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

The overall goal of the following experiment is to measure the efficiency of water transport through the leaf, also known as the leaf hydraulic conductance K leaf. This method allows simultaneous determination of the stoat conductance for a leaf at a known level of hydration status and irradiance. The K leaf is an important determinant of plant water use photos, synthetic rate, and growth, and ecological adaptation.

This measurement is based on excised shoots of three leaves, dehydrated to a given leaf water potential, which will be measured on two leaves of the chute using a pressure chamber as a second step. The remaining third leaf will be measured for K leaf using the evaporative flux method under a given irradiance first to determine the flow rate through the leaf. Next, once steady state flow is achieved, the flow rate is recorded and the leaf is placed in the pressure chamber to determine the water potential driving force corresponding to that flow rate.

This will enable us to calculate kle by dividing the flow rate by the water potential driving force. The S stomatal conductance can be obtained by dividing the flow rate by the vapor pressure difference between the leaf and air. Near the leaf Results are obtained that show that the decline of leaf hydraulic conductance and S somatic conductance with water potential depends on irradiance and differs across species, which has important implications for physiology, whole plant performance and ecology.

A major advantage of this technique over other methods for measuring leaf hydraulics such as the high pressure flow meter, the rehydration kinetics method or the vacuum pump method is that water movement through the leaf follows the natural pathways of transpiration. This method can help answer key questions in the fields of plant physiology and ecology, such as how are leaves designed to allow optimal plant performance in given environments and how sensitive species are to environmental changes such as drought. Collect the chutes in the evening or night and place them in the dark plastic bag filled with wet paper towels.

Then in the lab underwater recut at least two nodes from the end of every shoot, rehydrate overnight in pure water, or another solution to be used for kle measurements. Cut the leaf from the chute with a fresh razor blade under partially Degas flow solution. Then wrap pre-stretch param around the petal to ensure good seal between petal and tubing.

After releasing bubbles and opening the valve in the system, connect the leaf to silicone tubing under ultrapure water to prevent air entering the system. When the leaf is lifted above the source of water, then the water inside the tubing is under sub atmospheric pressure. If no air is drawn into the tubing, then the seal is good.

Alternatively, a compression fitting with rubber gasket can be used for peles with especially complex shapes or for grass blades, which can be wrapped around a plastic rod and sealed with the compression fitting. Note, the tubing from the leaf is running via a syringe with stop cock to a graduated cylinder on a balance that logs data every 30 seconds to a computer for the calculation of flow rate through the leaf face, the leaves add axial surface upwards in wood frames using fishing line, place the leaf and frame above a large box fan and under a light source providing photosynthetically active radiation position the leaves slightly above the level of the meniscus in the graduated cylinder. Because the leaf is placed above the water in the graduated cylinder, any uptake into the leaf is driven by transpiration.

Note that small containers filled with wet paper towels are inside the balance chamber to ensure the air in the balance is water saturated Before putting the leaf on to verify that no evaporation from the water source escapes to the atmosphere in the balance, halt the flow using a four-way stop cock valve in the tubing and check that the mass of the water cylinder on the balance does not decline. A clear Pyrex container placed above the leaf will absorb the heat of the lamp. Maintain leaf temperature between 23 and 28 degrees Celsius throughout the experiment by adding cold water to the Pyrex container.

Allow the leaf to transpire on the system at least 30 minutes, and then until the flow rate has stabilized with at least five measurements at 32nd flow intervals, verify a coefficient of variation of less than 5%It is essential for the flow rate you reach steady state. Because this method assumes stably fo potential discard any measurements if the flowin leaf changes. This could be due to stem metal closure leakage in the seal or blockage in the system by particles or air bubbles.

When the leaf has reached steady state, proceed to record leaf temperature with a thermocouple. Quickly remove the leaf from the tubing dab, dry the peole and place the leaf into a sealable bag that had been previously exhaled in to halt transpiration and place bag within a second bag. Let the leaf equilibrate in its bag for at least 20 minutes.

Average the final 10 flow rate measurements. Then measure the final leaf water potential with a pressure chamber. Also, measure the leaf area.

Now calculate the leaf hydraulic conductance and normalize by leaf area to correct for changes induced by temperature, dependence of water viscosity. Standardize K leaf values to 25 degrees Celsius using the vapor pressure deficit from the weather station. Determine the stoma conductance as detailed in the accompanying text.

Collect the chutes in the evening or night and placed them in a dark plastic bag filled with wet paper towels. Then in the lab underwater recut at least two nodes from the end of every shoot rehydrate overnight in pure water. Or another solution to be used for K leaf measurements.

For everynight, rehydration and measurement, we recommend water unless the impacts of different solutions are to be investigated as a factor. Under ultrapure water, cut the chutes into segments of at least three leaves. Then using a fan, dehydrate the chutes for different periods of time.

To obtain a range of leaf water potentials, exhale into sealable bags and place them around each leaf of the chute. Then cover the whole chute with a large sealable bag, including wet paper towels and equilibrate for at least 30 minutes. Excise the top and bottom leaf.

Measure the initial leaf water potential using a pressure chamber Discard to shoot. If the difference in water potential of the two leaves is greater than 0.1 mke for strongly dehydrated leaves, this range can be extended to 0.3 mega Pro. Proceed with the third leaf to determine leaf water potential and s stoma conductance.

Following the evaporative flux method described earlier, construct hydraulic vulnerability curves also plot the decline of stoma conductance. In response to leaf dehydration, We recommend obtaining at least six K leaf values per 0.5 megapascal interval of leaf water potential. This interval can be reduced to 0.25 megapascal for drought sensitive species.

Next, perform the Dixon Outlier test to remove outliers from the vulnerability curve. Because species differ in their dehydration responses of kle fit different functions for each species and select the maximum likelihood model. We recommend fitting at least four functions previously used in the literature The decline of leaf hydraulic conductance with D dehydration very strongly across species.

Under high irradiance drought sensitive species experience a stronger decline at less negative water potentials than more drought tolerant species. The water potential at 80%loss of hydraulic conductance for the music herb Hetus Annuus was three megapascals less negative than that of heteros are Beau Folia native to California Chaparral. Interestingly, heteros are beauti Folia responded linearly to the decline in water potential.

Whereas Heus Annuus showed a non-linear decline. Here, both the stoma conductance and leaf hydraulic conductance respond dramatically to irradiance. For opus indica, the stoma conductance decline strongly with dehydration for leaves measured under high irradiance and also under lab light mimicking conditions of well-lit leaves on intact plants.

At midday, the EFM data indicate that leaves placed under high irradiance and dehydrated to the same leaf water potential have similar okeoma conductance to leaves measured on intact plants with a parameter notable in well-hydrated leaves, the hydraulic conductance shows a stronger light response than stoat conductance at maximum hydration. The leaf hydraulic conductance was fourfold higher for leaves exposed to high irradiance compared to low under high irradiance, the leaf hydraulic conductance declined more strongly with dehydration. The water potential at 80%loss of hydraulic conductance was 0.93 megapascals less negative under high than low irradiance.

After watching this video, you should have a good understanding of how to measure maximum leaf hydraulic conductance and stem metal conductance, and how to construct lethal mobility curves. Using the EFM once mastered, this technique can be performed properly in less than one hour per leaf. Remember to keep the flow solution very clean and to avoid any air bubbles.

Also clean the system at least once per week, flush the entire system of tubing and containers with 10%bleach for 20 minutes, and then rinse and fill with the gassed flow solution. This technique allows plant physiologists to explore how and why species differ in leaf hydraulic conductance and its responses to the environment for a wide range of plant species.