Polyamine Oxidase is the Source of Hydrogen Peroxide

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

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

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

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

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)

The HR Generates Reactive Oxygen Species

ROS Are Molecular Oxygen Derivatives

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

Pathogens are not the Sole Elicitors of Oxidative Bursts

Major Sources of H 2 O 2 From Metabolic Reactions • In Plants – Photosynthetic Electron Transport Chain – NADPH Oxidases – Flavin-Oxidase – Oxalate Oxidase – Amine Oxidases

The Role of Polyamines in Plant Defense Responses • Polyamine catabolism has been correlated with H 2 O 2 induced defense responses (Schipper et. al. , 2000) • Polyamine oxidase (PAO) is a key enzyme involved in polyamine catabolism

PAO is Responsible for the Catabolism of Polyamines Spermidine

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

Research Objectives • Down-regulate PAO using si. RNA – Should result in decreased PAO activity, H 2 O 2, and PAO m. RNA levels • Demonstrate si. RNA can be introduced into intact plants using a basic infiltration method

Method of Infiltration Infiltrate si. RNA at two sites

PAO Activity (nmol/min/mg) 450 250 400 200 350 300 150 200 150 100 50 50 0 Hour 2 hour 4 hour 6 hour 8 hour 10 hour 12 Hour 14 hour 16 hour 18 hour 20 hour 22 hour Time PAO si. RNA - H 2 O 2 Random si. RNA - PAO Random si. RNA - H 2 O 2 Non-infiltrated Leaves Hydrogen Peroxide (RLUs/ug of Protein) Polyamine Oxidase Activity and Hydrogen Peroxide Levels in Picolinic Acid Treated Avena sativa

Discussion • Preliminary results indicate a successful downregulation of PAO by PAO si. RNA • Levels of H 2 O 2 also decreased in PAO si. RNA infiltrated leaves, indicating that the H 2 O 2 produced by PAO significantly contributes to the oxidative burst of HR • The proposed infiltration technique may serve as a more efficient way to introduce si. RNA into plant model systems

Further Research • RT-PCR to confirm knockdown of PAO m. RNA

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

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

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

Determining Most Effective PAO si. RNA 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 TGA GCA CAG-3’ PAOb anti sense 5’-AAC TGT GCT CAG 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’

Determining Ideal PAO si. RNA Concentration • Optimal concentration for down-regulating PAO using infiltration was determined (based on PAO activity levels) – PAO si. RNA vs. Random si. RNA – Three concentrations: 0. 1 M, 0. 5 M, and 1. 0 M

Percent Decrease in PAO Activity

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

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