Cation (Ca²⁺ and Mn²⁺) Partitioning Assays with Intact Arabidopsis Chloroplasts

Determination of the relative distribution of Ca²⁺ and Mn²⁺ is an important tool for analyzing mutants showing altered levels of calcium and/or manganese transporters in the chloroplast envelope or thylakoid membrane. The method described in this protocol allows quantitative analyses of the relative distribution of calcium and manganese ions between chloroplast stroma and thylakoids using the isotopes [⁴⁵Ca] and [⁵⁴Mn] as radioactive tracers. To avoid contaminations with non chloroplastidic membrane systems, the method is designed for isolating pure and intact chloroplasts of Arabidopsis thaliana. Intact chloroplasts are isolated via Percoll gradient centrifugation. Chloroplasts are then allowed to take up [⁴⁵Ca] or [⁵⁴Mn] during a light incubation step. After incubation, chloroplasts are either kept intact or osmotically/mechanically treated to release thylakoids. The amount of incorporated [⁴⁵Ca] or [⁵⁴Mn] can be determined by liquid scintillation counting and the relative distribution calculated.