Plant Biotechnology Journal (2005) 3, pp. 475–486 doi: 10.1111/j.1467-7652.2005.00138.x © 2005 Blackwell Publishing Ltd 475 Blackwell Publishing, Ltd. Biodegradation of atrazine in transgenic plants expressing a modified bacterial atrazine chlorohydrolase ( atzA ) gene Lin Wang 1 , Deborah A. Samac 2,3 , Nir Shapir 3,4,5 , Lawrence P. Wackett 1,3,4 , Carroll P. Vance 3,6 , Neil E. Olszewski 3,7 and Michael J. Sadowsky 1,3,5, * 1 BioTechnology Institute, University of Minnesota, St. Paul, MN 55108, USA 2 USDA-ARS-Plant Science Research Unit and Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108, USA 3 Center for Microbial and Plant Genomics, University of Minnesota, St. Paul, MN 55108, USA 4 Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, MN 55108, USA 5 Department of Soil, Water, and Climate, University of Minnesota, St. Paul, MN 55108, USA 6 Research Unit and Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108, USA 7 Department of Plant Biology, University of Minnesota, St. Paul, MN 55108, USA Summary Atrazine is one of the most widely used herbicides in the USA. Atrazine chlorohydrolase (AtzA), the first enzyme in a six-step pathway leading to the mineralization of atrazine in Gram-negative soil bacteria, catalyses the hydrolytic dechlorination and detoxification of atrazine to hydroxyatrazine. In this study, we investigated the potential use of transgenic plants expressing atzA to take up, dechlorinate and detoxify atrazine. Alfalfa, Arabidopsis thaliana and tobacco were transformed with a modified bacterial atzA gene, p-atzA, under the control of the cassava vein mosaic virus promoter. All transgenic plant species actively expressed p-atzA and grew over a wide range of atrazine concentrations. Thin layer chromatography analyses indicated that in planta expression of p-atzA resulted in the production of hydroxyatrazine. Hydroponically grown transgenic tobacco and alfalfa dechlorinated atrazine to hydroxyatrazine in leaves, stems and roots. Moreover, p-atzA was found to be useful as a conditional-positive selection system to isolate alfalfa and Arabidopsis transformants following Agrobacterium -mediated transformation. Our work suggests that the in planta expression of p -atzA may be useful for the development of plants for the phytoremediation of atrazine-contaminated soils and soil water, and as a marker gene to select for the integration of exogenous DNA into the plant genome. Received 3 January 2005; revised 6 April 2005; accepted 11 April 2005. * Correspondence (fax +612 625 2208; e-mail Sadowsky@umn.edu) Keywords: atrazine, atrazine chlorohydrolase, AtzA, phytoremediation, transgenic plants. Introduction Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5- triazine] is a widely used herbicide for the control of broad- leaf weeds in corn and sugarcane. It is the predominant member of a broad class of s -triazine herbicides, and approx- imately 76 –100 million pounds of atrazine are applied annu- ally in the USA. Atrazine is transformed relatively slowly in the environment (Erickson and Lee, 1989), with an average half- life ranging from 4 to 57 weeks (Anderson and Georgeson, 1989), and has been detected in soils as well as in ground and surface water in several countries (Belluck et al ., 1991). In Pseudomonas sp. strain ADP (Mandelbaum et al ., 1995), as in many other Gram-negative atrazine-degrading soil bacteria (de Souza et al ., 1998a; Sadowsky and Wackett, 2000; Wackett et al ., 2002), atrazine catabolism is initiated by a plasmid-borne atzA gene (Martinez et al ., 2001), encoding atrazine chlorohydrolase (AtzA). AtzA catalyses the hydrolytic dechlorination of atrazine, resulting in the production of hydro- xyatrazine as the first intermediate in the degradation path- way (Figure 1). Hydroxyatrazine is non-herbicidal (Gysin and Knuesli, 1960), more biodegradable (Goswami and Green, 1971), non-toxic and more strongly sorbed to soils than atra- zine (Clay and Koskinen, 1990). AtzA, which has been