The Role of External Carbonic Anhydrase in Inorganic Carbon Acquisition by Chlamydomonas reinhardii at Alkaline pH

Abstract

The role of external carbonic anhydrase in inorganic carbon acquisition and photosynthesis by Chiamydomonas reinhardii at alkaline pH (8.0) was studied. Acetazolamide (50 micromolar) completely inhibited external carbonic anhydrase (CA) activity as determined from isotopic disequilibrium experiments. Under these conditions, photosynthetic rates at low dissolved inorganic carbon (DIC) were far greater than could be maintained by CO2 supplied from the spontaneous dehydration of HC03-thereby showing that C. reinhardii has the ability to utilize exogenous HCO3-. Acetazolamide increased the concentration of DIC required to half-saturate photosynthesis from 38 to 80 micromolar, while it did not affect the maximum photosynthetic rate. External CA activity was also removed from the cell-wall-less mutant (CW-15) by washing. This had no effect on the photosynthetic kinetics of the algae while the addition of acetazolamide to washed cells (CW-15) increased the K?'c from 38 to 80 micromolar. Acetazolamide also caused a buildup of the inorganic carbon pool upon NaHCO3 addition, indicating that this compound partially inhibited internal CA activity. The effects of acetazolamide on the photosynthetic kinetics of C. reinhardii are likely due to the inhibition of internal rather than a consequence of the inhibition of external CA.Further analysis of the isotopic disequilibrium experiments at saturating concentration of DIC provided evidence consistent with active CO2 transport by C. reinhardii. The observation that C. reinhardii has the ability to take up both CO2 and bicarbonate throws into question the role of external CA in the accumulation of DIC in this alga.of carbon for photosynthesis (23). Findenegg (12) (26) then the function of external CA is called into question.Internal CA appears to facilitate the CO2 supply to Rubisco from the internal HCO3 pool (24, 25). Spalding et al. (24,25) showed that a mutant of C. reinhardii lacking internal CA exhibited high rates of photorespiration and accumulated large concentrations of intracellular inorganic carbon. Their conclusion was that an internal CA was required to facilitate CO2 supply to Rubisco from actively transported HCO3 . Proponents of the theory that CO2 is the only species of carbon to cross the plasmalemma suggest that active transport ofbicarbonate occurs at the chloroplast envelope and that the internal CA is located within the chloroplast (21,22).In this report we provide evidence that C. reinhardii has the ability to take up either CO2 or bicarbonate and that the magnitude of each flux is dependent upon the experimental conditions. Our results suggest that both CO2 and HCO3 accumulation are active and occur at the plasmalemma. We also provide evidence that external CA is not necessary for efficient photosynthesis at alkaline pH.

Active Transport of CO2 by the Cyanobacterium Synechococcus UTEX 625

“…In this context it is worth noting that there is no evidence that the cyanobacteria examined so far produce their own extracellular CA (2,3,8,9,13,20,21) while many green algae do (7,23). The function of the extracellular CA of green algae remains a subject of debate (30) and thus its ecological role is unknown. At the low cell densities of cyanobacteria that commonly exist in nature and at mildly alkaline pH it is probable that the dehydration rate of HCO3-to CO2 is not a limiting factor for CO2 uptake even in the absence of extracellular CA.…”

Active CO2 Transport by the Green Alga Chlamydomonas reinhardtii

“…In Chlamydomonas, HC03-is actively transported (4, 25, 29), but CO2 uptake has been considered to be passive (18,20). Carbon dioxide, however, is taken up from the medium faster than HCO3-by Chlamydomonas (13,28,29) and several authors (13,29) have considered the possibility of active CO2 transport.…”

“…In the case of cyanobacteria, both HCO3-and CO2 are substrates for active transport (2,3,6,7,14,15) with CO2 being selectively and preferentially used by the cells (2,6,16). In Chlamydomonas, HC03-is actively transported (4,25,29), but CO2 uptake has been considered to be passive (18,20). Carbon dioxide, however, is taken up from the medium faster than HCO3-by Chlamydomonas (13,28,29) and several authors (13,29) have considered the possibility of active CO2 transport.…”

“…In Chlamydomonas, HC03-is actively transported (4, 25, 29), but CO2 uptake has been considered to be passive (18,20). Carbon dioxide, however, is taken up from the medium faster than HCO3-by Chlamydomonas (13,28,29) and several authors (13,29) have considered the possibility of active CO2 transport.Studies on the DIC transport mechanism of green algae are complicated by their cellular compartmentation. Recently, it was shown that isolated chloroplasts of low C02 Chlamydomonas reinhardtii were able to accumulate DIC (19) and a model was presented where the only active DIC transport mechanism was located on the chloroplast envelope (18,19).…”

The development of a CO2‐concentrating mechanism in Emiliania huxleyi

“…Some microalgai spp. exhibit an apparent high affinity for CO, and a concomitant ability for CO., transport across the plasma membrane (Williams & Turpin, 1987;Munoz & Merrett, 1988;Sultemeyer et al, 1989;Gehl, Colman & Sposato, 1990). The external and internal enzymes of carbonic anhydrase are essential components of the CCM in some microalgae (Moroney, Husic & Tolbert, 1985).…”