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).