Biology Lab
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Does Leaf Structure Differ Within A Plant? INTRODUCTION
Environments may vary greatly at small scales in both time and space. Natural selection has equipped plants and animals with the means of dealing with environmental variations in light, temperature, relative humidity, nutrients and other factors. For animals whose adult stages are often highly mobile, one way of dealing with temperature change is to simply move to a more favorable place. However, this is not an option for plants whose adult stages are stationary. Yet, plants exhibit a range of adaptive strategies to survive in their particular environments that include protective spines as those exhibited by cacti or temperate deciduous trees that enter “dormancy” when seasonal conditions cannot sustain growth. Additionally, the local environment (microenvironment) surrounding an individual plant can vary from a few feet above the ground at its trunk compared to 30 feet above the ground at its crown. For example, light availability and wind exposure for leaves growing high and on the outside margins of the leaf canopy (also called sun leaves) are often exposed to a drastically different micro- environments compared to leaves growing lower and in the interior portion of a tree (also called shade leaves).
What determines the phenotype of a plant or any other organism? Yes, the organism genes or more specifically, the combination of those genes (genotype). However, genes do not exist in a vacuum, rather, genes are surrounded by a cellular environment and can be influenced by this environment. Interestingly, although all somatic cells in any organism have the same DNA, individual cells may develop different final phenotypes in response to environmental variation in a process called phenotypic plasticity (FIGURE 1).
FIGURE 1: Phenotypic plasticity is an outcome of the interactions between genotype and developmental environment. With respect to plants, leaves within an individual plant are all genetically similar because all are part of a plant that arose from a single fertilized egg, but, leaf phenotypes might differ depending on where they grow on the tree (Figure 2). However, plant species vary in their response to environmental conditions. How does a researcher distinguish between traits that are mostly effected by genes versus the by the environment? One way to begin to answer this questions is to compare leaves that have the same exact DNA, for instance, by studying many leaves from the same tree and from different locations on a tree.
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FIGURE 2: Red oak leaves from two different locations on the plant
OBJECTIVE: In today’s laboratory, you will determine whether or not the microenvironment
surrounding a desert plant effects leave phenotype. Specifically, you will address the question: Do plants respond to varied microenvironments by producing phenotypically (morphologically) different leaves within the same individual? Overview: To determine the presence of phenotypic plasticity, you will measure and examine phenotypic attributes of leaves taken from the same plant but which were growing in one of two different microenvironments on the plant:1) outer canopy (sun) or 2) inner canopy (shade). Thus, any phenotypic differences you measure among leaves taken from a single individual plant would be due to microenvironmental causes.
PART 1: Testing the Influences of Environment on Leaf Morphology Background Leaves are of interest to biologists because they are the chief organs of photosynthesis and gas exchange in plants and are linked to plant biological fitness. For this reason, intense natural selection has occurred on these important structures and has led to enormous variations in their size, color, shape, texture, surface area and arrangement found in the plant kingdom. For example, one finds that in many species, leaves are often arranged on the stem in such a fashion as to receive maximum sunlight but, at the same time cast minimum shadow on other leaves of the plant leading to more efficient photosynthesis. How is a leaf put together? Leaves are organs and thus, organs consist of integrated tissues consisting of a system of cells that vary in structure and function but which constitute the covering of the plant body called the epidermis (Figure 3). Cells within the leaf epidermis form the stomata. Two specialized epidermal cells called guard cells form and regulate surface openings or pores called stomata. Guard cells change in shape and thus, bring about the opening and closing of stomata. Guard cells are normally kidney-bean shaped and contain chloroplasts. They may be located on the upper leaf surface only, lower leaf surface only, or both the upper and lower leaf surfaces (Figure 1.) The functions of the stomata (including the guard cells) are exchange of gasses with the atmosphere and regulation of water vapor in an out of the leaf. The loss of water vapor out of leaf is known as stomatal transpiration. More than 90% of the water taken into a plant may be lost this process and can lead to plant dehydration and eventually death. However, transpiration may have a secondary affect of cooling the leaf through the process of evaporation. Depending on which plant surface is involved, three categories of transpiration are acknowledged:
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1) Cuticular transpiration - Transpiration taking place through the cuticle -outermost layer of stems, leaves is called cuticular transpiration. It accounts for 0.1% water loss. 2) Lenticular transpiration - Lenticels are small openings present in woody stems, twigs and fruits. Loss of water vapor through lenticels is called lenticular transpiration. It accounts for 1% water loss. 3) Stomatal transpiration - Stomata are minute pores present in the epidermis of leaves, young stems, etc. The loss of water vapor through stomata is called stomatal transpiration. About 93% of water loss takes place through the stomata only.
FIGURE 3. Cross section of a leaf showing stomatal openings
Additionally, a layer of wax-like material called cutin that usually covers the epidermal surfaces restricts water loss from leaf surfaces. This cuticle layer restricts transpiration and is variable in thickness depending on the environment in which the plant exists (i.e. desert vs rain forest). As a result, many plants living in habitats that vary in water availability reduce water loss by secreting a thick waxy cuticle, producing hairs (trichomes) and/or, by closing stomata during the hottest part of the day when evaporation is at its greatest.
• Watch this overview of water movement through a plant • Review Lab Powerpoint located in Canvas
Some Questions to answer: The following are a few questions that can be answered by reading the above portion of the lab, reviewing the corresponding lab powerpoint or additional resources associated with this lab that are posted in Canvas (if available). You will not turn in these questions, but this is information you should know and could appear on quizzes or practicals. What are two major functions of a leaf? What is the equation for photosynthesis? In addition to leaves, what are 3 other organs (and their) functions found in most plants? What are three forms of transpiration?
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What is transpiration?
What are the major cells of a leaf depicted in the figure above? How might leaves differ in morphology as a result of their location within a plant? Describe how one can determine if the one is looking a the upper portion of a leaf when examining microscope slide of a cross section? How do the microenvironments differ from outer to inner canopy of a plant with dense numbers of leaves? What are some hypothetical morphological differences of Sun vs Shade leaves? What path does water travel to get to the leaf from the roots? How are the functions of xylem and phloem different? Procedures: Overview: Outer (sun )and inner canopy (shade) leaves were collected from a single tree and you will determine if these locations differ in leaf mass. Mass is influenced by the amount of water and other cellular products, number of palisade cells and thickness of cuticle. All of these variables are, in turn, greatly influenced by leaf size (area). Thus, large leaves (leaves with the greatest area) would be heavier not because of any adaption to increased light conditions or wind due to location. One way to eliminate effect of size on mass is to standardize the area being weighed/measured (see below). What possible differences in mass do you expect between sun and shade leaves and why? METHODS Mass data was determined using the following procedures: PLANT NAME=_AZ ROSEWOOD_( Vauquelinia californica)
1. Each researcher collected 10 leaves from the outer canopy (sun leaves) and 10 leaves from the inner canopy (shade leaves).
2. Keeping sun leaves separate from shade leaves, researchers used a hand-held hole-punch to standardize leaf area by “punching” out identical sized pieces of plant material (disks). Researchers collected (hole-punched) 10 disks from each leaf. They avoided veins in the leaves. Researchers were careful in their counting and measuring of mass-- leaf disks are sneaky and like to stick together and stick to the hole-puncher.
3. All disks were weighed from their respective locations (sun vs shade) to determine total mass and placed in the data table located on the class canvas site. PART II: Testing the influence of microenvironment on numbers of leaf stomata. Background Evidence suggests many species have non-uniform stomatal opening under both laboratory and field conditions, for example, different areas of the same leaf seem capable of responding independently to microenvironmental conditions (Spence, 1987). For example, changes in the degree of stomatal opening (aperture) reflect the cumulative effect of many physiological responses by a leaf to its environment. Thus, the dimensions of stomatal pores have a big effect on the rate of gas exchange. Additionally, the numbers of stomata present on leaf surfaces can also influence the rate of water loss. Because of stomata’s role in plant survival,
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stomata are a reasonable dependent variables to explore. Objective. The purpose of this portion of the exercise is to determine and compare the relative abundance of stomata on lower leaf surfaces among leaves developing under different micro-environmental conditions. Overview: Sun and shade leaves from a single tree were collected and the number of stomata on the upper and lower surfaces were determine. Predictions: State some possible differences in stomata numbers you would expect to occur in sun and shade leaves and why? For example, to reduce water loss, plant leaves from exposed outer canopy areas might have fewer stomata per leaf area compared to more shade locations. PROCEDURE: 1. One sun leaf and one shade leaf were selected from the same plant used above. A layer of
nail polish (I have found that Wet-N-Wild clear nail polish works the best for this) to the lower surface of one sun and one shade leaf. The nail polish will make an impression of all stomata on the leaf surface.
2. After the nail polish had dried, a piece of double-sided clear tape was placed on the area of dried nail polish. The tape was peeled off with a resulting thin “peel” of a nail polish. This peel contains the stomata sample. The tape was placed onto a glass microscope slide with the nail polish peel (sample) facing upward.
3. The slide was placed on a light microscope. Using the scanning power (4x) objective first, the stomata were brought into focus. Next, the stomata were viewed under high power (40x).
4. Stomatal counts can now be made under the high power objective (40x). All stomata in the field of view were counted (even partial stomata). This was done for three different fields of vision on the same peel by moving the microscope slide. The average number of stomata out of three were determined and placed in a spreadsheet.
5. Calculating the number of stomata per centimeter: A ratio can be set up comparing the number of stomata counted per 0.0017 cm2 to the number of stomata per one square centimeter. Using the average number of stomata of the three counts, divide by the area of the field of vision under 40x which is 0.0017 square centimeters.
EXAMPLE: How many stomata do you count in the image above?_____________(yes,10) (please note for this example we will just use the number 10 as our average)
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Use the following equation to determine the number of stomata per cm2 average # stomata determined from microscope = X # of stomata 0.0017 cm2 1.0 cm2 average # of stomata calculated = 0.0017 (X # of stomata) then….. X # of stomata/ cm2 = (average # of stomata)/0.0017 cm2 Thus, using above count X # of stomata/ cm2 = 10/0.0017 cm2
X # of stomata/ cm2 = 5,882
Classroom Results: a classroom Excel spreadsheet file of leaf mass and stomata/cm has been uploaded to CANVAS for use in the assignment. Assignment: You will not turn the following in but the quiz and practical will ask you about the answers of the following: STEPS
1. Download the classroom data set after it has been posted 2. Calculate descriptive statistics 3. run a t-test (unpaired, assuming equal variances) comparing the mass of sun vs
shade leaves 4. run a t-test (unpaired, assuming equal variances) comparing the number of stomata/
cm2 of sun vs shade leaves. 5. Determine the two-tailed p values for each test 6. Determine from the p-value if mass significantly varied by microclimate location or if it did
not. Explain how you know this. 7. Determine from the p-value if # of stomata/ cm2 significantly differed by microclimate
location. Explain how you know this. 8. Review all terms, procedures and purpose of today’s lab 9. Next, go to Canvas and take the quiz.