Metabolite analysis by LC–MS/MS

Metabolite extraction and analysis was performed according to the method described previously (Guo et al. 2014). Leaves were harvested from 5-week-old seedlings and ground in liquid nitrogen immediately. Metabolites were extracted from about 0.1 g leaf powders or chloroplasts isolated from 0.2 g leaves with 3 mL methanol-chloroform (7:3, v/v) and incubated at − 20 ℃ for 2 h with occasional vortexing. A total of 0.9 µg 1,4-piperazinediethanesulfonic acid (PIPES) was added to each sample as internal standard. Water soluble metabolites were extracted by adding 2.4 mL ddH₂O and centrifuged at 500 g for 5 min. The upper phase was removed to a new glass tube. The upper phase was dried with gas nitrogen at room temperature, re-dissolved with 200 L ddH₂O and diluted by tenfold for metabolite analysis by LC–MS/MS (QTRAP® 6500 + , AB SCIEX, USA). The level of metabolites was determined by compared to PIPES.