Carbonic anhydrase activity in leaves as measured in vivo by 18O exchange between carbon dioxide and water

Abstract

Carbonic anhydrase activity of intact Commelina communis L. leaves was measured using mass spectrometry, by following the 18O-exchange kinetics between 18O-enriched carbon dioxide and water. A gas-diffusion model (Gerster, 1971, Planta 97, 155–172) was used to interpret the 18O-exchange kinetics and to determine two constants, one (k) related to the hydration of CO2 and the other (ke), related to the diffusion of 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 17200 +2200 ·min−1 (mean±SD, 12 experiments), i.e., about 8 600 times the uncatalyzed hydration of CO2 in pure water, and was specifically inhibited by ethoxyzolamide, a powerful inhibitor of carbonic anhydrases, half-inhibition occurring around 10−5 Methoxyzolamide. The diffusion constant (ke) was 1.18±0.28·min−1 (mean±SD, 12 experiments) and was only slightly inhibited (about 20%) by ethoxyzolamide. Carbonic anhydrase activity of stripped leaves was not affected by the leaf water status (up to 50% relative water deficits), was strongly inhibited by monovalent anions such as Cl− or NO3−, and decreased by about 50% when the photon flux density during growth was increased from 100 to 500 μmol photons·m −2·s−1. By studying the effect of ethoxyzolamide (10−4 M) on photosynthetic O2 exchange, measured using 18O2 and mass spectrometry, we found that inhibition of carbonic anhydrase activity by 92–95% had little effect on the response curves of net O2 evolution to increased CO2 concentrations. Ethoxyzolamide had no effect on the photosynthetic electron-transport rate, measured as gross O2 photosynthesis at high CO2 concentration (>350 μl·−1), but was found to increase both gross O2 photosynthesis and O2 uptake at lower CO2 levels. The chloroplastic CO2 concentration calculated from O2-exchange data was not significantly modified by ethoxyzolamide. We conclude from these results that, under normal conditions of photosynthesis, most of the carbonic anhydrase activity is not involved in CO2 assimilation. Measurement of carbonic anhydrase activity using 18O-isotope exchange therefore provides a suitable model to study the in-vivo regulation of this chloroplastic enzyme in plants submitted to various environmental conditions.