The explosive 2,4,6-trinitrotoluene (TNT) is a major worldwide military pollutant. The presence of this toxic and highly persistent pollutant, particularly at military sites and former manufacturing facilities, presents various health and environmental concerns. Due to the chemically resistant structure of TNT, it has proven to be highly recalcitrant to biodegradation in the environment. Here, we demonstrate the importance of two glutathione transferases (GSTs), GST-U24 and GST-U25, from Arabidopsis (Arabidopsis thaliana) that are specifically up-regulated in response to TNT exposure. To assess the role of GST-U24 and GST-U25, we purified and characterized recombinant forms of both enzymes and demonstrated the formation of three TNT glutathionyl products. Importantly, GST-U25 catalyzed the denitration of TNT to form 2-glutathionyl-4,6-dinitrotoluene, a product that is likely to be more amenable to subsequent biodegradation in the environment. Despite the presence of this biochemical detoxification pathway in plants, physiological concentrations of GST-U24 and GST-U25 result in only a limited innate ability to cope with the levels of TNT found at contaminated sites. We demonstrate that Arabidopsis plants overexpressing GST-U24 and GST-U25 exhibit significantly enhanced ability to withstand and detoxify TNT, properties that could be applied for in planta detoxification of TNT in the field. The overexpressing lines removed significantly more TNT from soil and exhibited a corresponding reduction in glutathione levels when compared with wild-type plants. However, in the absence of TNT, overexpression of these GSTs reduces root and shoot biomass, and although glutathione levels are not affected, this effect has implications for xenobiotic detoxification.The containment and cleanup of environmental pollutants is increasingly both a legal requirement and a responsible action in many developed countries. The most commonly used explosives in military weapons are 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and their continual use, along with production and decommissioning, are progressively contaminating millions of hectares of military land . Bioremediation of TNT is particularly challenging, as the electron-withdrawing properties of the three nitro groups render the aromatic ring particularly resistant to oxidative attack and ring cleavage by microbial oxygenases, which in the environment are normally central to the biodegradation of aromatic compounds (Qasim et al., 2007). In the United States, the Environmental Protection Agency and the military are addressing methods by which toxic TNT and RDX can be contained and detoxified on active military training ranges. One way this problem might be tackled is through the use of plants that are adapted to detoxify these compounds. This could be achieved either by traditional breeding programs or by genetic modification, as has been demonstrated previously for both RDX and TNT (Hannink et al., 2001;Rylott et al., 2006;Jackson et al., 2007).In t...

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Summary 26 The explosive 2,4,6-trinitrotoluene (TNT) is a significant, global environmental 27 pollutant that is both toxic and recalcitrant to degradation. Given the sheer scale, and 28 inaccessible nature of contaminated areas, phytoremediation may be a viable clean-up 29 approach. Here, we have characterised a Drosophila melanogaster (Meigen, 1830) 30 glutathione transferase (DmGSTE6) which has activity towards TNT. 31  Recombinantly-expressed, purified DmGSTE6 produces predominantly 2-32 glutathionyl-4,6-dinitrotoluene, and has a 2.5-fold higher V max , and 5-fold lower K m 33 than previously characterised TNT-active Arabidopsis thaliana (L. 2004), and many contaminated sites in Europe and Asia (Kalderis et al., 2011; Pichtel, 2012). 59)For example, the Werk Tanne former ammunition site in Germany, detonated in 1944, is 60 heavily contaminated with TNT (Eisentraeger et al., 2007). Increased environmental 61 awareness is now compelling governments to identify sites of explosives contamination and 62 put together remediation strategies (Lima et al., 2011). However, a major challenge to 63 cleaning-up these sites is the sheer scale and hazardous nature of many contaminated sites, 64 which rules-out many strategies such as excavation, land fill and off-site treatments, as 65 prohibitively expensive. Phytoremediation may be a viable alternative approach. 66TNT is not readily degraded in the environment due to the electron-withdrawing properties of 67 the three nitro groups of TNT which render the aromatic ring particularly resistant to 68 oxidative attack and ring cleavage (Qasim et al., 2009); the main route of aromatic 69 compounds by soil microbes. Instead microbial flora catalyse a series of reductive reactions, 70 producing predominantly hydroxylamino dinitrotoluene (HADNT) and amino dinitrotoluene 71 (ADNT) and further reduced derivatives (Rylott et al., 2011b). In plants, HADNT and ADNT 72 can be conjugated to sugars, for example, to glucose by UDP-glucosyltransferases (Gandia-73 Herrero et al., 2008), and it has recently been shown that glutathione transferases can 74 conjugate the TNT molecule directly (Gunning et al., 2014; Rylott et al., 2015). Two 75Arabidopsis thaliana (L.) Heynh (Arabidopsis) glutathione transferase (GST) genes, AtGST-76 U24 and AtGST-U25, are specifically upregulated in response to TNT exposure, and their 77 gene products catalyse the formation of three characterised TNT glutathionyl-products 78 (Gunning et al., 2014). The removal of a nitro group in one of the three products, 2-79 glutathionyl-4,6-dinitrotoluene, has the potential to be more amenable to subsequent 80 biodegradation in the environment, a property that could be applied in planta for the 81 5 detoxification of TNT in the field. Expression of AtGST-U24 and AtGST-U25 in Arabidopsis 82 conferred increased ability to take up and detoxify TNT; however, in the absence of TNT, 83 overexpression of these GSTs caused a reduction in plant biomass; an effect with deleterious 84 implications for xenobiotic detoxification...

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The explosive xenobiotic 2,4,6-trinitrotoluene (TNT) is a major worldwide environmental pollutant and its persistence in the environment presents health and environmental concerns. The chemical structure of TNT dictates that biological detoxification pathways follow predominantly reductive transformation of the nitro groups, and as a result, TNT is notoriously recalcitrant to mineralization in the environment. Plant-based technologies to remediate this toxic pollutant rely on a solid understanding of the biochemical detoxification pathways involved. Toward this, two Arabidopsis Tau class glutathione transferases, GSTU24 and GSTU25, have been identified that catalyze the formation of three TNT-glutathionylated conjugates. These two GSTs share 79% identity yet only GSTU25 catalyzes the substitution of a nitro group for sulfur to form 2-glutathionyl-4,6-dinitrotoluene. The production of this compound is of interest because substitution of a nitro group could lead to destabilization of the aromatic ring, enabling subsequent biodegradation. To identify target amino acids within GSTU25 that might be involved in the formation of 2-glutathionyl-4,6-dinitrotoluene, the structure for GSTU25 was determined, in complex with oxidized glutathione, and used to inform site-directed mutagenesis studies. Replacement of five amino acids in GSTU24 established a conjugate profile and activity similar to that found in GSTU25. These findings contribute to the development of plant-based remediation strategies for the detoxification of TNT in the environment.