In contrast, green mesophyll cells showed a biphasic light response in which the hyperpolarization and the acidification were preceded by a rapid depolarization of V_m and an alkalinization of the apoplast. Nevertheless, green and white tissues showed comparable growth promotions in response to light. The light response of the leaf mesophyll is a composite of two separate photosystems. The initial depolarization and alkalinization are mediated by photosynthesis and blocked by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The slower hyperpolarization, acidification and growth response, on the other hand, are clearly in response to light absorption by pigments other than chlorophyll. Received: 11 February 2000 / Accepted: 2 May 2000     

The effect of light on membrane potential, apoplastic pH and cell expansion in leaves of Pisum sativum L. var. Argenteum.

The connection between three light responses of green leaf cells-membrane potential (V_m), H⁺ net efflux and growth, was analyzed. Illumination of mesophyll cells in leaves from Argenteum peas caused two rapid responses: (i) a de- and repolarization of V_m and (ii) an alkalinization of the apoplast. The rapid responses were completely eliminated by the photosynthetic inhibitor 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) but not affected by ortho-vanadate, an inhibitor of the plasma membrane (PM) H⁺-ATPase. The rapid changes were followed by a set of delayed responses: (i) a slow, gradual hyperpolarization of V_m, (ii) a gradual acidification of the mesophyll apoplast and (iii) an increased rate of elongation. These three light responses persisted under DCMU but were completely eliminated by vanadate. The data show that the delayed (in contrast to the rapid) responses were due to a stimulation of PM H⁺ pumps which occurred independently of non-cyclic photosynthetic electron transport and the “dark” processes depending on it. When the rapid responses were blocked by DCMU, light-induced acidification, hyperpolarization of the membrane potential and growth proceeded simultaneously. A shared (4-min) lag phase indicated slower signal processing in mesophyll than in epidermal cells where light stimulation of PM H⁺ pumps was rapid. Received: 3 September 1998 / Accepted: 15 October 1998     

The membrane potential of Arabidopsis thaliana guard cells; depolarizations induced by apoplastic acidification

The apoplastic pH of guard cells probably acidifies in response to light, since light induces proton extrusion by both guard cells and epidermal leaf cells. From the data presented here, it is concluded that these apoplastic pH changes will affect K⁺ fluxes in guard cells of Arabidopsis thaliana (L.) Heynh. Guard cells of this species were impaled with double-barrelled microelectrodes, to measure the membrane potential (E_m) and the plasma-membrane conductance. Guard cells were found to exhibit two states with respect to their E_m, a depolarized and a hyperpolarized state. Apoplastic acidification depolarized E_m in both states, though the origin of the depolarization differed for each state. In the depolarized state, the change in E_m was the result of a combined pH effect on instantaneously activating conductances and on the slow outward rectifying K⁺ channel (s-ORC). At a more acidic apoplastic pH, the current through instantaneously activated conductances became more inwardly directed, while the maximum conductance of s-ORC decreased. The effect on s-ORC was accompanied by an acceleration of activation and deactivation of the channel. Experiments with acid loading of guard cells indicated that the effect on s-ORC was due to a lowered intracellular pH, caused by apoplastic acidification. In the hyperpolarized state, the pH-induced depolarization was due to a direct effect of the apoplastic pH on the inward rectifying K⁺ channel. Acidification shifted the threshold potential of the channel to more positive values. This effect was accompanied by a decrease in activation times and an increase of deactivation times, of the channel. From the changes in E_m and membrane conductance, the expected effect of acidification on K⁺ fluxes was calculated. It was concluded that apoplastic acidification will increase the K⁺-efflux in the depolarized state and reduce the K⁺-influx in the hyperpolarized state. Received: 28 April 1997 / Accepted: 10 November 1997     

Effect of cytoplasmic acidification on the membrane potential of T-lymphocytes: Role of trace metals

Summary The effect of lowering intracellular pH on the membrane potential (E _m ) of rat thymic lymphocytes was studied using the potential-sensitive dyebis-oxonol. Cells were acid loaded by addition of the electroneutral K⁺/H⁺ exchanging ionophore nigericin. Acidification to pH 6.3 in Na⁺-free solution resulted in a biphasic change inE _m : an early transient hyperpolarization followed by a sustained depolarization. These changes were associated with a rise in cytosolic free Ca²⁺ ([Ca²⁺] _i ). The hyperpolarization was eliminated when the change in [Ca²⁺] _i was prevented using BAPTA, an intracellular Ca²⁺ chelator. Moreover, a similar hyperpolarization was elicited by elevation of [Ca²⁺] _i at physiological pH _i using ionomycin, suggesting involvement of Ca²⁺-activated K⁺ channels. In contrast, the depolarization phase could not be mimicked by raising [Ca²⁺] _i with ionomycin. However, intracellular BAPTA effectively inhibited the acidificationinduced depolarization. Inhibition was also obtained by extracellular addition of EGTA or dithiothreitol, even when the external free Ca²⁺ concentration remained unaltered. These observations suggested a possible role of contaminating trace metals. Cytosolic acidification is envisaged to induce intracellular accumulation of one or more trace metals, which induces the observed changes inE _m . Accordingly, similar changes inE _m can be induced without acidification by the addition of small amounts of Cu²⁺ to the medium. The ionic basis of theE _m changes induced by acidification and the significance of these observations are discussed.     

Light-induced membrane potential changes of epidermal and mesophyll cells in growing leaves of Pisum sativum

Light transiently depolarizes the membrane of growing leaf cells. The ionic basis for changes in cell membrane electrical potentials in response to light has been determined separately for growing epidermal and mesophyll cells of the argenteum mutant of pea (Pisum sativum L.). In mesophyll cells light induces a large, transient depolarization that depends on the external Cl^– concentration, is unaffected by changes in the external Ca²⁺ or K⁺ concentration, is stimulated by K⁺-channel blockers tetraethylammonium (TEA⁺) and Ba²⁺, and is inhibited by 3-(3-4-dichlorophenyl)-1,1-dimethylurea (DCMU). In isolated epidermal tissue, light induces a small, transient depolarization followed by a hyperpolarization of the membrane potential. The depolarization is enhanced by increasing the external Ca²⁺ concentration and by addition of Ba²⁺, and is not sensitive to DCMU. Epidermal cells in contact with mesophyll display a depolarization resembling the response of the underlying mesophyll cells. The light-induced depolarization in mesophyll cells seems to be mediated by an increased efflux of Cl^– while the membrane-potential changes in epidermal strips reflect changes in the fluxes of Ca²⁺ and in the activity of the proton-pumping ATPase.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - CCCP carbonylcyanide m-chlorophenylhydrazone - DCMU 3-(3-4-dichlorophenyl)-1,1-dimethylurea - LID _e light-induced depolarization in epidermal cells - LID _m light-induced depolarization in mesophyll cells - LIH light-induced hyperpolarization - TEA⁺ tetraethylammonium Ecotrans paper #43. This research was supported by National Science Foundation grants DCB-8903744 and MCB-9220110 to E.V.     

Chloroplast-generated ROS dominate NaCl- induced K+ efflux in wheat leaf mesophyll

Mesophyll K⁺ retention ability has been recently reported as an important component of salinity stress tolerance in wheat. In order to investigate the role of ROS in regulating NaCl^-induced K⁺ efflux in wheat leaf mesophyll, a series of pharmacological experiments was conducted using MV (methyl viologen, superoxide radical inducer), DPI (an inhibitor of NADPH oxidase), H₂O₂ (to mimic apoplastic ROS), and EGCG ((−)-Epigallocatechin gallate, ROS scavenger). Mesophyll pre-treatment with 10 μM MV resulted in a significantly higher NaCl^-induced K⁺ efflux in leaf mesophyll, while 50 μM EGCG pre-treatment alleviated K⁺ leakage under salt stress. No significant change in NaCl^-induced K⁺ efflux in leaf mesophyll was found in specimens pre-treated by H₂O₂ and DPI, compared with the control. The highest NaCl^-induced H⁺ efflux in leaf mesophyll was also found in samples pre-treated with MV, suggesting a futile cycle between increased H⁺-ATPase activity and ROS-induced K⁺ leak. Overall, it is suggested that, under saline stress, K⁺ efflux from wheat mesophyll is mediated predominantly by non-selective cation channels (NSCC) regulated by ROS produced in chloroplasts, at least in bread wheat.     

Effect of external K+, Ca2+, and Ba2+ on membrane potential and ionic conductance in rat astrocytes

Summary 1. The purpose of this study was (a) to identify if astrocytes show a similar non-Nernstian depolarization in low K⁺ or low Ca²⁺ solutions as previously found in human glial and glioma cells, and (b) to analyze the influence of the K⁺ conductance on the membrane potential of astrocytes.2. The membrane potential (E_m) and the ionic conductance were studied with whole-cell patch-clamp technique in neonatal rat astrocytes (5–9 days in culture) and in human glioma cells (U-251MG).3. In 3.0 mM K⁺, E_m was –75 ± 1.0 mV (mean ± SEM,n=39) in rat astrocytes and –79 ± 0.7 mV (n=5) in U-251MG cells. In both cell types E_m changed linearly to the logarithm of [K⁺]₀ between 3.0 and 160 mM K⁺. K⁺ free medium caused astrocytes to hyperpolarize to –93 ± 2.7 mV (n=21) and U-251MG cells to depolarize to –27 ± 2.1 mV (n=3).4. The I-E curve did not show inward rectification in astrocytes at this developmental stage. The slope conductance (g) exhibited only a small decrease (–19%) in K⁺ free solution and no significant change in 160 mM K⁺.5. Ba²⁺ (1.0 mM) depolarized astrocytes to –45 ± 2.9 mV (n=11), decreasing the slope conductance (g) by 42.4 ± 8.3% (n=11). Ca²⁺ free solution depolarized astrocytes to –53 ± 3.4 mV (n=12) and resulted in a positive shift of the I-E curve, increasing g by 15.3 ± 8.2% (n=8).6. Calculations indicated that a block of K⁺ channels explains the depolarizing effect of Ba²⁺. The effects of K⁺ free or Ca²⁺ free solutions on E_m can be explained by a transformation of K⁺ channels to non-specific leakage channels. That astrocytes show a different reaction to low K⁺ than glioma cells can be related to the lack of inwardly rectifying K⁺ channels in astrocytes at this developmental stage.     

Extracellular charge adsorption influences intracellular electrochemical homeostasis in amphibian skeletal muscle

The membrane potential measured by intracellular electrodes, E_m, is the sum of the transmembrane potential difference (E₁) between inner and outer cell membrane surfaces and a smaller potential difference (E₂) between a volume containing fixed charges on or near the outer membrane surface and the bulk extracellular space. This study investigates the influence of E₂ upon transmembrane ion fluxes, and hence cellular electrochemical homeostasis, using an integrative approach that combines computational and experimental methods. First, analytic equations were developed to calculate the influence of charges constrained within a three-dimensional glycocalyceal matrix enveloping the cell membrane outer surface upon local electrical potentials and ion concentrations. Electron microscopy confirmed predictions of these equations that extracellular charge adsorption influences glycocalyceal volume. Second, the novel analytic glycocalyx formulation was incorporated into the charge-difference cellular model of Fraser and Huang to simulate the influence of extracellular fixed charges upon intracellular ionic homeostasis. Experimental measurements of E_m supported the resulting predictions that an increased magnitude of extracellular fixed charge increases net transmembrane ionic leak currents, resulting in either a compensatory increase in Na⁺/K⁺-ATPase activity, or, in cells with reduced Na⁺/K⁺-ATPase activity, a partial dissipation of transmembrane ionic gradients and depolarization of E_m.     

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.     

Boron induces hyperpolarization of sunflower root cell membranes and increases membrane permeability to k

Although many studies have alluded to a role for boron (B) in membrane function, there is little evidence for a direct effect of B on the plasmalemma of higher plant cells. These studies were conducted to demonstrate, by electrophysiological techniques, a direct effect of B on the membrane potential (E_m) of sunflower (Helianthus annuus [L.], cv Mammoth Grey Stripe) root tip cells and to determine if the response to B occurs rapidly enough to account for the previously observed effects of B on ion uptake. By inserting a glass microelectrode into an individual cell in the root tip, the E_m of the cell was determined in basal salt medium (BSM), pH 6.0. The perfusion solution surrounding the root tissue was then changed to BSM + 50 micromolar H₃BO₃, pH 6.0. The exposure to B induced a significant plasmalemma hyperpolarization in sunflower root cells within 20 minutes. After just 3 minutes of exposure to B, the change in E_m was already significantly different from the negligible change in E_m observed over time in root cells never exposed to B. Membrane hyperpolarization could be caused by a stimulation of the proton pump or by a change in the conductance of one or more permeable ions. Since B has been shown to affect K⁺ uptake by plants, the electrophysiological techniques described above were used to determine if B has an effect on membrane permeability to K⁺, and could thereby lead to an increased diffusion potential. When sunflower root tips were pretreated in 50 micromolar B for 2 hours, cell membranes exhibited a significantly greater depolarization with each 10-fold increase in external [K⁺] than minus-B cells. Subsequent studies demonstrated that the depolarization due to increased external [K⁺] was also significantly greater when tissue was exposed to B at the same time as the 10-fold increase in [K⁺], indicating that the effect of B on K⁺ permeability was immediate. Analysis of sunflower root tips demonstrated that treatment in 50 micromolar B caused a significantly greater accumulation of K⁺ after 48 hours. The B-induced increase in K⁺ uptake may cause a subsequent stimulation of the H⁺-ATPase (proton pump) and lead to the observed hyperpolarization of root cell membranes. Alternatively, B may stimulate the proton pump, with the subsequent hyperpolarization resulting in an increased driving force for K⁺ influx.     

Evidence for coupling between Na+ pump activity and TEA-sensitive K+ currents in Xenopus laevis oocytes

Using the two-microelectrode voltage clamp technique in Xenopus laevis oocytes, we estimated Na⁺-K⁺-ATPase activity from the dihydroouabain-sensitive current (I _DHO) in the presence of increasing concentrations of tetraethylammonium (TEA⁺; 0, 5, 10, 20, 40 mm), a well-known blocker of K⁺ channels. The effects of TEA⁺ on the total oocyte currents could be separated into two distinct parts: generation of a nonsaturating inward current increasing with negative membrane potentials (V _M) and a saturable inhibitory component affecting an outward current easily detectable at positive V _M. The nonsaturating component appears to be a barium-sensitive electrodiffusion of TEA⁺ which can be described by the Goldman-Hodgkin-Katz equation, while the saturating component is consistent with the expected blocking effect of TEA⁺ on K⁺ channels. Interestingly, this latter component disappears when the Na⁺-K⁺-ATPase is inhibited by 10 m DHO. Conversely, TEA⁺ inhibits a component of I _DHO with a k _d of 25±4 mm at +50 mV. As the TEA⁺-sensitive current present in I _DHO reversed at –75 mV, we hypothesized that it could come from an inhibition of K⁺ channels whose activity varies in parallel with the Na⁺-K⁺-ATPase activity. Supporting this hypothesis, the inward portion of this TEA⁺-sensitive current can be completely abolished by the addition of 1 mm Ba²⁺ to the bath. This study suggests that, in X. laevis oocytes, a close link exists between the Na-K-ATPase activity and TEA⁺-sensitive K⁺ currents and indicates that, in the absence of effective K⁺ channel inhibitors, I _DHO does not exclusively represent the Na⁺-K⁺-ATPase-generated current.     

Short-and long-term effects of cadmium on transmembrane electric potential (E m) in maize roots

In this study, the effects of Cd on root growth, respiration, and transmembrane electric potential (E _m) of the outer cortical cells in maize roots treated with various Cd concentrations (from 1 μM to 1 mM) for several hours to one week were studied. The E _m values of root cells ranged between −120 and −140 mV and after addition of Cd they were depolarized immediately. The depolarization was concentration-dependent reaching the value of diffusion potential (E _D) when the Cd concentration exceeded 100 μM. The values of E _D ranged between −65 to −68 mV (−66 ± 1.42 mV). The maximum depolarization of E _m was registered approx. 2.5 h after addition of Cd to the perfusion solution and in some cases, partial (Cd > 100 μM) or complete repolarization (Cd < 100 μM) was observed within 8–10 h of Cd treatment. In the time-dependent experiments (0 to 168 h) shortly after the maximum repolarization of E _m a continuous concentration-dependent decrease of E _m followed at all Cd concentrations. Depolarization of E _m was accompanied by both increased electrolyte leakage and inhibition of respiration, especially in the range of 50 μM to 1 mM Cd, with the exception of root cells treated with 1 and 10 μM Cd for 24 and 48 h. Time course analysis of Cd impact on root respiration revealed that at higher Cd concentrations (> 50 μM) the respiration gradually declined (∼ 6 h) and then remained at this lowest level for up to 24 h. All the Cd concentrations used in this experiment induced significant inhibition of root elongation and concentrations higher than 100 μM stopped the root growth within the first day of Cd treatment. Our results suggest that Cd does not cause irreversible changes in the electrogenic plasma membrane H⁺ ATPase because fusicoccin, an H⁺ ATPase activator diminished the depolarizing effect of Cd on the E _m. The depolarization of E _m in the outer cortical cells of maize roots was the result of a cumulative effect of Cd on ATP supply, plasmalemma permeability, and activity of H⁺ ATPase.     

Transport Properties of the Tomato Fruit Tonoplast : I. Identification and Characterization of an Anion-Sensitive H-ATPase

An anion-sensitive H⁺-translocating ATPase was identified in membrane vesicles isolated from mature green tomato (Lycopersicon esculentum) fruit. The H⁺-ATPase was associated with a low density membrane population having a peak density of 1.11 grams per cubic centimeter, and its activity was inhibited by NO₃⁻, N,N′-dicyclohexylcarbodiimide and diethylstilbestrol but not by vanadate, azide, molybdate, or oligomycin. This H⁺-ATPase has an unusual pH dependence indicating both a slightly acidic and a near neutral peak of activity. Chloride was found to be a potent stimulator of ATPase activity. The K_m for the H⁺-ATPase was approximately 0.8 millimolar ATP. The characteristics of this H⁺-ATPase are very similar to those described for a number of plant cell tonoplast H⁺-ATPases suggesting that the activity identified in tomato fruit membranes is tonoplast-associated. This report demonstrates the feasibility of isolating tonoplast vesicles from acidic fruit tissues for studies of transport activities associated with fruit development and maturation.     

Plasmalemma redox activity and h extrusion in roots of fe-deficient cucumber plants

Cucumber plants (Cucumis sativus L.) with incipient Fe deficiency showed increased root capacity to reduce chelated Fe³⁺ compared to Fe-sufficient plants. When Fe-ethylenediaminete-traacetate was added to the root medium of the Fe-deficient plants, the reductase activity was associated with acidification of the medium and an increase in the net apparent K⁺ efflux. In the presence of the H⁺-ATPase inhibitor N,N′-dicyclohexylcarbodiimide the net apparent H⁺ efflux was completely suppressed, though some reductase activity was preserved, and the net apparent K⁺ efflux was significantly increased. The inhibition of the reductase activity by N,N′-dicyclohexylcarbodiimide was similar whether the pH of the medium was buffered or not. Anoxia and the protonophore carbonyl cyanide m-chlorophenyl hydrazone also caused a similar inhibition of the reductase activity. It is proposed that this redox system transports electrons only and that its activity is inhibited by plasmamembrane depolarization and anoxia. The H⁺ and K⁺ efflux associated with the reductase activity may be a result of the plasmamembrane depolarization it causes.     

Impact of fusaproliferin on primary roots of maize cultivars differing in their susceptibility to <Emphasis Type="Italic">Fusarium</Emphasis>

Effects of fusaproliferin (FUS) on membrane potential (E _M), electrolyte leakage, enzymes activity and respiration of roots, were studied in two maize cultivars (Zea mays L.), differing in their susceptibility to this toxin. In short-term experiments (≤ 6 h), E _M has been rapidly and significantly depolarized by FUS. The rapidity of E _M depolarization in tolerant cv. Lucia was more expressive in comparison with susceptible cv. Pavla, but the extent of E _M depolarization was higher in cv. Pavla. In both maize cultivars, higher depolarization of E _M was registered in cells of root zone I. In long-term experiments after the first E _M depolarization, which occurred during the first 6 h of FUS treatment, gradual depolarization continued up to 24 h and was represented not only by the active component (E _P) but also by the passive component (E _D) of E _M. The decrease in E _M and E _D was followed by a loss of K⁺ ions from FUS treated roots of both cultivars. The leak of K⁺ ions from the root cells of both root zones as well as both maize cultivars increased with the time of FUS treatment and was significantly higher in susceptible cv. Pavla than in tolerant cv. Lucia. FUS treatment of maize roots resulted in a significant decrease of root respiration which was higher in susceptible cv. Pavla than in tolerant cv. Lucia.     

Electron transport across the plasmalemma of Lemna gibba G1

Lemna gibba L., grown in the presence or absence of Fe, reduced extracellular ferricyanide with a V _max of 3.09 mol · g^-1 fresh weight · h^-1 and a K _m of 115 M. However, Fe³⁺-ethylenediaminetetraacetic acid (EDTA) was reduced only after Fe-starvation. External electron acceptors such as ferricyanide, Fe³⁺-EDTA, 2,6-dichlorophenol indophenol or methylene blue induced a membrane depolarization of up to 100 mV, but electron donors such as ferrocyanide or NADH had no effect. Light or glucose enhanced ferricyanide reduction while the concomitant membrane depolarization was much smaller. Under anaerobic conditions, ferricyanide had no effect on electrical membrane potential difference (E_m). Ferricyanide reduction induced H⁺ and K⁺ release in a ratio of 1.16 H⁺+1 K⁺/2 e^- (in +Fe plants) and 1.28 H⁺+0.8 K⁺/2 e^- (in -Fe plants). Anion uptake was inhibited by ferricyanide reduction. It is concluded that the steady-state transfer of electrons and protons proceeds by separate mechanisms, by a redox system and by a H⁺-ATPase.Abbreviations E _m electrical membrane potential difference - EDTA ethylenediaminetetraacetic acid - DCPIP dichlorophenol indophenol - +Fe control plant - -Fe iron-deficient plant - FW fresh weight - H⁺ electrochemical proton gradient     

Effects of hyper-osmotic stress on K+ fluxes, H+ extrusion, transmembrane electric potential difference and comparison with the effects of fusicoccin

The stimulation of H⁺ extrusion by hyper-osmotic stress (0.2–0.3 M mannitol) in cultured cells of Arabidopsis thaliana (L.) Heynh. was shown to be associated with an inhibition of Cl^? efflux, whereas hypo-osmotic stress, inhibiting H⁺ extrusion, early and strongly stimulated Cl^? efflux. In this paper, we investigate the contribution of other factors [K⁺ transport and transmembrane electric potential difference (E_m)] to the hyper-osmotic-induced activation of the plasma membrane (PM) H⁺-ATPase. The effects of mannitol (MA) on K⁺ transport and on E_m were compared with those of fusicoccin (FC) since the modes of action of osmotica and of the toxin in stimulating H⁺-ATPase activity seem to differ at least in some steps. The changes in H⁺ extrusion induced by hyper- or hypo-osmotic stress were opposite and could be reversed by the application of the respective opposite stress. The effect of MA on H⁺ extrusion was dependent on the presence of K⁺ (or Rb⁺) similarly to that of FC, while Na⁺ and Li⁺, which also stimulated the FC effect, were ineffective on that of MA. The MA effect was independent of the anions (Cl^?, SO₄^2?, NO₃^?) accompanying K⁺. K⁺ net uptake and K⁺ influx were stimulated by both MA and FC. Tetraethylammonium (TEA⁺) and Cs⁺ inhibited both MA- and FC-induced H⁺ extrusion, suggesting the involvement of K⁺ channels. MA (0.2 M) induced a strong hyperpolarization of E_m both in the absence and in the presence of K⁺. The hyperpolarizing effect of MA was also found when the cells were already hyperpolarized by FC, and was rapidly reversed by removing the osmoticum from the medium. In the presence of the lipophilic cation tributylbenzylammonium (TBBA⁺), MA was no longer able to stimulate H⁺ extrusion, while FC still stimulated it. In cells pretreated with TBBA⁺, which strongly depolarized E_m, the subsequent addition of FC repolarized it, while the hyperpolarizing effect of MA was lacking. On the contrary, in cells pretreated with Erythrosine B (EB), E_m was strongly depolarized and the following addition of FC did not hyperpolarize it, while the hyperpolarizing effect of MA was still observed. These results suggest that the mechanism of MA in activating H⁺ extrusion and K⁺ uptake is different from that of FC. The rise in net K⁺ uptake seems to be driven by the activation of some hyperpolarizing system that does not seem to depend on a direct activation of PM H⁺-ATPase, but rather on the inhibition of Cl^? efflux induced by hyper-osmotic stress.     

Leaf responses to drought stress in Mediterranean accessions of Solanum lycopersicum: anatomical adaptations in relation to gas exchange parameters

In a previous study, important acclimation to water stress was observed in the Ramellet tomato cultivar (TR) from the Balearic Islands, related to an increase in the water‐use efficiency through modifications in both stomatal (g_s) and mesophyll conductances (g_m). In the present work, the comparison of physiological and morphological traits between TR accessions grown with and without water stress confirmed that variability in the photosynthetic capacity was mostly explained by differences in the diffusion of CO₂ through stomata and leaf mesophyll. Maximization of g_m under both treatments was mainly achieved through adjustments in the mesophyll thickness and porosity and the surface area of chloroplasts exposed to intercellular airspace (S_c). In addition, the lower g_m/S_c ratio for a given porosity in drought‐acclimated plants suggests that the decrease in g_m was due to an increased cell wall thickness. Stomatal conductance was also affected by drought‐associated changes in the morphological properties of stomata, in an accession and treatment‐dependent manner. The results confirm the presence of advantageous physiological traits in the response to drought stress in Mediterranean accessions of tomato, and relate them to particular changes in the leaf anatomical properties, suggesting specific adaptive processes operating at the leaf anatomical level.     

Functional Consequences of Various Leucine Mutations in the M3/M4 Loop of the Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase α-Subunit

Leucines were mutated within the sequence L³¹¹ILGYTWLE³¹⁹ of the extracellular loop flanking the third (M3) and fourth (M4) transmembrane segments (M3/M4 loop) of the Torpedo Na⁺,K⁺-ATPase α-subunit. Replacement of Leu³¹¹ with Glu resulted in a considerable loss of Na⁺,K⁺-ATPase activity. Replacement of Leu³¹³ with Glu shifted the equilibrium of E₁P and E₂P toward E₁P and reduced the rate of the E₁P to E₂P transition. The reduction of the transition rate and stronger inhibition of Na⁺,K⁺-ATPase activity by Na⁺ at higher concentrations together suggest that there is interference of Na⁺ release on the extracellular side in the Leu³¹³ mutant. Thus, Leu³¹³ could be in the pathway of Na⁺ exit. Replacement of Leu³¹⁸ with Glu yielded an enzyme with significantly reduced apparent affinity for both vanadate and K⁺, with an equilibrium shifted toward E₂P and no alteration in the transition rate. The reduced vanadate affinity is due to the lower rate of production of vanadate-reactive [K⁺ ₂]E₂ caused by inhibition of dephosphorylation through reduction of the K⁺ affinity of E₂P. Thus, Leu³¹⁸ may be a critical position in guiding external K⁺ to its binding site.     

Systemin transiently depolarizes the tomato mesophyll cell membrane and antagonizes fusicoccin-induced extracellular acidification of mesophyll tissue

The plant polypeptide signal systemin induces proteinase inhibitor synthesis in tomato leaves. We show here that systemin elicits a transient depolarization of the tomato mesophyll cell membrane. Furthermore it triggers a transient decrease in the external pH of the mesophyll tissue which is followed by a sustained pH increase. In the presence of fusicoccin (which has been shown to antagonize the synthesis of proteinase inhibitors) the depolarization and transient H⁺ efflux are attenuated whereas the slower phase of the sustained electroneutral H⁺ influx persists. These results suggest that systemin-induced changes in ion transport play a role in the early phases of systemin signal transduction.