## 3 Flavonoids
### Biosynthesis of Flavonoids

Table 3: _Arabidopsis thaliana_ genes encoding the key enzymes involved in synthesis of flavonoids. The key reaction in flavonoid biosynthesis is the condensation of activated coumaric acid (4-coumaroyl-CoA) with three molecules of activated malonic acid (malonyl-CoA). This reaction is catalyzed by chalcone synthase (CHS), which is the key and the rate-limiting enzyme in the flavonoid biosynthetic pathway. CHS activity leads to the formation of naringenin-chalcone [194]. Total flavonoid level decreased in tomato plants after RNAi-mediated _CHS_ gene suppression [195]. Malonyl-CoA and p-coumaroyl-CoA are also used in the synthesis of stilbenes catalyzed by stilbene's synthase (STS) [196]. The formation of stilbenes is the first branch of the flavonoid biosynthetic pathway that has only been found in a few plants, such as grapes, pine, sorghum, and peanuts [197; 198]. Naringenin-chalcone is the origin for other chalcones after hydroxylation of the ring B. Its formation is also a key branch point for the synthesis of several major classes of flavonoids: flavonones, flavonoids, and anthocyanins. Naringenin-chalcone is then exposed to dehydrodxylation by chalcone reductase (CHR), yielding isoliquiritigenin [199]. Chalcone isomerases (CHIs) utilize either naringenin-chalcone or isoliquiritigenin to generate naringenin and liquiritigenin, which are another class of flavonoids: flavanones. These flavanones, as well as pentahydroxylavanone and eriodictyl, are common substrates for the synthetic branches leading to flavones, isoflav, and phlobaphenes [200; 201]. Aurones, yellow plant pigments, which are also synthesized from chalcones [202; 203] are found in a relatively small number of plant species, such as snapdragon, sunflower, and coreopsis. Hydroxylation of naringenin by flavanone 3-hydroxylase (F3H) and flavonoid 3'-hydroxylase (F3H) produces dihydrofanvonols, from which dihydrofanvonol reductase (DFR) produces leucoanthocyanidins (Figure 4). The role of the carrot _F3H_ gene was tested on model purple calli. Targeted knockout of this _F3H_ gene using the CRISPR/Cas9 system led to color loss of these calli (Table 2) [148]. With the help of the flavonoid synthase (FLS) enzyme, dihydrofanvonols are converted into flavonoids (Flavan-3-ols) [204] with kaempferol synthesized first, followed by the subsequent formation of other flavonoids. The transgenic tobacco mutant containing the _FLS_ gene from _Camellia sinensis_ showed the accumulation of kaempferol and a decrease in the anthocyanin content in flowers [205]. The other transgenic tobacco mutant, overexpressing _FLS_ from _Allimum cepa_, possessed enhanced quercetin levels in roots [206]. Kaempferol is also important for biosynthesis of 4-HBA, one of the main precursors of UQ synthesis [47]. _A. thaliana_ plants with knocked-out genes encoding kaempferol 3-O-rhammosyltransferase and kaempferol 3-O-glucosyltransferaseaccumulated an increased amount of UQ [147]. This was due to the impaired glycosylation of kaempferol by these enzymes to form kaempferol 3-O-glycos that is a concurrent biochemical pathway for 4-HBA synthesis. This increase in UQ levels is attributed to the absence of glycosylation of kaempferol by the knocked-out enzymes, which normally form kaempferol 3-O-glycos. These glycosides are part of a parallel biochemical pathway that competes with the synthesis of 4-HBA, a precursor for UQ. F3'H is a cytochrome P450 monooxygenase responsible for the hydroxylation of the 3'-position on the B-ring of flavonoids. This enzyme can catalyze the conversion of dihydrofanvonol dihydrokaempferol to dihydroquercetin or kaempferol to quercetin [184]. A site-specific mutation of the _O_s_F3'_\(H\) gene in rice with black grains led to a significant decrease in flavonoid content and, accordingly, a change in grain color to ocher. The same color with the same decrease in flavonoid content was observed in _DFR_ gene knockout plants (Table 2) [149].