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.

Fig 1. Diagrams of the OX (EPSPS over-expression) and EV (empty vector, pB2WG7) constructs.

In the OX construct, the entry clone-EPSPS cDNA sequence was inserted between the attR1 and attR2 sites.

Fig 2. Photos of representative plants from OX, EV, and wild-type Arabidopsis lines in Experiment A, showing amount of glyphosate damage from 0.0x dose vs. 1.0x dose, 21 days after spraying the vegetative rosettes.

Plants with median damage levels in each line are shown.

Fig 3. Relative glyphosate resistance of OX, EV, and wild-type Arabidopsis lines, showing means ± 1 SE in Experiments A, B, and C at 21 days after spraying with glyphosate (1.0x or 0.5x).

Sample sizes and other details are summarized in Table 1. Above-ground biomass (grams fresh weight) is shown for Experiment A. Visual damage scores are based on a scale of 0–5, with 0 for plants that died, and 5 for plants that were mostly green and were developing new leaves. Means that do not share superscripts are significantly different at p<0.05 (Tukey tests). NA indicates “not applicable” for lines that were not used in Experiments A and C.

Fig 4. Relative normalized expression of EPSPS for OX, EV, and wild-type Arabidopsis lines that were used in Experiment C (see Fig 3).

Means (N = 3–4) ± 1 SE are shown for non-flowering (top) and flowering plants (bottom; one flowering line, OX1, had N = 2). Means that do not share superscripts are significantly different at p<0.05 (Tukey tests).