2.5. Analysis of ABA and IAA concentrations in plants tissues

Plant endogenous IAA and ABA concentrations were analyzed using high‐performance liquid chromatography‐electrospray‐high‐resolution accurate mass spectrometry (HPLC‐ESI‐HRMS). Indole‐3‐acetic acid (IAA) and cis,trans‐abscisic acid (ABA) reactants as well as the deuterium‐labeled internal standards ²H₅‐indole‐3‐acetic acid (D‐IAA) and ²H₆‐(+)‐cis,trans‐abscisic acid (D‐ABA) were purchased from OlChemin Ltd.

The extraction and purification of the hormones of plant samples were carried out using the following method: 0.25 g of frozen plant tissue (previously ground to a powder in a mortar with liquid N₂) was homogenized with 2.5 ml of precooled (−20°C) methanol:water:HCOOH (90:9:1, v/v/v, with 2.5 mM Na‐diethyldithiocarbamate) and 25 µl of a stock solution of 1,000 ng/ml of deuterium‐labeled internal standards in methanol. Extraction was performed by shaking the samples for 60 min at 2000 rpm at room temperature in a Multi Reax shaker. After extraction, solids were separated by centrifugation at 20.000 RCF for 10 min using a Sigma 4‐16K Centrifuge, followed again by re‐extraction with an additional 1.25 ml of extraction mixture by shaking for 20 min and centrifugation. About 2 ml of the pooled supernatants was separated and evaporated at 40°C using a RapidVap Evaporator. The residue was redissolved in 500 µl of methanol/0.133% acetic acid (40:60, v/v) and centrifuged at 20.000 RCF for 10 min before the injection into the HPLC‐ESI‐HRMS system.

Hormones were quantified using a Dionex Ultimate 3000 UHPLC device coupled to a Q Exactive Focus Mass Spectrometer (Thermo Fisher Scientific), equipped with an HESI(II) source, a quadrupole mass filter, a C‐Trap, a HCD collision cell, and an Orbitrap mass analyzer. A reverse‐phase column (Synergi 4 mm Hydro‐RP 80A, 150 × 2 mm; Phenomenex) was used. A linear gradient of methanol (A), water (B), and 2% acetic acid in water (C) was used: 38% A for 3 min, 38% to 96% A in 12 min, 96% A for 2 min, and 96% to 38% A in 1 min, followed by a stabilization time of 4 min. The percentage of C remained constant at 4%. The flow rate was 0.30 ml/min, the injection volume was 40 µl, and column and sample temperatures were 35 and 15°C, respectively. Ionization source working parameters were optimized and are reported in Table S2.

The detection and quantification of IAA and ABA were carried out using a Full‐MS experiment with MS/MS confirmation in the negative ion mode, employing multilevel calibration curves with the internal standards. MS¹ extracted from the Full‐MS spectrum is used for quantitative analysis, and MS² is used for the confirmation of targets identity. For Full‐MS, a m/z scan ranging from 62 to 550 was chosen, and the resolution was set at 70.000 FWHM, the automatic gain control (AGC) target at 1·e⁶, and the maximum injection time (IT) at 250 ms. A mass tolerance of 5 ppm was accepted. The MS/MS confirmation parameters are: resolution of 17.500 FWHM, isolation window of 3.0 m/z, AGC target of 2·e⁵, maximum IT of 60 ms, loop count of 1, and minimum AGC target of 3·e³. Instrument control and data processing were carried out by TraceFinder 3.3 EFS software. Accurate masses (m/z) for the phytohormones and their internal standards as well as for the principal fragments of these molecules are reported in Table S3.