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

Summary

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.

Abstract

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.

Introduction

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 TMM^{1,2,3,4,5,6,7,8,9,10}. 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 safranin^12,13,14. 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 materials^8,15; 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 peptides^3,7,16. 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 density^2,4,5, 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.

Protocol

1. Staining Arabidopsis cotyledons with TBO

1. Seed sterilization and growth conditions
   1. 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 seedlings grown on 1/2 Murashige and Skoog (MS) plates (2.16 g/L containing 0.8% agar [w/v]) without inducer (e.g., β-estradiol) as controls (Figure 1 and Figure 2).
   2. 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 μL of sterile 0.1% agar.
   3. Sow ~30 seeds per genotype on 1/2 MS agar plates or 1/2 MS agar plates containing an inducer (e.g., 10 µM β-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.
   4. Stratify the seeds by placing the plates at 4 °C without light for 3-5 days to synchronize the germination.
   5. After stratification, incubate the plates in a growth chamber at 22 °C under 120 µmol·m⁻²·s⁻¹ light with a photoperiod of 16 h of light and 8 h of dark for 10 days.
2. Sampling and TBO staining of Arabidopsis cotyledons
   1. 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.
   2. 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.
   3. 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.
   4. Repeat step 1.2.3 using 1 mL of 50% ethanol and then 20% ethanol.
   5. 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.
   6. Remove all the distilled water from the tube. Then, immediately add ~200 μL 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.
   7. 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.
3. Imaging and data analysis
   1. Take a microscope slide, and add a drop of 15% glycerol (~50 μL). 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.
   2. 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.
   3. For each genotype, calculate the epidermal phenotype using the following formulas described by Jangra et al.¹⁷:
      ​Stomatal index (%) = (Number of stomata/Total number of epidermal cells) × 100
      Stomatal density (mm⁻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.

2. Bioassays for stomatal peptides

NOTE: The procedure for bioassays is shown in Figure 3.

1. Seed sterilization and growth conditions
   1. 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).
   2. Stratify the seeds for 3 days at 4 °C in the dark.
   3. Take out each of the two plates at ~10 h intervals, and incubate the seeds on plates at 22 °C under 120 µmol·m⁻²·s⁻¹ light with a photoperiod of 16 h of light and 8 h of dark for 1 day.
2. Peptide treatment and imaging
   1. 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.
   2. Add either a buffer alone (50 mM Tris-HCl [pH 8.0]) or two different concentrations of the peptide (typically, 1 µM and 2.5 µM 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 −80 °C are stable for a couple of years, but freeze-thaw cycles should be avoided by storing the peptide solution in small aliquots.
   3. 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 µmol·m⁻²·s⁻¹ light) for 5-7 days at 22 °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.
   4. 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.
   5. Take another clean microscope slide, and place on it a drop of propidium iodide solution (~25 μL, 2 mg/mL in H₂O).
   6. 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.
   7. 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 epf2^2,5 seedlings that were treated with buffer only (Figure 4A, B) or two batches of EPF2 peptide solution (Figure 4C-F) and were stored at −80 °C over 1 year.

Results

Various stomatal transgenic plants and mutants known to have less or more stomatal density and clustering (epf2^2,5, epf1 epf2^2,5, tmm¹², a STOMAGEN-silenced line⁴, and transgenic lines carrying the estradiol-inducible Est::EPF1 or Est::EPF2 overexpression construct⁷) were used to d...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
18 mm x 18 mm cover slip	VWR	16004-326
24-well sterile plates with lid	VWR	CA62406-183
3M Micropore surgical tape	Fisher Scientific	19-027-761	Microporous surgical paper tape used to seal MS plates
76 x 26 mm Microscope slide	TLG	GEW90-2575-03
Acetic acid, ≥99.8%	Fisher Scientific	A38-212
Agar	BioShop	AGR001.1
Bleech	Household bleach (e.g., Clorox)
Confocal microscope	Nikon		Nikon C2 operated by NIS-Elements
Ethanol	Greenfield	P210EAAN
FIJI	Open-srouce	(Fiji Is Just) ImageJ v2.1/1.5.3j	Downloaded from https://imagej.net/software/fiji/
Forceps	Sigma-Aldrich	F6521
Gamborg's vitamin mixture	Cassson Labs	GBL01-100ML	Store at 4 °C
Glycerol	Fisher Scientific	G33-4
Growth chambers	Conviron, model E15		16h light cycle, set at 21°C with a light intensity of 120 µmol·m-2·s-1.
Lights	HD Supply	25272	Fluorescent  lights in growth chambers, Sylvania F72T12/CW/VHO 72"T12 VHO 4200K
Microcentrifuge tube	Fisher Scientific	14-222-155	Tubes in which Arabidopsis thaliana seeds are placed to perform sterilization
Microscope	Nikon		Nikon Eclipse TiE equipped with a DsRi2 digital camera
Murashige and Skoog basal salts	Cassson Labs	MSP01-1LT	Store at 4 °C
Petri Dish 100 mm x 20 mm	Fisher Scientific	08-757-11Z	Petri dishes in which MS media is poured for the purpose of growing Arabidopsis thaliana
Propidium Iodide	VWR	39139-064
Scalpel	Fisher Scientific	08-916-5A
Sucrose	BioShop	SUC700.5
Toluidine blue O	Sigma-Aldrich	T3260-5G
Tris base	Sigma-Aldrich	T1503
Triton X-100	Sigma-Aldrich	T8787-100ML
β-Estradiol	Sigma-Aldrich	E2758