## 2 Results
### Targeted and Untargeted Molecular Responses: Comparison of DYE-Treated versus Untreated Berries
This effect did not evidence a substantial interference of the treatment, with the expression trends over time remaining a progressive downregulation towards ripening for genes belonging to group 4 and an upregulation for genes belonging to group 3. An enrichment analysis was performed for each group. Group 1 (including 225 DEGs) showed significant enrichments for the terms \"response to chitin\" and \"respiratory burst involved in defence response\" (Figure 8C). Group 2, probably due to a lower number of genes (41 DEGs), did not highlight any enrichment results. Group 3 (64 DEGs) evidenced the significantly enriched terms \"terpene synthase activity\" and \"terpenoid biosynthetic process\", \"lyase activity\", and \"magnesium ion binding\". Finally, group 4 was enriched for the term \"vacuolar membrane\". Figure 8: Results of the untargeted analysis considering the interaction of time per treatment on the modulation of ripening-related genes in the berry. (**A**) is a heatmap showing the clustering of four groups according to gene’s behavior throughout ripening from TP2 to H. (**B**) shows the dynamics of expression of each group of genes indicated in the corresponding clusters identified in (**A**). (**C**) reports the enrichment analysis made with G:Profiler for each group. Group 2 is not shown since no enrichment could be identified due to the low number of genes for this group. An in-depth analysis of the putative function of the DEGs belonging to the four groups pointed out a selective transcriptional modulation of several genes playing a role in abiotic and biotic stress responses and in specific hormonal metabolic and transduction processes in response to DYE treatments (listed in Table 1). In fact, two genes (_Vitvi08g02053_, _Vitvi08g02051_, group 1) were identified that encode proteins with homology to E3 Ubiquitin Ligases (U-box 23), are putatively involved in the regulation of responses to drought stress and to PAMP-triggered immunity [22; 23], and belong to the \"response to chitin\" and \"respiratory burst involved in defence response\" enriched categories of group 1. A third U-box protein encoding gene (U-box 17) (_Vitvi12g00139_, group 4), possibly involved in defenses against pathogens [24], appeared to be downregulated. Group 1 also included two WRKY transcription factors, namely WRKY30 (_Vitvi15g01003_) and WRKY33 (_Vitvi08g00793_), identified in Arabidopsis as positive regulators of abiotic responses and resistance to Botrytis, respectively [25; 26]. Remarkably and consistently, with the activation of biotic and abiotic stress responses, several genes involved in the modulation of ROS homeostasis were upregulated. These include one gene encoding a glutathione peroxidase (_Vitvi02g00332_, group 1), counteracting oxidative damage [27], one encoding a redox-responsive AP2/ERF transcription factor (ERF109) (_Vitvi03g00500_, group 1) required for activation of ROS quenching genes and proteins, and a respiratory burst oxidase (RBOHB) (_Vitvi14g00183_, group 2) encoding a gene required for the H\({}_{2}\)O\({}_{2}\)-dependent induction of thermotolerance through the stimulation of heat shock proteins [28; 29]. Accordingly, several abiotic stress- and ROS-inducible genes encoding heat shock proteins, with putative roles in thermotolerance, were upregulated (e.g., HSP 17.4 (Vitvi13g00491, group 1), HSP 70.1 (_Vitvi06g00443_, group 2; _Vitvi08g02189_, group 1) and HSP 90.1 (_Vitvi02g00025_ and _Vitvi16g01103_, group 2)) together with a heat shock transcription factor A2 (_Vitvi04g00092_, group 1), putatively involved in the activation of heat stress memory genes [30], a heat shock transcription factor A6B (_Vitvi07g00078_, group 1), involved in ABA inducible thermotolerance [31] and a histone chaperone protein (anti silencing function ASF1 (_Vitvi01g00372_, group 1)) responsible for the activation of heat stress responses through histone modifications [32] (Table 1).