Lab 3 – Plant Tissues, Roots, and Respiration Introduction This lab covers some of the tissues found in plants, plant roots, and the metabolic process of respiration. Tissues are collections of cells that have a similar function. One of the common tissues in plants is parenchyma. Parenchyma is commonly used to represent “typical” plant cells as they are found in many locations in plants. Epidermal tissue is found on the outside of plants and serves a protective function. Xylem is a tissue that conducts water and phloem conducts sugar and other organic molecules. We will study some tissues in this lab and continue our study in other labs as we look at different plant organs. Activity: Tissues - Parenchyma and Xylem The photograph below is of one of the “strings” on the inside peel of a banana. You will see the faint outline of parenchyma cells which look like microscopic clear plastic bags. Parenchyma cells are found in numerous plant organs such as leaves, stems, and fruits as seen in figure 1. Figure 1. Parenchyma cells Xylem is a water conducting tissue in plants (Xylos is Greek for wood). Vessel elements are one kind of cell in xylem and they look like coils or spirals that make a spring-like form (like a slinky) as seen in figure 2. These spirals can expand as the plant increases in length and they keep the cell open for water conduction. The following illustration is of a vessel element on the inside of a banana peel. Figure 2. Xylem Vessel Element. Roots We study roots first as plant organs, as they are relatively simple in terms of structure compared to other plant organs. Monocots, such as corn and lilies have fibrous roots (figure 3). There is no main root in these plants. Eudicots, such as carrots and oak trees, have tap roots with one main root and several lateral roots (or branch roots). Figure 3. Tap root (left) and fibrous roots (right) Activity: Examination of Longitudinal section of Zea (corn) Root We use Zea mays (corn) to look at a longitudinal section (l.s.) of the root (figure 4). Corn is common, inexpensive, and provides a great example of root structure. Corn is a monocot as it only has one seed leaf (known as a cotyledon). At the tip of the root is the root cap. It is a tough covering that allows roots to penetrate soils but keeps the delicate portions of the root safe. The root cap has starch grains in the cells that function to determine a plant’s position. If a plant is growing on a hill and the hill slides a bit, the roots may not be oriented in a downward direction anymore. The starch grains settle to the bottom of the root cap cells and the roots respond by the influence of hormones and grow down. This response is known as positive gravitropism (toward the gravitational force). This was first described by Charles Darwin. Negative gravitropism is seen in stems which grow upward, in the opposite direction of the pull of gravity. Just above the root cap is the root apical meristem. This is a lens-shaped region that has cells that actively divide, producing more cells. A meristem is a region in a plant that generates new tissue. Think of meristems as being the plant equivalent to animal stem cells. Frequently meristems can produce new root tissue (or in the case of stems, new stem tissue). Above the apical meristem is the zone of division. You can identify this region as the cells are cube-shaped. The region is named for actively dividing cells. You counted the phases of mitosis in the zone of division in lab 2. The cells above the zone of division are columnar and this is the zone of elongation. Here the cells lengthen driving the root deeper into the soil. Above the zone of elongation is the zone of maturation (also known as the zone of differentiation). Here is where root hairs are apparent. Root hairs are epidermal appendages. They are extensions of the epidermal cells and increase the surface area of the root for water and mineral absorption. The root has stopped elongating at this zone. In this section you may also see traces of newly forming xylem. Xylem conducts water from the root to the stem of the plant. Learn the parts of the root in the longitudinal section of root in figure 4 and note the features mentioned above. The entire root is a composite photograph taken from a couple of fields of view. Figure 4. Long Section of Root (left). Close-up of Zone of Maturation with Root Hairs and Xylem (right) Cross Section of Roots Activity: Zea cross section Examine a prepared slide of Zea mays roots in cross section (abbreviated xs) in figures 5 and 6. Find the epidermal tissue (epidermis) on the outside of the specimen. This tissue is made of epidermal cells. Inside the epidermis is ground tissue. The cortex is inside the epidermis, making up about half the outside diameter of the root, consisting of parenchyma cells and terminating at a ring of tissue called the endodermis. The endodermis is a single layer of cells and it has a casparian strip which is part of the cell wall of the endodermis that seals the spaces between the cells. The casparian strip contains suberin which is a waterproof material. Water in the soil contains many micro-organisms (some harmful to the plant) and by sealing the edges of the cells the casparian strip forces the water to go through the cell membrane instead of the spaces between the cells. By going through the cell membrane the water is filtered and micro-organisms are prevented from entering the xylem of the root and passing deeper into the plant. The endodermis can be distinguished by the thickened walls. Just inside the endodermis is the pericycle which is also a single layer of cells. This layer is responsible for the production of lateral roots. Lateral roots are rare in monocots. The cortex, endodermis and pericycle are all cells of the ground tissue. Inside the pericycle is ring of vascular tissue consisting of the waterconducting xylem and the sugar-conducting phloem outside of the xylem. Phloem in these plants has a sugar conducting sieve cell and a smaller companion cell. More ground tissue is found in the middle of the root and it is the pith consisting of parenchyma cells. Learn the details of the cross section of root in the following figures and note the features mentioned previously. Figure 5. Cross Section of Zea mays Root. Figure 6. Details of Zea mays Root xs. Activity: Cross Section of Eudicot Root We will use a prepared slide of Ranunculus (Buttercup) for this part of the lab (figure 7). Buttercups are members of a group of flowering plants known as eudicots as they have two cotyledons that emerge from the seed (remember monocots have only one). The pattern of root anatomy is a little different in eudicots than it is in monocots. In Ranunculus the outermost layer that you will see in the following photograph is the epidermis. Inside of the epidermis, and making up most of the diameter of the root, is the cortex. The word cortex comes from a Latin word meaning bark (as in tree bark). The cortex has parenchyma cells with starch grains. The starch grains here are storage structures (unlike those in the root cap where the starch grains are used to determine gravity). Many plants store their food underground for protection. Deep to the cortex is the endodermis with its casparian strip. The endodermis is a single layer of cells. The endodermis can be distinguished by the thickened walls. Locate the endodermis and casparian strip. Look for thin-walled passage cells in the endodermis which allow water to pass through their cell membranes. These passage cells are part of the endodermis but they do not have the thick casparian strip around them. Just inside the endodermis is another thin layer called the pericycle. As in monocots this is where lateral roots arise. At the very center of the root is a red cluster of cells that forms a cross. This is the xylem seen in cross section. The xylem seen in longitudinal section appears as a spiral or a spring. If you were to look at a spring end-on it would look like a circle. These circular structures stain red because of the lignin in the cells. In roots of vascular plants the xylem matures toward the center of the root. This pattern is known as exarch xylem which is the normal pattern in the roots of most vascular plants. The oldest xylem (protoxylem) is on the outside and the youngest (metaxylem) is on the inside. In the arms of the xylem you can find the phloem so the phloem is found between the xylem and the pericycle. Phloem conducts sugar and some hormones throughout the plant. The sieve cell and companion cell of phloem come from the same mother cell. The sieve cell has no nucleus so it could not survive very long without the companion cell which has a nucleus. The nucleus of the companion cell directs the activity and makes compounds for both itself and the sieve cell. Examine figure 7 for the terms listed previously. Figure 7. Cross Section of Eudicot Root. Upper figure, overview. Lower figure details of the stele (the central part). Activity: Radish Seedling Examine the surface features of a radish seedling in figure 8. These seedlings have been sprouted for 3-5 days. The fine fuzzy threads that you see are the root hairs. Find the root cap, zone of division, and zone of maturation in the seedling. The yellow oval structures on top are the cotyledons. Figure 8. Radish Seedling Plant Respiration Rates We will look at the metabolic process of respiration in this lab where tissues consume oxygen and release carbon dioxide. Plants are known for photosynthesis but they also respire just like animals do. Plants consume oxygen by the process of aerobic respiration. The chemical equation for this is: C6H12O6 + 6O2  6H2O + 6CO2 + 36 ATP Sugar and oxygen produce water, carbon dioxide, and energy carrying molecules of ATP. We can study the rate of respiration by measuring the reduction of the reactants (sugar and oxygen) or measuring the products (water, carbon dioxide, or ATP). As oxygen and carbon dioxide are of similar volumes we can estimate the volume of oxygen used if we measure the carbon dioxide (CO2) removed from the system. To do this we use potassium hydroxide (KOH)as a chemical that absorbs carbon dioxide. This is seen in the following formula: CO2 + 2KOH → K2CO3 + H2O Potassium hydroxide reacts with CO2 gas and forms potassium carbonate (K2CO3) a white precipitate. This removes carbon dioxide from the reaction vessel reducing the volume which is proportional to the amount of oxygen consumed. Oxygen use is variable among organisms. The metabolic rate varies between species, within species, and by age of the individual. For example the basal metabolic rate (BMR,) or the resting metabolic rate, of a rat is 15 mL/Kg/min (VO2). For a reptile it is about 5-10 times less than that of a mammal (1.5 – 3 mL/Kg/min). We measure the metabolic rate of reptiles as the standard metabolic rate (SMR) because they are ectothermic (cold blooded) and their metabolic rate varies with the external temperature. We know that some people have higher metabolic rates than others, but even within the lifespan of one individual the metabolic rate changes. From age 20 to age 60 the basal metabolic rate of a human commonly decreases by 15%. In this lab we measure the respiration rate in plant tissue. This is done with the use of a respirometer (figure 9). What you predict the respiration rate of plant tissue is compared to that of a rat or a reptile? Guess the plant rate relative to the rate of a rate or reptile and enter this in question 13 in the Review Section at the end of the lab. One of the problems we have in measuring the respiratory rates in plants is that it is masked by the photosynthetic rate during the day. We can either measure the plant’s respiratory rate in dark or we can measure the non-photosynthetic tissue such as that of the roots. We will examine both of these in this lab. There is another problem and that is the nature of gas in terms of measuring volume when there are potential changes in pressure or temperature. This is expressed by the Ideal Gas Law which is described as PV= nRT (where P = pressure, V = volume, n = the number of molecules of a gas, R is a constant and T = temperature.) We are close to sea level so we assume the pressure will be constant for the lab. Figure 9. Respirometer Activity: Respiration Rate 1. We have 4 test tubes with 2 pellets of KOH in each test tube (figure 9). 2. On top of the KOH there is 1/4 of a cotton ball which acts as a physical barrier to the KOH 3. Tube 1 has 20 germinating peas that weigh 12.6 grams. 4. Tube 2 has 12.6 grams of leaves. 5. Tube 3 has 12.6 grams of roots. 6. Tube 4 has 12.6 grams of stems 7. All tubes are gently but firmly stoppered. 8. A 1 mL pipette is inserted into each stopper. 9. All tubes are in a water bath with the pipette tips pointing up, to equilibrate (figure 10). 10. The tubes are inverted at the same time and the time is recorded for all samples when the tubes reach the 1 mL mark. Results Time for tube with leaves to reach the 1 mL mark: 31 minutes Time for tube with germinating pea seeds to reach the 1 mL mark was 18 minutes Time for tube with stems to reach the 1 mL mark was 52 min Time for tube with roots to reach the 1 mL mark was 37 minutes Calculate the respiratory rate as mL O2/Kg/min = (VO2) for all four samples and record your answers in Question 14 in the Review Section at the end of this lab. You calculation to get any credit for this lab assignment. must do this For example: Let’s say peas weigh 50 grams and it took 5 minutes to consume 1 mL of oxygen. 1. Convert grams to kilograms (50 grams = 0.05 kg) 2. Find out how much oxygen was used for 1 kg of material (1 mL O2/0.05 = 20 mL/kg) 3. Divide by the number of minutes (5 min: 20mL O2/kg/5 min = 4mL/kg/min) Figure 10. Respiration Apparatus Review 1. What is the most common tissue found in young roots such as Ranunculus? 2. What is the water conducting tissue in plants known as? 3. What is gravitropism? 4. What is the function of the root cap? 5. In what zone of the root are cells actively dividing? 6. What structure increases the surface area of roots for water absorption? 7. In what zone do you find root hairs in a longitudinal section of a root? 8. What is the function of the endodermis? 9. Ranunculus is a plant that lives for many years. Corn is an annual plant. Why are there no starch grains in the roots of Zea mays? 10. Why might it be important for the root not to be moving through the soil when there are root hairs developing? 11. Label the diagram of the root tip below with the terms you learned in this exercise. 12. Label the cross section of root below. Respiration Experiment Results 13. Guess the plant respiration rate relative to a rat or reptile. Select the choice below. More or the same as a mammal (rat) The same as a reptile Less than a reptile 14. Record your results for the experiment data in the following spaces. mL O2/Kg/min germinating peas mL O2/Kg/min Roots mL O2/Kg/min Stems mL O2/Kg/min Leaves 15. Provide an explanation for the metabolic differences in the roots, stems, or leaves as measured in this lab exercise. 16. What is the function of the potassium hydroxide (KOH) in this experiment? 17. Reflect on the outcome of the respiratory rate of your experiment in comparison with the rate of a rat or reptile. Explain why you think the rate might be similar or different. 18. How did this compare to your initial guess for the respiratory rate? Image sources All images by Eric Wise except for: Figure 8 - http://www.mcqplus.com/daily-answers/2017/6/7/root-hairs Figure 10 - https://www.cabarrus.k12.nc.us