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     

Regulation of photosynthesis in nitrogen deficient wheat seedlings

Nitrogen effects on the regulation of photosynthesis in wheat (Triticum aestivum L., cv Remia) seedlings were examined. Ribulose 1,5-bisphosphate carboxylase/oxygenase was rapidly extracted and tested for initial activity and for activity after incubation in presence of CO₂ and Mg²⁺. Freeze clamped leaf segments were extracted for determinations of foliar steady state levels of ribulose 1,5-bisphosphate, triose phosphate, 3-phosphoglycerate, ATP, and ADP. Nitrogen deficient leaves showed increased ATP/ADP and triose phosphate/3-phosphoglycerate ratios suggesting increased assimilatory power. Ribulose 1,5-bisphosphate levels were decreased due to reduced pentose phosphate reductive cycle activity. Nevertheless, photosynthesis appeared to be limited by ribulose 1,5-bisphosphate carboxylase/oxygenase, independent of nitrogen nutrition. Its degree of activation was increased in nitrogen deficient plants and provided for maximum photosynthesis at decreased enzyme protein levels. It is suggested that ribulose 1,5-bisphosphate carboxylase/oxygenase activity is regulated according to the amount of assimilatory power.     

Photosynthesis and Activation of Ribulose Bisphosphate Carboxylase in Wheat Seedlings : Regulation by CO(2) and O(2)

Photosynthetic carbon assimilation in plants is regulated by activity of the ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase. Although the carboxylase requires CO₂ to activate the enzyme, changes in CO₂ between 100 and 1,400 microliters per liter did not cause changes in activation of the leaf carboxylase in light. With these CO₂ levels and 21% O₂ or 1% or less O₂, the levels of ribulose bisphosphate were high and not limiting for CO₂ fixation. With high leaf ribulose bisphosphate, the K_act(CO₂) of the carboxylase must be lower than in dark, where RuBP is quite low in leaves. When leaves were illuminated in the absence of CO₂ and O₂, activation of the carboxylase dropped to zero while RuBP levels approached the binding site concentration of the carboxylase, probably by forming the inactive enzyme-RuBP complex.

The mechanism for changing activation of the RuBP carboxylase in the light involves not only Mg²⁺ and pH changes in the chloroplast stroma, but also the effects of binding RuBP to the enzyme. In light when RuBP is greater than the binding site concentration of the carboxylase, Mg²⁺ and pH most likely determine the ratio of inactive enzyme-RuBP to active enzyme-CO₂-Mg²⁺-RuBP forms. Higher irradiances favor more optimal Mg²⁺ and pH, with greater activation of the carboxylase and increased photosynthesis.

     

Glyoxylate inhibition of ribulosebisphosphate carboxylase/oxygenase activation in intact,lysed, and reconstituted chloroplasts

At bicarbonate concentrations equivalent to air levels of CO₂, activation of ribulosebisphosphate carboxylase/oxygenase (rubisco) was inhibited by micromolar concentrations of glyoxylate in intact, lysed, and reconstituted chloroplasts and in stromal extracts. The concentration of glyoxylate required for 50% inhibition of light activation in intact chloroplasts was estimated to be 35 micromolar. No direct inhibition by glyoxylate was observed with purified rubisco or rubisco activase at micromolar concentrations. Levels of ribulose 1,5-bisphosphate and ATP increased in intact chloroplasts following glyoxylate treatment. Results from experiments with well-buffered lysed and reconstituted chloroplast systems ruled out lowering of pH as the cause of inhibition. With intact chloroplasts, micromolar glyoxylate did not prevent activation of rubisco at high (10 mM) concentrations of bicarbonate, indicating that rubisco could be spontaneously activated in the presence of glyoxylate. These results suggest the existence of a component of the in vivo rubisco activation system that is not yet identified and which is inhibited by glyoxylate.Abbreviations PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - rubisco ribulosebisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate     

14CO2 fixation and glycolate metabolism in the dark in isolated maize (Zea mays L.) bundle sheath strands

Bundle sheath strands capable of assimilating up to 68 μmoles CO₂ per mg chlorophyll per hr in the dark have been isolated from fully expanded leaves of Zea mays L. This dark CO₂-fixing system is dependent on exogenous ribose-5-phosphate, ADP or ATP, and Mg²⁺ for maximum activity. The principal product of dark fixation in this system is 3-phosphoglycerate, indicating that the CO₂-fixing reaction is mediated by ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39). The rate of dark CO₂ uptake in the strands in the presence of saturating levels of ribose-5-phosphate plus ADP is inhibited by oxygen. The inhibitory effect of oxygen is rapidly and completely reversible, and is relieved by increased levels of CO₂. Glycolate is synthesized in this dark system in the presence of [U-¹⁴C]ribose-5-phosphate, ADP, oxygen, and an inhibitor of glycolate oxidase (EC 1.1.3.1). Glycolate formation is completely abolished by heating the strands, and the rate of glycolate synthesis is markedly reduced by either lowering the oxygen tension or increasing the level of CO₂.These results, obtained with intact cells in the absence of light, indicate that the direct inhibitory effect of oxygen on photosynthesis is associated with photosynthetic carbon metabolism, probably at the level of ribulose-1,5-bisphosphate carboxylase, and not with photophosphorylation or photosynthetic electron transport. Furthermore, the findings indicate that the synthesis of glycolate from exogenous substrate can readily occur in the absence of photosynthetic electron transport, an observation consistent with the ribulose-1, 5-bisphosphate “oxygenase” scheme for glycolate formation during photosynthesis.     

Reversible inhibition of the calvin cycle and activation of oxidative pentose phosphate cycle in isolated intact chloroplasts by hydrogen peroxide

Hydrogen peroxide (6x10^-4 M) causes a 90% inhibition of CO₂-fixation in isolated intact chloroplasts. The inhibition is reversed by adding catalase (2500 U/ml) or DTT (10 mM). If hydrogen peroxide is added to a suspension of intact chloroplasts in the light, the incorporation of carbon into hexose- and heptulose bisphosphates and into pentose monophosphates is significantly increased, whereas; carbon incorporation into hexose monophosphates and ribulose 1,5-bisphosphate is decreased. At the same time formation of 6-phosphogluconate is dramatically stimulated, and the level of ATP is increased. All these changes induced by hydrogen peroxide are reversed by addition of catalase or DTT. Additionally, the conversion of [¹⁴C]glucose-6-phosphate into different metabolites by lysed chloroplasts in the dark has been studied. In presence of hydrogen peroxide, formation of ribulose-1,5-bisphosphate is inhibited, whereas formation of other bisphosphates,of triose phosphates, and pentose monophosphates is stimulated. Again, DTT has the opposite effect. The release of ¹⁴CO₂ from added [¹⁴C]glucose-6-phosphate by the soluble fraction of lysed chloroplasts via the reactions of oxidative pentose phosphate cycle is completely inhibited by DTT (0.5 mM) and re-activated by comparable concentrations of hydrogen peroxide. These results indicate that hydrogen peroxide interacts with reduced sulfhydryl groups which are involved in the light activation of enzymes of the Calvin cycle at the site of fructose- and sedoheptulose bisphophatase, of phosphoribulokinase, as well as in light-inactivation of oxidative pentose phosphate cycle at the site of glucose-6-phosphate dehydrogenase.Abbreviations ADPG ADP-glucose - DHAP dihydroxyacetone phosphate - DTT dithiothreitol - FBP fructose-1,6-bisphosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HMP hexose monophosphates (fructose-6-phosphate, glucose-6-phosphate, glucose-1-phosphate) - 6-PGI 6-phosphogluconate - PMP pentose monophosphates (xylulose-5-phosphate, ribose-5-phosphate, ribulose-5-phosphate) - RuBP ribulose-1,5-bisphosphate - S7P sedoheptulose-7-phosphate - SBP sedoheptulose-1,7-bisphosphate Dedicated to Prof. Dr. W. Simonis on the occasion of his 70th birthday     

Exchange Properties of the Activator CO(2) of Spinach Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

The exchange properties of the activator CO₂ of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase were characterized both in vitro with the purified enzyme, and in situ within isolated chloroplasts. Carboxyarabinitol-1,5-bisphosphate, a proposed reaction intermediate analog for the carboxylase activity of the enzyme, was used to trap the activator CO₂ on the enzyme both in vitro and in situ. Modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in intact chloroplasts during a light/dark cycle was associated with a similar modulation in carboxyarabinitol-1,5-bisphosphate-trapped CO₂. The exchange kinetics of the activator CO₂ were monitored by activation of the enzyme to steady state in the presence of ¹²CO₂, followed by addition of ¹⁴CO₂ and determination of the amount of labeled CO₂ trapped on the enzyme by carboxyarabinitol-1,5-bisphosphate. Rate constants (K_obs) for exchange with both the purified enzyme (0.45 min⁻¹) and in illuminated chloroplasts (0.18 min⁻¹) were comparable to the observed rate constants for enzyme activation under the two conditions. A similar exchange of the activator CO₂ was not observed in chloroplasts in the dark. Kinetic analysis of the exchange properties of the purified enzyme were consistent with an equilibrium between active and inactive forms of the enzyme during steady state activation.     

Reversible heat-inactivation of the calvin cycle: A possible mechanism of the temperature regulation of photosynthesis

Photosynthetic CO₂ fixation rates in leaves and intact chloroplasts of spinach measured at 18°–20° C are substantially decreased by pretreatment at temperatures exceeding 20° C. Mild heating which causes 80% inhibition of CO₂ fixation does not affect phosphoglyceroacid reduction and causes increases in the ATP/ADP ratio and the light-induced transthylakoid proton gradient. The inactivation of the CO₂ fixation is completely reversible with half-times of recovery in the order of 15–20 min. Comparison of steady-state patterns of ¹⁴C labeled Calvin cycle intermediates of heat-treated and control samples reveals a large increase in the ribulose-1,5-bisphosphate/phosphoglyceroacid ratio and a large decrease in the phosphoglyceroacid/triosephosphate ratio. It is concluded that inactivation of CO₂ fixation occurring at elevated temperatures is caused by inhibition of the ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39). Measurements of light-induced light scattering changes of thylakoids and of the light-induced electrochromic absorption shift show that these signals are affected by mild heating in a way which is strictly correlated with the inactivation of the CO₂ fixation. It is proposed that the function of the ribulose-1,5-bisphosphate carboxylase in vivo requires a form of activation that involves properties of the thylakoid membrane which are affected by the heat treatment. The fact that these changes in thylakoid membrane properties and of ribulose-1,5-bisphosphate carboxylase activity are already affected at elevated temperatures which can still be considered physiological, and the reversible nature of these changes, suggest that they may play a role in temperature regulation of the overall photosynthetic process.Abbreviations 9-AA 9-aminoacridine - DMO 5,5-dimethyloxazolidine-2,4-dione - FBP fructose-1,6-bisphosphate - HEPES N-2-hydroxyethylpiperazine N-2-ethane sulfonic acid - HMP hexose monophosphates - PGA 3-phosphoglycerate - PMP pentose monophosphates - RuBP ribulose-1,5-bisphosphate - SBP seduheptulose-1,7-bisphosphate - TP triose monophosphates     

Enzyme activities in an artificial stroma medium

When spinach leaf tissue was subjected to evaporative dehydration, photosynthetic capacity at very high (5%) CO₂ concentration and saturating irradiance (300 W·m^-2), decreased in parallel to the relative water content (RWC). A 50% inhibition was observed at 60–40% RWC. In order to examine whether the inhibition was caused by increased solute concentrations in chloroplasts or cytoplasm, an artificial stroma medium (ASM) was set up containing all major osmotically relevant solutes measured in isolated intact spinach chloroplasts. Subsequently, the response of enzyme activities to normal and to increased concentrations of ASM was examined. Inhibition of enzymes by a concerted increase of all solutes was well correlated to the in-vivo response of photosynthesis to dehydration (60% inhibition at double-strength ASM). Inhibitory solutes were mainly divalent inorganic anions, such as sulfate and phosphate. Inhibition of ribulose-1,5-bisphosphate carboxylase by these ions as studied in more detail. Inhibition of the enzyme by sulfate and phosphate was competitive with respect to ribulose-1,5-bisphosphate, but not with respect to CO₂. The K_I for sulfate was 2.1 mmol·l^-1 and for phosphate 0.57 mmol·l^-1. Sugars and amino acids at the concentrations found in spinach chloroplasts did not prevent inhibition of enzymes by anions. The results indicate that increased anion concentrations in cells and organelles are responsible for primary, quickly reversible effects of moderate dehydration on plant tissues.Abbreviations ASM artificial stroma medium - RuBP ribulose 1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate-carboxylase/oxygenase - RWC relative water content     

Enzymic Fractionation of the Stable Carbon Isotopes of Carbon Dioxide by Ribulose-1,5-bisphosphate Carboxylase

The enzymic fractionation of the stable carbon isotopes of CO₂ (Δco₂) was determined using a purified preparation of ribulose-1,5-bisphosphate (RuBP) carboxylase isolated from cotton (a C₃ plant) leaves. The bicarbonate concentration in the reaction mixture saturated the enzyme and furnished an infinite pool of ¹²CO₂ and ¹³CO₂ for enzyme fractionation. The RuBP was 96 to 98% pure. The phosphoglycerate synthesized in the reaction mixtures was purified free of RuBP, phosphoglycolate, and other phosphate esters by column chromatography on Dowex 1-Cl⁻ resin. The average Δco₂ value of −27.1% was determined from five separate experiments. A discussion of the isotope fractionation associated with photosynthetic CO₂ fixation in plants shows that the enzymic fractionation of stable carbon isotopes of CO₂ by RuBP carboxylase is of major importance in determining the δ¹³C values of C₃ plants.     

The effect of divalent metal ions on the activity of Mg++ depleted ribulose-1,5-bisphosphate oxygenase

Ribulose-1,5-bisphosphate carboxylase-oxygenase is deactivated by removal of Mg⁺⁺. The enzyme activities can be restored to a different extent by the addition of various divalent ions in the presence of CO₂. Incubation with Mg⁺⁺ and CO₂ restores both enzyme activities, whereas, the treatment of the enzyme with the transition metal ions (Mn⁺⁺, Co⁺⁺, and Ni⁺⁺) and CO₂ fully reactivates the oxygenase: however, the carboxylase activity remains low. In experiments where CO₂-free conditions were conscientiously maintained, no reactivation of RuBP oxygenase was observed, although Mn⁺⁺ ions were present. Other divalent cations such as Ca⁺⁺ and Zn⁺⁺, restore neither the carboxylase nor the oxygenase reaction. Furthermore, the addition of Mn⁺⁺ to the Mg⁺⁺ and CO₂ preactivated enzyme significantly inhibited carboxylase reactions, but increased the oxygenase reaction.Abbreviation RuBP ribulose-1,5-bisphosphate. The enyme unit for RuBP carboxylase is defined as mol CO₂ fixed·min^-1 and for the RuBP oxygenase as mol O₂ consumed · min^-1     

Involvement of stromal ATP in the light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase in intact isolated chloroplasts

Light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) and stromal ATP content were measured in intact isolated spinach chloroplasts. Treatments which decreased stromal ATP, such as incubation with the ATP analog β,γ-methylene adenosine triphosphate or with the energy transfer inhibitor phloridzin inhibited the light activation of rubisco. In the absence of added inorganic phosphate (Pi), light activation of rubisco was inhibited, coincident with low stromal ATP. Addition of methyl viologen restored both stromal ATP and rubisco activity to levels observed in the presence of Pi. Activation of rubisco was inhibited in the presence of 2 millimolar dihydroxyacetone phosphate or 3-phosphoglycerate and stromal ATP was also decreased under these conditions. Both were partially restored by increasing the Pi concentration. The strong correlation between activation state of rubisco and stromal ATP concentration in intact chloroplasts under a wide variety of experimental conditions indicates that light activation of rubisco is dependent on ATP and proportional to the ATP concentration. These observations can be explained in terms of the rubisco activase protein, which mediates activation of rubisco at physiological concentrations of CO₂ and ribulose-1,5-bisphosphate and is dependent upon ATP.     

A facile method to determine the CO2/O2 specificity factor for ribulose bisphosphate carboxylase/oxygenase

A rapid method to determine the CO₂/O₂ specificity factor of ribulose 1,5-bisphosphate carboxylase/oxygenase is presented. The assay measures the amount of CO₂ and O₂ fixation at varying CO₂/O₂ ratios to determine the relative rates of each reaction. CO₂ fixation is measured by the incorporation of the moles of¹⁴CO₂ into 3-phosphoglycerate, while O₂ fixation is determined by subtraction of the moles of CO₂ fixed from the moles of RuBP consumed in each reaction. By analyzing the inorganic phosphate specifically hydrolyzed from RuBP under alkaline conditions, the amount of RuBP present before and after catalysis by rubisco can be determined.     

Influence of adenosine phosphates and magnesium on photosynthesis in chloroplasts from peas, sedum, and spinach

¹⁴CO₂ photoassimilation in the presence of MgATP, MgADP, and MgAMP was investigated using intact chloroplasts from Sedum praealtum, a Crassulacean acid metabolism plant, and two C₃ plants: spinach and peas. Inasmuch as free ATP, ADP, AMP, and uncomplexed Mg²⁺ were present in the assays, their influence upon CO₂ assimilation was also examined. Free Mg²⁺ was inhibitory with all chloroplasts, as were ADP and AMP in chloroplasts from Sedum and peas. With Sedum chloroplasts in the presence of ADP, the time course of assimilation was linear. However, with pea chloroplasts, ADP inhibition became progressively more severe, resulting in a curved time course. ATP stimulated assimilation only in pea chloroplasts. MgATP and MgADP stimulated assimilation in all chloroplasts. ADP inhibition of CO₂ assimilation was maximal at optimum orthophosphate concentrations in Sedum chloroplasts, while MgATP stimulation was maximal at optimum or below optimum concentrations of orthophosphate. MgATP stimulation in peas and Sedum and ADP inhibition in Sedum were not sensitive to the addition of glycerate 3-phosphate (PGA).

PGA-supported O₂ evolution by pea chloroplasts was not inhibited immediately by ADP; the rate of O₂ evolution slowed as time passed, corresponding to the effect of ADP on CO₂ assimilation, and indicating that glycerate 3-phosphate kinase was a site of inhibition. Likewise, upon the addition of AMP, inhibition of PGA-dependent O₂ evolution became more severe with time. This did not mirror CO₂ assimilation, which was inhibited immediately by AMP. In Sedum chloroplasts, PGA-dependent O₂ evolution was not inhibited by ADP and AMP. In chloroplasts from peas and Sedum, the magnitude of MgADP and MgATP stimulation of PGA-dependent O₂ evolution was not much larger than that given by ATP, and it was much smaller than MgATP stimulation of CO₂ assimilation. Analysis of stromal metabolite levels by anion exchange chromatography indicated that ribulose 1,5-bisphosphate carboxylase was inhibited by ADP and stimulated by MgADP in Sedum chloroplasts.

The appearance of label in the medium was measured when [U-¹⁴C] ADP-loaded Sedum chloroplasts were challenged with ATP, ADP, or AMP and their Mg²⁺ complexes. The rate of back exchange was stimulated by the presence of Mg²⁺. This suggests that ATP, ADP, and AMP penetrate the chloroplast slower than their Mg²⁺ complexes. A portion of the CO₂ assimilation and O₂ evolution data could be explained by differential penetration rates, and other proposals were made to explain the remainder of the observations.

     

Biochemical Correlates of the Circadian Rhythm in Photosynthesis in Phaseolus vulgaris

A circadian rhythm in photosynthesis occurs in Phaseolus vulgaris after transfer from a natural or artificial light:dark cycle to constant light. The rhythm in photosynthesis persists even when intercellular CO₂ partial pressure is held constant, demonstrating that the rhythm in photosynthesis is not entirely due to stomatal control over the diffusion of CO₂. Experiments were conducted to attempt to elucidate biochemical correlates with the circadian rhythm in photosynthesis. Plants were entrained to a 12-hour-day:12-hour-night light regimen and then monitored or sampled during a subsequent period of constant light. We observed circadian oscillations in ribulose-1,5-bisphosphate (RuBP) levels, and to a lesser extent in phosphoglyceric acid (PGA) levels, that closely paralleled oscillations in photosynthesis. However, the enzyme activity and activation state of the enzyme responsible for the conversion of RuBP to PGA, ribulose-1,5-bisphosphate carboxylase/oxygenase, showed no discernible circadian oscillation. Hence, we examined the possibility of circadian effects on RuBP regeneration. Neither ribulose-5-phosphate kinase activity nor the level of ATP fluctuated in constant light. Oscillations in triose-phosphate levels were out of phase with those observed for RuBP and PGA.     

Reversible light-activation of ribulose bisphosphate carboxylase/oxygenase in isolated barley protoplasts and chloroplasts

The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase displayed near-maximal activity in isolated, intact barley (Hordeum vulgare L. cv. Pennrad) mesophyll protoplasts. The carboxylase deactivated 40 to 50% in situ when protoplasts were dark-incubated 20 minutes in air-equilibrated solutions. Enzyme activity was fully restored after 1 to 2 minutes of light. Addition of 5 millimolar NaHCO₃ to the incubation medium prevented dark-inactivation of the carboxylase. There was no permanent CO₂-dependent activation of the protoplast carboxylase either in light or dark. Activation of the carboxylase from ruptured protoplasts was not increased significantly by in vitro preincubation with CO₂ and Mg²⁺. In contrast to the enzyme in protoplasts, the carboxylase in intact barley chloroplasts was not fully reactivated by light at atmospheric CO₂ levels. The lag phase in carbon assimilation was not lengthened by dark-adapting protoplasts to low CO₂ demonstrating that light-activation of the carboxylase was not involved in photosynthetic induction. Irradiance response curves for reactivation of the the carboxylase and for CO₂ fixation by isolated barley protoplasts were similar. The above results show that there was a fully reversible light-activation of the carboxylase in isolated barley protoplasts at physiologically significant CO₂ levels.     

Activation of ribulose bisphosphate carboxylase in intact chloroplasts by CO2 and light

The activity of ribulose 1,5-bisphosphate (RuBP) car?ylase in intact spinach chloroplasts is shown to depend on light and CO₂. This activity was measured upon lysis of chloroplasts and assay of the initial activity using nonlimiting substrate concentrations. Incubation of chloroplasts at 25 °C in the absence of CO₂ results in a gradual inactivation of the RuBP car?ylase. In the presence of CO₂ the initial activity is preserved or increased. CO₂ is also able to reactivate the chloroplast car?ylase previously inactivated in the absence of CO₂. Upon illumination of the chloroplasts, additional activation was observed. This light activation results from an increased affinity for CO₂ of the chloroplast car?ylase. At pH 7.8, the enzyme in dark-adapted chloroplasts required 112 μ m CO₂ for half activation, while in the light it required 24 μ m CO₂. The light activation was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, carbonylcyanide 3-chlorophenylhydrazone, or dl-glyceraldehyde. Part of the light activation is most likely due to increased Mg²⁺ in the stroma. dl-Glyceraldehyde inhibition also suggests that some intermediate of the photosynthetic carbon cycle is involved. These results suggest that photosynthetic CO₂ assimilation in the chloroplast depends upon the amount of activation of the RuBP car?ylase. This activation is regulated by CO₂ and light-induced changes in the chloroplast stroma such as pH, Mg²⁺, and intermediates of the photosynthetic carbon cycle.     

The effect of magnesium and other ions on the distribution of ribulose 1,5-bisphosphate carboxylase in chloroplast extracts

In an attempt to produce chloroplast extracts containing ribulose 1,5-bisphosphate (RuBP) carboxylase in its fully activated state, MgCl₂ and NaHCO₃ were included in the medium used to osmotically shock chloroplasts. Extracts prepared in this manner contained lower levels of the enzyme than those prepared in the absence of MgCl₂ and NaHCO₃. The difference in enzyme levels was found to be attributable to an association in the presence of Mg²⁺, between RuBP carboxylase and the thylakoids removed from the extract during its preparation. Some monovalent cations caused a similar association, although to a lesser extent. The trivalent cation Tris(ethylenediamine) cobalt(III) was more effective in causing this association, but was highly inhibitory to the enzyme. The results suggest that the attraction between thylakoids and RuBP carboxylase in the presence of certain ions is likely to be electrical in nature. The results are discussed in terms of the media used to isolate RuBP carboxylase.     

Metabolite levels in the stroma of spinach chloroplasts exposed to osmotic stress: Effects of the pH of the medium and exogenous dihydroxyacetone phosphate

The levels of stromal photosynthetic intermediates were measured in isolated intact spinach (Spinacia oleracea L.) chloroplasts exposed to reduced osmotic potentials. Stressed chloroplasts showed slower rates of metabolite accumulation upon illumination than controls. Relative to other metabolites sedoheptulose-1,7-bisphosphate (SBP) and fructose-1,6-bisphosphate (FBP) accumulated in the stroma in the stressed treatments. Under these conditions 3-phosphoglycerate (3-PGA) efflux to the medium was restricted. Chloroplasts previously incubated with [³²P]KH₂PO₄ and [³²P]dihydroxyacetone phosphate ([³²P]DAP) in the dark were characterized by very high FBP and SBP levels prior to illumination. Metabolism of these pools upon illumination increased with increasing pH of the medium but was consistently inhibited in osmotically stressed chloroplasts. The responses of stromal FBP and SBP pools under hypertonic conditions are discussed in terms of both inhibited light activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) and sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37), and likely increases in stromal ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) active-site concentrations.Abbreviations and symbols DAP dihydroxyacetone phosphate - FBP fructose-1,6-bisphosphate - PGA 3-phosphoglycerate - RuBP ribulose-1,5-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - _s osmotic potential     

Carbon dioxide fixation and related properties in sections of the developing green maize leaf

Light and dark ¹⁴CO₂ assimilation, pulse-chase (¹⁴CO₂ followed by ¹²CO₂) labeling experiments both in the light and in the dark, photorespiratory activity and some enzymes (ribulose 1,5-bisphosphate (RuBP) carboxylase, phosphoenolpyruvate (PEP) carboxylase, and NADP-malic enzyme) were followed in sections of 2.5 centimeters from the base (younger tissue) to the tip (oldest tissue) of the green maize leaf. Tissue was taken from the third leaf of 12- to 16-day-old plants consisting of sections 0 to 2.5 centimeters (base), 4.5 to 7.0 centimeters (center) and 9.0 to 11.5 centimeters (top) measured from the base. Some of these properties were also determined in the intact leaves of 4-day-old maize plants.