Amaryllidaceae alkaloids: identification and partial characterization of montanine production in Rhodophiala bifida plant

Andressa Reis,Jaume Bastida,Marina Scopel,Sophie Massot,Pascal Ratet,José A S Zuanazzi,Luciana R Tallini,Kevin Magne

doi:10.1038/s41598-019-44746-7

Abstract

Rhodophiala bifida (R. bifida) is a representative of the Amaryllidaceae plant family and is rich in montanine, an alkaloid with high pharmaceutical potential. Despite the interest in these compounds, many steps of the biosynthetic pathway have not been elucidated. In this study, we identified the alkaloids produced in different organs of R. bifida under different growth conditions, set up the conditions for in vitro R. bifida regeneration and initiated the molecular characterization of two R. bifida genes involved in alkaloids biosynthesis: the Norbelladine 4′-O-Methyltransferase (RbN4OMT) and the Cytochrome P450 (RbCYP96T). We show that montanine is the main alkaloid produced in the different R. bifida organs and developed a direct organogenesis regeneration protocol, using twin-scale explants cultivated on media enriched with naphthalene acetic acid and benzyladenine. Finally, we analyzed the RbN4OMT and RbCYP96T gene expressions in different organs and culture conditions and compared them to alkaloid production. In different organs of R. bifida young, adult and regenerated plants, as well as under various growing conditions, the transcripts accumulation was correlated with the production of alkaloids. This work provides new tools to improve the production of this important pharmaceutical compound and for future biotechnological studies.

Highlights

Rhodophiala bifida (R. bifida) is a representative of the Amaryllidaceae plant family and is rich in montanine, an alkaloid with high pharmaceutical potential
Because of the high biotechnological interest for the montanine produced by R. bifida, we identified the alkaloids produced in this plant, set up in vitro R. bifida regeneration conditions and cloned the RbN4OMT and RbCYP96T genes
Our analysis shows that montanine was the major alkaloid produced in R. bifida and that other molecules were found in low amount

Summary

Introduction

Rhodophiala bifida (R. bifida) is a representative of the Amaryllidaceae plant family and is rich in montanine, an alkaloid with high pharmaceutical potential. Plants belonging to the Amaryllidaceae family produce alkaloids that have been extensively studied because of their pharmaceutical properties. The genus Rhodophiala (Amaryllidaceae) is endemic in South America and contains more than 30 bulbous species showing ornamental potential due to their colorful and attractive flowers These plants usually grow in restricted areas and are geographically isolated[1]. Montanine has acetylcholinesterase inhibition, anti-reumatic, antimicrobial and antiproliferative effects These important pharmaceutical properties justify an increasing of interest towards this class of compounds[2,3,4,5,6,7,8,9,10]. Despite all the pharmaceutical interests of the galanthamine molecule and the search for new drugs derived from the Amaryllidaceae alkaloids (AmAl), the corresponding biosynthetic pathway is not fully characterized yet. Montanine is characterized by a unique 5,11-methanomorphanthridine skeleton[11]

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Conclusion