Comparative metabolism of atrazine and EPTC in proso millet (Panicum miliaceum L.) and corn

The purpose of this study was to examine the differential activities of proso millet (Panicum miliaceum L.) and corn (Zea mays L.) with respect to atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-S-triazine] and EPTC (S-ethyldipropyl thiocarbamate) metabolism. GSH-S-transferase was isolated from proso millet shoots and roots. When assayed spectrophotometrically using CDNB (1-chloro 2,4-dinitrobenzene) as a substrate, the shoot enzyme had only 10% of the activity of corn shoot enzyme while the root enzyme had 33% the activity of corn root enzyme. However, when proso millet shoot GSH-S-transferase was assayed in vitro using ¹⁴C-ring-labeled atrazine, it degraded the atrazine to water-soluble products at the same rate as the corn shoot enzyme. Incubation of excised proso millet and corn roots with [¹⁴C]EPTC indicated that uptake of EPTC was similar in both plants. However, proso millet metabolized the EPTC to water-soluble products at only half the rate of corn. Glutathione levels of proso millet roots were 35.9 μg GSH/g fresh wt, compared with 65.4 μg GSH/g fresh wt for corn. However, a 2.5-day pretreatment with R-25788 (N,N-diallyl-2-2-dichloroacetamide) elevated proso millet GSH levels to 62.7 μg GSH/g fresh wt. R-25788 did not elevate the activity of proso millet GSH-S-transferase, in contrast to its effects on corn. We conclude that differences in response to atrazine and EPTC in proso millet and corn are a result of their differential metabolism.     

Biochemical response of inbred and hybrid corn (Zea mays L.) to R-25788 and its distribution with EPTC in corn seedlings

The average endogenous GSH content of eight lines of inbred corn was almost twofold greater than ten varieties of hybrid corn. When inbred and hybrid corn lines were treated with R-25788, the average GSH content increased by 56 and 95%, respectively. R-25788 protected two special inbred corn lines, GT 112 (atrazine susceptible) and GT 112 RfRf (atrazine resistant) from EPTC injury by increasing the GSH content and GSH S-transferase activity in roots. Most of the radiolabel from [¹⁴C]R-25788-treated plants remained in the root tissues whereas the radiolabel in [¹⁴C]EPTC-treated plants was evenly distributed between foliar and root tissues. From radiolabel experiments, hybrid corn seedlings were found to absorb more R-25788 from soil than EPTC. There was no difference between inbred and hybrid corn in the amounts of R-25788 or EPTC taken up or in the enhancement of GSH S-transferase activity caused by R-25788.     

Effects of the herbicide EPTC and the protectant DDCA on incorporation and distribution of [2-14C]acetate into major lipid fractions of maize cell suspension cultures

The rapid effects of the herbicide EPTC (S-ethyl dipropylthiocarbamate) and the protectant DDCA (N,N-diallyl-2,2-dichloroacetamide) on [2-¹⁴C]acetate incorporation into lipids of maize cell cultures were studied in order to determine whether they act at similar sites of lipid synthesis. DDCA, at 0.05 mM and 0.1 mM, increased the incorporation of [2-¹⁴C]acetate into neutral lipids of a total lipid extract within 2 h. It had very little effect on the major polar lipid constituents. DDCA altered neither the distribution of label within the major lipid classes, nor turnover of the major lipids within 2 h. EPTC (0.1 mM) inhibited overall uptake of [2-¹⁴C]acetate into both neutral and polar lipids by about 30% after a 2-h incubation. The major polar lipid affected was an unidentified glycolipid. In addition to reducing the quantity of lipids synthesized, EPTC changed the lipid profile, altering the distribution of label, mainly within the neutral lipid fraction. A crude membrane fraction from maize cells contained both polar lipids and some neutral lipids. DDCA stimulated [2-¹⁴C]acetate incorporation into different lipid species. EPTC inhibited incorporation of [2-¹⁴C]acetate into both neutral and polar membrane lipids but altered significantly only its distribution into neutral lipids. DDCA (0.1 mM) given together with EPTC (0.2 mM) partially counteracted the effect of EPTC within the neutral lipid fraction. It is suggested that DDCA has a rapid effect on lipid synthesis, but it is probably not sufficient to account for the entire mode of action of the protectant.     

Rapid effects of a thiocarbamate herbicide and its dichloroacetamide protectant on macromolecular syntheses and glutathione levels in maize cell cultures

The rapid effects of the thiocarbamate herbicide S-ethyl dipropyl thiocarbamate (EPTC) and the herbicide protectant N,N-diallyl-2,2-dichloroacetamide (DDCA) on macromolecular syntheses and glutathione (GSH) levels in maize cell cultures were studied to determine whether stimulation of GSH could be the primary mechanism of action of DDCA. EPTC (0.5 and 1 mM) reduced incorporation of radioactive precursors within 1 hr after treatment, and affected incorporation of [³H]acetate into lipids more than incorporation of [³H]adenosine into acid-precipitable nucleic acids, or [¹⁴C]protein hydrolysate into protein. [¹⁴C]EPTC was rapidly biotransformed within 8 hr by maize cell suspensions. Measureable decreases in GSH levels following treatment with 1 mM EPTC occurred after 15 hr. DDCA stimulated incorporation of [³H]acetate into lipids within 4 hr but did not affect incorporation of [¹⁴C]protein hydrolysate into protein or [³H]adenosine incorporation into nucleic acids. Measureable increases in GSH following DDCA treatment began after 12 hr. Treatment with EPTC and DDCA in combination inhibited incorporation of [³H]acetate into lipids less than EPTC given alone. Increases in GSH levels could be observed following pretreatments with glutathione precursors, but no protectant activity could be detected, in contrast to treatments with DDCA. It is suggested that DDCA has an initial rapid effect on lipid metabolism followed by a slower effect involving increases in cellular GSH.     

Competition between a thiocarbamate herbicide and herbicide protectants at the level of uptake into maize cells in culture

The rapid interactions between the herbicide S-ethyl dipropyl thiocarbamate (EPTC) and the structurally similar herbicide protectant N,N-diallyl 2,2-dichloroacetamide (DDCA) at the level of herbicide uptake were examined in maize cell cultures. When the two compounds were given simultaneously, DDCA inhibited uptake of [¹⁴C]EPTC into maize cells measured for 30 min. A Lineweaver-Burk plot indicated this inhibition to be competitive. N,N-Diallyl 2-chloroacetamide (CDAA), a compound similar in structure to DDCA, inhibited uptake to a lesser extent. Other protectants having no similarity in structure to either DDCA or EPTC had no inhibitory effect on the uptake of EPTC. The data suggest that competition between DDCA and EPTC for a site of uptake may be related to their similarity in chemical structure. Experiments with metabolic inhibitors suggested that uptake of EPTC is not via an active transport mechanism. We suggest that competition for uptake between EPTC and DDCA may represent the first step in a complex series of interactions between the herbicide and its protectant that contributes to the protection of maize from herbicide injury.     

Dichloroacetamide antidotes enhance thiocarbamate sulfoxide detoxification by elevating corn root glutathione content and glutathione S-transferase activity

Glutathione (GSH) content and GSH S-transferase activity are consistently increased in corn roots on 24-hr exposure of corn seedlings to part per million levels of N,N-diallyl-2,2-dichloroacetamide (R-25788) and related antidotes for thiocarbamate herbicide injury in susceptible corn varieties. This combined enhancement of enzyme activity and cofactor level leads to rapid detoxification of thiocarbamate sulfoxides, which are proposed to be the active herbicidal compounds formed on metabolic sulfoxidation. S-(N,N-Dipropylcarbamyl)-GSH is formed by this enzyme-catalyzed detoxification of EPTC sulfoxide. This hypothesis on antidote mode of action is supported by studies on 32 dichloroacetamides and related compounds and on the concentration- and time-dependent relationships of R-25788 action. The liver GSH content is normal in mice injected with high doses of R-25788, but the content is reduced when EPTC or EPTC sulfoxide is administered. EPTC sulfoxide also carbamoylates the thiol group of coenzyme A in neutral aqueous medium.     

Gibberellin precursor biosynthesis inhibition by EPTC and reversal by R-25788

S-ethyl dipropylthiocarbamate (EPTC) inhibited gibberellic acid (GA) precursor biosynthesis in a cell-free enzyme preparation from unruptured, etiolated sorghum (Sorghum bicolor L. cv. G522 DR) coleoptiles. EPTC, 1 μM, inhibited incorporation of [¹⁴C]mevalonic acid into kaurene 60%, while 10 μM EPTC inhibited ¹⁴C incorporation into kaurene 90%. The precursor of kaurene cyclization (GGPP) increased in ¹⁴C content at both EPTC concentrations. R-25788 reversed the EPTC inhibition of kaurene synthesis. Kaurene oxidation was modified by both EPTC and R-25788. Hypothesized modes of action for EPTC and R-25788 are (a) inhibition of GA synthesis, (b) increased peroxidase activity resulting in increased lignification, (c) increased detoxification by sulfoxidation and carbamoylation, and (d) inhibition of fatty acid synthesis and/or desaturation. These hypotheses are discussed with three of them being incorporated into one working unit which correlates with EPTC and R-25788 symptom phenology. The fourth hypothesis could also fit into this general pattern.     

Inhibition of conversion of geranylgeranyl-chlorophyll to phytol-chlorophyll by S-ethyl dipropylthiocarbamate (EPTC)

The influence of EPTC (S-ethyl dipropylthiocarbamate) on the hydrogenation of geranylgeranylchlorophyll (GG-Chl) to phytol-Chl was studied during the greening (6-, 12-, 18-, 24-, and 48-hr incandescent light exposure) of etiolated wheat [Triticum aestivum (L.) cv “Stacy”] and sorghum [Sorghum bicolor (L.) Moench cv “G 522DR”] seedlings grown in nutrient solution containing ¹⁴C-labeled sodium acetate. Chloroplast pigment synthesis occurred and small quantities of GG-Chl were found in both Chl^?a and Chl^?b. When wheat seedlings were greened for 48 hr in an EPTC concentration series (1 nM to 100 μM), geranylgeraniol (GG) content increased from 11% (control) to 60% (100 μM EPTC) of the isoprenoid alcohol esterified to chlorophyllide a, but Chl-b contained ≤1% GG-Chl at all concentrations of EPTC. Sorghum seedlings greened for 48 hr in the same EPTC concentration series contained about 3% GG (control) while 100 and 40% GG esterified to chlorophyllide a and chlorophyllide b, respectively, after 48 hr exposure to 100 μM EPTC. Thus, EPTC prevented hydrogenation of GG-Chl to phytol-Chl on the Chl molecule more in sorghum than in wheat.     

Metabolism of EPTC by a pure bacterial culture isolated from thiocarbamate-treated soil

A bacterial strain has been isolated from an enhanced thiocarbamate degradation soil and identified as Corynebacterium sp. The strain was capable of rapidly metabolizing EPTC in a liquid culture where the herbicide was the sole source of carbon. Evolution of high quantities of [¹⁴C]carbon dioxide was coupled with a rapid decline of [¹⁴C]EPTC in the medium; after 12 h incubation these accounted for, respectively, 60% and 0% of the recoverable radioactivity. Radioactivity in the polar extract increased gradually up to 20% after 6 h of incubation and then declined slowly. TLC analysis and identification based on comparison to reference compounds showed that the polar extract consisted of EPTC sulfoxide and two conjugates, EPTC-GSH and EPTC-cysteine (1·8%, 3·4%, and 16%, respectively). Piperonyl butoxide and tetcyclasis, but not tridiphane, were found to be effective inhibitors of EPTC metabolism in the bacterial culture, suggesting that the breakdown of EPTC might be carried out by a cytochrome P-450 monooxygenase-type activity. The thiocarbamate extender, dietholate, also strongly inhibited the metabolism of EPTC in bacterial culture. Based on these results it was postulated that the bacteria metabolize EPTC mainly by hydroxylation of the α-propyl carbon finally to release [¹⁴C]carbon dioxide, while EPTC sulfoxidation appears to be a minor route.     

Localizing the action of two thiadiazolyl herbicidal derivatives

Enzymatically isolated leaf cells from navy beans (Phaseolus vulgaris L., cv. “Tuscola”) were used to study the effect of buthidazole (3-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-4-hydroxy-1-methyl-2-imidazolidinone) and tebuthiuron (N-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-N,N′-dimethylurea) on photosynthesis, protein, ribonucleic acid (RNA), and lipid synthesis. The incorporation of NaH¹⁴CO₃, [¹⁴C]leucine, [¹⁴C]uracil, and [¹⁴C]acetic acid as substrates for the respective metabolic process was measured. Time-course and concentration studies included incubation periods of 30, 60, and 120 min and concentrations of 0.1, 1, 10, and 100 μM of both herbicides. Photosynthesis was very sensitive to both buthidazole and tebuthiuron and was inhibited in 30 min by 0.1 μM concentrations. RNA and lipid syntheses were inhibited 50 and 87%, respectively, by buthidazole and 42 and 64%, respectively, by tebuthiuron after 120 min at 100 μM concentration. Protein synthesis was not affected by any herbicide at any concentration or any exposure time period. The inhibitory effects of buthidazole and tebuthiuron on RNA and lipid syntheses may be involved in the ultimate herbicidal action of these herbicidal chemicals.     

S-Ethyl dipropylthiocarbamate, N,N-Diallyl-2-chloroacetamide,and 2-chloroallyl diethyldithiocarbamate influence on sulfhydryl-dependent sucrose accumulation

l-[U-¹⁴C]sucrose accumulation by phloem sieve tube members (PSTM) of wheat (Triticum aestivum L. ‘Holley’) and sorghum (Sorghum bicolor L. ‘G522 DR’) was inhibited by the nonpermeant sulfhydryl inhibitor p-chloromercuribenzenesulfonic acid (PCMBS), and this inhibition was reversed by the permeant sulfhydryl protectants dithiothreitol (DTT) and dithioerythritol (DTE). S-Ethyl dipropylthiocarbamate (EPTC) (≤0.1 mM) did not inhibit [¹⁴C]sucrose accumulation by wheat or sorghum PSTM. N-N-Diallyl-2-chloroacetamide (CDAA) (1 mM) inhibited [¹⁴C]sucrose accumulation by sorghum but not by wheat PSTM. The inhibition of [¹⁴C]sucrose accumulation in sorghum PSTM by the membrane permeant CDAA was reversed by DTT. Sorghum growth was inhibited by <1 μM CDAA. Membrane permeant 2-chloroallyl diethyldithiocarbamate (CDEC) (0.1 mM) inhibited [¹⁴C]sucrose accumulation by PSTM of sorghum but not wheat. The inhibition of sucrose accumulation in sorghum PSTM by 0.1 mM CDEC was reversed by DDT.     

Accelerated degradation of thiocarbamate herbicides in Israeli soils following repeated use of vernolate

Accelerated degradation of vernolate, EPTC and butylate but not of cycloate was detected in soils from three locations in Israel which were treated annually with vernolate. Repeated application of EPTC to soils with and without a history of vernolate application, under laboratory conditions, resulted in a progressive increase in its rate of dissipation with each application. Accelerated degradation of EPTC was also rapidly induced by mixing small amounts (5%) of soil with a history of vernolate treatment with soil that had never received vernolate. Liberation of ¹⁴CO₂ from [¹⁴C]EPTC was more rapid in vernolate-treated soils than in untreated soils, indicating a development of microbial populations in soil capable of rapidly degrading the EPTC. Degradation of [¹⁴C]EPTC was faster in soil previously cropped with maize than in non-cropped soil, but slower in soils cropped with cotton or peanuts.     

In vitro metabolism of pentachloronitrobenzene to pentachloromethylthiobenzene by onion: Characterization of glutathione S-transferase,cysteine C-S lyase,and S-adenosylmethionine methyl transferase activities

Pentachloromethylthiobenzene (PCTA) was synthesized in vitro from pentachloronitrobenzene (PCNB) at pH 7.9 by an enzyme system from onion root that required dithiothreitol, glutathione, and S-adenosylmethionine. The soluble enzyme system was isolated from onion root by ammonium sulfate fractionation and differential centrifugation. The system contained glutathione S-transferase activity with PCNB, C-S lyase activity with S-(pentachlorophenyl)cysteine, S-adenosylmethionine methyl transferase activity with pentachlorothiophenol (PCTP), and presumably several peptidase activities. All activities were stable when the crude enzyme system was stored at ?25°C. Evidence for the following sequence of reactions in PCTA synthesis was presented: PCNB→¹S-(pentachlorophenyl)glutathione→²S-(pentachlorophenyl)-γ-glutamylcysteine→³S-(pentachlorophenyl)cysteine→⁴ PCTP→⁵ PCTA. The first reaction was studied with [¹⁴C]PCNB. Reactions 2–4 were studied with S-([¹⁴C]pentachlorophenyl)glutathione, S-([¹⁴C]pentachlorophenyl)cysteine, and peptide inhibitors. Reaction 5 was studied with [¹⁴C]PCTP, S-[¹⁴C]adenosylmethionine, and inhibitors. The possible use of the enzyme system in the characterization of other glutathione conjugates was discussed.     

Herbicide perfluidone (1,1,1-trifluoro-N-[2-methyl-4-(phenylsulfonyl)phenyl]methanesulfonamide): Its metabolism in rats and chickens

Rats and chickens were each given a single oral dose (10 or 100 mg/kg body wt) of 1,1,1-trifluoro-N-[2-methyl-4-(phenylsulfonyl)phenyl-¹⁴C(U)]methanesulfonamide ([¹⁴C]perfluidone). Depending on the size of the dose, from 8.4 to 36.2% of the [¹⁴C] was eliminated in the urine and from 36.4 to 85.4% was eliminated in the feces within 48 hr after dosing. Less than 1% of the [¹⁴C] given to laying hens as [¹⁴C]perfluidone was present in the eggs produced during the first 96 hr after dosing. The percentage of the administered [¹⁴C] that remained in these animals (body with G.I. tract and contents removed) varied from 0.34 (96 hr after dosing) to 1.68% (48 hr after dosing). ¹⁴C-labeled compunds in the urine and feces from the rats and chickens were purified by solvent extraction, column chromatography, and gas-liquid chromatography, and then identified by infrared and mass spectrometry. The parent compound was the major ¹⁴C-labeled component in the urine and feces of both animals. 1,1,1-Trifluoro-N-[2-methyl-4-(3-hydroxyphenylsulfonyl)phenyl]methanesulfonamide was present in the feces of both animals. The proposed structures of other metabolites were 1,1,1-trifluoro-N-hydroxy-N-[2-methyl-4-(phenylsulfonyl)phenyl]methanesulfonamide (rat urine) and 1,1,1-trifluoro-N-{2-methyl-4-[(methylsulfonyl)-phenylsulfonyl]phenyl}methanesulfonamide (chicken urine).     

Metabolism of methfuroxam (2,4,5-trimethyl-N-phenyl-3-furancarboxamide) in the rat and isolated rat hepatocytes

Isolated rat hepatocytes were incubated for 4 hr with [phenyl-U-¹⁴C]2,4,5-trimethyl-N-phenyl-3-furancarboxamide ([¹⁴C]methfuroxam). ¹⁴C-Labeled metabolites were isolated by solvent extraction, column chromatography, and high-pressure liquid chromatography, and were then characterized by analysis of infrared and mass spectra. Metabolism of [¹⁴C]methfuroxam by isolated hepatocytes included: (1) hydroxylation of the 2-, 4-, and 5-methyl groups on the furan ring; (2) hydroxylation at the para position of the benzene ring; (3) combinations of 1 and 2; (4) the addition of a sulfur-containing adjunct to the methylfuran moiety; and (5) conjugation of 1–4. Rats given a single intragastric dose of [¹⁴C]methfuroxam excreted 56% of the ¹⁴C in the urine and 42% in the feces within 54 hr. Metabolism of [¹⁴C]methfuroxam by the intact rats included: (1) hydroxylation of the methylfuran moiety; (2) hydroxylation of the benzene ring; (3) the addition of S-methyl, methyl sulfoxide, and other sulfur-containing groups to methfuroxam; (4) combinations of 1–3; and (5) conjugation of 1–4.     

Gibberellin influence on membrane disruption by thiocarbamate herbicides

Vernolate (0, 8, 16, 31, 62, 125.0, or 250.0 ppbw) incorporated into sand inhibited the growth of wheat (Triticum aestivum L. cv Holley) at 125.0 ppbw. These growth inhibition and morphological responses were virtually identical to wheat response to EPTC at 125 ppbw. ¹⁴C from vernolate (carbonyl labeled) (125.0 ppbw) was absorbed into wheat seedlings at approximately 1.8 μM on the presumption that the ¹⁴C present was [¹⁴C]vernolate. Since the response of wheat to the thiocarbamate herbicides resembles a gibberellic acid (GA) deficiency and cell enlargement requires the presence of functional plasmalemmas and tonoplasts, the question of membrane disruption by excessive concentrations of thiocarbamate herbicides and potential reversal thereof by GA₃ was studied by measuring the efflux of K⁺, Na⁺, and Mg²⁺. GA₃ (0.003 μM) stimulated lettuce leaf disc growth in diameter and fresh weight. This GA-stimulated increase in size and weight was reversed by 1 mM EPTC. Betacyanin efflux from beet leaf tonoplasts was increased by 1 mM EPTC and this efflux was not reversed by exogenous GA₃ (0.3 μM). This influence by supraoptimal EPTC concentrations was shown to be via membrane disruption, which obviated any possible GA influence by eliminating the functionality of the membranes requisite to the development of a GA response. It is concluded that viable mode-of-action studies must measure physiological responses consistent with the symptomology of herbicide responses normally observed with each herbicide at field concentrations.     

Comparative studies of safeners for the prevention of EPTC injury in maize

The herbicide safener N-dichloroacetyl-1-oxa-4-aza-spiro-4,5-decane (AD-67) is of similar efficiency as the extensively used N.N-diallyl-2,2-dichloroacetamide (R-25788) and the structurally related 3-(dichloroacetyl)-2,2-dimethyl-1,3-oxa-zolidine (AD-2) in reducing EPTC [S-ethyl-N,N-dipropyl (thiocarhamate)] injury to maize (Zea mays L. cv. KSC 360). EPTC treatment produces growth retardation and deformities and inhibits CO₂ fixation. It does not reduce epicuticular lipids appreciably but affects wax arrangement on the leaf surface. When EPTC is applied together with one of these safeners, these injuries are not observed. All three safeners act similarly. Each prevents the herbicide-induced aggregation of epicuticular wax of maize, thereby protecting the plants against the formation of areas where the underlying cuticle layers are exposed and increase in transpiration.     

Comparative effects of three isoxazole-urea herbicidal derivatives on the metabolism of enzymatically isolated soybean leaf cells*

The effects of the herbicide isouron and of its plant degradation products designated as metabolite l {N-[5-(1,1-dimethylethyl)-3-isoxazolyl]-N-methylurea} and metabolite 2 {N-[5-(1,1-dimethylethyl)-3-isoxazolyl]-urea} on the metabolism of enzymatically isolated leaf cells of soybean [Glycine max (L.) Merr., cv. Essex] were compared under laboratory conditions. Photosynthesis, protein synthesis, ribonucleic acid synthesis, and lipid synthesis were assayed by the incorporation of NaH¹⁴CO₃, [¹⁴C]-leucine, [¹⁴C]-uracil, and [¹⁴C]-acetate, respectively, into the isolated cells. Time-course and concentration studies included incubation periods of 30, 60, and 120 min and concentrations of 0.1, 1, 10 and 100 μM of the three herbicides. The urea derivative of isouron (metabolite 2) was the least active of the three compounds. The activity of the mono-methylated derivative of isouron (metabolite 1) was comparable to that of isouron and the sensitivity of the four processes to both chemicals decreased in the order: photosynthesis > ribonucleic acid synthesis > lipid synthesis > protein synthesis. The concentration of isouron that caused a 50% inhibition of photosynthesis of the isolated soybean leaf cells was calculated at 0.51 μM. The effects of isouron and metabolite 1 on photosynthesis, lipid and RNA synthesis appeared to be independent of incubation lime as maximal inhibition occurred within 30 min. Inhibition of protein synthesis by both chemicals was time-dependent, increasing in magnitude with concomitant increases in incubation time.     

Alachlor influence on sorghum growth and gibberellin precursor synthesis

Growth (14 days) of sorghum (Sorghum bicolor L. cv G522 DR) from seed planted in sand into which alachlor [2-chloro-2′,6′-diethyl-N-(methoxymethyl)acetanilide] was uniformly incorporated (0, 0.07, 0.14, 0.28, 0.56, 1.12, 2.24, or 4.48 kg/ha) was reduced by 0.14 kg/ha and severely inhibited (88%) by 0.56 kg/ha while cellular water cotent was not greatly influenced by 0.56 kg/ha. When added into the nutrient solution bathing the roots of 96-hr sorghum seedlings, alachlor (0, 0.0156, 0.0312, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, or 128 ppmw) was not lethal to 14-day-old sorghum at rates up to 32 ppmw (92% survival); however, shoot and root lengths were reduced 43 and 58%, respectively. Alachlor inhibition of sorghum growth appears to be closely associated with inhibition of cell enlargement; the coleoptile is the most susceptible stage of sorghum growth to alachlor. This situation closely resembles growth where gibberellic acid (GA) synthesis is inhibited. [2-¹⁴C]Mevalonic acid ([2-¹⁴C]MVA) incorporation into terpenoid GA precursors was evaluated using a cell-free enzyme system from etiolated sorghum coleoptiles. Alachlor did not inhibit total ¹⁴C incorporation but incorporation of ¹⁴C into kaurenol and sterols was decreased ca 80 and 75%, respectively, by 10^?6M alachlor. Analyses for [¹⁴C]geranylgeraniol (GG), [¹⁴C]farnesol, and [¹⁴C]geraniol contents showed accumulation of [¹⁴C]farnesol and [¹⁴C]GG, and decreased [¹⁴C]geraniol. When seeds to which CGA-43089 [α-(cyanomethoximino)-benzacetonitrile] was applied 8 weeks prior to planting were substituted for untreated seeds, incorporation of [2-¹⁴C]MVA into [¹⁴C]kaurenol was increased by alachlor while [¹⁴C]GG and [¹⁴C]farnesol accumulated and [¹⁴C]geraniol was absent at 10^?6M alachlor. Additionally, sterol content increased in “safened” systems but was still decreased by alachlor. These data demonstrate multiple sites of alachlor activity in the GA and terpenoid biosynthetic pathway.     

Metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide] inhibition of gibberellin precursor biosynthesis

[2-¹⁴C]Mevalonic acid incorporation into gibberellic acid precursors was measured in cell-free extracts from sorghum [Sorghum bicolor (L.) Moench var. G-522 DR] coleoptiles. ¹⁴C incorporation into ent-kaur-16-ene was inhibited ca. 90% by 10^?7 to 10^?4M metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide]. [¹⁴C]Geranylgeraniol (GG) content increased. [¹⁴C]Farnesol content was not altered and [¹⁴C]geraniol content decreased. Total ¹⁴C incorporation was decreased by metolachlor. In the safener [α-(cyanomethoximino)benacetonitrile]-treated sorghum seed coleoptile cell-free system, total ¹⁴C incorporation increased, [¹⁴C]kaurene and relative kaurence content increased 4× up to 10⁵M metolachlor, and [¹⁴C]farnesol, and [¹⁴C]GG contents increased while relative farnesol and relative GG contents were not influenced by metolachlor. Thus, the inhibition of kaurene synthesis by metolachlor was reversed by the safener. Since the biosynthetic processes are mevalonic acid → geraniol → farnesol → GG → copalylol → kaurene, these data corroborate a proposed gibberellic acid biosynthesis inhibition between GG and kaurene as well as a partial blockage between mevalonic acid and geraniol. Thus, a portion of metolachlor-induced growth inhibitions of sorghum could be explicable on the basis of gibberellic acid biosynthesis inhibitions.