Functional characterization of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) involved in the biosynthesis of triterpenoid saponins in Platycodon grandiflorum

Terpenoids represent an extensive class of natural compounds with significant biological activities. Consequently, identifying the genes implicated in their synthesis is crucial for advancements in agricultural and medicinal applications. To elucidate the regulatory role of the DXR gene in terpenoid biosynthesis, three genes encoding PgDXR ( PgDXR1–PgDXR3 ) were identified from Platycodon grandiflorus. Quantitative real-time PCR analysis revealed that PgDXR genes were widely expressed across various tissues, and PgDXR genes exhibit significant transcriptional responses to drought (PEG6000), abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA) treatments. Subcellular localization studies demonstrated that PgDXR proteins are localized to distinct subcellular compartments (PgDXR1 in chloroplasts; PgDXR2/3 in nucleus and cytoplasm). Phylogenetic analysis indicated that PgDXR1 may play a crucial role in triterpenoid saponin metabolism. Furthermore, both in situ hybridization and Western blot analysis indicated that PgDXR1 (mRNA and protein) expression is highest in the roots of two-year-old P. grandiflorum plants. We further analyzed the biological functions of PgDXR1 in triterpenoid saponin synthesis both in vitro and in vivo. Yeast one-hybrid (Y1H) assays demonstrated that MYB39 binds to the PgDXR1 promoter, while dual-luciferase (LUC) assays confirmed that MYB39 transcriptionally activates PgDXR1 . This study lays a foundation for further elucidating the molecular regulatory mechanism of terpenoid synthesis in P. grandiflorum and provides a reference for understanding the biological functions of the DXR . • PgDXR1 is involved in triterpenoid saponin metabolism in vivo and exhibits enzyme catalysis in vitro. • MYB39 regulate the biosynthesis of triterpenoid saponins by modulating the transcription of the target gene PgDXR1. • This study not only revealed the potential role of PgDXR in triterpenoid saponins, but also provided the basics for bioengineering triterpenoid in plants.