Plant Electrophysiology Volume 662 || Plant Response to Stress: Microelectrode Voltage-Clamp Studies

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<ul><li><p>Chapter 3Plant Response to Stress: MicroelectrodeVoltage-Clamp Studies</p><p>Franois Bouteau and Daniel Tran</p><p>Abstract Microelectrode voltage-clamp techniques have existed since more than60 years with the pioneering work on squid axons. In the past decades, the patch-clamptechnique became the most widely used voltage-clamp technique for recording mac-roscopic currents in plant cells and it largely improved the descriptions of the roles andproperties of various ion transport systems. However, the necessary removal of the cellwall limits its application to protoplasts. Microelectrode voltage-clamp techniquesallow circumventing this problem in working with cells with their intact walls. Withthese techniques, whole cell currents could be recorded from electrically isolated plantcell types challenged by various environmental stresses that could not be addressedwith the patch-clamp technique. In this chapter, we discuss findings concerning dif-ferent types of ion channel regulations recorded using microelectrode techniques oncells upon challenge by abiotic or biotic stresses; sustained ion channel activitiesleading to osmotic changes and transient ion channel regulations possibly involved inthe signaling process. We further highlight the complex regulations of ion channelsobserved in host and nonhost cells by harpins from plant pathogenic bacteria.</p><p>3.1 Introduction</p><p>Plants are sessile organisms that face multiple biotic and abiotic stresses in theirnatural environment. Abiotic stresses such as drought and salinity greatly affectplant growth and productivity worldwide resulting in billion dollar losses in crop</p><p>F. Bouteau (&amp;) D. TranLaboratoire dElectrophysiologie des Membranes, Institut de Biologie des Plantes,Universit Paris Diderot-Paris 7, Sorbonne Paris Cit, 91405 Orsay, Francee-mail:</p><p>A. G. Volkov (ed.), Plant Electrophysiology,DOI: 10.1007/978-3-642-29119-7_3, Springer-Verlag Berlin Heidelberg 2012</p><p>69</p></li><li><p>production around the globe. In addition, nearly one-third of crops and stored foodlosses are caused by plant pests, and agricultural crops can be injured whenexposed to high concentrations of various air pollutants. Injury can range fromvisible markings to reduced growth and yield, to premature death of the plant.Studying and revealing the mechanisms that plants have evolved to cope withbiotic and abiotic stresses is thus, a major issue for plant biologists.</p><p>Plants have an outstanding ability to display the optimal responses to stressfulenvironmental conditions. This is the result of an amazingly complex array ofphysiological mechanisms for signal perception and from multiple cross talksignaling pathways. These signaling mechanisms could include hormones(abscisic acid, salicylic acid, ethylene, jasmonic acid, etc.), chemicals (Ca2+,reactive oxygen species [ROS], etc.), or physical events (e.g. propagating elec-trical or hydrostatic pressure waves) (Spoel and Dong 2008; Zhang et al. 2008).Recent studies have revealed several molecules, including transcription factors andkinases, as promising candidates for common players that are involved in crosstalk between stress signaling pathways, and emerging evidence suggests that bothhormonal, as well as ROS signaling pathways play key roles in the cross talkbetween biotic and abiotic stress signaling that leads to plant responses (Fujitaet al. 2006). Ion flux regulations could also be observed in response to most of thestresses a plant could perceive. Such regulations could occur at different levels ofsignaling pathways or participate directly in the response. Due to its position at theinterface between the cytosol and the external medium, the plasma membrane isboth, a site of perception of external signals and one of the major vouchers of cellhomeostasis. It is therefore an initiation site of signaling pathways eliciting theplant response to biotic or abiotic stress (Jones et al. 1994; Dixon et al. 1996;Batistic and Kudla 2009; Kadono et al. 2010; Hauser et al. 2011), and forpathogens a target which allows alteration of cellular homeostasis for example bydisrupting the H+-ATPase activity (Zhou et al. 2000; Bunney et al. 2002), or byincreasing membrane permeability by creating pores (Hutchison et al. 1995;Goudet et al. 1999), or by activating ion channels (El-Maarouf et al. 2001; Errakhiet al. 2008a; Zhang et al. 2008; Jeworutzki et al. 2010).</p><p>Concerning biotic stress, a beam of physiological data suggests that pathogenattack is accompanied by an early deregulation of the ion fluxes of the target cell,particularly H+, Ca2+, K+, and anions such as NO3</p><p>- (Atkinson et al. 1990; Pikeet al. 1998; Wendehenne et al. 2002; Bouizgarne et al. 2006a; Reboutier et al.2007a, b; Errakhi et al. 2008a, b). The variation of the flow of ions through theplasma membrane could be induced within minutes or even seconds. So this is oneof the first events following perception. Given the magnitude of the inducedion efflux, in most cases, it is reasonable to attribute the response to the opening ofion channels (only carriers able to account for the high conductance required).Regulation of the channels following the attack may also represent a factor in thepathogenesis and/or a step in the defense response of the plant. Such events wereshown to be associated to other early induced responses (production of ROS,induction of MAP kinase) and are components of the transduction chainsleading to pathogenesis and defense responses (Scheel 1998; Somssich and</p><p>70 F. Bouteau and D. Tran</p></li><li><p>Hahlbrock 1998). In this context, the regulation of ion transport systems at theplasma membrane appears to be also an important factor in hypersensitiveresponse (HR) associated with plant programmed cell death (PCD) (Atkinson et al.1990; Jabs et al. 1997; del Pozo and Lam 1998; Scheel 1998; Somssich andHahlbrock 1998; Heath 2000; Wendehenne et al. 2002). Recent studies providedgenetic evidences positioning cyclic nucleotide-gated channels (CNGC) in thesignaling of pathogen attack and the induction of HR (Clough et al. 2000; Balagueet al. 2003). Also in vivo recording of CNGC remain scarce, they could beinvolved in the calcium influx that triggers mechanism of HR (Leng et al. 2002).</p><p>Regarding abiotic stress, the rapid regulation of ion channels was also observedin response to hyperosmotic stress such as salinity or nonionic hyperosmotic stressused to mimick dehydration (Teodoro et al. 1998; Zingarelli et al. 1999; Shabalaand Newman 2000; Shabala and Lew 2002; Shabala et al. 2007) or drought(Dauphin et al. 2001). In such osmotic stresses, because of the osmotic gradientgenerated, water flows out of the cell. In response to hypertonic stress, increase orrestoration of K+ uptake through K+ channels is the major mechanism used in cellosmotic adjustment as reported in various plant tissues (Teodoro et al. 1998;Zingarelli et al. 1999; Shabala and Newman 2000; Dauphin et al. 2001; Shabalaand Lew 2002). In response to drought, one of the well-studied systems at cellularlevel is the stomatal closure and its regulation by abscisic acid (ABA) (Sirichandraet al. 2009; Hauser et al. 2011). Among abiotic stresses, pollutant generatedstresses are more and more prevalent and could lead to loss of productivity inplants of agronomic interest. Recent studies highlighted the involvement of ionchannels in plant responses to pollutants (Kadono et al. 2006; Vahisalu et al. 2008;Kadono et al. 2010; Tran et al. 2010; Yukihiro et al. 2012). As regards CNGC,genetic evidences position the slow-type anion channel (SLAC1) during plantresponse to ozone (Vahisalu et al. 2008, 2010), and the guard cell outwardrectifying K+ channel (GORK) in response to oxidative stress (Demidchik et al.2010).</p><p>3.2 Microelectrode Voltage-Clamp Techniques</p><p>Much of what we know about the properties of ion channels was provided byvoltage-clamp studies developed on the squid giant axon (Cole 1949; Hodgkinet al. 1952). Since these pioneering studies, many derived techniques have evolvedand voltage-clamp analysis has been extended to a wide range of cell types,comprising plant cells. The voltage-clamp is a method that allows the measure-ment of ion flow through a membrane as an electric current. Conventionalvoltage-clamp using several electrodes has provided the foundations of plantelectrophysiology, (Findlay 1961; Gradmann et al. 1978; Beilby 1982). Thedevelopment of the patch-clamp studies (Neher and Sakmann 1976) has allowedmore wide-ranging studies comprising plant cell studies since the initial work ofSchroeder et al. (1984) on guard cell protoplasts. During the past decades, the</p><p>3 Plant Response to Stress: Microelectrode Voltage-Clamp Studies 71</p></li><li><p>patch-clamp technique has extended the application of voltage-clamp methods tothe recording of ionic currents flowing through single channels, and in its wholecell configuration has also become the most widely used method for recordingmacroscopic currents in plant cells. Electrophysiological studies have thereforegreatly improved the descriptions of both, the properties of various ion channelsand ion transport in nutrition, osmoregulation and signalization (Ward et al. 2009).Although the patch-clamp technique presents numerous advantages and notablycould in principle allow working on almost any plant cell type, a crucial step inpreparing the plant cells is the removal of the cell wall to allow giga-ohm seals.Hence, most patch-clamp studies on plant cells utilize protoplasts after enzymaticdigestion of the cell wall. Such protoplasts thus lack their plasma membrane/extracellular matrix connections, and non-physiological osmotic environment inthe application of patch-clamp techniques to most plant cells, are unavoidablefactors. As a matter of fact, removing the cell wall during protoplast preparation islikely to alter the capacity of the cells to respond to pathogens or elicitors(Klusener and Weiler 1999; Carden and Felle 2003; Humphrey et al. 2007).Moreover, the digestive enzymes used to remove the cell wall (Klusener andWeiler 1999), as cell wall fragments such as oligogalacturonides, could elicitdefense responses in plant cells, notably ion fluxes (Reymond et al. 1995), makingdifficult secondary analysis of pathogen-derived molecules on ion fluxes by patch-clamp. Laser microsurgery techniques have been described to expose the plasmamembrane to patch-clamp pipette (Taylor and Brownlee 1992; Kurkdjian et al.1993; Henriksen et al. 1996), but this approach is not well developed. The use ofmicroelectrode voltage-clamp techniques (MEVC) is an attractive alternative tothe whole-cell configuration of the patch-clamp technique. Indeed, with micro-electrode(s) the whole cell voltage-clamp measurements can be conducted in situon cells with an intact cell wall. It is further noteworthy that the voltage-clamptechnique allows studying the activities of ion channels in the environmentprimarily as physiological [composition not controlled, low perfusion from thepipette (Blatt and Slayman 1983)], which is not provided by the perfusion ofthe patch-clamp on the protoplasts. These conditions could be important since theactivity of ion channels may require cytosolic intermediates for their activationand/or their regulation (Lee et al. 2009). These more physiological conditions arethus best suited to record ion channel activities in cells challenged by environ-mental stimuli.</p><p>A voltage clamp with two electrodes (TEVC) was initially applied to plant cellslarge and robust enough to tolerate several intracellular electrodes (Findlay 1961;Gradmann et al. 1978; Beilby 1982). A significant advance in TEVC has been thedevelopment of double-barreled electrodes allowing working on the smallest plantcells. In this configuration, one barrel of the electrode measures voltage, whilecurrent is injected through the other barrel. Numerous studies were performed withTEVC method on stomatal guard cells (Blatt et al. 1987; Blatt 1991, 1992;Roelfsema and Prins 1997; Roelfsema et al. 2001) and on root hairs (Lew 1991;Meharg and Blatt 1995; Lew 1996; Blatt et al. 1997). An alternative to the TEVCtechnique is the discontinuous single electrode voltage-clamp (dSEVC) technique</p><p>72 F. Bouteau and D. Tran</p></li><li><p>or switch-clamp. The main advantage of using only one microelectrode is thelower impact of impalement on cell membrane. In this method, a single electrodeperforms the current injection and potential measurements, by rapid switchingbetween a current injection mode and potential measuring mode (Brennecke andLindemann 1974; Finkel and Redman 1984; Halliwell et al. 1994). This methodneeds microelectrodes with fast settling times and thus takes advantage of a slowertime constant of the plasma membrane compared with the electrode. The switchingfrequency between the current injection- and voltage measuring-mode should thusbe high enough to allow any voltage, associated with charging of the electrodecapacitance during the current injection cycle to decay before sampling ofmembrane potential. For a fuller description of the MEVC and their optimizationthe reader is referred to Finkel and Redman (1984) and Halliwell et al. (1994). ThedSEVC has been successfully applied to different plant cell types, Fucus eggs(Taylor and Brownlee 1992), laticifers (Bouteau et al. 1996), guard cells (Forestieret al. 1998; Raschke et al. 2003), root hairs (Bouteau et al. 1999), and culturedcells (Jeannette et al. 1999; El-Maarouf et al. 2001).</p><p>During voltage-clamp experiments, the membrane voltage is held at a definedvoltage by injecting into the cells, with a feedback amplifier, currents equal inamplitude, and opposite in sign to those that flow through the membrane. Most ofthe time, the voltage is forced to change from a holding potential (Vh) in a squarestep fashion as rapidly as possible from one steady state to another (Fig. 3.1a).Upon such protocols, different potential steps are swept allowing the acquisition ofinformation about the ion channel behaviors and their mode of gating (opening and</p><p>I (nA)</p><p>V (mV)</p><p>Time</p><p>Depolarizating potential step </p><p>Hyperpolarizatingpotential step</p><p>Outward currents: cation efflux oranion influx</p><p>Inward currents : cation influx oranion efflux</p><p>Protocol</p><p>Recorded current</p><p>Im</p><p>Vh = Vm</p><p>*</p><p>*</p><p>(a)</p><p>(b)</p><p>(c)</p><p>(d)</p><p>out</p><p>in</p><p>Fig. 3.1 a Voltage-clamp protocol: voltage changes in a square fashion from a holding potential(Vh). b Typical currents recorded from a voltage-clamped cell when the membrane potential isstepped as shown in a. Spikes of capacity current occur at the edges of the pulse (asterisk). c Thevoltage steps and recorded currents are then usually represented superimposed. d Currentvoltagerelationship corresponding to the values of current intensity at one defined time of the voltage-clamps (dashed line) plot to the corresponding voltages</p><p>3 Plant Response to Stress: Microelectrode Voltage-Clamp Studies 73</p></li><li><p>closure). Indeed, most ion channels are affected by the membrane potential:depolarizing voltage promoting outward currents when hyperpolarizing voltagefavoring inwa...</p></li></ul>


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