# Spatial Variability of Grape Berry Maturation Program at the Molecular Level
## 3 Results and Discussion
### Spatial Variability in the Expression of Key Genes Involved in Berry Maturation  
The vineyard in this study presented high phenotypic variability in vegetative and berry compositional parameters. The variability observed at harvest was the result of dissimilar developmental and metabolic processes occurring from fruit set to ripening at each block. Berry development is a process divided into two sigmoidal successive growth periods [46], during which berry morphology and composition undergo profound changes orchestrated by the berry maturation transcriptional program [27]. To assess the gene expression evolution in berries from plants at different vigor statuses, microarray analysis was applied at five time points from post-fruit set to harvest (Supplementary Dataset S2). We explored the transcriptomic dataset, looking for genes with key roles in berry ripening that showed distinct expression patterns between blocks classified as HVC, MVC, and LVC. Among these, three members of the NAC transcription factors family were included: _VviNAC26 (Vitvi01g01038)_, _VviNAC33 (Vitvi1 gg01484)_, and _VviNAC60 (Vitvi08g01843)_[47], which were previously defined as switch genes, namely master regulators of the berry transition from the vegetative to the mature phase [48]. These genes are characterized by drastic upregulation at or near veraison, which is correlated with the shutdown of primary metabolic pathways active during the vegetative growth of the berry [27]. In our results, the expression peak described for these genes was higher in LVC and likely determined an enhanced effect on the downstream ripening processes they control (Figure 3). _VviNAC26_ was demonstrated to undergo large expression variations under different abiotic stresses [47]; hence, it is reasonable that its expression level would be affected by vineyard variability. Figure 2: [PERSON]’s correlation coefficient matrix for parameters measured at each block during the growing seasons. Hierarchical cluster analysis was applied to the measured parameters using the average linkage method. Asterisks represent statistically significant correlations based on \(t\)-test (\(p\leq 0.05=\) ‘\(\ast\)’; \(p\leq 0.01=\) ‘\(\ast\)’; \(p\leq 0.001=\) ‘\(\ast\)\(\ast\)’). NDVI: Normalized difference vegetation index. LAI: Leaf area index. TSS: Total soluble solids. YAN: Yeast assimilable nitrogen. [PERSON] et al. [49] reported associations between a set of _VviNAC26_ polymorphisms and several berry size parameters. The authors suggested that specific polymorphisms reduce the expression of _VviNAC26_, resulting in lowered biosynthesis of the ripening phytohormone abscisic acid (ABA), in turn leading to larger berries. Our results support these indications, as low, medium, and high berry weights observed, respectively, under LVC, MVC, and HVC, were inversely related to the expression levels of _VviNAC26_ throughout berry maturation (Figures 3 and S1). To better visualize the intra-vineyard variability in _VviNAC26_ expression, a spatial variability map was generated by interpolating the expression data of the 14 blocks at veraison (0 DAV), the time point at which gene expression levels were most variable across the sampling blocks (Figure 4). The map does, indeed, show a generalized inversed gene expression level pattern compared to vine vigor and berry weight maps (Figure 1b,c). _VviNAC33_ promotes organ de-greening and represses organ growth during the vegetative-to-mature phase transition, whereas _VviNAC60_ is a regulator of whole plant senescence- and ripening-related processes [50; 51]. _VviNAC33_ and _VviNAC60_ showed the largest differences by vigor class when their expression peaked at 11 DAV (Figure 3), which was, in fact, the time point used for map generation. The _VviNAC60_ map mirrored both the vigor class and _VviNAC26_ maps, whereas the _VviNAC33_ map did not match as well (Figures 1b and 4). This could be attributed to the fact that the _VviNAC33_ gene showed less variability in the vineyard, in agreement with previous reports [50].