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The duplicated R and Sn genes regulate the maize anthocyanin biosynthetic pathway and encode tissue‐specific products that are homologous to helix‐loop‐helix transcriptional activators. As a consequence of their coupling in the genome, Sn is partially silenced. Genomic restriction analysis failed to reveal gross structural DNA alterations between the strong original phenotype and the weak derivatives. However, the differences in pigmentation were inversely correlated with differences in the methylation of the Sn promoter. Accordingly, treatment with 5‐azacytidine (AZA), a demethylating agent, restored a strong pigmentation pattern that was transmitted to the progeny and that was correlated with differential expression of the Sn transcript. Genomic sequencing confirmed that methylation of the Sn promoter was more apparent in the less pigmented seedlings and was greatly reduced in the AZA revertants. In addition, some methylcytosines were located in non‐symmetrical C sequences. These findings provide an insight into Sn and R interaction, a process that we have termed Reduced Expression of Endogenous Duplications (REED). We propose that increasing the copy number of regulatory genes by endogenous duplication leads to such epigenetic mechanisms of silencing. Further understanding of the REED process may have broader implications for gene regulation and may identify new levels of regulation within eukaryotic genomes.

SummaryThe pl1 gene encodes a MYB-related transcriptional activator committed to the regulation of anthocyanin biosynthesis in maize. Here, we report the genetic and molecular characterisation of pl-bol3, an Andean allele displaying features that make it different from all the known pl1 alleles. pl-bol3 has partial, lightindependent expression, and it is active mainly in the juvenile phase of growth. It has a complex molecular structure, containing multiple pl1 gene copies, thus being the ®rst complex locus discovered in the c1/pl1 family. Although the composite genes of the complex locus encode proteins identical to other functional PL1 proteins, the putative promoters of the pl-bol3 gene are different from the promoters of Pl-Rhoades (Pl-Rh) and pl1 sun-red alleles. The intensity and the tissue speci®city of anthocyanin production directed by pl-bol3 differ signi®cantly from that of Pl-Rh and the original pl-W22, and are speci®ed by the interaction of pl-bol3 with the different r1/b1 gene family members and the competence of pl-bol3 to different pigment tissues. This allele represents a natural example of gene duplication and diversi®cation of expression, giving rise to a signi®cant change in phenotype and, in this way, is analogous to the complex r1 locus in maize. Analysis of the pl-bol3 allele contributes to understanding the generation of diversity associated with multiple-copy genes and the molecular basis of allele-speci®c gene expression.

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