Phytohormones Extraction and Analysis

Phytohormones were extracted and analyzed as described elsewhere [6 (link)]. Briefly, ~120 mg of frozen tissue was extracted twice with 1 ml of 80% methanol/water and centrifuged at 20,000 g for 15 min at 4°C. The supernatant was passed through a C18 cartridge, and the samples were collected in a 5-ml tube for speed-Vac evaporation to dryness. The residue was resuspended in 1 ml of 20% methanol/water. Ten μl of filtrated extract as injected in a U-HPLC-MS system consisting of an Accela Series U-HPLC (ThermoFisher Scientific, USA) coupled to an Exactive mass spectrometer (ThermoFisher Scientific) using heated electrospray ionization (HESI) interface. Mass spectra were obtained using Xcalibur software version 2.2 (ThermoFisher Scientific). The auxin homeostasis metabolites were identified according to the molecular mass and retention time values in the total ion chromatograms from the phytohormone analysis. In each compound, the area obtained was used for comparisons.