BIO203 – Laboratory 4 Transpiration

Introduction

From the previous lab and lecture materials, you are familiar with the general anatomy of a vascular
plant, and know that water is taken up via the roots, travels up the stem, and exits the plant via openings
on the leaf known as stomata (singular: stoma). This process is known as transpiration. Plants
transpire 99% of the water that is taken up through the roots, with only 1% being used directly in
metabolic functions such as photosynthesis. This seemingly inefficient process is used to generate the
tension required to drive water up from the roots to the entire height of the plant.

The transport of water upward from

roots to shoots in the xylem is
governed by differences in water
potential, with water molecules
moving from an area of high
potential (higher free energy, more
water) to an area of low potential
(lower free energy, less water).
Transpiration creates a lower water
potential in the leaf, and the
cohesion-tension hypothesis
describes the forces that facilitate
water movement through the plant
(see Figure 1).

Tension applied to the column of

water between root and transpiring
leave depends upon hydrogen
bonds between water molecules
(cohesion) and with polar
substances (adhesion), such as
elements in the xylem walls. This
property creates a tiny column of
water in each xylem tube with the
tensile strength of a steel wire. If
that column of water remains
unbroken, water evaporating
through the stomata will generate a
negative pressure gradient right
down to the root. At the root, water
will be absorbed in response to this Figure 1. Transpiration: the cohesion-tension theory explaining water movement in the xylem. Image from
Pearson Education 2008.
stress on the column and follow the
water potential gradient.

Stomata must open to allow for the entry and egress of gases (CO2 used in photosynthesis and O2
produced in photosynthesis), however this also results in the evaporation of water. Consequently, a
balance must be maintained between the transport of CO2 and O2 and the loss of water. To maintain
homeostasis, plants must adjust their rates of transpiration in response to environmental conditions. This
is accomplished both with anatomical and physiological features that enable a given species to be better
adapted to survive in its particular habitat and climate (please review the differences between C3, C4
and CAM plants here).

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BIO203 – Laboratory 4 Transpiration

The controlled opening and closing of stomata allows the

plant to regulate both gas exchange and water balance. The
two small cells that border the stomatal pore are called
+
guard cells. An accumulation of solutes and/or ions (e.g. K )
causes an influx of water into guard cells increasing the
water pressure potential of the cells. This increase in turgor
pressure pushes apart the cells, thereby opening the
stomatal pore. As guard cells lose water and shrink, they
Figure 2. Opening and closing of stomata by guard cells.
straighten and close the stomatal pore (see Figure 2). (a) When guard cells absorb water and swell, they become
curved and open (b) When they lose water they shrink and
close. Image from Mauseth et al. 2014.
The location and density of stomata is strongly influenced by
genetics and niche adaptations. They commonly occur on both the upper (adaxial) and lower (abaxial)
leaf surfaces (though relative abundance may vary), but they can be found exclusively on the abaxial
surface in some tree species (e.g. oleander, aspen), exclusively on the adaxial surface in some plants
(e.g. water lily), and are absent altogether on submerged leaves of aquatic plants. (Can you explain why
this could be?)

In this lab, we are trying to establish a connection between form and function, i.e. we would like to test
whether there is a correlation between a physical characteristic (leaf surface area and/or stomatal
density) and the rate of transpiration. You will also perform an experiment examining the relationship
between different physiological adaptations and transpiration rates. Specifically, you will measure and
compare the rate of transpiration between C3, C4 and CAM plants (Part A). In Part B, you will learn to
use an image analysis program (ImageJ) to calculate the surface area of the leaves of the plant used in
Part A. Lastly (in Part C), you will make peels of the epidermal leaf surfaces to estimate stomatal density.

Objectives

! Determine the rate of transpiration for your plant (C3, C4 or CAM) per leaf surface area
2
! Determine stomatal density for your plant (C3, C4 or CAM) per mm and use this to infer
stomatal density per leaf
! Determine whether leaf surface area and/or stomatal density are related to the rate of
transpiration for your particular plants
! Examine the class data and compare transpiration rates, leaf area & stomatal denisty among C3,
C4 and CAM plants.

Thought Questions

When comparing transpiration rates among different types of plants (C3, C4 and CAM), you might wish
to consider...

1. Do all plants transpire at the same rate? Is there a relationship between the habitat to which
species have adapted and their rate of transpiration?
2. What physical and/or physiological adaptations might the different species have that would affect
their transpiration rates?
3. Are leaf surface area and the number of stomata correlated to the rate of transpiration?

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BIO203 – Laboratory 4 Transpiration

BIG IDEA 4
Part A: Measuring Transpiration Rate using a Potometer

Reagents, Supplies and Equipment

•
PREPARATION
Representative plant species: bean (C3 plant), corn (C4 plant) and kalanchoe (CAM plant)
• Clamp stand with 3 clamps • Vaseline
• Light source (max. setting)
Materials •andRazorEquipment
blade
• Representative plant species that are available in a particular region or seas
• 1mL pipette with attached clear plastic • Dissecting needle
as Impatiens (a moisture-loving plant), Coleus, oleander (more drought tol
tubing • Dissecting tray
Phaseolus vulgaris (bean seedlings), pea plants, varieties of Lycopersicon (to
• Syringe Kimwipes
peppers,•ferns, / paper towels
or even Wisconsin Fast Plants (If students plan to investiga
• 400 mL beaker transpiration in several different species of plants, you will have to purchas
of plants, or students can use cuttings from plants found on campus. Note
Procedure: plants can be used to study other biological concepts, such as plant evoluti
selection, genetics, adaptation, and plant reproduction.)
1. Fill both the 400 mL beaker and the dissecting tray halfway with water.
• Safety goggles,
2. Attach the syringe to the end of the 1 mL pipette-tubing apparatus.calculator,
Placemicroscope,
the free-endmicroscope
of the tubingslides, clear cellophane
nail polish, scissors
into the beaker. Using the syringe, pull water up into the pipette through the tubing.
• Graph
Watch for any air bubbles. If bubbles form, paper
you will need andtometric ruler
flush out as water
the neededand to determine
start over.leaf surface area
3. Fill the pipette approximately to the 0 mL mark• and leave the syringe attached to the pipette.
Potometer, which students assemble from clear plastic tubing, a ring stand
It doesn’t need to be exactly at the 0 mL, just make
clamp, andsure to record
a 0.1-mL the actual
or 1.0-mL pipette,starting value.
depending on the diameter of the s
4. Bend the apparatus into a U-shape, then attach the tubing-pipette connection in one clamp
is recommended that you have available clear plastic (Figuretubing of different siz
3), and place the free end of the tubing in the dissecting
accommodatetray. stems from different plants.) A syringe without needle can b
Make sure that the water is at the very endfilling
of thethe
tubing
tubing before placing
with water, oritthe
in tubing
the dissecting tray.using a water bottle
can be filled
should be able to make the observations that air bubbles in the tubing coul
VERY IMPORTANT: During the following steps, the with
cuttranspiration
end of the plant and that
mustwhen
always assembling
be under thewater
potometer, as shown in F
end of
and not exposed to air! If the end comes out of the water, a the
smallstemairmust
bubblebe immersed in the in
will be trapped water. If students are using a gas
the end
sensor, the
and the plant will not be able to draw up water as efficiently. tubing
If this is inserted
happens, you directly
must cut into
offthe
thedevice;
end no pipette is required
again about a centimeter up the stem.
Plant Calibrated
cutting pipette
5. Place the stem of your plant of interest in the dissecting tray. While

mm
0

keeping the lower stem of your plant under water in the dissecting
1

tray, cut off the stem at an approximately 45° angle with a razor.
6. Stick the stem of your plant into the free end of the tubing. Remove
2

Tight
from the dissecting tray and dry apparatus and plant with Kimwipes.
3

seal
Ensure that the water levels are the same on both sides, and 4

the water precisely extends to the end of the tubing. If 5

needed, adjust accordingly using the syringe.

7. Attach the tubing-plant connection in the other clamp of the
potometer, being careful not to crush the plant stem (see Figure 3).
8. Smear enough Vaseline to make a watertight seal where the plant’s Water-filled
stem and the tubing come together. tube
9. Turn on the light source and ensure it is set to the maximum setting.
10. Remove the syringe and watch the water level.
Figure 3. Potometer assembly. Image
If you have a watertight seal, the level of water in the pipette Potometer
from http://www.phschool.com/science/
should not change. If the water level drops, you must repeat this biology_place/labbench/lab9/design.html
Figure 2. Potometer Assembly
process (steps 1-9).
11. Read and record the water level in the pipette at time zero and continue recording readings every
For whole
minute for the first 5 minutes, and then every 5• minutes plant
until 60 transpiration, small potted
minutes has passed (seeplants
Tablewith 1). many green leaves
Impatiens, tomato seedlings),
! After 10 minutes of recording, start the surface peels described in Part C. the plastic container they come in, one-gallo
plastic food storage bags, and string
! After 60 minutes of recording, remove the plant from the tubing for use inBio_T_Lab11_02 (If
Part B. using this method, students place t
potted plant or root ball with dirt in the plastic bag.)

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BIO203 – Laboratory 4 Transpiration

Part B: Calculating Leaf Surface Area using ImageJ

Reagents, Supplies and Equipment

• Representative plant species (C3, C4 or CAM plants) from transpiration experiment
• Light source
• Ruler
• Digital camera
• ImageJ software

Procedure:
1. Count and record the total number of leaves on your plant.
2. Image capture of leaves:
! Arrange all leaves on a pink background in very bright-lighted conditions. Lay leaves out
around a ruler. This will serve as your reference for calculating area, so it should be clearly
displayed in the picture with no overlap of leaves. Also, fill in a pink slip of paper indicating
your group’s initials, the plant name/type and date.
Note that the quality of your picture will affect your results greatly, so ensure that you
minimize shadows and have clearly defined, non-overlapping leaves.
! Take a few pictures and save them with your group name (initials) and descriptive title (type
of plant) and import the picture from the digital camera to your computer.
3. Open the ImageJ program:
! This is listed under Course Applications, and then in the ImageJ folder. Open your leaf image
by clicking File → Open → your file name
4. Setting the scale from your reference:
! Draw a line over a 50 mm section of the ruler (click once to start the line and once again to
complete it), then select Analyze → Set Scale. In the Set Scale window, enter 50 into the
‘Known distance’ box and change the ‘Unit of length’ box to mm, check the box ‘Global’.

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5. Manually set the threshold setting to include entire leaf surface:

! First, convert your leaf image from colour to grayscale: Image → Type → 8-bit. Then, chose
Image → Adjust →Threshold and drag the sliders back and forth to get all of the leaf in red
(while minimizing background or shadow) and click ‘Apply’.

!
!

6. Calculate the area of the leaves:

! First, surround the leaves with the rectangular selection tool, then Analyze → Analyze
Particles. Enter 20 as the minimum particle size, select dropdown option ‘Show Outlines’,
check ‘Display results’ and ‘Include holes’ and click ‘OK’. The data window will give you the
area of each leaf surface examined – make sure that the program has recognized the correct
number of leaves. Record the values in Table 2.

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BIO203 – Laboratory 4 Transpiration

Part C: Calculating Stomatal Density using Surface Peels

Reagents, Supplies and Equipment

• Representative plant species (C3, C4 or CAM plants) from surface area experiment
• Compound microscope
• Microscope slides
• Clear nail polish
• Clear cellophane tape and scissors
• Ruler

Procedure:
1. Apply three patches (thin coat) of clear nail polish to both abaxial (lower) and adaxial (upper) leaf
surfaces. Leave to set.
Do NOT overlap the site of nail polish on the abaxial and adaxial surfaces – i.e. make sure
that the nail polish is on different parts of the leaf at the top and bottom
2. Label frosted slides (type of plant, and leaf surface: abaxial and adaxial).
3. When the peel is dry, cut a small piece of tape with the scissors and place it on the dried nail polish
patch. Press down on the tape to ensure good contact with the nail polish, and then pull on a corner
of the tape to remove the nail polish impression. Tape your impression to the appropriate
microscope slide, and use scissors to trim away any excess tape. You can
fit three peels onto one slide.
4. Examine the leaf impression under a compound microscope using the
40X objective lens (or 400X magnification).
! Search for areas where there are numerous stomata (see Figure 4)
and count all the stomata in the microscopic field. Record the
number in Table 3. Repeat counts for at least four other distinct
fields on the slide. Determine the average number of stomata.
5. From the average number per microscopic field calculate the average Figure 4. Impression of abaxial leaf
2 surface of Begonia rex showing numerous
number of stomata per mm (see Appendix A for calculating stomatal stomata. Image from Peterson et al. 2008.
density).
!
!
References

1. AP Biology Investigative Labs: An Inquiry-Based Approach. Investigation 11: Transpiration. 2012.

New York: The College Board. Retrieved from
http://apcentral.collegeboard.com/apc/members/courses/teachers_corner/218954.html
2. Cheung, K. 2013. Microscope calculations. Vancouver Community College Learning Centre.
Retrieved from library.vcc.ca/learningcentre/pdf/vcclc/MicroscopeCalculations.pdf
3. Holtzclaw, TK. 2008. The Biology Place: Transpiration lab. Pearson Education. Retrieved from
http://www.phschool.com/science/biology_place/labbench/lab9/intro.html
4. Krempels, DM. 2009. Botany laboratory: water movement through the xylem. Retrieved from
www.bio.miami.edu/dana/227/227_watermovement.pdf
th
5. Mauseth, JD. 2014. Botany: An Introduction to Plant Biology, 5 edition. Jones and Bartlett
Learning.
6. Peterson et al. 2008. Teaching plant anatomy through creative laboratory exercises. National
Research Council of Canada.

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Appendix A: Calculating Stomatal Density

When looking into a microscope, the distance across the center of the circle is referred to as the
diameter of field of view (dFOV).
1. You will need to determine the microscope magnification for all objective lenses. The magnification
is how many times the object has been enlarged. To calculate the total magnification, you will need
to multiply the magnification of the ocular lens with the magnification of the objective lens.
2. To calculate the dFOV, you will need to place a transparent ruler on
the microscope stage and measure the dFOV using the 4X
objective lens.
For example, for this particular microscope, we can see that
the dFOV at 40X magnification (4X objective lens x 10X ocular
lens) is 5 mm (see Figure 5).
3. However, you probably examined your leaf surface using the 40X
objective lens, and you cannot see the ruler markings well enough to Figure 5. Calculating dFOV using
millimetre ruler. Image from Cheung 2013.
calculate the dFOV directly for this magnification. To calculate the
dFOV for high magnifications, you will need to use the following equation:
dFOV #1 x magnification #1 = dFOV #2 x magnification #2
4. Now you have the diameter of your observed field of view. You can use this to calculate the average
number of stomata per mm , remembering that the area of a circle = πr .#
2 2

# #

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Data tables

Table 1 – Transpiration Rate (record type of plant here: ________________________________):

Time (minutes) Pipette value (mL)
0
1
2
3
4
5
10
15
20
25
30
35
40
45
50
55
60
Total water loss:

Table 2 – Leaf Surface Area for Representative Leaves:

2 2
Leaves Area (mm ) Leaves Area (mm )
Leaf 1 Leaf 9
Leaf 2 Leaf 10
Leaf 3
Leaf 4
Leaf 5
Leaf 6
Leaf 7
Leaf 8
Total

Table 3 – Average Number of Stomata per Microscopic Field of View (FOV):

Microscope
Adaxial surface Average Abaxial surface Average
FOV #
1 ! ! ! !
2 ! !
3 ! !
4 ! !
5 ! !

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