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-inducible gene (e.g., Est::EPF27) to use transgenic&#160;seedlings grown on 1/2 Murashige and Skoog (MS) plates (2.16 g/L containing 0.8% agar [w/v]) without inducer (e.g., &#946;-estradiol) as controls (Figure 1 and Figure 2).</li>
		<li>Remove the sterilization solution, wash the seeds four times with 1 mL of sterile water in a laminar flow hood, and resuspend them in ~200 &#956;L of sterile 0.1% agar.</li>
		<li>Sow ~30 seeds per genotype on 1/2 MS agar plates or 1/2 MS agar plates containing an inducer (e.g., 10 &#181;M &#946;-estradiol) for the chemical induction of the transgene (e.g., Est::EPF27) using a pipette, and seal with micropore tape. 
		NOTE: The seeds need to be evenly distributed on the plate to avoid placing them in close proximity, as this will prevent the seedlings from growing uniformly.</li>
		<li>Stratify the seeds by placing the plates at 4 &#176;C without light for 3-5 days to synchronize the germination.</li>
		<li>After stratification, incubate the plates in a growth chamber at 22 &#176;C under 120 &#181;mol&#183;m&#8722;2&#183;s&#8722;1 light with a photoperiod of 16 h of light and 8 h of dark for 10 days.</li>
	</ol>
	</li>
	<li>Sampling and TBO staining of Arabidopsis cotyledons
	<ol>
		<li>At 10 days post-germination, 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. 
		NOTE: Cotyledons are sampled from 10 day old seedlings because they do not have trichomes and immature epidermis cells, which simplifies the interpretation of the epidermal phenotypes.</li>
		<li>Place each cotyledon into a microcentrifuge tube containing 1 mL of a fixing solution (9:1 ethanol to acetic acid) using forceps, and leave the sample in the fixing solution overnight, at minimum, and at room temperature. 
		NOTE: The samples can then be stored for up to a couple of years in this condition. The image Figure 2E was taken from a cotyledon sample that was stored in fixing solution for over 3 years. It is important to keep the cotyledon in a fixing solution immediately after cutting and to place no more than five cotyledons in each microcentrifuge tube for homogeneous sample preparation later.</li>
		<li>Remove the fixing solution, and add 1 mL of 70% ethanol. After inverting the tube a couple of times, leave the tube containing the samples at RT for ~ 30 min.</li>
		<li>Repeat step 1.2.3 using 1 mL of 50% ethanol and then 20% ethanol.</li>
		<li>Replace the 1 mL of 20% ethanol with 1 mL of distilled water, and then leave the tube containing the cotyledon samples for ~30 min after inverting the tube a few times. 
		NOTE: The samples may stay in distilled water for more than 24 h, but this is not recommended for obtaining good-quality images (well-stained images for different epidermal cell types) for quantitative analysis.</li>
		<li>Remove all the distilled water from the tube. Then, immediately add ~200 &#956;L of TBO staining solution (0.5% TBO in H2O, filtered) for ~2 min. 
		NOTE: Make sure each cotyledon sample is evenly exposed to TBO solution by gently flicking the tubes during incubation. To prevent overstaining with TBO (the timing of the staining is essential and can be variable for each genotype), do not process more than six tubes containing samples at a time.</li>
		<li>Remove the TBO staining solution as thoroughly as possible, and then immediately wash the samples a couple of times by adding 1 mL of fresh distilled water.</li>
	</ol>
	</li>
	<li>Imaging and data analysis
	<ol>
		<li>Take a microscope slide, and add a drop of 15% glycerol (~50 &#956;L). Place the cotyledon with the abaxial side up into 15% glycerol on the slide using fine forceps. Then, gently cover it with a coverslip so that any air bubbles formed can be removed from the sample.</li>
		<li>Image the abaxial side of the cotyledon epidermis using a brightfield microscope (Figure 1 and Figure 2), and then examine the epidermal phenotype by counting the number of stomata and other epidermal cell types.</li>
		<li>For each genotype, calculate the epidermal phenotype using the following formulas described by Jangra et al.17: 
		&#8203;Stomatal index (%) = (Number of stomata/Total number of epidermal cells) &#215; 100 
		Stomatal density (mm&#8722;2) = Number of stomata/Area (mm2) 
		NOTE: Image at least eight cotyledons (N = 8) for each genotype, and document the epidermal phenotype of each genotype by comparing it to the phenotype of the wild-type plant and/or transgenic plants expressing a chemically-inducible transgene grown without an inducer.</li>
	</ol>
	</li>
</ol>
2. Bioassays for stomatal peptides
NOTE: The procedure for bioassays is shown in Figure 3.
<ol>
	<li>Seed sterilization and growth conditions
	<ol>
		<li>Sterilize ~25 seeds for each treatment as above, and sow approximately half of the seeds onto each of two 1/2 MS agar plates in a laminar flow hood. 
		NOTE: Use the genotype with high and/or low epidermal cells (e.g., epf22) instead of using a wild-type background (e.g., Col-0) to easily detect the biological activities of the peptides on the epidermal cell patterning and differentiation (Figure 4).</li>
		<li>Stratify the seeds for 3 days at 4 &#176;C in the dark.</li>
		<li>Take out each of the two plates at ~10 h intervals, and incubate the seeds on plates at 22 &#176;C under 120 &#181;mol&#183;m&#8722;2&#183;s&#8722;1 light with a photoperiod of 16 h of light and 8 h of dark for 1 day.</li>
	</ol>
	</li>
	<li>Peptide treatment and imaging
	<ol>
		<li>In a laminar flow hood, carefully transplant 10-12 one day old Arabidopsis seedlings from each of the two prepared plates into a 24-well plate containing 1.5 mL of 1/2 MS liquid medium in each well (~20 seedlings per well). 
		NOTE: The timing of the peptide treatment for the seedlings is critical to the phenotypic outcomes of the peptide treatment, so prepare the seeds on two separate plates to obtain two different seedling developmental stages for the treatment.</li>
		<li>Add either a buffer alone (50 mM Tris-HCl [pH 8.0]) or two different concentrations of the peptide (typically, 1 &#181;M and 2.5 &#181;M peptide in 50 mM Tris-HCl [pH 8.0]) to each well containing ~20 one day old Arabidopsis seedlings germinated on 1/2 MS agar plates. 
		NOTE: Remove the peptide solution aliquots from a freezer, and keep them at RT for a few minutes before the treatment. Peptides stored in a freezer at &#8722;80 &#176;C are stable for a couple of years, but freeze-thaw cycles should be avoided by storing the peptide solution in small aliquots.</li>
		<li>After gently mixing the seedlings with a buffer alone or a peptide solution using a pipette, seal the plate with micropore tape. Incubate the assay plate under long-day conditions (16 h photoperiod, 120 &#181;mol&#183;m&#8722;2&#183;s&#8722;1 light) for 5-7 days at 22 &#176;C. 
		NOTE: Prevent the seedlings from growing too close together to allow each seedling to have even exposure to the peptide solution. Gently rotate the plate for 2-3 h before incubating the plate in a growth chamber to increase aeration.</li>
		<li>Transfer each seedling from the well containing either buffer alone or peptide 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.</li>
		<li>Take another clean microscope slide, and place on it a drop of propidium iodide solution (~25 &#956;L, 2 mg/mL in H2O).</li>
		<li>Place one of the small pieces of cotyledon into a drop of propidium iodide solution using forceps, and gently place the coverslip. Apply additional propidium iodide solution on the edge of the coverslip to remove any air bubbles that have formed.</li>
		<li>Image the abaxial side of the cotyledon using a confocal microscope, and compare the images with the images from seedlings grown in 1/2 MS medium containing buffer only. 
		NOTE: The images presented in Figure 4 were taken from wild-type Col-0 or epf22,5 seedlings that were treated with buffer only (Figure 4A, B) or two batches of EPF2 peptide solution (Figure 4C-F) and were stored at &#8722;80 &#176;C over 1 year.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Hara, K., Kajita, R., Torii, K. U., Bergmann, D. 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. 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+signalling+peptide+EPFL9+is+a+positive+regulator+of+stomatal+development.">The signalling peptide EPFL9 is a positive regulator of stomatal development.</a> New Phytologist. 186 (3), 609-614 (2010).</li><li>Lee, J. 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=Direct+interaction+of+ligand-receptor+pairs+specifying+stomatal+patterning.">Direct interaction of ligand-receptor pairs specifying stomatal patterning.</a> Genes &amp; Development. 26 (2), 126-136 (2012).</li><li>Shpak, E. D., McAbee, J. M., Pillitteri, L. J., Torii, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomatal+patterning+and+differentiation+by+synergistic+interactions+of+receptor+kinases.">Stomatal patterning and differentiation by synergistic interactions of receptor kinases.</a> Science. 309 (5732), 290-293 (2005).</li><li>Nadeau, J. A., Sack, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+stomatal+distribution+on+the+Arabidopsis+leaf+surface.">Control of stomatal distribution on the Arabidopsis leaf surface.</a> Science. 296 (5573), 1697-1700 (2002).</li><li>Meng, X., 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=Differential+function+of+Arabidopsis+SERK+family+receptor-like+kinases+in+stomatal+patterning.">Differential function of Arabidopsis SERK family receptor-like kinases in stomatal patterning.</a> Current Biology. 25 (18), 2361-2372 (2015).</li><li>Lampard, G. R., Macalister, C. 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=Arabidopsis+stomatal+initiation+is+controlled+by+MAPK-mediated+regulation+of+the+bHLH+SPEECHLESS.">Arabidopsis stomatal initiation is controlled by MAPK-mediated regulation of the bHLH SPEECHLESS.</a> Science. 322 (5904), 1113-1116 (2008).</li><li>Yang, M., Sack, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+too+many+mouths+and+four+lips+mutations+affect+stomatal+production+in+Arabidopsis.">The too many mouths and four lips mutations affect stomatal production in Arabidopsis.</a> The Plant Cell. 7 (12), 2227-2239 (1995).</li><li>Theunissen, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+studying+grass+leaf+epidermis.">An improved method for studying grass leaf epidermis.</a> Stain Technology. 64 (5), 239-242 (1989).</li><li>Venkata, B. P., 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=crw1--A+novel+maize+mutant+highly+susceptible+to+foliar+damage+by+the+western+corn+rootworm+beetle.">crw1--A novel maize mutant highly susceptible to foliar damage by the western corn rootworm beetle.</a> PLoS One. 8 (8), 71296 (2013).</li><li>Tucker, M. 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. C., Petzold, H. E., Dickerman, A. W., Beers, E. 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>

No conflicts of interest were declared.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>18 mm x 18 mm cover slip</td>
			<td>VWR</td>
			<td>16004-326</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well sterile plates with lid</td>
			<td>VWR</td>
			<td>CA62406-183</td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>76 x 26 mm Microscope slide</td>
			<td>TLG</td>
			<td>GEW90-2575-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid, &#8805;99.8%&#160;</td>
			<td>Fisher Scientific</td>
			<td>A38-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>BioShop</td>
			<td>AGR001.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleech</td>
			<td>Household bleach (e.g., Clorox)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope&#160;</td>
			<td>Nikon&#160;</td>
			<td></td>
			<td>Nikon C2 operated by NIS-Elements&#160;</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Greenfield</td>
			<td>P210EAAN</td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td>Open-srouce</td>
			<td>(Fiji Is Just) ImageJ v2.1/1.5.3j</td>
			<td>Downloaded from https://imagej.net/software/fiji/</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Sigma-Aldrich</td>
			<td>F6521&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Gamborg&#39;s vitamin mixture</td>
			<td>Cassson Labs</td>
			<td>GBL01-100ML</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>G33-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth chambers</td>
			<td>Conviron,&#160;model E15</td>
			<td></td>
			<td>16h light cycle, set at 21&#176;C with a light intensity of 120 &#181;mol&#183;m-2&#183;s-1.</td>
		</tr>
		<tr>
			<td>Lights</td>
			<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>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Microscope&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td>Nikon Eclipse TiE equipped with a DsRi2 digital camera</td>
		</tr>
		<tr>
			<td>Murashige and Skoog basal salts&#160;</td>
			<td>Cassson Labs</td>
			<td>MSP01-1LT</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Propidium Iodide&#160;</td>
			<td>VWR</td>
			<td>39139-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Fisher Scientific</td>
			<td>08-916-5A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>BioShop</td>
			<td>SUC700.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine blue O</td>
			<td>Sigma-Aldrich</td>
			<td>T3260-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Estradiol</td>
			<td>Sigma-Aldrich</td>
			<td>E2758</td>
			<td></td>
		</tr>
	</tbody>
</table>

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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1.5K Views.  Concordia University. 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. 

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

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito.    This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed.  The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction1, boosting the immune response2, pheromone production3, as well as nutrition, including the synthesis of essential amino acids4, among others. &#160; &#160;
Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms.
To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing 13C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA5. The incorporation of 13C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled (12C) one. In the end, the 13C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the 12C-unlabeled similar one6. Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species.
Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The phylogenetic analysis of the DNA was done using pyrosequencing, which allowed high resolution and precision in the identification of insect gut bacterial community. As main substrate, 13C-labeled glucose was used in the experiments. The substrate was fed to the insects using an artificial diet.

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

The active bacterial community associated with the gut of Spodoptera littoralis, was determined by stable-isotope-probing (SIP) coupled to pyrosequencing. Using this methodology, identification of the metabolically active bacteria species within the community was done with high resolution and precision.

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Relating Stomatal Conductance to Leaf Functional Traits

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, morphological leaf traits have the potential to summarize plants strategies in terms of water use efficiency, growth pattern and nutrient use. The leaf economics spectrum (LES) is a recognized framework in functional plant ecology and reflects a gradient of increasing specific leaf area (SLA), leaf nitrogen, phosphorus and cation content, and decreasing leaf dry matter content (LDMC) and carbon nitrogen ratio (CN). The LES describes different strategies ranging from that of short-lived leaves with high photosynthetic capacity per leaf mass to long-lived leaves with low mass-based carbon assimilation rates. However, traits that are not included in the LES might provide additional information on the species&#39; physiology, such as those related to stomatal control. Protocols are presented for a wide range of leaf functional traits, including traits of the LES, but also traits that are independent of the LES. In particular, a new method is introduced that relates the plants&#8217; regulatory behavior in stomatal conductance to vapor pressure deficit. The resulting parameters of stomatal regulation can then be compared to the LES and other plant functional traits. The results show that functional leaf traits of the LES were also valid predictors for the parameters of stomatal regulation. For example, leaf carbon concentration was positively related to the vapor pressure deficit (vpd) at the point of inflection and the maximum of the conductance-vpd curve. However, traits that are not included in the LES added information in explaining parameters of stomatal control: the vpd at the point of inflection of the conductance-vpd curve was lower for species with higher stomatal density and higher stomatal index. Overall, stomata and vein traits were more powerful predictors for explaining stomatal regulation than traits used in the LES.

Unraveling how physiology and morphology are linked allows a deeper understanding of mechanistic functioning of plant leaves. We present both a procedure to derive parameters of stomatal regulation from stomatal conductance measurements and correlations with traditional functional leaf traits.

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...