Photosynthesis (In Depth)

Photosynthesis (In Depth)

info

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

1. Overview

1.1 Definition

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 ($\mathrm{NADPH}$).

1.2 The Two Stages

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

2. Chloroplast Structure

2.1 Adaptations for Photosynthesis

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

2.2 Evidence for the Endosymbiotic Theory

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.

3. Photosynthetic Pigments

3.1 Types of Pigment

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.

3.2 Absorption and Action Spectra

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.

3.3 Thin-Layer Chromatography of Pigments

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:

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

4. Light-Dependent Reactions

4.1 Photosystems

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

4.2 Non-Cyclic Photophosphorylation (Z-Scheme)

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 $\mathrm{H^+}$).

4.3 Cyclic Photophosphorylation

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).

5. The Calvin Cycle (Light-Independent Reactions)

5.1 Overview

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 $\mathrm{CO_2}$.

5.2 Detailed Steps

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.

5.3 Summary of the Calvin Cycle

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):

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

5.4 Fate of Triose Phosphate (G3P)

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).

5.5 Photorespiration

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).

warning

Common Pitfall Students often refer to the Calvin cycle as the "light-independent reactions" and state that they "do not require light." While the Calvin cycle itself does not directly use light, it is dependent on the products of the light-dependent reactions (ATP and $\mathrm{NADPH}$), which cease in the dark. In practice, the Calvin cycle stops within seconds of placing a plant in darkness because ATP and $\mathrm{NADPH}$ are rapidly depleted.

6. Limiting Factors

6.1 Principle of 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.

6.2 Light Intensity

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 $\mathrm{CO_2}$.

6.3 $\mathrm{CO_2}$ Concentration

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 (typically 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.

6.4 Temperature

Temperature affects photosynthesis because the Calvin cycle is enzyme-catalysed (primarily by Rubisco). The rate increases with temperature up to an optimum (typically $25$--$30\ ^\circ\mathrm{C}$ 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).

6.5 Interacting Factors: Graphical Analysis

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.

7. $\mathrm{C_3}$, $\mathrm{C_4}$, and CAM Plants

7.1 $\mathrm{C_3}$ Plants

$\mathrm{C_3}$ 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.

7.2 $\mathrm{C_4}$ Plants

$\mathrm{C_4}$ 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).

7.3 CAM Plants

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.

7.4 Comparison

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

warning

Common Pitfall Students often write that "$\mathrm{C_4}$ plants do not use the Calvin cycle." $\mathrm{C_4}$ plants do use the Calvin cycle -- it occurs in the bundle sheath cells, not in the mesophyll cells. The $\mathrm{C_4}$ pathway is a supplementary $\mathrm{CO_2}$-concentrating mechanism that feeds $\mathrm{CO_2}$ into the Calvin cycle. Both pathways are present and operate together.

8. Practical Investigations

8.1 Measuring the Rate of Photosynthesis

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.

8.2 Worked Example: Investigating Light Intensity

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.

Practice Problems

Details

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

Details

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

Details

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

Details

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

Details

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

9. Photosynthesis and the Environment

9.1 The Compensation Point

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

9.2 The Saturation Point

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.

9.3 Daily Patterns of Gas Exchange

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.

9.4 Seasonal Variation

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.

10. Photosynthesis and Crop Yield

10.1 Greenhouse Cultivation

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

10.2 Maximising Light Interception

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.

10.3 Calculating Gross and Net Photosynthesis

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.

11. Additional Photosynthetic Pigments

11.1 Accessory Pigments and the Antenna Complex

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.

11.2 The Absorption Spectrum and Action Spectrum

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.

11.3 Dissolved $\mathrm{CO_2}$ and Carbonate Chemistry

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.

12. Practical Skills in Photosynthesis Investigations

12.1 Using Hydrogencarbonate Indicator

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).

12.2 Controlling Variables

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

12.3 Worked Example: Light Intensity and Rate

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.

warning

Common Pitfall Students often plot rate against distance from the lamp rather than light intensity. Light intensity follows the inverse square law ($I \propto 1/d^2$), so the rate vs. distance graph is non-linear. Always convert distances to light intensity values before plotting.

13. The Global Carbon Cycle and Photosynthesis

13.1 Photosynthesis as a Carbon Sink

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.

13.2 The Carbon Balance

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.

14. Light Reactions: Advanced Mechanisms

14.1 The Z-Scheme

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 $\mathrm{NADPH}$.

14.2 Cyclic Photophosphorylation

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.

14.3 Photophosphorylation vs Oxidative Phosphorylation

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, $\mathrm{H_2O}$, $\mathrm{CO_2}$

14.4 Chemiosmosis in Chloroplasts

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.

15. The Calvin Cycle: Advanced Topics

15.1 Regulation of the Calvin Cycle

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.

15.2 Photorespiration: Detailed Mechanism

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 $\mathrm{CO_2}$.

15.3 Calculating the Cost of Photorespiration

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 $= \frac◆LB◆5 \times 3◆RB◆◆LB◆4◆RB◆ = 3.75$ 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.

16. Chromatography of Photosynthetic Pigments

16.1 Principle

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:

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

16.2 Expected Results

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

16.3 Worked Example

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.

17. Limiting Factors: Graphical Analysis

17.1 Interpreting Limiting Factor Graphs

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).

17.2 Worked Example: $\mathrm{CO_2}$ and Light Intensity Interactions

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.

17.3 Temperature and the Rate of Photosynthesis

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.

warning

Common Pitfall When asked "what is the limiting factor at point X on a graph," students often say "temperature" when the graph shows rate vs $\mathrm{CO_2}$. The limiting factor is the factor that is NOT on the x-axis -- the factor held constant. If the graph shows rate vs $\mathrm{CO_2}$ concentration and the curve has plateaued, the limiting factor is light intensity or temperature (whichever is held constant). Always identify which factor is being manipulated and which are held constant.

23. Investigating Factors Affecting the Rate of Photosynthesis

23.1 Using Hydrogencarbonate Indicator: Detailed Method

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.

23.2 Calculating Light Intensity

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.

23.3 Sources of Error

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

---

tip

Diagnostic Test

22. Photosynthesis and Agriculture: Maximising Crop Yield

22.1 The Light Compensation Point in Different Plants

The light compensation point varies between species:

Plant Type	Typical Compensation Point ($\mu\mathrm{mol\ photons\ m^{-2}\ s^{-1}}$)	Adaptation
Shade-tolerant (e.g., ferns, forest floor plants)	5--15	Efficient photosynthesis at low light; large, thin leaves with many chloroplasts
Shade-intolerant (e.g., sunflowers, maize)	30--50	Require high light; small, thick leaves; high photosynthetic capacity at high light
$\mathrm{C_4}$ plants (e.g., maize, sugarcane)	Lower than $\mathrm{C_3}$ at same temperature	$\mathrm{CO_2}$-concentrating mechanism reduces photorespiration

22.2 Greenhouse Gas Enrichment Calculations

Worked Example. A greenhouse operator enriches the $\mathrm{CO_2}$ concentration from 400 ppm (ambient) to 1000 ppm. The cost is $\pounds 0.50$ per $\mathrm{m^3}$ of $\mathrm{CO_2}$ gas. The greenhouse has a volume of $500\ \mathrm{m^3}$.

$\mathrm{CO_2}$ needed to raise from 400 ppm to 1000 ppm $= (1000 - 400) \times 10^{-6} \times 500\ \mathrm{m^3} = 600 \times 10^{-6} \times 500 = 0.30\ \mathrm{m^3}$.

Cost $= 0.30 \times \pounds 0.50 = \pounds 0.15$ per enrichment cycle.

However, this is a one-time enrichment. To maintain the elevated level, continuous $\mathrm{CO_2}$ supply is needed to replace $\mathrm{CO_2}$ consumed by photosynthesis. If the crop consumes $10\ \mathrm{g\ CO_2\ m^{-2}\ h^{-1}}$ and the greenhouse floor area is $200\ \mathrm{m^2}$:

$\mathrm{CO_2}$ consumed per hour $= 10 \times 200 = 2000\ \mathrm{g\ h^{-1}}$.

At standard conditions, 1 mole $\mathrm{CO_2} = 44\ \mathrm{g}$, and 1 mole of gas occupies approximately $24\ \mathrm{dm^3}$.

Volume of $\mathrm{CO_2}$ consumed per hour $= \frac{2000}{44} \times 24 = 1091\ \mathrm{dm^3\ h^{-1}} = 1.09\ \mathrm{m^3\ h^{-1}}$.

Cost per hour $= 1.09 \times \pounds 0.50 = \pounds 0.55$.

This cost is offset by the increased crop yield (typically 20--40% increase in biomass for $\mathrm{C_3}$ crops at 1000 ppm $\mathrm{CO_2}$).

22.3 Light and Plant Growth

Worked Example. A greenhouse uses LED grow lights providing 200\ \mu\mathrm{mol\ photons\ m^{-2}\ s^{-1} of PAR (photosynthetically active radiation, 400--700 nm). The greenhouse floor area is $200\ \mathrm{m^2}$. Electricity costs $\pounds 0.15$ per kWh.

Daily light integral (DLI) for a 16-hour photoperiod:

$\text{DLI} = 200 \times 16 \times 3600 \times 10^{-6} = 200 \times 57600 \times 10^{-6} = 11.52\ \mathrm{mol\ photons\ m^{-2}\ day^{-1}}$.

Total daily light $= 11.52 \times 200 = 2304\ \mathrm{mol\ photons\ day^{-1}}$.

Energy per photon (at 550 nm, average PAR wavelength): $E = \frac◆LB◆hc◆RB◆◆LB◆\lambda◆RB◆ = \frac◆LB◆6.626 \times 10^{-34} \times 3 \times 10^8◆RB◆◆LB◆550 \times 10^{-9}◆RB◆ = 3.61 \times 10^{-19}\ \mathrm{J}$.

Total energy per day $= 2304 \times 6.022 \times 10^{23} \times 3.61 \times 10^{-19} = 2304 \times 2.17 \times 10^5 = 5.0 \times 10^{8}\ \mathrm{J} = 139\ \mathrm{kWh}$.

Daily electricity cost $= 139 \times \pounds 0.15 = \pounds 20.85$.

---

tip

Diagnostic Test

18. The Chloroplast: Structure and Adaptations

18.1 Chloroplast Anatomy

Structure	Description	Function
Double membrane	Outer membrane (permeable) and inner membrane (selectively permeable)	Compartmentalisation; controls entry/exit of molecules
Thylakoid membrane	Internal membrane system forming flattened sacs (thylakoids)	Site of light-dependent reactions (PSII, cytochrome $b_6f$, PSI, ATP synthase)
Thylakoid lumen	Space inside the thylakoid	Proton accumulation site for chemiosmosis (low pH, approximately 5)
Grana	Stacks of thylakoids	Maximise surface area for light absorption
Lamellae	Thylakoids connecting grana	Allow connections between grana for electron and proton transport
Stroma	Fluid-filled space surrounding thylakoids	Site of the Calvin cycle (light-independent reactions); contains enzymes, DNA, ribosomes, starch granules

18.2 Chloroplast DNA and Endosymbiosis

Like mitochondria, chloroplasts contain their own circular DNA, 70S ribosomes, and reproduce by binary fission. They are thought to have originated from a photosynthetic cyanobacterium that was engulfed by a eukaryotic cell (primary endosymbiosis). Evidence:

• Chloroplast DNA is similar to cyanobacterial DNA.
• Ribosomes are 70S (prokaryotic size).
• Chloroplasts are surrounded by a double membrane.
• Chlorophyll a is found in both cyanobacteria and chloroplasts.

19. Environmental Factors Affecting Photosynthesis: Quantitative

19.1 Worked Example: Light Intensity and Rate

The relationship between light intensity and photosynthetic rate can be described by a rectangular hyperbola:

$P = \frac◆LB◆P_{\max} \times I◆RB◆◆LB◆K_m + I◆RB◆$

Where $P$ = photosynthetic rate, $P_{\max}$ = maximum rate (at saturation), $I$ = light intensity, $K_m$ = light intensity at which the rate is half of $P_{\max}$.

If $P_{\max} = 20\ \mu\mathrm{mol\ CO_2\ m^{-2}\ s^{-1}}$ and $K_m = 50\ \mu\mathrm{mol\ photons\ m^{-2}\ s^{-1}}$:

At $I = 50$: $P = \frac◆LB◆20 \times 50◆RB◆◆LB◆50 + 50◆RB◆ = \frac{1000}{100} = 10\ \mu\mathrm{mol\ CO_2\ m^{-2}\ s^{-1}}$ (half of $P_{\max}$, as expected).

At $I = 200$: $P = \frac◆LB◆20 \times 200◆RB◆◆LB◆50 + 200◆RB◆ = \frac{4000}{250} = 16\ \mu\mathrm{mol\ CO_2\ m^{-2}\ s^{-1}}$.

At $I = 1000$: $P = \frac◆LB◆20 \times 1000◆RB◆◆LB◆50 + 1000◆RB◆ = \frac{20000}{1050} = 19.0\ \mu\mathrm{mol\ CO_2\ m^{-2}\ s^{-1}}$ (approaching $P_{\max}$).

19.2 Temperature Optimum Curves

$\mathrm{C_3}$ plants have a temperature optimum of approximately 25--30 degrees C. Above this, the rate declines due to:

• Enzyme denaturation (Rubisco and other Calvin cycle enzymes).
• Increased photorespiration (oxygenase activity increases faster than carboxylase activity).
• Stomatal closure (reducing $\mathrm{CO_2}$ availability).

$\mathrm{C_4}$ plants have a higher temperature optimum (approximately 35--40 degrees C) because the $\mathrm{CO_2}$-concentrating mechanism in bundle sheath cells reduces photorespiration. This is why $\mathrm{C_4}$ grasses (maize, sorghum) dominate in tropical grasslands.

19.3 Water Stress and Photosynthesis

Water deficiency affects photosynthesis through several mechanisms:

1. Stomatal closure: ABA triggers stomatal closure, reducing $\mathrm{CO_2}$ uptake and limiting the Calvin cycle.
2. Reduced electron transport: water stress reduces the hydration of thylakoid membranes, impairing electron transport.
3. Rubisco inactivation: dehydration can cause conformational changes in Rubisco.
4. Reduced chlorophyll content: severe water stress causes chlorophyll degradation (leaves turn yellow).

The effect of water stress is most pronounced during the afternoon, when transpiration rates are highest and leaf water potential is lowest. This creates a midday depression in the photosynthetic rate even when light intensity is at its peak.

20. $\mathrm{C_4}$ and CAM Photosynthesis: Detailed Mechanisms

20.1 $\mathrm{C_4}$ Photosynthesis in Detail

Step 1 (mesophyll cells): $\mathrm{CO_2}$ is fixed by PEP carboxylase to form oxaloacetate (4C), which is converted to malate.

$\mathrm{PEP + CO_2 \xrightarrow{PEP\ carboxylase} oxaloacetate \to malate}$

Step 2 (bundle sheath cells): malate diffuses into the bundle sheath cells (through plasmodesmata) and is decarboxylated, releasing $\mathrm{CO_2}$.

$\mathrm{Malate \to pyruvate + CO_2}$

The released $\mathrm{CO_2}$ has a high concentration in the bundle sheath cells (up to 60 times atmospheric), which suppresses the oxygenase activity of Rubisco and minimises photorespiration.

Step 3: the $\mathrm{CO_2}$ enters the Calvin cycle in the bundle sheath cells.

Step 4: pyruvate diffuses back to the mesophyll cells and is converted to PEP using ATP (the energy cost of the $\mathrm{C_4}$ pathway).

20.2 Energetic Cost of $\mathrm{C_4}$

The $\mathrm{C_4}$ pathway uses 5 ATP per $\mathrm{CO_2}$ fixed (2 extra ATP per $\mathrm{CO_2}$ compared to $\mathrm{C_3}$). This extra cost is worthwhile at high temperature and low $\mathrm{CO_2}$ because it eliminates photorespiration, which wastes more energy than the $\mathrm{C_4}$ pathway consumes.

Condition	$\mathrm{C_3}$ Advantage	$\mathrm{C_4}$ Advantage
Low temperature ($< 25$ degrees C)	Less photorespiration; no extra ATP needed	Extra ATP wasted (photorespiration is minimal anyway)
Low light intensity	Lower ATP demand per $\mathrm{CO_2}$	Extra ATP demand not met by limited light
High temperature ($> 30$ degrees C)	Photorespiration reduces efficiency	$\mathrm{CO_2}$ concentration eliminates photorespiration
High light intensity	--	More ATP available to fuel the $\mathrm{C_4}$ pathway
Arid conditions	--	Higher water-use efficiency (fewer stomata needed)
$\mathrm{CO_2}$ levels $<$ current atmospheric	Photorespiration is severe	Efficient at low $\mathrm{CO_2}$

20.3 CAM Photosynthesis in Detail

CAM plants (e.g., Opuntia, Kalanchoe, pineapple) separate the two stages of $\mathrm{C_4}$ photosynthesis temporally rather than spatially:

Night (stomata open):

• $\mathrm{CO_2}$ enters through open stomata (minimal water loss because air is cooler and more humid at night).
• $\mathrm{CO_2}$ is fixed by PEP carboxylase to form malate.
• Malate is stored in large vacuoles.

Day (stomata closed):

• Malate is released from vacuoles and decarboxylated, releasing $\mathrm{CO_2}$.
• The $\mathrm{CO_2}$ enters the Calvin cycle (Rubisco operates at high internal $\mathrm{CO_2}$, minimising photorespiration).
• Stomata are closed, minimising water loss.

CAM plants have the highest water-use efficiency of all photosynthetic pathways, but the lowest photosynthetic rate (limited by the amount of malate that can be stored in vacuoles overnight).

21. Investigating Photosynthesis: Classic Experiments

21.1 Van Helmont's Experiment (1640s)

Van Helmont planted a willow tree in a pot with 91 kg of soil. After 5 years, the tree had gained 75 kg but the soil had lost only 57 g. He concluded that plants grow from water (not from soil). This was partially correct -- most of the mass comes from $\mathrm{CO_2}$ (not yet known), not water.

21.2 Priestley's Experiment (1771)

Priestley showed that a mint plant "restored" air that had been "injured" by a burning candle or a breathing mouse. He demonstrated that plants produce a substance (oxygen) that supports combustion and respiration.

21.3 Ingenhousz's Experiment (1779)

Ingenhousz showed that plants produce oxygen only in the presence of light (not in the dark). He also showed that only the green parts of the plant produce oxygen.

21.4 The Hill Reaction (1939)

Robert Hill demonstrated that isolated chloroplasts (in the absence of $\mathrm{CO_2}$) can produce oxygen when illuminated in the presence of an artificial electron acceptor (e.g., ferricyanide). This proved that oxygen production (photolysis of water) is separate from carbon fixation (the Calvin cycle), which requires $\mathrm{CO_2}$.

$2\mathrm{H_2O + 2\ \text{ferricyanide (oxidised)} \xrightarrow{\text{light, chloroplasts}} O_2 + 2\ \text{ferrocyanide (reduced)} + 4H^+}$

21.5 Calvin, Benson, and Bassham (1950s)

Using radioactive $\mathrm{^{14}CO_2}$ and two-dimensional paper chromatography, Calvin and colleagues traced the path of carbon through photosynthesis. By exposing algae to $\mathrm{^{14}CO_2}$ for varying lengths of time and identifying the radioactive compounds, they determined the sequence of the Calvin cycle:

• After 5 seconds: GP (glycerate-3-phosphate, 3C) was labelled.
• After 30 seconds: sugar phosphates (TP, hexose phosphates, RuBP) were labelled.
• The first labelled compound was GP, confirming that $\mathrm{CO_2}$ is incorporated into a 5-carbon acceptor (RuBP) to produce a 3-carbon compound.

22. Agricultural Applications of Photosynthesis Research

22.1 Improving Crop Yield

Crop yield depends on the efficiency of photosynthesis. Research has focused on:

1. Increasing Rubisco specificity: natural Rubisco has a low affinity for $\mathrm{CO_2}$ and also catalyses photorespiration (oxygenation of RuBP). Engineering Rubisco with higher $\mathrm{CO_2}$ specificity could increase photosynthetic efficiency by up to 30%.

2. Introducing C4 pathways into C3 crops: C4 plants (maize, sugarcane) concentrate $\mathrm{CO_2}$ around Rubisco, suppressing photorespiration. Attempts to introduce C4 anatomy and biochemistry into rice (the C4 Rice Project) aim to increase rice yield by 30--50%.

3. Optimising light harvesting: adjusting leaf angle, chlorophyll distribution, and antenna size to maximise light capture and minimise wasteful dissipation of excess light energy.

4. Reducing photorespiration: engineering alternative metabolic pathways to recycle the products of photorespiration without releasing $\mathrm{CO_2}$.

22.2 Greenhouse Gas Management

Photosynthesis is a key carbon sink. Understanding photosynthetic efficiency informs climate change mitigation:

• Global photosynthesis: terrestrial plants fix approximately $120\ \mathrm{Gt\ C\ yr^{-1}}$; marine phytoplankton fix approximately $50\ \mathrm{Gt\ C\ yr^{-1}}$.
• Carbon fertilisation effect: elevated atmospheric $\mathrm{CO_2}$ increases photosynthetic rate in C3 plants (up to a point, limited by other factors such as nitrogen availability).
• Limitations: the carbon fertilisation effect is limited by nutrient availability, temperature, and water stress. Tropical forests may become carbon sources under extreme drought.

22.3 Biofuels from Photosynthetic Organisms

• Bioethanol: produced by fermentation of sugars from C4 crops (maize, sugarcane). Energy return on investment (EROI) is relatively low ($\approx 1.3$--$1.6$).
• Biodiesel: from plant oils (soybean, oil palm, microalgae). Microalgae have much higher lipid content ($\approx 30$--$60\%$ dry weight) and higher photosynthetic efficiency than terrestrial plants.
• Photobiological hydrogen production: some photosynthetic bacteria and green algae (e.g., Chlamydomonas reinhardtii) can produce $\mathrm{H_2}$ under anaerobic conditions using hydrogenase enzymes. Currently inefficient; research ongoing.

23. Integration with Other Metabolic Pathways

23.1 Photosynthesis and Respiration: The Carbon Cycle

Process	Location	Inputs	Outputs
Photosynthesis (light reactions)	Thylakoid membrane	Light, $\mathrm{H_2O}$, ADP, $\mathrm{NADP^+}$	$\mathrm{O_2}$, ATP, $\mathrm{NADPH}$
Photosynthesis (Calvin cycle)	Stroma	$\mathrm{CO_2}$, ATP, $\mathrm{NADPH}$	TP, GP, RuBP, ADP, $\mathrm{NADP^+}$
Glycolysis	Cytoplasm	Glucose, ATP, $\mathrm{NAD^+}$	Pyruvate, ATP (net 2), $\mathrm{NADH}$
Link reaction	Mitochondrial matrix	Pyruvate, $\mathrm{NAD^+}$, CoA	Acetyl CoA, $\mathrm{CO_2}$, $\mathrm{NADH}$
Krebs cycle	Mitochondrial matrix	Acetyl CoA, $\mathrm{NAD^+}$, FAD	$\mathrm{CO_2}$, ATP, $\mathrm{NADH}$, $\mathrm{FADH_2}$
Oxidative phosphorylation	Inner mitochondrial membrane	$\mathrm{NADH}$, $\mathrm{FADH_2}$, $\mathrm{O_2}$, ADP	$\mathrm{H_2O}$, ATP

The products of photosynthesis (glucose, $\mathrm{O_2}$) are the reactants of respiration. The products of respiration ($\mathrm{CO_2}$, $\mathrm{H_2O}$) are the reactants of photosynthesis.

23.2 Fate of Triose Phosphate (TP)

TP produced in the Calvin cycle has several fates:

1. Converted to hexose sugars (glucose, fructose) by reverse glycolysis. Hexoses can be used for respiration.
2. Converted to starch (amylose and amylopectin) for storage in chloroplasts. Starch is the main storage carbohydrate in most plants.
3. Converted to sucrose for transport in the phloem. Sucrose is the main transport sugar in most plants because it is non-reducing (does not react with amino groups in proteins).
4. Converted to cellulose for cell wall synthesis. Cellulose is the main structural polysaccharide in plant cell walls.
5. Converted to lipids (triglycerides) for energy storage in seeds (e.g., sunflower, rapeseed).
6. Converted to amino acids by combining with nitrogen (from $\mathrm{NO_3^-}$ or $\mathrm{NH_4^+}$) in a process called nitrogen assimilation.

warning

Common Pitfall Students often think that the Calvin cycle directly produces glucose. In fact, the Calvin cycle produces TP (a 3-carbon compound), which must then be converted to glucose (6C) by joining two TP molecules. Only some TP molecules are exported from the chloroplast; the rest are used to regenerate RuBP. For every 6 molecules of $\mathrm{CO_2}$ fixed, the cycle produces 2 molecules of TP that can be exported, while 10 molecules of TP are used to regenerate 6 molecules of RuBP.

23.3 Calculating Net Primary Productivity (NPP)

$\mathrm{NPP} = \mathrm{GPP} - R$

Where:

• GPP = Gross Primary Productivity (total energy fixed by photosynthesis)
• R = Respiratory losses (energy used by the plant in respiration)
• NPP = Net Primary Productivity (energy available to herbivores and decomposers)

Example: A forest has a GPP of $20,000\ \mathrm{kJ\ m^{-2}\ yr^{-1}}$ and respiratory losses of $12,000\ \mathrm{kJ\ m^{-2}\ yr^{-1}}$.

$\mathrm{NPP} = 20,000 - 12,000 = 8,000\ \mathrm{kJ\ m^{-2}\ yr^{-1}}$

The NPP represents the energy available to the next trophic level (herbivores). Only approximately 10--20% of NPP is transferred to herbivores (the rest is lost as heat, uneaten material, or undigested waste).

24. Photosynthesis Under Stress Conditions

24.1 Water Stress

Water deficit affects photosynthesis in several ways:

1. Stomatal closure: water-stressed plants close stomata to reduce water loss, which also limits $\mathrm{CO_2}$ uptake, reducing the rate of the Calvin cycle.
2. Reduced electron transport: water is the electron donor in the light reactions; severe water stress limits electron supply.
3. Rubisco degradation: prolonged water stress can lead to oxidative damage and degradation of Rubisco.
4. Photoinhibition: if stomata are closed, light energy cannot be used for $\mathrm{CO_2}$ fixation, leading to accumulation of excited chlorophyll molecules and production of reactive oxygen species (ROS), which damage the photosynthetic apparatus.

24.2 Temperature Stress

Temperature Range	Effect on Photosynthesis
Below optimum	Enzyme activity decreases (Q10 effect); membrane fluidity decreases, affecting electron transport
Optimum (25--30 degrees C for C3; 30--40 degrees C for C4)	Maximum photosynthetic rate
Above optimum	Enzymes denature (especially Rubisco activase); photorespiration increases; membranes become too fluid
Extreme heat (> 40 degrees C)	Photosystem II is damaged; thylakoid membranes disrupted; irreversible photoinhibition

24.3 Light Stress

• High light intensity: can cause photoinhibition (damage to photosystem II). Plants protect themselves through non-photochemical quenching (NPQ), which dissipates excess light energy as heat.
• Low light intensity: limits the rate of the light reactions, reducing ATP and NADPH production, which in turn limits the Calvin cycle.
• Rapid fluctuations in light: plants in natural environments experience sunflecks (brief periods of direct sunlight under a canopy). The Calvin cycle takes time to activate (the "induction period"), so plants that experience frequent sunflecks may not achieve maximum photosynthetic efficiency.

25. Photosynthesis Practical Investigations

25.1 Measuring the Rate of Photosynthesis

Method	What is Measured	Procedure	Advantages	Limitations
Oxygen production (bubble count)	Number of $\mathrm{O_2}$ bubbles per minute	Place aquatic plant (e.g., Elodea) in a test tube of water; count bubbles under different conditions	Simple; qualitative comparison	Bubble size varies; not quantitative
Oxygen production (gas syringe)	Volume of $\mathrm{O_2}$ per unit time	Collect $\mathrm{O_2}$ from aquatic plant in an inverted measuring cylinder or gas syringe	More quantitative than bubble counting	Temperature must be controlled
Mass change of aquatic plant	Change in mass per unit time	Weigh plant before and after a period in light (blot dry)	Simple	Water adhering to plant affects mass; evaporation confounds results
Sensor (dissolved $\mathrm{O_2}$ probe)	Dissolved $\mathrm{O_2}$ concentration	Place probe in water with aquatic plant; record $\mathrm{O_2}$ concentration over time	Accurate; continuous data	Expensive equipment; probe drift
$\mathrm{CO_2}$ uptake	Change in $\mathrm{CO_2}$ concentration	Use $\mathrm{CO_2}$ sensor in a sealed chamber with plant	Direct measure of substrate use	Expensive; sealed chamber may alter conditions

25.2 Investigating the Effect of Light Intensity

Procedure:

1. Cut a piece of Elodea (approx 10 cm) and place it in a boiling tube of sodium hydrogen carbonate solution ($\mathrm{NaHCO_3}$, 0.5%) to provide a constant supply of dissolved $\mathrm{CO_2}$.
2. Place the boiling tube in a water bath at constant temperature (e.g., 25 degrees C).
3. Position a lamp at measured distances from the boiling tube (e.g., 5, 10, 15, 20, 25, 30 cm).
4. Record the distance and measure the rate of $\mathrm{O_2}$ production (bubble count or gas syringe) at each distance.
5. Calculate light intensity using the inverse square law: $\text{light intensity} \propto \frac{1}{d^2}$ (where $d$ is the distance from the lamp).

Expected results:

• At low light intensity, the rate of photosynthesis increases linearly with light intensity (light is the limiting factor).
• At higher light intensities, the rate plateaus (another factor becomes limiting -- usually $\mathrm{CO_2}$ concentration or temperature).
• The graph of rate vs light intensity shows a characteristic rectangular hyperbola.

25.3 Investigating the Effect of $\mathrm{CO_2}$ Concentration

Procedure:

1. Place Elodea in boiling tubes with different concentrations of $\mathrm{NaHCO_3}$ solution (e.g., 0.1%, 0.2%, 0.5%, 1.0%, 2.0%).
2. Maintain constant light intensity and temperature.
3. Measure the rate of $\mathrm{O_2}$ production at each concentration.

Expected results: similar rectangular hyperbola; rate increases with $\mathrm{CO_2}$ concentration until another factor becomes limiting.

25.4 Using a Chlorophyll Extract

Chlorophyll can be extracted from leaves by grinding them in a solvent (e.g., ethanol or acetone) and filtering. The extract can be used to:

• Separate photosynthetic pigments by paper chromatography or thin-layer chromatography (TLC).
• Measure absorbance using a colorimeter or spectrophotometer. Chlorophyll $a$ absorbs most strongly at approximately 430 nm (blue) and 662 nm (red). Chlorophyll $b$ absorbs most strongly at approximately 453 nm and 642 nm.

The absorption spectrum of chlorophyll closely matches the action spectrum of photosynthesis (the rate of photosynthesis at different wavelengths), supporting the hypothesis that chlorophyll is the primary photosynthetic pigment.

26. Chloroplast Structure and Function

26.1 Detailed Chloroplast Anatomy

Structure	Description	Function
Outer membrane	Smooth, freely permeable to small molecules	Contains porins for passive diffusion of molecules up to 10 kDa
Inner membrane	Selectively permeable; contains transport proteins	Controls movement of metabolites in and out of the chloroplast
Intermembrane space	Between outer and inner membranes	Similar composition to cytosol
Stroma	Fluid-filled interior of the chloroplast	Site of the Calvin cycle; contains DNA, ribosomes, starch granules, lipid droplets
Thylakoid membrane	Internal membrane system forming flattened sacs	Site of the light-dependent reactions; contains photosystems, ETC, ATP synthase
Thylakoid lumen	Interior of the thylakoid sac	Protons accumulate here during the light reactions, creating the proton gradient
Granum (plural: grana)	Stack of thylakoids	Increases surface area for light absorption
Lamellae (intergranal thylakoids)	Thylakoids connecting grana	Allow communication between grana; distribute products of light reactions
Starch granule	Stored in the stroma	Storage form of glucose (temporary)

26.2 Chloroplast DNA and Endosymbiosis

Chloroplasts contain their own circular DNA (approximately 120--160 kbp, encoding approximately 100--120 genes), ribosomes (70S, similar to bacterial ribosomes), and can replicate independently. This is consistent with the endosymbiotic theory: chloroplasts evolved from free-living photosynthetic bacteria (cyanobacteria) that were engulfed by a eukaryotic host cell approximately 1.5 billion years ago.

Evidence for endosymbiotic theory:

1. Chloroplasts have their own DNA, which is circular (like bacterial DNA).
2. Chloroplasts have 70S ribosomes (similar to prokaryotes, not the 80S ribosomes of eukaryotes).
3. Chloroplasts are surrounded by a double membrane (the inner membrane is the original bacterial membrane; the outer membrane is from the host cell's phagocytic vesicle).
4. Chloroplasts reproduce by binary fission (similar to bacteria).
5. The antibiotic chloramphenicol inhibits protein synthesis on 70S ribosomes (including chloroplast ribosomes) but not 80S ribosomes.
6. Molecular phylogenetics shows chloroplast DNA is most closely related to cyanobacteria.

26.3 Photosynthetic Pigments

Pigment	Colour	Absorption Peaks	Solubility	Location
Chlorophyll $a$	Blue-green	~430 nm (blue), ~662 nm (red)	Fat-soluble	Reaction centres of photosystems I and II
Chlorophyll $b$	Yellow-green	~453 nm (blue), ~642 nm (red)	Fat-soluble	Light-harvesting complexes (antenna pigments)
Carotenoids (e.g., $\beta$-carotene)	Orange	~450 nm (blue-violet)	Fat-soluble	Light-harvesting complexes; photoprotection (quench singlet oxygen)
Xanthophylls (e.g., lutein, violaxanthin)	Yellow	~450 nm (blue-violet)	Fat-soluble	Photoprotection; NPQ (non-photochemical quenching)
Phycobilins (e.g., phycoerythrin, phycocyanin)	Red/blue	~550 nm (green)	Water-soluble	Accessory pigments in red algae and cyanobacteria

Accessory pigments (chlorophyll $b$, carotenoids) absorb light at wavelengths where chlorophyll $a$ absorbs poorly (e.g., blue-green, green), extending the range of light that can be used for photosynthesis. Energy absorbed by accessory pigments is transferred to chlorophyll $a$ in the reaction centre by resonance energy transfer.

27. C4 and CAM Photosynthesis: Detailed Comparison

27.1 The C4 Pathway

C4 plants (maize, sugarcane, sorghum, millet) have a $\mathrm{CO_2}$ concentrating mechanism that suppresses photorespiration:

1. $\mathrm{CO_2}$ fixation: $\mathrm{CO_2}$ is initially fixed by PEP carboxylase in mesophyll cells. PEP carboxylase has a much higher affinity for $\mathrm{CO_2}$ than Rubisco and does not react with $\mathrm{O_2}$. $\mathrm{PEP + CO_2 \to oxaloacetate\ (4C)}$
2. Conversion: oxaloacetate is converted to malate (or aspartate) and transported to bundle sheath cells (which surround the vascular bundles).
3. Decarboxylation: malate is decarboxylated in bundle sheath cells, releasing $\mathrm{CO_2}$ at a high concentration around Rubisco.
4. Calvin cycle: the concentrated $\mathrm{CO_2}$ enters the Calvin cycle in the bundle sheath cells.
5. Regeneration: the 3-carbon product (pyruvate) returns to the mesophyll cells and is converted back to PEP (using ATP).

Advantages of C4 photosynthesis:

• Suppresses photorespiration (high $\mathrm{CO_2}$ concentration in bundle sheath cells outcompetes $\mathrm{O_2}$ for Rubisco).
• Higher photosynthetic efficiency at high temperatures and high light intensities.
• Higher water use efficiency (stomata can be partially closed while maintaining $\mathrm{CO_2}$ uptake).

Disadvantages:

• Requires additional ATP (2 extra ATP per $\mathrm{CO_2}$ fixed compared to C3).
• C4 pathway enzymes are inducible; C4 plants have a different leaf anatomy (Kranz anatomy).

27.2 CAM Photosynthesis

CAM (Crassulacean Acid Metabolism) plants (cacti, succulents, pineapple, Kalanchoe) separate the initial $\mathrm{CO_2}$ fixation and the Calvin cycle in time rather than in space:

Time	Process	What Happens
Night	Stomata open; $\mathrm{CO_2}$ fixation	$\mathrm{CO_2}$ enters through open stomata; PEP carboxylase fixes $\mathrm{CO_2}$ into oxaloacetate, which is converted to malic acid and stored in vacuoles. Water loss is minimised because temperatures are lower and humidity is higher at night.
Day	Stomata closed; Calvin cycle	Malic acid is released from vacuoles; decarboxylated to release $\mathrm{CO_2}$; $\mathrm{CO_2}$ enters the Calvin cycle. Light provides ATP and NADPH.

27.3 Comparison of C3, C4, and CAM

Feature	C3	C4	CAM
Examples	Wheat, rice, soybean, most plants	Maize, sugarcane, sorghum	Cacti, pineapple, Kalanchoe
Initial $\mathrm{CO_2}$ acceptor	RuBP (5C)	PEP (3C)	PEP (3C)
Initial $\mathrm{CO_2}$ fixing enzyme	Rubisco	PEP carboxylase	PEP carboxylase
Location of Calvin cycle	Mesophyll cells	Bundle sheath cells	Mesophyll cells (during day)
Photorespiration	Significant at high temperatures	Suppressed	Suppressed
Water use efficiency	Moderate	High	Very high
Temperature optimum	20--25 degrees C	30--40 degrees C	Very wide range
Light saturation	Moderate	High	Low (slow growth)
Growth rate	Moderate	High	Low

28. Light-Dependent Reactions: Detailed Mechanism

28.1 Photosystem II (PSII)

1. Light energy is absorbed by antenna pigments (chlorophyll $b$, carotenoids) and transferred to the reaction centre chlorophyll $a$ (P680) by resonance energy transfer.
2. P680 becomes excited (P680*) and donates an electron to the primary electron acceptor (pheophytin).
3. P680+ is a very strong oxidising agent. It extracts electrons from water by photolysis:

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

4. The electron passes through the PSII electron transport chain: pheophytin $\to$ plastoquinone (PQ) $\to$ cytochrome $b_6f$ complex.
5. As electrons pass through cytochrome $b_6f$, $\mathrm{H^+}$ ions are pumped from the stroma into the thylakoid lumen (contributing to the proton gradient).

28.2 Photosystem I (PSI)

1. Light energy is absorbed by PSI antenna pigments and transferred to the reaction centre P700.
2. P700 becomes excited (P700*) and donates an electron to ferredoxin (Fd), a soluble iron-sulphur protein.
3. The electron from PSII reaches PSI via plastocyanin (PC), a mobile copper protein in the thylakoid lumen.
4. Ferredoxin-NADP$^+$ reductase (FNR) transfers electrons from reduced ferredoxin to $\mathrm{NADP^+}$:

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

The $\mathrm{H^+}$ ions come from the stroma (not the lumen).

28.3 Chemiosmosis and ATP Synthesis

1. Photolysis of water releases $\mathrm{H^+}$ into the thylakoid lumen.
2. Electron transport through cytochrome $b_6f$ pumps additional $\mathrm{H^+}$ from stroma to lumen.
3. $\mathrm{NADP^+}$ reduction removes $\mathrm{H^+}$ from the stroma (contributing to the gradient).
4. The resulting proton gradient ($\mathrm{H^+}$ concentration higher in lumen than stroma) drives ATP synthesis by ATP synthase (also called CF$_0$-CF$_1$ ATPase).
5. $\mathrm{H^+}$ flows back through ATP synthase from lumen to stroma, driving rotation of the enzyme and phosphorylation of ADP to ATP.

Z-scheme summary: $\mathrm{H_2O \to PSII \to PQ \to cyt\ b_6f \to PC \to PSI \to Fd \to NADP^+}$

Products of the light reactions: ATP, $\mathrm{NADPH}$, $\mathrm{O_2}$.

28.4 Cyclic Photophosphorylation

When the Calvin cycle requires more ATP than $\mathrm{NADPH}$ (or when $\mathrm{NADP^+}$ is scarce), PSI can operate in cyclic mode:

1. Light excites P700, which donates an electron to ferredoxin.
2. Instead of reducing $\mathrm{NADP^+}$, the electron is transferred back to the cytochrome $b_6f$ complex (via ferredoxin-plastoquinone reductase, FQR).
3. The electron returns to P700 via plastocyanin.
4. $\mathrm{H^+}$ is pumped into the lumen, driving ATP synthesis.
5. No $\mathrm{NADPH}$ is produced; no $\mathrm{O_2}$ is evolved (water is not split).

29. The Calvin Cycle: Detailed Steps

29.1 Phase 1: Carbon Fixation

• Enzyme: Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), the most abundant protein on Earth.
• Reaction: $\mathrm{CO_2}$ (1C) + RuBP (5C, ribulose-1,5-bisphosphate) $\to$ 2 $\times$ GP (3C, glycerate-3-phosphate).
• For every 3 molecules of $\mathrm{CO_2}$ fixed, 6 molecules of GP are produced.

29.2 Phase 2: Reduction

• GP is phosphorylated by ATP (from the light reactions) and reduced by NADPH (from the light reactions) to form TP (triose phosphate, glyceraldehyde-3-phosphate, 3C).
• Reaction: GP + ATP + NADPH $\to$ TP + ADP + $\mathrm{NADP^+}$ + $\mathrm{P_i}$.

29.3 Phase 3: Regeneration of RuBP

• Of the 6 molecules of TP produced, 5 are used to regenerate 3 molecules of RuBP (requiring 3 ATP).
• Reaction: 5 TP (3C each, total 15C) + 3 ATP $\to$ 3 RuBP (5C each, total 15C) + 3 ADP.
• 1 molecule of TP (3C) is the net product of the cycle (this can be used to make glucose, other carbohydrates, lipids, or amino acids).

29.4 Summary: What Is Needed and Produced (per 3 $\mathrm{CO_2}$)?

Input	Quantity	Output	Quantity
$\mathrm{CO_2}$	3	TP (exported)	1
RuBP	3 (consumed); 3 (regenerated)	GP (intermediate)	6
ATP	6 (3 in reduction + 3 in regeneration)	ADP	6
NADPH	6	$\mathrm{NADP^+}$	6

29.5 Factors Affecting the Calvin Cycle

Factor	Effect	Explanation
$\mathrm{CO_2}$ concentration	Increased $\mathrm{CO_2}$ increases the rate of Calvin cycle (up to saturation)	More substrate for Rubisco
Temperature	Optimum at 25--30 degrees C; decreases above and below	Enzyme activity (Rubisco); denaturation at high temperature
Light intensity	Indirect effect: light is needed to produce ATP and NADPH	Without light reactions, the Calvin cycle stops due to lack of ATP and NADPH
$\mathrm{O_2}$ concentration	High $\mathrm{O_2}$ decreases net $\mathrm{CO_2}$ fixation (photorespiration)	Rubisco also acts as an oxygenase; photorespiration wastes energy and releases $\mathrm{CO_2}$

29.6 Photorespiration

When $\mathrm{O_2}$ concentration is high (or $\mathrm{CO_2}$ is low), Rubisco catalyses the oxygenation of RuBP:

$\text{RuBP} + \mathrm{O_2} \to \text{1 GP (3C)} + \text{glycolate (2C)}$

Glycolate is transported to peroxisomes, where it is converted to glycine (2C). Two glycines are converted to serine (3C) + $\mathrm{CO_2}$ + $\mathrm{NH_3}$ (in mitochondria). Serine is converted back to GP.

Consequences of photorespiration:

• Wastes carbon (one $\mathrm{CO_2}$ is released per 2 oxygenations).
• Consumes ATP and $\mathrm{NH_3}$.
• Reduces net photosynthetic efficiency by approximately 25% in C3 plants at 25 degrees C (more at higher temperatures).

30. Photosynthesis and the Carbon Cycle

30.1 The Global Carbon Cycle

Process	$\mathrm{CO_2}$ Flux ($\mathrm{Gt\ C\ yr^{-1}}$)	Direction
Photosynthesis (terrestrial)	$\approx -120$	Absorbs $\mathrm{CO_2}$
Photosynthesis (marine)	$\approx -50$	Absorbs $\mathrm{CO_2}$
Respiration (terrestrial)	$\approx +120$	Releases $\mathrm{CO_2}$
Respiration (marine)	$\approx +50$	Releases $\mathrm{CO_2}$
Fossil fuel combustion	$\approx +9.5$	Releases $\mathrm{CO_2}$
Deforestation	$\approx +1.5$	Releases $\mathrm{CO_2}$
Ocean uptake	$\approx -2.5$	Absorbs $\mathrm{CO_2}$ (dissolves in water)
Net flux to atmosphere	$\approx +5$	Accumulating (increasing atmospheric $\mathrm{CO_2}$)

30.2 Compensation Point and Saturation Point

Concept	Definition	Significance
Compensation point	The light intensity at which the rate of photosynthesis equals the rate of respiration (net gas exchange $= 0$)	Below this point, the plant respires more than it photosynthesises; above it, the plant has a net gain of organic compounds
Saturation point	The light intensity at which increasing light intensity no longer increases the rate of photosynthesis (another factor becomes limiting)	Above this point, $\mathrm{CO_2}$ concentration or temperature is the limiting factor

30.3 Daily Pattern of Gas Exchange

Time of Day	Net $\mathrm{CO_2}$ Exchange	Net $\mathrm{O_2}$ Exchange	Explanation
Night	$\mathrm{CO_2}$ released	$\mathrm{O_2}$ consumed	No photosynthesis (no light); only respiration occurs
Dawn	Net $\mathrm{CO_2}$ uptake begins	Net $\mathrm{O_2}$ release begins	Light intensity reaches compensation point; photosynthesis rate exceeds respiration rate
Midday	Maximum net $\mathrm{CO_2}$ uptake	Maximum net $\mathrm{O_2}$ release	Light intensity is at or above saturation point; temperature and $\mathrm{CO_2}$ may become limiting
Late afternoon	Net $\mathrm{CO_2}$ uptake decreases	Net $\mathrm{O_2}$ release decreases	Light intensity decreases below saturation point
Dusk	Net exchange $= 0$	Net exchange $= 0$	Light intensity drops to compensation point

30.4 Crop Yield and Greenhouse Management

Increasing crop yield:

1. Increase light intensity (artificial lighting in greenhouses; reflective mulches).
2. Increase $\mathrm{CO_2}$ concentration ($\mathrm{CO_2}$ enrichment in greenhouses to $\approx 1000\ \mathrm{ppm}$).
3. Optimise temperature (25--30 degrees C for C3 crops).
4. Optimise water supply (drip irrigation; hydroponics).
5. Use C4 crops in hot, dry environments (maize, sugarcane).
6. Select high-yielding varieties.
7. Control pests and diseases.

Greenhouse advantages for crop growth:

• Higher $\mathrm{CO_2}$ concentration than outside (due to plant respiration inside the enclosed space; can be supplemented).
• Higher temperature (glasshouse effect).
• Protection from wind and pests.
• Extended growing season.
• Control over light (artificial lighting during winter).

31. Dissolved Oxygen and Aquatic Photosynthesis

31.1 Measuring Dissolved Oxygen

Dissolved $\mathrm{O_2}$ (DO) concentration in water is measured using an oxygen probe or the Winkler method:

Winkler method:

1. Collect a water sample in a sealed bottle (no air bubbles).
2. Add manganese(II) sulphate and alkali (KOH/KI): $\mathrm{Mn^{2+}}$ is oxidised to $\mathrm{Mn^{3+}}$ (brown precipitate) by dissolved $\mathrm{O_2}$.
3. Add sulphuric acid: the precipitate dissolves, oxidising iodide to iodine ($\mathrm{I_2}$).
4. Titrate with sodium thiosulphate ($\mathrm{Na_2S_2O_3}$), using starch as an indicator (blue colour disappears at the endpoint).

The volume of thiosulphate used is proportional to the dissolved $\mathrm{O_2}$ concentration.

31.2 Factors Affecting DO in Water

Factor	Effect on DO
Temperature	Higher temperature $\to$ lower DO (gases are less soluble in warm water)
Photosynthesis rate	Higher photosynthesis $\to$ higher DO (during daylight hours)
Respiration rate	Higher respiration $\to$ lower DO (all organisms respire, including plants at night)
Organic pollution	Bacteria decompose organic matter, consuming $\mathrm{O_2}$ (increases BOD; decreases DO)
Turbulence/aeration	Increases DO (air dissolves in water at the surface)
Altitude	Higher altitude $\to$ lower atmospheric pressure $\to$ lower DO
Salinity	Higher salinity $\to$ lower DO (salt reduces $\mathrm{O_2}$ solubility)

31.3 Diurnal Variation in DO

In a pond or lake:

Time	DO Trend	Explanation
Dawn	Minimum DO	Respiration through the night has consumed $\mathrm{O_2}$; no photosynthesis in the dark
Morning	DO increasing	Photosynthesis rate exceeds respiration rate as light intensity increases
Mid-afternoon	Maximum DO	Peak photosynthesis rate; maximum $\mathrm{O_2}$ production
Evening	DO decreasing	Light intensity decreases; photosynthesis rate drops below respiration rate
Night	DO decreasing	Only respiration occurs; DO steadily decreases until dawn

32. Photosynthesis and Human Nutrition

32.1 Essential Minerals for Plant Growth

Plants require mineral ions for healthy growth:

Mineral Ion	Role in Plants	Deficiency Symptom
Nitrogen ($\mathrm{NO_3^-, NH_4^+}$)	Amino acid, protein, nucleotide synthesis	Stunted growth; chlorosis (yellowing of older leaves); reduced protein synthesis
Phosphorus ($\mathrm{PO_4^{3-}}$)	ATP, nucleic acids, phospholipids; root development	Poor root growth; dark green/purple leaves
Potassium ($\mathrm{K^+}$)	Osmoregulation; stomatal opening; enzyme activation	Wilting; brown leaf margins; weak stems
Magnesium ($\mathrm{Mg^{2+}}$)	Chlorophyll component; enzyme activator (Rubisco)	Chlorosis between leaf veins; reduced photosynthesis
Calcium ($\mathrm{Ca^{2+}}$)	Middle lamella formation (calcium pectate); cell signalling	Blossom end rot (tomatoes); poor cell wall formation
Iron ($\mathrm{Fe^{2+/3+}}$)	Chlorophyll synthesis; electron transport chain components	Interveinal chlorosis; severe chlorosis
Sulfur ($\mathrm{SO_4^{2-}}$)	Amino acid synthesis (cysteine, methionine); coenzymes	Yellowing of young leaves; general stunting

32.2 Hydroponics

Hydroponics is growing plants without soil, in a nutrient solution:

Advantages	Disadvantages
Precise control of nutrient concentrations	Expensive to set up and maintain
No soil-borne diseases	Requires technical knowledge; risk of system failure (power cuts, pump failure)
Higher yields per unit area	Not suitable for large-scale grain production
Water-efficient (recirculating systems)	Requires continuous monitoring of pH, EC (electrical conductivity), and nutrient levels
Can grow anywhere (urban environments, deserts)	Energy costs (artificial lighting, pumps, heaters)

33. Light-Independent Reactions: The Calvin Cycle in Detail

33.1 The Three Stages

Stage	What Happens	Enzyme	Key Molecules
Carbon fixation	$\mathrm{CO_2}$ combines with RuBP (5C) to form two molecules of GP (3C)	Rubisco	$\mathrm{CO_2}$ + RuBP $\to$ 2 $\times$ GP
Reduction	GP is reduced to TP (triose phosphate) using ATP and reduced NADP from the light-dependent reactions	No named enzyme (uses ATP and NADPH)	GP + ATP + NADPH $\to$ TP + NADP$^+$ + ADP + $P_i$
Regeneration of RuBP	5 out of every 6 TP molecules are used to regenerate RuBP (the remaining 1 TP is used to make glucose)	Series of enzymes (phosphogluconate pathway)	5 $\times$ TP (3C) $\to$ 3 $\times$ RuBP (5C); costs 3 ATP

33.2 ATP and NADP Requirements

For every 3 turns of the Calvin cycle (fixing 3 $\mathrm{CO_2}$):

Molecule	Number Required
$\mathrm{CO_2}$ fixed	3
RuBP consumed	3
GP produced	6
ATP consumed	9 (6 in reduction, 3 in regeneration)
Reduced NADP consumed	6 (in reduction only)
TP produced	6 (5 recycled, 1 exported)
Glucose produced (from 2 exported TP)	Requires 6 turns of the cycle

For one molecule of glucose ($\mathrm{C_6H_{12}O_6}$):

• 6 $\mathrm{CO_2}$ fixed
• 6 turns of the Calvin cycle
• 18 ATP consumed
• 12 reduced NADP consumed

34. Factors Limiting the Rate of Photosynthesis

34.1 Limiting Factors at Different Light Intensities

Light Intensity	Limiting Factor	Explanation
Low	Light intensity	Few photons reach chlorophyll; rate of light-dependent reactions limits overall rate
Moderate	$\mathrm{CO_2}$ concentration (or temperature)	Light is no longer limiting; $\mathrm{CO_2}$ supply to Rubisco becomes the bottleneck
High	Temperature (or $\mathrm{CO_2}$)	Light and $\mathrm{CO_2}$ are both saturating; enzyme activity (Rubisco) limits rate; temperature affects enzyme kinetics

34.2 Effects of Individual Limiting Factors

Factor	Effect on Rate	Explanation
Light intensity (increase)	Rate increases then plateaus	More light = more ATP and reduced NADP = faster Calvin cycle; plateaus when another factor is limiting
$\mathrm{CO_2}$ concentration (increase)	Rate increases then plateaus	More $\mathrm{CO_2}$ = faster carbon fixation by Rubisco; plateaus when another factor is limiting
Temperature (increase)	Rate increases to optimum then decreases	Enzyme kinetics: rate doubles per $10\degree\mathrm{C}$ rise (Q10); above optimum ($\sim$25--30$\degree\mathrm{C}$), enzymes denature; membranes become leaky
Water availability	Rate decreases	Water is a reactant in the light-dependent reactions; also causes stomatal closure (reducing $\mathrm{CO_2}$ uptake)

34.3 Greenhouse Gas Effects on Photosynthesis

Gas	Current Atmospheric Concentration	Effect on Plant Growth
$\mathrm{CO_2}$	~420 ppm (rising ~2.5 ppm/year)	Higher $\mathrm{CO_2}$ increases photosynthetic rate in C3 plants (up to a point); C4 plants already have a $\mathrm{CO_2}$-concentrating mechanism so benefit less
$\mathrm{O_3}$ (ozone)	Increasing near ground level (pollution)	Damages chlorophyll; reduces photosynthetic rate; damages stomatal function

35. Investigating the Rate of Photosynthesis

35.1 Common Practical Methods

Method	What Is Measured	How	Limitations
Oxygen production (pondweed)	Volume of $\mathrm{O_2}$ gas produced per unit time	Canadian pondweed (Elodea) in a test tube; count bubbles or collect gas in a graduated tube	Bubble size varies; not all $\mathrm{O_2}$ is collected; temperature may fluctuate
Oxygen probe	Dissolved $\mathrm{O_2}$ concentration in solution	$\mathrm{O_2}$ electrode placed in the solution with the plant	Expensive equipment; probe may be affected by other dissolved gases
Mass change	Increase in dry mass of plant over time	Harvest plants at intervals; dry to constant mass; weigh	Destructive (different plants each time); slow
Absorbance (DCPIP)	Rate of decolourisation of DCPIP	DCPIP is blue when oxidised and colourless when reduced (accepts electrons from the light-dependent reactions); time how long it takes for DCPIP to decolourise	DCPIP can act as an electron acceptor, potentially inhibiting normal electron flow; colour change can be subjective

35.2 Variables in Photosynthesis Experiments

Variable	Type	How to Control/Vary
Light intensity	Independent	Vary distance of lamp from plant (inverse square law); use a light meter to measure lux
$\mathrm{CO_2}$ concentration	Independent	Vary concentration of sodium hydrogen carbonate ($\mathrm{NaHCO_3}$) solution
Temperature	Control	Water bath; thermometer
Wavelength of light	Independent	Use coloured filters (red, blue, green)
Number/size of leaves	Control	Use same species, same age, same number of leaves
Time	Control	Use a stopwatch

36. Chloroplast Structure and Adaptations

36.1 Chloroplast Components

Component	Description	Function
Double membrane	Outer membrane (permeable to small molecules); inner membrane (selectively permeable; contains transport proteins)	Compartmentalisation; controls entry/exit of substances
Thylakoid membrane	Internal membrane system; flattened sacs (thylakoids) stacked into grana	Site of the light-dependent reactions; contains photosystems, ETC, ATP synthase
Thylakoid lumen	Space inside the thylakoid	Protons accumulate here during the light-dependent reactions; proton gradient drives ATP synthesis
Stroma	Fluid-filled matrix surrounding the thylakoids	Site of the Calvin cycle (light-independent reactions); contains enzymes (Rubisco), DNA, ribosomes, starch grains
Granum (plural: grana)	Stack of thylakoids	Increases surface area for the light-dependent reactions
Lamellae (intergranal thylakoids)	Thylakoid membranes connecting grana	Connect grana; provide additional membrane surface for light-dependent reactions
Starch grains	Stored in the stroma	Temporary storage of carbohydrate produced by photosynthesis

36.2 Adaptations of the Chloroplast

Adaptation	Benefit
Large surface area of thylakoid membranes	Maximises the number of photosystems and ETC components; increases light absorption and ATP production
Thylakoid membranes arranged in stacks (grana)	Pack many membranes into a small space; efficient use of space
Stroma contains its own DNA and ribosomes	Supports the endosymbiotic theory (chloroplasts were once free-living prokaryotes); allows some chloroplast proteins to be synthesised locally
Chlorophyll pigments in thylakoid membranes	Absorb light energy for photosynthesis
Thin thylakoid membranes	Short diffusion distance for protons and electrons

37. C3, C4, and CAM Photosynthesis Comparison

37.1 Key Differences

Feature	C3 Plants	C4 Plants	CAM Plants
First product of carbon fixation	3-carbon compound (GP / 3-phosphoglycerate)	4-carbon compound (oxaloacetate)	3-carbon compound (GP, at night) and 4-carbon compound (malate, at night)
Carbon-fixing enzyme	Rubisco (in mesophyll cells)	PEP carboxylase (in mesophyll cells); Rubisco (in bundle sheath cells)	PEP carboxylase (at night); Rubisco (during the day)
Photorespiration	Significant at high temperatures (Rubisco has oxygenase activity)	Minimal (PEP carboxylase has no affinity for $\mathrm{O_2}$)	Minimal (stomata closed during the day; $\mathrm{CO_2}$ released internally)
Stomatal behaviour	Open during the day	Open during the day	Closed during the day; open at night (reduces water loss)
Water use efficiency	Low (loses more water per unit $\mathrm{CO_2}$ fixed)	High	Very high
Temperature optimum	20--25$\degree\mathrm{C}$	30--40$\degree\mathrm{C}$	Hot, arid environments
Examples	Wheat, rice, oats, soybean, most temperate plants	Maize, sugarcane, sorghum, tropical grasses	Cacti, pineapple, jade plant, Kalanchoe

37.2 Why C4 Plants Are More Efficient in Hot Climates

At high temperatures:

1. Rubisco's oxygenase activity increases (fixes $\mathrm{O_2}$ instead of $\mathrm{CO_2}$) $\to$ photorespiration.
2. Stomata close to reduce water loss $\to$ less $\mathrm{CO_2}$ enters.
3. Internal $\mathrm{CO_2}$ concentration drops.
4. Rubisco fixes more $\mathrm{O_2}$ (vicious cycle).

C4 plants solve this by:

1. PEP carboxylase (in mesophyll) has high affinity for $\mathrm{CO_2}$ and no affinity for $\mathrm{O_2}$.
2. The 4-carbon compound (malate) is transported to bundle sheath cells.
3. Malate is decarboxylated, releasing $\mathrm{CO_2}$ at high concentration around Rubisco.
4. High $\mathrm{CO_2}$ concentration suppresses photorespiration.

38. The Light-Dependent Reactions in Detail

38.1 Photosystems

Feature	Photosystem II (PSII)	Photosystem I (PSI)
Primary pigment	P680 (chlorophyll a, absorbs at 680 nm)	P700 (chlorophyll a, absorbs at 700 nm)
Location	Thylakoid membrane (inner surface)	Thylakoid membrane (outer surface)
Function	Absorbs light energy; splits water (photolysis); passes electrons to the ETC	Absorbs light energy (second photon); re-energises electrons; passes them to NADP reductase
First in the chain	Yes (electrons enter the ETC here)	No (receives electrons from PSII via ETC)

38.2 Non-Cyclic Photophosphorylation (Z-Scheme)

Step	Location	What Happens
1	PSII	Light energy excites electrons in P680 to a higher energy level
2	PSII	Excited electrons are passed to the primary electron acceptor (pheophytin); P680 becomes oxidised ($\mathrm{P680^+}$)
3	PSII	$\mathrm{P680^+}$ is a very strong oxidising agent; it strips electrons from water (photolysis): $\mathrm{2H_2O \to 4H^+ + 4e^- + O_2}$
4	ETC	Electrons pass through a series of electron carriers (plastoquinone $\to$ cytochrome b6f complex $\to$ plastocyanin); energy released pumps protons into the thylakoid lumen
5	PSI	Light energy excites electrons in P700; electrons are passed to ferredoxin, then to NADP reductase
6	Stroma	NADP reductase reduces $\mathrm{NADP^+}$ to reduced NADP using the electrons and $\mathrm{H^+}$ from the stroma

38.3 Products of the Light-Dependent Reactions

Product	Used For
ATP (by chemiosmosis; ATP synthase uses the proton gradient)	Provides energy for the Calvin cycle (reduction of GP to TP)
Reduced NADP	Provides reducing power (electrons and $\mathrm{H^+}$) for the Calvin cycle
$\mathrm{O_2}$	Released as a by-product of photolysis; used by organisms for aerobic respiration

39. Chlorophyll and Light Absorption

39.1 Types of Chlorophyll and Accessory Pigments

Pigment	Colour	Absorption Peaks	Role
Chlorophyll a	Blue-green	430 nm (blue) and 662 nm (red)	Primary photosynthetic pigment; directly involved in the light-dependent reactions (P680 in PSII; P700 in PSI)
Chlorophyll b	Yellow-green	453 nm (blue) and 642 nm (red)	Accessory pigment; absorbs light at wavelengths that chlorophyll a absorbs less efficiently; passes energy to chlorophyll a
Carotenoids (carotene, xanthophyll)	Yellow, orange	400--500 nm (blue-violet)	Accessory pigments; absorb blue light; pass energy to chlorophyll a; also photoprotective (quench singlet oxygen, preventing photo-oxidative damage)
Phycobilins (phycocyanin, phycoerythrin)	Red, blue	500--650 nm	Accessory pigments in red algae and cyanobacteria

39.2 Why Leaves Are Green

• Chlorophyll absorbs red and blue light most effectively.
• Green light (500--600 nm) is reflected and transmitted (not absorbed).
• This is why leaves appear green.

39.3 Absorption and Action Spectra

Spectrum	What It Shows	How It Is Measured
Absorption spectrum	The range of wavelengths of light absorbed by each pigment	Extract pigments with solvent; pass white light through the extract; measure absorption at each wavelength using a spectrophotometer
Action spectrum	The rate of photosynthesis at each wavelength of light	Illuminate a plant with light of different wavelengths (using coloured filters); measure the rate of photosynthesis (e.g., $\mathrm{O_2}$ production) at each wavelength

The action spectrum closely matches the absorption spectrum of chlorophyll, confirming that chlorophyll is the primary photosynthetic pigment.

40. Plant Hormones (Plant Growth Substances)

40.1 Auxins (IAA, Indole-3-Acetic Acid)

Feature	Description
Site of production	Shoot tip (apical meristem); young leaves
Transport	Unidirectional (polar transport): basipetal (from shoot tip towards root); via auxin efflux carriers (PIN proteins)
Effects	Cell elongation (acid growth hypothesis: auxin activates proton pumps $\to$ cell wall becomes more acidic $\to$ cell wall-loosening enzymes (expansins) break cross-links $\to$ wall becomes extensible $\to$ cell expands); apical dominance (inhibits lateral bud growth); root initiation (cuttings); tropisms (phototropism, gravitropism)
Commercial use	Rooting powder (auxin in cuttings); weedkillers (synthetic auxins like 2,4-D cause uncontrolled growth and death in broad-leaved plants)

40.2 Gibberellins

Feature	Description
Site of production	Young leaves, roots, developing seeds
Effects	Stem elongation; seed germination (gibberellin stimulates production of amylase in the aleurone layer of barley seeds $\to$ starch is hydrolysed to glucose $\to$ provides energy for the growing embryo); bolting (rapid stem elongation in response to day length)
Commercial use	Spraying grapes with gibberellic acid to produce larger fruits; brewing industry (gibberellins used in malting barley)

40.3 Ethylene

Feature	Description
Type	Gas (diffuses easily through air and tissues)
Site of production	Ripening fruits; damaged tissues
Effects	Fruit ripening (converts starch to sugars; softens cell walls by breaking down pectin); leaf abscission (dropping of leaves); senescence (ageing)
Commercial use	Ripening bananas (expose unripe bananas to ethylene gas to trigger ripening)

40.4 Cytokinins

Feature	Description
Site of production	Root tips; transported upwards in the xylem
Effects	Promote cell division (cytokinesis); delay leaf senescence; promote shoot growth (work antagonistically with auxin)
Commercial use	Used in tissue culture (with auxin) to stimulate shoot formation

41. Photosynthesis and the Carbon Cycle

41.1 The Carbon Cycle

Process	$\mathrm{CO_2}$ Change	Description
Photosynthesis	$\mathrm{CO_2}$ removed from atmosphere	Plants, algae, and cyanobacteria convert $\mathrm{CO_2}$ to organic compounds (glucose)
Respiration	$\mathrm{CO_2}$ released into atmosphere	All living organisms respire; glucose is broken down to release $\mathrm{CO_2}$
Combustion (fossil fuels)	$\mathrm{CO_2}$ released into atmosphere	Burning coal, oil, natural gas releases $\mathrm{CO_2}$ that was previously locked underground for millions of years
Decomposition	$\mathrm{CO_2}$ released	Decomposers break down dead organic matter; respire and release $\mathrm{CO_2}$
Ocean absorption	$\mathrm{CO_2}$ absorbed from atmosphere	Oceans absorb ~25% of anthropogenic $\mathrm{CO_2}$; forms carbonic acid (ocean acidification)
Volcanic activity	$\mathrm{CO_2}$ released	Volcanoes release $\mathrm{CO_2}$ from the Earth's mantle; minor contribution compared to human activities
Deforestation	Reduced $\mathrm{CO_2}$ absorption	Fewer trees to absorb $\mathrm{CO_2}$; additionally, burning forests releases stored carbon
Limestone formation	$\mathrm{CO_2}$ removed (long-term)	Marine organisms (coral, foraminifera) use $\mathrm{CO_2}$ to form calcium carbonate shells; over geological time, this becomes limestone rock

42. Fertilisation and Seed Development

42.1 Fertilisation in Flowering Plants

Step	What Happens
1	Pollen grain lands on the stigma; germinates and grows a pollen tube down the style towards the ovule
2	The pollen tube carries two male gamete nuclei (from the generative nucleus) to the ovule
3	The pollen tube enters the ovule through the micropyle; tip bursts and releases the two male nuclei
4	Double fertilisation: one male nucleus fuses with the egg cell (2n zygote); the other fuses with two polar nuclei (3n endosperm)
5	The zygote develops into the embryo; the endosperm develops into a nutrient store; the ovule integuments develop into the seed coat (testa)

42.2 Seed Structure

Part	Origin	Function
Seed coat (testa)	Integuments of the ovule	Protection; physical barrier against desiccation and pathogens
Embryo	Zygote (2n)	Develops into the new plant; consists of plumule (shoot tip), radicle (root tip), and cotyledon(s)
Cotyledon(s)	Part of the embryo	Stores nutrients (in non-endospermic seeds, e.g., beans) or absorbs nutrients from the endosperm (in endospermic seeds, e.g., maize)
Endosperm	Triple fusion (3n)	Nutrient store for the developing embryo (in endospermic seeds)
Radicle	Part of embryo	Develops into the primary root
Plumule	Part of embryo	Develops into the shoot

43. Plant Disease and Defence

43.1 Physical Plant Defences

Defence	Description
Cellulose cell wall	Physical barrier against pathogens; thickened in many plants
Cuticle	Waxy layer on leaves; prevents entry of pathogens and reduces water loss
Lignin	Waterproof polymer in cell walls (especially in xylem); resistant to degradation by most enzymes
Callose	Polysaccharide deposited in cell walls at infection sites; blocks plasmodesmata (slowing spread of virus)
Bark	Outer protective layer on tree trunks and roots; physical barrier
Thorns and spines	Deter herbivores
Hairs (trichomes)	Physical barrier to insect herbivores; can secrete sticky substances or toxins

43.2 Chemical Plant Defences

Defence	Description	Example
Tannins	Phenolic compounds that bind to proteins; make plant tissues unpalatable and difficult to digest; inhibit herbivore enzymes	Oak leaves; tea
Alkaloids	Nitrogen-containing compounds with potent pharmacological effects on animals	Nicotine (tobacco); caffeine (coffee); cocaine (coca); atropine (deadly nightshade); morphine (opium poppy)
Terpenoids	Volatile organic compounds; repel herbivores and attract natural enemies of herbivores (parasitoid wasps)	Pyrethrins (chrysanthemum); menthol (mint); limonene (citrus)
Phytoalexins	Antimicrobial compounds synthesised in response to pathogen attack	Resveratrol (grapes); camalexin (Arabidopsis)
Salicylic acid	Plant hormone; activates systemic acquired resistance (SAR) -- whole-plant immunity	Released at infection site; travels through the plant; activates defence genes throughout the plant

44. Photorespiration

44.1 What Is Photorespiration?

Photorespiration is a wasteful process that occurs when Rubisco binds $\mathrm{O_2}$ instead of $\mathrm{CO_2}$:

Step	What Happens	Result
1	Rubisco binds $\mathrm{O_2}$ to RuBP (instead of $\mathrm{CO_2}$)	Produces one molecule of 3-phosphoglycerate (3C) and one molecule of 2-phosphoglycolate (2C)
2	2-phosphoglycolate is converted to glycine in the peroxisome	Uses $\mathrm{O_2}$; releases $\mathrm{CO_2}$
3	Glycine is converted to serine in the mitochondria	Releases $\mathrm{NH_3}$
4	Serine is converted back to a Calvin cycle intermediate	Costs ATP

44.2 Why Photorespiration Is Wasteful

Problem	Description
Wastes $\mathrm{CO_2}$	$\mathrm{CO_2}$ is released (opposite of carbon fixation)
Wastes ATP	Energy is consumed to recover the carbon
Wastes $\mathrm{O_2}$	$\mathrm{O_2}$ is consumed without producing useful energy
Reduces yield	Fewer carbohydrates are produced per unit of light energy
Estimated loss	May reduce photosynthetic efficiency by 20--50% in C3 plants under hot, dry conditions

44.3 Conditions That Increase Photorespiration

Condition	Why It Increases Photorespiration
High temperature	Rubisco's affinity for $\mathrm{O_2}$ increases relative to its affinity for $\mathrm{CO_2}$
High light intensity	Increases $\mathrm{O_2}$ production from photolysis (more $\mathrm{O_2}$ available to compete with $\mathrm{CO_2}$)
Stomatal closure (drought)	Less $\mathrm{CO_2}$ enters the leaf; internal $\mathrm{CO_2}$ concentration drops; Rubisco is more likely to bind $\mathrm{O_2}$

45. Measuring the Rate of Photosynthesis

45.1 Common Methods

Method	Principle	Measurement
Oxygen electrode	Measures $\mathrm{O_2}$ concentration in solution using a Clark-type electrode	Rate of $\mathrm{O_2}$ production (\mu\mathrm{mol\ O_2\ m^{-2}\ s^{-1})
Audus microburette	Collects $\mathrm{O_2}$ bubbles from an aquatic plant (e.g., Elodea) in a capillary tube	Volume of $\mathrm{O_2}$ produced per unit time
Mass spectrometry	Measures uptake of $\mathrm{^{13}CO_2}$ and production of $\mathrm{^{13}C}$-labelled sugars	Rate of carbon fixation
Chlorophyll fluorimeter	Measures fluorescence emitted by chlorophyll when light is shone on a leaf	Efficiency of photosystem II ($F_v/F_m$ ratio)
Infrared gas analysis (IRGA)	Measures $\mathrm{CO_2}$ uptake and $\mathrm{H_2O}$ release from a leaf in a sealed chamber	Net photosynthetic rate; transpiration rate; stomatal conductance

45.2 Practical Considerations

Factor	How to Control/Measure
Light intensity	Use a lamp at measured distances; use a light meter (lux); place heat filter between lamp and plant to prevent heating
Temperature	Water bath (for aquatic plants); temperature-controlled chamber
$\mathrm{CO_2}$ concentration	Sodium hydrogencarbonate solution (for aquatic plants); IRGA chamber with controlled $\mathrm{CO_2}$
Chlorophyll concentration	Extract chlorophyll with acetone; measure absorbance at 663 nm and 645 nm using a colorimeter

46. C3, C4, and CAM Plants Compared

46.1 Summary Comparison

Feature	C3 Plants	C4 Plants	CAM Plants
First product of carbon fixation	3-phosphoglycerate (3C compound)	Oxaloacetate (4C compound)	Oxaloacetate (4C compound)
Enzyme for initial fixation	Rubisco (in mesophyll)	PEP carboxylase (in mesophyll); Rubisco (in bundle sheath)	PEP carboxylase (at night); Rubisco (during the day)
Photorespiration	Significant (especially at high temperature and low $\mathrm{CO_2}$)	Negligible ($\mathrm{CO_2}$ is concentrated around Rubisco)	Low (stomata closed during the day; internal $\mathrm{CO_2}$ is recycled)
Water use efficiency	Low (stomata open during the day)	High ($\mathrm{CO_2}$ pump allows stomata to be partially closed)	Very high (stomata open only at night)
Growth rate	Fast in cool, moist conditions	Fast in hot, sunny conditions	Slow; adapted to arid conditions
Examples	Wheat, rice, oats, soybean, most trees	Maize, sugarcane, sorghum, Amaranthus	Cactus, pineapple, Kalanchoe, succulents
Leaf anatomy	No bundle sheath around veins	Kranz anatomy (prominent bundle sheath cells around veins)	No special anatomy (all reactions occur in the same mesophyll cell, but at different times)

47. Plant Mineral Ions

47.1 Essential Mineral Ions

Ion	Function	Deficiency Symptom
Nitrogen ($\mathrm{NO_3^-}$)	Required for amino acids, proteins, nucleotides, chlorophyll	Chlorosis (yellowing of older leaves first); stunted growth
Magnesium ($\mathrm{Mg^{2+}}$)	Central atom in chlorophyll molecule; activates enzymes	Chlorosis (yellowing of leaves); leaves may turn red/purple
Phosphorus ($\mathrm{PO_4^{3-}}$)	Component of ATP, nucleic acids (DNA/RNA), phospholipids	Poor root growth; dark green/purple leaves
Potassium ($\mathrm{K^+}$)	Involved in stomatal opening/closing; activates enzymes; maintains turgor	Yellow leaves with dead spots; wilting
Calcium ($\mathrm{Ca^{2+}}$)	Component of middle lamella (calcium pectate); cell signalling	Stunted growth; meristem death
Iron ($\mathrm{Fe^{2+/3+}}$)	Required for chlorophyll synthesis (cofactor for enzymes)	Chlorosis of young leaves (interveinal chlorosis -- veins remain green)

47.2 Hydroponics

Feature	Description
What it is	Growing plants in a nutrient solution without soil
Advantages	Precise control of mineral ion concentrations; no soil-borne diseases; can grow crops in areas with poor soil; higher yields
Use in research	Used to investigate the effect of specific mineral deficiencies by omitting individual ions from the nutrient solution and comparing plant growth

48. Limiting Factors of Photosynthesis

48.1 The Concept of Limiting Factors

The principle of limiting factors states that the rate of a process is limited by the factor that is in shortest supply. At any given moment, only one factor limits the rate of photosynthesis.

48.2 Effects of Individual Limiting Factors

Factor	Effect on Rate	Graph Shape
Light intensity	Rate increases proportionally at low light; levels off at high light (light saturation point) as another factor becomes limiting	Plateau curve
$\mathrm{CO_2}$ concentration	Rate increases as $\mathrm{CO_2}$ increases; plateaus at high $\mathrm{CO_2}$	Plateau curve
Temperature	Rate increases with temperature (up to an optimum, ~25--35$^\circ$C); above the optimum, enzymes denature and the rate falls sharply	Bell-shaped curve

48.3 Interpreting Graphs

Scenario	Interpretation
Rate increases with light, then levels off	Light was initially limiting; at the plateau, $\mathrm{CO_2}$ or temperature is now limiting
Two curves at different $\mathrm{CO_2}$ concentrations: the higher $\mathrm{CO_2}$ curve reaches a higher plateau	$\mathrm{CO_2}$ concentration limits the maximum rate; more $\mathrm{CO_2}$ allows a higher maximum rate
Rate decreases at high temperature	Temperature is above the optimum; enzymes (especially Rubisco) are denaturing

---

tip

Diagnostic Test

::: :::