Phytodetoxification of the explosive 2,4,6-trinitrotoluene

The explosive 2,4,6-trinitrotoluene (TNT) has become an extensive global pollutant over the last 100 years and there are mounting concerns over the toxicity of TNT to biological systems. During World War I and II the toxic effects of TNT were discovered during large scale production, with 475 fatalities and over 17,000 TNT poisoning cases reported at manufacturing facilities. TNT has been shown to severely impact the diversity of soil microbial communities and the establishment of vegetation. In the U.S. alone it is estimated that some 10 million hectares of military land is contaminated with munitions constituents. Unlike similar situations where the environment has become contaminated with toxic agrochemicals and their use subsequently banned, the huge demand for military explosives means that TNT will continue to be manufactured and used globally on a massive scale for the foreseeable future.

Because of the scale of explosives pollution, particularly on military training ranges, there is considerable interest in developing plant based remediation strategies. Plants offer a low cost sustainable solution to containing and remediating explosives pollution. However, a fundamental understanding of the phytotoxicity of explosives, and the enzyme systems plants use to detoxify these compounds, and the rate-limiting steps, are required to enable the development of robust plant systems to contain and remediate explosives pollution effectively in situ. In plants, the majority of TNT remains in the roots, where it inhibits growth and development reducing whole plant biomass.

We have recently discovered that the mitochondria- and plastid-targeted enzyme monodehydroascorbate reductase 6 (MDHAR6) reduces TNT by one electron, forming a nitro radical which reacts with atmospheric oxygen, generating highly reactive superoxide. This futile catalytic cycle only requires catalytic quantities of TNT to continuously generate damaging reactive oxygen species in the mitochondria. We have demonstrated that mutants in MDHAR6 have dramatically enhanced TNT tolerance, and we propose that this reaction accounts almost entirely for TNT toxicity in plants.

The major goal of this research programme is to rigorously and quantitatively establish the fate and effects of TNT on plants. In order to achieve this objective we propose to study mechanisms of TNT toxicity and fully elucidate TNT induced detoxification pathways that include glucosylation, glutathionylation and oxidative activity by cytochromes P450. The fate of the TNT metabolites produced by these enzymes will then be established. We have previously demonstrated that the TNT active glutathione transferase GST-U25 results in the removal of a nitro group which could render the aromatic ring more amenable to biodegradation. We now have detailed structural information on GST-U25 that will allow us to engineer and improve the specificity and activity towards TNT. We hope to use the knowledge gained from this study to develop improved plant systems that will clean up polluted sites and prevent explosives pollution from contaminating water sources.

Our goal is to elucidate the mechanisms of 2,4,6-trinitrotoluene (TNT) detoxification and, to ultimately, use this information to generate improved plant systems for remediating TNT contaminated sites. Specifically:

1) We will use plant liquid culture based techniques to identify TNT metabolites. 13C-Labelled TNT will be used to increase sensitivity for LC/MS analysis.
2) In previous studies we have identified enzyme systems involved in TNT detoxification including nitroreductases (NR), glucosyltransferases (UGT) and glutathione transferases (GST). The further processing and fate of the TNT metabolites is not known nor has the metabolic flux of TNT through these routes been established. We will investigate the sequestration and localisation of these metabolites using engineered plant lines, including lines depleted in TNT active UGTs and GSTs using CRISPR-Cas9 gene editing techniques and NR and GST overexpressing lines. Metabolite profiling of the plant vacuoles will be performed using 13C-TNT and LC/MS.
3) Structural and mechanistic characterisation will be performed on GST-U25 to gain insight into the specificity of U25 for TNT. Rational mutagenesis will be performed to improve TNT-GST performance and increased production of a desirable TNT-gutathionyl product.
4) We will investigate the role of cytochromes P450 in TNT detoxification. In underpinning studies we have demonstrated that three P450s are likely to play a key role in TNT detoxification. The P450s will be deleted using CRISPR-Cas9 and mutant lines tested for increased sensitivity towards TNT. The P450s will be recombinantly expressed and activity towards TNT determined.
5) We have previously shown that the enzyme monodehydroascorbate reductase 6 (MDHAR6) mediates TNT toxicity in plants. We plan to establish an understanding of the physiological roles of all MDHARs in different subcellular locations and how each contributes to TNT toxicity.
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The project will have a very positive environmental impact since it promotes the use of plant systems to remove toxic pollutants from the environment. With population growth placing pressure on existing agricultural soils it is becoming ever-more necessary to remediate contaminated land. Plants offer a low cost sustainable alternative to expensive ex situ remediation processes and much greater public acceptance, especially compared to destructive processes such as removing polluted soil and incinerating it or sending it to landfill. Our work could, therefore, have long-term benefits for the environment and society at large.

Key beneficiaries in the short term will be the military who are interested in the development of plant based systems to prevent off site migration of explosives pollutants and subsequent contamination of water sources. The project will most likely lead to new research grants with the U.K. MoD and the U.S. DoD.

In addition, we are committed to seeing that the impacts of the work are maximised. The PI and researcher Co-I have a good record in terms of public outreach and communication. Beneficiaries include schools and the public through outreach activities, highlighting the use of plants for environmental remediation.

We will use proven processes to protect IP and publish results in scientific journals and at conferences. We will also use existing UK networks (eg the Bioscience KTN, the NNFCC, BBSRC NIBBs) to communicate progress through their events and web-based or printed media. When appropriate, discoveries will be disseminated by the University to the general media through press releases. To ensure professional management of intellectual property, CNAP operates regular IP reviews of all projects. CNAP has an outstanding track record in commercialisation of strategic research through on-going collaborations with companies throughout the Industrial Biotechnology sector. Potential impacts include new patent filings and license agreements that will have a commercial value.

The programme will provide researchers with wide-ranging skills relevant to the establishment of a vibrant industrial biotechnology and innovation-led industrial sector in the U.K. We will encourage the researchers to attend networking meetings organised by the BBSRC's NIBBs and other relevant networks that may be established through the new BBSRC new initiatives in industrial biotechnology.