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Carbonic anhydrase activity in leaves as measured in vivo by18O exchange between carbon dioxide and water

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Abstract

Carbonic anhydrase activity of intactCommelina communis L. leaves was measured using mass spectrometry, by following the18O-exchange kinetics between18O-enriched carbon dioxide and water. A gas-diffusion model (Gerster, 1971, Planta97, 155–172) was used to interpret the18O-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 inCommelina 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 NO −3 , 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 using18O2 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 using18O-isotope exchange therefore provides a suitable model to study the in-vivo regulation of this chloroplastic enzyme in plants submitted to various environmental conditions.

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Abbreviations

CA:

carbonic anhydrase

Ccc:

chloroplastic CO2 concentration

Ce :

external CO2 concentration

EZA:

ethoxyzolamide

k:

CO2 hydration rate constant

ke :

CO2 diffusion rate constan

PPFD:

photosynthetic photon flux density

Rubisco:

ribulose-1,5 bisphosphate carboxylase oxygenase

RWD:

relative water deficit

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Authors and Affiliations

  1. CEA, Département de Physiologie Végétale et Ecosystèmes, Centre de Cadarache, F-13108, Saint-Paul-lez-Durance, France

    Gilles Peltier, Laurent Cournac, Valérie Despax, Bernard Dimon, Laurent Fina & Dominique Rumeau

  2. Laboratoire d'Ecologie Végétale, CNRS URA 1492, Université Paris Sud Bât 362, F-91405, Orsay, France

    Bernard Genty

Authors
  1. Gilles Peltier
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  2. Laurent Cournac
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  3. Valérie Despax
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  4. Bernard Dimon
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  5. Laurent Fina
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  6. Bernard Genty
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  7. Dominique Rumeau
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Corresponding author

Correspondence to Gilles Peltier.

Additional information

The authors wish to thank P. Carrier for technical assistance with mass-spectrometric experiments and Dr. P. Thibault for helpful suggestions and comments. Dr. A. Vavasseur is gratefully acknowledged for supplyingCommelima communis. cultures. P.C., P.T. and A.V. are all from the CEA, Département de Physiologie Végétale et Ecosystèmes, Cadarache, France.

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Peltier, G., Cournac, L., Despax, V. et al. Carbonic anhydrase activity in leaves as measured in vivo by18O exchange between carbon dioxide and water. Planta 196, 732–739 (1995). https://doi.org/10.1007/BF01106768

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