Sample collection and untargeted metabolomics
Four leaves in the third position from the branch tip were harvested for
untargeted metabolomics at 43 DAT (31 August 2017) and at 131 DAT (27
November 2017). The leaves were flash-frozen in liquid nitrogen and
stored at -80 °C until subsequent metabolomic analysis.
An untargeted metabolomics approach was conducted in the UHPLC 1290
chromatographic system coupled to a hybrid quadrupole-time-of-flight
(Q-TOF) G6550 mass spectrometer (UHPLC/Q-TOF) (Agilent Technologies,
Santa Clara, CA, USA). A Waters Acquity UPLC® BEH C18
column (100 × 2.1 mm i.d., 1.7 μm) (Waters Corp., Milford, MA, USA) was
used for reverse-phase chromatographic separation. The binary gradient
consisted of water and acetonitrile and the Riken Plasma method was
followed (Tsugawa et al., 2019). The injection volume was 2 μL and the
mass spectrometer was run in positive polarity and SCAN mode (range:
100–1,700 m/z; extended dynamic range setting). Quality controls (QC)
were prepared by pooling 10-μL samples. Five QCs were acquired in
data-dependent mode (auto MS/MS) at 1 Hz, 10 precursors/cycle, collision
energies of 10 V, 30 V, and 50 V), and in iterative mode with active
exclusion to increase the number of compounds targeted for tandem MS
fragmentation.
Alignment, blank filtration, and identification were performed in MSDIAL
v. 4.0 (Riken, Tokyo, Japan) using the publicly available library MoNA
(Mass Bank of North America) and an internal standard compound library
as specified in the Supplementary Table 1. Compounds lacking
experimental MS/MS spectra were annotated with MSFINDER (Riken, Tokyo,
Japan) following the procedure described in Blaženović et al. (2019).
The alternatives were filtered by retention time prediction (Bonini et
al., 2020). MSI (metabolomics standards initiative) levels for each
identified compound are listed in Supplementary Table 1.