Maathuis F J, May S T, Graham N S, Bowen H C, Jelitto T C, Trimmer P, Bennett M J, Sanders D, White P J
Department of Biology, University of York, UK.
Plant J. 1998 Sep;15(6):843-51. doi: 10.1046/j.1365-313x.1998.00256.x.
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.
植物细胞质膜上的离子转运过程通常通过将膜片钳电压钳（膜片钳）电生理技术应用于分离的原生质体来进行研究。由于植物由许多组织和细胞类型组成，并且每种组织和细胞类型可能专门执行特定功能并拥有独特的转运蛋白组合，因此确定用于膜片钳研究的原生质体的解剖学来源很重要。本文描述了一种通用的分子遗传学方法，用于标记特定细胞类型以便后续进行膜片钳研究，并给出了一个具体例子：比较拟南芥根的皮层细胞和中柱细胞原生质体中的钾离子电流。构建了转基因拟南芥，其中维多利亚水母绿色荧光蛋白（GFP）的表达由CaMV 35S启动子驱动（mGFP3系）。在转基因mGFP3系的根中，可见荧光仅限于中柱。从mGFP3系的根中制备原生质体，并使用膜片钳技术测定非荧光（皮层/表皮）和荧光（中柱）原生质体中的钾离子电流。结果发现，与中柱细胞相比，皮层/表皮细胞原生质体中观察到内向整流钾离子通道（IRC）活性的频率以及IRC相对于外向整流钾离子通道的相对发生率均显著较低。GFP的存在不影响钾离子通道的发生率或生物物理特性。得出的结论是，以细胞特异性方式表达GFP的转基因拟南芥的产生是一种方便且可靠的方法，可用于标记来自不同细胞类型的原生质体，以便后续进行膜片钳研究。
