CO2 Response Curves

Why Measure CO2 Response?

An A-Ci curve (assimilation rate plotted against intercellular CO2 concentration) can provide a number of insights into the biochemistry of a leaf or plant1:

  • CO2 compensation point
  • The value of Ci where photosynthesis and respiration are in balance.
  • Carboxylation efficiency
  • The initial slope provides an in vivo measure of the activity of Rubisco in the leaf. This is sometimes called the mesophyll conductance.
  • Stomatal limitations
  • Stomatal limitation of photosynthesis can be separated from mesophyll limitations.
  • Carboxylation limitations
  • Within the mesophyll, carboxylation limitations can be separated from electron transport limitations.

Operational Considerations

Some things to consider when doing a CO2 response curve.


Even the 6400-02 LED (red only) source will work just fine for CO2 response curves, since the goal is to maintain constant light during the measurement. Stomatal behavior, which the blue light controls, is not as important for this measurement, provided the stomata stay reasonably open. Differential closing (“patchiness”) can be a problem, however.


Speed is important, not precise, predetermined, in-chamber values. There- fore, use the mixer in the constant reference mode. If you wish to eliminate the time that the system takes to lock in on a particular reference value, you could also run the mixer in the constant control signal mode (option C). If you do, you’ll be entering the target values for the AutoProgram in mV instead of μmol mol-1.

In what order should the curve be measured? There are a couple of constraints to consider. One is that high CO2 concentrations may induce some stomatal closure, so if you are including high CO2, they should be done last. The other constraint is that if too much time is spent near the CO2 compensation point, enzyme deactivation may occur. A suggested measurement scheme is to start at ambient, go down to the compensation point, return to ambient, then increase to the upper limit.


The response curve should be measured under constant temperature conditions. Operate the coolers at a constant leaf temperature.

Humidity Control

Operate the flow control for constant water mole fraction. Expect higher conductances and transpiration rates at the low CO2 values, so choose a mole fraction target that gives a flow rate that has room to increase (e.g. 500 or 600 μmol s-1).


Since the concentrations of CO2 are covering a large range, match before each reading.

With OPEN version 3.2 and above, you are asked for a matching threshold (the absolute value of the ΔCO2 value). Thus, you don’t have to decide whether to match before each observation or not; it will match on the ones with ΔCO2 smaller than your threshold, and skip the rest.


This can be a problem for A-Ci curves, since there can be a large concentration gradient between the chamber and ambient. See Diffusion Leaks.


Here’s how to make an automatic CO2 response curve. It uses the AutoProgram “A-CiCurve”, described on page 9-32 in the instruction manual.

  1. Set the chamber conditions
  2. Light: Set the desired value. If not using the LED source, note that constant light is critical for this measurement. It should typically be saturating light (usually > 1500 μmol m-2 s-1 for C3 plants).
  3. Flow: Fixed at 300 μmol s-1. You want 50% RH or higher in the chamber. CO2: Constant reference CO2, at about 40 or 50 μmol mol-1 above ambient.
  4. Clamp onto the leaf
  5. Set the humidity control
  6. Note the value of H2OS_mml. Then change the flow control to constant mole fraction, and target this value. The flow should be 300 μmol s-1 or so. We’ll need room for it to increase, because the conductance will likely increase during the measurement as the chamber CO2 decreases.
  7. Set the temperature
  8. Set the temperature control for constant leaf temperature.
  9. Open a log file
  10. Make sure you have the computations, prompts, log list, log options, etc. that you need.
  11. Area and Stomatal Ratio
  12. Are they correct?
  13. Real time graphics
  14. Set up a screen for plotting A-Ci. (Photo on the Y axis, Ci on the X). If there already is one, clear its data.
  15. Match the IRGAs
  16. Be sure CO2S_μml is stable before you do this.
  17. Launch the “A-CiCurve” Autoprogram
  18. Press 5 then f1. Pick “A-CiCurve” from the list.
  19. When asked “Append to current data file?” Press Y.
  20. When asked “Enter the desired values?”, edit the entries until they are what you want. For example, use 400 300 200 100 50 400 400 600 800. (If it’s a C4 plant, use 0 instead of 50). Notice there are two 400’s in a row after the low value. That is not an error, but a trick to give the leaf some recovery time after the low CO2 measurement. Later on, we can discard the first of those readings, if it doesn’t fit.
  21. When asked “Minimum wait time”, enter the desired value. 60 seconds is usually adequate. This is the time after each CO2 level change that the system will wait before checking stability to see if it can log.
  22. When asked “Maximum wait time”, enter the desired time, such as 120. This is the longest any one point will take.
  23. When asked “Match if |ΔCO2| < ppm”, enter 15.
  24. When asked “Stability Definition OK? (Y/N)”, respond as needed. The experiment will then start automatically.
  25. Watch the curve develop
  26. Press 4 then f3, and watch the curve develop.
  27. When it’s done...
  28. Once the curve is done, you may want to set the CO2 back to the starting value, to let the leaf recover. Or just take the leaf out of the chamber if you are done with it.
  29. After the fact analysis
  30. Before you close the data file, you may want to access GraphIt (press View File (f2 level 1) in New Measurements mode). If the axes are not defined for an A-Ci curve, press QuikPik Config (f1) and select “A-Ci Curve”.
  31. Answer these questions by plotting the appropriate data. Did the sample cell humidity stay constant? Did leaf temperature stay constant?
  32. Exit GraphIt, and close the file
  33. Press escape until you get back to New Measurements mode, then press CLOSE_FILE (f3 level 1) to close the file.