Example: Measuring the CO2 response

The aim of this protocol is to discover the CO2 effects on A, gsw and gm. The CO2 response curve should begin at ambient light and CO2 then proceed to low CO2 in as few of steps as possible to describe the response and then step up to high CO2. Time should be given at each CO2 level for gsw and gm acclimation. Typically, gsw is the slowest to acclimate, requiring 10-30 minutes at each CO2 level.

From Tazoe et al, 2011.
Figure 12‑55. Results published in Tazoe, et al., 2011 [7].

  1. First, go through the suggested warmup procedure (Suggested warmup procedure).

    This process can take more than an hour, so be patient.

  2. (Optional, but recommended) After running the LI-6800 warm-up tests (Step 3), do a TGA zero and optionally, a 1-point span if span gas is available.

  3. After calibrating the TGA, proceed to step 4 in Suggested warmup procedure, to do an isotopic calibration (after reaching a suitably low H2O concentration in the TGA flow path). See Isotopic calibrations.

  4. Check Constants > Gas Exchange.

    • Set leaf area if the leaf does not fill the chamber.
    • Check stomatal ratio.

  5. Check Constants > Leaf Light.

    • Check the absorptance constants being used for each color of the light source installed is appropriate for your species.
    • Check C13_discrim_consts and C3_gm_consts.
    • Use defaults.

  6. Set Environment > Flow.

    • For 6×6 chamber: Set flow to 1,000 µmol s-1.
    • For 3×3 chamber: Set flow to 600 µmol s-1.
    • For 6 cm2 fluorometer: Set pump to medium and flow to 300 µmol s-1.

  7. Set Environment > H2O.

    Control VPD_leaf between 1-1.5 kPa.

  8. Set Environment > CO2.

    Control CO2_s at 420 µmol mol-1 to begin.

  9. Set Environment > Fan.

    Control at 10,000 rpm.

  10. Set Environment > Temperature.

    Control Txchg at 25 °C (or ambient T).

  11. Set Environment > Light.

    • Control at saturating PPFD (or nearly saturating).
    • Set color spec to at least 10% blue.
    • For the fluorometer, limit blue to 40 µmol m-2 s-1.

  12. Set TGA > Status > Settings.

    • Averaging: Use 60 to 120 s.
    • Stability: Use 0.2 to 0.3 µmol mol-1 min-1.
    • Timeout: Set for 2x Averaging, e.g., 120 to 240 seconds.

  13. Set Log Setup > Stability.

    Tap Load and select the file named C13_discrim. It checks for TGA stability as you’ve defined it, and also that ∆_obs is a reasonable value. (No statistics on ∆_obs, since it is already an averaged TGA-derived value.)

    • ΔCO2: Period 20 s, slope limit 0.5 µmol mol-1 min-1.
    • ΔH2O: Period 20 s, slope limit 0.05 mmol mol-1 min-1.
    • gsw: Period 20 s, slope limit 0.005 mol m-2 min-1.
    • Stable (TGA_status): Custom, Test: f(x) = x.
    • Δ_obs (C13_dscrm): Custom, Test: f(x,m,s,d) = x > 0 and x < 44, compute stats period 30 s.

  14. Set Log Setup > Row/Column Options.

    • Make sure the C13_dscrm_consts and C3_gm_consts are all checked to log as a row.
    • Include TGA_stats and any other optional groups you wish.

  15. Set Log Setup > Matching Options.

    • CO2 match: Only match If Time > 10 mins since last match and CO2 has changed more than 5 ppm since last match.
    • H2O match: Only match if Time > 10 mins since last match.
    • Open a Log File.

  16. Set Programs > Programs > AutoLog-GenLoop.

    • Edit Sequence to CO2_r parameter and set the CO2 sequence to 420, 340, 250, 150, 50, 420, 420, 700, 1000, 1500 µmol mol-1.
    • Log interval 60 seconds.
    • Step duration 20 minutes (this may not be long enough for gsw for all species, but it is a good starting point).

  17. Start the program.

  18. Monitor the Measurements tab.

    If you haven’t already configured grid and graphs for the TGA, load the example file TGA-25 (or TGA-25F if using the fluorometer). See Grid and graph settings for TGA Support. This will do the following:

    • Grid 4 is set with common TG25 related parameters and calculations.
    • Graph E shows real-time δC13 reference and sample values.
    • Graph F shows real-time observed, simple C3 model, and infinite gm discrimination values.
    • Graph H is already setup with A vs. Ci, if the fluorometer is installed ETR is also shown.

  19. Monitor Program.

    The program progress is shown on the Programs tab.

  20. After the program is complete, go to Log Setup > Logging to > and close the file.

    An *.xlsx file will be created on close.

  21. Download the files and analyze the data using your favorite software.

    A variety of modern tools are listed at Curve fitting tools.