1. Analysis of PsbS Protein Levels in Response to Environmental Stress in Balsam Fir (Abies balsamea) and Pea (Pisum sativum) Michelle Hackner 2006

2. Background

3. The Xanthophyll Cycle

4. Previous Research Li, et al. (2000)  The role of PsbS in the xanthophyll cycle R. W. Pearcy (1998)  Photoprotective protein concentrations  Chlorophyll a : chlorophyll b ratios  Ratio of D1 reaction centers: light-harvesting complexes B1 and B2

5. Goals 1.Confirm that samples show characteristics typical of sun- and shade-acclimated plants 2.Compare PsbS concentration in sun-acclimated versus shade-acclimated pea (Pisum sativum) 3.Compare PsbS concentration in balsam fir (Abies balsamea) needles collected during the winter versus those collected during the summer

6. Hypotheses 1.There will be a higher concentration of PsbS protein in sun-acclimated pea plants than in those grown in the shade 2.There will be a higher concentration of PsbS protein in balsam fir samples collected during the winter than in those collected during the summer

7. Collection and Analysis of Samples Pea and balsam fir samples were collected and the thylakoids isolated by centrifuging Samples were analyzed with spectrophotometer:  Chlorophyll analysis: absorbance taken at 663.6 and 646.6 nm  Protein analysis (w/ Bio-Rad protein dye): absorbance taken at 595 nm

8. Electrophoresis Acrylamide gels were run at 40 mA for one hour

9. Gel Transfer Gels were run at 300 mA for one hour to transfer to PVDF membranes

10. Western Blotting Antibody wash ratios ProteinPrimary antibodySecondary antibody PsbSAnti-PsbS: 1:5000, 2 μL of antibody in 10 mL 2% milk in PBS-T Anti-chicken: 1:5000, 2 μL antibody in 10 mL 2% milk in PBS-T LhcB1 and LhcB2 Anti-LhcB1 and anti-LhcB2: 1:5000, 2 μL of EACH antibody in 10 mL 2% milk in PBS-T (both antibodies in one wash) Anti-chicken: 1:5000, 2 μL antibody in 10 mL 2% milk in PBS-T D1Anti-D1: 1:5000, 2 μL of antibody in 10 mL 2% milk in PBS-T Anti-rabbit: 1:5000, 2 μL antibody in 10 mL 2% milk in PBS-T

11. Transfer to Film and O.D. Analysis

12. Results: Lhcb1 and Lhcb2 vs. D1 D1: Lhcb1 and Lhcb2 ratios were higher in sun- acclimated plants than in shade-acclimated plants Type of Analysis LhcB1 and LhcB2 D1 Reaction Centers P-value Equal- Protein 28.891% less in shade 54.10% less in shade 0.0430 Equal- Chlorophyll 31.14% less in shade 68.46% less in shade 0.0832

13. Results: Chlorophyll p = 0.0000103 Sun-acclimated pea chlorophyll Trial 646.6 (nm) 663.6 (nm) Chlorophyll a (Chl a) Chlorophyll b (Chl b) Chl a: Chl b 10.68371.537616.936.8582.468 20.59311.346914.845.8822.524 30.64991.460516.086.5242.464 Average15.956.4222.485 Shade-acclimated pea chlorophyll Trial 646.6 (nm) 663.6 (nm) Chlorophyll a (Chl a) Chlorophyll b (Chl b) Chl a: Chl b 10.11620.23012.4951.3251.883 20.11950.23832.5861.3541.901 30.09620.19022.0621.0981.877 Average2.3811.2591.890

14. PsbS Concentration Gradient Figure 3: PsbS Concentration Gradient

15. Results: Equal-Chlorophyll Shade-acclimated pea plants have an average 54.55% less PsbS protein than sun-acclimated pea plants (p = 0.05373) PsbS in sun- and shade- acclimated pea plants, relative to chlorophyll

16. Results: Equal-Protein Shade-acclimated pea plants had an average of 55.54% less PsbS protein than sun-acclimated pea plants (p = 0.00985) PsbS in sun- and shade-acclimated pea plants, relative to protein

17. Results: Balsam Fir Winter-collected balsam fir was shown to have more PsbS protein than summer-collected balsam fir PsbS in balsam fir  Sun-acclimated plants: 59.98% less  Shade-acclimated plants: 54.62% less

18. Conclusions Relative to chlorophyll, sun-acclimated pea plants had a higher PsbS concentration than shade-acclimated plants (p = 0.05373) When compared relative to protein, sun-acclimated pea plants had a higher PsbS concentration than shade- acclimated pea plants (p = 0.00985) Winter-collected balsam fir samples had a higher PsbS concentration than summer-collected balsam fir samples

19. Future Work Further research on other plant species:  Balsam fir and other conifers  Moss and similar species Biological engineering of plants for survival in a broader range of climates:  Protection of species from climate changes  Crop species

20. Acknowledgements Dr. Amy Verhoeven Laura Suurmeyer, Angela Osmolak, and Paul Morales Ms. Lois Fruen Dr. Jacob Miller Ms. Denise Pivarnik-Nova My research classmates

21. Michelle Hackner 2006 Analysis of PsbS Protein Levels in Response to Environmental Stress in Balsam Fir (Abies balsamea) and Pea (Pisum sativum)