In grasses, residues homologous to residues Ile-1,781 and Ile-2,041 in the carboxyl-transferase (CT) domain of the chloroplastic acetyl-coenzyme A (CoA) carboxylase (ACCase) from the grass weed black-grass (Alopecurus myosuroides [Huds.]) are critical determinants for sensitivity to two classes of ACCase inhibitors, aryloxyphenoxypropionates (APPs) and cyclohexanediones. Using natural mutants of black-grass, we demonstrated through a molecular, biological, and biochemical approach that residues Trp-2,027, Asp-2,078, and Gly-2,096 are also involved in sensitivity to ACCase inhibitors. In addition, residues Trp-2,027 and Asp-2,078 are very likely involved in CT activity. Using three-dimensional modeling, we found that the side chains of the five residues are adjacent, located at the surface of the inside of the cavity of the CT active site, in the vicinity of the binding site for APPs. Residues 1,781 and 2,078 are involved in sensitivity to both APPs and cyclohexanediones, whereas residues 2,027, 2,041, and 2,096 are involved in sensitivity to APPs only. This suggests that the binding sites for these two classes of compounds are overlapping, although distinct. Comparison of three-dimensional models for black-grass wild-type and mutant CTs and for CTs from organisms with contrasted sensitivity to ACCase inhibitors suggested that inhibitors fitting into the cavity of the CT active site of the chloroplastic ACCase from grasses to reach their active sites may be tight. The three-dimensional shape of this cavity is thus likely of high importance for the efficacy of ACCase inhibitors.

A 3,300-bp DNA fragment encoding the carboxyl-transferase domain of the multidomain, chloroplastic acetyl-coenzyme A carboxylase (ACCase) was sequenced in aryloxyphenoxypropionate (APP)-resistant and -sensitive Alopecurus myosuroides (Huds.). No resistant plant contained an Ile-1,781-Leu substitution, previously shown to confer resistance to APPs and cyclohexanediones (CHDs). Instead, an Ile-2,041-Asn substitution was found in resistant plants. Phylogenetic analysis of the sequences revealed that Asn-2,041 ACCase alleles derived from several distinct origins. Allele-specific polymerase chain reaction associated the presence of Asn-2,041 with seedling resistance to APPs but not to CHDs. ACCase enzyme assays confirmed that Asn-2,041 ACCase activity was moderately resistant to CHDs but highly resistant to APPs. Thus, the Ile-2,041-Asn substitution, which is located outside a domain previously shown to control sensitivity to APPs and CHDs in wheat (Triticum aestivum), is a direct cause of resistance to APPs only. In known multidomain ACCases, the position corresponding to the Ile/Asn-2,041 residue in A. myosuroides is occupied by an Ile or a Val residue. In Lolium rigidum (Gaud.), we found Ile-Asn and Ile-Val substitutions. The Ile-Val change did not confer resistance to the APP clodinafop, whereas the Ile-Asn change did. The position and the particular substitution at this position are of importance for sensitivity to APPs.

Summary• The geographical structure of resistance to herbicides inhibiting acetyl-coenzyme A carboxylase (ACCase) was investigated in the weed Alopecurus myosuroides (black-grass) across its geographical range to gain insight into the process of plant adaptation in response to anthropogenic selective pressures occurring in agricultural ecosystems.• We analysed 297 populations distributed across six countries in A. myosuroides' main area of occupancy. The frequencies of plants resistant to two broadly used ACCase inhibitors and of seven mutant, resistant ACCase alleles were assessed using bioassays and genotyping, respectively.• Most of the resistance was not endowed by mutant ACCase alleles. Resistance and ACCase allele distribution patterns were characterized by mosaicism. The prevalence of resistance and of ACCase alleles differed among countries.• Resistance clearly evolved by redundant evolution of a set of resistance alleles or genes, most of which remain unidentified. Resistance in A. myosuroides was shaped by variation in the herbicide selective pressure at both the individual field level and the national level.

Summary• Effective herbicide resistance management requires an assessment of the range of spatial dispersion of resistance genes among weed populations and identification of the vectors of this dispersion.• In the grass weed Alopecurus myosuroides (black-grass), seven alleles of the acetyl-CoA carboxylase ( ACCase ) gene are known to confer herbicide resistance. Here, we assessed their respective frequencies and spatial distribution on two nested geographical scales (the whole of France and the French administrative district of Côte d'Or) by genotyping 13 151 plants originating from 243 fields.• Genetic variation in ACCase was structured in local populations at both geographical scales. No spatial structure in the distribution of resistant ACCase alleles and no isolation by distance were detected at either geographical scale investigated.• These data, together with ACCase sequencing and data from the literature, suggest that evolution of A. myosuroides resistance to herbicides occurred at the level of the field or group of adjacent fields by multiple, independent appearances of mutant ACCase alleles that seem to have rather restricted spatial propagation. Seed transportation by farm machinery seems the most likely vector for resistance gene dispersal in A. myosuroides .

We investigated the molecular bases for resistance to several classes of herbicides that bind tubulins in green foxtail (Setaria viridis L. Beauv.). We identified two a-and two b-tubulin genes in green foxtail. Sequence comparison between resistant and sensitive plants revealed two mutations, a leucine-to-phenylalanine change at position 136 and a threonine-to-isoleucine change at position 239, in the gene encoding a2-tubulin. Association of mutation at position 239 with herbicide resistance was demonstrated using near-isogenic lines derived from interspecific pairings between green foxtail and foxtail millet (Setaria italica L. Beauv.), and herbicide sensitivity bioassays combined with allele-specific PCR-mediated genotyping. Association of mutation at position 136 with herbicide resistance was demonstrated using herbicide sensitivity bioassays combined with allele-specific PCR-mediated genotyping. Both mutations were associated with recessive cross resistance to dinitroanilines and benzoic acids, no change in sensitivity to benzamides, and hypersensitivity to carbamates. Using three-dimensional modeling, we found that the two mutations are adjacent and located into a region involved in tubulin dimer-dimer contact. Comparison of three-dimensional a-tubulin models for organisms with contrasted sensitivity to tubulin-binding herbicides enabled us to propose that residue 253 and the vicinity of the side chain of residue 251 are critical determinants for the differences in herbicide sensitivity observed between organisms, and that positions 16, 24, 136, 239, 252, and 268 are involved in modulating sensitivity to these herbicides.