Chemical analyses
For chemical analyses of GSLs, immediately after detaching leaves for
the bioassays, all the remaining leaves were flash-frozen in liquid
nitrogen and ground to powder using mortars and pestles in liquid
nitrogen. A 100 mg aliquot was weighed for GLS extraction, and added
with the extraction solvent (1.0 ml methanol: H2O: formic acid
(70:29.5:0.5, v/v)) along with 5 glass beads, shaken in a tissuelyser
(Retsch GMBH, Haan, Germany) for 4 min at 30 Hz, and centrifuged at
12800 rpm for 3 min. The supernatant was diluted 20 times with 70%
methanol and transferred to an HPLC vial. GLS identification and
quantification was performed using an Acquity ultra-high pressure liquid
chromatography (UHPLC) from Waters (Milford, MA) interfaced to a Synapt
G2 quadrupole time-of-flight mass spectrometry (QTOF) from Waters with
electrospray ionization, using the method as described in (Glauseret al. 2012).