Expression analysis of Cell wall invertase under abiotic stress conditions influencing specialized metabolism in Catharanthus roseus

M J Nishanth,Sudarshan Singh Rathore,S Srinidhi,Bindu Simon,S A Sheshadri

doi:10.1038/s41598-018-33415-w

M J Nishanth, Sudarshan Singh Rathore + Show 3 more

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https://doi.org/10.1038/s41598-018-33415-w

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Journal: Scientific Reports	Publication Date: Oct 10, 2018
Citations: 20	License type: open-access

Affiliation: SASTRA University

Abstract

Catharanthus roseus is a commercial source for anti-cancer terpenoid indole alkaloids (TIAs: vincristine and vinblastine). Inherent levels of these TIAs are very low, hence research studies need to focus on enhancing their levels in planta. Since primary metabolism provides precursors for specialized-metabolism, elevating the former can achieve higher amounts of the latter. Cell Wall Invertase (CWIN), a key enzyme in sucrose-metabolism catalyses the breakdown of sucrose into glucose and fructose, which serve as carbon-skeleton for specialized-metabolites. Understanding CWIN regulation could unravel metabolic-engineering approaches towards enhancing the levels of TIAs in planta. Our study is the first to characterize CWIN at gene-expression level in the medicinal plant, C. roseus. The CWINs and their inter-relationship with sucrose and TIA metabolism was studied at gene and metabolite levels. It was found that sucrose-supplementation to C. roseus leaves significantly elevated the monomeric TIAs (vindoline, catharanthine) and their corresponding genes. This was further confirmed in cross-species, wherein Nicotiana benthamiana leaves transiently-overexpressing CrCWIN2 showed significant upregulation of specialized-metabolism genes: NbPAL2, Nb4CL, NbCHS, NbF3H, NbANS, NbHCT and NbG10H. The specialized metabolites- cinnamic acid, coumarin, and fisetin were significantly upregulated. Thus, the present study provides a valuable insight into metabolic-engineering approaches towards augmenting the levels of therapeutic TIAs.

Highlights

Cell Wall Invertase (CWIN, EC: 3.2.1.26), a key enzyme in sucrose-metabolism catalyses the irreversible breakdown of sucrose into glucose and fructose
Homology based analysis of the C. roseus coding sequences revealed the presence of three CWIN isoforms; CRO_T000083 (CrCWIN1), CRO_T031716 (CrCWIN2), and CRO_T020329 (CrCWIN3)
It is known that CWIN from Sugarcane, SoCIN1 encodes a protein 577 amino acids in length[25] and Arabidopsis thaliana CWIN with seven exons and six introns encodes 584 amino acids with mass 66.280kDa27, highlighting the molecular similarities among CWINs from C. roseus and other plants

Summary

Introduction

Cell Wall Invertase (CWIN, EC: 3.2.1.26), a key enzyme in sucrose-metabolism catalyses the irreversible breakdown of sucrose into glucose and fructose It has several pleiotropic roles such as stress-response, sugar-signalling, flower, fruit and seed development[1,2,3]. Industrial production of vincristine and vinblastine is achieved by chemical coupling of more abundant[15] monomeric precursors-vindoline and catharanthine[16]. Understanding the interplay between primary and specialized-metabolisms at molecular level involving the important genes and enzymes could unravel novel ways to enable manipulation of specialized-metabolites biosynthesis in planta. The gene-expression results were further supported by metabolite analysis (includes monomeric TIAs: vindoline, catharanthine; bis-indole TIA: vinblastine). To study the effect of C. roseus CWIN on specialized-metabolism in cross-species, the full-length CWIN isoform (CrCWIN2) was transiently overexpressed in N. benthamiana leaves, followed by gene-expression and metabolite analyses

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