In this protocol, we describe how to quantify starch in guard cells of Arabidopsis thaliana using the fluorophore propidium iodide and confocal laser scanning microscopy. This simple method enables monitoring, with unprecedented resolution, the dynamics of starch in guard cells.
Ultrasonic irradiation of Vicia faba L. epidermal strips for 2 min preferentially disrupts the epidermal cells but does not impair guard cell movements.
Maximal opening induced by fusicoccin requires that K(+) be provided to the guard cells from external sources. A mobile organic anion is not required.
Procedures for obtaining epidermal strips with only the guard cells alive have been developed but they are either tedious, require considerable experience or may damage the guard cells (Meidner and Mansfield, 1968; Squire and Mansfield, 1972b; Allaway and Hsiao, 1973). Such material is, however, very useful for studies on some aspects of the mechanism of guard cell movements. In this paper a simple, rapid and reproducible technique using ultrasonic irradiation to preferentially disrupt epidermal cells is reported which largely overcomes these drawbacks. ll[aterials
A simple method for reproducibly peeling leaf epidermis tissue is described. Epidermis was obtained from eight species using the method, and its properties of value in research on stomatal physiology evaluated.
Effects of the method of peeling on cell viability and mesophyll contamination were quantified, and a comparison was made between the effects of peeling and subsequent treatments on a plant with morphologically distinct subsidiary cells, Commelina communis, and one without, Vicia faba.
The results indicate that artefacts in experiments on stomatal physiology involving leaf epidermis could arise not only from the peeling method, but also the plant species chosen.
Earlier on in the year I perfected a non-invasive method to look at stomatal density and guard cell size of herbarium specimens. After preliminary tests using clear nail varnish proved unsuccessful, I found a method online (http://www.saps.org.uk/secondary/teaching-resources/299-measuring-stomatal-density-) that involved using Germolene liquid plaster on the herbaria specimens to make an imprint of the leaf surface. For each individual, a photo was taken marking leaves 1-5 with a number so later on I know which leaves have been analysed, and I can account for leaf area, length and width when determining the stomatal density / guard cell size of individuals.
1.) Spread germolene, liquid skin over surface of leaf in a 1cm2 patch and allow to dry for 5-10 minutes so it is no longer tacky on the leaf.
2.) The germolene will dry transparent, and you will need to gently pry the surface of the germolene until it goes opaque. I was using dental tools for this, the ‘dental explorer’ tool (pictured below) with a right-angled head was perfect for this. Run the outside edge of right angle (not the sharp end) gently over the surface of the germolene, you will start to see the germolene turning opaque as it detached from the leaf surface.
3.) Once the whole surface has turned opaque, use the sharp end of the tool to gently pry at the edge of the germolene until it starts to peel off, use fine tweezers to remove gently without damaging the leaf.
4.) Once you have your germolene imprint, mount onto a microscope slide and place a cover slip over the top of the sample.
*** for the species I was working with Impatiens glandulifera I took one measurement from the left hand side of the abaxial surface 1cm from the leaf vein.