Identifying Genes Involved in alkaloid Biosynthesis in Through Transcriptomics and Gene Co-Expression Analysis.

The lesser periwinkle accumulates numerous monoterpene indole alkaloids (MIAs) including the vasodilator vincamine. While the biosynthetic pathway of MIAs has been largely elucidated in other Apocynaceae such as , the counterpart in remains mostly unknown, especially for reactions leading to MIAs specific to this plant. As a consequence, we generated a comprehensive transcriptome elaborated from eight distinct samples including roots, old and young leaves exposed to low or high light exposure conditions. This optimized resource exhibits an improved completeness compared to already published ones. Through homology-based searches using genes as bait, we predicted candidate genes for all common steps of the MIA pathway as illustrated by the cloning of a tabersonine/vincadifformine 16--methyltransferase (Vm16OMT) isoform. The functional validation of this enzyme revealed its capacity of methylating 16-hydroxylated derivatives of tabersonine, vincadifformine and lochnericine with a Km 0.94 ± 0.06 µM for 16-hydroxytabersonine. Furthermore, by combining expression of fusions with yellow fluorescent proteins and interaction assays, we established that Vm16OMT is located in the cytosol and forms homodimers. Finally, a gene co-expression network was performed to identify candidate genes of the missing biosynthetic steps to guide MIA pathway elucidation.