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,<....}

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....

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