3200 q trap hybrid triple quadrupole linear ion trap mass spectrometer

Hormone analysis was carried out on four samples, each of which contained all seedlings from six of the 24 wells or from the four-week-old A. thaliana three leaf discs from every single plant were sampled, three individual plants were sampled as one sample. Plant hormone levels were determined as described by^{40 (link)}. Briefly, samples were homogenized in tubes with 1.3 mm silica beads using a FastPrep-24 instrument (MP Biomedicals, USA). The samples were then extracted with a methanol/H₂O/formic acid (15:4:1, v:v:v) mixture, which was supplemented with stable isotope-labeled phytohormone internal standards (10 pmol per sample) in order to check recovery during purification and validate the quantification. The clarified supernatants were subjected to solid phase extraction using Oasis MCX cartridges (Waters Co., USA). The eluates were evaporated to dryness and the generated solids dissolved in 30 μl of 15% (v/v) acetonitrile in water. Quantification was performed on an Ultimate 3000 high-performance liquid chromatograph (Dionex, USA) coupled to a 3200 Q TRAP hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems, USA) as described by^{41 (link)}. Metabolite levels were expressed in pmol/g fresh weight (FW).

Frozen samples (ca 10 mg FW) were homogenized and extracted with cold (−20 °C) methanol/water/formic acid (15/4/1, v/v/v) as described in Dobrev and Kaminek [16 (link)] and Dobrev and Vankova [17 (link)]. The following isotope-labelled internal standards (10 pmol/sample) were then added ²H₅-tZ, ²H₅-tZR, ²H₅-tZRMP, ²H₅-tZ7G, ²H₅-tZ9G, ²H₅-tZOG, ²H₅-tZROG, ²H₃-DZ, ²H₃-DZR, ²H₃-DZ9G, ²H₃-DZRMP, ²H₇-DZOG, ²H₆-iP, ²H₆-iPR, ²H₆-iP7G, ²H₆-iP9G, ²H₆-iPRMP (Olchemim, CR, Olomouc, Czech Republic). Phytohormones were separated with a reverse phase-cation exchange SPE column (Oasis-MCX, Waters, Milford, MA, USA) into the acid fraction by elution with methanol and into the basic fraction by elution with 0.35 M NH₄OH in 60% methanol which was used for CK determination. The latter fraction was analyzed using HPLC (Ultimate 3000, Dionex, Sunnyvale, CA, USA) coupled to 3200 Q TRAP hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems, Waltham, MA, USA). Hormone quantification was performed by the isotope dilution method with multilevel calibration curves (r² > 0.99). Data processing was performed with the Analyst 1.5 software package (Applied Biosystems, Waltham, MA, USA).

The youngest fully expanded leaf samples (ca 50 mg FW) were purified and analyzed according to Dobrev and Kamínek (2002), Dobrev and Vankova (2012) and Svačinova et al. [94 –96 ]. Frozen samples were homogenized and extracted with cold (− 20 °C) methanol/water/formic acid (15/4/1, v/v/v). The following isotope-labelled internal standards (10 pmol/sample) were added: ¹³C₆-IAA (Cambridge Isotope Laboratories); ²H₄-SA (Sigma-Aldrich); ²H₃-PA, ²H₃-DPA (NRC-PTI); ²H₆-ABA, ²H₅-JA, ²H₅-transZ, ²H₅-transZR, ²H₅-transZ7G, ²H₅-transZ9G, ²H₅-transZOG, ²H₅-transZROG, ²H₅-transZRMP, ²H₃-DZ, ²H₃-DZR, ²H₃-DZ9G, ²H₆-iP, ²H₆-iPR, ²H₆-iP7G, ²H₆-iP9G, ²H₆-iPRMP (Olchemim). Phytohormones were separated with a reverse phase-cation exchange SPE column (Oasis-MCX, Waters) into the acid fraction by elution with methanol [auxins, abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA)], and into the basic fraction by elution with 0.35 M NH₄OH in 60% methanol [cytokinins (CKs)]. Fractions were analyzed using HPLC (Ultimate 3000, Dionex) coupled to a 3200 Q TRAP hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems). Hormone quantification was performed by the isotope dilution method with multilevel calibration curves (r² > 0.99). Data processing was performed with the Analyst 1.5 software package (Applied Biosystems). Raw data are included in the Supplemented Table 3.