Stimulation of Weak Acid Uptake and Increase in Cell Sap pH as Evidence for Fusicoccin- and K-Induced Cytosol Alkalinization

In maize root segments fusicoccin induced a consistent increase in cell sap pH (taken as representative of vacuolar pH). This effect was markedly enhanced by the presence of K⁺ in the medium, whereas in the absence of fusicoccin K⁺ did not significantly influence cell sap pH. Treatment with a weak acid at 2 mm concentration inhibited the uptake of a different (¹⁴C-labeled) weak acid fed at a lower concentration, thus suggesting that acidification of the cytoplasm inhibits weak acid uptake. Fusicoccin and K⁺ increased the rate of uptake of 5,5-dimethyloxazolidine-2,4-dione, butyric acid, or isobutyric acid slightly when fed separately, strongly when fed in combination. The synergism between fusicoccin and K⁺ in stimulating weak acid uptake was parallel to that observed for the stimulation of H⁺ extrusion. Application of the weak acid distribution method to a condition of `quasi-equilibrium' indicated that fusicoccin induces a cytosolic pH increase of about 0.14 unit. These results are interpreted as providing circumstantial evidence that fusicoccin- and K⁺- induced stimulation of H⁺ extrusion led to an alkalinization of the cytosol, and that other early metabolic responses, such as an increase in malate level, are a consequence of the increase in cytosolic pH.     

Potassium Transport and Regulation of Intracellular pH in Elodea densa Leaves

The effects of extracellular K⁺ concentration ([K⁺]_o) on the pH of cell sap, “bulk cytoplasm” and vacuole have been investigated in Elodea densa leaves under conditions of either low or high activity of the plasmalemma electrogenic H⁺ pump. Cell sap pH was evaluated directly in the cell sap expressed after freezing and thawing. Cytoplasmic and vacuolar pH were calculated by the weak base and weak acid distribution method, DMO and benzylamine appearing to be a suitable acid and base, respectively, for this purpose in this material. When added to the basal medium (no rapidly permeating ions present), 5 mM K⁺ induced an increase in intracellular pH, larger for the cell sap and the vacuole (about 0.2 units), and smaller but still significant for the cytoplasm (0.07 units). This alkalinizing effect of K⁺ was thus associated with a significant decrease in the pH difference across the tonoplast. The alkalinizing effect of K⁺ was markedly and synergistically enhanced by the presence of fusicoccin, a condition inducing a marked activation of H⁺ extrusion and of K⁺ uptake. The correlation between these effects of [K⁺]_o on intracellular pH and those on H⁺ extrusion indicates that changes in extracellular K⁺ concentration, and thus in K⁺ influx, can influence cytoplasmic and vacuolar pH by modulating the rate of H⁺ extrusion by the plasmalemma H⁺ pump.     

Dissociation of H + extrusion from K+ uptake by means of lipophilic cations

Abstract Dissociation of active H⁺ extrusion (?ΔH⁺) from K⁺ uptake in pea and maize root segments was attempted by substituting K⁺ in the incubation medium with lipophilic cations assumed to enter the cell by passive, non-specific, permeation through the lipid component of the plasmalemma. Among the compounds tested, tributylbenzylammonium significantly stimulated ?ΔH⁺ in the absence of other monovalent cations in the medium. This effect was much more evident when the experiment was carried out in the presence of fusicoccin, which strongly stimulates proton extrusion and monovalent cation uptake, and hyperpolarizes the trans-membrane electric potential in these materials. Also the lipophilic cations tetraphenylphosphonium, dimethyldibenzylammonium and hexylguanidine markedly stimulated FC-promoted ?ΔH⁺. Octylguanidine at a low concentration induced an early stimulation followed by a strong inhibition of ?ΔH⁺. A complete lack of additivity was observed between the effects of lipophilic cations and that of K⁺ on H⁺ extrusion. Lipophilic cations severely inhibited K⁺ uptake. These data are interpreted as supporting the view of an electric, rather than a chemical, (namely, involving the same carrier system) nature of the coupling of active H⁺ extrusion with K⁺ influx.     

Inhibition of fusicoccin-stimulated K+/H+ transport in root tips from maize seedlings pretreated with Chlorsulfuron

Abstract Fusicoccin (FC)-stimulated K⁺ (₈₆Rb) uptake and proton extrusion of maize (Zea mays) root apical segments were inhibited by pretreatment of 4-day-old seedlings with the herbicide Chlorsulfuron. In the range of Chlorsulfuron concentrations 0.01-10 mmol m^?3, the percentage of inhibition was 15% at 0.01 mmol m^?3 and progressively increased with Chlorsulfuron concentration up to 60% at 10 mmol m^?3. At the maximum concentration tested (10 mmol m^?3), the inhibition was evident after 1.5 h of pre-treatment. The binding of FC to microsomal fractions of root segments from Chlorsulfuron-pretreated seedlings was inhibited by 30%. It is suggested that Chlorsulfuron causes an alteration at the plasmalemma level involving the FC binding sites. The ineffectiveness of Chlorsulfuron in inhibiting FC-stimulaled K⁺ uptake when administered to excised segments, while inhibiting the enzyme acetolactate synthase, pointed out by Ray (1984) as the site of action of Chlorsulfuron in pea plants, suggests that the observed inhibition of K⁺ uptake and H⁺ extrusion is not induced by Chlorsulfuron inhibition of this enzyme. An alternative site of action of Chlorsulfuron is hypothesized in maize plants.     

Effect of Exogenous Putrescine, Spermidine, and Spermine on K Uptake and H Extrusion through Plasmamembrane in Maize Root Segments

The action of exogenous polyamines (putrescine, spermidine, and spermine) on `washing' and fusicoccin-stimulated K⁺ uptake and H⁺ extrusion through the plasmamembrane in maize (Zea mays L., hybrid line Plenus S 516) root apical segments was studied. The results showed that polyamines inhibit the washing-stimulated K⁺ influx and H⁺ extrusion without interfering with K⁺ uptake and H⁺ extrusion stimulated by fusicoccin. Spermidine appeared to be the most effective in inhibiting K⁺ uptake and H⁺ extrusion while putrescine showed a smaller inhibiting action with respect to the others. The analysis of kinetic constants indicated that the polyamines behave as competitive inhibitors with respect to K⁺.     

Potassium and Phosphate Uptake in Corn Roots: Further Evidence for an Electrogenic H/K Exchanger and an OH/Pi Antiporter

Evidence is presented that K⁺ uptake in corn root segments is coupled to an electrogenic H⁺/K⁺ -exchanging plasmalemma ATPase while phosphate uptake is coupled to an OH⁻/Pi antiporter. The plasmalemma ATPase inhibitor, diethylstilbestrol, or the stimulator, fusicoccin, altered K⁺ uptake directly and phosphate uptake indirectly. On the other hand, mersalyl, an OH⁻/Pi antiporter inhibitor, inhibited phosphate uptake instantly but only slightly affected K⁺ uptake. Collapse of the proton gradient across the membrane by (p-trifluoromethoxy) carbonyl cyanide phenylhydrazone resulted in immediate inhibition of K⁺ uptake but only later inhibited phosphate uptake. Changing the pH of the absorption solution had opposite effects on K⁺ and phosphate uptake. In addition, a 4-hour washing of corn root tissue induced a 5-fold increase in the rate of K⁺ uptake with little or no lag, but only a 2- to 3-fold increase in phosphate uptake with a 30- to 45-minute lag. Collectively these differences strongly support the coupling of an electrogenic H⁺/K⁺ -exchanging ATPase to an OH⁻/Pi antiporter in corn root tissue.     

Ion Fluxes and Phytochrome Protons in Mung Bean Hypocotyl Segments: II. Fluxes of Chloride, Protons, and Orthophosphate in Apical and Subhook Segments

The active form of phytochrome (Pfr) decreased CI⁻ uptake by subhypocotyl hook segments of Phaseolus aureus Roxb. and increased uptake by apical segments. Pfr had similar effects on Pi [³²Pi] uptake. Modulations of Pi [³²Pi] uptake were detectable 10 minutes following photoconversion. Pfr may modulate Pi influx across the plasmalemma. Pfr inhibited H⁺ extrusion by subhook segments and enhanced extrusion by apical hook segments. No rapid effects on H⁺ extrusion were found. Phytochrome may regulate a K⁺ -H⁺ exchange process. The differential responses of the two regions of the hypocotyl are discussed with respect to Pfr-mediated changes in growth and development.     

Potassium Transport in Corn Roots : III. Perturbation by Exogenous NADH and Ferricyanide

It has recently been reported that plasmalemma electron transport may be involved in the generation of H⁺ gradients and the uptake of ions into root tissue. We report here on the influence of extracellular NADH and ferricyanide on K⁺ (⁸⁶Rb⁺) influx, K⁺ (⁸⁶Rb⁺) efflux, net apparent H⁺ efflux, and O₂ consumption in 2-centimeter corn (Zea mays [A632 × Oh43]) root segments and intact corn roots. In freshly excised root segments, NADH had no effect on O₂ consumption and K⁺ uptake. However, after the root segments were given a 4-hour wash in aerated salt solution, NADH elicited a moderate stimulation in O₂ consumption but caused a dramatic inhibition of K⁺ influx. Moreover, net apparent H⁺ efflux was significantly inhibited following NADH exposure in 4-hour washed root segments.

Exogenous ferricyanide inhibited K⁺ influx in a similar fashion to that caused by NADH, but caused a moderate stimulation of net H⁺ efflux. Additionally, both reagents substantially altered K⁺ efflux at both the plasmalemma and tonoplast.

These complex results do not lend themselves to straightforward interpretation and are in contradiction with previously published results. They suggest that the interaction between cell surface redox reactions and membrane transport are more complex than previously considered. Indeed, more than one electron transport system may operate in the plasmalemma to influence, or regulate, a number of transport functions and other cellular processes. The results presented here suggest that plasmalemma redox reactions may be involved in the regulation of ion uptake and the `wound response' exhibited by corn roots.

     

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.     

Irpexil (Methyl-acethydroxamoilacetate)-induced changes in some membrane functions and transmembrane potential in wheat seedling root segments

Abstract

A strong K⁺-dependent, fusicoccin-sensitive increase in acidic extrusion paralleled by a corresponding increase in net K⁺ uptake and hyperpolarization of transmembrane electrical potential was observed in the root segments of wheat seedlings cultured in diluite CaSO₄+0.27 mM Methyl-acethydroxamoilacetate. Moreover, K⁺-induced depolarization was also increased up to three times.     

Abscisic acid effects on intracellular pH and ion fluxes in barley leaf segments

Changes in intracellular pH and in H⁺, K⁺ and Cl^? fluxes were evaluated in different experimental conditions in leaf segments of barley (Hordeum vulgare cv. Georgie) incubated in the dark, at pH 5.5, in the presence or absence of abscisic acid (ABA), and a comparison was made between the effects of ABA and those of erythrosin B (EB), a plasmalemma H⁺-pump inhibitor. In all conditions tested, ABA induced a cell sap acidification, an alkalinization of the external medium, a decrease in K⁺ intracellular contents, and an increase in the contents of Cl^?. The ABA-induced decrease in K⁺ content was chiefly due to the inhibition of K⁺ influx. On the contrary, ABA did not influence the uptake of Cl^?, but inhibited Cl^? efflux, the inhibition satisfactorily accounting for the larger Cl^? content observed in the presence of the hormone. The intracellular acidification and the decrease in apparent outward net transport of H⁺ observed with ABA were seemingly not associated with the activity of the proton pump, the transmembrane electrical potential difference, or K⁺ transport. On the contrary, a correlation was evident with the changes in Cl^? content. These results and, in particular, the similarity between the effects of ABA and those induced by 4,4 -diisothiocyano-2,2-disulfonic acid stilbene (DIDS), a Cl^? channel-blocking agent, suggest that the ABA-induced changes in intracellular pH and in H⁺ transport might depend on the capability of ABA to inhibit Cl^? efflux, more than on a primary inhibition of the H⁺ pump, and propose an important role for ABA in regulating the Cl^? channels.     

Close coupling between extrusion of H+ and uptake of K+ by barley roots

Extrusion of H⁺ by intact barley (Hordeum vulgare L.) roots was automatically titrated. Simultaneously, uptake of K⁺ into the roots, transport of K⁺ through the roots, and (as a residual term) accumulation of K⁺ within the root tissue were determined. When no monovalent cation was present in the medium the steady rate of H⁺ release was close to zero. Addition of K⁺ stimulated H⁺ extrusion within less than 1 min. The stimulation of H⁺ release was apparently limited only by the movement of K⁺ through the apoplast of the roots. The steady rate of H⁺ extrusion depended on the availability of external K⁺ and saturated at a K⁺ concentration of about 100 mol· dm^-3. Half-maximum rates of net K⁺ uptake and H⁺ extrusion were reached at a K⁺ concentration of about 10 mol·dm^-3. With (slowly absorbable) sulfate as the only anion present, the stoichoimetry between H⁺ release and net K⁺ uptake was one. In conclusion, the uptake of K⁺ across the plasmalemma of the cells of the root cortex is electrically coupled to H⁺ extrusion.     

Suitability of Arabidopsis for studies on intracellular pH regulation: correlation between H+ pump activity, cytosolic pH and malate level in wild-type and in a mutant partially insensitive to fusicoccin

Data are presented on the suitability of Arabidopsis thaliana seedlings for studies on intracellular pH regulation. In this material, grown in the dark in liquid medium, the determination of weak acid distribution at equilibrium provides an adequate method for calculating cytosolic pH values, in spite of the failure of benzylamine as a vacuolar pH probe. The stimulation of the H⁺ pump by K⁺ or K⁺ and fusicoccin (FC) is associated with a marked alkalinization of both cytosol and cell sap, and with a strong increase in malate level, whereas its inhibition by erythrosin B (EB) leads to the opposite effects. A good quantitative correlation is evident between the changes in net H⁺ extrusion and those in intracellular pH and malate content, in particular, with FC+K⁺. Cell sap buffer capacity is strongly influenced by the different treatments, its changes being substantially accounted for by changes in malate level. A comparison between the values of intracellular pH and malate level in wt and in the 5-2 mutant shows that in the mutant the cytosolic pH is always more acidic, and the intracellular alkalinization induced by FC+K⁺ and also by K⁺ alone is significatively lower. These results support the view that the partial insensitivity of 5-2 to FC is due to a reduced functionality of the H⁺-extruding system on which FC acts, and that the depression of the H⁺ pump activity in the mutant does not depend on a possible regulation by constitutively higher cytosolic pH values.     

Relationship of Transplasmalemma Redox Activity to Proton and Solute Transport by Roots of Zea mays

Transplasmalemma redox activity, monitored in the presence of exogenous ferricyanide stimulates net H⁺ excretion and inhibits the uptake of K⁺ and α-aminoisobutyric acid by freshly cut or washed, apical and subapical root segments of corn (Zea mays L. cv “Seneca Chief”). H⁺ excretion is seen only following a lag of about 5 minutes after ferricyanide addition, even though the reduction of ferricyanide occurs before 5 minutes and continues linearly. Once detected, the enhanced rate of H⁺ excretion is retarded by the ATPase inhibitors N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and vanadate. A model is presented in which plasmalemma redox activity in the presence of ferricyanide involves the transport only of electrons across the plasmalemma, resulting in a depolarization of the membrane potential and activation of an H⁺-ATPase. Such a model implies that this class of redox activity does not provide an additional and independent pathway for H⁺ transport, but that the activity may be an important regulator of H⁺ excretion. The 90% inhibition of K⁺ (⁸⁶Rb⁺) uptake within 2 minutes after ferricyanide addition can be contrasted with the 5 to 15% inhibition of uptake of α-aminoisobutyric acid. The possibility exists that a portion of the K⁺ and most of the α-aminoisobutyric acid uptake inhibitions are related to the ferricyanide-induced depolarization of the membrane potential, but that the redox state of some component of the K⁺ uptake system may also regulate K⁺ fluxes.     

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

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

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.     

Fusicoccin receptors. Evidence for endogenous ligand

The binding of fusicoccin to the microsomal preparations of maize roots in vitro is increased several-fold when segments of the tissue are washed for 2 h in distilled water before homogenization. Addition of freeze-dried wash solution to microsomal preparations of spinach leaves or fresh roots, washed roots, or coleoptiles of maize inhibited the binding of fusicoccin to particulate fractions. The freeze-dried material also blocked fusicoccin-promoted H⁺ extrusion from maize root segments. Roots may contain one or more water-soluble compounds competing with fusicoccin at the receptor level; such ligands might play a physiological role as modulators of the H⁺/K⁺ exchange system in higher plants.Abbreviation FC Fusicoccin     

Effects of Cr on proton extrusion, potassium uptake and transmembrane electric potential in maize root segments

Abstract Proton extrusion of maize root Zea mays segments, was inhibited by the presence of Cr (o.n. + 6; present in solution as CrO₄^2-, Cr₂O₇^2-) in the incubation medium: the minimum inhibiting concentration was 2 × 10^?3 mol m^?3 and the inhibition progressively increased with Cr concentration. Cr inhibited proton extrusion. Also, when this activity was stimulated by the presence of K⁺ or fusicoccin (FC) in the incubation medium, the K⁺ and FC stimulating effect was still present when proton extrusion was inhibited by Cr. In addition, Cr inhibited K⁺ uptake. This inhibition was higher (50%) at K⁺ concentrations up to 1 mol m^?3 lower (15%) at higher K⁺ concentrations. This result indicates that the system responsible for K⁺ uptake operating at low K⁺ concentrations is more sensitive to Cr inhibition. Cr had no effect on transmembrane electric potential (PD). The depolarizing and hyper-polarizing effect of K+ and FC, respectively, were not affected by Cr; but Cr enhances the depolarizing effect of the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCP). These results indicate that Cr inhibited the proton translocating mechanism coupled with K⁺ uptake, but did not change the net transport of charges through the plasmalemma. The Cr effect is discussed, taking into account the possibility of a direct effect of Cr at the membrane level or, alternatively, of an effect on some metabolic processes controlling membrane function.     

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.     

Further Characterization on the Transport Property of Plasmalemma NADH Oxidation System in Isolated Corn Root Protoplasts

Recent experiments show that exogenous NADH increases the O₂ consumption and uptake of inorganic ions into isolated corn (Zea mays L. Pioneer Hybrid 3320) root protoplasts (Lin 1982, Proc Natl Acad Sci USA 79: 3773-3776). A mild treatment of protoplasts with trypsin released most of the NADH oxidation system from the plasmalemma (Lin 1982 Plant Physiol 70: 326-328). Further studies on this system showed that exogenous NADH (1.5 millimolar) tripled the proton efflux from the protoplasts thus generating a greater electrochemical proton gradient across the plasmalemma. Trypsin also released ubiquinone (11.95 nanomoles per milligrams protein) but not flavin or cytochrome from the system. Kinetic analyses showed that 1.5 millimolar NADH quadrupled V_max of the mechanism I (saturable) component of K⁺ uptake, while K_m was not affected. Diethylstibestrol and vanadate inhibited basal (ATPase-mediated) K⁺ influx and H⁺ efflux, while NADH-stimulated K⁺ uptake was not or only slightly inhibited. p-Chloromercuribenzene-sulfonic acid, N,N′-dicyclohexylcarbodiimide, ethidium bromide, and oligomycin inhibited both ATPase- and NADH-mediated H⁺ and K⁺ fluxes. A combination of 10 millimolar fusicoccin and 1.5 millimolar NADH gave an 11-fold increase of K⁺ influx and a more than 3-fold increase of H⁺ efflux. It is concluded that a plasmalemma ATPase is not involved in the NADH-mediated ion transport mechanism. NADH oxidase is a -SH containing enzyme (protein) and the proton channel is an important element in this transport system. Fusicoccin synergistically stimulates the effect of NADH on K⁺ uptake.