A role for mitochondrial carbonic anhydrase in limiting CO2 leakage from low CO2-grown cells of Chlamydomonas reinhardtii

A model is presented which quantifies a possible role for the carbonic anhydrase in the mitochondrial matrix of Chlamydomonas reinhardtii which incorporates the observation that the expression of this enzyme is increased under growth conditions in which the expression of the carbon dioxide-concentrating mechanism is increased. It is assumed that the inorganic carbon enters the cytosol from the medium, and leaves the cytosol to the plastids, as HCO₃⁻ and that there is negligible carbonic anhydrase activity in the cytosol. The role of the mitochondrial carbonic anhydrase is suggested to be the conversion to HCO₃^– of the CO₂ produced in the mitochondria in the light from tricarboxylic acid cycle activity and from decarboxylation of glycine in any photorespiratory carbon oxidation cycle activity which is not suppressed by the carbon concentrating mechanism. If there is a HCO₃⁻ channel in the inner mitochondrial membrane then almost all of the inorganic carbon leaves the mitochondria as HCO₃⁻, thus limiting the potential for CO₂ leakage through the plasmalemma. This mechanism could increase inorganic C supply to ribulose bisphosphate carboxylase-oxygenase by some 10% at the energetic expense of less than 1% of the total ATP generation by plastids plus mitochondria.     

Carbonic anhydrase is induced prior to arylsulfatase under the simultaneous deprivation of inorganic carbon and sulfate in Chlamydomonas reinhardtii (Volvocales, Chlorophyta)

The activity of periplasmic arylsulfatase (Ars), which catalyzes the cleavage of sulfate from aromatic sulfur compounds, was detected in cells acclimated to the sulfate-deficient conditions in a unicellular green alga Chlamydomonas reinhardtii Dangeard, but not in Chlorella, Scenedesmus, Dunaliella and Porphyridium. Upon the transfer of cells to sulfate-deficient autotrophic media under high-CO₂ conditions, the induction of Ars was observed only in the light, but not in the light with dichlorophenyldimethylurea (DCMU) nor in the dark. However, Ars was induced in the light with DCMU or in the dark when acetate was present as an organic carbon source, but not citrate. Under similar high-CO₂ conditions, high-CO₂ requiring mutants of cia-3 and cia-5, whose photosynthetic activities are greatly limited under low CO₂, showed much lower level of Ars activities than wild type cells. Under Iow-CO₂ conditions the induction of Ars was greatly suppressed even in wild type and no induction was observed in both mutants. These results suggest that the stimulation of photosynthetic or respiratory carbon metabolism are necessary for the induction of Ars. In contrast, the induction of periplasmic carbonic anhydrase (CA) which was synthesized de novo specifically under CO₂-limited conditions was strongly suppressed by the addition of organic carbon sources, such as acetate and citrate. When cells are subjected to CO₂-limitation and sulfate-deficiency simultaneously, the induction of CA was initiated immediately, while that of Ars was initiated following the completion of CA induction with an about 4-h lag. When the concentration of CO₂ was suddenly lowered during the induction of Ars, the induction of Ars ceased quickly, and the induction of CA was initiated instead. From these results the induction of CA was suggested to have priority over that of Ars under the dual stress of CO₂, and sulfate-deprivation.     

The CO2-concentrating mechanism is absent in the green alga Coccomyxa: a comparative study of photosynthetic CO2 and light responses of Coccomyxa, Chlamydomonas reinhardtii and barley protoplasts

Photosynthesis was characterized for the unicellular green alga Coccomyxa sp., grown at low inorganic carbon (C_i) concentrations, and compared with Chlamydomonas reinhardtii, which had been grown so that the CO₂ concentrating mechanism (CCM) was expressed, and with protoplasts isolated from the C₃ plant barley (Hordeum vulgare). Chlamydomonas had a significantly higher C_i-use efficiency of photosynthesis, with an initial slope of the C_i-response curve of 0.7 mol(gChl)⁻¹ h⁻¹ mmol C_im⁻³)⁻¹, as compared to 0.3 and 0.23 mol(gChl)⁻¹ h⁻¹ (mmol C_im⁻³)⁻¹ for Coccomyxa and barley, respectively. The affinity for C_i was also higher in Chlamydomonas, as the half maximum rate of photosynthesis [K_0.5 (C_i)] was reached at 0.18 mol m⁻³, as compared to 0.30 and 0.45 mol m⁻³ for Coccomyxa and barley, respectively. Ethoxyzolamide (EZ), an inhibitor of the enzyme carbonic anhydrase (CA) and the CCM, caused a 17-fold decrease in the initial slope of the photosynthetic Cj-response curve in Chlamydomonas, but only a 1.5- to two-fold decrease in Coccomyxa and barley. The photosynthetic light-response curve showed further similarities between barley and Coccomyxa. The rate of bending of the curve, described by the convexity parameter, was 0.99 (sharp bending) and 0.81–0.83 (gradual bending) for cells grown under low and high light, respectively. In contrast, the maximum convexity of Chlamydomonas was 0.85. The intrinsically lower convexity of Chlamydomonas is suggested to result from the diversion of electron transport from carbon fixation to the CCM. Taken together, these results suggest that Coccomyxa does not possess a CCM and due to this apparent lack of a CCM, we propose that Coccomyxa is a better cell model system for studying C₃ plant photosynthesis than many algae currently used.     

The role of extracellular carbonic anhydrase for accumulation of inorganic carbon in the green alga Chlamydomonas reinhardtii. A comparison between wild-type and cell-wall-less mutant cells

The role of extracellular carbonic anhydrase (CA_ex) for dissolved inorganic carbon (DIC) accumulation in the green alga Chlamydomonas reinhardtii was investigated. It was found that when algal cells were bubbled with ambient air, cell-wall-less mutant cells exhibited the same high photosynthetic affinity for CO₂ as wild-type cells despite a 10 times lower activity of CA^ex. It was also found that the affinity for CO₂ was further increased when the total DIC concentration of the algal medium was reduced from that in equilibrium with ambient air to even lower levels. This increased affinity was not correlated with any further increase in the CA_ex activity. Dextran-bound sulfonamide (DBS. 100 μM bound ligand) completely inhibited the activity of CA_ex in intact, low-DIC grown, wild-type cells, while photosynthesis at <2 μM CO₂(aq) proceeded at a far greater rate than could be maintained by CO₂ supplied from the spontaneous dehydration of HCO^?₃. DBS-inhibition of CA_ex, during the induction of the DIC-accumulating mechanism in previously high-DIC grown cells, only caused a 50% inhibition of photosynthesis at 10 μM CO₂(aq) after 1 h of low-DIC acclimation. It was also shown that 50 μM acetazolamide (AZ) inhibited photosynthesis at low DIC concentrations to a relatively higher degree than DBS, suggesting that AZ inhibited intracellular CA as well. Taken together, these results suggest that low-DIC grown cells of C. reinhardtii have the ability to transport HCO^?₃ across the plasma membrane in addition to the CA_ex-mediated, facilitated diffusion and/or transport of CO₂. It is also suggested that the relative importance of these two fluxes (CO₂ or HCO^?₃) is dependent on the growth and experimental conditions. Facilitated CO₂ uptake seems to be most prevalent, supported by HCO^?₃-transport under more or less extreme situations, such as a reduction of CO₂ to extremely low concentrations, leakage of CA_ex to the medium as in cultures of cell-wall-less mutant cells or when the activity of CA_ex has been artificially inhibited.     

Effect of CO2, CO2 and Diamox on photosynthesis and photorespiration in Chlamydomonas reinhardtii (green alga) and Anacystis nidulans (Cyanobacterium, blue-green alga)

Photorespiration by Chlamydomonas reinhardtii and Anacystis nidulans was measured as the oxygen inhibition of CO₂ uptake and the CO₂ compensation points. Net photosynthesis was oxygen dependent in Chlamydomonas grown in 5% CO₂, but CO₂ insensitive in cultures bubbled with air. Anacystis, even when cultured in 5% CO₂, exhibited an CO₂ insensitive net photosynthesis. The CO₂ compensation point of Chlamydomonas grown in cultures bubbled with air and Anacystis grown in 5% CO₂ enriched air, were reached shortly after the measurement was begun and the values were very low, less than 10 μl CO₂ 1^?1; while Chlamydomonas grown in 5% CO₂ enriched air for 4 days showed a high, but temporary CO₂ compensation point (60 μl CO₂ 1^?1). After a two hour adaptation in low CO₂, a stable, low CO₂ compensation point was reached. It seems that photorespiration can only be detected by the methods used in this study when the algae are cultured in high CO₂, but a mechanism exists which blocks photorespiration when the green algae are adapted to low CO₂ concentrations. When Chlamydomonas was treated with Diamox, an inhibitor of carbonic anhydrase, after cultivation in low CO₂ (air), the cells behaved as if they had been grown in high CO₂. They showed an oxygen sensitive net photosynthesis and a high CO₂ compensation point. This indicates that carbonic anhydrase plays an important role in the regulation of a measurable photorespiration in Chlamydomonas. The results are discussed in relation to previous observations of photorespiration measured by enzyme assay, metabolic products and gas exchange properties.     

The effects of pH and dissolved inorganic carbon on external carbonic anhydrase activity in Chlorella saccharophila

External carbonic anhydrase (CA) activity in Chlorella saccharophila is suppressed by growth at high dissolved inorganic carbon and at acid pH. External CA activity was shown to be suppressed by growth at pHs below 7.0, with total repression at pH5.0. Growth in the presence of the buffer 3-[N-Morpholino]propane-sulphonic acid (MOPS) between pH 7 and 8 suppressed CA activity. Cells grown at pH8.0 aerated at 6 dm³ h^?1 exhibited external CA activity of 5 units mg^?1 Chl once the dissolved inorganic carbon (DIC) was reduced to 300 mmol m^?3, and this increased to 30 units mg^?1 Chl over a period of 3d while the DIC dropped to 30mmol m^?3. Cells aerated at 180 dm³ h^?1 showed a similar trend in CA activity, although the onset was delayed by 1 d and the DIC did not drop below 300 mmol m^?3. Cells grown at pH 7.8 near an air equilibrium DIC of 300 mmol m^?3had no detectable external CA activity. It is probable that it is the CO² supply to the cell, and not total DIC or HCO^?₃ which controls external CA activity. Cells grown at pH 5.0 had no detectable activity, although they reduced the CO² concentration to 0.6 mmol m^?3. The loss of CA upon transfer of air-grown cells to 10 mmol mol^?1 CO² took place over 48 h and was light dependent, while the loss upon transfer from alkaline pH to acid pH look place over 12 h and was independent of light. The effects of pH are independent of the response to CO₂.     

CO2 is the main inorganic C species entering photosynthetically active leaf protoplasts of the freshwater macrophyte Ranunculus penicillatus ssp. pseudofluitans

Submerged aquatic macrophytes growing in water where free CO₂ is unavailable (above pH 8·2) must use mechanisms to supply external dissolved inorganic carbon in a form available to chloroplasts (CO₂). Active transport of HCO₃^– across the plasmalemma has not been proven to be widespread in aquatic macrophytes and catalytic conversion of HCO₃^– to CO₂ is the usual supply mechanism in submerged macrophytes. The interaction of leaf form and function in this respect was investigated in the linear, submerged leaves of Ranunculus penicillatus (Dumort.) Bab ssp. pseudofluitans (Syme) S.Webster. Viable protoplasts were isolated using a mixture of cell wall degrading enzymes optimized for this species. Protoplast viabilities greater than 80% after 5 h of isolation were achieved. Photosynthetic rates of isolated protoplasts were comparable with that of intact plant tissue. Results of carbon isotopic disequilibrium experiments showed that CO₂ was the preferred species of dissolved inorganic carbon for photosynthesis by protoplasts and that HCO₃^– which predominates in the plant’s natural environment mainly contributes by supplying CO₂ outside the cells.     

The molecular and biochemical analyses of CO2-concentrating mechanisms in cyanobacteria and microalgae

Abstract. The supply of dissolved inorganic carbon for photosynthesis in the aquatic environment is much more variable than that experienced by terrestrial plants. In response to this variability, cyanobacteria and many species of microalgae acclimate to rapid changes in the availability of dissolved inorganic carbon by the induction of high affinity/high capacity CO₂-concentrating systems (CCMs). Biochemical and molecular analyses of the acclimation response have recently identified several components that are required for efficient operation of the CCMs and CO₂ assimilation. This has been accomplished using in vivo labelling studies, and characterization of high inorganic carbon (C_i) requiring cyanobacterial and algal mutants. The identification and regulation of expression of polypeptides synthesized in response to limiting C_i concentrations, and the proposed role of the carboxysome and the pyrenoid in the functioning of the CCMs are examined.     

The regulation of photosynthetic rate and activation of extracellular carbonic anhydrase under CO2-limiting conditions in the marine diatom Skeletonema costatum

In the marine diatom Skeletonema costatum , carbonic anhydrase activity exterior to the plasma membrane (CA_ext) was detected only when the available CO₂ concentration was less than 5·0 mmol m^–3, this activity being unaffected by the total dissolved inorganic carbon concentration. The inhibition of CA_ext by dextran bound sulphonamide (DBS) demonstrated the key role of this enzyme in maintaining photosynthetic rate under CO₂-limited conditions. Treatment with trypsin followed by affinity chromatography on p-aminomethylbenzene-sulphamide agarose and subsequent SDS-PAGE analysis revealed a polypeptide from carbon-replete cells of identical molecular mass to the CA_ext released by trypsin from CO₂-limited cells. Redox activity in the plasma membrane of intact cells was measured by following the light-dependent reduction of ferricyanide or NADP, the greatest activity being shown by CO₂-limited cells. Overall the results suggest that high rates of redox activity under conditions of CO₂-limitation were required for the activation of CA_ext.     

Evaporation and carbonic anhydrase activity recorded in oxygen isotope signatures of net CO2 fluxes from a Mediterranean soil

The oxygen stable isotope composition (δ¹⁸O) of CO₂ is a valuable tool for studying the gas exchange between terrestrial ecosystems and the atmosphere. In the soil, it records the isotopic signal of water pools subjected to precipitation and evaporation events. The δ¹⁸O of the surface soil net CO₂ flux is dominated by the physical processes of diffusion of CO₂ into and out of the soil and the chemical reactions during CO₂–H₂O equilibration. Catalytic reactions by the enzyme carbonic anhydrase, reducing CO₂ hydration times, have been proposed recently to explain field observations of the δ¹⁸O signatures of net soil CO₂ fluxes. How important these catalytic reactions are for accurately predicting large‐scale biosphere fluxes and partitioning net ecosystem fluxes is currently uncertain because of the lack of field data. In this study, we determined the δ¹⁸O signatures of net soil CO₂ fluxes from soil chamber measurements in a Mediterranean forest. Over the 3 days of measurements, the observed δ¹⁸O signatures of net soil CO₂ fluxes became progressively enriched with a well‐characterized diurnal cycle. Model simulations indicated that the δ¹⁸O signatures recorded the interplay of two effects: (1) progressive enrichment of water in the upper soil by evaporation, and (2) catalytic acceleration of the isotopic exchange between CO₂ and soil water, amplifying the contributions of ‘atmospheric invasion’ to net signatures. We conclude that there is a need for better understanding of the role of enzymatic reactions, and hence soil biology, in determining the contributions of soil fluxes to oxygen isotope signals in atmospheric CO₂.     

Regulation of the induction of bicarbonate uptake by dissolved CO2 in the marine diatom, Phaeodactylum tricornutum

Physiological properties of photosynthesis were determined in the marine diatom, Phaeodactylum tricornutum UTEX640, during acclimation from 5% CO₂ to air and related to H₂CO₃ dissociation kinetics and equilibria in artificial seawater. The concentration of dissolved inorganic carbon at half maximum rate of photosynthesis (K_0·5[DIC]) value in high CO₂‐grown cells was 1009 mmol m ^{? 3} but was reduced three‐fold by the addition of bovine carbonic anhydrase (CA), whereas in air‐grown cells K_0·5[DIC] was 71 mmol m ^{? 3}, irrespective of the presence of CA. The maximum rate of photosynthesis (P_max) values varied between 300 and 500 μ mol O₂ mg Chl ^{? 1} h ^{? 1} regardless of growth pCO₂. Bicarbonate dehydration kinetics in artificial seawater were re‐examined to evaluate the direct HCO₃ ^? uptake as a substrate for photosynthesis. The uncatalysed CO₂ formation rate in artificial seawater of 31·65°/_oo of salinity at pH 8·2 and 25 °C was found to be 0·6 mmol m ^{? 3} min ^{? 1} at 100 mmol m ^{? 3} DIC, which is 53·5 and 7·3 times slower than the rates of photosynthesis exhibited in air‐ and high CO₂‐grown cells, respectively. These data indicate that even high CO₂‐grown cells of P. tricornutum can take up both CO₂ and HCO₃ ^? as substrates for photosynthesis and HCO₃ ^? use improves dramatically when the cells are grown in air. Detailed time courses were obtained of changes in affinity for DIC during the acclimation of high CO₂‐grown cells to air. The development of high‐affinity photosynthesis started after a 2–5 h lag period, followed by a steady increase over the next 15 h. This acclimation time course is the slowest to be described so far. High CO₂‐grown cells were transferred to controlled DIC conditions, at which the concentrations of each DIC species could be defined, and were allowed to acclimate for more than 36 h. The K_0·5[DIC] values in acclimated cells appeared to be correlated only with [CO_2(aq)] in the medium but not to HCO₃ ^? , CO₃^{2 ?} , total [DIC] or the pH of the medium and indicate that the critical signal regulating the affinity of cells for DIC in the marine diatom, P. tricornutum, is [CO_2(aq)] in the medium.     

The acquisition and accumulation of inorganic carbon by the unicellular green alga Chlorella ellipsoidea

Abstract. The uptake and accumulation of inorganic carbon has been investigated in Chlorella ellipsoidea cells grown at acid or alkaline pH. Carbonic anhydrase (CA) was detected in ceil extracts but not in intact cells and CA activity in acid-grown cells was considerably less than that in alkali-grown cells. Both cell types demonstrates low K_1/2 (CO₂) values in the range pH 7.0–8.0 and these were unaffected by O₂ concentration. The CO₂ compensation concentrations of acid- and alkali-grown cells suspended in aqueous media were not significantly different in the range of pH 6.0–8.0, but at pH 5.0, the CO₂ compensation concentrations of acid-grown cells (57.4cm³ m⁻³) were lower than those of alkali-grown cells (79.2cm³ m⁻³). The rate of photo-synthetic O₂ evolution in the range pH 7.5–8.0 exceeded the calculated rate of CO₂ supply two- to three-fold, in both acid- and alkali-grown cells, indicating that HCO₃⁻ was taken up by the cells. Accumulation of inorganic carbon was measured at pH 7.5 by silicone-oil centri-fugation, and the concentration of unfixed inorganic carbon was found to be 5.1 mol m⁻³ in acid-grown and 6.4mol m⁻³ in alkali-grown cells. These concentrations were 4.6- and 5.9-fold greater than in the external medium. These results indicate that photorespiration is suppressed in both acid- and alkali-grown cells by an intracellular accumulation of inorganic carbon due, in part, to an active uptake of bicarbonate.     

Blue-light requirement for the biosynthesis of an NO2− transport system in the Chlamydomonas reinhardtii nitrate transport mutant S10*

The blue-light requirement for the biosynthesis of nitrite reductase and an NO₂^– transport system was studied in Chlamydomonas reinhardtii mutant S10. The only oxidized nitrogen species that could be taken up by this mutant was NO₂^–, due to the presence of NO₂^– transport systems and the absence of high-affinity NO₃^– transporters. NH₄⁺-grown cells required illumination with blue light to recover the ability to take up NO₂^– when resuspended in an NO₂^–-containing NH₄⁺-deprived medium. This blue-light- dependent recovery, which took 1 h, could be suppressed by cycloheximide, indicating that protein biosynthesis was involved. The biosynthesis of nitrite reductase took place in cell suspensions irradiated with red light, even in the absence of NO₂^–, thus suggesting that the process requiring blue light was the biosynthesis of an NO₂^– transport system. Nitrite reductase-containing cells (pre-irradiated with red light) took 1 h to start consuming NO₂^– when they were additionally irradiated with blue light in the presence of this anion, and this process was also cycloheximide-sensitive. The NO₂^– transport system operated either under red plus blue light or red light only. Thus, in C. reinhardtii mutant S10 cells, blue light was only required for the biosynthesis of an NO₂^– transport system and not for its activity.     

Seventeen years of elevated CO2 exposure in a Chesapeake Bay Wetland: sustained but contrasting responses of plant growth and CO2 uptake

Increased atmospheric CO₂ concentration (Ca) produces a short‐term stimulation of photosynthesis and plant growth across terrestrial ecosystems. However, the long‐term response remains uncertain and is thought to depend on environmental constraints. In the longest experiment on natural ecosystem response to elevated Ca, we measured the shoot‐density, biomass and net CO₂ exchange (NEE) responses to elevated Ca from 1987 to 2003 in a Scirpus olneyi wetland sedge community of the Chesapeake Bay, MD, USA. Measurements were conducted in five replicated open‐top chambers per CO₂ treatment (ambient and elevated). In addition, unchambered control plots were monitored for shoot density. Responses of daytime NEE, Scirpus plant biomass and shoot density to elevated Ca were positive for any single year of the 17‐year period of study. Daytime NEE stimulation by elevated Ca rapidly dropped from 80% at the onset of the experiment to a long‐term stimulation average of about 35%. Shoot‐density stimulation by elevated Ca increased linearly with duration of exposure (r²=0.89), exceeding 120% after 17 years. Although of lesser magnitude, the shoot biomass response to elevated Ca was similar to that of the shoot density. Daytime NEE response to elevated Ca was not explained by the duration of exposure, but negatively correlated with salinity of the marsh, indicating that this elevated‐Ca response was decreased by water‐related stress. By contrast, circumstantial evidence suggested that salinity stress increased the stimulation of shoot density by elevated Ca, which highlights the complexity of the interaction between water‐related stresses and plant community responses to elevated Ca. Notwithstanding the effects of salinity stress, we believe that the most important finding of the present research is that a species response to elevated Ca can continually increase when this species is under stress and declining in its natural environment. This is particularly important because climate changes associated with elevated Ca are likely to increase environmental stresses on numerous species and modify their present distribution. Our results point to an increased resilience to change under elevated Ca when plants are exposed to adverse environmental conditions.     

Seasonal CO2 exchange patterns of developing peach (Prunus persica) fruits in response to temperature, light and CO2 concentration

CO₂ exchange rates per unit dry weight, measured in the field on attached fruits of the late-maturing Cal Red peach cultivar, at 1200 μmol photons m^?2S^?1 and in dark, and photosynthetic rates, calculated by the difference between the rates of CO₂ evolution in light and dark, declined over the growing season. Calculated photosynthetic rates per fruit increased over the season with increasing fruit dry matter, but declined in maturing fruits apparently coinciding with the loss of chlorophyll. Slight net fruit photosynthetic rates ranging from 0. 087 ± 0. 06 to 0. 003 ± 0. 05 nmol CO₂ (g dry weight)^?1 S^?1 were measured in midseason under optimal temperature (15 and 20°C) and light (1200 μmol photons m^?2 S^?1) conditions. Calculated fruit photosynthetic rates per unit dry weight increased with increasing temperatures and photon flux densities during fruit development. Dark respiration rates per unit dry weight doubled within a temperature interval of 10°C; the mean seasonal O₁₀ value was 2. 03 between 20 and 30°C. The highest photosynthetic rates were measured at 35°C throughout the growing season. Since dark respiration rates increased at high temperatures to a greater extent than CO₂ exchange rates in light, fruit photosynthesis was apparently stimulated by high internal CO₂ concentrations via CO₂ refixation. At 15°C, fruit photosynthetic rates tended to be saturated at about 600 μmol photons m^?2 S^?1. Young peach fruits responded to increasing ambient CO₂ concentrations with decreasing net CO₂ exchange rates in light, but more mature fruits did not respond to increases in ambient CO₂. Fruit CO₂ exchange rates in the dark remained fairly constant, apparently uninfluenced by ambient CO₂ concentrations during the entire growing season. Calculated fruit photosynthetic rates clearly revealed the difference in CO₂ response of young and mature peach fruits. Photosynthetic rates of younger peach fruits apparently approached saturation at 370 μl CO₂1^?2. In CO₂ free air, fruit photosynthesis was dependent on CO₂ refixation since CO₂ uptake by the fruits from the external atmosphere was not possible. The difference in photosynthetic rates between fruits in CO₂-free air and 370 μl CO₂ 1^?1 indicated that young peach fruits were apparently able to take up CO₂ from the external atmosphere. CO₂ uptake by peach fruits contributed between 28 and 16% to the fruit photosynthetic rate early in the season, whereas photosynthesis in maturing fruits was supplied entirely by CO₂ refixation.     

Inhibition by cupric ions of the hydration of CO2 catalyzed by carbonic anhydrase II

The inhibition by cupric ions of the hydration of CO₂ catalyzed by carbonic anhydrase II is interesting because of the results of Tuet al. obtained at chemical equilibrium, indicating that Cu²⁺ inhibits specifically a proton transfer in the catalytic pathway. We have measured this inhibition at steady state, using stopped-flow methods. The inhibition by Cu²⁺ of the hydration of CO₂ catalyzed by carbonic anhydrase II had aK_I near 1×10^–6 M atpH 7.0 and gave inhibition that is noncompetitive atpH 6.0 and mixed, but close to uncompetitive, atpH 6.8. ThepH dependence of this binding is consistent with a binding site for Cu²⁺ on the enzyme with apK_a near 7. The binding interaction between Cu²⁺ and the fluorescent inhibitor 5-dimethylaminonaphthalene-l-sulfonamide on carbonic anhydrase II was noncompetitive, indicating that the binding site for Cu²⁺ is distinct from the coordination sphere of zinc in which the actual interconversion of CO₂ and HCO₃^– and the binding of sulfonamides takes place.     

The turnover of carbon pools contributing to soil CO2 and soil respiration in a temperate forest exposed to elevated CO2 concentration

Soil carbon is returned to the atmosphere through the process of soil respiration, which represents one of the largest fluxes in the terrestrial C cycle. The effects of climate change on the components of soil respiration can affect the sink or source capacity of ecosystems for atmospheric carbon, but no current techniques can unambiguously separate soil respiration into its components. Long‐term free air CO₂ enrichment (FACE) experiments provide a unique opportunity to study soil C dynamics because the CO₂ used for fumigation has a distinct isotopic signature and serves as a continuous label at the ecosystem level. We used the ¹³C tracer at the Duke Forest FACE site to follow the disappearance of C fixed before fumigation began in 1996 (pretreatment C) from soil CO₂ and soil‐respired CO₂, as an index of belowground C dynamics during the first 8 years of the experiment. The decay of pretreatment C as detected in the isotopic composition of soil‐respired CO₂ and soil CO₂ at 15, 30, 70, and 200 cm soil depth was best described by a model having one to three exponential pools within the soil system. The majority of soil‐respired CO₂ (71%) originated in soil C pools with a turnover time of about 35 days. About 55%, 50%, and 68% of soil CO₂ at 15, 30, and 70 cm, respectively, originated in soil pools with turnover times of less than 1 year. The rest of soil CO₂ and soil‐respired CO₂ originated in soil pools that turn over at decadal time scales. Our results suggest that a large fraction of the C returned to the atmosphere through soil respiration results from dynamic soil C pools that cannot be easily detected in traditionally defined soil organic matter standing stocks. Fast oxidation of labile C substrates may prevent increases in soil C accumulation in forests exposed to elevated [CO₂] and may consequently result in shorter ecosystem C residence times.     

The effect of light quality on the induction of efficient photosynthesis under low CO2 conditions in Chlamydomonas reinhardtii and Chlorella pyrenoidosa

The effect of blue and red light on the adaptation to low CO₂ conditions was studied in high-CO₂ grown cultures of Chlorella Pyrenoidosa (82T) and Chlamydomonas reinhardtii(137⁺) by measuring O₂ exchange under various inorganic carbon (Cⁱ) concentrations. At equal photosynthetic photon flux density (PPFD), blue light was more favourable for adaptation in both species, compared to red light. The difference in photosynthetic oxygen evolution between cells adapted to low C_iunder blue and red light was more pronounced when oxygen evolution was measured under low C_i compared to high C_i conditions. The effect of light quality on adaptation remained for several hours. The different effects caused by blue and red light was observed in C. pyrenoidosa over a wide range of PPFD with increasing differences at increasing PPFD. The maximal difference was obtained at a PPFD above 1 500 μmol m^?2s^?1. We found no difference in the extracellular carbonic anhydrase activity between blue- and red light adapted cells. The light quality effect recorded under C_i-limiting conditions in C. reinhardtii cells adapted to air, was only 37% less when instead of pure blue light red light containing 12.5% of blue light (similar PPFD as blue light) was used during adaptation to low carbon. This indicates that in addition to affecting photosynthesis, blue light affected a sensory system involved in algal adaptation to low C_i conditions. Since the affinity for C_i of C. Pyrenoidosa and C. reinhardtii cells adapted to air under blue light was higher than that of cells adapted under red light, we suggest that induction of some component(s) of the C_i accumulating mechanism is regulated by the light quality.