Search

Rapid cytoplasmic streaming in characean algae mediates communications between remote cell regions exposed to uneven irradiance. The metabolites exported from brightly illuminated chloroplasts spread along the internode with the liquid flow and cause transient changes in chlorophyll fluorescence at cell areas that are exposed to dim light or placed shortly in darkness. The largest distance to which the photometabolites can be transported has not yet been determined. Neither is it known if lateral transport has an influence on the induction of chlorophyll fluorescence. In this study, the relations between spatial connectivity of anchored chloroplasts in characean internodes and fluorescence induction curves were examined. Connectivity between remote cell parts was pronounced upon illumination of a cell spot at a distance up to 10 mm from the area of fluorescence measurement, provided the spot was located upstream in the cytoplasmic flow. Spatial interactions between distant cell sites were also manifested in strikingly different slow stages of fluorescence induction caused by narrow- and wide-field illumination. Cytochalasin D, cooling of bath solution, and inactivation of light-dependent envelope transporters were used to disturb cyclosis-mediated spatial interactions. Although fluorescence induction curves induced by narrow- and wide-field illumination differed greatly under control conditions, they became similar after the inhibition of cyclosis with cytochalasin D. The results indicate that cytoplasmic streaming not only drives the lateral translocation of photometabolites but also promotes the export of reducing power from illuminated chloroplasts due to flushing the chloroplast surface and keeping sharp concentration gradients.

Induction changes in chlorophyll fluorescence are associated with photosynthetic electron transfer, generation of the transmembrane proton gradient, and production of carbohydrates in the CO2 fixation cycle. The reactions of photosynthesis are also accompanied by the outflow of photoproducts from illuminated chloroplasts and their long-distance transport. The exchange of metabolites across the chloroplast envelope membranes is carried out by transporters that are active in the light and cease to operate in darkness. Inactivation of light-dependent envelope transporters in Chara cells interrupts spatial signaling manifested as a transient fluorescence rise in response to illumination of a distant cell area. The dark adaptation was found to down-regulate the entry of metabolites from the streaming cytoplasm into shaded chloroplasts but had rather low influence on metabolite export from illuminated plastids. Fluorescence induction curves were quite sensitive to illumination or darkening of the sample area residing outside the region of photometric assay. The amplitude of slow fluorescence changes observed under dim illumination of the whole Chara internode was substantially larger than under narrow-field illumination of the fluorescence assay region. The results indicate that the slow increase in fluorescence during the induction period in characean cells results not only from photosynthetic activity of chloroplasts in the examined cell region but also from interactions between the analyzed and neighboring cell areas. When the cytoplasmic streaming was arrested by cytochalasin D, similar induction changes were induced by local and global illumination, indicating a disruption of long-range interactions. The results suggest that the liquid flow not only carries metabolites from illuminated to shaded cell parts but also facilitates the export of photometabolites from chloroplasts to the cytoplasm.

Illuminated giant cells of characean algae exhibit membrane excitability as well as the spatial patterns of photosynthesis and transmembrane H+ fluxes. The excitation of plasmalemma under these conditions results in the transient degradation of external alkaline and acid zones and inhibits photosynthesis in the alkaline zones. The generation of action potential in the patterned internodes is followed by cell hyperpolarization that peaks in 1 min and lasts up to 15 min. In order to exclude the influence of drifting resting potential on the chloroplast response to plasma membrane excitation, the voltage clamp mode was applied in this work, and chlorophyll fluorescence changes caused by a short depolarizing pulse were monitored. The depolarizing shift of membrane potential under voltage clamp conditions was found to induce a large depression of $$F_{{\text{m}}}^{{{'}}}$$ chlorophyll fluorescence and photosynthetic activity, provided that inward Ca2+ and Cl– currents were triggered and that a steady-state inward H+ flux (or OH– efflux) persisted before the application of an electric stimulus. The depolarization-induced ion currents measured in the alkaline and acidic cell regions under light and in darkness were found to differ significantly. The results are consistent with the notion that the massive inward H+ flow occurring in the alkaline cell regions under illumination is associated with the acidic shift of cytoplasmic pH. Divergent amplitudes of ionic currents in different cell parts can be partially determined by the presence of numerous plasmalemmal invaginations, charasomes specifically localized in the acid zones, as well as by sharp local changes in external pH in acid zones during the perforation of cell wall with a measuring microelectrode.

Measurements of OJIP–SMT patterns of fluorescence induction (FI) in Synechocystis sp. PCC 6803 (Synechocystis) cells on a time scale up to several minutes were mathematically treated within the framework of thylakoid membrane (T-M) model (Belyaeva et al., Photosynth Res 140:1–19, 2019) that was renewed to account for the state transitions effects. Principles of describing electron transfer in reaction centers of photosystems II and I (PSII and PSI) and cytochrome b6f complex remained unchanged, whereas parameters for dissipative reactions of non-radiative charge recombination were altered depending on the oxidation state of QB-site (neutral, reduced by one electron, empty, reduced by two electrons). According to our calculations, the initial content of plastoquinol (PQH2) in the total quinone pool of Synechocystis cells adapted to darkness for 10 min ranged between 20 and 40%. The results imply that the PQ pool mediates photosynthetic and respiratory charge flows. The redistribution of PBS antenna units responsible for the increase of Chl fluorescence in cyanobacteria (qT2 → 1) upon state 2 → 1 transition or the fluorescence lowering (qT1 → 2) due to state 1 → 2 transition were described in the model by exponential functions. Parameters of dynamically changed effective cross section were found by means of simulations of OJIP–SMT patterns observed on Synechocystis cells upon strong (3000 μmol photons m−2s−1) and moderate (1000 μmol photons m−2s−1) actinic light intensities. The corresponding light constant values kLΣAnt = 1.2 ms−1 and 0.4 ms−1 define the excitation of total antenna pool dynamically redistributed between PSII and PSI reaction centers. Although the OCP-induced quenching of antenna excitation is not involved in the model, the main features of the induction signals have been satisfactorily explained. In the case of strong illumination, the effective cross section decreases by approximately 33% for irradiated Synechocystis cells as compared to untreated cells. Under moderate light, the irradiated Synechocystis cells showed in simulations the same cross section as the untreated cells. The thylakoid model renewed with state transitions description allowed simulation of fluorescence induction OJIP–SMT curves detected on time scale from microseconds to minutes.

Giant-sized cells, such as the internodes of characean algae, demonstrate rapid (up to 100 μm/s) rotational cytoplasmic streaming that participates in long-distance intracellular interactions and coordinates the functional activity of organelles under a nonuniform light environment. The specific functions of intense cytoplasmic streaming remain poorly investigated. The lateral transport of photometabolites can be detected by combining pinhole illumination with measurements of chlorophyll fluorescence and external pH on microscopic cell regions located downstream of the locally illuminated area. The involvement of cyclosis in regulation of photosynthesis and plasmalemmal H+ transport was revealed by this means. In regions exposed to bright local light, the chloroplasts export reducing equivalents and triose phosphates into the streaming cytoplasm that spread over the cell and induce a transient rise of chlorophyll fluorescence in shaded cell areas located far from the site of photostimulus application. This review highlights the properties of cyclosis-mediated fluorescence changes, including the photoinduction of long-distance transmission, sensitivity to metabolic inhibitors, its nonuniform spatial distribution in illuminated cells, and gradual (1–5 min) inactivation of long-range signaling after transferring the cell to darkness. A stimulatory influence of the action potential on long-distance signal transmission is shown. The new method is suitable for studying the intercellular transport of metabolites and the permeability of plasmodesmata.

Primary physicochemical steps in microwounding of plants were investigated using electrochemical nano- and microprobes, with a focus on the role of oxygen in the wounding responses of individual plant cells. Electrochemical measurements of cell oxygen content were made with carbon-filled quartz micropipettes with platinum-coated tips (oxygen nanosensors). These novel platinum nanoelectrodes are useful for understanding cell oxygen metabolism and can be employed to study the redox biochemistry and biology of cells, tissues and organisms. We show here that microinjury of Chara corallina internodal cells with the tip of a glass micropipette is associated with a drastic decrease in oxygen concentration at the vicinity of the stimulation site. This decrease is reversible and lasts for up to 40 minutes. Membrane stretching, calcium influx, and cytoskeleton rearrangements were found to be essential for the localized oxygen depletion induced by cell wall microwounding. Inhibition of electron transport in chloroplasts or mitochondria did not affect the magnitude or timing of the observed response. In contrast, the inhibition of NADPH oxidase activity caused a significant reduction in the amplitude of the decrease in oxygen concentration. We suggest that the observed creation of localized anoxic conditions in response to cell wall puncture might be mediated by NADPH oxidase.

Export of reducing power from chloroplasts to cytoplasm serves to balance the NADPH/ATP ratio that is optimal for CO2 assimilation. Rapid cytoplasmic streaming in characean algae conveys the exported metabolites downstream towards the shaded plastids where envelope transporters may operate for the import of reducing power in accordance with the direction of concentration gradients. Import of reducing equivalents by chloroplasts in the analyzed area transiently enhances the pulse-modulated chlorophyll fluorescence F′ controlled by the redox state of photosystem II acceptor QA. When the microfluidic pathway was transferred to darkness while the analyzed cell area remained in dim background light, the amplitude of cyclosis-mediated F′ changes dropped sharply and then recovered within 5–10 min. The suppression of long-distance signaling indicates temporal depletion of transmitted metabolites in the streaming cytoplasm. The return to overall background illumination induced an exceptionally large F′ response to the first local light pulse admitted to a remote cell region. This indicates the appearance of excess reductants in the streaming cytoplasm at a certain stage of photosynthetic induction. The results suggest highly dynamic exchange of metabolites between stationary chloroplasts lining the microfluidic pathway and the streaming cytoplasm upon light–dark and dark–light transitions. Evidence is obtained that slow stages of chlorophyll fluorescence induction in algae with rapid cytoplasmic streaming directly depend on cyclosis-mediated long-distance delivery of metabolites produced far beyond the analyzed cell area.

Background The Characeae are multicellular green algae, which are closely related to higher plants. Their internodal cells are a convenient model to study membrane transport and organelle interactions. Results In this study we report on the effect of brefeldin A, an inhibitor of vesicle trafficking, on internodal cells of Chara australis. Brefeldin A induced the commonly observed agglomeration of Golgi bodies and trans Golgi network into "brefeldin compartments" at concentrations between 6 and 500 μM and within 30-120 min treatment. In contrast to most other cells, however, brefeldin A inhibited endocytosis and significantly decreased the number of clathrincoated pits and clathrincoated vesicles at the plasma membrane. Brefeldin A did not inhibit secretion of organelles at wounds induced by puncturing or local light damage but prevented the formation of cellulosic wound walls probably because of insufficient membrane recycling. We also found that brefeldin A inhibited the formation of alkaline and acid regions along the cell surface ("pH banding pattern") which facilitates carbon uptake required for photosynthesis; we hypothesize that this is due to insufficient recycling of ion transporters. During long-term treatments over several days, brefeldin A delayed the formation of complex 3D plasma membranes (charasomes). Interestingly, brefeldin A had no detectable effect on clathrin-dependent charasome degradation. Protein sequence analysis suggests that the peculiar effects of brefeldin A in Chara internodal cells are due to a mutation in the guanine nucleotide exchange factor GNOM required for recruitment of membrane coats via activation of ADP-ribosylation factor proteins. Conclusions and significance This work provides an overview on the effects of brefeldin A on different processes in Chara australis. It revealed similarities but also distinct differences in vesicle trafficking between higher plant and algal cells. It shows that characean internodal cells are a promising model to study interactions between seemingly distant metabolic pathways. This article is protected by copyright. All rights reserved.

After transferring the dark-acclimated cyanobacteria to light, flavodiiron proteins Flv1/Flv3 serve as a main electron acceptor for PSI within the first seconds because Calvin cycle enzymes are inactive in the dark. Synechocystis PCC 6803 mutant Δflv1/Δflv3 devoid of Flv1 and Flv3 retained the PSI chlorophyll P700 in the reduced state over 10 s (Helman et al., 2003; Allahverdiyeva et al., 2013). Study of P700 oxidoreduction transients in dark-acclimated Δflv1/Δflv3 mutant under the action of successive white light pulses separated by dark intervals of various durations indicated that the delayed oxidation of P700 was determined by light activation of electron transport on the acceptor side of PSI. We show that the light-induced redox transients of chlorophyll P700 in dark-acclimated Δflv1/Δflv3 proceed within 2 min, as opposed to 1–3 s in the wild type, and comprise a series of kinetic stages. The release of rate-limiting steps was eliminated by iodoacetamide, an inhibitor of Calvin cycle enzymes. Conversely, the creation with methyl viologen of a bypass electron flow to O2 accelerated P700 oxidation and made its extent comparable to that in the wild-type cells. The lack of major sinks for linear electron flow in iodoacetamide-treated Δflv1/Δflv3 mutant, in which O2- and CO2-dependent electron flows were impaired, facilitated cyclic electron flow, which was evident from the decreased steady-state oxidation of P700 and from rapid dark reduction of P700 during and after illumination with far-red light. The results show that the photosynthetic induction in wild-type Synechocystis PCC 6803 is largely hidden due to the flavodiiron proteins whose operation circumvents the rate-limiting electron transport steps controlled by Calvin cycle reactions.

Local illumination of the characean internode with a 30-s pulse of white light was found to induce the delayed transient increase of modulated chlorophyll fluorescence in shaded cell parts, provided the analyzed region is located downstream in the cytoplasmic flow at millimeter distances from the light spot. The fluorescence response to photostimulation of a remote cell region indicates that the metabolites produced by source chloroplasts in an illuminated region are carried downstream with the cytoplasmic flow, thus ensuring long-distance communications between anchored plastids in giant internodal cells. The properties of individual stages of metabolite signaling are not yet well known. We show here that the export of assimilates and/or reducing equivalents from the source chloroplasts into the flowing cytoplasm is largely insensitive to the direction of plasma-membrane H+ flows, whereas the events in sink regions where these metabolites are delivered to the acceptor chloroplasts under dim light are controlled by H+ fluxes across the plasma membrane. The fluorescence response to local illumination of remote cell regions was best pronounced under weak background light and was also observed in a modified form within 1–2 min after the transfer of cell to darkness. The fluorescence transients in darkened cells were suppressed by antimycin A, an inhibitor of electron transfer from ferredoxin to plastoquinone, whereas the fluorescence response under background light was insensitive to this inhibitor. We conclude that the accumulation of reduced metabolites in the stroma leads to the reduction of photosystem II primary quinone acceptor (QA) via two separate (photochemical and non-photochemical) pathways.

Immobile chloroplasts in Chara internodal cells release photometabolites into the streaming cytoplasm that distributes the exported solutes and provides metabolic connectivity between spatially remote plastids. The metabolite transmission by fluid flow is evident from chlorophyll fluorescence changes in shaded chloroplasts upon local illumination applied upstream of the analyzed area. The connectivity correlates with the pH pattern on cell surface: it is strong in cell regions with high H+-pump activity and is low in regions featuring large passive H+ influx (OH– efflux). One explanation for low connectivity under the alkaline bands is that H+ influx lowers the cytoplasmic pH, thus retarding metabolic conversions of solutes carried by the microfluidic transporter. The cessation of H+ influx across the plasma membrane by eliciting the action potential and by adding NH4Cl into the medium greatly enhanced the amplitude of cyclosis-mediated fluorescence transients. The transition from latent to the transmissive state after the dark pretreatment was paralleled by the temporary increase in chlorophyll fluorescence, reflecting changes in photosynthetic electron transport. It is proposed that the connectivity between distant chloroplasts is controlled by cytoplasmic pH.

Photosynthetic organisms adjust their activity to changes in irradiance by different ways, including the operation of cyclic electron flow around photosystem I (PSI) and state transitions that redistribute amounts of light energy absorbed by PSI and PSII. In dark-acclimated wild type cells of Synechocystis PCC 6803, linear electron transport was activated after the first 500 ms of illumination, while cyclic electron flow around PSI was long predominant in the mutant deficient in flavodiiron protein Flv3. Chlorophyll P700 oxidation associated with activation of linear electron flow extended in the Flv3− mutant to several tens of seconds and included a P700+ re-reduction phase. Parallel monitoring of chlorophyll fluorescence and the redox state of P700 indicated that, at low light intensity both in wild type and in the Flv3− mutant, the transient re-reduction step coincided in time with S-M fluorescence rise, which reflected state 2–state 1 transition (Kaňa et al., 2012). Despite variations in the initial redox state of plastoquinone pool, the oxidases-deficient mutant, succinate dehydrogenase-deficient mutant, and wild type cells did not show the S-M rise under high-intensity light until additional Flv3− mutation was introduced in these strains. Thus, the lack of available electron acceptor for PSI was the main cause for the appearance of S-M fluorescence rise under high light. It is concluded that the lack of Flv3 protein promotes cyclic electron flow around PSI and facilitates the subsequent state 2–state 1 transition in the absence of strict relation to the dark-operated pathways of plastoquinone reduction or oxidation.

A new Thylakoid model is presented, which describes in detail the electron/proton transfer reactions between membrane protein complexes including photosystems II and I (PSII, PSI), cytochrome (Cyt) b 6 f, mobile plastoquinone PQ pool in the thylakoid membrane, plastocyanin in lumen and ferredoxin in stroma, reduction of NADP via FNR and cyclic electron transfer. The Thylakoid model parameters were fitted both to Chl fluorescence induction data (FI) and oxido-reductions of P700 (ΔA 810) measured from 20 μs up to 20 s in pea leaves. The two-wave kinetics of FI and ΔA 810 (O(JI)PSM and OABCDE) were described quantitatively, provided that the values of membrane electrochemical potential components ΔΨ(t), pHL(t)/pHS(t) are in physiologically relevant ranges. The time courses on the time scale from nanoseconds to tens of seconds of oxido-reduction changes of ET components as well as concentrations of proton/ions (K+, Cl−) were calculated. We assume a low constant FNR activity over this period. Charge movements across the thylakoid membrane by passive leakage and active ATPase transport and proton buffer reactions are simulated. The dynamics of charge fluxes during photosynthetic induction under low light (PFD 200 μmol photons m−2 s−1) were analyzed. The initial wave of P700 oxidation within 20 ms during independent operation of PSI and PSII was followed after 50 ms by PSI donor-side reduction from reduced PQ pool via Cyt b 6 f site. The Cyt b 6 f reactions contribute to the stabilization of fluxes in the time range 1 s 10 s) would need the investigation of FNR activation effect in order to explain the transitions between cyclic and linear electron transport.

Symplastic interconnections of plant cells via perforations in adjoining cell walls (plasmodesmata) enable long-distance transport of photoassimilates and signaling substances required for growth and development. The pathways and features of intercellular movement of assimilates are often examined with fluorescent tracers whose molecular dimensions are similar to natural metabolites produced in photosynthesis. Chlorophyll fluorescence was recently found to be a sensitive noninvasive indicator of long-distance intracellular transport of physiologically produced photometabolites in characean internodes. The present work shows that the chlorophyll microfluorometry has a potential for studying the cell-to-cell transport of reducing substances released by local illumination of one internode and detected as the fluorescence increase in the neighbor internode. The method provides temporal resolution in the time frame of seconds and can be used to evaluate permeability of plasmodesmata to natural components released by illuminated chloroplasts. The results show that approximately one third of the amount of photometabolites released into the streaming cytoplasm during a 30-s pulse of local light permeates across the nodal complex with the characteristic time of ~ 10 s. The intercellular transport was highly sensitive to moderate elevations of osmolarity in the bath solution (150 mM sorbitol), which contrasts to the view that only transnodal gradients in osmolarity (and internal hydrostatic pressure) have an appreciable influence on plasmodesmal conductance. The inhibition of cell-to-cell transport was reversible and specific; the sorbitol addition had no influence on photosynthetic electron transport and the velocity of cytoplasmic streaming. The conductance of transcellular pores increased in the presence of the actin inhibitor cytochalasin d but the cell-to-cell transport was eventually suppressed due to the deceleration and cessation of cytoplasmic streaming. The results show that the permeability of plasmodesmata to low-molecular photometabolites is subject to upregulation and downregulation.

The dark-to-light transitions enable energization of the thylakoid membrane (TM), which is reflected in fast and slow (OJIPSMT or OABCDE) stages of fluorescence induction (FI) and P700 oxidoreduction changes (ΔA810). A Thylakoid Membrane model (T-M model), in which special emphasis has been placed on ferredoxin-NADP+-oxidoreductase (FNR) activation and energy-dependent qE quenching, was applied for quantifying the kinetics of FI and ΔA810. Pea leaves were kept in darkness for 15 min and then the FI and ΔA810 signals were measured upon actinic illumination, applied either directly or after a 10-s light pulse coupled with a subsequent 10-s dark interval. On the time scale from 40 µs to 30 s, the parallel T-M model fittings to both FI and ΔA810 signals were obtained. The parameters of FNR activation and the buildup of qE quenching were found to differ for dark-adapted and preilluminated leaves. At the onset of actinic light, photosystem II (PSII) acceptors were oxidized (neutral) after dark adaptation, while the redox states with closed and/or semiquinone QA(−)QB(−) forms were supposedly generated after preillumination, and did not relax within the 10 s dark interval. In qE simulations, a pH-dependent Hill relationship was used. The rate constant of heat losses in PSII antenna kD(t) was found to increase from the basic value kDconst, at the onset of illumination, to its maximal level kDvar due to lumenal acidification. In dark-adapted leaves, a low value of kDconst of ∼ 2 × 106 s−1 was found. Simulations on the microsecond to 30 s time scale revealed that the slow P-S-M-T phases of the fluorescence induction were sensitive to light-induced FNR activation and high-energy qE quenching. Thus, the corresponding time-dependent rate constants kD(t) and kFNR(t) change substantially upon the release of electron transport on the acceptor side of PSI and during the NPQ development. The transitions between the cyclic and linear electron transport modes have also been quantified in this paper.

The spectral composition of sunlight in natural aquatic habitats depends on the depth of submergence and the solar time. Changes in the fractional amount of far-red light (FRL) in the total photon flux are associated with the redistribution of light energy absorbed by the two photosystems of photosynthesis. Experiments with a culture of Scenedesmus quadricauda revealed the influence of FRL preillumination on photosynthetic electron transport and associated processes in microalgae. The plant efficiency analyzer M-PEA-2 was used to measure simultaneously the induction curves of prompt fluorescence, delayed fluorescence, and the redox transients of chlorophyll P700. The results are discussed in terms of the action of FRL preillumination on cyclic and noncyclic electron flows with account for relocation of the mobile light-harvesting complex of photosystem II during reversible State 1–State 2 transitions. Essential similarities and substantial distinctions have been revealed in the effects of FRL on photosynthetic electron flows in microalgae and higher plant leaves.

Proton flows across the plant cell membranes play a major role in electrogenesis and regulation of photosynthesis and ion balance. The profiles of external pH along the illuminated internodal cells of characean algae consist of alternating high- and low-pH zones that are spatially coordinated with the distribution of photosynthetic activity of chloroplasts underlying these zones. The results based on confocal laser scanning fluorescence microscopy, pH microsensors, and pulse-amplitude-modulated chlorophyll microfluorometry revealed that the coordination of H+ transport and photosynthesis is disrupted by the 2 different environmental cues (low light and wounding) and by a chemical, wortmannin interfering with the inositol phospholipid metabolism. On the one hand, the transition from moderate to low irradiance diminished the peaks in the profiles of photosystem II (PSII) quantum efficiency but did not remove the pH bands. On the other hand, the microwounding of the internode with a glass micropipette, impacting primarily the cell wall, resulted in a rapid local alkalinization of the external medium (by 2–2.5 pH units) near the cell surface, thus mimicking the appearance of natural pH bands. Despite their seeming similarity, the alkaline bands of intact cells were eliminated by wortmannin, whereas the wound-induced alkalinization was insensitive to this drug. Furthermore, the attenuation of natural pH bands in wortmannin-treated cells was accompanied by the enhancement in spatial heterogeneity of PSII efficiency and electron transport rates, which indicates the complexity of chloroplast–plasma membrane interactions. The results suggest that the light- and wound-induced alkaline areas on the cell surface are associated with different ion-transport systems.

Cytoplasmic streaming occurs in most plant cells and is vitally important for large cells as a means of long-distance intracellular transport of metabolites and messengers. In internodal cells of characean algae, cyclosis participates in formation of light-dependent patterns of surface pH and photosynthetic activity, but lateral transport of regulatory metabolites has not been visualized yet. Hydrogen peroxide, being a signaling molecule and a stress factor, is known to accumulate under excessive irradiance. This study was aimed to examine whether H2O2 produced in chloroplasts under high light conditions is released into streaming fluid and transported downstream by cytoplasmic flow. To this end, internodes of Chara corallina were loaded with the fluorogenic probe dihydrodichlorofluorescein diacetate and illuminated locally by a narrow light beam through a thin optic fiber. Fluorescence of dihydrodichlorofluorescein (DCF), produced upon oxidation of the probe by H2O2, was measured within and around the illuminated cell region. In cells exhibiting active streaming, H2O2 first accumulated in the illuminated region and then entered into the streaming cytoplasm, giving rise to the expansion of DCF fluorescence downstream of the illuminated area. Inhibition of cyclosis by cytochalasin B prevented the spreading of DCF fluorescence along the internode. The results suggest that H2O2 released from chloroplasts under high light is transported along the cell with the cytoplasmic flow. It is proposed that the shift of cytoplasmic redox poise and light-induced elevation of cytoplasmic pH facilitate the opening of H(+)/OH(-)-permeable channels in the plasma membrane.

Long-distance communications in giant characean internodal cells involve cytoplasmic streaming as an effective means for transportation of regulatory substances. The local illumination of Chara corallina Klein ex C.L.Willdenow internodal cells with an intense 30 s pulse of white light caused a transient increase of modulated chlorophyll fluorescence in cell regions positioned downstream the cytoplasmic flow after a delay whose duration increased with the axial distance from the light source. No changes in fluorescence were observed in cell regions residing upstream of the light spot. The transient increase in actual fluorescence Fʹ in cell areas exposed to constant dim illumination at large distances from the brightly lit area indicates the transmission of photosynthetically active metabolite between chloroplasts separated by 1–5 mm distances. The shapes of fluorescence transients were sensitive to retardation of cytoplasmic streaming by cytochalasin D and to variations in cyclosis velocity during gradual recovery of streaming after an instant arrest of cyclosis by elicitation of the action potential. Furthermore, the analysed fluorescence transients were skewed on the ascending or descending fronts depending on the position of light-modulated cytoplasmic package at the moment of streaming cessation with respect to the point of measurements. The observations are simulated in qualitative terms with a simplified streaming–diffusion model.

Activities of ion pumps and H+-conducting channels in the plasmalemma of illuminated characean algae are distributed inhomogeneously along the internode, which accounts for the shifts of surface pH up to 3.5 units between various cells regions. Spatial variations in cytoplasmic properties provide the basis for uneven distribution of photosynthetic activity along the cell length and might affect the operation of H+-transporting systems at the tonoplast. In order to visualize the longitudinal distribution of the vacuolar pH in Chara corallina internodal cells, the pH microelectrode was inserted into the vacuole and the cell sap was gradually displaced along the cell during intracellular perfusion with an artificial medium. Fluorescein was added to the perfusion medium as a fluorescent marker to detect the arrival of the replacing medium into the area of pH and fluorescence measurements. In light-adapted cells, nonuniform longitudinal pH profiles were observed, with pH shifts as large as 2–2.5 units. In dark-adapted cells, the pH shifts in longitudinal profiles did not exceed 0.5 pH units. The occurrence of large pH changes within the vacuole of individual internodes indicates the possibility of nonuniform distribution of the tonoplast H+-transporting systems in different regions of the illuminated cell.

The steady-state regime of linear photosynthetic electron transport implies concerted operation of photosystems I and II (PSI and PSII) in plant leaves. Acidification of the thylakoid lumen is known to cause down-regulation of PSII photochemical activity but it is not yet clear how the proton accumulation in the lumen affects the PSI activity and coordinated operation of the two photosystems in intact leaves. Chlorophyll fluorescence and absorbance of oxidized chlorophyll P700 in the near-infrared region ΔA 810–870 (ΔA 810) are convenient noninvasive indicators of the redox state of PSII and PSI components, respectively. Simultaneous measurements of chlorophyll fluorescence and ΔA 810 in pea leaves revealed that some kinetic stages in the induction curves occur synchronously both in dark-adapted and preilluminated leaves. After the treatment of leaves with ionophores promoting or inhibiting the light-induced thylakoid pH gradient (valinomycin, nigericin, monensin), the induction curves of ΔA 810 and chlorophyll fluorescence were consistently modified. The results suggest that characteristic stages of ΔA 810 induction curve, representing the second and the third waves of P700 photooxidation, are closely related to ΔpH generation, although the bases of ΔpH dependence differ for these two stages. The second wave of ΔA 810 depends presumably on stroma alkalinization as a precondition for photoactivation of electron flow from PSI to terminal acceptors. The third wave of ΔA 810 is apparently due to retardation of electron flow between PSII and PSI upon acidification of the lumen.

The polyphasic patterns of fluorescence induction rise in pea leaves in vivo and after the treatment with ionophores have been studied using a Plant Efficiency Analyzer. To analyze in detail photosystem II (PS II) electron transfer processes, an extended PS II model was applied, which included the sums of exponential functions to specify explicitly the light-driven formation of the transmembrane electric potential (ΔΨ(t)) as well as pH in the lumen (pHL(t)) and stroma (pHS(t)). PS II model parameters and numerical coefficients in ΔΨ(t), pHL(t), and pHS(t) were evaluated to fit fluorescence induction data for different experimental conditions: leaf in vivo or after ionophore treatment at low or high light intensity. The model imitated changes in the pattern of fluorescence induction rise due to the elimination of transmembrane potential in the presence of ionophores, when ΔΨ = 0 and pHL(t), pHS(t) changed to small extent relative to control values in vivo, with maximum ΔΨ(t) ∼ 90 mV and ΔΨ(t) ∼ 40 mV for the stationary state at ΔpH ≅ 1.8. As the light intensity was increased from 300 to 1200 μmol m−2 s−1, the heat dissipation rate constants increased threefold for nonradiative recombination of P680+Phe− and by ∼30% for P680+Q A − . The parameters ΔΨ, pHS and pHL were analyzed as factors of PS II redox state populations and fluorescence yield. The kinetic mechanism of fluorescence quenching is discussed, which is related with light-induced lumen acidification, when +Q A − and P680+ recombination probability increases to regulate the QA reduction.

Photosynthesizing cells of characean algae exposed to light are able to produce pH bands corresponding to alternate areas with dominant H+-pump activity and high H+-conductance of the cell membrane. The action potential generation temporally arrests the counter-directed H+ fluxes, which gives rise to opposite pH shifts in different cell regions and represents a suitable indicator for activities of the plasma membrane H+-transporting systems. Measurements of pH near the cell surface by means of microelectrodes and microspectrophotometry in the presence of pH-indicating dye thymol blue have shown that the treatment of cells with dithiothreitol (SH-group reducing agent) suppresses pH changes induced by the action potential generation in the alkaline cell areas and considerably increases the concurrent pH changes in the acid regions. Measurements of plasma membrane resistance in the alkaline zones revealed that dithiothreitol inhibits the light-dependent conductance of the resting cell and diminishes the conductance inactivation caused by the action potential generation. The data suggest that the reduction of accessible disulfide bonds results in the decrease of H+-conductance, whereas the activity of plasma membrane H+-pump remains unimpaired or is even enhanced.

Chlorophyll fluorescence Imaging and Microscopy PAM fluorometry were applied to study spatial dynamics of photosystem II quantum yield $$ \left( {\Delta F/F_m^\prime } \right) $$ and non-photochemical quenching (NPQ) in resting and electrically stimulated Chara corallina cells in the absence and presence of the hydrophilic electron acceptor methyl viologen (MV) in the external medium. Electrical excitation of the plasma membrane temporarily enhanced the heterogeneity of photosynthetic patterns under physiological conditions (in the absence of MV), but irreversibly eliminated these patterns in the presence of MV. These findings suggest that the action potential (AP) of the excitable plant cell affects the spatial patterns of photosynthesis and chlorophyll fluorescence through different pathways operated in the absence and presence of MV. Based on the extent of NPQ as an indicator of MV-dependent electron flow, it is supposed that MV cannot permeate into the chloroplasts of photosynthetically active “acid cell regions” but gains an immediate access to the stroma of these chloroplasts after triggering of an AP. The AP-triggered MV-dependent non-photochemical quenching in the chloroplasts of acidic cell regions was routinely observed at 0.1 mM Ca2+ in the medium but not at elevated (2 mM) external Ca2+ concentration. The results are interpreted in terms of competition between two permeant divalent ion species, Ca2+ and MV2+, for their passage through the voltage-gated calcium channels of the plasma membrane. It is proposed that the herbicidal activity of MV in characean cells, here serving as model object, can be manipulated by triggering AP and varying Ca2+ concentration in the environmental medium.

Differential absorbance measurements and fluorometry were applied to examine the impact of dicyclohexylcarbodiimide (DCCD, an inhibitor of H+ conductance in thylakoid membranes) and nigericin (a K+/H+ antiporter) on photoinduced redox state transients of chlorophyll P700 and the induction curves of chlorophyll fluorescence in pea (Pisum sativum L., cv. Premium) leaves. The treatment of leaves with DCCD strongly modified the kinetics of P700+ absorbance changes (ΔA810) by promoting rapid photooxidation of P700. These characteristic changes in ΔA810 induction kinetics and P700+ accumulation did not appear when the leaves were treated with DCCD in the presence of nigericin. In addition to opposite modifications of ΔA810 kinetics evoked by permeability-modifying agents, the fluorescence induction curves differed conspicuously depending on leaf incubation in DCCD solutions with or without nigericin. The observed modifications of fluorescence induction curves and ΔA810 indicate that DCCD suppresses electron transport from photosystem II (PSII) to P700, whereas this inhibition is removed by nigericin. The results suggest that slowing down of the electron transport rate in the presence of DCCD was caused by elevation of ΔpH in thylakoids. The prevention of pH gradient formation in the presence of protonophore lowered also the steady-state P700+ level in far-red irradiated leaves and accelerated the subsequent dark reduction of P700. These findings indicate that PSI-driven cyclic electron flow is accelerated after the removal of the pH gradient.

Redox transients of chlorophyll P700, monitored as absorbance changes Δ A 810 , were measured during and after exclusive PSI excitation with far-red (FR) light in pea ( Pisum sativum , cv. Premium) leaves under various pre-excitation conditions. Prolonged adaptation in the dark terminated by a short PSII + PSI− exciting light pulse guarantees pre-conditions in which the initial photochemical events in PSI RCs are carried out by cyclic electron transfer (CET). Pre-excitation with one or more 10 s FR pulses creates conditions for induction of linear electron transport (LET). These converse conditions give rise to totally different, but reproducible responses of P700 − oxidation. System analyses of these responses were made based on quantitative solutions of the rate equations dictated by the associated reaction scheme for each of the relevant conditions. These provide the mathematical elements of the P700 induction algorithm (PIA) with which the distinguishable components of the P700 + response can be resolved and interpreted. It enables amongst others the interpretation and understanding of the characteristic kinetic profile of the P700 + response in intact leaves upon 10 s illumination with far-red light under the promotive condition for CET. The system analysis provides evidence that this unique kinetic pattern with a non-responsive delay followed by a steep S-shaped signal increase is caused by a photoelectrochemically controlled suppression of the electron transport from Fd to the PQ-reducing Q r site of the cytb 6 f complex in the cyclic pathway. The photoelectrochemical control is exerted by the PSI-powered proton pump associated with CET. It shows strong similarities with the photoelectrochemical control of LET at the acceptor side of PSII which is reflected by release of photoelectrochemical quenching of chlorophyll a fluorescence.

Generation of action potential (AP) in plasma membranes of characean algae has a strong impact on photoreactions occurring in chloroplasts. Under physiological conditions, AP suppresses electron transport in alkaline and acidic regions, although to a different extent; these changes are transient and reversible. In the presence of the artificial electron acceptor, methyl viologen (MV2+), AP-induced changes in electron transport in photosystem II become irreversible. Incubation of Chara corallina internodal cells with MV2+ has no effect on the chlorophyll P700 photooxidation kinetics in photosystem I reaction centers, suggesting that MV2+ is inaccessible for interactions with photosystem I, because its permeation into chloroplasts of a resting cell is hindered by membrane barriers. At the same time, AP generation in the presence of MV2+ is accompanied by irreversible modification of P700 photooxidation kinetics, as can be evidenced from differences in absorption changes at 810 and 870 nm (ΔA810 signals). These findings suggest that MV2+ permeation into chloroplasts in situ is facilitated during or after the AP generation. Similar to the ΔA810 signals, light-induced changes in membrane potential do not depend on the presence of MV2+ in the external medium until the first excitatory stimulus is applied. Electric photoresponses of the cell are irreversibly modified by AP generated in the presence of MV2+ at the expense of non-cyclic photosynthetic electron transport redirected to the MV2+ reduction. It is concluded that AP effects on chloroplast photosynthesis in situ are complex and involve permeability changes for MV2+ in membrane barriers of the “plasmalemma-chloroplast envelope” system.

Photoinduced changes in the redox state of photosystem I (PSI) primary donor, chlorophyll P700 were studied by measuring differential absorbance changes of pea leaves at 810 nm minus 870 nm (ΔA810). The kinetics of ΔA810 induced by 5-s pulses of white light were strongly affected by preillumination. In dark-adapted leaves, the light pulse caused a transient oxidation of P700 and its subsequent reduction. An identical pulse, applied after 30-s preillumination with white light, induced sequential appearance of two peaks of P700 oxidation. These kinetic differences of ΔA810 reflect regulatory changes of electron flow on the donor and acceptor sides of PSI induced by illumination of leaf for 20–40 s. The amplitude of ΔA810 second peak depended nonmonotonically on the dark interval preceding illumination: it increased with the length of dark period in the range 3–10 s and decreased upon longer dark intervals. The second wave of ΔA810 disappeared after the treatment with combination of ionophores preventing ΔpH and electric potential formation at the thylakoid membrane. In leaves treated with monensin eliminating ΔpH only, the ΔA810 signals become incompletely reversible and were characterized by slow relaxation in darkness. The results indicate an important role of electrochemical proton gradient in generation of the second wave of light-induced P700 oxidation.

Characean cells exposed to illumination arrange plasma-membrane H(+) fluxes and photosynthesis in coordinated spatial patterns. The limited availability of CO(2) in alkaline bands accounts for the lower effective quantum yield of photosystem II (DeltaF/F(m)') in chloroplasts of these bands compared to acidic zones. The effect of electrically triggered action potential on the spatial distribution of photosynthetic parameters (DeltaF/F(m)' and non-photochemical quenching, NPQ) and extracellular pH was studied with fluorescence imaging and pH microelectrodes. In the resting cell at a range of light intensities, the periodic profile of extracellular pH is parallel to the profile of NPQ and antiparallel to that of DeltaF/F(m)'. After triggering the action potential, the pH banding temporarily disappeared, but in contrast, the differences in effective quantum yield and NPQ patterns became more apparent. The transient changes in pH-banding, effective quantum yield and non-photochemical quenching are discussed in relation to alterations in intracellular Ca(2+) and H(+) concentrations during and after the action potential.

Effects of inhibitors and cofactors of cyclic and noncyclic electron transport on nonphotochemical quenching of chlorophyll fluorescence induced by action potential (AP) was investigated in Chara corallina cells. Under control conditions, energy-dependent quenching (qE) develops upon the increase in photosynthetically active radiation (PAR); it also arises and reversibly disappears after AP generation at moderate irradiances. The treatment of cells with diuron (DCMU) completely eliminated qE established at high irradiances and qE induced by AP generation. The activation of cyclic electron transport by DCMU in combination with phenazine methosulfate restored qE at high irradiances but did not restore qE imposed after AP generation. The presence in the medium of a PSI acceptor, methyl viologen at concentrations from 100 μM to 0.83 mM had no effect on fluorescence and photosynthetic activity of chloroplasts until the application of a single excitatory stimulus. Once a single AP was generated in the presence of methyl viologen, it induced irreversible qE at a wide range of irradiances, which indicates the AP-triggered redirection of a part of electron flow from the main pathway to the artificial acceptor. It is concluded that AP generation opens access for permeation of a divalent cation methyl viologen from the medium to the chloroplast stroma across two membrane barriers, the plasmalemma and the inner membrane of the chloroplast envelope.

The F(0) fluorescence yield in intact photosystem II (PSII), Ca-depleted PSII (PSII(-Ca/NaCl)), and Mn-depleted PSII membranes was measured before and after dim light treatment (1-2 min), using flash-probe fluorescence and fluorescence induction kinetic measurements. The value of F(0) after the light treatment (F'(0)) was larger than F(0) in dark-adapted PSII membranes and depended on the appearance of the slowly relaxing, reduced plastoquinone pool (t(1/2) = 4 min) formed during preillumination, which was not totally reoxidized before the F'(0) measurement. In PSII(-Ca/NaCl) such a pool also appeared, but the F'(0) yield was even higher than in intact PSII membranes. In Mn-depleted PSII membranes, the pool did not form. Interestingly, the yield of F'(0) in Ca-depleted PSII membranes prepared using chelators (EGTA and citrate) or containing 5 mM EGTA was significantly lower than in PSII(-Ca/NaCl) samples prepared without chelators. These data indicate that chelators inhibit the reduction of Q(A) and Q(B) and formation of the slowly relaxing plastoquinone pool, or alternatively they increase the rate of its oxidation. Such an effect can be explained by coordination of the chelator molecule to the Mn cluster in PSII(-Ca/NaCl) membranes, rather than different amounts of residual Ca2+ in the membranes (with or without the chelator), since the remaining oxygen-evolving activity (approximately 15%) in PSII(-Ca/NaCl) samples did not depend on the presence of the chelator. Thus, chelators of calcium cations not only have an effect on the EPR properties of the S2 state in PSII(-Ca/NaCl) samples, but can also influence the PSII properties determining the rate of plastoquinone pool reduction and/or oxidation. The effect of some toxic metal cations (Cd, Cu, Hg) on the formation of the slowly relaxing pool in PSII membranes was also studied.

This study deals with effects of membrane excitation on photosynthesis and cell protection against excessive light, manifested in non-photochemical quenching (NPQ). In Chara corallina cells, NPQ and pericellular pH displayed coordinated spatial patterns along the length of the cell. The NPQ values were lower in H(+)-extruding cell regions (external pH approximately 6.5) than in high pH regions (pH approximately 9.5). Generation of an action potential by applying a pulse of electric current caused NPQ to increase within 30-60 s. This effect, manifested as a long-lived drop of maximum chlorophyll fluorescence (F(m)'), occurred at lower photosynthetic flux densities (PFD) in the alkaline as compared to acidic cell regions. The light response curve of NPQ shifted, after generation of an action potential, towards lower PFD. The release of NPQ by nigericin and the rapid reversal of action potential-triggered NPQ in darkness indicate its relation to thylakoid DeltapH. Generation of an action potential shortly after darkening converted the chloroplasts into a latent state with the F(m) identical to that of unexcited cells. This state transformed to the quenched state after turning on weak light that was insufficient for NPQ prior to membrane excitation of the cells. The ionophore, A23187, shifted NPQ plots similarly to the action potential effect, consistent with a likely role of a rise in the cytosolic Ca(2+) level in the action potential-induced quenching. The results suggest that a rapid electric signal, across the plasma membrane, might exert long-lived effects on photosynthesis and chlorophyll fluorescence through ion flux-mediated pathways.

Chlorophyll fluorescence methods were applied to probe in vivo photosystem II (PSII) function in Chlamydomonas reinhardtii grown in sulfur-depleted media under aerobic conditions. The rates of oxygen evolution and dark reduction decreased during a 24-h incubation in sulfur-deficient medium, while the respiration rate increased. The analysis of chlorophyll fluorescence induction curves suggests that electron transport was perturbed on both the acceptor and donor sides of PSII. Light-induced violaxanthin de-epoxidation and non-photochemical fluorescence quenching were suppressed, owing to dark accumulation of zeaxanthin. Also sulfur-deprived cells showed elevated concentrations of violaxanthin and lutein. Sulfur deprivation stimulated a pronounced (three- to four-fold) increase in chlorophyll a fluorescence intensity (parameters Fo and Fm), probably due to greater light absorption by carotenoids and changes in the excitation energy transfer and deactivation in PSII of C. reinhardtii.

When exposed to light, the cells of characean algae produce intermittent regions of H+ extrusion and H+ absorption, featuring different photosynthetic activities. Methods for local measurements of outer pH, O2 content, and photochemical activity of photosystem II (PSII) were applied to examine microscopic regions of Chara coralline Klein ex Willd. internodes. The results show that the functional spatial heterogeneity of these excitable cells is controlled not only by light but also by electric excitation of the plasma membrane. Generation of a single action potential (AP) induced a reversible transition to the state with homogenous pH distribution and had different effects on photosynthesis in cell regions producing alkaline and acid zones. The effective quantum yield of PSII primary processes and the maximal chlorophyll fluorescence decreased after AP in the alkaline cell regions but were almost unaffected in the acidic cell regions. The suppression of photosynthesis after AP was also evident in the decrease of photosynthetic O2 evolution. The results provide evidence that electric signals arising at the plasmalemma are transmitted to the level of thylakoid membranes. The effects of electric excitation on fluorescence and the quantum yield of PSII photochemistry were best pronounced at low light intensities and low level of nonphotochemical quenching. The sensitivity of chlorophyll fluorescence in resting and excited cells to light intensity and protonophores indicates that the AP-induced fluorescence changes derive from the increase in pH gradient at the thylakoid membrane. The temporal elimination of alkaline zones and inhibition of photosynthesis apparently arise from parallel operational sequences that have a common initial stage. A possible role of cytosolic Ca2+ rise in the mechanism of photosynthesis suppression after electric excitation of the plasma membrane is discussed.

Capillary microelectrodes and pulse amplitude-modulated microfluorometry were used to study light-triggered changes in cell membrane potential, chlorophyll fluorescence, and photochemical yield of PSII in chloroplasts of a hornwort Anthoceros sp. The action potential was generated by illuminating the plant sample for a few seconds. It was accompanied by a reversible decrease in quantum efficiency of PSII and by nonphotochemical quenching of fluorescence that continued as long as 10 min after the light stimulus. The presence of ammonium ions (2 mM) enhanced the amplitude and prolonged the duration of dark changes of fluorescence parameters in accordance with the reported increase in duration and amplitude of the light-triggered action potential in the presence of NH 4 + . A rapid retardation of PSII activity within the first seconds of illumination was also evident from absorbance changes at 810 nm reflecting the redox conversions of chlorophyll P700. The PSII-dependent stage of reduction in the induction curves of P700 absorbance was strongly suppressed, and the amplitudes of signals induced by white and far-red light (717 nm) differed insignificantly. It is concluded that a short-term irradiation triggers the generation of ΔpH at the thylakoid membranes, which is accompanied by inhibition of the plasma membrane H+ pump and by reversible inactivation of PSII due to increased thermal dissipation of chlorophyll excitations.

In Chara corallina cells exposed to continuous light, external pH (pHo) and photosystem II (PSII) photochemical yield show correlated banding patterns. Photosynthetic activity is low in cell regions producing alkaline zones and high in the acid regions. We addressed the question whether (and how) photosynthetic activity and plasma membrane (PM) H+-pumping and H+-conductance are coupled in the different bands. First, PM H+-pump activity was stimulated with fusicoccin. This resulted in a more acidic pH in the acid bands without disturbing the correlation of photosynthetic electron transport and H+ fluxes across the PM. Next, H+-pump activity was reduced through microinjection of a phosphorylated peptide matching the canonical 14-3-3 binding motif RSTpSTP in the acid cell region. Microinjection induced a rapid (~5 min) rise in pHo by ca. 1.0 unit near the injection site, whereas the injection of the non-phosphorylated peptide had no effect. This pH rise confirms the supposed inhibition of the H+-pump upon the detachment of 14-3-3 proteins from the H+-ATPase. However, the PSII yield in the cell regions corresponding to the new alkaline peak remained high, which violated the normal inverse relations between the pHo and PSII photochemical yield. We conclude that the injection of the competitive inhibitor of the H+ATPase disrupts the balanced operation of PM H+-transport and photosynthetic electron flow and promotes electron flow through alternative pathways.

Using pH microelectrodes and a Microscopy 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 (F(m)') in the alkaline cell regions, as well as the quantum yield of photosystem II photochemistry, without affecting F(m)' 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.

In experiments with the alga Chara corallina Klein ex Willd., the appearance of subcellular domains with different photosynthetic activities, as well as formation of alkaline and acid zones near the cell surface were monitored with pulse-amplitude modulated microfluorometry and pH microelectrodes. After transfer of a dark-adapted cell to actinic light, the effective yield of PSII photochemistry (ΔF/F m′) underwent different induction changes in cell regions where acid and alkaline zones were produced. The PSII effective yield decreased for 5–15 min of illumination in cell regions forming the alkaline bands but increased after the initial decline in the acid regions. The photoinduced decrease in ΔF/F m′ in the alkaline regions occurred faster than or concurrently with the change in local pH near the cell surface (pH0). The light-induced change in pH0 was manifested as a steep transition after a latent period of variable lengths. The kinetics of ΔF/F m′ and F m, specific for alkaline regions, were replaced by those typical of acid regions, when the illumination area was narrowed to 2 mm. The results show that the formation of subcellular domains with different photosynthetic activities is not strictly bound to particular cell regions but is a dynamic event determined by spatial coordination of photosynthesis in a long cylindrical cell.

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 (QA) 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 [Bioelectrochemistry 57 (2002) 123].

A scanning microprobe technique was used to study pH distribution near the cell surface of Chara corallina Klein ex Willd. under variations of light intensity, dark–light transitions, and local illumination of various cell parts. In darkness, the H+-transporting activity of plasmalemma was distributed homogeneously over the cell surface. However, after exposing the cell to weak light (irradiance 0.2–0.5 W/m2), individual alkaline peaks with ΔpH of 1–2 units were observed in the longitudinal pH profile. The peaks in the longitudinal pH profile became more numerous with the increase in light intensity. The plot of ΔpH as a function of light intensity included a steep transition from zero to its maximum amplitude. In strong light (100 W/m2), the pH bands alternated along the cell length with a periodic length of about 7 mm. It is shown that the light-induced formation of ring-shaped bands with H+-exporting and H+ sink activities is preceded by the appearance of irregularly located spots (patches). When small cell parts were illuminated and the light spot was suddenly shifted along the cell to another position, the alkaline bands reorganized in two ways. In some cases, this treatment was followed by a gradual shift in the band position (without attenuation in the peak height) toward the illuminated area. In other cases, the initial band disappeared after such treatment, and a new alkaline band emerged in the vicinity of the illuminated area. Despite the apparent similarity of regular bands in the longitudinal pH profiles, the properties of individual bands (variable sensitivity to light intensity changes, the ability of bands to move along the cell under external treatment) differ substantially. It is supposed that the light-induced formation of the pH profile is based on primary fluctuations of photosynthetic and transport activities in the chloroplast layer and the plasmalemma, as well as on further rearrangements of membrane domains that stabilize band locations.

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.

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.

Continuous profiles of local pH near the cell surface of Chara corallinawere recorded during uniform longitudinal movement of an internodal cell relative to a stationary pH microelectrode. Under illumination, the pH profile consisted of alternating acid and alkaline bands with a pH difference of up to 3 pH units. After darkening, the bands disappeared and pH became uniformly distributed along the cell length. Chlorophyll fluorescence of chloroplasts was measured by microfluorometry at different locations within one cell, and significant differences were observed in close relation to light-dependent pH banding. The chlorophyll fluorescence yield was lower in zones of low external pH than in alkaline zones both under actinic and saturating light. The fluorescence parameters Fand F" m and the quantum yield of photosystem II (PSII) displayed variations along the cell length in accordance with pH changes in unstirred layers of the medium. The results show that PSII photochemical efficiency and the rate of noncyclic electron transport are higher in the chloroplasts of acid zones (zones of H+extrusion from the cell) than in alkaline zones. The dependence of photosynthetic electron transport on local pH near the cell surface may result from different contents of CO2in acid and alkaline regions. The acid zones are enriched with CO2that readily permeates through the membrane providing the substrate for the Calvin cycle. Conversely, a poorly permeating form, HCO–3is predominant in alkaline zones, which may restrict the dark reactions and photosynthetic electron flow.

In patch-clamp experiments on isolated chloroplasts of Peperomia metallica Lind. et Rodig. (Piperaceae), the replacement of 50 mM KCl in a medium with 50 mM NH4Cl strongly influenced the parameters of photocurrent known to reflect the generation of electric potential in thylakoids. The addition of NH+4to the medium modified the induction curves of the photocurrent as well as the currents induced by single-turnover flashes in preilluminated chloroplasts. Under the action of a prolonged light pulse (∼1 s), the steady-state current was much higher in the ammonium-containing medium than in the presence of K+. Preillumination of a dark-adapted chloroplast with a 1-s light pulse suppressed the current induced by a single-turnover flash (6 μs) in the presence of K+ but caused an elevation (by 50–150%) of the flash-induced current and shortening of its relaxation time in the presence of NH+4. The origin of different induction kinetics for the photocurrent in K+ and NH+4 media is partly clear, because ammonium prevents generation of the pH gradient and, subsequently, eliminates the ΔpH-dependent suppression of the electron transport rate. However, this does not explain the origin of NH+4-dependent photostimulation of the current generated by single-turnover flashes. This phenomenon arises from the thylakoid swelling caused by the accumulation of NH+4 in the lumen and from the respective changes in the network resistances. The network element most sensitive to thylakoid swelling is the lateral resistance of the lumen: it decreases upon enlargement of the cross-section area. Stimulation of the flash-induced current by preillumination in the presence of NH+4 was accompanied by accelerated relaxation of the current, indicating that the phenomena observed are caused by the reduction of network resistance involved in the discharge of the membrane capacity. Thus, the light-induced structural changes in the thylakoid system have a marked effect on the currents measured with the patch-clamp technique.

A patch-clamp method was used for measuring flash-induced currents (photocurrents) in Peperomia metallica chloroplasts at different holding potentials ( V h ) in a ‘whole-thylakoid’ configuration. The applied holding potentials modified the kinetics of the photocurrents dramatically. A partially reversible, transient decrease in the magnitude of the holding current was observed with a minimum at 50–200 ms after the flash followed by a slow relaxation in 1–10 s. Under similar conditions the chloroplast input (seal) conductance was followed by phase-sensitive detection with sine-wave potential modulation. The seal conductance was found to decrease transiently by 0.3–5% after a single-turnover flash and much more upon continuous illumination or repetitive flashes. The kinetics of these conductance changes were comparable to the current profile as revealed by subtracting the current traces measured in the absence and presence, respectively, of a holding potential. A major part of the conductance changes are suggested to be intimately related to alterations in the electrical properties of the lateral conductance phases along the thylakoid lamellae. The changes are extremely sensitive to agents that modify ion transport and exchange processes in the membrane and its adjoining phases. The patch-clamp method is unique for quantifying these conductance changes in intact chloroplasts on a ms time scale.

Mechanical wounding of cell walls occurring in plants under the impact of pathogens or herbivores can be mimicked by cell wall incision with a glass micropipette. Measurements of pH at the surface of Chara corallina internodes following microperforation of cell wall revealed a rapid (10–30 s) localized alkalinization of the apoplast after a lag period of 10–20 s. The pH increase induced by incision could be as large as 3 pH units and relaxed slowly, with a halftime up to 20 min. The axial pH profile around the incision zone was bell-shaped and localized to a small area, extending over a distance of about 100 μm. The pH response was suppressed by lowering cell turgor upon the replacement of artificial pond water (APW) with APW containing 50 mM sorbitol. Stretching of the plasma membrane during its impression into the cell wall defect is likely to activate the Ca2 + channels, as evidenced from sensitivity of the incision-induced alkalinization to the external calcium concentration and to the addition of Ca2 +-channel blockers, such as La3 +, Gd3 +, and Zn2 +. The maximal pH values attained at the incision site (~ 10.0) were close to pH in light-dependent alkaline zones of Chara cells. The involvement of cytoskeleton in the origin of alkaline patch was documented by observations that the incision-induced pH transients were suppressed by the inhibitors of microtubules (oryzalin and taxol) and, to a lesser extent, by the actin inhibitor (cytochalasin B). The results indicate that the localized increase in apoplastic pH is an early event in mechanoperception and depends on light, cytoskeleton, and intracellular calcium.

Chlorophyll fluorescence induction curves induced by an actinic pulse of red light follow different kinetics in dark-adapted plant leaves and leaves preilluminated with far-red light. This influence of far-red light was abolished in leaves infiltrated with valinomycin known to eliminate the electrical (Δφ) component of the proton-motive force and was strongly enhanced in leaves infiltrated with nigericin that abolishes the ΔpH component. The supposed influence of ionophores on different components of the proton motive force was supported by differential effects of these ionophores on the induction curves of the millisecond component of chlorophyll delayed fluorescence. Comparison of fluorescence induction curves with the kinetics of P700 oxidation in the absence and presence of ionophores suggests that valinomycin facilitates a build-up of a rate-limiting step for electron transport at the site of plastoquinone oxidation, whereas nigericin effectively removes limitations at this site. Far-red light was found to be a particularly effective modulator of electron flows in chloroplasts in the absence of ΔpH backpressure on operation of the electron-transport chain.

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 NH 4 Cl and TMPD is observed and considered to be caused by a reversible release of current limitation in the interfacial conductance barriers in the lumen.

Pre-illumination of the thylakoid membrane of Peperomia metallica chloroplasts leads to a reversible suppression of the flash-induced electrical potential as measured either with the electrochromic bandshift (P515), microelectrode impalement or patch-clamp technique. The energization-dependent potential suppression was not observed in the presence of 1 μM nigericin suggesting the involvement of proton and/or cation gradients. Energization in the presence of 3-(3.4-dichlorophenyl)-1,1-dimethylurea (DCMU) and N,N,N',N'-tetramethylphenylenediamine (TMPD), i.e. cyclic electron flow around photosystem (PS) I, results in the accumulation of TMPD + in the thylakoid lumen. The reversible suppression of the flash-induced membrane potential was not observed in these conditions indicating that it is not a general cation-induced increase of membrane capacitance. Cyclic electron flow around PSI in the presence of DCMU and phenazine methosulfate (PMS) results in the accumulation of PMS + and H + in the thylakoid lumen. The absence of reversible suppression of the flash-induced membrane potential for this condition shows that accumulation of protons does not lead to (1) a reversible increase of membrane capacitance and (2) a reversible suppression of PSI-dependent electrogenesis. Reversible inactivation of PSII by a low pH in the thylakoid lumen is therefore proposed to be the cause for the temporary suppression of the flash-induced electrical potential. The flash-induced PSII-dependent membrane potential, as measured after major oxidation of P700 in far-red background light, was indeed found to be suppressed at low assay pH (pH 5) in isolated spinach (Spinacia oleracea) chloroplasts.