Specialized Metabolite Extraction and Analysis

Specialized metabolites were extracted as described in Sipari et al. [13 (link)] and analyzed with the same instrumentation as the lipophilic metabolites. Samples were analyzed both in positive (ESI +) and negative (ESI −) ionization mode. The mass range (m/z) was set to 100–1500 in positive and 100–1000 in negative mode. The compounds were separated on an Acquity UPLC BEH C18 column (1.7 µm, 50 × 2.1 mm, Waters, Ireland). The injection volume was 2 µL, oven temperature was 40 °C, and tray temperature was 10 °C. The mobile phases consisted of (A) H₂O and (B) acetonitrile (Chromasolv grade, Sigma-Aldrich, Steinheim, Germany) both containing 0.1% formic acid (Sigma-Aldrich, Steinheim, Germany). A linear gradient started from 95% A and proceeded to 10% in 5 min. The eluent composition was changed back to 95% at 5.1 min and left to equilibrate for 0.9 min, with a total analysis time of 6 min. Data processing with MassLynx and the identification of metabolites were performed as described for lipophilic metabolites. The relative levels of the specialized metabolites were calculated by normalizing the analyte peak area by the peak area of the internal standard, 4-methyl-umbelliferone, and the fresh weight of the sample.