Molecular cloning and metabolomic characterization of the 5-enolpyruvylshikimate-3-phosphate synthase gene from Baphicacanthus cusia

Jian Yu,Rui Zhang,Qi Ye,Lei Zhang,Xiaoli Ma,Qitao Bu,Ruibing Chen,Yihan Zhang,Daozhi Wei,Peimin Gong,Shuju Ning,Hexin Tan

doi:10.1186/s12870-019-2035-0

Abstract

BackgroundIndigo alkaloids, such as indigo, indirubin and its derivatives, have been identified as effective antiviral compounds in Baphicacanthus cusia. Evidence suggests that the biosynthesis of indigo alkaloids in plants occurs via the shikimate pathway. The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is involved in plant metabolism; however, its underlying putative mechanism of regulating the production of indigo alkaloids is currently unknown.ResultsOne gene encoding EPSPS was isolated from B. cusia. Quantitative real-time PCR analysis revealed that BcEPSPS was expressed at the highest level in the stem and upregulated by methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA) treatment. The results of subcellular localization indicated that BcEPSPS is mainly expressed in both the plastids and cytosol, which has not been previously reported. An enzyme assay revealed that the heterogeneously expressed BcEPSPS protein catalysed the generation of 5-enolpyruvyl shikimate-3-phosphate. The overexpression of BcEPSPS in Isatis indigotica hairy roots resulted in the high accumulation of indigo alkaloids, such as indigo, secologanin, indole and isorhamnetin.ConclusionsThe function of BcEPSPS in catalysing the production of EPSP and regulating indigo alkaloid biosynthesis was revealed, which provided a distinct view of plant metabolic engineering. Our findings have practical implications for understanding the effect of BcEPSPS on active compound biosynthesis in B. cusia.

Highlights

Indigo alkaloids, such as indigo, indirubin and its derivatives, have been identified as effective antiviral compounds in Baphicacanthus cusia
Random coils and alpha helices were the most abundant structural elements distributed throughout most parts of the BcEPSPS secondary structure, while beta turns and extend strands were intermittently distributed in the protein (Additional file 2: Figure S1B)
A phylogenetic tree was constructed for the enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein family by comparing B. cusia with 13 other species of plants by phylogenetic analysis, which indicated that EPSPS in Dicliptera chinensis appeared to be phylogenetically in the same clade as BcEPSPS (Fig. 1a)

Summary

Introduction

Indigo alkaloids, such as indigo, indirubin and its derivatives, have been identified as effective antiviral compounds in Baphicacanthus cusia. Baphicacanthus cusia (Nees) Bremek, is widely distributed in Fujian, Yunnan, Sichuan and Guangdong provinces in China and is an important medicinal plant. Indigo naturalis (Qingdai) is made from its leaves and stems, which is known as “Jian Qingdai” and is a famous regional drug in Fujian Province. The main compounds isolated and identified from B. cusia are indole alkaloids [6], terpenoid alkaloids [7], quinoline ketone alkaloids [8], sterols, flavonoids [9], lignans [10], amino acids, organic acids [11], and plant polysaccharides [9]. Indole alkaloids are the main components of indigo naturalis and the main active constituents of the B. cusia

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