Aryl sulphatase

GSs were extracted and analyzed as previously described with minor modifications (Guo et al., 2011 (link)). Samples (500 mg) were boiled in 3 mL water for 10 min. After transferring the supernatant to a new tube, the residues were washed with water (3 mL), and the combined aqueous extract was applied to a DEAE-Sephadex A-25 (30 mg) column (pyridine acetate form; Sigma, St. Louis, MO, USA). The column was washed three times with 20 mM pyridine acetate and twice with water. The glucosinolates were converted into their desulfo analogs by overnight treatment with 100 μL of 0.1% (1.4 units) aryl sulphatase (Sigma, St. Louis, MO, USA) added into the column, and the desulfoglucosinolates were collected by eluting with 2 × 0.5 mL water. HPLC analysis was performed using an HPLC system consisting of an Agilent HPLC series chromatograph (Agilent Technologies). The same C18 column and procedure was used as described in Guo et al. (2011 (link)). The peak was detected at 226 nm. Ortho-nitrophenyl-β-d-galactopyranoside (Sigma, St. Louis, MO, USA) was used as an internal standard. The glucosinolate content was calculated as μmol/g fresh weight.

Glucosinolate analysis was performed as described previously (van Dam et al., 2004 ). Lyophilized, finely ground above-ground tissue was dissolved in 1ml 70% MeOH in water (v/v) in a 2ml Eppendorf tube, vortexed, and immediately boiled for 5min to inactivate any remaining myrosinase. The tubes were placed in an ultrasonic bath for 15min and centrifuged (10min, 10 000rpm). The extraction was repeated for the pellet, but with the boiling step omitted. Both supernatants were combined per sample and applied to a DEAE-Sephadex A 25 column, desulphated with arylsulphatase (Sigma, St. Louis, IL, USA), and separated on a reversed phase C-18 column on HPLC with a CH₃CN–H₂O gradient. Glucosinolate detection was performed with a Photo Diode Array (PDA) detector (200–350nm) with 229nm as the integration wavelength. Sinigrin (2-propenylglucosinolate) was used as an external standard. The response factors at 229nm from three sources (Buchner, 1987 ; EC, 1990 ; Brown et al., 2003 (link)) were used to calculate the concentrations of the glucosinolates. Desulphoglucosinolate peaks were identified by comparing HPLC retention times and UV spectra with standards kindly provided by M. Reichelt (MPI Chemical Ecology, Jena, Germany) and a certified rape seed standard (Community Bureau of Reference, Brussels, code BCR-367R).

Quantitative analyses of GSs were performed as previously described (Miao et al. 2013; Xu et al. 2016). Briefly, freshly collected seedlings were boiled twice in 1 mL water for 10 min to extract GSs. Then the aqueous extract was applied to a DEAE‐Sephadex A‐25 column (30 mg, pyridine acetate form, Sigma) and treated by aryl sulphatase (Sigma) overnight. The desulphoglucosinolates were analyzed by an Angient 1260 Infinity II LC System equipped with a Spherisorb C18 column (5‐µm particle size, 4.6 mm × 250 mm). ONPG (Solarbio) was used as an internal standard.