Engineering tropane alkaloid production and glyphosate resistance by overexpressing AbCaM1 and G2-EPSPS in Atropa belladonna

Highlights

• AbCaM1 is a novel calmodulin regulating alkaloid biosynthesis in Atropa belladonna.

• The method using glyphosate as direct selection pressure is established to obtain engineered plants of A. belladonna.

• The plants of A. belladonna overexpressing AbCaM1 and G2-EPSPS have high-yield tropane alkaloids and glyphosate resistance.

Abstract

Atropa belladonna is an important industrial crop for producing anticholinergic tropane alkaloids (TAs). Using glyphosate as selection pressure, transgenic homozygous plants of A. belladonna are generated, in which a novel calmodulin gene (AbCaM1) and a reported EPSPS gene (G2-EPSPS) are co-overexpressed. AbCaM1 is highly expressed in secondary roots of A. belladonna and has calcium-binding activity. Three transgenic homozygous lines were generated and their glyphosate tolerance and TAs’ production were evaluated in the field. Transgenic homozygous lines produced TAs at much higher levels than wild-type plants. In the leaves of T2GC02, T2GC05, and T2GC06, the hyoscyamine content was 8.95-, 10.61-, and 9.96 mg/g DW, the scopolamine content was 1.34-, 1.50- and 0.86 mg/g DW, respectively. Wild-type plants of A. belladonna produced hyoscyamine and scopolamine respectively at the levels of 2.45 mg/g DW and 0.30 mg/g DW in leaves. Gene expression analysis indicated that AbCaM1 significantly up-regulated seven key TA biosynthesis genes. Transgenic homozygous lines could tolerate a commercial recommended dose of glyphosate in the field. In summary, new varieties of A. belladonna not only produce pharmaceutical TAs at high levels but tolerate glyphosate, facilitating industrial production of TAs and weed management at a much lower cost.

Introduction

Tropane alkaloids (TAs), including hyoscyamine, anisodamine, and scopolamine, are widely used in the treatment of motion sickness, Alzheimer's disease, and spasms due to their anticholinergic properties (Poupko et al., 2007; Sweta and Lakshmi, 2015; Yun et al., 1992). Atropine, the racemic hyoscyamine, is included in the Essential Drug Lists authorized by the World Health Organization. These pharmaceutical TAs are specifically produced in the medicinal plants of Solanaceae, including the plant genera of Atropa, Hyoscyamus, Datura, Brugmansia, Duboisia, Anisodus, and Scopolia (Yamada and Hashimoto, 1982). Of them, Atropa belladonna is one of the most important crops widely cultivated for industrially producing TAs. Unfortunately, TA production is very low in plants. Chemical synthesis of TAs is academically successful but has failed commercially due to its very high cost. To date, the medicinal plants of Solanaceae are the only source for commercially producing TAs. It has been a common goal for the TA industry to develop new varieties with high-yield pharmaceutical TAs. Breeders have used classical methods such as genetic breeding, polyploid breeding, and radiation breeding to develop new varieties of A. belladonna with high yields of TAs, but ultimately failed. Weeds are a big problem for crop plantation, because they compete with crops for space, light, water, and nutrients. Uncontrolled weed growth often leads to severely reduced crop harvests (Zimdahl, 2018). As a herbal plant, A. belladonna needs frequent weeding during plantation. Obviously, manual weeding is laborious and expensive. Therefore, it is also vital to develop new varieties of A. belladonna with herbicide resistance. Genetic engineering is a promising approach to develop new herbicide-resistant varieties of A. belladonna with high TA yields.

By virtue of recent research, the biosynthesis of TAs is now completely understood. Three TA biosynthesis genes, namely, ornithine decarboxylase (ODC), putrescine N-methyltransferase (PMT), and hyoscyamine 6β-hydroxylase (H6H), have been used in engineering TA biosynthesis in A. belladonna plants. ODC catalyzes decarboxylation of ornithine to produce putrescine that goes into the biosynthesis of polyamines and TAs. Overexpression of ornithine decarboxylase enhances the production of hyoscyamine and anisodamine, but does not alter the scopolamine production in transgenic plants of A. belladonna (Zhao et al., 2020). PMT is the committed enzyme converting putrescine to form N-methylputrescine that is a specific intermediate in TA biosynthesis. Overexpression of the PMT gene in A. belladonna plants significantly enhances the biosynthesis of N-methylputrescine, but does not increase TA production (Rothe et al., 2003; Sato et al., 2001). H6H catalyzes 6β-hydroxylation of hyoscyamine to yield anisodamine and subsequently converts anisodamine into scopolamine through epoxidation. Overexpression of the H6H gene of Hyoscyamus niger markedly elevates scopolamine production and dramatically reduces hyoscyamine production in transgenic plants of A. belladonna, due to H6H-mediated conversion of hyoscyamine into scopolamine (Wang et al., 2011; Xia et al., 2016; Yun et al., 1992). Notably, in the foregoing studies, neomycin phosphotransferase II (NPTII) was used as a selection marker. The NPTII gene is useless once transgenic plants are established using kanamycin as selection pressure. To date, there are no reports on developing varieties of A. belladonna with herbicide resistance, due to lack of transgenic technologies using non-antibiotics, such as glyphosate, as selection pressure.

Theoretically, regulatory genes might be employed to enhance the production of various metabolites. However, no such regulatory genes controlling TA biosynthesis have hitherto been reported. Calcium is a second messenger that regulates diverse physiological processes in plants including metabolite biosynthesis. Calmodulin (CaM) is the most important Ca²⁺-binding protein that functions in the transduction of Ca²⁺ signaling into a variety of metabolic pathways, including secondary metabolism (Mahady and Beecher, 1994; Yang and Poovaiah, 2003). Based on introducing calmodulin antagonists to plant cell cultures, it was found that Ca²⁺/CaM signaling regulated the biosynthesis of different kinds of secondary metabolites, such as resveratrol (Kiselev et al., 2013), ginsenoside Rb1 (Yue and Zhong, 2005), phytoalexin (Preisig and Moreau, 1994), sesquiterpenes (Vögeli et al., 1992), and rubber (Wititsuwannakul et al., 1990). The production of TAs in root cultures of Datura stramonium, Datura metel, and H. niger can be promoted by elevating calcium concentrations in media (Amdoun et al., 2010; Chaouch et al., 2009; Pudersell et al., 2003). When the ratio of calcium to potassium is increased in the medium, A. belladonna plants also produced TAs at higher levels (Smolenski et al., 1967). These previous studies suggest that Ca²⁺/CaM signaling could play a crucial role in regulating TA biosynthesis. Therefore, we propose that TA production might be enhanced through overexpressing the CaM gene in planta.

In this study, we develop homozygous lines of transgenic A. belladonna plants with overexpression of calmodulin identified from A. belladonna (AbCaM1) and G2 5-enolpyruvylshikimate-3-phosphate synthase identified from Pseudomonas fluorescens (G2-EPSPS) (Dun et al., 2007). AbCaM1 is a novel calmodulin gene highly expressed in secondary roots of A. belladonna, in which TAs are synthesized. G2-EPSPS has high catalytic activity and glyphosate tolerance (Dun et al., 2007) and it has been used to develop glyphosate-resistant crops, including maize, soybean and rice (Dong et al., 2017; Guo et al., 2015; Liu et al., 2015). The TA production and glyphosate resistance were investigated in the transgenic homozygous lines of A. belladonna grown in the field. These transgenic homozygous lines of A. belladonna, with co-expression of AbCaM1 and G2-EPSPS, were able to tolerate commercial usage of glyphosate and produce TAs at markedly high levels, indicating great potential for industrial production of pharmaceutical tropane alkaloids.