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

  • Mengmeng Zhu
  • Byeong Wook Jeon
  • Sisi Geng
  • Yunqing Yu
  • Kelly Balmant
  • Sixue Chen
  • Sarah M. AssmannEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1363)


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.

Key words

Electrophysiology Epidermal peels Guard cells Guard cell-enriched epidermal peels Protoplasting Reactive oxygen species (ROS) Stomatal movement 



Research on guard cell signaling in the Assmann laboratory is supported by BARD grant IS-4541-12 and by NSF grants IOS-1025837, MCB-1121612, MCB-1157921, and MCB-1412644 to S.M.A. Research on guard cell signaling in the Chen laboratory is supported by NSF grants MCB- 0818051, MCB-1158000, and MCB-1412547 to S.C.


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Mengmeng Zhu
    • 1
  • Byeong Wook Jeon
    • 1
  • Sisi Geng
    • 2
  • Yunqing Yu
    • 1
  • Kelly Balmant
    • 2
  • Sixue Chen
    • 2
  • Sarah M. Assmann
    • 1
    Email author
  1. 1.Biology DepartmentPenn State UniversityUniversity ParkUSA
  2. 2.Plant Molecular and Cellular Biology Program, Department of Biology, Genetics InstituteUniversity of FloridaGainesvilleUSA

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