Laboratories for Measuring Physiological
Processes in Plants and Animals

Measurement of CO₂-limited photosynthesis, carboxylation
efficiency and CO₂ compensation point.

Photosynthesis is the process by which plants use light energy to synthesize organic compounds. These organic compounds are made from CO₂ absorbed from the atmosphere and are used to feed most living organisms. The availability of CO₂ can greatly affect the photosynthetic process, either increasing the rate of CO₂ fixation into organic compounds when supplies are plentiful or severely reducing it when supplies become limiting. Thus the effect of changing levels of CO₂ on photosynthesis become more important as atmospheric CO₂ levels increase due to the burning of fossil fuels.

In this experiment you will demonstrate that CO₂ is required for photosynthesis, and that the rate of photosynthesis increases with CO₂ concentration in the atmosphere until a CO₂ saturation point is reached. At that point, photosynthetic rate is limited by the ability of the leaf to process the CO₂ that is delivered to it. Limitation may be caused by insufficient light energy to drive the maximum rate of photosynthesis, or by the rate at which enzymes catalyze the steps in photosynthetic CO₂ metabolism.

At very low concentrations of CO₂ the rate of CO₂ fixation in photosynthesis approaches the rate of CO₂ production in photorespiration. When these two opposing fluxes of CO₂ balance, the plant is at the CO₂ compensation point. You will be measuring the CO₂ compensation point in this experiment.

Materials

Leaves of a higher plant (either C3 or C4 species)
Qubit Systems CO₂ analysis package with an infra-red gas analyzer
Gas bags containing CO₂ concentrations of 10, 50, 100, 200, 434, 900, and 1200 ppm

Procedure

1. Arrange the components of the photosynthesis package as described in the General Introduction. Turn on the computer and select the CO₂ Analysis package.
   
   
2. Each 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; then hit return.
   
   
3. 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.
   
   
4. With Logger Pro running (start data collection by clicking on the Collect button at the top of the screen) attach the gas bag containing the highest CO₂ concentration to the inlet of the pump, and attach the outlet of the 500 ml flow restrictor to the inlet of the magnesium perchlorate drying column. Plug in the pump to start it running.
   
   
5. Attach the outlet of the magnesium perchlorate column to the inlet of the calibrated IRGA. If the IRGA is calibrated correctly, the stable CO₂ concentration shown on the digital display will match that shown numerically on the computer screen underneath the graph.
   
   
6. Record the CO₂ concentration in the gas bag. This is your "reference CO₂" concentration. Also record the air temperature.
   
   
   
7. 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.
   
   
   
8. To begin photosynthetic measurements 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 several minutes, especially if the leaf has been maintained in a low light environment (such as the laboratory bench) prior to the experiment. Typically, there will be a rapid decline in CO₂ concentration followed by a more gradual decline as the leaf responds to the increased light level by opening its stomata.
    
    
11. Record the CO₂ concentration when steady state conditions have been attained. This is your "analysis CO₂" concentration at maximum 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 (e.g. PS1200) and save it in your data folder.
    
    
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. Restart Logger Pro and attach the gas bag containing the next highest CO₂ concentration to the inlet of the pump. Attach the outlet of the flow restrictor to the inlet of the magnesium perchlorate drying column.
    
    
15. Record the CO₂ concentration in the gas bag as your new reference value.
    
    
16. Repeat procedures 8 to 12 above, and then repeat the entire sequence with the next lowest CO₂ concentration. Continue with the experiment until you have measured leaf CO₂ exchange at each CO₂ concentration provided to you.
    
    
17. 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 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.

 

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 µmoles 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 µmoles per liter thus:

   DCO₂/22.413 ([T+C]/T)

   where C is the temperature in °C and T is the absolute temperature (273 K). At a temperature of 20° C and a DCO₂ of 40 ppm, the DCO₂ would be equivalent to 1.66 µmoles 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 µmol/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. The photosynthetic rate in our example would therefore be 15.6 µmol CO₂/m²/sec which is a reasonable rate for a C3 species under ambient conditions.

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.

 

Results and Discussion

 

Leaf Area = _____ cm²

CO2 Concentration (ppm)	DCO2 (ppm)	Photosynthetic Rate (µmol CO2/m2/s)

 

When you have calculated rates of photosynthesis at each CO₂ concentration used in your experiment, present your data as a graph with photosynthesis plotted on the y axis and CO₂ concentration on the x axis.

A generalized photosynthetic CO₂ response curve is shown below. Note that at low CO₂ concentrations, photosynthesis increases almost linearly as CO₂ concentration is increased. This is because at these concentrations the rate of photosynthesis is limited by the availability of the CO₂ substrate. In C3 species CO₂ and O₂ compete for the active site of Rubisco, and as CO₂ concentration is reduced the oxygenation reaction of Rubisco increases at the expense of the carboxylation reaction. At higher CO₂ concentrations there is less of an increase in photosynthetic rate per unit increase in CO₂, and eventually photosynthesis reaches CO₂ saturation at the highest CO₂ concentration used in the experiment. Under these conditions, the carboxylation reactions of photosynthesis are maximized, and photosynthetic rate is limited either by the supply of light to the light reactions or by the turnover rate of the photosynthetic enzymes.

 

 

 

 From QUBIT SYSTEMS Inc

 

The photosynthetic CO₂ 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. Important aspects of the CO₂ response curve include:

The CO₂ Compensation Point. Extrapolate the linear portion of the CO₂ response curve to intercept the x axis at the point where photosynthetic rate is zero. The CO₂ concentration at this point is called the CO₂ compensation point and it represents the CO₂ concentration at which CO₂ consumption in photosynthesis is balanced by CO₂ production in photorespiration.

The Rate of Photorespiration. If the linear part of the CO₂ response curve is extrapolated to intercept the y axis at zero CO₂ concentration, the negative rate of photosynthesis at this point gives an estimate of photorespiration rate.

Carboxylation Efficiency. Carboxylation efficiency may be defined as the increase in photosynthetic rate achieved per unit increase in CO₂ at the site of CO₂ fixation. In your experiment, you did not measure the CO₂ concentration at the site of CO₂ fixation, but only the CO₂ concentration in the external atmosphere. However, a qualitative measurement of carboxylation efficiency may still be made by calculating the initial slope of the CO₂ response curve.

The CO₂ Saturation Point of Photosynthesis. The CO₂ concentration beyond which the CO₂ response curve plateaus is called the CO₂ saturation point of photosynthesis. At this point increases in CO₂ concentration do not cause increases in photosynthetic rate, so factors other than the supply of CO₂ must be limiting the photosynthetic process. These factors include:

      a) The supply of light to the leaf.

      b) The amount, and turn-over rate, of enzymes involved in the "dark reactions" of photosynthesis.

 

Estimate the CO₂ compensation point, the rate of photorespiration, carboxylation efficiency, and the CO₂ saturation point of photosynthesis from your graphs. Record the values below:

CO2 compensation point	=	______________
Rate of photorespiration	=	______________
Carboxylation efficiency	=	______________
CO2 saturation point	=	______________

 

1. Discuss all the factors that might influence the CO₂ concentration at the site of carboxylation and describe how you would change the design of the experiment to make more accurate measurements of carboxylation efficiency.
   
   
   
   
2. Did you measure the CO₂ saturation point in your experiment? If so do you think that light supply was the major factor limiting photosynthesis at this point? How would you test this?
   
   
   
   
3. Using the class data compare the carboxylation efficiencies you calculated for C3 and C4 plants and explain the basis for any differences you find.
   
   
   
   
4. Compare the CO₂ compensation points in the C3 and C4 species and explain the basis for any differences you find.
   
   
   
   
5. Compare the estimated rates of photorespiration in the C3 and C4 plants. What is the basis of any differences that you see?
   
   
   
   
6. Compare the CO₂ saturation point of photosynthesis in the C3 and C4 plants and compare photosynthetic rates under atmospheric CO₂ conditions (about 350 ppm CO₂). What is the basis of any differences that you see