Root Metabolome Analysis Protocol

Plant material for the analysis of the root metabolome was collected in the same experiment as that earlier used for pea leaf metabolome analysis [5 (link)]. The samples of plant material (0.2–0.3 g) were ground as described by Puzanskiy et al. [55 (link)] and subjected to a single-stage extraction with 2 mL methanol: chloroform: water (5:2:1) mixture. Tissue debris was removed by centrifugation at 12,000× g for 10 min at −5 °C. The supernatant was collected and evaporated in a vacuum evaporator (Eppendorf, Germany). The dried material was dissolved in pyridine with the internal tricosane standard (nC23, tricosane). The samples were then supplied with the sylilating agent BSTFA:TMCS 99:1 (Sigma-Aldrich) and derivatizated at 90 °C for 20 min.