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PLANT PHYSIOLOGY

Measurement of CO2-limited photosynthesis, carboxylationefficiency and CO2 compensation point

In this experiment you will demonstrate that CO2is required for photosynthesis, and

that the rate of photosynthesis increases with CO2concentration in the atmosphere until aCO2saturation point is reached. At that point, photosynthetic rate is limited by the ability ofthe leaf to process the CO2that is delivered to it. Limitation may be caused by insufficientlight energy to drive the maximum rate of photosynthesis, or by the rate at which enzymescatalyze the steps in photosynthetic CO2metabolism.

At very low concentrations of CO2the rate of CO2fixation in photosynthesisapproaches the rate of CO2production in photorespiration. When these two opposingfluxes of CO2balance, the plant is at the CO2compensation point. You will be measuringthe CO2 compensation point in this experiment.

Materials

Leaves of a higher plant (either C3 or C4 species)Qubit Systems CO2analysis package with an infra-red gas analyzerGas bags containing CO2 concentrations of 100, 200, 434, 900, and 1200 ppm

Procedure

1. Arrange the components of the photosynthesis package as described in the GeneralIntroduction. Turn on the computer and select the CO2Analysis package and theS151CO2 Logger Pro file.

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 themaximum value displayed and typing in 30.

3. With Logger Pro running (start data collection by clicking on the Collect button at thetop of the screen) attach the gas bag containing the highest CO2concentration to theinlet of the pump, and attach the outlet of the 500 ml flow restrictor to the inlet of themagnesium perchlorate drying column.

4. Attach the outlet of the magnesium perchlorate column to the inlet of the calibratedIRGA. If the IRGA is calibrated correctly, the stable CO2 concentration shown on thedigital display will match that shown numerically on the computer screen underneath

the graph.

5. Record the CO2 concentration in the gas bag. This is your "referenceCO2" concentration.

6. To observe CO2consumption, or evolution from the leaf, you will need to set theLogger Pro display so that the yaxis of the graph has a range of approximately 130ppm CO2, including values up to 30 ppm above the CO2concentration in the gasbag, and up to 100 ppm below the values in the "reference gas". For example, if the

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bag holds 1200 ppm CO2, adjust the yaxis so that the upper limit is 1230 ppm CO2and the lower limit is 1100 ppm CO2.

7. If the yaxis requires adjustment, stop data collection by clicking on the STOP buttonand adjust axes values. Restart data collection by clicking on the COLLECT button.If your trace goes off screen at any time during a run, you may use the slider controlat the right side of the graph to alter the range of the yaxis. Alternatively, you may

select VIEW from the main menu, and then 'Autoscale' to bring your trace back onscreen.

8. Seal a leaf inside the leaf chamber and place the LED light source directly above it.

9. The leaf chamber has four gas ports grouped in pairs. Each pair consists of a port onthe upper surface of the chamber located directly above a port on the lower surfaceof the chamber. These ports distribute gas to the upper and lower surface of the leafthrough an inlet manifold.

10. Gas leaving the leaf chamber enters an outlet manifold leading to the other pairedgas ports. 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 dryingcolumn. Attach the outlet of the drying column to the inlet of the IRGA.

11. Turn on the light and adjust output to maximum. Observe the decline in the CO2concentration of the gas leaving the leaf chamber as photosynthesis consumes theCO2delivered to the leaf in the reference gas.

12. Wait until a steady value of CO2is measured. This may take several minutes,especially if the leaf has been maintained in a low light environment (such as thelaboratory bench) prior to the experiment. Typically, there will be a rapid decline inCO2concentration followed by a more gradual decline as the leaf responds to theincreased light level by opening its stomata.

13. Record the CO2 concentration when steady state conditions have beenattained. This is your "analysis CO2" concentration at maximum CO2concentration.

14. Stop data collection by clicking on the STOP button. Save your data by selectingSave As . .in the File menu. Give your data an appropriate file name (e.g.PS1200) and save it in your data folder.

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

16. Restart Logger Pro and attach the gas bag containing the next highest CO2concentration to the inlet of the pump. Attach the outlet of the flow restrictor to theinlet of the magnesium perchlorate drying column.

17. Record the CO2concentration in the gas bag. As described in point 6 above, stopdata collection and adjust the yaxis of the CO2display so that the rangeencompasses values 30 ppm above and 100 ppm below, the new reference value.

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18. Repeat procedures 9 to 15 above, and then repeat the entire sequence with the nextlowest CO2concentration. Continue with the experiment until you have measuredleaf CO2exchange at each CO2 concentration provided to you.

19. If the leaf you are using completely filled the leaf chamber, the area enclosed wouldbe 9 cm2. If the leaf did not completely fill the chamber you will need to determine thearea 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 theleaf. 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. Sumthe results and divide the total by 4. The value you obtain is equal to the area of theleaf in cm2.

Calculation of CO2 Exchange Rate

Measurements of photosynthetic, photorespiratory, and respiratory rates in leaves areusually expressed as rates of CO2exchange per unit time per unit leaf area. The units

most commonly used are moles of CO2per m2per second. To express your data in

these units use the following calculations:

1. Calculate the difference between the CO2concentration in the reference andanalysis gases. For example, if the experiment was conducted in air of 350 ppmCO2, at a flow rate of 500 ml/min, the depletion of CO2due to leaf uptake inphotosynthesis at high light may result in an analysis gas CO2concentration of 310ppm. The difference between the reference and sample gas streams (dCO2) in thisexample would be 40 ppm.

2. Convert the dCO2 value from ppm into moles per liter thus:

dCO2/22.413 ([T+C]/T)

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

At a temperature of 20 C and a dCO2of 40 ppm, the dCO2would be equivalent to

1.66 moles CO2per liter.

3. Multiply the CO2value by the flow rate (in liters per second) used in your experimentto obtain a CO2 exchange rate per second. A flow rate of 500 ml/min is equivalent to0.0083 liters/sec. So the CO2 exchange rate in our example would be 0.014

mol/sec.

4. Express your CO2exchange rate on a leaf area basis by dividing the CO2 exchangerate per second by the leaf area in m2. If the leaf completely fills the chamber, thearea used in the calculation would be 9 cm2, equivalent to 0.0009 m2. The

photosynthetic rate in our example would therefore be 15.6 mol 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:

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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 Examinefrom the Analyze menu. A vertical line will appear on each of yourgraphs which can be moved along the data points on the graphs by moving themouse. Boxes will also appear on each graph showing data values and time valuesfor each run displayed. As you move the vertical line on a graph, the numericaldisplay in the box will change to show you the exact data values and time value at thepoint on each graph where the line is situated. If the box obscures any part of the traceclick 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

(mol CO2/m2/s)

When you have calculated rates of photosynthesis at each CO2 concentration used inyour experiment, present your data as a graph with photosynthesis plotted on the y axisand CO2concentration on the x axis.

A generalized photosynthetic CO2 response curve is shown on the opposite page. Notethat at low CO2 concentrations, photosynthesis increases almost linearly as CO2concentration is increased. This is because at these concentrations the rate ofphotosynthesis is limited by the availability of the CO

2

substrate. In C3 species CO2

andO2 compete for the active site of Rubisco, and as CO2 concentration is reduced theoxygenation reaction of Rubisco increases at the expense of the carboxylation reaction.At higher CO2concentrations there is less of an increase in photosynthetic rate per unitincrease in CO2, and eventually photosynthesis reaches CO2saturation at the highest CO2concentration used in the experiment. Under these conditions, the carboxylationreactions of photosynthesis are maximized, and photosynthetic rate is limited either by thesupply of light to the light reactions or by the turnover rate of the photosynthetic enzymes.

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0

5

10

15

20

0 250 500 750

CO2Compensation Point

Carboxylation Efficiency

Gradient =

CO2Saturation Point

External CO2Concentration (ppm)

*

Estimated rate ofPhotorespiration

Generalized PhotosyntheticCO2Response Curve

PhotosyntheticRate

(molCO2/m2/s

)

From QUBIT SYSTEMS Inc Laboratory Manual

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The photosynthetic CO2response 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 physiologyand ecophysiology of the plant. Important aspects of the CO2response curve include:

The CO2Compensation Point. Extrapolate the linear portion of the CO2 responsecurve to intercept the xaxis at the point where photosynthetic rate is zero. The CO2concentration at this point is called the CO2compensation point and it represents the CO2

concentration at which CO2 consumption in photosynthesis is balanced by CO2 productionin photorespiration.

The Rate of Photorespiration. If the linear part of the CO2response curve isextrapolated to intercept the yaxis at zero CO2concentration, the negative rate ofphotosynthesis at this point gives an estimate of photorespiration rate.

Carboxylation Efficiency. Carboxylation efficiency may be defined as the increase inphotosynthetic rate achieved per unit increase in CO2at the site of CO2fixation. In yourexperiment, you did not measure the CO2concentration at the site of CO2fixation, but onlythe CO2concentration in the external atmosphere. However, a qualitative measurementof carboxylation efficiency may still be made by calculating the initial slope of the CO2response curve.

The CO2 Saturation Point of Photosynthesis. The CO2 concentration beyondwhich the CO2response curve plateaus is called the CO2saturation point ofphotosynthesis. At this point increases in CO2concentration do not cause increases inphotosynthetic rate, so factors other than the supply of CO2must be limiting thephotosynthetic 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.

1. Estimate the CO2

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

CO2 compensation point = _ __ __ __ __ __ __ _

Rate of photorespiration = _ _ __ _ __ _ __ _ __ _

Carboxylation efficiency = _ _ __ _ __ _ __ _ __ _

CO2 saturation point = ______________

2 . Discuss all the factors that might influence the CO2 concentration at thesite of carboxylation and describe how you would change the design ofthe experiment to make more accurate measurements of carboxylationefficiency.

3 . Did you measure the CO2saturation point in your experiment? If so doyou think that light supply was the major factor limiting photosynthesisat this point? How would you test this?

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4 . Using the class data compare the carboxylation efficiencies youcalculated for C3 and C4 plants and explain the basis for anydifferences you find.

5 . Compare the CO2 compensation points in the C3 and C4 sp ec ie sand explain the basis for any differences you find.

6 . Compare the estimated rates of photorespiration in the C3 and C 4plants. What is the basis of any differences that you see?

7 . Compare the CO2 saturation point of photosynthesis in the C 3and C4 plants and compare photosynthetic rates underatmospheric CO2 conditions (about 350 ppm CO2). What is thebasis of any differences that you see?