Allele-specific methylation, and InDels of <i>PmMYB10.5b</i> induced by alternative splicing, participate in regulating the leaf color change in <i>Prunus mume</i> ‘Meiren’

Abstract Prunus mume ‘Meiren,’ a member of the Meiren cultivar group, is a valuable ornamental woody plant prized for its purple flowers and leaves. However, its leaf color exhibits instability during the growth and development, and the underlying mechanisms remain unclear. In our study, we conducted genome-wide methylation analysis on leaves at different developmental stages to investigate the role of methylation patterns and allele-specific methylation (ASM) in leaf color change. Results revealed a significant increase in CHH methylation during leaf development, suggesting its responsiveness to environmental factors and dynamic association with color changes. Notably, CG methylation was imbalanced between the ‘Meiren’ haplotype M (HM) and haplotype C (HC), with the HM subgenome showing higher methylation levels, particularly in promoter regions of key anthocyanin-related genes like PmMYB10.5, where ASM negatively correlated with allele-specific expression (ASE). Additionally, we identified two alternative splicing variants of PmMYB10.5b, named PmMYB10.5b1 (PmMYB10.5b△I24), and PmMYB10.5bP (PmMYB10.5b△D10), respectively. Both the InDel mutations altered the R2 domain structure of the MYB protein. Functional assays demonstrated that these variants lost transcriptional activation ability and failed to promote anthocyanin biosynthesis. Instead, they may compete with the PmMYB10.5b for binding to the PmbHLH3, disrupting regulatory complexes in the anthocyanin pathway and exerting inhibitory effects. These results augment our understanding of the epigenetic and genetic factors influencing leaf color change in ‘Meiren’ and provide novel insights into its regulatory mechanisms.