a role for glutathione transferases functioning as glutathione peroxidases in resistance to multiple...

The Plant Journal (1999) 18(3), 285–292

A role for glutathione transferases functioning asglutathione peroxidases in resistance to multiple herbicidesin black-grass

Ian Cummins1, David J. Cole2 and Robert Edwards1,*1Crop Protection Group, Department of BiologicalSciences, University of Durham, Durham DH1 3LE, UK,and2Rhone-Poulenc Agriculture Ltd, Ongar, Essex CM5 OHW,UK

Summary

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 gluta-

thione 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 peroxid-

izing 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.

Introduction

Resistance of grass weeds to multiple herbicides of dif-fering modes of action is increasingly occurring in arable

Received 7 December 1998; revised 15 March 1999; accepted 16 March 1999.*For correspondence (fax 144 191 3742417;e-mail [email protected]).

© 1999 Blackwell Science Ltd 285

crops around the world and can compromise selectivechemical weed control (Holt et al., 1993). Resistance toindividual classes of herbicides can often be attributed tospecific mutations resulting in target sites with reducedsensitivities to inhibition, as recently demonstrated withdinitroaniline resistance in the weed Eleusine indica, whichresulted from a single mutation in tubulin, the target siteof this herbicide type (Anthony et al., 1998). Resistance tomultiple herbicides in weeds is less well defined, althoughenhanced herbicide metabolism has been proposed as alikely mechanism (Holt et al., 1993). In plants, the mostimportant steps in herbicide detoxification are catalysedby cytochrome P450 mono-oxygenases (CYPs) and gluta-thione transferases (GSTs), with the contribution of eachof these enzyme systems dependent on the herbicidesubstrate (Cole, 1994). These detoxifying enzymes areencoded by multi-gene families in crops, with the CYPscatalysing oxidation reactions (Schuler, 1996) while GSTsconjugate electrophilic xenobiotics with glutathione (Marrs,1996). These detoxification systems are expressed bothconstitutively and induced in response to herbicidesafeners, compounds which increase herbicide toleranceof cereals relative to competing weeds (Hatzios and Wu,1996). In contrast, grass weeds normally express muchlower levels of CYPs and GSTs, and are more sensitive toherbicides because they metabolize them more slowly(Cole, 1994).

Selection for enhanced expression of detoxifyingenzymes in competing weeds following repeated applica-tions with selective herbicides has been suggested asa powerful mechanism for evolving cross resistance todiffering classes of herbicides (Holt et al., 1993). Herbicidecross-resistance is particularly problematic in grass weedsand has been well documented in black-grass (Alopecurusmyosuroides), a competitive weed of wheat in Europe (Hallet al., 1997). In the UK, the pristine Rothamsted black-grass population from Hertfordshire is sensitive to selectiveherbicides used in wheat, having never been exposed topesticides (Hall et al., 1997). In contrast, the populationsPeldon from Essex and Lincs E1 from Lincolnshire havebeen treated with herbicides over several years and showresistance to the herbicides fenoxaprop-ethyl, an aryloxy-phenoxypropionate inhibitor of acetyl CoA carboxylase, aswell as chlorotoluron, a phenylurea which disrupts electrontransport in photosystem II (Cummins et al., 1997b; Hallet al., 1997). The resistance of Lincs E1 and Peldon to theseherbicides of differing modes of action cannot be explained

286 Ian Cummins et al.

by reduced target site sensitivities, leading to the sugges-tion that enhanced detoxification is a probable cross-resistance mechanism (Hall et al., 1997). In the case ofchlorotoluron, as compared with Rothamsted plants, thePeldon population has already been demonstrated to con-tain enhanced activities of CYPs able to detoxify the herbi-cide (Hyde et al., 1996). However, CYPs cannot accountfor resistance to fenoxaprop-ethyl, which is detoxified byglutathione conjugation in wheat and competing weeds(Tal et al., 1993). Studies in wheat have demonstratedthat the glutathione conjugation of fenoxaprop-ethyl iscatalysed by specific GST isoenzymes (Cummins et al.,1997a), and when the activities towards the enzyme wereassayed in black-grass, both Peldon and Lincs E1 containedhigher conjugating activities towards the herbicide thandetermined in Rothamsted (Cummins et al., 1997b). How-ever, recent metabolism studies have shown that metabol-ism of fenoxaprop-ethyl cannot account for the high degreeof resistance to this herbicide determined in Lincs E1 (Hallet al., 1997).

These apparently contradictory results have led us toreassess the role of GSTs in herbicide resistance in black-grass. GSTs in plants are related to the theta-type GSTs inanimals and have assumed multiple functions in counter-acting biotic and abiotic stresses (Marrs, 1996). Within thediverse family of GSTs in plants, it is possible to groupthese enzymes into four major types based on similaritiesin sequence, with the type I and III isoenzymes being mostabundant in the plant species studies to date (Dixon et al.,1998b). Although the functions of GSTs in endogenousmetabolism remain undefined, there is a consensus thatcertain plant GSTs have secondary activities as glutathioneperoxidases (GPOXs) and are able to protect cells fromcytotoxicity by reducing organic hydroperoxides to theircorresponding less toxic alcohols (Dixon et al., 1998b;Marrs, 1996; Roxas et al., 1997). As lipid peroxidation is acommon consequence of herbicide action (Hassall, 1990),we became interested in the role of GSTs in protectingplants from the oxidative injury caused by herbicides andthe potential effect this would have on herbicide resistancein weeds. We now report on the characterization of type IGSTs from black-grass with high GPOX activity which arepresent in populations showing resistance to multipleherbicides and demonstrate that the expression of theseenzymes is induced in response to both oxidative stressand treatment with herbicide safeners.

Results

Identification of GSTs in black-grass populations showingresistance to multiple herbicides

We had previously demonstrated that black-grass con-tained polypeptides of the correct size for GST subunits

© Blackwell Science Ltd, The Plant Journal, (1999), 18, 285–292

Figure 1. Western blots of crude extracts from black-grass populationsdiffering in herbicide resistance traits using antisera raised to the type Imaize 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 recognizedby both antisera had a molecular mass of 25 kDa while the two upperpolypetides were 27 and 28 kDa, respectively.(b) Analysis of 10-day-old black-grass shoots using the anti-ZmGST I-IIserum, with samples in lanes 1, 2, 4, 6 and 7 derived from populationsshowing resistance to both phenyl urea and aryloxyphenoxypropionateherbicides; lane 5 corresponded to a population which showed resistanceto aryloxyphenoxypropionates based on insensitivity of the target site,acetyl CoA carboxylase; lanes 3 and 8 came from herbicide-sensitivepopulations. The immunodetected polypeptides had molecular masses of27 and 28 kDa.

which were specifically recognized by an antiserum raisedto the wheat (Triticum aestivum L.) type III GST homodimerTaGST1–1 (Cummins et al., 1997b). When assayed byWestern blotting using the anti-TaGST1–1 serum, theRothamsted, Peldon and Lincs populations of black-grassall contained a strongly reacting 25 kDa polypeptide, whilstadditional weakly recognized 27 and 28 kDa polypeptideswere only observed in the herbicide-resistant Peldon andLincs plants (Figure 1a). When an antiserum raised to themaize (Zea mays L.) type I GST heterodimer ZmGST I-II wasused, it selectively recognized 27 and 28 kDa polypeptidesexpressed at high levels in the herbicide-resistant, but notin the herbicide-susceptible plants (Figure 1a). When theanti-ZmGST I-II serum was used in Western blots of extractsfrom a wider panel of black-grass plants with differingherbicide resistance traits, the 27 and 28 kDa polypeptides

Glutathione transferases and herbicide resistance 287

were identified in all populations showing cross-resistanceto herbicides, but were absent in herbicide-susceptibleblack-grass and in plants showing herbicide resistance dueto modified target-site sensitivity (Figure 1b). These resultssuggested that the enhanced expression of GSTs immuno-gically related to ZmGST I-II in black-grass was associatedwith resistance to multiple herbicides.

Cloning and characterization of black-grass GSTs

In view of the difficulties of purifying GSTs to completehomogeneity for detailed characterization from plants con-taining multiple, but related, isoenzymes (Cummins et al.,1997a), cDNAs encoding the different types of GST subunitsin black-grass were cloned from seedlings of the herbicide-resistant Peldon population and the respective recombin-ant enzymes over-expressed in bacteria. Screening of theexpression library with antisera raised to TaGST1–1 identi-fied three very similar clones, which were termedAmGST1a, AmGST1b and AmGST1c. The AmGST1a andAmGST1b clones both encoded polypeptides of 235 aminoacid residues, with minor variations in sequence, whileAmGST1c contained several conservative substitutions andwas smaller than the other clones by six amino acidresidues. The AmGST1a/b clones encoded a polypeptideof predicted mass 25.5 kDa, showing 46% identity to themaize type III GST ZmGST V (Figure 2a). The threeAmGST1 clones were expressed as their respective β-galactosidase fusion proteins and all found to have identicalGST activities when assayed with the substrates shown inTable 1. Further characterization of this group of cloneswas limited to AmGST1a. Analysis of the recombinantfusion protein by SDS–PAGE and Western blotting con-firmed that AmGST1a was specifically recognized by theanti-TaGST1–1 serum (data not shown), and it was con-cluded that the AmGST1 clones encoded the 25 kDa GSTsubunit polypeptide determined in black-grass extracts.

When the cDNA library was screened with the anti-ZmGST I-II serum, four clones were identified, sequencedand termed AmGST2a, AmGST2b, AmGST2c andAmGST2d, respectively. On the basis of the predicted sizeof the respective polypeptides and differences in sequencesbetween residues 36–39, the four AmGST2 clones couldbe grouped into the pairs AmGST2a, AmGST2b andAmGST2c, AmGST2d. Both pairs most closely resembledthe type I maize GST ZmGST I (Figure 2b) showing 63%and 62% identity, respectively, at the predicted amino acidlevel. From the Western blotting studies with the anti-ZmGST I-II serum, black-grass apparently contained twoimmunologically related polypeptides of molecular masses27 and 28 kDa (Figure 1). However, exhaustive screeningwith this antiserum only identified the AmGST2 group ofcDNAs which encoded polypeptides of nearly identicalmolecular mass (24.8–24.9 kDa). To clarify the correspond-


Figure 2. Predicted amino acid sequences of AmGST clones showingalignment 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 residuesdiffering within the respective AmGST sequences are underlined.

Table 1. GST activities of pure recombinant AmGSTs towardmodel xenobiotic and herbicide (fluorodifen and fenoxaprop-ethyl) substrates together with GPOX activities towards organichydroperoxides

Mean enzyme activity (6 SE)(nkat mg–1 protein)

Substrate AmGST1 AmGST2

CDNB 680 6 25 670 6 36Crotonaldehyde 0 6 0 0 6 0Ethacrynic acid 0 6 0 3.69 6 0.30Benzyl isothiocyanate 0.57 6 0.10 7.92 6 0.51Fluorodifen 0.08 6 0.01 0.01 6 0.00Fenoxaprop-ethyl 0.06 6 0.01 0.01 6 0.00Cumene hydroperoxide 0 6 0 4.54 6 0.67Linoleic hydroperoxidea 0 6 0 16.74 6 1.74

Enzyme activities were determined in triplicate.a13-hydroperoxy-cis-9,trans-11-octadecadienoic acid.


Figure 3. Western blot of the recombinant AmGST2 polypeptides ascompared with the GST subunits expressed in the herbicide-resistantPeldon black-grass probed with the anti-ZmGST I-II serum following theirresolution by SDS–PAGE.Lane 1: crude extract from Peldon; lane 2: recombinant AmGST2a; lane 3:recombinant AmGST2c with the molecular mass indicated.

ence of clones with polypeptides, AmGST2a and AmGST2c,which represented the two subclasses of AmGST2 cDNAs,were subcloned into pET11 and the recombinant GSTsanalysed by SDS–PAGE and Western blotting (Figure 3).The polypeptides were indistinguishable from one anotherwhen probed with the anti-ZmGST I-II serum and co-migrated with the 28 kDa polypeptide observed in theherbicide-resistant black-grass. Subsequently, it was deter-mined that the immunologically related 27 kDa polypeptidepresent in the herbicide-resistant black-grass was probablya partial degradation product of AmGST2, as prolongeddialysis of crude extracts resulted in an increase in theabundance of the 27 kDa polypeptide and a concomitantdecrease in the 28 kDa polypeptide (data not shown).

The GSTs encoded by AmGST1 and AmGST2 wereanalysed by gel filtration chromatography and shown tobe active as the respective homodimers AmGST1–1 andAmGST2–2. The recombinant enzymes were then purifiedby affinity chromatography and assayed for activities withxenobiotic substrates including several herbicides(Table 1). AmGST1–1 showed a similar range of GST activi-ties to type III GSTs isolated from wheat (Cummins et al.,1997a), being highly active in conjugating the diphenylether herbicide fluorodifen. This GST also showed appre-ciable activity toward fenoxaprop-ethyl, which is usedas a herbicide to control black-grass. Significantly, theAmGST2–2 enzyme, which was enhanced in the herbicide-resistant populations, showed lower activities toward theherbicides, casting further doubt on their relativeimportance in protecting black-grass plants by acceleratingherbicide metabolism. Instead, the possibility that theblack-grass GSTs were performing an indirect cytoprotect-ive function resulting in herbicide resistance was consid-ered. GSTs were assayed for their ability to detoxify toxicmetabolites arising from oxidative injury (Table 1). Bothenzymes showed no detectable activities towards crotonal-dehyde, an alkenal substrate resembling cytotoxic α,β-unsaturated aldehydes derived from the peroxidation oflipids and nucleic acids (Berhane et al., 1994). However,


Figure 4. Resolution of GST and GPOX activities in herbicide-susceptibleand herbicide-resistant 30-day-old black-grass plants by anion-exchangechromatography.Crude plant extracts from Rothamsted (s) and Peldon (m) were appliedonto a Q-Sepharose column and bound protein eluted with the increasingconcentration of NaCl shown (–). Fractions were assayed for (a) GST activitytowards CDNB and (b) GPOX activity towards cumene hydroperoxide.

a major difference between the black-grass GSTs wasdetermined when they were assayed for glutathione peroxi-dase (GPOX) activity. While the constitutively expressedAmGST1–1 had no activity as a GPOX, AmGST2–2 wasvery active in catalysing the reduction of both cumenehydroperoxide and linoleic acid hydroperoxide (Table 1).

Relationship between GSTs functioning as GPOXs andherbicide resistance

The association of GPOX activity with the AmGST2–2isoenzyme enhanced in herbicide-resistant populationsindicated that specific GSTs may function to protect black-grass from toxic organic hydroperoxides formed as aconsequence of herbicide injury. When GST and GPOXisoenzymes in crude plant extracts were resolved by anionexchange chromatography, GST activity toward the modelsubstrate 1-chloro-2,4-dinitrobenzene (CDNB) predomi-nantly eluted in one peak, with the activities in the herbi-cide-resistant Peldon being fourfold greater than insusceptible Rothamsted (Figure 4a). In Peldon, this majorpeak of GST activity co-eluted with a peak of GPOX activitywhich was absent in the extracts from Rothamsted plants(Figure 3b). However, both populations contained identicalactivities of a GPOX which was not associated with GSTactivity eluting around fraction 10 (Figure 3b). Thus, only


Table 2. Hydroperoxide content in the shoots of herbicide-resistantand herbicide-susceptible black-grass treated with selective andnon-selective herbicides

Hydroperoxide content (µmol g–1 FW)

Herbicide Rothamsted Peldon Lincs E1

Control 1.14 6 0.17 0.75 6 0.08 0.91 6 0.10Selective herbicidesFenoxaprop-ethyla 2.23 6 0.14 0.80 6 0.10 0.75 6 0.16Clodinafop-propargyla 1.75 6 0.15 0.82 6 0.18 1.03 6 0.22Chlorotoluronb 1.93 6 0.02 0.71 6 0.04 1.45 6 0.33Non-selective herbicidesFluorodifenc 3.28 6 0.25 0.85 6 0.05 0.97 6 0.06Paraquatd 4.05 6 0.27 1.08 6 0.14 1.40 6 0.05

Values represent means of triplicate incubations 6 SE.aAcetyl CoA carboxylase inhibitor; bphotosystem I inhibitor;cprotoporphyrinogen oxidase inhibitor; dphotosystem II inhibitor.

those GPOXs with GST activities were enhanced in thePeldon population.

To determine the association between enhanced GPOXactivity and herbicide resistance, plants of the susceptibleRothamsted and resistant Peldon and Lincs populationswere analysed for hydroperoxide formation followingexposure to both non-selective herbicides and selectiveherbicides used to control black-grass in wheat (Table 2).All the herbicides tested caused a significant increase inhydroperoxide content in Rothamsted plants, with theincreases being greatest following treatment with the per-oxidizing herbicides paraquat, fluorodifen and chlorotolu-ron. In contrast, the herbicides caused considerably lesshydroperoxide formation in the Peldon and Lincs plants.The link between elevated AmGST2 expression, suppres-sion of herbicide-invoked hydroperoxide formation andherbicide resistance was also examined by determiningthe relative sensitivities of Rothamsted and Peldon plantsto paraquat, a herbicide known to cause phytotoxicity bygenerating reactive oxygen species and organic hydro-peroxides (Iturbe-Ormaetxe et al., 1998). When comparedwith untreated controls, a 48 h treatment with paraquatresulted in a much greater wilting and desiccation inRothamsted plants than in Peldon (Figure 5).

Mechanisms of enhanced GST expression in herbicide-resistant populations

Total RNA from herbicide-susceptible Rothamsted andherbicide-resistant Peldon plants was analysed by gel-blotanalysis, using RNA probes prepared against AmGST1 andAmGST2 (Figure 6). AmGST1 transcripts were detected inboth populations but were more abundant in Peldon.However, while the AmGST2 probe strongly recognized anmRNA in Peldon, this transcript was scarcely detectablein Rothamsted. This suggested that the enhancement in


Figure 5. The effect of a 48 h treatment with 0.1 mM paraquat on Rothamsted(2) and Peldon (4) black-grass, with the respective controls incubated inwater alone shown for Rothamsted (1) and Peldon (3).

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 ofRothamsted (b) or Peldon (c) using the anti-ZmGST I-II-serum following a24 or 48 h exposure to the herbicide safener fenchlorazole-ethyl (FCE),desiccation (DRY), 0.1 mM paraquat (PQ) or 0.1 mM 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 the28 kDa polypeptide being AmGST2 and the other polypeptides partialdegradation products.

AmGSTs observed in herbicide-resistant black-grass wasdue to increased expression of the respective genes. It wasthen of interest to determine whether other genes encodingenzymes with related anti-oxidant functions to the GPOXactivity of AmGST2 were also induced in herbicide-resistantplants. An RNA probe corresponding to a wheat phospho-


lipid hydroperoxide glutathione peroxidase (PHGPOX) wasgenerated. Plant PHGPOXs resemble the selenium-depend-ent GPOXs in animals and show different specificity towardhydroperoxides than GST–GPOXs (Eshdat et al., 1997). ThePHGPOX probe hybridized to a RNA species with similarintensity in both Peldon and Rothamsted (Figure 6a), dem-onstrating that only GSTs with GPOX activity were up-regulated in the herbicide-resistant plants, confirming theresults of Figure 4.

As type I GSTs resembling AmGST2 are enhanced inwheat in response to a diverse range of biotic stresses(Mauch and Dudler, 1993), a potential mechanism forelevated expression could be that the stress-induciblesignal(s) which regulate GST expression were permanentlyactivated in herbicide-resistant black-grass. To test thishypothesis, the expression of AmGST2 was monitored byWestern blotting in seedlings of Rothamsted and Peldonexposed to a range of chemical and environmental stresstreatments known to induce GSTs or impose oxidativestress in plants (Marrs, 1996; Yang et al., 1998). All thesetreatments greatly enhanced immunodetectable AmGST2subunits in Rothamsted, but this induction was only mar-ginal in Peldon (Figure 6b).

Discussion

Our results show a relationship between GSTs functioningto counteract oxidative stress and resistance to multipleherbicides with differing modes of action in black-grass. Arole for GSTs in reducing oxidative injury due to bioticstresses has been suggested for some time by disparateobservations that stimuli which impose oxidative stresson plants, such as infection and environmental stresses,induce the expression of selected GST genes (reviewed byMarrs, 1996). Furthermore, when a GST with GPOX activitywas over-expressed in tobacco, the transgenic plantsbecame more tolerant to chilling and saline conditions(Roxas et al., 1997), which are stresses associated withoxidative injury (Iturbe et al., 1998). However, a similarrole for GSTs in counteracting abiotic stress imposedby herbicides has received far less attention due to theprotective role of GSTs in catalysing the conjugation anddetoxification of many types of herbicides including thio-carbamates, chloro-s-triazines, chloroacetanilides anddiphenyl ethers (Cole, 1994). Potential roles in herbicidetolerance for enzymes, including GSTs, which preventoxidative stress have been proposed in the case of peroxid-izing herbicides such as oxyfluorfen (Knorzer et al., 1996),which kill plants due to the generation of reactive oxygenspecies (ROS). In black-grass, enhanced expression of theAmGST2–2 isoenzyme which showed GPOX activity wasassociated with a reduced accumulation of hydroperoxidesgenerated by the action of the ROS-generating herbicidesparaquat, chlorotoluron and fluorodifen. In the case of


paraquat, this reduction in peroxidation resulted in a signi-ficant increase in tolerance to the herbicide as comparedwith herbicide-susceptible Rothamsted plants. Interes-tingly, herbicides such as fenoxaprop-ethyl and clodinafop-propargyl which inhibit fatty acid synthesis also generatedhydroperoxides in herbicide-susceptible black-grass, pre-sumably as a consequence of disrupting primary metabol-ism. It is interesting to speculate that such peroxidationarising as a secondary consequence of the mode of actionof these herbicides may be a primary cause of toxicity,which may explain why enhanced expression of theAmGST2 GPOX resulted in resistance to arylphenoxy propi-onate inhibitors of fatty acid synthesis.

Enhanced AmGST2 expression in the herbicide-resistantblack-grass resulted from a major increase in the abund-ance of the respective transcript. However, AmGST1 tran-scripts were only modestly enhanced, while PHGPOX wasunaffected, demonstrating that the induction of AmGST2was specific. The causes of increased AmGST2 transcriptabundance were not investigated in detail, but Southernanalysis of genomic DNA from Rothamsted and Peldonblack-grass plants gave identical patterns and intensitiesof hybridization, suggesting that the AmGST2 gene hadnot been amplified in the herbicide-resistant populations(data not shown). Instead, the causes of AmGST2 inductionin the herbicide-resistant black-grass appeared to be linkedto the permanent up-regulation of the respective gene,which in the herbicide-susceptible populations wasresponsive to treatments which imposed oxidative stress,such as ROS-generating chemicals and drought stress.The herbicide safener fenchlorazole-ethyl also enhancedAmGST2 expression in black-grass, demonstrating thatGST induction by safeners is not exclusive to cereal crops(Hatzios and Wu, 1996). However, our studies have demon-strated that GST induction by herbicide safeners is notassociated with significant hydroperoxide formation(unpublished observation), suggesting that these com-pounds must activate GST expression either by a separatesignalling pathway to oxidative stress, or act downstreamof early oxidative signalling events. Safeners thereforeact to induce GSTs by a distinct mechanism from otherxenobiotics such as triiodobenzoic acid, which generateROS prior to GST enhancement (Flury et al., 1998). Signi-ficantly, the black-grass populations showing multipleherbicide resistance were far less responsive to both oxidat-ive stress and safener treatment, which is consistent withthe permanent activation of a gene which was normallyactivated in response to either of these stimuli.

Our results give a further insight into the functioningof GSTs as proteins with broad-ranging cytoprotectivefunctions in preventing oxidative stress in plants and otherorganisms. It is unlikely that AmGST2 constitutes the onlymechanism of multiple herbicide resistance in black-grass,as it has also been reported that the Peldon population


shows increased CYP-mediated detoxification of thephenylurea herbicide chlorotoluron (Hyde et al., 1996).However, it is possible that these co-ordinately regulatedGST and CYP xenobiotic detoxification systems are com-ponents of an anti-oxidant protective system, which havebeen selected for constitutive expression in crops as con-tributing towards stress tolerance, and are now appearingas traits in grass weeds as a result of selection resultingfrom unrotated herbicide usage. Significantly, GSTs resem-bling AmGST2 are known to be constitutively expressedin maize and other cereal crops to high levels, althoughtheir functions have yet to be determined (Dixon et al.,1998b). Characterization of the promoter region of theAmGST2 gene and identification of the factors whichregulate its expression will now provide further insightsinto the potential molecular mechanisms by which resist-ance to multiple herbicides can arise in weeds and suggeststrategies to counteract this problem. As a more immediatebenefit, immunorecognition of enhanced expression ofgenes encoding such GST–GPOXs could now be used todiagnose herbicide cross-resistance in black-grass.

Experimental procedures

Plant material and treatment with herbicides andsafeners

Seeds of black-grass populations of defined herbicide resistancetraits (Hall et al., 1997) were generously donated by Dr S. Moss(IACR-Rothamsted, Harpenden, UK) and grown for 20 days asdetailed previously (Cummins et al., 1997b). For studies withherbicide safener, cut shoots were placed in 32.5 µM fenchlorazole-ethyl dissolved in 0.1% acetone, or in 0.1% acetone alone, for72 h. For all herbicides except paraquat, cut shoots were placedin 1 mM treatment solutions prepared from 100 mM stock solutionsof the herbicides in acetone, with the control solutions consistingof 1% (v/v) acetone. For paraquat treatments, black-grass shootswere incubated in 0.1 mM paraquat for 24 h then transferred tofresh water and incubated for a further 24 h under constantillumination (150 µmol m–2 sec–1). Plants were treated with rosebengal (0.1 mM) as described for the experiments with paraquat.At harvest, plants were frozen in liquid nitrogen and storedat –80°C.

Assays for hydroperoxides

After a 24 h treatment with herbicide, tissue was ground to a finepowder under liquid nitrogen and extracted in 2 vol of 50 mM

potassium phosphate, pH 7.2, containing 1% (v/v) Triton X-100and 0.1% (w/v) butylhydroxytoluene. After extraction at roomtemperature for 30 min, samples were centrifuged (10 000 g,

5 min) and 10 µl of the supernatant added to 100 µl of the preparedPeroXOquant reagent (Pierce). After incubation for 20 min,samples were clarified by centrifugation and quantified from theirabsorbance at 560 nm relative to a standard curve prepared usingH2O2 as recommended by the manufacturer.


Cloning and expression of black-grass GSTs

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

Recombinant AmGST1 polypeptides were expressed as theirrespective β-galactosidase fusion proteins using the pBluescriptplasmid (Stratagene) in E. coli SOLR cells after a 16 h induction with1 mM IPTG. The AmGST2 inserts were excised from pBluescript,subcloned into pET11a and expressed in BL21 cells after inductionfor 3 h in the presence of 1 mM IPTG. Bacteria were harvested bycentrifugation and sonicated in 100 mM Tris–HCl pH 7.5 containing2 mM EDTA and 1 mM DTT. After centifugation, the proteins in thesupernatant were precipitated by the addition of ammoniumsulphate to 80% saturation. Recombinant GSTs were then purifiedby a combination of affinity chromatography on glutathione aga-rose and anion-exchange chromatography as described previouslyfor 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 plantand recombinant enzymes and their analysis by SDS–PAGE andWestern blotting using the antisera raised against ZmGST I-II orGST TaGST1–1 were as described previously (Cummins et al.,1997a). For RNA gel-blot analysis, total RNA (10 µg) was resolvedon a 1.2% agarose–formaldehyde gel, transferred to a nylonmembrane and incubated with digoxygenin-labelled AmGST1,AmGST2 or wheat PHGPOX RNA probes. Hybridization, washingand detection by enhanced chemiluminesence were as recom-mended by the manufacturer (Boehringer).

Acknowledgements

The work was supported by the LINK programme ‘Technologiesfor Sustainable Farming Systems’ through a grant jointly fundedby the Agrifood Directorate of the Biotechnology and BiologicalSciences Research Council and Rhone-Poulenc Agriculture Ltd.The authors acknowledge the expert advice and assistance of DrStephen Moss, IACR Rothamsted, in supplying herbicide-resistantblack-grass seed and resistance factor data.

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a role for glutathione transferases functioning as glutathione peroxidases in resistance to multiple...

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