LC-ESI full scan mass spectrometry
The metabolomic analysis was carried out with six biological replicates per treatment. Fifty milligrams of freeze‐dried leaf or root material were extracted at 4 °C with 1 ml of MeOH:H2O (10:90) containing 0.01% of HCOOH. After the centrifugation at full speed at 4 °C for 15 min, the supernatant was filtered through 0.2 μm cellulose filters (Regenerated Cellulose Filter, 0.20 μm, 13 mm D. pk/100; Teknokroma). 20 μl were injected into an Acquity UPLC system (Waters, Mildford, MA, USA) interfaced with a hybrid quadrupole time‐of‐flight instrument (QTOF MS Premier). Subsequently, a second fragmentation function was introduced into the TOF analyser to identify the signals detected. This function was programmed in a t‐wave ranging from 5 to 45 eV to obtain a fragmentation spectrum of each analyte (Gamir et al., 2014). Positive and negative electrospray signals were analysed independently to obtain a global view of the data conduct. To elute analytes, a gradient of methanol and water containing 0.01% HCOOH was used. Three independent biological replicates per treatment, each with three technical replicates, were randomly injected. The LC separation was performed using an UPLC Kinetex 2.6 μm particle size EVO C18 100 A, 50 x 2.1 mm (Phenomenex). Chromatographic conditions and solvent gradients and further were established as described by Gamir et al. (2014).