## 3 Flavonoids
### Biosynthesis of Flavonoids
The knockout of the _F3'H_ gene in _Euphoria pulcherrima_ resulted in a significant decrease in cyanidin levels and a consequent change in bract color from red to yellow-red (Table 2) [150]. Flavanones are also the substrates in the flavone, isoflavone, and phlobaphene biosynthetic pathways (Figure 4). F3H competes with flavone synthase (FNS), isoflavone synthase (IFS), and flavanone 4-reductase (FNR), respectively, for these common substrates [207]. FNS is subdivided into two classes: FNSI and FNSI. FNSI comprises NADPH- and oxygen-dependent cytochrome P450-membrane monooxygenases, widely distributed in higher plants [208; 209]. FNSI consists of soluble 2-oxoglutate- and Fe\({}^{2+}\)-dependent dioxy genases, which are predominantly found in Apiaceae [210]. Nevertheless, FNSI was also identified and characterized in maize and Arabidopsis [185]. The biosynthesis of flavones is similar in all higher plants, but in _Scutellaria bialensis_, which is a traditional medicinal plant in China and is rich in flavones, the additional flavone synthetic pathway was found [211]. In this pathway, cinnamic acid is converted directly by cinnamate-CoA ligase (CLL-7) to cinnamyl-CoA independently of C4H and 4 CL enzyme activity (Figure 4), with subsequent formation of flavones baialein and norwogonin. It has been shown that flavanones (naringenin and eriodictyol) are converted to flavan-4-ols upon inhibition of F3H activity due to the catalytic activity of FNR [212]. Flavan-4-ols are the immediate precursors of one more class of flavonoids, phlobaphenes [213]. Phlobaphenes are only synthesized in maize and other cereals [186; 214] and this way is controlled by the MYB (myeloblastosis)-type transcription factor (TF) P [215]. Anthocyanidin synthase (ANS), which is also called leucanthocyanidin dioxygenase (LDOX), converts leucanthocyanidin to anthocyanins. It was also shown that this enzyme takes part in the proanthocyanidin biosynthesis pathway, which is important for normal vacuole development in Arabidopsis [216]. Proanthocyanidin's (tannins) are synthesized from leucanthocyanidin and anthocyanins due to the activity of anthocyanin reductase (ANR) [13] and leucanthocyanidin reductase (LAR) [217]. LAR, which is responsible for reduction of leucanthocyanidin to catechin (trans-flavan-3-ol), has been reported in legumes [218], grapes [219] and _Populus trichocarpa_[220], but it has not been found in Arabidopsis. Flavonoid 3-O-glucosyltransferase (FGT) is involved in the glycosylation of unstable anthocyanins to stable anthocyanins [221]. Seven genes encoding this enzyme have been identified in the Arabidopsis genome [188; 189; 190; 191]. Their further modifications (acylation, glycosylation, and methylation) lead to the formation of various anthocyanins [222]. Flavonoid biosynthesis is under the control of transcriptional regulator complex MBW, composed of the basic helix-loop-helix (bHLH), MYB, and WD40 (tryptophan-aspartic acid (W-D) dipeptide) proteins. The family of bHLH proteins, which is involved in many essential biological processes, is very common in all eukaryotic organisms [223]. The ability of MYB TFs to bind to DNA and interact with other proteins is governed by a conserved MYB domain at their N-terminus [224]. According to the number and position of MYB domain repeats, MYB proteins can be divided into four groups (1R-, R2R3-, 3R-, and 4R-MYB), where the most important TFs are R2R3-MYB (for a review, see [225; 226]). In _A. thaliana_, the R2R3-MYB gene family is formed by the genes _MYB11_, _MYB12_, and _MYB111_[227]. In Arabidopsis, the knockout of _MYB12_ (_ATMYB12_) resulted in reduced amounts of quercetin and kaempferl in the seedlings, and the flavonoid content was increased in ATMYB12-OX plants. However, the plants with either overexpressed or knocked-out _ATMYB12_ gene did not show any significant difference in flavonoid content, and there were no obvious changes in their phenotype compared to wild-type plants.