Analysis of bHLH and MYB domain proteins: species‐specific regulatory differences are caused by divergent evolution of target anthocyanin genes

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Abstract
The regulatory anthocyanin loci, an1, an2, an4 and an11 of Petunia hybrida, and r and c1 from Zea mays, control transcription of different sets of target genes. Both an2 and c1 encode a MYB-type protein. This study reports the isolation of a P. hybrida gene, jaf13, encoding a basic helix-loop-helix protein that, on the basis of sequence homology and intron/exon structure, represents the P. hybrida orthologue of the Z. mays r genes. Ectopic expression of an2 and jaf13 is sufficient for activation of the dihydroflavonol 4-reductase-A (dfrA) promoter and enhanced pigment accumulation in P. hybrida. This indicates that an2 and jaf13 play a key role in determining the tissue-specific expression pattern of structural genes. However, because chalcone synthase (chs) and flavanone-3-hydroxylase (f3h) are not activated, the pattern of pigmentation is not fundamentally altered. Expression of an2 in Z. mays complements a mutation in pl, a c1 paralogue, indicating that an2 activates a wider set of target genes in this host. Transient expression assays in Z. mays and P. hybrida tissues showed that C1 and R or AN2 and JAF13 can activate the promoter of the c2 gene, encoding Z. mays CHS, but not the chsA promoter from P. hybrida. These results indicate that regulatory anthocyanin genes are conserved between species and that divergent evolution of the target gene promoters is responsible for the species-specific differences in regulatory networks.