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>

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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 using TBO-stained Arabidopsis cotyledons is that obtaining high-quality images with visual contrast for different epidermal cell types depends on the staining time and the unique characteristics of the tissue, which may be both species-dependent and genotype-dependent (Figure 1)18. Other phenotyping methods to observe epidermal cells using epidermal peels have the same challenges, and they also require more time and special training. Difficulties in obtaining properly stained epidermis images sufficient for data analysis can be avoided by staining cotyledons from genotypes with fewer stomata for longer periods of time and samples from genotypes with more stomata for shorter periods of time. The staining time for each genotype can also be adjusted by first checking only a few samples to optimize the staining time for genotypes with similar epidermal phenotypes.
Obtaining a sufficient amount of well-folded bioactive peptides, especially cysteine-rich peptides (which are ligands that have diverse functions in controlling plant development, including stomatal patterning), has been a challenge1,2,3,4,5,6,19,20,21,22,23. Furthermore, even if bioactive peptides are generated, the screening of peptides that have potential functions in specific biological processes is another challenging task because the bioactive peptides produced are often a mixture of well-folded, active, and inactive peptides. This paper presents an efficient bioassay method to discover and characterize potential stomatal peptides. This method has been successful in identifying various important peptides that control different aspects of stomatal development in Arabidopsis, as well as the grass Brachypodium3,4,7,17. However, the major limitation of this technique is the variability in the phenotypic responses, which most likely occurs due to the use of seedlings with slightly different developmental stages and peptide solutions containing different amounts of bioactive peptides. Early attempts at this method involved the placement of wild-type seeds directly in the wells of a plate containing a peptide solution after stratification. Without the use of genotypes that have more and/or less epidermal cells (e.g., epf22, Figure 4) and 1 day old Arabidopsis seedlings germinated on 1/2 MS agar plates, a lower number of seedlings are able to grow and show clear phenotypic responses after the peptide application. In this protocol, the variability issue was addressed by using ~20 one-day-old Arabidopsis seedlings with two different developmental stages and by using two different concentrations of peptide solution (1 &#956;M and 2.5 &#956;M).
The epidermal phenotypes of the peptide-treated samples can also be analyzed by the first phenotypic analysis method presented using TBO-stained samples. This method allows for relatively easy quantitative phenotypic analysis because a relatively large area of each sample can be analyzed. Additionally, this method offers flexibility in sample preparation after harvesting samples prepared in the same conditions for imaging. However, the images taken by this method are generally not of the highest quality. On the other hand, confocal imaging after propidium iodide staining gives higher-quality images with epidermal details. However, this method only allows for a small area of tissue to be focused on for analysis. In addition, only a certain number of the live tissues prepared can be analyzed at once.
In conclusion, the phenotypic analysis presented here can be used for the quick and effective examination of the potential genes controlling stomatal patterning and differentiation, thus improving the understanding of the mechanisms of stomatal development and peptide function. In addition, this bioassay protocol can be used to identify other small molecules that have a role in epidermal tissue patterning and as an initial screening method to determine the structure of well-folded bioactive peptides.

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

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 demonstrate the effectiveness of the two phenotypic analyses presented here, which aimed to identify and characterize the genes that have a role in stomatal development and patterning. In order to produce high-quality epidermis images without the need for epidermal peels to quantify the different types of epidermal cells for analysis, it is critical to pre-adjust the sample staining time with TBO for each genotype, as each one may have different epidermal phenotypes compared to that of the wild-type control (Col-0) (Figure 1A and Figure 2A). Based on experience, genotypes with fewer stomata require longer staining times with TBO (Figure 1B and Figure 2D,E), while genotypes with more stomata and clustering require shorter staining times (Figure 1C and Figure 2B,C). As varying amounts of properly folded peptides in the total peptide solution can mask the biological activity of the peptides in stomatal development, it is good practice to use additional, higher total concentrations of the peptide solution (e.g., 2.5 &#956;M EPF2), as well as genotypes that have less or more stomatal phenotypes (e.g., epf2), for the bioassays. The activity of EPF2 peptides, which have a role in inhibiting stomatal initiation, was easily detected by using epf2 mutants with more epidermal cells than Col-0, even if certain batches of the prepared EPF2 peptides had smaller amounts of the bioactive forms of the peptide (e.g., EPF2 peptide solution batch 1 in Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64899/64899fig01.jpg" /> 
Figure 1: The effect of the incubation time with TBO on the genotypes exhibiting different epidermal phenotypes. Abaxial cotyledon images from 10 day old seedlings of three Arabidopsis genotypes: (A)&#160;wild-type (Col-0), (B)&#160;a STOMAGEN-silenced line (STOMAGEN-ami), and (C) epf1 epf2 mutants. Col-0 represents the wild-type Arabidopsis control, and the STOMAGEN-silenced line and the epf1 epf2 mutants represent the genotypes with low and high numbers of stomata, respectively. The images were taken using an inverted microscope with a 20x objective lens (0.35 mm2 field of view). The quality of the images was variable, although all the cotyledon samples from the different genotypes were stained with TBO (0.5% TBO in H2O) for the same amount of time (2 min) before imaging. Therefore, to obtain well-stained images for analysis, it is important to predetermine the adequate TBO staining time for each genotype. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64899/64899fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64899/64899fig02.jpg" /> 
Figure 2: TBO-stained epidermis images without the use of epidermal peels for quantitative analysis. Representative epidermis images of cotyledons from 10 day old seedlings of (A) wild-type (Col-0), (B)&#160;tmm, and (C,D)&#160;transgenic lines carrying an estradiol-inducible EPF2 (Est::EPF2) or (E)&#160;EPF1 overexpression construct (Est::EPF1) can be used&#160;for the quantitative analysis of the epidermal phenotype.A cotyledon in a fixing solution for over 3 years was used to take the image presented in (E). The images were taken using an inverted microscope with a 20x objective lens (0.35 mm2&#160;field of view). The cells were outlined by TBO staining, and half the width of the full-size images is presented for display. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64899/64899fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64899/64899fig03.jpg" /> 
Figure 3: Procedure for the bioassays. (A) The seeds are sown on two 1/2 MS agar plates and taken out at 10 h intervals. (B) The 1 day old Arabidopsis seedlings are transplanted to 24-well plates containing 1/2 MS medium with either mock or two different peptide concentrations. (C) After 5-7 days, the seedlings are ready for imaging to determine the biological activity of the peptides in stomatal development and patterning. (D) A cotyledon slice is placed in a drop of propidium iodide solution on a microscope slide for imaging. <a href="https://www.jove.com/files/ftp_upload/64899/64899fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64899/64899fig04.jpg" /> 
Figure 4: Confocal microscopy of the abaxial epidermis of Arabidopsis cotyledons following peptide treatment. Representative confocal images of (A) wild-type and (B)&#160;epf2 cotyledon epidermis grown for 6-7 days in a buffer solution and an (C-F)&#160;epf2 cotyledon epidermis grown with two different batches of EPF2 peptides. The images were taken using a confocal microscope using a 40x objective lens (excitation of 561 nm and a 561 nm long-pass emission filter) to capture the propidium iodide staining and visualize the cell outlines. Each EPF2 peptide solution prepared has different amounts of properly folded (bioactive) and misfolded (inactive) forms of the peptides. Therefore, for the initial screening of peptides with a potential role in stomatal development, it is recommended to use two different concentrations of the total peptide solutions as indicated for the bioassays. Scale bar = 30 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64899/64899fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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Universidad Científica del Sur

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Biology

1.6K 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

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

Biochemistry

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a significant increase and decrease in guard cell volume, respectively. Because shuttle transport of ions and water occurs between guard cells and larger neighboring epidermal cells during stomatal movement, the spaced distribution of plant stomata is considered an optimal distribution for stomatal movement. Experimental systems for perturbing the spaced pattern of stomata are useful to examine the spacing pattern's significance. Several key genes associated with the spaced stomatal distribution have been identified, and clustered stomata can be experimentally induced by altering these genes. Alternatively, clustered stomata can be also induced by exogenous treatments without genetic modification. In this article, we describe a simple induction system for clustered stomata in Arabidopsis thaliana seedlings by immersion treatment with a sucrose-containing medium solution. Our method is easy and directly applicable to transgenic or mutant lines. Larger chloroplasts are presented as a cell biological hallmark of sucrose-induced clustered guard cells. In addition, a representative confocal microscopic image of cortical microtubules is shown as an example of intracellular observation of clustered guard cells. The radial orientation of cortical microtubules is maintained in clustered guard cells as in spaced guard cells in control conditions.

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

The goal of this protocol is to demonstrate how to induce clustered stomata in cotyledons of Arabidopsis thaliana seedlings by immersion treatment with a sugar-containing medium solution and how to observe intracellular structures such as chloroplasts and microtubules in the clustered guard cells using confocal laser microscopy.

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a ...

Environment

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

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

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

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.

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

Developmental Biology

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

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 ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

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

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

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

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.

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Automated Stomatal Response Measurement to Bacterial Invasion in Arabidopsis

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

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake for photosynthesis and transpirational water loss but also for restricting bacterial invasion. While plants close stomata upon recognition of microbes, pathogenic bacteria, such as Pseudomonas syringae pv. tomato DC3000 (Pto), reopen the closed stomata to gain access into the leaf interior. In conventional assays for assessing stomatal responses to bacterial invasion, leaf epidermal peels, leaf discs, or detached leaves are floated on bacterial suspension, and then stomata are observed under a microscope followed by manual measurement of stomatal aperture. However, these assays are cumbersome and may not reflect stomatal responses to natural bacterial invasion in a leaf attached to the plant. Recently, a portable imaging device was developed that can observe stomata by pinching a leaf without detaching it from the plant, together with a deep learning-based image analysis pipeline designed to automatically measure stomatal aperture from leaf images captured by the device. Here, building on these technical advances, a new method to assess stomatal responses to bacterial invasion in Arabidopsis thaliana is introduced. This method consists of three simple steps: spray inoculation of Pto mimicking natural infection processes, direct observation of stomata on a leaf of the Pto-inoculated plant using the portable imaging device, and automated measurement of stomatal aperture by the image analysis pipeline. This method was successfully used to demonstrate stomatal closure and reopening during Pto invasion under conditions that closely mimic the natural plant-bacteria interaction.

Author Spotlight: Advancing Stomatal Research with Automated Aperture Measurement

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.

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake ...

Immunology and Infection

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

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

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.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

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

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