Determination of Cytokinin Levels

CK purification was performed according to previously described methods (Svačinová et al., 2012) with some modifications (Šmehilová et al., 2016). Briefly, CKs were extracted from 30 mg of frozen powdered tissue in modified Bieleski buffer (methanol/water/formic acid, 15/4/1, v/v/v) containing a cocktail of stable isotope-labeled internal standards (0.25 pmol of CK bases, ribosides, and N-glucosides; 0.5 pmol of CK O-glucosides and nucleotides added per sample) and purified using two solid phase extraction columns. CK concentration was determined by UHPLC-MS/MS (ultra-high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer equipped with an electrospray interface). Quantification was performed with Masslynx software (v4.1; Waters) using a standard isotope dilution method. The ratio of endogenous CK to the appropriate labeled standard was determined and used to quantify the level of endogenous compounds in the original extract according to the known quantity of the added internal standard.