Preparation of Epidermal Peels and Guard Cell Protoplasts for Cellular, Electrophysiological, and -Omics Assays of Guard Cell Function

Abstract

Bioassays are commonly used to study stomatal phenotypes. There are multiple options in the choice of plant materials and species used for observation of stomatal and guard cell responses in vivo. Here, detailed procedures for bioassays of stomatal responses to abscisic acid (ABA) in Arabidopsis thaliana are described, including ABA promotion of stomatal closure, ABA inhibition of stomatal opening, and ABA promotion of reaction oxygen species (ROS) production in guard cells. We also include an example of a stomatal bioassay for the guard cell CO2 response using guard cell-enriched epidermal peels from Brassica napus. Highly pure preparations of guard cell protoplasts can be produced, which are also suitable for studies on guard cell signaling, as well as for studies on guard cell ion transport. Small-scale and large-scale guard cell protoplast preparations are commonly used for electrophysiological and -omics studies, respectively. We provide a procedure for small-scale guard cell protoplasting from A. thaliana. Additionally, a general protocol for large-scale preparation of guard cell protoplasts, with specifications for three different species, A. thaliana, B. napus, and Vicia faba is also provided.

The Arabidopsis heterotrimeric G‐protein β subunit, AGB1, is required for guard cell calcium sensing and calcium‐induced calcium release

“…Because agb1 mutants, like cas mutants , showed an impaired Ca o response, we therefore queried whether agb1 plants were, like cas , deficient in all three signaling events. The fluorescent probes H 2 DCF‐DA and DAF‐FM DA were used to measure H 2 O 2 and NO, respectively (Zhu et al ., ). At least under the conditions of our assay, we found that H 2 O 2 production and NO production in response to Ca o occurred identically in Col and agb1 mutants (Figure ).…”

“…Effects of external calcium (Ca o ) on stomatal movements were studied for both stomatal opening and closure by the single‐cell tracking method described in Zhu et al . (). Epidermal peels were mounted in a coverslip chamber with medical adhesive (Hollister, Libertyville, IL, USA).…”

“…For [Ca 2+ ] cyt imaging, samples were prepared following the method in Zhu et al . (). In brief, epidermal peels were mounted on a coverslip chamber with medical adhesive (Hollister, Libertyville, IL, USA) and incubated with 20 m m KCl, 5 m m Mes‐Tris, pH 6.15 under illumination (150 μmol m −2 sec −1 white light) for 3 h. The coverslip chamber was placed on the stage of the confocal microscope.…”

Jasmonate‐mediated stomatal closure under elevated CO2 revealed by time‐resolved metabolomics

“…Guard cell protoplasts (GCPs) have been successfully used as model systems for a wide range of studies ranging from patch‐clamping electrophysiology (Hashimoto et al ., ; Zhang et al ., ; Hu et al ., ; Tian et al ., ) to biochemical analyses, as well as systems biology such as proteomics (Zhu et al ., , ) and metabolomics (Jin et al ., ). We prepared stomata that were highly pure (> 90% with minimal impurities from mesophyll and epidermal cells on a cell‐count basis) and showed > 98% viability based on fluorescein diacetate staining (Zhu et al ., ). Compared with GCPs, stomata were well preserved in stomatal structure, which allowed for the monitoring of the stomatal movement and direct correlation with metabolomic changes over real‐time CO 2 treatment.…”

The G Protein β-Subunit, AGB1, Interacts with FERONIA in RALF1-Regulated Stomatal Movement

“…Total RNA was isolated from rosette leaves, two-week-old whole seedlings, siliques or highly 548 purified guard cell protoplasts (Zhu et al, 2016) using the NucleoSpin RNA Plant kit (Macherey-549 Nagel) and treated with RQ1 RNase-Free DNase (Promega) to remove DNA contamination 550 following the manufacturers' instructions. Two μg RNA was reverse-transcribed into cDNA using 551 the SuperScript® III Reverse Transcriptase kit (Invitrogen), and the cDNA was diluted 3 times 552 for use as a template in qRT-PCR.…”