Quantification of ABA and GA were performed according to the methods described in our previous studies (Chen et al., 2011 ; Shu et al., 2013 (link), 2016a (link)).
For ABA, dry and imbibed (6 h after sowing, 400 mg) soybean seeds were ground in liquid nitrogen and extracted for 24 h in methanol containing D6-ABA (OIChemIm Co. Ltd.) as an internal standard. Purification was performed using an Oasis Max solid-phase extract cartridge (Waters) and eluted with 5% formic acid in methanol. The elution was then dried and reconstituted, and injected into a LC–tandem MS system consisting of an Acquity ultra-performance LC (Acquity UPLC; Waters) and a triple-quadrupole tandem MS (Quattro Premier XE; Waters). Three biological replications were performed.
For GA, dry and imbibed (6 h after sowing, 400 mg) soybean seeds were ground in liquid nitrogen and extracted with 80% (v/v) methanol. GA d2 isotope standards were added to the samples before grinding. The crude extracts were purified by reversed-phase solid-phase extraction, ethyl-ether extraction, and derivatization. The resulting mixture was injected into a capillary electrophoresis-MS system (Agilent Technologies) for quantitative analysis. Three biological replications were performed.
For ABA, dry and imbibed (6 h after sowing, 400 mg) soybean seeds were ground in liquid nitrogen and extracted for 24 h in methanol containing D6-ABA (OIChemIm Co. Ltd.) as an internal standard. Purification was performed using an Oasis Max solid-phase extract cartridge (Waters) and eluted with 5% formic acid in methanol. The elution was then dried and reconstituted, and injected into a LC–tandem MS system consisting of an Acquity ultra-performance LC (Acquity UPLC; Waters) and a triple-quadrupole tandem MS (Quattro Premier XE; Waters). Three biological replications were performed.
For GA, dry and imbibed (6 h after sowing, 400 mg) soybean seeds were ground in liquid nitrogen and extracted with 80% (v/v) methanol. GA d2 isotope standards were added to the samples before grinding. The crude extracts were purified by reversed-phase solid-phase extraction, ethyl-ether extraction, and derivatization. The resulting mixture was injected into a capillary electrophoresis-MS system (Agilent Technologies) for quantitative analysis. Three biological replications were performed.
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