Regulation of photosynthetic electron transport and photophosphorylation in intact chloroplasts and leaves of Spinacia oleracea L.

Oxygen ist reduced by the electron transport chain of chloroplasts during CO₂ reduction. The rate of electron flow to oxygen is low. Since antimycin A inhibited CO₂-dependent oxygen evolution, it is concluded that cyclic photophosphorylation contributes ATP to photosynthesis in chloroplasts which cannot satisfy the ATP requirement of CO₂ reduction by electron flow to NADP and to oxygen. Inhibition of photosynthesis by antimycin A was more significant at high than at low light intensities suggesting that cyclic photophosphorylation contributes to photosynthesis particularly at high intensities. Cyclic electron flow in intact chloroplasts is under the control of electron acceptors. At low light intensities or under far-red illumination it is decreased by substrates which accept electrons from photosystem I such as oxaloacetate, nitrite or oxygen. Obviously, the cyclic electron transport pathway is sensitive to electron drainage. In the absence of electron acceptors, cyclic electron flow is supported by far-red illumination and inhibited by red light. The inhibition by light exciting photosystem II demonstrated that the cyclic electron transport pathway is accessible to electrons from photosystem II. Inhibition can be relieved by oxygen which appears to prevent over-reduction of electron carriers of the cyclic pathway and thus has an important regulatory function. The data show that cyclic electron transport is under delicate redox control. Inhibition is caused both by excessive oxidation and by over-reduction of electron carriers of the pathway.     

Oxygen requirement of photosynthetic CO2 assimilation

In the absence of electron acceptors and of oxygen a proton gradient was supported across thylakoid membranes of intact spinach chloroplasts by far-red illumination. It was decreased by red light. Inhibition by red light indicates effective control of cyclic electron flow by Photosystem II. Inhibition was released by oxygen which supported a large proton gradient. Oxygen appeared to act as electron acceptor simultaneously preventing over-reduction of electron carriers of the cyclic electron transport pathway. It thus has an important regulatory function in electron transport. Under anaerobic conditions, the inhibition of electron transport caused by red illumination could also be released and a large proton gradient could be established by oxaloacetate, nitrite and 3-phosphoglycerate, but not by bicarbonate. In the absence of oxygen, ATP levels remained low in chloroplasts illuminated with red light even when bicarbonate was present. They increased when electron acceptors were added which could release the over-reduction of the electron transport chain. Inhibition of electron transport in the presence of bicarbonate was relieved and CO₂-fixation was initiated by oxygen concentrations as low as about 10 μM. Once CO₂ fixation was initiated, very low oxygen levels were sufficient to sustain it. The results support the assumption that pseudocyclic electron transport is necessary to poise the electron transport chain so that a proper balance of linear and cyclic electron transport is established to supply ATP for CO₂ reduction.     

Changes in electron transport,superoxide dismutase and ascorbate peroxidase isoenzymes in chloroplasts and mitochondria of cucumber leaves as influenced by chilling

In order to clarify the relationship between chill-induced disturbance in photosynthetic, respiratory electron transport and the metabolism of reactive oxygen species (ROS), leaf gas exchange, chlorophyll fluorescence quenching, respiration, and activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) were investigated in chloroplasts and mitochondria of cucumber (Cucumis sativus) leaves subjected to a chill (8 °C) for 4 d. Chilling decreased net photosynthetic rate (P _N) and quantum efficiency of photosystem 2 (Φ_PS2), but increased the ratio of Φ_PS2 to the quantum efficiency of CO₂ fixation (ΦCO₂) and non-photochemical quenching (NPQ) in cucumber leaves. While chilling inhibited the activity of cytochrome respiration pathway, it induced an increase of alternative respiration pathway activity and the reduction level of Q-pool. Chilling also significantly increased O₂ ^• production rate, H₂O₂ content, and SOD and APX activities in chloroplasts and mitochondria. There was a more significant increase in SOD and APX activities in chloroplasts than in mitochondria with the increase of membrane-bound Fe-SOD and tAPX in chloroplasts being more significant than other isoenzymes. Taken together, chilling inhibited P _N and cytochrome respiratory pathway but enhanced the photosynthetic electron flux to O₂ and over-reduction of respiratory electron transport chain, resulting in ROS accumulation in cucumber leaves. Meanwhile, chilling resulted in an enhancement of the protective mechanisms such as thermal dissipation, alternative respiratory pathway, and ROS-scavenging mechanisms (SODs and APXs) in chloroplasts and mitochondria.     

Electron flow accompanying the ascorbate peroxidase cycle in maize mesophyll chloroplasts and its cooperation with linear electron flow to NADP+ and cyclic electron flow in thylakoid membrane energization

Potential roles for cyclic and pseudocyclic electron flow in C₄ plants are to provide ATP for the C₄ cycle and, under excess light, to down-regulate PS II activity through membrane energization. Intact mesophyll chloroplasts of maize were used to evaluate forms of electron transport including the Mehler peroxidase reaction (linear electron flow to O₂, formation of H₂O₂ which is reduced by ascorbate, and linear flow linked to reduction of oxidized ascorbate). Addition of H₂O₂ to isolated chloroplasts in the light in the presence of an uncoupler induced Photosystem (PS) II activity, as determined from increases in photochemical quenching of chlorophyll fluorescence (q_p) and the quantum yield of PS II. H₂O₂ also induced dissipation of energy by thylakoid membrane energization and non-photochemical fluorescence quenching (q_n), which was inhibited by addition of an uncoupler. These effects of H₂O₂ on q_p and q_n were inhibited by addition of KCN, an inhibitor of ascorbate peroxidase. The results suggest that H₂O₂ is reduced via ascorbate, and that the oxidized ascorbate is then reduced by linear electron flow contributing to photochemistry and thylakoid membrane energization. Evidence for function of pseudocyclic electron flow via the Mehler peroxidase reaction was obtained with only oxygen as an electron acceptor, as well as in the presence of oxaloacetate a natural electron acceptor in C₄ photosynthesis. KCN decreased q_p and PS II yield in the absence and presence of oxaloacetate and, in the former case, it severely reduced q_{_n}. KCN also decreased pH formation across the thylakoid membrane based on its decrease in the light-induced quenching of 9-aminoacridine fluorescence, particularly in the absence of oxaloacetate. Antimycin A, an inhibitor of cyclic electron flow, also diminished pH formation. These results provide evidence for shared energization of thylakoid membranes by the Mehler peroxidase reaction, cyclic electron flow, and linear electron flow linked to the C₄ pathway.     

Evidence for Cyclic Photophosphorylation during CO(2) Fixation in Intact Chloroplasts: Studies with Antimycin A, Nitrite, and Oxaloacetate

This study examines the effect of antimycin A and nitrite on ¹⁴CO₂ fixation in intact chloroplasts isolated from spinach (Spinacia oleracea L.) leaves. Antimycin A (2 micromolar) strongly inhibited CO₂ fixation but did not appear to inhibit or uncouple linear electron transport in intact chloroplasts. The addition of small quantities (40-100 micromolar) of nitrite or oxaloacetate, but not NH₄Cl, in the presence of antimycin A restored photosynthesis. Antimycin A inhibition, and the subsequent restoration of photosynthetic activities by nitrite or oxaloacetate, was observed over a wide range of CO₂ concentration, light intensity, and temperature. High O₂ concentration (up to 240 micromolar) did not appear to influence the extent of the inhibition by antimycin A, nor the subsequent restoration of photosynthetic activity by nitrite or oxaloacetate. Studies of O₂ exchanges during photosynthesis in cells and chloroplasts indicated that 2 micromolar antimycin A stimulated O₂ uptake by about 25% while net O₂ evolution was inhibited by 76%. O₂ uptake in chloroplasts in the presence of 2 micromolar antimycin A was 67% of total O₂ evolution. These results suggest that only a small proportion of the O₂ uptake measured was directly linked to ATP generation. The above evidence indicates that cyclic photophosphorylation is the predominant energy-balancing reaction during photosynthesis in intact chloroplasts. On the other hand, pseudocyclic O₂ uptake appears to play only a minimal role.     

Flexible coupling between light-dependent electron and vectorial proton transport in illuminated leaves of C3 plants. Role of photosystem I-dependent proton pumping

The role of cyclic electron transport has been re-examined in leaves of C₃ plants because the bioenergetics of chloroplasts (H⁺/e = 3 in the presence of a Q-cycle; H⁺/ATP = 4 of ATP synthesis) had suggested that cyclic electron flow has no function in C₃ photosynthesis. After light activation of pea leaves, the dark reduction of P700 (the donor pigment of PSI) following far-red oxidation was much accelerated. This corresponded to loss of sensitivity of P700 to oxidation by far-red light and a large increase in the number of electrons available to reduce P700⁺ in the dark. At low CO₂ and O₂ molar ratios, far-red light was capable of decreasing the activity of photosystem II (measured as the ratio of variable to maximal chlorophyll fluorescence, F_v/F_m) and of increasing light scattering at 535 nm and zeaxanthin synthesis, indicating formation of a transthylakoid pH gradient. Both the light-induced increase in the number of electrons capable of reducing far-red-oxidised P700 and the decline in F_v/F_m brought about by far-red in leaves were prevented by methyl viologen. Antimycin A inhibited CO₂-dependent O₂ evolution of pea leaves at saturating but not under limiting light; in its presence, far-red light failed to decrease F_v/F_m. The results indicate that cyclic electron flow regulates the quantum yield of photosystem II by decreasing the intrathylakoid pH when there is a reduction in the availability of electron acceptors at the PSI level (e.g. during drought or cold stresses). It also provides ATP for the carbon-reduction cycle under high light. Under these conditions, the Q-cycle is not able to maintain a H⁺/e ratio of 3 for ATP synthesis: we suggest that the ratio is flexible, not obligatory. Received: 23 February 1999 / Accepted: 19 August 1999     

Energetic and regulatory role of proton potential in chloroplasts

The review focuses on the energetic and regulatory role of proton potential in the activity of chloroplasts, the light energy-converting organelles of plant cells. Mechanisms of generation of the transmembrane difference of electrochemical potentials of hydrogen ions in the chloroplast thylakoid membranes are considered. Methods for measuring the intrathylakoid pH in chloroplasts are described. It is shown that under conditions of phosphorylation in chloroplasts, the pH of the intrathylakoid space decreases moderately (pH_in ⩾ 6.0–6.2, at the stroma pH_out ∼ 7.8–8.0), with a corresponding concentration component of equal to ΔpH ⩽ 1.6–2.0. On analyzing the energy and structural features of ATP synthase of chloroplasts, we conclude that the energy stored as the concentration component of the proton potential ΔpH is sufficient to sustain ATP synthesis. The mechanisms of pH-dependent regulation of electron transport in chloroplasts (photosynthetic control of electron transport, enhancement of non-photochemical quenching of chlorophyll excitation in the light-harvesting antenna, light-induced activation of the Calvin-Benson cycle reactions, activation of ATP synthase) are considered briefly.     

Formation of Hydrogen Peroxide and Oxygen Dependence of Photosynthetic CO2 Assimilation by Intact Chloroplasts

1. Hydrogen peroxide excretion by photosynthesizing intact spinachchloroplasts was determined. The rates were dependent on theoxygen concentration and on the ATP/NADPH requirement of thefinal electron acceptor. Upon CO₂ assimilation a maximum rateof 0.9 µmol H₂O₂/mg chlorophyll/hr and half saturationat 7.5 x 10^–5 M O₂ were found. Excretion of H₂O₂ was considerablyreduced upon photosynthetic reduction of glycerate 3-phosphateor oxaloacetate.
2. Light- and HCO₃-saturated CO₂ assimilationwas inhibited bymore than 50% by anaerobic conditions, whereuponquantum efficiencywas also drastically decreased. However,no anoxic influencewas detected with glycerate 3-phosphateas the terminal electronacceptor and the quantum requirementwith this acceptor wasnot increased by anaerobiosis. Thus theenhancing effect ofoxygen on CO₂ assimilation was ascribedto an improvement ofphotosynthetic ATP supply.
3. Since thestimulation of anaerobic photosynthetic CO₂ assimilationbyoxygen was markedly greater than the concomitant increaseinH₂O₂ evolution, photosynthetic oxygen reduction alone isnotsufficient to produce the required additional ATP for theobservedenhanced CO₂ assimilation. But it provides a meansto avoidthe over-reduction of photosynthetic electron carriersand thusenables aerobic cyclic photophosphorylation. This supportsthehypothesis that cyclic photophosphorylation is not an alternativeto ATP formation by "pseudocyclic" electron transport, but ratherthat it depends on the latter.

(Received January 5, 1981; Accepted March 9, 1981)     

Electron transport and transmembrane proton transfer in photosynthetic systems of oxygenic type in Silico

Using a mathematical model of light-induced stages of photosynthesis, which takes into account the key stages of pH-dependent regulation on the acceptor and donor sides of PS I, we analyzed electron and proton transport in chloroplasts of higher plants and in cyanobacterial cells. A comparison of computer simulations with experimental data showed that our model adequately described the complex nonmonotonic kinetics of the light-induced redox transients of P₇₀₀. Effects of atmospheric gases (CO₂ and O₂) on the kinetics of photooxidation of P₇₀₀ and generation of the transmembrane pH difference were studied. We also analyzed how cyclic electron transport influenced the kinetics of electron transfer, intrathylakoid pH, and ATP production. Within the framework of our model, we described the time courses of electron flow through PS II and distribution of electron fluxes on the acceptor side of PS I in chloroplasts and in cyanobacteria. It was demonstrated that contributions of cyclic electron transport and electron flow to O₂ (the Mehler reaction) were significant during the initial phase of the induction period, but diminished upon activation of the Calvin-Benson cycle.     

Co-operative activation of chloroplast fructose-1,6-bisphosphatase by reductant,pH and substrate

Intact chloroplasts capable of high rates of photosynthesis fail to reduce CO₂ when illuminated in the absence of oxygen. While anaerobiosis limits proton gradient formation leading to ATP deficiency (Ziem-Hanck, U. and Heber, U. (1980) Biochim. Biophys. Acta 591, 266–274), light activation of fructose-1,6-bisphosphatase was also inhibited by anaerobiosis, whereas light activation of NADP-malate dehydrogenase was stimulated by anaerobiosis, indicating that reductant was still available for light activation. The chloroplast pool of NADP was largely reduced during illumination under anaerobiosis and electron transport to oxaloacetate was not inhibited by anaerobic conditions. Significant light activation of fructose-bisphosphatase was observed in anaerobic chloroplasts with 3-phosphoglycerate as substrate, but not with dihydroxyacetone phosphate (3-phosphoglycerate supports electron transport and hence proton gradient formation). In the absence of added substrates, illumination of anaerobic chloroplasts resulted in some light activation of fructose-bisphosphatase when the pH of the medium was increased. Under these conditions, light activation was stimulated by dihydroxyacetone phosphate. Dihydroxyacetone phosphate added together with oxaloacetate allowed light activation of fructose-bisphosphatase in anaerobic chloroplasts, while neither substrate added alone was effective. Formation of a transthylakoid proton gradient can therefore substitute for an alkaline suspension medium by causing an alkaline shift of the stromal pH on illumination. The data are interpreted as indicating that fructose-bisphosphatase, but not NADP-malate dehydrogenase, requires an alkaline pH and the presence of substrate for rapid reductive light activation and they bear on the interpretation of the lag observed in photosynthesis in chloroplasts and leaves on illumination after a prolonged dark period.     

Effect of Oxygen on Photosynthetic 14CO2 Fixation in Chroomonas sp. (Cryptophyta) II. Effects of Inhibitors, Uncouplers and an Artificial Electron Mediator on the Inhibition of 14CO2 Fixation by Anaerobiosis

In "air-grown" Chroomonas sp. cells, low concentrations of DCMU(less than 0.1 µM) could prevent the inhibition of ¹⁴CO₂fixation by anaerobiosis under light-saturating conditions (morethan 40 W.m^–2), with phenazine methosulfate showing asimilar effect. Antimycin A, carbonyl cyanide m-chlorophenylhydrazone(CCCP), and N,N'-dicyclohexylcarbodiimide strongly inhibitedanaerobic photosynthesis at concentrations which did not significantlyinhibit the rate under 2% O₂ at high light intensity (200 W.m^–2),although 0.2 µM CCCP stimulated the rate under 2% O₂ tosome extent. On the other hand, KCN inhibited the rate muchmore strongly under 2% O₂ than N₂, although it inhibited therate very strongly at concentrations above 5 µM both underN₂ and 2% O₂. These results suggest that the inhibition of photosynthetic¹⁴CO₂ fixation by anaerobiosis in this alga result from ATPdeficiency caused by over-reduction of electron carriers ofthe cyclic electron flow and that oxygen can prevent the over-reduction.Cyclic electron flow seems to be necessary to provide additionalATP for CO₂ reduction under anaerobic conditions, although itseems to be less necessary under aerobic conditions. (Received July 21, 1983; Accepted January 23, 1984)     

The role of chloroplast NAD(P)H dehydrogenase in protection of tobacco plant against heat stress

The environmental temperature is one of the mainfactors affecting plant growth and development. Insummer, plants are frequently influenced by hightemperature. In recent years, global temperature wasremarkably elevated accompanied with the climaticchanges,…     

In vivo temperature dependence of cyclic and pseudocyclic electron transport in barley

The effect of temperature on the rate of electron transfer through photosystems I and II (PSI and PSII) was investigated in leaves of barley (Hordeum vulgare L.). Measurements of PSI and PSII photochemistry were made in 21% O₂ and in 2% O₂, to limit electron transport to O₂ in the Mehler reaction. Measurements were made in the presence of saturating CO₂ concentrations to suppress photorespiration. It was observed that the O₂ dependency of PSII electron transport is highly temperature dependent. At 10 °C, the quantum yield of PSII (ΦPSII) was insensitive to O₂ concentration, indicating that there was no Mehler reaction operating. At high temperatures (>25 °C) a substantial reduction in ΦPSII was observed when the O₂ concentration was reduced. However, under the same conditions, there was no effect of O₂ concentration on the ΔpH-dependent process of non-photochemical quenching. The rate of electron transport through PSI was also found to be independent of O₂ concentration across the temperature range. We conclude that the Mehler reaction is not important in maintaining a thylakoid proton gradient that is capable of controlling PSII activity, and present evidence that cyclic electron transport around PSI acts to maintain membrane energisation at low temperature. Received: 6 July 2000 / Accepted: 3 August 2000     

The role of chloroplast NAD(P)H dehydrogenase in protection of tobacco plant against heat stress

After incubation at 42°C for more than 48 h, brown damages occurred on the stems of tobacco (Nicotiana tabacum L.) ndhC-ndhK-ndhJ deletion mutant (ΔndhCKJ), followed by wilt of the leaves, while less the phenotype was found in its wild type (WT). Analysis of the kinetics of post-illumination rise in chlorophyll fluorescence indicated that the PSI cyclic electron flow and the chlororespiration mediated by NAD(P)H dehydrogenase (NDH) was significantly enhanced in WT under the high temperature. After leaf disks were treated with methyl viologen (MV), photosynthetic apparatus of ΔndhCKJ exhibited more severe photo-oxidative damage, even bleaching of chlorophyll. Analysis of P700 oxidation and reduction showed that the NDH mediated cyclic electron flow probably functioned as an electron competitor with Mehler reaction, to reduce the accumulation of reactive oxygen species (ROS). When leaf disks were heat stressed at 42°C for 6 h, the photochemical activity declined more markedly in ΔndhCKJ than in WT, accompanied with more evident decrease in the amount of soluble Rubisco activase. In addition, the slow phase of millisecond-delayed light emission (ms-DLE) of chlorophyll fluorescence indicated that NDH was involved in the building-up of transthylakoid proton gradient (ΔpH), while the consumption of ΔpH was highly inhibited in ΔndhCKJ after heat stress. Based on the results, we supposed that the cyclic electron flow mediated by NDH could be stimulated under the heat stressed conditions, to divert excess electrons via chlororespiration pathway, and sustain CO₂ assimilation by providing extra ΔpH, thus reducing the photooxidative damage.     

Regulation of the photosynthetic electron transport chain

The regulation of electron transport between photosystems II and I was investigated in the plant Silene dioica L. by means of measurement of the kinetics of reduction of P₇₀₀ following a light-to-dark transition. It was found that, in this species, the rate constant for P₇₀₀ reduction is sensitive to light intensity and to the availability of CO₂. The results indicated that at 25 °C the rate of electron transport is down-regulated by approximately 40–50% relative to the maximum rate achievable in saturating CO₂ and that this down-regulation can be explained by regulation of the electron transport chain itself. Measurements of the temperature sensitivity of this rate constant indicated that there is a switch in the rate-limiting step that controls electron transport at around 20 °C: at higher temperatures, CO₂ availability is limiting; at lower temperatures some other process regulates electron transport, possibly a diffusion step within the electron transport chain itself. Regulation of electron transport also occurred in response to drought stress and sucrose feeding. Measurements of non-photochemical quenching of chlorophyll fluorescence did not support the idea that electron transport is regulated by the pH gradient across the thylakoid membrane, and the possibility is discussed that the redox potential of a stromal component may regulate electron transport. Received: 4 March 1999 / Accepted: 25 May 1999     

Regulation of proton-to-electron stoichiometry in photosynthetic electron transport: physiological function in photoprotection

The primary stable products of photosynthetic electron flow are NADPH and ATP. Stoichiometry of their production depends on the ratio of protons pumped across the thylakoid membrane to electrons passed through the electron transport pathway (H⁺/e⁻ ratio). Flexible requirements of the ATP/NADPH ratio by various assimilatory reactions in chloroplasts must be fulfilled by the H⁺/e⁻ ratio during the electron flow. In addition to the well-known role of ΔpH during ATP synthesis, ΔpH also functions as a trigger of the down-regulation of photosystem II (PSII) photochemistry. Excessive light energy is safely dissipated as heat by this regulatory process to suppress the generation of toxic reactive oxygen species. Thus, regulation of the H⁺/e⁻ ratio may function in the photoprotection, as well as in the regulation of the ATP/NADPH production ratio. It has long been the consensus that the H⁺/e⁻ ratio can be controlled by regulating the proton-transporting Q-cycle in the cytochrome b ₆ f complex and by the cyclic electron flow around photosystem I (PSI). Despite the possible physiological importance and the long history of interest, the molecular identity of Q-cycle regulation and the cyclic electron flow around PSI have been remained unclear. The recent improvements in research tools, including the genetic approach using chlorophyll fluorescence imaging and establishment of the chloroplast transformation technique, are providing new insights into classical topics. In this review, we focus on regulation of the H⁺/e⁻ ratio especially from the view of photosynthetic regulation. Received: August 2, 2001 / Accepted: October 1, 2001     

Characterization of an Electron Transport Pathway Associated with Glucose and Fructose Respiration in the Intact Chloroplasts of Chlamydomonas reinhardtii and Spinach

The role of an electron transport pathway associated with aerobic carbohydrate degradation in isolated, intact chloroplasts was evaluated. This was accomplished by monitoring the evolution of ¹⁴CO₂ from darkened spinach (Spinacia oleracea) and Chlamydomonas reinhardtii chloroplasts externally supplied with [¹⁴C]fructose and [¹⁴C]glucose, respectively, in the presence of nitrite, oxaloacetate, and conventional electron transport inhibitors. Addition of nitrite or oxaloacetate increased the release of ¹⁴CO₂, but it was shown that O₂ continued to function as a terminal electron acceptor. ¹⁴CO₂ evolution was inhibited up to 30 and 15% in Chlamydomonas and spinach, respectively, by 50 μm rotenone and by amytal, but at 500- to 1000-fold higher concentrations, indicating the involvement of a reduced nicotinamide adenine dinucleotide phosphate-plastoquinone oxidoreductase. ¹⁴CO₂ release from the spinach chloroplast was inhibited 80% by 25 μm 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. ¹⁴CO₂ release was sensitive to propylgallate, exhibiting approximately 50% inhibition in Chlamydomonas and in spinach chloroplasts of 100 and 250 μm concentrations, respectively. These concentrations were 20- to 50-fold lower than the concentrations of salicylhydroxamic acid (SHAM) required to produce an equivalent sensitivity. Antimycin A (100 μm) inhibited approximately 80 to 90% of ¹⁴CO₂ release from both types of chloroplast. At 75 μm, sodium azide inhibited ¹⁴CO₂ evolution about 50% in Chlamydomonas and 30% in spinach. Sodium azide (100 mm) combined with antimycin A (100 μm) inhibited ¹⁴CO₂ evolution more than 90%. ¹⁴CO₂ release was unaffected by uncouplers. These results are interpreted as evidence for a respiratory electron transport pathway functioning in the darkened, isolated chloroplast. Chloroplast respiration defined as ¹⁴CO₂ release from externally supplied [1-¹⁴C]glucose can account for at least 10% of the total respiratory capacity (endogenous release of CO₂) of the Chlamydomonas reinhardtii cell.     

Control of electron flow in intact chloroplasts by the intrathylakoid pH,not by the phosphorylation potential

In the presence of nitrite or oxaloacetate, intact chloroplasts evolved oxygen at a significant rate for the initial 1 to 2 min of illumination. Subsequently, oxygen evolution was suppressed progressively. The suppressed oxygen evolution was stimulated strikingly by NH₄Cl. The results indicate that coupled electron flow in intact chloroplasts is controlled in the light, and the control is released by NH₄Cl. However, at low concentrations, NH₄Cl was not an effective uncoupler of photophosphorylation in intact chloroplasts. Intrachloroplast ATP levels and ATP/ADP ratios were not significantly influenced by NH₄Cl. In contrast, the quenching of 9-aminoacridine fluorescence, which can be used to indicate the intrathylakoid pH in intact chloroplasts, was reduced drastically even by low concentrations of NH₄Cl. This suggests that the chloroplast phosphorylation potential is not in equilibrium with the proton gradient. In coupled chloroplasts, the intrathylakoid pH was lower in the light with nitrite than with oxaloacetate as electron acceptor. Electron flow was also more effectively controlled in chloroplasts illuminated with nitrite than with oxaloacetate. It is concluded that the intrathylakoid pH, not the phosphorylation potential, is a factor in the control of the rate of electron flow in intact chloroplasts.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - OAA oxalo-acetate - MES 2-(N-morpholino)-ethanesulfonic acid - HEPES N-2-hyroxyethylpiperazine-N-2-ethanesulfonic acid Postal address     

pH regulation in apoplastic and cytoplasmic cell compartments of leaves

 The regulation of pH in the apoplast, cytosol and chloroplasts of intact leaves was studied by means of fluorescent pH indicators and as a response of photosynthesis to acid stress. The apoplastic pH increased under anaerobiosis. Aeration reversed this effect. Apoplastic responses to CO₂, HCl or NH₃ differed considerably. Whereas HCl and ammonia caused rapid acidification or alkalinization, the return to initial pH values was slow after cessation of fumigation. Addition of CO₂ either did not produce the acidification expected on the basis of known apoplastic buffering or even caused some alkalinization. Removal of CO₂ shifted the apoplastic pH into the alkaline range before the pH returned to initial steady-state levels. In the presence of vanadate, the alkaline shift was absent and the apoplastic pH returned slowly to the initial level when CO₂ was removed from the atmosphere. In contrast to the response of the apoplast, anaerobiosis acidified the cytosol or, in some species, had little effect on its pH. Acidification was rapidly reversed upon re-admission of oxygen. The CO₂-dependent pH changes were very fast in the cytosol. Considerable alkalinization was observed after removal of CO₂ under aerobic, but not under anaerobic conditions. Rates of the re-entry of protons into the cytosol during recovery from CO₂ stress increased in the presence of oxygen with the length of previous exposure to high CO₂. Effective pH regulation in the chloroplasts was indicated by the recovery of photosynthesis after the transient inhibition of photosynthetic electron flow when CO₂ was increased from 0.038% to 16% in air. As photosynthesis became inhibited under high CO₂, reduction of the electron transport chain increased transiently. The time required for recovery of photosynthesis from inhibition during persistent CO₂ stress was similar to the time required for establishing steady-state pH values in the cytosol under acid stress. The high capacity of leaf cells for the rapid re-attainment of pH homeostasis in the apoplast and the cytoplasm under acid or alkaline stress suggested the rapid activation or deactivation of membrane-localised proton-transporting enzymes and corresponding ion channel regulation for co-transport of anions or counter-transport of cations together with proton fluxes. Acidification of the cytoplasm appeared to activate energy-dependent proton export primarily into the vacuoles whereas apoplastic alkalinization resulted in the pumping of protons into the apoplast. Proton export rates from the cytosol into the apoplast after anaerobiosis were about 100 nmol (m² leaf area)⁻¹ s⁻¹ or less. Proton export under acid stress into the vacuole was about 1200 nmol m⁻² s⁻¹. The kinetics of pH responses to the addition or withdrawal of CO₂ indicated the presence of carbonic anhydrase in the cytosol, but not in the apoplast. Received: 19 July 1999 / Accepted: 29 December 1999     

Energy charge,phosphorylation potential and proton motive force in chloroplasts

Adenylate concentrations were measured in intact chloroplasts under a variety of conditions. Energy charge was significant in the dark and increased in the light, but remained far below values expected from observed phosphorylation potentials in broken chloroplasts, which were 80 000 M^?1 or more in the light. With nitrite as electron acceptor, phosphorylation potentials in intact chloroplasts were about 80 M^?1 in the dark and only 300 M^?1 in the light. Similar phosphorylation potentials were observed, when oxaloacetate, phosphoglycerate or bicarbonate were used as substrates. ΔG′_ATP was ?42 kJ/mol in darkened intact chloroplasts, ?46 kJ/mol in illuminated intact chloroplasts and ?60 kJ/mol in illuminated broken chloroplasts. Uncoupling by NH₄Cl, which stimulated electron transport to nitrite or oxaloacetate and decreased the proton gradient, failed to decrease the phosphorylation potential of intact chloroplasts. Also, it did not increase the quantum requirement of CO₂ reduction. It is concluded that the proton motive force as conventionally measured and phosphorylation potentials are far from equilibrium in intact chloroplasts. The insensitivity of CO₂ reduction and of the phosphorylation potential to a decrease in the proton motive force suggests that intact chloroplasts are over-energized even under low intensity illumination. However, such a conclusion is at variance with available data on the magnitude of the proton motive force.