Deuterium labeled hormones

Determination/quantification of the phytohormones ABA, indole-3-acetic acid (IAA), JA, SA, and the phytoalexin camalexin was performed after 9 days of co-cultivation according to Großkinsky et al. (2014 (link)) with slight modifications. Briefly, volatile exposed and control seedlings were frozen and ground in liquid nitrogen; 200 mg per sample were extracted with 80% methanol and internal standards were added for the quantification of camalexin (6-fluoroindole-3-carboxyaldehyde; Sigma-Aldrich, Steinheim, Germany) and the phytohormones (deuterium-labeled hormones; Olchemim Ltd, Olomouc, Czech Republic). The extracts were directly subjected to HPLC analysis for camalexin (Ultimate 3000; Dionex, Sunnyvale, USA). For phytohormone determination, methanol extracts were passed through Chromafix C18-columns (Macherey-Nagel, Düren, Germany), completely dried (Integrated SpeedVac® Concentrator System AES1000; Savant Instruments Inc., Holbrook, USA), resuspended in 20% methanol, passed through Chromafil PES-20/25 filters (Macherey-Nagel, Düren, Germany) and subjected to UHPLC-MS/MS (Thermo Scientific; Waltham, USA) analyses.

For ABA measurements, 50 mg of lyophilized fruit pericarp was ground and resuspended in 80% methanol—1% acetic acid solution containing internal standards (deuterium-labeled hormones; OlChemim Ltd., Olomouc, Czech Republic). The mix was shaken for one hour at 4 °C, and the extracted fraction was incubated overnight at −20 °C. The tissue extractions were conducted in triplicate for each developmental stage. Samples were centrifuged, and the supernatant was vacuum dried and dissolved in 1% acetic acid. A reverse-phase column (OasisHLB) was used, and the eluate was dried and dissolved in a solution of 5% acetonitrile and 1% acetic acid. An autosampler and reverse-phase UHPLC chromatography column, 2.6 μm Accucore RP-MS, 100 mm × 2.1 mm (Thermo Fisher Scientific, San Diego, CA, USA) was used. ABA was separated through a gradient of acetonitrile (2–55%) containing 0.05% acetic acid at a rate of 400 μL/min over 22 min and detected in a Q-Exactive mass spectrometer (Orbitrap detector; ThermoFisher Scientific; San Diego, CA, USA). Targeted Selected Ion Monitoring and Electrospray Ionization in the negative mode were used to detect ABA. Measurements were performed using external calibration curves with the Xcalibur 4.0 and TraceFinder 4.1 SP1.

ABA, IAA, GA_1, and GA₄ were measured in both varieties at 34, 38, and 44 DAFB in the 2018–2019 season. For the extraction, 10 mg of flesh- and peel-enriched tissue was freeze-dried, ground, and suspended in 80% methanol—1% acetic acid solution containing internal standards (deuterium-labeled hormones; OlChemim Ltd., Olomouc, Czech Republic). The mix was shaken for one hour at 4ºC, and the extracted fraction was maintained at − 20ºC overnight. The samples were centrifuged, and the supernatant was vacuum dried and then dissolved in 1% acetic acid. A reverse-phase column (OasisHLB) was used⁵⁸ , and the eluate was dried and dissolved in 5% acetonitrile—1% acetic acid. An autosampler and reverse-phase UHPLC chromatography column, 2.6 µm Accucore RP-MS, 100 mm × 2.1 mm (ThermoFisher Scientific, San Diego, CA, USA) were used. Then the hormones were separated using a gradient of acetonitrile (2%-55%) containing 0.05% acetic acid, at a rate of 400 µL/min over 22 min. ABA, IAA, GA_1, and GA₄ were detected in a Q-Exactive mass spectrometer (Orbitrap detector; ThermoFisher Scientific; San Diego, CA, USA). Targeted Selected Ion Monitoring and Electrospray Ionization in the negative mode were used to detect the hormones⁵⁸ . The quantifications were performed using external calibration curves with the Xcalibur 4.0 and TraceFinder 4.1 SP1.