Multi-omics analysis reveals new insights into the bicolor flower phenotype of Paeonia suffruticosa ‘Erqiao’

•Multi-omics (transcriptomics, proteomics, and metabolomics) were used to analyses the mechanism of flower color formation in bicolor P. suffruticosa ‘Erqiao’.•The 6 bp insertion mutation of PsF3′H existed only in ‘Erqiao’ and was stably expressed in the pink-white petals, not other P. suffruticosa cultivars or ‘Erqiao’ tissues.•The insertion mutation of PsF3′H gene which only stably expressed in pink-white petals reduced the synthesis of the PsF3′H protein and led to the obstruction of anthocyanin synthesis, which further changed the synthesis direction of the entire metabolic pathway. The bicolor phenotype is conducive to revealing the changes in plant flower color and an important scientific model for studying the regulatory mechanism of flower color. The flowers of Paeonia suffruticosa ‘Erqiao’ have two colors, pink-white and purple-red, and bicolor flowers are rare, possessing unique ornamental value. The distribution of the two colors in a bicolor ‘Erqiao’ flower is similar to a pie chart and can be divided into two distinct parts, with the percentages of the two parts varying. We analyzed the mechanism of flower color formation in ‘Erqiao’ using transcriptomics, proteomics, and metabolomics. The results showed that the differentially abundant metabolites (DAMs), differentially abundant proteins (DAPs), and differentially expressed genes (DEGs) in the two petal types were primarily enriched in phenylpropanoid and flavonoid biosynthesis. Compared with purple-red petals, the PsF3′H protein content in pink-white petals was lower than that in purple-red petals. The content of PsF3′H’s downstream metabolites decreased, while that of some metabolites in the collateral branch of phenylpropanoid biosynthesis increased. The PsF3′H sequence analysis of different P. suffruticosa cultivars and ‘Erqiao’ tissues revealed that the 6 bp insertion mutation of PsF3′H existed only in ‘Erqiao’ and was stably expressed in the pink-white petals. Using virus-induced gene silencing to silence PsF3′H in ‘Erqiao’ petals, the color of the petals faded significantly. Therefore, through multi-omics analysis, we found that the insertion mutation of PsF3′H reduced the synthesis of the PsF3′H protein and led to the obstruction of anthocyanin synthesis; furthermore, other branches and metabolic pathways of the upstream pathway were more active, thus changing the synthesis direction of the entire metabolic pathway. The 6-bp insertion mutation of PsF3′H may be a reason for the rare ap