Level of photosynthetic intermediates in isolated spinach chloroplasts

The level of intermediates of the photosynthetic carbon cycle was measured in intact spinach chloroplasts in an attempt to determine the cause of the induction lag in CO₂ assimilation. In addition, transient changes in the level of the intermediates were determined as affected by a light-dark period and by the addition of an excess amount of bicarbonate during a period of steady photosynthesis. Assayed enzymically were: ribulose 1,5-diphosphate, pentose monophosphates (mixture of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, hexose monophosphates (mixture of glucose 6-phosphate, glucose 1-phosphate, and fructose 6-phosphate), glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, glycerate acid 3-phosphate, a mixture of fructose 1,6-diphosphate and sedoheptulose 1,7-diphosphate, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP).     

Ribulose diphosphate carboxylase/oxygenase. III. Isolation and properties.

Similarities in properties of ribulose diphosphate carboxylase and oxygenase activities further substantiate the hypothesis that the same protein catalyzes both reactions. The Km (ribulose diphosphate) is 0.33 mM for the ribulose diphosphate oxygenase, when assayed in air with an oxygen electrode. Maximum activity is obtained with 10 to 35 mM MgCl2. Higher MgCl2 concentrations are inhibitory, but they shift the pH optimum from 9.3 or 9.4 to 8.7 or 9.0. MnCl2 is an effective cofactor of the oxygenase and some activity is obtained with CoCl2. Both the ribulose diphosphate carboxylase and oxygenase activity of the purified protein from spinach leaves are slowly inactivated by storage at 0 degrees and reactivated in 10 min at 50 degrees, provided both 25 mM MgCl2 and 1 mM dithiothreitol are present. The sulfhydryl groups of the enzyme which react rapidly with 5,5'-dithiobis(2-nitrobenzoic acid) are approximately 4 at pH 7.8 and 11 at pH 9.4. At both pH values ribulose diphosphate prevents two of these sulfhydryl groups from reacting with this reagent. About 50% inhibition of the oxygenase activity at pH 9.0 occurs with 50 mM bicarbonate in the presence of 3 mM ribulose diphosphate, and from variations in these parameters the inhibition is attributed to the CO2 species. The purified enzyme of acrylamide gels prevented the reduction of nitroblue tetrazolium in the presence of the superoxide radical, but the enzyme in solution did not react as a superoxide dismutase.     

Bicarbonate stabilization of ribulose 1,5-diphosphate carboxylase.

The carboxylase and oxygenase activities of purified soybean ribulose 1,5-di-P carboxylase (EC4.1.1.39) were unstable when reactions were initiated with enzyme. Time courses of carboxylase and oxygenase activities were curvilinear, approximating hyperbolas. Double reciprocal plots of amount of CO2 incorporated and P-glycolate produced vs. time were constructed to determine a constant representing the half-time of initial enzyme activity, K. K increased with increasing bicarbonate concentration but was independent of O2 tensions between 0.21 and 5 atm. When time courses of carboxylase and oxygenase activities were determined simultaneously, K was identical for both activities. Linear time courses were obtained py preincubation of the enzyme for 10 min in the absence of bicarbonate or by adding 46 mM MgCl2 to the reaction mixture. The observed bicarbonate-dependent decline in ribulose 1,5-di-P carboxylase activity with time is the probable cause for the anomalously high Km(CO2) values previously reported for this enzyme. In the experiments reported here, the apparent Km(CO2) at pH 8.5 increased from 6 muM CO2 at zero time to 78 muM CO2 at 10 min. The corresponding bicarbonate Km values ar 1;3 and 17 mM, respectively, The interaction between bicarbonate and enzyme may be important in the light activation of photosynthetic CO2 fixation in vivo.     

Regulation of Soybean Net Photosynthetic CO(2) Fixation by the Interaction of CO(2), O(2), and Ribulose 1,5-Diphosphate Carboxylase

Kinetic properties of soybean net photosynthetic CO₂ fixation and of the carboxylase and oxygenase activities of purified soybean (Glycine max [L.] Merr.) ribulose 1, 5-diphosphate carboxylase (EC 4.1.1.39) were examined as functions of temperature, CO₂ concentration, and O₂ concentration. With leaves, O₂ inhibition of net photosynthetic CO₂ fixation increased when the ambient leaf temperature was increased. The increased inhibition of CO₂ fixation at higher temperatures was caused by a reduced affinity of the leaf for CO₂ and an increased affinity of the leaf for O₂. With purified ribulose 1,5-diphosphate carboxylase, O₂ inhibition of CO₂ incorporation and the ratio of oxygenase activity to carboxylase activity increased with increased temperature. The increased O₂ sensitivity of the enzyme at higher temperature was caused by a reduced affinity of the enzyme for CO₂ and a slightly increased affinity of the enzyme for O₂. The similarity of the effect of temperature on the affinity of intact leaves and of ribulose 1,5-diphosphate carboxylase for CO₂ and O₂ provides further evidence that the carboxylase regulates the O₂ response of photosynthetic CO₂ fixation in soybean leaves. Based on results reported here and in the literature, a scheme outlining the stoichiometry between CO₂ and O₂ fixation in vivo is proposed.     

Inhibition of ribulose 1,5-diphosphate carboxylase by 6-phosphogluconate

6-Phosphogluconate is a much more effective inhibitor of the photosynthetic carboxylation enzyme, ribulose-1, 5-diphosphate carboxylase, than other sugar phosphates and sugar acids of the reductive and oxidative pentose phosphate cycles. The inhibition appears to be noncompetitive with ribulose 1,5-diphosphate. Since 6-phosphogluconate is unique to the oxidative cycle and inhibits at concentrations comparable to those found in vivo, it is proposed that its inhibition of the carboxylase may be a regulatory factor. If so, it would operate during darkness as a different control factor from those factors postulated to activate the carboxylase during photosynthesis.     

The glycolate pathway and photosynthetic competence in euglena

The development of glycolate pathway enzymes has been determined in relation to photosynthetic competence during the regreening of Euglena cultures. Phosphoglycolate phosphatase and glycolate dehydrogenase rapidly reached maximal levels of activity but the complete development of ribulose 1,5-diphosphate carboxylase and concomitant photosynthetic carbon dioxide fixation were not attained until 72 hours of illumination. Specific inhibitors of protein synthesis showed that the formation of ribulose 1,5-diphosphate carboxylase in both division-synchronized and regreening cultures was prevented by both cycloheximide and d-threo-chloramphenicol, whereas phosphoglycolate phosphatase formation was only inhibited by d-threo-chloramphenicol but not by l-threo-chloramphenicol or cycloheximide. Since cycloheximide prevented ribulose diphosphate carboxylase synthesis and photosynthetic carbon dioxide fixation without affecting phosphoglycolate phosphatase synthesis during regreening, it was concluded that photosynthetic competence was not necessary for the development of the glycolate pathway enzymes. The inhibition of phosphoglycolate phosphatase synthesis by d-threo-chloramphenicol but not by l-threo-chloramphenicol or cycloheximide shows that the enzyme was synthesized exclusively on chloroplast ribosomes, whereas protein synthesis on both chloroplast and cytoplasmic ribosomes was required for the formation of ribulose 1,5-diphosphate carboxylase. Although light is required for the development of both Calvin cycle and glycolate pathway enzymes during regreening it is concluded that the two pathways are not coordinately regulated.     

Inhibition of the formation of photosynthetic enzymes by inhibitors of photosynthesis

It has been shown previously that an increase in ribulose diphosphate carboxylase activity occurs upon brief illumination of leaves of dark-grown Zea mays plants; an increase in ribose 5-phosphate isomerase occurs after prolonged illumination. Both of these responses to illumination are inhibited by chloramphenicol.

The administration of p-chlorophenyldimethylurea, an inhibitor of photosynthesis, to etiolated maize does not affect the normal early rise in ribulose diphosphate carboxylase activity when the leaves are illuminated but does block the increase in ribose 5-phosphate isomerase. This pattern of response suggests that photosynthetic activity is required for the increase in isomerase—perhaps products of photosynthesis induce isomerase synthesis—but that the level of ribulose diphosphate carboxylase is controlled by other processes. Chlorophyll formation (as has been shown by others) is slightly suppressed by the inhibitor; levels of total soluble leaf protein appear to be unaffected.

Salicylaldoxime, which is a more general inhibitor of metabolism than p-chlorophenyldimethylurea, arrests the normally observed increases of ribulose diphosphate carboxylase, ribose 5-phosphate isomerase, and chlorophyll during illumination of dark-grown maize. The level of soluble leaf protein is also lower in leaves treated with this compound.

     

d-Ribulose 1,5-diphosphate carboxylase from the blue-green alga Aphanocapsa 6308

d-Ribulose 1,5-diphosphate carboxylase from extracts of the unicellular blue-green alga Aphanocapsa 6308 has been purified by ammonium sulphate precipitation and linear sucrose density gradient centrifugation. The molecular weight was estimated to be 525 000 and the enzyme consisted of two types of sub-unit of molecular weights 51 000 and 15 000. The small sub-units were not detected after purification involving acid precipitation but were observed if the acid precipitation step was omitted. The Michaelis constants for Mg²⁺ and CO₂, when tested under air, were 0.35 mM and 0.071 mM respectively. Oxygen acted as a competitive inhibitor with respect to CO₂, suggesting that the enzyme also acts as an oxygenase. This was confirmed by measuring ribulose diphosphate-dependent O₂ uptake. A 1:1 stoichiometry between ribulose diphosphate utilization and O₂ consumption was observed. 6-Phosphogluconate inhibited carboxylase activity both at high (20 mM) and low (1 mM) bicarbonate concentrations. The data are compared with the properties of ribulose diphosphate carboxylase from other autotrophic prokaryotes and from chloroplasts.Abbreviations RuDP d-Ribulose 1,5-diphosphate - EDTA ethylene diamine tetraacetic acid - GSH reduced glutathione - SDS sodium dodecyl sulphate - 6PGluc 6-phosphogluconate - STB supplemented Tris buffer     

Activation and regulation of ribulose bisphosphate carboxylase-oxygenase in the absence of small subunits.

Ribulose 1,5-bisphosphate carboxylase from Rhodospirillum rubrum requires CO2 and Mg2+ for activation of both CO2, both the carboxylase and oxygenase activities are stimulated by 6-phoshpo-D-gluconate, fructose 1,6-bisphosphate, 2-phosphoglycolate, 3-phosphoglycerate, NADPH, and fructose 6-phosphate. The carboxylase activity is not activated by ribose 5-phosphate. The substrate, ribulose bisphosphate, neither activates nor inhibits the CO2 and Mg2+ activation of this enzyme. Activation by CO2 and Mg2+ is rapid and results in increased susceptibility to active-site-directed protein modification reagents. Because the R. rubrum carboxylase-oxygenase is a dimer of large subunits and contains no small subunits, these results suggest that the effector binding sites of the higher plant enzyme may also be found on the large subunit.     

Changing Activities During Senescence and Sites of Synthesis of Photosynthetic Enzymes in Leaves of the Labiate, Perilla frutescens (L.) Britt.

The changing activities of several regulatory enzymes of thephotosynthetic carbon reduction cycle accompanying ageing ofthe third leaf pair of Perilla frutescens fall into two distinctcategories: firstly, enzymes which reach maximum activity priorto the completion of leaf expansion followed by a rapid decline(phosphoribulokinase, ribulose-l,5-diphosphate carboxylase,and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase);secondly, enzymes which maintain high activity beyond completionof leaf expansion and decline only at a late stage in senescence(phosphoglycerate kinase, NADH-linked glyceraldehyde-3-phosphatedehydrogenase, alkaline fructose-1,6-diphosphatase, and ribose-5-phosphateisomerase). The introduction of the ribosomal inhibitors D-threochloramphenicol, lincomycin, D-2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide, and cycloheximide to illuminated, detached shootsystems of 2-d-darkened Perilla plants has suggested that synthesisof ribulose diphosphate carboxylase, NADPH-dependent glyceraldehyde-3-phosphatedehydrogenase, and possibly phosphoribulokinase, is mediatedby 70 S-based chloroplastic ribosomes. A chloroplastic siteof synthesis of these three photosynthetic enzymes is consistentwith their early deterioration during leaf ageing.     

Effects of CO2, O2 and temperature on a high-affinity form of ribulose diphosphate carboxylase-oxygenase from spinach

A high-affinity form of ribulose diphosphate carboxylase, observed transiently in spinach-leaf extracts soon after extraction, was inhibited by O₂ competitively with respect to CO₂. Analogously, the ribulose diphosphate oxygenase activity for this form was inhibited by CO₂, competitively with respect to O₂. For each gas, the K_m for the reaction in which it was a substrate was similar to its K_i for the reaction it inhibited. The Arrhenius activation energy for the oxygenase reaction was 1.5 times that of the carboxylase. These characteristics are consistent with ribulose diphosphate oxygenase being the enzymatic reaction responsible for synthesizing the substrate for photorespiration and with the concept that the balance between photosynthesis and photorespiration of leaves is a reflection of the ratio between the two activities of this bi-functional enzyme.     

Ribulose bisphosphate carboxylase/oxygenase in toluene-permeabilized Rhodospirillum rubrum.

Toluene-permeabilized Rhodospirillum rubrum cells were used to study activation of and catalysis by the dual-function enzyme ribulose bisphosphate carboxylase/oxygenase. Incubation with CO2 provided as HCO3-, followed by rapid removal of CO2 at 2 degrees C and subsequent incubation at 30 degrees C before assay, enabled a determination of decay rates of the carboxylase and the oxygenase. Half-times at 30 degrees C with 20 mM-Mg2+ were 10.8 and 3.7 min respectively. Additionally, the concentrations of CO2 required for half-maximal activation were 56 and 72 microM for the oxygenase and the carboxylase respectively. After activation and CO2 removal, inactivation of ribulose bisphosphate oxygenase in the presence of 1 mM- or 20mM-Mn2+ was slower than that with the same concentrations of Co2+ or Mg2+. Only the addition of Mg2+ supported ribulose bisphosphate carboxylase activity, as Mn2+, Co2+ and Ni2+ had no effect. A pH increase after activation in the range 6.8-8.0 decreased the stability of the carboxylase but in the range 7.2-8.0 increased the stability of the oxygenase. With regard to catalysis. Km values for ribulose 1,5-bisphosphate4- were 1.5 and 67 microM for the oxygenase and the carboxylase respectively, and 125 microM for O2. Over a broad range of CO2 concentrations in the activation mixture, the pH optima were 7.8 and 8-9.2 for the carboxylase and the oxygenase respectively. The ratio of specific activities was constant (9:1 for the carboxylase/oxygenase) of ribulose bisphosphate carboxylase/oxygenase in toluene-treated Rsp. rubrum. Below concentrations of 10 microM-CO2 in the activation mixture, this ratio increased.     

The inhibition of ribulose 1,5-bisphosphate carboxylase oxygenase by several 2-carboxyhexitol 1- and 6-phosphates

The condensation of D-fructose 6-phosphate or 1-phosphate with cyanide has been used to synthesize 2-carboxyhexitol 6-phosphates and 1-phosphates. The products have been characterized in terms of their action on ribulose bisphosphate carboxylase/oxygenase. The reaction of D-fructose 6-phosphate with cyanide is four times as fast (at 22°C) at pH 7.5 than at pH 11.5 and the primary products of condensation are more easily isolated by anion exchange chromatography. Two minor chromatographic peaks (I and II) for diastereomeric 2-carboxyhexitol 6-phosphates are isolated in addition to two major peaks, III and IV, which are lactones. The lactones are those of 2-C-carboxy-D-glucitol 6-phosphate (CG6P) in peak III and 2-C-carboxy-D-mannitol 6-phosphate (CM6P) in peak IV, as established after dephosphorylation by the relative rates of oxidation by periodate and by gas chromatographic retention times of the acetates. Analogous methodology has been used to synthesize the diastereomeric 2-carboxy-hexitol 1-phosphates (CG1P and CM1P) and their lactones from D-fructose 1-phosphate. The four carboxylates inhibit ribulose bisphosphate carboxylase/oxygenase from spinach or Pseudomonas oxalaticus in the following decreasing order of potency: CG6P, CM6P, CG1P, CM1P. The inhibition pattern suggests that the binding of the 5-phosphate moiety of the intermediate in the reaction catalyzed by ribulose bisphosphate carboxylase/oxygenase may be stronger by an order of magnitude than the binding of the 1-phosphate group.     

Effect of glycidate, an inhibitor of glycolate synthesis in leaves, on the activity of some enzymes of the glycolate pathway

Under conditions where glycolate synthesis was inhibited at least 50% in tobacco (Nicotiana tabacum L.) leaf discs treated with glycidate (2,3-epoxypropionate), the ribulose diphosphate carboxylase activity in extracts and the inhibition of the activity by 100% oxygen were unaffected by the glycidate treatment. [1-¹⁴C]Glycidate was readily taken into leaf discs and was bound to leaf proteins, but the binding occurred preferentially with proteins of molecular weight lower than ribulose diphosphate carboxylase. Glycidate added to the isolated enzyme did not inhibit ribulose diphosphate carboxylase activity or affect its inhibition by 100% O₂. Thus, glycidate did not inhibit glycolate synthesis by a direct effect on ribulose diphosphate carboxylase/oxygenase.     

The isolation of factor V from bovine plasma

1. The activity per unit of chlorophyll of certain carboxylases, and of other enzymes involved in photosynthesis, was determined in leaf extracts of the tropical grasses, sugar-cane, maize and sorghum, and compared with the activities for wheat, oat and silver-beet. Maximum rates of photosynthetic carbon dioxide uptake were also measured for comparison with enzyme activities. 2. Phosphopyruvate carboxylase activity was about 60 times greater in the tropical grasses than in wheat, oat and silver-beet and was severalfold higher than the rates of photosynthetic carbon dioxide uptake. Most of the enzyme was located in the chloroplast fraction of cell extracts. 3. Phosphopyruvate carboxylase was apparently the major photosynthetic carbon dioxide-fixing enzyme in the tropical grasses, although malic enzyme may contribute to a lesser extent. 4. Tropical grasses contained less than one-tenth of the ribulose diphosphate carboxylase activity present in wheat, oat and silver-beet. For the tropical grasses this activity, determined with a saturating concentration of bicarbonate, was approx. 10% of the rate of photosynthesis. 5. The fraction-1 protein content of leaf extracts paralleled the ribulose diphosphate carboxylase activity. 6. In contrast, the activity of several other enzymes of the Calvin cycle was similar in the different species examined.     

Regulation of glucose-6-phosphate dehydrogenase in spinach chloroplasts by ribulose 1,5-diphosphate and NADPH/NADP+ ratios.

The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) FROM SPINACH CHLOROPLASTS IS STRONGLY REGULATED BY THE RATIO OF NADPH/NADP+, with the extent of this regulation controlled by the concentration of ribulose 1,5-diphosphate. Other metabolites of the reductive pentose phosphate cycle are far less effective in mediating the regulation of the enzyme activity by NADPH/NADP+ ratio. With a ratio of NADPH/NADP+ of 2, and a concentration of ribulose 1,5-diphosphate of 0.6 mM, the activity of the enzyme is completely inhibited. This level of ribulose 1,5-diphosphate is well within the concentration range which has been reported for unicellular green algae photosynthesizing in vivo. Ratios of NADPH/NADP+ of 2.0 have been measured for isolated spinach chloroplasts in the light and under physiological conditions. Since ribulose 1,5-diphosphate is a metabolite unique to the reductive pentose phosphate cycle and inhibits glucose-6-phosphate dehydrogenase in the presence of NADPH/NADP+ ratios found in chloroplasts in the light, it is proposed that regulation of the oxidative pentose phosphate cycle is accomplished in vivo by the levels of ribulose 1,5-diphosphate, NADPH, and NADP+. It already has been shown that several key reactions of the reductive pentose phosphate cycle in chloroplasts are regulated by levels of NADPH/NADP+ or other electron-carrying cofactors, and at least one key-regulated step, the carboxylation reaction is strongly affected by 6-phosphogluconate, the metabolic unique to the oxidative pentose phosphate cycle. Thus there is an interesting inverse regulation system in chloroplasts, in which reduced/oxidized coenzymes provide a general regulatory mechanism. The reductive cycle is activated at high NADPH/NADP+ ratios where the oxidative cycle is inhibited, and ribulose 1,5-diphosphate and 6-phosphogluconate provide further control of the cycles, each regulating the cycle in which it is not a metabolite.     

The role of enzyme activation state in limiting carbon assimilation under variable light conditions

The mechanisms regulating transient photosynthesis by soybean (Glycine max) leaves were examined by comparing photosynthetic rates and carbon reduction cycle enzyme activities under flashing (saturating 1 s lightflecks separated by low photon flux density (PFD) periods of different durations) and continuous PFD. At the same mean PFD, the mean photosynthetic rates were reduced under flashing as compared to continuous light. However, as the duration of the low PFD period lengthened, the CO₂ assimilation attributable to a lightfleck increased. This enhanced lightfleck CO₂ assimilation was accounted for by a greater postillumination CO₂ fixation occurring after the lightfleck. The induction state of photosynthesis, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fructose 1,6-bisphosphatase (FBPase) and ribulose 5-phosphate kinase (Ru5P kinase) activities all responded similarly and were all lower under flashing as compared to constant PFD of the same integrated mean value. However, the fast phase of induction and FBPase and Ru5P kinase activities were reduced more than were the slow phase of induction and rubisco activity. This was consistent with the role of the former enzymes in the fast induction component that limited RuBP regeneration. Competition for reducing power between carbon metabolism and thioredoxin-mediated enzyme activation may have resulted in lower enzyme activation states and hence lower induction states under flashing than continuous PFD, especially at low lightfleck frequencies (low mean PFD).Abbreviations FBPase fructose 1,6-bisphosphatase (EC 3.1.3.11) - LUE lightfleck use efficiency - P-glycerate 3-phosphoglycerate - PICF post-illumination CO₂ fixation - Ru5P kinase ribulose 5-phosphate kinase (EC 2.7.1.19) - RuBP ribulose 1,5-bisphosphate - rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - SBpase sedoheptulose 1,7-bisphosphatase (EC 3.1.3.37)     

Changing ribulose diphosphate carboxylase/oxygenase activity in ripening tomato fruit

Tomato fruit (Lycopersicum esculentum Mill) from green, pink, and red stages were assayed for changes in the activity of ribulose diphosphate carboxylase and oxygenase, phosphoenolpyruvate carboxylase, changes in the levels of glycolate and respiratory gas exchange. The ribulose diphosphate carboxylase activity decreased as the fruit ripened. By comparison, the ribulose diphosphate oxygenase activity increased during the transition from the green to the pink stage, and declined afterward. The changes in the endogenous glycolate levels and the respiratory gas exchange, as observed at different stages of ripening, resembled the changes in the ribulose diphosphate oxygenase activity. The utilization of glycolate in further metabolic activity may result in the formation of peroxidases required for the onset of ripening.     

Effect of Disalicylidenepropanediamine on the Light-dependent Reduction of Carbon Dioxide and Glycerate 3-Phosphate in Intact Spinach Chloroplasts

Disalicylidenepropanediamine (DSPD) at 0.1 to 1 mm levels inhibited light-dependent (14)CO(2) assimilation in intact spinach chloroplasts about 50 to 80%, and this inhibition was accompanied by an increased ratio of (14)C-glycerate 3-phosphate to (14)C-glyceraldehyde 3-phosphate. Enzymatic analysis established that DSPD also inhibited the light-dependent reduction of glycerate 3-phosphate in intact spinach chloroplasts. DSPD at 0.5 mm did not inhibit ribose 5-phosphate isomerase, ribulose 5-phosphate kinase, glycerate 3-phosphate kinase, NADP(+)-linked glyceraldehyde 3-phosphate dehydrogenase or ribulose 1,5-diphosphate carboxylase. The inhibition of chloroplast (14)CO(2) assimilation by DSPD appeared to be related to the inhibition of the photosynthetic electron transport chain. These observations are consistent with experimental results which demonstrated that DSPD inhibited directly the chloroplast lamellar membrane-mediated, light-dependent reduction of ferredoxin (Trebst, A. and M. Burba, 1967, Z. Pflanzenphysiol. 57: 419-433 and Ben-Amotz, A. and M. Avron, 1972, Plant Physiol. 49: 244-248).     

Modelling C3 photosynthesis: A sensitivity analysis of the photosynthetic carbon-reduction cycle

A model of the C ₃ photosynthetic system is developed which describes the sensitivity of the steadystate rate of carbon dioxide assimilation to changes in the activity of several enzymes of the system. The model requires measurements of the steady-state rate of carbon dioxide assimilation, the concentrations of several intermediates in the photosynthetic system, and the concentration of the active site of ribulose 1,5-bisphosphate carboxyalse/oxygenase (Rubisco). It is shown that in sunflowers (Helianthus annuus L.) at photon flux densities that are largely saturating for the rate of photosynthesis, the steady-stete rate of carbon dioxide assimilation is most sensitive to Rubisco activity and, to a lesser degree, to the activities of the stromal fructose, 6-bisphosphatase and the enzymes catalysing sucrose synthesis. The activities of sedoheptulose 1,7-bisphosphatase, ribulose 5-phosphate kinase, ATP synthase and the ADP-glucose pyrophosphorylase are calculated to have a negligible effect on the flux under the high-light conditions. The utility of this analysis in developing simpler models of photosynthesis is also discussed.Abbreviations c _i intercellular CO₂ concentration - C _infP ^supJ control coefficient for enzyme P with respect to flux J - DHAP dihydroxyacetonephosphate - E4P erythrose 4-phosphate - F6P fructose 6-phosphate - FBP fructose 1,6-bisphosphate - FBPase fructose 1,6-bisphosphatase - G3P glyceraldehyde 3-phosphate - G1P glucose 1-phosphate - G6P glucose 6-phosphate - Pi inorganic phosphate - PCR photosynthetic carbon reduction - PGA 3-phosphoglyceric acid - PPFD photosynthetically active photon flux density - R _n ^J response coefficient for effector n with respect to flux J - R5P ribose 5-phosphate - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - Ru5P ribulose 5-phosphate - RuBP ribulose 1,5-bisphosphate - S7P sedoheptulose 7-phosphate - SBP sedoheptulose 1,7-bisphosphate - SBPase sedoheptulose 1,7-bisphosphatase - SPS sucrose-phosphate synthase - Xu5P xylulose 5-phosphate - _n ^P elasticity coefficient for effector n with respect to the catalytic velocity of enzyme P This research was funded by an Australian Research Council grant to I.E.W. and was undertaken during a visity by K.A.M. to the James Cook University of North Queensland. The expert help of Glenys Hanley and Mick Kelly is greatly appreciated.