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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 TMM1,2,3,4,5,<....

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-indu.......

Representative Results

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.......

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
18 mm x 18 mm cover slipVWR16004-326
24-well sterile plates with lidVWRCA62406-183
3M Micropore surgical tapeFisher Scientific19-027-761Microporous surgical paper tape used to seal MS plates
76 x 26 mm Microscope slideTLGGEW90-2575-03
Acetic acid, ≥99.8% Fisher ScientificA38-212
AgarBioShopAGR001.1
BleechHousehold bleach (e.g., Clorox)
Confocal microscope Nikon Nikon C2 operated by NIS-Elements 
EthanolGreenfieldP210EAAN
FIJIOpen-srouce(Fiji Is Just) ImageJ v2.1/1.5.3jDownloaded from https://imagej.net/software/fiji/
ForcepsSigma-AldrichF6521 
Gamborg's vitamin mixtureCassson LabsGBL01-100MLStore at 4 °C
GlycerolFisher ScientificG33-4
Growth chambersConviron, model E1516h light cycle, set at 21°C with a light intensity of 120 µmol·m-2·s-1.
LightsHD Supply25272Fluorescent  lights in growth chambers, Sylvania F72T12/CW/VHO 72"T12 VHO 4200K 
Microcentrifuge tubeFisher Scientific14-222-155Tubes in which Arabidopsis thaliana seeds are placed to perform sterilization
Microscope NikonNikon Eclipse TiE equipped with a DsRi2 digital camera
Murashige and Skoog basal salts Cassson LabsMSP01-1LTStore at 4 °C
Petri Dish 100 mm x 20 mm Fisher Scientific08-757-11ZPetri dishes in which MS media is poured for the purpose of growing Arabidopsis thaliana
Propidium Iodide VWR39139-064
ScalpelFisher Scientific08-916-5A
SucroseBioShopSUC700.5
Toluidine blue OSigma-AldrichT3260-5G
Tris baseSigma-AldrichT1503
Triton X-100Sigma-AldrichT8787-100ML
β-EstradiolSigma-AldrichE2758

References

  1. Hara, K., Kajita, R., Torii, K. U., Bergmann, D. C., Kakimoto, T. The secretory peptide gene EPF1 enforces the stomatal one-cell-spacing rule. Genes & Development. 21 (14), 1720-1725 (2007).
  2. Hara, K., et al.

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