The ability of plants to uptake and transform 2,4,6-trinitrotoluene (TNT) was investigated using the aquatic plant Myriophyllum spicatum, axenic Myriophyllum aquaticum, and Catharanthus roseus hairy root cultures. Studies demonstrate that Myriophyllum, with or without its periphyton, and C. roseus transform TNT. Low concentrations of aminated nitrotoluenes (2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene) were observed in the extracellular medium and tissue extracts. Primary products of transformation were not identified, and mineralization was not observed. Mass balances demonstrate that a large percentage of the unknown TNT transformation products were associated with the plant. This fraction could be at least partially recovered from the plant tissue with methanol extraction. A soluble fraction was also present in the medium. The absence of periphyton had little impact on the results observed. Medium concentrations of 4-amino-2,6-dinitrotoluene were greater in systems in which the periphyton was not removed. For the first time, the intrinsic ability of plants to transform TNT has been confirmed. The formation of soluble, uncharacterized transformation products is a concern for potential phytoremediation applications.

This paper examines processes in plants for the formation of fate products of TNT beyond its aminated reduction products, 2-amino-4,6-dinitrotoluene and 4-amino-2,6dinitrotoluene. TNT metabolites were isolated and characterized in combination with temporal analyses of product profiles and 14 C distribution, in microbe-free, axenic root cultures of Catharanthus roseus. Four unique TNTderived compounds were isolated. Using evidence from 1 H NMR, mass spectroscopy, HPLC, acid hydrolysis, and enzymatic hydrolysis with β-glucuronidase and β-glucosidase, they were established as conjugates formed by reactions of the amine groups of 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene. From the mass spectral evidence, at least a six-carbon unit from the plant intracellular millieu was involved in conjugate formation. Mass balance analysis indicated that, by 75 h after TNT amendment of the initial TNT radiolabel, extractable conjugates comprised 22%, bound residues comprised another 29%, 2-amino-4,6dinitrotoluene was 4%, and the rest remained unidentified. Isolates from TNT-amended roots versus monoaminodinitrotoluene-amended roots were not identical, suggesting numerous possible outcomes for the plant-based conjugation of 2-amino-4,6-dinitrotoluene or 4-amino-2,6-dinitrotoluene. This study is the first direct evidence for the involvement of the primary reduction products of TNTs2-amino-4,6dinitrotoluene and 4-amino-2,6-dinitrotoluenesin conjugation processes in plant detoxification of TNT.

Hairy root cultures of Catharanthus roseus were established by infection of seedlings with Agrobacterium rhizogenes 15834. Approximately 150 transformants from four different. C. roseus cultivars were screened for desirable traits in growth and indole alkaloid production. Five hairy root clones grew well in liquid culture with doubling times similar to those reported for cell suspensions. Fast growing clones had similar morphologies, characterized by thin, straight, and regular branches with thin tips. The levels of key alkaloids, ajmalicine, serpentine, and catharanthine, in these five clones, also compared well with literature data from cell suspensions, yet HPLC and GC-MS data indicate the presence of vindoline in two clones at levels over three orders of magnitude greater than the minute amounts reported in cell culture. These results suggest that further optimization may result in hairy roots as a potential source of vindoline and catharanthine, the two monomers necessary to synthesize that antineoplastic drug, vinblastine.

Studies were conducted to isolate and identify polar and oxygen-sensitive intermediates of 2,4,6-trinitrotoluene (TNT) transformation by Clostridium acetobutylicum. Studies conducted in anaerobic cell extracts demonstrated that a polar product formed from the transformation of 2,4-dihydroxylamino-6-nitrotoluene by a mechanism known as the Bamberger rearrangement. The product was stabilized by derivatization with acetic anhydride, and the structure was confirmed by mass spectroscopy, 1H NMR, and IR spectroscopy techniques. The reaction occurred in the presence of cell extract and H2 but did not occur in cell extract-free controls. From spectroscopic data, the product of 2,4-dihydroxylamino-6-nitrotoluene rearrangement was identified as either 2-amino-4-hydroxylamino-5-hydroxyl- 6-nitrotoluene (4-amino-6-hydroxylamino-3-methyl-2-nitrophenol) or 2-hydroxylamino-4-amino-5-hydroxyl-6-nitrotoluene (6-amino-4-hydroxylamino-3-methyl-2-nitrophenol). Acid-catalyzed rearrangement of 2,4-dihydroxylamino-6-nitrotoluene resulted in a single product, which after derivatization, was identical to a derivatized product from cell extracts. Acid-catalyzed Bamberger rearrangement oc curs with the hydroxyl addition para to the participating hydroxylamine, indicating that the 2-amino-4-hydroxylamino-5-hydroxyl-6-nitrotoluene (4-amino-6-hydroxylamino-3-methyl-2-nitrophenol) was the product isolated form cell extracts. This product was also confirmed in whole cell systems that had been fed TNT. Following derivatization of the culture broth, a product was isolated that was identical to those isolated from crude cell extracts and acid catalysis experiments.

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