Relationship between the Level of Adenine Nucleotides and the Carboxylation Activity of Illuminated Isolated Spinach Chloroplasts: A Study with Antimycin A

The changes in the levels of intact spinach (Spinacia oleracea L.) chloroplast adenine nucleotides during the time course of light-dependent CO₂ fixation were determined with respect to the effect of antimycin A. This study demonstrated that antimycin A lowered the rate of ATP formation during the induction period of carboxylation. While the steady state levels of ATP and the energy-charge value also decreased in the presence of antimycin, the concomitant increase of the CO₂ fixation activities insured higher ATP turnover rates. Changes in the labeling of CO₂ fixation products during the lag phase suggested a stepwise activation of the Calvin cycle, with fructose 1,6-diphosphate, and ribulose 5-phosphate kinase being activated before ribulose 1,5-diphosphate carboxylase. The possible mechanisms of the enhancement of CO₂ fixation activity by antimycin A in relation to its action on photophosphorylation during the lag phase are 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.     

Effect of antimycin a on photosynthesis of intact spinach chloroplasts

Low concentrations (0.5-10 μm) of antimycin A were shown to increase the rate of CO₂ fixation, O₂ evolution and inorganic phosphate esterification in intact spinach (Spinacia oleracea) chloroplasts. The increase was highest when the light intensity was saturating. Stimulation was independent of the bicarbonate concentration and was accompanied by an enhancement in the synthesis of glycerate 3-phosphate with a decrease in dihydroxyacetone phosphate. The antibiotic decreased the Michaelis constant of the chloroplast but not of ribulose 1,5-diphosphate carboxylase for bicarbonate. It was suggested that antimycin A is affecting that portion (outer envelope) of the intact chloroplast which contains the enzyme mechanism for controlling the pace of CO₂ fixation.     

Uptake of bicarbonate ion in darkness by isolated chloroplast envelope membranes and intact chloroplasts of spinach

Bicarbonate uptake by isolated chloroplast envelope membranes and intact chloroplasts of spinach (Spinacia oleracea L. var. Viroflay) in darkness exhibited a similar dependency upon temperature, pH, time, and concentrations of isolated or attached envelope membranes. This similarity in uptake properties demonstrates the usefulness of the envelope membranes for the study of chloroplast permeability. Maximal rates for dark HCO₃^- uptake by isolated envelope membranes and intact chloroplasts were more than sufficient to account for the maximal rates of photosynthetic CO₂ fixation observed with intact chloroplasts. The active species involved in the uptake process was found to be HCO₃^- and not CO₂. The significance of HCO₃^- uptake and its relationship to carbonic anhydrase and ribulose diphosphate carboxylase is discussed. Conditions for maximal HCO₃^- uptake in darkness by intact chloroplasts were found to be similar to those required for maximal photosynthetic CO₂ fixation, suggesting that HCO₃^- uptake by the envelope membrane may regulate photosynthetic CO₂ fixation.     

The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO2 fixation in the light and dark

1. The pH in the stroma and in the thylakoid space has been measured in a number of chloroplast preparations in the dark and in the light at 20 °C. Illumination causes a decrease of the pH in the thylakoid space by 1.5 and an increase of the pH in the stroma by almost 1 pH unit.2. CO₂ fixation is shown to be strongly dependent on the pH in the stroma. The pH optimum was 8.1, with almost zero activity below pH 7.3. Phosphoglycerate reduction, which is a partial reaction of CO₂ fixation, shows very little pH dependency.3. Low concentrations of the uncoupler m-chlorocarbonylcyanide phenylhydrazone (CCCP) inhibit CO₂ fixation without affecting phosophoglycerate reduction. This inhibition of CO₂ fixation appears to be caused by reversal of light induced alkalisation in the stroma by CCCP.4. Methylamine has a very different effect compared to CCCP. Increasing concentrations of methylamine inhibit CO₂ fixation and phosphoglycerate reduction to the same extent. The light induced alkalisation of the stroma appears not to be significantly inhibited by methylamine, but the protons in the thylakoid space are neutralized. The inhibition of CO₂ fixation by higher concentrations of methylamine is explained by an inhibition of photophosphorylation. It appears that methylamine does not abolish proton transport.5. It is shown that intact chloroplasts are able to fix CO₂ in the dark, yielding 3-phosphoglycerate. This requires the addition of dihydroxyacetone phosphate as precursor of ribulosemonophosphate and also to supply ATP, and the addition of oxaloacetate for reoxidation of the NADPH in the stroma.6. Dark CO₂ fixation in the presence of dihydroxyacetone phosphate and oxaloacetate has the same pH dependency as CO₂ fixation in the light. This demonstrates that CO₂ fixation in the dark is not possible, unless the pH in the medium is artificially raised to pH 8.8.7. It is shown that pH changes occurring in the stroma after illumination are sufficient to switch CO₂ fixation from zero to maximal activity. This offers a mechanism for light control of CO₂ fixation, avoiding wasteful CO₂ fixation in the dark.     

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.     

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.     

Fatty Acid Synthesis in Hydrilla Chloroplasts

The rates of carboxylation, photophosphorylation and acetate incorporation have been compared in the intact and broken chloroplasts of Hydrilla verticillata Royle leaves in the presence and absence of certain inhibitors and metabolites. The intact chloroplasts showed low rates of photophosphorylation, high rates of carboxylation, and exhibited normal capacity for fatty acid biosynthesis. In broken chloroplasts a drastic decrease was observed in the rates of carboxylation and acetate incorporation. However, the rate of photophosphorylation was considerably increased. In the presence of light, inhibitors such as iodoacetamide, arsenite and sodium azide decreased the photophosphorylation rate. F-1,6-di-P and PGA stimulated CO₂ fixation rate. In the absence of artificial light, inhibitors such as sodium arsenite, gluconate-6-phosphate, sodium azide and iodoacetamide decreased the rate of CO₂ fixation. CoA, ATP, G-6-P, F-1,6-di-P Stimulated the synthesis of fatty acids. Exogenous supply of ADP. NADH, NADP and NADPH did not stimulate fatty acid biosynthesis probably because these compounds could not gain entry into the chloroplasts. Light was necessary for the in vitro fatty acid biosynthesis.     

On the Participation of Phosphoribulokinase in the Light Regulation of CO(2) Fixation

CO₂ fixation by a suspension of isolated spinach chloroplasts was terminated by turning off the light, and changes of metabolite levels in the chloroplast stroma and the surrounding medium were assayed. Whereas CO₂ fixation comes to a total stop within 15 seconds, a conversion of triose phosphates to heptose, hexose, and pentose monophosphates is found to occur for 1 to 2 minutes afterwards. It seems from these data that an inactivation of fructose and sedoheptulose bisphosphatases proceeds with a lag period. In contrast, the conversion of pentose monophosphates to ribulose 1,5-bisphosphate is inhibited immediately after the stop of illumination. As the stromal level of freely available ATP was not depleted under this condition, these data demonstrate that ribulose 5-phosphate kinase was very rapidly inactivated after darkening of the chloroplasts. Essentially, the same effect is also observed when CO₂ fixation is partially inhibited by addition of moderate concentrations of m-chlorocarbonyl phenylhydrazone, partially uncoupling photophosphorylation. It appears from these results, that the activity of ribulose 5-phosphate kinase is not only regulated by light through the mediation of reduced carriers like thioredoxin but also by alternative parameters, e.g. stromal metabolite levels.     

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.

     

Metabolism of separated leaf cells: I. Preparation of photosynthetically active cells from tobacco

Suspensions of mesophyll cells, prepared from tobacco leaves by treatment with pectinase, fixed CO₂ by photosynthesis. The products of carbon assimilation were similar for both cells and intact tissue. The cells sustained a constant fixation rate for 20 to 25 hours. For optimal CO₂ fixation, enzymatic maceration of the tissue was accomplished in 0.8 m sorbitol, but photosynthesis was optimal in 0.6 m sorbitol at pH 7 to 7.5. A hypertonic environment during maceration, which results in cell plasmolysis, is essential to maintain intact plasmalemmas and hence photosynthetically active cells. For sustained CO₂ fixation, light intensities below 500 foot-candles were required. Higher light intensities (to 1000 foot-candles) gave high initial rates of CO₂ fixation, but the cells bleached and were inactive on prolonged incubation. At pH 7.0 the bicarbonate concentration at maximal velocity of CO₂ fixation was about 1.5 mm and the apparent Km for bicarbonate was 0.2 mm.     

Effects of inhibitors of catalase on photosynthesis and on catalase activity in unwashed preparations of intact chloroplasts

The catalase activity of unwashed preparations containing intact spinach (Spinacia oleracea L.) chloroplasts is inhibited both by cyanide and by azide at concentrations which also cause inhibition of photosynthetic CO₂- dependent O₂ evolution.

Aminotriazole can also be used to inhibit this contaminant catalase, and in this case inhibition of catalase can be achieved at aminotriazole concentrations which have little effect on the rate of photosynthetic CO₂ fixation. Aminotriazole may be used as a specific inhibitor of catalase in order to demonstrate inhibition of photosynthesis by added H₂O₂.

It is therefore concluded that inhibition of photosynthesis by cyanide and azide does not necessarily result from inhibition of catalase in the chloroplast preparation, and that intact chloroplasts do not produce inhibitory concentrations of H₂O₂ under the best experimental conditions for CO₂ fixation.

     

Mechanism of glycolate transport in spinach leaf chloroplasts

The incorporation of ¹⁴CO₂ into glycolate by intact spinach leaf (Spinacia oleracea L. var. Kyoho) chloroplasts exposed to ¹⁴CO₂ (NaH¹⁴CO₃, 1 millimolar) in the light was determined as a function of O₂ concentrations in the reaction media. A hyperbolic saturation curve was obtained, apparent K_m (O₂) of 0.28 millimolar, indicating that glycolate is produced predominantly by ribulose-1,5-bisphosphate carboxylase/oxygenase. A concentration gradient of glycolate was invariably observed between chloroplast stroma and the outside media surrounding chloroplasts during photosynthetic ¹⁴CO₂ fixation under an O₂ atmosphere.     

Cyclic Photophosphorylation in Vivo and its Relation to Photosynthetic CO(2)-Fixation

Salicylaldoxime (2 × 10⁻³m and less) inhibits cyclic photophosphorylation in intact Chlorella cells severely whereas photosynthetic O₂-evolution and ¹⁴CO₂-fixation is hardly affected. Cyclic photophosphorylation in vivo was measured by following anaerobic light dependent glucose uptake. A similar difference in susceptibility has been observed with carbonylcyanide-p-trifluoromethoxyphenylhydrazone. Various controls exclude the possibility that the difference in inhibition was caused by differing experimental conditions or, in the case of glucose assimilation, by an inhibition of a reaction other than photophosphorylation.     

Isolation of Intact Chloroplasts from Spinach Leaf by Centrifugation in Gradients of the Modified Silica “Percoll”

The isolation of the photosynthetically competent chloroplast preparations was undertaken by means of the density gradient centrifugation on the modified silica sol “Percoll.” A clear separation of the intact chloroplast sustaining the high photosynthetic activities (light dependent CO₂ fixation ca. 130μmol/mg Chl·hr) was established. The contamination of mitochondria and peroxisomes was estimated to be less than 3% by measuring the activities of their marker enzymes. The chloroplasts were proved to be free from endoplasmic reticulum and cytosol. The photosynthetic CO₂ fixation of the isolated chloroplast preparations was saturated by illumination of the light intensity of 20,000 Lux (12 mW/cm², 400~750 nm).     

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.     

Nomenclature for Isolated Chloroplasts

THE nomenclature used to describe the “intactness” or degree of breakage of isolated chloioplasts has been most confusing in the past few years. This resulted chiefly from reports that chloroplasts should be isolated carefully and rapidly to obtain high rates of CO₂ fixation (50–250 µmol/mg chlorophyll/h). This type of chloroplast cannot translocate NADP, ferricyanide or ADP through its intact limiting membrane or envelope. “Class I” chloroplasts, as seen by phase contrast microscopy, had previously been considered to be similar to the in vivo situation. Unfortunately, in vitro they showed only low rates of CO₂ a fixation and easily transported NADP, ferricyanide and ADP into the chloroplast.     

O(2)-insensitive photosynthesis in c(3) plants : its occurrence and a possible explanation

Leaves of C₃ plants which exhibit a normal O₂ inhibition of CO₂ fixation at less than saturating light intensity were found to exhibit O₂-insensitive photosynthesis at high light. This behavior was observed in Phaseolus vulgaris L., Xanthium strumarium L., and Scrophularia desertorum (Shaw.) Munz. O₂-insensitive photosynthesis has been reported in nine other C₃ species and usually occurred when the intercellular CO₂ pressure was about double the normal pressure. A lack of O₂ inhibition of photosynthesis was always accompanied by a failure of increased CO₂ pressure to stimulate photosynthesis to the expected degree. O₂-insensitive photosynthesis also occurred after plants had been water stressed. Under such conditions, however, photosynthesis became O₂ and CO₂ insensitive at physiological CO₂ pressures. Postillumination CO₂ exchange kinetics showed that O₂ and CO₂ insensitivity was not the result of elimination of photorespiration.

It is proposed that O₂ and CO₂ insensitivity occurs when the concentration of phosphate in the chloroplast stroma cannot be both high enough to allow photophosphorylation and low enough to allow starch and sucrose synthesis at the rates required by the rest of the photosynthetic component processes. Under these conditions, the energy diverted to photorespiration does not adversely affect the potential for CO₂ assimilation.

     

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.     

Isolation of intact chloroplasts with high CO2 fixation capacity from sugarbeet leaves containing calcium oxalate

Intact chloroplasts were isolated from sugarbeet leaves by the mechanical disruption technique normally used for spinach. The chloroplast pellet contained a ring of white irregularly shaped crystals which were identified as calcium oxalate. The chloroplasts were greater than 90% intact yet good rates of CO₂ fixation were only obtained when inorganic pyrophosphate or 3-phosphoglycerate were added to the assay medium. Chloroplasts free of calcium oxalate were prepared by purification on a three step Percoll gradient. These purified chloroplasts were highly intact and showed high rates of CO₂ fixation without adding inorganic pyrophosphate or 3-phosphoglycerate. With optimal assay conditions (0.2 mM orthophosphate and pH 8.0) rates of 110–130 μ mole per milligram chlorophyll per hour were routinely obtained. It is concluded that intact chloroplasts capable of high rates of CO₂ fixation can be prepared from sugarbeet leaves using a simple three step Percoll gradient.