This piece introduces a simple technique for examining the stomata of leaves, in the context of changes in atmospheric composition. It is followed by a description of some recent research on plant responses to rising levels of carbon dioxide. The technique can be used in studying plant response to many kinds of stress.
The following procedures and commentary are from a GL network message by Brad Williamson and Steve Case, teachers at Olathe, KS.
"We now have examined hundreds of leaves for leaf stomata counts. The leaves that we have been using are mostly from the cottonwood (Populus deltoides). A limitation of the technique is that smooth, non-hairy leaves must be used. We have tried this technique with about 120 rookie students and have been able to have each student count the stomata on at least two leaves by the end of the period even though they share microscopes."
Clear plastic (cellophane) tape
clear nail polish
1. Collect your leaves. These can be dried or the leaves can actually be left on the tree and sampled live. We record the tree's identity, and also number the leaves tested.
2. Paint a 1 x 2 cm oval of clear fingernail polish on the leaf, avoiding ribbed veins. We test both the top of the leaves and the undersides, where most of the stomata are. We usually paint the top and bottom at the same time to speed up the process. Often we will paint 10 leaves at a time.
3. After the polish has dried it can be peeled off quite easily. Use clear cellophane tape to make a quick and foolproof mount as follows: Simply place the tape on the polish still on the leaf and lift the polish replica off of the leaf and tape to the slide. By placing the taped replicas perpendicular to the long axis of the slide we have been able to place up to 4 replicas on one slide. This greatly speeds the counting. It is surprising, but the tape's adhesive and obvious optical limitations don't seem to make much of a difference. Every teacher and most students express quite a bit of pure joy and amazement at their first view of stomata replicas.
4. We have been counting all of the stomata visible in one field of view at a magnification of 400x. We find that for most plants counting at 100x yields a count of about 100 to 200 stomata per view. This is too high a number for kids to keep track of by silent counting without a grid. By going to the higher magnification the counts are more manageable at about 12 to 30 stomata per field of view. We have been making 5 counts per replica but have found that 3 would be adequate. There is not a lot of variation on one replica. All counts should be converted to number of stomata per square mm to account for variation in microscopes.
5. In trying to determine the average count on a tree we have found that at least ten leaves need to be sampled because of the large amount of variation even in one tree. Of course this alone could make an excellent project. We have also tried to count the stomata on at least 10 trees in a given area to deal with the variation between trees.
Alfalfa might be an excellent indicator species since it has nearly global distribution. A study in Israel involving alfalfa demonstrated seasonal differences in stomatal counts as well as differences between irrigated and non-irrigated fields.
Teacher's Notes: Using this protocol in the class
Grade level: Grades 7-12
Summary of Activities: This activity is designed to stimulate student research. The number of stomata on a leaf can be easily determined using clear nail polish, tape, and a glass slide. The number of stomata are counted under high power (400X) and the stomatal density is calculated. The students develop research projects that investigate environmental factors that may affect the number of stomata on a leaf or the amount of time the stomata are open.
Rationale: Many times the lab work we do in science class is demonstration work. We discuss a principle in class and perform a laboratory "experiment" to illustrate that principle. The problem with this approach is that students assume we always know how experiments turn out. However, science is not based on a cookbook and the results are not known in advance. The joy and excitement of science is the exploration of the unknown. Students who participate in science discover this excitement.
Source: The original technique for stomata counting came from The American Biology Teacher, January 1990.
1) The structure and function of stomata in leaves.
Topics to cover:
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 must occur between water loss and carbon dioxide uptake.
2) Environmental factors that may cause a variation in the number of stomata on a leaf's surface. This discussion will lead to possible project ideas. The following are some suggestions that may get students thinking.
The age of the tree and the number of stomata found on the leaf.
The side 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.
Undertake a cline study to study stomata variations 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.
Proceeding to student research projects
As the discussion proceeds ,the students should be thinking of ways to measure, test, or simulate these environmental conditions. Remember these are only suggestions. The students will be most excited about projects that they generate.
Research projects may be conducted by the whole class, by teams, or by individuals. They need to think about their initial question, and about the data they will need to collect. The requirement of a final report should focus concern about keeping track of things, analysis, and presentation. Leave room for failure students can look at other research project reports as a way to think about their own ideas.
Carbon dioxide in the atmosphere
Carbon dioxide and plant response
Leaf Stomata background
Activity: Leaf stomata protocol and teachers notes
Carbon Dioxide in the atmosphere
Carbon dioxide (CO2) is a minor component of Earth's atmosphere. The present concentration is about 350 parts per million (ppm). This concentration, however, is notably higher than the concentration estimated from the last century in the 1850s, the figure was about 275 ppm. It is clear that the principal cause for the increase in atmospheric CO2 is the burning of fossil fuels (coal and petroleum products). (Fossil fuel combustion adds about 5.6 x 1015 grams (5.6 gigatons) of carbon to the air annually, while deforestation and related activities adds somewhere between 0.5 and 2.0 gigatons.)
Although CO2 is a trace gas in the atmosphere, the increase over the past century is cause for concern, for at least two reasons.
1. CO2 is a "greenhouse gas," one of the components of the air that is effective at absorbing solar radiation and releasing it as heat. This has been good for life, on the whole, since it has kept the average temperature of the Earth within limits tolerable to life. (If there were no greenhouse gases, the global average temperature would be around -18°C.) Scientists predict that the recent abrupt rise in CO2 is causing global warming, though the exact amount is debated. (For more about this, and ways to explore how the atmospheric system might behave under various possible conditions in the future, see the "Modelling" paper in this volume.)
2. Carbon is a fundamental component of all living things, and plants and other photosynthetic organisms are the principal agents that make carbon available to other organisms, by turning atmospheric CO2 into plant tissue and other products, through photosynthesis. Plants have evolved to function in the CO2-poor atmosphere that has obtained through geologic time, and the limited availability of CO2 is one major factor controlling the abundance of life. What happens if there is a lot more CO2 in a rather short time? Even if there is no resulting global warming, the increasing carbon-richness of the atmosphere might have important effects on the earth's vegetation, and thus on all ecosystems around the world.
Carbon dioxide and plant response
How might plants respond to a higher level of CO2? In the debates about CO2 emissions and global warming, policy makers have seized on a well-established fact of horticulture: for years, greenhouses have increased the level of CO2 as a fertilizer for many plants. The results have been larger, more productive plants, and some people hope that this is evidence of the way plants will respond to the global experiment the human race is taking part in. There are some considerations that make this hopeful view seem incomplete. In discussing some of these complications, we can see also what kinds of plant response we could be looking for in observation, modelling, and experimentation over the next couple of decades.
It should be noted that the effects vary from species to species, and that very few species have been studied. Another variable is that a certain proportion of plants (perhaps 25%) have a variant on the photosynthetic process that makes them more efficient users of both water and CO2; these plants are referred to as C4 plants, while most other plants have what is called the C3 pathway. The C4 plants show much less response to CO2 enrichment than do C3 plants.
Physiological effects. Researchers have reported several physiological responses to CO2 fertilization. These experiments control for other factors. The plants have the same, unlimited amounts of light, water, and other nutrients as the control specimens, so that the only difference between the experimental plants and the control plants is the amount of CO2 surrounding them as they grow.
Plants may grow faster, at least for a short period of time, though this apparently does not mean that the plant in the end is larger. They photosynthesize more rapidly, fixing more carbon. This results in more plant tissue, such as leaves, stem, and roots. Some plants use water more efficiently (measured as the ratio between the increase in dry weight per amount of water used). The number of stomata per unit area may decrease in the presence of more CO2 (see below). Finally, the chemical composition of the leaves changes (as well as other parts of the plant). The quantity most often studied is the ratio of carbon to nitrogen (C:N ratio) in the tissue. In all cases studied so far, the ratio has grown; that is, the proportion of nitrogen has shrunk.
These experimental findings have the following implications (among others):
1. In a natural setting, where CO2 is increasing but no one is providing the extra nitrogen, water, or other nutrients needed to accompany the extra growth, the increase in growth may be much less than reported from experimental studies. We can not, therefore, expect major increases in crop yields, for example, just because of CO2 enrichment. It is hard to predict just what the effects will be on wild plants.
2. Plants make sugar in the leaves during photosynthesis, but then the sugar ("photosynthate") gets deployed where the plant needs it. Some goes locally, some as stem, some as flower, some as root. If a plant has an opportunity to photosynthesize faster than usual, it will have more photosynthate to allocate. On the other hand, it will need more nutrients of other kinds, and a common response to such a need is to grow more roots, to forage for nitrogen, phosphorus, etc. This implies that in some plants at least we might get extra growth in the roots, rather than in other areas -- an important issue for farmers.
3. If there is a smaller proportion of nitrogen in leaves, that means that herbivores will be getting less protein per bite of leaf. This means that they will probably need to eat more leaves , not a cheerful prospect for the farmer!
Reproductive effects. Some plants produce larger seeds, fruits, or flowers under CO2 fertilization. Larger seeds might be good in some ways (giving the seedling a better start, for example), but might interfere with seed dispersal, or make the seed more attractive to seed predators. Many plants have flowered earlier when CO2 fertilized; in at least one case, flowering was delayed. In the wild, this might have unexpected repercussions, if it disrupts the timing of the plant's flowering in relation to the readiness of its pollinator. The plants might not be pollinated, and the pollinator might lack an important element in its annual cycle as well.
Community effects. If CO2 enrichment speeds growth in some species but not others, if it affects a plant's water-use efficiency and allocation of resources, including the size of fruits, and if it can affect the timing of flowering and other aspects of life-style, then we can expect that plant communities will change, perhaps radically. Plant competition for light, for water and other soil resources, and for space for new seedlings will change. Some studies have explored this in controlled conditions, but there is a lot of room for more experimentation.
Leaf stomata background
The stomata of leaves are the channels through which the plant interacts with the atmosphere directly: gases diffuse inward and outward, water is lost to the air. The stomata open and close in response to various stimuli and physiological states of the plant, including internal vs. external gas concentrations, water stress, heat stress, and pollutants. Because the stomata are so responsive, their behavior is a good focus for experimentation. Recent research has shown that, in addition to the openness of the stomata, another kind of plant response to stress is seen in the number of stomata per unit area of leaf surface. This is of interest because it is not a response to one stressful incident, but like the plant's phenology is a response to a combination of growing conditions over an extended period of time.
Usually, when speaking of stomatal responses to environmental stress, we refer to the opening or closing of the stomata. For example, when a plant is experiencing water-stress during drought, the stomata close part-way or completely, which slows or prevents transpiration. The same response occurs when the plant is surrounded by pollutants, such as smog (especially ozone) or SO2. All of these responses shield the plant from damage to some degree; at the same time, the closing of the stomata affects photosynthetic rates and other metabolic processes, since it limits or prevents the uptake of CO2, and also limits the loss of water through transpiration.
Recent work confirms that there are other effects that may result from changes in the climate, including changes in the concentration of atmospheric gases. In particular, the density of stomata on a leaf (that is, the number of stomata per square millimeter), may be responsive to CO2 levels. The first suggestive evidence that this might be so came from an analysis of ecotypes of plants that have a wide altitudinal spread. (An ecotype is a local variant of a species that is particularly well adapted to local conditions. A plant species with a wide range of possible habitats, such as apple (Malus) or tobacco (Nicotiana), may have ecotypes that are adapted for living at high altitudes or at low altitudes, or are more resistant to water stress or ozone pollution, etc.)
It turns out that in many species, high-altitude ecotypes have a higher stomatal density than their lowland ecotypes. This can correlate with many differences between the two habitats, including temperature, exposure, water conditions, and variations in atmospheric composition. Some experimentation by F. I. Woodward and F. A. Bazzaz has demonstrated that the relevant variable is CO2 partial pressure the proportion of CO2 in the atmosphere. In their experiments, the clearest result was that a reduction of CO2 led to an increase in stomatal density. Further work suggests that CO2 and stomatal density are inversely correlated -- more CO2, fewer stomata.
Confirmation from preserved specimens
Now, Woodward and others (Woodward 1987, Woodward and Bazzaz 1988, Penuelas and Matamala 1990) have analyzed preserved specimens from a range of species to analyze for stomatal density and also chemical composition. Their results confirm the field and experimental results, so that it is likely that this is a real response: plants have been responding to the historical trends of the past few centuries. There is some variation in response from species to species, and ecotypes grown at high altitude seem more sensitive to increases in CO2 than their lowland kin. This suggests that though there is genetic variability in the response (some individuals are more responsive than others), there is a real response which all individuals show to some degree.
It is not clear just how these effects are produced by the growing plant. It is likely that the developmental response is similar in important ways to other stress responses. Stomatal density changes in response to other stresses, such as drought or shade-stress; in these cases, it seems clear that the primary effect is on leaf expansion. For example, in drought, the leaves will not expand as much, and this will result in a high density of stomata By contrast, leaves in the shade (for species that grow in both sun and shade) are usually wider and thinner than the leaves that grow in higher light levels, and their stomata are less densely packed.
The density of stomata has been shown in many cases to reflect important changes in atmospheric composition, as well as other kinds of environmental stresses. Because the responses have been reported for a relatively small number of species, and because the phenomena are related to many important issues of plant physiology and ecology, the simple protocol outlined below can be used in many useful experiments or field studies.
Garbutt, K., W.E. Williams, and F.A. Bazzaz (1990) "Analysis of the differential response of five annuals to elevated CO2 during growth." Ecology 71(3).1185-1194.
LaMarche, V.C. Jr. et al. (1984) "Increasing atmospheric carbon dioxide: Tree-ring evidence for growth enhancement in natural vegetation." Science 225.1019-21
Penuelas, Josep and Roser 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 41.1119-24.
Raven, Peter, R. Evert, and S. Eichorn. (1986) The Biology of Plants (4th ed.) New York: Worth Publishers.
Woodward, F.I. (1987) "Stomatal numbers are sensitive to increases in CO2 from pre-industrial levels." Nature 327.617-8.
Woodward, F.I. and F.A. Bazzaz (1988) "The Responses of Stomatal Density to CO2 Partial Pressure." Journal of Experimental Botany 39.1771-81.