Transgenic plants and wild plants of Arabidopsis at the flowering stages were used for quantification of the plant endogenous hormone with 1 g/plant. Leaves of Arabidopsis were ground with 10 mL isopropanol/hydrochloric acid and shaken at 4°C for 30 min (You et al., 2016 (link)). There were 9 transgenic plants and 9 wild plants. Subsequently, 20 mL dichloromethane was added. The mixture was shaken at 4°C for 30 min and centrifuged at 13,000 rpm at 4°C for 5 min. The organic fraction was separated and then dried under nitrogen in darkness. The solid residue was re-suspended in 400 μL methanol/0.1% methanoic acid. The sample was filtered with a 0.22 μm filter membrane before HPLC-MS/MS analysis.
HPLC analysis was performed using a poroshell 120 SB-C18 (Agilent, United States) column (150 × 2.1 mm × 2.7 μm). The mobile phase A solvents consisted of methanol + 0.1% methanoic acid and the mobile phase B solvents consisted of ultrapure water + 0.1% methanoic acid. The injection volume was 2 μL. MS conditions were as follows: the spray voltage was 4,500 V; the pressure of the air curtain, nebulizer, and aux gas were 15, 65, and 70 psi, respectively, and the atomizing temperature was 400°C.
HPLC analysis was performed using a poroshell 120 SB-C18 (Agilent, United States) column (150 × 2.1 mm × 2.7 μm). The mobile phase A solvents consisted of methanol + 0.1% methanoic acid and the mobile phase B solvents consisted of ultrapure water + 0.1% methanoic acid. The injection volume was 2 μL. MS conditions were as follows: the spray voltage was 4,500 V; the pressure of the air curtain, nebulizer, and aux gas were 15, 65, and 70 psi, respectively, and the atomizing temperature was 400°C.
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