Stoichiometry in the assay of ribulose bisphosphate oxygenase and carboxylase

Complete stoichiometry of the reaction catalyzed by ribulose 1,5-bisphosphate (RuBP) oxygenase from spinach and Rhodospirillum rubrum has been determined. Before initiation and after termination, RuBP has been measured either by release of equimolar orthophosphate at 25°C in the presence of 1 n NaOH or by complete carboxylation using ¹⁴CO₂ and RuBP carboxylase. The RuBP-dependent oxygen consumption has been measured continuously with an oxygen electrode. After termination of catalysis, 3-phosphoglycerate production has been determined spectrophotometrically using phosphoglycerokinase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, α-glycerophosphate dehydrogenase, ATP, and NADH. To measure phosphoglycolate, this product was first hydrolyzed with alkaline phosphatase and the resultant glycolate oxidized by glycolate oxidase. Attendant H₂O₂ formation catalyzed by peroxidase has then been measured colorimetrically. Interference by ribulose in the measurement of glycolate can be easily corrected. Procedures are rapid and do not require separation of reactants and products. Results are in excellent accord with the expected stoichiometry for catalysis by RuBP oxygenase and also enable an estimate of competing catalysis by RuBP carboxylase.     

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.     

Anomalous nonstoichiometry detected by dual label analysis of ribulose 1,5-bisphosphate carboxylase

When ribulose-1,5-bisphosphate carboxylase is assayed under N₂ using [³H]ribulose 1,5-bisphosphate and ¹⁴CO₂, [³H]3-phosphoglycerate and [¹⁴C]3-phosphoglycerate are produced in nonstoichiometric amounts in a ratio which approaches 7 at low concentrations of CO₂ (2 micromolar) assuming a 1:1 ratio at V_max (280 micromolar). The log of the molar ratio varies as a linear function of log[CO₂]. Nonstoichiometry could be explained by CO₂ contaminatio of the reactants or tritium contamination of the products. However, the magnitude of CO₂ contamination required (18 ± 4 micromolar) is far in excess of controlled CO₂ (<0.1 micromolar), and the required tritium contaminant would have to vary from 30 to 85% of the purified 3-phosphoglycerate at the 58 and 2 micromolar CO₂ assay levels, respectively. This contrasts with detectable tritium contamination which is only 1 to 4% and correctable. Nonstoichiometry is evident using either 1 or 5 labeled [³H]ribulose 1,5-bisphosphate. When 3-phosphoglycerate is reisolated as glycerate the ³H/¹⁴C ratio remains unchanged.     

A fluorimetric study of substrate and effector binding of D-ribulose-1,5-biphosphate carboxylase/oxygenase from spinach

2-p-toluidino-naphthalene-6-sulfonate (TNS) is a sensitive fluorescent reporter group for the detection of the events at the reaction centres of the ribulose biphosphate carboxylase/oxygenase from spinach. The formation of binary complexes of the carboxylase with substrates and effectors is associated with significant changes (ΔF) of the fluorescence emission of the enzyme-TNS-complex. This indicates substrate and effector induced conformational changes of the enzyme. From the concentration dependence of ΔF the following dissociation constants for ribulose biphosphate (RuBP) and Mg²⁺ were determined: K(RuBP) = 0,5 μM and K(Mg²⁺) = 1 mM. Sugar phosphates, e.g. 6-phosphogluconate, which show regulatory effects in the carboxylation and oxygenation of RuBP, function antagonistically to RuBP, presumably by competition with RuBP for its allosteric binding site.     

Mild water stress effects on carbon-reduction-cycle intermediates, ribulose bisphosphate carboxylase activity, and spatial homogeneity of photosynthesis in intact leaves

We have examined the effect of mild water stress on photosynthetic chloroplast reactions of intact Phaseolus vulgaris leaves by measuring two parameters of ribulose bisphosphate (RuBP) carboxylase activity and the pool sizes of RuBP, 3-phosphoglycerate (PGA), triose phosphates, hexose monophosphates, and ATP. We also tested for patchy stomatal closure by feeding ¹⁴CO₂. The k_cat of RuBP carboxylase (moles CO₂ fixed per mole enzyme per second) which could be measured after incubating the enzyme with CO₂ and Mg²⁺ was unchanged by water stress. The ratio of activity before and after incubation with CO₂ and Mg²⁺ (the carbamylation state) was slightly reduced by severe stress but not by mild stress. Likewise, the concentration of RuBP was slightly reduced by severe stress but not by mild stress. The concentration of PGA was markedly reduced by both mild and severe water stress. The concentration of triose phosphates did not decline as much as PGA. We found that photosynthesis in water stressed leaves occurred in patches. The patchiness of photosynthesis during water stress may lead to an underestimation of the effect of stomatal closure. We conclude that reductions in whole leaf photosynthesis caused by mild water stress are primarily the result of stomatal closure and that there is no indication of damage to chloroplast reactions.     

Simultaneous kinetic analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase activities

An assay was developed for simultaneous kinetic analysis of the activities of the bifunctional plant enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase [EC 4.1.1.39]. [1-¹⁴C,5-³H]Ribulose 1,5-bisphosphate (RuBP) was used as the labeled substrate. Tritium enrichment of the doubly labeled 3-phosphoglycerate (3-PGA) product, common to both enzyme activities, may be used to calculate V_c/V_o ratios from the expression A/(B-A) where A and B represent the ³H/¹⁴C isotope ratios of doubly labeled RuBP and 3-PGA, and V_c and V_o represent the activities of carboxylase and oxygenase, respectively. Doubly labeled substrate was synthesized from [2-¹⁴C]glucose and [6-³H]glucose using the enzymes of the pentose phosphate pathway coupled with phosphoribulokinase.     

Changes in Levels of Intermediates of the C(4) Cycle and Reductive Pentose Phosphate Pathway under Various Light Intensities in Maize Leaves

The rate of CO₂ assimilation and levels of metabolites of the C₄ cycle and reductive pentose phosphate pathway in attached leaves of maize (Zea mays L.) were measured over a range of light intensity from 0 to 1,900 microEinsteins per square meter per second under a saturated CO₂ concentration of 350 microliters per liter and a limiting CO₂ concentration of 133 microliters per liter. The level of ribulose 1,5-bisphosphate (RuBP) stayed almost constant (around 60 nanomoles per milligram chlorophyll [Chl]) from low to high light intensities under 350 microliters per liter. Levels of 3-phosphoglycerate (PGA) increased from 100 to 650 nanomoles per milligram Chl under 350 microliters per liter CO₂ with increasing light intensity. The calculated RuBP concentration of 6 millimolar (corresponded to 60 nanomoles per milligram Chl) was about two times above the estimated RuBP binding-site concentration on ribulose bisphosphate carboxylase-oxygenase (Rubisco) of ~2.6 millimolar in maize bundle sheath chloroplasts in the light. The ratio of RuBP/PGA increased with decreasing light intensity under 350 microliters per liter CO₂. These results suggest that RuBP carboxylation is under control of light intensity possibly due to a limited supply of CO₂ to Rubisco through the C₄ cycle whose activity is highly dependent on light intensity. Pyruvate level increased with increasing light intensity as long as photosynthesis rate increased. A positive relationship between levels of PGA and those of pyruvate during steady-state photosynthesis under various conditions suggests that an elevated concentration of PGA increases the carbon input into the C₄ cycle through the conversion of PGA to PEP and consequently the level of total intermediates of the C₄ cycle can be raised to mediate higher photosynthesis rate.     

Photometric method for routine determination of kcat and carbamylation of rubisco

Ribulose bisphosphate carboxylase (rubisco) is the first enzyme in photosynthetic CO₂ assimilation. It is also the single largest sink for nitrogen in plants. Several parameters of rubisco activity are often measured including initial activity upon extraction, degree of carbamylation, catalytic constant of the enzyme (k_cat), and the total amount of enzyme present in a leaf. We report here improvements of the photometric assay of rubisco in which rubisco activity is coupled to NADH oxidation which is continuously monitored in a photometer. The initial lag usually found in this assay was eliminated by assaying rubisco activity at pH 8.0 instead of 8.2, using a large amount of phosphoglycerate kinase, and adding monovalent cations to the assay buffer. We found that when using the photometric assay, the ratio of activity found initially upon extraction divided by the activity after incubating with CO₂ and Mg²⁺ reflects the degree of carbamylation as determined by ¹⁴carboxyarabinitol bisphosphate/¹²carboxyarabinitol bisphosphate competition. We developed methods for measuring the catalytic constant of rubisco as well as the total amount of enzyme present using the photometric assay and carboxyarabinitol 1,5-bisphosphate. We believe that the photometric assay for activity will prove more useful than the ¹⁴CO₂ assay in many studies.Abbreviations CA1P 2-carboxyarabinitol 1-phosphate - GAP glyceraldehyde 3-phosphate - OD optical density - PGA 3-phosphoglycerate - rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate     

Influence of leaf age on photosynthesis, enzyme activity, and metabolite levels in wheat

The rate of photosynthesis under high light (1000 micromole quanta per square meter per second) and at 25°C was measured during development of the third leaf on wheat plants and compared with the activity of several photosynthetic enzymes and the level of metabolites. The rate of photosynthesis reached a maximum the 7th day after leaf emergence and declined thereafter. There was a high and significant correlation between the rate of photosynthesis per leaf area and the activities of the enzymes ribulose 5-phosphate kinase (r = 0.91), ribulose 1,5-bisphosphate (RuBP) carboxylase (r = 0.94), 3-phosphoglycerate (PGA) kinase (r = 0.82), and fructose 1,6-bisphosphatase (r = 0.80) per leaf area. There was not a significant correlation of photosynthesis rate with chlorophyll content. The rate of photosynthesis was strongly correlated with the level of PGA (r = 0.85) and inversely correlated with the level of triose phosphate (dihydroxyacetone phosphate and glyceraldehyde 3-phosphate) (r = 0.92). RuBP levels did not change much during leaf development; therefore photosynthesis rate was not correlated with the level of RuBP. The rate of photosynthesis was at a maximum when the ratio of PGA/triose phosphate was high, and when the ratio of RuBP/PGA was low. Although several enzymes change in parallel with leaf development, the metabolite changes suggest the greatest degree of control may be through RuBP carboxylase. The sucrose content of the leaf was highest under high rates of photosynthesis. There was no evidence that later in leaf development, photosynthesis (measured under high light and at 25°C) was limited by utilization of photosynthate.     

Limiting Factors in Photosynthesis: VI. Regeneration of Ribulose 1,5-Bisphosphate Limits Photosynthesis at Low Photochemical Capacity

Earlier work (SE Taylor, N Terry [1984] Plant Physiol 75: 82-86) has shown that the rate of photosynthesis may be colimited by photosynthetic electron transport capacity, even at low intercellular CO₂ concentrations. Here we monitored leaf metabolites diurnally and the activities of key Calvin cycle enzymes in the leaves of three treatment groups of sugar beet (Beta vulgaris L.) plants representing three different in vivo photochemical capacities, i.e. Fe-sufficient (control) plants, moderately Fe-deficient, and severely Fe-deficient plants. The results show that the decrease in photosynthesis with Fe deficiency mediated reduction in photochemical capacity was through a reduction in ribulose 1,5-bisphosphate (RuBP) regeneration and not through a decrease in ribulose 1,5-bisphosphate carboxylase/oxygenase activity. Based on measurements of ATP and NADPH and triose phosphate/3-phosphoglycerate ratios in leaves, there was little evidence that photosynthesis and RuBP regeneration in Fe-deficient leaves were limited directly by the supply of ATP and NADPH. It appeared more likely that photochemical capacity influenced RuBP regeneration through modulation of enzymes in the photosynthetic carbon reduction cycle between fructose-6-phosphate and RuBP; in particular, the initial activity of ribulose-5-phosphate kinase was strongly diminished by Fe deficiency. Starch and sucrose levels changed independently of one another to some extent during the diurnal period (both increasing in the day and decreasing at night) but the average rates of starch or sucrose accumulation over the light period were each proportional to photochemical capacity and photosynthetic rate.     

A sensitive,simultaneous analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase efficiencies: Graphical determination of the CO2/O2 specificity factor

A simple approach to determine CO₂/O₂ specificity factor () of ribulose 1,5-bisphosphate carboxylase/oxygenase is described. The assay measures the amount of CO₂ fixation at varying [CO₂]/[O₂] ratios after complete consumption of ribulose 1,5-bisphosphate (RuBP). Carbon dioxide fixation catalyzed by the carboxylase was monitored by directly measuring the moles of ¹⁴CO₂ incorporated into 3-phosphoglycerate (PGA). This measurement at different [CO₂]/[O₂] ratios is used to determine graphically by several different linear plots the total RuBP consumed by the two activities and the CO₂/O₂ specificity factor. The assay can be used to measure the amounts of products of the carboxylase and oxygenase reactions and to determine the concentration of the substrate RuBP converted to an endpoint amount of PGA and phosphoglycolate. The assay was found to be suitable for all [CO₂]/[O₂] ratios examined, ranging from 14 to 215 micromolar CO₂ (provided as 1–16 mM NaHCO₃) and 614 micromolar O₂ provided as 50% O₂. The procedure described is extremely rapid and sensitive. Specificity factors for enzymes of highly divergent values are in good agreement with previously published data.Abbreviations HEPPS N-(2-hydroxyethyl)piperazine-N-(3-propanesulfonic acid) - L large subunit of rubisco - PGA 3-phosphoglyceric acid - rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP d-ribulose 1,5-bisphosphate - S small subunit of rubisco - XuBP d-xylulose 1,5-bisphosphate     

The Regulation of Photosynthesis in Leaves of Field-Grown Spring Wheat (Triticum aestivum L., cv Albis) at Different Levels of Ozone in Ambient Air

Wheat (Triticum aestivum L. cv Albis) was grown in open-top chambers in the field and fumigated daily with charcoal-filtered air (0.015 microliters per liter O₃), nonfiltered air (0.03 microliters per liter O₃), and air enriched with either 0.07 or 0.10 microliters per liter ozone (seasonal 8 hour/day [9 am-5 pm] mean ozone concentration from June 1 until July 10, 1987). Photosynthetic ¹⁴CO₂ uptake was measured in situ. Net photosynthesis, dark respiration, and CO₂ compensation concentration at 2 and 21% O₂ were measured in the laboratory. Leaf segments were freeze-clamped in situ for the determination of the steady state levels of ribulose 1,5-bisphosphate, 3-phosphoglycerate, triose-phosphate, ATP, ADP, AMP, and activity of ribulose, 1,5-bisphosphate carboxylase/oxygenase. Photosynthesis of flag leaves was highest in filtered air and decreased in response to increasing mean ozone concentration. CO₂ compensation concentration and the ratio of dark respiration to net photosynthesis increased with ozone concentration. The decrease in photosynthesis was associated with a decrease in chlorophyll, soluble protein, ribulose bisphosphate carboxylase/oxygenase activity, ribulose bisphosphate, and adenylates. No decrease was found for triose-phosphate and 3-phosphoglycerate. The ratio of ATP to ADP and of triosephosphate to 3-phosphoglycerate were increased suggesting that photosynthesis was limited by pentose phosphate reductive cycle activity. No limitation occurred due to decreased access of CO₂ to photosynthetic cells since the decrease in stomatal conductance with increasing ozone concentration did not account for the decrease in photosynthesis. Ozonestressed leaves showed an increased degree of activation of ribulose bisphosphate carboxylase/oxygenase and a decreased ratio of ribulose bisphosphate to initial activity of ribulose bisphosphate carboxylase/oxygenase. Nevertheless, it is suggested that photosynthesis in ozone stressed leaves is limited by ribulose bisphosphate carboxylation possibly due to an effect of ozone on the catalysis by ribulose bisphosphate carboxylase/oxygenase.     

Photosynthesis and Ribulose 1,5-Bisphosphate Carboxylase in Rice Leaves: Changes in Photosynthesis and Enzymes Involved in Carbon Assimilation from Leaf Development through Senescence

Changes in photosynthesis and the ribulose 1,5-bisphosphate (RuBP) carboxylase level were examined in the 12th leaf blades of rice (Oryza sativa L.) grown under different N levels. Photosynthesis was determined using an open infrared gas analysis system. The level of RuBP carboxylase was measured by rocket immunoelectrophoresis. These changes were followed with respect to changes in the activities of RuBP carboxylase, ribulose 5-phosphate kinase, NADP-glyceraldehyde 3-phosphate dehydrogenase, and 3-phosphoglyceric acid kinase.

RuBP carboxylase activity was highly correlated with the net rate of photosynthesis (r = 0.968). Although high correlations between the activities of other enzymes and photosynthesis were also found, the activity per leaf of RuBP carboxylase was much lower than those of other enzymes throughout the leaf life. The specific activity of RuBP carboxylase on a milligram of the enzyme protein basis remained fairly constant (1.16 ± 0.07 micromoles of CO₂ per minute per milligram at 25°C) throughout the experimental period.

Kinetic parameters related to CO₂ fixation were examined using the purified carboxylase. The K_m(CO₂) and V_max values were 12 micromolar and 1.45 micromoles of CO₂ per minute per milligram, respectively (pH 8.2 and 25°C). The in vitro specific activity calculated at the atomospheric CO₂ level from the parameters was comparable to the in situ true photosynthetic rate per milligram of the carboxylase throughout the leaf life.

The results indicated that the level of RuBP carboxylase protein can be a limiting factor in photosynthesis throughout the life span of the leaf.

     

Long- and short-term effects of decreased sink demand on carbohydrate levels and photosynthesis in wheat leaves

Wheat (Triticum aestivum L.) ears were removed to investigate long-term regulation of photosynthesis by sink demand at ambient CO₂ and 22 °C. The CO₂ level was also increased to 660 μmol mol^?1 and temperature was lowered to 5 °C to examine short-term responses of photosynthesis to low sink demand. Sink removal inhibited photosynthesis and increased leaf levels of glucose, fructose and ribulose-1, 5-bisphosphate (RuBP), and the glucose-6-phosphate (G6P)/fructose-6-phosphate (F6P) and RuBP/3-phosphoglycerate (PGA) ratios under growth conditions, but had no effect on the activity and activation state of ribulose-1, 5-bisphosphate carboxylase oxygenase (Rubisco) either under growth or short-term conditions, suggesting an inhibition of photosynthesis by decreased in vivo catalysis of Rubisco. Photosynthesis increased similarly in eared and earless shoots after a rise in CO₂ concentration, and the ratio of triose-phosphates (glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, TP) to PGA was similar or higher for removed than intact ears, suggesting that feedback inhibition of photosynthesis was not caused by a limitation of ATP synthesis in chloroplasts. Under short-term conditions (660 μmol mol^?1 CO₂, 5 °C), TP and RuBP levels and the TP/PGA and TP/RuBP ratios were increased by sink removal, indicating an additional limitation of photosynthesis by the rate of RuBP regeneration.     

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.

     

Leaf Phosphate Status, Photosynthesis and Carbon Partitioning in Sugar Beet: II. Diurnal Changes in Sugar Phosphates, Adenylates, and Nicotinamide Nucleotides

Sugar Beets (Beta vulgaris L. cv F58-554H1) were cultured hydroponically in growth chambers. Leaf orthophosphate (Pi) levels were varied nutritionally. The effect of decreased leaf phosphate (low-P) status was determined on the diurnal changes in the pool sizes of leaf ribulose 1,5-bisphosphate (RuBP), 3-phosphoglycerate (PGA), triose phosphate, fructose 1,6-bisphosphate, fructose-6-phosphate, glucose-6-phosphate, adenylates, nicotinamide nucleotides, and Pi. Except for triose phosphate, low-P treatment caused a marked reduction in the levels of leaf sugar phosphates (on a leaf area basis) throughout the diurnal cycle. Low-P treatment decreased the average leaf RuBP levels by 60 to 69% of control values during the light period. Low-P increased NADPH levels and NADPH/NADP⁺ ratio but decreased ATP; the ATP/ADP ratio was unaffected. Low P treatment caused a marked reduction in RuBP regeneration (RuBP levels were half the RuBP carboxylase binding site concentration) but did not depress PGA reduction to triose phosphate. These results indicate that photosynthesis in low-P leaves was limited by RuBP regeneration and that RuBP formation in low-P leaves was not limited by the supply of ATP and NADPH. We suggest that RuBP regeneration was limited by the supply of fixed carbon, an increased proportion of which was diverted to starch synthesis.     

RuBP Limitation of Photosynthetic Carbon Fixation during NH(3) Assimilation : Interactions between Photosynthesis, Respiration, and Ammonium Assimilation in N-Limited Green Algae

The effects of ammonium assimilation on photosynthetic carbon fixation and O₂ exchange were examined in two species of N-limited green algae, Chlorella pyrenoidosa and Selenastrum minutum. Under light-saturating conditions, ammonium assimilation resulted in a suppression of photosynthetic carbon fixation by S. minutum but not by C. pyrenoidosa. These different responses are due to different relationships between cellular ribulose bisphosphate (RuBP) concentration and the RuBP binding site density of ribulose bisphosphate carboxylase/oxygenase (Rubisco). In both species, ammonium assimilation resulted in a decrease in RuBP concentration. In S. minutum the concentration fell below the RuBP binding site density of Rubisco, indicating RuBP limitation of carboxylation. In contrast, RuBP concentration remained above the binding site density in C. pyrenoidosa. Compromising RuBP regeneration in C. pyrenoidosa with low light resulted in an ammonium-induced decrease in RuBP concentration below the RuBP binding site density of Rubisco. This resulted in a decrease in photosynthetic carbon fixation. In both species, ammonium assimilation resulted in a larger decrease in net O₂ evolution than in carbon fixation. Mass spectrometric analysis shows this to be a result of an increase in the rate of mitochondrial respiration in the light.     

Ribulose bisphosphate carboxylase: altered genetic expression in tall fescue

A decaploid tall fescue (Festuca arundinacea Schreb) genotype has been found which exhibits net photosynthetic rates of 32 to 41 mg CO₂/dm²·hour as opposed to a mean of 22 mg CO₂/dm²·hour for 10 hexaploid genotypes. The decaploid genotype exhibited a ribulose 1,5-bisphosphate (RuBP) carboxylase specific activity 1.3- to 2-fold higher than typical tall fescue genotypes. Specific activities of photorespiratory enzymes and nitrate reduction enzymes were lower in the decaploid than the hexaploid genotypes. Results suggest that genetic expression of RuBP carboxylase activity may have been altered to increase the net photosynthesis rate in the decaploid genotype.     

Photosynthesis and ribulose-1,5-bisphosphate carboxylase/oxygenase in rice leaves from emergence through senescence. Quantitative analysis by carboxylation/oxygenation and regeneration of ribulose 1,5-bisphosphate

Changes in gas-exchange rates during the life span of the leaves of rice (Oryza sativa L.) were analyzed quantitatively by measuring changes in the carboxylation/oxygenation and regeneration of ribulose 1,5-bisphosphate (RuBP) at photon fluence rates of 2000 (saturating) and 500 (subsaturating) μmol quanta·m^-2·s^-1 under ambient air conditions. The RuBP levels were always higher than the active-site concentrations of RuBP carboxylase (EC 4.1.1.39), irrespective of the irradiance supplied. Analysis of the CO₂-assimilation rate as a function of intercellular CO₂ concentration indicated that RuBP regeneration does not limit CO₂ assimilation. The estimated RuBP-carboxylase/oxygenase activity in vivo was linearly correlated to the rate of CO₂ assimilation at each level of irradiance. This enzyme activity was just enough to account for the rate of CO₂ assimilation at the saturating irradiance and was 35% more than the rate of CO₂ assimilation at the subsaturating irradiance. Analysis of the assimilation rate at subsaturating irradiance as a function of intercellular CO₂ concentration indicated that a limitation caused by enzyme activation comes into play. The results indicate that the rate of CO₂ assimilation in rice leaves under ambient air conditions is limited during their entire life span by the RuBP-carboxylation/oxygenation capacity.     

Effects of growth and measurement light intensities on temperature dependence of CO2 assimilation rate in tobacco leaves

Effects of growth light intensity on the temperature dependence of CO₂ assimilation rate were studied in tobacco (Nicotiana tabacum) because growth light intensity alters nitrogen allocation between photosynthetic components. Leaf nitrogen, ribulose 1·5‐bisphosphate carboxylase/oxygenase (Rubisco) and cytochrome f (cyt f) contents increased with increasing growth light intensity, but the cyt f/Rubisco ratio was unaltered. Mesophyll conductance to CO₂ diffusion (g_m) measured with carbon isotope discrimination increased with growth light intensity but not with measuring light intensity. The responses of CO₂ assimilation rate to chloroplast CO₂ concentration (C_c) at different light intensities and temperatures were used to estimate the maximum carboxylation rate of Rubisco (V_cmax) and the chloroplast electron transport rate (J). Maximum electron transport rates were linearly related to cyt f content at any given temperature (e.g. 115 and 179 µmol electrons mol^?1 cyt f s^?1 at 25 and 40 °C, respectively). The chloroplast CO₂ concentration (C_trans) at which the transition from RuBP carboxylation to RuBP regeneration limitation occurred increased with leaf temperature and was independent of growth light intensity, consistent with the constant ratio of cyt f/Rubisco. In tobacco, CO₂ assimilation rate at 380 µmol mol^?1 CO₂ concentration and high light was limited by RuBP carboxylation above 32 °C and by RuBP regeneration below 32 °C.