Effects of temperature on the regulation of photosynthetic carbon assimilation in leaves of maize and barley

The aim of this work was to examine the effect of temperature in the range 5 to 30 ° C upon the regulation of photosynthetic carbon assimilation in leaves of the C₄ plant maize (Zea mays L.) and the C₃ plant barley (Hordeum vulgare L.). Measurements of the CO₂-assimilation rate in relation to the temperature were made at high (735 bar) and low (143 bar) intercellular CO₂ pressure in barley and in air in maize. The results show that, as the temperature was decreased, (i) in barley, pools of phosphorylated metabolites, particularly hexose-phosphate, ribulose 1,5-bisphosphate and fructose 1,6-bisphosphate, increased in high and low CO₂; (ii) in maize, pools of glycerate 3-phosphate, triose-phosphate, pyruvate and phosphoenolpyruvate decreased, reflecting their role in, and dependence on, intercellular transport processes, while pools of hexose-phosphate, ribulose 1,5-bis phosphate and fructose 1,6-bisphosphate remained approximately constant; (iii) the redox state of the primary electron acceptor of photosystem II (Q_A) increased slightly in barley, but rose abruptly below 12° C in maize. Non-photochemical quenching of chlorophyll fluorescence increased slightly in barley and increased to high values below 20 ° C in maize. The data from barley are consistent with the development of a limitation by phosphate status at low temperatures in high CO₂, and indicate an increasing regulatory importance for regeneration of ribulose 1,5-bisphosphate within the Calvin cycle at low temperatures in low CO₂. The data from maize do not show that any steps of the C₄ cycle are particularly cold-sensitive, but do indicate that a restriction in electron transport occurs at low temperature. In both plants the data indicate that regulation of product synthesis results in the maintenance of pools of Calvin-cycle intermediates at low temperatures.Abbreviations Glc6P glucose-6-phosphate - Fru6P fructase-6-phosphate - Frul,6bisP fructose-1,6-bisphosphate - PGA glycerate-3-phosphate - p _i intercellular partial pressure of CO₂ - RuBP ribulose-1,5-bisphosphate - triose-P sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate We thank the Agricultural and Food Research Council, UK (Research grant PG50/67) and the Science and Engineering Research Council, UK for financial support. C.A.L. was supported by the British Council, by the Conselho Nacional de Desenvolvimento Cientiflco e Tecnologico (CNPq), Brazil and by an Overseas Research Student Award. We also thank Mark Stitt (Bayreuth, FRG) and Debbie Rees for helpful discussions.     

The relationship between contents of photosynthetic metabolites and the rate of photosynthetic carbon assimilation in leaves of Amaranthus edulis L.

The relationship between the gas-exchange characteristics of attached leaves of Amaranthus edulis L. and the contents of photosynthetic intermediates was examined in response to changing irradiance and intercellular partial pressure of CO₂. After determination of the rate of CO₂ assimilation at known intercellular CO₂ pressure and irradiance, the leaf was freeze-clamped and the contents of ribulose-1,5-bisphosphate, glycerate-3-phosphate, fructose-1,6-bisphosphate, glucose-6-phosphate, fructose-6-phosphate, triose phosphates, phosphoenolpyruvate, pyruvate, oxaloacetate, aspartate, alanine, malate and glutamate were measured. A comparison between the sizes of metabolite pools and theoretical calculations of metabolite gradients required for transport between the mesophyll and the bundle-sheath cells showed that aspartate, alanine, glycerate-3-phosphate and triose phosphates were present in sufficient quantities to support transport by diffusion, whereas pyruvate and oxaloacetate were not likely to contribute appreciably to the flux of carbon between the two cell types. The amounts of ribulose-1,5-bisphosphate were high at low intercellular partial pressures of CO₂, and fell rapidly as the CO₂-assimilation rate increased with increasing intercellular partial pressures of CO₂, indicating that bundle-sheath CO₂ concentrations fell at low intercellular partial pressures of CO₂. In contrast, the amount of phosphoenolpyruvate and of C₄-cycle intermediates declined at low intercellular partial pressures of CO₂. This behaviour is discussed in relation to the co-ordination of carbon assimilation between the Calvin and C₄ cycles.Abbreviations PEP phosphoenolpyruvate - PGA glycerate-3-phosphate - p _i intercellular CO₂ pressure - RuBP ribulose-1,5-bisphosphate - triose-P triose phosphates     

Regulation of Photosynthesis in Triazine-Resistant and -Susceptible Brassica napus

The response of photosynthetic carbon assimilation and chlorophyll fluorescence quenching to changes in intercellular CO₂ partial pressure (C_i), O₂ partial pressure, and leaf temperature (15-35°C) in triazine-resistant and -susceptible biotypes of Brassica napus were examined to determine the effects of the changes in the resistant biotype on the overall process of photosynthesis in intact leaves. Three categories of photosynthetic regulation were observed. The first category of photosynthetic response, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-limited photosynthesis, was observed at 15, 25, and 35°C leaf temperatures with low C_i. When the carbon assimilation rate was Rubisco-limited, there was little difference between the resistant and susceptible biotypes, and Rubisco activity parameters were similar between the two biotypes. A second category, called feedback-limited photosynthesis, was evident at 15 and 25°C above 300 microbars C_i. The third category, photosynthetic electron transport-limited photosynthesis, was evident at 25 and 35°C at moderate to high CO₂. At low temperature, when the response curves of carbon assimilation to C_i indicated little or no electron transport limitation, the carbon assimilation rate was similar in the resistant and susceptible biotypes. With increasing temperature, more electron transport-limited carbon assimilation was observed, and a greater difference between resistant and susceptible biotypes was observed. These observations reveal the increasing importance of photosynthetic electron transport in controlling the overall rate of photosynthesis in the resistant biotype as temperature increases. Photochemical quenching of chlorophyll fluorescence (q_P) in the resistant biotype never exceeded 60%, and triazine resistance effects were more evident when the susceptible biotype had greater than 60% q_P, but not when it had less than 60% q_P.     

Photosynthetic characteristics of coccoid marine cyanobacteria

Two strains of marine Synechococcus possessed a much greater potential for photorespiration than other marine algae we have studied. This conclusion was based on the following physiological and biochemical characteristics: a) a light-dependent O₂ inhibition of photosynthetic CO₂ assimilation at atmospheric O₂ concentrations. The degree of inhibition was dependent on the relative concentrations of dissolved O₂ and CO₂, being greatest at 100% O₂ with no extra bicarbonate added to the medium; b) actively photosynthesizing cells had high levels of ribulose-1,5-bisphosphate carboxylase compared with phosphoenolpyruvate carboxylase; ribulose-1,5-bisphosphate oxygenase activities were three times greater than ribulose-1,5-bisphosphate carboxylase activities; c) cells photosynthesizing in 21% O₂, showed significant ¹⁴C-labelling of phosphoglycolate and glycolate and the percentage of total carbon-14 incorporated into these two compounds increased when the O₂ concentration was 100%; d) at 100% O₂, there was a post-illumination enhanced rate of O₂ consumption, which was three times greater than dark respiration, and the rate declined with increasing bicarbonate concentrations. The inhibitory effect of O₂ on photosynthesis did not appear to be solely due to photorespiration, since O₂ inhibition of photosynthetic O₂ evolution was much greater than that of photosynthetic CO₂ assimilation. Also, O₂ inhibition of photosynthetic O₂ evolution declined only slightly with decreasing light intensities, while the inhibition of CO₂ assimilation declined rapidly with decreasing light intensity.     

The relationship between steady-state gas exchange of bean leaves and the levels of carbon-reduction-cycle intermediates

The relationship between the gas-exchange characteristics of attached leaves of Phaseolus vulgaris L. and the pool sizes of several carbon-reduction-cycle intermediates was examined. After determining the rate of CO₂ assimilation at known intercellular CO₂ pressure, O₂ pressure and light, the leaf was rapidly killed (<0.1 s) and the levels of ribulose-1,5-bisphosphate (RuBP), 3-phosphoglyceric acid (PGA), fructose-1,6-bisphosphate, fructose-6-phosphate, glucose-6-phosphate, glyceraldehyde-3-phosphate, and dihydroxyacetone phosphate were measured. In 210 mbar O₂, photosynthesis appeared RuBP-saturated at low CO₂ pressure and RuBP-limited at high CO₂ pressure. In 21 mbar (2%) O₂, the level of RuBP always appeared saturating. Very high levels of PGA and other phosphate-containing compounds were found with some conditions, especially under low oxygen.Abbreviations and symbols C₁ intercellular CO₂ pressure - PGA 3-phosphoglyceric acid - RuBP ribulose-1,5-bisphosphate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase     

Carbon assimilation by different developmental stages of Laminaria saccharina

Various stages of the life cycle of the marine brown alga Laminaria saccharina (L.) Lamour. (Laminariales, Phaeophyta) including male and female gametophytes, female gametes, zygotes and young sporophytes of different age were investigated for their potentials of carbon dioxide (¹⁴CO₂) fixation. Rates of photosynthesis attain the same order of magnitude in all stages. Photosynthetic ¹⁴CO₂-fixation is accompanied by a substantial light independent carbon assimilation. This is confirmed by rate determinations of the equivalent carboxylating enzymes present in the plants, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and phosphoenolpyruvate carboxokinase (EC 4.1.1.32) as well as by chromatographic analyses of the appropriate [¹⁴C]-assimilate patterns.Abbreviations RuBP-C ribulose-1,5-bisphosphate carboxylase - PEP-CK phosphoenolpyruvate carboxykinase - PEP phosphoenolpyruvate - PS photosynthesis - DF dark fixation     

Seasonal Changes of Selected Parameters of CO2 Fixation Biochemistry of Norway Spruce Under the Long-Term Impact of Elevated CO2

Twelve-year-old Norway spruce (Picea abies [L.] Karst.) trees were exposed to ambient (AC) or elevated (EC) [ambient + 350 μmol(CO₂) mol^-1] CO₂ concentrations in open-top-chamber (OTC) experiment under the field conditions of a mountain stand. Short-term (4 weeks, beginning of the vegetation season) and long-term (4 growing seasons, end of the vegetation season) effects of this treatment on biochemical parameters of CO₂ assimilation were evaluated. A combination of gas exchange, fluorescence of chlorophyll a, and application of a mathematical model of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) activity was used. The analysis showed that the depression of photosynthetic activity by long-term impact of elevated CO₂ was mainly caused by decreased RuBPCO carboxylation rate. The electron transport rate as well as the rate of ribulose-1,5-bisphosphate (RuBP) formation were also modified. These modifications to photosynthetic assimilation depended on time during the growing season. Changes in the spring were caused mainly by local deficiency of nitrogen in the assimilating tissue. However, the strong depression of assimilation observed in the autumn months was the result of insufficient carbon sink capacity. This revised version was published online in June 2006 with corrections to the Cover Date.     

A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species

Various aspects of the biochemistry of photosynthetic carbon assimilation in C₃ plants are integrated into a form compatible with studies of gas exchange in leaves. These aspects include the kinetic properties of ribulose bisphosphate carboxylase-oxygenase; the requirements of the photosynthetic carbon reduction and photorespiratory carbon oxidation cycles for reduced pyridine nucleotides; the dependence of electron transport on photon flux and the presence of a temperature dependent upper limit to electron transport. The measurements of gas exchange with which the model outputs may be compared include those of the temperature and partial pressure of CO₂(p(CO₂)) dependencies of quantum yield, the variation of compensation point with temperature and partial pressure of O₂(p(O₂)), the dependence of net CO₂ assimilation rate on p(CO₂) and irradiance, and the influence of p(CO₂) and irradiance on the temperature dependence of assimilation rate.Abbreviations RuP₂ ribulose bisphosphate - PGA 3-phosphoglycerate - C=p(CO₂) partial pressure of CO₂ - O=p(O₂) partial pressure of O₂ - PCR photosynthetic carbon reduction - PCO photorespiratory carbon oxidation     

Regulation of photosynthetic electron-transport in Phaseolus vulgaris L., as determined by room-temperature chlorophyll a fluorescence

The regulation of photosystem II (PSII) by light-, CO₂-, and O₂-dependent changes in the capacity for carbon metabolism was studied. Estimates of the rate of electron transport through PSII were made from gas-exchange data and from measurements of chlorophyll fluorescence. At subsaturating photon-flux density (PFD), the rate of electron transport was independent of O₂ and CO₂. Feedback on electron transport was observed under two conditions. At saturating PFD and low partial pressure of CO₂, p(CO₂), the rate of electron transport increased with p(CO₂). However, at high p(CO₂), switching from normal to low p(O₂) did not affect the net rate of photosynthetic CO₂ assimilation but the rate of electron-transport decreased by an amount related to the change in the rate of photorespiration. We interpret these effects as 1) regulation of ribulose-1,5-bisphosphatecarboxylase (RuBPCase, EC 4.1.1.39) activity to match the rate of electron transport at limiting PFD, 2) regulation of electron-transport rate to match the rate of RuBPCase at low p(CO₂), and 3) regulation of the electron-transport rate to match the capacity for starch and sucrose synthesis at high p(CO₂) and PFD. These studies provide evidence that PSII is regulated so that the capacity for electron transport is matched to the capacity for other processes required by photosynthesis, such as ribulose-bisphosphate carboxylation and starch and sucrose synthesis. We show that at least two mechanisms contribute to the regulation of PSII activity and that the relative engagement of these mechanisms varies with time following a step change in the capacity for ribulose-bisphosphate carboxylation and starch and sucrose synthesis. Finally, we take advantage of the relatively slow activation of deactivated RuBPCase in vivo to show that the activation level of this enzyme can limit the rate of electron transport as evidenced by increased feedback on PSII following a step change in p(CO₂). As RuBPCase as activated, the feedback on PSII declined.Abbreviations and symbols J_C electron-transport rate calculated from CO₂-assimilation measurements - J_F electron-transport rate calculated from fluorescence parameters - PFD photon-flux density - q_E energy-dependent quenching - PSII photosystem II - q_Q Q-dependent quenching - QY quantum yield - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) C.I.W. publication No. 1015     

Some relationships between contents of photosynthetic intermediates and the rate of photosynthetic carbon assimilation in leaves of Zea mays L.

The relationship between the gas-exchange characteristics of attached leaves of Zea mays L. and the contents of photosynthetic intermediates was examined at different intercellular partial pressure of CO₂ and at different irradiances at a constant intercellular partial pressure of CO₂. (i) The behaviour of the pools of the C₄-cycle intermediates, phosphoenolpyruvate and pyruvate, provides evidence for light regulation of their consumption. However, light regulation of phosphoenolpyruvate carboxylase does not influence the assimilation rate at limiting intercellular partial pressures of CO₂. (ii) A close correlation between the pools of phosphoenolpyruvate and glycerate-3-phosphate exists under many different flux conditions, consistent with the notion that the pools of C₄ and C₃ cycles are connected via the interconversion of glycerate-3-phosphate and phosphoenolpyruvate. (iii) The ratio of triose-phosphate to glycerate-3-phosphate is used as an indicator of the availability of ATP and NADPH. Changes of this ratio with CO₂ and with irradiance are compared with results obtained in C₃ leaves and indicate that the mechanism of regulation of carbon assimilation by light in leaves of C₄ plants may differ from that in C₃ plants. (iv) The behaviour of the ribulose-1,5-bisphosphate pool with CO₂ and irradiance is contrasted with the behaviour of these pools measured in leaves of C₃ plants.Abbreviations P _i intercellular CO₂ pressure - RuBP ribulose-1,5-bisphosphate - PEP phosphoenolpyruvate - triose-P triose phosphates - PGA glycerate-3-phosphate     

Feedback-limited photosynthesis and regulation of sucrose-starch accumulation during cold acclimation and low-temperature stress in a spring and winter wheat

Regulation of sucrose-starch accumulation and its effect on CO₂ gas exchange and electron transport were studied in low-temperature-stressed and cold-acclimated spring (Katepwa) and winter (Monopol) cultivars of wheat (Triticum aestivum L.). Low-temperature stress of either the spring or winter cultivar was associated with feedback-limited photosynthesis as indicated by a 50–60% reduction in CO₂ assimilation rates, twofold lower ATP/ADP ratio, and threefold lower electron transport rate than 20°C-grown control plants. However, no limitations were evident at the level of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) in low-temperature-stressed plants. Cold acclimation of the spring cultivar resulted in similar feedback-limited photosynthesis observed during low-temperature stress. In contrast, cold acclimation of the winter cultivar resulted in an adjustment of CO₂ assimilation rates to that of control plants. However, we show, for the first time, that this capacity to adjust CO₂ assimilation still appeared to be associated with limited triose phosphate utilisation, a twofold lower ATP/ADP ratio, a reduction in electron transport rates but no restriction at the level of Rubisco compared to controls grown at 20°C. Thus, contrary to previous suggestions, we conclude that cold-acclimated Monopol appears to exhibit feedback limitations at the level of electron transport characteristic of cold-stressed plants despite the maintenance of high rates of CO₂ assimilation. Furthermore, the differential capacity of the winter cultivar to adjust CO₂ assimilation rates was associated with higher levels of sucrose accumulation and a threefold higher sucrose-phosphate synthase activity despite an apparent limitation in triose phosphate utilisation.Abbreviations AGPase ADP-glucose pyrophosphorylase - FBPase fructose-1,6-bisphosphatase - Fru 6-P fructose 6-phosphate - Fru 1,6-BP fructose 1,6-bisphosphate - Glc 6-P glucose 6-phosphate - PGA 3-phosphoglyceric acid - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - RuBP ribulose 1,5-bisphosphate - SPS sucrose-phosphate synthase - Triose-P triose phosphate     

Effect of temperature on the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the rate of respiration in the light

Responses of the rate of net CO₂ assimilation (A) to the intercellular partial pressure of CO₂ (p _i ) were measured on intact spinach (Spinacia oleracea L.) leaves at different irradiances. These responses were analysed to find the value of p _i at which the rate of photosynthetic CO₂ uptake equalled that of photorespiratory CO₂ evolution. At this CO₂ partial pressure (denoted ), net rate of CO₂ assimilation was negative, indicating that there was non-photorespiratory CO₂ evolution in the light. Hence was lower than the CO₂ compensation point, . Estimates of were obtained at leaf temperatures from 15 to 30°C, and the CO₂/O₂ specificity of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (E.C. 4.1.1.39) was calculated from these data, taking into account changes in CO₂ and O₂ solubilities with temperature. The CO₂/O₂ specificity decreased with increasing temperature. Therefore we concluded that temperature effects on the ratio of photorespiration to photosynthesis were not solely the consequence of differential effects of temperature on the solubilities of CO₂ and O₂. Our estimates of the CO₂/O₂ specificity of RuBP carboxylase/oxygenase are compared with in-vitro measurements by other authors. The rate of nonphotorespiratory CO₂ evolution in the light (R _d ) was obtained from the value of A at . At this low CO₂ partial pressure, R _d was always less than the rate of CO₂ evolution in darkness and appeared to decrease with increasing irradiance. The decline was most marked up to about 100 mol quanta m^-2 s^-1 and less marked at higher irradiances. At one particular irradiance, however, R _d as a proportion of the rate of CO₂ evolution in darkness was similar in different leaves and this proportion was unaffected by leaf temperature or by [O₂] (ambient and greater). After conditions of high [CO₂] and high irradiance for several hours, the rate of CO₂ evolution in darkness increased and R _d also increased.Abbreviations and symbols A rate of net CO₂-assimilation - CO₂ compensation point - CO₂ compensation point in the absence of R _d - p _i intercellular partial pressure of CO₂ - R _d (day respiration) rate of non-photorespiratory CO₂ evolution in the light - R _n (night respiration) rate of CO₂ evolution in darkness - RuBP ribulose-1,5-bisphosphate - Rubisco RuBP carboxylase/oxygenase     

Factors influencing the capacity for photosynthetic carbon assimilation in barley leaves at low temperatures

The aim of this work was to examine the effect upon photosynthetic capacity of short-term exposure (up to 10 h) to low temperatures (5° C) of darkened leaves of barley (Hordeum vulgare L.) plants. The carbohydrate content, metabolite status and the photosynthetic rate of leaves were measured at low temperature, high light and higher than ambient CO₂. Under these conditions we could detect whether previous exposure of leaves to low temperature overcame the limitation by phosphate which occurs in leaves of plants not previously exposed to low temperatures. The rates of CO₂ assimilation measured at 8° C differed by as much as twofold, depending upon the pretreatment. (i) Leaves from plants which had previously been darkened for 24 h had a low content of carbohydrate, had the lowest CO₂-assimilation rates at low temperature, and photosynthesis was limited by carbohydrate, as shown by a large stimulation of photosynthesis by feeding glucose, (ii) Leaves from plants which had previously been illuminated for 24 h and which contained large carbohydrate reserves showed an accumulation of phosphorylated intermediates and higher CO₂-assimilation rates at low temperature, but nevertheless remained limited by phosphate, (iii) Maximum rates of CO₂ assimilation at low temperature were observed in leaves which had intermediate reserves of carbohydrate or in leaves which were rich in carbohydrate and which were also fed phosphate. It is suggested that carbohydrate reserves potentiate the system for the achievement of high rates of photosynthesis at low temperatures by accumulation of photosynthetic intermediates such as hexose phosphates, but that this potential cannot be realised if, at the same time, carbohydrate accumulation is itself leading to feedback inhibition of photosynthesis. This work was supported by the Agricultural and Food Research Council, UK (Research grant PG50/67) and by the Science and Engineering Reserach Council, UK. C.A.L. was supported by the British Council, by an Overseas Research Student Award and by the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil.     

Influences of leaf temperature on photosynthetic carbon metabolism in wheat

Net photosynthetic assimilation rate (A), extractable activities of three photosynthetic enzymes, and the concentrations of six metabolites were determined for wheat (Tricum aestivum L.) leaves as leaf temperature was varied under photorespiring (350 microliters per liter CO₂ and 21% O₂) and under nonphotorespiring conditions (800 microliters per liter CO₂ and 2% O₂). The extractable activity of ribulose-1,5-bisphosphate carboxylase (Rubisco) and fructose-1,6-bisphosphatase declined with increasing leaf temperature from 15 to 45°C. Leaf concentrations of ribulose-1,5-bisphosphate (RuBP) declined slightly between 15 and 25°C but increased to a level which is 4 to 5 times the binding site concentration of Rubisco at leaf temperatures of 35 and 45°C. Leaf concentrations of 3-phosphoglycerate, fructose-6-phosphate, and glucose-6-phosphate all declined with increasing leaf temperature. Outside of the limitations imposed by photorespiration, it is proposed that under high light and at suboptimal temperatures, A is limited by rate of utilization of triose phosphate; at optimal temperatures, by the availability of substrate (CO₂ and RuBP) under photorespiring conditions or utilization of triose phosphate under nonphotorespiring conditions; and at supraoptimal temperatures, by the activation state of Rubisco.     

Role of intracellular carbonic anhydrase in inorganic-carbon assimilation by Porphyridium purpureum

Air-grown cells of Porphyridium purpurem contain appreciable carbonic-anhydrase activity, comparable to that in air-grown Chlamydomonas reinhardtii, but activity is repressed in CO₂-grown cells. Assay of carbonic-anhydrase activity in intact cells and cell extracts shows all activity to be intracellular in Porphyridium. Measurement of inorganic-carbon-dependent photosynthetic O₂ evolution shows that sodium ions increase the affinity of Porphyridium cells for HCO ₃ ^- . Acetazolamide and ethoxyzolamide were potent inhibitors of carbonic anhydrase in cell extracts but at pH 5.0 both acetazolamide and ethoxyzolamide had little effect upon the concentration of inorganic carbon required for the half-maximal rate of photosynthetic O₂ evolution (K_0.5[CO₂]). At pH 8.0, where HCO ₃ ^- is the predominant species of inorganic carbon, the K_0.5 (CO₂) was increased from 50 M to 950 M in the presence of ethoxyzolamide. It is concluded that in air-grown cells of Porphyridium. HCO ₃ ^- is transported across the plasmalemma and intracellular carbonic anhydrase increases the steady-state flux of CO₂ from inside the plasmalemma to ribulose-1,5-bisphosphate carboxylase-oxygenase by catalysing the interconversion of HCO ₃ ^- and CO₂ within the cell.Abbreviations AZ acetazolamide - EZ ethoxyzolamide - K_0.5[CO₂] half-maximal rate of photosynthetic O₂ evolution     

The in-vivo response of the ribulose-1,5-bisphosphate carboxylase activation state and the pool sizes of photosynthetic metabolites to elevated CO2 inPhaseolus vulgaris L.

The short-term, in-vivo response to elevated CO₂ of ribulose-1,5-bisphosphate carboxylase (RuBPCase, EC 4.1.1.39) activity, and the pool sizes of ribulose 1,5-bisphosphate, 3-phosphoglyceric acid, triose phosphates, fructose 1,6-bisphosphate, glucose 6-phosphate and fructose 6-phosphate in bean were studied. Increasing CO₂ from an ambient partial pressure of 360–1600 bar induced a substantial deactivation of RuBPCase at both saturating and subsaturating photon flux densities. Activation of RuBPCase declined for 30 min following the CO₂ increase. However, the rate of photosynthesis re-equilibrated within 6 min of the switch to high CO₂, indicating that RuBPCase activity did not limit photosynthesis at high CO₂. Following a return to low CO₂, RuBPCase activation increased to control levels within 10 min. The photosynthetic rate fell immediately after the return to low CO₂, and then increased in parallel with the increase in RuBPCase activation to the initial rate observed prior to the CO₂ increase. This indicated that RuBPCase activity limited photosynthesis while RuBPCase activation increased. Metabolite pools were temporarily affected during the first 10 min after either a CO₂ increase or decrease. However, they returned to their original level as the change in the activation state of RuBPCase neared completion. This result indicates that one role for changes in the activation state of RuBPCase is to regulate the pool sizes of photosynthetic intermediates.Abbreviations and symbols A net CO₂ assimilation rate - C_a ambient CO₂ partial pressure - C_i intercellular CO₂ partial pressure - CABP 2-carboxyarabinitol 1,5-bisphosphate - k_cat catalytic turnover rate per RuBPCase molecule - PFD photon flux density (400 to 700 nm on an area basis) - PGA 3-phosphoglyceric acid - P_i orthophosphate - RuBP ribulose 1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39)     

Stimulation of photosynthesis by 2% oxygen at low temperatures is restored by phosphate

The effect of phosphate feeding on the influence of low (2%) oxygen on photosynthetic carbon assimilation has been investigated in leaf discs of spinach (Spinacia oleracea L.) at 12°C. The following observations were made. First, after the transition from 20% O₂ to 2% O₂, the rate of CO₂ uptake was inhibited at CO₂ concentrations between about 250 and about 800 l CO₂·l^-1. Second, phosphate feeding stimulated the rate of CO₂ uptake in 20% O₂ at higher concentrations of CO₂ (500–900 l·l^-1). Third, phosphate feeding stimulated the rate of CO₂ uptake in 2% O₂ at all but the highest (900 l·l^-1) and lowest 74 (l·l^-1) concentrations of CO₂ employed. Phosphate thereby restored the stimulation of photosynthesis by 2% O₂ and it did so over a wide range of lower temperatures. Fourth, oscillatory behaviour, however generated, was dampened by phosphate feeding, even at very low concentrations of CO₂. Contents of leaf metabolites were measured during the transition to 2% O₂ in control and phosphate-fed leaf discs. During this period the ratio glycerate-3-phosphate/triose phosphate rose steeply, but fell again only in the phosphate-treated leaf discs. These data, taken together with measured ATP/ADP ratios, showed that assimilatory power, the ratio [ATP]·[NAD(P)H]/[ADP]·[Pi]·[NAD(P)], decreased when leaves were exposed to 2% O₂, but that this decrease was minimised by previous feeding of phosphate. The mechanism of phosphate limitation is discussed in the light of the results.Abbreviations C_i intercellular concentration of CO₂ - RuBP ribulose-1,5-bisphosphate     

Specific reduction of chloroplast glyceraldehyde-3-phosphate dehydrogenase activity by antisense RNA reduces CO2 assimilation via a reduction in ribulose bisphosphate regeneration in transgenic tobacco plants

The reduction of 3-phosphoglycerate (PGA) to triose phosphate is a key step in photosynthesis linking the photochemical events of the thylakoid membranes with the carbon metabolism of the photosynthetic carbon-reduction (PCR) cycle in the stroma. Glyceraldehyde-3-phosphate dehydrogenase: NADP oxidoreductase (GAPDH) is one of the two chloroplast enzymes which catalyse this reversible conversion. We report on the engineering of an antisense RNA construct directed against the tobacco (Nicotiana tabacum L.) chloroplastlocated GAPDH (A subunit). The construct was integrated into the tobacco genome by Agrobacterium-mediated transformation of leaf discs. Of the resulting transformants, five plants were recovered with reduced GAPDH activities ranging from 11 to 24% of wild-type (WT) activities. Segregation analysis of the kanamycin-resistance character in self-pollinated T₁ seed from each of the five transformants revealed that one plant (GAP-R) had two active DNA inserts and the others had one insert. T₁ progeny from GAP-R was used to generate plants with GAPDH activities ranging from WT levels to around 7% of WT levels. These were used to study the effect of variable GAPDH activities on metabolite pools for ribulose1,5-bisphosphate (RuBP) and PGA, and the accompanying effects on the rate of CO₂ assimilation and other gasexchange parameters. The RuBP pool size was linearly related to GAPDH activity once GAPDH activity dropped below the range for WT plants, but the rate of CO₂ assimilation was not affected until RuBP levels dropped to 30–40% of WT levels. That is, the CO₂ assimilation rate fell when RuBP per ribulose-1,5-biphosphate carboxylase-oxygenase (Rubisco) site fell below 2 mol·(mol site)^–1 while the ratio for WT plants was 4–5 mol·m(mol site)^–1. Leaf conductance was not reduced in leaves with reduced GAPDH activities, resulting in an increase in the ratio of intercellular to ambient CO₂ partial pressure. Conductance in plants with reduced GAPDH activities was still sensitive to CO₂ and showed a normal decline with increases in CO₂ partial pressure. Although PGA levels did not fluctuate greatly, the effect of reduced GAPDH activity on RuBP-pool size and assimilation rate can be interpreted as being due to a blockage in the regeneration of RuBP. Concomitant gas-ex change and chlorophyll a fluorescence measurements indicated that photosynthesis changed from being Rubisco-limited to being RuBP-regeneration-limited at a lower CO₂ partial pressure in the antisense plants than in WT plants. Photosynthetic electron transport was down-regulated by the build-up of a large proton gradient and the electron-transport chain did not become over-reduced due to a shortage of NADP. Plants with severely reduced GAPDH activity were not photoinhibited despite the continuous presence of a large thylakoid proton gradient in the light. Along with plant size, Rubisco activity, leaf soluble protein and chlorophyll content were reduced in plants with the lowest GAPDH activities. We conclude that chloroplastic GAPDH activity does not appear to limit steady-state photosynthetic CO₂ assimilation at ambient CO₂. This is because WT leaves maintain the ratio of RuBP per Rubisco site about twofold higher than the level required to achieve a maximal rate of CO₂ assimilation.Abbreviations and Symbols bp base pairs - DHAP dihydroxy-acetone phosphate - GAPDH glyceraldehyde-3-phosphate dehy-drogenase - PCR photosynthetic carbon reduction - PGA 3-phosphoglycerate - p_i intercellular CO₂ partial pressure - q_NP non-photochemical fluorescence quenching - q_Q photochemicalfluorescence quenching - PSII quantum efficiency of electronflow through PSII - Rubisco ribulose-1,5-bisphosphate carboxy-lase-oxygenase - RuBP ribulose-1,5-bisphosphate - WT wild type We thank Karin Harrison, Prue Kell, Anne Gallagher and Barbara Setchell for excellent technical assistance. G.D.P. and S.V.C. acknowledge support from QE II Research Fellowships (Australian Research Council).     

Ribulose-1,5-bis-phosphate carboxylase activity in altitudinal populations of Espeletia schultzii Wedd

The ribulose-1,5-bis-phosphate (RBPC) ¹⁴CO₂ fixation rate was measured at four different temperatures, 5°, 15°, 25° and 35° C, in three populations of Espeletia schultzii at different altitudes, 3100, 3550 and 4200 ma.s.l. The fixation rate increased with temperature increase in the populations studied. The population at 4200 m showed the higher rate at any temperature, followed by those at 3550 and 3100 m. The Km(CO₂) increased with temperature increase, but the values were similar among populations. The V _max values increased with temperature and were higher for the 4200-m population. These results suggest that the RBPC enzyme is more activated in the highland population and that the enzyme kinetics are not similar among populations.     

The CO2/O2 specificity of ribulose 1,5-bisphosphate carboxylase/oxygenase

The substrate specificity factor, V _cK_o/V_oK_c, of spinach (Spinacia oleracea L.) ribulose 1,5-bisphosphate carboxylase/oxygenase was determined at ribulosebisphosphate concentrations between 0.63 and 200 M, at pH values between 7.4 and 8.9, and at temperatures in the range of 5° C to 40° C. The CO₂/O₂ specificity was the same at all ribulosebisphosphate concentrations and largely independent of pH. With increasing temperature, the specificity decreased from values of about 160 at 5° C to about 50 at 40° C. The primary effects of temperature were on K _c [Km(CO₂)] and V _c [Vmax (CO₂)], which increased by factors of about 10 and 20, respectively, over the temperature range examined. In contrast, K _o [Ki (O₂)] was unchanged and V _o [Vmax (O₂)] increased by a factor of 5 over these temperatures. The CO₂ compensation concentrations () were calculated from specificity values obtained at temperatures between 5° C and 40° C, and were compared with literature values of . Quantitative agreement was found for the calculated and measured values. The observations reported here indicate that the temperature response of ribulose 1,5-bisphosphate carboxylase/oxygenase kinetic parameters accounts for two-thirds of the temperature dependence of the photorespiration/photosynthesis ratio in C₃ plants, with the remaining one-third the consequence of differential temperature effects on the solubilities of CO₂ and O₂.Abbreviations RuBPC/O(ase) ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - CO₂ compensation concentration