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.

Shikimic acid/quinic acid hydroxy cinnamyl transferase (HCT) is one of the key enzymes in the phenylpropanoid pathway. However, the role of the HCT gene in chlorogenic acid (CGA) biosynthesis in peach fruit remains unclear. For this, we identified the accumulation pattern of CGA in four peach cultivars, cloned and characterized 11 PpHCT gene members, and further analyzed the expression patterns of these PpHCT genes during fruit development. The contents of CGAs in the four peach cultivars all exhibited a trend of increasing and then decreasing during the fruit growth and development. Moreover, the contents of CGAs in the peel and flesh were tissue-specific. Gene structure analysis indicated that the PpHCT genes were highly conserved, containing two exons and one intron. The protein structure analysis demonstrated that the PpHCT proteins contained two conserved motifs (HXXXD, DFGWG) and a transferase domain (PF02458), which belonged to the BAHD acyltransferase family. The cis-acting element analysis suggested that the promoters of PpHCT genes contained many light-related, hormone-related, stress-related, tissue-specific, and circadian-related elements, and they could participate in a variety of biological processes. Phylogenetic analysis showed that the HCT proteins of peach were closely related to the HCT proteins of plum and had a close evolutionary relationship. The qRT-PCR analysis indicated that the expression levels of PpHCT1 and PpHCT2 showed an opposite trend to the accumulation of CGA, whereas the expression levels of PpHCT4, PpHCT5, PpHCT7, PpHCT8, and PpHCT11 demonstrated the same trend as CGA accumulation. It was worth noting that only PpHCT4 and PpHCT5 were highly expressed in the two high-CGA cultivars but showed low levels of expression in the two low-CGA cultivars. Therefore, it was hypothesized that these two genes might be key genes to the synthesis of CGA in peach fruit. Those findings provide a theoretical basis for further study on the biological functions of the HCT gene and help to reveal the molecular mechanism of CGA.