Copyright © Physiologia Plantarum 2001PHYSIOLOGIA PLANTARUM 113: 477–485. 2001Printed in Ireland—all rights reser�ed ISSN 0031-9317

Biochemical characterisation of esterases active in hydrolysingxenobiotics in wheat and competing weeds

Ian Cumminsa, MichaeI Burnetb,1 and Robert Edwardsa,*

aDepartment of Biological Sciences, Uni�ersity of Durham, Durham, DH1 3LE, UKbSyngenta, Jealott’s Hill International Research Centre, Bracknell, RG42 6ET, UK1Present address: Sympore GmbH, Auf der Morgenstelle 1, D-72076 Tubingen, Germany*Corresponding author, e-mail: robert.edwards@durham.ac.uk

Received 16 February 2001; revised 19 June 2001

Esterase activities toward model xenobiotic substrates (p-ni- Resolved esterase activities in the weeds were distinct fromthose observed in the Tritcum species. However, unlike thetrophenyl acetate, naphthyl acetate) and pesticide esters (di-case with other classes of xenobiotic-metabolising enzymes,clofop methyl, bromoxynil octanoate, binapacryl) have beenthe complement of esterases in the Peldon and Rothamstedcompared in crude extracts from wheat (Triticum aesti�um L.)populations of black-grass appeared to be identical. In alland Triticum progenitors of wheat. Esterase activities were

also determined in the weeds, wild oat (A�ena fatua) and two species, the more basic esterases (�pI 5.0) were sensitive toinhibition by organophosphate and carbamate insecticides,populations of black-grass (Alopecurus myosuroides), one of

which (Rothamsted) is susceptible to herbicides, while the suggesting that they were B-class esterases. In contrast, theother (Peldon) shows cross-resistance to multiple classes of acidic wheat esterase (pI 4.6) with the greatest activity toward

�-naphthyl acetate was insensitive to insecticides. This wheat-herbicides. Esterase activity toward the model substrates wasspecific esterase was purified 7000-fold by a combination ofhighest in wheat, while the weeds were more active in hy-

drolysing the pesticides. Using isoelectric focussing (pH 4–8), hydrophobic interaction chromatography, gel filtration and13 proteins with esterase activity toward �-naphthyl acetate anion-exchange chromatography. The purified esterase be-

haved as a monomeric 45-kDa protein showing high activitycould be resolved in hexaploid wheat (genome AABBDD). Thetoward p-nitrophenyl acetate and �-naphthyl acetate, butpattern of these activities was most similar to that of thelimited activity toward the pesticides.diploid progenitor T. tauschii (DD), excepting a major acidic

esterase (pI 4.6), which originated from T. urartu (AA).

remarkably little is known about these simple reactions inplants. While in the abiotic environment synthetic estersundergo chemical hydrolyses, the available literature wouldsuggest that, in plants, these reactions are catalysed byesterases, as is the case in animals (Hassall 1990). In bothinsects and mammals, a good deal is known about esterasesactive in the metabolism of xenobiotics due to their impor-tance in drug and insecticide metabolism (Heymann 1980).Based on classical early studies, the esterases present inanimals were classified into A and B classes based on theiractivity toward organophosphate insecticides (Aldridge1953). Class-A esterases (EC 3.1.1.2) actively hydrolyseorganophosphates, while class-B esterases (EC 3.1.1.1) bindorganophosphates and are then inactivated by them due to

Introduction

Many pesticides and pollutants enter the leaves of plants inthe form of hydrophobic esters, which pass readily throughthe waxy cuticle (Hassall 1990). Following absorption, esterhydrolysis is then a major route of metabolism and this canlead to the inactivation of the xenobiotic or the release of abiologically active acid or alcohol (Bounds and Hutson2000). As examples of bioactivation by hydrolysis in plants,the fungicide binapacryl and the herbicide bromoxynil oc-tanoate are hydrolysed to release their bioactive alcoholmoieties, while the herbicide diclofop methyl is activated todiclofop acid (Fig. 1). In contrast, the herbicide thiazopyr isinactivated by esterase action (Feng et al. 1995). Despite theimportance of these hydrolyses in controlling thebioavailability of pollutants and crop protection agents,

Abbre�iations – APP, aryloxyphenoxypropionate; IEF, isoelectric focussing; HPLC, high-performance liquid chromatography.

Physiol. Plant. 113, 2001 477

irreversible phosphorylation of a serine residue at the activesite.

Too little is known to define whether or not this A/Bclassification system applies in plants or how many types ofesterases are involved in hydrolysing pesticides or otherxenobiotic compounds in crops and weeds. The best-charac-terised hydrolytic activity toward a pesticide in plants is thearyl acylamidase-mediated detoxification of the herbicidepropanil (3,4-dichloropropionanilide) in rice, originally de-scribed over 30 years ago (Frear and Still 1968). The mostrecent studies demonstrate that this hydrolase is a 180-kDamembrane-associated enzyme and appears to be of theB-class, being strongly inhibited by both organophosphateand carbamate insecticides (Leah et al. 1994). A B-classesterase, which hydrolysed the pyrethroid insecticidecyfluthrin, was also identified in tomato cell suspensioncultures, though this 32-kDa soluble enzyme was clearlyquite distinct from the aryl acylamidase (Preiss et al. 1988).

In the course of our investigations into the mechanismsunderpinning the selectivity of herbicides in wheat (Triticumaesti�um L.) and competing weeds, we are interested in therole of esterases in determining pesticide metabolism andbioavailability. In wheat and competing grass weeds, esterhydrolysis is an important initial reaction in the metabolismof the aryloxyphenoxypropionate (APP) herbicides, such asdiclofop methyl (Fig. 1). The APPs are used to control wild

grasses in wheat, with the relative rates of metabolism incrops and weeds being a major determinant of selectivity(Owen 2000). Significantly, it has been reported that theAPP benzoylprop ethyl was hydrolysed to the bioactivebenzoylprop acid more rapidly in the sensitive grass weedwild oat (A�ena fatua L.) than in tolerant wheat, suggestingthat bioactivation through hydrolysis contributed to theselectivity of the herbicide (Jeffcoat and Harries 1973). A60-kDa esterase showing specificity for benzoylprop ethylwas subsequently partially purified from leaves of wild oat,but the corresponding enzyme activity could not be demon-strated in wheat (Hill et al. 1978).

Wheat has been reported to hydrolyse a variety of estersof 2,4-dichlorophenoxyacetic acid and the related compoundtriclopyr, though the respective esterases were not character-ised (Lewer and Owen 1987). When crude extracts fromwheat cell suspension-cultured cells were assayed with themodel substrate �-naphthyl acetate, 12 esterase activitiescould be resolved using non-denaturing gel electrophoresis(Krell and Sandermann 1984). A 38-kDa esterase composedof 22-kDa polypeptides, which hydrolysed the plasticiserbis(2-ethyl-hexyl)phthalate, was subsequently purified 280-fold from wheat cell cultures (Krell and Sandermann 1984)and 10-fold from wheat plants (Krell and Sandermann1985). The esterase was selectively active toward ester sub-strates with alkyl chains of 6–8 carbon atoms and wasinhibited by chemicals, which selectively inactivate serinehydrolases. In view of the importance of esterases in con-trolling the metabolism and bioavailability of pesticides, wenow report on the spectrum of esterase activities towardxenobiotics and pesticides in wheat and the competing grassweeds wild oat (A. fatua) and black-grass (Alopecurusmyosuroides). In the case of black-grass, we have examinedthe esterases in two biotypes, termed Rothamsted and Pel-don, which differ in their resistance to herbicides. Thebiotype Rothamsted is sensitive to all classes of selectivegraminicides used to control black-grass, while the biotypePeldon shows cross-resistance to phenylurea and APP herbi-cides (Hall et al. 1997). Herbicide cross-resistance in Peldonblack-grass has previously been linked to an increased ca-pacity to detoxify herbicides (Hall et al. 1997). In turn, thishas been linked to the elevated expression of enzymes, suchas cytochrome P450 monooxygenases (Hyde et al. 1996) andglutathione transferases (Cummins et al. 1999). It was there-fore also of interest to determine whether or not esteraseactivities toward herbicides were similar in herbicide-sensi-tive and herbicide-resistant black-grass. Having identifieddifferences in the esterase complement of crop and weeds,we have then purified and characterised a major esterasefound exclusively in wheat and identified the origins of thediffering esterases present in hexaploid wheat by examiningthe isoenzyme content of progenitor Triticum species.

Materials and methods

Plant material

Seeds of hexaploid bread wheat (Triticum aesti�um L.genome AABBDD) cv. Hunter were obtained from Plant

Fig. 1. Structures of model substrates and pesticide esters used inthis study.

Physiol. Plant. 113, 2001478

Breeding International, Cambridge, UK with seeds of theprogenitor species Triticum dicoccum (AABB), Triticumtauschii (DD) and Triticum urartu (AA) kindly provided byMr M. Ambrose, John Innes Centre, Norwich, UK. Seedsof wild oat (A�ena fatua) were purchased from Herbiseed(Wokingham, UK), while seeds from the Rothamsted andPeldon populations of black-grass (Alopecurus myosuroides)were obtained as described previously (Cummins et al.1999). Plants were grown in environmental growth cham-bers and the foliage harvested, frozen in liquid nitrogen andstored at −80°C until required (Cummins et al. 1997).

Enzyme purification

Unless otherwise stated, all purification steps were carriedout on ice or at 4°C. Frozen 7-day-old wheat shoots wereground to a powder using a mortar and pestle prior to theaddition of 3 volumes of 0.1 M Tris-HCl (pH 7.5), 2 mMEDTA and 5% w/v polyvinylpolypyrrolidone. After centrifu-gation (10000 g, 15 min), (NH4)2SO4 was added to thesupernatant to 40% saturation and the extract was recen-trifuged (10000 g, 15 min). Proteins remaining in the super-natant were recovered by adding (NH4)2SO4 to 80%saturation. The 40–80% pellet was suspended in 20 mMKH2PO4/K2HPO4 buffer (pH 7.2) containing 1 M(NH4)2SO4 and applied to a 100-ml column of phenyl-Sep-harose using a Gradi-Frac chromatography system (Amer-sham Pharmacia Biotech UK Ltd., Little Cholfont, UK).After washing to remove unbound protein, the column waseluted at 4 ml min−1 with a linearly decreasing gradient of(NH4)2SO4 ending with 20 mM KH2PO4/K2HPO4 (pH 7.2)in a total volume of 200 ml. Remaining hydrophobic proteinswere then eluted with a linearly increasing gradient (totalvolume 100 ml) of ethylene glycol at 2 ml min−1 ending in20 mM KH2PO4/K2KPO4 (pH 7.2):ethylene glycol (1:1 v/v).Fractions (10 ml) were collected and samples of interest werepooled, dialysed against 20 mM KH2PO4/K2HPO4 (pH 7.2)and precipitated with 85% (NH4)2SO4. Protein pellets wereresuspended in 20 mM KH2PO4/K2HPO4 (pH 7.2) contain-ing 0.15 M NaCl and applied at 0.5 ml min−1 to a Superdex200 HR 10/30 column coupled to a Bio-Rad HRLC system.The column was eluted with loading buffer and fractions (1ml) showing activity pooled and desalted using PharmaciaPD-10 Sephadex columns into 20 mM Bis-Tris (pH 6.0).Samples were applied (0.25 ml min−1) to a 1.2-ml Uno-Qanion-exchange high-performance liquid chromatography(HPLC) column (Bio-Rad, Hampstead, UK) and proteinsresolved with a linearly increasing concentration (total vol-ume 30 ml) of NaCl (0.15 M to 0.5 M). Active fractions werepooled and after desalting reapplied to the Uno-Q columnunder identical conditions. During purification, fractionswere routinely assayed for activity using p-nitrophenyl ace-tate. Fractions of interest were analysed by SDS-PAGE andisoelectric focussing (IEF).

Esterase assays

Crude protein extracts were prepared for assay by desaltingon Sephadex G-25 columns (Pharmacia PD-10) using 100

mM KH2PO4/K2HPO4 buffer (pH 7.2) and the proteincontent determined using a dye-binding reagent (Bio-Rad)with �-globulin as the reference protein. Esterase activitywith p-nitrophenyl esters and �-naphthyl acetate substrateswere monitored colorimetrically (Krell and Sandermann1984). Activity toward pesticide esters was determined byquantifying the hydrolysis products using reversed-phaseHPLC. Pesticide esters were obtained from Syngenta (Berk-shire, UK) or were purchased from Greyhound ChemicalCo. (Merseyside, UK). Hydrolysis products were identifiedby co-chromatography with authentic standards whereavailable. Alternatively, the parent esters were incubatedwith 50 �g ml−1 porcine esterase (Sigma, Poole, UK) in 100mM KH2PO4/K2HPO4 (pH 7.2) at 37°C for 1 h. Afterstopping the reaction with 1 volume HPLC-grade methanol,the precipitated protein was removed by centrifugation(10000 g, 5 min) and 50 �l of the supernatant analysed byHPLC. Reaction products were resolved on a C18 column(250 mm×4.6 mm, 5 �m packing, Phenomenex, Mac-clesfield, UK) using 1% orthophosphoric acid (solvent A)and linearly increasing proportions of acetonitrile (solventB) as the running solvents. Elution at 0.8 ml min−1 wasinitiated with 5% B increasing to 10% B (0–10 min) fol-lowed by an increase to 100% B (10–40 min). The columnwas then washed with 100% B (40−50 min) prior tore-equilibration with 5% B. The column eluate was moni-tored for UV absorbance at 264 nm and the amounts ofhydrolysed pesticide present calculated from a standardcurve prepared following complete hydrolyses of knownamounts of parent compound using porcine esterase.

Electrophoresis

For IEF, samples were first desalted in 5 mM KH2PO4/K2HPO4, pH 7.2, using Sephadex G-25 spun columns andtheir protein content determined. Samples were then appliedto 6% polyacrylamide slab gels supported on GelBond PAGfilm (FMC, Sigma), prepared with 2.6% cross-linker, con-taining 2% (v/v) ampholyte, pH 3–10, 2% (v/v) ampholyte,pH 4–7, and 10% (v/v) glycerol. Electrophoresis was per-formed using a water-cooled Multiphor flatbed system(Pharmacia) with solutions of 1 M orthophosphoric acid atthe anode and 1 M NaOH at the cathode. Electrophoresisconditions were 200 V for 1 h prior to sample application,sequentially followed by 200 V (1 h), 500 V (1 h) and 1000V (2 h). For calibration, horse heart myoglobin, (pI 6.8,7.2), carbonic anhydrase II (pI 5.9), �-lactoglobulin A, (pI5.1) and glucose oxidase (pI 4.2) were used and visualised bystaining for 1 h with Gelcode blue reagent (Pierce &Wariner, Chester, UK) after overnight fixation in 12% w/vtrichioroacetic acid with 5% w/v sulphosalicylic acid. Toidentify proteins with esterase activity, after electophoresis,gels were immersed in 0.1 M KH2PO4/K2HPO4 (pH 7.2)containing Fast Blue RR (0.5 mg ml−1), �-naphthyl acetate(0.1 mg ml−1), 0.01% (v/v) formaldehyde and 1 mM acet-azolamide. Active esterases were visualised by monitoringthe appearance of coloured bands and the reaction stoppedby rinsing the gel with 5% (v/v) acetic acid. For SDS-PAGE,10% gels were used and polypeptides visualised using either

Physiol. Plant. 113, 2001 479

Table 1. Esterase activity (mean�SE, n=3) toward p-nitrophenyl-acetate and pesticide substrates in 40–80% ammonium sulphate pelletsfrom seedlings of wheat, Triticum progenitor species and competing grass weeds. No esterase activity was detected in 0–40% ammoniumsulphate pellets. Minimal activity was detected in 80–100% ammonium sulphate pellets. For the Triticum species the genome type is shownin parentheses. ND, no activity detected.

Species p-Nitrophenyl acetate BinapacrylDiclofop methyl Bromoxynil octanoate(pkat mg−1)(nkat mg−1) (pkat mg−1) (pkat mg−1)

T. aesti�um l.30�0.07 94�6l.0�0.0 4.0�0.3(AABBDD)T. dicoccum 0.28+0.06 ND 57�22.1�0.2(AABB)T. urartu 0.88�0.02 37�30.9�0.0 2.5�0.4(AA)T. tauschii 0.30�0.03 67�3ND 3.5�0.4(DD)A. fatua 0.46�0.03 116�102.4�0.1 8.2�0.3A. myosuroides 101�90.68�0.08 12.0�0.3 5.1�1.1‘Rothamsted’A. myosuroides 0.67�0.05 80�1010.0�0.0 7.2�1.0‘Peldon’

silver staining or Gelcode blue reagent (Cummins et al.1997).

Results

Esterase activity toward xenobiotics and pesticides inTriticum species and grass weeds

Esterase activities toward a diverse range of pesticides weredetermined in crude preparations from wheat seedlings us-ing HPLC-based assays to quantify hydrolysis products.Activity was detected with the herbicides diclofop methyland bromoxynil octanoate and the fungicide binapacryl(Table 1). However, no activity was determined with thefungicide metalaxyl, the insecticides pirimiphos methyl, per-methrin and cyhalothrin or the herbicides 2,4-dichlorophe-noxyacetyl sec butyrate, chlorpropham, imazamethabenzmethyl, flamprop isopropyl, benzoylprop ethyl orchiorimuron ethyl (data not shown). In contrast, a commer-cially available porcine liver preparation readily hydrolysedmost of the tested compounds, with the exception of chlor-propham, metalaxyl, pirimiphos methyl and chlorimuronethyl (data not shown).

Esterase activities toward binapacryl, bromoxynil oc-tanoate and diclofop methyl and the model substrate p-ni-trophenyl acetate were compared in wheat and itsprogenitor Triticum species, together with wild oat and theblack-grass biotypes Rothamsted and Peldon (Table 1).Withall substrates tested, esterase activity was higher inhexaploid wheat than in the related progenitor Triticumspecies. The activity toward diclofop methyl was only ob-served in hexaploid wheat (genome AABBDD) and T.urartu (AA), suggesting this activity was specific to the AAgenome. However, the diclofop methyl-hydrolysing activitywas absent in the AA-containing tetraploid T. dicoccum(AABB). While wheat showed higher esterase activity to-ward p-nitrophenyl acetate than the weeds, wild oat andblack-grass were more efficient in hydrolysing the 3 pesti-cides, particularly the APP herbicide diclofop methyl (Table1).

Having demonstrated differences in esterase activities inthe Triticum species and grass weeds, the respective esteraseactivities were then resolved using IEF non-denaturing gelelectrophoresis. Esterases were visualised by incubating thegels with �-naphthyl acetate, with the hydrolysed �-naph-thol forming an insoluble coloured dye in the presence oftetrazolium Fast Blue salt (Schaller and von Deimling 1979).Crude extracts from wheat (T. aesti�um) shoots containedmultiple active esterases with pI values in the range pH4.6–8 (Fig. 2). The exact number of esterases identifiedvaried depending on incubation conditions, but typically 13active enzymes could be visualised. Identical patterns of

Fig. 2. Esterase activity toward �-naphthyl acetate following reso-lution by non-denaturing IEF of crude protein extracts (100 �g)from the shoots of 1, T. aesti�um (genome AABBDD); 2, T.dicoccum (AABB); 3, T. tauschii (DD); 4, T. urartu (AA); 5, A.fatua ; 6, A. myosuroides biotype Peldon; 7, A. myosuroides biotypeRothamsted. The positions of pI marker proteins are arrowed.

Physiol. Plant. 113, 2001480

Fig. 3. Inhibition of esterase activitytoward �-naphthyl acetate followingexposure of crude plant extracts (100�g protein) to either acetone (A), 0.1mM paraoxon (Po), 0.1 mMparathion (Pt) or 0.1 mM carbaryl(Ca) for 1 h followed by resolutionby IEF. From left to right, the plantextracts analysed were derived fromblack-grass biotype Peldon,black-grass biotype Rothamsted,wild-oat, wheat, T. urartu, T.tauschii and T. dicoccum. Thepositions of pI marker proteins arearrowed.

activity were seen with the alternative substrate �-naphthylacetate (data not shown). When compared with the esteraseprofile of the Triticum progenitor species (Fig. 2), it wasapparent that the esterase banding pattern seen in wheat(hexaploid genome AABBDD) was most similar to that ofthe diploid progenitor T. tauschii (DD). In contrast, themost visible esterase activities in the tetraploid progenitor T.dicoccum (AABB) were largely the minor activities presentin wheat. On first inspection this suggested that the esterasesactive in �-naphthyl acetate hydrolysis had been predomi-nantly derived from the DD genome. However, analysis ofextracts from T. urartu (AA) demonstrated that this genomewas the likely source of the acidic isoenzyme (pI 4.6), whichrepresented the major enzyme activity detected in wheatusing this assay (Fig. 2).

Shoot extracts from the weeds showed distinct activityprofiles as compared with the Triticum species with 13 bandsvisible in A�ena fatua and 11 bands in Alopecurusmyosuroides. The most striking difference between the re-solved activities was the absence in the weeds of the majoracidic isoenzyme present in wheat. Interestingly, the profileof �-naphthyl acetate-hydrolysing esterases in the herbicide-resistant black-grass Peldon was identical to the profiledetermined in the herbicide-susceptible Rothamsted. Thisobservation confirmed the results obtained with p-nitro-phenyl acetate and the pesticide ester substrates (Table 1),namely that both biotypes had identical esterase activities.

It was then of interest to determine whether or not theseesterases could be tentatively classified as A or B classenzymes based on their sensitivity to inhibition byorganophosphate and carbamate insecticides. Protein prepa-rations of total crude extracts from wheat leaves wereassayed for esterase activity toward p-nitrophenyl acetateand �-naphthyl acetate in the presence of 0.1 mM insecti-cide. The organophosphate paraoxon reduced activity to-ward p-nitrophenyl acetate by 18% and toward �-naphthylacetate by 75%, with the carbamate carbaryl reducing thetwo activities by 28% and 38%, respectively. To determinewhich esterases were sensitive to inhibition by insecticides,the crude wheat extracts were incubated with either 0.1 mMparaoxon, 0.1 mM parathion or 0.1 mM carbaryl for 60 mmprior to IEF and activity staining (Fig. 3). In wheat,paraoxon effectively inhibited all esterases with isoelectricpoints above pH 5, though the more acidic isoenzymes,notably the esterase of pI 4.6, were unaffected. Carbaryl

showed a similar spectrum of inhibitory activity toparaoxon, but was less effective in reducing the activity ofthe esterases. To confirm the specificity of this inhibition,parathion, which is the inactive phosphorothionate precur-sor of paraoxon, was used. As expected, parathion wasineffective in inhibiting any of the esterases (Fig. 3). Therelative sensitivity of wheat esterases to inhibition by theinsecticides helped to confirm the sources of these enzymesin the progenitor species. Thus, the relatedness of the es-terases in T. tauschii and wheat was further demonstrated bytheir similarities in activity staining in the presence of carba-ryl (Fig. 3). The exception to this was the major activity ofthe acidic wheat esterase (pI 4.6), which was derived from T.urartu (AA) and was insensitive to inhibition by carbaryland paraoxon.

As determined in wheat, the majority of esterases in wildoat and black-grass were also strongly inhibited byparaoxon and to a lesser extent by carbaryl (Fig. 3). Thepatterns of inhibition by paraoxon and carbaryl in theextracts from the Peldon and Rothamsted black-grass plantsfurther confirmed earlier observations concerning the simi-larities in the esterase complement in the herbicide-sensitive(Rothamsted) and herbicide-resistant (Peldon) biotypes.

Purification and characterisation of a p-nitrophenylacetate-hydrolysing esterase from wheat shoots

In order to characterise the esterases involved in the hydrol-ysis of pesticides and other xenobiotics in greater detail, theenzyme responsible for the majority of p-nitrophenyl acetatehydrolysis in wheat seedlings was purified from the shoots.The purification strategy adopted was to use a combinationof classical chromatographic methods, based on resolutionby hydrophobicity, molecular mass and net acidic charge,respectively. A summary of the purification obtained isgiven in Table 2. Crude protein preparations were firstprecipitated with 40–80% (NH4)2SO4 and then applied to aphenyl-Sepharose hydrophobic interaction column. Usingp-nitrophenyl acetate as the enzyme substrate, a small pro-portion of the activity was eluted with decreasing salt con-tent (Fig. 4A). However, the majority of the activity wasrecovered after the addition of ethylene glycol in one majorpeak, followed by a minor peak. These two peaks wereanalysed by IEF with the major peak containing a similarprofile of esterases to that determined present in the crude

Physiol. Plant. 113, 2001 481

preparation (Fig. 5). The second minor hydrophobic frac-tion contained several basic isoenzymes, which represented aminor proportion of the total activity present on the activitygels and this fraction was not purified further (data notshown). The proteins present in the major hydrophobic peakwere further purified on a calibrated high-performance gelfiltration chromatography column. The majority of esteraseactivity, representing 70% of the recovered activity, eluted asone peak, which co-eluted with ovalbumin (43 kDa), with 3minor peaks with estimated masses of 95, 60 and 25 kDa,respectively, also observed (Fig. 4B). Although gel filtrationchromatography gave good purification, some 90% of thetotal activity in the enzyme preparation was lost in this step(Table 2). The major esterase activity, which had an esti-mated relative molecular mass of 45 kDa, was then sub-jected to two rounds of high-performance anion-exchangechromatography, with the final enzyme activity eluting as adefined shoulder on a UV-absorbing peak (Fig. 4C). Asdetermined from its specific activity, this final esterasepreparation was apparently purified 7300-fold in 6% yield(Table 2). When analysed by IEF, it was clear that thepurified preparation contained a single active esterase of pI4.6, corresponding to the dominant acidic esterase seen inthe crude wheat shoot extracts (Fig. 5). When analysed bySDS-PAGE the purified preparation was enriched with a45-kDa polypeptide (Fig. 6), which matched the expectedmass of the esterase predicted from gel filtration. Furtherproof that the 45-kDa polypeptide represented the esterasewas obtained by monitoring the relative abundance of thepolypeptide in the active fractions eluted from the finalion-exchange run (Fig. 4C). Only the relative content of the45-kDa polypeptide mirrored the esterase activity towardp-nitrophenyl acetate. After allowing for losses in activityduring purification, this 45-kDa esterase could theoreticallyaccount for all the activity toward p-nitrophenyl acetateobserved in the crude wheat extract. However, other minorfractions with this activity had been resolved from the45-kDa esterase during the course of the purification (Fig.4), suggesting that several wheat esterases could hydrolysep-nitrophenyl acetate.

The final preparation of the 45-kDa esterase was esti-mated to be 90% pure based on densitometric analysis ofsilver-stained SDS-PAGE gels. The final preparation wasthen assayed for activity toward model and pesticide es-terase substrates (Table 3). With p-nitrophenyl acetate assubstrate, the purified enzyme showed Michaelis-Mentenkinetics, with an apparent Km of 4.2 mM and a Vmax of 267nkat �g−1 pure protein. With other p-nitrophenol esters,activity declined with the increasing chain length of the acid

moiety, such that activity with the butyrate (C4) ester wasonly 5% of that determined with the acetate (C2) ester, withno activity seen with the caprylate (C8) ester. The purifiedesterase was also highly active toward both �-naphthylacetate and �-naphthyl acetate, but showed limited activitytoward pesticide esters, being able to hydrolyse binapacryland bromoxynil octanoate, but unable to hydrolyse diclofopmethyl or other APP herbicides (data not shown). Even withbinapacryl and bromoxynil octanoate, after correcting forrecoveries, it could be calculated that the 45-kDa esteraseaccounted for only 0.3 and 0.4%, respectively, of the totalactivity toward these pesticides present in the crude wheatextracts. The enzyme showed no hydrolysing activity towardthe organophosphate insecticides parathion or paraoxon orthe carbamate carbaryl, indicating that the enzyme couldnot be classified as a classic A-class esterase. To determine ifthe enzyme was a B-class esterase, it was pre-incubated with0.5 mM solutions of serine hydrolase inhibitors and thenassayed for activity toward p-nitrophenyl acetate. Phenyl-methylsulphonyl fluoride, parathion and paraoxon had noeffect on activity, while carbaryl reduced activity by 45%.However, with 0.1 mM carbaryl, no inhibition was observedand it was concluded that the 45-kDa enzyme was not aclassical B-class esterase.

Discussion

Although esterases have long been used in plants asbiomarkers for genotyping in zymogen analysis of crops andweeds (Wouters and Booy 2000) and markers for cell viabil-ity (Steward et al. 1999), remarkably little is known aboutthe functions of these enzymes in planta. Similarly, the genesencoding esterases active in pesticide metabolism have notbeen identified in plants. This is in contrast to the situationin animals and insects where their importance in protectingagainst the toxicity of drugs and insecticides has made themthe object of extensive investigation (Oakeshott et al. 1999).Our results confirm earlier observations concerning the pres-ence of multiple esterases with activity toward �-naphthylacetate in wheat (Krell and Sandermann 1984). IEF clearlyresolved 13 esterases with activity toward �-naphthyl acetatein wheat shoots. However, analysis of the esterases presentin the Triticum precursors suggested that the IEF analysisprobably underestimates the true number of esterase isoen-zymes present, with multiple forms potentially co-chro-matographing in hexaploid wheat (Fig. 2). The inhibitorstudies with organophosphate and carbamate insecticidesdemonstrated that the majority of the esterases with activitytoward �-naphthyl acetate and isoelectric points in the

Table 2. Purification of acidic esterase with activity toward p-nitrophenyl acetate from wheat shoots.

PurificationTotal activity YieldTotal protein (mg) Specific activityPurification step(nkat mg−1)(nkat) (-fold) (%)

Crude extract 10010.16131584150.463800Ammonium sulphate 1749 2.9 1332.41420600Phenyl-Sepharose pool 10815.0

Gel filtration pool 3.6 150 41.6 260 11.47.81596255.4102Anion-exchange 1 0.4

Anion-exchange 2 0.07 81.8 1168 7300 6.2

Physiol. Plant. 113, 2001482

Fig. 4. Chromatograms showing the sequential purification ofwheat esterase with UV absorbance at 280 nm shown with theunbroken line and associated activity toward p-nitrophenyl acetateshown in the bar chart. A, hydrophobic interaction chromatogra-phy, with the decreasing ammonium sulphate and subsequent in-creasing content of ethylene glycol shown with the dashed line; B,high-performance gel filtration chromatography; C, second roundof high-performance anion-exchange chromatography with the in-creasing content of NaCl shown with the dashed line.

In addition to showing activity toward p-nitrophenylacetate, the purified 45-kDa esterase could hydrolyse, andthereby bioactivate, the non-selective herbicide bromoxyniloctanoate and the fungicide binapacryl. However, the en-zyme represented only a small proportion of the total activ-ity toward these pesticides present in whole wheat plantsand showed no activity toward diclofop methyl or otherAPP herbicides used in wheat. Interestingly the 45-kDaesterase had its origins in the progenitor T. tauschii, whichwas also the sole donor of the hydrolysing activity towardsAPP herbicides in wheat (Table 1). Significantly, T. tauschiihas also been shown to be an important donor of glu-tathione transferases active in herbicide metabolism to mod-ern bread wheat (Riechers et al. 1997).

Our results demonstrate that the esterase complement ofwild oat and black-grass is very different to that observed inwheat with esterase activity toward the APP herbicide diclo-fop methyl being higher in the weeds than in the crop. Theresults of our study therefore give support to earlier obser-vations concerning the rapid bioactivation by hydrolysis ofAPPs in grass weeds, which then contributes to their sensi-tivity to these herbicides (Jeffcoat and Harries 1973). In thecase of the black-grass biotypes, the resistance of Peldon to

Fig. 5. Non-denaturing IEF gel showing esterase activity toward�-naphthyl acetate associated with the sequential purification of themajor esterase activity toward p-nitrophenyl acetate following 1,precipitation with 40–80% ammonium sulphate (100 �g protein); 2,major active fraction from hydrophobic interaction chromatogra-phy eluted with ethylene glycol (50 �g protein); 3, high-performancegel filtration chromatography (20 �g protein); 4, second round ofhigh-performance anion-exchange chromatography (100 ngprotein). Positions of pI marker proteins are arrowed.

range pH 5–7 were B-class esterases, being irreversiblyinhibited by these compounds (Fig. 3). However, our resultsdemonstrated that the highly active acidic esterase of pI 4.6present in wheat, which accounted for a large proportion ofthe activity toward �-naphthyl acetate and most of theactivity toward p-nitrophenyl acetate, was insensitive toinhibition by organophosphate insecticides and could not bea B-esterase. This was confirmed by purifying the corre-sponding 45-kDa esterase and demonstrating that the en-zyme was insensitive to organophosphates or otherinhibitors of serine hydrolases. However, by classical defini-tion, the enzyme was not an A-esterase, being unable tohydrolyse organophosphates. The 45-kDa esterase purifiedin the current study was very different from the 38-kDaesterase with activity toward bis(2-ethyl-hexyl)phthalate pre-viously isolated from wheat cell cultures (Krell and Sander-mann 1984). The plasticiser-hydrolysing enzyme wascomposed of 22-kDa polypeptides and was inhibited byparaoxon and PMSF (Krell and Sandermann 1984). In turn,neither the 45- nor 38-kDa esterases appear to be directlyrelated to other plant hydrolases, such as lipases, active inhydrolysing natural products (Mohamed et al. 2000).

Physiol. Plant. 113, 2001 483

Fig. 6. Composite image of silver-stained polypeptides resolved bySDS-PAGE following sequential purification by 40–80% ammo-nium sulphate precipitation (lane 2, 30 �g protein), hydrophobicinteraction chromatography (lane 3, 30 �g protein), high-perfor-mance gel filtration chromatography (lane 4, 20 �g protein). Inlanes 5, 6, 7 (approximately 100 ng protein), fractions from thesecond round of high-performance anion-exchange chromatographyare analysed showing the co-incidence in abundance of the 45-kDapolypeptide with enzyme activities toward p-nitrophenyl acetate of156 nkat mg−1 protein (lane 5), 687 nkat mg−1 protein (lane 6)and 1168 nkat mg−1 protein (lane 7). The molecular weight mark-ers are shown in lane 1.

appear to be indistinguishable in terms of their esteraseactivities. Similarly, it has been reported in metabolismstudies in vivo that several APP herbicides are hydrolysed tothe respective free acids at similar rates in Peldon andRothamsted (Hall et al. 1997). The similarity in expressionof esterases in the two populations of black-grass is inmarked contrast to the expression of other pesticide-metabolising enzymes, such as glutathione transferases(Cummins et al. 1997) and cytochrome P450 monooxyge-nases (Hyde et al. 1996), which are expressed at higher levelsin Peldon as compared with Rothamsted. Thus, the herbi-cide-resistant and herbicide-susceptible biotypes will havesimilar abilities to de-esterify and bioactivate the APP herbi-cides, but only the resistant plants have an enhanced capac-ity to degrade the bioactive acid (Hall et al. 1997) anddetoxify products of phytotoxic injury (Cummins et al.1999).

Our results demonstrate that there are a large number ofdiverse proteins in wheat with hydrolysing activities towardpesticides and other xenobiotics and that these activitiesdiffer form those seen in competing weeds. It will now be ofinterest to identify further types of esterase present in cropsand weeds and determine their role in regulating the activityof pesticides.

Acknowledgements – Ian Cummins and Robert Edwards acknowl-edge the support of Syngenta and the Biotechnology and BiologicalSciences Research Council (grant 12/SAP 13249), which has beenco-ordinated through the Ministry of Agriculture Fisheries andFood LINK programme ‘Sustainable Arable Production throughPrecision Input Optimisation’. The authors thank Dr TorquilFraser, Zeneca Agrochemicals Ltd., and Dr Jane Townson for theirsupport and expert advice.

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Edited by P. Bo� ger

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