A2 13 Photosynthesis | A Level Biology 9700

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🎯What you need to be able to do

Relate chloroplast structure to function and state where the light-dependent and light-independent stages occur.
Explain that ATP and reduced NADP from the light-dependent stage are used in the Calvin cycle.
Describe the roles of chlorophyll a, chlorophyll b, carotene and xanthophyll, and interpret absorption and action spectra.
Use chromatography to separate chloroplast pigments and calculate R_f values.
Explain cyclic and non-cyclic photophosphorylation and the role of the oxygen-evolving complex.
Explain photophosphorylation by chemiosmosis across the thylakoid membrane.
Outline the three stages of the Calvin cycle and the role of rubisco, and state what the intermediates are used for.
Explain the effects of light intensity, carbon dioxide concentration and temperature as limiting factors, and describe investigations using DCPIP and whole plants.

📚The biology

The chloroplast

A double membrane encloses the stroma, a fluid matrix. Within it, thylakoids — flattened sacs with an internal thylakoid space — are stacked into grana.

The thylakoids are the site of the light-dependent stage. Stacking them into grana gives a very large surface area for the pigments and electron carriers, and the thylakoid space is a small enclosed compartment in which a proton gradient builds quickly.
The stroma is the site of the light-independent stage (the Calvin cycle) and contains the enzymes for it, plus starch grains, lipid droplets, circular DNA and 70S ribosomes.

The light-dependent stage supplies the light-independent stage with ATP and reduced NADP; the light-independent stage uses them to make complex organic molecules from carbon dioxide. That is the connection between the two, and it is why the Calvin cycle stops within seconds of the light going off even though it does not use light itself.

Pigments

Four pigments are named: chlorophyll a, chlorophyll b, carotene and xanthophyll. All absorb light in the thylakoids and pass the energy to chlorophyll a at the reaction centre. Having several pigments with different absorption peaks lets the plant use a wider range of wavelengths than chlorophyll a alone could.

An absorption spectrum plots how much light of each wavelength a pigment absorbs.
An action spectrum plots the rate of photosynthesis at each wavelength.

The two curves have similar shapes — both peak in the blue-violet and the red, and both dip in the green, which is reflected rather than absorbed and is why leaves look green. That correspondence is the evidence that these pigments are the ones driving photosynthesis. The action spectrum is slightly broader than the absorption spectrum of chlorophyll a alone, because the accessory pigments contribute.

Chromatography and R_f

Pigments are extracted in a solvent, spotted onto chromatography paper, and separated as the solvent rises. Each pigment travels a characteristic distance, and is identified by its R_f value:

\[ R_{\mathrm{f}} = \frac{\text{distance moved by the pigment}}{\text{distance moved by the solvent front}} \]

Both distances are measured from the origin (the pencil line where the spot was applied) to the centre of the pigment spot. R_f has no units and is always between 0 and 1. It is constant for a given pigment in a given solvent, which is why published values must be compared only against the same solvent system.

The light-dependent stage

Non-cyclic photophosphorylation — involves both photosystem I and photosystem II:

Light is absorbed and photoactivation of chlorophyll occurs: electrons are raised to a higher energy level and leave the chlorophyll.
The oxygen-evolving complex catalyses the photolysis of water, splitting it to replace the lost electrons. This releases protons and oxygen — the oxygen is a by-product and is the source of all atmospheric oxygen.
Electrons pass along a chain of carriers, releasing energy that is used to make ATP.
Electrons finally reduce NADP (together with protons), forming reduced NADP.

Cyclic photophosphorylation — involves only photosystem I. Photoactivation occurs, the electron passes along carriers and returns to the same chlorophyll. ATP is synthesised, but no reduced NADP and no oxygen are produced, because no water is split and no electron leaves the system. Its purpose is to top up ATP when the Calvin cycle needs proportionally more ATP than reduced NADP.

In both, ATP is made by chemiosmosis: energy from the electrons is used to transfer protons across the thylakoid membrane into the thylakoid space, and the protons return to the stroma by facilitated diffusion through ATP synthase, driving ATP synthesis. The mechanism is identical to that in mitochondria — only the membrane and the direction differ.

The Calvin cycle

Three stages, in the stroma:

Fixation. The enzyme rubisco catalyses the combination of carbon dioxide with ribulose bisphosphate (RuBP, 5C), yielding two molecules of glycerate 3-phosphate (GP, 3C).
Reduction. GP is reduced to triose phosphate (TP) using reduced NADP and ATP from the light-dependent stage.
Regeneration. Most TP is used to regenerate RuBP, in reactions that use ATP, so the cycle can continue.

The remaining TP leaves the cycle. Calvin cycle intermediates are used to make other molecules: GP can be used to make some amino acids, and TP to make carbohydrates, lipids and amino acids. Six turns of the cycle fix six carbon dioxide molecules, enough for one hexose.

Predicting concentration changes when a factor is removed. These questions are answered by asking which step is blocked and what therefore accumulates. Remove carbon dioxide: fixation stops, so RuBP is no longer used up — RuBP rises and GP falls. Remove light: no ATP or reduced NADP, so GP cannot be reduced — GP rises and RuBP falls, because regeneration needs ATP and TP. The two cases give opposite answers, so read which one you have been given.

Limiting factors

Light intensity, carbon dioxide concentration and temperature are limiting factors. At any moment the rate is set by whichever is in shortest supply. On a graph, the rising portion shows the plotted factor limiting; the plateau shows that something else has become limiting.

Light intensity — more light means more photoactivation, so more ATP and reduced NADP. Plateaus when another factor limits, or when all pigments are saturated.
Carbon dioxide concentration — more CO₂ means more substrate for rubisco and faster fixation. Atmospheric CO₂ is only about 0.04%, so it is very often the limiting factor in the field, and raising it in a glasshouse increases yield.
Temperature — affects the enzyme-controlled reactions of the Calvin cycle, so the rate rises to an optimum then falls as enzymes including rubisco denature. The light-dependent reactions are much less temperature-sensitive, being photochemical.

To identify the limiting factor from a graph, look at the plateau: increasing the plotted variable no longer helps, so something else is limiting — and if a second curve at a higher level of another factor plateaus higher, that second factor was the limiting one.

Investigations

Isolated chloroplasts with DCPIP. DCPIP is an artificial electron acceptor: it takes the electrons that would have reduced NADP, and is blue when oxidised, colourless when reduced. The rate of decolourisation therefore measures the rate of the light-dependent stage. Use it to compare light intensities or wavelengths; read with a colorimeter for numerical data. Keep the chloroplast suspension cold and use it quickly.
Whole aquatic plants. Count bubbles of oxygen, or better, collect and measure the gas volume, from pondweed under different light intensities, carbon dioxide concentrations (varying sodium hydrogencarbonate) or temperatures. Light intensity is varied by moving a lamp, with a heat shield of water between lamp and plant so that temperature stays constant — otherwise two variables change at once.

✏️Worked example

A leaf pigment extract was separated by chromatography. The solvent front travelled 88 mm from the origin. Four spots were seen, with their centres at 15 mm (yellow), 40 mm (blue-green), 51 mm (yellow-green) and 84 mm (orange). (a) Calculate the R_f value of each pigment. (b) The orange pigment is carotene. Suggest why it travelled furthest. (c) A student obtains R_f values differing from published ones. Give two reasons that do not involve measurement error.

(a) R_f is the distance moved by the pigment divided by the distance moved by the solvent front, both measured from the origin to the centre of the spot:

yellow: \( 15/88 = 0.17 \)

blue-green: \( 40/88 = 0.45 \)

yellow-green: \( 51/88 = 0.58 \)

orange: \( 84/88 = 0.95 \)

All lie between 0 and 1, as they must, and R_f has no units. By convention these are usually quoted to two decimal places.

(b) Carotene is the most soluble in the solvent used and the least strongly attracted to the chromatography paper. A pigment moves as far as the balance between those two attractions allows: the more soluble it is in the moving solvent and the less it adsorbs to the stationary paper, the further it is carried before the solvent front stops. Carotene is a non-polar hydrocarbon, so it dissolves readily in an organic solvent, whereas the chlorophylls have polar groups that bind to the paper.

(c) First, a different solvent. R_f is constant only for a stated pigment in a stated solvent, because the value depends on the relative attraction of the pigment for solvent and paper; change the solvent and every value changes. Second, a different stationary phase — a different grade of chromatography paper, or a thin-layer plate instead of paper — alters adsorption in the same way. Temperature and the degree of saturation of the tank with solvent vapour also affect the values, which is why the tank is kept sealed.

Check it. Every R_f must be less than 1, because no pigment can travel further than the solvent carrying it. A value greater than 1 means the two distances have been divided the wrong way round, or the solvent front was measured before the run finished. Check also that the order of the values matches the order of the spots up the paper — 0.17, 0.45, 0.58, 0.95 ascends as the distances do, which confirms no pair has been swapped.

Measuring to the leading edge of the spot, or from the bottom of the paper. Both distances must be measured from the origin — the pencil line where the extract was applied — and to the centre of each spot. Measuring from the bottom edge of the paper adds a constant to both numbers and distorts every ratio; measuring to the leading edge systematically inflates every R_f. Note too that the origin line must be drawn in pencil and must sit above the solvent level, or the pigments dissolve away into the solvent instead of running up the paper.

📝Practise

Work through these, then reveal the answer. Each question targets a different objective from the list above.

1. A plant in bright light is suddenly placed in darkness. Predict and explain what happens to the concentrations of GP and RuBP.

GP rises and RuBP falls. In darkness the light-dependent stage stops, so no ATP and no reduced NADP are produced. GP is still being formed, because rubisco continues to fix carbon dioxide onto the RuBP already present, but GP can no longer be reduced to triose phosphate, since that step requires reduced NADP and ATP. GP therefore accumulates. RuBP falls because it continues to be used up in fixation while its regeneration from triose phosphate, which requires ATP, has stopped. Both changes level off once RuBP is exhausted. Note that removing carbon dioxide instead gives the opposite pattern: fixation stops, so RuBP accumulates and GP falls.

2. Explain why plants contain several photosynthetic pigments rather than chlorophyll a alone.

Each pigment has its own absorption spectrum — it absorbs some wavelengths strongly and others hardly at all. Chlorophyll a absorbs mainly in the blue-violet and red regions. Chlorophyll b, carotene and xanthophyll are accessory pigments that absorb at somewhat different wavelengths and pass the absorbed energy on to chlorophyll a at the reaction centre. Having several pigments therefore allows the plant to absorb and use a wider range of wavelengths of the available light, so more of the incident energy is harvested and the rate of photosynthesis is higher, particularly in shaded or filtered light where the spectrum is altered. This is why the action spectrum for photosynthesis is broader than the absorption spectrum of chlorophyll a alone.

3. Compare cyclic and non-cyclic photophosphorylation.

Non-cyclic: involves both photosystem I and photosystem II; electrons lost from photosystem II are replaced by the photolysis of water, catalysed by the oxygen-evolving complex, which releases oxygen as a by-product and protons; electrons travel a one-way path and finally reduce NADP; the products are ATP and reduced NADP. Cyclic: involves photosystem I only; the excited electron travels along carriers and returns to the same chlorophyll molecule; no water is split, so no oxygen is released and no electron is available to reduce NADP; the only product is ATP. Both involve photoactivation of chlorophyll and both make ATP by chemiosmosis across the thylakoid membrane. Cyclic photophosphorylation supplements ATP supply when the Calvin cycle requires more ATP than reduced NADP.

4. A graph shows the rate of photosynthesis against light intensity at two carbon dioxide concentrations, 0.04% and 0.4%. Both curves rise then plateau, but the 0.4% curve plateaus at a higher rate. Explain.

On the rising part of each curve, light intensity is the limiting factor: increasing it increases the rate, because more photoactivation means more ATP and reduced NADP for the Calvin cycle. At the plateau, further light makes no difference, so something other than light has become limiting. That the 0.4% curve plateaus higher shows the factor limiting the 0.04% curve was carbon dioxide concentration — supplying more of it raised the ceiling, which it could not have done had CO₂ not been limiting. More carbon dioxide means more substrate for rubisco and a faster rate of fixation of CO₂ onto RuBP. The 0.4% curve is itself limited at its plateau by something else again, most likely temperature or the concentration of enzymes. Commercially, this is the justification for raising carbon dioxide levels in glasshouses.

5. Describe how you would use DCPIP and a suspension of chloroplasts to compare the rate of the light-dependent stage at two light intensities.

Extract chloroplasts by grinding leaves in an ice-cold isotonic buffer, filtering and centrifuging, keeping the suspension cold throughout to preserve activity. Add equal volumes of chloroplast suspension and DCPIP solution to each of two tubes. DCPIP is an artificial electron acceptor, taking the electrons that would otherwise reduce NADP; it is blue when oxidised and colourless when reduced. Place one tube at each light intensity, measured with a light meter, keeping temperature constant with a water bath and a heat shield. Record the time taken to decolourise, or better, take absorbance readings at fixed intervals in a colorimeter and calculate the rate of change. Faster decolourisation indicates a faster rate of the light-dependent stage. Include a control kept in darkness and one with boiled chloroplasts, both of which should stay blue, showing that the colour change requires light and functional chloroplasts.

6. Explain why the light-independent stage stops within seconds of the light being switched off, even though it does not require light.

The light-independent stage is not driven by light directly, but it depends on two products of the light-dependent stage: ATP and reduced NADP. Both are needed for the reduction of GP to triose phosphate, and ATP is also needed for the regeneration of RuBP. Neither is stored in quantity — the pool of each in the stroma is very small and is turned over continuously. When the light goes off, photoactivation and photolysis stop immediately, so no more ATP or reduced NADP is made, and the existing pool is exhausted within seconds. GP accumulates because it cannot be reduced, RuBP is used up and not regenerated, and the cycle halts. The name ‘light-independent’ refers only to the fact that light is not a direct reactant; it does not mean the stage can run in the dark.

🔗Go deeper — other people’s work

These are external resources, not mine. If one stops working, tell me and everything above it on this page still stands.

SAPS (Science and Plants for Schools) — the standard chloroplast extraction and DCPIP protocol, with the practical detail that makes it work
Any interactive Calvin cycle animation — watching the carbon atoms move makes the ‘six turns per hexose’ arithmetic obvious
Published absorption and action spectra plotted on the same axes — the correspondence between them is the single most useful figure in this topic