Laboratories for Measuring Physiological
Processes in Plants and Animals

Carbon Dynamics - Part 1

Question: How do organisms acquire energy?

All organisms need energy to live. Photosynthetic organisms, such as plants and some bacteria, can capture energy in the form of light and use it to convert CO₂ into carbon compounds that can serve as a source of food and as a form of stored energy. These organisms are known as autotrophs or "self-feeders". Non-photosynthetic organisms, such as animals, fungi and other types of bacteria, must get their energy pre-packaged in the form of carbon compounds made previously by autotrophs. These organisms are called heterotrophs, or "other-feeders".

Both autotrophs and heterotrophs must break down carbon compounds to release the energy they contain to sustain their own metabolism. This breakdown process is called respiration and it occurs at all times in all living cells of all organisms, plants, animals and bacteria. The process of photosynthesis is thus the opposite of respiration, taking energy from light and using it to convert CO₂ and H₂O into organic carbon compounds, such as sugars, while respiration breaks down these organic carbon compounds to CO₂ and H₂O, releasing energy in the process. These competing processes are illustrated in the diagram below:

Please note the relative position of CO₂ and glucose on the energy scale denoting that glucose contains much more energy than CO₂ because light energy has been used to fuel its synthesis.

Photosynthetic pigments, such as the chlorophylls and carotenoids, absorb light and convert its energy into chemical energy in the form of ATP and NADPH₂. These compounds can then be used to convert CO₂ molecules into sugar molecules. In the overall process of photosynthesis CO₂ molecules are absorbed by the leaf and O₂ molecules are released as a waste product of the process, as seen in the equation below:

Light
energy

6 CO₂ + 12 H₂O → C₆H₁₂O₆ + 6 O₂ + 6 H₂O

The process of respiration reverses this equation and breaks down sugars using O₂ and releasing energy for the organism to use for its metabolic activities.

This laboratory is designed to introduce you to one method of measuring photosynthesis and respiration by using CO₂ gas exchange. Changes in the concentration of CO₂ in the air passing over a leaf can determine if the leaf is taking up CO₂ during photosynthesis or producing CO₂ during respiration and what the rate of these processes is. You will be using an infra-red gas analyzer (IRGA) that can detect changes in CO₂ concentrations in parts per million (ppm), a very sensitive method of measurement. In preparation for this laboratory please review information about the equipment and its use at www.grinnell.edu/courses/bio/qubitmanual under Measurement of Photosynthesis Using CO₂ Analyzer.

Materials

Leaves of C₃ and C₄ plants

Qubit Systems CO₂ analysis package with an infra-red gas analyzer

Gas bags containing room air

Procedures – Part I – Photosynthesis and Respiration Measurements

1. This part of your experiment should take approximately 30 minutes to complete. Adjust the time axis on the computer display to show this value by clicking on the maximum value displayed and typing in 30. Hit return to enter the new value.

2. Seal a leaf inside the leaf chamber and place the LED light source directly above it. Turn on the light source to full intensity. Do NOT hook up the leaf chamber to the system at this time.

3. Attach the gas bag containing room air to the inlet of the pump, attach the black (500 ml/min) flow restrictor to the outlet of the pump and attach the outlet of the flow restrictor to the inlet (stopper end) of the magnesium perchlorate drying column. Plug in the pump to start it running.

4. Attach the outlet of the magnesium perchlorate column to the inlet of the calibrated IRGA. Start data collection by clicking on the Collect button at the top of the screen). If the IRGA is calibrated correctly, the stable CO₂ concentration shown on the digital display in the IRGA will match that shown numerically on the computer screen underneath the graph. Do not stop the computer program at any time during the experiment, only when you have completed your last measurement.

5. Record CO₂ concentration in room air until a stable baseline is established, approximately 2 to 3 minutes.

5. Record the stable CO₂ concentration in the gas bag in the Results section of this handout. This is your "reference CO₂" concentration or baseline concentration. Also record the air temperature.

6. If your trace goes off screen at any time during a run, you may use the slider control at the left side of the graph to alter the range of the y axis. Alternatively, you may select VIEW from the main menu, and then 'Autoscale' to bring your trace back on screen.

7. To begin photosynthetic measurements attach the outlet of the pump with the flow restrictor attached to the inlet of the leaf chamber and attach the outlet of the leaf chamber to the inlet of the temperature/humidity sensor and the outlet of this sensor to the inlet of the drying column. Attach the outlet of the drying column to the inlet of the IRGA.

8. Observe the change in the CO₂ concentration of the gas leaving the leaf chamber as photosynthesis consumes the CO₂ delivered to the leaf in the reference gas. Which direction would you expect the trace to move relative to the baseline during photosynthesis? during respiration?

9. Wait until a steady value of CO₂ is measured. This may take several minutes, especially if the leaf has been maintained in a low light environment (such as the laboratory bench) prior to the experiment.

10. Record the CO₂ concentration when steady state conditions have been attained. This is your "analysis CO₂-light" concentration.

11. To make respiration measurements, turn off the light and wait until a new steady state value of CO₂ is measured in the dark. Record this value as "analysis CO₂-dark". If this value appears to be lower than the baseline you established at the beginning of the experiment, remove the leaf chamber and temp/humidity sensors from the flow path and establish a new baseline or reference CO₂ concentration.

12. Stop data collection by clicking on the Stop button. Save your data by selecting Save As . . in the File menu. Give your data an appropriate file name and save it in the Bio. 251 data folder designated for your lab section.

13. Detach the gas bag from the inlet of the pump and seal it. Detach the outlet of the pump from the inlet of the leaf chamber.

14. If the leaf you are using completely filled the leaf chamber, the area enclosed would be 9 cm². If the leaf did not completely fill the chamber you will need to determine the area of the leaf in the chamber. To do this remove the LED light source from the chamber, place the acetate grid on the surface of the chamber so that it covers the leaf. Count the number of interstices (points where two lines intersect) completely enclosed by the area of the leaf. Any interstices falling exactly on the leaf margin should be given a value of 0.5. Sum the results and divide the total by 4. The value you obtain is equal to the area of the leaf in cm².

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 it appeared when you saved it at the end of the experiment.

·        Select Examine from the Analyze menu. A vertical line will appear on each of your graphs which can be moved along the data points on the graphs by moving the mouse. Boxes will also appear on each graph showing data values and time values for each run displayed. As you move the vertical line on a graph, the numerical display in the box will change to show you the exact data values and time value at the point on each graph where the line is situated. If the box obscures any part of the trace click and drag with the mouse to place the box in a convenient location.

Calculation of CO₂ Exchange Rate

Measurements of photosynthetic, photorespiratory, and respiratory rates in leaves are usually expressed as rates of CO₂ exchange per unit time per unit leaf area. The units most commonly used are mmoles of CO₂ per m² per second. To express your data in these units use the following calculations:

1. Calculate the difference between the CO₂ concentration in the reference and analysis gases. For example, if the experiment was conducted in air of 350 ppm CO₂, at a flow rate of 500 ml/min, 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 (DCO₂) in this example would be 40 ppm.

2. Convert the DCO₂ value from ppm into mmoles per liter as follows:

DCO₂

22.4 [(T+C)/T]

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

Note: Recall that 1 mole of an ideal gas occupies 22.4 liters at standard conditions (273 K, 1 atm). Since your measurements were not made at 273 K (i.e. 0° C) you must correct the gas volume for this by determining the ratio between the actual temperature and the standard temperature. You then multiply the volume of gas by this correction factor.

At a temperature of 20° C and a DCO₂ of 40 ppm, the DCO₂ would be equivalent to 1.66 mmoles CO₂ per liter.

3. Multiply the CO₂ value by the flow rate (in liters per second) used in your experiment to obtain a CO₂ exchange rate per second. A flow rate of 500 ml/min is equivalent to 0.0083 liters/sec. So the CO₂ exchange rate in our example would be 0.014 mmol/sec.

4. Express your CO₂ exchange rate on a leaf area basis by dividing the CO₂ exchange rate per second by the leaf area in m². If the leaf completely fills the chamber, the area used in the calculation would be 9 cm², equivalent to 0.0009 m² (9 X 10^-4 m²). The photosynthetic rate in our example would therefore be 15.6 mmol CO₂/m²/sec which is a reasonable rate for a C3 species under ambient conditions.

Calculate rates of both photosynthesis and respiration for your leaf using the instructions above.

Results

Plant Species ______________________

Leaf Area = ________ cm² Air Temperature ________ ° C

Reference CO₂ concentration = ________________ ppm

Analysis CO₂ - light = ________________________ ppm

Analysis CO₂ - dark = ________________________ ppm

Show calculations, with units, below:

Procedures – Part II – Light Response Curves

1. This part of your experiment should take approximately 60 minutes to complete. Adjust the time axis on the computer display to show this value by clicking on the maximum value displayed and typing in 60. Hit return to enter the new value.

2. Seal a leaf inside the leaf chamber and place the LED light source directly above it. Turn on the light source to full intensity. Do NOT hook up the leaf chamber to the system at this time. Determine the maximum output of the LED light source and record this value in mmoles quanta/m²/sec in the Results section of this handout.

3. Attach a full gas bag containing room air to the inlet of the pump, attach the black (500 ml/min) flow restrictor to the outlet of the pump and attach the outlet of the flow restrictor to the inlet (stopper end) of the magnesium perchlorate drying column. Plug in the pump to start it running.

4. Attach the outlet of the magnesium perchlorate column to the inlet of the calibrated IRGA. Start data collection by clicking on the Collect button at the top of the screen). If the IRGA is calibrated correctly, the stable CO₂ concentration shown on the digital display in the IRGA will match that shown numerically on the computer screen underneath the graph. Do not stop the computer program at any time during the experiment, only when you have completed your last measurement.

5. Record the CO₂ concentration in the gas bag in the Results section of this handout. This is your "reference CO₂" concentration or baseline concentration.

6. If your trace goes off screen at any time during a run, you may use the slider control at the left side of the graph to alter the range of the y axis. Alternatively, you may select VIEW from the main menu, and then 'Autoscale' to bring your trace back on screen.

7. Adjust the output of the LED light source to approximately 1% of its maximum and proceed to measure photosynthesis under these conditions.

8. To begin photosynthetic measurements attach the outlet of the pump with the flow restrictor attached to the inlet of the leaf chamber and attach the outlet of the leaf chamber to the inlet of the temperature/humidity sensor and the outlet of this sensor to the inlet of the drying column. Attach the outlet of the drying column to the inlet of the IRGA.

9. Observe the decline in the CO₂ concentration of the gas leaving the leaf chamber as photosynthesis consumes the CO₂ delivered to the leaf in the reference gas.

10. Wait until a steady value of CO₂ is measured. This may take 2 to 3 minutes. At this low light intensity you may detect little or no photosynthetic activity (i.e. the CO₂ level may be the same as or higher than the baseline reading) because respiration may be as great or greater than photosynthetic rate.

11. Record the CO₂ concentration when steady state conditions have been attained. This is your analysis CO₂ concentration at 1% illumination.

12. Now adjust the LED light source to 2.5% of its maximum output and wait until a new steady value of CO₂ is measured. Record this value as your analysis CO₂ concentration at 2.5% illumination.

13. Repeat this process with light intensities of 5%, 10%, 20%, 40%, 60%, 80% and 100 % of the maximum output of the LED light source.

14. When you have completed all of these measurements stop data collection by clicking on the Stop button. Save your data by selecting Save As . . in the File menu. Give your data an appropriate file name and save it in the Bio. 251 data folder designated for your lab section.

15. Detach the gas bag from the inlet of the pump and seal it. Detach the outlet of the pump from the inlet of the leaf chamber.

16. If the leaf you are using completely filled the leaf chamber, the area enclosed would be 9 cm². If the leaf did not completely fill the chamber you will need to determine the area of the leaf in the chamber. To do this remove the LED light source from the chamber, place the acetate grid on the surface of the chamber so that it covers the leaf. Count the number of interstices (points where two lines intersect) completely enclosed by the area of the leaf. Any interstices falling exactly on the leaf margin should be given a value of 0.5. Sum the results and divide the total by 4. The value you obtain is equal to the area of the leaf in cm².

Results

 

Plant species ________________________________

Reference CO₂ concentration = _____________ ppm

Maximum intensity of LED light source = _______________ mmoles quanta/m²/sec

Show a sample calculation, with units, in the space below:

When you have calculated rates of photosynthesis at each light intensity used in your experiment, present your data for both C3 and C4 plants as a graph with photosynthetic rate (mmol CO₂/m²/sec) plotted on the y axis and light intensity (mmol quanta/m²/sec) on the x axis. This will produce a photosynthetic light response curve. An example of such a curve for a generalized leaf is shown in the graph below.

 

From QUBIT SYSTEMS Inc Laboratory Manual

The photosynthetic light response curve of a particular plant is influenced by many factors, and a study of the components of the curve can tell us a great deal about the physiology and ecophysiology of the plant. The following are important aspects of such a curve:

The Light Compensation Point. Extrapolate the linear portion of your light response curve to intercept the x axis at the point where photosynthetic rate is zero. The light intensity at this point is called the light compensation point.

1) What does this point represent in terms of the physiology of the plant?

2) Compare the light compensation points in the C₃ and C₄ plants and suggest reasons for any of the differences that you see.

The Rate of Dark Respiration. If the linear part of the light response curve is extrapolated to intercept the y axis at zero light intensity, the negative rate of photosynthesis at this point gives an estimate of "dark" respiration rate.

3) What metabolic processes are included in "dark" respiration?

4) Do the rates of dark respiration differ in C₃ and C₄ plants? If so, suggest reasons for this difference.

5) Criticize this method for estimating dark respiration and suggest a way in which it may be measured directly.

Photochemical Efficiency. Photochemical efficiency may be defined as the increase in photosynthetic rate achieved per unit increase in light absorbed by the leaf.

6) What part of the curve would you examine to determine photochemical efficiency?

7) Compare the photochemical efficiencies of C₃ and C₄ plants and explain any differences that you observe. C₄ species are usually restricted to high light environments. Do your data indicate any reason for this?

8) In your experiment, you did not measure light absorbed by the leaf, but only the amount of light incident upon the leaf. Describe how you would change the design of the experiment to make more accurate measurements of photochemical efficiency.

The Light Saturation Point of Photosynthesis. The light intensity beyond which the light response curve plateaus is called the light saturation point of photosynthesis. At this point increases in light intensity do not cause increases in photosynthetic rate, so other factors apart from the supply of light must be limiting the photosynthetic process.

9) What might these factors be?

10) Did you measure the light saturation point in your experiment? If not, why do you think the light saturation point was not reached?

If the light saturation point had been reached, what do you think would be the most likely factor limiting photosynthesis at this point? How would you test this?

11) Your experiment involved a C₃ dicotyledonous plant and a C₄ monocotyledonous plant. Do you think this experiment allows you to make generalized conclusions about the photosynthetic characteristics of C₃ and C₄ species? How would you improve the experiment to make comparisons more informative?

12) Remove a leaf from the C₃ and C₄ plants and hold them up to the light. What do you notice about the veins of each leaf? How does this anatomy correlate with the mechanisms of carbon fixation in each species?