# Color Mutations Alter the Biochemical Composition in the San Marzano Tomato Fruit
## 3 Results
### Targeted Analysis of Volatile Compounds  
In order to give a more specific characterization of the flavor, volatile composition of each of the mutated lines in comparison with the wild-type SM was carried out. The selected analytical strategy allowed the relative quantification of 68 VOCs unequivocally identified by both mass spectra and retention index with those of authentic standards (Table S3). Overall, eight compounds were related to benzenoids (B), ten to branched-chain amino acid-relatives (BCAA), nine to apocarotonoids (C), two to esters (E), twenty-four to fatty acids derivatives (L), four to phenylalanine derivatives (Phe), two to sulfur compounds (S) and six to monoterpenoids (T). PCA of the volatile composition revealed that the first two components explained about the 54% of the total variance; the score plot showed the position of the double mutants, related to their parental lines, and with respect to SM (Figure 2a). Indeed, \(r\) and \(y\_r\) were co-located in the same dial, in agreement with the VOC untargeted metabolome plot; conversely, \(r\_gf\) placed halfway between \(r\) and \(gf\) according to PC1 (Figure 2a). Moreover, PC1 kept the mutants \(r\) and \(y\_r\) separated from the other lines. PC2 placed \(y\_r\) and its parental lines in the upper side of the graph, together with SM. The corresponding loading plot was able to identify groups of metabolites, often belonging to the same metabolic pathways, as apocarotonoids (C, in red) and lipids (L, in light blue), terpenids (T, in green blue) and branched-chain amino acid derivatives (BCAA, in blue; Figure 2b). A comparison between the score and the loading plots revealed the overall compositional differences between the mutated lines. Indeed, one of the most obvious features was that all the lines harboring the _yellow flesh_ mutation (\(r\), \(y\_r\) and \(r\_gf\)) and, in a lesser extent, also the double mutant \(y\_gf\), were characterized by producing lower levels of volatile apocarotonoids, which was particularly dramatic in the case of some linear apocarotonoids such as 6-methyl-5-hepten-2-one (Figure 3). In the case of the mutants for _yellow flesh_ this was in accordance with the scarcity of their carotenoid precursors. These lines also showed lower levels of several phenylalanine derivatives. A characteristic feature of the lines \(y\), \(r\) and \(y\_r\) was the high production of fatty acid derivatives together with low levels of branched-chain amino acid-related volatiles, conversely to double mutant lines \(y\_gf\) and \(r\_gf\), which showed the opposite pattern. Finally, \(y\) and \(gf\) lines were characterized by higher levels of apocarotonoids and terpenids (Figure 2b). Figure 2: PCA of log2 values of 68 volatile compounds measured by a solid-phase micro-extraction gas-chromatography coupled to mass spectrometry (HS-SPME/GC-MS). (**a**) PC1 × PC2 score plot of the six mutated lines plus San Marzano (SM). (**b**) PC1 × PC2 loading plot. Line (a) and metabolite class (b) abbreviations are explained in Tables 1 and 2 respectively. To further investigate the volatile compounds content of each of the mutated line in comparison with their original parental SM, a \(t\)-test analysis was performed. Out of 68 VOCs identified, the line with the highest number of compounds significantly different from SM was \(y\), mainly because of differences in fatty acid derivatives, among other VOCs (Table 2). The lines containing the \(r\) mutation strongly differed for apocarotenoid volatiles, a group of metabolites considered to be involved in tomato flavor [37], representing the metabolic pathways most notably altered in these ILs (Figure 3). Some volatiles, such as the benzenoid eugenol, had higher levels in all mutant lines (Figure 3a). Mutants carrying _yellow flesh_ had lower levels of the apocarotenoid \(\beta\)-ionone (Figure 3b), as well as 6-methyl-5-hepten-2-one, where \(y\_gf\) acquired lower levels too (Figure 3c).