Figure 1. Basic structure of the leaf, with a close up of the leaf surface showing Stomata and Guard cells.

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1 BIOL100 Laboratory Assignment 3: Analysis of Stomata Name: Stomata (singular=stoma) are the respiratory control structures in plants (see Figure 1 below). They are essentially small holes in the surface of the leaf, surrounded by a pair of guard cells. These cells open the hole when their water level is high, and close when they become dehydrated, which controls the rate of gas exchange in the leaf there are 3 important gasses that can be transferred between the leaf and the atmosphere, all of them linked to the process of photosynthesis. The summary chemical equation for photosynthesis is: 6CO 2 + 6H 2 O C 6 H 12 O 6 + 6O 2. Oxygen (O 2 ) is a byproduct of photosynthesis, and sometimes needs to be released from the leaf. If oxygen concentrations build up in the leaf, photorespiration can occur (This is essentially photosynthesis running backwards) which results in a net loss of energy to the plant, so O 2 must be removed from the leaf. However, since aerobic cellular respiration requires and consumes oxygen, photorespiration is a relatively rare occurrence. Carbon dioxide (CO 2 ) is a necessary ingredient for photosynthesis, so it needs to be brought into the leaf from the atmosphere. Water (H 2 O) is brought into the leaf from the roots via xylem, but in the leaf it tends to evaporate and be lost through open stomata. The final product of photosynthesis, Glucose (C 6 H 12 O 6 ) is not a gas but a carbohydrate (i.e., a sugar). Glucose is dissolved in water and either taken to other parts of the plant by the phloem, used to manufacture more complex compounds in the leaf itself, or used in the process of cellular respiration to provide energy to the plant cells. Figure 1. Basic structure of the leaf, with a close up of the leaf surface showing Stomata and Guard cells. Plants have a problem with gas exchange in the leaf (Figure 2). They need to get carbon dioxide into the leaf. Diffusion, the tendency of particles to move from areas of 1

2 high concentration to areas of low concentration, can handle this just fine. Photosynthesis uses carbon dioxide, keeping its concentration low, so carbon dioxide has a natural tendency to diffuse into the leaf. The problem is that the plant needs a lot of water, and not only for photosynthesis. Water vapor tends to be high in concentration within the leaf (ideally at 100% relative humidity), so water has a natural tendency to diffuse out of the leaf and it diffuses faster the warmer it is. The job of the stomata is to control diffusion of the gases in the leaf. If the plant keeps the stomata closed all the time, water will not be lost, but carbon dioxide will depleted until photosynthesis stops. If the stomata stay open all of the time, then oxygen and carbon dioxide and water diffuse freely, and water loss can become a major problem. Water is used for a large variety of processes in the leaf, and water loss can lead to cell death. Water loss occurs faster when it is warm, or when the air is particularly dry. When it is both hot and dry as in a desert water is generally the limiting resource for plants and animals. Plants have many strategies for managing this tradeoff between carbon dioxide uptake and water loss. These can involve the number, placement and distribution of stomata on the plant, and/or the timing of when they open and close. For this reason, the location and number of stomata often vary with various factors in a plant s environment. Today s lab will look at one such relationship between environmental factors and stomata. Leaf Interior: CO 2 Low O 2 High H 2 O High Stoma Atmosphere CO 2 High O 2 Low H 2 O Low Figure 2. Normal relative concentrations of gasses between a leaf and the atmosphere in the presence of light. Which way will each gas tend to diffuse if the stoma is open? 2

3 Lab Activity: Stomata The question we will address in today s lab is: A) Are stomata more dense on leaves from the outside (exterior) of a plant than leaves nearer the center (interior) of the plant? Before collecting any data, answer these questions: 1) Why might you expect the interior leaves to have more stomata than exterior leaves? 2) Why might you expect exterior leaves to have more stomata then interior leaves? 3) Why might you expect there to be no difference between the numbers of stomata? 4) Your answers to the questions above provide you with a total of three alternative hypotheses, each of which makes a different prediction. Which of the alternative hypotheses you developed above do you think is most likely to be true? Method: 1. Working in groups of 2, take 1 leaf from the each of the groups (interior and exterior). 2. Paint a small portion (<1 cm 2 ) of the bottom of each leaf with nail polish. Do not paint over a main vein of the leaf (usually running down the center) to collect your sample; paint only the flattest part of the leaf. 3. Allow Nail Polish to dry for approximately 3 minutes. 4. Place a small piece of clear tape (ex. packing tape) over the nail polish on one leaf and then gently peel it off: This will make an impression of the leaf surface. (Try not to touch the sticky part of the tape with your fingers.) 5. Tape the impressions to a single clean slide, trimming excess with scissors if needed. You should be able to easily fit 2 impressions on a single slide. Label each impression. 6. Focus the microscope and make sure you can identify a stoma. To clearly view the stomata, you will need to use the minimum amount of light possible; adjust the rheostat on the lamp and the iris diaphragm on the condenser to restrict incoming light, maximizing contrast. Randomly select an area and count all stomata visible in a single field of view at 400X power. PLEASE NOTE: if you are over a large vein, there will be no stomata. Ensure that you are not including a vein in your field of view, 3

4 as this will dramatically reduce the number of stomata and skew your data. 7. Repeat step 6 for 3 randomly selected fields of view for each leaf. 8. Calculate the average this is the average number of stomata per field of view. As long as all students use the same power, this gives a rough measure of stomata density, measured as stomata per field of view. An actual measure of density would be expressed as Stomata per cm 2. Cleanup: 1. Strip off the tape from the slide and discard; the tape should have the impression of the leaf attached. 2. Clean the glass slide and return to the box; these slides are recycled, not discarded. Formal Laboratory Research Report. As listed in the syllabus and discussed on the first day of class each of you will be involved in writing scientific research report. You will be given instructions on the exact format for that report and what is to be included at a later date. The report will be based on an experiment you design based on stomatal density (i.e. the experiment you just performed). Observation: The density of stomata varies on different leaves. The above observation requires the formation of an observation based question and hypothesis: What factors might affect the distribution and density (number of stomata in a given area) of stomata on leaves? The following are some possible questions: A) Are stomata denser on the top or bottom surfaces of leaves? C) Are stomata found at different densities on larger versus smaller leaves? D) Are stomata found at different concentrations on different colored leaves? As a group you will formulate a question and a hypothesis that you would like to test. You may choose one of the questions above or choose one of your own (with your instructors approval). The experiment will be conducted during week 7 (May 11/12). Answer the questions below about the experiment you are going to perform. 4

5 BIOL100 Laboratory Assignment 3: Analysis of Stomata/Experimental Design Name: Answer the following questions regarding the experiment you just performed in class. 1. What was the QUESTION being asked? 2. Clearly state your HYPOTHESIS. 3. What is the DEPENDENT VARIABLE? 4. What is the INDEPENDENT VARIABLE? 5. Clearly state your PREDICTION. 6. Present the results in form of a table. (Go back to the first lab handout if you need help!) 7. What is your interpretation of the results? (Do you reject or fail to reject your hypothesis?) 5

6 Answer the following questions regarding the experiment you plan to perform for your group project. 1. What is the QUESTION being asked? 2. Clearly state your HYPOTHESIS. 3. What idea/concepts lead you to this hypothesis? 4. Clearly state your PREDICTION. 6

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