The maize Bronze2 (Bz2) gene catalyzes the last defined step in anthocyanin synthesis, acting after Bz1, a glucosyl transferase that catalyzes formation of cyanidin 3-glucoside (C3G). C3G is the most abundant anthocyanin "parent" molecule in maize. In bz2 mutants, C3G accumulates in the cytoplasm, suggesting that BZ2 has a role in vacuolar sequestration. Based on similarity to a few proven plant Glutathione S-transferase (GST) genes, we predicted that BZ2 catalyzed glutathione (GSH) conjugation with anthocyanin as a pre-requisite for vacuolar compartmentation. Thus pigment synthesis would parallel steps in xenobiotic detoxification: hydroxylation by cytochrome P450 mono-oxygenases, glycosylation, glutathionation, and vacuolar sequestration. Biochemical evidence indicates that BZ2 is a carrier protein, not an enzyme. In Petunia hybrida, a Phi-type GST that shares only 11% amino acid identity with BZ2 performs the same role; maize GST3, a stress-induced Phi-type GST, can weakly complement bz2 mutants. We have constructed bz2 gst3 double mutants, and also bz2 gst1 gst3 triple mutants; the mutants are viable, pointing out the redundancy of function within this large gene family. We propose that BZ2 has been selected for flavonoid binding to ferry cargo from synthesis sites in or associated with endomembranes to an ATP Binding Cassette transporter (ABC pump) located in the tonoplast. Anthocyanin sequestration is vanadate-sensitive, a hallmark of directly energenized ABC transporters. Based on a genetic analysis of anthocyanin diffusion in tissues, we concluded that the relevant ABC transporter should be controlled by the same transcription factors that regulate the structural genes of the biosynthetic pathway. MRP29 is a maize ABC protein that is regulated by the R transcription factor, and it is highly expressed in purple tissues. Plants expressing antisense-MRP29 and Mu-induced mutants are viable, and they are being used to analyze function in the anthocyanin and general flavonoid metabolism.

Key words: Bronze2, glutathione S-transferase, multidrug transporter, Petunia hybrida, Zea mays