Polyamines Interact with Hydroxyl Radicals in

Activating Ca2+ and K+ Transport across the Root

Epidermal Plasma Membranes1[W]
Isaac Zepeda-Jazo2,3, Ana Mara Velarde-Buenda2, Rene Enrquez-Figueroa, Jayakumar Bose,
Sergey Shabala, Jesus Muniz-Murgua, and Igor I. Pottosin*
Centro Universitario de Investigaciones Biomedicas, Universidad de Colima, 28045 Colima, Mexico (I.Z.-J.,
A.M.V.-B., R.E.-F., J.M.-M., I.I.P.); and School of Agricultural Science, University of Tasmania, Hobart,
Tasmania 7001, Australia (J.B., S.S.)

Reactive oxygen species (ROS) are integral components of the plant adaptive responses to environment. Importantly, ROS
affect the intracellular Ca 2+ dynamics by activating a range of nonselective Ca2+-permeable channels in plasma membrane
(PM). Using patch-clamp and noninvasive microelectrode ion flux measuring techniques, we have characterized ionic currents
and net K+ and Ca2+ fluxes induced by hydroxyl radicals (OH ) in pea (Pisum sativum) roots. OH , but not hydrogen peroxide,
activated a rapid Ca2+ efflux and a more slowly developing net Ca2+ influx concurrent with a net K+ efflux. In isolated protoplasts, OH evoked a nonselective current, with a time course and a steady-state magnitude similar to those for a K+ efflux in
intact roots. This current displayed a low ionic selectivity and was permeable to Ca2+. Active OH -induced Ca2+ efflux in roots was
suppressed by the PM Ca2+ pump inhibitors eosine yellow and erythrosine B. The cation channel blockers gadolinium, nifedipine,
and verapamil and the anionic channel blockers 5-nitro-2(3-phenylpropylamino)-benzoate and niflumate inhibited OH -induced
ionic currents in root protoplasts and K+ efflux and Ca2+ influx in roots. Contrary to expectations, polyamines (PAs) did not inhibit
the OH -induced cation fluxes. The net OH -induced Ca2+ efflux was largely prolonged in the presence of spermine, and all PAs
tested (spermine, spermidine, and putrescine) accelerated and augmented the OH -induced net K+ efflux from roots. The latter
effect was also observed in patch-clamp experiments on root protoplasts. We conclude that PAs interact with ROS to alter
intracellular Ca2+ homeostasis by modulating both Ca2+ influx and efflux transport systems at the root cell PM.
d

Increased reactive oxygen species (ROS) production

is a common denominator of plant adaptive responses
to a large number of abiotic and biotic stresses (Mittler,
2002). ROS are produced by cell wall-associated peroxidases, apoplastic diamine and polyamine oxidases,
plasma membrane (PM) NADPH oxidase, and oxidases
and peroxidases in mitochondria, chloroplasts, and
peroxisomes (Mahalingam and Fedoroff, 2003). A combination of increasing ROS production and limited
energy resources to replenish the antioxidant activity
results in ROS accumulation (Taylor et al., 2004). The
most abundant types of ROS in plants are hydrogen
peroxide (H2O2) and the two free oxygen radicals,
namely superoxide radical ( O22) and hydroxyl radical
d

This work was supported by Consejo Nacional de Ciencia y

Tecnologa (grant no. CB 82913 to I.I.P. and fellowships to I.Z.-J. and
A.M.V.-B.), University of Tasmania Visiting Fellowship to I.I.P., and
the Australian Research Council (grant no. DP1094663 to S.S.).
2
These authors contributed equally to the article.
3
Present address: Instituto de Biotecnologa, Universidad Nacional Autonoma de Mexico, Cuernavaca, Morelos 62210, Mexico.
* Corresponding author; e-mail pottosin@ucol.mx.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Igor I. Pottosin (pottosin@ucol.mx).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.111.179671

(OH ), the latter being the most reactive with biomolecules and structures. The lifetime of the OH is only
1029 s, which implies that it acts within 1 nm from the
point of its formation and does not cross the membrane
(Mori and Schroeder, 2004). Generation of the OH in
roots, via the activity of intrinsic peroxidases, is a
prerequisite for cell wall loosening and normal root
growth (Schopfer et al., 2002; Liszkay et al., 2004). The
extent of ROS accumulation is ultimately a determinant
of whether ROS production is part of a signal mechanism (at low levels) or a harmful event (at high levels)
for plants (Foyer and Noctor, 2005; Miller et al., 2010).
Thus, stress-specific modulation of ROS production
and scavenging is crucial. Up-regulation of ROSresponsive transcripts under osmotic stress was confined almost exclusively to shoots, whereas during
salinity, these changes were observed almost exclusively in roots (Davletova et al., 2005a, 2005b; Miller
et al., 2010).
Ion channels have long been considered as potential
ROS targets. In the PM, H2O2 activates the hyperpolarization-activated nonselective Ca2+-permeable
channels (Pei et al., 2000; Demidchik et al., 2007) and
inhibits outward- and inward-rectifying potassium
channels (Kohler et al., 2003). The induction of Ca2+
influx by H2O2 in guard cells mediates elicitor- or
abscisic acid-induced stomatal closure (Lee et al.,
1999; Pei et al., 2000; Schroeder et al., 2001). OH has
d

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Zepeda-Jazo et al.

activated a nonselective Ca2+-permeable current in the

mature zone of Arabidopsis (Arabidopsis thaliana)
roots, whereas external H2O2 does not evoke any
current in this tissue (Demidchik et al., 2003, 2007).
The OH -induced current was implicated in elongation via Ca2+ signaling (Foreman et al., 2003). Also, in
Arabidopsis roots, OH was shown to activate the
constitutively expressed outward-rectifying K+ current, as part of a programmed cell death scenario
(Demidchik et al., 2010). Therefore, it appears that
different ROS may have rather different spectra of
physiological activities and differ in their effects on
the PM ion channels.
The accumulation of the polyamines (PAs) putrescine
(Put2+), spermidine (Spd3+), and spermine (Spm4+) is
another common component of stress responses of
plants and correlates with plant stress resistance
(Bouchereau et al., 1999; Walters, 2003). The up- or
down-regulation of genes involved in the biosynthesis
and degradation of PAs was reported to modulate plant
sensitivity to drought, salt, osmotic, oxidative, and cold
stresses (Kusano et al., 2007a, 2007b; Rhee et al., 2007;
Alcazar et al., 2010; Takahashi and Kakehi, 2010). Under
stress conditions, PAs can act either directly, as chemical
chaperones for DNA and other macromolecules, or
indirectly, as positive regulators of stress response
genes (Rhee et al., 2007). PAs normally reduce the
membrane leakage induced by abiotic stresses in plants.
In addition to unspecific increase of the membrane
rigidness, due to the immobilization of negatively
charged phospholipids, more specific effects could be
in work, such as activation of the antioxidant enzymes
by PAs (Groppa and Benavides, 2008; Gill and Tuteja,
2010). In plants, PAs may play a dual role, as free radical
scavengers and antioxidant enzyme activators but also
as a source of H2O2 (Takahashi and Kakehi, 2010). If the
latter mechanism prevails, instead of preventing it, PAs
could increase the oxidative damage (Mohapatra et al.,
2009, and refs. therein).
Among immediate molecular targets for PAs, ion
channels and receptors are receiving growing attention. In animal cells, PAs block a variety of K+ and
other cation-selective channels (Drouin and Hermann,
1994; Ficker et al., 1994; Lopatin et al., 1994; Bahring
et al., 1997; Lu and Ding, 1999). In plants, PAs inhibit
PM Shaker-type K+ channels in guard, cortical, epidermal, and xylem parenchyma cells (Liu et al., 2000;
Zhao et al., 2007), nonselective cation channels in
mesophyll and root PM (Shabala et al., 2007; Zhao
et al., 2007), as well as nonselective cation fast and
slow vacuolar channels (Bruggemann et al., 1998;
Dobrovinskaya et al., 1999a, 1999b). PAs are the only
organic polycations that are present in sufficient quantities under stress to play the role of channels blockers
without compromising cell metabolism (Alcazar et al.,
2010). At the same time, PAs could act as cofactors in
the activation of PM H+ pumps (Reggiani et al., 1992;
Liu et al., 2005b; Garufi et al., 2007).
If the blockage of PM K+ and nonselective cation
channels were the dominant effect of PAs, it would
d

2168

assist the retention of intracellular K+ and the reduction of Na+ influx under salt stress, thus ameliorating
its detrimental effects on plant ionic homeostasis
(Zepeda-Jazo et al., 2008). However, recent results by
Pandolfi et al. (2010) suggested that PAs, depending on
growth conditions or particular root zone, could either
suppress the salt-induced K+ efflux or stimulate it.
Such stimulation may take place via cross talk between
PAs and ROS, while the latter are produced in the
apoplast via PA catabolization by amino oxidases, as
already mentioned. Indeed, it was shown recently that
PAs may be actively exported to the apoplast and be
oxidized there, generating ROS; the latter will activate
Ca2+ entry across the PM. Such a pathway was shown
to mediate abscisic acid-induced stomatal closure in
Vicia faba guard cells (An et al., 2008), the production of
volatile terpenoids in lima bean (Phaseolus lunatus)
leaves (Ozawa et al., 2009), and to control pollen tube
growth (Wu et al., 2010). Importantly, salt stress also
provokes the exodus of PAs into the apoplast, with its
further oxidation and ROS production. Depending on
the conditions, this may result in either a tolerance
response or lead to programmed cell death (Moschou
et al., 2008a). A coproduction of PAs and ROS and their
interplay under stresses, therefore, may influence PM
ion conductance in different ways, affecting in particular Ca2+ signaling.
In this work, we have studied ROS-induced Ca2+
and K+ fluxes and currents in pea (Pisum sativum) roots
and tested the effects of PAs on their kinetics. Our
results suggest a novel mechanism, where PAs act as
cofactors in the ROS induction of PM Ca2+-permeable
nonselective current and as inducers of the active Ca2+
efflux across the PM.
RESULTS
Kinetics and Pharmacology of the OH -Induced K+ and
Ca2+ Fluxes in Pea Roots
d

OH was generated by the application of copper

ascorbate (Cu/A; Biaglow et al., 1997; Halliwell and
Gutteridge, 1999). Briefly, being reduced by ascorbate,
copper catalyzes a sequence of one-electron reduction steps from molecular oxygen to O22, from O22 to
H2O2, and finally, from H2O2 to OH . The last step is
analogous to the classical Fenton reaction, where reduced Fe2+ is normally used instead of Cu+ (Biaglow
et al., 1997).
Net Ca2+ and K+ fluxes induced by 1 mM Cu/A
were measured by the noninvasive microelectrode
ion flux measuring (MIFE) technique from pea root
epidermis (for details, see Materials and Methods).
The concentration of 1 mM Cu/A was selected here to
make our results comparable with previously published studies on roots (Demidchik et al., 2003, 2010;
Cuin and Shabala, 2007). This treatment provoked a
long-lasting (more than 40-min) net K+ efflux from
pea roots (Fig. 1A), while the Ca2+ flux showed a more
complex kinetics, undergoing a switch from a rapidly
d

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Polyamines Potentiate OH -Induced Ion Fluxes

developed net Ca2+ efflux immediately after Cu/A

application to a net Ca2+ influx 10 to 15 min later (Fig.
1B). The application of ascorbate (1 mM) alone did not
have any significant (P , 0.05) effect on net ion fluxes,
whereas 1 mM Cu2+ evoked a relatively small K+ and
Ca2+ efflux (Fig. 1, C and D). As Cu2+ alone acts as a
superoxide radical scavenger (Schopfer et al., 2002),
the observed flux stimulation may be related to OH
production, due to the reduction of Cu2+ added by
intrinsic apoplastic ascorbate. Ca2+ and K+ flux responses were observed also at lower Cu/A concentrations (see below).
The identity of ion flux components evoked by 1 mM
Cu/A was subjected to a pharmacological analysis.
Pretreatment with the nonspecific cation channel
blocker gadolinium (Gd3+; 0.1 mM) strongly diminished the development of the OH -induced K+ efflux
and abolished Ca2+ influx, favoring net Ca2+ efflux
(Fig. 1A). Eosine yellow (EY; 0.5 mM), a specific inhibitor of the PM Ca2+ pump (Romani et al., 2004;
Beffagna et al., 2005), almost completely suppressed
the OH -induced Ca 2+ efflux (Fig. 1B) without a significant effect on the OH -induced K+ efflux (Fig. 1A).
Pretreatment with another fluorescein derivative,
erythrosine B (0.5 mM), caused a decrease of the OH induced Ca2+ efflux by 77% (n = 10) without significant
effects on Ca2+ influx or K+ efflux (P , 0.05). Thus, at
least two transport systems appear to mediate the
observed Ca2+ fluxes. One of them is an active Ca2+
efflux system. This system is rapidly activated by OH
and is sensitive to EY (Fig. 1B), suggesting the Ca2+ATPase as a possible candidate (White and Broadley,
2003). The second (slower) transport component appears to be passive, it is sensitive to Gd3+, and it could
mediate both Ca2+ influx and K+ efflux (Fig. 1, A and B).
d

Slower (steady-state) Ca2+ and K+ flux components

were subjected to a further analysis, where pharmacological agents were added directly to the experimental
chamber after 30 min of OH treatment. Figure 2 shows
that the application of 0.1 mM Gd3+, the Ca2+ channel
blockers nifedipine and verapamil, which also block
nonselective cation channels activated by OH (Demidchik et al., 2003; Demidchik and Maathuis, 2007), and
5-nitro-2(3-phenylpropylamino)-benzoic acid (NPPB)
and niflumic acid, which block several anionic channels in plants (Roberts, 2006), all cause significant (P ,
0.05) inhibition of K+ and Ca2+ fluxes. The substantial
inhibition of Ca2+ influx by Gd3+, nifedipine, and
niflumate unmasked a continuing Ca2+ efflux (Fig.
2B). Therefore, both OH -induced Ca2+ efflux and influx
appeared to occur at steady state, but at high (1 mM)
Cu/A concentration, the influx dominates over the
efflux, resulting in a net Ca2+ influx.
d

PAs Potentiate the OH -Induced Ion Fluxes in Pea Roots

In pea roots, the concentration of free PAs ranged

from submillimolar (Spm4+ and Spd3+) to low millimolar (up to 4 mM for Put2+; Shen and Galston, 1985).
Therefore, we have tested the effects of Put2+, Spd3+, or
Spm4+ at 1 mM concentration on the OH -induced K+
and Ca2+ fluxes. Similar concentrations, justified by
natural PA contents, were tested previously against
PM channels in different plant tissues (Liu et al., 2000;
Shabala et al., 2007; Zhao et al., 2007). Each of three
PAs added in combination with 1 mM Cu/A augmented and accelerated the OH -induced K+ efflux
(Fig. 3). Simultaneous application of Cu/A with either
1 mM Put2+ or Spd3+ suppressed the Ca2+ influx and
slightly diminished the peak Ca2+ efflux. At the same
d

Figure 1. Effects of Gd3+ and EY on the OH induced K+ and Ca2+ fluxes in pea roots. OH
radicals were generated by mixing 1 mM CuCl2
with 1 mM sodium ascorbate in the bath at the
times indicated by arrows. A, K+ fluxes were
strongly suppressed in the presence of Gd3+ (0.1
mM; triangles) but were insensitive to EY (0.5 mM;
squares). B, Gd3+ at 0.1 mM suppressed the OH induced Ca2+ influx, whereas the presence of 0.5
mM EY in the bath abolished the Ca2+ efflux. The
sign convention efflux negative applies to all
MIFE measurements. Data are means 6 SE; n = 6,
4, and 4 individual roots for control conditions
(1 mM Cu/A only), Gd3+, and EY treatments,
respectively. C and D, Control experiments,
where 1 mM CuCl2 (Cu; white triangles) or sodium
ascorbate (A; black circles) was applied separately. Data are means 6 SE; n = 3 or 4 individual
roots, respectively.
d

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Zepeda-Jazo et al.

in the presence of Spm4+ or Put2+. Spm4+ alone did

not provoke a significant K+ efflux (3 6 5 nmol m22 s21),
whereas Put2+ induced a K+ efflux of 41 6 5 nmol
m22 s21. However, even in the latter case, the K+ efflux
produced by a combination of 0.01 to 0.1 mM Cu2+ (+0.1
mM ascorbate) and 1 mM Put2+ was twice as high
compared with the sum of fluxes produced by Cu/A
and Put2+ separately. The synergistic effect of PAs with
ROS, albeit less pronounced in relative terms, is conserved up to the highest (1 mM) Cu/A concentration
tested.
At the same time, Cu/A-induced Ca2+ efflux reached
its maximum level already at the copper concentration
of 0.1 mM (Fig. 5B). This implies a lower threshold for
the activation of the PM Ca2+ efflux system as compared
with the induction of passive cation permeability, mediating both K+ efflux and Ca2+ influx. However, the
addition of Put2+ or Spm4+ alone also provoked Ca2+

Figure 2. Pharmacology of the steady-state OH -induced K+ and Ca2+

fluxes in pea roots. Drugs (0.1 mM Gd3+, nifedipine, verapamil, NPPB,
or niflumic acid) were introduced to the bath 30 min after the induction
of the ion fluxes by 1 mM Cu/A. A, Net K+ fluxes. B, Net Ca2+ fluxes. Ion
fluxes were averaged over 5-min periods immediately before (as a
control) and after drug addition. Fluxes are plotted as means 6 SE, with
the number of individual roots for each treatment indicated in parentheses.

time, in the presence of Spm4+, the OH -induced Ca2+

efflux was greatly prolonged (Fig. 4). The effects of
Put2+ or Spm4+ alone and on the background of variable concentrations of Cu/A are described below.
d

Physiological Concentrations of Redox-Active Transient

Metals Catalyze the Activation of K+ and Ca2+ Fluxes

The apoplastic space normally contains submillimolar concentrations of ascorbate (Pignocchi and Foyer,
2003; Kukavica et al., 2009). In legume nodules, copper
concentration is 10 to 50 mM, whereas the concentration
of iron, another redox-active metal, is between 0.1 and
0.7 mM (Becana and Klucas, 1992). Similar contents of
catalytically active copper and iron, equivalent to
approximately 30 mM and 0.3 mM, were found in pea
root cell walls (Kukavica et al., 2009). The production
of OH by the Cu/A mixture depends only weakly
on the ascorbate concentration, within a range of 0.1 to
4.0 mM, but strongly on the copper concentration
(Biaglow et al., 1997). In light of the above, we have
tested the effects of different copper concentrations on
the induction of K+ and Ca2+ fluxes using a fixed (0.1
mM) concentration of ascorbate. Results shown in
Figure 5A indicate that at physiological concentrations
(0.010.1 mM) of Cu2+, the Cu/A mixture evoked
a relatively small K+ efflux (1020 nmol m22 s21);
this, however, was strongly (up to 10-fold) potentiated
d

2170

Figure 3. Natural PAs potentiate OH -induced K+ efflux in pea roots.

Roots were treated either by 1 mM Cu/A alone (circles) or in a
combination with 1 mM Put2+ (A), Spd3+ (B), or Spm4+ (C). Data are
means 6 SE; n = 6 to 7 individual roots assayed for each treatment.
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Polyamines Potentiate OH -Induced Ion Fluxes

d

Patch-Clamp Characterization of the OH -Induced Ion

Currents in Pea Root Protoplasts

Most (approximately 80%) protoplasts prepared

from the pea root mature zone displayed only small
(less than 20 pA) and unspecific leak currents when
assayed in the whole-cell mode. In a few cases, protoplasts expressed either outward-rectifying K+-selective
or weakly voltage-dependent nonselective cation currents, classified on the basis of their reversal potentials,
approximately 270 and approximately 220 mV, respectively, as compared with EK = 272 mV and ECl =
+57 mV. These currents were rapidly abolished by the
application of 1 mM Cu/A (Fig. 7). After a delay of a
few minutes, a new current started to develop. Nor-

Figure 4. Effects of PAs on the OH -induced Ca2+ fluxes in pea roots.

Fluxes were evoked either by the addition of 1 mM Cu/A alone (circles)
or in the presence of 1 mM Put2+ (A), Spd3+ (B), or Spm4+ (C). Data are
means 6 SE; n = 6 to 7 for each treatment, except Spm4+ (n = 14).

transient efflux, very similar in peak magnitude and

duration to the maximal one produced by Cu/A.
Steady-state Ca2+ influx was observed only at the
highest (1 mM) Cu2+ concentration; it was greatly
diminished in the presence of Put2+ and converted to
a net efflux in the presence of Spm4+ (Fig. 5C).
As compared with copper, iron is a 10 times more
abundant transient metal ion in pea roots, and it also
has a higher midpoint redox potential, so that even in
the absence of ascorbate it would be approximately
half reduced and capable, therefore, of converting
H2O2 to OH via the Fenton reaction (Becana and
Klucas, 1992; Biaglow et al., 1997; Kukavica et al.,
2009). Figure 6 shows that 0.5 mM Fe2+ provoked a substantial Ca2+ efflux and less pronounced K+ efflux.
These could be induced by Fe2+-catalyzed conversion of the intrinsic H2O2 to the OH . H2O2 alone does
not provoke any significant K+ or Ca2+ flux, but once
supplemented with 0.5 mM Fe2+, it caused a massive K+
efflux and Ca2+ influx.
d

Figure 5. Effects of Put2+ and Spm4+ on the OH -induced K+ and Ca2+

fluxes in pea roots as a function of Cu2+ concentration. K+ (A) and Ca2+
(B and C) fluxes are shown with 1, 0.1, and 0.01 mM CuCl2 (+0.1 mM
sodium ascorbate) only (white circles) or in a combination with 1 mM
Spm4+ (black squares) or Put2+ (white triangles). Ca2+ fluxes were
measured as the average response in the first 5 min after the application
of treatment (peak values in B) or 30 min after the application of
treatment (steady state in C). Arrows indicate the condition of zero Cu/A
added (1 mM Spm4+ or Put2+ only). Data are means 6 SE; n = 4 to
7 individual roots for each treatment.

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Zepeda-Jazo et al.

the current that developed in a response to ROS grew

up faster and reached a larger amplitude as compared
with the application of 1 mM Cu/A alone (Fig. 8B;
Supplemental Fig. S1). PAs (Put2+ and Spm4+) greatly
reduced the delay in the development of OH -induced
current and augmented two or four times, respectively,
the maximal current magnitude (Fig. 8C).
To check whether the OH -induced current conducts
Ca2+, we increased the bath CaCl2 concentration to 20
mM. If the current conducts cations (Ca2+ in this case)
better than anions (Cl2), one should expect an increase
of the inward current due to the increased Ca2+ influx,
without or with a little increase of the outward one. On
the contrary, if the current is carried mainly by anions,
an increased influx of Cl2 needs to be reflected by an
increase of the outward current. As can be seen from
the data presented in Figure 9, A and B, it was the
inward current that increased by up to 100% at 2160
mV, and this could only be explained by the fact that
OH -induced current mediates a substantial calcium
influx. However, the OH -induced current could also
conduct other cations, even as large as tetraethylammonium (TEA+; Fig. 9B). It should be noted that the
increase of the external salt (Ca2+ or TEA+ plus Cl2)
concentration had little effect on the reversal potential
of the OH -induced current, which was close to zero in
all cases. This behavior is consistent with a weak
preference between cations and anions for their entrance into the pore from either membrane side, indicating a small difference in their relative permeability.
Yet, cations appear to be conducted easier across the
membrane (i.e. they display a higher absolute permeability), reflected by a larger increment of the respective current, inward versus outward, upon the
increase of salt concentration in the bath (for the bases
of selective permeability, see Hille, 2001). To verify the
hypothesis of a dual cation and Cl2, permeability of
d

Figure 6. H2O2 alone is unable to induce K+ and Ca2+ fluxes in pea

roots. K+ (A) and Ca2+ (B) fluxes were recorded after the application of
5 mM H2O2 (white squares), 0.5 mM FeSO4 (white circles), or 5 mM
H2O2 after 0.5 mM FeSO4 (black circles). Data are means 6 SE; n = 5 to
6 individual roots for each treatment.

mally, this current became apparent at about 10 min

after the application of Cu/A and reached a steadystate level after 30 to 40 min of incubation (Fig. 8A).
The addition of 1 mM Put2+ or Spm4+ alone did not
evoke any current (data not shown). When 1 mM Put2+
or Spm4+ was added simultaneously with 1 mM Cu/A,

Figure 7. OH inhibits ionic currents constitutively expressed in pea root protoplasts. After the achievement and stabilization of
whole-cell configuration, a control record of currents as a response to a standard voltage steps protocol (depicted at the top) was
undertaken. Original records from the two distinct protoplasts are presented. A, Protoplast expressing outward-rectifying
potassium current. B, Another protoplast expressing a nonselective cation current. Bottom traces show currents recorded in the
same protoplast as above 3 min after the application of 1 mM Cu/A into the bath. Bath contained 5 mM and pipette contained 100
mM KCl; for detailed solution compositions, see Materials and Methods. Dashed lines indicate zero current levels.
2172

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Polyamines Potentiate OH -Induced Ion Fluxes

blocker quinine caused a significant block only at

higher (0.5 mM) concentration (Fig. 10). Verapamil and
quinine did not significantly affect outward currents,
implying a voltage-dependent block. No significant
(P , 0.05) effects on the magnitude of OH -induced
currents were found after the application of ruthenium
red (0.1 mM; n = 3), 4,4-diisothiocyanostilbene-2,2disulfonate (0.5 mM; n = 4), or amiloride (1 mM; n = 3).
d

DISCUSSION
d

OH But Not H2O2 Induces Nonselective Currents

Mediating K+ Efflux and Ca2+ Influx and Activates the
Ca2+ Pumping in the Root Mature Zone
d

OH was shown to activate nonselective cation channels, mediating Ca2+ influx protoplasts isolated from
different root zones (Demidchik et al., 2003; Foreman
et al., 2003). These channels are likely to be responsible
for the OH -induced Ca2+ influx and, at least partly, for
the K+ efflux measured in living roots (Demidchik et al.,
d

Figure 8. OH -induced ionic currents in pea root protoplasts. Typical

current records for two individual protoplasts, taken before the treatment and at different times after its application, are shown. A, Protoplast was treated with 1 mM Cu/A alone. B, Protoplast was treated with
1 mM Cu/A plus 1 mM Put2+. Ionic conditions are as in Figure 7C. PAs
potentiate the induction of ion currents by OH . Specific (pA pF21)
currents were measured at 2160 mV as a function of time with 1 mM
Cu/A alone (control; circles) in the presence of either 1 mM Put2+
(triangles) or Spm4+ (squares). Data are means 6 SE; n = 6 to 7
individual protoplasts assayed for each treatment.
d

the OH -induced currents, we substituted 100 mM KCl

in the pipette with 100 mM K-HEPES. This resulted in a
shift of the reversal potential to 234 mV. This value
was intermediate between EK and ECl. The observed
3-fold decrease of the inward current with no significant change of the outward one (Fig. 9C) reflects a
decrease in the Cl2 efflux, caused by its substitution
with HEPES. This experiment also shows that the OH induced current has little, if any, permeability to large
anions like HEPES. Although the OH -induced current
was sensitive to the cation channels blockers Gd3+,
nifedipine, and verapamil, the anionic channel pore
blockers niflumate and NPPB were also efficient (Figs.
9, A and D, and 10). The nonspecific cation channel
d

Figure 9. Ionic selectivity of the OH -induced currents in pea root

protoplasts. A, Typical recording of OH -induced currents measured
with a standard bath (5 mM KCl plus 2 mM CaCl2) at elevated (20 mM)
CaCl2 (n = 6) and after a subsequent application of Gd3+ (0.1 mM) to the
bath. B, Current-voltage relationships obtained for a standard bath
(circles; n = 10) or a bath containing 20 mM CaCl2 (squares; n = 6) or
TEA-Cl (diamonds; n = 5). C, Mean current-voltage relationship
obtained for a standard bath, but in the pipette 100 mM KCl was
substituted for 100 mM K-HEPES (triangles; n = 7). Equilibrium potentials for K+ and Cl2 for these conditions are indicated by arrows. For a
comparison, the current-voltage curve for standard (control) conditions
is redrawn from B. D, OH -induced current is sensitive to anionic
channel blocker. Ionic conditions are as in C. The dashed line indicates
the zero current level.
d

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Zepeda-Jazo et al.

increase of the inward current by 50% to 100% (between 2120 and 2160 mV; Fig. 9B). This implies that,
initially, the Ca2+ fraction was about 5% to 10% of the
total current, which is comparable to the relation
between K+ and Ca2+ flux magnitudes in MIFE experiments (Fig. 1). Such a relation is to be expected for a
nonselective channel, whose relative conductance for
different ions is determined by the relation of their
concentrations in experimental media, in this case,
more than 1 order of magnitude higher for K+ as
compared with Ca2+. Finally, the inhibition pattern of
the OH -induced current in patch-clamp experiments
(Fig. 10) was qualitatively similar to that obtained by
MIFE for the K+ efflux in intact roots (Fig. 2A), with
Gd3+ being the most potent blocker. One may note that
positively charged compounds, Gd3+ and verapamil,
were more potent in MIFE experiments on intact roots
than in patch-clamp experiments on isolated protoplasts. A similar quantitative difference was reported
before by Demidchik and coworkers (2003). This difference may be expected due to a lower ionic strength
in MIFE experiments, which tends to increase the
negative surface potential and the local concentration
of cations; surface potential also may be reduced due
to the removal of cell walls upon protoplast isolation.
In studies on plant cells, no selective blockers were
developed against any type of ion channel. Moreover,
some blockers that were previously considered to be
relatively selective (e.g. dihydropyridines such as nifedipine or phenylalkylamines such as verapamil,
which block voltage-dependent Ca2+-selective channels in animal cells) have different targets in plant
cells. These block several types of nonselective Ca2+permeable channels and even some outward-rectifying
K+ channels (Demidchik and Maathuis, 2007). Therefore, one may not rely on a single blocker and needs to
test a variety of broad-spectrum inhibitors.
The effects of TEA+, Gd3+, and verapamil on the
OH -induced current reported here were qualitatively
similar to those reported for the Arabidopsis root
mature epidermis (Demidchik et al., 2003), although
with somewhat lower affinity. An apparent lack of
cation/anion selectivity of the OH -induced current
forced us to test additionally some anionic pore
blockers; these were proved to be equally efficient
(Figs. 2 and 10). Yet, a stilbene derivative, 4,4-diisothiocyanostilbene-2,2-disulfonate, irreversibly modifying and inhibiting some anion channels and
transporters, was inefficient in our case. No significant
inhibition of OH -induced current was found for either
ruthenium red (a known inhibitor of Ca2+ uniporter
and a variety of Ca2+ and Ca2+-permeable channels) or
amiloride (an inhibitor of cation transporters and some
nonselective cation channels).
A poor ion selectivity of the OH -induced current
and its sensitivity to both cation and anion channel
inhibitors (Figs. 9 and 10) seems surprising at first
glance. Yet, there are multiple reports on plant PM
channels with a low cation-to-anion selectivity (for
review, see Demidchik et al., 2002; for experimental
d

Figure 10. Pharmacology of the OH -induced ion currents in pea root

protoplasts. Relative ion currents in the presence of Gd3+ (0.1 mM),
nifedipine (0.1 mM), verapamil (0.1 mM), quinine (0.5 mM), NPPB (0.1
mM), or niflumate (0.1 mM) in the bath are shown. Negative (black) and
positive (white) bars (means 6 SE; n = 38 protoplasts for each
treatment) correspond to currents measured at 2160 mV and +80
mV, respectively; control-specific currents (n = 25) at these potentials
were 229 6 1 and 12 6 1 pA pF21, respectively.

2003, 2010). To verify this relation, we have analyzed

the kinetics and pharmacology of OH -induced Ca2+
and K+ fluxes in pea roots, applying a noninvasive
MIFE technique. The results presented in Figures
1 and 2 suggest that there are at least two kinetic
components of the OH -induced fluxes: (1) a relatively
slow K+ efflux paralleled by approximately 20-fold
smaller Ca2+ influx; and (2) a rapidly developed Ca2+
efflux. This tentative component separation was further confirmed by a pharmacological analysis, as the
active Ca2+ efflux was sensitive to the Ca2+ pumpspecific inhibitors EY (Fig. 1B) and erythrosine B.
Fluorescein derivatives EY and erythrosine B also
inhibit the PM H+ pump, but at 1,000-fold higher
concentrations (De Michelis et al., 1993). On the other
hand, both supposedly passive processes, K+ efflux
and Ca2+ influx, were blocked by Gd3+, nifedipine,
verapamil, niflumate, or NPPB (Fig. 2). We suggested,
therefore, that both K+ efflux and Ca2+ influx are
mediated by a nonselective passive conductance. Several lines of evidence are consistent with this suggestion. First, the kinetics of the OH -induced current
measured in the whole-cell mode in patch experiments on individual root protoplasts was similar to
that for K+ efflux and Ca2+ influx in MIFE experiments
on intact roots. Second, the steady-state magnitude
of the outward (mainly carried by K+) OH -induced
current, 12 pA pF21 at +80 mV, is equivalent to 1,200
nmol m22 s21, fairly comparable to the OH -induced
K+ efflux reported by the MIFE technique (Fig. 1).
Third, a 10- fold increase of external Ca2+ provoked an
d

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Polyamines Potentiate OH -Induced Ion Fluxes

evidence, see Zepeda-Jazo et al., 2008). When it comes

to OH -induced currents in the root PM, it should be
noted that they were never rigorously tested for cation
over anion selectivity, as these currents were only
assayed under pseudosymmetric ionic conditions, in
the absence of steep salt gradients across the membrane. Under these ionic conditions, reversal potential
values were close to zero, which was a compromise
between equilibrium potential values for Cl2, monovalent, and divalent cations (Foreman et al., 2003). This
observation might imply a significant anion permeability of the OH -induced currents, not only a weak
discrimination between different cations. Indeed, under the ionic conditions of the experiment presented in
Figure 9C, the reversal potential of the whole-cell
current was substantially different from zero, but
again, it was in the middle between equilibrium potentials for K+ and Cl2. Besides, substitution of the 90%
of intracellular Cl2 with a large nonpermeable anion
(HEPES) unraveled a contribution of the Cl2 influx to
the inward current, which dramatically decreased
without a significant change of the outward one. On
the contrary, the H2O2-activated Ca2+-permeable channels in guard cells and the root elongation zone
display a somewhat better selectivity among cations
(e.g. lacking permeability for TEA+) and a clear preference of cation over Cl2 (Pei et al., 2000; Demidchik
et al., 2007). As shown previously (Demidchik et al.,
2007) and in this paper (Fig. 6), H2O2-activated channels are not present in the mature root zone, at least
in the plant species studied. Thus, two different ROS
species, OH and H2O2, activate distinct Ca2+-permeable
channels in the root PM.
d

Physiological Concentrations of Copper and Iron Can

Catalyze OH Production, Which Induces a Substantial
K+ Leak
d

The ion fluxes and currents discussed so far were

induced by high (1 mM) Cu/A concentration. Although even higher Cu/A concentrations were used
to demonstrate the role of OH in cell wall loosening
and stretching during the elongation process (Schopfer,
2001) and equivalent concentrations were applied for
the activation of Ca2+-permeable channels in roots
(Demidchik et al., 2003; Foreman et al., 2003), the
effects of lower Cu/A concentrations need to be studied to reveal the thresholds for OH -activated currents.
Demidchik and coworkers (2010), using long-living
OH -specific spin-trap 5,5-dimethyl-1-pyrroline-Noxide, have shown that production of OH by 1 mM
Cu/A in pure solution without plants was approximately equivalent to that produced by intact Arabidopsis roots subjected to 100 mM NaCl. However, in
the presence of living roots naturally producing H2O2,
the generation of OH induced by 1 mM Cu/A increased severalfold and was equivalent to 3- or 5-fold
of its production upon the application of 250 or 100 mM
NaCl, respectively. Extreme (several hundred micro-

moles) and lethal OH production occurs in illuminated photosynthetic tissues treated with herbicides
(paraquat) or upon the inhibition of ascorbate peroxidase (Babbs et al., 1989; Burkhard and Heber, 1996).
However, even a much lower physiological (10 mM)
concentration of copper reduced by ascorbate is able to
generate 2 mmol of OH within 10 min (Biaglow et al.,
1997). In biological systems, not only total OH generation but also the location of the production site with
respect to the target molecules and scavenging mechanisms existing within this locality is important. Our
data imply that at up to 0.1 mM Cu/A, only small K+
efflux resulted from the mature zone of pea roots
(Fig. 5A). However, this efflux was strongly (up to
1 order of magnitude) potentiated by PAs, reaching
100 to 200 nmol m22 s21 (see discussion of the mechanism of PA action below). On the other hand, iron
(another redox-active transient metal capable of
generating OH via the Fenton reaction) is present at
pea cell walls at 10-fold higher concentrations than
copper, mainly as a part of peroxidase reaction centers
(Becana and Klucas, 1992; Kukavica et al., 2009).
Moreover, on an equimolar iron basis, the peroxidase
iron is a 1 to 2 orders of magnitude better Fenton
reaction catalyst as compared with the inorganic iron
or Fe-EDTA (Chen and Schopfer, 1999). In our experiments, inorganic iron alone at 0.5 mM concentration
provoked a relatively large (up to 100 nmol m22 s21) K+
efflux, which grew severalfold upon the application of
millimolar H2O2 (Fig. 6A). For a comparison, K+ efflux
from the root mature zone, induced by 50 to 100 mM
NaCl, was (in nmol m22 s21) 100 to 200 for Arabidopsis, 100 for maize (Zea mays; Pandolfi et al., 2010), 100
to 400 for barley (Hordeum vulgare) varieties different
in their salt tolerance (Chen et al., 2007), and about 50
on average for different wheat (Triticum aestivum)
cultivars (Cuin et al., 2008). Therefore, physiological
concentrations of copper (supplemented by PAs) and
iron are capable of catalyzing OH production sufficient to induce quite a significant K+ leak from pea
roots persisting tens of minutes.
d

PAs Potentiate the OH -Induced PM

Passive Conductance
d

The kinetics of the OH -induced cation currents and

K fluxes across the PM were further modulated by
PAs. PAs are present at high, submillimolar to millimolar, concentrations in pea roots. Spm4+ and Spd3+
are more abundant near root apices, whereas Put2+
is almost equally distributed between the root base
and apex (Shen and Galston, 1985). When it comes to
the known effects of PAs on passive membrane conductance, only the inhibitory effects, either direct or
indirect, on K+-selective and nonselective cation channels were documented for plants so far (Bruggemann
et al., 1998; Dobrovinskaya et al., 1999a, 1999b; Liu
et al., 2000; Shabala et al., 2007; Zhao et al., 2007). Thus,
beneficial roles of PAs during stresses were discussed
+

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Zepeda-Jazo et al.

in this context. As an example, the PA contribution to

salt stress tolerance in plants was explained on the
basis of their inhibition of Na+ influx, reducing the
consequent membrane depolarization and K+ efflux
(Shabala et al., 2007; Zhao et al., 2007; Zepeda-Jazo
et al., 2008). However, our recent study (Pandolfi et al.,
2010) showed that PAs, in particularly Spm4+, depending on the growing condition and root zone, can
reduce NaCl-induced K+ efflux, cause no change, or
even stimulate it. Clearly, for explanations of the
effects of externally applied PAs in vivo, one needs
to consider not only the effects of PAs per se but also of
PA metabolization associated with ROS generation as
well as their roles in ROS scavenging as early effects.
In particular, all natural PAs are powerful OH scavengers, at concentrations above 1.5 mM eliminating
virtually all OH produced by a mixture of 0.2 mM
H2O2 with 0.04 mM iron (Das and Misra, 2004). On the
other hand, oxidation of Spm4+ and Spd3+ by polyamine oxidase, and Put2+ by diamine oxidase, leads to
the formation of H2O2 (Moschou et al., 2008b), which
could be further converted to OH . External application of PAs to roots mimics their export to apoplast,
which occurred in response to different environmental cues, and is associated with a stimulation of Ca2+
influx by ROS, generated as a result of PA oxidation
(An et al., 2008; Moschou et al., 2008a; Ozawa et al.,
2009; Wu et al., 2010). In addition to the Ca2+ influx,
overall PM leakage could increase, also leading to K+
loss. Thus, catabolization of PAs and ROS generation may outweigh their roles as ROS scavengers,
activators of the antioxidant enzymes, and microsomal NADPH oxidase inhibitors (Papadakis and
Roubelakis-Angelakis, 2005; Mohapatra et al., 2009;
Takahashi and Kakehi, 2010). Increase of ROS production due to PA catabolization may indeed take
place in intact pea roots. Indirect evidence for this is
that Put2+ alone provoked a much higher K+ efflux as
compared with Spm4+ (Fig. 5A), which may reflect a
much higher apoplast expression of diamine oxidase
as compared with polyamine oxidase, in the apoplast
of dicots, particularly pea (Moschou et al., 2008b). The
contribution of such a mechanism should be ruled out,
however, in the case of isolated protoplasts, because
apoplastic amine oxidases are washed out upon the
protoplast isolation procedure (Kaur-Sawhney et al.,
1981). Besides, we have not observed any membrane
current stimulation by treatment with PAs alone. Still,
PAs may act as OH scavengers, but this could only
preclude or handicap the development of OH induced ionic currents. Therefore, exogenous PAs
could stimulate the induction of ionic currents in
protoplasts by OH only if PAs are acting as cofactors
in this process. To the best of our knowledge, this
possibility has not been considered in the literature so
far. However, it was reported that PAs may stabilize
the binding of other cofactors essential for the ion
channel activity, such as PIP2 for Kir and plant Shaker
K+ channels (Liu et al., 2005a; Xie et al., 2005). Thus, the
precise biophysical mechanisms of this synergism
d

2176

between PAs and ROS interaction warrant a separate

investigation.
PAs and OH Activate Ca2+ Pumping in Intact Roots
d

Another intriguing finding of this study is the activation of active Ca2+ efflux across the root epidermis
by OH and PAs (Figs. 1B, 4, and 5). This flux component has a substantially lower threshold for its activation by OH as compared with the K+ efflux (Fig. 5,
A and B). Stimulation of the active EY-sensitive Ca2+
efflux across the PM by ROS was reported in a single
study on an Arabidopsis cell culture (Romani et al.,
2004). It was shown that ROS generated by oligogalacturonide treatment assisted Ca2+-ATPase activation
by calmodulin. However, in that paper, ROS induced
net Ca2+ influx first, and net Ca2+ efflux was measured
only in 10 to 20 min; this is exactly the opposite order
compared with the OH -induced Ca2+ flux kinetics in
our work (Figs. 1B and 4). On the other hand, there is a
large body of data on animal PM and sarcoplasmic
reticulum Ca2+ pumps showing their inhibition by
ROS, which may originate from protein cross-linking,
lipid peroxidation, and concurrent inhibition by oxidized forms of the calmodulin (for review, see Waring,
2005). These effects develop slowly (hours), which
may preclude their observation under the conditions
of our study.
At the same time, Put2+ and Spm4+ evoked Ca2+
efflux that was very similar to that induced by OH .
One possibility is that this efflux was actually caused
by ROS, generated during PA catabolization. Alternatively/additionally, PAs in principle can activate PM
Ca2+-ATPase in one of the ways they affect another
P-type pump, H+-ATPase. These involve the PAinduced promotion of the interaction of 14-3-3 proteins
with the autoinhibitory domain at the H+-ATPase C
terminus (Garufi et al., 2007) and the activation of the H+
pump via a nitric oxide-dependent pathway (Tun et al.,
2006; Arasimowicz-Jelonek et al., 2009; Zandonadi et al.,
2010). Finally, if PAs activate the H+ pump, they could
cause the stimulation of Ca2+ pumping across the PM
via a coupled mechanism, as PM Ca2+-ATPase exports
two Ca2+ ions in the exchange for the two imported H+
ions (Beffagna et al., 2005).
d

Implications of PA and OH Effects on Intracellular

Ca2+ Homeostasis

Irrespective of the precise mechanism of ROS and

PA early effects on Ca2+ fluxes across the root epidermis PM, some consequences for Ca2+ homeostasis
and signaling could be drawn from our experimental data. Moderate OH production results in a transient increase of Ca2+ pumping, outweighing the
OH -induced passive Ca2+ influx (Fig. 5, B and C).
The resultant depletion of the intracellular Ca2+ pool
should reduce the Ca2+-dependent PM NADPH oxidase activity in a feedback manner (Takeda et al.,
2008), thus diminishing ROS generation. Membraned

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Polyamines Potentiate OH -Induced Ion Fluxes

bound NADPH oxidase is an electrogenic enzyme that

transports an electron from the interior to the exterior
of the cell upon oxygen reduction, and it is stimulated
by membrane hyperpolarization. The latter may be
generated by an increased PM pump activity. As an
example, activation of the H+-ATPase by fusicoccin
results in higher OH production (Liszkay et al., 2004).
However, activation of the nonselective ion conductance by OH (Fig. 9) would tend to depolarize the
membrane, although opposed by enhanced H+ and
Ca2+ pumping as a response to the depolarization. The
PM H+ pump is also activated by hyperosmotic stress
or salt. This increases cytosolic pH, which leads to a
lower synthesis of NADPH, thus decreasing its availability for NADPH oxidase (Beffagna et al., 2005).
And, as mentioned in a previous section, the H+ pump
may be activated by PAs. As Ca2+-ATPase operates as a
1:1 Ca2+:H+ exchanger, it might be of secondary relevance which enzyme, H+- or Ca2+-ATPase, is the
primary target for OH / PA, while the activity of
both pumps will be mutually coupled via the H+
circuit. Thus, most likely, under conditions of moderate OH production, the joint activity of H+ and Ca2+
pumps would tend to diminish ROS production by
the PM NADPH oxidase. Oscillations of intracellular
H+ and Ca2+, in parallel with oscillations of ROS
production levels, may also be expected under these
conditions.
At strong oxidative stress, here experimentally provoked by 1 mM Cu/A, in the absence of PAs, net Ca2+
influx was observed at steady state (after 30 min
of incubation; Figs. 1B and 5C). However, on the
background of PAs (Figs. 4 and 5C), it is either largely
diminished (Put2+) or reverted to an efflux (Spm4+).
The direction of the net Ca2+ flux at any time is defined
by the sign of the algebraic sum of Ca2+ efflux and
influx, which are generated by distinct PM transport
systems. As can be seen from Figures 3 and 8C, at
longer incubation times, Put2+ stimulates the OH induced passive K+ efflux (and, presumably, also the
associated Ca2+ influx) more than Spm4+ does. At the
same time, active Ca2+ efflux observed in the presence
of Put2+ and Spm4+ is approximately equal (Fig. 5B).
Thus, at longer incubation times, one may expect a
relatively higher net Ca2+ efflux in the presence of
Spm4+ as compared with Put2+, which is indeed the
case (Fig. 4). Thus, the ratio between different PAs is
important to define the direction of a net Ca2+ flux
across the PM. In the pea root mature zone, Put2+ is
accumulated at a higher concentration than the sum
of Spd3+ and Spm4+ concentrations (Shen and Galston,
1985), although in the apoplast of pea and other dicots,
the activity of diamine oxidase dominates over the
activity of polyamine oxidase (Moschou et al., 2008b).
PA concentration patterns are known to be not only
species, tissue, and age specific but also stress specific
(Alcazar et al., 2010). As an example, salt stress shifts
the distribution in favor of higher PAs, Spm4+ and
Spd3+, at the expense of Put2+, even though the total
PA concentration changes are not very dramatic. Yet,
d

plants opposing this tendency (i.e. maintaining a

higher Put2+/Spm4+ ratio) turn out to be more salt
tolerant and, importantly, to better accumulate cations, including Na+, as required for the osmotic
adjustment (Zapata et al., 2008). Among other explanations, this might be related to the fact that Put2+
could support a longer lasting potentiation of the OH induced passive conductance (Fig. 3A) and could also,
in contrast to Spm4+ and Spd3+, allow some net Ca2+
influx at steady state (Fig. 4).
Summarizing, our study demonstrates that lower
OH concentrations induce Ca2+ pumping, while higher
OH levels activate also a passive Ca2+ uptake across the
root PM. The overall direction of the net Ca2+ flux is
further modulated by natural PAs in a species-specific
manner. Thus, stress-induced changes in OH production and in the levels of different PAs may be translated
into intracellular Ca2+ changes.
d

MATERIALS AND METHODS

Plant Material
Pea (Pisum sativum Greenfeast) seeds were surface sterilized (full-strength
commercial bleach for 30 min) and thoroughly rinsed with distilled water.
Seeds were germinated in a dark growth cabinet at +24C in two layers of wet
paper in petri dishes for 2 to 3 d. Uniformly germinated seedlings were
selected and transferred to a bubbled hydroponic culture unit comprising a
3-L plastic container over which seedlings were suspended on a plastic grid so
that their roots were almost completely immersed in the growth solution (0.5
mM KCl and 0.1 mM CaCl2). Aeration was provided by one aquarium air pump
via flexible plastic tubing. Seedlings were grown under constant (+24C)
conditions in a lighted growth cabinet until 5 d old. Roots of 8 to 10 cm long
were used for current and flux measurements.

Measurements of Ion Fluxes

Net K+ and Ca2+ fluxes were measured using the noninvasive MIFE
technique (University of Tasmania innovation). The principles of the MIFE
measurements, microelectrode fabrication, and calibration are available in
previous publications (Shabala et al., 2001, 2007), and the theory of the MIFE
measurements is available elsewhere (Newman, 2001; Shabala et al., 2006a).
During experiments, pea seedlings were placed in 30-mL measuring chambers. Their roots were immobilized in a horizontal position as described
elsewhere (Cuin and Shabala, 2005) and preincubated in a new solution
containing 0.5 mM KCl, 0.1 mM CaCl2, 5 mM MES, and 2 mM Tris base, pH 6.0,
for 1 h. Normally, two ion-selective microelectrodes, one for K+ and another
for Ca2+, were used in the same experiment. During measurements, electrodes
were moved between positions M1 and M2, 50 and 150 mm from the root
surface, respectively,
in the mature zone (approximately 1520 mm from the
d
tip). OH was generated by the addition of CuCl2 (0.011 mM) plus sodium
ascorbate (0.11 mM) into the chamber. PAs (Spm4+, Spd3+, and Put2+) were
added simultaneously with Cu/A mixture, or individually. Net K+ and Ca2+
fluxes were measured for over 40 min after the treatment. Different blockers
were added 10 min before the Cu/A treatment (EY, erythrosine B, Gd3+) or 30
min later (Gd3+, nifedipine, NPPB, niflumate) as indicated.

Isolation of Pea Protoplasts from the Root Mature Zone

The isolation of root protoplasts was developed based on the previously
described protocols used for mesophyll protoplasts (Demidchik and Tester,
2002; Shabala et al., 2006b) using the same modifications as described by Chen
et al. (2007). Exodermal cylinders of root segments of pea seedlings, after
mechanical separation of steles, were placed into 3 mL of the enzyme solution
containing 2% (w/v) cellulase (Yakult Honsha), 1.2% (w/v) cellulysin
(Calbiochem), 0.1% (w/v) pectolyase, 0.1% (w/v) bovine serum albumin, 10
mM KCl, 10 mM CaCl2, and 2 mM MgCl2, pH 5.7, adjusted with 2 mM MES, and

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Zepeda-Jazo et al.

osmolality was adjusted to 750 mosmol with sorbitol. After 40 min of

incubation in the enzyme solution (in the dark at 30C; agitated on a 90-rpm
rotary shaker), root segments were transferred to the so-called wash solution
(as above, minus enzymes) and thoroughly washed for another 2 min.
Segments were then transferred into the measuring chamber filled with a
solution containing 5 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2, and 2 mM MESKOH, pH 5.7, with osmolality of 650 mosmol adjusted by D-sorbitol. By gently
shaking, protoplasts were released into the measuring chamber used for
patch-clamp experiments (see next section). Once the protoplasts were released, we washed them with EDTA-bath solution (the same as release-bath
solution plus 5 mM EDTA) to clean the cell membrane surface and then
washed them again with release-bath solution alone. The origin of released
protoplasts was verified by size distribution, as within a mature root zone the
average cortical cell is about twice as large in diameter as an epidermal one
(Rost et al., 1988). The mean diameter for protoplasts used in patch-clamp
experiments was 11.5 mm, which coincided with a position of a peak
corresponding to a fraction of the smallest protoplasts in our preparation
(mean diameter of 11.2 mm, close to the value reported for pea protoplasts
from the root epidermis); due to some overlapping of peaks corresponding to
different protoplast fractions (Supplemental Fig. S2), one may not exclude
some contribution of cortical protoplasts into those studied by patch clamp,
with a probability of more than 60% that the patched protoplasts originated
from the epidermis.

Patch-Clamp Experiments
Measurements on individual pea root protoplasts were made using an
Axopatch 200 patch-clamp amplifier (Axon Instruments) in the conventional
whole-cell configuration as described by Shabala et al. (2006b). The basic
pipette solution contained (in mM) 100 KCl, 3 MgCl2, 0.8 CaCl2, 2 K2EGTA, and
5 HEPES-KOH, pH 7.4, osmolality of 650 mosmol adjusted with D-sorbitol.
Membrane potentials were clamped at 2100 mV throughout the experiments,
and voltage pulses were applied in 20-mV steps, from 2140 or 2160 mV to
+40 or +80 mV. A typical access resistance was between 12 and 30
MV, compensated by 60% to 70% using the Axopatch 200 compensation
circuit, and the whole-cell capacitance
was in the range between 2 and 6 pF. To
d
stimulate the production of OH , 1 mM CuCl2 and sodium ascorbate were
mixed directly in the experimental chamber. PAs (Spm4+ or Put2+) were added
directly into the bath solution to give a final concentration of 1 mM. In
pharmacological assays, the inhibitors Gd3+ (0.1 mM), nifedipine (0.1 mM),
verapamil (0.1 mM), quinine (0.5 mM), amiloride (1 mM), ruthenium red (0.1
mM), and NPPB and niflumate (both 0.1 mM) were added directly into the
bath. In selectivity measurements, external solutions with 20 mM CaCl2 or
TEA-Cl were applied by bath perfusion. Anion (Cl2) permeability was
verified by substitution of 100 mM Cl2 in the patch pipette with 100 mM
K-HEPES (100 mM KOH plus 244 mM HEPES, pH 7.4). In the latter case, the
necessary correction for liquid junction potential between the pipette and the
bath was made using the JPCalc program by P.H. Barry (University of New
South Wales). All chemicals were of analytical grade, purchased from Sigma.

Supplemental Data
The following materials are available in the online version of this article.
d

Supplemental Figure S1. Development of the OH -induced current is

accelerated in the presence of spermine.
Supplemental Figure S2. Smallest, mostly of epidermal origin, protoplasts
are selected for patch-clamp assays.
Received May 6, 2011; accepted October 3, 2011; published October 6, 2011.

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