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

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.

Overexpressing Exogenous 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) Genes Increases Fecundity and Auxin Content of Transgenic Arabidopsis Plants

“…In addition, we did not find significant differences in fitness-related traits between the EV and parental lineages. These results support previous findings that enhanced fitness/fecundity (as shown in (Su et al, 2008 ; Wang et al, 2014 ; Yang et al, 2017a ),b) is not the consequence of an insertion or position effect (process of transgenesis), but due to the action of the transgene itself. We therefore confirm that overexpression of EPSPS in GE plants with a proper level can result in overproduction of their EPSPS and increased glyphosate tolerance.…”

“…Increased tolerance to glyphosate in our transgenic plants overexpressing an EPSPS gene is presumably due to the sufficiently surplus EPSPS that can bind glyphosate (Rogers et al, 1983 ), as reported in P. hybrida (Shah et al, 1986 ), tobacco plants (Jones et al, 1996 ), and Arabidopsis (Klee et al, 1987 ; Yang et al, 2017a ). Thus, these results confirm the strategy of overproducing EPSPS driven by a strong promoter (e.g., pCaMV35S for dicots and pUbiquitin for monocots) to be effective in increase GE crops' tolerance to glyphosate, regardless of exogenous (as in this study) or endogenous (Klee et al, 1987 ; Su et al, 2008 ; Yang et al, 2017a ). The application of an endogenous transgene overproducing EPSPS at a proper level to develop GE glyphosate-tolerant crops may have particular commercial values.…”

“…In contrast, none of the Arabidopsis plants in the corresponding transgene-absent lineages, EV, and parent controls survived at this dosage (0.4 m M ). Increased glyphosate tolerance has also been observed in other GE plants overexpressing EPSPS transgenes, such as Petunia hybrida (Shah et al, 1986 ), rice (Su et al, 2008 ; Lu et al, 2014a , b ), tobacco plants (Jones et al, 1996 ), and A. thaliana (Klee et al, 1987 ; Yang et al, 2017a ), regardless of the origins (endogenous or exogenous) of EPSPS genes. All these results indicate that the transfer of an overexpressing EPSPS gene into plants, including the model plant Arabidopsis in this study and other studies (Klee et al, 1987 ; Yang et al, 2017a ), can increase glyphosate tolerance of the transgenic plants due to overproduction of EPSPS.…”

“…This strategy includes developing GE plants with multiple copies of the EPSPS gene (Rogers et al, 1983 ; Goldsbrough et al, 1990 ; Shyr et al, 1992 ) and increasing basal levels of the EPSPS enzyme by fusing an EPSPS gene with a strong promotor (Klee et al, 1987 ; Su et al, 2008 ). Notably, certain GE glyphosate-tolerant crops overexpressing EPSPS not only have increased glyphosate tolerance (Klee et al, 1987 ; Su et al, 2008 ; Vats, 2015 ; Yang et al, 2017a ), but unexpectedly also showed increased yield (Zhou et al, 2003 ; Owen et al, 2010 ) and other fitness traits. The increased yield can also be manifest as increased fecundity in GE hybrid progeny with weedy ( O. sativa f. spontanea , Wang et al, 2014 ) and wild ( O. rufipogon , Yang et al, 2017b ) rice overexpressing EPSPS , even under glyphosate-free conditions.…”

An intragenic approach to confer glyphosate resistance in chile (Capsicum annuum) by introducing an in vitro mutagenized chile EPSPS gene encoding for a glyphosate resistant EPSPS protein

“…It is difficult to explain why the EPSPS protein from petunia and chile which share ~90% sequence similarity show differences in glyphosate-resistance but we cannot rule out the possibility that the EPSPS protein from this petunia cell line was resistant to glyphosate, notwithstanding that this line was deemed resistant due to EPSPS gene amplification (36). More recently, it has been shown that Arabidopsis plants transformed with an Arabidopsis EPSPS gene driven by the CaMV35S promoter was tolerant to glyphosate [ 37 ]. In another study, it was shown that a an EPSPS gene from Convolvulus arvensis driven by the CaMV35S promoter also conferred resistance to glyphosate in Arabidopsis [ 38 ].…”