# Color Mutations Alter the Biochemical Composition in the San Marzano Tomato Fruit
## 3 Results
### Targeted Analysis of Polar Metabolites  
The relative quantification of 128 polar metabolites allowed to complete the metabolomics characterization of the six mutants under study. P metabolites were divided into different metabolic classes, including 19 amino acids (AA), 17 acids (AC), four amines (AM), two lipids (LI), one nucleic acid (NU), 15 sugars and polyols (SAP), 11 alkaloids (ALK), 55 phenylpropanoids (PHE) and three vitamins (VIT; Table S5). The score plot of the first two components explained about the 52% of the total variance, with PC1 that particularly separated \(r\) and \(r\_\)_gf_ from all the other lines, and PC2 that identified a group including _gf_ and \(r\_\)_gf_ together with SM (Figure 4c). Interestingly, P untargeted and targeted metabolites differently separated the mutants under study, providing clues about a large extent of distinct metabolic components contributing to their chemical profiles. The loading plot grouped metabolites belonging to the same metabolic pathway (Figure 4d). By the comparison of the score and the loading plots, the position of some metabolites in relation to the lines studied was highlighted; indeed, many kaempfers and quercetins were in the PHE group corresponding to the area of the \(y\) mutants. On the contrary, many naringenins grouped in the opposite side. Most AAs were grouped together, matching with \(r\) and in opposition to the SAP group (Figure 4d). To investigate the P metabolite content of each of the mutated lines in comparison with SM, a \(t\)-test analysis was performed (Table 2; Table S5), giving emphasis to nutritional- and sensorial attribute-related molecules. The line with the highest number of differentially accumulated polar  
Figure 5: Relative log2 variation in (**a**) phytoene, (**b**) \(\beta\)-carotene, (**c**) lycopene, (**d**) all-trans-neoxanthin, (**e**) chlorophyll b, (**f**) plastoquinone, (**g**) \(\delta\)-tocopherol, (**h**) \(\beta\)-\(\gamma\)-tocopherol, of 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. compounds was the \(y\) single mutant, with a preponderance of down-regulated metabolites, as expected; the PHE group mostly contributed to this diversity (Table 2). On the contrary, _gf_ was the line more like SM. Notably, lines containing \(r\) showed a higher number of AA over SM (Table 2). As already highlighted in the corresponding loading plot (Figure 4d), lines carrying \(y\) had lower levels of the PHE naringenin chalcone glucoside (Figure 6a), and conversely higher levels of kaempfer-hexose deoxyhexose-pentose compared to SM, a biochemical phenotype also observed in _gf_ and \(r\_\_\)_gf_ too for the latter (Figure 6b). Regarding the ALK group, calcysteine B1 resulted statistically lower in all the lines, with exception of _gf_ and \(y\_\_\)_gf_, which however displayed reduced amounts compared to SM (Figure 6c). In addition, a series of primary metabolites were characterized by higher levels in the mutants under study: for example, the AA proline in \(r\) and \(y\_r\) (Figure 6d) and the VIT nicotinamide in _gf_, \(r\) and \(y\_r\) (Figure 6e). Similarly, the SAP glucoheptulose was higher in _gf_, whereas all the other lines were more similar to SM (Figure 6f). The LI phosphocoline displayed lower levels in lines carrying the \(y\) mutation (Figure 6g), while the AC sinapinic acid was higher in all lines, with the only exception of \(r\) (Figure 6h). Figure 6: Relative log2 variation in (**a**) naringenin chalcone glucoside, (**b**) kaempfer-hexose-deoxyhexose-pentose, (**c**) calcysteine B1, (**d**) proline, (**e**) nicotinamide, (**f**) glucoheptulose, (**g**) phosphocoline, (**h**) sinapinic acid, of six fruit mutant lines in San Marzano (SM) background compared with the recurrent parent.