Functional Characterization of aroA from Rhizobium leguminosarum with Significant Glyphosate Tolerance in Transgenic Arabidopsis

Abstract

Glyphosate is the active component of the top-selling herbicide, the phytotoxicity of which is due to its inhibition of the shikimic acid pathway. 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the shikimic acid pathway. Glyphosate tolerance in plants can be achieved by the expression of a glyphosate-insensitive aroA gene (EPSPS). In this study, we used a PCR-based two-step DNA synthesis method to synthesize a new aroA gene (aroA R. leguminosarum) from Rhizobium leguminosarum. In vitro glyphosate sensitivity assays showed that aroA R. leguminosarum is glyphosate tolerant. The new gene was then expressed in E. coli and key kinetic values of the purified enzyme were determined. Furthermore, we transformed the aroA gene into Arabidopsis thaliana by the floral dip method. Transgenic Arabidopsis with the aroA R. leguminosarum gene was obtained to prove its potential use in developing glyphosate-resistant crops.

Quantitative Proteomic Analysis of Wheat Seeds during Artificial Ageing and Priming Using the Isobaric Tandem Mass Tag Labeling

“…The enzyme 5-enolpyruvylshikimate- 3-phosphate synthase, encoded by aroA (3-phosphoshikimate 1-carboxyvinyltransferase), is the sixth enzyme in the shikimate pathway and catalyzes the conversion of phosphoenol-pyruvate and shikimate-3-phosphate into 5-enolpyruvylshikimate 3-phosphate. Meanwhile, chorismate synthase, the seventh enzyme in the shikimate pathway catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate to the last common precursor, chorismate, in the biosynthesis of numerous aromatic compounds in bacteria, fungi, and plants [ 72 , 80 , 82 ]. The up-regulated proteins that participated in phenylalanine, tyrosine, and tryptophan biosynthesis are shown in S11 Fig .…”

Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Pantoea sp. 5-Enolpyruvylshikimate-3- Phosphate Synthase

“…In addition, the glyphosate resistance level of the transgenic Arabidopsis was 2-fold higher than that of the agricultural application level. We also compared our results with previous studies, which showed that the glyphosate tolerance level of our transgenic A. thaliana and tobacco is 10 mM and 5 mM [30,31], respectively. The 20 mM glyphosate resistance of our transgenic Arabidopsis is obviously higher.…”

“…In E. coli, amino acid residues G96, T97, and P101 mutations (corresponding to G97A, T98I, and P102S in aroA that also increased glyphosate resistance have been studied [15]. These amino acid mutations cause the displacement of the nearby amino acid residues around S3P/PEP [29,30]. Although G96, T97, and P101 mutations at the S3P/PEP active sites have been widely studied, no reports concerning the glyphosate resistance of three-site mutants have been done.…”

Growth Inhibition Test of Glyphosate Herbicide For Glyphosate-Degrading-Bacteria Screening

“…Another previous study reported that the consortium of Pseudomonas putida, Pseudomonas aeruginosa, and Acetobacter faecalis did not accelerate the biodegradation process, but they could reduce lag time from 24 hours to 12 hours [30] .The higher IC 50 value of consortium culture from unexposed soil could be caused by high diversity of bacteria in unexposed soil. Based on previous studies, 50% growth inhibition of P. fluorescens AFT36, Escherichia coli, Bacillus subtilis, B. jabonicum, and P. aeruginosa, P. putida W1616 were caused by glyphosate concentration of 3.0 μM, 75 μM, 174 μM, 1.1 mM, 1.1 mM, and 3 mM, respectively [31] [4] .Several previous studies have also been carried out to determine IC50 value of the EPSPS enzyme of some bacteria like E. coli(0,055 mM), Ochrobacterium anthropi(0,61 mM) [28]; Rhizobium leguminosarum(1,025 mM) [18] ,S. aureus(1,6 mM) [32] ; Agrobacterium tumefaciens CP4 (11 mM) [33] . Molecular mechanism of EPSP synthase in natural glyphosatetolerant microorganisms had not been studied intensively [33] .…”

“…Microbial resistance towards glyphosate is related to the sensititivity of EPSP (5-enolpyruvylshikimate-3-phosphate synthase) which is encoded by the gene aroA and has the important role in shikimate pathway. EPSP synthases are divided into two classes: 1) Class I EPSPsynthases, found in all plants and in many Gram-negative bacteria, which are generallysensitive to glyphosate and 2) Class II EPSP synthases which are found in naturally glyphosate tolerant microbes.These two enzyme classes have amino acid identity less than 30% [18] . The objective of this study was to observe the effect of glyphosate herbicide towards consortium culture from glyphosate-contaminated soil and uncontaminated soil by determining IC 50 parameter.…”