Introduction (written for students):

      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 a pair of banana-shaped cells called guard cells (see figure above). When open, stomata allow CO2 to enter the leaf for synthesis of glucose, and also allow for water, H2O, and free oxygen, O2, to escape. In addition to opening and closing the stomata (stomata behavior), plants may exert control over their gas exchange rates 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 greatly amplify the potential for behavioral control over water loss rate and CO2 uptake.

red maple leaf

stomata viewed at 400x in nail polish
impression from leaf underside
© Marc Brodkin, 2000
full size image

       But why, you might ask, might it be adaptive for a plant to control its rates of water loss and CO2 uptake? One answer can be found in the sun. Generally, plant photosynthetic apparati are only designed to function well over a rather narrow range of temperatures. When heated, cytochromes, pigments, and membranes critical to phosphorylation and carbon fixation rapidly denature (i.e., they cook). To avoid this, an individual plant may open its stomata and evaporate water which will lower the 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.

       But, on the other hand, if water is not available, such as under drought conditions, excessive evaporation might lead to desiccation 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.



Materials and Methods (written for faculty):

Study Sites

       Plant samples for this lab are to be collected from plants on campus within a few minutes walking distance of class. Despite that Widener University is in urban Chester, PA, and that we do this lab in mid-winter, it is not a problem for students to find plants with green leaves for their studies. This includes ornamental evergreen ground cover plants, shrubs, and trees (such as grasses and weedy perennial forbs, hollys, yews, and conifers). The activity does not work well on dried plant material, because it is a bit tricky, but not impossible, to obtain the stomata samples (see below).

Overview of Data Collection and Analysis Methods.

Week 1.

       Envision an environmental difference that might affect stomata density and formulate an hypothesis about which way you would expect stomata density to vary and WHY. Discuss these in detail with your lab instructor PRIOR to taking any data. Next, decide on a place anywhere within about 10 minutes walking time where you intend to collect leaf samples in the environmental types of interest, and go and get them. Bring your leaf samples back to lab and count their stomata densities (see Methods for Obtaining Stomata Impressions below). Lastly, submit your co-authored research proposal with your partner. This document should fit on one page and should contain three sections according to the Guidelines for Stomata Research Proposal below.

Week 2.

       Next week, bring all of your data to class, finish counting stomata (if you have not already have done so), and your instructor will help you with the statistical analyses, and computer graphics generation of your stomata data to test your hypothesis (see Guidelines for Data Analysis below). In addition, you should begin to produce your oral and written reports which are due the following week.

Week 3.

       The entire lab period this week will be devoted to a symposium of presentations of your research results to your peers. You and your research partner will make a 12 minute oral report to your peers using visual aids (such as an overhead projector and/or video projector for a PowerPoint presentation, see Guidelines for Oral Presentations below). Also on this date, your co-authored written report is due (see Guidelines for Written Reports below) as well as your disk copy of your data (see Guidelines for Data Management below). Your individually written critical review of this multiweek lab activity is due the following week (see Guidelines for Reflective Reviews of Lab Activities below).


Methods for Obtaining Stomata Impressions.


Detailed Guidelines Are Available in the Links Below.



Questions for Further Thought and Discussion:

  1. How exactly do stomata open and close? How do guard cells work? Specifically explain the roles of ions and any plant hormones.

  2. As you will see in this lab activity, plants confronted with different environmental conditions vary the number of stomata per unit area by quite a lot. Yet, in theory the same result due to having more stomata could be attained by simply having bigger stomata with no difference in stomata number - however, plants vary stomata number and not stomata size. Why? Given your answer to question #1, why might plants vary stomata density rather than stomata size?

  3. Why might it be adaptive for stomata to occur mostly (if not entirely) on the undersides of leaves? What plants show the reverse pattern for which stomata are only on the upper leaf surface?

  4. Some cacti thrive in some of the hottest deserts on earth where water is extremely scarce for most of the year. To deal with the scarcity of water, cacti have evolved an unusual set of adaptations including a remarkable capacity to soak up water into fleshy stems when it rains and hold onto this water during drought. One way cacti have to hold onto water is to ONLY open their stomata at night when it is cooler and more humid. However, if CO2 is only allowed into these plants at night how are cacti able to synthesize sugar with it via photosynthesis during the day many hours later?

  5. Diagram and describe some of the physical aspects of leaf design that would reduce water loss in a dry environment. Specifically address how leaf size, shape, orientation to the sun, color, fuzziness, thickness, water-proofing, stomata design, stomata density, etc., might vary from a wet to a dry environment.

  6. Climate change due to the rapidly increasing levels of greenhouse gases (particularly CO2) in our atmosphere is a serious current global concern. How might stomata density serve as a bioindicator for monitoring the response of plants to changes in greenhouse gas concentrations in the future? (Hint: what do the data say for how stomata density varies with CO2 concentration?)

  7. As a related question to the one above, how might stomata density serve as a bioindicator for estimating CO2 concentrations in the past (paleoclimates)? Find and and summarize two instances of research on this topic in the literature. (Hint: see references below, [F. Wagner’s in particular].)

  8. Given your knowledge of the tradeoffs plant leaves face between carbon dioxide uptake and evaporative water loss, speculate upon the “behavioral” features in stomata you would expect to evolve in plants adapted to dry environments with variable and unpredictable water supply. Research your answer and provide support (with references) for any mechanism(s) that has(have) been identified as a way for stomata to “behave” in response to humidity and water availability.

  9. Among bryophytes, stomata are restricted to the sporophyte life stage (found in mosses and some hornworts). Why? Why might it be adaptive for only the sporophyte and not the gametophyte stage in the life cycle to possess stomata?

  10. Plant tissues are extremely sensitive to damage by the powerfully oxidizing effects of ozone (O3. What effects would you expect this to have on urban-rural gradients in stomata density, and how would this effect interact with other urban-rural gradient effects on plants? What are the implications of these issues to urban agriculture?

  11. Many bacteria and fungi that are parasitic of plants face the daunting task of finding and infecting a new host by airborne spore dispersal followed by germinating upon and then penetrating the leaf surface of their host. What are some of the specific adaptations possessed by some of these parasites to gain access to leaf tissue by entering through stomata thereby evading the plant leaf cuticle? (Hint: search on rust fungi, Uromyces.)

  12. What role do stomata play in the solution to the problem of getting water up to the leaves from the roots of woody plants (which for a tall tree such as a redwood can be over 350 feet up!)? Using a little system diagram, sketch and describe the role of stomata in water uptake.

*** Note: Answers to many of these questions and numerous other comments by the contributing author can be found in the "NOTES TO FACULTY" page.



References and Links:



Tools for Assessment of Student Learning Outcomes:

       Below is a graphic to illustrate the relationships among the laboratory objectives, activities, and assessment instruments. Click on this graphic to see the complete system diagram for this lab activity.

click to view *.pdf system diagram of Objectives/ Activities/ Assessments for this lab.

       Below we explain how we have designed these assessment instruments for our classes. However, instructors should modify, omit, and/or add their own assessment instruments to meet the needs of your students. Keep in mind that:


Scoring Rubric for Questions for Thought.

       We ask our students to submit written answers to any 4 of the questions. Students should use their texts or any other written references to answer these questions - but they must cite the complete and exact source of any text, web, or other outside material that they used. We strongly recommend that you read out loud to your students your course policy on plagiarism (which should be in your syllabus), and if you do not have one GET ONE!

       Each citation of a research article or book should have: Author(s). Year. Title of paper. Journal. Volume: Pages. Each citation of an internet resource page should have: Author(s) if known. Specific Title of the Page. General Title of “Home” page/ Organization Name for the Site. Full “http” address. Date of Your Download.

       Answers should be word processed, single spaced, 12 point, 1” margins, minimum 1/2 page in length, and in some cases including a well-documented Table or Figure.

       Our Scoring Rubric for Answers to Questions can be found below.


Scoring Rubric for Stomata Research Proposals.

       As described in the "Guidelines for Stomata Research Proposal" there are three parts to this assignment: Introduction, Methods, and Possible Results. In addition, students must generate an hypothetical graph of what their results would look like that would show an answer to their hypothesis about stomata variation.

       Our Scoring Rubrics for Stomata Research Proposals can be found below.


Scoring Rubrics for Written Reports.

       As is described in the “Guidelines for Written Reports”, there are seven sections for your reports: Abstract, Introduction, Methods, Results, Discussion, Literature Cited (if any), and an Appendix. The maximum number of points for each section varies from 5-15 points with a total of 40 points.

       Our Scoring Rubrics for Written Reports for each section closely follow these guidelines and can be found below.

       In our experience, for the students’ first drafts it is more consistent on our part to read and apply the scoring rubrics for all of the students’ Introductions, all Methods, all Results, all Discussions, and then all Abstracts, rather than read each report all the way through and have to re-think out each rubric. Typically, we have about 12-16 students in each lab section, and two lab sections, which translates to 12-16 papers.

       After we return the first drafts of our students’ written reports, our students have two weeks to revise and re-submit (and many are sent to Widener’s Writing Center for consultation sessions). The scoring rubrics above apply equally to their first drafts and revisions, and students base their revisions on their section scores and our miscellaneous written comments directly on their manuscripts. We also ask them to turn back in their original submission at the same time as their revision, which although it introduces some bias in our grading of their revision, such bias is offset by our ability to compare their old and new versions and thereby quickly perceive their effort and allocation in their revision. In addition, we feel it is not entirely fair to them to “mark them down” for major new problems we discover in their revision that we should have caught in their first draft (however, this policy does not apply to spelling or grammatical errors that should have been caught by better proofreading on their part to begin with).


Scoring Rubric for Oral Presentation.

       As described in the “Guidelines for Oral Presentations", there are four sections for your reports: Introduction, Methods, Results and Specific Discussion, and General Discussion. The maximum number of points for each section is 10 points with a total of 40 points. Our Scoring Rubrics for Oral Presentations for each section closely follow these guidelines and can be found below.

       In addition, during the Stomata Research Symposium, students should be rewarded for participation. This can be problematic if your expectations are not made crystal clear to then beforehand. We offer 10 points max for this facet of the activity, which represents 5% of the total grade. Our Scoring Rubrics for Symposium Participation can also be found below.


Scoring Rubric for Assessing Data Management.

       As described in the “Guidelines for Data Management”, there are three computer files that constitute this part of the assignment: the manuscript, the presentation/graphics, and the spreadsheet data files. Our Scoring Rubric follows these “Guidelines” closely:


Scoring Rubric for Assessing the Student Reflective Reviews of the Lab Activities.

       As described in the "Guidelines for Reflective Reviews of Lab Activities", each student submits an individual review. Our Scoring Rubrics for Critical Reviews of the Lab follows these guidelines closely and can be found below.


Summary Table of Point Totals and Links to Detailed Scoring Rubrics:.

Rubric for Answers to Questions 40 pointsindividual
Rubric for Stomata Research Proposals 20 pointsgroup
Scoring Rubric for Oral Presentations 40 pointsgroup
Scoring Rubrics for Written Reports (1st version) 40 pointsgroup
Scoring Rubrics for Written Reports (revision) 40 pointsgroup
Scoring Rubric for Data Management 20 pointsgroup
Scoring Rubric for Symposium Participation 10 pointsindividual
Scoring Rubric for Reflective Reviews of Lab Activities 20 pointsindividual


230 points

75% group

              …which is 20% of the total course grade.

              In the extremely rare case that group participation is inequitable, or some other group cooperation issues arise, we will require individual submissions for the Written Report revision and Data Management. We has only been necessary a couple of times in the last 5 years.



Tools for Formative Evaluation of this Experiment:

       In our "Guidelines for Reflective Reviews of Lab Activities" we describe an activity that collects information from students to evaluate the general design and specific events that occurred during this lab activity. This is a very useful assignment, and in our experience students enjoy being constructive participants in the design of their curriculum. Many comments are insightful and very helpful in our year-to-year revisions. We use a Scoring Rubric for Reflective Reviews of Lab Activities to grade these on a 20 point scale.

       Extensive notes on how to conduct formative evaluation are in the Teaching Resources sector of TIEE in an ESSAY ON EVALUATION.