Photosynthetic carbon metabolism in isolated pea chloroplasts: metabolite levels and enzyme activities

We report here that enzyme activation precedes the rise in metabolite levels, which appear to limit photosynthetic CO2 fixation during induction in pea leaf chloroplasts. Therefore light activation may be required for the build-up of photosynthetic intermediates and hence for photosynthesis in isolated chloroplasts. Analysis of metabolite levels and the known kinetic properties of the chloroplast enzymes indicates that the reductive pentose phosphate cycle is subject to control which fluctuates between several points during induction and when CO2 fixation is maximal. The transketolase-aldolase-catalyzed reactions around sedoheptulose-biphosphatase appear to provide a simple and effective primary control for photosynthetic CO2 fixation. When substrate levels and enzyme active site concentrations are taken into account, there is insufficient glyceraldehyde 3-phosphate dehydrogenase, aldolase, and transketolase activity to support photosynthetic CO2 fixation at observed rates. These results suggest that there may be direct transfer of glyceraldehyde 3-phosphate among these enzymes in the pea chloroplast.     

Photosynthetic control by isolated pea chloroplasts

Isolated pea chloroplasts undergo both cyclic and non-cyclic electron flow. Both processes are coupled to photophosphorylation. During non-cyclic flow the rate of oxygen production showed ADP-governed ;photosynthetic control' analogous to respiratory control of isolated mitochondria. Measurements of ADP/O and photosynthetic control ratios yielded values of 1-1.3 and 2-5.7 respectively. ;Photosynthetic control' was shown to be dependent on the intactness of the chloroplasts.     

Photosynthetic carbon metabolism in isolated maize bundle sheath strands

Photosynthetically active bundle sheath strands capable of assimilating up to 8 micromoles CO₂ per milligram chlorophyll per hour have been isolated from fully expanded leaves of Zea mays L. Mesophyll cell contamination of the preparations was negligible, as evidenced by light and electron microscopy and by a high ratio of chlorophyll a to chlorophyll b in the strands. Ribose 5-phosphate markedly stimulated the rate of photosynthetic ¹⁴CO₂ fixation by the isolated strands. In contrast, both pyruvate and phosphoenolpyruvate had a comparatively small stimulatory effect on bundle sheath ¹⁴CO₂ fixation. After 5 minutes of photosynthesis in ¹⁴C-bicarbonate, 95% of the incorporated ¹⁴C was found in compounds other than C₄-dicarboxylic acids, most notably in 3-phosphoglycerate and sugar phosphates. A similar distribution of ¹⁴C was observed in the presence of exogenous ribose 5-phosphate. Extracts of bundle sheath strands contained high specific activities of “malic” enzyme, phosphoglycolate phosphatase, hydroxypyruvate reductase, and ribulose 1,5-diphosphate carboxylase, whereas the specific activities of NADP⁺-malate dehydrogenase and phosphopyruvate carboxylase were extremely low. These results indicate that the Calvin cycle occurs in the bundle sheath cells of maize.     

Inhibition of photosynthetic carbon metabolism in isolated chloroplasts by iodoacetol phosphate

Carbon dioxide-dependent and 3-phosphoglycerate (PGA)-dependent O₂ evolution by isolated chloroplasts of wheat is inhibited by micromolar levels of iodoacetol phosphate (IAP). Loss of the activity is time-dependent and a higher concentration of PGA increases the half-time for inhibition (e.g. at 40 micromolar IAP the half-time is about 0.5 minutes at 1 millimolar PGA compared to 1.5 minutes at 10 millimolar PGA). A marked inhibition of NADP glyceraldehyde-3-P dehydrogenase was observed when chloroplasts were pretreated with micromolar levels of IAP, osmotically shocked, and several stromal enzymes assayed.     

Photosynthetic activity of isolated chloroplasts from Euglena gracilis

Functional chloroplasts from photoheterotrophic Euglena gracilis can be isolated in isoosmotic gradients of 10–80% Percoll. The chloroplasts display rates of CO₂ dependent O₂ evolution and CO₂ fixation of 30–50 mol mg^-1 chlorophyll h^-1 or 25–35% of the net O₂ evolution by the whole cells and appear to be strikingly different from spinach chloroplasts in several respects: 1. tolerance to high concentration of orthophosphate in the assay medium; 2. inability to support oxaloacetate-dependent O₂ evolution; 3. ability to support only low to moderate rates of 3-phosphoglycerate-dependent O₂ evolution; 4. an apparent absence of a phosphate translocator in the terms described by Heldt and Rapley ([1970] FEBS Lett. 10, 143–148).University of California, Dept. of Plant and Soil Biology, 108 Hilgard Hall, Berkeley, CA 94720 USA     

Assimilation of carbon dioxide in mesophyll chloroplasts and bundle sheath strands mechanically isolated from corn leaves

Mesophyll chloroplasts capable of assimilating 1.2 µmolesCO₂ per milligram chlorophyll per hour were isolated from 7-day-oldcorn (Zea mays, Nagano No. 1) leaves. Addition of phosphoenolpyruvateincreased the rate of CO₂ fixation in light up to 22 µmolesper milligram chlorophyll per hour, whole exogenously addedribose 5-phosphate and adenosine triphosphate brought aboutonly small increases. The CO₂ fixation products were mostlymalate and aspartate. Bundle sheath strands isolated from the same plants were capableof assimilating 3–26 µmoles CO₂ per milligram chlorophyllper hour. The fixation rate increased 3- to 5-fold on additionof ribose 5-phosphate and adenosine triphosphate, while exogenousphosphoenolpyruvate had no effect. The bulk of early productsof light-induced CO₂ fixation were phosphate esters. These results indicate that corn mesophyll chloroplasts initiallyfix CO₂ by phoenolpyruvate carboxylase and that reductive pentosephosphate cycle occurs in corn bundle sheath cells, but notin the mesophyll chloroplasts. (Received January 25, 1974; )     

Regulation of photosynthetic carbon metabolism during phosphate limitation of photosynthesis in isolated spinach chloroplasts

Intact chloroplasts isolated from spinach were illuminated in the absence of inorganic phosphate (Pi) or with optimum concentrations of Pi added to the reaction medium. In the absence of Pi photosynthesis declined after the first 1–2 min and was less than 10% of the maximum rate after 5 min. Export from the chloroplast was inhibited, with up to 60% of the ¹⁴C fixed being retained in the chloroplast, compared to less than 20% in the presence of Pi. Despite the decreased export, chloroplasts depleted of Pi had lower levels of triose phosphate while the percentage of total phosphate in 3-phosphoglycerate was increased. Chloroplast ATP declined during Pi depletion and reached dark levels after 3–4 min in the light without added Pi. At this point, stromal Pi concentration was 0.2 mM, which would be limiting to ATP synthesis. Addition of Pi resulted in a rapid burst of oxygen evolution which was not initially accompanied by net CO₂ fixation. There was a large decrease in 3-phosphoglycerate and hexose plus pentose monophosphates in the chloroplast stroma and a lesser decrease in fructose-1,6-bisphosphate. Stromal levels of triose phosphate, ribulose-1,5-bisphosphate and ATP increased after resupply of Pi. There was an increased export of ¹⁴-labelled compounds into the medium, mostly as triose phosphate. Light activation of both fructose-1,6-bisphosphatase and ribulose-1,5-bisphosphate carboxylase was decreased in the absence of Pi but increased following Pi addition.It is concluded that limitation of Pi supply to isolated chloroplasts reduced stromal Pi to the point where it limits ATP synthesis. The resulting decrease in ATP inhibits reduction of 3-phosphoglycerate to triose phosphate via mass action effects on 3-phosphoglycerate kinase. The lack of Pi in the medium also inhibits export of triose phosphate from the chloroplast via the phosphate transporter. Other sites of inhibition of photosynthesis during Pi limitation may be located in the regeneratige phase of the reductive pentose phosphate pathway.Abbreviations FBP Fructose-1,6-bisphosphate - FBPase Fructose-1,6-bisphosphatase - MP Hexose plus pentose monophosphates - PGA 3-phosphoglycerate - Pi inorganic orthophosphate - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase - TP Triose Phosphate     

Photosynthetic characteristics of chloroplasts isolated fromMesembryanthemum crystallinum L., a halophilic plant capable of Crassulacean acid metabolism

Photosynthetically highly active chloroplasts were routinely obtained by rupture of leaf protoplasts from the halophyteMesembryanthemum crystallinum which exhibited the photosynthetic characteristics of either a C₃ plant when grown with 20 mmol l^-1 NaCl in the rooting medium, or a Crassulacean-acid-metabolism (CAM) plant when grown with 400 mmol l^-1 NaCl. Photosynthesis rates of C₃ and CAM chloroplasts were 150–250 and 90–150 μmol mg^-1 chlorophyll h^-1, respectively. Because of osmotic adjustment, CAM chloroplasts required higher sorbitol concentrations (0.7–0.8 mol l^-1) in the assay medium than C₃ chloroplasts (0.3–0.4 mol l^-1) for optimum activity. Substitution of sorbitol by NaCl as the osmoticum strongly reduced photosynthesis of CAM chloroplasts. Rates of electron transport (ferricyanide reduction, uncoupled) remained unaffected over a range of sorbitol concentrations (0 to 1 mol l^-1). Sensitivity of electron transport to increasing levels of NaCl was less pronounced than the NaCl-sensitivity of CO₂ fixation by intact chloroplasts. The CAM chloroplasts showed a broad pH optimum of photosynthesis between pH 7.0 and 8.2; photosynthesis of C₃ chloroplasts dropped markedly below pH 7.6. The CAM chloroplasts maintained a higher transenvelope proton gradient than C₃ chloroplasts both in the light and dark. External pyruvate (5 mmol l^-1) inhibited photosynthesis of CAM chloroplasts, but not of C₃ chloroplasts. Inhibition was reduced by increased external concentrations of orthophosphate.     

Light-enhanced carbon dioxide fixation in isolated chloroplasts

Preillumination of leaves of spinach, soybean and maize in theabsence of CO₂ greatly enhanced the capacity for fixing CO₂in an immediately following dark period. Lightenhanced darkCO₂-fixation was further observed in isolated chloroplasts ofspinach and soybean. When isolated chloroplasts were illuminated,CO₂-fixing capacity in the subsequent dark period increasedrapidly at first and later more slowly attaining a stationaryvalue in about 20 min. When the light was turned off at thisstage, the capacity decreased very rapidly becoming zero inabout 10 min. The magnitude of the enhanced dark fixation andits decay in the dark were not influenced by the presence orabsence of atmospheric oxygen. In both leaves and isolated chloroplasts,no significant change in oxygen (21%) occurred in distributionpatterns of radioactivity in products fixed by photosynthetic,or light-enhanced, dark, ¹⁴CO₂-fixation. In preilluminated leaves¹⁴C was incorporated into sucrose in the subsequent dark period,indicating that the photosynthetic carbon reduction cycle isoperating in light-enhanced dark fixation in higher plants. ¹Present address: Noda Institute for Scientific Research, Noda,Chiba Prefecture (Received August 10, 1970; )     

Photosynthetic control and photophosphorylation in photosystem II of isolated spinach chloroplasts

Two cycles of photosynthetic control have been observed in isolated spinach chloroplasts in the presence of lipophilic class III electron acceptors, which may accept electrons at PS II. $\text{ADP}\text{O}$ ratios of 0.8 to 0.9 were recorded;rates of oxygen evolution were stimulated by phosphorylating reagents and uncouplers. Addition of the plastoquinone antagonist DBMIB decreased photosynthetic control, oxygen evolution and photophosphorylation. We believe that there is a coupling site associated with PSII which can be rate limiting. Comparison of the $\text{P}\text{2e}$ ratios observed with class I and class III electron acceptors leads us to propose that more than 0.6 and possibly approaching one molecule of ATP can be formed for every pair of electrons transported from water to PSII acceptors.     

Photosynthetic carbon metabolism of a marine grass

The δ¹³C value of a tropical marine grass Thalassia testudinum is −9.04‰. This value is similar to the δ¹³C value of terrestrial tropical grasses. The δ¹³C values of the organic acid fraction, the amino acid fraction, the sugar fraction, malic acid, and glucose are: −11.2‰, −13.1‰, −10.1‰, −11.1‰, and −11.5‰, respectively. The δ¹³C values of malic acid and glucose of Thalassia are similar to the δ¹³C values of these intermediates in sorghum leaves and attest to the presence of the photosynthetic C₄-dicarboxylic acid pathway in this marine grass. The inorganic HCO₃⁻ for the growth of the grass fluctuates between −6.7 to −2.7‰ during the day. If CO₂ fixation in Thalassia is catalyzed by phosphoenolpyruvate carboxylase (which would result in a −3‰ fractionation between HCO₃⁻ and malic acid), the predicted δ¹³C value for Thalassia would be −9.7 to −5.7‰. This range is close to the observed range of −12.6 to −7.8‰ for Thalassia and agree with the operation of the C₄-dicarboxylic acid pathway in this plant. The early products of the fixation of HCO₃⁻ in the leaf sections are malic acid and aspartic acid which are similar to the early products of CO₂ fixation in C₄ terrestrial plants.     

Photosynthetic oxygen reduction in isolated intact chloroplasts and cells in spinach

The time course of light-induced O(2) exchange by isolated intact chloroplasts and cells from spinach was determined under various conditions using isotopically labeled O(2) and a mass spectrometer. In dark-adapted chloroplasts and cells supplemented with saturating amounts of bicarbonate, O(2) evolution began immediately upon illumination. However, this initial rate of O(2) evolution was counterbalanced by a simultaneous increase in the rate of O(2) uptake, so that little net O(2) was evolved or consumed during the first approximately 1 minute of illumination. After this induction (lag) phase, the rate of O(2) evolution increased 3- to 4-fold while the rate of O(2) uptake diminished to a very low level. Inhibition of the Calvin cycle, e.g. with dl-glyceraldehyde or iodoacetamide, had negligible effects on the initial rate of O(2) evolution or O(2) uptake; both rates were sutained for several minutes, and about balanced so that no net O(2) was produced. Uncouplers had an effect similar to that observed with Calvin cycle inhibitors, except that rates of O(2) evolution and photoreduction were stimulated 40 to 50%.These results suggest that higher plant phostosynthetic preparations which retain the ability to reduce CO(2) also have a significant capacity to photoreduce O(2). With near-saturating light and sufficient CO(2), O(2) reduction appears to take place primarily via a direct interaction between O(2) and reduced electron transport carriers, and occurs principally when CO(2)-fixation reactions are suboptimal, e.g. during induction or in the presence of Calvin cycle inhibitors. The inherent maximum endogenous rate of O(2) reduction is approximately 25 to 50% of the maximum rate of noncyclic electron transport coupled to CO(2) fixation. Although the photoreduction of O(2) is coupled to ion transport and/or phosphorylation, this process does not appear to supply significant amounts of ATP directly during steady-state CO(2) fixation in strong light.     

Glyoxylate and glutamate effects on photosynthetic carbon metabolism in isolated chloroplasts and mesophyll cells of spinach

Addition of millimolar sodium glyoxylate to spinach (Spinacia oleracea) chloroplasts was inhibitory to photosynthetic incorporation of ¹⁴CO₂ under conditions of both low (0.2 millimolar or air levels) and high (9 millimolar) CO₂ concentrations. Incorporation of ¹⁴C into most metabolites decreased. Labeling of 6-P-gluconate and fructose-1,6-bis-P increased. This suggested that glyoxylate inhibited photosynthetic carbon metabolism indirectly by decreasing the reducing potential of chloroplasts through reduction of glyoxylate to glycolate. This hypothesis was supported by measuring the reduction of [¹⁴C]glyoxylate by chloroplasts. Incubation of isolated mesophyll cells with glyoxylate had no effect on net photosynthetic CO₂ uptake, but increased labeling was observed in 6-P-gluconate, a key indicator of decreased reducing potential. The possibility that glyoxylate was affecting photosynthetic metabolism by decreasing chloroplast pH cannot be excluded. Increased ¹⁴C-labeling of ribulose-1,5-bis-P and decreased 3-P-glyceric acid and glycolate labeling upon addition of glyoxylate to chloroplasts suggested that ribulose-bis-P carboxylase and oxygenase might be inhibited either indirectly or directly by glyoxylate. Glyoxylate addition decreased ¹⁴CO₂ labeling into glycolate and glycine by isolated mesophyll cells but had no effect on net ¹⁴CO₂ fixation. Glutamate had little effect on net photosynthetic metabolism in chloroplast preparations but did increase ¹⁴CO₂ incorporation by 15% in isolated mesophyll cells under air levels of CO₂.     

Photosynthetic formation of the aspartate family of amino acids in isolated chloroplasts

The metabolism of ¹⁴C-labeled aspartic acid, diaminopimelic acid, malic acid and threonine by isolated pea (Pisum sativum L.) chloroplasts was examined. Light enhanced the incorporation of [¹⁴C] aspartic acid into soluble homoserine, isoleucine, lysine, methionine and threonine and protein-bound aspartic acid plus asparagine, isoleucine, lysine, and threonine. Lysine (2 millimolar) inhibited its own formation as well as that of homoserine, isoleucine and threonine. Threonine (2 millimolar) inhibited its own synthesis and that of homoserine but had only a small effect on isoleucine and lysine formation. Lysine and threonine (2 millimolar each) in combination strongly inhibited their own synthesis as well as that of homoserine. Radioactive [1,7-¹⁴C]diaminopimelic acid was readily converted into [¹⁴C]threonine in the light and its labeling was reduced by exogenous isoleucine (2 millimolar) or a combination of leucine and valine (2 millimolar each). The strong light stimulation of amino acid formation illustrates the point that photosynthetic energy is used in situ for amino acid and protein biosynthesis, not solely for CO₂ fixation.     

Glycolate and glyoxylate metabolism by isolated peroxisomes or chloroplasts

Chloroplasts, mitochondria, and peroxisomes from leaves were separated by isopycnic sucrose density gradient centrifugation. The peroxisomes converted glycolate-¹⁴C or glyoxylate-¹⁴C to glycine, and contained a glutamate: glyoxylate aminotransferase as indicated by an investigation of substrate specificity. The pH optimum for the aminotransferase was between 7.0 and 7.5, and the Km for l-glutamate was 3.6 mm and for glyoxylate, 4.4 mm. The reaction of glutamate plus glyoxylate was not reversible. The isolated peroxisomes did not convert glycine to glyoxylate nor glycine to serine.     

Photosynthetic control in isolated spinach chloroplasts with endogenous and artificial electron acceptors

Spinach chloroplasts, isolated rapidly in isotonic media will reproducibly give photosynthetic control rates (State 3/State 4) of 4–6, and ADP/O ratios (equivalent to ATP/2e⁻) of 1.4 – 2.1 when assayed in slightly hypotonic media. Photosynthetic control can be followed as oxygen evolution with either ferricyanide or NADP as electron acceptors, or as oxygen uptake in the presence of azide, which blocks chloroplast catalase, either alone (endogenous catalyst) or with added methyl viologen. This control can be triggered either by added ADP or by added Pi in all cases. Optimum concentrations of Mg, Pi and EDTA are required; the pH is also critical. Excess EDTA results in an inhibition of electron transport on addition of ADP.     

Energetic factors affecting carbon dioxide fixation in isolated chloroplasts

Light- and HCO₃⁻-saturated (10 millimolar) rates of O₂ evolution (120 to 220 micromoles O₂ per milligram chlorophyll per hour), obtained with intact spinach chloroplasts, are decreased up to 3-fold by changes in assay conditions such as omission of catalase from the medium, the use of high (≥1 millimolar) inorganic phosphate, inclusion of NO₂⁻ as an electron acceptor, or bright illumination at low partial pressures of O₂. These inhibitions may be reversed by addition of uncoupling levels of NH₄Cl or of antimycin concentrations that partially block cyclic electron transfer between cytochrome b₆ and cytochrome f. Measurements of the pH gradient across the thylakoid membrane with the fluorescent probe, 9-aminoacridine, indicate that changes in ΔpH are sufficient to account for both the inhibited and restored rates of electron transport. It follows that the rate of HCO₃⁻-saturated photosynthesis may be restricted by a proton gradient back pressure under these conditions.