Erin Atkinson NASA Goddard Space Flight Center Summer 2004

1. Exploring the metabolic pathways of Synechococcus and Dunaliella through fluorescence decay kinetics Erin Atkinson NASA Goddard Space Flight Center Summer 2004

2. Ocean Biogeochemistry Laboratories • Laboratory for Hydrospheric Processes, Oceans and Ice Branch, Code 971 • study the biologically mediated flux of oceanic carbon through a combination of remote sensing data and field and laboratory measurements • Primary productivity modeling • Carbon fixation modeling • Phytoplankton physiology and optical properties

3. Ocean productivity algorithm Net primary production is a function of: biomass incident solar flux light utilization efficiency NPP = f ( Chl,Zeu,Io, Pb)

4. Biomass (Chl, Zeu) and incident solar flux (Io) are satellite derived terms, while light utilization efficiency (Pb) is a empirically derived physiological term. • Biomass and light utilization efficiency both vary over approximately two orders of magnitude • Play an equal role in calculating estimates of global ocean productivity • Current productivity estimates derived from satellite ocean color data place heavy emphasis biomass term, while less attention has been given to establishing a better understanding and application of the physiological term.

5. Journal of Phycology Review Behrenfeld et al, 2004 • Positive correlation between the light limited slope (b) and light-saturated rate (Pbmax) of photosynthesis • Ek-dependent variability • Parallel changes in b and Pbmax • No change in light saturation index (Ek= Pbmax/ b) • Results from photoacclimation • Ek-independent variability • Changes in light saturation index • Physiological basis unknown Behrenfeld et al, 2004

6. Ek-independent variability Proposed mechanism = Alternative metabolic pathway preferred under nutrient stress • As nutrient stress increases and growth rate decreases, photosynthetically generated reductants are increasingly used for simple ATP generation through a fast respiratory pathway that skips the carbon reduction cycle (Behrenfeld et al, 2004). Behrenfeld et al, 2004

7. Project objectives • To generate improved understanding of phytoplankton physiology, identify and investigate evidence of alternative metabolic pathways by using inhibitors to simulate conditions of nutrient stress. • Demonstrate the use of fluorescence decay experiments as a viable method for exploring metabolic pathways in phytoplankton.

8. Metabolic Electron Transport • Capture and transport of electrons via photosynthetic and respiratory reactions generates a proton gradient necessary for the production of ATP. • In prokaryotes, photosynthesis and respiration both occur on the thylakoid membrane. • In eukaryotes, photosynthesis takes place in the chloroplast and respiration takes place in the mitochondria.

9. Prokaryotic pathway ATP NADPH O2 H2O ATPase Cyt b6f aa3 Oxid e- PSI PSII PQ e- e- c553 H2O O2

10. Eukaryotic pathway Cyt b6f e- e- PSII PQ PSI O2 H2O e- Chloroplast O2 H2O ATP CAC ETC Mitochondria

11. Empty Filled Fluorescence • Occurs when the photon receptor sights of the PSII reaction membrane become filled and additional incoming photons bounce off • Measured via fast repetition rate (FRR) fluorometer PSII

12. FRR Profile

13. Previous studies • Berry et al, 2002 • Examined electron transport pathways in Synechocystis (freshwater prokaryote) using fluorescence induction • Inhibitors used to isolate an alternative oxidase pathway within the thylakoid membrane • Behrenfeld in New Zealand • Performed fluorescence decay inhibition experiments on Synechocystis • Consistent with the findings of Berry et al.

14. Synechococcus • Marine prokaryote • Cells approximately 1.5uM • Very abundant in the oceans • Major primary producers on the global scale

15. Dunaliella • Marine eukaryote • Cells approximately 10uM • Two equally long, anteriorly directed flagella

16. Inhibitors • Used to simulate the metabolic effects of nutrient stress on the phytoplankton • By blocking electron transport, inhibitors cause a lack of fluorescence decay (PSII remains saturated).

17. DCMU DBMIB Darkness TOBC Prokaryotic inhibitors O2 H2O Cyt b6f aa3 Oxid PSI PSII PQ H2O O2 c553

18. Eukaryotic inhibitors DCMU TOBC Cyt b6f DBMIB PSII PQ PSI Darkness O2 H2O Chloroplast O2 H2O CAC ETC Mitochondria

19. DCMU

20. TOBC

21. DBMIB

22. DCMU DBMIB Darkness TOBC Synechococcus O2 H2O Cyt b6f aa3 Oxid PSI PSII PQ H2O O2 c553

23. Dunaliella DCMU TOBC Cyt b6f DBMIB PSII PQ PSI Darkness O2 H2O O2 H2O Unspecified alternate pathway CAC ETC

24. Conclusions • Analyzing variability in fluorescence decay is an effective method for the study of electron transport pathways. • Thus far, no evidence of an alternative pathway that would account for Ek-independent variability in Synechococcus. • Preliminary evidence of an alternative pathway after cytochrome b6f, but before PSI in Dunaliella that could account for the proposed Ek-independent variability due to nutrient stress.

25. Future Work • Supplement fluorescence decay data with O2 evolution data to quantitatively characterize changes in photosynthesis and respiration rates as a result of metabolic inhibition. • Experimentation with additional inhibitors to define alternative pathways more precisely. • Conduct similar experiments with other prokaryotic and eukaryotic species of phytoplankton.

26. Acknowledgements Mike Behrenfeld Kirby Worthington Ondrej Prasil Antonio Mannino