Discovery and Mode-of-Action Characterization of a New Class of Acetolactate Synthase-Inhibiting Herbicides.

Herbicides are effective tools to manage weeds and enable food production and sustainable agriculture. Corteva Agriscience R&D has recently discovered new diphenyl-ether compounds displaying excellent postemergent efficacy on important weed species along with corn safety. Here, we describe the chemistry, biology, biochemistry, and computational modeling research that led to the discovery and elucidation of the primary mode of action for these compounds. The target protein was found to be acetolactate synthase (ALS), a key enzyme in the biosynthesis of branched chain amino acids (valine, leucine, and isoleucine). While weed resistance evolution to ALS herbicides is widespread, the molecular interaction of the diphenyl-ether compounds at the active site of the ALS enzyme differs significantly from that of some commercial ALS inhibitors. The unique biochemical profile of these molecules along with their excellent herbicidal activity and corn selectivity make them a noteworthy development in the pursuit of novel, safe, and sustainable weed control solutions.

A New Class of Diaryl Ether Herbicides: Structure-Activity Relationship Studies Enabled by a Rapid Scaffold Hopping Approach.

We report on the development of a novel class of diaryl ether herbicides. After the discovery of a phenoxybenzoic acid with modest herbicidal activity, optimization led to several molecules with improved control of broadleaf and grass weeds. To facilitate this process, we first employed a three-step combinatorial approach, then pivoted to a one-step Ullmann-type coupling that provided faster access to new analogs. After determining that the primary target site of our benchmark diaryl ethers was acetolactate synthase (ALS), we further leveraged this copper-catalyzed methodology to conduct a scaffold hopping campaign in the hope of uncovering an additional mode of action with fewer documented cases of resistance. Our comprehensive and systematic investigation revealed that while the herbicidal activity of this area seems to be exclusively linked to ALS inhibition, our molecules represent a structurally distinct class of Group 2 herbicides. The structure-activity relationships that led us to this conclusion are described herein.

Biochemical Basis for the Time-of-Day Effect on Glufosinate Efficacy against Amaranthus palmeri.

Glufosinate, a glutamine synthetase (GS) inhibitor, often provides variable weed control depending on environmental conditions such as light, temperature and humidity at the time of application. Midday applications normally provide improved efficacy compared to applications at dawn or dusk. We investigated the biochemical basis for the time-of-day effect on glufosinate efficacy in Amaranthus palmeri. GS1/GS2 gene expression and GS1/GS2 protein abundance were assessed in different parts (young leaves, old leaves, and roots) of plants incubated in the dark compared to those in the light. The turnover of GS total activity was also evaluated overtime following glufosinate treatment at midday compared to dusk application. The results suggest that GS in A. palmeri is less expressed and less abundant in the dark compared to in the light. Midday application of glufosinate under intense light conditions following application provide full control of A. palmeri plants. Consequently, these plants are unable to recover GS activity by de novo protein synthesis. Full activity of GS is required for complete inhibition by the irreversible inhibitor glufosinate. Therefore, glufosinate applications should always be performed in the middle of the day when sunlight is intense, to prevent weed escapes from the herbicide treatment.

Monitoring Glyphosate- and Chlorimuron- resistant Conyza spp. Populations in Brazil.

Conyza species are important weeds in global agriculture, especially due to their capacity to evolve resistance to multiple herbicide mechanisms of action. We aimed to evaluate the frequency and distribution of resistance to glyphosate and chlorimuron-ethyl in Conyza spp. populations from Brazil. Seed samples were collected from grain production areas across nine Brazilian states over five consecutive years (2014 to 2018). Prior to resistance monitoring trials, dose-response assays were conducted to determine a single dose of glyphosate or chlorimuron-ethyl to discriminate resistant and susceptible populations. Resistance monitoring based on plant responses to the application of discriminatory doses of glyphosate (960 g ha-1) or chlorimuron-ethyl (20 g ha-1). Populations were classified as resistant, moderately resistant, or susceptible to either herbicide. While glyphosate resistance was highly frequent (71.2%) in all the five years, chlorimuron-ethyl resistant populations occurred at 39.8% of the total. The frequency of multiple resistance to both herbicides (35.3%) was proportional to the occurrence of chlorimuron-ethyl resistance (39.6%). Resistance to glyphosate and to chlorimuron-ethyl were found across all states evaluated.

Evolution of EPSPS double mutation imparting glyphosate resistance in wild poinsettia (Euphorbia heterophylla L.).

The evolution of glyphosate resistance (GR) in weeds is an increasing problem. Glyphosate has been used intensively on wild poinsettia (Euphorbia heterophylla L.) populations for at least 20 years in GR crops within South America. We investigated the GR mechanisms in a wild poinsettia population from a soybean field in southern Brazil. The GR population required higher glyphosate doses to achieve 50% control (LD50) and 50% dry mass reduction (MR50) compared to a glyphosate susceptible (GS) population. The ratio between the LD50 and MR50 of GR and GS resulted in resistance factors (RF) of 6.9-fold and 6.1-fold, respectively. Shikimate accumulated 6.7 times more in GS than in GR when leaf-discs were incubated with increasing glyphosate concentrations. No differences were found between GR and GS regarding non-target-site mechanisms. Neither population metabolized glyphosate to significant levels following treatment with 850 g ha-1 glyphosate. Similar levels of 14C-glyphosate uptake and translocation were observed between the two populations. No differences in EPSPS expression were found between GS and GR. Two target site mutations were found in all EPSPS alleles of homozygous resistant plants: Thr102Ile + Pro106Thr (TIPT-mutation). Heterozygous individuals harbored both alleles, wild-type and TIPT. Half of GR individuals were heterozygous, suggesting that resistance is still evolving in the population. A genotyping assay was developed based on the Pro106Thr mutation, demonstrating high efficiency to identify homozygous, heterozygous or wild-type EPSPS sequences across different plants. This is the first report of glyphosate-resistant wild-poinsettia harboring an EPSPS double mutation (TIPT) in the same plant.

Glufosinate-ammonium: a review of the current state of knowledge.

Glufosinate is a key herbicide to manage glyphosate-resistant weeds mainly because it is a broad-spectrum herbicide, and transgenic glufosinate-resistant crops are available. Although glufosinate use has increased exponentially over the past decade, the treated area with this herbicide is far less than that with glyphosate. This is because glufosinate often provides inconsistent performance in the field, which is attributed to several factors including environmental conditions, application technology, and weed species. Glufosinate is also highly hydrophilic and does not translocate well in plants, generally providing poor control of grasses and perennial species. In the soil, glufosinate is rapidly degraded by microorganisms, leaving no residual activity. While there have been concerns regarding glufosinate toxicology, its proper use can be considered safe. Glufosinate is a fast-acting herbicide that was first discovered as a natural product, and is the only herbicide presently targeting glutamine synthetase. The mode of action of glufosinate has been controversial, and the causes for the rapid phytotoxicity have often been attributed to ammonia accumulation. Recent studies indicate that the contact activity of glufosinate results from the accumulation of reactive oxygen species and subsequent lipid peroxidation. Glufosinate disrupts both photorespiration and the light reactions of photosynthesis, leading to photoreduction of molecular oxygen, which generates reactive oxygen species. The new understanding of the mode of action provided new ideas to improve the herbicidal activity of glufosinate. Finally, a very few weed species have evolved glufosinate resistance in the field, and the resistance mechanisms are generally not well understood requiring further investigation. © 2020 Society of Chemical Industry.

A novel insight into the mode of action of glufosinate: how reactive oxygen species are formed.

Glufosinate targets glutamine synthetase (GS), but its fast herbicidal action is triggered by reactive oxygen species (ROS). The relationship between GS inhibition and ROS accumulation was investigated in Amaranthus palmeri. Glufosinate's fast action is light-dependent with no visual symptoms or ROS formation in the dark. Inhibition of GS leads to accumulation of ammonia and metabolites of the photorespiration pathway, such as glycolate and glyoxylate, as well as depletion of other intermediates such as glycine, serine, hydroxypyruvate, and glycerate. Exogenous supply of glycolate to glufosinate-treated plants enhanced herbicidal activity and dramatically increased hydrogen peroxide accumulation (possibly from peroxisomal glycolate oxidase activity). Glufosinate affected the balance between ROS generation and scavenging. The activity of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase increased after glufosinate treatment in an attempt to quench the nascent ROS burst. Low doses of atrazine and dinoseb were used to investigate the sources of ROS by manipulating photosynthetic electron transport and oxygen (O2) evolution. ROS formation depended on electron flow and O2 evolution in photosystem II (PSII). Inhibition of GS disrupted photorespiration, carbon assimilation, and linear electron flow in the light reactions. Consequently, the antioxidant machinery and the water-water cycle are overwhelmed in the presence of light and glufosinate. The O2 generated by the splitting of water in PSII becomes the acceptor of electrons, generating ROS. The cascade of events leads to lipid peroxidation and forms the basis for the fast action of glufosinate.

Trp2027Cys mutation evolves in Digitaria insularis with cross-resistance to ACCase inhibitors.

Sourgrass (Digitaria insularis) is one of the most problematic weeds in South America because glyphosate resistance is widespread across most crop production regions. Acetyl coenzyme A carboxylase (ACCase)-inhibiting herbicides have been intensively used to manage D. insularis, which substantially increased selection pressure for this class of herbicides. We confirmed resistance to ACCase herbicides in a D. insularis population from Brazil and characterized its molecular basis. Resistant plants showed high level of resistance to haloxyfop (resistance factor, RF = 613-fold), low level of resistance to pinoxaden (RF = 3.6-fold), and no resistance to clethodim. A target-site mutation, Trp2027Cys, was found in the ACCase sequence from resistant plants. A protein homology model shows that the Trp2027Cys mutation is near the herbicide-binding pocket formed between two ACCase chains, and is predicted to obstruct the access of aryloxyphenoxypropionates (FOP) herbicides to the binding site. A qPCR-based single nucleotide polymorphism genotyping method was validated to discriminate susceptible (wild-type Trp2027) and resistant (mutant Cys2027) alleles. All resistant plants were homozygous for the mutation and the assay could be used for early detection of resistance in D. insularis field samples with suspected resistance to ACCase inhibitors.

Physiological Factors Affecting Uptake and Translocation of Glufosinate.

Glufosinate is considered a contact herbicide because of its fast activity and limited translocation in plants. We used Palmer amaranth (Amaranthus palmeri S. Watson) as a model species to study plant-related factors affecting glufosinate uptake and translocation. Glufosinate uptake increased rapidly during the initial 24 h, achieving maximum uptake from this time on. The rate of uptake saturated with doses higher than 250 µM glufosinate, suggesting the involvement of a membrane transporter. When glufosinate concentrations were higher (>1 mM), uptake was a simple diffusion process in favor of a concentration gradient between the inside and the outside of the cells. Glufosinate uptake was inhibited by the presence of glutamine. The fast action of glufosinate did not limit its own translocation. Because glufosinate is highly water soluble, it translocates mostly through the apoplast or the xylem system. Consequently, old leaves tend to accumulate more herbicide than young meristematic leaves.

A novel TIPT double mutation in EPSPS conferring glyphosate resistance in tetraploid Bidens subalternans.

BACKGROUND: Bidens subalternans (greater beggarticks) is a tetraploid and troublesome weed infesting annual crops in most tropical regions of the world. A glyphosate-resistant (GR) B. subalternans biotype was detected in a soybean field from Paraguay. A series of physiological and molecular analyses were conducted to elucidate its resistance mechanisms. RESULTS: The GR biotype had a high level of resistance (> 15-fold LD50 ), relative to a glyphosate-susceptible (GS) biotype. Shikimate accumulation was up to ten-fold greater for GS compared with GR. We found no differences in sensitivity when plants were treated and kept under lower (10/4 °C) or higher temperatures (25/20 °C). GS and GR had the same relative EPSPS gene copy number, and similar glyphosate absorption and translocation rates. Neither biotype metabolized glyphosate. A double amino acid substitution (TIPT - Thr102Ile and Pro106Thr) was found in only one EPSPS allele from one of the two EPSPS homoeologs present in tetraploid GR B. subalternans. CONCLUSION: This is the first report of a TIPT double mutation conferring high levels of glyphosate resistance in a weed species. The presence of both wild-type and TIPT mutant EPSPS on the polyploid genome of GR B. subalternans may offset a potential fitness cost, requiring additional research to confirm the absence of deleterious effects. © 2019 Society of Chemical Industry.

Physiological and molecular analysis of glyphosate resistance in non-rapid response Ambrosia trifida from Wisconsin.

BACKGROUND: We previously identified a glyphosate-resistant A. trifida phenotype from Wisconsin USA that showed a non-rapid response to glyphosate. The mechanism of glyphosate resistance in this phenotype has yet to be elucidated. We conducted experiments to investigate non-target-site resistance and target-site resistance mechanisms. The roles of glyphosate absorption, translocation, and metabolism in resistance of this phenotype have not been reported previously, nor have EPSPS protein abundance or mutations to the full-length sequence of EPSPS. RESULTS: Whole-plant dose-response results confirmed a 6.5-level of glyphosate resistance for the resistant (R) phenotype compared to a susceptible (S) phenotype. Absorption and translocation of 14 C-glyphosate were similar between R and S phenotypes over 72 h. Glyphosate and AMPA concentrations in leaf tissue did not differ between R and S phenotypes over 96 h. In vivo shikimate leaf disc assays confirmed that glyphosate EC50 values were 4.6- to 5.4-fold greater for the R than S phenotype. Shikimate accumulation was similar between phenotypes at high glyphosate concentrations (>1000 µM), suggesting that glyphosate entered chloroplasts and inhibited EPSPS. This finding was supported by results showing that EPSPS copy number and EPSPS protein abundance did not differ between R and S phenotypes, nor did EPSPS sequence at Gly101, Thr102, and Pro106 positions. Comparison of full-length EPSPS sequences found five nonsynonymous polymorphisms that differed between R and S phenotypes. However, their locations were distant from the glyphosate target site and, therefore, not likely to affect enzyme-glyphosate interaction. CONCLUSION: The results suggest that a novel mechanism confers glyphosate resistance in this A. trifida phenotype. © 2019 Society of Chemical Industry.

Predicting herbicide movement across semi-permeable membranes using three phase partitioning.

Herbicide efficacy depends on herbicides crossing cell and organelle membranes. We evaluated an artificial membrane system to understand how herbicides cross biological membranes. This understanding aids in predicting herbicide behavior in planta and, consequently, efficacy, mode of action, and whether active transporter-based herbicide resistance mechanisms may be possible. Five herbicides with different log Kow and pKa values were assessed: glyphosate, 2,4-D, clopyralid, sulfentrazone and glufosinate. The artificial membrane apparatus included four semipermeable membranes containing buffers with pH 2.7, 5 and/or 7.4, floating in a bath of diethyl ether. These conditions were based on the pH from different cellular compartments and the pKa for these herbicides. Changes in herbicide concentration due to movement were measured using radioactivity or liquid chromatography mass spectrometry. In general, herbicide behavior followed the pattern predicted by their calculated pKa and log Kow. Herbicides added to an acidic phase (pH 2.7) were more mobile than when they were added to the more basic phase (pH 7.4), except when herbicide's pKa was lower than the pH of the starting phase. Clopyralid, 2,4-D, and sulfentrazone showed significant acid trapping behavior due to their weak acid functional groups. Sulfentrazone and 2,4-D had a high affinity for the nonpolar, diethyl ether bath, especially when they were protonated at low pH. Our findings illustrate the robustness of the system to provide predictions about herbicide behavior at the subcellular level.

Role of Glutamine Synthetase Isogenes and Herbicide Metabolism in the Mechanism of Resistance to Glufosinate in Lolium perenne L. spp. multiflorum Biotypes from Oregon.

Glufosinate-resistant Lolium perenne L. spp. multiflorum biotypes from Oregon exhibited resistance levels up to 2.8-fold the field rate. One resistant biotype (MG) had an amino acid substitution in glutamine synthetase 2 (GS2), whereas the other (OR) exhibited the wild-type genotype. We hypothesized that the amino acid substitution in GS2 is involved in the resistance mechanism in MG and that non-target site resistance mechanisms are present in OR. OR metabolized glufosinate faster than the other two biotypes, with >75% of the herbicide metabolized in comparison to 50% in MG and the susceptible biotype. A mutation in GS2 co-segregating with resistance in MG did not reduce the enzyme activity, with results further supported by our enzyme homology models. This research supports the conclusion that a metabolism mechanism of glufosinate resistance is present in OR and that glufosinate resistance in MG is not due to an altered target site.

Reactive oxygen species trigger the fast action of glufosinate.

MAIN CONCLUSION: Glufosinate is primarily toxic to plants due to a massive light-dependent generation of reactive oxygen species rather than ammonia accumulation or carbon assimilation inhibition. Glutamine synthetase (GS) plays a key role in plant nitrogen metabolism and photorespiration. Glufosinate (C5H12NO4P) targets GS and causes catastrophic consequences leading to rapid plant cell death, and the causes for phytoxicity have been attributed to ammonia accumulation and carbon assimilation restriction. This study aimed to examine the biochemical and physiological consequences of GS inhibition to identify the actual cause for rapid phytotoxicity. Monocotyledonous and dicotyledonous species with different forms of carbon assimilation (C3 versus C4) were selected as model plants. Glufosinate sensitivity was proportional to the uptake of herbicide between species. Herbicide uptake also correlated with the level of GS inhibition and ammonia accumulation in planta even with all species having the same levels of enzyme sensitivity in vitro. Depletion of both glutamine and glutamate occurred in glufosinate-treated leaves; however, amino acid starvation would be expected to cause a slow plant response. Ammonia accumulation in response to GS inhibition, often reported as the driver of glufosinate phytotoxicity, occurred in all species, but did not correlate with either reductions in carbon assimilation or cell death. This is supported by the fact that plants can accumulate high levels of ammonia but show low inhibition of carbon assimilation and absence of phytotoxicity. Glufosinate-treated plants showed a massive light-dependent generation of reactive oxygen species, followed by malondialdehyde accumulation. Consequently, we propose that glufosinate is toxic to plants not because of ammonia accumulation nor carbon assimilation inhibition, but the production of reactive oxygen species driving the catastrophic lipid peroxidation of the cell membranes and rapid cell death.