Donald L. Wyse

Allelic mutations in acetyl-coenzyme A carboxylase confer herbicide tolerance in maize

SummaryThe genetic relationship between acetyl-coenzyme A carboxylase (ACCase; EC 6.4.1.2.) activity and herbicide tolerance was determined for five maize (Zea mays L.) mutants regenerated from tissue cultures selected for tolerance to the ACCase-inhibiting herbicides, sethoxydim and haloxyfop. Herbicide tolerance in each mutant was inherited as a partially dominant, nuclear mutation. Allelism tests indicated that the five mutations were allelic. Three distinguishable herbicide tolerance phenotypes were differentiated among the five mutants. Seedling tolerance to herbicide treatments cosegregated with reduced inhibition of seedling leaf ACCase activity by sethoxydim and haloxyfop demonstrating that alterations of ACCase conferred herbicide tolerance. Therefore, we propose that at least three, and possible five, new alleles of the maize ACCase structural gene (Acc1) were identified based on their differential response to sethoxydim and haloxyfop. The group represented by Acc1-S1, Acc1-S2 and Acc1-S3 alleles, which had similar phenotypes, exhibited tolerance to high rates of sethoxydim and haloxyfop. The Acc1-H1 allele lacked sethoxydim tolerance but was tolerant to haloxyfop, whereas the Acc1-H2 allele had intermediate tolerance to sethoxydim but was tolerant to haloxyfop. Differences in tolerance to the two herbicides among mutants homozygous for different Acc1 alleles suggested that sites on ACCase that interact with the different herbicides do not completely overlap. These mutations in maize ACCase should prove useful in characterization of the regulatory role of ACCase in fatty acid biosynthesis and in development of herbicide-tolerant maize germplasm.

Inhibition of corn acetyl-CoA carboxylase by cyclohexanedione and aryloxyphenoxypropionate herbicides

Abstract Cyclohexanedione (sethoxydim, clethodim) and aryloxyphenoxypropionate (haloxyfop, diclofop, fluazifop, quizalofop) herbicides exhibit selective herbicidal activity on grasses. Dicots are tolerant to these herbicide classes. The effects of haloxyfop and sethoxydim on fatty acid biosynthesis in corn ( Zea may L.) and pea ( Pisum sativum ) seedling chloroplasts were examined. The incorporation of [ 14 C]acetate or [ 14 C]pyruvate into fatty acids in isolated corn seedling chloroplasts was inhibited 90% or greater by 10 μ M sethoxydim or 1 μ M haloxyfop. The incorporation of [ 14 C]acetate into fatty acids in isolated pea chloroplasts was not inhibited by 100 μ M sethoxydim or 10 μ M haloxyfop. The effect of these herbicides on fatty acid synthetase from disrupted corn chloroplasts was assayed using [ 14 C]malonyl-CoA. Sethoxydim (10 μ M ) and haloxyfop (1 μ M ) stimulated incorporation of [ 14 C]malonyl-CoA into fatty acids by approximately 50%. Acetyl-CoA carboxylase from disrupted corn seedling chloroplasts was inhibited by sethoxydim and haloxyfop with I 50 values of 4.7 and 0.5 μ M , respectively. Other aryloxyphenoxypropionate (diclofop, fluazifop, quizalofop) and cyclohexandione (clethodim) herbicides also inhibited this enzyme. Acetyl-CoA carboxylase activity from pea seedling chloroplasts was not inhibited by 1 m M sethoxydim or 0.1 m M haloxyfop. Thus, cyclohexanedione and aryloxyphenoxypropionate herbicides are potent inhibitors of acetyl-CoA carboxylase in corn, a susceptible species; whereas the enzyme from pea, a tolerant species, was tolerant to the herbicides.

Kinetics of inhibition of acetyl-coenzyme A carboxylase by sethoxydim and haloxyfop☆

The mechanism of inhibition of acetyl-CoA carboxylase by sethoxydim and haloxyfop was examined using a semipurified enzyme preparation extracted from Black Mexican Sweet Maize (Zea mays L.) suspension-culture cells. As determined by SDS-PAGE and Western blotting, the enzyme preparation contained a major biotin-containing polypeptide (Mr 222,000) and a minor biotincontaining polypeptide (Mr 73,400). The kinetics of enzyme inhibition by sethoxydim and haloxyfop were determined for the substrates MgATP, HCO3−, and acetyl-CoA. Sethoxydim and haloxyfop were linear, noncompetitive inhibitors for the three substrates, and the pattern of inhibition was similar for both herbicides. The Kis values for sethoxydim were 1.9, 5.6, and 13.3 μM for acetyl-CoA, HCO3−, and MgATP, respectively. The Kis values for haloxyfop were 0.36, 0.87, and 2.89 μM for acetyl-CoA, HCO3−, and MgATP, respectively. For both herbicides, Kis < Kii for acetyl-CoA, whereas Kii < Kis for MgATP and HCO3−. The kinetic data suggest that the transcarboxylation reaction catalyzed by acetyl-CoA carboxylase (acetyl-CoA → malonyl-CoA) is more sensitive to inhibition than is the biotin carboxylation reaction. Kinetic analysis also indicated that sethoxydim and haloxyfop are reversible, mutually exclusive inhibitors of acetyl-CoA carboxylase.

Purification and Characterization of Acetyl-Coenzyme A Carboxylase from Diclofop-Resistant and -Susceptible Lolium multiflorum

Acetyl-coenzyme A carboxylase (ACCase) was purified =100-fold (specific activity 3.5 units mg-1) from leaf tissue of diclofopresistant and -susceptible biotypes of Lolium multiflorum. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified fractions from both biotypes contained a single 206-kD biotinylated polypeptide. The molecular mass of the native enzyme from both biotypes was approximately 520 kD. In some cases the native dimer from both biotypes dissociated during gel filtration to form a subunit of approximately 224 kD. The inclusion of 5% (w/v) polyethylene glycol 3350 (PEG) in the elution buffer prevented this dissociation. Steady-state substrate kinetics were analyzed in both the presence and absence of 5% PEG. For ACCase from both biotypes, addition of PEG increased the velocity 22% and decreased the apparent Km values for acetyl-coenzyme A (acetyl-CoA), but increased the Km values for bicarbonate and ATP. In the presence of PEG, the Km values for bicarbonate and ATP were approximately 35% higher for the enzyme from the susceptible biotype compared with the resistant enzyme. In the absence of PEG, no differences in apparent Km values were observed for the enzymes from the two biotypes. Inhibition constants (Ki app) were determined for CoA, malonyl-CoA, and diclofop. CoA was an S-hyperbolic (slope replots)-I-hyperbolic (intercept replots) noncompetitive inhibitor with respect to acetyl-CoA, with Ki app values of 711 and 795 [mu]M for enzymes from the resistant and susceptible biotypes, respectively. Malonyl-CoA competitively inhibited both enzymes (versus acetyl-CoA) with Ki app values of 140 and 104 [mu]M for ACCase from resistant and susceptible biotypes, respectively. Diclofop was a linear noncompetitive inhibitor of ACCase from the susceptible biotype and a nonlinear, or S-hyperbolic-I-hyperbolic, noncompetitive inhibitor of ACCase from the resistant biotype. For ACCase from the susceptible biotype the slope (Kis) and intercept (Kii) inhibition constants for diclofop versus acetyl-CoA were 0.08 and 0.44 [mu]M, respectively. ACCase from the resistant biotype had a Ki app value of 6.5 [mu]M. At a subsaturating acetyl-CoA concentration of 50 [mu]M, the Hill coefficients for diclofop binding were 0.61 and 1.2 for ACCase from the resistant and susceptible biotypes, respectively. The Hill coefficients for diclofop binding and the inhibitor replots suggest that the resistant form of ACCase exhibits negative cooperativity in binding diclofop. However, the possibility that the nonlinear inhibition of ACCase activity by diclofop in the enzyme fraction isolated from the resistant biotype is due to the presence of both resistant and susceptible forms of ACCase cannot be excluded.

Effects of Acetyl-Coenzyme A Carboxylase Inhibitors on Root Cell Transmembrane Electric Potentials in Graminicide-Tolerant and -Susceptible Corn (Zea mays L.).

Herbicidal activity of aryloxyphenoxypropionate and cyclohexanedione herbicides (graminicides) has been proposed to involve two mechanisms: inhibition of acetyl-coenzyme A carboxylase (ACCase) and depolarization of cell membrane potential. We examined the effect of aryloxyphenoxypropionates (diclofop and haloxyfop) and cyclohexanediones (sethoxydim and clethodim) on root cortical cell membrane potential of graminicide-susceptible and -tolerant corn (Zea mays L.) lines. The graminicide-tolerant corn line contained a herbicide-insensitive form of ACCase. The effect of the herbicides on membrane potential was similar in both corn lines. At a concentration of 50 [mu]M, the cyclohexanediones had little or no effect on the membrane potential of root cells. At pH 6, 50 [mu]M diclofop, but not haloxyfop, depolarized membrane potential, whereas both herbicides (50 [mu]M) dramatically depolarized membrane potential at pH 5. Repolarization of membrane potential after removal of haloxyfop and diclofop from the treatment solution was incomplete at pH 5. However, at pH 6 nearly complete repolarization of membrane potential occurred after removal of diclofop. In graminicide-susceptible corn, root growth was significantly inhibited by a 24-h exposure to 1 [mu]M haloxyfop or sethoxydim, but cell membrane potential was unaffected. In gramincide-tolerant corn, sethoxydim treatment (1 [mu]M, 48 h) had no effect on root growth, whereas haloxyfop (1 [mu]M, 48 h) inhibited root growth by 78%. However, membrane potential was the same in roots treated with 1 [mu]M haloxyfop or sethoxydim. The results of this study indicate that graminicide tolerance in the corn line used in this investigation is not related to an altered response at the cell membrane level as has been demonstrated with other resistant species.