Relating Stomatal Conductance to Leaf Functional Traits

The overall goal of this procedure is to relate eco physiological to morphological properties of leaves to better understand ecosystem functioning in diverse plant communities. This is accomplished by first measuring stoma conductance in daily courses, and then extracting relevant parameters that describe stoma control in the different species. The second step is to measure stoma size and density using a microscope.

Next, the leaf vein traits are assessed. The final step is to link the morphological and anatomical features to the eco physiological properties of the plant species. This allows a deeper mechanistic understanding of plant leaves as seen here for the example of somatic closure, when air becomes drier for different species with different leaf traits.

The general idea of our study is to use easily measurable leaf traits as proxies for key plant functions that require large effort if measured directly. The main objective of this protocol is to connect several valley assessed leaf traits to some regulation, which is a key aspect in a plant strategy to balance water use and growth With method can help answering key questions in the field of functional biodiversity research as to how eco physiological processes of different species in a diverse tree community contribute to ecosystem functioning. Choose leaves of different species and individuals according to a reproducible pattern.

For example, choose leaves that have the same height, same exposure, same position within the plant if possible, only from the same node and only from one category such as sun or shade.Leaves. Only measure leaves in healthy and fully developed condition. Mark the leaves on the individuals to ensure that the repeated measurements are done on the same leaf.

Using a steady state parameter, prepare for measurements of stoate conductance only on the leaf surface while avoiding the mid rib and strong leaf veins. Start measurements in the early morning hours before sunrise. Taking five to 10 repeated measurements until s stoma conductance values shows a clear decline at noon.

A daily course of measurements will deliver good data for analyzing the relationships between vapor pressure deficit and STO metal conductance. With each dooma conductance measurement, record temperature and relative humidity, preferably with portable loggers to directly measure the conditions at the possession of the same leaf. Use the August Roche Magnus formula for calculating the vapor pressure deficit.

Now, plot species wise, all stomal conductance data against vapor pressure deficit. Combining all daily courses of individual leaves into one analysis per species. Extract the maximum value observed from the stomal conductance data by searching for the maximum value to scale the model.

For species wise comparability. Divide the observed values through the maximum value observed for that species. For each species regress the logics of the stomate conductance to vapor pressure deficit, and the quadratic term of the vapor pressure deficit using a generalized linear model with a binomial error distribution.

Next, extract the parameter of stoate regulation for every species by calculating the absolute modeled maximum stomate conductance or GS max values. To do this, calculate the vapor pressure deficit at maximum stomal conductance by setting the first derivative of this curve to zero, which gives A VPD GS max fit equals minus B over two. A insert VPD GS max fit into the formula of the curve and raise it to the power of E to obtain max fit.

Calculate the mean of all conductance measurements per species to calculate relative values from the scaled model. Extract nooma conductance conductance and vapor pressure deficit values for the following two points, the stomal, conductance and vapor pressure deficit values at the maximum of the model and the vapor pressure deficit at the second point of inflection of the curve. Multiply these values by GS max to obtain absolute stoat conductance values.

For these points. Take samples preferably from exactly the same leaves that have been used for the measurements of stoate conductance. If this is not possible, apply the same selection procedure that was applied to choose the leaves for the S somatic conductance measurements, preferably on the same individuals.

If the samples cannot be processed immediately, store them in 70%alcohol. Apply a thin layer of colorless quickly drying nail polish to a fresh sample. After the nail polish has out, gently peel the impression from the leaf and proceed for the microscopic analysis.

As with a normal leaf sample, connect a camera to an optical microscope capable of magnifications between 40 times to 400 times after the camera is connected to the microscope. Match the pictures taken to the optical magnification and resolution of the picture with the help of a scale employing open source image processing software like Image J.Analyze these pictures. Draw a shape with the shape tool from the image analysis tool on the image in an area with no dirt, thumbprints, damaged areas or large leaf veins.

Count the S stoma in this area as well as in at least 50, 000 square microns per sample. Measure the S stomatal guard cell length and poor length. Calculate the number of S per square millimeter to bleach the leaves.

Leave them at least 72 hours in a 50%solution of de colorizer, heating the solution up to 30 degrees Celsius. Rinse the leaves several times in water afterwards. Adapt the bleaching process for the specific species depending on their leaf characteristics to color the leaves.

Place them in 99%ethanol color them for two to 30 minutes in a 1%sine solution. After coloring, rinse the leaves several times in water. If the leaves are too deeply stained, some additional time.

An ethanol or de colorizer may help to analyze the samples. Scan the leaves with a backlight scanner at a resolution of approximately 1, 200 dots per inch. Match the scans taken to the resolution of the picture with the help of a scale to ensure that pixel length can be traced back to absolute length measures of the scanned leaf.

Measure the area circumference length and width of the leaves. Calculate several indices such as the ratio of length to width or the ratio of circumference to area. Then cut a one to one centimeter rectangle out of the middle of the picture.

Measure the diameter of the veins of first and second order. Measure the length of all veins of first order. In this quadrant, shown here are fitted model graphs for the stomal conductance data to vapor pressure deficit regression for all species.

The different colored lines represent each leaf habit with black lines representing evergreen species and red lines representing deciduous species. Exploring the links between patterns of stoat conductance regulation and leaf traits reveal that the vapor pressure deficit at the point of inflection decreased with s soad density and stoat index and increased with leaf carbon content. In contrast, no parameter of somatic conductance showed a clear relationship to leaf habit.

The high variation between the two groups of leaf habit shows that different regulatory mechanisms exist both within the group of evergreen and assiduous leaf habits. When applying this procedure, it's important to adjust details of the treatments to the specific leaf properties. Since leaf traits show a large variation among species Following this procedure, our morphological leaf traits can also be linked to other eco physiological characteristics such as xim.

Sensitivity to cavitation or specific hydraulic connectivity, again, helps to understand planned responses to drought. We hope that our video will inspire the use of morphological leaf traits and functional biodiversity research with the aim to predict ecosystem functioning from easily measurable traits.