Ionic Nature of Burn-Induced Variation Potential in
Wheat Leaves
Lyubov Katicheva, Vladimir Sukhov, Elena Akinchits and Vladimir Vodeneev*
Variation potential (VP) in higher plants cells is a transitory
depolarization of the plasma membrane occurring in response to external damage. The effects of VP on different
physiological processes are actively studied, but little is
known about their ionic nature, which limits the interpretation of VP-induced functional changes. It is thought that
VP generation is based on transient inactivation of plasma
membrane proton pumps and is not connected to passive
ionic fluxes. To study burn-induced VP in wheat seedlings,
we measured membrane electric potential and cell input
resistance. Cell input resistance decreased during VP generation, indicating that ionic channels were activated. In addition, VP amplitude decreased when the extracellular
calcium concentration was lowered. When anion channels
were blocked by ethacrynic acid addition, the VP had poor
depolarization speed and amplitude. A decrease in the
chlorine gradient by extracellular chlorine concentration
shift leads to lowering of the VP amplitude and depolarization speed. This result indicates the role of chlorine efflux in
depolarization phase formation. The VP repolarization is
connected to potassium ion efflux, that is confirmed by
repolarization suppression under addition of the potassium
channel blocker tetraethylammonium (TEA) and an increase
in the extracellular potassium concentration. We also
showed that the addition of a proton pump inhibitor
leads to membrane potential depolarization and inhibition
of VP generation. These results suggest that the VP may be
formed not only by transient suppression of proton pumps
but also by passive ionic fluxes through the membrane.
Keywords: Cell input resistance Microelectrode technique
Passive ionic fluxes Proton pump Variation potential Wheat.
Abbreviations: AP, action potential; APW, artificial pond
water; Avp, amplitude of variation potential; EA, ethacrynic
acid; Em, transmembrane electrical potential; Emr, transmembrane electrical potential at rest; Rin, cell input resistance;
TEA, tetraethylammonium; VD, velocity of depolarization;
VP, variation potential; VR, velocity of repolarization; WS,
wound signal.
Introduction
Electrical signals in higher plants have been reported to play a
key role in stress sensing and systemic response development.
Plants show two types of electrical reactions depending on the
nature of the stimulation: action potential (AP) and variation
potential (VP). A plethora of physiological responses induced
by electrical signals have been observed in plants, including
changes in photosynthetic processes, increase in the respiration
rate and gene expression activation (Trebacz et al. 2006, Fromm
and Lautner 2007, Baluska and Mancuso 2009, Grams et al.
2009, Sukhov et al. 2012). Though little is known about the
mechanisms of induction of functional changes by AP or VP,
it is considered that ionic concentration shifts in cells during
electrical reaction development play the main role in such
transformation (Trebacz et al. 2006, Pyatygin et al. 2008). The
ionic nature of the AP is currently better described; its formation starts with Ca2+ influx and the opening of chlorine channels, which depolarizes the plasma membrane. Shortly after Cl
efflux has begun, the AP peaks, and K+ efflux starts processes
leading to repolarization (Trebacz et al. 2006, Fromm and
Lautner 2007).
Unlike APs, VPs are not self-propagated electrical signals.
The prevalent hypothesis of VP propagation relates to their
induction by localized damaged hydraulic waves, which cause
generation of an electrical response in plant cells. The possibility
of such a propagation mechanism has been proved experimentally in a number of studies (Malone et al. 1994, Stahlberg and
Cosgrove 1997, Mancuso, 1999). However, according to another
hypothesis, suggested by Ricca (1916), VPs might also be
induced by migration of a wound chemical substance from
the zone of damage (Rhodes et al. 1999, Vodeneev et al. 2012).
The mechanism of VP generation also differs from that of
APs and seems to reflect only a transient inactivation of proton
pumps at the plasma membrane without a contribution from
passive ionic fluxes (Julien et al. 1991, Stahlberg et al. 2006).
Proton pump participation in VP generation was identified
by modulation of H+-ATPase activity and membrane permeability to H+, and measurement of apoplastic pH changes
during VP generation (Julien et al. 1991, Stahlberg et al. 2006,
Vodeneev et al. 2011). In some studies, the absence of passive
Regular Paper
Department of Biophysics, Lobachevsky State University of Nizhni Novgorod, Gagarina Av., 23, Nizhni Novgorod, Russia
*Corresponding author: E-mail, [email protected]; Fax, 8(831) 462-32-02.
(Received July 18, 2013; Accepted June 9, 2014)
Plant Cell Physiol. 55(8): 1511–1519 (2014) doi:10.1093/pcp/pcu082, available online at www.pcp.oxfordjournals.org
! The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Plant Cell Physiol. 55(8): 1511–1519 (2014) doi:10.1093/pcp/pcu082 ! The Author 2014.
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L. Katicheva et al.
ionic fluxes through membrane channels during VP development was shown by several parameters: stable membrane resistance at the VP (Stahlberg and Cosgrove 1996, Stahlberg et al.
2006), no measurable changes in membrane permeability to K+,
Cl–, Ca2+ and Na+ during VP development (Julien et al. 1991)
and no changes in VP parameters with ionic channel activity
inhibitors (Stahlberg and Cosgrove 1996, Rousset et al. 2002).
Conversely, the complex kinetics of VPs with fast potential
changes at the VP front slope and the occurrence of impulses
may be related to the activation of ion channels during VP formation, and the involvement of passive ionic fluxes in VP generation is still under discussion. Calcium and chlorine ion
concentration shifts during development of the electrical reaction (Zimmerman and Felle 2009) are in good agreement with a
possible role for passive ionic fluxes in the VP. It has also been
thought that calcium ions may cause an efflux of anions into the
apoplast to induce fast depolarization during VPs (Vodeneev
et al. 2011). Moreover, theoretical analysis of VP generation
mechanisms also indicates the possibility that passive ionic
fluxes participate in this process (Sukhov et al. 2013).
The aim of the present study was to analyze the possible
involvement of passive ionic fluxes, i.e. Ca2+, Cl and K+, in
the generation of VPs. For the recognition of the ionic diffusion
during VPs, cell input resistance was recorded simultaneously
with electrical activity. Changes in VP parameters with the addition of different inhibitors served as indicators of the participation of calcium, chlorine or potassium channels in VP generation.
Results
VP characteristics
The transmembrane electrical potential (Em) of parenchymal
cells recorded without leaf tip burning (Emr) was 98 ± 3 mV.
Leaf tip burning led to the generation of a VP; transient membrane depolarization with an amplitude (AVP) of 75 ± 4 mV, and
speeds of membrane depolarization (VD) of 9.09 ± 0.09 mV s 1
and repolarization (VR) of 0.54 ± 0.09 mV s 1 (Fig. 2). Changes
in these parameters under various inhibitors and ionic concentrations were used to identify the ionic flows that participate in
VP generation.
VP generation and resistance changes
The stability of Em shifts induced by an applied current was
checked before VP initiation. As seen in Fig. 3A, without perturbation the amplitude of Em shifts was constant, i.e. current
pulses with the chosen parameters did not affect voltagedependent processes at the plasma membrane and cell input
resistance (Rin). Fig. 3B shows a typical recording of VP induced
by leaf burning with simultaneous measurement of resistance
changes. It should be noted that the recorded VPs had parameters close to those of one recorded without the application of
current pulses. As seen in Fig. 3B, there was a decrease in Rin
during VP generation. Rin dropped at the beginning of depolarization and slowly recovered in the repolarization phase. Such
1512
changes in Rin may indicate the appearance of passive ionic
fluxes through activated channels.
Inhibitor and ion concentration effects on VP
generation
To reveal the transport systems taking part in VP formation,
metabolic inhibitors and channels blockers were used.
The role of H+-ATPase in VP generation was studied by the
addition of sodium orthovanadate (0.5 mM). As seen in Fig. 4A,
the suppression of the proton pump activity led to a membrane
depolarization of 32 ± 0.8 mV and the leaf injury resulted only
in a very weak electrical reaction without an expressed repolarization phase.
The participation of calcium ions in VP generation was estimated by the addition of the calcium chelator EGTA (1 mM) to
the artificial pond water (APW). The absence of free calcium had
no effect on Em values but AVP decreased dramatically (Fig. 4B).
Fig. 4C shows that the addition of an anion channels blocker
[ethacrynic acid (EA), 0.5 mM] also led to VP amplitude decrease. Moreover, the depolarization speed was also decreased.
Analysis of potassium ion participation in VP generation was
performed by adding a potassium channel blocker [tetraethylammonium (TEA), 0.5 mM] to the medium. The electric reaction to the leaf injury in the presence of TEA exhibited different
behavior to that of the control VP; in particular, the amplitude of
the depolarization and membrane repolarization decreased at a
slower rate (Fig. 4D). Moreover, the addition of both TEA and EA
to the medium resulted in a small Em depolarization and considerable suppression of the electric reaction to burning (Fig. 4E).
In order to provide additional validation of the VP experiments carried out using ionic channel blockers, we performed
measurements of the changes in VP vs. ionic concentration in
the extracellular space, with the results summarized in Table 1.
Both the amplitude and depolarization speed were increased, as
the extracellular calcium molar concentration was increased in
a range from 0.05 to 5 mM, which we interpret to be due to the
increase of the calcium influx to the cell. At the same time, the
increase of the extracellular chlorine concentration in the range
of 1–10 mM resulted in a notable drop in the decrease of the VP
amplitude and depolarization speed, as the efflux of chlorine
ions from the cell was suppressed. At the same time, the increase of the potassium ion concentration suppressed the repolarization speed and had a negligible effect on the amplitude
and speed of depolarization of VP. No essential changes in Emr
were detected following 1 h incubation of the plant in the solutions with different ionic concentrations. The presented evidence indicates that the passive ion fluxes play an important
role in VP generation, as discussed in the following section.
Changes of membrane resistance under the effect
of inhibitors on VP
To identify the ionic channels whose activation results in resistance changes, the VP and Rin were recorded simultaneously
after the addition of channels blockers.
Plant Cell Physiol. 55(8): 1511–1519 (2014) doi:10.1093/pcp/pcu082 ! The Author 2014.
Ionic nature of variation potential
Microelectrode current/voltage clamp amplifier,
PC
E
Er
Objective
Cuvette with:
artificial pond water (APW)
or APW with inhibitors
Microscope stage
Wheat leaf
Wheat roots
Burn
Light
source
E
E
Er
Er
I = 40pA
V, mV
Wheat leaf
in
out
Wheat leaf
in
Rin =
out
Fig. 1 Scheme of the experimental system for measuring Em and Rin. A wheat leaf is placed on the microscope stage, part of it is fixed in the
cuvette, and the measuring electrode is inserted in a parenchymal cell. The site of burning is indicated by the arrow. The separate scheme on the
left illustrates registration of the membrane potential (Em); the separate scheme on the right illustrates registration of the cell input resistance
(Rin). For the Rin measurement, current pulses with an amplitude of 40 pA alternated with frequencies of 2 Hz injected through the electrode to
cells. The amplitude of the membrane potential deflections was then used to calculate the Rin by Ohm’s law equation. E, measuring electrode; Er,
reference electrode; I, amplitude of the current pulses injected into the cells; V, amplitude of the membrane potential deflections.
Fig. 2 Variation potential in wheat induced by leaf tip burning (arrow). The measuring electrode was located in the leaf parenchymal cell of the
second to third layers. The reference electrode was immersed in APW. The distance between the burning site and recording zone was
approximately 5 cm. (A) A typical record of the variation potential (VP). (B) The parameters of the electric reaction: Emr, membrane voltage
at rest (mV); AVP, amplitude of the VP (mV); VD, speed of VP depolarization (mV s 1); VR, speed of VP repolarization (mV s 1).
Plant Cell Physiol. 55(8): 1511–1519 (2014) doi:10.1093/pcp/pcu082 ! The Author 2014.
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L. Katicheva et al.
Fig. 3 Changes in cell input resistance during VP generation. For calculation of Rin, current clamp mode was used and square current pulses with
frequencies of 2 Hz and an amplitude of 40 pA were applied. (A) A typical record of the membrane voltage with transient induction by current
pulses and calculated Rin at rest. (B) Changes in membrane voltage with transient induction by current pulses and calculated membrane
resistance after leaf tip burning (arrow). 1, Em; 2, Rin.
%
%
100s
100
100
80
Em, mV
0
A
-20
Em, mV
0
60
40
-20
20
B
0
Em, mV
-10
-30
C
-50
-70
40
%
100
80
0
Em1r A2VP V
3D 4
Em, mV
0
-60
-80
%
-100
100
E
60
80
Em, mV
0
60
-20
20
D
0
Em1r A2VP 3
VD 4
-40
-60
20
0
r 2
3 4
E1
m AVP VD
40
-80
20
40
-20
100
80
60
40
%
-60
-80
60
20
-40
2 V3D 4
Em1r AVP
-40
80
0
-40
-100
Em1r A2
VP V
3D 4
-60
-90
-80
-110
-100
Fig. 4 Changes in parameters of VP induced by leaf tip burning (arrow) with addition of inhibitors. Inhibitors were added separately into the
cuvette where the 4 cm area of the leaf was fixed. Plants were incubated in the presence of the inhibitors for at least 1 r. In the diagrams, the
measured VP parameters are presented as a percentage of the control values. (A) The effect of proton pump inactivation by sodium
orthovanadate (0.1 mM) on VP parameters. (B) The effect of the calcium chelator EGTA (0.5 mM) on VP parameters. (C) The effect of the
anion channel blocker EA (0.5 mM) on VP parameters. (D) The effect of the potassium channel blocker TEA (0.5 mM) on VP parameters. (E) The
effect of addition of both EA (0.5 mM) and TEA (0.5 mM) on VP parameters.
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Plant Cell Physiol. 55(8): 1511–1519 (2014) doi:10.1093/pcp/pcu082 ! The Author 2014.
Ionic nature of variation potential
Table 1 Effect of the concentration of ion on parameters of VP, induced by wheat leaf burning
Composition
Component tested for effect on parameters of VP
Ca2+ (0.05–5)
K+ (1–10)
Cl– (2–10)
CaCl2
0.05
0.5
5
0.5
0.5
0.5
0.5
0.5
0.5
NaCl
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
KCl
1
1
1
1
1
1
1
5
10
Choline chloride
10
9
–
–
3
8
9
4
–
Concentration
Ca
2+
Cl
–
K
+
0.05
0.5
5a
2a
5
10
1a
5
10
Amplitude of VP ± SD (%)
60 ± 9
78 ± 9
100 ± 6
100 ± 9
72 ± 10
75 ± 12
100 ± 13
96 ± 4
98 ± 6
Velocity of depolarization ± SD (%)
49 ± 12
68 ± 15
100 ± 8
100 ± 12
57 ± 15
66 ± 20
100 ± 14
88 ± 25
81 ± 8
Velocity of repolarization ± SD (%)
46 ± 9
50 ± 10
100 ± 29
100 ± 27
69 ± 28
84 ± 26
100 ± 14
49 ± 10
18 ± 4
a
Concentrations by which the average VP parameters were maximal are indicated as 100%
Fig. 5 Membrane resistance measurement with the addition of ionic channels blockers. (A) Changes in membrane voltage with transient
induction by current pulses and calculated membrane resistance after leaf tip burning (arrow) in the presence of EA (0.5 mM). (B) Changes in
membrane voltage with transient induction by current pulses and calculated membrane resistance after leaf tip burning (arrow) in the presence
of TEA (0.5 mM). 1, Em; 2, Rin.
Rin dynamics during the development of the electrical reaction changed when channel blockers were added. With EA,
there were no changes in Rin on VP generation (Fig. 5A).
Suppression of potassium channel activity by TEA led to
changes in Rin dynamics on VP generation; a fast Rin recovery
at the repolarization phase (Fig. 5B).
Discussion
VP is thought to be based on transient inactivation of H+ATPase (Julien et al. 1991, Stahlberg et al. 2006). According to
our results (Fig. 4A), under selective suppression of H+-ATPase
by sodium orthovanadate, the electrical reaction to local injury
is almost absent, which means that proton pumps participate
in formation of the VP. However, VP inhibition may also be
connected to the indirect influence of orthovanadate on passive ionic flows by Vm depolarization, which leads to a lowering
of the electrochemical gradients of ions.
A drop in the cell input resistance during development of
the electrical reaction (Fig. 3B) provides evidence that VP generation is connected to activation of ionic channels and the
passive ionic flows. It is thought that the generation of another
type of electrical reaction—the AP—is connected to activation
of calcium, chlorine and potassium channels and corresponding ionic flows (Trebacz et al. 2006). The same channels and
ionic fluxes might play a role in formation of the VP. Ionic
mechanism of VP generation may be analyzed by the characterization of the VP parameters vs. the extracellular ionic concentrations that is widely accepted for analysis of the nature of
the generation of the reaction (Hodick and Sievers 1988, Julien
et al. 1991, Rousset et al. 2002). Fig. 4B shows that chelating of
Plant Cell Physiol. 55(8): 1511–1519 (2014) doi:10.1093/pcp/pcu082 ! The Author 2014.
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L. Katicheva et al.
calcium ions leads to VP suppression. An increase in the extracellular calcium concentration from 0.05 to 5 mM gives rise to
the increase in the VP amplitude and depolarization speed, as
shown in Table 1. It should also be noted that a decrease in the
extracellular calcium concentration leads to lowering of the
repolarization speed (Fig. 4B; Table 1). Such a result might
be connected to an ability of calcium ions on the extracellular
side to activate the proton pump (Qiu and Su 1998). Generally
these results convincingly prove that the calcium flux is essential for VP generation in wheat leaves and corroborate the
results reported for other plants (Julien et al. 1991, Vodeneev
et al. 2011).
For identification of participation of ionic channels in electric
reactions, inhibition of transport system activity is also widely
used (Tyerman 1992, Shimmen 2002, Lautner et al. 2005). In
particular, EA was used as chlorine channels blocker in many
studies on higher plants and algae (Marten et al. 1992, Johannes
et al. 1998, Shimmen 2002, Tavares et al. 2011). The effect of TEA
on potassium channels in higher plant cells was also widely
shown previously (Stahlberg and Cosgrove 1992, Grabov et al.
1997, Lautner et al. 2005, Demidchik et al. 2010). The results we
obtained when channels blockers were added indicate the possibility that VP is formed by ionic channels (Fig. 4). In particular,
the recorded drop in VP amplitude with EA addition indicates
the participation of anion channels in the development of the
burn-induced electrical reaction. The anions that make a contribution to depolarization could be represented by chlorine
ions. This supposition is based on the significant concentration
gradient of the chlorine ions and their participation in the AP
depolarization (Opritov et al. 1991, Trebacz et al. 2006). A decrease in the chlorine gradient by an extracellular chlorine concentration shift leads to AVP and depolarization speed lowering
(Table 1). Moreover, changes of apoplastic pCl which corresponds to chlorine ion efflux to the apoplast during VP generation, were noticed (Zimmerman and Felle 2009, Vodeneev et al.
2011). Hence, chlorine ions make a contribution to the membrane depolarization of VP.
The repolarization phase of VP in the wheat leaf is possibly
connected not only with proton pump reactivation but also
with potassium ion efflux. A decrease of potassium gradients by
an increase in extracellular potassium concentration decreases
the repolarization phase (Table 1). Addition of the potassium
channel blocker TEA also leads to a decrease in repolarization
speed (Fig. 4D). Under TEA, a small decrease in depolarization
speed was also registered (Fig. 4D) that may be connected to a
non-specific effect of the channel blocker.
It is known that changes in membrane resistance of the AP
correspond exactly to activation of chlorine and potassium channels (Samejima and Sibaoka 1982). The fast drop in Rin at the
start of VP generation may also be connected to chlorine channel
activation, and the low values of Rin at repolarization may be
connected to potassium channel activation. This was supported
by the stable Rin values on VP generation when an anion channel
blocker was added and by the fast Rin value recovery at the
repolarization phase with TEA addition (Fig. 5A).
1516
Our results show the participation of not only proton
pumps, but also of passive ionic fluxes during VP generation.
However, these results do not agree with previous studies in
which the absence of passive ionic flows on VP generation
was reported. There are data showing stable levels of membrane resistance during VP formation (Stahlberg and
Cosgrove 1992, Stahlberg and Cosgrove 1996). It is worth
noting that the authors do not exclude the participation
of passive ionic flows in the development of VP; in their
experiments, membrane resistance may have remained
stable because the opening of ionic channels was compensated for by the closure of other channels (Stahlberg and
Cosgrove 1992). There are also results indicating that there
are no measurable changes in membrane permeability to
Ca2+, Cl– or K+ during VP development (Julien et al.
1991). It should be noted that these permeability changes
were recorded during the slow repolarization phase and,
according to our results, membrane resistance changes
from the very beginning of the electrical reaction.
There are also some contradictions with our data on the
influence of channel blockers on VP generation. Our results
show dramatic changes of VP parameters with the addition
of inhibitors, whereas in other studies channel blockers had
no influence on VP (Stahlberg and Cosgrove 1996). At the
same time, in several works, the possible participation of calcium ions in VP generation has been shown by the reduced AVP
in calcium-free extracellular medium (Julien et al. 1991,
Stahlberg and Cosgrove 1996) and with inactivation of calcium
channels by lanthanum (Julien et al. 1991).
Based on our results and data from the literature, the
following sequence of events might occur during VP generation (Fig. 6). External damage induces formation of a
wound signal (WS) which might be manifested as a hydraulical wave (Malone et al. 1994, Mancuso 1999) or a chemical
agent (Rhodes et al. 1996, Vodeneev et al. 2012). The WS
triggers stretch-activated (Stahlberg et al. 2006) or ligandoperated (Sukhov et al. 2013) calcium channels, and calcium
ions enter the cell. It is known that calcium ions can induce
proton pump inactivation, that causes proton influx, and
affect Ca2+-dependent chlorine channels and chlorine ion
efflux (Fromm and Lautner 2007, Vodeneev et al. 2011).
Both processes lead to membrane depolarization. The start
of the membrane repolarization is probably based on potassium ion diffusion out of the cell. Such consequent activation of anionic and potassium channels leads to a transient
decrease in the cell input resistance, which has been registered in our work. The reactivation of proton pumps makes
a significant contribution to the subsequent repolarization,
because a slow repolarization phase develops under more
negative voltage values than the Nernst potential for potassium ions (Stahlberg et al. 2006).
This suggested mechanism is similar to the model for AP
generation in higher plants (Vodeneev et al. 2006). The main
difference is probably in the initial activation of different types
of calcium channels; ligand-operated or stretch-activated in VP
Plant Cell Physiol. 55(8): 1511–1519 (2014) doi:10.1093/pcp/pcu082 ! The Author 2014.
Ionic nature of variation potential
Cl–
out
+
K
Wheat leaf
2+
Ca
in
Em
Ca2+
[Ca]in
WS
H+
+
H
WS
Burn
Fig. 6 Scheme of the possible mechanism of the variation potential generation in wheat leaf. WS, wound signal; Em, membrane potential; [Ca]in,
concentration of intracellular calcium.
instead of voltage-dependent in AP (Stahlberg et al. 2006,
Sukhov et al. 2013). This difference might result in a more
continuous increase in the concentration of calcium ions in
cells, causing a slower electrical reaction in comparison with
the development of AP.
Materials and Methods
Plant materials
Seedlings of wheat (Triticum aestivum L.) were cultivated
hydroponically in a Binder KBW 240 (Germany) plant growth
chamber at 24 C under a 16/8 h (light/dark) photoperiod.
Seedlings that were 14–21 d old with a leaf blade length of
15–20 cm were used in experiments (Vodeneev et al. 2012).
Electrical measurements
For measurements of the intracellular membrane potential
(Em) and resistance (Rin) dynamics, the microelectrode technique [Olympus BX51microscope; Luigs and Neumann automatic manipulators; Multiclamp 700B amplifier (Axon Inst.;
Molecular Devices); and a PC] was used. The entire plant was
placed at the side of the microscope, and the leaf was fixed
horizontally. Part of the leaf was placed in a cuvette filled with
APW, which comprised 0.1 mL NaCl, 0.1 mL KCl and 0.5 mL
CaCl2 (Fig. 1). For separate experiments, APW in the cuvette
was changed to a solution with inhibitors or different ionic
compositions as described below.
Single-barreled microelectrodes were fabricated from
1.2 mm (OD) borosilicate glass tubing. Capillaries were pulled
in a horizontal pipette puller and filled with 100 mM KCl. The
tip diameter was approximately 0.5 mm. The electrode was
moved by motorized manipulators and its location was
checked visually using a microscope. Electrical measurements
were carried out in parenchymal cells of the second to third
layer. The reference electrode was immersed into a cuvette with
APW. The VP was induced by open-flame burning of the leaf tip
(apical 2–3 mm) for 2 s (Vodeneev et al. 2012). The distance
between the registration zone and the wounded area equaled
5 cm. VP parameters such as amplitude (AVP), and depolarization (VD) and repolarization (VR) speeds were estimated. The
VR was measured as the average speed of the membrane potential recovering from the VP peak to the potential values at
half of the AVP.
Plant Cell Physiol. 55(8): 1511–1519 (2014) doi:10.1093/pcp/pcu082 ! The Author 2014.
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L. Katicheva et al.
For the measurement of Rin, changes in the membrane
voltage under applied current pulses were estimated. Current
pulses with an amplitude of 40 pA alternated with frequencies
of 2 Hz depolarized cells by a few milliVolts. The amplitude of
the membrane potential deflections was then used to calculate
the Rin (Stahlberg and Cosgrove 1992). A scheme of the experimental system for Em and Rin measurement is shown in Fig. 1.
Calculated Rin includes membrane resistance and the resistance
of the electrode, which remains constant during the
measurements.
Inhibition of transport system activity
This analysis was based on measurements of VP parameter
changes under the influence of inhibitors. A direct inhibitor
of the plasma membrane H+-ATPase activity, sodium orthovanadate (Lew 1991), a calcium chelator, EGTA (Julien et al. 1991,
Stahlberg and Cosgrove 1992), an anion channel blocker, EA
(Tyerman 1992, Tavares et al. 2011), and a potassium channel
blocker, TEA (Stahlberg and Cosgrove 1992, Lautner et al. 2005),
were used. The concentrations of the chemical agents are indicated in the Results and in the figure legends. Plants were
incubated in the presence of the inhibitors for at least 1 h.
The measured parameters of variation potentials are presented
as percentages of the control values.
Change in the ionic composition
Parameters of VP were measured under different ionic concentrations in extracellular medium (Hodick and Sievers
1988). For the preparation of different compositions, CaCl2,
NaCl, KCl and choline chloride were used in several combinations of concentrations. To define the effect of each separate ion by its concentration variation, the concentrations of
the other active ions were maintained constant (Table 1).
Plants were incubated in the compositions for at least 1 h.
The final concentrations of each tested ions are presented in
Table 1.
Statistical analysis
Each series of experiments comprised 5–8 repetitions, and
every measurement was performed on a separate plant.
Representative records obtained in individual measurements,
mean values and standard errors are presented.
Funding
This work was supported by the Ministry of Education and
Science of the Russian Federation [contract No 6.2050.2014/
K] and by the Russian Foundation for Basic Research [research
project No 13-04-97152-r-a.].
Disclosures
The authors have no conflicts of interest to declare.
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