Measuring CO(2) and [Formula: see text] permeabilities of isolated chloroplasts using a MIMS-(18)O approach

To support photosynthetic CO(2) fixation by Rubisco, the chloroplast must be fed with inorganic carbon in the form of CO(2) 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 (18)O-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 CO(2) 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 CO(2) 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 CO(2) permeability was reduced 50% in the mutant compared with the wild-type reference.