Giss c18

Analysis was performed using liquid chromatography–mass Spectrometer (LC‐MS/MS, Model Shimadzu‐8050) coupled with ESI system and QQQ mass analyzer. Ultra‐fast liquid chromatography (LC; Model‐ Shimadzu Prominence) system contained column oven (Model‐ CTO‐10AC), auto sampler (Model‐ SIL‐20AC HT), and shim‐pack GISS C18 reversed‐phase column (250 × 4.6 mm i.d.; particle size 5 µm). N₂ gas (drying & nebulizing gas), air (heating gas), and Ar gas (collision gas) were used for sample analysis.

The leaves were grounded in liquid nitrogen and dissolved in an appropriate amount of 50% acetonitrile. The samples were ultrasonically crushed for 30 min, and then centrifuged at 15,000g for 10 min at 4 °C. The supernatant was filtered (0.22 micron) and collected for analysis. For metabolome analysis, samples were analyzed using a HPLC-based targeted method [89 (link)]. The analytical conditions were shown as follows. HPLC: column, shim-pack GISS C18 (pore size 1.9 µm, length 2.1 × 100 mm); solvent system, A, water (0.04% acetic acid), B, acetonitrile (0.04% acetic acid); gradient program, 0 min, 5% B; 12.0 min, 95% B; 13.2 min, 95% B; 13.3 min, 5% B; 15.0 min, 5% B; flow rate, 0.4 mL min^–1; temperature, 40 °C; injection volume: 2 µL. The targeted metabolic profiling analysis was conducted by using a scheduled multiple reaction monitoring (MRM) via LC-ESI-QQQ-MS/MS system (LCMS-8060, SHIMADZU, Japan). The ESI source operation parameters were listed as follows: nebulizing gas flow, 3 L min^–1; heating gas flow, 10 L min^–1; interface temperature, 500 °C; DL temperature, 250 °C; heat block temperature, 400 °C; drying gas flow, 10 L min^–1. The recorded data were handled with LabSolutions 5.91 software. Standards ATP, ADP and AMP saltsslat were purchased from Aladdin (CHN).