Identification and transcription analysis of the phenolic acid biosynthesis pathway in Pseudotaxus chienii

Abstract Pseudotaxus chienii, an endangered relic conifer endemic to China, possesses significant medicinal potential, yet its phenolic acid biosynthesis and glycosylation mechanisms remain unelucidated. This study integrated metabolomics, mass spectrometry imaging (MSI), transcriptomics, and molecular biology to systematically characterize the phenolic acid pathway in P. chienii. Untargeted metabolomics identified distinct phenolic acid accumulation patterns between P. chienii and Taxus mairei. Matrix-Assisted Laser Desorption/Ionization with Laser Post-Ionization Mass Spectrometry Imaging‌（MALDI-2 MSI）visualized ten phenolic acids, confirming leaves as the primary accumulation site. Genome-wide analysis revealed a complete phenolic acid biosynthesis pathway with key enzyme-encoding genes (4 phenylalanine ammonia-lyase, 5 cinnamoyl-CoA reductase, 2 ferulate 5-hydroxylase, etc.), while hydroxycinnamoyl‑CoA:quinate hydroxycinnamoyl transferase (HQT) was absent, indicating loss of chlorogenic acid synthesis. We identified 504 glycosyltransferase (GT) genes, with 48 leaf-specific ones (predominantly GT1 subfamily). Promoter and correlation analyses highlighted WRKY‑domain‑containing transcription factor (PichiChr12G332970.1) and Ethylene‑Responsive Factor (ERF) (PichiChr3G086100.1) as core transcription factors. Electrophoretic mobility shift assay and dual-luciferase assays validated WRKY (PichiChr12G332970.1) directly activating three GT genes. This study clarifies the molecular basis and transcriptional regulation of phenolic acid glycosylation in P. chienii, providing a framework for exploiting its medicinal resources and guiding conservation-oriented breeding.