Cell marking in Arabidopsis thaliana and its application to patch–clamp studies

• Published in: The Plant journal : for cell and molecular biology Vol. 15; no. 6; pp. 843 - 851
• Main Authors: Maathuis, Frans J. M., May, Sean T., Graham, Neil S., Bowen, Helen C., Jelitto, Till C., Trimmer, Paul, Bennett, Malcolm J., Sanders, Dale, White, Philip J.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-09-1998; Blackwell Science
• Subjects: Arabidopsis - cytology; Arabidopsis - physiology; Arabidopsis thaliana; Biological and medical sciences; Biological Transport; Cell Membrane - physiology; Cell physiology; Electrophysiology; Fundamental and applied biological sciences. Psychology; Genes, Reporter; Green Fluorescent Proteins; Luminescent Proteins - biosynthesis; Luminescent Proteins - genetics; Microscopy, Confocal; Patch-Clamp Techniques; Plant physiology and development; Plants, Genetically Modified; Plasma membrane and permeation; Potassium Channels - physiology; Spectrometry, Fluorescence; Plant tissue; Fluorescent tracer; Cell labelling; Tissue specificity; Ion transport; Patch clamp method; Gene expression; Arabidopsis thaliana; Cruciferae; Dicotyledones; Protoplast; Angiospermae; Plasma membrane; Spermatophyta; Transgenic plant; Experimental plant
• DOI: 10.1046/j.1365-313X.1998.00256.x
• EISSN: 1365-313X
• ISSN: 0960-7412

Summary
Ion transport processes at the plasma membrane of plant cells are frequently studied by applying membrane‐patch voltage‐clamp (patch–clamp) electrophysiological techniques to isolated protoplasts. As plants are composed of many tissues and cell types, and each tissue and cell type may be specialized to a particular function and possess a unique complement of transport proteins, it is important to certify the anatomical origin of the protoplasts used for patch–clamp studies. This paper describes a general molecular genetic approach to marking specific cell types for subsequent patch–clamp studies and presents a specific example: a comparison of the K+ currents in protoplasts from cortical and stelar cells of
Arabidopsis
roots. Transgenic
Arabidopsis
were generated in which the expression of green fluorescent protein (GFP) from
Aequoria victoria
was driven by the CaMV 35S promoter (line mGFP3). In roots of the transgenic mGFP3 line, visible fluorescence was restricted to the stele. Protoplasts were generated from roots of the mGFP3 line and K+ currents in non‐fluorescent (cortical/epidermal) and fluorescent (stelar) protoplasts were assayed using patch–clamp techniques. It was found that both the frequency of observing inward rectifying K+ channel (IRC) activity and the relative occurrence of IRC compared to outward rectifying K+ channels were significantly lower in protoplasts from cortical/epidermal cells compared to cells of the stele. The presence of GFP did not affect the occurrence or biophysical properties of K+ channels. It is concluded that the generation of transgenic
Arabidopsis
expressing GFP in a cell‐specific fashion is a convenient and reliable way to mark protoplasts derived from contrasting cell types for subsequent patch–clamp studies.