Endogenous ABA assay
ABA content was determined by HPLC-electrospray ionization-tandem mass spectrometry method (Chen et al. 2011). Internal ABA was extracted according to the method and made some modulation. Plant samples were harvest, processed by vacuum freeze drying by a lyophilizer (FDU-1110, EYELA, Tokyo, Japan) and ground in liquid nitrogen, then the samples were weighed and transferred to a 50 mL centrifuge tube. Added 15 mL methanol containing 20% ddH2O (v/v) at 4℃ for 12 h for extraction. [2H6] ABA (50 ng/g) was added to plant samples as internal standard (IS) prior to grinding. After centrifugation at 10,000 rpm at 4℃ for 20 min, the supernatant was collected and passed through a C-18 (100 mg) SPE cartridge, which was preconditioned with 8 mL ddH2O, 8 mL methanol, and 8 mL methanol containing 20% ddH2O (v/v). The elution was pooled, dried with nitrogen gas and re-dissolved with 2 mL ddH2O. The solution was acidified with 240 µL, 0.1 mol/L HCL and extracted with ethyl ether (4 × 1 mL). The organic phases were combined, dried by nitrogen gas and re-dissolved with 80 µL acetonitrile (ACN). To the resulting solution, 10 µL trimethylamine (TEA, 20 µmol·mL-1) and 10 µL 3-bromoactonyltrimethylammonium bromide (BTA, 20 µmol·mL-1) were added. The reaction solution was vortexed for 30 min to mix up and dried with nitrogen gas and re-dissolved with 200 µL ACN containing 20% (v/v) ddH2O. 20 µL of the solution was subjected to HPLC-electrospray ionization-tandem mass spectrometry analysis. Each treatment contained at least 3 biological repeats and each repeat contained 3 shoots or roots samples. Each testing sample contained 3 replicates.