Full-length transcriptome and light-regulated analyses identify candidate genes involved in rhynchophylline and isorhynchophylline biosynthesis in Uncaria rhynchophylla

Abstract Rhynchophylline (RIN) and isorhynchophylline (IRN) are the two most important monoterpene indole alkaloids (MIAs) in Uncaria rhynchophylla , and serve as the material basis for its diverse pharmacological activities. However, the molecular mechanisms underlying their biosynthesis remain poorly understood. To elucidate these mechanisms, high-performance liquid chromatography (HPLC) was employed to analyze the accumulation patterns of RIN and IRN in different organs. A combination of Illumina RNA-Seq and single-molecule real-time (SMRT) sequencing was applied to U. rhynchophylla , generating a total of 93,338 transcripts, from which 4,013 long non-coding RNAs (lncRNAs) and 3,762 transcription factors (TFs) were predicted. A total of 129 genes encoding enzymes potentially involved in the upstream biosynthesis of RIN and IRN were identified. RNA-Seq data revealed that most of these genes exhibited higher expression levels in roots. Furthermore, two ABC transporters and seven MATE transporters were identified, which showed close evolutionary relationships with known alkaloid transporters and were considered as candidate transporters for RIN and IRN. Through comparative transcriptomic analysis, full-length transcripts of 11 light-responsive candidate genes involved in the RIN and IRN biosynthetic pathway were identified. Shading and re-illumination experiments further demonstrated that the expression patterns of Ur8HGO , UrSTR1 , and UrSGD1 were consistent with the accumulation trends of RIN and IRN. Functional characterization of UrSTR1 and UrSGD1 was performed using virus-induced gene silencing (VIGS), and the results showed that silencing these two genes significantly reduced RIN and IRN contents, indicating their involvement in RIN and IRN biosynthesis. These findings, based on a hybrid sequencing platform, provide valuable genetic information for U. rhynchophylla . The combined transcriptomic and metabolomic analyses contribute to a better understanding of the biosynthesis of RIN and IRN.