Focus on Weed Control: Tandem Amplification of a Chromosomal Segment Harboring 5-Enolpyruvylshikimate-3-Phosphate Synthase Locus Confers Glyphosate Resistance in Kochia scoparia

Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations.Glyphosate [N-(phosphonomethyl) Gly] is the most widely used agricultural pesticide globally (Duke and Powles, 2008). Originally, being a nonselective herbicide, its use was limited to vegetation management in noncrop areas; however, introduction of glyphosate-resistant (GR) crops in the late 1990s, coupled with their exceptional adoption, led to accelerated use totaling approximately 128 million ha worldwide in 2012 (James, 2012). GR crop technology has made a significant contribution to global agriculture and the environment, as it not only increased farm income by $32.2 billion (Brookes and Barfoot, 2013), but also moderated the negative environmental impacts of mechanical weed management practices (Gardner and Nelson, 2008; Bonny, 2011). Glyphosate offers a simple, effective, and economic weed management option in GR crops. In addition, it provides immense value in no-till crop production systems by enabling soil and moisture conservation. However, due to intensive glyphosate selection pressure, several weed populations globally have evolved resistance through a variety of mechanisms. Globally, herbicide resistance, in particular the recent proliferation of glyphosate resistance in weed species, is a major crop protection threat; nearly two dozen GR weed species have been reported in the last 15 years (Heap, 2014).Glyphosate, an aminophosphonic analog of the natural amino acid Gly, nonselectively inhibits 5-Enolpyruvylshikimate-3-Phosphate synthase (EPSPS) in plants, preventing the biosynthesis of the aromatic amino acids Phe, Tyr, and Trp (Steinrücken and Amrhein, 1980), resulting in the death of glyphosate-sensitive individuals. In plants, EPSPS is one of the key enzymes in the shikimate pathway (Herrmann and Weaver, 1999), and glyphosate inhibits EPSPS by binding to EPSPS-shikimate-3-P binary complex forming an EPSPS-shikimate-3-P-glyphosate complex (Alibhai and Stallings, 2001). Bradshaw et al. (1997) hypothesized against the likelihood of weeds evolving resistance to glyphosate, primarily because of its complex biochemical interactions in the shikimate pathway and also due to the absence of known glyphosate metabolism in plants. Nonetheless, several cases of glyphosate resistance, as a result of difference in glyphosate translocation (Preston and Wakelin, 2008) or mutations in the EPSPS, were confirmed (Baerson et al., 2002). More importantly, duplication/amplification of the EPSPS appears to be the basis for glyphosate resistance in several weeds (Sammons and Gaines, 2014). Here, we use duplication to refer to the formation of first repetition of a chromosomal segment and amplification to refer to increase in number of the repetitions (more than two repetitions of a chromosomal segment) under positive selection. The first case of EPSPS amplification as a basis for glyphosate resistance was reported in an Amaranthus palmeri population from GA (Gaines et al., 2010). In this A. palmeri population, there is a massive increase (>100-fold relative to glyphosate-susceptible [GS] plants) in EPSPS copies, and these copies are dispersed throughout the genome (Gaines et al., 2010).Field-evolved GR Kochia scoparia populations were first reported in western Kansas in 2007 (Heap, 2014). We previously determined that evolution of GR populations of K. scoparia in the U.S. Great Plains is also due to amplification of the EPSPS (A. Wiersma and P. Westra, unpublished data). Unlike in GR A. palmeri, we found relative EPSPS:acetolactate synthase (ALS) copies ranging from three to nine in GR K. scoparia populations. While it quickly became widespread in the region, its presence was reported in another five Great Plains states by 2013 (Heap, 2014). GR K. scoparia populations we tested were 3- to 11-times resistant (population level) to glyphosate compared with a GS population (Godar, 2014), and EPSPS expression positively correlated with genomic EPSPS copy number (A. Wiersma and P. Westra, unpublished data). Here, we reveal the genomic organization of the amplified EPSPS copies in two GR K. scoparia populations, an alternative mechanism of gene amplification to that reported in GR A. palmeri.     

Involvement of facultative apomixis in inheritance of EPSPS gene amplification in glyphosate-resistant Amaranthus palmeri

The inheritance of glyphosate resistance in two Amaranthus palmeri populations (R1 and R2) was examined in reciprocal crosses (RC) and second reciprocal crosses (2RC) between glyphosate-resistant (R) and -susceptible (S) parents of this dioecious species. R populations and Female-R × Male-S crosses contain higher 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene copy numbers than the S population. EPSPS expression, EPSPS enzyme activity, EPSPS protein quantity, and level of resistance to glyphosate correlated positively with genomic EPSPS relative copy number. Transfer of resistance was more influenced by the female than the male parent in spite of the fact that the multiple copies of EPSPS are amplified in the nuclear genome. This led us to hypothesize that this perplexing pattern of inheritance may result from apomictic seed production in A. palmeri. We confirmed that reproductively isolated R and S female plants produced seeds, indicating that A. palmeri can produce seeds both sexually and apomictically (facultative apomixis). This apomictic trait accounts for the low copy number inheritance in the Female-S × Male-R offsprings. Apomixis may also enhance the stability of the glyphosate resistance trait in the R populations in the absence of reproductive partners.     

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.     

No fitness cost of glyphosate resistance endowed by massive <Emphasis Type="Italic">EPSPS</Emphasis> gene amplification in <Emphasis Type="Italic">Amaranthus palmeri</Emphasis>

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.     

Evolution of a Double Amino Acid Substitution in the 5-Enolpyruvylshikimate-3-Phosphate Synthase in Eleusine indica Conferring High-Level Glyphosate Resistance

Glyphosate is the most important and widely used herbicide in world agriculture. Intensive glyphosate selection has resulted in the widespread evolution of glyphosate-resistant weed populations, threatening the sustainability of this valuable once-in-a-century agrochemical. Field-evolved glyphosate resistance due to known resistance mechanisms is generally low to modest. Here, working with a highly glyphosate-resistant Eleusine indica population, we identified a double amino acid substitution (T102I + P106S [TIPS]) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant individuals. This TIPS mutation recreates the biotechnology-engineered commercial first generation glyphosate-tolerant EPSPS in corn (Zea mays) and now in other crops. In E. indica, the naturally evolved TIPS mutants are highly (more than 180-fold) resistant to glyphosate compared with the wild type and more resistant (more than 32-fold) than the previously known P106S mutants. The E. indica TIPS EPSPS showed very high-level (2,647-fold) in vitro resistance to glyphosate relative to the wild type and is more resistant (600-fold) than the P106S variant. The evolution of the TIPS mutation in crop fields under glyphosate selection is likely a sequential event, with the P106S mutation being selected first and fixed, followed by the T102I mutation to create the highly resistant TIPS EPSPS. The sequential evolution of the TIPS mutation endowing high-level glyphosate resistance is an important mechanism by which plants adapt to intense herbicide selection and a dramatic example of evolution in action.Modern herbicides make major contributions to global food production by easily removing weeds while maintaining sustainable soil conservation practices. However, persistent herbicide selection of huge weed numbers across vast areas has resulted in the widespread evolution of herbicide-resistant weed populations. Worldwide, there are currently more than 449 unique cases of herbicide resistance, with about 11 new cases reported annually, on average (Heap, 2015). Target site resistance due to target gene mutation is one of the major mechanisms enabling resistance evolution (Gressel, 2002; Powles and Yu, 2010).The most important and globally used herbicide in crop fields is glyphosate (Duke and Powles, 2008). Glyphosate disrupts the shikimate pathway by specifically inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; Steinrücken and Amrhein, 1980). Glyphosate resistance was initially considered to be unlikely to evolve in nature based on the facts that intentional selection for glyphosate tolerance using whole plants and cell/tissue culture was unsuccessful, and laboratory-generated highly resistant EPSPS mutants displayed undesirable enzyme kinetics (Bradshaw et al., 1997; for review, see Pline-Srnic, 2006). This seemed to be true, as resistance was not found during the first 15 years of glyphosate use, primarily as a nonselective herbicide. However, unprecedented intensive glyphosate use for controlling large numbers of weeds over massive areas, especially after the introduction of glyphosate-resistant transgenic crops, imposed high selection pressure on weeds, resulting in the evolution of glyphosate resistance in populations of 32 weed species (Heap, 2015). Since the first identification of a resistance-endowing EPSPS point mutation, P106S, in a glyphosate-resistant Eleusine indica population (Baerson et al., 2002), several other resistance-endowing single-amino acid substitutions at P106 (P106T, P106A, and P106L) have been reported in glyphosate-resistant weeds (e.g. Ng et al., 2004; Yu et al., 2007; Kaundun et al., 2011; for review, see Sammons and Gaines, 2014). These single-codon EPSPS resistance mutations only endow low-level glyphosate resistance (2- to 3-fold the recommended rates). This is not surprising, because glyphosate is a competitive inhibitor of the second substrate, phosphoenolpyruvate (PEP; Boocock and Coggins, 1983), and is considered a transition state mimic of the catalyzed reaction course (Schönbrunn et al., 2001). Indeed, highly glyphosate-resistant EPSPS variants (e.g. mutants at G101 or T102) have greatly increased K_m values (decreased affinity) for PEP when expressed in Escherichia coli (Eschenburg et al., 2002; Funke et al., 2009; for review, see Sammons and Gaines, 2014). In contrast, P106 substitutions confer weak glyphosate resistance but preserve adequate EPSPS functionality (Healy-Fried et al., 2007; for review, see Sammons and Gaines, 2014). Aside from P106 EPSPS gene mutations, there are other glyphosate resistance mechanisms, including EPSPS gene amplification and nontarget-site reduced glyphosate translocation/nontarget-site increased vacuole sequestration (Lorraine-Colwill et al., 2002; Gaines et al., 2010; Ge et al., 2010; for review, see Powles and Preston, 2006; Shaner, 2009; Powles and Yu, 2010; Sammons and Gaines, 2014). Generally, each of these mechanisms endows moderate-level (4- to 8-fold the recommended rates) glyphosate resistance.Low-level glyphosate resistance due to the EPSPS P106 mutations was reported in Malaysian E. indica (Baerson et al., 2002; Ng et al., 2004). Recently, we reported a highly (more than 10-fold the recommended rates) glyphosate-resistant Malaysian E. indica population (Jalaludin et al., 2015). This paper investigates the high-level glyphosate resistance in this population, and is, to our knowledge, the first to reveal the sequential evolution of a double amino acid substitution in EPSPS.     

Transformation and segregation of GFP fluorescence and glyphosate resistance in horseweed (<Emphasis Type="Italic">Conyza canadensis</Emphasis>) hybrids

The goal of this research was to generate a breeding population of horseweed segregating for glyphosate resistance. In order to generate a marker to select between hybrids of glyphosate resistant (GR) and glyphosate susceptible (GS) horseweed, a GR horseweed accession from western Tennessee was transformed with a green fluorescent protein (GFP) transgene. The GFP marker allowed for the simple and accurate determination of GR hybrid plants by visual observation. GR plants were shown to be transgenic via the green fluorescence under UV light, and resistant to glyphosate when sprayed with the field-use-rate 0.84 kg acid equivalent ha⁻¹ of glyphosate (i.e. Roundup^TM) herbicide. An in vitro screen for glyphosate resistance in seedlings was developed, and a 5 μM glyphosate concentration was found to reduce dry weight in GS seedlings but not in GR seedlings. The GR plants containing GFP were then hand-crossed with GS plants from eastern Tennessee under greenhouse conditions, with GS plants acting as the pollen acceptor. Resulting seed was collected and germinated for GFP fluorescence screening. Seedlings that exhibited the transgenic GFP phenotype were selected as F₁ hybrids between GR and GS horseweed. Thirty GS×GR hybrids were produced on the basis of a green-fluorescent GFP phenotype of GR plants. GS×GFP/GR F₁ hybrids produced F₂ seeds, and F₂ plants were shown to segregate for GFP fluorescence and glyphosate resistance independently. Both traits segregated at a Mendelian 3:1 ratio, indicating a single gene is responsible for each phenotype.     

Assessment of gene flow from glyphosate-resistant transgenic soybean to conventional soybean in China

Glyphosate-resistant (GR) transgenic soybean has never been cultivated commercially in China. It is essential to develop the separation measures required to prevent out-crossing between GR and conventional soybean (Glycine max (L.) Merr.) by characterizing the transgene flow before GR soybean is released. In this study, the transgene flow from a GR transgenic soybean AG5601 to conventional soybeans was characterized. First, natural out-crossing rate was evaluated using 36 conventional soybean varieties interplanted with GR soybean AG5601 transformed with a cp4 EPSPS gene conferring the resistance to herbicide glyphosate in the field in 2007 and 2008 in China. Second, drift distance of cp4 EPSPS gene from GR soybean AG5601 to soybean cv. Zhonghuang13 was evaluated using the progenies harvested from eight directions at different distance. Third, the relationship of gene flow of GR soybean AG5601 with flowering synchronization days or insect pollinators of each variety was analyzed using regression analysis. Thirty-two of 36 tested conventional soybean varieties had surviving progenies after two times of sprays of glyphosate, and 49 of 41,679 progenies were verified to be glyphosate-tolerant heterozygous offspring. The out-crossing rates in positive varieties (having surviving offspring after two times of sprays of glyphosate) ranged from 0.039 to 0.934 %. The farthest distance (drift distance) between soybean AG5601 and cv. Zhonghuang13 at which out-pollinating was still able to be observed was 15 m, with an out-crossing rate of 0.012 %. Regression analysis showed that there was a positive relationship between cross-pollination frequency and flowering synchronization days or pollinator insects. Therefore, when GR soybean is released to the field, it should be critically separated with the conventional soybean in space and cultivation time with efficient insect control during flowering.     

Investigating the mechanisms of glyphosate resistance in <Emphasis Type="Italic">Lolium multiflorum</Emphasis>

Evolved resistance to the herbicide glyphosate has been reported in eleven weed species, including Lolium multiflorum. Two glyphosate-resistant L. multiflorum populations were collected, one from Chile (SF) and one from Oregon, USA (OR), and the mechanisms conferring glyphosate resistance were studied. Based on a Petri dish dose–response bioassay, the OR and the SF populations were two and fivefold more resistant to glyphosate when compared to the susceptible (S) population, respectively; however, based on a whole-plant dose–response bioassay, both OR and SF populations were fivefold more resistant to glyphosate than the S population, implying that different resistance mechanisms might be involved. The S population accumulated two and three times more shikimic acid in leaf tissue 96 h after glyphosate application than the resistant OR and SF populations, respectively. There were no differences between the S and the glyphosate-resistant OR and SF populations in ¹⁴C-glyphosate leaf uptake; however, the patterns of ¹⁴C-glyphosate translocation were significantly different. In the OR population, a greater percentage of ¹⁴C-glyphosate absorbed by the plant moved distal to the treated section and accumulated in the tip of the treated leaf. In contrast, in the S and in the SF populations, a greater percentage of ¹⁴C-glyphosate moved to non-treated leaves and the stem. cDNA sequence analysis of the EPSP synthase gene indicated that the glyphosate-resistant SF population has a proline 106 to serine amino acid substitution. Here, we report that glyphosate resistance in L. multiflorum is conferred by two different mechanisms, limited translocation (nontarget site-based) and mutation of the EPSP synthase gene (target site-based).     

Co‐expression of GR79 EPSPS and GAT yields herbicide‐resistant cotton with low glyphosate residues

Glyphosate‐resistant (GR) crops have been adopted on a massive scale by North and South American farmers. Currently, about 80% of the 120 million hectares of the global genetically modified (GM) crops are GR crop varieties. However, the adoption of GR plants in China has not occurred at the same pace, owing to several factors including, among other things, labour markets and the residual effects of glyphosate in transgenic plants. Here, we report the co‐expression of codon‐optimized forms of GR79 EPSPS and N‐acetyltransferase (GAT) genes in cotton. We found five times more resistance to glyphosate with 10‐fold reduction in glyphosate residues in two pGR79 EPSPS‐pGAT co‐expression cotton lines, GGCO2 and GGCO5. The GGCO2 line was used in a hybridization programme to develop new GR cottons. Field trials at five locations during three growing seasons showed that pGR79‐pGAT transgenic cotton lines have the same agronomic performance as conventional varieties, but were USD 390‐495 cheaper to produce per hectare because of the high cost of conventional weed management practices. Our strategy to pyramid these genes clearly worked and thus offers attractive promise for the engineering and breeding of highly resistant low‐glyphosate‐residue cotton varieties.     

Glyphosate reduces shoot concentrations of mineral nutrients in glyphosate-resistant soybeans

Although glyphosate-resistant (GR) technology is used in most countries producing soybeans (Glycine max L.), there are no particular fertilize recommendations for use of this technology, and not much has been reported on the influence of glyphosate on GR soybean nutrient status. An evaluation of different cultivar maturity groups on different soil types, revealed a significant decrease in macro and micronutrients in leaf tissues, and in photosynthetic parameters (chlorophyll, photosynthetic rate, transpiration and stomatal conductance) with glyphosate use (single or sequential application). Irrespective of glyphosate applications, concentrations of shoot macro- and micronutrients were found lower in the near-isogenic GR-cultivars compared to their respective non-GR parental lines Shoot and root dry biomass were reduced by glyphosate with all GR cultivars evaluated in both soils. The lower biomass in GR soybeans compared to their isogenic normal lines probably represents additive effects from the decreased photosynthetic parameters as well as lower availability of nutrients in tissues of the glyphosate treated plants.     

Effects of glyphosate on the movement of assimilates of two Lolium perenne L. populations with differential herbicide sensitivity

Glyphosate applications trigger the depletion of aromatic amino acid pools and the decrease of photosynthesis that results in changes in carbon metabolism. The aim of this work was to determine the effect of glyphosate on the export of ¹⁴C from ¹⁴C-glucose to the main sinks, by comparing a glyphosate-resistant Lolium perenne population with a susceptible one. Untreated plants of the two populations grown in hydroponics were labeled with ¹⁴C-glucose applied at the youngest expanded leaf at the tillering stage. Similar ¹⁴C-glucose absorption and ¹⁴C distribution patterns were recorded in both populations. In another experiment, half of the plants of each population were treated with glyphosate, whereas the other half was sprayed with water (controls). Glucose absorption did not vary under glyphosate treatment, regardless of the sensitivity of each population to the herbicide. However, the translocation of ¹⁴C and its distribution patterns were significantly affected by glyphosate within 1 day in the susceptible population. The treated susceptible plants showed 57% higher ¹⁴C retention at the labeled area than their controls. The lower ¹⁴C movement significantly affected the unexpanded leaves and the apical meristem on the labeled tiller. Moreover, the ¹⁴C released from roots was significantly decreased by glyphosate only in the susceptible plants. Glyphosate did not influence leaf absorption, translocation, or release of ¹⁴C-labeled glucose plus radiolabeled metabolites in the resistant population.     

Glyphosate tolerance by Clitoria ternatea and Neonotonia wightii plants involves differential absorption and translocation of the herbicide

Glyphosate tolerance by Clitoria ternatea, Neonotonia wightii and Amaranthus hybridus was studied in whole plants from Mexico. Experiments in a controlled growth chamber showed both legumes to be highly tolerant of glyphosate, with and ED₅₀ values of 600.18 g ae ha^?C1 for C. ternatea and 362.94 g ae ha^?C1 for N. wightii. On the other hand, A. hybridus was highly susceptible to the herbicide (ED₅₀?=?42.22 g ae ha^?C1). Shikimate accumulation peaked 96 h after treatment in the tolerant plants and the susceptible weed under 500 g ae ha^?C1 glyphosate. The shikimic acid content of whole leaves was 4.0 and 5.0 times higher in the susceptible weed than in N. wightii and C. ternatea, respectively. ¹⁴C-glyphosate absorption and translocation tests showed A. hybridus to absorb 30% more herbicide than the legumes 24 h after glyphosate foliar application. ¹⁴C-glyphosate translocation as measured by quantified autoradiography revealed increased translocation of the herbicide to untreated leaves and roots in A. hybridus relative to the two legumes. The cuticular surface of A. hybridus exhibited very low wax coverage relative to the epicuticular surface of N. wightii and, especially, C. ternatea. No significant degradation of glyphosate to aminomethylphosphonic acid and glyoxylate metabolites was detected among the tolerant leguminous plants or the susceptible weed population. These results indicate that the high glyphosate tolerance of Clitoria ternatea and Neonotonia wightii is mainly a result of poor penetration and translocation of the herbicide to apical growing points in their plants.     

Mutation by DNA shuffling of 5‐enolpyruvylshikimate‐3‐phosphate synthase from Malus domestica for improved glyphosate resistance

A new 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate‐tolerant plants. However, wild‐type MdEPSPS is not suitable for the development of transgenic glyphosate‐tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA shuffling on MdEPSPS, and one highly glyphosate‐resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site‐directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single‐site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate‐resistant crops.     

Expression of a bacterial <Emphasis Type="Italic">aroA</Emphasis> mutant, <Emphasis Type="Italic">aroA-M1</Emphasis>, encoding 5-enolpyruvylshikimate-3-phosphate synthase for the production of glyphosate-resistant tobacco plants

Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants. We have previously reported a strategy for engineering glyphosate-resistant class I EPSPS based on staggered-PCR technology. Selected mutant enzymes exhibited high K_i[glyphosate] and low K_m[PEP] values compared to the parental enzymes from Escherichia coli (EcaroA) and Salmonella typhimurium (StaroA). One mutant, aroA-M1, was further engineered with a tobacco chloroplast leader sequence, and then placed in the binary vector pCAMBIA1300 for Agrobacterium-mediated gene transfer to tobacco (Nicotiana tabacum cv. Xanthi). Transgenic plants with increased resistance to glyphosate were generated.     

Occurrence of an herbicide‐resistant plant trait in agricultural field margins

Agricultural environments allow study of evolutionary change in plants. An example of evolution within agroecological systems is the selection for resistance to the herbicide glyphosate within the weed, Conyza canadensis. Changes in survivorship and reproduction associated with the development of glyphosate resistance (GR) may impact fitness and influence the frequency of occurrence of the GR trait. We hypothesized that site characteristics and history would affect the occurrence of GR C. canadensis in field margins. We surveyed GR occurrence in field margins and asked whether there were correlations between GR occurrence and location, crop rotation, GR crop trait rotation, crop type, use of tillage, and the diversity of herbicides used. In a field experiment, we hypothesized that there would be no difference in fitness between GR and glyphosate‐susceptible (GS) plants. We asked whether there were differences in survivorship, phenology, reproduction, and herbivory between 2 GR and 2 GS populations of C. canadensis in agrestal and ruderal habitats. We found that geographic location was an important factor in the occurrence of GR C. canadensis in field margins. Although not consistently associated with either glyphosate resistance or glyphosate susceptibility, there were differences in phenology, survivorship, and herbivory among biotypes of C. canadensis. We found equal or greater fitness in GR biotypes, compared to GS biotypes, and GR plants were present in field margins. Field margins or ruderal habitats may provide refugia for GR C. canadensis, allowing reproduction and further selection to occur as seeds recolonize the agrestal habitat. Agricultural practices may select for ecological changes that feed back into the evolution of plants in ruderal habitats.     

Expression of glyphosate resistance in carrot somatic hybrid cells through the transfer of an amplified 5-enolpyruvylshikimic acid-3-phosphate synthase gene

Summary A Daucus carota cell line selected as resistant to N-(phosphonomethyl)-glycine (glyphosate) was found to have increased levels of 5-enolpyruvylshikimic acid-3-phosphate synthase (EPSPS) activity of 5.5 times over wild-type carrot and an EPSPS protein level increase of 8.7 times as confirmed by Western hybridization analysis. Southern blot hybridization using a petunia EPSPS probe showed increases in the number of copies of EPSPS genes in the glyphosate-resistant line which correlated with the higher levels of the EPSPS enzyme. The mechanism of resistance to glyphosate is therefore due to amplification of the EPSPS gene. To examine the stability of the amplified genes, cloned lines selected as doubly resistant to Dl-5-methyltryptophan (5MT) and azetidine-2-carboxylate (A2C) were fused with the amplified EPSPS glyphosate-resistant cell line. Somatic hybrids expressed resistances to 5MT in a semidominant fashion while A2C and glyphosate resistance was expressed as dominant, or semi-dominant traits, in a line-specific manner. The hybrid lines possessed additive chromosome numbers of the parental lines used and no double minute chromosomes were observed. The glyphosate-resistant parental line and most somatic hybrids retained the amplified levels of EPSPS in the absence of selection pressure over a 3-year period.     

Morphological responses of different eucalypt clones submitted to glyphosate drift

This work aimed to evaluate the effects of simulated glyphosate drift on leaf growth and micromorphology of Eucalyptus spp. clones, using subdoses. A factorial scheme consisting of three clones, Eucalyptus urophylla, E. grandis and the hybrid E. urophylla × E. grandis (E. urograndis) and five sub-rates (0; 43.2; 86.4; 172.8 and 345.6 g e.a. ha⁻¹ of glyphosate) were used in a randomized block design, with four repetitions. The herbicide was applied on the plants so as not to reach the superior third, 23 days after seedling planting. At 7 and 15 days after application (DAA), the leaves collected from the first basal branch of the plants were processed according to the conventional methodology used for micromorphological studies. The effects of glyphosate drift were proportional to the rates tested, with E. urophylla being more tolerant to the herbicide than E. grandis and E. urograndis. Glyphosate symptoms were the same for the different clones tested, being characterized by wilting, chlorosis and leaf curling, and, at higher rates, by necrosis, foliar senescence and death of the eucalypt plants. Plants submitted to 172.8 and 345.6 g ha⁻¹ of glyphosate had severe injuries in the aerial part, affecting their development, resulting in reduced height, stem diameter and dry mass at 50 DAA. The micromorphological damages occurred prior to the appearance of visible symptoms, with erosion of the epicuticular waxes and fungal hypha infestation in plants exposed to glyphosate drift being observed in the three clones. No marked difference in leaf micromorphology was observed that could explain the differential tolerance among the three clones studied. The results show that further studies on wax and cuticle constitution of Eucalyptus spp. are needed for the elucidation of the mechanisms of differential tolerance of eucalypt species and clones to glyphosate.     

Inheritance of evolved glyphosate resistance in <Emphasis Type="Italic">Conyza canadensis</Emphasis> (L.) Cronq.

N-(phosphonomethyl)glycine (glyphosate) resistance was previously reported in a horseweed [Conyza (=Erigeron) canadensis (L.) Cronq.] population from Houston, DE (P ₀ ^R ). Recurrent selection was performed on P ₀ ^R , since the population was composed of susceptible (5%) and resistant (95%) phenotypes. After two cycles of selection at 2.0 kg ae glyphosate ha^–1, similar glyphosate rates that reduced plant growth by 50%, glyphosate rates that inflicted 50% mortality in the population, and accumulations of half of the maximum detectable shikimic acid concentration were observed between the parental P ₀ ^R and the first (RS₁) and second (RS₂) recurrent generations. In addition, RS₁ and RS₂ did not segregate for resistance to glyphosate. This suggested that the RS₂ population comprised a near-homozygous, glyphosate-resistant line. Whole-plant rate responses estimated a fourfold resistance increase to glyphosate between RS₂ and either a pristine Ames, IA (P ₀ ^P ) or a susceptible C. canadensis population from Georgetown, DE (P ₀ ^S ). The genetics of glyphosate resistance in C. canadensis was investigated by performing reciprocal crosses between RS₂ and either the P ₀ ^P or P ₀ ^S populations. Evaluations of the first (F₁) and second (F₂) filial generations suggested that glyphosate resistance was governed by an incompletely dominant, single-locus gene (R allele) located in the nuclear genome. The proposed genetic model was confirmed by back-crosses of the F₁ to plants that arose from achenes of the original RS₂, P ₀ ^P , or P ₀ ^S parents. The autogamous nature of C. canadensis, the simple inheritance model of glyphosate resistance, and the fact that heterozygous genotypes (F₁) survived glyphosate rates well above those recommended by the manufacturer, predicted a rapid increase in frequency of the R allele under continuous glyphosate selection. The impact of genetics on C. canadensis resistance management is discussed.