Leaf-level O₂ and CO₂ measurements with the LI-6800 and Picarro G2207-i

The LI-6800 Portable Photosynthesis System probes the carbon fixation reactions of photosynthesis by calculating fluxes for CO₂ (Assimilation) from the difference in measured concentrations of CO₂ and H₂O entering and exiting a leaf cuvette. With the 6800-01A Multiphase Flash Fluorometer, additional information on the light reactions of photosynthesis, including quantum yield of photosystem II (Φ_PSII) and electron transport rate (J) can also be calculated. The source of electrons for redox reactions in the chloroplast is H₂O, resulting in the evolution of Oxygen (O₂). While the general stoichiometry of these reactions is 1 mol O₂ evolved per mol CO₂ fixed (von Caemmerer 2000), net fluxes of O₂ can depend on environmental conditions, as O₂ is produced solely during the light reactions, but can be consumed by at least three separate reactions (Canvin et al., 1980). Simultaneous measurements of O₂ exchange, CO₂ exchange and chlorophyll fluorescence can provide unique information regarding leaf biochemistry.

Oxygen concentrations in the atmosphere are two to three orders of magnitude larger than CO₂, yet the leaf fluxes are similar in magnitude. Significant challenges occur when measuring small changes in O₂ concentration on a large nominal background concentration, resulting in potentially larger errors in the O₂ fluxes relative to the CO₂ fluxes. With proper attention to the setup and signal averaging, physiologically significant data can be collected.

Here we describe measurements using a Picarro G2207-i O₂ gas concentration analyzer coupled to the LI-6800 Portable Photosynthesis System to quantify both CO₂ and O₂ fluxes using the 9968-210 subsample kit. Best practices for plumbing the instruments together, correction for dilution by foreign gases, and calculation of O₂ fluxes are discussed. A discussion of relative uncertainties between CO₂ and O₂ fluxes and a few example data-sets are also included.

Plumbing

The first consideration for an open flow-through gas exchange measurement should be the concentration stability of the air supply to the measurement cuvette, in this case the air source supplied via the LI-6800 console. In the open atmosphere O₂ concentration is fairly constant over the short-term, but can vary significantly inside a poorly ventilated lab space. In order to achieve a constant reference O₂ concentration, a source of constant O₂ is preferred. For the measurements described here, we used a tank of CO₂-free compressed air (N₂/O₂ mix) supplied to the LI-6800 console.

A recommended plumbing configuration for the LI-6800 and G2207-i is shown in Figure 1‑1. To supply sample air to the G2207-i, the LI-6800 provides subsample outlets on both the reference and sample air streams. In the G2207-i, these sample and reference air streams must be sequentially sampled. A needle valve is placed on each of the lines to maintain constant, controlled airflow from the subsample ports. Due to the comparatively low flow rate of the G2207-i (80-110 sccm or 50 – 75 µmol s^-1), diverting 100 μmol s^-1 from each channel is sufficient for the O₂ measurements. Continuous flow from each line even when not being sampled helps to minimize dead volumes and keep the air-lines purged. The flow meters at the exhaust ports of the LI-6800 gas analyzers can be used to adjust the needle valve on each subsample line to ensure the proper flow rate. Initial setup of the flow can be done by closing the reference or sample needle valve and monitoring the respective IRGA exhaust flow meter. Then the needle valve can be opened until 100 μmol s^-1 is being diverted. This flow rate should be continuously monitored throughout the experiments.

By continuously venting the output of both reference and sample air streams, there will be minimal dead volumes present in the system. On the G2207-i side of the valve, it is necessary to include an open split, as the LI-6800 airstream will supply a larger flow rate than required by the G2207-i. The open flow split and identical flow rate in the two airstreams is important for the G2207-i. the G2207-i’s vacuum pump acts to maintain cavity pressure by adjusting flow through the system. A change in flow rate through the G2207-i will cause pressure changes up-stream including scrub tubes present in the flow path. Both H₂O and CO₂ should be removed from the airstream (see Box 1 on foreign gas dilution), therefore changes in the pressure can cause differential scrubbing between sample and reference lines. In our experiments, the pressure effect was on the order of 30 – 50 ppm H₂O (0.03 – 0.05 mmol mol^-1), enough to cause significant errors in the O₂ measurements (see Appendix A: Foreign Gas Dilution on O2). The order of the scrub tubes is also important. If using soda lime to scrub CO₂ and Drierite® to scrub H₂O, air should pass through the soda lime first. Soda lime must contain moisture to be effective and releases water vapor as a result. Placing Drierite® before the soda lime will not result in a dry airstream for O₂ measurements and will shorten the effective life of the soda lime.

Oxygen Evolution calculations

The flux calculations for O₂ evolution will depend upon the scrubbing scheme chosen. We recommend scrubbing H₂O at all times, but it is up the you to choose whether or not to scrub CO₂. The flux calculation for O₂ evolution will depend on what foreign gases are being scrubbed. A full derivation for either scenario is given in Appendix B: Mass balance equations for CO2 and O2 flux calculation. In the case of scrubbing both H₂O and CO₂ the calculation for O₂ evolution (O_E) is:

1‑1

Where s is measured leaf area (m²), u_i is the flow rate entering the leaf cuvette (mol s^-1), O_R and O_S are the O₂ concentrations measured in reference and sample, respectively in ppm, (or mol O₂ mol ^-1).

If scrubbing H₂O only, the equation for O₂ evolution is:

1‑2

where C_R and C_S are CO2_r and CO2_s respectively, from the LI-6800. Note that the above equations have been derived to report positive values for O₂ evolution and negative values for O₂ consumption.

Uncertainty in Calculated Fluxes for CO₂ and O₂

The uncertainty in a differential measurement, as computed from two absolute measurements, is the sum of the uncertainties in each absolute measurement. In general, for leaf-level measurements, the expected Δ in gas concentrations between sample and reference air streams in O₂ will be similar to the Δ measured for CO₂. For any given flux value, the Δ in concentration is determined by the flow rate to the cuvette and the amount of leaf material being measured. Figure 1‑2A shows the expected Δ as a function of the flux rate for two different cases – the large 6×6 cuvette encompassing 36 cm² of leaf area, and the fluorometer with 6 cm² of leaf area. In both scenarios, a cuvette flow rate of 300 μmol s^-1 was assumed, to allow 100 μmol s^-1 flowing to the O₂ analyzer and maintaining 200 μmol s^-1 through the LI-6800 sample gas analyzer. For very low fluxes, for example a respiration rate of 1 μmol m^-2 s^-1, the Δ generated is 2 μmol mol^-1 and 12 μmol mol^-1 for a leaf area of 6 cm² and 36 cm², respectively.

The uncertainty in calculated flux is a function of the uncertainty in the measured Δ between the reference and sample airstreams. Here, we will use the 1-σ standard deviation specification (e.g., 68% confidence interval) provided by the manufacturer (0.1 μmol mol^-1 on CO₂ for the LI-6800 with 4-second averaging and 2 μmol mol^-1 on O₂ for the G2207-i at 300 second averaging). The uncertainty in calculated fluxes is calculated as a function of the 1-σ standard deviation and expected Δ. The calculated uncertainty in fluxes is shown in Figure 1‑2B, as % uncertainty from the “true” value. In the case of the fluorometer, with a Δ of 2 μmol mol^-1, the uncertainty in CO₂ Assimilation in the LI-6800 is 5%, while in the G2207-i it is > 100%. However, when using the larger 6x6 cuvette with a Δ of 12 μmol mol^-1, uncertainties decrease for both measurements, <1% for the LI-6800 and < 20% for the G2207-i.

While good measurements can be made using any of the LI-6800 cuvettes, caution should be taken when measuring small leaf areas as the errors in the O₂ measurements can become considerable. Best practice is to use the largest leaf area as possible and to operate at low flow rates. This will increase the Δ and reduce uncertainty. Additionally, when leaf area and/or fluxes are small, it is critical to use longer averaging times in the G2207-i (up to 5 minutes) to increase precision.

Example Data

We performed several experiments to validate combined measurements with the LI-6800 and the G2207-i. In order to reduce uncertainties, we used low flow rates and large leaf areas.

Light response curves were performed on Phaseolus vulgaris (bean) leaves (Figure 1‑3). Our results are in line with the expected 1:1 stoichiometry for CO₂:O₂ (von Caemmerer 2000). While not studied in detail, the φO₂ in the linear portion of the light response curve is higher than that for φCO₂, agreeing with theoretical expectations (Singsass et al., 2001). The light response curves were performed using the 6x6 cuvette with 36 cm² of leaf area. At higher light intensities, ΔO₂ is greater than 100 μmol mol^-1, and uncertainty in the measurements is reduced. Near the light compensation point, uncertainty in both CO₂ and O₂ measurements will be high. With the large leaf area in this cuvette, ΔO₂ is reasonably large even during respiration measurements (~9 μmol mol^-1, see Figure 1‑2B).

Additionally, CO₂ response curves were performed on the C4 species Zea mays (corn) using the 6800-01A MultiPhase Flash Fluorometer (Figure 1‑4). This cuvette allows for the combined simultaneous measurement of CO₂ gas exchange, O₂ gas exchange and chlorophyll fluorescence. The LI-6800 fluorometer can measure up to 6 cm² leaf area. In this experiment, electron transport rate (J) can be calculated from three different measurements: 1) Fluorescence, or , where ΦPS_II is measured by chlorophyll fluorescence, Q is incident light intensity, α is leaf absorptance here assumed to be 0.84 and f_II is fraction of photons absorbed by photosystem II, here assumed to be 0.5; 2) Gross CO₂ exchange , where A_G is Gross CO₂ Assimilation, or net CO₂ Assimilation + dark respiration; and 3) Gross O₂ exchange, , where O_EG is gross O₂ evolution (net O₂ evolution + dark O₂ consumption).

The results of these comparisons are shown in Figure 1‑5. Slopes of ~1 in all cases agree with theoretical expectations and with previous results using isotopic techniques to measure O₂ exchange (Ruuska et al., 2000).

Conclusions

O₂ measurements can be combined with CO₂ and chlorophyll fluorescence to provide unique information for leaf-level physiological research. Issues to consider prior to performing experiments include plumbing the system, dealing with dilution by foreign gases and expected uncertainty in the O₂ flux measurements relative to CO₂ flux uncertainty.

Appendix A: Foreign Gas Dilution on O₂

In a non-reactive mixture of multiple gas species at constant temperature and pressure, the addition of one species results in a commensurate decrease in the mole fraction of all other gas species in the mixture. We refer to this effect as dilution. An equation can be written to describe this dilution effect for O₂ (see Hupp, 2011 for derivation of the dilution correction). First, let’s consider the dilution impacts of adding only H₂O on O₂ mole fraction (all units in mol mol^-1):

1‑3

where O_meas is the mole fraction measured, W_meas is the water mole fraction in the airstream and O_dry is the dilution-corrected O₂ concentration. The high abundance of O₂ in the atmosphere leads to a large dilution effect. For example, at typical atmospheric concentration of 21% (210,000 μmol mol^-1), an increase of 5 ppm (0.005 mmol mol^-1) water vapor leads to a ~1 μmol mol^-1 dilution of O₂.

In a leaf-level gas exchange system, incoming air must be humidified to prevent stomatal closure during measurements, and the leaf will add H₂O to the airstream. The sample airstream can have significantly larger water mole fraction than the reference airstream. For this reason, the H₂O dilution effect cannot be ignored. The precision of the LI-6800 H₂O gas analyzer is 0.01 mmol mol^-1 (10 ppm) at 10 mmol mol-1 (1,000 ppm). From equation 1‑3 then, at best the uncertainty in a dilution-corrected O₂ measurement near 21% is 2 μmol mol^-1 simply due to uncertainty in the H₂O measurement. This uncertainty can be as large as the induced O₂ change in some cases. Additionally, water sorption on cuvette surfaces and tubing will cause further errors. For these reasons, a dilution corrected O₂ flux measured in wet air streams unacceptably uncertain. To make high precision O₂ measurements it is necessary to calculate small O₂ fluxes, and the H₂O must be scrubbed prior to entering the G2207-i O₂ analyzer physically eliminating the need for an H₂O dilution correction.

The analysis above justifies the necessity of scrubbing H₂O from the airstream before making the O₂ measurements. CO₂ will also differ significantly between reference and sample air streams, enough to require a dilution correction. Just as for H₂O, a 5 μmol mol^-1 change in CO₂ will cause a 1 μmol mol^-1 in O₂ near 21% O₂ concentrations. However, for CO₂, the LI-6800 measurement precision is better (<0.1 μmol mol^-1). Uncertainties in the CO₂ concentration measurement result in an uncertainty of ~0.02 μmol mol^-1 in the CO₂ dilution-corrected O₂ concentration. However, if CO₂ is not scrubbed, then the choice of chemicals for scrubbing H₂O becomes more limited, as the H₂O scrubber must not interact with CO₂. In our experience, the chemical of choice for this scenario is magnesium perchlorate (Mg(ClO₄)₂), which comes with certain drawbacks; the final state of magnesium perchlorate is a liquid, which needs to be prevented from entering the analyzer and causing damage. Magnesium perchlorate can also be quite expensive. For these reasons, scrubbing both H₂O and CO₂ from the airstream prior to the G2207-i is often the best solution. A single set of chemical columns placed directly in front of the G2207-i, can be used to scrub both sample and reference lines (Figure 1‑1).

Table 1‑1. Useful part numbers.

Part Number	Description
300-157121 Included in the subsampling kit part # 9968-210	Hose barb (metric; M5×0.8 to 1/8" ID)
300-10471a	Needle Valve
300-07385a	¼” Quick-connect “T” fitting
300-03367	¼” Quick-connect “Y” fitting
300-03123	¼” Quick-connect straight union
8150-250	Bev-a-line tubing (15m roll)
300-01961	Balston air Filter
300-07025	Solenoid valve 6 PSI 5V
314-07215	25 pin D -ub connector for LI-6800 console
9960-093	Scrub Tube with ¼” hose barb fittings

Appendix B: Mass balance equations for CO₂ and O₂ flux calculation

The LI-6800 system measures both CO₂ and H₂O and corrects for the mole fraction dilution of CO₂ by H₂O. Uncertainties in H₂O measurements as well as the kinetics of H₂O surface interactions make using the dilution correction problematic (see Appendix A: Foreign Gas Dilution on O2). Thus, the options are to scrub only water vapor and correct the O₂ concentrations for dilution by CO₂, or to additionally scrub CO₂ and H₂O. We will provide mass balances and final flux calculations for both calculations here.

Scrub H₂O and CO₂

The mass balance for O₂ in an open-path gas exchange system can be written as:

1‑4

where s is leaf area (m²), O_E is O₂ evolution (μmol m^-2 s^-1), u_i and u_o are molar flow rates (mol s^-1), entering and exiting the leaf cuvette, respectively, and O_R and O_S are O₂ concentrations (μmol O₂ mol^-1) entering and exiting the leaf cuvette, respectively. When scrubbing both H₂O and CO₂ from the airstream prior to measurement by the Picarro G2207-i O₂ analyzer, the output flow rate u_o is altered only by the addition of O₂:

1‑5

Combining equations 1‑4 and 1‑5 and solving for sO_E yields the flux equation

1‑6

Scrub only H₂O

The mass balance for O₂ in the LI-6800 system when H₂O is scrubbed but CO₂ is not, is identical to equation 1‑4

1‑7

However, in this case the flow exiting the cuvette is altered by both the uptake of CO₂ and evolution of O₂

1‑8

where A is CO₂ Assimilation (μmol CO2 mol^-1 s^-1) and other variables as in equation 1‑4. Combining equations 1‑5 and 1‑4 and solving for sO_E yields

1‑9

A similar equation can be written for the mass balance of CO₂

1‑10

Substituting the expression for sA into equation 1‑9 allows us to solve for sO_E in terms of the concentrations. This form of the equation can be used to calculate O₂ evolution in the case where CO₂ is not being scrubbed.

1‑11

References

Canvin, David T, Joseph A Berry, Murray R Badger, Heinrich Fock, and C Barry Osmond. “Oxygen Exchange in Leaves in the Light.” Plant Physiology 66, no. 2 (1980): 302–7.

Hupp, J.R. 2011. The Importance of Water Vapor Measurements and Corrections. LI-COR, Inc., Application Note 129.

Ruuska, Sari A, Murray R Badger, T John Andrews, and Susanne Von Caemmerer. “Photosynthetic Electron Sinks in Transgenic Tobacco with Reduced Amounts of Rubisco: Little Evidence for Significant Mehler Reaction.” Journal of Experimental Botany 51, no. suppl\_1 (2000): 357–68.

Singsaas, Eric L, Donald R Ort, and Evan H DeLucia. “Variation in Measured Values of Photosynthetic Quantum Yield in Ecophysiological Studies.” Oecologia 128, no. 1 (2001): 15–23.

Von Caemmerer, Susanne. Biochemical Models of Leaf Photosynthesis. Csiro publishing, 2000.