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Monoterpene indole alkaloids (MIAs) are valuable metabolites produced in numerous medicinal plants from the Apocynaceae family such as Alstonia scholaris , which synthesizes strictamine, a MIA displaying neuropharmacological properties of a potential importance. To get insights into the MIA metabolism in A. scholaris , we studied here both the spatial and transcriptional regulations of MIA genes by performing a robust transcriptomics analysis of the main plant organs, leaf epidermis but also by sequencing RNA from leaves transiently overexpressing the master transcriptional regulator MYC2. These transcriptomic studies notably demonstrated that the first steps of the MIA pathway are successively distributed in the internal phloem associated parenchyma and epidermis, and that MYC2 exerts a remarkable transcriptional effect by modulating the expression of around 1000 genes. By combining these distinct datasets, we initiated the search for MIA-related genes encoding CYP71, based on the similarity of expression compared to already known MIA genes. Transient expression of these candidates in Nicotiana benthamiana leaves and yeast notably led to the identification of a related isoform of rhazimal synthase (RHS) capable of converting the MIA precursor geissoschizine into akuammicine, strictamine and 16- epi -pleiocarpamine. Investigating its catalytic mechanism revealed that strictamine results from rhazimal deformylation and that a similar mechanism may also explain 16- epi -pleiocarpamine synthesis. This prompted us to rename these enzymes geissoschizine cyclase due to their capacity of cyclizing geissoschizine into three different MIA scaffolds and to form both C-C and C-N bonds. This identification thus illustrates the potential of integrating spatial and transcriptional regulation analysis for MIA gene identification. • A. scholaris and C. roseus may share similar organization in the route to MIA. • Combining epidermis and MYC2 transcriptomes identifies MIA candidate genes. • CYP71 geissoschizine cyclase catalyzes the formation of C-C and C-N bonds. • Geissoschizine cyclase produces akuammilan, strychnos and mavacuran alkaloids. • Strictamine is synthesized through geissoschizine cyclase activity.
Méteignier et al. (Thu,) studied this question.