A novel 5-enolpyruvoylshikimate-3-phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide

Abstract

Understanding evolutionary interactions among crops and weeds can facilitate effective weed management. For example, gene flow from crops to their wild or weedy relatives can lead to rapid evolution in recipient populations. In rice (Oryza sativa), transgenic herbicide resistance is expected to spread to conspecific weedy rice (Oryza sativa f. spontanea) via hybridization. Here, we studied fitness effects of transgenic over-expression of a native 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) gene developed to confer glyphosate resistance in rice. Controlling for genetic background, we examined physiological traits and field performance of crop-weed hybrid lineages that segregated for the presence or absence of this novel epsps transgene. Surprisingly, we found that transgenic F2 crop-weed hybrids produced 48-125% more seeds per plant than nontransgenic controls in monoculture- and mixed-planting designs without glyphosate application. Transgenic plants also had greater EPSPS protein levels, tryptophan concentrations, photosynthetic rates, and per cent seed germination compared with nontransgenic controls. Our findings suggest that over-expression of a native rice epsps gene can lead to fitness advantages, even without exposure to glyphosate. We hypothesize that over-expressed epsps may be useful to breeders and, if deployed, could result in fitness benefits in weedy relatives following transgene introgression.

Keywords: Oryza sativa; epsps; fitness; genetically engineered; glyphosate-resistant; introgression; risk assessment; weedy rice.

Fig 1

Crossing procedure for obtaining crop–weed (Oryza sativa f. spontanea and O. sativa) hybrid lineages that segregated for the 5-eno-lpyruvoylshikimate-3-phosphate synthase (epsps) transgene. Genetically engineered (GE) refers to plants with one (+ −) or two (+ +) copies of the epsps transgene. F₁ and F₂ plants were used in field experiments to test for differences between GE and non-GE controls in growth and fecundity; F₂ plants also were used to compare gene expression and EPSPS protein levels; the F₃ generation was used to test for differences in tryptophan concentration, photosynthetic rate, and per cent seed germination.

Fig 2

Increased expression of the 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) gene and EPSPS protein content in transgenic F₂ crop–weed (Oryza sativa f. spontanea and O. sativa) hybrids. The four crop–weed hybrid lineages are designated WH1, WH2, WH3, and WH4. Means are shown with 1 ± SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (t-tests with Bonferroni corrections). (a) Gene expression from epsps (arrowhead, left side) and actin control (full arrow, right side) in genetically engineered (GE) (WH1+) and non-GE (WH1−) plants; M indicates the DL2000 DNA marker; n = 3. (b) epsps gene expression in GE (upper lines) and non-GE (lower lines) WH1 plants detected by real-time PCR. Rn represents the amount of PCR produced by the epsps transgene against that by a reference gene (action) as measured based on fluorescence intensity, shown with the number of PCR cycles. The inserted bar chart represents the relative levels of gene expression when WH1− was set as 1. The y-axis shows the logarithm-transformed (log₁₀) gene expression level; n = 3. (c) Mean percentage of relative EPSPS protein content of GE (green symbols) and non-GE plants (red symbols), determined by enzyme-linked immunosorbent assay (ELISA); n = 9.

Fig 3

Increases in (a) tryptophan (Trp) concentration and (b) photosynthetic rates of transgenic F₃ plants from four crop–weed (Oryza sativa f. spontanea and Oryza sativa) hybrid lineages. Closed columns indicate genetically engineered (GE) plants and open columns indicate non-GE plants. The four crop–weed hybrid lineages are designated WH1, WH2, WH3, and WH4. Means and SE are based on n = 3 leaf samples for Trp and n = 3 plants for photosynthesis. +, P < 0.1; *, P < 0.05; **, P < 0.01 (t-tests with Bonferroni corrections). Error bars represent ± SE.

Fig 4

Increased per cent germination of F₃ seeds with the 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) transgene in each crop–weed (Oryza sativa f. spontanea and Oryza sativa) hybrid lineage. The four crop–weed hybrid lineages are designated WH1, WH2, WH3, and WH4. Closed columns indicate genetically engineered (GE) and open columns indicate non-GE plants. n = 4 and the error bars represent ± SE. *, P < 0.05; ***, P < 0.001 (t-tests with Bonferroni corrections).

Fig 5

Increased growth and fecundity of F₂ crop–weed (Oryza sativa f. spontanea and Oryza sativa) hybrid progeny with the 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) transgene in field experiments. The number of panicles (flowering shoots) and seeds per plant for each of the four crop–weed lineages are shown, with means and SE (n = 6). Closed columns indicate genetically engineered (GE) plants and open columns indicate non-GE plants. (a, c) Results from pure cultivation of either GE or non-GE plants in each plot (compared using t-tests with Bonferroni corrections); (b, d) results from mixed cultivation of GE and non-GE plants competing in the same plot (compared using paired t-tests with Bonferroni corrections). **, P < 0.01; ***, P < 0.001; see Table 2 for ANOVAs and Supporting Information Table S3 for a summary of these and other fitness-related traits.