Cell marking inArabidopsis thaliana andits application to patch–clamp studies

Abstract

SummaryIon 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

pH control of the plant outwardly‐rectifying potassium channel SKOR

“…These data provide the first characterisation at the single channel level of a cloned shaker‐like outwardly‐rectifying plant K + channel. Interestingly, the SKOR single channel conductance (23 pS in 10 mM external K + ) is similar (taking into account the different K + concentration used) to that of Kout characterised in stelar cells of Arabidopsis (15.5 pS [28]) where SKOR is expressed [9]. The functional similarities between SKOR and Kout channels characterised in vivo [10,29] suggest that tight structural relationships exist between the various members of the plant family of shaker‐like outwardly‐rectifying channels.…”

Compatible solutes reduce ROS‐induced potassium efflux in Arabidopsis roots

“…Arabidopsis root plasma membrane depolarizationactivated K + channels have been widely reported (Maathuis & Sanders 1995;Maathuis et al 1998). To test whether a OH • -induced membrane depolarization was responsible for the massive K + efflux observed ( Fig.…”

“…A decrease in membrane potential associated with an oxidative burst has been described in many pathogen-elicited (Mathieu et al 1991;Simon-Plas, Elmayan & Blein 2002) and wounded (Orozco-Cárdenas, Narvaez-Vasquez & Ryan 2001) plants, and has been usually attributed to the general effect of ROS on biological membranes (Stark 2005). Such a ROS-induced depolarization may activate depolarizationactivated K + channels (Maathuis & Sanders 1995;Maathuis et al 1998), causing a substantial K + efflux, similar to that previously reported under salt (Shabala, Shabala & Van Volkenburgh 2003) or acid-stress (Babourina et al 2001) conditions. Supporting evidence for such a scenario can be found in the fact that a clear dose-dependent effect was found for both OH • -induced membrane depolarization ( Fig.…”

AtHKT1;1 Mediates Nernstian Sodium Channel Transport Properties in Arabidopsis Root Stelar Cells

“…An Arabidopsis thaliana control line, E2586, that expresses GFP in stelar cells was used in the present study [23], [52], [53]. This enhancer trap line was crossed into the athkt1;1-4 loss-of-function mutant background and homozygous athkt1;1-4 X E2586 seeds were isolated (Columbia-0 background).…”

“…In the present study we used enhancer trap lines [23], [52], [53] that GFP label root stele cells to investigate ionic currents in wild-type and AtHKT1;1 knock-out plants. Direct patch clamping allowed us to address several key questions on the functional and biophysical properties of an HKT transporter in its native cells, including:…”