2h5 ja

Root samples were collected from ten-days old plants. Five root samples (150 mg each) were collected from Hs-Tyr-expressing plants (Line 2.3) or Col-0. Root samples were purified and analysed as mentioned previously^{43 (link), 44 (link)}. Briefly, samples were homogenized with a ball mill (MM301, Retsch) and extracted in cold (−20 °C) methanol/water/formic acid (15/4/1 v/v/v). The following labelled internal standards (10 pmol/sample) were added: ¹³C₆-IAA (Cambridge Isotope Laboratories); ²H₄-SA (Sigma-Aldrich); ²H₂-OxIAA and ²H₅-JA(Olchemim). Extracts were purified using SPE-C18 column (SepPak-C18, Waters) and a mixed mode reverse phase–cation exchange SPE column (Oasis-MCX, Waters). Hormone metabolites were analysed using HPLC (Ultimate 3000, Dionex) coupled to a hybrid triple quadrupole/linear ion trap mass spectrometer (3200 Q TRAP, Applied Biosystems). Quantification of hormones was done using the isotope dilution method with multilevel calibration curves (r² > 0.99). Data processing was carried out with Analyst 1.5 software (Applied Biosystems). Data are presented as mean ± standard error.

For hormone analysis A. thaliana and E. salsugineum leaf samples were collected at midday. Extraction and analysis were performed according to Dobrev and Kamínek (2002) (link) and Dobrev and Vankova (2012) (link). Briefly, approximately 100 mg fresh samples were homogenized and extracted with methanol/water/formic acid (15/4/1, v/v/v). The following labeled internal standards (10 pmol per sample) were added: ²H₆-ABA, ²H₃-PA, ²H₄-SA, ²H₅-JA (Olchemim, Czech Republic). Extracts were purified using SPE-C18 column (SepPak-C18, Waters, Milford, MA, USA) and separated on a reverse phase-cation exchange column (Oasis-MCX, Waters). The hormone fraction was eluted with methanol, separated by HPLC (Ultimate 3000, Dionex/Thermo Fisher Scientific, Austria) and the hormones were quantified using a hybrid triple quadrupole/linear ion trap mass spectrometer (3200 Q TRAP, Applied Biosystems/MDS SCIEX, Foster City, CA, USA). The analyses were carried out in three biological replicates. The analysis of ABA and its catabolites DPA (dihydrophaseic acid) and PA (phaseic acid); ACC (1-aminocyclopropane-1-carboxylic acid), SA (salicylic acid), jasmonic acid (JA) and jasmonate precursor cis-OPDA [cis-(+)-12-oxo-phytodienoic acid] and jasmonoyl-L-isoleucine (JA-Ile) was performed.