The overall goal of this procedure is to collect flom exudate from Arabidopsis or other plants to analyze its contents or confirm the presence of compounds within the flom stream. This is accomplished by first cutting and incubating the petal in potassium EDTA to prevent sealing of the sea element. The second step is to thoroughly wash the petal to remove any remaining EDTA that can interfere with the subsequent analysis and also damage cells during long-term exposure.
Next, the flow EM exudates are collected into water. Her the final step is to prepare samples for subsequent analysis by L-C-M-S-G-C-M-S gel, electrophoresis, and thin layer chromatography. Ultimately, the EDTA facilitated phem exudation can be used to analyze and confirm phem contents like metabolites and lipids, proteins, or RNAs.
Plants cannot escape adverse conditions. As a result, they require very efficient and fast systems to transmit environmental signals throughout the plants and respond by changing the development. One of these signaling pathways is the plant flow, which is quite complex to understand its composition.
We use the EDTA facilitated flow sedation for the collection and analysis of flow contents. It was originally developed in Perilla by king and but can easily be modified for other species. Demonstrating the procedure will be Elena and US both undergrad students from my lab Prior to starting this procedure.
Grow Arabidopsis plans for four to six weeks in growth chambers with a 12 hour photo period on the day of the collection. Diluted previously prepared 100 millimolar potassium EDTA solution with water to give a final concentration of 20 millimolar. Next, fill two seven to 15 centimeter diameter glass dishes halfway with the 20 millimolar potassium EDTA solution.
When finished, fill 1.7 milliliter reaction tubes with 1.4 milliliters of the 20 millimolar potassium EDTA solution. Then fill an equal number of reaction tubes with 1.4 milliliters of millipore water. After removing four to six week old plants from the growth chambers, harvest the rosette leaves by cutting them with a razor blade at the base of the petal close to the center of the rosette.
Immediately place the leaves in the dishes containing 20 millimolar potassium EDTA with the cut end of the peole submerged in the solution. Once 15 leaves have been collected from three to four plants, gently stack them on top of each other such that the cut petals are aligned with each other. Recut the base of the petals and gently roll the leaves.
Following this gently insert the rolled leaves into one of the reaction tubes containing 20 millimolar potassium EDTA for exudate collection. In the dark line the bottom of a large shallow dish with wet paper towels. Position the rack with the leaf filled reaction tubes in the dish and place it into a black plastic bag with slits for aeration.
Then place the entire setup in a cabinet for exudate collection. In the light line the bottom of a clear plexiglass container with wet paper towels. Place the rack with the leaf filled reaction tubes in the container and close it.
After removing the rack from the container, one hour later, take the leaves outta the reaction tubes. Wash the leaves thoroughly with distilled water to remove all of the EDTA. Immediately transfer the leaves into the reaction tubes containing millipore water and return them to the humidified containers for the collection of exudates following removal of the rack from the humidified container.
Five to eight hours later, take the leaves out of the tubes, then freeze the flow exudates in liquid nitrogen and store them in a minus 80 degrees Celsius freezer. For further analysis on the following day, thaw the samples in preparation for lipid analysis. Then pull two to three samples and add an equal amount of chloroform and methanol to the combined solution.
Vortex the mixture for a few seconds. Next, centrifuge the sample for four minutes at 400 gs. When finished, collect the bottom organic phase in a glass tube after repeating the partition with the top phase, three more times, dry the combined organic phases under a stream of nitrogen and submit to thin layer chromatography and LCMS analysis.
This method allows the harvest of enough phem exudates to identify phem localized proteins and the changes in their level in response to development or stress. In some cases, researchers may want to use proteinase inhibitors to prevent degradation of proteins. As shown here, proteins are in very low abundance, yet their level is high enough for subsequent proteomics experiments.
Using LCMS or for western blot analysis findings in cucurbits suggest that the cutting of the STEM leads to a disruption of the water potential balance and a subsequent influx of water and possible contaminants from the alast. However, collection for times ranging from one to eight hours do not affect the flow M protein profile and composition InOpSys. The amount of protein collected and the pattern of proteins found varies between species and treatments.
As a result, protein signals can be visualized, identified and followed. mRNA and microRNAs can also be identified in flom exudates by this method. However, treatment with RNAs inhibitor is necessary to prevent degradation of the MR.NA during the extended exudation time.
Since the C elements do not contain functional chloroplasts, rubis, small or large subunit mRNAs can be used as negative controls while known pH flu m localized mRNA like ubiquitin conjugating enzyme may be used as a positive control. Similarly, sugars or small metabolites can be analyzed using either GCMS or LCMS. It should be mentioned that the phem exudates contain a large number of functional enzymes, including almost the entire glycolytic pathway.
Hence, using several time points may reveal metabolic processes during the exudate collection. In all exudates, sucroses is the most abundant metabolite. This is most obvious after a collection for one hour.
However, after five hours, the sucrose to fructose ratio is slightly reduced. If the same exudate is collected for one hour and then left on the bench for an additional four hours, the sucrose to fructose ratio is reduced to a much larger extent. Suggesting that active enzymes in the phem exudates lead to degradation of sucrose at room temperature.
In addition, the fact that continuous collection for five hours shows only a slight reduction in the sucrose to fructose ratio is consistent with findings that phem loading and transport of molecules from companion cells into the sea elements may continue during exudation until the system loses its vitality. Lipids in the phem exudates and by extension long distance lipid signaling are a fairly recent field of interest in plant science. Due to their hydrophobic nature, lipids are only present in low concentrations and may be bound to other molecules for solubilization.
Yet the EDTA facilitated exudation allows for the collection of sufficient material to visualize and identify flow and lipids from several plant species. As shown here, it is possible to separate several lipid species using thin layer chromatography. LCMS allows one to identify different lipid species and to monitor changes within the lipid profiles of different genotypes or treatments.
This allows for the study of the role of lipids during plant development and stress response. Once mastered, this technique can be done and as little as three hours if it is performed properly. However, for most samples, we would suggest a collection time of five to eight hours.
In this case, an early morning start of the leaf harvest is recommended. This procedure can be modified for other plants and can lead to the discovery or confirmation of novel flow compounds. While attempting this procedure, it is important to remember to watch the PDLs thoroughly after the first hour to remove the EDTA and to not squeeze the leaves to prevent contamination with content of other cells.
In addition, wearing gloves also protects your samples from contaminants that you could introduce from your skin To prevent contamination, use the appropriate controls for protein RNA or metabolite samples. Suggested controls are listed in the text protocol. This method has allowed us to gain new insights into the composition of the plant flow.
We were able to identify several lipids within the form exudate, which were not expected in the aqueous environment of the floor. This led us and others to introduce the concept of long distance lipid signaling, which has now developed into exciting new field implant research.
