Soluble sugars were extracted and derivatized sequentially with methoxamine hydrochloride and N-methyl-N-trimethylsilyl-trifluoroacetamide, as described previously (Hu et al., 2018 (link)). The metabolites were analyzed with a Shimadzu GCMS-2010 SE (Shimadzu Corp., Kyoto, Japan). Values were calculated based on their corresponding standard curves and internal standards.
Amino acids were extracted and measured as described previously (Huo et al., 2020b (link)). Briefly, 200 mg of frozen leaf samples were extracted in 2 ml 50% ethanol (including 0.1 M HCl) and centrifuged at 13,000 g for 10 min. The supernatant was added to methanol at a final volume of 10 ml. The samples were filtered through a 0.22 μm filter to analyze the metabolites with a liquid chromatography-mass spectrometry system (QTRAP5500; SCIEX, Concord, ONT, Canada) equipped with an Inertsil ODS-4 C18 column (4.6 × 250 mm, 5 μm) at a flow rate of 0.3 ml/min. The solvent system consisted of water containing 0.1% (v/v) formic acid (A) and acetonitrile (B). Data were quantified by comparing the peak surface areas with those obtained using standard amino acids (Sigma-Aldrich, St. Louis, MO, United States).
Amino acids were extracted and measured as described previously (Huo et al., 2020b (link)). Briefly, 200 mg of frozen leaf samples were extracted in 2 ml 50% ethanol (including 0.1 M HCl) and centrifuged at 13,000 g for 10 min. The supernatant was added to methanol at a final volume of 10 ml. The samples were filtered through a 0.22 μm filter to analyze the metabolites with a liquid chromatography-mass spectrometry system (QTRAP5500; SCIEX, Concord, ONT, Canada) equipped with an Inertsil ODS-4 C18 column (4.6 × 250 mm, 5 μm) at a flow rate of 0.3 ml/min. The solvent system consisted of water containing 0.1% (v/v) formic acid (A) and acetonitrile (B). Data were quantified by comparing the peak surface areas with those obtained using standard amino acids (Sigma-Aldrich, St. Louis, MO, United States).
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