Dissolved inorganic carbon utilization and the development of extracellular carbonic anhydrase by the marine diatom Phaeodactylum tricornutum

Abstract

The presence of extracellular carbonic anhydrase (CA) in relation to medium composition was investigated using cultures of the marine diatom Phaeodactylum tricornutum Bohlin. Large-volume cultures, with low initial cell inocula were grown on ASP-2 (no dissolved inorganic carbon (DIC), 550 μM NO₃⁻), f/2 (2·0 mM DIC, 880 μM NO₃⁻) and modified f/2 (2·0 mM DIC, 20 μM NO₃⁻ media. Cells growing on ASP-2 showed extracellular CA in the early stages of growth, whereas extracellular CA was not detected until partial depletion of total DIC in the stationary phase for cultures on f/2 or modified f/2. Both HCO₃⁻ and CO₂ were important in carbon limitation, extracellular CA being present when the free-CO₂ concentration fell below 5 μM, but the HCO₃⁻ concentration needed to be below 1 mM. When carbon-replete cells were transferred to carbon-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 activity. The membrane-impermeable inhibitor of carbonic anhydrase, dextran-bound sulphonamide (DBS) was used to inhibit extracellular CA activity in relation to photosynthetic rate in carbon-replete and carbon-limited cells. At the lowest DIC concentration (0·10 mM), for cells with maximum external CA activity, DBS gave over 80% inhibition of the photosynthetic 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 activity 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 inadequate to support the photosynthetic rate attained by a carbon-replete chloroplast at optimum photon flux density, extracellular CA is activated. This mechanism would explain the observed interaction of CO₂ and HCO₃⁻ in the regulation of extracellular CA activity.