2.3 Metabolome extraction and analysis
We extracted and analyzed all samples according to Ristok et al.(2019) with slight changes. We extracted 20 mg dried and ground plant tissue of each sample in 1 mL of extraction buffer (methanol / 50 mM acetate buffer, pH 4.8; 50 / 50 [v/v]). All samples were homogenized for 5 min at 30 Hz using a Retsch mixer mill MM 400, and subsequently centrifuged for 10 min at 20,000 g and 4°C. We collected the supernatant in a 2 mL Eppendorf tube, repeated the extraction procedure with the remaining pellet, and combined both supernatants. Lastly, we centrifuged (20,000 g, 5 min, 4°C) all extracts, transferred 200 µL to an HPLC vial, and added 800 µL extraction buffer, resulting in a 1:5 dilution.
We performed chromatographic separation of all diluted extracts by injecting 2 µL on a Thermo Scientific Dionex UltiMate 3000 (Thermo Scientific Dionex, Sunnyvale, USA) UPLC unit, equipped with a C18 column (Acclaim RSLC 120 C18, 2.2 µm, 120 Å, 2.1 x 150 mm, Thermo Fisher Scientific). We applied the following binary elution gradient at a flow rate of 0.4 mL min-1 and a column temperature of 40°C: 0 – 2 min, 95% A (water and 0.05% formic acid), 5% B (acetonitrile and 0.05% formic acid); 2 – 12 min, 5 to 50% B; 12 – 13 min, 50 to 95% B; 13 – 15 min, 95% B; 15 – 16 min, 95 to 5% B; 16 – 20 min, 5% B.
Metabolites were analyzed on a liquid chromatography quadrupole time-of-flight mass spectrometer (LC-qToF-MS; Bruker maXis impact HD; Bruker Daltonik, Bremen, Germany) with an electrospray ionization source operated in negative mode. Instrument settings were as follows: capillary voltage, 2500 V; nebulizer, 2.5 bar; dry gas temperature, 220°C; dry gas flow, 11 L min-1; scan range, 50 – 1400 m/z; acquisition rate, 3 Hz. We used sodium formate clusters (10 mM solution of NaOH in 50 / 50% [v/v] isopropanol / water containing 0.2% formic acid) to perform mass calibration.