Sucrose synthase (SUS) is a pivotal enzyme bridging primary carbon metabolism and secondary biosynthesis in plants. In Panax notoginseng, we demonstrate that sucrose synthase 1 (PnSUS1) serves as a metabolic bottleneck for saponin glycosylation by supplying UDP-glucose. PnWRKY38 was identified as a WRKY transcription factor whose expression correlated with both PnSUS1 and saponin accumulation. Overexpression of PnWRKY38 could up-regulate PnSUS1 expression by 3.5-fold, increase SUS enzyme activity by 2.8-fold, and elevate UDP-glucose pools by 68%. Consequently, the total content of ginsenosides Rg1, Rb1, and Rd rose by 2.1–2.4-fold. Conversely, PnSUS1 or PnWRKY38 suppression reduced UDP-glucose available and saponin biosynthesis by >50%. Y1H and luciferase assays indicated that PnWRKY38 directly activated PnSUS1 expression by binding to W-box motifs in its promoter. These results not only illustrate the specific function that PnSUS1 executes in UDPG biosynthesis but also reveal a new WRKY transcriptional regulatory module regulating notoginsenosides production.

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