The two phenotypic analysis methods presented here offers high-quality images that are sufficient for conducting quantitative phenotypic analysis and for demonstrating phenotypic responses of peptide treatment with epidermal details. Consequently, these two methods can be used for understanding epidermal cell patterning and development. These two methods are relatively easy and reliable techniques as they do not require any toxic chemicals or epidermal piece that are widely used for epidermal phenotypic analysis.
However, the techniques are complicated and they require specialized training. To begin, carefully select and cut out one of the cotyledons from the individual seedlings that are growing uniformly with other seedlings on the plate to limit variability. Place each cotyledon into a microcentrifuge tube containing one milliliter of a fixing solution using forceps and leave the sample in the fixing solution overnight at room temperature.
Remove the fixing solution and add one milliliter of 70%ethanol. Invert the tube a couple of times and leave the tube containing the samples at room temperature for around 30 minutes. Repeat this step using one milliliter of 50%ethanol and then 20%ethanol.
Replace the one milliliter of 20%ethanol with one milliliter of distilled water, invert the tube containing the cotyledon samples a few times and leave it for around 30 minutes. Remove all the distilled water from the tube and immediately add around 200 microliters of Toluidine Blue O or TBO staining solution for around two minutes. Remove the TBO staining solution as thoroughly as possible and then immediately wash the samples a couple of times by adding one milliliter of fresh distilled water.
In a laminar flow hood, carefully transplant 10 to 12 one-day-old Arabidopsis seedlings from each of the two prepared plates into a 24-well plate containing 1.5 milliliters of half MS liquid medium in each well. Add either 50 millimolar tris HCL buffer alone or two different concentrations of the peptide to each well containing the seedlings germinated on half MS agar plates. Gently mix the seedlings with the buffer alone or a peptide solution using a pipette and seal the plate with micropore tape.
Incubate the assay plate under long day conditions for five to seven days at 22 degrees Celsius. Transfer seedling from the well on a cover slide and dissect the cotyledon of the seedling. Place the abaxial side of the cotyledon up using forceps and cut it into small pieces.
Take another clean microscope slide and place a drop of propidium iodide solution on it. Place one of the small pieces of cotyledon into the drop using forceps and gently place the coverslip. Apply additional propidium iodide solution on the edge of the coverslip to remove any air bubbles that are formed.
Image the abaxial side of the cotyledon using a confocal microscope and compare the images with the images from seedlings grown in half MS medium containing buffer only. Abaxial cotyledon images from 10-day-old seedlings of three Arabidopsis genotypes, wild type, a STOMAGEN silenced line, and epf1 epf2 mutants are shown here. Here, the STOMAGEN silenced line represents the genotypes with low numbers of stomata and the epf1 epf2 mutants represent the genotypes with high numbers of stomata.
The representative epidermis images of cotyledons from 10-day-old seedlings of wild type, tmm, and transgenic lines carrying an estradiol inducible epf2 or epf1 overexpression construct were used for the quantitative analysis of the epidermal phenotype. The representative confocal images of wild type and epf2 cotyledon epidermis grown for six to seven days in a buffer solution and an epf2 cotyledon epidermis grown with two different batches of epf2 peptides are shown in this figure. By performing this procedure, make sure to cut one of the cotyledons from an individual seedling that is already growing uniformly with the other seedlings.
Also choose a seedling that do not touch the MS media to limit variability. Another point to remember is that do not place more than five cotyledons in a single microcentrifuge tube. Also, gently flick the tube to ensure even distribution of the TBO stains around the cotyledons to stain them good.
Considering multiple structural peptides exist in a peptide solution, this bioassay method will be very useful for the identification of properly folded and bioactive forms of peptide and their biological functions.
