## 3 Results and Discussion  
_E. sativa_ is rich in GSLs, making it an interesting plant species for the development of bioproducts. Salinity stress is one of the abiotic stresses that showed a correlation with GSLs content in Brassicaceae [46] and, according to [PERSON] et al., 2009 and [PERSON] et al., 2012 [47, 48], AGs are the most effective osmolytes in plants salt stress activated response. Broccoli exposed to salinity stress exhibit different effects on GSL synthesis and accumulation in different cultivars, with variations in both AGs and indole GSLs, while in _B. rapa_ salt treatments, and, in particular, Na\({}_{2}\)SO\({}_{4}\), caused an increase in the concentration of aliphatic, indole and aromatic GSLs, as well as the expression of key AG biosynthetic genes like _CYP79F1_[31, 32]. Furthermore, a mild salt stress (34 mM NaCl) positively impacts on glucoerucin, 4-methylthiobutyl GSL, (GER) and glucoarphanin, 4-methylsulfinylbutyl GSL, (GRA) content in _E. sativa_ shoots [36], while no significitive differences in glucosinolate hydrolysis products in _Eruca sativa_ leaves were evidenced at higher NaCl levels (65 and 130 mM) [49]. In the present study, we investigated Na\({}_{2}\)SO\({}_{4}\) elicitation as a strategy for AGs modulation in different organs of _E. sativa_ examined at the flowering stage, and the modulation effect on the expression profile of selected putative key biosynthetic and regulatory genes was also evaluated. Starting from the roots upwards, an increasing concentration of total AGs was observed, ranging from about 15-20 \(\upmu\)mol/g in roots to 25-50 \(\upmu\)mol/g in apical leaves and more than 60 umol/g in flowers, with a different composition, GER being the main one in roots, dimeric 4-mercaptoethanolGSL (dimeric 4-MBGSL) in leaves and GRA in flowers. The full characterization of desulfated dimeric 4-MBGSL is reported in Supplementary file (Figures S2 and S4; Tables S2 and S3). In roots, AGs were 94-98% of the total GSL content in all samples: GER was the main GSL, followed by GRA (Figure 2a,d); while indolic GSLs (4-hydroxyglucobrassicin and neoglucobrassicin) were detected in traces. GER increased upon the highest dose of Na2 SO4 in roots by 2.5 times (Figure 2a). Interestingly, at transcriptional level, the elicitation with Na2 SO4 at the highest concentration determined also a significant upregulation of the _BCAT4_ gene (Figure 2d). However, in the same organ and conditions, the other biosynthetic genes examined in the current study did not show changes or were downregulated. Regarding the regulatory TFs, only _MYC4_ was found slightly, but significantly upregulated upon salt treatment at 25 mM and _MYC3_ slightly downregulated at the highest concentration (Figure 2d). GSL profiles of leaves were characterized by the presence of GRA, GER and dimeric 4-MBGSL, with dimeric 4-MBGSL being the main GSL (Figure 2b). Traces of 4-(-\(\beta\)-D-glucopyranosyldisulfanyl)butyl GSL were also detected by HPLC and identified in comparison to the retention times and UV spectra of the standard purified as described in \"Glucosinolate extraction and analysis\" section and reported in Supplementary file (Figures S1, S3 and S4; Tables S2 and S3). In apical leaves, the concentration of dimeric 4- MBGSL roughly doubled due to the treatment with Na2 SO4 (Figure 2b). Interestingly, upon salt treatment, _BCAT4_, _MAMI_ and _CYP83A1_ genes were downregulated, and the GER content was also lower, less than 50% compared to the control (Figure 2b,e) suggesting a role in AG synthesis in _E. sativa_ yet to be elucidated. A decrease in GER in basal leaves, which have the lowest content of GSLs (15-20 umol/g) compared to the other epigeal organs (Figure S5), but a higher total biomass, could suggest a possible role in the increase in dimeric 4-MBGSL in apical leaves not of de novo synthesis, but of translocation of GSLs, which are known to move upwards to seeds during maturation [50], and of non-enzymatic dimerization [51].