Phytohormones quantification. LC-ESI tandem mass spectrometry
Plant samples were stored at −80 °C, freeze dried and powdered for subsequent analysis. Fifty milligrams of dry powder were used for hormonal extraction. Ultrapure water (Millipore, www.merckmillipore.com) was added containing a pool of internal standards abscisic acid‐d6 (ABA‐d6), salicylic acid‐d5 (SA‐d5) and JA‐Ile‐d6. Precise quantification was performed by using external calibration curves with each pure chemical compound. The content of the tube was vortexed and left at 4 °C in order to hydrate the plant sample. Five glass beads (2 mm Ø) were added into each microtube and the extraction was performed in a mixer mill at a frequency of 30 Hz for 3 min. Tubes were centrifuged at 13,000 rpm for 30′, and supernatant was recovered and placed into a new tube. A second extraction was then conducted, and the supernatant was added to the previous one. The pH was adjusted to 2.5–2.7 with acetic acid and the extraction was partitioned twice against diethyl ether. The two organic fractions were concentrated until dryness in a centrifugal evaporator (Speed vac) at room temperature. Samples were resuspended in 1 mL of H2O/MeOH (90:10) with 0.01% of HCOOH leading to a final concentration of internal standards of 100 ng/mL. The chromatographic separation was carried out by injection of 20 μl on an UPLC Kinetex 2.6 μm particle size EVO C18 100 A, 50 x 2.1 mm (Phenomenex). The quantification of the plant hormones was done in an Acquity ultraperformance liquid chromatography system (UPLC; Waters, Mildford, MA, USA), which was connected to a triple quadrupole mass spectrometer (TQD, Waters, Manchester, UK). The chromatographic and mass spectrometry conditions were those published by Gamir et al. (2012). Masslynx v 4. 1(Waters, Manchester, UK) software was used to process the quantitative data obtained from calibration standards and samples.