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.

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.

Chlorophyll fluorescence images demonstrate variable pathways in the effects of plasma membrane excitation on electron flow in chloroplasts of Chara cells.

Chlorophyll fluorescence Imaging and Microscopy PAM fluorometry were applied to study spatial dynamics of photosystem II quantum yield (ΔF/F'(m)) 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 Ca(2+) in the medium but not at elevated (2 mM) external Ca(2+) concentration. The results are interpreted in terms of competition between two permeant divalent ion species, Ca(2+) and MV(2+), 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 Ca(2+) concentration in the environmental medium.

Transient removal of alkaline zones after excitation of Chara cells is associated with inactivation of high conductance in the plasmalemma.

The action potential (AP) of excitable plant cells is a multifunctional physiological signal. Its generation in characean algae suppresses the pH banding for 15-30 min and enhances the heterogeneity of spatial distribution of photosynthetic activity. This suppression is largely due to the cessation of H(+) influx (OH(-) efflux) in the alkaline cell regions. Measurements of local pH and membrane conductance in individual space-clamped alkaline zones (small cell areas bathed in an isolated pool of external medium) showed that the AP generation is followed by the transient disappearance of alkaline zone in parallel with a large decrease in membrane conductance. These changes, specific to alkaline zones, were only observed under continuous illumination following a relaxation period of at least 15 min after previous excitation. The excitation of dark-adapted cells produced no conductance changes in the post-excitation period. The results indicate that the origin of alkaline zones in characean cells is not due to operation of electroneutral H(+)/HCO(3)(-) symport or OH(-)/HCO(3)(-) antiport. It is concluded that the membrane excitation is associated with inactivation of plasmalemma high conductance in the alkaline cell regions.

Action potential in Chara cells intensifies spatial patterns of photosynthetic electron flow and non-photochemical quenching in parallel with inhibition of pH banding.

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.

Action potential in a plant cell lowers the light requirement for non-photochemical energy-dependent quenching of chlorophyll fluorescence.

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.

Excitation-induced dynamics of external pH pattern in Chara corallina cells and its dependence on external calcium concentration.

The influence of cell excitation and external calcium level on the dynamics of light-induced pH bands along the length of Chara corallina cells is studied in the present paper. Generation of an action potential (AP) transiently quenched these pH patterns, which was more pronounced at 0.05-0.1 mM Ca2+ than at higher concentrations of Ca2+ (0.6-2 mM) in the medium. After transient smoothing of the pH bands, some alkaline peaks reemerged at slightly shifted positions in media with low Ca2+ concentrations, while at high Ca2+ concentrations, the alkaline spots reappeared exactly at their initial positions. This Ca2+ dependency has been revealed by both digital imaging and pH microelectrodes. The stabilizing effect of external Ca2+ on the locations of recovering alkaline peaks is supposedly due to formation of a physically heterogeneous environment around the cell owing to precipitation of CaCO3 in the alkaline zones at high Ca2+ during illumination. The elevation of local pH by dissolving CaCO3 facilitates the reappearance of alkaline spots at their initial locations after temporal suppression caused by cell excitation. At low Ca2+ concentrations, when the solubility product of CaCO3 is not attained, the alkaline peaks are not stabilized by CaCO3 dissolution and may appear at random locations.