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