Branson 2510 ultrasonic cleaner

Abscisic acid levels were determined by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) as described previously (Müller and Munné-Bosch, 2011 (link)). In short, 100 mg per sample was extracted with 200 µl methanol/isopropanol/acetic acid 50:49:1 (v/v/v) using ultrasonication and vortexing (Branson 2510 ultrasonic cleaner, Bransonic, Danbury, CT, USA) for 30 min. Deuterium-labeled ABA was then added, and after centrifugation at 600×g for 15 min at 4°C, the pellet was re-extracted using the same procedure. Supernatants were pooled and filtered through a 0.22-µm polytetrafluoroethylene (PTFE) filter (Waters, Milford, MA, USA) before analyses. ABA levels were analyzed using UHPLC-ESI-MS/MS as described in Müller and Munné-Bosch (2011) (link). Quantification was performed considering recovery rates for each sample by using a deuterium-labeled internal standard. Three replicates were conducted for each treatment.

Plant hormones, including different jasmonates (OPDA, JA and the conjugated form JA‐Ile), ABA and the cytokinin t‐Z were extracted and quantified by ultrahigh‐performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS). Ground leaves (50 mg) and freeze‐dried roots (30 mg) were extracted with 250 μl of a methanol:2‐propanol:glacial acetic acid (50:49:1) mix and deuterium‐labeled standards (d5‐JA, d6‐ABA, and d5‐tZ). The extracts were subjected to ultrasonication (Branson 2510 ultrasonic cleaner, Bransonic) and vortexing for 30 min, followed by a 10 min centrifugation at 15,000 g (PrismR, Labnet International Inc.). The supernatant was collected, and the pellet was reextracted. Both supernatants were merged and filtered through a hydrophobic 0.22 μm filter (Phenomenex). Hormone levels were analyzed by UHPLC‐ESI‐MS/MS as described by Müller and Munné‐Bosch (2011 (link)). The UHPLC was coupled to a triple quadrupole mass spectrometer (QTRAP 4000, AB Sciex). A LUNA C18 column (Phenomenex, 1.6 μm, 100 × 2.1 mm) was used. Solvent A was water with 0.05% acetic acid and solvent B was acetonitrile with 0.05% acetic acid. Flow rate was set at 0.6 ml min⁻¹. Quantification was made considering recovery rates for each sample by using the deuterium‐labeled internal standards. Calibration curves for each analyte were generated using MultiQuantTM 3.0.1 software.

Hormonal profiling was performed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC/ESI-MS/MS) as described by Müller and Munné-Bosch (2011) (link). ABA, GA₁ and GA₃, IAA, JA, and SA were analyzed using negative ion mode and the CKs, IPA, tZ, and tZR using positive ion mode. Extracts were performed using 100 mg of well powdered fresh leaf with a mixture of methanol and acetic acid (99:1, v/v) as a solvent. Deuterium-labeled plant hormones were added to the extract and 250 mm³ of the final mixture was vortexed and ultra-sonicated for 30 min (Branson 2510 ultrasonic cleaner, Bransonic, Danbury, CT, United States). Then the extract was vortexed again and centrifuged for 10 min at 4°C and 200 g. The supernatant was collected, filtered using a 0.22 μm PTFE filter (Waters, Milford, MA, United States) and injected into the HPLC/ESI-MS/MS system.