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.     

Hydrogenase-Mediated Activities in Isolated Chloroplasts of Chlamydomonas reinhardii

Isolated intact chloroplasts of Chlamydomonas reinhardii were found to catalyze photoreduction of CO₂ in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea when adapted under an atmosphere of H₂ demonstrating the association of a hydrogenase and anaerobic adaptation system with these plastids. The specific activity of photoreduction was approximately one third that detected in cells and protoplasts. Photoreduction was found to have a lower osmoticum optimum relative to aerobically maintained chloroplasts (50 millimolar versus 120 millimolar mannitol). 3-Phosphoglycerate (3-PGA) stimulated photoreduction up to a peak at 0.25 millimolar beyond which inhibition was observed. In the absence of 3-PGA, inorganic phosphate had no effect on photoreduction but in the presence of 3-PGA, inorganic phosphate also stimulated the reaction. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone inhibited photoreduction but inhibition by the former could be partially overcome by exogenously added ATP. The intact plastid can also catalyze photoevolution of H₂ while lysed chloroplast extracts catalyzed the reduction of methyl viologen by H₂. Both reactions occurred at rates approximately one-third of those found in cells. The oxyhydrogen reaction in the presence or absence of CO₂ was not detected.     

Photosynthetic oxygen exchange in isolated cells and chloroplasts of c(3) plants

Photosynthetic O₂-production and photorespiratory O₂-uptake were measured, using stable isotope techniques, in isolated intact leaf cells of the C₃ plant Xanthium strumarium L., and isolated intact chloroplasts of Spinacia oleracea L (var. Yates 102). Considerable light dependent O₂-uptake was observed in both systems, a proportion of which could be suppressed by CO₂ (63% suppression in chloroplasts by 50 micromolar CO₂, 58% in cells by 100 micromolar CO₂ and 250 micromolar O₂). At low O₂, O₂-uptake was CO₂ insensitive. At high CO₂ up to 19% of total electron flow was to O₂ in cells and up to 14% in chloroplasts. O₂-uptake showed inhibition by KCN (61% in cells, 35% in chloroplasts by 0.2 millimolar KCN). O₂-uptake half saturated at 75 to 85 micromolar O₂ in cells and 50 to 65 micromolar O₂ in chloroplasts, at low CO₂. The results are discussed in terms of the RuP₂-oxygenase reaction and direct photoreduction of O₂ via a Mehler reaction.     

Effects of glyoxylate on photosynthesis by intact chloroplasts

Because glyoxylate inhibits CO₂ fixation by intact chloroplasts and purified ribulose bisphosphate carboxylase/oxygenase, glyoxylate might be expected to exert some regulatory effect on photosynthesis. However, ribulose bisphosphate carboxylase activity and activation in intact chloroplasts from Spinacia oleracea L. leaves were not substantially inhibited by 10 millimolar glyoxylate. In the light, the ribulose bisphosphate pool decreased to half when 10 millimolar glyoxylate was present, whereas this pool doubled in the control. When 10 millimolar glyoxylate or formate was present during photosynthesis, the fructose bisphosphate pool in the chloroplasts doubled. Thus, glyoxylate appeared to inhibit the regeneration of ribulose bisphosphate, but not its utilization.

The fixation of CO₂ by intact chloroplasts was inhibited by salts of several weak acids, and the inhibition was more severe at pH 6.0 than at pH 8.0. At pH 6.0, glyoxylate inhibited CO₂ fixation by 50% at 50 micromolar, and glycolate caused 50% inhibition at 150 micromolar. This inhibition of CO₂ fixation seems to be a general effect of salts of weak acids.

Radioactive glyoxylate was reduced to glycolate by chloroplasts more rapidly in the light than in the dark. Glyoxylate reductase (NADP⁺) from intact chloroplast preparations had an apparent K_m (glyoxylate) of 140 micromolar and a V_max of 3 micromoles per minute per milligram chlorophyll.

     

Salicylhydroxamic Acid (SHAM) Inhibition of the Dissolved Inorganic Carbon Concentrating Process in Unicellular Green Algae

Rates of photosynthetic O₂ evolution, for measuring K_0.5(CO₂ + HCO₃⁻) at pH 7, upon addition of 50 micromolar HCO₃⁻ to air-adapted Chlamydomonas, Dunaliella, or Scenedesmus cells, were inhibited up to 90% by the addition of 1.5 to 4.0 millimolar salicylhydroxamic acid (SHAM) to the aqueous medium. The apparent K₁(SHAM) for Chlamydomonas cells was about 2.5 millimolar, but due to low solubility in water effective concentrations would be lower. Salicylhydroxamic acid did not inhibit oxygen evolution or accumulation of bicarbonate by Scenedesmus cells between pH 8 to 11 or by isolated intact chloroplasts from Dunaliella. Thus, salicylhydroxamic acid appears to inhibit CO₂ uptake, whereas previous results indicate that vanadate inhibits bicarbonate uptake. These conclusions were confirmed by three test procedures with three air-adapted algae at pH 7. Salicylhydroxamic acid inhibited the cellular accumulation of dissolved inorganic carbon, the rate of photosynthetic O₂ evolution dependent on low levels of dissolved inorganic carbon (50 micromolar Na-HCO₃), and the rate of ¹⁴CO₂ fixation with 100 micromolar [¹⁴C] HCO₃⁻. Salicylhydroxamic acid inhibition of O₂ evolution and ¹⁴CO₂-fixation was reversed by higher levels of NaHCO₃. Thus, salicylhydroxamic acid inhibition was apparently not affecting steps of photosynthesis other than CO₂ accumulation. Although salicylhydroxamic acid is an inhibitor of alternative respiration in algae, it is not known whether the two processes are related.     

Uptake of inorganic carbon by isolated chloroplasts from air-adapted dunaliella

Neither Dunaliella cells grown with 5% CO₂ nor their isolated chloroplasts had a CO₂ concentrating mechanism. These cells primarily utilized CO₂ from the medium because the K_(0.5) (HCO₃⁻) increase from 57 micromolar at pH 7.0 to 1489 micromolar at pH 8.5, where as the K_(0.5) CO₂ was about 12 micromolar over the pH range. After air adaptation for 24 hours in light, a CO₂ concentrating mechanism was present that decreased the K_0.5 (CO₂) to about 0.5 micromolar and K_0.5 (HCO₃⁻) to 11 micromolar at pH 8. These K_0.5 values suggest that air-adapted cells preferentially concentrated CO₂ but could also use HCO₃⁻ from the medium. Chloroplasts isolated from air-adapted cells had a K_(0.5) for total inorganic carbon of less than 10 micromolar compared to 130 micromolar for chloroplasts from cells grown on high CO₂. Chloroplasts from air-adapted cells, but not CO₂-grown cells, concentrate inorganic carbon internally to 1 millimolar in 60 seconds from 240 micromolar in the medium. Maximum uptake rates occurred after preillumination of 45 seconds to 3 minutes. The CO₂ concentrating mechanism by chloroplasts from air-adapted cells was light dependent and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or flurocarbonyl-cyamidephenylhydrazone (FCCP). Phenazine-methosulfate at 10 micromolar to provide cyclic phosphorylation partially reversed the inhibition by DCMU but not by FCCP. One to 0.1 millimolar vanadate, an inhibitor of plasma membrane ATPase, inhibited inorganic carbon accumulation by isolated chloroplasts. Vanadate had no effect on CO₂ concentration by whole cells, as it did not readily cross the cell plasmalemma. Addition of external ATP to the isolated chloroplast only slightly stimulated inorganic carbon uptake and did not reverse vanadate inhibition by more than 25%. These results are consistent with a CO₂ concentrating mechanism in Dunaliella cells which consists in part of an inorganic carbon transporter at the chloroplast envelope that is energized by ATP from photosynthetic electron transport.     

Glucose Respiration in the Intact Chloroplast of Chlamydomonas reinhardtii

Chloroplastic respiration was monitored by measuring ¹⁴CO₂ from ¹⁴C glucose in the darkened Chlamydomonas reinhardtii F-60 chloroplast. The patterns of ¹⁴CO₂ evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phosphoenolpyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The K_m for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of ¹⁴CO₂ was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO₂ evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1, C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO₂ evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH₄Cl and KCl (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phosphoenolpyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to CO₂ and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism.     

Evidence for Endogenous Cyclic Photophosphorylation in Intact Chloroplasts: CO(2) Fixation with Dihydroxyacetone Phosphate

This study examines the capacity of intact spinach (Spinacia oleracea L.) chloroplasts to fix ¹⁴CO₂ when supplied with Benson-Calvin cycle intermediates in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Under these conditions, substantial ¹⁴CO₂ fixation occurred in the light but not in the dark when either dihydroxyacetone phosphate, ribulose 5-phosphate, fructose 6-phosphate, or fructose bisphosphate was added. The highest rate of ¹⁴CO₂ fixation (20-40 micromoles per milligram chlorophyll per hour) was obtained with dihydroxyacetone phosphate. In contrast, no ¹⁴CO₂ fixation occurred when 3-phosphoglycerate was used. ¹⁴CO₂ fixation in the presence of dihydroxyacetone phosphate and DCMU was inhibited by carbonylcyanide m-chlorophenylhydrazone, dl-glyceraldehyde, and pyridoxal 5′-phosphate. Low concentrations of O₂ (25-50 micromolar) stimulated ¹⁴CO₂ fixation, but the activity decreased with increasing O₂ concentrations. The fixation of ¹⁴CO₂ in the presence of DCMU and dihydroxyacetone phosphate was also observed in maize bundle sheath cells. These results provide direct evidence for cyclic photophosphorylation in intact chloroplasts. The activity measured is adequate to support all the extra ATP requirements for maximum rates of photosynthesis in these intact chloroplasts.     

Reduced sulfur and nitrogen compounds and molecular hydrogen as electron donors for anaerobic CO2 photoreduction in Anacystis nidulans

Photosynthesis by Anacystis nidulans was studied in presence of reduced sulfur or nitrogen compounds, or of hydrogen. O₂ evolution and CO₂ fixation were depressed by sulfide, sulfite, cysteine, thioglycollate, hydroxylamine and hydrazine. Sulfite, cysteine and hydrazine inhibited O₂ evolution much more strongly than CO₂ fixation, indicating ability to supply electrons for CO₂ photoreduction; DCMU suppressed these photoreductions. In contrast, some anoxygenic photosynthetic CO₂ fixation insensitive to DCMU was found with sulfide, thiosulfate and hydrogen. Emerson enhancement studies confirmed that sulfite, cysteine and hydrazine acted on photosystem II, while photoreduction supported by sulfide, thiosulfate and hydrogen needed photosystem I only.Sulfite was photooxidized to sulfate, sulfide to elemental sulfur, and thiosulfate to sulfate plus elemental sulfur; the sulfur accumulated inside the cells. Results on the stoichiometries of the photoreductions were consistent with the photooxidation products determined. Inhibitor studies suggested photosynthetic CO₂ fixation through the Calvin cycle.While photoreduction by all reductants used was found to be constitutive in Anacystis, the process was stimulated by anaerobic preincubation with the reductants only in the cases of hydrogen and thiosulfate; this adaptation was prevented by chloramphenicol and by O₂. Anaerobic photoautotrophic growth of Anacystis was, however, not observed; the increase in dry weight with H₂ and thiosulfate was not accompanied by cell multiplication or by an increase in chlorophyll content. Parallel short-term experiments with Chlorella did not reveal any constitutive photoreduction in this eukaryotic alga.Abbreviations CAP chloramphenicol - CCCP carbonyl cyanide m-chlorophenylhydrazone - DBMIB dibromothymoquinone - DCMU dichlorophenyl dimethyl urea - DSPD disalicylidenepropane diamine-(1,3) - EDAC 1-ethyl-3(3-dimethylaminopropyl-) carbodiimide     

Photoinhibition of CO(2)-Dependent O(2) Evolution by Intact Chloroplasts Isolated from Spinach Leaves

Intact spinach (Spinacia oleracea L.) chloroplasts, when pre-illuminated at 4 millimoles quanta per square meter per second for 8 minutes in a CO₂-free buffer at 21% O₂, showed a decrease (30-70%) in CO₂-dependent O₂ evolution and ¹⁴CO₂ uptake. This photoinhibition was observed only when the O₂ concentration and the quantum fluence rate were higher than 4% and 1 millimole per square meter per second, respectively. There was only a small decrease in the extent of photoinhibition when the CO₂ concentration was increased from 0 to 25 micromolar during the treatment, but photoinhibition was abolished when the CO₂ concentration was increased to 30 micromolar. Addition of small quantities of P-glycerate (40-200 micromolar) or glycerate (160 micromolar) was found to prevent photoinhibition. Other intermediates of the Calvin cycle (fructose-6-P, fructose-1,6-P, ribose-5-P, ribulose-5-P) also prevented photoinhibition to various extents. Oxaloacetate was not effective in preventing photoinhibition in these chloroplasts. The amount of O₂ evolved during treatments with 3-P-glycerate or glycerate was no more than 65% of that measured in the presence of low CO₂ concentrations (9-12 micromolar) which did not prevent photoinhibition. In all cases, the extent to which photoinhibition was prevented by these metabolites was not correlated to the amount of O₂ evolved during the photoinhibitory treatment. It is concluded that in these chloroplasts the prevention of the O₂-dependent photoinhibition of light saturated CO₂ fixation capacity is not linked to the dissipation of excitation energy via the photosynthetic electron transport nor to ATP utilization. The requirement of O₂ for photoinhibition of CO₂ fixation capacity in isolated chloroplasts may be explained by an effect of O₂ in allowing metabolic depletion of Calvin cycle intermediates.     

Coupling of Carbon Dioxide Fixation to the Oxyhydrogen Reaction in the Isolated Chloroplast of Chlamydomonas reinhardtii

The oxyhydrogen reaction (the reduction of O₂ to water by H₂) in the presence of CO₂ was studied in the isolated Chlamydomonas reinhardtii chloroplast by monitoring the rate of ¹⁴CO₂ incorporation into acid-stable products in the dark. The endogenous rate of CO₂ uptake (50-125 nmol/mg chlorophyll per h) was increased about 3- to 4-fold by ATP and additionally when combined with glucose, ribose-5-phosphate, and glycerate-3-phosphate. The rate was diminished 50 to 75%, respectively, when H₂ was replaced by N₂ or by air. Decrease in CO₂ uptake by dl-glyceraldehyde was taken to indicate that the regenerative phase and complete Calvin cycle turnover were involved. Diminution of CO₂ incorporation by rotenone, antimycin A, and 2,5-dibromo-3-methyl-6-isopropanol-p-benzoquinone was attributed to an inhibition of the oxyhydrogen reaction, resulting in an elevated NADPH/NADP ratio. If so, then the diminished CO₂ uptake could have been by “product inhibition” of the carbon metabolic network. Our data are consistent with the proposal (H. Gaffron [1942] J Gen Physiol 26: 241-267) that CO₂ fixation coupled to the oxyhydrogen reaction is dependent to some extent on exchloroplastic metabolism. This support is primarily ATP provided by mitochondrial respiration.     

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.     

Kinetics of the Oxyhydrogen Reaction in the Presence and Absence of Carbon Dioxide in Scenedesmus obliquus

The oxyhydrogen reaction in the presence and absence of CO₂ was studied in H₂-adapted Scenedesmus obliquus by monitoring the initial rates of H₂, O₂, and ¹⁴CO₂ uptake and the effect of inhibitors on these rates with gas-sensing electrodes and isotopic techniques. In the presence of 0.02 atmosphere O₂, the pH₂ was varied from 0 to 1 atmosphere. Whereas the rate of O₂ uptake increased by only 30%, the rate of H₂ uptake increased severalfold over the range of pH₂ values. At 0.1 atmosphere H₂ and 0.02 atmosphere O₂, rates for H₂ and O₂ uptake were between 15 and 25 micromoles per milligram chlorophyll per hour. As the pH₂ was changed from 0 to 1 atmosphere, the quotient H₂:O₂ changed from 0 to roughly 2. This change may reflect the competition between H₂ and the endogenous respiratory electron donors. Respiration in the presence of glucose and acetate was also competitive with H₂ uptake. KCN inhibited equally respiration (O₂ uptake in the absence of H₂) and the oxyhydrogen reaction in the presence and absence of CO₂. The uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone accelerated the rate of respiration and the oxyhydrogen reaction to a similar extent. It was concluded that the oxyhydrogen reaction both in the presence and absence of CO₂ has properties in common with components of respiration and photosynthesis. Participation of these two processes in the oxyhydrogen reaction would require a closely linked shuttle between mitochondrion and chloroplast.     

Carbon dioxide and nitrite photoassimilatory processes do not intercompete for reducing equivalents in spinach and soybean leaf chloroplasts

Previously, C Baysdorfer and JM Robinson (1985 Plant Physiol 77: 318-320) demonstrated that, in a reconstituted spinach chloroplast system, NADP photoreduction functioning at most maximal rate and reductant demand, was the successful competitor with NO₂⁻ photoreduction for reduced ferredoxin. This resulted in a repression of NO₂⁻ reduction until all NADP available had been almost totally reduced. Further experiments, employing isolated, intact spinach leaf plastids and soybean leaf mesophyll cells, were conducted to examine competition for reductant between CO₂ and NO₂⁻ photoassimilation, in situ. In isolated, intact plastid preparations, regardless of whether the demand for reductant by CO₂ photoassimilation was high (5 millimolar `CO<sub>2</sub>') with rates of CO<sub>2</sub> fixation in the range 40 to 90 micromoles CO<sub>2</sub> fixed per hour per milligram chlorophyll, low (0.5 millimolar `CO₂') with rates in the range 5 to 8 micromoles CO₂ per hour per milligram chlorophyll, or zero (no `CO<sub>2</sub>'), NO<sub>2</sub><sup>−</sup> photoreduction displayed equal rates in the range of 8 to 22 micromoles per hour per milligram chlorophyll. In the absence of `CO₂', but in the presence of saturating white light, 3-phosphoglycerate photoreduction at rates of 82 to 127 micromoles per hour per milligram chlorophyll did not repress, and occasionally stimulated concomitant rates of NO₂⁻ reduction which ranged from 23.4 to 38.5. Conversely, in plastid preparations, NO₂⁻ at levels of 50 to 100 micromolar, stimulated plastid CO₂ fixation when `CO<sub>2</sub>' was saturating with respect to carboxylation. Further, levels of NO<sub>2</sub><sup>−</sup> in the range 250 to 2500 micromolar, stimulated soybean leaf mesophyll cell net CO<sub>2</sub> fixation as much as 1.5-fold if `CO₂' was saturating with respect to CO₂ fixation. It appeared likely that, in high light in vivo, CO₂ and NO₂⁻ photoassimilatory processes are not forced to intercompete for reduced ferredoxin in the intact chloroplast.     

Carbon dioxide fixation in the light and in the dark by isolated spinach chloroplasts

Factors affecting CO₂ fixation in the spinach (Spinacia oleracea) chloroplast were investigated. Free magnesium ions are shown to be highly inhibitory for photosynthetic CO₂ fixation in isolated intact spinach chloroplasts. The pH optimum for CO₂ fixation is about 8.5 but is dependent upon the reaction medium. Conditions are defined under which chloroplasts illuminated in the absence of CO₂ accumulate ribulose 1,5-diphosphate, and fix CO₂ in a subsequent dark period when high magnesium ion concentrations are provided. The regulation of photosynthetic CO₂ assimilation by these factors is discussed.     

Response of carbon dioxide fixation to water stress: parallel measurements on isolated chloroplasts and intact spinach leaves

Application of water stress to isolated spinach (Spinacia oleracea) chloroplasts by redutcion of the osmotic potentials of CO₂ fixation media below −6 to −8 bars resulted in decreased rates of fixation regardless of solute composition. A decrease in CO₂ fixation rate of isolated chloroplasts was also found when leaves were dehydrated in air prior to chloroplast isolation. An inverse response of CO₂ fixation to osmotic potential of the fixation medium was found with chloroplasts isolated from dehydrated leaves—namely, fixation rate was inhibited at −8 bars, compared with −16 or −24 bars.     

Inhibition of photosynthetic carbon dioxide fixation in isolated spinach chloroplasts exposed to reduced osmotic potentials

Reduced osmotic potentials inhibited the rate of CO₂ fixation by isolated intact spinach (Spinacia oleracea) chloroplasts. This inhibition was observed immediately after transfer of chloroplasts from a solution containing 0.33 m sorbitol to higher sorbitol concentrations, and the depressed rate remained constant. The inhibited CO₂ fixation could not be attributed to a decreased rate of photosynthetic electron transport, since NADP reduction was unaffected by subjecting the chloroplasts to low potentials. It could also not result from restricted permeability to CO₂, as CO₂ concentrations had no effect on the relative inhibition induced by the lowered potential.     

Active Transport of Inorganic Carbon Increases the Rate of O(2) Photoreduction by the Cyanobacterium Synechococcus UTEX 625

Chlorophyll a fluorescence of Synechococcus UTEX 625 was quenched during the transport of inorganic carbon, even when CO₂ fixation was inhibited by iodoacetamide. Measurements with a pulse modulation fluorometer showed that at least 75% of the quenching was due to oxidation of Q_a, the primary acceptor of photosystem II. Mass spectrometry revealed that transport of inorganic carbon increased the rate of O₂ photoreduction. Hence, O₂ could serve as an electron acceptor to allow oxidation of Q_a even in the absence of CO₂ fixation.     

The Reducing Power and the CO2 Fixation Activity of Isolated Choroplasts

Antimycin A, which stimulates the Co₂ fixation activity of isolated intact spinach (Spinacia oleracea L) chloroplasts, was shown to stimulate also the oxygen evolution in the presence of oxaloacetate, as do the uncouplers. Some uncoupers (tetramethlethylenediamine, Nh₄CL) were shown to stimulate the CO₂ fixation activity, and to decrease the intrachloroplastic ATP levels, like antimycin A. The intrachloroplastic NADPH levels were not affected by the inhibitors studied.     

Glycolaldehyde Inhibits CO(2) Fixation in the Cyanobacterium Synechococcus UTEX 625 without Inhibiting the Accumulation of Inorganic Carbon or the Associated Quenching of Chlorophyll a Fluorescence

When studying active CO₂ and HCO₃⁻ transport by cyanobacteria, it is often useful to be able to inhibit concomitant CO₂ fixation. We have found that glycolaldehyde was an efficient inhibitor of photosynthetic CO₂ fixation in Synechococcus UTEX 625. Glycolaldehyde did not inhibit inorganic carbon accumulation due to either active CO₂ or HCO₃⁻ transport. When glycolaldehyde (10 millimolar) was added to rapidly photosynthesizing cells, CO₂ fixation was stopped within 15 seconds. The quenching of chlorophyll a fluorescence remained high (≤ 82% control) when CO₂ fixation was completely blocked by glycolaldehyde. This quenching was relieved upon the addition of a glucose oxidase oxygentrap. This is consistent with our previous finding that q-quenching in the absence of CO₂ fixation was due to O₂ photoreduction. Photosynthetic CO₂ fixation was also inhibited by d,l,-glyceraldehyde but a sixfold higher concentration was required. Glycolaldehyde acted much more rapidly than iodoacetamide (15 seconds versus 300 seconds) and did not cause the onset of net O₂ evolution often observed with iodoacetamide. Glycolaldehyde will be a useful inhibitor when it is required to study CO₂ and HCO₃⁻ transport without the complication of concomitant CO₂ fixation.