Materials and reagents

Joining two LI-6800

Two cuvette flanges (in-house designed, schematics in Supplement 1)
Lower chamber lid (in-house designed, schematics in Supplement 2)
Sealed top chamber lid (in-house designed)
Note: Sealed top lid design is similar to the lower chamber lid design except for the thermocouple holder apertures. The sealed top chamber lid does not have thermocouple holder apertures.
Sealed flange (in-house designed, schematics in Supplement 3)
Flexible stainless-steel tube, 12 mm diameter
O-rings and screws according to the schematics
Thermocouple extension cable with 3.5 mm Jack connectors
(Recommended) LI-6800 Custom Chambers Manifold (LI-COR, catalog number: 6800-19)
Bev-A-line IV tube

Before taking measurements

Polycarbonate sheet 2–3 mm thick, big enough to cover the surface of the chamber and seals

Boundary layer conductance estimation

Glass microfibre filters GF/A 9 cm (Whatman, catalog number: WHA1820090)
PEEK tube Orange 1/16 × 0.02 (IDEX, catalog number: 1532)
250 mL glass beaker
Pure water (reverse osmosis or distilled)

Equipment

Two photosynthesis systems LI-6800 (LI-COR Biosciences, Lincoln, Nebraska)

Software

Add-on script for LI-6800 to use double chamber (in-house coded, download from: https://github.com/PlantPhysiologist/MSF_UpperLower)

Procedure

Joining two LI-6800

Connect the flanges and tubing as shown in Figure 1 and 2. The upper and lower jaws holding the cuvettes should remain connected to LI-6800 #1 so that the lever for opening and closing the chamber remains operational. Place the thermocouple from the LI-6800 #1 to touch the leaf and position #2 to measure the air temperature of the lower cuvette. Keep the light of the LI-6800 #2 switched off to avoid extra heat in the system.

Figure 2. Flanges- and tubing-connected LI-6800 #1 (A) and #2 (B)

Alternatively, reference and sample gases from LI-6800 #2 (Figure 3) can be separated using a Custom Chambers Manifold from LI-COR (part number 6800-19, not included when buying an LI-6800), and then connecting the mixing volume and the reference gas in one tube going to the chamber and leaving the second tube connected to the sample gas inlet only. This last setup is recommended, though it is not used in Márquez et al. (2021) as it was developed after publication.

Figure 3. LI-6800 manifold in the head of the LI-6800 (A) and the connection for the Custom Chambers Manifold (B). Reference gas output (red), sample gas inlet (blue), and mixing fan volume (yellow). Connections are Bev-A-line IV tubes. Output and input tubing from the Custom Chamber Manifold are connected to Flexible stainless-steel tubes of 12 mm diameter.

Before taking measurements

Installing/loading the add-on script
1. Using a thumb drive or the Ethernet port, copy the add-on script file to the folder config.
2. Load the configuration Double cuvette from the display of your LI-6800 in the tab Startup/Configuration.

Warming up the instruments
1. As usual when using an LI-6800, put new chemicals in the water and CO₂ scrub cylinders, refill the water in the humidifier, and install a new CO₂ cartridge.
2. Turn off the light on both LI-6800s.
3. Place the polycarbonate sheet to isolate the gas from each cuvette and close the chamber.
4. Check for leaks by blowing CO₂ (breath is a suitable mix) around the chamber and connections while checking the CO₂ readings (they should not change).
  Note: Correct any leak before continuing with measurements or warm up.
5. Run the automatic warm up of the instrument as usual.
6. Remove the polycarbonate sheet.

Boundary layer conductance estimation

Prepare the evaporative surface (Figure 4)
1. Fill the beaker with distilled water and submerge one end of the PEEK tube in it.
2. Suck through the PEEK tube to fill it with water.
3. Damp two microfibre filter sheets.
4. Put the PEEK tube between the two damped microfibre filter sheets.
5. Place the filter in the LI-6800 chamber and close it (the tube must sit in the middle of the chamber between the filters).
6. Place the beaker such that the water surface is a few centimetres higher than the filters, creating a siphon link, and taking care not to overflow the filter paper.
7. Ensure that the thermocouple in LI-6800 #1 is touching the microfibre filter and that #2 is not touching it.
  
  Figure 4. Schematic of the setup to measure boundary layer conductance

Measuring boundary layer conductance at different fan speeds
1. Set the parameters:
  Both LI-6800s to a flow rate of 600 μmol s^-1; sample CO₂ concentration at 400 μmol mol^-1; and lights off.
  LI-6800 #1 to an air temperature of 25 °C and air saturation deficit (ASD) at 1.2 kPa.
  LI-6800 #2 to a leaf temperature (thermocouple) of 25 °C and vapour pressure difference (VPD) at 1.2 kPa.
2. Take measurements from both cuvettes varying the fan revolutions (rpm) from 5,000 up to 13,000 rpm in steps of 1,000 rpm (Figure 5).
  Note: The flow rate also affects the estimated boundary layer conductance, but it is usually a minor effect for the measurements taken with an LI-6800.
  
  Figure 5. Measurements of boundary layer conductance

Taking measurements

Placing a leaf in the chamber
1. The leaf must cover the whole area of the chamber.
2. Verify that only the thermocouple from LI-6800 #1 is touching the leaf.
3. Set the overpressure in both LI-6800s to 0.1 kPa. Always keep the same overpressure in both cuvettes to avoid mass flow across the leaf.
4. Start the protocol for your measurements and open a file to save your data.

Data analysis

Data analysis and adding the new boundary layer estimation to the LI-6800s
1. Extract the Excel log files from both LI-6800s.
2. In the data file from LI-6800 #2, replace the values given for leaf temperature with those given in the file from LI-6800 #1.
3. Calculate the boundary layer conductance to water (g_bw).
  
  where E is transpiration, w_sat is vapour saturation mole fraction at filter temperature, and w_a is water content in the cuvette air. Note that this is identical to the equation for total conductance (g_tw) in the LI-6800 results spreadsheet.
4. Extract the gbw data and fit a quadratic equation (g_bw = ax²+ bx + c) using fan speed per thousand (rpm/1,000) as the x input.
  1. Polynomial of second order trendline from Excel is a suitable tool to fit the equation.
  2. We use fan speed per thousand in the equation to adjust a, b, and c to four significant figures in the script.
5. Using the LI-6800 display, insert the values for the variables a, b, and c in the tab Constants/User.
6. If the tubing and manifolds in the connections are not modified, the equation for estimating g_bw does not need to be recalculated.

Post-processing data
Replace the Tair values with the Tleaf from the LI-6800 #2 spreadsheet, and the Tleaf values of the #2 spreadsheet for those in #1. The new calculations included by the add-on will appear clustered in the first columns of the results spreadsheet but will not replace the normal ones. Thus, the user should be careful not to mix the data when retrieving it.

Notes

Work with flow rates of 500 μmol s^-1 or higher on each LI-6800 when possible.
Set the same overpressure on both LI-6800s to avoid mass flow across the leaf.
The PEEK tube may be replaced by a piece of microfibre filter with one end in the beaker and the other between the two microfibre filters, but take extra precautions to prevent the microfibre outside the chamber from drying during the measurements.
Set the same air temperature on both cuvettes to keep the cuvette condition identical when modifying water parameters (notice that the thermocouple is measuring the air temperature in LI-6800 #2).

Acknowledgments

We thank P. Groeneveld for technical support and building the LI-6800 connector. We also thank ANID Doctorado, Becas Chile/2015 Folio 72160160 for funding part of the research and ARC support in the form of a Discovery Grant (no. DP210103186). The method here adapted was first published in Márquez et al. (2021) Nature Plants DOI: 10.1038/s41477-021-00861-w.

Competing interests

The authors declare no competing interests.

References

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