**3. Biosynthetic pathway and regulation**

Biosynthesis of the flavone backbone is originated from the phenylpropanoid pathway fol‐ lowed by the flavonoid biosynthetic branch (**Figure 1**). The phenylalanine ammonia-lyase (PAL) deaminates the phenylalanine, being converted into trans-cinnamate, after that a hydroxyl group is introduced on the phenyl ring by cinnamic acid 4-hydroxylase (C4H), being the trans-cinnamate converted into 4-coumarate. The carboxyl group of p-4-coumarate is then activated to form 4-coumarate-CoA (by a thioester bond), catalyzed by 4-coumarate-CoA ligase (4CL). This product, 4-coumarate-CoA, is substrate for different enzymes, so it represents a branching point of the pathway to either stilbenes of flavonoids. In this case, 4 coumarate-CoA is then condensed with three units of malonyl-CoA by the chalcone synthase (CHS, first enzyme of the flavonoid pathway), forming the naringenin chalcone (flavonone), which is transformed into naringenin by the chalcone synthase (CHI). Naringenin is hydrox‐ ylated by flavonone-3-hydroxylase (F3H) being converted in dihydrokaempferol, which is then hydroxylated by flavonoid-3′-hydroxylase (F3′H) and transformed in dihydroquercetin or by flavonoid-3′-5′-hydroxylase (F3′5′H) to form dihydromyricetin. Flavonols are synthe‐ sized at this point by the flavonol synthase (FLS), which introduces a double bond between C2 and C3 in either of the three above-mentioned molecules forming kaempferol, quercetin, or myricetin, respectively. Dihydroquercetin is reduced by dihydroflavonol reductase (DFR) to obtain leucocyanidin; similarly, dihydrokaempferol is transformed in leucopelargonidin and dihydromyricetin in leucodelphinidin. Anthocyanins are synthetized at this point by the anthocyanidin synthase (ANS) obtaining cyanidin, pelargonidin, or delphinidin, respectively. Catechins include (+)-catechin and (−)-epicatechin; (+)-catechin is obtained when leucocyani‐ din reductase (LAR) reduces leucocyanidin, and (−)-epicatechin is obtained when anthocyani‐ din reductase (ANR) reduces cyanidin [1, 7, 8].

Flavonols have a 3-hydroxyflavone (IUPAC name: 3-hydroxy-2-phenylchromen-4-one) as the main structure. The diversity of these compounds is derived from the different positions of the hydroxyl groups of the phenolic ring that are usually glycosylated and can undergo fur‐ ther modifications like acylations; in this group, the three main families are derived from

Anthocyanins are mainly glycosylated as well, being the aglycon the anthocyanin molecule. The chemical structure of this aglycone is the flavylium ion (2-phenyl-benzopirilo) that has a benzopyran aromatic ring, and a phenolic ring. There are six different families within this group, namely cyanidin, pelargonidin, delphinidin, malvinidin, peonidin, and petunidin. As in the case of flavonoids, the greatest source of chemical diversity is the number and position of sugars for glycosylation. Acylation is another main biochemical mechanism leading to diverse anthocyanin molecules in *Arabidopsis* [14, 15]. Up to date, several enzymes have been character‐ ized to catalyze these acylation reactions, using either malonyl-CoA or *p*-coumaroyl-CoA as substrates to transfer the malonyl or *p*‐coumaroyl groups to cyanin structures [16]. Diversity can be further increased transferring sinapoyl groups to cyanins to form sinapoylated cyanins [17]. Catechins have two benzene rings (A-, B-) and a dihydropyran heterocyclic ring (C) with a hydroxyl group over carbon 3. As a result of this structure, catechins have four diasteroiso‐ mers, two with *trans* configuration called catechin ((+)-catechin and (−)-catechin), and two with *cis* configuration called epicatechin ((+)-epicatechin and (−)-epicatechin). These catechins can further polymerize to form proanthocyanins, in which the diversity of structures relies on

the number of monomers that polymerize and the type of bonds that stabilize them.

Biosynthesis of the flavone backbone is originated from the phenylpropanoid pathway fol‐ lowed by the flavonoid biosynthetic branch (**Figure 1**). The phenylalanine ammonia-lyase (PAL) deaminates the phenylalanine, being converted into trans-cinnamate, after that a hydroxyl group is introduced on the phenyl ring by cinnamic acid 4-hydroxylase (C4H), being the trans-cinnamate converted into 4-coumarate. The carboxyl group of p-4-coumarate is then activated to form 4-coumarate-CoA (by a thioester bond), catalyzed by 4-coumarate-CoA ligase (4CL). This product, 4-coumarate-CoA, is substrate for different enzymes, so it represents a branching point of the pathway to either stilbenes of flavonoids. In this case, 4 coumarate-CoA is then condensed with three units of malonyl-CoA by the chalcone synthase (CHS, first enzyme of the flavonoid pathway), forming the naringenin chalcone (flavonone), which is transformed into naringenin by the chalcone synthase (CHI). Naringenin is hydrox‐ ylated by flavonone-3-hydroxylase (F3H) being converted in dihydrokaempferol, which is then hydroxylated by flavonoid-3′-hydroxylase (F3′H) and transformed in dihydroquercetin or by flavonoid-3′-5′-hydroxylase (F3′5′H) to form dihydromyricetin. Flavonols are synthe‐ sized at this point by the flavonol synthase (FLS), which introduces a double bond between C2 and C3 in either of the three above-mentioned molecules forming kaempferol, quercetin, or myricetin, respectively. Dihydroquercetin is reduced by dihydroflavonol reductase (DFR) to obtain leucocyanidin; similarly, dihydrokaempferol is transformed in leucopelargonidin

**3. Biosynthetic pathway and regulation**

kaempferol (4′OH), quercetin (3′, 4′, 5′OH) and rutin (3′, 4′OH).

134 Flavonoids - From Biosynthesis to Human Health

All these aglycons are highly apolar, so they are immediately glycosylated to increase polar‐ ity, in order to be stored in vacuoles or translocated throughout the plant, hence glycosiltrans‐ ferases are very important for glycosylation as well as transport mechanisms. In *Arabidopsis*, three genes, *TT12*, *TT19*, and *AHA10*, have been functionally characterized to be associated with the transport of anthocyanins. However, these enzymes show different levels of specific‐ ity for flavonoids [18]. Anthocyanins are stored in the central vacuole of cells, so they need to be transported from the cytosol to the vacuole. Two major hypotheses have been proposed to solve this transport: either transporter-mediated or vesicle-mediated transport [19–21].

Plants have depicted a system in which all these enzymes are extremely well organized in the different compartments within cells, in order to improve efficiency of these natural products' synthesis. Successive enzymes are arranged in imaginary units termed metabolons, anchored to the ER membrane, ensuring channeling of the intermediate precursors in the complex with‐ out diffusing to the cytosol, avoiding metabolic interferences [18, 22].

*A. thaliana* is a good model species for the identification of genes controlling flavonoid metab‐ olism (**Table 3**) [23], because all pathway core genes of anthocyanins have been molecularly, genetically, and biochemically characterized in this plant. On the other hand, it is amenable to both molecular and classical genetic analysis [24, 25].

On the other hand, homologues to all core genes in the flavonol-anthocyanin pathway have been identified in *Rubus* sp. Var Loch Ness [26]. Also, most genes corresponding to the MYB transcrip‐ tion factors have also been identified with similar functions. Interestingly, MYB12 [26] that was originally identified as a key flavonol-specific transcriptional activator in *A. thaliana* [27] and in


**Table 3.** List of the flavonol-anthocyanin pathway core and regulatory genes in *A. thaliana* [23].

other plant species such as tomato [28] has not been found in *Rubus* [26, 29] suggesting a differ‐ ent control mechanism of the flavonol-anthocyanin pathway in this plant species.

Currently, dihydrokaempferol and dihydroquercetin are the only two dihydroflavo‐ nol molecules identified in *Arabidopsis* [18]. Flavonoids have been analyzed using liquid chromatography-mass spectrometry (LC-MS) and/or nuclear magnetic resonance (NMR). Briefly, anthocyanins and glycosylated kaempferol flavonols are mostly found in leaves [17], whereas seeds contain epicatechin, PAs, and larger amounts of glycosylated quercetin flavo‐ nols [30, 31]. Interestingly, arabidopsis seeds contain large amounts of PAs similar to those present in other crop seeds or fruits (**Table 4**) [32–34].


**Table 4.** Compounds identified by LC-MS-MS in seed extracts of wild type *A. thaliana* [31].

The metabolic profile of flavonols and anthocyanins in blackberry fruits is formed by the fla‐ vonols kempferol and quercetin and their respective derivatives, while cyanidin derivatives are the unique anthocyanidins present. Interestingly, catechins and epicatechins are also pres‐ ent, especially upon fruit ripening [26, 35]. The specific composition for blackberries obtained from *Rubus spp*. Var Loch Ness appears in **Table 5**.


**Table 5.** Compounds identified in *Rubus* spp. Var. Loch Ness fruit by LC-MS-IT-ToF [26].

other plant species such as tomato [28] has not been found in *Rubus* [26, 29] suggesting a differ‐

Currently, dihydrokaempferol and dihydroquercetin are the only two dihydroflavo‐ nol molecules identified in *Arabidopsis* [18]. Flavonoids have been analyzed using liquid chromatography-mass spectrometry (LC-MS) and/or nuclear magnetic resonance (NMR). Briefly, anthocyanins and glycosylated kaempferol flavonols are mostly found in leaves [17], whereas seeds contain epicatechin, PAs, and larger amounts of glycosylated quercetin flavo‐ nols [30, 31]. Interestingly, arabidopsis seeds contain large amounts of PAs similar to those

> Quercetin-hexoside-rhamnoside Quercetin-3-O-rhamnoside Quercetin‐rhamnoside dimer 1 Quercetin‐rhamnoside dimer 2 Quercetin‐rhamnoside dimer 3 Quercetin‐rhamnoside dimer 4 Quercetin‐di‐rhamnoside Quercetin-3-O-glucoside

Kaempferol

Kaempferol-rhamnoside

Procyanidin trymer Procyanidin tetramer Procyanidin pentamer Procyanidin hexamer Procyanidin heptamer

Kaempferol-rhamnoside-hexoside Kaempferol-3, 7-di-O-rhamnoside

Isorhamnetin-hexoside-rhamnoside Isorhamnetin-di-rhamnoside Isorhamnetin-rhamnoside

Kaempferol-3-O-glucoside-7-O-rhamnoside

ent control mechanism of the flavonol-anthocyanin pathway in this plant species.

present in other crop seeds or fruits (**Table 4**) [32–34].

Anthocyanins Procyanidin dimer

Catechins Epicatechin

**Table 4.** Compounds identified by LC-MS-MS in seed extracts of wild type *A. thaliana* [31].

Flavonols Quercetin-rhamnoside-hexoside

**Group Compound**

136 Flavonoids - From Biosynthesis to Human Health
