†††† In this laboratory you will apply what you learned about water potential from the Diffusion and Osmosis laboratory to the movement of water within plants.You will measure transpiration under different laboratory conditions and also study the organization of the plant stem as it relates to the movement of water.





At the completion of this laboratory you should be able to:


        Describe how differences in water potential affect the transport of water from roots to stems to leaves.

        Relate transpiration to the overall process of water transport in plants.

        Discuss the importance of the properties of water, including hydrogen bonding, adhesion, and cohesion, to the transport of water in plants.

        Quantitatively demonstrate the effects of different environmental conditions on the rate of transpiration in plants.

        Identify the vascular tissues of the plant stem and describe their functions.





†† The amount of water needed daily by plants for growth and maintenance of tissues is small in comparison to the amount that is lost through the process of transpiration, the evaporation of water from the plant surface, and guttation, the loss of liquids from the ends of vascular tissues at the margins of leaves.If the water that is lost from aerial plant parts is not replaced by water transported up from plant roots, the plant will wilt and eventually die.


†† The transport of water up from the roots in the xylem tissue is governed by differences in water potential.These differences account for water movement, not only from cell to cell, but over long distances within the plant.In a root, minerals transported from the soil accumulate in the xylem vessels of the vascular tissues of the stele.This, in addition to the negative pressure (tension) in the xylem tissues, lowers the water potential of the xylem.Thus, water will move into the xylem by osmosis, forcing fluid up the xylem vessels.This upward movement results in root pressure, but this pressure can only move water a short distance up the xylem.Rather than being pushed up from below by root pressure, the water and dissolved minerals in the xylem (xylem sap) are pulled upward as a result of transpiration.



†† The stomatal openings of a leaf open into the air spaces that surround the mesophyll cells of the leaf.The moist air in these spaces has a higher water potential than the air outside the leaf and water tends to evaporate from the leaf surface moving from an area of higher water potential to an area of lower water potential.The moisture in the air spaces is replaced by water from the mesophyll cells, lowering their water potential (solute in the mesophyll cells becomes more concentrated when less water is present - recall that increasing solute concentration lowers water potential).Water will then move into the mesophyll cells by osmosis from surrounding cells with higher water potentials, including those of the xylem.


†† The gradient in water potential between the xylem and the air outside the leaf that is caused by transpiration results in the transpirational pull of water from the xylem into the leaf mesophyll cells and eventually into the leaf spaces.Cohesion of water molecules to one another due to hydrogen bond formation causes this pull to transmit throughout the column of water in the xylem, all the way from the leaves to the roots. Adhesion of water molecules to the walls of the xylem cells offsets the effects of gravity.


†† The upward transpirational pull on the fluid in the xylem causes a tension (negative pressure) to form in the xylem, pulling the walls of the xylem inward.Recall that increased pressure causes water potential to become more positive.Tension, however, because it is the opposite of pressure, causes water potential in the xylem to decrease.This decrease, transmitted through the column of fluid in the xylem all the way to the roots, causes water to move from the soil across the cortex of the root and into the xylem of the stele.A root structure called the Casparian Strip located in the root endodermis tissue prevents the backflow of water from the xylem tissues into the soil.


†† Since the opening of stomates, which allows transpiration to occur, is also required for the entry of CO2 used in photosynthesis, a balance must be maintained between the two processes.Plants accomplish this by regulating the opening and closing of stomates on the leaf surface.



Transpiration in the Bush Bean Phaseolus vulgaris


†† Many environmental conditions, including those conditions that influence the opening and closing of stomates, will affect the rate of transpiration.Increases in temperature, light intensity, and the presence of dry air currents can increase transpiration rates.A humid environment usually slows the rate of transpiration.In this exercise you will measure the rate of transpiration per unit area of leaf tissue using a potometer.




Work in groups of 2-4 students.


A. Preparation of the plants.


1.      Use a sharp razor blade to cut the stem of a 2-3 week old bush bean plant, Phaseolus vulgaris, about 2 cm above the soil line.Place the pot to one side for later observations of the cut stump. (leaves of Geranium plants may be substituted).

2.      Place the plant stem in a water trough and recut the stem underwater to prevent air bubbles from blocking the xylem. Keep the cut surface of the stem underwater.

3.      Attach the pipette to a piece of clear plastic tubing.Attach a small syringe to the pipette. Fill the plastic tubing and the pipette with water from the water trough by pulling out the plunger on the syringe.The syringe will keep the water under negative tension and keep it inside the tubing and pipette while you work.

4.      Insert several centimeters of the cut plant stem into one end of the tubing.Seal the plant into the tube with petroleum jelly.


†††† Caution - the plant stem will crush if too much pressure is used to apply the petroleum jelly.


5.      Gently bend the plastic tubing into a "U" and lift from the water trough.

6.      This apparatus is called the potometer.Attach the plant/tube to clamps on a ring stand to maintain the "U" shape of the plastic tube.

7.      When the plant/tube is in place, gently remove the syringe from the pipette.Some water may flow from the plant end of the tube.Check the space at the cut plant stem for air bubbles.Insert the stem further into the tubing if necessary and reposition the petroleum jelly seal.It is OK if the water level drops in the pipette, but there must be an adequate amount of water in the tube.


†††† Caution - There must NOT be an air bubble at the surface of the cut stem.Do not get any of†††††††††††††††††††††† the petroleum jelly over the cut stem surface.


8.      Repeat steps 1-6 until the plants assigned to your group (2 or 3) have been prepared.Allow the plants to equilibrate for approximately 5 minutes.

9.      After all plants are positioned and equilibrated, carefully add a drop of dark dye at the tip of the pipette.As the water is transpired from the plant, the dye should move down the pipette.

10.  Record the total amount of water (in ml) that is transpired over time at 5-minute intervals for 30 minutes.If the dye moves below the calibrated level of the pipette, add another drop at the tip and continue recording.


B. Environmental Treatments.


Each group will be assigned one of the following treatments:††


A) Control - room conditions of light and wind.

B) Light - place a floodlight 1 meter from the plants.

C) Wind - place a fan on low speed 1 meter from the plants to create a gentle breeze.

†† Caution - be sure the breeze doesn't hit other plants.


D) Humidity - encase the plants in a clear plastic bag.Mist the leaves with water and leave the††††††††††††††††††††††††††† bottom of the bag open.

C. Experiment Conclusion.


At the end of the experiment, conduct the following steps:


1.      Cut the leaves off your bean plant.Blot off all excess water and weigh them.

2.      Estimated the total leaf surface area for each plant using the following formula:


††††† Leaf Surface Area (m2) =Weight of leaves (grams)

†††††††††††††††††††††††††† ††††††††††††††††††††††††160 grams/m2


3.      Give your leaf surface area data and the transpiration data for each plant to the instructor for the preparation of a class data set.

4.      Examine the bean stem stumps in the pot of soil.Record your observation.



Analysis of the Results


†† The class data set will be used in reporting the results of the experiment.The average loss of water (pressure change) over time for each treatment can be calculated as follows:


1.      Calculate the accumulated amount of water transpired at each time interval (5, 10, 15, 20, 25, 30).Divide this figure by the leaf surface area of the plant. To get the amount of water (ml) per surface area (m2).

2.      Repeat step 1 for each plant.

3.      Calculate an average value within a treatment for each time interval.

4.      Create a graph that shows the average amount of water lost per leaf surface area over time.Place the independent variable (time) on the X-axis and the dependent variable (average water lost/surface area) on the Y-axis.Be sure to label the axis and give the units. Place all four treatments on the same graph and give a key (color, symbol, line type etc.) to identify each treatment.



Answer the following questions on a separate sheet of paper:


1.         Write a hypothesis for the experiment.What were the expected results for each treatment?

2.         Give a written explanation of the results as shown in your graph.Explain how each of the conditions caused a change in transpiration as compared to the control (donít forget to tell what the control did too).

3.         Write a discussion comparing the hypothesis to the results.Are any of results suspect?What were possible problems in the experiment?

4.         What is the advantage to a plant of closed stomata when water is in short supply?What are the disadvantages?

5.      Describe several adaptations that enable plants to reduce water loss from their leaves.††††††††††

†††††† Include both structural and physiological adaptations.

6.      What was observed on the cut stumps at the end of the experiment?What process does this represent?

Structure of Plant Vascular Tissue


†† The movement of fluids and nutrients through the plant body occurs in the vascular tissue: the xylem and phloem of the roots, stems, and leaves.In this exercise we will study the structure of the plant vascular tissues.




1.      A stalk of celery has been placed in a water solution containing a water-soluble dye.The "stalk" is actually the petiole of a leaf.

2.      Examine the stalk for evidence of movement of the dye into the xylem.Record your observations.

3.      Use a razor blade to make a free-hand slice of the celery stalk.A small thinly cut wedge of the stalk will suffice.Place the slide on a microscope slide.Is there any evidence of the dye in the cut stalk?Record your observations.

4.      Place a drop of water to the celery slice and add a coverslip. Examine the slice with a microscope under the low power (4X) objective.Is there any evidence of the dye?†† Record your observations.

5.      Examine the celery slice under a higher power objective.Is there any evidence of the dye?Make a sketch to show the cells where the dye is found.

6.      Slice the celery stalk longitudinally (longways) and look for evidence of the dye.Record your observations.



Answer the following questions on a separate sheet of paper.


1.††† Summarize your observations of the presence of the dye in the celery stalk.

2.††† Give a representative drawing of the cells that contained the dye.What is the name of these†††††††† cells?

3.††† Explain how the dye was able to move throughout the celery stalk.

5.         If the dye were added to the soil around a celery plant, would you expect the dye to move

into the leaves?Explain.