HPLC analysis of salicylic acid

SA was extracted from 3–4 biological replicates per genotype and analyzed by HPLC as previously described (Zhang et al., 2017). Data were corrected for recovery using samples spiked with o-anisic acid as an internal control. Pure SA and o-anisic acid (Sigma-Aldrich) were used as standards. SA and o-anisic acid content was determined by fluorescence (SA, excitation 301 nm, emission 412 nm; o-anisic acid, excitation 301 nm, emission 365 nm) after separation on a C18 reverse-phase HPLC column (ZORBAX SB-C18, Agilent Technologies, TN, USA) with the Agilent Technologies 1200/1100 series LC system. The column was maintained at 25 °C, and methanol:0.5% glacial acetic acid (60:40, v/v) was flowed through at a rate of 1.25 ml min^–1 for ~20 min.