An intronless tau class glutathione transferase detoxifies several herbicides in flufenacet resistant ryegrass.

Resistance to pre-emergence herbicides, e.g., inhibitors of the biosynthesis of very-long-chain fatty acids (VLCFAs), is evolving in response to increased use of these compounds. Grass weeds such as ryegrasses (Lolium spp.) have accumulated resistance to various herbicide modes of action. Here, an RNA-Seq analysis was conducted using three ryegrass populations resistant to the VLCFA biosynthesis inhibitor flufenacet to investigate this phenomenon. Besides various transcripts, including putative long non-coding RNAs (lncRNAs), a single putatively functional tau class glutathione transferase (GST) was constitutively differentially expressed. It was further induced by herbicide application. This GST was expressed as a recombinant protein in Escherichia coli along with other GSTs and detoxified flufenacet rapidly in vitro. Detoxification rates of other herbicides tested in vitro were in accordance with cross-resistance patterns previously determined in vivo. A genome-wide GST analysis revealed that the candidate GST was located in a cluster of three intronless GSTs. Their intronless nature possibly results from the retroposition of cellular mRNAs followed by tandem duplication and may affect gene expression. The large number of GSTs (≥ 195) in the genome of rigid ryegrass (L. rigidum) compared to other plant organisms is likely a key factor in the ability of this weed to evolve resistance to different herbicide chemistries. However, in the case of flufenacet resistance, a single upregulated GST with high affinity for the substrate flufenacet possibly contributes over-proportionally to rapid herbicide detoxification in planta. The regulation of this gene and the role of differentially expressed transcripts, including various putative lncRNAs, require further investigation.

Current status of community resources and priorities for weed genomics research.

Weeds are attractive models for basic and applied research due to their impacts on agricultural systems and capacity to swiftly adapt in response to anthropogenic selection pressures. Currently, a lack of genomic information precludes research to elucidate the genetic basis of rapid adaptation for important traits like herbicide resistance and stress tolerance and the effect of evolutionary mechanisms on wild populations. The International Weed Genomics Consortium is a collaborative group of scientists focused on developing genomic resources to impact research into sustainable, effective weed control methods and to provide insights about stress tolerance and adaptation to assist crop breeding.

Genome-wide study of glutathione transferases and their regulation in flufenacet susceptible and resistant black-grass (Alopecurus myosuroides Huds.).

BACKGROUND: Glutathione transferases (GSTs) are enzymes with a wide range of functions, including herbicide detoxification. Up-regulation of GSTs and their detoxification activity enables the grass weed black-grass (Alopecurus myosuroides Huds.) to metabolize the very-long-chain fatty acid synthesis inhibitor flufenacet and other herbicides leading to multiple herbicide resistance. However, the genomic organization and regulation of GSTs genes is still poorly understood. RESULTS: In this genome-wide study the location and expression of 115 GSTs were investigated using a recently published black-grass genome. Particularly, the most abundant GSTs of class tau and phi were typically clustered and often followed similar expression patterns but possessed divergent upstream regulatory regions. Similarities were found in the promoters of the most up-regulated GSTs, which are located next to each other in a cluster. The binding motif of the E2F/DP transcription factor complex in the promoter of an up-regulated GST was identical in susceptible and resistant plants, however, adjacent sequences differed. This led to a stronger binding of proteins to the motif of the susceptible plant, indicating repressor activity. CONCLUSIONS: This study constitutes the first analysis dealing with the genomic investigation of GST genes found in black-grass and their transcriptional regulation. It highlights the complexity of the evolution of GSTs in black-grass, their duplication and divergence over time. The large number of GSTs allows weeds to detoxify a broad spectrum of herbicides. Ultimately, more research is needed to fully elucidate the regulatory mechanisms of GST expression. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

RNA and protein biomarkers for detecting enhanced metabolic resistance to herbicides mesosulfuron-methyl and fenoxaprop-ethyl in black-grass (Alopecurus myosuroides).

BACKGROUND: The evolution of non-target site resistance (NTSR) to herbicides leads to a significant reduction in herbicide control of agricultural weed species. Detecting NTSR in weed populations prior to herbicide treatment would provide valuable information for effective weed control. While not all NTSR mechanisms have been fully identified, enhanced metabolic resistance (EMR) is one of the better studied, conferring tolerance through increased herbicide detoxification. Confirming EMR towards specific herbicides conventionally involves detecting metabolites of the active herbicide molecule in planta, but this approach is time-consuming and requires access to well-equipped laboratories. RESULTS: In this study, we explored the potential of using molecular biomarkers to detect EMR before herbicide treatment in black-grass (Alopecurus myosuroides). We tested the reliability of selected biomarkers to predict EMR and survival after herbicide treatments in both reference and 27 field-derived black-grass populations collected from sites across the UK. The combined analysis of the constitutive expression of biomarkers and metabolism studies confirmed three proteins, namely, AmGSTF1, AmGSTU2 and AmOPR1, as differential biomarkers of EMR toward the herbicides fenoxaprop-ethyl and mesosulfuron in black-grass. CONCLUSION: Our findings demonstrate that there is potential to use molecular biomarkers to detect EMR toward specific herbicides in black-grass without reference to metabolism analysis. However, biomarker development must include testing at both transcript and protein levels in order to be reliable indicators of resistance. This work is a first step towards more robust resistance biomarker development, which could be expanded into other herbicide chemistries for on-farm testing and monitoring EMR in uncharacterised black-grass populations. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The bZIP transcription factor BIP1 of the rice blast fungus is essential for infection and regulates a specific set of appressorium genes.

The rice blast fungus Magnaporthe oryzae differentiates specialized cells called appressoria that are required for fungal penetration into host leaves. In this study, we identified the novel basic leucine zipper (bZIP) transcription factor BIP1 (B-ZIP Involved in Pathogenesis-1) that is essential for pathogenicity. BIP1 is required for the infection of plant leaves, even if they are wounded, but not for appressorium-mediated penetration of artificial cellophane membranes. This phenotype suggests that BIP1 is not implicated in the differentiation of the penetration peg but is necessary for the initial establishment of the fungus within plant cells. BIP1 expression was restricted to the appressorium by both transcriptional and post-transcriptional control. Genome-wide transcriptome analysis showed that 40 genes were down regulated in a BIP1 deletion mutant. Most of these genes were specifically expressed in the appressorium. They encode proteins with pathogenesis-related functions such as enzymes involved in secondary metabolism including those encoded by the ACE1 gene cluster, small secreted proteins such as SLP2, BAS2, BAS3, and AVR-Pi9 effectors, as well as plant cuticle and cell wall degrading enzymes. Interestingly, this BIP1 network is different from other known infection-related regulatory networks, highlighting the complexity of gene expression control during plant-fungal interactions. Promoters of BIP1-regulated genes shared a GCN4/bZIP-binding DNA motif (TGACTC) binding in vitro to BIP1. Mutation of this motif in the promoter of MGG_08381.7 from the ACE1 gene cluster abolished its appressorium-specific expression, showing that BIP1 behaves as a transcriptional activator. In summary, our findings demonstrate that BIP1 is critical for the expression of early invasion-related genes in appressoria. These genes are likely needed for biotrophic invasion of the first infected host cell, but not for the penetration process itself. Through these mechanisms, the blast fungus strategically anticipates the host plant environment and responses during appressorium-mediated penetration.

Designing New Protoporphyrinogen Oxidase-Inhibitors Carrying Potential Side Chain Isosteres to Enhance Crop Safety and Spectrum of Activity.

There are several methods to control weeds, which impose particular challenges for farmers in all parts of the world, although applying small molecular compounds still remains the most efficient technology to date. However, plants can evolve to become resistant toward active ingredients which is also the case for protoporphyrinogen oxidase (PPO) inhibitors, a class of highly effective herbicides in use for more than 50 years. Hence, it is essential to continuously discover and develop new herbicidal PPO inhibitors with enhanced intrinsic activity, an improved resistance profile, enhanced crop safety, favorable physicochemical properties, and a clean toxicological profile. By modifying structural key features from known PPO inhibitors such as tiafenacil, inspired by isostere and mix&match concepts in combination with modeling investigations based on a wild-type Amaranthus crystal structure, we have found new promising lead structures showing strong activity in vitro and in vivo against several notorious dicotyledon and monocotyledon weeds with emerging resistance (e.g., Amaranthus palmeri, Amaranthus tuberculatus, Lolium rigidum, and Alopecurus myosuroides). While several phenyl uracils carrying an isoxazoline motif in their thio-linked side chain showed promising resistance-breaking potential against different Amaranthus species, introducing a thioacrylamide side chain afforded outstanding efficacy against resistant grass weeds.

Recombinant glutathione transferases from flufenacet-resistant black-grass (Alopecurus myosuroides Huds.) form different flufenacet metabolites and differ in their interaction with pre- and post-emergence herbicides.

BACKGROUND: Black-grass (Alopecurus myosuroides Huds.) has become a problematic weed in cereals in Europe. Besides resistance to post-emergent herbicides becoming increasingly widespread, enhanced metabolism of inhibitors of the synthesis of very-long-chain fatty acids (VLCFAs), such as flufenacet, is evolving. Yet, cross-resistance patterns and evolution of this resistance remains poorly understood. RESULTS: The cDNA sequences of five glutathione transferases (GSTs) upregulated in flufenacet resistant black-grass were identified and used for recombinant protein expression. Moderate to slow detoxification of flufenacet was verified for all candidate GSTs expressed in E. coli, and the most active protein produced flufenacet-alcohol instead of a glutathione conjugate, in the presence of reduced glutathione (GSH). Moreover, cross-resistance to other VLCFA-inhibitors e.g., acetochlor and pyroxasulfone and the ACCase inhibitor fenoxaprop was verified in vitro. Various other herbicides of different modes of action including VLCFA-inhibitors were not detoxified by the candidate GSTs. CONCLUSIONS: As several in planta upregulated GSTs detoxified flufenacet in vitro, the shift in sensitivity observed in black-grass populations, is likely a result of an additive effect. The polygenic character and the relatively low turnover rate of the individual GSTs may explain the slow evolution of flufenacet resistance. In addition, flufenacet resistance was accompanied by cross-resistance with some, but not all, herbicides of the same mode of action, and furthermore to the ACCase inhibitor fenoxaprop-ethyl. Hence, not only the rotation of herbicide modes of action, but also of individual active ingredients is important for resistance management. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Design, synthesis and screening of herbicidal activity for new phenyl pyrazole-based protoporphyrinogen oxidase-inhibitors (PPO) overcoming resistance issues.

BACKGROUND: Whilst there are several methods to control weeds, which continuously plague farmers around the globe, the application of small molecular compounds is still the most effective technology to date. Plants can evolve to become resistant to PPO-inhibitors, a class of herbicides in commercial use since the 1960s. It is therefore essential to continuously develop new herbicides based on this mode-of-action with enhanced intrinsic activity, an improved resistance profile and favourable physicochemical properties. Based on an Amaranthus PPO crystal structure and subsequent modelling studies, halogen-substituted pyrazoles have been investigated as isosteres of uracil-based PPO-inhibitors. RESULTS: By combining structural features from the commercial PPO-inhibitors tiafenacil and pyraflufen-ethyl and by investigating receptor-binding properties, we identified new promising pyrazole-based lead structures showing strong activity in vitro and in vivo against economically important weeds of the Amaranthus genus: A. retroflexus, and resistant A. palmeri and A. tuberculatus. CONCLUSION: The present work covers a series of novel PPO-inhibiting compounds that contain a pyrazole ring and a substituted thioacetic acid sidechain attached to the core phenyl group. These compounds show good receptor fit in line with excellent herbicidal activity against weeds that plague corn and rice crops with low application rates. This, in combination with promising selectivity in corn, have the potential to mitigate and affect weeds that have become resistant to some of the current market standards. Remarkably, some of the novel PPO-inhibitors outlined herein show efficacies against economically important weeds that were superior to recently commercialized and structurally related tiafenacil. © 2023 Society of Chemical Industry.

The blackgrass genome reveals patterns of non-parallel evolution of polygenic herbicide resistance.

Globally, weedy plants are a major constraint to sustainable crop production. Much of the success of weeds rests with their ability to rapidly adapt in the face of human-mediated management of agroecosystems. Alopecurus myosuroides (blackgrass) is a widespread and impactful weed affecting agriculture in Europe. Here we report a chromosome-scale genome assembly of blackgrass and use this reference genome to explore the genomic/genetic basis of non-target site herbicide resistance (NTSR). Based on our analysis of F2 seed families derived from two distinct blackgrass populations with the same NTSR phenotype, we demonstrate that the trait is polygenic and evolves from standing genetic variation. We present evidence that selection for NTSR has signatures of both parallel and non-parallel evolution. There are parallel and non-parallel changes at the transcriptional level of several stress- and defence-responsive gene families. At the genomic level, however, the genetic loci underpinning NTSR are different (non-parallel) between seed families. We speculate that variation in the number, regulation and function of stress- and defence-related gene families enable weedy species to rapidly evolve NTSR via exaptation of genes within large multi-functional gene families. These results provide novel insights into the potential for, and nature of plant adaptation in rapidly changing environments.

Genetic architecture underlying HPPD-inhibitor resistance in a Nebraska Amaranthus tuberculatus population.

BACKGROUND: Amaranthus tuberculatus is a problematic weed species in Midwest USA agricultural systems. Inhibitors of 4-hydroxyphenylpyruvate dioxygenase (HPPD) are an important chemistry for weed management in numerous cropping systems. Here, we characterize the genetic architecture underlying the HPPD-inhibitor resistance trait in an A. tuberculatus population (NEB). RESULTS: Dose-response studies of an F1 generation identified HPPD-inhibitor resistance as a dominant trait with a resistance factor of 15.0-21.1 based on dose required for 50% growth reduction. Segregation analysis in a pseudo-F2 generation determined the trait is moderately heritable (H2  = 0.556) and complex. Bulk segregant analysis and validation with molecular markers identified two quantitative trait loci (QTL), one on each of Scaffold 4 and 12. CONCLUSIONS: Resistance to HPPD inhibitors is a complex, largely dominant trait within the NEB population. Two large-effect QTL were identified controlling HPPD-inhibitor resistance in A. tuberculatus. This is the first QTL mapping study to characterize herbicide resistance in a weedy species.

Cytochrome P450 CYP81A10v7 in Lolium rigidum confers metabolic resistance to herbicides across at least five modes of action.

Rapid and widespread evolution of multiple herbicide resistance in global weed species endowed by increased capacity to metabolize (degrade) herbicides (metabolic resistance) is a great threat to herbicide sustainability and global food production. Metabolic resistance in the economically damaging crop weed species Lolium rigidum is well known but a molecular understanding has been lacking. We purified a metabolic resistant (R) subset from a field evolved R L. rigidum population. The R, the herbicide susceptible (S) and derived F2 populations were used for candidate herbicide resistance gene discovery by RNA sequencing. A P450 gene CYP81A10v7 was identified with higher expression in R vs. S plants. Transgenic rice overexpressing this Lolium CYP81A10v7 gene became highly resistant to acetyl-coenzyme A carboxylase- and acetolactate synthase-inhibiting herbicides (diclofop-methyl, tralkoxydim, chlorsulfuron) and moderately resistant to hydroxyphenylpyruvate dioxygenase-inhibiting herbicide (mesotrione), photosystem II-inhibiting herbicides (atrazine and chlorotoluron) and the tubulin-inhibiting herbicide trifluralin. This wide cross-resistance profile to many dissimilar herbicides in CYP81A10v7 transgenic rice generally reflects what is evident in the R L. rigidum. This report clearly showed that a single P450 gene in a cross-pollinated weed species L. rigidum confers resistance to herbicides of at least five modes of action across seven herbicide chemistries.

Coexpression Clusters and Allele-Specific Expression in Metabolism-Based Herbicide Resistance.

In the last decade, Amaranthus tuberculatus has evolved resistance to 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-hydroxyphenylpyruvate dioxygenase inhibitors in multiple states across the midwestern United States. Two populations resistant to both mode-of-action groups, one from Nebraska (NEB) and one from Illinois (CHR), were studied using an RNA-seq approach on F2 mapping populations to identify the genes responsible for resistance. Using both an A. tuberculatus transcriptome assembly and a high-quality grain amaranth (A. hypochondriacus) genome as references, differential transcript and gene expression analyses were conducted to identify genes that were significantly over- or underexpressed in resistant plants. When these differentially expressed genes (DEGs) were mapped on the A. hypochondriacus genome, physical clustering of the DEGs was apparent along several of the 16 A. hypochondriacus scaffolds. Furthermore, single-nucleotide polymorphism calling to look for resistant-specific (R) variants, and subsequent mapping of these variants, also found similar patterns of clustering. Specifically, regions biased toward R alleles overlapped with the DEG clusters. Within one of these clusters, allele-specific expression of cytochrome  P450  81E8 was observed for 2,4-D resistance in both the CHR and NEB populations, and phylogenetic analysis indicated a common evolutionary origin of this R allele in the two populations.

Publisher Correction: Evolution of generalist resistance to herbicide mixtures reveals a trade-off in resistance management.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Evolution of generalist resistance to herbicide mixtures reveals a trade-off in resistance management.

Intense selection by pesticides and antibiotics has resulted in a global epidemic of evolved resistance. In agriculture and medicine, using mixtures of compounds from different classes is widely accepted as optimal resistance management. However, this strategy may promote the evolution of more generalist resistance mechanisms. Here we test this hypothesis at a national scale in an economically important agricultural weed: blackgrass (Alopecurus myosuroides), for which herbicide resistance is a major economic issue. Our results reveal that greater use of herbicide mixtures is associated with lower levels of specialist resistance mechanisms, but higher levels of a generalist mechanism implicated in enhanced metabolism of herbicides with diverse modes of action. Our results indicate a potential evolutionary trade-off in resistance management, whereby attempts to reduce selection for specialist resistance traits may promote the evolution of generalist resistance. We contend that where specialist and generalist resistance mechanisms co-occur, similar trade-offs will be evident, calling into question the ubiquity of resistance management based on mixtures and combination therapies.

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.

Uptake, translocation, and metabolism of glyphosate, glufosinate, and dicamba mixtures in Echinochloa crus-galli and Amaranthus palmeri.

BACKGROUND: Echinochloa crus-galli (L.) Beauv. and Amaranthus palmeri S. Wats are two common and problematic weeds prevalent across the Midsouth of the USA. Herbicide absorption, translocation, and metabolism were investigated as potential sources of herbicide antagonism on A. palmeri and E. crus-galli using 14 C-labeled herbicides. Three 14 C-labeled herbicides, glyphosate, glufosinate, and dicamba, were utilized individually in separate experiments. RESULTS: Uptake of 14 C-glyphosate in E. crus-galli was 15% of the total applied radioactivity for glyphosate/glufosinate (897 + 595 g a.i./a.e. ha-1 ) compared to 25% for glyphosate alone. Similarly, uptake of 14 C-glyphosate in A. palmeri reduced by 10% when applied with glufosinate. Applying glyphosate/dicamba (897/560 g a.e. ha-1 ) reduced 14 C-glyphosate uptake in both species. In the 14 C-glufosinate experiment, both species absorbed less 14 C-glufosinate when mixed with glyphosate compared to glufosinate alone. No metabolic degradation of glyphosate was observed in either species. E. crus-galli metabolized dicamba 23 times faster than A. palmeri. When glufosinate was applied with dicamba, metabolic degradation of 14 C-dicamba was limited in both species. For example, 99.9% of the applied radioactivity was recovered in A. palmeri as the parent compound when 14 C-glufosinate dicamba was applied with glufosinate, compared to 95.7% for dicamba alone. CONCLUSION: These findings demonstrate absorption, translocation, or metabolism of dicamba, glufosinate, and glyphosate can be affected by mixing with another herbicide. As mixing two herbicides is often a critical component of resistance management, careful investigation into the performance of these mixtures in the field is needed. © 2020 Society of Chemical Industry.

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.

Omics Potential in Herbicide-Resistant Weed Management.

The rapid development of omics technologies has drastically altered the way biologists conduct research. Basic plant biology and genomics have incorporated these technologies, while some challenges remain for use in applied biology. Weed science, on the whole, is still learning how to integrate omics technologies into the discipline; however, omics techniques are more frequently being implemented in new and creative ways to address basic questions in weed biology as well as the more practical questions of improving weed management. This has been especially true in the subdiscipline of herbicide resistance where important questions are the evolution and genetic basis of herbicide resistance. This review examines the advantages, challenges, potential solutions, and outlook for omics technologies in the discipline of weed science, with examples of how omics technologies will impact herbicide resistance studies and ultimately improve management of herbicide-resistant populations.

Alterations in Life-History Associated With Non-target-site Herbicide Resistance in Alopecurus myosuroides.

The evolution of resistance to herbicides is a classic example of rapid contemporary adaptation in the face of a novel environmental stress. Evolutionary theory predicts that selection for resistance will be accompanied by fitness trade-offs in environments where the stress is absent. Alopecurus myosuroides, an autumn-germinating grass weed of cereal crops in North-West Europe, has evolved resistance to seven herbicide modes-of-action, making this an ideal species to examine the presence and magnitudes of such fitness costs. Here, we use two contrasting A. myosuroides phenotypes derived from a common genetic background, one with enhanced metabolism resistance to a commercial formulation of the sulfonylurea (ALS) actives mesosulfuron and iodosulfuron, and the other with susceptibility to these actives (S). Comparisons of plant establishment, growth, and reproductive potential were made under conditions of intraspecific competition, interspecific competition with wheat, and over a gradient of nitrogen deprivation. Herbicide dose response assays confirmed that the two lines had contrasting resistance phenotypes, with a 20-fold difference in resistance between them. Pleiotropic effects of resistance were observed during plant development, with R plants having a greater intraspecific competitive effect and longer tiller lengths than S plants during vegetative growth, but with S plants allocating proportionally more biomass to reproductive tissues during flowering. Direct evidence of a reproductive cost of resistance was evident in the nitrogen deprivation experiment with R plants producing 27% fewer seed heads per plant, and a corresponding 23% reduction in total seed head length. However, these direct effects of resistance on fecundity were not consistent across experiments. Our results demonstrate that a resistance phenotype based on enhanced herbicide metabolism has pleiotropic impacts on plant growth, development and resource partitioning but does not support the hypothesis that resistance is associated with a consistent reproductive fitness cost in this species. Given the continued difficulties associated with unequivocally detecting costs of herbicide resistance, we advocate future studies that adopt classical evolutionary quantitative genetics approaches to determine genetic correlations between resistance and fitness-related plant life history traits.

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.