Reinvestigation of auxin and fusicoccin stimulation of the plasma-membrane H+-ATPase activity

In vivo treatment of maize (Zea mays L.) coleoptile segments with auxin (indole-3-acetic acid; IAA) and fusicoccin (FC) followed by plasma-membrane isolation was used to characterize the effects of these treatments on the plasma-membrane H⁺-ATPase. Both IAA and FC increased H⁺ extrusion and elongation rate of the coleoptile segments, FC more strongly than IAA. Plasma membranes isolated after in-vivo treatment with FC showed a twofold stimulation of ATP hydrolysis and a several-fold stimulation of H⁺ pumping, whereas no effect was observed after IAA treatment, irrespective of whether the plasma membranes were prepared by two-phase partitioning or sucrose-gradient centrifugation. A more detailed investigation of the kinetic properties and pH dependence of the enzyme showed that FC treatment led to a twofold increase in V _max, a decrease in K _m for ATP from 1.5 mM to 0.24 mM, and a change in pH dependence resulting in increased activity at physiological pH levels. Again, IAA treatment showed no effects. Quantitation of the H⁺-ATPase by immunostaining using four different antibodies revealed no difference between IAA-and FC-treated material, and controls. From these data we conclude that (i) neither IAA nor FC gives rise to an increase in the amount of H⁺ -ATPase molecules in the plasma membrane that can be detected after membrane isolation, and (ii) if the H⁺-ATPase is activated by IAA, this activation is, in contrast to FC activation, not detectable after membrane isolation.Abbreviations BTP 1,3-bis(tris[hydroxymethyl]methylamino)-propane - FC fusicoccin - lyso-PC lysophosphatidylcholine - Mes 2-(N-morpholino)ethanesulfonic acid This paper is dedicated to Prof. Dieter Klämbt on the occasion of his 65th birthdayWe thank Ann-Christine Holmström and Adine Karlsson for excellent technical assistance, Professor Ramón Serrano (Instituto de Biologia Molecular y Celular de Plantas, UPV-CSIC, Universidad Politecnica, Valencia, Spain) for a generous gift of antisera to the H⁺-ATPase and Professor Wolfgang Michalke (Institut für Biologie III, Albert-Ludwigs-Universität, Freiburg, Germany) for kindly providing the monoclonal antibody to the H⁺-ATPase. This work was supported by the Swedish Natural Science Research Council, the Deutsche Agentur für Raumfahrtangelegenheiten (DARA, Bonn) via AGRAVIS (Bonn) and by the Ministerium für Wissenschaft und Forschung (MWF, Düsseldorf). Thomas Jahn received scholarships from the Deutsche Graduiertenförderung des Landes Nordrhein-Westfalen and the Deutscher Akademischer Austauschdienst (DAAD, Bonn).     

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.     

Factors affecting the binding between fusicoccin and plasma membranes from maize roots

Plasma membranes have been purified from roots of maize (Zea mays L.) using a two-phase aqueous polymer system, dextran-polyethylene glycol. The plant material was homogenized in the presence of a mixture of natural protease inhibitors from potato (Solanum tuberosum L.); these inhibitors have been shown to be more effective than phenylmethylsulfonyl fluoride in suppressing the endogenous proteases in maize roots. Inhibition of proteolysis in the homogenization medium markedly increased (about tenfold) the number of lowaffinity binding sites for fusicoccin (FC). In addition, storage of plasma membranes at −20° C decreased both the number of the low-affinity sites and their dissociation constant (K_D); this effect was in all probability caused by lipid peroxidation. The presence of EDTA throughout isolation and storage of the plasma membranes stabilized the parameters of FC binding to the membranes. The kinetics of binding of [³H]dihydroFC and the competition between [³H]dihydroFC and FCs A, C, J, and H were determined for the low-affinity sites. It was found that (i) the rate constant of association between FC and the low-affinity binding sites is about two orders of magnitude lower than that for the high-affinity sites; (ii) different FCs can be arranged in the order of decreasing avidity for the low-affinity FCbinding site: FC A>FC C>FC J>FC H. The authors are indebted to Dr. L.M. Krasnopolskaya (Institute of Agricultural Biotechnology, Moscow, Russia) for fusicoccins A, C, J, and H, and to Dr. A.V. Galkin (Institute of Agricultural Biotechnology, Moscow, Russia) for valuable comments and ren dering the paper into English.     

Proton translocation in corn coleoptiles: ATPase or redox chain?

The O₂ dependence of net H⁺ efflux of maize coleoptiles has been investigated. Below 100 M O₂, H⁺ efflux in young (1 cm long) coleoptiles is markedly decreased while old (7 cm long) coleoptiles show a decline only at 10 M O₂. Old coleoptiles show the same decrease in net H⁺ efflux as young ones if treated with fusicoccin. The ratio of alteration of CO₂ production to the change in net proton efflux is about 1:1 at 40–80 M O₂ but not at 10 M O₂. An influx can be observed at 10 M O₂ in young as well as in old coleoptiles if the H⁺ concentration is held at values below pH 6.5. Lower O₂ concentrations lead to an increase of net H⁺ efflux, which might be caused by leaching of organic acids resulting from anaerobic processes, but CO₂ production is not significantly changed at these values. It is proposed that more than one system is responsible for proton translocation across the plasmalemma. One of the systems has a high sensitivity to reduced O₂ concentration which is within the same range as the high K_m of the alternative path.Abbreviation FC fusicoccin     

Phloem loading in Vicia faba leaves: Effect of N-ethylmaleimide and parachloromercuribenzenesulfonic acid on H+ extrusion,K+ and sucrose uptake

The effects of a penetrating (NEM) and a non-penetrating (PCMBS) sulfhydryl-specific reagent on proton extrusion, ⁸⁶Rb and [U-¹⁴C]sucrose uptake by Vicia faba leaves have been studied. Proton extrusion was strongly or completely inhibited by 0.1 mM NEM. ⁸⁶Rb and [U-¹⁴C]sucrose uptake were markedly reduced by NEM concentrations equal to or higher than 0.5 mM. Under our experimental conditions, PCMBS (1 mM) exerted a strong inhibition on [¹⁴C]sucrose uptake but did not inhibit proton extrusion and ⁸⁶Rb uptake. The sensitivity of phloem loading to PCMBS is thought to be a consequence of sugar-carrier blockage and not of inhibition of the proton pump.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - DES diethylstilbestrol - DCCD dicyclohexylcarbodiimide - FC Fusicoccin - NEM N-ethylmaleimide - PCMBS p-chloromercuribenzenesulfonic acid     

Lack of immunological cross reactivity between the transport enzymes (Na+ + K+)-ATPase and (K+ + H+)-ATPase

Goat antisera against (Na⁺ + K⁺)-ATPase and its isolated subunits and against (K⁺ + H⁺)-ATPase have been prepared in order to test for immune cross-reactivity between the two enzymes, whose catalytic subunits show great chemical similarity. None of the (Na⁺ + K⁺)-ATPase antisera cross-reacted with (K⁺ + H⁺)-ATPase or inhibited its enzyme activity. The same was true for the (K⁺ + H⁺)-ATPase antiserum with regard to (Na⁺ + K⁺)-ATPase and its subunits and its enzyme activity. So not withstanding the chemical similarity of their subunits, there is no immunological cross-reactivity between these two plasma membrane ATPases.Number LIII in the series Studies on (Na⁺ + K⁺)-Activated ATPase.     

Arbuscular mycorrhizal fungi induce differential activation of the plasma membrane and vacuolar H+ pumps in maize roots

Roots undergo multiple changes as a consequence of arbuscular mycorrhizal (AM) interactions. One of the major alterations expected is the induction of membrane transport systems, including proton pumps. In this work, we investigated the changes in the activities of vacuolar and plasma membrane (PM) H(+) pumps from maize roots (Zea mays L.) in response to colonization by two species of AM fungi, Gigaspora margarita and Glomus clarum. Both the vacuolar and PM H(+)-ATPase activities were inhibited, while a concomitant strong stimulation of the vacuolar H(+)-PPase was found in the early stages of root colonization by G. clarum (30 days after inoculation), localized in the younger root regions. In contrast, roots colonized by G. margarita exhibited only stimulation of these enzymatic activities, suggesting a species-specific phenomenon. However, when the root surface H(+) effluxes were recorded using a noninvasive vibrating probe technique, a striking activation of the PM H(+)-ATPases was revealed specifically in the elongation zone of roots colonized with G. clarum. The data provide evidences for a coordinated regulation of the H(+) pumps, which depicts a mechanism underlying an activation of the root H(+)-PPase activity as an adaptative response to the energetic changes faced by the host root during the early stages of the AM interaction.     

Distribution of growth and proton efflux in gravireactive roots of maize (Zea mays L.)

Roots of Zea mays were maintained in a vertical orhorizontal position and the local elongation rate and H⁺ fluxes were measured using Sephadex beads containing a pH indicator. When the roots were kept horizontally, the growth of the lower side was strongly inhibited and that of the upper side slightly stimulated as compared with vertical roots. The H⁺ extrusion, which was greatest in the elongation zone, was strongly inhibited on the lower side and slightly stimulated on the upper side as compared with vertical roots.     

Maize tonoplast PP(i)-dependent H(+)/Ca(2+) exchange: two K(s) for Ca(2+) and inhibition by thapsigargin

Maize root tonoplasts are able to accumulate Ca(2+) using the energy derived from the H(+) gradient formed during PP(i) hydrolysis. Oxalate increases 6- to 10-fold the amount of Ca(2+) accumulated by tonoplast. Two apparently different K(s) values for Ca(2+) with values of 0.36 and 4.70 microM were detected when oxalate was included in the medium and the free Ca(2+) concentration in the medium was buffered with the use of EGTA. Binding of Ca(2+) to the outer surface of tonoplasts inhibits the outflow of Ca(2+) previously accumulated by the tonoplast, half-maximal inhibition being observed in presence of 1 microM Ca(2+). Thapsigargin, a specific inhibitor of Ca(2+)-ATPase, inhibits the Ca(2+) uptake driven by H(+) gradient but does not inhibit the hydrolysis of PP(i) nor the formation of a H(+) gradient.     

Essential sulfhydryl groups in the catalytic center of the tonoplast H+-ATPase from coleoptiles ofZea mays L. as demonstrated by the biotin-streptavidin-peroxidase system

Functional properties and the localization of essential SH-groups of the tonoplast H⁺-ATPase fromZea mays L. were studied. In contrast to the pyrophosphate-dependent H⁺-translocation activity of the tonoplast, the H⁺-ATPase activity was inhibited by SH-blocking agents, such as N-ethylmaleimide and iodoacetic acid. In the case ofp-hydroxymercuribenzoate, HgCl₂ and oxidized glutathione, the inhibition could be reversed by adding reduced glutathione or dithiothreitol. Incubation of tonoplast vesicles with oxidized glutathione or N-ethylmaleimide in the presence of Mg·ADP—a competitive inhibitor of the ATP-dependent H⁺ pump—avoided the inhibition of the H⁺-pumping activity. This effect is an indication for the occurrence of essential SH-groups at the catalytic site of the H⁺-ATPase. In order to characterize the active center these thiols were specifically labeled with maleimidobutyrylbiocytin. Subsequently, the membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to an immobilizing membrane. The maleimidobutyrylbiocytin-labeled active-center protein was detected by a biotin-streptavidin-peroxidase staining system and was shown to be a 70-kDa subunit of the tonoplast H⁺-ATPase. It is suggested that the oxidation state of the critical sulfhydryl groups within the active center of the enzyme and their reversible blocking by endogenous compounds might be of great importance for the regulation of the enzyme activity in vivo.     

Mechanisms of fusicoccin action: evidence for concerted modulations of secondary K+ transport in a higher plant cell

Fusicoccin (FC) has long been known to promote K⁺ uptake in higher plant cells, including stomatal guard cells, yet the precise mechanism behind this enhancement remains uncertain. Membrane hyperpolarization, thought to arise from primary H⁺ pumping stimulated in FC, could help drive K⁺ uptake, but the extent to which FC stimulates influx and uptake frequently exceeds any reasonable estimates from Constant Field Theory based on changes in the free-running membrane potential (V _m) alone; furthermore, unidirectional flux analyses have shown that in the toxin K⁺ (⁸⁶Rb⁺) exchange plummets to 10% of the control (G.M. Clint and E.A.C. MacRobbie 1984, J. Exp. Bot.35 180–192). Thus, the activities of specific pathways for K⁺ movement across the membrane could be modified in FC. We have explored a role for K⁺ channels in mediating these fluxes in guard cells ofVicia faba L. The correspondence between FC-induced changes in chemical (⁸⁶Rb⁺) flux and in electrical current under voltage clamp was followed, using the K⁺ channel blocker tetraethylammonium chloride (TEA) to probe tracer and charge movement through K⁺-selective channels. Parallel flux and electrical measurements were carried out when cells showed little evidence of primary pump activity, thus simplifying analyses. Under these conditions, outward-directed K⁺ channel current contributed appreciably to charge balance maintainingV _m, and adding 10 mM TEA to block the current depolarized (positive-going)V _m; TEA also reduced⁸⁶Rb⁺ efflux by 68–80%. Following treatments with 10 M FC, both K⁺ channel current and⁸⁶Rb⁺ efflux decayed, irreversbly and without apparent lag, to 10%–15% of the controls and with equivalent half-times (approx. 4 min). Fusicoccin also enhanced⁸⁶Rb⁺ influx by 13.9-fold, but the influx proved largely insensitive to TEA. Overall, FC promotednet cation uptake in 0.1 mM K⁺ (Rb⁺), despite membrane potentials which were 30–60 mVpositive of the K⁺ equilibrium potential. These results tentatively link (chemical) cation efflux to charge movement through the K⁺ channels. They offer evidence of an energy-coupled mechanism for K⁺ uptake in guard cells. Finally, the data reaffirm early suspicions that FC alters profoundly the K⁺ transport capacity of the cells, independent of any changes in membrane potential.Abbreviations and symbols E _K equilibrium potential for K⁺ - FC fusicoccin - Hepes 4-(2-hydroxyethyl)-1-piperazineeth-anesulfonic acid - G _m membrane (slope) conductance atV _m - I-V current-voltage (relationship) - apparent rate constant for exchange - K _i ⁺ , K ₀ ⁺ intracellular, extracellular K⁺ (concentration) - TEA tetraethylammonium chloride - V _m free-running membrane potential (difference)     

K+ current stimulation by Cl- in the midgut epithelium of tobacco hornworm (Manduca sexta)

Summary Chloride-stimulated K⁺ secretion by Manduca sexta midgut (5th-instar larvae) was measured as K⁺-carried short-circuit current of the tissue mounted in an Ussing chamber. Microscopic parameters, such as single-channel current and channel density for the rate-determining passive transport step across the basolateral goblet cell membrane (i.e. K⁺ channels), were estimated by means of current-fluctuation analysis of the K⁺ channel blockade by haemolymph-side Ba²⁺ ions. Ba²⁺ was equally effective with Cl^- or gluconate (Glu^-) as the principal ambient anion. The Ba²⁺-induced K⁺ channel conduction noise is reflected by a Lorentzian, or relaxation, noise component in the power spectrum of the K⁺ current fluctuations. A reduced Lorentzian plateau value, but an unchanged corner frequency, were observed when Cl^- was replaced by Glu^-. The results from the analysis of a two-state model of K⁺ channel block by Ba²⁺, with respect to the anion-replacement effects, suggest that the observed changes in K⁺ current and Lorentzian plateau value mirror a complex change of the underlying parameters: Cl^- omission reduces single channel current but increases channel density so that the product of single channel current and channel density is smaller in Glu^- than in Cl^-. It seems likely that basolateral K⁺ channels (1) are subject to anionic gating ligands, and (2) depend on anions with respect to the rate of K⁺ transfer through and open K⁺ channel.Abbreviations a.c. alternating current - single-channel conductance - E _K K⁺ Nernst potential - f frequency contained in current noise - f _c corner frequency - Glu^- gluconate - G _t transepithelial conductance - I _sc short-circuit current - I _K K⁺ current - I _K(max) maximal K⁺ current - i single-channel current - K _Ba barium inhibition constant - K _m Michaelis constant of saturating K⁺ current - k ₀₁ and k ₁₀ barium association and dissociation rate constant, respectively - M K⁺ channel density - S _f power density - S _o Lorentzian plateau value - P _o channel-open probability - P _K K⁺ permeability - V _sc cellular potential at short-circuit These results have already been presented in part, at the 1989 joint meeting of the German and Israel Physiological Societies in Jerusalem (Zeiske et al. 1990).     

Characterization of the plasma-membrane H+-ATPase from Vicia faba guard cells

Stomatal movement is controlled by external and internal signals such as light, phytohormones or cytoplasmic Ca²⁺. Using Vicia faba L., we have studied the dose-dependent effect of auxins on the modulation of stomatal opening, mediated through the activity of the plasma-membrane H⁺-ATPase. The patch-clamp technique was used to elucidate the electrical properties of the H⁺-ATPase as effected by growth regulators and seasonal changes. The solute composition of cytoplasmic and extracellular media was selected to record pump currents directly with high resolution. Proton currents through the ATPase were characterized by a voltage-dependent increase in amplitude, positive to the resting potential, reaching a plateau at more depolarized values. Upon changes in extracellular pH, the resting potential of the cell shifted with a non-Nernst potential response (±21 mV), indicating the contribution of a depolarizing ionic conductance other than protons to the permeability of the plasma membrane. The use of selective inhibitors enabled us to identify the currents superimposing the H⁺-pump as carried by Ca²⁺. Auxinstimulation of this electroenzyme resulted in a rise in the outwardly directed H⁺ current and membrane hyperpolarization, indicating that modulation of the ATPase by the hormone may precede salt accumulation as well as volume and turgor increase. Annual cycles in pump activity (1.5–3.8 μA · cm^-2) were expressed by a minimum in pump current during January and February. Resting potentials of up to -260 mV and plasmamembrane surface area, on the other hand, did not exhibit seasonal changes. The pump activity per unit surface area was approximately 2- to 3-fold higher in guard cells than in mesophyll cells and thus correlates with their physiological demands.     

The changing sensitivity to auxin of the plasma-membrane H+-ATPase: Relationship between plant development and ATPase content of membranes

The auxin sensitivity of the plasma-membrane H⁺-ATPase from tobacco leaves (Nicotiana tabacum L. cv. Xanthi) depends on the physiological state of the plant (Santoni et al., 1990, Plant Sci. 68, 33–38). Results based on the study of auxin sensitivity according to culture conditions which accelerate or delay tobacco development demonstrate that the highest auxin sensitivity is always associated with the end of the period of induction to flowering. Auxin stimulation of H⁺-translocation activity corresponds to an increase of the apparent ATPase affinity for ATP. The plasma-membrane H⁺-ATPase content, measured with an enzyme-linked immunosorbent assay using a specific anti-H⁺-ATPase antibody, varies according to plant development, and was found to increase by 100% during floral induction. The specific molecular ATPase activity also changes according to plant development; more particularly, the decrease in molecular ATPase activity upto and during the floral-induction period parallels the increase of sensitivity to indole-3-acetic acid.Abbreviations ELISA enzyme-linked immunosorbent assay - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate Authors are grateful to Mrs. Grosclaude (Lab. Virologie, INRA, Jouy-en-Josas, France) and Mrs. Boudon (Lab. Mycoplasmes, INRA, Dijon, France) for support and advice in the preparation of antibodies. This work was supported by grants No. 89/512/6 from the E.P.R of Bourgogne and No. 89 C 0662 from M.R.T.     

Simultaneous influx of ammonium and potassium into maize roots: kinetics and interactions

The interaction between ammonium and potassium during influx was examined in roots of dark-grown decapitated corn seedlings (Zea mays L., cv. Pioneer 3369A). Influx was measured during a 10-min exposure to either (¹⁵NH₄)₂SO₄ ranging from 10 to 200 M NH ₄ ⁺ with and without 200 M K(⁸⁶Rb)Cl or to K(⁸⁶Rb)Cl ranging from 10 to 200 M K⁺ with and without 200 M NH ₄ ⁺ as (¹⁵NH₄)₂SO₄. The simple Michaelis-Menten model described the data well only for potassium influx in the presence of ambient ammonium. For the other three instances, the data were improved by assuming that a second influx mechanism became operative as the low-concentration phase approached saturation. Two distinct mechanisms are thus indicated for both ammonium and potassium influx within the range of 10 to 200 M.The influx mechanism operating at low concentrations showed greater affinity for potassium than for ammonium, even though the capacity for ammonium transport was twice as large as that for potassium. It is suggested that this phase involved a common transport system for the two ions and that localized low acidity next to the internal surface, following H⁺ extrusion, favored ammonium deprotonation and dissociation from the transport system-ammonium complex. Parallel decreases in V _max and increases in K_m of the low-concentration saturable phase occurred for ammonium influx when ambient potassium was present and for potassium influx when ambient ammonium was present. The data support a mixed-type inhibition in each case. Simultaneous measurement of potassium and ammonium influx showed that they were highly negatively correlated at the lower concentrations, indicating that the extent to which influx of the inhibited ion was restricted was associated with influx of the inhibitor ion. Presence of ambient ammonium eliminated the second phase of potassium influx. In contrast, the presence of ambient potassium decreased the concentration at which the second phase of ammonium influx was initiated but did not restrict the rate.Paper no. 11131 of the Journal Series of the North Carolina Agricultural Research ServiceThe use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned     

Inhibition of elongation and H+ and K+ transport by the phosphodiesterase inhibitor aminophylline in plant tissue

Aminophylline, an inhibitor of cyclic nucleotide phosphodiesterase (EC 3.1.4.17), inhibits elongation and correlated H⁺ and K⁺ transport in embryos of Haplopappus gracilis and in pea internode segments. Moreover, the drug strongly inhibits the stimulation of these processes by fusicoccin and indole-3-acetic acid and reduces passive permeability of the membrane. The possible mechanisms of action of aminophylline are discussed.Abbreviations cAMP adenosine 3:5-cyclic monophosphate - FC fusicoccin - IAA indole-3-acetic acid - MES 2-N-morpholinoethanesulfonic acid - PDE cyclic nucleotide phosphodiesterase     

Effects of spermidine and spermine levels on salt tolerance associated with tonoplast H+-ATPase and H+-PPase activities in barley roots

The effects of polyamines (Putrescine— Put; Spermidine—Spd; and Spermine—Spm) on␣salt tolerance of seedlings of two barley (Hordeum vulgare L.) cultivars (J4, salt-tolerant; KP7, salt-sensitive) were investigated. The results showed that, the salt-tolerant cultivar J4 seedlings accumulated much higher levels of Spd and Spm and lower Put than the salt-sensitive cultivar KP7␣under salt stress. At the same time, the dry weight of KP7 decreased significantly than that of␣J4. After methylglyoxal bis(guanylhydrazone) [MGBG, an inhibitor of S-adenosylmethionine decarboxylase (SAMDC)] treatment, Spd and Spm levels together with the dry weight of both cultivars were reduced, but the salt-caused dry weight reduction in two cultivars could be reversed by the concomitant treatment with Spd. MGBG decreased the activities of tonoplast H⁺-ATPase and H⁺-PPase too, but the experiments in vitro indicated that MGBG was not able to affect the above two enzyme activities. However, the polyamines, especially Spd, promoted their activities obviously. These results suggested that the conversion of Put to Spd and Spm and maintenance of higher levels of Spd and Spm were necessary for plant salt tolerance.