Quantifying Plant Hormones in Xylem Sap

In sap, IAA, ABA, SA, JA and JA-Ile were analyzed according to Albacete et al. [95 (link)], with some modifications. Briefly, xylem sap samples from eight different plants per treatment were filtered through 13 mm diameter Millex filters with a 0.22 µm pore size nylon membrane (Millipore, Bedford, MA, USA). Then, 10 µL of filtrated extract was 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), using a heated electrospray ionization (HESI) interface. Mass spectra were obtained using Xcalibur software version 2.2 (ThermoFisher Scientific). For quantification of the plant hormones, calibration curves were constructed for each analyzed component (1, 10, 50 and 100 µg L⁻¹).