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
Common = ubiquitous in all living organisms.
Notice the positive charge on these polyamines.

3. Polyamines
Free polyamines interact electrostatically with polyanionic macromolecules:
Stabilize DNA
DNA-protein interactions
Also interact with other acidic substrates (membrane phospholipids)
The positive backbone of polyamines allows…
Increase melting point …
Membrane phospholipids…increase rigidity.

4. The Roles of Polyamines in Plants
Cell division/Embryogenesis
Root Formation
Plant Hormone Regulation
Senescence
Plant Defense Responses
These interactions promote other cell processes such as…

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)
These plant defense responses are the focus of my research.
This phenomenon as a model to study ROS.

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
Leads to the question:
What is the source of H2O2 that triggers this response.

10. Major Sources of H2O2 From 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
Proportionate increase in Hydrogen peroxide.

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

17. 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

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

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

20. 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 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

23. 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’

24. 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

25. Percent Decrease in PAO Activity

26. 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

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