Untargeted metabolomic profiling and phytohormone analysis
Tomato seedlings were flash-frozen in liquid nitrogen fourteen days after transplantation (24 h after the second PA treatment). Roots and shoots (from cotyledons upwards) were ground in a liquid-nitrogen chilled mortar. Six samples (3 plants per sample) were analyzed per treatment. 100 mg powder was extracted with 2 ml cold methanol (30 minutes, 1000 rpm, 25°C) and centrifuged (10 minutes, 19 330g, RT). The supernatant was dried in a vacuum concentrator (Labconco CentriVap) at 35°C, while the residue was air-dried (24 h, 60°C) and weighed. Metabolites were dissolved in 200 µl 1:1 miliQ-water-cyclohexane (15 minutes, 1200 rpm, RT) and centrifuged (5 minutes, 19 330g, RT).
Seventy µl aqueous phase was filtered through a 0.2 µm filter (PAL AcroPrep) and 3 µl filtrate was analyzed by reversed-phase UPLC (Acquity UPLC I-class, Waters) coupled to a quadrupole-time-of-flight mass spectrometer (Vion IMS QToF, Waters) via an electrospray ionization source in negative ionization mode. Chromatography and MS conditions were as in De Meester et al. (2018). Pooled control samples, prepared by combining 3 µl of each sample, were used for chromatogram alignment and quality control.
Data were normalized to sample dry weight, and features were filtered using three criteria: (a) non-zero intensity in all replicates of at least one treatment, (b) mean MS intensity > 5 in at least one treatment and (c) relative SD < 30% in pooled control samples. Features were quantile-normalized (Bolstad, Irizarry, Åstrand & Speed 2003), centered and generalized logarithm-transformed (Durbin, Hardin, Hawkins & Rocke 2002) using MetaboAnalyst v. 5.0 (Chong, Wishart & Xia 2019). Differentially abundant features were identified using FDR-adjusted heteroscedastic t-tests with a significance threshold of P < 0.05 and absolute log2-fold change > 1. Pathway analysis was performed using Mummichog v. 2.0 (Li et al. 2013) as implemented in MetaboAnalyst v. 5.0, with following parameters: mass tolerance 5.0 ppm, retention time included, primary ions enforced, P-value cut-off 0.05, A. thaliana pathway library.
For the targeted quantification of phytohormones, plant material was collected in the same manner as for metabolome profiling and 100 mg of this material was used for analysis using the methodology and instrumentation described in (Haeck et al. 2018).