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.     

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.     

Influence of glycerate on photosynthesis by wheat chloroplasts

Glycerate was found to effect photosynthetic O2 evolution in wheat chloroplasts by its conversion to triose phosphate and by influencing the rate of photosynthesis through the reductive pentose phosphate pathway. In the absence of bicarbonate, the photosynthetic O2 evolution with glycerate was low (10 to 25 mumol mg chlorophyll-1 h-1), and only about 15% of the rate of bicarbonate-dependent O2 evolution under optimum conditions. This corresponds to a rate of glycerate conversion to triose phosphate of 20 to 50 mumol mg chlorophyll-1 h-1, which appears sufficient to accommodate flux through the glycolate pathway in vivo. Pi was required for this glycerate-dependent O2 evolution; rates remained relatively constant between 0.1 and 40 mM Pi, and proceeded with little lag upon illumination (less than 0.5 min). Evidence for O2 evolution due to glycerate conversion to triose phosphate could be conclusively demonstrated by addition of glycolaldehyde, an inhibitor of the regenerative phase of photosynthesis, which prevents CO2 fixation. The effect of glycerate on photosynthesis in the presence of bicarbonate was determined by measuring both photosynthetic O2 evolution and 14CO2 fixation at varying Pi concentrations. Low concentrations of glycerate (micro- to millimolar levels) prevented inhibition of photosynthesis by Pi. With 1 mM bicarbonate and pH 8.2, which is favorable for glycolate synthesis, maximum rates of photosynthesis were obtained at low Pi (25 microM), whereas strong inhibition of photosynthesis occurred at only 0.2 mM Pi. Addition of glycerate relieved the inhibition of photosynthesis by Pi, indicating the possible importance of glycerate metabolism in the chloroplast under photorespiratory conditions. The initiation of photosynthesis by glycerate at inhibitory Pi levels occurred with little reduction in the ratio of CO2 fixed/O2 evolved, and the main effect of glycerate was on carbon assimilation. While the basis for the beneficial effect of glycerate on CO2 assimilation under moderate to high Pi levels is uncertain, it may increase the concentration of 3-phosphoglycerate (PGA) in the chloroplast, and thus make conditions more favorable for induction of photosynthesis and reduction of PGA to triose phosphate.     

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 degrees 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 phosphoglycerate 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.     

Inhibition of photophosphorylation by ATP and the role of magnesium in photophosphorylation.

ATP and pyrophosphate at high concentration (greater than 1 mM) inhibited photophosphorylation of isolated spinach chloroplasts in the normal salt medium and did not cause stimulation of electron transport. The inhibition of photophosphorylation by ATP or pyrophosphate was shown to be abolished by the addition of excess MgCl2, ADP and phosphate. It has been demonstrated that the rates of photophosphorylation in the absence and presence of ATP or pyrophosphate are determined similarly by the concentrations of magnesium-ADP (Mg - ADP-) and magnesiumphosphate (Mg - Pi) complexes. It is highly probable that Mg - ADP- and Mg - Pi, but not free ADP and free phosphate, are the active form of the substrates of photophosphorylation. This is in support of the view that ATP inhibits photophosphorylation by decreasing the concentration of Mg2+ which is available for the formation of the complex with ADP and phosphate.     

The effect of dihydroxyacetone phosphate and 3-phosphoglycerate on O2 evolution and on the levels of ATP,ADP and Pi in isolated intact chloroplasts

Addition of dihydroxyacetone phosphate (2.5 mM) or 3-phosphoglycerate (2.5 mM) to a suspension of isolated intact chloroplasts, which contains P_i only in low concentrations (0.2 mM) leads to a competitive inhibition of P_i uptake in the light. In consequence, the ATP/ADP ratio is strongly decreased. The rate of O₂ evolution is also reduced under these conditions, but the degree of inhibition is much higher after addition of dihydroxyacetone phosphate than after addition of 3-phosphoglycerate. Therefore, besides the competitive inhibition of P_i uptake, additional effects of dihydroxyacetone phosphate and 3-phosphoglycerate on O₂ evolution and CO₂ fixation of isolated intact chloroplasts must occur, which are discussed.     

CO(2) Assimilation and Malate Decarboxylation by Isolated Bundle Sheath Chloroplasts from Zea mays

Conditions for optimal CO₂ fixation and malate decarboxylation by isolated bundle sheath chloroplasts from Zea mays were examined. The relative rates of these processes varied according to the photosynthetic carbon reduction cycle intermediate provided. Highest rates of malate decarboxylation, measured as pyruvate formation, were seen in the presence of 3-phosphoglycerate, while carbon fixation was highest in the presence of dihydroxyacetone phosphate; only low rates were measured with added ribose-5-phosphate. Chloroplasts exhibited a distinct phosphate requirement and this was optimal at a level of 2 millimolar inorganic phosphate in the presence of 2.5 millimolar 3-phosphoglycerate, dihydroxyacetone phosphate, or ribose-5-phosphate. Malate decarboxylation and CO₂ fixation were stimulated by additions of AMP, ADP, or ATP with half-maximal stimulation occurring at external adenylate concentrations of about 0.15 millimolar. High concentrations (>1 millimolar) of AMP were inhibitory. Aspartate included in the incubation medium stimulated malate decarboxylation and CO₂ assimilation. In the presence of aspartate, the apparent Michaelis constant (malate) for malate decarboxylation to pyruvate by chloroplasts decreased from 6 to 0.67 millimolar while the calculated V_max for this process increased from 1.3 to 3.3 micromoles per milligram chlorophyll. Aspartate itself was not metabolized. It was concluded that the processes mediating the transport of phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate transport on the one hand, and also of malate might differ from those previously described for chloroplasts from C₃ plants.     

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.     

Activation of fructose 1,6-bisphosphatase in darkened intact chloroplasts by NADPH

When intact chloroplasts are incubated in the dark with dihydroxyacetone phosphate, an increase in fructose 1,6-bisphosphatase activity occurs which resembles the reductive activation observed in illuminated chloroplasts. Under optimum conditions, the activity increases to about 150 μmol · h^?1 · mg^?1 chlorophyll within 60 min. The dark activation of the enzyme is reversed by electron acceptors such as oxaloacetate, nitrite, and 3-phosphoglycerate plus ATP. Activation is most marked under strictly anaerobic conditions, being strongly inhibited by O₂. It is concluded that NADPH, generated from dihydroxyacetone phosphate in situ in the reaction catalyzed by NADP⁺-dependent glyceraldehyde phosphate dehydrogenase, can provide electrons for the reductive activation of fructose 1,6-bisphosphatase in the dark.     

Analysis of the role of the phosphate translocator and external metabolites in steady-state chloroplast photosynthesis

The role of the phosphate translocator and the importance of the extrachloroplastic concentrations of phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate in steady-state photosynthesis is examined with a kinetic model. The steady-state stromal concentrations of these compounds are calculated as a function of the rate of the various partial reactions of photosynthesis, at various external concentrations which span those likely to occur in vivo. It is shown how the net transport requirements of the various reactions necessitate different adjustments in the stromal concentrations of these compounds, away from the equilibrium values expected in the absence of metabolism. Under most circumstances, the high exchange capacity of the phosphate translocator relative to the transport requirements of CO₂ fixation limits the extent of these displacements, but conditions when the phosphate translocator is limiting photosynthesis are observed and discussed. The model provides a basis for a more quantitative understanding of the role of the phosphate translocator and the external concentrations of phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate in photosynthesis.     

Inhibited light activation of fructose and sedoheptulose bisphosphatase in spinach chloroplasts exposed to osmotic stress

The light activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) and sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37) was inhibited in isolated intact spinach (Spinacia oleracea L.) chloroplasts exposed to reduced osmotic potentials. Decreases in the velocity and magnitude of light activation correlated with the overall reduction in CO₂ fixation rates. Responses of osmotically stressed chloroplasts to both varying pH and exogeous dihydroxyacetone phosphate (DHAP) or 3-phosphoglycerete (PGA) were examined. In the presence of DHAP, the absolute rate of CO₂ fixation was increased and this increase was most pronounced at alkaline pH. Enhanced light activation of these enzymes was also observed under these conditions. However, in the presence of PGA, similar increases in photosynthetic rate and enzyme activation were not evident. Light-dependent stromal alkalization was unaffected by the stress treatments. Inhibition of light activation under hypertonic conditions is discussed in terms of substrate availability, possible alterations of the redox state of ferredoxin and associated electron carriers, and inhibited enzyme-enzyme or enzyme-substrate interactions involved in the light activation process.Abbreviations and symbols DHAP dihydroxyacetone phosphate - PGA 3-phosphoglycerate - _s osmotic potential     

Cyclic and Non-cyclic Photophosphorylation as Energy Sources for Active K Influx in Hydrodictyon africanum

Under anaerobic conditions in the light, active K influx inHydrodictyon africanum is supported by cyclic photophosphorylation.The use of selective inhibitors shows that, in the presenceof CO₂, a considerable portion of the ATP used by the K pumpis supplied by noncyclic photophosphorylation. The rest of theATP in these conditions comes from cyclic photophosphorylation.This is true under light-limiting as well as light-saturatedconditions. If non-cyclic photophosphorylation is inhibited (by removalof carbon dioxide, by the addition of cyanide which interfereswith the carboxylation reaction, or by inhibition of photosystemtwo with DCMU or supplying only far-red light), the K influxat low light intensities is stimulated, and its characteristicsbecome those of a process powered by cyclic photophosphorylationalone. These results are interpreted in terms of a competitionfor ATP between K influx and CO₂ fixation. Implicit in thisexplanation is a requirement for a switch of excitation energyabsorbed by photosystem one from cyclic photophosphorylationto non-cyclic photophosphorylation whenever conditions (presenceof CO₂and photosystem two activity) allow CO₂ fixation to occur. Further evidence for such a switch of excitation energy absorbedby photosystem one was obtained in experiments in which redand far-red light were applied separately and together. It wasfound that CO₂ fixation showed the Emerson enhancement effect,while K influx (in the presence of CO₂) shows a ‘de-enhancement’.This suggests that far-red light alone powers cyclic photophosphorylation;if red light is also present, some of the far-red quanta arediverted to non-cyclic photophosphorylation. The nature of the interaction between cyclic and non-cyclicphotophosphorylation is discussed in relation to these and otherpublished results.     

Uptake of ATP analogs by isolated pea chloroplasts and their effect on CO2 fixation and electron transport.

1. The ATP analog, adenylyl-imidodiphosphate rapidly inhibited CO2-dependent oxygen evolution by isolated pea chloroplasts. Both alpha, beta- and beta, gamma-methylene adenosine triphosphate also inhibited oxygen evolution. The inhibition was relieved by ATP but only partially relieved by 3-phosphoglycerate. Oxygen evolution with 3-phosphoglycerate as substrate was inhibited by adenylyl-imidodiphosphate to a lesser extent than CO2-dependent oxygen evolution. The concentration of adenylylimidodiphosphate required for 50% inhibition of CO2-dependent oxygen evolution was 50 micronM. 2. Although non-cyclic photophosphorylation by broken chloroplasts was not significantly affected by adenylyl-imidodiphosphate, electron transport in the absence of ADP was inhibited by adenylyl-imidodiphosphate to the same extent as by ATP, suggesting binding of the ATP analog to the coupling factor of phosphorylation. 3. The endogenous adenine nucleotides of a chloroplast suspension were labelled by incubation with [14C]ATP and subsequent washing. Addition of adenylyl-imidodiphosphate to the labelled chloroplasts resulted in a rapid efflux of adenine nucleotides suggesting that the ATP analog was transported into the chloroplasts via the adenine nucleotide translocator. 4. It was concluded that uptake of ATP analogs in exchange for endogenous adenine nucleotides decreased the internal ATP concentration and thus inhibited CO2 fixation. Oxygen evolution was inhibited to a lesser extent in spinach chloroplasts which apparently have lower rates of adenine nucleotide transport than pea chloroplasts.     

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 microM. 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.     

Photosynthesis by isolated chloroplasts. Inhibition by dl-glyceraldehyde of carbon dioxide assimilation

Photosynthetic carbon assimilation and associated CO(2)-dependent O(2) evolution by chloroplasts isolated from pea shoots and spinach leaves is almost completely inhibited by 10mm-dl-glyceraldehyde. The inhibitor is without appreciable effect on photosynthetic electron transport, photophosphorylation, the carboxylation of ribulose 1,5-diphosphate or the reduction of 3-phosphoglycerate, but apparently blocks the conversion of triose phosphate into ribulose 1,5-diphosphate.     

Phenylmereuric Acetate and Phosphate Control of Light-Dependent Shrinkage and Reactions in Chloroplasts

An investigation of the action of phenylmereuric acetate (PMA) and phosphate on light-induced shrinkage (measured by light scattering and Coulter Counter techniques) and on photosynthetic reactions in spinach chloroplasts led to the following conclusions:

• 1) PMA stimulated light-induced shrinkage (under conditions of cyclic and non-cyclic electron flow) at concentrations which completely inhibited cyclic and non-cyclic photophosphorylation and nicotinamide adenine dinucleotide phosphate (NADP) reduction, though ferricyanide reduction was activated. Although PMA inhibited NADP reduction (probably because this sulfhydryl reagent interfered with the ferredoxin-NADP rednetase) it ean also be considered an uncoiipler (when ferricyanide is the electron acceptor).
• 2) Phosphate maximized light-induced shrinkage (under conditions of cyclic and non cyclic electron flow) at concentrations which did not affect ferricyanide reduction but caused a 40 to 50 per cent inhibition of NADP reduction.
• 3) The pattern of the light scattering response to these two compounds was quite different. In the presence of PMA, the forward (light on) and hack (light off) reactions went to completion rapidly. In the presence of phosphate, the back reaction was rapid but, in the light-induced reaction, three phases were discernible.
• 4) Compared with uncouplers such as NH₄Cl, carbonyl cyanide m-chlorophenyl-hydrazone, pentachlorophenol, and dicoumarol, all of which inhibited both photophosphorylation and conformational changes in chloroplasts, PMA (like quinacrine) had a specific action since it inhibited photophosphorylation while shrinkage was stimulated.
• 5) It appeared that PMA acted at a site beyond the formation of high energy inter-mediates and that, in the absence of photophosphorylation, more energy was diverted to mechanical work (shrinkage). It would seem that, in a cyclic electron flow system, in which ATP synthesis is blocked at a late step (e.g. by PMA), shrinkage may be an indirect method for measuring electron flow.

     

The C4-dicarboxylic acid pathway of photosynthesis. Identification of intermediates and products and quantitative evidence for the route of carbon flow

1. When leaves with the C(4)-dicarboxylic acid pathway of photosynthesis are exposed to (14)CO(2) the major labelled compounds formed, in order of labelling, are dicarboxylic acids, 3-phosphoglycerate, bexose phosphates and sucrose. During the present studies several quantitatively minor intermediates were identified and their labelling behaviour is described. 2. The pattern of labelling of dihydroxyacetone phosphate, fructose 1,6-diphosphate and ribulose di- and mono-phosphates during radiotracer pulse-chase experiments was consistent with their operation as intermediates in the pathway of carbon dioxide fixation. 3. Serine, glycine, alanine and glutamate had labelling patterns typical of products secondary to the main flow of carbon. 4. The mechanism of the transfer of label from C-4 of dicarboxylic acids to C-1 of 3-phosphoglycerate was also examined. Evidence consistent with pyruvate being derived from C-1, C-2 and C-3 of oxaloacetate, and for a relationship between ribulose 1,5-diphosphate and the acceptor for the C-4 carboxyl group, was obtained. 5. Evidence is provided that, under steady-state conditions, essentially all the label incorporated from (14)CO(2) into C-1 of 3 phosphoglycerate enters via C-4 of the dicarboxylic acids. These and other studies indicated that the route via dicarboxylic acids is essentially the sole route for entry of carbon into 3-phosphoglycerate.     

Effects of glycidate on carbon dioxide fixation with isolated spinach chloroplasts

Glycidate (2,3-epoxypropionate) stimulated CO₂ fixation in isolated spinach chloroplasts up to 100%. In the presence of glycidate the initial lag phase was abolished and the chloroplasts exported mainly 3-phosphoglycerate instead of dihydroxyacetone phosphate.     

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.     

Effects of the relative extrachloroplastic concentrations of inorganic phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate on the rate of starch synthesis in isolated spinach chloroplasts

The effect of external inorganic phosphate (Pi) on starch synthesis in isolated spinach (Spinacia oleracea American Hybrid No. 424) chloroplasts in the presence of millimolar concentrations of 3-phosphoglycerate (PGA) and/or dihydroxyacetone phosphate (DAP) was examined. Whereas CO₂ fixation was relatively constant as the ratio of the external phosphate to the PGA + DAP varied from 1:3 to 3:1, starch synthesis varied from 17% to 2% of the CO₂ fixation rate. With DAP alone, maximal starch synthesis was about 10% of the CO₂ fixation rate. The data demonstrate that the Pi/(PGA + DAP) ratio in the cytoplasm of plant cells could serve to regulate the flow of newly fixed carbon into starch without alterations in the rate of CO₂ fixation.