Enzymatic Transformations of Chloroacetanilide Herbicides and Explosive Chemicals by Plants: In Vivo and In Vitro Studies

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Thursday, July 1, 2004
Melissa P. Mezzari
Journal Title: 
PhD Thesis

Understanding metabolic routes and catabolic enzymes involved in the transformation of xenobiotics in plants are of great importance to phytoremediation purpose. Studies were conducted to explore transformation reactions of acetochlor (AOC), metolachlor (MOC), 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazine (HMX) in hybrid poplars and Arabidopsis. Here, we report the use of (1) spectrophotometric techniques for enzyme activities; (2) immuno-localization studies using an antibody developed against MOC; (3) confocal microscopy to study the in vivo conjugation reactions with reduced glutathione in root cells of poplar and Arabidopsis; (4) molecular techniques for gene isolation and quantification of three GST isoforms and 12-oxophytodienoate reductase (OPR) in Arabidopsis; (5) modeling of radiolabeled RDX and HMX in poplar cells based on the “green liver” concept. Results from MOC immunolocalization indicate that this compound is found in vascular tissues, which correspond to the localization of the parent compound without further transformations in the cytoplasm of the plant cell. Spectrophotometer assay results support variable induced activities from all enzymes tested in all chemicals. It was also observed that GRs are induced by TNT and RDX, which are enzymes that are thought to be involved in reduction reactions of these explosive compounds. Molecular analysis confirmed the induction of GST and OPR genes in Arabidopsis when exposed to TNT, AOC and MOC. RDX did not show significant increase in gene expression, and therefore GSTs and OPRs are enzymes that may not be involved in the transformation of this compound. In vivo conjugation reactions using multiphoton and confocal microscopy also confirm that glutathione conjugation reactions do not occur in plants exposed to RDX and TNT. Conjugation reactions with glutathione were observed only in plants exposed to AOC and MOC, which is a common detoxification pathway for these chemicals. Studies with three mathematical models imply high correlation between the collected and simulated data from plant culture exposed to RDX and HMX. Results show that the final fate of these compounds is binding to cell wall. The simplest model developed (model I) is the best model description of these particular results.


Mezzari, Melissa Paola. Enzymatic transformations of chloroacetanilide herbicides and explosive chemicals by plants: In vivo and in vitro studies. The University of Iowa, 2004.