[Show abstract][Hide abstract] ABSTRACT: A range of marine photosynthetic picoeukaryote phytoplankton species grown in culture were screened for the presence of extracellular carbonic anhydrase (CAext), a key enzyme in inorganic carbon acquisition under carbon- limiting conditions in some larger marine phytoplankton species. Of the species tested, extracellular carbonic anhydrase was detected only in Micromonas pusilla Butcher. The rapid, light-dependent development of CAext when cells were transferred from carbon-replete to carbon-limiting conditions was regulated by the available free- CO2 concentration and not by total dissolved inorganic carbon. Kinetic studies provided support for a CO2- concentrating mechanism in that the K0.5[CO2] (i.e. the CO2 concentration required for the half-maximal rate of photosynthesis) was substantially lower than the Km[CO2] of Rubisco from related taxa, whilst the intracellular carbon pool was at least seven fold greater than the extracellular DIC concentration, for extracellular DIC values 1.0 mm.
It is proposed that when the flux of CO2 into the cell is insufficient to support the photosynthetic rate at an optimum photon irradiance, the development of CAext increases the availability of CO2 at the plasma membrane. This ensures rapid acclimation to environmental change and provides an explanation for the central role of M. pusilla as a carbon sink in oligotrophic environments.
[Show abstract][Hide abstract] ABSTRACT: The relationships among inorganic carbon transport, bicarbonate availability, intracellular pH, and culture age were investigated in high-calcifying cultures of Emiliania huxleyi (Lohmann) Hay & Mohler. Measurement of inorganic carbon transport by the silicone-oil centrifugation technique demonstrated that gadolinium, a potential Ca2+ channel inhibitor, blocked intracellular inorganic carbon uptake and photosynthetic 14CO2+ fixation in exponential-phase cells. In stationary-phase cells, the intracellular inorganic carbon concentration was unaffected by gadolinium. Gadolinium was also used to investigate the link between bicarbonate and Ca2+ transport in high-calcifying cells of E. huxleyi. Bicarbonate availability had significant and rapid effects on pHi in exponential- but not in stationary-phase cells. 4′, 4′-Diisothiocyanostilbene-2,2′-disulfonic acid did not block bicarbonate uptake from the external medium by exponential-phase cells. Inorganic carbon utilization by exponential- and stationary-phase cells of Emiliania huxleyi was investigated using a pH drift technique in a closed system. Light-dependent alkalization of the medium by stationary-phase cells resulted in a final pH of 10.1 and was inhibited by dextran-bound sulphonamide, an inhibitor of external carbonic anhydrase. Exponential-phase cells did not generate a pH drift. Overall, the results suggest that for high-calcifying cultures of E. huxleyi the predominant pathway of inorganic carbon utilization differs in exponential and stationary phase cells of the same culture.
[Show abstract][Hide abstract] ABSTRACT: The contribution of bicarbonate to total dissolved inorganic carbon (DIC) utilization was investigated using 18 marine phytoplankton species, including members of Bacillariophyceae, Dinophyceae, Prymnesiophyceae, and Raphidophyceae, under carbon-replete or -limited conditions. Extracellular carbonic anhydrase (CA) was assayed as an indicator of extracellular CA-catalyzed HCO−3 utilization. For some species, extracellular CA was constitutive, in others activity was detected under conditions of carbon limitation, and in others, even under carbon-limited conditions, activity was not detected. In species without extracellular CA, direct HCO−3 uptake was investigated using a pH drift technique in a closed system, DIC measurements, and the use of the anion exchange inhibitor 4′4′-diisothiocyanatostilbene-2,2-disulfonic acid (DLDS). Three of these species (Chaetoceros compressus, Thalassiosira pseudonana, and Glenodinium foliaceum) gave a pH drift not inhibited by DIDS, but cultures of Chrysochromulina kappa, Gephrocapsa oceanica, and Coccolithus pelagicus, in which DLDS inhibited DIC uptake, did not give a pH drift. This result shows that direct HCO3− transport may occur by an anion exchange-type mechanism in some species but not others. Of the eighteen species investigated, only Heterosigma akashiwo did not have the potential for direct uptake or extracellular CA-catalyzed HCO−3 utilization.
[Show abstract][Hide abstract] ABSTRACT: The relationship between photosynthesis and calcification was investigated in a high-calcifying strain of Emiliania huxleyi. At pH 8.3 and a photon flux density of 50 μmol m−2 s−1 calcification and photosynthetic 14CO2 fixation were carbon saturated at 1 mM DIC (dissolved inorganic carbon) but at a photon flux density of 300 μmol m−2 s−1 calcification and photosynthetic 14CO2 fixation were not saturated at the DIC concentration of sea-water, 2 mM. When HCO3− provides the bulk of inorganic carbon the stoichiometry between photosynthetic 14CO2 fixation and calcification was 1:1. In the high-calcifying strain of E. huxleyi the stoichiometry between photosynthetic 14CO2 fixation and O2 evolution was 2:1 but in a low calcifying strain the stoichiometry was 1:1. High nitrate concentrations, i.e. 1000 μM were required to inhibit calcification. The optimum pH for calcification and photosynthetic 14CO2 fixation was 7.8. From the results a model is proposed in which a molecule of HCO3− is the precursor of calcite in the coccolith vesicle with the extrusion of H+ into the cytosol, while another HCO3− provides CO2 in the chloroplast with the extrusion of OH−. The interaction of these processes maintains cytoplasmic pH near neutrality.
[Show abstract][Hide abstract] ABSTRACT: ABSTRACTHCO3− utilization by the marine microalga Nannochloropsis oculata was investigated using a pH drift technique in a closed system. Light-dependent alkalization of the medium resulted in a final pH of 10.5, confirming substantial HCO3− use by this alga. Alkalinity remained constant throughout the pH drift. Measurement of dissolved inorganic carbon (DIC) or the uptake of H14CO3− showed that nearly 50% of the total DIC remained external to the plasma membrane on completion of a pH drift. The rate of light-driven alkalization was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and thus was dependent on photosynthesis. Light-driven alkalization was not inhibited by a membrane-impermeable inhibitor of carbonic anhydrase (CA), dcxtran-bound sulphonamide (DBS), indicating that external CA was not involved in HCO3− utilization. The anion-cxchangc inhibitor 4′,4′-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) completely inhibited light-driven alkalization of the medium and H14CO3− uptake, providing unequivocal support for a direct uptake of H14CO3−. Chloride ions were essential for DIC-dependent photosynthetic oxygen evolution, suggesting that bicarbonate transport occurs by HCO3−/CI− exchange.
[Show abstract][Hide abstract] ABSTRACT: The relationships between extra- and intra-cellular carbonic anhydrase, calcification rate, utilization of dissolved inorganic carbon (DIG) and culture age were investigated in a high-calcifying strain of Emiliania huxleyi. The detection of carbonic anhydrasc was dependent on culture age; neither the extra-cellular activity of intact cells nor activity in crude homogenates was detected until the stationary phase. By the stationary phase DIC in the medium was totally depleted and the calcification rate had decreased by 60°0. Extra- and intra-cellular carbonic anhydrase was stimulated by Na+but not Cl ions. The isolation of intact organelles by isopycnic gradient centrifugal ion from exponential and stationary phase cultures of high calcifying cells showed high carbonic anhydrase activity in the chloroplast fraction but carbonic anhydrase was not detected in low-calcifying cells even after gradient centrifugation. Although 50/IM ethoxyzolamide was an effective inhibitor of carbonic anhydrase activity in vitro, photosynthetic 14CO2 fixation was only inhibited 30%, calcification rate 10%, and the internal inorganic carbon pool unaffected in intact cells. It is proposed that chloroplast carbonic anhydrase maintains the steady state flux of COj, from the chloroplast envelope to Rubisco, much of the CO2 arising by release from HCO3−in the cytosol, required to maintain cytoplasmic pH near neutrality in high-calcifying cells.
[Show abstract][Hide abstract] ABSTRACT: The relationship between inorganic-carbon dependent photosynthetic oxygen evolution and calcification was investigated in high- and low-calcifying strains of Emiliania huxleyi showing a ten-fold difference in calcification rate. Unlike the low-calcifying strain calcifying cultures showed a four-fold increase in inorganic carbon (1 mM) dependent photosynthetic oxygen over the pH range 5-8.3 resulting in a 20 fold difference in photosynthetic rate between the two strains at pH 8.3. Calcifying cells have a high affinity for HCO3−, the concentration of dissolved inorganic carbon [DIC] required for half-maximal rate of photosynthetic O2 evolution (K0.5[DIC]) being 200 μM at pH 8-3. In mid-exponential phase cultures the stoichiometry between 14CO2 fixation and calcification was 1 : 1 so it is likely that the high photosynthetic rate at pH 8.3 is sustained by 14CO2, released from H 14CO2 during calcification.
Measurement of bicarbonate transport by the silicone-oil-layer centrifugal filtering technique demonstrated a rapid uptake and achievement of equilibrium (less than 3 s) between the intracellular and external inorganic carbon concentrations in low and high-calcifying cells. Subsequent metabolism of the 14C intracellular inorganic carbon pool did not occur in low-calcifying cells suggesting the block in calcification occurs either in transport into or within the coccolith vesicle.
[Show abstract][Hide abstract] ABSTRACT: In the marine diatom Skeletonema costatum, carbonic anhydrase activity exterior to the plasma membrane (CAext) 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 CAext 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 CAext 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 CAext.
[Show abstract][Hide abstract] ABSTRACT: In the marine phytoplankton species tested, a possible link between exofacial ferricyanide reduction at the plasma membrane of intact cells, inorganic carbon status of the cells, and extracellular carbonic anhydrase (CA) activity is proposed. In species with no extracellular CA activity under carbon-limited or carbon-replete conditions, barely detectable ferricyanide reduction was observed. Species in which extracellular CA was only detected under carbon-limited conditions showed high rates of exofacial ferricyanide reduction, as shown previously for Skeletonema costatum. Immunological analysis has demonstrated that the CA protein was present in both carbon-limited and carbon-replete cells, even though the CA activity could only be detected when inorganic carbon was limiting. Incubation of the inactive extracellular CA protein from carbon-replete cells with DTT caused activation of the enzyme. It is proposed that CA limitation in the light promotes proton extrusion and increased plasma membrane redox activity, which result in the protonation and activation of the extracellular CA.
[Show abstract][Hide abstract] ABSTRACT: This study investigated inorganic carbon accumulation in relation to photosynthesis in the marine dinoflagellate Prorocentrum micans. Measurement of the internal inorganic carbon pool showed a 10-fold accumulation in relation to external dissolved inorganic carbon (DIC). Dextran-bound sulfonamide (DBS), which inhibited extracellular carbonic anhydrase, caused more than 95% inhibition of DIC accumulation and photosynthesis. We used real-time imaging of living cells with confocal laser scanning microscopy and a fluorescent pH indicator dye to measure transient pH changes in relation to inorganic carbon availability. When steady-state photosynthesizing cells were DIC limited, the chloroplast pH decreased from 8.3 to 6.9 and cytosolic pH decreased from 7.7 to 7.1. Re-addition of HCO3- led to a rapid re-establishment of the steady-state pH values abolished by DBS. The addition of DBS to photosynthesizing cells under steady-state conditions resulted in a transient increase in intracellular pH, with photosynthesis maintained for 6 s, the amount of time needed for depletion of the intracellular inorganic carbon pool. These results demonstrate the key role of extracellular carbonic anhydrase in facilitating the availability of CO2 at the exofacial surface of the plasma membrane necessary to maintain the photosynthetic rate. The need for a CO2-concentrating mechanism at ambient CO2 concentrations may reflect the difference in the specificity factor of ribulose-1,5 bisphosphate carboxylase/oxygenase in dinoflagellates compared with other algal phyla.
[Show abstract][Hide abstract] ABSTRACT: Calcification in coccolithophorids requires major intracellular fluxes of inorganic carbon and calcium. This paper summarises the major cellular fluxes of substrates and products of calcification described in a simple four compartment model (cytosol, Golgi, coccolith vesicle and chloroplast). Measurements of the cytosolic and intra-coccolith vesicle pH and electrical potentials across the plasma membrane and coccolith vesicle membrane allow calculations of the proton electrochemical gradients across these membranes and estimates of the free carbonate and calcium concentrations in the coccolith vesicle. Calcification may provide a relatively low cost route for elevating the concentration of carbon dioxide in the chloroplast. This may have benefits in terms of the nutrient requirements for photosynthesis and growth. In particular, a close relationship appears to exist between calcification and the availability of phosphorus which may correlate with the occurrence of large scale blooms of Emiliania huxleyi in the North Atlantic.
Journal of Marine Systems 10/1996; 9(1-9):45-56. DOI:10.1016/0924-7963(96)00015-2 · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: summaryThe development of an inorganic carbon concentrating mechanism (CCM) in relation to the expression of extracellular carbonic anhydrase (CAext) in cells from the stationary phase of Emiliania huxleyi cultures was investigated. Unlike exponential phase cells, those in the stationary phase showed a high affinity for CO2: the K0.5[CO2] (i.e. the CO2 concentration required for half-maximal rate of photosynthesis) was 1.6μM at pH 8.3. Measurement of the internal inorganic carbon concentration by the silicone oil centrifugation technique showed that, with 1 mM external dissolved inorganic carbon (DIC), the intracellular inorganic carbon concentration was sixfold greater at pH 7.5 and twofold greater at pH 8.3, than the external concentration. The potent CAext inhibitor, dextran-bound sulphonamide (DBS), gave an 80% inhibition of DIC-dependent photosynthetic oxygen evolution. Similarly, DBS decreased the intracellular DIC concentration and inhibited photosynthetic 14CO2 fixation. The lipid-permeable CA inhibitor, ethoxyzolamide (EZ), increased the intracellular DIC concentration but inhibited photosynthetic 14CO2 fixation. The observed intracellular DIC concentrations indicated that the stationary phase cells have the capacity to transport CO2 actively against a concentration gradient.
New Phytologist 07/1996; 133(3):383-389. DOI:10.1111/j.1469-8137.1996.tb01905.x · 7.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Growth of a high-calcifying strain of Emiliania huxleyi (Lohmann) Hay & Mohler was investigated in cultures aerated with varying concentrations of CO2 in air and compared with growth in 0.03 % (V/V) CO2. Cultures aerated with 0.1 % (V/V) CO2/air under identical conditions resulted in approximately 40 % reduction in cell number and final cell yield. A concentration of 0.5 % (v/v) CO2 completely inhibited growth. In the virtual absence of CO2, cells could grow to the same levels as in those cultures aerated with air-equilibrated levels Of CO2, i.e. 0.03 % (V/V) CO2. Measurement of internal pH (pH(i)) gave comparable results using either the 5,5-dimethyl-2[C-14]oxazalidine-2,4-dione (DMO) method or a fluorescent probe technique (BCECF-AM). At external pH 8.3, intracellular pH of cells aerated with air was 6.9 whilst pH(i) of cells aerated with 0.1 % (V/V) CO2 was 6.4. Lowering external pH decreased growth rate for cultures aerated with 0.03 % (V/V) CO2. A 30 % reduction in cell number and final cell yield occurred at pH 7.8 increasing to almost 60 % at pH 7.0; pH(i) decreased at more acidic external pH down to 6.38 at pH 7.0. Carbon dioxide concentration and external pH appear to be equally important in the growth of high-calcifying cells. The significance of these results is considered in relation to the development of mesoscale blooms of E. huxleyi.
[Show abstract][Hide abstract] ABSTRACT: Ethanolic dehydration (20% to 70%) of the thylakoid membranes of Chlorogloeopsis fritschii resulted in an 8% to 58% loss of glutamine synthetase activity. In Chlorella pyrenoidosa, hydroxypyruvate reductase and fumarase, marker enzymes of the peroxisomes and mitochondria, respectively, diffused from the organelles on dehydration.
World Journal of Microbiology and Biotechnology 03/1994; 10(2):187-190. DOI:10.1007/BF00360884 · 1.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High-calcifying cells of Emiliania huxleyi were grown on a synthetic seawater medium and the effect of nitrate (NO- 3) concentration on growth, calcite accumulation, calcification rate and DIC (dissolved inorganic carbon) utilisation determined. The stoichiometry between NO- 3 utilisation and calcite production was 1:6·5 (mol/mol). Calcification and growth were tightly coupled: calcite production ceased when cultures entered the stationary phase due to NO- 3 depletion, but by adding a pulse of NO- 3 growth and calcification were restored. The initial C/N ratio in the medium was important in relation to calcification rate. At 20 µM NO- 3 the total DIC (2 mM) was rapidly depleted, the calcification rate subsequently declining, whereas at 5 and 10 µM NO- 3 rates of calcification were constant at 20 g carbon cell-1 × 1014·h-1 throughout culture growth, excess DIC being present relative to the available NO- 3. Calcite production per unit NO- 3 was similar for isolates of E. huxleyi from neritic, oligotrophic and nitrate-rich waters. In laboratory cultures, where the photon flux density is optimised for growth, the initial NO- 3 concentration is a reliable indicator of final calcite yield.
European Journal of Phycology 11/1993; 28(4-4):243-246. DOI:10.1080/09670269300650351 · 1.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The relationship between calcite production and dissolved inorganic carbon (DIC) utilization was investigated using high- and low-calcifying strains of Emiliania huxleyi aerated with either air (0.03 % v/v CO2) or CO2-free air. With cultures grown on air, calcite production was eight-fold greater in the high-calcifying culture, but growth rates for high- and low-calcifying cultures were similar. Growth and calcite production were accompanied by a concomitant decrease in DIC and free CO2 in the high-calcifying culture, showing that HCO3− provides inorganic carbon for calcite synthesis. In low-calcifying cultures DIC and free CO2 were relatively constant, confirming that cells acquire inorganic carbon mainly by the diffusive entry of free CO2.
When cultures were aerated with CO2-free air the free CO2 concentration was below the K0.5[CO2] for high- and low-calcifying cells and the low-calcifying cells were unable to grow. Growth of high-calcifying cells was observed, and the calcite yield was little changed from cultures gassed with air (0.03 % v/v CO2). At these minimal CO2concentrations HCO3− may provide inorganic carbon for calcite synthesis and CO2 for photosynthesis, allowing growth to occur. Calcite synthesis by E. huxleyi decreases DIC and carbonate alkalinity in cultures not in equilibrium with the gas phase.
New Phytologist 01/1993; 123(4):679-684. DOI:10.1111/j.1469-8137.1993.tb03777.x · 7.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: summaryInorganic carbon-dependent photosynthetic oxygen evolution was saturated at a photon flux density of 100 μmol m−2s−1for air-grown cells of a low calcifying strain of Emittania huxleyi (Lohmann) Kamptner. Measurement of photosynthetic oxygen evolution at constant inorganic carbon concentration but varying pH showed that exogenous bicarbonate was not a major carbon source for photosynthesis. At pH 8.0 the concentration of dissolved inorganic carbon (DIC) required for the half-maximal rate of photosynthetic O2 evolution (K0.5[DIC]) was 2.86 mm; the rate of non-enzymic dehydration of HCO3 greatly exceeding the rate of CO2 fixation. Carbon dioxide uptake occurs by diffusive entry as shown by the K0.5 [DIC] of 12.5 μM at pH 5.0.Bicarbonate uptake, measured by the silicone-oil-layer centrigual filtering technique, did not show Michaelis-Menten type kinetics. The electrical membrane potential difference was determined from the distribution of the lipophilic cation tetra[3H]phenylphosphonium (TPP+) between cells and the media. Cells grown at pH 8.0 exhibited a negative membrane potential (inside of cell relative to outside) of about −60 mV.
[Show abstract][Hide abstract] ABSTRACT: Four species of freshwater cyanobacteria (A&M cyZidricu, Anucystis n&Zans, Nostoc canilta and Nostoc muscorum) contained as major lipid classes monogalactosyldiacylglycerols, digalactosyldiacylglycerols, sulphoquinovosyldiacylglycerols and phosphatidylglycerols. Unlike photosynthetic eukaryotes, cyanobacteria incubated for 7 d in the dark suffered no decrease in the concentrations of these classes, except for N. museorum. Growth, photosynthesis and nitrogen fixation were 3040% lower after dark incubation. The nitrogen-fixing cyanobacteria, Anabaena cyliirdric, N. cdnu and N. museorurn, contained alcohol glycosides and a highly-polar unknown glycolipid at high concentrations. The proportion of these two lipid classes decreased in the dark in N. muscorum alone. Extracts from Anacystis nidulans and N. caninu, and to a lesser extent N. muscorum, contained sterols, whose concentration increased after dark incubation. Anubuenu cylindricu contained considerable concentrations of linolenic acid in its total lipid, which did not decrease on dark incubation, and was not present mainly in monogalactosyldiacylglycerols as in photosynthetic eukaryotes. Palmitoleic acid, which is primarily confined to phosphatidylglycerols in photosynthetic eukaryotes, was distributed among the major lipid classes of N. eanina.
Journal of general microbiology 10/1990; 136(10):2043-2048. DOI:10.1099/00221287-136-10-2043
[Show abstract][Hide abstract] ABSTRACT: Homogenates of Dunaliella primolecta, D. salina and D. tertiolecta were assayed for glycollate oxidase and glycollate dehydrogenase. Both D. primolecta and D. salina but not D. tertiolecta showed substantial glycollate-dependent O2-uptake which is characteristic of glycollate oxidase. L-Lactate was an alternative substrate and both glycollate- and L-lactate-dependent O2 uptake were insensitive to 2 mM cyanide. Glycollate dehydrogenase, measured by following the glycollate-dependent reduction of 2,6-dichlorophenolindophenol under aerobic conditions, was present in D. primolecta, D. salina and D. tertiolecta. In the presence of glycollate and D-lactate, rates were additive so both glycollate and D-lactate dehydrogenases are present in the homogenates. Glycollate and D-lactate oxidation were both inhibited by 2 mM cyanide. Organelles released from phototrophically grown cells of D. primolecta were separated by isopycnic centrifugation on sucrose gradients. Glycollate oxidase was present in the peroxisome fraction at an equilibrium density of 1.25 g/cm(3), while the major peak of glycollate dehydrogenase activity was in the mitochondrial fraction at an equilibirium density of 1.22 g/cm(3).