Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

Summary

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

Abstract

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is often difficult to isolate and thus studying them often provides a challenge.

This study establishes a stomatal guard cell enrichment method. The protocol utilizes Scotch tape to isolate guard cells and extract proteins from the cells. This method improves the integrity and yield of guard cell during isolation and provides a reliable sample for the study of cell signaling processes and how they correlate to stomatal movement phenotypes. In this method, the plant leaves were partitioned between two portions of tape and peeled apart to remove the abaxial side. This protocol was applied to Arabidopsis leaf tissue to isolate guard cells. The cells were treated with fluorescein diacetate (FDA) to determine viability and with abscisic acid (ABA) to assess stomata movement in response to ABA. In conclusion, this protocol proves useful for preparing enriched stomatal guard cells and isolating proteins. The quality of the cells and proteins obtained here enable physiological and other biological studies.

Introduction

Stomata play an important role in the overall physiology and fitness of plants. These tiny plant specific structures are formed by two specialized epidermal cells known as guard cells and are found most abundantly on the abaxial surface of leaves. Stomata are necessary for the exchange of gasses between the plant and the atmosphere, as well as the control of water loss through transpiration. Guard cells regulate stomata aperture through the integration of different external (e.g., light, humidity, pathogen contact, carbon dioxide levels) and internal (e.g., endogenous hormones) stimuli^1,2. In....

Protocol

1. Growing Plants

1. Germinate the seeds in potting mixture. Grow plants in a growth chamber with a light intensity of 140 µmol photons m⁻² s ⁻¹ and a photoperiod of 8 h light at 22 °C and 16 h dark at 18 °C for 2 weeks.
2. Transplant seedlings of similar sizes individually into 4" diameter pots containing the soil and grow for an additional 3 weeks in the same conditions described in step 1.1.
   NOTE: Water plants with tap water twice a week to keep the soil moist.

2. Preparation of Enriched Stomata

1. Remove the leaves of 5-week old A. thaliana

Results

A representative image of Arabidopsis guard cells before and after digestion of the mesophyll and epidermal cells is shown in Figure 1. Guard cell viability before and after the removal of mesophyll and epidermal cells can be observed using FDA to measure enzymatic activity and cell membrane integrity (Figure 1B and 1D). Figure 2 illustrates stomatal movement of Arabidopsis in respon.......

Discussion

Guard cells are a model system for studying signal transduction mechanisms in plants and it is important to ensure that the preparation of samples used in the study is the most appropriate to answering biological questions. Despite the increasing interest in guard cells within the plant research community, there is no universal method on how to prepare stomatal guard cells that would allow both the stomatal movement and physiology as well as the molecular changes to be studied in one system. The challenge to isolate them.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Stainless Steel Surgical Scalpel	Feather	NC9999403
Scotch Tape (3M)	ULINE	S-9781
Petri Dish	Sigma-Aldrich	BR455701
Cellulase (Onozuka R-10)	Yakult Pharmaceutical Industry Co., Ltd.	21560003-3
Macerozyme R-10	Yakult Pharmaceutical Industry Co., Ltd.	21560003-4
DM6000B Microscope	Leica Microsystems
Protease and Phosphatase Inhibitor Cocktails	Thermo Fisher Scientific	PI78443
Oak Ridge High-Speed PPCO Centrifuge Tubes	Thermo Fisher Scientific	3119-0030
EZQ Protein Quantitation Kit	Thermo Fisher Scientific	R33200
Fluorescein Diacetate (FDA)	Thermo Fisher Scientific	F1303
Abscisic Acid	Sigma-Aldrich	A4906
Metro Mix 500	BWI Companies	TX-500
Laemmli Sample Buffer	Bio-Rad	161-0737
Bio-Safe Comassie (G-250)	Bio-Rad	161-0786
Microcentrifuge Tube (2mL)	USA Scientific, Inc.	1620-2700
ImageJ software	National Institute of Health
Tweezers	Sigma-Aldrich	F4017-1EA
12% Mini-PROTEAN TGX Precast Gel	Bio-Rad	456-1043
Microscope slides	Fisherbrand	12-550-A3