3.2.2. Flavonoids

Flavonoids are a large class of phenolic compounds, over 6000 have been identified so far (Metabolimics). They are among the best characterized class of natural compounds. Decades of work has been done on the biosynthesis, regulation and natural variation (Dooner et al. 1991; Mol et al. 1996; Grotewold 2006) . They consist of several subclasses, including anthocyanins, flavonols, flavones, chalcones, and aurones, based on their core structure: the aglycone. Flavonoids are important for plant growth and development, such as pathogen resistance, pigment production, UV light protection, pollen growth, and seed coat development. Anthocyanins are the best-known subclass of flavonoids, because they are, besides carotenoids, also responsible for the coloration of flowers and fruits. They are stored in the vacuole and exists in the range from red, pink, mauve, purple, blue, and violet. The pH of the vacuole and presence of co-pigments can also modify the colour. The famous Litmus test is based on the colour shift from red to blue that anthocyanins undergo when changing from low pH to high pH. Other known flavonoids are the flavonols kaempferol and quercetin, that can protect plants against UV-B radiation. Another important class is the proanthocyanins, or tannins, that add a bitter taste or astringency to certain plant tissues (e.g. unripe banana and raspberries) and thereby can act as anti-feedants. This can be for herbivore insects, but also for animals including humans (Palo and Robbins 1991; Ament 2004). In addition to flavonoids, other phenolic compounds can add to the quality of food. They determine fragrance, flavour, colour, and antioxidant status (Craig 1999; Verdonk et al. 2003; Petersen et al. 2009).

In plants, flavonoids are synthesized in the cytosol, and transported to the vacuole. The pH of the vacuole determines the colour, in combination co-pigments and metal ions (Tanaka et al. 2008). They are important for the attraction of pollinators and seed dispersers, through their distinctive colours. Another biological interaction that is facilitated by flavonoids is the legume-rhizobium bacteria interaction. During low nitrogen conditions, legume plants secrete specific flavonoids (isoflavonoids, e.g. genistein and daizein), which are recognized by the bacteria (Liu and Murray 2016). The oligomeric proanthocyanins can protect plants against microorganisms above-ground, for example strawberries can keep Botrytis in a quiescent state (Jersch et al. 1989; Amil-Ruiz et al. 2011).

The biosynthesis of flavonoids branches off from the general phenylpropanoid pathway (another class of phenolic compounds, including cinnamic acid and caffeic acid), which is fed by the aromatic amino acids phenylalanine and tyrosine. The shikimate pathway, best known for being the target for Roundup (Glyphosate), provides the aromatic amino acids (Figure 3.3A) (Herrmann and Weaver 1999; Maeda and Dudareva 2012) . The phenylpropanoid pathway provides precursors for many important plant secondary metabolites, lignin, benzenoids, coumarins, and stilbenes (Buchanan-Wollaston et al. 2002). However, the flavonoid branch is considered the most important side branch of the phenylpropanoid pathway (Figure 3.3B). The first step in the flavonoid pathway, is the conversion of coumaroyl-CoA together with 3 Malonyl-CoA molecules into naringin chalcone by chalcone synthase (CHS). This molecule is immediately isomerized into naringenin by chalcone isomerase (CHI). From this precursor, dihydrokaempferol (DHK) is synthesized by hydroxylation of the 3-position of the C ring (Figure 3.3C). DHK can be hydroxylated on the 3-position of the B ring to form dihydroquercetin, and on both the 3 and the 5 position of the C ring to form dihydromyrcetin by F3’H and F3’5’H respectively. The dihydroflavonols are reduced to the leucoanthocyanidins leucocyanidin, leucopelargonidin and leucodelphinidin by dihydroflavonols 4-reductase (DFR). The final step to the anthocyanidins is performed by ANS, anthocyanidin synthase, resulting in cyanidin (base aglycone for red compounds), pelargonidin (orange), and delphinidin (violet/blue) (Fig 3.3B). These aglycones are subsequently ‘decorated’ by a 3-glucosyl transferase (3GT) to yield an anthocyanin3-glucoside. Further down the pathway substitution by 5-glucosyl- (5GTs), rhamnosyl- (RTs), acyl- (ATs) and/or methyltransferases (MTs), results in the ‘decorated’ anthocyanins with different colours(Koes et al. 2005). The absence of specific colour in important horticultural crops, such as violet/blue in rose and chrysanthemum, and orange in petunia is due to the absence of specific enzymes in the pathway, or lacking substrate specificity (reviewed in Tanaka et al. 2008)). Anthocyanidins can be modified by acyltransferases, methyltransferases and glycosyltransferases. The A- and C- ring are modified by these enzymes which are less specific to the exact anthocyanin structure of the B-ring. Instead these enzymes are specific for the modification and the donor substrates (Tanaka et al. 2008). Proanthocyanins are oligomeric flavonoids, or oligomeric proanthocyanidins (OPC), dimer and trimer polymerizations of catechin and/or epicatechin. Catechin and epicatechin are derived from leucocyanidin and cyanidin by LAR (leucoanthocyanidin reductase), and ANR (anthocyanidin reductase), respectively (Figure 3.3B). The enzymes and genes for most of these steps in the pathway were characterized through work on coloured mutant in maize and petunia. Manipulating flower colour by targeting various enzymatic steps and genes in flavonoid biosynthesis has been quite successful, particularly in petunia (Reviewed in Koes et al. 2005).

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Figure 3.3: Flavonoids and anthocyanin biosynthesis. A: Simplified model of the biosynthesis pathway towards phenylpropanoid precursors for flavonoids (see also Maeda and Dudareva, 2012). B: Simplified model of the anthocyanin biosynthetic pathway, (Koes et al., (2005); Abdou and Verdonk, unpublished). C: Basic aglycone flavonoids structure, with rings named and positions numbered (Harborne and Baxter 1999). Abbreviations: ANS: Anthocyanin synthase; CHS: Chalcone synthase; CHI: Chalcone isomerase; DAHP: 3-Deoxy-D-arabino-heptulosonate-7-phosphate; DFR: dihydroflavonol reductase; DHQ: 3-Dehydroquinate; E4-P: Erythrose 4-phosphate; EPSP: 5-enolpyruvylshikimate-3-phosphate; EPSPS: EPSP synthase; F3H: flavonoid 3-hydroxylase; F3’H: flavonoid 3’-hydroxylase; F3’5’H: flavonoid 3’5’hydroxylase; PEP:  phospho enol-pyruvate; 1: DAHP synthase; 2: DHQ synthase; 3: DHQ dehydratase/Shikimate dehydrogenase; 4: Shikimate kinase; 5: Chorismate Synthase; 6: anthranilate synthase for Trp/Chorismate mutase/prephenate aminotransferase; 7: arogenate dehydratase for Phe/ arogenate dehydrogenase for Tyr; 8: Tyrosine aminotransferase; 9: HPP dioxygenase/homogentisate phytyltransferase; 10: cinnamate-4-hydroxylase; 11: 4-Coumarate-CoA ligase; 12: caffeoyl-coenzyme A O-methyltransferase; 13: Ferulic acid hydroxylase; 14: caffeoyl-coenzyme A O-methyltransferase.