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Aryloxyphenoxypropionate (fop), cyclohexadione (dim) and phenylpyrazoline (pinoxaden) herbicides targeting plastid acetyl-CoA carboxylase (ACCase) have been widely used to control grassy weeds. The successful application of these herbicides is significantly hampered by herbicide-resistant weed populations of 42 species (Heap, 2012). Eleven mutations affecting ACCase herbicide sensitivity have been identified (Table 1): I1781L and I1781V, W1999C and W1999L, W2027C, I2041N and I2041V, D2078G, C2088R, and G2096A and G2096S (amino acid residue numbering as in the Alopecurus myosuroides ACCase) with nontarget-site resistance further aggravating the problem (Delye, 2005; Delye et al., 2005, 2008; Zhang & Powles, 2006a,b; Liu et al., 2007; Yu et al., 2007; Kaundun, 2010; Petit et al., 2010; Powles & Yu, 2010; Collavo et al., 2011; Cruz-Hipolito et al., 2011; Scarabel et al., 2011; Beckie & Tardif, 2012).
Table 1. Amino acid residues (G, A, I, L, S & V) involved in herbicide resistance (shaded in green) and binding (conservative substitutions are indicated by orange shading)
All of the known herbicide-resistant mutations occur in the carboxyl transferase (CT) domain of the multidomain plastid ACCase found in grasses (Fig. 1). Plastids of dicotyledonous plants contain herbicide-resistant multisubunit ACCases (Tong, 2012). The multidomain eukaryotic ACCases (plant cytosolic, yeast, human) are mostly resistant to fops and dims. The herbicide-binding sites have been determined by X-ray crystallography of their complexes with the yeast CT domain (Zhang et al., 2004; Xiang et al., 2009; Yu et al., 2010). Fourteen of the 32 amino acid residues found in the partially overlapping binding sites are conserved in all species. Conservative substitutions are allowed for the remaining residues (Table 1). The fitness penalty for mutating the conserved binding-site residues could be high, if not lethal, decreasing ACCase activity below the threshold level required for de novo fatty acid synthesis and plant growth. Only three herbicide-resistant mutations are found for the binding-site residues: residue 1781 in the binding sites of all three classes of herbicides, and 1999 and 2041 in the fop-binding site (Table 1). The remaining four residues that give rise to herbicide resistance are located in the immediate vicinity of 1781, 1999 or 2041 (Fig. 1). The herbicide-binding sites overlap to different degrees with the acetyl-CoA binding site in the CT active site.
Figure 1. Herbicide-binding sites in the carboxyl transferase (CT) domain of yeast ACCase. Position of the herbicide-resistant mutations in multidomain ACCase is shown with amino acid residues numbered according to the Alopecurus myosuroides sequence. In this structure, wildtype residues found in sensitive grasses are shown as gray spheres; colored spheres show major changes introduced by the resistant mutations. Green, loss of the carboxylic group; orange, addition of hydrophilic side chain; pink, loss of methyl group; red, addition of methyl group. In the model, the cysteine sulfur atoms of C1999 and C2027 occupy the same positions as the centers of the tryptophan aromatic ring systems of the corresponding wildtype Trp resides. They are not visible in the orientation shown. The common atoms for each herbicide class are shown as blue spheres and the variable atoms are shown as sticks. An additional carbon atom in sethoxydim, not present in the other dims, is shown as a dark blue sphere. Dotted spheres show residues in the 4Å-herbicide binding pocket (Table 1). Models were prepared using PyMol based on PDB structures 1UYR, 1UYS, 3K8X, 3PGQ and 1OD2 (Zhang et al., 2003, 2004; Xiang et al., 2009; Yu et al., 2010).
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The herbicide-resistant mutations have been studied in several grass species, both in planta and using ACCase enzymatic assays (Delye, 2005; Delye et al., 2005, 2008; Zhang & Powles, 2006a,b; Liu et al., 2007; Yu et al., 2007; Kaundun, 2010; Petit et al., 2010; Powles & Yu, 2010; Collavo et al., 2011; Cruz-Hipolito et al., 2011; Scarabel et al., 2011). Although a link between the mutations and plant herbicide resistance has been established, analysis of plant resistance was often complicated by a lack of proper characterization of the resistant plant populations. Several studies have addressed these problems (Liu et al., 2007; Yu et al., 2007; Delye et al., 2008; Kaundun, 2010; Petit et al., 2010; Scarabel et al., 2011). Homozygous mutants have a higher degree of resistance than heterozygous mutants, leading to an increased survival rate at higher herbicide doses. Multiple resistance mutations in a single population or individual plant affect both the response to specific herbicides and their application rates in field tests. Sequences of all ACCase homoeologs have to be analyzed for possible mutations. ACCase sensitivity to inhibitors has been established as the major molecular determinant of the plant response to herbicide application in the field, but there are many steps that affect the agricultural outcome in a species-dependent manner, such as effective herbicide concentration and formulation, foliar uptake and penetration, translocation and transport to plastids, and finally chemical stability, modification and detoxification in plants.
Here we report the effect of six of the 11 mutations on the sensitivity of wheat (Triticum aestivum) plastid ACCase towards five fops, three dims and pinoxaden, studied by the comparison of IC50 shifts (resistance indices, RIs) determined using yeast ACCase gene-replacement strains to avoid the complexity inherent in the whole-plant experiments.
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Our findings are in agreement with the consensus pattern of herbicide resistance derived from earlier studies (Table 2). With the wide dynamic range of the IC50 shift measurements (better than 100-fold for most herbicides), we can correlate herbicide structure (Fig. S1) with the location of the mutated amino acid residues relative to the herbicide-binding site (Fig. 1) and RI imposed by a particular mutation (Table 2).
The ACCase RI range underlying plant resistance observed in whole plants and in the field is extremely broad. Biotypes with the I1781L mutation exhibit resistance to fops, dims and pinoxaden (Zagnitko et al., 2001; Christoffers et al., 2002; Delye, 2005; White et al., 2005; Liu et al., 2007; Beckie & Tardif, 2012). Their RIs (Table 2) vary from 6 to 17. For the I1781V mutant, RIs vary from 8 to 35. The twofold increase in IR for sethoxydim and tralkoxydim, the only difference compared with I1781L (Table 2), may be sufficient to explain the higher resistance of Phalaris paradoxa carrying the I1781V mutation to dims (Collavo et al., 2011). At the other end of the spectrum, all of the RIs for D2078G exceed 100 (Table 2). Biotypes with this mutation are resistant to all three classes of herbicides (Delye, 2005; Liu et al., 2007; Yu et al., 2007; Kaundun, 2010; Collavo et al., 2011; Cruz-Hipolito et al., 2011; Beckie & Tardif, 2012).
I1781 is part of the binding site of all three classes of herbicides, which explains the cross-resistance and the low RIs caused by replacement of Ile with Leu or Val. D2078 is not a part of either herbicide-binding site, but it is located next to and in contact with I1781. Substituting Asp with Gly at position 2078 can be expected to affect binding of all herbicides with larger RIs as a result of a more substantial structural change.
The W1999C mutation has been reported to affect sensitivity to fenoxaprop (Liu et al., 2007; Beckie & Tardif, 2012), consistent with the RI of 86 (Table 2). Quizalofop resistance has not been tested on W1999C mutant plants, but we recorded an RI of 122 (Table 2). W1999 contributes to the fop-binding site. Fenoxaprop and quizalofop have large two-ring aromatic moieties (Fig. S1) pointing towards W1999 (Fig. 1), explaining why the W1999C mutation affects their binding. Sensitivity to clodinafop is not affected and resistance to diclofop is only modest (RI = 6; Table 2). The W1999C mutation increases sensitivity to haloxyfop (RI = 0.3; Table 2), which differs from clodinafop and diclofop primarily by the presence of a 3-fluoromethyl group (Fig. S1), which is in close proximity to W1999 (Fig. 1). RIs for the three dims are low (2–5) (Table 2), which is consistent with the position of their binding site at a distance from W1999 (Fig. 1). The pinoxaden-binding site is also further away from W1999; the W1999C mutation leads to only moderate resistance to pinoxaden (RI = 7; Table 2).
The W2027C mutation has the strongest effect on sensitivity to fops, with RI of 39 for haloxyfop and over 200 for the remaining fops (Table 2), consistent with the high resistance of W2027C mutant biotypes to fops. RIs for dims and pinoxaden are much lower (8–15; Table 2), consistent with the greater distance between their binding sites and the W1999/W2027 stack (Fig. 1). W2027C mutant biotypes have been reported to be resistant to pinoxaden but either resistant or sensitive to dims (Delye, 2005; Liu et al., 2007; Yu et al., 2007).
A similar pattern of resistance is observed for I2041N: RIs > 100 for fops, 13 for pinoxaden and 5–7 for dims (Table 2). Residue I2041 contributes to the fop-binding site, which explains the strong effect of the Ile to Asn substitution on sensitivity to these herbicides. I2041N mutant biotypes are resistant to fops but either resistant or sensitive to pinoxaden and dims (Delye, 2005; Zhang & Powles, 2006b; Liu et al., 2007; Petit et al., 2010; Cruz-Hipolito et al., 2011; Beckie & Tardif, 2012).
An additive effect of amino acid substitutions at residues critical for herbicide binding explains sensitivity/resistance of other eukaryotic multidomain ACCase. Two amino acid differences, I1781L and D2078G (Table 1), are sufficient to explain resistance of wheat cytosolic ACCase to most of the herbicides tested. Based on the yeast test, wheat cytosolic ACCase (strain C100) is sensitive to only tepraloxydim and modestly to pinoxaden (Table S1).
Toxoplasma gondii ACC1 (apicoplast ACCase) differs from the sensitive grass ACCases at only two of the binding site/resistance residues: Leu and Met, at residues equivalent to wheat plastid ACCase residues 1781 and 2088, respectively (Table 1). The IC50 for T. gondii ACC1 inhibition with haloxyfop and clodinafop is c. 5 μM (Zuther et al., 1999; Jelenska et al., 2002), significantly higher than the corresponding IC50 for wheat plastid ACCase, but similar to the IC50 of the I1781L mutant, 3.2 and 7.5 μM, respectively (Table S1). Met at the residue equivalent to 2088 is clearly not critical for fop sensitivity, but may contribute to T. gondii ACC1 resistance to dims. Arg at position 2088 causes resistance to all three classes of herbicides (Powles & Yu, 2010).
The yeast ACCase is resistant to all three classes of herbicides, as indicated by the lack of growth inhibition of wildtype yeast up to 100 μM (Joachimiak et al., 1997; Nikolskaya et al., 1999) and their weak binding to the yeast CT domain (Zhang et al., 2003, 2004; Xiang et al., 2009; Yu et al., 2010). Yeast ACCase contains three resistance-causing residues, L1781, V2041 and A2096 (Table 1). We have not studied I2041V and G2096A mutants in our yeast system. These mutations were reported to cause resistance to fops but not dims or pinoxaden (Powles & Yu, 2010). Met found in yeast at a residue equivalent to grass residue 2088 probably adds to dim resistance, as suggested by the properties of T. gondii ACC1 (see earlier). Finally, human ACC1 (cytosolic ACCase) and ACC2 (mitochondrial ACCase), which have Ile, Val and Met at positions equivalent to residues 1781, 2041 and 2088, respectively, are resistant to fops and dims (Marjanovic et al., 2010).
Many mutations in the herbicide-binding site or in its immediate vicinity cause different degrees of cross-resistance among and between the three classes of inhibitors. At least some degree of resistance was detected for 51 of 54 herbicide–mutation combinations (Table 2). Resistant populations that arose in many areas of the globe as a result of extensive use of fops and/or dims may be difficult to combat with new chemical classes of herbicides targeting the same enzyme with the same mode of action, as the pinoxaden case illustrates (Kaundun, 2010; Petit et al., 2010; Collavo et al., 2011; Cruz-Hipolito et al., 2011; Scarabel et al., 2011; Beckie & Tardif, 2012). Pinoxaden was introduced in 2006, some 30 yr after introduction of fops and dims. It belongs to a different chemotype, but has the same mode of action. Many weed populations harbor mutant ACCases resistant to pinoxaden with RI in the range of 7–120, for the six mutations reported here (Table 2). Heavy use of pinoxaden, in turn, will accelerate selection of multiple mutants and the spread of resistance to fops and dims. The utility of new herbicides targeting ACCase will depend on their ability to overcome the high degree of cross-resistance already existing in weed populations. Inhibitors targeting ACCase parts other than the CT domain have been developed (Marjanovic et al., 2010; Tong, 2012). Our yeast system can facilitate the discovery and analysis of new herbicides targeting ACCase.