Jasmonate Quantification

Jasmonate quantification was performed according to de Ollas et al. (2013). A sample of 0.4 g of frozen plant material was extracted in 5 mL of distilled water, after spiking with 100 ng dihydrojasmonic acid as internal standard. After centrifugation at 4000 g at 4°C, supernatants were recovered and pH adjusted to 3.0 with 30% acetic acid. The acidified water extract was partitioned twice against 3 mL of di-ethyl ether. The organic layer was recovered and evaporated under vacuum in a centrifuge concentrator. The dry residue was then resuspended in 1 mL of a 10% MeOH solution by gentle sonication. The resulting solution was filtered and directly injected into a HPLC system (Acquity SDS UPLC, Waters Corp., Milford, MA, United States). Separations were carried out on a C18 column (C18 Gravity, 1.8 μm particle size, 50 × 2.1 mm, Macherey-Nagel, Germany) using a linear gradient of MeOH and water supplemented with 0.1% acetic acid at a flow rate of 300 μl min⁻¹. Jasmonates (12-oxophytodienoic acid, OPDA; jasmonic acid, JA and jasmonoyl isoleucine, JA-Ile) were quantified with a TQS triple quadrupole mass spectrometer (Micromass Ltd., Manchester, United Kingdom) connected online to the output of the column through an orthogonal Z-spray electrospray ion source. Transitions for JA/DHJA (209 > 59/211 > 59), OPDA (291 > 165), and JA-Ile (322 > 130) were monitored in negative ionization mode. Quantitation of plant hormones was carried out by external calibration using standards of known concentration. Processing of chromatograms, integration of peaks and quantitation was performed with Masslynx 4.1 software (Micromass Ltd., Manchester, United Kingdom).