2005 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, 2004 through September 30, 2005
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:

This project is a follow-on to R829479C013. The objectives of this research project are the production of dicamba-tolerant soybean plants and characterization of the dicamba O-demethylase enzyme responsible for the degradation of dicamba by Pseudomonas maltophilia, strain DI-6.

Progress Summary:

Previously, we had isolated the dicamba O-demethylase from P. maltophilia, strain DI-6, and performed initial enzyme characterization. This led to the discovery that the enzyme was composed of three separate components, an oxygenase (now named dicamba monooxygenase, DMO), a ferredoxin, and a reductase. We demonstrated requirements for dicamba, oxygen, nicotinamide adenine dinucleotide (NADH), amd Mg2+ for the reaction in which dicamba is converted into the herbicidally inactive compound, 3,6-dichlorosalicylic acid (DCSA). Each of the three components was purified to near-homogeneity and subjected to amino acid sequence analysis by Edman degradation procedures. The peptide sequences obtained allowed for preparation of degenerate oligonucleotide probes, which were used to select bacterial clones carrying each of the genes encoding the three dicamba O-demethylase components.

Each of the genes encoding the three components of dicamba O-demethylase were genetically engineered for expression in higher plants. Agrobacterium tumefaciens-based transformation protocols were utilized to place the genes in regenerated, transgenic tobacco plants. Spraying the transgenic plants with dicamba revealed that only the presence of the genetically modified DMO gene was needed to provide plants with protection against treatment with dicamba. Further analyses demonstrated that high levels of tolerance to dicamba could be obtained. That is, while control, nontransgenic tobacco plants were killed quickly after treatment with dicamba at 0.5 lb/acre. Several transgenic tobacco plants expressing the genetically engineered DMO gene were tolerant to treatments with at least 5 lbs/acre (i.e., 10 times the rate used by farmers to kill broadleaf weeds in their field). These experiments were repeated using tomatoes. Results were nearly identical to those obtained with tobacco.

The next step was to determine if the modified DMO gene could be inserted into soybean plants and provide this valuable crop with resistance to dicamba. In brief, these experiments were successful. Several lines of transgenic soybean plants fully tolerant to levels of dicamba used by farmers were obtained. Plants were tested under field conditions where they also showed high levels of tolerance to treatment with dicamba.

We also have moved forward with analysis of the biochemical properties of DMO and its associated ferredoxin and reductase. These studies have demonstrated that DMO is a member of the Rieske nonheme-iron monooxygenase family of enzymes. The kinetic and physical properties of DMO, ferredoxin and reductase components have been determined. Data regarding the characterization of the three enzymatic components of dicamba O-demethylase have been published along with a manuscript describing the cloning and characterization of the genes encoding three components of dicamba O-demethylase.

Future Activities:

We will continue our characterization of the enzymatic activities of the three components of dicamba O-demethylase. Likewise, we will endeavor to make changes to the genetically engineered DMO gene to determine if better enzymatic activity and herbicide tolerance can be obtained in transgenic plants.


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:

    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, heavy metal, environmental cleanup, dicamba, dicamba O-demethylase, dicamba monooxygenase, DMO, transgenic plants, dicamba ­tolerance,, 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

    Relevant Websites:

    http://www.cpbr.org Exit

    Progress and Final Reports:

    Original Abstract
  • 2006 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