Chloroplast Isolation and Fractionation

Three weeks old Arabidopsis thaliana wild‐type (Col‐0 ecotype) or cmt1 (SALK_129037c, grown for 6 weeks) plants were grown onto soil under 12 h light and 12 h dark periods (120 μmol photons s⁻¹·m⁻² light intensity).
When isolating intact chloroplasts, the foliar tissue was ground in a kitchen blender with Isolation buffer (330 mm sorbitol, 50 mm HEPES pH 7.5, 2 mm EDTA, 1 mm MgCl₂, 5 mm ascorbic acid) and filtered through one layer of gauze. This solution was centrifuged for 5 min at 1500 g and 4 °C to rescue the pellet and resuspend it in Washing buffer (330 mm sorbitol, 50 mm HEPES pH 7.5), which was loaded on percoll gradients of 40% percoll solution (330 mm sorbitol, 50 mm HEPES pH 7.6, 40% percoll) and 80% percoll solution (330 mm sorbitol, 50 mm HEPES pH 7.6, 80% percoll) and centrifuged for 5 min at 8000 g and 4 °C and the intact chloroplasts rescued to be washed with washing buffer.
For the separation of chloroplast into stroma and membrane fractions, the procedure skipped the percoll gradient and continued with resuspension of crude chloroplasts in osmotic shock buffer (0.6 m sucrose, 1 mm EDTA, 10 mm Tricine pH 7.9). The sample was then incubated on ice for 30 min under darkness and centrifuged for 1 h at 100 000 g and 4 °C to separate the soluble fraction (stroma) from the pellet (membranes).