ANALYSIS OF BHLH AND MYB DOMAIN PROTEINS - SPECIES-SPECIFIC REGULATORY DIFFERENCES ARE CAUSED BY DIVERGENT EVOLUTION OF TARGET ANTHOCYANIN GENES

The regulatory anthocyanin loci, an1, an2, an4 and an11 of Petunia hyb rida, and r and c1 from Zea mays, control transcription of different s ets of target genes. Both an2 and cl encode a MYB-type protein. This s tudy reports the isolation of a P. hybrida gene, jaf13, encoding a bas ic helix-loop-helix protein that, on the basis of sequence homology an d internation structure, represents the P hybrida orthologue of the Z. mays r genes. Ectopic expression of an2 and jaf13 is sufficient for a ctivation of the dihydroflavonol 4-reductase-A (dfrA) promoter and enh anced pigment accumulation in P hybrida. This indicates that an2 and j af13 play a key role in determining the tissue-specific expression pat tern of structural genes. However, because chalcone synthase (chs) and flavanone-3-hydroxylase (f3h) are not activated, the pattern of pigme ntation is not fundamentally altered. Expression of an2 in Z. mays com plements a mutation in pl, a c1 paralogue, indicating that an2 activat es a wider set of target genes in this host. Transient expression assa ys in Z. mays and P. hybrida tissues showed that C1 and R or AN2 and J AF13 can activate the promoter of the c2 gene, encoding Z. mays CHS, b ut not the chsA promoter from P hybrida. These results indicate that r egulatory anthocyanin genes are conserved between species and that div ergent evolution of the target gene promoters is responsible for the s pecies-specific differences in regulatory networks.