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First published online July 15, 2009; 10.1104/pp.109.141598 Plant Physiology 151:253-261 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
The Role of Oxophytodienoate Reductases in the Detoxification of the Explosive 2,4,6-Trinitrotoluene by Arabidopsis1,[W],[OA]Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5YW, United Kingdom
The explosive 2,4,6-trinitrotoluene (TNT) is a significant environmental pollutant that is both toxic and recalcitrant to degradation. Phytoremediation is being increasingly proposed as a viable alternative to conventional remediation technologies to clean up explosives-contaminated sites. Despite the potential of this technology, relatively little is known about the innate enzymology of TNT detoxification in plants. To further elucidate this, we used microarray analysis to identify Arabidopsis (Arabidopsis thaliana) genes up-regulated by exposure to TNT and found that the expression of oxophytodienoate reductases (OPRs) increased in response to TNT. The OPRs share similarity with the Old Yellow Enzyme family, bacterial members of which have been shown to transform explosives. The three predominantly expressed forms, OPR1, OPR2, and OPR3, were recombinantly expressed and affinity purified. Subsequent biochemical characterization revealed that all three OPRs are able to transform TNT to yield nitro-reduced TNT derivatives, with OPR1 additionally producing the aromatic ring-reduced products hydride and dihydride Meisenheimer complexes. Arabidopsis plants overexpressing OPR1 removed TNT more quickly from liquid culture, produced increased levels of transformation products, and maintained higher fresh weight biomasses than wild-type plants. In contrast, OPR1,2 RNA interference lines removed less TNT, produced fewer transformation products, and had lower biomasses. When grown on solid medium, two of the three OPR1 lines and all of the OPR2-overexpressing lines exhibited significantly enhanced tolerance to TNT. These data suggest that, in concert with other detoxification mechanisms, OPRs play a physiological role in xenobiotic detoxification.
Large amounts of land and water are heavily contaminated by explosives, mainly as a result of the manufacture and military use of munitions. The high financial cost associated with cleaning up these contaminated sites largely precludes the use of many existing remediation technologies, such as soil excavation and incineration or disposal to landfill. There is a great deal of work documenting the global contamination, general toxicity, and microbial metabolism of 2,4,6-trinitrotoluene (TNT) in the environment; however, relatively little is known about the enzymes mediating the detoxification of TNT in plants (for review, see Rylott and Bruce, 2009  ).
Phytoremediation, the use of plants to remove environmental pollutants, offers a low-cost, sustainable alternative to conventional remediation technologies and is attracting considerable attention as a means to clean up sites contaminated with explosives. While TNT is a potent phytotoxin, plants are able to detoxify low levels of TNT. In an effort to determine how plant tolerance could be further improved, we are investigating the biochemistry and enzymology underlying the innate ability of plants to detoxify TNT. The detoxification of xenobiotics has been loosely categorized into three phases (Sandermann, 1992
Data from both our microarray experiments (Gandia-Herrero et al., 2008 ) and other expression studies (Ekman et al., 2003; Mezzari et al., 2005) have found that members of the small gene family of oxophytodienoate reductases (OPRs) in Arabidopsis (Arabidopsis thaliana) are up-regulated following exposure to TNT. The Arabidopsis genome contains three characterized OPRs: OPR1, OPR2, and OPR3. In addition, there are three as yet uncharacterized putative OPRs, named here as OPR4, OPR5, and OPR6, with OPR4 and OPR5 being identical. The physiological functions of the OPRs remain obscure, with the exception of OPR3, which is involved in jasmonic acid biosynthesis, converting (9S,13S)-12-oxophytodienoic acid to 3-2(2'(Z)-pentyl)cyclopentane-1-octanoic acid in the peroxisome (Sanders et al., 2000; Stintzi and Browse, 2000; for review, see Wasternack, 2007). OPR3 is located on chromosome II within the Arabidopsis genome and contains a C-terminal Ser-Arg-Leu type 1 peroxisome-targeting sequence. The remaining OPRs are all located on chromosome I and do not possess any known organelle-targeting sequences. The spatial expression of OPR1 and OPR2 across root cells where TNT accumulates (Biesgen and Weiler, 1999; Baerenfaller et al., 2008) favors a role in detoxification.
The OPRs share similarity with the Old Yellow Enzyme family, a group of flavoenzymes that has been repeatedly associated with the transformation of explosives (Binks et al., 1996
Pentaerythritol tetranitrate reductase, an Old Yellow Enzyme homolog isolated from Enterobacter cloacae (Binks et al., 1996 The OPR4/5 protein is predicted to have reduced activity toward TNT, compared with the other OPRs, owing to a C-terminal truncation that removes residues thought to be important in binding the cofactor NADH, Thus, we investigated OPR1, -2, and -3 as likely candidates for the TNT nitroreduction activity in Arabidopsis.
Expression of OPRs in Arabidopsis in Response to TNT
To identify genes up-regulated in response to TNT treatment, we performed a microarray experiment (Gandia-Herrero et al., 2008
Transformation of TNT by Recombinant OPRs To determine whether OPR1, OPR2, and OPR3 were capable of producing reduced TNT products and hydride Meisenheimer complexes, OPR1, -2, and -3 were cloned into pET-15b expression vectors and transformed into Escherichia coli. Following expression optimization steps, soluble, active recombinant protein was obtained for OPR1, OPR2, and OPR3. The recombinant proteins were purified by affinity chromatography (Fig. 3A ), and the identities of these proteins were confirmed using trypsin digestion and matrix-assisted laser-desorption ionization time of flight/time of flight (MALDI-TOF/TOF) tandem mass spectrometry.
The NADH-dependent transformation of TNT by the purified OPRs was followed by HPLC over a period of 3 h. An NADH-dependent alcohol dehydrogenase was employed to keep the levels of NADH constant during the course of the assay. Figure 3 shows that all three OPRs were able to transform TNT to HADNT intermediates. No ADNTs were observed in the time scale monitored, although the decrease in HADNT concentration in the OPR2 and OPR3 assays (Fig. 3, D and E) suggests further transformation. The accumulation of an additional peak, more polar than HADNT, was observed in both OPR2 and -3 assays, which potentially represents dihydroxylamino-nitrotoluene (data not shown). Reduction of TNT by OPR1 resulted in the formation of products with retention times of 3.7, 7.3, and 9.2 min and spectra with absorbance maxima at approximately 500 nm (Fig. 3C). These were not seen in reaction mixtures catalyzed by OPR2 and OPR3 and were identified as dihydride Meisenheimer TNT complexes by reference to characterized standards generated by pentaerythritol tetranitrate reductase (Williams et al., 2004 ). 2H–-TNT adducts have been shown to yield four isomers in dynamic equilibrium. These are accounted for as the 1,3- and 3,5-hydride additions to the TNT molecule, with the respective tautomers forming protonated nitroalkanes (Williams et al., 2004). Figure 3C shows that levels of 2H–-TNT 3.7 and 9.2 decreased after 40 and 140 min, respectively, but no corresponding increases in any detected compounds were seen. The purified preparation of OPR1 showed the presence of a significant level of a contaminating protein (Fig. 3A). Trypsin digestion and MALDI-TOF/TOF tandem mass spectrometry analysis revealed this to be an E. coli 2-deoxy-D-gluconate 3-dehydrogenase. Although deemed unlikely to catalyze the observed reaction with TNT, we assayed cell extracts of NovaBlue (DE3) E. coli, containing pET-15b with and without OPR1. Only cell extract containing OPR1 was able to transform TNT to yield the dihydride-Meisenheimer TNT adducts, confirming that the observed activity with TNT was due to OPR1.
To dissect the individual contributions of the OPRs toward TNT detoxification in Arabidopsis, T-DNA insertional mutants opr1 and opr2 (Alonso et al., 2003
The OPR1, OPR2, and OPR3 genes were overexpressed using the 35S promoter, with the C-terminal Ser-Arg-Leu type 1 peroxisome-targeting sequence present in OPR3 conserved in the overexpressing OPR3 construct. As a control to monitor the detection of ADNT and dihydride Meisenheimer TNT complexes in our experiments, the onr gene for the bacterial Old Yellow Enzyme homolog, pentaerythritol tetranitrate reductase, was transformed into Arabidopsis. Transformed lines were generated, and four independent lines were characterized for each gene. As predicted from our earlier studies on tobacco transformed with the same construct, all four 35S-onr lines exhibited enhanced tolerance to TNT. The expression of line 35S-onr1 is shown in Figure 4B, and this line was used as a control in our liquid culture and solid medium experiments.
To investigate the tolerance of the knockout, knockdown, and overexpressing lines to TNT, these lines were grown on solid medium containing 2 µM TNT. Arabidopsis seedlings expressing onr had root lengths 2-fold longer than wild-type plants when grown on 2 µM TNT (Fig. 5A
). This enhanced tolerance to TNT agrees with that previously observed in transgenic tobacco (French et al., 1999
TNT Transformation in Liquid Culture
As expected from earlier results (French et al., 1999 After 7 d of growth in medium containing 200 µM TNT, seedlings of the 35S-onr line, OPR1-overexpressing lines 1.1, and 9.4, and OPR2-overexpressing line 8.4 were less chlorotic and had greater biomasses than wild-type seedlings (Fig. 6 ). Biomass was not significantly altered from the wild type in the control flasks without TNT; however, in the absence of TNT, it was noted that the OPR-overexpressing lines showed enhanced survival under hydroponic conditions (data not shown).
In the flasks containing the knockdown OPR1,2 lines grown in liquid culture with 300 µM TNT, TNT was taken up more slowly than in the wild type by all three knockdown lines in the first 2 d. Thereafter, no more TNT was removed from the medium by the knockdown OPR1,2 lines, and significantly less ADNT and Meisenheimer complex compounds were released into the medium than from the wild type (Fig. 7 ).
We have cloned and purified three recombinant Arabidopsis OPRs, OPR1, OPR2, and OPR3, which exhibit significant (43%, 44%, and 36%, respectively) identity with bacterial pentaerythritol tetranitrate reductase, a member of the Old Yellow Enzyme family that is able to transform TNT. The greatest degree of similarity, both structurally and in regulation, exists between OPR1 and OPR2, and both are up-regulated in the root on exposure to TNT (Biesgen and Weiler, 1999 ). However, OPR1 had the greatest increase in expression in response to TNT treatment, suggesting a more dominant role in detoxification. The mechanism of TNT uptake and the intracellular site of accumulation are not known; however, while OPR1 and OPR2 are predicted to be active in the cytosol, OPR3 is targeted to the peroxisome, and this localization is likely to reduce accessibility toward TNT in planta.
The in vitro activity data clearly show that OPR1, OPR2, and OPR3 all have activity toward TNT. In liquid culture, the overexpressing OPR1 lines 1.1 and 9.4 had increased TNT uptake and produced more TNT transformation products than the wild type, suggesting increased TNT metabolism. Conversely, the knockdown OPR1,2 lines removed less TNT from liquid culture and produced fewer TNT transformation products. When grown on solid medium, two of the three OPR1-overexpressing lines and all of the OPR2-overexpressing lines exhibited significantly enhanced tolerance to TNT (as measured by root growth). This agrees with previous data in tobacco expressing onr, which showed that enhanced TNT transformation directly increased TNT tolerance (French et al., 1999
Expression analysis (Fig. 4) showed that in both the OPR-overexpressing and knockdown OPR1,2 transgenic lines, only the targeted OPR expression levels were altered; the expression levels of the remaining OPR homologs remained unchanged. While this result shows that OPR transcript levels are regulated independently from each other, it is possible that posttranscriptional regulation inhibited the generation of higher levels of active protein in the overexpressing lines. Previous studies using promoter β-glucuronidase fusions with Arabidopsis OPR1 and OPR2 showed that although transcript levels transiently increased in response to local and systemic wounding, UV-C illumination, and coldness, no subsequent changes in protein levels were detected (Biesgen and Weiler, 1999
A wide range of
In summary, we demonstrate that, in agreement with characterized bacterial Old Yellow Enzyme homologs, OPR1, -2, and -3 display activities toward TNT and OPRs contribute to the detoxification of TNT in the roots of Arabidopsis. A more detailed understanding of the biochemical mechanism of TNT detoxification in plants will allow opportunities to select or breed robust plant phenotypes in a rational way for field applications. This work further supports the view that members of the Old Yellow Enzyme family of flavoproteins play a role in the general detoxification of xenobiotic compounds in plants as well as microorganisms.
Biochemicals TNT was kindly provided by the Defence Science and Technology Laboratory at Fort Halstead, Kent, United Kingdom.
OPR1, OPR2, and OPR3 were cloned from Arabidopsis (Arabidopsis thaliana ecotype Columbia-0 [Col-0]) cDNA into pET-15b (Novagen). OPR1 and OPR3 were expressed in NovaBlue (DE3) Escherichia coli (Novagen), and OPR2 was expressed in Rosetta-gami B (DE3) E. coli (Novagen). Cells were grown to mid-log phase at 37°C and induced with 0.4 mM isopropylthio-β-galactoside. After 20 h, cells were spun at 12,000g for 10 min at 4°C, washed in buffer A (50 mM potassium phosphate buffer, pH 7), sonicated, and centrifuged at 4°C and 12,000g for 30 min. Soluble extract of OPR1 was applied to a Mimetic Orange 1 affinity chromatography column, and OPR2 and -3 were applied to a Mimetic Blue 1 column (Prometic Biosciences). Columns were washed with buffer A, then protein was eluted using a sodium chloride gradient, concentrated on 10-kD spin filters (Perbio Science), and dialyzed into buffer A. Mimetic Orange 2 matrix was added to the OPR1 fraction to bind and remove contaminants. Protein identities were confirmed following SDS-PAGE and trypsin digest, by MALDI-TOF peptide mass fingerprinting, immediately followed by more detailed protein characterization using MALDI-TOF/TOF tandem mass spectrometry on the same prepared sample.
Assays and HPLC analysis were performed as described previously (Williams et al., 2004
Wild-type Arabidopsis ecotypes Col-0 and Wassilewskija were obtained from the Nottingham Arabidopsis Stock Centre at the University of Nottingham. The opr1 (SALK_145355) and opr2 (SALK_014855) mutants were obtained from T-DNA express (Alonso et al., 2003
The 35S overexpression lines were generated using the pART27 binary cassette system (Gleave, 1992
The knockdown OPR1,2 chimera was constructed as follows. OPR1 and OPR2 sequences were amplified using kdOPR1 and kdOPR2 primers, and the fragments were digested with BfaI and ligated together. The OPR1-OPR2 ligated sequence was then reamplified using kdOPR1F and kdOPR2R primers, sequenced, and cloned into pHellsgate 8 (Wesley et al., 2001 All constructs was transformed into Arabidopsis Col-0 using Agrobacterium tumefaciens strain GV3101. Transformants and homozygous lines were selected using kanamycin resistance. The T-DNA border regions of opr1 and opr2 were amplified for sequencing using T-DNA left border primer LBa1 in combination with OPR1F for opr1 and OPR2-seq for opr2. Insertion sequences are shown in Supplemental Figure S1. Endogenous OPR1 and OPR2 were amplified using OPR1F/OPR1R and OPR2F/OPR2R primers. All primer sequences are described in Supplemental Table S1.
The DNase-treated total RNA was isolated from 14-d-old liquid culture-grown plants using the RNeasy kit (Qiagen). The synthesis of cDNA was primed with oligo(dT)12-18 using SuperScript II reverse transcriptase (Invitrogen) containing RNasin (Promega) at 42°C for 90 min, before inactivation at 70°C for 15 min. For quantitative RT-PCR, cDNA was purified using a Promega Wizard SV gel and PCR cleanup system and quantified. The rtOPR primers and probe sets (Supplemental Table S1), specific for each OPR gene, were tested for primer efficiency, then TaqMan quantitative PCR was performed using 50 ng of cDNA in an Applied Biosystems 7300 real-time PCR machine with a cycle of 50°C for 2 min, 95°C for 10 min, and then 40 cycles of 95°C for 15 s, 60°C for 1 min using FAM (5') and TAMRA (3') fluorescent dyes. The level of expression was normalized against ACT2 (GenBank accession no. NM_180280). Data were analyzed using ABI software according to the manufacturer's recommendations. Semiquantitative RT-PCR was performed on the overexpressing OPR lines using serial dilutions of cDNA and sqOPR primers. Reactions were denatured at 94°C for 3 min, cycled 25 times at 94°C for 15 s, 55°C for 30 s, 1 min for 72°C, and then held at 72°C for 10 min. For reactions with OPR2 primers, the annealing step was 60°C. ACT2 gene expression using sqACT primers was used as a reference. Primer sequences are shown Supplemental Table S1. Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: OPR1 (NM_202428), OPR2 (NM_106319), OPR3 (NM_126619), OPR4 (NM_101664), OPR5 (NM_179352), and OPR6 (NM_100810).
The following materials are available in the online version of this article.
We are grateful to Prof. John Browse for kindly providing the opr3 mutant. We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants and Nottingham Arabidopsis Stock Centre for distributing plant lines. Received May 15, 2009; accepted July 8, 2009; published July 15, 2009.
1 This work was supported by the Strategic Environmental Research and Development Program of the U.S. Department of Defense, by a Burgess studentship to E.R.B., and by a Biotechnology and Biological Sciences Research Council studentship to Z.C.S.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Neil C. Bruce (ncb5{at}york.ac.uk).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.141598 * Corresponding author; e-mail ncb5{at}york.ac.uk.
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/β-unsaturated ketones and aldehydes, including vinyl ketones, smaller molecules such as acrolein, and those with more extended