Phytohormone levels
Entire plant shoots (comprising leaves, stipules and stems) that were
fed on by A. pisum were harvested and metabolism was immediately
stopped by immersion in liquid nitrogen. Samples were stored at
-80oC before freeze drying. Five replicates per line
(wild type, rms3-1 and rms4-1 mutant pea) were analysed
for cytokinins (trans -zeatin, tZ, zeatin riboside, ZR, and
isopentenyl adenine, iP), gibberellins (GA1,
GA3{gibberellic acid}, and GA4),
indole-3-acetic acid (IAA), abscisic acid (ABA), salicylic acid (SA),
jasmonic acid (JA), and the ethylene precursor
1-aminocyclopropane-1-carboxylic aid (ACC) according to Albaceteet al. (2008) with some modifications. Freeze-dried tissue (50 mg
dry weight, DW) was extracted overnight at -20°C using a
methanol/water/formic acid solution (15/4/1 by volume, pH 2.5). Then, 10
μL of internal standard mix, comprising deuterated phytohormones
([2H5]tZ,
[2H5]tZR,
[2H6]iP,
[2H2]GA1,
[2H2]GA3,
[2H2]GA4,
[2H5]IAA,
[2H6]ABA,
[2H4]SA,
[2H6]JA,
[2H4]ACC, Olchemim Ltd, Olomouc,
Czech Republic) each at 1 μg·mL−1, was added to the
extraction homogenate. Solids were then separated by centrifugation (20,
000 g) for 15 mins, and extracted again for 30 mins at 4°C in an
additional 0.5 mL of the same extraction solution. The pooled
supernatants were filtered through a Sep-Pak Plus C18 cartridge (SepPak
Plus, Waters, USA) to remove interfering lipids and plant pigments, and
evaporated at 40ºC under a vacuum either to near dryness or until
organic solvent was removed. Any remaining residue was dissolved in 1 mL
methanol/water (20/80, v/v) in an ultrasonic bath. The dissolved samples
were filtered through 13 mm diameter Millex filters with 0.22 μm pore
diameter nylon membrane (Millipore, Bedford, MA, USA).
Ten μL of filtered extract were injected into a U-HPLC-MS system
comprising 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 ionisation
(HESI) interface. Xcalibur software version 2.2 (ThermoFisher
Scientific, Waltham, MA, USA) was used to obtain mass spectra. To
quantify the plant hormones, calibration curves were constructed for
each analysed component (1, 10, 50, and 100 μg·L−1)
and corrected for 10 μg·L−1 deuterated internal
standards. Recovery percentages ranged between 90 and 95%.