Effects of (22S,23S)-Homobrassinolide and Related Compounds on Membrane Potential and Transport of Egeria Leaf Cells

(22S,23S)-Homobrassinolide was tested for its effect on the electric cell potential, proton extrusion, ferricyanide reduction, and amino acid and sucrose uptake of leaves of Egeria densa Planchon. In the light, (22S,23S)-homobrassinolide and its derivative, 2α-3α-dihydroxy-5α-stigmast-22-en-6-one, were similar to each other and similar to fusicoccin in causing hyperpolarization and proton extrusion, whereas stigmasterol was less effective. In darkness, the three sterols showed comparable effects. (22S,23S)-Homobrassinolide slightly stimulated ferricyanide reduction and promoted uptake of sucrose and α-aminoisobutyric acid. The results are compatible with a stimulation of an electrogenic proton pump mechanism at the plasmalemma by (22S,23S)-homobrassinolide.     

Transmembrane electrical potentials in growing maize roots

The anti-auxin 4-chlorophenoxyisobutyric acid (PCIB) applied at a concentration of 10^-2 mol m^-3 to maize root segments was found to induce a transmembrane electrical potential of up to-130 mV (pd of 30 mV). The kinetics of this response were comparable to the time scale for PCIB-stimulated H⁺-extrusion. Both effects are eliminated by the addition of p-fluoromethoxycarbonyl cyanide phenylhydrazone (FCCP). Treatment with fusicoccin (FC) and PCIB together does not result in a hyperpolarization greater than with FC alone. Benzoic acid (10^-2 mol m^-3) had no effect on the transmembrane electrical potentials. These results are discussed in relation to a possible electrogenic proton pump which may be regulated by perturbations in the cellular auxin content or activity.Abbreviations ATPase adenosine triphosphatase - FC fusicoccin - FCCP p-fluoromethoxy carbonylcyanide phenylhydrazone - IAA indole-3yl-acetic acid - NAA naphthyl-lylacetic acid - PCIB 4-chlorophenoxyisobutyric acid - PD potential difference     

Proton extrusion by leaf discs ofVicia faba L.: light- and ion-stimulated H(su+) release†

Previous electrophysiological and tracer kinetic studies indicated that the uptake of neutral amino acids took place by means of the proton cotransport mechanism in the leaf tissue of broad bean plants. The present investigations were designed to characterize the origin of the driving force for this process, and the proton pumping activity of leaf cells ofVicia. This activity is known to be revealed when peeled broad been leaf discs, floated on a bathing solution in the light or in darkness acidify the medium. White light caused the strongest acidification. The presence of K⁺ and Na⁺ in the external solution increased the H⁺ secretion significantly, whereas addition of Ca⁺⁺caused only an insignificant enhancement of proton extrusion. The inhibitors of photosynthetic electron transport DCMTJ (50 μM) and nitrofen (50 μM) eliminated the light-enhanced H⁺ release indicating the dependence on photosynthesis. The involvement of a proton pump was evidenced by the effects of the uneoupler CCCP, the SH reagent HgCl₂ and the ATPase inhibitor orthovanadate. The experimental results support the conclusion that H⁺ extrusion byVicia leaf cells is an active electrogenic process requiring metabolic energy. In the light this energy requirement is suppliedvia photosynthetic electron transport. Dedicated to Prof. Dr. F. Jacob on the occasion of his 60th birthday     

Rapid response of the plasma-membrane potential in oat coleoptiles to auxin and other weak acids

We have compared the effects of the auxin, indole-3-acetic acid (IAA) with that of other weak acids on the plasma-membrane potential of oat (Avena sativa L.) coleoptile cells. Cells treated with 1 M IAA at pH 6 depolarize 20–25 mV in 10–12 min, but they then repolarize, until by 20–25 min their potentials are about 25 mV more negative than the initial value. Similar concentrations of benzoic and butyric acids cause the initial depolarization, but not the subsequent hyperpolarization. The hyperpolarization is therefore specific to IAA. All the weak acids, including IAA, evoke a rapid hyperpolarization when their concentrations are raised to 10 mM. This result indicates that at high concentrations, the uptake of undissociated weak acids activates electrogenic proton pumping, most likely by lowering cytoplasmic pH. In contrast, the hyperpolarization observed with concentrations of IAA four orders of magnitude lower appears to be a specific hormonal effect. This specific, auxin-induced hyperpolarization occurs at the same time as the initiation of net proton secretion and supports the hypothesis that auxin initiates extension growth by increasing proton pumping.Abbreviations FC fusicoccin - IAA indole-3-acetic acid     

Regulation of electrogenic proton pumping by auxin and fusicoccin as related to the growth of Avena coleoptiles

The temporal relations between early responses to indoleacetic acid (IAA), proton secretion, hyperpolarization of the membrane potential, and growth change during the incubation of segments of oat (Avena sativa L.) coleoptiles in a low salt medium. When IAA is added after pretreatment of several hours, proton secretion increases after a latency of 7 minutes and reaches its maximum 10 to 15 minutes later. This timing coincides with both the increase in growth of the segments and the hyperpolarization of the membrane potential of parenchyma cells, consistent with the hypothesis that the change in membrane voltage reflects the activity of an electrogenic proton pump. The extent of IAA-induced hyperpolarization is substantially reduced by elevating [KCl]₀, most likely because this increases the passive conductance of the membrane. Neither growth nor proton secretion is affected by high [KCl]₀ (30 millimolar), indicating that neither process is limited by the magnitude of the membrane potential. These results are consistent with the acid growth hypothesis. Following short incubation times, however, IAA-induced hyperpolarization and growth are detected within 10 minutes, while acidification of the medium is delayed for more than 40 minutes. This result is seemingly in conflict with the acid growth hypothesis, but in freshly cut tissue, the pH of the external medium may not reflect the pH of the epidermal cell walls. The temporal coincidence of auxin-induced growth and hyperpolarization suggests that in freshly isolated segments the hyperpolarization is a more sensitive indication of proton secretion than is acidification of the external aqueous environment.     

Effect of temperature on growth,proton extrusion and membrane potential in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) coleoptile segments

The effects of temperature (5–45°C) on endogenous growth, growth in the presence of either indoleacetic acid (IAA) or fusicoccin (FC), and proton extrusion in maize coleoptile segments were studied. In addition, membrane potential changes at some temperatures were also determined. It was found that in this model system endogenous growth exhibits a clear maximum at 30°C, whereas growth in the presence of IAA and FC shows the maximum value in the range 30–35°C and 35–40°C, respectively. Simultaneous measurements of growth and external medium pH indicated that FC at stressful temperatures was not only much more active in the stimulation of growth, but was also more effective in acidifying the external medium than IAA. Also the addition of either IAA or FC to the bathing medium at 30 and 40°C did not change the kinetic characteristic of membrane potential changes observed for both substances at 25°C. However, the increased temperature significantly decreased IAA and FC-induced membrane hyperpolarization. IAA in the incubation medium, at 10°C, brought about additional membrane depolarization (apart from the one induced by low temperature). In contrast to IAA, FC at 10°C caused gradual repolarization of membrane potential, which correlated with both FC-induced growth and FC-induced proton extrusion. A plausible interpretation for temperature-induced changes in growth of maize coleoptile segments is that, at least in part, these changes were mediated via a PM H⁺-ATPase activity.     

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.     

Membrane potentials of vallisneria leaf cells and their relation to photosynthesis

A study has been made of the effects of the inhibitors carbonylcyanide m-chlorophenylhydrazone (CCCP), 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), and of anoxia on the light-sensitive membrane potential of Vallisneria leaf cells. The present results are compared with the known effects of these inhibitors on ion transport and photosynthesis (Prins 1974 Ph.D thesis). The membrane potential is composed of a diffusion potential plus an electrogenic component. The electrogenic potential is about −13 millivolts in the dark and −80 millivolts in the light. The inhibitory effect of DCMU and CCCP on the electrogenic mechanisms strongly depends on the light intensity used, the inhibition being less at a higher light intensity. This is of significance in view of the often conflicting results obtained with these inhibitors. With ion transport in Vallisneria the electrogenic pump derives its energy from phosphorylation; however, the process which causes the initial light-induced hyperpolarization and the process that keeps the membrane potential at a steady hyperpolarized state in the light have different energy requirements. The action of photosystem I alone is sufficient to induce the initial hyperpolarization. For continuous operation in the light the activity of photosystem II also is needed.     

Photosynthetic control of the plasma membrane H+-ATPase in Vallisneria leaves. I. Regulation of activity during light-induced membrane hyperpolarization

In mesophyll cells of the aquatic angiosperm Vallisneria gigantea Graebner, red, blue, or blue plus far-red light induced a typical membrane hyperpolarization, whereas far-red light alone had little effect. Both N,N'-dicyclohexylcarbodiimide, a potent inhibitor of H+-ATPase, and carbonylcyanide m-chlorophenylhydrazone, an uncoupler, produced a considerable membrane depolarization in the dark-adapted cells and a complete suppression of the light-induced hyperpolarization. Although 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosynthetic electron transport, did not affect the membrane potential in darkness, it completely inhibited the light-induced membrane hyperpolarization. In vivo illumination of the leaves with red light caused a substantial decrease in the Km for ATP, not only of the vanadate-sensitive ATP-hydrolyzing activity in leaf homogenate, but also of the ATP-dependent H+-transporting activity in plasma membrane (PM) vesicles isolated from the leaves by aqueous polymer two-phase partitioning methods. The effects of red light were negated by the presence of DCMU during illumination. In vivo illumination with far-red light had no effect on the Km for ATP of H+-transporting activity. These results strongly suggest that an electrogenic component in the membrane potential of the mesophyll cell is generated by the PM H+-ATPase, and that photosynthesis-dependent modulation of the enzymatic activity of the PM H+-ATPase is involved in the light-induced membrane hyperpolarization.     

Diverse effects of insulin-induced hyperpolarization on 3-O-methyl-D-glucose (3-O-MG) transport in frog skeletal muscles

It has been suggested that the insulin-induced hyperpolarization might be a mediator of the stimulatory action of insulin on glucose transport. The purpose of the present study was to investigate the relationship between the insulin-induced hyperpolarization and the stimulatory action of insulin on glucose transport in skeletal muscle. Satorius muscles dissected from bullfrogs (Rana catesbeiana) were used. Insulin induced a hyperpolarization of the membrane and an increase in the 3-O-Methyl-D-glucose (3-O-MG) uptake and extrusion. In the presence of valinomycin, insulin had no significant effect on the membrane potential. Insulin still had the stimulatory action on both the 3-O-MG uptake and extrusion even in the presence of valinomycin, under whose condition insulin had no significant effect on the membrane potential. The magnitude of the stimulatory action of insulin on the 3-O-MG uptake in the presence of valinomycin was smaller than that in the absence of valinomycin. The magnitude of the stimulatory action of insulin on the 3-O-MG extrusion was, on the contrary, larger than that in the absence of valinomycin. The abolishment of the insulin-induced hyperpolarization decreased the 3-O-MG uptake and increased the 3-O-MG extrusion. The observation in the present study concludes that insulin has two different actions on glucose transport. One of them is developed through the insulin-induced hyperpolarization, which increases the 3-O-MG uptake and decreases the 3-O-MG extrusion. The other action is irrelevant of the insulin-induced hyperpolarization and stimulates both the 3-O-MG uptake and extrusion.     

Erythrosin B as an effective inhibitor of electrogenic H+ extrusion

Abstract In 24-h-genninaled radish seedlings erythrosin B (EB), an effective inhibitor of microsomal as well as of partially purified vanadate-sensitive ATPase markedly inhibited the basal and the FC-stimulated proton extrusion, and induced a rapid depolarization of FC-hyperpolarized trans-membrane electric potential (PD) without causing any significant change of ATP level. The effects of EB on H⁺ extrusion were partially additive with those of vanadatc, another inhibitor of plasma membrane H⁺-ATPase. These effects are interpreted as due to a direct inhibition by EB on plasma membrane H⁺-ATPase involved in H⁺ electrogenic transport in the higher plants.     

Relationship between transpiration and amino acid accumulation in Brassica leaf discs treated with cytokinins and fusicoccin

Both cytokinins and fusicoccin (FC) stimulated the transpirationand the amino acid accumulation in leaf discs of Brassica campestrisvar. komatsuna. Enhancement effects were of the same magnitude.Both the accumulation and the transpiration were similarly inhibitedwhen vaseline was smeared on the leaf surface. Abscisic acid(ABA) also inhibited those cytokinin-induced effects. The accumulationof amino acid-¹⁴C was at the cytokinin- or FC-treated site unlessthe leaf surface was smeared with vaseline. These facts suggestthat cytokinin- or FC-induced amino acid accumulation in leafis caused by the stimulation of transpiration. Present address: ¹ Department of Environmental Studies, Collegeof Integrated Arts & Sciences, Hiroshima University, Higashisenda-machi,Hiroshima 730, Japan. Present address: ² Mitsui Memorial Hospital, 1-Kanda-Izumicho,Chiyoda-ku, Tokyo 101, Japan. (Received May 26, 1977; )     

Effects of a brassinosteroid on growth and electrogenic proton extrusion in Azuki bean epicotyls

Brassinolide, a plant hormone newly isolated from pollen, promotes growth of the stem of a number of plant species. Similar effects are induced by a brassinosteroid (BR), the synthetic 24-epibrassinolide. In this paper the effects of BR on acid secretion and transmembrane electrical potential difference in Azuki bean ( Vigna angularis , Ohwi and Ohashi cv. Takara) epicotyls were determined in short term experiments and compared with the effects on growth. At concentrations between 10^-7 to 10^-5 M , BR stimulates, similarly to indole-3-acetic acid (IAA), growth and H⁺ extrusion and hyperpolarizes the transmembrane electric potential (PD). These effects of BR, as well as those of IAA, are suppressed by inhibitors of RNA and protein synthesis. All these effects of BR and IAA appear roughly additive, even when both hormones are present at their optimal concentrations. The data are interpreted as showing that the action of BR on growth is at least in part mediated by its capability to activate electrogenic proton extrusion. The additivity of the effects of BR and IAA suggests that the primary mechanism of action of the two hormones is different.     

Protein phosphorylation in intact cultured sycamore (Acer pseudoplatanus) cells and its response to fusicoccin.

Fusicoccin (FC), a natural diterpene glucoside able to stimulate electrogenic H+ extrusion in higher plants, has been shown to stimulate the phosphorylation of a polypeptide of molecular mass approx. 33 kDa in intact cultured cells of sycamore (Acer pseudoplatanus). The effect is specific, rapid and insensitive to cycloheximide. The presence of the 33 kDa polypeptide and the stimulation by FC have been observed in SDS-containing cell homogenates and in the microsomal and soluble fractions after cell fractionation.     

Evidence for activation of an active electrogenic proton pump in Ehrlich ascites tumor cells during glycolysis

Summary The addition of glucose to a suspension of Ehrlich ascites tumor cells results in rapid acidification of the extracellular medium due to lactic acid production. The nature of the H⁺ efflux mechanism has been studied by measuring the time course of the acidification, the rate of proton efflux, the direction and relative magnitude of the H⁺ concentration gradient, and the voltage across the membrane. Using the pH-sensitive dye acridine orange, we have established that after addition of 10mm glucose an outward-directed H⁺ concentration gradient develops. As the rate of glycolysis slows, the continued extrusion of H⁺ reverses the direction of the H⁺ concentration gradient. Changes in absorbance of the voltagesensitive dye diethyloxadicarbocyanine iodide (DOCC), and changes in the distribution of the lipid permeant cation tetraphenyl phosphonium, showed a dramatic and persistent hyperpolarization of the membrane voltage after glucose addition. The hyperpolarization was prevented by the protonophore tetrachlorosalicylanalide (TCS) and by valinomycin, but not by the neutral-exchange ionophore nigericin. Inhibitors of lactate efflux were found to reduce the rate of acidification after glucose addition but they had no effect on the magnitude of the resulting hyperpolarization. On the basis of these and other data we suggest that an active electrogenic pump mechanism for H⁺ efflux may be activated by glucose and that this mechanism operates independently of the lactate carrier system.     

Demonstration of the root surface electrogenic ion pump activity revealed from the seasonal inversion in the phase relation between electro-radicogram and the diurnal oscillation of air temperature in a field tree (Diospyros kaki)

Re-examination of the electro-radicogram (ERG) obtained during past 10 years research (Masaki and Okamoto in Trees (Berl) 21:433–442, 2007) enabled us to discriminate the excess activity of the electrogenic ion pump in the root surface cell membrane over that of the xylem pump during most of the foliate phase. The trans-root electric potential (TRP) is defined as the difference between V _ps (electric potential difference between symplast and bulk water phase surrounding the root) and V _px (electric potential difference between symplast and xylem apoplast). The diurnal oscillation of TRP followed that of the air temperature and/or light intensity with a delay of several hours during defoliate phase. This means the superiority of the electrogenic activity of the xylem pump over that of the root surface pump. However, after leaf expansion, TRP began to oscillate inversely with the temperature change with a short delay, indicating the superiority of the electrogenic activity of the surface ion pump over that of the xylem pump. An experimental lumbering of the surroundings of the kaki tree in foliate phase prominently increased the ERG amplitude, keeping the inverted phase relation, with the increase in transpiration caused by the increased illumination. An incidental sudden fall of the temperature and illumination caused an inverse reaction.     

Effect on Membrane Potential and Electrical Activity of Adding Sodium to Sodium-Depleted Cardiac Purkinje Fibers

Canine cardiac Purkinje fibers exposed to Na-free solutions containing 128 mM TEA and 16 mM Ca show resting potentials in the range -50 to -90 mV; if the concentration of Na in the perfusate is raised from 0 to 4 to 24 mM, hyperpolarization follows. If the initial resting potential is low, the hyperpolarization tends to be greater; the average increase in the presence of 8 mM Na is 14 mV. Such hyperpolarization is not induced by adding Na to K-free solutions, is not seen in cooled fibers, or in fibers exposed to 10^-3 M ouabain, nor is it induced by adding Li and thus may result from electrogenic sodium extrusion. Fibers exposed to Na-free solutions are often spontaneously active; if they are quiescent they often show repetitive activity during depolarizing pulses. Such spontaneous or repetitive activity is suppressed by the addition of Na. This suppression may or may not be related to the hyperpolarization.     

Effects of ophiobolin B on cell enlargement and H+/K+ exchange in maize coleoptile tissues

Ophiobolin B (OPH B), a sesterpene metabolite of Helminthosporium oryzae, inhibits proton extrusion from maize coleoptiles. Moreover OPH B counteracts the biological activity of fusicoccin (FC), another terpenoid toxin produced by Fusicoccum amygdali having a similar basic chemical structure: OPH B inhibits FC-promoted proton extrusion, potassium uptake and cell enlargement.The findings suggest that the effect of OPH B in stimulating electrolites, glucose and aminoacid leakage, reported in a previous paper, can be explained by the capacity of the toxin to inhibit proton extrusion.Abbreviations FC fusicoccin - OPH B ophiobolin B     

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

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

Fusicoccin effects on maize roots: relationship between malate accumulation and elongation

Abstract In maize root segments malate accumulation, measured by gas chromatography, was found to be strongly promoted by fusicoccin (FC). The effects of FC on root elongation and proton extrusion were also analysed. Growth rates and extrusion rates were found to be proportional to malate content. Other organic acids were analysed, but no significant changes were found following FC treatment. The effects of FC are discussed in relation to the regulation of metabolism and growth.