Plant hormone extraction and analysis
The main classes of plant hormones, cytokinins [trans- zeatin
(t-Z), zeatin riboside (ZR) and isopentenyladenine (iP)], gibberellin
A3 (GA3), indole acetic acid (IAA), abscisic acid (ABA),
jasmonic acid (JA), salicylic acid (SA) and the ethylene precursor
1-aminocyclopropane-1-carboxylic acid (ACC), as well as the ABA
catabolites, dihydrophaseic acid (DPA) and phaseic acid (PA) were
extracted and analysed as described previously in Albacete et al. (2008)
with some modifications. Fresh plant material (0.1 g FW of leaf or root)
was homogenized in liquid nitrogen and incubated in 1 mL of cold (-20°C)
extraction mixture of methanol/water (80/20, v/v) for 30 min at 4ºC.
Solids were separated by centrifugation (20,000 g, 15 min at 4ºC) and
re-extracted for another 30 min at 4ºC with 1 mL of extraction solution.
Pooled supernatants were passed through Sep-Pak Plus C18 cartridges
(previously conditioned with 3 mL of extraction buffer) to remove
interfering lipids and some plant pigments. The supernatant was
collected and evaporated under vacuum at 40ºC. The residue was dissolved
in 1 mL methanol/water (20/80, v/v) solution using an ultrasonic bath.
The dissolved samples were filtered through 13 mm diameter Millex
filters with 0.22 µm pore size nylon membrane (Millipore, Bedford, MA,
USA) and placed into opaque microcentrifuge tubes.
Ten µL of filtered extract (xylem, leaf or root) were injected in a
U-HPLC-MS system consisting of an Accela Series U-HPLC (ThermoFisher
Scientific, Waltham, MA, USA) coupled to an Exactive mass spectrometer
(ThermoFisher Scientific, Waltham, MA, USA) using a heated electrospray
ionization (HESI) interface. Mass spectra wereobtained using the
Xcalibur software version 2.2 (ThermoFisher Scientific, Waltham, MA,
USA). To quantify the plant hormones, calibration curves were
constructed for each analysed component (0, 1, 10, 50, and 100 µg
L-1). ABA catabolites were identified by extracting
the exact mass of the target catabolite from the full scan chromatogram
obtained in the negative mode, adjusting a mass tolerance of ≤ 1 ppm.
The concentrations were semi-quantitatively determined from the
extracted area of the derivative peak by using the calibration curve of
ABA.