Arabidopsis Glutathione Transferases U24 and U25 Exhibit a Range of Detoxification Activities with the Environmental Pollutant and Explosive, 2,4,6-Trinitrotoluene      

Abstract

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...

Phytoremediation of Explosive-Contaminated Soils

“…One of the three processes reported was the direct conjugation of TNT through nucleophilic substitution of the nitro group to form 2-glutathionyl-4,6-dinitrotoluene. The other two products were C-glutathionylated HADNT isomers at the methyl group of TNT [42] (Fig. 1).…”

“…Collectively, the results do not indicate the involvement of glutathione conjugates. However, glutathione conjugates were identified in a recent study investigating the GSTs, GSTU24, and GSTU25 [42]. These GSTs were chosen for their high activity to TNT and were studied in transgenic Arabidopsis.…”

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

“…4 Previously, GSTs had been shown to be upregulated in response to TNT Keywords: Arabidopsis thaliana, environmental pollutant, Glutathione transferases, TNT, xenobiotic detoxification, 2, 4, 6-trinitrotoluene treatment, and their involvement in TNT detoxification hypothesized. [8][9][10][11][12] Our study 13 investigated GST-U24 and GST-U25, the 2 Arabidopsis GSTs most highly upregulated in response to TNT under our treatment conditions. We demonstrated that Arabidopsis plants overexpressing GST-U24 or GST-U25 removed more TNT from soil and were more resistant to TNT toxicity than wild type plants, due to the direct glutathionylation of TNT.…”

“…GST-U24 produced predominantly conjugate 1 (C-glutathionylated 4-HADNT), whereas GST-U25 produced conjugates 1, 2 (C-glutathionylated 2-HADNT) and 3 (2-glutathionyl-4,6-dinitrotoluene). 13 Conjugate 3 is of particular interest due to the denitration of TNT; the nitro groups offer stability to the ring through resonance.…”

“…At pH 6.5 to 7.0, production of conjugate 3 by GST-U25 is preferred over the other 2 conjugates. That only conjugate 3 was detected in the root extracts from GST-U25 overexpressing plants fed TNT suggests that the root cytosol pH is favorable for conjugate 3 production 13 . Further studies are now required to investigate whether conjugate 3 is a step closer to the catabolism of TNT into intermediates of general metabolism, or possibly mineralization, of this organic pollutant.…”