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Lev65484_ch05.qxd 6/4/01 2:59 PM Page 53 5 LABORATORY TOPIC Plants Do It All— Photosynthesis, Respiration, and Transpiration BACKGROUND Physiology is the branch of biology that focuses on the function of structures and the variety of dynamic processes that allow an organism to live. Understanding all the processes, such as photosynthesis or protein synthesis, can help us understand the organism. This understanding can also help us adjust conditions for practical purposes, such as to increase crop yields or control pests. This laboratory topic introduces three important processes in the lives of plants—photosynthesis, respiration, and transpiration. Unfortunately, this is only a glimpse into the fascinating inner workings of a live plant. This lab is divided into four exercises. The first explores the flow of carbon dioxide related to photosynthesis and respiration. The second investigates factors affecting the production of starch via photosynthesis. The third and fourth exercises explore the flow of water out of plants via transpiration; Exercise C examines transpiration for individual leaves and plants, and Exercise D demonstrates what happens when plants act together in a community. LEARNING OBJECTIVES After completing this laboratory topic, students should be able to: 1. Better understand the factors that can affect the physiology of plants. 2. Relate the processes of photosynthesis and respiration to plant growth. 3. Better understand the process of transpiration in plants. 4. Explore the factors that affect the rate of transpiration in plants, including anatomical features such as stomata and environmental conditions such as temperature. 5. Understand the processes of scientific inquiry by posing questions, designing investigations, collecting empirical data, testing hypotheses, and communicating the results. EXERCISE A: The Ins and Outs of CO 2 The common statement “Animals respire, plants photosynthesize” is only partially correct. Although it is true that plants are capable of producing their own organic compounds through the process of photosynthesis, plants are so versatile that they can also respire just like animals. Photosynthesis is a complex series of reactions involving the capture of light energy, conversion to chemical energy, and finally the synthesis of carbohydrates. It is one of the main biosynthetic processes by which energy and carbon enter the network of living organisms. A summary of the ultimate reactants and products of photosynthesis can be stated as: EQUATION 1: Light + CO2 + H2O Æ (CH2O)n + O2 carbon dioxide Water Sugar Oxygen This equation is deceptive in its simplicity because photosynthesis involves numerous intermediate steps. Photosynthesis can be divided into two series of reactions. The first series, light harvesting, results in the capturing of light energy and the temporary storage of this energy in ATP and NADPH. The second series of reactions, carbon fixation, uses the energy in the ATP and NADPH to synthesize sugars from CO2. Light harvesting starts with the absorption of light energy by the pigment chlorophyll embedded in the membranes of the chloroplasts within plant cells. The energy excites electrons within the chlorophyll molecule to a higher energy state. At this energy state, the electrons are easily transferred to other chemicals that accept the electrons. The electrons are transferred from one chemical to the next much like the way buckets of water are passed from one person to the next in a bucket brigade. Eventually, the electrons are transferred to an electron acceptor called NADP+ (nicotinamide adenine dinucleotide phosphate). To replace the lost electrons of the chlorophyll molecules, water (H2O) is split to produce electrons, protons (H+), and oxygen gas (O2). In addition, a proton gradient is generated that is used to produce ATP (adenosine triphosphate) from ADP 53 Lev65484_ch05.qxd 6/4/01 2:59 PM Page 54 54 APPLIED BOTANY (adenosine diphosphate) and Pi (inorganic phosphate). ATP is a temporary energy carrier. The NADP+ is converted to NADPH by the adding of two electrons and one proton (H+). At the end of the light-harvesting phase, NADPH, ATP, and O2 are produced. A summary of the light-harvesting phase can be stated as: EQUATION 2: Light + H2O + ADP + Pi + NADP+ Æ ATP + NADPH + H+ + 1⁄2O2 In the carbon fixation phase of photosynthesis, the energy and electrons of ATP and NADPH are used to form carbon–carbon bonds. Carbon dioxide (CO2) enters a cyclic series of reactions (the Calvin cycle), and eventually sugars (CH2O)n are produced. The name of the enzyme that catalyzes the fusion of CO2 to the first chemical in the cycle (ribulose bisphosphate) is rubisco. This is worth mentioning since rubisco is the most abundant protein on our globe. The first sugar to exit the cycle is a three-carbon sugar, G3P (glyceraldehyde 3-phosphate). G3P can be used to generate other sugars, such as glucose (C6H12O6). The summary of the carbon-fixation phase of photosynthesis can be stated as: EQUATION 3: ATP + NADPH + CO2 Æ ADP + Pi + NADP+ + (CH2O)n Take some time to examine the three equations. Equation 1 is the summary of equations 2 and 3. What information is lost by only looking at equation 1? Which phase requires light? Which phase is not directly dependent on light, yet needs the products of the light-dependent phase? ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ The two phases of photosynthesis actually occur in two different locations within the chloroplasts. The light-harvesting phase occurs on the membranes of the thylakoids, whereas the carbon-fixation phase occurs in the stroma, the space between the thylakoids. Given this, what compounds cycle back and forth between the two phases? Does carbon enter the system in both phases? All animals, including humans, ultimately depend on plants to produce the oxygen gas (O2) they need. O2 is actually a by-product of photosynthesis. We are lucky that plants produce oxygen. For example, an average hectare of corn produces enough oxygen per day to support 325 people. Where do the atoms that make up the O2 come from? If you remove a plant from the light, what happens to its oxygen production? What is the fate of the products of photosynthesis? G3P can easily be converted into various six-carbon sugars, such as glucose or fructose, or stored as starch, a polysaccharide formed as a chain of glucose molecules. When these basic sugars are combined with other elements, such as nitrogen or phosphorus, all the other organic compounds in a plant, such as proteins, nucleic acids, lipids, or alkaloids, can be formed. In this way, plants make all their basic building blocks. And since animals are incapable of carbon fixation themselves, plants make the basic building blocks for animals as well. Animal food has to come from other organisms, and ultimately most of it comes from plants. In Laboratory Topic 10, you will relate your own needs for energy to production of chemical forms of energy by plants. Of course, the plants could probably care less about the welfare of animals. Respiration is another fundamental process of living organisms. Before we proceed, it is important to compare two definitions of respiration. Many of you may consider respiration the process of breathing air in and out of your body. Mammals like yourself pull air into their lungs by contraction of the diaphragm and exhale the air as the diaphragm relaxes. The inhalation and exhalation of air is one valid definition of respiration. Humans even use breathing as a sign of life itself. Biologists often use another definition of respiration to describe what happens to some of the components of the air, particularly CO2 and O2, at the cellular level. Cellular respiration is defined as the process by which cells release energy from organic compounds to generate ATP through a series of chemical reactions involving the transfer of electrons. In aerobic respiration, oxygen (O2) is the final electron acceptor. In anaerobic respiration, or fermentation, some other chemical is the final electron acceptor. The main results of cellular respiration are organic compounds broken down to simpler compounds, with some energy becoming available for use in other metabolic steps. The overall process of aerobic cellular respiration can be stated as: ____________________________________________ EQUATION 4: (CH2O)n + O2 Æ CO2 + H2O + ____________________________________________ Compare equation 4 with equation 1. On the surface, respiration appears to be merely the reverse of photosynthesis. But in reality, aerobic respiration is another complex series of reactions that can be divided into three phases: glycolysis, the Krebs cycle, and the electron transport chain. ____________________________________________ ____________________________________________ energy (ATP and heat) Lev65484_ch05.qxd 6/4/01 2:59 PM Page 55 PLANTS DO IT ALL—PHOTOSYNTHESIS, RESPIRATION, In glycolysis, a molecule of the six-carbon sugar glucose is oxidized to two molecules of the three-carbon pyruvate, and some of the energy is recaptured in the production of ATP. The Krebs cycle completes the oxidation of pyruvate to produce carbon dioxide (CO2) and reduced electron carriers. In the electron transport chain, a proton (H+) gradient drives the production of even more ATP and is coupled with the transfer of electrons to oxygen (O2), producing water (H2O). After the entire process of respiration is complete, much of the energy released from the glucose is recaptured in the production of ATP. Since no conversion of energy is 100% efficient, some of the energy is lost as entropy and is no longer available to the organism. The ATP, however, can be used for all the other normal processes of life, such as synthesis of new tissue, response to external stimuli, or movement of materials throughout the body. Just like animals, plants use aerobic respiration to recapture the energy held in the sugar molecules produced during photosynthesis. Just like animals, plants use glycolysis, the Krebs cycle, and the electron transport chain to produce ATP. Plants, however, do not need to consume preformed organic compounds as animals must. They produce their own organic compounds via photosynthesis. For this reason, some people say, “Plants make their own food.” Again we emphasize, plants photosynthesize and respire. Animals only respire. As long as the net production of new material via photosynthesis exceeds the breakdown of molecules to produce ATP via respiration (P > R), a plant will grow and increase in biomass. But sometimes, plants need to rely heavily on the energy stored in sugars, and respiration can exceed net gain from photosynthesis (R > P). This is common when photosynthetic tissues are not yet available, such as in a germinating seed or regrowth of buds in the tips of branches each spring. What do you suppose happens when a plant is kept in the dark for a long time or at night? Take a look at equations 1 and 4 again. Notice how on the surface, carbon dioxide is consumed by photosynthesis, yet produced by respiration (fig. 5.1). If you had an experimental system with only plants, the balance between photosynthesis and respiration could be determined by monitoring the levels of carbon dioxide around a plant. In this activity, we will use a CO2 sensor to measure the concentration of CO2 in the air surrounding plants in various conditions and see what happens. Relate what you observe to what could be happening biologically. Materials Needed for Exercise A Bell jar Cellulose acetate or diluted clear nail polish (see Instructors’ Notes at the end of this manual for preparation) Coleus (coleus) plants Cotton balls AND TRANSPIRATION 55 Light energy Carbon dioxide Oxygen Carbon dioxide Water vapor Oxygen Water Food produced and sent to other parts of plant Food converted to energy to be used by all parts of plant Photosynthesis Respiration FIGURE 5.1 COMPARISON OF PHOTOSYNTHESIS AND RESPIRATION IN PLANTS. Dry peas, beans, or other seeds Gas sampling bottle, 250 ml Glass plate Glass terrarium or jar with lid Logger Pro software Microwave oven PC or Macintosh computer Peas, beans, or other seeds soaked for 24 hours Petroleum jelly (Vaseline) Photoflood lamp Potato, apple, or other plant organ void of chlorophyll Ring stand Sodium pyrogallate (see Instructors’ Notes for preparation) Split rubber stopper Universal lab interface Various plants, such as Pelargonium (Geranium) or Zebrina Vernier CO2 gas sensor Water-filled heat shield to control temperature generated from lamp Procedure for Exercise A The Vernier CO2 gas sensor monitors the concentration levels of CO2 gas in air. In this exercise, you will use the Lev65484_ch05.qxd 6/4/01 2:59 PM Page 56 56 APPLIED BOTANY gas sensor to monitor changes in the levels within a closed chamber as your test plant either consumes CO2 via photosynthesis or produces CO2 via respiration. When the gas sensor is connected to a computer, you can easily observe the changes in levels over time and record your results. Once you are familiar with the system, you will be able to pose your own questions, formulate your own hypotheses, design experiments, predict the outcomes, collect data, and interpret the results. 1. Detecting net photosynthesis. Connect the CO2 gas sensor to your computer according to the manufacturer’s instructions. Place a large plant in a glass terrarium and insert the gas sensor probe. Cover the terrarium with a lid to prevent the air inside the chamber from mixing with the air outside. Arrange the lamp to shine light on the plant. Since photoflood lamps often generate intense heat, you may need to place a water-filled heat shield between the lamp and the terrarium. Monitor the levels of CO2 for 10–60 minutes. Note the change in the levels and the rate of change. What is happening? What does this tell you about what is happening biologically? Record your results in worksheet 5-1 at the end of this laboratory topic. Try changing the light intensity by moving the lamp further from the plant or inserting filters. What happens to the rate of change? What happens if you turn off the light entirely? Record your results in worksheet 5-1. 2. Detecting respiration in plants. Cut a fresh potato into 1 cm3 cubes and place approximately ten of them in a 250-ml sampling bottle. Connect the CO2 gas sensor with the split rubber stopper. Monitor the change in the levels of CO2. What do you observe? Is the potato respiring? Is the potato alive? ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ Now place dry peas, beans, or other seeds in the sampling bottle. Monitor the CO2 levels. Compare these results with peas, beans, or seeds soaked in water overnight. Is there any difference? What is happening within the seed? ____________________________________________ ____________________________________________ ____________________________________________ Try other plants, or change the conditions, such as temperature. You may try to start with a higher initial CO2 level by exhaling into the sample chamber. Pose your own questions and design your own experiments. What affects photosynthesis? When is the consumption of CO2 the greatest? ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ Cut another fresh potato into 1 cm3 cubes. This time, microwave the cubes just long enough to kill any living tissue (approximate 30–40 seconds). Place the cubes in the sampling bottle and monitor the CO2 levels as before. Now what do you observe? How do these results compare with those of the fresh potato cubes? ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ Try other tissues, such as cut pieces of an apple or a carrot. What do you observe? Record your results in worksheet 5-1. 3. Do plants need oxygen? We mentioned that plants are aerobic, but is this really true? What would happen if plants were deprived of oxygen? Do seeds need oxygen to germinate, grow, and survive? Lev65484_ch05.qxd 6/4/01 2:59 PM Page 57 PLANTS DO IT ALL—PHOTOSYNTHESIS, RESPIRATION, Bell jar To computer Ring stand Cotton Wire mesh platform CO2 sensor Inverted beaker AND TRANSPIRATION 57 Remove the CO2 sensors, and place each setup in a warm location in the lab. Cover the jars with paper, or place them in the dark. Observe the seeds after several days. What do you see? What is the effect of O2 on germination? Speculate on the correlation between germination and respiration. Why was it important to keep the seeds in the dark? ____________________________________________ Soaked seeds Na pyrogallate Culture dish ____________________________________________ Glass plate ____________________________________________ ____________________________________________ For this activity, we will use sodium pyrogallate to remove oxygen from our test chamber. The setup is illustrated in figure 5.2. Make a platform out of wire mesh, and place it in a 15-cm-diameter culture dish. Cover the platform with moist cotton. Place seeds (peas or beans) that have been soaked in water overnight on the moist cotton. Add 200 ml of freshly prepared sodium pyrogallate in the bottom of the culture dish below the platform. Suspend the CO2 sensor over the seeds with a ring stand. Cover the whole setup with a bell jar sealed to a bottom glass plate with petroleum jelly. The cord to the sensor must extend under the lip of the bell jar. Monitor the CO2 levels over time. As a control, establish a similar setup with another set of soaked seeds on moist cotton. Instead of sodium pyrogallate, fill the bottom of the culture dish with water. Again monitor the CO2 levels. What do you observe? What is the correlation between CO2 production and the availability of O2? From what you observe, do you think the seeds need O2 for respiration? ____________________________________________ Record your results in worksheet 5-1. 4. How does CO2 enter leaves? Where does CO2 enter a leaf? Does it enter through the epidermal cells, or does it enter through the stomata? a. Remove a healthy leaf from a Coleus plant. Make an epidermal peel of the lower surface of the leaf by barely scoring the surface with a razor blade and peeling the outer layer of cells off. Mount with a drop of water on a microscope slide and examine under the microscope. Look for stomata, openings in the surface surrounded by a pair of cells called guard cells (see fig. 3.2). Count the number of stomata per cm2. Make another epidermal peel of the upper surface of a leaf. Count the number of stomata per cm2 for this surface and compare. Which surface has more stomata? (Usually in Coleus one surface has nearly all of the stomata. Is this the case for your plant? Which surface, upper or lower, has more?) b. An alternative way to view the stomata on the surface of a leaf is to make an impression of the surface with cellulose acetate. Paint the surface of the leaf with cellulose acetate, and let dry. Peel off the impression and observe with the microscope. If you have difficulty removing the impression, try wilting the leaf in a warm (not hot) oven first and then remove the peel. ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ Petroleum jelly FIGURE 5.2 APPARATUS TO DETERMINE RELATIONSHIP OF OXYGEN TO RESPIRATION IN GERMINATING SEEDS. Lev65484_ch05.qxd 6/4/01 2:59 PM Page 58 58 APPLIED BOTANY c. Set up four Coleus plants for this experiment. For the first plant, cover the upper surfaces of all the leaves with petroleum jelly. Place the plant in the test chamber, insert the CO2 sensor, and monitor the levels of CO2 for approximately 10–120 minutes. For the second plant, cover the lower surfaces of all the leaves and monitor the CO2 in the test chamber. For the third plant, cover both the upper and lower surfaces and monitor the CO2. Finally, for the fourth plant, leave all the leaves uncovered and monitor the CO2. This is your control. Place all four plants under high light intensity to stimulate photosynthesis. What do you observe? What is the relationship between the relative rates of CO2 uptake and the number of stomata on the open surface? Record your observations in worksheet 5-1. From your experiment, do you conclude that most of the CO2 enters the leaf through the epidermis or the stomata? (Chlorophytum)—one plant grown for 4 days in the dark and one grown in a greenhouse or under grow lights Procedure for Exercise B ____________________________________________ 1. You will test for starch in the leaves of four plants. Two of the plants have variegated leaves, and the other two have solid green leaves. One of the variegated plants and one of the solid green plants were grown for at least 4 days in the dark. The other two plants (one of each type) were grown in the light, either in the greenhouse or under grow lights. 2. Take a leaf from each plant. Mark the leaves from plants grown in the light with a notch so you can identify them later. Place each leaf in boiling water to remove any anthocyanins (purple or red pigments). Then place each leaf in hot ethanol to remove the chlorophyll. This should be done in a well-ventilated area. Finally, soak each leaf in iodine solution to detect starch. 3. Draw an outline of each leaf on worksheet 5-2 at the end of this laboratory topic. Indicate where starch is detected. What do you conclude is the effect of light on the production of starch? What is the relationship between the pattern of variegation and the detection of starch? Why do you suppose this relationship exists? If a leaf has little to no starch, how does the plant stay alive? ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ EXERCISE B: Saving for Another Day—Storing Starch Some of the first carbohydrates produced via photosynthesis are simple sugars such as glucose. Glucose is highly soluble in water, so many plants convert it to starch, the insoluble storage polysaccharide. Laboratory Topic 13 explores starch in a variety of food crops. In this activity, we will examine where starch is stored in a leaf after photosynthesis. Materials Needed for Exercise B Beaker of boiling water Beaker of hot ethanol in a double boiler (Caution: ethanol ignites easily.) Beaker of Lugol’s iodine solution Plants with solid green leaves, such as wandering jew (Tradescantia), geranium (Pelargonium), or ivy (Hedera)—one plant grown for 4 days in the dark and one grown in a greenhouse or under grow lights Plants with variegated leaves, such as Coleus, variegated geranium (Pelargonium), or spider plant ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ EXERCISE C: Transpiration Another important process in plants is the movement of water from the roots to the leaves. Transpiration of water from the surface of the leaves is an important part of this movement. Do plants transpire water at the same rates under the same conditions? What conditions increase the transpiration of water? When should you be more concerned about watering your houseplants or garden plants? When should you water more, or less? In this exercise, we will detect water vapor as it evaporates from leaves. Then we will compare plants under different conditions to see what variables affect the transpiration of water. Finally, we will look at the relative number of stomata in a given area of leaf and relate this factor to transpiration rates.