# Pigment-Related Mutations Greatly Affect Berry Metabolome in San Marzano Tomatoes
## 3 Results
### Univariate Analysis of NP, P, and VOC Metabolites in the Four Mutated Lines  
To investigate in detail which compounds varied in the mutated lines in comparison with the WT, a _t_-test analysis was performed, focusing on molecules with recognized nutritional and sensory value. Regarding NPs, lines were enriched in QUIs, such as plastoquinone and ubiquinone-10 (Figure 4a) and TOCs, such as \(\alpha\)-tocopherol and \(\beta/\gamma\)-tocopherol (Figure 4b). In the _pd_ line, tocopherol levels were lower than in the WT, but this line, interestingly, was enriched in some xanthophylls (all-trans-neoxanthin and luteoxanthin; Table S2). _hp-2, pd_, and _Aft_atv_ showed higher contents in PHE metabolites, such as quercetin; naringenin chalcone-dihexose was higher than WT in _hp-2_ and _pd_ but lower in _Aft_atv_ (Figure 4c). For VIT P compounds, _hp-2_ and _pd_ showed a remarkable increase in nicotinamide (Figure 4d). Finally, higher levels in almost all the lines were found for compounds belonging to the B (Figure 4e) and L (Figure 4f) VOC classes. A group of metabolites displayed a similar tendency in the different mutants, such as naringenin dihexose I-II and phloretin-di-C-hexose and kaempfer-dihexose-deoxyhexose and 1-nitro-2-phenylethane, showing higher and lower contents compared to the WT, respectively. A second group of compounds conversely showed a divergent attitude of metabolic changes, such as kaempfer-hexose, coniferyl alcohol beta-d-glucoside, and naringenin (Figure 5). Similarly, PP precursors were characterized by a general increase, except for phenylalanine and ferulic acid-hexose I-III, reduced in _pd_ and _lp-1_, respectively. Notably, kaempfer glycosides and derivatives displayed a more variegated attitude, with dihydrokaempfer and dihydrokaempfer-7-O-glucose reduced in, respectively, _Aft_atv_ and _hp-1_/_Aft_atv_, and kaempfer-glucosyl-glucose-rhannoside with lower levels in _hp-1_ and _hp-2_, together with the aforementioned kaempfer-dihexose-deoxyhexose, decreased in all mutants under study. On the contrary, all quercetin derivatives were characterized by higher contents, except for quercetin-deoxyhexose-hexose-deoxyhexose and quercetin-dihexose-pentose-deoxyhexose, which were reduced in _hp-2_. Interestingly, compounds related to salicylic acid, a well-renewed plant hormone, also exhibited altered levels: More specifically, methyl salicate was lower in all mutants except in _hp-2_, ethyl salicate increased in _hp-1_ and _Aft_atv_, and, in the same mutants, salicaldehyde had significantly lower and higher levels compared to the WT. Figure 4: Fold change in each line compared to the WT of selected NP (**a**,**b**), P (**c**,**d), and VOC (**e**,**f**) compounds. *, **, and *** indicate means significantly lower and higher than WT for \(p\leq 0.05\), 0.01, and 0.001 after Student’s \(t\)-test, respectively. Line abbreviations are explained in Materials and Methods. In addition to PP metabolism, we used the metabolic mapping approach to depict additional sections of the primary and secondary metabolism: In Figure S1 sugar metabolism is represented, together with some related NP and VOC compounds. Again, many metabolites changed similarly in the four lines, and others showed a dissimilar behavior. Specifically, primary sugars were generally decreased in the introgressed lines in comparison with the WT, as well as the alkaloids a-tomatine and calysteinges A3/B1 and some amino acids (alanine, lysine, valine), whereas proline, nicotinamide, and quinones were increased. Other metabolites, such as BCAA VOCs, were generally increased in one genotype (_Aff_atv_) and decreased in another (_pd_; Figure S1). Notably, a series of molecules with distinct pro-nutritional properties showed contrasting changes within the mutants under study.