Weed resistance to herbicides has increased exponentially during the past 30 to 40 yr, consequently reducing the number of effective products available to control certain species and populations. Future efforts should target not only the discovery of new protein binding sites and the development of new molecules, but also the revival of old molecules with reduced efficacy due to widespread herbicide resistance. The addition of herbicide synergists that inhibit metabolic pathways or enhance intrinsic plant stress is a possible solution to ameliorate the negative effects caused by the lack of new herbicide chemistries. Glutathione S-transferase (GST) enzymes are involved with numerous herbicide detoxification reactions and plant stress responses. This review approaches the potential use of natural and synthetic GST inhibitors to enhance herbicidal activity or induce crop safety to provide effective, sustainable weed management strategies in the future.

Organophosphate insecticides, which have the capacity to inhibit specific herbicide-degrading (cytochrome P450) enzymes, have been used to explore metabolic herbicide-resistance mechanisms in weeds. This study investigates the response of seven field-selected rigid ryegrass (Lolium rigidum Gaudin) populations to herbicides from three different sites of action in the presence or absence of the P450 inhibitor phorate. Phorate antagonized the thiocarbamate herbicides triallate and prosulfocarb (8-fold increase in LD50) in multiple resistant L. rigidum populations with resistance to three different site-of-action herbicides. In contrast, phorate synergized trifluralin and propyzamide in some populations, reducing the LD50 by 50%. Conversely, treatment with phorate had no significant effect on the LD50 for S-metolachlor or pyroxasulfone (inhibitors of very-long-chain fatty-acid synthesis). Phorate has diverse effects that are herbicide and population dependant in field-selected L. rigidum, suggesting P450 involvement in the metabolism of trifluralin and failure to activate thiocarbamate herbicides in these populations. This research highlights the need for implementation of diverse approaches other than herbicide alone as part of a long-term integrated strategy to reduce the likelihood of metabolism-based resistance to PPI herbicides in L. rigidum.

Microsomes (100,000 g pellet containing mixed membrane fractions but primarily endoplasmic reticulum) were isolated from shoots of corn, shattercane, and woolly cupgrass grown from naphthalic anhydride treated or untreated seed to determine if metabolism of bentazon, nicosulfuron, and primisulfuron could be demonstrated in the preparations. Corn is tolerant of all three herbicides, shattercane is tolerant of bentazon, and woolly cupgrass is tolerant of bentazon and primisulfuron. Naphthalic anhydride treatment was required for detectable bentazon, nicosulfuron, and primisulfuron hydroxylation in corn microsomes and for bentazon hydroxylation in woolly cupgrass microsomes. Bentazon hydroxylation was low, but detectable, in microsomes from shattercane shoots without naphthalic anhydride treatment. Naphthalic anhydride-treated corn microsomes hydroxylated 292, 120, and 52 pmol mg−1 protein min−1 of bentazon, nicosulfuron, and primisulfuron, respectively. Primisulfuron (19 pmol mg−1 protein min−1), but not nicosulfuron, was hydroxylated in woolly cupgrass microsomes. Neither nicosulfuron nor primisulfuron was hydroxylated in shattercane microsomes. Bentazon and primisulfuron inhibited nicosulfuron hydroxylation in corn microsomes. Bentazon, but not nicosulfuron, also inhibited primisulfuron hydroxylation in the corn microsomes. This indicates that the three herbicides can interact at the same cytochrome P-450(s) in corn. Primisulfuron hydroxylation was not inhibited by either bentazon or nicosulfuron in woolly cupgrass microsomes. This suggests that the cytochrome P-450(s) for primisulfuron hydroxylation are different between corn and woolly cupgrass. Also, bentazon hydroxylation in corn and shattercane microsomes was inhibited by the cytochrome P-450 inhibitor tetcyclasis, while that in woolly cupgrass was not. Again, this suggests a difference in the cytochrome P-450(s) responsible for bentazon metabolism among the species. Although absolute conclusions comparing in vitro microsomal activities to whole plant herbicide tolerance cannot be made because it is unknown whether the same cytochrome P-450(s) are studied in microsomes from naphthalic anhydride-treated tissue as are responsible for in vivo herbicide metabolism, there was a broad correlation between metabolism of a particular herbicide in microsomes of a species and the species' tolerance of that herbicide.

Long-term absorption of 14C-atrazine [2-chloro-4 - (ethylamino) - 6 - (isopropylamino) - s - triazine] by excised velvetleaf (Abutilon theophrasti Medic.) roots was investigated. A rapid, initial phase of absorption was followed by a long-term, linear phase of absorption, which resulted in atrazine accumulation in the tissue to concentrations above the external solution. This accumulation did not result from metabolism of the atrazine. Aging of the excised tissue appeared to be responsible for the accumulation. Dinitrophenol, but not cyanide, inhibited accumulation. Atrazine accumulation decreased as the temperature during absorption was decreased. The Q10 the absorption rate was ca. 2.5 between 15 and 25 C. Efflux of atrazine from the tissue occurred in two phases. At 25 C, loss of 80% of the atrazine occurred in 20 min. At 4C, the rate of this efflux was slower and the phase lasted longer. At both 4 and 25 C a second, slower phase of efflux occurred through at least 6 h and resulted in loss of equal amounts of atrazine at the two temperatures. These experiments show that, in addition to rapid absorption of atrazine to a passive equilibrium, excised velvetleaf roots accumulate atrazine by a metabolically dependent process over long periods of time.

Excised roots of corn (Zea mays L. ‘B73 × Mo 17′) and oat (Avena sativa L. ‘Goodfield’) and potato (Solanum tuberosum L. ‘Kennebec’) tuber slices absorbed 14C-atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine] in a similar manner during short (1 to 30 min) absorption periods. Within 30 min, atrazine concentrations in tissues and ambient solution were equal. After 24 h of absorption, corn roots contained the most 14C, followed in order by velvetleaf (Abutilon theophrasti Medic.) roots, oat roots, and potato tuber slices. Corn and velvetleaf accumulated 14C to concentrations above the ambient solution. Efflux of 14C was slower for corn than oat, potato, and velvetleaf. Metabolism of atrazine to hydroxyatrazine [2-hydroxy-4-(ethylamino)-6-(isopropylamino)-s-triazine] accounted for accumulation and slow efflux of 14C in corn. These experiments show that initial, rapid absorption of atrazine is comparable, but long-term accumulation differs among these tissues.

A mathematical model describing the behavior of 14C-haloxyfop {2-[4-[(3-chloro-5-(trifluoromethyl)-2-pyridinyl] oxy] phenoxy] propanoic acid} and its methyl and ethoxyethyl esters on yellow foxtail [Seteria glauca (L.) Beauv. #3 SETLU] has been developed to evaluate volatility, penetration, ester transformation, metabolism, and transport of the chemical in the plant system. The effects of crop oil concentrate, temperature, structure of the molecule, and the addition of bentazon [3-(1-methylethyl)-(1H)-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide] to the application solution on the kinetic rate constants have been studied. Crop oil concentrate was found to dramatically increase penetration at an optimal level of approximately 0.3% (v/v). Significant increases in rate constants were observed at higher temperatures. Esters were rapidly converted to the acid in the plant with conversion of the ethoxyethyl ester being approximately twice as fast as the methyl ester. Subsequent metabolism of the acid proceeded at a moderate rate which was independent of the original ester form of the molecule. All kinetic processes were described by first-order rate constants with the exception of transport which appeared to slow a few hours after application even though acid levels in the plant were high. When bentazon was applied in combination with haloxyfop-methyl, penetration of the ester was somewhat inhibited, while transport of radioactivity associated with the molecule to other plant parts was dramatically reduced.

14C-labeled 4-amino-6-(1,1-dimethylethyl)-3-(ethylthio)-1,2,4-triazin-5(4H)-one (ethiozin)3 was metabolized more rapidly in tolerant than in sensitive wheat, Triticum aestivum L., cultivars. After a 6-h herbicidal pulse, the main metabolites were conjugates at all incubation times up to 48 h. The levels of deaminated and dethioethylated metabolites never exceeded 4% of the extractable radioactivity and also did not differ between tolerant and sensitive plants. On the contrary, 92% of the extractable radioactivity was in conjugates after 24 h in the leaves of tolerant plants compared to 25% in the leaves of sensitive plants. The differently sensitive wheat cultivars conjugated metribuzin, 4-amino-6-(1,1-dimethylethyl)-3-(methylthio)-1,2,4-triazin-5(4H)-one, at about half the rate that was observed with ethiozin. This finding may explain the fact that most wheat cultivars are more sensitive to metribuzin compared with ethiozin.

Phytotoxicity of chlorsulfuron {2-chloro-N-[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino] carbonyl] benzenesulfonamide} in cell cultures of Canada thistle [Cirsium arvense (L.) Scop. # CIRAR] was approximately 30-fold greater than in leafy spurge (Euphorbia esula L. # EPHES) cultures. Differences in chlorsulfuron phytotoxicity in these two species were attributed to large differences in cellular metabolism of the herbicide. Leafy spurge cells metabolized all of the applied 14C-chlorsulfuron within 72 h of treatment, while Canada thistle metabolized less than 2%. Acid hydrolysis of the metabolic products isolated from leaf disks and cell cultures of leafy spurge yielded a radioactive product that cochromatographed with a 2-chlorobenzenesulfonamide standard. Therefore, metabolic transformation occurred on the heterocyclic portion of the molecule. A major metabolite was further characterized as 2-chloro-N-[[4-(hydroxymethyl)-6-methoxy-1,3,5-triazin-2-yl]aminocarbonyl]-benzenesulfonamide by cochromatography with the authentic standard.

Photosynthesis in corn (Zea mays L. ‘Pioneer 3369A’) was inhibited 25% by atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine] after a 2-h root exposure in nutrient solution. Photosynthesis of the treated plants had completely recovered 21.4 h after removal from the treatment solution. Recovery of photosynthesis from second and third successive atrazine treatments required only 10.3 h and 4.0 h, respectively. Atrazine metabolism rates in corn plants after 1, 2, or 3 successive 4-h treatment periods, where each treatment period was followed by a 12-h recovery period, showed increased rates of metabolism of atrazine during each recovery time. An increased level of glutathione-S-atrazine was found following the first 4-h atrazine treatment and 12-h recovery. The enhanced production of GS-atrazine was maintained throughout subsequent exposures and recoveries. An inverse correlation (r2 = 0.992) was found between the increase in GS-atrazine production following each 4-h atrazine exposure and the time required for corn plants to recover from atrazine-induced photosynthesis inhibition. Enhanced detoxification of atrazine by corn plants pretreated with atrazine was indicated. No enhanced tolerance to other herbicides was observed from atrazine pretreatment. This phenomenon is similar to that described for the enhanced detoxification of several insecticides in animal systems.

Behavior of 14C-haloxyfop-methyl {methyl 2-[4-[(3-chloro-5-(trifluoromethyl)-2-pyridinyl] oxy] phenoxy] propanoate} was examined in intact plants of soybean [Glycine max (L.) Merr. ‘Mead’], shattercane [Sorghum bicolor (L.) Moench. ♯ SORVU], and yellow foxtail [Setaria glauca (L.) Beauv. ♯ SETLU] and in cell cultures of soybean and yellow foxtail. Foliar absorption of 14C-haloxyfo-methyl by intact plants was almost complete within 48 h. Significant translocation occurred within 48 h of treatment, with soybean and shattercane plants translocating more 14C than yellow foxtail. Metabolism of 14C-haloxyfop-methyl in intact plants was similar among species. Haloxyfop-methyl was rapidly hydrolyzed to haloxyfop in treated leaves and then translocated to metabolically active areas within the plants. Significant quantities of polar products were also isolated. Soybean cells in suspension culture contained higher levels of 14C than did yellow foxtail cells. Most of the 14C in soybean cells was haloxyfop-methyl, while yellow foxtail contained mostly haloxyfop.

Sorghum [Sorghum bicolor (L.) Moench. ‘G-623 GBR’] bioassays indicated that shoot growth was more susceptible to metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide] and more responsive to the antidote CGA-92194 {α-[(1,3-dioxolan-2-yl-methoxy)-imino] benzeneacetonitrile} than root growth. Seed treatment with CGA-92194 increased seedling shoot tolerance to metolachlor approximately tenfold. CGA-92194 seed treatment enhanced shoot absorption of 14C-metolachlor approximately twofold. Metolachlor was initially metabolized to the glutathione conjugate in untreated shoots and those treated with CGA-92194. However, CGA-92194 seed treatment caused accelerated metolachlor metabolism in the shoot, decreasing metolachlor content and increasing formation of the glutathione conjugate. Cyometrinil {(Z)-α[(cyanomethoxy)imino] benzeneacetonitrile}, flurazole [phenylmethyl 2-chloro-4-(trifluoromethyl)-5-thiazolecarboxylate], naphthalic anhydride (1H,3H-naphtho[1,8-cd]-pyran-1,3-dione), and dichlormid (2,2-dichloro-N,N-di-2-propenylacetamide) also protected sorghum from metolachlor injury and enhanced metolachlor absorption and metabolism. The degree of protection conferred by a particular antidote was correlated with its ability to enhance metabolism of metolachlor in shoot tissue. These results are consistent with the hypothesis that the above antidotes protect sorghum from metolachlor injury by inducing rapid detoxification of metolachlor through conjugation with glutathione.

Antidotes were evaluated under greenhouse conditions for their ability to prevent injury to corn and sorghum from imazethapyr. Corn was more tolerant to imazethapyr and more effectively protected from imazethapyr toxicity than sorghum. Naphthalic anhydride (NA)3, CGA 92194, or flurazole treatment of corn seed reduced plant injury from preemergence applications of imazethapyr. Corn injury from postemergence applications of imazethapyr was decreased by seed treatment with NA or CGA 92194. Sorghum injury from preemergence applications of imazethapyr was not reduced by seed treatment with the antidotes. NA treatment of sorghum seed was the most effective antidote treatment for decreasing injury from postemergence applications of imazethapyr but the level of sorghum protection was much less than that achieved with corn. Corn seedlings grown from NA-treated and untreated seed absorbed equal amounts of 14C into the roots from nutrient solution containing 14C-imazethapyr. The NA-treated corn plants translocated less of the absorbed 14C to the shoots than the untreated plants. NA treatment of corn seeds increased the rate of imazethapyr conversion to soluble metabolites 2-and 10-fold in the corn seedling roots and shoots, respectively. The increased rate of imazethapyr metabolism in corn following NA seed treatment may be responsible for the protection from imazethapyr toxicity.

Concentrations of clomazone in nutrient solution causing 50% reduction in shoot fresh weights were 325 and 2 μM for soybean and velvetleaf, respectively. Root uptake of 14C–clomazone from nutrient solution and translocation to shoots were similar for the two species. Soybean and velvetleaf contained similar amounts of 14C per plant; however, because velvetleaf had lower tissue weights, concentration of 14C was higher in velvetleaf. Clomazone did not accumulate in roots but was rapidly translocated to shoots of both species. No more than 30% of absorbed clomazone remained in roots at any time in the experiment. Soybean and velvetleaf leaves contained 4.2 and 1.7 nmol clomazone g−1 fresh weight, respectively, and 3.7 and 6.1 nmol clomazone metabolites g−1 fresh weight, respectively. Thus, velvetleaf metabolized clomazone more rapidly than soybean did. Treatment of clomazone metabolites with β-glucosidase demonstrated that metabolism occurred by oxidative cleavage and conjugation of the benzyl moiety in both plant species. One aglycone cochromatographed with 2–chlorobenzylalcohol. The results suggest that reduced absorption or translocation or increased detoxication of clomazone by soybean seedlings is probably not responsible for their tolerance to this herbicide.

Research was conducted to elucidate mechanism(s) of nicosulfuron and primisulfuron selectivity in corn, woolly cupgrass, and shattercane. Corn absorbed less than one half the nicosulfuron and primisulfuron that woolly cupgrass and shattercane absorbed. Acetolactate synthase (ALS) isolated from three species responded similarly to nicosulfuron and primisulfuron. Values for 50% (I50) inhibition of ALS for nicosulfuron were 36.9, 37.2 and 29.5 nM for corn, woolly cupgrass and shattercane, respectively. Primisulfuron I50 values were 13.8, 11.5 and 10.0 nM for corn, woolly cupgrass and shattercane, respectively. Shattercane's I50 was different from corn's but not from that of woolly cupgrass. Corn rapidly metabolized nicosulfuron and primisulfuron, with a half-life of less than 4 h. Shattercane metabolized the herbicides more slowly, with a half-life greater than 72 h for nicosulfuron and 36 h for primisulfuron. Nicosulfuron and primisulfuron half-lives were greater than 72 h and less than 4 h, respectively, in woolly cupgrass. Therefore, selectivity with nicosulfuron and primisulfuron is likely based on metabolism to nonphytotoxic compounds. Corn tolerance to nicosulfuron and primisulfuron was also attributed to reduced herbicide penetration and translocation below the treated leaf.

Translocation and metabolism of 14C-2,4-D [(2,4-dichlorophenoxy)acetic acid] and effects of 2,4-D on protein synthesis were compared in ‘T–68’ (2,4-D tolerant) and ‘Viking’ (susceptible) birdsfoot trefoil (Lotus corniculatus L.) in an attempt to elucidate some tolerance mechanisms. After 14C-2,4-D was applied to upper trifoliate leaves, significantly less 2,4-D was found in stems, in leaves below the treated leaves, and in roots of T–68 compared to Viking. More 2,4-D was bound to alcohol-insoluble cellular constituents of T–68 leaves, stems, and roots. When alcohol-soluble components were fractionated, slightly more 14C water-soluble compounds were found in T–68, indicating further inactivation by glycosylation. No amino acid-2,4-D conjugates were found. The rate of 14CO2 evolution from 14C-2,4-D treated seedlings in T–68 was five times that in Viking. Protein synthesis appeared to be more rapid in T–68 but the relationship to 2,4-D was not clear. In part, 2,4-D resistance in T–68 may result from its ability to inactivate 2,4-D by differential binding and conjugation and by side chain breakdown as indicated by 14CO2 release.

Resistance to imazethapyr was identified in a population of common sunflower that had been treated with imazethapyr for seven consecutive years. The imazethapyr-resistant biotype of common sunflower was approximately 170 times more resistant to imazethapyr than the susceptible biotype based on the rate required for 25% control. Resistance was due to altered acetolactate synthase (ALS) that is less sensitive to imazethapyr. The imazethapyr concentration required to inhibit in vitro ALS activity by 25% was 210–fold higher in the resistant biotype than in the susceptible biotype. Differences in absorption, translocation, and metabolism of imazethapyr in common sunflower biotypes were not sufficient to explain the resistance to imazethapyr.

Phenmedipham metabolism in leaf tissue of sugarbeet (tolerant) and rapeseed (sensitive) was compared. Sugarbeet leaf discs metabolized phenmedipham much more rapidly than rapeseed leaf discs, forming two metabolites of relatively low polarity. The less polar of these (metabolite 21) was a precursor to the other (metabolite 11), and its properties indicate derivation from phenmedipham by a single hydroxylation and monoglycosylation. Synthetic N-hydroxyphenmedipham was converted by both species into a compound that cochromatographs with metabolite 21. Purified metabolite 21 was much less inhibitory to light-driven oxygen evolution by isolated thylakoids of both species than was phenmedipham. Hydroxylation/glycosylation without prior carbamate hydrolysis appears to be a major factor in the tolerance of sugarbeet to phenmedipham.

Chloroacetamide and thiocarbamate herbicides have many properties in common: both herbicide classes are effective only as preemergence herbicides; they inhibit early seedling growth and cause similar injury symptoms in susceptible species; they are detoxified in plants by glutathione conjugation; they have a similar spectrum of selectivity; they can be applied safely in certain susceptible grass crops when applied with antidotes; and they can inhibit the synthesis of lipids, isoprenoids, and other metabolic processes requiring coenzyme A. It can be hypothesized that these similarities are due to the ability of the chloroacetamides and the sulfoxide of thiocarbamates to bind covalently to enzymes, coenzymes, or metabolic intermediates containing sulfhydryl (-SH) groups.

Greenhouse experiments were conducted to determine if the inheritance of bentazon susceptibility in the corn inbred ‘GA209’ is a recessive single gene trait Bentazon was applied at 4.4 kg ai ha-1 plus 1% by vol crop oil concentrate in all experiments. This treatment caused 96 and 30% visual injury and 86 and 50% dry weight loss for corn inbreds GA209 and ‘Ky226,’ respectively. Corn inbreds ‘B73,’ ‘T61,’ Mo17,’ ‘Pa91,’ and ‘CI66’ showed less than 18% injury and 10% dry weight loss and were considered tolerant of bentazon. Single crosses and reciprocal single crosses of GA209 with the other inbreds were considered bentazon tolerant with approximately 10% stunting and tissue necrosis and 20% dry weight reduction resulting from bentazon. Bentazon susceptibility was not maternally inherited. Apparent single recessive gene control of bentazon susceptibility of GA209 was observed for F2 and backcross segregations. However, subsequent analyses of F3 and selfed-backcross populations indicated that two genes, probably located on the same chromosome, controlled the bentazon susceptibility. Duplicate dominant epistasis provided the best fit for injury and dry weight reduction segregations in populations derived from the cross of inbreds GA209 and B73.

Metabolism and phytotoxicity of bentazon by suspension-cultured cells of soybean and velvetleaf were compared. Growth of suspension cells of both species was reduced when the cells were exposed to increasing concentrations of bentazon. However, soybean plants were tolerant and velvetleaf plants were susceptible to postemergence applications of bentazon. After incubation with 1 μM 14C-bentazon for 6 h, soybean and velvetleaf cells in the log phase of the culture growth cycle contained similar levels of 14C (6 nmol/g fresh weight). Of the total 14C in the soybean cells, 57 to 92% was present as the glucosyl conjugates of 6-OH- and 8-OH-bentazon with the remainder present as bentazon; the percentage depended on the phase of the culture growth cycle. Bentazon metabolism was greatest in the stationary phase of growth. Thin, transverse sections of soybean hypocotyl metabolized bentazon to the same two metabolites as soybean suspension cells did. The ratio of 6-O-glucosyl-bentazon to 8-O-glucosyl-bentazon was always greater than 1:1 for both the hypocotyl sections and the suspension cells. Bentazon metabolites were not detected in the velvetleaf cells, the velvetleaf hypocotyl sections, or the media of either species. Soybean suspension-cultured cells appear to be a valid and advantageous system for studying the hydroxylation and glucosylation of bentazon, the primary reactions believed to be responsible for detoxication of the herbicide in tolerant plants.