Carotenoids, as natural tetraterpenes, play a pivotal role in the yellow coloration of peaches and contribute to human dietary health. Despite a relatively clear understanding of the carotenoid biosynthesis pathway, the regulatory mechanism of miRNAs involved in carotenoid synthesis in yellow peaches remain poorly elucidated. This study investigated a total of 14 carotenoids and 40 xanthophyll lipids, including six differentially accumulated carotenoids: violaxanthin, neoxanthin, lutein, zeaxanthin, cryptoxanthin, and (E/Z)-phytoene. An integrated analysis of RNA-seq, miRNA-seq and degradome sequencing revealed that miRNAs could modulate structural genes such as PSY2, CRTISO, ZDS1, CHYB, VDE, ZEP, NCED1, NCED3 and the transcription factors NAC, ARF, WRKY, MYB, and bZIP, thereby participating in carotenoid biosynthesis and metabolism. The authenticity of miRNAs and target gene was corroborated through quantitative real-time PCR. Moreover, through weighted gene coexpression network analysis and a phylogenetic evolutionary study, coexpressed genes and MYB transcription factors potentially implicated in carotenoid synthesis were identified. The results of transient expression experiments indicated that mdm-miR858 inhibited the expression of PpMYB9 through targeted cleavage. Building upon these findings, a regulatory network governing miRNA-mediated carotenoid synthesis was proposed. In summary, this study comprehensively identified miRNAs engaged in carotenoid biosynthesis and their putative target genes, thus enhancing the understanding of carotenoid accumulation and regulatory mechanism in yellow peach peel and expanding the gene regulatory network of carotenoid synthesis.

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