## 5 Modern Technologies for Ascorbic Acid Biofortification in Tomatoes
### Biofortification through Novel Regulatory Genes and Transcription Factors  
As the manipulation of structural genes from the AsA biosynthetic and recycling pathways has only had limited success so far (Table 2), it is becoming evident that other key components of AsA accumulation, including transcription factors and regulatory proteins, also co-exist in model and crop species. In tomatoes, RNA-Seq studies revealed that several transcription factors, including MYB, NAC, and ZIF, may control the expression of AsA-biosynthetic genes, and further correlate with fruit AsA content [159], whilst a weighted gene correlation network analysis study demonstrated that the AsA biosynthetic genes had weak connectivity to AsA accumulation in ripening tomatoes [65]. In light of this observation, overexpressing the _SIHZ24_ transcription factor that binds the promoters of _GMP_, _GME_, and _GGP_ in tomatoes, resulted in a 1.5-fold and 1.2-fold increase of leaf or fruit AsA content, respectively [136]. Similarly, the tomato _basic helix-loop-helix 59_ (_bHLH59_) gene, that co-localizes with an AsA-related QTL, regulates the expression levels of several genes of the l-galactose pathway, such as _PMM_, _GMP_, and _GME_[122]. Overexpression this gene increased the leaf AsA content 1.5-fold, while RNAi lines had only 65% of WT AsA levels. The effect of _bHLH59_ on AsA accumulation is due to nucleotide differences in the promoter region ofHLH59. Overexpression of the regulatory factor _SIZF3_ significantly enhanced AsA content in tomato leaves (Table 2) [123]. _SIZF3_ promotes the accumulation of AsA by preventing _GMP_ degradation and ubiquitination through binding COP9 signalosome subunit 5B (CSN5B), a key component of the photomorphogenic COP9 signalosome, and thus inhibiting the binding of CSN5B to _GMP_. The DNA-binding with one finger (Dof) proteins are plant-specific transcription factors with diverse biological functions, such as large gene families in most plant species, including tomato with 34 gene members [160]. The _SIDo22_ factor has been demonstrated to negatively regulate AsA levels and transcription of AsA biosynthetic and recycling genes in tomato [137]. In particular, leaves and fruits of the _SIDo22_ RNAi transgenic tomato lines had 1.3- and 1.6-fold higher AsA levels, respectively, compared to WT plants. Furthermore, the expression of _GGP_, _GalDH_, _GLDH_, _MDHAR_, cytosolic _APX_, and _GR_ was induced in transgenic plants. Nonetheless, knockdown of the _SIDof22_ gene also resulted in a decreased tolerance to salinity, which was associated with the downregulation of the _salt overly sensitive 1_ gene. In total, the tomato genome contains 43 NBS-LRR (NL) subfamily resistance proteins, whose function is largely unknown. Knock-down of _SINL3_3 gene raised the AsA content of leaves and fruits 2.7- and 1.3-fold, respectively, in _SINL3_3-RNAi transgenic lines compared to WT plants [138]. The accumulation of AsA in the transgenic plants has been attributed to the high transcript levels of nearly all the structural AsA-related genes, such as _GMP_, _GME_, _GGP_, _GPP_, _GalDH_, _GLDH_, _MIOX_, _APX_, _MDHAR_, and _DHAR_. Although the exact mode of interaction between AsA metabolism and _NL3_3 is still unclear, the suppressed expression of _SINL3_3 also promoted tolerance to methyl viologen and gray mold infection by _Botrytis cinerea_, probably via enhancing the ROS scavenging capacity through the AsA-GSH cycle. The tomato CCAAT-box transcription factor (_SINFYA10_) has been recently demonstrated to be the first CCAAT-binding factor negatively controlling AsA synthesis at multiple sites, as it can bind to the promoter of _GME_ and _GGP1_[125]. Transgenic tomato lines overexpressing _SINFYA10_ contained significantly lower AsA contents in their leaves and fruits, as a result of the decreased abundance of both genes. d-Arabinono-1,4-lactone oxidase (_ALO_), the yeast analog GLDH, catalyzes the conversion of d-arabinono-1,4-lactone to erythroscorbate [161].