Leaf Stomata as Bioindicators of Environmental Change

By Steve Case

Introduction

Leaf stomatal densities can be determined by a simple laboratory technique and yet have wide application in understanding environmental change. Several researchers have evidence which indicates that stomatal densities change in response to changing atmospheric levels of carbon dioxide. Stomata may also vary in response to the amount of annual rainfall in different localities. Because this investigation involves climatic variations, it requires that many geographically dispersed sites collect and share data.

In this experiment, leaves from two species of trees will be collected and the stomatal index on the upper and lower epidermis of each leaf will be determined.

Background Information

Leaves are the primary photosynthetic organs of most plants. Leaf surfaces are equipped with small openings or pores called stomata which allows carbon dioxide to enter the leaf and oxygen to escape to facilitate photosynthesis. In addition, water is lost through stomata during a process called transpiration. It is estimated that approximately 99% of the water absorbed by the roots of the plant is lost by the leaves in transpiration.

The number of stomata on leaf surfaces varies widely among different species of plants. Generally, the lower epidermis of the leaf tends to have a higher total than the upper surface. Botanists have made stomatal counts for many species. Their data indicates that the number of stomata may vary from zero on the upper epidermis of an apple leaf to as high as 58,140 per square centimeter on the lower epidermis of black oak leaves.

Stomatal densities can be determined by a simple laboratory technique and yet have wide application in understanding environmental change. Several researchers have evidence which indicates that stomatal densities change in response to changing atmospheric levels of carbon dioxide. Stomata may also vary in response to the amount of annual rainfall in different localities. Because this investigation involves climatic variations, it requires that many geographically dispersed sites collect and share data.

Leaves from two species of trees will be collected and the stomatal index on the upper and lower epidermis of each leaf will be determined. The number of stomata on a leaf will be determined using clear nail polish, tape, and a glass slide. The number of stomata and epidermal cells will be counted under high power (400X). A stomatal index will be determined for the leaf. The Stomatal Index (I) =[S / (E+S)] * 100, where S is the number of stomata per unit area, and E is the number of epidermal cells per same unit area. The data form individual leaves will be combined and a mean will be calculated for each tree sampled. This data will be shared with other geographically dispersed sites that are studying these trees.

Procedure

Background reading and material on the structure and function of stomata in leaves should be readily available. Topics to be explored should include:

• Leaf structure and function.
• Stomata's role in carbon dioxide intake.
• Stomata's role in oxygen release.
• Stomata's role in water release.
• The balance that the plant must achieve between water loss and carbon dioxide uptake.

Materials

1. Clear Nail Polish
2. clear cellophane tape
3. a glass slide
4. microscope

Protocol for the Activity

1. Students should be divided into teams of two. 10 leaves from each of 10 trees should be collect should be collected from Aspen (scientific name) trees and from Eastern Cottonwood (scientific name) trees if these trees grow locally. Data about the location of the tree should be recorded on the data form. Leaves should be collected from the south side of the tree, from approximately shoulder height, and from the same position on the twig. Leaves should be labeled with a tree number and a leaf number. A small piece of masking tape on the point of the leaf works well.
   
   
2. Each leaf should be painted with clear fingernail polish between the 2nd and 3rd vein on each surface (figure 1). An oval spot approximately one half by one centimeter is sufficient to provide enough leaf surface to make the count.
   
   
3. Allow the fingernail polish to dry completely.
   
   
4. Firmly press a short strip of clear Scotch tape (not frosted) over the dried nail polish on the lower epidermis. Carefully peel the tape from the leaf and affix it to a clean microscope slide. Place the tape toward one end of the slide perpendicular to the lang axis of the slide (cross-wise). Label the tape with a sharpie pen to identify it as being from the lower epidermis of the leaf and the tree number the leaf came from. Example Tree 1, Leaf 1, Lower epidermis would be labeled on the slide as T1L1L. Repeat this procedure for the upper epidermis of the same leaf, placing the tape on the other end of the same slide. This tape would be labeled T1L1U.
   
   
5. Repeat this procedure for each of the collected leaves. Be sure to label each leaf and each slide. Organization of data is critical.
   
   
6. Trace the leaves on a piece of acetate with 1mm grids mark on it and measure the leaf surface area.
   
   
7. Place the leaves in a plant press. After drying, remove the leaves and store for possible future reference.
   
   
8. Each group of two students should count the stomata and the epidural cells on the leaf casts of two leaves. Count the stomata on the leaf casts at high power, about 400X although the exact magnification is not important. To successfully count the number of stomata in a field of view you will need to focus, using the fine adjustment, up and down to bring different planes into focus. Both members of the group should count the number of leaf stomata and the number of epidural cells in the first field of view. They should then compare their counts for the same field of view to insure the accuracy of their count. If there are differences then the two counters should discuss which cells they counted and which ones they did not count. Some stomata and epidural cells will be partially in or out of the field of view. You can adjust to this by dividing the field of view into imaginary quarter segments. Include in your counts the stomata and epidural cells touching to edge of the field for the upper right and the lower left quarters. Do not include in your counts the stomata touching the edge of the field in the lower right and upper left quarter. Each group should count 5 randomly selected field of views per leaf cast and enter the counts on to the data tables.
   
   
9. Complete the data form with all data, Latitude, Longitude, rainfall this year from January to September 1, and the stomata and epidural cell counts.
   
   
10. Send the data, using the form in the Access Excellence Online Research area. If you do not have access to the World Wide Web, request the form on a floppy disk and send your data on the 3.5 inch floppy disk to Steve Case at the address below.
    
    
11. All collect data will be available in the Online Research area. Students should begin reviewing and interpreting data. Data analysis should be posted in the Online Research area.
    
    

At this point the students should be lead in a discussion of environmental factors that may cause a variation in the number of stomata on a leaf's surface. This discussion will lead to individual project ideas. The following are some suggestions that may get students thinking.

• What is the normal variation found on a plant?
  
  
• The age of the tree and the number of stomata found on the leaf.
  
  
• The side, compass direction, of the tree the leaf comes from and the number of stomata found on the leaf.
  
  
• The direction of the slope the tree is growing on and the number of stomata found on the leaf.
  
  
• The difference in stomata on leaves grown in a carbon dioxide enriched environment to those grown under normal atmospheric conditions.
  
  
• Is there a clinal variation along a rainfall gradient.
  
  
• Study the variation in numbers of stomata that occur between plants growing in similar habitats but using different photosynthetic pathways, C3, C4, and CAM.
  
  

As the discussion proceeds the students should be thinking of ways to measure, test, and/or simulate these environmental conditions. Individual research projects should be sent in for posting on the Web. These projects will increase our understanding of leaf stomata as bioindicators.

Additional References

The original technique for stomata counting came from "The American Biology Teacher", January 1990. "Is there a Correlation Between Rainfall Amounts and the Number of Stomata in Cottonwood Leaves". Neill, Robert L., David M. Neill, Bernard F. Frye.

Stomatal Index - The Botanical Review Vol. 40, January - March 1974

Stomatal numbers are sensitive to increases in CO2 from pre-industrial levels, F. I. Woodard, Nature June 1987 : 617-619.

Van Der Burgh, Johan , Jenk Visscher, David Dilcher, Wolfram M. Kurschner. "Paleoatmospheric Signatures in Neogene Fossil Leaves." Science, Vol. 260, June 18 1993, 1788-1790.

Garbutt, K., W. E. Williams, and F. A. Bazzaz (1990) "Analysis of differential response of five annuals to elevated CO2 during growth. " Ecology 71(3).1185-1194.

Bazzaz, F. A. "The Response of Natural Ecosystems to the Rising Global CO2 Levels." Annual Review of Ecology Systematic, 1990, 21: 167-196.

Penuelas, Josep, Riser Matamala. (1990) "Changes in N and S Leaf Content, Stomatal Density and Specific leaf Area of 14 Plant Species during the Last Three Centuries of CO2 Increase." Journal of Experimental Botany 41.1119-1124.

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