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.

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.

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.

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'.

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 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.

Single-walled carbon nanotubes protect photosynthetic reactions in Chlamydomonas reinhardtii against photoinhibition.

Single-walled carbon nanotubes (SWCNTs) are among the most exploited carbon allotropes in nanosensing, bioengineering, and photobiological applications, however, the interactions of nanotubes with the photosynthetic process and structures are still poorly understood. We found that SWCNTs are not toxic to the photosynthetic apparatus of the model unicellular alga Chlamydomonas reinhardtii and demonstrate that this carbon nanomaterial can protect algal photosynthesis against photoinhibition. The results show that the inherent phytotoxicity of the nanotubes may be overcome by an intentional selection of nanomaterial characteristics. A low concentration (2 µg mL-1) of well-dispersed, purified and small SWCNTs did not alter the growth and pigment accumulation of the cultures. Indeed, under the photoinhibitory conditions of our experiments, SWCNT-enriched samples were characterized by a lower rate of PSII inactivation, reduced excitation pressure in PSII, a higher rate of photosynthetic electron transport, and an increased non-photochemical quenching in comparison with the controls. In addition, SWCNTs change the distribution of energy between the photosystems in favour of PSII (state 1). The underlying mechanism of this action is not yet understood but possibly, electrons or energy can be exchanged between the redox active nanotubes and photosynthetic components, and probably other redox active intra-chloroplast constituents. Alternatively, nanotubes may promote the formation of an NPQ conformation of PSII. Our results provided evidence for such electron/energy transfer from photosynthetic structures toward the nanotubes. The discovered photoprotective effects can potentially be used in photobiotechnology to maintain the photosynthetic activity of microorganisms under unfavourable conditions.

Microfluidic interactions involved in chloroplast responses to plasma membrane excitation in Chara.

Adaptation of plants to environmental changes involves the mechanisms of long-distance signaling. In characean algae, these mechanisms comprise the propagation of action potential (AP) and the rotational cytoplasmic streaming acting in cooperation with light-dependent exchange of ions and metabolites across the chloroplast envelope. Both excitability and cyclosis exert conspicuous effects on photosynthetic activity of chloroplasts but possible influence of cyclosis arrest on the coupling of AP stimulus to photosynthetic performance remained unexplored. In this study, fluidic interactions between anchored chloroplasts were allowed or restricted by illuminating the whole internode or a confined cell area (2 mm in diameter), respectively. Measurements of chlorophyll fluorescence parameters (F' and Fm') in cell regions located close to calcium crystal depositions revealed that the AP generation induced long-lasting Fm' oscillations that persisted in illuminated cells. The AP generation often induced the F' oscillations, whose number diminished upon the transfer of internodal cells from total to local background light. The results indicate that the AP-induced changes in photosynthetic parameters, F' in particular, have a complex origin and comprise the internal processes caused by the elevation of stromal Ca2+ concentration in the analyzed chloroplasts and the stages related to ion and metabolite exchange mediated by cytoplasmic streaming. It is supposed that the composition of flowing cytoplasm is heterogeneous due to the spatial alteration of calcified and noncalcified cell sites, but this heterogeneity is enhanced and can be visualized after the transient cessation and restoration of cytoplasmic streaming.

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.

Deficiency in flavodiiron protein Flv3 promotes cyclic electron flow and state transition under high light in the cyanobacterium Synechocystis sp. PCC 6803.

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 (Kana 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.

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.

Transient depletion of transported metabolites in the streaming cytoplasm of Chara upon shading the long-distance transmission pathway.

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.

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.

PH-dependent cell-cell interactions in the green alga Chara.

Characean internodal cells develop alternating patterns of acid and alkaline zones along their surface in order to facilitate uptake of carbon required for photosynthesis. In this study, we used a pH-indicating membrane dye, 4-heptadecylumbiliferone, to study the kinetics of alkaline band formation and decomposition. The differences in growth/decay kinetics suggested that growth occurred as an active, autocatalytic process, whereas decomposition was due to diffusion. We further investigated mutual interactions between internodal cells and found that their alignment parallel to each other induced matching of the pH banding patterns, which was mirrored by chloroplast activity. In non-aligned cells, the lowered photosynthetic activity was noted upon a rise of the external pH, suggesting that the matching of pH bands was due to a local elevation of membrane conductance by the high pH of the alkaline zones of neighboured cells. Finally, we show that the altered pH banding pattern caused the reorganization of the cortical cytoplasm. Complex plasma membrane elaborations (charasomes) were degraded via endocytosis, and mitochondria were moved away from the cortex when a previously acid region became alkaline and vice versa. Our data show that characean internodal cells react flexibly to environmental cues, including those originating from neighboured cells.

Interchloroplast communications in Chara are suppressed under the alkaline bands and are relieved after the plasma membrane excitation.

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.

Cyclosis-mediated intercellular transmission of photosynthetic metabolites in Chara revealed with chlorophyll microfluorometry.

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.

Photoinduction of electron transport on the acceptor side of PSI in Synechocystis PCC 6803 mutant deficient in flavodiiron proteins Flv1 and Flv3.

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.

Long-range interactions of Chara chloroplasts are sensitive to plasma-membrane H+ flows and comprise separate photo- and dark-operated pathways.

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.

Cyclosis-mediated long distance communications of chloroplasts in giant cells of Characeae.

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 30s 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 F' 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 1-5mm 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.

Pathways for external alkalinization in intact and in microwounded Chara cells are differentially sensitive to wortmannin.

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.