Name: identification of the genes involved in stomatal development via epidermal phenotype scoring
Uploaded: 2023-01-20T13:05:00
Description: Watch this Scientific Journal Video about Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring at JoVE.com

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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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>
			<td>VWR</td>
			<td>16004-326</td>
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			<td>24-well sterile plates with lid</td>
			<td>VWR</td>
			<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>
			<td></td>
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			<td>Acetic acid, &#8805;99.8%&#160;</td>
			<td>Fisher Scientific</td>
			<td>A38-212</td>
			<td></td>
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			<td>Agar</td>
			<td>BioShop</td>
			<td>AGR001.1</td>
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			<td>Bleech</td>
			<td>Household bleach (e.g., Clorox)</td>
			<td></td>
			<td></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>Petri Dish 100 mm x 20 mm&#160;</td>
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			<td>Propidium Iodide&#160;</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>Tris base</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 ...

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