Stomatal movement, initiated by specialized epidermal cells known as guard cells (GCs), plays a pivotal role in plant gas exchange and water use efficiency. Despite protocols existing for isolating GCs through proplasting for carrying out biochemical, physiological, and molecular studies, protocols for isolating GCs with their cell walls still intact have been lacking in the literature. In this paper, we introduce a method for the isolation of complete GCs from Vicia faba and show their membrane to remain impermeable through propidium iodide staining. This methodology enables further in-depth analyses into the cell wall composition of GCs, facilitating our understanding of structure-function relationship governing reversible actuation within cells.

iGC yields were determined by calculating the dry weight percentages of remaining plant cells after isolation. Measurements were taken on the same day that leaves were collected and GCs were isolated. First, the harvested leaves were sorted by size (small, medium, and large) into approximately three equal piles. In order to document the size of leaves used to isolate GCs, leaf surface areas were measured using pixel counts in ImageJ (S1 Table). However, surface area measurements were not used in calculating 109
the final yield of iGCs.

To calculate the final percentage of iGCs, dry weights from three different isolation experiments for the initial leaves and final iGCs were used. Dry weight of the leaves was determined by first measuring the wet weight of the three piles of leaves with a SECURA324-1S analytical balance. Their moisture content was then measured with a Mettler Toldeo Halogen Moisture Analyzer HC103 (115V). A standard drying program was run at 60 °C with 5 (1 mg/140 s) switch-off criterion. Using the wet weight and moisture content of each pile, the dry weight was calculated using Equation 1. Similar to the dry weight of the leaves, the dry weight of the iGCs was determined by first measuring the wet weight and then the moisture content.

Mass dried (g) = Mass original − ((Moisture level x Mass original)/100)