## 5 Modern Technologies for Ascorbic Acid Biofortification in Tomatoes
### Biofortification through Enhancing Ascorbate Biosynthesis  
Over the last decades, a broad number of successful and less successful biotechnological approaches have tried to enhance AsA biosynthesis in tomato plants overcoming specific rate-limiting steps of the L-galactose pathway [3; 8; 141]. In spite of the fact that the early, non-specific for AsA synthesis, genes of the L-galactose pathway have not been employed in tomato plants, several structural genes from the following steps such as _GMP_[128; 129], _GME_[18; 114], _GGP_[126; 130], and _GLDH_[131], have been successfully overexpressed or down-regulated, affecting the AsA leaf and fruit pool size to different extents (Table 2). In line with the fact that _GMP_ has not been correlated with AsA levels in ripening tomatoes [13; 14], its overexpression resulted in a moderate (1.5- to 1.7-fold) [126] or limited (up to 1.5-fold) [129] increase in AsA content in the leaves and fruits, respectively. Similarly, modifications of _GME_ expression had a relatively limited effect on AsA content in either leaf or fruit tissues in tomatoes [18; 114]. Several lines of evidence reinforce the initial hypothesis that manipulation of _GGP_, the enzyme that catalyzes the first committed step of the main biosynthetic pathway, could serve as a valuable strategy to enhance AsA levels in several crop species, including tomato, as its transcripts levels significantly correlated with AsA changes [14]. Indeed, overexpression of the kiwi _GGP_ has led to an up to six-fold increase in AsA content in tomato fruit, and less, but still significant, in strawberry (two-fold), or potato (three-fold) [126]. This modification of _GGP_ expression in tomato fruit also resulted in the loss of seeds and of the jelly of locule tissue, highlighting the putative involvement of AsA in inhibiting seed formation, although the exact mode of action is still unclear. Overexpression of _GGP_ in a fruit-specific manner, resulted in a three-fold increase of AsA in ripe tomato fruit, whereas a similar approach for _GPP_ did not result in a similar increase of fruit AsA ([PERSON] et al., unpublished data). More recently, using combined forward genetics with mapping-by-sequencing approaches, the impaired pollen fertility that resulted in seedless tomato fruits has been related to the over-accumulation of AsA (up to 5-fold WT level) in AsA-enriched mutants [27]. In a less successful effort, [PERSON] et al. [130] achieved a nearly 50% decrease in leaf AsA content, by down-regulating a tomato gene coding _GGP_. Interestingly, increased co-expression of both _GGP_ and _GME_ in _Arabidopsis_, resulted in an up to seven-fold increase in leaf AsA [26; 142], indicating that these two genes operate synergistically to govern leaf AsA pool size [26; 126; 142; 143]. This finding is consistent with the co-regulation of the transcript levels of _GGP_ and _GME_, at least in tomatoes. In particular, overexpression of _GME_ significantly reduced _GGP_ transcripts [114], repression of _GGP_ increased _GME_ transcripts [130], and diminution of _GME_ enhanced _GGP_ transcripts in the transgenic lines [18], probably in an effort to maintain a stable AsA pool size. A few years ago, [PERSON] and co-authors [26] shed some further light on the feedback regulation of AsA levels via suppression of _GGP_ translation under high AsA levels, which probably explains the relatively limited success in single transgenic interventions so far. The authors demonstrated that in a wide range of species including tomato, the 5\({}^{\prime}\)-untranslated region (UTR) of _GGP_ contains a highly-conserved upstream open reading frame (uORF), that encodes a peptide that can inhibit its translation [26; 144]. According to this model, when AsA accumulation is enhanced, the uORF is translated and inhibits _GGP_ translation, whilst the uORF is disabled and _GGP_ is translated when AsA levels are low.