In a transpiration experiment, the amount of water lost from a plant is measured to investigate how the upper and lower leaf surfaces, stomata, light, temperature, humidity, wind, and other factors affect the transpiration rate.
Most of the water absorbed by the roots is transported through the xylem to the leaves, where it becomes water vapor and is released into the surrounding air. This phenomenon is called transpiration.
Transpiration occurs mainly through the stomata of leaves, although a small amount of water is also lost through the leaf surface, including the cuticle. Because stomatal density often differs between the upper and lower surfaces of a leaf, an experiment in which the leaf surfaces are coated with petroleum jelly can be used to compare which surface contributes more to transpiration.
This article assumes a mass-loss method using a leafy branch and presents the introduction, precautions, items to record, reference experimental values, calculations, sources of error, an example results section, and discussion examples in that order.
Note:
This article is educational reference material intended to help students discuss results obtained in biology experiments at schools, universities, and similar institutions. The experimental conditions and measured values shown here are illustrative examples and are not actual experimental results.
If a utility knife or pruning shears are used to cut branches, never point the blade toward yourself or anyone nearby, and take care to avoid cuts.
When applying petroleum jelly to leaves, avoid contact with the eyes and mouth, and wash your hands after the experiment. Follow the laboratory manual and your instructor’s directions for the actual procedure.
Basics of Transpiration
Where Transpiration Occurs
Leaves contain intercellular air spaces. Water evaporating from the surfaces of mesophyll cells enters these spaces as water vapor. The vapor then diffuses into the outside air mainly through the stomata.
Stomatal Transpiration
This is transpiration through the stomata. Most of the water lost from leaves is generally lost by stomatal transpiration. The transpiration rate changes greatly depending on whether the stomata are open or closed.
Cuticular Transpiration
This is transpiration through the cuticle covering the leaf epidermis. It is generally less than stomatal transpiration, but it does not fall completely to zero even when the stomata are closed.
Roles of Transpiration
- Promotes the movement of water from roots to leaves
- Helps transport mineral salts
- Helps prevent excessive increases in leaf temperature
- Contributes to maintaining cell turgor
Main Items to Record in the Results
- Plant species used
- Number of branches and leaves
- Total leaf area
- Leaf-surface treatment conditions
- Surface to which petroleum jelly was applied
- Mass at the start of the experiment
- Mass at the end of the experiment
- Mass loss
- Measurement time
- Transpiration amount per unit time
- Transpiration rate per unit leaf area
- Difference from the control
- Inhibition rate
- Room temperature
- Humidity
- Presence or absence of wind, or wind speed
- Light conditions
- Number of measurements
- Mean
- Standard deviation
- Wilting and other changes in leaf appearance
Reference Experimental Conditions
| Item | Reference Condition |
|---|---|
| Plant material | Camellia branch with leaves |
| Number of leaves | 5 leaves per condition |
| Branch length | Approximately 15 cm |
| Container | Small flask containing water |
| Water-surface treatment | Covered with liquid paraffin to prevent evaporation from the water surface |
| Leaf-surface treatment | Untreated, upper surface only, lower surface only, or both surfaces coated with petroleum jelly |
| Measurement time | 60 min |
| Temperature | 25 ℃ |
| Relative humidity | 50% |
| Light condition | Light condition |
| Replicates | 3 per condition |
Reference Experimental Values
Leaf-Surface Treatment and Mass Loss
| Treatment | Initial Mass (g) | Final Mass (g) | Mass Loss (g/60 min) |
|---|---|---|---|
| Untreated | 128.42 | 127.82 | 0.60 |
| Petroleum jelly on upper surface only | 127.96 | 127.48 | 0.48 |
| Petroleum jelly on lower surface only | 128.15 | 127.98 | 0.17 |
| Petroleum jelly on both surfaces | 128.08 | 128.01 | 0.07 |
| Leafless control | 122.30 | 122.28 | 0.02 |
Example Replicate Measurements
| Treatment | Trial 1 (g/60 min) | Trial 2 (g/60 min) | Trial 3 (g/60 min) | Mean (g/60 min) |
|---|---|---|---|---|
| Untreated | 0.58 | 0.61 | 0.60 | 0.597 |
| Upper surface only | 0.46 | 0.49 | 0.48 | 0.477 |
| Lower surface only | 0.16 | 0.18 | 0.17 | 0.170 |
| Both surfaces | 0.06 | 0.08 | 0.07 | 0.070 |
Environmental Conditions and Transpiration
| Condition | Temperature (℃) | Relative Humidity (%) | Mass Loss (g/60 min) |
|---|---|---|---|
| Dark, no wind | 25 | 50 | 0.24 |
| Light, no wind | 25 | 50 | 0.60 |
| Light, airflow | 25 | 50 | 0.88 |
| Light, high humidity | 25 | 80 | 0.31 |
| Light, high temperature | 35 | 50 | 0.82 |
Reference Values Corrected for Leaf Area
| Treatment | Leaf Area (cm²) | Transpiration (g/60 min) | Transpiration Rate (mg/cm²·h) |
|---|---|---|---|
| Untreated | 120 | 0.60 | 5.00 |
| Upper surface only | 118 | 0.48 | 4.07 |
| Lower surface only | 121 | 0.17 | 1.40 |
| Both surfaces | 119 | 0.07 | 0.59 |
Calculation Methods
Mass Loss
If the initial mass is 128.42 g and the final mass is 127.82 g, the calculation is as follows.
Mass loss = Initial mass − Final mass
= 128.42 − 127.82
= 0.60 g
Transpiration per Hour
If the mass decreases by 0.30 g in 30 minutes, the calculation is as follows.
Transpiration rate = Mass loss ÷ Measurement time
= 0.30 g ÷ 0.5 h
= 0.60 g/h
Correction Using a Leafless Control
If the mass loss in the untreated group is 0.60 g and that in the leafless control is 0.02 g, the calculation is as follows.
Corrected transpiration = Mass loss in test group − Mass loss in leafless control
= 0.60 − 0.02
= 0.58 g/60 min
Transpiration Rate per Unit Leaf Area
If the leaf area is 120 cm² and the transpiration over 1 hour is 0.60 g, first convert 0.60 g to 600 mg.
Transpiration rate = Transpiration ÷ Leaf area ÷ Time
= 600 mg ÷ 120 cm² ÷ 1 h
= 5.00 mg/(cm²·h)
Transpiration Inhibition Rate
If the untreated group is 0.60 g and the lower-surface petroleum-jelly group is 0.17 g, the calculation is as follows.
Inhibition rate (%) = (Untreated group − Treated group) ÷ Untreated group × 100
= (0.60 − 0.17) ÷ 0.60 × 100
≈ 71.7%
Comparison of Contributions from the Upper and Lower Surfaces
Assuming that coating a surface with petroleum jelly suppresses transpiration from that surface, the apparent contributions can be compared using the differences from the untreated group.
Decrease caused by coating the upper surface = 0.60 − 0.48 = 0.12 g
Decrease caused by coating the lower surface = 0.60 − 0.17 = 0.43 g
In this example, the decrease is greater when the lower surface is coated, suggesting that more transpiration occurred from the lower surface of the leaf.
Mean
If the measurements for the untreated group are 0.58, 0.61, and 0.60 g, the calculation is as follows.
Mean = (0.58 + 0.61 + 0.60) ÷ 3
= 0.597 g/60 min
Standard Deviation
s = √{Σ(xi − x̄)2/(n − 1)}
The standard deviation calculated from 0.58, 0.61, and 0.60 g for the untreated group is approximately 0.015 g.
Percentage Increase Between Conditions
If the transpiration rate is 0.60 g/h under light with no wind and 0.88 g/h under light with airflow, the calculation is as follows.
Percentage increase (%) = (0.88 − 0.60) ÷ 0.60 × 100
≈ 46.7%
Main Sources of Error
| Source of Error | Effect on Measurement | Likely Effect on Results | Improvement |
|---|---|---|---|
| Evaporation from the water surface | Adds water loss not caused by the plant | Overestimates transpiration | Cover the water surface with liquid paraffin or a similar material |
| Insufficient sealing of the container | Allows water to evaporate through gaps | Overestimates transpiration | Seal with a stopper or Parafilm |
| No leafless control | Prevents correction for apparatus-related mass loss | Produces a value greater than the true transpiration | Measure a leafless control at the same time |
| Different leaf areas | Changes the area available for transpiration | Samples with more leaf area show larger values | Standardize leaf area or correct for leaf area |
| Different leaf ages or conditions | Changes stomatal density and opening | Increases variation among samples | Use leaves from similar positions on the same plant |
| Air entering the xylem during cutting | Interferes with water uptake | Causes wilting and lowers transpiration | Recut the stem under water |
| Uneven application of petroleum jelly | Fails to cover stomata sufficiently | Underestimates the treatment effect | Apply a thin, even layer |
| Petroleum jelly on the petiole or branch | Affects normal water movement | Causes an abnormally low transpiration value in the treated group | Apply only to the specified leaf surface |
| Mass of petroleum jelly | Makes initial mass differ among conditions | Makes simple comparisons difficult | Use the mass after application as the initial value |
| Time differences during weighing | Transpiration continues during measurement | Shifts the starting time among conditions | Measure quickly in a consistent order |
| Insufficient balance precision | Makes small mass differences difficult to detect | Increases error under low-transpiration conditions | Use a balance with an appropriate minimum readability |
| Room-temperature fluctuations | Changes evaporation rate and stomatal opening | Values vary with measurement time | Measure under constant-temperature conditions |
| Humidity fluctuations | Changes the water-vapor concentration gradient between the leaf and air | Lower humidity tends to increase transpiration | Measure and standardize humidity |
| Differences in airflow | Changes the boundary layer at the leaf surface | Samples exposed to stronger wind show larger values | Standardize wind speed and direction |
| Differences in light intensity | Changes stomatal opening | Transpiration may increase under light conditions | Standardize illuminance and direction of illumination |
| Measurement time too short | Produces a small mass difference and increases relative error | Increases variability | Use an appropriate measurement duration |
| Measurement time too long | Water shortage causes stomata to close | Transpiration rate decreases later in the experiment | Measure intermediate time points as well |
| Individual differences in stomatal distribution | Changes transpiration capacity among leaves | Increases variation in upper/lower surface differences | Increase the number of replicates |
Why Mass Loss Is Large in the Untreated Group
In the untreated group, water is lost through stomata and the cuticle on both the upper and lower leaf surfaces. Therefore, transpiration is greater than in groups whose leaf surfaces are coated with petroleum jelly.
Why Coating the Lower Surface Greatly Reduces Transpiration
In many terrestrial plants, stomata are more abundant on the lower surface of the leaf. Applying petroleum jelly to the lower surface covers many stomata and obstructs the release of water vapor, greatly reducing transpiration.
Why Mass Loss Does Not Reach Zero Even When Both Surfaces Are Coated
Mass loss may not become completely zero because of uneven petroleum-jelly application, slight evaporation from the water surface or container, water loss from the branch or petiole, weighing error, and other factors.
Why Wind Increases Transpiration
A layer of still, humid air forms at the leaf surface. This is called the boundary layer. Wind thins this layer and maintains the water-vapor concentration gradient between the inside of the leaf and the surrounding air, thereby promoting transpiration.
Why High Humidity Reduces Transpiration
When the humidity of the surrounding air is high, the difference in water-vapor concentration between the inside of the leaf and the outside air becomes smaller. This lowers the diffusion rate of water vapor and reduces transpiration.
Why Transpiration Increases under Light Conditions
In many plants, light promotes stomatal opening. When stomata open, water vapor can diffuse outward more easily, so transpiration may be greater than under dark conditions.
Example of How to Write the Results
Using leafy camellia branches, the effect of petroleum-jelly treatment of the leaf surfaces on transpiration was investigated from the mass loss over 60 minutes.
Mass loss was 0.60 g in the untreated group, 0.48 g when only the upper surface was coated, 0.17 g when only the lower surface was coated, and 0.07 g when both surfaces were coated.
The untreated group showed the greatest mass loss, whereas the group with both surfaces coated with petroleum jelly showed the smallest mass loss.
In addition, transpiration decreased more when only the lower surface was coated than when only the upper surface was coated.
The leafless control showed a mass loss of 0.02 g, so the corrected transpiration of the untreated group was 0.58 g/60 min.
Points to Connect the Results to the Discussion
- How did transpiration differ between the untreated and petroleum-jelly-treated groups?
- Which surface, upper or lower, contributed more to transpiration?
- How is this difference related to stomatal distribution?
- Why did transpiration not become zero even when both surfaces were coated?
- Why is a leafless control necessary?
- How did light, temperature, humidity, and wind affect the transpiration rate?
- How were differences in leaf area corrected?
- How did the method of cutting the branch affect the results?
- What kind of error can uneven petroleum-jelly application cause?
- Could the stomata have closed during the measurement?
- Can all mass loss be regarded as being caused by transpiration?
- What physiological roles does transpiration play?
Discussion Example
In this experiment, petroleum jelly was applied to the surfaces of camellia leaves, and transpiration was compared using mass loss over 60 minutes.
The untreated group showed the greatest mass loss at 0.60 g. The mass loss was 0.48 g when only the upper surface was coated, 0.17 g when only the lower surface was coated, and 0.07 g when both surfaces were coated.
Petroleum jelly covers the leaf surface and obstructs the escape of water vapor through the stomata and cuticle. Therefore, transpiration was considered to decrease as a larger portion of the leaf surface was treated.
Coating only the upper surface reduced mass loss by 0.12 g compared with the untreated group, whereas coating only the lower surface reduced it by 0.43 g. This suggests that the plant used in this experiment transpired more from the lower leaf surface.
In many terrestrial plants, stomata are more abundant on the lower surface of leaves. Applying petroleum jelly to the lower surface therefore covered many stomata and strongly suppressed water-vapor diffusion, resulting in a marked decrease in transpiration.
Even when both surfaces were coated, a mass loss of 0.07 g was observed. Possible causes include uneven application of petroleum jelly, water loss from branches or petioles, evaporation through gaps in the container or from the water surface, and measurement error of the balance.
The leafless control also showed a mass loss of 0.02 g, indicating that evaporation from the apparatus was not completely prevented. The corrected transpiration of the untreated group was 0.58 g/60 min. Including a control therefore allows water loss attributable to the plant to be estimated more appropriately.
Transpiration was greater under light conditions than under dark conditions. This was likely because light promoted stomatal opening, allowing water vapor to diffuse more readily out of the leaf.
Transpiration increased under airflow compared with still-air conditions. Wind removed the humid boundary layer at the leaf surface and maintained the water-vapor concentration gradient between the inside of the leaf and the surrounding air.
Transpiration decreased under high-humidity conditions. Because the amount of water vapor in the surrounding air increased, the water-vapor concentration gradient between the inside of the leaf and the outside air became smaller, reducing the diffusion rate of water vapor.
Transpiration increased under high-temperature conditions. The higher temperature increased molecular motion and promoted evaporation. However, at still higher temperatures, water shortage may cause stomata to close and transpiration to decrease.
In this comparison, the leaf areas of the samples were not completely identical, so comparisons based only on mass loss include the effect of differences in leaf size.
For the untreated group with a leaf area of 120 cm² and transpiration of 0.60 g/h, the transpiration rate per unit leaf area was 5.00 mg/(cm²·h). Correcting for leaf area makes samples of different sizes easier to compare.
One possible source of error was air entering the xylem when the branch was cut. If air bubbles form in the xylem, water uptake is impeded, the leaves wilt, the stomata close, and transpiration decreases.
To prevent this, the cut end of the branch should be recut under water so that air does not enter the xylem.
Uneven application of petroleum jelly can leave some stomata uncovered and cause transpiration in the treated group to be overestimated. Conversely, if petroleum jelly adheres to the petiole or branch, it may interfere with water movement and reduce transpiration more than intended.
If the measurement period is too long, water shortage in the plant may cause the stomata to close, so the transpiration rate may decrease over time. It is therefore desirable to measure mass at regular intervals and check whether transpiration changes linearly with time.
To improve measurement accuracy, it is important to standardize leaf area and leaf age, keep temperature, humidity, wind speed, and light intensity constant, prevent evaporation from the water surface, include a leafless control, and perform multiple measurements for each condition.
These results indicate that transpiration from leaves occurs mainly through the stomata and that, in the plant used here, more transpiration occurred from the lower leaf surface. The results also show that the transpiration rate changes with environmental conditions such as light, temperature, humidity, and wind.
Discussion Example When the Difference Between Untreated and Treated Groups Is Small
A small difference between the untreated and petroleum-jelly-treated groups may result from uneven application of petroleum jelly, stomata that were not widely open, low overall transpiration under high-humidity or dark conditions, or a measurement period that was too short.
Discussion Example When Coating the Upper Surface Causes a Greater Decrease
If transpiration decreases greatly when the upper surface is coated, the plant used may also have many stomata on the upper surface. However, it is also necessary to check whether the amount of petroleum jelly, leaf area, leaf condition, and coated area were standardized.
Discussion Example When Mass Decreases Greatly in the Both-Surfaces Treatment
If mass still decreases greatly when both surfaces are coated, possible causes include uneven application of petroleum jelly, evaporation from the water surface or container, insufficient sealing, and water loss from the branch or petiole.
Discussion Example When Measurements Show Large Variation
Large variation among measurements may have resulted from differences among samples in leaf area, leaf age, stomatal opening, branch-cutting condition, amount of petroleum jelly applied, temperature, humidity, or airflow.
Summary
Transpiration is the process by which water inside a plant is lost to the surrounding air as water vapor, mainly through the stomata of leaves.
Applying petroleum jelly to leaf surfaces suppresses water loss through stomata and the cuticle, thereby reducing mass loss.
If transpiration decreases greatly when the lower surface is coated, this suggests that stomata are more abundant on the lower surface and that stomatal transpiration from the lower surface was the major pathway.
The transpiration rate changes with light, temperature, humidity, wind, leaf area, stomatal opening, and other factors.
In the discussion, it is important to relate differences between upper- and lower-surface petroleum-jelly treatments, leaf-area correction, the leafless control, environmental conditions, evaporation from the water surface, uneven coating, and air entering the xylem during cutting to differences and variation in the measured values.
