Transmembrane movements of artificial redox mediators in relation to electron transport and ionic currents in chloroplasts

Electrochemical data obtained with TMPD⁺-sensitive electrodes indicate that ammonium-uncoupled chloroplasts retain TMPD (N,N,N',N'-tetramethyl- p -phenylenediamine) mainly in the reduced form during illumination, whereas uncoupled DCMU-treated chloroplasts accumulate TMPD in the oxidized form (TMPD⁺). This observation indicates that the reduced plastoquinol is the preferred electron donor for photosystem I (PSI) and TMPD can only compete efficiently when plastoquinone reduction is blocked. After adding DCMU the formation of a transmembrane gradient for TMPD⁺ is reflected by a slow-down of the electrogenic electron transport and by the emerging of the overshoot of the membrane current in the light-off response. A light-dependent increase in photoelectric current generated by chloroplasts in the presence of NH₄Cl and TMPD is observed and considered to be caused by a reversible release of current limitation in the interfacial conductance barriers in the lumen.     

Transmembrane movements of artificial redox mediators in relation to electron transport and ionic currents in chloroplasts

Electrochemical data obtained with TMPD⁺-sensitive electrodes indicate that ammonium-uncoupled chloroplasts retain TMPD (N,N,N',N'-tetramethyl- p -phenylenediamine) mainly in the reduced form during illumination, whereas uncoupled DCMU-treated chloroplasts accumulate TMPD in the oxidized form (TMPD⁺). This observation indicates that the reduced plastoquinol is the preferred electron donor for photosystem I (PSI) and TMPD can only compete efficiently when plastoquinone reduction is blocked. After adding DCMU the formation of a transmembrane gradient for TMPD⁺ is reflected by a slow-down of the electrogenic electron transport and by the emerging of the overshoot of the membrane current in the light-off response. A light-dependent increase in photoelectric current generated by chloroplasts in the presence of NH₄Cl and TMPD is observed and considered to be caused by a reversible release of current limitation in the interfacial conductance barriers in the lumen.     

Light-dependent changes of the Mg concentration in the stroma in relation to the Mg dependency of CO2 fixation in intact chloroplasts

(1) Light-dependent changes of the Mg²⁺ content of thylakoid membranes were measured at pH 8.0 and compared with earlier measurements at pH 6.6. In a NaCl and KCl medium, the light-dependent decrease in the Mg²⁺ content of the thylakoid membranes at pH 8.0 is found to be 23 nmol Mg²⁺ per mg chlorophyll, whereas in a sorbitol medium it is 83 nmol Mg₂₊ per mg chlorophyll.

(2) A light dependent increase in the Mg²⁺ content of the stroma was detected when chloroplasts were subjected to osmotic shock, amounting to 26 nmol/mg chlorophyll. Furthermore, a rapid and reversible light-dependent efflux of Mg²⁺ has been observed in intact chloroplasts when the divalent cation ionophore A 23 187 was added, indicating a light-dependent transfer of about 60 nmol of Mg²⁺ per mg chlorophyll from the thylakoid membranes to the stroma.

(3) CO₂ fixation, but not phosphoglycerate reduction, could be completely inhibited when A 23 187 was added to intact chloroplasts in the absence of external Mg²⁺. If Mg²⁺ was then added to the medium, CO₂ fixation was restored. Half of the maximal restoration was achieved with about 0.2 mM Mg²⁺, which is calculated to reflect a Mg²⁺ concentration in the stroma of 1.2 mM. The further addition of Ca²⁺ strongly inhibits CO₂ fixation.

(4) The results suggest that illumination of intact chloroplasts causes an increase in the Mg²⁺ concentration of 1–3 mM in the stroma. Compared to the total Mg²⁺ content of chloroplasts, this increase is very low, but it appears to be high enough to have a possible function in the light regulation of CO₂ fixation.     

Uptake of Magnesium by Chromaffin Granules In Vitro: Role of the Proton Electrochemical Gradient

Abstract: The chromaffin granule membrane in vitro is impermeable to protons as well as to Mg²⁺; however, when granules are incubated in the presence of the proton ionophore carbonyl cyanide p -trifluoromethoxy-phenylhydrazone or an inhibitor of the granule membrane Mg²⁺-dependent ATPase, the metal ion is accumulated inside the granules. This accumulation is dependent upon the granule transmembrane potential. The simultaneous presence of the ATPase inhibitor and the proton ionophore markedly increases metal ion incorporation. Mg²⁺ incorporation is also promoted by nigericin in the presence of potassium or sodium ions, indicating that Mg²⁺ accumulation is also dependent upon the transmembrane pH gradient. Concomitant with the Mg²⁺ accumulation, there is a significant loss of endogenous catecholamines. It is concluded that Mg²⁺ accumulation is determined by the electrochemical gradient maintained across the membrane. Once the metal ion has accumulated into the granules it displaces catecholamines from their storage sites.     

Na+-independence of the stability under alkaline conditions of the membrane of an alkalophilic Bacillus

Abstract The stability under alkaline conditions of the membrane of the alkalophile was studied. By an alkaline treatment in the absence of Na⁺ or Li⁺, the abilities of the membrane vesicles, when energized with ascorbate plus tetramethylphenylenediamine, to produce a membrane potential (negative, inside) and transmembrane pH gradient (outside > inside) were rapidly lost. The activity of cytochrome oxidase was not affected by the alkaline treatment irrespective of the presence of Na⁺. It is likely that the membrane structure is sensitive to an alkaline pH and maintained specifically by the presence of Na⁺ (or Li⁺) in the alkaline medium.     

Enhancement of the light-triggered electrical response in plant cells following their de-energization with uncouplers

Light-triggered membrane potential changes in cells of a liverwort Anthoceros are greatly enhanced by the ionophorous uncouplers nigericin and monesin. Stimulation of the light-triggered electrical response (LTER) by nigericin occurred concomitantly with inhibition of a slow decline in the chlorophyll fluorescence, which suggests that the transmembrane pH gradient in thylakoids is not essential for generation of LTER at the plasma membrane. The extent of monensin-stimulated LTER remained high under a diminished driving force for the ionophore-induced proton-cation exchange across the plasma membrane (elevation of the external Na⁺ concentration from 1 to 50 m M ), which indicates that energy uncoupling in chloroplasts is more related to the electric response enhancement than the induction of the H⁺/K⁺(Na⁺) exchange at the plasma membrane. Enhancement of LTER by ionophores occurs in parallel with stimulation of light-triggered pH changes (alkalinization) in the vicinity of the cell surface, which suggests an association of trans-membrane H⁺ fluxes with LTER. The results are consistent with the hypothesis that illumination produces a temporary inhibition of the plasma membrane H⁺ pump with a subsequent activation of gated channels and transient rapid depolarization of the cell.     

Effect of dicyclohexylcarbodiimide on the proton conductance of thylakoid membranes in intact chloroplast

The effects of dicyclohexylcarbodiimide, a potent inhibitor of chloroplast ATPase, on the light-induced electric potential changes in intact chloroplasts of Peperomia metallica and of a hornwort Anthoceros sp. were investigated by means of glass microcapillary electrodes. The characteristics of potential changes induced by flashes or continuous light in chloroplasts of both species are similar except for the phase of potential rise in continuous light, which is clearly biphasic in Anthoceros chloroplasts. Dicyclohexylcarbodiimide at concentration 5 · 10⁻⁵ M completely abolishes the transient potential undershoot in the light-off reaction but has little effect on the peak value of the photoelectric response. The membrane conductance in the light and in the dark was tested by measuring the decay kinetics of flash-generated potential in dark-adapted and preilluminated chloroplasts. In the absence of dicyclohexylcarbodiimide, preillumination causes a significant acceleration of the potential decay. The light-induced changes in the decay kinetics of flash-induced responses were abolished in the presence of dicyclohexylcarbodiimide, whereas the rate of potential decay in dark-adapted chloroplasts was not altered by dicyclohexylcarbodiimide. The results are consistent with the notion that dicyclohexylcarbodiimide diminishes H⁺ conductance of energized thylakoid membranes by interacting with the H⁺ channel of ATPase. The occurrence of a lag (approx. 300 ms) on the plot of potential undershoot (diffusion potential) versus illumination time might suggest the increase in H⁺ permeability coefficient of thylakoid membrane during illumination.     

Sulfide-induced oxygen uptake by isolated spinach chloroplasts catalyzed by photosynthetic electron transport

In addition to an inhibitory effect on the photoreduction of NADP⁺ by isolated spinach chloroplasts ( Spinacea oleracea L. cv. Melody Hybrid), sulfide initiated oxygen uptake by chloroplasts upon illumination, both in presence and absence of an electron acceptor. Sulfide-induced oxygen uptake was sensitive to DCMU demonstrating the involvement of photosynthetic electron transport. Addition of superoxide dismutase to the chloroplast suspension prevented the sulfide-induced oxygen uptake, which indicated that sulfide may be oxidized by the chloroplast, its oxidation being initiated by superoxide formed upon illumination (at the reducing side of PSI). Tris-induced inhibition of NADP⁺ photo-reduction could not be abolished by sulfide, which indicated that sulfide could not act as an electron donor for PSI.     

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.     

Changes in the photosynthetic reaction centre II in the diatom Phaeodactylum tricornutum result in non-photochemical fluorescence quenching

Diatoms are an important group of primary producers in the aquatic environment. They are able to acclimate to fast changes in the light intensity by various mechanisms including a rise in non-photochemical fluorescence quenching (NPQ). The latter has been attributed to the xanthophyll cycle (XC) following activation of diadinoxanthin de-epoxidase by the acidification of the thylakoid lumen. To examine whether fluorescence quenching in the diatom Phaeodactylum tricornutum depends on the ΔpH generated by the photosynthetic electron transport, we arrested the latter by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU). This treatment hardly affected the NPQ or XC, even when methylviologen was present. Dissipation of the ΔpH by 2,4-dinitrophenol inhibited the XC but did not alter NPQ. Similar results, i.e. inhibition of the XC but normal fluorescence quenching, were observed when the experiments were performed at 3°C. Measurements of thermoluminescence showed that excess light treatment caused a marked decline in the signals obtained as a result of recombination of Q_B^- with the S₃ state of the Mn cluster; this was also observed in cells treated with DCMU (recombination of Q_A^- with S₂). Light treatment also diminished the Q_A^- re-oxidation signals. The data suggest that changes in PSII core centre itself due to exposure to excess light conditions play an important part in the acclimation of P. tricornutum to the changing light conditions.