CARBONIC-ANHYDRASE ACTIVITY IN LEAVES AS MEASURED IN-VIVO BY O-18 EXCHANGE BETWEEN CARBON-DIOXIDE AND WATER

Carbonic anhydrase activity of intact Commelina communis L. leaves was measured using mass spectrometry, by following the O-18-exchange kine tics between O-18-enriched carbon dioxide and water. A gas-diffusion m odel (Gerster, 1971, Planta 97, 155-172) was used to interpret the O-1 8-exchange kinetics and to determine two constants, one (k) related to the hydration of CO2 and the other (k(e)), related to the diffusion o f CO2. Both constants were determined in Commelina communis L. leaves after stripping the lower epidermis to remove any stomatal influence. The hydration constant (k) was 17 200 +/- 2 200 . min(-1) (mean +/- SD , 12 experiments), i.e., about 8 600 times the uncatalyzed hydration o f CO2 in pure water, and was specifically inhibited by ethoxyzolamide, a powerful inhibitor of carbonic anhydrases, half-inhibition occurrin g around 10(-5) M ethoxyzolamide. The diffusion constant (k(e)) was 1. 18 +/- 0.28 . min(-1) (mean +/- SD, 12 experiments) and was only sligh tly inhibited (about 20%) by ethoxyzolamide. Carbonic anhydrase activi ty of stripped leaves was not affected by the leaf water status (up to 50% relative water deficits), was strongly inhibited by monovalent an ions such as Cl- or NO3-, and decreased by about 50% when the photon f lux density during growth was increased from 100 to 500 mu mol photo-n s . m(-2). s(-1). By studying the effect of ethoxyzolamide (10(-4) M) on photosynthetic O-2 exchange, measured using O-18(2) and mass spectr ometry, we found that inhibition of carbonic anhydrase activity by 92- 95% had little effect on the response curves of net O-2 evolution to i ncreased CO2 concentrations. Ethoxyzolamide had no effect on the photo synthetic electron-transport rate, measured as gross O-2 photos but wa s found to increase both gross at CO2 concentration (> 350 mu l . 1(-1 )), but was found to increase both gross O-2 photosynthesis and O-2 up take at lower CO2 levels. The chloroplastic CO2 concentration calculat ed from O-2-exchange data was not significantly modified by ethoxyzola mide. We conclude from these results that, under normal conditions of photosynthesis, most of the carbonic anhydrase activity is not involve d in CO2 assimilation. Measurement of carbonic anhydrase activity usin g O-18-isotope exchange therefore provides a suitable model to study t he in-vivo regulation of this chloroplastic enzyme in plants submitted to various environmental conditions.