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

Learning Objective

Students will be able to describe and carry out investigations using redox indicators (DCPIP and methylene blue) and a suspension of isolated chloroplasts to determine how light intensity and light wavelength affect the rate of photosynthesis.

Key Concepts

• Photosynthetic electron transport reduces NADP⁺ and oxidises water, producing O₂.
• Redox indicators change colour when they are reduced or oxidised, providing a visual measure of electron flow.
• Limiting factors (e.g., light intensity, wavelength) alter the rate at which electrons are transferred.

Redox Indicators

• DCPIP (2,6‑dichlorophenol‑indophenol): blue in oxidised form, colourless when reduced.
• Methylene blue: blue when oxidised, colourless when reduced.

Principle of the Experiments

The rate of reduction of the indicator is proportional to the rate of photosynthetic electron transport. By measuring the time taken for a fixed amount of colour change, the relative photosynthetic rate can be calculated.

Rate of photosynthesis (using an indicator) can be expressed as:

\$\text{Rate} = \frac{\Delta C}{\Delta t}\$

where \$\Delta C\$ is the change in concentration of the reduced indicator (or the amount of colour lost) and \$\Delta t\$ is the time taken.

Experimental Design Overview

1. Prepare a suspension of isolated chloroplasts in a suitable buffer.
2. Add a known concentration of DCPIP or methylene blue.
3. Expose the mixture to light of defined intensity and wavelength.
4. Record the time taken for the indicator to change colour completely.
5. Repeat for different light intensities and wavelengths.

Materials

• Fresh spinach leaves (or other green plant material)
• Homogenisation buffer (e.g., 0.33 M sucrose, 50 mM Tris‑HCl, pH 7.8)
• Centifuge and tubes
• DCPIP solution (0.5 mM)
• Methylene blue solution (0.1 mM)
• Light source with adjustable intensity (e.g., LED lamp)
• Filters or monochromator to select wavelengths (e.g., 450 nm, 550 nm, 650 nm)
• Stopwatch
• Cuvettes or clear test tubes
• Thermostated water bath (maintain 25 °C)

Procedure

Isolation of Chloroplasts

1. Weigh 10 g of fresh spinach leaves and grind in 50 mL cold homogenisation buffer using a mortar and pestle.
2. Filter the homogenate through cheesecloth into a centrifuge tube.
3. Centrifuge at 1 000 g for 5 min at 4 °C to pellet intact chloroplasts.
4. Discard supernatant and gently resuspend the pellet in 10 mL cold buffer. This suspension is the chloroplast stock.

Assay with DCPIP

1. In a cuvette, mix 2 mL chloroplast suspension with 2 mL 0.5 mM DCPIP solution.
2. Place the cuvette in the water bath at 25 °C.
3. Expose to light of a chosen intensity and wavelength.
4. Start the stopwatch when the light is switched on.
5. Observe the colour change from blue to colourless; record the time (\$t\$) when the solution becomes colourless.
6. Repeat three times for each light condition and calculate the mean time.

Assay with Methylene Blue

1. Prepare a mixture of 2 mL chloroplast suspension and 2 mL 0.1 mM methylene blue.
2. Proceed as in steps 2‑5 above, recording the time for the blue colour to disappear.

Data Recording Tables

Data Analysis

• Plot mean time (or calculated rate) against light intensity for each wavelength.
• Identify the intensity at which the rate no longer increases – the light‑saturation point.
• Compare rates at different wavelengths to determine the action spectrum of the chloroplast suspension.

Safety Considerations

• Wear gloves and goggles when handling plant material and chemicals.
• DCPIP and methylene blue are irritants; avoid skin contact and inhalation.
• Use caution with high‑intensity light sources to prevent eye damage.
• Dispose of chemical waste according to school laboratory guidelines.

Possible Extensions

• Investigate the effect of temperature on the rate using the same set‑up.
• Replace chloroplast suspension with intact leaf discs and measure O₂ evolution with a gas syringe.
• Use a spectrophotometer to quantify the decrease in absorbance of the indicator for more precise rate calculations.

Summary

By using DCPIP and methylene blue as visual redox indicators, students can quantitatively assess how light intensity and wavelength limit the rate of photosynthetic electron transport in isolated chloroplasts. The experiments reinforce concepts of limiting factors, the action spectrum, and the relationship between light energy and biochemical processes in photosynthesis.