Tomato fruit fortification by gene editing of Glutathione S-transferase (GST) loci

Tomato (S. lycopersicum) is a worldwide cultivated food crop and its global yield has increased over the past decade. Its fruit contain many health-promoting compounds that have been involved in the prevention of several chronic diseases and dysfunctions (Frusciante et al., 2007. In addition to its economic and nutritional importance, tomato is an important model plant for scientific research on fruit development and quality. Since its first release in 2012, the tomato genome sequence has been widely used as a reference genome for scientific research and biotechnology assisted breeding approaches. In order to control the overall level of reactive oxygen species (ROS) and prevent cellular damage and lipid peroxidation, the plant metabolic processes deploy a plethora of bioactive compounds with antioxidant activity as defense shield against oxidative stress. The antioxidant system consists of several metabolites and enzymatic proteins such as glutathione S-transferase (GSTs). GSTs are phase II metabolic isozymes that catalyze the conjugation of the tripeptide (γ-Glu-Cys-Gly) glutathione (GSH) to a variety of substrates such as endobiotic and xenobiotic compounds for the detoxification. GSH is a key player in the plant response to the oxidative stress. In fact, GSH biosynthesis is stimulated when the cell faces stress conditions and builds up its defense capability. Also, GSH collaborates with ascorbate and NADPH in the Foyer-Halliwell–Asada cycle (Potters et al., 2002) for H2O2 detoxification preserving cells from damages brought about by exceeding levels of ROS. In plants, GSTs exist as a multigene superfamily and can be grouped in cytosolic, mitochondrial and microsomal. Based on their sequence plant GSTs are categorized in distinctive classes, that are tau (U), phi (F), theta (T), zeta (Z), lambda (L), dehydroascorbate reductase (DHAR), γ-subunit of the eukaryotic translation elongation factor 1B (EF1Bγ), tetrachlorohydroquinone dehalogenase (TCHQD), metaxin, Ure2p, hemerythrin (H), iota (I), microsomal prostaglandin E-synthase type 2 (mPGES-2) and glutathionyl hydroquinone reductase (GHR) (Ref.). Phi and tau are the largest plant GST classes. Within the tomato genome 90 GST genes unevenly distributed across the 13 synthetic chromosomes has been previously identified (Islam et al. 2017). Among others, chromosomes 7 and 9 harbor the highest number of GST genes, 23 and 12, respectively. Moreover, GST genes are grouped in 10 clusters and the two major tau cluster are located on chromosomes 7 and 9. Previous research carried out in our laboratory allowed us to associate in tomato GST up-regulation with fruit accumulation of phenolic compounds (Di Matteo et al., 2013). However, the use of GST genes to improve the nutritional quality of the fruit remains largely unexplored given the complexity of this gene family with a high degree of redundancy and duplication that limit the specificity of many metabolic engineering technologies and the high homeostatic strength that allows the system to react and debunk or cancel any effect of externally-induced unbalance. To functionally characterize candidate genes and breed tomato for enhanced fruit nutritional quality, genome editing technologies relay on DNA repair mechanisms that occur in cells either by non-homologous end-joining or homology directed repair systems (Steinert et al., 2016). Editing technologies, can efficiently achieve site-directed mutations of target sequences and further removal of foreign DNA by segregation in order to deal with regulatory and public acceptance issues.