Abstract
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.
Keywords: Arabidopsis, Chloroplast, Ion transport, Thylakoid membrane, Envelope membrane, Photosynthesis
Background
Calcium and manganese are structural components of photosystem II and form the inorganic Mn4CaO5 cluster, where water oxidation takes place with the outcome of electrons, protons and molecular oxygen. Ca2+ and Mn2+ fluxes across the chloroplast envelope membrane and the thylakoid membrane are fundamental processes enabling the plant cell to meet the high demand of PSII for these cations. In a previous study, a Ca2+/H+ antiport activity was analyzed using isolated thylakoid membranes from pea plants (Ettinger et al., 1999). In the model plant Arabidopsis thaliana hardly any Ca2+/H+ antiport activity is detectable in isolated thylakoid membranes (Schneider et al., 2016), thus a protocol which allows the thylakoid membrane system to reside in its naturally physiological environment, namely the chloroplast is presented. Furthermore, this protocol permits the relative distribution of Ca2+ and Mn2+ in chloroplasts to be determined. The protocol given here has been tested with Arabidopsis as well as with pea plants.
Materials and Reagents
Equipment
Procedure
Data analysis
Notes
Arabidopsis growth can be performed as described in Steinberger et al., 2015. This protocol is also suitable for two-week old pea plants. For isolation of intact chloroplasts, it is important to avoid any starch granules, which accumulate during the light period. Starch granules inside chloroplasts might prevent accumulation of intact chloroplasts at the interface of the two Percoll layers. Therefore, dark adapt plants for at least 16 h; during the night starch degradation takes place.
Recipes
Acknowledgments
We thank Gabi Burkhard for excellent technical assistance. This work was carried out in the laboratory of Prof. Dario Leister (Biozentrum der LMU München, Department Biologie I, Munich, Germany) and supported by funds from the Deutsche Forschungsgesellschaft. This protocol is adapted from Schneider et al., 2016.
References
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