Overexpression of a fatty acid amide hydrolase compromisesinnate immunity in Arabidopsis

Li Kang1, Yuh-Shuh Wang1, Srinivasa Rao Uppalapati1, Keri Wang1, Yuhong Tang1, Vatsala Vadapalli2, Barney J. Venables2,

Kent D. Chapman2, Elison B. Blancaflor1 and Kirankumar S. Mysore1,*

1Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK, USA, and2Center for Plant Lipid Research, Department of Biological Sciences, University of North Texas, Denton, TX, USA

Received 29 January 2008; revised 19 May 2008; accepted 5 June 2008; published online 29 July 2008.*For correspondence (fax 001 580 224 6692; e-mail ksmysore@noble.org).

Summary

N-acylethanolamines are a group of lipid mediators that accumulate under a variety of neurological and

pathological conditions in mammals. N-acylethanolamine signaling is terminated by the action of diverse

hydrolases, among which fatty acid amide hydrolase (FAAH) has been well characterized. Here, we show that

transgenic Arabidopsis lines overexpressing an AtFAAH are more susceptible to the bacterial pathogens

Pseudomonas syringae pv. tomato and P. syringae pv. maculicola. AtFAAH overexpressors also were highly

susceptible to non-host pathogens P. syringae pv. syringae and P. syringae pv. tabaci. AtFAAH overexpressors

had lower amounts of jasmonic acid, abscisic acid and both free and conjugated salicylic acid (SA), compared

with the wild-type. Gene expression studies revealed that transcripts of a number of plant defense genes, as

well as genes involved in SA biosynthesis and signaling, were lower in AtFAAH overexpressors than wild-type

plants. Our data suggest that FAAH overexpression alters phytohormone accumulation and signaling which in

turn compromises innate immunity to bacterial pathogens.

Keywords: N-acylethanolamine, FAAH, fatty acid amide hydrolase, salicylic acid, non-host resistance.

Introduction

N-acylethanolamines (NAEs) comprise a class of fatty acid

amides found in trace amounts in animal tissues. One type

of NAE, namely anandamide (N-arachidonoylethanolamine)

functions in the endocannabinoid signaling pathway by

binding to a G-protein-coupled receptor, the cannabinoid

receptor 1 (CB1), in the brain. The same receptor also binds

to D9-tetrahydrocannabinol (THC), the active component of

marijuana (Cravatt et al., 2004; Devane et al., 1992).

Although this type of retrograde signaling by anandamide

has been the major function ascribed to NAEs in mammals,

NAEs are now gaining prominence not only in neuro-

transmission but also in other important physiological pro-

cesses such as immune signaling (Munro et al., 1993). One

significant breakthrough in NAE research was the cloning of

the enzymes that degrades NAEs. One of the enzymes,

called fatty acid amide hydrolase (FAAH), hydrolyzes NAE

into its corresponding free fatty acid and ethanolamine.

As more functions for NAEs in animals were being

discovered, parallel work showed that these fatty acid

amides were also endogenous constituents of plant tissues.

It appears that plants display striking similarities to mam-

malian systems in terms of their accumulation and overall

metabolism of NAEs (Blancaflor and Chapman, 2006;

Chapman, 2000, 2004). For instance, levels of NAEs in

tobacco leaves were shown to rise 10- to 50-fold when they

were challenged with fungal elicitors (Tripathy et al., 1999).

This elevation of endogenous NAEs in plants subjected to

biotic stress mirrors somewhat the accumulation of NAEs

under a variety of neurological and pathological conditions

in mammals (Berdyshev et al., 2000, 2001; Berger et al.,

2004; Schmid et al., 2002). Other biological effects of NAEs

in plants include remodeling of the cytoskeleton, alteration

of seedling growth, inhibition of phospholipase D activity,

modulation of elicitor-induced alkalinization response,

induction of defense gene expression and interaction with

ABA signaling (Austin-Brown and Chapman, 2002;

Blancaflor et al., 2003; Komis et al., 2006; Teaster et al.,

2007; Tripathy et al., 1999). Although such observations

have positioned NAEs as novel metabolites that could

govern important regulatory and signaling pathways in

336 ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd

The Plant Journal (2008) 56, 336–349 doi: 10.1111/j.1365-313X.2008.03603.x

plants (Lopez-Bucio et al., 2006), much remains to be

learned regarding their precise roles in mediating various

plant physiological processes.

The case for NAEs as potential lipid mediators in plants

has been strengthened recently by the identification of an

Arabidopsis thaliana FAAH gene (AtFAAH, At5g64440)

based upon the similarity of the amidase signature (AS)

domain and conservation of several key catalytic residues of

AtFAAH with mammalian FAAH (Shrestha et al., 2003).

When expressed as recombinant proteins in Escherichia

coli, plant FAAHs were capable of hydrolyzing NAEs in vitro

(Shrestha et al., 2003; Wang et al., 2006b). Consistent with

their in vitro NAE hydrolytic activity, seeds and seedlings of

Arabidopsis faah knockouts had elevated levels of endoge-

nous NAEs, while AtFAAH overexpressors had reduced

NAEs (Wang et al., 2006b). AtFAAH overexpressors exhib-

ited enhanced seedling growth, suggesting that the timely

depletion of NAEs might be involved in seed germination

and early seedling establishment (Wang et al., 2006b). The

availability of plants with altered FAAH expression has now

paved the way for more detailed studies of NAE metabolism

and how it affects plant development and responses to the

environment. Because of the increasing role for NAEs in

mammalian immune signaling (Buckley et al., 2006), one

logical area to probe for potential functions for NAEs in

plants would be plant immunity and defense responses,

where NAEs were first suggested to be involved (Tripathy

et al., 1999, 2003).

Here we report that overexpression of AtFAAH, a gene

involved in NAE metabolism, compromises the innate

immunity of Arabidopsis against several host and non-host

pathogens. Collectively, our data indicate that AtFAAH

intersects with plant defense pathways by altering phyto-

hormone signaling. Our results reinforce the notion that

plants, like animals, utilize fatty acid amides in mediating

immunity to pathogens and add to a growing list of

important plant processes that are modulated by NAE. This

study provides genetic evidence that altering the expression

of a gene involved in NAE metabolism can affect plant

defense responses and supports earlier pharmacological

studies implicating NAEs in plant defense signaling.

Results

Overexpressing AtFAAH compromised basal resistance in

Arabidopsis

To investigate the role of FAAH in plant disease resistance,

the Arabidopsis wild-type Columbia-0 (Col-0), a vector con-

trol line, three independent AtFAAH overexpressor lines

(OE2a-1, OE7a-1, OE11a-1) and two AtFAAH T-DNA knockout

lines (faah, salk_095108 and salk_118043), described in

Wang et al. (2006b), were used. When sprayed or infiltrated

with the virulent bacterial strains Pseudomonas syringae pv.

tomato DC3000 (P. s. tomato) and P. syringae pv. maculicola

(P. s. maculicola), all AtFAAH overexpressor lines exhibited

enhanced susceptibility manifested as larger areas of

chlorotic spots when compared with Col-0 and the vector

control line (Figure 1a and Figure S1). The faah mutant

(salk_095108) displayed a similar degree of susceptibility as

Col-0 and the vector control lines (Figure 1a). The enhanced

susceptibility of the AtFAAH overexpressors was even more

evident when considering the time in which visible symp-

toms developed. For instance, visible chlorotic lesions star-

ted to develop on leaves of Col-0, the vector control and two

knockout lines 3 days post-inoculation (dpi) with bacterial

suspension cells at 106 colony-forming units (CFU) ml)1.

However, symptoms developed on all tested AtFAAH over-

expressors as early as 2 dpi. Consistent with these symp-

toms, the bacterial population was about 10 times higher in

the leaves of AtFAAH overexpressors than in Col-0 or faah

(Figure 1b).

AtFAAH overexpressors were susceptible to a few non-host

pathogens

Results from several studies demonstrate that basal resis-

tance is often associated with non-host resistance in plants

(Bais et al., 2005; He et al., 2006). Therefore, we next exam-

ined whether the reduced basal resistance of the AtFAAH

overexpressors had any impact on non-host disease resis-

tance. Five days after infiltration with a non-host bacterial

strain, P. syringae pv. syringae (P. s. syringae), at

5 · 106 CFU ml)1 clear chlorotic lesions formed around the

leaf edges of AtFAAH overexpressors while Col-0 and faah

remained mostly symptomless (Figure 2a). Symptoms

developed on overexpressors as early as 2 days after inoc-

ulation and continued to progress while Col-0 and faah were

free of symptoms. Consistent with these observations, the

bacterial population accumulated to levels more than

50-fold higher in AtFAAH overexpressors when compared

with Col-0 and faah lines (Figure 2b). Similar results were

obtained upon inoculation with another non-host pathogen,

P. syringae pv. tabaci (P. s. tabaci; Figure 2a). The P. s. tabaci

population in AtFAAH overexpressors was almost 100-fold

higher than in Col-0 and faah lines at 3 dpi (Figure 2b).

AtFAAH overexpressors did not exhibit compromised

resistance when challenged with several other non-host

pathogens, including P. syringae pv. phaseolicola NPS3121,

P. syringae pv. glycinea, Xanthomonas campestris pv.

campestris and X. campestris pv. vesicatoria (data not

shown).

R-gene mediated resistance was not affected in AtFAAH

overexpressors

Common signaling components have been reported

between host and non-host resistance pathways (Mysore

FAAH overexpression compromises innate immunity 337

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 56, 336–349

(a)

(b)

Figure 1. Overexpression of AtFAAH compro-

mised basal resistance in Arabidopsis.

(a) Disease symptoms on Arabidopsis wild-type

Col-0, faah knockout and AtFAAH overexpressors

(OE7a-1 and OE11a-1) were photographed

3 days post-inoculation (dpi) with Pseudomonas

syringae pv. tomato (P. s. tomato) and Pseudo-

monas syringae pv. maculicola (P. s. maculicola)

at 106 colony-forming units (CFU) ml)1. Inocu-

lated leaves are indicated by arrows.

(b) Growth of the bacteria at 3 dpi in Col-0, faah,

OE7a-1 and OE11a-1. Error bars are standard

errors from four biological replicates. Letters on

the graph donates statistically different groups

by Turkey’s group test (P > 0.005).

(a)

(b)

Figure 2. Non-host resistance was compro-

mised in AtFAAH overexpressors.

(a) Symptoms at 5 days post-inoculation (dpi)

and (b) growth (3 dpi) in Col-0, faah, OE7a-1 and

OE11a-1 after inoculation with Pseudomonas

syringae pv. syringae (P. s. syringae) and Pseu-

domonas syringae pv. tabaci (P. s. tabaci).

Leaves were infiltrated with bacterial suspen-

sions at concentration of 5 · 106 colony-forming

units (CFU) ml)1 for symptom development and

106 CFU ml)1 for growth assays. Inoculated

leaves are indicated by arrows. Letters on the

graph donates statistically different group by

Turkey’s group test (P > 0.005). Error bars are

standard errors from four biological replicates.

338 Li Kang et al.

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 56, 336–349

and Ryu, 2004; Peart et al., 2002). We therefore investigated

whether R-gene mediated resistance to P. s. tomato carrying

the corresponding avr gene is affected in AtFAAH overex-

pressors. The ecotype Col-0 has several R genes including

RPS2, RPM1 and RPS4. Upon inoculation with an avirulent

pathogen, P. s. tomato expressing avrRpt2 (an avirulent gene

for RPS2), the hypersensitive response (HR) on AtFAAH

overexpressors developed slightly faster than wild-type Col-0

and faah knockout. The differences were particularly clear

with a lower concentration of pathogen (Figure S2a). How-

ever, the bacterial populations were not significantly

different in any tested plant (Figure S2b). Similarly, P. s.

tomato expressing avrB (an avirulent gene for RPM1) and P.

s. tomato expressing avrRPS4 (an avirulent gene for RPS4)

caused the HR to develop faster on AtFAAH overexpressors,

but the bacterial growth was comparable to those in wild-

type plants (data not shown). These data suggest that

overexpression of AtFAAH did not compromise gene-for-

gene mediated resistance, at least for the three R-gene-

mediated pathways tested.

NAE profiles were not altered substantially in the leaves of

AtFAAH overexpressors

Previously, it was shown that several lines of Arabidopsis

AtFAAH overexpressors had a lower NAE content in seeds

and seedlings than the wild-type (Wang et al., 2006b). The

NAE profiles in leaves of 4-week-old AtFAAH overexpressors

and wild-type plants were examined to see if there was a

change in the NAE content or composition (Figure S3).

Surprisingly, the overall NAE contents in mature leaves of

AtFAAH overexpressor, OE7a-1, were similar to wild-type

leaves, particularly on a fresh weight basis (Figure S3).

Inoculation with non-host pathogen P. s. syringae or buffer

did not appear to significantly affect the overall NAE content

in the leaves of the wild-type or OE7a-1. These results

indicate that the steady-state profiles of the NAE pools in

mature leaves were not significantly altered by AtFAAH

overexpression.

AtFAAH overexpressors had low basal levels of salicylic

acid, jasmonic acid and abscisic acid

Overwhelming evidence indicates that endogenous salicylic

acid (SA) in plants is one of the key signaling molecules that

activates plant defense responses against invading patho-

gens (Durrant and Dong, 2004; Shah, 2003). We therefore

assessed whether the enhanced susceptibility of AtFAAH

overexpressors to the tested pathogens is associated with

changes in endogenous SA content. Total SA was mea-

sured in Col-0 and OE7a-1 plants before and 12 h post-

inoculation (hpi) with buffer, P. s. tomato or P. s. syringae.

Buffer-inoculated (12 hpi) or uninoculated OE7a-1 had

about two-fold less free SA and also less conjugated SA

than Col-0 (Figure 3a). Upon pathogen inoculation, the

amount of conjugated SA was still lower in OE7a-1 than Col-0

at 12 hpi whereas free SA was about two-fold higher in OE7a-

1 when compared with Col-0 (Figure 3a, lower panel).

We speculated that the initial lower SA content in AtFAAH

overexpressors might be one of the factors contributing to

their hyper-susceptibility to the tested host and non-host

pathogens. To test this hypothesis we treated OE7a-1 and

Col-0 plants with a SA analogue, benzo-(1,2,3)-thiadiazole-7-

carbothioic acid S-methyl ester (BTH), 3 days prior to

bacterial infection. It has been shown that BTH treatment

resulted in SA accumulation in plants that led to systemic

acquired resistance (SAR; Lawton et al., 1996). Our results

showed that after BTH treatment, the AtFAAH overexpres-

sor, OE7a-1, was no longer susceptible to the non-host

pathogens P. s. tabaci and P. s. syringae (Figure 3b).

Furthermore, the bacterial population in OE7a-1 was

reduced to near undetectable levels at 3 dpi and no signif-

icant differences were observed in bacterial populations

between OE7a-1 and Col-0 (data not shown). In contrast, the

virulent bacterium P. s. tomato remained 10-fold higher in

OE7a-1 than Col-0 even after BTH treatment (Figure 3c)

although the bacterial growth was slightly reduced in BTH-

treated plants. These results suggest that reduced levels of

SA may contribute to the enhanced susceptibility phenotype

of AtFAAH overexpressors.

The SA signaling pathway is reported to be antagonistic to

jasmonic acid (JA) signaling in plants (Clarke et al., 2000;

Dong, 1998). We therefore measured endogenous JA in Col-0

and OE7a-1 plants before and 24 hpi with buffer, P. s. tomato

or P. s. syringae. Buffer-inoculated (24 hpi) or uninoculated

OE7a-1 had about three-fold less JA (Figure S4a). Upon

inoculation with non-host pathogen, the amount of JA was

still lower in OE7a-1 than Col-0 at 24 hpi (Figure S4a).

Recently, it has been shown that NAE metabolism interacts

with ABA signaling in Arabidopsis seedlings (Teaster et al.,

2007). We therefore measured endogenous ABA in Col-0

and OE7a-1 plants at 24 hpi with buffer or P. s. syringae.

Buffer-inoculated (24 hpi) OE7a-1 had about 2.5-fold less

ABA (Figure S4b). Upon inoculation with non-host patho-

gen the amount of ABA was reduced in Col-0 but not in

OE7a-1 plants (Figure S4b).

Numerous plant defense-related genes were downregulated

in AtFAAH overexpressors

To investigate further the molecular mechanisms underly-

ing the susceptibility of AtFAAH overexpressors to non-

host pathogens, we used an Affymetrix ATH1 whole

genome microarray GeneChip to examine transcriptional

changes in AtFAAH overexpressor OE7a-1 and Col-0 at 0, 6

and 12 hpi with the non-host pathogen, P. s. syringae. We

used two approaches to interpret the microarray results.

The first approach aimed to identify the genes differentially

FAAH overexpression compromises innate immunity 339

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 56, 336–349

expressed over the time course of inoculation by the non-

host pathogen P. s. syringae in both Col-0 and OE7a-1. The

expression profile from Col-0 or OE7a-1 at 6 and 12 hpi was

compared with those at 0 hpi of its own genotype to

determine the global changes caused by inoculation of

P. s. syringae (Figure S5a). Large numbers of genes were

differentially expressed between 0 and 6 or 12 dpi in both

Col-0 and OE7a-1 after bacterial infection. Interestingly, as

shown in Table 1, the number of genes that were differ-

entially expressed in OE7a-1 was significantly smaller than

those differentially expressed in Col-0. Many of the

defense-related genes that were induced in Col-0 in

response to P. s. syringae inoculation were not induced in

OE7a-1. These results suggest that the ability of AtFAAH

overexpressors to mount a global transcriptional response

against P. s. syringae infection is compromised.

In the second approach, gene expression at the same time

point was compared between the AtFAAH overexpressor and

the wild-type in order to identify genes intrinsically differen-

tially expressed or genes that responded in a different

(a) (b)

(c)

Figure 3. AtFAAH overexpressor had less

endogenous salicylic acid (SA) and benzo-

(1,2,3)-thiadiazole-7-carbothioic acid S-methyl

ester (BTH) treatment restored its resistance to

non-host pathogens.

(a) Salicylic acid quantification in uninoculated

Col-0 and AtFAAH overexpressor (OE7a-1) and

12 h after inoculation with buffer or Pseudomo-

nas syringae pv. tomato (P. s. tomato) or Pseu-

domonas syringae pv. syringae (P. s. syringae) at

5 · 106 colony-forming units (CFU) ml)1. Error

bars represent the standard error. The SA anal-

ysis represents an average of three independent

biological replicates.

(b) Treatment with BTH restored the resistance of

OE7a-1 to non-host pathogens Pseudomonas

syringae pv. tabaci (P. s. tabaci) and P. s. syrin-

gae. Infiltrated leaves were photographed at

5 days post-inoculation (dpi). Plants were

sprayed with buffer or BTH at 150 mM concen-

tration 72 h before infiltrating P. s. tabaci or P. s.

syringae at 5 · 106 CFU ml)1 or (c) with P. s.

tomato at 106 CFU ml)1. The graph shows

growth of P. s. tomato at 3 dpi.

Table 1 Comparison of expression profilesof Col-0 and OE7a-1 between 0 h and 6 or12 h after inoculation with the non-hostpathogen Pseudomonas syringae pv.syringae

Data set

Total no. ofgenesdifferentiallyexpressed

No. genes that wereinduced afterinoculation

No. genes that weresuppressed afterinoculation

Unique Common Unique Common

Col-0 6 h:0 h 4913 1178 1321 884 1530OE7a-1 6 h:0 h 3969 561 557Col-0 12 h:0 h 5830 1320 1615 1390 1505OE7a-1 12 h:0 h 4150 415 615

340 Li Kang et al.

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 56, 336–349

manner to P. s. syringae infection between OE7a-1 and Col-0.

At 0 hpi, 507 genes (223 higher and 284 lower in OE7a-1) were

intrinsically differentially expressed between OE7a-1 and

Col-0. The number of differentially expressed genes nearly

tripled at 6 hpi to 1432 differentially expressed genes (479

higher and 953 lower in OE7a-1). At 12 hpi, we observed an

even higher number of differentially expressed genes (2063)

between OE7a-1 and Col-0, with 883 higher and 1180 lower in

OE7a-1. These differentially expressed genes were classified

based on their functional annotation (Figure S6).

Using ratios of transcript levels from OE7a-1 and Col-0, a

heat map of 3126 genes differentially expressed in OE7a-1 at

any of the three time points was generated (Figure S5b). The

number of downregulated genes was slightly greater among

all the differentially expressed genes between OE7a-1 and

Col-0. Upon close examination of the microarray data, we

observed that transcripts of majority of the differentially

expressed defense-related genes (�200 genes) were less

abundant in OE7a-1 when compared with Col-0, especially at

6 and 12 hpi (Figure S5s, Table S1). Strikingly, transcripts of

around 64 genes that encode proteins with Toll and inter-

leukin-1 receptor/coiled-coil (TIR/CC) and/or nucleotide bind-

ing site (NBS) and/or leucine-rich repeat (LRR) domains

(typically present in R genes; DeYoung and Innes, 2006)

were significantly less abundant in OE7a-1 when compared

with Col-0 (Table S2).

A high degree of consistency from two independent

experiments indicates that the results generated from the

study were reproducible (Figure S5a). The correlation coef-

ficients from two independent experiments were 0.98/0.98,

0.98/0.99 and 0.98/0.99 for Col-0/OE7a-1 at 0-, 6- and 12-h

time points, respectively. To further validate the microarray

data, we selected 24 genes, along with the AtFAAH and two

reference genes, for real-time quantitative RT-PCR (qRT-

PCR) study. Despite some variations in absolute fold-differ-

ences, the qRT-PCR data were consistent with the micro-

array data (Table S3).

AtFAAH overexpressors are compromised in SA-mediated

signaling

As indicated earlier, the AtFAAH overexpressor had rela-

tively smaller amounts of total SA content than the wild-

type before and 12 h after challenging with pathogens

(Figure 3a). Consistent with these results, our microarray

data showed that transcripts of several genes involved in SA

biosynthesis (ICS1, PAL1 and PAL2) were less abundant in

the AtFAAH overexpressor (OE7a-1) upon pathogen inocu-

lation when compared with Col-0 (Figure 4, Table S4). Even

though these genes were induced in both OE7a-1 and Col-0

upon bacterial inoculation, the scale of induction was much

lower in OE7a-1 when compared with Col-0. Furthermore,

transcripts of many genes involved in SA-mediated defense

signaling, such as PR1, PR2, PR5, EDS1, EDS5, PAD4, etc.,

were also less abundant in OE7a-1 when compared with Col-0

(Figure 4, Table S4).

To further confirm the microarray results and the role of

AtFAAH in SA-mediated signaling, the expression profiles of

several genes involved in SA biosynthesis or signaling, a

jasmonic acid (JA) signaling gene and a representative R

gene were examined using qRT-PCR in two separate exper-

iments with independent biological replicates for OE7a-1

and Col-0 plants treated with either buffer (control), P. s.

tomato or P. s. syringae. As expected, we observed larger

amounts of AtFAAH transcripts in OE7a-1 when compared

with Col-0 (Figure 5). It is noteworthy that the AtFAAH

expression in Col-0 was slightly induced in all treatments.

Typical marker genes for SA-mediated defense signaling,

PR1a and PR2, were highly induced in Col-0 at 12 hpi. These

genes were considerably less abundant in OE7a-1 at 6 and

12 hpi. One of the SA biosynthetic genes (ICS1) that was

tested was highly induced in Col-0 at both 6 and 12 hpi.

Consistent with the microarray data, ICS1 was not induced in

OE7a-1 except at 12 hpi with P. s. tomato. However, the

induction of ICS1 at 12 hpi in OE7a-1 was not as high as in

Col-0. The expression profiles of the WRKY transcription

factor genes (WRKY 46 and WRKY 70) and EDS5 were also

similar to that of ICS1 in all treatments in both Col-0 and

OE7a-1. The expression profiles of an RPP13-like and an

EDS-1-like genes were different from that of ICS1 and the

WRKY genes; these genes were constitutively very low in

OE7a-1 when compared with Col-0, and their expression

profiles did not change in response to mock or pathogen

inoculation in OE7a-1. On the other hand the amounts of

transcript for PDF1.2a, a marker gene for JA and ethylene

signaling, was slightly higher in OE7a-1 compared with Col-0

in most cases except at the 12-h time point in plants infected

with P. s. tomato. However, transcripts of several genes

involved in JA biosynthesis (OPR3, AOC2, AOC4, LOX1 and

LOX2) were less abundant in OE7a-1 compared with Col-0 at

one or more time points after inoculation with P. s. tomato

(Table S1). This was consistent with our observation that

OE7a-1 had less JA than Col-0 (Figure S4a).

Despite differential expression of PR genes, expression

profiles of TGA transcription factors that are involved in the

activation of PR gene expression (Zhang et al., 1999, 2003)

were not significantly different between OE7a-1 and Col-0.

However, a number of MYB and WRKY transcription factors

were expressed at much lower levels in OE7a-1 than those in

Col-0 at various sampling points (Tables S4 and S5). These

data argue for the existence of a novel mechanism through

which AtFAAH negatively affects the SA-mediated defense

signaling pathway.

AtFAAH is associated with membranes

Like mammalian FAAH, AtFAAH contains a putative

transmembrane domain near the N-terminus that predicts

FAAH overexpression compromises innate immunity 341

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 56, 336–349

localization to cellular membranes (Shrestha et al., 2003). To

confirm the location of AtFAAH in the cell, we generated

AtFAAH–GFP translational fusions under the control of the

CaMV 35S promoter and generated Arabidopsis plants sta-

bly expressing this construct. Consistent with our NAE

amidohydrolase activity assays (Shrestha et al., 2002), the

Figure 4. Heat map of salicylic acid (SA)-responsive, biosynthesis and signaling genes that were differentially expressed between Col-0 and OE7a-1 at any of 0, 6,

and 12 h after inoculation with a non-host pathogen Pseudomonas syringae pv. syringae (P. s. syringae) at 5 · 106 colony-forming units (CFU) ml)1.

Ratios of these genes between OE7a-1 and Col-0 were used for hierarchical clustering using Ward’s method available in Spotfire DecisionSite 8.2.1. The color

scheme of ratios is shown on the map with red representing higher expression and green representing lower expression in OE7a-1 when compared with Col-0. The

lower panel shows expression of PDF1.2a, PDF1.2b [markers for jasmonic acid (JA) signaling] and AtFAAH in the experiment.

Figure 5. Relative expression profiles determined by real-time quantitative RT-PCR of selected genes at 0, 6 and 12 h in Col-0 and AtFAAH overexpressor (OE7a-1)

after inoculation with either buffer or Pseudomonas syringae pv. tomato (P. s. tomato) or Pseudomonas syringae pv. syringae (P. s. syringae).

The treatments are indicated at the bottom of the graphs and the sampling time points are indicated on the x-axes. The y-axes donate relative gene expression levels

to Col-0 buffer treatment at 0 h (value as 1). Numbers above the bars are the actual ratios between OE7a-1 and Col-0. Error bars were derived from three technical

replicates. Experiments were repeated twice with samples from two independent experiments and the results were similar.

342 Li Kang et al.

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 56, 336–349

FAAH overexpression compromises innate immunity 343

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35S::AtFAAH–GFP fusion localized primarily to the endo-

plasmic reticulum (ER) in leaf epidermal cells and cotyle-

dons. The pattern of AtFAAH–GFP localization mirrored that

of a construct fused to the HDEL motif, a recognition signal

that directs proteins to the ER (Figure 6a–c). Leaves from

plants expressing the 35::AtFAAH–GFP fusion were count-

erstained with the plasma-membrane dye FM4-64. Single

optical confocal sections at the median plane of the cells

show strong GFP labeling along the periphery of the

epidermal cells. The GFP signal coincides with regions of

FM4-64 staining indicating that in addition to the ER, AtFAAH

is localized to the plasma membrane (overlay image in

Figure 6d). Similar results were observed in four indepen-

dent transgenic lines. We then looked to see if the localiza-

tion of AtFAAH was altered after pathogen inoculation,

but the membrane localization of AtFAAH–GFP was unal-

tered 3 days after inoculation with either host (P. s. tomato)

or non-host pathogen (P. s. syringae) tested (data not

shown).

Extracts of AtFAAH–GFP fusions expressed in E. coli still

exhibited NAE hydrolase activity in vitro but activity was

about 10-fold less than AtFAAH without GFP. Furthermore,

plants expressing the 35S::AtFAAH–GFP fusion had lower

NAE hydrolase activity than plants expressing 35S::AtFAAH,

despite roughly similar levels of FAAH protein in the

transgenic plants (Sang-Chul Kim and KDC, University of

Texas, Denton, TX, unpublished data). Despite the lower

activity of the AtFAAH–GFP fusion, this construct was able to

rescue the faah line from hypersensitivity to exogenous

NAE12:0 (Wang et al., 2006b). The faah line transformed

with the 35S::AtFAAH–GFP construct exhibited enhanced

resistance to NAE levels that would typically be strongly

inhibitory to the AtFAAH knockouts (Figure 6e). However,

35S::AtFAAH–GFP expressed in the faah background resul-

ted in smaller seedlings and shorter roots compared with

35S::AtFAAH plants when grown on exogenous NAE12:0

(Figure 6e). These differences could be explained by the

lower NAE hydrolase activity of AtFAAH–GFP.

Discussion

N-acylethanolamines have diverse physiological functions

in mammals. Of particular interest here is the emerging role

of NAEs in mammalian cellular stress responses and the

immune function (Salzet et al., 2000). While there was initial

evidence that NAEs affect how plants respond to pathogens

(Chapman et al., 1998; Tripathy et al., 1999), we still lack an

appreciable understanding of the molecular mechanisms

underlying the effects of NAEs on plant immunity. To begin

to address this issue, we conducted a detailed analysis on

(a)

(d)

(e)

(b) (c) Figure 6. Localization of AtFAAH–GFP.

Confocal micrographs of epidermal cells from

Arabidopsis leaves expressing 35S::GFP (a),

35S::GFP–HDEL (b) and 35S::AtFAAH–GFP (c).

The reticulate pattern of 35S::AtFAAH–GFP is

similar to 35S::GFP–HDEL, which targets to the

endoplasmic reticulum (ER). The inset shows a

pair of guard cells. The perinuclear pattern in

AtFAAH–GFP is typical of ER localization.

(d) Single confocal optical sections of epidermal

cells from leaves of Arabidopsis plants express-

ing 35S::AtFAAH–GFP. The leaf was counter-

stained with FM4-64, which labels the plasma

membrane. The overlay image shows that the

GFP signal around the cell periphery co-localizes

with FM4-64 (yellow areas), indicating that FAAH

is also localized to the plasma membrane.

(e) The growth of faah knockouts on 30 lM

N-acylethanolamine (NAE) 12:0 is significantly

inhibited while AtFAAH overexpressors are not.

344 Li Kang et al.

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 56, 336–349

Arabidopsis plants with altered expression of the NAE

hydrolyzing enzyme FAAH.

Here we provided evidence that Arabidopsis plants over-

expressing FAAH have compromised immune responses to

a number of pathogens including non-host bacterial patho-

gens that typically do not cause disease in Arabidopsis.

However, resistance was not compromised against all non-

host pathogens tested, suggesting that this phenomenon

was specific for certain interactions and was not due to a

general reduction in plant fitness. In fact it should be pointed

out that AtFAAH overexpressors actually exhibited

enhanced growth phenotypes (Wang et al., 2006b). Whereas

AtFAAH overexpressors exhibited modified responses to

pathogens, faah knockouts did not show altered sensitivity

to any host or non-host pathogens tested. This observation

is consistent with our previous findings in seedlings of faah

knockouts, which did not show altered growth phenotype

other than an increased sensitivity to exogenous NAE (Wang

et al., 2006b). The lack of any obvious phenotype in the faah

knockout could be attributed to alternative competing

pathways by which NAE is catabolized in the plant. In

mammalian systems, a number of other enzymes have been

shown to participate in the degradation of NAEs, including

an acid amidase (Tsuboi et al., 2005), a second FAAH (Wei

et al., 2006), lipoxygenases (Ueda et al., 1995) and cycloox-

ygenases (Kozak et al., 2002). In this regard, bioinformatics

analyses revealed seven amidase genes in the Arabidopsis

genome that have a conserved amidase signature (AS)

domain (Kilaru et al., 2007) and may encode an alternative

FAAH. These reasons may explain why NAEs are still

degraded in the absence of AtFAAH and also explain the

lack of any visible phenotype in faah knockouts.

The NAE levels were similar between faah and Col-0

seedlings, although in seeds faah knockout plants contained

about 30% more NAE than Col-0 (Wang et al., 2006b).

Consistent with these results, faah behaved similarly to

wild-type Col-0 in response to inoculations with host and

non-host pathogens. Surprisingly, changes in NAE content

and composition in response to non-host pathogens was not

significantly different in mature leaves of FAAH overexpres-

sors compared with the wild-type (Figure S3), contrary to

what might be expected from the increased capacity for NAE

turnover in these FAAH-overexpressing plants (Wang et al.,

2006b). However, it may be that changes in NAEs are highly

localized to specific cells or subcellular compartments and

any changes may be difficult to detect relative to the bulk

steady-state levels of NAEs in leaves, given the current

methodology. This is particularly difficult given the low

levels of endogenous NAEs in mature leaves (<20 ng g)1

fresh weight for all types summed together). Infiltration and

spray application of NAEs to mature Arabidopsis plants did

not alter the resistance to pathogens, nor did it activate the

SA signaling pathway (data not shown). Moreover, FAAH

may function in ways additional to its catalytic activity to

mediate defense responses, perhaps via protein–protein

interactions. Indeed, in other work with ABA signaling in

seedlings, the action of FAAH was not entirely explained by

its effect on NAE content or composition (Teaster et al.,

2007). Overexpression of FAAH led to increased capacity for

NAE turnover, but not to significant alterations in overall

NAE content, despite a clear hypersensitivity of FAAH

overexpressors to exogenous ABA. The regulation of NAE

metabolism by FAAH, and the function of FAAH outside the

NAE pathway appear to be complex and are only beginning

to be addressed. Nonetheless it is clear that the alteration of

FAAH expression leads to marked changes in plant growth

(Wang et al., 2006b), phytohormone signaling (Teaster

et al., 2007) and the capacity for plant defense against

pathogens.

Numerous reports have put SA at center of the resistance

network against pathogens. The hyper-susceptibility of

AtFAAH overexpressors prompted us to ask whether there

was any change of SA in AtFAAH overexpressors which

would lead to compromised resistance to pathogens. Sur-

prisingly, we found that endogenous SA was at least two-

fold less in AtFAAH overexpressors when compared with

Col-0. The hyper-susceptible phenotype of AtFAAH overex-

pressors to non-host bacterial pathogens could be reversed

by treating plants with an SA analogue, BTH (Figure 3b).

This indicates that the lower SA levels in AtFAAH overex-

pressors contributed at least partially to the heightened

sensitivity to pathogens. AtFAAH overexpressors did not

compromise R-gene-mediated resistance (Figure S2), prob-

ably due to greater accumulation of SA during the resistance

response. Consistent with the reduced SA levels in AtFAAH

overexpressors was a global downregulation of SA biosyn-

thesis and SA-mediated defense signaling genes.

Recent studies have reported that lipid-based molecules

are signals for immunity in plants (Weber, 2002). Genetic

screens for Arabidopsis mutants with altered defense

responses have identified mutations in several genes puta-

tively involved in lipid metabolism (Shah, 2005). Also, work

with the Arabidopsis ssi2 (suppressor of SA-insensitivity 2)

mutant showed that deficit of 18:1 oleic acid led to constit-

utive activation of the SA-mediated resistance pathway

while JA signaling was impaired (Kachroo et al., 2001).

The enhanced susceptibility phenotype accompanied by

suppression of the SA pathway in AtFAAH overexpressors is

the reverse of the ssi2 mutant, and continues to support the

strong connection between lipid metabolism and plant

defense.

Changes in hormone levels were induced by NAE20:4 and

THC and were reported to affect spermatogenesis, fertiliza-

tion and embryo development in animals (Wang et al.,

2006a). Here, we found that overexpressing AtFAAH affected

hormone levels in plants. As mentioned above, levels of

endogenous free SA were at least two-fold lower in AtFAAH

overexpressors when compared with Col-0 (Figure 3). Sur-

FAAH overexpression compromises innate immunity 345

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 56, 336–349

prisingly, after pathogen infection, the free SA levels

increased by more than two-fold in the AtFAAH overexpres-

sor when compared with Col-0. However, the levels of

conjugated and total SA still remained lower in the AtFAAH

overexpressor (Figure 3). Even though it has been proposed

that free SA is the mobile form of SA (Enyedi et al., 1992a,b),

it is still not clear whether conjugated SA also plays a role in

plant defense. Jasmonic acid is another phytohormone that

is known to play a role in plant defense. It has been well

documented that in plants, JA- and SA-mediated defense

responses act antagonistically (Clarke et al., 2000; Dong,

1998). Consistent with these earlier observations, we found

that transcripts of plant defensin genes PDF1.2a and PDF1.2b

(marker genes for JA signaling) were, constitutively and at

6 hpi with a non-host pathogen, more abundant in the

AtFAAH overexpressor when compared with Col-0 (Figure 5,

Table S3). However, we found that transcripts of several JA

biosynthetic genes were less abundant in the AtFAAH

overexpressor when compared with Col-0, and this was

consistent with our observation that AtFAAH overexpressors

had less JA (Figure S4). It has been observed that AtFAAH

overexpressors were hypersensitive to the phytohormone

ABA, and exogenous application of NAE12:0 induced

expression of a number of ABA-responsive genes in

Arabidopsis (Teaster et al., 2007). Recent literature suggests

a role for the ABA signaling pathway in susceptibility of

plants to disease caused by the host pathogen P. s. tomato

(Mauch-Mani and Mauch, 2005; Robert-Seilaniantz et al.,

2007; de Torres-Zabala et al., 2007). We found that the ABA

levels in AtFAAH overexpressors were about two-fold less

than in Col-0 (Figure S4B). Interestingly, ABA levels were

about two-fold less in Col-0 treated with a non-host patho-

gen when compared with buffer-treated Col-0, suggesting a

role for ABA in non-host resistance. An earlier report by

Cahill and Ward (1989) has shown that decrease in ABA

concentration occurred only in the incompatible soybean–

Phytophthora megasperma f. sp. glycinea interactions. As

reported earlier (Wang et al., 2006b), AtFAAH overexpres-

sors have enhanced vegetative growth compared with Col-0

plants, suggesting a possible involvement of growth

hormones such as auxin and cytokinin. All these data

together suggest an intricate role for AtFAAH and NAEs in

the phytohormone signaling network.

Apart from genes involved in phytohormone synthesis

and signaling, transcript profiling revealed that transcripts of

several other plant defense-related genes were found to be

less abundant in the AtFAAH overexpressor when compared

with Col-0. Plants encode a large number of R proteins that

contain a region of LRRs, a putative nucleotide binding site,

an N-terminal leucine zipper and coiled-coil or Toll and

interleukin-1 receptor domains (Martin et al., 2003). Tran-

scripts of a large number of putative and well-characterized

R genes or their homologs were less abundant in the

AtFAAH overexpressor compared with Col-0 (Table S2,

Figure 5). Further investigation is needed to understand

the biological implications of downregulation of these R

genes in AtFAAH overexpressors.

Similar to its mammalian counterpart, AtFAAH–GFP

fusion proteins were associated with the cell membrane

(Figure 6c,d). The membrane localization of FAAH is

required to facilitate channeling of fatty acid amide (FAA)

through membrane, and is considered to be important for its

function (McKinney and Cravatt, 2005). In the future, we will

attempt to express AtFAAH under the control of the native

promoter since overexpression using the 35S promoter

could lead to mislocalization of the protein. Nonetheless, our

observation that AtFAAH–GFP is localized to the ER is

consistent with localization studies of mammalian FAAH

(Oddi et al., 2005). The conservation of the function and

subcellular localization of FAAH in plants and animals

suggested the possibility of a similar signal transduction

system. Identification of the NAE receptors will lead to

further understanding of the role of AtFAAH and NAEs in

plant defense.

In conclusion, we have presented data showing compro-

mised immune responses of plants overexpressing AtFAAH.

The hypersensitivity of AtFAAH overexpressors to a number

of host and non-host pathogens can be explained in part by

alterations in phytohormone synthesis and signaling.

Although there are obviously a number of other molecular

targets through which NAE and FAAH exert their effects, our

study presents an important entry point for probing further

into the mechanisms of functioning of NAE in plant immu-

nity and adds to a growing list of plant processes that are

clearly influenced by this understudied group of lipids.

Experimental procedures

Plant material and bacterial pathogens

Arabidopsis thaliana wild-type Col-0 and transgenic 35S::AtFAAHlines (OE2a-1, OE7a-1 and OE11a-1) were grown in growthchambers at 20�C under short-day condition (8 h light). Four- tofive-week-old plants were used for inoculation in all experiments.Bacterial pathogens were grown in King’s B (KB) medium contain-ing appropriate antibiotics at 30�C.

Pathogen inoculation and RNA isolation

For bacterial inoculation, bacteria were grown in KB medium withappropriate antibiotics for 16 h and were pelleted by centrifugationat 3000 g. Bacterial pellets were washed three times and suspendedin sterile water. Leaves were inoculated with a needle-less syringefor symptom development and growth assay. Leaf disks frominoculated leaves were ground in water and serial dilutions of eachsample were plated on a KB agar plate for quantification of patho-gens in plant tissues. Leaves were inoculated by spray inoculationfor some experiments. To reduce variation in infection, vacuuminfiltration was used for experiments involving microarray analysisand quantification of SA/JA/ABA. A final concentration of 0.01% of

346 Li Kang et al.

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 56, 336–349

Silwet L-77 (OSI, Danbury, CT) was added to facilitate infection inspraying and vacuum infiltration experiments. Leaf tissues col-lected from at least 10 individual plants at designated times werepooled for RNA extraction. Total RNA was extracted and purifiedusing the RNeasy MinElute Kit (Qiagen, http://www1.qiagen.com/),according to the manufacturer’s instructions.

Quantification of NAE

An average of 4–5 g of mature leaf material was collected from eachof the control (0 h) and infiltrated plants. The treated plants werevacuum infiltrated with buffer control or P. s. syringae at5 · 106 CFU ml)1. To reduce variations, only fully expanded matureleaves were harvested for analysis. The NAE extraction, enrichmentand identification/quantification by gas chromatography/massspectrometry (GC/MS) were performed as described (Venableset al., 2005) with a few modifications (see Appendix S1).

Quantification of SA, JA and ABA

Free SA was extracted from Arabidopsis leaves (�300 mg) and wasanalyzed as described (Schmelz et al., 2003; Appendix S1). The SAand JA were separated on a RTx-5 column (30 m · 0.25 mm ·0.25 mm, Restek, http://www.restek.com/) using conditions des-cribed earlier (Uppalapati et al., 2005). Free SA was quantified fromthe methylene chloride:1 propanol layer. The remaining (top) waterphase was used for the extraction of conjugated SA. To quantifyconjugated SA, the water phase was acid hydrolyzed (37% con-centrated HCl) for 1 h at 95�C to release free SA. The sample wascooled to room temperature, and after adding an internal standardwas re-extracted and quantified for free SA as described (Schmelzet al., 2003). The SA and JA quantification experiments wererepeated at least three times with independent samples using threetechnical replicates during each experiment. The ABA was extractedusing methods similar to those described by Walker-Simmonset al., 2000; Appendix S1).

Microarray analyses

The Affymetrix GeneChip� Arabidopsis ATH1Genome Array(Affymetrix, http://www.affymetrix.com/) was used in the expres-sion analysis. For microarray experiments, total RNA was extractedfrom leaf tissues from two independent biological replicates. Foreach replicate, inoculated leaves from eight plants were pooled forRNA isolation, and 10 lg of total RNA was used for microarraylabeling reaction. Labeling, hybridization and scanning wereperformed according to the manufacturer’s instructions (Affyme-trix). For every chip, the signal level for each gene was obtainedfrom 11 probe sets and data were collected and normalized againstbackground using MAS5 implemented in GCOS1.4 (Affymetrix).Data normalization between chips was conducted using robustregression (Dozmorov and Centola, 2003). To select genes differ-entially expressed between treatment conditions, an expressionratio of 2 and above with a cut-off P-value (Student’s t-test) of 0.05were used as the selection criteria. The ‘presence/absence’ calldetermined by the MAS5 algorithm for each gene signal was alsoused as a criterion for selection, and only genes whose signals weretruly present in the samples were selected.

For clustering analyses, ratios of selected genes were used.Hierarchical clusters were generated for genes using Ward’s method(Ward, 1963) available in Spotfire DecisionSite 8.2.1 (Spotfire Inc.,http://spotfire.tibco.com/). Gene ontology analysis for selected

genes was obtained using web-based tools at TAIR for functioncategory (http://arabidopsis.org/tools/bulk/go/index.jsp).

Real-time quantitative RT-PCR analysis

The RT reaction was performed with 2 lg of total RNA and 0.5 lg ofpoly (dT)16 primer at 37�C using Omniscript RT Kit (Qiagen) andRNase inhibitor (Promega, http://www.promega.com/) in a finalvolume of 20 ll. Gene-specific primers for PCR were designed usingthe PRIMEREXPRESS software (Applied Biosystems, http://www3.appliedbiosystems.com/) and synthesized by Integrated DNATechnologies (http://eu.idtdna.com/). Primer sequences are listed inTable S5.

Quantitative RT-PCR was performed with an ABI PRISM 7900(Applied Biosystems) using PowerSYBR Green master mix (AppliedBiosystems). Relative quantification of PCR products was per-formed via a standard curve method using Arabidopsis ElongationFactor 1a as an endogenous control. The relative expression levelsof designated calibrator (Col-0, buffer treated at 0 h) were analyzedaccording to the manufacturer’s instructions (user’s bulletin no. 2;Applied Biosystems).

Localization of AtFAAH–GFP

Arabidopsis plants (ecotype Col-0) and faah knockouts were trans-formed with 35S::AtFAAH-GFP construct (see Appendix S1) usingthe floral dip method (Clough and Bent, 1998). Green fluorescentprotein from the epidermal cells of leaves from 4-week-old plantswas imaged with a Leica TCS SP2 AOBS laser confocal scanningmicroscope (Leica Microsystems, http://www.leica-microsys-tems.com/) using the 488-nm line of an Ar laser and emission wasdetected at 520 nm. Leaves were counterstained with membranedye FM4-64 and imaged using the 543 nm line of a He/Ne laser.

Acknowledgements

We thank Drs Jian-Min Zhou and Xiaoyan Tang for various bacterialstrains, Dr Carol Bender for the GC column and internal standardsfor SA quantification and Drs Jyoti Shah and Marilyn Roossinck forcritical review of the manuscript. This work was supported by theSamuel Roberts Noble Foundation and in part by the Department ofEnergy (DOE) Biosciences (DE-FG02-05ER15647).

Supporting Information

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Spray inoculation symptoms.Figure S2. RPS2-AvrRpt2-mediated resistance is not affected inAtFAAH overexpressors.Figure S3. Quantification of N-acylethanolamine in AtFAAH over-expressor and Col-0.Figure S4. Quantification of jasmonic acid and ABA in AtFAAHoverexpressor and Col-0.Figure S5. Heat maps of differentially expressed genes.Figure S6. Functional category of differentially expressed genes.Table S1. Differentially expressed defense related genes.Table S2. Toll and interleukin-1 receptor/coiled-coil (TIR/CC)–nucle-otide binding site (NBS) – leucine-rich repeat (LRR) type of R genesdifferentially expressed.Table S3. Comparison of microarray and real-time qRT-PCR data.Table S4. Expression values of list of salicylic acid-related genes.

FAAH overexpression compromises innate immunity 347

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 56, 336–349

Table S5. Primer sets used for real-time quantitative RT-PCR.Appendix S1. Supplementary methods.Please note: Wiley-Blackwell Publishing are not responsible for thecontent or functionality of any supporting materials supplied by theauthors. Any queries (other than missing material) should bedirected to the corresponding author for the article.

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