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1 Biology 3460 Week 12 1 Biology Plant Physiology - Lab Exercise 10 Effect of Environmental Changes on Stomatal Size and Density Introduction The size and density of stomata are characteristics that can change dramatically as plants acclimate to environmental changes. The size of stomata influences how fast they can respond to environmental changes (smaller size faster response). Stomatal size also influences the total number of stomata that can be packed into a given area of leaf surface. Therefore, both the size and density of stomata usually change simultaneously as plants act to adjust gas exchange between the atmosphere and the leaf internal air spaces based on the environmental conditions. Stomatal size and density are known to vary in response to changes in atmospheric CO 2 concentration, and other characteristics of their growth environments such as light intensity, air temperature and soil water availability. In this lab exercise you will test for significant changes to stomatal size and density in bean plants grown in contrasting environments. Protocol: Demonstration of the Scanning Electron Microscope (SEM) A demonstration will be provided to illustrate the use of the SEM for observing and capturing images of the surface of leaves from plants grown under different environmental conditions. An example image of a leaf surface is shown below. Leaf surface images will be used to measure the density and size of stomata. Counting stomata and estimating stomatal density Open each image in free software tool ImageJ (http://rsbweb.nih.gov/ij/) and use the Multi- Point Selection tool to count the number of stomata. Stomata at the edges which are only partially in the field of view should be included if half or more is visible. Each image contains a scale bar which can be used to calculate the area of the image. In ImageJ select the Line tool, and create a line the length of the scale bar. Select Analyze Set Scale and enter the units (mm) and the known distance (convert m to mm). Create a new line the width of the image and select Analyze Measure the length will be displayed in a Results window popup on the right-hand side of the box. Repeat this process for the image height, and then calculate the image area. For an image captured at 400x, the area is mm 2. Determine the stomatal density (expressed as number/mm 2 ) for two leaves for each of two treatments. Bean plants were grown in two growth chambers with the following conditions: 14 hour photoperiod, 25 ºC day/ 20 ºC night temperatures for both chambers; one chamber had high light intensity (photon flux density, nm) of 700 mol m -2 s -1, and the other chamber had low light intensity of 300 mol m -2 s -1. Do this for both the adaxial (top) and abaxial (bottom) surfaces. Record your data below. Density High Light Low Light Abaxial surface (#/mm 2 ) Adaxial surface (#/mm 2 )

2 2 Measuring stomatal length Use the Set Scale procedure outlined above and then measure the lengths of three randomly selected stomata in each image. Use the average of the three measurements as the value for the stomatal length of that image. Repeat the measurements for two images for each treatment and both leaf surfaces. For these purposes stomatal length is defined as the distance between the edges of the cuticular ridges which overarch each stomatal pore. Record your data below. Length High Light Low Light Abaxial surface ( m) Adaxial surface ( m) Assignment Make graphs that compare (i) the density of stomata, and (ii) the length of stomata on both the adaxial (top) and abaxial (bottom) leaf surfaces of bean plants grown in two treatments. Conduct the appropriate statistical tests to analyze (i) the light intensity effect and (ii) the differences between leaf surfaces.

3 Biology 3460 Week 12 3 Leaf stomatal characteristics have been altered over evolutionary time as plants have adapted to changes in atmospheric CO 2 concentration as shown in the graphs above (Franks and Beerling, 2009). As stomatal density increases stomatal size decreases - this is necessary to fit more stomata onto the leaf surface Reduction in stomatal size is associated with: - more efficient stomata, with faster opening and closing responses Smaller size and higher density of stomata: - results in a shorter path length for gas diffusion and higher max. stomatal conductance

4 Biology 3460 Week 12 1 Biology Plant Physiology - Lab Exercise 11 Plant Secondary Products: Flavonoids Anthocyanins Introduction Flavonoids are compounds synthesized from products of the shikimic acid pathway. They are considered secondary plant products because their production and metabolism is not associated with the acquisition and metabolism of the primary plant materials during photosynthesis, water relations and mineral nutrition. Anthocyanins are one sub-group of flavonoids that are responsible for the colours of many plant parts, particularly flowers. The colour of anthocyanins depends on a number of factors including the ph of the cell vacuole (see attached research report materials). Variation in the characteristics of soils that plants are growing in can affect the colour of flowers because of differences in soil ph. The influence of ph on the colour of anthocyanins can be demonstrated in a simple experiment described below. Protocol: Work as a group of two or three students to complete this exercise. 1. Make a crude extract of red cabbage. First chop the cabbage into fine pieces. Add approximately 300 ml of chopped cabbage to about 400 ml of distilled water and mix them in a blender at high speed. 2. Filter the mixture from the blender through 4 layers of cheesecloth into a beaker. The solution should be a bright purple colour. If the purple colour is too dark, add more water to dilute the solution. 3. Pour approx. 15 ml of the cabbage extract into 5 small beakers (30 50 ml size). 4. Add approx. 20 ml of one the 5 different ph solutions provided (ranging from strong acid to strong base) to a beaker containing the cabbage extract. The colour of the cabbage extract will change depending on the ph of the solution that was added, as shown in the table below. Table 1. Cabbage extract colour indicators for ph of tested solution. ph Colour 0 Red 2 Rose 4 Pink 6 Lavender 7 Purple 8 Blue 10 Green-Brown 12 Green 14 Yellow For future reference, if you would like to present this experiment to student groups etc.: Vinegar (5% acetic acid) can be used as a weak acid (ph about 5), a 0.1 M solution of sodium bicarbonate (baking soda) can be used as a weak base (ph about 9). Low concentrations (0.1 M solution) of hydrochloric or sulfuric acid work well as strong acids (ph about 0). In addition, low concentrations (0.1 M solution) of sodium hydroxide or ammonium hydroxide work well as

5 2 strong bases (ph about 12). Some household cleaning products can also be used as strong bases. The ph of the solutions can be measured with litmus paper. Appendix A; Further Reading on Flavonoids PLANT BIOLOGY: A Question of Color Petal color is a key morphological trait of flowers that influences reproductive success. It is controlled by the pigments produced by the plant, and by the ph at which the pigments are stored. In plants, the anthocyanins--the main class of flower pigments--reside in the vacuoles, an intracellular acidic subcompartment. Because the color of a pigment is often ph-sensitive, variations in petal color have been used to identify mutations in ph regulation. In petunias, Verweij et al. now identify PH5 as a P-type proton pump localized to the vacuolar membrane. Similar P-type proton ATPases have previously been found to reside on the cell surface, not on intracellular membranes, which contain their own vacuolar-type proton ATPases. In PH5 mutants, vacuolar acidification is reduced without changing the expression of the anthocyanin pigments or other coloration-related processes, leading to blue flower coloration (shown to the right of a wild-type flower), which is never observed in natural habitats. Expression of the PH5 gene is linked to the same transcription regulation involved in anthocyanin production. This coordination of pigment production and ph regulation of the pigment-containing compartment is an important aspect in maintaining flower and also seed coloration, which also requires PH5 activity during pigment accumulation. -- SMH Nat. Cell Biol. 10, /ncb1805 (2008).

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