Photosynthesis: Light-Independent Reactions
This lesson is mapped to AQA 7402 Section 3.5.2 — Photosynthesis: light-independent reactions (Calvin cycle) (refer to the official AQA specification document for exact wording). The light-independent reactions — universally known as the Calvin cycle or the Calvin–Benson–Bassham (CBB) cycle — are the second stage of photosynthesis. They take place in the stroma of the chloroplast and do not require light directly. However, they consume the two products of the light-dependent reactions — ATP and NADPH — so they cannot continue indefinitely in darkness; in a leaf placed in the dark, the Calvin cycle halts within a few minutes once the stromal ATP and NADPH pools are exhausted.
The cycle was elucidated between 1946 and 1956 by Melvin Calvin, Andrew Benson, and James Bassham at the University of California, Berkeley. Their classic "lollipop experiment" used the green alga Chlorella in a thin, illuminated, lollipop-shaped flask. The cells were given a brief pulse of ¹⁴CO₂, then plunged into hot methanol at progressively longer intervals to halt enzymes. Two-dimensional paper chromatography followed by autoradiography revealed where the radioactive ¹⁴C had ended up at each time-point — and the first stable radioactive product, after just a few seconds of illumination, was the 3-carbon compound glycerate-3-phosphate (GP). Calvin won the 1961 Nobel Prize in Chemistry for this work (paraphrased). Subsequent contributions by Benson and Bassham established the full cyclical character of the pathway.
Key Definition: The light-independent reactions (Calvin cycle) use ATP and NADPH from the light-dependent reactions to fix CO₂ into organic molecules in the stroma of the chloroplast. The net product per three CO₂ fixed is one molecule of triose phosphate (GALP).
The Calvin Cycle: Overview
The cycle has three discrete stages:
- Carbon fixation — CO₂ is added to the 5C acceptor RuBP, catalysed by RuBisCO.
- Reduction — the resulting 3C compound (GP) is reduced to triose phosphate (GALP), using ATP and NADPH.
- Regeneration of RuBP — five out of every six GALP molecules are used to regenerate the 5C acceptor, ensuring the cycle continues.
The net carbon balance is straightforward: for every 3 CO₂ that enter, 1 triose phosphate (3C) exits as the net product. The remaining carbon is recycled through the regeneration phase.
graph TD
A["CO₂ (1C)"] -->|"RuBisCO<br/>carbon fixation"| B["RuBP (5C)"]
B --> C["Unstable 6C intermediate"]
C --> D["2 × GP (3C)"]
D -->|"+ ATP → ADP+Pi<br/>+ NADPH → NADP⁺<br/>reduction"| E["2 × GALP (3C)"]
E -->|"5/6 GALP<br/>+ 3 ATP per 3 CO₂"| F["RuBP regeneration"]
F --> B
E -.->|"1/6 GALP (net product)"| G["Glucose, sucrose, starch<br/>cellulose, lipids, amino acids"]
style B fill:#3498db,color:#fff
style D fill:#e67e22,color:#fff
style E fill:#27ae60,color:#fff
style G fill:#9b59b6,color:#fff
Stage 1: Carbon Fixation
- Carbon dioxide (CO₂) from the atmosphere diffuses into the leaf through stomata, then through the air spaces of the spongy mesophyll, into the photosynthetic mesophyll cells, and finally into the chloroplast stroma.
- CO₂ combines with a 5-carbon (5C) acceptor molecule, ribulose-1,5-bisphosphate (RuBP).
- The reaction is catalysed by RuBisCO (ribulose bisphosphate carboxylase/oxygenase) — the most abundant enzyme on Earth, constituting up to 50% of soluble leaf protein and an estimated 0.7 Gt globally.
- The immediate product is an unstable 6C intermediate that splits immediately into two molecules of glycerate-3-phosphate (GP), each a 3C compound.
CO₂ + RuBP (5C) → 2 × GP (3C)
Key Definition: Carbon fixation is the incorporation of inorganic CO₂ into an organic molecule (GP). It is the first step in the Calvin cycle and is catalysed by RuBisCO.
The fact that the first stable product is a 3C compound rather than the 6C glucose textbook predictions might have suggested was the great surprise of Calvin's experiments and required a rethink of how photosynthesis works.
Stage 2: Reduction of GP to GALP
- Each molecule of GP (3C) is reduced to glyceraldehyde-3-phosphate (GALP), also called G3P or triose phosphate (TP). GALP is a 3C sugar phosphate.
- The reduction is a two-step enzyme-catalysed sequence requiring:
- ATP (from the light reactions) — provides the energy by phosphorylating GP to 1,3-bisphosphoglycerate.
- NADPH (from the light reactions) — provides the reducing power (the hydrogen atoms) for the subsequent reduction of 1,3-BPG to GALP.
- ATP is hydrolysed to ADP + Pi; NADPH is oxidised to NADP⁺. Both ADP and NADP⁺ are recycled to the thylakoid membrane for re-use in the light reactions, creating a tight cycle between stroma and thylakoid.
GP (3C) + ATP + NADPH → GALP (3C) + ADP + Pi + NADP⁺
Exam Tip: The reduction of GP to GALP is the step that requires both ATP and NADPH. If either runs out (e.g. in darkness, or under stress conditions that disrupt the light reactions), GP accumulates and GALP decreases — a classic graph-interpretation question.
Stage 3: Regeneration of RuBP
- For every 3 CO₂ fixed, 6 GALP (3C) are produced.
- 5 out of 6 GALP molecules are used to regenerate 3 RuBP (5C) — ensuring the cycle can continue with no net loss of acceptor.
- Regeneration requires ATP (but not NADPH).
- The rearrangement of five 3C molecules into three 5C molecules involves several enzyme-catalysed steps (transketolase, aldolase, ribose-5-phosphate isomerase, phosphoribulokinase — the latter is the final phosphorylation step that converts ribulose-5-phosphate into RuBP).
5 × GALP (3C) + 3 ATP → 3 × RuBP (5C) + 3 ADP + 3 Pi
The mathematics is elegant: 5 × 3C = 15 carbons in; 3 × 5C = 15 carbons out. The carbon balance is conserved within the cycle; only the net product (one GALP per three CO₂) leaves it.
The Fate of GALP: The Net Product of Photosynthesis
The remaining 1 out of every 6 GALP (i.e. the net product of the cycle) is exported from the chloroplast (via the triose phosphate/phosphate antiporter in the inner chloroplast envelope) and used as a building block for larger organic molecules. At A-Level you should be able to give named examples of each:
Glucose and Other Carbohydrates
- Two GALP (3C) can be combined to form glucose (6C) — a hexose sugar.
- Glucose can then be used to make:
- Sucrose — the main transport sugar in plants (glucose + fructose joined by an α(1→2) glycosidic bond); transported in the phloem from source to sink (AQA 7402 Section 3.3 transport in plants).
- Starch — the main storage polysaccharide in plants (polymer of α-glucose); accumulates in chloroplasts during the day and is hydrolysed at night.
- Cellulose — a structural polysaccharide forming plant cell walls (polymer of β-glucose joined by β(1→4) bonds; AQA 7402 Section 3.1.2).
- Fructose — another hexose sugar, especially abundant in fruits and nectar.
Amino Acids
- GALP can be converted into Krebs-cycle intermediates (e.g. α-ketoglutarate, oxaloacetate) which are transaminated with nitrogen from absorbed nitrate ions (NO₃⁻ → NH₄⁺ in the plant via nitrate reductase and nitrite reductase).
- Amino acids are used for protein synthesis (AQA 7402 Section 3.4.1).
Lipids (Fatty Acids and Glycerol)
- GALP provides the carbon skeleton for fatty acid synthesis (via conversion to acetyl-CoA in the plastid).
- Glycerol (a 3C molecule) is directly derived from GALP.
- Fatty acids and glycerol combine to form triglycerides and phospholipids.
Nucleotides
- The ribose and deoxyribose sugars of nucleotides are derived from GALP via the pentose phosphate pathway.
Exam Tip: In exam questions about the fate of the products of photosynthesis, give specific named examples: glucose for respiration, starch for storage, cellulose for cell walls, amino acids for protein synthesis, lipids for membranes. Vague "used to make organic molecules" answers lose marks.
Stoichiometry of the Calvin Cycle
For the fixation of 3 CO₂ (the minimum needed to produce one net GALP):
| Input | Quantity |
|---|
| CO₂ | 3 molecules |
| ATP | 9 molecules (6 for reduction of GP, 3 for regeneration of RuBP) |
| NADPH | 6 molecules (for reduction of GP to GALP) |
| Output | Quantity |
|---|
| GALP (net product) | 1 molecule (3C) |
| ADP + Pi | 9 molecules (recycled to thylakoid) |
| NADP⁺ | 6 molecules (recycled to thylakoid) |
To produce one molecule of glucose (6C), the cycle must fix 6 CO₂, requiring 18 ATP and 12 NADPH.
Effect of Environmental Changes on the Calvin Cycle
Examiners frequently test understanding of the cycle by asking how the concentrations of GP, GALP, and RuBP change when light or CO₂ availability is altered. The trick is to identify which step is affected and trace the consequences forward and backward through the cycle.
In the Dark (No Light)
- Light-dependent reactions stop → no ATP or NADPH produced.
- GP cannot be reduced to GALP → GP accumulates.
- GALP and RuBP concentrations decrease: GALP is no longer being produced; RuBP is still consumed by carbon fixation but is not being regenerated (regeneration requires ATP).
- Carbon fixation eventually slows as RuBP runs out; the cycle approaches a halt.
Low CO₂ Concentration
- Less CO₂ available for carbon fixation by RuBisCO.
- RuBP is not consumed as fast → RuBP accumulates.
- Less GP is produced → GP concentration decreases.
- Less GP means less GALP → GALP concentration decreases.
High CO₂ Concentration
- More CO₂ is fixed → GP increases initially.
- More GP is reduced to GALP (provided ATP and NADPH are still available from the light reactions).
- RuBP concentration decreases initially (it is consumed faster than it is regenerated until the cycle re-equilibrates).
- A new steady state is reached at higher overall cycle flux.
Low Light Intensity (but Some Light)
- Less ATP and NADPH produced.
- Reduction of GP slows → GP accumulates moderately.
- GALP decreases; RuBP regeneration slows; cycle flux decreases overall but does not halt.
Exam Tip: Draw a quick mental flowchart whenever asked one of these "what happens to [X] if [Y] changes" questions. Identify which step is directly affected, then trace the consequences: a step slows or stops, its substrate accumulates, its product decreases, and downstream steps slow.
RuBisCO: The Key Enzyme
- Full name: Ribulose-1,5-bisphosphate carboxylase/oxygenase.
- It is the most abundant protein on Earth — constituting up to 50% of soluble leaf protein, with a total global mass estimated at ~0.7 gigatonnes.
- It is comparatively slow: a catalytic rate of ~3–10 reactions per second per active site (compare with most enzymes at 10²–10⁵ per second). This is why so much of it is needed; the leaf compensates for slow turnover with sheer enzyme abundance.
- RuBisCO has a dual catalytic activity:
- Carboxylase activity — fixes CO₂ to RuBP (the productive Calvin cycle reaction).
- Oxygenase activity — fixes O₂ to RuBP instead of CO₂, producing one molecule of GP and one of phosphoglycolate (a wasteful 2C compound that must be salvaged via the energetically expensive photorespiration pathway). Photorespiration becomes more pronounced at high temperatures (where O₂ solubility is favoured relative to CO₂) and at low CO₂. Photorespiration is beyond the AQA A-Level specification proper but is essential context for the C₄ and CAM adaptations (also beyond spec).
- RuBisCO requires Mg²⁺ as a cofactor and is activated by RuBisCO activase (an ATP-dependent regulator) — providing another indirect coupling between the light and dark reactions.
Synoptic Links
This lesson connects to:
- AQA 7402 Section 3.5.2 — Light-dependent reactions: the Calvin cycle absolutely depends on ATP and NADPH from the thylakoid; the two stages are tightly coupled via the stroma–thylakoid exchange of ADP/ATP and NADP⁺/NADPH.
- AQA 7402 Section 3.7.5 — Energy transfer through ecosystems: the net product GALP supports gross primary production (GPP), which after subtracting plant respiration gives net primary production (NPP) — the energy base of all food chains.
- AQA 7402 Section 3.1.2 — Carbohydrates: the polysaccharides starch and cellulose are downstream products of GALP, providing storage and structural roles respectively.
Common Errors and Mark-Loss Patterns