Download Receptor-Like Activity Evoked by Extracellular ADP in Arabidopsis

Receptor-Like Activity Evoked by Extracellular ADP in
Arabidopsis Root Epidermal Plasma Membrane1
Vadim Demidchik, Zhonglin Shang, Ryoung Shin, Renato Colaço, Anuphon Laohavisit,
Sergey Shabala, and Julia M. Davies*
Department of Biology, University of Essex, Colchester CO4 3SQ, United Kingdom (V.D.); College of Life
Science, Hebei Normal University, Shijiazhang 050016, Hebei, China (Z.S.); Riken Plant Science Center,
Yokohama City, Kanagawa 230–0045, Japan (R.S.); Department of Plant Sciences, University of Cambridge,
Cambridge CB2 3EA, United Kingdom (R.C., A.L., J.M.D.); and School of Agricultural Science, University of
Tasmania, Hobart, Tasmania 7001, Australia (S.S.)
Extracellular purine nucleotides are implicated in the control of plant development and stress responses. While extracellular
ATP is known to activate transcriptional pathways via plasma membrane (PM) NADPH oxidase and calcium channel
activation, very little is known about signal transduction by extracellular ADP. Here, extracellular ADP was found to activate
net Ca2+ influx in roots of Arabidopsis (Arabidopsis thaliana) and transiently elevate cytosolic free Ca2+ in root epidermal
protoplasts. An inward Ca2+-permeable conductance in root epidermal PM was activated within 1 s of ADP application and
repeated application evoked a smaller current. Such response speed and densitization are consistent with operation of
equivalents to animal ionotropic purine receptors, although to date no equivalent genes for such receptors have been identified
in higher plants. In contrast to ATP, extracellular ADP did not evoke accumulation of intracellular reactive oxygen species.
While high concentrations of ATP caused net Ca2+ efflux from roots, equivalent concentrations of ADP caused net influx.
Overall the results point to a discrete ADP signaling pathway, reliant on receptor-like activity at the PM.
Extracellular purine nucleotides have been shown
to be involved in the regulation of plant cell viability,
membrane permeability, immunity, symbiosis, stress
responses, and growth (Lew and Dearnaley, 2000; Tang
et al., 2003; Chivasa et al., 2005, 2009; Kim et al., 2006,
2009; Roux and Steinebrunner, 2007; Wu et al., 2007;
Riewe et al., 2008a; Wu and Wu, 2008; Yi et al., 2008;
Demidchik et al., 2009; Govindarajulu et al., 2009; Kim
et al., 2009; Clark et al., 2010a, 2010b; Tanaka et al.,
2010a, 2010b; Terrile et al., 2010; Tonón et al., 2010).
Purine nucleotide release from plant cells may occur
through wounding, exocytosis, or through the activity
of plasma membrane (PM) ATP-binding cassette transporters (Thomas et al., 2000; Kim et al., 2006). Growth
points are hotspots of extracellular ATP (Kim et al.,
2006) while touch-stimulated ATP release is mediated
by heterotrimeric G-protein activity (Weerasinghe et al.,
2009). Levels of extracellular ATP and ADP can be regulated by the concerted action of ecto-apyrases, phos1
This work was supported by the Leverhulme Trust (grant no.
F/09 741/C), the Biotechnology and Biological Sciences Research
Council, the University of Cambridge Frank Smart Trust and
Brookes Fund, the Royal Society (China Incoming Fellowship), the
Australian Research Council, and The Monsanto Company.
* Corresponding author; e-mail [email protected].
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:
Julia M. Davies ([email protected]).
www.plantphysiol.org/cgi/doi/10.1104/pp.111.174722
phatases, and adenosine nucleosidases with retrieval of
adenine (the ultimate hydrolytic product) by PM purine
permeases (Komoszyński, 1996; Steinebrunner et al.,
2003; Wu et al., 2007; Riewe et al., 2008a, 2008b;
Govindarajulu et al., 2009; Clark et al., 2010b; Liang
et al., 2010). Manipulation of ecto-apyrase levels results in abnormal growth in Arabidopsis (Arabidopsis
thaliana), cotton (Gossypium hirsutum), and potato (Solanum tuberosum; Wu et al., 2007; Riewe et al., 2008a;
Clark et al., 2010a, 2010b) while nodulation by Bradyrhizobium japonicum is impaired in ecto-apyrase-deficient
soybean (Glycine max; Govindarajulu et al., 2009).
Current models suggest that poise of the cell’s state
between death, stress adaptation, and growth involves
signaling governed by the level of extracellular ATP
(Chivasa et al., 2005, 2009; Roux and Steinebrunner,
2007; Wu et al., 2007; Wu and Wu, 2008; Kim et al.,
2009; Clark et al., 2010a, 2010b; Terrile et al., 2010;
Tonón et al., 2010).
The mechanism of purine nucleotide perception is
unclear as higher plant genomes do not encode for
homologs of animal PM purinoceptors (Kim et al.,
2006; Fountain et al., 2008). The latter are either coupled to heterotrimeric G proteins or are ionotropic
receptors (activated by nanomolar or even 0.1 mM
ATP) that permit Ca2+ influx to transduce the purine
nucleotide signal (for review, see Khakh and North,
2006). It remains possible that plants evolved structurally different systems for purine sensing to animals
but antagonists of animal purinoceptors are effective
against plant extracellular ATP responses. Despite the
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Demidchik et al.
apparent absence of classical purinoceptors in higher
plants, extracellular purine nucleotides can cause transient elevations of cytosolic free Ca2+ ([Ca2+]cyt), in
common with animals (Demidchik et al., 2003a, 2009;
Jeter et al., 2004; Tanaka et al., 2010b). In Arabidopsis roots, extracellular ATP most likely triggers intracellular Ca2+ release, resulting in stimulation of PM
NADPH oxidase activity (Demidchik et al., 2009). The
resultant reactive oxygen species (ROS) activate a PM
Ca2+ influx pathway that governs a transcriptional
response. In Arabidopsis root hairs, extracellular ATP
also causes intracellular ROS accumulation by activating NADPH oxidases (Clark et al., 2010b). Leaves also
support extracellular ATP-induced transcription via
PM NADPH oxidases (Song et al., 2006). Nitric oxide
is now also established as a product of extracellular
purine nucleotide perception. Nitric oxide evolution
by plants has been observed on application of extracellular ATP or extracellular ADP and linked to influx
of Ca2+ or production of phosphatidic acid (Foresi
et al., 2007; Wu and Wu, 2008; Reichler et al., 2009;
Clark et al., 2010b; Sueldo et al., 2010; Tonón et al.,
2010).
In this study, the effects of extracellular ADP on ion
transport at the plant PM have been examined. Extracellular ADP elevates [Ca2+]cyt in both roots and leaves
of Arabidopsis and it can be equally or more effective
than extracellular ATP (Demidchik et al., 2003a, 2009;
Jeter et al., 2004; Tanaka et al., 2010b). In common with
extracellular ATP, extracellular ADP can elicit oscillations of [Ca2+]cyt in Arabidopsis seedlings (Tanaka
et al., 2010b). At the physiological level, extracellular
ADP stimulates nodule development (Govindarajulu
et al., 2009) and was shown to stimulate root hair
elongation (Lew and Dearnaley, 2000). In common
with extracellular ATP, it is now known that extracellular ADP has a biphasic effect on cotton fiber and root
hair elongation with high concentrations being inhibitory (Clark et al., 2010a, 2010b). Arabidopsis root
epidermal cells have been used here as these are well
characterized electrophysiologically and are among
the first cell types to sense the pathogen attack and
environmental change that could involve purine signaling. By using a fast-perfusion system in patch-clamp
electrophysiology experiments, extracellular ADP has
been delivered to the extracellular PM face of protoplasts within 1 s, permitting the fastest resolution to
date of rapid activation of plant ion transport events
by an extracellular purine nucleotide. These events appear to be independent of heterotrimeric G proteins.
Epidermal protoplasts expressing the [Ca 2+ ] cyt reporter aequorin have been used to explore further the
ability of extracellular ADP to evoke [Ca2+]cyt elevation.
In further contrast to extracellular ATP, extracellular
ADP does not elicit intracellular ROS accumulation.
Using whole roots, it has also been possible to resolve
extracellular ADP-induced net Ca2+ and K+ fluxes; high
concentrations of ATP but not ADP evoke net Ca2+ efflux.
Overall the results show rapid activation of PM Ca2+—
and K + —conductances by extracellular ADP, with
initial events supported by PM ionotropic receptorlike activity. The differential responses of intracellular
ROS and net Ca2+ flux to extracellular ADP and extracellular ATP could provide the basis for distinct
signaling outcomes.
RESULTS
Extracellular ADP Activates a PM Ca2+
Influx Conductance
To establish whether the PM could respond to extracellular ADP, protoplasts from mature epidermal root
cells were assayed using the whole-cell patch-clamp
configuration to resolve activity of channel populations
as a function of applied membrane voltage and extracellular agonist. Application of 20 mM extracellular ADP
to protoplasts (using conventional peristaltic pump
delivery to the recording chamber) transiently activated
a hyperpolarization-activated (i.e. activated by increasingly negative voltage) Ca2+-permeable conductance
(Fig. 1A), very similar to that characterized in response
to 20 mM extracellular ATP in this cell type (Demidchik
et al., 2009). The concentration used is representative of
wounding levels (Song et al., 2006). The increase in
inwardly directed current at hyperpolarized (inside
negative) voltage was observed 1 to 3 min after extracellular ADP addition (a similar time course to the extracellular ATP response reported by Demidchik et al.,
2009; Shang et al., 2009) and was evident over the
physiological voltage range for this cell type (Maathuis
and Sanders, 1993). Mean 6 SE current-voltage (IV)
relationships are shown in Figure 1B; negative voltages
are physiologically relevant. At 2200 mV, extracellular
ADP induced a peak mean 6 SE current increase from
2163 6 36 pA to 2382 6 63 pA at 2200 mV (n = 4);
current then declined over a 5 to 15 min period.
Negative current signifies influx of positive charge into
the cytosol or efflux of negative charge. The peak current response is not significantly different (Student’s
t test) to that found for 20 m M extracellular ATP
(Demidchik et al., 2009).
Rapid, Reversible, and Desensitizing Response to
Extracellular ADP at the PM
Having found that the PM was electrically responsive
to extracellular ADP on slow perfusion and long-term
exposure, a fast-perfusion system was used to deliver
extracellular ADP to the PM within 1 s. In contrast to
experiments using peristaltic pump delivery of the
agonist to the recording chamber (with a waiting time
for flow from the chamber edge to the protoplast), the
fast-perfusion system used a primed multichannel delivery pipette placed near the protoplast (described
further in “Materials and Methods”). In these experiments, the pipette solution (PS; equivalent to the cytosol) contained a nonhydrolyzable GDP analog (0.5 mM
GDPbS) to minimize G-protein-mediated channel activation (Li and Assmann, 1993). In preliminary experi-
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Receptor-Like Activity Evoked by Extracellular ADP
configuration was disturbed or lost by distortion of the
protoplast. Given the high failure rate, a higher concentration of extracellular ADP (0.3 mM) was used in
these trials to ensure activation. In the remaining 15
trials in which seal resistance was unaffected by fast
perfusion, extracellular ADP evoked a rapid and reversible change in PM conductance (Fig. 2A). With the
PM clamped at a holding potential (HP) of 290 mV,
fast perfusion with control bathing solution (BS) was
without effect (Fig. 2A, top trace). Addition of 0.3 mM
extracellular ADP caused a downward current deflection, consistent with entry of positive charge into the
protoplast (Fig. 2A, middle trace). Current activation
occurred immediately after exposure to extracellular
ADP. Fast removal of extracellular ADP resulted in
immediate recovery to control conditions. As can be
seen from the middle trace of Figure 2A, a repeated
application of 0.3 mM extracellular ADP to the same
protoplast again caused rapid and reversible current
activation. However in this case, recovery to baseline
current began in the presence of extracellular ADP.
This was typical of all protoplasts where extracellular
ADP activation was observed.
Figure 1. Extracellular ADP activates a PM influx conductance in root
epidermal protoplasts. A, Effect of 20 mM extracellular ADP on wholecell currents from a representative protoplast, recording conditions
were as used previously by Demidchik et al. (2009). B, Mean 6 SE IV
relationships of control and peak ADP peak response (n = 4). Control BS
was as described in “Materials and Methods” (20 mM CaCl2, 0.1 mM
KCl, and Na+ equimolar to test BS, pH 6). PS comprised (mM): 40
K-gluconate, 10 KCl, 1 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’tetraacetic acid (free Ca2+ adjusted to 100 nM with 0.4 CaCl2), pH 7.2
adjusted with Tris/MES.
ments, this increased the K+ efflux current, as described
by Li and Assmann (1993), indicative of effective
G-protein inhibition. It has been proposed that extracellular purine nucleotides can relay a signal via heterotrimeric G proteins in plants (Song et al., 2006; Roux
and Steinebrunner, 2007; Wu and Wu, 2008), so their
inhibition could delineate possible direct extracellular
ADP effects on a receptor-like channel. Experiments were
performed within the first 20 to 30 min of recording so
that extracellular ADP-activated currents could be distinguished from the time-dependent development
of the constitutive hyperpolarization-activated Ca2+permeable conductance. The latter becomes the dominant
Ca2+ conductance in this cell type after approximately
1 h (Demidchik et al., 2002).
Fast perfusion proved problematic as in approximately 80% of trials (65 out of 85), the recording
Figure 2. Rapid activation of epidermal PM cation conductances by
extracellular ADP. The results are from whole-cell recordings on
protoplasts from the root mature epidermis. A, Top trace: two sequential applications of control BS (green bars) to a protoplast from the
mature epidermis failed to elicit a current; HP across the PM was 290
mV inside negative. Middle trace (same protoplast): two sequential
applications to the same protoplast of 0.3 mM ADP (red bars) by fast
perfusion evoked inwardly directed current (downward deflection).
Baseline current was recovered on removal of the ADP stimulus.
Bottom trace (different protoplast); inhibiting effect of 0.3 mM Gd3+,
added together with ADP in the second application. BS was as in Figure
1. Test BS included 0.3 mM NaADP 6 0.3 mM GdCl3 as indicated. PS
included 0.5 mM GDPbS. B, Current in response to voltage ramp (40-s
long started from +100 mV) recorded from a single protoplast, approximately 1 min after the addition of control BS (green), 0.3 mM ADP (red),
or 0.3 mM ADP with 0.3 mM Gd3+ (blue). Solutions as in A.
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Demidchik et al.
Mean 6 SE values of extracellular ADP-evoked
currents at HP = 290 mV are given in Table I. In
experimental set A, the first application of 0.3 mM
extracellular ADP (event 1) caused a mean 89% increase in inwardly directed current. After removal of
the stimulus and recovery to baseline current, a second
application of 0.3 mM extracellular ADP (event 2)
caused a mean 54% increase. All differences are statistically significant (Student’s t test; P , 0.05). In
experimental set B, event 1 (0.3 mM extracellular ADP)
evoked a mean 101% increase in inwardly directed current
at HP = 290 mV; in event 2, application of 0.3 mM extracellular ADP and 0.3 mM Gd3+ to the same protoplasts only
caused a mean 11% increase. Thus, the greater part of the
extracellular ADP-induced current was sensitive to the
cation channel blocker, Gd3+. To explore cation channel
activity further, IV relationships were determined.
Extracellular ADP Increases PM Ca2+ Influx and K+
Efflux Conductance
As Figure 2B shows, fast delivery of 0.3 mM extracellular ADP increased both inwardly and outwardly
directed currents across the PM of a single protoplast,
resulting in a positive shift of reversal potential (Erev)
away from the equilibrium potentials for K+ and Cl2
(EK = 2158 mV; ECl = 236 mV) and toward that for Ca2+
(ECa = 155 mV). The mean Erev of the extracellular ADPinduced current (estimated by subtracting the control
IV from that determined in the presence of extracellular
ADP; Fig. 3, inset) was 4.7 6 8.4 mV (n = 4), indicating
Ca2+ permeation. Extracellular ADP approximately
doubled the Gd3+-sensitive inward current (Figs. 2B
and 3) over the physiological voltage range for this cell
type (2100 to 2150 mV; Maathuis and Sanders, 1993);
thus, Ca2+ could be delivered to the cell to act as a signal
Table I. The effect of sequential extracellular ADP addition on PM
current
Control values were taken 1 to 3 s immediately preceding the
application of ADP. Event 1 refers to the first application of ligand and
Event 2 to the subsequent application to the same protoplast. Recording conditions as in Figure 1A. HP = 290 mV.
Procedure
Mean 6
SE
Maximal Currents
mA m22
Set A
Event 1 (n = 4)
Control
0.3 mM ADP
Event 2 (n = 3)
Control
0.3 mM ADP
Set B
Event 1 (n = 3)
Control
0.3 mM ADP
Event 2 (n = 3)
0.3 mM ADP
0.3 mM ADP + 0.3 mM Gd3
240.63 6 3.45
276.59 6 4.95
236.74 6 2.00
256.51 6 5.3
248.36 6 19.83
297.01 6 12.90
243.4 6 15.23
248.33 6 21.30
Figure 3. Extracellular ADP activates Ca2+ influx and K+ efflux in
epidermal protoplasts. Mean 6 SE IV relationships for control conditions and in the presence of 0.3 mM extracellular ADP, n = 4; solutions
as in Figure 2. Current values were taken from voltage ramps applied
1 to 3 min after application of control BS or activation by ADP. Insert:
difference IV relationship (control currents were subtracted from the
values obtained after ADP activation).
in response to the extracellular purine nucleotide. For
example, from the mean response shown in Figure 3, at
2120 mV inward Ca2+ current increased from 269 6 23
mA m22 to 2159 6 25 mA in response to 0.3 mM
extracellular ADP (n = 4; mean 6 SE). This large Gd3+sensitive Ca2+ influx current helps explain an extracellular ADP-induced elevation in whole root and root
epidermal protoplast [Ca2+]cyt found in aequorin luminometry assays (Demidchik et al., 2003a, 2009). In our
experimental conditions only K+ efflux (50 mM K+ in the
PS) could mediate the outward current (Shabala et al.,
2006). Both inward and outward currents were also
sensitive to the cation channel blocker Gd3+. It may be
that both Ca2+ influx and K+ efflux conductances were
caused by the activation of one cation channel type or
that Ca2+ influx activated K+ efflux.
Extracellular ADP Elevates Root Epidermal [Ca2+]cyt
Epidermal protoplasts were isolated from the mature epidermis of plants constitutively expressing
cytosolic apoaequorin to examine extracellular ADPinduced [Ca2+]cyt elevation. Previously, such protoplasts were found to sustain transient [Ca2+]cyt elevations
in response to 0.1 m M extracellular ATP, its poorly
hydrolyzable analog adenosine 5#-(a,b-methylene)
triphosphate (abATP), and ADP. All these were inhibited by Gd3+ and the purinoceptor antagonist, suramin
(Demidchik et al., 2009). AMP (as an ADP breakdown
product) up to 1 mM has been shown to be ineffective
(Demidchik et al., 2009). A test concentration of 0.1 mM
ADP was used in this study as it is a feasible concentration to simulate a wound response (Song et al.,
2006) and was found to cause Ca2+ current activation
in four out of nine whole-cell patch-clamp trials using slow perfusion (data not shown). Application of
0.1 mM extracellular ADP at pH 6.0 caused a transient
increase in [Ca2+]cyt (Fig. 4A). Overall the mean peak
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Receptor-Like Activity Evoked by Extracellular ADP
[Ca2+]cyt increase caused by this extracellular ADP concentration was 0.43 6 0.02 mM [Ca2+]cyt (n = 52), a value
comparable to that reported previously for protoplasts
from this cell type in response to 0.1 mM ADP or ATP
(Demidchik et al., 2009). Extracellular ADP was added
as its Na+ salt; NaCl as a control is not effective as an
elicitor in this assay (Demidchik et al., 2009). The extracellular ADP-induced increase depended on Ca2+
influx as depletion of external Ca2+ from 1 mM to 5 mM
in a paired test reduced the extracellular ADP-induced
increase by 84% (0.1 mM extracellular ADP, 1 mM Ca2+;
0.38 6 0.04 mM [Ca2+]cyt, n = 3: 0.1 mM extracellular ADP,
5 mM Ca2+; 0.06 6 0.05 mM [Ca2+]cyt, n = 4). An application of 0.1 mM extracellular ADP was used as the
standard stimulus in further trials.
The peak [Ca2+]cyt response to extracellular ADP
was insensitive to the G-protein inhibitor pertussis
toxin (200 ng mL21; Alloisio et al., 2004; extracellular
ADP; 0.34 6 0.04 mM [Ca2+]cyt, n = 6: extracellular ADP +
pertussis toxin; 0.34 6 0.02 mM [Ca2+]cyt, n = 11). The
cation channel blocker, Gd 3+, is already known to
inhibit the extracellular ADP-induced elevation of
[Ca2+]cyt in these epidermal protoplasts (Demidchik
et al., 2009). TEA+ (10 mM), a K+ channel blocker, significantly inhibited the [Ca2+]cyt elevation by 25% (extracellular ADP; 0.63 6 0.02 mM [Ca2+]cyt, n = 6: ADP +
TEA+; 0.47 6 0.03 mM [Ca2+]cyt, n = 6; P , 0.001, ANOVA).
This suggests that K+ channels may be involved in
changing membrane voltage regulating driving force
for the Ca2+ influx. Depolarizing the PM with 80 mM
K+ (Demidchik et al., 2002) significantly reduced the
[Ca2+]cyt response by 55%, consistent with reduction of
Ca2+ influx at depolarized voltage in patch-clamp trials
(1 mM K+; 0.55 6 0.03 mM [Ca2+]cyt, n = 5: 80 mM K+;
0.25 6 0.01 mM [Ca2+]cyt, n = 4; P , 0.001). The effect of
pH was tested against the standard application of
0.1 mM extracellular ADP, revealing a clear neutral pH
optimum (0.57 6 0.02 mM [Ca2+]cyt, n = 4) and a significantly enhanced response at alkaline pH compared
to physiological apoplastic pH 5 to 6 (Fig. 4B; P ,
0.001). Nevertheless, at pH 5, elevation of [Ca2+]cyt remained significant (control [Ca2+]cyt baseline pH 5,
0.11 6 0.02 mM; [Ca2+]cyt peak, 0.44 6 0.03 mM, n = 5; P ,
0.001). In separate tests (using a plate reader rather
than a luminometer), although absolute values of peak
[Ca2+]cyt were lower, the pattern of a greater response
at neutral pH rather than pH 6 was retained for 0.1 mM
ADP and also 0.1 mM ATP (Fig. 4C, n = 4; P , 0.05).
Response to ATP was significantly greater than to ADP at
both pH. The response to the poorly hydrolyzable ADP
analog ADPbS [adenosine 5#-O-(2-thiodiphosphate)]
was not significantly different to the ADP response
while abATP evoked significantly lower responses than
ATP at both pH (n = 4; P , 0.05). Thus part of the ATP
response may involve hydrolysis products but the ADP
response does not.
Extracellular ADP Does Not Increase Intracellular Levels
of ROS
In Arabidopsis roots, extracellular ATP causes accumulation of intracellular ROS via activation of the
PM NADPH oxidase AtRBOHC (Demidchik et al.,
2009). At the whole-root level, extracellular ADP was
applied under the same conditions used by Demidchik
et al. (2009) for intracellular ROS imaging [using esterloaded 5-(and-6-)-chloromethyl-2’,7’-dichlorohydrofluorscein diacetate] in response to extracellular ATP.
Solutions were designed to ensure that extracellular
[Ca2+] remained constant at 0.1 mM throughout experimental treatments as purine nucleotides are potent
Ca2+ chelators. Chelation of extracellular Ca2+ can trigger extracellular superoxide anion production by roots
(Mortimer et al., 2008). Here, extracellular ATP was
found again to cause intracellular ROS accumulation
(Fig. 5A). In contrast, extracellular ADP did not result
in significant accumulation of intracellular ROS (Fig. 5,
A and B). Previously a response was seen by 15 s after
Figure 4. Transient [Ca2+]cyt elevation by extracellular ADP in epidermal protoplasts. A, Representative trace of [Ca2+]cyt response in mature
epidermal protoplasts evoked by 0.1 mM ADP,
recorded using a luminometer. Discharge denotes
the addition of 2 M CaCl2 and 20% (v/v) ethanol
to enable [Ca2+]cyt estimation. B, pH dependence
of the epidermal protoplast [Ca2+]cyt response.
Mean 6 SE peak [Ca2+]cyt response to 0.1 mM ADP
with variable extracellular pH. Results are from
four to five independent trials. C, pH dependence
of the epidermal protoplast mean 6 SE peak [Ca2+]cyt
response in separate trials using a plate reader.
The effects of 0.1 mM ADP, ATP, ADPbS, or abmeATP were tested at pH 6 or 7 (n = 4).
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Figure 5. Extracellular ADP does not cause intracellular ROS accumulation. Arabidopsis roots were ester loaded with 5-(and-6)chloromethyl-2#,7#-dichlorodihydrofluorescein (CM-H2DCF) and exposed to extracellular ADP or ATP. A, Top sections, roots
bathed in medium (0.1 mM KCl, 0.1 mM CaCl2, pH 6.0) with 0, 0.1 mM, 1 mM extracellular ADP, or 0.1 mM extracellular ATP with
fluorescence false color mapping applied using the same scaling used for the positive extracellular ATP response reported by
Demidchik et al. (2009). Bottom sections, corresponding bright-field images. Scale bar = 2 mm. B, Mean 6 SE fluorescence per
pixel for control (n = 23), 0.01 mM extracellular ADP (n = 24), 0.1 mM extracellular ADP (n = 25), and 1 mM extracellular ADP (n =
23) exposure. C, In separate trials, roots were exposed to 1 mM ADP or ATP but with Ca2+ maintained at 1 mM. Scale bar = 0.2
mm, fluorescence false color-mapping scale is shown on the right. D, Mean 6 SE fluorescence per pixel for 0.1 mM Ca2+ buffer
(n = 8), 1 mM Ca2+ buffer (n = 17), 1 mM ADP with 1 mM Ca2+ (n = 11), 1 mM ATP with 1 mM Ca2+ (n = 11).
extracellular ATP application (0.01–1 mM) with 0.1 mM
extracellular ATP causing over a 3-fold increase in
signal intensity (Demidchik et al., 2009). Here, the
same concentration range for extracellular ADP under
identical conditions did not evoke intracellular ROS
production, even over an extended observation period. Under control conditions, mean 6 SE fluorescence
intensity of single primary roots was 19 6 0.9 fluorescence per pixel (n = 23) and there were no significant
increases on application of extracellular ADP (Tukey’s
multiple comparison test; Fig. 5B). Separate trials
using a different imaging system (Leica) and with
extracellular Ca2+ maintained at 0.1 or 1 mM resulted in
higher control values than recorded previously, showing the variable nature of ROS accumulation (Fig. 5, C
and D). Extracellular ATP (1 mM) caused an increase in
fluorescence intensity with 1 mM Ca2+ but 1 mM ADP
did not (Fig. 5, C and D; n = 11). Fluorescence intensity
with ADP was significantly lower than with ATP
(Student’s t test; P , 0.02). Overall the results suggest
that the extracellular ADP/[Ca2+]cyt pathway is independent of intracellular ROS.
Extracellular ADP Regulates Net Ca2+ and K+ Fluxes from
Excised Roots
To test whether extracellular ADP can activate cation fluxes in intact cells, we monitored net fluxes at the
epidermis of excised roots using the MIFE system for
noninvasive microelectrode Ca2+- and K+-flux measurements. This permits fine spatio-temporal mapping
of net fluxes. Constancy of extracellular Ca2+ (0.1 mM
Ca2+) ensured that changes in flux were due to extracellular ADP perception alone (rather than changes in
[Ca2+]) and also aided electrode function. Application
of extracellular ADP (Na+ salt) to the elongation zone
epidermis caused transient increases in net Ca2+ influx
(i.e. more positive flux values) in a dose-dependent
manner (Fig. 6, A and B; n = 3–6 for 1 mM to 1 mM
extracellular ADP). A significant increase above basal
level was first detected at 3 mM (P , 0.02; Student’s
t test). Duration of transients was up to 4 min. Control
flux levels were recovered. In control experiments
for the effect of Na+, 4 mM NaCl had no effect on net
flux (n = 4).
Net Ca2+ influx in response to extracellular ADP was
less pronounced in mature zone epidermis and [ADP]
below 10 mM did not evoked significant changes (Fig.
6C, n = 3–7). The mature zone has been found previously to be less receptive to exogenous ROS than the
elongation zone (Demidchik et al., 2003b, 2009). Differences between elongation zone and mature zone
were clearer for extracellular ADP-induced K+ efflux
(Fig. 6D), with elongation zone epidermis (Fig. 6E, n =
3–6) supporting greater net K+ efflux than the mature
zone (Fig. 6F, n = 3–7) across the concentration range
tested. Significant changes in K+ flux relative to basal
were observed at 1 mM ADP in both zones. Extracel-
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Receptor-Like Activity Evoked by Extracellular ADP
Figure 6. Extracellular ADP-induced elevation of net Ca2+ influx and K+ efflux at the root epidermis. A, Response of elongation
zone epidermal net Ca2+ flux to 3 mM, 100 mM, or 1 mM ADP, representative traces superimposed. Positive values signify influx. B,
Mean 6 SE net Ca2+ influx at the elongation zone epidermis (n = 3–6). C, Mean 6 SE net Ca2+ influx at the mature epidermis (n =
3–7). D, Response of mature and elongation zone epidermis to 300 mM ADP, representative traces superimposed. Negative values
signify efflux. E, Mean 6 SE net K+ efflux at the elongation zone epidermis (n = 3–6). F, Mean 6 SE net K+ efflux at the mature
epidermis (n = 3–7). Basal assay solution comprised 0.1 mM KCl, 0.1 mM CaCl2, pH 6.0.
lular AMP was found previously to be a very weak
inducer of root Ca2+ influx (Demidchik et al., 2009) and
seedling [Ca2+]cyt elevation (Tanaka et al., 2010b). Here,
AMP did not elicit a K+ efflux response even at 1 mM,
in either the elongation zone or mature epidermis
(elongation zone control; 237 6 34 nmol m22 s21, n = 3:
elongation zone + AMP, 231 6 26 nmol m22 s21, n = 3;
mature zone control; 219 6 13 nmol m22 s21, n = 3: AMP,
210 6 18 nmol m22 s21, n = 3). This suggests that
breakdown of extracellular ADP to AMP would help
terminate the cell’s response.
Extracellular ATP and its poorly hydrolyzable analog adenosine 5#-(a,b-methylene)triphosphate (abATP)
were also tested. The pattern of greater net Ca2+ and K+
fluxes at the elongation zone was retained for both
agonists (Figs. 7 and 8). In the elongation zone, both
extracellular ATP and abATP up to 100 mM caused
transient net Ca2+ influx comparable to that evoked by
extracellular ADP (Fig. 7, A–D). The threshold of the
detectable ATP response lay between 0.3 and 1 mM (n =
6). However while at 300 mM and 1 mM extracellular
ADP promoted net Ca2+ influx, ATP and abATP caused
net Ca2+ efflux (n = 5–17; Fig. 7, C and D). The mean
peak net Ca2+ efflux caused by 1 mM abATP was almost
double that caused by 1 mM ATP (Fig. 7, C and D). At
the mature epidermis, while 1 mM ADP caused net Ca2+
influx, both 1 mM ATP and abATP again caused net
Ca2+ efflux with abATP eliciting the greater response
(Fig. 7E; 1 mM ATP, 25 6 21 nmol m22 s21, n = 8). Thus
ATP hydrolysis appears necessary to limit the Ca2+
efflux response. At both elongation zone and mature
epidermis, both extracellular ATP and abATP caused
dose-dependent net K+ efflux in common with ADP
(Fig. 8). Thus while at high concentrations of ADP
net Ca 2+ influx and K + efflux appear as a coupled
response, net Ca2+ flux is uncoupled from K+ efflux at
high concentrations of ATP or abATP.
DISCUSSION
Evidence is now accumulating that extracellular
ATP is a regulator of plant physiology and development (Lew and Dearnaley, 2000; Tang et al., 2003;
Chivasa et al., 2005, 2009; Kim et al., 2006, 2009; Roux
and Steinebrunner, 2007; Wu et al., 2007; Riewe et al.,
2008a, 2008b; Wu and Wu, 2008; Yi et al., 2008;
Demidchik et al., 2009; Reichler et al., 2009; Clark
et al., 2010a, 2010b; Tanaka et al., 2010a, 2010b; Terrile
et al., 2010; Tonón et al., 2010). In common with ATP, it
is likely that ADP is released on wounding and could
perhaps also be released with ATP by exocytosis or the
activity of ATP-binding cassette transporters (Thomas
et al., 2000; Kim et al., 2006; Tanaka et al., 2010a).
Extracellular ATP hotspots have been found in elongating root cells (Kim et al., 2006) and ADP may also
be released there. This would be consistent with the
finding here that the Arabidopsis root elongation zone
epidermis supported a greater flux response to extracellular ADP than the mature zone. Such data support
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Demidchik et al.
Figure 7. Extracellular ATP- and abme-ATP-induced net Ca2+fluxes at
the root epidermis. A, Response of elongation zone epidermis to 1 to
1,000 mM ATP, representative traces superimposed. B, Mean 6 SE net
Ca2+ influx at the elongation zone epidermis in response to 0.3 to 100
mM ATP (n = 6–18). C, Mean 6 SE net Ca2+ flux at the elongation zone
epidermis in response to 100 to 1,000 mM ATP (n = 7–18). D, Mean 6 SE
net Ca2+ flux at the elongation zone epidermis in response to abmeATP (n = 3–7). E, Mean 6 SE net Ca2+ flux at the mature epidermis in
response to abme-ATP (n = 3–5). Recording conditions as Figure 6.
the premise that the sensitivity of different cells to
extracellular purine nucleotides differs (Tanaka et al.,
2010b). Suramin (a purinoceptor antagonist) inhibits
Arabidopsis root elongation and extracellular ADPinduced [Ca2+]cyt elevation (Demidchik et al., 2009).
Moreover, there is a dose-dependent effect of extracellular ADP on Arabidopsis root hair elongation
(Clark et al., 2010b) further implicating extracellular
ADP in growth regulation. Plant extracellular apyrases can hydrolyze ADP to regulate its extracellular
levels and so terminate any signal it could invoke
(Komoszyński, 1996; Steinebrunner et al., 2003; Riewe
et al., 2008a, 2008b; Tanaka et al., 2010a, 2010b).
Results here show that the epidermis is a contributory cell type to the whole-root elevation of [Ca2+]cyt by
extracellular ADP observed by Demidchik et al.
(2003a) and the whole-seedling response, thought to
be generated largely by the root, observed by Tanaka
et al., (2010b). Moreover, that protoplasts could sustain
the [Ca2+]cyt elevation demonstrates that the wall is not
critical for the response of this cell type. Previously, it
had been suggested that wall proteins capable of
phosphorylation intercept extracellular purine nucleotides to evoke a cellular response (Chivasa et al.,
2005). The results also show that extracellular ADP can
generate a [Ca2+]cyt signal in the mature epidermis
seemingly independently of heterotrimeric G-protein
activity. Further studies on the epidermis now need to
focus on G-protein mutants.
The response speed of the PM to extracellular ADP in
the fast-perfusion patch-clamp trials here is consistent
with the receptor-like activity suggested by Lew and
Dearnaley (2000) to explain extracellular ADP-induced
depolarization of Arabidopsis root hair PM voltage.
Previously, extracellular ADP-induced rapid elevation
of [Ca2+]cyt in root epidermal protoplasts was inhibited
by 92% with the same concentration (0.3 mM) of Gd3+
used here to block PM influx currents (Demidchik et al.,
2009). The very rapid response of the PM inward Ca2+
conductance to extracellular ADP (fast perfusion) and
its block by 0.3 mM Gd3+ raises the possibility that Ca2+
influx is an initial event. In common with animal
purinceptor responses (Roberts and Evans, 2007), extracellular ADP-induced current decayed in the continued presence of the agonist and repeated application of
extracellular ADP to the epidermal PM resulted in a
lower level of current activation (Fig. 2A; Table I). Thus,
a [Ca2+]cyt signal evoked by extracellular ADP release
could be diminished by desensitization and terminated
by apyrase or phosphatase activity. This helps explain
the transient nature of the [Ca2+]cyt response and perhaps also that of the epidermal Ca2+ and K+ fluxes.
Efflux of K+ could readily counter the depolarizing
effect of purine nucleotide-induced Ca2+ influx on PM
voltage and help return this membrane to a hyperpolarized state. The greater magnitude of the K+ efflux
compared to Ca2+ influx suggests that K+ efflux may
also serve other purposes.
Previously, extracellular ATP was found to stimulate production of extracellular superoxide anion by
Arabidopsis etiolated hypocotyls (Tonón et al., 2010),
and leaves (Song et al., 2006). In leaves, this was
through activation of PM NADPH oxidases (Song
et al., 2006). Extracellular ADP stimulated leaf and
root extracellular superoxide anion production by an
unknown mechanism (Song et al., 2006; Demidchik
et al., 2009). In roots of Arabidopsis, Medicago truncatula, and Phaseolus vulgaris extracellular ATP also
resulted in accumulation of intracellular ROS, essentially hydrogen peroxide (H 2O2; Kim et al., 2006;
Cárdenas et al., 2008; Demidchik et al., 2009; Clark
et al., 2010b). In this study, and in contrast to extracellular ATP, extracellular ADP did not trigger accumulation of intracellular ROS. This is in agreement with
the findings of Kim et al. (2006) in which extracellular
ATP but not extracellular ADP triggered intracellular
ROS accumulation in M. truncatula root hairs. Certainly, there is a precedent for triggers of Ca2+ influx
resulting in distinct ROS responses. In Arabidopsis cell
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Receptor-Like Activity Evoked by Extracellular ADP
Figure 8. Extracellular ATP- and abme-ATP-induced net K+ fluxes at the root epidermis. A, Response of elongation zone
epidermis to 0.3 to 1,000 mM ATP, representative traces superimposed. B, Mean 6 SE net K+ efflux at the elongation zone
epidermis in response to 0.3 to 1,000 mM ATP (n = 6–37). C, Mean 6 SE net K+ efflux at the mature epidermis in response to 10 to
1,000 mM ATP (n = 4–13). D, Response of elongation zone and mature epidermis to 300 mM abme-ATP, representative traces
superimposed. E, Mean 6 SE net K+ efflux at the elongation zone in response to 3 to 1,000 mM abme-ATP (n = 3–7). F, Mean 6 SE
net K+ efflux at the mature epidermis in response to 3 to 1,000 mM abme-ATP (n = 3–6). Recording conditions as Figure 6.
suspension cells, both hyper- and hypoosmotic stress
trigger Ca2+ influx but only hypoosmotic stress results
in extracellular H2O2 accumulation (Beffagna et al.,
2005). Extracellular superoxide anions (resulting from
perception of extracellular purine nucleotides) could
generate H 2O 2 that could then be translocated to
the cytosol by PM aquaporins (Bienert et al., 2007;
Dynowski et al., 2008). One possible explanation for
the disparity between the intracellular H2O2 accumulation response to extracellular ATP and extracellular
ADP is that in the extracellular ADP pathway, H2O2
translocation is blocked. This could be achieved by
closure and/or retrieval of H2O2-permeable aquaporins (Boursiac et al., 2008). Alternatively, extracellular
ADP perception could inhibit an intracellular ROSgenerating system, stimulate an intracellular antioxidant activity, or extracellular ADP perception in the
apoplast could result in suppression of extracellular
ROS production downstream of superoxide anions.
Given the importance of ROS in plant cell signaling,
these possibilities merit further investigation particularly in the context of nodule development. Extracellular ADP has been found to regulate this process that
is known to involve complex spatio-temporal changes
in ROS (Chang et al., 2009; Govindarajulu et al., 2009).
Whether ATP-dependent intracellular ROS accumulation is the cause of net Ca2+ efflux from the root
epidermis at high [ATP] now needs to be determined
as this may be relevant to the suppression of defense
responses noted when exogenous ATP is increased
(Chivasa et al., 2009).
The pH dependence of animal ionotropic purinoreceptors varies with isoform (King et al., 1997; Wildman
et al., 1999; Wirkner et al., 2008). Here, the extracellular
ADP-induced [Ca2+]cyt response was operative over a
wide pH range but activity at more neutral to alkaline
pH suggests that extracellular ADP (and ATP) signaling could operate during stress responses in which
the cytosol acidifies and apoplastic pH elevates above
its normal acid value, such as hypoosmotic stress or
elicitor challenge (Mathieu et al., 1996; Pugin et al.,
1997; Beffagna et al., 2005). Additionally, pH dependence of ADP- or ATP-induced Ca2+ and K+ flux
could be relevant at growth points, such as the root hair
apex, at which apoplastic pH oscillates and pH oscillations are linked to nitrogen availability (Monshausen
et al., 2007; Bloch et al., 2011). Overall, extracellular ADP
is competent to act as a trigger of [Ca2+]cyt-dependent
signaling with the potential to evoke responses distinct to those produced by ATP. Its initial perception
at the PM appears reliant on ionotropic receptor-like
activity.
CONCLUSION
Perception of extracellular ADP at the root epidermal PM is consistent with the operation of functional
equivalents of ionotropic purine nucleotide receptors.
In contrast to extracellular ATP, downstream events do
not include accumulation of intracellular ROS thus
indicating disparate signaling pathways.
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Demidchik et al.
MATERIALS AND METHODS
Plant Growth
Arabidopsis (Arabidopsis thaliana Columiba-0; Columbia-0 expressing
apoaequorin under a 35s promoter; Demidchik et al., 2009) from laboratory
stock was grown vertically on full-strength Murashige and Skoog (Duchefa;
www.duchefa.com) medium with 1% (w/v) Suc and 0.3% (w/v) Phytagel
(Sigma; www.sigmaaldrich.com). Growth conditions were 22°C and 16 h day
length, 100 mmol m22 s21 irradiance.
Patch-Clamp Electrophysiology
Protoplasts were isolated from root mature epidermis (10–15 d plants)
using the protocol described by Demidchik et al. (2003b). Protoplasts used for
patch clamping were 20 6 1.5 mm in diameter. The PS contained (mM): 40
K-gluconate, 10 KCl, 1 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic
acid (free Ca2+ adjusted to 100 nM with 0.4 CaCl2), pH 7.2 adjusted with Tris/
MES. The G-protein antagonist GDPbS (0.5 mM) was incorporated into the PS
as indicated. Control BS comprised (mM): 20 CaCl2, 0.1 KCl, 0.3 NaCl (varied
to supply equimolar Na+ to that in purine-containing BS), pH 6 with 2 Tris/4
MES. BS containing purine agonist was freshly prepared directly before
application and the pH of the solution was adjusted where necessary (Tris). BS
and PS were adjusted to 290 to 300 mOsM with D-sorbitol. Delivery of BS +
ADP to the recording chamber was either by gravity feed or by a fast-flow
system comprising an AutoMate perfusion system with four pinch valves,
ValveBank controller, and four-barrel delivery pipette (barrel diameter 0.1
mm; AutoMate Scientific Inc.). This system allowed switching to test BS
in 0.01 s. Average delivery time was less than 1 s. The delivery pipette was
placed at 30 to 40 mm from the protoplast surface and delivery speed was 0.5
to 1 mL s21. Two barrels of the delivery pipette were used—one for control BS
and another for purine-containing BS. Solution changes were controlled by the
programmable ValveBank controller. Recording equipment was as described
in Demidchik et al. (2003b). Ion activities were calculated using Geochem
(Parker et al., 1995). Liquid junction potentials were measured and corrected
as described previously (Demidchik et al., 2003b).
[Ca2+]cyt Measurement
The protocol described in Demidchik et al. (2007) was used to isolate
protoplasts from root mature epidermis of Arabidopsis (10–15 d old) expressing cytosolic (apo)aequorin (cauliflower mosaic virus 35S promoter) with the
modification that incubation with protoplasting enzymes was increased from
50 to 60 min. Protoplasts were suspended for 3 to 4 h (dark, 28°C) in recording
medium (mM: 1 CaCl2, 1 KCl, 1 MgCl2, 10 Glc; pH 6.0 adjusted by 2 Tris/4
MES; 290–300 mOsM with D-sorbitol) with 10 mM coelenterazine (NanoLight
Technology; www.nanolight.com) to match the protocol used by Demidchik
et al. (2009) for ATP and ADP studies. After washing, recording medium
(without coelentrazine) and standard luminometry procedures were used.
The discharging solution enabling estimation of [Ca2+]cyt contained 2 M CaCl2
and 20% (w/v) ethanol. Luminescence was recorded using a luminometer or
FLUOstar plate reader.
Imaging of ROS
Imaging of intracellular ROS used 50 mM 5-(and-6-)-chloromethyl-2’,7’dichlorohydrofluorscein diacetate, acetyl ester (Molecular Probes; www.
invitrogen.com) with dye loading as described previously (Demidchik et al.,
2009). Assay medium was as described by Demidchik et al. (2009) and
comprised (mM): 0.1 KCl, 0.1 or 1 CaCl2, 4 MES, and 2 Tris base (pH 6.0).
Images of 5-d-old roots were acquired either with a Nikon SMZ 1500
microscope and a QImaging Retiga cooled 12-bit camera or a Leica M165
FC microscope and a Leica DFC310 FX 1.4-megapixel camera. Ca2+ was
maintained at 0.1 or 1 mM in the presence of purines by appropriate additions
of CaCl2 (calculated using MaxChelator and Geochem programs; Parker et al.,
1995; Patton et al., 2004).
MIFE Recordings of Net Cation Fluxes
Excised roots (5–15 d old) were fixed into a recording chamber by an agar
drop and immersed in 0.5 mL control solution comprising (in mM) 0.1 KCl, 0.1
CaCl2, 4 MES, 2 Tris base (pH 6.0; Demidchik et al., 2009). Chamber design
permitted rapid mixing of test solution and resumption of flux recording 20 to
30 s after addition of 0.5 mL test solution. Ca2+ was maintained at 0.1 mM in the
presence of purines by appropriate additions of CaCl2. Levels were confirmed
experimentally in a cell-free assay using a Ca2+-selective microelectrode.
Purines were added as Tris, Na+, or Li+ salts. NaCl or LiCl at equimolar
concentrations did not perturb K+ efflux (tested experimentally). Measurements of mature epidermal flux were taken 1 to 1.5 mm from the root apex and
for the elongation zone, 100 to 150 mm from the apex (Demidchik et al., 2007).
Net Ca2+ influx and K+ efflux were determined using the noninvasive slowly
vibrating microelectrode (MIFE) technique. K+- and Ca2+-selective microelectrodes (external tip diameter approximately 3 mm) were made and calibrated
as described previously (Shabala and Shabala, 2002). Electrodes were calibrated before and after use. Agonists (tested up to 1 mM) had no effect on
calibration or electrode response time. The microelectrode was placed 20 mm
above the root surface and moved between two positions, 20 and 50 mm above
the root surface in a square wave manner (5-s half-cycle).
ACKNOWLEDGMENTS
We thank Daniel Schachtmann for advice and Adeeba Dark for help with
plant cultivation.
Received February 18, 2011; accepted May 6, 2011; published May 11, 2011.
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