In maize there are a number of regulatory genes that mediate transcriptional activation of the anthocyanin biosynthesis pathway genes (see Selinger and Chandler (1999) for a discussion of these regulatory loci). For example, R has been shown to regulate 3 enzymes involved in anthocyanin biosynthesis; chalcone synthase [EC 2.3.1.74] encoded by C2, dihydroflavonol 4-reductase (dihydroquercitin reductase) (DFR) [EC 1.1.1.219] encoded by A1, and flavonol 3-O-glucosyltransferase (UFGT or 3GT) [EC 2.4.1.91] encoded by bronze1 (Bz1). Regulation of the anthocyanin pathway in maize requires two classes of transcription factors; one class (B and R) contains a bHLH motif, and the other (C1 and P1) contains a Myb domain. To activate the genes of the anthocyanin pathway, a protein from each class must be expressed; neither alone is sufficient for induction (Lesnick and Chandler, 1998; and references cited therein). Cell lines of maize engineered to express the C1 and R accumulate two cyanidin derivatives that are similar to the predominant anthocyanin found in differentiated tissues. In contrast, expression of P causes accumulation of 3-deoxy flavonoids (Grotewold et al, 1998).

In Arabidopsis dihydroflavonol 4-reductase (DFR) [EC 1.1.1.219] is encoded by the tt3 locus. Flavonol synthase (FLS) may be encoded by tt6, chalcone synthase (CHS) [EC 2.3.1.74] by tt4, and flavonoid 3'-hydroxylase (F3'H) [EC 1.14.13.21] by tt7 (where tt refers to transparent testa mutants) (Pelletier et al, 1997).

The A2 locus in maize may encode a dioxygenase; thus anthocyanidin synthase (ANS) may actually represent a leucoanthocyanidin dioxygenase (LDOX). The LDOX of Arabidopsis responsible for converting leucopelargonidin to pelargonidin and leucocyanidin to cyanidin, has recently been cloned (Pelletier et al, 1997). UDP-flavonol 3-O-glucosyltransferase (UFGT or 3GT) [EC 2.4.1.91] would then further elaborate pelargonidin and cyanidin to pelargonidin-3-glucoside and cyanidin-3-glucoside, respectively.

The Bronze2 (Bz2) gene in maize encodes a glutathione S-transferase [EC 2.5.1.18] that performs the last genetically defined step in anthocyanin biosynthesis -- tagging cyanidin-3-glucoside with glutathione, allowing for transport to the vacuole via a tonoplast Mg-ATP-requiring GS-X pump (Marrs and Walbot, 1997; Lu et al, 1998; Alfenito et al, 1998). The equivalent locus to Bz2 in petunia is Anthocyanin9 (An9), although the maize Bz2 gene encodes a type III GST while the petunia An9 locus encodes a type I GST (Alfenito et al, 1998).

The 3-deoxyanthocyanidin phytoalexins (apigeninidin and leuteolinidin) accumulated by Sorghum bicolor in response to inoculation with the fungus Cochliobolus heterostrophus, are thought to be derived from naringenin. Fungal inoculation leads to repression of the transcription of the genes encoding F3H, DFR and ANS, leading to inhibition of light-induced accumulation of anthocyanin. In contrast, PAL and CHS are induced. This presumably diverts metabolic flux away from anthocyanin synthesis towards naringenin and 3-deoxyanthocyanidin synthesis to meet immediate biochemical needs for plant defense (Lo and Nicholson, 1998).

The flavan 3,4-diols produced by the action of DFR are not only the precursors of anthocyanins (whose synthesis is mediated by ANS and 3GT), but also catechins and condensed tannins, synthesized by flavan 3,4-diol reductase (FDR) and condensing and polymerizing enzymes. Consequently, antisense-DFR plants of birdsfoot trefoil exhibit reduced condensed tannin levels (Robbins et al, 1998).

Alfenito MR, Souer E, Goodman CD, Buell R, Mol J, Koes R, Walbot V 1998 Functional complementation of anthocyanin sequestration in the vacuole by widely divergent glutathione S-transferases. Plant Cell 10: 1135-1149.

Holton TA, Cornish EC 1995 Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7: 1071-1083.

Grotewold E, Chamberlin M, Snook M, Siame B, Butler L, Swenson J, Maddock S, St. Clair G, Bowen B 1998 Engineering secondary metabolism in maize cells by ectopic expression of transcription factors. Plant Cell 10: 721-740.

Lesnick ML, Chandler VL 1998 Activation of the maize anthocyanin gene a2 is mediated by an element conserved in many anthocyanin promoters. Plant Physiol. 117: 437-445.

Lo SCC, Nicholson RL 1998 Reduction of light-induced anthocyanin accumulation in inoculated sorghum mesocotyls. Implications for a compensatory role in the defense response. Plant Physiol. 116: 979-989.

Lu YP, Li ZS, Drozdowicz YM, Hortensteiner S, Martinoia E, Rea PA 1998 AtMRP2, an Arabidopsis ATP binding cassette transporter able to transport glutathione S-conjugates and chlorophyll catabolites. Functional comparisons with AtMRP1. Plant Cell 10: 267-282.

Marrs KA, Walbot V 1997 Expression and RNA splicing of the maize glutathione S-transferase Bronze2 gene is regulated by cadmium and other stresses. Plant Physiol. 113: 93-102.

Pelletier MK, Murrell JR, Shirley BW 1997 Characterization of flavonol synthase and leucoanthocyanidin dioxygenase genes in Arabidopsis. Further evidence for differential regulation of "early" and "late" genes. Plant Physiol. 113: 1437-1445.

Robbins MP, Bavage AD, Strudwicke C, Morris P 1998 Genetic manipulation of condensed tannins in higher plants. II. Analysis of birdsfoot trefoil plants harboring antisense dihydroflavonol reductase constructs. Plant Physiol. 116: 1134-1144.

Selinger DA, Chandler VL 1999 A mutation in the pale aleurone color1 gene identifies a novel regulator of the maize anthocyanin pathway. Plant Cell 11: 5-14.