Transcriptome and Metabolome Profiling Provide Insights into Flavonoid Biosynthesis and the Mechanism of Color Formation in Zanthoxylum bungeanum Maxim.

The color of <i>Zanthoxylum bungeanum</i> Maxim. (<i>Z. bungeanum</i>) is a key quality indicator and a factor limiting the development of its industry. However, the underlying mechanisms governing color formation remain largely unexplored. In this study, an integrative analysis of transcriptome and metabolome profiles was conducted across four developmental stages to elucidate the color formation mechanism in <i>Z. bungeanum</i>. A total of 137 flavonoids were identified as the fruits ripened, with high levels of differentially accumulated metabolites (DAMs), including tricetin and (-)-epigallocatechin, which were strongly associated with color formation. This suggests their significant contribution to the pigmentation process. Nine differentially expressed genes (DEGs) were identified as candidate genes involved in color development. Additionally, 15 transcription factors (TFs) (12 MYB and 3 bHLH) exhibited expression patterns similar to those of structural genes in the flavonoid biosynthetic pathway, indicating their role in regulating flavonoid synthesis. The bioinformatics analysis of three key flavonoid synthesis genes-<i>ZbCHI</i>, <i>ZbFLS</i>, and <i>ZbANR</i>-revealed that all three proteins exhibit hydrophobic structures without transmembrane domains. Among them, <i>ZbANR</i> possesses signal peptide regions, whereas <i>ZbCHI</i> and <i>ZbFLS</i> do not. Subcellular localization predictions suggest that <i>ZbCHI</i> is most likely localized in the chloroplast, <i>ZbFLS</i> in the cytoplasm, and <i>ZbANR</i> in the membrane. Functional analyses revealed that their transient expression in <i>Nicotiana benthamiana</i> (<i>N. benthamiana</i>) increased the flavonoid content, with <i>ZbANR</i> overexpression producing a distinct white phenotype in the plants. This study enriches transcriptomic data and provides a comprehensive understanding of flavonoid metabolism and the molecular basis of color formation in <i>Z. bungeanum</i>, offering a valuable theoretical foundation for future breeding programs.