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									          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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