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

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Presentation on theme: "Analysis of PsbS Protein Levels in Response to Environmental Stress in Balsam Fir (Abies balsamea) and Pea (Pisum sativum) Michelle Hackner 2006."— Presentation transcript:

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 and 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 % less in shade 54.10% less in shade Equal- Chlorophyll 31.14% less in shade 68.46% less in shade

13 Results: Chlorophyll p = Sun-acclimated pea chlorophyll Trial (nm) (nm) Chlorophyll a (Chl a) Chlorophyll b (Chl b) Chl a: Chl b Average Shade-acclimated pea chlorophyll Trial (nm) (nm) Chlorophyll a (Chl a) Chlorophyll b (Chl b) Chl a: Chl b Average

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 = ) 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 = ) 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 = ) When compared relative to protein, sun-acclimated pea plants had a higher PsbS concentration than shade- acclimated pea plants (p = ) 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)


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