## 3 Flavonoids
### Genetic Approaches for Boosting Flavonoid Production in Plants
#### 3.3.2 Regulation of Transcription Factor Activity to Enhance Flavonoid Biosynthesis  
The regulation of flavonoid biosynthesis, which involves the participations of transcription factors bHLH and the proteins of MYB family is described above. The genes of the R2R3-MYB subfamily of transcription factors are actively used as targets for genome editing to increase the level of flavonoids in plants [247; 248]. Overexpression of _AtMYB12_ in Arabidopsis simultaneously increases the expression of _AtCHS_, _AtCHI_, _AtF3H_, and _AtFLS_ (Table 3) [247; 248]. The resulting transgenic Arabidopsis plants exhibited enhanced resistance to drought and salinity (25% PEG6000 or 200 mM NaCl for 2 weeks) [248]. The levels of H\({}_{2}\)O\({}_{2}\) and malondialdehydedehyde (MDA) in the transgenic plants were 40-60% lower compared to the wild-type plants, while the activities of SOD and peroxidase were 30-40% higher [248]. Transgenic tobacco plants overexpressing _AtMYB12_ also demonstrated increased resistance to biotic stress caused by insect pests, such as _Spodoptera litura_ and _Helicoverap armigera_[10]. The enhanced resistance of transgenic plants is likely associated with the accumulation of the flavonoid rutin, which is toxic to insects. PAP1 (Production of Anthocyanin Pigment 1) is another transcription factor, a typical representative of the R2R3-MYB transcription factors. Overexpression of _AtPAP1_ in tobacco led to increased accumulation of flavonoids in all parts of the plant [249]. In these transgenic tobacco plants, the expression of genes _PALs_, _CHS_, _CHI_, _F3H_, _ANS_, and _DFR_ (Table 3, Figure 4) was significantly higher compared to control tobacco plants. Furthermore, these transgenic plants also demonstrated increased resistance to insect pests, such as _S. litura_[249]. Arabidopsis double mutant _WOX1_, which simultaneously overexpresses both MYB12 and PAP1 TFs, contained up to 20 times more anthocyanins in all parts of the plants, although the overall flavonoid content only increased two-fold [235]. The _WOX1_ plants accumulated more anthocyanins and total flavonoids than the plants that individually overexpress either MYB12 or PAP1. These double mutants demonstrated greater radical scavenging activity and increased resistance to oxidative stress induced by methyl viologen, approximately three-fold compared to the wild-type plants [235]. Additionally, the double mutants showed enhanced drought resistance (no watering for 20 days) compared to the wild-type plants. Simultaneous expression of two maize transcription factors, MYB type C1 and MYC type LC, under the control of the fruit-specific tomato E8 promoter led to an increase in the content of kaempfer (approximately 60-fold), naringenin (2-fold), and quercetin (3-fold) in tomato fruits [250]. In _C1/LC_ transgenic tomato plants, the expression of _CHS_, _F3H_, and _DFR_ increased by over 100-fold, while the expression of _FLS_ and _ANS_ was also higher in transgenic plants, by 5 to 15 times compared to the wild-type plants [250]. The tomato genome contains four homologous R2R3-MYB transcription factors: SIAN2, SIANT1, SIANT1-like, and SIAN2-like/Aft. Dark purple tomato plants were obtained by inserting the 35S CaMV promoter into the promoter region of the _SIANT1_ gene using the TALENs and CRISPR/Cas9 systems, and the bean yellow dwarf virus (BeYDV) vector for producing donor DNA. This strategy increased the expression level of the TFs, which resulted in an increased expression level of genes responsible for flavonoid synthesis [158]. Insertion of the CaMV 35S into the promoter region of the _SIANT1_ gene using CRISPR/LbCpf1-based HDR resulted in pronounced plant pigmentation and allowed visual selection of edited plants [159]. Tomato _SIAN2-like_ mutants generated by CRISPR/Cas9 accumulated less amounts of flavonoids and were dysregulated in the expression of many flavonoid biosynthesis genes [160]. The _SIAN2_ mutation introduced using CRISPR/Cas9 into purple tomatoes cv.