# Lab 3: Transpiration in High Light/High Wind and Low Light/Low Wind Habitats

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## **Objectives**

- Determine the rates of water loss for four plant species under different environmental conditions.
- Learn how to calculate the water vapor concentrations inside and outside leaves
- Learn how to calculate transpiration and total resistance rates.
- Use ANOVAs to compare these rates statistically

## **Lab 3 Overview**

I. Group oral presentations of key characteristics of experimental plants

- II. Formulate testable hypotheses/experimental questions about Plant Transpiration Rates
- a. Document Greenhouse Environmental Conditions

- b. Document Rates of Plant Water Loss

- c. Measure Total Leaf Area

- a. Calculate water vapor concentrations inside and outside leaves

- b. Calculate Mean Rates of Transpiration and Resistance to Transpiration

- c. Use t-tests and ANOVAs to compare means of Transpiration and Resistance rates

- Well-watered, labeled plants have been placed in two different experimental conditions before the start of the lab. The non-living evaporative surfaces have been sealed off with plastic bags.
- Carefully weigh each individual plant and record the weight in grams (g).
- Return the plants to the appropriate environment.
- Re-weigh the plants at 10-min intervals and record the weight change in your data sheet. Continue this for 40 min.
- Copy down the TOTAL LEAF AREA value for each plant in your lab notebook. It will not be saved.
- When you are finished please leave the scanners ON.
- Be sure all leaves have been removed. Discard the leaves in the composting/disposal container provided in the lab and please return the empty envelopes to the instructor.

- a. transpiration rates in light versus dark environments

- b. resistance rates in light versus dark environments

- a. high light / high wind : transpiration rates

- b. low light / low wind : transpiration rates

- c. high light / high wind : resistance rates

- d. low light / low wind : resistance rates

- III. Test hypotheses/experimental questions about plant Transpiration Rates

- IV. Data Analysis and Presentation

## **Plant Transpiration: Background**

Stomata are pores that allow the exchange of gases in the plant with atmospheric gases. For the plant the most important of these exchanges focuses on the uptake of CO_{2} for photosynthesis and the movement of water from root to leaf during transpiration. While excessive loss of water vapor is a hazard of plant life, transpiration is a vital function that provides the pressure differential that moves water up from the roots bringing minerals for biosynthesis to the cells, drives phloem transport, and also cools the leaf. In this lab you will investigate the physiological response of four different plants to particular environmental conditions. Studies that examine anatomical and physiological mechanisms as they relate to physical and biological environments are part of the study of ecophysiology. The class will determine the transpiration rates of the four plant species under two different environmental conditions (high wind/high light and low wind/low light).

Leaves have evolved physical barriers that prevent excessive water loss. For example, a waxy cuticle is very effective in preventing the loss of water through a leaf's epidermis. That means that the diffusion of CO_{2}, O_{2}, and H_{2}O into and out of the leaf is restricted to the stomata. In most cases ~ 90 % of the gas exchange occurs through the stomata. Plants can open their stomata to different degrees or completely close them and thus control the resistance to transpiration. Also, as you will document in Lab 4, the number of stomata can vary significantly between different species. For most leaves the number of stomata and the stomatal aperture play the most important role in regulating the leaf’s gas exchange by influencing the stomatal resistance to water loss.

Another resistance to diffusion is the boundary layer resistance of a leaf. The boundary layer is an unstirred layer of air next to the leaf surface. Because the air in this layer does not directly mix with the bulk atmosphere all gas molecules that must traverse this layer must do so only by the random motion of individual molecules. The thicker the boundary layer, the greater the boundary layer resistance and the slower the diffusion. The thickness of the boundary layer is controlled primarily by wind speed and plant structure. Higher wind speed leads to thinner boundary layers and thus to lower resistance.

Yet another resistance to diffusion is the internal pathway resistance, which is the resistance water molecules face when diffusing through the air spaces inside a leaf to reach the stomatal pores. The thicker the leaf the higher the internal resistance. Also leaves that have very tightly packed mesophyll cells with few air spaces in between the cells have a higher internal resistance to water loss.

Using an expression that has been developed to describe the flow of electricity (Ohm's law), scientists are able to quantify the rates of transpiration under different conditions. This expression states that the flow of electrons through a circuit is directly proportional to the voltage difference (driving force) but inversely proportional to the resistance. A similar mathematical expression can be used to describe the behavior of water diffusing out of a leaf. The flux of water or transpiration (J_{wv}) is directly proportional to the driving force (water concentration difference or ∆c_{wv}), and inversely proportional to the resistance (R_{wv}):

**Transpiration rate = (c**

_{wv}int - c_{wv}ext)/ R_{wv}In this circumstance the leaf’s total resistance to water vapor transfer, R_{wv}, is equal to the sum of the individual resistances noted above:

**R**

_{wv}= R stomatal + R boundary layer + R internal pathway## **Document Greenhouse Environmental Conditions**

Draft a data table in your lab notebook to document environmental conditions in each of the two greenhouse environments (high light/high wind and low light/low wind). Variables include light intensity (μmol photons m^{-2}s^{-1}), wind speed (m s^{-1}, air temperature (°C), leaf surface temperature (°C), and relative humidity (RH, %).

**Internal Water Vapor Concentration**

The internal environment of a plant leaf is assumed to be completely saturated with water. That is, the air inside the leaf holds the maximum amount of water (100% relative humidity, RH) possible at a given temperature. Consequently, knowing the leaf temperature, the internal water vapor concentration can be read directly from a Water Vapor Concentration Table . Determine the leaf temperatures (T_{L}) of several different leaves of each plant using the thermocouple thermometer while the plants are still in their respective environments. Look up the saturation water vapor concentration (c_{wv}) that corresponds to these average temperatures in the Water Vapor Concentration Table .

**External Water Vapor Concentration**

Water Vapor Concentration in the ambient air at any given relative humidity can be calculated by multiplying relative humidity times the temperature dependent Saturation Water Vapor Concentration value.

**c**

_{wv}at temperature T = (saturation c_{wv}at temperature T)· RHLook up the saturation water vapor concentration (c_{wv}) that corresponds to the air temperature that you have recorded in each environment in the Water Vapor Concentration Table . Multiply this value by the RH of each environment, and record your calculated External c_{wv} values in your lab notebook.

## **Document Rates of Plant Water Loss**

Three replicates of each species will be investigated, which means that each group will have 6 plants of one species to work with in this part of the experiment.

Draft a data table in your lab notebook to record the weight (g) of each plant at the following times (min): 0, 10, 20, 30, 40, for a total of five data points per plant. Variables include time (min), and weight (g) for each plant.

Now calculate the rate (slope) of water loss for each plant over time:
Create a scatter plot in Excel of the weight of each plant (g) versus time (min) using your data (one graph for each individual plant). See: Statistics and Graphing: making a linear regression in Excel. Fit a linear regression line through your data points and choose the option that displays the equation and its R^{2} value on the graph. This equation will have a **y = mx + b** format, where "m", the slope of the line, equals the rate of water loss in g min^{-1}. Use the absolute value of the slope.

Convert your rates for each plant from g min^{-1} to μg sec^{-1} and record these values in your lab notebook.

**rate in μg sec**

^{-1}= (g min^{-1}) * (1 min/60 sec) * (10^{6}μg/g)Assume that the slope of your regression line is 0.1 g min^{-1}, then (0.1 g min^{-1}) * (1min/60 s)* (10^{6}μg/g)= 1.66 x 10^{3}μg sec^{-1}

## **Measure Total Leaf Area**

We will be using Adobe Photoshop, and ImageJ OS10 software to determine Total Leaf Area. Directions and the exact programs to be used this term will be provided.

Draft a data table in your lab notebook to document leaf area and transpiration rate for each of your plants. Variables include plant number, treatment (environment), leaf area (cm^{2}), and transpiration rate (μg cm^{-2} s^{-1}).

Cut off all leaves (no petioles) from each plant and save them in the appropriately labeled envelope. There is a different envelope for each plant. If you are working with Rhoeo, avoid contact with the sap by wearing gloves.

In the lab, arrange the leaves on the protective plastic covering the scanner bed so they are not touching. If all the leaves do not fit you will need to scan twice and add the 2 surface areas together. Carefully close the scanner lid, and follow the instructions posted on the scanner bed cover.

## **Calculate Mean Transpiration Rates and Resistance to Transpiration**

**Transpiration Rates**

To compare plants of different sizes and different leaf areas, it is necessary to express the transpiration rate on an equal area basis of 1 cm^{2}. For each plant, divide water loss rate calculated above by its total leaf area.

**Transpiration rate = Water Loss rate (µg s**

^{-1}) / Total leaf area (cm^{2})Let's assume you determined that the leaf area of your plant was 263.2 cm^{2}. If you then divide the water loss of the entire plant (1.66 x 10^{3} µg s^{-1}) by the total leaf area (263.2 cm^{2}) you obtain the value of 6.3 µg cm^{-2} s^{-1} which is the transpiration rate per unit area.

**Resistance to Transpiration.**

Total resistance to transpiration (R_{wv}^{tot}) is the sum of internal air, stomatal, and boundary resistances for your experimental plants.

Calculate the total resistance by dividing the water vapor concentration difference between the inside and the outside of the leaf by the transpiration rate.

**Resistance = (c**

_{wv}internal - c_{wv}external) / Transpiration rateIf the transpiration rate of your plant was 6.3 µg cm^{-2} s^{-1}, the external water vapor (c_{wv} ext) in the greenhouse was 6.92 µg cm^{-3}, and the value for the c_{wv} internal of your plant was 30.37 µg cm^{-3}, your calculation would be:

**R**

_{wv}^{tot}(s cm^{-1}) = [30.37 µg cm^{-3}- 6.92 µg cm^{-3}] / 6.3 µg cm^{-2}s^{-1}= 3.72 s cm^{-1}.## **Ungraded Assignment: Process the Data using JMP: Use t-tests and ANOVAs to Compare Means of Transpiration and Resistance Rates**

To process and evaluate today's data, compare your results statistically (See Statistics and Graphing for Tutorials):

- 1. Conduct two t-tests using data for

__your species__only:

- 2. Conduct four ANOVAS to evaluate differences among all four species

## **Graded Assignment-Writing for Science: Results Figure Design**

An expanded version of this first assignment in the plant series might provide helpful details.

1. Prepare **a figure with a caption** comparing the mean transpiration rates (± SD) of the 4 plants in both environmental conditions as described by your instructor.

2. Perform the appropriate statistics and cite these within a paragraph of **results text** that describes the trends in transpiration rates supported by your data.

Link to the Statistics and Graphing for instructions on how to construct a figure comparing means ± SD and perform the statistical analyses described by your instructor. See the Science Writing Guidelines for a description of what should be contained in a results section, how to format figures, the figure caption, and how to cite statistical results in text.

3. Take Pre-Lab 4 quiz.

## **Other Labs in this Section**

Lab 4: Plant Anatomy

Lab 5: Measurement of Chlorophyll Concentrations and Rates of Photosynthesis in Response to Increasing Light Intensity

Lab 6: Group Oral Presentations