Overexpression of ORCA3 and G10H in Catharanthus roseus plants regulated alkaloid biosynthesis and metabolism revealed by NMR-metabolomics.

Qifang Pan,Kexuan Tang,Quan Wang,Yuesheng Tian,Guofeng Wang,Fang Yuan,Robert Verpoorte,Young Hae Choi,Jingya Zhao,Shihai Xing,Leandro Peña

doi:10.1371/journal.pone.0043038

Abstract

In order to improve the production of the anticancer dimeric indole alkaloids in Catharanthuse roseus, much research has been dedicated to culturing cell lines, hairy roots, and efforts to elucidate the regulation of the monoterpenoid indole alkaloid (MIA) biosynthesis. In this study, the ORCA3 (Octadecanoid-derivative Responsive Catharanthus AP2-domain) gene alone or integrated with the G10H (geraniol 10-hydroxylase) gene were first introduced into C. roseus plants. Transgenic C. roseus plants overexpressing ORCA3 alone (OR lines), or co-overexpressing G10H and ORCA3 (GO lines) were obtained by genetic modification. ORCA3 overexpression induced an increase of AS, TDC, STR and D4H transcripts but did not affect CRMYC2 and G10H transcription. G10H transcripts showed a significant increase under G10H and ORCA3 co-overexpression. ORCA3 and G10H overexpression significantly increased the accumulation of strictosidine, vindoline, catharanthine and ajmalicine but had limited effects on anhydrovinblastine and vinblastine levels. NMR-based metabolomics confirmed the higher accumulation of monomeric indole alkaloids in OR and GO lines. Multivariate data analysis of 1H NMR spectra showed change of amino acid, organic acid, sugar and phenylpropanoid levels in both OR and GO lines compared to the controls. The result indicated that enhancement of MIA biosynthesis by ORCA3 and G10H overexpression might affect other metabolic pathways in the plant metabolism of C. roseus.

Highlights

In the 1950s, the Canadian scientists Robert Noble and Charles Beer first isolated and characterized the alkaloid vinblastine from Catharanthus roseus leaves [1]
After three rounds of kanamycin (90 mg L21) selection, a total of 16 independent plantlets transformed with the construct of ORCA3 (OR) and 39 independent plantlets transformed with the construct of G10H and ORCA3 (GO) survived
As a key transcription factor in the JA-induced monoterpenoid indole alkaloids (MIA) pathway, ORCA3 overexpression resulted in the up-regulation of several alkaloid-related biosynthetic genes in C. roseus cell cultures [28]

Summary

Introduction

In the 1950s, the Canadian scientists Robert Noble and Charles Beer first isolated and characterized the alkaloid vinblastine from Catharanthus roseus leaves [1]. More than 130 monoterpenoid indole alkaloids (MIA) have been found in different parts of C. roseus [2]. Of the MIAs, dimeric indole (bisindole) alkaloids vincristine and vinblastine are being used clinically as anticancer agents. Despite their importance, sources of the compounds are still limited. C. roseus is the main source of MIAs but the low yields have been an obstacle to production of the compounds. The high medical value and extremely low yields from C. roseus [3] of vinblastine and vincristine have motivated extensive studies to elucidate MIA biosynthesis and regulation

Methods

Results

Conclusion