2006 Progress Report: Development of Herbicide-Tolerant Energy and Biomass Crops

EPA Grant Number: R829479C022
Subproject: this is subproject number 022 , 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: Development of Herbicide-Tolerant Energy and Biomass Crops
Investigators: Weeks, Donald P.
Institution: University of Nebraska at Lincoln
EPA Project Officer: Lasat, Mitch
Project Period: October 1, 2004 through September 30, 2007 (Extended to December 31, 2007)
Project Period Covered by this Report: October 1, 2005 through September 30, 2006
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text |  Recipients Lists
Research Category: Hazardous Waste/Remediation , Targeted Research

Objective:

The objectives are:

  1. Genetically engineer biomass and energy crops for resistance to the herbicide, dicamba.
  2. Characterize the biochemical and molecular basis for dicamba resistance.
  3. Conduct a detailed analysis of dicamba O-demethylase and its biochemistry.
  4. Provide undergraduate research training through the SURE program.

Progress Summary:

Genetically Engineer Biomass and Energy Crops for Resistance to the Herbicide, Dicamba

We have placed a genetically engineered version of the oxygenase component of dicamba O-demethylase, dicamba monooxygenase (DMO), i n several broadleaf plants and crops, including soybean plants. All of these transgenic plants have displayed significant levels of tolerance (resistance) to treatment with dicamba at concentrations well above those used in normal agricultural practices. We now have 3 years of field test data demonstrating that the dicamba resistance trait is genetically stable and that there is no loss of resistance over time. Likewise, the agronomic performance of the DMO-containing plants has been equivalent to that of nontransgenic plants of the same variety.

Characterize the Biochemical and Molecular Basis for Dicamba Resistance and Conduct a Detailed Analysis of Dicamba O-Demethylase and Its Biochemistry

We have overproduced DMO in Escherichia coli cells and purified large quantities of the enzyme to homogeneity. This has allowed us to carry out extensive biochemical characterizations including enzyme kinetics with a variety of potential substrates and/or inhibitors. In addition, we have been successful in obtaining crystals of DMO that diffract well in initial studies with an X-ray defractometer. Complete structural analyses of the DMO enzyme are planned. These should allow us to define the substrate binding pocket, the active site of the enzyme at which dicamba is converted to 3,6-dichlorosalicylic acid (DCSA) and, hopefully, gain knowledge regarding the mechanisms of oxygen activation and its interaction with the O-methyl group of dicamba.

Site-directed mutants of the DMO molecule have been produced and analyzed for their abilities to carry out oxidation of the dicamba molecule. Interestingly, mutants with both greater and lesser amounts of enzyme activity have been uncovered and await more complete biochemical characterization.

Provide Undergraduate Research Training Through the SURE Program

We have hosted five undergraduate students (including one b lack student and three female students) in our laboratory during the past year. They have been directly involved in planning, performing, and analyzing experiments in the laboratory. One project in particular should be mentioned. One student has isolated, purified, and cloned DNA segments of the several megaplasmids of Pseudomonas maltophilia, strain DI-6, the bacterium from which the DMO gene was isolated. He has obtained DNA sequence for over 1.7 million base pairs of these megaplasmids. He is now identifying specific genes carried by these megaplasmids and will attempt to reconstruct the entire pathway by which this bacterium metabolizes dicamba and utilizes it as both a sole carbon source and a sole energy source.

Future Activities:

We will continue field trials of energy and biomass crops that contain the dicamba-resistance gene trait to determine their performance characteristics under a variety of environmental conditions.  Likewise, we will test this gene in additional crop species to determine if the performance of the resistance gene is every bit as good as in the plants tested to date.

We will complete the determination of the structure of the DMO enzyme by X-ray crystallography and compare this with the structures of the mutant DMO enzymes we have created by site-directed mutagenesis.  Likewise, we will complete our analyses of the enzymatic properties of our various mutant DMO enzymes.

Predictions and testing of proposed pathways for dicamba degradation will be conducted.  Artificial plasmids containing genes encoding the entire pathway will be constructed to determine if the proposed pathway is correct and to determine if it is sufficiently efficient to allow growth of the host bacterium on dicamba as sole sources of carbon and energy.


Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other subproject views: All 2 publications 2 publications in selected types All 2 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 Chakraborty S, Behrens M, Herman PL, Arendsen AF, Hagen WR, Carlson DL, Wang XZ, Weeks DP. A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6. Archives of Biochemistry and Biophysics 2005;437(1):20-28. R829479C022 (2005)
R829479C022 (2006)
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  • Journal Article Herman PL, Behrens M, Chakraborty S, Chrastil BM, Barycki J, Weeks DP. A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6:gene isolation, characterization, and heterologous expression. Journal of Biological Chemistry2005;280(26):24759-24767. R829479C022 (2005)
    R829479C022 (2006)
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  • Supplemental Keywords:

    dicamba, dicamba O-demethylase, dicamba monooxygenase, DMO, megaplasmid, genetic engineering, soybean, energy, biomass,, RFA, Scientific Discipline, INTERNATIONAL COOPERATION, Waste, TREATMENT/CONTROL, Remediation, Environmental Chemistry, Technology, Hazardous Waste, Biochemistry, Ecology and Ecosystems, Hazardous, hazardous waste treatment, microbial degradation, reaction system, transgenic plants, computational fluid dynamics, biomass, biotechnology, agriculture, phytoremediation

    Progress and Final Reports:

    Original Abstract
  • 2005 Progress Report
  • 2007
  • 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