The explosive 2,4,6-trinitrotoluene (TNT) is a significant environmental pollutant that is both toxic and recalcitrant to degradation. Phytoremediation is being increasingly proposed as a viable alternative to conventional remediation technologies to clean up explosives-contaminated sites. Despite the potential of this technology, relatively little is known about the innate enzymology of TNT detoxification in plants. To further elucidate this, we used microarray analysis to identify Arabidopsis (Arabidopsis thaliana) genes up-regulated by exposure to TNT and found that the expression of oxophytodienoate reductases (OPRs) increased in response to TNT. The OPRs share similarity with the Old Yellow Enzyme family, bacterial members of which have been shown to transform explosives. The three predominantly expressed forms, OPR1, OPR2, and OPR3, were recombinantly expressed and affinity purified. Subsequent biochemical characterization revealed that all three OPRs are able to transform TNT to yield nitro-reduced TNT derivatives, with OPR1 additionally producing the aromatic ring-reduced products hydride and dihydride Meisenheimer complexes. Arabidopsis plants overexpressing OPR1 removed TNT more quickly from liquid culture, produced increased levels of transformation products, and maintained higher fresh weight biomasses than wild-type plants. In contrast, OPR1,2 RNA interference lines removed less TNT, produced fewer transformation products, and had lower biomasses. When grown on solid medium, two of the three OPR1 lines and all of the OPR2-overexpressing lines exhibited significantly enhanced tolerance to TNT. These data suggest that, in concert with other detoxification mechanisms, OPRs play a physiological role in xenobiotic detoxification.

The explosive 2,4,6-trinitrotoluene (TNT) is a highly toxic and persistent environmental pollutant. Due to the scale of affected areas, one of the most cost-effective and environmentally friendly means of removing explosives pollution could be the use of plants. However, mechanisms of TNT phytotoxicity have been elusive. Here, we reveal that phytotoxicity is caused by reduction of TNT in the mitochondria, forming a nitro radical that reacts with atmospheric oxygen, generating reactive superoxide. The reaction is catalyzed by monodehydroascorbate reductase 6 (MDHAR6), with Arabidopsis deficient in MDHAR6 displaying enhanced TNT tolerance. This discovery will contribute toward the remediation of contaminated sites. Moreover, in an environment of increasing herbicide resistance, with a shortage in new herbicide classes, our findings reveal MDHAR6 as a valuable plant-specific target.