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.     

The effect of O2 and CO2 concentration on photosynthesis and glycolate accumulation in bean leaves treated with α-hydroxy-2-pyridinemethanesulfonic acid (α-HPMS), the glycolate oxidase inhibitor

¹⁴CO₂ assimilation, ¹⁴C incorporation into glycolate and glycolate accumulation in -HPMS treated bean leaves at various O₂ and CO₂ concentrations were studied. In 1% CO₂ oxygen concentration had no significant effect on glycolate accumulation and ¹⁴C incorporation into glycolate. In the CO₂ concentration range of 0.03% to 0.01%, increased oxygen concentration decreased not only ¹⁴CO₂ assimilation but also glycolate accumulation and ¹⁴C incorporation into glycolate. In 1% and 0.1% CO₂, no matter what O₂ concentration was supplied, and in 0.03% CO₂ with 2% and 21% O₂, all of the glycolate accumulated was formed from newly assimilated carbon. In 0.01% CO₂ and 2%, 21% and 100% O₂, and in 0.03% CO₂ with 100% O₂, a substantial portion of the glycolic acid that accumulated in leaves originated from endogenous unlabelled substrates. These findings are discussed in terms of possible changes in the ratio of RuBP carboxylation to RuBP oxygenation and of changes of RuBP pool size, induced by changing O₂ and CO₂ concentrations.This work was supported by the Polish Academy of Sciences, Contract No. 10.2.10.     

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.     

Characterization of a Photosynthesizing Reconstituted Spinach Chloroplast Preparation : REGULATION BY PRIMER, ADENYLATES, FERREDOXIN, AND PYRIDINE NUCLEOTIDES

A particulate preparation (MgP) capable of photosynthetic CO₂ assimilation without the addition of stromal protein was obtained by rupturing whole spinach (Spinacia oleracea var. America) chloroplasts in 15 millimolar MgCl₂ buffered with Tricine at pH 8.5. This CO₂ assimilation was dependent upon light, inorganic phosphate, ferredoxin, ADP, NAD or NADP, and primer. Excepting glycolate, the products of CO₂ fixation by MgP were similar to those found with whole chloroplasts.     

Glycolate synthesis by intact chloroplasts: Studies with inhibitors of photophosphorylation

Intact spinach chloroplasts, capable of evolving O₂ in response to CO₂ at rates greater than 70 μmol/h · mg of chlorophyll, synthesize glycolate from added dihydroxyacetone phosphate, ribose 5-phosphate, or xylulose 5-phosphate, when illuminated in the presence of O₂. The synthesis of glycolate from these compounds is dependent upon photophosphorylation and is inhibited by each of the three classes of photophosphorylation inhibitors [Izawa, S., and Good, N. E. (1972) in Methods in Enzymology, Vol. 24, Part B, pp. 355–377)]: an uncoupler, carbonylcyanide-4-trifluoromethoxyphenylhydrazone (FCCP), an energy transfer inhibitor, Dio-9, and a phosphate analog, arsenate. Neither arsenate nor Dio-9 causes the collapse of the light-generated proton gradient between thylakoid and stroma compartments of the chloroplasts, so that the inhibition of glycolate synthesis by these compounds cannot be ascribed to an inactivation of Calvin cycle enzymes thought to be associated with the collapse of such a proton gradient. The dependency of glycolate synthesis upon photophosphorylation indicates that an ATP-consuming reaction(s) is involved in the conversion of the substrates (triose and pentose monophosphates) to glycolate. The formation of dihydroxyethylthiamine pyrophosphate, the “active glycolaldehyde” intermediate of the transketolase reaction, from triose and pentose monophosphates has no known requirements for ATP. On the other hand, the conversion of both triose and pentose monophosphates to ribulose 1,5-bisphosphate, the substrate for the ribulose 1,5-bisphosphate oxygenase reaction, requires ATP. It is concluded that glycolate synthesis by intact isolated chloroplasts is primarily the result of ribulose 1,5-bisphosphate oxygenase activity. No substantial role in glycolate synthesis can be attributed to the oxidation of dihydroxyethylthiamine pyrophosphate, the intermediate of the transketolase reaction.     

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.     

Dependence of the membrane potential on intracellular ATP concentration in tonoplast-free cells of Nitellopsis obtusa

Intact chloroplasts were obtained from mesophyll protoplasts isolated from Mesembryanthemum crystallinum in the C₃ or Crassulacean acid metabolism (CAM) photosynthetic mode, and examined for the influence of inorganic phosphate (Pi) on aspects of bicarbonate-dependent O₂ evolution and CO₂ fixation. While the chloroplasts from both modes responded similarly to varying Pi, some features appear typical of chloroplasts from species capable of CAM, including a relatively high capacity for photosynthesis in the absence of Pi, a short induction period, and resistance to inhibition of photosynthesis by high levels of Pi. In the absence of Pi the chloroplasts retained 75–85% of the ¹⁴CO₂ fixed and the total export of dihydroxyacetone phosphate was low compared with the rate of photosynthesis. In CAM plants the ability to conduct photosynthesis and retain most of the fixed carbon in the chloroplasts at low external Pi concentrations may enable storage of carbohydrates which are essential for providing a carbon source for the nocturnal synthesis of malic acid. At high external Pi concentrations (e.g. 10 25 mM), the amount of total dihydroxyacetone phosphate exported to the assay medium relative to the rate of photosynthesis was high while the products of ¹⁴CO₂ fixation were largely retained in the chloroplasts which indicates starch degradation is occurring at high Pi levels. Starch degradation normally occurs in CAM plants in the dark; high levels of Pi may induce starch degradation in the light which has the effect of limiting export of the immediate products of photosynthesis and thus the degree of Pi inhibition of photosynthesis with the isolated chloroplast.     

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.     

Photoassimilation of CO2 by Isolated Bundle-Sheath Strands of Zea mays I. Stimulation of CO2 Assimilation by Adding Various Intermediates of the Photosynthetic Cycle; Evidence for a Deficient Photosystem II Activity

Isolated bundle-sheath (BS) strands from leaves of mature maize plants show enhanced rates of CO₂ fixation in the presence of reduced intermediates of the photosynthetic cycle (R5P, DHAP, FruDP.) 3PGA is the major labelled product of ¹⁴CO₂ fixation whatever the substrate added. CO₂ fixation is much lower with PGA than with reduced intermediates, suggesting a limited capacity of the cells to regenerate RuDP (the CO₂-acceptor) from PGA. These two experimental facts, which are characteristic features of bundle-sheath photosynthesis for maize (a species with agranal bundle-sheath chloroplasts) indicate that phaotosystem II activity is a limiting factor for the evolution of the bundle-sheath photosynthetic process. Nevertheless, a reducing capacity arises as proved by sensitivity of CO₂ fixation to DCMU, particularly when PGA is added to the bundle-sheath. PGA synthesis occurs, in the presence of non-limiting amounts of CO₂, according to the equation: RuDP + CO₂→ 2 PGA; the oxygen effect on ¹⁴CO₂ fixation, at lower CO₂ concentration, is interpreted as a dilution effect of the internal pool of ¹⁴CO₂ by unlabelled CO₂ generated by photorespiration.     

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.     

Inhibition of photorespiration and increase of net photosynthesis in isolated maize bundle sheath cells treated with glutamate or aspartate

Net photosynthetic ¹⁴CO₂ fixation by isolated maize (Zea mays) bundle sheath strands was stimulated 20 to 35% by the inclusion of l-glutamate or l-aspartate in the reaction mixture. Maximal stimulation occurred at a 7.5 mm concentration of either amino acid. Since the photosynthetic rate and the glutamate-dependent stimulation in the rate were equally sensitive to a photosynthetic electron transport inhibitor, 3-(p-chlorophenyl)-1,1-dimethylurea, it was concluded that glutamate did not stimulate CO₂ fixation by supplying needed NADPH (NADH) through glutamate dehydrogenase. Treatment of the bundle sheath strands with glutamate inhibited glycolate synthesis by 59%. Photorespiration in this tissue, measured as the O₂ inhibition of CO₂ fixation (the Warburg effect), was inhibited by treatment with glutamate. The stimulation in net photosynthetic CO₂ fixation probably results from the decrease in photorespiratory CO₂ loss. This metabolic regulation of the rate of glycolate synthesis and photorespiration observed with isolated bundle sheath strands could account for the inability to detect rapid photorespiration in the mature intact maize leaf.     

Influence of pH upon the Warburg Effect in Isolated Intact Spinach Chloroplasts: I. Carbon Dioxide Photoassimilation and Glycolate Synthesis

The influence of pH upon the O₂ inhibition of ¹⁴CO₂ photoassimilation (Warburg effect) was examined in intact spinach (Spinacia oleracea) chloroplasts. With conditions which favored the Warburg effect, i.e. rate-limiting CO₂ and 100% O₂, O₂ inhibition was greater at pH 8.4 to 8.5 than at pH 7.5 to 7.8. At pH 8.5, as compared with 7.8, there was an enhanced ¹⁴C-labeling of glycolate, and a decrease of isotope in some phosphorylated Calvin cycle intermediates, particularly triose-phosphate. The ¹⁴C-labeling of starch was also more inhibited by O₂ at higher pH. The enhanced synthesis of glycolate during ¹⁴CO₂ assimilation at higher pH resulted in a diminution in the level of phosphorylated intermediates of the Calvin cycle, and this was apparently a causal factor of the increased severity of the Warburg effect.     

Glycolate biosynthesis by Scenedesmus and Chlorella in the presence or absence of NaHCO3

Summary Both Scenedesmus and Chlorella excreted comparable quantities of glycolate. Glycolate formation was dependent upon light and oxygen, but occured in the absence of added CO₂ or NaHCO₃ for net photosynthesis. In an environment of 3000 ft. c. light and an atmosphere of oxygen, about 35 g glycolate were excreted per hour per milliliter 1% (v/v) algae without NaHCO₃ or CO₂. Upon addition of NaHCO₃ the rate increased to about 55 g. Glycolate formation in the light in the absence of CO₂ may result from photometabolism of algal polysaccharides.Glycolate excretion by Scenedesmus occurred at all pH values between 6.5 and 9.5 and was not related to utilization of bicarbonate. Scenedesmus obliquus excreted glycolate when existing in plates of four or eight cells, but not when present as small individual cells.At pH 9 ¹⁴C fixation by Scenedesmus was faster than fixation by Chlorella. There was no significant difference in products of ¹⁴C fixation formed by Scenedesmus at pH values between 6.5 and 9.5.For unknown reasons -hydroxy-2-pyridinemethanesulfonate stimulated CO₂ fixation by Scenedesmus by at least 100%. This sulfonate had no effect on glycolate excretion nor upon the distribution of ¹⁴C among the products of ¹⁴CO₂ fixation by Scenedesmus.Supported in part by NSF Grant GB-4154 and published with the approval of the Director of the Michigan Agricultural Experiment Station as journal article No. 3946. The research was initiated during the period when N. E. Tolbert was supported in part by a National Institutes of Health Senior Fellowship at the Biochemisches Institut, Universität, Freiburg/Br., Germany.     

Photosynthetic and Photorespiratory Carbon Metabolism in Mesophyll Protoplasts and Chloroplasts Isolated from Isogenic Diploid and Tetraploid Cultivars of Ryegrass (Lolium perenne L.)

Photosynthetic ¹⁴CO₂ fixation, [¹⁴C]glycolate formation, and the decarboxylation of [1-¹⁴C]glycolate and [1-¹⁴C]glycine by leaf mesophyll protoplasts isolated from isogenic diploid and tetraploid cultivars of ryegrass (Lolium perenne L.) were examined. The per cent O₂ inhibition of photosynthesis in protoplasts from the tetraploid cultivar was less than that of the diploid line at both 21 and 49% O₂. Kinetic studies revealed that the K_m (CO₂) for photosynthesis by the diploid protoplasts was about twice that of the tetraploid line. In contrast, the K_i (O₂) for protoplast photosynthesis was similar in both cultivars, as was the potential for oxidizing glycolate and glycine to CO₂ via the photorespiratory carbon oxidation cycle. Although the maximal rates of glycolate accumulation by the isolated protoplasts in the presence of 21% O₂ and a glycolate oxidase inhibitor were similar in the two cultivars, the percentage of total fixed ¹⁴C entering the [¹⁴C]glycolate pool and the ratio of the rate of [¹⁴C]glycolate formation to ¹⁴CO₂ fixation at 21% O₂ and low pCO₂ were about two times greater in protoplasts and intact chloroplasts isolated from the diploid line compared to the tetraploid. These results fully support the recent observation that a doubling of ploidy in various ryegrass cultivars reduced the K_m (CO₂) of purified ribulose bisphosphate carboxylase-oxygenase by about one-half without affecting the K_i (O₂) (Garrett 1978 Nature 274: 913-915).     

Fixation of O(2) during Photorespiration: Kinetic and Steady-State Studies of the Photorespiratory Carbon Oxidation Cycle with Intact Leaves and Isolated Chloroplasts of C(3) Plants

Mass spectrometric techniques were used to trace the incorporation of [¹⁸O]oxygen into metabolites of the photorespiratory pathway. Glycolate, glycine, and serine extracted from leaves of the C₃ plants, Spinacia oleracea L., Atriplex hastata, and Helianthus annuus which had been exposed to [¹⁸O]oxygen at the CO₂ compensation point were heavily labeled with ¹⁸O. In each case one, and only one of the carboxyl oxygens was labeled. The abundance of ¹⁸O in this oxygen of glycolate reached 50 to 70% of that of the oxygen provided after only 5 to 10 seconds exposure to [¹⁸O]oxygen. Glycine and serine attained the same final enrichment after 40 and 180 seconds, respectively. This confirms that glycine and serine are synthesized from glycolate.

The labeling of photorespiratory intermediates in intact leaves reached a mean of 59% of that of the oxygen provided in the feedings. This indicates that at least 59% of the glycolate photorespired is synthesized with the fixation of molecular oxygen. This estimate is certainly conservative owing to the dilution of labeled oxygen at the site of glycolate synthesis by photosynthetic oxygen. We examined the yield of ¹⁸O in glycolate synthesized in vitro by isolated intact spinach chloroplasts in a system which permitted direct sampling of the isotopic composition of the oxygen at the site of synthesis. The isotopic enrichment of glycolate from such experiments was 90 to 95% of that of the oxygen present during the incubation.

The carboxyl oxygens of 3-phosphoglycerate also became labeled with ¹⁸O in 20- and 40-minute feedings with [¹⁸O]oxygen to intact leaves at the CO₂ compensation point. Control experiments indicated that this label was probably due to direct synthesis of 3-phosphoglycerate from glycolate during photorespiration. The mean enrichment of 3-phosphoglycerate was 14 ± 4% of that of glycine or serine, its precursors of the photorespiratory pathway, in 10 separate feeding experiments. It is argued that this constant dilution of label indicates a constant stoichiometric balance between photorespiratory and photosynthetic sources of 3-phosphoglycerate at the CO₂ compensation point.

Oxygen uptake sufficient to account for about half of the rate of ¹⁸O fixation into glycine in the intact leaves was observed with intact spinach chloroplasts. Oxygen uptake and production by intact leaves at the CO₂ compensation point indicate about 1.9 oxygen exchanged per glycolate photorespired. The fixation of molecular oxygen into glycolate plus the peroxisomal oxidation of glycolate to glyoxylate and the mitochondrial conversion of glycine to serine can account for up to 1.75 oxygen taken up per glycolate.

These studies provide new evidence which supports the current formulation of the pathway of photorespiration and its relation to photosynthetic metabolism. The experiments described also suggest new approaches using stable isotope techniques to study the rate of photorespiration and the balance between photorespiration and photosynthesis in vivo.

     

14CO2 Fixation and Compartmentation of Carbon Metabolism in a Recombined Chloroplast- 'Cytoplasm' System

The Compartmentation of ¹⁴CO₂ fixation and concomitant metabolism of ^l4C-iabelled products in a recombined system, composed of isolated intact spinach (Spinacia oleracea) chloroplasls and a ‘cytoplasm’ fraction, has been studied. Addition of ‘cytoplasm’ to chloroplasts fixing ¹⁴CO₂ increased the label in hexoae monophosphates outside the chloroplasts at the expense of excreted dihydroxyacetone phosphate. The label in ammo acids was increased both inside and outside the chloroplasts. The results support the view that chloroplasls are not able to make 2-oxoacids for amino acid synthesis directly from fixed CO₂, but have to co-operate with the cytoplasm and other organelles. The results also show that recombined systems can be useful for studies on the compartmen tation of carbon metabolism in pholosynthesizing plant tissues.     

The role of calcium ions and the thioredoxin system in regulation of spinach chloroplast fructosebisphosphatase

Illumination of isolated type A spinach chloroplasts causes a rapid increase in their activity of fructosebisphosphatase, as assayed at physiological substrate and Mg²⁺ concentrations. Activation is accelerated by addition of dihydroxyacetone phosphate to the chloroplasts and decreased by inorganic phosphate concentrations greater than those optimal for CO₂ fixation. At all times, measured fructosebisphosphatase activity was greater than was necessary to account for the observed rates of CO₂ fixation. Activation of purified fructosebisphosphatase $\text{in vitro}$ by dithiothreitol or reduced thioredoxin is extremely slow, but is greatly accelerated in the presence of physiological concentrations of Mg²⁺ and fructosebisphosphate if Ca²⁺ ions are present. Increased concentrations of fructosebisphosphate greatly increase the rate and extent of activation whereas in the absence of fructosebisphosphate Ca²⁺ ions have no effect. Neither inorganic phosphate nor dihydroxyacetone phosphate significantly affect the rate of activation. Ca²⁺ ions strongly inhibit the activity of the activated form of fructosebisphosphatase. It is proposed that free Ca²⁺ ions within chloroplasts are involved in preventing fructosebisphosphatase from functioning in the dark, and that free and/or bound Ca²⁺ facilitates the rapid reductive activation of this enzyme when the light is turned on again.     

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.     

On the light dependence of Fatty Acid synthesis in spinach chloroplasts

The capacity of intact chloroplasts to synthesize long chain fatty acids from acetate depends on the stroma pH in Spinacia oleracea, U. S. hybrid 424. The pH optimum is close to 8.5. Lowering of the stroma pH leads to a reduction of acetate incorporation but does not suffice to eliminate fatty acid synthesis completely. Chain elongation from palmitic to oleic acid shows the same pH dependence. Fatty acid synthesis is activated in the dark upon the simultaneous addition of dihydroxyacetone phosphate and orthophosphate supplying ATP and oxaloacetate for reoxidation of NADPH in the stroma. Under these conditions both dark fatty acid synthesis and synthesis of oleate from palmitate show the same pH dependence as in the light. Dark fatty acid synthesis is further stimulated by increasing the stromal Mg²⁺ concentration with the ionophore A 23187. In contrast to CO₂ fixation, dark fatty acid synthesis is considerably reduced by dithiothreitol (DTT). This observation may be due to an acetyl-CoA deficiency, caused by a nonenzymic acylation of DTT, and a competition for ATP between DTT-activated CO₂ fixation and fatty acid synthesis. Because d,l-glyceraldehyde as inhibitor of CO₂ fixation compensates the DTT effect on dark fatty acid synthesis, reducing equivalents may be involved in the light dependence of acetate activation.