Decades of military activity on live-fire training ranges have resulted in the contamination of land and groundwater by recalcitrant high explosives, in particular, 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). While the transformation products of TNT, and TNT itself, are highly toxic, they tend to bind strongly to organic matter and clay in soil and so remain contained at the site of contamination. RDX however, is a major concern due to its high soil mobility. Contamination of RDX on training ranges, and subsequent contamination of groundwater poses a significant threat to drinking water sources. There are currently no cost-effective processes to remediate large areas of contaminated vegetated land on training ranges, or methods to contain the RDX. In previous SERDP-funded studies (ER-1318 & ER-1498), plants were genetically modified through the insertion of two genes, Rhodococcus rhodochrous 11Y (xplA) and Rhodococcus rhodochrous 11Y genome (xplB), to degrade RDX from soils. The objective for this project was to demonstrate and evaluate, through the use of field-scale testing, the ability of XplAB-expressing grasses to contain and degrade RDX from explosives-contaminated soil in situ.

The project team has shown that the unique cytochrome P450 enzyme XplA in combination with its partnering reductase XplB, from the soil bacterium Rhodococcus rhodochrous 11Y, metabolize RDX to produce nitrite, formaldehyde and the ring degradation products, 2-nitro-2,4-diazabutanal (2-nitro-2,4-diazabutanal; aerobic conditions) or methylenedinitramine (MEDINA; anaerobic conditions) (Jackson et al. 2007). In this report, plants genetically modified to express XplAB were assessed for their ability to remediate RDX-impacted soil.

TNT is often a co-contaminant alongside RDX, and is highly toxic, meaning that for the XplAB-expressing plants to remediate RDX, they also need demonstrative resistance to TNT. The bacterial gene nfsI encodes a nitroreductase (NR) which transforms TNT to the relatively unstable and non-toxic hydroxylamino dinitrotoluene (HADNT). This HADNT, and subsequent intermediate amino dinitrotoluene are both then conjugated to sugars (Gandia-Herrero et al. 2008) and 14C-labelling studies reveal subsequent incorporation into plant biomass (Brentner et al. 2010; Sens et al. 1999; Sens et al. 1998).

Initial work was conducted with Arabidopsis, understood genetically, but unsuitable for military land conditions. Thus, switchgrass (Panicum virgatum), western wheatgrass (Pascopyrum smithii) and creeping bentgrass (Agrostis stolonifera) were all transformed with the genes required to express XplA, XplB, and NR.

References

Brentner, L., S. Mukherji, S. Walsh, and J. Schnoor. 2010. Localization of Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-Trinitrotoluene (TNT) in Poplar and Switchgrass Plants using Phosphor Imager Autoradiography. Environmental Pollution, 158(2):470-475.

Gandia-Herrero, F., A. Lorenz, T. Larson, I.A. Graham, D.J. Bowles, E.L. Rylott, and N.C. Bruce. 2008. Detoxification of the Explosive 2,4,6-Trinitrotoluene in Arabidopsis: Discovery of Bifunctional O- and C-glucosyltransferases. The Plant Journal, 56(6):963-974.

Jackson, R.G., E.L. Rylott, D. Fournier, J. Hawari, and N.C. Bruce. 2007. Exploring the Biochemical Properties and Remediation Applications of the Unusual Explosive-degrading P450 System XplA/B. Proceedings of the National Academy of Science of the United State of America, 104(43):16822-16827.

Sens, C., P. Scheidemann, A. Klunk, and D. Werner. 1998. Distribution of14C-TNT and Derivatives in Different Biochemical Compartments of Phaseolus vulgaris. Environmental Science and Pollution Research, 5:202-208.

Sens, C. P. Scheidemann, and D. Werner. 1999. The Distribution of 14C-TNT in Different Biochemical Compartments of the Monocotyledonous Triticum aestivum. Environmental Pollution, 104:113-119.

The performance of the transgenic materials was tested through four quantitative and three qualitative performance objectives. While the project team was not able to produce viable transgenic seeds nor significantly reduce soil levels of RDX, they did meet all the other performance objectives for the project.

Currently, the cost to implement the technology is heavily weighted to the requirements of the Animal Plant Health Inspection Service (APHIS) permit and monitoring process and the production of transgenic transplants. However, these costs will drop significantly if the materials can be deregulated and/or if viable transgenic seeds can be produced.

The main issue preventing the future implementation of XplAB-expressing switchgrass is regulatory, and specifically due to the transgenic nature of the plant. The switchgrass is currently limited to small, monitored testing under APHIS regulation 7 CFR part 340. APHIS may be petitioned to show that a regulated article, such as XplAB-expressing switchgrass, is unlikely to pose a plant pest risk, and therefore should no longer be regulated under the plant pest provisions of the Plant Protection Act or the regulations at 7 CFR part 340. Deregulating the XplAB-expressing switchgrass will allow for both larger scale testing and widespread implementation. (Project Completion 2020).

Johnston, E.J., E.L. Rylott, E. Beynon, A. Lorenz, V. Chechik, and N.C. Bruce. 2015. Monodehydroascorbate Reductase Mediates TNT Toxicity in Plants. Science, 349:1072-1075.

Rylott, E.L., V. Gunning, K. Tzafestas, H. Sparrow, E. J. Johnston, A.S. Bretnall, J.R. Potts, and N.C. Bruce. 2015. Phytodetoxification of the environmental pollutant and explosive 2,4,6-trinitrotoluene. Plant Signaling & Behavior, 10(1):e977714.

Rylott, E.L., E.J. Johnston, and N.C. Bruce. 2015. Advances in Transgenic Phytoremediation Technologies for Persistent Organic Pollutants. Journal of Experimental Botany, 66:6519-33.

Rylott, E.L., and N.C. Bruce. 2019. Right on Target: Using Plants and Microbes to Remediate Explosives. International Journal of Phytoremediation. https://doi.org/10.1080/15226514.2019.1606783.

Sabir, D.K., N. Grosjean, E.L. Rylott, and Bruce, N.C. 2017. Investigating Differences in the Ability of XplA/B-Containing Bacteria to Degrade the Explosive Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). FEMS Microbiology Letters, 364:fnx144. https://doi.org/10.1093/femsle/fnx144.

Tzafestas, K., M.M. Razalan, I. Guylev, A.M.A. Mazari, B. Mannervik, E.L. Rylott, and N.C. Bruce. 2017. Expression of a Drosophila Glutathione Transferase in Arabidopsis Confers the Ability to Detoxify the Environmental Pollutant, and Explosive, 2,4,6-Trinitrotoluene. New Phytologist, 214:294-303.

Tzafestas, K., L. Ahmad, M.P. Dani, G. Grogan, E.L. Rylott, and N.C. Bruce. 2018. Structure-guided Mechanisms Behind the Metabolism of 2,4,6-Trinitrotoluene by Glutathione Transferases U25 and U24 that Lead to Alternate Product Distribution. Frontiers in Plant Science, 9:1846. https://doi.org/10.3389/fpls.2018.01846.

Zhang, L., R. Routsong, Q. Nguyen, E.L. Rylott, N.C. Bruce, and S.E. Strand. 2016. Expression in Grasses of Multiple Transgenes for Degradation of Munitions Compounds on Live-fire Training Ranges. Plant Biotechnology Journal, 15:624-633.

Zhang, L., E.L. Rylott, N.C. Bruce, and S.E. Strand. 2017. Phytodetoxification of TNT by Transplastomic Tobacco (Nicotiana tabacum) Expressing a Bacterial Nitroreductase. Plant Molecular Biology, 95(1-2):99-109.

Zhang, L., E.L. Rylott, N.C. Bruce, and S.E. Strand. 2019. Genetic Modification of Western Wheatgrass (Pascopyrum smithii) for the Phytoremediation of RDX and TNT. Planta, 249:1007-1015.