## 5 Modern Technologies for Ascorbic Acid Biofortification in Tomatoes
### Biofortification through Enhancing Ascorbate Biosynthesis
Considering the ubiquity of this uORF in _GGP_ genes from mosses to angiosperms, it is evident that regulation of _GGP_ mRNA translation without the need for gene transcription modification points at an attractive biotechnological target to adjust the AsA pool in plant tissues in response to altering environmental conditions that may need to bypass transcriptional regulation [143]. Notwithstanding the fact that _GPP_ transcript levels have been correlated with AsA content in ripening tomatoes or in response to ethylene, wounding, cold, and post-anoxic conditions [13], it is unlikely that this step alone serves as the key rate-limiting factor forAsA biosynthesis, at least in tomato fruit. Consistently, overexpressing _GPP_ in tomato resulted in a 1-7-fold increase in leaf AsA content, but no change in fruit AsA content [116]. However, when _GGP_ and _GPP_ were overexpressed simultaneously in _Arabidopsis_, a four-fold change in leaf AsA levels was recorded [135], highlighting the importance of the co-expression of structural genes to ensure the meaningful boost in the flux of the pathway. In this regard, tomato hybrids resulting from separate transgenic lines expressing the _GGP_ and _GPP_ genes in a fruit-specific manner did not exhibit increased levels of fruit AsA contents ([PERSON] et al., unpublished data). As for the last two enzymes of the L-galactose pathway, _GalDH_ and _GLDH_, none of them were found to exert a noteworthy effect on AsA levels of tomato tissues [131] exerting nearly null fold change compared to WT plants, while the effect of these genes may be more pronounced in other species, including rice [145; 146], _Arabidopsis_[135], and tobacco [147]. Gene pyramiding is a classical method in plant breeding according to which desirable genes are assembled into a single genotype [148], which has gained considerable attention in order to improve the synthesis of certain metabolites, involved in carotenoid [149] and anthocyanin accumulation [150]. However, the application of gene pyramiding for AsA-related genes was not conducted until recently [116]. According to this study, AsA biofortification in tomato was achieved by pyramiding _GMP_\(\times\)_GME_, _GGP_\(\times\)_GPP_, and _GMP_\(\times\)_GME_\(\times\)_GGP_\(\times\)_GPP_ by the conventional crossing of individual transgenic lines. Results showed that the increase in AsA levels was comparable between single-gene transgenic lines and pyramiding-lines, clearly demonstrating that AsA cannot be enhanced beyond a level due to the feedback inhibition loop of AsA accumulation and homeostasis. Further studies are required to determine AsA accumulation by pyramiding genes from different regulatory pathways, to avoid the side-effects from feedback inhibition occurring when co-expressing genes from single metabolic routes. Among the alternative biosynthetic routes for AsA biosynthesis, the one through d-galacturonic acid, which has a dual role involved in both cell wall pectins and AsA biosynthesis, has given the most promising results in tomato fruits. In particular, overexpression of _GalUR_ from strawberry resulted in a two-fold increase in AsA levels of hairy roots [132], 1.2- to 2.5-fold change in fruits [115; 133; 134], and 1.3- to 1.6-fold change in leaves [133; 134] (Table 2). Regarding theulose biosynthetic route from the animal-like pathway, overexpressing the rat _GuLDH/GLOSE_ showed moderate AsA levels in tomato fruits [151], similar to potato tubers [152], despite the encouraging findings in other species such as tobacco (seven-fold increase) or lettuce (four-fold increase) [153]. The contribution of the D-gluconate pathway or _myo_-inositol pathway to AsA biosynthesis is highly controversial in plant tissues, although it can affect carbon incorporation into the cell wall [15; 36]. Overexpression of _Arabidopsis myo_-inositol oxygenase (_AtMIMX_), the enzyme catalyzing the conversion of _myo_-inositol to D-glucuronate, increased AsA contents 1.4-fold in green fruits [128].