Roles of ApcD and orange carotenoid protein in photoinduction of electron transport upon dark-light transition in the Synechocystis PCC 6803 mutant deficient in flavodiiron protein Flv1.

Flavodiiron proteins Flv1/Flv3 accept electrons from photosystem (PS) I. In this work we investigated light adaptation mechanisms of Flv1-deficient mutant of Synechocystis PCC 6803, incapable to form the Flv1/Flv3 heterodimer. First seconds of dark-light transition were studied by parallel measurements of light-induced changes in chlorophyll fluorescence, P700 redox transformations, fluorescence emission at 77 K, and OCP-dependent fluorescence quenching. During the period of Calvin cycle activation upon dark-light transition, the linear electron transport (LET) in wild type is supported by the Flv1/Flv3 heterodimer, whereas in Δflv1 mutant activation of LET upon illumination is preceded by cyclic electron flow that maintains State 2. The State 2-State 1 transition and Orange Carotenoid Protein (OCP)-dependent non-photochemical quenching occur independently of each other, begin in about 10 s after the illumination of the cells and are accompanied by a short-term re-reduction of the PSI reaction center (P700+). ApcD is important for the State 2-State 1 transition in the Δflv1 mutant, but S-M rise in chlorophyll fluorescence was not completely inhibited in Δflv1/ΔapcD mutant. LET in Δflv1 mutant starts earlier than the S-M rise in chlorophyll fluorescence, and the oxidation of plastoquinol (PQH2) pool promotes the activation of PSII, transient re-reduction of P700+ and transition to State 1. An attempt to induce state transition in the wild type under high intensity light using methyl viologen, highly oxidizing P700 and PQH2, was unsuccessful, showing that oxidation of intersystem electron-transport carriers might be insufficient for the induction of State 2-State 1 transition in wild type of Synechocystis under high light.

Intercellular permeation and cyclosis-mediated transport of a fluorescent probe in Characeae.

Intercellular communication and transport is the essential prerequisite for the function of multicellular organisms. Simple diffusion as a transport mechanism is often inefficient in sustaining the effective exchange of metabolites, and other active transport mechanisms become involved. In this paper, we use the giant cells of characean algae as a model system to explore the role of advection and diffusion in intercellular transport. Using fluorescent dye as a tracer, we study the kinetics of the permeation of the fluorophore through the plasmodesmata complex in the node of tandem cells and its further distribution across the cell. To explore the role of cytoplasmic streaming and the nodal cell complex in the transport mechanism, we modulate the cytoplasmic streaming using action potential to separate the diffusive permeation from the advective contribution. The results imply that the plasmodesmal transport of fluorescent probe through the central and peripheral cells of the nodal complex is differentially regulated by a physiological signal, the action potential. The passage of the probe through the central cells of the nodal complex ceases transiently after elicitation of the action potential in the internodal cell, whereas the passage through the peripheral cells of the node was retained. A diffusion-advection model is developed to describe the transport kinetics and extract the permeability of the node-internode cell wall from experimental data.

Electrical Signals at the Plasma Membrane and Their Influence on Chlorophyll Fluorescence of Chara Chloroplasts in vivo.

Action potentials of plant cells are engaged in the regulation of many cell processes, including photosynthesis and cytoplasmic streaming. Excitable cells of characean algae submerged in a medium with an elevated K+ content are capable of generating hyperpolarizing electrical responses. These active responses of plasma membrane originate upon the passage of inward electric current comparable in strength to natural currents circulating in illuminated Chara internodes. So far, it remained unknown whether the hyperpolarizing electrical signals in Chara affect the photosynthetic activity. Here, we showed that the negative shift of cell membrane potential, which drives K+ influx into the cytoplasm, is accompanied by a delayed decrease in the actual yield of chlorophyll fluorescence F' and the maximal fluorescence yield Fm' under low background light (12.5 µmol m-2 s-1). The transient changes in F' and Fm' were evident only under illumination, which suggests their close relation to the photosynthetic energy conversion in chloroplasts. Passing the inward current caused an increase in pH at the cell surface (pHo), which reflected high H+/OH- conductance of the plasmalemma and indicated a decrease in cytoplasmic pH due to the H+ entry into the cell. The shifts in pHo arising in response to the first hyperpolarizing pulse disappeared upon repeated stimulation, thus indicating the long-term inactivation of plasmalemmal H+/OH- conductance. Suppression of plasmalemmal H+ fluxes did not abolish the hyperpolarizing responses and the analyzed changes in chlorophyll fluorescence. These results suggest that K+ fluxes between the extracellular medium, cytoplasm, and stroma are involved in the functional changes of chloroplasts reflected by transients of F' and Fm'.

Effects of chloroplast-cytoplasm exchange and lateral mass transfer on slow induction of chlorophyll fluorescence in Characeae.

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.

Brefeldin A inhibits clathrin-dependent endocytosis and ion transport in Chara internodal cells.

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 (BFA), an inhibitor of vesicle trafficking, on internodal cells of Chara australis. BFA 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, BFA inhibited endocytosis and significantly decreased the number of clathrin-coated pits and clathrin-coated vesicles at the plasma membrane. BFA 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 BFA inhibited the formation of alkaline and acid regions along the cell surface ('pH banding pattern') which facilitates carbon uptake required for photosynthesis; we hypothesise that this is due to insufficient recycling of ion transporters. During long-term treatments over several days, BFA delayed the formation of complex 3D plasma membranes (charasomes). Interestingly, BFA had no detectable effect on clathrin-dependent charasome degradation. Protein sequence analysis suggests that the peculiar effects of BFA 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 BFA on different processes in C. 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.

Prolonged oxygen depletion in microwounded cells of Chara corallina detected with novel oxygen nanosensors.

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.

Inhibition of endosomal trafficking by brefeldin A interferes with long-distance interaction between chloroplasts and plasma membrane transporters.

The huge internodal cells of the characean green algae are a convenient model to study long-range interactions between organelles via cytoplasmic streaming. It has been shown previously that photometabolites and reactive oxygen species released by illuminated chloroplasts are transmitted to remote shaded regions where they interfere with photosynthetic electron transport and the differential activity of plasma membrane transporters, and recent findings indicated the involvement of organelle trafficking pathways. In the present study, we applied pulse amplitude-modulated microscopy and pH-sensitive electrodes to study the effect of brefeldin A (BFA), an inhibitor of vesicle trafficking, on long-distance interactions in Chara australis internodal cells. These data were compared with BFA-induced changes in organelle number, size and distribution using fluorescent dyes and confocal laser scanning microscopy. We found that BFA completely and immediately inhibited endocytosis in internodal cells and induced the aggregation of organelles into BFA compartments within 30-120 min of treatment. The comparison with the physiological data suggests that the early response, the arrest of endocytosis, is related to the attenuation of differences in surface pH, whereas the longer lasting formation of BFA compartments is probably responsible for the acceleration of the cyclosis-mediated interaction between chloroplasts. These data indicate that intracellular turnover of membrane material might be important for the circulation of electric currents between functionally distinct regions in illuminated characean internodes and that translational movement of metabolites is delayed by transient binding of the transported substances to organelles.

Photoregulation of photosystem II activity mediated by cytoplasmic streaming in Chara and its relation to pH bands.

Chloroplasts in vivo exposed to strong light export assimilates and excess reducing power to the cytoplasm for metabolic conversions and allocation to neighboring and distant organelles. The cytoplasmic streaming, being particularly fast in characean internodes, distributes the exported metabolites from brightly illuminated cell spots to light-limited regions, which is evident from the transient increase in chlorophyll fluorescence of shaded areas in response to illumination of distant cell regions situated upstream the liquid flow. It is not yet known whether long-distance communications between anchored chloroplasts are interfered by pH banding that commonly arises in characean internodes under the action of continuous or fluctuating light. In this study, microfluorometry, pH-microsensors, and local illumination were combined to examine long-distance transport and subsequent reentry of photosynthetic metabolites, including triose phosphates, into chloroplasts of cell regions producing external alkaline and acid bands. The lateral transmission of metabolic signals between distant chloroplasts was found to operate effectively in cell areas underlying acid zones but was almost fully blocked under alkaline zones. The rates of linear electron flow in chloroplasts of these regions were nearly equal under dim background light, but differed substantially at high light when availability of CO2, rather than irradiance, was the rate-limiting factor. Different productions of assimilates by chloroplasts underlying CO2-sufficient acid and CO2-deficient alkaline zones were a cause for contrasting manifestations of long-distance transport of photosynthetic metabolites. Nonuniform cytoplasmic pH in cells exhibiting pH bands might contribute to different activities of metabolic translocators under high and low pH zones.

Photoinduction of cyclosis-mediated interactions between distant chloroplasts.

Communications between chloroplasts and other organelles based on the exchange of metabolites, including redox active substances, are recognized as a part of intracellular regulation, chlororespiration, and defense against oxidative stress. Similar communications may operate between spatially distant chloroplasts in large cells where photosynthetic and respiratory activities are distributed unevenly under fluctuating patterned illumination. Microfluorometry of chlorophyll fluorescence in vivo in internodal cells of the alga Chara corallina revealed that a 30-s pulse of localized light induces a transient increase (~25%) in F' fluorescence of remote cell parts exposed to dim background light at a 1.5-mm distance on the downstream side from the illuminated spot in the plane of unilateral cytoplasmic streaming but has no effect on F' at equal distance on the upstream side. An abrupt arrest of cytoplasmic streaming for about 30s by triggering the action potential extended either the ascending or descending fronts of the F' fluorescence response, depending on the exact moment of streaming cessation. The response of F' fluorescence to localized illumination of a distant cell region was absent in dark-adapted internodes, when the localized light was applied within the first minute after switching on continuous background illumination of the whole cell, but it appeared in full after longer exposures to continuous background light. These results and the elimination of the F' response by methyl viologen known to redirect electron transport pathways beyond photosystem I indicate the importance of photosynthetic induction and the stromal redox state for long-distance communications of chloroplasts in vivo.

Convoluted Plasma Membrane Domains in the Green Alga Chara are Depleted of Microtubules and Actin Filaments.

Charasomes are convoluted plasma membrane domains in the green alga Chara australis. They harbor H(+)-ATPases involved in acidification of the medium, which facilitates carbon uptake required for photosynthesis. In this study we investigated the distribution of cortical microtubules and cortical actin filaments in relation to the distribution of charasomes. We found that microtubules and actin filaments were largely lacking beneath the charasomes, suggesting the absence of nucleating and/or anchoring complexes or an inhibitory effect on polymerization. We also investigated the influence of cytoskeleton inhibitors on the light-dependent growth and the darkness-induced degradation of charasomes. Inhibition of cytoplasmic streaming by cytochalasin D significantly inhibited charasome growth and delayed charasome degradation, whereas depolymerization of microtubules by oryzalin or stabilization of microtubules by paclitaxel had no effect. Our data indicate that the membrane at the cytoplasmic surface of charasomes has different properties in comparison with the smooth plasma membrane. We show further that the actin cytoskeleton is necessary for charasome growth and facilitates charasome degradation presumably via trafficking of secretory and endocytic vesicles, respectively. However, microtubules are required neither for charasome growth nor for charasome degradation.

Strong alkalinization of Chara cell surface in the area of cell wall incision as an early event in mechanoperception.

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 (10-30s) localized alkalinization of the apoplast after a lag period of 10-20s. The pH increase induced by incision could be as large as 3 pH units and relaxed slowly, with a halftime up to 20min. The axial pH profile around the incision zone was bell-shaped and localized to a small area, extending over a distance of about 100µm. The pH response was suppressed by lowering cell turgor upon the replacement of artificial pond water (APW) with APW containing 50mM sorbitol. Stretching of the plasma membrane during its impression into the cell wall defect is likely to activate the Ca(2+) channels, as evidenced from sensitivity of the incision-induced alkalinization to the external calcium concentration and to the addition of Ca(2+)-channel blockers, such as La(3+), Gd(3+), and Zn(2+). 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.

Oscillations of chlorophyll fluorescence after plasma membrane excitation in Chara originate from nonuniform composition of signaling metabolites in the streaming cytoplasm.

Excitable cells of higher plants and characean algae respond to stressful stimuli by generating action potentials (AP) whose regulatory influence on chlorophyll (Chl) fluorescence and photosynthesis extends over tens of minutes. Unlike plant leaves where the efficiency of photosystem II reaction (YII) undergoes a separate reversible depression after an individual AP, characean algae exhibit long-lasting oscillations of YII after firing AP, provided that Chl fluorescence is measured on microscopic cell regions. Internodal cells of charophytes feature an extremely fast cytoplasmic streaming that stops immediately during the spike and recovers within ~10 min after AP. In this study a possibility was examined that multiple oscillations of YII and Chl fluorescence parameters (F', Fm') result from the combined influence of metabolic rearrangements in chloroplasts and the cyclosis cessation-recovery cycle induced by the Ca2+ influx during AP. It is shown that the AP-induced Fm' and YII oscillations disappear when the fluidic communications between the analyzed area (AOI) and surrounding cell regions are restricted or eliminated. The microfluidic signaling was manipulated in two ways: by narrowing the illuminated cell area and by arresting the cytoplasmic streaming with cytochalasin D (CD). The inhibition of Fm' and YII oscillations was not caused by the loss of cell excitability, since CD-treated cells retained the capacity of AP generation. The mechanism of AP-induced oscillations of YII and Chl fluorescence seems to involve the lateral microfluidic transport of signaling substances in combination with the distribution pattern of these substances that was enhanced during the period of streaming cessation.

Instant rerouting of photosynthetic electron transport to O2 reduction after the plasma membrane excitation of Chara in the presence of methyl viologen.

-Action potential (AP) of excitable plant cells is an important signaling event that can differentially alter physicochemical and physiological processes in various parts of the same cell. In giant cells of characean algae, the AP propagation has minor effect on photosynthetic electron transport in areas with high activity of plasmalemmal H+-pump but inhibits linear electron flow in regions featuring high passive H+/OH- conductance of the plasma membrane (PM). Uneven spatial distributions of local periplasmic and cytoplasmic pH facilitate the operation of distinct (CO2-dependent and O2-mediated) pathways of photoinduced electron flow, which presumably accounts for differential influence of AP on photosynthesis. The excitation of Chara australis cell in the presence of methyl viologen (MV), a redox mediator with the prooxidant action, provides a convenient model system to clarify the influence of voltage-dependent ion fluxes across PM on photosynthetic activity of chloroplasts. This study shows that permeation of MV to their target sites in chloroplasts is restricted by PM in resting cells, but MV easily passes through ionic channels opened during the PM depolarization. This gated permeation of MV gives rise to strong non-photochemical quenching, decrease in the effective quantum yield of linear electron flow, apparent O2 uptake, and, finally, the enhanced ROS production, as detected by the fluorescent probe dichlorofluorescein. Taken together, the results indicate that the AP generation in the presence of MV acts as trigger for instant redirection of photosynthetic linear electron flow from CO2-dependent route to the path of O2 reduction with the eventual formation of H2O2 as a dominant and most stable ROS form.

Antimycin A effect on the electron transport in chloroplasts of two Chlamydomonas reinhardtii strains.

The effects of antimycin A on the redox state of plastoquinone and on electron donation to photosystem I (PS I) were studied in sulfur-deprived Chlamydomonas reinhardtii cells of the strains cc406 and 137c. We found that this reagent suppresses cyclic electron flow around PS I in the cc406 strain, whereas this inhibitory effect was completely absent in the 137c strain. In the latter strain, antimycin A induced rapid reduction of plastoquinone in the dark and considerably enhanced the rate of electron donation to P700 (+) in the dark. Importantly, neither myxothiazol, an inhibitor of mitochondrial respiration, FCCP, a protonophore, nor propyl gallate, an inhibitor of the plastid terminal oxidase, induced such a strong effect like antimycin A. The results indicate that in the chloroplast of the 137c strain, antimycin A has a site of action outside of the machinery of cyclic electron flow.

Effects of far-red light on fluorescence induction in infiltrated pea leaves under diminished ΔpH and Δφ components of the proton motive force.

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.

Propagation of photoinduced signals with the cytoplasmic flow along Characean internodes: evidence from changes in chloroplast fluorescence and surface pH.

Emerging evidence suggests that cytoplasmic streaming can regulate the plasma-membrane H(+) transport and photosynthetic electron flow. Microfluorometric and surface pH measurements on Chara corallina internodes revealed the transmission of photoinduced signals by the cytoplasmic flow for a distance of few millimeters from the site of stimulus application. When a 30-s pulse of bright light was locally applied, the downstream cell regions responded with either release or enhancement of non-photochemical quenching of chlorophyll fluorescence, depending on the background irradiance of the analyzed cell area. Under dim background irradiance (<20 µmol m(-2) s(-1)), the arrival of the distant signal from the brightly illuminated 400-µm-wide zone elevated the maximal fluorescence F m (') in the analyzed downstream area, whereas at higher background irradiances it induced strong quenching of F m (') . At intermediate irradiances the increase and decrease in F m (') appeared as two successive waves. The transition between the F m (') responses of opposite polarities occurred at a narrow threshold range of irradiances. This indicates that inevitable slight variations in irradiance at the bottom chloroplast layer combined with the cyclosis-transmitted signals may contribute to the formation of a photosynthetic activity pattern. The rapid cyclosis-mediated release of non-photochemical quenching, unlike the delayed response of opposite polarity, was associated with opening of H(+) (OH(-))-conducting plasma membrane channels, as evidenced by the concurrent alkaline pH shift on the cell surface. It is proposed that the initial increase in F m (') after application of a distant photostimulus is determined, among other factors, by the wave of alkaline cytoplasmic pH.

Effects of cell excitation on photosynthetic electron flow and intercellular transport in Chara.

Impact of membrane excitability on fluidic transport of photometabolites and their cell-to-cell passage via plasmodesmata was examined by pulse-modulated chlorophyll (Chl) microfluorometry in Chara australis internodes exposed to dim background light. The cells were subjected to a series of local light (LL) pulses with a 3-min period and a 30-s pulse width, which induced Chl fluorescence transients propagating in the direction of cytoplasmic streaming along the photostimulated and the neighboring internodes. By comparing Chl fluorescence changes induced in the LL-irradiated and the adjoining internodes, the permeability of the nodal complex for the photometabolites was assessed in the resting state and after the action potential (AP) generation. The electrically induced AP had no influence on Chl fluorescence in noncalcified cell regions but disturbed temporarily the metabolite transport along the internode and caused a disproportionally strong inhibition of intercellular metabolite transmission. In chloroplasts located close to calcified zones, Chl fluorescence increased transiently after cell excitation, which indicated the deceleration of photosynthetic electron flow on the acceptor side of photosystem I. Functional distinctions of chloroplasts located in noncalcified and calcified cell areas were also manifested in different modes of LL-induced changes of Chl fluorescence, which were accompanied by dissimilar changes in efficiency of PSII-driven electron flow. We conclude that chloroplasts located near the encrusted areas and in the incrustation-free cell regions are functionally distinct even in the absence of large-scale variations of cell surface pH. The inhibition of transnodal transport after AP generation is probably due to Ca2+-regulated changes in plasmodesmal aperture.

Plasma membrane-chloroplast interactions activated by the hyperpolarizing response in characean cells.

Signaling pathways in plant cells often comprise electrical phenomena developing at the plasma membrane. The action potentials in excitable plants like characean algae have a marked influence on photosynthetic electron transport and CO2 assimilation. The internodal cells of Characeae can also generate active electrical signals of a different type. The so called hyperpolarizing response develops under the passage of electric current whose strength is comparable to physiological currents circulating between nonuniform cell regions. The plasma membrane hyperpolarization is involved in multiple physiological events in aquatic and terrestrial plants. The hyperpolarizing response may represent an unexplored tool for studying the plasma membrane-chloroplast interactions in vivo. This study shows that the hyperpolarizing response of Chara australis internodes whose plasmalemma was preliminary converted into the K+-conductive state induces transient changes in maximal (Fm') and actual (F') fluorescence yields of chloroplasts in vivo. These fluorescence transients were light dependent, suggesting their relation to photosynthetic electron and H+ transport. The cell hyperpolarization promoted H+ influx that was inactivated after a single electric stimulus. The results indicate that the plasma membrane hyperpolarization drives transmembrane ion fluxes and modifies the ionic composition of cytoplasm, which indirectly (via envelope transporters) affects the pH of chloroplast stroma and chlorophyll fluorescence. Remarkably, the functioning of envelope ion transporters can be revealed in short-term experiments in vivo, without growing plants on solutions with various mineral compositions.

Effects of cyclosis on chloroplast-cytoplasm interactions revealed with localized lighting in Characean cells at rest and after electrical excitation.

Cytoplasmic streaming in Characean internodes enables rapid intracellular transport and facilitates interactions between spatially remote cell regions. Cyclosis-mediated distant interactions might be particularly noticeable under nonuniform illumination, in the vicinity of light-shade borders where metabolites are transported between functionally distinct cell regions. In support of this notion, chlorophyll fluorescence parameters assessed on a microscopic area of Chara corallina internodal cells (area of inspection, AOI) responded to illumination of nearby regions in asymmetric manner depending on the vector of cytoplasmic streaming. When a beam of white light was applied through a 400-µm optic fiber upstream of AOI with regard to the direction of cytoplasmic streaming, non-photochemical quenching (NPQ) developed after a lag period in AOI exposed to moderate intensity light. Conversely, no NPQ was induced in the same cell area when the beam position was shifted to an equal distance downstream of AOI. Light-response curves for the efficiency of photosystem II electron transport in chloroplasts differed markedly depending on the illumination pattern (whole-cell versus small area illumination) but these differences were eliminated after the inhibition of cytoplasmic streaming with cytochalasin B. Localized illumination promoted chloroplast fluorescence responses to electrical plasmalemma excitation at high light intensities, which contrasts to the requirement of low to moderate irradiances for observation of the stimulus-response coupling under whole-cell illumination. The results indicate that different photosynthetic capacities of chloroplasts under general and localized illumination are related to lateral transport of nonevenly distributed cytoplasmic components between the cell parts with dominant photosynthetic and respiratory metabolism.

Far-red light-regulated efficient energy transfer from phycobilisomes to photosystem I in the red microalga Galdieria sulphuraria and photosystems-related heterogeneity of phycobilisome population.

Phycobilisomes (PBS) are the major photosynthetic antenna complexes in cyanobacteria and red algae. In the red microalga Galdieria sulphuraria, action spectra measured separately for photosynthetic activities of photosystem I (PSI) and photosystem II (PSII) demonstrate that PBS fraction attributed to PSI is more sensitive to stress conditions and upon nitrogen starvation disappears from the cell earlier than the fraction of PBS coupled to PSII. Preillumination of the cells by actinic far-red light primarily absorbed by PSI caused an increase in the amplitude of the PBS low-temperature fluorescence emission that was accompanied by the decrease in PBS region of the PSI 77 K fluorescence excitation spectrum. Under the same conditions, fluorescence excitation spectrum of PSII remained unchanged. The amplitude of P700 photooxidation in PBS-absorbed light at physiological temperature was found to match the fluorescence changes observed at 77 K. The far-red light adaptations were reversible within 2-5min. It is suggested that the short-term fluorescence alterations observed in far-red light are triggered by the redox state of P700 and correspond to the temporal detachment of the PBS antenna from the core complexes of PSI. Furthermore, the absence of any change in the 77 K fluorescence excitation cross-section of PSII suggests that light energy transfer from PBS to PSI in G. sulphuraria is direct and does not occur through PSII. Finally, a novel photoprotective role of PBS in red algae is discussed.