Extracellular ATP is a central signaling moleculein plant stress responsesYangrong Cao1, Kiwamu Tanaka1, Cuong T Nguyen and Gary Stacey2

Available online at www.sciencedirect.com

ScienceDirect

Because of their sessile nature, plants have developed a

number of sophisticated signaling systems to adapt to

environmental changes. Previous research has shown that

extracellular ATP is an important signaling molecule used by

plants and functions in a variety of processes, including growth,

development, and stress responses. Recently, DORN1 was

identified as the first plant purinoceptor, essential for the plant

response to ATP. The identification of the receptor is a

milestone for our overall understanding of various physiological

events regulated by extracellular ATP. In this review, we will

discuss the possible roles of DORN1 providing future direction

for research into the role of extracellular ATP in plants.

Addresses

Divisions of Plant Sciences and Biochemistry, National Center for

Soybean Biotechnology, C.S. Bond Life Sciences Center, University of

Missouri, Columbia, MO 65211, USA

Corresponding author: Stacey, Gary (staceyg@missouri.edu)1 These authors contributed equally to this work.2 Permanent address: 217E C.S. Bond Life Sciences Center, 1201

Rollins Street, University of Missouri, Columbia, MO 65211, USA.

Current Opinion in Plant Biology 2014, 20:82–87

This review comes from a themed issue on Biotic interactions

Edited by Makoto Hayashi and Martin Parniske

http://dx.doi.org/10.1016/j.pbi.2014.04.009

1369-5266X/Elsevier Ltd

IntroductionAdenosine 50-triphosphate (ATP) serves as a universal

energy source for all organisms. ATP is maintained at a

very high concentration (�mM) inside of the cell [1].

However, wounding (e.g. due to herbivory) or stimulation

of the cell with the appropriate stimuli (e.g. touch) can

cause the release of ATP into the extracellular matrix

where it can be recognized by plasma membrane loca-

lized purinoceptors. Animals have two general classes of

purinoceptors: P2X ligand-gated ion channels and P2Y G

protein-coupled receptors [2]. Extracellular ATP has

been shown to play a variety of roles in animals, including

muscle contraction, inflammation, neurotransmission, cell

growth and death [3].

In contrast to animals, the role of extracellular ATP as a

signal in plants has received considerably less attention.

Current Opinion in Plant Biology 2014, 20:82–87

Several reviews have been published in last few years [4–7] surveying evidence that extracellular ATP is a signal in

plants. However, relatively few laboratories have focused

on extracellular ATP reflecting the skepticism that exists

about its function in plants. A similar situation was pre-

sent in the animal research community for many years.

This lasted from the initial reports on extracellular ATP

to the first identification of specific purinoceptors [8,9],

which seemed to convince people that this signaling

pathway did indeed exist in animals [10,11]. Therefore,

the hope is that the recent identification of the first plant

purinoceptor DORN1 [12��] will stimulate greater in-

terest in the role of extracellular signaling in plants.

DORN1 unveils an extracellular ATP signalingpathway in plantsSequence based searches failed to identify animal-like

P2X and P2Y receptors in plants [4]. Therefore, Choi

et al. [12��] utilized a forward genetic screen to identify

Arabidopsis thaliana mutants that failed to show an intra-

cellular calcium response upon the addition of ATP. This

led to the identification of several mutants defective in the

DORN1 gene, which encodes LecRK-I.9 (At5g60300), a L-

type lectin receptor-like kinase. A variety of assays were

used to show that dorn1 mutant plants are insensitive to

ATP, as well as other nucleotides, with the exception of

pyrimidine nucleotides (such as CTP). The extracellular

lectin domain of DORN1 directly binds to ATP with high

affinity (Kd = 45.7 nM) and with the same relative selectiv-

ity as shown by the plant to nucleotides. For example, as

expected, dorn1 mutant plants still respond to CTP [12��].

Therefore, it is likely that Arabidopsis has a separate re-

ceptor for CTP. This is not surprising given that animals

also possess several purinoceptors that can vary with regard

to the ligand specificity. For example, some animal P2Y

receptors recognize other nucleotides (e.g. ADP and UTP)

in addition to ATP [3,13].

Given the accepted nomenclature for purinoceptors in

animals (i.e. P2Y, P2X), Choi et al. [12��] proposed

that DORN1 be considered the founding member in a

new family of purinoceptors, P2K (where K refers to

kinase). Computer based homology searches indicate that

lectin-receptor kinases are only found in plants [14] and,

therefore, the P2K family appears to be plant-specific.

Within the plant kingdom, L-type LecRKs are found in

primitive plants (e.g. moss) through higher plants, but not

in algae [12��]. Indeed, a P2X receptor-like receptor was

previously identified in green algae but appears to act in

the cytoplasm, not as a plasma membrane receptor [15].

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Extracellular ATP in plant stress responses Cao et al. 83

In Arabidopsis, there are 45 genes encoding L-type

LecRK [16], some of which have been implicated in

plant innate immunity [17��,18�,19�,20], response to

wounding [21], salt stress [22,23], osmotic stress

[23,24], and hormone signaling [23–25], suggesting that

the L-type LecRK gene family plays an important role in

mediating plant response to diverse stresses. It is con-

ceivable that some LecRKs function as P2K purinocep-

tors, functionally redundant to DORN1 in specific tissues

and/or growth or developmental stages. Given that dorn1mutant plants still respond normally to pyrimidine

nucleotides, it is also possible that other LecRKs are

pyrimidine receptors.

Extracellular ATP plays a fundamental role inmediating plant environmental responsesExogenous application of ATP can rapidly trigger

elevation of cellular Ca2+ concentration, production of

nitric oxide (NO), reactive oxygen species (ROS), and

phosphatidic acid, and activation of mitogen-activated

protein kinase (MPK) phosphorylation [26�,27–29,30�,31–33]. Many of these same responses (e.g.

Ca2+, NO, and ROS) also exemplify the response of

Figure 1

Defence response(GO:0006952)

Response to stress(GO:0006950)

N-terminmodi(GO:0

Protein(GO:0

Regula-tion of

defenseresponse(GO:0031

347)Protein lipida

(GO:00064

Salt stressresponse(GO:00096

51) Lipoprotemetabolis

(GO:00421

Response to chitin

(GO:0010200)

Immune response(GO:0006955)

Otherorganismsresponse

(GO:0051707)

Biotic stimuliresponse

(GO:0009607)

Signaltransduction(GO:0007165)

Functional categorization of ATP-upregulated genes using GO term enrichmen

322 ATP-upregulated genes by AgriGO (http://bioinfo.cau.edu.cn/agriGO/). The

rectangle represents a cluster of related terms labeled according to representa

Size of the rectangle reflects the p-value. Rectangles are grouped in superclus

immune response (green), protein modification (red), and programmed cell dea

Expression Omnibus.

www.sciencedirect.com

animal cells to extracellular ATP. These responses ulti-

mately lead to the induction of gene expression. For

example, Choi et al. [12��] showed that the expression

of �600 genes in Arabidopsis responded to the addition of

ATP but none of these genes responded in dorn1 mutant

plants. A total of 322 genes were upregulated by ATP

addition [12��]. A comparison of this list of ATP-induced

genes to those responding to a variety of other stresses

showed a significant overlap. Examples are shown in

Figure 1. Many ATP-induced genes are classified into

categories known to respond to biotic or abiotic stresses.

Consistent with this result, various biotic and abiotic

stimuli induce ATP release [30�,34–39]. Therefore, an

obvious hypothesis is that ATP, released from plants as a

result of stresses, acts as an intermediate signal to activate

stress–responsive pathways.

Role of extracellular ATP in plant response towoundingMany stimuli including chemical, heat, pathogen attack,

and other stresses can trigger ATP release from cells, but

the levels of ATP released are usually very low (as

summarized in [4,5]). For example, nM levels of ATP

al proteinfication031365)

acylation043543)

Programmedcell death

(GO:0012501)

Death(GO:0016267)

Immune systemprocess

(GO:0002376)

Photo-synthesis(GO:0015

979)

Carbohy-drate

biosyn-thesis

(GO:0016051)

Response tostimuli

(GO:0050896)

Plastidorgani-zation

(GO:0009657)

tion97) Peptide

modifi-cation

(GO:0018193)in

m57)

Current Opinion in Plant Biology

t test based on Biological Processes. A list of GO terms was generated for

list was summarized and visualized by REVIGO (http://revigo.irb.hr/). Each

tive GO terms and accession number categories shown in the rectangles.

ters with the same color based on the SimRel semantic similarity measure:

th (dark blue). The accession number of the gene list is GSE52610 in Gene

Current Opinion in Plant Biology 2014, 20:82–87

84 Biotic interactions

were released into the surrounding medium when the

Arabidopsis root was mechanically stimulated [38]. How-

ever, given the high affinity of DORN1 for ATP, these

levels of extracellular ATP may be sufficient to trigger

receptor activation. In contrast, ATP levels released at the

site of wounding can reach �40 mM [30�]. Indeed, Choi

et al. [12��] found that �60% of the genes induced by ATP

addition to Arabidopsis were also induced by wounding.

The majority (90%) of these overlapping genes

responded very early to wounding [12��]. Transgenic

lines ectopically expressing DORN1 at a high level

showed a much stronger gene induction in response to

both ATP and wound treatments; whereas expression was

markedly reduced in the dorn1 mutant plants. These data

suggest that extracellular ATP is released during wound-

ing as a damage associated molecular pattern (DAMP)

signal, which is then recognized by the DORN1 receptor

(Figure 2). ATP is well known as a DAMP signal in

animals where it contributes to the wound-induced

inflammatory response which is an important defense

against short-term pathogen infection [40,41]. Although

comparable data is lacking for other biotic and abiotic

stresses, it seems likely that extracellular ATP signaling,

mediated through DORN1, is also important in the plant

response to a variety of stresses.

Role of extracellular ATP signaling in plantinnate immunityIn addition to the response to wounding, there is also

some evidence to suggest that extracellular ATP plays an

important role in plant immune responses. The function

Figure 2

[ATP]e

[ATP]i(mM level)

Wound Exocytosis Transport

An overview of ATP signaling pathway in plants. Cytosolic ATP is discharge

transport. The released ATP binds to the extracellular lectin domain of the D

domain. Receptor activation ultimately leads to a variety of cellular respons

expression.

Current Opinion in Plant Biology 2014, 20:82–87

of extracellular ATP in regulating plant immune

responses seems to be dose-dependent and time-depend-

ent. Short-term treatment (minutes to several hours) with

ATP induced defense-related gene expression and respir-

atory burst oxidase homolog D and F (RBOHD and

RBOHF)-dependent ROS production in Arabidopsis[30�]. Exposure to high levels of ATP (�mM) was shown

to trigger programmed cell death in Populus euphratica[42�]. Similar treatments also led to stomatal closure in

Arabidopsis [43]. Both program cell death and stomatal

closing are viewed as positive indicators of a plant innate

immunity response. Recently, ATP addition was reported

to trigger calcium increase in endoplasmic reticulum [44]

and mitochondria [45], which might link to the induction

of apoptosis [46]. Suppression of apyrases (AtAPY1 and

AtAPY2), which leads to a slight increase of extracellular

ATP levels, induced the expression of several genes, most

of which are involved in biotic stress responses [47��].These data suggest that short-term treatment with ATP

or exposure to higher levels of ATP can enhance plant

defense. However, other conflicting data are available,

perhaps reflecting differences in experimental design.

For example, Clark et al. [43] reported that treatment

with low levels of ATP resulted in stomatal opening

[43,48], which, although not tested, would allow more

efficient pathogen invasion. Prolonged exposure (�days)

to exogenous ATP was shown to suppress hypersensitive

cell death induced by a pathogen-derived mycotoxin [49]

and decrease salicylic acid production, pathogenesis-

related (PR) gene expression, and resistance to bacterial

pathogens and tobacco mosaic virus [50,51], suggesting

[Ca2+ ]i

LectinAPY

Kinase

MPKKK?MPKK?MPK3/6

Ca2+

Ca2+

Binds to ATP receptor

DORN1Limit [ATP]e

Current Opinion in Plant Biology

d outside the cell via cell lysis (i.e. wounding), exocytosis, or active

ORN1 receptor, which in turn activates the intracellular DORN1 kinase

es, including increased cytosolic Ca2+, MAPK activation, and gene

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Extracellular ATP in plant stress responses Cao et al. 85

that prolonged ATP treatment or exposure to low levels

of ATP can suppress plant immune responses.

Plant innate immunity is composed of two successive

layers. The initial plant response is often mediated by

recognition of microbe-associated molecular patterns

(MAMP); that is, conserved molecular signatures on

the microbe that plants have evolved to recognize. This

recognition results in MAMP-triggered immunity (MTI).

However, pathogens adapted to their host by synthesizing

and secreting into the host cell specific proteins, termed

effectors, which actively suppress innate immunity.

Plants can counter this suppression through the pro-

duction of resistance proteins (R proteins) that, either

directly or indirectly, recognize specific effectors. This R

protein mediated immunity is termed effector-triggered

immunity (ETI).

There is considerable overlap between genes induced by

ATP and those also induced by a variety of pathogens

(Figure 1), which might reflect an overlap between the

plant DAMP response (to ATP) and MTI. Indeed, the

addition of chitin mixture or yeast extract is known to

induce ATP release in the root tissues of Medicago trun-catula and Salvia miltiorrhiza [35,39]. Previously, LecRK-

I.9 (DORN1) was shown to be a potential target of IPI-O

[52], a RXLR-dEER effector protein secreted by the

pathogen Phytophthora. Indeed, mutant plants defective

in LecRK-I.9 showed enhanced susceptibility to the

oomycete pathogen P. brassicae [17��]. Strong ectopic

expression of LecRK-I.9 in Arabidopsis, potato, and Nicoti-ana benthamiana plants resulted in increased resistance to

Phytophthora [17��,53�], suggesting the possibility of using

DORN1 to improve disease resistance in crop plants.

Therefore, the data suggest a central role for ATP in

mediating the plant innate immunity response, as well as

the DORN1 receptor, which pathogens have evolved to

target with effector proteins, presumably to suppress

disease resistance.

A model for ATP actionThe identification of the first plant purinoceptor should

encourage greater attention to the role of ATP as an

important signal in plants [12��]. A possible model for

the action of ATP is shown in Figure 2. In this model,

cytosolic ATP is released by physical wounding or other

stresses. Since brefeldin A, an inhibitor of vesicular

transport, blocks ATP release upon chitin treatment,

it is likely that vesicle fusion with the plasma mem-

brane is one mode of ATP release [39]. Such a path for

ATP release has been well documented in animal

systems [54]. Recently plasma membrane-located

ATP transporters were identified, which could transport

ATP into the plant apoplast [55]. The released ATP

directly binds to DORN1 which causes intracellular

signaling by activating DORN1’s kinase activity. Sub-

sequent signaling steps, undefined at this point, would

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lead to induction of calcium influx, ROS and NO

production, MPK phosphorylation, and gene expres-

sion. This pathway would activate those plant processes

necessary to protect the plant against detrimental

environmental change.

Conclusion and future directionsA wealth of published data strongly supports the existence

of extracellular ATP in plants and it role as a vital extra-

cellular signal. The identification of the DORN1 purino-

ceptor and studies of plants lacking this receptor indicate

that extracellular ATP is a central signal involved in the

plant response to a variety of stresses. It seems likely that

these stresses induce the release of ATP and responses,

previously attributed strictly to the stress, are actually in

direct response to extracellular ATP. Therefore, in order to

fully understand the mechanisms by which plants recog-

nize and respond to stress, it is essential that we understand

the critical and central role of extracellular ATP. Many,

many questions remain to be answered. For example, work

on the structure and function of DORN1 is just beginning.

Similar to other receptors, it is likely that DORN1 acts in

conjunction with other proteins, which likely facilitate

downstream signaling. Examples would include those

proteins that are targets of DORN1 kinase activity.

Animals possess a number of P2X and P2Y receptors

and, therefore, one would expect that other P2K receptors

will be found in plants. Choi et al. [12��] found that

mutations in DORN1 eliminated all of the responses that

they could measure in response to extracellular ATP

addition. Therefore, other Arabidopsis P2K receptors likely

act in specific tissues or during specific growth or devel-

opmental stages. However, the Arabidopsis receptor that

mediates the response to CTP remains to be identified and

may be a P2K family member.

AcknowledgementsThis work was supported by the Division of Chemical Sciences,Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S.Department of Energy (grant no. DE-FG02-08ER15309) and the Next-Generation BioGreen 21 Program Systems and Synthetic AgrobiotechCenter, Rural Development Administration, Republic of Korea (grant no.PJ009068).

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53.�

Bouwmeester K, Han M, Blanco-Portales R, Song W, Weide R,Guo LY, van der Vossen EA, Govers F: The Arabidopsis lectinreceptor kinase LecRK-I.9 enhances resistance toPhytophthora infestans in Solanaceous plants. PlantBiotechnol J 2014, 12:10-16.

The authors demonstrate that strong ectopic expression of LecRK-I.9(DORN1) in transgenic plants increases plant resistance to Phytophthorainfection.

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Current Opinion in Plant Biology 2014, 20:82–87