Mitotically heritable epigenetic modifications of CmMYB6 control anthocyanin biosynthesis in chrysanthemum

Flower color, which is determined by various chemical pigments, is a vital trait for ornamental plants, in which anthocyanin is a major component. However, the epigenetic regulation of anthocyanin biosynthesis remains poorly understood. During chrysanthemum cultivation, we found a heterochromatic chrysanthemum accession (YP) whose progeny generated by asexual reproduction contained both yellow-flowered (YP-Y) and pink-flowered (YP-P) plants. In this study, we aimed to elucidate the epigenetic mechanisms of different flower colors in the YP plant progeny. Metabolome and transcriptome analyses revealed that the difference in flower color between YP-Y and YP-P was caused by expression variation of the anthocyanin biosynthesis gene CmMYB6. Bisulfite sequencing revealed that methylation at the CmMYB6 promoter, especially in the CHH context, was higher in YP-Y than YP-P. After demethylation of the CmMYB6 promoter using the dCas9-TET1cd system, the flower color returned from yellow to pink. Furthermore, the methylation status of the CmMYB6 promoter was higher in YP-Y over three consecutive generations, indicating that this methylation status was heritable mitotically. Finally, investigation of other chrysanthemum cultivars showed that the methylation of CmMYB6 decreased gradually with the increase in anthocyanin content. These results lay an epigenetic foundation for the improvement of flower color in horticultural plants.