Quantifying Plant Hormone Levels

Plant hormones of free SA and JA productions were extracted according to a previously described method [63 (link), 64 (link)]. SA and JA were extracted and quantified according to the method of Liu et al. with appropriate modifications [65 (link)]. Briefly, twig samples (0.5 g for each sample) were immediately frozen in liquid nitrogen and ground with pestle and mortar. The ground samples were extracted with 500 μL modified Bieleski solvent (methanol/H₂O, 80/20, v/v) at 4 °C for 12 h. The solutions of SA and JA were prepared as internal standards at a concentration of 1 μg/mL in 100% methanol. All nano-LC experiments were performed on a Shimadzu Prominence nano-flow liquid chromatography system (Kyoto, Japan) with two LC-20 AD nano pumps, two vacuum degassers, a LC-20AB HPLC pump, a SIL-20 AC HT autosampler, and a FCV nano valve. The analytical column of poly (MAA-co-EDMA) monolithic column (100 μm i.d., 360 μm o.d., 30-cm long, purchased from Weltech Co., Ltd., Wuhan, China) was connected to the nano-LC system and conditioned with the mobile phase (ACN/H₂O, 50/50, v/v) at a flow rate of 600 μL/min for 30 min.