Evidence for the H+-co-transport of d-alanine by the digestive glands of Dionaea muscipula Ellis

Abstract. The net uptake of ¹⁴C-D-alanine by the adaxial surfaces of the trap lobes of Dionaea muscipula Ellis (Venus's flytrap) is positively correlated with the rate of H⁺ efflux from the trap lobes. Effectors that stimulate H⁺ efflux (secretion elicitor, fusicoccin and auxin) enhance D-alanine uptake whereas effectors that inhibit H⁺ efflux (diethylstilbestrol) retard uptake. It is proposed that amino acid uptake by the digestive glands of the trap lobes proceeds via a mechanism which mediates the co-transport of H⁺ ions and amino acids. A model is presented in which a positively charged ternary complex of carrier plus amino acid plus H⁺ is transported down an inwardly directed H⁺ electrochemical potential difference generated by the secretion-elicitor-provoked discharge of HCl into the trap cavity.     

Chilling induces a decrease in pyrophosphate-dependent H+-accumulation associated with a DeltapH(vac)-stat in mung bean, a chill-sensitive plant

Chilling leads to cytoplasmic acidification in chill-sensitive plants. A possible explanation for this observation is that a ΔpH-stat between the cytosol and vacuole (ΔpH_vac-stat) is perturbed by chilling. To understand the nature of this ΔpH_vac-stat, the effect of temperature, between 20 and 0 °C, on pyrophosphate (PPi)- or ATP-dependent acidification of vacuolar vesicles, isolated from mung bean hypocotyls, was determined. Over the temperature range investigated, the H⁺-influx mediated by PPase was balanced with the H⁺-efflux, which was PPi-dependently suppressed, and consequently a constant pH in vesicles (pH_in) of ca. 5 was maintained against temperature changes. However, the ΔpH_in driven by ATP decreased as the temperature dropped. Thus, the PPi-dependent H⁺-accumulation may function as an essential factor to form a ΔpH_vac-stat against temperature changes. Next, to study the chilling sensitivity of PPi-dependent H⁺-accumulation, vacuolar vesicles were isolated from control seedlings or from seedlings chilled at 0 °C for 1 d. Chilling treatment resulted in a decrease in the H⁺-accumulation rate and in the steady-state ΔpH_in formed by PPi, the causes of which were enhanced by PPi-dependent H⁺-efflux and reduced by H⁺-influx driven by PPase. Together, the results suggest that the decrease of PPi-dependent H⁺-accumulation associated with the ΔpH_vac-stat could result in cytoplasmic acidification.     

Synergistic activation of plasma membrane H+– ATPase in Arabidopsis thaliana cells by turgor decrease and by fusicoccin

The regulation of the H⁺-ATPase of plasma membrane is a crucial point in the integration of transport processes at this membrane. In this work the regulation of H⁺-ATPase activity induced by changes in turgor pressure was investigated and compared with the stimulating effect of fusicoccin (FC). The exposure of cultured cells of Arabidopsis thaliana L. (ecotype Landsberg 310–14-2) to media containing mannitol (0. 15 or 0. 3 M ) or polyethylene glycol 6000 (PEG) (15. 6% or 22% w/v) resulted in a decrease in the turgor pressure of the cells and in a strong stimulation of H⁺ extrusion in the incubation medium. The osmotica-induced H⁺ extrusion was (1) inhibited by the inhibitor of plasma membrane H⁺-ATPase, erythrosin B (EB), (2) dependent on the external K⁺ concentration, (3) associated with a net K⁺ influx, and (4) lead to an increase of cellular malate content. These results show that the reduction of external osmotic potential stimulates the activity of plasma membrane H⁺-ATPase
The effect of mannitol was only partially inhibited by treatments with cycloheximide (CH) and cordycepin, which block protein and mRNA synthesis, respectively. All the effects of osmotica were qualitatively and quantitatively similar to those induced by 5 μ M FC. However, when FC and mannitol (or PEG) were fed together, their effects on H⁺ extrusion appeared synergistic, irrespective of whether FC was present at suboptimal or optimal concentrations. This behaviour suggests that the modes of action of FC and of the osmotica on H⁺-ATPase activity differ at least in some step(s)     

pH homeostasis and bioenergetic work in alkalophiles

Abstract A Na⁺/H⁺ antiporter catalyses coupled Na⁺ extrusion and H⁺ uptake across the membranes of extremely alkalophilic bacilli. This exchange is electrogenic, with H⁺ translocated inward > Na⁺ extruded. It is energized by the Δψ ² component of the ΔμH⁺ that is established during primary proton pumping by the alkalophile respiratory chain complexes. These complexes abound in the membranes of extreme alkalophiles. Combined activity of the respiratory chain, the antiporter, and solute transport systems that are coupled to Na⁺ re-entry, allow the alkalophiles to maintain a cytoplasmic pH that is several pH units more acidic than optimal external pH values for growth. There is no compelling evidence for a specific and necessary role for any ion other than sodium in pH homeostasis, and although there is very high cytoplasmic buffering capacity in the alkaline range, active mechanisms for pH homeostasis are crucial. Energization of the antiporter as well as the proton translocating F ₁ F ₀-ATPase that catalyses ATP synthesis in the extreme alkalophiles must accommodate the problem of the low net ΔμH⁺ and the very low concentrations of protons, per se, in the external medium. This problem is by-passed by other bioenergetic work functions, such as solute uptake or motility, that utilize sodium ions for energy-coupling in the place of protons.     

Demonstration of a K+/H+ exchange activity in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

Abstract Washed cells of Rhodopseudomonas sphaeroides forma sp. denitrificans , grown under photodenitrifying conditions, exhibited K⁺ uptake dependent on the transmembrane proton gradient (Δ pH). These cells also acidified the suspension medium in response to K⁺ pulses both aerobically and anaerobically in light and in the dark. The results indicate that the photodenitrifier has a reversible K⁺/H⁺ exchange activity which reflects its role in regulating the intracellular K⁺ concentration, as well as intracellular pH. The acidification of the external medium resulting from K⁺ pulses was inhibited by carbonyl cyanide- m -chlorophenylhydrazone (CCCP) indicating that the antiporter is energy-dependent. Addition of KCl to washed cells depolarized the membrane potential (Δψ) with a concomitant increase in ΔpH, indicating that the K⁺/H⁺ antiporter was electrogenic.     

The tonoplast H+-pyrophosphatase of radish seedlings: biochemical characteristics

The activity of the H⁺-pyrophosphatase (H⁺-PPase) was characterized in microsomes from 24-h-old radish ( Raphanus sativus L., ev. Tondo Rosso Quarantino) seedlings, which are virtually devoid of the tonoplast H⁺-ATPase. The H⁺-PPase was localized to membranes which roughly comigrated with the plasma membrane in a sucrose density gradient, but clearly separated from plasma membrane when microsomes were partitioned in an aqueous dextran-polyethylene glycol two-phase system. The H⁺-PPase activity was strictly dependent on Mg²⁺ and on the presence of a monovalent cation (K⁺=Rb⁺=NH₃⁺Cs⁺≫Na⁺Li⁺) and was insensitive to anions such as Cl−, Br−, NO₃− and SO₄^2-. It was inhibited by F−, imidodiphosphate and Ca²⁺. It had a pH optimum between pH 7.5 and 8.5 and was saturated by low concentrations of pyrophosphate (half saturation at 30 μ M pyrophosphate). All of these characteristics are identical to those reported for the tonoplast H⁺-PPase from various plant materials. The functional molecular weight of the H⁺-PPase, measured with the radiation-inactivation technique was 96 kDa.     

Tonoplast H+ pumps are activated during callus formation of tuber tissues of Jerusalem artichoke (Helianthus tuberosus)

Changes in tonoplast H⁺-ATPase (EC 3.6.1.3) and H⁺–PPase (EC 3.6.1.1) activities were examined during the early period of callus formation in tuber tissues of Jerusalem artichoke ( Helianthus tuberosus L.). In callus-forming tissues cultured on a medium containing 2,4-D, the ATP-dependent H⁺-translocation activity of tonoplast vesicles increased 3-fold after a 2-day lag phase, while the ATP-hydrolytic activity and amount of tonoplast H⁺-ATPase protein were relatively constant after the lag phase. In the control tissue disks cultured on a medium free of 2,4-D, large declines in ATP-hydrolytic and ATP-dependent H⁺-translocation activities were observed. By contrast, the PP-dependent H⁺-translocation activity of tonoplast vesicles increased about 8-fold during the first 3 days of culture without any lag phase, and regardless of the presence of 2,4-D in the culture medium. However, the PP-hydrolytic activity and amount of H⁺-PPase protein did not change during the culture period, independently of callus formation. Transfer of the control tissue disks to the 2,4-D-containing medium, however, resulted in a further rapid stimulation of PP-dependent H⁺-translocation as well as an activation of ATP-dependent H⁺-translocation. These results suggest that both tonoplast H⁺ pumps are involved in callus formation of tuber tissues of Jerusalem artichoke.     

Uptake of neutral and acidic amino acids by Lemna gibba correlated with the H+-electrochemical gradient at the plasmalemma

Uptake rates of L-alanine, L-serine and L-aspartate and trans-membrane electrical potentials (Δψ) were determined for a pH range in the external medium between 3.5 and 9.0. The proton electrochemical gradients (     ) were calculated from Δψ, pH of the medium, and an assumed cytoplasmic pH of 7.5. At external amino-acid concentrations of 0.1 mol m⁻³, where carrier-mediated uptake dominates total uptake, a linear correlation between uptake rates and     is obtained, which extrapolates to zero uptake at zero     . This corroborates the contention that neutral and acidic amino acids are taken up by Lemna gibba L. by H⁺-cotransport.     

H+/PPi stoichiometry of a membrane‐bound pyrophosphatase of plant mitochondria

The H⁺/PP_i stoichiometry of the mitochondrial H⁺‐PP_iase from pea ( Pisum sativum L.) stem was determined by two kinetic approaches, and compared with the H⁺/substrate stoichiometries of the mitochondrial H⁺‐ATPase, and the vacuolar H⁺‐PP_iase and H⁺‐ATPase. Using sub‐mitochondrial particles or preparations enriched in vacuolar membranes, the rates of substrate‐dependent H⁺‐transport were evaluated: by a mathematical model, describing the time‐course of H⁺‐gradient (ΔpH) formation; or by determining the rate of H⁺‐leakage following H⁺‐pumping inhibition by EDTA at the steady‐state ΔpH. When the H⁺‐transport rates were divided by those of PP_i or ATP hydrolysis, measured under identical conditions, apparent stoichiometries of ca 2 were determined for the mitochondrial H⁺‐PP_iase and H⁺‐ATPase, and for the vacuolar H⁺‐ATPase. The stoichiometry of the vacuolar H⁺‐PP_iase was found to be ca 1. From these results, it is suggested that the mitochondrial H⁺‐PP_iase may, in theory, function as a primary H⁺‐pump poised towards synthesis of PP_i and, therefore, acting in parallel with the main H⁺‐ATPase.     

Substrate stabilization of lysophosphatidylcholine-solubilized plasma membrane H+-ATPase from oat roots

Plasma membrane vesicles with H⁺-ATPase activity were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots using an aqueous polymer two-phase system. Of several detergents tested, only lysophosphatidylcholine solubilized the H⁺-ATPase in an active form. Solubilization of the H⁺-ATPase with lysophosphatidylcholine was possible in the absence of glycerol, but the ATPase activity decreased about 4–5 times as rapidly in the absence as in the presence of 30% (w/v) glycerol. The solubilized enzyme was further stabilized by ATP and protons. Addition of 1 m M ATP to the plasma membranes halted inactivation of the H⁺-ATPase. Even in the absence of polyol compounds and ATP, the enzyme was stable for hours at relatively low pH with an optimum around pH 6.7 at room temperature. The curve for the stability of soluble H⁺-ATPase as a function of pH closely resembles the pH curve for the activity of the H⁺-ATPase. This suggests that binding of protons to transport sites may stabilize the soluble H⁺-ATPase in an enzymatically active form.     

Inhibition of Glutamate Uptake and Proton Pumping in Synaptic Vesicles by S-Nitrosylation

Abstract: Nitric oxide (NO; including NO^•, NO⁺, and NO⁻) was found to inhibit glutamate uptake by isolated synaptic vesicles of rat brain. This was observed when two unrelated NO donors, S -nitrosogluthathione and S -nitroso- N -acetylpenicillamine, were used. The primary target of NO is the H⁺-ATPase found in the synaptic vesicles, which leads to dissipation of the electrochemical proton gradient and inhibition of glutamate uptake. Oxyhemoglobin (12 µ M ) and, to a much lesser extent, methemoglobin protected the vacuolar H⁺-ATPase from inhibition. Inhibition of H⁺ pumping by NO was reversed by addition of 0.5 m M dithiothreitol. The results indicate that the vacuolar H⁺-ATPase from synaptic vesicles is inhibited by NO by a mechanism that involves S -nitrosylation of critical sulfhydryl groups in the enzyme. The interaction of NO with synaptic vesicles might be of importance for the understanding of the multiple effects of NO in neurotransmission.     

In vivo and in vitro effects of boron on the plasma membrane proton pump of sunflower roots

The effect of boron excess and deficiency on H⁺ efflux from excised roots from sunflower ( Heliarahus annuus L. cv. Enano) seedlings and on plasma membrane H⁺-ATPase (EC 3.6.1.35) in isolated KI-washed microsomes has been investigated. When seedlings were grown in media with toxic levels of H₃BO₃ (5 m M ) or without added boron and exposed to light conditions, an inhibition of the capacity for external acidification by excised roots was observed as compared to roots from seedlings grown with optimal H₃BO₃ concentration (0.25 m M ). Toxic and deficient boron conditions also inhibited the vanadate-sensitive H⁺-ATPase of microsomes isolated from the roots. The mechanism of boron toxicity was investigated in vitro with microsorne vesicles. A strong effect of boron on the vanadate-sensitive, ATP-dependent H⁺ transport was found, but the vanadate-sensitive phospho-bydrolase activity was not affected. These results suggest that boron could exert an effect on the plasma membrane properties, directly or indirectly regulating, proton transport.     

Plasma membrane ATPase activity during pepino (Solanum muricatum) ripening

Plasma membrane-enriched samples were extracted from pepino fruit (cv. El Camino) by phase partitioning. H⁺-ATPase (EC 3.6.1.35) activity in these samples increased during late fruit development (immediately before the onset of ripening) and western blotting confirmed there was an increase in enzyme abundance at this time. H⁺-ATPase activity decreased during early ripening and then increased again in the final phase of ripening. Immunolocalisation showed the plasma membrane H⁺-ATPase was most abundant in the outer cell layers of the fruit, which are considered to have a major role in determining fruit texture. Fruit softening was not accelerated by harvest and there was no stimulation of H⁺-ATPase activity by harvest. An in vitro tensile test using fruit rings showed tissue softening proceeded faster at low apoplastic pH (4.5) than at pH 6.5; and tissue buffered at pH 6.5 softened less than unbuffered rings. Erythrosin B, an inhibitor of the plasma membrane H⁺-ATPase, also retarded softening in vitro. These data suggest that plasma membrane H⁺-ATPase activity may contribute to the onset of pepino softening through a reduction in apoplastic pH.     

Effect of K+ and H+ stress and role of Ca2+ in the regulation of intracellular K+ concentration in mung bean roots

The effects of external K⁺, H⁺ and Ca2⁺ concentrations on the intracellular K+ concentration, [K⁺]i, and the K⁺-ATPase activity in 2-day-old mung bean roots [ Vigna mungo (L.) Hepper] were investigated. [K⁺]i, in mung bean roots was markedly decreased by external K⁺ or H⁺ stress and did not recover the initial value even after the stress was removed. This decrease in [K⁺]i, gradually disappeared with the addition of (Ca2⁺. Ca2⁺ may offset the harmful effects of ion stress. Ca2⁺ seems to have two effects on K+ transport; control of K⁺ permeability and activation of K⁺ uptake, although K⁺-ATPase activity was inhibited by Ca2⁺ concentrations higher than 10–4 M. We suggest that Ca2⁺ activates K⁺ uptake indirectly through the acidification of the cytoplasm.     

Effects of desiccation on the 77°K fluorescence properties of the liverwort Porella navicularis and the isolated lichen green alga Trebouxia pyriformis

The uptake of the auxin type herbicide 2,4-D into rice seedlings ( Oryza sativa L. cv. Dunghan Shali) and its effects on the K⁺, NH⁺₄ and NO₃ ion uptake and the K⁺ content were investigated at different pH values. A short incubation of the roots in 0.01 m M 2,4-D caused a marked ion uptake inhibition only at low pH. The non-auxin type herbicide benthiocarb did not produce such an inhibitory effect. Lowering of the pH in the external medium led to an increased 2,4-D uptake by the roots. These results can be explained by the increased H⁺ permeability of the membranes, allowing a more rapid entrance of 2,4-D into the root cells, thereby inhibiting the active ion uptake. Rice roots not subjected to 2,4-D treatment responded to H⁺ stress with an increased anomalous K⁺ uptake and a decreased K⁺ content. With reference to the effects of pH changes on the ion and 2,4-D uptake, possible transport mechanism of NH⁺₄ and 2,4-D are briefly discussed.     

Cation-stimulated H+ efflux by intact roots of barley

Abstract. Rates of proton extrusion and potassium (⁸⁶Rb) influx by intact roots of barley ( Hordeum vulgare cvs . Fergus, Conquest and Betzes) plants were simultaneously measured in short-term (15min) experiments. The nature and extent of apparent coupling between these ion fluxes was explored by manipulating conditions of temperature, pH and cation composition and concentration during flux determinations. In addition, the influence of salt status upon these fluxes was examined. At low K⁺ concentrations (0.01 to 1 mol m⁻³), H⁺ efflux and K⁺ influx were strongly correlated in both low- and high-K⁺ roots, although K⁺: H⁺ exchange stoichiometries were almost consistently greater than 2:1. At higher concentrations (1 to 5 mol m⁻³), H⁺ efflux was either reduced or remained unchanged while K⁺ influxes increased. In the presence of Na₂SO₄, rates of H⁺ extrusion demonstrated similar cation dependence, although below 10 mol m⁻³ Na₂SO₄, H⁺ fluxes were generally 50% lower than in equivalent concentrations of K₂SO₄. These observations are considered in the context of current hypotheses regarding the mechanisms of k⁺/H⁺ exchange.     

Adaptation of plasma membrane H+-ATPase of rice roots to low pH as related to ammonium nutrition

The preference of paddy rice for NH₄⁺ rather than NO₃^- is associated with its tolerance to low pH since a rhizosphere acidification occurs during NH₄⁺ absorption. However, the adaptation of rice root to low pH has not been fully elucidated. This study investigated the acclimation of plasma membrane H⁺-ATPase of rice root to low pH. Rice seedlings were grown either with NH₄⁺ or NO₃^-. For both nitrogen forms, the pH value of nutrient solutions was gradually adjusted to pH 6.5 or 3.0. After 4 d cultivation, hydrolytic H⁺-ATPase activity, V _max, K _m, H⁺-pumping activity, H⁺ permeability and pH gradient across the plasma membrane were significantly higher in rice roots grown at pH 3.0 than at 6.5, irrespective of the nitrogen forms supplied. The higher activity of plasma membrane H⁺-ATPase of adapted rice roots was attributed to the increase in expression of OSA1, OSA3, OSA7, OSA8 and OSA9 genes, which resulted in an increase of H⁺-ATPase protein concentration. In conclusion, a high regulation of various plasma membrane H⁺-ATPase genes is responsible for the adaptation of rice roots to low pH. This mechanism may be partly responsible for the preference of rice plants to NH₄⁺ nutrition.     

Calcium transport and ATPase activity in a microsomal vesicle fraction from corn roots

Abstract. An investigation has been made of methods for isolating membrane vesicles from corn ( Zea mays L.) roots active in calcium transport and K⁺-stimulated ATPase. Pretreating and grinding the roots at room temperature with EGTA and fusicoccin increases basal ATPase activity. Improvement in Ca²⁺ uptake requires isolation of a scaled vesicle fraction by the method of Sze(1980). Sorbitol is superior to sucrose as an osmoticant. The pH optimum for Ca²⁺ uptake is 7.5. whereas that for associated ATPase activity is 6.5. Calmodulin strongly stimulates Ca²⁺ uptake in a process little affected by uncouplers and ATPase inhibitors, but blocked by chlorpromazine. Fusicoccin gives less stimulation of Ca²⁺ uptake which is sensitive to uncouplers, and is dependent upon isolation with fusicoccin present. It appears that the sealed vesicle fraction may possess two Ca²⁺ transport systems: a calmodulin-activated Ca²⁺-transporting ATPase, and a Ca²⁺/H⁺ antiport coupled through the protonmotive force to a fusicoccin-stimulated H⁺-ATPase.     

NaCl-induced accumulation of tonoplast and plasma membrane H+-ATPase message in tomato

NaCl-induced changes in the accumulation of message for the 70 kDa subunit of the tonoplast H⁺-ATPase and plasma membrane H⁺-ATPase were studied in hydroponically grown plants of Lycopersicon esculentum Mill. cv. Large Cherry Red. There was increased accumulation of message for the 70 kDa (catalytic) subunit of the tonoplast H⁺-ATPase in expanded leaves of tomato plants 24 h after final NaCl concentrations were attained. This was a tissue-specific response; levels of this message were not elevated in roots or in young, unexpanded leaves. The NaCl-induced accumulation of this message was transient in the expanded leaves and returned to control levels within 7 days. The temporal and spatial patterns of NaCl-induced accumulation of message for the plasma membrane H⁺-ATPase differed from the patterns associated with the 70 kDa subunit of the tonoplast H⁺-ATPase. NaCl-induced accumulation of the plasma membrane H⁺-ATPase message occurred in both roots and expanded leaves. Initially accumulation of the plasma membrane H⁺-ATPase message was greater in root tissue than in expanded leaves, but increased to higher levels in expanded leaves after 7 days. These results suggest that increased expression of the tonoplast H⁺-ATPase is an early response to salinity stress and may be associated with survival mechanisms, rather than with long-term adaptive processes.     

A Phosphopeptide Corresponding to the Cytosolic Stretch Connecting Transmembrane Segments 8 and 9 of the Plasma Membrane H+-ATPase Binds 14-3-3 Proteins and Inhibits Fusicoccin-Induced Activation of the H+-ATPase

Abstract: A putative consensus domain for binding of 14-3-3 proteins to the plasma membrane (PM) H⁺-ATPase was identified in the highly-conserved sequence RSR(p)SWSF [where (p)S is Ser776 of the maize isoform MHA2], localized in the cytosolic stretch connecting transmembrane segments 8 and 9. A 15 amino acid biotinylated phosphopeptide comprising this motif: i) bound a recombinant 14-3-3 protein, ii) inhibited fusicoccin-induced stimulation of the PM H⁺-ATPase activity both in PM isolated from germinating radish ( Raphanus sativus L.) seedlings and in ER isolated from Saccharomyces cerevisiae expressing AHA1 (an isoform of Arabidopsis thaliana PM H⁺-ATPase), and iii) inhibited fusicoccin binding to PM isolated from germinating radish seedlings. The corresponding non-phosphorylated peptide was inactive in all the performed assays. Together, these results suggest that the cytosolic strand connecting transmembrane segments 8 and 9 of the PM H⁺-ATPase is a 14-3-3 binding site which might cooperate with the C-terminal domain of the'enzyme in generating a stable association between the H⁺-ATPase and 14-3-3 protein.