Intracellular carbonic anhydrase activities in Dunaliella tertiolecta (Butcher) and Chlamydomonas reinhardtii (Dangeard) in relation to inorganic carbon concentration during growth: further evidence for the existence of two distinct carbonic anhydrases associated with the chloroplasts

Using mass-spectrometric measurements of ¹⁸O exchange from ¹³C¹⁸O₂ intracellular carbonic anhydrase (CA) activity was investigated in the unicellular green algae Dunaliella tertiolecta and Chlamydomonas reinhardtii which were either grown on air enriched with 5% CO₂ (high-C_i cells) or on air (low-C_i cells). In D. tertiolecta high- and low-C_i cells had detectable levels of internal CA activity when measured under in-vivo conditions and this activity could be split up into three distinct forms. One CA was not associated with the chloroplasts, while two isozymes were found to be located within the plastids. The activities of all intracellular CAs were always about twofold higher in low than in high-C_i cells of D. tertiolecta and the chloroplastic enzymes were completely induced within 4 h of adaptation to air. One of the chloroplastic CAs was found to be soluble the other was insoluble. In addition to the physical differences, MgSO₄ in vitro caused a more than twofold stimulation of the soluble activity while the insoluble form of CA remained rather unaffected. In C. reinhardtii, MgSO₄ increased the soluble CA activity by 346% and the concentration of MgSO₄ required for half-maximum stimulation was between 10 and 15 mM. Again, the insoluble CA activity was not affected by MgSO₄. Furthermore, the soluble isoenzyme was considerably more sensitive to ethoxyzolamide, a potent inhibitor of CA, than the insoluble enzyme. The concentration of inhibitor causing 50% inhibition of soluble CA activity was 110 and 85 μM ethoxyzolamide for D. tertiolecta and C. reinhardtii, respectively. From these data we conclude that the two chloroplast-associated CAs are distinct enzymes.     

Induction of intracellular carbonic anhydrases during the adaptation to low inorganic carbon concentrations in wild-type and ca-1 mutant cells of Chlamydomonas reinhardtii

Mass-spectrometric measurements of ¹⁸O exchange from ¹³C¹⁸O₂ were used to follow changes in the intracellular carbonic anhydrase (CA) activity of cells of Chlamydomonas reinhardtii Dang, wild type and the ca-1 mutant during adaptation to air. With intact cells as well as with crude homogenates total intracellular CA activity in wild-type cells increased six to tenfold within 4 h after transferring cells from 5% CO₂ (high inorganic carbon, C_i) to ambient air (air adapted). After that time the activity slowly declined to a level similar to that observed with cells which had been continuously grown in air (low-C_i grown). In the ca-1 mutant, total CA was induced to a similar extent during 4 h of adaptation; however, absolute activities were two to three times lower in ca-1 than in the wild type regardless of the CO₂ supply. When crude extracts from wild-type cells were separated into soluble and insoluble fractions, each fraction contained about half of the internal CA activity. Within 4 h of adaptation, both forms of CA activity were simultaneously enhanced by nine to tenfold, reaching levels similar to those found in low-C_igrown cells. In contrast, in the ca-1 mutant the soluble CA activity was only enhanced by about eightfold while the level of insoluble CA was very low even in low-C_i cells. After isolation of intact chloroplasts from wild-type cells and further subfractionation, around 70–80% of total chloroplastic CA activity was found to be in the insoluble fraction while 17–20% remained in the soluble fraction. Both chloroplastic CA activities were inducible within the first 4 h of adaptation to air, with each of them being eight to ten times higher than in high-C_i algae. After that time their activities were similar to the corresponding CA values in low-C_i-grown cells. In contrast, plastids from high-C_i cells of the ca-1 mutant showed 40% less insoluble-CA activity compared to the wild type and this insoluble-CA activity was not increased at all by transferring algae to air. In addition, no soluble-CA activity was detected in chloroplasts from high-C_i and air-adapted ca-1 cells. These results indicate the presence of three intracellular CA activities in high-C_i air-adapted and low-C_i cells of the wild type and that two of them are associated with the chloroplasts. All three activities are completely induced within the first 4 h of adaptation to air in wild-type cells. In contrast, it was not possible to induce any of the chloroplastic CA activities in the ca-1 mutant. The possibility that the soluble chloroplastic CA represents a pyrenoid-located CA is discussed.This work is dedicated to Professor A. Wild on the occasion of his 65th birthday     

Evidence for the contribution of pseudocyclic photophosphorylation to the energy requirement of the mechanism for concentrating inorganic carbon in Chlamydomonas

Mass-spectrometric measurements of ¹⁶O₂ and ¹⁸O₂ were made to compare the rates of light-dependent O₂ evolution and uptake by Chlamydomonas reinhardtii Dang. grown in air (0.035% CO₂; low-C_i cells) or CO₂-enriched air (5% CO₂; high-C_i cells) at pH 5.5 and 8.0. While at pH 5.5, no differences were observed in the isotopic O₂-gas exchange of high- and low-C_i cells, at pH 8.0 the rates of true O₂ evolution and uptake were considerably higher in low-C_i than in high-C_i cells. The enhanced rates of O₂ uptake and evolution by low-C_i cells were completely inducible within 6 h after transferring high-C_i cells to ambient air. At pH 8.0, O₂ uptake in the light was inhibited by 2 M 3-(3,4-dichlorophenyl)-1,1 dimethylurea in both types of alga, but this effect was more pronounced in low-C_i than in high-C_i cells.When the cells were grown at pH 5.5 the activities of the superoxide-radical-degrading enzymes, superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase, were similar regardless of the CO₂ concentration provided during growth. At pH 8.0, however, the activities of these enzymes were 4 to 20 times higher in low-C_i than in high-C_i cells. When high-C_i cells were allowed to acclimate to ambient air for 6 h at pH 8.0, the activities of superoxide dismutase, ascorbate peroxidase and monodehydroascorbate dehydrogenase increased to more than 50% of the level observed with low-C_i cells. These results are consistent with an enhanced operation of O₂ photoreduction which could provide energy to the inorganic-carbon-concentrating mechanism via pseudo-cyclic photophosphorylation.     

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 and inorganic carbon fluxes in low- and high-C1 cells of Chlamydomonas reinhardtü and Scenedesmus obliquus

Carbonic anhydrase (CA) activity associated with high- and low-dissolved inorganic carbon (C₁) grown cells was examined in whole cells by measuring ¹⁸O exchange from doubly labeled CO₂ (¹³C¹⁸O¹⁸O). Both algal species showed the presence of extracellular (periplasmic) as well as intracellular CA activity, which were both greatly increased in low-C₁ cells. The periplasmic CA activity was at least 40-fold higher in lowcompared to high-C₁ cells in both C. reinhardtii and S. obliquus. while low-C₁ cells of S. obliquus showed the highest activity of internal CA. The CA inhibitor ethoxyzolamide showed a strong inhibition of the C₁ uptake process in both C. reinhardtii and S. obliquus as in cyanobacteria. which may indicate that the nature of the primary uptake process is similar in both green algae and cyanobacteria. By using a mass spectrometnc disequilibrium technique it was possible to separate the C₁ fluxes of net HCO^?₃-uptake and net CO₂-uptake during steady-state photosynthesis in high- and Sow-C₁ grown cells of Chlamydomonas reinhardtii (WT. 2137+) and Scenedesmus obliquus (WT. D3). It was found that both high- and low-C₁ cells of the two algae can utilize both CO₂ and HCO^?₃ for photosynthesis, although low-C₁ cells have a higher affinity for the uptake of both C₁ species. Induction at low-C₁ causes an increase in the affinity of both species for HCO^?₃ and CO₂; changes in net CO₂-uptake were, however, significantly greater.     

Effect of external CO2 concentrations on protein synthesis in the green algae Scenedesmus obliquus (Turp.) Kütz and Chlorella vulgaris (Kosikov)

Unicellular algae grown under low-CO₂ conditions (0.03% CO₂) have developed a means of concentrating CO₂ at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase. Cells with the CO₂-concentrating mechanism (CCM) acquire the ability to accumulate inorganic carbon to a level higher than that obtained by simple diffusion. To identify proteins which are involved in the organization of the CCM, cells of Scenedesumus obliquus and Chlorella vulgaris grown in high CO₂ (5% CO₂ in air) were transferred to low-CO₂ (0.03%) conditions in the presence of ³⁵SO _inf4 ^sup2? and, thereafter, polypeptides labeled with ³⁵S were detected. Under low-CO₂ conditions the inducton of 36-, 39-, 94- and 110- to 116kDa polypeptides were particularly observed in S. obliquus and 16-, 19-, 27-, 36-, 38- and 45-kDa polypeptides were induced in C. vulgaris. Western blots with antibodies raised against 37-kDa subunits of the periplasmic carbonic anhydrase (CA) of Chlamydomonas reinhardtii showed immunoreactive bands with the 39-kDa polypeptide in the whole-cell homogenates from S. obliquus and with 36 and 38-kDa polypeptides in both high- and low-CO₂grown cells of C. vulgaris. Anti-pea-chloroplast CA antibodies cross-reacted with a single polypeptide of 30 kDa in the whole-cell homogenates but not with thylakoid membranes. The CA activity was associated with soluble and membrane-bound fractions, except thylakoid membranes.     

The induction of the CO2-concentrating mechanism is correlated with the formation of the starch sheath around the pyrenoid of Chlamydomonas reinhardtii

The pyrenoid is a prominent proteinaceous structure found in the stroma of the chloroplast in unicellular eukaryotic algae, most multicellular algae, and some hornworts. The pyrenoid contains the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase and is sometimes surrounded by a carbohydrate sheath. We have observed in the unicellular green alga Chlamydomonas reinhardtii Dangeard that the pyrenoid starch sheath is formed rapidly in response to a decrease in the CO₂ concentration in the environment. This formation of the starch sheath occurs coincidentally with the induction of the CO₂-concentrating mechanism. Pyrenoid starch-sheath formation is partly inhibited by the presence of acetate in the growth medium under light and low-CO₂ conditions. These growth conditions also partly inhibit the induction of the CO₂-concentrating mechanism. When cells are grown with acetate in the dark, the CO₂-concentrating mechanism is not induced and the pyrenoid starch sheath is not formed even though there is a large accumulation of starch in the chloroplast stroma. These observations indicate that pyrenoid starch-sheath formation correlates with induction of the CO₂-concentrating mechanism under low-CO₂ conditions. We suggest that this ultrastructural reorganization under lowCO₂ conditions plays a role in the CO₂-concentrating mechanism C. reinhardtii as well as in other eukaryotic algae.     

Photorespiratory ammonium assimilation in chloroplasts of Chlamydomonas reinhardtii

The effect of CO₂ concentration on the rate of photorespiratory ammonium excretion and on glutamine synthetase (GS) and carbonic anhydrase (CA) isoenzymes activities has been studied in Chlamydomonas reinhardtii cw-15 mutant (lacking cell wall) and in the high CO₂-requiring double mutant cia-3/cw-15 (lacking cell wall and chloroplastic carbonic anhydrase). In cw-15 cells, both the extracellular (CA_ext) and chloroplastic (CA_chl) CA activities increased after transferring cells from media bubbled with 5% CO₂ in air (v/v, high-C_i cells) to 0.03% CO₂ (low-C_i cells), whereas in cia-3/cw-15 cells only the CA_ext was induced after adaptation to low-C_i conditions and the CA_chl activity was negligible. During adaptation to low-C_i conditions in the presence of 1 mM of l-methionine-D,L-sulfoximine (MSX), a specific inhibitor of GS activity, both mutant strains excreted photorespiratory ammonium into nitrogen free medium. In addition, the ammonium excretion rate by cw-15 in the presence of MSX was lower in cells grown and kept at 5% CO₂ than in high-C_i cells adapted to 0.03% CO₂. The double mutant cia-3/cw-15 excreted photorespiratory ammonium at a higher rate than did cw-15. Total GS activity (GS-1 plus GS-2) increased during adaptation to 0.03% CO₂ in both strains of C. reinhardtii. However, only the activity GS-2, which is located in the chloroplast, increased during the adaptation to low CO₂, whereas the cytosolic GS-1 levels remained similar in high and low-C_i cells. We conclude that: (1) cia-3/cw-15 cells lack chloroplastic CA activity; (2) in C. reinhardtii photorespiratory ammonium is refixed in the chloroplasts through the GS-2/GOGAT cycle; and (3) chloroplastic GS-2 concentration changes in response to the variation of environmental CO₂ concentration.     

Absorption characteristics and kinetics of CO2 capture into N-methyldiethanolamine aqueous solution catalyzed by the immobilized carbonic anhydrase

Abstract

Carbonic anhydrase (CA) is the most effective CO₂ hydratase catalyst, but the poor storage stability and repeatability of CA limit its development. Therefore, CA was immobilized on the epoxy magnetic composite microspheres to enhance the CO₂ absorption into N-methyldiethanolamine (MDEA) aqueous solution in this work. In the presence of immobilized CA, the CO₂ absorption rate of MDEA solution (10?wt%) (0.63?mmol·min^?1) was greatly improved by almost 40%, and their reaction equilibrium time was shortened from 150?min to 90?min compared with that into MDEA solution. The results indicated that the absorption of CO₂ into MDEA solution had been significantly enhanced by using CA. After the 7th reuse recycle, the activity of the immobilized CA was still closed to its initial value at 313.15?K. Moreover, enzyme catalytic kinetics of immobilized CA was investigated using the p-nitrophenyl acetate (p-NPA) as substrate. The values of Michaelis–Menten constant (K_m) and the maximum velocity (V_max) of the immobilized CA were calculated to be 27.61?mmol/L and 20.14?×?10^?3?mmol·min^?1·mL^?1, respectively. Besides, the kinetics of CO₂ reaction into MDEA with or without CA were also compared. The results showed that CO₂ absorption into CA/MDEA aqueous solution obeyed the pseudo first order regime and the second order kinetics rate constant (k₂) was calculated to be 929?m³·kmol^?1·s^?1, which was twice higher than that of MDEA aqueous solution without immobilized CA (k₂=414 m³·kmol^?1·s^?1) at 313.15?K.     

Evidence for 18O labeling of photorespiratory CO2 in photoautotrophic cell cultures of higher plants illuminated in the presence of 18O2

The ¹⁸O-enrichment of CO₂ produced in the light or during the post-illumination burst was measured by mass spectrometry when a photoautotrophic cell suspension of Euphorbia characias L. was placed in photorespiratory conditions in the presence of molecular ¹⁸O₂. The only ¹⁸O-labeled species produced was C¹⁸O¹⁶O; no C¹⁸O¹⁸O could be detected. Production of C¹⁸O¹⁶O ceased after addition of two inhibitors of the photosynthetic carbon-oxidation cycle, aminooxyacetate or aminoacetonitrile, and was inhibited by high levels of CO₂. The average enrichment during the post-illumination burst was estimated to be 46 ± 15% of the enrichment of the O₂ present during the preceding light period. Addition of exogenous carbonic anhydrase, by catalyzing the exchange between CO₂ and H₂O, drastically diminished the ¹⁸O-enrichment of the produced CO₂. The very low carbonio-anhydrase level of the photoautotrophic cell suspension probably explains why the ¹⁸O labeling of photorespiratory CO₂ could be observed for the first time. These data allow the establishment of a direct link between O₂ consumption and CO₂ production in the light, and the conclusion that CO₂ produced in the light results, at least partially, from the mitochondrial decarboxylation of the glycine pool synthesized through the photosynthetic carbon-oxidation cycle. Analysis of the C¹⁸O¹⁶O and CO₂ kinetics provides a direct and reliable way to assess in vivo the real contribution of photorespiratory metabolism to CO₂ production in the light.     

Complementation analysis of the inorganic carbon concentrating mechanism of Chlamydomonas reinhardtii

Summary Six independently isolated mutants of Chlamydomonas reinhardtii that require elevated CO₂ for photoautotrophic growth were tested by complementation analysis. These mutants are likely to be defective in some aspect of the algal concentrating mechanism for inorganic carbon as they exhibit CO₂ fixation and inorganic carbon accumulation properties different from the wild-type. Four of the six mutants defined a single complementation group and appear to be defective in an intracellular carbonic anhydrase. The other two mutations represent two additional complementation groups.Abbreviations HS high salt medium which has 13 mM phosphate at pH 6.8 - HSA high salt plus 36 mM acetate medium - YA high salt medium with 4 g yeast extract per L and 36mM acetate - Arg arginine - cia^- CO₂ accumulation mutants that cannot grow on low CO₂ - C_i inorganic carbon (CO₂+HCO _- ³ ) - CA carbonic anhydrase - mt mating type Supported in part by the McKnight Foundation and by NSF grant PCM 8005917 and published as journal article 11924 from the Michigan State Agriculatural Experiment Station     

Historical perspective on microalgal and cyanobacterial acclimation to low- and extremely high-CO2 conditions

Reports in the 1970s from several laboratories revealed that the affinity of photosynthetic machinery for dissolved inorganic carbon (DIC) was greatly increased when unicellular green microalgae were transferred from high to low-CO₂ conditions. This increase was due to the induction of carbonic anhydrase (CA) and the active transport of CO₂ and/or HCO₃ ⁻ which increased the internal DIC concentration. The feature is referred to as the `CO₂-concentrating mechanism (CCM)'. It was revealed that CA facilitates the supply of DIC from outside to inside the algal cells. It was also found that the active species of DIC absorbed by the algal cells and chloroplasts were CO₂ and/or HCO₃ ⁻, depending on the species. In the 1990s, gene technology started to throw light on the molecular aspects of CCM and identified the genes involved. The identification of the active HCO₃ ⁻ transporter, of the molecules functioning for the energization of cyanobacteria and of CAs with different cellular localizations in eukaryotes are examples of such successes. The first X-ray structural analysis of CA in a photosynthetic organism was carried out with a red alga. The results showed that the red alga possessed a homodimeric β-type of CA composed of two internally repeating structures. An increase in the CO₂ concentration to several percent results in the loss of CCM and any further increase is often disadvantageous to cellular growth. It has recently been found that some microalgae and cyanobacteria can grow rapidly even under CO₂ concentrations higher than 40%. Studies on the mechanism underlying the resistance to extremely high CO₂ concentrations have indicated that only algae that can adopt the state transition in favor of PS I could adapt to and survive under such conditions. It was concluded that extra ATP produced by enhanced PS I cyclic electron flow is used as an energy source of H⁺-transport in extremely high-CO₂ conditions. This same state transition has also been observed when high-CO₂ cells were transferred to low CO₂ conditions, indicating that ATP produced by cyclic electron transfer was necessary to accumulate DIC in low-CO₂ conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

A model of carbon dioxide assimilation in Chlamydomonas reinhardii

A simple model of photosynthetic CO₂ assimilation in Chlamydomonas has been developed in order to evaluate whether a CO₂-concentrating system could explain the photosynthetic characteristics of this alga (high apparent affinity for CO₂, low photorespiration, little O₂ inhibition of photosynthesis, and low CO₂ compensation concentration). Similarly, the model was developed to evaluate whether the proposed defects in the CO₂-concentrating system of two Chlamydomonas mutants were consistent with their observed photosynthetic characteristics. The model treats a Chlamydomonas cell as a single compartment with two carbon inputs: passive diffusion of CO₂, and active transport of HCO ₃ ^- . Internal inorganic carbon was considered to have two potential fates: assimilation to fixed carbon via ribulose 1,5-bisphosphate carboxylase-oxygenase or exiting the cell by either passive CO₂ diffusion or reversal of HCO ₃ ^- transport. Published values for kinetic parameters were used where possible. The model accurately reproduced the CO₂-response curves of photosynthesis for wild-type Chlamydomonas, the two mutants defective in the CO₂-concentrating system, and a double mutant constructed by crossing these two mutants. The model also predicts steady-state internal inorganic-carbon concentrations in reasonable agreement with measured values in all four cases. Carbon dioxide compensation concentrations for wild-type Chlamydomonas were accurately predicted by the model and those predicted for the mutants were in qualitative agreement with measured values. The model also allowed calculation of approximate energy costs of the CO₂-concentrating system. These calculations indicate that the system may be no more energy-costly than C₄ photosynthesis.Abbreviations Chl chlorophyll - RuBPC/O ribulose 1,5-bisphosphate carboxylase-oxygenase - CA carbonic anhydrase     

Regulation of the low-CO2-inducible polypeptides in Chlamydomonas reinhardtii

Polypeptides of 21, 36 and 37 kDa are induced in the unicellular green alga Chlamydomonas reinhardtii Dang. when cells are transferred from high (2%) to low (0.03%) CO₂ concentrations. The synthesis of these polypeptides is correlated with the induction of the CO₂-concentrating mechanism. In this work we studied the effect of the growth conditions on the synthesis of these polypeptides with the aim of clarifying whether the induction of all three of these low-CO₂-inducible polypeptides requires the same environmental factor. Our results showed that induction of the 21- and 36-kDa polypeptides under low-CO₂ conditions occurred only in the light, while the 37-kDa periplasmic carbonic anhydrase (EC 4.2.1.1) was induced in light, darkness, and in both synchronous and asynchronous cultures. In addition, induction of these polypeptides appeared to be determined more by the O₂/CO₂ ratio than by the CO₂ concentrations. None of these polypeptides could be induced in either of two different mutants of C. reinhardtii, one lacking ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and the other with inactive enzyme. Our results indicate that the 21- and 36-kDa polypeptides are regulated by a mechanism different from that controlling the 37-kDa polypeptide.Abbreviations pCA (periplasmic) carbonic anhydrase - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - TAP Trisacetate phosphate medium The authors thank Prof. M. Spalding (Iowa State University, USA) for providing antisera to LIP-21 and LIP-36. We thank Prof. S. Bartlett and Dr. J. Moroney (Louisiana State University, USA) for providing antibodies to C. reinhardtii, Rubisco and 37-kDa pCA, respectively. This work was supported by the Instituto Tecnologico de Canarias.     

Low-CO2-inducible protein synthesis in the green alga Dunaliella tertiolecta

In the green marine alga Dunaliella tertiolecta, a CO₂-concentrating mechanism is induced when the cells are grown under low-CO₂ conditions (0.03% CO₂). To identify proteins induced under low-CO₂ conditions the cells were labelled with ³⁵SO₄ ^2–, and seven polypeptides with molecular weights of 45, 47, 49, 55, 60, 68 and 100 kDa were detected. The induction of these polypeptides was observed when cells grown in high CO₂ (5% CO₂ in air) were switched to low CO₂, but only while the cultures were growing in light. Immunoblot analysis of total cell protein against pea chloroplastic carbonic anhydrase polyclonal antibodies showed immunoreactive 30-kDa bands in both high- and low-CO₂-grown cells and an aditional 49-kDa band exclusively in low-CO₂-grown cells. The 30-kDa protein was shown to be located in the chloroplast. Western blot analysis of the plasmamembrane fraction against corn plasma-membrane AT-Pase polyclonal antibodies showed 60-kDa bands in both high- and low-CO₂ cell types as well as an immunoreactive 100-kDa band occurring only in low-CO₂-grown cells. These results suggest that there are two distinct forms of both carbonic anhydrase and plasma-membrane ATPase, and that one form of each of them can be regulated by the CO₂ concentration.Abbreviations CA carbonic anhydrase - DIC dissolved inorganic carbon (CO₂+ HCO₃ ^–) - CCM CO₂-concentrating mechanism - low CO₂ air containing 0.03% CO₂ - high CO₂ air supplemented with 5% CO₂ (v/v) We thank Prof. John Coleman for providing antibodies raised against pea chloroplast CA, Dr. James V. Moroney for providing antibodies raised against the 37-kDa periplasmic carbonic anhydrase of CO₂ Chlamydomonas reinhardtii, and Prof. Leonard T. Robert for a gift of corn plasma-membrane 100-kDa ATPase antibodies. We thank Dr. Jeanine Olsen (University of Groningen, the Netherlands) for style comments. This work was supported by the Institute Tecnológico de Canarias (Spain).     

Luminescence decay kinetics in relation to quenching and stimulation of dark fluorescence from high and low CO2 adapted cells of Scenedesmus obliquus and Chlamydomonas reinhardtii

Two green algal species, Chlamydomonas reinhardtii and Scenedesmus obliquus, exhibited a relative maximum during the decay of luminescence, when adapted to low CO₂ conditions that was not observed in high CO₂ adapted cells.From the kinetics of transient changes in the level of dark fluorescence, after illumination and parallel to the luminescence maxima, it was concluded that the maximum in Scenedesmus was mainly related to a decrease in nonphotochemical quenching, whereas in Chlamydomonas the maximum was mainly related to a dark reduction of the primary PS II acceptor Q_A.ATP/ADP ratios from low CO₂ adapted Scenedesmus showed transient high levels after a dark/light transition that was not observed in high CO₂ adapted cells. After 30 s of illumination the ATP/ADP ratios however stabilized at the same steady state level as in high CO₂ adapted cells.Dark addition of HCO₃ ^- to low CO₂ adapted cells of Chlamydomonas resulted in a rapid transient quenching of luminescence that was not observed in low CO₂ adapted cells of neither species.It is concluded that the luminescence maxima present in both low CO₂ adapted Scenedesmus and Chlamydomonas reflect adaptation of the cells to low CO₂ conditions. It is further suggested that the difference in mechanistic origin of luminescence maxima in the two species reflects differences in adaptation.Abbreviations ADP adenosine-diphosphate - ATP adenosine-triphosphate - C_i inorganic carbon - F_D dark fluorescence recorded under dark adapted conditions - F₀ fluorescence with all reaction centers open - FV variable fluorescence - PS I photosystem I - PS II photosystem II - Q_A the first quinone acceptor of PS II     

The CO2-concentrating mechanism in a starchless mutant of the green unicellular alga Chlorella pyrenoidosa

The CO₂-concentrating mechanism (CCM) was induced in the green unicellular alga Chlorella when cells were transferred from high (5% CO₂) to low (0.03%) CO₂ concentrations. The induction of the CCM correlated with the formation of a starch sheath specifically around the pyrenoid in the chloroplast. With the aim of clarifying whether the starch sheath was involved in the operation of the CCM, we isolated and physiologically characterized a starchless mutant of Chlorella pyrenoidosa, designated as IAA-36. The mutant strain grew as vigorously as the wild type under high and low CO₂ concentrations, continuous light and a 12 h light/12 h dark photoperiod. The CO₂ requirement for half-maximal rates of photosynthesis [K_0.5(CO₂)] decreased from 40 μM to 2–3 μM of CO₂ when both wild type and mutant were switched from high to low CO₂. The high affinity for inorganic carbon indicates that the IAA-36 mutant is able to induce a fully active CCM. Since the mutant does not have the pyrenoid starch sheath, we conclude that the sheath is not involved in the operation of the CCM in Chlorella cells.     

Isolation and Characterization of High CO(2)-Requiring-Mutants of the Cyanobacterium Synechococcus PCC7942 : Two Phenotypes that Accumulate Inorganic Carbon but Are Apparently Unable to Generate CO(2) within the Carboxysome

A total of 24 high CO₂-requiring-mutants of the cyanobacterium Synechococcus PCC7942 have been isolated and partially characterized. These chemically induced mutants are able to grow at 1% CO₂, on agar media, but are incapable of growth at air levels of CO₂. All the mutants were able to accumulate inorganic carbon (C_i) to levels similar to or higher than wild type cells, but were apparently unable to generate intracellular CO₂. On the basis of the rate of C_i release following a light (5 minutes) → dark transition two extreme phenotypes (fast and slow release mutants) and a number of `intermediate' mutants (normal release) were identified. Compared to wild-type cells, Type I mutants had the following characteristics: fast C_i release, normal internal C_i pool, normal carbonic anhydrase (CA) activity in crude extracts, reduced internal exchange of ¹⁸O from ¹⁸O-labeled CO₂, 1% CO₂ requirement for growth in liquid media, normal affinity of carboxylase for CO₂, and long, rod-like carboxysomes. Type II mutants had the following characteristics: slow C_i release, increased internal C_i pool, normal CA activity in crude extracts, normal internal ¹⁸O exchange, a 3% CO₂ requirement for growth in liquid media, high carboxylase activity, normal affinity of carboxylase for CO₂, and normal carboxysome structure but increased in numbers per cell. Both mutant phenotypes appear to have genetic lesions that result in an inability to convert intracellular HCO₃⁻ to CO₂ inside the carboxysome. The features of the type I mutants are consistent with a scenario where carboxysomal CA has been mistargeted to the cytosol. The characteristics of the type II phenotype appear to be most consistent with a scenario where CA activity is totally missing from the cell except for the fact that cell extracts have normal CA activity. Alternatively the type II mutants may have a lesion in their capacity for H⁺ import during photosynthesis.     

Carbonic Anhydrase Activity Associated with the Cyanobacterium Synechococcus PCC7942

Intact cells and crude homogenates of high (1% CO₂) and low dissolved inorganic carbon (C_i) (30-50 microliters per liter of CO₂) grown Synechococcus PCC7942 have carbonic anhydrase (CA)-like activity, which enables them to catalyze the exchange of ¹⁸O from CO₂ to H₂O. This activity was studied using a mass spectrometer coupled to a cuvette with a membrane inlet system. Intact high and low C_i cells were found to contain CA activity, separated from the medium by a membrane which is preferentially permeable to CO₂. This activity is most apparent in the light, where ¹⁸O-labeled CO₂ species are being taken up by the cells but the effluxing CO₂ has lost most of its label to water. In the dark, low C_i cells catalyze the depletion of the ¹⁸O enrichment of CO₂ and this activity is inhibited by both ethoxyzolamide and 2-(trifluoromethoxy)carbonyl cyanide. This may occur via a common inhibition of the C_i pump and the C_i pump is proposed as a potential site for the exchange of ¹⁸O. CA activity was measurable in homogenates of both cell types but was 5- to 10-fold higher in low C_i cells. This was inhibited by ethoxyzolamide with an I₅₀ of 50 to 100 micromolar in both low and high C_i cells. A large proportion of the internal CA activity appears to be pelletable in nature. This pelletability is increased by the presence of Mg²⁺ in a manner similar to that of ribulose bisphosphate carboxylase-oxygenase activity and chlorophyll (thylakoids) and may be the result of nonspecific aggregation. Separation of crude homogenates on sucrose gradients is consistent with the notion that CA and ribulose bisphosphate carboxylase-oxygenase activity may be associated with the same pelletable fraction. However, we cannot unequivocally establish that CA is located within the carboxysome. The sucrose gradients show the presence of separate soluble and pelletable CA activity. This may be due to the presence of separate forms of the enzyme or may arise from the same pelletable association which is unstable during extraction.     

Membrane-Associated Polypeptides Induced in Chlamydomonas by Limiting CO(2) Concentrations

Chlamydomonas reinhardtii and other unicellular green algae have a high apparent affinity for CO₂, little O₂ inhibition of photosynthesis, and reduced photorespiration. These characteristics result from operation of a CO₂-concentrating system. The CO₂-concentrating system involves active inorganic carbon transport and is under environmental control. Cells grown at limiting CO₂ concentrations have inorganic carbon transport activity, but cells grown at 5% CO₂ do not. Four membrane-associated polypeptides (M_r 19, 21, 35, and 36 kilodaltons) have been identified which either appear or increase in abundance during adaptation to limiting CO₂ concentrations. The appearance of two of the polypeptides occurs over roughly the same time course as the appearance of the CO₂-concentrating system activity in response to CO₂ limitation.