explain that energy transferred as ATP and reduced NADP from the light-dependent stage is used during the light-independent stage (Calvin cycle) of photosynthesis to produce complex organic molecules

Photosynthesis as an Energy‑Transfer Process

Learning objective (AO1 / AO2)

Explain how the energy captured as ATP and reduced NADP⁺ (NADPH) during the light‑dependent reactions is used in the light‑independent reactions (Calvin cycle) to synthesise complex organic molecules, and relate this to experimental investigations of limiting factors.

1. Syllabus mapping (Cambridge International AS & A Level Biology 9700 – Topic 13)

2. Overview of Photosynthesis

1. Light‑dependent reactions – occur in the thylakoid membranes; convert photon energy into the chemical energy carriers ATP and NADPH and evolve O₂.
2. Light‑independent reactions (Calvin cycle) – occur in the stroma; use ATP and NADPH to fix CO₂ into carbohydrate precursors and ultimately into glucose, starch, cellulose, fatty acids, amino acids and nucleotides.

3. Light‑dependent Reactions – Detailed Mechanism

3.1 Chloroplast structures relevant to the light reactions

• Thylakoid membrane – houses photosystems I & II, the cytochrome b₆f complex, plastoquinone (PQ), plastocyanin (PC), ferredoxin (Fd) and ATP synthase.
• Thylakoid lumen – site of proton accumulation; O₂ released from water‑splitting diffuses out of the lumen to the chloroplast interior and then to the atmosphere.
• Stroma – location of NADP⁺ reduction to NADPH and the Calvin cycle.

3.2 Photosystem II (PS II) – entry point for electrons

1. Absorption of a photon by chlorophyll a (P680) excites the reaction centre to P680*.
2. P680* donates an electron to the primary quinone acceptor Q_A, then to the secondary quinone Q_B, forming plastoquinol (PQH₂).
3. The oxygen‑evolving complex (OEC) extracts four electrons from two H₂O molecules:
   

   2 H₂O → O₂ + 4 H⁺ + 4 e⁻
   

   The four protons are released into the lumen, contributing to the proton motive force.

4. PQH₂ carries the electrons to the cytochrome b₆f complex.

3.3 Linear (non‑cyclic) electron flow

1. PQH₂ donates electrons to the cytochrome b₆f complex, which pumps additional H⁺ from the stroma into the lumen.
2. Electrons are passed to plastocyanin (PC) and then to Photosystem I (PS I).
3. Absorption of a photon by chlorophyll a (P700) excites it to P700*; the electron is transferred to ferredoxin (Fd).
4. Fd reduces NADP⁺ + H⁺ → NADPH (catalysed by ferredoxin‑NADP⁺ reductase, FNR).
5. Overall: H₂O + 2 H₂O + 8 photons → O₂ + NADPH + ATP (see section 4 for ATP).

3.4 Cyclic photophosphorylation (only PS I)

• Electrons from reduced Fd are redirected back to the cytochrome b₆f complex instead of reducing NADP⁺.
• This route generates additional ATP without producing NADPH or O₂ – essential when the Calvin cycle’s ATP demand exceeds NADPH supply.

3.5 Electron carriers and redox potentials (useful for AO2)

3.6 Photophosphorylation – ATP synthesis

• The proton gradient (ΔpH) generated by electron transport drives ATP synthase (CF₀CF₁).
• Overall reaction (non‑cyclic flow):
  

  ADP + Pᵢ + 4 photons → ATP + H₂O

4. Energy Carriers Produced

5. Light‑independent Reactions (Calvin Cycle)

5.1 Overall stoichiometry (per three CO₂ fixed)

3 CO₂ + 9 ATP + 6 NADPH + 5 H₂O → G3P + 9 ADP + 9 Pᵢ + 6 NADP⁺ + 8 H⁺

5.2 Three phases and key enzymes

5.3 Rubisco oxygenase activity (photo‑respiration)

• When O₂ competes with CO₂ for Rubisco’s active site, the reaction
  

  RuBP + O₂ → 1‑phosphoglycolate + 3‑PGA occurs.

• Photo‑respiration releases CO₂ and consumes ATP without producing G3P, reducing overall efficiency – an important A‑Level point.

5.4 C₃, C₄ and CAM pathways (advanced box)

Only the C₃ pathway is required for the core syllabus, but a brief comparison helps with higher‑order questions:

• C₃ – CO₂ fixed directly by Rubisco in the mesophyll (e.g., wheat, rice).
• C₄ – CO₂ first fixed to oxaloacetate in mesophyll cells (PEP carboxylase), then shuttled to bundle‑sheath cells where the Calvin cycle operates (e.g., maize, sugarcane).
• CAM – Temporal separation; CO₂ fixed at night, stored as malate, and released for the Calvin cycle during daylight (e.g., pineapple, cactus).

5.5 Energy accounting for one glucose molecule

To produce one C₆H₁₂O₆ (6 CO₂ fixed) the cycle must turn twice (2 × 3 CO₂):

• ATP required = 2 × 9 = 18 ATP
• NADPH required = 2 × 6 = 12 NADPH
• Net G3P produced = 2 G3P (one incorporated into glucose, one recycled)

6. Integration of Light‑dependent and Light‑independent Stages

• ATP and NADPH diffuse from the thylakoid lumen into the stroma where the Calvin cycle operates.
• Continuous consumption of ATP and NADPH in the stroma maintains the proton gradient that drives further photophosphorylation.
• O₂ released from the OEC exits the chloroplast to the atmosphere; CO₂ taken up from the air balances the carbon skeletons formed.
• When ATP demand exceeds NADPH supply, cyclic photophosphorylation is up‑regulated.

7. Production of Complex Organic Molecules

Glyceraldehyde‑3‑phosphate (G3P) is the direct output of the Calvin cycle. It can be channelled into several biosynthetic routes:

• Sugars – G3P → glucose‑6‑phosphate → glucose, sucrose, starch (polymer in chloroplasts) or cellulose (polymer in cell walls).
• Fatty acids – G3P provides the carbon backbone for acetyl‑CoA, the precursor of fatty‑acid synthesis.
• Amino acids – Carbon skeletons of many amino acids (e.g., serine, glycine, cysteine) are derived from 3‑PGA or G3P.
• Nucleotides – Ribose‑5‑phosphate, generated via the pentose‑phosphate branch of the Calvin cycle, is the sugar component of nucleotides.

Thus the high‑energy phosphate bonds of ATP and the reducing power of NADPH become embedded in the C‑H bonds of these organic compounds.

8. Investigation of Limiting Factors (AO2 / AO3)

8.1 Practical outline – leaf‑disc assay

1. Collect uniform leaf discs (≈ 5 mm diameter) from a fresh spinach leaf.
2. Place discs in a 10 mL syringe containing 0.02 M NaHCO₃ solution.
3. Vary one factor while keeping the others constant:

   • Light intensity – neutral‑density filters (10 %, 30 %, 60 % of full intensity).
   • CO₂ concentration – bubble air, 5 % CO₂, or pure CO₂ through the solution.
   • Temperature – water baths at 15 °C, 25 °C, 35 °C.
   • Wavelength – monochromatic LEDs (red ≈ 660 nm, blue ≈ 450 nm, green ≈ 540 nm).

4. Seal the syringe, invert it and record the time taken for each disc to rise (oxygen bubbles provide buoyancy).
5. Calculate the rate of O₂ evolution (disc · min⁻¹) and plot against the varied factor. Discuss the plateau in terms of ATP/NADPH availability.

8.2 Sample data‑analysis question (AO2)

“The graph below shows the rate of O₂ evolution versus light intensity. Explain why the rate plateaus at high intensities and relate this to the availability of ATP and NADPH.”

• At low‑to‑moderate light, the rate rises because more photons drive higher electron flow, producing more ATP and NADPH.
• Beyond a certain intensity, the Calvin cycle’s capacity to consume ATP and NADPH becomes limiting; excess photons cannot increase the rate further, leading to a plateau.
• Additional factors such as saturation of PS II reaction centres and photoinhibition may also contribute.

9. Quantitative Measurements of Photosynthetic Activity (AO2 / AO3)

• DCPIP assay – 2,6‑dichlorophenol‑indophenol (blue) is reduced by electrons from the photosynthetic electron transport chain, causing a colour change to colourless. The decrease in absorbance at 600 nm (ΔA · min⁻¹) is proportional to the rate of electron transport.
• Clark‑type oxygen electrode – measures the rise in dissolved O₂ in a sealed chamber containing isolated chloroplasts. The slope of the O₂‑vs‑time plot gives the photosynthetic rate in µmol O₂ min⁻¹.
• Safety & accuracy notes

  • Wear gloves and goggles when handling DCPIP (strong oxidiser).
  • Calibrate the oxygen electrode with air‑saturated water before each experiment.
  • Maintain temperature within ±0.5 °C to avoid artefacts from enzyme kinetics.

10. Cross‑topic Linkages (Key Concepts)

• Energy & Respiration – ATP generated in photosynthesis fuels cellular processes; NADPH is analogous to NADH in oxidative phosphorylation.
• Cell Structure – The thylakoid membrane exemplifies the fluid‑mosaic model; the stromal matrix is a specialised cytosol.
• Biological Molecules – Pigment absorption spectra (chlorophyll a, b, carotenoids) illustrate structure‑function relationships.
• Enzyme Regulation – Rubisco activity is modulated by CO₂/O₂ competition, carbamylation, and activation by RuBP‑binding protein (RUBP).

11. Key Equations to Remember

• Overall photosynthetic reaction (light‑driven):
  

  6 CO₂ + 6 H₂O → C₆H₁₂O₆ + 6 O₂

• Photophosphorylation (non‑cyclic):
  

  ADP + Pᵢ + 4 photons → ATP + H₂O

• NADPH formation:
  

  NADP⁺ + 2e⁻ + H⁺ + 2 photons → NADPH

• Carbon fixation (first Calvin‑cycle step):
  

  RuBP + CO₂ → 2 3‑PGA (catalysed by Rubisco)

• Reduction of 3‑PGA:
  

  3‑PGA + ATP → 1,3‑BPGA (PGK)
  

  1,3‑BPGA + NADPH → G3P + NADP⁺ (GAPDH)

• Regeneration of RuBP:
  

  5 G3P + 3 ATP → 3 RuBP + 3 ADP + 3 Pᵢ (PRK, transketolase, aldolase)

• Overall stoichiometry for one glucose molecule:
  

  6 CO₂ + 18 ATP + 12 NADPH + 6 H₂O → C₆H₁₂O₆ + 18 ADP + 18 Pᵢ + 12 NADP⁺ + 6 O₂