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).