Effects of over-expressing a native gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) on glyphosate resistance in Arabidopsis thaliana.

Xiao Yang,Wanying Zhao,Zachery T Beres,Lin Jin,Allison A Snow,David Mackey,Jason T Parrish

doi:10.1371/journal.pone.0175820

Abstract

Widespread overuse of the herbicide glyphosate, the active ingredient in RoundUp®, has led to the evolution of glyphosate-resistant weed biotypes, some of which persist by overproducing the herbicide’s target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS is a key enzyme in the shikimic acid pathway for biosynthesis of aromatic amino acids, lignin, and defensive compounds, but little is known about how overproducing EPSPS affects downstream metabolites, growth, or lifetime fitness in the absence of glyphosate. We are using Arabidopsis as a model system for investigating phenotypic effects of overproducing EPSPS, thereby avoiding confounding effects of genetic background or other mechanisms of herbicide resistance in agricultural weeds. Here, we report results from the first stage of this project. We designed a binary vector expressing a native EPSPS gene from Arabidopsis under control of the CaMV35S promoter (labelled OX, for over-expression). For both OX and the empty vector (labelled EV), we obtained nine independent T3 lines. Subsets of these lines were used to characterize glyphosate resistance in greenhouse experiments. Seven of the nine OX lines exhibited enhanced glyphosate resistance when compared to EV and wild-type control lines, and one of these was discarded due to severe deformities. The remaining six OX lines exhibited enhanced EPSPS gene expression and glyphosate resistance compared to controls. Glyphosate resistance was correlated with the degree of EPSPS over-expression for both vegetative and flowering plants, indicating that glyphosate resistance can be used as a surrogate for EPSPS expression levels in this system. These findings set the stage for examination of the effects of EPSPS over-expression on fitness-related traits in the absence of glyphosate. We invite other investigators to contact us if they wish to study gene expression, downstream metabolic effects, and other questions with these particular lines.

Highlights

enolpyruvylshikimate-3-phosphate synthase (EPSPS) (5-enolpyruvoylshikimate-3-phosphate synthase, EC2.5.1.19) is a key enzyme in the shikimic acid pathway, which accounts for more than ~30% of carbon fixed by photosynthesis in vascular plants [1,2]
Overproduction of EPSPS can occur due to mutations that result in extra copies of the EPSPS gene, as reported in Amaranthus palmeri, A. tuberculatus, A. spinosus, Lolium multiflorum, Kochia scoparia, Eleusine indica, and Bromus diandrus [8,12,13]
Overproduction of EPSPS has been reported for some glyphosate-resistant biotypes of Conyza canadensis [20,21], gene amplification has not been identified as the cause

Summary

Introduction

EPSPS (5-enolpyruvoylshikimate-3-phosphate synthase, EC2.5.1.19) is a key enzyme in the shikimic acid pathway, which accounts for more than ~30% of carbon fixed by photosynthesis in vascular plants [1,2]. Millions of hectares of farmland are planted with transgenic, glyphosate-tolerant crops [7] Most of these crop varieties have an EPSPS gene from Agrobacteria sp. Due to heavy reliance on glyphosate worldwide, more than 35 weed species have evolved resistance to this herbicide through a variety of mechanisms (e.g., [8,9]). Overproduction of EPSPS has been reported for some glyphosate-resistant biotypes of Conyza canadensis [20,21], gene amplification has not been identified as the cause. Overproduction of EPSPS was reported for cell lines of carrot, tobacco, and petunia after many cycles of glyphosate-induced selection pressure in the laboratory [3,22,23]

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Results