PbAGO4‐dependent hypermethylation of the PbNAC71 promoter regulates anthocyanin synthesis in pear

Bud mutations in perennial fruit trees often involve epigenetic variation, but the underlying mechanisms remain unclear. We identified a spontaneous green bud mutant of the red-skinned pear "Aohong No. 1" that fails to accumulate anthocyanins under light. Integrated methylome and transcriptome analyses revealed that in the mutant, hypermethylation of a 148-bp light-responsive enhancer in the PbNAC71 promoter, maintained by PbAGO4, represses PbNAC71 expression. In the wild type, this enhancer remains hypomethylated, whereas in the mutant, it is hypermethylated; this differential methylation is detectable at the young fruit stage and stably maintained at the post-harvest stages examined, representing a pre-set epigenetic state. Silencing of PbNAC71 downregulated PbMYB10 and PbCHI1, and attenuated PbMYB10-mediated activation of PbCHS1 and PbDFR3, thereby suppressing anthocyanin accumulation. Both PbAGO4 knockout in callus and PbAGO4 silencing in fruit peel reduced enhancer methylation, and increased PbNAC71 expression and anthocyanin accumulation. Together, these results reveal a mechanism by which PbAGO4-dependent methylation of a light-responsive enhancer establishes a stable pre-set state that controls how environmental light signals regulate anthocyanin synthesis.