Copyright © Physiologia Plantarum 2000PHYSIOLOGIA PLANTARUM 108: 101–109. 2000Printed in Ireland—all rights reser6ed ISSN 0031-9317

Molecular cloning and characterization of a type III glutathioneS-transferase from cell suspension cultures of opium poppy treated witha fungal elicitor

Min Yu and Peter J. Facchini*

Department of Biological Sciences, Uni6ersity of Calgary, Calgary, Alberta, T2N 1N4, Canada*Corresponding author, e-mail: pfacchin@ucalgary.ca

Received 19 August 1999; revised 18 October 1999

Glutathione S-transferases (GSTs) catalyze the conjugation nant enzyme exhibited strong GST activity in bacterialof electrophilic compounds to glutathione (GSH), and are protein extracts, using 1-chloro-2,4-dinitrobenzene as the sub-

strate. Recombinant GST2 activity was inhibited by p-cou-involved in the detoxification of xenobiotics in plants. How-maroyl- and feruloyl-CoA esters, and p-coumaric- andever, little is known about the endogenous substrates of plant

GSTs, or their role in normal cellular processes. A cDNA ferulic-acid amides of tyramine, but not by free hydroxycin-namic acids or aromatic amines. Although type III GSTs inlibrary was constructed with poly(A)+ RNA isolated from cell

suspension cultures of opium poppy (Papa7er somniferum L.) opium poppy appear to bind select endogenous phenolics, thein vitro conjugation of GSH to these compounds was nottreated with a fungal elicitor. A putative type III GST

fragment was amplified by PCR from the cDNA library using detected. Genomic DNA gel blot-hybridization analysis con-degenerate primers designed from conserved domains found in firmed the existence of a small family of type III GST genes

in opium poppy. RNA gel blot-hybridization analysis showedseveral plant GSTs. The cDNA library was screened with thethat type III GST transcripts were most abundant in the rootsPCR product and 3 homologous clones were isolated. Deduced

amino acid sequences from these clones displayed extensive of mature opium poppy plants, and were induced in developingseedlings and in cell suspension cultures treated with a fungalhomology to other plant type III GSTs, especially within theelicitor. Relative transcript levels were generally consistentN-terminal domain. One full-length cDNA, designatedwith the levels of total GST enzyme activity in each tissue.pGST2, was expressed in Escherichia coli, and the recombi-

associated with resistance to a broad spectrum of herbicidesdue to the diverse substrate specificity of the multitude ofGSTs found in plants.

In contrast to the well-established function of GSTs in theacute detoxification of xenobiotics, little is known abouttheir role in normal plant cellular or physiological processes.GSTs have been implicated in a variety of stress responsessuch as dehydration (Kiyosue et al. 1993), wounding (Kimet al. 1994), pathogen challenge (Dudler et al. 1991), andexposure to heavy metals (Czarnecka et al. 1988), and havebeen shown to be induced by active oxygen (Bartling et al.1993), herbicide safeners (Marrs 1996), and the hormonesauxin (Takahashi et al. 1989) and ethylene (Zhou and

Introduction

Glutathione S-transferases (GSTs; EC 2.5.1.18) are solubledimeric proteins that catalyze the covalent conjugation ofvarious xenobiotics to the tripeptide glutathione (g-glu-tamyl-cysteinyl-glycine; GSH) in mammals, insects, bacteria,and plants (Marrs 1996). GSH acts as a cellular nucleophilethrough the thiol group of a cysteinyl residue that attacksthe electrophilic centers of hydrophobic, and typically cyto-toxic, compounds (Mannervik and Danielson 1988). Inplants, glutathione conjugates are selectively imported intovacuoles for further processing or degradation (Marrs 1996).Plant GSTs have been extensively studied due to their rolein the detoxification of several types of selective herbicides(Marrs 1996). Variable levels of detoxification are often

Abbre6iations – BME, b-mercaptoethanol; CDNB, 1-chloro-2,4-dinitrobenzene; DCNB, 1,2-dichloro-4-nitrobenzene; GST, glutathioneS-transferase; IPTG, isopropyl-b-D-thiogalactopyranoside.

Physiol. Plant. 108, 2000 101

Goldsbrough 1993). Several flavonoid and phenyl-propanoid natural products have been proposed as en-dogenous substrates for plant GSTs (Marrs 1996, Li et al.1997). Although the direct role of GSTs in the conjuga-tion of these compounds with GSH remains controversial,glutathionation has been suggested as a necessary step be-fore vacuolar sequestration can occur via a glutathionepump located in the tonoplast membrane (Li et al. 1997).

GSTs are encoded by an ancient and diverse gene fam-ily. Mammalian GSTs have been divided into 5 groups (a,m, p, s, and u) based on sequence similarity, immunologi-cal cross-reactivity, and substrate specificity. All cytosolicGSTs in plants belong to the archaic u class (Meyer et al.1991, Pemble and Taylor 1992) and are subdivided into 3types (I, II, and III) based on a combination of sequenceconservation, immunological cross-reactivity, and intron/exon organization within the corresponding gene (Drooget al. 1995, Marrs 1996). Type I GSTs have 2 introns andare often transcriptionally and translationally inducible;type II GSTs have 9 introns, but the only reported se-quence is from carnation (Meyer et al. 1991); type IIIGSTs have only 1 intron and are also transcriptionallyand translationally inducible. Collectively, GSTs areamong the most abundant nonphotosynthetic enzymes inmany domesticated plants comprising, for example, up to1% of the soluble protein in a maize leaf (Marrs 1996).

Treatment of opium poppy cell suspension cultures witha fungal elicitor results in the activation of an arsenal ofbiochemical defenses. These include the accumulation ofthe antimicrobial benzylisoquinoline alkaloid sanguinarineand the incorporation of hydroxycinnamic acid amides inthe cell wall (Facchini 1998). Sanguinarine and its precur-sors are endogenous cytotoxins; thus, metabolic sequestra-tion to the vacuole is an important aspect of alkaloidbiosynthesis in plant cells. Hydroxycinnamic acid amidesare synthesized in the cytosol, but are oxidatively cross-linked to the cell wall as a means of cellular reinforce-ment. The mechanisms involved in the translocation ofsecondary metabolites, including the extracellular secretionof amides and the vacuolar localization of alkaloids, arepoorly understood.

In maize, mutations in the anthocyanin biosyntheticgene Bronze-2 (BZ-2) affect the proper localization of theanthocyanin precursor cyanidin-3-glucoside (C3G) to thevacuole (Marrs 1996). Accumulation of C3G in the cyto-sol results in poor vigor, cell necrosis, and a bronze pig-mentation. The BZ-2 protein was shown to be a type IIIGST, which is involved in the vacuolar deposition of C3G(Marrs et al. 1995). Transport of C3G into acidic vacuolesallows the anthocyanin to display its red-purple color. Theapparent function of the BZ-2 GST in the vacuolar local-ization of anthocyanins prompted us to consider the possi-bility that other type III GSTs might play a role in thetranslocation of benzylisoquinoline alkaloids and/or hy-droxycinnamic acid amides of tyramine in opium poppy.In this paper, we report the molecular cloning and charac-terization of type III GST cDNAs from opium poppy cellsuspension cultures treated with a fungal elicitor.

Materials and methods

Maintenance of plants and cell suspension cultures

Opium poppy (Papa6er somniferum L. cv. Marianne) plantswere grown under greenhouse conditions at day/night tem-peratures of 20/18°C. Seedlings were grown at 23°C insterile Petri plates on Phytagar (Gibco, Burlington, ON,Canada) containing Gamborg B5 salts and vitamins under aphotoperiod of 16 h using standard cool white fluorescenttubes (Sylvania Gros-Lux Wide Spectrum, Mississauga, ON,Canada) with a fluence rate of 35 mmol m−2 s−1. Beforegermination, the seeds were surface sterilized with 20% (v/v)sodium hypochlorite for 15 min and thoroughly rinsed withsterile water. Opium poppy cell suspension cultures weremaintained in the dark at 23°C on 1B5C medium consistingof Gamborg B5 salts and vitamins plus 550 mM myo-inosi-tol, 1 g l−1 hydrolyzed casein, 60 mM sucrose, and 4.5 mM2,4-dichlorophenoxyacetic acid (2,4-D). Cells were subcul-tured every 6 days using a 1:4 dilution of inoculum to freshmedium.

Elicitor treatment of cell suspension cultures

Fungal elicitor was prepared from Botrytis spp. according toFacchini (1998). A section (1 cm2) of mycelia cultured onpotato dextrose agar was grown in 50 ml 1B5C medium,including supplements but lacking 2,4-D, on a gyratoryshaker (120 rpm) at 22°C in the dark for 6 days. Mycelia (ca10 g fresh weight) and remaining medium (ca 40 ml) werehomogenized at maximum speed for 10 min with a Polytron(Brinkmann, Westbury, NY, USA), autoclaved (121°C) for20 min, and subsequently centrifuged under sterile condi-tions with the supernatant serving as elicitor. Elicitor treat-ments were initiated by the addition of 0.5 ml of fungalhomogenate to 50 ml of cultured cells in rapid growth phase(2–3 days after subculture). Cells were collected by vacuumfiltration and stored at −80°C.

GST enzyme assay

Plant tissues were frozen under liquid nitrogen, ground to afine powder with a mortar and pestle, and extracted in 100mM Tris-HCl, pH 7.8. Bacterial pellets were extracted in100 mM Tris-HCl, pH 7.8, by sonication. In both cases,debris was removed by centrifugation, and supernatantswere desalted on PD-10 columns (Pharmacia, Uppsala, Swe-den). Published spectrophotometric assays were used todetermine GST activity with the following electrophilic sub-strates: CDNB, DCNB, 4-nitrobenzyl chloride, 4-phenyl-3-buten-2-one, ethacrynic acid (Holt et al. 1995), and benzylisothiocyanate (Kolm et al. 1995). Standard reactions con-tained 100 mM potassium phosphate, pH 6.5 (pH 7.5 forDCNB), 1.0 mM GSH (0.25 mM for 4-phenyl-3-buten-2-one and ethacrynic acid), 1.0 mM electrophilic substrate (0.2mM for benzyl isothiocyanate and ethacrynic acid; 0.05 mMfor 4-phenyl-3-buten-2-one), 1% (v/v) ethanol, and 100 mlprotein extract in a total volume of 1.0 ml. The reaction wasinitiated by the addition of the electrophilic substrate, andthe change in absorption at the appropriate wavelength was

Physiol. Plant. 108, 2000102

monitored for 180 s. Initial reaction velocities were cor-rected for the spontaneous non-enzymatic reaction. One unitof activity is defined as the formation of 1 mmol productmin−1 at 25°C, with the product quantified according topublished extinction coefficients. Protein concentration wasdetermined using the Bradford assay (Bio-Rad, Hercules,CA, USA) with bovine serum albumin (BSA) as thestandard.

GST inhibition assays were performed with 1 mM GSHand 1 mM CDNB as described above, except that variouspotential inhibitors were added to the reactions, at severalconcentrations up to 500 mM, before the addition ofenzyme.

Nucleic acid isolation and analysis

Total RNA was isolated according to Logemann et al.(1987) and poly(A)+ RNA was selected by oligo(dT) cellu-lose chromatography. For gel blot analysis, 15 mg of totalRNA was fractionated on 1.0% (w/v) agarose gels, contain-ing 7% (v/v) formaldehyde, before transfer to nylon mem-branes. Poppy leaf genomic DNA (20 mg) was isolated,digested with restriction endonucleases, electrophoresed on1.0% agarose gels, and transferred to nylon membranes.Blots were hybridized to the random-primer-[32P]-labeledfull-length pGST2 insert at 65°C (high stringency) or 55°C(low stringency) in 0.25 M sodium phosphate, pH 8.0, 7%(w/v) SDS, 1% (w/v) BSA, and 1 mM EDTA. Blots werewashed at 65°C (high stringency) or 55°C (low stringency),twice with 2× SSC containing 0.1% (w/v) SDS, and twicewith 0.2× SSC containing 0.1% (w/v) SDS (1× SSC=0.15 M NaCl and 0.015 M sodium citrate, pH 7.0). Theblots were autoradiographed on Kodak X-OMAT film at−80°C. Double-stranded DNAs were sequenced using thedideoxynucleotide chain-termination method. The ClustalXsoftware package was used to generate basic sequence align-ments, which were then optimized manually.

Library construction and screening

A unidirectional oligo(dT)-primed cDNA library was con-structed in lUni-ZAPII XR, according to the manufactur-er’s instructions (Stratagene, La Jolla, CA, USA), usingpoly(A)+ RNA isolated from opium poppy cell suspensioncultures treated for 10 h with fungal elicitor. The primarylibrary contained 1×107 phage and was screened with a270-bp random-primer-[32P]-labeled fragment of opiumpoppy GST cDNAs amplified by PCR using degenerateoligonucleotide primers [sense primer=5%-TGGCCN(A/T)(G/C)NCCNTT(T/C)G-3%; antisense primer=5%-GT-NGCCCA(A/G)AANC(G/T)NGC-3%]. The primers weredesigned from 2 highly conserved domains found in type IIIGSTs from Aegilops squarrosa (GenBank accession no.AF004353), Arabidopsis thaliana (AC000348), Carica papaya(AJ000923), Eucalyptus globulus (U80615), Glycine max(AF048978, P32110, X68819), Nicotiana tabacum (Q03663),Mesembryanthemum crystallinum (AF079511), Oryza sati6a(AF050102), Picea mariana (AF051214), Vigna radiata(U20809), and Zea mays (U14599, Y12862). The sense

primer corresponded to the motif WPSPF[V/G] (residues13–17 in the A. squarrosa sequence), and the antisenseprimer to the motif ARFWA[D/K] (residues 98–103 in theA. squarrosa sequence). Thirty PCR cycles were performedat an annealing temperature of 40°C using Taq DNA poly-merase and approximately 1×105 phage from the primarycDNA library as template. The phage were lysed at 100°Cfor 10 min before PCR was initiated. Hybridization condi-tions were identical to those described above. Plasmids wererescued from phage that produced a positive signal using theR408 helper phage.

Heterologous expression of GST2 from opium poppy inEscherichia coli

The coding region from opium poppy pGST2 was amplifiedby 30 PCR cycles using oligonucleotides that introducedEcoRI and BamHI restriction sites at the 5%- and 3%-ends,respectively [sense primer=5%-ATATATGAATTCTTATG-GCAGGATCAGGAAGTGA-3%; antisense primer=5%-ATATATGGATCCTTTCTAGTTTGAAGGAGGAG-3%].PCR was performed at an annealing temperature of 50°Cusing opium poppy pGST2 as template. The PCR productwas digested with EcoRI and BamHI and ligated into thecorresponding restriction sites of the pT7-7 expression vec-tor to yield the pT7-GST2 construct. E. coli XL-1 Blue cellswere transformed with pBS-GST2, which consisted of afull-length GST2 cDNA fused in frame to the first 38codons from the lacZ gene in pBluescript. E. coliBL21(DE3)physS cells were transformed with pT7-GST2,which encoded a translational fusion between the GST2polypeptide and 5 spurious N-terminal amino acids. Bothbacterial strains were grown to A600=0.5 at 30°C in Luria-Bertani media containing 0.5 mM IPTG. After centrifuga-tion, the cell pellets were either solubilized in SDS samplebuffer and subjected to SDS-PAGE on a 10% (w/v) poly-acrylamide gel, or extracted for soluble protein. As controls,E. coli XL-1 Blue cells transformed with pBluescript, andBL21(DE3)physS cells transformed with pT7-7, were pro-cessed as described above.

Results

Cloning of type III GST cDNAs from elicited opium poppycell cultures

A 270-bp PCR product amplified from an opium poppyelicitor-treated cell suspension culture cDNA library usingdegenerate primers was used to screen the same cDNAlibrary at high stringency. Three different full-length cDNAs(pGST1, pGST2, and pGST3) were identified among theisolated clones. Comparison of amino acid sequences de-duced from the longest open reading frame on each clonewith proteins in the GenBank, EMBL, and Swiss-Prot data-bases revealed extensive homology with type III GSTs froma variety of plant species.

The nucleotide and deduced amino acid sequences of thepGST2 cDNA from opium poppy are shown in Fig. 1. The947-bp clone consisted of an open reading frame of 699 bp,

Physiol. Plant. 108, 2000 103

flanked by a 26-bp 5%-leader sequence and a 192-bp 3%-un-translated region, and followed by a polyadenylate tract.The deduced translation product initiated at the first in-frame ATG was comprised of 233 amino acids with apredicted molecular mass of 26 kDa, and a predictedisoelectric point of 4.8. The opium poppy pGST1 andpGST3 cDNAs showed 94 and 95% nucleotide identity,respectively, to pGST2. At the amino acid level, GST1 andGST3 exhibited 100 and 97% identity to GST2, since mostdivergent nucleotides were found in untranslated regions.These opium poppy GST cDNA clones have been depositedin the GenBank nucleotide sequence database under theaccession numbers AF118924 (pGST1), AF118925 (pGST2),and AF118926 (pGST3).

Alignment of the deduced amino acid sequence frompGST2 with clones in the GenBank database showed thatthe isolated opium poppy cDNAs belonged to the superfam-ily of type III GSTs (Droog et al. 1995). Overall, the GST2sequence from opium poppy was most similar to other typeIII GSTs within the N-terminal domain, whereas the C-ter-minal region showed more variation. As shown in Fig. 2,opium poppy GST2 shared the highest (46–48%) overall

Fig. 2. Alignment of the deduced amino acid sequence of opiumpoppy GST2 with other members of the type III GST gene familyfrom a variety of plant species: A. thaliana T7N9.15 (GenBankaccession number AC000348); A. squarrosa (AF004353); P. marianaprobable GST (AF051214); Z. mays Bronze-2 [BZ-2] (U14599); andG. max Gm-HSP26A (P32110). Shaded boxes indicate residues thatare identical in at least 50% of the aligned proteins. Gaps intro-duced into sequences to maximize alignments are shown by dots.

Fig. 1. Nucleotide and predicted amino acid sequence of the GST2cDNA from opium poppy cell suspension cultures treated with afungal elicitor. Underlined sequences indicate the location ofHindIII restriction endonuclease sites.

amino acid identity with probable type III GSTs fromArabidopsis, wheat (A. squarrosa), and black spruce (P.mariana). Extensive (50%) amino acid identity in the C-ter-minal region was also found in a cotton (Gossypium hirsu-tum) clone (AF064201), but the partial sequence wasmissing ca 80 amino acids from the N-terminus. Other typeIII GSTs, notably BZ-2 from maize (Z. mays) and thewell-characterized Gm-HSP26A auxin-induced protein fromsoybean (G. max), displayed lower (40%) overall amino acididentity with opium poppy GST2 (Fig. 2). The reducedidentity in BZ-2 and Gm-HSP26A, compared with type IIIGSTs from Arabidopsis, wheat, and black spruce, reflects agreater sequence divergence in the C-terminal region.

Physiol. Plant. 108, 2000104

Organization of the type III GST gene family in opiumpoppy

Gel blot-hybridization analysis of genomic DNA digestedwith various restriction endonucleases and probed with thefull-length GST2 cDNA at high stringency, showed that thefamily of type III GST genes is restricted to 3 or 4 membersin opium poppy (Fig. 3). Rehybridization of the same blotshown in Fig. 3 with the GST2 probe at low stringencyrevealed an additional 3 to 4 bands in each lane, suggestingthe presence of other genes with limited homology to theisolated GST cDNAs (data not shown). The ca 300-bpHindIII fragment revealed by the GST2 probe (Fig. 3) is inagreement with the 2 HindIII sites found at positions 454and 764 in the pGST2 nucleotide sequence (Fig. 1). Ho-mologous HindIII sites were also found in pGST1 andpGST3.

Heterologous expression of opium poppy GST2 in E. coli

A full-length pGST2 clone (pBS-GST2) was identified inwhich the open reading frame was fused in-frame to the first38 codons of the lacZ gene, followed by 9 spurious ‘codons’from the pGST2 5%-untranslated region. The resulting fusionprotein consisted of 47 amino acids from the N-terminus ofb-galactosidase and the 5%-leader sequence of the opium

Fig. 4. Heterologous expression of the opium poppy GST2 cDNAin E. coli using 2 different expression vectors. pBS-GST2 is com-prised of the first 30 codons of b-galactosidase fused in-frame to the5%-leader sequence and coding region from GST2 in pBluescript.pT7-GST2 consists of 5 spurious N-terminal codons fused to theGST2 coding region in pT7-7. (A) Total bacterial proteins (100 mg)fractionated by SDS-PAGE on a 10% (w/v) polyacrylamide gel.Arrowheads indicate the GST2 fusion proteins resulting from eachvector. Numbers on the right refer to standard protein size markersin kDa. (B) Specific GST enzyme activity in total soluble proteinextracts from E. coli transformed with the pBluescript or pT7-7vectors, or the pBS-GST2 or pT7-GST2 constructs.

Fig. 3. Southern blot analysis of opium poppy genomic DNAhybridized to the full-length GST2 cDNA. The ca 300-bp HindIIIfragment (arrowhead) is in agreement with the position of HindIIIsites in pGST2. Opium poppy genomic DNA (20 mg) was digestedwith BamHI, EcoRI, HindIII, or XbaI, fractionated in a 1.0%agarose gel, transferred to a nylon membrane, and hybridized athigh stringency to the 32P-labeled GST2 cDNA. Numbers on theright refer to standard DNA size markers in kilobases (kb).

poppy GST2 cDNA, fused to the 233 amino acids of GST2.The apparent molecular mass, determined by SDS-PAGE,of the pBS-GST2 fusion protein expressed in E. coli wasconsistent with the predicted molecular mass of 31.1 kDa(Fig. 4A). Soluble GST activity, measured with GSH andCDNB as substrates, was more than 10-fold higher inbacteria harboring pBS-GTS2 compared with bacteriatransformed with pBluescript (Fig. 4B). The opium poppyGST2 coding region was subcloned into the pT7-7 expres-sion vector to eliminate the extensive N-terminal polypep-tide fusion contained within the original pGST2 cDNA. ThepT7-GST2 construct directed the expression of recombinantGST2 with only 5 spurious amino acids (MARIL) fused toits N-terminus. The apparent molecular mass, determinedby SDS-PAGE, of the pT7-GST2 fusion protein expressedin E. coli was consistent with the predicted molecular massof 26.5 kDa (Fig. 4A). Soluble GST activity, determinedwith GSH and CDNB as substrates, was approximately20-fold higher in bacteria harboring pT7-GST2 comparedwith bacteria transformed with pT7-7 (Fig. 4B).

Physiol. Plant. 108, 2000 105

Table 1. Specific activity of total soluble protein extracts from bacteria transformed with pT7-GST2 toward a variety of xenobioticsubstrates. Values are the mean9SD of 3 independent experiments. a In all cases, 1 mM GSH was used except in combination withethacrynic acid (0.25 mM) and trans-4-phenyl-3-buten-2-one (0.25 mM). b Change in absorption was monitored for 180 s at the wavelengthindicated. ND, not detected.

Specific activity (nmol mg−1 protein min−1)Substrate Concentrationa (mM) Wavelengthb (nm)

1-Chloro-2,4-dinitrobenzene 56981 3401,2-Dichloro-4-nitrobenzene 1 0.3890.043454-Nitrobenzyl chloride 1.0490.251 310Ethacrynic acid 1 ND270

NDtrans-4-Phenyl-3-buten-2-one 1 290Benzyl isothiocyanate 0.2 274 ND

Recombinant opium poppy GST2, produced in bacteriatransformed with pT7-GST2, was assayed for GST activityusing GSH and a variety of xenobiotic substrates (Table 1).The highest activity was found with the model GST sub-strate, CDNB. Weak GST activity was detected towardDCNB and 4-nitrobenzyl chloride, whereas 4-phenyl-3-buten-2-one, benzyl isothiocyanate, and ethacrynic acidwere not accepted as substrates. The latter 2 substrates weretested because they contain electrophilic groups similar tothose found in alkenals which accumulate during oxidativestress in animals (Berhane et al. 1994, Kolm et al. 1995).

Recombinant GST2 activity was inhibited by p-cou-maroyl- and feruloyl-CoA esters, and p-coumaric- and fer-ulic-acid amides of tyramine (Table 2). Feruloyl-CoA wasthe most effect inhibitor, followed by p-coumaroyl-CoA,feruloyltyramine, and p-coumaroyltyramine. In contrast,opium poppy GST2 activity was not inhibited by freehydroxycinnamic acids, coenzyme A, or aromatic amines atconcentrations up to 500 mM (Table 2). Sanguinarine wasalso not effective as an inhibitor of GST2 activity.

Developmental and inducible expression of type III GSTgenes in opium poppy

Expression of type III GSTs in opium poppy plants,seedlings, and elicitor-treated cell suspension cultures wasdetermined by RNA gel blot-hybridization analysis usingGST2 as the probe under high stringency conditions (Fig.5). Type III GST transcripts were most abundant in matureopium poppy roots and root tips (Fig. 5A). Lower transcriptlevels were detected in young and mature leaves and inflower buds. The lowest relative levels of expression oc-curred in young and mature stems. In whole opium poppyseedlings, type III GST transcripts were detected within 1day after imbibition (Fig. 5B). Transcript levels graduallyincreased up to 13 days after imbibition, which corre-sponded to the initiation of apical meristem development. Inopium poppy cell suspension cultures, type III GST tran-scripts were abundant in control cultures, but increasedapproximately 2-fold within 2–5 h after treatment withfungal elicitor (Fig. 5C). Subsequently, transcript levels re-turned to near control levels over the next 24 h.

Specific GST activity levels, measured with GSH andCDNB as substrates, were generally consistent with type IIIGST transcript levels in opium poppy tissues (Fig. 6). In theplant, GST activity was highest in mature roots and roottips, and lowest in flower buds and leaves (Fig. 6A). How-ever, GST activity in stems, especially mature stems, was

higher than in any other organ except roots, despite some-what lower type III GST transcript levels (Fig. 5A). GSTactivity in whole seedlings increased from the lowest level 1day after imbibition to the highest detected level 13 daysafter imbibition (Fig. 6B), in agreement with the inductionof type III GST transcripts in opium poppy seedlings (Fig.5B). Similarly, GST activity increased in elicitor-treated cellsuspension cultures from 190 nmol mg−1 protein min−1 incontrol cultures to a maximum level of approximately 330nmol mg−1 protein min−1 5 h after the initiation of elicitortreatment (Fig. 6C), which is also consistent with the elici-tor-mediated induction of type III GST transcripts (Fig.5C). It must be emphasized, however, that Fig. 6 representsthe total activity of all GSTs in the various opium poppytissues, whereas Fig. 5 only shows the relative transcriptlevels of type III GSTs with extensive sequence identity toopium poppy GST2.

Discussion

A small family of highly homologous type III GST cloneswas isolated from an opium poppy elicitor-treated cell sus-pension culture cDNA library. Gel blot-hybridization analy-sis of genomic DNA showed that the immediate gene familyis comprised of only 3 to 4 members (Fig. 3). However,re-hybridization of the blot at low stringency revealed sev-eral other homologous regions in the opium poppy genome,

Table 2. Inhibition of GST2 activity in total soluble protein ex-tracts from bacteria transformed with pT7-GST2 by various en-dogenous metabolites. The standard GST assay, performed using 1mM glutathione and 1 mM 1-chloro-2,4-dinitrobenzene, exhibiteda specific activity of 56 nmol mg−1 protein min−1 in the absence ofinhibitors. Values are the mean9SD of 3 independent experiments.IC50 is the concentration of each compound at which GST specificactivity in the standard assay was reduced by 50%. NI, no inhibi-tion at 500 mM.

Compound IC50 (mM)

Cinnamic acid NINIp-Coumaric acid

Ferulic acid NICoenzyme A NI

7994p-Coumaroyl-CoAFeruloyl-CoA 4893

375912p-CoumaroyltyramineFeruloyltyramine 12095

NITyramineNIDopamine

Sanguinarine NI

Physiol. Plant. 108, 2000106

Fig. 5. Levels of type III GST mRNAs in: (A) different organs ofmature opium poppy plants; (B) whole opium poppy seedlings ondifferent days after imbibition; and (C) opium poppy cell suspen-sion cultures at different times after treatment with a fungal elicitor.Total RNA was extracted and 15 mg was fractionated on 1.0% (v/v)formaldehyde agarose gels, transferred to nylon membranes, andhybridized at high stringency with the 32P-labeled GST2 cDNA.Gels were stained with ethidium bromide prior to blotting to ensureequal loading.

1992, Reinemer et al. 1996). It has been suggested that thevariability in the C-terminal domain is due to the diversityof electrophilic compounds recognized as GST substrates(Marrs 1996). The diverse substrate affinity exhibited by

Fig. 6. Specific GST enzyme activity toward CDNB in: (A) differ-ent organs of mature opium poppy plants; (B) opium poppyseedlings on different days after imbibition; and (C) opium poppycell suspension cultures at different times after treatment with afungal elicitor.

suggesting the existence of a limited number of relatedGSTs. An extended gene family comprised of GST homo-logues in opium poppy is not unexpected, since the currentlist of Arabidopsis expressed sequence tags (ESTs) includesmore than 200 different ESTs with homology to GSTs.However, since a motif of only about 15 amino acids isconserved in sequence and position among the more than 40GSTs isolated from plants (Marrs 1996), we cannot rule outthe possibility that other GSTs occur in opium poppy.

Alignment of opium poppy type III GSTs to other plantGST sequences reveals extensive conservation within theN-terminal domain (Fig. 2); thus, 2 conserved characteristicsof all GSTs (i.e. recognition of GSH and the ability todimerize) are localized to this region. X-ray crystallographicanalysis of GSTs from both plants and animals has verifiedthe N-terminal domain as the site of catalytic GSH bindingand protein dimerization, and the variable C-terminal do-main as the site of substrate recognition (Pemble and Taylor

Physiol. Plant. 108, 2000 107

various GSTs suggests that these enzymes might work col-lectively to scavenge toxic chemicals, comprised of bothendogenous metabolites and exogenous xenobiotics, in thecytosol. The relative similarity, especially in the C-terminaldomain, between opium poppy type III GSTs and thosefrom Arabidopsis (T7N9.15), wheat, and black spruce (Fig.2) suggests that these enzymes might exhibit a conservedspecificity for xenobiotics and/or endogenous substrates.

The pT7-GST2 construct was assembled to remove thespurious ca 5 kDa N-terminal polypeptide from the b-galac-tosidase-GST2 fusion protein (Fig. 4). The almost nativerecombinant GST2 protein resulting from pT7-GST2 wasexpected to exhibit the same catalytic properties as nativeGST2 from opium poppy. Recombinant GST2 and severalother plant GSTs display similar specificities toward variousmodel substrates (Table 1), with CDNB as the preferredelectrophile (Marrs 1996). The lower activity levels of GST2toward DCNB and 4-nitrobenzyl chloride are similar to thesubstrate specificity of recombinant maize GST V-V (Dixonet al. 1998) and purified sorghum GSTs (Gronwald andPlaisance 1998), among others (Marrs 1996 and referencestherein). In contrast to certain other plant GSTs, however,ethacrynic acid and benzyl isothiocyanate were not ac-cepted. In pea, GST activity toward ethacrynic acid wasinducible by treatment with GSH and fungal elicitor (Ed-wards 1996), whereas tobacco auxin-induced GSTs wereinhibited by ethacrynic acid (Droog et al. 1995).

Despite the numerous GSTs that have been isolated froma variety of plants, the normal physiological function ofonly one specific GST has been identified: BZ-2 is involvedin the vacuolar deposition of anthocyanins in maize (Marrset al. 1995). A characteristic of plant GSTs that has prohib-ited the identification of their cellular and physiologicalfunctions, is the apparent lack of GST activity towardputative endogenous substrates when the proteins are pro-duced in heterologous systems (Coleman et al. 1997, Alfen-ito et al. 1998). For example, the An9 gene from petunia,which encodes a type I GST, compliments the BZ-2 muta-tion in maize, resulting in C3G accumulation in the vacuole.However, the AN9 protein produced in electroporatedmaize cells, in recombinant E. coli, or in vitro failed toconjugate GSH to C3G, which has been suggested as an invivo substrate for BZ-2 and AN9 (Alfenito et al. 1998).Heterologously expressed BZ-2 and AN9 have both beenshown to conjugate GSH and model GST substrates, suchas CDNB (Marrs et al. 1995, Alfenito et al. 1998).

Similarly, heterologously expressed opium poppy type IIIGSTs show strong GSH-conjugation activity toward CDNB(Fig. 4). This activity was effectively and selectively inhibitedby p-coumaroyl- and feruloyl-CoA esters and p-coumaric-and ferulic-acid amides of tyramine (Table 2). However, wewere unable to detect a GST activity that resulted in theconjugation of GSH to these phenolic compounds. Thepossibility that anthocyanins, such as C3G, might also causethe inhibition of heterologous BZ-2 and AN9 activity to-ward model GST substrates must be tested. Inhibition of invitro GST activity by C3G, in the absence of GSH conjuga-tion, would be consistent with a putative role for BZ-2 andAN9 as ‘carrier’ proteins involved in the vacuolar localiza-tion of anthocyanins (Marrs et al. 1995, Alfenito et al.

1998). Although genetic evidence for the in vivo function ofopium poppy type III GSTs is not available, our datasuggest that these proteins possess the ability to bind CoA-esters and tyramine-derived amides of select phenolic acids;thus, opium poppy type III GSTs might play a role in themetabolism and/or intra- or extra-cellular translocation ofselect phenolics, in a manner similar to the still poorlyunderstood function of BZ-2 in anthocyanin transport. Incontrast, sanguinarine did not inhibit recombinant GST2activity; thus, sanguinarine metabolism or translocation donot appear to involve GST2.

Type III GSTs from opium poppy were expressed at alldevelopmental stages and in all organs, but transcript levelswere more abundant in roots (Fig. 5) suggesting that GSTsplay a specific role in development, localized metabolism,and/or xenobiotic detoxification. The abundance of GST2transcripts and enzyme activity in opium poppy roots issimilar to the developmental expression pattern of type IGSTs, such as GST I and GST IV in maize (Holt et al.1995). GST transcript accumulation (Fig. 5) and enzymeactivity (Fig. 6) rapidly increased following germination,again suggesting a developmental or protective function.The increase in GST transcript levels (Fig. 5) in elicitor-treated cell suspension cultures was at least partially respon-sible for the concomitant increase in GST activity (Fig. 6).GST transcripts have also been reported to accumulate inresponse to infection or treatment with fungal elicitors inwheat (Dudler et al. 1991), soybean (Levine et al. 1994),potato (Hahn and Strittmatter 1994), and pea (Edwards1996). Based on our data, it is compelling to speculate thatopium poppy type III GSTs are involved in the metabolismand intra- or extra-cellular translocation of hydroxycin-namic acid-CoA esters and cell-wall bound hydroxycin-namic acid amides, as part of the normal developmentalphysiology and defense responses of the plant.

Acknowledgements – Research was supported by grants from theNatural Sciences and Engineering Research Council (NSERC) ofCanada and the Alberta Agricultural Research Institute (AARI) toPJF.

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Edited by I. M. Mo*ller

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