External Carbonic Anhydrase Research Articles

Effect of Heavy Metals on the Activity of External Carbonic Anhydrase of Microalga Chlamydomonas reinhardtii and Microalgae from Karst Lakes

Effect of Heavy Metals on the Activity of External Carbonic Anhydrase of Microalga Chlamydomonas reinhardtii and Microalgae from Karst Lakes

Photosynthetic use of exogenous inorganic carbon in the agarophyte Gracilaria lemaneiformis (Rhodophyta)

Oxygen evolution characteristics of the agarophyte Gracilaria lemaneiformis (Bory) Weber-van Bosse (Rhodophyta) were examined to establish the mechanism of photosynthetic use of exogenous inorganic carbon (Ci). Oxygen evolution in seawater of pH 8.0 was sharply reduced with the increase of pH. However, the rates of O 2 evolution at pH 8.0 and above exceeded the theoretical calculated maximum rates supported solely by the CO 2 supply from the spontaneous dehydration of HCO 3 − in the bulk seawater. In addition, G. lemaneiformis exhibited a high pH compensation point (9.58). These data indicate that G. lemaneiformis was able to use HCO 3 − as a source of Ci for photosynthesis. Both external and internal carbonic anhydrase (CA) activities, which could be detected potentiometrically, were essential for photosynthesis at alkaline pH values (8.0 and 9.0), because O 2 evolution was inhibited with the addition of CA inhibitors acetazolamide (AZ) and ethoxyzolamide (EZ). By contrast, the anion exchanger inhibitor, 4,4′-diisothiocyano-stilbene-2,2′-disulfonate (DIDS) had no inhibitory effects on photosynthetic O 2 evolution at neither acidic nor alkaline pH values. Therefore, the experiments with CA inhibitors would suggest that the mechanism of extracellular dehydration of HCO 3 − into CO 2 mediated by external CA activity was involved in HCO 3 − use by G. lemaneiformis. The relationship between photosynthetic O 2 evolution rates and Ci concentrations showed that the normal Ci composition in natural seawater could fully saturate photosynthesis of G. lemaneiformis at pH 8.0 or lower. However, the affinity for Ci and the carbon-saturated maximum photosynthesis were dramatically reduced at pH 9.0.

Seasonal and tidal variability of environmental carbon related physico-chemical variables and inorganic C acquisition in Gracilariopsis longissima and Enteromorpha intestinalis from Los Toruños salt marsh (Cádiz Bay, Spain)

The seasonal and tidal variability of inorganic C acquisition mechanisms, photosynthesis, internal composition and growth were studied in two co-occurring macroalgae in Los Toruños salt marsh (Cádiz Bay), Gracilariopsis longissima and Enteromorpha intestinalis. This variability was monitored together with physico-chemical variables affecting carbon availability, photosynthesis, and growth. The environmental variables, such as light, temperature, pH, salinity, oxygen, alkalinity, dissolved inorganic carbon (DIC) and CO 2, displayed not only an expected seasonal cycle but also a daily (tidal) variability, with abrupt and rapid changes influenced by biological activities, physical variables, tidal state and tidal timing. In contrast to environmental variables, photosynthesis, pigments and C:N composition were affected by seasonal changes but not by tidal regimes, as organisms integrated these short-term fluctuations in physico-chemical variables. Photosynthesis, pigments and internal N composition were maximal in autumn and minimal in summer for both species. Growth showed a seasonal trend, displaying a summer drop with negative values. This response can be the result of extreme values of environmental variables (temperature, light, pH, nutrients, and the shortage of DIC) in summer, in comparison with higher growth rates in September onwards. The use of inhibitors of carbon acquisition in situ at normal DIC concentrations (2.2. mM) revealed species-dependent differences. While the external carbonic anhydrase (CA) activity showed a constitutive character in G. longissima, it showed little effect in E. intestinalis, which relies on internal CA activity. The 4, 4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS)-sensitive bicarbonate transport in G. longissima was effective in winter. In contrast, DIDS stimulated photosynthesis in summer, and relieved AZ inhibition. This response could suggest a stimulation of a H + extrusion mediated-CO 2 transport in periods of low CO 2 availability.

SOURCE OF INORGANIC CARBON FOR PHOTOSYNTHESIS IN TWO MARINE DINOFLAGELLATES1

Inorganic carbon uptake was investigated in two marine dinoflagellates, Amphidinium carterae Hulburt and Heterocapsa oceanica Stein. Mass spectrometric and potentiometric assays indicated that both species lacked external carbonic anhydrase (CA). The presence of internal CA was demonstrated by potentiometric assay and by the inhibition of photosynthesis upon the addition of 500 μM ethoxyzolamide a membrane‐permeable inhibitor of CA. The capacity for bicarbonate transport was investigated by comparing the calculated rate of spontaneous CO2 formation at pH 8.2 and 25°C with the rate of photosynthesis after the addition of 100 μM NaHCO3. Both species appeared to have a very limited capacity for direct bicarbonate uptake. Monitoring of CO2 and O2 fluxes in both species by mass spectrometry demonstrated a rapid uptake of CO2 on illumination, to concentrations below the CO2 equilibrium concentration, indicating an effective selective uptake of CO2. This dependence of photosynthesis on free CO2 alone suggests that these species are CO2 limited in their natural environment because the CO2 concentration of seawater is very low.

PHOTOSYNTHETIC UTILIZATION OF INORGANIC CARBON IN THE ECONOMIC BROWN ALGA, HIZIKIA FUSIFORME (SARGASSACEAE) FROM THE SOUTH CHINA SEA1

The mechanism of inorganic carbon (Ci) acquisition by the economic brown macroalga, Hizikia fusiforme (Harv.) Okamura (Sargassaceae), was investigated to characterize its photosynthetic physiology. Both intracellular and extracellular carbonic anhydrase (CA) were detected, with the external CA activity accounting for about 5% of the total. Hizikia fusiforme showed higher rates of photosynthetic oxygen evolution at alkaline pH than those theoretically derived from the rates of uncatalyzed CO2 production from bicarbonate and exhibited a high pH compensation point (pH 9.66). The external CA inhibitor, acetazolamide, significantly depressed the photosynthetic oxygen evolution, whereas the anion‐exchanger inhibitor 4,4′‐diisothiocyano‐stilbene‐2,2′‐disulfonate had no inhibitory effect on it, implying the alga was capable of using HCO3− as a source of Ci for its photosynthesis via the mediation of the external CA. CO2 concentrations in the culture media affected its photosynthetic properties. A high level of CO2 (10,000 ppmv) resulted in a decrease in the external CA activity; however, a low CO2 level (20 ppmv) led to no changes in the external CA activity but raised the intracellular CA activity. Parallel to the reduction in the external CA activity at the high CO2 was a reduction in the photosynthetic CO2 affinity. Decreased activity of the external CA in the high CO2 grown samples led to reduced sensitiveness of photosynthesis to the addition of acetazolamide at alkaline pH. It was clearly indicated that H. fusiforme, which showed CO2‐limited photosynthesis with the half‐saturating concentration of Ci exceeding that of seawater, did not operate active HCO3− uptake but used it via the extracellular CA for its photosynthetic carbon fixation.

Use of light and inorganic carbon acquisition by two morphotypes of Zostera noltii Hornem

The affinity for dissolved inorganic carbon (DIC) and the mechanisms to use HCO 3 − as a source of DIC for photosynthesis were investigated in two morphotypes of Zostera noltii Hornem. Both morphotypes were collected at Ria Formosa lagoon (Southern Portugal) at two different levels in the intertidal. Affinity for DIC at saturating photon fluence rate (PFR), estimated as photosynthetic conductance for CO 2 ( g p(CO 2) ), was reduced by 75% in the Z. noltii plants adapted to shade conditions (lower intertidal) in comparison to the sun morphotype (45×10 −6 and 182×10 −6 m s −1, respectively), indicating that the plants acclimated to sun conditions (higher intertidal) had a higher capacity to use HCO 3 − as DIC source for photosynthesis. Since external carbonic anhydrase activity was negligible and a large inhibitory effect was produced by Tris buffer addition, this HCO 3 − use was attributed to the operation of H + ATPases creating low pH zones in periplasmic space. The photosynthetic CO 2-flux supported for this mechanism was calculated to be 53 μmol O 2 m −2 s −1 in sun morphotype, about 80% out of maximum photosynthesis rate. In order to determine the possible photosynthetic energy cost of the HCO 3 − use, the effect of decreasing light on photosynthetic rates and g p(CO 2) was estimated. Photosynthetic conductance decreased in both morphotypes at non-saturating PFR. This dependence of g p(CO 2) on PFR indicated the existence of a positive interactive effect between DIC and PFR which was more pronounced in the shade morphotype since the ascending slope of O 2 evolution vs. PFR curves at limiting PFRs was reduced from 7.2 to 2.3 mmol O 2 mol photon −1 at 4 and 0.5 mol m −3 of DIC, respectively.

Inorganic carbon acquisition in two species of marine prymnesiophytes

Inorganic carbon acquisition has been investigated in the marine prymnesiophytes Dicrateria inornata and Ochrosphaera neapolitana. The presence of external and internal carbonic anhydrase (CA) was detected in air-grown cells of D. inornata by potentiometric assay and corroborated by mass spectrometry. Growth on 3% CO2 repressed external CA activity. Extracts of air-grown cells of O. neapolitana showed considerable internal CA activity, but no external CA activity was detected by mass spectrometry. Illumination of air-grown cells of D. inornata incubated with 100 μM H13CO3 and treated with the CA inhibitor acetazolamide caused a rapid drop in the extracellular CO2 concentration to a level below its equilibrium concentration indicating that cells were actively taking up CO2 from the medium. The rate of O2 evolution at the CO: compensation concentration was higher than could be supported by the uncatalysed formation of CO2 from HCO3, . demonstrating that the cells were taking up HCO3 −directly. Similar results were obtained with air-grown cells of O. neapolitana. Active CO2 transport in both species was not repressed by growth under high levels of CO2. The photosynthetic electron transport inhibitor 3-(3′ 4′-dichlorophenyl)-l, 1-dimethylurea (DCMU) inhibited CO2 uptake in air-grown cells of both species, demonstrating that the energy to support active CO2 transport was derived from photosynthetic linear electron transport. These results show that the physiological characteristics of the CO2 concentrating mechanism in related species do not appear to be correlated with the availability of CO2 in the surrounding medium.

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The affinity for dissolved inorganic carbon (DIC) and the mechanisms to use HCO3− as a source of DIC for photosynthesis were investigated in two morphotypes of Zostera noltii Hornem. Both morphotypes were collected at Ria Formosa lagoon (Southern Portugal) at two different levels in the intertidal. Affinity for DIC at saturating photon fluence rate (PFR), estimated as photosynthetic conductance for CO2 (gp(CO2)), was reduced by 75% in the Z. noltii plants adapted to shade conditions (lower intertidal) in comparison to the sun morphotype (45×10−6 and 182×10−6 m s−1, respectively), indicating that the plants acclimated to sun conditions (higher intertidal) had a higher capacity to use HCO3− as DIC source for photosynthesis. Since external carbonic anhydrase activity was negligible and a large inhibitory effect was produced by Tris buffer addition, this HCO3− use was attributed to the operation of H+ ATPases creating low pH zones in periplasmic space. The photosynthetic CO2-flux supported for this mechanism was calculated to be 53 μmol O2 m−2 s−1 in sun morphotype, about 80% out of maximum photosynthesis rate. In order to determine the possible photosynthetic energy cost of the HCO3− use, the effect of decreasing light on photosynthetic rates and gp(CO2) was estimated. Photosynthetic conductance decreased in both morphotypes at non-saturating PFR. This dependence of gp(CO2) on PFR indicated the existence of a positive interactive effect between DIC and PFR which was more pronounced in the shade morphotype since the ascending slope of O2 evolution vs. PFR curves at limiting PFRs was reduced from 7.2 to 2.3 mmol O2 mol photon−1 at 4 and 0.5 mol m−3 of DIC, respectively.

THE INVOLVEMENT OF A PLASMA MEMBRANE H+‐ATPase IN THE BLUE‐LIGHT ENHANCEMENT OF PHOTOSYNTHESIS IN LAMINARIA DIGITATA (PHAEOPHYTA)1

Many brown algae, including the kelp Laminaria digitata (Huds.) Lamour., exhibit enhanced photosynthesis when they are given a small amount of blue‐light in addition to a background of saturating red light. This blue light effect is correlated with an increased uptake of carbon. In the present study, we tested the hypothesis that blue light acts by increasing the activity of a plasma membrane H+‐ATPase, thereby promoting an active carbon uptake across the plasma membrane. Photosynthetic carbon uptake was studied in pH‐drift experiments under illumination with red and blue light and using different inhibitors. Vanadate, an inhibitor of plasma membrane H+‐ATPases, had a minor inhibitory effect on carbon uptake rates under saturating red light conditions, but inhibited the blue‐light enhancement by approximately 60%. An inhibitor of external carbonic anhydrase, acetazolamide, decreased the carbon uptake in both red light and in red plus blue light by 48% and 68%, respectively. These results suggest that photosynthetic carbon uptake depends on an external carbonic anhydrase under both red and red plus blue light conditions, and that blue light induces an increased activity of a P‐type H+‐ATPase in the plasma membrane. The proton buffer Tris, which has a buffering capacity similar to vanadate in seawater, had no inhibitory effect on carbon uptake rates neither in red light nor in red plus blue light, showing that the inhibitory effect of vanadate is not caused by its effect as a buffer. The blue‐light enhancement was also abolished by a protein kinase inhibitor (H‐7), suggesting that the transduction of the blue‐light signal involves a protein kinase, which activates the plasma membrane H+‐ATPase by phosphorylation.

Acquisition and use of bicarbonate by Emiliania huxleyi.

• Bicarbonate acquisition mechanisms and the kinetics of dissolved inorganic carbon (DIC) use in photosynthesis and calcification were investigated in Emiliania huxleyi. • Photosynthesis was measured using O2 evolution and 14 C incorporation and calcification was measured with 14 C. Noncalcifying (coccolith-free) cells were produced from calcifying (coccolith-bearing) cells of the same strain of E. huxleyi, so that photosynthesis could be monitored independently from calcification. • Neither photosynthesis nor calcification was saturated at the ambient DIC concentration of seawater. In coccolith-bearing cells, both processes showed biphasic kinetics with DIC concentration, with a hiatus located at 1mM. The same biphasic pattern and similar rates of photosynthesis were found in the coccolith-free cells. Inhibitor experiments showed that E. huxleyi acquires bicarbonate mainly by an anion exchange protein, but external carbonic anhydrase can be activated at low concentrations of DIC. • We conclude that the biphasic kinetics of photosynthesis and calcification are caused by the presence of two bicarbonate acquisition mechanisms and also, since calcification does not enhance photosynthesis in this coccolithophore, we question the current view that the two processes are tightly coupled.

ACQUISITION OF INORGANIC CARBON BY THE MARINE HAPTOPHYTE ISOCHRYSIS GALBANA (PRYMNESIOPHYCEAE)1

Inorganic carbon acquisition has been investigated in the marine haptophyte Isochrysis galbana. External carbonic anhydrase (CA) was present in air‐grown (0.034% CO2) cells but completely repressed in high (3%) CO2‐grown cells. External CA was not inhibited by 1.0 mM acetazolamide. The capacity of cells to take up bicarbonate was examined by comparing the rate of photosynthetic O2 evolution with the calculated rate of spontaneous CO2 supply; at pH 8.2 the rates of O2 evolution exceeded the CO2 supply rate 14‐fold, indicating that this alga was able to take up HCO3−. Monitoring CO2 concentrations by mass spectrometry showed that suspensions of high CO2‐grown cells caused a rapid drop in the extracellular CO2 in the light and addition of bovine CA raised the CO2 concentration by restoring the HCO3−‐CO2 equilibrium, indicating that cells were maintaining the CO2 in the medium below its equilibrium value during photosynthesis. A rapid increase in extracellular CO2 concentration occurred on darkening the cells, indicating that the cells had accumulated an internal pool of unfixed inorganic carbon. Active CO2 uptake was blocked by the photosynthetic electron transport inhibitor 3‐(3′,4′‐dichlorphenyl)‐1,1‐dimethylurea, indicating that CO2 transport was supported by photosynthetic reactions. These results demonstrate that this species has the capacity to take up HCO3− and CO2 actively as sources of substrate for photosynthesis and that inorganic carbon transport is not repressed by growth on high CO2, although external CA expression is regulated by CO2 concentration.

Sources of inorganic carbon for phytoplankton in the eastern Subtropical and Equatorial Pacific Ocean

We present the results of a field study examining inorganic carbon utilization by phytoplankton assemblages in the eastern Subtropical and Equatorial Pacific Ocean. Data from isotope disequilibrium experiments demonstrate that HCO3- was the principal form of inorganic carbon taken up by all of the in situ phytoplankton populations we sampled. In a cyanobacteria‐dominated assemblage, HCO3- uptake occurred chiefly through a direct transmembrane transport mechanism. Diatom‐dominated assemblages expressed extracellular carbonic anhydrase and transported CO2 derived from the catalyzed dehydration of HCO3-. Direct HCO3- transport by the diatoms may have also occurred. In a 3‐d incubation experiment, we observed the CO2‐dependent regulation of inorganic C uptake in diatom‐dominated phytoplankton assemblages. Phytoplankton assemblages grown at 150 ppm CO2 possessed external carbonic anhydrase activity and took up HCO3- following its dehydration to CO2. In contrast, the assemblages cultured with 750 ppm CO2 appeared to lack external carbonic anhydrase activity and rely solely on CO2 as an exogenous source of carbon for photosynthesis. The CO2 effect on inorganic C utilization occurred in the absence of a detectable difference in phytoplankton growth rates between the 150 and 750 ppm CO2 treatments. Our field data provide compelling evidence that HCO3- utilization is prevalent in natural marine phytoplankton communities and is regulated by ambient CO2 concentrations. We discuss the ecological and biogeochemical implications of these results.

Dissolved inorganic carbon concentration mechanism in Chlamydomonas moewusii

The dissolved inorganic carbon concentrating mechanism(s) of Chlamydomonas moewusii CC 55 was compared with C. reinhardtii strain 137. C. moewusii is similar to C. reinhardtii with respect to maximal rates of photosynthetic oxygen evolution, CO 2 fixation, respiration, and the ability to efficiently concentrate inorganic carbon. C. moewusii has a low, but measurable amount of external carbonic anhydrase (CA) that was not inhibited by acetazolamide (AZ), an inhibitor of periplasmic carbonic anhydrase (pCA) in C. reinhardtii. The K 0.5(CO 2) for air-grown C. moewusii is about 1 μM and the algal cells accumulated dissolved inorganic carbon (DIC) to a level of about 1 mM in 60 s. AZ did not inhibit CO 2 fixation and the DIC accumulation by air-grown cells of C. moewusii. The K 0.5(CO 2) for both species remains constant from pH 6.5 to 9.5 while K 0.5(HCO 3 -) increased logarithmically, which indicates that CO 2 is the apparent inorganic carbon species that enters the cells in both algae. Antiserum prepared against the 37 kDa peptide of pCA from C. reinhardtii was immunoreactive with polypeptides of 26, 28, and 32 kDa in C. moewusii. The periplasmic carbonic anhydrase (pCA) activity is a part of the dissolved inorganic carbon concentrating mechanism in C. reinhardtii, but C moewusii accomplished inorganic carbon accumulation without an AZ-sensitive pCA.

Inorganic carbon acquisition and its energization in eustigmatophyte algae.

The eustigmatophyceans are primitive unicellular algae that represent the most basal group of ochrophytes. They are believed to be obligate photoautotrophs, occurring mainly in freshwater and soil but with some marine representatives. The freshwater eustigmatophytes Monodus subterraneus and Vischeria stellata, and the marine eustigmatophyte Nannochloropsis gaditana, have been studied by mass spectrometry with respect to their characteristics for inorganic carbon (Ci) uptake. A CO2 concentrating mechanism was found in all three, but an external carbonic anhydrase (CA) was not detected. The acquisition of Ci from the external medium was based on the active transport of HCO3-, CO2, or both. In particular, N. gaditana was able to use HCO3- exclusively as an exogenous carbon source for photosynthesis, with this HCO3- being subsequently converted to CO2 by an intracellular CA for photosynthetic fixation. A unique characteristic of this species was its capacity to transport HCO3- during prolonged periods of time in the dark. In contrast, M. subterraneus utilized CO2 alone through an active transport process, whereas V. stellataexhibited the capacity to transport both HCO3- and CO2. The uptake of CO2 also continued in the dark in V. stellatacells. Regardless of the Ci species taken up, transport was abolished by anoxia and by inhibitors of mitochondrial respiration. These results indicate that that the supply of Ci for photosynthetic CO2 fixation is partly dependent upon mitochondrial activity in these primitive eukaryotes.

Dinoflagellate symbioses: strategies and adaptations for the acquisition and fixation of inorganic carbon.

Dinoflagellates exist in symbiosis with a number of marine invertebrates including giant clams, which are the largest of these symbiotic organisms. The dinoflagellates (Symbiodinium sp.) live intercellularly within tubules in the mantle of the host clam. The transport of inorganic carbon (Ci) from seawater to Symbiodinium (=zooxanthellae) is an essential function of hosts that derive the majority of their respiratory energy from the photosynthate exported by the zooxanthellae. Immunolocalisation studies show that the host has adapted its physiology to acquire, rather than remove CO2, from the haemolymph and clam tissues. Two carbonic anhydrase (CA) isoforms (32 and 70 kDa) play an essential part in this process. These have been localised to the mantle and gill tissues where they catalyse the interconversion of HCO3- to CO2, which then diffuses into the host tissues. The zooxanthellae exhibit a number of strategies to maximise Ci acquisition and utilisation. This is necessary as they express a form II Rubisco that has poor discrimination between CO2 and O2. Evidence is presented for a carbon concentrating mechanism (CCM) to overcome this disadvantage. The CCM incorporates the presence of a light-activated CA activity, a capacity to take up both HCO3-and CO2, an ability to accumulate an elevated concentration of Ci within the algal cell, and localisation of Rubisco to the pyrenoid. These algae also express both external and intracellular CAs, with the intracellular isoforms being localised to the thylakoid lumen and pyrenoid. These results have been incorporated into a model that explains the transport of Ci from seawater through the clam to the zooxanthellae.

Analysis of Chlamydomonas mutants with abnormal expression of CO2 and HCO3- uptake systems.

Eukaryotic microalgae such as Chlamydomonas reinhardtii possess an inducible CO2 concentrating mechanism that operates as a very close interaction between pyrenoid-based Rubisco, various carbonic anhydrases (CAs), and inorganic carbon (Ci) transport systems. While external and internal CAs have been characterised to the molecular level, the biochemistry and molecular biology of Ci uptake mechanisms have not been elucidated. Both Ci species, CO2 and HCO3-, are taken up by the cells and chloroplasts during steady-state photosynthesis. After acclimation to limiting Ci, CO2 and HCO3- transport, measured in whole cells or chloroplasts, change their kinetic characteristics from a constitutive low-affinity state to an inducible high-affinity state. In order to learn more about the genes involved in the signal transduction pathway and in the Ci transport systems, we performed insertional mutagenesis using the arg7 gene as a selectable marker. Application of aqueous membrane inlet mass spectrometry allowed discrimination between CO2 and HCO3- uptake. Data is presented on two mutants, M46 and M21, which show severe damage to the constitutive Ci uptake systems and which are unable to induce a high-affinity state. The mutations might be either in the signal transduction pathway or in the transporters themselves. In addition, we present data that shows a very close connection between high-affinity HCO3- uptake and high-affinity NO3- uptake in cells of C. reinhardtii.

The diversity of inorganic carbon acquisition mechanisms in eukaryotic microalgae.

Eukaryotic microalgae have developed CO2concentrating mechanisms to maximise the concentration of CO2 at the active site of Rubisco in response to the low CO2 concentrations in the external aquatic medium. In these organisms, the modes of inorganic carbon (Ci) uptake are diverse, ranging from diffusive CO2 uptake to the active transport of HCO3 -and CO2 and many have an external carbonic anhydrase to facilitate HCO3- use. There is unequivocal evidence for the mechanisms of Ci uptake in only about 25 species of microalgae of the chlorophyte, haptophyte, rhodophyte, diatom, and eustigmatophyte groups. Most of these species take up both CO2 and HCO3-, but the rates of uptake of each of these substrates varies with the algal species. A few species take up only one of the two forms of Ci, an adaptation that is not necessarily correlated with their ecological distribution. Evidence is presented for the active uptake of HCO3- and CO2 in two marine haptophytes,Isochrysis galbana Parke and Dicrateria inornata Parke, and for active transport of CO2 but lack of HCO3- uptake in two marine dinoflagellates, Amphidinium carteraeHulburt and Heterocapsa oceanica Stein.

Evidence for active CO2 uptake by a CO2-ATPase in the acidophilic green alga Eremosphaera viridis

We examined the mechanism(s) responsible for active uptake of dissolved inorganic carbon (DIC) during photosynthesis in the green alga Eremosphaera viridis De Bary. O2 electrode measurements of algal oxygen evolution and CO2 fluxes as a function of DIC availability indicate that E. viridis actively imports only CO2 during photosynthesis, and does not possess external carbonic anhydrase (CA). The K0.5[CO2] was 14.2 and 10.1 µM at pH 5.0 and 8.0, respectively. Both membrane potential and cellular resistance were measured under controlled conditions of [CO2] at either 2 or 15 µM. Active CO2 uptake was electrically silent, suggesting that CO2 uptake might be mediated by a CO2-ATPase. Comparison of ATPase activity in microsomal preparations at low (0 µM) and high (15 µM) [CO2] indicated a 1.25-fold increase in ATP hydrolysis in high [CO2]. The CO2-ATPase activity was inhibited by the broad-acting inhibitors diethylstilbestrol (DES) and N',N'-dicyclohexylcarbodiimide (DCCD) but unaffected by vanadate, fluoride, and nitrate. The K0.5[CO2] of the ATPase activity was 22.5 µM, a value very similar to the K0.5[CO2] for CO2 uptake by whole algal cells. These results suggest the existence of a CO2-ATPase as the major importer of DIC for photosynthesis in the microalga E. viridis.Key words: chlorophyte, CO2 transport, CO2-ATPase, photosynthesis, electrical potential, mass spectrometry.

The inhibition of the carbon concentrating mechanism of the green alga Chlorella saccharophila by acetazolamide.

The effects of the carbonic anhydrase (CA) inhibitors acetazolamide (AZ) and dextran-bound sulfonamide (DBS) on HCO3--dependent O2 evolution in Chlorella saccharophila were evaluated. Addition of 4 µM AZ or 0.4 mg ml-1 DBS to photosynthesizing cells reduced the O2 evolution rate at low dissolved inorganic carbon (DIC) concentration, decreased the size of the intracellular acid-labile carbon pool, and decreased the apparent affinity of the cells for DIC. Measurement of the whole-cell affinity of cells for CO2 and HCO3- in the presence and absence of inhibitors indicated that active HCO3- transport was inhibited by AZ and DBS. The inhibition of HCO3- transport was independent of the inhibition of external and internal CA. These results suggest that the active uptake of HCO3- occurs initially by the interaction of HCO3- and a CA-like transporter.

Photosynthesis might be limited by light, not inorganic carbon availability, in three intertidal Gelidiales species.

• The interaction between incident photon fluence density (PFD) and dissolved inorganic carbon (DIC) availability on photosynthesis is reported for three species of Gelidiales (Rhodophyta) from the Canary Islands. • Photosynthetic O2 evolution in response to both DIC concentration and PFD, and external carbonic anhydrase activity were measured in thalli of Gelidium canariensis, Gelidium arbuscula and Pterocladiella capillacea maintained in a controlled-environment room. • No detectable external carbonic anhydrase activity, high sensitivity to alkaline pH and moderate values of photosynthetic conductance for DIC indicated that the three species had a low capacity for using the external pool of HCO3 - ; therefore photosynthetic rates were dependent on CO2 availability. The seawater concentration of DIC was insufficient to saturate photosynthesis at high PFD; photosynthesis vs PFD was not affected by DIC concentration at 0.07-0.04mol O2 mol-1 photon. ANOVA revealed that incident PFD had a greater effect than DIC availability on photosynthesis rates in G. canariensis. • Photosynthesis is probably limited by incident light rather than DIC concentration despite the apparent low affinity for HCO3 - in G. canariensis, G. arbuscula and P. capillacea in their natural habitats.