Extraction, Purification, Identification and Quantification of Brassinosteroids.

The extraction and purification of various BRs were performed according to Oklestkova et al. (2017), with some modifications. Samples of root parts of control, water-stressed and BL-treated seedlings (about 5 mg DW) were extracted in ice-cold acetonitrile (60%) and then centrifuged. The supernatant was supplemented with a BRs internal standard mixture containing 25 pmol of 2H3-labelled BRs (OlChemIm Ltd. Olomouc, Czech Republic). The samples were purified through Discovery DPA-6 S SPE columns (Supelco, Bellefonte, PA USA), evaporated to dryness and then reconstructed in 40 µl of methanol. The analysis of BRs was performed by liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS; ACQUITY UPLC® I-Class System, Waters, Milford, MA, USA) with the use of triple quadrupole mass spectrometer Xevo™ TQ-S MS (Waters MS Technologies, Manchester, UK) according to Oklestkova et al. (2017) and Tarkowská et al. (2016). The analyses were performed in five replicates. BR concentrations are listed in pmol/g DW.