*2.3.1.8 Anthocyanin and Leucoanthocyanidin Biosynthesis*

Major enzyme in flavonoid metabolism in the anthocyanidin and proanthocyanidin pathways is dihydroflavonol-4-reductase (DFR). A hydroxyl group is produced at C-4 position of ring C by the NADPH-dependent reductase known as DFR [60–62]. Dihydroflavonols, DHQ, DHK, and DHM are reduced by DFR to produce leucocyanidin, leucopelargonidin, leucoanthocyanidins, and leucodelphinidin [63]. DFR, for example, transforms DHK to leucopelargonidin in *Vitis vinifera* [64]. The direct synthetic precursor of anthocyanidin and proanthocyanidin. Leucoanthocyanidin, is a crucial intermediary by-product in the flavonoid pathway. The colorless leucopelargonidin, leucocyanidin, and leucodelphinidin are converted into the equivalent anthocyanidins under the catalysis of anthocyanidin synthase (ANS) (the colored pelargonidin, cyanidin, and delphinidin) [65, 66]. An alternative name for ANS is leucoanthocyanidin dioxygenase (LDOX). Similar to FNSI, F3H, and FLS, ANS/ LDOX is a FeII/2-oxoglutarate-dependent dioxygenase that stimulates the dehydroxylation of C-4 and formation of a double bond in ring C [67]. In Strawberries, anthocyanin content has been found to get enhanced when ANS is overexpressed [68].

#### *2.3.1.9 Proanthocyanidin biosynthesis*

Condensed tannins, also known as proanthocyanidins, are a form of flavonoid made up of leucoanthocyanidins and anthocyanidins [69]. The primary proanthocyanidin units are cis-flavan-3-ols, trans-flavan-3-ols, and flavan-3-ols. Proanthocyanidins are produced when flavan-3-ols are polymerized (or condensed) [70, 71]. To make colored tannins (yellow to brown), polyphenol oxidase (PPO)

converts colorless proanthocyanidins into plant vacuoles [72]. The major and ratelimiting enzymes in proanthocyanidin production are leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR). Studies have revealed that overexpression of putative leucoanthocyanidin reductase gene (*PtrLAR3)* significantly elevates proanthocyanidin levels in *Populus tomentosa* [73]. Additionally, in alfalfa (*Medicago sativa*), overexpression of *OvBAN*, an ANR gene obtained from *Onobrychis viviaefolia*, increases the concentration of proanthocyanidin and the activity of the ANR enzyme [74]. However, proanthocyanidin and anthocyanin biosynthesis pathways have a competitive relationship since they utilize the same substrates [75].

#### **2.4 Flavonoid biosynthesis in plants is regulated by transcriptional regulation**

In the modification of flavonoid production, transcriptional control is crucial. The major transcriptional regulator in flavonoid biosynthesis is the MBW complex, which consists of WD40, bHLH, and MYB. The MYB domain at the N-terminus of MYB transcription factors (TFs) is needed for DNA binding and interaction with other proteins [76]. According to the amount and location of MYB domain repeats, MYB proteins is categorized into four groups: 3R*-*MYB, 4R*-*MYB, R2R3*-*MYB, and 1R*-*MYB*/*MYB-related. Among the four, R2R3*-*MYB members are mostly engaged in flavonoid metabolism regulation [21].

#### **2.5 Plasticity of flavonoid pathway**

Flavonoids have been discovered in epidermal cells such as trichomes, palisade, and spongy mesophyll. Moreover, flavonoids are found intracellularly in numerous cell compartments such as chloroplasts, vacuoles, and the nucleus [77–79]. The shikimate, phenylpropanoid, flavonoid, anthocyanin, and lignin pathways produce plant phenolics. The aromatic amino acids, including phenylalanine, are produced through the shikimate pathway, and the flavonoids are formed by a series of elongation and cyclization stages. Flavonoids get divided into numerous 15-carbon families, including flavanone, flavonol, flavone, flavan-3-ol, anthocyanidin, and isoflavone. It is evident that the level of B-ring hydroxylation is the sole difference between most of the main molecules [80].

#### *2.5.1 Anthocyanin-proanthocyanidin pathway cross-talk*

Despite the fact that the anthocyanin and proanthocyanidin routes use identical biochemical intermediates, they are the most and least studied flavonoid processes, respectively. Both branches include the formation of precursors from 4-coumaroyl-CoA and malonyl-CoA. ANS, DFR, and a variety of anthocyanidin-modifying enzymes transform dihydromyricetins, dihydroquercetins, and dihydrokaempferols into anthocyanins. Anthocyanidin rhamnosyltransferases, UDP-glucuronosyl/ UDP-glycosyltransferases, methyltransferases, glutathione transferases, and Glycosyltransferases are among the anthocyanidin-modifying enzymes. On the other hand, the family of DFR, LAR, and ANR enzymes convert dihydroflavonols to trans- and cis-epimeric forms of gallocatechins, catechins, and afzelechins in the proanthocyanidin-specific pathway [19, 72, 81, 82].

In a number of plant species, cross-talk between members of the flavonoid pathways' anthocyanin- and proanthocyanidin-specific branches has been seen. Studies have revealed that overexpression of the *ANR* gene in tobacco has suppressed anthocyanin production and induced proanthocyanidin biosynthesis in flower petals. Meanwhile, upregulation of ANR has caused a subset of leaf cells in *Medicago truncatula* plants to produce three times more proanthocyanidin and cut anthocyanin synthesis by half [83, 84].

#### *2.5.2 Lignin-flavonoid pathway cross-talk*

Chemical scaffolds of lignin polymers originated and evolved to offer mechanical support to plants, shield them from UV damage and pathogen invasion, as well as increase the hydrophobicity of their vasculature. As a result, these metabolites have played a critical role in the development of land plants and also, in the colonization of various geographical and ecological environments. Similar to flavonoids, this route assisted the manufacture of H and G lignin in early terrestrial plants by enlisting enzymes from primary metabolism [85].

Redirecting metabolic fluxes between the lignin and flavonoid pathways showed molecular and metabolic cross-talks in a variety of plants with down-regulated genes implicated in the phenylpropanoid, lignin, and flavonoid processes. The flow from feruloyl-CoA to G and S units is lessened when the *CCR* gene is silenced in tobacco, tomato, and poplar, which resulted in a decrease in the amount of phenolic chemicals which are particular to lignin [86, 87]. The quantities and composition of several stress-related flavonoid intermediates and derivatives, on the other hand, were significantly increased in these transgenic lines.
