Study Photosynthesis with the Floating Leaf Disk Assay

Every living organism needs energy to survive, to grow, and to reproduce. Humans and animals eat foods with carbohydrates, proteins, and fats to produce the energy they need to survive. But plants do not eat. They make their own energy source in the form of energy-rich carbohydrates (sugars) through a process called photosynthesis. Photosynthesis is a multi-step, enzyme-mediated process that converts light energy into chemical energy. During photosynthesis, plant cells use light energy (such as light emitted from the Sun), water (H₂O), and carbon dioxide (CO₂) as reactants to produce sugar molecules (C₆H₁₂O₆) and oxygen (O₂) (Figure 1):

Figure 1. During photosynthesis, plants convert water (H₂O), carbon dioxide (CO₂), and light into oxygen (O₂) and sugars like glucose (C₆H₁₂O₆).

Photosynthesis takes place in the chloroplasts within the plant's cells. The chloroplasts contain special pigments that react to light. Chlorophyll is one of the pigments that can absorb light in the blue and red spectrum from the visible light spectrum. Chlorophyll does not absorb light in the green spectrum of light, but reflects it instead. This is why leaves with chlorophyll usually appear green. During the first part of photosynthesis—the light-dependent reaction—chlorophyll and other pigments harness the light energy to produce NADPH and ATP, which are two types of energy-carrier molecules. At the same time, water is split into oxygen (O₂) and protons (H⁺). The next stage is light-independent and is often referred to as the dark reaction. In this step, the two energy-carrier molecules NADPH and ATP are utilized in a series of chemical reactions called the Calvin cycle. In the Calvin cycle, the plants take carbon dioxide (CO₂) from the air and use it to ultimately make sugars such as glucose or sucrose. These sugars can be stored for later use by the plant as an energy source to fuel its metabolism and growth.

Photosynthesis is responsible for replenishing Earth's atmosphere with oxygen that we breathe. Thus, it is not only crucial for plants, but also for all organisms that rely on oxygen for their survival. Many factors affect how quickly plants are able to conduct photosynthesis. Without enough light or water, for example, a plant cannot photosynthesize very quickly. Similarly, the concentration of carbon dioxide—another reactant in photosynthesis—affects how fast photosynthesis can occur. Temperature also plays a significant role, as photosynthesis is an enzyme-mediated reaction. This is because at high temperatures, enzymes can get damaged and thus become inactivated. Other factors that affect the rate of photosynthesis are the light intensity, the amount of chlorophyll or other color pigments in a plant, and the color of light.

Similar to any other chemical reaction, the rate of photosynthesis can be determined by either measuring the decrease of its reactants or the increase of its products. You could, for example, measure the consumption of carbon dioxide or the production of oxygen over time. Without the use of extensive laboratory equipment, the rate of photosynthesis can be determined indirectly by conducting a floating leaf disk assay to measure the rate of oxygen production (Figure 2). In the floating leaf disk assay, 10 or more leaf disk samples are punched out of a leaf. In the next step, a vacuum is used to replace the air pockets within the leaf structure with a baking soda (bicarbonate) solution. The dissolved baking soda provides the carbon dioxide that the leaf needs for photosynthesis. The leaf disks are then sunk in the baking soda solution and exposed to light. As the plant leaf photosynthesizes, oxygen is produced that accumulates as oxygen gas bubbles at the outside of the leaf disk. The attached oxygen gas changes the buoyancy of the leaf disk and once enough oxygen has been produced the leaf disk will rise to the surface of the baking soda solution. The time from exposure to light until the leaf disk rises to the top of the solution is a measure of how much oxygen has been produced and thus a proxy for the rate of photosynthesis.

Figure 2. The floating leaf disk assay allows to indirectly determine the rate of photosynthesis.

In this lesson plan, students will place 10 disks in the baking soda solution at the same time. A good way to collect data is to count the number of floating disks at the end of a fixed time interval; for example, after every minute until all disks are floating. The time required for 50% of the leaves to float represents the Effective Time (ET₅₀). ET₅₀ can be determined by timing when the fifth leaf floats, or by graphing the number of disks floating over time, as shown in Figure 3. An ET₅₀ of 11.5 minutes (min), for example, as shown in Figure 3, would mean that after 11.5 min 50% of the leaves (5 out of the 10) floated on top of the baking soda solution. In the context of oxygen production, you could also say that an ET₅₀ value of 11.5 min means that it took 11.5 min to produce enough oxygen to make 50% of the leaf disks float.

The x-axis shows time in minutes. The y-axis shows the number of floating leaf disks. After 7 minutes the first leaf disk floats, after 11 minutes 4 leaf disks float, at 12 minutes 7 leaf disks float, at 13 minutes 8 leaf disks float, and after 14 minutes all 10 leaf disks float. A red line indicates at what time 50% (5) leaf disks float (at about 11.5 minutes). This time is labeled Effective Time ET50.

Figure 3. Example results for the floating leaf disk assay. The graph shows the time on the x-axis and the number of floating leaves on the y-axis. The Effective Time (ET₅₀) represents the time required for 50% of the leaves to float. By extrapolating from the graph, the 50% floating point in this graph is about 11.5 min.

Reaction rates are usually expressed as the concentration of reactant consumed or the concentration of product formed per unit of time. As mentioned above, we can use the ET₅₀ as a proxy for how much oxygen has been produced to make half of the leaf disks float. This means that the ET₅₀ value is proportional to the inverse of the rate of oxygen production, or proportional to the inverse of the rate of photosynthesis. The reciprocal of ET₅₀ or 1/ET₅₀ can thus be used as a simple measure of the rate of photosynthesis.

This lesson plan has two parts. In the first part, students use the leaf disk assay to explore how plants make energy using photosynthesis. In the second part, students design and conduct experiments that use the leaf disk assay to investigate several variables that have the potential to affect the rate of photosynthesis. This allows students to apply and review scientific concepts involved in photosynthesis, such as cell structure and function, enzyme activity, energy use and storage, and reaction rates.