ABA and ABA-GE Quantification by Liquid Chromatography (LC)/Mass Spectrometry (MS)

A modification of the method of Park et al. (2016) was used to determine ABA and ABA-GE contents. Briefly, 8-day-old seedlings grown on ½ MS media (not containing sucrose) were transferred to 5 μM ABA-containing media and treated for 12, 24, 48, 72, or 120 h, or transferred onto DMSO, or transferred onto 10, 50, or 100 μM ABA-containing medium and grown for an additional 5 or 11 days. A total of 0.1-0.2 g of frozen fresh sample was ground in liquid nitrogen with a small steel ball in a 2 mL vial. Following the addition of 1.0 mL of ethyl acetate, homogenates were mixed in a Geno/Grinder homogenizer. After centrifugation at 15,200 ×g for 10 min at 4°C, the supernatant was transferred to a 2 mL Eppendorf tube. After the second extraction by adding 0.5 mL of ethyl acetate without internal standards, the combined extracts were vacuum-dried in a concentrator at 30°C. The dried extracts were dissolved in 100 μL of 70% methanol, vortexed for 20 min, and then centrifuged at 15,200 ×g for 10 min at 4°C. The supernatant was transferred to 1.5 mL LC vials, and then injected into the liquid chromatography (LC)/mass spectrometry (MS) system.