Background

Stomata are microscopic pores located in the epidermis of aerial tissues of most land plants, delimited by specialized cells known as guard cells. Given that the plant epidermis is covered by the cuticle, an external impermeable layer, almost 95% of gas exchange occurs through stomatal pores. Thus, the regulation of the stomatal pore area controls the uptake and release of CO₂ and O₂ and the maintenance of hydric homeostasis by the regulation of H₂O vapor loss, through the transpiration stream [1].

Guard cells continuously sense internal and external cues and integrate them into a complex signaling network that produces modifications in guard cell volume to control the stomatal pore width [2,3]. The isolation of epidermal peels, cellular monolayers mainly formed by functional guard cells, has become a widely used and validated experimental system to study signaling mechanisms in plants [4].

Several protocols have been developed in order to study guard cell physiology [5–7]. Although all of them converge in the isolation of epidermal peels, there are different approaches including the use of adhesive tape [8–10], resin imprints where leaf surface is copied [11–13], blender and filters [14–16], tape and razor blade [17–19], and striping with fine-tip tweezers [20–22].

For the last twenty years, our research group has reproducibly employed fine-tip tweezers to prepare epidermal strips, where almost 90% of living epidermal cells are guard cells (Figure 1A and B) [20,23–27]. Isolation of epidermal peels allowed us to perform stomatal aperture assays and in vivo measurements of endogenously encoded fluorescent proteins (biosensors) that specifically detect hydrogen peroxide (H₂O₂,), the glutathione redox potential (E_GSH), and the biologically relevant portion of adenosine 5'-triphosphate (MgATP^2-) [25,27]. In addition, the methodological procedure presented here has been employed to obtain guard cells–enriched RNA (Figure 1B) to analyze transcript levels of different target genes [23,26]. Although peeling produces mechanical stress, evidenced by an increase in reactive oxygen species (ROS) in guard cells, we set up the conditions to recover the strips to basal redox status levels without affecting guard cells’ viability or their physiological responses to different signals [27]. In this protocol, we describe how to prepare isolated epidermal peels excised from the abaxial side of Arabidopsis thaliana leaves and how to perform different experiments using this biological system to study real-time and single-cell physiological processes.

Figure 1. Living cells in epidermal peels. A. Arabidopsis thaliana wild-type ecotype Col-0 epidermal peels were incubated in opening buffer (OB, 5 mM MES pH6.1, 50 mM KCl) under light for 30 min and then loaded with 5 μM of the viability fluorescent dye fluoresce in diacetate (FDA) for 5 min. Peels were washed three times with OB and then mounted on a coverslip, and analyzed by epifluorescence microscopy. Fluorescent cells were quantified, and the total cell/fluorescent cell percentage was calculated for each cell type. PC: pavement cells, GC: guard cells. B. Representative image of an epidermal peel stained with FDA taken with a 40 × objective. Scale bar: 10 μm. C- RT-PCR analysis using guard cell–specific gene ECIREFERUM2 (CER2) (930 bp) and mesophyll cell–specific gene carbonic anhydrase 1 (Canh1) (1,000 bp) was performed in RNA prepared from guard cell–enriched (GC-e) or mesophyll cell–enriched (MC-e) extracts. The amplification of ACTIN (651 bp) transcripts under identical conditions was used as a constitutive expression control. Data from Scuffi et al. [26].