Red fruit peel is an attractive target for pineapple breeding. Various pineapple accessions with distinct red coloration patterns exist; however, the precise molecular mechanism accounting for these differences remains unknown, which hinders the pineapple breeding process from combining high fruit quality with red peel. In this study, we characterized a transcription factor, AcMYB266, which is preferentially expressed in pineapple peel and positively regulates anthocyanin accumulation. Transgenic pineapple, Arabidopsis, and tobacco plants overexpressing AcMYB266 exhibited significant anthocyanin accumulation. Conversely, transient silencing of this gene led to decreased anthocyanin accumulation in pineapple red bracts. In-depth analysis indicated that variations of AcMYB266 sequences in the promoter instead of the protein-coding region seem to contribute to different red coloration patterns in peels of three representative pineapple varieties. In addition, we found that AcMYB266 was located in a cluster of four MYB genes exclusive to and conserved in Ananas species. Of this cluster, each was proved to regulate anthocyanin synthesis in different pineapple tissues, illustrating an interesting case of gene subfunctionalization after tandem duplication. In summary, we have characterized AcMYB266 as a key regulator of pineapple red fruit peel and identified an MYB cluster whose members were subfunctionalized to specifically regulate the red coloration of different pineapple tissues. The present study will assist in establishing a theoretical mechanism for pineapple breeding for red fruit peel and provide an interesting case for the investigation of gene subfunctionalization in plants.

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