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Development of transgenic tobacco plants overexpressing maize glutathione S-transferase I for chloroacetanilide herbicides phytoremediation
Biomolecular Engineering 22 (2005) 121–128 www.elsevier.com/locate/geneanabioeng
Development of transgenic tobacco plants overexpressing maize glutathione S-transferase I for chloroacetanilide herbicides phytoremediation Margarita Karavangeli b, Nikolaos E. Labrou a,*, Yannis D. Clonis a, Athanasios Tsaftaris b,c a
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, Greece b Laboratory of Genetics and Plant Breeding, Aristotle University of Thessaloniki, Thessaloniki GR-54124, Greece c Institute of Agrobiotechnology, CERTH, 6th km Charilaou-Thermis Road, P.O. Box 361, Thermi GR-57001, Greece Received 14 February 2005; received in revised form 25 February 2005; accepted 1 March 2005
Abstract Glutathione S-transferases (GSTs, EC 2.5.1.18) are a multigene family of detoxification enzymes that biotransform a wide variety of endogenous and exogenous electrophilic substrates, including herbicides. The isozyme GST I from maize exhibits significant catalytic activity for the chloroacetanilide herbicide alachlor and appears to be involved in its detoxifying process. To establish the in planta ability of GST I to detoxify from alachlor, transgenesis studies were carried out. The gene gstI–6His, which encodes for 6His-tagged GST I, was used for the construction of a binary vector suitable for genetic engineering of tobacco plants (Nicotiana tabacum). Through biolistic method transgenic tobacco plants were obtained. Integration of gstI–6His gene in transgenic tobacco plants genome was confirmed by polymerase chain reaction and Southern blot hybridization. The expression of active GST I was established by Western blot analysis, using anti-6His antibody, and by direct purification of 6-His tagged GST I on Ni–NTA agarose. Primary transformed plants harboring the gstI–6His gene were transferred to MS medium supplemented with alachlor and their phenotype was evaluated. The transgenic plants showed substantially higher tolerance to alachlor compared to non-transgenic plants in terms of root, leaves and vigorous development. These transgenic plants are potentially useful biotechnological tools for the development of phytoremediation system for the degradation of herbicide pollutants in agricultural fields. # 2005 Elsevier B.V. All rights reserved. Keywords: Alachlor; Herbicide detoxification; Phytoremediation; Transgenic tobacco; Xenobiotics
1. Introduction Plants contain a complex array of enzymes which are able to detoxify xenobiotics including esterases and cytochrome P450-dependent oxygenases (phase 1 metabolising enzymes), glutathione S-transferases (GSTs), glucosyl transferases (GTs) and malonyl transferases (phase 2 metabolising enzymes) and ATP-driven vacuolar transporters (phase 3 enzymes). GSTs catalyse the Abbreviations: Alachlor, 2-Cl-N-[2,6-diethylphenyl]-N-[methoxymethyl]acetamide; GSH, glutathione; GST, glutathione S-transferase; gstI–6His: 6His-tagged glutathione S-transferase I gene; nptII, neomycin phosphotransferase II gene; Ni–NTA, Ni2+–nitrilotriacetic acid * Corresponding author. Tel.: +30 210 5294308; fax: +30 210 5294308. E-mail address: [email protected] (N.E. Labrou). 1389-0344/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bioeng.2005.03.001
nucleophilic attack of the sulphur atom of glutathione (gGlu–Cys–Gly, GSH) on electrophilic groups of a variety of hydrophobic substrates, including herbicides, insecticides and carcinogens [1–4]. Plant GSTs gained particular attention with respect to the detoxification from herbicides that belong mainly to two classes, chloroacetanilide and triazines [5–8]. Chloroacetanilide and triazine herbicides are widely used for the control of annual grasses and broad-leaf weeds in a variety of major crops such as maize and soybeans [9]. Alachlor is a preemergence herbicide and is applied to young plants. It is absorbed through the roots and transferred to the upper parts of the plant through the apoplast. It represses the elongation of the root system and the development of the shoots of young plants [9].
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Cultivation systems of a variety of crops have tended to rely increasingly on the use of a number of agrochemicals for the maintenance of regular agricultural production. However, their residues sometimes affect the ecosystems and resulted in the pollution of crops. Therefore it is important to develop a system of rapid degradation of chemicals in the agriculture environment after use. Phytoremediation is an emerging new technology that uses plants to remove or degrade various pollutants from soils [10]. Although a number of plant species are able to accumulate high amounts of heavy metals or to degrade various organic soil pollutants, their remedial capacity can be significantly increased by genetic manipulation [11]. For example, transgenic yellow poplar plants overexpressing the bacterial gene encoding mercuric reductase were explored for the phytoremediation of mercury pollution [12]. Recently, poplar and tobacco transgenic plants overexpressing g-glutamylcysteine synthase were developed for remediation of cadmium and alachlor [13]. GSTs are considered as candidates for the development of such transgenic systems for the rapid degradation of herbicide pollutants in the agricultural fields. So far, four approaches have been reported for the generation of transgenic plants overexpressing GST isoenzymes. In all cases bifunctional isoenzymes were employed possessing both glutathione S-transferase and peroxidase activities (e.g. ZmGST27, an engineered form of ZmGSTU1, and NtGST107) [14–16]. The derived transgenic plants showed stress [16] and herbicide tolerance [14,15]. However, due to the bifunctional activities it was impossible to establish whether herbicide tolerance was exclusively due to the GSH-conjugation activity. Inspite of this uncertainty, GSTs may prove to be useful tools for phytoremediation. Before this to happen, questions need to be addressed with regards the ability of transgenic tobacco plants, expressing maize GST I, to tolerate alachlor. Therefore, an in planta characterisation needs to be established prior any further investigation or application of GST I.
were obtained from New England Biolabs, USA. Reduced GSH was obtained from Sigma, USA. Alachlor was obtained from Agan Chemical Manufactured (Israel) or by Rieden-deHaen (Germany). HYBOND-NX nylon membranes were purchased from Amersham-LifeScience, (UK). DNA purification kits were obtained from Qiagen (Germany). Pfu DNA polymerase and all other molecular biology reagents were obtained from Promega (U.K.).
3. Methods 3.1. Cloning, expression and purification of maize GST I Cloning of maize GST I into a pQE70 expression vector to yield the pQEGST expression plasmid was described by Labrou et al. [17]. Expression and purification of recombinant GST I were performed according to Labrou et al. [17]. 3.2. Assay of enzyme activity and protein Routine enzyme assays were performed by monitoring the formation of the conjugate of CDNB (1 mM) and GSH (2.5 mM) at 340 nm (e = 9.6 mM1 cm1) at 30 8C according to a published method [18]. Enzyme assays for the herbicides alachlor, metolachlor, atrazine, and chlorimuron ethyl were performed as described in [19,20]. Enzyme assays for cumene hydroperoxide were performed as described in [21]. Observed reaction velocities were corrected for spontaneous reaction rates when necessary. All enzyme assays velocities were determined in triplicate in buffers equilibrated at constant temperature. Protein concentration was determined by the method of Bradford [22] using bovine serum albumin (fraction V) as standard. 3.3. Construction of a transformation vector
2. Materials and methods 2.1. Materials Tobacco (Nicotiana tabacum) seeds from the cultivar Basmas Xanthi were obtained from Greek Institute of Tobacco. Seeds were surface sterilized in 2.5% NaOCl solution for 15 min, rinsed in sterile distilled water and finally placed for germination in sterile petri dishes containing MS basal medium supplemented with 30 g/l sucrose, 0.8% agar and adjusted to pH 5.8 before autoclaving. The plants were grown at 25 8C under a 16 h photoperiod using artificial light. Helios GeneGun was obtained from BioRad. DNTPs, PCR DIG Probe synthesis kit and Western blot kit were obtained from Roche, UK. Restriction enzymes
Standard recombinant methods were adopted for the construction of the plant transformation vector pART27– GSTI–6His that was used for tobacco transformations [23]. The maize gstI–6His gene was excised from the plasmid pGSTI [18], and subcloned in the EcoRI restriction site of the pART27 vector. This latter vector carries the CaMV 35S promoter and the 30 untranslated region of the octopine synthase (ocs) gene as a terminator. The entire CaMV35S–gstI-ocs cassette was digested by NotI restriction enzyme, and the resulted cassette was inserted into in the T-DNA region of the pART27 binary vector. The pART27 vector posses the nptII gene, which encodes for neomycin phopshotransferase II and confers resistance to the antibiotic kanamycin. The nptII gene is under the control of the nopaline synthase (nos) gene promoter.
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3.4. Transformation procedure Leaf discs from young tobacco plants were used as explants for transformation. Gold particles were coated with the transformation vector (plasmid DNA) and leaf discs were bombarded with those gold particles using a helium pressure-based apparatus (Helios1 Gene Gun, Bio-Rad). Bombarded leaf discs were maintained on MS medium with growth regulators (0.1 mg/l NAA, 1 mg/l BAP) for 2 days and then transferred to the same medium supplemented with 100 mg/l kanamycin for regeneration. Regenerated plantlets were transferred to MS medium lacking plant growth regulators and containing the same concentration of the antibiotic, for rooting. The explants were cultured at 25 8C with a 16 h photoperiod using artificial light. 3.5. DNA analysis Total genomic DNA was isolated from leaf tissue of primary transformants following an extraction procedure according to the instructions of the DNeasy Plant Mini Kit1 (Qiagen, Germany). PCR analysis was carried out with 100– 200 ng genomic DNA in a 12 ml total reaction volume containing 1 reaction buffer, 0.75 mM MgCl2, 0.2 mM dNTPs, 1 mM from each of the primers and 0.2–0.5 units of Taq polymerase. Different PCR reactions were carried out for the amplification of nptII, gstI genes and for a part of the gstI cassette. The primers that used for the amplification of the nptII gene were 50 -GAGGCTATTCGGCTATGACTG-30 and 50 -ATCGGGAGCGGCGATACCGTA-30 , for the gstI gene were 50 -CCACCGCCGAGCACAAGAGC-30 and 50 TAGGGCGTAGCGAACAGGCAGAGA-30 . For the amplification of the cassette the primers that were used was one of the promoter region CaMV-35S, 50 -AGGAGCATCGTGGAAAAAGAAGAC-30 , and the reverse primer was the same that used for the gstI gene. For Southern blot analysis, aliquots of 10 mg DNA were digested with EcoRI and fractionated by 0.8% agarose gel electrophoresis. DNA was then transferred to nylon membranes (HYBOND-NX1), and the subsequent hybridization reactions were carried out according to standard procedures [23]. The probe that was used for the hybridization was part of the nptII gene labeled with the digoxygenin molecule. The probe prepared using PCR technique, during which the DIG11-dUTP was incorporated into the amplification product, using the PCR DIG Probe Synthesis Kit (Roche, UK). The hybridization and detection procedures were conducted using the DIG DNA Labeling Kit (Roche, UK) according to the manufacturer’s instructions. Non-transgenic plants were used as negative controls. 3.6. Western blot analysis and purification of His6tagged GST I expressed in transgenic tobacco leaves Detection of 6His-tagged GST I expressed in transgenic tobacco was accomplished using an Anti-6His antibody
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(mouse monoclonal antibody) and as a secondary antibody anti-mouse-Ig-POD as follows: samples from transformed and untransformed tobacco leaves were homogenised with mortal and paste in Tris/HCl buffer (50 mM, pH 7.0) and subjected to SDS-PAGE on 12.5% gels. The protein was electrophoretically transferred to PVDF membranes. Nonspecific binding sites were blocked by incubating membranes for 1–2 h at room temperature in 1% (w/v) blocking solution in Tris-buffered saline (50 mM Tris–HCl, 150 mM NaCl, pH 7.5). The membrane was probed with Anti-6His antibody for 30–45 min at room temperature in Tris-buffered saline. Blots were washed four times with 100 ml of Tris-buffered saline containing 0.1% Tween-20, for 15–25 min each. Anti-6His antibody-reactive bands were detected using anti-mouse-IgPOD and the luminescence substrate luminol according to the manufacturer’s instructions. Purification of 6His-tagged GST I from transgenic tobacco leaves was achieved by metal chelate affinity chromatography on Ni–NTA column as follows: samples from transformed and untransformed tobacco leaves were homogenised with mortal and paste in potassium phosphate buffer (50 mM, pH 8.0) containing sodium chloride (0.3 M) and the resulted plant extract (1 ml, total GST activity 2.5 U/ ml, 0.85 mg total protein) was loaded to a column of Ni– NTA adsorbent (0.5 ml), which was previously equilibrated with potassium phosphate buffer (50 mM, pH 8.0) containing sodium chloride (0.3 M). Non-adsorbed protein was washed off with 10 ml equilibration buffer, followed by 20 ml of potassium phosphate buffer (50 mM, pH 6.2) containing sodium chloride (0.3 M) and glycerol (10%, v/v). Bound GST I was eluted with equilibration buffer containing 0.2 M imidazole (2 ml). 3.7. Herbicide tolerance studies Plants from seven different transgenic lines of transformed tobacco plants were transferred to MS media supplemented with two different concentrations of the herbicide alachlor (0.01 g/l and 0.015 g/l). Non-transgenic plants were used as negative controls (one non-transgenic plant per line). Transgenic and non-transgenic plants were followed by measuring their development at the end of 20 days exposure period. Each experiment was performed by two replicate plants per treatment. The significant differences between mean values were evaluated by Student’s t-test. Differences were considered to be significant at P = 0.05. The plants were cultured at 25 8C with a 16 h photoperiod under artificial light.
4. Results and discussion 4.1. Analysis of substrate specificity of maize GST I Metabolic detoxification is probably the most general major mechanism involved in plant tolerance to herbicides. In particular, the GSH/GST system appears to be widespread
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among plants and to confer protection against toxic chemicals by catalysing the conjugation of glutathione to an electrophile center of hydrophobic molecules by means of the –SH group [2–4]. Species tolerant or susceptible to a wide spectrum of herbicides (triazines, chloroacetanilides, thiocarbamates) are characterized by high and low levels of GST, respectively, and an increase in specific GST activity in response to some herbicide treatment has been reported in a few cases [7,8]. Thus the system appears to be involved in the determination of plant tolerance to these classes of molecules. Alachlor is one of the most widely used herbicide that is used for selective weed control in agricultural crops [9]. The biotransformation of alachlor in plants has not been fully characterised. However, recent studies suggest Phase 2-mediated biotransformation by GSTs. In maize (Zea mays L.), conjugation with GSH is a major route of metabolism of the chloroacetanilide herbicides [8,15,24]. The isoenzyme GST I from maize has been the subject of intense research because of its ability to detoxify a wide range of xenobiotics. It belongs to class phi and to type I of GSTs, according to Edwards et al. [4] and McGonigle et al. [20] classifications, respectively. GST I is a homodimer protein of 214 amino acids, and exhibits wide substrate specificity towards several classes to herbicides (Fig. 1). Two features have been reported, based on the crystal structure [25,26] and on extensive mutagenesis studies [17,18,27,28], to contribute to wide substrate specificity and high catalytic efficiency towards electrophile substrates: the size and shape of the H-site and its high flexibility. These features are determined, at atomic level, by the hydrophobic residues Trp12, Phe35, Ile118, and the flexibility in the upper part of the H-site is modulated by Gly123 and Gly124 [25–28]. Table 1 shows the specific activity of the enzyme exhibited for atrazine, alachlor, metolachlor, chlorimuron ethyl, and cumene hydroperoxide. The enzyme exhibits substantial catalytic activity towards alachlor, moderate activity towards atrazine and chlorimuron ethyl, but little activity toward metolachlor. In addition the enzyme was tested for its ability to act as glutathione peroxidase using the synthetic model substrate cumene hydroperoxide. GSTs have been shown to have GSH peroxidase activity and reduce organic hydroperoxides of fatty acids and nucleic acids to the corresponding monohydroxyalcohols [2,16]. As shown in Table 1, GST I does not exhibit any detectable glutathione peroxidase activity (Table 1). 4.2. Transformation and analysis of tobacco plants The plant expression vector containing the gstI–6His gene was constructed and introduced into leaf discs of
Fig. 1. Structures of selected herbicides that metabolized by the GSH/GST system. (A) Atrazine; (B) alachlor; (C) metolachlor, and (D) chlorimuron ethyl.
tobacco cv. Basmas Xanthi. DNA was delivered to leaf discs by particle bombardment using a hand gene gun device using helium gas for the delivery of the particles. Bombarded leaf discs were cultured in regeneration medium in the presence of the antibiotic kanamycin. Nineteen plantlets that rooted in the presence of antibiotic were selected. Genomic DNA from leaves of transformed tobacco plants was purified and analyzed by PCR. Reactions were carried out using two different pair of primers. The first pair was designed to amplify a 700 bp internal fragment of the nptII gene, whereas the second was used to amplify an internal fragment of gstI coding region, sized 430 bp.
Table 1 Activity of the GST I (nmol min1 mg1) with selected herbicides, assayed as described in the text Enzyme
Alachlor
Metolachlor
Atrazine
Chlorimuron ethyl
Cumene hydroperoxide
GSTI
80.5 4.6
ND
0.75 0.2
0.4 0.1
ND
ND: no significant activity detected.
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Fig. 3. Western blot hybridization. Western blot analysis of total protein fractions extracted from the leaf tissue of primary transformants. Proteins were resolved by SDS-PAGE and probed with anti-6His antibody. Lane 1: purified recombinant GSTI–6His (0.5 mg) expressed in E. coli [18]. Lane 2: total protein extract (50 mg) from transformed 20 plants; Lane 3: total protein extract from untransformed plants (50 mg). Molecular weight marker is shown by the arrow.
Fig. 2. DNA analysis of tobacco plants. (A) PCR analysis of genomic DNA from transformed with pART27–gstI–6His tobacco plants. Lanes A and B: molecular markers 100 bp and 1 Kb, respectively. Lanes 1–5: amplification of the nptII gene from transformed (lanes 1 and 2) and untransformed (line 3) tobacco plants; Lane 4: PCR analysis in the absence of genomic DNA (white control); Lane 5: amplification of the nptII gene from the plasmid pART27–gstI–6His (positive control). (B) PCR analysis of genomic DNA from transformed with pART27–gstI–6His tobacco plants for the amplification of gstI gene. Lanes A: molecular marker 100 bp. Lanes 1–8: amplification part of the gstI gene of transformed (lanes 1–6) and untransformed (Lane 7) tobacco plants; Lane 8: PCR analysis in the absence of genomic DNA (white control); Lane 9: amplification of the gstI gene from the plasmid pART27–gstI–6His (positive control). (C) PCR analysis of genomic DNA from transformed with pART27–gstI–6His tobacco plants for the amplification of CaMV35S–gstI-ocs 30 cassette. Lane A: molecular marker 100 bp; lanes 1–7: amplification of the cassette from transformed (lanes 1–3) and untransformed (lane 4) tobacco plants; Lane 5: PCR analysis in the absence of genomic DNA (white control); Lane 6: plasmid pART27–GSTI–6His (positive control).
Finally, a part of the cassette CaMV35S–gstI–6His-ocs 30 that consisted of the promoter and the coding region was amplified in order to check the integrity of the expression cassette. The same PCR reactions were also carried out using DNA
isolated from wild-type plants and plasmid pART27–gstI– 6His DNA as a negative and positive control, respectively. The results of PCR analysis are illustrated in Fig. 2. Southern blot analysis was performed on genomic DNA from leaves of putative transgenic plants and from untransformed control plants. Genomic DNA was digested with EcoRI and analyzed using as a probe part of the nptII gene labeled with digoxygenin. The results of the Southern blot confirmed the presence of the nptII gene only in the genomic DNA from leaves of putative transgenic plans (data not shown). 4.3. Expression of active 6His tagged GST I in transgenic tobacco plants Western blot analysis was also performed using total protein extract from leaves of transformed and untransformed plants, employing monoclonal antibody against the 6His tag (Fig. 3). The results provide direct evidence that transgenic tobacco plants express maize GST I. The extra six histidine residues tagged on the N-terminus of the enzyme, enable GST I to be rapidly purified from transgenic leaf extract by metal chelate affinity chromatography on Ni–NTA column. On the basis of at least 80% yield of the purification procedure, the level of GST I expression in transgenic tobacco plants may be estimated approximately as 0.03% of total soluble leaf proteins. Notably, affinity chromatography on Ni–NTA column failed to purify any GST activity from leaf extract of untransformed plants. Kinetic analysis of the purified enzyme gave kinetic constants (Km for GSH of 1.2 0.1 mM and Km for CDNB of 1.6 0.1 mM) which are indistinguishable from
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Fig. 4. The influence of herbicide alachlor in non-transformed tobacco plants after 20 days, at the apical meristem (A) and root system (B). On the left is tobacco plant in medium without herbicide, in the middle the concentration of the herbicide is 0.01 g/l and on the right is 0.015 g/l.
that exhibited by the recombinant E. coli expressed enzyme [18]. Taking into account that heterodimerization strongly influence the catalytic and kinetic properties of the enzyme [29] one can conclude that GST I is expressed as a fully active homodimer, presumably without subunit heterodimerization with endogenous tobacco’s GSTs subunits. 4.4. Alachlor tolerance studies The tobacco Basmas Xanthi cultivar that was used in the transformation experiments is sensitive to alachlor. Primary transformed plants from seven different transformation events harboring the gstI gene were tested for their tolerance
to the present of the herbicide. Transgenic as well as nontransgenic tobacco plants with six leaves were transferred to MS medium supplemented with alachlor at concentrations of 0.01 or 0.015 g/l, and their phenotype was evaluated. Twenty days post-alachlor treatment the development of non-transgenic plants was completely inhibited (Fig. 4). Root development was almost completely repressed while in the upper part of the plants, decreased vigor and inability of the apical meristem to develop new leaves were observed, compared to the control plants. Transgenic plants showed substantially higher tolerance to alachlor compared to nontransgenic plants (Fig. 5). For example, transgenic plants 20 days post-alachlor treatment, showed 4.9 0.3 and
Fig. 5. The influence of herbicide alachlor on the apical meristem of transformed and non-transformed tobacco plants. On the left is an untransformed tobacco plant in medium without herbicide, in the middle and right lanes are transformed and untransformed plants, respectively, in MS medium where the concentration of the herbicide was 0.015 g/l. All plants were developed for 20 days.
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4.6 0.4 new leaves at 0.01 or 0.015 g/l alachlor concentrations, respectively, whereas the development of new leaves of non-transgenic plants was completely inhibited. These results show that the transgenic lines are more tolerant to alachlor than wild-type plants. The isoenzyme GST I, although exhibits wide substrate specificity, shows very low catalytic efficiency towards other herbicides and pollutants (Table 1). Therefore, we do not expect the transgenic tobacco plants to show substantially higher tolerance against other herbicides and pollutants. So far, four approaches have been reported for the generation of transgenic plants overexpressing GST isoenzymes [14–16,30]. The derived transgenic plants showed stress [16] and herbicide tolerance [14,15,30]. In the report describing gst transformation in tobacco plant with ZmGST II [30] Western blot analysis and activity assay against metolachlor were used to determine the level of expression of heterologous GST. The derived transgenic plants were showed tolerance against metolachlor [30]. Certain theta, phi and tau GSTs have been shown to have GSH peroxidase activity and reduce organic hydroperoxides of fatty acids and nucleic acids to the corresponding monohydroxyalcohols [2,16]. This functionality in GSTs has been demonstrated to be important in herbicide crossresistance in black-grass [5]. However, GST I does not exhibit any detectable GSH peroxidase activity and hence our results show that this activity should not directly connected with herbicide tolerance.
5. Conclusions In this report we addressed questions regarding the in planta functional role of GST I. We established that the expression of a gstI gene from maize, encoding for an enzyme lacking peroxidase activity, provides significant protection against the chloroacetanilide herbicide alachlor. The results of the present work have practical significance since may provide the basis for potentially useful biotechnological tools for the development of phytoremediation system for the degradation of herbicide pollutants in agricultural fields.
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Development of transgenic tobacco plants overexpressing maize glutathione S-transferase I for chloroacetanilide herbicides phytoremediation Margarita Karavangeli b, Nikolaos E. Labrou a,*, Yannis D. Clonis a, Athanasios Tsaftaris b,c a
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, Greece b Laboratory of Genetics and Plant Breeding, Aristotle University of Thessaloniki, Thessaloniki GR-54124, Greece c Institute of Agrobiotechnology, CERTH, 6th km Charilaou-Thermis Road, P.O. Box 361, Thermi GR-57001, Greece Received 14 February 2005; received in revised form 25 February 2005; accepted 1 March 2005
Abstract Glutathione S-transferases (GSTs, EC 2.5.1.18) are a multigene family of detoxification enzymes that biotransform a wide variety of endogenous and exogenous electrophilic substrates, including herbicides. The isozyme GST I from maize exhibits significant catalytic activity for the chloroacetanilide herbicide alachlor and appears to be involved in its detoxifying process. To establish the in planta ability of GST I to detoxify from alachlor, transgenesis studies were carried out. The gene gstI–6His, which encodes for 6His-tagged GST I, was used for the construction of a binary vector suitable for genetic engineering of tobacco plants (Nicotiana tabacum). Through biolistic method transgenic tobacco plants were obtained. Integration of gstI–6His gene in transgenic tobacco plants genome was confirmed by polymerase chain reaction and Southern blot hybridization. The expression of active GST I was established by Western blot analysis, using anti-6His antibody, and by direct purification of 6-His tagged GST I on Ni–NTA agarose. Primary transformed plants harboring the gstI–6His gene were transferred to MS medium supplemented with alachlor and their phenotype was evaluated. The transgenic plants showed substantially higher tolerance to alachlor compared to non-transgenic plants in terms of root, leaves and vigorous development. These transgenic plants are potentially useful biotechnological tools for the development of phytoremediation system for the degradation of herbicide pollutants in agricultural fields. # 2005 Elsevier B.V. All rights reserved. Keywords: Alachlor; Herbicide detoxification; Phytoremediation; Transgenic tobacco; Xenobiotics
1. Introduction Plants contain a complex array of enzymes which are able to detoxify xenobiotics including esterases and cytochrome P450-dependent oxygenases (phase 1 metabolising enzymes), glutathione S-transferases (GSTs), glucosyl transferases (GTs) and malonyl transferases (phase 2 metabolising enzymes) and ATP-driven vacuolar transporters (phase 3 enzymes). GSTs catalyse the Abbreviations: Alachlor, 2-Cl-N-[2,6-diethylphenyl]-N-[methoxymethyl]acetamide; GSH, glutathione; GST, glutathione S-transferase; gstI–6His: 6His-tagged glutathione S-transferase I gene; nptII, neomycin phosphotransferase II gene; Ni–NTA, Ni2+–nitrilotriacetic acid * Corresponding author. Tel.: +30 210 5294308; fax: +30 210 5294308. E-mail address: [email protected] (N.E. Labrou). 1389-0344/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bioeng.2005.03.001
nucleophilic attack of the sulphur atom of glutathione (gGlu–Cys–Gly, GSH) on electrophilic groups of a variety of hydrophobic substrates, including herbicides, insecticides and carcinogens [1–4]. Plant GSTs gained particular attention with respect to the detoxification from herbicides that belong mainly to two classes, chloroacetanilide and triazines [5–8]. Chloroacetanilide and triazine herbicides are widely used for the control of annual grasses and broad-leaf weeds in a variety of major crops such as maize and soybeans [9]. Alachlor is a preemergence herbicide and is applied to young plants. It is absorbed through the roots and transferred to the upper parts of the plant through the apoplast. It represses the elongation of the root system and the development of the shoots of young plants [9].
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Cultivation systems of a variety of crops have tended to rely increasingly on the use of a number of agrochemicals for the maintenance of regular agricultural production. However, their residues sometimes affect the ecosystems and resulted in the pollution of crops. Therefore it is important to develop a system of rapid degradation of chemicals in the agriculture environment after use. Phytoremediation is an emerging new technology that uses plants to remove or degrade various pollutants from soils [10]. Although a number of plant species are able to accumulate high amounts of heavy metals or to degrade various organic soil pollutants, their remedial capacity can be significantly increased by genetic manipulation [11]. For example, transgenic yellow poplar plants overexpressing the bacterial gene encoding mercuric reductase were explored for the phytoremediation of mercury pollution [12]. Recently, poplar and tobacco transgenic plants overexpressing g-glutamylcysteine synthase were developed for remediation of cadmium and alachlor [13]. GSTs are considered as candidates for the development of such transgenic systems for the rapid degradation of herbicide pollutants in the agricultural fields. So far, four approaches have been reported for the generation of transgenic plants overexpressing GST isoenzymes. In all cases bifunctional isoenzymes were employed possessing both glutathione S-transferase and peroxidase activities (e.g. ZmGST27, an engineered form of ZmGSTU1, and NtGST107) [14–16]. The derived transgenic plants showed stress [16] and herbicide tolerance [14,15]. However, due to the bifunctional activities it was impossible to establish whether herbicide tolerance was exclusively due to the GSH-conjugation activity. Inspite of this uncertainty, GSTs may prove to be useful tools for phytoremediation. Before this to happen, questions need to be addressed with regards the ability of transgenic tobacco plants, expressing maize GST I, to tolerate alachlor. Therefore, an in planta characterisation needs to be established prior any further investigation or application of GST I.
were obtained from New England Biolabs, USA. Reduced GSH was obtained from Sigma, USA. Alachlor was obtained from Agan Chemical Manufactured (Israel) or by Rieden-deHaen (Germany). HYBOND-NX nylon membranes were purchased from Amersham-LifeScience, (UK). DNA purification kits were obtained from Qiagen (Germany). Pfu DNA polymerase and all other molecular biology reagents were obtained from Promega (U.K.).
3. Methods 3.1. Cloning, expression and purification of maize GST I Cloning of maize GST I into a pQE70 expression vector to yield the pQEGST expression plasmid was described by Labrou et al. [17]. Expression and purification of recombinant GST I were performed according to Labrou et al. [17]. 3.2. Assay of enzyme activity and protein Routine enzyme assays were performed by monitoring the formation of the conjugate of CDNB (1 mM) and GSH (2.5 mM) at 340 nm (e = 9.6 mM1 cm1) at 30 8C according to a published method [18]. Enzyme assays for the herbicides alachlor, metolachlor, atrazine, and chlorimuron ethyl were performed as described in [19,20]. Enzyme assays for cumene hydroperoxide were performed as described in [21]. Observed reaction velocities were corrected for spontaneous reaction rates when necessary. All enzyme assays velocities were determined in triplicate in buffers equilibrated at constant temperature. Protein concentration was determined by the method of Bradford [22] using bovine serum albumin (fraction V) as standard. 3.3. Construction of a transformation vector
2. Materials and methods 2.1. Materials Tobacco (Nicotiana tabacum) seeds from the cultivar Basmas Xanthi were obtained from Greek Institute of Tobacco. Seeds were surface sterilized in 2.5% NaOCl solution for 15 min, rinsed in sterile distilled water and finally placed for germination in sterile petri dishes containing MS basal medium supplemented with 30 g/l sucrose, 0.8% agar and adjusted to pH 5.8 before autoclaving. The plants were grown at 25 8C under a 16 h photoperiod using artificial light. Helios GeneGun was obtained from BioRad. DNTPs, PCR DIG Probe synthesis kit and Western blot kit were obtained from Roche, UK. Restriction enzymes
Standard recombinant methods were adopted for the construction of the plant transformation vector pART27– GSTI–6His that was used for tobacco transformations [23]. The maize gstI–6His gene was excised from the plasmid pGSTI [18], and subcloned in the EcoRI restriction site of the pART27 vector. This latter vector carries the CaMV 35S promoter and the 30 untranslated region of the octopine synthase (ocs) gene as a terminator. The entire CaMV35S–gstI-ocs cassette was digested by NotI restriction enzyme, and the resulted cassette was inserted into in the T-DNA region of the pART27 binary vector. The pART27 vector posses the nptII gene, which encodes for neomycin phopshotransferase II and confers resistance to the antibiotic kanamycin. The nptII gene is under the control of the nopaline synthase (nos) gene promoter.
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3.4. Transformation procedure Leaf discs from young tobacco plants were used as explants for transformation. Gold particles were coated with the transformation vector (plasmid DNA) and leaf discs were bombarded with those gold particles using a helium pressure-based apparatus (Helios1 Gene Gun, Bio-Rad). Bombarded leaf discs were maintained on MS medium with growth regulators (0.1 mg/l NAA, 1 mg/l BAP) for 2 days and then transferred to the same medium supplemented with 100 mg/l kanamycin for regeneration. Regenerated plantlets were transferred to MS medium lacking plant growth regulators and containing the same concentration of the antibiotic, for rooting. The explants were cultured at 25 8C with a 16 h photoperiod using artificial light. 3.5. DNA analysis Total genomic DNA was isolated from leaf tissue of primary transformants following an extraction procedure according to the instructions of the DNeasy Plant Mini Kit1 (Qiagen, Germany). PCR analysis was carried out with 100– 200 ng genomic DNA in a 12 ml total reaction volume containing 1 reaction buffer, 0.75 mM MgCl2, 0.2 mM dNTPs, 1 mM from each of the primers and 0.2–0.5 units of Taq polymerase. Different PCR reactions were carried out for the amplification of nptII, gstI genes and for a part of the gstI cassette. The primers that used for the amplification of the nptII gene were 50 -GAGGCTATTCGGCTATGACTG-30 and 50 -ATCGGGAGCGGCGATACCGTA-30 , for the gstI gene were 50 -CCACCGCCGAGCACAAGAGC-30 and 50 TAGGGCGTAGCGAACAGGCAGAGA-30 . For the amplification of the cassette the primers that were used was one of the promoter region CaMV-35S, 50 -AGGAGCATCGTGGAAAAAGAAGAC-30 , and the reverse primer was the same that used for the gstI gene. For Southern blot analysis, aliquots of 10 mg DNA were digested with EcoRI and fractionated by 0.8% agarose gel electrophoresis. DNA was then transferred to nylon membranes (HYBOND-NX1), and the subsequent hybridization reactions were carried out according to standard procedures [23]. The probe that was used for the hybridization was part of the nptII gene labeled with the digoxygenin molecule. The probe prepared using PCR technique, during which the DIG11-dUTP was incorporated into the amplification product, using the PCR DIG Probe Synthesis Kit (Roche, UK). The hybridization and detection procedures were conducted using the DIG DNA Labeling Kit (Roche, UK) according to the manufacturer’s instructions. Non-transgenic plants were used as negative controls. 3.6. Western blot analysis and purification of His6tagged GST I expressed in transgenic tobacco leaves Detection of 6His-tagged GST I expressed in transgenic tobacco was accomplished using an Anti-6His antibody
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(mouse monoclonal antibody) and as a secondary antibody anti-mouse-Ig-POD as follows: samples from transformed and untransformed tobacco leaves were homogenised with mortal and paste in Tris/HCl buffer (50 mM, pH 7.0) and subjected to SDS-PAGE on 12.5% gels. The protein was electrophoretically transferred to PVDF membranes. Nonspecific binding sites were blocked by incubating membranes for 1–2 h at room temperature in 1% (w/v) blocking solution in Tris-buffered saline (50 mM Tris–HCl, 150 mM NaCl, pH 7.5). The membrane was probed with Anti-6His antibody for 30–45 min at room temperature in Tris-buffered saline. Blots were washed four times with 100 ml of Tris-buffered saline containing 0.1% Tween-20, for 15–25 min each. Anti-6His antibody-reactive bands were detected using anti-mouse-IgPOD and the luminescence substrate luminol according to the manufacturer’s instructions. Purification of 6His-tagged GST I from transgenic tobacco leaves was achieved by metal chelate affinity chromatography on Ni–NTA column as follows: samples from transformed and untransformed tobacco leaves were homogenised with mortal and paste in potassium phosphate buffer (50 mM, pH 8.0) containing sodium chloride (0.3 M) and the resulted plant extract (1 ml, total GST activity 2.5 U/ ml, 0.85 mg total protein) was loaded to a column of Ni– NTA adsorbent (0.5 ml), which was previously equilibrated with potassium phosphate buffer (50 mM, pH 8.0) containing sodium chloride (0.3 M). Non-adsorbed protein was washed off with 10 ml equilibration buffer, followed by 20 ml of potassium phosphate buffer (50 mM, pH 6.2) containing sodium chloride (0.3 M) and glycerol (10%, v/v). Bound GST I was eluted with equilibration buffer containing 0.2 M imidazole (2 ml). 3.7. Herbicide tolerance studies Plants from seven different transgenic lines of transformed tobacco plants were transferred to MS media supplemented with two different concentrations of the herbicide alachlor (0.01 g/l and 0.015 g/l). Non-transgenic plants were used as negative controls (one non-transgenic plant per line). Transgenic and non-transgenic plants were followed by measuring their development at the end of 20 days exposure period. Each experiment was performed by two replicate plants per treatment. The significant differences between mean values were evaluated by Student’s t-test. Differences were considered to be significant at P = 0.05. The plants were cultured at 25 8C with a 16 h photoperiod under artificial light.
4. Results and discussion 4.1. Analysis of substrate specificity of maize GST I Metabolic detoxification is probably the most general major mechanism involved in plant tolerance to herbicides. In particular, the GSH/GST system appears to be widespread
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among plants and to confer protection against toxic chemicals by catalysing the conjugation of glutathione to an electrophile center of hydrophobic molecules by means of the –SH group [2–4]. Species tolerant or susceptible to a wide spectrum of herbicides (triazines, chloroacetanilides, thiocarbamates) are characterized by high and low levels of GST, respectively, and an increase in specific GST activity in response to some herbicide treatment has been reported in a few cases [7,8]. Thus the system appears to be involved in the determination of plant tolerance to these classes of molecules. Alachlor is one of the most widely used herbicide that is used for selective weed control in agricultural crops [9]. The biotransformation of alachlor in plants has not been fully characterised. However, recent studies suggest Phase 2-mediated biotransformation by GSTs. In maize (Zea mays L.), conjugation with GSH is a major route of metabolism of the chloroacetanilide herbicides [8,15,24]. The isoenzyme GST I from maize has been the subject of intense research because of its ability to detoxify a wide range of xenobiotics. It belongs to class phi and to type I of GSTs, according to Edwards et al. [4] and McGonigle et al. [20] classifications, respectively. GST I is a homodimer protein of 214 amino acids, and exhibits wide substrate specificity towards several classes to herbicides (Fig. 1). Two features have been reported, based on the crystal structure [25,26] and on extensive mutagenesis studies [17,18,27,28], to contribute to wide substrate specificity and high catalytic efficiency towards electrophile substrates: the size and shape of the H-site and its high flexibility. These features are determined, at atomic level, by the hydrophobic residues Trp12, Phe35, Ile118, and the flexibility in the upper part of the H-site is modulated by Gly123 and Gly124 [25–28]. Table 1 shows the specific activity of the enzyme exhibited for atrazine, alachlor, metolachlor, chlorimuron ethyl, and cumene hydroperoxide. The enzyme exhibits substantial catalytic activity towards alachlor, moderate activity towards atrazine and chlorimuron ethyl, but little activity toward metolachlor. In addition the enzyme was tested for its ability to act as glutathione peroxidase using the synthetic model substrate cumene hydroperoxide. GSTs have been shown to have GSH peroxidase activity and reduce organic hydroperoxides of fatty acids and nucleic acids to the corresponding monohydroxyalcohols [2,16]. As shown in Table 1, GST I does not exhibit any detectable glutathione peroxidase activity (Table 1). 4.2. Transformation and analysis of tobacco plants The plant expression vector containing the gstI–6His gene was constructed and introduced into leaf discs of
Fig. 1. Structures of selected herbicides that metabolized by the GSH/GST system. (A) Atrazine; (B) alachlor; (C) metolachlor, and (D) chlorimuron ethyl.
tobacco cv. Basmas Xanthi. DNA was delivered to leaf discs by particle bombardment using a hand gene gun device using helium gas for the delivery of the particles. Bombarded leaf discs were cultured in regeneration medium in the presence of the antibiotic kanamycin. Nineteen plantlets that rooted in the presence of antibiotic were selected. Genomic DNA from leaves of transformed tobacco plants was purified and analyzed by PCR. Reactions were carried out using two different pair of primers. The first pair was designed to amplify a 700 bp internal fragment of the nptII gene, whereas the second was used to amplify an internal fragment of gstI coding region, sized 430 bp.
Table 1 Activity of the GST I (nmol min1 mg1) with selected herbicides, assayed as described in the text Enzyme
Alachlor
Metolachlor
Atrazine
Chlorimuron ethyl
Cumene hydroperoxide
GSTI
80.5 4.6
ND
0.75 0.2
0.4 0.1
ND
ND: no significant activity detected.
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Fig. 3. Western blot hybridization. Western blot analysis of total protein fractions extracted from the leaf tissue of primary transformants. Proteins were resolved by SDS-PAGE and probed with anti-6His antibody. Lane 1: purified recombinant GSTI–6His (0.5 mg) expressed in E. coli [18]. Lane 2: total protein extract (50 mg) from transformed 20 plants; Lane 3: total protein extract from untransformed plants (50 mg). Molecular weight marker is shown by the arrow.
Fig. 2. DNA analysis of tobacco plants. (A) PCR analysis of genomic DNA from transformed with pART27–gstI–6His tobacco plants. Lanes A and B: molecular markers 100 bp and 1 Kb, respectively. Lanes 1–5: amplification of the nptII gene from transformed (lanes 1 and 2) and untransformed (line 3) tobacco plants; Lane 4: PCR analysis in the absence of genomic DNA (white control); Lane 5: amplification of the nptII gene from the plasmid pART27–gstI–6His (positive control). (B) PCR analysis of genomic DNA from transformed with pART27–gstI–6His tobacco plants for the amplification of gstI gene. Lanes A: molecular marker 100 bp. Lanes 1–8: amplification part of the gstI gene of transformed (lanes 1–6) and untransformed (Lane 7) tobacco plants; Lane 8: PCR analysis in the absence of genomic DNA (white control); Lane 9: amplification of the gstI gene from the plasmid pART27–gstI–6His (positive control). (C) PCR analysis of genomic DNA from transformed with pART27–gstI–6His tobacco plants for the amplification of CaMV35S–gstI-ocs 30 cassette. Lane A: molecular marker 100 bp; lanes 1–7: amplification of the cassette from transformed (lanes 1–3) and untransformed (lane 4) tobacco plants; Lane 5: PCR analysis in the absence of genomic DNA (white control); Lane 6: plasmid pART27–GSTI–6His (positive control).
Finally, a part of the cassette CaMV35S–gstI–6His-ocs 30 that consisted of the promoter and the coding region was amplified in order to check the integrity of the expression cassette. The same PCR reactions were also carried out using DNA
isolated from wild-type plants and plasmid pART27–gstI– 6His DNA as a negative and positive control, respectively. The results of PCR analysis are illustrated in Fig. 2. Southern blot analysis was performed on genomic DNA from leaves of putative transgenic plants and from untransformed control plants. Genomic DNA was digested with EcoRI and analyzed using as a probe part of the nptII gene labeled with digoxygenin. The results of the Southern blot confirmed the presence of the nptII gene only in the genomic DNA from leaves of putative transgenic plans (data not shown). 4.3. Expression of active 6His tagged GST I in transgenic tobacco plants Western blot analysis was also performed using total protein extract from leaves of transformed and untransformed plants, employing monoclonal antibody against the 6His tag (Fig. 3). The results provide direct evidence that transgenic tobacco plants express maize GST I. The extra six histidine residues tagged on the N-terminus of the enzyme, enable GST I to be rapidly purified from transgenic leaf extract by metal chelate affinity chromatography on Ni–NTA column. On the basis of at least 80% yield of the purification procedure, the level of GST I expression in transgenic tobacco plants may be estimated approximately as 0.03% of total soluble leaf proteins. Notably, affinity chromatography on Ni–NTA column failed to purify any GST activity from leaf extract of untransformed plants. Kinetic analysis of the purified enzyme gave kinetic constants (Km for GSH of 1.2 0.1 mM and Km for CDNB of 1.6 0.1 mM) which are indistinguishable from
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Fig. 4. The influence of herbicide alachlor in non-transformed tobacco plants after 20 days, at the apical meristem (A) and root system (B). On the left is tobacco plant in medium without herbicide, in the middle the concentration of the herbicide is 0.01 g/l and on the right is 0.015 g/l.
that exhibited by the recombinant E. coli expressed enzyme [18]. Taking into account that heterodimerization strongly influence the catalytic and kinetic properties of the enzyme [29] one can conclude that GST I is expressed as a fully active homodimer, presumably without subunit heterodimerization with endogenous tobacco’s GSTs subunits. 4.4. Alachlor tolerance studies The tobacco Basmas Xanthi cultivar that was used in the transformation experiments is sensitive to alachlor. Primary transformed plants from seven different transformation events harboring the gstI gene were tested for their tolerance
to the present of the herbicide. Transgenic as well as nontransgenic tobacco plants with six leaves were transferred to MS medium supplemented with alachlor at concentrations of 0.01 or 0.015 g/l, and their phenotype was evaluated. Twenty days post-alachlor treatment the development of non-transgenic plants was completely inhibited (Fig. 4). Root development was almost completely repressed while in the upper part of the plants, decreased vigor and inability of the apical meristem to develop new leaves were observed, compared to the control plants. Transgenic plants showed substantially higher tolerance to alachlor compared to nontransgenic plants (Fig. 5). For example, transgenic plants 20 days post-alachlor treatment, showed 4.9 0.3 and
Fig. 5. The influence of herbicide alachlor on the apical meristem of transformed and non-transformed tobacco plants. On the left is an untransformed tobacco plant in medium without herbicide, in the middle and right lanes are transformed and untransformed plants, respectively, in MS medium where the concentration of the herbicide was 0.015 g/l. All plants were developed for 20 days.
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4.6 0.4 new leaves at 0.01 or 0.015 g/l alachlor concentrations, respectively, whereas the development of new leaves of non-transgenic plants was completely inhibited. These results show that the transgenic lines are more tolerant to alachlor than wild-type plants. The isoenzyme GST I, although exhibits wide substrate specificity, shows very low catalytic efficiency towards other herbicides and pollutants (Table 1). Therefore, we do not expect the transgenic tobacco plants to show substantially higher tolerance against other herbicides and pollutants. So far, four approaches have been reported for the generation of transgenic plants overexpressing GST isoenzymes [14–16,30]. The derived transgenic plants showed stress [16] and herbicide tolerance [14,15,30]. In the report describing gst transformation in tobacco plant with ZmGST II [30] Western blot analysis and activity assay against metolachlor were used to determine the level of expression of heterologous GST. The derived transgenic plants were showed tolerance against metolachlor [30]. Certain theta, phi and tau GSTs have been shown to have GSH peroxidase activity and reduce organic hydroperoxides of fatty acids and nucleic acids to the corresponding monohydroxyalcohols [2,16]. This functionality in GSTs has been demonstrated to be important in herbicide crossresistance in black-grass [5]. However, GST I does not exhibit any detectable GSH peroxidase activity and hence our results show that this activity should not directly connected with herbicide tolerance.
5. Conclusions In this report we addressed questions regarding the in planta functional role of GST I. We established that the expression of a gstI gene from maize, encoding for an enzyme lacking peroxidase activity, provides significant protection against the chloroacetanilide herbicide alachlor. The results of the present work have practical significance since may provide the basis for potentially useful biotechnological tools for the development of phytoremediation system for the degradation of herbicide pollutants in agricultural fields.
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