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

PESTICIDE Biochemistry & Physiology

Pesticide Biochemistry and Physiology 88 (2007) 57–65 www.elsevier.com/locate/ypest

Role of transporters in paraquat resistance of horseweed Conyza canadensis (L.) Cronq. } a, Emil Pa´ldi b, Bala´zs Jo´ri a, Vilmos Soo´s a,b, Do´ra Szego a a,* Zolta´n Szigeti , Ilona Ra´cz , Demeter La´sztity a a

Department of Plant Physiology and Molecular Plant Biology, Eo¨tvo¨s Lora´nd University, 1117 Budapest, Pa´zma´ny Pe´ter se´ta´ny 1/C, Hungary b Agricultural Research Institute of the Hungarian Academy of Sciences, 2460 Martonva´sa´r, Hungary Received 12 April 2006; accepted 29 August 2006 Available 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 carrying paraquat to a metabolically inactive compartment. The uptake and intracellular localisation of paraquat, the effect of transporter inhibitors on resistance, and paraquat-induced gene expression were studied to obtain a better understanding of the mechanism of resistance. Investigations proved that paraquat entered the cells of both resistant and susceptible biotypes, approached the maximum within the first hour in chloroplasts, and then declined in all organelle fractions. In the resistant biotype paraquat was located in the vacuoles a day after treatment. Selective transporter inhibitors blocked the sequestration of paraquat, suggesting the participation of not directly energized transporters. Four EST fragments were identified that were expressed in response to paraquat. Two of them are thought to play a role in the general stress response (Ferr2, Myb). The others exhibit a similarity to transporters (EmrE, CAT) and could conceivably 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 haloid salts as the active agent in contact herbicides for total weed control in vineyards, orchards and gardens and for the defoliation and desiccation of field crops. Pq exerts its phytotoxic effect by diverting electrons from the photosynthetic electron transport chain on the reducing side of PSI at the FeSx level, while itself taking up an electron to form a cation radical. When this reverts to the Pq cation form by reacting with molecular oxygen, it generates a superoxide anion radical, which reacts with Fe3+ or H+ ions in the

*

Corresponding author. Fax: +36 13812164. E-mail address: [email protected] (I. Ra´cz).

0048-3575/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2006.08.013

Fenton reaction, while the paraquat cation radical generates hydroxyl radicals in the presence of hydrogen peroxide in the Winterbourn reaction. The reactive oxygen species formed during these reactions, particularly the superoxide and hydroxyl radicals, damage membranes through lipid peroxidation and the destruction of the double bonds in the side chains of fatty acids, thus leading to plant mortality [1–4]. The frequent, repeated use of paraquat has resulted in the development of resistant species, which at present number more than twenty, including both monocot and dicot weeds over a wide range of geographical locations. Since the appearance of resistant species, several hypotheses have been proposed to explain the mechanism of resistance in various species, but these have only been partially confirmed, if at all, experimentally. It became clear in the course of resistance studies that Pq does not decompose

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in the plants. Only in Rehmannia glutinosa, which exhibits natural resistance, could metabolisation (by converting Pq into a bound, inactive compound) be detected indirectly [5,6]. One of the most popular theories, for which evidence has been found in a number of paraquat-tolerant plants with lower resistance factors, explains resistance as the increased activity of antioxidant, protective enzyme systems [7]. The activity of these enzymes does indeed rise in certain species as the result of Pq or other oxidative stress, but since the Pq cation radical functions catalytically, it is clear that the plant will be unable to cope in the long term with protective enzyme systems alone. The largest body of evidence has been found to support theories suggesting that resistance is caused by the fact that only a limited amount of Pq reaches its site of action in the chloroplasts (limited penetration has been detected in Hordeum glaucum and Conyza bonariensis) or, if it does penetrate the chloroplasts, it is rapidly transferred to a metabolically inactive compartment by means of a sequestration mechanism [8–13]. Paraquat-resistant weeds were first observed in Hungary in the 1980s in an atrazine-resistant population (PqAr) of horseweed, Conyza canadensis [14], while later biotypes only resistant to paraquat (PqR) were also found [15]. The activity of enzymes detoxifying reactive oxygen species was not higher in the resistant plants [16,17]. There was a reduction in the functional activity characterised by Fv/Fm in these biotypes, demonstrating that Pq was able to penetrate the chloroplasts, where it caused a transitory inhibition, but within a few hours the functional activity started to return to normal. The recovery process, which is a light-dependent, induced mechanism, could be inhibited by cycloheximide, an inhibitor of eukaryotic protein synthesis [18,19]. It thus appeared probable that resistance involved a protein, or proteins, induced by paraquat and responsible for transporting Pq to a metabolically inactive compartment [20]. In both prokaryotes and eukaryotes special transporters are involved in the removal of xenobiotics, including paraquat, from cells or in their transfer to metabolically inactive compartments. The most widespread of these are various families of multidrug transporters, many members of which have been shown to be capable of transporting paraquat in organisms ranging from Escherichia coli through yeasts to human beings, and which result in paraquat resistance when overexpressed [21–23]. Among the multidrug transporters the ABC transporters are the best known. These are large membrane proteins with a complex structure, which use ATP directly for the active transport of various compounds. A further group consists of smaller antiporters, which exchange protons for some other molecule, thus exploiting the proton electrochemical potential gradient for transport. Bacteria contain antiporters responsible for resistance to antibiotics, while in humans neurotransmitters, including some capable of Pq transport, belong in this group. Even very small membrane proteins

may be capable of xenobiotic–proton/ion antiport, such as the EmrE protein in E. coli, responsible for resistance to paraquat and other toxins (e.g. ethidium). The structure of this protein is similar to that of the Fo subunit localised in the membrane of H+-ATPases [24,25]. Transporters responsible for the transport of molecules with a chemical structure or charge distribution similar to that of paraquat (e.g. polyamines, amino acids) may also play a role in resistance, as data in the literature confirm that they are capable of transporting paraquat [26–28]. In yeast, for example, a positive correlation was found between the expression of polyamine transporters and paraquat tolerance [29], in maize putrescine was observed to inhibit Pq uptake [30] while in Arctotheca calendula spermidine and cadaverine were effective in reducing paraquat translocation in susceptible biotype [31]. In our earlier studies putrescine was also found to provide protection against the inhibition of variable fluorescence by Pq in susceptible C. canadensis plants [27]. In the course of the present work studies were performed to elucidate the mechanism of Pq resistance. Susceptible and resistant biotypes of C. canadensis were used to investigate the uptake of Pq into the cells and its intracellular localisation, the effect on Pq resistance of inhibitors that selectively inhibit transporters enabling Pq to enter cells or various cell organelles, and genes expressed as the result of Pq treatment. The aim was to clarify the possible role in Pq resistance of transporters that transport or sequester Pq and to obtain a better understanding of the mechanism 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 Hoagland solution under laboratory conditions (illumination 130 lEm2 s1, 16 h light period, 22–25 C). Plants at the rosette stage were used in the experiments. For kinetic experiments plants were sprayed with 1 kg active ingredients/hectare (a.i./ha) paraquat as a 1% (v/v) solution (5 · 104 mol L1) of Gramoxone. Transporter inhibitors, Na-orthovanadate as a 104 mol L1, verapamil as a 5 · 105 mol L1, ethyleneglycol-bis-N,N1-tetraacetic acid (EGTA) as a 5 · 104 and KNO3 as a 104 mol L1 concentration solution, were also sprayed on the leaves of plants alone or in combination with Pq. Plants were treated with inhibitors 2 h before or after the Pq treatment. In the case of KNO3 treatment, leaves were spray-treated with the KNO3 solution every 60 min. Solutions of 0.5 · 104 mol L1 menadione and 1% (w/v) bengal rose, the agents generating reactive oxygen species, were also sprayed on the leaves. Functional activity of leaves was characterized by variable fluorescence (Fv/Fm) and expressed as percentage of the initial control values. Fluorescence parameters were determined by PAM fluorometer

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(Walz, Effeltrich, Germany). In kinetic experiments 10 leaves of 8–10 individual plants were used as parallel samples at every measuring point during the time course of Fv/ Fm changes. The experiments were repeated independently 9–12 times. 2.2. Cell fractionation and photometric determination of Pq content Cell fractionation of leaves of the S and the PqR biotypes of horseweed was carried out by differential centrifugation as described earlier [28] using 10 g fresh weight of leaves for each sample. Since the plants were spray treated with Pq, the intact leaves were washed with water three times for 1 minute before cell disruption and fractionation to remove residual extracellular Pq from the water free spaces (WFS) of the cell walls. The Pq content of the individual fractions (cell wall + nuclei, chloroplasts, mitochondria, cytosol + vacuole) was determined using 3–5 parallel samples. The cell fractionation procedure and the determination of the Pq contents of the fractions were carried out in three independent experiments. The total intracellular Pq content was determined using 5 · 0.1 g fresh weight of leaves. The experiments were repeated five times. The Pq content was determined using a modified version of the method of Kesary et al. [32]. After homogenising 0.1 g fresh weight of plant sample in 1 ml of 0.2% Na2EDTA solution, the samples were heat-treated for 3 min at 100 C, followed by centrifugation at 10,000g for 7 min. After adding 200 ll of 2 mol L1 NaOH and 200 ll 0.5% glucose to 600 ll supernatant, the samples were kept at 100 C for 1 min. The precipitant was removed by centrifugation at 10,000g for 3 min, after which the absorbance was recorded at 603 nm. The Pq content was determined using a calibration curve prepared from 1,1 0 -dimethyl-4,4 0 -bipyridinium dichloride. For comparison, a calibration curve was also prepared using Gramoxone (calculating the paraquat content on the basis of the active ingredient given by the manufacturer). The calibration curves were comparable, proving that the additional components of the herbicide Gramoxone did not interfere with the determination of Pq content. 2.3. DDRT-PCR and DNA sequencing The mRNA content of three-week-old plants was isolated with a GenoPrep DirectmRNA Kit according to the instructions of the manufacturer (GenoVision). cDNA synthesis was performed using a RevertAid First Strand PCR Synthesis Kit (MBI Fermentas) and H-Arbitrary universal primers (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 CTT TGG 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 -AAG CTT CGA CCA T-3 0 , HAP 7: 5 0 -AAG CTT AAC GAG G-3 0 , HAP 8: 5 0 -AAG CTT TTA CCG C-3 0 ). Following PCR amplification 1 ll of formamide loading dye was

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mixed with 7 ll of each sample. These were loaded onto a 6% urea, TBE, polyacrylamide sequencing gel (acrylamide–bis-acrylamide ratio 24:1). Electrophoresis was carried out at 55 W constant power for 2 h. The gel was then recovered and stained with silver nitrate according to the instructions of the manufacturer (Promega SILVER SEQUENCE DNA Sequencing System). The fragments of interest were excised with a scalpel and eluted by rinsing (1 h) and boiling (20 min) in 100 ll Milli-Q water. After precipitating and washing with ethanol the eluted DNA (4 ll) was re-amplified directly under conditions identical to those used for the initial PCR, with the corresponding H-T11-M (5 0 HTT-TTT TTT TTT M-3 0 ) and H-Arbitrary universal primers. The amplified PCR products were then sequenced using an ABI BigDye Terminator Cycle Sequencing v3.1 kit (Applied Biosystems). Sequences which recurred at least four times were considered for further analysis. The EST sequences were compared with sequences deposited in the databases (GenBank, EMBL, PDB), and translated nucleotid sequences were compared with protein sequences in databases (GenBank CDS Translations, PDB, and SwissProt) using the BLAST 2.2.8. algorithm [33]. Similar sequences were aligned and the sequence data were analysed using the ClustalW built-in BioEdit (version 5.0.9) package [34]. Graphical display was made with GeneDoc 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 by Fv/Fm in our earlier studies suggested that paraquat was able 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 chemical determination of both the changes in total intracellular Pq content following herbicide treatment, and the changes in its distribution among the cell compartments obtained by cell fractionation. In the leaves of spray-treated plants, from which the residual Pq in the cell walls was removed by washing prior to Pq determination, Pq appeared in the cells within 10 min of treatment and an hour after uptake near-maximum values were recorded in both susceptible and resistant plants (Fig. 1A and B). In PqR plants the intracellular Pq values were still close to maximum four hours after spraying. Even 24 h after spraying, when the PqR plants had recovered their functional activity, they still contained a considerable amount of intracellular Pq. A month after treatment with herbicide, when the plants were still in the rosette stage of development, the intracellular Pq concentration remained close to this level (Fig. 1C). In S plants, where the Fv/Fm values indicated a considerable reduction in physiological activity within 4 h and morphology also

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Fig. 1. Changes in intracellular paraquat content in susceptible and resistant biotypes of C. canadensis after Pq treatment. Leaves were spray-treated with 5 · 104 mol L1 paraquat. (A) Susceptible biotype (S); (B) resistant biotype (PqR); (C) changes in Pq content in S and PqR biotypes.

showed the plants to be wilting, there was a reduction in the Pq content (Fig. 1C). The decline in the Pq content measured in susceptible plants 4 h after treatment can be attributed to the fact that washing for 1 min removed Pq not only from the cell walls but also from cells with a damaged plasmalemma. Susceptible plants wilted within 24 h. The time course of changes in the Pq content of various cell fractions (nucleus + cell wall, chloroplasts, mitochondria, cytosol + vacuole) obtained by differential centrifugation was also determined in separate studies on the intracellular localisation of Pq. The results (Table 1) indicated that an hour after Pq treatment about 9% of the total Pq in the cell was localised in the chloroplasts in susceptible plants. This ratio declined to around 7–8% by the end of the second hour and close to 6% by the end of the fourth hour. In the chloroplasts of resistant plants, on the other hand, the maximum Pq content was somewhat lower, being at about 5–6% after 1 h and dropping to 5% by the end of the fourth hour (Table 1). Organelles in the PqR biotype did not contain Pq either 24 h or a month after the treatment. Although the intracellular Pq content remained high,

it was localised mostly in the cytosol + vacuole fraction. As the resistant plants were not destroyed by the treatment, but gradually recovered their physiological activity from the fourth hour onwards, the reduction in Pq content in the chloroplast fractions appears to be the result of the activation of a mechanism for moving Pq from the cytoplasm into a metabolically inactive compartment, probably the vacuoles, as suggested by the high Pq content of the cytosol + vacuole fraction. In the present work the cytoplasm and the vacuoles were not separated into separate fractions due to the difficulty of isolating vacuoles from weed plants, but the recovery of functional activity in resistant plants can only be explained by the transfer of Pq to a metabolically inactive compartment, since it is not decomposed 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 biotype of horseweed in Hungary is based on the activation

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Table 1 Changes in the paraquat content of different cell fractions in susceptible and resistant biotypes following Pq treatment, based on the photometric determination of Pq Time after Pq treatment (h)

Cell wall + nucleus Pq content nM/g fresh weight

Chloroplast Pq content nM/g fresh weight

Mitochondrium Pq content nM/g fresh weight

Cytosol + vacuolum Pq content nM/g fresh weight

S biotype 1 2 4

0.105 ± 0.029 0.140 ± 0.024 0.125 ± 0.018

0.202 ± 0.032 0.176 ± 0.027 0.130 ± 0.034

0.107 ± 0.022 0.177 ± 0.024 0.133 ± 0.019

1.767 ± 0.050 1.85 ± 0.0417 1.848 ± 0.037

PqR biotype 1h 2h 4h 24 h 1 month

0.123 ± 0.029 0.130 ± 0.018 0.131 ± 0.018 0.105 ± 0.028 0.066 ± 0.029

0.161 ± 0.032 0.155 ± 0.014 0.130 ± 0.012 — —

0.119 ± 0.021 0.130 ± 0.021 0.119 ± 0.013 — —

1.870 ± 0.063 1.863 ± 0.051 1.905 ± 0.038 2.147 ± 0.043 2.190 ± 0.067

of a mechanism whereby Pq is moved from its site of action to a metabolically inactive cell compartment, the vacuole. In the course of transportation the active agent, either in its original form or as a conjugate, has to pass through at least two membranes. To confirm this, studies were made on agents inhibiting transport processes by effectively blocking the proton pump ATPases (which supply the energy required for transportation) localised in the plasmalemma or the vacuolar membrane (nitrate, carbonyl-cyanide-m-chlorophenylhydrazone (CCCP) and N4N1dicyclohexylcarbodiimide (DCCD)), the ABC transporters (vanadate, verapamil) or special carrier molecules involved in the transport of xenobiotics (tetraphenylphosphoniumchloride (TPP)). The effect of transporter inhibitors on Pq resistance was investigated by monitoring variable fluorescence (Fv/Fm) values to detect changes in functional activity. To determine whether the various inhibitors exerted their effect primarily on transporters involved in moving Pq into inactive compartments or on those required for Pq to enter the cells, the plants were sprayed with inhibitors 2 h before or after the Pq treatment. In previous experiments investigating the effect of Pq treatment on the recovery of functional activity, i.e. on the transporters involved in sequestration, it was found that among the inhibitors added after Pq treatment, recovery was inhibited by DCCD, which blocks membrane-localised (Fo) parts of ion channels and by TPP, which specifically inhibits the low-molecular-weight EmrE protein responsible for Pq resistance in E. coli and homologous to the Fo region of the transporters [20]. In the present experiment nitrate, which inhibits the vacuolar proton pump ATPases, proved the most effective in blocking recovery in resistant biotypes. If nitrate was added an hour after Pq treatment the recovery process suffered approx. 50% inhibition (data not shown). The recovery process was completely obstructed by repeated spraying with nitrate (1, 2, 3 and 4 h after Pq treatment) (Fig. 2) or by floating Pq-treated leaves in nitrate solution. As nitrate selectively blocks ATPases in the vacuoles, responsible for energy supplies to vacuolar membranes, the results

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 104 mol L1 every 60 min after Pq treatment.

suggest that Pq sequestration uses energy from the proton gradient. Earlier data showed that in the susceptible biotype the application of DCCD or TPP 2 h after Pq treatment slightly increased the inhibition caused by Pq [20]. The present experiments indicated that nitrate also aggravated the effect of Pq in the susceptible biotype (data not shown). As no recovery can be detected in susceptible plants, it appears that all the mechanisms influenced by the blockers participate for a limited period in the development of the general stress response of susceptible plants and in Pq transport. Sodium orthovanadate, which inhibits the P-type plasmalemma proton pump ATPases and the high-molecularweight, ‘‘full size’’ ABC transporters, but has no effect on the vacuolar H+-ATPases in plants, had no substantial effect on either recovery or the development of Pq inhibition in the resistant biotype. Verapamil, which blocks the Ca2+ channels and inhibits the ABC transporters, proved to be ineffective, as did the complex-forming EGTA (data not shown). This again suggests that relatively small transporter proteins are involved in recovery, rather than ABC transporters with a complex structure. The three inhibitors

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mentioned above did not have any significant effect on the development 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 mechanism detoxifying oxygen radicals, the PqR biotype should be resistant not only to paraquat, but also to other oxidative agents. Tests were therefore conducted on the resistance of the S and PqR biotypes to bengal rose and menadione, which also generate toxic oxygen forms. The results indicated that both the PqR and S biotypes of C. canadensis were susceptible to bengal rose and to menadione (Fig. 3). These results confirm the conclusions drawn from enzyme activity studies, that resistance could not be attributed to the detoxification of reactive oxygen radicals.

effect, and to distinguish which genes are activated by Pq and which by superoxide or the general stress response, changes in gene expression were studied using the DDRT-PCR technique in susceptible and resistant plants treated with Pq and menadione (Fig. 4). The expression profile reveals the appearance of a number of new expressed sequence tags (EST) in both the susceptible and the resistant biotype in response to both Pq and menadione. After purifying and sequencing, ESTs were compared with known genes in databases, leading to the detection of four identified partial sequences induced only by Pq. Two of these exhibited increased expression in both the susceptible and the resistant biotype. One of them, a sequence of 191 nucleotides in length, was identified as a fragment exhibiting homology with an Myb transcription factor, while the other, a sequence containing 361 nucleotides, exhibited high similarity with the AtFerr2 gene. The complete sequence of the latter was determined by the authors

3.4. Changes in gene expression induced by paraquat and menadione To decide whether the up-regulation of the genes specifically responsible for resistance can be attributed to Pq itself or to the reactive oxygen species induced by the Pq

Fig. 3. Effect of bengal rose (A) and menadione (B) on the functional activity of susceptible and resistant biotypes of C. canadensis characterized by Fv/Fm values. Leaves were spray-treated with 0.5 · 104 mol L1 menadione and 1% (w/v) bengal rose.

Fig. 4. Differential display patterns of susceptible and paraquat resistant biotypes of C. canadensis on polyacrylamide sequencing gel. Isolated bands are labelled 1, susceptible control; 2, susceptible treated with 5 · 104 mol L1 paraquat; 3, susceptible treated with 5 · 104 mol L1 menadione; 4, resistant control; 5, resistant treated with 5 · 104 mol L1 paraquat; 6, resistant treated with 5 · 104 mol L1 menadione and 7, molecular weight marker.

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

(GeneBank Accession No. AJ786262) [35]. The Myb factor may be a transcription factor regulating the expression of further Pq-induced genes, while ferritin2 may contribute to a reduction in the initial damaging effect of Pq by binding free Fe reserves. The other two genes identified, which exhibited a far greater increase in expression in resistant than in susceptible plants as the result of Pq treatment, are thought to be the genes of transporters. One of them is homologous with an amino acid transporter (CAT4) capable of transporting cationic amino acids and polyamines [36] and with a PotE transporter [37] responsible for Pq resistance. The CAT4 transporter, which – judging by its sequence characteristics – is localised in the tonoplast and contains a domain responsible for binding amino acids, is probably able to transport Pq due to the similar charge distribution in the polyamine putrescine and in the Pq molecule. Identities between the CAT4 and the EST sequence are 88%, (Fig. 5A) and at the amino acid level 98% (Fig. 5B), consequently they seem to be homologues. No other gene exhibited such high degree of similarity with this EST. Fig. 5 emphasizes the degree of conservation in sequences PotE from E. coli, CAT4 from A. thaliana, and CAT EST from C. canadensis. The other EST, which exhibits far greater expression in the resistant biotype in response to Pq, is also a fragment of a low-molecular-weight putative transporter. This sequence seems to be homologous with the EmrE protein responsible for Pq resistance in E. coli [25] and the membrane localised subunit C of DET3 vacuolar H+-ATPase from A. thaliana [40]. 4. Discussion The results achieved so far in wide-ranging studies on the mechanism of Pq resistance in C. canadensis plants in Hungary indicate that Pq is able to enter the chloroplasts in the resistant biotype, causing transitional inhibition that

can be clearly detected on the basis of fluorescence quenching, followed within a few hours by the normalisation of functional activity characterized by Fv/Fm. This recovery has proved to be a light-induced mechanism that could be inhibited by a eukaryotic protein synthesis inhibitor. As Pq is not metabolised in Pq-resistant higher plants it was assumed that resistance could be attributed to the induction of proteins that inactivate Pq and/or transport it to metabolically inactive compartments. In the present work, investigations on Pq uptake and on its intracellular localisation directly proved that it was able to enter the cells within a short time in both susceptible and resistant biotypes and could be detected chemically in cell organelles. In both susceptible and resistant biotypes its quantity in the chloroplasts, the site of action, approached the maximum within the first hour; this was less than 10% of the total Pq content. After this peak, the Pq content in the chloroplast (and organelle) fractions was observed to decline. Despite the fact that the total Pq content of the cell exhibited practically no change after the fourth hour, no Pq could be detected in the organelles in surviving plants of the resistant biotype a day after Pq treatment (except in the cellwall + nucleus fraction, which contained cell-wall debris capable of passing through a Miracloth filter and also contained 2–3% Pq). In the susceptible biotype the intracellular Pq content decreased by the fourth hour after treatment and plants died off some 6 h after treatment. In the surviving resistant biotype the intracellular quantity of Pq exhibited practically no decline even a month after treatment, though the chloroplasts no longer contained any Pq. Measurements indicated that over 97% of the Pq was located in the cytosol + vacuole fraction; in other words, as there was no detectable physiological effect, it must be localised in the vacuoles, which are metabolically inactive. In experiments using selective transporter inhibitors to clarify the uptake of Pq into the cell and intracellular transport processes, it was found that the recovery of functional

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activity after Pq treatment, i.e. the transport of the xenobiotic to a metabolically inactive compartment, could be inhibited in resistant plants using the selective inhibitors DCCD and TTP. DCCD inhibited the Fo membrane-integrated part of H+-ATPase, resulting in the inhibition of the transmembrane proton gradient that provides energy for transport. A number of data in the literature suggest that the uptake of Pq, aliphatic amines and amino acids through the tonoplast is dependent on ATP but not on MgATP. This means that ATP does not provide the energy for these processes directly. Neither vanadate, which inhibits ABC transporters without influencing the functioning of vacuolar H+-ATPase, nor verapamil, which also inhibits ABC transporters and blocks the Ca2+ channels, nor the complex-forming EGTA had any effect on changes in the Pq-dependent functional activity in resistant plants. The ineffectiveness of vanadate confirms the suggestion that resistance is due not to the complex ABC transporters, but to small membrane-integrated transporter molecules that exploit the electrochemical potential gradient produced by proton pumps rather than obtaining energy for xenobiotic transport by cleaving ATP directly. In addition to Pq, which exerts its primary effect by generating toxic oxygen forms, tests were also made on resistance to menadione and bengal rose, agents which also generate reactive oxygen radicals. Both the resistant and susceptible biotypes of C. canadensis were found to be susceptible to these compounds, suggesting that Pq itself is the compound that induces resistance and confirming earlier results demonstrating that the enzymes that detoxify reactive oxygen species were not responsible for resistance, since no increased activity could be detected for these enzymes following Pq treatment. It is in any case difficult to conceive of such a high level of resistance as that recorded for the PqR biotype of horseweed in Hungary based purely on the detoxification mechanism, since Pq has a catalytic effect in the chloroplasts, continuously generating new reactive oxygen species. To obtain a closer knowledge of the proteins involved in the Pq resistance mechanism, changes in gene expression were monitored in response to Pq in susceptible and resistant biotypes. To distinguish between genes induced by Pq alone from those induced by oxidative stress, examinations were made on differences in gene expression after Pq and menadione treatment. Based on sequence homology with known genes, four EST fragments were identified that were only expressed in response to Pq, two of which exhibited enhanced expression in both susceptible and resistant biotypes. Of these, one was homologous with an Myb factor and the other with the AtFerr2 gene. The products of these genes probably play a role in the general stress response induced 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 radicals will be formed. The increase in expression in the other two ESTs in response to Pq was far greater in the resistant biotype than

in susceptible plants. These codes for proteins that are homologous to transporters and could conceivably be involved in the intracellular transport of Pq. One of them, a protein homologous with EmrE, exhibits similarity to a transporter responsible for Pq resistance in E. coli [25]. In addition to Pq, this molecule is also active in the transport of many other xenobiotics. This fragment is also similar to DET3 vacuolar H+-ATPase subunit C of A. thaliana [40] and can possibly take part in the development of proton gradient and provide energy for Pq transport. The other protein, CAT4, transports cationic amino acids and polyamines, so it is presumably able to transport Pq, which has similar charge distribution within the molecule. Recent data in the literature have proved that a high degree of resistance may be caused by a single gene. The introduction of the pqrA gene from Ochrobactrum anthropi led to the development of tobacco with resistance similar to that exhibited by C. canadensis to Pq, while the plants remained susceptible to other agents generating reactive oxygen species. This gene of a membrane localised transporter was alone responsible for the high level of resistance in the transgenic tobacco plants [38]. The analysis of sequence identity revealed that the protein coded by the pqrA gene contained a sequence homologous with the putrescinebinding region of the CAT4 transporter. This confirmed the suggestion, indirectly proved by the present experiments, that the resistance of C. canadensis could be explained by a similar mechanism, possibly involving a change in the structure or expression of a transporter. The CAT protein from horseweed, which exhibits great similarity with the CAT4 amino acid transporter, could be a candidate for this role, based on the properties so far detected. Work is currently underway in our laboratory to determine the full sequence of the Pq-induced genes and to confirm their direct participation in the resistance mechanism. Acknowledgment Thanks are due to Barbara Harasztos for revising the manuscript linguistically. References [1] K. Asada, Ascorbate-dehydrogenase a hydrogen-peroxide scavenging enzyme in plants, Physiol. Plantarum 85 (1982) 235–241. [2] C.H. Foyer, B. Halliwell, The presence of glutathione and glutathione-reductase in chloroplasts: a proposed role in ascorbic acid metabolism, Planta 133 (1976) 15–21. [3] Z. Szigeti, I. Ra´cz, D. La´sztity, Paraquat resistance of weeds—the case of Conyza canadensis (L.) Cronq., Z. Naturforsch. 65c (2001) 319–328. [4] E. Lehoczki, G. Laskay, I. Gaa´l, Z. Szigeti, Mode of action of paraquat in leaves of paraquat-resistant Conyza canadensis (L.) Cronq. Plant Cell Environ. 15 (1992) 531–539. [5] S.S. Kim, Y.O. Son, J.C. Chun, S.E. Kim, G.H. Chung, K.J. Hwang, J.C. Lee, Antioxidant property of an active component purified from the leaves of paraquat-tolerant Rehmannia glutinosa, Redox Rep. 10 (2005) 311–318.

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