Structural genes and enzymes

The identification and isolation of genes involved in flavonoid biosynthesis has benefited from the fact that many of the flavonoids are colored compounds. Mutant phenotypes are therefore often easily identifiable based on variation in color. In Arabidopsis many of the genes involved in flavonoid bioysnthesis have been uncovered based on the change in seed coat (testa) color. Wild-type Arabidopsis seeds have a brown color, and mutations in flavonoid biosynthetic genes result in yellow or pale brown color because the underlying cotyledons are visible. These mutants are referred to as transparent testa (tt) mutants. A total of 21 of these mutants have been identified, resulting from either chemical mutagenesis with EMS, or ionizing radiation (X-ray or fast neutrons). This includes 19 tt mutants, and two transparent testa glabra (ttg) mutants, which have pale seeds but also lack trichomes (leaf hairs) (reviewed by Winkel-Shirley, 2001). The absence of flavonoids in the seed coat reduces seed dormancy, and some of the tt mutants were actually identified based on their reduced dormancy, as opposed to the seed coat color.

Maize mutants with altered flavonoid metabolism can also be identified based on variation in color, either of the seeds, the vegetative parts of the plant, or the floral structures (anthers and silks). Petunia (Petunia hybrida) and snapdragon (Antirrhinum majus) have also been widely used as model species for the elucidation of flavonoid biosynthesis (reviewed by Winkel-Shirley, 2001). In the description of genes involved in flavonoid biosynthesis presented in this section, the emphasis will be on maize and Arabidopsis.

Flavonoid biosynthesis (Figure 3-7) is initiated from the condensation of p-coumaroyl-CoA (3.31) with three molecules malonyl-CoA (3.48), which is catalyzed by the enzyme chalcone synthase (CHS), and gives rise to 4,2',4',6' tetrahydroxychalcone (3.49). This compound can undergo a number of reactions that give rise to the different classes of compounds described in Section 3.6 of Chapter 1.

CHS in maize is encoded by the Colorless2 (C2) gene. Mutations in this gene result in yellow kernels as a result of a colorless aleurone (Reddy and Coe, 1962). The aleurone is the cell layer under the pericarp (the hard outer cell layer of the maize kernel). The C2 gene was cloned via transposon tagging with the Spm transposon by Wienand et al. (1986). Niesback-Klosgen et al. (1987) further characterized the gene. In Arabidopsis CHS is encoded by the TT4 locus (Feinbaum and Ausubel, 1988).

The product generated by CHS, 4,2',4',6' tetrahydroxychalcone (3.49) is the substrate for aurones, yellow pigments common in the petals of flowers, that contain a five-member ring and additional hydroxyl groups on the B-ring. An example is aureusidin (4,6,3',4'-tetrahydroxyaurone; 3.50). Aureusidin synthase, the enzyme responsible for the formation of aureusidin (3.50) was isolated from 32 kg of yellow snapdragon buds via a series of biochemical separations (Nakayama et al., 2000). Oligonucleotide primers based on partial amino acid sequence obtained from the isolated protein enabled the isolation of the corresponding cDNA clone. The cDNA was shown to encode a 64 kDa protein with similarity to polyphenoloxidases from various plant species. The mature aureusidin synthase can be produced after cleavage of a 10 kDa N-terminal transit peptide, and a 15 kDa C-terminal peptide of unknown function. The mature aureusidin synthase is a 39 kDa copper-containing glycoprotein that catalyzes both the hydroxy lation of the B-ring and the oxidative cyclization of the 5-member ring characteristic for aurones. The compound 3,4,2',4',6' pentahydroxychalcone was shown to be a better substrate for the production of aureusidin (3.50) and the aurone bracteatin (4,6,3',4',5'-pentahydroxyaurone). Given the similarity of aureusidin synthase to polyphenol oxidases (PPO's), Nakayama et al. (2001) investigated the specificity of the enzyme, and concluded it was a highly specific PPO with substrate specificity for chalcones with a 4-mono or 3,4-dihydroxy substitution pattern.

The formation of a six-member ring from 4,2',4',6' tetrahydroxychalcone (3.49), catalyzed by chalcone isomerase (CHI), results in the flavanone naringenin (3.51). In Arabidopsis CHI is encoded by the TT5 locus (Shirley et al., 1992) Naringenin is subsequently converted by flavanone 3-hydroxylase (F3H) to yield the flavanonol dihydrokaempferol (3.52). This compound can be converted by flavone synthase (FLS) to the flavone kaempferol (3.53). Alternatively, the B-ring of dihydrokaempferol can be substituted with additional hydroxyl groups by flavonoid 3'-hydroxylase (F3'H) or flavonoid 3',5'-hydroxylase (F3'5'H) to produce dihydroquercetin (3.54) and dihydromyricetin (3.55), respectively. These latter two compounds can subsequently be converted by FLS to their corresponding flavones, quercetin (3.56) and myricetin (3.57). F3H and F3'H are encoded by the Arabidopsis TT6 and TT7 genes, respectively (Pelletier and Shirley, 1996; Schoenbohm et al., 2000).

The flavanonols can also give rise to anthocyanins. This involves a multi-step process whereby they are first reduced to leucoanthocyanidins -leucopelargonidin (3.58), leucocyanidin (3.59) and leucodelphinidin (3.60) -by the enzyme dihydroflavonol 4-reductase (DFR) (Figure 3-7), followed by dehydration and glycosylation (Figure 3-8). The enzyme DFR is encoded by the maize Anthocyaninlessl (Al) gene, based on the colorless aleurone layer in the seed, and lack of pigmentation of the green tissues of the plant in the al mutant. This gene was cloned by O'Reilly et al. (1985) using transposon tagging, and its sequence was analyzed by Schwarz-Sommer et al. (1987). In Arabidopsis DFR is encoded by the TT3 locus (Shirley et al., 1992).

Anthocyanidin synthase (ANS) is the enzyme that dehydrates the leucoanthocyanidins (Figure 3-8). While this enzyme had been postulated to exist, and cDNA's encoding the putative ANS had been identified (e.g. A2 in maize; Messen et al., 1990), in vitro ANS activity was only recently demonstrated in extracts from the beefsteak plant (Perilla frutescens (L.) Britt; Saito et al., 1999).

ANS is a 2-oxoglutarate-dependent oxygenase that is thought to abstract a hydrogen radical from C2 of leucoanthocyanidin (3.61) to yield the radical (3.62) (Figure 3-8). Following a second hydrogen abstraction at C3, the 2-flaven-3,4-diol (3.63) is formed. The reaction can also occur in the reverse order, i.e. abstraction of the hydrogen at C3 followed by the one on C2. The colorless 2-flavene-3,4-diol is hydrated to 3-flavene-2,3-diol (3.64), which under acid conditions can give rise to anthocyanidin (3.65). The glycosylation of anthocyanidins results in the formation of anthocyanins

(3.66) and is catalyzed by UDP-glucose:flavonoid 3-O-glucosyltransferase, also referred to as anthocyanidin 3-glycosyl transferase (3GT). In maize this enzyme is encoded by the Bronzel (Bz1) gene (Dooner and Nelson, 1977; Larson and Coe, 1977), and mutations in this gene result in bronze aleurone in the seed and brown vegetative tissues. The Bz1 gene was cloned via transposon-tagging with Ac by Federoff et al. (1984). The maize bronze2 (bz2) mutant looks very similar to the bzl mutant. The Bz2 gene encodes a glutathione S-transferase required for tagging anthocyanins, synthesized as a result of Bz1 activity, with glutathione. This modification appears necessary for transfer of anthocyanins into the vacuole. The Bz2 gene was cloned via transposon-tagging with Mu and Ds by McLaughlin and Walbot (1987) and Theres et al. (1987), respectively.

Condensed tannins (3.68) arise from polymerization of flavonoids. Polymerization starts with the condensation of a 2,3-cis-flavonol residue

(3.67) onto a 2,3-trans--flavonol 'starter' residue, after which additional 2,3-cis-flavonol residues polymerize. The biosynthesis of the monomers of condensed tannins was poorly understood until the discovery of the Arabidopsis banyuls mutant. This mutant, named after a French wine, displays transparent testa as a result of accumulation of red anthocyanins, and loss of condensed tannins in the seed coat (Albert et al., 1997). The BANYULS gene was cloned and shown to encode an enzyme with similarity to DFR (Devic et al., 1999). Thus, it was initially proposed that the BANYULS gene encoded leucoanthocyanidin reductase (LAR), which reduces leucoanthocyanidins to the 2,3-trans-flavonol 'starter' residue. This was subsequently disproved, as the product of the BANYULS gene did not show any activity towards leucoanthocyanidins, but instead was shown to use anthocyanidins as a substrate (Xie et al., 2003). The resulting products are 2,3-cis-flavonols (3.67). The biosynthetic origin of the 2,3-trans-flavonols remains to be elucidated, but several uncharacterized DFR-like genes

Figure 3-7. Flavonoid biosynthesis (this page and next page). The enzymes involved in this pathway are: (a) chalcone synthase (E.C. 2.3.1.73), (b) aureusidin synthase (E.C. 1.21.3.6), (c) chalcone isomerase (E.C. 5.5.1.6), (d) flavanone 3-hydroxylase (E.C. 1.14.11.9), (e) flavone synthase (E.C. 1.14.11.22), f) flavonoid 3'-hydroxylase (E.C. 1.14.13.21),

Figure 3-7. Flavonoid biosynthesis (this page and next page). The enzymes involved in this pathway are: (a) chalcone synthase (E.C. 2.3.1.73), (b) aureusidin synthase (E.C. 1.21.3.6), (c) chalcone isomerase (E.C. 5.5.1.6), (d) flavanone 3-hydroxylase (E.C. 1.14.11.9), (e) flavone synthase (E.C. 1.14.11.22), f) flavonoid 3'-hydroxylase (E.C. 1.14.13.21),

have been identified in the Arabidopsis genome, one or more of which may encode LAR.

A detailed analysis of the Arabidopsis tt12 mutant revealed that the vacuole in the endothelial cells (the innermost layer of the testa) accumulated lower levels of condensed tannins, and that instead the cytosol contained higher levels of these compounds. The TT12 gene was cloned using T-DNA tagging. Sequence analysis revealed that the TT12 protein shows similarity to a multidrug secondary transporter of the MATE (multidrug and toxic compound extrusion) family. Based on the phenotype and the sequence similarity, the most likely role of the TT12 protein is that it functions as a vacuolar transporter for the precursors of condensed tannins (leucocyanidin and catechin), as it is very unlikely that the polymeric procyanidin itself may be handled by a transporter. Consequently, the pale brown color of the tt12 seeds may be the result of the accumulation of precursors of condensed tannins in the cytoplasm (Debeaujon et al., 2001).

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