Involvement of plasma membrane calcium influx in bacterial induction of the k/h and hypersensitive responses in tobacco

An early event in the hypersensitive response of tobacco to Pseudomonas syringae pv syringae is the initiation of a K⁺/H⁺ response characterized by specific plasma membrane K⁺ efflux, extracellular alkalinization, and intracellular acidification. We investigated the role of calcium in induction of these host responses. Suspension-cultured tobacco cells exhibited a baseline Ca²⁺ influx of 0.02 to 0.06 micromole per gram per hour as determined from ⁴⁵Ca²⁺ uptake. Following bacterial inoculation, uptake rates began to increase coincidently with onset of the K⁺/H⁺ response. Rates increased steadily for 2 to 3 hours, reaching 0.5 to 1 micromole per gram per hour. This increased Ca²⁺ influx was prevented by EGTA and calcium channel blockers such as La³⁺, Co²⁺, and Cd²⁺ but not by verapamil and nifedipine. Lanthanum, cobalt, cadmium, and EGTA inhibited the K⁺/H⁺ response in both suspension-cultured cells and leaf discs and prevented hypersensitive cell death in leaf discs. We conclude that increased plasmalemma Ca²⁺ influx is required for the K⁺/H⁺ and hypersensitive responses in tobacco.     

Transient Activation of Plasmalemma K Efflux and H Influx in Tobacco by a Pectate Lyase Isozyme from Erwinia chrysanthemi

A purified pectate lyase isozyme derived from Erwinia chrysanthemi induced rapid net K⁺ efflux and H⁺ influx in suspension-cultured tobacco cells. Comparable fluxes of other ions (Na⁺, Cl⁻) were not observed. The K⁺ efflux/H⁺ influx response began within 15 minutes after addition of enzyme to cell suspensions and continued for approximately 1 hour after which cells resumed the net H⁺ efflux exhibited prior to enzyme treatment. The response was not prolonged by a second enzyme dose 1 hour after the first. The K⁺/H⁺ response was characterized by saturation at low enzymic activity (2 × 10⁻³ units per milliliter), and inhibition by the protonophore, carbonyl cyanide m-chlorophenylhydrazone, and was not associated with membrane leakiness caused by structural cell wall damage. The total K⁺ loss and H⁺ uptake induced by enzyme was one-fourth to one-third that induced by Pseudomonas syringae pv. pisi and did not reduce cell viability. These results indicate that pectate lyase induces a K⁺ efflux/H⁺ influx response in tobacco similar to but of shorter duration than that induced by P. syringae pv. pisi during the hypersensitive response. Pectate lyase or other cell wall degrading enzymes may therefore influence the induction of hypersensitivity.     

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.     

H fluxes in excised samanea motor tissue : I. Promotion by light

Previous investigators revealed that white light-promoted leaflet opening in Samanea saman (Jacq) Merrill depends upon K⁺ uptake by extensor cells and efflux from flexor cells of the pulvinus, while dark-promoted closure depends upon K⁺ fluxes in the opposite directions. We now monitored H⁺ fluxes during pulvinar movement to test a model proposing coupled H⁺/K⁺ fluxes. H⁺ fluxes were monitored by measuring changes in the pH of a weakly buffered solution (initial pH = 5.5) bathing excised strips of extensor or flexor tissue. White light at hour 3 of the usual dark period promoted pulvinar opening, H⁺ efflux from extensor cells and uptake by flexor cells, while darkness at hours 2 to 4 of the usual light period promoted pulvinar closure, H⁺ uptake by extensor cells and efflux from flexor cells. The following conditions altered H⁺ fluxes during dark-promoted closure. (a) Light reversed the directions of the fluxes in both extensor and flexor cells. (b) Anoxia increased the rate of H⁺ uptake by extensor cells and promoted H⁺ uptake (rather than efflux) by flexor cells, consistent with an outwardly directed H⁺ pump. KCN showed similar effects initially, but they were transient. (c) Increase in external pH from 5.5 to 6.7 promoted H⁺ efflux (rather than uptake) by extensor cells and increased the rate of H⁺ efflux from flexor cells, presumably by decreasing the rate of inward diffusion. (d) Change in external K⁺ did not alter H⁺ fluxes by extensor cells, but removal of external K⁺ decreased the rate of H⁺ efflux from flexor cells by 70%. These observations support a model for coupled H⁺/K⁺ fluxes in pulvinar cells during light-and dark-promoted leaflet movements.     

Fluxes of h and k in corn roots : characterization and stoichiometries using ion-selective microelectrodes

We report here on an experimental system that utilizes ion-selective microelectrodes to measure the electrochemical potential gradients for H⁺ and K⁺ ions within the unstirred layer near the root surface of both intact 4-day-old corn seedlings and corn root segments. Analysis of the steady state H⁺ and K⁺ electrochemical potential gradients provided a simultaneous measure of the fluxes crossing a localized region of the root surface. Net K⁺ influx values obtained by this method were compared with unidirectional K⁺ (⁸⁶Rb⁺) influx kinetic data; at any particular K⁺ concentration, similar values were obtained by either technique. The ionspecific microelectrode system was then used to investigate the association between net H⁺ efflux and net K⁺ influx. Although the computed H⁺:K⁺ stoichiometry is dependent upon the choice of diffusion coefficients, the values obtained were extremely variable, and net K⁺ influx rarely appeared to be charge-balanced by H⁺ efflux. In contrast to earlier studies, we found the cortical membrane potential to be highly K⁺ sensitive within the micromolar K⁺ concentration range. Simultaneous measurements of membrane potential and K⁺ influx, as a function of K⁺ concentration, revealed similar K_m values for the depolarization of the potential (K_m 6-9 micromolar K⁺) and net K⁺ influx (K_m 4-7 micromolar K⁺). These data suggest that K⁺ may enter corn roots via a K⁺-H⁺ cotransport system rather than a K⁺/H⁺ antiporter.     

Light quality and osmoregulation in vicia guard cells : evidence for involvement of three metabolic pathways

Osmoregulation in opening stomata of epidermal peels from Vicia faba L. leaves was investigated under a variety of experimental conditions. The K⁺ content of stomatal guard cells and the starch content of guard cell chloroplasts were examined with cobaltinitrite and iodine-potassium iodide stains, respectively; stomatal apertures were measured microscopically. Red light (50 micromoles per square meter per second) irradiation caused a net increase of 3.1 micrometers in aperture and a decrease of −0.4 megapascals in guard cell osmotic potential over a 5 hour incubation, but histochemical observations showed no increase in guard cell K⁺ content or starch degradation in guard cell chloroplasts. At 10 micromoles per square meter per second, blue light caused a net 6.8 micrometer increase in aperture over 5 hours and there was a substantial decrease in starch content of chloroplasts but no increase in guard cell K⁺ content. At 25 micromoles per square meter per second of blue light, apertures increased faster (net gain of 5.7 micrometers after 1 hour) and starch content decreased. About 80% of guard cells had a higher K⁺ content after 1 hour of incubation but that fraction decreased to 10% after 5 hours. In the absence of KCl in the incubation medium, stomata opened slowly in response to 25 micomoles per square meter per second of blue light, without any K⁺ gain or starch loss. In dual beam experiments, stomata irradiated with 50 micomoles per square meter per second of red light for 3 hours opened without detectable starch loss or K⁺ gain; addition of 25 micomoles per square meter per second of blue light caused a further net gain of 4.4 micometers in aperture accompanied by substantial K⁺ uptake and starch loss. Comparison of K⁺ content in guard cells of opened stomata in epidermal peels with those induced to open in leaf discs showed a substantially higher K⁺ content in the intact tissue than in isolated peels. These results are not consistent with K⁺ (and its counterions) as the universal osmoticum in guard cells of open stomata under all conditions; rather, the data point to sugars arising from photosynthesis and from starch degradation as additional osmotica. Biochemical confirmation of these findings would indicate that osmoregulation during stomatal opening is the result of three key metabolic processes: ion transport, photosynthesis, and sugar metabolism.     

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

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

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

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

Effect of ouabain upon diuretic-sensitive K+ transport in cultured cells evidence for separate modes of operation of the transporter

(1) Unidirectional K⁺ (⁸⁶Rb) influx and efflux were measured in subconfluent layers of MDCK renal epithelial cells and HeLa carcinoma cells. (2) In both MDCK and HeLa cells, the furosemide-inhibitable and chloride-dependent component of K⁺ influx/efflux was stimulated 2-fold by a 30 min incubation in 1 · 10^?3 M ouabain. (3) Measurements of net K⁺ loss and Na⁺ gain in ouabain-treated cells at 1 h failed to show any diuretic sensitive component, confirming the exchange character of the diuretic-sensitive fluxes. (4) Prolonged incubations for 2.5 h in ouabain revealed a furosemide- and anion-dependent K⁺ (Cl^?) outward net flux uncoupled from net Na⁺ movement. Net K⁺ (Cl^?) outward flux was half-maximally inhibited by 2 μM furosemide. (5) After 2.5 h ouabain treatment, the anion and cation dependence of the diuretic-sensitive K⁺ influx/efflux were essentially unchanged when compared to untreated controls.     

Cell potentials, cell resistance, and proton fluxes in corn root tissue: effects of dithioerythritol

Studies were made of the effect of dithioerythritol on net proton flux, potassium influx and efflux, cell potential, and cell resistance in fresh and washed corn (Zea mays L. WF9XM14) root tissue. Dithioerythritol induces equal proton influx and potassium efflux rates, decreases membrane resistance, and hyperpolarizes the cell potential. Greater effects on H⁺ and K⁺ fluxes are secured at pH 7 than at pH 5. Other sulfhydryl-protecting reagents produced the same responses. No evidence could be found that dithioerythritol affected energy metabolism or membrane ATPase, and proton influx was induced in the presence of uncoupling agents.     

Increased Potassium Absorption Confers Resistance to Group IA Cations in Rubidium-Selected Suspension Cells of Brassica napus

Cell lines of suspension cultures of Brassica napus cv. Jet Neuf were identified for their ability to tolerate 100 millimolar Rb⁺, a level which was double the normally lethal concentration. Ten spontaneous isolates were obtained from approximately 5 × 10⁷ cells, one of which was reestablished as a cell suspension. This cell line, JL5, was also resistant to the other group IA cations— Li⁺, Na⁺, K⁺, and Cs⁺—and this trait was stable for at least 30 cell generations in the absence of Rb⁺ selection pressure. The growth characteristics were similar to those of sensitive cells under nonselective conditions. The selected JL5 cells were shown by analysis to have effected more net accumulation of K⁺ and Rb⁺ and less of Na⁺ than did the unselected cells. JL5 and unselected cells after 14 days of culture in basal medium contained 597.2 and 258.2 micromoles of K per gram dry weight, respectively. Michaelis-Menten kinetic analysis of K⁺ influx showed that JL5 possessed an elevated phase 1 V_max, but there was no alteration in its K_m. This is the first time that a plant mutation has been shown to have both increased influx and net absorption of a major essential cation.     

Reactions of corn root tissue to calcium

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

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

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

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

     

The effect of the uncoupler carbonyl cyanide m-chlorophenylhydrazone on K+ transport,ATP level and intracellular pH of Chlorella fusca

1. Low concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) induced net K⁺ uptake by Chlorella fusca, optimal concentrations being 3 μM CCCP in the light and 1 μM CCCP in the dark. Higher concentrations increasingly stimulated K⁺ release. 2. Measurements of the unidirectional K⁺ fluxes showed that CCCP-induced net K⁺ uptake in the light was mainly a consequence of an inhibition of efflux. In the dark, influx was slightly stimulated in addition. 3. In conditions of CCCP-induced net K⁺ uptake, the ATP level was decreased by less than 10%. With higher CCCP concentrations it fell drastically. 4. By means of the 5,5-dimethyloxazolidine-2,4-dione distribution technique, an acidification of the cell interior on the addition of CCCP was found. 5. It is concluded that uncoupler-induced net K⁺ uptake is due to an enhanced proton leakage into the cell across the plasmalemma. Intracellular acidification by this process stimulates ATP-dependent K⁺/H⁺ exchange which, in itself, is not affected at low uncoupler concentrations.     

Isolation and transport properties of protoplasts from cortical cells of corn roots

A procedure was developed for the enzymic isolation of large quantities of protoplasts from the cortex of Zea mays L. WF9 × MO 17 roots. Cortex was separated from the primary root, sectioned, and the cell walls digested for 3.5 hours in 2% (w/v) Cellulysin, 0.1% Pectolyase Y-23, 1 millimolar CaCl₂, 0.05% bovine serum albumin, 0.5 millimolar dithiothreitol in 0.6 molar mannitol (pH 5.6). Cortical cell protoplasts were collected by centrifugation and purified by flotation in a Ficoll step gradient. The yield of protoplasts was approximately 650 × 10³/gram fresh tissue. To obtain maximum yield it was essential to include an effective pectinase (Pectolyase Y-23) and protectants (bovine serum albumin and dithiothreitol) in the digestion medium.

Cortical cell protoplasts exhibited energy-dependent uptake of K⁺ (⁸⁶Rb), H₂³²PO₄⁻, and ³⁶Cl⁻ as well as net H⁺ extrusion. Ion fluxes were sustained for at least 3 hours. Influx of K⁺ was highest between pH 7.5 and 8.0, whereas the influx of H₂PO₄⁻ was greatest between pH 4.0 and 5.0. K⁺ and H₂PO₄⁻ influx and net H⁺ efflux were inhibited by respiratory poisons such as cyanide (0.1 millimolar) and oligomycin (5 micrograms per milliliter), and by inhibitors of plasma membrane ATPase such as diethylstilbestrol (50 micromolar). Calculated flux for Cl⁻ was low, but not greatly different from that observed for other plant cells. K⁺ flux was somewhat high, probably because the K⁺ concentration in the cortical cells was below steady-state. The results indicate that isolated cortical cell protoplasts retain transport properties which are similar to those of root tissue.

     

Proton/Phosphate Stoichiometry in Uptake of Inorganic Phosphate by Cultured Cells of Catharanthus roseus (L.) G. Don

Upon absorption of phosphate, cultured cells of Catharanthus roseus (L.) G. Don caused a rapid alkalinization of the medium in which they were suspended. The alkalinization continued until the added phosphate was completely exhausted from the medium, at which time the pH of the medium started to drop sharply toward the original pH value. Phosphate exposure caused the pH of the medium to increase from pH 3.5 to values as high as 5.8, while the rate of phosphate uptake was constant throughout (10-17 micromoles per hour per gram fresh weight). This indicates that no apparent pH optimum exists for the phosphate uptake by the cultured cells. The amount of protons cotransported with phosphate was calculated from the observed pH change up to the maximum alkalinization and the titration curve of the cell suspension. Proton/phosphate transport stoichiometry ranged from less than unity to 4 according to the amount of phosphate applied. At low phosphate doses, the stoichiometries were close to 4, while at high phosphate doses, smaller stoichiometries were observed. This suggests that, at high phosphate doses, activation of the proton pump is induced by the longer lasting proton influx acidifying the cytoplasm. The increased H⁺ efflux due to the proton pump could partially compensate protons taken up via the proton-phosphate cotransport system. Thus, the H⁺/H₂PO₄⁻ stoichiometry of the cotransport is most likely to be 4.     

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

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

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.

     

Effects of Temperature on H Secretion and Uptake by Excised Flexor Cells during Dark-Induced Closure of Samanea Leaflets

Previous studies reveal that dark-induced closure of Samanea leaflets is accompanied by H⁺ secretion from flexor motor cells. We now report that flexor tissue excised in the light, incubated in a weakly buffered bathing solution, and then darkened at different temperatures (18°C-30°C) acidified the medium (indicating net H⁺ efflux) at all temperatures tested, but most rapidly at the highest temperature. However, pH changes reversed direction after 20 to 70 minutes; the lower the temperature, the later pH reversal occurred, and the lower the pH at reversal and after 45 minutes. These data provide a basis for the previously reported promotive effect of low temperature on dark-induced leaflet closure, assuming net H⁺ and K⁺ fluxes are opposite in direction. Net H⁺ efflux at all temperatures tested was greater when the impermeant molecule iminodiacetate replaced small permeant anions in the bathing solution, suggesting that H⁺ uptake is coupled to anion uptake, probably via a H⁺/anion symport system. When permeant anions were deficient, the amount of malate in the tissue increased, presumably by new synthesis. Malate synthesis would substitute for H⁺/anion uptake in charge balance and in providing H⁺ for cytoplasmic pH regulation.     

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.     

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

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