ATPase activity assay

Intact chloroplasts were isolated from 4-week-old plants according to Mao et al.²³. The ATPase activity was determined by measuring by the amount of inorganic phosphate (Pi) in the reaction using an ATPase Activity Assay Kit (Sigma–Aldrich). Isolated chloroplast suspension (10 ul) was incubated for 30 min at room temperature in 30 ul assay buffer containing 40 mM Tis-HCl (pH 7.5), 80 mM NaCl, 8 mM MgAc₂, 1 mM EDTA and 1 mM ATP. The reaction was stopped by adding 200 ul Reagent and the released Pi was monitored by a microcolorimetric method after incubated for additional 30 min at room temperature.