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.