TNT phytotransformation in native and genetically modified species: significance of aromatic hydroxylamines in the metabolic pathway

The presence and role of hydroxylamines in TNT transformation were ascertained in two axenic (microbe-free) hydroponic plant systems through high TNT feeding experiments and the use of different analytical detection schemes. Hydroxylamines were formed within 10 hours of TNT addition, and usually disappeared within 20 hours, displaying a high turnover rate due to their instability and high reactivity. Hydroxylamines were shown to add biomolecules to their functional group and thereby directly form conjugates. The 4-isomer of the hydroxylamine was detected in very high levels (up to 30% of initial TNT added) in a transgenic tobacco system that had a bacterial nitroreductase inserted into its genome. It was shown that the bacterial enzyme was responsible for the formation of hydroxylamine, which disappeared rapidly from the media. These transgenic tobacco seedlings transformed TNT efficiently and were more resistant to high levels of TNT than the wild-type seedlings. A radiolabeled TNT mass balance was completed on Arabidopsis to delineate all branches of the TNT transformation pathway. It was observed that TNT ended up as intracellular-bounds, polymerized and bound to the plant biomass. The proportion of bounds reached greater than 80% by 168 hours representing complete TNT transformation. Other aspects of the Arabidopsis TNT transformation were also probed, and some specific characteristics of the pathway such as preference for 4-substituted metabolites were observed. Pseudo-first order rate constant estimates were obtained for various branches of the transformation pathway to reveal the rate-limiting steps of TNT transformation. Arabidopsis mutants were also analyzed to detect if their mutations had altered their metabolite profile. These mutants had demonstrated resistance to TNT toxicity in mutant library screening studies. The mutants were found to have greater resistance to TNT than wild-type seedlings, but did not appear to possess any specific advantages in the rate of TNT transformation. This seemed to indicate that the mutations did not target the TNT transformation biochemical pathway; rather they allowed these mutants to be more resistant to TNT. Based on all these experiments, a better understanding of TNT metabolism and the role of its transformation pathway in improving rates of phytoremediation have been obtained