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