A novel 5-enolpyruvylshikimate-3-phosphate synthase shows high glyphosate tolerance in Escherichia coli and tobacco plants.

Gaoyi Cao,Shengxue Zhang,Wei Lu,Guoying Wang,Yuwen Zhang,Xuewen Yang,Min Lin,Yunjun Liu,Yan Liu,Rongrong Chen,Jianhua Wang,Alejandra Bravo

doi:10.1371/journal.pone.0038718

Abstract

A key enzyme in the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the primary target of the broad-spectrum herbicide glyphosate. Identification of new aroA genes coding for EPSPS with a high level of glyphosate tolerance is essential for the development of glyphosate-tolerant crops. In the present study, the glyphosate tolerance of five bacterial aroA genes was evaluated in the E. coli aroA-defective strain ER2799 and in transgenic tobacco plants. All five aroA genes could complement the aroA-defective strain ER2799, and AM79 aroA showed the highest glyphosate tolerance. Although glyphosate treatment inhibited the growth of both WT and transgenic tobacco plants, transgenic plants expressing AM79 aroA tolerated higher concentration of glyphosate and had a higher fresh weight and survival rate than plants expressing other aroA genes. When treated with high concentration of glyphosate, lower shikimate content was detected in the leaves of transgenic plants expressing AM79 aroA than transgenic plants expressing other aroA genes. These results suggest that AM79 aroA could be a good candidate for the development of transgenic glyphosate-tolerant crops.

Highlights

Glyphosate (N-phosphonomethyl glycine), an important and potent herbicide, is widely used to control weeds in agricultural fields
Since the 1980s, researchers have begun to isolate glyphosate-insensitive enolpyruvylshikimate-3phosphate synthase (EPSPS) from bacteria or plants, and numerous promising enzymes have been identified by microbial screening and selective evolution [4,13,28,29]
The Agrobacterium sp. strain CP4 is a naturally occurring, glyphosate-tolerant microbe in environments contaminated with high concentration of glyphosate, and the CP4 EPSPS has been commercially used in genetically modified crops [30]

Summary

Introduction

Glyphosate (N-phosphonomethyl glycine), an important and potent herbicide, is widely used to control weeds in agricultural fields. Glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3phosphate synthase (EPSPS;EC 2.5.1.19), which converts phosphoenolpyruvate (PEP) and shikimate-3-phosphate (S3P) to 5enolpyruvylshikimate-3-phosphate (EPSP) and shuts down the shikimate pathway, leading to plant death. Type I EPSP synthases have been identified mainly in plants and bacteria, and type II EPSP synthases have been identified in some forms of bacteria. Type I EPSP synthases are naturally sensitive to glyphosate, whereas type II EPSP synthases are tolerant of glyphosate [2,3,4]. After the primary target of glyphosate was identified as EPSPS in the 1980s [5], EPSPS became the top choice for the development of transgenic glyphosate-tolerant crops. Over-expression of most wild EPSPS in transgenic plants can not confer plants with glyphosate tolerance [6]

Methods

Results

Conclusion