Studies of the Uptake of Nitrate in Barley : IV. Electrophysiology

Transmembrane electrical potential differences (Δψ) of epidermal and cortical cells were measured in intact roots of barley (Hordeum vulgare L. cv Klondike). The effects of exogenous NO₃⁻ on Δψ (in the concentration range from 100 micromolar to 20 millimolar) were investigated to probe the mechanisms of nitrate uptake by the high-affinity (HATS) and low-affinity (LATS) transport systems for NO₃⁻ uptake. Both transport systems caused depolarization of Δψ, demonstrating that the LATS (like the HATS) for NO₃⁻ uptake is probably mediated by an electrogenic cation (H⁺?) cotransport system. Membrane depolarization by the HATS was “inducible” by NO₃⁻, and saturable with respect to exogenous [NO₃⁻]. By contrast, depolarization by the LATS was constitutive, and first-order in response to external [NO₃⁻]. H⁺ fluxes, measured in 200 micromolar and in 5 millimolar Ca(NO₃)₂ solutions, failed to alkalinize external media as anticipated for a 2 H⁺:1 NO₃⁻ symport. However, switching from K₂SO₄ solutions (which were strongly acidifying) to KNO₃ solutions at the same K⁺ concentration caused marked reductions in H⁺ efflux. These observations are consistent with NO₃⁻ uptake by the HATS and the LATS via 2 H⁺:1 NO₃⁻ symports. These observations establish that the HATS for nitrate uptake by barley roots is essentially similar to those reported for Lemna and Zea mays by earlier workers. There are, nevertheless, distinct differences between barley and corn in their quantitative responses to external NO₃⁻.     

Extra- and Intracellular pH and Membrane Potential Changes Induced by K, Cl, H(2)PO(4), and NO(3) Uptake and Fusicoccin in Root Hairs of Limnobium stoloniferum

Short-term ion uptake into roots of Limnobium stoloniferum was followed extracellularly with ion selective macroelectrodes. Cytosolic or vacuolar pH, together with the electrical membrane potential, was recorded with microelectrodes both located in the same young root hair. At the onset of chloride, phosphate, and nitrate uptake the membrane potential transiently decreased by 50 to 100 millivolts. During Cl⁻ and H₂PO₄⁻ uptake cytosolic pH decreased by 0.2 to 0.3 pH units. Nitrate induced cytosolic alkalinization by 0.19 pH units, indicating rapid reduction. The extracellular medium alkalinized when anion uptake exceeded K⁺ uptake. During fusicoccin-dependent plasmalemma hyperpolarization, extracellular and cytosolic pH remained rather constant. Upon K⁺ absorption, FC intensified extracellular acidification and intracellular alkalinization (from 0.31 to 0.4 pH units). In the presence of Cl⁻ FC induced intracellular acidification. Since H⁺ fluxes per se do not change the pH, recorded pH changes only result from fluxes of the stronger ions. The extra- and intracellular pH changes, together with membrane depolarization, exclude mechanisms as K⁺/A⁻ symport or HCO₃⁻/A⁻ antiport for anion uptake. Though not suitable to reveal the actual H⁺/A⁻ stoichiometry, the results are consistent with an H⁺/A⁻ cotransport mechanism.     

Effects of Light on the Membrane Potential of Protoplasts from Samanea saman Pulvini : Involvement of K Channels and the H -ATPase

Rhythmic light-sensitive movements of the leaflets of Samanea saman depend upon ion fluxes across the plasma membrane of extensor and flexor cells in opposing regions of the leaf-movement organ (pulvinus). We have isolated protoplasts from the extensor and flexor regions of S. saman pulvini and have examined the effects of brief 30-second exposures to white, blue, or red light on the relative membrane potential using the fluorescent dye, 3,3′-dipropylthiadicarbocyanine iodide. White and blue light induced transient membrane hyperpolarization of both extensor and flexor protoplasts; red light had no effect. Following white or blue light-induced hyperpolarization, the addition of 200 millimolar K⁺ resulted in a rapid depolarization of extensor, but not of flexor protoplasts. In contrast, addition of K⁺ following red light or in darkness resulted in a rapid depolarization of flexor, but not of extensor protoplasts. In both flexor and extensor protoplasts, depolarization was completely inhibited by tetraethylammonium, implicating channel-mediated movement of K⁺ ions. These results suggest that K⁺ channels are closed in extensor plasma membranes and open in flexor plasma membranes in darkness and that white and blue light, but not red light, close the channels in flexor plasma membranes and open them in extensor plasma membranes. Vanadate treatment inhibited hyperpolarization in response to blue or white light, but did not affect K⁺ -induced depolarization. This suggests that white or blue light-induced hyperpolarization results from activation of the H⁺ -ATPase, but this hyperpolarization is not the sole factor controlling the opening of K⁺ channels.     

Evidence for Cotransport of Nitrate and Protons in Maize Roots : II. Measurement of NO(3) and H Fluxes with Ion-Selective Microelectrodes

We report here on an investigation of net nitrate and proton fluxes in root cells of maize (Zea mays L.) seedlings grown without (noninduced) and with (induced) 0.1 millimolar nitrate. A microelectrode system described previously (IA Newman, LV Kochian, MA Grusak, WJ Lucas [1987] Plant Physiol 84: 1177-1184) was utilized to quantify net ionic fluxes from the measurement of electrochemical potential gradients for NO₃⁻ and H⁺ within the unstirred layer at the root surface. The nitrate-inducibility, pH dependence, and concentration dependence of net NO₃⁻ uptake correlated quite closely with the electrical response of maize roots to nitrate under the same experimental conditions (as described in PR McClure, LV Kochian, RM Spanswick, JE Shaff [1990] Plant Physiol 93: 281-289). Additionally, it was found that potential inhibitors of the plasmalemma H⁺-ATPase (vandate, diethylstilbestrol), which were shown to abolish the electrical response to NO₃⁻ (in PR McClure, LV Kochian, RM Spanswick, JE Shaff [1990] Plant Physiol 93: 281-289), dramatically inhibited NO₃⁻ absorption. These results strongly indicate that the NO₃⁻ electrical response is due to the operation of a NO₃⁻ transport system in the plasmalemma of maize root cells. Furthermore, the results from the H⁺-ATPase inhibitor studies indicate that the NO₃⁻ transport system is linked to the H⁺-ATPase, presumably as a NO₃⁻/H⁺ symport. This is further supported by the pH response of the NO₃⁻ transport system (inhibition at alkaline pH values) and the change in net H⁺ flux from a moderate efflux in the absence of NO₃⁻, to zero net H⁺ flux after exposing the maize root to exogenous nitrate. Although these results can be explained by other interpretations, the simplest model that fits both the electrical responses and the NO₃⁻/H⁺ flux data is a NO₃⁻/H⁺ symport with a NO₃⁻:H⁺ flux stoichiometry >1, whose operation results in the stimulation of the H⁺-ATPase due to the influx of protons through the cotransport system.     

Bioelectrical response of the Nitella flexilis cell to illumination: A new possible state of plasmalemma in a plant cell

Transient hyperpolarization of the external cytoplasmatic membrane may be observed on rapid illumination of the Nitella flexilis cell. Several important properties of that response make the latter similar to a considerable degree to the excitation response.The condition for transient hyperpolarization is the normal functioning of the electron transport chain conjugated with non-cyclic photophosphorylation.The value of the membrane potential at the moment of hyperpolarization of the external cytoplasmic membrane, is determined by the difference in the electrochemical potential of HCO₃^? or H⁺. This state of the plasmalemma supplements the two other known states: normal and depolarized (excited), when the main ions determining membrane potential are K⁺ and Cl^?.     

The inhibitory effect of N,N′-dicyclohexylcarbodiimide in active sugar uptake by Rhodotorula glutinis

Cells of the yeast Rhodotorula glutinis on treatment with N,N′-dicyclohexylcarbodiimide (DCCD) at a concentration of about 0.5 mM fail to accumulate d-xylose, cause efflux of accumulated sugar and do not exhibit H⁺/sugar symport. The results are interpreted as being due to depolarization of the membrane potential by DCCD.     

NaCl Induces a Na/H Antiport in Tonoplast Vesicles from Barley Roots

Evidence was found for a Na⁺/H⁺ antiport in tonoplast vesicles isolated from barley (Hordeum vulgare L. cv California Mariout 72) roots. The activity of the antiport was observed only in membranes from roots that were grown in NaCl. Measurements of acridine orange fluorescence were used to estimate relative proton influx and efflux from the vesicles. Addition of MgATP to vesicles from a tonoplast-enriched fraction caused the formation of a pH gradient, interior acid, across the vesicle membranes. EDTA was added to inhibit the ATPase, by chelating Mg²⁺, and the pH gradient gradually dissipated. When 50 millimolar K⁺ or Na⁺ was added along with the EDTA to vesicles from control roots, the salts caused a slight increase in the rate of dissipation of the pH gradient, as did the addition of 50 millimolar K⁺ to vesicles from salt-grown roots. However, when 50 millimolar Na⁺ was added to vesicles from salt-grown roots it caused a 7-fold increase in the proton efflux. Inclusion of 20 millimolar K⁺ and 1 micromolar valinomycin in the assay buffer did not affect this rapid Na⁺/H⁺ exchange. The Na⁺/H⁺ exchange rate for vesicles from salt-grown roots showed saturation kinetics with respect to Na⁺ concentration, with an apparent K_m for Na⁺ of 9 millimolar. The rate of Na⁺/H⁺ exchange with 10 millimolar Na⁺ was inhibited 97% by 0.1 millimolar dodecyltriethylammonium.     

Electrogenic h-pumping pyrophosphatase in tonoplast vesicles of oat roots

A H⁺-translocating inorganic pyrophosphatase (H⁺-PPase) was associated with low density membranes enriched in tonoplast vesicles of oat roots. The H⁺-PPase catalyzed the electrogenic transport of H⁺ into the vesicles, generating a pH gradient, inside acid (quinacrine fluorescence quenching), and a membrane potential, inside positive (Oxonol V fluorescence quenching). Transport activity was dependent on cations with a selectivity sequence of Rb⁺ = K⁺ > Cs⁺; but it was inhibited by Na⁺ or Li⁺. Maximum rates of transport required at least 20 millimolar K⁺ and the K_m for this ion was 4 millimolar. Fluoride inhibited both ΔpH formation and K⁺-dependent PPase activity with an I₅₀ of 1 to 2 millimolar. Inhibitors of the anion-sensitive, tonoplast-type H⁺-ATPase (e.g. a disulfonic stilbene or NO₃⁻) had no effect on the PPase activity. Vanadate and azide were also ineffective. H⁺-pumping PPase was inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide, but its sensitivity to N,N′-dicyclohexylcarbodiimide was variable. The sensitivity to ions and inhibitors suggests that the tonoplast H⁺-PPase and the H⁺-ATPase are distinct activities and this was confirmed when they were physically separated after Triton X-100 solubilization and Sepharose CL-6B chromatography. H⁺ pumping activity was strongly affected by Mg²⁺ and pyrophosphate (PPi) concentrations. At 5 millimolar Mg²⁺, H⁺ pumping showed a K_maPP for PPi of 15 micromolar. The rate of H⁺ pumping at 60 micromolar PPi was often equivalent to that at 1.5 millimolar ATP. The results suggest PPi hydrolysis could provide another source of a proton motive force used for solute transport and other energy-requiring processes across the tonoplast and other membranes with H⁺-PPase.     

The effect of some ammonium salts on nitrate reductase level,onin vivo nitrate reduction and on nitrate content in excisedpisum sativum roots

The effect of some ammonium salts on nitrate reductase (NR) level, onin vivo nitrate reduction and on nitrate content was followed in the presence of nitrate in the medium, under changing experimental conditions, in excisedPisum sativum roots, and their effect was compared with that of KNO₃, Ca(NO₃)₂ and NaNO₃ at 15 mM NO₃ ^- concentration, i.e. at a concentration which considerably exceeded the level of saturation with nitrate with respect to nitrate reductase. The effect of ammonium salts on NR level is indirect and changes from a positive one to a strongly negative one which is dependent on the time of action of the salt, on the presence of other cations, on pH of the solution of the ammonium salt and on the nature of the anion of the ammonium salt. A positive effect on the enzyme level can be observed in the presence of other cations than NH₄ ⁺ at suitable concentrations of those ammonium salts, the solutions of which have their pH values in the acid region (i.e. NH₄H₂PO₄, (NH₄)₂SO₄ and NH₄NO₃). However their positive effect is independent of the presence of NH₄ ⁺ ions, and it is obviously the result of an increased concentration of H⁺ ions. A clear-cut negative effect on NR level can be observed after 24 h in one-salt NH₄NO₃ solution where NH₄ ⁺ is not balanced with other cations and thus certainly can adversely influence many metabolic processes, and in the solutions containing neutral (pH 6.2) and dibasic ammonium phosphates in which dissolved undissociated ammonia [(NH₃). (H₂O) which can also affect many metabolic processes incl. proteosynthesis] probably has a toxic influence. Thein vivo nitrate reduction is always depressed in excised pea roots in the presence of ammonium salts in the medium, regardless of the level of nitrate reductase. Under the described conditions, no relationship could be established between the enzyme level and the so-called metabolic NO₃ ^- pool (i.e. NO₂ ^- production under anaerobic conditions), nor between NR level and the total nitrate content in the roots. One-salt solutions of NaNO₃, Ca(NO₃)₂ and KNO₃ exert different effects on the level of nitrate reductase and on the content of NO₃ ^- in the roots, but the in vivo NO₃ ^- reduction shows the same trend as NR level in the roots influenced by these salts. Cl^- ions, supplied in NH₄C1, depress both NR level and NO₃ ^- content in the roots at higher concentrations, but they do not significantly affect the in vivo nitrate reduction in comparison with other ammonium salts. These results indicate that NR level,in vivo nitrate reduction, and nitrate uptake can be regulated in pea roots independently of each other.     

Characterization of potassium-dependent currents in protoplasts of corn suspension cells

Protoplasts obtained from corn (Zea mays) suspension cells were studied using the whole cell patch-clamp technique. One time-independent current, as well as two time-dependent currents were identified. All three currents were reduced by tetraethylammonium (9 millimolar), a K⁺ channel blocker. The time-independent current had a nearly linear current-voltage relationship and its reversal potential, defined as the voltage at which there is zero current, was highly dependent on the extracellular potassium concentration. One of the two time-dependent currents was activated, with rapid kinetics, by membrane hyperpolarization to potentials more negative than −100 millivolts. The second time-dependent current was activated with a sigmoidal time course by membrane depolarization to potentials more positive than −60 millivolts. It exhibited no inactivation and was carried primarily by potassium ions. These characteristics suggest that this latter current is caused by the voltage-dependent opening of delayed-rectifier K⁺ channels. These three currents, which are not generated by the plasmalemma H⁺-ATPase, are likely to assist in the regulation of the cellular K⁺ fluxes and membrane potential.     

Evidence for a Na/H Antiporter in Membrane Vesicles Isolated from Roots of the Halophyte Atriplex nummularia

The ATP-dependent establishment of a positive membrane potential (measured as S¹⁴CN⁻-accumulation) in membrane vesicles isolated from the roots of Atriplex nummularia Lindl. was not inhibited by NaMes and KMes at concentrations up to 140 millimolar. On the other hand, the formation of ΔpH (measured as ¹⁴C-methylamine accumulation or quenching of quinacrine fluorescence), was depressed by NaMes concentrations as low as 30 millimolar. Supply of NaMes after the ΔpH had been established brought about partial dissipation within 30 seconds. Extent of dissipation of ΔpH increased with NaMes concentration over the range tested (up to 180 millimolar). The H⁺/Na⁺ exchange indicated by these results was not due to the creation of a Na⁺ diffusion potential. Formation of ΔpH in these vesicles was stable to NO₃⁻ up to 100 millimolar; further, the dissipating effect of Na⁺ supply was apparent on a ΔpH formed in the presence of 30 millimolar NO₃⁻. Additional evidence that the origin of the membrane vesicles observed in this investigation was not the tonoplast and was probably the plasmalemma included the vanadate sensitivity of the establishment of the membrane potential.     

Putrescine-induced wounding and its effects on membrane integrity and ion transport processes in roots of intact corn seedlings

Interactions between putrescine and membrane function were examined with the use of a recently developed microelectrode system that enables us simultaneously to quantify membrane potentials and net K⁺ fluxes associated with individual cells at the root surface of an intact corn (Zea mays L.) seedling. In contrast to the results of others, our analyses indicate that exogenous putrescine (0.5 millimolar), in the absence of calcium, does not maintain membrane stability. In addition, putrescine caused a wound response characterized by a gradual depolarization of the membrane potential and a considerable net efflux of K⁺ from the root. In the presence of calcium, both short term (20 minutes) and long term (24 hours) exposure to a high concentration of exogenous putrescine (5 millimolar) also caused a reduction in the resting membrane potential and a significant K⁺ efflux. However, preincubating corn roots in a solution containing the antioxidant ascorbate ameliorated the wounding effects of putrescine and slightly increased potassium uptake. A similar preincubation in the absence of calcium did not protect membranes against putrescine-induced damage. The ameliorating effect of ascorbate on putrescine-induced membrane damage suggests that the wounding response of high putrescine levels in corn roots involves the catabolism of the polyamine by a cell wall diamine oxidase, with the concomitant production of hydrogen peroxide and free radicals resulting in peroxidative damage of the plasmalemma.     

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     

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.     

Correlated induction of nitrate uptake and membrane polypeptides in corn roots

Induction of corn (Zea mays L.) seedling root membrane polypeptides was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis in relation to induction of nitrate uptake. When nitrate uptake was studied using freshly harvested roots from 4-day old corn seedlings, a steady state rate of uptake was achieved after a lag of 2 to 3 hours. The plasma membrane fraction from freshly harvested roots (uninduced) and roots pretreated in 5 millimolar nitrate for 2.5 or 5 hours (induced) showed no differences in the major polypeptides with Coomassie blue staining. Autoradiography of the ³⁵S-methionine labeled proteins, however, showed four polypeptides with approximate molecular masses of 165, 95, 70, and 40 kilodaltons as being induced by both 2.5 and 5-hour pretreatment in 5 millimolar nitrate. All four polypeptides appeared to be integral membrane proteins as shown by Triton X-114 (octylphenoxypolyethoxyethanol) washing of the membrane vesicles. Autoradiography of the two-dimensional gels revealed that several additional low molecular weight proteins were induced. A 5-hour pretreatment in 5 millimolar chloride also induced several of the low molecular weight polypeptides, although a polypeptide of about 30 kilodaltons and a group of polypeptides around 40 kilodaltons appeared to be specifically induced by nitrate. The results are discussed in relation to the possibility that some of the polypeptides induced by nitrate treatment may be directly involved in nitrate transport through the plasma membrane.     

The electrical changes accompanying fertilization and cortical vesicle secretion in the medaka egg

The electrical properties of the egg of the medaka, Oryzias latipes, were studied before, during, and after fertilization. The resting potential of the unfertilized egg averaged ?39 ± 9 mV in Yamamoto's Ringers (Y. Ringers), but 20% of the values were between ?50 and ?60 mV. Fertilization triggers a small depolarization of 4 ± 3 mV in 10% Y. Ringers with an average duration of 20 ± 10 sec. The amplitude of this depolarization is independent of [Na⁺]_o, [Ca²⁺]_o, and [Cl^?]_o, so it appears to be due to a nonspecific leak triggered by sperm-egg fusion. The depolarization is followed by a longer hyperpolarizing phase with an average amplitude of 31 ± 12 mV. Recovery from this hyperpolarization has a fast phase lasting 155 ± 18 sec, followed by a slower phase which reaches a steady average membrane potential of ?19 ± 1 mV by 9 min after fertilization. The membrane resistance falls 10-fold during the first 2 min after fertilization, from 40 (1520 kΩ-cm²) to 3 MΩ. This is largely due to an increase in the K⁺ conductance. At the peak of the hyperpolarization, the membrane potential exhibits a 28 mV/decade [K⁺]_o dependence and a 6 mV/decade [Na⁺]_o dependence. The membrane resistance slowly recovers over the next 8 min to a value about 30% larger than before fertilization. The relation of current vs voltage was linear before, during, and after fertilization and indicated a reversal potential of ?98 ± 20 mV for the hyperpolarization peak. The egg's capacitance averaged 0.04 ± 0.01 μF (0.9 μF/cm²) before fertilization and approximately doubles within 90 sec after fertilization. It then decreases over a 9-min period, reaching a value 25% smaller than before fertilization.     

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.     

Membrane Potential and Proton Cotransport of Alanine and Phosphate as Affected by Permeant Weak Acids in Lemna gibba

The treatment of Lemna gibba plants with the weak acids (trimethylacetic acid and butyric acid), used as tools to decrease intracellular pH, induced a hyperpolarization of membrane potential, dependent on the concentration of the undissociated permeant form of the weak acid and on the value of the resting potential. Measurements were carried out both with `high potential' and `low potential' plants and the maximum values af acid induced hyperpolarizations were about 35 and 71 millivolts, respectively. Weak acids influenced also the transient light-dark membrane potential changes, typical for photosynthesizing material, suggesting a dependence of these changes on an acidification of cytoplasm. In the presence of the weak acids, the membrane depolarization induced by the cotransport of alanine and phosphate with protons was reduced; the maximum reduction (about 90%) was obtained with alanine during 2 millimolar trimethylacetic acid perfusion at pH 5. A strong inhibition of the uptake rates (up to 48% for [¹⁴C]alanine and 68% for ³²P-phosphate) was obtained in the presence of the weak acids, both by decreasing the pH of the medium and by increasing the concentration of the acid. In these experimental conditions, the ATP level and O₂ uptake rates did not change significantly. These results constitute good evidence that H⁺/solute cotransport in Lemna, already known to be dependent on the electrochemical potential difference for protons, is also strongly regulated by the cytoplasmic pH value.     

Perturbation of Chara Plasmalemma Transport Function by 2[4(2',4'-Dichlorophenoxy)phenoxy]propionic Acid

Electrophysiological measurements on internodal cells of Chara corallina Klein ex Willd., em. R.D.W. revealed that in the presence of (2-[4-(2′,4′-dichlorophenoxy)phenoxy]propionic acid) (diclofop) the membrane potential was very sensitive to the pH of the bathing medium. At pH 5.7, 100 micromolar diclofop caused a slow reduction in the electrogenic component of the membrane potential to the value of −123 ± 5 millivolts. Membrane resistance initially decreased, recovered transiently, then stabilized at approximately 65% of the control value. At pH 7.0, the potential appeared to plateau around −200 millivolts before rapidly declining to −140 ± 4 millivolts; removal of diclofop resulted in recovery of the electrogenic component. Diclofop reduced cytoplasmic ATP levels by 96.4% and 36.6% at pH 5.7 and 7.0, respectively. At pH 8.2, diclofop did not change the ATP concentration significantly, but induced a hyperpolarization of the membrane potential to near −250 millivolts, and also reduced or inhibited the dark-induced hyperpolarization; the light-induced depolarization was reduced to a lesser extent. DCMU applied in the light elicited the same response at the plasmalemma as placing cells in the dark. When K⁺ channels were opened and cells depolarized with 10 millimolar K⁺, diclofop induced a further depolarization of approximately 30 millivolts. Cells decoupled with HPO₄⁻² were still sensitive to diclofop. Currents associated with OH⁻ efflux and HCO₃⁻ influx, as measured with a vibrating probe technique, became spatially destabilized and reduced in magnitude in the presence of diclofop. After 60 minutes, most of the cell surface was engaged in a low level of OH⁻ efflux activity. The results indicate that diclofop may be a proton ionophore at pH 7.0 and 5.7. At pH 8.2, diclofop may inhibit the operation of the H⁺-ATPase and OH⁻ efflux systems associated with HCO₃⁻ transport by perturbing the control processes that integrate the two, without a reduction in ATP concentration.     

Simultaneous Measurements of Cytoplasmic K Concentration and the Plasma Membrane Electrical Parameters in Single Membrane Samples of Chara corallina

The electrophysiological properties of cytoplasm-rich fragments (single membrane samples) prepared from internodal cells of Chara corallina were explored in conjunction with K⁺-sensitive microelectrode and current-voltage (I-V) measurements. This system eliminated the problem of the inaccessible cytoplasmic layer, while preserving many of the electrical characteristics of the intact cells. In 0.1 millimolar external K concentration (K_o⁺), the resting conductance (membrane conductance G_m, 0.85 ± 0.25 Siemens per square meter (±standard error)) of the single membrane samples, was dominated by the proton pump, as suggested by the response of the near-linear I-V characteristic to changes in external pH. Initial cytoplasmic K⁺ activities (a_K+), judged most reliable, gave values of 117 ± 67 millimolar; stable a_K+ values were 77 ± 31 millimolar. Equilibrium potentials for K⁺ (Nernst equilibrium potential) (E_K) calculated, using either of these data sets, were near the mean membrane potential (V_m). On a cell-to-cell basis, however, E_K was generally negative of the V_m, despite an electrogenic contribution from the Chara proton pump. When K_o⁺ was increased to 1.0 millimolar or above, G_m rose (by 8- to 10-fold in 10 millimolar K_o⁺), the steady state I-V characteristics showed a region of negative slope conductance, and V_m followed E_K. These results confirm previous studies which implicated a K_o⁺-induced and voltage-dependent permeability to K⁺ at the Chara plasma membrane. They provide an explanation for transitions between apparent K_o⁺-insensitive and K_o⁺-sensitive (`K⁺ electrode') behavior displayed by the membrane potential, as recorded in many algae and higher plant cells.