PpMYB and PpbHLH transcription factors activate PpHCT5 to regulate chlorogenic acid biosynthesis in peach fruit

As the abundant phenolic substance in peach fruit, chlorogenic acid (CGA) has various biological activities and is an important target for improving the nutritional and resistance qualities. Nevertheless, the gene functions and regulatory mechanisms of CGA biosynthesis in peach fruit are poorly understood. Using gene expression patterns from different developmental stages and different peach cultivars, one structural gene PpHCT5 was identified as being involved in CGA biosynthesis. An in vitro enzyme activity assay confirmed that the PpHCT5 protein catalyzed the biosynthesis of caffeoyl CoA and quinic acid into CGA. The in vivo functional validation confirmed that PpHCT5 overexpression increased CGA accumulation in transgenic plants, including peach, tomato, and tobacco. Furthermore, upstream transcription factors of PpHCT5 were identified. PpMYB62 and PpbHLH14 activated PpHCT5 expression by directly binding promoter MBS or G-box elements, thereby inducing CGA accumulation in peach fruit. Additionally, PpMYB308 interactions with PpbHLH14 promoted the activating effect of PpbHLH14 on PpHCT5, thereby positively regulating CGA biosynthesis. Thus, a regulatory network of PpMYB and PpbHLH transcription factors to modulate the PpHCT5-mediated CGA biosynthesis pathway was constructed. It will be useful for elucidating the complex mechanisms underlying CGA biosynthesis and provide a theoretical basis for the development of resistance and increased CGA content breeding in peach.