LC–MS quantification of abscisic acid

For ABA analysis, leaves were subjected as previously described (Frusciante et al. 2022) with some modifications. Briefly, 10 mg of freeze-dried leaf powder was resuspended in 750 µL of 75% (v/v) cold methanol spiked with 0.5 µg/mL formononetin (Sigma–Aldrich, Cat. No. 47752-25MG-F). Semi-polar metabolites were extracted by vigorous agitation for 30 min at 25 °C. Samples were then centrifuged at 20,000 rcf for 20 min, the supernatant was collected, filtered with HPLC PTFE filter tubes (0.22 µm pore size), and subjected to LC–PDA–HRMS analysis as described (Demurtas et al. 2018). ABA was quantified by LC–MS in HESI negative ionization mode, integrating the area of the M-H ion of m/z 263.1289 at the retention time of 12.10 min (normalizing to the internal standard formononetin) and using a calibration curve established for the ABA standard (Sigma–Aldrich, Cat. No. A1049-100MG).