CO2 reduction by intact chloroplasts under a diminished proton gradient

9-Aminoacridine has been used to monitor the intrathylakoid pH of photo-synthetically competent intact chloroplasts. Values obtained from 9-aminoacridine accumulation in the chloroplasts must be corrected for light-dependent binding of 9-aminoacridine to the thylakoid membranes. During nitrite reduction by intact chloroplasts, the intrathylakoid proton concentration increased. It decreased somewhat during CO₂ reduction. However, low concentrations of uncoupling amines such as NH₃ or cyclohexylamine, which rapidly penetrated the chloroplast envelope and decreased the intrathylakoid proton concentration, failed to reduce, and actually stimulated, rates of CO₂-dependent oxygen evolution even under rate-limiting light. In contrast, low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or nigericin, which inhibited CO₂ reduction, even appeared to increase the intrathylakoid proton concentration. As indicated by measurements of the 515 nm signal of the chloroplasts, the light-induced membrane potential was not much affected by low concentrations of the uncoupling amines, but was decreased by FCCP and by high concentrations of the amines. Even in the presence of high concentrations of NH₄Cl, ATP/ADP ratios of illuminated chloroplasts remained far above the ratios observed in the dark. In contrast, low concentrations of FCCP were sufficient to reduce ATP/ADP ratios to the dark value even under high intensity illumination. The observations are difficult to explain within the framework of the chemiosmotic hypothesis as presently discussed.     

Rates and properties of endogenous cyclic photophosphorylation of isolated intact chloroplasts measured by CO2 fixation in the presence of dihydroxyacetone phosphate

1. Dihydroxyacetone phosphate in concentrations ? 2.5 mM completely inhibits CO₂-dependent O₂ evolution in isolated intact spinach chloroplasts. This inhibition is reversed by the addition of equimolar concentrations of P_i, but not by addition of 3-phosphoglycerate. In the absence of P_i, 3-phosphoglycerate and dihydroxyacetone phosphate, only about 20% of the ¹⁴C-labelled intermediates are found in the supernatant, whereas in the presence of each of these substances the percentage of labelled intermediates in the supernatant is increased up to 70–95%. Based on these results the mechanism of the inhibition of O₂ evolution by dihydroxyacetone phosphate is discussed with respect to the function of the known phosphate translocator in the envelope of intact chloroplasts.2. Although O₂ evolution is completely suppressed by dihydroxyacetone phosphate, CO₂ fixation takes place in air with rates of up to 65μ mol · mg^?1 chlorophyll · h^?1. As non-cyclic electron transport apparently does not occur under these conditions, these rates must be due to endogenous pseudocyclic and/or cyclic photophosphorylation.3. Under anaerobic conditions, the rates of CO₂ fixation in presence of dihydroxyacetone phosphate are low (2.5–7 μmol · mg^?1 chlorophyll · h^?1), but they are strongly stimulated by addition of dichlorophenyl-dimethylurea (e.g. 2 · 10^?7 M) reaching values of up to 60 μmol · mg^?1 chlorophyll · h^?1. As under these conditions the ATP necessary for CO₂ fixation can be formed by an endogenous cyclic photophosphorylation, the capacity of this process seems to be relatively high, so it might contribute significantly to the energy supply of the chloroplast. As dichlorophenyl-dimethylurea stimulates CO₂ fixation in presence of dihydroxyacetone phosphate under anaerobic but not under aerobic conditions, it is concluded that only under anaerobic conditions an “overreduction” of the cyclic electron transport system takes place, which is removed by dichlorophenyl-dimethylurea in suitable concentrations. At concentrations above 5 · 10^?7 M dichlorophenyl-dimethylurea inhibits dihydroxyacetone phosphate-dependent CO₂ fixation under anaerobic as well as under aerobic conditions in a similar way as normal CO₂ fixation. Therefore, we assume that a properly poised redox state of the electron transport chain is necessary for an optimal occurrence of endogenous cyclic photophosphorylation.4. The inhibition of dichlorophenyl-dimethylurea-stimulated CO₂ fixation in presence of dihydroxyacetone phosphate by dibromothymoquinone under anaerobic conditions indicates that plastoquinone is an indispensible component of the endogenous cyclic electron pathway.     

Fructose-and sedoheptulosebisphosphatase. The sites of a possible control of CO2 fixation by light-dependent changes of the stromal Mg2+ concentration

1. The enzymatic steps of the CO₂ fixation cycle responsible for the overall inhibition of CO₂ fixation caused by the lowering of the Mg²⁺ concentration in the stroma were investigated. For this the Mg²⁺ concentration in the stroma was decreased by addition of the ionophore A 23187, and the levels of the intermediates of the CO₂ fixation cycle in the stroma of intact chloroplasts were assayed by ion exchange chromatography.2. The addition of the ionophore caused an increase of NADPH, ATP, fructose- and sedoheptulosebisphosphate and a dramatic decrease of phosphoglycerate in the stroma. These changes were reversed by the addition of Mg²⁺ and again affected by a subsequent addition of Ca²⁺. Ribulosebisphosphate and pentosemonophosphate levels in the stroma were only a little affected under these different conditions.3. The increase of the NADPH and ATP reflects the decreased utilization of these compounds due to the overall inhibition of CO₂ fixation. As phosphoglycerate and triosephosphate appear to be in near equilibrium with NADPH and ATP, the decrease of phosphoglycerate seems to be a consequence of the changes in the nucleotide levels.4. The rapid increase of fructose- and sedoheptulosebisphosphate after the addition of the ionophore A 23187 clearly demonstrates that the overall inhibition of CO₂ fixation caused by lowering the stromal Mg²⁺ is due to the inhibition of the hydrolysis of these sugar bisphosphates. It is concluded that the activities of fructose- and sedoheptulosebisphosphatase can be controlled by light dependent changes of the stromal Mg²⁺ concentration.     

CO2 dark fixation in the halophytic species mesembryanthemum crystallinum

The time course of ¹⁴CO₂ dark fixation was studied in leaves of the facultatively halophytic plant species Mesembryanthemum crystallinum cultivated with and without 400 mM NaCl in the nutrient medium. It is generally known from the literature that plants grown under saline conditions incorporate ¹⁴C predominately into amino acids. By contrast in leaves of M. crystallinum grown on NaCl and exposed to ¹⁴CO₂ in the dark, relatively more radioactivity is incorporated in the organic acids (especially malate) than in amino acids. The data obtained are discussed in relation to the NaCl induced Crassulacean acid metabolism in M. crystallinum reported earlier.     

Photosynthetic CO2 fixation in spinach chloroplasts inhibited by pine chloroplasts or extracts of pine chloroplasts

Chloroplasts isolated from pine needles were found to be inactive with respect to CO₂ fixation. Since it was suspected that pine needles may contain substances inhibitory to photosynthesis, studies were carried out using photosynthetically active isolated spinach chloroplasts and chloroplasts isolated from pine needles. When isolated pine chloroplasts were suspended in buffer and were added to isolated spinach chloroplasts they inhibited photosynthetic CO₂ fixation. When the pine chloroplasts were separated from the medium by centrifugation, the separated pine chloroplasts severely inhibited CO₂ fixation by isolated spinach chloroplasts, but the supernatant solution from the pine chloroplasts was not inhibitory. As little as 5% pine chloroplasts (based on chlorophyll content) produced 50% inhibition of CO₂ fixation by the spinach chloroplasts. Studies of fixation of ¹⁴C-labelled CO₂ by spinach chloroplasts were carried out in which after 5 min photosynthesis the pine chloroplasts were added. It was found that the subsequent inhibition of spinach CO₂ fixation was neither due to any effect on the rate of export of photosynthetic metabolites from the chloroplasts to the medium, nor to a direct effect on the RUBP carboxylase reaction. The principal effect was found to be an inhibition of the conversion of fructose-1,6-bisphosphate and sedoheptulose-1,7-bisphosphate to the respective monophosphates and inorganic phosphate. From this finding it was concluded that a principal effect of the inhibition by pine chloroplasts is probably an inhibition either directly or indirectly of the bisphosphatase enzymes in the spinach chloroplasts. Based on its distribution between organic and aqueous acidic or neutral solutions, the inhibitory factor of the pine chloroplasts must be lipophilic. Most of the factor could be transferred to an aqueous phase in a strongly alkaline solution. Following subsequent acidification of the aqueous phase the activity could be completely transferred back into the organic phase. This procedure allowed for separation of the inhibitory factor from most of the pigments and other lipophilic substances present in the pine chloroplasts and yielded a preparation which could be subsequently fractionated by thin layer chromatography. UV absorption was found in two fast moving spots and at the origin. The fastest running spot from the thin layer chromatography plate was found to be the one containing most of the inhibitory activity.     

The response of Synechococcus PCC7942 (Cyanophyta) to changes in CO2 supply in relation to the acclimation of the CO2-concentrating mechanism. I: physiological study

Distinct types of carboxysomes were distinguished in Synechococcus PCC 7942: electron-clear, electron-intermediate, carboxysomes with internal electron-clear areas, typical electron-dense and bar-shaped carboxysomes. Immunogold location with antibodies against the Rubisco large subunit showed specific label in all carboxysomes. The positive correlation between electron-density, the density of immunogold label, and the percentage of labeled structures within each type support a model of carboxysome biogenesis whereby electron-clear evolve to electron-intermediate and then to electron-dense carboxysomes by the progressive sequestering of Rubisco molecules. Cells responded to limitation in CO₂ supply by increasing carboxysome frequency and the proportion of typical electron-dense carboxysomes, the extent of the response depending on the degree of limitation. The time course of carboxysome expression during transfers between different conditions of CO₂ supply indicated that, under our experimental conditions, there were different levels of response, depending on the degree of limitation. The first level occured at atmospheric levels of CO₂ and involved changes in the affinity of the CCM and in carboxysome, which occurred simultaneously. More severe limitation of CO₂ supply affected carboxysomes exclusively, without further improvement in the affinity of the CCM.     

Stem respiration of Norway spruce trees under elevated CO2 concentration

Measurements of stem respiration were conducted for a period of four years (1999–2002) in 14-year old Norway spruce (Picea abies [L.] Karst) trees exposed to ambient (CA) and elevated CO₂ concentration (CE; ambient plus 350 μmol mol⁻¹). Stem respiration measurements of six trees per treatment were carried out 2–3 times per month during the growing season. Stem respiration in CE treatment was higher (up to 16 %) than in CA treatment. Temperature response of stem respiration (Q₁₀) for the whole experimental period ranged between 1.65–2.57 in CA treatment and 2.24–2.56 in CE treatment. The mean stem respiration rate normalized to 10 °C (R₁₀) in CA and CE treatments ranged between 1.67–1.95 and 2.19–2.72 μmol(CO₂) m⁻² s⁻¹, respectively. Seasonal variations in stem respiration were related to temperature and tree growth.     

Identification of the photosynthetic CO2 fixation inhibitors in isolated pine chloroplasts as resin acids

Thus far all attempts to isolate CO, fixing chloroplasts from pine have failed. In this paper it is proposed that resin acids present in pine needles partition into membranes during chloroplast isolation and interfere with specific reactions of the Calvin cycle. CO, fixation by isolated spinach chloroplasts was strongly inhibited by the introduction of a suspension of chloroplasts isolated from Pinus sylvestris L. A partially purified organic extract obtained from chloroplasts of this pine species also strongly inhibited CO, fixation by the spinach chloroplasts. The major inhibitory compounds from the organic extract were identified as a mixture of resin acids by gas-liquid chromatography and mass spectrometry. Two resin acids, abietic acid and dehydroabietic acid, were tested for inhibitory activity. Both resin acids were potent inhibitors of photosynthetic CO₂fixation, with dehydroabietic acid being about three times more potent than abietic acid.     

Growth characteristics of Botryococcus braunii 765 under high CO2 concentration in photobioreactor

To understand the potential of cultivating Botryococcus braunii with flue gas (normally containing high CO₂) for biofuel production, growth characteristics of B. braunii 765 with 2-20% CO₂ aeration were investigated. The results showed that the strain could grow well without any obvious inhibition under all tested CO₂ concentrations with an aeration rate of 0.2 vvm, even without any culture pH adjustment (ranged from 6.0 to 8.0). The maximum biomass among all conditions was 2.31 g L⁻¹ on 25th day at 20% CO₂. Hydrocarbon content and algal colony size increased with the increase of CO₂ concentration. A negative correlation between algal biomass and culture total phosphorus was observed (from −0.828 to −0.911, P < 0.01). Additionally, 2% sodium hypochlorite solution was used for photobioreactor sterilization to cultivate B. braunii.     

Gas exchange of Ginkgo biloba leaves at different CO2 concentration levels

One and a half year-old Ginkgo saplings were grown for 2 years in 7 litre pots with medium fertile soil at ambient air CO₂ concentration and at 700 μmol mol⁻¹ CO₂ in temperature and humidity-controlled cabinets standing in the field. In the middle of the 2nd season of CO₂ enrichment, CO₂ exchange and transpiration in response to CO₂ concentration was measured with a mini-cuvette system. In addition, the same measurements were conducted in the crown of one 60-year-old tree in the field. Number of leaves/tree was enhanced by elevated CO₂ and specific leaf area decreased significantly.CO₂ compensation points were reached at 75–84 μmol mol⁻¹ CO₂. Gas exchange of Ginkgo saplings reacted more intensively upon CO₂ than those of the adult Ginkgo. On an average, stomatal conductance decreased by 30% as CO₂ concentration increased from 30 to 1000 μmol mol⁻¹ CO₂. Water use efficiency of net photosynthesis was positively correlated with CO₂ concentration levels. Saturation of net photosynthesis and lowest level of stomatal conductance was reached by the leaves of Ginkgo saplings at >1000 μmol mol⁻¹ CO₂. Acclimation of leaf net CO₂ assimilation to the elevated CO₂ concentration at growth occurred after 2 years of exposure. Maximum of net CO₂ assimilation was 56% higher at ambient air CO₂ concentration than at 700 μmol mol⁻¹ CO₂.     

Photosynthetic CO2 fixation by chloroplast populations isolated by a polymer two-phase technique

Preparations of spinach chloroplasts, essentially free from contamination by other cellular material or whole cells, incorporated ¹⁴C almost entirely into glycolate, a polyglucan (probably starch) and intermediates of the Calvin cycle and starch synthesis. About 70% of the ¹⁴C was found in dihydroxyacetone phosphate, 3-phosphoglycerate and glycolate. Only small amounts were found in sucrose and amino acids.

On the other hand, preparations consisting of particles containing chloroplasts surrounded by a membrane-bound cytoplasmic layer including mitochondria and microbodies, gave a much broader spectrum of ¹⁴C-labelled products. Much less of the ¹⁴C was found in dihydroxyacetone phosphate and 3-phosphoglycerate. Instead, sucrose, malate, aspartate, alanine and some other amino acids contained about 40% of the ¹⁴C incorporated. It is concluded that sucrose is synthesized by cooperation between the chloroplast and the surrounding layer.     

Dark reduction of QA in intact barley leaves as an effect of lowered CO2 concentration monitored by chlorophyll a luminescence and chlorophyll a F0 dark fluorescence

When the CO₂ concentration in the atmosphere above an intact barley leaf was lowered in the dark after illumination, chlorophyll a luminescence and chlorophyll a dark fluorescence were stimulated. The stimulation was induced by lowered levels of CO₂ in a wide concentration range including concentrations well above that saturating photosynthesis. The stimulation of luminescence by lowered CO₂ concentrations was more pronounced after far-red excitation than after white light excitation. The difference in response to lowered CO₂ concentrations after white/far-red excitation was less pronounced for fluorescence than for luminescence. Stimulation of luminescence was more pronounced when the CO₂ concentration was lowered in an O₂-containing atmosphere than under anaerobic conditions. It is concluded that lowering of the CO₂ concentration in the dark after illumination causes a partial reduction of the primary Photosystem II acceptor Q_A.     

Change in the peripheral CO2 chemoreflex from rest to exercise

A single-breath CO₂ test of peripheral chemosensitivity has recently been described, and elaborated based on model simulations. This study was designed to measure the peripheral CO₂ chemoreflex at rest and during heavy exercise to see if carotid chemosensitivity to CO₂ increased. Ten healthy, adult males performed an incremental exercise test to determine their ventilatory anaerobic threshold (VAT), and 20 minutes of steady-state exercise at a pre-determined power output above VAT. Arterialized venous blood was obtained during each minute of incremental exercise to verify development of metabolic acidosis. Carotid chemosensitivity was tested repeatedly at rest and in steady-state exercise by the ventilatory response to a single breath of 13% CO₂ in air. The peripheral chemoreflex for CO₂ for the group of subjects doubled from rest to exercise (mean 0.0961 · s^–1 · kPa^–1) with all subjects showing an increase. We conclude that the gain of the carotid CO₂ chemoreflex increases from rest to exercise at work above the VAT.     

Reactions of the ferri-ferrocytochrome-c system with superoxide/oxygen and CO2/CO2 studied by fast pulse radiolysis

The reduction of ferricytochrome c by O₂⁻ and CO₂⁻ was studied in the pH range 6.6–9.2 and Arrhenius as well as Eyring parameters were derived from the rate constants and their temperature dependence. Ionic effects on the rate indicate that the redox process proceeds through a multiply-positively charged interaction site on cytochrome c. It is shown that the reaction with O₂⁻ and correspondingly with O₂ of ferrocytochrome c) is by a factor of approx. 10³ slower than warranted by factors such as redox potential. Evidence is adduced to support the view that this slowness is connected with the role of water in the interaction between O₂⁻/O₂ and ferri-ferrocytochrome c in the positively charged interaction site on cytochrome c in which water molecules are specifically involved in maintaining the local structure of cytochrome c and participate in the process of electron equivalent transfer.     

H2O2 destruction by ascorbate-dependent systems from chloroplasts

Washed lamellae from isolated spinach chloroplasts exhibited peroxidative activity with 3,3′-diaminobenzidine or ascorbate as electron donors. By heat treatment or by incubation of the chloroplasts with pronase a heat-labile enzymic activity (system A) and a heat-stable non-enzymic peroxidative activity (system B) could be differentiated.System A is membrane-bound, reacts with 3,3′-diaminobenzidine and with ascorbate as electron donors, shows a sharp pH optimum between 7.5 and 8.0 with both substrates and is inhibited competitively by cyanide.The heat-stable factor can be extracted from the chloroplast lamellae by heat treatment, reacts only with ascorbate as electron donor, shows increasing activity with higher pH values but no optimum and is not inhibited by cyanide.Both peroxidative systems in connection with a relatively high concentration of ascorbate in chloroplasts should represent an important tool for the detoxification of H₂O₂ which is produced in these organelles by photosynthetic O₂ reduction.     

Fermentative products and dark CO2 fixation during germination of seeds of Cicer arietinum

During the germination of Cicer arietinum L. the amounts of ethanol, lactate and malate reached their highest values at 24 hr, the concentration of ethanol being about 4 times that of lactate and twice that of malate. The activities of phosphoenolpyruvate carboxylase and malic enzyme seem to be correlated with the ability of cotyledons to fix CO₂ from NaH¹⁴CO₃ into malate and with the further decrease in this metabolise from 36 hr onwards.     

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.