RuBPCO kinetics and the mechanism of CO2 entry in C3 plants

Abstract. The CO₂ partial pressure in the chloroplasts of intact photosynthetic C₃ leaves is thought to be less than the intercellular CO₂ partial pressure. The intercellular CO₂ partial pressure can be calculated from CO₂ and H₂O gas exchange measurements, whereas the CO₂ partial pressure in the chloroplasts is unknown. The conductance of CO₂ from the intercellular space to the chloroplast stroma and the CO₂ partial pressure in the chloroplast stroma can be calculated if the properties of photosynthetic gas exchange are compared with the kinetics of the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPCO). A discrepancy between gas exchange and RuBPCO kinetics can be attributed to a deviation of CO₂ partial pressure in the chloroplast stroma from that calculated in the intercellular space. This paper is concerned with the following: estimation of the kinetic constants of RuBPCO and their comparison with the CO₂ compensation concentration; their comparison with differential uptake of ¹⁴CO₂ and ¹²CO₂; and their comparison with O₂ dependence of net CO₂ uptake of photosynthetic leaves. Discrepancy between RuBPCO kinetics and gas exchange was found at a temperature of 12.5 °C, a photosynthetic photon flux density (PPFD) of 550 μmol quanta m^?2 s^?1, and an ambient CO₂ partial pressure of 40 Pa. Consistency between RuBPCO kinetics and gas exchange was found if CO₂ partial pressure was decreased, temperature incresed and PPFD decreased. The results suggest that a discrepancy between RuBPCO kinetics and gas exchange is due to a diffusion resistance for CO₂ across the chloroplast envelope which decreases with increasing temperature. At low CO₂ partial pressure, the diffusion resistance appears to be counterbalanced by active CO₂ (or HCO₃) transport with high affinity and low maximum velocity. At low PPFD, CO₂ partial pressure in the chloroplast stroma appears to be in equilibrium with that in the intercellular space due to low CO₂ flux.     

Photosynthesis and the influence of CO2-enrichment on δ13C values in a C3 halophyte

Abstract Shifts in ?¹³C of the graminaceous C₃ halophyte Puccinellia nuttalliana (Schultes) Hitch. can be induced by salinization. To investigate this phenomenon, three approaches were taken: assay of carboxylases, CO₂-enrichment studies, and gas exchange analysis. Although ribulose-1,5-bisphosphate carboxylase activity decreased with salinity, phosphoenolpyruvate carboxylase activity did not increase and its levels were not atypical of C₃ plants. When plants were grown at four NaCl concentrations under atmospheres of 310 and 1300 cm³ m^?3 CO₂, the CO₂-enrichment enhanced the effects of salinity on ?¹³C. This is consistent with a biophysical explanation for salt-induced shifts in ?¹³C, whereby there is a steepening of the CO₂ diffusion gradient into the leaf. Gas exchange analysis indicated that intercellular CO₂ concentrations were depressed in the leaves of salt-affected plants. This resulted from a greatly decreased stomatal conductance coupled with only small effects on intrinsic photosynthetic capacity. Water-use efficiency was enhanced.     

Altered night-time CO2 concentration affects the growth, physiology and biochemistry of soybean

Soybean plants (Glycine max (L.) Merr. c.v. Williams) were grown in CO₂ controlled, natural-light growth chambers under one of four atmospheric CO₂ concentrations ([CO₂]): (1) 250 μmol mol^–1 24 h d^–1[250/250]; (2) 1000 μmol mol^–1 24 h d^–1[1000/1000]; (3) 250 μmol mol^–1 during daylight hours and 1000 μmol mol^–1 during night-time hours [250/1000] or (4) 1000 μmol mol^–1 during daylight hours and 250 μmol mol^–1 during night-time hours [1000/250]. During the vegetative growth phase few physiological differences were observed between plants exposed to a constant 24 h [CO₂] (250/250 and 1000/1000) and those that were switched to a higher or lower [CO₂] at night (250/1000 and 1000/250), suggesting that the primary physiological responses of plants to growth in elevated [CO₂] is apparently a response to daytime [CO₂] only. However, by the end of the reproductive growth phase, major differences were observed. Plants grown in the 1000/250 regime, when compared with those in the 1000/1000 regime, had significantly more leaf area and leaf mass, 27% more total plant dry mass, but only 18% of the fruit mass. After 12 weeks of growth these plants also had 19% higher respiration rates and 32% lower photosynthetic rates than the 1000/1000 plants. As a result the ratio of carbon gain to carbon loss was reduced significantly in the plants exposed to the reduced night-time [CO₂]. Plants grown in the opposite switching environment, 250/1000 versus 250/250, showed no major differences in biomass accumulation or allocation with the exception of a significant increase in the amount of leaf mass per unit area. Physiologically, those plants exposed to elevated night-time [CO₂] had 21% lower respiration rates, 14% lower photosynthetic rates and a significant increase in the ratio of carbon gain to carbon loss, again when compared with the 250/250 plants. Biochemical differences also were found. Ribulose-1,5-bisphosphate carboxylase/ oxygenase concentrations decreased in the 250/ 1000 treatment compared with the 250/250 plants, and phosphoenolpyruvate carboxylase activity decreased in the 1000/250 compared with the 1000/1000 plants. Glucose, fructose and to a lesser extent sucrose concentrations also were reduced in the 1000/250 treatment compared with the 1000/1000 plants. These results indicate that experimental protocols that do not maintain elevated CO₂ levels 24 h d^–1 can have significant effects on plant biomass, carbon allocation and physiology, at least for fast-growing annual crop plants. Furthermore, the results suggest some plant processes other than photosynthesis are sensitive to [CO₂] and under ecologically relevant conditions, such as high night-time [CO₂], whole plant carbon balance can be affected.     

On the significance of C3—C4 intermediate photosynthesis to the evolution of C4 photosynthesis

Abstract Evidence is drawn from previous studies to argue that C₃—C₄ intermediate plants are evolutionary intermediates, evolving from fully-expressed C₃ plants towards fully-expressed C₄ plants. On the basis of this conclusion, C₃—C₄ intermediates are examined to elucidate possible patterns that have been followed during the evolution of C₄ photosynthesis. An hypothesis is proposed that the initial step in C₄-evolution was the development of bundle-sheath metabolism that reduced apparent photorespiration by an efficient recycling of CO₂ using RuBP carboxylase. The CO₂-recycling mechanism appears to involve the differential compartmentation of glycine decarboxylase between mesophyll and bundle-sheath cells, such that most of the activity is in the bundlesheath cells. Subsequently, elevated phosphoenolpyruvate (PEP) carboxylase activities are proposed to have evolved as a means of enhancing the recycling of photorespired CO₂. As the activity of PEP carboxylase increased to higher values, other enzymes in the C₄-pathway are proposed to have increased in activity to facilitate the processing of the products of C₄-assimilation and provide PEP substrate to PEP carboxylase with greater efficiency. Initially, such a ‘C₄-cycle’ would not have been differentially compartmentalized between mesophyll and bundlesheath cells as is typical of fully-expressed C₄ plants. Such metabolism would have limited benefit in terms of concentrating CO₂ at RuBP carboxylase and, therefore, also be of little benefit for improving water- and nitrogen-use efficiencies. However, the development of such a limited C₄-cycle would have represented a preadaptation capable of evolving into the leaf biochemistry typical of fully-expressed C₄ plants. Thus, during the initial stages of C₄-evolution it is proposed that improvements in photorespiratory CO₂-loss and their influence on increasing the rate of net CO₂ assimilation per unit leaf area represented the evolutionary ‘driving-force’. Improved resourceuse efficiency resulting from an efficient CO₂-concentrating mechanism is proposed as the driving force during the later stages.     

Carbon: terrestrial C4 plants

The carbon isotope composition of terrestrial C₄ plants depends on the primary carboxylation of phosphoenolpyruvate (PEP) and on the diffusion of CO₂ to the carboxylation sites, but is also influenced by the final carboxylation of ribulose-1,5-bisphosphate (RuBP). Several models have been used for reproducing this complex situation. In the present review, a particular model is applied as a means to interpret the effects of environmental and genetically determined factors on carbon isotope discrimination during C₄ photosynthesis. As a new feature, the model considers four types of limitation of the overall CO₂ assimilation rate. Both carboxylation reactions are assumed to be limited by either maximum enzyme activity or maximum substrate regeneration rate. The model is applied to experimental data on the effects of CO₂, irradiance and water stress on short-term discrimination by leaves of several C₄ species measured simultaneously with CO₂ gas exchange characteristics. In particular, different patterns of the influence of low irradiances on carbon isotope discrimination are interpreted as due to variations in that irradiance at which a transition from limitation by PEP regeneration rate and RuBP carboxylase activity to limitation by the regeneration rates of both substrates occurs. After discussing literature data on the effects of environmental conditions on carbon isotope discrimination by C₄ plants seasonal and developmental changes in carbon isotope composition, studies on the systematic and geographic distribution of C₄ plants, evolutionary and genetical aspects, and some ecological implications are reviewed.     

CO2 and intracellular pH

Abstract The experimental determination of cytoplasmic and vacuolar pH values is discussed. Despite variation in these values evidence indicates that intracellular pH values are normally regulated within narrow limits. The regulatory mechanisms proposed involve the metabolic consumption of OH^& and the active efflux of H ⁺. The evidence for intracellular pH modification in response to CO₂ hydration and the production of HCO^?₃ and H⁺ is examined. Theoretical calculations and experimental data indicate that CO₂ concentrations as high as 5% will lower intracellular pH. Conversely, variation in CO₂ levels around atmospheric concentrations is unlikely to perturb intracellular pH. High CO₂ levels are found in bulky tissues, and flooded root systems. Evidence is presented that the slow diffusion of dissolved CO₂ compared to gaseous CO₂ results in its accumulation. It is proposed that the accumulation of respiratory CO₂ may reduce intracellular pH values when plant tissues, cells or protoplasts are maintained in a liquid culture medium. Finally, the possible role of dark CO₂ fixation and organic acid synthesis in the regulation of intracellular pH is examined.     

Products of photosynthetic 14CO2 fixation and related enzyme activities in fruiting structures of chickpea

Fruiting structures of a number of legumes including chickpea are known to carry out photosynthetic CO₂ assimilation, but the pathway of CO₂ fixation and particularly the role of phosphoenolpyruvate carboxylase (EC 4.1.1.31) in these tissues is not clear. Activities of some key enzymes of the Calvin cycle and C₄ metabolism, rates of ¹⁴CO₂ fixation in light and dark, and initial products of photosynthetic ¹⁴CO₂ fixation were determined in podwall and seedcoat (fruiting structures) and their subtending leaf in chickpea (Cicer arietinum L.). Compared to activities of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and other Calvin cycle enzyme, viz. NADP⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13), NAD⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and ribulose-5-phosphate kinase (EC 2.7.1.19), the levels of phosphoenolpyruvate carboxylase and other enzymes of C₄ metabolism viz. NADP⁺-malate dehydrogenase (EC 1.1.1.82), NAD⁺-malate dehydrogenase (EC 1.1.1.37), NADP⁺ malic enzyme (EC 1.1.1.40), NAD⁺-malic enzyme (EC 1.1.1.39), glutamate oxaloacetate transaminase (EC 2.6.1.1) and glutamate pyruvate transaminase (EC 2.6.1.2), were generally much higher in podwall and seedcoat than in the leaf. Podwall and seedcoat fixed ¹⁴CO₂ in light and dark at much higher rates than the leaf. Short-term assimilation of ¹⁴CO₂ by illuminated fruiting structures produced malate as the major labelled product with less labelling in 3-phosphoglycerate, whereas the leaf showed a major incorporation into 3-phosphoglycerate. It seems likely that the fruiting structures of chickpea utilize phosphoenolpyruvate carboxylase for recapturing the respired carbon dioxide.     

Carbon isotope discrimination in C3-C4 intermediates

Carbon isotope discrimination in C₃–C₄ intermediates is determined by fractionations during diffusion and the biochemical fractionations occurring during CO₂ fixation. These biochemical fractionations in turn depend on the fractionation by Rubisco in the mesophyll, the amount of CO₂ fixation. These biochemical fractionations in turn depend on the fractionation by Rubisco in the mesophyll, the amount of CO₂ fixation occurring in the bundle sheath, the extent of bundle-sheath leakiness and the contribution which C₄-cycle activity makes to the CO₂ pool there. In most instances, carbon isotope discrimination in C₃–C₄ intermediates is C₃-like because only a small fraction of the total carbon fixed is fixed in the bundle sheath. In particular, this must be the case for Flaveria intermediates which initially fix substantial amounts of CO₂ into C₄-acids. In C₃–C₄ intermediates that refix photorespiratory CO₂ alone, it is possible for carbon isotope discrimination to be greater than in C₃-species, particularly at low CO₂ pressures or at high leaf temperatures. Short-term measurements of carbon isotope discrimination and gas exchange of leaves can be used to study the photosynthetic pathways of C₃-C₄ intermediates and their hybrids as has recently been done for C₃ and C₄ species.     

C4 photosynthesis at low temperatures

Abstract. C₄ plants grown in optimum conditions are, by comparison to C₃, capable of higher maximum dry-matter yields and greater efficiencies of water and nitrogen use, yet they are rare outside the subtropics. Both latitudinal and altitudinal limits of C₄ distributions correlate most closely with a mean minimum temperature of 8-10°C during the period of active growth. The possibility that the C₄ process is inherently incapable of functioning at low temperatures is examined. The reversible effects of chilling on the quantum efficiency of C₄ photosynthesis and the functioning of the individual steps in the C₄ cycle are examined. Chilling also produces an irreversible loss of capacity to assimilate CO₂ which is directly proportional to the light received during chilling. It is suggested that the reversible reduction in capacity to assimilate CO₂ and the lack of an alternative pathway for the utilization of lightgenerated reducing power may make C₄ species more prone to chilling-dependent photoinhibition. Laboratory studies and limited field observations suggest that this damage would be most likely to occur during photosynthetic induction at the temperatures and light levels encountered on clear, cool mornings during the spring and early summer in cool climates. Even those C₄ species occurring naturally in cool climates do not appear fully capable of tolerating these conditions; indeed their growth patterns suggest that they may be adapted by avoiding 'rather than enduring' such conditions.     

Elevated CO2 stimulates associative N2 fixation in a C3 plant of the Chesapeake Bay wetland

In this study, the response of N₂ fixation to elevated CO₂ was measured in Scirpus olneyi, a C₃ sedge, and Spartina patens, a C₄ grass, using acetylene reduction assay and ¹⁵N₂ gas feeding. Field plants grown in PVC tubes (25 cm long, 10 cm internal diameter) were used. Exposure to elevated CO₂ significantly (P < 0·05) caused a 35% increase in nitrogenase activity and 73% increase in ¹⁵N incorporated by Scirpus olneyi. In Spartina patens, elevated CO₂ (660 ± 1 μ mol mol ^{− 1}) increased nitrogenase activity and ¹⁵N incorporation by 13 and 23%, respectively. Estimates showed that the rate of N₂ fixation in Scirpus olneyi under elevated CO₂ was 611 ± 75 ng ¹⁵N fixed plant ^{− 1} h ^{− 1} compared with 367 ± 46 ng ¹⁵N fixed plant ^{− 1} h ^{− 1} in ambient CO₂ plants. In Spartina patens, however, the rate of N₂ fixation was 12·5 ± 1·1 versus 9·8 ± 1·3 ng ¹⁵N fixed plant ^{− 1} h ^{− 1} for elevated and ambient CO₂, respectively. Heterotrophic non-symbiotic N₂ fixation in plant-free marsh sediment also increased significantly (P < 0·05) with elevated CO₂. The proportional increase in ¹⁵N₂ fixation correlated with the relative stimulation of photosynthesis, in that N₂ fixation was high in the C₃ plant in which photosynthesis was also high, and lower in the C₄ plant in which photosynthesis was relatively less stimulated by growth in elevated CO₂. These results are consistent with the hypothesis that carbon fixation in C₃ species, stimulated by rising CO₂, is likely to provide additional carbon to endophytic and below-ground microbial processes.     

D1 and D2 Dopaminergic Regulation of Acetylcholine Release from Striata of Freely Moving Rats

The effects of selective D1 and D2 dopaminergic agents on the extracellular acetylcholine (ACh) content in striata of freely moving rats were determined by the microdialysis technique. LY 171555, a selective D2 agonist, reduced ACh output by approximately 30% within 20 min at the dose of 0.2 mg/kg, i.p., whereas the D2 antagonists (-)-remoxipride (10 mg/kg, s.c.) and L-sulpiride (50 mg/kg, i.p.) induced maximal increases of approximately 50% within 10 and 20 min, respectively. In contrast, the D1 antagonist SCH 23390 (0.25 mg/kg, s.c.) decreased the extracellular ACh content by approximately 30% in 20 min, but lower doses--0.025 and 0.05 mg/kg--had no such effect. The stimulation of ACh release by LY 171555 was prevented by (-)-remoxipride but not by SCH 23390 (0.25 mg/kg, s.c.). In addition, the D1 agonist SKF 38393 failed to modify the ACh increasing effect of (-)-remoxipride. Thus, the D1 and D2 receptors subserve opposing functions on ACh release. The D1/D2 dopaminergic agonist R-apomorphine, at the does of 1 mg/kg, i.p., reduced ACh output by approximately 35% only when D1 receptors were blocked by SCH 23390 (0.025 mg/kg, s.c.). The results provide clear in vivo evidence of the tonic inhibition exerted by dopaminergic nigrostriatal input on the cholinergic system of the basal ganglia through D1 and D2 receptors.     

The effects of SO2 and O3 on the foliar nutrition of Scots pine, Norway spruce and Sitka spruce in the Liphook open-air fumigation experiment

Foliar elements were analysed in Scots pine, Sitka spruce and Norway spruce over a 6 year period before and during continuous exposure to SO₂ and O₃ in an open-air fumigation experiment. Sulphur dioxide treatment elevated foliar sulphur concentration in all species, and there were increases in foliar nitrogen in the two spruce species but not in pine. The concentrations of cations were frequently increased by SO₂ treatment, but there was no correlation between the sulphur concentration of needles and their total cation charge. SO₂-related elevations of foliar magnesium were correlated with the concentration of this element in soil solution, but the mechanism by which other cations were enhanced remains unclear. The only consistent effects on nutrient ratios were for SO₂ treatments to increase sulphur/cation ratios.     

SO2 and assimilatory sulfate reduction in beech leaves

The effect of SO₂ on the extractable activity of ATP sulfurylase (EC 2.7.7.4.). adenosine 5'-phosphosulfate sulfotransferase, ribulosebisphosphate carboxylase, chlorophyll, protein, sulfate, and amino acids was examined in leaves of potted grafts of beech ( Fagus sylvatica L.) treated in outdoor fumigation chambers. Addition of 0.025 and 0.075 μl SO₂ 1⁻¹ to unfiltered ambient air caused a decrease in the extractable activity of adenosine 5'-phosphosulfate sulfotransferase to about 20 to 30% of the controls. Neither the extractable activity of ATP sulfurylase and ribulosebisphosphate carboxylase nor the content in chlorophyll, total amino acids and protein were significantly affected by SO₂, but there was an increase in the sulfate content. Leaves treated with 0.075 μl SO₂ 1⁻¹ contained more alanine and cysteine and less serine than the controls. After transfer of the SO2-treated beech trees to control chambers there was an increase in adenosine 5'-phosphosulfate sulfotransferase activity, but no significant decrease in SO₂₋⁴-sulfur.