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 sh...

为支持核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）介导的光合二氧化碳（CO₂）固定过程，叶绿体需以二氧化碳或碳酸氢盐的形式获取无机碳源。然而，这两种物质（气体状的CO₂与带电的碳酸氢盐）快速穿过叶绿体被膜的具体机制尚未完全阐明。本文报道了一种实验方法，可借助膜进样质谱仪与氧-18标记的无机碳，测定这两种分子透过叶绿体被膜的通透系数。我们证实，叶绿体基质内的碳酸酐酶活性不会对该实验方法产生限制，并精准测定了多种植物来源叶绿体的表面积，以及CO₂与碳酸氢盐的通透系数。本次测定覆盖了多个植物物种的叶绿体，其中CO₂的通透系数范围为2.3×10⁻⁴ m·s⁻¹至8×10⁻⁴ m·s⁻¹，碳酸氢盐的通透系数为1×10⁻⁸ m·s⁻¹。我们将本方法应用于拟南芥水通道蛋白突变体的叶绿体样本，而此……