Effect of growth regulators on proton transport through cytoplasmic membrane of potato tuber cells

Effects of the growth regulators epibrassinolide-694 (EB), gibberellic acid (GA), and abscisic acid (ABA) on the ATP-dependent translocation of H+ through the membranes of plasma membrane vesicles of potato (Solanum tuberosum L.) tuber cells were studied. The ATP-dependent accumulation of H+ in the plasma membrane vesicles from dormant tubers was inhibited by EB and ABA and stimulated by GA. After the break of dormancy, the stimulatory effect of GA increased, the inhibitory effect of ABA decreased, and EB stimulated the accumulation of H+ in the vesicles. The data suggest that the plasma membrane H+ ATPase is a target of phytohormones that regulate the dormancy of potato tubers.     

Effect of phytohormones on the transmembrane translocation of protons in plasma membrane vesicles from tubers of Solanum tuberosum L

Effects of phytohormones gibberellic acid (GA) and abscisic acid (ABA) on the ATP-dependent transmembrane transport of protons were studied in plasma membrane vesicles (PMVs) from non-dormant potato tubers. The uptake of H+ into PMVs was assessed by the fluorescence quenching of acridine orange (AO) after the addition of ATP to the incubation medium. Addition of ATP to the incubation medium led to the instantaneous rise of the AO fluorescence intensity followed by its decrease. The fluorescence quenching was not observed in the presence of either protonophore CCCP or inhibitors of the membrane-bound H+-ATPase. It is concluded that the ATP-induced quenching of the AO fluorescence resulted from the accumulation of protons in PMVs due to the function of the plasma membrane-bound H+-ATPase. Depending on their concentrations, GA and ABA either inhibited or stimulated the ATP-driven H+ translocation across the vesicle membrane. The growth-stimulating hormone GA at concentrations of 10(-9)-10(-5) M increased the initial rate of the fluorescence quenching, whereas 10(-4) M GA slightly inhibited the H+ translocation. The growth inhibitor ABA at a concentration of 10(-9) M slightly increased the rate of the proton accumulation in PMVs; at higher concentrations (10(-8)-10(-4) M), ABA inhibited the H+ translocation. Acetic acid, which has pK similar to pK of GA and ABA, did not influence the ATP-dependent H+ accumulation in PMVs, suggesting the hormone-specific action of GA and ABA on the H+-ATPase activity. In the presence of DCC, which completely inhibited the accumulation of H+, GA and ABA did not affect the passive proton efflux from PMVs. It is proposed that the mechanisms of the regulatory effects of phytohormones may involve modification of H+-ATPase activity leading to changes in the electrochemical gradient of H+ across the plasma membrane.     

Effect of salicylic acid on the proton translocation activity of plasmalemma of potato tuber cells

Action of salicylic acid (SA) on the activity of membrane bound H⁺-ATPase and passive proton permeability of plasmalemma membrane vesicles (PMV) from parenchyma cells of potato tubers was detected. A correlation between SA action on germination of tubers and activity of plasmalemma H⁺-ATPase was revealed: the application of growth-stimulating concentrations of SA (10⁻¹⁰–10⁻⁸ M) in the system in vitro resulted in activation of plasmalemma H⁺-ATPase, while the use of growth-inhibiting concentrations (10⁻⁴, 10⁻⁵ M) provoked inhibition of the enzyme activity. Addition of jasmonic acid (JA) to the incubation mix resulted in increase of SA effect on the accumulation of H⁺ in PMV.     

Phytohormone Interactions in the Control of Proton Translocation Activity of Plant Cell Plasmalemmata

The effects of phytohormones (abscisic acid, gibberellic acid, and jasmonic acid) and ambiol (a synthetic growth regulator) on processes of H⁺ transport across the plasmalemma were studied in membrane vesicles isolated from the parenchyma of potato (Solanum tuberosum L.) tubers. Phytohormones and ambiol were tested either individually or in combinations. Each of the substances tested changed the initial rate of H⁺ uptake by the vesicles. Two signaling substances added to the incubation medium simultaneously modified the activity of each other. It is suggested that the interaction of a signaling substance with components of the plasmalemma alters the responses of the membrane to other signaling molecules.     

The effect of thaumatin gene overexpression on the properties of H+-ATPase from the plasmalemma of potato tuber cells

The introduction of the thaumatin gene into potato plants was accompanied by a decrease in the activity of H⁺-ATPase in the plasmalemma (PL) of tuber cells. When tubers were released from dormancy, the enzyme was activated in the tuber cells of both the original and transgenic plants. Experiments performed in vitro demonstrated that sensitivities to ambiol (AM) and jasmonic acid (JA) of H⁺-ATPase in the PL of tubers from the original plants were lower after the release from a period of deep dormancy. In preparations from the tubers of transgenic plants, the situation was reversed. The differences between the activities of H⁺-ATPase in the PL preparations produced from the original and transgenic tubers that sprouted under the action of AM and JA were detected. Thus, the overexpression of the thaumatin gene in potato plants changed the properties of H⁺-ATPase from PL.     

Effect of jasmonic acid on Ca<Superscript>+2</Superscript> transport through the plasmalemma of potato tuber cells

The rate of Ca²⁺ accumulation in plasmalemma vesicles isolated from quiescent and sprouting potato (Solanum tuberosum L.) tubers and the effect of 10^?5–10^?10 M jasmonic acid on the accumulation of Ca⁺² in plasmalemma vesicles and its efflux were studied. It was found that potato tuber plasmalemma contains a Ca⁺²,Mg⁺²-ATPase whose activity decreases upon the transition from forced quiescence to growth. The direction of the effect of jasmonic acid on Ca⁺²,Mg⁺²-ATPase (stimulation or suppression) depends on the physiological state of tubers and the phytohormone concentration.     

Brassinolide Effect on Growth of Apical Meristems, Ethylene Production, and Abscisic Acid Content in Potato Tubers

Treatment of intact potato (Solanum tuberosum L., cv. Nevskii) tubers with 24-epibrassinolide (EB) resulted in prolonged deep dormancy, increased production of ethylene and higher contents of free and bound abscisic acid (ABA) in buds. EB at the most efficient concentration 0.021 mg dm^–3, applied immediately after tuber harvest, inhibited sprouting by 36 – 38 d, increased ethylene formation after 1 and 7 d of storage by almost 300 and 150%, respectively, and increased the content of both free and bound ABA during the whole period of storage (on average by about 80%). Electron microscopic and morphometric studies showed that EB brings about a decrease in cell volume in tunica and all types of meristems and an increase in the number of vacuoles, accompanied by a decrease in their volume.     

Reduction of abscisic acid content and induction of sprouting in potato,Solanum tuberosum L., by thidiazuron

The effect of [(N-phenyl-N-1,2,3-thidiazol-5-ylurea)] (thidazuron) on sprouting of potato (Solanum tuberosum L.) tubers and the role of ABA in bud break and subsequent bud growth were studied. Abscisic acid (ABA) was quantitated by enzyme-linked immunosorbent assay (ELISA) from the peel of potato tubers. The ELISA results were also validated by gas chromatography-electron capture detector and confirmed by gas chromatography-mass spectrometry and by a lettuce hypocotyl bioassay. The degree of rest in the tubers was associated with ABA content in the peel. Basal portion (where tuber was attached to mother plant) contained the highest amount of ABA. Thidiazuron reduced ABA content and induced potato tuber sprouting. Exogenously applied ABA stimulated growth of buds that had emerged from dormancy.On leave from the Department of Horticulture, South China Agricultural University, Guangzhou, People's Republic of China.     

Melafen effect on ATP-dependent accumulation of calcium in plasma membrane vesicles from cells of potato tubers

Synthetic growth regulator melafen (10⁻⁵–10⁻¹⁰ M) was tested for aneffect on the Ca²⁺ accumulation in plasma membrane vesicles (PMVs) isolated from potato Solanum tuberosum L. tubers at forced rest and sprouting. Melafen proved to regulate the Ca²⁺ accumulation in PMVs by changing the activity of Ca²⁺, Mg²⁺-ATPase of the plasma membrane, while no effect was observed with respect to Ca²⁺ outflow from vesicles. The melafen effect on Ca²⁺, Mg²⁺-ATPase activity depended on the physiological condition of tubers and the melafen concentration.     

Abscisic Acid Suppresses the Hypersensitive Necrosis of Potato Tubers and Reverses the Arachidonic Acid-Induced Inhibition of Plasma Membrane-Bound ATPase

Abscisic acid (ABA) fully suppressed the development of the hypersensitive necrosis of potato tuber slices to crude elicitor from Phytophthora infestans and arachidonic acid (AA) if supplied before or up to 6 to 8 h after elicitor treatments. Later application of ABA only reduced the necrotic response. Added to highly purified plasma membrane preparation from tuber tissue, ABA eliminated the AA-induced inhibition of the plasma membrane-bound (K⁺+ Na⁺+ Mg²⁺) ATPase. It is suggested that ABA counteracts AA in the membrane.     

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

H-pumping driven by the plasma membrane ATPase in membrane vesicles from radish: stimulation by fusicoccin

The effect of fusicoccin on Mg:ATP-dependent H⁺-pumping in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings was investigated by measuring the initial rate of decrease in the absorbance of the ΔpH probe acridine orange. Fusicoccin stimulated Mg:ATP-dependent H⁺-pumping when the pH of the assay medium was in the range 7.0 to 7.6 while no effect of fusicoccin was detected between pH 6.6 and pH 6.0. Both basal and fusicoccin-stimulated H⁺-pumping were completely inhibited by vanadate and almost unaffected by nitrate. Fusicoccin did not change membrane permeability to protons and fusicoccin-induced stimulation of Mg:ATP-dependent H⁺-pumping was not affected by changes in the buffer capacity of the incubation medium. Deacetylfusicoccin stimulated H⁺-pumping as much as fusicoccin, while the physiologically inactive derivative 8-oxo-9-epideacetylfusicoccin did not. Stimulation of H⁺-pumping was saturated by 100 nanomolar fusicoccin. These data indicate that fusicoccin activates the plasma membrane H⁺-ATPase by acting at the membrane level independently of the involvement of other cell components. The percent stimulation by fusicoccin was the same at all ATP concentrations tested (0.5-5.0 millimolar), thus suggesting that with fusicoccin there is an increase in V_max of the plasma membrane H⁺-ATPase rather than a decrease in its apparent K_m for Mg:ATP.     

Separation of two types of electrogenic h-pumping ATPases from oat roots

Microsomal vesicles of oat roots (Avena sativa var Lang) were separated with a linear dextran (0.5-10%, w/w) or sucrose (25-45%, w/w) gradient to determine the types and membrane identity of proton-pumping ATPases associated with plant membranes. ATPase activity stimulated by the H⁺/K⁺ exchange ionophore nigericin exhibited two peaks of activity on a linear dextran gradient. ATPase activities or ATP-generated membrane potential (inside positive), monitored by SCN⁻ distribution, included a vanadate-insensitive and a vanadate-sensitive component. In a previous communication, we reported that ATP-dependent pH gradient formation (acid inside), monitored by quinacrine fluorescence quenching, was also partially inhibited by vanadate (Churchill and Sze 1983 Plant Physiol 71: 610-617). Here we show that the vanadate-insensitive, electrogenic ATPase activity was enriched in the low density vesicles (1-4% dextran or 25-32% sucrose) while the vanadate-sensitive activity was enriched at 4% to 7% dextran or 32% to 37% sucrose. The low-density ATPase was stimulated by Cl⁻ and inhibited by NO⁻₃ or 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS). The distribution of Cl⁻-stimulated ATPase activity in a linear dextran gradient correlated with the distribution of H⁺ pumping into vesicles as monitored by [¹⁴C]methylamine accumulation. The vanadate-inhibited ATPase was mostly insensitive to anions or DIDS and stimulated by K⁺. These results show that microsomal vesicles of plant tissues have at least two types of electrogenic, proton-pumping ATPases. The vanadate-insensitive and Cl⁻-stimulated, H⁺-pumping ATPase may be enriched in vacuolar-type membranes; the H⁺-pumping ATPase that is stimulated by K⁺ and inhibited by vanadate is most likely associated with plasma membrane-type vesicles.     

Possible Involvement of Abscisic Acid in Increases in Activities of Two Vacuolar H+-Pumps in Barley Roots under Aluminum Stress

Levels of abscisic acid (ABA) in barley roots increased upontreatment with AlCl₃. Treatment with AlCl₃ or ABA increasedboth ATP-dependent and PP_i-dependent H⁺-pumping activities intonoplast-enriched membrane vesicles. Increase in the H⁺-pumpingactivities caused by aluminum stress could result from increasedlevels of ABA. ¹Present address: Department of Botany, Faculty of Science,Hirosaki University, Hirosaki, Aomori, 036 Japan     

Mechanism of cAMP-induced H+-efflux ofDictyostelium cells: a role for fatty acids

AggregatingDictyostelium cells release protons when stimulated with cAMP. To find out whether the protons are generated by acidic vesicles or in the cytosol, we permeabilized the cells and found that this did not alter the cAMP-response. Proton efflux in intact cells was inhibited by preincubation with the V-type H⁺ ATPase inhibitor concanamycin A and with the plasma membrane H⁺ ATPase blocker miconazole. Surprisingly, miconazole also inhibited efflux in permeabilized cells, indicating that this type of H⁺ ATPase is present on intracellular vesicles as well. Vesicular acidification was inhibited by miconazole and by concanamycin A, suggesting that the acidic vesicles contain both V-type and P-type H⁺ ATPases. Moreover, concanamycin A and miconazole acted in concert, both in intact cells and in vesicles. The mechanism of cAMP-induced Ca²⁺-fluxes involves phospholipase A₂ activity. Fatty acids circumvent the plasma membrane and stimulate vesicular Ca²⁺-efflux. Here we show that arachidonic acid elicited H⁺-efflux not only from intact cells but also from acidic vesicles. The target of regulation by arachidonic acid seemed to be the vesicular Ca²⁺-relase channel.     

Plasma membrane ATPase and H+ transport activities in microsomal membranes from mycorrhizal tomato roots

ATPase activity, ATP-dependent H⁺ transport and the amount of antigenic tomato plasma membrane H⁺-APTase have been analysed in membrane vesicles isolated from Glomus mosseae- or Glomus intraradices-colonized roots and from non-mycorrhizal tomato roots. Microsomal protein content was higher in mycorrhizal than in control roots. The specific activity of the plasma membrane H⁺-ATPase was not affected by mycorrhizal colonization, although this activity increased in membranes isolated from mycorrhizal roots when expressed on a fresh weight basis. Western blot analysis of microsomal proteins using antibodies raised against the Arabidopsis thaliana plasma membrane H⁺ - ATPase showed that mycorrhizal colonization did not change the relative amount of tomato plasma membrane ATPase in the microsomes. However, on a fresh weight basis, there was a greater amount of this protein in roots of mycorrhizal plants. In addition, mycorrhizal membranes showed a higher specific activity of the vanadate-sensitive ATP-dependant H⁺ transport than membranes isolated from control roots. These results suggest that mycorrhiza might regulate the plasma membrane ATPase by increasing the coupling efficiency between H⁺ transport and ATP hydrolysis. The observed effects of mycorrhizal colonization on plasma membrane H⁺-ATPase were independent of the AM fungal species colonizing the root system.     

Low molecular weight humic substances stimulate H+-ATPase activity of plasma membrane vesicles isolated from oat (Avena sativa L.) roots

The effect of <5 KDa (low molecular weight, LMW) and >5 KDa (high molecular weight, HMW) humic fractions on transport activities of isolated plasma membrane vesicles was studied. The K⁺-stimulated component of the ATP-hydrolyzing activity was considerably increased by LMW humic substances at concentrations ranging from 0.075 mg org CL^-1 to 1 mg org CL^-1. The stimulation was still evident when the detergent Brij-35 was added in the assay mixture, indicating a direct effect of LMW humic substances on plasma membrane ATPase activity. The LMW humic fraction stimulated ATP-dependent intravesicular H⁺-accumulation with a pattern similar to that recorded for ATP hydrolysis. LMW humic substances induced also an increase in passive membrane permeability to protons, as revealed by following the dissipation of an artificially imposed pH gradient. Membrane permeability to anions, as measured by the anion-dependent active proton accumulation was affected by LMW humic substances. In the presence of NO₃ ^- these molecules clearly enhanced proton transport, while Cl^--dependent activity was almost unaffected, thus suggesting a specific action of LMW humic fraction on transmembrane NO₃ ^- fluxes. On the other hand, HMW humic substances decreased the passive permeability to protons and reduced the anion-dependent intravesicular H⁺-accumulation. The results suggest that the stimulatory effect of soil humic substances on plant nutrition and growth might be, at least in part, explained on the basis of both direct action of LMW humic molecules on plasma membrane H⁺-ATPase and specific modification of cell membrane permeability.     

Purification of Functionally Sealed Cytoplasmic Side-out Plasma Membrane Vesicles from Saccharomyces cerevisiae

Highly purified plasma membrane vesicles were prepared from yeast protoplasts by a combination of osmotic lysis, differential centrifugation, and separation in an aqueous dextran/polyethylene glycol two-phase system. The vesicles were predominantly (85-90%) of cytoplasmic side-out orientation and displayed large ATP-dependent proton pumping activity which was inhibited by vanadate (100 μM) but not by bafilomycin or nitrate. The preparation presented a distinct polypeptide profile with respect to the total membrane fraction and was enriched in the 110-kDa polypeptide corresponding to the plasma membrane H⁺-ATPase. This preparation of native plasma membranes vesicles is especially suitable for functional studies in vitro.     

Interaction of phytohormones and synthetic growth regulator melafen in the control of Ca2+ translocation across the plasma membrane of potato (Solanum tuberosum L.) tuber cells

Interference of phytohormones (jasmonic, gibberellic, and abscisic acids) and synthetic growth regulator melafen on Ca²⁺ translocation across the membrane of plasma membrane vesicles prepared from dormant potato (Solanum tuberosum L.) tubers was studied. The activity of plasma membrane Ca²⁺, Mg²⁺-ATPase was stimulated by melafen and jasmonic and gibberellic acids and suppressed by abscisic acid. These substrances did not change the passive membrane permeability for Ca²⁺. The pattern of the effect of melafen on the activity of Ca²⁺,Mg²⁺-ATPase depended on the presence of phytohormones in incubation medium. When melafen and each phytohormone were simultaneously added to incubation medium, their effects were not additive, which indicates that the effects of the tested compounds on the Ca²⁺ uptake into the plasma membrane vesicles are interdependent. Apparently, the interaction between the phytohormones and plasma membrane components modulates the response to melafen.     

Na+-transporting ATPase in the plasma membrane of halotolerant microalga Dunaliella maritima operates as a Na+ uniporter

A fraction of inside-out membrane vesicles enriched in plasma membranes (PM) was isolated from Dunaliella maritima cells. Attempts were made to reveal ATP-driven Na⁺-dependent H⁺ efflux from the PM vesicles to external medium, as detected by alkalization of the vesicle lumen. In parallel experiments, ATP-dependent Na⁺ uptake and electric potential generation in PM vesicles were investigated. The alkalization of the vesicle lumen was monitored with an impermeant pH-sensitive optical probe pyranine (8-hydroxy-1,3,6-pyrenetrisulfonic acid), which was loaded into vesicles during the isolation procedure. Sodium uptake was measured with ²²Na⁺ radioactive label. The generation of electric potential in PM vesicles (positive inside) was recorded with a voltage-sensitive probe oxonol VI. Appreciable Na⁺-and ATP-dependent alkalization of vesicle lumen was only observed in the presence of a protonophore CCCP (carbonyl cyanide-chlorophenylhydrazone). In parallel experiments, CCCP accelerated the ATP-dependent ²²Na⁺ uptake and abolished the electric potential generated by the Na⁺-ATPase at the vesicle membrane. A permeant anion NO^? ₃ accelerated ATP-dependent ²²Na⁺ uptake and promoted dissipation of the electric potential like CCCP did. At the same time, NO^? ₃ inhibited the ATP-and Na⁺-dependent alkalization of the vesicle lumen. The results clearly show that the ATP-and Na⁺-dependent H⁺ efflux from PM vesicles of D. maritima is driven by the electric potential generated at the vesicle membrane by the Na⁺-ATPase. Hence, the Na⁺-transporting ATPase of D. maritima carries only one ion species, i.e., Na⁺. Proton is not involved as a counter-ion in the catalytic cycle of this enzyme.