Abstract
The lesser periwinkle Vinca minor 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 Catharanthus roseus, the counterpart in V. minor remains mostly unknown, especially for reactions leading to MIAs specific to this plant. As a consequence, we generated a comprehensive V. minor 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 C. roseus 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-O-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 V. minor biosynthetic steps to guide MIA pathway elucidation.
Highlights
Monoterpene indole alkaloids (MIAs) are a class of specialized plant metabolites found in Gentianale order families; Apocynaceae, Rubiaceae and Loganiaceae
A total of eight libraries (Figure 2) were prepared from RNA extracted from four different tissue types including, mature leaves exposed to light (OL), young leaves exposed to light (YL), young leaves grown under low illumination conditions (YS) and adventitious roots (AR) developing from cut-stems immersed in water
Since the accumulation of many MIAs is stimulated by light, including vindoline in C. roseus [54,58], N,β-d-glucopyranosyl vincosamide in Psychotria leiocarpa [90] and camptothecin in Camptotheca acuminata [91], we exposed Vinca minor plants to low and high light exposure conditions, in order to explore whether a similar transcriptional regulation could be detected in V. minor
Summary
Monoterpene indole alkaloids (MIAs) are a class of specialized plant metabolites found in Gentianale order families; Apocynaceae, Rubiaceae and Loganiaceae. MIAs have important roles in plant defense [1,2,3] and constitute a large chemical diversity of pharmacophores with a variety of potent biological activities These activities make MIAs attractive pharmaceutical drugs with high commercial value, such as the anticancer drugs vinblastine and vincristine from Catharanthus roseus, the antiarrhythmic agent ajmaline from Rauvolfia serpentina and the vasodilator vincamine, from Vinca minor, used to treat cerebrovascular disorders. MIA producing plants vary a lot in biomass production and alkaloid content, prompting research efforts towards biosynthetic pathway elucidation This knowledge has the potential to stimulate alternative engineering approaches, including synthetic biology and metabolic engineering, for the upscaled commercial production of these valuable natural products [4,5,6]. Some alkaloids are specific to V. minor leaves, including vincarubine, picrinine, eburnamine, eburnamonine, minovincinine, minovine, vincadifformine and strictamine [7,8,12,16]
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