Determination of phytohormones
From each tree four leaves were harvested and immediately frozen in liquid nitrogen and stored at -20 °C. The leaves of each tree were pooled and 250 mg (per sample and two samples for each tree) were homogenized using a Geno/Grinder® (Spex SamplePrep, Stanmore, UK) at 1100 rpm for 1 min and extracted in 1.5 ml methanol containing 60 ng D4-SA (Santa Cruz Biotechnology, USA), 60 ng D6-JA (HPC Standards GmbH, Germany), 60 ng D6-ABA (Santa Cruz Biotechnology, USA), 12 ng D6-JA-Ile (HPC Standards GmbH), and D5-indolacetic acid (D5-IAA, OlChemIm s.r.o., Olomouc, Czech Republic) as internal standards. Samples were agitated on a horizontal shaker at room temperature for 10 min. The homogenate was mixed for 30 min and centrifuged at 13,000 rpm for 20 min at 4 °C and the supernatant was collected. The homogenate was re-extracted with 500μl methanol, mixed and centrifuged and the supernatants were pooled. The combined extracts were evaporated under reduced pressure at 30 °C and dissolved in 500μl methanol.
Phytohormone analysis was performed by LC-MS/MS as in Heyer et al. (2018) on an Agilent 1260 series HPLC system (Agilent Technologies) with the modification that a tandem mass spectrometer QTRAP 6500 (SCIEX, Darmstadt, Germany) was used. Details of the instrument parameters and response factors for quantification can be found in Table S2.
Indolacetic acid was quantified using the same LC-MS/MS system with the same chromatographic conditions but using positive mode ionization with an ion spray voltage at 5500 eV. Multiple reaction monitoring (MRM) was used to monitor analyte parent ion → product ion fragmentations as follows: m/z 176 →130 (collision energy (CE ) 19 V; declustering potential (DP) 31 V) for indolacetic acid (IAA); m/z 181 →133 + m/z 181 →134 + m/z 181 →135 (CE 19 V; DP 31 V) for D5-indolacetic acid.