What you'll learn
- How chloroplast structure links to the two stages of photosynthesis.
- How light energy is used to make ATP and reduced NADP.
- How the Calvin cycle fixes carbon dioxide into carbohydrate.
- How to interpret practical data from photosynthesis investigations.
The big picture: two linked reaction sets
Photosynthesis is not “plants making glucose in one step”. It is a sequence of enzyme-controlled reactions that transfer energy from light into chemical stores.
Photosynthesis
Photosynthesis is the process by which light energy is used to convert carbon dioxide and water into organic molecules, with oxygen released as a by-product.
The overall summary equation is:
6CO2+6H2O→C6H12O6+6O26\text{CO}_2 + 6\text{H}_2\text{O} \to \text{C}_6\text{H}_{12}\text{O}_6 + 6\text{O}_26CO2+6H2O→C6H12O6+6O2In reality, the first carbohydrate made is usually triose phosphate rather than glucose. Triose phosphate can then be used to make glucose, sucrose, starch, cellulose, amino acids and lipids.
The two stages
The light-dependent reaction transfers light energy into ATP and reduced NADP. The light-independent reaction, also called the Calvin cycle, uses ATP and reduced NADP to reduce carbon dioxide into carbohydrate.
Chloroplast structure: where the reactions happen
A chloroplast is an organelle found in photosynthetic eukaryotic cells. It has:
- a double membrane surrounding it
- flattened sacs called thylakoids
- stacks of thylakoids called grana
- fluid called the stroma, which contains enzymes for the Calvin cycle
The thylakoid membrane contains chlorophyll, photosystems, electron carriers and ATP synthase. This is where the light-dependent reaction happens. The stroma is where the Calvin cycle happens.
ATP is a short-term energy carrier. Reduced NADP, often written as NADPH, carries hydrogen and electrons from the light-dependent reaction to the Calvin cycle.
The light-dependent reaction
The light-dependent reaction happens on the thylakoid membranes. Keep an eye on the membrane in the diagram: the key idea is that excited electrons move along proteins, and this movement helps build a hydrogen ion gradient used to make ATP.
1. Light is absorbed by chlorophyll
Chlorophyll is a photosynthetic pigment that absorbs light. A photon is a packet of light energy.
Chlorophyll molecules are arranged in photosystems, which are protein-pigment complexes in the thylakoid membrane. There are two main photosystems involved in non-cyclic photophosphorylation:
- Photosystem II, which acts first
- Photosystem I, which acts second
When chlorophyll absorbs light, electrons become excited and leave the chlorophyll molecule. This is called photoionisation.
2. Water is split by photolysis
The lost electrons from photosystem II must be replaced. This is done by photolysis, which means splitting water using light energy:
2H2O→O2+4H++4e−2\text{H}_2\text{O} \to \text{O}_2 + 4\text{H}^+ + 4\text{e}^-2H2O→O2+4H++4e−The electrons replace those lost from photosystem II. The hydrogen ions contribute to the proton gradient. Oxygen is released as a waste product.
Oxygen does not come from carbon dioxide
The oxygen released in photosynthesis comes from the splitting of water, not from carbon dioxide.
3. Electron transport builds a proton gradient
Excited electrons pass along an electron transport chain, a series of carrier proteins in the thylakoid membrane.
As electrons move through the chain, energy is released. This energy is used to pump hydrogen ions from the stroma into the thylakoid lumen, creating a proton gradient: a difference in hydrogen ion concentration across the membrane.
Hydrogen ions then diffuse back into the stroma through ATP synthase, an enzyme that makes ATP from ADP and inorganic phosphate. This process is called chemiosmosis.
Making ATP using light energy is called photophosphorylation.
4. NADP is reduced
Electrons are re-energised by light at photosystem I. They then combine with hydrogen ions and NADP to form reduced NADP.
In non-cyclic photophosphorylation, electrons flow from water to photosystem II, then to photosystem I, then to NADP. The products are ATP, reduced NADP and oxygen.
In cyclic photophosphorylation, electrons from photosystem I return to the electron transport chain instead of reducing NADP. This produces ATP only; no reduced NADP or oxygen is made.
Predicting the effect of blocked ATP synthase
If ATP synthase in the thylakoid membrane is inhibited:
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Hydrogen ions can no longer diffuse efficiently through ATP synthase from the thylakoid lumen into the stroma, so ATP production by chemiosmosis decreases.
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Hydrogen ions accumulate in the thylakoid lumen, making it harder for the electron transport chain to pump in more hydrogen ions, so electron flow tends to slow.
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The light-independent reaction receives less ATP, so carbon dioxide cannot be converted into carbohydrate as quickly.
The light-independent reaction: the Calvin cycle
The light-independent reaction happens in the stroma. It does not use light directly, but it depends on ATP and reduced NADP from the light-dependent reaction.
The diagram shows how carbon dioxide is fixed, reduced and then used to regenerate the starting molecule, ribulose bisphosphate.
Calvin cycle
The Calvin cycle is the light-independent stage of photosynthesis in which carbon dioxide is fixed and reduced to form triose phosphate, using ATP and reduced NADP.
1. Carbon fixation
Carbon fixation means incorporating inorganic carbon dioxide into an organic molecule.
Carbon dioxide combines with ribulose bisphosphate, usually shortened to RuBP. RuBP is a five-carbon compound.
This reaction is catalysed by the enzyme rubisco. The unstable six-carbon compound formed immediately splits into two molecules of glycerate 3-phosphate, usually shortened to GP. Each GP molecule has three carbon atoms.
2. Reduction of GP to TP
GP is converted into triose phosphate, usually shortened to TP.
This step needs:
- ATP, which provides energy
- reduced NADP, which provides hydrogen and electrons
Reduced NADP is oxidised back to NADP, and ATP becomes ADP and inorganic phosphate.
3. Regeneration of RuBP
Some TP leaves the cycle and is used to make useful organic molecules.
Most TP remains in the cycle and is rearranged to regenerate RuBP. This regeneration requires ATP. Regenerating RuBP is essential because RuBP must be available to accept more carbon dioxide.
Light-independent does not mean dark
The Calvin cycle is sometimes called the “dark reaction”, but that name is misleading. It does not need light directly, but it soon stops in darkness because ATP and reduced NADP run out.
Tracking carbon through the Calvin cycle
For three carbon dioxide molecules entering the Calvin cycle:
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Three 1-carbon carbon dioxide molecules combine with three 5-carbon RuBP molecules, giving six 3-carbon GP molecules.
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The six GP molecules are reduced to six TP molecules, using six ATP and six reduced NADP.
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One 3-carbon TP molecule can leave the cycle. The other five TP molecules, containing 15 carbon atoms in total, are rearranged to regenerate three 5-carbon RuBP molecules, using three more ATP.
For one hexose sugar, such as glucose, the cycle needs six carbon dioxide molecules. This gives two TP molecules that can combine to form one six-carbon sugar. Overall, this uses 18 ATP and 12 reduced NADP.
Linking the two reactions
The two stages form a cycle of carriers:
- the light-dependent reaction makes ATP and reduced NADP
- the Calvin cycle uses ATP and reduced NADP
- the Calvin cycle returns ADP, inorganic phosphate and NADP to the light-dependent reaction
This is why changes in one stage affect the other.
Predicting the effect of low carbon dioxide
If carbon dioxide concentration suddenly falls while light intensity stays high:
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Rubisco has less carbon dioxide available, so carbon fixation of RuBP slows down.
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ATP and reduced NADP are still being produced, so existing GP can still be converted into TP and used to regenerate RuBP.
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RuBP concentration rises because it is regenerated but not used up as quickly; GP concentration falls because less new GP is being made.
Investigating photosynthesis using DCPIP
A useful practical method investigates the light-dependent reaction using isolated chloroplasts and DCPIP, a redox indicator.
A redox indicator changes colour when it gains or loses electrons. DCPIP is blue when oxidised and becomes colourless when reduced.
In the light-dependent reaction, DCPIP can accept electrons instead of NADP. So, the faster DCPIP loses its blue colour, the faster electron transport is happening.
A good method controls variables such as chloroplast concentration, DCPIP volume, temperature, pH, light intensity and exposure time. A colorimeter can measure absorbance, and repeats help identify uncertainty and anomalous results.
The rate can be estimated using:
rate=ΔAt\text{rate} = \frac{\Delta A}{t}rate=tΔAwhere AAA is absorbance and ttt is time in seconds.
Calculating the rate of DCPIP reduction
In red light, absorbance falls from 0.80 to 0.32 in 120 s. In green light, absorbance falls from 0.80 to 0.68 in 120 s.
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Calculate the change in absorbance for each wavelength: red light gives ΔA=0.80−0.32=0.48\Delta A = 0.80 - 0.32 = 0.48ΔA=0.80−0.32=0.48, while green light gives ΔA=0.80−0.68=0.12\Delta A = 0.80 - 0.68 = 0.12ΔA=0.80−0.68=0.12.
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Substitute into the rate equation: red light gives 0.48120 s=0.0040 s−1\frac{0.48}{120\ \text{s}} = 0.0040\ \text{s}^{-1}120 s0.48=0.0040 s−1, while green light gives 0.12120 s=0.0010 s−1\frac{0.12}{120\ \text{s}} = 0.0010\ \text{s}^{-1}120 s0.12=0.0010 s−1.
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Compare the rates: 0.00400.0010=4.0\frac{0.0040}{0.0010} = 4.00.00100.0040=4.0, so DCPIP was reduced four times faster in red light than in green light.
In the exam
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Be precise about locations: light-dependent reactions happen on thylakoid membranes; the Calvin cycle happens in the stroma.
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Link products and reactants clearly: ATP and reduced NADP are made in the light-dependent reaction and used in the Calvin cycle.
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For application questions, track what is being made and used up. If a substrate is not available, the molecule that uses it tends to accumulate.
Check yourself
- Why does blocking photolysis reduce oxygen production?
- What happens to GP and RuBP concentrations if carbon dioxide concentration falls?
- Why can the Calvin cycle not continue for long in darkness?
