GSL analysis by HPLC-UV
Fresh seedlings (20 to 100 mg) were harvested, weighted and freeze-dried
until constant weight and ground to fine powder. GSLs were extracted
with 1 mL of 80% methanol solution containing 0.05 mM of Sinalbin as
internal standard. After centrifugation, 700 µL of extract was loaded
onto DEAE Sephadex A 25 columns and treated with arylsulfatase for
desulfation (Sigma-Aldrich). The eluted desulfo-GSLs were separated
using high performance liquid chromatography (Agilent 1100 HPLC system,
Agilent Technologies) on a reversed phase column (Nucleodur Sphinx RP,
250 x 4.6 mm, 5 µm, Macherey-Nagel, Düren, Germany) with a water (A) -
acetonitrile (B) gradient: 0 - 1.0 min, 1.5% B; 1.0 - 6.0 min, 1.5-5%
B; 6.0 - 8.0 min, 5 - 7% B; 8.0 - 18.0 min, 7 - 21% B; 18.0 - 23.0
min, 21 - 29% B; 23.0 - 23.1 min, 29 - 100% B; 23.1 - 24.0 min 100% B
and 24.1 - 28.0 min 1.5% B; flow 1.0 mL min-1.
Detection was performed with a photodiode array detector and peaks were
integrated at 229 nm. Desulfated GSLs were identified by comparison of
their retention time and UV spectra to those of purified standards
previously extracted from A. thaliana(Brownet al. , 2003). We used the following molar response factors for
quantification of individual GSL relative to the internal standard
Sinalbin: 2.0 for aliphatic GSLs and 0.5 for indolic GSLs
(Burowet al. , 2006).