This research was funded through the Natural Resources and Engineering Research Council of Canada (NSERC) Discovery program and Concordia University. K.B. is supported by the National Overseas Scholarship from India.

1. Staining Arabidopsis cotyledons with TBO
<ol>
	<li>Seed sterilization and growth conditions
	<ol>
		<li>Sterilize ~30 Arabidopsis seeds per genotype in a microcentrifuge tube by adding 1 mL of a seed sterilization solution (33% commercial bleach, 0.1% Triton X-100), and gently rock for 10-12 min at room temperature (RT). 
		NOTE: Sterilize ~30 seeds of the wild-type Arabidopsis accession Columbia (Col-0) and/or ~60 seeds of transgenic plants carrying a chemically-indu...

<ol>
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C., Kakimoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secretory+peptide+gene+EPF1+enforces+the+stomatal+one-cell-spacing+rule.">The secretory peptide gene EPF1 enforces the stomatal one-cell-spacing rule.</a> Genes &amp; Development. 21 (14), 1720-1725 (2007).</li><li>Hara, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+cell+density+is+autoregulated+via+a+secretory+peptide,+EPIDERMAL+PATTERNING+FACTOR+2+in+Arabidopsis+leaves.">Epidermal cell density is autoregulated via a secretory peptide, EPIDERMAL PATTERNING FACTOR 2 in Arabidopsis leaves.</a> Plant &amp; Cell Physiology. 50 (6), 1019-1031 (2009).</li><li>Kondo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomatal+density+is+controlled+by+a+mesophyll-derived+signaling+molecule.">Stomatal density is controlled by a mesophyll-derived signaling molecule.</a> Plant &amp; Cell Physiology. 51 (1), 1-8 (2010).</li><li>Sugano, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomagen+positively+regulates+stomatal+density+in+Arabidopsis.">Stomagen positively regulates stomatal density in Arabidopsis.</a> Nature. 463 (7278), 241-244 (2010).</li><li>Hunt, L., Gray, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+signaling+peptide+EPF2+controls+asymmetric+cell+divisions+during+stomatal+development.">The signaling peptide EPF2 controls asymmetric cell divisions during stomatal development.</a> Current Biology. 19 (10), 864-869 (2009).</li><li>Hunt, L., Bailey, K. J., Gray, J. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+small+RNA+pathways+promote+the+mitotic+events+of+megagametogenesis+during+female+reproductive+development+in+Arabidopsis.">Somatic small RNA pathways promote the mitotic events of megagametogenesis during female reproductive development in Arabidopsis.</a> Development. 139 (8), 1399-1404 (2012).</li><li>Ohki, S., Takeuchi, M., Mori, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NMR+structure+of+stomagen+reveals+the+basis+of+stomatal+density+regulation+by+plant+peptide+hormones.">The NMR structure of stomagen reveals the basis of stomatal density regulation by plant peptide hormones.</a> Nature Communications. 2, 512 (2011).</li><li>Jangra, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplicated+antagonistic+EPF+peptides+optimize+grass+stomatal+initiation.">Duplicated antagonistic EPF peptides optimize grass stomatal initiation.</a> Development. 148 (16), (2021).</li><li>Zhao, C., Craig, J. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+xylem+and+phloem+transcriptomes+from+secondary+tissues+of+the+Arabidopsis+root-hypocotyl.">The xylem and phloem transcriptomes from secondary tissues of the Arabidopsis root-hypocotyl.</a> Plant Physiology. 138 (2), 803-818 (2005).</li><li>Uchida, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+inflorescence+architecture+by+intertissue+layer+ligand-receptor+communication+between+endodermis+and+phloem.">Regulation of inflorescence architecture by intertissue layer ligand-receptor communication between endodermis and phloem.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (16), 6337-6342 (2012).</li><li>Tameshige, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+secreted+peptide+and+its+receptors+shape+the+auxin+response+pattern+and+leaf+margin+morphogenesis.">A secreted peptide and its receptors shape the auxin response pattern and leaf margin morphogenesis.</a> Current Biology. 26 (18), 2478-2485 (2016).</li><li>Abrash, E. B., Davies, K. A., Bergmann, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+signaling+specificity+in+Arabidopsis+by+spatially+restricted+buffering+of+ligand-receptor+interactions.">Generation of signaling specificity in Arabidopsis by spatially restricted buffering of ligand-receptor interactions.</a> The Plant Cell. 23 (8), 2864-2879 (2011).</li><li>Uchida, N., Tasaka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+plant+vascular+stem+cells+by+endodermis-derived+EPFL-family+peptide+hormones+and+phloem-expressed+ERECTA-family+receptor+kinases.">Regulation of plant vascular stem cells by endodermis-derived EPFL-family peptide hormones and phloem-expressed ERECTA-family receptor kinases.</a> Journal of Experimental Botany. 64 (17), 5335-5343 (2013).</li><li>Kosentka, P. Z., Overholt, A., Maradiaga, R., Mitoubsi, O., Shpak, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EPFL+Signals+in+the+boundary+region+of+the+SAM+restrict+its+size+and+promote+leaf+initiation.">EPFL Signals in the boundary region of the SAM restrict its size and promote leaf initiation.</a> Plant Physiology. 179 (1), 265-279 (2019).</li></ol>

The two phenotypic analysis methods for identifying and characterizing the genes controlling stomatal patterning and differentiation presented here are convenient and reliable assays since the protocols do not require the use of epidermal peels and specialized equipment (which are time-consuming and require special training for sample preparation) but do produce high-quality images for the quantitative analysis of epidermal phenotypes.
A limitation of this technique for phenotypic analysis usi...

Proper patterning and differentiation of the plant stomata are critical for their function in two fundamental biological processes, photosynthesis and transpiration, and are enforced by EPF peptide signaling pathways. In Arabidopsis, three secreted cysteine-rich peptides, EPF1, EPF2, and STOMAGEN/EPFL9, control different aspects of stomatal development and are perceived by cell-surface receptor components, including ERECTA-family receptor kinases (ER, ERL1, and ERL2), SERKs, and TMM1,2,3,4,5,6,7,8,9,10. This recognition then leads to the downregulation of the transcription factors that promote stomatal differentiation by a MAPK-dependent process11. The discovery of these core stomatal genes is primarily achieved by the phenotypic screening of mutants exhibiting epidermal defects. This paper presents relatively simple and efficient phenotyping methods for visualizing the stomata and other epidermal cells, which are required to identify and characterize the potential genes controlling stomatal patterning and differentiation.
The observation of the details of the plant epidermis has typically been achieved by using epidermal peels with or without staining with a dye such as toluidine blue O (TBO) or safranin12,13,14. However, the main challenge of these methods is that they require specialized training to peel the leaf epidermis without tearing the tissues and to carefully observe and analyze the patterning data while avoiding the images taken from different parts of the leaf. Chemical treatments to clear the tissue samples with reagents such as chloral hydrate-based clearing solutions have also been widely used for a various range of biological materials8,15; these treatments do generate a great deal of phenotypic information by providing high-quality images but also require the use of dangerous chemicals (e.g., formaldehyde, chloral hydrate). This paper first presents a relatively easy and convenient phenotyping method that produces images sufficient for quantitative analysis but does not require the use of dangerous chemicals and epidermal leaf peels for the sample preparation. A TBO-stained cotyledon epidermis is also ideal for the study of stomatal development because the lack of trichomes and the smaller developmental gradient in cotyledons allow for the simple and tractable interpretation of the epidermal phenotypes.
Stomatal EPF peptides belong to the group of plant-specific, cysteine-rich peptides that have relatively large mature sizes and intramolecular disulfide bonds between conserved cysteine residues. Correct conformational folding is critical for their biological function, but cysteine-rich peptides, which are produced by either chemical synthesis or a heterologous recombination system, can be inactive and are a mixture of both properly folded and unfolded peptides3,7,16. Thus, the screening of bioactive peptides that have a role in controlling stomatal development has been a very challenging task. This manuscript additionally describes a bioassay for the better identification and characterization of bioactive stomatal peptides. In this method, Arabidopsis seedlings are grown in a multi-well plate containing media with and without potential peptides for 6-7 days. Then, the cotyledon epidermis is visualized using a confocal microscope. In general, to clearly visualize the biological activity of potential peptides in stomatal development, the genotypes that produce more and/or less stomatal lineage cells, such as the epf2 mutant, which produces more epidermal cells, and the STOMAGEN-ami line, which confers reduced epidermal cell density2,4,5, are used in addition to the wild-type Arabidopsis control (Col-0) for the bioassays.
Overall, the two protocols presented here can be used for the quick and efficient assessment of various epidermal phenotypes and for screening small peptides and hormones that have a role in controlling stomatal patterning and development.

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			<td>18 mm x 18 mm cover slip</td>
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			<td>16004-326</td>
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			<td>24-well sterile plates with lid</td>
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			<td>CA62406-183</td>
			<td></td>
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			<td>3M Micropore surgical tape</td>
			<td>Fisher Scientific</td>
			<td>19-027-761</td>
			<td>Microporous surgical paper tape used to seal MS plates</td>
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			<td>76 x 26 mm Microscope slide</td>
			<td>TLG</td>
			<td>GEW90-2575-03</td>
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			<td>Acetic acid, &#8805;99.8%&#160;</td>
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			<td>Agar</td>
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			<td>AGR001.1</td>
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			<td>Household bleach (e.g., Clorox)</td>
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			<td>Confocal microscope&#160;</td>
			<td>Nikon&#160;</td>
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			<td>Nikon C2 operated by NIS-Elements&#160;</td>
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			<td>Downloaded from https://imagej.net/software/fiji/</td>
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			<td>F6521&#160;</td>
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			<td>Gamborg&#39;s vitamin mixture</td>
			<td>Cassson Labs</td>
			<td>GBL01-100ML</td>
			<td>Store at 4 &#176;C</td>
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			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>G33-4</td>
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			<td>Growth chambers</td>
			<td>Conviron,&#160;model E15</td>
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			<td>16h light cycle, set at 21&#176;C with a light intensity of 120 &#181;mol&#183;m-2&#183;s-1.</td>
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			<td>HD Supply</td>
			<td>25272</td>
			<td>Fluorescent&#160; lights in growth chambers, Sylvania F72T12/CW/VHO 72&#34;T12 VHO 4200K&#160;</td>
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			<td>Microcentrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>14-222-155</td>
			<td>Tubes in which Arabidopsis thaliana seeds are placed to perform sterilization</td>
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			<td>Microscope&#160;</td>
			<td>Nikon</td>
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			<td>Nikon Eclipse TiE equipped with a DsRi2 digital camera</td>
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			<td>Murashige and Skoog basal salts&#160;</td>
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			<td>Store at 4 &#176;C</td>
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			<td>Petri Dish 100 mm x 20 mm&#160;</td>
			<td>Fisher Scientific</td>
			<td>08-757-11Z</td>
			<td>Petri dishes in which MS media is poured for the purpose of growing Arabidopsis thaliana</td>
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			<td>Propidium Iodide&#160;</td>
			<td>VWR</td>
			<td>39139-064</td>
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			<td>Scalpel</td>
			<td>Fisher Scientific</td>
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			<td>Sucrose</td>
			<td>BioShop</td>
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			<td>Toluidine blue O</td>
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			<td>&#946;-Estradiol</td>
			<td>Sigma-Aldrich</td>
			<td>E2758</td>
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identification of the genes involved in stomatal development via epidermal phenotype scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant productivity and survival. As such, understanding the mechanisms by which stomata develop and pattern has tremendous agronomic value. This paper describes two phenotypic methods using Arabidopsis cotyledons that can be used to characterize the genes controlling stomatal development and patterning. Presented first are procedures for analyzing the stomatal phenotypes using toluidine blue O-stained cotyledons. This method is fast and reliable and does not require the use of epidermal peels, which are widely used for phenotypic analyses but require specialized training. Due to the presence of multiple cysteine residues, the identification and generation of bioactive EPF peptides that have a role in stomatal development have been challenging. Thus, presented second is a procedure used to identify stomatal ligands and monitor their biological activity by bioassays. The main advantage of this method is that it produces reproducible data relatively easily while reducing the amount of peptide solution and the time required to characterize the role of the peptides in controlling stomatal patterning and development. Overall, these well-designed protocols enhance the efficiency of studying the potential stomatal regulators, including cysteine-rich secretory peptides, which require highly complex structures for their activity.

Various stomatal transgenic plants and mutants known to have less or more stomatal density and clustering (epf22,5, epf1 epf22,5, tmm12, a STOMAGEN-silenced line4, and transgenic lines carrying the estradiol-inducible Est::EPF1 or Est::EPF2 overexpression construct7) were used to d...

Watch this Scientific Journal Video about Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring at JoVE.com

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

This paper describes two phenotyping methods without the use of epidermal peels to characterize the genes controlling stomatal development. The first method demonstrates how to analyze a stomatal phenotype using a toluidine blue O-stained plant epidermis. The second method describes how to identify stomatal ligands and monitor their biological activities.

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant ...

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.

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1.5K vistas.  Concordia University. Los dos métodos de análisis fenotípico presentados aquí ofrecen imágenes de alta calidad que son suficientes para realizar análisis fenotípicos cuantitativos y para demostrar las respuestas fenotípicas del tratamiento con péptidos con detalles epidérmicos. En consecuencia, estos dos métodos se pueden utilizar para comprender el patrón y el desarrollo de las células epidérmicas. Estos dos métodos son técnicas relativamente fáciles y confiables, ya que no requieren ningún prod...