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Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene

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Abstract

The widespread use of atrazine and other s-triazine herbicides to control weeds in agricultural production fields has impacted surface and groundwater in the United States and elsewhere. We previously reported the cloning, sequencing, and expression of six genes involved in the atrazine biodegradation pathway of Pseudomonas sp. strain ADP, which is initiated by atzA, encoding atrazine chlorohydrolase. Here we explored the use of enhanced expression of a modified bacterial atrazine chlorohydrolase, p-AtzA, in transgenic grasses (tall fescue, perennial ryegrass, and switchgrass) and the legume alfalfa for the biodegradation of atrazine. Enhanced expression of p-AtzA was obtained by using combinations of the badnavirus promoter, the maize alcohol dehydrogenase first intron, and the maize ubiquitin promoter. For alfalfa, we used the first intron of the 5′-untranslated region tobacco alcohol dehydrogenase gene and the cassava vein mosaic virus promoter. Resistance of plants to atrazine in agar-based and hydroponic growth assays was correlated with in vivo levels of gene expression and atrazine degradation. The in planta expression of p-atzA enabled transgenic tall fescue to transform atrazine into hydroxyatrazine and other metabolites. Results of our studies highlight the potential use of transgenic plants for bioremediating atrazine in the environment.

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Acknowledgments

This study was supported, in part, from Grants from The Consortium for Plant Biotechnology Research, and Syngenta Crop Protection. We would like to thank Melinda Dornbusch for watering plants in the greenhouse and William Koskinen and Brian Barber for help with HPLC and LC–MS analyses. The mention of a trade-mark, proprietary product or vendor does not constitute a guarantee or warranty of the product by the United States Department of Agriculture (USDA), and does not imply its approval to the exclusion of other products and vendors that might also be suitable.

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  1. Andrew W. Vail

    Present address: , 3M Corporate Headquarters, 3M Center, St. Paul, MN, 55144-1000, USA

  2. Hirotaka Uefuji

    Present address: Jectas Innovators Company Ltd, University of the Ryukyus, Okinawa, 903-0129, Japan

Authors and Affiliations

  1. BioTechnology Institute, University of Minnesota, 1479 Gortner Ave., 140 Gortner Labs, St. Paul, MN, 55108, USA

    Andrew W. Vail, Ping Wang, Hirotaka Uefuji, Lawrence P. Wackett & Michael J. Sadowsky

  2. USDA-ARS-Plant Science Research Unit, St. Paul, MN, USA

    Deborah A. Samac & Carroll P. Vance

  3. Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, MN, USA

    Lawrence P. Wackett

  4. Department of Soil, Water, and Climate, University of Minnesota, St. Paul, MN, USA

    Michael J. Sadowsky

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Corresponding author

Correspondence to Michael J. Sadowsky.

Electronic supplementary material

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11248_2014_9851_MOESM1_ESM.tif

Construction of vectors pSAM1b (A) and pUAM1b (B). These vectors were usedin biolistic-mediated plant transformation of tall fescue (TIFF 108 kb)

11248_2014_9851_MOESM2_ESM.tif

Construction of vectors pSAM1a (A) and pUAM1a (B). These vectors were used inAgrobacterium-mediated plant transformation of perennial ryegrass, switchgrass, and tall fescue (TIFF 117 kb)

11248_2014_9851_MOESM3_ESM.tif

Construction of vector pPW1Plus. This vector was used in Agrobacteriummediatedplant transformation of alfalfa (TIFF 83 kb)

11248_2014_9851_MOESM4_ESM.tif

In vitro activity of p-AtzA from cell-free extracts from leaf, stem, and root samplesof transgenic line AP-44 and wild-type (WT) alfalfa. Atrazine dechlorination was analyzed using14C-UL-ring-atrazine. Lanes: (1) control without cell extract; (2) cell-free extract from E. coli(pMD4); (3-5) cell-free extracts from WT alfalfa leaves, stems, and roots, respectively; (6-8) cellfree-extracts from AP-44 leaves, stems, and roots, respectively (TIFF 152 kb)

Supplementary material 5 (DOCX 14 kb)

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Vail, A.W., Wang, P., Uefuji, H. et al. Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene. Transgenic Res 24, 475–488 (2015). https://doi.org/10.1007/s11248-014-9851-7

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