Photosynthesis – Energy Transfer Process (Cambridge International AS & A Level Biology 9700)
Assessment‑Objective (AO) Mapping
| Learning Objective | AO1 – Knowledge & Understanding | AO2 – Application & Analysis | AO3 – Practical Skills & Evaluation |
|---|
| State the overall photosynthetic equation. | ✓ | | |
| Describe the light‑dependent reactions (PS II & PS I, electron flow, ATP/NADPH formation). | ✓ | ✓ (explain why non‑cyclic flow produces O₂) | |
| Describe the Calvin‑Benson (light‑independent) cycle. | ✓ | ✓ (calculate ATP/NADPH consumption per CO₂ fixed) | |
| Identify the major chloroplast pigments and interpret their absorption spectra. | ✓ | ✓ (match peaks to pigments, discuss antenna function) | |
| Carry out paper chromatography to separate chloroplast pigments and use Rf values for identification. | | ✓ (analyse chromatograms, calculate Rf) | ✓ (design, execute, evaluate the practical) |
| Explain how light intensity, CO₂ concentration, temperature and water availability limit photosynthetic rate. | ✓ | ✓ (predict direction of change, interpret data) | |
| Design and evaluate simple experiments to measure photosynthetic rate (O₂ evolution or CO₂ uptake). | | | ✓ |
| Link photosynthesis to cellular respiration, describe photo‑respiration and calculate respiratory quotient (RQ). | ✓ | ✓ (interpret RQ values, discuss energy balance) | |
1. Overall Photosynthetic Equation
6 CO2 + 6 H2O + light energy → C6H12O6 + 6 O2
Photons are captured by pigment molecules in the thylakoid membranes; the energy is stored in ATP and NADPH, which then drive CO₂ fixation in the Calvin‑Benson cycle.
2. Chloroplast Structure (relevant to the syllabus)
- Double‑membrane envelope – encloses the organelle.
- Stroma – fluid matrix where the Calvin‑Benson cycle occurs; contains DNA, ribosomes and enzymes.
- Thylakoid system
- Stacked discs = grana; sites of the light‑dependent reactions.
- Unstacked thylakoids = stroma lamellae; connect grana and house ATP synthase.
- Thylakoid membrane houses photosystems, electron carriers and the oxygen‑evolving complex.
3. Light‑Dependent Reactions
- Location: Thylakoid membranes (grana & stroma lamellae).
- Key components: PS II (P680), plastoquinone (PQ), cytochrome b6f, plastocyanin (PC), PS I (P700), ferredoxin (Fd), NADP⁺‑reductase, ATP synthase.
- Non‑cyclic electron flow (most common):
- Light excites P680 → electron ejection.
- H₂O is split (photolysis) → O₂ + 2H⁺ (lumen) + 2e⁻.
- e⁻ travel via PQ → cytochrome b₆f → PC → P700.
- Second photon excites P700 → e⁻ reduce NADP⁺ → NADPH.
- Proton gradient (H⁺) drives ATP synthesis (photophosphorylation).
- Cyclic photophosphorylation (PS I only): Excited e⁻ from P700 return to PQ, generating extra ATP but no NADPH or O₂.
4. Light‑Independent Reactions (Calvin‑Benson Cycle)
| Phase | Key Steps | Products |
|---|
| Carbon fixation | Rubisco catalyses CO₂ + RuBP → 2 × 3‑PGA | 2 × 3‑PGA |
| Reduction | 3‑PGA + ATP + NADPH → G3P | G3P (some leaves cycle, some forms glucose) |
| Regeneration | 5 G3P + 3 ATP → 3 RuBP | RuBP restored for the next turn |
For every 3 CO₂ fixed the cycle consumes 9 ATP and 6 NADPH, producing one G3P that can be converted to glucose.
4.1. C₃, C₄ and CAM Pathways (brief comparison)
| Feature | C₃ | C₄ | CAM |
|---|
| Initial CO₂ fixation enzyme | Rubisco (carboxylase) | PEP carboxylase (mesophyll) | PEP carboxylase (night) |
| Primary site of Calvin cycle | Mesophyll chloroplasts | Bundle‑sheath chloroplasts | Mesophyll chloroplasts (day) |
| Adaptation to | Temperate, moderate light | High light, high temperature, low CO₂ | Arid, water‑limited environments |
| Limiting factor under drought | Stomatal closure → photo‑respiration | CO₂ concentration in bundle sheath maintained | Stomata open at night → water loss reduced |
5. Chloroplast Pigments, Absorption & Action Spectra
- Pigments absorb specific wavelengths and transfer excitation energy to the reaction‑centre chlorophyll a.
- Accessory pigments broaden the usable spectrum and protect against excess light.
5.1. Typical Absorption Peaks (nm)
| Pigment | Peak(s) | Role |
|---|
| Chlorophyll a | 430, 662 | Primary donor in both photosystems |
| Chlorophyll b | 453, 642 | Extends absorption; funnels energy to chlorophyll a |
| β‑Carotene | 450‑500 | Absorbs blue‑green; transfers energy; quenches triplet chlorophyll |
| Lutein / Violaxanthin / Neoxanthin (xanthophylls) | 470‑500 | Photoprotection (xanthophyll cycle) & supplemental harvesting |
5.2. Interpreting an Absorption Spectrum (example box)
Task: A graph shows absorbance vs wavelength with peaks at 430 nm, 452 nm, 662 nm and a broad band 470‑500 nm.
- 430 nm → chlorophyll a (blue region)
- 452 nm → chlorophyll b (blue‑green region)
- 662 nm → chlorophyll a (red region)
- 470‑500 nm → carotenoids (β‑carotene & xanthophylls)
Students should state which pigments are present and explain how their combined absorption matches the action spectrum of photosynthesis (maximal activity ≈ 430‑660 nm).
6. Paper Chromatography of Chloroplast Pigments
6.1. Principle
Pigments are separated on cellulose paper (stationary phase) by a solvent (mobile phase). The distance a pigment travels relative to the solvent front is expressed as the retardation factor (Rf).
Rf = (distance travelled by pigment) ÷ (distance travelled by solvent front)
Each pigment has a characteristic Rf range under standard conditions, allowing identification.
6.2. Materials (Cambridge standard)
- Whatman No. 1 chromatography paper (or equivalent)
- Solvent: petroleum ether : acetone (90 : 10 v/v)
- Fresh spinach (or other green plant material)
- Mortar & pestle, 80 % (v/v) acetone for extraction
- Capillary tubes or micropipettes
- Pencil, ruler, sealed chromatography chamber
6.3. Step‑by‑Step Procedure
- Cut paper into strips 2 cm × 15 cm.
- Draw a faint pencil line 2 cm from the bottom (origin).
- Grind ~0.5 g fresh spinach with 5 ml 80 % acetone to a uniform green paste.
- Spot 1–2 µl of the extract onto the origin line; let dry. Repeat 2–3 times to concentrate.
- Place the strip in the chamber so that the solvent level is ~0.5 cm below the origin. Seal the chamber.
- Allow the solvent to ascend the paper until it is ~1 cm from the top (≈15 min). Do not disturb.
- Remove the strip, immediately mark the solvent front, and dry in a fume cupboard.
- Measure distances from origin to each pigment band and to the solvent front; calculate Rf values.
6.4. Typical Rf Values (Petroleum ether : acetone = 90 : 10)
| Pigment | Visible colour | Typical Rf range | Primary role |
|---|
| β‑Carotene | Orange | 0.85 – 0.90 | Absorbs blue‑green; photoprotection |
| Lutein | Yellow‑orange | 0.75 – 0.80 | Accessory pigment; xanthophyll cycle |
| Violaxanthin | Yellow‑orange | 0.70 – 0.75 | Photoprotection (xanthophyll cycle) |
| Chlorophyll b | Blue‑green | 0.55 – 0.60 | Extends absorption; transfers energy to chlorophyll a |
| Chlorophyll a | Dark blue‑green | 0.45 – 0.50 | Primary pigment for charge separation |
6.5. Interpreting the Chromatogram (AO2 + AO3)
- Match each measured Rf with the reference range (±0.02 acceptable).
- Order of migration reflects polarity: non‑polar carotenes travel farthest; the most polar pigment (chlorophyll a) travels least.
- Band intensity gives a qualitative estimate of relative pigment abundance – useful when discussing antenna size.
- Unexpected bands may indicate minor pigments (e.g., neoxanthin) or experimental errors (over‑loading, solvent impurity).
- For quantitative work, densitometry can be used to calculate the proportion of each pigment; relate this to light‑harvesting efficiency.
7. Photo‑respiration
- Cause: Rubisco can act as an oxygenase, fixing O₂ instead of CO₂.
- Pathway: O₂ + RuBP → 1 × 3‑PGA + 1 × 2‑phosphoglycolate → (peroxisome, mitochondrion) → CO₂ + NH₃ (energy‑costly).
- Consequences:
- Loss of previously fixed carbon (CO₂ release).
- Consumption of ATP and NADPH without net carbohydrate gain.
- Reduced photosynthetic efficiency, especially at high temperature and low CO₂.
- Relevance to the syllabus: Explains why C₃ plants are more affected by drought and high temperature than C₄ plants.
8. Linking Pigment Composition to Energy‑Transfer Efficiency
- Accessory pigments increase the quantum yield by harvesting photons that chlorophyll a cannot absorb.
- The “antenna size” (total pigment per reaction centre) can be estimated from band intensities on a chromatogram; larger antennas improve light capture under low irradiance but increase heat loss under excess light.
- Carotenoids (β‑carotene, xanthophylls) protect the photosystems by dissipating excess energy as heat (non‑photochemical quenching).
- Students may calculate the % contribution of each pigment (e.g., β‑carotene ≈ 30 % of total absorbance) and discuss how a shift in this proportion would affect the rate of ATP/NADPH production.
9. Limiting Factors in Photosynthesis (Syllabus 13.2)
| Factor | Effect on Rate | Typical Experimental Test |
|---|
| Light intensity | Rate rises until light‑saturation; then plateaus. | Vary distance of a lamp from leaf discs; measure O₂ evolution. |
| CO₂ concentration | Rate increases to a saturation point; limited by Rubisco activity. | Inject known volumes of CO₂ into a sealed chamber with leaf material. |
| Temperature | Rate rises to an optimum (~25 °C for many C₃ plants) then falls due to enzyme denaturation. | Place leaf samples in water baths at different temperatures; record O₂ evolution. |
| Water availability | Stomatal closure reduces CO₂ entry; severe drought damages the photosynthetic apparatus. | Expose plants to progressive drought; measure gas exchange. |
10. Measuring Photosynthetic Rate (AO3 – Practical Skills)
10.1. Oxygen‑Evolution (Water‑Displacement) Method
- Insert a leaf disc (or a small spinach piece) into a sealed syringe filled with water.
- Expose the leaf to a known light intensity.
- O₂ produced displaces water; record the volume change over time.
- Calculate rate (mL O₂ min⁻¹) and, if required, convert to µmol O₂ g⁻¹ FW min⁻¹.
10.2. Carbon‑Dioxide Uptake Using an Infrared Gas Analyzer (IRGA)
- Clamp a leaf in a sealed cuvette connected to the IRGA.
- Measure the decline in CO₂ concentration under controlled light and temperature.
- Express the rate as µmol CO₂ m⁻² s⁻¹.
10.3. Qualitative Starch Test
- Expose leaves to light for a set period.
- Boil in water, de‑colourise with ethanol, then add iodine solution.
- Blue‑black colour = starch (photosynthetic activity); colourless = no starch.
11. Link to Cellular Respiration & Respiratory Quotient (RQ)
- During daylight, photosynthesis supplies ATP and NADPH; respiration consumes O₂ and releases CO₂.
- Respiratory Quotient (RQ) = CO₂ produced ÷ O₂ consumed.
- Carbohydrate oxidation: RQ ≈ 1.0 (C₆H₁₂O₆ + 6 O₂ → 6 CO₂ + 6 H₂O).
- Fat oxidation: RQ ≈ 0.7 (more O₂ required per CO₂).
- Example calculation: If a leaf disc consumes 0.30 mmol O₂ min⁻¹ and produces 0.28 mmol CO₂ min⁻¹, RQ = 0.28 ÷ 0.30 ≈ 0.93, indicating predominately carbohydrate metabolism.
- Comparing RQ values under light and dark conditions helps students understand the balance between photosynthetic production and respiratory consumption of gases.
12. Summary of Key Points
- Photosynthesis converts light energy into chemical energy (ATP, NADPH) and stores it as carbohydrate.
- Light‑dependent reactions occur in thylakoid membranes; the Calvin‑Benson cycle occurs in the stroma.
- Chlorophyll a is the primary pigment; chlorophyll b and carotenoids extend the absorption range and protect the system.
- Paper chromatography separates pigments; identification is based on characteristic Rf values (β‑carotene ≈ 0.88, lutein ≈ 0.78, chlorophyll b ≈ 0.58, chlorophyll a ≈ 0.48).
- Photo‑respiration reduces efficiency, especially in C₃ plants under high temperature/low CO₂.
- Limiting factors (light, CO₂, temperature, water) can be investigated experimentally; results are interpreted using AO2‑style data analysis.
- Linking photosynthesis to respiration via RQ provides a quantitative view of plant energy balance.