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     

Role of intracellular carbonic anhydrase in inorganic-carbon assimilation by Porphyridium purpureum

Air-grown cells of Porphyridium purpurem contain appreciable carbonic-anhydrase activity, comparable to that in air-grown Chlamydomonas reinhardtii, but activity is repressed in CO₂-grown cells. Assay of carbonic-anhydrase activity in intact cells and cell extracts shows all activity to be intracellular in Porphyridium. Measurement of inorganic-carbon-dependent photosynthetic O₂ evolution shows that sodium ions increase the affinity of Porphyridium cells for HCO ₃ ^- . Acetazolamide and ethoxyzolamide were potent inhibitors of carbonic anhydrase in cell extracts but at pH 5.0 both acetazolamide and ethoxyzolamide had little effect upon the concentration of inorganic carbon required for the half-maximal rate of photosynthetic O₂ evolution (K_0.5[CO₂]). At pH 8.0, where HCO ₃ ^- is the predominant species of inorganic carbon, the K_0.5 (CO₂) was increased from 50 M to 950 M in the presence of ethoxyzolamide. It is concluded that in air-grown cells of Porphyridium. HCO ₃ ^- is transported across the plasmalemma and intracellular carbonic anhydrase increases the steady-state flux of CO₂ from inside the plasmalemma to ribulose-1,5-bisphosphate carboxylase-oxygenase by catalysing the interconversion of HCO ₃ ^- and CO₂ within the cell.Abbreviations AZ acetazolamide - EZ ethoxyzolamide - K_0.5[CO₂] half-maximal rate of photosynthetic O₂ evolution     

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     

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.     

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     

Photosynthetic inorganic carbon uptake and accumulation in two marine diatoms

Some physiological characteristics of photosynthetic inorganic carbon uptake have been examined in the marine diatoms Phaeodactylum tricornutum and Cyclotella sp. Both species demonstrated a high affinity for inorganic carbon in photosynthesis at pH7.5, having K_1/2(CO₂) in the range 1.0 to 4.0mmol m^?3 and O_2? and temperature-insensitive CO₂ compensation concentrations in the range 10.8 to 17.6 cm³ m^?3. Intracellular accumulation of inorganic carbon was found to occur in the light; at an external pH of 7.5 the concentration in P. tricornutum was twice, and that in Cyclotella 3.5 times, the concentration in the suspending medium. Carbonic anhydrase (CA) was detected in intact Cyclotella cells but not in P. tricornutum, although internal CA was detected in both species. The rates of photosynthesis at pH 8.0 of P. tricornutum cells and Cyclotella cells treated with 0.1 mol m^?3 acetazolamide, a CA inhibitor, were 1.5- to 5-fold the rate of CO₂ supply, indicating that both species have the capacity to take up HCO₃^? as a source of substrate for photosynthesis. No Na⁺ dependence for HCO₃^? could be detected in either species. These results indicate that these two marine diatoms have the capacity to accumulate inorganic carbon in the light as a consequence, in part, of the active uptake of bicarbonate.     

Effect of photon flux density on inorganic carbon accumulation and net CO2 exchange in a high-CO2-requiring mutant of Chlamydomonas reinhardtii

The effect of photon flux density on inorganic carbon accumulation and photosynthetic CO₂ assimilation was determined by CO₂ exchange studies at three, limiting CO₂ concentrations with a ca-1 mutant of Chlamydomonas reinhardiii. This mutant accumulates a large internal inorganic carbon pool in the light which apparently is unavailable for photosynthetic assimilation. Although steady-state photosynthetic CO₂ assimilation did not respond to the varying photon flux densities because of CO₂ limitation, components of inorganic-carbon accumulation were not clearly light saturated even at 1100 mol photons m^-2 s^-1, indicating a substantial energy requirement for inorganic carbon transport and accumulation. Steady-state photosynthetic CO₂ assimilation responded to external CO₂ concentrations but not to changing internal inorganic carbon concentrations, confirming that diffusion of CO₂ into the cells supplies most of the CO₂ for photosynthetic assimilation and that the internal inorganic carbon pool is essentially unavailable for photosynthetic assimilation. The estimated concentration of the internal inorganic carbon pool was found to be relatively insensitive to the external CO₂ concentration over the small range tested, as would be expected if the concentration of this pool is limited by the internal to external inorganic carbon gradient. An attempt to use this CO₂ exchange method to determine whether inorganic carbon accumulation and photosynthetic CO₂ assimilation compete for energy at low photon flux densities proved inconclusive.     

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.     

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.     

Interactions of glycolate-, HCO 3 - -, Cl--, and H+-balance of Scenedesmus obliquus

Excretion and absorption of glycolate by young cells of Scenedesmus obliquus (Turp.) Krüger strain D₃ grown synchronously with 2% CO₂ was compared after no pretreatment with air (CO₂-adapted) or after a 2 h adaptation to normal air (0.03% CO₂) (air-adapted). At 21% O₂, excretion occurred only from CO₂-adapted cells at high pH (pH 8.0). Under conditions where no excretion occurred, external glycolate (0.2 mM) was taken up by both air-and CO₂-adapted cells at a much faster rate at pH 5 than at pH 8. The uptake was accompanied by an apparent stoichiometric uptake of H⁺. CO₂-adapted algae exhibited high uptake rates that were even higher in the dark than in the light. Air-adapted algae showed high uptake rates in the light but only minimal uptake in the dark. The uptake rate was decreased to about 1/3 with 5% CO₂, except with CO₂-adapted cells in the light, in which a slight stimulation occurred. Cl^- ions inhibited glycolate uptake by air-adapted cells in the light; conversely, light-stimulated Cl^- uptake of these cells was inhibited by glycolate. A hypothesis is discussed according to which the internal pH regulates the uptake and release of Cl^-, HCO ₃ ^- , and glycolate.Abbreviations DCMU 3-(3,4 dichlorophenyl)-1, 1-dimethyl urea - FCCP carbonyl cyanide p-trifluoro-methoxyphenylhydrazone - HEPES 2-(4-(2-hydroxyethyl)-piperazinyl) ethanesulfonic acid - HPMS -hydroxypyridinemethanesulfonate - MES 2-morpholinoethanesulfonic acid - PCV packed cell volume     

The role of carbon dioxide in glucose metabolism of Bacteroides fragilis

The effect of CO₂ concentration on growth and glucose fermentation of Bacteroides fragilis was studied in a defined mineral medium. Batch culture experiments were done in closed tubes containing CO₂ concentrations ranging from 10% to 100% (with appropriate amounts of bicarbonate added to maintain the pH at 6.7). These experiments revealed that CO₂ had no influence on growth rate or cell yield when the CO₂ concentration was above 30% CO₂ (minimum available CO₂–HCO ₃ ^- , 25.5 mM), whereas a slight decrease in these parameters was observed at 20% and 10% CO₂ (available CO₂–HCO ₃ ^- , 17 and 8.5 mM, respectively). If CO₂–HCO ₃ ^- concentrations were below 10 mM, the lag phase lengthened and a decrease in maximal growth rate and cell yield were observed. The amount of acetate made decreased, while d-lactate concentration increased. A net production of CO₂ allowed growth under conditions of extremely low concentrations of added CO₂.When B. fragilis was grown in continuous culture with 100% CO₂ or 100% N₂, the dilution rate influenced the concentrations of acetate, succinate, propionate, d-lactate, l-malate and formate formed. Decreasing the dilution rate favored propionate and acetate production under both conditions. When the organism was grown with 100% N₂, the amount of propionate formed was greater than the amount of succinate formed at all dilution rates. Except at slow dilution rates the reverse was true when 100% CO₂ was used. B. fragilis was unable to grow at dilution rates faster than 0.154 h^-1 when grown with 100% N₂; the Y _glc ^max was 67.9 g DW cells/mol glucose and m _s was 0.064 mmol glucose/g DW·h. If the gas atmosphere was 100% CO₂ the organism was washed out of the culture when the dilution rate exceeded 0.38 h^-1; the Y _glc ^max was 59.4 g DW cells/mol glucose and m _s was 0.094 mmol glucose/g DW·h.Measurement of the phosphoenolpyruvate (PEP) carboxykinase (E.C. 4.1.1.49) with whole, permeabilized cells of B. fragilis showed an increase of specific enzyme activity with decreasing CO₂ concentrations. The mechanisms used by B. fragilis to adjust to low levels of CO₂ are discussed.     

Active uptake of CO2 during photosynthesis in the green alga Eremosphaera viridis is mediated by a CO2-ATPase

Mass spectrometry was used to investigate the uptake of CO₂ in Eremosphaera viridis DeBary. Upon illumination, cells preincubated at pH 7.5 with 100 M dissolved inorganic carbon (DIC) rapidly depleted almost all the free CO₂ from the medium. Rapid equilibrium between HCO ₃ ^- and CO₂ occurred upon addition of bovine carbonic anhydrase (CA) to the medium, showing that CO₂ depletion resulted from a selective uptake of CO₂ rather than an uptake of all inorganic carbon species. Glycolaldehyde (10 mM) completely inhibited CO₂ fixation but had little effect on CO₂ transport. Transfer of glycolaldehyde-treated cells to the dark caused a rapid efflux of CO₂ from the unfixed intracellular DIC pool which was found to be at least threeto sixfold higher in concentration than that of the external medium. These results indicate that E. viridis actively transports CO₂ against a concentration gradient. No external CA was detected in these cells either by potentiometric or mass-spectrometric assay. In the absence of external CA, the rate of photosynthetic O₂ evolution in the pH range 7.5 to 8.0 did not exceed the calculated rate of CO₂ supply, indicating a limited capacity for HCO₂ uptake in these cells. Electrophysiological measurements indicate that CO₂ uptake is electrically silent and thus is not a consequence of H⁺-CO₂ symport activity. Microsomal membranes isolated from Eremosphaera showed ATPase activity which was enhanced by CO₂. These results indicate that active CO₂ uptake is mediated by an ATPase.Abbreviations BTP 1,3-bis[tris(hydroximethyl)-methylamino]-propane - CA carbonic anhydrase - Chl chlorophyll - DIC dissolved inorganic carbon - [¹⁴C]DMO 5,5-dimethyl-[2-¹⁴C]-oxaz-didine-2,4-dione - WA Wilbur-Anderson units This work was supported by grants to B.C. and R.R.L. from the Natural Sciences and Engineering Research Council of Canada. We thank the Department of Biology, Queen's University, Kingston, Ontario for the use of the mass-spectrometer facility. We are indebted to A.G. Miller for his expert advice on operating the mass spectrometer and to Ms. Shahebina Samji for running the Bradford assays.     

Effect of dissolved inorganic carbon on the expression of carboxysomes,localization of Rubisco and the mode of inorganic carbon transport in cells of the cyanobacterium Synechococcus UTEX 625

In the cyanobacterium Synechococcus UTEX 625, the extent of expression of carboxysomes appeared dependent on the level of inorganic carbon (CO₂+HCO _inf3 ^sup- ) in the growth medium. In cells grown under 5% CO₂ and in those bubbled with air, carboxysomes were present in low numbers (<2 · longitudinal section^-1) and were distributed in an apparently random manner throughout the centroplasm. In contrast, cells grown in standing culture and those bubbled with 30 l CO₂ · 1^-1 possessed many carboxysomes (>8 · longitudinal section^-1). Moreover, carboxysomes in these cells were usually positioned near the cell periphery, aligned along the interface between the centroplasm and the photosynthetic thylakoids. This arrangement of carboxysomes coincided with the full induction of the HCO _inf3 ^sup- transport system that is involved in concentrating inorganic carbon within the cells for subsequent use in photosynthesis. Immunolocalization studies indicate that the Calvin cycle enzyme ribulose bisphosphate carboxylase was predominantly carboxysome-localized, regardless of the inorganic carbon concentration of the growth medium, while phosphoribulokinase was confined to the thylakoid region. It is postulated that the peripheral arrangement of carboxysomes may provide for more efficient photosynthetic utilization of the internal inorganic carbon pool in cells from cultures where carbon resources are limiting.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon (CO₂+HCO _inf3 ^sup- +CO _inf3 ^sup2- ) - PRK phosphoribulokinase - RuBP ribulose 1,5-bisphosphate - Rubisco LS large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase     

pH Regulation in tissue-cultured bovine lens epithelial cells

Summary The intracellular pH (pH _i ) of tissue-cultured bovine lens epithelial cells was measured in small groups of 6 to 10 cells using the trapped fluorescent dye 2,7-bis-(2-,carboxyethyl)-5 (and 6)carboxyfluorescein (BCECF). When perifused at 35°C with artificial aqueous humour solution (AAH) containing 16 mM HCO ₃ ^- and 5% CO₂, pH 7.25, pH _i was 7.19±0.02 (sem, n = 95). On removing HCO ₃ ^- and CO₂ there was an initial transient alkalinization followed by a fall in pH to a steady value of 6.97±0.03 (sem, n = 54). Addition of 0.25 mM 4,4-diisothiocyanatostilbene2, 2-disulfonic acid (DIDS) to AAH containing HCO ₃ ^- and CO₂ led to a rapid and pronounced fall in pH. Exposure to Na⁺-free AAH again led to a marked fall in pH _i , but in this case the addition of DIDS did not produce a further fall. Substitution of the impermeant anion gluconate for Cl^– in the presence of HCO ₃ ^- led to a rise in pH _i , while substitution in the absence of HCO ₃ ^- led to a fall in pH _i . The above data indicate a significant role for a sodium-dependent Cl^–-HCO ₃ ^- exchange mechanism in the regulation of pH _i . Addition of 1 mM amiloride to control AAH in both the presence and absence of HCO ₃ ^- led to a marked fall in pH _i , indicating that a Na⁺/H⁺ exchange mechanism also has a significant role in the regulation of pH _i . There is evidence for a lactic acid transport mechanism in bovine lens cells, as addition of lactate to the external medium produced a rapid fall in pH _i . Larger changes in pH _i were observed in control compared to HCO ₃ ^- -free AAH and in the latter case a pronounced alkalinizing overshoot was obtained on removing external lactate. Tissue-cultured bovine lens cells thus possess at least three membrane transport mechanisms that are involved in pH regulation. The buffering capacity of the lens cells was measured by perturbing pH _i with either NH ₄ ⁺ or procaine. The values obtained were similar in both cases and the intrinsic buffering capacity measured in the absence of external HCO ₃ ^- was 5 mm/pH unit (procaine). However, in the presence of HCO ₃ ^- and CO₂ the buffer capacity increases approximately fourfold, indicating that HCO ₃ ^- is the principal intracellular buffer.We acknowledge financial support from the Wellcome Trust and the Humane Research Trust for this project. M.R. Williams was in receipt of a Science & Engineering Research Council studentship.     

Stomatal responses to carbon dioxide of isolated epidermis from a C3 plant,the Argenteum mutant of Pisum sativum L., and a crassulacean-acid-metabolism plant Kalanchoë daigremontiana Hamet et Perr

The response of stomata in isolated epidermis to the concentration of CO₂ in the gaseous phase was examined in a C₃ species, the Argenteum mutant of Pisum sativum, and a crassulacean-acid-metabolism (CAM) species, Kalanchoë daigremontiana. Epidermis from leaves of both species was incubated on buffer solutions in the presence of air containing various volume fractions of CO₂ (0 to 10000·10^–6). In both species and in the light and in darkness, the effect of CO₂ was to inhibit stomatal opening, the maximum inhibition of opening occurring in the range 0 to 360·10^–6. The inhibition of opening per unit change in concentration was greatest between volume fractions of 0 and 240·10^–6. There was little further closure above the volume fraction of 360·10^–6, i.e. approximately ambient concentration of CO₂. Thus, although leaves of CAM species may experience much higher internal concentrations of CO₂ in the light than those of C₃ plants, this does not affect the sensitivity of their stomata to CO₂ concentration or the range over which they respond. Stomatal responses to CO₂ were similar in both the light and the dark, indicating that effects of CO₂ on stomata occur via mechanisms which are independent of light. The responses of stomata to CO₂ in the gaseous phase took place without the treatments changing the pH of the buffered solutions. Thus it is unlikely that CO₂ elicited stomatal movement by changing either the pH or the HCO ₃ ^– /CO ₃ ^2- equilibria. It is suggested that the concentration of dissolved unhydrated CO₂ may be the effector of stomatal movement and that its activity is related to its reactivity with amines.     

Effects of [CO2] and nitrogen fertilization on soils planted with ponderosa pine

The effects of six years treatment with elevated [CO₂] (350, 525, and 700 μl l^-1) and nitrogen (N) (0, 10, and 20 g N m^-2 yr^-1) on soils, soil solution, and CO₂ efflux in an open-top chamber study with ponderosa pine (Pinus ponderosa Laws.) are described. The clearest [CO₂] effect was in year 6, when a pattern of lower soil N concentration and higher C/N ratio with elevated [CO₂] emerged. Statistically significant effects of elevated [CO₂] on soil total C, extractable P, exchangeable Mg²⁺, exchangeable Ca²⁺, base saturation, and soil solution HCO₃ ^- and NO₃ ^- were also found in various treatment combinations and at various times; however, these effects were inconsistent among treatments and years, and in many cases (P, Mg²⁺, Ca²⁺, base saturation) reflected pre-treatment differences. The use of homogenized buried soil bags did not improve the power to detect changes in soil C and N or help resolve the inconsistencies in soil C patterns. Nitrogen fertilization had the expected negative effects on exchangeable Ca²⁺, K⁺, and Mg²⁺ in year 6, presumably because of increased NO₃ ^- leaching, but had no consistent effect on soil C, N, or extractable P. This revised version was published online in June 2006 with corrections to the Cover Date.     

Differential responses to copper in rainbow trout (Oncorhynchus mykiss) acclimated to sea water and brackish water

Rainbow trout, Oncorhynchus mykiss, acclimated to 33% sea water (12 mg·ml^-1 salinity) experienced significant (10 meq·1^-1) increases in plasma [Na⁺] and [Cl^-] within 5 h of exposure to 6.3 mol copper·1^-1 indicating severe impairment of branchial ionoregulatory capacity. All plasma ion levels subsequently stabilised once the transbranchial [Na⁺] gradient was reduced to zero. The similar ionic strength of the external medium and their body fluids appeared to protect trout maintained in 33% sea water from further ionoregulatory stress and any secondary physiological disturbances during exposure to copper. Despite three- and fourfold greater transbranchial [Na⁺] and [Cl^-] gradients, trout acclimated to full-strength sea water (35 mg·ml^-1 salinity) suffered no major changes in plasma Na⁺, Cl^-, K⁺, or Ca²⁺, blood gases or haematology during 24 h exposure to 6.3 mol copper·1^-1. This reduction in toxicity in full strength sea water cannot be explained by differences in copper speciation. We suggest that during acute exposure to waterborne copper, active NaCl extrusion is unaffected due to the basolateral location of the gill Na⁺/K⁺-ATPase, but that ionoregulatory disturbances can occur due to gill permeability changes secondary to the displacement of surface-bound Ca²⁺. However, in full strength sea water the three-fold higher ambient [Ca²⁺] and [Mg²⁺] appear to be sufficient to prevent any detrimental permeability changes in the presence of 6.3 mol copper·1^-1. Plasma [NH ₊ ⁴ ] and [HCO _- ³ ] were both significantly elevated during exposure to copper, indicating that some aspects of gill ion transport (specifically the apical Na⁺/NH ₊ ⁴ and Cl^-/HCO _- ³ exchanges involved in acid/base regulation and nitrogenous waste excretion) are vulnerable to inhibition in the presence of waterborne copper.Abbreviations C _aO₂ arterial oxygen content - Hb haemoglobin - Hct haematocrit - MABP mean arterial blood pressure - MCHC mean cell haemoglobin content - MO₂ rate of oxygen consumption - P _a CO₂ arterial carbon dioxide tension - P _aO₂ arterial oxygen partial pressure - S salinity - SW sea water - T _Amm total ammonia (=NH₃+NH ₊ ⁴ ) - T _{CO 2} total carbon dioxide - TEP transepithelial potential - TOC total organic carbon - %Hb-O₂ percentage of haemoglobin saturated with oxygen     

The optimal growth conditions for the biomass production of Isochrysis galbana and the effects that phosphorus, Zn2+, CO2, and light intensity have on the biochemical composition of Isochrysis galbana and the activity of extracellular CA

The effects of phosphorus, Zn²⁺, CO₂, and light intensity on growth, biochemical composition, and the activity of extracellular carbonic anhydrase (CA) in Isochrysis galbana were investigated. A significant change was observed when the concentration of phosphorus in the medium was increased from 5 μmol/L to 1000 μmol/L affecting I. galbana’s cell density, biochemical composition, and the activity of extracellular CA. Phosphorous concentration of 50 μmol/L to 500 μmol/L was optimal for this microalgae. The Zn²⁺ concentration at 10 μmol/L was essential to maintain optimal growth of the cells, but a higher concentration of Zn²⁺ (≥ 1000 μmol/L) inhibited the growth of I. galbana. High CO₂ concentrations (43.75 mL/L) significantly increased the cell densities compared to low CO₂ concentrations (0.35 mL/L). However, the activity of extracellular CA decreased significantly with an increasing concentration of CO₂. The activity of extracellular CA at a CO₂ concentration of 43.75 mL/L was approximately 1/6 of the activity when the CO₂ concentration was at 0.35 mL/L CO₂. Light intensity from 4.0 mW/cm² to 5.6 mW/cm² was beneficial for the growth, biochemical composition and the activity of extracellular CA. The lower and higher light intensity was restrictive for growth and changed its biochemical composition and the activity of extracellular CA. These results indicate that phosphorus, Zn²⁺, CO₂, and light intensity are important factors that impact growth, biochemical composition and the activity of extracellular CA in I. galbana.