Photosynthesis (In Depth)

Info: Board Coverage AQA Paper 1 | Edexcel A Paper 1 | OCR (A) Paper 1 | CIE Paper 4

Photosynthesis is the process by which photoautotrophs convert light energy into chemical energy stored in organic molecules. The overall equation for photosynthesis:

$6\mathrm{CO_2} + 6\mathrm{H_2O} \xrightarrow{\text{light}} \mathrm{C_6H_{12}O_6} + 6\mathrm{O_2}$

$\Delta G = +2870\ \mathrm{kJ\ mol^{-1}}$

Photosynthesis is endergonic — energy is required. This energy is supplied by light, captured by photosynthetic pigments and converted to chemical energy in the form of ATP and reduced NADP ($\mathrm{NADPH}$).

Feature	Light-Dependent Reactions	Light-Independent Reactions (Calvin Cycle)
Location	Thylakoid membranes of chloroplasts	Stroma of chloroplasts
Light required?	Yes (directly)	No (but requires the products of light reactions)
Inputs	Light, $\mathrm{H_2O}$, $\mathrm{NADP^+}$ADP, $P_i$	$\mathrm{CO_2}$ATP, $\mathrm{NADPH}$RuBP
Outputs	$\mathrm{O_2}$ATP, $\mathrm{NADPH}$	$\mathrm{C_6H_{12}O_6}$ (via G3P), $\mathrm{NADP^+}$ADP, $P_i$
Main products	Energy carriers (ATP, NADPH)	Triose phosphate (G3P), which can form glucose

Chloroplasts are double-membraned organelles (approximately $4$—$10\ \mu\mathrm{m}$ in length, $1$—$5\ \mu\mathrm{m}$ in diameter) with a highly organised internal structure adapted to maximise the efficiency of photosynthesis.

Key structural features:

• Outer membrane: permeable to small molecules and ions.
• Inner membrane: selectively permeable; contains transport proteins.
• Stroma: the fluid-filled matrix inside the inner membrane. Contains the enzymes of the Calvin cycle, circular DNA, $70\mathrm{S}$ ribosomes, and starch granules.
• Thylakoids: flattened, membrane-bound sacs. The thylakoid membrane contains photosynthetic pigments, electron carriers, and ATP synthase. The thylakoid membrane encloses the thylakoid lumen (interior space).
• Grana (singular: granum): stacks of thylakoids. The stacked arrangement maximises the surface area for light absorption and the density of photosystems.
• Lamellae (intergranal thylakoids): thylakoid membranes connecting grana, allowing communication between them.
• Starch granules: temporary storage of carbohydrate produced by the Calvin cycle.

Structural Feature	Adaptation for Photosynthesis
Large surface area	Maximises light absorption
Thylakoid membranes	Provide a large surface for photosystems and electron transport chain
Grana stacks	Concentrate photosystems and increase the density of light-harvesting complexes
Thylakoid lumen	Small compartment allows rapid proton accumulation for chemiosmosis
Stroma	Contains high concentration of Calvin cycle enzymes
Transparent outer regions	Allow light to penetrate to inner thylakoids

Chloroplasts, like mitochondria, possess circular DNA, $70\mathrm{S}$ ribosomes, and a double membrane. These features support the endosymbiotic theory: chloroplasts were originally free-living photosynthetic prokaryotes (similar to modern cyanobacteria) that were engulfed by a eukaryotic cell.

Photosynthetic pigments absorb specific wavelengths of light and transfer the energy to the photosynthetic reaction centres.

Pigment	Absorption Peaks	Colour Reflected	Location
Chlorophyll a	$\approx 430\ \mathrm{nm}$ (blue), $\approx 660\ \mathrm{nm}$ (red)	Green	Reaction centre (PSI and PSII)
Chlorophyll b	$\approx 455\ \mathrm{nm}$ (blue), $\approx 640\ \mathrm{nm}$ (red)	Yellow-green	Antenna complex (light-harvesting)
Carotenoids	$\approx 450$—$500\ \mathrm{nm}$ (blue-green)	Orange, yellow	Antenna complex; photoprotection
Xanthophyll	$\approx 450\ \mathrm{nm}$	Yellow	Antenna complex; photoprotection

Chlorophyll a is the primary photosynthetic pigment — it is found at the reaction centres of both photosystems and directly participates in the light-dependent reactions. Chlorophyll b, carotenoids, and xanthophylls are accessory pigments that absorb light at wavelengths where chlorophyll a absorbs poorly and transfer the energy to chlorophyll a. This broadens the range of wavelengths that can be used for photosynthesis.

Carotenoids also have a photoprotective role: they absorb excess light energy and dissipate it as heat, preventing the formation of reactive oxygen species that would damage the thylakoid membrane.

An absorption spectrum shows the wavelengths of light absorbed by a pigment (or mixture of pigments). It is measured using a spectrophotometer.

An action spectrum shows the rate of photosynthesis at different wavelengths. It is measured by placing a plant under light of different wavelengths and measuring $\mathrm{O_2}$ production or $\mathrm{CO_2}$ uptake.

The action spectrum of photosynthesis closely matches the absorption spectrum of chlorophyll a (with some contributions from accessory pigments), confirming that chlorophyll a is the primary photosynthetic pigment.

The separation of photosynthetic pigments can be demonstrated using thin-layer chromatography (TLC) or paper chromatography:

1. Extract pigments by grinding leaves in solvent (e.g., acetone).
2. Apply the extract as a spot on a chromatography plate (or paper).
3. Place the plate in a solvent (e.g., a mixture of petroleum ether and propanone).
4. The solvent rises by capillary action, carrying the pigments at different rates.
5. More soluble pigments travel further; less soluble pigments remain closer to the origin.

The resulting chromatogram shows separated bands: carotenoids (top, most soluble, yellow-orange), xanthophyll (below carotenoids, yellow), chlorophyll a (blue-green), chlorophyll b (yellow-green, lowest, least soluble).

The $R_f$ value (retention factor) can be calculated:

$R_f = \frac{\text{Distance travelled by pigment}}{\text{Distance travelled by solvent front}}$

Photosystems are protein-pigment complexes embedded in the thylakoid membrane. Each consists of:

• An antenna complex (light-harvesting complex): hundreds of accessory pigment molecules (chlorophyll b, carotenoids) and chlorophyll a molecules that absorb light and transfer the energy to the reaction centre.
• A reaction centre: a special pair of chlorophyll a molecules that undergo a redox reaction when excited, donating electrons to an electron acceptor.

Two photosystems operate in series:

Feature	Photosystem II (PSII)	Photosystem I (PSI)
Primary pigment	P680 (chlorophyll a absorbing at $680\ \mathrm{nm}$)	P700 (chlorophyll a absorbing at $700\ \mathrm{nm}$)
Location	Inner surface of thylakoid membrane	Outer surface of thylakoid membrane
Function	Splits water; feeds electrons into ETC	Boosts electrons to $\mathrm{NADP^+}$
Electron acceptor	Plastoquinone (PQ)	Ferredoxin

Non-cyclic photophosphorylation involves both photosystems operating in sequence, producing both ATP and $\mathrm{NADPH}$And releasing $\mathrm{O_2}$.

Step 1: Light absorption by PSII. A photon of light is absorbed by the antenna complex of PSII and the energy is transferred to P680. P680 becomes excited ($\mathrm{P680^*}$) and donates an electron to the primary electron acceptor (pheophytin). P680 is now oxidised ($\mathrm{P680^+}$), a very strong oxidising agent.

Step 2: Photolysis of water. The strong oxidising power of $\mathrm{P680^+}$ is used to split water molecules in a process called photolysis:

$\mathrm{2H_2O \to 4H^+ + 4e^- + O_2}$

The electrons replace those lost by P680. The $\mathrm{O_2}$ is released as a by-product (all atmospheric $\mathrm{O_2}$ comes from photosynthesis). The $\mathrm{H^+}$ ions contribute to the proton gradient.

Step 3: Electron transport through the ETC. Electrons pass from PSII through a series of carriers:

• Pheophytin $\to$ plastoquinone (PQ) $\to$ cytochrome $b_6f$ complex $\to$ plastocyanin (PC) $\to$ PSI.

At the cytochrome $b_6f$ complex, protons are pumped from the stroma into the thylakoid lumen, contributing to the proton gradient.

Step 4: Light absorption by PSI. Electrons arrive at PSI and another photon is absorbed, re-exciting P700. P700 donates the electron to ferredoxin (Fd) via ferredoxin-NADP reductase (FNR).

Step 5: $\mathrm{NADPH}$ production. The enzyme ferredoxin-NADP reductase (FNR) transfers electrons from ferredoxin to $\mathrm{NADP^+}$Reducing it to $\mathrm{NADPH}$:

$\mathrm{NADP^+ + 2e^- + H^+ \to NADPH}$

Step 6: ATP synthesis by chemiosmosis. The proton gradient across the thylakoid membrane (high $\mathrm{H^+}$ in the lumen, low in the stroma) drives ATP synthesis by ATP synthase. Protons flow from the lumen to the stroma through ATP synthase, which phosphorylates ADP to ATP.

The proton gradient is generated by three sources:

1. Photolysis of water (releases $\mathrm{H^+}$ into the lumen).
2. Pumping by the cytochrome $b_6f$ complex (translocates $\mathrm{H^+}$ from stroma to lumen).
3. Removal of $\mathrm{H^+}$ from the stroma by $\mathrm{NADPH}$ production (reducing $\mathrm{NADP^+}$ consumes stroma $\mathrm{H^+}$).

Cyclic photophosphorylation involves only PSI and produces ATP but no $\mathrm{NADPH}$ and no $\mathrm{O_2}$:

1. Light is absorbed by PSI, exciting P700.
2. P700 donates electrons to ferredoxin.
3. Instead of passing to $\mathrm{NADP^+}$Electrons are passed back to the cytochrome $b_6f$ complex, then to plastocyanin, and back to PSI.
4. Protons are pumped at the cytochrome $b_6f$ complex, creating a proton gradient for ATP synthesis.

Cyclic photophosphorylation generates additional ATP when the Calvin cycle requires more ATP than $\mathrm{NADPH}$ (the Calvin cycle uses 3 ATP per 2 $\mathrm{NADPH}$But non-cyclic photophosphorylation produces them in approximately equal amounts).

The Calvin cycle (also called the Calvin-Benson cycle or the $\mathrm{C_3}$ cycle) occurs in the stroma and uses ATP and $\mathrm{NADPH}$ from the light-dependent reactions to fix $\mathrm{CO_2}$ into organic molecules.

The cycle turns three times to produce one molecule of G3P (triose phosphate, 3-carbon), from which glucose (6-carbon) can be synthesised. Each turn fixes one molecule of $\mathrm{CO_2}$.

Step 1: Carbon fixation. $\mathrm{CO_2}$ diffuses into the stroma and is fixed by the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which catalyses the reaction of $\mathrm{CO_2}$ with ribulose-1,5-bisphosphate (RuBP, 5-carbon) to produce an unstable 6-carbon intermediate. This immediately splits into two molecules of glycerate-3-phosphate (GP, also called 3-phosphoglycerate, 3-PGA), each with 3 carbons:

$\mathrm{CO_2 + RuBP\ (5C) \to 2\ GP\ (3C)}$

Rubisco is the most abundant protein on Earth. It is also a relatively slow enzyme (turnover number $\approx 3\ \mathrm{s^{-1}}$), which limits the rate of photosynthesis.

Step 2: Reduction of GP to triose phosphate (TP). GP is phosphorylated by ATP (from the light-dependent reactions) and then reduced by $\mathrm{NADPH}$:

$\mathrm{GP + ATP \to 1,3\text{-}bisphosphoglycerate + ADP}$

$\mathrm{1,3\text{-}BPG + NADPH + H^+ \to triose\ phosphate\ (TP,\ G3P) + NADP^+ + P_i}$

This step requires 1 ATP and 1 $\mathrm{NADPH}$ per molecule of GP (2 ATP and 2 $\mathrm{NADPH}$ per $\mathrm{CO_2}$ fixed).

Step 3: Regeneration of RuBP. For every 6 molecules of TP produced (from 3 turns of the cycle fixing 3 $\mathrm{CO_2}$), 5 molecules are used to regenerate 3 molecules of RuBP (5-carbon). The remaining 1 molecule of TP is the net product, which can be used to synthesise glucose, other carbohydrates, lipids, or amino acids.

The regeneration of RuBP involves a complex series of reactions (3-, 4-, 5-, 6-, and 7-carbon sugar phosphates) catalysed by several enzymes, consuming 3 ATP per turn.

For every 3 turns (fixing 3 $\mathrm{CO_2}$):

Input	Output
3 $\mathrm{CO_2}$	1 TP (G3P, net product)
9 ATP (3 per turn)	9 ADP + 9 $P_i$
6 $\mathrm{NADPH}$ (2 per turn)	6 $\mathrm{NADP^+}$ + 6 $\mathrm{H^+}$
5 TP (recycled)	3 RuBP (regenerated)

The net equation for the Calvin cycle (per glucose):

$6\mathrm{CO_2} + 18\ ATP + 12\ NADPH + 12\ H_2O \to C_6H_{12}O_6 + 18\ ADP + 18\ P_i + 12\ NADP^+$

The TP produced by the Calvin cycle can be used to synthesise:

• Glucose (and other hexose sugars): 2 TP molecules combine to form glucose ($\mathrm{C_6H_{12}O_6}$).
• Sucrose: the main transport sugar in plants, synthesised in the cytoplasm from glucose and fructose.
• Starch: the main storage carbohydrate in plants, synthesised from glucose in the stroma.
• Cellulose: a structural polysaccharide synthesised from glucose at the plasma membrane.
• Amino acids and lipids: TP can be converted to glycerate (for amino acid synthesis) or acetyl CoA (for fatty acid synthesis).

Rubisco has a dual activity: it can catalyse both the carboxylation of RuBP (fixing $\mathrm{CO_2}$Productive) and the oxygenation of RuBP (fixing $\mathrm{O_2}$Wasteful). When $\mathrm{O_2}$ combines with RuBP, one molecule of GP (3-carbon) and one molecule of phosphoglycolate (2-carbon) are produced. Phosphoglycolate is converted to glycolate, which enters a salvage pathway in peroxisomes and mitochondria that releases $\mathrm{CO_2}$ and consumes ATP and $\mathrm{NADH}$ — a net loss of energy and fixed carbon.

Photorespiration is favoured by:

• High temperature (increases Rubisco”s affinity for $\mathrm{O_2}$ over $\mathrm{CO_2}$).
• High $\mathrm{O_2}$ concentration.
• Low $\mathrm{CO_2}$ concentration.

Photorespiration can reduce the efficiency of photosynthesis by $25$—$50\%$ in $\mathrm{C_3}$ plants under hot, dry conditions. $\mathrm{C_4}$ and CAM plants have evolved mechanisms to minimise photorespiration (see Section 7).

## 6. Limiting Factors

The rate of photosynthesis is determined by the factor that is in shortest supply (the limiting factor). At any given moment, only one factor is truly limiting; increasing other factors will not increase the rate.

The three main limiting factors are: light intensity, $\mathrm{CO_2}$ concentration, and temperature.

At low light intensity, the rate of photosynthesis is proportional to light intensity (the graph is a straight line through the origin). Light is the limiting factor because it drives the light-dependent reactions.

As light intensity increases, the rate plateaus at the light saturation point, where another factor ($\mathrm{CO_2}$ concentration or temperature) becomes limiting. The rate no longer increases because all available chlorophyll molecules are excited simultaneously and the Calvin cycle enzymes are operating at maximum rate.

Compensation point: the light intensity at which the rate of photosynthesis exactly equals the rate of respiration. There is no net gas exchange. Below the compensation point, respiration exceeds photosynthesis and the plant has a net consumption of $\mathrm{O_2}$ and net release of $\mathrm{CO_2}$.

At low $\mathrm{CO_2}$ concentration (close to the atmospheric concentration of $\approx 0.04\%$Or $400\ \mathrm{ppm}$), $\mathrm{CO_2}$ is often the limiting factor. Increasing $\mathrm{CO_2}$ concentration increases the rate of photosynthesis up to a plateau ( at approximately $0.5$—$1.0\%$), where another factor (light or temperature) becomes limiting.

The initial rise is because more $\mathrm{CO_2}$ is available for Rubisco, increasing the rate of carbon fixation. Commercial greenhouse growers supplement $\mathrm{CO_2}$ to approximately $1000\ \mathrm{ppm}$ to increase crop yields.

Temperature affects photosynthesis because the Calvin cycle is enzyme-catalysed (primarily by Rubisco). The rate increases with temperature up to an optimum ( $25$—$30\ ^\circ\mathrm{C}$ for $\mathrm{C_3}$ plants), then decreases sharply as enzymes denature.

Temperature does not directly affect the light-dependent reactions (which are photochemical, not enzymatic), but it does affect:

• The activity of Calvin cycle enzymes.
• The fluidity of the thylakoid membrane (affecting electron transport).
• The solubility of $\mathrm{CO_2}$ (higher temperatures reduce $\mathrm{CO_2}$ solubility).
• The rate of photorespiration (increases with temperature).

When plotting the rate of photosynthesis against one factor at different levels of another:

• Rate vs. Light intensity at two $\mathrm{CO_2}$ concentrations: both curves plateau, but the higher $\mathrm{CO_2}$ curve plateaus at a higher rate. The higher $\mathrm{CO_2}$ curve levels off at a higher light intensity.
• Rate vs. $\mathrm{CO_2}$ concentration at two temperatures: both curves plateau, but the higher temperature curve plateaus at a higher rate (up to the optimum temperature).
• Rate vs. Temperature at two light intensities: the higher light curve plateaus at a higher rate and at a higher temperature before denaturation occurs.

Worked Example. A student measures the rate of photosynthesis (as $\mathrm{O_2}$ production in $\mu\mathrm{mol\ m^{-2}\ s^{-1}}$) at different light intensities and two $\mathrm{CO_2}$ concentrations:

Light intensity (arbitrary units)	5	10	20	40	80
Rate at $0.04\%\ \mathrm{CO_2}$	2	4	7	9	10
Rate at $0.10\%\ \mathrm{CO_2}$	2	5	10	14	16

At low light intensity (5 units), both curves give the same rate (2) — light is the limiting factor. At higher light intensities, the $0.10\%\ \mathrm{CO_2}$ curve gives a higher rate, indicating that $\mathrm{CO_2}$ was limiting at the lower concentration. The $0.10\%\ \mathrm{CO_2}$ curve continues to rise beyond where the $0.04\%$ curve plateaus.

$\mathrm{C_3}$ plants (the majority of plants, including wheat, rice, soybean, and most trees) fix $\mathrm{CO_2}$ directly into GP (a 3-carbon compound) via Rubisco in the Calvin cycle. They have no special mechanism to concentrate $\mathrm{CO_2}$ and are therefore susceptible to photorespiration.

$\mathrm{C_3}$ plants are most efficient in cool, moist environments with moderate light intensity, where photorespiration is minimal.

$\mathrm{C_4}$ plants (e.g., maize, sugarcane, sorghum) have a $\mathrm{C_4}$ carbon fixation pathway that concentrates $\mathrm{CO_2}$ in bundle sheath cells, minimising photorespiration and maximising photosynthetic efficiency at high temperatures.

Mechanism:

1. Mesophyll cells: $\mathrm{CO_2}$ is fixed by PEP carboxylase (which has a much higher affinity for $\mathrm{CO_2}$ than Rubisco and does not react with $\mathrm{O_2}$) by combining with phosphoenolpyruvate (PEP, 3-carbon) to form oxaloacetate (4-carbon, hence "$\mathrm{C_4}$").

$\mathrm{PEP + CO_2 \to oxaloacetate}$

2. Oxaloacetate is converted to malate (or aspartate, another 4-carbon acid).

3. Malate is transported to bundle sheath cells (which surround the vascular bundles), where it is decarboxylated, releasing $\mathrm{CO_2}$ at high concentration.

4. The released $\mathrm{CO_2}$ enters the Calvin cycle (via Rubisco) in the bundle sheath cells. The high $\mathrm{CO_2}$ concentration suppresses photorespiration by outcompeting $\mathrm{O_2}$ for Rubisco’s active site.

5. The 3-carbon product (pyruvate) is transported back to the mesophyll cells and converted back to PEP using ATP (the $\mathrm{C_4}$ cycle costs 2 extra ATP per $\mathrm{CO_2}$ fixed, but this is offset by the reduced photorespiration).

CAM (Crassulacean Acid Metabolism) plants (e.g., pineapple, cacti, orchids, succulents) are adapted to very arid conditions. They minimise water loss by opening stomata at night (when temperatures are lower and humidity is higher) and closing them during the day.

Mechanism:

1. Night: stomata open; $\mathrm{CO_2}$ enters and is fixed by PEP carboxylase into malate (stored in vacuoles).
2. Day: stomata close; malate is released from vacuoles and decarboxylated, releasing $\mathrm{CO_2}$ for the Calvin cycle. The light-dependent reactions provide ATP and $\mathrm{NADPH}$.

CAM plants have very low rates of photosynthesis (because the amount of $\mathrm{CO_2}$ stored at night is limited) but extremely high water-use efficiency.

Feature	$\mathrm{C_3}$ Plants	$\mathrm{C_4}$ Plants	CAM Plants
Initial fixation	Rubisco ($\mathrm{C_3}$ compound)	PEP carboxylase ($\mathrm{C_4}$ compound)	PEP carboxylase ($\mathrm{C_4}$ at night)
First product	GP (3C)	Oxaloacetate/malate (4C)	Malate (4C, stored at night)
Leaf anatomy	No Kranz anatomy	Kranz anatomy (bundle sheath cells)	No Kranz anatomy
Photorespiration	Significant at high temperature	Minimal ($\mathrm{CO_2}$ concentrated)	Minimal (stomata closed during the day)
Water use efficiency	Moderate	High	Very high
Habitat	Cool, moist environments	Hot, sunny environments	Arid, desert environments
ATP cost per $\mathrm{CO_2}$	3 ATP	5 ATP	5 ATP
Examples	Wheat, rice, soybean, trees	Maize, sugarcane, sorghum	Cacti, pineapple, orchids, aloe vera

## 8. Practical Investigations

Common methods include:

1. Oxygen production: measuring the volume of $\mathrm{O_2}$ bubbles released from an aquatic plant (e.g., Elodea/pondweed) at different light intensities. The plant is placed in a test tube of water with a light source at varying distances. A gas syringe or inverted measuring cylinder can collect the $\mathrm{O_2}$.

2. pH change indicator: using a $\mathrm{pH}$ indicator (e.g., hydrogencarbonate indicator) to detect $\mathrm{CO_2}$ uptake. As $\mathrm{CO_2}$ is absorbed for photosynthesis, the pH increases, changing the colour of the indicator from orange-red to purple.

3. Chlorophyll extraction: measuring the absorbance of light by chlorophyll extracts at different wavelengths using a spectrophotometer to produce an absorption spectrum.

A student measures the volume of $\mathrm{O_2}$ produced by Elodea at different distances from a lamp:

Distance from lamp (cm)	5	10	20	30	50
$\mathrm{O_2}$ volume ($\mathrm{mm^3\ min^{-1}}$)	45	42	32	22	10

Light intensity is inversely proportional to the square of the distance (inverse square law):

$I \propto \frac{1}{d^2}$

Relative light intensities: $d = 5 \Rightarrow I = 400$; $d = 10 \Rightarrow I = 100$; $d = 20 \Rightarrow I = 25$; $d = 30 \Rightarrow I = 11.1$; $d = 50 \Rightarrow I = 4$.

Plotting rate against relative light intensity gives a curve that rises steeply at low intensities and begins to plateau at higher intensities, consistent with the expected pattern for photosynthesis.

Problem 1

Describe how the light-dependent reactions of photosynthesis convert light energy into chemical energy. In your answer, explain the roles of photosystems I and II, photolysis of water, and chemiosmosis. (6 marks)

Answer. Light energy is absorbed by photosynthetic pigments in the antenna complexes of PSII and PSI and transferred to the reaction centres. In PSII, light excites P680, which donates an electron to the electron transport chain. The oxidised P680 is a strong oxidising agent that extracts electrons from water (photolysis: $\mathrm{2H_2O \to 4H^+ + 4e^- + O_2}$), releasing $\mathrm{O_2}$ as a by-product. Electrons pass from PSII through plastoquinone, the cytochrome $b_6f$ complex (which pumps protons into the thylakoid lumen), and plastocyanin to PSI. In PSI, light excites P700, which donates electrons to ferredoxin. Ferredoxin-NADP reductase transfers electrons to $\mathrm{NADP^+}$Reducing it to $\mathrm{NADPH}$. The proton gradient across the thylakoid membrane (generated by photolysis, proton pumping, and $\mathrm{NADPH}$ production) drives ATP synthesis as protons flow through ATP synthase (chemiosmosis). The products are ATP, $\mathrm{NADPH}$And $\mathrm{O_2}$.

If you get this wrong, revise: Non-Cyclic Photophosphorylation

Problem 2

Explain the role of Rubisco in the Calvin cycle. Why is photorespiration a problem for plants, and how do $\mathrm{C_4}$ plants overcome this problem? (5 marks)

Answer. Rubisco catalyses the fixation of $\mathrm{CO_2}$ by combining it with RuBP (5-carbon) to form two molecules of GP (3-carbon). This is the first step of the Calvin cycle and the sole route by which inorganic carbon enters the biosphere. Rubisco has a dual activity: it can also catalyse the oxygenation of RuBP (combining it with $\mathrm{O_2}$), producing one molecule of GP and one of phosphoglycolate. This is photorespiration, a wasteful process that releases $\mathrm{CO_2}$Consumes ATP, and reduces the net yield of photosynthesis. Photorespiration increases at high temperatures and low $\mathrm{CO_2}$ concentrations because Rubisco’s affinity for $\mathrm{O_2}$ increases relative to $\mathrm{CO_2}$ under these conditions. $\mathrm{C_4}$ plants overcome this by using PEP carboxylase (which has no affinity for $\mathrm{O_2}$) to fix $\mathrm{CO_2}$ into a 4-carbon acid in mesophyll cells. This acid is transported to bundle sheath cells, where it is decarboxylated to release $\mathrm{CO_2}$ at high concentration around Rubisco. The high $\mathrm{CO_2}$ concentration suppresses photorespiration by outcompeting $\mathrm{O_2}$ for Rubisco’s active site.

If you get this wrong, revise: Photorespiration and C4 Plants

Problem 3

A student investigates the effect of temperature on the rate of photosynthesis in a $\mathrm{C_3}$ plant. The results show that the rate increases from $10$ to $30\ ^\circ\mathrm{C}$Plateaus between $30$ and $35\ ^\circ\mathrm{C}$And then decreases above $35\ ^\circ\mathrm{C}$. Explain these results. (5 marks)

Answer. Between $10$ and $30\ ^\circ\mathrm{C}$The rate of photosynthesis increases because increasing temperature increases the kinetic energy of molecules, leading to more frequent collisions between enzyme molecules (Rubisco and other Calvin cycle enzymes) and their substrates. This increases the rate of the enzyme-catalysed reactions in the Calvin cycle. The light-dependent reactions are less affected by temperature because they are photochemical rather than enzymatic. Between $30$ and $35\ ^\circ\mathrm{C}$The rate plateaus because another factor (likely $\mathrm{CO_2}$ concentration or light intensity) becomes limiting. Above $35\ ^\circ\mathrm{C}$The rate decreases because the high temperature causes denaturation of Calvin cycle enzymes, particularly Rubisco. The active site changes shape, reducing the enzyme’s ability to catalyse carbon fixation. Additionally, the solubility of $\mathrm{CO_2}$ decreases at higher temperatures, and photorespiration increases, further reducing net photosynthesis.

If you get this wrong, revise: Temperature and Limiting Factors

Problem 4

For the Calvin cycle, calculate the number of ATP and $\mathrm{NADPH}$ molecules required to produce one molecule of glucose ($\mathrm{C_6H_{12}O_6}$). (4 marks)

Answer. Glucose has 6 carbon atoms. Each turn of the Calvin cycle fixes 1 $\mathrm{CO_2}$ (1 carbon), so 6 turns are needed to produce one glucose molecule (from 2 molecules of G3P/TP). Per turn: 3 ATP are consumed (1 for GP phosphorylation in step 2, and 2 for RuBP regeneration in step 3) and 2 $\mathrm{NADPH}$ are consumed (in step 2). Therefore, for 6 turns: ATP required $= 6 \times 3 = 18$ ATP. $\mathrm{NADPH}$ required $= 6 \times 2 = 12\ \mathrm{NADPH}$. Note that 6 turns produce 6 TP, of which 2 are used to make glucose and 4 are recycled to regenerate 3 RuBP.

If you get this wrong, revise: Summary of the Calvin Cycle

Problem 5

Compare and contrast $\mathrm{C_4}$ and CAM plants in terms of their adaptations to reduce photorespiration and their water-use strategies. (4 marks)

Answer. Both $\mathrm{C_4}$ and CAM plants use PEP carboxylase to initially fix $\mathrm{CO_2}$ into a 4-carbon acid (oxaloacetate/malate), concentrating $\mathrm{CO_2}$ around Rubisco and reducing photorespiration. Both pathways cost additional ATP per $\mathrm{CO_2}$ fixed compared to $\mathrm{C_3}$ plants. However, they differ in their spatial and temporal separation: $\mathrm{C_4}$ plants separate the two pathways spatially — initial fixation occurs in mesophyll cells, and the Calvin cycle occurs in bundle sheath cells (Kranz anatomy). CAM plants separate them temporally — initial fixation occurs at night (when stomata are open to reduce water loss), and the Calvin cycle operates during the day (when stomata are closed). $\mathrm{C_4}$ plants are adapted to hot, sunny environments (e.g., tropical grasslands) and have higher overall photosynthetic rates; CAM plants are adapted to arid environments (e.g., deserts) and have very high water-use efficiency but low photosynthetic rates.

If you get this wrong, revise: C4 and CAM Plants

The compensation point is the light intensity at which the rate of photosynthesis exactly equals the rate of respiration. At this point, there is no net exchange of gases.

Below the compensation point, respiration exceeds photosynthesis: the plant has a net consumption of $\mathrm{O_2}$ and a net release of $\mathrm{CO_2}$.

Above the compensation point, photosynthesis exceeds respiration: the plant has a net production of $\mathrm{O_2}$ and a net uptake of $\mathrm{CO_2}$.

The compensation point varies between species:

Plant Type	Typical Compensation Point (arbitrary units)	Reason
Shade-tolerant	Low (5—15)	Efficient photosynthesis at low light; adapted to grow under a canopy
Shade-intolerant	Higher (30—50)	Require high light; grow rapidly in open habitats
C4 plants	Lower than $\mathrm{C_3}$ plants at the same temperature	More efficient at low $\mathrm{CO_2}$; compensate at lower light

The saturation point is the light intensity at which the rate of photosynthesis plateaus. Beyond this point, increasing light intensity has no further effect because another factor ($\mathrm{CO_2}$ concentration or temperature) is limiting.

C4 plants generally have a higher saturation point than $\mathrm{C_3}$ plants because PEP carboxylase is so efficient at fixing $\mathrm{CO_2}$ that the Calvin cycle can operate at maximum rate even at relatively low $\mathrm{CO_2}$ concentrations.

During a 24-hour period, the net gas exchange varies:

• Night: no photosynthesis (no light); only respiration occurs. Net $\mathrm{CO_2}$ release, net $\mathrm{O_2}$ consumption.
• Dawn: light intensity increases, photosynthesis begins. At the compensation point, net gas exchange switches from respiration-dominated to photosynthesis-dominated.
• Midday: photosynthesis rate peaks; maximum net $\mathrm{O_2}$ production and net $\mathrm{CO_2}$ uptake.
• Dusk: light intensity decreases; photosynthesis rate declines.
• Night: compensation point is reached again; net respiration resumes.

In temperate deciduous forests, photosynthetic activity varies seasonally:

• Spring: buds open, leaves expand, photosynthesis increases.
• Summer: maximum leaf area; peak photosynthetic rate.
• Autumn: leaves senesce; chlorophyll is broken down (revealing yellow/orange carotenoids, hence autumn colours); photosynthetic rate declines.
• Winter: deciduous trees are leafless; photosynthesis ceases; only respiration continues.

Commercial growers manipulate the environment to maximise crop yield:

Factor	How It Is Manipulated	Effect on Photosynthesis
$\mathrm{CO_2}$ enrichment	Supplying $\mathrm{CO_2}$ gas to greenhouses	Increases rate; raises the saturation point; shifts compensation point to lower light
Temperature control	Heating/cooling systems	Optimises enzyme activity; avoids enzyme denaturation
Light supplementation	Artificial lighting (e.g., LED grow lights)	Extends the photoperiod; increases total photosynthate
Water and nutrients	Irrigation, fertiliser (nitrate, phosphate)	Ensures raw materials are not limiting
Spacing and pruning	Optimising plant density and leaf area	Maximises light interception per unit area

The rate of photosynthesis in a crop canopy depends on how much light the leaves intercept:

• Leaf area index (LAI): the total area of leaves per unit ground area. Higher LAI means more light interception, up to the point where lower leaves are shaded.
• Canopy architecture: the arrangement of leaves affects light penetration. A planophile canopy (horizontal leaves) intercepts light efficiently but shades lower leaves. An erectophile canopy (steeply angled leaves) allows light to penetrate deeper, improving overall canopy photosynthesis.
• Intercropping: growing two crops together (e.g., maize and beans) increases total LAI and light interception compared to monoculture.

Gross photosynthesis is the total rate of $\mathrm{CO_2}$ fixation, including $\mathrm{CO_2}$ released by respiration.

Net photosynthesis is the rate of $\mathrm{CO_2}$ uptake minus the rate of $\mathrm{CO_2}$ release from respiration:

$\text{Net photosynthesis} = \text{Gross photosynthesis} - \text{Respiration}$

Worked Example. A plant’s leaves fix $\mathrm{CO_2}$ at a rate of $12.0\ \mu\mathrm{mol\ m^{-2}\ s^{-1}}$ (gross photosynthesis) and release $\mathrm{CO_2}$ at a rate of $2.0\ \mu\mathrm{mol\ m^{-2}\ s^{-1}}$ (respiration).

Net photosynthesis $= 12.0 - 2.0 = 10.0\ \mu\mathrm{mol\ m^{-2}\ s^{-1}}$.

If the respiration rate increases to $4.0\ \mu\mathrm{mol\ m^{-2}\ s^{-1}}$ (e.g., at higher temperature), net photosynthesis decreases to $12.0 - 4.0 = 8.0\ \mu\mathrm{mol\ m^{-2}\ s^{-1}}$.

This illustrates that increasing temperature can either increase or decrease net photosynthesis, depending on whether the effect on gross photosynthesis (through enzyme kinetics) or the effect on respiration is greater.

The light-harvesting antenna complex consists of several hundred pigment molecules (chlorophyll a, chlorophyll b, carotenoids, xanthophylls) arranged around a reaction centre containing two special chlorophyll a molecules. Energy is transferred from pigment to pigment by resonance energy transfer (Forster mechanism):

1. A pigment molecule absorbs a photon and is excited to a higher energy state.
2. The excitation energy is transferred to a neighbouring pigment molecule by dipole-dipole interaction.
3. This process repeats, with the energy passing through the antenna complex like a Mexican wave until it reaches the reaction centre.

The efficiency of energy transfer is very high ($>95\%$), and the energy is always transferred “downhill” in energy (from shorter wavelength to longer wavelength, from blue to red) because the antenna pigments are arranged in order of their absorption maxima.

The absorption spectrum of a pigment solution shows the wavelengths of light absorbed. Chlorophyll a has absorption peaks at $\approx 430\ \mathrm{nm}$ (blue/violet) and $\approx 660\ \mathrm{nm}$ (red), with a gap in the green region (which is why chlorophyll reflects green light).

The action spectrum shows the rate of photosynthesis at each wavelength. For isolated chloroplasts, the action spectrum closely matches the absorption spectrum of chlorophyll a, confirming that chlorophyll a is the primary photosynthetic pigment. However, for whole leaves, the action spectrum is broader, extending into the blue-green region ($450$—$500\ \mathrm{nm}$), reflecting the contribution of accessory pigments (chlorophyll b and carotenoids) to light absorption.

Aquatic plants face a unique challenge: $\mathrm{CO_2}$ dissolves in water and forms carbonic acid:

\mathrm{CO_2 + H_2O \rightleftharpoons H_2CO_3 \rightleftharpoons H^+ + HCO_3^- \rightleftharpoons 2H^+ + CO_3^{2-}

This reduces the concentration of freely available $\mathrm{CO_2}$ (as dissolved $\mathrm{CO_2}$), potentially limiting photosynthesis. Aquatic plants have adaptations:

• $\mathrm{CO_2}$-concentrating mechanisms in some aquatic plants (analogous to $\mathrm{C_4}$).
• Thin leaves to reduce diffusion distance.
• Large surface area to maximise gas exchange.
• Efficient use of bicarbonate ($\mathrm{HCO_3^-}$) as a carbon source.

The pH of water also affects photosynthesis: lower pH (more acidic) shifts the equilibrium towards $\mathrm{CO_2}$ and $\mathrm{H_2CO_3}$Reducing the availability of bicarbonate. Some aquatic plants (e.g., Elodea) are acid-tolerant and can photosynthesise effectively at lower pH than other species.

Hydrogencarbonate indicator (phenol red) changes colour with pH:

pH / $\mathrm{CO_2}$ level	Colour	Meaning
High pH (low $\mathrm{CO_2}$)	Purple	Photosynthesis exceeds respiration
Intermediate	Orange-red	Near compensation point
Low pH (high $\mathrm{CO_2}$)	Yellow	Respiration exceeds photosynthesis

In a photosynthesis investigation using hydrogencarbonate indicator:

1. Place a piece of aquatic plant (e.g., Elodea) in a boiling tube with hydrogencarbonate indicator.
2. Place the tube at different distances from a light source (different light intensities).
3. Record the time taken for the indicator to change from red/orange to purple.
4. Shorter time = faster photosynthesis (faster $\mathrm{CO_2}$ uptake, raising pH).

Variable	How to Control
Light intensity	Use a lamp at measured distances; use a light meter to measure intensity; keep other light sources off
Temperature	Water bath at constant temperature; allow the plant to equilibrate before starting
$\mathrm{CO_2}$	Use the same volume of indicator solution for each tube
Plant size	Use the same length of the same species from the same plant
Time	Start timing simultaneously for all tubes; use a stopwatch

A student measures the time for hydrogencarbonate indicator to change from red to purple at different light intensities:

Light intensity ($\mu\mathrm{mol\ photons\ m^{-2}\ s^{-1}}$)	10	30	60	100	200	400
Time to colour change (minutes)	25	14	8	5.5	3.2	3.0

The rate of photosynthesis is proportional to $1/\text{time}$:

Light intensity	Rate (1/time, $\mathrm{min^{-1}}$)	Rate ($\mu\mathrm{mol\ m^{-2}\ s^{-1}}$)
10	0.040	0.40
30	0.071	0.71
60	0.125	1.25
100	0.182	1.82
200	0.313	3.13
400	0.333	3.33

The rate increases with light intensity up to approximately 200 $\mu\mathrm{mol\ photons\ m^{-2}\ s^{-1}}$Then plateaus. At very high intensities (400), the rate barely increases, indicating that another factor ($\mathrm{CO_2}$ concentration or temperature) has become limiting.

## 13. The Global Carbon Cycle and Photosynthesis

Photosynthesis is the primary mechanism by which atmospheric $\mathrm{CO_2}$ is converted to organic carbon. Globally, photosynthesis fixes approximately $120 \times 10^{15}\ \mathrm{g\ C\ yr^{-1}}$ (gross primary production, GPP). Of this, approximately $60 \times 10^{15}\ \mathrm{g\ C\ yr^{-1}}$ is lost through plant respiration (R), giving a net primary production (NPP) of approximately $60 \times 10^{15}\mathrm{g\ C\ yr^{-1}}$.

Human activities release approximately $9.5 \times 10^{15}\ \mathrm{g\ C\ yr^{-1}}$ as $\mathrm{CO_2}$ (fossil fuel combustion, cement production, deforestation). The net imbalance means atmospheric $\mathrm{CO_2}$ is increasing at approximately $2\ \mathrm{ppm\ yr^{-1}}$Driving global warming.

For a stable atmospheric $\mathrm{CO_2}$ concentration:

$\text{Photosynthetic fixation} = \text{Respiration} + \text{Combustion} + \text{Decomposition}$

Currently, anthropogenic $\mathrm{CO_2}$ emissions exceed the capacity of natural sinks (photosynthesis and ocean absorption), leading to accumulation. Deforestation reduces photosynthetic capacity, further worsening the imbalance. Reforestation and increasing photosynthetic efficiency (e.g., through genetic engineering of $\mathrm{C_4}$ pathways into $\mathrm{C_3}$ crops) are potential mitigation strategies.

The “Z-scheme” describes the flow of electrons through Photosystems II and I, so named because the redox potential traces a Z-shape when plotted:

1. $\mathrm{H_2O}$ is split by PSII (photolysis), releasing $\mathrm{O_2}$, $\mathrm{H^+}$And electrons. The electrons have a relatively low energy (high redox potential, approximately $+0.8\ \mathrm{V}$).
2. Electrons pass through the electron transport chain (plastoquinone, cytochrome $b_6f$Plastocyanin), losing energy at each step. This energy is used to pump $\mathrm{H^+}$ into the thylakoid lumen.
3. The electrons reach PSI (low redox potential, approximately $-1.2\ \mathrm{V}$) and are re-energised by a second photon of light.
4. The re-energised electrons are transferred to ferredoxin and then to NADP reductase, which reduces $\mathrm{NADP^+}$ to $\mathrm{NADPH}$.

In addition to the non-cyclic (linear) electron flow described above, plants can also carry out cyclic photophosphorylation, which uses only PSI:

1. Electrons from PSI are transferred to ferredoxin.
2. Instead of being passed to NADP reductase, the electrons are passed back to the cytochrome $b_6f$ complex and then to plastocyanin, returning to PSI.
3. As electrons cycle through the ETC, $\mathrm{H^+}$ is pumped into the thylakoid lumen, generating a proton gradient that drives ATP synthesis.
4. No $\mathrm{NADPH}$ is produced and no $\mathrm{O_2}$ is evolved (water is not split).

Cyclic photophosphorylation is thought to operate when the Calvin cycle requires more ATP than NADPH (the ATP:NADPH ratio required by the Calvin cycle is 3:2, but non-cyclic electron flow produces them in approximately a 2.7:2 ratio). Cyclic flow makes up the ATP deficit.

Feature	Photophosphorylation	Oxidative Phosphorylation
Location	Thylakoid membranes (chloroplasts)	Inner mitochondrial membrane
Energy source	Light (photons)	Organic molecules (NADH, $\mathrm{FADH_2}$)
Electron donor	Water (photolysis)	NADH, $\mathrm{FADH_2}$
Final electron acceptor	$\mathrm{NADP^+}$	Oxygen ($\mathrm{O_2}$)
Proton gradient	Across thylakoid membrane ($\mathrm{H^+}$ accumulates in lumen)	Across inner mitochondrial membrane ($\mathrm{H^+}$ accumulates in intermembrane space)
ATP synthase	CF1CF0-ATP synthase (CF1 head in stroma)	F1F0-ATP synthase (F1 head in matrix)
Products	ATP, \mathrm{NADPH}$$\mathrm{O_2}	ATP, \mathrm{H_2O}$$\mathrm{CO_2}

The proton gradient across the thylakoid membrane has three contributors:

1. Photolysis of water: releases $\mathrm{H^+}$ into the lumen.
2. $\mathrm{Q}$ cycle (cytochrome $b_6f$ complex): pumps $\mathrm{H^+}$ from the stroma to the lumen (similar to Complex III in mitochondria).
3. NADP reductase: consumes $\mathrm{H^+}$ in the stroma when reducing $\mathrm{NADP^+}$ to $\mathrm{NADPH}$Increasing the $\mathrm{H^+}$ gradient.

The proton motive force drives ATP synthesis as $\mathrm{H^+}$ flows back to the stroma through ATP synthase. The pH of the lumen can reach approximately 5.0 (compared to pH 8.0 in the stroma), giving a $\Delta\mathrm{pH}$ of approximately 3.0.

The Calvin cycle is regulated by the light reactions through two mechanisms:

1. pH effect: light-driven proton pumping acidifies the thylakoid lumen but alkalinises the stroma (pH rises from approximately 7.0 in the dark to approximately 8.0 in the light). Key Calvin cycle enzymes (Rubisco, fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase) have higher activity at higher pH.

2. Thioredoxin system: light reduces ferredoxin, which reduces thioredoxin via ferredoxin-thioredoxin reductase. Reduced thioredoxin reduces disulphide bonds in Calvin cycle enzymes, activating them. In the dark, the enzymes are oxidised (inactive).

This ensures that the Calvin cycle only runs when the light reactions are producing the ATP and NADPH it requires.

Rubisco is a dual-function enzyme. It can catalyse either:

• Carboxylation: $\mathrm{RuBP + CO_2 \to}$ 2 molecules of GP (productive; feeds the Calvin cycle).
• Oxygenation: $\mathrm{RuBP + O_2 \to}$ 1 molecule of GP + 1 molecule of phosphoglycolate (wasteful).

The relative rates of carboxylation vs oxygenation depend on the ratio of $\mathrm{CO_2}$ to $\mathrm{O_2}$ at the active site of Rubisco. At high temperature, the solubility of $\mathrm{CO_2}$ decreases faster than that of $\mathrm{O_2}$And Rubisco’s affinity for $\mathrm{O_2}$ increases relative to $\mathrm{CO_2}$. This is why photorespiration is more significant at high temperatures.

Photorespiration consumes $\mathrm{O_2}$ and releases $\mathrm{CO_2}$ (effectively “undoing” photosynthesis), and uses ATP without producing sugar. It reduces the efficiency of photosynthesis by approximately 25% in $\mathrm{C_3}$ plants at warm temperatures.

The phosphoglycolate produced by oxygenation is salvaged through the photorespiratory pathway (involving peroxisomes and mitochondria), which converts it back to a Calvin cycle intermediate (glycerate-3-phosphate) at a cost of 1 ATP and loss of $\mathrm{CO_2}$.

In a $\mathrm{C_3}$ plant at $25$ degrees C, for every 4 carboxylation reactions, there is approximately 1 oxygenation reaction (25% photorespiration).

For every 5 turns of the Calvin cycle:

• 4 turns fix 4 $\mathrm{CO_2}$ (net gain: 4 carbon).
• 1 turn is “wasted” on oxygenation (no net carbon gain, but ATP and $\mathrm{NADPH}$ are still consumed).

Effective ATP cost per net $\mathrm{CO_2}$ fixed $= \frac{5 \times 3}{4} = 3.75$ ATP per $\mathrm{CO_2}$ (instead of 3 ATP in the absence of photorespiration).

At 35 degrees C, the ratio may increase to 1 oxygenation per 2 carboxylations, making photorespiration even more costly.

Chromatography separates mixtures based on differential partitioning between a mobile phase (solvent) and a stationary phase (paper or TLC plate).

Paper chromatography of plant pigments:

1. Extract pigments by grinding leaves in solvent (e.g., acetone).
2. Apply a spot of the extract to a pencil line near the bottom of a chromatography paper.
3. Place the paper in a solvent (mobile phase) with the spot above the solvent level.
4. The solvent moves up the paper by capillary action, carrying pigments with it.
5. Different pigments travel at different rates depending on their:

• Solubility in the mobile phase (more soluble = travels further).
• Affinity for the stationary phase (greater affinity = travels less far).

6. Calculate the retention factor ($R_f$) for each pigment:

$R_f = \frac{\text{Distance travelled by pigment}}{\text{Distance travelled by solvent front}}$

Pigment	Approximate $R_f$ Value	Colour	Solubility in Solvent
Carotene	$\approx 0.95$	Orange-yellow	Most soluble (non-polar); travels furthest
Xanthophyll	$\approx 0.71$	Yellow	Less soluble than carotene (contains oxygen)
Chlorophyll a	$\approx 0.59$	Blue-green	Intermediate
Chlorophyll b	$\approx 0.42$	Yellow-green	Least soluble (most polar); travels least far

A student carries out chromatography of leaf pigments. The solvent front travels $12.0\ \mathrm{cm}$. The distances travelled by each pigment spot are: carotene $= 11.4\ \mathrm{cm}$Xanthophyll $= 8.5\ \mathrm{cm}$Chlorophyll a $= 7.1\ \mathrm{cm}$Chlorophyll b $= 5.0\ \mathrm{cm}$.

$R_f$ values: carotene $= \frac{11.4}{12.0} = 0.95$; xanthophyll $= \frac{8.5}{12.0} = 0.71$; chlorophyll a $= \frac{7.1}{12.0} = 0.59$; chlorophyll b $= \frac{5.0}{12.0} = 0.42$.

These values match the expected range, confirming the identity of the pigments.

When plotting the rate of photosynthesis against one factor while keeping others constant:

• At low values of the manipulated factor, the rate increases linearly (the manipulated factor is limiting).
• The curve then begins to level off as another factor becomes limiting.
• The plateau represents the maximum rate achievable under the given conditions (the limiting factor is the one held constant).

A student investigates the effect of $\mathrm{CO_2}$ concentration on the rate of photosynthesis at two light intensities:

$\mathrm{CO_2}$ concentration (%)	Rate at low light ($\mu\mathrm{mol\ CO_2\ m^{-2}\ s^{-1}}$)	Rate at high light ($\mu\mathrm{mol\ CO_2\ m^{-2}\ s^{-1}}$)
0.01	2	4
0.03	4	10
0.05	6	16
0.10	8	24
0.20	8	24
0.40	8	24

At low light intensity, the rate plateaus at $8\ \mu\mathrm{mol\ CO_2\ m^{-2}\ s^{-1}}$ (at 0.10% $\mathrm{CO_2}$). Above this $\mathrm{CO_2}$ concentration, the rate does not increase, indicating that light intensity has become the limiting factor.

At high light intensity, the rate plateaus at $24\ \mu\mathrm{mol\ CO_2\ m^{-2}\ s^{-1}}$ (at 0.10% $\mathrm{CO_2}$). The higher plateau indicates that with more light available, a higher maximum rate is achievable before $\mathrm{CO_2}$ becomes limiting.

At low temperature (below the optimum of approximately 25—30 degrees C for $\mathrm{C_3}$ plants), the rate of photosynthesis is limited by enzyme activity (kinetic energy is low, fewer enzyme-substrate collisions). As temperature increases, enzyme activity increases, and the rate rises.

Above the optimum, the rate declines because:

• Rubisco and other enzymes begin to denature.
• Photorespiration increases (Rubisco’s oxygenase activity increases faster than its carboxylase activity).
• Stomata close to reduce water loss, reducing $\mathrm{CO_2}$ uptake.

## 23. Investigating Factors Affecting the Rate of Photosynthesis

Aim: to investigate the effect of light intensity on the rate of photosynthesis.

Method:

1. Cut 5 pieces of healthy Elodea (Canadian pondweed), each approximately 5 cm long.
2. Place each piece in a boiling tube with 10 cm$^3$ of hydrogencarbonate indicator (orange-red at approximately pH 7).
3. Seal each tube with a bung and ensure no air bubbles are trapped.
4. Place each tube at a known distance from a light source (e.g., 5, 10, 20, 40, 80 cm).
5. Measure the light intensity at each distance using a light meter.
6. Start a stopwatch and record the time taken for the indicator to change from orange-red to purple (pH $\approx 8.5$).
7. Repeat 3 times at each distance and calculate a mean time.

Controls:

• All tubes must have the same volume of indicator solution.
• All pieces of Elodea must be from the same plant, similar length, and similar mass.
• Temperature must be kept constant (use a water bath).
• The light source must be the same type and wattage throughout.

Light intensity follows the inverse square law:

$I \propto \frac{1}{d^2}$

If the light intensity at 10 cm is $400\ \mu\mathrm{mol\ photons\ m^{-2}\ s^{-1}}$Then at 40 cm:

$I = 400 \times \frac{10^2}{40^2} = 400 \times \frac{100}{1600} = 25\ \mu\mathrm{mol\ photons\ m^{-2}\ s^{-1}}$.

Important: always plot rate (1/time) against light intensity (not distance), because the relationship between rate and distance is non-linear.

Error	Effect	How to Minimise
Elodea pieces of different sizes	Different photosynthetic rates	Measure and match pieces by mass/length
Temperature fluctuations	Affects enzyme activity	Water bath at constant temperature
Light from other sources	Increases light intensity at all positions	Carry out in a dark room; use a blackout box
Evaporation from the indicator	Changes concentration	Seal tubes properly; use narrow tubes
Colour change is gradual and subjective	Timing errors	Use a colorimeter for more precise measurement; use the same person to judge the endpoint

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## Cross-References

• Biology
• Cells
• Genetics and DNA