Enhanced transformation of tnt by tobacco plants expressing a bacterial nitroreductase

The manufacture, disposal, and detonation of explosives have resulted in the pollution of large tracts of land and groundwater. Historically, 2,4,6-trinitrotoluene (TNT) is the most widely used military explosive and is toxic to biological systems and recalcitrant to degradation. To examine the feasibility of enhancing the ability of plants to detoxify the explosive TNT, we created transgenic tobacco (Nicotiana tabacum) constitutively expressing the nsfI nitroreductase gene from Enterobacter cloacae. The product of TNT reduction by the nitroreductase was found to be 4-hydroxylamino-2,6-dinitrotoluene (4-HADNT). Characterization of the transgenic lines in sterile, aqueous conditions amended with TNT demonstrated that these plants were able to remove all of the TNT from the medium at an initial concentration of 0.5 mM (113 mg L(-1)) TNT. In contrast, growth was suppressed in wild-type plants at 0.1 mM (23 mg L(-1)). Following uptake, transgenic seedlings transformed TNT predominantly to 4-HADNT and its high levels appeared to correlate with enhanced tolerance and transformation of TNT. Transformation products of TNT were subsequently conjugated to plant macromolecules to a greater degree in transgenic tobacco, indicating enhanced detoxification compared to the wild type.     

12-oxo-phytodienoic acid-glutathione conjugate is transported into the vacuole in Arabidopsis

While exogenous toxic compounds such as herbicides are thought to be sequestered into vacuoles in the form of glutathione (GSH) conjugates, little is understood about natural plant products conjugated with GSH. To identify natural products conjugated with GSH in plants, metabolites in the Arabidopsis γ-glutamyl transpeptidase (ggt) 4 knockout mutants that are blocked in the degradation of GSH conjugates in the vacuole were compared with those in wild-type plants. Among the metabolites identified, one was confirmed to be the 12-oxo-phytodienoic acid (OPDA)-GSH conjugate, indicating that OPDA, a precursor of jasmonic acid (JA), is transported into the vacuole as a GSH conjugate.     

Phytodetoxification of the environmental pollutant and explosive 2,4,6-trinitrotoluene

Our recent study highlights the role of 2 glutathione transferases (GSTs) in the detoxification of the environmental pollutant, 2,4,6-trinitrotoluene (TNT) in Arabidopsis thaliana. TNT is toxic and highly resistant to biodegradation in the environment, raising both health and environmental concerns. Two GSTs, GST-U24 and GST-U25, are upregulated in response to TNT treatment, and expressed predominantly in the root tissues; the site of TNT location following uptake. Plants overexpressing GST-U24 and GST-U25 exhibited significantly enhanced ability to withstand and detoxify TNT, and remove TNT from contaminated soil. Analysis of the catalytic activities of these 2 enzymes revealed that they form 3 TNT-glutathionyl products. Of particular interest is 2-glutathionyl-4,6-dinitrotoluene as this represents a potentially favorable step toward subsequent degradation and mineralization of TNT. We demonstrate how GSTs fit into what is already known about pathways for TNT detoxification, and discuss the short and longer-term fate of TNT conjugates in planta.     

Functional importance of the family 1 glucosyltransferase UGT72B1 in the metabolism of xenobiotics in Arabidopsis thaliana

The Arabidopsis type 1 UDP-glucose-dependent glucosyltransferase UGT72B1 is highly active in conjugating the persistent pollutants 3,4-dichloroaniline (DCA) and 2,4,5-trichlorophenol (TCP). To determine its importance in detoxifying xenobiotics in planta, mutant plants where the respective gene has been disrupted by T-DNA insertion have been characterized. Extracts from the knockout ugt72B1 plants showed radically reduced conjugating activity towards DCA and TCP and the absence of immunodetectable UGT72B1 protein. In contrast, activities towards phenolic natural products were unaffected. When aseptic root cultures were fed [14C]-DCA, compared with wild types, the ugt72B1 plants showed a reduced rate of uptake of the xenobiotic and very little metabolism to soluble DCA-glucose or associated polar conjugates. Instead, the knockouts accumulated non-extractable radioactive residues, most probably associated with lignification. When the feeding studies were carried out with [14C]-TCP, rates and routes of metabolism were identical in the wild type and knockouts, with TCP-glucoside a major product in both cases. Similar differential effects on the metabolism of DCA and TCP were obtained in whole plant studies with wild type and ugt72B1 mutants, demonstrating that while UGT72B1 had a central role in metabolizing chloroanilines in Arabidopsis, additional UGTs could compensate for the conjugation of TCP in the knockout. TCP was equally toxic to wild type and ugt72B1 plants, while surprisingly, the knockouts were less sensitive to DCA. From this it was concluded that the glucosylation of DCA may not be as effective in xenobiotic detoxification as bound-residue formation.     

TNT Biotransformation and Detoxification by a Pseudomonas Aeruginosa Strain

Successful microbial-mediated remediation requires transformationpathways that maximize metabolism and minimize the accumulation of toxic products. Pseudomonas aeruginosa strain MX, isolated from munitions-contaminated soil, degraded 100 mg TNT L^-1 in culture medium within 10 h under aerobic conditions. The major TNT products were 2-amino-4,6-dinitrotoluene (2ADNT, primarily in the supernatant) and 2,2'-azoxytoluene (2,2'AZT, primarily in the cell fraction), which accumulated as major products via the intermediate2-hydroxylamino-4,6-dinitrotoluene (2HADNT). The 2HADNT and2,2'AZT were relatively less toxic to the strain than TNT and 2ADNT. Aminodinitrotoluene (ADNT) production increased when yeast extract was added to the medium. While TNT transformation rate was not affected by pH, more HADNTs accumulated at pH 5.0 than at pH 8.0 and AZTs did not accumulate at the lower pH. The appearance of 2,6-diamino-4-nitrotoluene (2,6DANT) and 2,4-diamino-6-nitrotoluene (2,4DANT); dinitrotoluene (DNT) and nitrotoluene (NT); and 3,5-dinitroaniline (3,5DNA) indicated various routes of TNT metabolism and detoxification by P. aeruginosa strain MX.     

3,4-Dichloroaniline is detoxified and exported via different pathways in Arabidopsis and soybean

The metabolic fate of [UL-14C]-3,4-dichloroaniline (DCA) was investigated in Arabidopsis root cultures and soybean plants over a 48 h period following treatment via the root media. DCA was rapidly taken up by both species and metabolised, predominantly to N-malonyl-DCA in soybean and N-glucosyl-DCA in Arabidopsis. Synthesis occurred in the roots and the respective conjugates were largely exported into the culture medium, a smaller proportion being retained within the plant tissue. Once conjugated, the DCA metabolites in the medium were not then readily taken up by roots of either species. The difference in the routes of DCA detoxification in the two plants could be explained partly by the relative activities of the respective conjugating enzymes, soybean containing high DCA-N-malonyltransferase activity, while in Arabidopsis DCA-N-glucosyltransferase activity predominated. A pre-treatment of plants with DCA increased DCA-N-malonyltransferase activity in soybean but not in Arabidopsis, indicating differential regulation of this enzyme in the two plant species. This study demonstrates that DCA can undergo two distinct detoxification mechanisms which both lead to the export of conjugated metabolites from roots into the surrounding medium in contrast to the vacuolar deposition more commonly associated with the metabolism of xenobiotics in plants.     

The Role of Oxophytodienoate Reductases in the Detoxification of the Explosive 2,4,6-Trinitrotoluene by Arabidopsis

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): activation, conjugation, and compartmentation. The proposed route of TNT detoxification follows these phases. The electron-withdrawing properties of the nitro groups of TNT make the aromatic ring electron deficient. This favors reductive transformation reactions in plants, and the TNT molecule is most commonly activated by the reduction of a nitro group to give hydroxylamino and then amino derivatives (Fig. 1, pathway A). Following the introduction of a functional group, more hydrophilic molecules such as Glc are conjugated to the activated TNT molecule (Gandia-Herrero et al., 2008), facilitating transport and subsequent compartmentation or sequestration.Open in a separate windowFigure 1.The transformation of TNT by pentaerythritol tetranitrate reductase. Pathway A shows the transformation of the nitro group to nitroso-dinitrotoluene (NODNT), HADNT, and then ADNT products. Pathway B shows the reduction of the aromatic ring to form hydride and dihydride Meisenheimer complexes, then chemical condensation with HADNT to form diarylamines.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; Schaller and Weiler, 1997; Snape et al., 1997; Basran et al., 1998; French et al., 1998; Blehert et al., 1999; Pak et al., 2000; Fitzpatrick et al., 2003; Williams et al., 2004). Studies also indicate that Old Yellow Enzyme homologs function as antioxidants, detoxifying the breakdown products of lipid peroxidation and other toxic electrophilic compounds (Kohli and Massey, 1998; Williams and Bruce, 2002; Fitzpatrick et al., 2003; Trotter et al., 2006). This oxidative stress could result from exposure to xenobiotics including TNT, wounding, or pathogen attack.Pentaerythritol tetranitrate reductase, an Old Yellow Enzyme homolog isolated from Enterobacter cloacae (Binks et al., 1996), possesses two catalytic activities toward TNT (Fig. 1): nitroreduction of TNT to form hydroxylamino-dinitrotoluene (HADNT) and then amino-dinitrotoluene (ADNT), and aromatic ring reduction of TNT to yield hydride and dihydride (2H⁻-TNT) Meisenheimer TNT adducts (French et al., 1998; Williams et al., 2004). The TNT ring-reduced compounds condense via a nonenzymatic reaction with HADNTs to form diarylamines, with the liberation of nitrite (Wittich et al., 2008). Expression of pentaerythritol tetranitrate reductase in tobacco (Nicotiana tabacum) confers both resistance to, and the ability to transform, TNT (French et al., 1999). OPR1, OPR2, and OPR3 share 43%, 44%, and 36% identity, respectively, with pentaerythritol tetranitrate reductase, and all possess the conserved active site amino acids crucial for TNT transformation by pentaerythritol tetranitrate reductase and other members of the Old Yellow Enzyme family (Snape et al., 1997; French et al., 1998; Khan et al., 2004), suggesting that they are capable of transforming TNT.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.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> glucosyltransferases with activities toward both endogenous and xenobiotic substrates

Arabidopsis thaliana Heynh. harbors UDP-glucose-dependent glucosyltransferase (UGT; EC 2.4.1.-) activities that are able to glucosylate xenobiotic substrates as a crucial step in their detoxification, similar to other plants. However, it has remained elusive whether side-activities of UGTs acting on endogenous substrates could account for that property. Therefore, seven recombinantly expressed A. thaliana enzymes were tested using the phytotoxic xenobiotic model compound 2,4,5-trichlorophenol (TCP) as a substrate. The enzymes were selected from the large Arabidopsis UGT gene family because their previously identified putative endogenous substrates comprised both carboxylic acid, and phenolic and aliphatic hydroxyl moieties as biochemical targets. In addition, UGT75D1, which was shown to accept the endogenous flavonoid kaempferol as a substrate, was included. All enzymes tested, except the sterol-conjugating UGT80A2, glucosylated TCP as a parallel activity. The K(m) values for TCP ranged from 0.059 to 1.25 mM. When tested at saturating concentrations of the native substrates the glucosylation of TCP by the glucose-ester-forming UGT84A1 and UGT84A2 was suppressed by p-coumaric acid and sinapic acid, respectively. In contrast, the activities of UGT72E2 and UGT75D1 toward their phenolic native substrates and the xenobiotic TCP were mutually inhibited. TCP was a competitive inhibitor of sinapyl alcohol glucosylation by UGT72E2. These overlapping in vitro activities suggest cross-talk between the detoxification of xenobiotics and endogenous metabolism at the biochemical level, depending on the presence of competing substrates and enzymes.     

Tolerance to, and uptake and degradation of 2,4,6-trinitrotoluene (TNT) are enhanced by the expression of a bacterial nitroreductase gene in Arabidopsis thaliana

Arabidopsis thaliana was transformed with a gene encoding a nitroreductase (NTR, E.C.1.6.99.7) with activity against a wide range of nitroaromatic compounds. The gene was transferred from Escherichia coli by an Agrobacterium-mediated in planta method. The obtained seeds were sowed to produce T1 plants, and they were assayed for the integration of the transgene in the plant genome. Transgenic plants that were positive with the PCR analysis were self-pollinated to produce T2 generation plants. Seven lines obtained were assayed for the NTR activity. While the non-transformed wild-type plants showed no detectable NTR activity, the enzyme activity of the transgenic plant lines was approx. 20 times higher. Using the line with the highest NTR activity, the phytoremediation characteristics of plants against 2,4,6-trinitrotoluene (TNT) was investigated. While the wild-type plants did not grow in the presence of 0.1 mM TNT, the transgenic plants grew almost normally in this condition. The uptake of TNT by seedlings of transgenic plants increased by 7 to 8 times when theywere floated on TNT solution. HPLC analysis showed that the peak due to TNT taken upinto plant body was much smaller in the transgenic plants as compared with that of the wild type, and that a number of peaks attributable to the degradation products of TNT, including 4-amino-2,6-dinitrotoluene, were detected in the extract from the transgenic plants. This indicates that the expression of bacterial NTR improved the capability of plants to degrade TNT.     

Plant processes important for the transformation and degradation of explosives contaminants

Environmental contamination by explosives is a worldwide problem. Of the 20 energetic compounds, 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) are the most powerful and commonly used. Nitroamines are toxic and considered as possible carcinogens. The toxicity and persistence of nitroamines requires that their fate in the environment be understood and that contaminated soil and groundwater be remediated. This study, written as a minireview, provides further insights for plant processes important for the transformation and degradation of explosives. Plants metabolize TNT and the distribution of the transformation products, conjugates, and bound residues appears to be consistent with the green liver model concept. Metabolism of TNT in plants occurs by reduction as well as by oxidation. Reduction probably plays an important role in the tolerance of plants towards TNT, and, therefore a high nitroreductase capacity may serve as a biochemical criterion for the selection of plant species to remediate TNT. Because the activities and the inducibilities of the oxidative enzymes are far lower than of nitroreductase, reducing processes may predominate. However, oxidation may initiate the route to conjugation and sequestration leading ultimately to detoxification of TNT, and, therefore, particularly the oxidative pathway deserves more study. It is possible that plants metabolize RDX also according to the green liver concept. In the case of plant metabolism of HMX, a conclusion regarding compliance with the green liver concept was not reached due to the limited number of available data.     

AtPDR12 contributes to lead resistance in Arabidopsis

Arabidopsis (Arabidopsis thaliana) contains about 130 ATP-binding cassette (ABC) proteins, which are likely to contribute to the transport of diverse materials, including toxic substances. However, the substrates of ABC transporters remain unknown in most cases. We tested which ABC transporter is involved in detoxification of lead [Pb(II)]. Among the many tested, we found that the message level of only AtPDR12 increased in both shoots and roots of Pb(II)-treated Arabidopsis, suggesting that it may be involved in the detoxification of Pb(II). AtPDR12-knockout plants (atpdr12) were used to further test this possibility. In Pb(II)-containing medium, atpdr12 plants grew less well and had higher Pb contents than those of wild-type plants. In contrast, AtPDR12-overexpressing Arabidopsis plants were more resistant to Pb(II) and had lower Pb contents than wild-type plants. The mutant phenotypes and their Pb contents, as well as the localization of the GFP:AtPDR12 fusion protein at the plasma membrane, suggest that AtPDR12 functions as a pump to exclude Pb(II) and/or Pb(II)-containing toxic compounds from the cytoplasm. Inhibition of glutathione synthesis by addition of buthionine sulfoximine to the growth medium exacerbated the Pb(II)-sensitive phenotype of atpdr12 plants, consistent with a glutathione-dependent detoxification mechanism operating in parallel with an AtPDR12-dependent mechanism. Thus, we propose that AtPDR12 is an ABC transporter that contributes to Pb(II) resistance in Arabidopsis.     

Engineering plants for the phytoremediation of RDX in the presence of the co-contaminating explosive TNT

The explosive compounds hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-trinitrotoluene (TNT) are widespread environmental contaminants commonly found as co-pollutants on military training ranges. TNT is a toxic carcinogen which remains tightly bound to the soil, whereas RDX is highly mobile leaching into groundwater and threatening drinking water supplies. We have engineered Arabidopsis plants that are able to degrade RDX, whilst withstanding the phytotoxicity of TNT. Arabidopsis thaliana (Arabidopsis) was transformed with the bacterial RDX-degrading xplA, and associated reductase xplB, from Rhodococcus rhodochrous strain 11Y, in combination with the TNT-detoxifying nitroreductase (NR), nfsI, from Enterobacter cloacae. Plants expressing XplA, XplB and NR remove RDX from soil leachate and grow on soil contaminated with RDX and TNT at concentrations inhibitory to XplA-only expressing plants. This is the first study to demonstrate the use of transgenic plants to tackle two chemically diverse organic compounds at levels comparable with those found on contaminated training ranges, indicating that this technology is capable of remediating concentrations of RDX found in?situ. In addition, plants expressing XplA and XplB have substantially less RDX available in aerial tissues for herbivory and potential bioaccumulation.     

Potential detoxification of gossypol by UDP-glycosyltransferases in the two Heliothine moth species Helicoverpa armigera and Heliothis virescens

The cotton bollworm Helicoverpa armigera and the tobacco budworm Heliothis virescens are closely related generalist insect herbivores and serious pest species on a number of economically important crop plants including cotton. Even though cotton is well defended by its major defensive compound gossypol, a toxic sesquiterpene dimer, larvae of both species are capable of developing on cotton plants. In spite of severe damage larvae cause on cotton plants, little is known about gossypol detoxification mechanisms in cotton-feeding insects. Here, we detected three monoglycosylated and up to five diglycosylated gossypol isomers in the feces of H. armigera and H. virescens larvae fed on gossypol-supplemented diet. Candidate UDP-glycosyltransferase (UGT) genes of H. armigera were selected by microarray studies and in silico analyses and were functionally expressed in insect cells. In enzymatic assays, we show that UGT41B3 and UGT40D1 are capable of glycosylating gossypol mainly to the diglycosylated gossypol isomer 5 that is characteristic for H. armigera and is absent in H. virescens feces. In conclusion, our results demonstrate that gossypol is partially metabolized by UGTs via glycosylation, which might be a crucial step in gossypol detoxification in generalist herbivores utilizing cotton as host plant.     

Phylogenomic analysis of UDP‐dependent glycosyltransferases provides insights into the evolutionary landscape of glycosylation in plant metabolism

Glycosylated metabolites generated by UDP‐dependent glycosyltransferases (UGTs) play critical roles in plant interactions with the environment as well as human and animal nutrition. The evolution of plant UGTs has previously been explored, but with a limited taxon sampling. In this study, 65 fully sequenced plant genomes were analyzed, and stringent criteria for selection of candidate UGTs were applied to ensure a more comprehensive taxon sampling and reliable sequence inclusion. In addition to revealing the overall evolutionary landscape of plant UGTs, the phylogenomic analysis also resolved the phylogenetic association of UGTs from free‐sporing plants and gymnosperms, and identified an additional UGT group (group R) in seed plants. Furthermore, lineage‐specific expansions and contractions of UGT groups were detected in angiosperms, with the total number of UGTs per genome remaining constant generally. The loss of group Q UGTs in Poales and Brassicales, rather than functional convergence in the group Q containing species, was supported by a gene tree of group Q UGTs sampled from many species, and further corroborated by the absence of group Q homologs on the syntenic chromosomal regions in Arabidopsis thaliana (Brassicales). Branch‐site analyses of the group Q UGT gene tree allowed for identification of branches and amino acid sites that experienced episodic positive selection. The positively selected sites are located on the surface of a representative group Q UGT (PgUGT95B2), away from the active site, suggesting their role in protein folding/stability or protein–protein interactions.