# Color Mutations Alter the Biochemical Composition in the San Marzano Tomato Fruit
## 4 Discussion
### Biochemical Changes in Fruits of Yellow Flesh and of Its Combinations with \(y\) and \(gf\)  
Mutants containing \(r\) were clearly separated from the other lines by NP targeted metabolites, mainly due to the compositional differences of carotenoids. The double mutant \(r\_gf\) mapped in a distinct position compared with \(r\) and \(y\_r\), indicating partially additive and partially synergistic effects of this combination. The primary effect of the \(r\) mutation in the carotenoid pathway was to strongly reduce phytoene, and the colored carotenoids \(\beta\)-carotene and lycopene, as expected [42]. Phytofluene was also reduced in \(r\) genotypes, as previously described in tomato accessions [43] and in ripening fruits of \(Psy1\) knockout lines [44]. In the latter case, a concomitant decrease in the volatile apocarotenoids was also observed [44]. In literature, the study of \(r\) metabolites was often limited to the analysis of carotenoids, including the xanthophyll lutein, whose levels, in line with our results, did not vary [11]. Intriguingly, we evidenced that all-trans-neoxanthin, the last xanthophylls in the carotenoid pathway, in \(r\) mutants showed levels higher than SM; it is possible that this represents a cellular strategy implemented to compensate for the decrease of other carotenoids. Indeed, xanthophylls act in flowing the energy through the photosynthetic apparatus and protecting organisms against damage caused by photosynthesis itself [45]. In addition, both violaxanthin and neoxanthin are key substrates in ABA biosynthesis, and their variation can influence fruit ripening and the attitude towards abiotic stresses [46]. In this context, enhanced levels of xanthophylls could provide enough metabolic flux to guarantee an adequate ABA production. The same hypothesis can be extended to the analysis of quinones, such as plastoquinone and plastoquinol-9, whose levels increased in _yellow flesh_ mutants. The importance of quinones in basic metabolic processes, such as respiration and photosynthesis, has been established [47] and an increase in their content is plausible, in a scenario where it is necessary to compensate for the lack of carotenoids. Important metabolic changes occurred in the \(r\) mutants for P compounds; several amino acids positively varied, as proline in the single mutant and in combination with \(y\), and valine/norvaline in the single mutant or combined with \(gf\). Proline and valine have significant functions in plant cells as plant stress sensor, ABA and polyamines interactors, and precursors of BCAA-derived volatiles [48; 49]. Similarly, the vitamin nicotinamide, that plays a primary role in pyridine metabolism [50], also increased in \(r\), alone or with \(y\). Carotenoid-derived volatiles have an important role in tomato flavor, as their levels positively correlate with tomato acceptability [51]. Loss-of-function of \(Psy1\) in _yellow flesh_ mutants led to the lack of substrates for apocarotenoid production [52], thus justifying the low amount of C VOCs such as \(\beta\)-ionone [53] and 6-methyl-5-hepten-2-one [54]. Conversely, the L 1-penten-3-one and (\(E\))-2-pentenal increased in \(r\) and \(r\_y\), suggesting a role for the lipoxygenase-linoleate (LOX) enzyme that catalyzes the oxidation of polyunsaturated fatty acids by molecular oxygen with the formation of unstable hydroperoxides which in turn oxidize carotenoid pigments [55]. In summary, compensating the depletion of the principal carotenoids, lines carrying the \(r\) mutation offer peculiar nutraceuticalal properties, for an increased content of amino acids, vitamins, xanthophylls and quinones. Xanthophylls have been highlighted in recent studies for their positive contribution towards total dietary carotenoid intake [56], whereas quinones showed positive properties in treating cardiovascular diseases, chronic gingivitis and periodontitis and a favorable impact on cancer treatment and human reproductive health [57].