Untargeted metabolomics analysis
Arabidopsis roots were extracted using a homogeniser-assisted extraction in 80% methanol solution with 0.1% (v/v) formic acid, centrifuged and filtered through 0.22 µm cellulose filters. The phytochemical profile of roots was investigated through ultra-high-pressure liquid chromatography (UHPLC) coupled with quadrupole-time-of-flight (QTOF) mass spectrometry, as previously reported by Senizza et al. , 2021. Briefly, the mobile phase consisted of a mixture of water and acetonitrile (both LC-MS grade, VWR, Milan, Italy) acidified with 0.1% (v/v) formic acid, with a gradient from 6 to 94% of acetonitrile in 35 min. An injection volume of 6 μl and a pentafluorophenylpropyl column (2.0 × 100 mm, 3 µm - Agilent Technologies, Santa Clara, CA, USA) were used. The mass spectrometer acquired ions in the range 100-1200 m/z in positive scan mode (ESI+) at a rate of 0.8 spectra/s (40,000 FWHM, absolute peak height threshold 5000 counts).
For raw data processing, the software Profinder B.07 (Agilent Technologies) was used, considering monoisotopic mass (5-ppm tolerance for mass accuracy), isotope spacing and ratio according to the “find-by-formula” algorithm. Mass and retention time alignment, as well as compound filtering, were performed before compounds annotation. The database PlantCyc was used as a reference for annotations (Hawkinset al. 2021). Only the compounds identified in 100% of replications within at least one condition were retained in the dataset and used further. According to COSMOS Metabolomics Standards Initiative, the annotation process corresponded to a Level 2 of identification (i.e., putatively annotated compounds) (Salek et al. 2015).