Mg-chelatase activity assay

In-vitro Mg-chelatase activity assays were performed according to Hansson et al. (1999). WT and xan-h.chli-1 seeds were sown in vermiculite and grown in the dark for 10 days. Etiolated seedlings were then homogenized in 0.4 M mannitol, 20 mM Tricine-NaOH pH 9 and 1 mM DTT. Intact chloroplasts were enriched by 15-min centrifugation at 3000 g and loaded onto a 40% (vol/vol) Percoll cushion in homogenization buffer. Gradients were centrifuged for 15 min at 13,000 g. After washing steps in homogenization buffer, chloroplasts were resuspended in 200 μL of lysis buffer (20 mM Tricine-NaOH pH 9, 1 mM DTT, 1 mM PMSF). After a centrifugation step at 11,000 g for 5 min, the recovered supernatants containing the Mg-chelatase subunits were adjusted to the same protein concentration. The enzymatic assay was carried out by adding 1 μL of the reaction cocktail (50 mM ATP, 250 mM creatine phosphate, 250 mM MgCl₂, and 0.06 mM deuteroporphyrin). Reactions were stopped by adding 1 mL acetone/water/25% ammonia (80/20/1, vol/vol/vol) and 200 µL heptane was added to remove chlorophyll from samples. To measure the relative amount of Mg-deuteroporphyrin, the emission spectrum of the acetone phase was recorded from 550 to 600 nm using an excitation wavelength of 408 nm. Excitation and emission slits were set to 5 nm.