G Model

PSL 8977 1–8 ARTICLE IN PRESS

Plant Science xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Plant Science
journal homepage: www.elsevier.com/locate/plantsci

1 Review

2 Salicylic acid signaling in disease resistance

3 Q1 Dhirendra Kumar ∗
4 Department of Biological Sciences, East Tennessee State University, Box 70703, Johnson City, TN 37614, USA
5

6
17 a r t i c l e i n f o a b s t r a c t
7
8 Article history: Salicylic acid (SA) is a key plant hormone that mediates host responses against microbial pathogens.
9 Available online xxx Identification and characterization of SA-interacting/binding proteins is a topic which has always excited
10 scientists studying microbial defense response in plants. It is likely that discovery of a true receptor for
11 Keywords: SA may greatly advance understanding of this important signaling pathway. SABP2 with its high affinity
12 Salicylic acid (SA) for SA was previously considered to be a SA receptor. Despite a great deal work we may still not have
13 Systemic acquired resistance (SAR)
true a receptor for SA. It is also entirely possible that there may be more than one receptor for SA. This
14 SA-binding protein 2 (SABP2)
scenario is more likely given the diverse role of SA in various physiological processes in plants including,
15 NPR1
16 NPR3
modulation of opening and closing of stomatal aperture, flowering, seedling germination, thermotoler-
ance, photosynthesis, and drought tolerance. Recent identification of NPR3, NPR4 and NPR1 as potential
SA receptors and ␣-ketoglutarate dehydrogenase (KGDHE2), several glutathione S transferases (GSTF)
such as SA binding proteins have generated more interest in this field. Some of these SA binding proteins
may have direct/indirect role in plant processes other than pathogen defense signaling. Development
and use of new techniques with higher specificity to identify SA-interacting proteins have shown great
promise and have resulted in the identification of several new SA interactors. This review focuses on SA
interaction/binding proteins identified so far and their likely role in mediating plant defenses.
© 2014 Published by Elsevier Ireland Ltd.

18 Contents

19 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
20 2. SA biosynthesis and metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
21 3. SA-binding proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
22 4. NPR1, NPR3 and NPR4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
23 5. Concluding remarks and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
24 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
25 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
26

27 1. Introduction aspirin, exhibited increased resistance against tobacco mosaic virus 36

(TMV) [4,5]. Treatment with SA and its derivative induced expres- 37

28 Salicylic acid (SA), a simple phenolic compound is well studied sion of pathogenesis-related proteins [6–8]. SA is required for the 38
29 for its role in activating plant defenses especially systemic acquired activation of robust SAR and is marked by the increased expres- 39
30 resistance (SAR) [1,2]. SA and its derivative (aspirin: acetyl SA) sion of many defense proteins including pathogenesis-related (PR) 40
31 have been widely used for years as an anti-inflammatory drug. Ini- proteins. Plants defective in SA synthesis/accumulation exhibit 41
32 tially SA was discovered as a major component in bark extract of enhanced susceptibility to pathogens [9,10]. Besides SA, other plant 42
33 willow (Salix) tree. Aspirin became the first synthetic drug to be hormones known for their direct/indirect role in plant signaling 43
34 used for anti-inflammatory agent [3]. The role of SA in plants was are jasmonic acid (JA), ethylene (ET), abscisic acid (ABA), aux- 44
35 recorded for the first time in 1987 [4]. Tobacco plants treated with ins, gibberellins (GA), brassinosteroids, and cytokinins (CKs) [11]. 45

Many of these hormone mediated signaling pathways are also 46

known to crosstalk resulting in an antagonistic or synergistic 47

∗ Tel.: +1 423 439 6928; fax: +1 423 439 5958. interaction [12]. JA pathway when activated in response to her- 48

E-mail addresses: kumard@etsu.edu, KUMARD@mail.etsu.edu bivory or wounding triggers a systemic response similar to SAR. 49

http://dx.doi.org/10.1016/j.plantsci.2014.04.014
0168-9452/© 2014 Published by Elsevier Ireland Ltd.

Please cite this article in press as: D. Kumar, Salicylic acid signaling in disease resistance, Plant Sci. (2014),
http://dx.doi.org/10.1016/j.plantsci.2014.04.014
 G Model
PSL 8977 1–8 ARTICLE IN PRESS
2 D. Kumar / Plant Science xxx (2014) xxx–xxx

50 Treatment of plants with SA is known to suppress JA induced redundant proteins with overlapping functions is one reason why 111

51 wounding response [13,14]. Some pathogen like Pseudomonas T-DNA insertion may not be suitable for the identification of SA 112

52 syringe induces activation of both SA and JA pathways [13]. Addi- interacting/binding proteins. 113

53 tional studies have shown that SA-induced defense mostly acts The SABP, catalase was the first soluble plant protein found to 114

54 against biotrophs while JA activated defense is targeted toward physically bind SA [48]. SABP was identified and purified using 115

55 wounding and necrotropic pathogens [15,16]. Activation of an 14 C-SA [49,50]. In plants, catalases are known to detoxify H O 116
2 2
56 immune pathway against biotrophic pathogens suppress defense produced during various metabolic processes. Binding of SA to 117

57 against necrotropic pathogens [17,18]. Arabidopsis plants treated the catalase resulted in inhibition of its H2 O2 hydrolyzing activ- 118

58 with low concentrations of JA and SA exhibited a synergistic effect ity [49]. It was hypothesized that inhibition of catalases by SA 119

59 on the expression of PR1 and PDF1.2 genes while treatment with could potentially lead to accumulation of toxic H2 O2 which then 120

60 higher concentrations resulted in an antagonistic effect [19]. Muta- activates expression of defense genes and systemic acquired resis- 121

61 tion in a fatty acid desaturase (ssi2) resulted in the upregulation tance (SAR). Supporting this hypothesis, another SAR inducer, 122

62 of the SA pathway and suppression of the JA mediated pathway 2,6-dichloroisonicotinic acid (INA), has been shown to inhibit cata- 123

63 [20,21]. ET also shows extensive crosstalk with SA–JA signaling lase activity in tobacco [51]. Transgenic tobacco plants expressing a 124

64 pathways. ET potentiates expression of SA dependent PR1 gene yeast catalase gene (CAT1) accumulated less H2 O2 around Tobacco 125

65 expression in Arabidopsis and in tobacco plants it is required for mosaic virus (TMV) induced necrotic lesions compared to control 126

66 activation of the SAR response [22,23]. ABA, an important hormone plants. TMV induced necrotic lesions were larger compared to con- 127

67 in signaling abiotic stress has recently emerged as a key component trol plants both in primary inoculated and secondary inoculated 128

68 of plant immune signaling [24]. ABA antagonizes the SA mediated upper leaves, suggesting that catalase has a role in inducing disease 129

69 plant defense responses at multiple steps [11,25]. Overall, molecu- resistance. Increased levels of catalase in these transgenic plants 130

70 lar details of ET, JA and SA interactions are still poorly understood more likely detoxified H2 O2 resulting in a decrease in its availability 131

71 and require further investigations. for activating resistance [52]. The transgenic tobacco with reduced 132

catalase activity developed necrotic lesions and induced expres- 133

sion of PR genes only under high light conditions, suggesting that 134
72 2. SA biosynthesis and metabolism
SA inhibition of catalase may not be required for the induction of the 135

defense response. Later studies showed that SA binds to many iron 136
73 In plants, SA is synthesized in plastids via two routes from cho-
containing enzymes, e.g. aconitase, catalase, lipoxidase and peroxi- 137
74 rismate, a product of the shikimate pathway. One route is through
dase suggesting that SA binding to catalase was not specific [53]. SA 138
75 isochorismate synthase (ICS), which is believed to be responsible
bound to SABP with a moderate affinity (Kd = 14 ␮M)[48]. To search 139
76 for >90% of SA synthesized during activation of stress response
for high affinity SA-binding proteins, a ligand with higher affinity 140
77 [10]. The other route uses the phenylalanine ammonia-lyase (PAL)
(3 H-SA) was synthesized and used for identification of additional 141
78 mediated pathway [26]. SA is readily modified to its many deriva-
SA-binding proteins [54]. 142
79 tives (via glucosylation, methylations, amino acid conjugation,
SA-binding protein 3 (SABP3) was identified as a stroma local- 143
80 sulphonation, hydroxylation, etc.) but most are not active com-
ized carbonic anhydrase. It has moderate affinity Kd = 3.7 ␮M for SA 144
81 pounds [27]. Most of the SA produced in plants is glucosylated (SAG)
compared to SABP2 (Kd = 90 nM) [55] (Table 1). But unlike SABP, SA 145
82 and believed to be the main storage form with the potential to be
binding has no effect on the carbonic anhydrase activity of SABP3 146
83 converted back to SA through enzymatic reactions catalyzed by a
[55]. Carbonic anhydrase in animals helps to transport CO2 out of 147
84 SA ␤-glucosidase [28,29]. A methylated derivative (MeSA; methyl
muscle cells and provide bicarbonate to mitochondria for gluco- 148
85 salicylate) is also inactive but is volatile and could readily diffuse
neogenesis [56]. In C4 plants, cytosol localized carbonic anhydrase 149
86 through membranes. Volatilization of SA through MeSA synthe-
catalyzes the conversion of CO2 into bicarbonate, which is used 150
87 sis could help plants excrete SA outside of the cell in which it is
during carbon fixation by the C4 enzyme, phosphoenolpyruvate 151
88 synthesized for eventual diffusion out of the plant [30,31]. This
carboxylase [57]. In contrast, antisense carbonic anhydrase tobacco 152
89 mechanism may help plants to reduce the accumulation of SA and
plants with 99% reduction in activity had little or no effect on 153
90 its resulting toxic effects, which has the potential to cause cell death
photosynthesis or general fitness of the plant [58]. Recent stud- 154
91 [32]. Besides plant immune signaling, SA also serves as an impor-
ies using a T-DNA insertion mutant of a plastid localized carbonic 155
92 tant signaling molecule in various physiological responses such as
anhydrase in Arabidopsis showed a reduction in seedling estab- 156
93 drought [33], thermogenesis [34–36], stomatal closure [37], seed
lishment compared to wild type plants at ambient CO2 levels 157
94 germination [38], flowering [39–42], salt stress [43], ozone [44],
[59]. Overexpression of carbonic anhydrase in chloroplast led to 158
95 and chilling [45]. A recent study suggests a role for SA in clathrin-
an increase in Rubisco activity [58]. Virus-induced gene silencing 159
96 mediated endocytic protein trafficking [46]. The main focus of this
of a SABP3 homolog in Nicotiana benthamina led to the suppres- 160
97 review is to discuss major SA interacting/binding proteins identi-
sion of the Pto:avrPto mediated hypersensitive response [55]. In 161
98 fied to date and their role in understanding of SA signaling pathway
yet another study, carbonic anhydrase transcripts were shown to 162
99 in disease resistance. There are a number of excellent reviews
be upregulated in compatible reactions while down regulated in 163
100 describing other aspects of SA signaling [2,27,47].
incompatible reactions at the 12 h time point [60]. By 24–28 h 164

carbonic anhydrase transcripts were completely downregulated. 165

101 3. SA-binding proteins Only by 72 h, where carbonic anhydrase transcripts upregulated 166

again [60]. Silencing of carbonic anhydrase in N. benthamiana 167

102 To identify cellular proteins which physically interact and bind allowed increased growth of Phytophthora infestans [60]. These 168

103 to SA, a combination of biochemical and traditional column chro- results suggest that SABP3/carbonic anhydrase is needed for pos- 169

104 matography was used. Proteins from tobacco plants which bound itive regulation of defense responses in plants. SABP3 is a target 170

105 to SA labeled with 14 C or 3 H were identified, purified and char- for modification via S-nitrosylation during later stages of R-gene 171

106 acterized for their role in SA-mediated plant defense response. mediated protection against avirulent plant pathogens [61]. S- 172

107 Several tobacco proteins were identified as SA-binding proteins. nitrosylation is the covalent attachment of nitric oxide moiety to a 173

108 Meanwhile a genetic approach using Arabidopsis mutants identi- cysteine thiol of a protein to form S-nitrosothiol [62]. Modification 174

109 fied a number of key components of the SA signaling pathway but by S-nitrosylation at Cys280 renders SABP3 unable to bind to SA 175

110 did not directly identify any SA-binding proteins. The presence of and lose its carbonic anhydrase activity [63]. SABP3 is a positive 176

Please cite this article in press as: D. Kumar, Salicylic acid signaling in disease resistance, Plant Sci. (2014),
http://dx.doi.org/10.1016/j.plantsci.2014.04.014
 G Model
PSL 8977 1–8 ARTICLE IN PRESS
D. Kumar / Plant Science xxx (2014) xxx–xxx 3

Table 1
SA-interacting proteins.

SA-interacting protein Identity Source Ligand Binding affinity to SA (Kd )

SABPa Catalase Nicotiana tabacum 14

C-SA 14.0 ␮M
SABP2b Methyl esterase Nicotiana tabacum 3
H-SA 0.09 ␮M
SABP3c Carbonic anhydrase Nicotiana tabacum 3
H-SA 3.70 ␮M
NPR1d A protein with ankyrin repeat and BTB/POZ domain Arabidopsis thaliana Genetics/T-DNA mutant 0.14 ␮M
NPR3e CUL3 adapter protein Arabidopsis thaliana Genetics/NPR1 paralog 0.98 ␮M
NPR4f CUL3 adapter protein Arabidopsis thaliana Genetics/NPR1 paralog 0.046 ␮M
Ascorbate peroxidaseg Ascorbate peroxidase Nicotiana tabacum 3
H-SA ?
KGDHE2h E2 subunit of ␣-ketoglutarate dehydrogenase 2 Arabidopsis thaliana 4-azido SA, SPR ?
GSTF2i Glutathione S-transferase 2 Arabidopsis thaliana 4-azido SA, SPR ?
GSTF8j Glutathione S-transferase 8 Arabidopsis thaliana 4-azido SA, SPR ?
GSTF10k Glutathione S-transferase 10 Arabidopsis thaliana 4-azido SA, SPR ?
GSTF11l Glutathione S-transferase 11 Arabidopsis thaliana 4-azido SA, SPR ?
a
SABP [48].
b
SABP2 [54].
c
SABP3 [55].
d
NPR1[106].
e
NPR3 [105].
f
NPR4 [105].
g
Ascorbate peroxidase [90].
h
KGDHE2 [92].
i
GSTF2 [92].
j
GSTF8 [92].
k
GSTF10 [92].
l
GSTF11 [92].

177 regulator of plant disease resistance in Arabidopsis and its modifi- mediated silencing of SABP2 made transgenic tobacco plants less 217

178 cation via S-nitrosylation affects its ability to influence immune resistant and defective in mounting a robust SAR response, sug- 218

179 response [64]. This appears to be a host mechanism to quickly gesting an important role for SABP2 in disease resistance [66]. This 219

180 dampen defense responses and bring the cell back to normal state. indicates that SABP2 is a likely a bona fide SA receptor. 220

181 At this point, it is unclear if pathogens hijack this mechanism to To verify if the SAR defective phenotype of transgenic tobacco 221

182 suppress defense responses or if it is merely a component of a neg- plants (1–2) silenced in SABP2 expression was specifically caused 222

183 ative feedback loop of the immune response. Interestingly, plant by silencing of SABP2 or was a result of off-target silencing of other 223

184 defense regulator NPR1 is also S-nitrosylated at Cys156 and this gene/s, a synthetic gene complementation approach using a syn- 224

185 favors its oligomerization in cytoplasm during the resting phase thetic version of native (nat) tobacco SABP2 was undertaken [69]. 225

186 of the plant cells [65], while SA induces an oligomer to monomer The synthetic (syn) version of SABP2 was designed and manufac- 226

187 change through the activity of thioredoxins [65]. tured to maximize differences in the DNA sequence of the native 227

188 The SABP2, a high affinity SA-binding protein was identified tobacco gene by taking advantage of the availability of alternate 228

189 using 3 H-SA as a ligand [54]. Using conventional chromatogra- codons for each amino acid. Syn SABP2 was 24% different, with 229

190 phy methods it was purified 24,000-fold from uninduced tobacco no more than nine bases of exact match in a stretch with native 230

191 leaves [66]. During initial stages of purification SABP (catalase) tobacco SABP2. Both nat and syn SABP2 coded for the exact same 231

192 was fractionated out in a 0–50% ammonium sulfate fraction while amino acids. Both control (C3; N. tabacum Xanthi NN plants trans- 232

193 both SABP3 (carbonic anhydrase) and SABP2 (the 29 kDa protein) formed with empty vector) and SABP2 silenced (1–2 transgenic) 233

194 co-fractionated in 50–75% fraction. During the next purification plants were stably transformed with nat and syn version of SABP2 234

195 step of size exclusion chromatography using Sephadex 100, SABP3 [66]. Plants challenged with TMV were assessed for SAR develop- 235

196 and SABP2 eluted in different fractions. Through a combination of ment. SABP2 silenced plants expressing synthetic SABP2 regained 236

197 partial peptide sequencing, RT-PCR and 5 -RACE, the cDNA cor- the wild type phenotype and exhibited SAR. This showed that the 237

198 responding to SABP2 was amplified, cloned and sequenced [66]. SAR defective phenotype of SABP2 silenced (1–2) plants was due to 238

199 This ∼29 kDa protein consisted of 260-amino acids. Subsequent specific silencing of SABP2 and not due to off-target gene silencing. 239

200 sequence analysis showed that it is member of the ␣/␤-hydrolase Further analysis also showed that control C3 plants transformed 240

201 fold protein superfamily [67]. Members of this family of proteins with syn SABP2 showed stronger SAR than C3 plants [69]. 241

202 have diverse biochemical activities such as hydrolases and other The use of a synthetic gene for the generation of transgenic 242

203 enzymes (lipase, esterases, thioesterases, cutinase, epoxy hydro- plants overexpressing a protein of interest may be a significant 243

204 lase, etc.) that are involved in the activation of H2 O2 , or HCN biotechnological tool. It is important to note that attempts to gener- 244

205 (␣-hydroxynitrile lyases, haloperoxides) and which share a com- ate transgenic tobacco plants constitutively overexpressing SABP2 245

206 mon and conserved catalytic triad of amino acids, Ser, Asp and have been unsuccessful (Kumar and Klessig, unpublished results). 246

207 His [68]. The recombinant ∼29 kDa protein expressed with a N- Attempts to overexpress NtSABP2 (tobacco SABP2 gene) in Arabidop- 247

208 terminal 6xHis tag, bound specifically to SA and its active analogs, sis succeeded partially but only for the first generation (Kumar 248

209 e.g. 5-CSA, 2.6-DHBA (does induce PR expression and resistance) and Klessig, unpublished results). By the second generation, the 249

210 but did not bind to inactive analogs, e.g. 2,5-DHBA, 4-HBA (does expression of NtSABP2 in transgenic Arabidopsis plants was com- 250

211 not induce PR proteins and resistance). Recombinant SABP2 exhib- pletely lost (Kumar and Klessig, unpublished results). This shows 251

212 ited lipase/esterase-like activity with artificial substrates, but a that plants may have mechanisms to efficiently regulate the expres- 252

213 subsequent search for the plant based substrate led to the identifi- sion of SABP2. This may be needed to regulate local levels of SA 253

214 cation of methyl salicylic acid as a potential substrate [66,67]. Other that protect the cell against toxic effects of SA. Substantially mod- 254

215 methylated plant hormones, e.g. MeJA (methyl jasmonic acid) and ified SABP2 via synthetic gene technology may offer a mechanism 255

216 MeIAA (methylindoleacetic acid) were poor substrates [67]. RNAi to overcome such endogenous control mechanisms that suppress 256

Please cite this article in press as: D. Kumar, Salicylic acid signaling in disease resistance, Plant Sci. (2014),
http://dx.doi.org/10.1016/j.plantsci.2014.04.014
 G Model
PSL 8977 1–8 ARTICLE IN PRESS
4 D. Kumar / Plant Science xxx (2014) xxx–xxx

257 overexpression of genes like SABP2. It is important to note that the Two homologs (PtSABP2-1 and PtSABP2-2) of NtSABP2 were 323

258 complementation experiments described above were conducted identified from woody poplar plants with 77% identity [83]. Poplar 324

259 by expressing various constructs from an extradiol inducible pro- is one of the fastest growing tree species and is considered an ideal 325

260 moter using pER8 vector and not through constitutive expression candidate crop for a bioenergy production. Both PtSABP2-1 and 326

261 [70]. PtSABP2-2 were highly similar (98% identity) to each other and 327

262 Identification of MeSA as an endogenous substrate for SABP2 showed methyl salicylate esterase activity. The major differences 328

263 and inhibition of its catalytic activity by SA binding indicated an were in their promoter region and in their response to stress sig- 329

264 important role for MeSA in SA mediated plant defenses [67]. MeSA nals. PtSABP2-1 not induced upon treatment with SA, MeJA or by 330

265 was identified as a volatile plant compound synthesized by tobacco wounding, while PtSABP2-2 was highly induced by SA, MeJA and 331

266 plants infected with TMV [30]. MeSA is synthesized from SA in upon wounding. Poplar has higher endogenous levels of SA com- 332

267 plants and has been suggested to act by converting into SA (Fig. 1) pared to tobacco and Arabidopsis, as do potato and rice also [44,84]. 333

268 [30]. MeSA being volatile, travels as an airborne signal to neigh- Using full length tobacco SABP2 (NtSABP2) amino acid sequence to 334

269 boring plants where it induces expression of PR proteins as well search, a number of Arabidopsis proteins (total of 18) were iden- 335

270 as in the healthy tissue of the infected plants. Later studies showed tified as potential homologs (At-MES; Arabidopsis thaliana methyl 336

271 high level accumulation of MeSA in plant tissues infected with TMV estrase #1–18). These proteins were 32–57% identical to tobacco 337

272 or Pseudomonas syringae pv. phaseolicola [71]. Using a combination NtSABP2 [85]. AtMES1 was most similar while AtMES18 was least 338

273 of grafting experiments involving SABP2 silenced (1–2) transgenic similar to NtSABP2 [85]. To identify a true ortholog, these proteins 339

274 tobacco plants and biochemical analysis it was shown that MeSA were further characterized for their esterase activity using MeSA as 340

275 is a phloem mobile signal for SAR in plants [72]. Active SABP2 is a substrate. AtMES1, 2, 4, 7 and 9 showed SA-inhibitable esterase 341

276 required in systemic uninfected tissues to convert phloem mobile activity [85]. AtMES1, 7 and 9 when expressed in SABP2 silenced 342

277 MeSA to SA in order to induce resistance. This observation argues tobacco plants, rescued its SAR defect phenotype [85]. Individual 343

278 that SABP2 is a receptor for MeSA [73]. Binding of SA to SABP2 to Arabidopsis T-DNA insertion mutants were not compromised in 344

279 inhibit its esterase activity in primary tissue was required for SAR SAR response while simultaneous silencing of AtMES1, 2, 7, and 345

280 induction. Timely inhibition of SABP2 esterase activity in primary 9 resulted in a SAR defective phenotype. This shows that several 346

281 tissue was required to ensure accumulation of MeSA in primary tis- Arabidopsis proteins are redundant for methyl esterase activity. 347

282 sue and its phloem mediated transport to systemic healthy tissue. StMES1, a potato homolog of NtSABP2 was identified and cloned 348

283 Synthesis of MeSA is catalyzed by SA-methyl transferase (SAMT). [86]. StMES1 showed 74% identity and 85% similarity to NtSABP2. 349

284 Plants lacking this SA-methyl transferase activity in tobacco, potato At the enzymatic level it catalyzed the conversion of MeSA to SA. 350

285 and Arabidopsis were SAR defective [74]. Recent studies using T- Similar to NtSABP2, SA inhibited its esterase activity. Expression 351

286 DNA insertion mutants suggests that MeSA or jasmonic acid may of StMES1 in SABP2 silenced transgenic tobacco plants, comple- 352

287 not be the mobile signal for SAR in Arabidopsis plants [75]. Levels of mented its local and SAR defective phenotype showing that it is a 353

288 MeSA increase in pathogen infected tobacco and Arabidopsis plants true ortholog of NtSABP2 [86]. This approach is being used in sweet 354

289 [30,71]. It is produced through a reaction catalyzed by an SAMT like orange to confer resistance to bacterial diseases through incorpo- 355

290 enzyme using S-adenosine-l-methionine as a methyl donor [76]. ration of the tobacco SABP2 gene [87,88]. Citrus fruits are prone 356

291 Coronatine, a Pseudomonas syringae virulence factor enhances the to many devastating diseases and this effort to express tobacco 357

292 production of MeSA (by activating SAMT activity) which is likely to SABP2 in phloem uses a phloem specific Arabidopsis SUC2 promoter 358

293 be lost as a volatile compound, thereby reducing levels of SA [77]. [89]. 359

294 This mechanism appears to help P. syringae in attenuation of SA- Ascorbate peroxidase is another SA interacting protein whose 360

295 mediated plant defense responses [75]. This could also be a strategy activity is shown to be inhibited by SA [90]. Ascorbate perox- 361

296 of P. syringae for protecting the host plant against potential damage idases are known to detoxify cells of H2 O2 that is constantly 362

297 caused by accumulation of SA to high concentrations. Premature produced as by product of photosynthesis, photorespiration, oxida- 363

298 damage to plant tissue may not in the best interest of pathogen tive phosphorylation, and fatty acid ␤-oxidation [91]. Ascorbate 364

299 multiplication and growth. peroxidases are localized in the chloroplast and cytosoplasm. 365

300 A discovery by Attaran et al. [75] suggested that MeSA is not Ascorbate peroxidase is inhibited only by active analogs of SA, e.g. 2, 366

301 required for SAR development in Arabidopsis plants prompted 6-dichloroisonicotinic acid (INA) and not by inactive analogs. It was 367

302 Klessig and colleagues to examine reasons for the discrepancy hypothesized that SA mediated inhibition of ascorbate peroxidases 368

303 between their observation that MeSA is required for SAR devel- led to increased levels of H2 O2 in the cell which in turn triggered a 369

304 opment in Arabidopsis. Out of several factors which are likely to resistance response [90]. 370

305 influence SAR, light appears to play critical role in development of Use of 14 C and 3 H labeled SA coupled with column chromatog- 371

306 full or partial defense responses. It is suggested that the extent of raphy led to the identification of SABP, SABP3 and SABP2, but these 372

307 light exposure following primary pathogen infection determines proteins do not appear to be bonafide SA-receptors. To identify true 373

308 if MeSA is required for SAR development or not [78]. In wild type SA receptors, a combination of photoaffinity labeling (light based 374

309 tobacco and Arabidopsis plants inoculations early in the day fol- covalent cross-linking of interacting partner with the ligand) and 375

310 lowed by long exposure to light, induced a strong SAR response. In surface plasmon resonance-based (PMR) techniques were used. 376

311 contrast, mutant Arabidopsis bsmt1 (defective in SA methyl trans- These methods resulted in the identification of several new SA- 377

312 ferase activity, fail to accumulate MeSA), exhibited a different binding proteins [92]. The newly identified proteins include the E2 378

313 response and requirement for MeSA depending on the time of inoc- subunit of ␣-ketogutarate dehydrogenase and several glutathione 379

314 ulations during the day. When inoculated in morning hours and S-transferases (GSTF2, GSTF8, GSTF10 and GSTF11). SA inhibited 380

315 exposed to light for long hours (more than 3.5 h), the plants did not enzymatic activity of all these newly identified SABPs. Interest- 381

316 obligately require MeSA for SAR development [78]. Inoculation of ingly these new SABPs showed little or no SA-binding activity using 382

317 these mutants in late afternoon/evening with little exposure to light 3 H-SA as used in the identification and characterization of SABP3 383

318 (less than 3.5 h), obligately required MeSA for SAR development. [55] and SABP2 [66] showing that this new technique with its 384

319 The importance of light in plant immunity is well documented ability to detect weak and transient interactions has the poten- 385

320 [21,79–82]. SABP2 homologs have now been identified and char- tial to identify high specificity SA-binding proteins. Further work 386

321 acterized from a number of plant species including Arabidopsis, using this technology is likely to identify additional SA-binding 387

322 poplar, potato and tomato. proteins.

Please cite this article in press as: D. Kumar, Salicylic acid signaling in disease resistance, Plant Sci. (2014),
http://dx.doi.org/10.1016/j.plantsci.2014.04.014
 G Model
PSL 8977 1–8 ARTICLE IN PRESS
D. Kumar / Plant Science xxx (2014) xxx–xxx 5

Fig. 1. A model showing activation of SA signaling in a plant cell. Pathogen attack results in increased biosynthesis of SA via ICS/PAL pathway in plastids. SA methyl transferase
(SAMT) catalyzes conversion of SA to MeSA which diffuses into the cytoplasm where it is converted back to SA by the esterase activity of SABP2. Increased SA levels in the
cytoplasm disrupts the oligomeric NPR1 into its monomers which then migrate to the nucleus to activate transcription of SA responsive defense genes including PR genes.
NPR1 is phosphorylated upon its interaction with transcription factors. Proteosome mediated degradation of phosphorylated NPR1 trigger’s expression SA responsive genes.

388 4. NPR1, NPR3 and NPR4 NPR3 and NPR4 directly bind to SA. Interestingly, the binding affin- 419

ity of NPR3 (Kd = 981 nM) and NPR4 (Kd = 46 nM) are very different, 420

389 NPR1 (Nonexpresser of Pathogenesis-Related protein 1) acts suggesting a differential role based on the intracellular concentra- 421

390 downstream of SA and is a key regulator of the SA-dependent path- tions of SA [105] (Table 1). Also, the SA-binding affinities of NPR4 422

391 way [93]. Overexpression of the NPR1 gene resulted in enhanced (Kd = 46 nM) and SABP2 (Kd = 90 nM) are similar. In yet another 423

392 disease resistance in several diverse plant species, e.g. carrot [94], recent study, NPR1 itself was claimed to be a receptor for SA [106] 424

393 rice [95,96], tobacco [97], tomato [98], wheat [99], apple [100]. (Table 1). It is now suggested that the SA-NPR1 complex is highly 425

394 Since, NPR1 did not physically bind to 3 H-SA, it was not con- labile and difficult to detect which may explain why it took so long 426

395 sidered to be a SA receptor [101]. Changes in cytosolic levels of for this interaction to be discovered and why regular assays using 427

396 SA results in breakdown of cytosol localized oligomeric NPR1into 3 H-SA for the detection of NPR1 binding to SA were unsuccessful 428

397 monomers and are followed by the migration of monomeric NPR1 [106]. Using an equilibration dialysis method, it was determined 429

398 to the nucleus [65]. Levels of NPR1 in the nucleus are critical for that NPR1 interacted with [14 C]SA with a Kd of 140 nM (Table 1). 430

399 inducing disease resistance. In the nucleus, NPR1 monomers inter- This binding of NPR1 to SA was through the copper ion associated 431

400 act with transcription factors including TGAs (basic leucine zipper with Cys521/529. Association of copper is crucial for NPR1 bind- 432

401 transcription factors) to induce expression of disease resistance ing to SA [106]. Removal of copper by chelation abolished NPR1 433

402 genes including PR genes (Fig. 1) [102]. Plants lacking NPR1 are binding to SA. Furthermore, the direct interaction of SA with NPR1 434

403 defective in SA-induced transcriptional reprogramming and fail is critical for the functioning of its C-terminal activation domain. 435

404 to activate SAR [93,103]. In a resting plant cell nucleus, NPR1 is This C-terminal activation domain remains inhibited because of the 436

405 quickly degraded to minimize basal expression of defense genes auto-inhibitory activity of the N-terminal BTB/POZ domain. Inter- 437

406 [104]. Upon activation of an immune response, i.e. SAR, NPR1 enters action with SA releases the C-terminal activation domain from the 438

407 the nucleus in large numbers, binds to transcription factors and is BTB-POZ domain. 439

408 phosphorylated by kinase activity associated with the transcription

409 initiation complex. This is followed by ubiquitination of phospho- 5. Concluding remarks and perspectives 440
410 rylated NPR1, a process that appears to be critical for activation of
411 SAR in plants [104]. It is interesting to note that any NPR1 monomer The SA pathway is one of the most extensively studied path- 441
412 entering the nucleus is eventually degraded but only the degrada- ways in plant disease resistance. Great progress has been made 442
413 tion of phosphorylated NPR1 results in the activation of SAR while over the last two decades in understanding the biochemistry of 443
414 degradation of unphosphorylated NPR1 (in resting cells) does not this system. The role of SA in plant immune response has been 444
415 result in induction of defense genes and SAR [105]. Recent stud- known for the last 35 yrs. Academia and industry have both shown 445
416 ies have indicated that NPR3 and NPR4 (paralogs of NPR1) have a tremendous interests in learning more about this pathway. Sev- 446
417 role in mediating degradation of NPR1 [105]. The npr3npr4 double eral synthetic compounds mimicking SA and its resistance inducing 447
418 mutant is defective in induction of SAR. In contrast to NPR1, both activity have been made available to agriculture for crop protection. 448

Please cite this article in press as: D. Kumar, Salicylic acid signaling in disease resistance, Plant Sci. (2014),
http://dx.doi.org/10.1016/j.plantsci.2014.04.014
 G Model
PSL 8977 1–8 ARTICLE IN PRESS
6 D. Kumar / Plant Science xxx (2014) xxx–xxx

449 BTH/ASM (acibenzolar S-methyl) is one good example. But under- [10] M.C. Wildermuth, J. Dewdney, G. Wu, F.M. Ausubel, Isochorismate synthase 514

450 standing the biochemical pathways and its target components is required to synthesize salicylic acid for plant defence, Nature 414 (2001) 515
562–565. 516
451 is crucial for designing and developing effective crop protection [11] C.M. Pieterse, D. Van der Does, C. Zamioudis, A. Leon-Reyes, S.C. Van Wees, 517
452 agents. Availability of Arabidopsis genome sequence and T-DNA Hormonal modulation of plant immunity, Annu. Rev. Cell Dev. Biol. 28 (2012) 518
453 insertion mutants has greatly helped in dissecting this pathway. 489–521. 519
[12] J. Mundy, H.B. Nielsen, P. Brodersen, Crosstalk, Trends Plant Sci. 11 (2006) 520
454 A biochemical approach to identify and characterize SA interact- 63–64. 521
455 ing/binding proteins has also provided useful information on many [13] S.H. Spoel, A. Koornneef, S.M. Claessens, J.P. Korzelius, J.A. Van Pelt, M.J. 522
456 important components. Still, our understanding of this pathway is Mueller, A.J. Buchala, J.P. Metraux, R. Brown, K. Kazan, L.C. Van Loon, X. Dong, 523
C.M. Pieterse, NPR1 modulates cross-talk between salicylate- and jasmonate- 524
457 far from complete. More sophisticated and sensitive technologies
dependent defense pathways through a novel function in the cytosol, Plant 525
458 are needed to further probe this important pathway. As we have Cell 15 (2003) 760–770. 526
459 seen in case of SA-binding proteins, the availability of higher speci- [14] S.C. van Wees, M. Luijendijk, I. Smoorenburg, L.C. van Loon, C.M. Pieterse, 527
Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is 528
460 ficity ligands helped in the discovery of new SA interacting/binding
not associated with a direct effect on expression of known defense-related 529
461 proteins. It is interesting to note that binding affinity to SA for genes but stimulates the expression of the jasmonate-inducible gene Atvsp 530
462 SABP2 (Kd = 90 nM), NPR3 (Kd = 46 nM) and NPR1 (Kd = 140 nM) are upon challenge, Plant Mol. Biol. 41 (1999) 537–549. 531

463 in a similar range (Table 1). Besides plant immune responses, SA [15] B.N. Kunkel, D.M. Brooks, Cross talk between signaling pathways in pathogen 532
defense, Curr. Opin. Plant Biol. 5 (2002) 325–331. 533
464 has been implicated in development, photosynthesis, flowering, [16] A. Verhage, S.C. van Wees, C.M. Pieterse, Plant immunity: it’s the hormones 534
465 thermogenesis, abiotic stress, and other important aspects of plant talking, but what do they say? Plant Physiol. 154 (2010) 536–540. 535

466 physiology. The role of SABPs in these physiological processes in [17] S.H. Spoel, J.S. Johnson, X. Dong, Regulation of tradeoffs between plant 536
defenses against pathogens with different lifestyles, Proc. Natl. Acad. Sci. 537
467 plants is not studied and need attention. Identification of new U.S.A. 104 (2007) 18842–18847. 538
468 SABPs, e.g. KGDHE2, supports earlier observations that SA inhibits [18] A. Koornneef, A. Leon-Reyes, T. Ritsema, A. Verhage, F.C. Den Otter, L.C. 539
469 mitochondrial respiration and has opened further avenues of inves- Van Loon, C.M. Pieterse, Kinetics of salicylate-mediated suppression of jas- 540
monate signaling reveal a role for redox modulation, Plant Physiol. 147 (2008) 541
470 tigation. In animals, SA mediated inhibition of KGDHE2 led to the 1358–1368. 542
471 accumulation of ROS (Reactive Oxygen Species), a key signaling [19] L.A. Mur, P. Kenton, R. Atzorn, O. Miersch, C. Wasternack, The outcomes 543
472 molecule in plants and animals [107]. Several newly identified of concentration-specific interactions between salicylate and jasmonate 544
signaling include synergy, antagonism, and oxidative stress leading to cell 545
473 SABPs are GSTs (Table 1) and are known scavengers of ROS. SA binds
death, Plant Physiol. 140 (2006) 249–262. 546
474 and inhibits their activity, which suggests that in plants it may help [20] P. Kachroo, J. Shanklin, J. Shah, E.J. Whittle, D.F. Klessig, A fatty acid desaturase 547
475 to accumulate ROS as a pathway to cell death. In humans, SA and its modulates the activation of defense signaling pathways in plants, Proc. Natl. 548
Acad. Sci. U.S.A. 98 (2001) 9448–9453. 549
476 derivatives are well known for their anti-inflammatory, antipyretic
[21] P. Kachroo, A.C. Chandra-Shekara, D.F. Klessig, Plant signal transduction and 550
477 and analgesic properties. AMPK is activated by increased cellular defense against viral pathogens, Adv. Virus Res. 66 (2006) 161–191. 551
478 levels of AMP resulting in stimulation of fat utilization in ani- [22] M.C. Verberne, J. Hoekstra, J.F. Bol, H.J. Linthorst, Signaling of systemic 552

479 mals. Recent observation that SA binds to and activates a cellular acquired resistance in tobacco depends on ethylene perception, Plant J. 35 553
(2003) 27–32. 554
480 metabolic regulator, AMP-activated protein kinase (AMPK) has pro- [23] M. De Vos, W. Van Zaanen, A. Koornneef, J.P. Korzelius, M. Dicke, L.C. Van Loon, 555
481 vided a different insight into energy metabolism in animals [108]. C.M. Pieterse, Herbivore-induced resistance against microbial pathogens in 556

482 Overall, given the importance of SA in a wide range of processes Arabidopsis, Plant Physiol. 142 (2006) 352–363. 557
[24] F.Y. Cao, K. Yoshioka, D. Desveaux, The roles of ABA in plant–pathogen inter- 558
483 both in animals and plants, it is essential to have a better under- actions, J. Plant Res. 124 (2011) 489–499. 559
484 standing of this hormone and its actions. [25] M. Yasuda, A. Ishikawa, Y. Jikumaru, M. Seki, T. Umezawa, T. Asami, A. 560
Maruyama-Nakashita, T. Kudo, K. Shinozaki, S. Yoshida, H. Nakashita, Antag- 561
onistic interaction between systemic acquired resistance and the abscisic 562
acid-mediated abiotic stress response in Arabidopsis, Plant Cell 20 (6) (2008) 563
485 Acknowledgments 1678–1692. 564
[26] H.I. Lee, J. Leon, I. Raskin, Biosynthesis and metabolism of salicylic acid, Proc. 565
Natl. Acad. Sci. U.S.A. 92 (1995) 4076–4079. 566
Q2
486 The author wishes to thank Dr Tom Laughlin, East Tennessee [27] D.A. Dempsey, A.C. Vlot, M.C. Wildermuth, D.F. Klessig, Salicylic Acid biosyn- 567
487 State University and reviewers for their comments. Author of this thesis and metabolism, Arabidopsis Book/Am. Soc. Plant Biol. 9 (2011) 568
Q3 review is supported by a grant from NSF (MCB 1022077) and a RDC
488 e0156. 569
[28] H.I. Lee, I. Raskin, Purification, cloning, and expression of a pathogen inducible 570
489 grant from East Tennessee State University. Author apologizes to UDP-glucose:salicylic acid glucosyltransferase from tobacco, J. Biol. Chem. 571
490 all those work could not be covered in this review. 274 (1999) 36637–36642. 572
[29] H.I. Lee, I. Raskin, Glucosylation of salicylic acid in Nicotiana tabacum Cv. 573
Xanthi-nc, Phytopathology 88 (1998) 692–697. 574
[30] V. Shulaev, P. Silverman, I. Raskin, Airborne signalling by methyl salicylate in 575
491 References plant pathogen resistance, Nature 385 (1997) 718–721. 576
[31] R. Xu, F. Song, Z. Zheng, OsBISAMT1, a gene encoding S-adenosyl- 577
492 [1] J. Shah, The salicylic acid loop in plant defense, Curr. Opin. Plant Biol. 6 (2003) l-methionine: salicylic acid carboxyl methyltransferase, is differentially 578
493 365–371. expressed in rice defense responses, Mol. Biol. Rep. 33 (2006) 223–231. 579
494 [2] A.C. Vlot, D.A. Dempsey, D.F. Klessig, Salicylic acid, a multifaceted hormone [32] M.J. Cronje, I.E. Weir, L. Bornman, Salicylic acid-mediated potentiation of 580
495 to combat disease, Annu. Rev. Phytopathol. 47 (2009) 177–206. Hsp70 induction correlates with reduced apoptosis in tobacco protoplasts, 581
496 [3] G. Weissmann, Aspirin, Sci. Am. 264 (1991) 84–90. Cytometry A 61 (2004) 76–87. 582
497 [4] R.F. White, Acetylsalicylic acid (aspirin) induces resistance to tobacco mosaic [33] A. Chini, J.J. Grant, M. Seki, K. Shinozaki, G.J. Loake, Drought tolerance estab- 583
498 virus in tobacco, Virology 99 (1979) 410–412. lished by enhanced expression of the CC-NBS-LRR gene, ADR1, requires 584
499 [5] J.F. Antoniw, A.M. Dunkley, R.F. White, J. Wood, Soluble leaf proteins of virus- salicylic acid, EDS1 and ABI1, Plant J. 38 (2004) 810–822. 585
500 infected tobacco (Nicotiana tabacum) cultivars [proceedings], Biochem. Soc. [34] J.F. Dat, C.H. Foyer, I.M. Scott, Changes in salicylic acid and antioxidants dur- 586
501 Trans. 8 (1980) 70–71. ing induced thermotolerance in mustard seedlings, Plant Physiol. 118 (1998) 587
502 [6] T. Gaffney, L. Friedrich, B. Vernooij, D. Negrotto, G. Nye, S. Uknes, E. Ward, H. 1455–1461. 588
503 Kessmann, J. Ryals, Requirement of salicylic acid for the induction of systemic [35] H.T. Liu, W.D. Huang, Q.H. Pan, F.H. Weng, J.C. Zhan, Y. Liu, S.B. Wan, Y.Y. 589
504 acquired resistance, Science 261 (1993) 754–756. Liu, Contributions of PIP(2)-specific-phospholipase C and free salicylic acid 590
505 [7] J. Malamy, J.P. Carr, D.F. Klessig, I. Raskin, Salicylic acid a likely endogenous to heat acclimation-induced thermotolerance in pea leaves, J. Plant Physiol. 591
506 signal in the resistance response of tobacco to viral infection, Science 250 163 (2006) 405–416. 592
507 (1990) 1002–1004. [36] C.Y. Yoo, K. Miura, J.B. Jin, J. Lee, H.C. Park, D.E. Salt, D.J. Yun, R.A. Bressan, 593
508 [8] J.P. Metraux, H. Signer, J. Ryals, E. Ward, M. Wyss-Benz, J. Gaudin, K. Raschdorf, P.M. Hasegawa, SIZ1 small ubiquitin-like modifier E3 ligase facilitates basal 594
509 E. Schmid, W. Blum, B. Inverardi, Increase in salicylic acid at the onset of thermotolerance in Arabidopsis independent of salicylic acid, Plant Physiol. 595
510 systemic acquired resistance in cucumber, Science 250 (1990) 1004–1006. 142 (2006) 1548–1558. 596
511 [9] T.P. Delaney, S. Uknes, B. Vernooij, L. Friedrich, K. Weymann, D. Negrotto, T. [37] I.C. Mori, R. Pinontoan, T. Kawano, S. Muto, Involvement of superoxide gener- 597
512 Gaffney, M. Gut-Rella, H. Kessmann, E. Ward, J. Ryals, A central role of salicylic ation in salicylic acid-induced stomatal closure in Vicia faba, Plant Cell Physiol. 598
513 acid in plant disease resistance, Science 266 (1994) 1247–1250. 42 (2001) 1383–1388. 599

Please cite this article in press as: D. Kumar, Salicylic acid signaling in disease resistance, Plant Sci. (2014),
http://dx.doi.org/10.1016/j.plantsci.2014.04.014
 G Model
PSL 8977 1–8 ARTICLE IN PRESS
D. Kumar / Plant Science xxx (2014) xxx–xxx 7

600 [38] S. Lee, S.G. Kim, C.M. Park, Salicylic acid promotes seed germination under [66] D. Kumar, D.F. Klessig, High-affinity salicylic acid-binding protein 2 is required 686
601 high salinity by modulating antioxidant activity in Arabidopsis, New Phytol. for plant innate immunity and has salicylic acid-stimulated lipase activity, 687
602 188 (2010) 626–637. Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 16101–16106. 688
603 [39] J.B. Jin, Y.H. Jin, J. Lee, K. Miura, C.Y. Yoo, W.Y. Kim, M. Van Oosten, Y. Hyun, [67] F. Forouhar, Y. Yang, D. Kumar, Y. Chen, E. Fridman, S.W. Park, Y. Chiang, 689
604 D.E. Somers, I. Lee, D.J. Yun, R.A. Bressan, P.M. Hasegawa, The SUMO E3 ligase, T.B. Acton, G.T. Montelione, E. Pichersky, D.F. Klessig, L. Tong, Structural and 690
605 AtSIZ1, regulates flowering by controlling a salicylic acid-mediated floral pro- biochemical studies identify tobacco SABP2 as a methyl salicylate esterase 691
606 motion pathway and through affects on FLC chromatin structure, Plant J. 53 and implicate it in plant innate immunity, Proc. Natl. Acad. Sci. U.S.A. 102 692
607 (2008) 530–540. (2005) 1773–1778. 693
608 [40] C.F. Cleland, A. Ajami, Identification of the flower-inducing factor isolated [68] N. Lenfant, T. Hotelier, E. Velluet, Y. Bourne, P. Marchot, A. Chatonnet, ESTHER, 694
609 from aphid honeydew as being salicylic acid, Plant Physiol. 54 (1974) the database of the alpha/beta-hydrolase fold superfamily of proteins: tools 695
610 904–906. to explore diversity of functions, Nucleic Acids Res. 41 (2013) D423–D429. 696
611 [41] C.F. Cleland, O. Tanaka, Effect of daylength on the ability of salicylic acid to [69] D. Kumar, C. Gustafsson, D.F. Klessig, Validation of RNAi silencing specificity 697
612 induce flowering in the long-day plant Lemna gibba G3 and the short-day using synthetic genes: salicylic acid-binding protein 2 is required for innate 698
613 plant Lemna paucicostata 6746, Plant Physiol. 64 (1979) 421–424. immunity in plants, Plant J. 45 (2006) 863–868. 699
614 [42] C. Martinez, E. Pons, G. Prats, J. Leon, Salicylic acid regulates flowering time [70] J. Zuo, Q.W. Niu, N.H. Chua, Technical advance: an estrogen receptor-based 700
615 and links defence responses and reproductive development, Plant J. 37 (2004) transactivator XVE mediates highly inducible gene expression in transgenic 701
616 209–217. plants, Plant J. 24 (2000) 265–273. 702
617 [43] O. Borsani, V. Valpuesta, M.A. Botella, Evidence for a role of salicylic acid in [71] M. Seskar, V. Shulaev, I. Raskin, Endogenous methyl salicylate in pathogen- 703
618 the oxidative damage generated by NaCl and osmotic stress in Arabidopsis inoculated tobacco plants, Plant Physiol. 116 (1998) 387–392. 704
619 seedlings, Plant Physiol. 126 (2001) 1024–1030. [72] S.W. Park, E. Kaimoyo, D. Kumar, S. Mosher, D.F. Klessig, Methyl salicylate is 705
620 [44] J.R. Koch, R.A. Creelman, S.M. Eshita, M. Seskar, J.E. Mullet, K.R. Davis, Ozone a critical mobile signal for plant systemic acquired resistance, Science 318 706
621 sensitivity in hybrid poplar correlates with insensitivity to both salicylic acid (2007) 113–116. 707
622 and jasmonic acid. The role of programmed cell death in lesion formation, [73] D. Kumar, D.F. Klessig, The search for the salicylic acid receptor led to discov- 708
623 Plant Physiol. 123 (2000) 487–496. ery of the SAR signal receptor, Plant Signal. Behav. 3 (2008) 691–692. 709
624 [45] I.M. Scott, S.M. Clarke, J.E. Wood, L.A. Mur, Salicylate accumulation inhibits [74] D.A. Dempsey, D.F. Klessig, SOS – too many signals for systemic acquired 710
625 growth at chilling temperature in Arabidopsis, Plant Physiol. 135 (2004) resistance? Trends Plant Sci. 17 (2012) 538–545. 711
626 1040–1049. [75] E. Attaran, T.E. Zeier, T. Griebel, J. Zeier, Methyl salicylate production and 712
627 [46] Y. Du, R. Tejos, M. Beck, E. Himschoot, H. Li, S. Robatzek, S. Vanneste, J. Friml, jasmonate signaling are not essential for systemic acquired resistance in Ara- 713
628 Salicylic acid interferes with clathrin-mediated endocytic protein trafficking, bidopsis, Plant Cell 21 (2009) 954–971. 714
629 Proc. Natl. Acad. Sci. U.S.A. 110 (2013) 7946–7951. [76] J.R. Ross, K.H. Nam, J.C. D’Auria, E. Pichersky, S-Adenosyl-l- 715
630 [47] A. Kachroo, G.P. Robin, Systemic signaling during plant defense, Curr. Opin. methionine:salicylic acid carboxyl methyltransferase, an enzyme involved 716
631 Plant Biol. 16 (2013) 527–533. in floral scent production and plant defense, represents a new class of plant 717
632 [48] Z. Chen, D.F. Klessig, Identification of a soluble salicylic acid-binding pro- methyltransferases, Arch. Biochem. Biophys. 367 (1999) 9–16. 718
633 tein that may function in signal transduction in the plant disease-resistance [77] X.Y. Zheng, N.W. Spivey, W. Zeng, P.P. Liu, Z.Q. Fu, D.F. Klessig, S.Y. He, X. Dong, 719
634 response, Proc. Natl. Acad. Sci. U.S.A. 88 (1991) 8179–8183. Coronatine promotes Pseudomonas syringae virulence in plants by activating a 720
635 [49] Z. Chen, H. Silva, D.F. Klessig, Active oxygen species in the induction of signaling cascade that inhibits salicylic acid accumulation, Cell Host Microbe 721
636 plant systemic acquired resistance by salicylic acid, Science 262 (1993) 11 (2012) 587–596. 722
637 1883–1886. [78] P.P. Liu, C.C. von Dahl, D.F. Klessig, The extent to which methyl salicylate is 723
638 [50] Z. Chen, J.W. Ricigliano, D.F. Klessig, Purification and characterization of a required for signaling systemic acquired resistance is dependent on exposure 724
639 soluble salicylic acid-binding protein from tobacco, Proc. Natl. Acad. Sci. U.S.A. to light after infection, Plant Physiol. 157 (2011) 2216–2226. 725
640 90 (1993) 9533–9537. [79] L.C. Roden, R.A. Ingle, Lights, rhythms, infection: the role of light and the 726
641 [51] U. Conrath, Z. Chen, J.R. Ricigliano, D.F. Klessig, Two inducers of plant defense circadian clock in determining the outcome of plant–pathogen interactions, 727
642 responses 2,6-dichloroisonicotinec acid and salicylic acid, inhibit catalase Plant Cell 21 (2009) 2546–2552. 728
643 activity in tobacco, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 7143–7147. [80] S. Kangasjarvi, J. Neukermans, S. Li, E.M. Aro, G. Noctor, Photosynthesis, pho- 729
644 [52] A. Talarczyk, M. Krzymowska, W. Borucki, J. Hennig, Effect of yeast CTA1 gene torespiration, and light signalling in defence responses, J. Exp. Bot. 63 (2012) 730
645 expression on response of tobacco plants to tobacco mosaic virus infection, 1619–1636. 731
646 Plant Physiol. 129 (2002) 1032–1044. [81] S. Karpinski, H. Gabrys, A. Mateo, B. Karpinska, P.M. Mullineaux, Light per- 732
647 [53] M. Ruffer, B. Steipe, M.H. Zenk, Evidence against specific binding of salicylic ception in plant disease defence signalling, Curr. Opin. Plant Biol. 6 (2003) 733
648 acid to plant catalase, FEBS Lett. 377 (1995) 175–180. 390–396. 734
649 [54] H. Du, D.F. Klessig, Identification of a soluble, high-affinity salicylic acid- [82] A.C. Chandra-Shekara, M. Gupte, D. Navarre, S. Raina, R. Raina, D. Klessig, P. 735
650 binding protein in tobacco, Plant Physiol. 113 (1997) 1319–1327. Kachroo, Light-dependent hypersensitive response and resistance signaling 736
651 [55] D.H. Slaymaker, D.A. Navarre, D. Clark, O. del Pozo, G.B. Martin, D.F. Klessig, against Turnip Crinkle Virus in Arabidopsis, Plant J. 45 (2006) 320–334. 737
652 The tobacco salicylic acid-binding protein 3 (SABP3) is the chloroplast car- [83] N. Zhao, J. Guan, F. Forouhar, T.J. Tschaplinski, Z.M. Cheng, L. Tong, F. Chen, Two 738
653 bonic anhydrase, which exhibits antioxidant activity and plays a role in poplar methyl salicylate esterases display comparable biochemical properties 739
654 the hypersensitive defense response, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) but divergent expression patterns, Phytochemistry 70 (2009) 32–39. 740
655 11640–11645. [84] A.M. Morse, T.J. Tschaplinski, C. Dervinis, P.M. Pijut, E.A. Schmelz, W. Day, 741
656 [56] R.P. Henry, Multiple roles of carbonic anhydrase in cellular transport and J.M. Davis, Salicylate and catechol levels are maintained in nahG transgenic 742
657 metabolism, Annu. Rev. Physiol. 58 (1996) 523–538. poplar, Phytochemistry 68 (2007) 2043–2052. 743
658 [57] M.D. Hatch, J.N. Burnell, Carbonic anhydrase activity in leaves and its role in [85] A.C. Vlot, P.P. Liu, R.K. Cameron, S.W. Park, Y. Yang, D. Kumar, F. Zhou, T. 744
659 the first step of c(4) photosynthesis, Plant Physiol. 93 (1990) 825–828. Padukkavidana, C. Gustafsson, E. Pichersky, D.F. Klessig, Identification of likely 745
660 [58] N. Majeau, M.A. Arnoldo, J.R. Coleman, Modification of carbonic anhydrase orthologs of tobacco salicylic acid-binding protein 2 and their role in systemic 746
661 activity by antisense and over-expression constructs in transgenic tobacco, acquired resistance in Arabidopsis thaliana, Plant J. 56 (2008) 445–456. 747
662 Plant Mol. Biol. 25 (1994) 377–385. [86] P.M. Manosalva, S.W. Park, F. Forouhar, L. Tong, W.E. Fry, D.F. Klessig, Methyl 748
663 [59] F.J. Ferreira, C. Guo, J.R. Coleman, Reduction of plastid-localized carbonic esterase 1 (StMES1) is required for systemic acquired resistance in potato, 749
664 anhydrase activity results in reduced Arabidopsis seedling survivorship, Plant Mol. Plant Microbe Interact. 23 (2010) 1151–1163. 750
665 Physiol. 147 (2008) 585–594. [87] M. Dutt, G. Ananthakrishnan, M.K. Jaromin, R.H. Brlansky, J.W. Grosser, Eval- 751
666 [60] S. Restrepo, K.L. Myers, O. del Pozo, G.B. Martin, A.L. Hart, C.R. Buell, W.E. Fry, uation of four phloem-specific promoters in vegetative tissues of transgenic 752
667 C.D. Smart, Gene profiling of a compatible interaction between Phytophthora citrus plants, Tree Physiol. 32 (2012) 83–93. 753
668 infestans and Solanum tuberosum suggests a role for carbonic anhydrase, Mol. [88] M. Dutt, G. Barthe, J.W. Grosser, Transformation with SAR genes for resistance Q4 754
669 Plant Microbe Interact. 18 (2005) 913–922. to Huanglingbing and Canker, in: Proceedings of the International Society of 755
670 [61] A. Feechan, E. Kwon, B.W. Yun, Y. Wang, J.A. Pallas, G.J. Loake, A central role Citriculture, pp. 289–294. 756
671 for S-nitrosothiols in plant disease resistance, Proc. Natl. Acad. Sci. U.S.A. 102 [89] Y. Zhao, Q. Liu, R.E. Davis, Transgene expression in strawberries driven by a 757
672 (2005) 8054–8059. heterologous phloem-specific promoter, Plant Cell Rep. 23 (2004) 224–230. 758
673 [62] D. Spadaro, B.W. Yun, S.H. Spoel, C. Chu, Y.Q. Wang, G.J. Loake, The redox [90] J. Durner, D.F. Klessig, Inhibition of ascorbate peroxidase by salicylic acid and 759
674 switch: dynamic regulation of protein function by cysteine modifications, 2,6-dichloroisonicotinic acid, two inducers of plant defense responses, Proc. 760
675 Physiol. Plant. 138 (2010) 360–371. Natl. Acad. Sci. U.S.A. 92 (1995) 11312–11316. 761
676 [63] Y.Q. Wang, A. Feechan, B.W. Yun, R. Shafiei, A. Hofmann, P. Taylor, P. Xue, [91] S. Shigeoka, T. Ishikawa, M. Tamoi, Y. Miyagawa, T. Takeda, Y. Yabuta, K. 762
677 F.Q. Yang, Z.S. Xie, J.A. Pallas, C.C. Chu, G.J. Loake, S-nitrosylation of AtSABP3 Yoshimura, Regulation and function of ascorbate peroxidase isoenzymes, J. 763
678 antagonizes the expression of plant immunity, J. Biol. Chem. 284 (2009) Exp. Bot. 53 (2002) 1305–1319. 764
679 2131–2137. [92] M. Tian, C.C. von Dahl, P.P. Liu, G. Friso, K.J. van Wijk, D.F. Klessig, The com- 765
680 [64] M. Yu, B.W. Yun, S.H. Spoel, G.J. Loake, A sleigh ride through the SNO: regu- bined use of photoaffinity labeling and surface plasmon resonance-based 766
681 lation of plant immune function by protein S-nitrosylation, Curr. Opin. Plant technology identifies multiple salicylic acid-binding proteins, Plant J. (2012), 767
682 Biol. 15 (2012) 424–430. http://dx.doi.org/10.1111/tpj.12016. 768
683 [65] Y. Tada, S.H. Spoel, K. Pajerowska-Mukhtar, Z. Mou, J. Song, C. Wang, J. Zuo, X. [93] H. Cao, J. Glazebrook, J.D. Clarke, S. Volko, X. Dong, The Arabidopsis NPR1 gene 769
684 Dong, Plant immunity requires conformational changes [corrected] of NPR1 that controls systemic acquired resistance encodes a novel protein containing 770
685 via S-nitrosylation and thioredoxins, Science 321 (2008) 952–956. ankyrin repeats, Cell 88 (1997) 57–63. 771

Please cite this article in press as: D. Kumar, Salicylic acid signaling in disease resistance, Plant Sci. (2014),
http://dx.doi.org/10.1016/j.plantsci.2014.04.014
 G Model
PSL 8977 1–8 ARTICLE IN PRESS
8 D. Kumar / Plant Science xxx (2014) xxx–xxx

772 [94] O. Wally, J. Jayaraj, Z.K. Punja, Broad-spectrum disease resistance to [101] M. Moreau, M. Tian, D.F. Klessig, Salicylic acid binds NPR3 and NPR4 to regu- 794
773 necrotrophic and biotrophic pathogens in transgenic carrots (Daucus carota late NPR1-dependent defense responses, Cell Res. 22 (2012) 1631–1633. 795
774 L.) expressing an Arabidopsis NPR1 gene, Planta 231 (2009) 131–141. [102] M. Kinkema, W. Fan, X. Dong, Nuclear localization of NPR1 is required for 796
775 [95] H.A. Fitzgerald, M.S. Chern, R. Navarre, P.C. Ronald, Overexpression of activation of PR gene expression, Plant Cell 12 (2000) 2339–2350. 797
776 (At)NPR1 in rice leads to a BTH- and environment-induced lesion-mimic/cell [103] H. Cao, S.A. Bowling, A.S. Gordon, X. Dong, Characterization of an Arabidopsis 798
777 death phenotype, Mol. Plant Microbe Interact. 17 (2004) 140–151. mutant that is nonresponsive to inducers of systemic acquired resistance, 799
778 [96] M. Chern, H.A. Fitzgerald, P.E. Canlas, D.A. Navarre, P.C. Ronald, Overex- Plant Cell 6 (1994) 1583–1592. 800
779 pression of a rice NPR1 homolog leads to constitutive activation of defense [104] S.H. Spoel, Z. Mou, Y. Tada, N.W. Spivey, P. Genschik, X. Dong, Proteasome- 801
780 response and hypersensitivity to light, Mol. Plant Microbe Interact. 18 (2005) mediated turnover of the transcription coactivator NPR1 plays dual roles in 802
781 511–520. regulating plant immunity, Cell 137 (2009) 860–872. 803
782 [97] T. Srinivasan, K.R. Kumar, G. Meur, P.B. Kirti, Heterologous expression of Ara- [105] Z.Q. Fu, S. Yan, A. Saleh, W. Wang, J. Ruble, N. Oka, R. Mohan, S.H. Spoel, Y. 804
783 bidopsis NPR1 (AtNPR1) enhances oxidative stress tolerance in transgenic Tada, N. Zheng, X. Dong, NPR3 and NPR4 are receptors for the immune signal 805
784 tobacco plants, Biotechnol. Lett. 31 (2009) 1343–1351. salicylic acid in plants, Nature 486 (2012) 228–232. 806
785 [98] W.C. Lin, C.F. Lu, J.W. Wu, M.L. Cheng, Y.M. Lin, N.S. Yang, L. Black, S.K. Green, [106] Y. Wu, D. Zhang, J.Y. Chu, P. Boyle, Y. Wang, I.D. Brindle, V. De Luca, C. Despres, 807
786 J.F. Wang, C.P. Cheng, Transgenic tomato plants expressing the Arabidopsis The Arabidopsis NPR1 protein is a receptor for the plant defense hormone 808
787 NPR1 gene display enhanced resistance to a spectrum of fungal and bacterial salicylic acid, Cell Rep. 1 (2012) 639–647. 809
788 diseases, Transgenic Res. 13 (2004) 567–581. [107] L. Tretter, V. Adam-Vizi, Generation of reactive oxygen species in the reac- 810
789 [99] R. Makandar, J.S. Essig, M.A. Schapaugh, H.N. Trick, J. Shah, Genetically tion catalyzed by alpha-ketoglutarate dehydrogenase, J. Neurosci. 24 (2004) 811
790 engineered resistance to Fusarium head blight in wheat by expression of 7771–7778. 812
791 Arabidopsis NPR1, Mol. Plant Microbe Interact. 19 (2006) 123–129. [108] S.A. Hawley, M.D. Fullerton, F.A. Ross, J.D. Schertzer, C. Chevtzoff, K.J. Walker, 813
792 [100] M. Malnoy, Q. Jin, E.E. Borejsza-Wysocka, S.Y. He, H.S. Aldwinckle, Overex- M.W. Peggie, D. Zibrova, K.A. Green, K.J. Mustard, B.E. Kemp, K. Sakamoto, G.R. 814
793 pression of the apple MpNPR1 gene confers increased disease resistance in Steinberg, D.G. Hardie, The ancient drug salicylate directly activates AMP- 815
Malus × domestica, Mol. Plant Microbe Interact. 20 (2007) 1568–1580. activated protein kinase, Science 336 (2012) 918–922. 816

Please cite this article in press as: D. Kumar, Salicylic acid signaling in disease resistance, Plant Sci. (2014),
http://dx.doi.org/10.1016/j.plantsci.2014.04.014