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Polyamine Oxidase is the Source of Hydrogen Peroxide in Plants During the Hypersensitive Response

Polyamine Oxidase is the Source of Hydrogen Peroxide in Plants During the Hypersensitive Response. Savannah A. Benally, Glenn D. Kuehn. Polyamines. Polyamines are aliphatic cations that can occur freely or in conjugated states Uncommon: E.g. Homospermidine ,

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Polyamine Oxidase is the Source of Hydrogen Peroxide in Plants During the Hypersensitive Response

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  1. Polyamine Oxidase is the Source of Hydrogen Peroxide in Plants During the Hypersensitive Response Savannah A. Benally, Glenn D. Kuehn

  2. Polyamines • Polyamines are aliphatic cations that can occur freely or in conjugated states • Uncommon: E.g. Homospermidine, norspermidine, and norspermine • Common: Putrescine, Spermidine, and Spermine

  3. Polyamines • Free polyamines interact electrostatically with polyanionic macromolecules: • Stabilize DNA • DNA-protein interactions • Also interact with other acidic substrates (membrane phospholipids)

  4. The Roles of Polyamines in Plants • Cell division/Embryogenesis • Root Formation • Plant Hormone Regulation • Senescence • Plant Defense Responses

  5. Pathogens Induce Plant Defense Responses • Known as the hypersensitive response (HR) • Characterized by an “oxidative burst” • Under pathogen-induced stress conditions, plants have the capability of inducing defense responses (Lamb et. al., 1997)

  6. The HR Generates Reactive Oxygen Species

  7. ROS Are Molecular Oxygen Derivatives

  8. ROS Are Implicated in Various Events • Hydrogen peroxide possesses many roles: • Direct protective agent • Cell-wall oxidative cross-linking • Induces apoptosis • Guanine → 8-oxoguanine • Caspase-3 • Diffusible, influencing neighboring cells

  9. Pathogens are not the Sole Elicitors of Oxidative Bursts

  10. Major Sources of H2O2From Metabolic Reactions • In Plants • Photosynthetic Electron Transport Chain • NADPH Oxidases • Flavin-Oxidase • Oxalate Oxidase • Amine Oxidases

  11. The Role of Polyamines in Plant Defense Responses • Polyamine catabolism has been correlated with H2O2 induced defense responses (Schipper et. al., 2000) • Polyamine oxidase (PAO) is a key enzyme involved in polyamine catabolism

  12. PAO is Responsible for the Catabolism of Polyamines Spermidine

  13. Research Hypothesis Through the catabolism of polyamines, PAO is the main contributor to the elevated levels of hydrogen peroxide observed during the oxidative burst in plants

  14. Research Objectives • Down-regulate PAO using siRNA • Should result in decreased PAO activity, H2O2, and PAO mRNA levels • Demonstrate siRNA can be introduced into intact plants using a basic infiltration method

  15. Method of Infiltration Infiltrate siRNA at two sites

  16. Discussion • Preliminary results indicate a successful down-regulation of PAO by PAO siRNA • Levels of H2O2 also decreased in PAO siRNA infiltrated leaves, indicating that the H2O2 produced by PAO significantly contributes to the oxidative burst of HR • The proposed infiltration technique may serve as a more efficient way to introduce siRNA into plant model systems

  17. Further Research • RT-PCR to confirm knockdown of PAO mRNA

  18. Acknowledgements • Dr. Glenn D. Kuehn • Kuehn Lab (Kristina, Antonia, Salvador, Alex, and Murphy) • Dr. Champa Sengupta-Gopalan • Dr. Suman Bagga

  19. Polyamine Oxidase is the Source of Hydrogen Peroxide in Plants During the Hypersensitive Response Savannah A. Benally, Glenn D. Kuehn

  20. Initial Research • Oats were grown and treated with α-picolinic acid at six days of age • Leaves were collected hourly for 24 hours • Assayed for • Protein concentrations: Bradford assay • PAO activity: Colorimetric assay • Hydrogen peroxide: Chemiluminescent assay

  21. Determining Most Effective PAO siRNA PAO zero sense - 5’-AAT GTA CAC GGC CAT GTC GAA-3’ PAO zero anti sense 5’-AAT TCG ACA TGG CCG TGT ACA-3’ PAOa sense - 5’-AAA CCC TTC TCC TTG TAG ATG-3’ PAOa anti sense - 5’-AAC ATC TAC AAG GAG AAG GGT-3’ PAOb sense - 5’-AAC ATC TTC TTC TGA GCA CAG-3’ PAOb anti sense - 5’-AAC TGT GCT CAG AAG AAG ATC-3’ PAOc sense - 5’-AAG ACG TGG TAT TTG CAC ATC-3’ PAOc anti sense - 5’-AAG ATG TGC AAA TAC CAC GTC-3’ PAOd sense - 5’-AAC TGA AAC CTC TAC TCA TAC-3’ PAOd anti sense - 5’-AAG TAT GAG TAG AGG TTT CAG-3’

  22. Determining Ideal PAO siRNA Concentration • Optimal concentration for down-regulating PAO using infiltration was determined (based on PAO activity levels) • PAO siRNA vs. Random siRNA • Three concentrations: 0.1 M, 0.5 M, and 1.0 M

  23. Percent Decrease in PAO Activity

  24. Does Down-regulation of PAO Affect Hydrogen Peroxide Levels? • Oat seedlings were infiltrated with: • PAO siRNA (1 µM): Sprayed with picolinic acid • Random siRNA (1 µM): Sprayed with picolinic acid • Control oat seedlings were not infiltrated and not sprayed with picolinic acid

  25. Does Down-regulation of PAO Affect Hydrogen Peroxide Levels? • Crude extract was assayed for: • Protein concentration • PAO activity • Hydrogen peroxides levels

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