WGT-aware analysis reveals increased complexity in the yohimbane biosynthesis pathway of Rauvolfia tetraphylla

Arising from Stander et al. Communications Biology https://doi.org/10.1038/s42003-023-05574-8 (2023) Rauvolfia tetraphylla (Apocynaceae), distributed across Mexico, Tropical America, and Southeast Asia, is known to produce pharmacologically important monoterpene indole alkaloids such as ajmaline, reserpiline, yohimbine, and heteroyohimbanes1. Elucidating the enzymatic pathways2 underlying their biosynthesis is therefore essential for enhancing our understanding of MIA metabolism and enabling their industrial production. In a recent edition of Communication Biology, Stander et al. [STAN23]3 presented a high-quality assembly for R. tetraphylla and uncovered the yohimbane biosynthesis pathway. Based on their proteomics and transcriptomic analysis, two major findings emerged: (1) a medium chain dehydrogenase/reductase (MDR), yohimbane synthase (YOS), produces four yohimbane diastereomers, and (2) three MDR transcripts (MDRT), MSTRG.5530, MSTRG.5531, and MSTRG.5534, along with geissoschizine synthase (GS, MSTRG.5528), can generate a mixture of yohimbane isomers. However, our reanalysis of the raw sequencing reads incorporating haplotype phasing and expression profiling revealed that most candidate genes exist as three homeologous copies with distinct tissue-specific expression. Furthermore, we demonstrate preferential interactions among specific MDRT and GS homeologs, indicating complex cross-subchromosomal enzymatic organisation overlooked in STAN23. Furthermore, several reported candidate genes are chimeric, indicating that incorrect estimation of whole-genome triplication and lack of haplotype phasing can severely limit robust identification and functional characterisation of the yohimbane biosynthetic pathway.