Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

1. Measuring Leaf Hydraulic Conductance for Hydrated Leaves (K_max)

1. Collect shoots preferably in the evening or night such that xylem tensions are low, cut in air and place in dark plastic bag filled with wet paper towels. In the lab, recut at least two nodes from the end of every shoot under water. Let shoot rehydrate overnight in pure water, or other solution to be used for K_leaf measurements. Ultrapure water is recommended as a flow solution unless the impacts of different solutions are to be investigated as a factor.
2. Cut the leaf from the shoot with a fresh razor blade under partially degassed flow solution. Ultrapure water is recommended as a flow solution unless the impacts of different solutions are to be investigated as a factor. We recommend degassing the solution overnight in a flask using a vacuum pump, but other methods may be employed.
3. Wrap pre-stretched parafilm around the petiole to ensure a good seal between petiole and tubing. This step is especially helpful for non-round petioles, as wrapping extra parafilm will help round the petiole to fit inside the tubing. Dental paste can be applied to the petiole before wrapping the parafilm to fill grooves in petioles.
4. Connect the leaf to silicone tubing under ultrapure water to prevent air entering the system. As the leaf is lifted above the source of water, the water in the tubing is under subatmospheric pressure, and thus the seal is good if no air is drawn into the tubing during the measurement. Alternatively to sealing the petiole directly into silicone tubing, a compression fitting with rubber gasket can be used (e.g., (Omnifit A2227 bore adaptor; Omnifit, Cambridge, UK); this approach is useful for petioles with especially complex shapes, or for grass blades, which can be wrapped around a glass rod and sealed with the compression fitting. When feasible, sealing the petiole directly into silicone tubing has the advantage of being rapid and leaving a petiole-tubing junction that is transparent, allowing bubbles to be more easily seen. The tubing connects the leaf to hard tubing running to a graduated cylinder on a balance (±10 μg resolution), which logs data every 30 sec to a computer (e.g. using “Balance Link” Software; Mettler-Toledo GmbH, Greifensee, Switzerland) for the calculation of flow rate through the leaf.
5. Leaves should be held adaxial surface upwards in wood frames strung with fishing line above a large box fan and under a light source ( > 1,000 μmol • m² • s^-1 photosynthetically active radiation for high-irradiance measurements), slightly above the level of the meniscus in the graduated cylinder. The leaf should be placed above the water in the graduated cylinder to ensure that water moving to the leaf is driven by transpiration, and not entering the leaf under pressure due to gravity, as would be the case if the leaf were placed below the meniscus of the water in the cylinder. Small containers filled with wet paper towel are placed inside the balance chamber to ensure the air in the balance is water-saturated, thus avoiding evaporation from the water source to the atmosphere in the balance. This possibility can be checked by halting flow into the leaf using a four-way stopcock valve in the tubing, and checking that the mass of the water cylinder on the balance does not decline.
6. Place a clear Pyrex container filled with water above the leaf to absorb the heat of the lamp.
7. Maintain leaf temperature between 23 °C and 28 °C throughout the experiment by adding more cold water to the Pyrex container such that the heat lamp does not heat the Pyrex container sufficiently to warm the air around the leaf below.
8. Allow the leaf to transpire on the apparatus for at least 30 min, thus giving sufficient time to acclimate to high irradiance, and additionally until flow rate has stabilized, with no upward or downward trend, and with a coefficient of variation <5% for at least 5-10 measurements made at 30 sec flow intervals. It is essential for the flow rate to reach a steady state because this method assumes a stable Ψ_leaf. Previous studies found these criteria to be sufficient for stabilization of flow rate (E), Ψ_leaf and K_leaf; tests with longer measurement periods after stable flow was established showed no relationship of K_leaf to measurement time for each of seven species of a wide range of leaf capacitance^17,18 . Stabilization generally takes 30 min or less, but in some species may require more than an hour for some species.

Note: When flow rate is very low (<8 μg s^-1), stability can be determined using the running average of the last five 30 sec intervals.

9. Discard measurements if the flow suddenly changes, either due to sudden stomatal closure, or apparent leakage from the seal or blockage in the system by particles or air bubbles.
10. When flow stabilizes, record leaf temperature with a thermocouple.
11. Quickly remove the leaf from the tubing, dab dry the petiole and place the leaf into a sealable bag which had been previously exhaled in, to halt transpiration.
12. Let the leaf equilibrate in its bag for at least 20 min.
13. Average the final 10 flow rate measurements. This will be your E measurement.
14. When the leaf is equilibrated, measure the final leaf water potential (Ψ_final) with a pressure chamber.
15. Measure leaf area with a flatbed scanner and image processing software, or with a leaf area meter.
16. K_leaf is calculated as E / -ΔΨ_leaf (where ΔΨ_leaf = Ψ_final – 0 MPa) and further normalized by leaf area. Units are in mmol m^-2 s^-1 MPa^-1. Because the leaf is positioned just above the level of water in the graduated cylinder, the effect of gravity in reducing the pressure of water entering the leaf would be negligible, especially relative to the Ψ_final, which is always > -0.1 MPa.
17. To correct for changes in K_leaf induced by temperature dependence of water viscosity^15,19,20 , standardize K_leaf values to 25 °C by using the following equation:

18. Stomatal conductance (g_s) can be determined as E divided by the leaf-to-air vapor pressure difference, equivalent to the vapor pressure deficit (VPD), determined using a temperature and relative humidity sensor adjacent to the leaf. VPD is calculated using the following equation²¹

where RH is the relative humidity, VP is the saturation vapor pressure of the air (in kPa; a function of temperature, according to the Arden Buck equation²²), and AP the atmospheric pressure (in kPa).

2. Measuring Leaf Hydraulic Conductance for Dehydrated Leaves

1. Collect shoots preferably in the evening or night such that xylem tensions are low, cut in air and place in dark plastic bags filled with wet paper towels. In the lab, recut at least two nodes from the end of every shoot under water. Let shoot rehydrate overnight in pure water, or other solution to be used for K_leaf measurements. Ultrapure water is recommended as a flow solution unless the impacts of different solutions are to be investigated as a factor.
2. Cut shoots into segments with at least three leaves under ultrapure water and dehydrate shoots with a fan for different periods of time to a range of Ψ_leaf values.
3. Once shoots are dehydrated, place sealable bags (Whirl-Pak; Nasco, Fort Atkinson, WI, USA) which have been previously exhaled in) around each leaf of the shoot, and then place the whole shoot in a large sealable bag with wet paper towel.
4. Let the shoot equilibrate for at least 30 min (for strongly dehydrated shoots longer equilibration times may be necessary).
5. After equilibration, excise the top and bottom leaf and measure initial Ψ_leaf (=Ψ_o) using a pressure chamber.
6. Discard the shoot if the difference in the Ψ_leaf of those two leaves is greater than 0.1 MPa (for strongly dehydrated leaves, this range can be extended to 0.3 MPa).
7. The third leaf is then used to determine K_leaf and g_s with the EFM, following steps 1.2 -1.18 above. Notably, steady state E can be obtained even for leaves that have been severely dehydrated leaves, as stomata still open and generate g_s values substantially higher than cuticular conductance¹⁷.
8. To construct hydraulic vulnerability curves, calculate K_leaf using the Ψ_final as in step 1.16, and then plot K_leaf values against whichever is lowest, Ψ_o or Ψ_final, i.e., the strongest dehydration experienced by the leaf during the experiment (=Ψ_lowest). Similarly, the decline of g_s in response to leaf dehydration can be determined by plotting g_s against Ψ_lowest. Previous work has shown that dehydration induces at least partially irreversible declines in K_leaf and g_s^14,16.
9. We recommend obtaining at least 6 K_leaf values per 0.5 MPa interval of Ψ_lowest (this interval can be reduced to 0.25 MPa if the species you are working with is really vulnerable to dehydration).
10. Perform Dixon outlier test on K_leaf values for each 0.5 MPa water potential interval of Ψ_lowest to remove outliers from your vulnerability curve²³.
11. Because species differ in their responses of K_leaf and g_s to dehydration^14,16, fit different functions for each species and select the maximum likelihood (lowest AIC value; see ¹⁴ for details). We recommend fitting at least four functions previously used in the literature: linear (), sigmoidal (), logistic () and exponential ().

3. Representative Results

The decline of leaf hydraulic conductance with dehydration varies strongly across species (Fig. 1). Drought sensitive species experience a stronger decline at less negative water potentials than more drought tolerant species, when measured under high irradiance (PAR > 1,000 μmol • m^-2 • s^-1). The water potential at 80% loss of hydraulic conductance (P₈₀) for mesic herb Helianthus annuus was 3 MPa less negative than that of Heteromeles arbutifolia, native to California chaparral. Heteromeles arbutifolia responded linearly to the decline in water potential, whereas Helianthus annuus showed a non-linear decline.

Both stomatal conductance (g_s) and leaf hydraulic conductance (K_leaf) can also respond dramatically to irradiance (Fig. 2). For Raphiolepis indica, the stomatal conductance declined strongly with dehydration for leaves measured under high irradiance (PAR > 1,000 μmol • m^-2 • s^-1) and under lab light (PAR < 6 μmol • m^-2 • s^-1). Under high irradiance, leaves measured with the EFM that had been dehydrated to the water potential that is typical for well-lit leaves on intact plants at mid-day showed similar g_s as for leaves measured on intact plants with a porometer (star in Fig. 2). Notably, for leaves at maximum hydration, K_leaf was four-fold higher for leaves exposed to high than low irradiance (11 vs 2.8 mmol m^-2 s^-1 MPa^-1), and therefore K_leaf showed a stronger light response in well hydrated leaves than g_s. Further, under high irradiance, K_leaf declined more strongly with dehydration. The P₈₀ was 0.93 MPa less negative under high than low irradiance.

Figure 1. Leaf hydraulic vulnerability curves for a drought sensitive (top) and a drought tolerant (bottom) species (from ^14,24 ). Each point represents a single measurement of leaf hydraulic conductance (K_leaf) on a detached leaf using the evaporative flux method (EFM). The vertical bars represent the water potential at which the species lost 80% of initial leaf hydraulic conductance (P₈₀). Best fit functions using maximum likelihood are plotted for both species.

Figure 2. The response of stomatal conductance (g_s) and of leaf hydraulic conductance (K_leaf) to declining leaf water potential for Raphiolepis indica measured under high vs low irradiance (from ¹⁶). (PAR > 1,000 and < 6 mmol • m^-2 • s^-1). Each point represents a single measurement on a detached leaf using the evaporative flux method (EFM), with white and black dots representing leaves measured under high and low irradiance respectively. Equations for stomatal conductance curves in high vs low irradiance: and . Equations for vulnerability curves in high vs. low irradiance: and . The grey star in the upper panel represents the mean ± standard error for g_s and leaf water potential measured on leaves of intact plants at mid-day with a porometer. Grey and black vertical bars in the lower panel represent the water potential at which the species lost 80% of initial leaf hydraulic conductance (P₈₀) under high and low irradiance respectively.