Objectives:
1) To explore the transpiration-cohesion-tension theory of how water moves in plants.
2) To determine how different environmental factors, e.g. humidity and wind, affect the rate of sap flow in plants.
I. BACKGROUND
Everyone is familiar with the animal heart, and it’s function of moving fluids bearing nutrients and dissolved gasses through the animal body. Plants also must move nutrients, but have no similar pump. This lab will explore the most commonly accepted model for how movement of fluids is accomplished in plants, the transpiration-cohesion-tension theory.
Using this theory, we will explore how different environmental conditions affect the transpiration rate. Remember that transpiration occurs when the stomates open and water evaporates from the leaf surface. Transpiration of water from spongy mesophyll cells creates a difference in the osmotic pressure of the cell solution in these cells relative to their neighboring cells, an osmotic gradient that causes water to diffuse into the mesophyll from their neighbors. Since these neighboring cells include xylem cells, transpiration at the leaf surface causes the movement of water into the living cells of the leaf from the xylem cells, which are dead. It is this movement that establishes the flow of water in the xylem because water molecules cohere to each other, then as one molecule evaporates it pulls the next water molecule with it.
Thus, transpiration provides a “pull” on water from the leaves of the plant. The evaporation of water from spongy mesophyll cells is sufficient to power the lifting of water in trees. Careful experimentation has shown that the great cohesive properties of water molecules (that is their ability to hang together because of hydrogen bonds in spite of the tension created by lifting the water in the xylem cells) plays a critical role in making the ascent of sap possible in tall plants.
The stomates play a critical role in controlling this evaporation process. If the stomates are closed, the interior leaf space quickly becomes saturated with water and diffusion out of the mesophyll cells stops. When the stomates are open, the water molecules suspended in the interior leaf space (that is the water vapor) diffuse out into the environment, because water-vapor concentration is lower outside of the leaf. Because the stomates control the diffusion of water vapor out of the leaf interior, they control the transpiration process.
The transpiration rate, however, is not something directly measurable so we need something to substitute for the transpiration rate. One good measure of transpiration rate is sap flow since the rate of flow, by the theory, is directly correlated with the rate water transpires from the leaf surface.
The term “sap” refers to the aqueous solution of mineral ions and dissolved carbohydrates that move in the xylem of plants. The real interest in sap flow comes from looking up at very tall trees and asking the question, how does the water get to the top of the tree? Plant biologists with an engineering background knew that a regular suction pump can only lift water 34 feet up a pipe, so how do trees - which can reach almost 400 feet in height (redwoods and eucalyptus) do it?
Sorting out the possibilities leads to the following set of ideas. To begin with, the water can be pushed, pulled, or pushed and pulled both. We can identify three general locations from which force can be applied:
i) in the roots (providing “push”),
ii) in the vascular system itself (the xylem), or
iii) in the leaves outside of the xylem (providing “pull”).
Environmental conditions affect transpiration rates when the stomates are open. For instance, strong winds may cause water to evaporate from the surface of the leaf, which in turn would increase the transpiration rate. But if the winds become too strong, stomates may close and thus decrease, or stop transpiration. Increased levels of carbon dioxide have been shown to be one factor that triggers the closing of stomates, which in turn would also decrease, or stop transpiration.
II. FORMING HYPOTHESES
Observation: For water and nutrients to get from the roots to the leaves, sap needs to move through the xylem.
Questions: 1) Why does sap move? 2) How do environmental condition affect sap flow?
Hypotheses:
1) Leaves are sufficient to draw water through stems.
2) Changes in environmental conditions affect water movement.
Experiment: Using a transpiration rig, we will measure the rate of flow of water into a plant stem at room temperature, with increased wind, and increased humidity conditions.
Predictions: Develop explicit predictions for the rate of water flow for each of the three conditions:
1) ambient conditions
2) increased wind
3) increased humidity
Results: Record your results in graphs. Does the rate of sap flow increase under wind conditions? What is the affect of humidity on the sap flow rate?
III. METHODS
A. PROCEDURE FOR SETTING UP TRANSPIRATION RIG
First you need to build your transpiration rig. See Figure 1 below for reference.
1. Fasten four clamps along a ring stand so that they are well apart. The top clam must be a ring clamp
2. Rest the funnel in the ring clamp. Fit one of the two shorter hose ends from the three-way hose onto the funnel.
3. Wrap tape around the narrow end of the 1/10 in 1/100 ml pipette so that it will fit snugly into the second short hose end. Fasten the jaws of the second clamp from the top over the cork on the pipette so that the narrow end is down. Finally, place the second short hose end over the narrow end of the pipette.
Figure 1. Example of a “transpiration rig” for measuring the flow of water into a plant stem.
4. Put a white plastic or metal hose clamp in place, but not tightly shut, on both short hoses, and the long hose. These are used to stop the flow of water through the hoses between trials. Submerge the long third hose end in about three inches of water in the washbasin.
5. Pour water from a beaker into the funnel to fill the hose system. Drain water out of the long hose into the wash basin until no bubbles are visible in the hoses. You may have to work bubbles along the hose with your fingers. Make sure that the third hose end remains submerged in the washbasin.
6. Make sure both hose clamps on the short hoses are completely tightened down when you have gotten rid of all the air.
7. Take a tree shoot from the supply. Tightly wrap a piece of parafilm around the stem very near the bottom to make a watertight seal in the hose.
8. Hold the end of the stem underwater and take a clean slice off the base with a razor blade or clippers.
9. Be sure there is an adjustable metal hose clamp over the end of the long hose and then insert the base of the stem into the hose UNDERWATER. If either the hose or the plant emerges, let the water in from the funnel to force air out of the tube, make a new cut in the stem, and reinsert the stem. Be sure that the parafilm you wrapped the stem with makes a watertight seal with the hose end, and then tighten the hose clamp around the tip of the hose where the stem emerges. Be sure that you do not over tighten the hose clamp and crush the stem which will reduce water flow through it.
10. Clamp the stem between the jaws of the lowest clamp on the stand. Dry the leaves with a towel if they are wet.
11. Attach a thermometer to the remaining clamp on the stand. Be sure to record temperatures during each experimental trial that you conduct.
B. MEASUREMENT OF SAP FLOW UNDER VARIOUS CONDITIONS
NOTE: Before you start measuring anything, you may want to jump to Section IV Analysis to see what data you need to collect and make the appropriate data sheets.
11. Determine the sap flow rate under control lab conditions. Open the screw clamp leading to the pipette and adjust the water level so you can measure its drop in the pipette. Measure the amount of water that has flowed into the stem over ten minutes, minute by minute or measure the amount of time it takes to empty the pipette, or some similar measure. You and your teaching fellow need to decide based on how fast the water is taken up by the stem.
12. Determine the effect of wind on the system with a small fan, which is also available in the lab. Measure rate of water flow into the stem the same way as for controlled lab conditions.
13. Determine the effect of increased humidity by covering the whole shoot with an airtight transparent plastic bag, close with masking tape, and wait five minutes. Then measure the rate of water that flowed from your pipette as you did for the previous two trials.
14. For each of your three treatments, ambient, bag, and wind, graph your results using either graph paper or a graphing program like Excel.
IV. Analysis
15. Working together within your group, we will now analyze the data. Start by looking back to the hypotheses and the predictions you generated. Using the chart below, calculate a set of average flow values for each treatment.
a. Rate of Sap Flow
Table 1. Sap flow measurements for three trials under different conditions.
Trial #1 / Trial #2 / Trial #3 / AverageSap Flow
Ambient
Elevated Wind
Elevated Humidity
b. Decide with your teaching fellow how to summarize your results in order to compare treatments.
16. Evaluate the hypotheses
a. Using your results, are these results consistent with the description of transpiration at the beginning of the lab? And are they consistent with the other groups in the class?
b. Are the hypotheses supported or refuted by the data collected?
c. Discuss possible explanations for the differences or similarities among treatments in terms of what you have learned about the flow of water out of leaves via stomates.
d. Finally, what aspects of the transpiration theory are left unaddressed by these experiments? What experiments would you design to test the remaining ideas?
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