1.

NINE TRANSPIRATIO N

OVERVIEW
In this lab your students will:
1.apply what they have learned about water potential from Lab 1 (Diffusion and Osmosis) to
the movement of water within the plant,
2.measure transpiration under different lab conditions, and
3.study the organization of the plant stem and leaf as it relates to these processes by observing
sections of tissue.

TIME RE QUIREMENTS
Exercise 9A requires a 50- to 60-minute class period for setup and data collection, but can
give excellent results with fewer readings as long as a full 10 minutes are used
for equilibration; 15-30 minutes for data analysis.
Exercise 9B requires one 45-minute lab period.

STUDE NT MATERIALS AND EQUIPMENT

Exerci se 9A

two-week-old plant seedlings 8-12

(Phaseolus vulgaris, or other plant)
O.I-mL pipettes in 0.01 divisions 1 12
electric fan
2-3
atomizer 2-3
100-watt floodlight 2-3
1
transparent plastic bags
3
clear plastic tubing (Tygon" or something similar)
(1/8" inner diameter and 1/16" wall thickness)
lO-mL syringe (to fill potometer) 40cm 5 meters
ring stands 1 12
extension clamps 1 12
petroleum jelly (tube or jar) 2 24
shallow tray of tap water 1
1
stopwatch or classroom clock
1 12
1 12 or 1
Exe rcise 98

nut-and-bolt micro tomes 3mL 12

paraffin wax 1 50mL
single-edge razor blades lOmL 12
glycerin 100mL
lOmL
50% ethanol
lOmL 100mL
distilled water
toluidine blue ° lOmL
3
100mL
100mL
small petri dishes 36
1
slides and cover slips
1 12
compound microscope
12

PREPARATION OF MATERIALS A ND SOLUTIONS

To melt the paraffin wax, put it in a beaker and place that beaker in a second beaker of water.
Heat the water until the wax just melts; do not boil.

50% ethanol (ETOH):

Add 45 mL of distilled water to 50 mL of 95% ETOH (if absolute or 100% alcohol is used, mix
1: 1 with water).

Toluidine blue 0:
2.Prepare phosphate buffer as follows:
a.To 4.24 g Na2HP04 add distilled Hp to 200 mL.
b.To 4.13 g NaH2HP04 add distilled Hp to 200 mL.
c.Mix 51 mL of a. (immediately above) with 49 mL of b. to pH 6.8.
2.Add 0.025 g toluidine blue ° to 100 mL buffer.
3.Mix 1: 1 with distilled water before use.
Note: Adjust volumes for the number of students in your class.

PREPARATI ON SUGGESTIONS
Exercise 9A:
Any potometer setup can be used for this experiment; the one used here is designed to be
simple and to yield quick results. Any plant can be used although, for sectioning purposes, a
soft herbaceous stem is preferable. For wind conditions, set the fan on low. For humidity
conditions, place a fairly heavy transparent plastic bag over the entire plant (but do not close off
the bag by taping the open end shut). Be sure to leave the pipette free for reading.

Exercise 9B:
Toluidine blue ° stain can be used to study the anatomy of many different stem types or roots.
Have students bring in common flora and determine whether the specimens are monocots or
dicots. Instead of petri dishes, cut-off paper cups can be used for staining and later discarded.
•Remind students to wait until the paraffin is completely solidified before making sections.
•Obtain the large nuts and bolts from a local hardware store.
14.a. Independent variable: Time (min)
b. Dependent variable: Water Loss (ml.zm')
Title: The Effects of Various Treatments on Water Loss in Plants
EXERCISE 9A: Transpiration
Note: In this sample experiment, the increase in leaf
temperature in
SAMPLE RESULTS the light
Table 9 .3: Class Average Cumulative Water Loss in mL/ m 2 treatment and air
(from page 103 in the student manual) movement in the
fan treatment
0.001 4-= Leaf Surface Area (m") resulted in a

Time (minutes)

Treatment 0 10 20 30

Room 0 2.19 4-.56 6.57

Light 0 4-.16 7.57 11.73

Fan 0 4- .50 7.58 11.00

Mist 0 1· 30 2·44 3.65

h
Sample data are from lO-minute intervals. You may have your students use 3-minute intervals. i
gher rate of water evaporation from the leaves.
Graph s.a However, if leaf temperature or air movement increased
(from page 104 in the student manual) even further, the rate of water loss from
the leaves might actually have decreased, due to the closing
of stomates as a mechanism for
1
decreasing very high Treatment b
2
rates of water loss.
I/ I Treatment c
ANALYSI S I/V OF
10 V
RESULTS
(from pages 105-106 in the
student 8 manual)

1.Answers Water Treatment a

will vary Loss 6
with v student data.
For best (ml.zm') V
v results, have
students l take their
/
data for 4 1 the
/ Treatment d
r

-
/
rate r '
'- " "
calculations
2 r "'
from Graph 9.l.
v

-
2.Light Condition - 1 I- Absorption of
light resulted in an ,.. increase in leaf
"
temperature; since o I":: :':: :
oevaporation 10 20 increases as
the
30temperature
rate of water
increases, the increase in leaf Time (min) temperature
results in an increased rate of leaf water loss.
Fan Condition - An increase in wind speed results in an increase in the rate of leaf water
loss because increased wind decreases the boundary layer of still air at the leaf surface. This
 boundary layer acts to slow leaf water loss. Increased wind also causes the rapid removal of
evaporating water molecules from the leaf surface. This results in a low water potential in
the air immediately surrounding the leaf, which leads to increased rates of water loss from
the leaf.
Mist Condition - Increased humidity in the air surrounding the leaf decreases the water
potential gradient between the saturated air in the leaf air spaces and the air surrounding the
leaf, resulting in a decreased rate of leaf water loss. However, when the humidity of the air
surrounding the leaf is very low, the water potential of the air is low. Therefore, the water
potential gradient between the air spaces of the leaf and the surrounding air is high, and the
rate of leaf water loss increases.
3.At each step of transpiration, water moves from an area of higher water potential to an area
of lower water potential. Students may give details in various plant structures: root hair,
xylem, leaf mesophyll, leaf air space, and stomata.
4.Advantage: Closed stomata prevent excessive water loss. This prevents the plant from
wilting and/or dying. Lack of water in the air spaces of the leaf is a limiting factor in the
light dependent reactions of photosynthesis.
Disadvantages: Closed stomata prevent CO2 from entering and 02 limits the light
independent reactions of photosynthesis. A decrease in the CO2 coupled with an increase in
02 in the air spaces of the leaf can result in the cells converting to photorespiration.
5.Adaptations to reduce leaf water loss include a reduced number of stomates, an increase in
the thickness of the leaf cuticle, a decrease in leaf surface area, and adaptations that decrease
air movements around stomates, such as dense hairs and sunken stomates. (Students may
also mention C-4 or CAM photosynthesis.)
6.Because leaves are all different in size, reporting the water loss without considering a unit
area would provide noncomparable data.
Further Investi gat ions
1.You may also wish to prepare an epidermal peel of a leaf to investigate the structure of the
stomata. Thi can be done by applying a small strip of nitrocellulose paper to a microscope
slide using acetone. Apply pressure to make the nitrocellulose paper stick. Cut out several
discs of leaf tissue using a cork borer and dip them in a 70% solution of acetone-ethyl
alcohol (70 mL acetone, 30 mL ethanol). Apply them to the nitrocellulose strip on the slide
and apply pressure with a small cork. Let the slide dry and remove the leaf disc. Examine
using the microscope. You may wish to stain the preparation.
2.An alternative preparation for investigating the stomata structure involves painting the lower
surface of a leaf with clear fingernail polish. Peel and mount the dried polish on a slide to
view stomatal structure and distribution. Compare numbers and distribution on top versus
bottom surfaces of leaves.
3.The transpiration rate of a plant can be followed by connecting a computer interfaced
pressure sensor to the tube of water. As the plant loses water, the air bubble in the tube will
increase and the pressure will decline. This method requires only one petroleum jelly seal
and quickly shows any system leaks.
4.Leaves from various plant species can be placed on a bulletin board and observations can
be made on the length of time needed for them to dehydrate. Structural modifications to
prevent water loss (heavy cuticle, etc.) can be related to the rate of water loss.
5.The structure of the stem can be studied by placing stalks of celery in water containing red
food dye. The food dye moves quickly up the stalk and leaves a red-colored trace of its
movement. The celery stalks are firm enough that free-hand razor sections can be made for
microscopic analysis. The stalks can also be split lengthwise to show the pathway of xylem
up the stalk.

NOTES
• The concept of reaction rates is also covered in Labs 2, 4, 5, and 12.
ο The movement of water can be linked to osmotic potentials in Lab 1.
ο Very large bean plants in the humidity treatment may actually show a gain of water to the
tube rather than a loss. Large plants have a great deal of water in their stems and leaves
under the pull of gravity. When transpiration is reduced, as in the high humidity treatment,
and the roots have been removed to prevent backflow (by the casparian strip), water may
move from the stem and leaves into the tube. This observation is a nice way to tie together
plant structure and water movement.
ο The light from an overhead projector can be aimed on a table top. This light is usually bright
enough to increase photosynthesis and transpiration without causing the plants to overheat.
ο An alternative method is to wrap the root balls of small well-watered plants (impatiens) in
plastic bags, mass each plant, and heat according to directions (light, fan, misted with plastic
bag, etc.). Mass should be recorded for five consecutive days.
ο Plants that also do well in this lab include vinca, chrysanthemum, and coleus.