Fig. 5. Shikimate pathway
The biosynthesis of flavonoids starts with the condensation of one p-coumaroyl-CoA molecule (shikimate-derived, B ring) with three molecules of malonyl-CoA (polyketid origin, A ring) to give chalcone (2’4’6’4-tetrahydroxychalcone) catalyzed by chalcone synthase (CHS) enzymes. Chalcones are subsequently isomerized to flavanone by chalcone flavanone isomerase (CHI). The pathway diverges from these central intermediates into several side branches, each providing a different class of flavonoids.
The initial steps in this pathway are referred to as the general phenylpropanoid pathway [26]. In phenylpropanoid pathway, an aromatic amino acid, phenylalanine first converted to p-coumaroyl-CoA through the activity of phenylalanine ammonia lyase (PAL), cinnamic acid 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL). PAL catalyzed the deamination of phenylalanine to trans-cinnamic acid in the first committed step called as deamination of phenylalanine to trans-cinnamic acid [27]. In the phenylpropanoid pathway, the second step involves the activity of C4H, a cytochrome P450 monooxygenase, in plants. This enzyme catalyzes the hydroxylation of trans-cinnamic acid and generates p-coumaric acid. This was the first oxidation reaction [28]. The third step in the general phenylpropanoid pathway involves the activation of p-coumaroyl-CoA by the action of 4-courmarate: CoA ligase. In plants, the activity of 4CL is positively correlated with anthocyanin and flavonol content in response to stress [29], whereas PAL, C4H, and 4CL are generally coordinately expressed [30]. Chalcone synthase (CHS) catalyzes the stepwise condensation of three acetate units to yield naringenin chalcones. In vitro, naringenin chalcone is transformed into naringenin by chalcone isomerase (CHI) [31]. Naringenin is a very important flavonoid frame catalyzed by FNS I and FNS II (flavone synthases I and II) and IFS (isoflavone synthase) to form flavones and isoflavones respectively [32]. Naringen can also be catalyzed by flavanone-3-hydroxylase (F3H), flavonol-3’-hydroxylase (F3’H) and flavonol-3’5’-hydroxylase (F3’5’H) to synthesized dihydro-myricetin, dihydro-kaempferol and dihydro-quercetin, respectively [33]. The flavonol synthase (FLS) converted dihydro flavonols into flavonols (like kaempferol, quercetin and myricetin), which further was catalyzed by dihydroflavonol 4-reductase (DFR) to generate leucoanthocyanidins [34], further catalyzed by leucoanthocyanidin dioxygenase (LDOX) to produce anthocyanidins [35]. anthocyanidins and leucoanthcyanidins were again converted to proanthocyanidins catalyzed by leucoanthocyanidin reductase (LAR) and anthocyanidine reductase (ANR), repectively [36]. variations in anthocyanins is then responsible for the stabilization of vacuolar anthocyanins which includes glycosylation, methylation and acylation [37].
In general phenylpropanoid pathway shown in figure 6 is common to all the downstream metabolites like flavonoids and lignin. The metabolic pathway continues through a series of enzymatic modifications to yield flavonols, dihydroflavonols and anthocyanins.