ABA quantification

Seedlings were grown for 7 d in continuous light. The ground tissue (about 150 mg of frozen seedlings) was resuspended in 80% (v/v) methanol-1% (v/v) acetic acid containing the internal standard deuterium-labeled hormone (²H₆-ABA) and mixed by shaking during 1 h at 4°C. The extract was kept at −20°C overnight. After centrifugation, the supernatant was dried in a vacuum evaporator. The dry residue was dissolved in 1% (v/v) acetic acid and passed through a reverse phase Oasis HLB column. The final residues were dissolved in 5% (v/v) acetonitrile-1% (v/v) acetic acid. ABA hormone was then separated using an autosampler and reverse-phase Ultra High Performance Liquid Chromatography (2.6 µm Accucore RP-MS column, 50 mm length × 2.1 mm i.d.; ThermoFisher Scientific) with a 5%–50% (v/v) acetonitrile gradient containing 0.05% (v/v) acetic acid, at 400 µL·min⁻¹ over 14 min. The ABA was analyzed with a Q-Exactive mass spectrometer (Orbitrap detector; ThermoFisher Scientific) by targeted selected ion monitoring. The concentrations of ABA in the extracts were determined using embedded calibration curves and the Xcalibur 4.0 and TraceFinder 4.1 SP1 programs.