Plant hormone extraction and analysis
The main classes of plant hormones, cytokinins [trans- zeatin (t-Z), zeatin riboside (ZR) and isopentenyladenine (iP)], gibberellin A3 (GA3), indole acetic acid (IAA), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA) and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), as well as the ABA catabolites, dihydrophaseic acid (DPA) and phaseic acid (PA) were extracted and analysed as described previously in Albacete et al. (2008) with some modifications. Fresh plant material (0.1 g FW of leaf or root) was homogenized in liquid nitrogen and incubated in 1 mL of cold (-20°C) extraction mixture of methanol/water (80/20, v/v) for 30 min at 4ºC. Solids were separated by centrifugation (20,000 g, 15 min at 4ºC) and re-extracted for another 30 min at 4ºC with 1 mL of extraction solution. Pooled supernatants were passed through Sep-Pak Plus C18 cartridges (previously conditioned with 3 mL of extraction buffer) to remove interfering lipids and some plant pigments. The supernatant was collected and evaporated under vacuum at 40ºC. The residue was dissolved in 1 mL methanol/water (20/80, v/v) solution using an ultrasonic bath. The dissolved samples were filtered through 13 mm diameter Millex filters with 0.22 µm pore size nylon membrane (Millipore, Bedford, MA, USA) and placed into opaque microcentrifuge tubes.
Ten µL of filtered extract (xylem, leaf or root) were injected in a U-HPLC-MS system consisting of an Accela Series U-HPLC (ThermoFisher Scientific, Waltham, MA, USA) coupled to an Exactive mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) using a heated electrospray ionization (HESI) interface. Mass spectra wereobtained using the Xcalibur software version 2.2 (ThermoFisher Scientific, Waltham, MA, USA). To quantify the plant hormones, calibration curves were constructed for each analysed component (0, 1, 10, 50, and 100 µg L-1). ABA catabolites were identified by extracting the exact mass of the target catabolite from the full scan chromatogram obtained in the negative mode, adjusting a mass tolerance of ≤ 1 ppm. The concentrations were semi-quantitatively determined from the extracted area of the derivative peak by using the calibration curve of ABA.