Resistance to post-emergent herbicides is becoming common for grass weeds on New Zealand wheat and barley farms.

To estimate the prevalence of herbicide-resistant weeds, 87 wheat and barley farms were randomly surveyed in the Canterbury region of New Zealand. Over 600 weed seed samples from up to 10 mother plants per taxon depending on abundance, were collected immediately prior to harvest (two fields per farm). Some samples provided by agronomists were tested on an ad-hoc basis. Over 40,000 seedlings were grown to the 2-4 leaf stage in glasshouse conditions and sprayed with high priority herbicides for grasses from the three modes-of-action acetyl-CoA carboxylase (ACCase)-inhibitors haloxyfop, fenoxaprop, clodinafop, pinoxaden, clethodim, acetolactate synthase (ALS)-inhibitors iodosulfuron, pyroxsulam, nicosulfuron, and the 5-enolpyruvyl shikimate 3-phosphate synthase (EPSPS)-inhibitor glyphosate. The highest manufacturer recommended label rates were applied for the products registered for use in New Zealand, often higher than the discriminatory rates used in studies elsewhere. Published studies of resistance were rare in New Zealand but we found weeds survived herbicide applications on 42 of the 87 (48%) randomly surveyed farms, while susceptible reference populations died. Resistance was found for ALS-inhibitors on 35 farms (40%) and to ACCase-inhibitors on 20 (23%) farms. The number of farms with resistant weeds (denominator is 87 farms) are reported for ACCase-inhibitors, ALS-inhibitors, and glyphosate respectively as: Avena fatua (9%, 1%, 0% of farms), Bromus catharticus (0%, 2%, 0%), Lolium spp. (17%, 28%, 0%), Phalaris minor (1%, 6%, 0%), and Vulpia bromoides (0%, not tested, 0%). Not all farms had the weeds present, five had no obvious weeds prior to harvest. This survey revealed New Zealand's first documented cases of resistance in P. minor (fenoxaprop, clodinafop, iodosulfuron) and B. catharticus (pyroxsulam). Twelve of the 87 randomly sampled farms (14%) had ALS-inhibitor chlorsulfuron-resistant sow thistles, mostly Sonchus asper but also S. oleraceus. Resistance was confirmed in industry-supplied samples of the grasses Digitaria sanguinalis (nicosulfuron, two maize farms), P. minor (iodosulfuron, one farm), and Lolium spp. (cases included glyphosate, haloxyfop, pinoxaden, iodosulfuron, and pyroxsulam, 9 farms). Industry also supplied Stellaria media samples that were resistant to chlorsulfuron and flumetsulam (ALS-inhibitors) sourced from clover and ryegrass fields from the North and South Island.

Parallel and nonparallel genomic responses contribute to herbicide resistance in Ipomoea purpurea, a common agricultural weed.

The repeated evolution of herbicide resistance has been cited as an example of genetic parallelism, wherein separate species or genetic lineages utilize the same genetic solution in response to selection. However, most studies that investigate the genetic basis of herbicide resistance examine the potential for changes in the protein targeted by the herbicide rather than considering genome-wide changes. We used a population genomics screen and targeted exome re-sequencing to uncover the potential genetic basis of glyphosate resistance in the common morning glory, Ipomoea purpurea, and to determine if genetic parallelism underlies the repeated evolution of resistance across replicate resistant populations. We found no evidence for changes in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), glyphosate's target protein, that were associated with resistance, and instead identified five genomic regions that showed evidence of selection. Within these regions, genes involved in herbicide detoxification-cytochrome P450s, ABC transporters, and glycosyltransferases-are enriched and exhibit signs of selective sweeps. One region under selection shows parallel changes across all assayed resistant populations whereas other regions exhibit signs of divergence. Thus, while it appears that the physiological mechanism of resistance in this species is likely the same among resistant populations, we find patterns of both similar and divergent selection across separate resistant populations at particular loci.

Glyphosate in livestock: feed residues and animal health1.

Glyphosate is a nonselective systemic herbicide used in agriculture since 1974. It inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, an enzyme in the shikimate pathway present in cells of plants and some microorganisms but not human or other animal cells. Glyphosate-tolerant crops have been commercialized for more than 20 yr using a transgene from a resistant bacterial EPSP synthase that renders the crops insensitive to glyphosate. Much of the forage or grain from these crops are consumed by farm animals. Glyphosate protects crop yields, lowers the cost of feed production, and reduces CO2 emissions attributable to agriculture by reducing tillage and fuel usage. Despite these benefits and even though global regulatory agencies continue to reaffirm its safety, the public hears conflicting information about glyphosate's safety. The U.S. Environmental Protection Agency determines for every agricultural chemical a maximum daily allowable human exposure (called the reference dose, RfD). The RfD is based on amounts that are 1/100th (for sensitive populations) to 1/1,000th (for children) the no observed adverse effects level (NOAEL) identified through a comprehensive battery of animal toxicology studies. Recent surveys for residues have indicated that amounts of glyphosate in food/feed are at or below established tolerances and actual intakes for humans or livestock are much lower than these conservative exposure limits. While the EPSP synthase of some bacteria is sensitive to glyphosate, in vivo or in vitro dynamic culture systems with mixed bacteria and media that resembles rumen digesta have not demonstrated an impact on microbial function from adding glyphosate. Moreover, one chemical characteristic of glyphosate cited as a reason for concern is that it is a tridentate chelating ligand for divalent and trivalent metals; however, other more potent chelators are ubiquitous in livestock diets, such as certain amino acids. Regulatory testing identifies potential hazards, but risks of these hazards need to be evaluated in the context of realistic exposures and conditions. Conclusions about safety should be based on empirical results within the limitations of model systems or experimental design. This review summarizes how pesticide residues, particularly glyphosate, in food and feed are quantified, and how their safety is determined by regulatory agencies to establish safe use levels.

Glyphosate perturbs the gut microbiota of honey bees.

Glyphosate, the primary herbicide used globally for weed control, targets the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme in the shikimate pathway found in plants and some microorganisms. Thus, glyphosate may affect bacterial symbionts of animals living near agricultural sites, including pollinators such as bees. The honey bee gut microbiota is dominated by eight bacterial species that promote weight gain and reduce pathogen susceptibility. The gene encoding EPSPS is present in almost all sequenced genomes of bee gut bacteria, indicating that they are potentially susceptible to glyphosate. We demonstrated that the relative and absolute abundances of dominant gut microbiota species are decreased in bees exposed to glyphosate at concentrations documented in the environment. Glyphosate exposure of young workers increased mortality of bees subsequently exposed to the opportunistic pathogen Serratia marcescens Members of the bee gut microbiota varied in susceptibility to glyphosate, largely corresponding to whether they possessed an EPSPS of class I (sensitive to glyphosate) or class II (insensitive to glyphosate). This basis for differences in sensitivity was confirmed using in vitro experiments in which the EPSPS gene from bee gut bacteria was cloned into Escherichia coli All strains of the core bee gut species, Snodgrassella alvi, encode a sensitive class I EPSPS, and reduction in S. alvi levels was a consistent experimental result. However, some S. alvi strains appear to possess an alternative mechanism of glyphosate resistance. Thus, exposure of bees to glyphosate can perturb their beneficial gut microbiota, potentially affecting bee health and their effectiveness as pollinators.

Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Pantoea sp. 5-Enolpyruvylshikimate-3-Phosphate Synthase.

Glyphosate inhibits the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway. A mutant of EPSPS from Pantoea sp. was identified using site-directed mutagenesis (SDM). The mutant significantly improved glyphosate resistance. The mutant had mutations in three amino acids: Gly97 to Ala, Thr 98 to Ile and Pro 102 to Ser. These mutation sites in E.coli have been studied as significant active sites of glyphosate resistance. However, in our research they were found to jointly contribute to the improvement of glyphosate tolerance. In addition, the level of glyphosate tolerance in transgenic Arabidopsis confirmed the potentiality of the mutant in breeding glyphosate-resistant plants.

Glyphosate Resistance and EPSPS Gene Duplication: Convergent Evolution in Multiple Plant Species.

One of the increasingly widespread mechanisms of resistance to the herbicide glyphosate is copy number variation (CNV) of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. EPSPS gene duplication has been reported in 8 weed species, ranging from 3 to 5 extra copies to more than 150 extra copies. In the case of Palmer amaranth (Amaranthus palmeri), a section of >300 kb containing EPSPS and many other genes has been replicated and inserted at new loci throughout the genome, resulting in significant increase in total genome size. The replicated sequence contains several classes of mobile genetic elements including helitrons, raising the intriguing possibility of extra-chromosomal replication of the EPSPS-containing sequence. In kochia (Kochia scoparia), from 3 to more than 10 extra EPSPS copies are arranged as a tandem gene duplication at one locus. In the remaining 6 weed species that exhibit EPSPS gene duplication, little is known about the underlying mechanisms of gene duplication or their entire sequence. There is mounting evidence that adaptive gene amplification is an important mode of evolution in the face of intense human-mediated selection pressure. The convergent evolution of CNVs for glyphosate resistance in weeds, through at least 2 different mechanisms, may be indicative of a more general importance for this mechanism of adaptation in plants. CNVs warrant further investigation across plant functional genomics for adaptation to biotic and abiotic stresses, particularly for adaptive evolution on rapid time scales.

Target-site EPSPS Pro-106 mutations: sufficient to endow glyphosate resistance in polyploid Echinochloa colona?

BACKGROUND: This study confirms and characterises glyphosate resistance in two polyploid Echinochloa colona populations from north-eastern Australia. RESULTS: Glyphosate dose response revealed that the two resistant populations were marginally (up to twofold) resistant to glyphosate. Resistant plants did not differ in non-target-site foliar uptake and translocation of (14) C-glyphosate, but contained the known target-site 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) mutation Pro-106-Thr and/or Pro-106-Leu. Although plants carrying either a single or two EPSPS mutations were glyphosate resistant relative to the susceptible population, they were still controlled at the field rate of glyphosate (450 g a.e. ha(-1) ) when treated under warm conditions (25/20 °C). However, when treated in hot conditions (35/30 °C), most mutant resistant plants (68%) can survive the field rate, and an increase (2.5-fold) in glyphosate LD50 was found for both the R and S populations. CONCLUSIONS: This study shows that one or two EPSPS Pro-106 mutations are insufficient to confer field-rate glyphosate resistance in polyploidy E. colona at mild temperatures. However, control of these mutant plants at the glyphosate field rate is poor at high temperatures, probably owing to reduced glyphosate efficacy. Therefore, glyphosate should be applied during relatively mild (warm) temperature periods in the summer growing season to improve E. colona control.

Possible glyphosate tolerance mechanism in pitted morningglory (Ipomoea lacunosa L.).

Natural tolerance of Ipomoea lacunosa to glyphosate has made it problematic in the southeastern U.S. since the adoption of glyphosate-resistant crops. Experiments were conducted to determine (i) the variability in tolerance to glyphosate among accessions, (ii) if there is any correlation between metabolism of glyphosate to aminomethylphosponic acid (AMPA) or sarcosine and the level of tolerance, and (iii) the involvement of differential translocation in tolerance to glyphosate. Fourteen I. lacunosa accessions had GR50 values ranging from 58 to 151 grams of acid equivalent per hectare (ae/ha) glyphosate, a 2.6-fold variability in tolerance to glyphosate. There was no evidence of the most tolerant (MT) accession metabolizing glyphosate to AMPA more rapidly than the least tolerant (LT) accession. Metabolism to sarcosine was not found. (14)C-glyphosate absorption was similar in the two accessions. LT accession translocated more (14)C-glyphosate than MT accession at 24 and 48 h after treatment. Differential translocation partly explains glyphosate tolerance in MT accession.

Characterization of a class II 5-enopyruvylshikimate-3-phosphate synthase with high tolerance to glyphosate from Sinorhizobium fredii.

5-Enopyruvylshikimate-3-phosphate synthase (EPSP synthase) is an important enzyme in the shikimate pathway mediating the biosynthesis of aromatic compounds in plants and microorganisms. A novel class II EPSP synthase AroA S. fredii from Sinorhizobium fredii NGR234 was overexpressed in Escherichia coli BL21. It was purified to homogeneity and its catalytic properties were studied. The enzyme exhibited optimum catalytic activity at pH 8.0 and 50 °C. It was stable below 40 °C, and over a broad range of pH 5.0-9.0. The EPSP synthase was increasingly activated by 100 mM of the chlorides of NH4 (+), K(+), Na(+) and Li(+). Kinetic analysis of AroA S. fredii suggested that the enzyme exhibited a high glyphosate tolerance and high level of affinity for phosphoenolpyruvate, which indicates the enzyme with a high potential for structural and functional studies and its potential usage for the generation of transgenic crops resistant to the herbicide.

Characterization and site-directed mutagenesis of a novel class II 5-enopyruvylshikimate-3-phosphate (EPSP) synthase from the deep-sea bacterium Alcanivorax sp. L27.

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroAA.sp. A phylogenetic analysis revealed that AroAA.sp (1317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The KM for PEP and IC50 [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroAA.sp were 78 µM and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC50 [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type AroAA.sp. The specific activity of the wild-type AroAA.sp, the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity.

A red and far-red light receptor mutation confers resistance to the herbicide glyphosate.

Glyphosate is a widely applied broad-spectrum systemic herbicide that inhibits competitively the penultimate enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) from the shikimate pathway, thereby causing deleterious effects. A glyphosate-resistant Arabidopsis mutant (gre1) was isolated and genetic analyses indicated that a dysfunctional red (R) and far-red (FR) light receptor, phytochrome B (phyB), caused this phenotype. This finding is consistent with increased glyphosate sensitivity and glyphosate-induced shikimate accumulation in low R:FR light, and the induction of genes encoding enzymes of the shikimate pathway in high R:FR light. Expression of the shikimate pathway genes exhibited diurnal oscillation and this oscillation was altered in the phyB mutant. Furthermore, transcript analysis suggested that this diurnal oscillation was not only dependent on phyB but was also due to circadian regulatory mechanisms. Our data offer an explanation of the well documented observation that glyphosate treatment at various times throughout the day, with their specific composition of light quality and intensity, results in different efficiencies of the herbicide.

EPSPS amplification in glyphosate-resistant spiny amaranth (Amaranthus spinosus): a case of gene transfer via interspecific hybridization from glyphosate-resistant Palmer amaranth (Amaranthus palmeri).

BACKGROUND: Amaranthus spinosus, a common weed of pastures, is a close relative of Amaranthus palmeri, a problematic agricultural weed with widespread glyphosate resistance. These two species have been known to hybridize, allowing for transfer of glyphosate resistance. Glyphosate-resistant A. spinosus was recently suspected in a cotton field in Mississippi. RESULTS: Glyphosate-resistant A. spinosus biotypes exhibited a fivefold increase in resistance compared with a glyphosate-susceptible biotype. EPSPS was amplified 33-37 times and expressed 37 times more in glyphosate-resistant A. spinosus biotypes than in a susceptible biotype. The EPSPS sequence in resistant A. spinosus plants was identical to the EPSPS in glyphosate-resistant A. palmeri, but differed at 29 nucleotides from the EPSPS in susceptible A. spinosus plants. PCR analysis revealed similarities between the glyphosate-resistant A. palmeri amplicon and glyphosate-resistant A. spinosus. CONCLUSIONS: Glyphosate resistance in A. spinosus is caused by amplification of the EPSPS gene. Evidence suggests that part of the EPSPS amplicon from resistant A. palmeri is present in glyphosate-resistant A. spinosus. This is likely due to a hybridization event between A. spinosus and glyphosate-resistant A. palmeri somewhere in the lineage of the glyphosate-resistant A. spinosus plants. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

In-silico analysis and expression profiling implicate diverse role of EPSPS family genes in regulating developmental and metabolic processes.

BACKGROUND: The EPSPS, EC 2.5.1.19 (5-enolpyruvylshikimate -3-phosphate synthase) is considered as one of the crucial enzyme in the shikimate pathway for the biosynthesis of essential aromatic amino acids and secondary metabolites in plants, fungi along with microorganisms. It is also proved as a specific target of broad spectrum herbicide glyphosate. RESULTS: On the basis of structure analysis, this EPSPS gene family comprises the presence of EPSPS I domain, which is highly conserved among different plant species. Here, we followed an in-silico approach to identify and characterize the EPSPS genes from different plant species. On the basis of their phylogeny and sequence conservation, we divided them in to two groups. Moreover, the interacting partners and co-expression data of the gene revealed the importance of this gene family in maintaining cellular and metabolic functions in the cell. The present study also highlighted the highest accumulation of EPSPS transcript in mature leaves followed by young leaves, shoot and roots of tobacco. In order to gain the more knowledge about gene family, we searched for the previously reported motifs and studied its structural importance on the basis of homology modelling. CONCLUSIONS: The results presented here is a first detailed in-silico study to explore the role of EPSPS gene in forefront of different plant species. The results revealed a great deal for the diversification and conservation of EPSPS gene family across different plant species. Moreover, some of the EPSPS from different plant species may have a common evolutionary origin and may contain same conserved motifs with related and important molecular function. Most importantly, overall analysis of EPSPS gene elucidated its pivotal role in immense function within the plant, both in regulating plant growth as well its development throughout the life cycle of plant. Since EPSPS is a direct target of herbicide glyphosate, understanding its mechanism for regulating developmental and cellular processes in different plant species would be a great revolution for developing glyphosate resistant crops.

Circular permutation of E. coli EPSP synthase: increased inhibitor resistance, improved catalytic activity, and an indicator for protein fragment complementation.

We performed the first circular permutation analysis for E. coli 5-enolpyruvylshikimate-3-phosphate synthase, and identified one circular permutant with notably increased resistance to its specific inhibitor and several others with moderately improved catalytic activity. Valid circular permutation sites can be used as effective split sites of protein fragment complementation.

Genome-wide transcriptional responses of Escherichia coli to glyphosate, a potent inhibitor of the shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase.

The shikimate pathway enzymes offer attractive targets for the development of antimetabolites. Glyphosate is an effective antimetabolite that inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase in the shikimate pathway, thereby resulting in a shortage of the chorismate-derived essential aromatic amino acids. However, little is known about the genome-wide transcriptional responses of bacteria to glyphosate shock. In the current study, a transcriptome analysis of Escherichia coli (E. coli) exposed to glyphosate identified the differential expression of 1040 genes, which represent 23.2% of the genome. The differentially expressed genes are primarily involved in amino acid metabolism, cell motility, and central carbon metabolism, indicating that the impact of glyphosate on the shikimate pathway also extends to other metabolic pathways. Expectedly, almost all genes encoding the proteins for the shikimate and specific aromatic amino acid pathways were downregulated after the addition of glyphosate. Furthermore, the expression of many energy- and metabolism-related genes was repressed. In contrast, glyphosate treatment induced the coordinated upregulation of at least 50 genes related to cell motility and chemotaxis. The reverse transcription-quantitative real-time PCR (RT-qPCR) data showed that the expression profiles of selected genes from the referred pathways were found to be consistent with the microarray data. The results suggest that the presence of glyphosate during growth induces metabolic starvation, an energy drain and other non-target effects.

Transition state analysis of enolpyruvylshikimate 3-phosphate (EPSP) synthase (AroA)-catalyzed EPSP hydrolysis.

Proton transfer to carbon atoms is a significant catalytic challenge because of the large intrinsic energetic barrier and the frequently unfavorable thermodynamics. The main catalytic challenge for enolpyruvylshikimate 3-phosphate synthase (EPSP synthase, AroA) is protonating the methylene carbon atom of phosphoenolpyruvate, or EPSP, in the reverse reaction. We performed transition state analysis using kinetic isotope effects (KIEs) on AroA-catalyzed EPSP hydrolysis, which also begins with a methylene carbon (C3) protonation, as an analog of AroA's reverse reaction. As part of this analysis, an inorganic phosphate scavenging system was developed to remove phosphate which, though present in microscopic amounts in solution, is ubiquitous. The reaction was stepwise, with irreversible C3 protonation to form an EPSP cation intermediate; that is, an AH(‡)*AN mechanism. The large experimental 3-(14)C KIE, 1.032 ± 0.005, indicated strong coupling of C3 with the motion of the transferring proton. Calculated 3-(14)C KIEs for computational transition state models revealed that the transition state occurs early during C3-H(+) bond formation, with a C3-H(+) bond order of ≈0.24. The observed solvent deuterium KIE, 0.97 ± 0.04, was the lowest observed to date for this type of reaction, but consistent with a very early transition state. The large 2-(14)C KIE reflected an "electrostatic sandwich" formed by Asp313 and Glu341 to stabilize the positive charge at C2. In shifting the transition state earlier than the acid-catalyzed reaction, AroA effected a large Hammond shift, indicating that a significant part of AroA's catalytic strategy is to stabilize the positive charge in the EPSP cation. A computational model containing all the charged amino acid residues in the AroA active site close to the reactive center showed a similar Hammond shift relative to the small transition state models.

The combination of quantitative PCR and western blot detecting CP4-EPSPS component in Roundup Ready soy plant tissues and commercial soy-related foodstuffs.

UNLABELLED: With the widespread use of Roundup Ready soy (event 40-3-2) (RRS), the comprehensive detection of genetically modified component in foodstuffs is of significant interest, but few protein-based approaches have been found useful in processed foods. In this report, the combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different RRS plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing both meat and plant protein concentrates. The validity of the 2 methods was confirmed first. We also showed that the CP4-EPSPS protein existed in different RRS plant tissues. In certain cases, the results from the western blot and the qPCR were not consistent. To be specific, at least 2 degraded fragments of CP4-EPSPS protein (35.5 and 24.6 kDa) were observed. For dried bean curd crust and deep-fried bean curd, a degraded protein fragment with the size of 24.6 kDa appeared, while cp4-epsps gene could not be traced by qPCR. In contrast, we found a signal of cp4-epsps DNA in 3 foodstuffs, including soy-containing ham cutlet product, meat ball, and sausage by qPCR, while CP4-EPSPS protein could not be detected by western blot in such samples. Our study therefore concluded that the combination of DNA- and protein-based methods would compensate each other, thus resulting in a more comprehensive detection from nucleic acid and protein levels. PRACTICAL APPLICATION: The combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different Roundup Ready soy (event 40-3-2) plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing a combination of both meat and plant protein concentrates. This study indicated that the combination of DNA- and protein-based methods would supplement each other for genetically modified detection from nucleic acid and protein levels. Accordingly, qPCR and western blot could be used in CP4-EPSPS detection in a wide variety of soy-related foodstuffs.

Molecular basis of glyphosate resistance-different approaches through protein engineering.

Glyphosate (N-phosphonomethyl-glycine) is the most widely used herbicide in the world: glyphosate-based formulations exhibit broad-spectrum herbicidal activity with minimal human and environmental toxicity. The extraordinary success of this simple, small molecule is mainly attributable to the high specificity of glyphosate for the plant enzyme enolpyruvyl shikimate-3-phosphate synthase in the shikimate pathway, leading to the biosynthesis of aromatic amino acids. Starting in 1996, transgenic glyphosate-resistant plants were introduced, thus allowing application of the herbicide to the crop (post-emergence) to remove emerged weeds without crop damage. This review focuses on mechanisms of resistance to glyphosate as obtained through natural diversity, the gene-shuffling approach to molecular evolution, and a rational, structure-based approach to protein engineering. In addition, we offer a rationale for the means by which the modifications made have had their intended effect.

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.