Name: Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring (Video) | JoVE Journal
Uploaded: 2023-01-20T13:05:00.000+00:00
Duration: 5 min 22 s
Description: Learn phenotypic analysis methods for stomatal development using Arabidopsis seedlings and toluidine blue O staining. These reliable techniques enable quantitative assessment of epidermal phenotypes without toxic chemicals.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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/pubmed?term=((The+secretory+peptide+gene+EPF1+enforces+the+stomatal+one-cell-spacing+rule%5BTitle%5D)+AND+%22Genes+%26+Development%22%5BJournal%5D)">The secretory peptide gene EPF1 enforces the stomatal one-cell-spacing rule.</a> Genes & Development. 21 (14), 1720-1725 (2007).</li>
<li>Hara, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidermal+cell+density+is+autoregulated+via+a+secretory+peptide%2C+EPIDERMAL+PATTERNING+FACTOR+2+in+Arabidopsis+leaves%5BTitle%5D)+AND+%22Plant+%26+Cell+Physiology%22%5BJournal%5D)">Epidermal cell density is autoregulated via a secretory peptide, EPIDERMAL PATTERNING FACTOR 2 in Arabidopsis leaves.</a> Plant & Cell Physiology. 50 (6), 1019-1031 (2009).</li>
<li>Kondo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stomatal+density+is+controlled+by+a+mesophyll-derived+signaling+molecule%5BTitle%5D)+AND+%22Plant+%26+Cell+Physiology%22%5BJournal%5D)">Stomatal density is controlled by a mesophyll-derived signaling molecule.</a> Plant & Cell Physiology. 51 (1), 1-8 (2010).</li>
<li>Sugano, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stomagen+positively+regulates+stomatal+density+in+Arabidopsis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((The+signaling+peptide+EPF2+controls+asymmetric+cell+divisions+during+stomatal+development%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">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/pubmed?term=((The+signalling+peptide+EPFL9+is+a+positive+regulator+of+stomatal+development%5BTitle%5D)+AND+%22New+Phytologist%22%5BJournal%5D)">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/pubmed?term=((Direct+interaction+of+ligand-receptor+pairs+specifying+stomatal+patterning%5BTitle%5D)+AND+%22Genes+%26+Development%22%5BJournal%5D)">Direct interaction of ligand-receptor pairs specifying stomatal patterning.</a> Genes & 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/pubmed?term=((Stomatal+patterning+and+differentiation+by+synergistic+interactions+of+receptor+kinases%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Control+of+stomatal+distribution+on+the+Arabidopsis+leaf+surface%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Differential+function+of+Arabidopsis+SERK+family+receptor-like+kinases+in+stomatal+patterning%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">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/pubmed?term=((Arabidopsis+stomatal+initiation+is+controlled+by+MAPK-mediated+regulation+of+the+bHLH+SPEECHLESS%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((The+too+many+mouths+and+four+lips+mutations+affect+stomatal+production+in+Arabidopsis%5BTitle%5D)+AND+%22The+Plant+Cell%22%5BJournal%5D)">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/pubmed?term=((An+improved+method+for+studying+grass+leaf+epidermis%5BTitle%5D)+AND+%22Stain+Technology%22%5BJournal%5D)">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/pubmed?term=((crw1--A+novel+maize+mutant+highly+susceptible+to+foliar+damage+by+the+western+corn+rootworm+beetle%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Somatic+small+RNA+pathways+promote+the+mitotic+events+of+megagametogenesis+during+female+reproductive+development+in+Arabidopsis%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((The+NMR+structure+of+stomagen+reveals+the+basis+of+stomatal+density+regulation+by+plant+peptide+hormones%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((Duplicated+antagonistic+EPF+peptides+optimize+grass+stomatal+initiation%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((The+xylem+and+phloem+transcriptomes+from+secondary+tissues+of+the+Arabidopsis+root-hypocotyl%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">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/pubmed?term=((Regulation+of+inflorescence+architecture+by+intertissue+layer+ligand-receptor+communication+between+endodermis+and+phloem%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((A+secreted+peptide+and+its+receptors+shape+the+auxin+response+pattern+and+leaf+margin+morphogenesis%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">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/pubmed?term=((Generation+of+signaling+specificity+in+Arabidopsis+by+spatially+restricted+buffering+of+ligand-receptor+interactions%5BTitle%5D)+AND+%22The+Plant+Cell%22%5BJournal%5D)">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/pubmed?term=((Regulation+of+plant+vascular+stem+cells+by+endodermis-derived+EPFL-family+peptide+hormones+and+phloem-expressed+ERECTA-family+receptor+kinases%5BTitle%5D)+AND+%22Journal+of+Experimental+Botany%22%5BJournal%5D)">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/pubmed?term=((EPFL+Signals+in+the+boundary+region+of+the+SAM+restrict+its+size+and+promote+leaf+initiation%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">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><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, &amp;ge;99.8%&amp;nbsp;</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&amp;nbsp;</td><td>Nikon&amp;nbsp;</td><td></td><td>Nikon C2 operated by NIS-Elements&amp;nbsp;</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&amp;nbsp;</td><td></td></tr><tr><td>Gamborg&#039;s vitamin mixture</td><td>Cassson Labs</td><td>GBL01-100ML</td><td>Store at 4 &amp;deg;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,&amp;nbsp;model E15</td><td></td><td>16h light cycle, set at 21&amp;deg;C with a light intensity of 120 &amp;micro;mol&amp;middot;m&lt;sup&gt;-2&lt;/sup&gt;&lt;sup&gt;&amp;middot;&lt;/sup&gt;s&lt;sup&gt;-1&lt;/sup&gt;.</td></tr><tr><td>Lights</td><td>HD Supply</td><td>25272</td><td>Fluorescent&amp;nbsp; lights in growth chambers, Sylvania F72T12/CW/VHO 72&quot;T12 VHO 4200K&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Cassson Labs</td><td>MSP01-1LT</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Petri Dish 100 mm x 20 mm&amp;nbsp;</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&amp;nbsp;</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>&amp;beta;-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

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...

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.

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 ...

identification-genes-involved-stomatal-development-via-epidermal

Research

JoVE Journal

Biology

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

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.

Relating Stomatal Conductance to Leaf Functional Traits

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

Here, we present a simple method for direct observation and automated measurement of stomatal responses to bacterial invasion in Arabidopsis thaliana. This method leverages a portable stomatal imaging device, together with an image analysis pipeline designed for leaf images captured by the device.

Direct Observation and Automated Measurement of Stomatal Responses to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana

This protocol presents a comparative method to assess stomatal and epidermal traits in two distinct species using epidermal peels, clearing techniques, and AI-assisted image analysis. The goal is to enable high-throughput, reproducible quantification of cellular structures underlying stomatal function for agricultural applications and broad plant research.

Characterizing Stomatal and Epidermal Traits Using Peels, Clearing, and AI-Based Image Analysis

Learn phenotypic analysis methods for stomatal development using Arabidopsis seedlings and toluidine blue O staining. These reliable techniques enable quantitative assessment of epidermal phenotypes without toxic chemicals.

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

Explore More Videos