Systems Biology at Washington University

FIBR


Redox Changes Related to Photosystem II : Treatment with DCMU

Correlate Redox changes within cyanobacterial cells by selective inhibition of Photosystem II (PSII) and related electron flow at the level of plastoquinone B (QB) by using DCMU, an herbicidal inhibitor of PSII.

Inhibition of electron flow during photosynthesis
Assay of Photosynthetic activity
Transcription profiling reflect adaptive changes in Cellular Redox Environment
Co-expression network contained thirteen subnetworks with hub-and-spoke architecture. Composition of the largest subnetwork



Microarray Experiments : Design and Data


Inhibition of Electron Flow During Photosynthesis

The activity passing through Photosynthetic electron transport pathways determine the redox potential of the electron carriers as well as their determining the redox environment within a cell that is reflected in the relative concentrations of reduced versus oxidized forms of the components in the pathway. Metabolic changes in these component concentrations, reduced vs oxidized shift the redox potential within the cell. The change in redox environment initiates changes in related metabolic and electron transfer pathways to maintain homeostasis. Thus, we can project that changes in environmental conditions effect cellular electrochemistry. We have utilized a specific inhibitor of Photosystem II, DCMU. DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a very specific and sensitive inhibitor of photosynthesis. It blocks the plastoquinone binding site of photosystem II, disallowing the electron flow from where it is generated, in photosystem II, to plastoquinone. This interrupts the photosynthetic electron transport chain in photosynthesis and thus blocks the ability of the oxygenic photosynthetic cells to turn light energy into chemical energy (ATP and reductant potential). DCMU only blocks electron flow from Photosystem II, it has no effect on photosystem I or other reactions in photosynthesis, such as light absorption or carbon fixation in the Calvin cycle. We used a partial (20%) inhibition of the flux through Photosystem II to damp down the photosynthetic system so that the cell responds to the changes rather than shutting down entirely. This is an experimental approximation of environmental changes that would modulate photosynthetic systems in the real environment. Additional redox effects within the cell occur because PS I absorbs electrons that are oxidized from water in PS II. Thus, the electron "hole" of PS I cannot be satisfied, effectively turning down photosynthesis by preventing the reduction of NADP+ to NADPH, and the cyclic photosynthetic pathway since electron shuttling is associated with proton pumping across the membrane into the lumen.
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Assay of Photosynthetic Activity

Oxygen-evolution in intact cyanobacteria (Synechocystis 6803) directly assays the activity of Photosystem II (PSII). As photosystem II absorbs light energy, electrons in the reaction-centre chlorophyll are excited to a higher energy level and are trapped by the primary electron acceptors. To replenish the deficit of electrons, electrons are extracted from water and supplied to the chlorophyll. This process of electron extraction results in the net hydrolysis of 2 molecules of water (H₂O) to produce one molecule of O₂ and related protons (H+). Measurements of flash-induced variable fluorescence shows that DCMU inhibited the decay of variable fluorescence by blocking the oxidation of the PSII-reduced primary electron acceptor, Q-A, by the PSII secondary electron acceptor, QB, by displacing QB from the D1 protein. This partial inhibition is maintained over time as reflected by a parallel inhibition of O₂ evolution.
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Transcription Profiling Reflect Adaptive Changes in Cellular Redox Environment

Microarray measurement of differential transcription of genes reflect differences between perturbed (experimental) and unperturbed (control) states within a cell. The partial inhibition of photosynthetic electron transfer is the perturbed state which is reflected in the transcription profile with in the cell. We have followed this perturbation over time to attempt to determine: how the cell responds over time, “Is the initial response the same as long term compensatory responses?”, and how do the “Redoxin”s, proteins that are known to respond to intracellular changes in redox potential such as Glutaredoxins, Peroxiredoxins, Thioredoxins, and related proteins respond to redox perterbations
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Co-Expression Network


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Microarray Experiments : Design and Data