The role of the intra- and extracellular protons in the photosynthetic response induced by the variation potential in pea seedlings

Local burning induces generation and propagation of variation potential (VP) in higher plants. VP induces transient inactivation of photosynthesis, which is possibly connected with proton signal in plant cell. Analysis of the role of changes in intracellular and extracellular pH in the VP-induced photosynthetic response in pea seedlings was the aim of this work. It was shown that local burning induced VP propagation, which was accompanied with a decrease of intracellular pH and increase of extracellular pH. VP induced photosynthesis inactivation that included an increase in the nonphotochemical fluorescence quenching and a decrease in the CO₂ assimilation rate. Analysis of photosynthetic responses under control and low external CO₂ concentration and changes in pH showed that there were two components in the responses. The first component appeared as a fast decrease of the CO₂ assimilation and increase of nonphotochemical quenching. It depended on the activity of the dark stage of photosynthesis and was connected with apoplast alkalization. The second component was presented as a slow increase of nonphotochemical quenching. It weakly depended on a dark stage and was connected with a decrease of intracellular pH.     

Influence of variation potential on resistance of the photosynthetic machinery to heating in pea

Electrical signals [action potentials (APs) and variation potentials (VPs)] induced by local stimuli are a mechanism that underlies rapid plant response to environmental factors. Such signals induce a number of functional responses, including changes in photosynthesis. Ultimately, these responses are considered to increase plant resistance to stress factors, but this question has been poorly investigated. We studied the influence of VP on photosynthesis and resistance of the photosynthetic machinery to heating in leaves of pea (Pisum sativum). Localized burning induced a VP that decreased photosynthesis parameters [CO₂ assimilation rate and quantum yields of photosystem I (PSI) and photosystem II (PSII)]. The photosynthetic response was initiated by a decrease in photosynthesis dark‐stage activity, which in turn increased resistance of PSI to heating. Three results supported this hypothesized mechanism: (1) the magnitude of VP‐induced decrease in CO₂ assimilation and enhanced PSI resistance to heating were highly correlated; (2) the VP influence on PSI resistance to heating was suppressed under a low external CO₂ concentration and (3) decreasing external CO₂ concentration imitated the VP‐induced photosynthetic response and increased PSI resistance to heating.     

Aluminum‐dependent dynamics of ion transport in Arabidopsis: specificity of low pH and aluminum responses

Low‐pH and Al³⁺ stresses are the major causes of poor plant growth in acidic soils. However, there is still a poor understanding of plant responses to low‐pH and Al³⁺ toxicity. Low‐pH or combined low‐pH and Al³⁺ stress was imposed in order to measure rhizosphere pH, ion fluxes, plasma membrane potential and intracellular H⁺ concentration in distal elongation and mature zones (MZs) along the longitudinal axis of Arabidopsis thaliana roots. Low‐pH stress facilitated H⁺ influx into root tissues and caused cytoplasmic acidification; by contrast, combined low‐pH/Al³⁺ treatment either decreased H⁺ influx in the distal elongation zone (DEZ) or induced H⁺ efflux in the MZ, leading to cytoplasmic alkalinization in both zones. Low‐pH stress induced an increase in rhizosphere pH in the DEZ, whereas combined low‐pH/Al³⁺ stress resulted in lower rhizosphere pH in both root zones compared with the low‐pH treatment alone. Low‐pH stress facilitated K⁺ efflux; the presence of Al³⁺ diminished K⁺ efflux or favored K⁺ influx into root tissues. In both zones, low‐pH treatment induced plasma membrane (PM) depolarization, which was significantly diminished (P≤ 0.05) when combined stresses (low‐pH/100 µM Al³⁺) were imposed. After 60 min of exposure, low pH caused PM depolarization, whereas low pH/100 µM Al³⁺ caused PM hyperpolarization. Thus, low pH and Al³⁺ toxicity differentially affect root tissues and, consequently, the rhizosphere, which might underpin the differential mechanisms of plant adaptation to these abiotic stresses.     

H Cotransports in Corn Roots as Related to the Surface pH Shift Induced by Active H Excretion

The surface pH shift induced by active H⁺ excretion in corn (Zea mays L.) roots was estimated using acetic acid influx as a pH probe (H Sentenac, C Grignon 1987 Plant Physiol 84: 1367-1372). At constant bulk pH, buffering the medium strongly reduced the magnitude of the surface pH shift. This was used to study the effect of surface pH shift on H⁺ cotransports. In the absence of buffers, the surface pH shift increased with the bulk pH. Buffers decreased ³²Pi influx and this effect was stronger at pH 7.2 than at pH 5.8, and stronger in the absence than in the presence of an inhibitor of the proton pump (vanadate). Buffers exerted a similar depressive and pH-dependent effect on net NO₃⁻ uptake. They hyperpolarized the cell membrane, and stimulated ⁸⁶Rb⁺ influx, K⁺:H⁺ net exchange, and malate accumulation. These results are consistent with the hypothesis that H⁺ accumulation at the cell surface is effective in driving H⁺ reentry. We concluded that the surface pH shift due to proton pump activity is involved in the energetic coupling of H⁺ cotransports.     

Simultaneous measurements of H+ and O2 fluxes in Zostera marina and its physiological implications

Zostera marina (eelgrass) is an important ecological component of many shallow, temperate lagoons. Evidence suggests that Z. marina has a high bicarbonate utilization capability, which could be promoted by possible proton extrusion and the consequent formation of an ‘acid zone’ in the apoplastic space (unstirred layer) of its leaves. It has been found that 50 mM of the buffer Tris significantly inhibited the photosynthetic O₂ evolution of Z. marina and it was proposed that this was because of Tris's ability to bond with protons outside the cell wall. To investigate if H⁺ played an important role in the photosynthetic carbon utilization of Z. marina, it is very important to simultaneously monitor the photosynthesis status and possible H⁺ fluxes. However, probably because of the lack of suitable techniques, this has never been attempted. In this study, experiments were undertaken on Z. marina by monitoring H⁺ and O₂ fluxes and the relative electron transport rates during light–dark transition. During stable photosynthesis, in addition to an obvious O₂ outflow, there was a significant net H⁺ influx connected to Z. marina photosynthesis. The inhibitory effects of both Tris and respiration inhibitors on apparent O₂ evolution of Z. marina were confirmed. However, evidence did not support the proposed Tris inhibition mechanism.     

UPTAKE OF INORGANIC CARBON BY CLADOPHORA GLOMERATA (CHLOROPHYTA) FROM THE BALTIC SEA1

Carbon uptake in the green macroalga Cladophora glomerata (L.) Kütz. from the brackish Baltic Sea was studied by recording changes in pH, alkalinity, and inorganic carbon concentration of the seawater medium during photosynthesis. The use of specific inhibitors identified three uptake mechanisms: 1) dehydration of HCO₃ ^? into CO₂ by periplasmic carbonic anhydrase, followed by diffusion of CO₂ into the cell; 2) direct uptake of HCO₃ ^? via a 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulfonate‐sensitive mechanism; and 3) uptake of inorganic carbon by the involvement of a vanadate‐sensitive P‐type H ⁺ ‐ATPase (proton pump). A decrease in the alkalinity of the seawater medium during carbon uptake, except when treated with vanadate, indicated a net uptake of the ionic species contributing to alkalinity (i.e. HCO₃ ^? , CO₃^{2 ?} , and OH ^? ) from the medium, where OH ^? influx is equivalent to H ⁺ efflux. This would suggest that the proton pump is involved in HCO₃ ^? transport. We also show that the proton pump can be induced by carbon limitation. The inducibility of carbon uptake in C. glomerata may partly explain why this species is so successful in the upper littoral zone of the Baltic Sea. Usually, carbon limitation is not a problem in the upper littoral of the sea. However, it may occur frequently within dense Cladophora belts with high photosynthetic rates that create high pH and low carbon concentrations in the alga's microenvironment.     

Sodium Dependency of the Photosynthetic Electron Transport in the Alkaliphilic Cyanobacterium Arthrospira platensis

Arthrospira (Spirulina) platensis (A. platensis) is a model organism for investigation of adaptation of photosynthetic organisms to extreme environmental conditions: the cell functions in this cyanobacterium are optimized to high pH and high concentration (150–250 mM) of Na⁺. However, the mechanism of the possible fine-tuning of the photosynthetic functions to these extreme conditions and/or the regulation of the cellular environment to optimize the photosynthetic functions is poorly understood. In this work we investigated the effect of Na-ions on different photosynthetic activities: linear electron transport reactions (measured by means of polarography and spectrophotometry), the activity of photosystem II (PS II) (thermoluminescence and chlorophyll a fluorescence induction), and redox turnover of the cytochrome b ₆ f complex (flash photolysis); and measured the changes of the intracellular pH (9-aminoacridine fluorescence). It was found that sodium deprivation of cells in the dark at pH 10 inhibited, within 40 min, all measured photosynthetic reactions, and led to an alkalinization of the intracellular pH, which rose from the physiological value of about 8.3–9.6. These were partially and totally restored by readdition of Na-ions at 2.5–25 mM and about 200 mM, respectively. The intracellular pH and the photosynthetic functions were also sensitive to monensin, an exogenous Na⁺/H⁺ exchanger, which collapses both proton and sodium gradients across the cytoplasmic membrane. These observations explain the strict Na⁺-dependency of the photosynthetic electron transport at high extracellular pH, provide experimental evidence on the alkalization of the intracellular environment, and support the hypothesized role of an Na⁺/H⁺ antiport through the plasma membrane in pH homeostasis (Schlesinger et al. (1996). J. Phycol. 32, 608–613). Further, we show that (i) the specific site of inactivation of the photosynthetic electron transport at alkaline pH is to be found at the water splitting enzyme; (ii) in contrast to earlier reports, the inactivation occurs in the dark and, for short periods, without detectable damage in the photosynthetic apparatus; and (iii) in contrast to high pH, Na⁺ dependency in the neutral pH range is shown not to originate from PSII, but from the acceptor side of PSI. These data permit us to conclude that the intracellular environment rather than the machinery of the photosynthetic electron transport is adjusted to the extreme conditions of high pH and high Na⁺ concentration.     

Cellular energization protects the photosynthetic machinery against salt-induced inactivation in Synechococcus

The effects of the energization of cells by light and by exogenous glucose on the salt-induced inactivation of the photosynthetic machinery were investigated in the cyanobacterium Synechococcus sp. PCC 7942. The incubation of the cyanobacterial cells in a medium supplemented with 0.5 M NaCl induced a rapid decline with a subsequent slow decline, in the oxygen-evolving activity of Photosystem (PS) II and in the electron-transport activity of PSI. Light and exogenous glucose each protected PSII and PSI against the second phase of the NaCl-induced inactivation. The protective effects of light and glucose were eliminated by an uncoupler of phosphorylation and by lincomycin, an inhibitor of protein synthesis. Light and glucose had similar effects on the NaCl-induced inactivation of Na⁺/H⁺ antiporters. After photosynthetic and Na⁺/H⁺-antiport activities had been eliminated by the exposure of cells to 0.5 M NaCl in the darkness, both activities were partially restored by light or exogenous glucose. This recovery was prevented by lincomycin. These observations suggest that cellular energization by either photosynthesis or respiration, which is necessary for protein synthesis, is important for the recovery of the photosynthetic machinery and Na⁺/H⁺ antiporters from inactivation by a high level of NaCl.     

Age-dependent changes of photosynthetic responses induced by electrical signals in wheat seedlings

Electrical signals in plants, namely, the action potential (AP) and variation potential (VP) alter the activity of many processes, including photosynthesis. The functional responses induced by electrical signals vary in direction and amplitude, which might be determined by variable conditions of plants prior to stimulation, by the development stage in particular. In this work, the parameters of VP-induced photosynthetic responses were analyzed at various stages of wheat seedling development. Local wounding of the second leaf in wheat plants induced the propagation of VP and altered the activity of photosynthesis at a distance from the wound location. The amplitude of VP was enlarged when the seedling age increased from 11 to 18 days. The VP-induced photosynthetic response changed with age both qualitatively and quantitatively. The amplitude of VP-induced changes in CO₂ assimilation and nonphotochemical quenching (NPQ) increased with age, which might be due to the increase in VP amplitude and associated changes in Ca²⁺ and H⁺ concentrations. The quantum yield of photosystem II photoreaction was subject to age-dependent changes: the photochemical quantum yield (γ(PSII)) was found to increase after VP in young leaves, whereas the decline in γ(PSII) was observed after the VP propagation in mature leaves. The results may explain the diversity of photosynthetic responses caused by the electrical signals.     

Cell potentials, cell resistance, and proton fluxes in corn root tissue: effects of dithioerythritol

Studies were made of the effect of dithioerythritol on net proton flux, potassium influx and efflux, cell potential, and cell resistance in fresh and washed corn (Zea mays L. WF9XM14) root tissue. Dithioerythritol induces equal proton influx and potassium efflux rates, decreases membrane resistance, and hyperpolarizes the cell potential. Greater effects on H⁺ and K⁺ fluxes are secured at pH 7 than at pH 5. Other sulfhydryl-protecting reagents produced the same responses. No evidence could be found that dithioerythritol affected energy metabolism or membrane ATPase, and proton influx was induced in the presence of uncoupling agents.     

Sucrose and proton cotransport in Ricinus cotyledons

In Ricinus cotyledons, evidence for proton extrusion came from observation of direct acidification of the medium in the presence of potassium salts. Increasing K⁺ influx with increasing pH suggested a link between K⁺ influx and H⁺ efflux by an H⁺ pump. The kinetics of K⁺ influx and H⁺ efflux were consistent with a 1:1 stoichiometry K⁺:H⁺, which may indicate either electrical coupling or carrier mediated exchange. The results were consistent with an H⁺ pump setting up an electrochemical potential gradient which provides the driving force for an H⁺-sucrose cotransport and the movement of K⁺. With reference to this, a model for phloem loading is suggested.     

Mycochromone decreases ATP-driven proton translocation in plant plasma membrane- and tonoplast-enriched vesicles

Abstract Mycochromone, a metabolite produced by Mycosphaerella rosigena, inhibits the ATP-dependent proton translocation and the ATP-generated electrical potential in pea stem tonoplast-enriched vesicles, without affecting the H⁺/K⁺ exchange induced by nigericin or an artificially imposed proton gradient. The inhibition is dependent on the time of pre-incubation and mycochromone concentration. In addition, mycochromone inhibits the ATP-dependent proton translocation in radish plasma membrane-enriched vesicles, though it does not alter ATPase activity (evaluated by hydrolysis of ATP) in either type of plant vesicle. Mycochromone seems to act on the H⁺ channels for proton translocation of the H⁺-pumping ATPase localized on plasmalemma and tonoplast, without affecting the catalytic site of ATP hydrolysis.     

Proton extrusion by leaf discs ofVicia faba L.: light- and ion-stimulated H(su+) release†

Previous electrophysiological and tracer kinetic studies indicated that the uptake of neutral amino acids took place by means of the proton cotransport mechanism in the leaf tissue of broad bean plants. The present investigations were designed to characterize the origin of the driving force for this process, and the proton pumping activity of leaf cells ofVicia. This activity is known to be revealed when peeled broad been leaf discs, floated on a bathing solution in the light or in darkness acidify the medium. White light caused the strongest acidification. The presence of K⁺ and Na⁺ in the external solution increased the H⁺ secretion significantly, whereas addition of Ca⁺⁺caused only an insignificant enhancement of proton extrusion. The inhibitors of photosynthetic electron transport DCMTJ (50 μM) and nitrofen (50 μM) eliminated the light-enhanced H⁺ release indicating the dependence on photosynthesis. The involvement of a proton pump was evidenced by the effects of the uneoupler CCCP, the SH reagent HgCl₂ and the ATPase inhibitor orthovanadate. The experimental results support the conclusion that H⁺ extrusion byVicia leaf cells is an active electrogenic process requiring metabolic energy. In the light this energy requirement is suppliedvia photosynthetic electron transport. Dedicated to Prof. Dr. F. Jacob on the occasion of his 60th birthday     

Electrochromism: a useful probe to study algal photosynthesis

In photosynthesis, electron transfer along the photosynthetic chain results in a vectorial transfer of protons from the stroma to the lumenal space of the thylakoids. This promotes the generation of an electrochemical proton gradient (Δμ _H ⁺ ), which comprises a gradient of electric potential (ΔΨ) and of proton concentration (ΔpH). The Δμ _H ⁺ has a central role in the photosynthetic process, providing the energy source for ATP synthesis. It is also involved in many regulatory mechanisms. The ΔpH modulates the rate of electron transfer and triggers deexcitation of excess energy within the light harvesting complexes. The ΔΨ is required for metabolite and protein transport across the membranes. Its presence also induces a shift in the absorption spectra of some photosynthetic pigments, resulting in the so-called ElectroChromic Shift (ECS). In this review, we discuss the characteristic features of the ECS, and illustrate possible applications for the study of photosynthetic processes in vivo.     

Fine‐tuned regulation of the K+/H+ antiporter KEA3 is required to optimize photosynthesis during induction

KEA3 is a thylakoid membrane localized K⁺/H⁺ antiporter that regulates photosynthesis by modulating two components of proton motive force (pmf), the proton gradient (?pH) and the electric potential (?ψ). We identified a mutant allele of KEA3, disturbed proton gradient regulation (dpgr) based on its reduced non‐photochemical quenching (NPQ) in artificial (CO₂‐free with low O₂) air. This phenotype was enhanced in the mutant backgrounds of PSI cyclic electron transport (pgr5 and crr2‐1). In ambient air, reduced NPQ was observed during induction of photosynthesis in dpgr, the phenotype that was enhanced after overnight dark adaptation. In contrast, the knockout allele of kea3‐1 exhibited a high‐NPQ phenotype during steady state in ambient air. Consistent with this kea3‐1 phenotype in ambient air, the membrane topology of KEA3 indicated a proton efflux from the thylakoid lumen to the stroma. The dpgr heterozygotes showed a semidominant and dominant phenotype in artificial and ambient air, respectively. In dpgr, the protein level of KEA3 was unaffected but the downregulation of its activity was probably disturbed. Our findings suggest that fine regulation of KEA3 activity is necessary for optimizing photosynthesis.     

Ascorbate accumulation during sulphur deprivation and its effects on photosystem II activity and H2 production of the green alga Chlamydomonas reinhardtii

In nature, H₂ production in Chlamydomonas reinhardtii serves as a safety valve during the induction of photosynthesis in anoxia, and it prevents the over‐reduction of the photosynthetic electron transport chain. Sulphur deprivation of C. reinhardtii also triggers a complex metabolic response resulting in the induction of various stress‐related genes, down‐regulation of photosynthesis, the establishment of anaerobiosis and expression of active hydrogenase. Photosystem II (PSII) plays dual role in H₂ production because it supplies electrons but the evolved O₂ inhibits the hydrogenase. Here, we show that upon sulphur deprivation, the ascorbate content in C. reinhardtii increases about 50‐fold, reaching the mM range; at this concentration, ascorbate inactivates the Mn‐cluster of PSII, and afterwards, it can donate electrons to tyrozin Z⁺ at a slow rate. This stage is followed by donor‐side‐induced photoinhibition, leading to the loss of charge separation activity in PSII and reaction centre degradation. The time point at which maximum ascorbate concentration is reached in the cell is critical for the establishment of anaerobiosis and initiation of H₂ production. We also show that ascorbate influenced H₂ evolution via altering the photosynthetic electron transport rather than hydrogenase activity and starch degradation.     

Ethanol-Induced Activation of ATP-Dependent Proton Extrusion in Elodea densa Leaves

In Elodea densa leaves, ethanol up to 0.17 m stimulates H⁺ extrusion activity. This effect is strictly dependent on the presence of K⁺ in the medium and is suppressed by the presence of the plasmalemma H⁺-ATPase inhibitor vanadate. Stimulation of H⁺ extrusion is associated with (a) a decrease in cellular ATP level, (b) a marked hyperpolarization of transmembrane electrical potential, and (c) an increase in net K⁺ influx. These results suggest that ethanol-induced H⁺ extrusion is mediated by an activation of the plasma membrane ATP-dependent, electrogenic proton pump. This stimulating effect is associated with an increase of cell sap pH and of the capacity to take up the weak acid 5,5-dimethyloxazolidine-2,4-dione, which is interpretable as due to an increase of cytosolic pH. This indicates that the stimulation of H⁺ extrusion by ethanol does not depend on a cytosolic acidification by products of ethanol metabolism. The similarity of the effects of ethanol and those of photosynthesis on proton pump activity in E. densa leaves suggests that a common metabolic situation is responsible for the activation of the ATP-dependent H⁺-extruding mechanism.     

The role of plasma membrane H+‐ATPase in jasmonate‐induced ion fluxes and stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) elicits stomatal closure in many plant species. Stomatal closure is accompanied by large ion fluxes across the plasma membrane (PM). Here, we recorded the transmembrane ion fluxes of H⁺, Ca²⁺ and K⁺ in guard cells of wild‐type (Col‐0) Arabidopsis, the CORONATINE INSENSITIVE1 (COI1) mutant coi1‐1 and the PM H⁺‐ATPase mutants aha1‐6 and aha1‐7, using a non‐invasive micro‐test technique. We showed that MeJA induced transmembrane H⁺ efflux, Ca²⁺ influx and K⁺ efflux across the PM of Col‐0 guard cells. However, this ion transport was abolished in coi1‐1 guard cells, suggesting that MeJA‐induced transmembrane ion flux requires COI1. Furthermore, the H⁺ efflux and Ca²⁺ influx in Col‐0 guard cells was impaired by vanadate pre‐treatment or PM H⁺‐ATPase mutation, suggesting that the rapid H⁺ efflux mediated by PM H⁺‐ATPases could function upstream of the Ca²⁺ flux. After the rapid H⁺ efflux, the Col‐0 guard cells had a longer oscillation period than before MeJA treatment, indicating that the activity of the PM H⁺‐ATPase was reduced. Finally, the elevation of cytosolic Ca²⁺ concentration and the depolarized PM drive the efflux of K⁺ from the cell, resulting in loss of turgor and closure of the stomata.     

Chloroplastic ATP synthase builds up a proton motive force preventing production of reactive oxygen species in photosystem I

Over‐reduction of the photosynthetic electron transport (PET) chain should be avoided, because the accumulation of reducing electron carriers produces reactive oxygen species (ROS) within photosystem I (PSI) in thylakoid membranes and causes oxidative damage to chloroplasts. To prevent production of ROS in thylakoid membranes the H⁺ gradient (ΔpH) needs to be built up across the thylakoid membranes to suppress the over‐reduction state of the PET chain. In this study, we aimed to identify the critical component that stimulates ΔpH formation under illumination in higher plants. To do this, we screened ethyl methane sulfonate (EMS)‐treated Arabidopsis thaliana, in which the formation of ΔpH is impaired and the PET chain caused over‐reduction under illumination. Subsequently, we isolated an allelic mutant that carries a missense mutation in the γ‐subunit of chloroplastic CF₀CF₁‐ATP synthase, named hope2. We found that hope2 suppressed the formation of ΔpH during photosynthesis because of the high H⁺ efflux activity from the lumenal to stromal side of the thylakoid membranes via CF₀CF₁‐ATP synthase. Furthermore, PSI was in a more reduced state in hope2 than in wild‐type (WT) plants, and hope2 was more vulnerable to PSI photoinhibition than WT under illumination. These results suggested that chloroplastic CF₀CF₁‐ATP synthase adjusts the redox state of the PET chain, especially for PSI, by modulating H⁺ efflux activity across the thylakoid membranes. Our findings suggest the importance of the buildup of ΔpH depending on CF₀CF₁‐ATP synthase to adjust the redox state of the reaction center chlorophyll P700 in PSI and to suppress the production of ROS in PSI during photosynthesis.