Western International University Material



Associate Level Material

LeafLab

Before beginning LeafLab:

1. Print out these lab experiment instructions. A printed copy of these instructions will aid in completing the lab accurately and effectively, because you will not need to switch back and forth between computer screens.

2. Disable your pop-up blocker. LeafLab and the LeafLab Online Notebook will open in new browser windows. If you have a pop-up blocker, they will be blocked.

3. Read the online introduction and background information related to this lab

The experiment is divided into two sections: Self-Check Experiment and Exploration Experiment.

The Self-Check Experiment is designed to help you become familiar with the lab. The answers to the Self-Check Experiment questions are given to you (in red text). Completing the Self-Check Experiment and checking your answers will help you verify that you are completing the experiments correctly.

The Exploration Experiment is the experiment you will be conducting and turning in to your instructor for credit. You will report your findings for the Exploration Experiment in the LeafLab report.

Getting to Know LeafLab

This assignment is designed to help you become familiar with the operation of LeafLab.

The first screen that appears in LeafLab takes you to a virtual lab that contains all of the equipment you will need to carry out your experiments.

1. Click on each piece of equipment to learn about its purpose.

For each experiment you conduct, you must understand the experimental setup you will be using and manipulating. The basic experimental design begins with a lamp as a source of visible light. The intensity of visible light produced by this lamp can be increased or decreased, and the wavelengths of visible light released by the lamp can be altered using different filters that will allow only certain wavelengths of visible light to strike the leaf you are studying. To prevent the leaf sample from drying out or burning due to heat from the lamp, a reservoir of water is placed between the lamp and the leaf. LeafLab allows you to choose from several different leaf samples.

The leaf is contained in a sealed chamber. In addition to manipulating the quantity and quality of light striking the leaf in the chamber, you can manipulate various environmental conditions of the leaf chamber, such as gas flow, temperature, and carbon dioxide (CO2 ) concentration. When collecting data, the concentration of CO2 in the leaf chamber will be measured using an infrared gas analyzer (IRGA). Because water vapors that can affect the accuracy of the IRGA will be produced in the chamber when light strikes the leaf, air leaving the chamber is passed through a drying column prior to entering the IRGA. The IRGA measures the amount of infrared radiation absorbed by CO2 in the air coming from the leaf chamber. Analyzing data on the amount of CO2 consumed by your leaf will enable you to study several different parameters of photosynthesis.

Are you comfortable with the purpose of each piece of equipment in LeafLab? Be sure that you understand the experimental design before continuing.

Self-Check Experiment: Light Intensity and Photosynthetic Rates in Tomato Leaves

Based on what you already know about photosynthesis, develop a testable hypothesis to explain the influence of an increase in light intensity on the photosynthetic rate in tomato leaves.

You should hypothesize that an increase in light intensity will increase photosynthetic rate in tomato leaves.

Choosing a Leaf and Measuring Leaf Surface Area

1. To set up each experiment, first choose the leaf you are interested in studying. Leaves from six different plants are available in LeafLab. The plants available to you are tomato (C3 plant), corn (C4 plant), two different clones of goldenrod (one favors sunny conditions, the other favors shade), and two types of fescue grass that differ by their number of chromosomes.

2. Click on the Choose Leaf button on the left side of the screen. For this experiment, we will use a leaf from a tomato plant. A dark box should appear around the tomato leaf in the left corner of this screen indicating that the tomato plant has already been selected (tomato is the default plant).

3. Read the legend about tomato plants that appears on the right side of the screen. Similar legends will appear for each plant that you select. Also notice that the bottom three panels of this screen show graphics of the whole plant, leaf, and fruit structures for the tomato plant. Similar legends and graphics will appear for each plant you select.

Because the calculations of photosynthetic rate that this simulation will generate as data are expressed as a value per unit of leaf surface area, you must begin each experiment by determining the total surface area of the leaf you have chosen for your experiment.

4. Click on the Measure Area button on the left side of the screen. A view of a tomato leaf will appear, overlaid by a series of grid squares. Click on one of the squares. It will now be shaded green, indicating that you have measured this area of the leaf.

• A tally of the number of squares selected and the total surface area (in cm2) for all squares selected is provided on the right side of the screen. The scale for measuring area is different for certain plants. Notice that the scale for tomato plants is set up so that each square is 0.1 cm2 in area.

• Continue to select squares until you have measured the entire surface area of this leaf. When you get to the edges of the leaf, notice that some of the grid boxes are not completely filled by the leaf. For these boxes, double-click on each box. The box will now be shaded a lighter green than the other boxes. Notice that these boxes are scored as one-half of the area of the other boxes to adjust your area measurement for incompletely filled boxes. Hint: You can measure surface area quickly by using your mouse to click and drag across several boxes.

5. The surface area value that you just measured should be recorded in your lab notebook for future reference as follows:

• Click on the Add to Notes button at the lower left of the screen to record this value in your notebook.

• Important: After you have recorded the value, do not close your lab notebook. To continue with your experiment, minimize your notebook.

For each plant in LeafLab, only one size leaf will appear; therefore, once you have measured leaf area for a particular plant you will not have to measure the area of this leaf again unless you exit LeafLab and then return. The area measurement will be available to you even if you switch plants to perform another experiment. If you do not measure leaf surface area at the beginning of an experiment, LeafLab will not let you run the experiment.

Once you have defined the total surface area of the leaf, you are almost ready to run an experiment. Before you can begin collecting data, you must know what environmental parameters in the leaf chamber you can manipulate and the types of data you will be collecting.

6. Click on the Collect Data button on the left side of the screen. The screen that appears is labeled Input Controls.

• For this first experiment, many of the Input Controls will be left at their default value.

• At the bottom of the screen, locate the box labeled Expt #. This box will identify each experiment that you perform so you can return to this experiment if you need.

• This first experiment should be indicated by a "1" in this box.

Input Controls

The Input Controls view allows you to change several conditions in the chamber.

• Gas flow is measured in milliliters of gas entering the chamber per minute (ml/min). The default value for gas flow is "off."

• Temperature is measured in degrees Celsius (°C). The default value for temperature is set at 25°C, the temperature of a warm room (80.6°F), which is a fairly comfortable temperature for most plants living in a typical greenhouse.

• Carbon dioxide concentration is measured in parts per million (ppm). The default value is 350.0 ppm. This value approximates the normal atmospheric concentration of carbon dioxide.

• Light intensity is measured in micromoles of light photons released per square meter per second ([pic]mol/m2/s). The default value is that the light is turned off (0 [pic]mol/m2/s). Unless you are performing an experiment to measure photosynthetic rates under darkness, you must always adjust light intensity before you can collect data.

• The light filters control allows you to determine which wavelengths of light you would like the leaf to be exposed to. The default is no filter; thus, white (visible) light is striking the leaf.

1. Find the input controls for each of the parameters mentioned above, then set up the conditions for this experiment as follows:

• Set gas flow to a medium value by clicking on the medium button.

• Notice that gas flow has now changed from 0 ml/min to 500 ml/min.

• Also, notice that the measurement of CO2 output has started.

Gas flow is a parameter that you will change depending on the size of the leaf you are working with. For tomato and goldenrod leaves, medium gas flow is appropriate. For corn, a high gas flow is necessary. For fescue, the small blades of grass require a low gas flow. If you try to run an experiment without gas flowing into the leaf chamber, no data will be generated when the lamp is turned on.

Leave temperature, carbon dioxide concentration, and the light filter at their default values.

2. This experiment begins by leaving lamp intensity at 0mol/m2/s.

• Note: An intermediate value of approximately 1000 [pic]mol/m2/s is close to representing a typical value of sunlight on a sunny day. Light intensity can be changed either by using the slider for each parameter or by entering a value into the text box that appears to the right of each slider and then pressing the Enter key.

3. Locate the numerical value for CO2 output in the leaf chamber.

• The IRGA is determining CO2 output by comparing the amount of CO2 entering the chamber with the amount of CO2 in the air leaving the chamber. Remember that the leaf is consuming CO2 as its cells perform photosynthesis, but these cells are also undergoing cell respiration to produce ATP--a process that produces CO2 as a waste product. Therefore, the IRGA is recording CO2 output as a measure of net photosynthetic rate--the difference between CO2 consumption during photosynthesis and CO2 production during cell respiration. Note: It is important to realize that you are not measuring O2 production in these experiments.

4. You will be taking measurements of the CO2 output in this experiment. Before recording any measurements, always make sure that the line on the chart recording is horizontal and not wavy before taking a measurement. This is especially important after you have changed an input parameter, because you must wait for the experimental conditions in the leaf chamber to "settle down" to your desired input setting(s).

5. Click on the Record button to take a recording of CO2 output. Notice that when you click the Record button, a solid black line appears on the chart recording, indicating that a measurement was taken.

6. Set the lamp to an intensity of 200mol/m2/s by entering the value into the text box that appears to the right of the slider and then pressing the Enter key. Notice that once you turn up the lamp intensity, a chart recording of CO2 output, as determined by the IRGA, will change in the box just above the chart recording.

7. Continue this experiment by increasing light intensity by increments of approximately 200mol/m2/s to 400, 600, 800, 1000, 1200, 1400, 1600, 1800, and 2000 [pic]mol/m2/s.

• It is easy to make these changes by typing these values into the text box to the right of the light intensity slider and then pressing the Enter key

8. After each change in light intensity, wait for the chart recording to flatten out to a horizontal line and then record your data by clicking the Record button

9. Note: It is important to change only one parameter (variable) at a time when collecting data. In this experiment, all parameters are kept the same, except for light intensity.

Analyzing Data

To prepare your data for analysis, switch to the Prepare Data view.

1. Switch to the Prepare Data view by clicking on the Prepare Data button at the left of the screen.

The purpose of the Prepare Data function is to use the data that you recorded to calculate photosynthetic rate. In this view, the table at the bottom of the screen contains values for all of the input parameters of each experiment that you conducted. This table also indicates the concentration of CO2 entering the leaf chamber (C-in) and the concentration of CO2 leaving the leaf chamber (C-out). The calculated value for photosynthetic rate (P) will appear in the far right column of the table.

The four equations at the top of this screen will be used to calculate photosynthetic rate. The photosynthetic rate will be calculated for you, however, review the following descriptions of each equation:

• Equation 1: Calculates CO2 consumption by subtracting C-out from C-in and reports this value as the change in CO2 concentration ([pic]CO2).

• Equation 2: Converts [pic]CO2 concentration from parts per million to [pic]mol/liter, based on the temperature of the leaf flask.

• Equation 3: Calculates the rate of CO2 that is available for exchange between the leaf and the flask by multiplying [pic]CO2 concentration against gas flow.

• Equation 4: Net photosynthetic rate (P) is calculated by dividing the CO2 available for exchange by the total surface area of the leaf being studied. This value is reported as the number of micromoles of CO2 released per square meter of leaf surface area per second ([pic]mol/m2/s).

2. To calculate photosynthetic rate, start by looking at the table and reviewing the information contained in each column.

3. Click on the row for the experiment that you want to analyze. In this case, experiment 1. The selected row will now be highlighted in green.

4. Select the whole table by clicking on the top row (0 light data) and dragging the mouse to the bottom of the table.

5. To perform the calculations, click on the Compute button, located on the right side of the screen just above the P column in the table.

6. Net photosynthetic rate will now be calculated and added to the table.

Plotting Data

Plotting data from the calculations that you generate is important for understanding the results of your experiments.

1. To do this, use the Plot Data function of LeafLab. This function will produce a scatter plot of your data.

• Click on the Plot Data button on the left side of the screen.

• Click in the title box at the top of this screen and title this first plot "PS Rates vs. Light in Tomato.”

• You can express your data on these plots in two different ways. On the x-axis you can choose to plot either "light intensity" (this is the default) or CO2 input (C-in). On the y-axis you will plot photosynthetic rate (P) values. You can also change the symbol and symbol color that will appear on the plot.

2. Prepare a plot of light intensity versus P values as follows:

• Select the whole table by clicking on the top row (0 light data) and dragging the mouse to the bottom of the table.

• Leave the x-axis at its default value of light intensity.

• Click the Plot Selected Data button to plot the data. A plot of your data should appear.

• Notice that the horizontal line at the center of the plot indicates a "0" value of light intensity.

• Add the data from this plot to your notebook by clicking on the Add to Notes button on the Data tab at the lower left of the screen.

Interpreting Your Data Plots

Although basic trends in your data can sometimes be estimated by simply looking at the data points on your scatter plots, quantitative measures of the effects you are studying can only be determined by fitting a curve to your data.

Curve fitting involves producing a statistically derived best-fit line of data points on the graph; not a hand-drawn or estimated line connecting data points.

Once you have plotted your data, a Plot # tab will appear at the top of the Plot Data screen. Clicking on this tab will take you to the curve-fitting functions of LeafLab and allow you to switch between plots that you generate.

1. Click on the Plot 1 tab to enter the curve-fitting view.

• An enlarged view of the plot should now appear with a series of curve-fitting controls to the left of the plot.

2. The purpose and instructions for manipulating each control are described in following steps:

• Curve: generates a best-fit curve based on the data points selected. You will be generating a best-fit curve by following the steps listed next.

• y–Intercept: indicates the rate of dark respiration (light compensation point)

o To input the y-intercept: return to the data table by clicking on the Data tab. Look at the zero light measurement in the table and use the P value for this measurement as the initial measurement of the intercept.

o Return to the curve-fitting view and enter this P value directly into the intercept box.

• Slope (of the line): photochemical efficiency; indicates the rate at which photosynthesis increases as light intensity increases.

o To manipulate the slope: click on the up arrow next to the slope function (you will see the line rise up and begin to form a curve).

o Increase the slope of the line until the curve looks like it is matching (fitting) the data points.

• Asymptote (where the curve forms a straight line indicating that the data has leveled off): indicates photosynthetic saturation (maximum rate of photosynthesis).

o Look at the plot line and estimate where the data levels off. This is the asymptote.

o Click on the line, next to the data point that you think represents the asymptote. Two sets of numbers in parentheses will appear. The first number is light intensity and the second number is photosynthetic rate (P).

o Enter this P value into the asymptote box.

• Error SS Value: is an expression of the calculation of the distances of each data point from the fit line. The lower the Error SS value, the more accurate the line in representing the data.

o To find the lowest possible Error SS value, use the up or down arrows to adjust each parameter: the slope, asymptote, or intercept.

o Start with the slope; adjust the slope until you have the Error SS value number as low as possible. Stop when your adjustments cause the Error SS value number to increase.

o Then, follow the same process with the asymptote and the intercept.

o Continue to adjust the parameters until you get the absolute lowest possible Error SS value. The values that give the smallest Error SS value produce the best-fit line for your data points. Note: this value could be as low as 0.2.

3. Save your plot by clicking on the Export Graph button at the left of the screen. A separate window will now open showing your plot and a table with the intercept, slope, asymptote, and Error SS values. You can save this page by going to File and using the Save As feature of your browser.

Summary: What Did This Experiment Tell Us?

The experiment you just performed is representative of other experiments that you will conduct. A lot of information can be learned from studying the curves that you generate.

Study the curve of Photosynthetic Rate vs. Light in Tomato to answer the questions below.

The following is a representative plot and table from this experiment:

[pic]

|Series |Intercept |Slope |Asymptote |Error Sum of Squares |

|1 |-1.3 |0.038 |17.1 |0.198 |

1. Answer the following questions:

• What is the relationship between an increase in light intensity and photosynthetic rate in tomato leaves?

• Does this relationship support the hypothesis that you formulated?

An increase in light intensity increases photosynthetic rate, supporting the hypothesis.

• Photosynthetic saturation is the maximum rate of photosynthesis. What was the value for photosynthetic saturation in tomato leaves?

• What value of light intensity produced photosynthetic saturation in tomato leaves?

• Based on what you know about photosynthesis, provide possible reasons for what causes photosynthetic saturation (these cannot be determined from the plot).

Photosynthetic saturation in tomato leaves will occur at a light intensity of approximately 1600 [pic]mol/m2/s. At saturation, photosynthetic rate is approximately 15-17 (as indicated by the asymptote).

Exploration Experiment: Light Intensity and Photosynthetic Rates in Corn

1. Follow the steps detailed in the first experiment to test the effects of an increase in light intensity on photosynthetic rates in corn (a C4 plant).

• The only modification to the experiment is that you will need to use a high rate of gas flow. Keep all other parameters the same as you did for tomato.

2. When calculating P and plotting your data, make sure that you select only those values that you recorded for corn and not previously recorded values for tomato.

3. Plot photosynthetic rate versus light intensity and fit a curve to the data as you did for tomato.

4. Add your graph to the LeafLab Report by clicking on Export Graph.

5. Copy and paste your graph under the Data section of the LeafLab Report.

6. Answer the following questions in the LeafLab Report:

• What is the relationship between an increase in light intensity and photosynthetic rate in leaves from a corn plant? How does this relationship compare with what you observed for tomato plants?

• Photosynthetic saturation is the maximum rate of photosynthesis. What value of light intensity produced photosynthetic saturation in corn leaves?

References

LeafLab assignments and answers were adapted with permission from Pearson Education, Inc.

Biology Labs On-Line is a collaboration between the California State University system and Benjamin Cummings.

© 2002 California State University and Benjamin Cummings, an imprint of Pearson Education, Inc. Development was partially supported by a grant from the U.S. National Science Foundation.

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