地质封存二氧化碳与深地微生物相互作用研究进展

地质封存将工业和能源相关领域生产活动产生的二氧化碳（CO₂）进行捕集并注入到深部地下岩石构造中,以实现长期储存的目标,是降低温室气体排放、实现CO₂长期封存的重要可行性手段之一。向深部地下地质构造中注入大量CO₂会导致深地环境发生显著变化,进而引起原生微生物活性及群落结构发生明显改变。因此,地质封存CO₂能够直接或间接影响深地微生物驱动的生物地球化学过程。同时,微生物在短期和长期的超临界CO₂（scCO₂）胁迫作用下,也会通过不同的适应性进化方式影响CO₂在地下环境中的迁移、转化和赋存形态。本文介绍了国内外二氧化碳捕获与封存发展现状以及地质封存CO₂影响条件下的scCO₂-水-微生物-矿物的相互作用领域的最新科研进展,并展望了利用深地微生物强化CO₂固定以及将其转化为高附加值产物的潜力。     

Characterisation of carbon dioxide and bicarbonate transport during steady-state photosynthesis in the marine cyanobacterium Synechococcus strain PCC7002

Net O₂ evolution, gross CO₂ uptake and net HCO _inf3 ^su– uptake during steady-state photosynthesis were investigated by a recently developed mass-spectrometric technique for disequilibrium flux analysis with cells of the marine cyanobacterium Synechococcus PCC7002 grown at different CO₂ concentrations. Regardless of the CO₂ concentration during growth, all cells had the capacity to transport both CO₂ and HCO _inf3 ^su– ; however, the activity of HCO _inf3 ^su– transport was more than twofold higher than CO₂ transport even in cyanobacteria grown at high concentration of inorganic carbon (C_i = CO₂ + HCO _inf3 ^su– ). In low-C_i cells, the affinities of CO₂ and HCO _inf3 ^su– transport for their substrates were about 5 (CO₂ uptake) and 10 (HCO _inf3 ^su– uptake) times higher than in high-C_i cells, while air-grown cells formed an intermediate state. For the same cells, the intracellular accumulated C_i pool reached 18, 32 and 55 mM in high-C_i, air-grown and low-C_i cells, respectively, when measured at 1 mM external C_i. Photosynthetic O₂ evolution, maximal CO₂ and HCO _inf3 ^su– transport activities, and consequently their relative contribution to photosynthesis, were largely unaffected by the CO₂ provided during growth. When the cells were adapted to freshwater medium, results similar to those for artificial seawater were obtained for all CO₂ concentrations. Transport studies with high-C_i cells revealed that CO₂ and HCO _inf3 ^su– uptake were equally inhibited when CO₂ fixation was reduced by the addition of glycolaldehyde. In contrast, in low-C_i cells steady-state CO₂ transport was preferably reduced by the same inhibitor. The inhibitor of carbonic anhydrase ethoxyzolamide inhibited both CO₂ and HCO _inf3 ^su– uptake as well as O₂ evolution in both cell types. In high-C_i cells, the degree of inhibition was similar for HCO _inf3 ^su– transport and O₂ evolution with 50% inhibition occurring at around 1 mM ethoxyzolamide. However, the uptake of CO₂ was much more sensitive to the inhibitor than HCO _inf3 ^su– transport, with an apparent I₅₀ value of around 250 M ethoxyzolamide for CO₂ uptake. The implications of our results are discussed with respect to C_i utilisation in the marine Synechococcus strain.Abbreviations Chl chlorophyll - C_i inorganic carbon (CO₂ + HCO _inf3 ^su– ) - CA carbonic anhydrase - CCM CO₂-concentrating mechanism - EZA ethoxyzolamide - GA glycolaldehyde - K_1/2 concentration required for half-maximal response - Rubisco ribulose-1,5,-bisphosphate carboxylase-oxygenase D.S. is a recipient of a research fellowship from the Deutsche Forschungsgemeinschaft (D.F.G.). In addition, we are grateful to Donald A. Bryant, Department of Molecular and Cell Biology and Center of Biomolecular Structure Function, Pennsylvania State University, USA, for sending us the wild-type strain of Synechococcus PCC7002.     

Inhibition of the growth of two blue-green algae species (Microsystis aruginosa and Anabaena spiroides) by acidification treatments using carbon dioxide

The effect of pH adjusted by aeration with carbon dioxide (CO₂) on the growth of two species of blue-green algae, Microcystis aeruginosa and Anabaena spiroides, was investigated. Three conditions (pH 5.5, 6.0 and 6.5) were found to have significant inhibitory effects on the growth of the two algae species when acidification treatment was conducted during the logarithmic phase. Differences in the inhibition effect of acidification existed between the two species algae. The tolerance of M. aeruginosa to these conditions was also investigated. The results indicated that M. aeruginosa was inhibited significantly, but not dead at pH 6.5, whereas death occurred at pH 5.5 and 6.0. The greatest inhibitory effect of acidification treatment conducted during the stable breeding phase of M. aeruginosa occurred at pH 5.5, while no inhibitory effect was found at pH 6.5.     

Quantitative aspects of oxygen and carbon dioxide exchange through the lungs in Ocypode ceratophthalmus (Crustacea: Decapoda) during rest and exercise in water and air

Ghost crabs Ocypode ceratophthalmus were exercised in air and water to measure CO₂ and O₂ exchange rates using the method of instantaneous measurements of oxygen consumption rate (MO₂) where applicable. Average heart rate increased from 100 to nearly 400 pulses per minute after five minutes of exercise on a treadmill at a run rate of 0.133 m s^?1. It took less than a minute for oxygen taken up through the lung epithelium from the air inside the branchial cavity to reach the maximal oxygen consumption rate of 26.1 mmol O₂ kg^?1 h^?1. Resting MO₂ was 4.06 mmol O₂ kg^?1 h^?1 in air, but decreased to 3.37 mmol O₂ kg^?1 h^?1 in seawater. Radioactive CO₂ from injected l-lactate is released linearly by the lung. The percent accumulated 14-CO₂ in exhaled air, plotted against time, intersects zero time on the x -axis, indicating rapid gas exchange at the lung surface. The P ₅₀ values for native haemocyanin of 4.89 mm Hg before exercise, and 8.99 mm Hg after exercise, are typical of a high-affinity haemocyanin usually associated with terrestrial crabs. The current notion that Ocypode ceratophthalmus drown when submerged in seawater was not substantiated by our experiments. MO₂ in seawater increased from 3.37 mmol O₂ kg^?1 h^?1 for resting crabs to 5.72 mmol O₂ kg^?1 h^?1 during exercise. When submerged by wave-seawater in the natural environment and during exercise in respirometer-seawater O. ceratophthalmus do not swim but, having a specific density of 1.044, float nearly weightless with a minimum of body movements.     

Reassimilation of carbon dioxide by Flaveria (Asteraceae) species representing different types of photosynthesis

The capability to reassimilate CO₂ originating from intracellular decarboxylating processes connected with the photorespiratory glycolate pathway and-or decarboxylation of C₄ acids during C₄ photosynthesis has been investigated with four species of the genus Flaveria (Asteraceae). The C₃-C₄ intermediate species F. pubescens and F. anomala reassimilated CO₂ much more efficiently than the C₃ species F. cronquistii and, with respect to this feature, behaved similarly to the C₄ species F. trinervia. Therefore, under atmospheric conditions the intermediate species photorespired with rates only between 10–20% of that measured with F. cronquistii. At low oxygen concentrations (1,5%) the reassimilation potential of F. anomala approached that of F. trinervia and was distinct from that found with F. pubescens. The data are discussed with respect to a possible sequence of events during evolution of C₄ photosynthesis. If compared with related data for C₃-C₄ intermediate species from other genera they support the hypothesis that, during evolution of C₄ photosynthesis, an efficient capacity for CO₂ reassimilation evolved prior to a CO₂-concentrating mechanism.Abbreviations C₃, C₄ assimilated CO₂ initially found in 3-phosphoglycerate (C₃) or malate and aspartate (C₄) - D reassimilation coefficient - R _n , R _t net, total CO₂ evolution as measured with 0.03 and 3% CO₂, respectively - RuBP ribulose-1,5-bisphosphate - TPS true photosynthesis     

The relationship between the redox state of Q A and photosynthesis in leaves at various carbon-dioxide,oxygen and light regimes

The response of chlorophyll fluorescence elicited by a low-fluence-rate modulated measuring beam to actinic light and to superimposed 1-s pulses from a high-fluence-rate light source was used to measure the redox state of the primary acceptor Q _A of photosystem II in leaves which were photosynthesizing under steady-state conditions. The leaves were exposed to various O₂ and CO₂ concentrations and to different energy fluence rates of actinic light to assess the relationship between rates of photosynthesis and the redox state of Q _A. Both at low and high fluence rates, the redox state of Q _A was little altered when the CO₂ concentration was reduced from saturation to about 600 l·l^-1 although photosynthesis was decreased particularly at high fluence rates. Upon further reduction in CO₂ content the amount of reduced Q _A increased appreciably even at low fluence rates where light limited CO₂ reduction. Both in the presence and in the absence of CO₂, a more reduced Q _A was observed when the O₂ concentration was below 2%. Q _A was almost fully reduced when leaves were exposed to high fluence rates under nitrogen. Even at low fluence rates, Q _A was more reduced in shade leaves of Asarum europaeum and Fagus sylvatica than in leaves of Helianthus annuus and Fagus sylvatica grown under high light. Also, in shade leaves the redox state of Q _A changed more during a transition from air containing 350 l·l^-1 CO₂ to CO₂-free air than in sun leaves. The results are discussed with respect to the energy status and the CO₂-fixation rate of the leaves.Abbreviations and symbols L 1,2 first and second actinic light beam - Q _A primary acceptor of photosystem II - q _Q Q-quenching     

Quantum yields for uptake of carbon dioxide in C3 vascular plants of contrasting habitats and taxonomic groupings

The maximum quantum yields (_a,c) for CO₂ uptake in low-oxygen atmospheres were determined for 11 species of C₃ vascular plants of diverse taxa, habitat and life form using an Ulbricht-sphere leaf chamber. Comparisons were also made between tissues of varied age within species. The species examined were Psilotum nudum (L.) P. Beauv., Davallia bullata Wall. ex Hook., Cycas revoluta Thunb., Araucaria heterophylla (Salisb.) Franco, Picea abies (L.) Karst., Nerium oleander L., Ruellia humilis Nutt., Pilea microphylla (L.) Karst., Beaucarnea stricta Lem., Oplismenus hirtellus (L.) P. Beauv. and Poa annua L. Quantum yields were calculated from the initial slopes of the response of CO₂ uptake to the quantity of photons absorbed in conditions of diffuse lighting. Regression analysis of variance of the initial slopes of the response of CO₂ uptake to photon absorption failed to show any statistically significant differences between age classes within species or between the mature photosynthetic organs of different species. The constancy of _a,c was apparent despite marked variation in the light-saturated rates of CO₂ uptake within and between species. The mean _a,c was 0.093±0.003 for 11 species. By contrast, surface absorptance varied markedly between species from 0.90 to 0.60, producing proportional variation in the quantum yield calculated on an incidentlight basis. The ratio of variable to maximum fluorescence emission at 695 nm for the same tissues also failed to show any statistically significant variation between species, with a mean of 0.838±0.008. Mean values of _a,c reported here for C₃ species, in the absence of photorespiration, are higher than reported in previous surveys of vascular plants, but consistent with recent estimates of the quantum yields of O₂ evolution.Abbreviations and Symbols A rate of CO₂ uptake per unit projected area (mol · m^–2 · s^–1) - F_m the maximum fluorescence emission at 695 nm in saturating excitation light when closure of PSII reaction centres is maximal (relative units) - F_o the ground fluorescence at 695 nm when all PSII reaction centres are assumed open (relative units) - F_v the difference between F_m and F_o - J_Q rate of CO₂ uptake by the sample (nmol · s^–1) - J_Q rate of photon absorption by the sample (nmol · s^–1) - Q absorbed photon flux per unit of projected area (nmol · m^–2 · s^–1) - ₁ the light absorptance of photosynthetic organs (dimensionless) - s₁ and s'₁ the total and projected surface areas of the photosynthetic organs examined (m²) - _a,c and _i,c the quantum yields for CO₂ uptake on an absorbed- and incident-light basis, respectively (dimensionless) - _a,o the quantum yield for O₂ evolution on an absorbed-light basis (dimensionless) This work was supported by grant PI7179-BIO, FWF, Austria to H.B-N. and by a British Council travel award to S.P.L. This work was completed under the auspices of U.S. Department of Energy under Contract No. DE-AC02-76CH00016. We also thank Dr. K.J. Parkinson of PP Systems, Hitchin, UK for the loan of a prototype of a commercial integrating-sphere leaf chamber developed from our design.     

Oxygen and carbon dioxide exchanges in crassulacean-acid-metabolism-plants

The 24 h O₂ uptake and release together with the CO₂ balance have been measured in two CAM plants, one a non-succulent Sempervivum grandifolium, the other a succulent Prenia sladeniana. The O₂ uptake was estimated by the use of ¹⁸O₂. It was found that the mean hourly O₂ uptake in the light was 7 times that in the dark for Sempervivum and 5 times that for Prenia, after correction for the lightdark temperature difference. It was estimated that oxygen uptake in the light was 2.4 times greater than oxygen release (=net photosynthesis) in Sempervivum and 1.4 times greater in Prenia. In both plants there was a positive carbon balance over the 24 h period under the experimental conditions. It was estimated that malate formed during the night could, if completely oxidized to CO₂ and water, account for 74% of the light phase O₂ uptake in Sempervivum. In Prenia the O₂ uptake was more than sufficient to account for a full oxidation of malate.Abbreviations CAM Crassulacean acid metabolism - PAR photosynthetically active radiation - PEP phosphoenolpyruvate - RrBP ribulose-1,5-bisphosphate - TCA tricarboxylic acid cycle     

Oxygen inhibition of photosynthesis in the C4 species Amaranthus graecizans L.

In the C₄ plant, Amaranthus graecizans, increasing [O₂] from 2% up to 100% inhibited photosynthesis, quantum yield, and the carboxylation efficiency, and increased the CO₂ compensation point () from 2 to about 12 l/l. The O₂ inhibition of photosynthesis was fully reversible. When changing from 2.5 to 40% O₂ and vice versa, about 1 h was required for full equilibration with an O₂ inhibition of 18%; whereas in wheat, a C₃ species, inhibition of photosynthesis and its reversal occurs within minutes after changing [O₂], resulting in 63% inhibition of photosynthesis by 45% O₂. These differences in O₂ inhibition between a C₄ and C₃ species can be explained by high diffusive resistance across bundle-sheath cells of C₄ plants and the increased CO₂/O₂ ratio in bundle-sheath cells which is the consequence of the C₄ cycle. In A. graecizans, increased with increasing [O₂] but tended to reach a maximum at relatively high O₂ levels. The lack of a linear increase in as previously observed for C₃ species indicates that a considerable amount of photorespired CO₂ may be re-fixed with increasing levels of O₂. In comparison to previous reports with other C₄ species, photosynthesis of A. graecizans shows greater sensitivity to O₂, with a noticeable inhibition occurring with shifts from 2 to 21% O₂. A. graecizans has characteristics of other C₄ species with respect to Kranz anatomy, localization of PEP carboxylase in mesophyll cells and RuBP carboxylase in bundle-sheath cells, and little fractionation among carbon isotopes during CO₂ fixation. The basis for the higher sensitivity of photosynthesis of A. graecizans to O₂ may be based upon a lower diffusive resistance of gases across bundle-sheath cells than in some other C₄ species.Abbreviations CE carboxylation efficiency - RuBP ribulose-1,5-bisphosphate - CO₂ compensation point     

Atherosclerosis: the carbonic anhydrase, carbon dioxide, calcium concerted theory

Atherosclerosis is a dynamic multifaceted disease which affects the aorta and its major branches, characterized by the presence of lesions called atheromatous plaques. The plaque is a focal thickening of the intima caused by proliferation of smooth muscle cells, and the deposition of cholesterol, other lipids, hydroxyapatite and fibrous connective tissue. It is proposed that the determinant step of the process which leads to the disease atherosclerosis is the calcium precipitation which traps cholesterol in the plaque precursor matrix which contains lipoproteins, calcium carbonate, hydroxyapapatite, triglycerides, albumin, calmodulin and other proteins. The bear, a species which does not contract the disease is used as an example in support of the hypothesis. The bear's ability to regulate calcium levels and the regulation of acid base balance via regulation of carbon dioxide levels permits the control of the determinant step of plaque formation, that is calcification of the plaque.     

Legume presence increases photosynthesis and N concentrations of co-occurring non-fixers but does not modulate their responsiveness to carbon dioxide enrichment

Legumes, with the ability to fix atmospheric nitrogen (N), may help alleviate the N limitations thought to constrain plant community response to elevated concentrations of atmospheric carbon dioxide (CO₂). To address this issue we assessed: (1) the effects of the presence of the perennial grassland N₂ fixer, Lupinus perennis, on biomass accumulation and plant N concentrations of nine-species plots of differing plant composition; (2) leaf-level physiology of co-occurring non-fixing species (Achillea millefolium, Agropyron repens, Koeleria cristata) in these assemblages with and without Lupinus; (3) the effects of elevated CO₂ on Lupinus growth and symbiotic N₂ fixation in both monoculture and the nine-species assemblages; and (4) whether assemblages containing Lupinus exhibit larger physiological and growth responses to elevated CO₂ than those without. This study was part of a long-term grassland field experiment (BioCON) that controls atmospheric CO₂ at current ambient and elevated (560 µmol mol^–1) concentrations using free-air CO₂ enrichment. Nine-species plots with Lupinus had 32% higher whole plot plant N concentrations and 26% higher total plant N pools than those without Lupinus, based on both above and belowground measurements. Co-occurring non-fixer leaf N concentrations increased 22% and mass-based net photosynthetic rates increased 41% in plots containing Lupinus compared to those without. With CO₂ enrichment, Lupinus monocultures accumulated 32% more biomass and increased the proportion of N derived from fixation from 44% to 57%. In nine-species assemblages, Lupinus N derived from fixation increased similarly from 43% to 54%. Although Lupinus presence enhanced photosynthetic rates and leaf N concentrations of co-occurring non-fixers, and increased overall plant N pools, Lupinus presence did not facilitate stronger photosynthetic responses of non-fixing species or larger growth responses of overall plant communities to elevated CO₂. Non-fixer leaf N concentrations declined similarly in response to elevated CO₂ with and without Lupinus present and the relationship between net photosynthesis and leaf N was not affected by Lupinus presence. Regardless of the presence or absence of Lupinus, CO₂ enrichment resulted in reduced leaf N concentrations and rates of net photosynthesis.     

The relationship between carbon dioxide fixation and chlorophyll a fluorescence during induction of photosynthesis in maize leaves at different temperatures and carbon dioxide concentrations

The rate of CO₂ fixation (F_c) and 680 nm chlorophyll fluorescence emission (F680) were measured simultaneously during induction of photosynthesis in Zea mays L. leaves under varying experimental conditions in order to assess the validity of fluorescence as an indicator of in vivo photosynthetic carbon assimilation. Z. mays leaves showed typical Kautsky fluorescence induction curves consisting of a fast rise in emission (O to P) followed by a slow quenching via a major transient (S-M) to a steady-state (T). After an initial lag, net CO₂ assimilation commenced at a point corresponding to the onset of the S-M transient on the F680 induction curve. Subsequently, F_c and F680 always arrived at a steady-state simultaneously. Decreasing the dark-adaption period increased the rate of induction of both parameters. Alteration of leaf temperature produced anti-parallel changes in induction characteristics of F_c and F680. Reducing the CO₂ level to below that required for saturation of photosynthesis also produced anti-parallel changes during induction, however, at CO₂ concentrations tenfold greater than the atmospheric level the rate of F680 quenching from P to T was appreciably reduced without a similar change in the induction of F_c. Removal of CO₂ at steady-state produced only a small increase in F680 and a correspondingly small decrease in F680 occurred when CO₂ was re-introduced. The complex relationship between chlorophyll fluorescence and carbon assimilation in vivo is discussed and the applicability of fluorescence as an indicator of carbon assimilation is considered.Abbreviations F_c rate of CO₂ fixation - F680 fluorescence emission at 680 nm     

Optimization of the bare platinum electrode as an oxygen measurement system in photosynthesis

This paper is concerned with the definition of the standard conditions required for optimum operation of the bare platinum electrode with photosynthetic samples. Experimental evidence shows the following: 1) Polarization circuits should have zero resistance; 2) The electrolyte layer between the electrodes should have a conductance higher than 54×10^{–6 –1} per mm² of platinum electrode area; 3) The electrodes should be polarized just before taking the measurements. All these facts can be interpreted in terms of phenomena occurring on the electrode: The adsorption of hydrogen on the electrode imposes the need for low resistances in the system, and oxygen consumption by the electrode is minimized by polarizing the electrodes as late as possible. This investigation increases the reliability of the bare platinum electrode and gives a basis for a comparison of the results from different experiments. Demonstrations of the pertinence of these conditions are made in our lab with the algae Dunaliella Tertiolecta.     

Influence of enhanced CO2 on growth and photosynthesis of the red algaeGracilaria sp. andG. chilensis

The influence of elevated CO₂ concentrations on growth and photosynthesis ofGracilaria sp. andG. chilensis was investigated in order to procure information on the effective utilization of CO₂. Growth of both was enhanced by CO₂ enrichment (air + 650 ppm CO₂, air + 1250 ppm CO₂, the enhancement being greater inGracilaria sp. Both species increased uptake of NO₃ ^– with CO₂ enrichment. Photosynthetic inorganic carbon uptake was depressed inG. chilensis by pre-culture (15 days) with CO₂ enrichment, but little affected inGracilaria sp. Mass spectrometric analysis showed that O₂ uptake was higher in the light than in the dark for both species and in both cases was higher inGracilaria sp. The higher growth enhancement inGracilaria sp. was attributed to greater depression of photorespiration by the enrichment of CO₂ in culture.     

Carboxylating enzymes and pathway of photosynthetic carbon assmilation in different marine algae—Evidence for the C4-pathway?

Experiments on short-term photosynthesis in H¹⁴CO₃ ^- (2–5 s) using various species of different algal classes resulted in predominant ¹⁴C-labelling (>90% of total ¹⁴C-incorporation) of phosphorylated compounds. The percentage of malate and aspartate usually accounts for distinctly less than 10% of the total ¹⁴C-labelling. These findings are consistent with data from enzymatic analyses, since 97–100% of the carboxylation capacity is due to ribulose-1.5-biphosphate carboxylase (EC 4.1.1.39) in Rhodophyceae and Chlorophyceae. Phaeophyceae are generally characterized by considerable activity of phosphoenolpyruvate carboxykinase (EC 4.1.1.32): at least 10% of carboxylation is confined to this enzyme. Similar ratios are obtained when rates of photosynthesis and of light-independent CO₂-fixation are compared. Activity of phosphoenolpyruvate carboxylase (EC 4.1.1.31) could not be detected in the species investigated. The results are discussed with emphasis on the pathway of photosynthetic carbon assimilation in marine algae.Abbreviations PEP-CK phosphoenolpyruvate carboxykinase (EC 4.1.1.32) - PEP-C phosphoenolpyruvate carboxylase (EC 4.1.1.31) - RubP-C ribulose-1.5-biphosphate carboxylase (EC 4.1.1.39) Dedicated to Professor H. Fischer, Bonn, on his 65th birthday     

Efflux of carbon dioxide from snow-covered forest floors

The release of CO₂ from the snow surface in winter and the soil surface in summer was directly or indirectly measured in four cool-temperate deciduous broadleaved and evergreen needle forests. The closed chamber method (CC-method) and Fick's diffusion model (DM-method) were used for the direct and indirect measurements, respectively. The winter soil temperatures from the soil surface to 10 cm depth were between 0 and 2°C. The concentration of CO₂ within snowpack increased linearly with increasing snow depth. The average effluxes of CO₂ calculated from the gradients of CO₂ concentration in the snow using the DM-method ranged from 20 to 75 mg CO₂ m⁻² h⁻¹, while the CC-method showed the average effluxes of 20 to 50 mg CO₂m⁻²h⁻¹. These results reveal that the snow thermally insulates the soil, allowing CO₂ production to continue at soil temperatures a little above freezing throughout the winter. Carbon dioxide formed in the soil can move across snowpack up to the atmosphere. The winter/summer ratio of CO₂ emission was estimated to be higher than 7%. Therefore, the snow-covered soil served as a source of CO₂ in the winter and the effluxes represent an important part of the annual CO₂ budget in snowy regions.     

Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves

A series of experiments is presented investigating short term and long term changes of the nature of the response of rate of CO₂ assimilation to intercellular p(CO₂). The relationships between CO₂ assimilation rate and biochemical components of leaf photosynthesis, such as ribulose-bisphosphate (RuP₂) carboxylase-oxygenase activity and electron transport capacity are examined and related to current theory of CO₂ assimilation in leaves of C₃ species. It was found that the response of the rate of CO₂ assimilation to irradiance, partial pressure of O₂, p(O₂), and temperature was different at low and high intercellular p(CO₂), suggesting that CO₂ assimilation rate is governed by different processes at low and high intercellular p(CO₂). In longer term changes in CO₂ assimilation rate, induced by different growth conditions, the initial slope of the response of CO₂ assimilation rate to intercellular p(CO₂) could be correlated to in vitro measurements of RuP₂ carboxylase activity. Also, CO₂ assimilation rate at high p(CO₂) could be correlated to in vitro measurements of electron transport rate. These results are consistent with the hypothesis that CO₂ assimilation rate is limited by the RuP₂ saturated rate of the RuP₂ carboxylase-oxygenase at low intercellular p(CO₂) and by the rate allowed by RuP₂ regeneration capacity at high intercellular p(CO₂).