Antagonistic Interaction between Systemic AcquiredResistance and the Abscisic Acid–Mediated AbioticStress Response in Arabidopsis W

Michiko Yasuda,a,b Atsushi Ishikawa,c Yusuke Jikumaru,d Motoaki Seki,e Taishi Umezawa,f Tadao Asami,g

Akiko Maruyama-Nakashita,c,h Toshiaki Kudo,i Kazuo Shinozaki,f,j Shigeo Yoshida,j and Hideo Nakashitaa,i,1

a Plant Acquired Immunity Research Unit, Advanced Science Institute, RIKEN, Wako, Saitama 351-0198, Japanb Mayekawa MFG. Co., Moriya, Ibaraki 302-0118, Japanc Department of Bioscience, Fukui Prefectural University, Fukui 910-1195, Japand Growth Regulation Research Group, RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japane Plant Functional Genomics Research Group, RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japanf Gene Discovery Research Group, RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japang Department of Applied Biological Chemistry, University of Tokyo, Tokyo 113-8657, Japanh Metabolic Function Research Group, RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japani Environmental Molecular Biology Laboratory, Discovery Research Institute, RIKEN, Wako, Saitama 351-0198, Japanj RIKEN Plant Science Center, RIKEN Yokohama Institute, Yokohama, Kanagawa 230-0045, Japan

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is effective against a broad range of

pathogens. SAR development in dicotyledonous plants, such as tobacco (Nicotiana tabacum) and Arabidopsis thaliana, is

mediated by salicylic acid (SA). Here, using two types of SAR-inducing chemicals, 1,2-benzisothiazol-3(2H)-one1,1-dioxide

and benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester, which act upstream and downstream of SA in the SAR

signaling pathway, respectively, we show that treatment with abscisic acid (ABA) suppresses the induction of SAR in

Arabidopsis. In an analysis using several mutants in combination with these chemicals, treatment with ABA suppressed SAR

induction by inhibiting the pathway both upstream and downstream of SA, independently of the jasmonic acid/ethylene-

mediated signaling pathway. Suppression of SAR induction by the NaCl-activated environmental stress response proved to

be ABA dependent. Conversely, the activation of SAR suppressed the expression of ABA biosynthesis–related and ABA-

responsive genes, in which the NPR1 protein or signaling downstream of NPR1 appears to contribute. Therefore, our data

have revealed that antagonistic crosstalk occurs at multiple steps between the SA-mediated signaling of SAR induction and

the ABA-mediated signaling of environmental stress responses.

INTRODUCTION

Plants suffer various types of inevitable exogenous stresses,

such as pathogen attacks, insect herbivory, and abiotic environ-

mental stress. To survive such unfavorable conditions, plants

have evolved unique hormonally regulated self-protection sys-

tems. Salicylic acid (SA), jasmonic acid (JA), and ethylene (ET)

contribute to responses against biotic stresses by influencing

various signaling pathways that have complex networks of

synergistic and antagonistic interactions (Kunkel and Brooks,

2002). By contrast, abscisic acid (ABA) plays important roles in

both plant development and adaptation to abiotic stresses, such

as drought, salinity, and low temperature (Xiong et al., 2002;

Shinozaki et al., 2003). In addition, recent studies have shown

that ABA also plays important roles in disease susceptibility,

resistance to pathogen infection, and interaction with other

hormone-mediated biotic stress responses (Mauch-Mani and

Mauch, 2005; Melotto et al., 2006; de Torres-Zabala et al., 2007).

At infection sites, plants protect themselves from invading

microorganisms using pathogen-associated molecular pattern–

activated basal resistance, which is effective against general

microorganisms and by a resistance (R) gene–mediated defense

system that is effective even against pathogenic microorganisms

(McDowell and Dangl, 2000; Belkhadir et al., 2004; Pozo et al.,

2004). In incompatible interactions between plants and patho-

genic microorganisms, plants recognize the avirulence gene

products of individual pathogens using specific receptors, the

R gene products. This interaction causes, at the infection site, a

burst of reactive oxygen species (ROS), the rapid induction of a

hypersensitive response (HR) involving regulated cell death, and

the expression of pathogenesis-related (PR) genes (Durrant and

Dong, 2004). Subsequent to these events in the infected leaves,

the uninoculated leaves exhibit an increased level of PR gene

expression and usually develop long-lasting enhanced resis-

tance to further attacks by pathogens, termed systemic acquired

1 Address correspondence to nakashi@riken.jp.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Hideo Nakashita(nakashi@riken.jp).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.107.054296

The Plant Cell, Vol. 20: 1678–1692, June 2008, www.plantcell.org ª 2008 American Society of Plant Biologists

resistance (SAR) (Chester, 1933; Durner et al., 1997). SAR is

effective against a broad range of pathogens, including fungi,

bacteria, and viruses. SA has been identified as a signaling

molecule that acts during SAR development in dicotyledonous

plants, such as tobacco (Nicotiana tabacum) and Arabidopsis

thaliana, a phenomenon confirmed by the lack of SAR develop-

ment in NahG transgenic plants, which express the bacterial

SA-degrading enzyme salicylate hydroxylase (Gaffney et al.,

1993; Delaney et al., 1994).

Genetic approaches to unraveling plant defense signaling

pathways performed in Arabidopsis have identified various types

of mutants defective in the signal transduction that leads to SAR

induction. NPR1 has been identified and characterized as a key

regulatory component that functions downstream of SA in the

signal transduction cascade that mediates SAR induction. Recent

studies have indicated that the reducing environment induced by

SA signaling is required for the activation of NPR1 during SAR

induction (Mou et al., 2003). Several mutants defective in SA

production have been identified: enhanced disease susceptibil-

ity1 (eds1) (Parker et al., 1996) and eds5 (Nawrath et al., 2002;

Wiermer et al., 2005), phytoalexin-deficient4 (Zhou et al., 1998),

and SA induction-deficient2 (sid2) (Wildermuth et al., 2001). EDS5

shows similarity to members of the MATE (multidrug and toxin

extrusion) transporter family and is necessary for the accumula-

tion of SA and the PR-1 transcript after pathogen inoculation,

suggesting that EDS5 acts upstream of SA in SAR signaling.

SID2, which encodes ISOCHORISMATE SYNTHASE1 (ICS1), is

required for SA biosynthesis in the development of local and SAR.

These studies have revealed the detailed mechanisms of SAR

induction, but most of the other mechanisms that regulate SAR

remain to be clarified.

Treatment with SA induces resistance to pathogens and the

expression of a set of PR genes (Ward et al., 1991). In addition,

some synthetic compounds elicit SAR-related events, such as

the induction of broad disease resistance and the expression of

SAR marker genes, but have very little or no antibiotic activity.

Among these chemicals, 2,6-dichloroisonicotinic acid (Metraux

et al., 1990; Uknes et al., 1992), benzo(1,2,3)thiadiazole-7-

carbothioic acid S-methyl ester (BTH) (Friedrich et al., 1996;

Gorlach et al., 1996; Lawton et al., 1996), N-cyanomethyl-2-

chloroisonicotinamide (Yoshida et al., 1990; Nakashita et al.,

2002a; Yasuda et al., 2003a), 3-chloro-1-methyl-1H-pyrazole-

5-carboxylic acid (Nishioka et al., 2003; Yasuda et al., 2003b),

and N-(3-chloro-4-methylphenyl)-4-methyl-1,2,3-thiadiazole-5-

carboxamide (Yasuda et al., 2004) induce SAR by acting at the

point of SA accumulation or downstream in the SAR signal

transduction pathway. By contrast, probenazole and its deriva-

tive, 1,2-benzisothiazol-3(2H)-one1,1-dioxide (BIT), induce SAR

by stimulating the SAR signaling pathway upstream of SA

(Watanabe et al., 1979; Yoshioka et al., 2001; Nakashita et al.,

2002b). Some of these chemicals have been used to manage

diseases in crops. However, crops sometimes suffer heavy dam-

age from diseases despite the application of these chemicals.

This could be caused by both pathogen activity and the phys-

iological condition of the plant, which is affected by environmen-

tal factors.

Several studies have indicated that plant responses to envi-

ronmental stresses, such as drought and low temperature, have

some effects on their responses to pathogens. In Arabidopsis, a

short period of drought stress significantly increased the in

planta growth of the avirulent bacterium Pseudomonas syringae

pv tomato (Pst) 1065 relative to its growth in unstressed plants

(Mohr and Cahill, 2003). In rice (Oryza sativa) plants, low tem-

perature suppressed the resistance to infection by Magnaporthe

grisea (Koga et al., 2005). However, very little is known about

the molecular mechanisms underlying these phenomena

(Mauch-Mani and Mauch, 2005). Similarly, the involvement of

ABA in plant–pathogen interactions is ambiguous. Increased

endogenous levels of ABA were observed in response to infec-

tion by viruses, bacteria, and fungi (Steadman and Sequeira,

1970; Whenham et al., 1986; Kettner and Dorffling, 1995). The

application of ABA induced callose deposition, resulting in

resistance against necrotic fungi (Ton and Mauch-Mani, 2004;

Mauch-Mani and Mauch, 2005). In addition, ABA was shown to

function positively in basal resistance. Recognizing the invasion

of a bacterial or fungal pathogen induced closure of stomata,

restricting further invasion (Melotto et al., 2006). By contrast, the

application of ABA was also reported to increase the suscepti-

bility of plants to fungal pathogens (Henfling et al., 1980; Ward

et al., 1989; MacDonald and Cahill, 1999). Unlike many studies

focusing on plant–microbe interactions, the effects of environ-

mental stresses on the SAR have not been examined in detail.

Thus, we analyzed the effects of environmental stresses on

SAR using SAR-inducing chemicals in Arabidopsis. Arabidopsis

is a good model plant with which to investigate the mechanisms

underlying the regulation of SAR because various types of SAR-

related mutants and genes are available. SAR-inducing chem-

icals are also useful for the analysis of SAR signal transduction

because they allow the timing of the SAR induction to be con-

trolled. We used two types of SAR inducers: BIT, which induces

SAR by activating the pathway upstream of SA; and BTH, which

induces SAR without SA accumulation by activating the path-

way downstream of SA. Here, we demonstrate the suppression

of SAR by ABA-mediated environmental stresses. In addition,

we provide evidence of antagonistic interactions between SA-

mediated signaling leading to SAR induction and ABA-mediated

signaling leading to responses to environmental stressors.

RESULTS

Effect of Exogenous ABA on SAR Induction in Arabidopsis

To determine the effects of environmental stress on SAR, we

analyzed the effects of ABA treatment on chemically induced

SAR in Arabidopsis. We used a soil-drenching method for the

pathogen infection assay to avoid the dilution of the chemicals

that would have been caused by dipping the plants in bacterial

solutions. Treatment of Arabidopsis ecotype Columbia (Col-0)

plants with BIT or BTH induced resistance against the virulent

bacterial pathogen Pst DC3000. By 3 d postinoculation, BIT- or

BTH-treated plants contained 10-fold lower bacterial titers than

did water-treated control plants (Figure 1A). By contrast, in ABA-

pretreated plants, treatment with BIT or BTH did not reduce

bacterial growth; the bacterial levels were similar to those in

control or ABA-treated plants (Figure 1A). This indicates that ABA

suppressed the effects of BIT and BTH, suggesting that either

Crosstalk between SAR and ABA Signaling 1679

SAR induction or the disease resistance mechanism of SAR was

inhibited by ABA-mediated signaling.

Some PR genes are coordinately expressed in Arabidopsis

during the induction and maintenance of SAR and are also

expressed during SAR induced by chemical plant activators

such as BIT and BTH (Uknes et al., 1992; Lawton et al., 1996;

Yoshioka et al., 2001). Real-time PCR analysis indicated that

foliar treatment with BIT induced the expression of acidic PR-1 in

plants (Figure 1B). However, pretreatment with ABA suppressed

the BIT-induced expression of PR-1 in a dose-dependent man-

ner, while at the same time, inducing the ABA-responsive gene

RAB18 dose dependently (Figure 1B). The BIT-induced expres-

sion of PR-2 and PR-5 was also suppressed by pretreatment

of ABA (see Supplemental Figure 1A online). This negative effect

of ABA was also observed for BTH-induced expression of PR

genes (Figure 1B; see Supplemental Figure 1A online). We

confirmed the effect of ABA on the expression of PR-1 under

the pathogen infection assay conditions by applying the chem-

icals as a soil drench (see Supplemental Figure 1B online).

Suppression of PR-1 gene expression by infiltration of leaves

with BIT or BTH indicated that the actions, but not the uptake, of

SAR inducers were inhibited by pretreatment with ABA (see

Supplemental Figure 1C online). The effect of ABA on PR gene

expression was also assessed by histological analysis using

plants transformed with the PR-2:GUS (for b-glucuronidase)

construct (Cao et al., 1994). PR-2:GUS plants treated with BIT or

BTH by foliar spraying exhibited GUS activity in their leaves and

petioles (Figure 1C). However, pretreatment with ABA clearly and

dramatically suppressed the PR-2 expression induced by BIT or

BTH (Figure 1C). This result indicates that ABA suppresses the

induction of SAR by inhibiting signal transduction downstream of

BTH in the SAR signaling pathway.

Figure 1. Pretreatment with ABA Suppressed Chemically Induced SAR

in Arabidopsis.

(A) Growth of Pst DC3000 in Arabidopsis leaf tissues. Four-week-old

plants were pretreated with water (Control) or ABA (0.1 mg/pot) and

treated with BIT (1 mg/pot) or BTH (0.2 mg/pot) by soil drenching 5 d prior

to inoculation of Pst DC3000 (2 3 105 CFU/mL). Leaves were homog-

enized at 3 d postinoculation. The number of colony-forming units (CFUs)

was estimated by their growth on nutrient broth agar plates. Values

presented are the average 6 SD from six sets of three leaves each.

Different letters indicate statistically significant differences between

treatments (Student–Newman–Kuels [SNK] test, P < 0.05). The experi-

ment was repeated three times with similar results.

(B) Real-time PCR analysis of PR-1 and RAB18 expression in Arabidop-

sis treated with BIT or BTH in combination with ABA. Four-week-old

plants were foliar sprayed with 0, 4, 40, or 400 mM ABA 12 h prior to

treatment with 2 mM BIT or 0.5 mM BTH. Leaves were collected 2 d after

BIT or BTH treatment. Transcript levels were normalized to the expres-

sion of UBQ2 measured in the same samples. Relative mRNA levels

between treatments in each gene are presented. Each value is shown as

the average of three independent experiments 6 SD. Different letters

indicate statistically significant differences in the analysis of each gene

(SNK test, P < 0.05).

(C) Histochemical analysis of defense-related gene expression. Four-

week-old PR-2:GUS plants were sprayed with water (Control) or 400 mM

ABA 12 h prior to foliar treatment with 2 mM BIT or 0.5 mM BTH.

Histochemical GUS staining was performed 48 h after treatment with BIT

or BTH. Each experiment was performed with 12 plants and repeated

twice with similar results.

1680 The Plant Cell

A biologically active ABA analog, 1-(3-carboxyl-5-methylphenyl)-

1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexene (RCA-7a) (Asami

et al., 1998), strongly induced the ABA-responsive gene RAB18

(see Supplemental Figure 2A online). Real-time PCR analysis

indicated that the levels of BIT-induced PR-1 transcripts were

lower in plants pretreated with RCA-7a than in nontreated plants

(see Supplemental Figure 2A online). Pretreatment with RCA-7a

also significantly suppressed BIT-induced SA accumulation (see

Supplemental Figure 2B online). These results strongly suggest

that the suppressive effect of ABA on SAR induction occurs via

the hormonal action of ABA.

To confirm the mechanism of the suppression of SAR by ABA,

we assessed the effect of ABA on the SAR signaling pathway

by activating only the portion of the pathway downstream of SA. In

the SA biosynthesis-deficient mutants sid2-1 (Wildermuth et al.,

2001) and eds5-1 (Nawrath et al., 2002), the SAR signaling pathway

downstream of SA can be activated by treatment with BTH without

affecting the steps upstream of SA. ABA pretreatment suppressed

BTH-induced PR-1 gene expression in both mutants, confirming

that signal transduction downstream of SA is inhibited by ABA

(Figure 2A). This finding suggests that some regulatory mechanism

downstream of SA plays an important role in the crosstalk between

the SA- and ABA-mediated signaling pathways.

In nature, the onset of SAR is associated with increased levels

of SA both at the infection site and systemically (Malamy et al.,

Figure 2. Effects of ABA Downstream and Upstream of SA in the SAR

Signaling Pathway.

(A) Real-time PCR analysis of PR-1 and RAB18 expression induced by

BTH in SA signaling mutants. Four-week-old sid2-1 or eds5-1 mutants

were pretreated with 0, 40, or 400 mM ABA by foliar spraying 12 h prior to

foliar treatment with 0.5 mM BTH. Leaves were collected 2 d after BTH

treatment. Transcript levels were normalized to the expression of UBQ2

measured in the same samples. Relative mRNA levels between treat-

ments in each gene are presented. Each value is shown as the average of

three independent experiments 6 SD. Different letters indicate statisti-

cally significant differences in the analysis of each gene (SNK test, P <

0.05).

(B) Accumulation of free and total SA in wild-type plants. Plants were

treated with water and 2 mM BIT by foliar spraying 12 h after pretreat-

ment with water or 400 mM ABA. Leaves were harvested at 0, 1, 3, or 6 d

after treatment with BIT, and free and total SA (free SA þ SAG) levels

were quantified using HPLC. Closed squares, water treatment; closed

circles, BIT treatment; open squares, ABA pretreatment; open circles,

ABA pretreatment followed by BIT treatment. Different letters indicate

statistically significant differences 6 d after treatment (SNK test, P <

0.05). The values at 6 d after treatment were not significantly different

among three repeats of the experiment (analysis of variance, P < 0.05),

and similar results were obtained in a 5-d time-course experiment (see

Supplemental Figure 3 online). FW, fresh weight.

(C) Real-time PCR analysis of ICS1 expression. Wild-type plants were

treated with 2 mM BIT by foliar spraying 12 h after pretreatment with 0, 4,

40, or 400 mM ABA. Mock-treated plants (Control) were prepared by

pretreatment with ABA followed by treatment with water. Leaves were

collected 48 h after treatment and analyzed. Transcript levels were

normalized to the expression of UBQ2 measured in the same samples.

Relative mRNA levels between treatments in each gene are presented.

Each value is shown as the average of three independent experiments 6

SD. Different letters indicate statistically significant differences in the

analysis of each gene (SNK test, P < 0.05).

Crosstalk between SAR and ABA Signaling 1681

1990), suggesting that SA biosynthesis is one of the essential

regulating steps in SAR development. Thus, we examined the

effect of ABA on SA accumulation in plants. Application of BIT,

which activates the SAR signaling pathway upstream of SA,

gradually increased the levels of free and total (freeþ glycoside-

conjugated) SA in wild-type plants (Figure 2B; see Supplemental

Figure 3 online). However, in ABA-pretreated plants, the accu-

mulation of free and total SA was dramatically suppressed after

BIT application (Figure 2B). We also examined the effect of ABA

on expression of the SA biosynthesis-related gene ICS1. BIT,

similarly to pathogen infection, induced the expression of ICS1,

but the expression was suppressed by pretreatment with ABA in

a dose-dependent manner (Figure 2C). These results indicate

that the application of ABA suppressed the induction of SAR by

inhibiting signal transduction both upstream and downstream of

SA accumulation in the SAR signaling pathway. Therefore, the

SAR signaling pathway contains more than two regulatory com-

ponents, located upstream and downstream of SA, that are

negatively regulated by ABA signaling.

Suppression of SAR Development by ABA Is Independent

of the JA/ET Signaling Pathway

ABA and JA are involved in responses to wounding (Leon et al.,

2001). In ABA-deficient mutants of tomato (Solanum lycopersi-

cum) and potato (Solanum tuberosum), wound-inducible genes

are not activated upon wounding but are induced by the treat-

ment of undamaged tissues with ABA or JA (Pena-Cortes et al.,

1995). This suggests that ABA and JA mediate the wound

response and JA acts downstream of ABA in the signal trans-

duction pathway. By contrast, antagonistic interactions occur

between the SA and JA signaling pathways (Feys and Parker,

2000; Spoel et al., 2003). Thus, it is possible that exogenous ABA

activates the JA signaling pathway, resulting in the suppression

of the SAR signaling pathway. To determine the involvement

of other defense-related hormones, such as ET and JA, in the

suppressive effect of ABA on SAR induction, the expression of

PR-1 following BIT or BTH treatment was analyzed in the

JA-insensitive mutant coi1-1 and the ET-insensitive mutant

ein2-1 (Guzman and Ecker, 1990; Xie et al., 1998). Levels of the

PR-1 transcript similar to those in wild-type plants were detected

in BIT- or BTH-treated coi1-1 and ein2-1mutants (Figures 1B

and 3). By contrast, pretreatment with ABA suppressed PR-1

expression in both the mutants and the wild type (Figures 1B and

3). This result indicates that the suppressive effect of ABA on

SAR is regulated by unknown mechanisms, independent of the

JA/ET-mediated signaling pathway.

The Suppression of SAR Induction by Environmental

Stress Is Dependent upon ABA

ABA is synthesized in response to stress, such as drought or high

salinity (Xiong et al., 2002), and functions to induce physiological

changes and the expression of various genes to protect plants.

To determine whether environmental stresses do in fact provoke

the suppression of SAR, we examined the effect of NaCl treat-

ment on the induction of SAR. In wild-type plants, the addition of

NaCl (10 mM in pots) had no effect on the numbers of Pst

DC3000 in the inoculated leaves (Figure 4A). However, the NaCl

treatment significantly suppressed BIT- and BTH-induced dis-

ease resistance (Figure 4A). Further analysis revealed that

the NaCl treatment suppressed both BIT- and BTH-induced

PR-1 expression, while RAB18 expression was induced in NaCl-

treated plants (Figure 4B). These results indicated that signal

transduction downstream of SA was affected under these con-

ditions. In addition, the accumulation of SA with BIT treatment

was suppressed by the NaCl treatment (Figure 4C), indicating

that signal transduction upstream of SA was also inhibited.

Measurement of the levels of endogenous ABA revealed that

NaCl-treated wild-type plants contained ;3.6-fold higher levels

of ABA than the control plants (Table 1), confirming that NaCl

treatment activated the ABA-mediated signaling pathway. Thus,

the NaCl treatment suppressed the induction of SAR by

Figure 3. Induction of PR-1 Gene Expression by BIT or BTH in the

Signaling Mutants coi1-1 and ein2-1.

Four-week-old plants were foliar treated with water (Control), 2 mM BIT,

or 0.5 mM BTH 12 h after pretreatment with 400 mM ABA by foliar

spraying. Leaves were harvested 2 d after treatment and analyzed by

real-time PCR. The effect of ABA was confirmed by monitoring the

expression of the RAB18 gene. Transcript levels were normalized to the

expression of UBQ2 measured in the same samples. Relative mRNA

levels between treatments in each gene are presented. Each value is

shown as the average of three independent experiments 6 SD. Different

letters indicate statistically significant differences in the analysis of each

gene (SNK test, P < 0.05).

1682 The Plant Cell

Figure 4. Suppression of SAR Development by NaCl in Wild-Type Plants and CYP707A3ox.

Crosstalk between SAR and ABA Signaling 1683

activating ABA-mediated signal transduction, suggesting that re-

sponses to environmental stresses suppress the SA-dependent

signaling pathway of SAR induction.

The upregulation of ABA-mediated signal transduction by the

application of ABA or NaCl suppressed the development of SAR,

suggesting that endogenous ABA levels are important in defense

signaling. Although the ABA biosynthesis mutant aba1-1 has

been reported to exhibit low susceptibility to the virulent oomy-

cete Peronospora parasitica, but not to Pst DC3000 (Mohr and

Cahill, 2003), little effort has been made to determine the effects

of ABA deficiency on the signal transduction that causes induced

disease resistance. To determine whether ABA deficiency has

any effect on SAR, we compared SAR induction between the

wild type and an ABA-deficient transgenic line by examining

their resistance to Pst DC3000. The Arabidopsis cytochrome

P450 CYP707A promotes ABA degradation by converting ABA

to 89-hydroxy ABA (Kushiro et al., 2004). CYP707A3ox plants,

which constitutively express CYP707A3 using the 35S promoter,

exhibited low growth retardation after ABA application, high

transpiration, and reduced levels of endogenous ABA under both

normal and dehydrated conditions (Umezawa et al., 2006).

Measurement of endogenous ABA levels revealed that under

our experimental conditions, the ABA levels in CYP707A3ox

plants were 60% lower than those found in wild-type plants and

did not increase in response to NaCl treatment (Table 1). Al-

though the levels of bacterial growth in BIT- and BTH-treated

wild-type plants were 13 and 8%, respectively, of those found in

the water-treated control plants, bacterial levels in BIT- and BTH-

treated CYP707A3ox plants were only 2 and 1%, respectively, of

those found in control CYP707A3ox plants (Figure 4A). These

results indicate that the application of SAR inducers is more

effective in CYP707A3ox plants than in the wild type. The

enhancement of SAR by ABA deficiency was also observed in

wild-type plants treated with abamine, a specific ABA biosyn-

thesis inhibitor, and the ABA biosynthesis–deficient mutant aao3

(see Supplemental Figure 4 online) (Seo et al., 2000, 2004; Han

et al., 2004). To clarify the mechanisms of enhanced SAR

induction in CYP707A3ox plants, the effects of ABA deficiency

on PR-1 expression and SA accumulation were examined. The

PR-1 transcript levels in BIT- and BTH-treated wild-type plants

were 38- and 44-fold higher, respectively, than in nontreated

plants. By contrast, levels in BIT- and BTH-treated CYP707A3ox

plants were 57- and 59-fold higher, respectively, than in non-

treated plants (Figure 4B). The SA accumulation induced by BIT

was twice as high in CYP707A3ox than in the wild type (Figure

4C). These results indicate that the steps in the SAR signaling

pathway both upstream and downstream of SA can be enhanced

by ABA deficiency.

The development of SAR was antagonistically regulated by both

ABA-mediated environmental stress responses and endogenous

ABA levels. To clarify the detailed mechanisms regulating SAR

development, we investigated whether ABA accumulation is re-

quired to elicit the suppressive effect of environmental stress

responses on SAR. In CYP707A3ox plants, pretreatment with

NaCl did not suppress the BIT- or BTH-induced disease resis-

tance (Figure 4A), indicating the requirement for ABA for the

suppressive effect on SAR. Further analysis revealed that NaCl

pretreatment in CYP707A3ox plants failed to induce RAB18

expression or to suppress the PR-1 expression and SA accumu-

lation triggered by SAR-activating chemicals (Figures 4B and 4C),

indicating that the suppressive effects both upstream and down-

stream of SA were ABA dependent. These results strongly suggest

that ABA is a crucial molecule in the suppressive effect of

environmental stress responses on SAR induction.

Suppressive Effect of SAR on ABA Signaling

The observed suppression of SAR development by ABA-mediated

signal transduction led us to ask whether the activation of SAR

suppresses the ABA signaling pathway. We first analyzed the

effect of the activation of SAR using the Arabidopsis lesion mimic

Figure 4. (continued).

(A) Growth of Pst DC3000 in Arabidopsis leaf tissues. Pretreatment with water (Control) or NaCl (10 mM in pot) by soil drenching was performed on

4-week-old wild-type and CYP707A3ox plants 12 h prior to treatment with 1 mg/pot BIT or 0.2 mg/pot BTH. Challenge inoculation with Pst DC3000 was

performed 5 d after treatment with BIT or BTH. Leaves were harvested 3 d after inoculation, and in planta bacterial growth was estimated. Each value is

shown as the average 6 SD. Different letters indicate statistically significant differences between treatments (SNK test, P < 0.05).

(B) Real-time PCR analysis of PR-1 and RAB18 expression in water-, BIT-, BTH-, NaCl-, or a combination of BIT-NaCl– or BTH-NaCl–treated wild-type

and CYP707A3ox plants. Four-week-old plants were treated with water, 2 mM BIT, or 0.5 mM BTH by foliar spraying 12 h after pretreatment with NaCl

(10 mM in pot) by soil drenching. Leaves were collected 2 d after chemical treatment. Transcript levels were normalized to the expression of UBQ2

measured in the same samples. Relative mRNA levels between treatments in each gene are presented. Each value is shown as the average of three

independent experiments 6 SD. Different letters indicate statistically significant differences in the analysis of each gene (SNK test, P < 0.05).

(C) Accumulation of free and total SA in wild-type and CYP707A3ox plants. Plants were treated with 2 mM BIT by spraying 12 h after pretreatment with

water (Control) or NaCl (10 mM in pot) by soil drenching. Leaves were harvested 5 d after treatment, and free and total SA (free SA þ SAG) levels were

quantified using HPLC. Each value presented is the average 6 SD from four or five sets of ;25 plants. Different letters indicate statistically significant

differences between treatments (SNK test, P < 0.05).

Table 1. ABA Content in Wild-Type and CYP707A3ox Plants

Plants and Treatment ABA (ng/gFW)a

Wild type 5.05 6 0.43*

Wild type þNaCl 12 h 18.36 6 7.41**

CYP707A3ox 1.99 6 0.26*

CYP707A3ox þ NaCl 12 h 2.55 6 0.40*

a Values presented are the average of ABA contents 6 SD, each from

four or six sets of 25 to 30 plants.

Asterisks indicate statistically significant differences (SNK test, P <

0.05). Similar results were obtained in repeated experiments.

1684 The Plant Cell

mutant len3, which constitutively expresses PR genes and

accumulates SA (Ishikawa et al., 2006). The len3 npr1 double

mutant does not exhibit the cell death phenotype or PR-1 gene

expression, whereas significant amounts of SA accumulation

and the faint expression of the PR-2 and PR-5 genes are still

observed (Ishikawa et al., 2006). Microarray experiments were

performed as a screen to find potentially interesting effects on

different classes of genes in these mutants and the wild type. The

data suggested that some ABA signaling–related genes might be

affected in the different genetic backgrounds.

Thus, we used real-time PCR to monitor the transcript levels of

ABA biosynthesis–related genes (ABA1, ABA2, NCED3, and

AAO3) and ABA-responsive genes (RAB18, COR15A, MYC2,

and RD22) in the wild type and the npr1 mutant following NaCl

treatment. The transcript abundance of all of the genes examined

was higher in NaCl-treated plants than in control or BIT-treated

plants, although ABA2 exhibited a low response (Figures 5A and

5B). By contrast, pretreatment with BIT strongly suppressed the

effect of NaCl on the expression of these genes, except for ABA2

and AAO3. Pretreatment with BIT for 5 d induced the accumu-

lation of PR-1 gene transcripts, the levels of which were unal-

tered 6 h after the NaCl treatment (Figure 5C). These results

demonstrate a suppressive effect of SAR signaling on ABA-

mediated signal transduction, conclusively indicating that an-

tagonistic interactions occur between SAR and ABA-mediated

environmental stress responses. All of the examined ABA

biosynthesis–related genes were also induced by NaCl treat-

ment in the npr1 mutant (Figures 5A and 5B). Whereas BIT

pretreatment had significant suppressive effects on the NaCl-

induced expression of ABA1, COR15A, and RD22 in wild-type

plants, it did not in the npr1 mutant. By contrast, the NaCl-

induced expression of NCED3, RAB18, and MYC2 was sup-

pressed by pretreatment with BIT in both the npr1 mutant and the

wild-type plant. Significant expression of PR-1 was not detected

in the npr1 mutant under this experimental condition (Figure 5C).

These results suggest that the NPR1 protein, or signaling down-

stream of NPR1, partly contributes to the suppressive effect of

SAR signaling on ABA-mediated signal transduction.

DISCUSSION

Antagonistic Interaction between ABA- and

SA-Mediated Signaling

Our results demonstrate an antagonistic interaction between SA

signaling leading to SAR induction and ABA signaling in the

environmental stress response. Analyses using various mutants

as well as chemical activators and inhibitors of signal transduc-

tion have revealed multiple antagonistic crosstalk points be-

tween these signaling pathways (summarized in Figure 6). The

biosynthesis and accumulation of ABA are enhanced by salt,

drought, and, to some extent, cold stress (Xiong et al., 2002),

suggesting that drought, and probably cold stress, would fit this

model. Several reports have indicated that ABA modulates SA-

dependent defense responses to some pathogens (Audenaert

et al., 2002; Mauch-Mani and Mauch, 2005; Mohr and Cahill,

2007); however, the influences of ABA on SAR induction remain

to be determined. This study indicates that ABA plays an impor-

tant role in the regulation of defense mechanisms not only at the

site of pathogen infection but also in systemic signaling for the

enhancement of the plant innate immunity system.

Previous reports have shown that exogenous ABA increases

plant susceptibility to various pathogens, especially fungi, which

generally activate JA/ET-mediated defense signaling. Treatment

with ABA increased the susceptibility of potato tuber slices to the

fungal pathogens Phytophthora infestans and Cladosporium

cucumerinum (Henfling et al., 1980). The susceptibility of to-

bacco to blue mold disease caused by Peronospora tabacina

was also increased by treatment with ABA (Salt et al., 1986).

Similarly, ABA treatment reduced the resistance of plants to

various pathogens, such as the rice blast fungus M. grisea in rice

(Matsumoto et al., 1980; Koga et al., 2005) and Phytophthora

megasperma f. sp glycinea in soybean (Glycine max) (Ward et al.,

1989). Some of these phenomena have been explained with

biochemical evidence. In soybean, inoculation with P. mega-

sperma f. sp glycinea induced the gene expression and enzy-

matic activity of Phe ammonia lyase, which was suppressed by

ABA treatment (Ward et al., 1989). In a tobacco cell culture, ABA

treatment downregulated the accumulation of b-1,3-glucanase,

an antifungal protein, but not chitinase (Rezzonico et al., 1998),

probably because of the presence of the negative-acting ABA-

responsive element TAACAAA in the promoter region of the

b-1,3-glucanase gene (Giraudat et al., 1994). By contrast, there

have been very few studies of the effects of ABA on bacterial

infections that activate SA-mediated defense signaling. ABA

treatment increased the susceptibility of Arabidopsis to the

oomycete P. parasitica and the avirulent bacteria Pst 1065 but

not to the virulent bacteria Pst DC3000 (Mohr and Cahill, 2003).

On the other hand, recently, it was reported that bacterial

colonization of Pst DC3000 in Arabidopsis was enhanced by

the activation of ABA signaling, whether the signaling was

activated by the bacterial type III effector or by ABA treatment

(de Torres-Zabala et al., 2007).

Despite these findings, the involvement of ABA in disease

resistance seems to be complex and may depend on the type of

pathogen. Little effort has been made to clarify the effect of ABA

on SAR because it is difficult to investigate SA signaling by

excluding the effects of other events, such as HR, when using

pathogen infection. We successfully demonstrated the suppres-

sive effect of ABA signaling on SA-mediated signaling in SAR

induction using two SAR-inducing chemicals, BIT and BTH, in

combination with various mutants. During SAR development, the

positive regulator protein NPR1 and some TGA transcription

factors need to be reduced via cellular redox changes caused by

SA-mediated signaling (Despres et al., 2003; Mou et al., 2003).

Thus, ABA could have some effects on the redox change, an idea

that is currently being studied.

The effects of SA on environmental stress responses have also

been reported. In maize (Zea mays), treatment with SA sup-

pressed drought tolerance (Nemeth et al., 2002). In Arabidopsis,

the growth of wild-type plants was suppressed by SA accumu-

lation below 58C. However, NahG plants, which are unable to

accumulate SA, can grow better than wild-type plants under

these conditions (Scott et al., 2004). Similarly, NahG plants are

more resistant to NaCl treatment (Borsani et al., 2001). These

results suggest that SA plays a role in inhibiting environmental

Crosstalk between SAR and ABA Signaling 1685

Figure 5. Suppressive Effect of SAR Induction on the Expression of NaCl-Inducible Genes in Wild-Type Plants and npr1-1 Mutants.

1686 The Plant Cell

stress responses, which supports our finding that the activation

of SAR suppresses environmental stress responses.

Possible Mechanism of Crosstalk between ABA- and

SA-Mediated Signaling

Our analyses indicated that the suppressive effects of ABA on

the SA signaling pathway did not require JA/ET-mediated sig-

naling (Figure 3). Recently, it was reported that ABA has an

antagonistic interaction at the gene expression level with the JA/

ET-dependent signaling pathway, which plays an important role

in responses to pathogens and wounding (Anderson et al., 2004).

These studies suggest that both the SA- and JA-dependent

signaling pathways, which are activated in response to biotro-

phic and necrotrophic pathogens, respectively, can be sup-

pressed by ABA-mediated signaling. On the other hand, in the

complex network of regulatory interactions that occur during

plant stress responses, an antagonistic relationship between the

SA- and JA-mediated signaling pathways is evident (Doares

et al., 1995; Niki et al., 1998; Petersen et al., 2000; Kunkel and

Brooks, 2002). In conclusion, three-sided antagonistic interac-

tions among SA-, JA-, and ABA-mediated signaling appear

to function in the regulation of responses to exogenous biotic

and abiotic stresses. In the SA-JA antagonism, NPR1, a key

component of SAR signaling downstream of SA, likely plays a

critical role in modulating the SA-mediated suppression of JA-

responsive genes (Spoel et al., 2003), while this study has

revealed that NPR1 also takes part in the suppressive effect of

SAR signaling on ABA-mediated signaling.

How important are these antagonistic interactions for plants

that experience various biotic and abiotic stresses in nature?

Because the response reactions to both disease and environ-

mental stresses require significant amounts of energy for gene

expression and metabolic changes, plants need to effectively

regulate the strength of these responses to survive (Heil et al.,

2000; Tian et al., 2003; Dietrich et al., 2005). Since these

antagonistic interactions occur in a complex manner in multiple

steps, there should be several mechanisms functioning in this

crosstalk. One possible mechanism is the regulation of the

generation of ROS. ROS are speculated to act as a systemic

signal for SAR induction (Alvarez et al., 1998). In addition, it has

been reported in barley (Hordeum vulgare) that ABA treatment

increases catalase activity, which plays an important role in

scavenging H2O2 (Fath et al., 2001). However, further investiga-

tion is necessary to elucidate the involvement of ROS and to

identify the molecules that function in the crosstalk between ABA

and SA signaling. Unlike in SAR, treatment with non-protein

amino acid b-aminobutyric acid, a known priming-effect inducer,

simultaneously enhances disease resistance and salt and

drought stress tolerance in Arabidopsis (Jakab et al., 2005; Ton

et al., 2005), suggesting that not all disease resistance mecha-

nisms are affected by ABA signaling. To date, several other

induced disease resistance mechanisms have been identified.

However, it remains to be determined whether ABA suppresses

responses such as rhizobacteria-mediated induced disease

resistance and brassinosteroid-mediated disease resistance

induced by pathogen infection (Pieterse et al., 1998; Nakashita

et al., 2003).

The Function of Endogenous ABA in Plant

Disease Resistance

Several reports have indicated that endogenous ABA levels or

signaling affect disease development. The ABA-deficient tomato

sitiens mutant is more resistant to the necrotic fungus Botrytis

Figure 5. (continued).

Real-time PCR analysis of expression of the ABA biosynthesis-related genes ABA1, ABA2, NCED3, and AAO3 (A), ABA-responsive genes RAB18,

COR15A, MYC2, and RD22 (B), and SAR marker gene PR-1 (C). Plants were treated with NaCl (20 mM in pot) by soil drenching 5 d after pretreatment

with water (Control) or 2 mM BIT by foliar spraying. Leaves were collected 6 h after NaCl treatment. The effect of BIT was confirmed by expression of the

PR-1 gene. Transcript levels were normalized to the expression of UBQ2 measured in the same samples. Relative mRNA levels between treatments in

each gene are presented. Each value is shown as the average of three independent experiments 6 SD. Different letters indicate statistically significant

differences in the analysis of each gene (SNK test, P < 0.05).

Figure 6. Proposed Model for the Crosstalk between Biotic and Abiotic

Stress Responses.

SAR is induced through the SA-mediated signaling pathway, which

is activated by the HR to avirulent biotrophic pathogens. The ABA-

mediated signaling pathway is activated by environmental stressors,

such as salinity, drought, and, to some extent, by cold. Crosstalk

between these signaling pathways, shown as broken lines, was shown

to antagonistically regulate each other. Arrows indicate positive regula-

tion, and blunt ends denote negative regulation. White arrows indicate

the activation site of SAR signaling pathway by the chemicals BIT and

BTH. Some genes functioning in the signaling pathways are also shown.

Crosstalk between SAR and ABA Signaling 1687

cinerea than is the wild type (Audenaert et al., 2002). In rice, the

suppression of MAPK5, a MAP kinase that functions in ABA-

mediated environmental stress responses, results in lowered

tolerance to abiotic stress but enhanced disease resistance

(Xiong and Yang, 2003). The Arabidopsis aba1-1 mutant, defec-

tive in ABA biosynthesis, is less susceptible to the biotrophic

pathogen P. parasitica than is the wild type, although the ABA-

insensitive mutant abi1-1 exhibits a wild-type level of suscepti-

bility to the same pathogen (Mohr and Cahill, 2003). By contrast,

both the ABA-deficient aba2-1 and ABA-insensitive myc2 mu-

tants show enhanced resistance to the necrotic fungal pathogen

Fusarium oxysporum (Anderson et al., 2004). Some saprophytic

or parasitic fungal species, such as Cercospora rosicola and

B. cinerea, synthesize ABA (Crocoll et al., 1991; Nambara and

Marion-Poll, 2005). These phenomena suggest that endogenous

ABA levels in plants are an important factor in disease develop-

ment and that some pathogens may use ABA to invade plant

tissues. Further investigation of the crosstalk between ABA and

defense-related hormones may help to clarify these points.

METHODS

Plant Materials and Treatments

Arabidopsis thaliana Col-0 and eight different mutants and two trans-

formants of the same ecotype were used: sid2-1 (Wildermuth et al., 2001),

eds5-1 (Nawrath et al., 2002), coi1-1 (Xie et al., 1998), ein2-1 (Guzman and

Ecker, 1990), aao3-4 (Seo et al., 2004), npr1-1 (Cao et al., 1994), len3

mutants, the len3 npr1 double mutant (Ishikawa et al., 2006), and PR-2:

GUS (Cao et al., 1994) and CYP707A3ox (Umezawa et al., 2006) trans-

formants. Plants were grown in sterilized potting soil (Kureha) in plastic

pots (5 cm 3 5 cm 3 5 cm) inside a growth chamber under a 16 h:8 h

light:dark regimen at 228C with 60% humidity.

ABA [(þ)-isomer; Sigma-Aldrich], BIT (Nakashita et al., 2002b), BTH

(Syngenta Japan), abamine (Han et al., 2004), and NaCl were dissolved in

sterilized distilled water. RCA-7a (Asami et al., 1998) solution was

prepared by dilution of a stock solution (25 mM in acetone) with sterilized

distilled water to a concentration of 500 mM. Four-week-old plants were

treated with chemicals as indicated in the figures. Identification of

homozygous coi1-1 plants was performed as previously described (Abe

et al., 2008).

Pathogen Infection Assay

Pseudomonas syringae pv tomato (Pst) DC3000 was cultured in nutrient

broth medium (Eiken Chemical) for 24 h at 288C, and a bacterial suspen-

sion was prepared in 10 mM MgCl2 (2 3 105 colony-forming units per mL).

Challenge inoculation was performed by dipping the plants in bacterial

solution. After incubation for 3 d at 228C, the leaves were harvested from

the inoculated plants. Three to four leaves from different plants were

combined, weighed, and then homogenized in 10 mM MgCl2; the ho-

mogenate was then plated on nutrient broth agar containing rifampicin

(50 mg/L) at appropriate dilutions. After incubation for 2 d at 288C, the

number of rifampicin-resistant bacterial colonies was counted. More than

six homogenized samples were prepared for each experiment.

Measurement of SA Levels

Leaf tissues (0.5 g) were homogenized and extracted with 10 mL of 90%

methanol and then with 10 mL of 100% methanol. These two extracts

were combined, and 2 mL of this extract was dried at 408C. The dried

residue was extracted with 4 mL of water at 808C for 10 min, and the same

aliquot (1 mL) amounts was used for free and total SA analyses. One

aliquot was extracted with 2.5 mL ethyl acetate-cyclohexane (1:1) fol-

lowing the addition of 50 mL of concentrated HCl, and the upper layer was

dried and dissolved in 1 mL of 20% methanol in 20 mM sodium acetate

buffer, pH 5. This was subjected to HPLC analysis as the free SA sample.

Then, a 1-mL b-glucosidase (Sigma-Aldrich) solution (3 units/mL) was

added to the other aliquot and incubated for 6 h at 378C. This was

extracted with 2.5 mL ethyl acetate-cyclohexane (1:1) following the

addition of 50 mL of concentrated HCl, and an HPLC sample was

prepared following the method for free SA. SA analysis was performed

using the 8000 series HPLC system (Japan Spectroscopic) with a TSK-gel

ODS 80 column (4.6 3 150 mm; Toso) run with 20% methanol in a 20 mM

sodium acetate buffer, pH 5, at a flow rate of 1 mL/min. SA was detected

and quantified fluorometrically (295-nm excitation and 370-nm emission).

GUS Staining

Four-week-old PR-2:GUS plants were foliar treated with 2 mM BIT or

0.5 mM BTH 12 h after pretreatment with 400 mM ABA by foliar spraying.

Plants were harvested 3 d after treatment, and GUS staining was

performed by incubating whole plants in a GUS staining buffer containing

50 mM sodium phosphate, pH 7.0, 0.5 mM potassium ferrocyanide,

0.5 mM potassium ferricyanide, 10 mM EDTA, 0.1% (v/v) Triton X-100,

2% (v/v) dimethyl sulfoxide, and 0.5 mM 5-bromo-4-chloro-3-indolyl-b-

D-glucuronide (Nacalai Tesque) for 24 h at 378C (Jefferson et al., 1987).

Plants were then washed in a graded ethanol series (30, 50, 70, and 100%

[v/v]) for ;30 min in each solution.

Measurement of ABA Levels

ABA was extracted and purified as previously described (Saika et al.,

2007). ABA measurements were performed using the LC (ACQUITY

UPLC system; Waters) MS/MS (Q-Tof premier; Micromass) system. The

LC, equipped with an ACQUITY UPLC BEH C18 column (2.1 3 50 mm,

1.7 mm; Waters), was used with a binary solvent system composed of

acetonitrile containing water (A) and 0.05% acetic acid (B). Separation

was performed at a flow rate of 0.2 mL/min with a linear gradient of solvent

B from 3 to 98% in 10 min. The retention time of ABA and d6-ABA was

4.18 min. MS/MS conditions were as follows: capillary (kV) ¼ 2.8, source

temperature (8C)¼ 80, desolvation temperature (8C)¼ 400, cone gas flow

(L/h) ¼ 0, desolvation gas flow (L/h) ¼ 500, collision energy ¼ 8.0, and

MS/MS transition (m/z): 263/153 for unlabeled ABA and 269/159 for

D6-ABA. The calibration curve was calculated using spectrometer soft-

ware (MassLynx v. 4.1; Micromass) and the data from seven standard

solutions, each containing 10, 20, 50, 100, 200, 500, or 1000 pg/mL of

unlabeled ABA and 100 pg/mL of d6-ABA.

Real-Time PCR

Total RNA was extracted using Sepasol-RNA I super reagent (Nacalai

Tesque) and treated with DNaseI (Invitrogen) followed by phenol chloro-

form purification, according to the manufacturer’s instructions. Total RNA

(1 mg) was converted into cDNA with the Omniscript reverse transcriptase

kit (Qiagen) using oligo(dT)12-18 primers (GE Healthcare Bio-Sciences)

according to the manufacturer’s instructions, yielding 20 mL of cDNA

solution. Quantitative RT-PCR was performed using the GeneAmp SDS

7300 sequence detection system (Applied Biosystems). The amplification

reaction mixture contained 2 mL of 10-fold diluted-cDNA template, 2 mL of

primer solution (containing 5 mM [5 pmol/mL] of each forward and reverse

primer), 8 mL Milli Q water, and 12 mL 23 SYBR Green PCR Master Mix

(Applied Biosystems). The primer pairs used in the real-time PCR are

listed in Supplemental Table 1 online. Thermal cycling conditions con-

sisted of 2 min at 508C, 10 min at 958C, and 40 cycles of 15 s at 958C and

1688 The Plant Cell

1 min at 608C. Real-time PCR results were captured and analyzed using

the sequence detection software SDS version 1.2 (Applied Biosystems).

Transcript levels were normalized to the expression of UBQ2 measured in

the same samples (Maruyama-Nakashita et al., 2004).

Statistics

One-way analysis of variance was used to compare the averages of the

different results, and the SNK test was used as a post-test for pairwise

comparisons.

RNA Extraction and Gel Blot Analysis

Total RNA was extracted from frozen leaf samples using Sepasol-RNA I

super reagent (Nacalai Tesque) according to the manufacturer’s instruc-

tions. DNA fragments of coding regions for PR-1, PR-2, PR-5, and RD29B

genes were amplified by PCR from cDNA prepared from SA-treated

Arabidopsis. The PCR products were cloned into plasmid pCR 2.1

(Invitrogen), and the nucleotide sequences of these were confirmed.32P-labeled cDNA probes were synthesized by random priming of these

DNA fragments using Ready-To-Go DNA labeling beads (GE Healthcare).

Total RNA samples were subjected to 1.2% agarose–1.1% formalde-

hyde gel electrophoresis and then transferred to a nylon membrane

(Hybond Nþ; GE Healthcare Biosciences). After the transfer, RNA was

cross-linked to the membrane using a UV linker (GS Gene Linker; Bio-

Rad). Prehybridization and hybridization in 0.5 M sodium-phosphate

buffer, pH 7.2, containing 7% SDS, 1 mM EDTA, and 10 mg/mL BSA were

performed at 688C for 1 h or longer and for 8 h or longer, respectively. The

membrane was washed twice with 23 SSC containing 0.1% SDS for

30 min at 688C and then washed twice with 0.13 SSC containing 0.1%

SDS for 15 min at 688C. Detection was performed with a BAS2500 image

analyzer (Fuji Photo Film).

Microarray Experiments

Wild-type, len3, and len3npr1 plants were grown on soil for 5 weeks under

a 9 h:15 h light:dark regimen at 228C with 60% humidity. Total RNA was

isolated using Concert reagent (Invitrogen). mRNA was prepared using

the MACS mRNA isolation kit (Miltenyi Biotec). Preparation of fluorescent

probes, microarray hybridization, scanning, and data analysis were per-

formed as previously described (Seki et al., 2002; Sakuma et al., 2006).

Cy3- and Cy5- labeled cDNA probes were prepared from mRNAs isolated

from the len3 or len3 npr1 mutant and control wild-type plants, respec-

tively, grown under unstressed control conditions. The probes were

hybridized with the cDNA microarray, and the expression profiles of the

;7000 genes were analyzed. To assess the reproducibility of the micro-

array, we repeated the experiment three times with the same mRNA

samples. GeneSpring version 5.1 software was used for screening the

genes exhibiting interesting expression patterns.

Accession Numbers

Arabidopsis Genome Initiative locus identifiers for the genes mentioned in

this article are as follows: ABA1 (At5g67030), ABA2 (At1g52340), NCED3

(At3g14440), AAO3 (At2g27150), MYC2 (At1g32640), RD22 (At5g25610),

RAB18 (At5g66400), COR15A (At2g42540), RD29B (At5g52300), PR-1

(At2g14610), PR-2 (At3g57260), PR-5 (At1g57040), ICS1 (At1g74710), and

UBQ2 (At2g36170). The complete set of microarray data has been

deposited in the European Bioinformatics Institute ArrayExpress database

under accession number E-MEXP-1084 (www.ebi.ac.uk/arrayexpress/).

Supplemental Data

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

Supplemental Figure 1. Pretreatment with ABA Suppressed the

Expression of SAR Marker Genes by BIT or BTH.

Supplemental Figure 2. The Active ABA Analog RCA-7a Suppressed

SAR Induction.

Supplemental Figure 3. Accumulation of Free and Total SA in Wild-

Type Plants.

Supplemental Figure 4. SAR induction in ABM-Treated Wild-Type

Plants and ABA Biosynthesis-Deficient Mutant aao3.

Supplemental Table 1. Primer Pairs Used for Real-Time PCR.

ACKNOWLEDGMENTS

We thank B. Staskawicz (University of California, Berkeley), F. Ausubel

(Massachusetts General Hospital, Boston, MA), C. Nawrath (University

of Lausanne, Switzerland), and M. Seo (Growth Regulation Research

Group, RIKEN Plant Science Center) for providing the Pseudomonas

strain, PR2:GUS, the sid2 mutant, and the aao3 mutant, respectively.

We thank J. Ishida and M. Satou (Plant Functional Genomics Research

Group, RIKEN Plant Science Center) for their technical assistance in the

microarray analysis, M. Sekimoto (Growth Regulation Research Group,

RIKEN Plant Science Center) for supporting ABA measurements, and M.

Kusajima, M. Kawai, H. Masaki, and M. Yamada (RIKEN Discovery

Research Institute) for supporting plant cultures. We also thank N.

Miyauchi, T. Kushiro, H. Goda, M. Fujiwara, N. Kitahata, M. Uramoto,

K. Akutsu, H, Takahashi, and I. Yamaguchi for valuable discussions and

comments and E. Nambara and Y. Kamiya for critical reading of the

manuscript and stimulating discussions.

Received July 17, 2007; revised May 26, 2008; accepted June 6, 2008;

published June 27, 2008.

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1692 The Plant Cell

DOI 10.1105/tpc.107.054296; originally published online June 27, 2008; 2008;20;1678-1692Plant Cell

Akiko Maruyama-Nakashita, Toshiaki Kudo, Kazuo Shinozaki, Shigeo Yoshida and Hideo NakashitaMichiko Yasuda, Atsushi Ishikawa, Yusuke Jikumaru, Motoaki Seki, Taishi Umezawa, Tadao Asami,

ArabidopsisAbiotic Stress Response in Mediated−Antagonistic Interaction between Systemic Acquired Resistance and the Abscisic Acid

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