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

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(Determination of Chlorophyll Concentration)
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Design a figure to examine the correlation of maximum CO<sub>2</sub> consumption with total chlorophyll.  Discuss the design and interpretation of this figure. <br>
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Design a figure to examine the correlation of maximum CO<sub>2</sub> consumption with total chlorophyll.  Discuss the design and interpretation of this figure and be ready to discuss this results and their implications during your presentation. <br>
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Revision as of 14:09, 21 June 2011

Contents

Objectives

Lab 5 Overview

I. Formulate hypotheses about photosynthesis rates and chlorophyll concentrations

II. Test hypotheses about photosynthesis rates
a. Measure the rate of photosynthesis
b. Calculate the CO2 exchange rate
c. 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 per g of leaf tissue

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. This is called 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 the youngest leaf that is big enough to 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 leaf, and thus of the leaf that fills it, is 9 cm2.

  1. Check that both gas bag 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 hyacinthe (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, C02 Concentration with Leaf in the Chamber (ppm), ΔCO2 concentration (ppm), and Chamber CO2 Exchange Rate (µmol CO2 m-2 s-1).


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 it appeared when you saved it 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 over time for their plant (see "Assignments" below). These data are not suitable for statistical tests. Why might this be so?

To compare CO2 consumption rates, we will compare data for each plant species to that collected by other students in BISC 111 over multiple years. 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 from last lab containing 2 leaf punches.
  2. Make sure the leaf punches and any large debris settle back down.
  3. Prepare the blank tube (containing in 80% acetone:20% distilled water).
  4. Follow the steps to read absorbance in the Spectrophotometer. Spec. 20 tutorial.
  5. Read the absorbance for each tube at A665 (Chl a) and A645 (Chl b). FYI: Spec. 20 readings between 0.3 and 0.85 are preferable.
A665 Chl a __________ A645 Chl a __________


A665 Chl b __________ A645 Chl a __________


Calculating Chlorophyll concentration

Calculate the Chl a and b concentrations in the extract using these 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(A@665) - 2.79(A@645) X 5ml = ______total μg Chl a

Chl b = 21.5(A@645) - 5.1(A@665) X 5ml = ______total μg Chl b


Sum Chl a and Chl b = __________ μg in one leaf disk

Total Chlorophyll a+b in two leaf disks = ____________ μg


Calculate the area of 2 leaf disks.

Radius of one disk = 3 millimeter = 0.003 meters Area=πr2


Total leaf disc area = ______________ mm2 = _____________m2 X 2 = ________ m2


Relate the Amount of chlorophyll to Photosynthetic rate

[[Determine the amount of chlorophyll in 0.009 m2 leaf surface]]

Total chl in two leaf disks = _______ X 15.9 (conversion factor: # of disks in 9mm2 leaf section) = ______________ Total Chlorophyll per μg/m2

Calculate the photosynthetic rate per μg chlorophyll per sec

Photosynthetic rate μMol CO2 m-2 s-1 = _____________

divided by

Total chlorophyll μg/m-2
= ______________

= Photosynthetic rate μMol CO2/μg chlorophyll/sec = ___________

Pool the class data.


Draft a data table showing Maximum CO2 uptake, Total Chl a and b, and Photosynthetic rate/μg chlorophyll/sec for all 4 plants.

Image:111PhotoTablefin3.jpg

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 be ready to discuss this results and their implications during your presentation.


Assignments

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 spreadsheets from which the figures were made.

2. In groups of two, draft a new figure of your own choosing that demonstrates the presence or absence of a relationship between data collected on two different lab days. Incorporate your figure into your presentation next week (see below).

3. Prepare for oral presentations in Lab 6. Each pair of students will select an article from the primary literature that address concepts similar to those of the figure designed in Assignment 2. Read the article carefully, relate its content to your class experiments, prepare a PowerPoint presentation for your classmates, and present it orally.

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
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