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

Abstract

Transgenic glyphosate-tolerant plants overproducing EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) may exhibit enhanced fitness in glyphosate-free environments. If so, introgression of transgenes overexpressing EPSPS into wild relative species may lead to increased competitiveness of crop-wild hybrids, resulting in unpredicted environmental impact. Assessing fitness effects of transgenes overexpressing EPSPS in a model plant species can help address this question, while elucidating how overproducing EPSPS affects the fitness-related traits of plants. We produced segregating T₂ and T₃Arabidopsis thaliana lineages with or without a transgene overexpressing EPSPS isolated from rice or Agrobacterium (CP4). For each of the three transgenes, we compared glyphosate tolerance, some fitness-related traits, and auxin (indole-3-acetic acid) content in transgene-present, transgene-absent, empty vector (EV), and parental lineages in a common-garden experiment. We detected substantially increased glyphosate tolerance in T₂ plants of transgene-present lineages that overproduced EPSPS. We also documented significant increases in fecundity, which was associated with increased auxin content in T₃ transgene-present lineages containing rice EPSPS genes, compared with their segregating transgene-absent lineages, EV, and parental controls. Our results from Arabidopsis with nine transgenic events provide a strong support to the hypothesis that transgenic plants overproducing EPSPS can benefit from a fecundity advantage in glyphosate-free environments. Stimulated biosynthesis of auxin, an important plant growth hormone, by overproducing EPSPS may play a role in enhanced fecundity of the transgenic Arabidopsis plants. The obtained knowledge is useful for assessing environmental impact caused by introgression of transgenes overproducing EPSPS from any GE crop into populations of its wild relatives.

Keywords: Arabidopsis thaliana; abiotic stress; fitness; glyphosate-tolerance; growth hormone; indole-3-acetic acid; seed germination; transgenic plant.

Figure 1

A schematic pedigree to illustrate the production of transgenic (T) Arabidopsis progeny containing genes overexpressing the 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS). A T₀ transgenic Arabidopsis plant was self-pollinated to produce the T₁ progeny that contained transgene-homozygote (+ +), transgene-heterozygote (+ –), and transgene-absent (– –) plants. The isogenic transgene-homozygote (+ +) and transgene-absent (– –) lineages generated from T₁ plants through self-pollination (selfing) and molecular identification were retained for the experiments of glyphosate tolerance (T₂), and EPSPS transgene expression, biomass and auxin (IAA) content, and fitness assessment (T₃).

Figure 2

Average % seed germination of three Arabidopsis transgenic events under heat (A–C) and drought (D–F) stresses in T₃ transgene-present, transgene-absent, empty vector (EV), and parent (P) lineages in the glyphosate-free environment. Different letters above the columns indicate significances at P < 0.05 based on Duncan's multiple range test (n = 18). E+: EPSPS transgene-present lineages, E–: EPSPS transgene-absent lineages, Em+: mutant EPSPS transgene-present lineages, Em–: mutant EPSPS transgene-absent lineages; C+: CP4 transgene-present lineages, C–: CP4 transgene-absent lineages. Bars represent standard errors.

Figure 3

Average silique (A–C) and seed production (D–F) of three Arabidopsis transgenic events in T₃ transgene-present, transgene-absent, empty vector (EV), and parent (P) lineages in the glyphosate-free environment. Different letters above the columns indicate significances at P < 0.05 based on Duncan's multiple range test (n = 18). E+: EPSPS transgene-present lineages, E–: EPSPS transgene-absent lineages, Em+: mutant EPSPS transgene-present lineages, Em–: mutant EPSPS transgene-absent lineages; C+: CP4 transgene-present lineages, C–: CP4 transgene-absent lineages. Bars represent standard errors.

Figure 4

Average relative leaf-area (A–C) and plant height (D–F) of three Arabidopsis transgenic events in T₃ transgene-present, transgene-absent, empty vector (EV), and parent (P) lineages in the glyphosate-free environment. Different letters above the columns indicate significances at P < 0.05 based on Duncan's multiple range test (n = 18). Differences between transgene-present and transgene-absent lineages were compared based on the independent t-test after Bonferroni correction (n = 18). +P <0.1, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. E+: EPSPS transgene-present lineages, E–: EPSPS transgene-absent lineages, Em+: mutant EPSPS transgene-present lineages, Em–: mutant EPSPS transgene-absent lineages; C+: CP4 transgene-present lineages, C–: CP4 transgene-absent lineages. Bars represent standard errors.

Figure 5

Differences in biomass (A) and auxin (IAA) content (B) of 30-day plants between T₃ transgene-present and transgene-absent Arabidopsis lineages based on the independent t-test (n = 5). E+: EPSPS transgene-present lineage, E–: EPSPS transgene-absent lineages (event E/2); Em+: mutant EPSPS transgene-present lineages, Em–: mutant EPSPS transgene-absent lineages (event Em/3); C+: CP4 transgene-present lineages, C–: CP4 transgene-absent lineages (event C/2). ^**P < 0.01, ^***P < 0.001. Bars represent standard errors.