Quantifying Jasmonates and Salicylates

JA and JA-IL levels were quantified using an ethyl acetate extraction method in conjunction with HPLC/MS similar to that described in Chung et al. (2008 (link)). Briefly, samples (approximately 150 mg tissue) were frozen in liquid nitrogen and hormones were extracted using 1 mL of extraction solvent (80:20 methanol:water + 0.1% formic acid) for 18 h at −20 C. Extracts were then centrifuged (10,000 × g for 10 min at 4°C) and the supernatant was transferred to autosampler vials. Five μL of each supernatant were injected into a Waters UPLC BEH C18 column (2.1 × 50 mm; 1.7 μm particles) held at 50°C on a Waters (Milford, MA, USA) Acquity ultraperformance liquid chromatography (UPLC) system that was coupled to a Waters Quattro Premier XE tandem quadrupole mass spectrometer. Separation was performed using a linear gradient based upon 0.15% aqueous formic acid (A) and methanol (B) over a 3-min program using a total flow rate of 0.4 mL/min. Quantification of JA and SA was performed using electrospray ionization in negative-ion mode using multiple reaction monitoring (MRM), using m/z 209 ≥ 59 for JA, m/z 322 ≥ 130 for JA-IL (Chung et al., 2008 (link)), and m/z 137 ≥ 93 for SA (Zeng et al., 2011 (link)). Peak areas were integrated, and calibration curves generated, using Waters QuanLynx software.