## 3 Discussion
The latter analysis allowed the identification of four different groups of DEGs clustered according to their ripening specific expression profiles and significantly affected by the treatment in terms of either an increased or decreased expression level (Figure 8). For one group of genes (Group 1, Figure 8), the DYE treatment, besides resulting in a significant stimulation of transcription at both TP2 and H, reverted the trend of expression along ripening from a downregulation into an upregulation. Indeed, one of the main findings of this work was the identification of several genes involved in abiotic and biotic stress responses, highlighted as differentially expressed in later analyses. The identification of the term \"response to chitin\" in the upregulated DEG enrichment strategy suggests the elicitor-like mode of action of DYE, as well as the enrichment terms \"responseto heat\", \"response to hypoxia\", and \"response to pathogens and effector triggered immunity\" clearly indicated the stimulation by DYE of responses that recalled those typical of abiotic and biotic stresses (Figures 7 and 8). Since chitin is a ubiquitous cell wall structural component of fungi and is contained in DYEs, plants have chitin-specific receptors that activate defense mechanisms, relying on phenolics, terpenes, and ROS production [104; 105]. Our RNA-seq data support the hypothesis that DYE affected ROS homeostasis in the berries, as testified by the upregulation of a gene encoding glutathione peroxidase (_Vitroi2g00332_, group 1), responsible for the transformation of glutathione (GSH) into its oxidized form glutathione disulfide (GSSG) to scavenge H\({}_{2}\)O\({}_{2}\) in excess. The GSH:GSSG ratio is in fact an important bioindicator of cellular oxidative stress [27; 106]. The DYE treatment also stimulated sulfate metabolism by activating the transcription of the gene encoding APS (adenosine phosphosulfate), which is involved in the production of cysteine, which in turn contributes to GSH levels. This is in accordance with the tendentially higher (even though not significantly higher) contents of cysteine observed in treated samples in the 2020 vintage (supporting data, Table S1). Furthermore, as reported above, the concomitant upregulation of several genes involved in the modulation of ROS homeostasis, such as that encoding the Redox Responsive AP2/ERF transcription factor ERF109 (_Vitroi03g00500_, group 1) (activator of ROS quenching genes) and a respiratory burst oxidase (RBOHB) (_Vitroi14g00183_, group 2) generating H\({}_{2}\)O\({}_{2}\), led us to conclude that DYE is able to trigger an augmented ROS biosynthesis/scavenging loop and, consequently, an improved abiotic stress tolerance of berries (Table 1). This protective effect could depend on the downstream upregulation of a subset of ROS-inducible genes encoding proteins involved in controlling thermotolerance, such as heat shock proteins that were previously shown to be ROS-inducible (e.g., HSP 17.4, 70.1, and 90.1) (_Vitroi13g00491_, _Vitroi08g02189_, _Vitroi09g00045_, group 1; _Vitroi06g00443_, _Vitroi02g00025_, _Vitroi16g01103_, group 2) [36; 38; 40], NO-inducible (heat shock transcription factor A2) (_Vitroi01g01846_, group 1) [30], ABA-inducible (A6B) (_Vitroi07g00078_, group 1), or heat stress-inducible (anti silencing function ASF1) (_Vitroi1g00372_, group 1) [42]. These genes share a common transcriptional responsiveness in the presence of ROS, so this may be considered a downstream consequence of the above-mentioned increased ROS homeostatic levels evoked by the DYE treatment. In addition, the same genes are involved in the long-term regulation of heat stress responses and thermotolerance through the activation of heat stress memory genes and histone modifications [32]. Considering these data together, our findings provide for the first time significant molecular evidence for the previously hypothesized ([PERSON] et al., 2020) [14] elicitor-like mode of action of DYE on grapevine berries.