Light-induced pH changes and changes in absorbance of pH indicators in Rhodospirillum rubrum chromatophores.

1. The light-induced pH change of chromatophore suspensions from Rhodospirillum rubrum was stimulated significantly and similarly by KCl, NaCl, LiCl, RbCl, CsCl, MgCl2, MnCl2, and CaCl2. In the dark, the pH of chromatophore suspensions decreased immediately and markedly on adding these salts. 2. The light-induced pH change stimulated by KCl plus valinomycin was inhibited by LiCl and NaCl, but not by RbCl. 3. The optimum pH values for light-induced pH change and photosynthetic ATP formation were around 5 and 8, respectively. The amount of chromatophore-bound ubiquinone-10 reduced in the light was independent of pH from 5 to 9. At pH 8, the number of protons incorporated into chromatophores in the light was one-half of the number of ubiquinone-10 molecules reduced in the light. 4. Among several pH indicators tested, bromothymol blue (BTB) and neutral red (NR) showed absorbance changes on illumination of chromatophores. Although the pH change indicated by the absorbance change was opposite to the light-induced pH change of the medium, the effect of KCl on the absorbance changes of BTB and NR, and the effect of valinomycin on that of NR, but not on that of BTB, were similar to those on the light-induced pH change. 5. The light-induced absorbance change of BTB was significantly inhibited by NR, whereas that of NR was hardly influenced by BTB. 6. Oligomycin stimulated the light-induced absorbance change of BTB under either non-phosphorylating or phosphorylating conditions. On the other hand, that of NR under phosphorylating conditions was 50% of that under non-phosphorylating conditions, and was increased by oligomycin.     

Preparation and properties of chloroplasts depleted of chloroplast coupling factor 1 by sodium bromide treatment

Chloroplasts were treated with 2 m sodium bromide. The resulting particles lost their ATPase activity and chloroplast coupling factor 1 subunits were detected in the supernatant by means of gel electrophoresis and specific antibodies. The chloroplast coupling factor 1 depleted particles show high rates of Hill reaction with pH optimum shifted toward lower pH. The sodium bromide treatment also abolished the light-induced proton uptake. In the presence of N-methylphenazonium methosulfate light-induced proton release, insensitive to uncouplers, was observed. Addition of dicyclohexylcarbodiimide reversed the light-induced pH changes to the normal proton uptake and increased the pH optimum of the Hill reaction.     

Light-induced changes of cytosolic pH in Eremosphaera viridis: recordings and kinetic analysis

Short-term light-induced pH changes were measured by means of pH-selective microelectrodes in the uni-cellular green alga Eremosphaera viridis. Cytosolic pH changes were always transient and reversible within 1-3 min. Taking into account the low-pass filtering by the experimental set-up, the light-dependent cytosolic pH changes could be described by a sum of two exponential functions. By mathematical analysis it is demonstrated that the transient nature of light-dependent pH changes was due to a consecutive pH-stat regulation and not to a parallel light-triggered reaction. The short-term pH regulation depended linearly on the pH-deviation with no indication of a feedback control loop. Vacuolar light-dependent pH changes were measured to investigate further which pH-stat mechanisms are responsible for the back regulation of the light-dependent cytosolic pH changes. Vacuolar pH changes were too small to explain the recovery of the cytosolic pH after darkening or illumination by H⁺ fluxes across the tonoplast.Keywords: Eremosphaera viridis, ion-selective microelectrodes, light-induced pH-changes, pH-regulation, vacuolar pH.      

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.     

Regulation of rhizosphere acidification by photosynthetic activity in cowpea (Vigna unguiculata L. walp.) seedlings

In contrast to cereals or other crops, legumes are known to acidify the rhizosphere even when supplied with nitrates. This phenomenon has been attributed to N2 fixation allowing excess uptake of cations over anions; however, as we have found previously, the exposure of the shoot to illumination can cause rhizosphere acidification in the absence of N2 fixation in cowpea (Vigna unguiculata L. Walp). In this study, we examined whether the light-induced acidification can relate to photosynthetic activity and corresponding alterations in cation-anion uptake ratios. The changes of rhizosphere pH along the root axis were visualized using a pH indicator agar gel. The intensity of pH changes (alkalization/acidification) in the rhizosphere was expressed in proton fluxes, which were obtained by processing the images of the pH indicator agar gel. The uptake of cations and anions was measured in nutrient solution. The rhizosphere was alkalinized in the dark but acidified with exposure of the shoots to light. The extent of light-induced acidification was increased with leaf size and intensity of illumination on the shoot, and completely stopped with the application of photosynthesis inhibitor. Although the uptake of cations was significantly lower than that of anions, the rhizosphere was acidified by light exposure. Proton pump inhibitors N,N'-dicyclohexyl carbodimide and vanadate could not stop the light-induced acidification. The results indicate that light-induced acidification in cowpea seedlings is regulated by photosynthetic activity, but is not due to excess uptake of cations.     

Light-Induced Polar pH Changes in Leaves of Elodea canadensis: II. Effects of Ferricyanide: Evidence for Modulation by the Redox State of the Cytoplasm

The effect of an extracellular electron acceptor, ferricyanide, on the light-induced polar leaf pH changes of the submerged angiosperm Elodea canadensis in light and in darkness was determined. The rate of transmembrane ferricyanide reduction was stimulated by increased light intensity and was inhibited by inorganic carbon, indicating that changes in the redox state of the chloroplast were reflected at the plasma membrane. The addition of ferricyanide inhibited the light-induced polar leaf pH reaction. This effect could be balanced by increasing the light intensity. In the dark, the acidification induced by ferricyanide was not influenced by diethylstilbestrol at concentrations that completely inhibited the polar leaf pH changes. This indicates that the ferricyanide-induced H⁺ extrusion and the H⁺ transport during the polar reaction were mediated by different mechanisms.     

Light-induced conductivity changes in purple membrane suspensions.

Small light-induced changes in the conductivity of light-adapted purple membrane suspended in strong electrolyte solutions were detected. The method used involved modulated light and a phase sensitive detector and it allowed us to detect accurately changes as small as 0.0001% in the conductivity of the suspension. The light-induced conductivity changes turned out to be composed of at least two different event: a small fast increase in conductivity (tau approximately 2 ms) followed by a slower and larger decrease in this parameter (tau = 70 ms-80 ms). The effects of pH and temperature on these changes were studied. Both events reached maximal values around neutral pH and approached zero at both high and low pH's. Heating the suspension decreased the photoconductivity change and Arrhenius plots of the data showed breaks around 31 degrees C. It is suggested that the conductivity changes reflect changes in the surface charge of the membrane and can be used to follow the kinetics of the conformational changes occurring in the system.     

Light-induced conductivity changes in purple membrane suspensions

Small light-induced changes in the conductivity of light-adapted purple membrane suspended in strong electrolyte solutions were detected. The method used involved modulated light and a phase sensitive detector and it allowed us to detect accurately changes as small as 0.0001% in the conductivity of the suspension. The light-induced conductivity changes turned out to be composed of at least two different events: a small fast increase in conductivity (t ∼ 2 ms) followed by a slower and larger decrease in this parameter (Τ=70 ms-80 ms). The effects of pH and temperature on these changes were studied. Both events reached maximal values around neutral pH and approached zero at both high and low pH's. Heating the suspension decreased the photoconductivity change and Arrhenius plots of the data showed breaks around 31‡ C. It is suggested that the conductivity changes reflect changes in the surface charge of the membrane and can be used to follow the kinetics of the conformational changes occuring in the system.     

Light-induced cytoplasmic pH changes and their interrelation to the activity of the electrogenic proton pump in Riccia fluitans

In green thallus cells of the aquatic liverwort Riccia fluitans light-induced pH changes have been measured, using a turgor-resistant pH-sensitive microelectrode. (1) Light-off/-on causes oscillations of the cytoplasmic pH (pH_c), as well as of the membrane potential difference across the plasmalemma (ψ). Beside the well-known ψ_m changes, the first detectable pH_c change following light-off is a transient acidification of about 0.3 pH units, whereas light-on causes a transient alkalinization of roughly 0.4 pH units. (2) 1 μM DCMU eliminates these transients. (3) In the presence of 0.2 mM procaine, which alkalizes the cytoplasm to over pH 8, the light-induced ψ_m transients are enhanced, but are almost absent, if pH_c is acidified to 6.9 by 1 mM acetate. It is suggested that the transient light-induced changes in pH_c are caused by light-dependent proton translocation across the thylakoid membranes, and it is concluded that the subsequent changes in ψ_m are essentially the result of altered activities of the electrogenic proton pump in the plasmalemma, due to the observed fluctuations of its substrate, the proton.     

Structural changes of sensory rhodopsin I and its transducer protein are dependent on the protonated state of Asp76

Sensory rhodopsin I (SRI) functions in both positive and negative phototaxis in complex with halobacterial transducer protein I (HtrI). Orange light activation of SRI results in deprotonation of the retinylidene chromophore of SRI to produce the S 373 photocycle intermediate, the signaling state for positive phototaxis. In this study, we observed pH dependence on structural coupling between the two molecules upon the formation of the S 373 intermediate by means of Fourier transform infrared spectroscopy. At alkaline pH, where Asp76 (one of the counterions of the protonated retinylidene Schiff base) is deprotonated, HtrI-dependent alteration of the light-induced difference spectra is limited to reduction of amide I bands at 1661 (+)/ 1647 (-) cm (-1), and perturbation of one of the protonated carboxylic acid bands occurs at 1734 (-) cm (-1) (which appears to become ionized only when complexed with HtrI). However, at acidic pH, HtrI-complexed SRI exhibits not only light-induced reduction of the amide I changes but a wider range of spectral alterations including the appearance of several new amide I bands, perturbation of the chromophore-related vibrational modes, and other additional changes characteristic of tyrosine, glutamate, and aspartate residues. Since such pH dependence of structural changes was not observed in the complex of the D76N mutant of SRI, which behaves much like HtrI-complexed SRI in acidic conditions, we conclude that extensive orange light-induced conformational coupling between SRI and HtrI occurs only when Asp76 is neutralized.     

The Necessity for beta-Carotene in the 518 Nanometer Absorbance Change

The identity of the pigment responsible for the light-induced 518 nanometer absorbance change was investigated by extraction and reconstitution of spinach chloroplasts. Heptane extraction of carotene and quinones from lyophilized chloroplasts removes absorbance changes at 518 and 475 nanometers activated by both laser flash and continuous illumination. Some electron transport activity is always present, even in carotene- and quinone-depleted chloroplasts, but the light-induced pH increase disappears following the first extraction step. Readdition of pure β-carotene partially restores the 518 and 475 nanometer absorbance changes.     

Ultrastructural and Photometric Evidence for Light-Induced Changes in Chloroplast Structure in vivo

Photometric evidence for a reversible, red-light induced transmission decrease in excised leaf tissue or the thalli of certain marine algae has been obtained under conditions which correspond to the occurrence of a light-induced shrinkage of chloroplasts within the cells. Evidence supporting this conclusion is: A) The kinetics of the nonspecific transmission changes are similar to those observed in chloroplasts in vitro. B) The magnitude of the response is larger than could be accounted for by any known pigment which absorbs at 546 mμ. C) The light-induced transmission changes are optimal at pH 5.5 to 6.5 in the presence of electron flow cofactors and weak acid anions, conditions which are optimal for light-induced chloroplast shrinkage in isolated chloroplasts. D) Examination of chloroplast ultrastructure in dark incubated and illuminated chloroplasts reveals a flattening of the chloroplast structure and shrinkage.     

Evidence for the rate of the final step in the bacteriorhodopsin photocycle being controlled by the proton release group: R134H mutant

Light absorbed by bacteriorhodopsin (bR) leads to a proton being released at the extracellular surface of the purple membrane. Structural studies as well as studies of mutants of bR indicate that several groups form a pathway for proton transfer from the Schiff base to the extracellular surface. These groups include D85, R82, E204, E194, and water molecules. Other residues may be important in tuning the initial state pK(a) values of these groups and in mediating light-induced changes of the pK(a) values. A potentially important residue is R134: it is located close to E194 and might interact electrostatically to affect the pK(a) of E194 and light-induced proton release. In this study we investigated effects of the substitution of R134 with a histidine on light-induced proton release and on the photocycle transitions associated with proton transfer. By measuring the light-induced absorption changes versus pH, we found that the R134H mutation results in an increase in the pK(a) of the proton release group in both the M (0.6 pK unit) and O (0.7 pK unit) intermediate states. This indicates the importance of R134 in tuning the pK(a) of the group that, at neutral and high pH, releases the proton upon M formation (fast proton release) and that, at low pH, releases the proton simultaneously with O decay (slow proton release). The higher pK(a) of the proton release group found in R134H correlates with the slowing of the rate of the O --> bR transition at low pH and probably is the cause of this slowing. The pH dependence of the fraction of the O intermediate is altered in R134H compared to the WT but is similar to that in the E194D mutant: a very small amount of O is present at neutral pH, but the fraction of O increases greatly upon decreasing the pH. These results provide further support for the hypothesis that the O --> bR transition is controlled by the rate of deprotonation of the proton release group. These data also provide further evidence for the importance of the R134-E194 interaction in modulating proton release from D85 after light has led to its being protonated.     

Two possible roles of bacteriorhodopsin; a comparative study of strains of Halobacterium halobium differing in pigmentation.

The light-induced changes in pH and ATP level were compared for cell suspensions between strains of Halobacterium halobium differing in pigmentation after growth under the same conditions. Upon illumination, red cells which contained no detectable amount of bacteriorhodopsin showed only a pH increase, which, in the case of purple cells containing bacteriorhodopsin, was followed by a spontaneous pH decrease during illumination. Pre-incubation of cells at 75° for 5 min depressed the pH increase in both cells. Pre-illumination of cells with hydroxylamine depressed the pH decrease in purple cells. Whenever the pH increase was observed, the cellular ATP level increased. The presence of a bacteriorhodopsin different from that in the purple membrane is postulated.     

Effect of a light-induced pH gradient on purple-to-blue and purple-to-red transitions of bacteriorhodopsin

Bacteriorhodopsin-containing vesicles that were able to alkalize the extravesicular medium by greater than 1.5 pH units under illumination, i.e., inside-out vesicles, were reconstituted by reverse-phase evaporation with Halobacterium halobium polar lipids or exogenous phospholipids. Acid titration of a dark-adapted sample was accompanied by a color change from purple to blue (pKa = 2.5-4.5 in 0.15 M K2SO4), and alkali titration resulted in the formation of a red species absorbing maximally at 480 nm (pKa = 7 to greater than 9), the pKa values and the extents of these color changes being dependent on the nature of lipid. When a vesicle suspension at neutral or weakly acidic pH was irradiated by continuous light so that a large pH gradient was generated across the membrane, either a purple-to-blue or a purple-to-red transition took place. The light-induced purple-to-red transition was significant in an unbuffered vesicle suspension and correlated with the pH change in the extravesicular medium. The result suggests that the purple-to-red transition is driven from the extravesicular side, i.e., from the C-terminal membrane surface. In the presence of buffer molecules outside, the dominant color change induced in the light was the purple-to-blue transition, which seemed to be due to a large decrease in the intravesicular pH. But an apparently inconsistent result was obtained when the extravesicular medium was acidified by a HCl pulse, which was accompanied by a rapid color change to blue. We arrived at the following explanation: The two bR isomers, one containing all-trans-retinal and the other 13-cis-retinal, respond differently to pH changes in the extravesicular and the intravesicular medium. In this relation, full light adaptation was not achieved when the light-induced purple-to-blue transition was significant; i.e., only the 13-cis isomer is likely to respond to a pH change at the N-terminal membrane surface.     

The effects of dithiothreitol on violaxanthin de-epoxidation and absorbance changes in the 500-nm region

The effects of dithiothreitol on absorbance changes at 505 and 515 nm in isolated lettuce chloroplasts were investigated. Dithiothreitol inhibited the ascorbate-dependent 505-nm change that is due to the de-epoxidation of violaxanthin to zeaxanthin. Dithiothreitol was effective for both light-induced de-epoxidation at pH 7 and dark de-epoxidation at pH 5. Titration of de-epoxidase activity with dithiothreitol resulted in complete inhibition at about 5 μmoles dithiothreitol per mg chlorophyll. Removal of dithiothreitol restored de-epoxidase activity. These results are consistent with the view that dithiothreitol inhibits violaxanthin de-epoxidation and the corresponding 505-nm change by reducing a disulfide that is required for de-epoxidase activity.

Dithiothreitol was effective in resolving absorbance changes due to violaxanthin de-epoxidation and other changes that were superimposed under some conditions. At 515 nm and in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), phenazine methosulfate, and ascorbate, dithiothreitol inhibited the large, slow and irreversible change which was due to de-epoxidation but not the fast and reversible so-called 515-nm change. At 505 nm and under similar conditions, dithiothreitol revealed the presence of a slow reversible change in addition to the one from de-epoxidation. Results with dithiothreitol showed that the absorbance change at 505 nm in the presence of DCMU, 2,6-dichlorophenolindophenol and ascorbate was due entirely to de-epoxidation. Similarly, absorbance changes at 515 nm also appeared to be mainly from de-epoxidation but with the presence of a small transient change due to some other components. It is suggested that dithiothreitol may be useful in resolving complex light-induced absorbance changes in other photosynthetic systems as well as in enabling new studies on reversible absorbance changes in the 500-nm region.     

Light-induced pH and K+ changes in the apoplast of intact leaves

The K⁺-sensitive fluorescent dye benzofuran isophthalate (PBFI) and the pH-sensitive fluorescein isothiocyanate dextran (FITC-Dextran) were used to investigate the influence of light/dark transitions on apoplastic pH and K⁺ concentration in intact leaves of Vicia faba L. with fluorescence ratio imaging microscopy. Illumination by red light led to an acidification in the leaf apoplast due to light-induced H⁺ extrusion. Similar apoplastic pH responses were found on adaxial and abaxial sides of leaves after light/dark transition. Stomatal opening resulted only in a slight pH decrease (0.2 units) in the leaf apoplast. Gradients of apoplastic pH exist in the leaf apoplast, being about 0.5–1.0 units lower in the center of the xylem veins as compared with surrounding cells. The apoplastic K⁺ concentration in intact leaves declined during the light period. A steeper light-induced decrease in apoplastic K⁺, possibly caused by higher apoplastic K⁺, was found on the abaxial side of leaves concentration. Simultaneous measurements of apoplastic pH and K⁺ demonstrated that a light-induced decline in apoplastic K⁺ concentration indicative of net K⁺ uptake into leaf cells occurs independent of apoplastic pH changes. It is suggested that the driving force that is generated by H⁺ extrusion into the leaf apoplast due to H⁺-ATPase activity is sufficient for passive K⁺ influx into the leaf cells. Received: 7 March 2000 / Accepted: 12 May 2000     

Light-induced membrane potential and pH gradient in Halobacterium halobium envelope vesicles.

Illumination of envelope vesicles prepared from Halobacterium halobium cells causes translocation of protons from inside to outside, due to the light-induced cycling of bacteriorhodopsin. This process results in a pH gradient across the membranes, an electrical potential, and the movements of K+ and Na+. The electrical potential was estimated by following the fluorescence of a cyanine dye, 3,3'-dipentyloxadicarbocyanine. Illumination of H. halobium vesicles resulted in a rapid, reversible decrease of the dye fluorescence, by as much as 35%. This effect was not seen in nonvesicular patches of purple membrane. Observation of maximal fluorescence decreases upon ilumination of vesicles required an optimal dye/membrane protein ratio. The pH optimum for the lightinduced fluorescence decrease was 6.0. The decrease was linear with actinic light intensity up to about 4 X 10(5) ergs cn-2 s-1. Valinomycin, gramicidin, and triphenylmethylphosphonium ion all abolished the fluorescence changes. However, the light-induced pH change was enhanced by these agents. Conversely, buffered vesicles showed no pH change but gave the same or larger fluorescence changes. Thus, we have identified the fluorescence decrease with a light-induced membrane potential, inside negative. By using valinomycin-K+-induced membrane potentials, we calibrated the fluorescence decrease with calculated Nernst diffusion potentials. We found a linear dependence between potential and fluorescence decrease of 3 mV/%, up to 90 mV. When the envelope vesicles were illuminated, the total proton-motive force generated was dependent on the presence of Na+ and K+ and their concentration gradients across the membrane. In general, K+ appeared to be more permeable than Na+ and, thus, permitted development of greater pH gradients and lower electrical potentials. By calculating the total proton-motive force from the sum of the pH and potential terms, we found that the vesicles can produce proton-motive forces near--200 mV.     

Reversible effect of intensive light on photobiochemical properties of Rhodospirillum rubrum chromatophores

The effect of high intensity (photosynthesis-saturating) light on the optical properties of the bacteriochlorophyll and the light-induced H+ uptake by R. rubrum chromatophores was studied. It was shown that under aerobic conditions illumination causes reversible inhibition (in the dark) of the chromatophore ability for the light-induced uptake of H+, a reversible inhibition of the photosynthetical reaction center function and irreversible bleaching of the antennal bacteriochlorophyll. A kinetic comparison of spectral effects and reversible changes in pH as well as the effects of atmospheric oxygen and exogenous electron donors suggests that inhibition of photoactivity of the chromatophores upon illumination is due to accumulation of oxidized bacteriochlorophyll in the reaction center.