## 3 Discussion
Conversely, GSH content slightly decreased after DYE treatment, even though no significant differences were observed. In plants, glutathione contributesto sulfur metabolism, redox control, and detoxification, and in SB berries, it contributes to the biosynthesis of the above-mentioned 3 MH precursors by glutathionylation of trans-2-hexenal, a product of the LOX pathway [18]. Gamma glutamyl transpeptidase (GTT) is responsible for the conversion of GSH-MH into Cys-MH [12]. Indeed, the higher and lower expression, respectively, of the _VvGST3_ and _VvGGT_ encoding genes (Figure 5), and the above-described levels of the respective metabolites, may support the hypothesis of a stimulation of GSH conjugation with trans-2-hexenal in response to DYE. LOX pathway-derived products are often described as having an herbaceous, leafy \"green\" aroma in wine [97], thus the stimulation of their conjugation with GSH may also result in a reduced herbaceous character. Thus, our findings suggest for the first time that DYE treatment may increase the activity of the LOX pathway and induce a faster turnover of 2-hexenal and GSH through the regulation of GST and GGT. The analysis of the complete profiles of amino acids showed that the treatment with DYE overall did not strongly interfere with the evolution trends of the amino acid contents during ripening, which showed a progressive increase towards later ripening stages (from TP2 to H) for valine, methionine, proline, and tryptophan and a progressive decrease for arginine, asparagine, and GABA (Figure 3). When analyzing the single compounds, arginine and proline remained the most representative amino acids, accounting for 67% of total amino acids both in control and treated berries at ripening, although with different percentages in the two theses (Figure 3B). Nevertheless, DYE induced a delay in arginine decrease as well as in proline increase, resulting in a higher arginine-to-proline ratio at H. A higher arginine-to-proline ratio means a balanced proportion of the yeast-assimilable amino acids that are required to carry on a satisfying fermentation, particularly when a yeast strain with high nitrogen requirements is used [98]. Similarly, valine was more accumulated at most time points after DYE spraying during both years (2020 and 2021). According to [PERSON] et al. (2015) [99], valine is degraded into isobutyl alcohol, an aliphatic alcohol that is fermented by yeast, giving a desired fruity and floral aroma to wines [99]. The increase of this amino acid in grapes after DYE treatment may result in an improvement in wine aroma and is consistent with the effects suggested by [PERSON] et al. (2016) [15], reporting specifically on the effects exerted by DYE on berry amino acidic composition and its potential consequences on yeast fermentation [15]. Similarly, benzeneacetaldayide, which is synthesized from phenylalanine and appreciated in the sensory profile of wines, providing sweet flowery notes [100; 101], significantly increased in response to DYE. The downregulation of phenylalanine in one vintage may be explained because of its transformation into benzeneacetaldahyde. Proline has been suggested to play an adaptive role in plant stress tolerance, acting as a ROS scavenger, stabilizing proteins' structures, and signaling stress [102]. The observed reduction in proline levels induced by DYE treatment may be an indication of reduced berry stress. Similarly, GABA is a molecule associated with responses to abiotic and biotic stress factors [35; 103]. The decrease in GABA may further confirm the above findings and suggest that DYE could improve the overall health of the vine. These hypotheses were also supported by the untargeted RNA-Seq molecular analyses carried out on samples of the 2020 vintage, through: (1) the comparison of treated versus untreated berries at each time-point (TP2 and H); and (2) the evaluation of the DYE effects on the dynamics of ripening-associated genes.