What you'll learn
Photosynthesis: light and dark reactions is one of the two great energy-converting processes in biology, and at CAPE level it is studied at the biochemical detail of individual reaction stages. Photosynthesis converts light energy into the chemical energy of organic molecules, and it does so in two connected stages that occur in different parts of the chloroplast and depend on one another. By the end of this topic you should be able to relate chloroplast structure to function, describe the light-dependent reactions including both photophosphorylation routes, describe the Calvin cycle and the fate of its products, explain the evidence for limiting factors and interpret the associated graphs, describe chromatography of photosynthetic pigments, and explain how the two stages depend on each other.
Key terms and definitions
Photosynthesis — the conversion of light energy into chemical energy in organic molecules
Chlorophyll — the principal photosynthetic pigment, absorbing mainly red and blue light
Photosystem — a cluster of pigment molecules in the thylakoid membrane that funnels energy to a reaction centre
Photolysis — the splitting of water by light energy, releasing electrons, protons and oxygen
Photophosphorylation — the synthesis of ATP using light energy
Non-cyclic photophosphorylation — the route involving both photosystems, producing ATP, reduced NADP and oxygen
Cyclic photophosphorylation — the route involving photosystem I only, producing ATP alone
NADP — the coenzyme that carries hydrogen and electrons to the light-independent stage
Calvin cycle — the cycle of light-independent reactions fixing carbon dioxide
Rubisco — the enzyme catalysing the fixation of carbon dioxide onto ribulose bisphosphate
Ribulose bisphosphate — the five-carbon acceptor of carbon dioxide, abbreviated RuBP
Glycerate 3-phosphate — the first product of fixation, a three-carbon compound, abbreviated GP
Triose phosphate — the three-carbon sugar produced by reduction of GP, abbreviated TP
Limiting factor — the factor in shortest supply, which determines the rate
Core concepts
Chloroplast structure and function
The chloroplast is bounded by a double membrane forming the chloroplast envelope, which controls what enters and leaves.
Inside, an extensive system of thylakoid membranes is stacked into structures called grana. These membranes hold the photosynthetic pigments arranged into photosystems, and the stacking provides a very large surface area for light absorption. The thylakoid membrane also holds the electron carriers and the ATP synthase enzymes.
The stroma is the fluid surrounding the grana. It contains the enzymes of the Calvin cycle, including rubisco, and is where the light-independent reactions occur. It also contains circular DNA and 70S ribosomes, allowing the chloroplast to make some of its own proteins, and starch grains and lipid droplets storing the products of photosynthesis.
The separation of the two stages between thylakoid and stroma is not incidental: it allows the proton gradient needed for ATP synthesis to be maintained across the thylakoid membrane.
Photosynthetic pigments
Chlorophyll a is the primary pigment, present at the reaction centre of each photosystem. Chlorophyll b, carotene and xanthophyll are accessory pigments, absorbing light at wavelengths chlorophyll a absorbs poorly and passing the energy to it. Having several pigments widens the range of wavelengths that can be used.
Chlorophyll absorbs strongly in the red and blue-violet regions and reflects green, which is why leaves appear green.
An absorption spectrum plots the light absorbed by a pigment against wavelength. An action spectrum plots the rate of photosynthesis against wavelength. The close correspondence between the two is the evidence that the pigments absorbing the light are the ones driving photosynthesis, and questions often ask candidates to draw exactly that conclusion.
Pigments are separated by chromatography. A concentrated spot of leaf extract is placed on a chromatography paper above the solvent level, the solvent rises, and the pigments separate according to their relative solubility in the solvent and attraction to the paper. Rf values identify each pigment. Carotene, being most soluble in the organic solvent, travels furthest.
The light-dependent reactions
These occur on the thylakoid membranes and require light. They produce ATP and reduced NADP for the next stage, and oxygen as a by-product.
Light energy is absorbed by the pigments of photosystem II and funnelled to the reaction centre, raising a pair of electrons to a higher energy level so that they leave the chlorophyll. The chlorophyll is now positively charged and must be replenished.
Photolysis supplies the replacement. Light energy splits water into electrons, protons and oxygen. The electrons replace those lost from photosystem II, the protons accumulate in the thylakoid space, and the oxygen is released as a waste product or used in respiration. All the oxygen released in photosynthesis comes from water, not from carbon dioxide — a point established experimentally with isotopic labelling and frequently examined.
The excited electrons pass along a chain of electron carriers in the thylakoid membrane, losing energy at each transfer. This energy is used to pump protons from the stroma into the thylakoid space, creating a proton gradient.
The protons flow back into the stroma through ATP synthase, and the energy of this flow drives the synthesis of ATP from ADP and inorganic phosphate. This mechanism is chemiosmosis, and it is the same mechanism used in respiration.
The electrons finally arrive at photosystem I, where further light absorption raises their energy again. They are then accepted, together with protons from the stroma, by NADP, forming reduced NADP.
This whole route is non-cyclic photophosphorylation, and it produces ATP, reduced NADP and oxygen.
Cyclic photophosphorylation involves photosystem I alone. Excited electrons from photosystem I pass along carriers and return to photosystem I rather than to NADP. ATP is generated, but no reduced NADP and no oxygen, because no photolysis occurs. It provides additional ATP when the Calvin cycle needs more ATP than reduced NADP.
The light-independent reactions
These occur in the stroma and do not require light directly, but they depend entirely on the ATP and reduced NADP supplied by the light-dependent stage. They are therefore better described as light-independent than as dark reactions, since they stop within minutes in darkness once the supply runs out.
Carbon dioxide diffuses into the stroma and combines with ribulose bisphosphate, a five-carbon compound, in a reaction catalysed by rubisco. The resulting six-carbon compound is unstable and immediately splits into two molecules of glycerate 3-phosphate, each of three carbons.
Glycerate 3-phosphate is reduced to triose phosphate. This reduction requires both ATP, which supplies energy, and reduced NADP, which supplies hydrogen. The NADP is regenerated and returns to the thylakoid membrane.
Most of the triose phosphate is used to regenerate ribulose bisphosphate, which requires further ATP, so that the cycle can continue. Only a small proportion leaves the cycle to be used in synthesis.
For every six turns of the cycle, six molecules of carbon dioxide are fixed and twelve molecules of triose phosphate are produced, of which ten are used to regenerate six molecules of ribulose bisphosphate and two combine to form one molecule of a six-carbon sugar such as glucose.
The triose phosphate leaving the cycle is used to make glucose and then starch for storage, sucrose for transport, cellulose for cell walls, lipids by conversion to glycerol and fatty acids, and amino acids by combination with nitrogen from nitrate ions.
Limiting factors
The rate of photosynthesis is affected by light intensity, carbon dioxide concentration and temperature, and at any moment one of these limits the rate.
A graph of rate against light intensity rises steeply at first, as light is limiting, then levels off as some other factor becomes limiting. Repeating at a higher carbon dioxide concentration produces a higher plateau, which demonstrates that carbon dioxide was the limiting factor on the original plateau.
Temperature behaves differently from the other two. The light-dependent reactions are driven by light rather than by enzymes and are relatively unaffected, but the Calvin cycle is enzyme-catalysed. The rate therefore rises with temperature to an optimum and then falls sharply as the enzymes, particularly rubisco, are denatured. A temperature graph has a peak rather than a plateau.
The practical application is the commercial greenhouse, where growers supply artificial lighting, raise carbon dioxide concentration with burners, and control temperature, balancing the increased yield against the cost of providing each factor.
Evidence linking the two stages
A classic set of observations establishes the dependence of one stage on the other, and these are examinable.
If light is removed, the concentration of glycerate 3-phosphate rises and that of triose phosphate and ribulose bisphosphate falls. The reason is that without light there is no ATP or reduced NADP, so glycerate 3-phosphate cannot be reduced and accumulates, while ribulose bisphosphate cannot be regenerated and is used up by continued fixation.
If carbon dioxide is removed, the concentration of ribulose bisphosphate rises and that of glycerate 3-phosphate falls. Ribulose bisphosphate accumulates because it has nothing to combine with, while glycerate 3-phosphate continues to be converted to triose phosphate but is not replaced.
Being able to reason through these two cases from the cycle itself, rather than memorising them, is what the question is testing.
Worked examples
Example 1: Explaining a limiting factor graph (5 marks)
A graph of photosynthesis rate against light intensity is plotted at 0.04 per cent carbon dioxide and again at 0.4 per cent. Both rise and level off, the second at a higher rate. Explain.
On the rising section of each curve, light intensity is the limiting factor. Increasing it increases the rate because more light energy is absorbed by the photosystems, so more ATP and reduced NADP are produced for the Calvin cycle.
On the plateau, further increases in light intensity produce no increase in rate, so light is no longer limiting and another factor has become limiting.
Because raising the carbon dioxide concentration raises the height of the plateau, carbon dioxide must have been the limiting factor on the original plateau. With more carbon dioxide available, more ribulose bisphosphate can be carboxylated per unit time, so a higher rate is achieved before another factor, such as temperature or the quantity of rubisco, becomes limiting.
Example 2: Predicting concentration changes (5 marks)
A photosynthesising plant is suddenly placed in darkness. Predict and explain what happens to the concentrations of glycerate 3-phosphate and ribulose bisphosphate.
The concentration of glycerate 3-phosphate rises. In darkness the light-dependent reactions stop, so no ATP or reduced NADP is produced. Glycerate 3-phosphate cannot be reduced to triose phosphate because that reduction requires both, so it accumulates.
The concentration of ribulose bisphosphate falls. For a short time carbon dioxide continues to be fixed onto the ribulose bisphosphate already present, using it up. However, ribulose bisphosphate is regenerated from triose phosphate using ATP, and with no ATP available regeneration stops. Since it is being consumed but not replaced, its concentration falls.
Example 3: Explaining the source of oxygen (4 marks)
Describe the evidence and the mechanism showing that the oxygen released in photosynthesis comes from water rather than carbon dioxide.
Experiments using water labelled with the heavy oxygen isotope showed that the oxygen released carried the label, whereas when the carbon dioxide was labelled instead the released oxygen did not. This demonstrates that the oxygen originates in water.
The mechanism is photolysis. Light energy absorbed by photosystem II splits water molecules into electrons, protons and oxygen. The electrons replace those lost from the chlorophyll of photosystem II when light excited them, the protons contribute to the gradient across the thylakoid membrane and are later accepted by NADP, and the oxygen is released as a by-product.
Common mistakes and how to avoid them
The most frequent error is calling the second stage the dark reaction and stating that it occurs at night. It occurs in light or darkness but depends on products of the light-dependent stage, so it ceases shortly after light is removed.
Students often state that the oxygen released comes from carbon dioxide. It comes from the photolysis of water.
Another common slip is confusing the roles of ATP and reduced NADP in the Calvin cycle. ATP supplies energy; reduced NADP supplies hydrogen for the reduction. Both are needed for the reduction of glycerate 3-phosphate, and ATP alone for regeneration of ribulose bisphosphate.
Many candidates forget that most triose phosphate is used to regenerate ribulose bisphosphate rather than to make glucose. Only a small fraction leaves the cycle.
In limiting factor questions, answers frequently name a factor without explaining the graph. The shape is the evidence: rising means that factor is limiting, level means another is.
Finally, candidates sometimes state that cyclic photophosphorylation produces oxygen. It involves photosystem I only, with no photolysis, so it produces ATP alone.
Exam technique for "Photosynthesis: light and dark reactions"
State where each stage occurs before describing it — thylakoid membranes for light-dependent, stroma for light-independent. The location is often a mark in itself.
Track the three products of the light-dependent stage separately: ATP and reduced NADP pass to the Calvin cycle, oxygen is released. Keeping them separate prevents most confusion.
For prediction questions, reason from the cycle itself rather than recalling the answer. Ask what each substance is made from and what it is converted into, and the direction of change follows.
Use the abbreviations RuBP, GP and TP only after defining them once, and be sure to state the number of carbon atoms in each, since that is frequently credited.
In graph questions, identify the limiting factor for each section and justify it from the shape rather than from general knowledge.
Quick revision summary
Chloroplasts have a double envelope, thylakoid membranes stacked into grana carrying the photosystems for the light-dependent reactions, and a stroma containing Calvin cycle enzymes, circular DNA and 70S ribosomes. Chlorophyll a is the primary pigment with accessory pigments widening the absorbed spectrum, and the match between absorption and action spectra is the evidence that these pigments drive photosynthesis. In non-cyclic photophosphorylation, light excites electrons from photosystem II, photolysis of water replaces them and releases oxygen, electron transfer pumps protons to create a gradient, chemiosmosis through ATP synthase makes ATP, and photosystem I re-energises the electrons which reduce NADP. Cyclic photophosphorylation uses photosystem I alone and makes ATP only. In the Calvin cycle, rubisco fixes carbon dioxide onto five-carbon ribulose bisphosphate, giving two molecules of three-carbon glycerate 3-phosphate, which is reduced to triose phosphate using ATP and reduced NADP; most triose phosphate regenerates ribulose bisphosphate using further ATP, and the remainder forms sugars, starch, cellulose, lipids and amino acids. Light intensity, carbon dioxide concentration and temperature act as limiting factors, with temperature producing a peak because the Calvin cycle is enzyme-catalysed.