1. Phytotransformation Pathways of the Energetic Material TNT Murali Subramanian 1, Hangsik Moon 2, Sarah Rollo 1, David Oliver 2, Jacqueline V Shanks 1 1 Dept. of Chemical Engineering; 2 Dept. of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa Background: With over 40 explosive (specifically 2,4,6- trinitrotoluene) contaminated sites in the US, and many more worldwide, there exists, in these areas, a real potential for ecosystem damage and groundwater contamination. Phytoremediation, broadly defined as the application of plants of plant-biomass to clean up pollutant wastes (Figure 1), is an inexpensive, self-sustaining, ecologically harmonious treatment technology that may be suitable for prevention of contamination. Abstract: Plants have shown the capacity to take up and transform TNT, amongst other energetic materials, in the lab and at a field scale. Axenic (microbe-free) plant systems used in these studies have elucidated the unique role of plants in TNT transformation. Based on numerous experiments and analytical compound identification, a pathway depicting TNT transformation has been developed. The pathway conforms to a “green-liver” mode of xenobiotic transformation. C. roseus and Arabidopsis have been studied extensively for TNT transformation metabolite formation and kinetics. Hydroxylamines have been shown to form as the primary metabolites, capable of shifting the transformation pathway in various directions. Radiolabeled 14 C TNT-feed studies in these systems show the eventual binding of a modified parent molecule to the plant, thereby limiting its bioavailability. Recent studies have concentrated on determining the genes and enzymes involved in TNT transformation by screening mutant populations. Figure 3: A proposed TNT transformation pathway describes the uptake and transformation of TNT to various metabolites and conjugates (adapted from Subramanian and Shanks, in “Phytoremediation”, edited by Steve McCutcheon and Jerry Schnoor, p389-408). TNT is completely removed from the system within 120 hours. A combination of HPLC-PDA, MS and NMR were used to identify the structure of the metabolites formed. This pathway, based on the “green liver model”, shows the initial reduction of TNT to the hydroxylamines- 2- hydroxylamine-4,6-dinitrotoluene and its isomer the 4-hydroxylamine-2,6,- dinitrotoluene. The hydroxylamines are subsequently completely reduced to the monoamines- 2-amino-4,6-dinitrotoluene and 4-amino-2,6,-dinitrotoluene. Alternatively, the hydroxylamines can also be oxidized or isomerized to other metabolite branches. These reduced, oxidized and isomerized metabolites are then subjected to a plant conjugation mechanism, wherein plant biomolecules like sugars are attached to their functional groups. These transformation steps serve to reduce the toxicity of the parent TNT and polarize the compound. The conjugated metabolites are then polymerized by the plant enzymes and attached to the plant biomass, often irreversibly. Since these compounds have a final fate of being “bound” to the plant, they are not immediately bioavailable in the ecosystem. Figure 1:Phenomena included in phytoremediation: Phytoextraction (uptake and direct concentration), phytodegradation (uptake and biological transformation), phytovolatization, phytostabilization (immobilization of the contaminant in the soil) and rhizodegradation (action of microbes in the rhizosphere). Figure 2: While field-scale studies are very significant, basic metabolism-scale studies are important too as they help isolate natural mutants and generate hybrids to efficiently remove wastes. In addition, the toxicity and end-points of phytoremediation are fully understood. Figure 5: A complete mass balance for TNT transformation by C. roseus hairy roots, wherein uniformly labeled 14 C TNT was fed to the roots, and the radioactivity levels in the media and biomass were measured. Figure 6. Screening of Arabidopsis mutants to isolate strains with higher resistance to TNT. Contrast in germination of wild-type and mutants at 25 mg/L TNT, three weeks after plating. The mutant shows near complete germination, while the wild-type shows none. Acknowledgements: This research is supported in part by the U.S. Department of Defense, through the Strategic Environmental Research and Development Program (SERDP), Project CU-1319. The authors would also like to acknowledge Dr. Beom-Seok Seo for his initial efforts in the TNT screening procedure. The pathway figure shown in this poster is a cumulative of work by many researchers in this field. Basics Pathway Studies Genetic Studies Figure 4: TNT transformation by 2-week old axenic Arabidopsis seedlings. Extracellular levels of TNT fall rapidly at low to medium concentrations of TNT feed. At higher concentrations, phytotoxic effects exerted by TNT prevent its removal from the system. Figure 7. Comparison of TNT removal capacities of wild-type and mutant Arabidopsis seedlings. 110 mg/L of initial TNT killed the wild-type seedlings, but the mutant did not die. Significant phytotoxic effects were observed on the mutant, however. Metabolite and genetic analysis on the mutant can potentially reveal the genes and enzymes in TNT phytotransformation. GTA LB AAT Figure 8. An example of a map of the T-DNA insertion in an enhancer trap mutant line. T-DNA was inserted in the 4 th exon of the gene in chromosome 1 in this mutant. The arrow indicates the orientation of the gene, and ATG and TAA are the start and stop codons of the gene, respectively. Chromosome 1 RB Gene : At1g49560, myb family transcription factor.