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Understanding the function of detoxifying enzymes in plants toward xenobiotics is of major importance for phytoremediation applications. In this work, Arabidopsis (Arabidopsis thaliana; ecotype Columbia) seedlings were exposed to 0.6 mM acetochlor (AOC), 2 mM metolachlor (MOC), 0.6 mM 2,4,6-trinitrotoluene (TNT), and 0.3 mM hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). In vivo glutathione (GSH) conjugation reactions of AOC, MOC, RDX, and TNT were studied in root cells using a multiphoton microscope. In situ labeling with monochlorobimane, used as a competitive compound for conjugation reactions with GSH, confirmed that AOC and MOC are conjugated in Arabidopsis cells. Reverse transcription-PCR established the expression profile of glutathione S-transferases (GSTs) and nitroreductases enzymes. Genes selected for this study were AtGSTF2, AtGSTU1, AtGSTU24, and two isoforms of 12-oxophytodienoate reductase (OPR1 and OPR2). The five transcripts tested were induced by all treatments, but RDX resulted in low induction. The mRNA level of AtGSTU24 showed substantial increase for all chemicals (23-fold induction for AOC, 18-fold for MOC, 5-fold for RDX, and 40-fold for TNT). It appears that GSTs are also involved in the conjugation reactions with metabolites of TNT, and to a lesser extent with RDX. Results indicate that OPR2 is involved in plant metabolism of TNT (11-fold induction), and in oxidative stress when exposed to AOC (7-fold), MOC (9-fold), and RDX (2-fold). This study comprises gene expression analysis of Arabidopsis exposed to RDX and AOC, which are considered significant environmental contaminants, and demonstrates the importance of microscopy methods for phytoremediation investigations.Uptake is a necessary prerequisite for close contact between the pollutant and the detoxifying enzymes of plants that are localized in the cytosol of living cells. The presence and activity of this complex array of enzymes is crucial for degradation of chemicals under consideration for phytoremediation (Coleman et al., 1997a). However, very little is known about the exact number of plant enzymes potentially involved in the metabolism of xenobiotic compounds and their capacity to bind and metabolize them (Schalk et al., 1997;Scwitzguebel and Vanek, 2003).The sequential metabolic steps of xenobiotics in plant metabolism are grouped in three main phases known as phase I (conversion), phase II (conjugation), and phase III (compartmentation;Sandermann, 1994;Ohkawa et al., 1999). Initial transformations in phase I include a number of oxidation, reduction, and hydrolysis reactions. Oxidation is the most often observed, while certain organics, such as nitroaromatics, tend to favor reduction processes (Hannink et al., 2002). Conjugations of xenobiotics represent phase II transformation reactions, which are usually mediated by a sugar moiety or tripeptide, such as malonic acid, D-Glc, glutathione, Cys, and other amino acids (Sandermann, 1994). Conjugation reactions result in the formation of products that are generally much less toxic,...

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