A. A. Bulychev

Effect of a single excitation stimulus on photosynthetic activity and light-dependent pH banding in Chara cells.

Using pH microelectrodes and a Micro-scopy PAM (pulse-amplitude modulated) chlorophyll fluorometer, it is shown that a propagation of an action potential in Chara corallina leads to transient suppression of spatially periodic pH profiles along the illuminated cell. The suppression was manifested as a large pH decrease in the alkaline zones and a slight pH increase in the acid zones. The propagating action potential diminished the maximum yield of chlorophyll fluorescence (Fm′) in the alkaline cell regions, as well as the quantum yield of photosystem II photochemistry, without affecting Fm′ in the acid cell regions. The results indicate an interference of membrane excitation in the mechanisms responsible for pH banding patterns in Characean algae. Apparently, the electrical excitation of the plasma membrane in the alkaline cell regions initiates a pathway that can modulate membrane events at the thylakoid membrane.

Measurements of local pH changes near bilayer lipid membrane by means of a pH microelectrode and a protonophore-dependent membrane potential. Comparison of the methods

Shifts of pH near the bilayer lipid membrane (BLM) were measured in the absence of pH difference between bulk solutions by two methods, i.e. pH microelectrode and membrane potential recordings in the presence of a protonophore. A quantitative agreement of the results of both methods was obtained. The kinetics of the generation of potential induced by the addition of ammonium chloride was accounted for by the time of the diffusion through the unstirred layers. The thickness of the unstirred BLM layers was determined in the experiment.

The effect of cations and membrane permeability modifying agents on the dark kinetics of the photoelectric response in isolated chloroplasts

The kinetics of the photoelectric response induced by saturating light pulses were studied in isolated chloroplasts of Peperomia metallica as a function of K+- and Mg2+-concentrations in the medium in the absence and presence of ionophores for K+ and divalent cations. The dark decay of the potential generated in the light is found to be accelerated upon an increase in K+- or Mg2+-concentrations in the presence of valinomycin and A23187. An acceleration of the decay phase in the flash-induced response is also observed immediately after preillumination of the chloroplast. It is concluded that the dark kinetics of the potential decay after short and long light exposures are controlled by two different processes with rate constants of about 20 and 0.2s-1, respectively.

Comparative study on photosynthetic activity of chloroplasts in acid and alkaline zones of Chara corallina

A novel experimental approach has been applied to investigate the relationship between pH banding in Chara cells and photosynthetic activity of chloroplasts located in cell regions adjacent to acid and alkaline bands. The combination of pH microelectrode technique with pulse amplitude modulation (PAM) microfluorimetry enabled parallel measurements of longitudinal pH profiles and chlorophyll fluorescence yield in acid and alkaline zones of individual Chara cells. The scanning with a pH-microelectrode along the cell length revealed the light-dependent pH pattern, i.e., alternating acid and alkaline bands with pH differences as large as 2 - 3 pH units. In parallel, measurements of chlorophyll fluorescence yield under actinic light were performed using PAM microfluorometry. It was found that the effective photochemical yield of photosystem II is substantially higher in acid than in alkaline zones. The results clearly show that the banding pattern is not confined solely to the plasmalemma but is also exhibited in alternating photosynthetic performance of the underlying chloroplast layer. Apparently, the acid regions enriched with CO2 ensure sufficient flow of this substrate to the Calvin cycle reactions, thus promoting the photosynthetic rate, whereas the alkaline zones devoid of CO2 favor radiative losses of absorbed solar energy in chloroplasts.

Changes in the electrical potential across the thylakoid membranes of illuminated intact chloroplasts in the presence of membrane-modifying agents

Abstract The kinetics of the light-induced changes in the electrical potential across the thylakoid membranes of intact chloroplasts of Peperomia metallica were measured in the presence of ionophores A23187, volinomycin and nigericin, by means of micro-cappillary glass electrodes. A slow increase in potential occurs in the light in the presence of A23187 and of volinomycin after the completion of the well-known phase 1 and phase 2 potential response. This increase is inhibited by nigericin. The results are discussed to be due to a membrane diffusion potential associated with a K+-concentration gradient across the thylakoid membrane. This gradient is likely to be formed due to the passive efflux of K+-ions occurring across the thylakoid membranes in conjunction with the electrogenic proton influx.

Modulation of photosystem II chlorophyll fluorescence by electrogenic events generated by photosystem I

In an attempt to uncover electric field interactions between PS I and PS II during their functioning, fluorescence induction curves were measured on hydroxylamine-treated thylakoids of Chenopodium album under conditions ensuring low and high levels of photogenerated membrane potentials. In parallel experiments with Peperomia metallica chloroplasts, the photocurrents were measured with patch-clamp electrodes and served as indicator of electrogenic activity of thylakoid membranes in continuous light. Inhibition of linear electron flow at PS II donor side by hydroxylamine (0.1 mM) eliminated a slow rise of chlorophyll fluorescence to a peak level and suppressed photoelectrogenesis. Activation of PS I-dependent electron transport using cofactors of either cyclic (phenazine methosulfate) or noncyclic electron transport (reduced TMPD or DCPIP in combination with methyl viologen) restored photoelectrogenesis in hydroxylamine-treated chloroplasts and led to reappearance of slow components in the fluorescence induction curve. Exposure of thylakoids to valinomycin reduced the peak fluorescence in the presence of KCl but not in the absence of KCl. Combined application of valinomycin and nigericin in the presence of KCl exerted stronger suppression of fluorescence than valinomycin alone but was ineffective in the absence of KCl. In samples treated with hydroxylamine and PS I cofactors (DCPIP/ascorbate and methyl viologen), preillumination with a single-turnover flash or a multiturnover pulse shifted the induction curves of both membrane potential and chlorophyll fluorescence to shorter times, which confirms the supposed influence of PS I-generated electrical field on PS II fluorescence. A model is presented that describes modulating effect of the membrane potential on chlorophyll fluorescence and roughly simulates the fluorescence induction curves measured at low and high membrane potentials.

Photo-electrochemical control of photosystem II chlorophyll fluorescence in vivo.

The effect of electric field on chlorophyll fluorescence is considered on the basis of the reversible radical pair model. The hypothesis is presented that the electric fields generated by photosynthetic charge separation in reaction centers and propagated laterally through the thylakoid lumen are associated with changes in chlorophyll fluorescence yield.

Electro-induced changes of chlorophyll fluorescence in individual intact chloroplasts

Abstract The intensity of chlorophyll fluorescence in isolated chloroplasts of Peperomia metallica and in situ chloroplasts of Anthoceros sp. was found to change by 5–10% on passing the electric current through the microelectrode inserted in the intrathylakoid space. The dependence of fluorescence deflections on the direction of a current flow and the absence of electro-induced fluorescence changes in chloroplasts made ion-permeable with gramicidin indicate that the emission was affected by the membrane potential created during the passage of current. A hyperpolarizing displacement of the membrane potential, positive on the inside, produced an increase in the emission in the absence of inhibitors, but has no effect on the fluorescence in chloroplasts treated with DCMU. A stimulation of a variable fluorescence by a positive membrane potential was interpreted as due to slowing down of charge separations and a respective decrease of excitation trapping in Photosystem II. A displacement of the membrane potential to the negative direction resulted in a decrease of chlorophyll fluorescence in both the absence and presence of DCMU. The fluorescence emission seems to be sensitive to variations of the membrane potential of about 100 mV or even less. Possible effects of the light-induced membrane potential on charge separations and the fluorescence emission in Photosystem II are discussed.

Photoelectric effects on chlorophyll fluorescence of photosystem II in vivo. Kinetics in the absence and presence of valinomycin

Fluorescence induction curves (F(t)) in low intensity 1s light pulses have been measured in leaf discs in the presence and absence of valinomycin (VMC). Addition of VMC causes: (i) no effect on the initial fluorescence level Fo and the initial (O-J) phase of F(t) in the 0.01-1 ms time range. (ii) An approximately 10% decrease in the maximal fluorescence Fm in the light reached at the P level in the O-J-I-P induction curve. (iii) Nearly twofold increase in the rate and extent of the F(t) rise in the J-I phase in the 1-50 ms time range. (iv) A 60-70% decrease in the rise (I-P phase) in the 50-1000 ms time range with no appreciable effect, if at all, on the rate. System analysis of F(t) in terms of rate constants of electron transfer at donor and acceptor sides have been done using the Three State Trapping Model (TSTM). This reveals that VMC causes: (i) no, or very little effect on rate constants of e-transfer reactions powered by PSII. (ii) A manifold lower rate constant of radical pair recombination (k(-1)) in the light as compared to that in the control. The low rate constant of radical pair recombination in the reaction center (RC) in the presence of VMC is reflected by a substantial increase in the nonzero trapping efficiency in RCs in which the primary quinone acceptor (Q(A)) is reduced (semi-open centers). This causes an increase in their rate of closure and in the overall trapping efficiency. Data suggest evidence that membrane chaotropic agents like VMC abolish the stimulation of the rate constant of radical pair recombination by light. This light stimulation that becomes apparent as an increase in Fo has been documented before [Biophys. J. 79 (2000) 26]. It has been ascribed to effects of (changes in) local electric fields in the vicinity of the RC. The decrease of the I-P phase is attributed to a decrease in the photoelectric trans-thylakoid potential in the presence of VMC. Such effects have been hypothesized and illustrated.

Patch-clamp studies of light-induced currents across the thylakoid membrane of isolated chloroplasts

Abstract Light-induced currents through thylakoid membrane in isolated chloroplasts of Peperomia metallica were measured with suction electrodes under clamped membrane potential (MP). Comparison of the photocurrents and MP photoresponses on the same chloroplast indicated that the recording pipette had a low resistance access to a thylakoid lumen. Two components in the membrane photocurrent were distinguished. The fast inward current rose to an initial peak and declined within seconds to a steady level of about 50 pA. This component was not significantly affected by MP change in the range from −10 to +20 mV and seemed to reflect a light driven H ∗ influx into thylakoids. The slow component was only evident under certain conditions, e.g., in the presence of 30 nM nigericin or substrate amounts of ATP or ADP with phosphate. It was directed either inward or outward depending on the sign of a set MP. With increasing length of light pulses both outward and inward currents increased in extent and exhibited a slowing down of decay kinetics. It is concluded that slow currents, irrespective of their direction, are carried through the same pathway by the same ion species. An apparent similarity was observed between photocurrents and MP photoresponses measured under voltage clamp and current clamp conditions. A strong sensitivity of photocurrent kinetics to variations in MP suggested that the baseline dark MP is an important factor affecting the time-course of the MP formation during illumination.