Energetic materials (RDX, TNT, DNTs) are possible sources of groundwater and surface soil contamination at DoD training and testing sites. RDX, in particular, is more mobile than the other compounds in groundwater. Phytoremediation is an inexpensive, self-sustaining treatment technology that may be suitable for prevention of contamination. Phytoremediation of energetic materials requires basic knowledge of the transformation pathways of the materials for several purposes. Selection of high-performing native plants, engineering plants with enhanced transformation capabilities, identifying the fate of transformation products in the plants, and designing the external variables to operate a more effective phytoremediation process are all dependent on a knowledge base of the genetic structure, enzymatic structure, and biochemical reaction pathways.

The objective of this project was to construct a genetic and biochemical knowledge base for the transformation pathways of RDX, TNT, and DNTs by exploiting the fact that these chemicals are phytotoxic ~5 ppm (TNT) to ~20 ppm (RDX). Specific objectives were to:

1. Screen mutagenized populations of the model plant, Arabidopsis thaliana, to isolate mutants resistant to RDX, TNT, and the DNTs, due to under or over-expression of individual genes.
2. Genetically analyze mutants.
3. Use metabolite analyses to select final mutants and characterize function of mutants.

This project used Arabidopsis thaliana as a model plant system to perform genetic and biochemical studies to assist in the identification of the genes, enzymes, and pathway structure of metabolism of energetic materials. The strategy involved selection of mutants resistant to the energetic compound, and then genetic and metabolic characterization of the mutants. However, as a prerequisite to this strategy, two types of Arabidopsis thaliana mutant libraries, T-DNA insertion and Enhancer (4X)-trap, were generated and screening assays for resistance to TNT and RDX needed to be designed and implemented. Furthermore, uptake and metabolic fate studies of Arabidopsis thaliana exposed to RDX, TNT, and DNTs were necessary as controls to compare with the mutants. Thus, these prerequisite studies alone contributed to the tool and knowledge base on transformation of energetic materials in this genetically powerful plant system.

Genetic and biochemical studies in Arabidopsis of RDX, TNT, and DNTs provide the scientific and engineering communities with a knowledge base needed to better understand plant detoxification of these compounds, thus enabling phytoremediation and natural attenuation processes. Arabidopsis plants appeared able to withstand higher levels of RDX than TNT and the DNTs as evidenced by screening assays, biomass and root assays, and fate and transformation studies in liquid culture. Mineralization of RDX by plant tissue was determined to be plausible, given tissue culture studies and preliminary results in Arabidopsis. Native species with fast transpiration and growth rates potentially could be selected to process RDX without toxicity to the plant. In contrast, TNT and the DNTs were found to be much more phytotoxic, suggesting that transgenic approaches would be necessary for phytoremediation of these compounds at significant levels. Furthermore, the DNTs were not as readily transformed as TNT. Since the similar green-liver pathways appear to be followed, strategies successful for TNT should be tested on these compounds, but they may require increased enzyme activity for detoxification. 

This project developed screening strategies and generated mutants resistant to TNT. These mutants were resistant to TNT but did not differ in the profile of transformation products observed. In other words, it is likely a change in plant metabolism not directly associated with the transformation pathways of TNT occurred; the change was one that reduced phytotoxicity but not the transformation of the parent compound. This type of change has traditionally been very difficult for molecular biologists to study, since in this case, the metabolism of the whole plant needed to be analyzed to determine the change.

The SALK mutant studies implied that resistance phenotype of the activation-tagged mutant lines were not caused by interruption of a gene, but possibly by an enhancement of a gene up-regulated by the 4X 35S enhancer element in the T-DNA. The cloning of the enhancer trap mutants provided a resource for the research community to probe genes upstream or downstream of the insertions sites that may be important in phytotoxicity. 

Furthermore, testing of gene targets identified from gene expression studies also may provide clues to the toxicity response of plants. As shown in the gst mutant studies, genes that were induced in response to xenobiotics from a quantitative gene expression study did not necessarily warrant the in vivo involvement of these genes in the detoxification pathway or in their involvement in tolerance to the xenobiotic by plants. However, several other candidate genes remain to be tested, and can be accomplished with the current availability of Arabidopsis mutants.

The results of this investigation provided a knowledge base in the genetics, pathway structure, and operation of metabolism of energetic materials in plants. Results facilitated selection and/or natural breeding of beneficial plant species, metabolic engineering of transgenic plants for phytoremediation, environmental impact assessment of suspected energetic materials-contaminated media, and design of phytoremediation processes for the treatment of contaminated media.