title: 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.
authors.affiliation: Biology Department, Penn State University, 208 Mueller Laboratory, University Park, PA, 16802, USA.
authors.forename: Mengmeng
authors.initials: M
authors.lastname: Zhu
authors.affiliation: Biology Department, Penn State University, 208 Mueller Laboratory, University Park, PA, 16802, USA.
authors.forename: Byeong Wook
authors.initials: BW
authors.lastname: Jeon
authors.affiliation: Plant Molecular and Cellular Biology Program, Department of Biology, Genetics Institute, University of Florida, 2033 Mowry Road, Gainesville, FL, 32610, USA.
authors.forename: Sisi
authors.initials: S
authors.lastname: Geng
authors.affiliation: Biology Department, Penn State University, 208 Mueller Laboratory, University Park, PA, 16802, USA.
authors.forename: Yunqing
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Plant Molecular and Cellular Biology Program, Department of Biology, Genetics Institute, University of Florida, 2033 Mowry Road, Gainesville, FL, 32610, USA.
authors.forename: Kelly
authors.initials: K
authors.lastname: Balmant
authors.affiliation: Plant Molecular and Cellular Biology Program, Department of Biology, Genetics Institute, University of Florida, 2033 Mowry Road, Gainesville, FL, 32610, USA.
authors.forename: Sixue
authors.initials: S
authors.lastname: Chen
authors.affiliation: Biology Department, Penn State University, 208 Mueller Laboratory, University Park, PA, 16802, USA. sma3@psu.edu.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Assmann
mesh_terms: D001681:Biological Assay / Q000379:methods; D002458:Cell Fractionation / Q000379:methods*; D002470:Cell Survival; D058948:Cell Tracking / Q000379:methods; D008856:Microscopy, Fluorescence; D019441:Plant Epidermis / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D054046:Plant Stomata / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D011523:Protoplasts / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods; D059008:Time-Lapse Imaging
chemical_list: D017382:Reactive Oxygen Species
keywords: Electrophysiology; Epidermal peel s; Guard cell-enriched epidermal peels; Guard cells; Protoplasting; Reactive oxygen species (ROS); Stomatal movement