A role for glutathione transferases functioning as glutathione peroxidases in resistance to multiple herbicides in black-grass


*For correspondence (fax +44 191 3742417;
e-mail Robert.Edwards@durham.ac.uk).
The Plant Journal (1999) 18(3), 285–292


Black-grass (Alopecurus myosuroides) is a major weed of wheat in Europe, with several populations having acquired resistance to multiple herbicides of differing modes of action. As compared with herbicide-susceptible black-grass, populations showing herbicide cross-resistance contained greatly elevated levels of a specific type I glutathione transferase (GST), termed AmGST2, but similar levels of a type III GST termed AmGST1. Following cloning and expression of the respective cDNAs, AmGST2 differed from AmGST1 in showing limited activity in detoxifying herbicides but high activities as a glutathione peroxidase (GPOX) capable of reducing organic hydroperoxides. In contrast to AmGST2, other GPOXs were not enhanced in the herbicide-resistant populations. Treatment with a range of herbicides used to control grass weeds in wheat resulted in increased levels of hydroperoxides in herbicide-susceptible populations but not in herbicide-resistant plants, consistent with AmGST2 functioning to prevent oxidative injury caused as a primary or secondary effect of herbicide action. Increased AmGST2 expression in black-grass was associated with partial tolerance to the peroxidizing herbicide paraquat. The selective enhancement of AmGST2 expression resulted from a constitutively high expression of the respective gene, which was activated in herbicide-susceptible black-grass in response to herbicide safeners, dehydration and chemical treatments imposing oxidative stress. Our results provide strong evidence that GSTs can contribute to resistance to multiple herbicides by playing a role in oxidative stress tolerance in addition to detoxifying herbicides by catalysing their conjugation with glutathione.


Resistance of grass weeds to multiple herbicides of differing modes of action is increasingly occurring in arable crops around the world and can compromise selective chemical weed control ( Holt et al. 1993 ). Resistance to individual classes of herbicides can often be attributed to specific mutations resulting in target sites with reduced sensitivities to inhibition, as recently demonstrated with dinitroaniline resistance in the weed Eleusine indica, which resulted from a single mutation in tubulin, the target site of this herbicide type ( Anthony et al. 1998 ). Resistance to multiple herbicides in weeds is less well defined, although enhanced herbicide metabolism has been proposed as a likely mechanism ( Holt et al. 1993 ). In plants, the most important steps in herbicide detoxification are catalysed by cytochrome P450 mono-oxygenases (CYPs) and glutathione transferases (GSTs), with the contribution of each of these enzyme systems dependent on the herbicide substrate ( Cole 1994). These detoxifying enzymes are encoded by multi-gene families in crops, with the CYPs catalysing oxidation reactions ( Schuler 1996) while GSTs conjugate electrophilic xenobiotics with glutathione ( Marrs 1996). These detoxification systems are expressed both constitutively and induced in response to herbicide safeners, compounds which increase herbicide tolerance of cereals relative to competing weeds ( Hatzios & Wu 1996). In contrast, grass weeds normally express much lower levels of CYPs and GSTs, and are more sensitive to herbicides because they metabolize them more slowly ( Cole 1994).

Selection for enhanced expression of detoxifying enzymes in competing weeds following repeated applications with selective herbicides has been suggested as a powerful mechanism for evolving cross resistance to differing classes of herbicides ( Holt et al. 1993 ). Herbicide cross-resistance is particularly problematic in grass weeds and has been well documented in black-grass (Alopecurus myosuroides), a competitive weed of wheat in Europe ( Hall et al. 1997 ). In the UK, the pristine Rothamsted black-grass population from Hertfordshire is sensitive to selective herbicides used in wheat, having never been exposed to pesticides ( Hall et al. 1997 ). In contrast, the populations Peldon from Essex and Lincs E1 from Lincolnshire have been treated with herbicides over several years and show resistance to the herbicides fenoxaprop-ethyl, an aryloxyphenoxypropionate inhibitor of acetyl CoA carboxylase, as well as chlorotoluron, a phenylurea which disrupts electron transport in photosystem II ( Cummins et al. 1997b ; Hall et al. 1997 ). The resistance of Lincs E1 and Peldon to these herbicides of differing modes of action cannot be explained by reduced target site sensitivities, leading to the suggestion that enhanced detoxification is a probable cross-resistance mechanism ( Hall et al. 1997 ). In the case of chlorotoluron, as compared with Rothamsted plants, the Peldon population has already been demonstrated to contain enhanced activities of CYPs able to detoxify the herbicide ( Hyde et al. 1996 ). However, CYPs cannot account for resistance to fenoxaprop-ethyl, which is detoxified by glutathione conjugation in wheat and competing weeds ( Tal et al. 1993 ). Studies in wheat have demonstrated that the glutathione conjugation of fenoxaprop-ethyl is catalysed by specific GST isoenzymes ( Cummins et al. 1997a ), and when the activities towards the enzyme were assayed in black-grass, both Peldon and Lincs E1 contained higher conjugating activities towards the herbicide than determined in Rothamsted ( Cummins et al. 1997b ). However, recent metabolism studies have shown that metabolism of fenoxaprop-ethyl cannot account for the high degree of resistance to this herbicide determined in Lincs E1 ( Hall et al. 1997 ).

These apparently contradictory results have led us to reassess the role of GSTs in herbicide resistance in black-grass. GSTs in plants are related to the theta-type GSTs in animals and have assumed multiple functions in counteracting biotic and abiotic stresses ( Marrs 1996). Within the diverse family of GSTs in plants, it is possible to group these enzymes into four major types based on similarities in sequence, with the type I and III isoenzymes being most abundant in the plant species studies to date ( Dixon et al. 1998b ). Although the functions of GSTs in endogenous metabolism remain undefined, there is a consensus that certain plant GSTs have secondary activities as glutathione peroxidases (GPOXs) and are able to protect cells from cytotoxicity by reducing organic hydroperoxides to their corresponding less toxic alcohols ( Dixon et al. 1998b ; Marrs 1996; Roxas et al. 1997 ). As lipid peroxidation is a common consequence of herbicide action ( Hassall 1990), we became interested in the role of GSTs in protecting plants from the oxidative injury caused by herbicides and the potential effect this would have on herbicide resistance in weeds. We now report on the characterization of type I GSTs from black-grass with high GPOX activity which are present in populations showing resistance to multiple herbicides and demonstrate that the expression of these enzymes is induced in response to both oxidative stress and treatment with herbicide safeners.


Identification of GSTs in black-grass populations showing resistance to multiple herbicides

We had previously demonstrated that black-grass contained polypeptides of the correct size for GST subunits which were specifically recognized by an antiserum raised to the wheat (Triticum aestivum L.) type III GST homodimer TaGST1–1 ( Cummins et al. 1997b ). When assayed by Western blotting using the anti-TaGST1–1 serum, the Rothamsted, Peldon and Lincs populations of black-grass all contained a strongly reacting 25 kDa polypeptide, whilst additional weakly recognized 27 and 28 kDa polypeptides were only observed in the herbicide-resistant Peldon and Lincs plants ( Fig. 1a). When an antiserum raised to the maize (Zea mays L.) type I GST heterodimer ZmGST I-II was used, it selectively recognized 27 and 28 kDa polypeptides expressed at high levels in the herbicide-resistant, but not in the herbicide-susceptible plants ( Fig. 1a). When the anti-ZmGST I-II serum was used in Western blots of extracts from a wider panel of black-grass plants with differing herbicide resistance traits, the 27 and 28 kDa polypeptides were identified in all populations showing cross-resistance to herbicides, but were absent in herbicide-susceptible black-grass and in plants showing herbicide resistance due to modified target-site sensitivity ( Fig. 1b). These results suggested that the enhanced expression of GSTs immunogically related to ZmGST I-II in black-grass was associated with resistance to multiple herbicides.

Figure 1.

Western blots of crude extracts from black-grass populations differing in herbicide resistance traits using antisera raised to the type I maize GST ZmGST I-II and the type III wheat GST TaGST1–1.

(a) Analysis of duplicate protein samples from 30-day-old Rothamsted (Roth), Peldon (Pel) and Lincs E1 (Lincs). The lower polypeptide recognized by both antisera had a molecular mass of 25 kDa while the two upper polypetides were 27 and 28 kDa, respectively.

(b) Analysis of 10-day-old black-grass shoots using the anti-ZmGST I-II serum, with samples in lanes 1, 2, 4, 6 and 7 derived from populations showing resistance to both phenyl urea and aryloxyphenoxypropionate herbicides; lane 5 corresponded to a population which showed resistance to aryloxyphenoxypropionates based on insensitivity of the target site, acetyl CoA carboxylase; lanes 3 and 8 came from herbicide-sensitive populations. The immunodetected polypeptides had molecular masses of 27 and 28 kDa.

Cloning and characterization of black-grass GSTs

In view of the difficulties of purifying GSTs to complete homogeneity for detailed characterization from plants containing multiple, but related, isoenzymes ( Cummins et al. 1997a ), cDNAs encoding the different types of GST subunits in black-grass were cloned from seedlings of the herbicide-resistant Peldon population and the respective recombinant enzymes over-expressed in bacteria. Screening of the expression library with antisera raised to TaGST1–1 identified three very similar clones, which were termed AmGST1a, AmGST1b and AmGST1c. The AmGST1a and AmGST1b clones both encoded polypeptides of 235 amino acid residues, with minor variations in sequence, while AmGST1c contained several conservative substitutions and was smaller than the other clones by six amino acid residues. The AmGST1a/b clones encoded a polypeptide of predicted mass 25.5 kDa, showing 46% identity to the maize type III GST ZmGST V ( Fig. 2a). The three AmGST1 clones were expressed as their respective β-galactosidase fusion proteins and all found to have identical GST activities when assayed with the substrates shown in Table 1. Further characterization of this group of clones was limited to AmGST1a. Analysis of the recombinant fusion protein by SDS–PAGE and Western blotting confirmed that AmGST1a was specifically recognized by the anti-TaGST1–1 serum (data not shown), and it was concluded that the AmGST1 clones encoded the 25 kDa GST subunit polypeptide determined in black-grass extracts.

Figure 2.

Predicted amino acid sequences of AmGST clones showing alignment with the most similar GST sequences determined in crop plants.

(a) AmGST1 sequences aligned with the type III maize GST ZmGST V (accession Y12862).

(b) AmGST2 sequences aligned with the type I maize GST ZmGST I (X06754).

Residues present in all sequences are shown with an asterisk while residues differing within the respective AmGST sequences are underlined.

Table 1.  GST activities of pure recombinant AmGSTs toward model xenobiotic and herbicide (fluorodifen and fenoxaprop-ethyl) substrates together with GPOX activities towards organic hydroperoxides
Mean enzyme activity (± SE) (nkat mg–1 protein)
SubstrateAmGST1 AmGST2
  • Enzyme activities were determined in triplicate.

  • a

    13-hydroperoxy-cis-9,trans-11-octadecadienoic acid.

CDNB 680 ± 25 670 ± 36
Crotonaldehyde 0 ± 0 0 ± 0
Ethacrynic acid 0 ± 0 3.69 ± 0.30
Benzyl isothiocyanate0.57 ± 0.10 7.92 ± 0.51
Fluorodifen0.08 ± 0.01 0.01 ± 0.00
Fenoxaprop-ethyl0.06 ± 0.01 0.01 ± 0.00
Cumene hydroperoxide 0 ± 0 4.54 ± 0.67
Linoleic hydroperoxide a 0 ± 016.74 ± 1.74

When the cDNA library was screened with the anti-ZmGST I-II serum, four clones were identified, sequenced and termed AmGST2a, AmGST2b, AmGST2c and AmGST2d, respectively. On the basis of the predicted size of the respective polypeptides and differences in sequences between residues 36–39, the four AmGST2 clones could be grouped into the pairs AmGST2a, AmGST2b and AmGST2c, AmGST2d. Both pairs most closely resembled the type I maize GST ZmGST I ( Fig. 2b) showing 63% and 62% identity, respectively, at the predicted amino acid level. From the Western blotting studies with the anti-ZmGST I-II serum, black-grass apparently contained two immunologically related polypeptides of molecular masses 27 and 28 kDa ( Fig. 1). However, exhaustive screening with this antiserum only identified the AmGST2 group of cDNAs which encoded polypeptides of nearly identical molecular mass (24.8–24.9 kDa). To clarify the correspondence of clones with polypeptides, AmGST2a and AmGST2c, which represented the two subclasses of AmGST2 cDNAs, were subcloned into pET11 and the recombinant GSTs analysed by SDS–PAGE and Western blotting ( Fig. 3). The polypeptides were indistinguishable from one another when probed with the anti-ZmGST I-II serum and co-migrated with the 28 kDa polypeptide observed in the herbicide-resistant black-grass. Subsequently, it was determined that the immunologically related 27 kDa polypeptide present in the herbicide-resistant black-grass was probably a partial degradation product of AmGST2, as prolonged dialysis of crude extracts resulted in an increase in the abundance of the 27 kDa polypeptide and a concomitant decrease in the 28 kDa polypeptide (data not shown).

Figure 3.

Western blot of the recombinant AmGST2 polypeptides as compared with the GST subunits expressed in the herbicide-resistant Peldon black-grass probed with the anti-ZmGST I-II serum following their resolution by SDS–PAGE.

Lane 1: crude extract from Peldon; lane 2: recombinant AmGST2a; lane 3: recombinant AmGST2c with the molecular mass indicated.

The GSTs encoded by AmGST1 and AmGST2 were analysed by gel filtration chromatography and shown to be active as the respective homodimers AmGST1–1 and AmGST2–2. The recombinant enzymes were then purified by affinity chromatography and assayed for activities with xenobiotic substrates including several herbicides ( Table 1). AmGST1–1 showed a similar range of GST activities to type III GSTs isolated from wheat ( Cummins et al. 1997a ), being highly active in conjugating the diphenyl ether herbicide fluorodifen. This GST also showed appreciable activity toward fenoxaprop-ethyl, which is used as a herbicide to control black-grass. Significantly, the AmGST2–2 enzyme, which was enhanced in the herbicide-resistant populations, showed lower activities toward the herbicides, casting further doubt on their relative importance in protecting black-grass plants by accelerating herbicide metabolism. Instead, the possibility that the black-grass GSTs were performing an indirect cytoprotective function resulting in herbicide resistance was considered. GSTs were assayed for their ability to detoxify toxic metabolites arising from oxidative injury ( Table 1). Both enzymes showed no detectable activities towards crotonaldehyde, an alkenal substrate resembling cytotoxic α,β-unsaturated aldehydes derived from the peroxidation of lipids and nucleic acids ( Berhane et al. 1994 ). However, a major difference between the black-grass GSTs was determined when they were assayed for glutathione peroxidase (GPOX) activity. While the constitutively expressed AmGST1–1 had no activity as a GPOX, AmGST2–2 was very active in catalysing the reduction of both cumene hydroperoxide and linoleic acid hydroperoxide ( Table 1).

Relationship between GSTs functioning as GPOXs and herbicide resistance

The association of GPOX activity with the AmGST2–2 isoenzyme enhanced in herbicide-resistant populations indicated that specific GSTs may function to protect black-grass from toxic organic hydroperoxides formed as a consequence of herbicide injury. When GST and GPOX isoenzymes in crude plant extracts were resolved by anion exchange chromatography, GST activity toward the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) predominantly eluted in one peak, with the activities in the herbicide-resistant Peldon being fourfold greater than in susceptible Rothamsted ( Fig. 4a). In Peldon, this major peak of GST activity co-eluted with a peak of GPOX activity which was absent in the extracts from Rothamsted plants ( Fig. 3b). However, both populations contained identical activities of a GPOX which was not associated with GST activity eluting around fraction 10 ( Fig. 3b). Thus, only those GPOXs with GST activities were enhanced in the Peldon population.

Figure 4.

Resolution of GST and GPOX activities in herbicide-susceptible and herbicide-resistant 30-day-old black-grass plants by anion-exchange chromatography.

Crude plant extracts from Rothamsted (○) and Peldon (▴) were applied onto a Q-Sepharose column and bound protein eluted with the increasing concentration of NaCl shown (–). Fractions were assayed for (a) GST activity towards CDNB and (b) GPOX activity towards cumene hydroperoxide.

To determine the association between enhanced GPOX activity and herbicide resistance, plants of the susceptible Rothamsted and resistant Peldon and Lincs populations were analysed for hydroperoxide formation following exposure to both non-selective herbicides and selective herbicides used to control black-grass in wheat ( Table 2). All the herbicides tested caused a significant increase in hydroperoxide content in Rothamsted plants, with the increases being greatest following treatment with the peroxidizing herbicides paraquat, fluorodifen and chlorotoluron. In contrast, the herbicides caused considerably less hydroperoxide formation in the Peldon and Lincs plants. The link between elevated AmGST2 expression, suppression of herbicide-invoked hydroperoxide formation and herbicide resistance was also examined by determining the relative sensitivities of Rothamsted and Peldon plants to paraquat, a herbicide known to cause phytotoxicity by generating reactive oxygen species and organic hydroperoxides ( Iturbe-Ormaetxe et al. 1998 ). When compared with untreated controls, a 48 h treatment with paraquat resulted in a much greater wilting and desiccation in Rothamsted plants than in Peldon ( Fig. 5).

Table 2.  Hydroperoxide content in the shoots of herbicide-resistant and herbicide-susceptible black-grass treated with selective and non-selective herbicides
Hydroperoxide content (μmol g–1 FW)
HerbicideRothamstedPeldonLincs E1
  • Values represent means of triplicate incubations ± SE.

  • a

    Acetyl CoA carboxylase inhibitor;

  • b

    b photosystem I inhibitor;

  • c

    c protoporphyrinogen oxidase inhibitor;

  • d

    d photosystem II inhibitor.

Control1.14 ± 0.170.75 ± 0.080.91 ± 0.10
Selective herbicides
Fenoxaprop-ethyl a2.23 ± 0.140.80 ± 0.100.75 ± 0.16
Clodinafop-propargyl a1.75 ± 0.150.82 ± 0.181.03 ± 0.22
Chlorotoluron b1.93 ± 0.020.71 ± 0.041.45 ± 0.33
Non-selective herbicides
Fluorodifen c3.28 ± 0.250.85 ± 0.050.97 ± 0.06
Paraquat d4.05 ± 0.271.08 ± 0.141.40 ± 0.05
Figure 5.

The effect of a 48 h treatment with 0.1 m m paraquat on Rothamsted (2) and Peldon (4) black-grass, with the respective controls incubated in water alone shown for Rothamsted (1) and Peldon (3).

Mechanisms of enhanced GST expression in herbicide-resistant populations

Total RNA from herbicide-susceptible Rothamsted and herbicide-resistant Peldon plants was analysed by gel-blot analysis, using RNA probes prepared against AmGST1 and AmGST2 ( Fig. 6). AmGST1 transcripts were detected in both populations but were more abundant in Peldon. However, while the AmGST2 probe strongly recognized an mRNA in Peldon, this transcript was scarcely detectable in Rothamsted. This suggested that the enhancement in AmGSTs observed in herbicide-resistant black-grass was due to increased expression of the respective genes. It was then of interest to determine whether other genes encoding enzymes with related anti-oxidant functions to the GPOX activity of AmGST2 were also induced in herbicide-resistant plants. An RNA probe corresponding to a wheat phospholipid hydroperoxide glutathione peroxidase (PHGPOX) was generated. Plant PHGPOXs resemble the selenium-dependent GPOXs in animals and show different specificity toward hydroperoxides than GST–GPOXs ( Eshdat et al. 1997 ). The PHGPOX probe hybridized to a RNA species with similar intensity in both Peldon and Rothamsted ( Fig. 6a), demonstrating that only GSTs with GPOX activity were up-regulated in the herbicide-resistant plants, confirming the results of Fig. 4.

Figure 6.

Differences in expression of black-grass GSTs in herbicide-susceptible (Rothamsted) and herbicide-resistant (Peldon) black-grass.

(a) Northern blot of identical amounts of total RNA from Rothamsted (R) and Peldon (P), probed with (1) AmGST1, (2) AmGST2, (3) TaPHGPOX. Western blots of identical amounts of total shoot protein from shoots of Rothamsted (b) or Peldon (c) using the anti-ZmGST I-II-serum following a 24 or 48 h exposure to the herbicide safener fenchlorazole-ethyl (FCE), desiccation (DRY), 0.1 m m paraquat (PQ) or 0.1 m m rose bengal (RB). Controls (C) consisted of incubating the shoots for 48 h in 0.1% (v/v) acetone. The molecular masses of the polypeptides are indicated, with the 28 kDa polypeptide being AmGST2 and the other polypeptides partial degradation products.

As type I GSTs resembling AmGST2 are enhanced in wheat in response to a diverse range of biotic stresses ( Mauch & Dudler 1993), a potential mechanism for elevated expression could be that the stress-inducible signal(s) which regulate GST expression were permanently activated in herbicide-resistant black-grass. To test this hypothesis, the expression of AmGST2 was monitored by Western blotting in seedlings of Rothamsted and Peldon exposed to a range of chemical and environmental stress treatments known to induce GSTs or impose oxidative stress in plants ( Marrs 1996; Yang et al. 1998 ). All these treatments greatly enhanced immunodetectable AmGST2 subunits in Rothamsted, but this induction was only marginal in Peldon ( Fig. 6b).


Our results show a relationship between GSTs functioning to counteract oxidative stress and resistance to multiple herbicides with differing modes of action in black-grass. A role for GSTs in reducing oxidative injury due to biotic stresses has been suggested for some time by disparate observations that stimuli which impose oxidative stress on plants, such as infection and environmental stresses, induce the expression of selected GST genes (reviewed by Marrs 1996). Furthermore, when a GST with GPOX activity was over-expressed in tobacco, the transgenic plants became more tolerant to chilling and saline conditions ( Roxas et al. 1997 ), which are stresses associated with oxidative injury ( Iturbe et al. 1998 ). However, a similar role for GSTs in counteracting abiotic stress imposed by herbicides has received far less attention due to the protective role of GSTs in catalysing the conjugation and detoxification of many types of herbicides including thiocarbamates, chloro-s-triazines, chloroacetanilides and diphenyl ethers ( Cole 1994). Potential roles in herbicide tolerance for enzymes, including GSTs, which prevent oxidative stress have been proposed in the case of peroxidizing herbicides such as oxyfluorfen ( Knörzer et al. 1996 ), which kill plants due to the generation of reactive oxygen species (ROS). In black-grass, enhanced expression of the AmGST2–2 isoenzyme which showed GPOX activity was associated with a reduced accumulation of hydroperoxides generated by the action of the ROS-generating herbicides paraquat, chlorotoluron and fluorodifen. In the case of paraquat, this reduction in peroxidation resulted in a significant increase in tolerance to the herbicide as compared with herbicide-susceptible Rothamsted plants. Interestingly, herbicides such as fenoxaprop-ethyl and clodinafop-propargyl which inhibit fatty acid synthesis also generated hydroperoxides in herbicide-susceptible black-grass, presumably as a consequence of disrupting primary metabolism. It is interesting to speculate that such peroxidation arising as a secondary consequence of the mode of action of these herbicides may be a primary cause of toxicity, which may explain why enhanced expression of the AmGST2 GPOX resulted in resistance to arylphenoxy propionate inhibitors of fatty acid synthesis.

Enhanced AmGST2 expression in the herbicide-resistant black-grass resulted from a major increase in the abundance of the respective transcript. However, AmGST1 transcripts were only modestly enhanced, while PHGPOX was unaffected, demonstrating that the induction of AmGST2 was specific. The causes of increased AmGST2 transcript abundance were not investigated in detail, but Southern analysis of genomic DNA from Rothamsted and Peldon black-grass plants gave identical patterns and intensities of hybridization, suggesting that the AmGST2 gene had not been amplified in the herbicide-resistant populations (data not shown). Instead, the causes of AmGST2 induction in the herbicide-resistant black-grass appeared to be linked to the permanent up-regulation of the respective gene, which in the herbicide-susceptible populations was responsive to treatments which imposed oxidative stress, such as ROS-generating chemicals and drought stress. The herbicide safener fenchlorazole-ethyl also enhanced AmGST2 expression in black-grass, demonstrating that GST induction by safeners is not exclusive to cereal crops ( Hatzios & Wu 1996). However, our studies have demonstrated that GST induction by herbicide safeners is not associated with significant hydroperoxide formation (unpublished observation), suggesting that these compounds must activate GST expression either by a separate signalling pathway to oxidative stress, or act downstream of early oxidative signalling events. Safeners therefore act to induce GSTs by a distinct mechanism from other xenobiotics such as triiodobenzoic acid, which generate ROS prior to GST enhancement ( Flury et al. 1998 ). Significantly, the black-grass populations showing multiple herbicide resistance were far less responsive to both oxidative stress and safener treatment, which is consistent with the permanent activation of a gene which was normally activated in response to either of these stimuli.

Our results give a further insight into the functioning of GSTs as proteins with broad-ranging cytoprotective functions in preventing oxidative stress in plants and other organisms. It is unlikely that AmGST2 constitutes the only mechanism of multiple herbicide resistance in black-grass, as it has also been reported that the Peldon population shows increased CYP-mediated detoxification of the phenylurea herbicide chlorotoluron ( Hyde et al. 1996 ). However, it is possible that these co-ordinately regulated GST and CYP xenobiotic detoxification systems are components of an anti-oxidant protective system, which have been selected for constitutive expression in crops as contributing towards stress tolerance, and are now appearing as traits in grass weeds as a result of selection resulting from unrotated herbicide usage. Significantly, GSTs resembling AmGST2 are known to be constitutively expressed in maize and other cereal crops to high levels, although their functions have yet to be determined ( Dixon et al. 1998b ). Characterization of the promoter region of the AmGST2 gene and identification of the factors which regulate its expression will now provide further insights into the potential molecular mechanisms by which resistance to multiple herbicides can arise in weeds and suggest strategies to counteract this problem. As a more immediate benefit, immunorecognition of enhanced expression of genes encoding such GST–GPOXs could now be used to diagnose herbicide cross-resistance in black-grass.

Experimental procedures

Plant material and treatment with herbicides and safeners

Seeds of black-grass populations of defined herbicide resistance traits ( Hall et al. 1997 ) were generously donated by Dr S. Moss (IACR-Rothamsted, Harpenden, UK) and grown for 20 days as detailed previously ( Cummins et al. 1997b ). For studies with herbicide safener, cut shoots were placed in 32.5 μm fenchlorazole-ethyl dissolved in 0.1% acetone, or in 0.1% acetone alone, for 72 h. For all herbicides except paraquat, cut shoots were placed in 1 m m treatment solutions prepared from 100 m m stock solutions of the herbicides in acetone, with the control solutions consisting of 1% (v/v) acetone. For paraquat treatments, black-grass shoots were incubated in 0.1 m m paraquat for 24 h then transferred to fresh water and incubated for a further 24 h under constant illumination (150 μmol m–2 sec–1). Plants were treated with rose bengal (0.1 m m) as described for the experiments with paraquat. At harvest, plants were frozen in liquid nitrogen and stored at –80°C.

Assays for hydroperoxides

After a 24 h treatment with herbicide, tissue was ground to a fine powder under liquid nitrogen and extracted in 2 vol of 50 m m potassium phosphate, pH 7.2, containing 1% (v/v) Triton X-100 and 0.1% (w/v) butylhydroxytoluene. After extraction at room temperature for 30 min, samples were centrifuged (10 000 g, 5 min) and 10 μl of the supernatant added to 100 μl of the prepared PeroXOquant reagent (Pierce). After incubation for 20 min, samples were clarified by centrifugation and quantified from their absorbance at 560 nm relative to a standard curve prepared using H2O2 as recommended by the manufacturer.

Cloning and expression of black-grass GSTs

cDNA was prepared from poly(A)+ RNA isolated from the shoots of 20 day-old Peldon plants and cloned into λZAPII (Stratagene). The cDNA library was then immunoscreened on duplicate lifts using antisera raised in rabbits to ZmGST I-II ( Dixon et al. 1998a ) and TaGST1–1 ( Cummins et al. 1997a ). Using these antisera, two groups of clones termed AmGST1 and AmGST2 were identified on the basis of restriction analysis and sequenced on both strands using an ABI automated DNA sequencer. Homology searches were conducted using the BLAST program.

Recombinant AmGST1 polypeptides were expressed as their respective β-galactosidase fusion proteins using the pBluescript plasmid (Stratagene) in E. coli SOLR cells after a 16 h induction with 1 m m IPTG. The AmGST2 inserts were excised from pBluescript, subcloned into pET11a and expressed in BL21 cells after induction for 3 h in the presence of 1 m m IPTG. Bacteria were harvested by centrifugation and sonicated in 100 m m Tris–HCl pH 7.5 containing 2 m m EDTA and 1 m m DTT. After centifugation, the proteins in the supernatant were precipitated by the addition of ammonium sulphate to 80% saturation. Recombinant GSTs were then purified by a combination of affinity chromatography on glutathione agarose and anion-exchange chromatography as described previously for GSTs isolated from plants ( Cummins et al. 1997a ).

GST and GPOX analysis

GST and GPOX activities were extracted from black-grass plants ( Cummins et al. 1997b ) and isoenzymes resolved by anion-exchange chromatography using methods described previously ( Cummins et al. 1997a ). The GST and GPOX activities of plant and recombinant enzymes and their analysis by SDS–PAGE and Western blotting using the antisera raised against ZmGST I-II or GST TaGST1–1 were as described previously ( Cummins et al. 1997a ). For RNA gel-blot analysis, total RNA (10 μg) was resolved on a 1.2% agarose–formaldehyde gel, transferred to a nylon membrane and incubated with digoxygenin-labelled AmGST1, AmGST2 or wheat PHGPOX RNA probes. Hybridization, washing and detection by enhanced chemiluminesence were as recommended by the manufacturer (Boehringer).


The work was supported by the LINK programme ‘Technologies for Sustainable Farming Systems’ through a grant jointly funded by the Agrifood Directorate of the Biotechnology and Biological Sciences Research Council and Rhône-Poulenc Agriculture Ltd. The authors acknowledge the expert advice and assistance of Dr Stephen Moss, IACR Rothamsted, in supplying herbicide-resistant black-grass seed and resistance factor data.


  1. EMBL, GenBank, DDBJ. databases accession numbers, AJ, 010448 (AmGST1a), AJO, 010449 (AmGST1b), AJO, 10450 (AmGST1c), AJ, 010451 (AmGST2a), AJ, 010452 (AmGST2b), AJ, 010453 (AmGST2c), AJ, 010454 (AmGST2d) & AJ. 010455 (phospholipid glutathione peroxidase).