Phytohormones Extraction and Quantification

Tubes containing 100 mg of fresh and ground plant material were kept at −80°C, and transferred to liquid nitrogen before the extraction. The samples were removed from the liquid nitrogen and 1 mL of extraction solution containing the internal standards (d5-JA, d6-ABA, d5-IAA, and d4-SA), prepared as described in Preparation of Standards Solutions, were directly added. The samples were briefly mix with a vortex, and spiked with phytohormones standards as described in Method Validation to generate the calibration curve and quality control (QC) samples. The spiked samples were shaken for 30 min in the Starlab shaker and centrifuged at 16,000 g and 4°C for 5 min. The supernatant was transferred into a new 1,5 micro-centrifuge tube and dried in speed vac. After drying, 100 μL of MeOH were added to each sample, vortexed and centrifuged at 16,000 g and 4°C for 10 min. The supernatant was analyzed by HPLC-MS/MS.