DISSOLVED INORGANIC CARBON UTILIZATION AND THE DEVELOPMENT OF EXTRACELLULAR CARBONIC-ANHYDRASE BY THE MARINE DIATOM PHAEODACTYLUM-TRICORNUTUM

The presence of extracellular carbonic anhydrase (CA) in relation to m edium composition was investigated using cultures of the marine diatom Phaeodactylum tricornutum Bohlin. Large-volume cultures, with low ini tial cell inocula were grown on ASP-2 (no dissolved inorganic carbon ( DIC), 550 mu M NO3-), f/2 (2.0 mM DIC, 880 mu M NO3-) and modified f/2 (2.0 mM DIC, 20 mu M NO3- media. Cells growing on ASP-2 showed extrac ellular CA in the early stages of growth, whereas extracellular CA was not detected until partial depletion of total DIC in the stationary p hase for cultures on f/2 or modified f/2. Both HCO3- and CO2 were impo rtant in carbon limitation, extracellular CA being present when the fr ee-CO2 concentration fell below 5 mu M, but the HCO3- concentration ne eded to be below 1 mM. When carbon-replete cells were transferred to c arbon-limited conditions, extracellular CA was recorded within minutes , the process being light-dependent and completely inhibited by 3,3,4- dichlorophenyl-1,1-dimethylurea (DCMU). The addition of DIC to carbon- limited cells resulted in a rapid decrease in extracellular CA activit y. The membrane-impermeable inhibitor of carbonic anhydrase, dextran-b ound sulphonamide (DBS) was used to inhibit extracellular CA activity in relation to photosynthetic rate in carbon-replete and carbon-limite d cells. At the lowest DIC concentration (0.10 mM), for cells with max imum external CA activity, DBS gave over 80% inhibition of the photosy nthetic rate, demonstrating the key role of external CA in maintaining high photosynthetic rate under conditions of carbon limitation. It is proposed that the key factor in the regulation of extracellular CA ac tivity is the total flux of inorganic carbon (C-i) into the cell. This determines the C-i flux into the chloroplast and when this is inadequ ate to support the photosynthetic rate attained by a carbon-replete ch loroplast at optimum photon flux density, extracellular CA is activate d. This mechanism would explain the observed interaction of CO2 and HC O3- in the regulation of extracellular CA activity.