Photosynthesis happens in chloroplasts in two coupled stages. The first stage uses light energy to produce ATP and reduced NADP, splitting water and releasing oxygen as a by-product. The second stage uses those products to fix carbon dioxide into sugar through a three-step cycle.
Read this topic as an analogy.
Imagine a factory with two shifts working in the same building. The day shift runs on the assembly floor; the night shift runs in the warehouse. The two shifts hand off to each other across the building wall, and that handoff is the whole topic in one image.
The day shift runs on sunlight. Solar panels line the assembly floor. When sunlight strikes a panel, it knocks an electron out of the panel and into the wiring; the panel is left short of an electron until a fresh one arrives. The fresh electrons come from cracking open water bottles stored next to the panels: each cracked bottle releases two electrons (which go back to refill the panels), two protons (which get pumped into an overhead tank above the floor), and a bit of exhaust gas (oxygen) that gets vented out through a pipe.
Meanwhile, the original knocked-out electrons run through the wiring. At each station along the wiring, energy released by the electron's voltage drop is used to push more protons up into the overhead tank. By the end of the wiring, the tank above the assembly floor is full to bursting. The only way for the protons to come back down is through a waterwheel mounted in the floor, and that waterwheel turns a generator that charges a stockpile of batteries. Those batteries are the day shift's first deliverable.
Past the waterwheel, the electrons get re-energised by a second strike of sunlight and pass to a chemical-bottling station. There, the electrons combine with protons drawn from the room and a delivery substance, and the bottling station produces sealed bottles of a reducing chemical. Those bottles are the day shift's second deliverable.
Two deliverables, then: charged batteries and sealed bottles of reducer. Both get carried across the wall into the warehouse, where the night shift takes over.
The night shift cannot run without the day shift's deliveries. In the warehouse, half-built reaction chassis are waiting. A catalyst takes each incoming molecule of carbon dioxide and fastens it onto a chassis. The fastened chassis is unstable and immediately splits into two short sub-units. Each sub-unit gets charged up using a battery and reduced using a bottle of reducer, becoming a finished three-carbon component. Most of those finished components go straight back to be rebuilt into fresh half-built chassis (so the warehouse can catch the next CO₂); only a small fraction comes off the line as the day's actual finished product, which condenses into sugar.
The factory has three things that can slow it down. Insufficient sunlight on the panels (low light intensity). Insufficient carbon dioxide arriving at the warehouse (low CO₂). And the wrong working temperature for the catalysts (too cold and they move sluggishly; too hot and they fall apart). Any one of these three sets the overall production rate.
A quality-control bench off to the side handles two laboratory checks. The first uses a coloured indicator clipped onto the day-shift wiring; the indicator is blue when no current flows through it and turns colourless when current arrives. If the indicator stays blue, no electrons are reaching it, and the day shift is not running. The second check uses paper chromatography to identify which solar-panel pigments the factory has on stock; pigments dissolve into a solvent at different rates and separate into bands on the paper.
The "night shift" name is a useful image, but the warehouse does not literally run at night. It runs whenever the day shift has supplied it. Sunlight is what drives both shifts; the warehouse just runs slightly behind.
Mapping back to formal vocabulary. In an exam answer, the factory is the chloroplast. The assembly floor is the grana; the warehouse is the stroma. The day shift is the light-dependent reaction; the night shift is the Calvin cycle. Sunlight knocking electrons out of solar panels is photoionisation of chlorophyll. The water-cracking step is photolysis of water, which releases the oxygen by-product. The overhead tank is the proton gradient across the thylakoid membrane; the waterwheel is ATP synthase, and the batteries it charges are ATP. The sealed bottles of reducer are reduced NADP. In the warehouse, rubisco fastens carbon dioxide onto RuBP; the chassis splits into GP; the GP is reduced to triose phosphate using ATP and reduced NADP. Most triose phosphate regenerates RuBP so the cycle continues. The blue indicator is DCPIP. Drop the factory vocabulary when you write the answer; use the formal terms.
The chloroplast's structure matches the two stages of photosynthesis.
The chloroplast is the site of photosynthesis. Its internal architecture is built around the two sequential stages: the light-dependent reaction on the thylakoid membranes inside the grana, and the Calvin cycle in the stroma. The two compartments are tightly coupled; products of the first stage diffuse a short distance into the second.
Granum and stroma compared.
| Compartment | Location within chloroplast | Stage that runs here | Key features |
|---|---|---|---|
| Granum | Stacks of flattened thylakoid discs | Light-dependent reaction | Chlorophyll, electron carriers, ATP synthase, selectively permeable membrane |
| Stroma | Aqueous matrix around the grana | Calvin cycle | Rubisco and other Calvin enzymes, circular DNA, ribosomes |
Write
stroma of the chloroplastfor the Calvin cycle's location. Don't writestoma(that is a leaf pore),cytoplasm, ormitochondrion. The mark scheme distinguishes all four.
The granal stacking gives a large surface area for the chlorophyll, electron carriers, and ATP synthase channels of the light-dependent reaction. The thylakoid membrane is selectively permeable, so a proton gradient can build up in the lumen without leaking back across. The gradient is what powers ATP synthesis.
The light-dependent reaction produces ATP, reduced NADP, and oxygen.
Four events occur on the thylakoid membrane: photoionisation at photosystem II, electron transport with proton pumping, water photolysis, and NADP reduction at photosystem I. The three outputs are ATP (from chemiosmosis), reduced NADP (from PSI's reduction step), and oxygen (a by-product of water photolysis).
- Chlorophyll in photosystem II absorbs light. The light energy excites electrons in the chlorophyll molecule to a higher energy level.
- The excited electrons are lost from chlorophyll, and chlorophyll becomes positively charged.
Photoionisation is two events on chlorophyll only: light absorbed, electrons lost. Don't describe water photolysis, proton release, or oxygen release in a photoionisation answer; those are different events that earn marks elsewhere, not here.
Light energy splits water molecules in the thylakoid lumen. Each water molecule yields two electrons (which refill the chlorophyll positive charge), two protons (which add to the lumen pool), and half a molecule of oxygen (which is released as a by-product). Photolysis supplies the replacement electrons that keep the light-dependent reaction running.
The electron transport chain pumps protons from the stroma into the thylakoid lumen, creating a steep gradient. Protons can only return through ATP synthase channels in the granal membrane. Proton flow through ATP synthase drives the phosphorylation of ADP and inorganic phosphate to ATP. The product is photophosphorylation; the mechanism is chemiosmosis.
The electrons that crossed the first chain arrive at photosystem I, where a second photon re-excites them to high energy. The re-energised electrons reduce NADP: NADP combines with protons drawn from the stroma and the electrons from PSI to form reduced NADP. ATP, reduced NADP, and oxygen leave the light-dependent reaction together.
Write
reduced NADP(orNADPH). Don't writereduced NADorNADH; NAD is the respiration coenzyme. Substituting NAD for NADP is the most consistent language error in this topic and is rejected every time.
The Calvin cycle fixes carbon dioxide into triose phosphate through three stages.
The Calvin cycle uses the ATP and reduced NADP from the light-dependent reaction to incorporate carbon dioxide into organic molecules. It takes place in the stroma of the chloroplast and has three named stages: carbon fixation, reduction, and RuBP regeneration.
- Carbon fixation. Carbon dioxide combines with RuBP (a five-carbon molecule) in a reaction catalysed by rubisco. The unstable six-carbon intermediate immediately splits into two molecules of glycerate-3-phosphate (GP), each a three-carbon molecule.
- Reduction. Each GP is converted into triose phosphate (TP), a three-carbon sugar. ATP supplies the energy for the reaction; reduced NADP supplies the hydrogen that does the actual reduction.
- RuBP regeneration. Most of the triose phosphate is rearranged through a series of enzyme-catalysed steps back into RuBP, ready to fix the next carbon dioxide molecule. ATP is consumed in this stage too. A small fraction of TP exits the cycle to form glucose, lipids, amino acids, or glycerol.
Write
triose phosphatein full before using the abbreviation TP. BareTPon first use is rejected. Writeglycerate-3-phosphateorGP;glucose phosphateandglucose-3-phosphateare different compounds and score zero.
ATP supplies energy; reduced NADP supplies the hydrogen that does the reduction. Don't write
phosphate from ATP; phosphate transfer is not the credited mechanism here. Don't writereduced NADP oxidises GP; that reverses the direction of the reaction.
Pitfall — The four-step LDR-to-Calvin chain ends at RuBP
When the light-dependent reaction slows, the Calvin-cycle consequence is a four-step chain. Stopping at step one caps your answer at one mark.
Step one: less ATP and less reduced NADP are produced. Step two: less GP is reduced to triose phosphate. Step three: less triose phosphate is available to regenerate RuBP. Step four: less RuBP is available to react with carbon dioxide. The chain must end at RuBP for full credit. Only 1.68% of students scored all four marks on the 2022 photoionisation-inhibition question.
When the rate is reduced, write
lessat every step of the chain. Don't writeno ATPorno RuBP; the rate is lower, not zero. The chain describes a reduction in supply, not a complete failure of supply.
Three limiting factors set the rate of photosynthesis.
When one variable is below the level needed to sustain the maximum rate, that variable limits the overall rate regardless of the others. AQA examines three limiting factors: light intensity, carbon dioxide concentration, and temperature.
Higher light intensity raises the rate of photoionisation in PSII, increasing electron supply, water photolysis, and the production of ATP and reduced NADP. At low light intensities, the entire downstream chain (Calvin cycle included) is throttled. The credited variable name is light intensity, not amount of light and not sunlight.
Higher CO₂ raises the rate of the rubisco-catalysed carboxylation reaction. The low-CO₂ Explain answer is two marks: less CO₂ reacts with RuBP; less GP is formed. Rubisco denaturation belongs in temperature answers, not CO₂ answers; describing denaturation here is observational, not mechanistic.
Rising temperature raises the kinetic energy of molecules and the rate of enzyme-catalysed reactions up to the enzyme's optimum. Beyond optimum, the tertiary structure of rubisco and the other Calvin enzymes is disrupted, and the rate falls sharply. Temperature affects both stages, but its mark-scheme weight sits on the Calvin enzymes.
For an investigation that varies one factor, the other two must be stated as controlled variables. Don't jump to denaturation when a rate falls over time; the substrate may be being consumed, or the product may be inhibiting the enzyme. Read the data before committing to a mechanism.
For controlled variables, write
light intensityandcarbon dioxide concentration.Amount of light,amount of CO₂, andsunlightare rejected as uncontrollable phrasings; sunlight is rejected because it cannot be controlled in the laboratory.
Multiple pigments capture different wavelengths; chromatography separates them.
Plants use a suite of pigments, including chlorophyll a, chlorophyll b, carotene, and xanthophyll, that absorb light across different regions of the visible spectrum. Having multiple pigments raises the rate of the light-dependent reaction by harvesting a broader range of wavelengths. Blue and red are absorbed strongly; green is absorbed at a lower rate, but not at zero.
- Draw a pencil baseline near the bottom of the chromatography paper. Pencil graphite is insoluble in the solvent; ink would dissolve in the organic solvent and displace the origin line.
- Spot a concentrated leaf extract on the baseline. Let it dry, then spot again on the same point to build a strong sample.
- Place the paper in a beaker so that the solvent level is below the pencil baseline. If the solvent rises above the baseline, the pigment spots dissolve into the solvent reservoir and never travel.
- Allow the organic solvent to ascend the paper, carrying each pigment at a rate set by its solubility in the solvent. Remove the paper before the solvent front reaches the top of the paper.
- Calculate the Rf value of each pigment: the distance the pigment has travelled divided by the distance the solvent front has travelled, both measured from the baseline.
Different pigments have different solubilities in the organic solvent; more soluble pigments travel further with the solvent front, and less soluble pigments stay closer to the baseline.
Separation is due to different solubilities in the organic solvent.
Different polarities,different molecular sizes, anddifferent molecular massesare all rejected. On pigment-absorption graphs, name all three colours: blue, red, and green. Omitting green (because green is absorbed less, not absorbed at zero) is the most-flagged error.
DCPIP shows electrons flowing from the light-dependent reaction; the control tube has two functions.
DCPIP is an artificial electron acceptor: blue when oxidised, colourless when reduced. Isolated chloroplasts in a buffer with DCPIP are exposed to light. As the light-dependent reaction transfers electrons through its chain, DCPIP intercepts them and is reduced. The blue colour fades to colourless. The rate of colour change measures the rate of the light-dependent reaction.
Function one: a tube containing DCPIP and buffer but no chloroplasts shows that light alone does not reduce DCPIP. Function two: the contrast with the experimental tube shows that chloroplasts are required for the colour change. Both functions are independent mark points. It is a control or for comparison earns one mark at most.
DCPIP is reduced, not oxidised. The electrons reduce DCPIP;
oxidation of DCPIPreverses the chemistry. In a chloroplast osmosis context, writechloroplast;cell bursts,turgid,flaccid, andplasmolysedare rejected because the experiment uses isolated chloroplasts, not whole cells.
Key terms
- light-dependent reaction
- Calvin cycle
- chloroplast
- stroma
- reduced NADP
- ATP
- RuBP
- rubisco
- GP
- triose phosphate
- DCPIP
- light intensity