ACCase Gene Research Articles

A SNaPshot assay for the rapid and simple detection of known point mutations conferring resistance to ACCase‐inhibiting herbicides in Lolium spp.

SummaryEvolution of resistance to herbicides in weeds is becoming an increasing problem worldwide. To develop effective strategies for weed control, a thorough knowledge of the basis of resistance is required. Although non‐target‐site‐based resistance is widespread, target site resistance, often caused by a single nucleotide change in the gene encoding the target enzyme, is also a common factor affecting the efficacies of key herbicides. Therefore, fast and relatively simple high‐throughput screening methods to detect target site resistance mutations will represent important tools for monitoring the distribution and evolution of resistant alleles within weed populations. Here, we present a simple and quick method that can be used to simultaneously screen for up to 10 mutations from several target site resistance‐associated codons in a single reaction. As a proof of concept, this SNaPshot multiplex method was successfully applied to the genotyping of nine variable nucleotide positions in the CT domain of the chloroplastic ACCase gene from Lolium multiflorum plants from 54 populations. A total of 10 nucleotide substitutions at seven of these nine positions (namely codons 1781, 1999, 2027, 2041 2078, 2088 and 2096) are known to confer resistance to ACCase‐inhibiting herbicides. This assay has several advantages when compared with other methods currently in use in weed science. It can discriminate between different nucleotide changes at a single locus, as well as screening for SNPs from different target sites by pooling multiple PCR products within a single reaction. The method is scalable, allowing reactions to be carried out in either 96‐ or 384‐well plate formats, thus reducing work time and cost.

Herbicide resistance-endowing ACCase gene mutations in hexaploid wild oat (Avena fatua): insights into resistance evolution in a hexaploid species

Many herbicide-resistant weed species are polyploids, but far too little about the evolution of resistance mutations in polyploids is understood. Hexaploid wild oat (Avena fatua) is a global crop weed and many populations have evolved herbicide resistance. We studied plastidic acetyl-coenzyme A carboxylase (ACCase)-inhibiting herbicide resistance in hexaploid wild oat and revealed that resistant individuals can express one, two or three different plastidic ACCase gene resistance mutations (Ile-1781-Leu, Asp-2078-Gly and Cys-2088-Arg). Using ACCase resistance mutations as molecular markers, combined with genetic, molecular and biochemical approaches, we found in individual resistant wild-oat plants that (1) up to three unlinked ACCase gene loci assort independently following Mendelian laws for disomic inheritance, (2) all three of these homoeologous ACCase genes were transcribed, with each able to carry its own mutation and (3) in a hexaploid background, each individual ACCase resistance mutation confers relatively low-level herbicide resistance, in contrast to high-level resistance conferred by the same mutations in unrelated diploid weed species of the Poaceae (grass) family. Low resistance conferred by individual ACCase resistance mutations is likely due to a dilution effect by susceptible ACCase expressed by homoeologs in hexaploid wild oat and/or differential expression of homoeologous ACCase gene copies. Thus, polyploidy in hexaploid wild oat may slow resistance evolution. Evidence of coexisting non-target-site resistance mechanisms among wild-oat populations was also revealed. In all, these results demonstrate that herbicide resistance and its evolution can be more complex in hexaploid wild oat than in unrelated diploid grass weeds. Our data provide a starting point for the daunting task of understanding resistance evolution in polyploids.

Isolation and expression of genes for acetolactate synthase and acetyl‐CoA carboxylase in Echinochloa phyllopogon, a polyploid weed species

Target-site resistance is the major cause of herbicide resistance to acetolactate synthase (ALS)- and acetyl-CoA carboxylase (ACCase)-inhibiting herbicides in arable weeds, whereas non-target-site resistance is rarely reported. In the Echinochloa phyllopogon biotypes resistant to these herbicides, target-site resistance has not been reported, and non-target-site resistance is assumed to be the basis for resistance. To explore why target-site resistance had not occurred, the target-site genes for these herbicides were isolated from E. phyllopogon, and their expression levels in a resistant biotype were determined. Two complete ALS genes and the carboxyltransferase domain of four ACCase genes were isolated. The expression levels of ALS and ACCase genes were higher in organs containing metabolically active meristems, except for ACC4, which was not expressed in any organ. The differential expression among examined organs was more prominent for ALS2 and ACC2 and less evident for ALS1, ACC1 and ACC3. E. phyllopogon has multiple copies of the ALS and ACCase genes, and different expression patterns were observed among the copies. The existence of three active ACCase genes and the difference in their relative expression levels could influence the occurrence of target-site resistance to ACCase inhibitors in E. phyllopogon.

Molecular bases for resistance to acetyl‐coenzyme A carboxylase inhibitor in Japanese foxtail (Alopecurus japonicus)

Haloxyfop-R-methyl is a widely used herbicide to control Poaceae weeds. Alopecurus japonicus, a widespread annual grass, can no longer be controlled by haloxyfop-R-methyl after continuous use of this herbicide for several years. Dose-response experiments have established that the Js-R biotype of A. japonicas has evolved resistance to aryloxyphenoxypropionates (APPs). Target-site enzyme sensitivity experiments have established that the haloxyfop (free acid) rate causing 50% inhibition of acetyl-CoA carboxylase (ACCase) activity (I(50)) for the resistant (Js-R) biotype is 11 times higher than that for the susceptible (Js-S) biotype. In many cases, resistance to ACCase-inhibiting herbicides is due to a resistant ACCase enzyme. Full-length DNA and mRNA sequences of the plastidic ACCase gene were amplified. Eight single-nucleotide differences were detected in this region. Four of the nucleotide changes were silent mutations. However, the other four nucleotide mutations caused four amino acid substitutions, replacing Arg-1734 with Gly, Met-1738 with Leu, Thr-1739 with Ser and Ile-2041 with Asn in the R biotype respectively; the substitution at position 2041 had been reported, while the other three had not. The ACCase in the Js-R biotype was less susceptible to haloxyfop-R-methyl than that in the Js-S biotype. Moreover, the amino acid substitution of Ile-2041 with Asn might confer resistance to haloxyfop-R-methyl in A. japonicas.

Resistance to spiromesifen in Trialeurodes vaporariorum is associated with a single amino acid replacement in its target enzyme acetyl‐coenzyme A carboxylase

Spiromesifen is a novel insecticide and is classed as a tetronic acid derivative. It targets the insects' acetyl-coenzyme A carboxylase (ACCase) enzyme, causing a reduction in lipid biosynthesis. At the time of this publication, there are no reports of resistance to this class of insecticides in insects although resistance has been observed in several mite species. The greenhouse whitefly Trialeurodes vaporariorum (Westwood) is a serious pest of protected vegetable and ornamental crops in temperate regions of the world and spiromesifen is widely used in its control. Mortality rates of UK and European populations of T. vaporariorum to spiromesifen were calculated and up to 26-fold resistance was found. We therefore sought to examine the molecular mechanism underlying spiromesifen resistance in this important pest. Pre-treatment with piperonyl butoxide did not synergize spiromesifen, suggesting a target-site resistance mechanism. The full length ACCase gene was sequenced for a range of T. vaporariorum strains and a strong association was found between spiromesifen resistance and a glutamic acid substitution with lysine in position 645 (E645K) of this gene. A TaqMan allelic discrimination assay confirmed these findings. Although this resistance is not considered sufficient to compromise the field performance of spiromesifen, this association of E645K with resistance is the first report of a potential target site mechanism affecting an ACCase inhibitor in an arthropod species.

Genetic Resistance to Acetyl‐Coenzyme A Carboxylase‐Inhibiting Herbicides in Grain Sorghum

ABSTRACTWeed management is one of the most important considerations impacting sorghum [Sorghum bicolor (L.) Moench] production in the United States today. The objectives of this study were to evaluate the level of resistance, type of inheritance, and causal mutation of wild sorghum accessions that are resistant to acetyl‐coenzyme A carboxylase (ACCase)‐inhibiting herbicides compared to susceptible grain sorghum seed parents. Acetyl‐coenzyme A carboxylase‐inhibiting herbicides used in this study were fluazifop‐P and quizalofop‐P from the aryloxyphenoxypropionate (APP) family and clethodim and sethoxydim from the cyclohexanedione (CHD) family. Dose–response studies indicated the level of resistance was very high for APP herbicides but low to nonexistent for CHD herbicides. Estimates of the resistance factors to APP herbicides based on herbicide rate required for 50% growth reduction (GR50) were 54 to 64 for homozygous plants and 9 to 19 for heterozygous plants. Resistance to CHD herbicides was very low with resistance factors ranging from one to about five. Genetic segregation studies indicated a single gene is the cause of resistance to APP herbicides. Polymerase chain reaction (PCR) amplicons from the carboxyl transferase (CT) domain of the chloroplast directed ACCase gene were sequenced and a single point mutation that results in a cysteine replacing tryptophan (conversion from tryptophan at amino acid position 2027 to cysteine [Trp‐2027‐Cys]) was discovered. Given the high level of herbicide resistance and simple inheritance, these sources of APP resistance may provide useful herbicide resistance traits for use in sorghum.

Allelic variation of the ACCase gene and response to ACCase‐inhibiting herbicides in pinoxaden‐resistant Lolium spp.

The repeated use of acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicides to control grass weeds has selected for resistance in Lolium spp. populations in Italy. The efficacy of pinoxaden, a recently marketed phenylpyrazoline herbicide, is of concern where resistance to ACCase inhibitors has already been ascertained. ACCase mutations associated with pinoxaden resistance were investigated, and the cross-resistance pattern to clodinafop, haloxyfop, sethoxydim, clethodim and pinoxaden was established on homo/heterozygous plants for four mutant ACCase alleles. Seven different mutant ACCase alleles (1781-Leu, 1999-Leu, 2041-Asn, 2041-Val, 2078-Gly, 2088-Arg and 2096-Ala) and 13 combinations with two types of mutation were detected in the pinoxaden-resistant plants. The 1781-Leu allele appears to confer a dominant resistance to pinoxaden, clodinafop, haloxyfop, sethoxydim and clethodim at 60 g AI ha(-1) . The 2041-Asn and 2041-Val alleles are associated with dominant or partially dominant resistance to FOPs, no substantial resistance to DIMs and a moderate resistance to pinoxaden. The 2088-Arg allele endows a partially dominant resistance to clodinafop, sethoxydim and most likely to pinoxaden. In addition, non-target-site resistance mechanisms seem to be involved in pinoxaden resistance. Almost all the ACCase mutations selected in the field by other ACCase inhibitors are likely to confer resistance to pinoxaden. Although pinoxaden is sometimes able to control FOP-resistant populations, it should not be considered as a sustainable ACCase resistance management tool. The presence of non-ACCase-based resistance mechanisms that could confer resistance to herbicides with different modes of action further complicates the resistance management strategies.

Characterisation and management of Phalaris paradoxa resistant to ACCase-inhibitors

Phalaris paradoxa is a competitive grass commonly found in durum wheat crops of central and southern Italy. Among the 85 populations screened from 1998 to 2008 for resistance to ACCase-inhibitors and graminicide sulfonylureas, 17 resulted as being resistant to at least one ACCase inhibitor while none of the populations showed resistance to sulfonylureas. ACCase resistance in hood canary-grass seems to be spreading rather slowly in Italy. Out of the 17 populations, seven were characterised through outdoor dose-response pot experiments to investigate resistance levels and cross-resistance patterns to ACCase-inhibitors and multiple resistance to other mode of action. Molecular bases of resistance to the recently introduced DEN herbicide pinoxaden were also investigated. Six populations were confirmed to be ACCase-resistant with various cross-resistance patterns. Two populations were resistant to all tested ACCase herbicides, with pinoxaden resistance indexes (RI) based on survival ranging from 22 to 50. The two populations have been molecularly characterised for resistance to pinoxaden. A single point-mutation in the ACCase gene was identified in each population, causing the amino-acid substitutions of Ile1781Val and Asp2078Gly in 0478L and 0025, respectively. The results suggest that resistance of P. paradoxa to pinoxaden is due to an altered target site and different mutations cause different resistance levels. The biological characteristics of the species, mainly self-pollinated, and the absence of multiple resistance allow herbicides with different modes of action to be used for controlling ACCase-resistant populations. Chemical tools should be carefully used within integrated weed management strategies.

Isolation and Characterization of Putative Acetyl-CoA Carboxylases in Arachis hypogaea L.

Candidate genes of multisubunit (MS) and multifunctional (MF) acetyl-CoA carboxylase (ACCase) including accB1, accB2, accC, accA, accD, and MF-ACCase were isolated from peanut by sequencing a full-length cDNA library and homology-based cloning. Primary structures of peanut ACCases were highly conserved, especially biochemical function domains, compared with other higher plants and Escherichia coli. The numbers of homologs of each ACCase gene expressed in developing seed of peanut varied from two to five. Two editing sites of peanut accD were identified by comparing genomic DNA and its corresponding cDNAs. The MS-ACCase of Arachis hypogaea and Arabidopsis thaliana shared a conservative structure of gene organization. Semiquantitative reverse transcription polymerase chain reaction analysis indicated that MS-ACCase and MF-ACCase genes were expressed in all peanut tissues examined, but their mRNA expression level were quite different. In peanut seed development, accC, accA, accD, and MF-ACCase mRNAs were expressed strongly at 60 days after pegging.

Distinct non-target site mechanisms endow resistance to glyphosate, ACCase and ALS-inhibiting herbicides in multiple herbicide-resistant Lolium rigidum

This study investigates mechanisms of multiple resistance to glyphosate, acetyl-coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS)-inhibiting herbicides in two Lolium rigidum populations from Australia. When treated with glyphosate, susceptible (S) plants accumulated 4- to 6-fold more shikimic acid than resistant (R) plants. The resistant plants did not have the known glyphosate resistance endowing mutation of 5-enolpyruvylshikimate-3 phosphate synthase (EPSPS) at Pro-106, nor was there over-expression of EPSPS in either of the R populations. However, [(14)C]-glyphosate translocation experiments showed that the R plants in both populations have altered glyphosate translocation patterns compared to the S plants. The R plants showed much less glyphosate translocation to untreated young leaves, but more to the treated leaf tip, than did the S plants. Sequencing of the carboxyl transferase domain of the plastidic ACCase gene revealed no resistance endowing amino acid substitutions in the two R populations, and the ALS in vitro inhibition assay demonstrated herbicide-sensitive ALS in the ALS R population (WALR70). By using the cytochrome P450 inhibitor malathion and amitrole with ALS and ACCase herbicides, respectively, we showed that malathion reverses chlorsulfuron resistance and amitrole reverses diclofop resistance in the R population examined. Therefore, we conclude that multiple glyphosate, ACCase and ALS herbicide resistance in the two R populations is due to the presence of distinct non-target site based resistance mechanisms for each herbicide. Glyphosate resistance is due to reduced rates of glyphosate translocation, and resistance to ACCase and ALS herbicides is likely due to enhanced herbicide metabolism involving different cytochrome P450 enzymes.

The response of ACCase‐resistant Phalaris paradoxa populations involves two different target site mutations

Summary Phalaris paradoxa (awned canary‐grass) is an aggressive annual winter weed in wheat and other arable crops that is controlled mainly by ACCase‐inhibiting herbicides: cyclohexanediones (DIMs), aryloxyphenoxypropionates (FOPs) and phenylpyrazolines (DENs, e.g. pinoxaden). The selection pressure imposed on the weed populations by repeated use of these herbicides has resulted in the evolution of increased numbers of ACCase‐resistant populations of P. paradoxa in Israel and other countries. Two populations, Revadim (RV) and Mishmar Ha’emek (MH) that were exposed to differing weed and crop management tactics were investigated. Both populations were highly resistant to all FOPs, pinoxaden and cycloxydim, but responded differently to some DIMs. RV plants exhibited much higher resistance to tralkoxydim than MH plants, while showing similar low levels of resistance to tepraloxydim and clethodim. Both populations were equally susceptible to graminicides with other modes of action. The mutations responsible for the observed resistance were identified using PCR‐RFLP and by sequencing the carboxyl transferase domain of the chloroplastic ACCase gene. RV plants possess a substitution of Asp2078 to Gly, whereas in MH population a mixture of Ile2041 to Asn or Asp2078 to Gly was found. Our study demonstrates that lack of herbicide and crop rotation may result in the evolution of diverse target site mutations and differential response of the whole plant to ACCase inhibitors.

Genetic variation and population structure in black‐grass (Alopecurus myosuroides Huds.), a successful, herbicide‐resistant, annual grass weed of winter cereal fields

Black-grass (Alopecurus myosuroides) is an allogamous grass weed common in cereal fields of northern Europe, which developed resistance to a widely used family of herbicides, the ACCase-inhibiting herbicides. Resistance is caused by mutations at the ACCase gene and other, metabolism-based, mechanisms. We investigated the genetic structure of 36 populations of black-grass collected in one region of France (Côte d'Or), using 116 amplified fragment length polymorphism (AFLP) loci and sequence data at the ACCase gene. The samples were characterized for their level of herbicide resistance and genotyped for seven known ACCase mutations conferring resistance. All samples contained herbicide-resistant plants, and 19 contained ACCase mutations. The genetic diversity at AFLP loci was high (H(T) = 0.246), while differentiation among samples was low (F(ST) = 0.023) and no isolation by distance was detected. Genetic diversity within samples did not vary with the frequency of herbicide resistance. A Bayesian algorithm was used to infer population structure. The two genetic clusters inferred were not associated with any geographical structure or with herbicide resistance. A high haplotype diversity (H(d) = 0.873) and low differentiation (G(ST) = 0.056) were observed at ACCase. However, haplotype diversity within samples decreased with the frequency of ACCase-based resistance. We suggest that the genetic structure of black-grass is affected by its recent expansion as a weed. Our data demonstrate that the strong selection imposed by herbicides did not modify the genome-wide genetic structure of an allogamous weed that probably has large effective population sizes. Our study gives keys to a better understanding of the evolution of successful, noxious weeds in modern agriculture.

Six amino acid substitutions in the carboxyl‐transferase domain of the plastidic acetyl‐CoA carboxylase gene are linked with resistance to herbicides in a Lolium rigidum population

The molecular basis of an acetyl-CoA carboxylase (ACCase) target-based resistant Lolium rigidum population (WLR 96) was studied here. The carboxyl-transferase domain of the plastidic ACCase gene from resistant individuals was amplified by PCR and sequenced. The DNA sequences were aligned and compared with a susceptible population. Six amino acid substitutions were identified in the resistant population. The substitution Ile-2041-Asn, known to confer resistance to ACCase-inhibiting herbicides aryloxyphenoxypropionate (APP) in Alopecurus myosuroides, was identified in most resistant plants but it is always linked with other amino acid substitutions. This was confirmed by a cleaved amplified polymorphism (CAP) marker and an allele-specific PCR. The sole amino acid substitution Ile-2041-Asn was not found in this population. It is likely this mutation evolved later among individuals already possessing the other substitutions. Three haplotypes were identified from the resistant population based on the six amino acid combinations, and two are linked with herbicide resistance in this population. The multiple amino acid substitutions including the Ile-2041-Asn form the molecular basis endowing a high degree of resistance to ACCase-inhibiting herbicides in this L. rigidum population.

Differences in the molecular basis of resistance to the cyclohexanedione herbicide sethoxydim in Lolium multiflorum

SummaryResistance to the cyclohexanedione (CHD) herbicide sethoxydim was investigated in two UK Lolium multiflorum populations, Yorks A2 and PYL. Resistance screening experiments demonstrated a qualitative difference in the responses of the two populations to sethoxydim, suggesting that the molecular basis of resistance between them was different. After treatment, Yorks A2 plants were either alive (78% of sample tested) or dead (22% tested) but plants of the PYL population showed two further intermediate categories of response. The level of acetyl‐CoA carboxylase (ACCase) insensitivity directly correlated with the degree of resistance at the whole plant level, indicating that the molecular basis of resistance is associated with differences in ACCase sensitivity in each population. Direct sequencing of the carboxyl transferase domain of the ACCase gene showed that an Ile‐418‐Leu substitution in the L. multiflorum chloroplastic ACCase (GenBank accession number AY710293) confers resistance to sethoxydim in Yorks A2. This corresponds to amino acid residue 1781 in the Alopecurus myosuroides full ACCase sequence. This is the first report of this mutation in this L. multiflorum, which has also been reported in four other grass‐weeds, including L. rigidum. However, no amino acid substitutions were found to be specifically associated with the resistant phenotypes in the PYL population and the molecular basis of resistance in this population remains to be resolved.

The molecular bases for resistance to acetyl co-enzyme A carboxylase (ACCase) inhibiting herbicides in two target-based resistant biotypes of annual ryegrass (Lolium rigidum)

Acetyl-CoA carboxylase (ACCase) (EC.6.4.1.2) is an essential enzyme in fatty acid biosynthesis and, in world agriculture, commercial herbicides target this enzyme in plant species. In nearly all grass species the plastidic ACCase is strongly inhibited by commercial ACCase inhibiting herbicides [aryloxyphenoxypropionate (APP) and cyclohexanedione (CHD) herbicide chemicals]. Many ACCase herbicide resistant biotypes (populations) of L. rigidum have evolved, especially in Australia. In many cases, resistance to ACCase inhibiting herbicides is due to a resistant ACCase enzyme. Two ACCase herbicide resistant L. rigidum biotypes were studied to identify the molecular basis of ACCase inhibiting herbicide resistance. The carboxyl-transferase (CT) domain of the plastidic ACCase gene was amplified by PCR and sequenced. Amino acid substitutions in the CT domain were identified by comparison of sequences from resistant and susceptible plants. The amino acid residues Gln-102 (CAG codon) and Ile-127 (ATA codon) were substituted with a Glu residue (GAG codon) and Leu residue (TTA codon), respectively, in both resistant biotypes. Amino acid positions 102 and 127 within the fragment sequenced from L. rigidum corresponded to amino acid residues 1756 and 1781, respectively, in the A. myosuroides full ACCase sequence. Allele-specific PCR results further confirmed the mutations linked with resistance in these populations. The Ile-to-Leu substitution at position 1781 has been identified in other resistant grass species as endowing resistance to APP and CHD herbicides. The Gln-to-Glu substitution at position 1756 has not previously been reported and its role in herbicide resistance remains to be established.

Nucleotide variability at the acetyl coenzyme A carboxylase gene and the signature of herbicide selection in the grass weed Alopecurus myosuroides (Huds.).

Acetyl coenzyme A carboxylase (ACCase) is the target of highly effective herbicides. We investigated the nucleotide variability of the ACCase gene in a sample of 18 black-grass (Alopecurus myosuroides [Huds.]) populations to search for the signature of herbicide selection. Sequencing 3,396 bp encompassing ACCase herbicide-binding domain in 86 individuals revealed 92 polymorphisms, which formed 72 haplotypes. The ratio of nonsynonymous versus synonymous substitutions was very low, in agreement with ACCase being a vital metabolic enzyme. Within black grass, most nonsynonymous substitutions were related to resistance to ACCase-inhibiting herbicides. Differentiation between populations was strong, in contrast to expectations for an allogamous, annual plant. Significant H tests revealed recent hitchhiking events within populations. These results were consistent with recent and local positive selection. We propose that, although they have only been used since at most 15 black-grass generations, ACCase-inhibiting herbicides have exerted a positive selection targeting resistant haplotypes that has been strong enough to have a marked effect upon ACCase nucleotide diversity. A minimum-spanning network of nonrecombinant haplotypes revealed multiple, independent apparitions of resistance-associated mutations. This study provides the first evidence for the signature of ongoing, recent, pesticide selection upon variation at the gene encoding the targeted enzyme in natural plant populations.

An isoleucine to leucine mutation in acetyl-CoA carboxylase confers herbicide resistance in wild oat.

Wild oat (Avena fatua L.) populations resistant to herbicides that inhibit acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) represent an increasingly important weed control problem. The objective of this study was to determine the ACCase mutation responsible for herbicide resistance in a well-studied wild oat biotype (UMI). A 2039-bp region encompassing the carboxybiotin and acetyl-CoA binding domains of multifunctional plastidic ACCase was analyzed. DNA sequences representing three plastidic ACCase gene loci were isolated from both the resistant UMI and a herbicide-susceptible biotype, consistent with the hexaploid nature of wild oat. Only one nonsynonymous point mutation was found among the resistant wild oat sequences, inferring an isoleucine to leucine substitution. The position of this substitution corresponds to residue 1769 of wheat (Triticum aestivum L.) plastidic ACCase (GenBank accession No. AF029895). Analysis of an F2 population derived from a cross between a herbicide-resistant and a susceptible biotype confirmed co-segregation of herbicide resistance with the mutated ACCase. We conclude that the isoleucine to leucine mutation is responsible for herbicide resistance in UMI wild oat based on a comparison of the substitution site across species and ACCase types. While isoleucine is conserved among plastidic ACCases of herbicide-susceptible grasses, leucine is found in plastidic and cytosolic forms of multifunctional herbicide-resistant ACCase.

An isoleucine to leucine substitution in the ACCase of Alopecurus myosuroides (black-grass) is associated with resistance to the herbicide sethoxydim

The development of herbicide resistance in weeds poses a severe threat to weed control and crop production. For the aryloxyphenoxypropionates and cyclohexanediones which act on the plastidic ACCase of grass weeds, resistance usually occurs by alteration of the target site protein. In three species, Setaria viridis, Lolium rigidum, and Avena fatua, this has been shown to be associated with a mutation in the ACCase gene, resulting in an isoleucine to leucine substitution in the enzyme. Here we report the same amino acid substitution in the ACCase of sethoxydim-resistant Alopecurus myosuroides. The presence of the leucine correlates with a resistant phenotype and the presence of an insensitive ACCase. In one population, plants heterozygous for the mutation showed different levels of sensitivity and in some cases it was demonstrated that both alleles were expressed in the same plant. The fact that the leucine present in the insensitive plastidic ACCases of several graminaceous species with evolved resistance is also present in the naturally occurring insensitive plastidic ACCase of dicotyledons suggests that the selectivity of these herbicides also results from the same amino acid differences.

Phylogenetic analysis of the acetyl-CoA carboxylase and 3-phosphoglycerate kinase loci in wheat and other grasses.

We have applied a two-gene system based on the sequences of nuclear genes encoding multi-domain plastid acetyl-CoA carboxylase (ACCase) and plastid 3-phosphoglycerate kinase (PGK) to study grass evolution. Our analysis revealed that these genes are single-copy in most of the grass species studied, allowing the establishment of orthologous relationships between them. These relationships are consistent with the known facts of their evolution: the eukaryotic origin of the plastid ACCase, created by duplication of a gene encoding the cytosolic multi-domain ACCase gene early in grass evolution, and the prokaryotic (endosymbiont) origin of the plastid PGK. The major phylogenetic relationships among grasses deduced from the nucleotide sequence comparisons of ACCase and PGK genes are consistent with each other and with the milestones of grass evolution revealed by other methods. Nucleotide substitution rates were calculated based on multiple pairwise sequence comparisons. On a relative basis, with the divergence of the Pooideae and Panicoideae subfamilies set at 60 million years ago (MYA), events leading to the Triticum/Aegilops complex occurred at the following intervals: divergence of Lolium (Lolium rigidum) at 35 MYA, divergence of Hordeum (Hordeum vulgare) at 11 MYA and divergence of Secale (Secale cereale) at 7 MYA. On the same scale, gene duplication leading to the multi-domain plastid ACCase in grasses occurred at 129 MYA, divergence of grass and dicot plastid PGK genes at 137 MYA, and divergence of grass and dicot cytosolic PGK genes at 155 MYA. The ACCase and PGK genes provide a well-understood two-locus system to study grass phylogeny, evolution and systematics.

Plastid-localized acetyl-CoA carboxylase of bread wheat is encoded by a single gene on each of the three ancestral chromosome sets.

5'-End fragments of two genes encoding plastid-localized acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) of wheat (Triticum aestivum) were cloned and sequenced. The sequences of the two genes, Acc-1,1 and Acc-1,2, are 89% identical. Their exon sequences are 98% identical. The amino acid sequence of the biotin carboxylase domain encoded by Acc-1,1 and Acc-1,2 is 93% identical with the maize plastid ACCase but only 80-84% identical with the cytosolic ACCases from other plants and from wheat. Four overlapping fragments of cDNA covering the entire coding region were cloned by PCR and sequenced. The wheat plastid ACCase ORF contains 2,311 amino acids with a predicted molecular mass of 255 kDa. A putative transit peptide is present at the N terminus. Comparison of the genomic and cDNA sequences revealed introns at conserved sites found in the genes of other plant multifunctional ACCases, including two introns absent from the wheat cytosolic ACCase genes. Transcription start sites of the plastid ACCase genes were estimated from the longest cDNA clones obtained by 5'-RACE (rapid amplification of cDNA ends). The untranslated leader sequence encoded by the Acc-1 genes is at least 130-170 nucleotides long and is interrupted by an intron. Southern analysis indicates the presence of only one copy of the gene in each ancestral chromosome set. The gene maps near the telomere on the short arm of chromosomes 2A, 2B, and 2D. Identification of three different cDNAs, two corresponding to genes Acc-1,1 and Acc-1,2, indicates that all three genes are transcriptionally active.