Polyamine Oxidase is the Source

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Polyamine Oxidase is the Source of Hydrogen
Peroxide in Plants During the Hypersensitive
Response
Savannah A. Benally, Glenn D. Kuehn
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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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Polyamine Oxidase is the Source of Hydrogen
Peroxide in Plants During the Hypersensitive
Response
Savannah A. Benally, Glenn D. Kuehn
21
Initial Research

• Oats were grown and treated with a-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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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