2005 Progress Report: Transgenic Plants for Bioremediation of Atrazine and Related Herbicides

EPA Grant Number: R829479C019
Subproject: this is subproject number 019 , established and managed by the Center Director under grant R829479
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program
Center Director: Schumacher, Dorin
Title: Transgenic Plants for Bioremediation of Atrazine and Related Herbicides
Investigators: Sadowsky, Michael J. , Samac, Deborah A. , Vance, Carroll P. , Wackett, Lawrence P.
Institution: University of Minnesota
EPA Project Officer: Lasat, Mitch
Project Period: January 1, 2004 through December 31, 2004 (Extended to December 31, 2005)
Project Period Covered by this Report: January 1, 2005 through December 31,2005
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text |  Recipients Lists
Research Category: Targeted Research , Hazardous Waste/Remediation

Objective:

The objectives of this research project are to: (1) further evaluate transgenic alfalfa, tobacco plants, and other plants transformed by p-atzA for their ability to degrade atrazine and the related compounds transformed by p-atzA; (2) produce transgenic tall fescue, switchgrass, and ryegrass that have the ability to degrade and detoxify atrazine by incorporation of the p-atzA gene using Agrobacterium and biolistic transformation strategies; (3) modify initial vectors used for plant transformation by inclusion of a 5’ untranslated region of alcohol dehydrogenase gene, which has been shown to act as an enhancer of translation; and (4) quantify plant growth and atrazine degradation ability by transformed plant lines growing in soil.

Progress Summary:

Our overall objective is to produce transgenic plants that bioremediate atrazine contaminated soil and soil water, and prevent atrazine movement into waterways. To achieve this objective, we transformed alfalfa, Arabidopsis, and tobacco plants with a bacterial atrazine chlorohydrolase gene and examined transformants for their ability to degrade atrazine. Initial alfalfa transformation studies conducted using wild-type atzA downstream of the CMV 35S promoter indicated that the bacterial gene failed to express in planta. The atrazine chlorohydrolase gene used in the subsequent studies, p-atzA, was, modified from the original wild-type bacterial atzA by changing 359 nucleotides (representing changes to 312 codons) to promote efficient translation, relative to codon usage in alfalfa. The p-atzA also was modified to contain a plant-like consensus sequence, ACC, before the translation start codon, XbaI and BamHI restriction enzyme sites, and a TAA stop codon. The modified atzA gene was directionally cloned downstream of the CsVMV promoter to produce pPW1. Polymerase chain reaction (PCR) analyses doneusing primers specific for p-atzA indicated that 13 of 18 (72%), and 27 of 28 (96%) of T0 tobacco and alfalfa plants, respectively, contained p-atzA. Only two of 10 Arabidopsis seed lines (AR4 and AR10) that were generated from the Agrobacterium-mediated floral-dip transformation method germinated and survived on agar medium containing kanamycin. Both tested positive for p-atzA by PCR. Expression analyses, using real-time polymerase chain reaction (RT-PCR), indicated that 69 percent (26 of 27), and 100 percent (9 of 13), 96 percent (2 of 2) of the transgenic T0 tobacco, alfalfa, and Arabidopsis lines, respectively, expressed p-atzA in planta. Western blot analysis of soluble proteins from tobacco T0 plant lines indicated that the atzA protein was present in crude protein extracts, but in low abundance. The transgenic lines were able to tolerate a significantly higher concentration of atrazine relative to the wild-type parent plants. While the best transgenic T0 lines of tobacco, alfalfa, and Arabidopsis were able to survive and grow in the presence of 15, 10, and 5 µg/mL atrazine, respectively, wild-type tobacco, alfalfa, and Arabidopsis plants survived only in medium containing 0.4, 0.2, and 0.1 µg/mL atrazine, respectively. This represents a 38-, 50-, and 50-fold increase in atrazine tolerance for tobacco, alfalfa, and Arabidopsis, respectively, relative to wild-type plants.

To link the growth responses of transgenic plants to detoxification of atrazine, we investigated whether cell-free extracts obtained from transgenic T1 seedlings from tobacco line P10 plants growing on an agar medium would dechlorinate 14C-UL-ring-atrazine to hydroxyatrazine. Results of thin layer chromatography (TLC) analyses showed that cell-free extracts from leaves, stems, and roots of transgenic tobacco plants were capable of dechlorinating atrazine to hydroxyatrazine. Transgenic tobacco (P9 and P10) and alfalfa (A1 and A3) were grown hydroponically in the presence of 0.5 µg/mL 14C-UL-ring-atrazine, and evaluated for their ability to take-up and dechlorinate atrazine to hydroxyatrazine. Both transgenic alfalfa and tobacco plants had the ability to grow in the presence of 0.5 µg/mL atrazine, whereas the wild-type parent plants died (data not shown). Moreover, TCL and liquid scintillation studies conducted using leaf, stem, and root extracts from hydroponically-grown transgenic tobacco (A1 and A3) and alfalfa (P9 and P10) lines indicated that p-atzA activity was present in all plant samples examined. Densitometric analyses of TLC plates and liquid scintillation counting of extracts and plant growth solutions revealed that hydroxyatrazine accounted for 98.1 ± 0.3, 98.6 ± 0.1, and 82.9 ± 0.3 percent of the accumulated counts in tobacco leaves, stems, and roots, respectively, and 94 ± 1.5, 91.4 ± 0.8, and 85.7 ± 1.4 percent of the accumulated counts in alfalfa leaves, stems, and roots, respectively. Growth of Arabidopsis seeds on agar medium containing atrazine, or spraying plantlets with atrazine following growth in soil, were effective methods for the selection of Arabidopsis transformants following Agrobacterium-mediated transformation with p-atzA, about 68-76 percent of the seeds from transformed lines were capable of germinating and growing in the presence of atrazine, whereas no resistant plants were detected when wild-type seeds were examined.

Future Activities:

Because atrazine degradation in planta is governed by enzymatic activity, the translocation of atrazine into the plant, and its eventual movement to the plant roots, stems, and leaves by transpiration activity, we hypothesize that plants having larger root area and high transpiration rates, such as grasses, will more rapidly remove atrazine from soil and water. To achieve our goals, we will transform grass species with the p-atzA gene using Agrobacterium and biolistic-based transformation systems. Atrazine degradation by transgenic plants will be assessed in laboratory media, in hydroponically gown plant culture and in soils by using high performance liquid chromatography (HPLC) and 14C-atrazine coupled with TLC analyses.


Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other subproject views: All 2 publications 1 publications in selected types All 1 journal articles
Other center views: All 211 publications 48 publications in selected types All 44 journal articles
Type Citation Sub Project Document Sources
Journal Article Wang L, Samac DA, Shapir N, Wackett LP, Vance CP, Olszewski NE, Sadowsky MJ. Biodegradation of atrazine in transgenic plants expressing a modified bacterial atrazine chlorohydrolase (atzA) gene. Plant Biotechnology Journal 2005;3(5):475-486. R829479C019 (2005)
  • Abstract from PubMed
  • Other: USDA
  • Supplemental Keywords:

    sustainable industry, waste, agricultural engineering, bioremediation, environmental engineering, new technology, innovative technology, bioaccumulation, biodegradation, bioenergy, bioengineering, biotechnology, phytoremediation, plant biotechnology, bioremediation, carcinogen, contamination, phytodegradation, pollutant, toxicity,, Scientific Discipline, Waste, TREATMENT/CONTROL, Sustainable Industry/Business, Treatment Technologies, Environmental Chemistry, Technology, Bioremediation, New/Innovative technologies, Environmental Engineering, Agricultural Engineering, plant-based remediation, bioengineering, biodegradation, transgenic plants, biotechnology, plant biotechnology, remediation, atrazine, bioacummulation, phytoremediation

    Relevant Websites:

    http://www.cpbr.org exit EPA

    Progress and Final Reports:

    Original Abstract
  • 2004 Progress Report
  • Final

  • Main Center Abstract and Reports:

    R829479    The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R829479C001 Plant Genes and Agrobacterium T-DNA Integration
    R829479C002 Designing Promoters for Precision Targeting of Gene Expression
    R829479C003 aka R829479C011 Biological Effects of Epoxy Fatty Acids
    R829479C004 Negative Sense Viral Vectors for Improved Expression of Foreign Genes in Insects and Plants
    R829479C005 Development of Novel Plastics From Agricultural Oils
    R829479C006 Conversion of Paper Sludge to Ethanol
    R829479C007 Enhanced Production of Biodegradable Plastics in Plants
    R829479C008 Engineering Design of Stable Immobilized Enzymes for the Hydrolysis and Transesterification of Triglycerides
    R829479C009 Discovery and Evaluation of SNP Variation in Resistance-Gene Analogs and Other Candidate Genes in Cotton
    R829479C010 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals
    R829479C011 Biological Effects of Epoxy Fatty Acids
    R829479C012 High Strength Degradable Plastics From Starch and Poly(lactic acid)
    R829479C013 Development of Herbicide-Tolerant Energy and Biomass Crops
    R829479C014 Identification of Receptors of Bacillus Thuringiensis Toxins in Midguts of the European Corn Borer
    R829479C015 Coordinated Expression of Multiple Anti-Pest Proteins
    R829479C016 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
    R829479C017 Molecular Improvement of an Environmentally Friendly Turfgrass
    R829479C018 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals. II.
    R829479C019 Transgenic Plants for Bioremediation of Atrazine and Related Herbicides
    R829479C020 Root Exudate Biostimulation for Polyaromatic Hydrocarbon Phytoremediation
    R829479C021 Phytoremediation of Heavy Metal Contamination by Metallohistins, a New Class of Plant Metal-Binding Proteins
    R829479C022 Development of Herbicide-Tolerant Energy and Biomass Crops
    R829479C023 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
    R829479C024 Development of Vectors for the Stoichiometric Accumulation of Multiple Proteins in Transgenic Crops
    R829479C025 Chemical Induction of Disease Resistance in Trees
    R829479C026 Development of Herbicide-Tolerant Hardwoods
    R829479C027 Environmentally Superior Soybean Genome Development
    R829479C028 Development of Efficient Methods for the Genetic Transformation of Willow and Cottonwood for Increased Remediation of Pollutants
    R829479C029 Development of Tightly Regulated Ecdysone Receptor-Based Gene Switches for Use in Agriculture
    R829479C030 Engineered Plant Virus Proteins for Biotechnology