Quantifying Plant Hormone Profiles

Plant hormones, including different jasmonates (OPDA, JA and the conjugated form JA‐Ile), ABA and the cytokinin t‐Z were extracted and quantified by ultrahigh‐performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS). Ground leaves (50 mg) and freeze‐dried roots (30 mg) were extracted with 250 μl of a methanol:2‐propanol:glacial acetic acid (50:49:1) mix and deuterium‐labeled standards (d5‐JA, d6‐ABA, and d5‐tZ). The extracts were subjected to ultrasonication (Branson 2510 ultrasonic cleaner, Bransonic) and vortexing for 30 min, followed by a 10 min centrifugation at 15,000 g (PrismR, Labnet International Inc.). The supernatant was collected, and the pellet was reextracted. Both supernatants were merged and filtered through a hydrophobic 0.22 μm filter (Phenomenex). Hormone levels were analyzed by UHPLC‐ESI‐MS/MS as described by Müller and Munné‐Bosch (2011 (link)). The UHPLC was coupled to a triple quadrupole mass spectrometer (QTRAP 4000, AB Sciex). A LUNA C18 column (Phenomenex, 1.6 μm, 100 × 2.1 mm) was used. Solvent A was water with 0.05% acetic acid and solvent B was acetonitrile with 0.05% acetic acid. Flow rate was set at 0.6 ml min⁻¹. Quantification was made considering recovery rates for each sample by using the deuterium‐labeled internal standards. Calibration curves for each analyte were generated using MultiQuantTM 3.0.1 software.