Pulse-modulated photoacoustic measurements reveal strong gas-uptake component at high CO2-concentrations

The effect of high CO₂-concentration on photoacoustic signals from tobacco leaves is studied by means of a recently developed pulse modulation method which provides simultaneous information on photothermal and photobaric components in the millisecond time domain. High CO₂-concentrations are found to induce large gas-uptake signals. Simultaneous measurements of chlorophyll fluorescence suggest that the uptake signals are correlated with energy-dependent fluorescence quenching. Very similar CO₂-concentration dependencies are found in the absence and presence of methylviologen, which is known to catalyze O₂-reduction, and in the presence of glyceraldehyde, which blocks Calvin cycle and photorespiration. It is suggested that the CO₂-enhanced uptake signal is likely to reflect O₂-uptake in the Mehler reaction. However, it is not ruled out that also rapid CO₂-solubilisation or CO₂-binding caused by light-induced stroma alkalisation are involved. Strong uptake is also induced when the CO₂-concentration in the closed photoacoustic chamber increases due to dark-respiration. The consequences of these findings with respect to the interpretation of photoacoustic data (e.g., low-light effect) and to the regulatory role of O₂-dependent electron flow are discussed.     

Computer-controlled pulse modulation system for analysis of photoacoustic signals in the time domain

A newly developed photoacoustic system for measurement of photosynthetic reactions in intact leaves is described. The system is based on pulsed light-emitting diodes, the pulse program and pulse response analysis being computer controlled. Separation of various components in the overall photoacoustic signal is achieved by curve fitting analysis of the responses following individual measuring light pulses in the millisecond time domain. This procedure is in distinction to the conventionally used analysis in the frequency domain, with the advantage that various signal components are obtained by on-line deconvolution, yielding simultaneous recordings of photothermal (complement of energy storage) and photobaric (evolution and uptake) signals. The basic components of the new system are described by block diagrams and the principal steps for deconvolution of the overall photoacoustic response are outlined. An example of application with simultaneous recording of chlorophyll fluorescence is given. It is apparent that the photobaric uptake component represents a significant part of the overall signal, particularly during induction of photosynthesis after dark-adaptation. This component probably contains not only O₂-uptake but uptake of CO₂ as well.Abbreviations PA photoacoustic - LED light-emitting-diode - RAM random access memory     

Active CO(2) Transport by the Green Alga Chlamydomonas reinhardtii

Mass spectrometric measurements of dissolved free ¹³CO₂ were used to monitor CO₂ uptake by air grown (low CO₂) cells and protoplasts from the green alga Chlamydomonas reinhardtii. In the presence of 50 micromolar dissolved inorganic carbon and light, protoplasts which had been washed free of external carbonic anhydrase reduced the ¹³CO₂ concentration in the medium to close to zero. Similar results were obtained with low CO₂ cells treated with 50 micromolar acetazolamide. Addition of carbonic anhydrase to protoplasts after the period of rapid CO₂ uptake revealed that the removal of CO₂ from the medium in the light was due to selective and active CO₂ transport rather than uptake of total dissolved inorganic carbon. In the light, low CO₂ cells and protoplasts incubated with carbonic anhydrase took up CO₂ at an apparently low rate which reflected the uptake of total dissolved inorganic carbon. No net CO₂ uptake occurred in the dark. Measurement of chlorophyll a fluorescence yield with low CO₂ cells and washed protoplasts showed that variable fluorescence was mainly influenced by energy quenching which was reciprocally related to photosynthetic activity with its highest value at the CO₂ compensation point. During the linear uptake of CO₂, low CO₂ cells and protoplasts incubated with carbonic anhydrase showed similar rates of net O₂ evolution (102 and 108 micromoles per milligram of chlorophyll per hour, respectively). The rate of net O₂ evolution (83 micromoles per milligram of chlorophyll per hour) with washed protoplasts was 20 to 30% lower during the period of rapid CO₂ uptake and decreased to a still lower value of 46 micromoles per milligram of chlorophyll per hour when most of the free CO₂ had been removed from the medium. The addition of carbonic anhydrase at this point resulted in more than a doubling of the rate of O₂ evolution. These results show low CO₂ cells of Chlamydomonas are able to transport both CO₂ and HCO₃⁻ but CO₂ is preferentially removed from the medium. The external carbonic anhydrase is important in the supply to the cells of free CO₂ from the dehydration of HCO₃⁻.     

The Main Photoacoustic Gas Uptake Signal Reflects Light-Induced CO2-Uptake Associated with Stroma Alkalisation: New Evidence Based on Carbonic-Anhydrase-Antisense Transgenic Tobacco Plants

The photoacoustic gas uptake signal correlates with carbonicanhydrase activity in transgenic tobacco plants, various levelsof which were induced by antisense RNA [Price et al. (1994)Planta 193: 339]. This is taken as evidence that the uptakesignal is caused by C0₂-solubilisation upon light-driven stromaalkalisation rather than by (O₂-uptake. (Received December 8, 1997; Accepted January 31, 1998)     

Effects of Carbonic Anhydrase Inhibitors on Proton Exchange and Photosynthesis in Pea Protoplasts

At concentrations of 100–200 M, ethoxyzolamide, a lipophilic inhibitor of carbonic anhydrase, considerably (by 60%) inhibited light-induced CO₂-dependent oxygen evolution in pea protoplasts at the optimum concentration of inorganic carbon (100 M CO₂) in the medium. At the same concentrations of the inhibitor, electron transport in isolated pea thylakoids was inhibited only by 6–9%. Acetazolamide, a water-soluble inhibitor of carbonic anhydrase, affected neither the rate of CO₂-dependent O₂evolution in protoplasts nor electron transport in thylakoid membranes. A light-dependent proton uptake by protoplasts was demonstrated. At pH 7.2, the induction kinetics and the rate of proton uptake were similar to those for CO₂-dependent O₂evolution. The rate of proton uptake was decreased twofold by 1 mM acetazolamide. This fact agrees with the notion that a membrane-bound carbonic anhydrase is operative in the plasma membrane of higher plant cells. A mechanism of its functioning is suggested. Possible functions of carbonic anhydrases in the cells of C₃-plants are discussed.     

Photosynthesis of Ivy Leaves (Hedera helix) after Heat Stress I. CO2-Gas Exchange and Diffusion Resistances

For an analysis of the inhibition of the photosynthetic CO₂-uptake after heat stress attached leaves of Hedera helix L. were heat-stressed for 30 min at various temperatures. Subsequently their photosynthetic CO₂-uptake, transpiration, respiration in light and darkness, and CO₂-compensation concentration were measured under optimal conditions. After heat stress the stomatal resistance increased only corresponding to the raised CO₂-concentration inside the leaves (due to the reduced CO₂-uptake). The physical resistance between the mesophyll cell walls and the chloroplasts remained unchanged after heat stress. A non-stomatal inhibition of the CO₂-uptake is indicated by a strong increase of the CO₂-compensation concentration after heat stress. This is hardly due to a stimulation of the respiration in light, as the CO₂-evolution into CO₂-free air in light was even reduced. Therefore, it must be concluded that the photosynthetic process itself is impaired after heat stress.     

Carbonic Anhydrase-Deficient Mutant of Chlamydomonas reinhardii Requires Elevated Carbon Dioxide Concentration for Photoautotrophic Growth

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated which is deficient in carbonic anhydrase (EC 4.2.1.1) activity. This mutant strain, designated ca-1-12-1C (gene locus ca-1), was selected on the basis of a high CO₂ requirement for photoautotrophic growth. Photosynthesis by the mutant at atmospheric CO₂ concentration was very much reduced compared to wild type and, unlike wild type, was strongly inhibited by O₂. In contrast to a CO₂ compensation concentration of near zero in wild type at all O₂ concentrations examined, the mutant exhibited a high, O₂-stimulated CO₂ compensation concentration. Evidence of photorespiratory activity in the mutant but not in wild type was obtained from the analysis of photosynthetic products in the presence of ¹⁴CO₂. At air levels of CO₂ and O₂, the mutant synthesized large amounts of glycolate, while little glycolate was synthesized by wild type under identical conditions. Both mutant and wild type strains formed only small amounts of glycolate at saturating CO₂ concentration. At ambient CO₂, wild type accumulated inorganic carbon to a concentration several-fold higher than that in the suspension medium. The mutant cells accumulated inorganic carbon internally to a concentration 6-fold greater than found in wild type, yet photosynthesis was CO₂ limited. The mutant phenotype was mimicked by wild type cells treated with ethoxyzolamide, an inhibitor of carbonic anhydrase activity. These observations indicate a requirement for carbonic anhydrase-catalyzed dehydration of bicarbonate in maintaining high internal CO₂ concentrations and high photosynthesis rates. Thus, in wild type cells, carbonic anhydrase rapidly converts the bicarbonate taken up to CO₂, creating a high internal CO₂ concentration which stimulates photosynthesis and suppresses photorespiration. In mutant cells, bicarbonate is taken up rapidly but, because of a carbonic anhydrase deficiency, is not dehydrated at a rate sufficiently rapid to maintain a high internal CO₂ concentration.     

CO(2) and O(2) Exchanges in the CAM Plant Ananas comosus (L.) Merr: Determination of Total and Malate-Decorboxylation-Dependent CO(2)-Assimilation Rates; Study of Light O(2)-Uptake

Photosynthesis and light O₂-uptake of the aerial portion of the CAM plant Ananas comosus (L.) merr. were studied by CO₂ and O₂ gas exchange measurements. The amount of CO₂ which was fixed during a complete day-night cycle was equal to the amount of total net O₂ evolved. This finding justifies the assumption that in each time interval of the light period, the difference between the rates of net O₂-evolution and of net light atmospheric CO₂-uptake give the rates of malate-decarboxylation-dependent CO₂ assimilation. Based upon this hypothesis, the following photosynthetic characteristics were observed: (a) From the onset of the light to midphase IV of CAM, the photosynthetic quotient (net O₂ evolved/net CO₂ fixed) was higher than 1. This indicates that malate-decarboxylation supplied CO₂ for the photosynthetic carbon reduction cycle during this period. (b) In phase III and early phase IV, the rate of CO₂ assimilation deduced from net O₂-evolution was 3 times higher than the maximum rate of atmospheric CO₂-fixation during phase IV. A conceivable explanation for this stimulation of photosynthesis is that the intracellular CO₂-concentration was high because of malate decarboxylation. (c) During the final hours of the light period, the photosynthetic quotient decreased below 1. This may be the result of CO₂-fixation by phosphoenolpyruvate-carboxylase activity and malate accumulation. Based upon this hypothesis, the gas exchange data indicates that at least 50% of the CO₂ fixed during the last hour of the light period was stored as malate. Light O₂-uptake determined with ¹⁸O₂ showed two remarkable characteristics: from the onset of the light until midphase IV the rate of O₂-uptake increased progressively; during the following part of the light period, the rate of O₂-uptake was 3.5 times higher than the maximum rate of CO₂-uptake. When malate decarboxylation was reduced or suppressed after a night in a CO₂-free atmosphere or in continuous illumination, the rate of O₂-uptake was higher than in the control. This supports the hypothesis that the low rate of O₂-uptake in the first part of the light period is due to the inhibition of photorespiration by increased intracellular CO₂ concentration because of malate decarboxylation. In view of the law of gas diffusion and the kinetic properties of the ribulose-1,5-bisphosphate carboxylase/oxygenase, O₂ and CO₂ gas exchange suggest that at the end of the light period the intracellular CO₂ concentration was very low. We propose that the high ratio of O₂-uptake/CO₂-fixation is principally caused by the stimulation of photorespiration during this period.     

CO2 exchange characteristics during dark-light transitions in wild-type and mutant Chlamydomonas reinhardii cells

A burst of net CO₂ uptake was observed during the first 3–4 min after the onset of illumination in both wild-type Chlamydomonas reinhardii in which carbonic anhydrase was chemically inhibited with ethoxyzolamide and in a mutant of C. reinhardii (ca-1-12-1C) deficient in carbonic anhydrase activity. The burst was followed by a rapid decrease in the CO₂ uptake rate so that net evolution often occurred. After a 2–3 min period of CO₂ evolution, net CO₂ uptake again increased and ultimately reached a steady-state, positive rate. From [¹⁴CO₂]-tracer studies it was determined that CO₂ fixation proceeded at a nearly linear rate throughout the period of illumination. Thus, prior to reaching a steady state, there was a rapid accumulation of inorganic carbon inside the cells which apparently reached a supercritical concentration and the excess was excreted, causing a subsequent efflux of CO₂. A post illumination burst of net CO₂ efflux was also observed in ethoxyzolamide-inhibited wild type and ca-1 mutant cells, but not in the unihibited wild type. [¹⁴CO₂]-tracer experiments revealed that this burst was the result of a collapse of a large internal inorganic carbon pool at the onset of darkness rather than a photorespiratory post-illumination burst. These results indicate that upon illumination, chemical or genetic inhibition of carbonic anhydrase initially causes an accumulation of excess inroganic carbon in C. reinhardii cells, and that unknown regulatory mechanisms correct for this imbalance by first excreting the excess inorganic carbon and then, after several dampened oscillations, achieving an equilibrium between bicarbonate uptake, bicarbonate dehydration, and CO₂ fixation.     

Carboxysomal carbonic anhydrases: Structure and role in microbial CO2 fixation

Cyanobacteria and some chemoautotrophic bacteria are able to grow in environments with limiting CO₂ concentrations by employing a CO₂-concentrating mechanism (CCM) that allows them to accumulate inorganic carbon in their cytoplasm to concentrations several orders of magnitude higher than that on the outside. The final step of this process takes place in polyhedral protein microcompartments known as carboxysomes, which contain the majority of the CO₂-fixing enzyme, RubisCO. The efficiency of CO₂ fixation by the sequestered RubisCO is enhanced by co-localization with a specialized carbonic anhydrase that catalyzes dehydration of the cytoplasmic bicarbonate and ensures saturation of RubisCO with its substrate, CO₂. There are two genetically distinct carboxysome types that differ in their protein composition and in the carbonic anhydrase(s) they employ. Here we review the existing information concerning the genomics, structure and enzymology of these uniquely adapted carbonic anhydrases, which are of fundamental importance in the global carbon cycle.     

Evidence That an Internal Carbonic Anhydrase Is Present in 5% CO(2)-Grown and Air-Grown Chlamydomonas

Inorganic carbon (C_i) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO₂. Both air-grown cells, that have a CO₂ concentrating system, and 5% CO₂-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (C_i) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO₂-grown cells also accumulated some C_i, although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO₂ fixation by high CO₂-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO₂-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase.     

Carbon dioxide and senescence in cotton plants

Glandless cotton plants (Gossypium hirsutum L. cv. Coker 100) were subjected to the influence of high CO₂-bicarbonate. The content of protein decreased with no accompanying increase in its degraded products. The decrease in protein was correlated with the low content of chlorophyll and also with the reduced activity of carbonic anhydrase. The initiation of these correlations coincided with the time when the control leaves contained the highest enzyme activity during leaf growth. The high concentration of bicarbonate directly restricted the rate of photophosphorylation and that of the Hill reaction in isolated chloroplasts. The amount of ATP in leaves treated in vivo also diminished. High CO₂ as bicarbonate, however, did not directly inhibit the activity of carbonic anhydrase in vitro.     

Zinc deficiency, carbonic anhydrase, and photosynthesis in leaves of spinach

A shortage in the zinc supply to spinach (Spinacia oleracea L.) drastically reduced carbonic anhydrase levels with little effect on net CO₂ uptake per unit leaf area, except with the most severe zinc stresses. Under these conditions, carbonic anhydrase was below 10% and photosynthesis 60 to 70% of the control levels. When photosynthesis was measured at a range of CO₂ supply levels, zinc-deficient leaves were less efficient at 300 to 350 microliters per liter CO₂ and above, but the same as controls at lower CO₂ levels. This suggests that carbonic anhydrase does not affect the diffusion of CO₂, and that the effect of zinc deficiency was on the photosynthetic process itself. Our evidence does not support the hypothesis that carbonic anhydrase has some role in facilitating the supply of CO₂ to the sites of carboxylation within the chloroplast.     

Uptake of Inorganic Carbon by Isolated Chloroplasts of the Unicellular Green Alga Chlorella ellipsoidea

Chloroplasts, isolated from protoplasts of the green alga, Chlorella ellipsoidea, were estimated to be 99% intact by the ferricyanide-reduction assay, and gave CO₂ and PGA-dependent rates of O₂ evolution of 64.5 to 150 micromoles per milligram of chlorophyll per hour, that is 30 to 70% of the photosynthetic activity of the parent cells. Intact chloroplasts showed no carbonic anhydrase activity, but it was detected in preparations of ruptured organelles. Rates of photosynthesis, measured in a closed system at pH 7.5, were twice the calculated rate of CO₂ supply from the uncatalyzed dehydration of HCO₃⁻ indicating a direct uptake of bicarbonate by the intact chloroplasts. Mass spectrometric measurements of CO₂ depletion from the medium on the illumination of chloroplasts indicate the lack of an active CO₂ transport across the chloroplast envelope.     

Effects of Acetazolamide (Diamox), Ethoxzolamide and High Levels of CO2 on Carbonic Anhydrase, Photosystem Activity, and Oxygen Evolution in Euglena gracilis

Investigations using steady-state culture conditions indicate that carbonic anhydrase activity is correlated to the photosynthetic rate in Euglena in some but not all circumstances. When cultures grown with 5% CO₂ were changed to air growth, the photosynthetic rate was independent of the carbonic anhydrase activity. While experiments using the inhibitor acetazolamide indicated a close correlation between photosynthetic capacity and carbonic anhydrase activity, the inhibitor was found to be nonspecific. Acetazolamide altered photosystem activities directly as measured by the photoreduction of DCPIP in chloroplast preparations, whole-cell fluorescence transients of chlorophyll a, and by whole chain photoelectron flow. Ethoxzolamide, another inhibitor of carbonic anhydrase, was also found to inhibit photosystem activities, i.e., the photoreduction of DCPIP, and in vivo photoelectron flow, at high concentrations. Cells grown in 5% CO₂ were less sensitive to the effects of acetazolamide than cells exposed to air. The rate of electron flow in chloroplasts from cells grown with 5% CO₂ and exposed to 10 mM acetazolamide was 2.5-fold faster than that of chloroplasts from air-grown cells exposed to the same concentration of inhibitor. The whole cell chlorophyll a fluorescence transients of cultures grown with high CO₂ were completely different from those of air-grown cells and also showed fewer effects on exposure to acetazolamide. These results suggest a reevaluation of the hypothesis that carbonic anhydrase activity regulates photosynthesis. It is also apparent that results from air-grown and 5% CO₂-grown cultures cannot be directly compared in such studies.     

Carbon Oxysulfide Inhibition of the CO(2)-Concentrating Process of Unicellular Green Algae

Carbonyl sulfide (COS), a substrate for carbonic anhydrase, inhibited alkalization of the medium, O₂ evolution, dissolved inorganic carbon accumulation, and photosynthetic CO₂ fixation at pH 7 or higher by five species of unicellular green algae that had been air-adapted for forming a CO₂-concentrating process. This COS inhibition can be attributed to inhibition of external HCO₃⁻ conversion to CO₂ and OH⁻ by the carbonic anhydrase component of an active CO₂ pump. At a low pH of 5 to 6, COS stimulated O₂ evolution during photosynthesis by algae with low CO₂ in the media without alkalization of the media. This is attributed to some COS hydrolysis by carbonic anhydrase to CO₂. Although COS had less effect on HCO₃⁻ accumulation at pH 9 by a HCO₃⁻ pump in Scenedesmus, COS reduced O₂ evolution probably by inhibiting internal carbonic anhydrases. Because COS is hydrolyzed to CO₂ and H₂S, its inhibition of the CO₂ pump activity and photosynthesis is not accurate, when measured by O₂ evolution, by NaH¹⁴CO₃ accumulation, or by ¹⁴CO₂ fixation.     

Arabidopsis thaliana carbonic anhydrase: cDNA sequence and effect of CO2 on mRNA levels

A full-length cDNA clone encoding carbonic anhydrase was isolated from an Arabidopsis thaliana (Columbia) leaf library. Comparison of the derived amino acid sequence obtained from this clone with those of pea and spinach reveals a considerable degree of identity. The carbonic anhydrase cDNA was used to probe the level of RNA encoding this protein in the leaves of plants grown in elevated CO₂ (660 ppm). We have found that under these conditions the steady-state level of carbonic anhydrase mRNA was increased in comparison with control plants grown in normal atmospheric concentrations of CO₂ (330 ppm). This raises the intruiging possibility that there exists in higher plants a mechanism for perceiving and responding to changes in environmental CO₂ concentrations at the genetic level.     

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.     

Effect of Carbonic Anhydrase Inhibitors on Inorganic Carbon Accumulation by Chlamydomonas reinhardtii

Membrane-permeable and impermeable inhibitors of carbonic anhydrase have been used to assess the roles of extracellular and intracellular carbonic anhydrase on the inorganic carbon concentrating system in Chlamydomonas reinhardtii. Acetazolamide, ethoxzolamide, and a membrane-impermeable, dextran-bound sulfonamide were potent inhibitors of extracellular carbonic anhydrase measured with intact cells. At pH 5.1, where CO₂ is the predominant species of inorganic carbon, both acetazolamide and the dextran-bound sulfonamide had no effect on the concentration of CO₂ required for the half-maximal rate of photosynthetic O₂ evolution (K_0.5[CO₂]) or inorganic carbon accumulation. However, a more permeable inhibitor, ethoxzolamide, inhibited CO₂ fixation but increased the accumulation of inorganic carbon as compared with untreated cells. At pH 8, the K_0.5(CO₂) was increased from 0.6 micromolar to about 2 to 3 micromolar with both acetazolamide and the dextran-bound sulfonamide, but to a higher value of 60 micromolar with ethoxzolamide. These results are consistent with the hypothesis that CO₂ is the species of inorganic carbon which crosses the plasmalemma and that extracellular carbonic anhydrase is required to replenish CO₂ from HCO₃⁻ at high pH. These data also implicate a role for intracellular carbonic anhydrase in the inorganic carbon accumulating system, and indicate that both acetazolamide and the dextran-bound sulfonamide inhibit only the extracellular enzyme. It is suggested that HCO₃⁻ transport for internal accumulation might occur at the level of the chloroplast envelope.     

Carbonic anhydrase activity in leaves as measured in vivo by18O exchange between carbon dioxide and water

Carbonic anhydrase activity of intactCommelina communis L. leaves was measured using mass spectrometry, by following the¹⁸O-exchange kinetics between¹⁸O-enriched carbon dioxide and water. A gas-diffusion model (Gerster, 1971, Planta97, 155–172) was used to interpret the¹⁸O-exchange kinetics and to determine two constants, one (k) related to the hydration of CO₂ and the other (k_e), related to the diffusion of CO₂. Both constants were determined inCommelina communis L. leaves after stripping the lower epidermis to remove any stomatal influence. The hydration constant (k) was 17200 +2200 ·min^–1 (mean±SD, 12 experiments), i.e., about 8 600 times the uncatalyzed hydration of CO₂ in pure water, and was specifically inhibited by ethoxyzolamide, a powerful inhibitor of carbonic anhydrases, half-inhibition occurring around 10^–5 Methoxyzolamide. The diffusion constant (k_e) was 1.18±0.28·min^–1 (mean±SD, 12 experiments) and was only slightly inhibited (about 20%) by ethoxyzolamide. Carbonic anhydrase activity of stripped leaves was not affected by the leaf water status (up to 50% relative water deficits), was strongly inhibited by monovalent anions such as Cl^– or NO ₃ ^– , and decreased by about 50% when the photon flux density during growth was increased from 100 to 500 mol photons·m^–2·s^–1. By studying the effect of ethoxyzolamide (10^–4 M) on photosynthetic O₂ exchange, measured using¹⁸O₂ and mass spectrometry, we found that inhibition of carbonic anhydrase activity by 92–95% had little effect on the response curves of net O₂ evolution to increased CO₂ concentrations. Ethoxyzolamide had no effect on the photosynthetic electron-transport rate, measured as gross O₂ photosynthesis at high CO₂ concentration (>350 l·^–1), but was found to increase both gross O₂ photosynthesis and O₂ uptake at lower CO₂ levels. The chloroplastic CO₂ concentration calculated from O₂-exchange data was not significantly modified by ethoxyzolamide. We conclude from these results that, under normal conditions of photosynthesis, most of the carbonic anhydrase activity is not involved in CO₂ assimilation. Measurement of carbonic anhydrase activity using¹⁸O-isotope exchange therefore provides a suitable model to study the in-vivo regulation of this chloroplastic enzyme in plants submitted to various environmental conditions.Abbreviations CA carbonic anhydrase - C_cc chloroplastic CO₂ concentration - C_e external CO₂ concentration - EZA ethoxyzolamide - k CO₂ hydration rate constant - k_e CO₂ diffusion rate constan - PPFD photosynthetic photon flux density - Rubisco ribulose-1,5 bisphosphate carboxylase oxygenase - RWD relative water deficit The authors wish to thank P. Carrier for technical assistance with mass-spectrometric experiments and Dr. P. Thibault for helpful suggestions and comments. Dr. A. Vavasseur is gratefully acknowledged for supplyingCommelima communis. cultures. P.C., P.T. and A.V. are all from the CEA, Département de Physiologie Végétale et Ecosystèmes, Cadarache, France.