## 4. ascorbate and Glutathione
### The Approaches for Boosting Ascorbate and Glutathione Production  
Describing the discovery of enzymes involved in Asc biosynthesis, we have already reported examples of the successful creation of mutants with its increased synthesis. One more approach to obtain an increased production of the metabolites of interest in plants is to regulate the target gene expression at the translational level. For this purpose, the targeted mutations of nucleotide sequences are introduced in the region of upstream open reading frames (uORFs) of the genes of interest. UORFs are cis elements located in the 5\({}^{\prime}\) untranslated regions (UTR) of mRNA; they promote ribosome stalling and dissociation during mRNA translation, thus acting as translation repressors. Introducing mutations into the uORF regions can eliminate this suppressive effect, leading to an increased expression of the gene [317]. This strategy was successfully tested on the uORF of the _LsGGP2_ gene, encoding GDP-L-galactose phosphorylase, which is a key enzyme in the biosynthesis of Asc through the Smirnoff-Wheeler pathway. Editing of the uORF led to an increase in Asc content in lettuce (_Lactuca sativa_ L.) leaves by approximately 150% and an increased resistance to oxidative stress (Table 2) [173]. A similar study was done with tomato plants. The mutations in the uORF region of the _SIGGP1_ gene in two independent CRISPR-edited lines led to an increase in the Asc level in fruits (Table 2) [169]. In mutant tomatoes edited in the uORF-_GGP1_ (single nucleotide transversion from C to T) region, the amount of Asc increased by 2-5 times compared to the wild-type plants. However, these mutations led to impaired flower fertility, resulting in the production of homozygous mutants with the fruits having a small number of non-viable seeds or being completely seedless [169]. Another approach to increase the Asc level, which was confirmed experimentally, was the editing of the transcription factors of genes encoding proteins involved in its synthesis. The EIL2 protein controls carotenoid metabolism and Asc biosynthesis in tomatoes (_Solanum lycopersicum_). As previously described, the fruits of the CRISPR/Cas9 _eil2_ mutants had changes in fruit color and also showed an increase in ascorbic acid content. The authors showed that _SIEIL2_ repressed the expression of the gene _L-GALACTOSE-1-PHOSPHATASE 3 (SIGPP3)_ and _MYO-INOSTOL OXYGENASE 1 (SIMIX1)_ at the transcriptional level, resulting in a 1.6-fold increase in Asc synthesis through the L-galactose and myo-inositol pathways in the mutants (Table 2) [67]. To increase the level of Asc in plants, genes that regulate its metabolism were proposed as candidates for genome editing. These are the _ascorbic acid mannose pathway regulator 1_ (_AMR1_), _COP9 Signalosome Subunit 5B_ (_CSN5B_) and \(8\) (_CSN8_), and _NBS-LRR 33_ (_NL33_). This strategy is supported by data from insertionsal mutagenesis and RNA interference, demonstrating that reduced expression of these genes led to a higher concentration of Asc and enhanced plant stress resistance [318]. Plants with elevated Asc content were obtained by overexpressing monodehydroascorbate reductase (MDHAR), an enzyme that maintains the reduced Asc pool (Figure 5). Tobacco plants overexpressing the _MDHAR_ gene from acerola accumulated approximately 1.6 to 2 times more Asc and exhibited greater resistance to salt stress [319]. The content of MDA in the transgenic plants subjected to stress was about two times lower compared to wild-type plants [319]. The co-overexpression of MDHAR and DHAR from _Brassica rapa_ in Arabidopsis resulted in an increased glutathione content and an enhanced resistance to freezing (16 h at \(-5\)\({}^{\circ}\)C) [320]. Regulating the activity of GR, GST, and GPX, the enzymes directly affecting the levels of reduced/oxidized glutathione ratio, is an effective approach to increase the content of glutathione and the resistance of plants.