## 2 Results
### Targeted and Untargeted Molecular Responses: Comparison of DYE-Treated versus Untreated Berries  
Since DYE treatments result in the enhancement of some aroma components, the transcriptional expression of the main genes involved in berry aroma development [1; 12; 18] was determined through a targeted approach (RT-qPCR) on berries collected from both  
Figure 4: Aroma precursors determined in DYE-treated and untreated control Sauvignon Blanc’s berries. (**A**) shows bound aroma precursors and glutathione quantitated at harvest in the two years 2020 and 2021, while (**B**) represents free aroma precursors determined at all time points in samples collected in 2020. The significant differences were determined with a \(t\)-test with \(p\)-value \(<\) 0.1 (*) and with \(p\)-value \(<\) 0.05 (**). years to gain a first characterization of DYE molecular effects. Several genes (in total eight genes) were studied, including those encoding _VvGGT_ (Gamma-Glutamyl Transferase) (one gene) and _VvGST_ (Glutathione S-transferase) (one gene), selected on the basis of their role in the biosynthesis and metabolization of GSH- and cysteine-bound thiol precursors [12; 18]. _VviCCD_ (Carotenoid Cleavage Dioxygenase) (one gene) was studied for its role in the biosynthesis of norisoprenoids precursors from apocarotonoids [1; 19]. Among these, some displayed a specific expression trend and responsiveness to DYE treatment (shown in Figure 5) (all expression data are reported in Table S4). All the considered genes showed an increasing level of expression along with the progression of ripening, reaching a maximum at H. Some of them displayed further increases or decreases of expression in response to DYE, but no interferences with the overall expression dynamics were noticed. In fact, in treated grapes, the transcript levels of the _VvGST3_ gene (involved in the biosynthesis of GSH-MH) [18] appeared tendentially higher than those found in control grapes for both years. Conversely, _VvGGT_ (responsible for the conversion of GSH-3 MH into cys-3 MH) [12] showed always more abundant transcripts in control samples, even though more evidently at H in 2021, and _VviCCD4a_ was more abundantly transcribed in treated grapes at H in both years. Figure 5: Normalized expression of genes encoding enzymes involved in the biosynthesis of berry aromas or of aroma precursors. The gene expression was evaluated in both years in treated and untreated control samples at subsequent developmental ripening stages (EL-36 to EL-38) and time points. _VvGGT_ (Gamma-Glutamyl transferase) and _VvGST_ (Glutathione S-transferase) are involved in glutathione metabolism, while _VviCCD4a_ (Carotenoid Cleavage Dioxygenase) is involved in carotenoid metabolism and in the generation of norisoprenoids. Significant differences (*) (_p_-value < 0.1) and very significant differences (**) (_p_-value < 0.05) were determined with a _t_-test. To have a comprehensive, untargeted overview of the transcriptional profiles induced or repressed by the treatment with DYE, RNA-Seq analysis was performed at TP2 and H in berries collected from the year 2020 (Table S5). The selection of the samples to analyze was based on the more prominent effects exerted by the DYE treatment on the overall amino acid (Figures 2 and 3) and aroma precursor (Figure 4) profiles in this vintage. At first, a very stringent analysis was carried out, comparing treated versus control samples at each time point taken independently, to highlight the effects of the treatment without considering the effects of time (ripening). This analysis showed that there were more differentially expressed genes (DEGs) between treated and control samples at H (448 DEGs) rather than at TP2 (118 DEGs), and most DEGs at H were upregulated (301 DEGs) by the treatment, while at TP2 the number of up- or down-regulated genes was similar (54 and 64, respectively) (Figure 6). The majority of DEGs showed differences in the order of \(0.6<\log 2<1\) (upregulated) and \(-1<\log 2<-0.6\) (downregulated).