Plant extracellular ATP signalling by plasma membrane NADPH oxidase and Ca 2+ channels

Plant extracellular ATP signalling by plasma membraneNADPH oxidase and Ca2+ channels

Vadim Demidchik1,†,‡, Zhonglin Shang1,†,‡, Ryoung Shin2,†,‡, Elinor Thompson1, Lourdes Rubio1,†, Anuphon Laohavisit1,

Jennifer C. Mortimer1, Stephen Chivasa3, Antoni R. Slabas3, Beverley J. Glover1, Daniel P. Schachtman2,

Sergey N. Shabala4 and Julia M. Davies1,*

1Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK,2Donald Danforth Plant Science Center, St Louis, MO 63132, USA,3School of Biological and Biomedical Sciences, University of Durham, Durham DH1 3LE, UK, and4School of Agricultural Sciences, University of Tasmania, Hobart, Tas. 7001, Australia

Received 10 October 2008; revised 31 December 2008; accepted 15 January 2009.

*For correspondence: (fax +44 (0) 1223 333 953; e-mail [email protected]).†Present addresses: VD; Department of Biology, University of Essex, Colchester, CO43SQ, United Kingdom. ZS; College of Life Science, Hebei Normal University,

Yuhua East Road, Shijiazhang 050016, Hebei, China. RS; Riken Plant Science Center, Yokohama City, Kanagawa 230-0045, Japan. LR; Departamento de Biologıa

Vegetal, Facultad de Ciencias, Universidad de Malaga, Campus de Teatinos, 29071 Malaga, Spain.‡These authors contributed equally to this work.

SUMMARY

Extracellular ATP regulates higher plant growth and adaptation. The signalling events may be unique to higher

plants, as they lack animal purinoceptor homologues. Although it is known that plant cytosolic free Ca2+ can be

elevated by extracellular ATP, the mechanism is unknown. Here, we have studied roots of Arabidopsis thaliana

to determine the events that lead to the transcriptional stress response evoked by extracellular ATP. Root cell

protoplasts were used to demonstrate that signalling to elevate cytosolic free Ca2+ is determined by ATP

perception at the plasma membrane, and not at the cell wall. Imaging revealed that extracellular ATP causes

the production of reactive oxygen species in intact roots, with the plasma membrane NADPH oxidase

AtRBOHC being the major contributor. This resulted in the stimulation of plasma membrane Ca2+-permeable

channels (determined using patch-clamp electrophysiology), which contribute to the elevation of cytosolic free

Ca2+. Disruption of this pathway in the AtrbohC mutant impaired the extracellular ATP-induced increase in

reactive oxygen species (ROS), the activation of Ca2+ channels, and the transcription of the MAP kinase3 gene

that is known to be involved in stress responses. This study shows that higher plants, although bereft of

purinoceptor homologues, could have evolved a distinct mechanism to transduce the ATP signal at the plasma

membrane.

Keywords: adenosine triphosphate, calcium, channel, MAP kinase, reactive oxygen species.

INTRODUCTION

ATP is a ubiquitous intracellular energy source, but, in ani-

mals, ATP also acts at the extracellular face of the plasma

membrane as a paracrine or autocrine signalling agent

(Khakh and North, 2006). There, extracellular ATP is involved

(together with extracellular ADP and adenosine) in the reg-

ulation of a wide range of cellular processes, including

neurotransmission, immune response, and cell growth and

death (Khakh and North, 2006). Cellular responses to extra-

cellular purines are mediated by specific ‘purinoceptors’ that

comprise the P1 family (heterotrimeric G-protein-coupled

adenosine receptors) and P2 family (P2X, ligand-gated ion

channels; P2Y, G-protein-coupled receptors). The elevation

of intracellular cytosolic free Ca2+, [Ca2+]cyt, as a second

messenger is a common outcome of purinoceptor activation

(Khakh and North, 2006).

It is now over 30 years since the first observations on

the effects of extracellular ATP in plants, namely Venus

flytrap closure (Jaffe, 1973), induction of endonucleases in

oat leaves (Udvardy and Farkas, 1973) and stimulation of

K+ uptake (Luttge et al., 1974), were made. During this

period, the molecular mechanisms underpinning purine

signalling in animal cells have been elucidated, whereas

ª 2009 The Authors 1Journal compilation ª 2009 Blackwell Publishing Ltd

The Plant Journal (2009) doi: 10.1111/j.1365-313X.2009.03830.x

scant attention has been paid to plant systems. Recently,

it has become clear that extracellular ATP regulates plant

viability (Chivasa et al., 2005), growth (Kim et al., 2006;

Roux and Steinebrunner, 2007; Wu et al., 2007a), gravit-

ropism (Tang et al., 2003) and stress responses (Thomas

et al., 2000; Jeter et al., 2004; Song et al., 2006). That ATP

operates in such processes places it alongside better

established regulators such as ABA, in terms of

fundamental importance. The way in which extracellular

ATP functions as a plant cell regulator must now be

established.

ATP is released from plant cells by plasma membrane

ABC transporters (Thomas et al., 2000; Roux and Steineb-

runner, 2007), vesicular efflux (Kim et al., 2006) and wound-

ing (Jeter et al., 2004). The levels of extracellular ATP are

regulated by apyrases (extracellular nucleotide phosphohy-

drolases) that catalyse its breakdown (Thomas et al., 2000;

Wu et al., 2007a). Physiological studies point to roles for

extracellular ATP in cell viability, defence and growth.

Experimental depletion of extracellular ATP triggers plant

cell death in a range of species, and hence it has been

proposed to act as a suppressor of a default cell death

pathway (Chivasa et al., 2005). Commitment to cell death in

the model plant Arabidopsis thaliana caused by the myco-

toxin fumonisin B1 (an elicitor of death and defence

responses) correlates with a loss of extracellular ATP

(probably through hydrolysis), and can be reversed by its

application (Chivasa et al., 2005).

In roots, extracellular ATP levels are highest in the

elongation zone and at the root hair apex (Kim et al.,

2006), implicating ATP in growth regulation. The depletion

of extracellular ATP (by application of apyrase) severely

inhibits Medicago root hair elongation (Kim et al., 2006),

whereas the application of extracellular ADP results in

increased elongation of Arabidopsis root hairs (Lew and

Dearnaley, 2000). The current model assumes that extra-

cellular ATP must be tightly regulated, as too little ATP

results in cessation of growth and death, but too much

ATP also arrests growth, possibly by impairing auxin

transport (Tang et al., 2003; Kim et al., 2006; Roux and

Steinebrunner, 2007; Wu et al., 2007a). In Arabidopsis

seedlings and suspension cells, high extracellular ATP

concentrations cause the transcription of genes involved

in stress responses (Jeter et al., 2004; Song et al., 2006),

suggesting the involvement of extracellular ATP in the

switch from growth to stress adaptation (Roux and

Steinebrunner, 2007).

How plants perceive extracellular ATP remains unknown.

There are no equivalents to animal purinoceptors evident in

higher plant genomes (Kim et al., 2006; Fountain et al.,

2008). Although a P2X receptor has recently been identified

in the unicellular green alga Ostreococcus tauri, it does not

function at the plasma membrane (Fountain et al., 2008). In

Arabidopsis, extracellular ATP transiently elevates [Ca2+]cyt

in whole organs, possibly by increasing Ca2+ influx at the

plasma membrane (Demidchik et al., 2003a; Jeter et al.,

2004; Kim et al., 2006), but the underlying mechanisms of

extracellular ATP sensing and Ca2+ flux have not yet been

identified. Plasma membrane Ca2+-permeable channels

would be capable of mediating the influx of Ca2+. In

contrast to animals, the plasma membrane may not even

be the site of ATP recognition and initiation of signalling.

The plant extracellular matrix contains a suite of secreted

proteins capable of being phosphorylated by ATP (Chivasa

et al., 2002; Ndimba et al., 2003), and it has been suggested

that these could relay a signal to the cell (Chivasa et al.,

2005).

At present there is no direct evidence for channel activa-

tion in response to extracellular ATP, or even for the ATP

‘target’ residing at the plasma membrane, rather than in the

extracellular matrix. Here, we have aimed to resolve the site

of extracellular ATP perception in Arabidopsis and to

delineate an ATP-regulated stress signalling pathway. To

do so, we have refined the study of ATP effects in plants by

moving away from whole-organ studies to a defined cell

type: the root epidermis. Using this cell type, we have

determined that extracellular ATP is sensed at the plasma

membrane rather than in the wall. The perception of

extracellular ATP causes the production of reactive oxygen

species (ROS) through the activation of a specific plasma

membrane NADPH oxidase. ROS, in turn, activate plasma

membrane hyperpolarization-activated Ca2+ influx channels,

thereby mediating [Ca2+]cyt elevation. Extracellular ATP-

induced ROS accumulation, Ca2+ channel activation and

transcription of the stress-signalling mitogen-activated pro-

tein kinase MPK3 (Kovtun et al., 2000; Rentel et al., 2004) are

impaired in a loss-of-function NADPH oxidase mutant. This

indicates that extracellular ATP controls [Ca2+]cyt-dependent

signalling in plants, acting via redox-activated Ca2+ chan-

nels, but with receptors that are structurally distinct from

those of animals.

RESULTS

Extracellular ATP increases Ca2+ influx at the root epidermis

Extracellular ATP effects on plant Ca2+ have previously

been determined only at the whole-organ level of roots

and leaves (Demidchik et al., 2003a; Jeter et al., 2004).

Here, to elucidate the mechanistic basis of ATP responses,

we have examined a single cell type. The mature, fully

expanded root epidermis of Arabidopsis is ideal for

determining ATP stress signalling pathways, as it should

be capable of sensing environmental change, and ATP

responses here should be unrelated to the growth mech-

anism. To discriminate between ATP perception at the cell

wall or plasma membrane, we first checked for the ATP

activation of Ca2+ influx in the mature epidermis, and

2 Vadim Demidchik et al.

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03830.x

then removed cell walls enzymatically and tested the

protoplasts for responsiveness.

The influx of Ca2+ at the mature epidermis of excised roots

was measured using a vibrating Ca2+-selective microelec-

trode, and, under control conditions, the mean (� SE) net

Ca2+ influx was 0.53 � 1.6 nmol m)2 sec)1 (n = 97). The

extracellular Ca2+ concentration was maintained at 0.1 mM

to aid electrode function. A modification of the chamber

design allowed solution exchange, and a resumption of

recording after 20–30 sec rather than after minutes, as was

possible previously (Shabala et al., 1997). Thus early

responses to test agents could be recorded. In preliminary

trials an H+-selective microelectrode was used to confirm

that the addition of purines caused no change in extracel-

lular pH, and, as was found previously (Demidchik et al.,

2007), the addition of control buffer alone (no purines and

acting as a control for mechanical disturbance) did not cause

a change in the net Ca2+ flux (n = 4). Application of 30 lM

ATP (a concentration generated by wounding; Song et al.,

2006) or its stable analogue adenosine 5¢-(a,b-methylene)

triphosphate (abme-ATP) caused transient net Ca2+ influx

at the mature and fully-expanded epidermis (Figure 1a),

approximately 1 min after the addition of agonist. The peak

net influx in response to 30 lM ATP was 13 � 9 nmol m)2

sec)1, n = 5. In response to 30 lM abme-ATP, the peak net

influx was 7 � 2 nmol m)2 sec)1, n = 4. AMP was far less

potent, requiring an application of 1 mM to evoke a transient

increase in net Ca2+ influx of 3 � 3 nmol m)2 sec)1 (n = 4).

As free external Ca2+ activity was constant during the

application of purine, the increased influx was unlikely to

be a homeostatic response to chelation of extracellular Ca2+

by ATP.

Extracellular ATP is sensed at the plasma membrane

Enzymatic wall removal from the mature epidermis still

permitted ATP responses. Extracellular ATP caused the

transient and dose-dependent elevation of [Ca2+]cyt in

mature epidermal protoplasts (detected using cells that

constitutively express aequorin; Figure 1b,c). The resting

level of [Ca2+]cyt under control conditions (1 mM CaCl2, pH 6)

was 107 � 2 nM (n = 521). In these experiments the level of

extracellular Ca2+ was higher than that used previously, in

order to increase the driving force for Ca2+ and to maximize

the resultant signal. The peak [Ca2+]cyt response occurred

approximately 1 min after the application of the agonist.

Agonists were tested over a concentration range from 1 lM

to 1 mM. At the lowest concentration tested (1 lM), ATP

caused a peak increase in [Ca2+]cyt of 20 � 14 nM (n = 9),

representing a 19% increase above the basal level. The

duration of the transient [Ca2+]cyt elevation was dependent

on agonist concentration; e.g. the duration of the transient

induced by 100 lM and 1 mM ATP was 5.5 � 0.5 min (n = 9)

and 11 � 0.4 min (n = 3), respectively. Basal levels of

[Ca2+]cyt were always fully recovered after agonist challenge.

Extracellular abme-ATP and ADP also caused the transient

dose-dependent elevation of protoplast [Ca2+]cyt (Figure 1c).

As the Li+ salt of abme-ATP was used, the effect of Li+ on

[Ca2+]cyt was also tested. The addition of up to 2 mM LiCl did

not induce significant [Ca2+]cyt transients (n = 9). ADP was

added as its Na+ salt: NaCl as a control was not effective as

an elicitor, and even 1 mM only raised [Ca2+]cyt by

0.01 � 0.01 lM (n = 4). That ADP was an effective agonist

indicates that hydrolysis of the terminal phosphate of ATP

was not necessary for the elevation of [Ca2+]cyt. Protoplasts

(a) (b)

(d)(c)

Figure 1. Extracellular ATP increases the net

Ca2+ influx and elevates the intracellular cyto-

solic free Ca2+, [Ca2+]cyt, in the root epidermis.

(a) Extracellular adenosine 5¢-(a,b-methylene)tri-

phosphate (30 lM; abme-ATP) caused a transient

increase in the net Ca2+ influx to fully-expanded

Arabidopsis root epidermis. Extracellular Ca2+

was maintained at 0.1 mM.

(b) The transient increase in [Ca2+]cyt in protop-

lasts of fully-expanded Arabidopsis root epider-

mal cells, caused by treatment with 100 lM ATP,

and its inhibition by 0.5 mM suramin.

(c) Mean � SE peak transient [Ca2+]cyt increase in

protoplasts evoked by ATP, abme-ATP, ADP or

AMP (n = 9–12).

(d) Effect of 300 lM Gd3+ or 0.5 mM suramin

on the mean � SE peak transient protoplast

[Ca2+]cyt increase evoked by 100 lM ATP, abme-

ATP or ADP (n = 4–12).

(b–d) Extracellular 1 mM CaCl2.

Plant ATP signalling 3


exhibited a low sensitivity to AMP in terms of response

threshold and magnitude (Figure 1c).

Lowering extracellular Ca2+ from 1 mM to 5 lM caused an

85% reduction in the peak [Ca2+]cyt response to 100 lM

abme-ATP. The mean � SE was 200 � 40 nM in 1 mM Ca2+,

and 30 � 10 nM in 5 lM Ca2+ (n = 4), which is indicative of

the plasma membrane Ca2+ influx generating the majority

of the [Ca2+]cyt response. Gd3+ (a blocker of root cell

Ca2+-permeable channels; Demidchik et al., 2007; Foreman

et al., 2003) is known to inhibit the elevation of extracellular

ATP-induced [Ca2+]cyt in Arabidopsis roots and seedlings

(Demidchik et al., 2003a; Jeter et al., 2004). Here, 300 lM

Gd3+ inhibited the elevation of protoplast [Ca2+]cyt in

response to 100 lM ATP, abme-ATP or ADP by 88, 75 and

92%, respectively (n = 9–12; Figure 1(d)). Responses were

also inhibited by 0.5 mM suramin (100 lM ATP, 82%; 100 lM

abme-ATP, 97%; 100 lM ADP, 95%; n = 3–9; Figure 1d), an

antagonist of animal purinoceptors known to inhibit the

ATP-induced elevation of root [Ca2+]cyt (Demidchik et al.,

2003a). Suramin also significantly inhibited root elongation

(control, 5.6 � 0.02 mm day)1; 50 lM suramin, 1.5 � 0.002

mm day)1; n = 30; 6–8-day growth interval; P < 0.001,

Student’s t-test), supporting the premise that normal

growth requires the perception of extracellular ATP (Kim

et al., 2006; Roux and Steinebrunner, 2007; Wu et al., 2007a).

Overall, the results demonstrate that extracellular ATP is

sensed at the plasma membrane.

ATP evokes a plasma membrane Ca2+ conductance

Having resolved the extracellular ATP regulation of plant

[Ca2+]cyt at the single cell-type level, we then addressed

the underlying transport reactions. The inhibition of ATP-

induced elevation of [Ca2+]cyt by channel blockers implied

that plasma membrane Ca2+-permeable channels mediate

Ca2+ influx. To investigate such channels, protoplasts

from mature root epidermal cells were patch clamped.

This technique relies on complete wall removal, and

therefore its successful use verifies the protoplasting

protocol used in the aequorin studies. Application of

20 lM extracellular ATP to protoplasts (in ‘whole cell’

recording mode) transiently activated a plasma membrane

hyperpolarization-activated Ca2+ channel conductance

(Figure 2a), similar to those characterized previously in

this and other cell types, including guard cells (Pei et al.,

2000; Foreman et al., 2003; Demidchik et al., 2007; Wu

et al., 2007b). An increase in inwardly-directed current

was observed 1–3 min after the addition of ATP, and was

evident over the physiological voltage range for this cell

type (Maathuis and Sanders, 1993). At )200 mV, the cur-

rent increased approximately twofold from )164 � 42 pA

to a peak of )362 � 65 pA after 5–20 min exposure

(n = 6), after which the current declined. The ATP ana-

logue 2¢-O-(4-benzoylbenzoyl) adenosine 5¢-triphosphate

(BzBzATP) caused comparable activation of channel

(a)

(d)

(b) (c)

Figure 2. Extracellular ATP evokes a plasma

membrane Ca2+ conductance in root epidermal

protoplasts.

(a) Effect of 20 lM extracellular ATP. Whole-cell

currents are shown above mean � SE current–

voltage relationships (n = 6).

(b) 2¢-O-(4-Benzoylbenzoyl) adenosine 5¢-triphos-

phate (30 lM; BzBzATP) transiently increased the

whole-cell conductance (G, s), and 100 lM Gd3+

inhibited the response (d; n = 7).

(c) The ATP-activated conductance was variously

permeable to Ca2+, Ba2+, Mg2+ and Zn2+, and was

inhibited by 100 lM Gd3+ (n = 3 for all trials;

20 mM external divalent cation). The control

current–voltage relationship was recorded in

external Ca2+ with no ATP; Gd3+ was applied in

the Ca2+ + ATP test.

(d) Suramin (0.5 mM) prevented the activation of

the Ca2+ conductance by 20 lM ATP (n = 6).



conductance, which was fully inhibited by 100 lM Gd3+

(Figure 2b; 30 lM, n = 7).

The ATP-activated conductance was highly permeable to

Ba2+ and Mg2+, but was only weakly permeable to Zn2+

(100 lM ATP; Figure 2c), consistent with the operation of

Ca2+ channels, rather than K+ channels (Hirsch et al., 1998;

Foreman et al., 2003). The permeability sequence (deter-

mined from conductance values; Demidchik et al., 2003b)

was Ba2+ (1.21 � 0.14)>Ca2+ (1.00) > Zn2+ (0.19 � 0.05);

n = 3. The ATP-induced Ca2+ conductance was blocked by

100 lM Gd3+ (Figure 2c; n = 3), and activation was prevented

by 0.5 mM suramin (control I at )200 mV, )217 � 38 pA,

n = 6; suramin, )198 � 43 pA, n = 6; Figure 2d). These data

indicate that the hyperpolarization-activated Ca2+ conduc-

tance is required for the ATP-activated elevation of [Ca2+]cyt

observed in epidermal protoplasts and whole roots

(Demidchik et al., 2003a).

A 19-pS Ca2+ channel is activated in response to

extracellular ATP

We next determined the identities of the channels contrib-

uting to this transport reaction. At the single-channel level, a

4–5 pS channel (typical of the non-selective cation channels

found previously in this cell type (Demidchik et al., 2002),

was sometimes observed prior to the application of ATP (or

BzBzATP) to the extracellular face of excised outside-out

membrane patches. Its activity was insensitive to agonists.

The application of 30 lM ATP (or BzBzATP) stimulated a

transient burst of inwardly-directed positive current, which

occurred up to 1 min after the estimated arrival of agonist at

the membrane (ATP, three out of six patches, Figure 3(a);

BzBzATP, seven out of 11 patches; Figure 3b). The burst

duration was 17 � 2 min with ATP (n = 3), and 21 � 1 min

with BzBzATP (n = 7), consistent with the time course of

current activation measured from the entire plasma mem-

brane. Up to four, single, seemingly identical, open channel

states were observed early in the burst. The channel ampli-

tude increased on the hyperpolarization of the membrane

voltage (Figure 3c). The single channel conductances

derived from the first open state were 19 � 0.8 and

20 � 1.9 pS, for ATP and BzBzATP, respectively (Figure 4a).

The extrapolated reversal potentials were 19 � 11 and

33 � 4 mV, for ATP and BzBzATP, respectively. The

displacement of the reversal potential away from the equi-

librium potential for Cl) ()36 mV), and towards that for Ca2+

(+154 mV), is indicative of Ca2+ permeability, in addition to

possible Cl) permeability. The open probability (Popen,

estimated for the first open state at the height of the

purine-activated burst) decreased over time (Figure 4b).

Hyperpolarization increased Popen (Figure 4c), whereas

100 lM Gd3+ decreased it (Figure 4d). The Ca2+ permeability,

voltage dependence, transient activation and pharmaco-

logical profile are fully consistent with this channel’s

contributing to the ATP-activated Ca2+ conductance.

Extracellular ATP increases AtrbohC NADPH oxidase

activity

Extracellular ATP causes the accumulation of ROS in roots

(Kim et al., 2006) and leaves (Song et al., 2006). ROS

increase plasma membrane hyperpolarization-activated

Ca2+-permeable channel (HACC) activity in root and guard

cells (Pei et al., 2000; Demidchik et al., 2003b, 2007; Foreman

et al., 2003). Therefore, it was reasoned that the Ca2+ con-

ductance was activated by ATP via the production of ROS.

(a) (b)

(c)

Figure 3. Extracellular ATP activates single channels in excised outside-out plasma membrane patches from root epidermal protoplasts.

(a) Burst of channel opening in response to 30 lM ATP, with 20 mM external CaCl2; HP, holding potential.

(b) Burst of channel opening in response to 30 lM 2¢-O-(4-benzoylbenzoyl) adenosine 5¢-triphosphate (BzBzATP).

(c) The channel amplitude increased as the voltage was hyperpolarized (30 lM BzBzATP).



In a preliminary study to test whether ROS production in the

root could respond to purine nucleotides, extracellular

superoxide anion production was quantified through the

reduction of the soluble tetrazolium dye, sodium 3¢-[1-

(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-

nitro) benzene-sulfonic acid hydrate (TXX; Sutherland and

Learmouth, 1997; Able et al., 1998). This probe has been

used previously to examine Ca2+-stimulated NADPH oxidase

activity in Arabidopsis roots (Mortimer et al., 2008). Here,

superoxide anion production by excised Arabidopsis roots

was found to nearly double in response to 100 lM ADP

(control, 28 � 8 nmol superoxide lg)1 protein, n = 6; ADP,

45 � 6 nmol superoxide lg)1 protein, n = 6). However, this

approach could not be applied to single roots, and did not

permit the spatial resolution of ROS production. Therefore,

ROS imaging was used instead.

The application of ATP to individual Arabidopsis roots

resulted in the rapid (response observed by 15 sec),

dose-dependent (from 10 lM to 1 mM) accumulation of

intracellular ROS (Figure 5a), detected by ester-loaded

5- and 6-chloromethyl-2¢,7¢-dichlorodihydrofluorescein

(CM-H2DCF; Foreman et al., 2003; Shin and Schachtman,

2004). The accumulation of ROS occurred primarily in the

mature root (Figure 5a). The fluorescence intensity over the

length of single primary roots was 32 � 3 fluorescence per

pixel under control conditions, with a peak response of

117 � 4 fluorescence per pixel evoked by 100 lM ATP

(n = 27; P < 0.05, Tukey’s multiple comparison test). The

response to 100 lM ATP was significantly inhibited by the

NADPH oxidase inhibitor diphenylene iodonium (DPI;

Bolwell and Wojtaszek, 1997; Foreman et al., 2003) (20 lM

DPI; 15 � 1 fluorescence per pixel, n = 27; P < 0.05;

Figure 5a). Of the ten A. thaliana respiratory burst oxidase

homologs (AtRBOH) encoding NADPH oxidases, AtRBOHC

is expressed in the epidermis, and its loss-of-function

mutant (root hair defective 2, rhd2) fails to produce ROS to

activate channel-mediated Ca2+ influx in this tissue (Fore-

man et al., 2003). Basal levels of ROS in rhd2/AtrbohC roots

were lower than that in the wild type (WT; 25 � 1 fluores-

cence per pixel, n = 27), and the mutant failed to accumulate

(a)

(d)

(b)

(c)

Figure 4. A 19-pS Ca2+-permeable channel is activated in response to

extracellular ATP.

(a) Single-channel conductance of the first open state in response to 30 lM

ATP and 30 lM 2¢-O-(4-benzoylbenzoyl) adenosine 5¢-triphosphate (BzBzATP).

(b) The open probability (Popen) declines in the continued presence of 30 lM

ATP.

(c) The mean � SE increases in Popen upon hyperpolarization (30 lM

BzBzATP).

(d) The mean � SE Popen at )100 mV is decreased by 100 lM extracellular

Gd3+ (with the addition of 30 lM BzBzATP for activation). All trials were

performed in the presence of 20 mM external CaCl2.

(a) (b)

Figure 5. The rhd2/AtrbohC NADPH oxidase mutant shows an impaired accumulation of reactive oxygen species (ROS) in response to extracellular ATP.

(a) Accumulation of ROS in a representative wild-type (WT) root in response to 100 lM ATP. Upper panels, bright-field and false-colour mapped images of the WT

control. The mean � SE level was 32 � 3 fluorescence per pixel (n = 27). Middle panels, WT response to 100 lM ATP, recorded 30 sec after application. The

mean � SE level was 117 � 4 fluorescence per pixel (n = 27). Lower panels, WT response to 100 lM ATP with 20 lM diphenylene iodonium (DPI), which is an

NADPH oxidase inhibitor (Bolwell and Wojtaszek, 1997; Foreman et al., 2003). The mean � SE level was 15 � 1 fluorescence per pixel (n = 27).

(b) The accumulation of ROS evoked by 100 lM ATP was impaired in rhd2/AtrbohC. The mean � SE level was 45 � 2 fluorescence per pixel (n = 27). Scale bars for

all panels: 2 mm.



ROS to WT levels in response to 100 lM ATP (45 � 2 fluo-

rescence per pixel, n = 27; P < 0.05; Figure 5b). The increase

observed in the mutant is compatible with the known

presence of AtRBOHD or AtRBOHF in the roots (Kwak et al.,

2003), which in leaves can support the induction of ROS by

ATP (Song et al., 2006), or the activation of alternative

generators of ROS. AtRBOHC is also activated by increased

[Ca2+]cyt (Takeda et al., 2008). Blocking the influx of Ca2+ with

300 lM Gd3+ caused a 52% inhibition of the WT ROS

accumulation evoked by 100 lM ATP (56 � 3 fluorescence

per pixel, n = 27; P < 0.05). Although Gd3+ was a very

effective blocker of the ATP-induced Ca2+ conductance

(Figures 2c and 4d), it still allowed some ATP-induced

increase in [Ca2+]cyt in protoplasts (Figure 1c). This suggests

that ATP causes an increase in [Ca2+]cyt through intracellular

Ca2+ release, which could activate AtRBOHC to generate

ROS, which in turn activate Ca2+ influx through the HACC

conductance. This channel-mediated Ca2+ influx could then

potentiate AtRBOHC activity.

The Ca2+ conductance lies downstream of ATP-activated

AtRBOHC

Patch-clamp electrophysiology was used to determine

whether the HACC conductance lies downstream of

AtRBOHC. Under control conditions, the Ca2+ conductance

in rhd2/AtrbohC root mature epidermal protoplasts was not

significantly different to that in WT (whole-cell I at )200 mV:

rhd2/AtrbohC, )203 � 56 pA, n = 6; WT, )217 � 38 pA,

n = 6). However, unlike in the WT, the Ca2+ conductance in

rhd2/AtrbohC did not increase in response to extracellular

ATP (Figure 6a; I at )200 mV; )195 � 48 pA, n = 6). This

suggests that the WT channel activation was caused by

increasingly oxidative conditions, which is consistent with

ATP-induced ROS production by RHD2/AtRBOHC. Reducing

conditions, imposed by 1 mM DTT at both membrane faces,

caused WT mature epidermal protoplasts to phenocopy

rhd2/AtrbohC, thereby preventing the increase in Ca2+ con-

ductance by extracellular ATP (Figure 6b; n = 6). The results

are consistent with a signalling pathway in the mature epi-

dermis in which the stress levels of ATP activate AtRBOHC,

and hence the hyperpolarization-activated Ca2+ channel

conductance.

ATP-induced transcription requires AtRBOHC

No difference in root cell death kinetics (Chivasa et al.,

2005) was observed between the WT and rhd2/AtrbohC

(data not shown), suggesting that this response is not

governed by the ATP/AtRBOHC pathway. However, extra-

cellular ATP in leaves stimulates the transcription of genes

involved in stress signalling, such as AtMPK3 (Song et al.,

2006). This ATP effect is dependent on Ca2+ influx (Song

et al., 2006). Root AtMPK3 transcription is also upregulated

by stress-related ROS (Kovtun et al., 2000; Rentel et al.,

2004). As stress conditions such as wounding, osmotic

stress and touch cause ATP release (Song et al., 2006; Roux

and Steinebrunner, 2007), and as extracellular ATP causes

NADPH oxidase-dependent ROS production, we reasoned

that root AtMPK3 transcription lies downstream of the ATP

activation of RHD2/AtRBOHC and the HACC. Application of

ATP to rhd2/AtrbohC roots failed to cause increased AtMPK3

transcription (Figure 6c), confirming this premise.

DISCUSSION

The possibility that extracellular ATP has a regulatory role to

play in plants has received relatively little attention since the

initial studies were published. It is now clear that ATP is

involved in plant growth and stress responses, but mecha-

nistically our understanding of ‘how’ has remained largely at

the black-box level. Whereas there are parallels with animal

cells for mechanisms of ATP release and regulation of its

extracellular concentration (Kim et al., 2006; Roux and Stei-

nebrunner, 2007), a fundamental question of whether the

wall is involved in plant ATP signalling has only now been

answered. In the mature root epidermis, the wall is redun-

dant in the elevation of [Ca2+]cyt, and ATP is sensed at the

plasma membrane. This does not preclude the activity of cell

(a) (b)

(c)

Figure 6. Ca2+ channel activity and transcription are impaired in rhd2/

AtrbohC.

(a) ATP (20 lM) did not evoke Ca2+ channel activity in epidermal protoplasts

from rhd2/AtrbohC roots (with the same recording conditions as described in

Figure 2a). The whole-cell currents are shown above mean � SE current–

voltage relationships (n = 6).

(b) Reducing conditions (1 mM DTT in bath and pipette solutions) prevented

Ca2+ channel activation by 20 lM ATP in wild-type (WT) root epidermal

protoplasts (n = 6).

(c) Root MPK3 transcription did not increase in rhd2 upon 15 min of exposure

to 1 mM ATP. Primers produced a 959-bp transcript.



wall proteins in other cell types, and the possibility of their

involvement would certainly mark a distinctive split between

plant and animal ATP signalling systems.

This study has shown that plant extracellular ATP results

in the activation of plasma membrane NADPH oxidases.

These enzymes catalyse the formation of the extracellular

superoxide anion, and are implicated in plant development,

response to abiotic stress and defence against pathogens

(Kwak et al., 2003; Apel and Hirt, 2004; Carrol et al., 2005).

The control of rboh NADPH oxidases in Arabidopisis is also

exerted by ABA (Kwak et al., 2003); the relationship of ATP

and ABA in rboh-mediated growth and stress responses

must now be examined. In animal cells, plasma membrane

P2 ionotropic receptors bind extracellular ATP, causing

an influx of Ca2+, which acts as a second messenger to

activate NADPH oxidase, resulting in a respiratory oxidative

burst (Parvathenani et al., 2003). The application of La3+

(as a cation channel blocker) or the P2 receptor antago-

nist pyridoxalphosphate-6-azophenyl-2¢,4¢-disulfonic acid

(PPADS) prevented superoxide production by extracellular

ATP in Arabidopsis leaves (Song et al., 2006). As both

lanthanides and PPADS were found previously to inhibit

ATP-induced [Ca2+]cyt elevation in Arabidopsis roots (De-

midchik et al., 2003a) and leaves (Jeter et al., 2004), it has

been proposed that, as in animals, extracellular ATP acti-

vates a receptor in the plasma membrane that causes

channel-mediated Ca2+ influx, and subsequently the ele-

vated [Ca2+]cyt activates NADPH oxidases (Song et al., 2006).

However, the present study on roots shows that plasma

membrane Ca2+ channels operate downstream of an NADPH

oxidase, and that it is most likely that an ATP-induced

release of Ca2+ from internal stores lies at the beginning of

the [Ca2+]cyt elevation. Thus, PPADS is more likely to affect

an ATP-binding protein equivalent to a metabotropic, rather

than an ionotropic, receptor. The combination of the plasma

membrane AtRBOHC NADPH oxidase and its partner

ROS-activated plasma membrane Ca2+ channels would

increase the magnitude of the [Ca2+]cyt response, to trigger

transcription.

NADPH oxidase and plasma membrane Ca2+ channel

activity are involved in a range of plant developmental and

environmental response pathways (Pei et al., 2000; Foreman

et al., 2003; Apel and Hirt, 2004). The plasma membrane Ca2+

channels lying downstream of extracellular ATP are tran-

siently activated, voltage dependent and appear to be redox

sensitive. As they would open over the plasma membrane

voltage range reported for the mature epidermis (Maathuis

and Sanders, 1993), they would facilitate transient Ca2+

influx, and contribute to [Ca2+]cyt elevation. With a single-

channel conductance of 19–20 pS, these channels appear to

be distinct from the 15-pS Ca2+-permeable channel reported

previously in the plasma membrane of the mature root

epidermis, under an identical transmembrane Ca2+ gradient

(Demidchik et al., 2007). The 15-pS channel was also hyper-

polarization activated, and its Popen increased transiently in

response to intracellular H2O2 (Demidchik et al., 2007). The

identity of the ROS activating the 19–20-pS channel is

unknown at present, and it is also feasible that ROS

activation could be indirect.

The reliance of ATP-induced AtMPK3 transcription on the

AtRBOHC NADPH oxidase identifies a signalling pathway

that operates in stress adaptation (Figure 7). AtMPK3 is part

of a stress-responsive phosphorylation cascade initiated in

some cases by the activity of ANP1, a mitogen-activated

protein kinase kinase kinase (Kovtun et al., 2000). Touch, low

temperature and salinity responses involve AtMPK3

(Mizoguchi et al., 1996), and it functions downstream of

the oxidative signal-inducible 1 (OXI1) kinase involved in

root growth and basal resistance to pathogens (Rentel et al.,

2004). ATP is released by Arabidopsis in response to hyper-

and hypo-osmotic stress (Jeter et al., 2004). AtRBOHC is

implicated in the response to hypo-osmotic shock (Macph-

erson et al., 2008), and AtMPK3 is known to be involved

in hypo-osmotic and moderately hyperosmotic stress

responses, where its transcription is dependent on Ca2+

influx (Droillard et al., 2002). The results obtained here for

roots, and previously for the Ca2+ influx-dependent ATP

induction of AtMPK3 transcription in leaves (Song et al.,

2006), identify extracellular ATP, released during stress, as a

regulator of the MPK cascade. In the present study, roots

were grown on medium with an osmolarity of 115 mOsM,

and were then immersed in assay medium with an

Figure 7. Schematic model of a possible pathway for the induction of MPK3

transcription by extracellular ATP. The putative ATP-binding protein is

depicted as residing in the plasma membrane (PM). The perception of ATP

triggers (by an unknown mechanism) a release of intracellular Ca2+ from

stores such as the vacuole, endoplasmic reticulum or mitochondria. Elevated

intracellular cytosolic free Ca2+, [Ca2+]cyt, stimulates AtRBOHC NADPH

oxidase activity via its EF hands (Takeda et al., 2008). The superoxide anion

would then be produced extracellularly, and converted to other ROS, such as

H2O2 and hydroxyl radicals (Foreman et al., 2003). Peroxide could enter the

cytosol through PM aquaporins (Bienert et al., 2007; Dynowski et al., 2008)

and contribute to the accumulation of intracellular ROS shown in Figure 5.

The identity of the ROS activating the PM Ca2+-permeable channel are

unknown at present, but activation could be at the extra- or intracellular

membrane face (Demidchik et al., 2003b, 2007). Channel opening would

further increase [Ca2+]cyt, leading to further NADPH oxidase stimulation and

the induction of MPK3 transcription (Droillard et al., 2002; Song et al., 2006).

Intracellular ROS could also contribute to MPK3 transcription (Kovtun et al.,

2000; Rentel et al., 2004).



osmolarity of approximately 11 mOsM for non-invasive

slowly vibrating microelectrode (MIFE; microelectrode ion

flux estimate) determinations of net Ca2+ influx, ROS imag-

ing and transcript analysis. This represents a hypo-osmotic

stress that could have ‘primed’ the system, but at present,

the implications for the magnitude of the subsequent

response to applied ATP are unknown.

The identity of the plasma membrane receptor for ATP

remains enigmatic. Despite the recent breakthrough find-

ings of P2X equivalents in the amoeba Dictyostelium and the

unicellular green alga Ostreococcus tauri, neither of these

function at the plasma membrane (Fountain et al., 2007,

2008), and they offer no clues to the identity of higher plant

receptors. Higher plant genomes appear bereft of P2 homo-

logues. This study has shown that higher plants could have

evolved a distinct mechanism to transduce the ATP signal at

the plasma membrane. Isolation of the ATP receptor clearly

cannot rely on an animal or even algal paradigm, and

represents a challenging task. However, the strategies used

successfully to identify ligand–receptor interactions in plant

membranes (Yamaguchi et al., 2006) may yet prove fruitful.

EXPERIMENTAL PROCEDURES

Growth conditions and measurements

Arabidopsis thaliana (Col-0 and rhd2/AtrbohC from laboratorystock) was grown aseptically and vertically at 22�C for 5–15 days (16-h day, with 100 lmol m)2 sec)1 irradiance) on medium comprising0.3% (w/v) Phytagel (Sigma-Aldrich, http://www.sigmaaldrich.com),full-strength MS (Duchefa, http://www.duchefa.com) medium and1% (w/v) sucrose. The rhd2/AtrbohC mutant is defective in theAt5g1060 gene (GenBank accession number AF055355; TIGR, http://www.tigr.org/tdb/e2k1/ath1), and four mutant alleles have beendefined (Foreman et al., 2003). The rhd2-4 mutant is restored bycomplementation (Foreman et al., 2003), and was used here. Forgrowth determination, plants were grown on 5 mM KNO3, 5 mM

MgSO4, 5 mM 2-(N-morpholino)-ethanesulphonic acid (MES), 2 mM

NaCl, 1 mM CaCl2, 1 mM (NH4)2HPO4 and 1% (w/v) sucrose, sup-plemented with vitamins and micronutrients, and adjusted to pH 5.7with bis-tris propane. Free ion contents were estimated with GEO-CHEM (Parker et al., 1995) and Maxchelator (Patton et al., 2004). Theroot lengths were measured using ImageJ software (http://rsb.info.nih.gov/ij) at the 6–8-day interval. Test additions did not affectgermination.

Net Ca2+ fluxes

Net Ca2+ fluxes were measured using the MIFE technique (Shabalaet al., 1997). Excised roots (5–7 mm from the apex) were mounted ina perspex chamber by an agar drop, and immersed in 0.5 ml ofassay solution comprising 0.1 mM KCl, 0.1 mM CaCl2, 4 mM MESand 2 mM Tris base (pH 6.0). The root was viewed under an Olym-pus I·50 microscope (Olympus, http://www.olympus-global.com)using 200· magnification. Ion-selective microelectrodes with anexternal tip diameter of approximately 3 lm were manufacturedand calibrated as described previously (Shabala et al., 1997). Elec-trodes were calibrated before and after use. Purines (tested up to1 mM; Sigma-Aldrich) did not affect calibration values or electrode

response times. The microelectrode was placed 20 lm above theroot surface. During measurements, the electrode was movedbetween two positions, 20 and 50 lm above the root surface, in asquare-wave manner with a 5-sec half cycle. Measurements ofmature epidermal flux were taken 1–1.5 mm from the root apex. Thenet Ca2+ flux was measured for 5 min prior to the addition of 0.5 mlof assay solution; Ca2+ was maintained at 0.1 mM, estimated usingMAXCHELATOR (Patton et al., 2004), and confirmed using a Ca2+-selective microelectrode in root-free assay. Measurements wereresumed after 20–30 sec, when unstirred layer conditions werereached.

[Ca2+]cyt measurement

Protoplasts were isolated from the mature root epidermis ofArabidopsis expressing cytosolic (apo)aequorin (Cauliflower mo-saic virus, CaMV, 35S promoter) using the protocol described byDemidchik et al. (2007), with the modification that incubation withwall-degrading enzymes was increased from 50 to 60 min. Protop-lasts were held for 3–4 h (in the dark, at 28�C) in recording medium(1 mM CaCl2, 1 mM KCl, 1 mM MgCl2, 10 mM glucose, pH 6.0, 2 mM

Tris base/MES; 290–300 mOsM, with D-sorbitol) with 10 lM coelen-terazine (NanoLight Technology, http://www.nanolight.com). Afterwashing, recording medium (without coelentrazine) and standardluminometry procedures were used (Demidchik et al., 2007). Thedischarging solution contained 2 M CaCl2 and 20% (w/v) ethanol,and [Ca2+]cyt values were estimated according to the methoddescribed by Fricker et al. (1999).

Electrophysiology

Protoplasts used for patch clamping were 20 � 1.5 lm in diameter.For ATP studies, the pipette solution contained 40 mM K-gluconate,10 mM KCl and 1 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N¢,N¢-tetra-acetic acid (free Ca2+ to 100 nM with 0.4 mM CaCl2), pH 7.2(2 mM Tris/MES). For BzBzATP, Na-gluconate and NaCl replaced K-gluconate and KCl. The control bathing solution comprised 20 mM

CaCl2, 0.1 mM KCl and 0.3 mM NaCl (varied to supply equimolar Na+

to that in the purine-containing test), pH 6 (2 mM Tris/4 mM MES).Solutions were adjusted to 290–300 mOsM with D-sorbitol. Thepatch-clamp procedures were carried out as described previously(Demidchik et al., 2003b, 2007). The current–voltage relationshipswere recorded within 1 min.

Imaging and transcription

The ROS imaging with 50 lM 5- and 6-chloromethyl-2¢,7¢-dichloro-hydrofluorscein diacetate, acetyl ester (Molecular Probes, now partof Invitrogen, http://www.invitrogen.com) was performed asdescribed previously by Shin and Schachtman (2004). For imagingand transcription studies, WT and rhd2/AtrohC roots were treatedwith control and ATP-containing solutions, as used for determiningnet Ca2+ influx. DPI was purchased from Sigma-Aldrich. The imagesof roots were acquired with an SMZ 1500 microscope (Nikon, http://www.nikon.com) and a Retiga cooled 12-bit camera (Qimaging,http://www.qimaging.com). The preliminary study of superoxideanion production was performed according to the methoddescribed by Mortimer et al. (2008). Roots were excised fromapproximately 30 seedlings, and submerged in 10 mM phosphatebuffer with 0.5 mM CaCl2 and 500 lM XTT (Sigma-Aldrich) (Suth-erland and Learmouth, 1997; Able et al., 1998), pH 6.0, with orwithout 100 lM ADP. Roots were incubated at 21�C in the dark for



30 min and the sample absorbance at 470 nm was measured (1-cmpath length; Helios c spectrophotometer; Unicam, http://www.unicamgroup.com). Superoxide production was estimated usingthe XTT extinction co-efficient of 2.16 · 104 (mol L)1))1 cm)1. Rootswere snap-frozen in liquid nitrogen and homogenized with buffercomprising 50 mM Tris acetate, 100 mM potassium acetate, 1 mM

EGTA, 1 mM DTT and 20% (v/v) glycerol. The sample was clarifiedby centrifugation for 10 min at 4�C, and the supernatant wasassayed for protein content (Bio-Rad, http://www.bio-rad.com). Thexanthine oxidase/hypoxanthine (XO/HX) superoxide anion-gener-ating system (Suzuki, 2000) was used as a cell-free check on theability of ADP to perturb the XTT assay. XO (Sigma-Aldrich) wasfreshly prepared in experimental buffer at 0.5–5 U ml)1. HX (Sigma-Aldrich) was added to a final concentration of 500 lM with orwithout ADP.

RNA was extracted from excised roots (RNEasy; Qiagen, http://www.qiagen.com). cDNA was prepared using reverse transcriptase(BioScript; Bioline, http://www.bioline.com), and PCR was carriedout with the Arabidopsis actin 8 gene (At1g49240; forward primer,5¢-AGAAAGATGCGTATGTTGGTGA-3¢; reverse primer, 5¢-CTGC-TGGAAAGTGCTGAGGGAA-3¢). The number of cycles and levels ofcDNA required to amplify the product during the exponential phase,and at an equal level between all wild-type, mutant and ATP-treatedsamples, were determined. RT-PCR was then carried out for MPK3(At3g45640; forward primer, 5¢-TGTTGGATACGGAGACGA-3¢;reverse primer, 5¢-GCTGCACTTCTAACCGTA-3¢) for comparison ofMPK3 transcript levels between treatments. These primers produce a959-bp transcript.

ACKNOWLEDGEMENTS

The study was funded by the Leverhulme Trust, the BBSRC, theRoyal Society, the Australian Research Council, the FulbrightFoundation and the Monsanto Company.

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Plant extracellular ATP signalling by plasma membrane NADPH oxidase and Ca 2+ channels

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