The explosive 2,4,6-trinitrotoluene (TNT) is a persistent and toxic pollutant. High levels of TNT and the water-mobile co-pollutant Royal Demolition Explosive (RDX) have accumulated at manufacturing waste sites and military training ranges. Due to the scale of these sites, phytoremediation could be the most cost-effective and environmentally-friendly means of cleaning up the pollution. Plant lines which are both tolerant to, and able to degrade explosives pollution, are therefore being developed.
Prior to this PhD research, it was identified that Arabidopsis thaliana L. (Arabidopsis) plants deficient in MONODEHYDROASCORBATE REDUCTASE 6 (MDHAR6; At1g63940) have hugely enhanced TNT tolerance. In Chapter 3, the means behind this enhanced TNT tolerance is investigated. Enzymatic analysis identified that purified MDHAR6 reduces TNT by one electron, to a TNT nitro radical which autoxidises, generating superoxide. Reactive superoxide can oxidise and damage protein, DNA and lipids. This reaction could also be inhibitory to plant development, due to the futile use of NADH.
In Chapter 4, the organelle-targeting of MDHAR6 is investigated; MDHAR6 is unusual in that dual targeting to mitochondria or plastids appears to be dependent on the transcription start site used. A further understanding of MDHAR6 location would provide useful insight as to the endogenous role of this enzyme. Preliminary experiments indicate that MDHAR6 is more highly expressed in roots than leaves, and that a previously undescribed third transcription start site is dominant, encoding plastid-targeted MDHAR6.
The induction of detoxification genes following TNT treatment is explored in Chapter 5; the profile of detoxification genes induced following TNT treatment is similar to that following phytoprostane treatment, which requires class II TGACG-binding (TGA) factors. It is identified that induction of detoxification genes following TNT treatment also requires class II TGA factors, but the induction is not mediated by phytoprostanes.
