Photosynthesis

Photosynthesis is the process by which plants, algae, and some bacteria convert light energy into chemical energy stored in organic molecules. It is the fundamental process that sustains almost all life on Earth, providing both the organic compounds and the oxygen upon which heterotrophic organisms depend. The overall equation is:

6CO₂ + 6H₂O → C₆H₁₂O₆ + 6O₂

Key Definition: Photosynthesis is the process by which light energy is used to convert carbon dioxide and water into glucose and oxygen. It is an endergonic (energy-requiring) process in which light energy is converted to chemical energy in organic molecules.

Photosynthesis takes place in the chloroplasts and consists of two main stages: the light-dependent reactions (on the thylakoid membranes) and the light-independent reactions or Calvin cycle (in the stroma).

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Chloroplast Structure

A described diagram of a chloroplast would show a lens-shaped organelle approximately 5–10 µm long, bounded by a double membrane (envelope). Inside, stacks of flattened membrane-bound sacs called thylakoids are arranged in piles known as grana (singular: granum). The grana are connected by extensions of thylakoid membrane called intergranal lamellae (or stroma lamellae). The fluid-filled space surrounding the grana is the stroma, which contains enzymes, small circular DNA molecules, 70S ribosomes, lipid droplets, and starch grains. The interior space within each thylakoid is called the thylakoid lumen.

Structure	Function
Outer membrane	Permeable to small molecules; forms part of the chloroplast envelope
Inner membrane	Contains transport proteins; controls what enters/exits the stroma
Thylakoid membranes	Contain photosystems (I and II), electron carriers, and ATP synthase; site of light-dependent reactions
Grana (stacks of thylakoids)	Increase surface area for light absorption; contain high density of photosynthetic pigments
Thylakoid lumen	Enclosed space where H⁺ ions accumulate during chemiosmosis
Stroma	Fluid-filled matrix; site of the Calvin cycle; contains enzymes (including RuBisCO), DNA, 70S ribosomes
Intergranal lamellae	Connect thylakoids in different grana; maximise thylakoid surface area

Exam Tip: When asked to describe adaptations of the chloroplast for photosynthesis, always refer to: (1) the large surface area of thylakoid membranes for pigments and electron carriers, (2) the small volume of the thylakoid lumen for rapid H⁺ accumulation, and (3) the stroma containing all necessary Calvin cycle enzymes. Relate structure to function.

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Photosynthetic Pigments

• Chlorophyll a — the primary pigment; absorbs red (~680 nm) and blue-violet (~430 nm) light, reflects green.
• Chlorophyll b — an accessory pigment; absorbs slightly different wavelengths (peaks at ~500 nm and ~640 nm), broadening the absorption spectrum.
• Carotenoids (carotene, xanthophyll) — accessory pigments absorbing blue-violet light (400–500 nm); also protect chlorophyll from photooxidation by absorbing excess light energy.
• Pigments can be separated using chromatography (thin-layer or paper). The Rf value = distance moved by pigment ÷ distance moved by solvent front.

Absorption Spectra vs Action Spectra

Key Definition: An absorption spectrum shows the wavelengths of light absorbed by a pigment. An action spectrum shows the rate of photosynthesis at different wavelengths of light.

• The absorption spectrum of chlorophyll shows two main peaks — in the blue-violet region (~430 nm) and the red region (~680 nm) — with a trough in the green region (~550 nm), which is reflected.
• The action spectrum shows that the rate of photosynthesis is highest at the same wavelengths where chlorophyll absorbs most light (blue-violet and red), and lowest in the green region.
• The close correlation between the absorption spectrum and the action spectrum provides strong evidence that chlorophyll is the pigment responsible for photosynthesis. The slight differences between the two spectra (the action spectrum is slightly broader) are explained by the contribution of accessory pigments.
• Engelmann's experiment with Spirogyra and aerobic bacteria demonstrated this correlation: bacteria clustered around the parts of the alga illuminated by red and blue light, where the most O₂ was being produced.

A described graph comparing the two spectra would show wavelength of light (nm) on the x-axis (from 400 to 700 nm). Two curves are plotted: the absorption spectrum (showing peaks in the blue and red regions with a trough in the green) and the action spectrum (closely following the same pattern but slightly broader, indicating accessory pigments contribute at additional wavelengths).

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The Light-Dependent Reactions

These occur on the thylakoid membranes and require light energy.

Non-cyclic Photophosphorylation

1. Light energy is absorbed by photosystem II (PSII) (P680), exciting a pair of electrons to a higher energy level.
2. The excited electrons pass along an electron transport chain (ETC) of carrier proteins (including plastoquinone, cytochrome b6f complex, and plastocyanin), losing energy at each step.
3. This energy is used to pump H⁺ ions from the stroma into the thylakoid lumen, creating a proton gradient (chemiosmosis).
4. H⁺ ions flow back through ATP synthase into the stroma, driving the synthesis of ATP from ADP + Pi (photophosphorylation).
5. The electrons from PSII are passed to photosystem I (PSI) (P700), where they are re-excited by light.
6. The re-excited electrons are transferred via ferredoxin to NADP⁺ reductase, which combines them with H⁺ ions to reduce NADP⁺ to NADPH (reduced NADP).
7. The electrons lost from PSII are replaced by the photolysis of water: H₂O → 2H⁺ + ½O₂ + 2e⁻. Oxygen is released as a by-product.

flowchart LR
    W["H₂O
(Photolysis)"] -->|"2e⁻ + 2H⁺ + ½O₂"| PSII["Photosystem II
(P680)
Light excites e⁻"]
    PSII -->|"Excited e⁻"| ETC["Electron Transport Chain
(Plastoquinone → Cyt b6f → Plastocyanin)
Pumps H⁺ into thylakoid lumen"]
    ETC -->|"e⁻ passed to"| PSI["Photosystem I
(P700)
Light re-excites e⁻"]
    PSI -->|"e⁻ via Ferredoxin"| NADPR["NADP⁺ Reductase
NADP⁺ + H⁺ → NADPH"]
    ETC -.->|"H⁺ gradient drives"| ATP["ATP Synthase
ADP + Pi → ATP"]

Products of the light-dependent reactions: ATP, NADPH (reduced NADP), and O₂.

Cyclic Photophosphorylation

• Only PSI is involved.
• Excited electrons are passed along the ETC and return to PSI (hence "cyclic").
• Produces ATP only — no NADPH is formed, no photolysis occurs, no O₂ is released.
• Useful when the Calvin cycle demands more ATP than NADPH (the Calvin cycle requires 18 ATP but only 12 NADPH per glucose, so cyclic photophosphorylation makes up the shortfall).

Key Definition: Photophosphorylation is the synthesis of ATP from ADP and inorganic phosphate using light energy. It can be non-cyclic (involving both PSI and PSII, producing ATP and NADPH) or cyclic (involving only PSI, producing ATP only).

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The Light-Independent Reactions (Calvin Cycle)

These occur in the stroma and do not directly require light, but depend on the ATP and NADPH produced by the light-dependent reactions.

Steps of the Calvin Cycle

1. Carbon fixation — CO₂ combines with ribulose bisphosphate (RuBP, a 5C compound), catalysed by the enzyme RuBisCO (ribulose bisphosphate carboxylase-oxygenase), forming an unstable 6C intermediate that immediately splits into two molecules of glycerate-3-phosphate (GP), a 3C compound.
2. Reduction — GP is reduced to glyceraldehyde-3-phosphate (GALP / G3P / TP) using ATP (provides energy) and NADPH (provides hydrogen) from the light-dependent reactions. ATP is hydrolysed to ADP + Pi, and NADPH is oxidised to NADP⁺. Both are recycled back to the light-dependent reactions.
3. Regeneration of RuBP — Five out of every six GALP molecules are used to regenerate RuBP, requiring ATP. For every 3 turns of the cycle (3 CO₂ fixed), one net GALP molecule (3C) is produced.
4. The net GALP is used to synthesise glucose, amino acids, fatty acids, glycerol, and other organic molecules.

flowchart TD
    CO2["CO₂"] -->|"Carbon fixation
(RuBisCO)"| RuBP["RuBP (5C)"]
    RuBP --> unstable["Unstable 6C intermediate"]
    unstable --> GP["2x GP (3C)"]
    GP -->|"Reduction
uses ATP + NADPH"| GALP["2x GALP (3C)"]
    GALP -->|"5 out of 6 GALP
(uses ATP)"| RuBP
    GALP -->|"1 out of 6 GALP
(net product)"| Products["Glucose, amino acids,
fatty acids, glycerol"]

Worked Example 1 — Calvin Cycle Requirements per Glucose

For one molecule of glucose (C₆H₁₂O₆), 6 CO₂ molecules must be fixed. This requires 6 turns of the Calvin cycle.

Per turn: 1 CO₂ + 1 RuBP → 2 GP → 2 GALP (using 2 NADPH and 3 ATP for reduction and regeneration).

For 6 turns:

• CO₂ fixed: 6
• GP produced: 12
• GALP produced: 12
• NADPH used: 6 turns × 2 NADPH = 12 NADPH
• ATP used for reduction: 6 turns × 2 ATP = 12 ATP
• ATP used for regeneration of RuBP: 6 turns × 1 ATP = 6 ATP
• Total ATP used: 12 + 6 = 18 ATP

Of the 12 GALP produced, 2 GALP are used to make one glucose (2 × 3C = 6C), and the remaining 10 GALP are rearranged to regenerate 6 RuBP (10 × 3C = 30C → 6 × 5C = 30C).

Exam Tip: Learn the numbers 18 ATP and 12 NADPH per glucose. A common exam question asks you to explain why the Calvin cycle stops when light is removed — the answer is that ATP and NADPH from the light-dependent reactions are no longer being produced, so GP cannot be reduced and RuBP cannot be regenerated.

Effects of Changing Conditions on Calvin Cycle Intermediates

Condition Change	Effect on GP	Effect on RuBP	Explanation
Light intensity decreases	Increases	Decreases	Less ATP and NADPH produced → GP cannot be reduced → GP accumulates; less RuBP regenerated
CO₂ concentration decreases	Decreases	Increases	Less CO₂ to fix with RuBP → less GP formed → RuBP accumulates as it is still regenerated but not used
Temperature increases (to a point)	Decreases then increases	Variable	Enzymes work faster up to optimum; beyond optimum, RuBisCO denatures

Exam Tip: Questions on GP and RuBP levels are very common. Always think about what happens to the step that produces the molecule and the step that uses it up. If production stays the same but use decreases, the molecule accumulates.

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The Compensation Point

Key Definition: The compensation point is the light intensity (or CO₂ concentration) at which the rate of photosynthesis exactly equals the rate of respiration. At this point, there is no net gas exchange — CO₂ produced by respiration is used in photosynthesis, and O₂ produced by photosynthesis is used in respiration.

• Below the compensation point, the plant is a net consumer of O₂ and a net producer of CO₂ (respiration > photosynthesis).
• Above the compensation point, the plant is a net producer of O₂ and a net consumer of CO₂ (photosynthesis > respiration).
• Shade-tolerant plants have a lower compensation point than sun plants, meaning they can achieve net photosynthesis at lower light intensities.

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Limiting Factors of Photosynthesis

The rate of photosynthesis is determined by whichever factor is closest to its minimum value (law of limiting factors, proposed by F.F. Blackman).

Factor	Effect	Biological Explanation
Light intensity	Increases rate up to a plateau	Light provides energy for photolysis and excitation of electrons; at the plateau, all photosystems are saturated or another factor is limiting
CO₂ concentration	Higher CO₂ increases the rate until a plateau	CO₂ is the substrate for RuBisCO in carbon fixation; at the plateau, RuBisCO is saturated or another factor limits
Temperature	Increases rate up to an optimum (~25–30 °C); then rate drops sharply	Higher temperature increases kinetic energy of molecules and enzyme–substrate collisions; beyond the optimum, RuBisCO and other enzymes denature

A described graph of rate of photosynthesis against light intensity at two different CO₂ concentrations would show two curves, both rising steeply at low light intensity. The curve at higher CO₂ concentration continues to rise to a higher plateau than the curve at lower CO₂ concentration. Both plateau because at high light intensity, another factor (CO₂ or temperature) becomes limiting. The initial slopes of both curves are identical because at very low light intensity, light is the limiting factor regardless of CO₂ concentration.

Worked Example 2 — Interpreting a Rate-of-Photosynthesis Graph

A student investigates the effect of light intensity on the rate of photosynthesis using an aquatic plant (Elodea). She counts oxygen bubbles produced per minute at different light intensities:

Distance from lamp (cm)	Light intensity (arbitrary units, proportional to 1/d²)	Bubbles per minute
5	400	45
10	100	40
20	25	22
30	11	12
40	6.25	6

Note: Light intensity is proportional to 1/d², where d is the distance from the lamp.

At low light intensities (large distances), the rate increases steeply with light intensity — light is the limiting factor. At high light intensities (short distances), the rate begins to level off, suggesting that another factor (temperature or CO₂ concentration) is now limiting.

Exam Tip: Always state that light intensity is proportional to 1/d² (the inverse square law), not 1/d. This is a common error. When plotting a graph, plot rate against 1/d² (not distance) to obtain a meaningful relationship.

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Photosynthesis Practicals

The Photosynthometer (Audus Apparatus)

This apparatus measures the rate of photosynthesis by collecting and measuring the volume of oxygen gas released by an aquatic plant.

• A piece of Elodea (Canadian pondweed) is placed in a boiling tube of water containing sodium hydrogencarbonate (as a source of dissolved CO₂).
• The apparatus includes a capillary tube connected to the boiling tube. Oxygen released by the plant forms a gas bubble in the capillary tube.
• The distance the bubble moves in a given time is measured to calculate the rate of oxygen production (and hence photosynthesis).
• Variables can be changed systematically: light intensity (by changing the distance of the lamp), temperature (using a water bath), and CO₂ concentration (by varying the concentration of sodium hydrogencarbonate).

Counting Bubbles from Elodea

A simpler method involves counting the number of oxygen bubbles released by Elodea per unit time. This is less accurate because bubble size may vary, but it provides a quick estimate.

The Hill Reaction (DCPIP Experiment)

Key Definition: The Hill reaction demonstrates that the light-dependent reactions of photosynthesis can occur in isolated chloroplasts when provided with an artificial electron acceptor.

• Isolated chloroplasts are placed in a solution of DCPIP (dichlorophenolindophenol), a blue dye that turns colourless when reduced (i.e., when it accepts electrons).
• When illuminated, the chloroplasts carry out the light-dependent reactions: electrons from photolysis of water are passed along the electron transport chain and reduce DCPIP instead of NADP⁺.
• The rate of decolourisation of DCPIP can be measured using a colorimeter (measuring absorbance/transmission over time).
• A control tube kept in the dark should show no colour change, confirming that light is necessary.
• This experiment provides evidence that the light-dependent reactions occur in chloroplasts and involve electron transfer.

Worked Example 3 — Calculating Rf Values in Chromatography

A student separates photosynthetic pigments using thin-layer chromatography. The solvent front moves 12.0 cm from the origin. The pigments move the following distances:

Pigment	Distance moved (cm)	Rf value
Carotene	11.4	11.4 / 12.0 = 0.95
Xanthophyll	8.4	8.4 / 12.0 = 0.70
Chlorophyll a	6.0	6.0 / 12.0 = 0.50
Chlorophyll b	4.8	4.8 / 12.0 = 0.40

Carotene has the highest Rf value because it is the most soluble in the non-polar solvent (it is a non-polar hydrocarbon). Chlorophyll b has the lowest Rf value because it is the least soluble in the solvent (most polar of the four pigments). Rf values can be compared with reference values to identify unknown pigments.