Unformatted text preview: Lab 4: Measuring Stomatal Density Introduction Plants exchange gas and water with the atmosphere via specialized structures called ‘stomata’ ‘stomata’ means mouth in Greek. This exchange of gas with the atmosphere is necessary for the uptake of carbon dioxide, the source of carbon used in photosynthesis, and for the evaporation of water which is the driving force behind the process of transpiration. Stomata are microscopic pores on the surface plant leaves. They are formed by a pair of specialized epidermal cells called guard cells, which act as a turgor-pressure driven valve that open and close the aperture of the stomatal pore (i.e. the size of the opening), and thereby regulate the rate of gas exchange (CO2 uptake and water loss), in response to environmental conditions. In today’s lab, you will be taking varnish impressions of the epidermis of plant leaves that can be viewed under a microscope. You will be able to count the number of stomata and to calculate stomatal density. You will use these data to test hypotheses about stomatal placement and density. Functions Most of the plant epidermis (or “skin”) is composed of interlocking epidermal pavement cells covered with a waxy cuticle, that serve to protect the plant from desiccation. In order to facilitate the regulated exchange of gases between the plant and the atmosphere, stomatal pores are distributed across the surface of photosynthetic tissues like leaves and green stems. Stomata are most commonly found on the leaves, but they can also occur on the inflorescences of gymnosperms and angiosperms, fruits, herbaceous stems, petioles and tendrils. In many angiosperms and gymnosperms, stomata are found on both the adaxial (upper, proximal to plant the axis) and abaxial (lower, distal to the plant axis) leaf surfaces. The number and density of stomata are extremely variable even within a single species. (Ref: http://www.saps.org.uk/secondary/teaching-resources/299-measuring-stomatal-density) Experiment Requirements: Fresh plant leaves, microscope with micrometer, nail varnish, sticky tape, microscope slides. Protocol: 1. Identify the upper and lower surfaces of a leaf. 2. Spread a thin layer (this layer must be VERY thin) of nail varnish on the top side of the leaf. Let the nail varnish dry completely. 3. Press clear sticky tape over the dry varnish and press down to make a good connection with the nail polish. Peel the tape from the leaf surface. 4. Stick the impression to a clean slide. 5. Repeat the same procedure on the bottom surface of the leaf and place it on a different slide. 6. You may have to repeat this process a few times until you have a good specimen for examining. 7. Count the number of stomata in different fields of view (the area of the slide visible through the microscope at any one time). 8. Choose a magnification that gives a number of stomata you can keep track of when counting – too many cannot be accurately counted. 9. Count the stomata in at least 5 field of view (FOV) on each plant surface. 10. Record the results in the table given below. 11. Calculate the stomatal density for each FOV and calculate the average density for each surface. 12. Repeat this protocol so that you have data for both a monocot and dicot. Stomatal Density= Number of stomata in the FOV Area (mm2) To identify the area of the FOV make use of the following table Area (mm2) Magnification 40X 100X 400X 1000X 5 2 0.45 0.180 Magnification Monocot Abaxial surface Adaxial surface Dicot Abaxial surface Adaxial surface Calculations: 1 Stomatal count 2 3 4 5 Average Stomatal Density Questions: 1. Is the stomatal density on the top and bottom side of your leaf the same or different? What are the implications of this distribution for the rate of water loss (transpiration)? for carbon uptake (for photosynthesis)? What do the terms abaxial and adaxial mean? How could you calculate the total number of stomata for an entire leaf? (5pt) 2. What do you notice about the arrangement of stomata in grasses (monocotyledonous plants) compared to their arrangement in dicotyledonous plants? Were the stomata evenly distributed throughout the field of view? What are the functional consequences of this distribution? (3pt) 3. Every day, stomata open and close with a regular rhythm. The figure shows the times of day at which stomata are open and closed (line 1 C3 and C4 plants; line 2, CAM plants). What role do you think these rhythm play in regulating transpiration? Why is the rhythm inverted for CAM plants? (3pt) ...
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Stomata are responsible for allowing gas exchange between the inside of the leaf and the atmosphere. Stoma is the singular and stomata is the plural form. When viewed with a microscope, they often look like coffee beans. There are more than 32 stomata in the image of the Western Sword Fern leaf, to the right. Carbon dioxide (CO₂), oxygen (O₂) and water (H₂0) commonly move in or out via the stomata.
While gas exchange occurs, carbon (C) stays inside the leaf as a building block for the plant. Often, stomata are open during the day when photosynthesis is taking place and closed at night when it stops. By doing so, plants don’t lose too much water. If the stomata are open, gasses diffuse from areas of higher concentration to lower concentration. If photosynthesis is occurring the CO₂ higher concentration is outside the leaf. For H₂0 and O₂ the area of higher concentration is inside the leaf. This process is depicted in an animated clip prepared by Carnegie Institute for Science.
A common misconception that students have is that the stoma’s size can keep out large molecules and just let in the little molecules like CO₂ and H₂0. A stoma is on the order of 10-6m, while a CO₂ molecule is on the order of 10-10m. If we pretend that a stoma opening is one meter across, then the CO₂ molecule would be one tenth of a millimeter in size.
Each stoma is made of two guard cells. When these guard cells are swollen with water, they create an opening between them, the stomatal pore. Gas exchange occurs via the pore. When the guard cells are flaccid they lay close together, thus closing the stomatal pore. Plants that are “dicots” have kidney shaped guard cells and plants that are “monocots” have dumbbell shaped guard cells.
Normally stomata open in the morning and close during the night. However, not all plants open their stomata during the day. Some plants such as cacti and succulent plants open their stomata at night and close them during the day, in order to prevent losing too much water.
Stomata are usually found on both the top and the bottom of a leaf. Many plants have more stomata on the underside of the leaf. However there are exceptions, monocots, like grasses, have similar numbers on both the top and the bottom. Plants whose leaves rest on the surface of the water, like water lilies, often have very few stomata on the wet underside of their leaves.
Stomata are not the only way for plants to exchanges gases with the air. Plant roots, stems, bark, and fruits have lenticels on their outer surface. These allow oxygen in and carbon dioxide out, as the plant respires. They do not open and close, the way that stomata do. Examples of lenticels are the little spots on pears and the horizontal stripes on cherry tree bark.
Scientists make prints of stomata in order to easily see the surface of a leaf under the microscope. This video shows the process that we outline below. If you want to make the stomata or the locations of the stomata a surprise for your students, do not show it to the students before they begin, as it contains spoilers. The video also incorrectly states that the cells from the surface of the leaf are pulled off. Instead the nail polish is removed from the surface of the leaf. It is an impression of the leaf surface. The nail polish is just like plaster poured into a footprint in sand.
Some leaves work better than others for making prints. We find that smooth, sturdy leaves work well. We run into difficulty if leaves are very delicate or are covered by lots of hair. For this reason we suggest that you try your leaves out first or let students know that the method may not work for every leaf. You can also try this method with dried leaves.