Characterization of Two Proton Transport Systems in the Tonoplast of Plasmalemma-Permeabilized Nitella Cells

ATP-dependent and PP_i-dependent H⁺-transport systems of thetonoplast were characterized in plasmalemma-permeabilized Nitellacells, where direct access to the protoplasmic surface of thetonoplast was possible. Since H⁺ transport across the tonoplastcan be measured in situ, the identity of the membrane responsiblefor H⁺ pumping is unequivocal. H⁺ transport was evaluated bythe accumulation of neutral red. While both transport systemswere obligately dependent on Mg²⁺, the two transport systemsshowed completely different sensitivity to NO₃^– and K⁺,suggesting the presence of two types of H⁺-pumps in Nitellatonoplast. NO₃^– applied to the protoplasmic surface, completelyand reversibly inhibited ATP-dependent transport but had noeffect on PP_i-dependent transport. By contrast, NO₃^– appliedinto the vacuole by the vacuolar perfusion technique did notinhibit ATP-dependent or PP_i-dependent H⁺ transport. Replacementof K⁺ with the organic cation, BTP, inhibited PP_i-dependenttransport but not the ATP-dependent one, indicating that PP_i-dependenttransport is K⁺ dependent. The sensitivities of the H⁺ transportsystems found in the tonoplast of Nitella are quite similarto those of higher plant tonoplasts. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. (Received February 21, 1987; Accepted May 27, 1987)     

Coupling Ratios H+/e=3 versus H+/e=2 in Chloroplasts and Quantum Requirements of Net Oxygen Exchange during the Reduction of Nitrite, Ferricyanide or Methylviologen

Using intact and osmotically ruptured chloroplasts, ratios ofcoupling between deposition of protons in the intrathylakoidspace and light-dependent transport of electrons from waterto an external acceptor were determined. The data indicate couplingbetween proton and electron transport at a ratio of H⁺/e=3 withmethylviologen as electron acceptor in thylakoids and with nitriteas electron acceptor in intact chloroplasts. With ferricyanideas electron acceptor in thylakoids, values close to H⁺/e=2 wereobserved. Evidence is discussed that H⁺/e=3 is a fixed valuein intact chloroplasts at levels of thylakoid energization sufficientfor supporting effective carbon assimilation. In the presence of methylviologen and ascorbate, the minimumquantum requirement of oxygen uptake by thylakoids was about2.7 quanta of 675 nm light per O₂ indicating an e/O₂ ratio of1.33. In the absence of ascorbate, and with KCN present in additionto methylviologen, e/O₂ ratios up to 4 were observed. The minimumquantum requirement of oxygen evolution by thylakoids in thepresence of ferricyanide and by intact chloroplasts in the presenceof nitrite was about 8 quanta/O₂. (Received May 1, 1995; Accepted October 2, 1995)     

Proton/Pyruvate Cotransport into Mesophyll Chloroplasts of C4 Plants

Light-enhanced active pyruvate uptake into mesophyll chloroplastsof C₄ plants was reported to be mimicked by either of the twotypes of cation jump: H⁺-jump in maize and phylogenically relatedspecies (H⁺-type) and Na⁺-jump in all the other C₄ species tested(Na⁺-type) [Aoki, N., Ohnishi, J. and Kanai, R. (1992) PlantCell Physiol. 33: 805]. In this study, medium and stromal pH was monitored in the suspensionof C₄ mesophyll chloroplasts. Medium alkalization lasting for5 to 10 seconds after pyruvate addition was detected by a pHelectrode and observed only in the light and only in mesophyllchloroplasts from H⁺-type species, Zea mays L. and Coix lacryma-jobiL., but not in those from Na⁺-type species Panicum miliaceumL., Setaria italica (L.) Beauv. and Panicum maximum Jacq. Theinitial rate of H⁺ consumption showed good correlation with[¹⁴C]pyruvate uptake measured by silicone oil filtering centrifugation,both being inhibited by N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole to the same degree. The ratio of the rate of H⁺ uptaketo that of pyruvate uptake was always about 1. Pyruvate-inducedacidification of the stroma was observed in maize mesophyllchloroplasts. These results show one to one cotransport of H⁺and pyruvate anion into mesophyll chloroplasts of H⁺-type C₄species in the light. (Received January 5, 1994; Accepted May 6, 1994)     

Proton Transport and pH Control in Sinapis alba Root Hairs: A Study carried out with Double-Barrelled pH Micro-electrodes

Continuous measurements of cytoplasmic pH (pH_c) in Sinapis roothairs have been carried out with double-barrelled pH-micro-electrodesin order to gain information on translocation of protons acrossthe plasmalemma and cytoplasmic pH control. (i) The cytoplasmicpH of Sinapis (7–33 ? 0–12, standard conditions)changes no more than 0.1 pH_c, per pH_o-unit, regardless of whethercyanide is present or not. (ii) Weak acids rapidly acidify pH_cand hyperpolarize, while weak bases alkalize pH_c and depolarizethe cells, (iii) 1.0 mol M,³ NaCN acidifies the cytoplasm by0.4 to 0.7 pH-units, but alkalizes the vacuole. (iv) 20 mmolm^–3 CCCP has no significant effect on pH_c, if added atpH 9.6 or 7.2, but acidifies pH_c by 1.3 units at pH 4.3. Inthe presence of CCCP, cyanide acidifies the cytoplasm, (v) Chloridetransiently acidifies pH_c, while K⁺, Na⁺, and have no significant effects, (vi) Cytoplasmic buffer capacityforms a bell-shaped curve versus pH_c with an optimum of about50 mol m^–3 H⁺pH_c-unit. The modes of proton re-entry and the effects of active and passiveproton transport on cellular pH control are critically discussed.It is suggested that the proton leak, consisting of H⁺-cotransport(e.g. H⁺/Cl^–) rather than H⁺-uniport, is no threat topH_c. The proton export pump, although itself reacting to changesin pH_c, influences pH_c only to a minor extent. It is concludedthat buffer capacity and membrane transport play moderate rolesin pH_c control in Sinapis, while the interlocked H⁺-producingand -consuming reactions of cellular metabolism are the mainregulating factors. This makes pH control in Sinapis quite differentfrom bacterial and animal cells. Key words: Cytoplasmic pH, double-barrelled pH micro-electrode, pH control, proton transport, Sinapis     

Role for the Vacuolar H+-ATPase in Regulating the Cytoplasmic pH: An in Vivo Study Carried out in chl1, an Arabidopsis thaliana Mutant Impaired in NO-3 Transport

The effects of the growth in a medium containing NH₄NO₃ as nitrogensource were studied on cell sap pH, cytoplasmic pH and malatecontent in chl1, an Arabidopsis thaliana mutant impaired inchlorate and nitrate transport. In all the conditions testedthe pH of the cytoplasm in chl1 was more alkaline, and thatof the vacuole was more acidic as compared with those measuredin wt. Treatment with bafilomycin A1, a specific inhibitor ofthe vacuolar H⁺-ATPase, induced a small alkalinization of thevacuole, and a significant acidification of the cytoplasm, theseeffects being greater in chl1 than in wt. The greater responseof the mutant to bafilomycin Al suggests that, in the absenceof the inhibitor, the activity of the tonoplast H⁺-ATPase inchl1 is higher than in wt, this diversity being a possible reasonfor the differences in intracellular pH detected between thetwo strains. A possible role for the vacuolar H⁺-ATPase in regulatingthe cytoplasmic pH is discussed. (Received August 2, 1995; Accepted February 1, 1996)     

Quantitative Analysis of ATP-Dependent H+ Efflux and Pump Current Driven by an Electrogenic Pump in Nitellopsis obtusa

Internodal cells of Nitellopsis were made tonoplast-free byperfusion with a medium containing EGTA. Cytoplasmic concentrationsof solutes were controlled by a second perfusion with mediaof known composition. The electrogenic pump current (I_p), whichwas calculated from electrical data obtained from cells withand without ATP, was compared with the current carried by H⁺(I_H⁺) across the plasma membrane. A close correlation betweenI_p and I_H⁺ was found under various internal and external conditions.(1) I_p and I_H⁺ depended on the internal ATP and showed Michaelis-Mententype saturation curves. For I_p, K_m was 120 µM and themaximum current V_max was 15.1 mA m^–2, while for I_H⁺, K_mwas 160 µM and V_max was 16.6 mA m^–2. (2) I_p andI_H⁺ showed almost the same I_H²⁺ dependence. The Mg²⁺-dependentI_p was 19.5 mA m^–2, while the Mg²⁺-dependent I_H²⁺ was17.7 mA m^–2. (3) I_H²⁺ was maximal at an external pH of8 and decreased both in acidic and alkaline pH ranges. I_p wasnearly equal to I_H⁺ in the pH range between 8 and 5. (4) I_H⁺became maximal at an internal pH of 7.3, which is nearly thesame as the pH for maximal electrogenecity found by Mimura andTazawa (1984). All these facts support the idea proposed in our previous paper(Takeshige et al. 1985) that the electrogenic ion pump locatedin the plasma membrane of Nitellopsis is the H⁺ pump. ¹ Dedicated to Professor Dr. Erwin Bünning on the occasionof his 80th birthday. (Received June 21, 1985; Accepted December 20, 1985)     

Reappraisal of the Role of Sodium in the Light-Dependent Active Transport of Pyruvate into Mesophyll Chloroplasts of C4 Plants

The mechanism of light-dependent active transport of pyruvatein C₄ mesophyll chloroplasts has not been clarified, particularlyin Na⁺-type C₄ species, in which the pyruvate uptake into mesophyllchloroplasts is enhanced by illumination or by making a Na⁺gradient (Na⁺-jump) across the envelope in the dark. We re-investigatedhere the effect of Na⁺ on the active transport of pyruvate inmesophyll chloroplasts of Panicum miliaceum, a Na⁺-type C₄ species,by comparing the rate of pyruvate uptake at various externalpHs under four conditions; in the light and dark together with/withoutNa⁺-jump: (1) At neutral pH, the rate of pyruvate uptake inthe dark was enhanced by Na⁺-jump but scarcely by illumination.(2) While the enhancement effect by Na⁺-jump was independentof external pH, that by illumination increased greatly at pHover 7.4, and the effects of light and Na⁺ at the alkaline pHwere synergistic. (3) The light-enhanced pyruvate uptake wasrelated to stromal alkalization induced by illumination. Infact, pyruvate uptake was induced by H⁺-jump in the medium frompH 8.0 to 6.7. (4) Stromal pH was lowered by the addition ofK⁺-pyruvate and more by Na⁺-pyruvate into the medium at pH 7.8in the light. (5) However, the pH and ATP levels in the stromawere not affected by Na⁺-jump. Thus, we discussed possibility that besides pyruvate/Na⁺ cotransportat neutral pH in the medium, pyruvate/H⁺ cotransport enhancedby the presence of Na+ operates in mesophyll chloroplasts ofNa⁺-type C4 species at alkaline medium. ¹Present address: Biological Resources Division, Japan InternationalResearch Center for Agricultural Sciences (JIRCAS), Ministryof Agriculture, Forestry and Fisheries, 2-1 Ohwashi, Tsukuba,305 Japan     

Effect of Sudden Salt Stress on Ion Fluxes in Intact Wheat Suspension Cells

Although salinity is one of the major problems limiting agriculturalproduction around the world, the underlying mechanisms of highNaCl perception and tolerance are still poorly understood. Theeffects of different bathing solutions and fusicoccin (FC),a known activator of plasma membrane ATPase, on plasma membranepotential (E_m) and net fluxes of Na⁺, K⁺and H⁺were studied inwheat suspension cells (Triticum aestivum) in response to differentNaCl treatments. E_mof cells in Murashige and Skoog (MS) mediumwas less negative than in cells exposed to a medium containing10 mM KCl + 0.1 m M CaCl₂(KSM) and to a basic salt medium (BSM),containing 1 m M KCl and 0.1 m M CaCl₂. Multiphasic Na⁺accumulationin cells was observed, peaking at 13 min after addition of 120m M NaCl to MS medium. This time scale was in good agreementwith net Na⁺flux changes measured non-invasively by moving ion-selectivemicroelectrodes (the MIFE system). When 120 m M NaCl was addedto all media studied, a quick rise of Na⁺influx was reversedwithin the first 20 min. In both 120 and 20 m M NaCl treatmentsin MS medium, net Na⁺efflux was observed, indicating that activeNa⁺transporters function in the plant cell response to saltstress. Lower external K⁺concentrations (KSM and BSM) and FCpre-treatment caused shifts in Na⁺fluxes towards net influxat 120 m M NaCl stress. Copyright 2000 Annals of Botany Company Sodium, potassium, proton, membrane potential, fusicoccin, salt stress, wheat, Triticum aestivum     

Induction of the H+ Release from Thylakoid Membranes by Illumination in the Presence of Protonophores at High Concentrations

The generally observed light-induced uptake of protons intothe thylakoid lumen is diminished by adding protonophores. Insteadof the H⁺ uptake, the release of protons was observed duringillumination in the presence of various protonophores at highconcentrations, namely, 1 µM nigericin, 10 µM carbonylcyanidem-chlorophenylhydrazone or 30 µM gramicidin. An uncoupler,NH₄C1 (4 mM), and a detergent, Triton X-100 (0.02%), also inducedthe H⁺ release but a K⁺ ionophore, valinomycin, did not. Theamount of H⁺ released reached about 100 nmol H⁺ (mg Chl)^–1at pH 7.5 under continuous illumination. The rate of the H⁺release was similar to that of the conventional H⁺ uptake butits dark relaxation was much slower than that of the H⁺ uptake.We compared the H⁺ release in protonophore-added thylakoidswith the previously reported H⁺ release in coupling factor 1(CF₁-depleted thylakoids. The H⁺ release in thylakoids withnigericin showed similar characteristics to that in CF₁-depletedthylakoids in terms of their responses to pH, phenazine methosulfateand light intensity. Both types of H⁺ release were relativelyinsensitive to DCMU and were stimulated somewhat by DCMU atlow concentrations (around 200 nM). Nigericin did not inhibitthe superoxide dismutase activity of the membranes. These resultsindicate that the H⁺ release in protonophore-added thylakoidsand that in CF₁ depleted thylakoids involve the same mechanismand that water-derived protons from PS II that result from animpairment of the activity of superoxide dismutase, as previouslyproposed, are not involved. Judging from the rate of electronflow and the lumenal acidification under the illumination, weconclude that the H⁺ release is a light-dependent scalar processwhich can be observed in thylakoid membranes with high H⁺ permeability.The H⁺ release of this type was not observed in mitochondriafrom rat liver or in chromatophores from Rhodobacter sphaeroides. (Received November 29, 1990; Accepted June 27, 1991)     

Acidification and Alkalinization of Culture Medium by Catharanthus roseus cellsIs Anoxic Production of Lactate a Cause of Cytoplasmic Acidification?

Catharanthus roseus(L.) G. Don cells acidified Mura-shige-Skoogmedium rapidly. Upon transfer to fresh medium, the medium pH(initially5.3) dropped below 4 within 2 d. This acidificationwas reversed under hypoxic conditions. The cells induced a similaracidification in a simple medium consisting of CaCl₂, KCl, andglucose: medium pH dropped below 4 within 6 h. The acidificationwas accompanied by an influx of K⁺ at a H⁺(efflux)/K⁺ ratioof ca 0.6 as well as by an expansion of endogenous organic acidpool, in which malic and citric acids were the major components.Anoxia reversed all these processes: the direction of both K⁺and H⁺ fluxes reversed with a H⁺/K⁺ ratio of 1.70. Anoxia induceda cytoplasmic acidification from pH 7.6 (aerobic) to 7.4 asmeasured by 31P-NMR, accompanied by a rapid, long-lasting lactateaccumulation at expense of malic and citric acids. Evidencesuggested that accumulation of lactic acid was not a cause ofcytoplasmic acidification under anoxia, but a result of pH regulationby the biochemical pH-stat [Davies (1973) Symp. Soc. Exp. Biol.27: 513]. The anoxic acidification of the cytoplasm was ascribedto the influx of H⁺ from the medium. (Received April 18, 1997; Accepted July 8, 1997)     

Na+/H+ Antiporter in Tonoplast Vesicles from Rice Roots

The Na⁺/H ⁺ antiporter in vacuolar membranes transports Na⁺from the cytoplasm to vacuoles using a pH gradient generatedby proton pumps; it is considered to be related to salinitytolerance. Rice (Oryza sativa L.) is a salt-sensitive crop whosevacuolar antiporter is unknown. The vacuolar pH of rice roots,determined by ³¹P-nuclear magnetic resonance (NMR), increasedfrom 5.34 to 5.58 in response to 0.1 M NaCl treatment. Transportof protons into the tonoplast vesicles from rice roots was fluorometricallymeasured. Efflux of protons was accelerated by the additionof Na⁺. Furthermore, the influx of ²²Na⁺ into the tonoplastvesicles was accelerated by a pH gradient generated by proton-translocatingadenosine 5'-triphosphatase (H⁺-ATPase) and proton-translocatinginorganic pyro-phosphatase (H⁺-PPase). We concluded that thisNa⁺/H⁺antiporter functioned as a Na⁺ transporter in the vacuolarmembranes. The antiporter had a K_m of 10 mM for Na⁺ and wascompetitively inhibited by amiloride and its analogues. TheKⁱ values for 5-(N-methyl-N-isobutyl)-amiloride (MIA), 5-(N-ethyl-N-isopropyI)-amiloride(EIPA), and 5-(N, N-hexamethylene)-amiloride (HMA) were 2.2,5.9, and 2.9 µ M, respectively. Unlike barley, a salt-tolerantcrop, NaCl treatment did not activate the antiporter in riceroots. The amount of antiporter in the vacuolar membranes maybe one of the most important factors determining salt tolerance. ¹This work was supported by a grant from Bio-Media Project ofthe Japanese Ministry of Agriculture, Forestry and Fisheries(BMP96-III-1).     

Uncoupling Properties of the Herbicide N-n-Pentyl-N-Methyl-N'-(3,4-Dichlorophenyl)-Urea

The herbicide D₅ (N-n-pentyl-N-methyl-N'-(3, 4-dichlorophenyl)-ureacan uncouple oxidative phosphorylation in isolated plant mitochondria.This paper confirms that D₅ is an uncoupler that catalyzes thecollapse of the transmembrane potential gradient by inducinga movement of protons across the membrane. However, D₅ is notitself capable of transporting protons. D₅ gives complete uncouplingat 40 µM, a lower concentration than that required foruncoupling by the n-butyl homologue ‘neburon’. Analysisof the shape of the state 4 stimulation curve suggests thatD₅ might act as a dimer in the membrane. Attempts to demonstrate binding of D₅ to a membrane target gaveambiguous results, binding is not evident at 10 °C and 25°C but might occur at 15 °C and 20 °C. The calculatedherbicide concentration in the membrane (40 µM of which4–0 µM is as the dimer) is high and similar to thatof the major phospholipids. The calculated partition coefficientbetween medium and membrane (3.8 x 103) is in agreement withthe lipophilicity of the substituted urea herbicides. In the presence of a substrate, D₃ blocks both influx and effluxcalcium movement through the mitochondrial membrane but in theabsence of substrate, D₅ induces binding of calcium. Bindingrequires Mg⁺⁺ but not K⁺ or phosphate and leads to a releaseof H⁺. Ruthenium Red causes a partial inhibition of bindingbut no other reagent or ionophore tested had any effect. Sincebinding does not occur in turnip mitochondria which are unableto transport Ca⁺⁺ it is concluded that the effect is not directlylinked to the uncoupling action. The mechanism of action of D₅ is discussed and it is concludedthat D₅ probably acts as a dimer and perturbs membrane structure.The site of action is probably the lipid components of the membrane. Key words: Plant mitochondria, Herbicide, Substituted ureas, Calcium, Uncoupling     

Effect of Hormones on Sucrose Uptake and on ATPase Activity of Citrus sinensis L. Osbeck Leaves

Carbohydrate accumulation in young, fully expanded leaves ofCitrus sinensis L. Osbeck is affected by the presence of thefruitlet on the shoot. Previous work gave evidence that gibberellinsmay be involved in this 'fruit effect'. In the present workwe have studied the effect of gibberellic acid (GA₃) on ¹⁴C-sucroseuptake by leaf discs and whether its action could be due toa modulation of the plasma membrane ATPase, which maintainsthe H⁺ gradient that drives H⁺/sucrose co-transport. The effect of GA₃ on ¹⁴C-sucrose uptake depended on the osmolarityof the assay medium. At 300 mOsm a reduction in the uptake ratewas observed. The inhibitory effect of the hormone disappearedafter preincubating the leaf discs with para-chloromercuri-phenylsulphonicacid (PCMPS), a sulphydril binding inhibitor. ATPase activityof isolated plasma membrane vesicles was inhibited by IAA treatments,while GA₃ or ABA did not affect this enzyme, even after a 3h preincubation period. However, in the absence of a surfactantin the assay medium, GA₃, together with turgor pressure, modulatedplasma membrane ATPase activity, possibly through modificationsof membrane permeability. The hormone effect on ¹⁴ C-sucroseuptake may involve action on the sucrose carrier.Copyright 1994,1999 Academic Press Abscisic acid, Citrus sinensis, gibberellic acid, indoleacetic acid, orange, osmotic pressure, plasma membrane ATPase, ¹⁴C-sucrose uptake     

Proton Translocation Associated with Denitrification in a Photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

H⁺ translocation driven by NO₃^–, NO₂^– and N₂O reductionswith endogenous substrates in cells of Rhodopseudomonas sphaeroidesforma sp. denitrificans was investigated by the oxidant pulsemethod. Upon injection of nitrogenous oxides to anaerobic cellsin darkness, an alkaline transient in the external medium wasobserved, followed by acidification. The alkaline transientwas enhanced by carbonyl cyanide m-chlorophenylhydrazone. When a viologen dye was used as an electron donor in the presenceof 1 mM Af-ethylmaleimide and 0.1 mM 2-n-heptyl-4-hydroxyquinoline-N-oxideto preclude respiration-linked H⁺ extrusion, addition of KNO₃,KNO₂ and N₂O caused only a rapid alkalinization. The H⁺ consumptionstoichiometries, H⁺/2e^– ratios for NO₃^– reductionto NO₂^–, NO₂^– reduction to 1/2 N₂O and N₂O reductionto N₂ were –1.90, –3.18 and –2.04, respectively.These values agreed well with the fact that all reductions ofnitrogenous oxides in denitrification occur on the periplasmicside of the cytoplasmic membrane. When corrected for H⁺ consumption in the periplasm, the H⁺ extrusionstoichiometries, H⁺/2e^– ratios with endogenous substratesin the presence of K⁺/valinomycin for NO₃^– reduction toNO₂^–, NO₂^– reduction to 1/2 N₂O and N₂O reductionto N₂ were 4.05, 4.95 and 6.01, respectively. (Received August 4, 1982; Accepted January 13, 1983)     

Light-Induced H+ Accumulation in the Vacuole of Nitellopsis obtusa

The effects of light on the pH in the vacuole and the electricpotential difference across the plasmalemma and the tonoplastof Nitellopsis obtusa were investigated by means of conventionaland H⁺-specific glass or antimony microelectrodes. Illuminationis found to bring about a decrease in the pH of the vacuolarsap by 0.1–0.5 units concomitant with a depolarizationof the cell. The light-induced changes of the potential differenceand the vacuolar pH depend in different ways on the pH of theexternal medium (pH_o). At pH_o 9.0 cells exhibit great light-inducedpotential changes (up to 100 mV), but only small pH changesof the vacuolar sap. At neutral or slightly acidic pH_o valuesthe amplitude of the light-induced pH changes in the vacuoleincreases up to 0.3–0.5 pH units, but the amplitudes ofthe potential changes at the plasmalemma are relatively small.At pH_o 9.0 a transient acidification of the medium is observedupon illumination whereas at lower pH values light-induced alkalinizationwas only seen. Transfer of the cells from pH_o 9.0 to pH_o 7.5results in a cell hyperpolarization by 60–80 mV and adecrease of the vacuolar pH by 0.4–0.5 units under lightconditions but has no significant effect on the potential andthe vacuolar pH in the darkness. It is proposed that mechanismsof active H⁺ extrusion from the cytoplasm are located both inthe plasmalemma and the tonoplast. The observed acidificationin the vacuole appears to be determined by a light-induced increaseof the concentration of H⁺ in the cytoplasm. The H⁺ conductionof the plasmalemma seems to increase on illumination. The patternof the light-induced H⁺ fluxes across the tonoplast and theplasmalemma depends crucially on the extent of the light-inducedchanges in the H⁺ conductance and on the electrochemical gradientfor H⁺ at the plasmalemma.     

Portasomes as Coupling Factors in Active Ion Transport and Oxidative Phosphorylation

SYNOPSIS. We propose that particles, 7–15 nm in diameter,observed on the apical plasma membranes of cation transportingcells of insect midgut, salivary glands, and Malpighian tubulesare modified F₁-F₀ coupling complexes such as those found onphosphorylating membranes of mitochondria, chloroplasts, andbacteria. We suggest the generic term, portasome, to describeall of these particles and point out that they are located onthe side of the membrane which is electronegative and has thelow cation concentration, i.e., on the input side in each case.Biophysical evidence identifies the portasome bearing membraneas the ion transporting membrane in several insect epithelia,some of which exhibit ion modulated ATPase activity. The activityof a K⁺-modulated ATPase from Manduca sexta midgut is increasedin portasome enriched plasma membrane fractions. We proposethat portasomes orient the scalar hydrolysis of negatively chargedMgATP^2– to less negatively charged MgADP thereby eliminatingthe attraction of MgATP^2– to K⁺ with the result that theK⁺ ions are ejected to the opposite side of the portasome bearingmembrane. This mechanism explains the coupling of the scalarhydrolysis of ATP to the vectorial active transport of K⁺ whichleads to the establishment of a K⁺ electrochemical gradient.The reverse process, but with an H⁺ ionophore replacing a K⁺ionophore in the portasome, would provide a mechanism for couplingthe vectorial flow of H⁺, driven by a proton electrochemicalgradient, to scalar ATP synthesis and thereby provide a mechanismfor oxidative phosphorylation. Electrogenic active potassiumion transport would appear to have evolved from oxidative phosphorylation.     

Light-induced changes in chlorophyll a fluorescence and cytochrome f in intact spinach chloroplasts: The site of light-dependent regulation of electron transport

Dark-adapted intact spinach chloroplasts exhibited two peaks,P and M₁, at the early phase of fluorescence induction and atransient reduction of cytochrome f shortly after its initialphotooxidation and in parallel to the appearance of P. Analysisof the peak P and the transient reduction of cytochrome f indicatedthat electron transport in intact spinach chloroplasts was regulatedby light: electron transport was inactivated at the reducingside of photosystem I in the dark-adapted chloroplasts but rapidlyreactivated by illumination. The fluorescence peak M₁ was correlatedto the proton gradient formed across the thylakoid membrane. Effects on P and transient reduction of cytochromef of NO₂^–,3-phosphoglycerate (PGA) and oxalacetate (OAA), which can penetrateinto intact chloroplasts and accept electrons at different sitesafter photosystem I, were studied to determine the site of thelight regulation. NC₂^–, which receives electrons fromreduced ferredoxin, markedly diminished both P and the transientreduction of cytochrome.f, whereas PGA and OAA, the reductionsof which are NADP-dependent, failed to affect the two transients.The ineffectiveness of PGA and OAA could not be attributed tothe dark inactivation of glyceraldehyde-3-phosphate and malicdehydrogenases, because dark-adapted chloroplasts still retainedsufficiently high levels of the enzyme activities. The resultsindicate that electron transport in intact spinach chloroplastsis regulated by light after ferredoxin but before NADP, i.e.,at the reducing terminal of the electron transport chain. (Received May 29, 1980; )     

Bafilomycin A1 is a non-competitive inhibitor of the tonoplast H+-ATPase of maize coleoptiles

When microsomal membranes from maize (Zea mays L. cv. Clipper)coleoptiles were separated by isopyc-nic centrifugation on acontinuous 10–45% sucrose gradient, bafilomycin A1-inhibitedATPase activity co-localized with the activities of the tonoplastmarker-enzymes, nitrate-Inhibited ATPase and K⁺-dependent pyrophosphatase.Thus, bafilomycin A1 is a specific inhibitor of the vacuolarH⁺-ATPase of maize coleoptiles. Inhibition of the vacuolar H⁺-ATPaseby bafilomycin A1 was strictly dependent upon the concentrationof the enzyme present in the assay medium, suggesting a stoichiometricassociation between bafilomycin A1 and the vacuolar H⁺-ATPase.In tonoplast-enriched preparations, half-maximal inhibitionwas obtained at 43 pmol bafilomycin A1 mg^–1 protein. BafilomycinA1 inhibited the vacuolar H⁺-ATPase in a simple non-competitivemanner: increasing bafilomycin A1 concentrations reduced theV_max, of the H+ -ATPase, but had no effect on its K_m towardsATP. Key words: Bafilomycin A1, coleoptile, H⁺-ATPase (vacuolar), maize, Zea mays L     

Transient Cytoplasmic pH Changes in Correlation with Opening of Potassium Channels in Eremosphaera

Steigner, W. Khler, K., Simonis, W. and Urbach, W. 1988. Transientcytoplasmic pH changes in correlation with opening of potassiumchannels in Eremosphaera.—J. exp. Bot. 39: 23–36. The role of the cytoplasmic pH (pH_c) of Eremosphaera viridisin the signal transduction chain after light-off from the chloroplaststo the K⁺ channels in the plasmalemma of this unicellular algawas investigated. The temporary opening of K⁺ channels is indicatedby a transient hypcrpolarization (TP). To record rapid changesof pH_c, continuous measurements with pH sensitive micro-electrodeswere carried out. (i) Under normal conditions pH_c in the light(7·56 ±0·2) did not differ from pHc inthe dark (7·62 ±0·2). (ii) The vacuolepH ranged between 4·8 and 5·2. (iii) After light-offa rapid transient acidification of pHc O19±0·07occurred and a TP was released, (iv) In every case, the startof the transient acidification after light-off preceded thehyperpolarization by about 3s. (v) Light-on caused a rapid transientalkalinization but never a TP. (vi) Change to acid externalmedium (3.2) transiently acidified the cytoplasm and was ableto release a TP. (vii) After addition of NH₄Cl, pH_c again showeda rapid transient acidification and the release of a TP. The origin of the protons appearing in the cytoplasm after light-offis discussed critically with respect to the buffer capacity.Either direct or indirect translocation is a possible mechanismfor the movement of H⁺ from the chloroplasts into the cytoplasm.The intracellular acidification and its relation to the openingof potassium channels in the plasmalemma leads us to suggestthat a sudden change of pH_c is a potent internal signal factorin Eremosphaera viridis. Key words: Cytoplasmic pH, transient potential, K⁺–channels, Eremosphaera viridis     

Sodium Stimulates Regeneration of Phosphoenolpyruvate in Mesophyll Chloroplasts of Amaranthus tricolor

Mesophyll chloroplasts were isolated from leaves of a Na⁺-requiringNAD-malic enzyme type, dicotyledonous C₄ plant, Amaranthus tricolorL. The chloroplasts converted pyruvate to phosphoenolpyruvateunder illumination, and the conversion was stimulated by Na⁺.This observation may explain the requirement for Na⁺ of someC₄ plants. ² Present address: Institute for Life Science Research, NihonNohyaku Co., Ltd., Kawachi-Nagano, Osaka, 586 Japan