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Inspired by a question asked to me by a classmate, I have the following question about Light-independent (dark phase) reactions in photosynthesis:-

Let us suppose an algae sample was exposed to light for a considerable time so that maximum( if there is a limit) NADPH concentration was achieved. Now if the sample is placed in dark and radioactive ¹⁴CO₂ bubbled, will the cell be radiolabelled after some time of bubbling continuously?

I guess the answer depends on the active life of ATP and NADPH , the products of light reaction. If they are considerably stable, such that they are in sufficient concentration for executing Calvin cycle although their production (apart from respiratory ATP production) has ceased due to absence of light. If they are, then the ATP and NADPH produced during the initial exposure period will carry out carbon fixation with radioactive carbon and hence radio labelled sugars will be recoverable from the sample. If not, they will quickly degenerate (by hydrolysis, utilization or likewise) and they will be unable to carry out fixation after some fixed time after stopping the light.

What, under normal conditions, should be the time after which the light-reaction products are no longer capable of fixing CO₂ by Calvin cycle? And, ultimately, will the presence of radioactivity in sugars for the scenario above depend on the species of plant?

[I am ignoring all radioactivity due to dissolution of ¹⁴CO₂ in cytoplasm]

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It turns out that the so-called light independent reactions are not light-independent at all: there are several regulatory mechanisms in place to prevent the turning of the Calvin Cycle when there is no light energy available to produce ATP/NADPH. The hypothetical situation you described in your question demonstrates the necessity of such regulation. The cell cannot allow levels of ATP and NADPH to drop too low because those two factors are required for everything. So if you let the Calvin Cycle run when there is no means of fueling it (ultimately) from light energy, then the Calvin Cycle will run until ATP/NADPH is too low to sustain it further, at which point the cell dies. So several key enzymes in the Calvin cycle are activated indirectly by light.

One of the most well-studied regulatory mechanisms is the ferrodoxin/thioredoxin regulatory system. The enzyme Ferredoxin-Thioredoxin reductase takes electrons from ferredoxin (which is reduced by light energy via the photosystems) and transfers it to thioredoxin, a disulfide-based redox protein. Then thioredoxin reduces several key enzymes in the Calvin Cycle, which activates them (through conformational changes induced by disulfide bonds). The upshot is that when light is around to facilitate reduction, the metabolic enzymes of the Calvin cycle can work, but in the dark the cell oxidizes and these enzymes are turned off, switching off the Calvin cycle and arresting CO$_2$ fixation.

An orthogonal direction of light regulation of the "light-independent" reactions is RuBisCO Activase, which is required to modify the RuBisCO active site to be active. This requires ATP hydrolysis, which amplifies the sensitivity of the Calvin Cycle to ATP availability.

The takeaway is the light-independent reactions may not directly require light as an input, but they are regulated by light availability. I tried to look up how much time in the dark would be required to shift the redox balance such that the Calvin Cycle enzymes would be inactivated, but I couldn't find that data. People in the literature, however, seem to believe it would be quick (1).

There is some description of this at Wikipedia. If you want to look further, Bob Buchanan at Berkeley made a lot of seminal advances in this area; a review of his you might want to check out is (2).

(1): Ruelland E, Miginiac-Maslow M. (1999). Trends in Plant Science. 4(4): 136-141

(2): Buchanan BB. (1980). Annual Review of Plant Physiology. 31:341-374.

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+1 for the comprehensive answer:) On an imaginative side note, had these regulatory mechanisms been absent, what then would have been the time delay before the light reaction products at saturation would go below the minimum concentration required to execute Calvin cycle (just an order of magnitude, i.e. seconds? minutes? hours?)? Would ATP & NADPH utilisation be the only way of their depletion or would their natural decomposition (hydrolysis) also play a part? – Satwik Pasani Nov 21 '13 at 15:55
@Satwik I am not sure but I think before they would naturally dgrade, ATP and NADPH utilisation would limit them – biogirl Nov 22 '13 at 16:35

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