# Spatial Variability of Grape Berry Maturation Program at the Molecular Level
## 3 Results and Discussion
### Spatial Variability in Berry Ripening Transcriptomic Program
DEGs with significantly greater expression in LVS berries (207 genes) were functionally in phenylalanine metabolism and biosynthesis of secondary metabolites (Gene Ontology (GO) enrichment analysis; Figure S3). On the contrary, DEGs with higher expression in HVS berries (761 genes) were involved in photosynthesis mechanisms (Figure S4). The expression patterns of the 968 DEGs (\(t\)-test; \(p<0.05\); \(\mid\)FC\(\mid\)\(\geq\) 1.5) were represented by four main trends determined by cluster analysis (Figure 6; Supplementary Dataset S3). Clusters 1 and 2 were the most abundant, containing 311 and 406 genes, respectively. Cluster 1 included genes whose expression patterns showed a low peak before veraison followed by an upward trend thereafter in both vigor statuses. Based on their augmented expression from veraison, these genes can be considered among the players of late-ripening stages, in accordance with those similarly categorized in a transcriptomic survey that defined the molecular events marking the onset of grapevine berry ripening [27]. Two TFs were found in Cluster 1, _Vvi.BDla3_ (_Vitvi15g00735_) and _Vvi.NAC61_ (_Vitvi08g01841_). _Vvi.BDla3_ encodes for a LOB TF already reported to be a molecular marker of the vegetative to mature transition [48], whereas _VviNAC61_ is a NAC TF, recently proposed as a key regulator of late- and post-ripening related processes [58]. It was demonstrated that _VviNAC61_ governs stilbenoid metabolism, and, in fact, this gene appeared in Cluster 1 along with a stilbene synthase (_VviST343; Vitvi16g01480_) and two laccases (_Vitvi18g03027, Vitvi18g03356_) that are also thought to be putatively involved in stilbene oligomerization and recently were assigned to a stilbene-related gene regulatory network [59]. We observed that from veraison onwards, genes belonging to Cluster 1 reached a higher expression level in LVS. This finding is in line with the data shown in Figure 3 and supports the hypothesis that the expression of ripening-related genes is enhanced in LVS. Cluster 2 includes genes strongly expressed at fruit set (\(-30\) DAV) but rapidly downregulated during development. This cluster is the most populated, supporting the claim that transcriptomic rearrangements during ripening require switching off several genes, in chorus with the upregulation of the markers for veraison. The early/pre-veraison gene cluster defined in [PERSON] et al. [27] matched 52 genes belonging to Cluster 2, hence corroborating our finding (Supplementary Dataset S3). GO enrichment analysis revealed significant enrichment in genes related to primary (i.e., photosynthetic processes) and secondary metabolism (Figure S4). Unlike Cluster 1, no visible differences by degree of vigor could be observed for Cluster 2. Nevertheless, given the significance of the statistical analysis applied to extract said genes, we cannot rule out that even subtle changes in expression could have contributed to the phenotypic differences found by vine vigor. Genes belonging to Clusters 3 and 4 displayed an increased expression level in both vigor statuses before veraison, though their trends differed markedly by vigor status from veraison onward. The 101 genes belonging to Cluster 3 had a major expression peak in LVS, 11 DAV, which then dropped, whereas, in HVS, they maintained a constant expression level until maturity. Even though no enriched category was detected by GO enrichment analysis, many TFs and several transcripts involved in transport and signal transduction were noted in Cluster 3. The TFs included two WRKY factors (_Vitvi17g00102, VviWRKY52, Vitvi18g00742, VviWRKY54_) and the stilbene biosynthesis regulator _VviMYB15_. _Vvi.MYB15_ already drew attention as a marker of LVS, along with the two STS genes _VviSTST1_ and _VviSTST6_ (Figure 3). Consistently, _VviSTST1_ and _VviSTST6_ were also included in Cluster 3, with the addition of a third STS gene (_Vitvi10g01391, VviST55_). On the other hand, cluster 4 included 151 genes highly expressed in HVS from veraison onwards.