interpret absorption spectra of chloroplast pigments and action spectra for photosynthesis

Photosynthesis – Absorption & Action Spectra (Cambridge AS & A Level Biology 9700)

Plants harvest light energy with chloroplast pigments, convert it into chemical energy in the light‑dependent reactions, and then use that energy in the Calvin cycle. Two graphs are central to understanding this process:

• Absorption spectrum – how efficiently each pigment absorbs light of different wavelengths.
• Action spectrum – how efficiently the whole leaf (or isolated chloroplast) carries out photosynthesis at those wavelengths.

1. Chloroplast Structure and Functional Relevance

• Outer & inner membranes – regulate entry of metabolites.
• Stroma – site of the Calvin cycle; contains enzymes such as Rubisco.
• Thylakoid membrane – houses photosystems I & II, the electron‑transport chain (ETC), ATP synthase and the light‑harvesting pigment–protein complexes.
• Grana (stacks of thylakoids) – increase surface area for light capture; each disc is rich in PS II complexes.
• Lamellae (inter‑granal thylakoids) – connect grana and contain PS I complexes.

Because pigments are embedded in the thylakoid membrane, the combined absorption spectrum of a chloroplast reflects the sum of all individual pigment spectra and therefore determines which wavelengths of photosynthetically active radiation (PAR, 400–700 nm) can be used.

Typical schematic (text‑based) used in the Cambridge syllabus:

+-------------------+   PS II (P680)
|   Grana stack     |   ↓  H₂O → O₂ + 2e⁻
|   (many discs)   |   ↓
|   ↓ ↓ ↓ ↓ ↓ ↓    |   ↓  Plastoquinone (PQ)
+-------------------+   ↓  Cyt b₆f
|               ↓  Plastocyanin (PC)
|               ↓  PS I (P700)
|               ↓  Ferredoxin (Fd)
|               ↓  NADP⁺ → NADPH
V
Lamellae (unstacked)

2. Pigments, Their Absorption Maxima and Chromatography Rf Values

*Rf values are approximate; they depend on the exact solvent composition, paper quality and temperature. Always record the solvent front for each run.

3. Combined Absorption Spectrum of a Chloroplast

• Broad band covering most of the PAR region (400–700 nm).
• Two major peaks correspond to the λ_max of chlorophyll a (≈ 430 nm and ≈ 662 nm).
• A secondary set of peaks around 453 nm and 642 nm belong to chlorophyll b.
• Carotenoids add a shoulder in the blue‑violet region (≈ 450 nm) and protect the photosystems.
• In cyanobacteria and some algae, phycobilins contribute additional peaks near 560 nm.

Simple ASCII sketch (for quick visualisation):

Absorbance
|
1.0|          *                *
|          *                *
0.8|   *      *      *         *
|   *      *      *         *
0.6|   *      *      *   *     *
|   *      *      *   *     *
0.4|   *      *      *   *     *
|   *      *      *   *     *
0.2|   *      *      *   *     *
+--------------------------------- λ (nm)
400 450 500 550 600 650 700
^      ^      ^
|      |      |
Carotenoid  Chl b   Chl a

4. Action Spectrum for Photosynthesis

An action spectrum plots the rate of a photosynthetic process (e.g., O₂ evolution, CO₂ fixation, or electron flow) against wavelength, with photon flux kept constant. It therefore measures the *overall* efficiency of converting absorbed photons into chemical energy.

• Peaks line up with the absorption maxima of the pigments that feed electrons into the photosystems.
• The green region (≈ 550 nm) shows a trough because chlorophylls absorb poorly there, even though green light is abundant in sunlight.
• Differences between absorption and action spectra reveal losses such as fluorescence, heat dissipation, or non‑photochemical quenching.
• Peak quantum yields (ϕ) are typically ϕ_Chl a ≈ 0.85 and ϕ_Carotenoid ≈ 0.30. These values explain why a wavelength that is well‑absorbed by carotenoids contributes less to the overall photosynthetic rate.

5. Light‑Dependent Reactions (13.1)

1. Photolysis of water (PS II) – H₂O → 2 H⁺ + O₂ + 2e⁻.
2. Non‑cyclic photophosphorylation (linear electron flow) –

   H₂O → PS II (P680) → plastoquinone (PQ) → cytochrome b₆f → plastocyanin (PC) → PS I (P700) → ferredoxin (Fd) → NADP⁺ → NADPH. O₂ is released.

3. Cyclic photophosphorylation – Electrons from reduced ferredoxin return to the PQ pool, generating only ATP (no NADPH, no O₂).
4. ATP synthase (CF₁CF₀) – Uses the proton gradient (ΔpH) across the thylakoid membrane to synthesise ATP (photophosphorylation).
5. Photosystem II (P680) & Photosystem I (P700) – Primary reaction‑centre pigments (chlorophyll a) that absorb maximally at 680 nm and 700 nm respectively.

6. Light‑Independent (Calvin) Cycle (13.2)

1. Carbon fixation – RuBP + CO₂ → 2 3‑phosphoglycerate (3‑PGA) (enzyme: Rubisco).
2. Reduction phase – 3‑PGA + ATP + NADPH → Glyceraldehyde‑3‑phosphate (G3P).
3. Regeneration of RuBP – Most G3P is recycled, using ATP, to regenerate RuBP.
4. Net synthesis – For every 3 CO₂ fixed, 1 G3P exits the cycle and can be used to form glucose or other carbohydrates.

7. Linking Absorption and Action Spectra Quantitatively

The expected photosynthetic rate at a particular wavelength can be expressed as:

• A_i(λ) – Fraction of incident photons absorbed by pigment *i* at λ.
• φ_i – Quantum yield (photons used for photochemistry per photon absorbed). Typical values: φ_Chl a ≈ 0.85, φ_Carotenoid ≈ 0.30.
• I(λ) – Incident photon flux (kept constant in an action‑spectrum experiment).
• Because photon energy varies with wavelength (E = hc/λ), blue photons (shorter λ) carry more energy than red photons, influencing the amount of chemical energy that can be stored per photon.

8. Data‑Handling Practice (AO2)

Typical exam tasks

1. Plot an absorption spectrum (A vs. λ) and an action spectrum (O₂ evolution vs. λ) on the same wavelength axis.
2. Identify the two major peaks and relate them to pigment λ_max values.
3. Calculate the relative quantum yield at 460 nm and 660 nm using

   \$\phi = \frac{\text{Rate (µmol O₂ h⁻¹)}}{A \times I}\$

   (set I = 1 for a relative comparison).

4. Given a chromatography plate where the pigment spot travelled 6.2 cm and the solvent front 8.0 cm, compute the Rf value and decide which pigment it most likely represents using the table in section 2.

9. Summary of Key Points

• Chloroplast structure (thylakoid membranes, grana, lamellae, stroma) determines where light capture and carbon fixation occur.
• Chlorophyll a is the primary reaction‑centre pigment; chlorophyll b and accessory pigments (carotenoids, phycobilins) extend the usable wavelength range.
• The absorption spectrum shows what light a pigment can take in; the action spectrum shows how effectively that light drives photosynthesis after all downstream processes.
• Quantum yield (ϕ) and photon energy (E = hc/λ) explain why two wavelengths with similar absorbance can give different photosynthetic rates.
• Data‑handling skills required by the syllabus include constructing spectra from raw data, calculating Rf values, and estimating quantum yields.
• Understanding these concepts underpins experimental design (choice of growth LEDs, interpretation of pigment chromatography) and explains the characteristic green colour of most higher plants.

10. Sample Examination Question (Syllabus 13.1 & 13.2)

Question: A leaf is illuminated with monochromatic light at 660 nm and at 430 nm, each delivering the same photon flux. The rate of O₂ evolution at 660 nm is 80 % of the maximum rate observed at 430 nm. Explain this difference, referring to both the absorption spectrum and the action spectrum. Include in your answer:

• Why the two wavelengths are absorbed differently despite both being near pigment λ_max.
• The role of photon energy (E = hc/λ) in determining the amount of chemical energy that can be stored per photon.
• Any physiological processes that can reduce efficiency in the red region.

Answer outline (full marks):

1. Both 430 nm (blue) and 660 nm (red) correspond to absorption peaks of chlorophyll a (λ_max ≈ 430 nm and ≈ 662 nm). Hence the absorption of photons is high at both wavelengths.
2. Energy per photon is inversely proportional to wavelength (E = hc/λ). A 430 nm photon carries ≈ 2.9 eV, whereas a 660 nm photon carries ≈ 1.9 eV – about 35 % less energy.
3. The action spectrum therefore shows a lower rate at 660 nm because, although the same number of photons are absorbed, each photon provides less chemical energy to drive the light‑dependent reactions.
4. Additional factors in the red region: photosystems operate nearer to their saturation point, leading to a modest increase in non‑photochemical quenching (heat dissipation) and a slightly lower quantum yield (ϕ_red ≈ 0.80 vs ϕ_blue ≈ 0.85).
5. Consequently, the combined effect of lower photon energy and a small reduction in quantum efficiency accounts for the observed 80 % rate at 660 nm.