Cyanidin-3-O-glucoside Contributes to Leaf Color Change by Regulating Two bHLH Transcription Factors in Phoebe bournei

Anthocyanins produce different-colored pigments in plant organs, which provide ornamental value. Thus, this study was conducted to understand the mechanism of anthocyanin synthesis in ornamental plants. Phoebe bournei, a Chinese specialty tree, has high ornamental and economic value due to its rich leaf color and diverse metabolic products. Here, the metabolic data and gene expression of red P. bournei leaves at the three developmental stages were evaluated to elucidate the color-production mechanism in the red-leaved P. bournei species. First, metabolomic analysis identified 34 anthocyanin metabolites showing high levels of cyanidin-3-O-glucoside (cya-3-O-glu) in the S1 stage, which may suggest that it is a characteristic metabolite associated with the red coloration of the leaves. Second, transcriptome analysis showed that 94 structural genes were involved in anthocyanin biosynthesis, especially flavanone 3′-hydroxy-lase (PbF3′H), and were significantly correlated with the cya-3-O-glu level. Third, K-means clustering analysis and phylogenetic analyses identified PbbHLH1 and PbbHLH2, which shared the same expression pattern as most structural genes, indicating that these two PbbHLH genes may be regulators of anthocyanin biosynthesis in P. bournei. Finally, overexpression of PbbHLH1 and PbbHLH2 in Nicotiana tabacum leaves triggered anthocyanin accumulation. These findings provide a basis for cultivating P. bournei varieties that have high ornamental value.