describe and carry out investigations using redox indicators, including DCPIP and methylene blue, and a suspension of chloroplasts to determine the effects of light intensity and light wavelength on the rate of photosynthesis

Investigation of Limiting Factors in Photosynthesis (Cambridge IGCSE/A‑Level 9700 – Topic 13.2)

Learning Objectives (AO1‑AO3)

• AO1 – Knowledge: Describe chloroplast structure, the light‑dependent and light‑independent reactions, and the role of photosynthetic pigments.
• AO2 – Application: Explain how redox indicators (DCPIP, methylene blue) can be used to monitor electron transport in isolated chloroplasts.
• AO3 – Analysis & Evaluation: Design, carry out and critically evaluate investigations of the effects of light intensity, wavelength, CO₂ concentration, temperature and water availability on the rate of photosynthesis, using quantitative methods.

Key Concepts

• Photosynthesis occurs in chloroplasts. The thylakoid membranes (grana) host the light‑dependent reactions; the stroma hosts the Calvin cycle.
• Light‑dependent reactions generate ATP, NADPH and O₂ by transferring electrons from H₂O to NADP⁺ via the photosynthetic electron transport chain (PETC).
• Redox indicators change colour when reduced; the rate of colour loss is proportional to the rate of electron flow through the PETC.
• Limiting factors (light intensity, wavelength, CO₂, temperature, water) affect the speed of the light‑dependent reactions and therefore the overall rate of photosynthesis.

Chloroplast Structure (≈150 words)

Chloroplasts are double‑membrane organelles. The inner membrane encloses the stroma, which contains the Calvin‑cycle enzymes, ribosomes and DNA. Within the stroma are stacks of flattened thylakoid discs (grana) linked by stroma‑lamellae. The thylakoid membranes house photosystem II, photosystem I, the cytochrome b₆f complex, ATP synthase and the mobile electron carriers plastoquinone, plastocyanin and ferredoxin. Light absorbed by pigments (chlorophyll a, chlorophyll b, carotenoids) excites electrons in PS II; these electrons travel through the chain, reducing NADP⁺ to NADPH while water is split to release O₂. The spatial separation of the light‑dependent (thylakoid) and light‑independent (stroma) stages enables the use of isolated chloroplast suspensions to study electron transport directly.

Photosynthetic Pigments & Action Spectrum

Choosing monochromatic light at 450 nm (blue), 550 nm (green) and 650 nm (red) allows students to construct an action spectrum and compare it with the published pigment‑absorption spectrum.

Redox Indicators

Both indicators accept electrons directly from the PETC; the speed of de‑colourisation reflects the rate of electron flow.

Principle of the Experiments

The rate of reduction of the indicator (r) is proportional to the rate of photosynthetic electron transport (J).

\[

r = \frac{\Delta C}{\Delta t}

\]

• \(\Delta C\) = change in concentration of reduced indicator (or equivalent amount of colour lost).
• \(\Delta t\) = time taken for a defined colour change (e.g., from blue to colourless).

By measuring \(\Delta t\) under different experimental conditions, students obtain relative photosynthetic rates that can be compared directly.

Overall Experimental Design

1. Isolate intact chloroplasts from fresh spinach.
2. Prepare assay mixtures containing a redox indicator.
3. Expose the mixtures to light of known intensity and wavelength (or to other limiting factors).
4. Record the time for complete de‑colourisation.
5. Repeat each condition at least three times and calculate mean rates.
6. Analyse the data with appropriate graphs and evaluate the reliability of the method.

Materials

• Fresh spinach leaves (or any green plant material)
• Homogenisation buffer: 0.33 M sucrose, 50 mM Tris‑HCl, pH 7.8 (kept cold)
• Refrigerated centrifuge and 15 mL centrifuge tubes
• DCPIP solution (0.5 mM) and methylene blue solution (0.1 mM)
• LED light source with adjustable intensity (0–200 µmol m⁻² s⁻¹) and interchangeable filters or a monochromator (450 nm, 550 nm, 650 nm)
• Quantum sensor (optional but recommended) to record the actual photon flux (µmol m⁻² s⁻¹) for each intensity setting
• Thermostated water bath (maintain 25 °C ± 0.5 °C)
• Stopwatch, graduated pipettes, 10 mL cuvettes or clear test tubes
• 0.05 M sodium bicarbonate (NaHCO₃) for CO₂ variation
• pH‑stat or additional buffer (e.g., 10 mM phosphate, pH 7.8) to keep pH constant when CO₂ is added
• Thermometers or temperature probes for the temperature series
• Safety equipment: lab coat, goggles, nitrile gloves

Procedures

1. Isolation of Intact Chloroplasts

1. Weigh 10 g fresh spinach; chop finely.
2. Grind in 50 mL cold homogenisation buffer using a chilled mortar and pestle (≈ 30 s).
3. Filter through two layers of cheesecloth into a pre‑chilled centrifuge tube.
4. Centrifuge at 1 000 g for 5 min at 4 °C.
5. Discard the supernatant (broken cells, debris). Gently resuspend the green pellet in 10 mL cold buffer – this is the chloroplast stock (≈ 2 × 10⁶ chloroplasts mL⁻¹).
6. Keep the suspension on ice and use within 2 h.

2. Standard DCPIP Assay – Light Intensity & Wavelength

1. Label a cuvette “DCPIP – 450 nm”. Add 2 mL chloroplast suspension + 2 mL 0.5 mM DCPIP.
2. Place the cuvette in the water bath (25 °C). Position the light source at a fixed distance (e.g., 10 cm) from the cuvette for all measurements.
3. Choose a wavelength filter (450 nm, 550 nm or 650 nm) and set the lamp to the required intensity. Record the actual photon flux with a quantum sensor (if available) and note it in the data table.
4. Start the stopwatch simultaneously with the light.
5. Observe the colour change; stop the timer when the solution becomes colourless (no visible blue).
6. Record the time (s). Repeat three times and calculate the mean.
7. Repeat steps 1‑6 for the other two wavelengths and for at least three light intensities that span the linear‑to‑saturation region (e.g., 25 µmol m⁻² s⁻¹ ≈ low, 75 µmol m⁻² s⁻¹ ≈ mid, 150 µmol m⁻² s⁻¹ ≈ saturating).

3. Confirmatory Assay with Methylene Blue

1. Prepare a mixture of 2 mL chloroplast suspension + 2 mL 0.1 mM methylene blue.
2. Follow the same light‑exposure protocol as in the DCPIP assay (same wavelengths, same intensities, same distance).
3. Record the time for the blue colour to disappear; perform three replicates per condition.
4. Compare rates obtained with the two indicators to check consistency.

4. CO₂ Concentration Series (Substrate Limitation)

1. Prepare assay mixtures as in step 2, but replace part of the buffer with 0.05 M NaHCO₃ to give final dissolved CO₂ concentrations of 0, 5, 10 and 20 mM.
2. To avoid pH artefacts, add an additional 10 mM phosphate buffer (pH 7.8) to each mixture so that the overall pH remains constant (~7.8) across the series.
3. Keep light intensity and wavelength constant (e.g., 75 µmol m⁻² s⁻¹, 650 nm).
4. Measure de‑colourisation times as described above; repeat three times for each CO₂ level.

5. Temperature Series – Optimum for Light‑Dependent Reactions

1. Set the water bath to 10 °C, 20 °C, 30 °C and 40 °C (±0.5 °C).
2. Perform the DCPIP assay at a fixed light intensity and wavelength (e.g., 75 µmol m⁻² s⁻¹, 650 nm) for each temperature.
3. Record the colour‑loss times; repeat three replicates per temperature.
4. Plot rate vs. temperature to locate the optimum (usually 25–30 °C for spinach chloroplasts) and discuss enzyme‑kinetic and membrane‑fluidity effects.

6. Optional Extension – Pigment Chromatography

1. Extract pigments from a small amount of spinach in 80 % acetone (cold, protected from light).
2. Spot 5 µL of the extract on chromatography paper and develop in petroleum ether : acetone = 9 : 1.
3. After drying, measure Rf values for chlorophyll a, chlorophyll b and carotenoids.
4. Overlay the measured action spectrum (rate vs. wavelength) with a published absorption spectrum of the identified pigments to illustrate the relationship.

Data Recording Tables

Data Analysis

• Convert mean times to rates using the equation above.
• Light‑intensity graphs: Plot rate (y‑axis) against photon flux (x‑axis) for each wavelength. Identify the linear region and the light‑saturation point.
• Action‑spectrum graph: At a single, non‑saturating intensity (e.g., 75 µmol m⁻² s⁻¹), plot rate vs. wavelength. Overlay a published pigment‑absorption spectrum (or the chromatographic Rf data) to discuss why green light gives a low rate.
• CO₂ series: Plot rate vs. dissolved CO₂ concentration. Discuss why the curve plateaus (substrate saturation) and note any pH‑related deviations.
• Temperature series: Plot rate vs. temperature. Locate the optimum temperature and explain the decline at high temperatures (enzyme denaturation, membrane damage).
• Calculate %RSD (relative standard deviation) for replicates to assess precision.
• Identify random errors (timing, uneven illumination) and systematic errors (chloroplast damage, temperature drift, pH changes). Suggest specific improvements (e.g., use a magnetic stirrer to keep chloroplasts in suspension, calibrate the quantum sensor before each series).

Safety Considerations

• Wear lab coat, safety goggles and nitrile gloves throughout the experiment.
• DCPIP and methylene blue are irritants; avoid skin contact and inhalation of powders.
• Handle the centrifuge and cold buffers with care to prevent spills and frostbite.
• When using high‑intensity LEDs or xenon lamps, wear eye protection and avoid direct viewing of the beam.
• Dispose of chemical waste in clearly labelled containers according to school policy.

Evaluation Checklist (AO3)

Possible Extensions (Syllabus Links)

• Pigment chromatography – satisfies 13.2 b (identifying chlorophyll a, chlorophyll b and carotenoids).
• CO₂ variation – explores substrate limitation of the Calvin cycle (13.2 c).
• Temperature series – demonstrates the optimum for enzyme‑mediated light‑dependent reactions (13.2 d).
• Water‑availability experiment – repeat the DCPIP assay with osmotic solutions (0 %, 5 % sucrose) to illustrate the impact of turgor on chloroplast function (13.2 e).
• Respiration link – measure O₂ consumption in the dark using a gas syringe; calculate the respiratory quotient (RQ) and discuss competition between photosynthesis and respiration.
• Spectrophotometric quantification – record absorbance at 600 nm (DCPIP) or 660 nm (methylene blue) every 10 s with a spectrophotometer for more precise kinetic data.

Summary

Redox indicators such as DCPIP and methylene blue provide a rapid, visual method for quantifying electron flow through the light‑dependent reactions of isolated chloroplasts. By systematically varying light intensity, wavelength, CO₂ concentration, temperature and water availability, students can design investigations that meet all Cambridge 9700 requirements for limiting‑factor experiments, develop a clear understanding of structure‑function relationships in photosynthesis, and practise the full cycle of scientific enquiry (design, data collection, analysis, evaluation).