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

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Lab 5 Overview

I. Formulate hypotheses about photosynthesis rates and chlorophyll concentrations in plants from different environments

II. Test hypotheses about photosynthesis rates
a. Calculate the CO2 exchange rate
b. Q test of maximum CO2 consumption rate vs historical data
III. Test hypotheses about chlorophyll concentrations
a. Measure the concentration of Chl a and Chl b
IV. Data Analysis and Presentation
a. Scatter graph of CO2 consumption at different light intensities
b. Correlation of CO2 and total chlorophyll content

Photosynthesis: Background

Photosynthesis is the fundamental source of energy input for most terrestrial and aquatic ecosystems. As its name suggests, photosynthesis is a process by which photons (packets of light energy) are converted to chemical energy and used to synthesize metabolites within the organism. Photosynthesis involves the fixation of carbon dioxide into sugars with a simultaneous release of O2.

6 CO2 + 6 H20 + light → C6H12O6 + 6 O2

Remember that plants, like all other living organisms, need a source of organic carbon to grow, reproduce etc. The ultimate source of this organic carbon, at least in the terrestrial environment, is atmospheric carbon in the form of inorganic gaseous CO2 (one of the culprits in global warming). CO2 is needed in the chloroplasts, the sites of photosynthesis, where sunlight is used to transform the inorganic form of carbon (CO2) to a form of organic carbon such as glucose (C6H12O6), useful as a source of both energy and building materials. However, to enter a mesophyll cell and the chloroplast the atmospheric CO2 must dissolve in water and diffuse through moistened thin membranes to the sites of photosynthesis. This creates an obvious dilemma. How do plants expose thin moist membranes to the dry atmosphere without losing tremendous amounts of water? One could view the leaf as natural selection's answer to this problem. The moistened gas exchange surfaces (mesophyll cells) became internalized between a water proof skin (epidermis with cuticle) with tiny adjustable pores (stomata and guard cells) allowing CO2 to enter by diffusion, but at the same time retaining the capability of preventing excessive diffusional water loss. These morphological, anatomical, and physiological features were, of course, the focus of lab 3 and 4.

The process of photosynthesis involves the exchange of gases between the leaf and the environment. The leaf takes up CO2 and evolves O2, usually in a 1:1 ratio. Therefore, when we enclose the leaf in a lighted chamber, air passing through the chamber will become depleted in CO2 and enriched in O2. By measuring the rate at which these gases are exchanged, the rate of photosynthesis can be determined.

In the first part of lab you will explore how photosynthesis varies with light intensity in leaves of your plant species. You will measure CO2 consumption in plant leaves using an Infra Red Gas Analyzer (IRGA). This Qubit® Gas Analyzing system measures the concentration of CO2 in the gas leaving the leaf chamber. Calculation of the difference in CO2, with and without the presence of a leaf, and measurement of the flow rate of gas through the chamber, allows calculation of photosynthetic CO2 fixation rate.

At the end of this first experiment, we will make measurements after turning off the light, and thereby, arresting photosynthesis. Thus, we can measure respiration. You may be more familiar with cellular respiration in animals where energy is released from sugar with ATP and water as by-products when O2 is breathed in and CO2 is released. However, the process is much more complicated in plants. Plants respire at all times of the day, in light and dark. Many plants do not release this CO2 because it is used up right away in photosynthesis. O2 generated during photosynthesis in light conditions can be used by the plant in respiration before it is released. In the dark, when photosynthesis is not converting radiant into chemical energy, respiration is the dominant process, and therefore, more O2 will be used than created, resulting in a net increase in CO2.

Measuring the Rate of Photosynthsis

Setting up the Experiment
For your experiment, choose a young but fully mature leaf that will completely fill the leaf chamber. Carefully open the chamber, position the leaf as flat as possible, and seal the chamber tightly. The area of the chamber, and thus of the leaf that fills it, is 9 cm2.

  1. Check that both gas bags are filled and all connections are open. Set the flow meter for 200 ml min-1 (0.2 liters min-1).
  2. There is an incandescent light available to illuminate the whole plant outside the chamber. Keep the light at least 10 inches away from the plant to prevent leaf damage from the heat. DO NOT USE UNLESS NECESSARY.
  3. Open the 0-2000 setup.cmbl file, and a window will open listing 4 sensors: CO2 Analyzer, Temperature Sensor, LED Light, and Relative Humidity. Select each from the drop-down menu and click connect.

Fig. 5.1. Carbon dioxide exchange in a plant leaf measured with an InfraRed Gas Analyzer (IRGA). The leaf of a water hyacinth (Eichhornia crassipes, was placed in a Qubit® gas analyzing chamber and light intensity (irradiance) was increased in intervals.
        4. Immediately save your file by selecting "SAVE AS" and giving the file an appropriate name (e.g., Rhoeo Bench 2). This file is necessary for your data to be saved.
        5. Allow the leaf to acclimate in the sealed chamber for at least 20 minutes.
        6. Turn on the LED and adjust its illumination output to about 300 µE m-2 s-1. On the computer screen you will see 4 graphs (Fig 5.1): CO2 concentration (upper left), LED Irradiance (lower left),  Air Temperature (upper right), and Relative Humidity (lower right). Keep an eye on the irradiance and CO2 concentration.
        7. Set the recording so it will not turn off. To do this, go to Experiment: extend collection: and set it for 90 min or longer.
        8. Click: Collect (upper middle green button on the LoggerPro tool bar).

Draft a data table in your lab notebook to document the changing CO2 concentrations at different light intensities. Parameters should include Photon Flux, Reference CO2 Concentration, CO2 Concentration with Leaf in the Chamber (ppm), ΔCO2 concentration (ppm), and Chamber CO2 Exchange Rate (µmol CO2 m-2 s-1).111F2.PhotonFluxDataChart.png

Also be sure to record each of these parameters: Plant Name, Temperature (°C), Flow rate ( ml min-1), and Leaf Area (both cm2 and as converted to m2).

Running the Experiment
To run the experiment:

  1. Set the LED illumination back to 0 µE m-2 s-1. (Light is measured as electric current flows through the analyzer. There is a small amount of current running through even when the light is not on. The program sometimes translates this as being below zero.)
  2. Continue to “collect” until the recording stabilizes (5-20 minutes, depending on the plant).
  3. While you are waiting, adjust the y-axis by double clicking on the axis. A window will open with a tab labeled “Axes Options”. Click on the tab and enter an appropriate upper and lower limit for the y-axis. This will adjust the scale to emphasize the small changes in CO2.
  4. Check frequently to make sure you are recording. The system will sometimes stop recording if you manipulate the screen in any way, so avoid multitasking while collecting data. You can monitor the changes in data by holding the cursor over the graphed line, this will display the y-values in the left-hand corner of the window.
  5. Record the CO2 concentration in your notebook when STEADY STATE conditions are attained.
  6. Change the output of the light source from low light to high light in this order: 100, 300, 600, 800, 1000 µE m-2 s-1. Observe the stepwise decrease in the level of CO2 in the chamber. Why are the levels of CO2 decreasing? Typically, there is a rapid decrease in CO2 concentration followed by a more gradual decrease and then a leveling off as the leaf responds to increased light level.
  7. Turn the light down to 0 and observe that the CO2 concentration in the analysis gas increases again. Record this CO2 concentration when it reaches a steady state value.
  8. Finally remove the leaf from the chamber, reseal the chamber, and collect data until the level of CO2 stabilizes. This is your input or reference CO2 concentration (should be between 900-1100 ppm). It represents the concentration of CO2 in the gas bags. It should be slightly lower than the CO2 level when the light was off. Why? What effect would a leaf in the dark have on CO2 concentration?
  9. After you have completed measuring at all light levels, stop data collection by clicking on the STOP button on the computer screen. Save your data by clicking on “File” in the menu, and selecting “Save.” Or start over by selecting DATA: clear all data.

Calculating the CO2 Exchange Rate
Measurements of the rates of photosynthesis, and respiration in leaves are usually expressed as rates of CO2 exchange per unit time and leaf area. The units most commonly used are µmol CO2 m-2 s-1. To express your data in these units:

  1. Calculate the difference between the CO2 concentration in the reference and analysis gases. For example, if an experiment was conducted in air of 350 ppm CO2, at a flow rate of 500 mL min-1, the depletion of CO2 due to leaf uptake in photosynthesis at high light may result in an analysis gas CO2 concentration of 310 ppm. The difference between the reference and sample gas streams (∆CO2 = Reference CO2 - Sample CO2) in this example would be 40 ppm (350-310).
  2. Convert the ∆CO2 value from (ppm) into (µmol CO2 L-1):

∆CO2 in µmol CO2 L-1 = ∆CO2 in ppm / 22.415 [(T + C) / T]

                                                   where C is the temperature in °C and T is the absolute temperature (273 K).

EXAMPLE: At a temperature of 20°C and a ∆CO2 of 40 ppm, the ∆CO2 would be equivalent to 1.66 µmol CO2 L-1:

40 ppm ∆CO2 / 22.412 [ (273+20) / T] = 1.66 µmol CO2 L-1

      3. Multiply the ∆CO2 value by the flow rate (in L s-1) used in your experiment to obtain a CO2 exchange rate per second.           For example, a flow rate of 500 mL min-1 is equivalent to 0.0083 L s-1. So the CO2 exchange rate in our example would be           0.014 µmol CO2 s-1:

0.0083 L s-1*1.66 µmol CO2 L-1 = 0.014 µmol CO2 s-1

Express your CO2 exchange rate on a leaf area basis by dividing the CO2 exchange rate by the leaf area in m2. If the leaf completely filled your chamber, the area used in the calculation would be 9 cm2, equivalent to 0.0009 m2. The CO2 exchange rate in our example would therefore be 15.6 µmol CO2 m-2 s-1, which is a reasonable photosynthetic rate for a C3 species under ambient conditions.

0.014 µmol CO2 s-1 /0.0009 m2 = 15.6 µmol CO2 m-2 s-1

If you failed to record any of the essential data for your calculations during the experiment, you may retrieve the data from your saved file using the following procedure:

  1. Open the file containing your data. Your data will appear on the screen exactly as they appeared when you saved them at the end of the experiment.
  2. From the menu at the top of the screen select "Analyze" then "Examine". A vertical line will appear on the selected graph that moves horizontally with the mouse. A box will appear on the graph showing data and time values for each run displayed. As you move the cursor over the graph the numerical display in the box will change to show you the exact data point and time value. If the box obscures any part of the trace click on it and hold, then drag with the mouse to place the box in a convenient location.

Data Analysis and Presentation

Students from each bench will prepare a scatter graph of CO2 consumption (µmol CO2 m-2 s-1) at each light intensity for their plant. Examine the figure and select the maximum photosynthetic rate (notice at which light intensity the max is reached for your plant). Why are these data not suitable for statistical tests?

To compare maximum CO2 consumption rates, we will compare data for each plant species to that collected by other students in BISC 111 over multiple years. These data are separated by season; use the appropriate link to access the data. Fall cumulative data and Spring cumulative data. We will use Dixon's Q test to determine whether the values you recorded should be added to this data base, or whether your values represent outliers that should be disregarded.

  1. If your maximum CO2 consumption rate lies within the range of values of the data set provided, it can be added directly to the data set for your plant.
  2. If your value does not lie within the data set's range, arrange the values in the data set provided in order of increasing values, and then calculate the Q value where
Q = gap / range

  1. In this context, the "gap" is the difference between your value and the closest value to it, and the range is the difference between the lowest and highest value in the data set. Compare your calculated Q value with the table of limit values provided below. If your Q value is above the Q limit value for the number of observations in the data set at the 95% confidence level, label your value an outlier and exclude it from the data set.

Determination of Chlorophyll Concentration

Measuring Extract Absorbance

  1. Use the vortex to gently mix your tube(s) from last lab containing 2 leaf punches.
  2. Make sure the leaf punches and any large debris settle back down.
  3. Prepare a blank tube (containing 80% acetone:20% distilled water).
  4. Follow the steps to read absorbance in the Spectrophotometer. Spec. 20 tutorial.
  5. Read and record the absorbance of each tube at A665 and A645. FYI: Spec. 20 readings between 0.3 and 0.85 are preferable.

Draft a data table showing Maximum CO2 uptake (μMol CO2 m-2 s -1) and Total Chl a&b (mg m-2) for all 4 plants.


Calculate total chlorophyll in two leaf punches

Recall that, for each plant, the chlorophyll was extracted from two leaf punches in 5 ml of solvent. Calculate the total Chl a and b concentrations in the 2 leaf punch extract using the following chlorophyll determination equations based on Hartmut K. Lichtenthaler and Claus Buschmann's article in Current Protocols in Food Analytical Chemistry (2001) F4.3.1-F4.3.8 Copyright © 2001 by John Wiley & Sons, Inc..

Chl a = 12.25 A665 - 2.79 A645 = μg/ml in extract        Chl a = [12.25 A665 - 2.79 A645] * 5 = total μg in 2 leaf punches

Chl b = 21.5 A645 - 5.1 A665 = μg/ml in extract        Chl b = [21.5 A645 - 5.1 A665] * 5 = total μg in 2 leaf punches

Calculate the concentration of chlorophyll a+b per m2

For each leaf punch, radius = 3 mm = 0.003 m and surface area = π r2 = 3.14 * 0.0032 = 0.0000282 m2
Combined surface area of 2 leaf punches: 0.0000565 m2
Therefore, the total amount of Chl a+b (in μg) obtained from 2 leaf punches should be multiplied by 1/0.0000565 = 17699.1 to obtain the concentration of Chl a+b in μg m-2.
Convert to mg m-2.
Record the calculations in a class data sheet
Calculate the average concentration for each plant species
Pool class data.

Data Analysis

Design a figure to examine the correlation of maximum CO2 consumption with total chlorophyll. Discuss the design and interpretation of this figure and consider these results and their implications.


1. Draft a scatter graph comparing the rate of CO2 consumption plotted versus light intensity for each of the 4 plant species (one graph with all the data sets). Be sure that the figure legend is included, that the axes are labeled correctly, and that you include the units in which CO2 consumption and light are measured. Please do not include the raw data from which the graphs were plotted.

2. Prepare a new figure/table that shows the relationship between two or more plant characteristics (e.g. transpiration and photosynthesis rates). Incorporate your figure into your presentation next week (see below).

3. Select an article from the primary literature that addresses concepts relevant to the figure designed in question 2 above. Read the article carefully, relate its content to your class experiments, prepare a PowerPoint presentation for your classmates, and present it orally in Lab 6.

See the Statistics and Graphing link for a tutorial that shows how to prepare a scatter graph and other types of graphs.

Other Labs in this Section

Lab 3: Transpiration in High Light/High Wind and Low Light/Low Wind Habitats
Lab 4: Plant Anatomy

Lab 6: Group Oral Presentations