# Color Mutations Alter the Biochemical Composition in the San Marzano Tomato Fruit
## 3 Results
### Targeted Analysis of Non-Polar Metabolites  
To investigate changes at the NP specialized metabolome, LC-HRMS was used to determine the level of 54 known and previously validated compounds. They were divided in different metabolic classes, including 14 fatty acids (FA), one phospholipid (PHO), two sterols (STE), 15 carotenoids (CAR), eight chlorophylls (CHL), six quinones (QUI), and five tocochromanols (TOC; Table S4). The score plot of the first two PCA components explained about the 57% of the total variance, with double mutants differently spaced from their parental lines (Figure 4a). Indeed, PC1 kept the mutants carrying \(r\) separated from SM and the other lines. PC2 clearly separated \(y\_r\) and its parental lines from the three mutants carrying \(gf\) plus SM, partially confirming untargeted metabolomics results. The loading plot grouped metabolites belonging to the same metabolic pathways (Figure 4b). A comparison between the score and the loading plots revealed the compositional differences between the mutated lines. Indeed, the chlorophyll group in the upper side of PC2 characterized the _green flesh_ genotypes, while two quinones were in the lower side of PC2, in correspondence of the _yellow flesh_ genotypes. The carotenoid group was split into early carotenoids (up to lycopene) and xanthophylls at the opposite sides of PC1; they were respectively decreased and increased in the _yellow flesh_ mutant lines (Figure 4b).  
Figure 3: Relative log2 variation in selected volatiles involved in tomato flavor: (**a**) eugenol, (**b**) \(\beta\)-ionone, (**c**) 6-methyl-5-hepten-2-one, (**d**) (\(E\))-2-pentenal, (**e**) 1-penten-3-one, (**f**) 2-phenylethanol in the six fruit mutant lines in San Marzano (SM) background compared with the recurrent parent. Line symbols are reported in Table 1. Bars colored in grey and black indicate means significantly lower and higher than SM for \(p\leq 0.05\) after Student’s \(t\)-test, respectively.  
To further investigate the metabolic changes of each mutated line in comparison with the original parental SM, a \(t\)-test analysis was performed (Table 2; Table S4), and we particularly focused on metabolites with sensorial (color, taste) and health-related properties. The lines carrying \(r\) showed the highest number of NP compounds different from SM, mainly due, as expected, to differences in carotenoids (Table 2). The lower number of differences was shown by the single mutant \(y\), indicating that this genotype is more similar to SM for NP targeted compounds.  
\(r\), \(r\_\)_gf_ and \(y\_r\) were characterized by levels of phytoene (Figure 5a), \(\beta\)-carotene (Figure 5b) and lycopene (Figure 5c) lower than SM and the other lines, in agreement with previous reports; \(r\), \(r\_\)_gf_, \(y\_\)_gf_, and \(y\_r\) reported higher levels of the xanthophylls all-trans-neoxanthin (Figure 5d) and luteoxanthin (Table S4). At chlorophyll metabolism level, _gf_ was characterized by higher contents of both chlorophyll a (Table S4) and b, with the latter also higher in \(y\_\)_gf_ and \(r\_\)_gf_ (Figure 5e). Moreover, \(r\_\)_gf_ showed higher levels of pheophytin a and pheophorbide a (Table S4). Drawing the attention on quinones, plastoquinone increased in lines carrying \(r\) (Figure 5f) and plastoquinol-9 in \(r\) and \(y\_r\) (Table S4). Lastly, \(\delta\)-tocopherol amount was higher than SM only in \(r\_\)_gf_, while \(\gamma\)-tocopherol and \(\beta\)-tocopherol enhanced in \(r\_\)_gf_ and \(y\_\)_gf_ (Figure 5g,h).  
Figure 4: PCA of log2 values of 54 NP (**a**,**b**) and 128 P (**c**,**d**) metabolites measured by LC-HRMS. (**a**–**c**) PC1 \(\times\) PC2 score plot of the six mutated lines plus San Marzano (SM). (**b**–**d**) PC1 \(\times\) PC2 corresponding loading plots. Line (**a**,**c**) and metabolite class (**b**,**d**) abbreviations are explained in Tables 1 and 2 respectively.