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Pesticide Biochemistry and Physiology 88 (2007) 57–65

PESTICIDEBiochemistry & Physiology

Role of transporters in paraquat resistance of horseweed Conyzacanadensis (L.) Cronq.

Balazs Jori a, Vilmos Soos a,b, Dora Szeg}o a, Emil Paldi b,Zoltan Szigeti a, Ilona Racz a,*, Demeter Lasztity a

a Department of Plant Physiology and Molecular Plant Biology, Eotvos Lorand University, 1117 Budapest, Pazmany Peter setany 1/C, Hungaryb Agricultural Research Institute of the Hungarian Academy of Sciences, 2460 Martonvasar, Hungary

Received 12 April 2006; accepted 29 August 2006Available online 6 September 2006

Abstract

Paraquat (Pq) inducible transporters are presumed to play a role in the resistance mechanism of horseweed and to function by car-rying paraquat to a metabolically inactive compartment. The uptake and intracellular localisation of paraquat, the effect of transporterinhibitors on resistance, and paraquat-induced gene expression were studied to obtain a better understanding of the mechanism of resis-tance. Investigations proved that paraquat entered the cells of both resistant and susceptible biotypes, approached the maximum withinthe first hour in chloroplasts, and then declined in all organelle fractions. In the resistant biotype paraquat was located in the vacuoles aday after treatment. Selective transporter inhibitors blocked the sequestration of paraquat, suggesting the participation of not directlyenergized transporters. Four EST fragments were identified that were expressed in response to paraquat. Two of them are thought toplay a role in the general stress response (Ferr2, Myb). The others exhibit a similarity to transporters (EmrE, CAT) and could conceiv-ably be involved in the intracellular transport of paraquat and the mechanism of resistance.� 2006 Elsevier Inc. All rights reserved.

Keywords: Conyza canadensis; Paraquat resistance; Intracellular localisation of paraquat; Transporter inhibitors; Paraquat-induced gene expression

1. Introduction

Paraquat (1,1 0-dimethyl-4,4 0-bipiridyl) is a non-selective,post-emergence herbicide, widely used in the form of haloidsalts as the active agent in contact herbicides for total weedcontrol in vineyards, orchards and gardens and for thedefoliation and desiccation of field crops. Pq exerts its phy-totoxic effect by diverting electrons from the photosynthet-ic electron transport chain on the reducing side of PSI atthe FeSx level, while itself taking up an electron to forma cation radical. When this reverts to the Pq cation formby reacting with molecular oxygen, it generates a superox-ide anion radical, which reacts with Fe3+ or H+ ions in the

0048-3575/$ - see front matter � 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.pestbp.2006.08.013

* Corresponding author. Fax: +36 13812164.E-mail address: raczil@ludens.elte.hu (I. Racz).

Fenton reaction, while the paraquat cation radical gener-ates hydroxyl radicals in the presence of hydrogen peroxidein the Winterbourn reaction. The reactive oxygen speciesformed during these reactions, particularly the superoxideand hydroxyl radicals, damage membranes through lipidperoxidation and the destruction of the double bonds inthe side chains of fatty acids, thus leading to plant mortal-ity [1–4].

The frequent, repeated use of paraquat has resulted inthe development of resistant species, which at present num-ber more than twenty, including both monocot and dicotweeds over a wide range of geographical locations. Sincethe appearance of resistant species, several hypotheses havebeen proposed to explain the mechanism of resistance invarious species, but these have only been partially con-firmed, if at all, experimentally. It became clear in thecourse of resistance studies that Pq does not decompose

58 B. Jori et al. / Pesticide Biochemistry and Physiology 88 (2007) 57–65

in the plants. Only in Rehmannia glutinosa, which exhibitsnatural resistance, could metabolisation (by convertingPq into a bound, inactive compound) be detected indirectly[5,6].

One of the most popular theories, for which evidencehas been found in a number of paraquat-tolerant plantswith lower resistance factors, explains resistance as theincreased activity of antioxidant, protective enzyme sys-tems [7]. The activity of these enzymes does indeed rise incertain species as the result of Pq or other oxidative stress,but since the Pq cation radical functions catalytically, it isclear that the plant will be unable to cope in the long termwith protective enzyme systems alone.

The largest body of evidence has been found to supporttheories suggesting that resistance is caused by the fact thatonly a limited amount of Pq reaches its site of action in thechloroplasts (limited penetration has been detected inHordeum glaucum and Conyza bonariensis) or, if it doespenetrate the chloroplasts, it is rapidly transferred to a met-abolically inactive compartment by means of a sequestra-tion mechanism [8–13].

Paraquat-resistant weeds were first observed in Hungaryin the 1980s in an atrazine-resistant population (PqAr) ofhorseweed, Conyza canadensis [14], while later biotypesonly resistant to paraquat (PqR) were also found [15].The activity of enzymes detoxifying reactive oxygen specieswas not higher in the resistant plants [16,17]. There was areduction in the functional activity characterised byFv/Fm in these biotypes, demonstrating that Pq was ableto penetrate the chloroplasts, where it caused a transitoryinhibition, but within a few hours the functional activitystarted to return to normal. The recovery process, whichis a light-dependent, induced mechanism, could be inhibit-ed by cycloheximide, an inhibitor of eukaryotic proteinsynthesis [18,19]. It thus appeared probable that resistanceinvolved a protein, or proteins, induced by paraquat andresponsible for transporting Pq to a metabolically inactivecompartment [20].

In both prokaryotes and eukaryotes special transportersare involved in the removal of xenobiotics, including para-quat, from cells or in their transfer to metabolically inac-tive compartments. The most widespread of these arevarious families of multidrug transporters, many membersof which have been shown to be capable of transportingparaquat in organisms ranging from Escherichia coli

through yeasts to human beings, and which result in para-quat resistance when overexpressed [21–23]. Among themultidrug transporters the ABC transporters are the bestknown. These are large membrane proteins with a complexstructure, which use ATP directly for the active transportof various compounds. A further group consists of smallerantiporters, which exchange protons for some other mole-cule, thus exploiting the proton electrochemical potentialgradient for transport. Bacteria contain antiporters respon-sible for resistance to antibiotics, while in humans neuro-transmitters, including some capable of Pq transport,belong in this group. Even very small membrane proteins

may be capable of xenobiotic–proton/ion antiport, suchas the EmrE protein in E. coli, responsible for resistanceto paraquat and other toxins (e.g. ethidium). The structureof this protein is similar to that of the Fo subunit localisedin the membrane of H+-ATPases [24,25]. Transportersresponsible for the transport of molecules with a chemicalstructure or charge distribution similar to that of paraquat(e.g. polyamines, amino acids) may also play a role in resis-tance, as data in the literature confirm that they are capableof transporting paraquat [26–28]. In yeast, for example, apositive correlation was found between the expression ofpolyamine transporters and paraquat tolerance [29], inmaize putrescine was observed to inhibit Pq uptake [30]while in Arctotheca calendula spermidine and cadaverinewere effective in reducing paraquat translocation in suscep-tible biotype [31]. In our earlier studies putrescine was alsofound to provide protection against the inhibition of vari-able fluorescence by Pq in susceptible C. canadensis plants[27].

In the course of the present work studies were per-formed to elucidate the mechanism of Pq resistance. Sus-ceptible and resistant biotypes of C. canadensis were usedto investigate the uptake of Pq into the cells and its intra-cellular localisation, the effect on Pq resistance of inhibitorsthat selectively inhibit transporters enabling Pq to entercells or various cell organelles, and genes expressed as theresult of Pq treatment. The aim was to clarify the possiblerole in Pq resistance of transporters that transport orsequester Pq and to obtain a better understanding of themechanism of resistance.

2. Materials and methods

2.1. Plant material and treatments

Biotypes of C. canadensis resistant (PqR) or susceptible(S) to Pq were grown hydroponically in 1/4 strength Hoa-gland solution under laboratory conditions (illumination130 lEm2 s�1, 16 h light period, 22–25 �C). Plants at therosette stage were used in the experiments.

For kinetic experiments plants were sprayed with 1 kgactive ingredients/hectare (a.i./ha) paraquat as a 1% (v/v)solution (5 · 10�4 mol L�1) of Gramoxone. Transporterinhibitors, Na-orthovanadate as a 10�4 mol L�1, verapamilas a 5 · 10�5 mol L�1, ethyleneglycol-bis-N,N1-tetraaceticacid (EGTA) as a 5 · 10�4 and KNO3 as a 10�4 mol L�1

concentration solution, were also sprayed on the leaves ofplants alone or in combination with Pq. Plants were treatedwith inhibitors 2 h before or after the Pq treatment. In thecase of KNO3 treatment, leaves were spray-treated with theKNO3 solution every 60 min. Solutions of 0.5 ·10�4 mol L�1 menadione and 1% (w/v) bengal rose, theagents generating reactive oxygen species, were alsosprayed on the leaves. Functional activity of leaves wascharacterized by variable fluorescence (Fv/Fm) andexpressed as percentage of the initial control values. Fluo-rescence parameters were determined by PAM fluorometer

B. Jori et al. / Pesticide Biochemistry and Physiology 88 (2007) 57–65 59

(Walz, Effeltrich, Germany). In kinetic experiments 10leaves of 8–10 individual plants were used as parallel sam-ples at every measuring point during the time course of Fv/Fm changes. The experiments were repeated independently9–12 times.

2.2. Cell fractionation and photometric determination of Pq

content

Cell fractionation of leaves of the S and the PqR bio-types of horseweed was carried out by differential centrifu-gation as described earlier [28] using 10 g fresh weight ofleaves for each sample. Since the plants were spray treatedwith Pq, the intact leaves were washed with water threetimes for 1 minute before cell disruption and fractionationto remove residual extracellular Pq from the water freespaces (WFS) of the cell walls. The Pq content of the indi-vidual fractions (cell wall + nuclei, chloroplasts, mitochon-dria, cytosol + vacuole) was determined using 3–5 parallelsamples. The cell fractionation procedure and the determi-nation of the Pq contents of the fractions were carried outin three independent experiments. The total intracellularPq content was determined using 5 · 0.1 g fresh weight ofleaves. The experiments were repeated five times.

The Pq content was determined using a modified versionof the method of Kesary et al. [32]. After homogenising0.1 g fresh weight of plant sample in 1 ml of 0.2%Na2EDTA solution, the samples were heat-treated for3 min at 100 �C, followed by centrifugation at 10,000g for7 min. After adding 200 ll of 2 mol L�1 NaOH and200 ll 0.5% glucose to 600 ll supernatant, the samples werekept at 100 �C for 1 min. The precipitant was removed bycentrifugation at 10,000g for 3 min, after which the absor-bance was recorded at 603 nm. The Pq content wasdetermined using a calibration curve prepared from 1,1 0-di-methyl-4,4 0-bipyridinium dichloride. For comparison, acalibration curve was also prepared using Gramoxone (cal-culating the paraquat content on the basis of the activeingredient given by the manufacturer). The calibrationcurves were comparable, proving that the additional com-ponents of the herbicide Gramoxone did not interfere withthe determination of Pq content.

2.3. DDRT-PCR and DNA sequencing

The mRNA content of three-week-old plants was isolatedwith a GenoPrep �DirectmRNA Kit according to theinstructions of the manufacturer (GenoVision). cDNA syn-thesis was performed using a RevertAid First Strand PCRSynthesis Kit (MBI Fermentas) and H-Arbitrary universalprimers (HAP 1: 5 0-AAG CTT GAT TGC C-3 0, HAP 2:5 0-AAG CTT CGA CTG T-3 0, HAP 3: 5 0-AAG CTTTGG TCA G-3 0, HAP 4: 5 0-AAG CTT CTC AAC G-3 0,HAP 5: 5 0-AAG CTT AGT AGG C-3 0, HAP 6: 5 0-AAGCTT CGA CCA T-3 0, HAP 7: 5 0-AAG CTT AAC GAGG-3 0, HAP 8: 5 0-AAG CTT TTA CCG C-3 0). FollowingPCR amplification 1 ll of formamide loading dye was

mixed with 7 ll of each sample. These were loaded onto a6% urea, TBE, polyacrylamide sequencing gel (acrylam-ide–bis-acrylamide ratio 24:1). Electrophoresis was carriedout at 55 W constant power for 2 h. The gel was then recov-ered and stained with silver nitrate according to the instruc-tions of the manufacturer (Promega SILVER SEQUENCEDNA Sequencing System). The fragments of interest wereexcised with a scalpel and eluted by rinsing (1 h) and boiling(20 min) in 100 ll Milli-Q water. After precipitating andwashing with ethanol the eluted DNA (4 ll) was re-ampli-fied directly under conditions identical to those used forthe initial PCR, with the corresponding H-T11-M (5 0-HTT-TTT TTT TTT M-3 0) and H-Arbitrary universalprimers. The amplified PCR products were then sequencedusing an ABI BigDye Terminator Cycle Sequencing v3.1 kit(Applied Biosystems). Sequences which recurred at leastfour times were considered for further analysis.

The EST sequences were compared with sequencesdeposited in the databases (GenBank, EMBL, PDB), andtranslated nucleotid sequences were compared with proteinsequences in databases (GenBank CDS Translations, PDB,and SwissProt) using the BLAST 2.2.8. algorithm [33].Similar sequences were aligned and the sequence data wereanalysed using the ClustalW built-in BioEdit (version5.0.9) package [34]. Graphical display was made withGeneDoc Multiple Sequence Alignment Editor 2.6.002[39].

3. Results

3.1. Paraquat uptake and intracellular localisation in

susceptible and resistant biotypes of C. canadensis

The reduction in functional activity characterised byFv/Fm in our earlier studies suggested that paraquat wasable to penetrate into the cells and into the chloroplast,its site of action, in both susceptible and resistant plants.In the present work, this was confirmed by the direct chem-ical determination of both the changes in total intracellularPq content following herbicide treatment, and the changesin its distribution among the cell compartments obtainedby cell fractionation.

In the leaves of spray-treated plants, from which theresidual Pq in the cell walls was removed by washing priorto Pq determination, Pq appeared in the cells within 10 minof treatment and an hour after uptake near-maximum val-ues were recorded in both susceptible and resistant plants(Fig. 1A and B). In PqR plants the intracellular Pq valueswere still close to maximum four hours after spraying.Even 24 h after spraying, when the PqR plants had recov-ered their functional activity, they still contained a consid-erable amount of intracellular Pq. A month after treatmentwith herbicide, when the plants were still in the rosettestage of development, the intracellular Pq concentrationremained close to this level (Fig. 1C). In S plants, wherethe Fv/Fm values indicated a considerable reduction inphysiological activity within 4 h and morphology also

Fig. 1. Changes in intracellular paraquat content in susceptible and resistant biotypes of C. canadensis after Pq treatment. Leaves were spray-treated with5 · 10�4 mol L�1 paraquat. (A) Susceptible biotype (S); (B) resistant biotype (PqR); (C) changes in Pq content in S and PqR biotypes.

60 B. Jori et al. / Pesticide Biochemistry and Physiology 88 (2007) 57–65

showed the plants to be wilting, there was a reduction inthe Pq content (Fig. 1C). The decline in the Pq contentmeasured in susceptible plants 4 h after treatment can beattributed to the fact that washing for 1 min removed Pqnot only from the cell walls but also from cells with a dam-aged plasmalemma. Susceptible plants wilted within 24 h.

The time course of changes in the Pq content of variouscell fractions (nucleus + cell wall, chloroplasts, mitochon-dria, cytosol + vacuole) obtained by differential centrifuga-tion was also determined in separate studies on theintracellular localisation of Pq. The results (Table 1) indi-cated that an hour after Pq treatment about 9% of the totalPq in the cell was localised in the chloroplasts in susceptibleplants. This ratio declined to around 7–8% by the end ofthe second hour and close to 6% by the end of the fourthhour. In the chloroplasts of resistant plants, on the otherhand, the maximum Pq content was somewhat lower, beingat about 5–6% after 1 h and dropping to 5% by the end ofthe fourth hour (Table 1). Organelles in the PqR biotypedid not contain Pq either 24 h or a month after the treat-ment. Although the intracellular Pq content remained high,

it was localised mostly in the cytosol + vacuole fraction. Asthe resistant plants were not destroyed by the treatment,but gradually recovered their physiological activity fromthe fourth hour onwards, the reduction in Pq content inthe chloroplast fractions appears to be the result of theactivation of a mechanism for moving Pq from the cyto-plasm into a metabolically inactive compartment, probablythe vacuoles, as suggested by the high Pq content of thecytosol + vacuole fraction. In the present work the cyto-plasm and the vacuoles were not separated into separatefractions due to the difficulty of isolating vacuoles fromweed plants, but the recovery of functional activity in resis-tant plants can only be explained by the transfer of Pq to ametabolically inactive compartment, since it is not decom-posed in resistant plants.

3.2. Effect of transporter inhibitors on the resistance of the S

and PqR biotypes of C. canadensis

The results suggest that the resistance of the PqR bio-type of horseweed in Hungary is based on the activation

Table 1Changes in the paraquat content of different cell fractions in susceptible and resistant biotypes following Pq treatment, based on the photometricdetermination of Pq

Time after Pqtreatment (h)

Cell wall + nucleus Pqcontent nM/g fresh weight

Chloroplast Pqcontent nM/g fresh weight

Mitochondrium Pqcontent nM/g fresh weight

Cytosol + vacuolum Pqcontent nM/g fresh weight

S biotype

1 0.105 ± 0.029 0.202 ± 0.032 0.107 ± 0.022 1.767 ± 0.0502 0.140 ± 0.024 0.176 ± 0.027 0.177 ± 0.024 1.85 ± 0.04174 0.125 ± 0.018 0.130 ± 0.034 0.133 ± 0.019 1.848 ± 0.037

PqR biotype

1 h 0.123 ± 0.029 0.161 ± 0.032 0.119 ± 0.021 1.870 ± 0.0632 h 0.130 ± 0.018 0.155 ± 0.014 0.130 ± 0.021 1.863 ± 0.0514 h 0.131 ± 0.018 0.130 ± 0.012 0.119 ± 0.013 1.905 ± 0.03824 h 0.105 ± 0.028 — — 2.147 ± 0.0431 month 0.066 ± 0.029 — — 2.190 ± 0.067

Fig. 2. Changes in functional activity in the PqR biotype of C. canadensis,as revealed by Fv/Fm measurements. Leaves were sprayed with KNO3

solution at a concentration of 10�4 mol L�1 every 60 min after Pqtreatment.

B. Jori et al. / Pesticide Biochemistry and Physiology 88 (2007) 57–65 61

of a mechanism whereby Pq is moved from its site of actionto a metabolically inactive cell compartment, the vacuole.In the course of transportation the active agent, either inits original form or as a conjugate, has to pass throughat least two membranes. To confirm this, studies weremade on agents inhibiting transport processes by effectivelyblocking the proton pump ATPases (which supply theenergy required for transportation) localised in theplasmalemma or the vacuolar membrane (nitrate, carbon-yl-cyanide-m-chlorophenylhydrazone (CCCP) and N4N1-dicyclohexylcarbodiimide (DCCD)), the ABC transporters(vanadate, verapamil) or special carrier molecules involvedin the transport of xenobiotics (tetraphenylphosphonium-chloride (TPP)). The effect of transporter inhibitors onPq resistance was investigated by monitoring variable fluo-rescence (Fv/Fm) values to detect changes in functionalactivity. To determine whether the various inhibitors exert-ed their effect primarily on transporters involved in movingPq into inactive compartments or on those required for Pqto enter the cells, the plants were sprayed with inhibitors2 h before or after the Pq treatment.

In previous experiments investigating the effect of Pqtreatment on the recovery of functional activity, i.e. onthe transporters involved in sequestration, it was foundthat among the inhibitors added after Pq treatment, recov-ery was inhibited by DCCD, which blocks membrane-lo-calised (Fo) parts of ion channels and by TPP, whichspecifically inhibits the low-molecular-weight EmrE pro-tein responsible for Pq resistance in E. coli and homolo-gous to the Fo region of the transporters [20]. In thepresent experiment nitrate, which inhibits the vacuolar pro-ton pump ATPases, proved the most effective in blockingrecovery in resistant biotypes. If nitrate was added an hourafter Pq treatment the recovery process suffered approx.50% inhibition (data not shown). The recovery processwas completely obstructed by repeated spraying withnitrate (1, 2, 3 and 4 h after Pq treatment) (Fig. 2) or byfloating Pq-treated leaves in nitrate solution. As nitrateselectively blocks ATPases in the vacuoles, responsiblefor energy supplies to vacuolar membranes, the results

suggest that Pq sequestration uses energy from the protongradient.

Earlier data showed that in the susceptible biotype theapplication of DCCD or TPP 2 h after Pq treatment slightlyincreased the inhibition caused by Pq [20]. The presentexperiments indicated that nitrate also aggravated the effectof Pq in the susceptible biotype (data not shown). As norecovery can be detected in susceptible plants, it appears thatall the mechanisms influenced by the blockers participate fora limited period in the development of the general stressresponse of susceptible plants and in Pq transport.

Sodium orthovanadate, which inhibits the P-type plas-malemma proton pump ATPases and the high-molecular-weight, ‘‘full size’’ ABC transporters, but has no effect onthe vacuolar H+-ATPases in plants, had no substantialeffect on either recovery or the development of Pq inhibi-tion in the resistant biotype. Verapamil, which blocks theCa2+ channels and inhibits the ABC transporters, provedto be ineffective, as did the complex-forming EGTA (datanot shown). This again suggests that relatively small trans-porter proteins are involved in recovery, rather than ABCtransporters with a complex structure. The three inhibitors

62 B. Jori et al. / Pesticide Biochemistry and Physiology 88 (2007) 57–65

mentioned above did not have any significant effect on thedevelopment of the Pq effect in the susceptible biotype.

3.3. Sensitivity of the susceptible and Pq-resistant biotypes of

C. canadensis to agents generating reactive oxygen radicals

It was suggested that if resistance was caused by a mech-anism detoxifying oxygen radicals, the PqR biotype shouldbe resistant not only to paraquat, but also to other oxidativeagents. Tests were therefore conducted on the resistance ofthe S and PqR biotypes to bengal rose and menadione, whichalso generate toxic oxygen forms. The results indicated thatboth the PqR and S biotypes of C. canadensis were suscepti-ble to bengal rose and to menadione (Fig. 3). These resultsconfirm the conclusions drawn from enzyme activity studies,that resistance could not be attributed to the detoxificationof reactive oxygen radicals.

3.4. Changes in gene expression induced by paraquat and

menadione

To decide whether the up-regulation of the genes specif-ically responsible for resistance can be attributed to Pqitself or to the reactive oxygen species induced by the Pq

Fig. 3. Effect of bengal rose (A) and menadione (B) on the functionalactivity of susceptible and resistant biotypes of C. canadensis characterizedby Fv/Fm values. Leaves were spray-treated with 0.5 · 10�4 mol L�1

menadione and 1% (w/v) bengal rose.

effect, and to distinguish which genes are activated by Pqand which by superoxide or the general stress response,changes in gene expression were studied using theDDRT-PCR technique in susceptible and resistant plantstreated with Pq and menadione (Fig. 4). The expressionprofile reveals the appearance of a number of newexpressed sequence tags (EST) in both the susceptible andthe resistant biotype in response to both Pq and menadi-one. After purifying and sequencing, ESTs were comparedwith known genes in databases, leading to the detection offour identified partial sequences induced only by Pq. Twoof these exhibited increased expression in both the suscep-tible and the resistant biotype. One of them, a sequence of191 nucleotides in length, was identified as a fragmentexhibiting homology with an Myb transcription factor,while the other, a sequence containing 361 nucleotides,exhibited high similarity with the AtFerr2 gene. The com-plete sequence of the latter was determined by the authors

Fig. 4. Differential display patterns of susceptible and paraquat resistantbiotypes of C. canadensis on polyacrylamide sequencing gel. Isolatedbands are labelled 1, susceptible control; 2, susceptible treated with5 · 10�4 mol L�1 paraquat; 3, susceptible treated with 5 · 10�4 mol L�1

menadione; 4, resistant control; 5, resistant treated with 5 · 10�4 mol L�1

paraquat; 6, resistant treated with 5 · 10�4 mol L�1 menadione and 7,molecular weight marker.

Fig. 5. Alignement of fragments of PotE putrescine transport protein (gi:147330) from E. coli, CAT4 cationic amino acid transporter (gi: 30678908) fromA. thaliana and from putative C. canadensis CAT gene on nucleotide (A) and protein (B) level. Degree of conservation between the sequences is reflected bylevel of shading: black, 80–100%; grey, 60–80% and white, less than 60% conservation. Consensus residues are assigned based on the number ofoccurrences of the character in the column. A consensus line is built and displayed on either the upper or lower consensus line depending on occurrences. Ifthe number of occurrences of characters in a similarity group meets or exceeds the primary level, then the group number is built into the consensus line. Allother consensus line locations are cleared. Alignment was made with CLUSTALW default settings (34).

B. Jori et al. / Pesticide Biochemistry and Physiology 88 (2007) 57–65 63

(GeneBank Accession No. AJ786262) [35]. The Myb factormay be a transcription factor regulating the expression offurther Pq-induced genes, while ferritin2 may contributeto a reduction in the initial damaging effect of Pq by bind-ing free Fe reserves.

The other two genes identified, which exhibited a fargreater increase in expression in resistant than in suscepti-ble plants as the result of Pq treatment, are thought to bethe genes of transporters. One of them is homologous withan amino acid transporter (CAT4) capable of transportingcationic amino acids and polyamines [36] and with a PotEtransporter [37] responsible for Pq resistance. The CAT4transporter, which – judging by its sequence characteristics– is localised in the tonoplast and contains a domainresponsible for binding amino acids, is probably able totransport Pq due to the similar charge distribution in thepolyamine putrescine and in the Pq molecule. Identitiesbetween the CAT4 and the EST sequence are 88%,(Fig. 5A) and at the amino acid level 98% (Fig. 5B), conse-quently they seem to be homologues. No other gene exhib-ited such high degree of similarity with this EST. Fig. 5emphasizes the degree of conservation in sequences PotEfrom E. coli, CAT4 from A. thaliana, and CAT EST fromC. canadensis. The other EST, which exhibits far greaterexpression in the resistant biotype in response to Pq, is alsoa fragment of a low-molecular-weight putative transporter.This sequence seems to be homologous with the EmrEprotein responsible for Pq resistance in E. coli [25] andthe membrane localised subunit C of DET3 vacuolarH+-ATPase from A. thaliana [40].

4. Discussion

The results achieved so far in wide-ranging studies onthe mechanism of Pq resistance in C. canadensis plants inHungary indicate that Pq is able to enter the chloroplastsin the resistant biotype, causing transitional inhibition that

can be clearly detected on the basis of fluorescence quench-ing, followed within a few hours by the normalisation offunctional activity characterized by Fv/Fm. This recoveryhas proved to be a light-induced mechanism that couldbe inhibited by a eukaryotic protein synthesis inhibitor.As Pq is not metabolised in Pq-resistant higher plants itwas assumed that resistance could be attributed to theinduction of proteins that inactivate Pq and/or transportit to metabolically inactive compartments.

In the present work, investigations on Pq uptake and onits intracellular localisation directly proved that it was ableto enter the cells within a short time in both susceptible andresistant biotypes and could be detected chemically in cellorganelles. In both susceptible and resistant biotypes itsquantity in the chloroplasts, the site of action, approachedthe maximum within the first hour; this was less than 10% ofthe total Pq content. After this peak, the Pq content in thechloroplast (and organelle) fractions was observed todecline. Despite the fact that the total Pq content of the cellexhibited practically no change after the fourth hour, no Pqcould be detected in the organelles in surviving plants of theresistant biotype a day after Pq treatment (except in the cell-wall + nucleus fraction, which contained cell-wall debriscapable of passing through a Miracloth filter and also con-tained 2–3% Pq). In the susceptible biotype the intracellularPq content decreased by the fourth hour after treatment andplants died off some 6 h after treatment. In the survivingresistant biotype the intracellular quantity of Pq exhibitedpractically no decline even a month after treatment, thoughthe chloroplasts no longer contained any Pq. Measurementsindicated that over 97% of the Pq was located in the cyto-sol + vacuole fraction; in other words, as there was nodetectable physiological effect, it must be localised in thevacuoles, which are metabolically inactive.

In experiments using selective transporter inhibitors toclarify the uptake of Pq into the cell and intracellular trans-port processes, it was found that the recovery of functional

64 B. Jori et al. / Pesticide Biochemistry and Physiology 88 (2007) 57–65

activity after Pq treatment, i.e. the transport of the xenobi-otic to a metabolically inactive compartment, could beinhibited in resistant plants using the selective inhibitorsDCCD and TTP. DCCD inhibited the Fo membrane-inte-grated part of H+-ATPase, resulting in the inhibition of thetransmembrane proton gradient that provides energy fortransport. A number of data in the literature suggest thatthe uptake of Pq, aliphatic amines and amino acidsthrough the tonoplast is dependent on ATP but not onMgATP. This means that ATP does not provide the energyfor these processes directly. Neither vanadate, which inhib-its ABC transporters without influencing the functioning ofvacuolar H+-ATPase, nor verapamil, which also inhibitsABC transporters and blocks the Ca2+ channels, northe complex-forming EGTA had any effect on changesin the Pq-dependent functional activity in resistant plants.The ineffectiveness of vanadate confirms the suggestionthat resistance is due not to the complex ABC transporters,but to small membrane-integrated transporter moleculesthat exploit the electrochemical potential gradient pro-duced by proton pumps rather than obtaining energy forxenobiotic transport by cleaving ATP directly.

In addition to Pq, which exerts its primary effect by gen-erating toxic oxygen forms, tests were also made on resis-tance to menadione and bengal rose, agents which alsogenerate reactive oxygen radicals. Both the resistant andsusceptible biotypes of C. canadensis were found to be sus-ceptible to these compounds, suggesting that Pq itself is thecompound that induces resistance and confirming earlierresults demonstrating that the enzymes that detoxify reac-tive oxygen species were not responsible for resistance,since no increased activity could be detected for theseenzymes following Pq treatment. It is in any case difficultto conceive of such a high level of resistance as that record-ed for the PqR biotype of horseweed in Hungary basedpurely on the detoxification mechanism, since Pq has a cat-alytic effect in the chloroplasts, continuously generatingnew reactive oxygen species.

To obtain a closer knowledge of the proteins involved inthe Pq resistance mechanism, changes in gene expressionwere monitored in response to Pq in susceptible and resis-tant biotypes. To distinguish between genes induced by Pqalone from those induced by oxidative stress, examinationswere made on differences in gene expression after Pq andmenadione treatment. Based on sequence homology withknown genes, four EST fragments were identified that wereonly expressed in response to Pq, two of which exhibitedenhanced expression in both susceptible and resistant bio-types. Of these, one was homologous with an Myb factorand the other with the AtFerr2 gene. The products of thesegenes probably play a role in the general stress responseinduced by Pq, regulating the expression of other genes(Myb) or reducing the quantity of free intracellular iron(Ferr2) and thus making it less likely that hydroxyl radicalswill be formed.

The increase in expression in the other two ESTs inresponse to Pq was far greater in the resistant biotype than

in susceptible plants. These codes for proteins that arehomologous to transporters and could conceivably beinvolved in the intracellular transport of Pq. One of them,a protein homologous with EmrE, exhibits similarity to atransporter responsible for Pq resistance in E. coli [25]. Inaddition to Pq, this molecule is also active in the transportof many other xenobiotics. This fragment is also similar toDET3 vacuolar H+-ATPase subunit C of A. thaliana [40]and can possibly take part in the development of protongradient and provide energy for Pq transport. The otherprotein, CAT4, transports cationic amino acids and poly-amines, so it is presumably able to transport Pq, whichhas similar charge distribution within the molecule. Recentdata in the literature have proved that a high degree ofresistance may be caused by a single gene. The introductionof the pqrA gene from Ochrobactrum anthropi led to thedevelopment of tobacco with resistance similar to thatexhibited by C. canadensis to Pq, while the plants remainedsusceptible to other agents generating reactive oxygen spe-cies. This gene of a membrane localised transporter wasalone responsible for the high level of resistance in thetransgenic tobacco plants [38]. The analysis of sequenceidentity revealed that the protein coded by the pqrA genecontained a sequence homologous with the putrescine-binding region of the CAT4 transporter. This confirmedthe suggestion, indirectly proved by the present experi-ments, that the resistance of C. canadensis could beexplained by a similar mechanism, possibly involving achange in the structure or expression of a transporter.The CAT protein from horseweed, which exhibits greatsimilarity with the CAT4 amino acid transporter, couldbe a candidate for this role, based on the properties sofar detected. Work is currently underway in our laboratoryto determine the full sequence of the Pq-induced genes andto confirm their direct participation in the resistancemechanism.

Acknowledgment

Thanks are due to Barbara Harasztos for revising themanuscript linguistically.

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