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

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<center><font face="trebuchet ms" style="color:#2171B7" size="5">'''BISC 111/113: Introductory Organismal Biology'''</font><br>
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[[BISC 111/113 | <font face="trebuchet ms" style="color:#2171B7"> '''Introduction to Organismal Biology Lab''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
[[BISC 111/113:Calendar and Assignments | <font face="trebuchet ms" style="color:#2171B7"> '''Calendar and Assignments''' </font>]] 
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[[BISC 111/113:Statistics and Graphing | <font face="trebuchet ms" style="color:#2171B7"> '''Statistics and Graphing''' </font>]] 
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[[BISC 111/113:Science Writing Guidelines | <font face="trebuchet ms" style="color:#2171B7"> '''Science Writing Guidelines''' </font>]] 
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[[BISC 111/113:Lab 1: Biodiversity | <font face="trebuchet ms" style="color:#2171B7"> '''Lab 1: Biodiversity''' </font>]] 
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[[BISC 111/113:Lab 2: Population Growth | <font face="trebuchet ms" style="color:#2171B7"> '''Lab 2: Population Growth''' </font>]] 
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[[BISC 111/113:Labs 3-6: Plant Biology | <font face="trebuchet ms" style="color:#2171B7"> '''Labs 3-6: Plant Biology''' </font>]] 
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[[BISC 111/113:Labs 7-9: Animal Biology | <font face="trebuchet ms" style="color:#2171B7"> '''Labs 7-9: Animal Biology''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br>
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[[BISC 111/113:Lab 10: Laboratory Exam | <font face="trebuchet ms" style="color:#2171B7"> '''Lab 10: Laboratory Exam''' </font>]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
[[BISC 111/113:Lab 11: Population Growth 2 | <font face="trebuchet ms" style="color:#2171B7"> '''Lab 11: Population Growth 2''' </font>]] 
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[[BISC 111/113:Lab 12: Beetle Presentations | <font face="trebuchet ms" style="color:#2171B7"> '''Lab 12: Beetle Presentations''' </font>]] 
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=='''Objectives'''==

=='''Lab 5 Overview'''==
I. Formulate hypotheses about photosynthesis rates and chlorophyll concentrations in plants from different environments<br>
<dl>II. Test hypotheses about photosynthesis rates
<dd>a. Calculate the CO<sub>2</sub> exchange rate
<dd>b. Q test of maximum CO<sub>2</sub> consumption rate vs historical data
</dl>

<dl>III. Test hypotheses about chlorophyll concentrations
<dd>a. Measure the concentration of Chl a and Chl b 
</dl>

<dl>IV. Data Analysis and Presentation
<dd>a. Scatter graph of CO<sub>2</sub> consumption at different light intensities
<dd>b. Correlation of CO<sub>2</sub> and total chlorophyll content
</dl>

=='''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 O<sub>2</sub>.

<center>'''6 CO<sub>2</sub>  +  6 H<sub>2</sub>0  +  light  →  C<sub>6</sub>H<sub>12</sub>O<sub>6</sub>  +  6 O<sub>2</sub>'''</center>

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 CO<sub>2</sub> (one of the culprits in global warming).  CO<sub>2</sub> is needed in the chloroplasts, the sites of photosynthesis, where sunlight is used to transform the inorganic form of carbon (CO<sub>2</sub>) to a form of organic carbon such as glucose (C<sub>6</sub>H<sub>12</sub>O<sub>6</sub>), useful as a source of both energy and building materials.  However, to enter a mesophyll cell and the chloroplast the atmospheric CO<sub>2</sub> 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 CO<sub>2</sub> 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.<br><br>

The process of photosynthesis involves the exchange of gases between the leaf and the environment.  The leaf takes up CO<sub>2</sub> and evolves O<sub>2</sub>, 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 CO<sub>2</sub> and enriched in O<sub>2</sub>.  By measuring the rate at which these gases are exchanged, the rate of photosynthesis can be determined.<br><br>
	
In the first part of lab you will explore how photosynthesis varies with light intensity in leaves of your plant species.  You will measure CO<sub>2</sub> consumption in plant leaves using an Infra Red Gas Analyzer (IRGA).  This Qubit® Gas Analyzing system measures the concentration of CO<sub>2</sub> in the gas leaving the leaf chamber.  Calculation of the difference in CO<sub>2</sub>, with and without the presence of a leaf, and measurement of the flow rate of gas through the chamber, allows calculation of photosynthetic CO<sub>2</sub> fixation rate.<br><br>

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 O<sub>2</sub> is breathed in and CO<sub>2</sub> 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 CO<sub>2</sub> because it is used up right away in photosynthesis.  O<sub>2</sub> 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 O<sub>2</sub> will be used than created, resulting in a net increase in CO<sub>2</sub>.<br>

=='''Measuring the Rate of Photosynthsis'''==
'''Setting up the Experiment'''<br>
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 cm<sup>2</sup>.

#Check that both gas bags are filled and all connections are open. Set the flow meter for 200 ml min<sup>-1</sup>  (0.2 liters min<sup>-1</sup>).
#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.
#Open the '''0-2000 setup.cmbl''' file, and a window will open listing 4 sensors: CO<sub>2</sub> Analyzer, Temperature Sensor, LED Light, and Relative Humidity.  Select each from the drop-down menu and click connect.
<center>[[Image:111F11.PhotosynthesisDataImage.jpg|600px]]</center>
Fig. 5.1. Carbon dioxide exchange in a plant leaf measured with an InfraRed Gas Analyzer (IRGA). The leaf of a water hyacinth (<i>Eichhornia crassipes</i>, was placed in a Qubit® gas analyzing chamber and light intensity (irradiance) was increased in intervals.<br>
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4. Immediately save your file by selecting "SAVE AS" and giving the file an appropriate name (<i>e.g.</i>, Rhoeo Bench 2). This file is necessary for your data to be saved.<br>
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5. Allow the leaf to acclimate in the sealed chamber for at least 20 minutes.<br>
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;6. Turn on the LED and adjust its illumination output to about 300 µE m<sup>-2</sup> s<sup>-1</sup>. On the computer screen you will see 4 graphs (Fig 5.1):  CO<sub>2</sub> concentration (upper left), LED Irradiance (lower left),&nbsp; Air  Temperature (upper right), and Relative Humidity (lower right).  Keep an eye on the irradiance and CO<sub>2</sub> concentration.<br>
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;7. <u>Set the recording so it will not turn off.</u>  To do this, go to Experiment: extend collection: and set it for 90 min or longer.<br>
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;8. Click: Collect (upper middle green button on the LoggerPro tool bar).<br><br>

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Draft a data table in your lab notebook to document the changing CO<sub>2</sub> concentrations at different light intensities. Parameters should include Photon Flux, Reference CO<sub>2</sub> Concentration, CO<sub>2</sub> Concentration with Leaf in the Chamber (ppm), ΔCO<sub>2</sub> concentration (ppm), and Chamber CO<sub>2</sub> Exchange Rate (µmol CO<sub>2</sub> m<sup>-2</sup> s<sup>-1</sup>).[[Image:111F2.PhotonFluxDataChart.png|720px]]


Also be sure to record each of these parameters: '''Plant Name''', '''Temperature''' (°C), '''Flow rate''' ( ml min<sup>-1</sup>), and '''Leaf Area''' (both cm<sup>2</sup> and as converted to m<sup>2</sup>).
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'''Running the Experiment'''<br>
To run the experiment:
#Set the LED illumination back to 0 µE m<sup>-2</sup> s<sup>-1</sup>. (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.)
#Continue to “collect” until the recording stabilizes (5-20 minutes, depending on the plant).
#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 CO<sub>2</sub>.
#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.
#Record the CO<sub>2</sub> concentration in your notebook when STEADY STATE conditions are attained.
#Change the output of the light source from low light to high light in this order: 100, 300, 600, 800, 1000 µE m<sup>-2</sup> s<sup>-1</sup>. Observe the stepwise decrease in the level of CO<sub>2</sub> in the chamber.  Why are the levels of CO<sub>2</sub> decreasing? Typically, there is a rapid decrease in CO<sub>2</sub> concentration followed by a more gradual decrease and then a leveling off as the leaf responds to increased light level.
#Turn the light down to 0 and observe that the CO<sub>2</sub> concentration in the analysis gas increases again. Record this CO<sub>2</sub> concentration when it reaches a steady state value.
#Finally remove the leaf from the chamber, reseal the chamber, and collect data until the level of CO<sub>2</sub> stabilizes.  This is your input or reference CO<sub>2</sub> concentration (should be between 900-1100 ppm).  It represents the concentration of CO<sub>2</sub> in the gas bags.  It should be slightly lower than the CO<sub>2</sub> level when the light was off.  Why? What effect would a leaf in the dark have on CO<sub>2</sub> concentration?
#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.<br><br>

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

#Calculate the difference between the CO<sub>2</sub> concentration in the reference and analysis gases.  For example, if an experiment was conducted in air of 350 ppm CO<sub>2</sub>, at a flow rate of 500 mL min<sup>-1</sup>, the depletion of CO<sub>2</sub> due to leaf uptake in photosynthesis at high light may result in an analysis gas CO<sub>2</sub> concentration of 310 ppm.  The difference between the reference and sample gas streams (∆CO<sub>2</sub> = Reference CO<sub>2</sub> - Sample CO<sub>2</sub>) in this example would be 40 ppm (350-310).
#Convert the ∆CO<sub>2</sub> value from (ppm) into (µmol CO<sub>2</sub> L<sup>-1</sup>):<br><br>
<center>'''∆CO<sub>2</sub> in µmol CO<sub>2</sub> L<sup>-1</sup> = ∆CO<sub>2</sub> in ppm / 22.415 [(T + C) / T]'''</center>
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;where C is the temperature in °C and T is the absolute temperature (273 K).
<blockquote>
EXAMPLE: At a temperature of 20°C and a ∆CO<sub>2</sub> of 40 ppm, the ∆CO<sub>2</sub> would be equivalent to 1.66 µmol CO<sub>2</sub> L<sup>-1</sup>:
</blockquote>
<center>'''40 ppm ∆CO<sub>2</sub> / 22.412 [ (273+20) / T] = 1.66 µmol CO<sub>2</sub> L<sup>-1</sup>'''</center>

&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3. Multiply the ∆CO<sub>2</sub> value by the flow rate (in L s<sup>-1</sup>) used in your experiment to obtain a CO<sub>2</sub> exchange rate per second.  &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; For example, a flow rate of 500 mL min<sup>-1</sup> is equivalent to 0.0083 L s<sup>-1</sup>.  So the CO<sub>2</sub> exchange rate in our example would be &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 0.014 µmol CO<sub>2</sub> s<sup>-1</sup>:<br>

<center>'''0.0083 L s<sup>-1</sup>*1.66 µmol CO<sub>2</sub>  L<sup>-1</sup> = 0.014 µmol CO<sub>2</sub>  s<sup>-1</sup>'''</center><br>

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

<center> '''0.014 µmol CO<sub>2</sub>  s<sup>-1</sup> /0.0009 m<sup>2</sup> =  15.6 µmol CO<sub>2</sub> m<sup>-2</sup>  s<sup>-1</sup>'''</center><br>

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:
	
#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.
#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.<br><br>

=='''Data Analysis and Presentation'''==
Students from each bench will prepare a scatter graph of CO<sub>2</sub> consumption (µmol CO<sub>2</sub> m<sup>-2</sup> s<sup>-1</sup>) 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? <br><br>

To compare maximum CO<sub>2</sub> 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.  [https://spreadsheets.google.com/spreadsheet/ccc?key=0AqFkKnqqD7e7dEVOd29TRmU3a3RMbjZCMXJmQnVGdEE&hl=en_US Fall cumulative data] and [https://spreadsheets.google.com/spreadsheet/ccc?key=0AqFkKnqqD7e7dDM3clM1R3dlcUU0NWVFUzU1YV9qVEE&hl=en_US  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. 

#If your maximum CO<sub>2</sub> 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.
#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<br>
<center> '''Q = gap / range'''</center><br>
#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.<BR>
<center>[[Image:111F11.QTestChart.png|600px]]</center>
<BR>

=='''Determination of Chlorophyll Concentration'''==
'''Measuring Extract Absorbance'''
# Use the vortex to gently mix your tube(s) from last lab containing 2 leaf punches. <BR> 
# Make sure the leaf punches and any large debris settle back down.<BR>
# Prepare a blank tube (containing 80% acetone:20% distilled water).<BR>
# Follow the steps to read absorbance in the Spectrophotometer. [http://www.wellesley.edu/Biology/Concepts/Html/spec20instructions.html    Spec. 20 tutorial].<BR>
# Read and record the absorbance of each tube at A<sub>665</sub> and A<sub>645</sub>. FYI:  Spec. 20 readings between 0.3 and 0.85 are preferable. <BR>
        
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Draft a data table showing Maximum CO<sub>2</sub> uptake (μMol CO<sub>2</sub> m<sup>-2</sup> s <sup>-1</sup>) and Total Chl a&b (mg m<sup>-2</sup>) for all 4 plants. 

<center>[[Image:111PhotoTablefin3fin.jpg]]</center>

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'''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 [http://www.nshtvn.org/ebook/molbio/Current%20Protocols/CPFAC/faf0403.pdf  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.]. <BR><BR>

<center>Chl a = 12.25 A<sub>665</sub> - 2.79 A<sub>645</sub> = μg/ml in extract &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;    Chl a = [12.25 A<sub>665</sub> - 2.79 A<sub>645</sub>] * 5 = total μg in 2 leaf punches <BR><BR>
Chl b = 21.5 A<sub>645</sub> - 5.1 A<sub>665</sub> = μg/ml in extract &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;   Chl b = [21.5 A<sub>645</sub> - 5.1 A<sub>665</sub>] * 5 = total μg in 2 leaf punches </center><BR>

<BR>
'''Calculate the concentration of chlorophyll a+b per m<sup>2</sup>  '''  

For each leaf punch, radius = 3 mm = 0.003 m and   surface area = π r<sup>2</sup> = 3.14 * 0.003<sup>2</sup> = 0.0000282 m<sup>2</sup> <BR>
Combined surface area of 2 leaf punches: 0.0000565 m<sup>2</sup> <BR>
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<sup>-2</sup>. <BR>
Convert to mg m<sup>-2</sup>. <BR>
Record the calculations in a class data sheet<BR>
Calculate the average concentration for each plant species<BR>
Pool class data. <BR>

<BR>
'''Data Analysis'''

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 consider these results and their implications. <br>
<BR>

=='''Assignments'''==
1. Draft a scatter graph comparing the <u>rate</u> of CO<sub>2</sub> 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 CO<sub>2</sub> 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 [[BISC_111/113:Statistics_and_Graphing | 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]]<br>
[[Lab 4: Plant Anatomy]]<br>
[[Lab 6: Group Oral Presentations]]