Overexpression of improved EPSPS gene results in field level glyphosate tolerance and higher grain yield in rice

Abstract

Glyphosate is a popular, systemic, broad-spectrum herbicide used in modern agriculture. Being a structural analog of phosphoenolpyruvate (PEP), it inhibits 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) which is responsible for the biosynthesis of aromatic amino acids and various aromatic secondary metabolites. Taking a lead from glyphosate-resistant weeds, two mutant variants of the rice EPSPS gene were developed by amino acid substitution (T173I + P177S; TIPS-OsEPSPS and G172A + T173I + P177S; GATIPS-OsEPSPS). These mutated EPSPS genes were overexpressed in rice under the control of either native EPSPS or constitutive promoters (maize ubiquitin [ZmUbi] promoter). The overexpression of TIPS-OsEPSPS under the control of the ZmUbi promoter resulted in higher tolerance to glyphosate (up to threefold of the recommended dose) without affecting the fitness and related agronomic traits of plants in both controlled and field conditions. Furthermore, such rice lines produced 17%-19% more grains compared to the wild type (WT) in the absence of glyphosate application and the phenylalanine and tryptophan contents in the transgenic seeds were found to be significantly higher in comparison with WT seeds. Our results also revealed that the native promoter guided expression of modified EPSPS genes did not significantly improve the glyphosate tolerance. The present study describing the introduction of a crop-specific TIPS mutation in class I aroA gene of rice and its overexpression have potential to substantially improve the yield and field level glyphosate tolerance in rice. This is the first report to observe that the EPSPS has role to play in improving grain yield of rice.

Herbicide Resistance: Another Hot Agronomic Trait for Plant Genome Editing

“…The transformed genes containing double substitution mutation were overexpressed in rice, which developed significantly higher tolerance to glyphosate under the control of maize ubiquitin (ZmUbi) promoter. Interestingly, the transgenic rice plants presented 17 to 19% higher grain production along with greater tryptophan and phenylalanine contents in transgenic seeds compared to their wild type [ 92 ].…”

Increased Longevity and Dormancy of Soil-Buried Seeds from Advanced Crop–Wild Rice Hybrids Overexpressing the EPSPS Transgene

“…This result addressed our second question regarding the influences of the EPSPS transgene on dormancy of the GE crop-wild hybrid seeds, although our knowledge is still limited to confidently explain the underlying mechanisms connecting seed dormancy with the overexpressing EPSPS transgene in rice. However, we can explain the linkage between increased seed dormancy and overexpression of the EPSPS transgene well through increased tryptophan as reported by Wang et al and Achary et al [21,32] and increased IAA by Fang et al [31], both of which are supposedly responsible for seed dormancy [21,31,32,39]. Our finding perfectly agrees with the previous conclusions that overexpression of the endogenous EPSPS gene from cultivated rice enhances the performance of many fitness-related phenotypic characteristics of transgenic plants [15,21] and physiological and metabolic processes, including tryptophan, chlorophyll, and lignin syntheses [21,31,33,34].…”

“…Probably, the overproduction of EPSPS can accelerate the biosynthesis of tryptophan and then IAA in the shikimate pathway of the GE crop-wild hybrid descendants. Previous studies reported that overexpressing the EPSPS transgene significantly increased the tryptophan content in the GE-cultivated rice seeds [32] and in the crop-weed hybrid plants derived from an EPSPS transgenic rice (O. sativa) line (EP3) crossed with four weedy rice (O. sativa f. spontanea) accessions [21]. In addition, overexpression of the same rice EPSPS transgene also significantly increased the IAA content in GE Arabidopsis plants [31].…”

“…The rice line that was developed to tolerate the glyphosate herbicide contained a rice endogenous EPSPS (5-enolpyruvoylshikimate-3-phosphate synthase) transgene overexpressing the EPSP enzyme [30]. Further studies indicated that overexpressing the EPSPS transgene considerably increased seed production of GE hybrids under the glyphosate free conditions, in addition to increases in the content of chlorophyll, lignin, auxin (indole-3-acetic acid, IAA), and tryptophan [15,[31][32][33][34]. These results indicate that the transgene overexpressing EPSPS may cause considerable environmental impacts if it is incorporated into populations of crop wild relative species through pollen-mediated gene flow.…”

“…Studies on Arabidopsis and rice containing the transgene overexpressing EPSPS from different sources demonstrated substantial transgenic benefits, with only a slight position effect of gene insertion between different transgenic events [31,32]. However, no fitness effect was reported by the hygromycin resistant gene used as a selecting marker [31].…”