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 H2O2 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 H2O2 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 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

Method of Infiltration

Infiltrate siRNA at two sites

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

Further Research

• RT-PCR to confirm knockdown of PAO mRNA

Acknowledgements

• Dr. Glenn D. Kuehn• Kuehn Lab (Kristina, Antonia, Salvador, Alex,

and Murphy)• Dr. Champa Sengupta-Gopalan• Dr. Suman Bagga

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

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’

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

Percent Decrease in PAO Activity

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

Does Down-regulation of PAO Affect Hydrogen Peroxide Levels?

• Crude extract was assayed for:– Protein concentration – PAO activity– Hydrogen peroxides levels