Characteristics of light-induced H+ transport in spinach chloroplast at lower temperatures II. A possible role of structured water in H+ transport of chloroplast near 0{degrees}C

Light-induced H⁺ transport in spinach chloroplasts at lowertemperatures was investigated with a glass electrode. Half-decaytime of the H⁺ transport on turning off the illumination increasedwith lowering of the temperature. However, near the freezingpoint of bulk water, the H⁺ transport showed no dependency ontemperature. An Arrhenius plot of the apparent first-order rate constantof H⁺ transport showed a break at about 8°C. Below thatcritical temperature, activation energy was about 0 kcal/mole. The break temperature in the Arrhenius plot shifted by 2–3°Cto a higher temperature in the presence of 10% glycerol, aneffective water structure-enhancing agent. On the other hand,addition of 5 mM NaSCN, known as a potent water structure breaker,abolished the break and a constant activation energy was obtainedover the entire low temperature range. A similar effect wasobserved in the presence of 50 mM urea. On the basis of these results, we propose a mechanism for theH⁺ transport of chloroplasts at lower temperatures involvingstructured water, where rapid proton transfer between hydrogen-bondedstructured water molecules takes place. (Received September 11, 1975; )     

H+ Efflux and Trans-Root Potential Measured while Increasing the Temperature of Solutions Bathing Excised Roots of Zea mays

Kennedy, C. D. and Gonsalves, F. A. N. 1988. H⁺ efflux and trans-rootpotential measured while increasing the temperature of solutionsbathing excised roots of Zea mays.—J. exp. Bot. 39: 37–49. Novel temperature-ramp procedures have been used to measureH⁺ efflux and trans-root potential of excised roots of Zea mays(var. Fronica). Two types of experiment were performed: (1),increasing temperature from 17°C, and (2), pre-cooling theroots to 1°C before starting the temperature ramp. The ratesof increase of temperature for H⁺ efflux and trans-root potentialexperiments were 0·5 and 2·1°C min^–1respectively The H⁺ scans revealed strong sharp maxima at 30°C and 32°C,for non-pre-cooled and pre-cooled roots respectively, the latterbeing significantly smaller. The trans-root potential scansfor the pre-cooled roots showed a corresponding maximum at 30°C,which was inhibited by KCN (1-0 mmol dm^–3) with or withoutSHAM (10 mmol dm^–3), or Hg²⁺ (1, 10, 100 µmol dm^–3)in the bathing solutions. Some of the evidence suggests thatthese maxima are associated with electrogenic H⁺ pumping, mediatedby a plasma membrane-bound ATPase. However, no correspondingmaximum was observed in the trans-root potential scans for non-pre-cooledroots, the potential remaining at about — 75 m V from20°C to 35°C. As there is a 7-fold increase in H⁺ effluxbetween 20°C and 30°C, the relationship between netH+ efflux and electrogenic proton pumping in these roots isby no means clear. Some possibilities are considered here. Pre-cooled and non-pre-cooled roots show clear maxima in thetrans-root potential scans at about 46°C, at which temperaturethere is a slight net H⁺ influx. This, and other less prominentfeatures observed, are briefly discussed. Key words: H⁺ efflux, trans-root potential, temperature-ramp procedure, Zea mays, roots     

Light-induced H+ Efflux from Intact Cells of Cyanidium caldarium

Light-induced pH changes in suspensions of an acidophilic unicellularalga, Cyanidium caldarium Geitler, were studied as a functionof the pH of the medium. In the neutral pH region, alkalizationof the medium due to photosynthetic CO₂ uptake was observed.In the acidic pH region, illumination caused a significant decreasein the pH of the medium, indicating the efflux of H⁺ from thecells. Both the rate and extent of the pH decrease increasedas the pH of the medium was lowered to 3.0. The H⁺ efflux wasnot affected by 3-(3',4'-dichlorophenyl)-l,l-dimethylurea, butwas inhibited by phenylmercuric acetate. The fastest H⁺ effluxoccurred at 45°C, whereas its extent was almost constantfrom 25 to 50°C. The activity decreased at temperaturesabove 50°C and was inactivated completely at 60°C. Itsaction spectrum corresponded the spectrum for chlorophyll aabsorption. Results indicate that the light-induced H⁺ effluxis driven by photosystem I and is important in the maintenanceof the intracellular pH at the functional neutral region againsta steep pH gradient across the cell membrane. (Received May 6, 1981; Accepted August 8, 1981)     

Fatty acid composition of microsomal phospholipids and H+-ATPase activity in the roots of Scots pine seedlings grown at different root temperatures during flushing

The fatty acid composition of phospholipids in the microsomesand the vanadate-sensitive H⁺-ATPase activity of the roots ofone-year-old Scots pine (Pinus sylvestris L.) seedlings werestudied during flushing in spring. The seedlings in hydroponiccultures were subjected to different root temperatures (5, 12or 20°C). The shoot was maintained at 20/15° C (day/night)during the 35 d experiment. After 35 d at 5° C, root growthwas totally inhibited and shoot growth partly inhibited. In roots grown at 5° C the fatty acid composition of themicrosomal phospholipids and the degree of fatty acid unsaturation(bond index) were unchanged, while in roots grown at 12 and20° C the fatty acid composition changed and bond indexdecreased. At those root temperatures, the most obvious changewas a decline in the proportion of linolenic acid (C18:3). Inthe new white roots grown either at 12°C or 20°C theproportion of C18:2 was higher and the proportion of C18:3 lowerthan in 1-year-old roots. Independently of root temperature,H⁺-ATPase activity, determined on a fresh weight basis, declinedto half of the original activity during the experiment. Thedecline in H⁺ -ATPase activity was most rapid during the firstweek. In the old roots the decline in H⁺-ATPase activity followedclosely the decline in amount of membrane protein. In new rootsH⁺-ATPase activity was high and increased with increasing roottemperature. These results suggest that in the roots of Scotspine seedlings, vanadate-sensitive H⁺-ATPase activity is dependenton age, while changes in the microsomal fatty acid compositionof phospholipids are regulated mainly by root temperature. Key words: Fatty acids of phospholipids, microsomes, H⁺-ATPase, root temperature, Scots pine     

Effects of Growth and Assay Temperatures on Unidirectional K+ Fluxes in Roots of Rye (Secale cereale)

The effects of growth and assay temperature on unidirectionalK⁺ fluxes in excised roots of rye (Secale cereale cv. Rheidol)were studied using ⁸⁶Rb⁺ as a tracer. Both K⁺ influx to thevacuole, estimated as K⁺ uptake between 3 and 12 h after transferof unlabelled roots to radioactive solution, and movement ofK⁺ to the xylem were determined directly. Other fluxes weredetermined on excised roots of plants, which had been labelledwith ⁸⁶Rb⁺ since germination, by conventional triple exponentialefflux analysis. When assayed at 20°C, roots of plants previously grown at20°C(WG roots) had lower rates of net K⁺ uptake than rootsof low temperature-acclimated plants, grown with a temperaturediferential between roots (87°C) and shoots (20°C) eithersince germination (DG roots) or for 3 d prior to experiments(DT roots). This resulted from a greater unidirectional K⁺ effluxacross the plasma membrane and a reduced K⁺ flux to the xylemin WG roots, compared to DG or DT roots, rather than a decreasein unidirectional K⁺ influx or a decrease in the net K⁺ fluxto the vacuole. Indeed, although WG roots had lower rates ofK⁺ influx and K⁺ efflux across the tonoplast at 20°C thanDG or DT roots, roots of plants from all growth temperaturetreatments showed an equivalent net K⁺ flux to the vacuole. Although all unidirectional K⁺ fluxes in roots from plants grownunder all temperature regimes were reduced by lowering the temperatureof the root, these fluxes were differentially affected in rootsof plants from contrasting growth temperature treatments. Rapidcooling to 8°C of WG roots resulted in a lower rate of K+influx and a transient increase in K⁺ efflux across both theplasma membrane and tonoplast, compared to DG and DT roots.Furthermore, since the K⁺ flux to the xylem was lower in WGroots, the net K⁺ uptake at 8°C into WG roots was considerablyreduced compared to DG and DT roots. These results suggest thatlow temperature-acclimation of K⁺ fluxes in rye roots may involvea reduction in the temperature sensitivity of K⁺ influx anda curtailment of K⁺ efflux across both the plasma membrane andtonoplast at low temperatures. Key words: K⁺influx, K⁺ efflux, low temperature, potassium, rye (Secale cereale cv. Rheidol)     

Respiration-Dependent H+ Efflux from Intact Cells of Cyanidium caldarium

An active H⁺ efflux depending on respiration was found in anacidophilic unicellular alga, Cyanidium caldarium. Alkalizationof the medium due to passive H⁺ transport into the cells wasobserved when the respiratory activity was inhibited by addingrespiratory poisons, such as rotenone or antimycin A, or byintroducing pure nitrogen into the cell suspension. The extentof the H⁺ influx increased as the pH of the medium was loweredto 2.9, indicating that H⁺ leaks into the cells according tothe pH gradient across the plasma membrane. The medium pH whichhad increased under anaerobic condition returned to the originallevel with aeration of the cell suspension. This suggests thatan active H⁺ transport, related to respiration, pumps out theexcess H⁺ accumulated in the cells during anaerobic preincubation.The pH changes in the cell suspension were related to the intracellularATP level. From these results it was concluded that active H⁺efflux dependent upon oxidative phosphorylation functions inthe dark to maintain a constant intracellular pH against passiveH⁺ leakage through the plasma membrane. The light-induced H⁺ efflux and the respiration-dependent H⁺efflux were also compared in relation to the physiological roleof the active H⁺ efflux, especially with respect to the intracellularpH regulation in this alga. ¹The data in this paper are included in the Ph. D. dissertationsubmitted by M. Kura-Hotta to Tokyo Metropolitan University. (Received February 3, 1984; Accepted June 14, 1984)     

Characteristics of light-induced 515-nm absorbance change in spinach chloroplasts at lower temperatures II. Relationship between 515-nm absorbance change and rapid H+ uptake in chloroplasts after a short flash illumination

The absorbance change at 515 nm induced by a short (7.6 µsec)light flash in spinach chloroplasts was studied at sub-roomtemperatures in relation to rapid H⁺ uptake into chloroplasts. Lowering of temperature caused a marked decrease in the rateof recovery of 515-nm absorbance change after a flash illumination.Initial rate of rapid H⁺ uptake, measured with absorbance changeof bromcresol purple (BCP), was also reduced at lower temperatures,in a parallel fashion. Half-recovery time of the absorbancechange at 515 nm and rise-time of the pH-indicating absorbanceincrease of BCP coincided well at each temperature studied.Values of the calculated activation energy for these two processeswere almost the same. The parallelism between the 515-nm absorbance change and therapid H⁺ uptake after a single flash illumination was also observedwhen the electric field decay and/or H⁺ translocation were acceleratedby ionophorous antibiotics, carbonylcyanide m-chlorophenylhydrazoneor phenazine methosulfate. From these results, it is suggestedthat the rapid H⁺ uptake into chloroplast is chemically coupledto electron transfer and at the same time diffusion- (or transport-)controlled. Membrane potential, reflected in the 515-nm absorbancechange is dissipated with the rapid H⁺ influx. A model for theelectron-transfer-coupled H⁺ translocation involving a plastosemiquinoneloop is presented. Dissipation of the illumination-formed inside-positivemembrane potential by the influx of H⁺ is explained by the model. (Received September 17, 1976; )     

Electrogenic Pump Current and ATP-Dependent H+ Efflux Across the Plasma Membrane of Nitellopsis obtusa

The correlation between the pump current and the ATP-dependentH⁺ efflux was examined in internodal cells of Nitellopsis obtusa.To control the cytoplasmic pH and ATP concentration, the tonoplastwas removed by intracellular perfusion with an EGTA-containingmedium. Two groups of perfused cells were prepared, one with1 mM ATP (+ATP cells) and the other without ATP but with hexokinaseand glucose (–ATP cells). The ATP-dependent H⁺ effluxwas calculated as the difference in H⁺ efflux between the +ATPand –ATP cells. Based on an electrically equivalent circuitmodel of the plasma membrane, the pump current was calculatedfrom the membrane potentials and the membrane resistances ofboth +ATP and –ATP cells. When the membrane potentialwas not too high (–220 mV), the ATP-dependent H⁺ current(19 mA m^–2) was almost equal to the pump current (20 mAm^–2) calculated from the electrical data. This indicatesthat the electrogenic pump current across the plasma membraneof Nitellopsis obtuse was mostly carried by H⁺. But when themembrane potential was high (–280 mV), the H⁺ currentwas lower than the pump current. The possible cause of thisdiscrepancy is discussed. (Received November 5, 1984; Accepted February 28, 1985)     

Characterization of the vacuolar-type H+-ATPase from guard cell protoplasts of Commelina

Characteristics of the vacuolar-type (V-type) H⁺-ATPase fromguard cell protoplasts of Commelina communis L. were investigatedusing a linked enzyme assay and nitrate inhibition as a diagnosticindicator of the enzyme activity. ATPase activity was completelyinhibited by about 50 mol m^–3 nitrate and activity wasoptimal near pH 8.0. The temperature optimum for activity wasabout 37 C and an Arrhenius plot indicated changes in activationenergy for the ATPase at 15C and possibly at about 30 C. Theenzyme was stimulated by Cl^– while Ca²⁺ inhibited activity(l₅₀ = 1.5 mol m^–3). The apparent K_m (MgATP) was 0.62mol m^–3. Incubation of guard cell protoplasts for up to 5 h in 50 µMabscisic acid (ABA) or 25µM fusicoccin (FC) did not affectsubsequent ATPase activity. In vitro assays with FC or ABA alsodid not affect enzyme activity. Activity was not affected bylight or potassium ferricyanide, two factors which are knownto influence stomatal activity. Beticoline was a potent inhibitorof activity (l₅₀ = 50 µM) while DCCD was less effective(l₅₀ = 90µM). On chlorophyll, protein and protoplast bases, V-type ATPaseactivity was greater in guard cell protoplasts than mesophyllcell protoplasts by 66, 13.9 and 1.9, respectively. On atonoplast surface area basis the enzyme activity was 5.6 timeshigher in guard cell protoplasts than in mesophyll cell protoplasts Thus, although the characteristics of the V-type, H ⁺-ATPaseof GCP are very similar to those found in other cell types,rates of activity and probably tonoplast enzyme density aremuch greater in guard cell protoplasts than mesophyll cell protoplastsof C. communis which corresponds with the large and rapid ionfluxes across the tonoplast associated with stomatal movements Key words: Guard cell protoplasts, stomata, V-type H ⁺-ATPase     

Sodium gradient-dependent transport of magnesium in rat ventricular myocytes

Cytoplasmic concentration of Mg²⁺([Mg²⁺]_i) was measured with a fluorescentindicator furaptra in ventricular myocytes enzymatically dissociatedfrom rat hearts (25°C). To study Mg²⁺ transport acrossthe cell membrane, cells were treated with ionomycin inCa²⁺-free (0.1 mM EGTA) and high-Mg²⁺ (10 mM)conditions to facilitate passive Mg²⁺ influx. Rate of riseof [Mg²⁺]_i due to the net Mg²⁺influx was significantly smaller in the presence of 130 mMextracellular Na⁺ than in its absence. We also tested theextracellular Na⁺ dependence of the net Mg²⁺efflux from cells loaded with Mg²⁺. After[Mg²⁺]_i was raised by ionomycin and highMg²⁺ to the level 0.5-0.6 mM above the basal value(~0.7 mM), washout of ionomycin and lowering extracellular[Mg²⁺] to 1.2 mM caused rapid decline of[Mg²⁺]_i in the presence of 140 mMNa⁺. This net efflux of Mg²⁺ was completelyinhibited by withdrawal of extracellular Na⁺ and waslargely attenuated by imipramine, a known inhibitor of Na⁺/Mg²⁺ exchange, with 50% inhibition at 79 µM. The relation between the rate of net Mg²⁺ efflux andextracellular Na⁺ concentration([Na⁺]_o) had a Hill coefficient of 2 and[Na⁺]_o at half-maximal rate of 82 mM. Theseresults demonstrate the presence of Na⁺ gradient-dependentMg²⁺ transport, which is consistent withNa⁺/Mg²⁺ exchange, in cardiac myocytes.

     

Specific inhibition of photosystem II activity in chloroplasts by fumigation of spinach leaves with SO2

The phytotoxic effects of sulfur dioxide (SO₂) were investigatedby fumigating spinach plants with SO₂. Inhibition of 2,6-dichloroindophenol(DCIP) photoreduction was observed in spinach chloroplasts isolatedfrom fumigated leaves. NADP and DCIP photoreductions were inhibitedto a similar extent by fumigation with 2.0 ppm SO₂ but electronflow from reduced DCIP to NADP was not affected. When electronflow from H₂O to NADP was inhibited by 36%, a 39% inhibitionof non-cyclic photophosphorylation was observed. However, phenazinemethosulfate(PMS)-catalyzed cyclic photophosphorylation wasas active as in the control chloroplasts. Moreover, in the presenceof PMS, no significant suppression was observed in the extentof light-induced H⁺ uptake or in the rate of H⁺ efflux in chloroplasts.From these results, it can be concluded that SO₂ inhibits theelectron flow driven by photosystem II when plants have beenfumigated with SO₂. In spinach leaves fumigated with SO₂, the rate of photosyntheticO₂ evolution was reduced under light-limited conditions, whilethe rate of respiratory O₂ uptake changed slightly. (Received February 8, 1979; )     

Effect of Intracellular ATP Levels on the Light-Induced H+ Efflux from Intact Cells of Cyanidium caldarium

The light-induced H⁺ efflux observed at acidic pH in Cyanidiumcells was shown to be an active H⁺ transport depending on theintracellular ATP produced by cyclic photo-phosphorylation.Triton X-100 was found to act as an effective uncoupler in intactCyanidium cells without collapsing the pH gradient across theplasma membrane. Triton X-100 at 0.015% significantly reducedthe intracellular ATP levels, stimulated the p-BQ, Hill reactionand completely inhibited the light-induced H⁺ efflux. Inhibitionof the H⁺ efflux by Triton X-100 correlated well with the depressionof the apparent rale of light-induced ATP synthesis as wellas the decrease in the intracellular ATP level in light. The light-induced H⁺ efflux was completely inhibited by diethylstilbestrol,a specific inhibitor of plasma membrane ATPase, without anychanges in the intracellular ATP level, thereby suggesting theparticipation of the plasma membrane ATPase in the light-inducedH⁺ efflux. ¹The data in this paper are included in the Ph. D. dissertationsubmitted by M. Kura-Hotta to Tokyo Metropolitan University. (Received February 3, 1984; Accepted June 14, 1984)     

Coupling of Proton Fluxes in the Polar Leaves of Potamogeton lucens L

An attempt has been made to quantify the light-induced H⁺ effluxand influx observed in polar leaves of Potamogeton lucens.Theseproton fluxes are spatially separated. The H⁺ efflux, mediatedby a plasmalemma bound H⁺ –ATPase, occurs across theplasmamembrane at the morphological lower epidermis and is accompaniedby an H⁺ influx (or OH^– efflux) at the upper side oftheleaf. As a result, these leaves exhibit a remarkable pH–polarityin the light. The pH near the lower epidermis may drop to avalueas low as 3.5, while a pH of about 10.5 can be observed at theupper epidermis. Obviously this phenomenon requires theco–ordinationof transport processes in the different cell layers of the leaftissue. These observations led to quantitative studies oftherelation between the H⁺ fluxes at either plasmalemma. Thesefluxes were calculated from the pH values recorded at twodistancesfrom the leaf surface. Although the H⁺ influx always exceededthe efflux, a coupling between the transport processesacrosseither plasma membrane became evident from the time–coursesof the two fluxes. Key words: Potamogeton lucens, proton flux, flux coupling, pH–;polarity     

Light-induced Activation of Electrogenic H+ Extrusion and K+ Uptake in Elodea densa Depends on Photosynthesis and is Mediated by the Plasma membrane H+ ATPase

In Elodea densa leaves light strongly stimulates electrogenic,K ⁺-dependent, vanadate- and erythrosin B-sensitive H⁺ extrusionand hyperpolarizes the transmembrane electrical potential. Theseeffects of light are suppressed by treatment with DCMU, an inhibitorof photosynthesis, which has no effect on H⁺ extrusion in thedark. Light-induced H⁺ extrusion requires the presence of K⁺in the medium and is associated with increased K⁺ uptake andalkalinization of the cell sap. Light-induced H⁺ extrusion increaseswith increased CO₂ concentration. At constant CO₂ concentration(10⁴ parts 10^–6) the rate of H⁺ extrusion is stronglyenhanced by an increased light intensity up to 30 W m^–2.Different wavelengths, between 400 and 730 nm, induce a significantstimulation of both proton secretion and transmembrane potentialhyperpolarization. The stimulating effects of light on H⁺ extrusion, K⁺ uptakeand cell sap pH are very similar to those induced in the darkby fusicoccin, a toxin known to stimulate strongly ATP-driven,vanadate- and erythrosin B-sensitive H⁺ transport. In the light,the effects of fusicoccin are only partially additive to thoseof light, thus suggesting that the two factors influence thesame system. The identification of this system with the plasmamembrane H⁺-ATPase is indicated by the observed inhibition ofthe effects of either light or fusicoccin by the H⁺-ATPase inhibitorsvanadate and erythrosin B. These data indicate that the activation of electrogenic H⁺ extrusionand of K⁺ uptake by light is mediated by some products of photosynthesis.The mechanism and the possible physiological implications ofthis phenomenon are discussed. Key words: Photosynthesis, H⁺ pump, K⁺ uptake, Elodea densa     

The Influence of Ca2+ and K+ on H14CO3-Influx in Internodal Cells of Chara corallina

The influences of Ca²⁺-free solutions and increasing K⁺ concentrationson the H¹⁴CO^–₃ influx capacity of Chara corallina wereinvestigated. It was found that contact with Ca^2–freesolutions resulted in a gradual reduction in the H¹⁴CO^–₃influx capacity of these cells. Recovery of this influx capacity,following the return of Ca²⁺ to the experimental solution, followeda ‘mirror-image’ of the time course of decay. Potassium concentrations above a certain critical value (2 mM)induced a rapid reduction in H¹⁴CO^–₃ influx capacity.Normal activity was recovered within 60–90 min followingthe return to 0.2 mMK⁺ solutions. It was also shown that 10mM K⁺ can be used to determine the relative contribution of¹⁴C supplied by diffusion of ¹⁴CO₂ and transport of H¹⁴CO^–₃.The Ca²⁺ and K⁺ results are discussed in relation to the effectsof these treatments on the electrical properties of the plasmalemma.     

A Mathematical Model to Utilize the Logistic Function in Germination and Seedling Growth

Several models have been proposed to describe germination rates,but most are limited in statistical analysis and biologicalmeaning of indices. Therefore, a mathematical model is proposedto utilize the logistic function. The function was defined asan overall response including time, temperature, and the interactionbetween time and temperature. Cumulative germination percentagesover time were used to develop the model. Germination tests were conducted on indiangrass (Sorghastrumnutans (L.) Nash) strain ‘IG-2C-F1’, at constanttemperatures of 9, 12, 15, 20, 25, and 30 °C. The functionfitted the observed data over six temperatures at r² = 0.99.Time to reach 10% of final germination (Gt₁₀) increased from2.5 d at 30 °C to 44.0 d at 9 °C, and Gt₅₀ (time toreach 50% of final germination) increased from 3.6 d at 30 °Cto 53.8 d at 9 °C. True germination rate (% d^–1) foreach temperature was maximum at Gt₅₀. A linear model of 1/Gt₅₀versus temperature was used to estimate the base temperatureof 8.3 °C for germination. An Arrhenius plot indicated achange occurred between 20 °C and 25 °C for temperatureresponse of germination. Published data on hypocotyl growthof Cucumis melo L. were recalculated using the model. Absolutegrowth rates showed a temperature response similar to the publishedweighted-mean elongation rates. Base temperature for hypocotylgrowth of C. melo was estimated as 8.8 °C. The proposedmodel proved to be useful in calculating and interpreting germinationand growth kinetics. Key words: Indiangrass, Sorghastrum nutans (L.) Nash, Germination rate, Threshold temperature, Arrhenius plot, Growth rate, Cucumis melo L     

Photo-induced H+ Transport between Chloroplasts and the Cytoplasm in a Protoplasmic Droplet of Characeae

Svintitskikh, V. A., Andrianov, V. K. and Bulychev, A. A. 1985.Photo-induced H⁺ transport between chloroplasts and the cytoplasmin a protoplasmic droplet of Characeae.—J. exp. Bot. 36:1414–1429. The effects of light on the membrane potential and cytoplasmicpH of isolated droplets of protoplasm from Nitella have beenstudied using microcapillary electrodes and pH-sensitive antimonymicro-electrodes. Illumination of chloroplast-containing dropletscaused a change of the membrane potential with a concomitantacidification of both the cytoplasm and the outer medium, butit had no effect on the electrical resistance of the surfacemembrane. Treatment of protoplasmic droplets with uncouplers(NH₄Cl and CCCP) resulted in a complete inhibition of the light-inducedacidification of the cytoplasm, whereas the energy transferinhibitor DCCD had no effect. A correlation between the formationof a pH gradient across the thylakoid membrane and the acidificationof the cytoplasm was explicable in terms of the assumption ofrestricted spatial communication between the intra-thylakoidvolume and the cytoplasm in intact chloroplast. The photo-inducedacidification of the boundary layer of an external medium wasmarkedly stimulated under the action of inhibitors of H⁺-ATPaseDCCD and DES. These findings suggest that the active extrusionof H⁺ from the cytoplasm into the external medium is not drivenby an ATPase, although H+-conducting channels of membrane ATPaseprovide a pathway for a passive diffusion of protons from outsideinto the cytoplasm Key words: Transport of protons, protoplasmic droplet, intact chloroplasts, Characeae     

Syntaxin 1A has a specific binding site in the H3 domain that is critical for targeting of H+-ATPase to apical membrane of renal epithelial cells

H⁺ transport in the collecting duct is regulated by exocytic insertion of H⁺-ATPase-laden vesicles into the apical membrane. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptor (SNARE) proteins are critical for exocytosis. Syntaxin 1A contains three main domains, SNARE N, H3, and carboxy-terminal transmembrane domain. Several syntaxin isoforms form SNARE fusion complexes through the H3 domain; only syntaxin 1A, through its H3 domain, also binds H⁺-ATPase. This raised the possibility that there are separate binding sites within the H3 domain of syntaxin 1A for H⁺-ATPase and for SNARE proteins. A series of truncations in the H3 domain of syntaxin 1A were made and expressed as glutathione S-transferase (GST) fusion proteins. We determined the amount of H⁺-ATPase and SNARE proteins in rat kidney homogenate that complexed with GST-syntaxin molecules. Full-length syntaxin isoforms and syntaxin-1AC [amino acids (aa) 1–264] formed complexes with H⁺-ATPase and SNAP23 and vesicle-associated membrane polypeptide (VAMP). A cassette within the H3 portion was found that bound H⁺-ATPase (aa 235–264) and another that bound SNAP23 and VAMP (aa 190–234) to an equivalent degree as full-length syntaxin. However, the aa 235–264 cassette alone without the SNARE N (aa 1–160) does not bind but requires ligation to the SNARE N to bind H⁺-ATPase. When this chimerical construct was transected into inner medullary collecting duct cells it inhibited intracellular pH recovery, an index of H⁺-ATPase mediated secretion. We conclude that within the H3 domain of syntaxin 1A is a unique cassette that participates in the binding of the H⁺-ATPase to the apical membrane and confers specificity of syntaxin 1A in the process of H⁺-ATPase exocytosis. soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins; exocytosis; H⁺⁺ transport     

Effect of Root Zone Temperature on the Mineral Composition of Xylem Sap and Plasma Membrane K+-Mg++-ATPase Activity of Grafted-Cucumber and -Figleaf Gourd Root Systems

Cucumber (Cucumis sativus L.) seedlings were grafted onto cucumber-(CG) or figleaf gourd- (FG, Cucurbita ficifolia Bouché)seedlings in order to determine the effect of solution temperature(12, 22, and 32°C) on the mineral composition of xylem sapand the plasma membrane K⁺-Mg⁺⁺-ATPase activities of the roots.Low solution temperature (12°C) lowered the concentrationof NO^–₃ and H₂PO^–₄ in xylem sap of CG plants butnot of FG plants. Concentrations of K⁺, Ca⁺⁺ and Mg⁺⁺ in xylemsap were less affected than anions by solution temperature.The plasma membrane of FG plants grown in 12°C solutiontemperature showed the highest K⁺- Mg⁺⁺-ATPase activity at allATP concentrations up to 3 mM and at low reaction temperatureup to 12°C, indicating resistance of figleaf gourd to lowroot temperature. (Received December 27, 1994; Accepted March 10, 1995)     

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)