James D. Burton

Inhibition of plant acetyl-coenzyme a carboxylase by the herbicides sethoxydim and haloxyfop

Incorporation of [14C]acetate or [14C]pyruvate into fatty acids in isolated corn seedling chloroplasts was inhibited 90% or greater by 10 microM sethoxydim or 1 microM haloxyfop. At these concentrations, neither sethoxydim nor haloxyfop inhibited [14C]acetate incorporation into fatty acids in isolated pea chloroplasts. Sethoxydim (10 microM) and haloxyfop (1 microM) did not inhibit incorporation of [14C]malonyl-CoA into fatty acids in cell free extracts from corn tissue cultures. Acetyl coenzyme A carboxylase (EC 6.4.1.2) from corn seedling chloroplasts was inhibited by both sethoxydim and haloxyfop, with I50 values of 2.9 and 0.5 microM, respectively. This enzyme in pea was not inhibited by 10 microM sethoxydim or 1 microM haloxyfop.

CHANGES OVER TIME IN THE ALLELOCHEMICAL CONTENT OF TEN CULTIVARS OF RYE (Secale cereale L.)

Published studies focused on characterizing the allelopathy-based weed suppression by rye cover crop mulch have provided varying and inconsistent estimates of weed suppression. Studies were initiated to examine several factors that could influence the weed suppressiveness of rye: kill date, cultivar, and soil fertility. Ten cultivars of rye were planted with four rates of nitrogen fertilization, and tissue from each of these treatment combinations was harvested three times during the growing season. Concentrations of a known rye allelochemical DIBOA (2,4-dihydroxy-1,4-(2H)benzoxazine-3-one) were quantified from the harvested rye tissue using high performance liquid chromatography (HPLC). Phytotoxicity observed from aqueous extracts of the harvested rye tissue correlated with the levels of DIBOA recovered in harvested tissue. The amount of DIBOA in rye tissue varied depending on harvest date and rye cultivar, but was generally lower with all cultivars when rye was harvested later in the season. However, the late maturing variety ‘Wheeler’ retained greater concentrations of DIBOA in comparison to other rye cultivars when harvested later in the season. The decline in DIBOA concentrations as rye matures, and the fact that many rye cultivars mature at different rates may help explain why estimates of weed suppression from allelopathic agents in rye have varied so widely in the literature.

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.

The transport of an intact oligopeptide across adult mammalian jejunum.

The passage of an intact nonapeptide across adult rabbit jejunum mounted in an Ussing Chamber is demonstrated with an HPLC system which resolves the renin inhibitor Pro-His-Pro-Phe-His-Leu-Phe-Val-[3H]Phe from all labelled proteolytic cleavage products. Permeability of the peptide (0.016 cm hr-1) is approximately one-seventh that observed for the actively transported 3-0-methyl glucose (0.104 cm hr-1). Flux of the peptide is not changed by the absence of sodium. This study shows that adult mammalian intestine can transport intact oligopeptides and suggests that it will be feasible to develop orally active peptide drugs.

Absorption, translocation, and metabolism of foliar-applied CGA-362622 in purple and yellow nutsedge (Cyperus rotundus and C. esculentus)

Abstract Studies were conducted to evaluate the absorption, translocation, and metabolism of 14C–CGA-362622 when foliar-applied to purple and yellow nutsedge. Less than 53% of the herbicide was absorbed after 96 h. Both nutsedge species translocated appreciable amounts of herbicide (30%) out of treated leaves. Translocation was both acropetal and basipetal, with at least 25% transported basipetally. Neither nutsedge species translocated more than 4% of applied radioactivity to the tubers and roots. Most of the metabolites formed by the nutsedge species were more polar than 14C–CGA-362622 and averaged 69 and 61% of the radioactivity in purple and yellow nutsedge, respectively. The half-life of CGA-362622 was estimated at 4 h in both purple and yellow nutsedge. Nomenclature: CGA-362622, N-([4,6-dimethoxy-2-pyrimidinyl]carbamoyl)-3-(2,2,2,-trifluoroethoxy)-pyridin-2-sulfonamide sodium salt; purple nutsedge, Cyperus rotundus L. CYPRO; yellow nutsedge, Cyperus esculentus L. CYPES.

Inheritance of Evolved Glyphosate Resistance in a North Carolina Palmer Amaranth (Amaranthus palmeri) Biotype

Inheritance of glyphosate resistance in a Palmer amaranth biotype from North Carolina was studied. Glyphosate rates for 50% survival of glyphosate-resistant (GR) and glyphosate-susceptible (GS) biotypes were 1288 and 58 g ha−1, respectively. These values for F1 progenies obtained from reciprocal crosses (GR×GS and GS×GR were 794 and 501 g ha−1, respectively. Dose response of F1 progenies indicated that resistance was not fully dominant over susceptibility. Lack of significant differences between dose responses for reciprocal F1 families suggested that genetic control of glyphosate resistance was governed by nuclear genome. Analysis of F1 backcross (BC1F1) families showed that 10 and 8 BC1F1 families out of 15 fitted monogenic inheritance at 2000 and 3000 g ha−1 glyphosate, respectively. These results indicate that inheritance of glyphosate resistance in this biotype is incompletely dominant, nuclear inherited, and might not be consistent with a single gene mechanism of inheritance. Relative 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) copy number varied from 22 to 63 across 10 individuals from resistant biotype. This suggested that variable EPSPS copy number in the parents might be influential in determining if inheritance of glyphosate resistance is monogenic or polygenic in this biotype.

Absorption, Translocation, and Metabolism of Glufosinate in Transgenic and Nontransgenic Cotton, Palmer Amaranth (Amaranthus palmeri), and Pitted Morningglory (Ipomoea lacunosa)

Abstract Greenhouse studies were conducted to evaluate absorption, translocation, and metabolism of 14C-glufosinate in glufosinate-resistant cotton, nontransgenic cotton, Palmer amaranth, and pitted morningglory. Cotton plants were treated at the four-leaf stage, whereas Palmer amaranth and pitted morningglory were treated at 7.5 and 10 cm, respectively. All plants were harvested at 1, 6, 24, 48, and 72 h after treatment (HAT). Absorption of 14C-glufosinate was greater than 85% 24 h after treatment in Palmer amaranth. Absorption was less than 30% at all harvest intervals for glufosinate-resistant cotton, nontransgenic cotton, and pitted morningglory. At 24 HAT, 49 and 12% of radioactivity was translocated to regions above and below the treated leaf, respectively, in Palmer amaranth. Metabolites of 14C-glufosinate were detected in all crop and weed species. Metabolism of 14C-glufosinate was 16% or lower in nontransgenic cotton and pitted morningglory; however, metabolism rates were greater than 70% in glufosinate-resistant cotton 72 HAT. Intermediate metabolism was observed for Palmer amaranth, with metabolites comprising 20 to 30% of detectable radioactivity between 6 and 72 HAT. Nomenclature: Glufosinate; Palmer amaranth, Amaranthus palmeri S.Wats. AMAPA; pitted morningglory, Ipomoea lacunosa L. IPOLA; cotton, Gossypium hirsutum L.

Absorption, Translocation, and Metabolism of 14C-Glufosinate in Glufosinate-Resistant Corn, Goosegrass (Eleusine indica), Large Crabgrass (Digitaria sanguinalis), and Sicklepod (Senna obtusifolia)

Abstract Greenhouse studies were conducted to evaluate 14C-glufosinate absorption, translocation, and metabolism in glufosinate-resistant corn, goosegrass, large crabgrass, and sicklepod. Glufosinate-resistant corn plants were treated at the four-leaf stage, whereas goosegrass, large crabgrass, and sicklepod were treated at 5, 7.5, and 10 cm, respectively. All plants were harvested at 1, 6, 24, 48, and 72 h after treatment (HAT). Absorption was less than 20% at all harvest intervals for glufosinate-resistant corn, whereas absorption in goosegrass and large crabgrass increased from approximately 20% 1 HAT to 50 and 76%, respectively, 72 HAT. Absorption of 14C-glufosinate was greater than 90% 24 HAT in sicklepod. Significant levels of translocation were observed in glufosinate-resistant corn, with 14C-glufosinate translocated to the region above the treated leaf and the roots up to 41 and 27%, respectively. No significant translocation was detected in any of the weed species at any harvest timing. Metabolites of 14C-glufosinate were detected in glufosinate-resistant corn and all weed species. Seventy percent of 14C was attributed to glufosinate metabolites 72 HAT in large crabgrass. Less metabolism was observed for sicklepod, goosegrass, and glufosinate-resistant corn, with metabolites composing less than 45% of detectable radioactivity 72 HAT. Nomenclature: Glufosinate, goosegrass, Eleusine indica L. Gaertn., large crabgrass, Digitaria sanguinalis L.; sicklepod, Senna obtusifolia (L.) H.S. Irwin & Barneby.; corn, Zea mays L

Constitutive and inducible bentazon hydroxylation in shattercane (Sorghum bicolor) and Johnsongrass (S. halapense)

Abstract Bentazon hydroxylation was studied in excised shoots and microsomal preparations from shattercane ( Sorghum bicolor ) and Johnsongrass ( S. halapense ). Studies in vivo were conducted using excised shoots from untreated (constitutive) and naphthalic anhydride (NA)-treated (induced) etiolated seedlings. Both shattercane and Johnsongrass rapidly metabolized [ 14 C]bentazon forming two metabolites: A minor metabolite of 6-OH-bentazon and a major metabolite, tentatively identified as the glycosyl conjugate of 6-OH-bentazon. Pretreating the seeds of both species with naphthalic anhydride increased bentazon metabolism in etiolated shoots by twofold. The cytochrome P450 inhibitor tetcyclacis (50 μ M ) reduced bentazon metabolism in shoots from both NA-treated and untreated seedlings greater than 90%. Studies in vitro were conducted using microsomal preparations derived from NA-treated and untreated shattercance and Johnsongrass seedlings. Bentazon metabolism in microsomal preparations from both treated and untreated shattercane and Johnsongrass seedlings produced a single product: 6-OH-bentazon. Pretreatment of either shattercane or Johnsongrass with NA increased bentazon hydroxylation in vitro approximately twofold. Bentazon hydroxylation was nearly linear for 30 min in microsomal assays with both treated and untreated shattercane and Johnsongrass. Tetcyclacis (10 μ M ) and PBO (100 μ M ) inhibited bentazon hydroxylation in microsomes from treated and untreated seedlings from both species. Tridiphane (50 μ M ) did not affect bentazon hydroxylation. Substrate saturation assays demonstrated that bentazon hydroxylation in vitro was saturable. Kinetic constants calculated for bentazon hydroxylation with NA-treated and untreated shattercane were K m = 234 and 317 μ M , respectively, and V max = 3.54 and 2.00 nmol · (mg protein · min) −1 , respectively. Kinetic constants calculated for bentazon hydroxylation with NA-treated and untreated Johnsongrass were K m = 160 μM and 235 μ M , respectively, and V max = 1.25 and 0.68 nmol · (mg protein · min) −1 , respectively. These data suggest that bentazon hydroxylation in NA-treated and untreated shattercane and Johnsongrass is mediated by constitutive and inducible cytochrome P450 monooxygenase(s).