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Title:   Author  item
                                            WANG, LIN - UNIVERSITY OF MINNESOTAitem
                                            Samac, Deborah

                                                         - *Debby*item
                                            SHAPIR, NIR - UNIVERSITY OF MINNESOTAitem
                                            WACKETT, LAWRENCE - UNIVERSITY OF MINNESOTAitem
                                            Vance, Carrollitem
                                            OLSZEWSKI, NEIL - UNIVERSITY OF MINNESOTAitem
                                            SADOWSKY, MICHAEL - UNIVERSITY OF MINNESOTA
  Submitted to: Plant Biotechnology Journal
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 4/18/2005
                                    Publication  Date: 9/1/2005
                                    Citation: Wang, L., Samac, D.A., Shapir, N., Wackett, L.P., Vance, C.P., Olszewski, N.E., Sadowsky, M.J. 2005. Biodegradation of atrazine in transgenic plants expressing a modified bacterial atrazine chlorohydrolase (atzA) gene. Plant Biotechnology Journal. 3:475-486.Interpretive Summary: Weed control in corn, sorghum, and sugarcane fields relies heavily on the use of herbicides, particularly on the herbicide atrazine. Approximately 76-100 million pounds of atrazine are used annually in the United States for weed control. Atrazine can enter ground and surface water through accidental spills or by over-use and can persist in the environment because it degrades relatively slowly. Bacteria that degrade atrazine have been previously identified and the genes for enzymes to breakdown atrazine into non-herbicidal substances have been isolated from these bacteria. Using a series of six enzymes, the bacteria break down atrazine completely into its mineral components. We modified the first gene in this enzymatic pathway so that it can be used by plants to make an active atrazine-degrading enzyme. We found that all three plant species we developed with this gene, alfalfa, tobacco, and Arabidopsis, can use the gene effectively. In particular, alfalfa and tobacco plants with the modified bacterial gene took up atrazine and degraded it into the expected non-herbicidal compound within the plants. Tobacco plants with the modified bacterial gene had 38 times greater tolerance to atrazine than normal tobacco plants, and alfalfa plants with the gene had 50 times greater tolerance than normal alfalfa plants. Because these plants can withstand 5-30 times the normal application rate of atrazine, they could be used to clean up soil and water contaminated with atrazine. Such a remediation strategy, using plants to remove environmental contaminants, is more cost-effective and less environmentally damaging than current recommended remediation practices.
                                Technical Abstract: Atrazine is one of the most widely used herbicides for the control of broadleaf weeds in corn, sorghum, and sugarcane. The bacterial gene atzA encodes atrazine chlorohydrolase, the first enzyme in a six-step bacterial pathway leading to the complete mineralization of the herbicide atrazine in gram-negative and some gram-positive soil bacteria. AtzA catalyzes the hydrolytic dechlorination of atrazine to hydroxyatrazine, which is non-herbicidal. In the present study we investigated the potential use of transgenic plants expressing atzA to take-up, degrade, and detoxify atrazine. Three plant species, alfalfa (Medicago sativa), Arabidopsis thaliana, and tobacco (Nicotiana tabacum), were subjected to Agrobacterium tumefaciens-mediated transformation using a modified bacterial atrazine chlorohydrolase gene, p-atzA, whose expression was under control of the cassava vein mosaic virus (CsVMV) promoter. Reverse transcription-PCR indicated that transgenic plants of all three species expressed p-atzA. Transgenic plants of each species grew over a wide range of atrazine concentrations, whereas control, untransfomed plants were killed at much lower concentrations. Thin layer chromatographic analyses indicated that in planta expression of p-atzA resulted in the production of hydroxyatrazine. Hydroponically-grown transgenic tobacco and alfalfa lines took-up atrazine from the growth medium and degraded the herbicide to hydroxyatrazine in leaves, stems, and roots. Our work suggests that the in planta expression of p-atzA may be useful for developing plants for phytoremediation of atrazine-contaminated soils and soil-water.


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