title: Cell marking in Arabidopsis thaliana and its application to patch-clamp studies.
abstract: 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.
authors.affiliation: Department of Biology, University of York, UK.
authors.forename: F J
authors.initials: FJ
authors.lastname: Maathuis
authors.forename: S T
authors.initials: ST
authors.lastname: May
authors.forename: N S
authors.initials: NS
authors.lastname: Graham
authors.forename: H C
authors.initials: HC
authors.lastname: Bowen
authors.forename: T C
authors.initials: TC
authors.lastname: Jelitto
authors.forename: P
authors.initials: P
authors.lastname: Trimmer
authors.forename: M J
authors.initials: MJ
authors.lastname: Bennett
authors.forename: D
authors.initials: D
authors.lastname: Sanders
authors.forename: P J
authors.initials: PJ
authors.lastname: White
mesh_terms: D017360:Arabidopsis / Q000166:cytology / Q000502:physiology*; D001692:Biological Transport; D002462:Cell Membrane / Q000502:physiology; D004594:Electrophysiology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D018613:Microscopy, Confocal; D018408:Patch-Clamp Techniques; D030821:Plants, Genetically Modified; D015221:Potassium Channels / Q000502:physiology; D013050:Spectrometry, Fluorescence
chemical_list: D008164:Luminescent Proteins; D015221:Potassium Channels; D049452:Green Fluorescent Proteins