Determination of phytohormones
Four leaves were harvested of each tree, 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-indole-3-acetic 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; the
supernatant was collected. The homogenate was re-extracted with 500 μl
methanol, mixed and centrifuged; the supernatants were pooled. The
combined extracts were evaporated under reduced pressure at 30 °C and
dissolved in 500 μl 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.
Indole-3-acetic 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 IAA; m/z 181 →133 + m/z 181 →134
+ m/z 181 →135 (CE 19 V; DP 31 V) for D5-IES.