Measuring CO₂ and HCO₃− permeabilities of isolated chloroplasts using a MIMS-¹⁸O approach

To support photosynthetic CO₂ fixation by Rubisco, the chloroplast must be fed with inorganic carbon in the form of CO₂ or bicarbonate. However, the mechanisms allowing the rapid passage of this gas and this charged molecule through the bounding membranes of the chloroplast envelope are not yet completely elucidated. We describe here a method allowing us to measure the permeability of these two molecules through the chloroplast envelope using a membrane inlet mass spectrometer and ¹⁸O-labelled inorganic carbon. We established that the internal stromal carbonic anhydrase activity is not limiting for this technique, and precisely measured the chloroplast surface area and permeability values for CO₂ and bicarbonate. This was performed on chloroplasts from several plant species, with values ranging from 2.3 × 10^–4 m s^–1 to 8 × 10^–4 m s^–1 permeability for CO₂ and 1 × 10^–8 m s^–1 for bicarbonate. We were able to apply our method to chloroplasts from an Arabidopsis aquaporin mutant, and this showed that CO₂ permeability was reduced 50% in the mutant compared with the wild-type reference.