Download - Polyamine Oxidase is the Source of Hydrogen Peroxide in Plants During the Hypersensitive Response
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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
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Polyamines
• Free polyamines interact electrostatically with polyanionic macromolecules:
– Stabilize DNA– DNA-protein interactions– Also interact with other acidic substrates
(membrane phospholipids)
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The Roles of Polyamines in Plants
• Cell division/Embryogenesis• Root Formation• Plant Hormone Regulation• Senescence• Plant Defense Responses
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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)
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The HR Generates Reactive Oxygen Species
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ROS Are Molecular Oxygen Derivatives
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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
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Pathogens are not the Sole Elicitors of Oxidative Bursts
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Major Sources of H2O2 From Metabolic Reactions
• In Plants– Photosynthetic Electron Transport Chain– NADPH Oxidases– Flavin-Oxidase– Oxalate Oxidase– Amine Oxidases
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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
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PAO is Responsible for the Catabolism of Polyamines
Spermidine
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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
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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
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Method of Infiltration
Infiltrate siRNA at two sites
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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
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Further Research
• RT-PCR to confirm knockdown of PAO mRNA
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Acknowledgements
• Dr. Glenn D. Kuehn• Kuehn Lab (Kristina, Antonia, Salvador, Alex,
and Murphy)• Dr. Champa Sengupta-Gopalan• Dr. Suman Bagga
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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
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Hydrogen Peroxide and Polyamine Oxidase Activity In Picolonic Acid Treated Avena Sativa(Oat) Plants
Hydrogen Peroxide Activty Control PAO Enzymatic Activity
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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’
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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
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Percent Decrease in PAO Activity
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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
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Does Down-regulation of PAO Affect Hydrogen Peroxide Levels?
• Crude extract was assayed for:– Protein concentration – PAO activity– Hydrogen peroxides levels