C1.3 Photosynthesis
SL & HL+ AHL sectionsSL and HL, with AHL content · Molecules · Theme C
Big questions
- How does a leaf turn light into the chemical energy stored in sugar?
- What sets the upper limit on how fast a plant can photosynthesise?
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Photosynthesis: light energy into chemical energy
- Outer membrane
- Inner membrane (the two membranes form the double-membrane envelope)
- Stroma: site of the light-independent reactions (Calvin cycle)
- Granum: a stack of thylakoids
- Thylakoid membrane: site of the light-dependent reactions
- Thylakoid space (lumen) inside a thylakoid
- Lamella (intergranal thylakoid) joining grana
- Starch grain
- 70S ribosomes
- Naked circular DNA
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Text description of this visual
The first model is a chloroplast cut in half. It is bounded by two membranes (the envelope) with a narrow space between them. Inside is the stroma, a protein-rich fluid that contains starch grains, 70S ribosomes and a loop of naked DNA. Flattened sacs called thylakoids, each enclosing a thylakoid space (lumen), are stacked into grana, and neighbouring grana are joined by single unstacked thylakoids called lamellae. The light-dependent reactions take place in the thylakoid membranes and the light-independent reactions (the Calvin cycle) in the stroma. The second model (AHL) is a patch of thylakoid membrane with the stroma above and the thylakoid space below. In seven steps it shows light absorbed by photosystem II, water split on the lumen side, electrons passing along a chain of carriers while H⁺ is pumped into the lumen, light re-exciting the electrons in photosystem I, NADP reduced in the stroma, ATP made by ATP synthase as H⁺ diffuses back to the stroma, and cyclic electron flow around photosystem I.
Chloroplast
- Outer membrane
- The outer of the two membranes of the chloroplast envelope. It is freely permeable to small molecules such as CO₂, O₂ and water.
- Inner membrane
- The inner membrane of the envelope. It controls which substances enter and leave the stroma, for example letting triose phosphate out to the cytoplasm in exchange for phosphate.
- Stroma
- The fluid inside the inner membrane. It contains enzymes of the Calvin cycle, including large amounts of Rubisco, plus ATP, reduced NADP and the substrates they act on.
- Granum
- A stack of disc-shaped thylakoids (plural grana). Stacking packs a large area of thylakoid membrane, with its photosystems, into a small volume.
- Thylakoid
- A flattened membrane sac. Its membrane holds the photosystems, electron carriers and ATP synthase, so the light-dependent reactions happen here.
- Thylakoid space (lumen)
- The small volume of fluid enclosed by each thylakoid membrane. H⁺ ions accumulate here during the light-dependent reactions, so a steep proton gradient builds up quickly.
- Lamella (intergranal thylakoid)
- A single, unstacked thylakoid that joins grana together. Its lumen is continuous with the lumens of the thylakoids in the grana it connects.
- Starch grain
- Excess carbohydrate made in photosynthesis is stored here as insoluble starch, which has no osmotic effect. It is broken down again when sugar is needed, for example at night.
- 70S ribosomes
- Smaller ribosomes like those of prokaryotes. They make some of the chloroplast's own proteins; most chloroplast proteins are coded by nuclear genes and imported.
- Naked circular DNA
- The chloroplast's own small genome, a loop of DNA not associated with histones, like a bacterial chromosome. It is evidence for the endosymbiotic origin of chloroplasts.
- Light-dependent reactions
- Take place in the thylakoid membranes. Light energy is used to split water, release oxygen, make ATP and reduce NADP.
- Light-independent reactions
- The Calvin cycle takes place in the stroma. CO₂ is fixed by Rubisco and reduced to triose phosphate using ATP and reduced NADP from the thylakoids.
Thylakoid membrane (AHL)
- Stroma
- The fluid outside the thylakoid. Its H⁺ concentration stays low because protons are pumped out of it. NADP is reduced and ATP is made on this side.
- Thylakoid space (lumen)
- The fluid inside the thylakoid. Photolysis and proton pumping make its H⁺ concentration high, so a proton gradient develops across the thylakoid membrane.
- Thylakoid membrane
- A phospholipid bilayer that is impermeable to H⁺, so the proton gradient is kept. It holds the photosystems, electron carriers and ATP synthase in fixed positions.
- Light-harvesting (antenna) complex
- An array of many chlorophyll and accessory pigment molecules (green and orange) held by proteins. Each absorbs photons and passes the energy, pigment to pigment, towards the reaction centre.
- Reaction centre (P680)
- A special pair of chlorophyll a molecules in photosystem II. When energy reaches it, an electron is excited and emitted to an electron acceptor. It is refilled with electrons from water.
- Photosystem II
- A membrane protein complex holding an array of pigments and a reaction centre. It emits excited electrons into the electron transport chain and splits water.
- Water-splitting site
- Part of photosystem II on the lumen side (the oxygen-evolving complex, containing manganese). It splits water — 2H₂O → O₂ + 4H⁺ + 4e⁻ — releasing H⁺ into the lumen.
- Plastoquinone
- A small, mobile electron carrier that moves within the membrane. It carries electrons from photosystem II to the cytochrome complex, taking up H⁺ from the stroma as it does so.
- Cytochrome complex
- An electron carrier that uses energy released by the electrons to pump H⁺ from the stroma into the thylakoid space, building the proton gradient.
- Plastocyanin
- A small, mobile protein in the thylakoid space that carries electrons from the cytochrome complex to photosystem I.
- Photosystem I
- The second photosystem, with its own antenna of pigments. It is located where the thylakoid membrane faces the stroma and re-excites electrons that arrive from photosystem II.
- Reaction centre (P700)
- The reaction-centre chlorophyll of photosystem I. Absorbed energy excites an electron, which is emitted to ferredoxin; the electron is replaced by one arriving via plastocyanin.
- Ferredoxin
- An electron carrier on the stroma side of the membrane. It passes excited electrons from photosystem I to NADP reductase, or, in cyclic flow, back to the cytochrome complex.
- NADP reductase
- An enzyme on the stroma surface of the membrane. It transfers two electrons from photosystem I and one H⁺ from the stroma to NADP, forming reduced NADP.
- ATP synthase
- Lets H⁺ diffuse from the lumen back to the stroma. The flow turns part of the enzyme, which phosphorylates ADP to ATP on the stroma side (chemiosmosis).
- Light excites photosystem II. Photons (yellow) are absorbed by pigment molecules in the antenna complex. The energy is passed from pigment to pigment until it reaches the reaction centre chlorophyll, P680. An electron in P680 is raised to a higher energy level and emitted to an electron acceptor. This happens four times, so four excited electrons (purple) leave photosystem II.
- Photolysis of water. P680 has lost electrons, so it is a very strong oxidising agent. At the water-splitting site on the lumen side of photosystem II, two water molecules are split — 2H₂O → O₂ + 4H⁺ + 4e⁻. The electrons replace those lost by P680, the H⁺ (red) stay in the thylakoid space, and the oxygen is a waste product that diffuses away.
- Electron transport pumps protons. The excited electrons pass along a chain of carriers — plastoquinone, the cytochrome complex and plastocyanin — to photosystem I, losing energy at each step. The energy is used to pump H⁺ from the stroma into the thylakoid space. Together with H⁺ from photolysis this builds a steep proton gradient across the membrane.
- Light re-excites the electrons in photosystem I. Photons are absorbed by the antenna pigments of photosystem I and the energy is passed to its reaction centre, P700. Electrons are excited again and emitted to ferredoxin on the stroma side. The electrons that arrived from photosystem II via plastocyanin replace the ones P700 has lost.
- NADP is reduced in the stroma. Ferredoxin passes the electrons to NADP reductase on the stroma side of the membrane. Each NADP accepts two electrons and one H⁺ from the stroma, becoming reduced NADP. The four electrons from two water molecules reduce two NADP. Removing H⁺ from the stroma adds to the proton gradient.
- ATP synthesis by chemiosmosis. H⁺ is now much more concentrated in the thylakoid space than in the stroma, but the membrane is impermeable to it. H⁺ diffuses back into the stroma through ATP synthase, down its concentration gradient. The flow turns part of the enzyme, which joins ADP and inorganic phosphate (Pi) to make ATP in the stroma. Because the electrons came from photosystem II and end up in reduced NADP, this is non-cyclic photophosphorylation.
- Cyclic photophosphorylation. When the stroma needs extra ATP, excited electrons from photosystem I return via ferredoxin to the cytochrome complex instead of reducing NADP. They pass back to photosystem I through plastocyanin, pumping more H⁺ on the way, so ATP is made by ATP synthase. Photosystem II is not used, so no water is split, no oxygen is released and no NADP is reduced.
Photosynthesis is an energy transformation: light energy absorbed by pigments is converted into chemical energy in the bonds of carbon compounds such as glucose. Producers that photosynthesise supply this chemical energy to almost every food chain, so photosynthesis is the source of most of the energy used for life processes in ecosystems (see C4.2 Transfers of energy and matter).
The word equation
carbon dioxide + water → glucose + oxygen (light energy absorbed by chlorophyll)
The balanced equation is often written 6CO₂ + 6H₂O → C₆H₁₂O₆ + 6O₂.
Reducing carbon dioxide using hydrogen from water
Compare the molecules: carbon dioxide (CO₂) has one carbon to two oxygen atoms, while glucose (C₆H₁₂O₆) has one carbon to two hydrogens and one oxygen. To turn CO₂ into glucose, hydrogen must be added and oxygen removed. This is reduction.
The hydrogen comes from splitting water. Water is a very stable molecule, so splitting it needs energy, and this is supplied by light. Each water molecule provides hydrogen in the form of electrons and protons (H⁺), and its oxygen is released.
Oxygen is a by-product
Photosynthesis that splits water and releases oxygen is carried out by plants, algae and cyanobacteria. All of the oxygen released comes from water, not from carbon dioxide. Experiments in the 1940s with water labelled with the heavy oxygen isotope ¹⁸O showed that the labelled oxygen appeared in the O₂ given off.
Photosynthesising organisms use some of the oxygen in their own aerobic respiration, but in the light they produce far more than they use, and the rest diffuses out as waste. In aquatic plants it can be seen as bubbles; on land it diffuses unseen into the air through the stomata.
Separating photosynthetic pigments by chromatography
Leaves contain several photosynthetic pigments: chlorophyll a and b, and accessory pigments such as carotene and xanthophylls (together called carotenoids). They can be separated by paper chromatography or thin-layer chromatography (TLC).
Method in outline
- Grind leaf tissue with a little sand and a solvent such as propanone to extract the pigments.
- Draw a pencil origin line about 1 cm from the end of the strip (ink would dissolve and run).
- Build up a small, concentrated spot of extract on the origin by adding tiny drops and letting each dry.
- Stand the strip in a sealed tube with the running solvent below the origin, so the spot does not dissolve into the pool.
- Remove the strip before the solvent reaches the top and mark the solvent front at once, before it evaporates.
Each pigment moves at a different speed. A pigment that is more soluble in the running solvent, and less attracted to the stationary paper or TLC layer, travels further.
Calculating Rf
Rf = distance moved by the spot ÷ distance moved by the solvent
Measure both distances from the origin line, to the centre of the spot. For example, a blue-green spot 31 mm from the origin with a solvent front at 50 mm has Rf = 31 ÷ 50 = 0.62, which fits chlorophyll a.
Choose a spot to measure it from the origin line.
| Pigment | Colour | Rf |
|---|---|---|
| β-carotene | orange-yellow | 0.92 |
| Phaeophytin | grey | 0.78 |
| Chlorophyll a | blue-green | 0.62 |
| Chlorophyll b | yellow-green | 0.50 |
| Xanthophyll (e.g. lutein) | yellow | 0.38 |
| Xanthophyll (e.g. violaxanthin) | yellow | 0.27 |
Rf values depend on the solvent, the paper or TLC plate and the temperature, so the ones here are typical rather than fixed. Identify a pigment from its colour, its Rf and its position relative to the other spots. The order shown is for a typical non-polar solvent; a different solvent can change the order of the spots.
Text description of this simulation
A chromatography strip with a pencil origin line near the bottom where a concentrated spot of leaf extract was placed. A slider lets the running solvent rise up the strip. As it does, the pigments separate into spots: orange-yellow carotene runs nearly with the solvent front, followed by grey phaeophytin, blue-green chlorophyll a, yellow-green chlorophyll b and yellow xanthophylls lowest. Choosing a spot measures its distance from the origin and the distance moved by the solvent, calculates Rf = distance moved by spot ÷ distance moved by solvent, and identifies the pigment from a reference table of colours and typical Rf values.
Typical results, from the top of the strip: carotene (orange-yellow, Rf close to 1), sometimes grey phaeophytin, chlorophyll a (blue-green), chlorophyll b (yellow-green) and xanthophylls (yellow) lowest. This order is for the usual non-polar running solvent. Exact Rf values depend on the solvent, paper and temperature, and with a different solvent (or a different type of plate) the spots can come out in a different order, so identify pigments by both colour and Rf.
Absorption and action spectra
Why pigments absorb only some wavelengths
Sunlight reaching the ground includes the visible wavelengths 400–700 nm, the range used in photosynthesis. Shorter wavelengths (violet) carry more energy per photon than longer ones (red).
When a pigment absorbs a photon, the energy raises an electron in the molecule to a higher energy level: the electron becomes excited. This is how light energy starts to be converted into chemical energy. An electron can only jump between particular energy levels, so only photons with the right amount of energy (the right wavelengths) are absorbed. Other wavelengths are reflected or pass through.
Absorption spectra
An absorption spectrum plots the percentage of light absorbed by a pigment against wavelength. Label the x-axis with both the wavelength in nm and the colours of light.
- Chlorophylls absorb violet-blue and red light strongly and green light very little. Green light is reflected or transmitted, which is why leaves look green.
- Carotenoids absorb violet, blue and blue-green light, but not yellow, orange or red.
Action spectra
An action spectrum plots the rate of photosynthesis at each wavelength. Lighting a pondweed with one colour at a time (coloured LEDs or filters) and measuring the rate of oxygen production or CO₂ uptake gives the data.
- Chlorophyll a (absorption)
- 1%
- Chlorophyll b (absorption)
- 0%
- Carotenoids (absorption)
- 0%
- Action spectrum (rate)
- 25%
At 550 nm (green light): the chlorophylls and carotenoids absorb very little in solution, and much of this light is reflected, which is why leaves look green. Relative rate of photosynthesis: about 25% of the maximum. The rate is well above zero, and above the absorption curves, because in a leaf this light is scattered back and forth through many chloroplasts, so its long path means much of it is absorbed in the end; accessory pigments and the weak edges of the chlorophyll bands also absorb a little of it.
Show data table
| nm | Colour | Chl a | Chl b | Carotenoids | Action |
|---|---|---|---|---|---|
| 400 | violet | 33 | 3 | 3 | 44 |
| 420 | violet | 92 | 25 | 62 | 94 |
| 440 | violet | 68 | 74 | 84 | 98 |
| 460 | blue | 6 | 80 | 83 | 82 |
| 480 | blue | 0 | 7 | 82 | 56 |
| 500 | green | 0 | 0 | 11 | 27 |
| 520 | green | 0 | 0 | 0 | 23 |
| 540 | green | 0 | 0 | 0 | 25 |
| 560 | green | 2 | 0 | 0 | 27 |
| 580 | yellow | 5 | 4 | 0 | 31 |
| 600 | orange | 8 | 6 | 0 | 33 |
| 620 | red | 11 | 4 | 0 | 33 |
| 640 | red | 8 | 32 | 0 | 44 |
| 660 | red | 68 | 7 | 0 | 72 |
| 680 | red | 14 | 0 | 0 | 25 |
| 700 | red | 0 | 0 | 0 | 6 |
Why is the action spectrum above zero in green light? The absorption curves are for pigments extracted into a solvent, where light passes through only once. In a leaf, light is scattered and reflected between cells, so it travels a long path through many chloroplasts and even weakly absorbed green light is largely absorbed in the end. Accessory pigments and the weak edges of the chlorophyll bands also absorb some green light and pass the energy to chlorophyll a. So photosynthesis in green light is slower than in blue or red, but not zero.
Approximate shapes for pigments dissolved in an organic solvent (each scaled to 100% at its own peak) and a typical action spectrum. Exact peak positions shift a little inside the leaf.
Text description of this simulation
A graph with wavelength from 400 to 700 nm on the x-axis, labelled with both numbers and the colours of light (violet, blue, green, yellow, orange, red). Chlorophyll a absorbs most strongly at about 430 nm and 662 nm, chlorophyll b at about 453 nm and 642 nm, and both absorb very little green light. Carotenoids absorb violet, blue and blue-green light only. The action spectrum, showing the rate of photosynthesis at each wavelength, has peaks in blue and red and a trough in green that does not fall to zero. A slider picks a wavelength and reports how strongly each pigment absorbs it and the relative rate of photosynthesis.
| Absorption spectrum | Action spectrum | |
|---|---|---|
| y-axis | % light absorbed by a pigment | rate of photosynthesis |
| Peaks | blue and red | blue and red |
| Green | very low for chlorophyll | low, but well above zero |
The two have peaks in the same places because the light absorbed by chlorophyll is the light used. Some green light is still used because accessory pigments absorb it and pass the energy to chlorophyll.
Calculating rates for an action spectrum
Rate = amount of O₂ produced (or CO₂ used) ÷ time. If a pondweed releases 1.8 cm³ of oxygen in 6 minutes under blue light, the rate is 1.8 ÷ 6 = 0.30 cm³ min⁻¹. Repeat for each wavelength, then plot rate (y-axis) against wavelength (x-axis), joining the points with a smooth curve or straight lines.
Limiting factors and how to investigate them
The rate of photosynthesis can be limited by light intensity, carbon dioxide concentration or temperature. At any moment, the limiting factor is the one that is furthest from its optimum: raising it increases the rate, while raising either of the others has no effect.
- Light intensity: at low intensity the rate rises in proportion to it, because the light-dependent reactions supply ATP and reduced NADP more quickly. Then the rate levels off (a plateau) as another factor becomes limiting.
- CO₂ concentration: there is no photosynthesis without CO₂; the rate rises with concentration and then plateaus.
- Temperature: the rate rises with temperature up to an optimum, because enzyme-catalysed reactions speed up. Above the optimum it falls steeply as enzymes such as Rubisco are denatured.
the plotted factor is limiting another factor is limiting (plateau)
Lamp at about 18 cm (100% at 10 cm; intensity ∝ 1/distance²)
- Rate
- 36 a.u.
- Pondweed
- ≈ 14 bubbles min⁻¹
- Limiting factor
- light intensity
Rate 36 (arbitrary units). The limiting factor is light intensity: increasing it would increase the rate.
Show data table
| Light intensity (%) | Rate (a.u.) |
|---|---|
| 0 | 0.0 |
| 10 | 12.0 |
| 20 | 24.0 |
| 30 | 35.8 |
| 40 | 46.4 |
| 50 | 54.0 |
| 60 | 58.1 |
| 70 | 59.9 |
| 80 | 60.6 |
| 90 | 61.0 |
| 100 | 61.1 |
Qualitative model with arbitrary units. Each factor sets a maximum possible rate and the lowest one wins; real curves bend more gradually, and the optimum temperature varies between species.
Text description of this simulation
A graph of the rate of photosynthesis against light intensity, CO₂ concentration or temperature (you choose the x-axis), with the other two factors fixed by sliders. Where the plotted factor is the limiting factor, the line is solid and the rate rises as the factor increases; where another factor is limiting, the line is dashed and the rate levels off. The temperature graph rises to an optimum and then falls steeply as enzymes are denatured. The panel names the limiting factor, estimates oxygen bubbles per minute from a pondweed, and converts light intensity into the lamp distance that would give it, using the inverse square law. On the CO₂ graph, lines mark pre-industrial, present-day and typical FACE enrichment concentrations.
Designing an experiment
Choose one factor as the independent variable and measure the rate as the dependent variable, keeping the other factors constant (controlled variables).
| Factor | How to vary it | How to keep it constant |
|---|---|---|
| Light intensity | move a lamp to different distances (intensity is proportional to 1/distance²) or use a light meter | fixed lamp distance; LED or heat shield so the water is not warmed |
| CO₂ concentration | add measured amounts of sodium hydrogencarbonate to boiled, cooled water | add excess hydrogencarbonate |
| Temperature | thermostatic water bath | water bath at, say, 25 °C |
The rate can be measured by counting oxygen bubbles from a cut pondweed stem, by collecting the gas and measuring its volume, by timing leaf discs as they float, or by measuring CO₂ uptake from the change in pH (hydrogencarbonate indicator).
A hypothesis is a provisional explanation that can be tested, for example: “The rate will increase with light intensity, because more light energy is available to excite electrons, until another factor limits it”. Hypotheses can be proposed before an experiment, based on theory, or after it, based on the data, and need repeated testing.
Carbon dioxide enrichment experiments
Atmospheric CO₂ has risen from about 280 ppm before industrialization to about 420 ppm today and will keep rising this century. Because CO₂ is often the limiting factor in bright light, scientists want to predict how future CO₂ levels will affect photosynthesis, crop yields and the carbon stored by forests.
Greenhouse experiments
In enclosed greenhouse experiments plants are grown in glasshouses or chambers with CO₂ raised to set levels. Temperature, light, water and minerals can be closely controlled, so the effect of CO₂ alone can be measured. Commercial growers already enrich greenhouse air with CO₂ to raise yields. However, plants in pots and enclosures may not behave like plants in fields and forests.
FACE experiments
Free-air carbon dioxide enrichment (FACE) experiments are done in the open, on crops, grassland and forest. Rings of pipes or towers release CO₂ on the upwind side, and computer-controlled sensors keep the concentration inside the ring at a target such as 550 ppm. Control rings are set up in the same way but release ordinary air, so the only difference is the CO₂ concentration. The first FACE experiments used crops and young tree plantations; later ones enriched mature, natural forests.
FACE results show that photosynthesis is faster at higher CO₂, but growth and yield increased less than many enclosed experiments had predicted, partly because other factors such as nitrogen and water then become limiting.
Control in the laboratory and in the field
Control of variables is easier in a laboratory or greenhouse, but some questions can only be answered in the field, in natural ecosystems. In FACE, weather and soil cannot be controlled, so ecologists use many replicate rings, choose plots with similar soils and vegetation, and monitor uncontrolled variables such as temperature and rainfall.
Photosystems: arrays of pigment molecules
Photosynthetic pigments do not work as separate molecules. They are organized into photosystems: large complexes of proteins holding many pigment molecules in fixed positions. Photosystems are always located in membranes: the thylakoid membranes of chloroplasts in plants and algae, and the thylakoid membranes in the cytoplasm of cyanobacteria.
Each photosystem has two parts:
- Light-harvesting (antenna) complexes: hundreds of chlorophyll a, chlorophyll b and carotenoid molecules. When one absorbs a photon, the excitation energy is passed from pigment to pigment, always towards the centre.
- A reaction centre: a special pair of chlorophyll a molecules. When the energy reaches it, an electron is excited enough to be emitted to an electron acceptor. This is the step that turns light energy into chemical energy.
There are two types, named in the order they were discovered: photosystem II, with reaction centre P680, and photosystem I, with reaction centre P700 (the numbers are the wavelengths in nm they absorb best).
Why an array is better than single pigments
- More photons are caught. Even in bright light one chlorophyll molecule absorbs only a few photons per second. Hundreds of antenna pigments funnel energy to one reaction centre, so it is supplied rapidly.
- More wavelengths are used. Different pigments absorb different wavelengths, so a greater proportion of the energy in sunlight is harvested.
- Energy is funnelled precisely. The proteins hold each pigment at the right distance and orientation for energy to pass towards the reaction centre.
- Electrons can be passed on. The reaction centre is next to the electron acceptors that take the excited electrons.
A single pigment molecule could not perform any part of photosynthesis. In solution, isolated chlorophyll simply releases absorbed energy as heat and red fluorescence, because there is no acceptor to take an excited electron.
Photolysis of water in photosystem II
When P680 emits an excited electron it becomes positively charged and is a very strong oxidising agent. It regains electrons by splitting water. This happens at a water-splitting site (the oxygen-evolving complex, which contains manganese) on the side of photosystem II facing the thylakoid space:
2H₂O → O₂ + 4H⁺ + 4e⁻
The splitting is called photolysis because it depends on light-driven loss of electrons from P680.
- The electrons replace those emitted by P680, so photosystem II can be excited again.
- The protons (H⁺) are released into the thylakoid space and add to the proton gradient used to make ATP.
- The oxygen is a waste product. It diffuses out of the chloroplast and the cell.
Consequences of oxygen production
Photolysis evolved in the ancestors of cyanobacteria and transformed the Earth:
- Oxygen accumulated in the oceans and atmosphere, starting about 2.4 billion years ago (the Great Oxidation Event).
- Dissolved iron in the oceans was oxidised and precipitated, forming the banded iron formations that are now major iron ores.
- Aerobic respiration became possible, releasing much more energy from food than anaerobic pathways; organisms that could not tolerate oxygen were restricted to anoxic habitats.
- An ozone layer formed in the upper atmosphere, absorbing ultraviolet radiation and making life on land safer.
ATP production by chemiosmosis
ATP is made in thylakoids by chemiosmosis, as in mitochondria (see C1.2 Cell respiration). Making ATP using light energy is called photophosphorylation.
- Electron transport: excited electrons pass along a chain of electron carriers in the thylakoid membrane — plastoquinone, the cytochrome complex and plastocyanin.
- Proton pumping: energy released as electrons pass along the chain is used to pump H⁺ from the stroma into the thylakoid space. Together with H⁺ from photolysis, this creates a high concentration of H⁺ in the small thylakoid space: a proton gradient.
- ATP synthase: the membrane is impermeable to H⁺, so the protons can only diffuse back to the stroma through ATP synthase. Their flow down the gradient makes part of the enzyme rotate, and it phosphorylates ADP to ATP on the stroma side.
Excited electrons can come from either photosystem:
| Non-cyclic photophosphorylation | Cyclic photophosphorylation | |
|---|---|---|
| Electrons from | photosystem II, then re-excited in photosystem I | photosystem I only |
| Electrons end up | reducing NADP | back in photosystem I (via ferredoxin and the cytochrome complex) |
| Products | ATP, reduced NADP and O₂ | ATP only |
| Water split? | yes | no |
Cyclic photophosphorylation tops up ATP, because the Calvin cycle uses more ATP than reduced NADP.
NADP reduction and the thylakoid as a system
Reduction of NADP by photosystem I
Electrons arriving at photosystem I from photosystem II have lost energy. Light absorbed by photosystem I re-excites them at P700. They are emitted to ferredoxin, a carrier on the stroma side of the membrane, and then passed to the enzyme NADP reductase. NADP is reduced when it accepts two electrons that came from photosystem I and one H⁺ from the stroma:
NADP + 2e⁻ + H⁺ → reduced NADP
Reduced NADP and ATP are the two useful products of the light-dependent reactions, and both are released into the stroma, where the Calvin cycle uses them.
- Photosystem II: antenna pigments pass energy to the reaction centre chlorophyll (P680), which emits excited electrons
- Plastoquinone: carries electrons (and H⁺ picked up from the stroma) through the membrane
- Cytochrome complex: electron carrier that uses energy from the electrons to pump H⁺ into the thylakoid space
- Plastocyanin: carries electrons through the thylakoid space to photosystem I
- Photosystem I: light re-excites electrons at its reaction centre (P700)
- Ferredoxin: accepts excited electrons from photosystem I on the stroma side
- NADP reductase: NADP + 2e⁻ + H⁺ (from the stroma) → reduced NADP
- ATP synthase: H⁺ diffuse back to the stroma through it (chemiosmosis), powering ADP + Pi → ATP
Thylakoids are systems
A thylakoid works as a system because its parts interact and depend on each other. Photosystem II supplies excited electrons to the carrier chain; the chain supplies energy to pump protons; the proton gradient drives ATP synthase; photosystem I supplies electrons to NADP reductase; photosystem II replaces photosystem I’s electrons; and water replaces photosystem II’s.
| Process | Where in the thylakoid |
|---|---|
| Photolysis of water | photosystem II, on the side facing the thylakoid space |
| Proton pumping | cytochrome complex: from stroma into thylakoid space |
| ATP synthesis | ATP synthase: H⁺ flows from thylakoid space to stroma; ATP made in the stroma |
| NADP reduction | NADP reductase, on the stroma side of the membrane |
Features of the thylakoid that make this work: the membrane is impermeable to H⁺; the thylakoid space is a small volume, so the H⁺ concentration rises quickly; the phospholipid bilayer holds the hydrophobic photosystems; and the components are held in the right order and orientation.
The Calvin cycle
The light-independent reactions form the Calvin cycle in the stroma. They do not use light directly, but they depend on ATP and reduced NADP from the thylakoids.
Carbon fixation by Rubisco
CO₂ diffuses into the stroma and is fixed by the enzyme Rubisco (ribulose bisphosphate carboxylase), which joins it to RuBP (ribulose bisphosphate, 5C). The six-carbon product is unstable and splits at once into two molecules of glycerate 3-phosphate (GP, 3C):
RuBP + CO₂ → 2 glycerate 3-phosphate
There is more Rubisco on the planet than any other enzyme. It makes up a large share of the protein in leaves because each molecule fixes only a few CO₂ molecules per second, and it binds CO₂ poorly when the CO₂ concentration is low. A plant therefore needs a very high concentration of Rubisco in the stroma to fix carbon at a useful rate.
Synthesis of triose phosphate
Glycerate 3-phosphate is an acid. It is reduced to triose phosphate (TP), a three-carbon sugar phosphate. Reduced NADP supplies the hydrogen and ATP supplies the energy, one of each per GP.
Regeneration of RuBP
For the cycle to continue, every RuBP used must be replaced. In a series of reactions (details not needed), five TP are converted into three RuBP (15 carbon atoms in each case), using ATP.
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Text description of this animation
A diagram of the Calvin cycle in the stroma, followed for three turns. Each dot is one carbon atom and the dark dots are carbon atoms just fixed from carbon dioxide. Three molecules of RuBP (5 carbon atoms each) each combine with one CO₂, catalysed by Rubisco, giving six molecules of glycerate 3-phosphate (3 carbon atoms each). These are reduced to six triose phosphates using six ATP and six reduced NADP. One triose phosphate leaves the cycle; the other five are converted, using three ATP, back into three RuBP. A final step totals six turns, which make two spare triose phosphates — enough for one glucose. Counters record the CO₂ fixed, the ATP and reduced NADP used and the triose phosphate leaving the cycle.
- CO₂ enters the stroma. Carbon dioxide diffuses into the stroma from the air spaces in the leaf. Three molecules of ribulose bisphosphate (RuBP), a five-carbon sugar phosphate, are waiting there.
- Carbon fixation by Rubisco. Rubisco joins each CO₂ to a RuBP (carboxylation). The six-carbon product is unstable and splits at once into two molecules of glycerate 3-phosphate (GP), each with three carbon atoms. Three CO₂ + three RuBP give six GP.
- GP is reduced to triose phosphate. Each GP is converted to triose phosphate (TP), a three-carbon sugar phosphate, using one ATP (energy) and one reduced NADP (which supplies the hydrogen). Six GP need six ATP and six reduced NADP; the ADP, Pi and NADP go back to the thylakoids to be recycled.
- One TP leaves the cycle. Only one of the six TP is a net gain — it contains the three carbon atoms fixed in these three turns. It can leave the cycle to be used for making glucose, sucrose, starch and other compounds.
- RuBP is regenerated using ATP. The other five TP (15 carbon atoms) are rearranged in a series of reactions into three RuBP (15 carbon atoms), using three more ATP. With RuBP regenerated, the cycle can fix CO₂ again. Totals for three turns are nine ATP and six reduced NADP.
- Six turns make one glucose. A glucose molecule (6C) needs two TP, so the cycle must turn six times — fixing six CO₂ and using 18 ATP and 12 reduced NADP. Of the 12 TP made, 10 (five-sixths) are recycled to regenerate six RuBP, and two are combined to form glucose.
| For each… | CO₂ fixed | GP made | ATP used | Reduced NADP used | TP made | TP recycled | TP available |
|---|---|---|---|---|---|---|---|
| 3 turns | 3 | 6 | 9 | 6 | 6 | 5 | 1 |
| 6 turns (one glucose) | 6 | 12 | 18 | 12 | 12 | 10 | 2 |
If glucose is the product, five-sixths of all the TP made must be converted back to RuBP.
Making other carbon compounds
All the carbon in the compounds of a photosynthesising organism was originally fixed in the Calvin cycle. Every other carbon compound is made by metabolic pathways that start from a Calvin cycle intermediate (pathway details are not needed).
- Carbohydrates: two TP can be combined to make glucose; glucose and fructose make sucrose for transport in phloem; glucose is built into starch for storage and cellulose for cell walls.
- Amino acids: made from intermediates such as glycerate 3-phosphate. They need nitrogen, absorbed by roots as nitrate or ammonium ions, and some also need sulfur, absorbed as sulfate.
- Lipids: fatty acids and glycerol are made from Calvin cycle products.
- Nucleotides, ATP and phospholipids need phosphorus, absorbed as phosphate ions.
These mineral nutrients supply all the elements other than carbon, hydrogen and oxygen. Magnesium, for example, is needed for every chlorophyll molecule. This is why plants grown without nitrate show stunted growth even in bright light and high CO₂.
Interdependence of the two stages
The light-dependent and light-independent reactions depend on each other:
- The Calvin cycle needs a continuous supply of ATP and reduced NADP from the thylakoids.
- The thylakoids need a continuous supply of ADP, Pi and NADP, which the Calvin cycle regenerates.
Lack of light. With no light, no electrons are excited, so the light-dependent reactions stop. The ATP and reduced NADP in the stroma are used up within seconds, so glycerate 3-phosphate can no longer be reduced to triose phosphate. GP accumulates while RuBP is used up and not regenerated, and the Calvin cycle stops.
Lack of CO₂. With no CO₂, Rubisco cannot fix carbon and the Calvin cycle stops, so reduced NADP and ATP are no longer used. Soon all the NADP is reduced, leaving photosystem I with no acceptor for its excited electrons. The electron carriers stay reduced, so photosystem II cannot pass its excited electrons on. Photolysis stops and no oxygen is released, even in bright light.
The interdependence also explains why the stroma and thylakoids are so close together in the chloroplast (see B3.1 Gas exchange for how leaves supply CO₂ and light to them).
Must-know definitions
- Photosynthesis (word equation)
- carbon dioxide + water → glucose + oxygen, using light energy absorbed by chlorophyll. Light energy is transformed into chemical energy in carbon compounds; the hydrogen used to reduce CO2 comes from splitting water.
- Oxygen as a by-product
- The oxygen released by plants, algae and cyanobacteria comes from the splitting of water, not from carbon dioxide. Some is used in the organism's own respiration; the rest diffuses out as waste.
- R<sub>f</sub> value
- In chromatography, Rf = distance moved by the spot ÷ distance moved by the solvent, both measured from the origin line. It is always between 0 and 1 and, with the colour, identifies a pigment.
- Absorption spectrum vs action spectrum
- An absorption spectrum shows how much light of each wavelength a pigment absorbs. An action spectrum shows the rate of photosynthesis at each wavelength. Both peak in blue and red; the action spectrum stays above zero in green because accessory pigments absorb some of it.
- Limiting factor
- The factor that is furthest from its optimum and so sets the rate of photosynthesis at that moment — light intensity, CO2 concentration or temperature. Increasing a non-limiting factor has no effect on the rate.
- FACE experiments
- Free-air carbon dioxide enrichment: CO2 is released around plots of crops or natural vegetation in the open to raise the concentration (often to about 550 ppm), compared with control plots in normal air, to predict plant growth in a future high-CO2 atmosphere.
- PhotosystemAHL
- A membrane-bound array of chlorophyll and accessory pigments held by proteins, which funnels absorbed energy to a special reaction-centre chlorophyll that emits an excited electron. Photosystem II's reaction centre is P680; photosystem I's is P700.
- Chemiosmosis (in thylakoids)AHL
- Electron carriers use energy from excited electrons to pump H+ from the stroma into the thylakoid space. H+ then diffuses back to the stroma through ATP synthase, which uses the energy to make ATP from ADP and Pi.
- Reduction of NADPAHL
- At the stroma side of the thylakoid membrane, NADP reductase transfers two electrons from photosystem I and one H+ from the stroma to NADP, forming reduced NADP.
- Carbon fixation by RubiscoAHL
- RuBP (5C) + CO2 → 2 glycerate 3-phosphate (3C), catalysed by Rubisco in the stroma. Leaves contain a great deal of Rubisco, because each molecule fixes CO2 slowly and copes badly when CO2 is scarce.
- Triose phosphate and RuBP regenerationAHL
- Glycerate 3-phosphate is reduced to triose phosphate using ATP and reduced NADP. Five of every six triose phosphates are converted, using ATP, back into RuBP (5 TP → 3 RuBP); one in six is available to make glucose and other compounds.
Common exam mistakes
Loses marks The oxygen released in photosynthesis comes from carbon dioxide.
Write instead It comes from water, which is split to provide hydrogen (electrons and H+). The oxygen atoms of CO2 end up in glucose and water.
Loses marks Plants look green because chlorophyll absorbs green light.
Write instead Chlorophyll absorbs little green light. Green wavelengths are mostly reflected or transmitted, so that is the light that reaches our eyes.
Loses marks The absorption spectrum and the action spectrum are the same graph.
Write instead An absorption spectrum shows light absorbed by a pigment; an action spectrum shows the rate of photosynthesis. They have similar peaks, but the action spectrum is higher in green because accessory pigments pass energy to chlorophyll.
Loses marks Measuring Rf from the bottom of the paper, or getting Rf values greater than 1.
Write instead Measure both distances from the origin line (where the spot was placed): to the centre of the spot and to the solvent front. A spot can never move further than the solvent, so Rf is between 0 and 1.
Loses marks Increasing light intensity always increases the rate of photosynthesis.
Write instead Only while light is the limiting factor. On the plateau another factor (CO2 concentration or temperature) limits the rate, and only increasing that factor raises it.
Loses marks Above the optimum temperature the rate falls because the light-dependent reactions stop.
Write instead The rate falls because enzymes, including Rubisco, start to denature, so the enzyme-catalysed reactions slow down. Light absorption itself is hardly affected by temperature.
Loses marks ATP synthase pumps H+ into the thylakoid space.
Write instead The electron carriers (cytochrome complex) pump H+ into the thylakoid space. ATP synthase lets H+ diffuse back out to the stroma, down the gradient, and uses that flow to make ATP.
AHLLoses marks Rubisco joins CO2 to RuBP to make a stable six-carbon sugar (or glucose).
Write instead The six-carbon product is unstable and immediately splits into two glycerate 3-phosphate (3C). Glucose is made later, from triose phosphate that leaves the cycle.
AHLLoses marks Mixing terms, e.g. "NADP is converted to NADPH" or "NADP+ becomes reduced NADP".
Write instead Use one matching pair throughout an answer: NADP and reduced NADP, or NADP+ and NADPH.
AHLLoses marks Light-independent reactions happen at night, in the dark.
Write instead They need a continuous supply of ATP and reduced NADP from the light-dependent reactions, so they stop within seconds of the light being switched off. They are "light-independent" only because light is not used directly.
AHL
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Multiple-choice questions
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Write the word equation for photosynthesis.
carbon dioxide + water → glucose + oxygen, using light energy absorbed by chlorophyll.
What energy transformation happens in photosynthesis?
Light energy is transformed into chemical energy in carbon compounds such as glucose. This supplies most of the chemical energy used by life in ecosystems.
Where does the oxygen released in photosynthesis come from?
From water, which is split to provide the hydrogen used to reduce carbon dioxide. The oxygen is a by-product of plants, algae and cyanobacteria.
How is an Rf value calculated?
Rf = distance moved by the spot ÷ distance moved by the solvent, both measured from the origin line. It lies between 0 and 1.
List the leaf pigments in order from the top of a typical chromatogram.
Carotene (orange-yellow, near the solvent front), then chlorophyll a (blue-green), chlorophyll b (yellow-green) and xanthophylls (yellow). Grey phaeophytin may appear between carotene and chlorophyll a. This is the order with a typical non-polar solvent; other solvents can change it.
Why do pigments absorb only certain wavelengths, and why do leaves look green?
An absorbed photon must have exactly the energy needed to raise an electron to a higher level, so only some wavelengths are absorbed. Chlorophyll absorbs little green light, which is reflected or transmitted.
Absorption spectrum vs action spectrum
Absorption — % of light absorbed by a pigment at each wavelength. Action — rate of photosynthesis at each wavelength. Both peak in blue and red; the action spectrum is above zero in green due to accessory pigments.
Name the three limiting factors and how each can be varied in a pondweed experiment.
Light intensity (lamp distance, intensity ∝ 1/d²), CO₂ concentration (different amounts of sodium hydrogencarbonate) and temperature (thermostatic water bath). Vary one; keep the other two constant.
What is a FACE experiment and what is its advantage over a greenhouse study?
Free-air CO₂ enrichment — CO₂ is released around open-air plots to keep it at a raised level (e.g. 550 ppm), with control plots receiving air. Plants grow in natural field conditions, though fewer variables can be controlled.
Describe a photosystem.
A membrane-bound array of chlorophyll and accessory pigments held by proteins (antenna complexes) around a reaction-centre chlorophyll that emits excited electrons. Found in thylakoid membranes of chloroplasts and cyanobacteria.
Give the equation for photolysis and the fate of each product.
2H₂O → O₂ + 4H⁺ + 4e⁻. Electrons replace those lost by P680 in photosystem II; H⁺ adds to the proton gradient in the thylakoid space; O₂ is waste.
Outline chemiosmosis in the thylakoid.
Electron carriers use energy from excited electrons to pump H⁺ from the stroma into the thylakoid space. H⁺ diffuses back to the stroma through ATP synthase, which makes ATP from ADP + Pi.
Cyclic vs non-cyclic photophosphorylation
Non-cyclic — electrons from PSII pass to PSI and then reduce NADP; water is split and O₂ released; makes ATP and reduced NADP. Cyclic — electrons from PSI return to PSI via the carrier chain; makes ATP only.
What does Rubisco catalyse, and why is so much of it needed?
RuBP + CO₂ → 2 glycerate 3-phosphate (carbon fixation). Each molecule fixes CO₂ slowly and binds it poorly when CO₂ is scarce, so the stroma must hold a very high concentration of Rubisco. No enzyme on Earth is more plentiful.
How is triose phosphate made, and what happens to it?
Glycerate 3-phosphate is reduced using reduced NADP and ATP. Five of every six TP regenerate RuBP (5 TP → 3 RuBP, using ATP); the rest is used to make glucose, sucrose, starch, amino acids and other compounds.
Why does a lack of CO₂ stop photosystem II working?
The Calvin cycle stops, so NADP is not regenerated. With all NADP reduced, PSI has no electron acceptor, the carriers stay reduced, and PSII cannot pass on its excited electrons — so photolysis and O₂ release stop.
Last reviewed 19 October 2026. Spotted a mistake? Report it.