Metabolite accumulation patterns in Zanthoxylum bungeanum exocarp during fruit maturation and identification of an...

This study investigates the metabolic changes in the exocarp of Zanthoxylum bungeanum during fruit maturation. HPLC-QTOF-MS/MS analysis identified 319 compounds, including 62 alkaloids, 33 amino acids and derives, 102 flavonoids, 61 benzene and phenolic compounds, 28 terpenoids and steroids, 27 fatty acids and derivatives, and 6 organic acid and saccharides at three stages (green, semired, and red). PCA and heatmap revealed distinct metabolic profiles, with significant differences observed between red exocarp and the earlier stages (green and semired). The total flavonoids in red exocarp is twice that in green and semired exocarp, driven by the enrichment of flavonoid biosynthesis. Gene expression showed the upregulation of ZbPAL2, Zb4CL2/3/4, and ZbHCT2/9 in red exocarp, which enhanced the metabolic flux from phenylalanine/tyrosine to downstream phenolic and flavonoid compounds. Notably, anthocyanins accumulation was strongly associated with the induction of ZbDFR, ZbGT1, and ZbGST4/5. Functional characterization of the anthocyanidin 3-O-glucosyltransferase ZbGT1 confirmed its catalytic activity in synthesizing cyanidin 3-O-glucoside. Structural modeling and mutagenesis revealed that key residues (His84, His151, Ser20/Ser21, Thr293, His363, and Asp387) in ZbGT1 are critical for substrate binding and enzymatic activity. Mutation of His84, His151, and Ser21 to Ala reduced enzyme activity by ∼75 %. Mutations of Thr293, His363, and Asp387 to Ala decreased ZbGT1 activity by ∼90 %, making it nearly undetectable. Mutating Gln348, Glu371, and Trp345 to Ala caused only a slight 10 % decrease in activity. These findings elucidate the metabolic and molecular basis of exocarp color transition in Z. bungeanum, providing insights for improving fruit quality and stress adaptation through biotechnology.