BIOL 128 Labs

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Pre-lab Activities

Read pages 106-109, 624, 628-629, 642-649 in your text (Reece et. al., 2009. Biology concepts and connections. 7th ed. Pearson Education, San Francisco.). Answer the following questions in your personal electronic lab notebook (don't post answers to your lab section notebook) prior to attending lab during the week of 19 September.

Answers must be posted to your electronic lab notebook at least 1 hour before the start of your lab to be admitted into the lab.

1. What is the purpose of photosynthesis?
2. What is the overall chemical equation for photosynthesis?
3. Where does the water come from that is used in the photosynthetic process?
4. How does water move through a plant?

Overview

The following experiments is derived from Grant, B.W. and Vatnick, I. 2004. Environmental correlates of leaf stomata density. Teaching Issues and Experiments in Ecology. Vol. 1.

Objectives

By the end of this two-week lab, students should be able to:

Explain the main functions of stomata in plants

Explain how and why stomata density may differ among leaves

Define what is meant by a "scientifically-oriented" question

Devise a study to test how an environmental factor affects stomata density in trees/shrubs

Create leaf impressions to observe stomata using a standard protocol

Use a microscope properly to identify and count stomata on a leaf surface

Interpret data about stomata density and draw conclusions

Reading

Stomata Study (this web site)

Textbook: see above

Schedule

Procedure

Your groups will define a testable question to investigate dealing with the effect of an environmental variable on the density of stomata in Mountain Laurel leaves. Each group will determine how to study the question, select leaves to collect, count stomata, and interpret the results. The methods for how to obtain leaf impressions are included in this handout.

Your group should answer the following before collecting leaves. Discuss any questions you have with your instructor:

1. How will you collect leaves to answer the question? The most important thing to think about is how you will try and keep all variables the same except the one that is being tested. From where on the tree will you collect leaves? Note that it is recommended that you should collect and measure at least 12 different leaves (replicates) from each treatment type. Why are replicates important?

2. What is your hypothesis (prediction) for the outcome of your study? Try to come up with more than one. Explain your reasoning for each. Draw one clearly labeled figure of hypothetical data that would support one of your hypotheses (i.e. what would a graph look like if one of your hypotheses were supported?).

Trees to be Sampled

Mountain Laurel.

Each lab section has been assigned to collect leaves from a specific patch of Mountain Laurel on campus. Please review the images below to determine where your lab section (e.g., A, B, C, D, F, G, or H) should obtain their samples.

Figure 1. Aerial photograph of the WSU campus with the locations of Mountain Laurel indicated by light green blocks and the assigned sampling patches for each lab section identified in red. We are currently in W215 denoted by the red X.

Figure 2. Topographic map of the WSU campus with the locations of Mountain Laurel indicated by light green blocks and the assigned sampling patches for each lab section identified in red. We are currently in W215 denoted by the red X.

Introduction

Leaf stomata are the principal means of gas exchange in vascular plants. Stomata are small pores, typically on the undersides of leaves, that are opened or closed under the control of cells called guard cells. When open, stomata allow CO₂ to enter the leaf for synthesis of glucose (sugar), and also allow for water, H₂O, and free oxygen, O₂, to escape.

Stomata viewed at 400x in nail polish impression from leaf underside. © Marc Brodkin. 2000.

In addition to opening and closing the stomata (stomata behavior), plants may exert control over gas exchange by varying stomata density in new leaves when they are produced (such as in the spring or summer). The more stomata per unit area (stomata density) the more CO₂ can be taken up, and the more water can be released. Thus, higher stomata density can increase a plant’s potential for behavioral control over water loss rate and CO₂ uptake.

But why, you might ask, might it be beneficial (or adaptive) for a plant to control its rates of water loss and CO₂ uptake? One answer can be found in the sun. The plant cell "machinery" that is responsible for photosynthesis functions well over a narrow range of temperatures. When heated, cytochromes, pigments, and membranes critical to the process of photosynthesis denature (i.e., they cook). To avoid this, an individual plant may open its stomata, allowing water to evaporate, which lowers leaf temperature. Thus, one may hypothesize that leaves in the sun should have higher stomata density than do leaves in the shade - all else being equal.

On the other hand, if water is not available, such as under drought conditions, excessive evaporation might lead to desiccation (i.e., drying out) and an equally severe disruption of photosynthetic function. Thus, one might expect plant leaves exposed to drought conditions to have fewer stomata in sunlit environments.

The above discussion illustrates a very important concept in experimental biology – there are often alternative hypotheses to explain variation in nature. In this case, stomata density may increase or decrease in response to environmental variation in sunlight and water availability.

Note that since you will not be measuring sunlight or water availability you should use caution in how you word your acceptance or rejection of your hypothesis for your plants.

Methods

Obtaining Leaves

Remove a leaf from a tree/shrub and place it into a labeled paper bag. Write down as much information about your samples as you think is important. Consider the following:

1. Group number
2. Group member names
3. Location of leaf on tree (e.g., north, south, east, west; lower branches, upper branches; etc.)
4. Tree species (Mountain Laurel)
5. Leaf Exposure (sun or shade)
6. Tree Number (1 if it is the first tree of this species, 2 if second, etc.)
7. Leaf Number (1-12)
8. Optional: Latitude (from portable GPS)
9. Optional: Longitude (from portable GPS)
10. Repeat for all additional leaves sampled.

Obtaining Stomata Impressions

1. Obtain the leaf upon which you wish to census stomata.
2. On the side you wish to census stomata (typically the leaf underside) paint a rather thick swath of clear nail polish.
3. After the nail polish has dried (several minutes), obtain a square of VERY CLEAR tape (such as package sealing tape, but do NOT use scotch tape). Stick your tape piece to the area that contains the dried nail polish swath.
4. GENTLY, peel your nail polish swath from the leaf completely. You will see a cloudy impression of the leaf surface now attached to your tape piece (hereafter referred to as your "leaf impression").
5. Tape your leaf impression to a VERY CLEAN microscope slide and use scissors to cut off the excess tape.
6. Use a pen and write some sort of unique ID code directly on the slide. Write this same code on the data sheet.
7. Place the leaf back in the bag and place the bag in the plant press (optional).
8. Focus your leaf impression under at least 100x power and observe the stomata (see
   image above). You may not get to this part until the second lab period.
9. Search around on your impression to find an area that subjectively appears to have a high density of stomata. That is, move the slide around until the field of view is away from the edge of the impression and so that there are no dirt blobs, no thumbprints, no damaged areas, and no big leaf vein impressions in view.
10. Count all stomata you see, divide that number by the field of view area ^*, and record the value neatly on the data sheet (see link below).
11. Repeat the previous two steps three times, and the highest number of the three will be your one datum from this impression.
12. Repeat all steps above for at least 12 different leaf impressions (e.g., 12 different leaves) in each treatment group.

^* Your instructor will demonstrate the use of a stage micrometer so that you may convert your data from units of stomata number per field of view at 100x to units of stomata per mm².

Recording Data

1. Use the attached datasheet (see below) to document all the information that you collected about your leaves, including the stomata density that you calculated.
2. Post you data to the Lab Notebook for your lab section.
3. Alternatively, you can aggregate your data in an online Stomatal Density web site.

Data Analysis

Your instructor will introduce some very basic analysis techniques to be used in the analysis of the stomatal density data. Please read the resources in the navigation bar at the left (e.g., R Commander, Graphing with Excel, and Computing with Excel) to get an overview of data analysis techniques. Also, please "Statistics with stomata data".

Project Report

Title

A title should convey to your audience both the general topic of interest that you will address as well as something specific about your particular research.

Introduction

Provide a brief description of the context of the lab project. What are you studying, how do stomata work, etc. This can be about a paragraph for this project and you can cite this handout as a reference.

Be sure to include the following:

1. The specific question that you attempted to answer. Remember that a good question is often in the form of: "What is the effect of X on Y?
2. Your hypothesis (prediction) for what the answer could be. You will probably have more than one in this lab.
3. Reasoning for your hypothesis. Why do you expect one answer or another?

Methods

Provide a description of how you went about answering your question. NOTE: You don’t need to repeat the stomata methods handout, but you must refer to it. Provide a description of how you addressed your group's particular question.

Results

This section should be a written description of the data that you documented. You should provide mean stomata density for each of your treatment groups as well as present the variation in the data. Include at least one table of data and one graph of your results, each having its own figure caption. Be sure to label all axes and include units on your graph. All of this should be computer generated. NOTE: This is NOT the place to interpret the meaning of your data. Save interpretation for the discussion section.

Discussion

This is the place to interpret the meaning of your results. Write this in paragraph form and address the following:

• Do the data support or reject your hypotheses? Discuss.
• What aspects of your study make you confident in the results?
• What aspects of your study make you question the reliability of the results?
• What additional studies could you do to better answer your question?

Format

Double space the text and use 12 point font. How long should it be? Enough to fully address the questions and communicate your findings thoroughly. You will have to make a judgment about what constitutes appropriate effort and quality, however 2 pages of text would be meager, 10 pages would be too much!

Assessment of report

A = Exceptional report, all requirements of a “B” are met and:

Text clearly demonstrates an understanding of the functions of stomata in plants and how stomata density might be influenced by environmental variables.

Discussion carefully evaluates the reliability of the study and offers alternative interpretations that are reasonable.

B = Good report, all components are present.

All guidelines are fully met.

All tables and graphs neatly constructed, graphs have labeled axes and units.

Mean values and variability of data displayed appropriately.

Text clearly describes the patterns apparent in the data tables and graphs.

Discussion evaluates whether the hypotheses are supported or rejected.

Discussion addresses the reliability of the study, including possible errors.

Text is clearly written, with very few or no errors in grammar or spelling.

C = Fair report, all or most components are present but errors occur in a few.

D = Incomplete or poor report, several components are missing, have substantial errors, and/or are not acceptable.

F = Absent or unacceptable report.

NOTE: Pluses and minuses are used to modify the above rubric.