1. TRANSCRIPTIONAL PROFILING OF ARABIDOPSIS THALIANA ROOT RESPONSES TO MUNITIONS Drew R. Ekman 3, W. Walter Lorenz 2, Alan E. Przybyla 1, N. Lee Wolfe 3, Steven C. McCucteon 3, Jeffrey F.D. Dean 2 1 Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA 2 Daniel B. Warnell School of Forest Resources, University of Georgia, Athens, GA 30602, USA 3 U.S. EPA, National Exposure Research Laboratory, Ecosystems Research Division, Athens, GA 30605, USA Introduction Over most of the last century, manufacturing, processing, and storage of the explosives, 2,4,6- trinitrotoluene (TNT) and hexahydro-1,3,5- trinitro-1,3,5-triazine (RDX), have been responsible for extensive contamination of soil, as well as ground and surface water, throughout the U.S. and Europe. Unlike many other organic compounds possessing nitro- moieties, such as pesticides and various feedstock chemicals, these explosives (Figure 1) are highly resistant to biological degradation, and are thus able to persist in the environment for long periods of time. In recent years it has been observed that certain plants have the ability to remove TNT and RDX from their surroundings, suggesting that plants may offer a potential solution to the environmental contamination problem presented by these compounds. Unfortunately, few of the plants species identified as having the ability to extract munitions from soil are robust enough to provide practical remediation of the extensive contamination common to many of the sites known today. In an attempt to address this limitation, researchers have designed transgenic plants with greatly enhanced abilities to tolerate and remove TNT from their environment (1,2). However, our current understanding of plant metabolism underpinning the processes of uptake and degradation of TNT and RDX is still rudimentary. Further elucidation of these metabolic pathways will facilitate specific engineering of plants for improved removal and degradation of TNT and RDX, as well as for tolerance and persistence in environments where these compounds prove toxic for normal plants. We have employed a functional genomics approach using serial analysis of gene expression (SAGE) (3) to identify the full range of genes in the model plant, Arabidopsis thaliana, that respond to TNT and RDX. Changes in root gene expression were observed in Arabidopsis plants grown in the presence of sub-lethal levels of either TNT or RDX. Identification of the genes responding to these munitions will enhance our understanding of the manner in which plants cope with TNT and RDX in particular, and may extend to other xenobiotic contaminants in the environment. Abstract Serial Analysis of Gene Expression (SAGE) was used to identify Arabidopsis thaliana genes that respond to TNT and RDX exposure. Root tissues from plants grown in sterile liquid medium and exposed to sublethal amounts of the two munitions were used to prepare SAGE libraries, which were characterized to a depth of approximately 30,000 tags each. Transcriptome-level responses to the two munitions were very different. The tag most highly induced by TNT (27-fold greater than in control tissues) represented a glutathione S- transferase transcript, suggesting predominance of a detoxification response. In contrast, the tag most highly induced by RDX represented an NPR1-like protein transcript, which may suggest involvement of a more generalized stress response. A large number of cytochrome P450 transcripts and an ABC transporter transcript were strongly induced in the root tissues treated with TNT, which strongly supports the multiphase-phase model of xenobiotic metabolism that has previously been proposed for plants exposed to this compound. Other tags highly induced by RDX, including those encoding DNAJ-like proteins, vacuolar- processing enzyme, and various transcription factors, clearly demonstrate that different metabolic pathways are brought to bear on these two munitions. To the extent that it facilitates establishment of plants on contaminated sites, better understanding of the genes and pathways involved in resistance and/or degradation of these munitions by plants should help increase the success of future phytoremediation strategies directed at waste munitions and other xenobiotics. This work was supported by a USEPA NNEMS Graduate Fellowship to D.R.E. Although this work was reviewed by EPA and approved for presentation, it may not necessarily reflect official Agency policy. Conclusions This SAGE study strongly supports the multi-phase process proposed for plant metabolism of TNT (5,6). In the first phase of this process, cytochrome P-450 enzymes and mixed function oxidases act to alter specific sites on the toxic compound, making them more amenable to conjugation with glutathione (GSH) or six-carbon sugars in the second phase of detoxification (Figure 2). These conjugation reactions are generally catalyzed by glutathione S- transferases (GST) and UDP:glucosyltransferases. In the third phase of detoxification the conjugated compound is either sequestered in plant storage organelles or secreted to the apoplasm. Finally, in the final phase of processing, the conjugate is rendered inactive through the addition of further substitutions or by degradation processes. The gene expression differences noted between the TNT and control SAGE libraries also point to an oxidative stress response elicited by the presence of toxic concentrations of TNT (Table 4). This stress may be caused by either the parent molecule or any of its reactive, oxidized derivatives. TNT is considered both a mutagen and a carcinogen due in part to its tendency to yield oxidative metabolites that can react and couple with cytosolic or nuclear components. The Arabidopsis transcriptome response to RDX reveals a drastically different metabolic mechanism for dealing with this explosive. In contrast to the TNT responses, the SAGE results did not indicate major involvement of oxidative stress enzymes and cytochrome P450s. Thus, while metabolism of TNT in plants probably requires the multiphase mechanism described previously (Ekman et al. 2003), and as noted in other plant species dosed with a variety of other organic compounds, this may not be the case for RDX. This will be a major consideration in the engineering of plants for phytoremediation of sites contaminated with RDX. For plants to be able to remediate both of these explosives simultaneously (both TNT and RDX are often found together at polluted sites) it may be necessary to introduce a suite of genes specific for metabolizing each of these munitions. Using the insights gained from these studies, we hope to provide a means by which more educated choices can be made in the development of transgenic plants and in the assessment of native plants for suitability in remediation schemes. Methods Arabidopsis Sensitivity to Explosives Arabidopsis plants were grown in sterile liquid Murashige and Skoog medium for two weeks before dosing with different concentrations of TNT or RDX. Seedlings were inspected visually for obvious signs of toxicity—specifically leaf chlorosis and necrosis. Plants growing in 15 mg/L TNT or 150 mg/L RDX displayed a toxic response, but appeared to retain viability. These concentrations were chosen for the SAGE analyses. Tissue Growth and Harvesting Plants were grown for two weeks before amending media with TNT (15 mg/L) or RDX (150 mg/L). Treatments were replicated in multiple flasks, as well as on different days. After 24 hours of exposure, plants were removed from the media and washed in dH2O. Plant roots were immediately excised and frozen in liquid nitrogen for storage prior to RNA extraction. RNA Extraction and Creation of SAGE Libraries Total root RNA was isolated using LiCl precipitation of Chang et. al (4). SAGE libraries were made according to the SAGE Detailed Protocol, Version 1.0c (Velculescu et al., 1997a). References 1.French, C.E., Rosser S.J., Davies, G.J., Nicklin, S., Bruce, N.C. (1999). Biodegradation of explosives by transgenic plants expression pentaerythritol tetranitrate reductase. nature biotechnology 17, 491-494. 2.Hannink, N., Rosser, S.J., French, C.E., Basran, A., Murray, J.A.H., Nicklin, S., & Bruce, N.C. (2001). 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