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Exploring the metabolic pathways of Synechococcus and Dunaliella through fluorescence decay kinetics. Erin Atkinson NASA Goddard Space Flight Center Summer 2004. Ocean Biogeochemistry Laboratories. Laboratory for Hydrospheric Processes, Oceans and Ice Branch, Code 971

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Erin atkinson nasa goddard space flight center summer 2004

Exploring the metabolic pathways of Synechococcus and Dunaliella through fluorescence decay kinetics

Erin Atkinson

NASA Goddard Space Flight Center

Summer 2004


Ocean biogeochemistry laboratories

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


Ocean productivity algorithm

Ocean productivity algorithm

Net primary production is a function of:

biomass

incident solar flux

light utilization efficiency

NPP = f ( Chl,Zeu,Io, Pb)


Erin atkinson nasa goddard space flight center summer 2004

  • 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.


Journal of phycology review

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


E k independent variability

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


Project objectives

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.


Metabolic electron transport

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.


Prokaryotic pathway

Prokaryotic pathway

ATP

NADPH

O2

H2O

ATPase

Cyt b6f

aa3 Oxid

e-

PSI

PSII

PQ

e-

e-

c553

H2O

O2


Eukaryotic pathway

Eukaryotic pathway

Cyt b6f

e-

e-

PSII

PQ

PSI

O2

H2O

e-

Chloroplast

O2

H2O

ATP

CAC

ETC

Mitochondria


Fluorescence

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


Frr profile

FRR Profile


Previous studies

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.


Synechococcus

Synechococcus

  • Marine prokaryote

  • Cells approximately 1.5uM

  • Very abundant in the oceans

  • Major primary producers on the global scale


Dunaliella

Dunaliella

  • Marine eukaryote

  • Cells approximately 10uM

  • Two equally long, anteriorly directed flagella


Inhibitors

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).


Prokaryotic inhibitors

DCMU

DBMIB

Darkness

TOBC

Prokaryotic inhibitors

O2

H2O

Cyt b6f

aa3 Oxid

PSI

PSII

PQ

H2O

O2

c553


Eukaryotic inhibitors

Eukaryotic inhibitors

DCMU

TOBC

Cyt b6f

DBMIB

PSII

PQ

PSI

Darkness

O2

H2O

Chloroplast

O2

H2O

CAC

ETC

Mitochondria


Erin atkinson nasa goddard space flight center summer 2004

DCMU


Erin atkinson nasa goddard space flight center summer 2004

TOBC


Dbmib

DBMIB


Synechococcus1

DCMU

DBMIB

Darkness

TOBC

Synechococcus

O2

H2O

Cyt b6f

aa3 Oxid

PSI

PSII

PQ

H2O

O2

c553


Dunaliella1

Dunaliella

DCMU

TOBC

Cyt b6f

DBMIB

PSII

PQ

PSI

Darkness

O2

H2O

O2

H2O

Unspecified alternate pathway

CAC

ETC


Conclusions

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.


Future work

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.


Acknowledgements

Acknowledgements

Mike Behrenfeld

Kirby Worthington

Ondrej Prasil

Antonio Mannino


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