## 2 Materials and Methods
### Glucosinolate Extraction and Analysis  
GSLs were extracted from finely powdered freeze-dried samples, i.e., basal and apical leaves (350 mg), flowers (200 mg) and roots (500 mg) (biological replicates \(n=3\)) using boiling 70% ethanol. The determination of GSLs was performed after desulfation following the ISO 9167:2019(en) [40] method by using a HPLC-UV system (HP 1100 Series Ag  
Figure 1: Simplified biosynthetic pathway of _Eruca sativa_ major aliphatic glucosinolates. The chemical structures of the key metabolites are provided. The substituent GSL is indicative of the invariable and characteristic core of the glucosinolates. ilent HPLC System, Agilent Technologies, Santa Clara, CA, USA) equipped with a diode array detector and a Pursuit RXs5 C18 (250 \(\times\) 3.0 mm, 5 \(\upmu\)m) (Agilent Technologies, Santa Clara, CA, USA) [41]. GRA and GER were identified with respect to retention times and UV spectra according to [PERSON] et al., 2016 [42], while 4-(-\(\beta\)-D-glucopyranosyldisulfanyl)butyl GSL and dimeric 4-mercaptoethanolGSL (dimeric 4-MBSGL) were first isolated as desalted GSLs in preparative HPLC and then recognized by nuclear magnetic resonance (NMR) and HPLC-MS analyses, according to the following procedures: 100 g of finely powdered freeze-dried _E. sativa_ leaves were extracted in 1 L boiling 30% aqueous ethanol. The extract was centrifuged, filtered and finally concentrated in rotavapor Laborota 4002-digital (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany) to 100 mL final volume. 50 mL of extract were divided among eight DEAE Sephadex A-25 mini-columns (6 mL DEAE Sephadex A-25 for each column) conditioned with 25 mM sodium acetate buffer (pH 5.6). After washing with the same buffer (10 mL), 0.2 U purified sulfatase (0.26 U/mL) was loaded onto each mini-column, which were left overnight at 20 \({}^{\circ}\)C. The desulfated GSLs were eluted in three steps of 2.5 mL water each, for each mini-column, pooled and tested for the presence of desulfated GSLs. As desulfated GSLs were detected only in the first two eluates, these were combined and freeze-dried for successive purification. The obtained powder was dissolved in 2 mL of water and filtered. For preparative HPLC, it was used a Pursuits XRs C18 column (250 \(\times\) 10.0 mm, 5 \(\upmu\)m) with a Pursuits XRs C18 guard column (50 \(\times\) 10.0 mm, 5 \(\upmu\)m) (Agilent Technologies, Santa Clara, CA, USA). The column was eluted at a flow rate of 3.5 mL/min with aqueous acetonitrile (ACN) (solvent A: water; solvent B: ACN) at 30 \({}^{\circ}\)C following the following program: 0-1 min isocratic 1% B; 1-28 min linear gradient 1-22% B; 28-37 min linear gradient 22-1% B; 37-40 min isocratic 1% B. The desulfated GSLs were detected by monitoring their absorbance at 229 nm and the fractions corresponding to the major AGs in _E. sativa_ leaves were collected from 10 runs (injection volume = 100 \(\upmu\)L). After removal of ACN by nitrogen evaporation the combined fractions were freeze-dried and subjected to HPLC-UV, HPLC-MS and to NMR spectroscopy for structural identification. The NMR spectra were recorded in D\({}_{2}\)O at 25 \({}^{\circ}\)C on a Varian Inova 600 spectrometer (Varian, Palo Alto, CA, USA) operating at 600 MHz (for 1H-NMR) and chemical shifts were measured in \(\delta\) (ppm) with reference to the solvent (\(\delta\) = 4.79 ppm). MS data were acquired using an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with a Raptor Biphenyl column (2.1 \(\times\) 100 mm, 2.7 \(\upmu\)m) (Restek, Bellfonte, PA, USA). The column was eluted with solvent A: 4 mM ammonium formate in water and solvent B: 4 mM ammonium formate in methanol, both containing 0.1% (\(v/v\)) formic acid, following the program: 0-2 min isocratic 2% B; 2-28 min linear gradient 2-40% B; 28-32 min linear gradient 2% B. MS spectra were acquired operating in full scan, positive polarity.