The overall goal of this experiment is to prepare high quality enriched stomata guard cells for physiological, biochemical, and molecular analyses, such as transcriptomics, proteomics, and metabolomics. Hello, my name is Sheldon Lawrence. I am a graduate student in Dr.Chen's lab in the Department of Biology at the University of Florida.
Today, I am going to show you how to isolate guard cells from plant leaf tissue that are useful material for studying both the physiology and biology of stomata. This method is considered a novel method for isolating guard cells. It specifically allows for the enrichment of intact guard cells and provides reliable samples for the study of stomata cell signaling processes.
The application of this method can extend to Brassicaceae plants and other plant species with only small modification to the protocol. The demonstration is important as the main step to enrich the guard cells can prove challenging. To begin, select mature, five week old Arabidopsis plants.
Carefully remove a leaf with a scalpel, cutting the leaf at the base. Take two pieces of scotch tape and place the excised leaf between them. Gently apply pressure to both sides while ensuring that the end of the tape pieces don't adhere.
This can be done by inserting a finger between them. Gently remove the two pieces of tape away from each other, and immediately float the peel taken from the abaxial side of the leaf into stomata opening buffer. After all the peels have been collected, place them under light.
Leave them there for two hours. Take several peels and place them on microscope slides. Using a light microscope, adjust the lenses to view the guard cells and ensure that they are open.
Place the peels into 50 mls of the cell wall digestion enzyme solution. Float and incubate them at 26 degrees Celsius. After 20 minutes, remove the peels from the enzyme solution and rinse twice briefly with deionized water, and place in fresh stomata opening buffer.
Grind the peels in a mortar with liquid nitrogen, with a pestle. Then perform phenol extraction as described in the text. After grinding the peels in a mortar with a pestle, transfer the extract, along with the peels to an Oak Ridge centrifuge tube, and agitate on a shaker for an hour at four degrees Celsius.
Precipitate the phenol extracted proteins by adding five volumes of ice-cold 0.1 mol ammonium acetate in 100%methanol. After precipitating the protein overnight at 20 degrees Celsius, harvest the protein by centrifugation and perform pellet washes as described in the text. Re-suspend the pellet in one milliliter of cold acetone, and transfer to a two-milliliter micro centrifuge tube.
After centrifugation, discard the acetone supernatant and dry the pellet for ten minutes. Dissolve the protein sample in a dissolution buffer. After vortex and centrifugation, collect the supernatant and measure the protein concentration.
Run a SDS-PAGE gel. Visualize the separate protein bands and observe relative protein quality. This is a representative image of Arabidopsis guard cells before and after enzyme digestion of the mesophyll and epidermal cells.
Guard cell viability before and after the removal of mesophyll and epidermal cells can be observed using fluorescein diacetate, which measures enzymatic activity and cell membrane integrity. As you can see, guard cells are still viable after peeling and enzyme digestion. This example illustrates stomata movement of Arabidopsis in response to ABA treatment.
Guard cells are responsive to treatment and stomata aperture decreases more than 50%after 30 minutes of treatment with ABA. Here is a time course of stomata movement. In addition, a representative image of the stomata aperture at each time point is shown.
Protein detection, separation, and relative abundance is observed by SDS-PAGE. Bands are clear and distinct. Four biological replicates are included to emphasize the reproducibility of protein extraction from this method.
We first had the idea for this method when we tried to correlate guard cell molecular changes in real time with stomata movement. Once mastered, a protein sample from stomata guard cells can be prepared within a few hours and can be used in multiple experiments. It is important to remember not to inflict a large amount of damage to the guard cells while peeling and handling.
Following this method, other techniques can be applied to investigate guard cell signaling, such as isobaric labeling to study redux response of proteins in plants in the normal and stress conditions. After watching this video, you should have a good understanding of how to isolate and enrich intact and viable guard cells, as well as extract high-quality protein appropriate for downstream studies.
