Salicylic acid signaling in disease resistance

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ARTICLE IN PRESSG ModelSL 8977 1–8

Plant Science xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Plant Science

j ourna l ho me pa ge: www.elsev ier .com/ locate /p lantsc i

eview

alicylic acid signaling in disease resistance

hirendra Kumar ∗

epartment of Biological Sciences, East Tennessee State University, Box 70703, Johnson City, TN 37614, USA

r t i c l e i n f o

rticle history:vailable online xxx

eywords:alicylic acid (SA)ystemic acquired resistance (SAR)A-binding protein 2 (SABP2)PR1PR3

a b s t r a c t

Salicylic acid (SA) is a key plant hormone that mediates host responses against microbial pathogens.Identification and characterization of SA-interacting/binding proteins is a topic which has always excitedscientists studying microbial defense response in plants. It is likely that discovery of a true receptor forSA may greatly advance understanding of this important signaling pathway. SABP2 with its high affinityfor SA was previously considered to be a SA receptor. Despite a great deal work we may still not havetrue a receptor for SA. It is also entirely possible that there may be more than one receptor for SA. Thisscenario is more likely given the diverse role of SA in various physiological processes in plants including,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 potentialSA 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 proteinsmay have direct/indirect role in plant processes other than pathogen defense signaling. Developmentand use of new techniques with higher specificity to identify SA-interacting proteins have shown greatpromise and have resulted in the identification of several new SA interactors. This review focuses on SAinteraction/binding proteins identified so far and their likely role in mediating plant defenses.
© 2014 Published by Elsevier Ireland Ltd.

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1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. SA biosynthesis and metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003. SA-binding proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004. NPR1, NPR3 and NPR4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 005. Concluding remarks and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

. Introduction

Salicylic acid (SA), a simple phenolic compound is well studiedor its role in activating plant defenses especially systemic acquiredesistance (SAR) [1,2]. SA and its derivative (aspirin: acetyl SA)ave been widely used for years as an anti-inflammatory drug. Ini-

aspirin, exhibited increased resistance against tobacco mosaic virus(TMV) [4,5]. Treatment with SA and its derivative induced expres-sion of pathogenesis-related proteins [6–8]. SA is required for theactivation of robust SAR and is marked by the increased expres-sion of many defense proteins including pathogenesis-related (PR)proteins. Plants defective in SA synthesis/accumulation exhibit

Please cite this article in press as: D. Kumar, Salicylic

http://dx.doi.org/10.1016/j.plantsci.2014.04.014

ially SA was discovered as a major component in bark extract ofillow (Salix) tree. Aspirin became the first synthetic drug to besed for anti-inflammatory agent [3]. The role of SA in plants wasecorded for the first time in 1987 [4]. Tobacco plants treated with

∗ Tel.: +1 423 439 6928; fax: +1 423 439 5958.E-mail addresses: [email protected], [email protected]

ttp://dx.doi.org/10.1016/j.plantsci.2014.04.014168-9452/© 2014 Published by Elsevier Ireland Ltd.

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enhanced susceptibility to pathogens [9,10]. Besides SA, other planthormones known for their direct/indirect role in plant signalingare jasmonic acid (JA), ethylene (ET), abscisic acid (ABA), aux-ins, gibberellins (GA), brassinosteroids, and cytokinins (CKs) [11].

acid signaling in disease resistance, Plant Sci. (2014),

Many of these hormone mediated signaling pathways are alsoknown to crosstalk resulting in an antagonistic or synergisticinteraction [12]. JA pathway when activated in response to her-bivory or wounding triggers a systemic response similar to SAR.

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dx.doi.org/10.1016/j.plantsci.2014.04.014


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ARTICLESL 8977 1–8

D. Kumar / Plant Sc

reatment of plants with SA is known to suppress JA inducedounding response [13,14]. Some pathogen like Pseudomonas

yringe induces activation of both SA and JA pathways [13]. Addi-ional studies have shown that SA-induced defense mostly actsgainst biotrophs while JA activated defense is targeted towardounding and necrotropic pathogens [15,16]. Activation of an

mmune pathway against biotrophic pathogens suppress defensegainst necrotropic pathogens [17,18]. Arabidopsis plants treatedith low concentrations of JA and SA exhibited a synergistic effect

n the expression of PR1 and PDF1.2 genes while treatment withigher concentrations resulted in an antagonistic effect [19]. Muta-ion in a fatty acid desaturase (ssi2) resulted in the upregulationf the SA pathway and suppression of the JA mediated pathway20,21]. ET also shows extensive crosstalk with SA–JA signalingathways. ET potentiates expression of SA dependent PR1 genexpression in Arabidopsis and in tobacco plants it is required forctivation of the SAR response [22,23]. ABA, an important hormonen signaling abiotic stress has recently emerged as a key componentf plant immune signaling [24]. ABA antagonizes the SA mediatedlant defense responses at multiple steps [11,25]. Overall, molecu-

ar details of ET, JA and SA interactions are still poorly understoodnd require further investigations.

. SA biosynthesis and metabolism

In plants, SA is synthesized in plastids via two routes from cho-ismate, a product of the shikimate pathway. One route is throughsochorismate synthase (ICS), which is believed to be responsibleor >90% of SA synthesized during activation of stress response10]. The other route uses the phenylalanine ammonia-lyase (PAL)

ediated pathway [26]. SA is readily modified to its many deriva-ives (via glucosylation, methylations, amino acid conjugation,ulphonation, hydroxylation, etc.) but most are not active com-ounds [27]. Most of the SA produced in plants is glucosylated (SAG)nd believed to be the main storage form with the potential to beonverted back to SA through enzymatic reactions catalyzed by aA �-glucosidase [28,29]. A methylated derivative (MeSA; methylalicylate) is also inactive but is volatile and could readily diffusehrough membranes. Volatilization of SA through MeSA synthe-is could help plants excrete SA outside of the cell in which it isynthesized for eventual diffusion out of the plant [30,31]. Thisechanism may help plants to reduce the accumulation of SA and

ts resulting toxic effects, which has the potential to cause cell death32]. Besides plant immune signaling, SA also serves as an impor-ant signaling molecule in various physiological responses such asrought [33], thermogenesis [34–36], stomatal closure [37], seedermination [38], flowering [39–42], salt stress [43], ozone [44],nd chilling [45]. A recent study suggests a role for SA in clathrin-ediated endocytic protein trafficking [46]. The main focus of this

eview is to discuss major SA interacting/binding proteins identi-ed to date and their role in understanding of SA signaling pathway

n disease resistance. There are a number of excellent reviewsescribing other aspects of SA signaling [2,27,47].

. SA-binding proteins

To identify cellular proteins which physically interact and bindo SA, a combination of biochemical and traditional column chro-

atography was used. Proteins from tobacco plants which boundo SA labeled with 14C or 3H were identified, purified and char-cterized for their role in SA-mediated plant defense response.



everal tobacco proteins were identified as SA-binding proteins.eanwhile a genetic approach using Arabidopsis mutants identi-

ed a number of key components of the SA signaling pathway butid not directly identify any SA-binding proteins. The presence of

PRESSxx (2014) xxx–xxx

redundant proteins with overlapping functions is one reason whyT-DNA insertion may not be suitable for the identification of SAinteracting/binding proteins.

The SABP, catalase was the first soluble plant protein found tophysically bind SA [48]. SABP was identified and purified using14C-SA [49,50]. In plants, catalases are known to detoxify H2O2produced during various metabolic processes. Binding of SA tothe catalase resulted in inhibition of its H2O2 hydrolyzing activ-ity [49]. It was hypothesized that inhibition of catalases by SAcould potentially lead to accumulation of toxic H2O2 which thenactivates expression of defense genes and systemic acquired resis-tance (SAR). Supporting this hypothesis, another SAR inducer,2,6-dichloroisonicotinic acid (INA), has been shown to inhibit cata-lase activity in tobacco [51]. Transgenic tobacco plants expressing ayeast catalase gene (CAT1) accumulated less H2O2 around Tobaccomosaic virus (TMV) induced necrotic lesions compared to controlplants. TMV induced necrotic lesions were larger compared to con-trol plants both in primary inoculated and secondary inoculatedupper leaves, suggesting that catalase has a role in inducing diseaseresistance. Increased levels of catalase in these transgenic plantsmore likely detoxified H2O2 resulting in a decrease in its availabilityfor activating resistance [52]. The transgenic tobacco with reducedcatalase activity developed necrotic lesions and induced expres-sion of PR genes only under high light conditions, suggesting thatSA inhibition of catalase may not be required for the induction of thedefense response. Later studies showed that SA binds to many ironcontaining enzymes, e.g. aconitase, catalase, lipoxidase and peroxi-dase suggesting that SA binding to catalase was not specific [53]. SAbound to SABP with a moderate affinity (Kd = 14 �M)[48]. To searchfor high affinity SA-binding proteins, a ligand with higher affinity(3H-SA) was synthesized and used for identification of additionalSA-binding proteins [54].

SA-binding protein 3 (SABP3) was identified as a stroma local-ized carbonic anhydrase. It has moderate affinity Kd = 3.7 �M for SAcompared to SABP2 (Kd = 90 nM) [55] (Table 1). But unlike SABP, SAbinding has no effect on the carbonic anhydrase activity of SABP3[55]. Carbonic anhydrase in animals helps to transport CO2 out ofmuscle cells and provide bicarbonate to mitochondria for gluco-neogenesis [56]. In C4 plants, cytosol localized carbonic anhydrasecatalyzes the conversion of CO2 into bicarbonate, which is usedduring carbon fixation by the C4 enzyme, phosphoenolpyruvatecarboxylase [57]. In contrast, antisense carbonic anhydrase tobaccoplants with 99% reduction in activity had little or no effect onphotosynthesis or general fitness of the plant [58]. Recent stud-ies using a T-DNA insertion mutant of a plastid localized carbonicanhydrase in Arabidopsis showed a reduction in seedling estab-lishment compared to wild type plants at ambient CO2 levels[59]. Overexpression of carbonic anhydrase in chloroplast led toan increase in Rubisco activity [58]. Virus-induced gene silencingof a SABP3 homolog in Nicotiana benthamina led to the suppres-sion of the Pto:avrPto mediated hypersensitive response [55]. Inyet another study, carbonic anhydrase transcripts were shown tobe upregulated in compatible reactions while down regulated inincompatible reactions at the 12 h time point [60]. By 24–28 hcarbonic anhydrase transcripts were completely downregulated.Only by 72 h, where carbonic anhydrase transcripts upregulatedagain [60]. Silencing of carbonic anhydrase in N. benthamianaallowed increased growth of Phytophthora infestans [60]. Theseresults suggest that SABP3/carbonic anhydrase is needed for pos-itive regulation of defense responses in plants. SABP3 is a targetfor modification via S-nitrosylation during later stages of R-genemediated protection against avirulent plant pathogens [61]. S-


nitrosylation is the covalent attachment of nitric oxide moiety to acysteine thiol of a protein to form S-nitrosothiol [62]. Modificationby S-nitrosylation at Cys280 renders SABP3 unable to bind to SAand lose its carbonic anhydrase activity [63]. SABP3 is a positive

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Table 1SA-interacting proteins.

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

SABPa Catalase Nicotiana tabacum 14C-SA 14.0 �MSABP2b Methyl esterase Nicotiana tabacum 3H-SA 0.09 �MSABP3c Carbonic anhydrase Nicotiana tabacum 3H-SA 3.70 �MNPR1d A protein with ankyrin repeat and BTB/POZ domain Arabidopsis thaliana Genetics/T-DNA mutant 0.14 �MNPR3e CUL3 adapter protein Arabidopsis thaliana Genetics/NPR1 paralog 0.98 �MNPR4f CUL3 adapter protein Arabidopsis thaliana Genetics/NPR1 paralog 0.046 �MAscorbate peroxidaseg Ascorbate peroxidase Nicotiana tabacum 3H-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

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GSTF10 [92].l GSTF11 [92].

egulator of plant disease resistance in Arabidopsis and its modifi-ation via S-nitrosylation affects its ability to influence immuneesponse [64]. This appears to be a host mechanism to quicklyampen defense responses and bring the cell back to normal state.t this point, it is unclear if pathogens hijack this mechanism touppress defense responses or if it is merely a component of a neg-tive feedback loop of the immune response. Interestingly, plantefense regulator NPR1 is also S-nitrosylated at Cys156 and thisavors its oligomerization in cytoplasm during the resting phasef the plant cells [65], while SA induces an oligomer to monomerhange through the activity of thioredoxins [65].

The SABP2, a high affinity SA-binding protein was identifiedsing 3H-SA as a ligand [54]. Using conventional chromatogra-hy methods it was purified 24,000-fold from uninduced tobacco

eaves [66]. During initial stages of purification SABP (catalase)as fractionated out in a 0–50% ammonium sulfate fraction while

oth SABP3 (carbonic anhydrase) and SABP2 (the 29 kDa protein)o-fractionated in 50–75% fraction. During the next purificationtep of size exclusion chromatography using Sephadex 100, SABP3nd SABP2 eluted in different fractions. Through a combination ofartial peptide sequencing, RT-PCR and 5′-RACE, the cDNA cor-esponding to SABP2 was amplified, cloned and sequenced [66].his ∼29 kDa protein consisted of 260-amino acids. Subsequentequence analysis showed that it is member of the �/�-hydrolaseold protein superfamily [67]. Members of this family of proteinsave diverse biochemical activities such as hydrolases and othernzymes (lipase, esterases, thioesterases, cutinase, epoxy hydro-ase, etc.) that are involved in the activation of H2O2, or HCN�-hydroxynitrile lyases, haloperoxides) and which share a com-

on and conserved catalytic triad of amino acids, Ser, Asp andis [68]. The recombinant ∼29 kDa protein expressed with a N-

erminal 6xHis tag, bound specifically to SA and its active analogs,.g. 5-CSA, 2.6-DHBA (does induce PR expression and resistance)ut did not bind to inactive analogs, e.g. 2,5-DHBA, 4-HBA (doesot induce PR proteins and resistance). Recombinant SABP2 exhib-

ted lipase/esterase-like activity with artificial substrates, but a



ubsequent search for the plant based substrate led to the identifi-ation of methyl salicylic acid as a potential substrate [66,67]. Otherethylated plant hormones, e.g. MeJA (methyl jasmonic acid) andeIAA (methylindoleacetic acid) were poor substrates [67]. RNAi

mediated silencing of SABP2 made transgenic tobacco plants lessresistant and defective in mounting a robust SAR response, sug-gesting an important role for SABP2 in disease resistance [66]. Thisindicates that SABP2 is a likely a bona fide SA receptor.

To verify if the SAR defective phenotype of transgenic tobaccoplants (1–2) silenced in SABP2 expression was specifically causedby silencing of SABP2 or was a result of off-target silencing of othergene/s, a synthetic gene complementation approach using a syn-thetic version of native (nat) tobacco SABP2 was undertaken [69].The synthetic (syn) version of SABP2 was designed and manufac-tured to maximize differences in the DNA sequence of the nativetobacco gene by taking advantage of the availability of alternatecodons for each amino acid. Syn SABP2 was 24% different, withno more than nine bases of exact match in a stretch with nativetobacco SABP2. Both nat and syn SABP2 coded for the exact sameamino acids. Both control (C3; N. tabacum Xanthi NN plants trans-formed with empty vector) and SABP2 silenced (1–2 transgenic)plants were stably transformed with nat and syn version of SABP2[66]. Plants challenged with TMV were assessed for SAR develop-ment. SABP2 silenced plants expressing synthetic SABP2 regainedthe wild type phenotype and exhibited SAR. This showed that theSAR defective phenotype of SABP2 silenced (1–2) plants was due tospecific silencing of SABP2 and not due to off-target gene silencing.Further analysis also showed that control C3 plants transformedwith syn SABP2 showed stronger SAR than C3 plants [69].

The use of a synthetic gene for the generation of transgenicplants overexpressing a protein of interest may be a significantbiotechnological tool. It is important to note that attempts to gener-ate transgenic tobacco plants constitutively overexpressing SABP2have been unsuccessful (Kumar and Klessig, unpublished results).Attempts to overexpress NtSABP2 (tobacco SABP2 gene) in Arabidop-sis succeeded partially but only for the first generation (Kumarand Klessig, unpublished results). By the second generation, theexpression of NtSABP2 in transgenic Arabidopsis plants was com-pletely lost (Kumar and Klessig, unpublished results). This showsthat plants may have mechanisms to efficiently regulate the expres-


sion of SABP2. This may be needed to regulate local levels of SAthat protect the cell against toxic effects of SA. Substantially mod-ified SABP2 via synthetic gene technology may offer a mechanismto overcome such endogenous control mechanisms that suppress

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D. Kumar / Plant Sc

verexpression of genes like SABP2. It is important to note that theomplementation experiments described above were conductedy expressing various constructs from an extradiol inducible pro-oter using pER8 vector and not through constitutive expression

70].Identification of MeSA as an endogenous substrate for SABP2

nd inhibition of its catalytic activity by SA binding indicated anmportant role for MeSA in SA mediated plant defenses [67]. MeSA

as identified as a volatile plant compound synthesized by tobaccolants infected with TMV [30]. MeSA is synthesized from SA inlants and has been suggested to act by converting into SA (Fig. 1)30]. MeSA being volatile, travels as an airborne signal to neigh-oring plants where it induces expression of PR proteins as wells in the healthy tissue of the infected plants. Later studies showedigh level accumulation of MeSA in plant tissues infected with TMVr Pseudomonas syringae pv. phaseolicola [71]. Using a combinationf grafting experiments involving SABP2 silenced (1–2) transgenicobacco plants and biochemical analysis it was shown that MeSAs a phloem mobile signal for SAR in plants [72]. Active SABP2 isequired in systemic uninfected tissues to convert phloem mobileeSA to SA in order to induce resistance. This observation argues

hat SABP2 is a receptor for MeSA [73]. Binding of SA to SABP2 tonhibit its esterase activity in primary tissue was required for SARnduction. Timely inhibition of SABP2 esterase activity in primaryissue was required to ensure accumulation of MeSA in primary tis-ue and its phloem mediated transport to systemic healthy tissue.ynthesis of MeSA is catalyzed by SA-methyl transferase (SAMT).lants lacking this SA-methyl transferase activity in tobacco, potatond Arabidopsis were SAR defective [74]. Recent studies using T-NA insertion mutants suggests that MeSA or jasmonic acid mayot be the mobile signal for SAR in Arabidopsis plants [75]. Levels ofeSA increase in pathogen infected tobacco and Arabidopsis plants

30,71]. It is produced through a reaction catalyzed by an SAMT likenzyme using S-adenosine-l-methionine as a methyl donor [76].oronatine, a Pseudomonas syringae virulence factor enhances theroduction of MeSA (by activating SAMT activity) which is likely toe lost as a volatile compound, thereby reducing levels of SA [77].his mechanism appears to help P. syringae in attenuation of SA-ediated plant defense responses [75]. This could also be a strategy

f P. syringae for protecting the host plant against potential damageaused by accumulation of SA to high concentrations. Prematureamage to plant tissue may not in the best interest of pathogenultiplication and growth.A discovery by Attaran et al. [75] suggested that MeSA is not

equired for SAR development in Arabidopsis plants promptedlessig and colleagues to examine reasons for the discrepancyetween their observation that MeSA is required for SAR devel-pment in Arabidopsis. Out of several factors which are likely tonfluence SAR, light appears to play critical role in development ofull or partial defense responses. It is suggested that the extent ofight exposure following primary pathogen infection determinesf MeSA is required for SAR development or not [78]. In wild typeobacco and Arabidopsis plants inoculations early in the day fol-owed by long exposure to light, induced a strong SAR response. Inontrast, mutant Arabidopsis bsmt1 (defective in SA methyl trans-erase activity, fail to accumulate MeSA), exhibited a differentesponse and requirement for MeSA depending on the time of inoc-lations during the day. When inoculated in morning hours andxposed to light for long hours (more than 3.5 h), the plants did notbligately require MeSA for SAR development [78]. Inoculation ofhese mutants in late afternoon/evening with little exposure to lightless than 3.5 h), obligately required MeSA for SAR development.



he importance of light in plant immunity is well documented21,79–82]. SABP2 homologs have now been identified and char-cterized from a number of plant species including Arabidopsis,oplar, potato and tomato.


Two homologs (PtSABP2-1 and PtSABP2-2) of NtSABP2 wereidentified from woody poplar plants with 77% identity [83]. Poplaris one of the fastest growing tree species and is considered an idealcandidate crop for a bioenergy production. Both PtSABP2-1 andPtSABP2-2 were highly similar (98% identity) to each other andshowed methyl salicylate esterase activity. The major differenceswere in their promoter region and in their response to stress sig-nals. PtSABP2-1 not induced upon treatment with SA, MeJA or bywounding, while PtSABP2-2 was highly induced by SA, MeJA andupon wounding. Poplar has higher endogenous levels of SA com-pared to tobacco and Arabidopsis, as do potato and rice also [44,84].Using full length tobacco SABP2 (NtSABP2) amino acid sequence tosearch, a number of Arabidopsis proteins (total of 18) were iden-tified as potential homologs (At-MES; Arabidopsis thaliana methylestrase #1–18). These proteins were 32–57% identical to tobaccoNtSABP2 [85]. AtMES1 was most similar while AtMES18 was leastsimilar to NtSABP2 [85]. To identify a true ortholog, these proteinswere further characterized for their esterase activity using MeSA asa substrate. AtMES1, 2, 4, 7 and 9 showed SA-inhibitable esteraseactivity [85]. AtMES1, 7 and 9 when expressed in SABP2 silencedtobacco plants, rescued its SAR defect phenotype [85]. IndividualArabidopsis T-DNA insertion mutants were not compromised inSAR response while simultaneous silencing of AtMES1, 2, 7, and9 resulted in a SAR defective phenotype. This shows that severalArabidopsis proteins are redundant for methyl esterase activity.StMES1, a potato homolog of NtSABP2 was identified and cloned[86]. StMES1 showed 74% identity and 85% similarity to NtSABP2.At the enzymatic level it catalyzed the conversion of MeSA to SA.Similar to NtSABP2, SA inhibited its esterase activity. Expressionof StMES1 in SABP2 silenced transgenic tobacco plants, comple-mented its local and SAR defective phenotype showing that it is atrue ortholog of NtSABP2 [86]. This approach is being used in sweetorange to confer resistance to bacterial diseases through incorpo-ration of the tobacco SABP2 gene [87,88]. Citrus fruits are proneto many devastating diseases and this effort to express tobaccoSABP2 in phloem uses a phloem specific Arabidopsis SUC2 promoter[89].

Ascorbate peroxidase is another SA interacting protein whoseactivity is shown to be inhibited by SA [90]. Ascorbate perox-idases are known to detoxify cells of H2O2 that is constantlyproduced as by product of photosynthesis, photorespiration, oxida-tive phosphorylation, and fatty acid �-oxidation [91]. Ascorbateperoxidases are localized in the chloroplast and cytosoplasm.Ascorbate peroxidase is inhibited only by active analogs of SA, e.g. 2,6-dichloroisonicotinic acid (INA) and not by inactive analogs. It washypothesized that SA mediated inhibition of ascorbate peroxidasesled to increased levels of H2O2 in the cell which in turn triggered aresistance response [90].

Use of 14C and 3H labeled SA coupled with column chromatog-raphy led to the identification of SABP, SABP3 and SABP2, but theseproteins do not appear to be bonafide SA-receptors. To identify trueSA receptors, a combination of photoaffinity labeling (light basedcovalent cross-linking of interacting partner with the ligand) andsurface plasmon resonance-based (PMR) techniques were used.These methods resulted in the identification of several new SA-binding proteins [92]. The newly identified proteins include the E2subunit of �-ketogutarate dehydrogenase and several glutathioneS-transferases (GSTF2, GSTF8, GSTF10 and GSTF11). SA inhibitedenzymatic activity of all these newly identified SABPs. Interest-ingly these new SABPs showed little or no SA-binding activity using3H-SA as used in the identification and characterization of SABP3[55] and SABP2 [66] showing that this new technique with its


ability to detect weak and transient interactions has the poten-tial to identify high specificity SA-binding proteins. Further workusing this technology is likely to identify additional SA-bindingproteins.

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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( re it isc the nuN media

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SAMT) catalyzes conversion of SA to MeSA which diffuses into the cytoplasm wheytoplasm disrupts the oligomeric NPR1 into its monomers which then migrate to

PR1 is phosphorylated upon its interaction with transcription factors. Proteosome

. NPR1, NPR3 and NPR4

NPR1 (Nonexpresser of Pathogenesis-Related protein 1) actsownstream of SA and is a key regulator of the SA-dependent path-ay [93]. Overexpression of the NPR1 gene resulted in enhancedisease resistance in several diverse plant species, e.g. carrot [94],ice [95,96], tobacco [97], tomato [98], wheat [99], apple [100].ince, NPR1 did not physically bind to 3H-SA, it was not con-idered to be a SA receptor [101]. Changes in cytosolic levels ofA results in breakdown of cytosol localized oligomeric NPR1intoonomers and are followed by the migration of monomeric NPR1

o the nucleus [65]. Levels of NPR1 in the nucleus are critical fornducing disease resistance. In the nucleus, NPR1 monomers inter-ct with transcription factors including TGAs (basic leucine zipperranscription factors) to induce expression of disease resistanceenes including PR genes (Fig. 1) [102]. Plants lacking NPR1 areefective in SA-induced transcriptional reprogramming and failo activate SAR [93,103]. In a resting plant cell nucleus, NPR1 isuickly degraded to minimize basal expression of defense genes104]. Upon activation of an immune response, i.e. SAR, NPR1 entershe nucleus in large numbers, binds to transcription factors and ishosphorylated by kinase activity associated with the transcription

nitiation complex. This is followed by ubiquitination of phospho-ylated NPR1, a process that appears to be critical for activation ofAR in plants [104]. It is interesting to note that any NPR1 monomerntering the nucleus is eventually degraded but only the degrada-ion of phosphorylated NPR1 results in the activation of SAR whileegradation of unphosphorylated NPR1 (in resting cells) does not



esult in induction of defense genes and SAR [105]. Recent stud-es have indicated that NPR3 and NPR4 (paralogs of NPR1) have aole in mediating degradation of NPR1 [105]. The npr3npr4 doubleutant is defective in induction of SAR. In contrast to NPR1, both

converted back to SA by the esterase activity of SABP2. Increased SA levels in thecleus to activate transcription of SA responsive defense genes including PR genes.ted degradation of phosphorylated NPR1 trigger’s expression SA responsive genes.

NPR3 and NPR4 directly bind to SA. Interestingly, the binding affin-ity of NPR3 (Kd = 981 nM) and NPR4 (Kd = 46 nM) are very different,suggesting a differential role based on the intracellular concentra-tions of SA [105] (Table 1). Also, the SA-binding affinities of NPR4(Kd = 46 nM) and SABP2 (Kd = 90 nM) are similar. In yet anotherrecent study, NPR1 itself was claimed to be a receptor for SA [106](Table 1). It is now suggested that the SA-NPR1 complex is highlylabile and difficult to detect which may explain why it took so longfor this interaction to be discovered and why regular assays using3H-SA for the detection of NPR1 binding to SA were unsuccessful[106]. Using an equilibration dialysis method, it was determinedthat NPR1 interacted with [14C]SA with a Kd of 140 nM (Table 1).This binding of NPR1 to SA was through the copper ion associatedwith Cys521/529. Association of copper is crucial for NPR1 bind-ing to SA [106]. Removal of copper by chelation abolished NPR1binding to SA. Furthermore, the direct interaction of SA with NPR1is critical for the functioning of its C-terminal activation domain.This C-terminal activation domain remains inhibited because of theauto-inhibitory activity of the N-terminal BTB/POZ domain. Inter-action with SA releases the C-terminal activation domain from theBTB-POZ domain.

5. Concluding remarks and perspectives

The SA pathway is one of the most extensively studied path-ways in plant disease resistance. Great progress has been madeover the last two decades in understanding the biochemistry ofthis system. The role of SA in plant immune response has been


known for the last 35 yrs. Academia and industry have both showntremendous interests in learning more about this pathway. Sev-eral synthetic compounds mimicking SA and its resistance inducingactivity have been made available to agriculture for crop protection.

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D. Kumar / Plant Sc

TH/ASM (acibenzolar S-methyl) is one good example. But under-tanding the biochemical pathways and its target componentss crucial for designing and developing effective crop protectiongents. Availability of Arabidopsis genome sequence and T-DNAnsertion mutants has greatly helped in dissecting this pathway.

biochemical approach to identify and characterize SA interact-ng/binding proteins has also provided useful information on manymportant components. Still, our understanding of this pathway isar from complete. More sophisticated and sensitive technologiesre needed to further probe this important pathway. As we haveeen in case of SA-binding proteins, the availability of higher speci-city ligands helped in the discovery of new SA interacting/bindingroteins. It is interesting to note that binding affinity to SA forABP2 (Kd = 90 nM), NPR3 (Kd = 46 nM) and NPR1 (Kd = 140 nM) aren a similar range (Table 1). Besides plant immune responses, SAas been implicated in development, photosynthesis, flowering,hermogenesis, abiotic stress, and other important aspects of planthysiology. The role of SABPs in these physiological processes inlants is not studied and need attention. Identification of newABPs, e.g. KGDHE2, supports earlier observations that SA inhibitsitochondrial respiration and has opened further avenues of inves-

igation. In animals, SA mediated inhibition of KGDHE2 led to theccumulation of ROS (Reactive Oxygen Species), a key signalingolecule in plants and animals [107]. Several newly identified

ABPs are GSTs (Table 1) and are known scavengers of ROS. SA bindsnd inhibits their activity, which suggests that in plants it may helpo accumulate ROS as a pathway to cell death. In humans, SA and itserivatives are well known for their anti-inflammatory, antipyreticnd analgesic properties. AMPK is activated by increased cellularevels of AMP resulting in stimulation of fat utilization in ani-

als. Recent observation that SA binds to and activates a cellularetabolic regulator, AMP-activated protein kinase (AMPK) has pro-

ided a different insight into energy metabolism in animals [108].verall, given the importance of SA in a wide range of processesoth in animals and plants, it is essential to have a better under-tanding of this hormone and its actions.

cknowledgments

The author wishes to thank Dr Tom Laughlin, East Tennesseetate University and reviewers for their comments. Author of thiseview is supported by a grant from NSF (MCB 1022077) and a RDCrant from East Tennessee State University. Author apologizes toll those work could not be covered in this review.

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