Regulation of carbonic-anhydrase activity,inorganic-carbon uptake and photosynthetic biomass yield inChlamydomonas reinhardtii

The regulation of carbonic anhydrase by environmental conditions was determined forChlamydomonas reinhardtii. The depression of carbonic anhydrase in air-grown cells was pH-dependent. Growth of cells on air at acid pH, corresponding to 10 m CO₂ in solution, resulted in complete repression of carbonic-anhydrase activity. At pH 6.9, increasing the CO₂ concentration to 0.15% (v/v) in the gas phase, corresponding to 11 M in solution, was sufficient to completely repress carbonic-anhydrase activity. Photosynthesis and intracellular inorganic carbon were measured in air-grown and high-CO₂-grown cells using a silicone-oil centrifugation technique. With carbonic anhydrase repressed cells limited inorganic-carbon accumulation resulted from non-specific binding of CO₂. With air-grown cells, inorganic-carbon uptake at acid pH, i.e. 5.5, was linear up to 0.5 mM external inorganic-carbon concentration whereas at alkaline pH, i.e. 7.5, the accumulation ratio decreased with increase in external inorganic-carbon concentration. It is suggested that in air-grown cells at acid pH, CO₂ is the inorganic carbon species that crosses the plasmalemma. The conversion of CO₂ to HCO ₃ ^- by carbonic anhydrase in the cytosol results in inorganic-carbon accumulation and maintains the diffusion gradient for carbon dioxide across the cell boundary. However, this mechanism will not account for energy-dependent accumulation of inorganic carbon when there is little difference in pH between the exterior and cytosol.     

Inorganic-carbon uptake by a small-celled strain of Stichococcus bacillaris

Air-grown cells of a marine, small-celled (2 m diameter) strain of Stichococcus bacillaris contained appreciable carbonic-anhydrase activity but this was repressed when cells were grown on air enriched with 5% (v/v) CO₂. Assay of carbonic-anhydrase activity using intact cells and cell extracts showed all activity was intracellular in this Stichococcus strain. Measurement of inorganic-carbon-dependent photosynthetic O₂ evolution at pH 5.0, where CO₂ is the predominant form of inorganic carbon, showed that the concentration of inorganic carbon required for half-maximal rate of photosynthetic O₂ evolution [K_0.5(CO₂)] was 4.0 M for both air- and CO₂-grown cells. At pH 8.3 the K_0.5(CO₂) was 0.3 mM for air-grown and 0.6 mM for CO₂-grown cells. Sodium ions did not enhance bicarbonate utilization. Measurement of the internal inorganic-carbon pool (HCO ₃ ^– +CO₂) by the silicone-oil-layer centrifugal filtering technique showed that air- and CO₂-grown cells were able to concentrate inorganic carbon up to 20-fold in relation to the external medium at pH 5.0 but not at pH 8.3. In this alga the high affinity for CO₂ and inorganic-carbon accumulation in CO₂- and air-grown cells results from active CO₂ transport that is not dependent on carbonic-anhydrase activity.Abbreviation Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid     

Inorganic-carbon uptake by the marine diatom Phaeodactylum tricornutum

Air-grown cells of the marine diatom Phaeodactylum tricornutum showed only 10% of the carbonic-anhydrase activity of air-grown Chlamydomonas reinhardtii. Measurement of carbonic-anhydrase activity using intact cells and cell extracts showed all activity was intracellular in Phaeodactylum. Photosynthetic oxygen evolution at constant inorganic-carbon concentration but varying pH showed that exogenous CO₂ was poorly utilized by the cells. Sodium ions increased the affinity of Phaeodactylum for HCO ₃ ^- and even at high HCO ₃ ^- concentrations sodium ions enhanced HCO ₃ ^- utilization. The internal inorganic-carbon pool (HCO ₃ ^- +CO₂] was measured using a silicone-oil-layer centrifugal filtering technique. The internal [HCO ₃ ^- +CO₂] concentration never exceeded 15% of the external [HCO ₃ ^- +CO₂] concentration even at the lowest external concentrations tested. It is concluded that an internal accumulation of inorganic carbon relative to the external medium does not occur in P. tricornutum.Abbreviation Hepes 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid     

Inorganic-carbon transport in some marine eukaryotic microalgae

Inorganic-carbon transport was investigated in the eukaryotic marine microalgaeStichococcus minor, Nannochloropsis oculata and aMonallantus sp. Photosynthetic O₂ evolution at constant inorganic-carbon concentration but varying pH showed thatS. minor had a greater capacity for CO₂ rather than HCO ₃ ^– utilization but forN. oculata andMonallantus HCO ₃ ^– was the preferred source of inorganic carbon. All three microalgae had a low affinity for CO₂ as shown by the measurement of inorganic-carbon-dependent photosynthetic O₂ evolution at pH 5.0. At pH 8.3, where HCO ₃ ^– is the predominant form of inorganic carbon, the concentration of inorganic carbon required for half-maximal rate of photosynthetic O₂ evolution [K _0.5 (CO₂)] was 53 M forMonallantus sp. and 125 M forN. oculata, values compatible with HCO ₃ ^– transport. Neither extra- nor intracellular carbonic anhydrase was detected in these three microalgal species. It is concluded that these microalgae lack a specific transport system for CO₂ but that HCO ₃ ^– transport occurs inN. oculata andMonallantus, and in the absence of intracellular carbonic anhydrase the conversion of HCO ₃ ^– to CO₂ may be facilitated by the internal pH of the cell.     

Role of external carbonic anhydrase in light-dependent alkalization byFucus serratus L. andLaminaria saccharina (L.) Lamour. (Phaeophyta)

It has been proposed that many marine macroalgae are able to utilize HCO ₃ ^– for photosynthesis and growth, and that energy-dependent ion pumping is involved in this process. We have therefore studied the light-dependent alkalization of the surrounding medium by two species of marine macroscopic brown algae,Fucus serratus L. andLaminaria saccharina (L.) Lamour. with the aim of investigating the role of extracellular carbonic anhydrase (EC 4.2.1.1.) in the assimilation of inorganic carbon from the seawater medium. In particular, the influence of membrane-impermeable or slowly permeable carbonic-anhydrase inhibitors on the rate of alkalization of the seawater has been investigated. Inhibition of the alkalization rate occurred in both species at an alkaline pH (pH 8.0) but no inhibition was observed at an acidic pH (pH 6.0). The alkalization was found to be light-dependent and inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and, thus, correlated with photosynthesis. Alkalization by macroalgae has previously been shown to be proportional to inorganiccarbon uptake. We suggest that alkalization of the medium at alkaline pH in both of the species examined is mainly the consequence of an extracellular reaction. The reaction is catalyzed by extracellular carbonic anhydrase which converts HCO ₃ ^– to OH^– and CO₂; CO₂ is then taken up through the plasmalemma. However, we do not exclude the involvement of other mechanisms of inorganic-carbon uptake.Abbreviations AZ acetazolamide - CA carbonic anhydrase - CAext extracellular carbonic anhydrase - C_i inorganic carbon - DBS dextran-bound sulfonamide - DCMU 3-(3,4-dichloro-phenyl)-1,1-dimethylurea - PPFD photosynthetic photon flux density This study was carried out with financial support by SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Trygger's Fund for Scientific Research (Sweden), SJFR (Swedish Council for Forestry and Agricultural Research) and CICYT (Spain). Z. Ramazanov is an invited professor of Ministerio de Educación y Ciencia, Spain.     

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.     

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.     

Photosynthesis and the intracellular inorganic carbon pool in the bluegreen alga Anabaena variabilis: Response to external CO2 concentration

The apparent photosynthetic affinity of A. variabilis to CO₂ is greatly affected by the CO₂ concentration in the medium during growth. Halfmaximal rate of photosynthetic O₂ evolution is achieved at 10 M and 100 M inorganic carbon (C_inorg) in cells grown at low-CO₂ (air) and high CO₂ (5% v/v CO₂ in air), respectively, whilst the maximum rate of photosynthesis is similar in both cases. Both high- and low-CO₂-grown Anabaena accumulate C_inorg within the cell; however, the rate of accumulation and the steady-state internal C_inorg concentration reached is much higher in low as compared with high-CO₂-grown cells. It is suggested that Anabaena cells actively accumulate C_inorg. Measurements of the kinetics of C_inorg transport indicate that the affinity of the transport mechanism for C_inorg is similar (K_m(C_inorg(150 M) in both high- and low-CO₂-grown cells. However, V _max is 10-fold higher in the latter case. It is suggested that this higher V _max for transport is the basis of the superior capability to accumulate C_inorg and the higher apparent photosynthetic affinity for external C_inorg in low-CO₂-grown Anabaena. Carbonic anhydrase activity was not detectable in Anabaena, yet both photosynthetic affinity to C_inorg in the medium (but not V _max) and the rate of accumulation of C_inorg were inhibited by the carbonic-anhydrase inhibitor ethoxyzolamide.Abbreviations C_inorg inorganic carbon - PEP phosphoenol pyruvate - RuBP ribulose-1,5-bisphosphate CIW-DPB Publication No. 682     

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.     

Role of carbonic anhydrase in photosynthesis and inorganic-carbon assimilation in the red alga Gracilaria tenuistipitata

The mechanism of inorganic-carbon (C_i) accumulation in the red seaweed Gracilaria tenuistipitata Zhang et Xia has been investigated. Extracellular and intracellular carbonic-anhydrase (CA) activities have been detected. Photosynthetic O₂ evolution in thalli and protoplasts of G. tenuistipitata were higher at pH 6.5 than at pH 8.6, where HCO ₃ ^– is the predominant form of C_i. Dextran-bound sulfonamide (DBS), a specific inhibitor of extracellular CA, reduced photosynthetic O₂ evolution at pH 8.6 and did not have any effect at pH 6.5. After inhibition with DBS, O₂ evolution was similar to the rate that could be supported by CO₂ from spontaneous dehydration of HCO ₃ ^– . The rate of photosynthetic alkalization of the surrounding medium by the algal thallus was dependent on the concentration of C_i and inhibited by DBS. We suggest that the general form of C_i that enters through the plasma membrane of G. tenuistipitata is CO₂. Bicarbonate is utilized mainly by an indirect mechanism after dehydration to CO₂, and this mechanism involves extracellular CA.Abbreviations C_i inorganic carbon (CO₂ + HCO ₃ ^– ) - CA carbonic anhydrase - DIC dissolved inorganic carbon (total) - DBS dextran-bound sulfonamide - EZ ethoxyzolamide - NSW natural seawater - PPFD photosynthetic photon flux density - REA relative enzyme activity - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This research was supported by the Deutsche Forschungsgemeinschaft (Bonn) as a programme of the Sonderforschungsbereich 251 der Universität Würzburg and by the Fonds der Chemischen Industrie (Frankfurt). Joint work in Würzburg was possible thanks to travel grants from the Chancellor of the University of Würzburg, Professor R. Günther, from the Australian National University under the auspices of its Overseas Studies Programme, and from the New Zealand — Federal Republic of Germany Scientific and Technological Exchange Programme, which are gratefully acknowledged. We thank Dr. A. Meyer and Ms. E. Kilian for untiringly conducting part of the experimental work, Ms. G. Theumer and Ms. D. Faltenbacher-Werner for their valuable assistance, and Mr. H. Walz (Walz Company, Effeltrich, FRG) for his skilled help with the calibration of our gas-exchange system for measurements with helox. The Department of Conservation, New Zealand, is thanked for permission to collect lichens.     

Uptake and efflux of inorganic carbon in Dunaliella salina

The apparent photosynthetic K_m (CO₂) of air-grown Dunaliella salina is 2 M as measured both by the filtering centrifugation technique and by O₂ electrode. These cells are capable of accumulating inorganic carbon (C_inorg) up to 20 times its concentration in the medium. It is suggested that air-grown Dunaliella cells are able to concentrate CO₂ within the cell. Analysis of the efflux of C_inorg from cells previously loaded with H¹⁴CO ₃ ^- demonstrated the existence of an internal pool which has an half-time of depletion of 2.5–7 min depending on the conditions of the experiment. This finding indicates that the internal C_inorg pool is not readily exchangeable with the external medium. Furthermore, the influence of the presence or absence of unlabelled C_inorg in the medium during the efflux experiment on the half-time observed indicate that efflux of C_inorg is not a simple diffusion process but is rather carrier-mediated.Abbreviation C_inorg inorganic carbon     

CO2 is the first species formed upon CO oxidation by CO dehydrogenase from Pseudomonas carboxydovorans

The active species of CO₂, i.e. CO₂ or HCO ₃ ^- , formed in the CO dehydrogenase reaction was determined using the pure enzyme from the carboxydotrophic bacterium Pseudomonas carboxydovorans. Employing an assay system similar to that used to test for carbonic anhydrase, data were obtained which are quite compatible with those expected if CO₂ is the first species formed. In addition, carbonic anhydrase activity was not detected in P. carboxydovorans.     

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.     

Photobiont-related differences in carbon acquisition among green-algal lichens

The photosynthetic properties of a range of lichens (eight species) containing green algal primary photobionts of either the genus Coccomyxa, Dictyochloropsis or Trebouxia were examined with the aim of obtaining a better understanding for the different CO₂ acquisition strategies of lichenized green algae. Fast transients of light/dark-dependent CO₂ uptake and release were measured in order to screen for the presence or absence of a photosynthetic CO₂-concentrating mechanism (CCM) within the photobiont. It was found that lichens with Trebouxia photobionts (four species) were able to accumulate a small pool of inorganic carbon (DIC; 70–140 nmol per mg chlorophyll (Chl)), in the light, which theoretically may result in, at least, a two to threefold increase in the stromal CO₂ concentration, as compared to that in equilibrium with ambient air. The other lichens (four species), which were tripartite associations between a fungus, a cyanobacterium (Nostoc) and a green alga (Coccomyxa or Dictyochloropsis) accumulated a much smaller pool of DIC (10–30 nmol·(mg Chl)^–1). This pool is most probably associated with the previously documented CCM of Nostoc, inferred from the finding that free-living cells of Coccomyxa did not show any signs of DIC accumulation. In addition, the kinetics of fast CO₂ exchange for free-living Nostoc were similar to those of intact tripartite lichens, especially in their responses to the CCM and the carbonic anhydrase (CA) inhibitor ethoxyzolamide. Trebouxia lichens had a higher photosynthetic capacity at low and limiting external CO₂ concentrations, with an initial slope of the CO₂-response curve of 2.6–3.9 mol·(mg Chl)^–1·h^–1·Pa^–1, compared to the tripartite lichens which had an initial slope of 0.5–1.1 mol-(mg Chl)^–1·h^–1·-Pa^–1, suggesting that the presence of a CCM in the photobiont affects the photosynthetic performance of the whole lichen. Regardless of these indications for the presence or absence of a CCM, ethoxyzolamide inhibited the steady-state rate of photosynthesis at low CO₂ in all lichens, indicating a role of CA in the photosynthetic process within all of the photobionts. Measurements of CA activity in photobiont-enriched homogenates of the lichens showed that Coccomyxa had by far the highest activity, while the other photobionts displayed only traces or no activity at all. As the CCM is apparently absent in Coccomyxa, it is speculated that this alga compensates for this absence with high internal CA activity, which may function to reduce the CO₂-diffusion resistance through the cell.Abbreviations CA carbonic anhydrase (EC 4.2.1.1) - CCM CO₂-concentrating mechanism - Chl chlorophyll - DIC dissolved inorganic carbon - EZ ethoxyzolamide or 6-ethoxy-2-benzo-thiazole-2-sulfonamide - GA glycolaldehyde - Hepps 4-(2-hydroxyethyl)-l-piperazinepropanesulfonic acid - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39) This research was supported by a grant from the Swedish Natural Sciences Resource Council to K.P.     

Inhibition of photosynthesis by intracellular carbonic anhydrase in microalgae under excess concentrations of CO2

When cells of Chlorococcum littorale that had been grown in air (air-grown cells) were transferred to extremely high CO₂ concentrations (>20%), active photosynthesis resumed after a lag period which lasted for 1–4 days. In contrast, C. littorale cells which had been grown in 5% CO₂ (5% CO₂-grown cells) could grow in 40% CO₂ without any lag period. When air-grown cells were transferred to 40% CO₂, the quantum efficiency of PS II (Φ_II) decreased greatly, while no decrease in Φ_II was apparent when the 5% CO₂-grown cells were transferred to 40% CO₂. In contrast to air-grown cells, 5% CO₂-grown cells showed neither extracellular nor intracellular carbonic anhydrase (CA) activity. Upon the acclimation of 5% CO₂-grown cells to air, photosynthetic susceptibility to 40% CO₂ was induced. This change was associated with the induction of CA. In addition, neither suppression of photosynthesis nor arrest of growth was apparent when ethoxyzolamide (EZA), a membrane-permeable inhibitor of CA, had been added before transferring air-grown cells of C. littorale to 40% CO₂. The intracellular pH value (pH_i) decreased from 7.0 to 6.4 when air-grown C. littorale cells were exposed to 40% CO₂ for 1–2 h, but no such decrease in pH_i was apparent in the presence of EZA. Both air- and 5% CO₂-grown cells of Chlorella sp. UK001, which was also resistant to extremely high CO₂ concentrations, grew in 40% CO₂ without any lag period. The activity of CA was much lower in air-grown cells of this alga than those in air-grown C. littorale cells. These results prompt us to conclude that intracellular CA caused intracellular acidification and hence inhibition of photosynthetic carbon fixation when air-grown C. littorale cells were exposed to excess concentrations of CO₂. No such harmful effect of intracellular CA was observed in Chlorella sp. UK001 cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ethoxyzolamide repression of the low photorespiration state in two submersed angiosperms

Net photosynthesis in the submersed angiosperms Myriophyllum spicatum L. and Hydrilla verticillata (L.f.) Royal was inhibited by 21% O₂, but the degree of inhibition was greater for plants in the high than in the low photorespiratory state. Increasing the CO₂ concentration from 50 through 2,500 l l^-1 decreased the O₂ inhibition of the high-photorespiration plants in a competitive manner, but it had no effect on the O₂ inhibition of plants in the low photorespiratory state. Carbonic-anhydrase activity increased by almost threefold with the induction of the low photorespiratory state. Ethoxyzolamide, an inhibitor of carbonic anhydrase, reduced the net photosynthesis of low-photorespiration Myriophyllum and Hydrilla plants by 40%, but their dark respiration was unaffected. This ethoxyzolamide inhibition of net photosynthesis exhibited a competitive response to CO₂ concentration, resulting in a decrease in the apparent affinity of photosynthesis for CO₂. The net photosynthesis of plants in the high photorespiratory state was inhibited only slightly by ethoxyzolamide, and this inhibition was independent of the CO₂ level. Ethoxyzolamide treatment caused an increase in the O₂ inhibition of net photosynthesis of plants in the low photorespiratory state. Ethoxyzolamide increased the low CO₂ compensation points of low-photorespiration Myriophyllum and Hydrilla, but the values for the high-photorespiration plants were unchanged. In comparison, the CO₂ compensation points of the terrestrial plants Sorghum bicolor (C₄), Moricandia arvensis (C₃-C₄ intermediate) and Nicotiana tabacum (C₃) were unaltered by ethoxyzolamide treatment. These data indicate that the low photorespiratory state in Myriophyllum and Hydrilla is repressed by ethoxyzolamide treatment, thus implicating carbonic anhydrase as a component of the photorespiration-reducing mechanism in these plants. The competitive interaction of CO₂ with ethoxyzolamide provides evidence that the low photorespiratory state in submersed angiosperms is the result of some type or types of CO₂ concentrating mechanism. In Myriophyllum it may be via bicarbonate utilization, but in Hydrilla it probably takes the form of an inducible C₄-type system.Abbreviations PEP phosphoenolpyruvate - RuBP ribulose bisphosphate     

Variable HCO 3 - affinity of Elodea canadensis Michaux in response to different HCO 3 - and CO2 concentrations during growth

Summary Elodea canadensis grows over a wide range of inorganic carbon, nutrient, and light conditions in lakes and streams. Affinity for HCO ₃ ^- use during photosynthesis ranged from strong to weak in Elodea collected from seven localities with different HCO ₃ ^- and CO₂ concentrations. The response to HCO ₃ ^- was also very plastic in plants grown in the laboratory at high HCO ₃ ^- concentrations and CO₂ concentrations varying from 14.8 to 2,200 M. Bicarbonate affinity was markedly reduced with increasing CO₂ concentrations in the growth medium so that ultimately HCO ₃ ^- use was not detectable. High CO₂ concentrations also decreased CO₂ affinity and induced high CO₂ compensation points (360M CO₂) and tenfold higher half-saturation values (800 M CO₂).The variable HCO ₃ ^- affinity is probably environmentally based. Elodea is a recently introduced species in Denmark, where it reproduces only vegetatively, leaving little opportunity for genetic variation. More important, local populations in the same water system had different HCO ₃ ^- affinities, and a similar variation was created by exposing one plant collection to different laboratory conditions.Bicarbonate use enabled Elodea to photosynthesize rapidly in waters of high alkalinity and enhanced the carbon-extracting capacity by maintaining photosynthesis above pH 10. On the other hand, use of HCO ₃ ^- represents an investment in transport apparatus and energy which is probably not profitable when CO₂ is high and HCO ₃ ^- is low. This explanation is supported by the findings that HCO ₃ ^- affinity was low in field populations where HCO ₃ ^- was low (0.5 and 0.9 m M) or CO₂ was locally high, and that HCO ₃ ^- affinity was suppressed in the laboratory by high CO₂ concentrations.Abbreviations DIC dissolved inorganic carbon (CO₂+ HCO ₃ ^- +CO ₃ ^- ) - CO₂ compensation point - K _1/2 apparent halfsaturation constant - P_{HCO 3} ^– interpolated photosynthesis in pure HCO ₃ ^- and zero CO₂ - P_max photosynthetic rate under carbon and light saturation     

Exogenous inorganic carbon sources for photosynthesis in seawater by members of the Fucales and the Laminariales (Phaeophyta): ecological and taxonomic implications

Summary Characteristics of inorganic carbon assimilation by photosynthesis in seawater were investigated in six species of the Fucales (five Fucaceae, one Cystoseiraceae) and four species of the Laminariales (three Laminariaceae, one Alariaceae) from Arbroath, Scotland. All of the algae tested could photosynthesise faster at high external pH values than the uncatalysed conversion of HCO ₃ ^- to CO₂ can occur, i.e. can use external HCO ₃ ^- . They all had detectable extracellular carbonic anhydrase activity, suggesting that HCO ₃ ^- use could involve catalysis of external CO₂ production, a view supported to some extent by experiments with an inhibitor of carbonic anhydrase. All of the algae tested had CO₂ compensation concentrations at pH 8 which were lower than would be expected from diffusive entry of CO₂ supplying RUBISCO as the initial carboxylase, consistent with the operation of energized entry of HCO ₃ ^- and / or CO₂ acting as a CO₂ concentrating mechanism. Quantitative differences among the algae examined were noted with respect to characteristics of inorganic C assimilation. The most obvious distinction was between the eulittoral Fucaceae, which are emersed for part of, or most of, the tidal cycle, and the other three families (Cystoseiraceae, Laminariaceae, Alariaceae) whose representatives are essentially continually submersed. The Fucaceae examined are able to photosynthesise at high pH values, and have lower CO₂ compensation concentrations, and lower K_1/2 values for inorganic C use in photosynthesis, at pH 8, than the other algae tested. Furthermore, the Fucaceae are essentially saturated with inorganic C for photosynthesis at the normal seawater concentration at pH 8 and 10°C. These characteristics are consistent with the dominant role of a CO₂ concentrating mechanism in CO₂ acquisition by these plants. Other species tested have characteristcs which suggest a less effective HCO ₃ ^- use and CO₂ concentrating mechanism, with the Laminariaceae being the least effective; unlike the Fucaceae, photosynthesis by these algae is not saturated with inorganic C in normal seawater. Taxonomic and ecological implications of these results are considered in relation to related data in the literature.     

Adaptations to a terrestrial existence by the robber crab,Birgus latro L.

Summary The activity of carbonic anhydrase (CA), which catalyses the equilibrium CO₂H⁺+HCO ₃ ^- , was investigated in various tissues implicated in the excretion of CO₂ by Birgus latro. Carbonic anhydrase was detected in the water-soluble fraction of gill tissue but also occurred in association with lipids (membrane bound). This is consistent with a CO₂ excretory role and an ion regulation function for the gills. In the lungs (branchial chamber lining) CA activity was found in the membrane bound fraction but was not detected in the soluble fraction, suggesting that the lung CA is not important for ion regulation. The specific CA activity of gill tissue homogenate (A=1.8±0.7·mg^-1) was higher than that measured for lung homogenates (A=0.4±0.2·mg^-1), but when the whole organ was considered the total CA activity in the lungs was not significantly different from total CA activity in the gills. In comparison to aquatic and amphibious crustaceans the specific activity of carbonic anhydrase in the lungs was high (25% cf. gill activity). This CA activity in the lungs could be correlated with significant CO₂ excretion by the lungs. CA may be retained in the branchial tissue as an adjunct to ion reabsorption by the gills.     

Effect of dissolved inorganic carbon on oxygen evolution and uptake by Chlamydomonas reinhardtii suspensions adapted to ambient and CO2-enriched air

Mass spectrometric measurements of ¹⁶O₂ and ¹⁸O₂ isotopes were used to compare the rates of gross O₂ evolution (E₀), O₂ uptake (U₀) and net O₂ evolution (NET) in relation to different concentrations of dissolved inorganic carbon (DIC) by Chlamydomonas reinhardtii cells grown in air (air-grown), in air enriched with 5% CO₂ (CO₂-grown) and by cells grown in 5% CO₂ and then adapted to air for 6h (air-adapted).At a photon fluence rate (PFR) saturating for photosynthesis (700 mol photons m^-2 s^-1), pH=7.0 and 28°C, U₀ equalled E₀ at the DIC compensation point which was 10M DIC for CO₂-grown and zero for air-grown cells. Both E₀ and U₀ were strongly dependent on DIC and reached DIC saturation at 480 M and 70 M for CO₂-grown and air-grown algae respectively. U₀ increased from DIC compensation to DIC saturation. The U₀ values were about 40 (CO₂-grown), 165 (air-adapted) and 60 mol O₂ mg Chl^-1 h^-1 (air-grown). Above DIC compensation the U₀/E₀ ratios of air-adapted and air-grown algae were always higher than those of CO₂-grown cells. These differences in O₂ exchange between CO₂- and air-grown algae seem to be inducable since air-adapted algae respond similarly to air-grown cells.For all algae, the rates of dark respiratory O₂ uptake measured 5 min after darkening were considerably lower than the rates of O₂ uptake just before darkening. The contribution of dark respiration, photorespiration and the Mehler reaction to U₀ is discussed and the energy requirement of the inducable CO₂/HCO₃ ^- concentrating mechanism present in air-adapted and air-grown C. reinhardtii cells is considered.Abbreviations DIC dissolved inorganic carbon - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - E₀ rate of photosynthetic gross O₂ evolution - PCO photosynthetic carbon oxidation - PFR photon fluence rate - PS I photosystem I - PS II photosystem II - U₀ rate of O₂ uptake in the light - MS mass spectrometer