Chapter 10. Photosynthesis: UNDERSTANDING PHOTOSYNTHESIS
Student Learning Outcome s
At the completion of this exercise, the student will be able to:
1) Describe the role of photosynthetic organisms.
2) Discuss the contributions of van Helmont, Priestley, and Ingenhousz on the history of
understanding photosynthesis.
3) Compare and contrast the visible and electromagnetic spectrum
4) Discuss the role of pigments in photosynthesis.
5) Discuss the internal anatomy of a leaf
6) Describe the role of the stomata in a leaf
7) Describe the function and organization of chloroplasts.
8) Discuss the location and products of the light – dependent and light – independent
reactions.
Overview:
It is time that plants and other photosynthetic organisms receive a much – deserved
“thank you”. Without them we would not exist. On the early Earth, primitive photosynthetic
bacteria and algae produced copious amounts of oxygen, changing the atmosphere forever and
making it conducive to the development of more complex life forms. It is hard to believe, but the
algae that inhabit primarily aquatic environments produce nearly 80% of the oxygen in the
atmosphere today and serve as the basis of many food chains. Plants such as grasses, shrubs, and
trees dominate terrestrial landscapes, providing habitat, food, and commercial products. Plants
are eloquent green machines that harness sunlight and produce carbohydrates and oxygen
necessary for the plants themselves, as well as other living things. This remarkable process is
termed photosynthesis. Keep in mind that photosynthesis is not the opposite counterpart to
respiration.
Early philosophers and scientists were intrigued by the mystery of plant growth. Noted
Greek scientist such as Aristotle and Theophrastus thought that plants obtained their essential,
nutrients exclusively from the soil. Although partially true, it took several centuries for the
mystery of plant growth to unfold. The first step in understanding the mystery was taken by a
Flemish physician Jan Baptista van Helmont, in the mid – 1600s. he grew a willow tree weighing
2.5 kg in a pot filled with 91 kg of rich soil. Van Helmont provided the tree with water
periodically. After 5 years, the tree weighed nearly 75 kg and the soil lost only 57 g of weight.
He concluded that the tree gained weight from the water, not the soil. Although he did not
understand the role of sunlight and carbon dioxide in plant growth, he initiated the scientific
study of plant biology.
In the 1770s, the English chemist Joseph Priestley performed a series of unique
experiments emphasizing the importance of atmospheric gases in plant growth. Eventually,
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Priestley was credited with the discovery of oxygen. Building upon the works of Priestley and
the “founder of modern chemistry” Antoine Lavoisier, the Dutch physician Jan Ingenhousz
hypothesized that plants utilize sunlight to split carbon dioxide and use the carbon for growth. He
also concluded that plants expelled oxygen as a waste product. As the result of his work,
Ingenhousz is credited with describing photosynthesis. In the mid 1800s, the German botanist
Julius von Sachs discovered chloroplasts and their role in photosynthesis. The light reaction, or
light – dependent reaction, of photosynthesis was described by Theodor Engelmann in the 1880s.
in 1940, Melvin Calvin traced the route of carbon throughout the entire process of
photosynthesis.
Sunlight powers photosynthesis. Using a prism, the English physicist Sir Isaac Newton
demonstrated that white light consists of a variety of color ranging from red at one end of the
visible spectrum to violet at the other end. In the mid 1800s, James Clerk Maxwell illustrated
that the visible spectrum was a minute portion of a continuous spectrum, or electromagnetic
spectrum, that includes radio waves, visible light, x – rays, and cosmic rays. Radiations of the
spectrum travel in waves measured in nanometers (1 nm = 10−9m). Radiations with longer
wavelengths (radio waves) have less energy, and those with shorter wavelengths (x –rays) have
more energy.
1. Observe the image of the electromagnetic spectrum above. What has more energy, a
radio wave signal or a gamma ray? ___________________________________________
2. Observe the image of the electromagnetic spectrum above. What has more energy, green
light of purple light? _______________________________________________________
3. If you go scuba diving, how do the colors shift with depth? Why? ___________________
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For an organism to utilize light energy, it has to be absorbed. In living systems, pigments
absorb light energy. Some pigments, such as melanin, absorb all wavelengths of light, and they
appear black. At the other end of the spectrum, many pigments absorb only certain wavelengths
of light and reflect the other wavelengths. The light absorption spectrum of a pigment
illustrates the wavelengths that are absorbed; for example, green leaves contain the pigment
chlorophyll which reflects the green portion of the spectrum.
Chlorophyll is the most important pigments in photosynthesis. Several types of
chlorophyll exist in nature. Chlorophyll a is the main photosynthetic pigment in some
cyanobacteria and in plants. Other pigments important in plants but not involved directly in
photosynthesis are called accessory pigments. Xanthophyll is a yellowish pigments. (i.e. fall
leaves), and carotene is an orange pigment (i.e. carrots). Chlorophyll b is considered an
accessory pigment in plants, broadening the spectrum that can be used in photosynthesis.
Leaves are the most conspicuous part of a plant. They vary tremendously in shape and
size, and some large trees have more than 100,000 leaves. One of the major functions of a leaf is
as a photosynthetic factory. The internal anatomy of a typical leaf is complex. A waxy cuticle
covers the upper side of the leaf, and an epidermis completes the upper and lower layers of a
typical leaf. Scattered primarily throughout the lower epidermis are stomata (sing, stoma) which
are tiny openings regulated by guard cells. The stomata allow the carbon dioxide from the
atmosphere to enter the leaf.
The center of the leaf consists of the mesophyll, composed of palisade parenchyma and
spongy parenchyma. The palisade parenchyma is columnar in shape and usually appears
beneath the upper epidermis. The spongy parenchyma is loosely packed and surrounded by
numerous air spaces. Most of the photosynthetic activity occurs in the cells of the palisade
parenchyma.
Chloroplasts reside within plant cells and serve as the organelles of photosynthesis. A
chloroplast consists of two outer membranes that surround a semifluid matrix called the stroma.
A third membrane system forms a series of flattened sacs called thylakoids. In some
chloroplasts, the thylakoids become stacked, forming a granum. Pigment molecules embedded
in the membrane of the thylakoids initiate photosynthesis. Sugars are synthesized in the stroma.
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The overall reaction for photosynthesis is:
This reaction is the result of a series of chemical reactions that are controlled and carried out by
specific enzymes. These reactions of photosynthesis are divided into two distinct metabolic
pathways:
1) In the light reaction, or light – dependent reactions, the pigments chlorophyll absorbs
light energy from sunlight and produces ATP, the coenzyme NADPH, and oxygen. The
light – independent reaction takes place in the thylakoid membrane of the chloroplasts.
2) The dark reaction, also known as the light – independent reaction or Calvin cycle,
takes place in the stroma of the chloroplasts. It is responsible for the fixing of a
carbohydrate (glucose).
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Photosynthetic organisms are estimated to produce as much as 200 billion metric tons of
carbohydrate yearly.
During aerobic respiration, plants and other organisms use the oxygen produced as a
byproduct of photosynthesis. The carbohydrates produced during photosynthesis are converted
into many plant products such as fiber, wood, and other structural materials. In addition, the
simple sugars the plant produces can be converted into disaccharides and polysaccharides, such
as starch for energy storage. Sugars also are utilized in the synthesis or amino acids to form
proteins and other cellular components. Life on Earth depends on the ability of photosynthetic
organisms to convert the radiant energy of the sun into ATP, oxygen, and other nutrients.
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EXTERNAL STRUCTURE OF A LEAF
Photosynthetic reactions usually take place in the leaves. Leaves generally consist of a blade and
a petiole. The petiole attaches the fattened blade to the stem. In this activity, you will examine a
typical dicot leaf.
PHOTOSYNTHESIS
Photosynthesis is the process by which light energy converts inorganic compounds to
organic substances with the subsequent release of elemental oxygen. It may very well be the
most important biological event sustaining life. Without it, most living things would starve, and
atmospheric oxygen would become depleted to a level incapable of supporting animal life.
Ultimately, the source of light energy is the sun, although on a small scale we can substitute
artificial light.
Nutritionally, two types of organisms exist in our world, autotrophs and
heterotrophs. Autotrophs (auto means self, troph means feeding) synthesize organic molecules
(carbohydrates) from inorganic carbon dioxide. The vast majority of autotrophs are the
photosynthetic organisms that you are familiar with – plants, as well as some protistans and
bacteria. There organisms use light energy to produce carbohydrates. (A few bacterial produce
their organic carbon compounds chemosynthetically, that is, using chemical energy)
By contrast, heterotrophs must rely directly or indirectly on autotrophs for, their
nutritional carbon and metabolic energy. Heterotrophs include animals, fungi, many protistans,
and most bacteria.
In both autotrophs and heterotrophs, carbohydrates originally produced by
photosynthesis are broken down by cellular respiration, releasing the energy captured from the
sun for metabolic needs.
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The photosynthetic reaction is often conveniently summarized by the equation:
6CO2 + 12H2O (+ light energy, enzymes and pigments)
C6H12O6 + 6H2O + 6O2
Although glucose is often produced during photosynthesis, it is usually converted to
another transport or storages compound unless it is used immediately for carbohydrates
metabolism. In plants and many protistans, the most common storage carbohydrate is starch, a
compound made up of numerous glucose units linked together. Starch is designated by the
chemical formula (C6H12O6). Most plants transport carbohydrate as sucrose. The following
experiments will give you a better understanding of the principles of photosynthesis.
PAPER CHROMATOGRAPHY
Plants have a variety of pigments that are necessary for photosynthesis to occur. These
pigments, chlorophyll a, chlorophyll b, and accessory pigments such as carotene and
xanthophyll, absorb light. The accessory pigments ultimately transmit the light energy they
absorb to chlorophyll a.
We can easily separate pigments by using a technique called paper chromatography.
Pigment is applied as a spot to one end of the paper; that end is put into a solvent (just below the
spot). The solvent moves up the paper, taking the pigment particles with it. The properties that
affect how far and how fast a particular pigment’s molecules will move up the paper with the
solvent are:
The Properties of Pigments
Absorption How well the pigment molecules stick to or absorb in the paper (the molecules that
absorb better tend to move slower)
The size of the pigment in the solvent (larger molecules tend to move more slowly
Size
than smaller ones)
Solubility Solubility of the pigment in the solvent (more – soluble molecules move faster and
farther)
We can identify each pigment by determining its Rf (ratio of fronts) value. The Rf value
describes the relationship between the distance moved by the pigment and the distance moved by
the solvent, and is calculated as follows:
𝑹𝑓 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑚𝑜𝑣𝑒 𝑏𝑦 𝑡ℎ𝑒 𝑝𝑖𝑔𝑚𝑒𝑛𝑡
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑚𝑜𝑣𝑒 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
Materials
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
Chromatography jar (containing one inch filled with the 9:1 solution of 10% Acetone in
Petroleum Ether)
Chromatography paper
 Hematocrit tube
Paintbrush (fine – tip)
 Color Pencils
Forceps
 Calculator
Chloroplast pigment solution
 Metric Ruler
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Procedure
1) Obtain a chromatography paper. [Warning: Use glove for this experiment because the oil from
your hands may interfere with the development of the chromatogram results.]
2) Using a metric ruler, draw a pencil line (DO NOT USE PEN) measuring 2cm starting from the
bottom but leave 0.5 cm away from each side of the chromatography paper as demonstrated on
the diagram shown below.
3) Next, draw another pencil line (once again DO NOT USE PEN) measuring 1cm beginning from
the top of the chromatography paper as demonstrated on the chromatogram shown below.
4) Using a small paint brush (fine tip) or a hematocrit tube paint a line of chloroplast pigment
solution across the paper chromatography on the 2 cm drawn line and let it dry. Repeat this step
10 times to obtain a better result. [NOTE: before applying a second application of pigment
solution, let pigment dry before applying another application of the chloroplast pigment solution.]
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5) Place your chromatography paper into a chromatography jar (containing one inch filled with the
9:1 solution of the 10% Acetone in Petroleum Ether) with the pigment at the bottom and close the
jar (see figures below). [Caution: the solvent and the pigment solution are highly flammable.
Keep them away from open flames.]
Accessory pigments
Accessory
pigments
6) Once the solvent reaches the 1 cm pencil marking, remove the paper chromatography with
forceps and place it on a paper towel.
7) Calculate the 𝑹𝒇 value for each pigment by measuring the distance of the solvent from its origin
to the highest point it traveled on the paper. Then measure the distance the different pigments
travel from the origin (2 cm pencil mark) to the center of each pigment band. Calculate the 𝑹𝒇
value by using the formula below. Record your measurement on table 10.1
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Band #
1
2
3
4
Pigment
Carotene
Xantophyll
Chlorophyll a
Chlorophyll b
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Solvent (9:1) Petroleum
Ether in Acetone
Color
Orange – Yellow
Yellow
Blue – green
Olive to
yellowish-green
Clear
Migration
Distance (mm)
𝑹𝒇 Value
1. What does a small 𝑅𝑓 value tell you about the characteristic of the moving molecules?
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2. Which is more soluble in the chromatography solvent, xanthophylls or chlorophyll a?
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3. Why did the plant extract appear green if yellow xanthophylls were present?
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4. Based on your observations during this experiment, what is responsible for the change in leaf
color during the fall?
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CARBON DIOXIDE UPTAKE DURING PHOTOSYNTHESIS
Recall that plants require carbon dioxide to produce carbohydrates (sugar0 during the light
independent reaction of the photosynthesis. To detect the uptake of carbon dioxide by the plant, you will
use a pH indicator solution. An indicator is a molecule that changes color depending on pH. In this
experiment you will be using a phenol red solution. Phenol red turns yellow when adding carbon dioxide
to its substance making it an acidic solution (pH < 7.0) and when red there is no presence of carbon
dioxide making it a neutral to basic solution (pH > 7.0)
In order to see this reaction take place, your breath will provide the carbon dioxide to a solution of phenol
red. This results in the following chemical reaction.
𝐇𝟐 𝐎 + 𝐂𝐎𝟐  𝐇𝟐 𝐂𝐎𝟑  𝐇 + + 𝐇𝐂𝐎𝟑
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A plant added to the solution can “fix” the carbon dioxide (that you just exhaled into) and this will cause
the pH to rise (become more alkaline). Your solution then turns red.
Materials
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2 Large test Tubes
Test tube Rack

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

Lamp
400 mL Beaker
Straw
10 cm of Elodea
Procedure
1) Professor will provide the students with the Phenol Red Solution in the 400 mL Beaker
2) Fill one test tube # 1 with Phenol red solution
3) With the straw, blow on the remaining phenol red until it turns yellow, then add the yellow
phenol red to test tube #2.
4) Insert a 10 cm Elodea on test tube #2.
5) Place both test tubes in front of a bright light for 60 – 90 minutes. [Note: This will ensure that
the plant has enough supplies of ATP and NADPH necessary for the light – independent
reaction.]
Test Tube 1 Phenol red (-) control.
Test Tube 2 Phenol red that turned
yellow (Co2) + Elodea. This is your
experimental control.
1. Has the color in either test tube changed? If so why? Explain.
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2. Explain. What is Carbon fixation?
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3. Did you see a change in color of the phenol red test tube with no elodea added? Why or why not?
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TEST FOR STARCH
As indicated by the overall formula of photosynthesis, one end product is a carbohydrate
(𝐂𝐇2 𝐎). But a number of different carbohydrates have the empirical formula𝐂𝐇2 𝐎. In this
experiment, you will perform a simple test to visually distinguish between two different
carbohydrates and water.
Materials
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

Bottle of Lugol’s solution
Bottle of double distilled water
Bottle of Starch solution


Bottle of Glucose solution
Three depression (spot) plates
Procedure
1) Place one drop of double distilled water, starch and glucose in the three depression (spot)
plate. Add one drop of Lugol’s solution in each spot.
2) Record your result for the presence of starch on the following table.
Solution
[(+/-) for the presence of starch]
Double distilled water
Starch
Glucose
RELATIONSHIP BETWEEN LIGHT AND PHOTOSYNTHESIS PRODUCTS
This experiment addresses the hypothesis that light is necessary for photosynthesis to proceed.
Materials
Per group:
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
Sharpie
1000 mL Beaker (fill with distilled
water and glass beads)
Hot plate
Long forceps
2 petri Dishes
Lugol’s Solution
Per Lab:
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


Hot Plate
1000 mL Beaker (fill with ethanol
and glass beads cover with foil
paper)
Light – grown geranium plant with
leaves cover or marks
Dark – grown geranium plant with
leaves cover or masks
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Procedures
1) Observe the two geranium plants available. One plant has been growing on bright light for several
hours. The other has been kept in the dark for a day or more. Bath leaves have an area cover with
a piece of foil paper.
2) Write a prediction regarding the presence of starch and the activity of photosynthesis for each
growing condition leaf in the following table.
Geranium Plant Growing
Condition
Light – Growing Plant
Dark – Growing Plant
Covered Area
Uncovered Area
3) Select a leaf from one of the two geranium plants. Pigment present in the pants must be removed
before a test for starch can be performed. Kill the leaf and extract the pigment.
a) Turn on the hot plate and set it to high setting. Allow the water to come to a boil.
b) With a sharpie, label one Petri dish light – grown plant and the other dark – grown plant
c) Remove a leaf from each condition plant and take it to your station.
d) Remove the piece of foul paper from both leaves.
e) Place one leaf in the beaker of boiling water for about a minute. This kills the tissue and breaks down
internal membranes (cell wall, plasma membrane, and vacuolar membrane)
f) Remove the wilted leaf from the water with the long forceps and place it on the Petri dish.
g) Place the wilted leaf in the beaker of boiling alcohol and cover it. Let it set for about a minute. This
will extract the photosynthetic pigments from the plant tissues. When the pigments have been
extracted, the liquid will appear green, and the leaf will appear to be mostly bleached.
h) Remove the leaf from the alcohol with forceps, and dip it back into the boiling water for about 15
seconds to soften and to remove excess alcohol.
4) Test the plant for the presence of starch:
a) Place killed leaf in a Petri dish and pour 2 mL of Iodine (Lugol’s) solution on top of the leaf.
b) Let it soak in the Iodine solution for a couple of minutes. Rinse the leaf and Petri dish with water to
remove the iodine solution in order to observe the pattern of staining.
c) Record your results in the given table
d) Repeat steps 3e – 4c for the other leaf.
Geranium Plant Growing
Condition
Light – Growing Plant
Dark – Growing Plant
Covered Area
Uncovered Area
1. What does the blue – black coloration of the leaf show?
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2. Why did the masked area fail to stain?
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3. Write the conclusion accepting or rejecting the hypothesis.
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ABSORPTION OF LIGHT BY CHLOROPLAST EXTRACT
People tend to think of sunlight as being white. However, as you will see in this experiment,
white light consists of continuum of wavelengths. If we see light of just one wavelength, the light will
appear colored.
When light hits a pigmented surface, some of the wavelengths are absorbed and others are
reflected or transmitted. In this experiment, you will discover which wavelength are absorbed,
transmitted, or reflected by particular pigments; among them the photosynthetic pigment chlorophyll.
Materials


Spectroscope
Colored filters (Blue, green and red)


Colored pencils
Small test tube containing pigment extract
Procedure
One way to separate light into its component parts is to view the light through a spectroscope.
The spectroscope contains a prism that causes a spectrum of color to form. A nanometer scale is
imposed on the spectrum to indicate the wavelength of each component of white light.
A) Observe the spectrum of white light given off by a light bulb through the spectroscope. With the
colors violet, blue, green, yellow, orange, and red record what you see on the given table.
B) Observe the spectrum produced by the three colored filters using the spectroscope.
1. Which color or colors are absorbed when the red filter is placed viewed through the spectroscope.
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2. Which color or colors are absorbed when the blue filter is placed viewed through the spectroscope.
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3. Which color or colors are absorbed when the green filter is placed viewed through the spectroscope.
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4. Make a general statement concerning the color of a pigment (filter) and absorption of light by that
pigment.
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C) Now obtain a small test tube containing spinach chloroplast pigment extract and place it
between the light source the spectroscope. By adjusting the height of the tube so that the upper
portion of the light passes through the pigment extract and the lower portion is white light, you
can compare the absorption spectrum of the pigment extract with the spectrum of white light.
An absorption spectrum is a spectrum of light waves absorbed by a particular pigment. By contrast, the
wavelengths that pass through the pigment extract and are visible in the spectroscope make up the
transmission spectrum of the pigment (Note: see picture below and picture on page 14).
D) Using the colored pencils, record the transmission spectrum of the chloroplast extract on the
following table.
1. How does the absorption spectrum of the chloroplast extract compare with the absorption
spectrum of the green filter?
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2. How might you explain the difference in absorption by the green filter and by the chloroplast
pigment extract? [Hint: see electromagnetic spectrum on page 14 and paper chromatography
table on page 10.]
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___________________________________________
Last Name, First Name [lab partner N0. 1]
____________________________________________
Last Name, First Name [lab partner N0. 2]
_______________________________
_______________________________
Last Name, First Name [lab
partner N0. 3]
___________________________
Last Name, First Name [lab
______________________
Section
partner N0. 4]
______________________
group #
Date
REVIEW QUESTIONS
1. Write the equation for photosynthesis
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2. Why photosynthesis is considered the most important chemical reaction of Earth?
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3. Compare and contrast the visible and electromagnetic spectrum.
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4. Name and describe three pigments found in the labs in this chapter.
(1) _______________________________________________________________________
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(2) _______________________________________________________________________
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(3) _______________________________________________________________________
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5. Sketch and label the basic internal anatomy of a leaf.
1.
2.
3.
4.
5. Describe the role of the stomata in leaves
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_____________________________________________________________________________
6. Sketch and label a chloroplast, including the membranes, stroma, thylakoids, central vacuole,
and nucleus.
6. grana.
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7. Describe the light – dependent and light – independent reactions of photosynthesis?
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8. How is chromatography used to study plant pigments?
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9. Discuss the succession of C3 and C4 plants in your yard or an open field through the four
seasons.
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10. Describe several products of photosynthesis that are important to humans.
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