Involvement of glutamine synthetase 2 (GS2) amplification and overexpression in Amaranthus palmeri resistance to glufosinate.

MAIN CONCLUSION: Amplification and overexpression of the target site glutamine synthetase, specifically the plastid-located isoform, confers resistance to glufosinate in Amaranthus palmeri. This mechanism is novel among glufosinate-resistant weeds. Amaranthus palmeri has recently evolved resistance to glufosinate herbicide. Several A. palmeri populations from Missouri and Mississippi, U.S.A. had survivors when sprayed with glufosinate-ammonium (GFA, 657 g ha-1). One population, MO#2 (fourfold resistant) and its progeny (sixfold resistant), were used to study the resistance mechanism, focusing on the herbicide target glutamine synthetase (GS). We identified four GS genes in A. palmeri; three were transcribed: one coding for the plastidic protein (GS2) and two coding for cytoplasmic isoforms (GS1.1 and GS1.2). These isoforms did not contain mutations associated with resistance. The 17 glufosinate survivors studied showed up to 21-fold increase in GS2 copies. GS2 was expressed up to 190-fold among glufosinate survivors. GS1.1 was overexpressed > twofold in only 3 of 17, and GS1.2 in 2 of 17 survivors. GS inhibition by GFA causes ammonia accumulation in susceptible plants. Ammonia level was analyzed in 12 F1 plants. GS2 expression was negatively correlated with ammonia level (r = - 0.712); therefore, plants with higher GS2 expression are less sensitive to GFA. The operating efficiency of photosystem II (ϕPSII) of Nicotiana benthamiana overexpressing GS2 was four times less inhibited by GFA compared to control plants. Therefore, increased copy and overexpression of GS2 confer resistance to GFA in A. palmeri (or other plants). We present novel understanding of the role of GS2 in resistance evolution to glufosinate.

Extrachromosomal DNA-mediated glyphosate resistance in Italian ryegrass.

BACKGROUND: An Italian ryegrass population from Arkansas, USA developed glyphosate resistance due to 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene amplification. The plants in this population with approximately 70 EPSPS copies were used in the present study for the physical mapping of amplified copies of EPSPS gene to determine the possible mechanism of EPSPS gene amplification conferring glyphosate resistance in Italian ryegrass. RESULT: Fluorescence in situ hybridization (FISH) analysis of glyphosate resistant (GR) Italian ryegrass plants with approximately 70 EPSPS copies displayed EPSPS hybridization signals randomly on most of the metaphase chromosomes. Glyphosate susceptible (GS) Italian ryegrass plants with one EPSPS copy displayed single prominent EPSPS hybridization signal, which was co-localized with 5S rDNA locus along with few additional signals on the outside of chromosomes. Pulsed-field gel electrophoresis (PFGE) followed by DNA blot using EPSPS gene as a probe identified a prominent EPSPS hybridization around the 400 kb region in GR DNA samples, but not in GS DNA samples. CONCLUSION: We report the extrachromosomal DNA-mediated glyphosate resistance in Italian ryegrass. Physical mapping of amplified copies of EPSPS gene in Italian ryegrass by FISH gives us a clue that the amplified copies of EPSPS gene may be present in the extrachromosomal DNA elements. Further analysis by PFGE followed by DNA blotting revealed that the extrachromosomal DNA containing EPSPS is approximately 400 kb similar in size with that of eccDNA replicon in Amaranthus palmeri. © 2023 Society of Chemical Industry.

Genomic revolution of US weedy rice in response to 21st century agricultural technologies.

Weedy rice is a close relative of cultivated rice that devastates rice productivity worldwide. In the southern United States, two distinct strains have been historically predominant, but the 21st century introduction of hybrid rice and herbicide resistant rice technologies has dramatically altered the weedy rice selective landscape. Here, we use whole-genome sequences of 48 contemporary weedy rice accessions to investigate the genomic consequences of crop-weed hybridization and selection for herbicide resistance. We find that population dynamics have shifted such that most contemporary weeds are now crop-weed hybrid derivatives, and that their genomes have subsequently evolved to be more like their weedy ancestors. Haplotype analysis reveals extensive adaptive introgression of cultivated alleles at the resistance gene ALS, but also uncovers evidence for convergent molecular evolution in accessions with no signs of hybrid origin. The results of this study suggest a new era of weedy rice evolution in the United States.

Field-Evolved ΔG210-ppo2 from Palmer Amaranth Confers Pre-emergence Tolerance to PPO-Inhibitors in Rice and Arabidopsis.

Resistance to protoporphyrinogen IX oxidase (PPO)-inhibitors in Amaranthus palmeri and Amaranthus tuberculatus is mainly contributed by mutations in the PPO enzyme, which renders herbicide molecules ineffective. The deletion of glycine210 (ΔG210) is the most predominant PPO mutation. ΔG210-ppo2 is overexpressed in rice (Oryza sativa c. 'Nipponbare') and Arabidopsis thaliana (Col-0). A foliar assay was conducted on transgenic T1 rice plants with 2× dose of fomesafen (780 g ha−1), showing less injury than the non-transgenic (WT) plants. A soil-based assay conducted with T2 rice seeds confirmed tolerance to fomesafen applied pre-emergence. In agar medium, root growth of WT rice seedlings was inhibited >90% at 5 µM fomesafen, while root growth of T2 seedlings was inhibited by 50% at 45 µM fomesafen. The presence and expression of the transgene were confirmed in the T2 rice survivors of soil-applied fomesafen. A soil-based assay was also conducted with transgenic A. thaliana expressing ΔG210-ppo2 which confirmed tolerance to the pre-emergence application of fomesafen and saflufenacil. The expression of A. palmeri ΔG210-ppo2 successfully conferred tolerance to soil-applied fomesafen in rice and Arabidopsis. This mutant also confers cross-tolerance to saflufenacil in Arabidopsis. This trait could be introduced into high-value crops that lack chemical options for weed management.

Can double PPO mutations exist in the same allele and are such mutants functional?

BACKGROUND: Resistance to protoporphyrinogen oxidase (PPO)-inhibiting herbicides is endowed primarily by target-site mutations at the PPX2 gene that compromise binding of the herbicide to the catalytic domain. In Amaranthus spp. PPX2, the most prevalent target mutations are deletion of the G210 codon, and the R128G and G339A substitutions. These mutations strongly affect the dynamic of the PPO2 binding pocket, resulting in reduced affinity with the ligand. Here we investigated the likelihood of co-occurrence of the most widespread target site mutations in the same PPX2 allele. RESULTS: Plants carrying R128G+/+ ΔG210+/-, where + indicates presence of the mutation, were crossed with each other. The PPX2 of the offspring was subjected to pyrosequencing and E. coli-based Sanger sequencing to determine mutation frequencies and allele co-occurrence. The data show that R128G ΔG210 can occur in one allele only; the second allele carries only one mutation. Double mutation in both alleles is less likely because of significant loss of enzyme activity. The segregation of offspring populations derived from a cross between heterozygous plants carrying ΔG210 G399A also showed no co-occurrence in the same allele. The offspring exhibited the expected mutation distribution patterns with few exceptions. CONCLUSIONS: Homozygous double-mutants are not physiologically viable. Double-mutant plants can only exist in a heterozygous state. Alternatively, if two mutations are detected in one plant, each mutation would occur in a separate allele. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

High Resistance to Quinclorac in Multiple-Resistant Echinochloa colona Associated with Elevated Stress Tolerance Gene Expression and Enriched Xenobiotic Detoxification Pathway.

Echinochloa colona and other species in this genus are a threat to global rice production and food security. Quinclorac, an auxin mimic, is a common herbicide for grass weed control in rice, and Echinochloa spp. have evolved resistance to it. The complete mode of quinclorac action and subsequent evolution of resistance is not fully understood. We analyzed the de novo transcriptome of multiple-herbicide-resistant (ECO-R) and herbicide-susceptible genotypes in response to quinclorac. Several biological processes were constitutively upregulated in ECO-R, including carbon metabolism, photosynthesis, and ureide metabolism, indicating improved metabolic efficiency. The transcriptional change in ECO-R following quinclorac treatment indicates an efficient response, with upregulation of trehalose biosynthesis, which is also known for abiotic stress mitigation. Detoxification-related genes were induced in ECO-R, mainly the UDP-glycosyltransferase (UGT) family, most likely enhancing quinclorac metabolism. The transcriptome data also revealed that many antioxidant defense elements were uniquely elevated in ECO-R to protect against the auxin-mediated oxidative stress. We propose that upon quinclorac treatment, ECO-R detoxifies quinclorac utilizing UGT genes, which modify quinclorac using the sufficient supply of UDP-glucose from the elevated trehalose pathway. Thus, we present the first report of upregulation of trehalose synthesis and its association with the herbicide detoxification pathway as an adaptive mechanism to herbicide stress in Echinochloa, resulting in high resistance.

Mechanism of Resistance to S-metolachlor in Palmer amaranth.

Herbicides are major tools for effective weed management. The evolution of resistance to herbicides in weedy species, especially contributed by non-target-site-based resistance (NTSR) is a worrisome issue in crop production globally. Glyphosate-resistant Palmer amaranth (Amaranthus palmeri) is one of the extremely difficult weeds in southern US crop production. In this study, we present the level and molecular basis of resistance to the chloroacetamide herbicide, S-metolachlor, in six field-evolved A. palmeri populations that had survivors at the recommended field-dose (1.1 kg ai ha-1). These samples were collected in 2014 and 2015. The level of resistance was determined in dose-response assays. The effective dose for 50% control (ED50) of the susceptible population was 27 g ai ha-1, whereas the ED50 of the resistant populations ranged from 88 to 785 g ai ha-1. Therefore, A. palmeri resistance to S-metolachlor evolved in Arkansas as early as 2014. Metabolic-inhibitor and molecular assays indicated NTSR in these populations, mainly driven by GSTs. To understand the mechanism of resistance, selected candidate genes were analyzed in leaves and roots of survivors (with 1 × S-metolachlor). Expression analysis of the candidate genes showed that the primary site of S-metolachlor detoxification in A. palmeri is in the roots. Two GST genes, ApGSTU19 and ApGSTF8 were constitutively highly expressed in roots of all plants across all resistant populations tested. The expression of both GSTs increased further in survivors after treatment with S-metolachlor. The induction level of ApGSTF2 and ApGSTF2like by S-metolachlor differed among resistant populations. Overall, higher expression of ApGSTU19, ApGSTF8, ApGSTF2, and ApGSTF2like, which would lead to higher GST activity in roots, was strongly associated with the resistant phenotype. Phylogenetic relationship and analysis of substrate binding site of candidate genes suggested functional similarities with known metolachlor-detoxifying GSTs, effecting metabolic resistance to S-metolachlor in A. palmeri. Resistance is achieved by elevated baseline expression of these genes and further induction by S-metolachlor in resistant plants.

Acclimation to cold stress reduces injury from low temperature and bispyribac-sodium on rice.

BACKGROUND: In subtropical areas, early planting exposes rice seedlings to cold stress, impairing seedling growth and making them more vulnerable to other stresses including herbicide injury. The objectives of this work were: to evaluate the effect of cold stress on bispyribac-sodium selectivity in rice; to determine the mechanisms of cold tolerance in sensitive ('Epagri 109') and tolerant ('IRGA 424') rice cultivars; and to ascertain that cold acclimatization influences bispyribac-sodium selectivity in rice. RESULTS: Prolonged cold stress caused high lipid peroxidation, increased rice injury, and stunted growth. Short-term acclimation with cold stress reduced rice injury with bispyribac-sodium. Total phenols were upregulated in rice exposed to cold stress. Prolonged cold stress increased the superoxide dismutase and catalase activity in IRGA 424. Antioxidant activity was higher in the cold-tolerant than in the cold-sensitive cultivar. Only catalase activity was responsive to bispyribac-sodium. OsRAN2, OsGSTL2, and CYP72A21 were upregulated by cold and herbicide stress in both cultivars. OsGSTL2 was upregulated more in IRGA 424 than in Epagri 109. OsFAD8 was upregulated in cold-sensitive rice exposed to short-duration cold stress but was not responsive to bispyribac-sodium. CONCLUSION: Cold stress reduces bispyribac-sodium selectivity in rice. Short-term acclimation to cold stress reduces the effect of cold stress and enhances bispyribac-sodium selectivity. The tolerance of rice (IRGA 424) to cold stress is due to differential induction of protection genes CYP72A21 and OsGSTL2 associated with herbicide metabolism, together with the accumulation of total phenols and higher activity of antioxidant enzymes.

Functional PPO2 mutations: co-occurrence in one plant or the same ppo2 allele of herbicide-resistant Amaranthus palmeri in the US mid-south.

BACKGROUND: Protoporphyrinogen IX oxidase 2 (PPO2) inhibitors are important for the management of glyphosate- and acetolactate synthase-resistant Palmer amaranth [Amaranthus palmeri (S.) Wats.]. The evolving resistance to PPO inhibitors is of great concern. We surveyed the evolution of resistance to fomesafen in the US Mid-south and determined its correlation with the known functional PPO2 target-site mutations (TSM). RESULTS: The 167 accessions analyzed were grouped into five categories, four resistant (147) and one susceptible (20). Arkansas accessions constituted 100% of the susceptible group while the Missouri accessions comprised 60% of the most resistant category. The majority of Mississippi accessions (88%) clustered in the high-survival-high-injury category, manifesting an early-stage resistance evolution. One hundred and fifteen accessions were genotyped for four known TSMs; 74% of accessions carried at least one TSM. The most common single TSM was ΔG210 (18% of accessions) and the predominant double mutation was ΔG210 + G399A (17%). Other mutations are likely less favorable, hence are rare. All TSMs were detected in three accessions. Further examination revealed that 9 and two individuals carried G399A + G210 and G399A + R128G TSM in the same allele, respectively. The existence of these combinations is supported by molecular modeling. CONCLUSIONS: Resistance to PPO inhibitors is widespread across the Mid-southern USA. Highly resistant field populations have plants with multiple mutations. G399A is the most prone to co-occur with other ppo2 mutations in the same allele. Mutation at R128 in the configuration of the PPO2 catalytic domain restrains the co-occurrence of R128G with ΔG210, making ΔG210 + G399A the most plausible, tolerable functional mutation combination to co-occur in the same ppo2 allele.

Snorkeling Strategy: Tolerance to Flooding in Rice and Potential Application for Weed Management.

Flooding is an important strategy for weed control in paddy rice fields. However, terrestrial weeds had evolved mechanisms of tolerance to flooding, resulting in new 'snorkeling' ecotypes. The aim of this review is to discuss the mechanisms of flooding tolerance in cultivated and weedy rice at different plant stages and the putative utility of this trait for weed management. Knowledge about flooding tolerance is derived primarily from crop models, mainly rice. The rice model informs us about the possible flooding tolerance mechanisms in weedy rice, Echinochloa species, and other weeds. During germination, the gene related to carbohydrate mobilization and energy intake (RAmy3D), and genes involved in metabolism maintenance under anoxia (ADH, PDC, and OsB12D1) are the most important for flooding tolerance. Flooding tolerance during emergence involved responses promoted by ethylene and induction of RAmy3D, ADH, PDC, and OsB12D1. Plant species tolerant to complete submersion also employ escape strategies or the ability to become quiescent during the submergence period. In weedy rice, the expression of PDC1, SUS3, and SUB1 genes is not directly related to flooding tolerance, contrary to what was learned in cultivated rice. Mitigation of flooding tolerance in weeds could be achieved with biotechnological approaches and genetic manipulation of flood tolerance genes through RNAi and transposons, providing a potential new tool for weed management.

Target-site mutation accumulation among ALS inhibitor-resistant Palmer amaranth.

BACKGROUND: Palmer amaranth (Amaranthus palmeri S. Wats) is one of the most common and troublesome weeds in the USA. Palmer amaranth resistance to acetolactate synthase (ALS) inhibitors is widespread in the USA, as in Arkansas. The cross-resistance patterns and mechanism of resistance are not known. Experiments were conducted to determine cross-resistance to ALS inhibitors and identify target-site mutations in 20 Palmer amaranth localities from 13 counties in Arkansas. RESULTS: All Palmer amaranth localities tested had plants cross-resistant to imazethapyr, flumetsulam, primisulfuron, pyrithiobac and trifloxysulfuron. The dose of trifloxysulfuron that caused 50% control was 21-56-fold greater for resistant accessions than for susceptible ones. All but three resistant plants analyzed had one or two relative copies of ALS; one plant had seven relative copies. All resistant plants tested (18 localities) carried the Trp574Leu mutation, which is known to confer broad resistance to ALS inhibitors, supporting the cross-resistance pattern observed. Besides the Trp574Leu mutation, 30% of localities had individuals with one additional resistance-conferring mutation including Ala122Thr, Pro197Ala or Ser653Asn. CONCLUSION: The Trp574Leu mutation in ALS is the primary mechanism of resistance to ALS inhibitors in Palmer amaranth from Arkansas, USA. In some localities, multiple mutations have accumulated in one plant. All localities tested contained plants with resistance to five families of ALS inhibitors. Localities with extremely high resistance to ALS inhibitors, and those outside the subset we studied, may harbor non-target site resistance mechanisms. ALS inhibitors are generally no longer effective on Palmer amaranth in these localities from the US mid-south. © 2018 Society of Chemical Industry.

A Novel Single-Site Mutation in the Catalytic Domain of Protoporphyrinogen Oxidase IX (PPO) Confers Resistance to PPO-Inhibiting Herbicides.

Protoporphyrinogen oxidase (PPO)-inhibiting herbicides are used to control weeds in a variety of crops. These herbicides inhibit heme and photosynthesis in plants. PPO-inhibiting herbicides are used to control Amaranthus palmeri (Palmer amaranth) especially those with resistance to glyphosate and acetolactate synthase (ALS) inhibiting herbicides. While investigating the basis of high fomesafen-resistance in A. palmeri, we identified a new amino acid substitution of glycine to alanine in the catalytic domain of PPO2 at position 399 (G399A) (numbered according to the protein sequence of A. palmeri). G399 is highly conserved in the PPO protein family across eukaryotic species. Through combined molecular, computational, and biochemical approaches, we established that PPO2 with G399A mutation has reduced affinity for several PPO-inhibiting herbicides, possibly due to steric hindrance induced by the mutation. This is the first report of a PPO2 amino acid substitution at G399 position in a field-selected weed population of A. palmeri. The mutant A. palmeri PPO2 showed high-level in vitro resistance to different PPO inhibitors relative to the wild type. The G399A mutation is very likely to confer resistance to other weed species under selection imposed by the extensive agricultural use of PPO-inhibiting herbicides.

High [CO2] and Temperature Increase Resistance to Cyhalofop-Butyl in Multiple-Resistant Echinochloa colona.

Changes in the environment, specifically rising temperature and increasing atmospheric carbon dioxide concentration [CO2], can alter the growth and physiology of weedy plants. These changes could alter herbicide efficacy, crop-weed interaction, and weed management. The objectives of this research were to quantify the effects of increased atmospheric [CO2] and temperature on absorption, translocation and efficacy of cyhalofop-butyl on multiple-resistant (MR) and susceptible (S) Echinochloa colona genotypes. E. colona, or junglerice, is a troublesome weed in rice and in agronomic and horticultural crops worldwide. Cyhalofop-butyl is a grass herbicide that selectively controls Echinochloa spp. in rice. Maximum 14C-cyhalofop-butyl absorption occurred at 120 h after herbicide treatment (HAT) with >97% of cyhalofop-butyl retained in the treated leaf regardless of [CO2], temperature, or genotype. Neither temperature nor [CO2] affected herbicide absorption into the leaf. The translocation of herbicide was slightly reduced in the MR plants vs. S plants either under elevated [CO2] or high temperature. Although plants grown under high [CO2] or high temperature were taller than those in ambient conditions, neither high [CO2] nor high temperature reduced the herbicide efficacy on susceptible plants. However, herbicide efficacy was reduced on MR plants grown under high [CO2] or high temperature about 50% compared to MR plants at ambient conditions. High [CO2] and high temperature increased the resistance level of MR E. colona to cyhalofop-butyl. To mitigate rapid resistance evolution under a changing climate, weed management practitioners must implement measures to reduce the herbicide selection pressure. These measures include reduction of weed population size through reduction of the soil seedbank, ensuring complete control of current infestations with multiple herbicide modes of action in mixture and in sequence, augmenting herbicides with mechanical control where possible, rotation with weed-competitive crops, use of weed-competitive cultivars, use of weed-suppressive cover crops, and other practices recommended for integrated weed management.

RNA-Seq transcriptome analysis of Amaranthus palmeri with differential tolerance to glufosinate herbicide.

Amaranthus palmeri (Amaranthaceae) is a noxious weed in several agroecosystems and in some cases seriously threatens the sustainability of crop production in North America. Glyphosate-resistant Amaranthus species are widespread, prompting the use of alternatives to glyphosate such as glufosinate, in conjunction with glufosinate-resistant crop cultivars, to help control glyphosate-resistant weeds. An experiment was conducted to analyze the transcriptome of A. palmeri plants that survived exposure to 0.55 kg ha-1 glufosinate. Since there was no record of glufosinate use at the collection site, survival of plants within the population are likely due to genetic expression that pre-dates selection; in the formal parlance of weed science this is described as natural tolerance. Leaf tissues from glufosinate-treated and non-treated seedlings were harvested 24 h after treatment (HAT) for RNA-Seq analysis. Global gene expression was measured using Illumina DNA sequence reads from non-treated and treated surviving (presumably tolerant, T) and susceptible (S) plants. The same plants were used to determine the mechanisms conferring differential tolerance to glufosinate. The S plants accumulated twice as much ammonia as did the T plants, 24 HAT. The relative copy number of the glufosinate target gene GS2 did not differ between T and S plants, with 1 to 3 GS2 copies in both biotypes. A reference cDNA transcriptome consisting of 72,780 contigs was assembled, with 65,282 sequences putatively annotated. Sequences of GS2 from the transcriptome assembly did not have polymorphisms unique to the tolerant plants. Five hundred sixty-seven genes were differentially expressed between treated T and S plants. Of the upregulated genes in treated T plants, 210 were more highly induced than were the upregulated genes in the treated S plants. Glufosinate-tolerant plants had greater induction of ABC transporter, glutathione S-transferase (GST), NAC transcription factor, nitronate monooxygenase (NMO), chitin elicitor receptor kinase (CERK1), heat shock protein 83, ethylene transcription factor, heat stress transcription factor, NADH-ubiquinone oxidoreductase, ABA 8'-hydroxylase, and cytochrome P450 genes (CYP72A, CYP94A1). Seven candidate genes were selected for validation using quantitative real time-PCR. While GST was upregulated in treated tolerant plants in at least one population, CYP72A219 was consistently highly expressed in all treated tolerant biotypes. These genes are candidates for contributing tolerance to glufosinate. Taken together, these results show that differential induction of stress-protection genes in a population can enable some individuals to survive herbicide application. Elevated expression of detoxification-related genes can get fixed in a population with sustained selection pressure, leading to evolution of resistance. Alternatively, sustained selection pressure could select for mutation(s) in the GS2 gene with the same consequence.