The role of extracellular carbonic anhydrase activity in inorganic carbon utilization of Phaeocystis globosa (Pyrmnesiophyceae): A comparison with other marine algae using isotopic disequilibrium technique (original) (raw)
Related papers
Physiologia Plantarum, 1994
Carbonic anhydrase activity and inorganic carbon fluxes in low-and high-Cj eells of Chiamydomonas reinhardtii and Scenedesmus ohliquus.-Physiol, Plant. 90; 537-547, Carbonic anhydrase iQ.\) activity associated with higb-and low-dissolved inorganic carbon (C.) grown cells was examined in whole cells by measuring '"O exchange from doubly labeled CO; ("C'*O"'O). Both aigal species showed the presence of extracellular (peripJasmic) as well as intracellular CA actiiily, which were both greatly increased in low-C, cells. The periplasmic CA activity was at least 40-fold higher in lowcompared to high-C, cells in both C, reinhardtii and 5, obliquus. while low-C, cells of S. ohiiquus showed the highest activity of intemai CA, The CA inhibitor ethoxyzolamide .showed a ,strong inhibition of the C, uptake process in both C, reinhardtii and S. ohiiquus as in eyanobacteria, which may indicate that the nature of the primary uptake process is similar in bolh green aigae and cyanobacteria. By using a mass spectrometric disequilibrium technique it w as possible to separate the C, fluxes of net HCO^-uptake and net CO;-uptake during steady-state photosynthesis in high-and low-C, grow'n cells of Chiamydomonas reinhardtii (WT, 2137-H and Scenedesmus ohiiquus (WT, D3), It was found that both high-and low-C; cells of the two algae ean utilize both CO; and HCOT for photosynthesis, although low-C, celis have a higher affinity for the uptake of both Ci species. Induction at iow-C; causes an increase in tbe affinity of both species for HCO; and CO;; changes in net CO;-uptake were, however, significantly greater.
PLANT PHYSIOLOGY, 2013
Many microalgae induce an extracellular carbonic anhydrase (eCA), associated with the cell surface, at low carbon dioxide (CO 2 ) concentrations. This enzyme is thought to aid inorganic carbon uptake by generating CO 2 at the cell surface, but alternative roles have been proposed. We developed a new approach to quantify eCA activity in which a reaction-diffusion model is fit to data on 18 O removal from inorganic carbon. In contrast to previous methods, eCA activity is treated as a surface process, allowing the effects of eCA on cell boundary-layer chemistry to be assessed. Using this approach, we measured eCA activity in two marine diatoms (Thalassiosira pseudonana and Thalassiosira weissflogii), characterized the kinetics of this enzyme, and studied its regulation as a function of culture pH and CO 2 concentration. In support of a role for eCA in CO 2 supply, eCA activity specifically responded to low CO 2 rather than to changes in pH or HCO 3 2 , and the rates of eCA activity are nearly optimal for maintaining cell surface CO 2 concentrations near those in the bulk solution. Although the CO 2 gradients abolished by eCA are small (less than 0.5 mM concentration difference between bulk and cell surface), CO 2 uptake in these diatoms is a passive process driven by small concentration gradients. Analysis of the effects of short-term and long-term eCA inhibition on photosynthesis and growth indicates that eCA provides a small energetic benefit by reducing the surface-to-bulk CO 2 gradient. Alternative roles for eCA in CO 2 recovery as HCO 3 2 and surface pH regulation were investigated, but eCA was found to have minimal effects on these processes.
Physiol Plant, 1994
Carbonic anhydrase activity and inorganic carbon fluxes in low-and high-Cj eells of Chiamydomonas reinhardtii and Scenedesmus ohliquus.-Physiol, Plant. 90; 537-547, Carbonic anhydrase iQ.\) activity associated with higb-and low-dissolved inorganic carbon (C.) grown cells was examined in whole cells by measuring '"O exchange from doubly labeled CO; ("C'*O"'O). Both aigal species showed the presence of extracellular (peripJasmic) as well as intracellular CA actiiily, which were both greatly increased in low-C, cells. The periplasmic CA activity was at least 40-fold higher in lowcompared to high-C, cells in both C, reinhardtii and 5, obliquus. while low-C, cells of S. ohiiquus showed the highest activity of intemai CA, The CA inhibitor ethoxyzolamide .showed a ,strong inhibition of the C, uptake process in both C, reinhardtii and S. ohiiquus as in eyanobacteria, which may indicate that the nature of the primary uptake process is similar in bolh green aigae and cyanobacteria. By using a mass spectrometric disequilibrium technique it w as possible to separate the C, fluxes of net HCO^-uptake and net CO;-uptake during steady-state photosynthesis in high-and low-C, grow'n cells of Chiamydomonas reinhardtii (WT, 2137-H and Scenedesmus ohiiquus (WT, D3), It was found that both high-and low-C; cells of the two algae ean utilize both CO; and HCOT for photosynthesis, although low-C, celis have a higher affinity for the uptake of both Ci species. Induction at iow-C; causes an increase in tbe affinity of both species for HCO; and CO;; changes in net CO;-uptake were, however, significantly greater.
Aquatic Botany, 2011
The effects on photosynthesis of acetazolamide (AZ, an inhibitor of the external carbonic anhydrase) and TRIS buffer at pH 8.7 were assessed in 24 species of red macroalgae. Only Palmaria palmata was unaffected by both substances. The rest of species were classified into three groups according to their sensitivity to TRIS and AZ. Photosynthesis of fourteen species was significantly inhibited by both TRIS and AZ. Inhibition by TRIS varied from almost 100% to 25% while AZ produced similar effects. Inhibition by TRIS was completely reverted by increasing the dissolved inorganic carbon concentration (DIC). This species group had half-saturation constants for photosynthesis (K m(DIC) ) ranging from 0.5 to 1.1 mM of DIC. TRIS produced a significant increase of K m(DIC) . Altogether, these results indicate that the algae sensitive to TRIS are capable of using HCO 3 − efficiently at pH 8.7. Furthermore, the buffering capacity of TRIS was responsible for its inhibitory effect on photosynthesis suggesting that HCO 3 − use was facilitated by excretion of protons outside the plasma membrane, which creates regions of low pH resulting in a higher-thanambient CO 2 concentration. In contrast, photosynthesis by two Porphyra species analysed was slightly stimulated by TRIS and completely inhibited by AZ, suggesting that the mechanism was different. In a third group of seaweeds, photosynthesis was insensitive to TRIS but it was significantly inhibited by AZ. These species had relatively high values of K m(DIC) indicating that they relied on purely diffusive entry of CO 2 generated by external carbonic anhydrase activity. Consequently, the results demonstrate that external carbonic anhydrase is widespread among red macroalgae since only P. palmata was insensitive to AZ. The functional significance of this enzyme was quite variable among the tested species.
Journal of Phycology, 2009
The role of carbonic anhydrase (CA) in inorganic carbon acquisition (dissolved inorganic carbon, DIC) was examined in Alboran Sea phytoplankton assemblages. The study area was characterized by a relatively high variability in nutrient concentration and in abundance and taxonomic composition of phytoplankton. Therefore, the relationship between environmental variability and capacity for using HCO 3 ) via external CA (eCA) was examined. Acetazolamide (AZ, an inhibitor of eCA) inhibited the primary productivity (PP) in 50% of the samples, with inhibition percentages ranging from 13% to 60%. The AZ effect was more prominent in the samples that exhibited PP >1 mg C AE m )3 AE h )1 , indicating that the contribution of eCA to the DIC photosynthetic flux was irrelevant at low PP. The inhibition of primary productivity by AZ was significantly correlated to the abundance of diatoms. However, there was no a relationship between AZ effect and CO 2 partial pressure (pCO 2 ) or nutrient concentration, indicating that the variability in the PP percentage supported by eCA was mainly due to differences in taxonomic composition of the phytoplankton assemblages. Ethoxyzolamide (EZ, an inhibitor of both external and internal CA) affected 13 of 14 analyzed samples, with PP inhibition percentages varying from 50% % to 95% %. The effects of AZ and EZ were partially reversed by doubling DIC concentration. These results imply that CA activity (external and ⁄ or internal) was involved in inorganic carbon acquisition in most the samples. However, EZ effect was not correlated with pCO 2 or taxonomic composition of the phytoplankton.
PLANT PHYSIOLOGY, 1988
The induction of a dissolved inorganic carbon (DIC) accumulating mechanism in the two algal species Scenedesmus obliquus (WT) and Chlamydomonas reinhardtii (137 c+) was physiologically characterized by monitoring DIC uptake kinetics at a low and constant DIC concentration (120-140 micromolar), after transfer from high-DIC culturing conditions. A potentiometric titration method was used to measure and calculate algal DIC uptake. Full acclimation to low-DIC conditions was obtained within a period of 90 min, after which time the DIC uptake had been increased 7 to 10 times. Experiments were also conducted in the presence of inhibitors against DIC accumulation. The inhibitor of extracellular carbonic anhydrase (CA), acetazolamide (50 micromolar), inhibited the adaptation partly, while the inhibitor of both extra- and intracellular CA, ethoxyzolamide (50 micromolar) totally inhibited the acclimation. Cycloheximide (10 micrograms per milliliter), which inhibits protein synthesis on cytoplasmic ribosomes, and vanadate (180 micromolar), which inhibits the plasmamembrane bound ATPase, also inhibited the acclimation totally. These results taken together suggest that the algae are dependent on intracellular CA, plasmamembrane bound ATPase, and de novo protein synthesis for DIC accumulation. Also, these components are more important than extracellular CA for the overall function of the DIC-accumulating mechanism.
Plant Physiol, 2008
The oceans globally constitute an important sink for carbon dioxide (CO 2) due to phytoplankton photosynthesis. However, the marine environment imposes serious restraints to carbon fixation. First, the equilibrium between CO 2 and bicarbonate (HCO 3 2) is pH dependent, and, in normal, slightly alkaline seawater, [CO 2 ] is typically low (approximately 10 mM). Second, the rate of CO 2 diffusion in seawater is slow, so, for any cells unable to take up bicarbonate efficiently, photosynthesis could become carbon limited due to depletion of CO 2 from their immediate vicinity. This may be especially problematic for those dinoflagellates using a form II Rubisco because this form is less oxygen tolerant than the usually found form I enzyme. We have identified a carbonic anhydrase (CA) from the free-living marine dinoflagellate Lingulodinium polyedrum that appears to play a role in carbon acquisition. This CA shares 60% sequence identity with d-class CAs, isoforms so far found only in marine algae. Immunoelectron microscopy indicates that this enzyme is associated exclusively with the plasma membrane. Furthermore, this enzyme appears to be exposed to the external medium as determined by whole-cell CA assays and vectorial labeling of cell surface proteins with 125 I. The fixation of 14 CO 2 is strongly pH dependent, suggesting preferential uptake of CO 2 rather than HCO 3 2 , and photosynthetic rates decrease in the presence of 1 mM acetazolamide, a non-membranepermeable CA inhibitor. This constitutes the first CA identified in the dinoflagellates, and, taken together, our results suggest that this enzyme may help to increase CO 2 availability at the cell surface.
PLANT PHYSIOLOGY, 2008
The oceans globally constitute an important sink for carbon dioxide (CO 2) due to phytoplankton photosynthesis. However, the marine environment imposes serious restraints to carbon fixation. First, the equilibrium between CO 2 and bicarbonate (HCO 3 2) is pH dependent, and, in normal, slightly alkaline seawater, [CO 2 ] is typically low (approximately 10 mM). Second, the rate of CO 2 diffusion in seawater is slow, so, for any cells unable to take up bicarbonate efficiently, photosynthesis could become carbon limited due to depletion of CO 2 from their immediate vicinity. This may be especially problematic for those dinoflagellates using a form II Rubisco because this form is less oxygen tolerant than the usually found form I enzyme. We have identified a carbonic anhydrase (CA) from the free-living marine dinoflagellate Lingulodinium polyedrum that appears to play a role in carbon acquisition. This CA shares 60% sequence identity with d-class CAs, isoforms so far found only in marine algae. Immunoelectron microscopy indicates that this enzyme is associated exclusively with the plasma membrane. Furthermore, this enzyme appears to be exposed to the external medium as determined by whole-cell CA assays and vectorial labeling of cell surface proteins with 125 I. The fixation of 14 CO 2 is strongly pH dependent, suggesting preferential uptake of CO 2 rather than HCO 3 2 , and photosynthetic rates decrease in the presence of 1 mM acetazolamide, a non-membranepermeable CA inhibitor. This constitutes the first CA identified in the dinoflagellates, and, taken together, our results suggest that this enzyme may help to increase CO 2 availability at the cell surface.
American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 2000
Symbiotic cnidarians absorb inorganic carbon from seawater to supply intracellular dinoflagellates with CO2for their photosynthesis. To determine the mechanism of inorganic carbon transport by animal cells, we used plasma membrane vesicles prepared from ectodermal cells isolated from tentacles of the sea anemone, Anemonia viridis. H14[Formula: see text] uptake in the presence of an outward NaCl gradient or inward H+gradient, showed no evidence for a Cl−- or H+- driven [Formula: see text] transport. H14[Formula: see text] and36Cl−uptakes were stimulated by a positive inside-membrane diffusion potential, suggesting the presence of [Formula: see text] and Cl−conductances. A carbonic anhydrase (CA) activity was measured on plasma membrane (4%) and in the cytoplasm of the ectodermal cells (96%) and was sensitive to acetazolamide (IC50= 20 nM) and ethoxyzolamide (IC50= 2.5 nM). A strong DIDS-sensitive H+-ATPase activity was observed (IC50= 14 μM). This activity was also highly sensitive t...
PLANT PHYSIOLOGY, 2001
A single intracellular carbonic anhydrase (CA) was detected in air-grown and, at reduced levels, in high CO 2 -grown cells of the marine diatom Phaeodactylum tricornutum (UTEX 642). No external CA activity was detected irrespective of growth CO 2 conditions. Ethoxyzolamide (0.4 mm), a CA-specific inhibitor, severely inhibited high-affinity photosynthesis at low concentrations of dissolved inorganic carbon, whereas 2 mm acetazolamide had little effect on the affinity for dissolved inorganic carbon, suggesting that internal CA is crucial for the operation of a carbon concentrating mechanism in P. tricornutum. Internal CA was purified 36.7-fold of that of cell homogenates by ammonium sulfate precipitation, and two-step column chromatography on diethylaminoethyl-sephacel and p-aminomethylbenzene sulfone amide agarose. The purified CA was shown, by SDS-PAGE, to comprise an electrophoretically single polypeptide of 28 kD under both reduced and nonreduced conditions. The entire sequence of the cDNA of this CA was obtained by the rapid amplification of cDNA ends method and indicated that the cDNA encodes 282 amino acids. Comparison of this putative precursor sequence with the N-terminal amino acid sequence of the purified CA indicated that it included a possible signal sequence of up to 46 amino acids at the N terminus. The mature CA was found to consist of 236 amino acids and the sequence was homologous to -type CAs. Even though the zinc-ligand amino acid residues were shown to be completely conserved, the amino acid residues that may constitute a CO 2 -binding site appeared to be unique among the -CAs so far reported.