Hormone content analysis

For each sample, 10 mg of freeze-dried powder were extracted with 0.8 mL of acetone/water/acetic acid (80/19/1 v:v:v). For each sample, 2 ng of each standard was added to the sample: abscisic acid, salicylic acid, jasmonic acid, and indole-3-acetic acid stable labeled isotopes used as internal standards were prepared as described previously [89]. The extract was vigorously shaken for 1 min, sonicated for 1 min at 25 Hz, shaken for 10 minutes at 4°C in a Thermomixer (Eppendorf), and then centrifuged (8000 g, 4°C, 10 min). The supernatants were collected, and the pellets were re-extracted twice with 0.4 mL of the same extraction solution, then vigorously shaken (1 min) and sonicated (1 min; 25 Hz). After the centrifugations, three supernatants were pooled and dried.

Each dry extract was dissolved in 140 μL of acetonitrile/water (50/50; v/v), filtered, and analyzed using a Waters Acquity ultra performance liquid chromatograph coupled to a Waters Xevo Triple quadrupole mass spectrometer TQS (UPLC-ESI-MS/MS). The compounds were separated on a reverse-phase column (Uptisphere C18 UP3HDO, 100 × 2.1 mm, 3 μm particle size; Interchim, France) using a flow rate of 0.4 mL·min^-1 and a binary gradient: (A) acetic acid 0.1% in water (v/v) and (B) acetonitrile with 0.1% acetic acid. For ABA, salicylic acid, jasmonic acid, the following binary gradients were used (time, % A): (0 min, 98%), (3 min, 70%), (7.5 min, 50%), (8.5 min, 5%), (9.6 min, 0%), (13.2 min, 98%), (15.7 min, 98%), and the column temperature was 40°C. Mass spectrometry was conducted in electrospray and multiple reaction monitoring scanning mode (MRM mode), in the negative ion mode. Relevant instrumental parameters were set as follows: capillary 1.5 kV (negative mode), source block and desolvation gas temperatures 130°C and 500°C, respectively. Nitrogen was used to assist the cone and desolvation (150 L·h^-1 and 800 L·h^-1, respectively), argon was used as the collision gas at a flow of 0.18 mL·min^-1. Samples were reconstituted in 140 μL of 50/50 acetonitrile/H₂O (v/v) per mL of injected volume. The limit of detection (LOD) and limit of quantification (LOQ) were extrapolated for each hormone from calibration curves and samples using Quantify module of MassLynx software, version 4.1.