## 3 Results and Discussion
Moreover, in apical leaves a downregulation of _MYC3-4_ was observed upon high doses of salt treatment, suggesting a direct involvement in _BCAT4_, _MAMI_ and _CYP83A1_ regulation, which is indeed, not affected by _MYB28a-b_ in line with the reported independent regulatory roles in aliphatic GSL synthesis [17]. Flowers represent the organ with the highest concentration of GSLs among the samples; the main GSL in flowers is GRA, which is the relevant chemical tag of health studies on Brassicaceae. The salt elicitation boosted an increase in GRA by 1.3 times (Figure 2c) and consequently of total GSLs. The biosynthetic machinery leading to AGs in _E. sativa_ was proven to present unique features in terms of gene families respect to other species of Brassicales, due to gene duplication event [34]. Indeed, the major regulatory gene _MYB28_ is present in three paralogous sequences in _E. sativa_ genome (_MYB28a_, _MYB28b_, _MYB28c_) although the ones correlated to AGs modulation are mainly _MYB28a_ and _MYB28b_. Taken as a whole, in the transcriptional profiling of the _MYB28a_ and _MYB28b_ genes, no significant modulations were observed, suggesting that the expression of the _MYB28c_ should also be evaluated. Moreover, in terms of experimental design, the ontological stage for elicitation treatments should also be considered, since previous studies evidence that differences in both the content and the spatial distribution of GSL together with a species--specific modulation of their profile can be observed [36]. Since in some cases the variability across biological replicates of the same organ and treatment was too high to highlight significant modulation in gene expression, with the aim of descriptively correlating biochemical quantification of AGs in _E. sativa_ plants and transcriptional profiling of selected biosynthetic and regulatory genes across organs, a correlation analysis was performed (Figure 3). At the metabolite level, GER negatively correlates to the dimeric 4-MBGSL, as expected, being GER a putative precursor 4-MBGSL. _MAM1_ and _SUR1_, which are involved in subsequent steps of AGs synthesis (Figure 1), positively correlate with _MYC3_, but _MAM1_ negatively correlates with _MYB28b_, which also negatively correlates with GRA. A positive correlation was also found between _MYC3_ and _MYC4_, even though it was not statistically significant. _CYP83A1_ appeared scarcely in correlation with both biosynthetic and regulatory genes; thus, other mechanisms yet to be investigated could be involved. Overall, these results show some independent correlations of both _MYC3_ and _MYB28b_ with biosynthetic genes and AGs, while there is no evidence of correlation for _MYBB28a_ and _MYC4_. Possible causal relations to AG modulation of _MYC3_ and _MYBB28b_ would deserve further investigation. Moreover, according to the _A. thaliana_ aliphatic GSL model [16], the lack of clear relationship between expression levels of biosynthetic genes and metabolite levels could suggest post-transcriptional mechanisms and translocation across organs, which would also be worthy of further studies in this species. Figure 2: Aliphatic glucosinolate content (**a–c**) and gene expression (**d–f**) relative to control after salt treatments in roots (**a,d**), apical leaves (**b,e**) and flowers (**c,f**). Data are expressed as mean of three independent samples and are reported on a dry weight basis. “*” indicates significant difference vs. control (ctrl) groups (\(p<0.05\)).