Quantitative Analysis of Phytohormones

Cytokinins (trans-zeatin), gibberellins (GA1, GA4, and GA3), indole-3-acetic acid, ABA, SA, jasmonic acid, and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) were analyzed in mature leaves according to Albacete et al. (2008) (link) with some modifications (Albacete et al., 2008 (link)). Ten microliters of extracted sample was injected in a Ultra high performance liquid chromatography (UHPLC)–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 were obtained using the Xcalibur software version 2.2 (ThermoFisher Scientific, Waltham, MA, USA). For quantification of the phytohormones, calibration curves were constructed for each analyzed component (1, 10, 50, and 100 μg/L) and corrected for 10 μg/L deuterated internal standards. Recovery percentages ranged between 92% and 95%. To examine responses to trans-zeatin treatment, the seeds of each genotype were surface-sterilized and plated on vertical MS plate (10 g/L agar) supplemented with indicated concentration of trans-zeatin.