Abstract
Have you ever seen a (non-carnivorous) plant eat? Probably not! Plants do not get the energy they need from food, but from the sunlight! In a process called photosynthesis, plants convert light energy, water, and carbon dioxide into oxygen and sugar. They can then use the sugar as an energy source to fuel their growth. Scientists have found an easy way to measure the rate of photosynthesis in plants. The procedure is called the floating leaf disk assay. In this plant biology project, you can use this procedure to investigate which factors affect the rate of photosynthesis. Do you think the light intensity, type of plant, or the temperature matter?Summary
Objective
To investigate the effect of different variables on photosynthesis using the floating leaf disk assay.
Introduction
Every living organism needs energy to grow and 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 and 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 production of oxygen or the consumption of carbon dioxide 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 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 on 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 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. Leaf disk assay picture.
In this project, 10 disks are placed 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 graphing the number of disks floating over time, as shown in Figure 3. An ET50 of 11.5 minutes, for example, as shown in Figure 3, would mean that after 11.5 minutes, 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 minutes means that it took 11.5 minutes 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.
An example can make this concept clear. If a glass of soda has 1,000 bubbles, and half of the bubbles (500 bubbles) pop in 5 min when the soda is at room temperature, the rate at which the bubbles pop is 500/5 min or 100/min at room temperature. Imagine you repeat the experiment, but with a glass of the same soda at refrigerator temperature and find that half of the bubbles (or 500 bubbles) pop in 10 min. The rate at refrigerator temperature is 500 bubbles in 10 min or 50 bubbles/minute. It is hard to count bubbles in soda, but if you only know that half of the bubbles pop in 5 min (room temperature) or 10 minutes (refrigerator temperature), you can use the reciprocal of these time measurements as indicators for the rate at which the bubbles pop. 1/ET50 is 1/(5 min) or 0.2/min at room temperature, and 1/(1 min) or 0.1/min at refrigerator temperature. Do you notice that the indicator for the rate at room temperature is still double the indicator for the rate at refrigerator temperature? That is why 1/ET50 is a good indicator of the rate of photosynthesis.
In this project, you will determine the Effective Time (ET50) under different environmental conditions to find out which variables affect the rate of photosynthesis. For example, you could change the light source, the brightness of the light, the color of the light, the temperature, the type of plant, or the color of the plant leaves.
Terms and Concepts
- Photosynthesis
- Enzyme
- Carbon dioxide
- Reactant
- Oxygen
- Chloroplast
- Chlorophyll
- Pigment
- Visible light spectrum
- NADPH
- ATP
- Calvin cycle
- Rate
- Floating leaf disk assay
- Buoyancy
- Effective Time (ET50)
- Reciprocal
Questions
- How do plants convert light energy into chemical energy?
- What happens during the floating leaf disk assay?
- What environmental factors do you think affect the rate of photosynthesis?
- How is photosynthesis connected to cellular respiration?
Bibliography
- Khan Academy. (n.d.). The light-dependent reactions, Retrieved October 7, 2020.
- Scitable. (n.d.). Photosynthetic Cells, from Nature Education. Retrieved October 7, 2020.
- Bitesize. (n.d.). What happens during photosynthesis?, BBC. Retrieved October 7, 2020.
Materials and Equipment
- Transparent cups, 6 or more (large enough to hold 300 mL)
- Measuring cup
- Water
- 1/8 or 1/4 teaspoon
- Baking soda
- Dish soap
- Plant leaves (spinach or ivy work best)
- Single hole puncher or sturdy straw
- Plastic syringe, 10-mL or bigger (without the needle)
- Permanent marker
- Light source (a bright light works best)
- Paper towels
- Timer
- Pencil or pen
- To investigate the following:
- Different light sources: LED, CFL, incandescent, halogen, sunlight
- Different light intensities: Ruler or measuring tape
- Different light colors: Colored cellophane sheets, tape
- Water temperature: Thermometer, microwave, ice cubes
- Different leaf colors: Yellow, red, and green leaves
- Different plant types: Leaves from different plant species
- Lab notebook
Experimental Procedure
Conducting the Floating Leaf Disk Assay
Before you start testing different variables, conduct the leaf disk assay once to get familiar with the procedure. The video Measure Photosynthesis with Floating Leaves demonstrates how to do each individual step. You will do all your experiments in triplicates after your test run; the following instructions are for the three trials of your experiment.
- Fill three cups with 300 mL water. Add about 1/8 teaspoon baking soda and 1 drop of liquid dish soap to each of the cups (if you are using a 1/4 teaspoon measure, fill it only halfway). Gently stir the solution until everything has dissolved. Try not to create too many bubbles. Note: The baking soda provides the plant leaves with carbon dioxide for photosynthesis.
- Punch out 30 leaf disks from the spinach or ivy leaves using the hole puncher or a straw. Avoid cutting through major leaf veins.
- Remove the plunger of the syringe and place 10 leaf disks into the syringe barrel.
- Place the plunger back into the syringe and push it down until only a small volume of air is left in the syringe. Be careful not to crush the leaf disks.
- Suck up a small volume of the baking soda solution from one of the cups into the syringe with the leaf disks and hold it vertically. The leaf disks should all float on the surface of the solution.
- Remove the air pockets within the leaves to make the leaf disks sink, as follows.
- Carefully push out all the air from the syringe.
- Close the opening of the syringe with a finger and pull back on the plunger to create a vacuum. Hold the vacuum for 10–15 seconds and swirl the leaf disks to suspend them in the solution.
- Stop pulling on the plunger and remove your finger from the syringe opening to release the vacuum.
- Repeat step 6 until all leaf disks have sunk to the bottom of the solution.
- Remove the plunger from the syringe and pour all 10 leaf disks with the solution into a fresh, clear plastic cup. Fill the cup with baking soda solution up to a depth of about 3 cm. Label this cup "1."
- Repeat steps 3–8 twice more, with 10 leaf disks each, to prepare the other two cups. Label the other cups "2" and "3."
- Place all three cups with the leaf disks under your light source. Make sure the light shines straight onto the cups from above. Each cup should be positioned so that they get an equal amount of light.
- Start a timer. Observe closely what happens to the leaf disks. Write your observations down in your lab notebook.
- At the end of each minute, record the number of floating disks for each cup in a data table like Table 1 in your lab notebook. Briefly swirl the cups to prevent the leaf disks from getting stuck to the bottom or sides of the cups.
- Continue the experiment until all of the leaf disks are floating.
| Time [minutes] | Number of Floating Leaf Disks | ||
|---|---|---|---|
| Cup 1 | Cup 2 | Cup 3 | |
| 0 | 0 | 0 | 0 |
| 1 | |||
| 2 | |||
| 3 | |||
| 4 | |||
| 5 | |||
| 6 | |||
| 7 | |||
| 8 | |||
| 9 | |||
| 10 | |||
| ... | |||
Analyzing Your Results
- For each cup, create a graph from your data table that shows the number of floating leaf disks over time. Plot the time on the x-axis and the number of floating leaf disks on the y-axis. An example graph for one cup is shown in Figure 4.
The x-axis shows time in minutes. The y-axis shows the number of floating leaf disks. After 3 minutes the first leaf disk floats, after 4 minutes 3 leaf disks float, after 6 minutes 4 leaf disks float, after 7 minutes 6 leaf disks float, after 8 minutes 7 leaf disks float, after 9 minutes 8 leaf disks float, and after 10 minutes all 10 leaf disks float.
Figure 4. Example graph that shows the results of a floating leaf disk assay. The x-axis shows the time, in minutes, the y-axis shows the number of floating leaf disks.
- Determine the median time from your data. The median time is the time it takes for 50% of the disks to float. This time is also called the Effective Time (ET50). Since you used 10 disks in each cup, you need to determine the time at which 5 disks floated in your experiment. You can extrapolate the ET50 from your graph as shown in Figure 3 in the Introduction. Note: Occasionally a disk fails to rise or takes a very long time to do so, so the median reflects the tendency of the leaf disk assay results better than the mean (average) does as it is more robust against outliers. The resource Stuck in the Middle-Mean vs. Median explains the difference between these two values.
- Calculate the average of all three ET50 values. Then use this average ET50 value to calculate the rate of photosynthesis for the conditions of your leaf disk assay. To do that, take the reciprocal of your average ET50 value, which means to calculate 1/ ET50. The unit of your photosynthesis rate will be 1/min or min-1.
Changing Variables to Investigate Photosynthesis
- Now that you are familiar with the leaf disk assay procedure and data analysis, you are ready to start your own investigations.
- Choose one variable that you want to investigate. You can find some suggestions in Table 2. The table also provides some possible variations for each variable that you can test.
| Variable | Possible Variations |
|---|---|
| Light intensity | Lightbulb 10 cm, 20 cm, 30 cm, 40 cm away from the cup |
| Light color | Use of green, red, blue, yellow, white cellophane filters |
| Type of light source | LED, CFL, incandescent, halogen lightbulbs, or sunlight |
| Water temperature | 10°C, 20°C, 30°C, 40°C |
| Baking soda concentration | 0g/100mL, 0.1g/100mL, 0.5g/100mL, 1g/100mL, 2g/100mL |
| Type of plant | A selection of different plant leaves |
| Leaf color | Red, yellow, green leaves from the same plant |
- Conduct the leaf disk assay for each condition or variation that you choose to test. Remember to record the number of floating leaf disks in a data table and do each experiment in triplicates. Note: It is very important that you only test one variation at a time in your experiment. To determine the effect of one variable, you can only vary this one variable and have to keep everything else the same.
- For every variation that you test, determine the ET50 value for each of your three trials. Then calculate the average of all three ET50 values.
- Calculate the reciprocal of your average ET50 values, 1/ ET50. The results will be the measure of the rate of photosynthesis. The goal is to compare the photosynthesis rates for each tested variation at the end, as shown in Table 3.
| Variation | Average ET50 [minutes] | 1/ET50 [minutes-1] |
|---|---|---|
| 0.5 g/100 mL | 20 | 0.05 |
| 1 g/100 mL | 16 | 0.0625 |
| 2 g/100 mL | 12.5 | 0.08 |
| 3 g/100 mL | 11 | 0.09 |
- Make a graph that shows the photosynthesis rate for each tested variation. Plot your variable on the x-axis and the rate of photosynthesis (or 1/ET50) on the y-axis. An example graph using the hypothetical data from Table 3 is shown in Figure 5. How do your photosynthesis rates compare?
The x-axis shows the baking soda concentration in grams per 100 mL. The y-axis shows the rate of photosynthesis, or 1/ET50 in minutes-1. The rate of photosynthesis is 0.05 minutes-1 at 0.5 g/100 mL baking soda, 0.0625 minutes-1 at 1 g/100 mL baking soda, 0.08 minutes-1at 2 g/100 mL baking soda, and 0.09 minutes-1at 3 g/100 mL baking soda.
Figure 5. Example graph that shows the calculated photosynthesis rates for different baking soda concentrations. The x-axis shows the baking soda concentration in g/100 mL, the y-axis shows the rate of photosynthesis or 1/ET50. Note: This data is hypothetical and does not reflect the results of real experiments.
- Look at your graph to interpret your data.
- How did your chosen variable affect the rate of photosynthesis?
- Do you see any trends in your data?
- Can you explain your results?
- Why does the rate of photosynthesis reach a plateau (only if you see that trend in your data)?
- If you would like, you can continue to test other variables from Table 2. You can also review other possibilities for further investigations in the Variations section.
Ask an Expert
Variations
- Find out how plants generate energy without sunlight in the dark! Once all your leaf disks are floating, cover the cup with aluminum foil. Then let it sit for 30 min to 1 h. What happens to the leaf disks in the cup? Can you explain your observations? Tip: The process that you are looking for is called cellular respiration. What happens during cellular respiration?
- Continue testing different variables from Table 2. Do you find variables that do not affect photosynthesis? Which variable has the biggest effect on the rate of photosynthesis?
- If you are interested in how plants grow and what they need to grow, consider doing Science Buddies' Hydroponics: Gardening Without Soil project.
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