The described method allows analysis of stomatal apertures with minimal leaf manipulation and usage of the same leaf for sequential measurements

Fig 1. Experimental setup for stomatal aperture measurements.(a) Schematic representation of the workflow; (b) epifluorescent microscopic picture of the Arabidopsis leaf stained with rhodamine 6G; (c) the same picture as in (b) after application of the option “sharpen” in ImageJ. Bars, 50 μm

A Rapid and Simple Method for Microscopy-Based Stomata Analyses

by Eisele J. F., Fässler F., Bürgel P. F., Chaban C. (2016)

Jochen F. Eisele, Florian Fäßler, Patrick F. Bürgel, Christina Chaban,

Department of Plant Physiology, Center for Plant Molecular Biology (ZMBP), University of Tübingen, Tübingen, Germany

===

In PLOS ONE https://doi.org/10.1371/journal.pone.0164576

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0164576

Fig 3. Visualization of stomatal apertures in intact leaves and epidermis peels.(a,b) Epifluorescent images of intact leaves mounted in water (upper panel) and in 30% glycerol (lower panel). (a) Staining with 1 μM rhodamine 6G; (b) staining with 10 μM rhodamine 6G. (c) Photograph of leaf epidermis peels. (d) Confocal image of cells in the peeled epidermis; λexc = 488 nm, λem = 505–545 nm (upper panel); λexc = 561 nm, λem = 600–640 nm (middle panel); bright field (lower panel). Bars, 50 μm.

Abstract

There are two major methodical approaches with which changes of status in stomatal pores are addressed: indirectly by measurement of leaf transpiration, and directly by measurement of stomatal apertures.

Application of the former method requires special equipment, whereas microscopic images are utilized for the direct measurements. Due to obscure visualization of cell boundaries in intact leaves, a certain degree of invasive leaf manipulation is often required. Our aim was to develop a protocol based on the minimization of leaf manipulation and the reduction of analysis completion time, while still producing consistent results.

We applied rhodamine 6G staining of Arabidopsis thaliana leaves for stomata visualization, which greatly simplifies the measurement of stomatal apertures. By using this staining protocol, we successfully conducted analyses of stomatal responses in Arabidopsis leaves to both closure and opening stimuli.

We performed long-term monitoring of living stomata and were able to document the same leaf before and after treatment. Moreover, we developed a protocol for rapid-fixation of epidermal peels, which enables high throughput data analysis.

The described method allows analysis of stomatal apertures with minimal leaf manipulation and usage of the same leaf for sequential measurements, and will facilitate the analysis of several lines in parallel.

Advertisements

Published by

Willem Van Cotthem

Honorary Professor of Botany, University of Ghent (Belgium). Scientific Consultant for Desertification and Sustainable Development.

Leave a Reply

Please log in using one of these methods to post your comment:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out /  Change )

Google photo

You are commenting using your Google account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s