Todd A. Gaines

Gene amplification confers glyphosate resistance in Amaranthus palmeri

The herbicide glyphosate became widely used in the United States and other parts of the world after the commercialization of glyphosate-resistant crops. These crops have constitutive overexpression of a glyphosate-insensitive form of the herbicide target site gene, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Increased use of glyphosate over multiple years imposes selective genetic pressure on weed populations. We investigated recently discovered glyphosate-resistant Amaranthus palmeri populations from Georgia, in comparison with normally sensitive populations. EPSPS enzyme activity from resistant and susceptible plants was equally inhibited by glyphosate, which led us to use quantitative PCR to measure relative copy numbers of the EPSPS gene. Genomes of resistant plants contained from 5-fold to more than 160-fold more copies of the EPSPS gene than did genomes of susceptible plants. Quantitative RT-PCR on cDNA revealed that EPSPS expression was positively correlated with genomic EPSPS relative copy number. Immunoblot analyses showed that increased EPSPS protein level also correlated with EPSPS genomic copy number. EPSPS gene amplification was heritable, correlated with resistance in pseudo-F2 populations, and is proposed to be the molecular basis of glyphosate resistance. FISH revealed that EPSPS genes were present on every chromosome and, therefore, gene amplification was likely not caused by unequal chromosome crossing over. This occurrence of gene amplification as an herbicide resistance mechanism in a naturally occurring weed population is particularly significant because it could threaten the sustainable use of glyphosate-resistant crop technology.

Glyphosate resistance: state of knowledge

Studies of mechanisms of resistance to glyphosate have increased current understanding of herbicide resistance mechanisms. Thus far, single-codon non-synonymous mutations of EPSPS (5-enolypyruvylshikimate-3-phosphate synthase) have been rare and, relative to other herbicide mode of action target-site mutations, unconventionally weak in magnitude for resistance to glyphosate. However, it is possible that weeds will emerge with non-synonymous mutations of two codons of EPSPS to produce an enzyme endowing greater resistance to glyphosate. Today, target-gene duplication is a common glyphosate resistance mechanism and could become a fundamental process for developing any resistance trait. Based on competition and substrate selectivity studies in several species, rapid vacuole sequestration of glyphosate occurs via a transporter mechanism. Conversely, as the chloroplast requires transporters for uptake of important metabolites, transporters associated with the two plastid membranes may separately, or together, successfully block glyphosate delivery. A model based on finite glyphosate dose and limiting time required for chloroplast loading sets the stage for understanding how uniquely different mechanisms can contribute to overall glyphosate resistance.

Mechanism of Resistance of Evolved Glyphosate-Resistant Palmer Amaranth (Amaranthus palmeri)

Evolved glyphosate resistance in weedy species represents a challenge for the continued success and utility of glyphosate-resistant crops. Glyphosate functions by inhibiting the plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). The resistance mechanism was determined in a population of glyphosate-resistant Palmer amaranth from Georgia (U.S.). Within this population, glyphosate resistance correlates with increases in (a) genomic copy number of EPSPS, (b) expression of the EPSPS transcript, (c) EPSPS protein level, and (d) EPSPS enzymatic activity. Dose response results from the resistant and an F(2) population suggest that between 30 and 50 EPSPS genomic copies are necessary to survive glyphosate rates between 0.5 and 1.0 kg ha(-1). These results further confirm the role of EPSPS gene amplification in conferring glyphosate resistance in this population of Palmer amaranth. Questions remain related to how the EPSPS amplification initially occurred and the occurrence of this mechanism in other Palmer amaranth populations and other glyphosate-resistant species.

No fitness cost of glyphosate resistance endowed by massive EPSPS gene amplification in Amaranthus palmeri

Abstract Amplification of the EPSPS gene has been previously identified as the glyphosate resistance mechanism in many populations of Amaranthus palmeri, a major weed pest in US agriculture. Here, we evaluate the effects of EPSPS gene amplification on both the level of glyphosate resistance and fitness cost of resistance. A. palmeri individuals resistant to glyphosate by expressing a wide range of EPSPS gene copy numbers were evaluated under competitive conditions in the presence or absence of glyphosate. Survival rates to glyphosate and fitness traits of plants under intra-specific competition were assessed. Plants with higher amplification of the EPSPS gene (53-fold) showed high levels of glyphosate resistance, whereas less amplification of the EPSPS gene (21-fold) endowed a lower level of glyphosate resistance. Without glyphosate but under competitive conditions, plants exhibiting up to 76-fold EPSPS gene amplification exhibited similar height, and biomass allocation to vegetative and reproductive organs, compared to glyphosate susceptible A. palmeri plants with no amplification of the EPSPS gene. Both the additive effects of EPSPS gene amplification on the level of glyphosate resistance and the lack of associated fitness costs are key factors contributing to EPSPS gene amplification as a widespread and important glyphosate resistance mechanism likely to become much more evident in weed plant species.

Identification of genetic elements associated with EPSPs gene amplification.

Weed populations can have high genetic plasticity and rapid responses to environmental selection pressures. For example, 100-fold amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene evolved in the weed species Amaranthus palmeri to confer resistance to glyphosate, the world’s most important herbicide. However, the gene amplification mechanism is unknown. We sequenced the EPSPS gene and genomic regions flanking EPSPS loci in A. palmeri, and searched for mobile genetic elements or repetitive sequences. The EPSPS gene was 10,229 bp, containing 8 exons and 7 introns. The gene amplification likely proceeded through a DNA-mediated mechanism, as introns exist in the amplified gene copies and the entire amplified sequence is at least 30 kb in length. Our data support the presence of two EPSPS loci in susceptible (S) A. palmeri, and that only one of these was amplified in glyphosate-resistant (R) A. palmeri. The EPSPS gene amplification event likely occurred recently, as no sequence polymorphisms were found within introns of amplified EPSPS copies from R individuals. Sequences with homology to miniature inverted-repeat transposable elements (MITEs) were identified next to EPSPS gene copies only in R individuals. Additionally, a putative Activator (Ac) transposase and a repetitive sequence region were associated with amplified EPSPS genes. The mechanism controlling this DNA-mediated amplification remains unknown. Further investigation is necessary to determine if the gene amplification may have proceeded via DNA transposon-mediated replication, and/or unequal recombination between different genomic regions resulting in replication of the EPSPS gene.

Herbicide-resistant weeds: from research and knowledge to future needs.

Synthetic herbicides have been used globally to control weeds in major field crops. This has imposed a strong selection for any trait that enables plant populations to survive and reproduce in the presence of the herbicide. Herbicide resistance in weeds must be minimized because it is a major limiting factor to food security in global agriculture. This represents a huge challenge that will require great research efforts to develop control strategies as alternatives to the dominant and almost exclusive practice of weed control by herbicides. Weed scientists, plant ecologists and evolutionary biologists should join forces and work towards an improved and more integrated understanding of resistance across all scales. This approach will likely facilitate the design of innovative solutions to the global herbicide resistance challenge.

Characterization of glyphosate resistance in Amaranthus tuberculatus populations.

The evolution of glyphosate-resistant weeds has recently increased dramatically. Six suspected glyphosate-resistant Amaranthus tuberculatus populations were studied to confirm resistance and determine the resistance mechanism. Resistance was confirmed in greenhouse for all six populations with glyphosate resistance factors (R/S) between 5.2 and 7.5. No difference in glyphosate absorption or translocation was observed between resistant and susceptible individuals. No mutation at amino acid positions G101, T102, or P106 was detected in the EPSPS gene coding sequence, the target enzyme of glyphosate. Analysis of EPSPS gene copy number revealed that all glyphosate-resistant populations possessed increased EPSPS gene copy number, and this correlated with increased expression at both RNA and protein levels. EPSPS Vmax and Kcat values were more than doubled in resistant plants, indicating higher levels of catalytically active expressed EPSPS protein. EPSPS gene amplification is the main mechanism contributing to glyphosate resistance in the A. tuberculatus populations analyzed.

Interspecific hybridization transfers a previously unknown glyphosate resistance mechanism in Amaranthus species

A previously unknown glyphosate resistance mechanism, amplification of the 5‐enolpyruvyl shikimate‐3‐phosphate synthase gene, was recently reported in Amaranthus palmeri. This evolved mechanism could introgress to other weedy Amaranthus species through interspecific hybridization, representing an avenue for acquisition of a novel adaptive trait. The objective of this study was to evaluate the potential for this glyphosate resistance trait to transfer via pollen from A. palmeri to five other weedy Amaranthus species (Amaranthus hybridus, Amaranthus powellii, Amaranthus retroflexus, Amaranthus spinosus, and Amaranthus tuberculatus). Field and greenhouse crosses were conducted using glyphosate‐resistant male A. palmeri as pollen donors and the other Amaranthus species as pollen recipients. Hybridization between A. palmeri and A. spinosus occurred with frequencies in the field studies ranging from <0.01% to 0.4%, and 1.4% in greenhouse crosses. A majority of the A. spinosus × A. palmeri hybrids grown to flowering were monoecious and produced viable seed. Hybridization occurred in the field study between A. palmeri and A. tuberculatus (<0.2%), and between A. palmeri and A. hybridus (<0.01%). This is the first documentation of hybridization between A. palmeri and both A. spinosus and A. hybridus.

Aminopyralid and Clopyralid Absorption and Translocation in Canada Thistle (Cirsium arvense)

Abstract Aminopyralid is a new auxinic herbicide that provides Canada thistle control at lower use rates than clopyralid. Studies were conducted to determine if differences in absorption, translocation, or metabolism account for aminopyralids greater biological activity. Radiolabeled aminopyralid and clopyralid were applied to individual leaves of rosette-stage Canada thistle plants. Nonionic surfactant was used for the absorption studies because it provided higher aminopyralid absorption than methylated seed oil or crop oil concentrate. Clopyralid was absorbed very rapidly, reaching 72% 24 h after treatment (HAT) and remaining near or above 80% during a 192-h time course. During the same time period, aminopyralid absorption increased from 34 to 60%. Clopyralid translocation out of the treated leaf was significantly higher than aminopyralid, 39% compared with 17%, respectively, 192 HAT. More of applied clopyralid translocated to aboveground tissue 192 HAT (27%) than to roots (12%), whereas aminopyralid translocation was similar in aboveground tissue (10%) and roots (7%) 192 HAT. Neither aminopyralid nor clopyralid was metabolized 192 HAT. Although aminopyralid is effective at lower use rates than clopyralid, clopyralid absorption and translocation were higher in Canada thistle. These results suggest that aminopyralids chemical structure may provide for greater biological activity at the target site than clopyralid. Nomenclature: Aminopyralid; clopyralid, Canada thistle, Cirsium arvense (L.) Scop. CIRAR

Jointed Goatgrass (Aegilops Cylindrica) by Imidazolinone-Resistant Wheat Hybridization under Field Conditions

Abstract Gene flow between jointed goatgrass and winter wheat is a concern because transfer of herbicide-resistance genes from imidazolinone-resistant (IR) winter wheat cultivars to jointed goatgrass could restrict weed-management options for this serious weed of winter wheat cropping systems. The objectives of this study were (1) to investigate the frequency of interspecific hybridization between IR wheat and jointed goatgrass in eastern Colorado, and (2) to determine the gene action of the IR acetolactate synthase (ALS) allele in IR wheat by jointed goatgrass and in IR wheat by imidazolinone-susceptible (IS) wheat backgrounds. Jointed goatgrass was sampled side-by-side with IR wheat and at distances up to 53 m away in both experimental plots and at commercial field study sites in 2003, 2004, and 2005. A greenhouse-screening method was used to identify IR hybrids in collected jointed goatgrass seed. The average percentage of hybridization across sites and years when IR wheat and jointed goatgrass were grown side-by-side was 0.1%, and the maximum was 1.6%. The greatest distance over which hybridization was documented was 16 m. The IR ALS allele contributed 25% of untreated ALS activity in jointed goatgrass by IR wheat F1 plants, as measured by an in vitro ALS assay. The hybridization rate between wheat and jointed goatgrass and the expression of the IR wheat ALS allele in hybrid plants will both influence trait introgression into jointed goatgrass. Nomenclature: Jointed goatgrass, Aegilops cylindrica Host AEGCY; hard red winter wheat, Triticum aestivum L. ‘Above’, ‘Bond’, ‘Prairie Red’, ‘Halt’.