Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states –...

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Transcript of Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states –...

Page 1: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.
Page 2: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

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

Page 3: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

Polyamines

• Free polyamines interact electrostatically with polyanionic macromolecules:

– Stabilize DNA– DNA-protein interactions– Also interact with other acidic substrates

(membrane phospholipids)

Page 4: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

The Roles of Polyamines in Plants

• Cell division/Embryogenesis• Root Formation• Plant Hormone Regulation• Senescence• Plant Defense Responses

Page 5: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

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)

Page 6: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

The HR Generates Reactive Oxygen Species

Page 7: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

ROS Are Molecular Oxygen Derivatives

Page 8: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

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

Page 9: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

Pathogens are not the Sole Elicitors of Oxidative Bursts

Page 10: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

Major Sources of H2O2 From Metabolic Reactions

• In Plants– Photosynthetic Electron Transport Chain– NADPH Oxidases– Flavin-Oxidase– Oxalate Oxidase– Amine Oxidases

Page 11: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

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

Page 12: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

PAO is Responsible for the Catabolism of Polyamines

Spermidine

Page 13: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

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

Page 14: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

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

Page 15: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

Method of Infiltration

Infiltrate siRNA at two sites

Page 16: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.
Page 17: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

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

Page 18: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

Further Research

• RT-PCR to confirm knockdown of PAO mRNA

Page 19: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

Acknowledgements

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

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

Page 20: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.
Page 21: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

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

Page 22: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

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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

Page 23: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

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’

Page 24: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

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

Page 25: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

Percent Decrease in PAO Activity

Page 26: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

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

Page 27: Polyamines Polyamines are aliphatic cations that can occur freely or in conjugated states – Uncommon: E.g. Homospermidine, norspermidine, and norspermine.

Does Down-regulation of PAO Affect Hydrogen Peroxide Levels?

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