A Repression‐Activation System Involving <scp>miR164a</scp> ‐ <scp> <i>NACs</i> </scp> Regulates Tissue‐Specific Anthocyanin Accumulation in Peach Fruit

Anthocyanins are often responsible for fruit colour, determining their commercial value and providing nutritional benefits. In this study, differential anthocyanin accumulation in peel, outer flesh near the peel (OF) and inner flesh around the stone (IF) was observed in two peach fruit cultivars. This was driven by the expression of the R2R3 MYB transcription factor (TF) gene PpMYB10.1. Transcriptome and degradome sequencing were used to identify the regulatory network upstream of PpMYB10.1. We identified and functionally verified that NAM/ATAF/CUC (NAC) TFs PpNAC22 and PpNAC100 are activators of anthocyanin accumulation. Protein-protein/DNA interaction assays were conducted, and it was observed that both PpNAC22 and PpNAC100 could form homodimers to directly activate the PpMYB10.1 promoter. Degradome analysis showed cleavage of PpNAC22 and PpNAC100 transcripts occurred via miR164a. Compared with peel and OF, a lower expression of miR164a in IF resulted in higher expression of PpNAC22 and PpNAC100. Functional characterisation showed that overexpression of miR164a significantly reduced anthocyanin accumulation driven by PpNAC22 and PpNAC100. Furthermore, another NAC TF, PpNAC29, was identified to be indirectly involved in the regulation of anthocyanin accumulation. PpNAC29 formed a heterodimer with PpNAC22, although it could not bind to the PpMYB10.1 promoter. PpNAC29 transcription could be activated by PpNAC22 and PpNAC100 and the transcript was not targeted by miR164a, revealing a miR164a-PpNAC22/100-PpNAC29 cascade. This miR164a-PpNAC22/100-PpMYB10.1 module, with the assistance of PpNAC29, explains contrasting anthocyanin accumulation in different fruit tissues of peach.