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Keywords:

  • salicylic acid (SA);
  • systemic acquired resistance;
  • NPR1;
  • crosstalk;
  • SA biosensor;
  • plant defense

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. SA Metabolism
  5. SA Signaling
  6. SA and Other Hormones
  7. Techniques for SA Quantification
  8. Future Perspectives
  9. Acknowledgements
  10. References

inline imageZhonglin Mou (Corresponding author)

The small phenolic compound salicylic acid (SA) plays an important regulatory role in multiple physiological processes including plant immune response. Significant progress has been made during the past two decades in understanding the SA-mediated defense signaling network. Characterization of a number of genes functioning in SA biosynthesis, conjugation, accumulation, signaling, and crosstalk with other hormones such as jasmonic acid, ethylene, abscisic acid, auxin, gibberellic acid, cytokinin, brassinosteroid, and peptide hormones has sketched the finely tuned immune response network. Full understanding of the mechanism of plant immunity will need to take advantage of fast developing genomics tools and bioinformatics techniques. However, elucidating genetic components involved in these pathways by conventional genetics, biochemistry, and molecular biology approaches will continue to be a major task of the community. High-throughput method for SA quantification holds the potential for isolating additional mutants related to SA-mediated defense signaling.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. SA Metabolism
  5. SA Signaling
  6. SA and Other Hormones
  7. Techniques for SA Quantification
  8. Future Perspectives
  9. Acknowledgements
  10. References

Salicylic acid (SA) is a secondary metabolite produced by a wide range of prokaryotic and eukaryotic organisms including plants. Chemically, it belongs to a group of phenolic compounds defined as substances that possess an aromatic ring bearing hydroxyl group or its functional derivative. Long before SA's regulatory role in multiple physiological processes of plants attracted people's attention, the pharmacological properties of salicylates (general name of SA and its derivatives) had been prized.

Salicylates have been known to possess medicinal properties since the 5th century B.C., when Hippocrates prescribed salicylate-rich willow leaf and bark for pain relief during childbirth (Rainsford 1984; Weissman 1991). However, the utilization of salicylate-containing plants for curing aches and fevers can be traced back to inhabitants of both the Old and New Worlds. The exploration of the chemical essence of the folk remedy began in the early 1800s (Raskin 1992a,1992b). In 1828, German scientist Johann A. Buchner purified a small quantity of the yellowish substance named salicin (a salicyl alcohol glucoside) from willow bark. Later, Raffaele Piria converted salicin into a sugar and an acid he named salicylic acid (SA). More natural sources of SA and other salicylates were identified, but the demand for SA as a pain reliever rapidly outstripped production capacity. In 1859, Hermann Kolbe and coworkers chemically synthesized SA. Subsequently, the synthetic process was improved, which led to the large-scale production of cheaply priced SA for greater medicinal use. In 1897, Felix Hoffmann rediscovered the synthetic derivative acetyl salicylic acid (ASA), a chemical originally created by Charles Frederic Gerhardt in 1853, which undergoes spontaneous hydrolysis to SA but produces less gastro-intestinal irritation and has similar therapeutic properties. In 1899, Bayer pharmaceutical company registered the trade name “Aspirin” for ASA. Today, Aspirin has become one of the most successful and widely used drugs worldwide.

The primary action of salicylates in mammals is attributed to disruption of eicosanoic acid metabolism (Mitchell et al. 1990), thereby altering the levels of prostaglandins and leukotrienes. SA also affects gene expression by altering the activity of multiple transcriptional factors and inducing signaling molecule nitric oxide (Kopp and Ghosh 1994). Further studies showed that SA appears to modulate signaling through nuclear factor-κB (NF-κB), a transcriptional factor that plays a central role in animal immunity (McCarty and Block 2006). In plants, exogenous application of SA or its derivates affects diverse plant processes such as thermogenesis (Raskin et al. 1987), seed germination (Rajou et al. 2006), seedling establishment (Alonso-Ramírez et al. 2009), cell growth (Vanacker et al. 2001), respiration (Norman et al. 2004), stomatal responses (Manthe et al. 1992; Lee 1998), senescence (Rao et al. 2001, 2002), thermotolerance (Clarke et al. 2004), and nodulation (Stacey et al. 2006). In addition, genetic mutant studies in Arabidopsis suggest that SA is involved in modulating cell growth (Rate et al. 1999), trichome development (Traw and Bergelson 2003), and leaf senescence (Morris et al. 2000). However, its effect on some of these processes may be indirect, because SA is heavily involved in crosstalk with other plant hormones (Robert-Seilaniantz et al. 2007; Pieterse et al. 2009).

The most well-established role of SA is as a signaling molecule in plant immune response (Vlot et al. 2009). Unlike animals, plants lack specialized immune cells and immunological memory. However, each plant cell has developed the capability of sensing pathogens and mounting immune responses. Recognition of pathogen-associated molecular patterns (PAMPs) results in PAMP-triggered immunity (PTI, formerly called basal resistance) that prevents pathogen colonization. However, during the arms race between pathogen and plants, pathogens have evolved effectors to dampen PAMP-triggered signals and host plants in turn have evolved resistance (R) proteins to detect the presence of pathogen effectors and induce effector-triggered immunity (ETI) (reviewed in Jones and Dangl 2006). Activation of defense signaling pathways (PTI or ETI) results in the generation of a mobile signal(s) that moves from local infected tissue to distal tissue, inducing systemic acquired resistance (SAR), which is a form of long-lasting immunity to a broad spectrum of pathogens. SA-mediated immune responses are important parts of both PTI and ETI (Tsuda et al. 2009), and also essential for the activation of SAR (reviewed in Durrant and Dong 2004). Studies in various plant species have shown that pathogen infection leads to SA accumulation not only in infected leaves but also in uninfected leaves that develop SAR (Malamy et al. 1990; Métraux et al. 1990), and that SA accumulation often parallels to or precedes the increase in expression of PR genes and development of SAR. Consistently, application of exogenous SA and its functional analogs, such as Aspirin, 2,6-dichloroisonicotinic acid (INA), and benzothiadiazole S-methyl ester (BTH), activates expression of PR genes and resistance against viral, bacterial, oomycete, and fungal pathogens in a variety of dicotyledonous (Malamy and Klessig 1992; Ryals et al. 1996; Shah and Klessig 1999) and monocotyledonous plants (Wasternack et al. 1994; Gorlach et al. 1996; Morris et al. 1998; Pasquer et al. 2005; Makandar et al. 2006). Conversely, blocking SA accumulation through expression of a bacterial salicylate hydroxylase, which converts SA to catechol, in transgenic tobacco and Arabidopsis compromises HR and abolishes SAR (Gaffney et al. 1993; Delaney et al. 1994). Similarly, mutation or application of inhibitor of enzymes involved in SA biosynthesis has been shown to enhance susceptibility to pathogen, yet the resistance can be restored through exogenous SA (Mauch-Mani and Slusarenko 1996; Nawrath and Métraux 1999; Wildermuth et al. 2001; Nawrath et al. 2002).

During the past two decades, significant progress has been made in understanding SA metabolism, signaling, and its interactions with other defense mechanisms, especially hormones. As much as these studies have provided insights into the functioning of SA in plant immunity, they also underscore how much remains unknown on the complexities of SA signaling. For example, how SA is synthesized in plants is still not fully defined. However, through systems biology methods, a better understanding of robust plant immunity network properties is emerging. New methods for SA quantification have been developed for large-scale mutant screening purposes. In this review, we will address recent advances and discuss the future perspectives in the above directions.

SA Metabolism

  1. Top of page
  2. Abstract
  3. Introduction
  4. SA Metabolism
  5. SA Signaling
  6. SA and Other Hormones
  7. Techniques for SA Quantification
  8. Future Perspectives
  9. Acknowledgements
  10. References

The importance of SA as a key signaling component in disease resistance and a regulator of other important physiological processes have stimulated considerable interest in its metabolism. By using the classic biochemical radiolabeling approach and mutant-based genetic analysis, two distinct enzymatic pathways for SA biosynthesis have been identified in plants (Lee et al. 1995; Chen et al. 2009). One is the phenylalanine ammonia lyase (PAL)-mediated phenylalanine pathway, and the other is the isochorismate synthase (ICS)-mediated isochorismate pathway. Both pathways require the primary metabolite chorismate, which is an intermediate of plant phenylpropanoid pathway (downstream of shikimic acid pathway leading to lignin, flavonoid, anthocyanin, and proanthocyanidin biosynthesis).

Biochemical studies using isotope feeding in the early 1960s have suggested two possible routes from phenylalanine to SA, which differ at the step involving hydroxylation of the aromatic ring. The common and initial enzymatic step of this pathway is the conversion of chorismate-derived L-phenylalanine to trans-cinnamic acid by PAL. Subsequently, trans-cinnamic acid is hydroxylated to form O-coumarate followed by oxidation of the side chain to yield SA. Alternatively, the side chain of trans-cinnamic acid can be initially oxidized to give benzoic acid, which is then hydroxylated to produce SA. Thus, the difference between the two routes is the hydroxylation of the aromatic ring before or after the chain-shortening reactions. Isotope feeding experiments indicate that SA is mainly formed from benzoic acid in some plant species such as tobacco, rice, potato, cucumber, sunflower, and pea (Klambt 1962; Yalpani et al. 1993; Leon et al. 1995; Silverman et al. 1995; Sticher et al. 1997), while other plant species can synthesize SA through the route of O-coumarate (Yalpani et al. 1993; Leon et al. 1995; Silverman et al. 1995). However, feeding of 14C-labeled phenylalanine, cinnamate, and benzoic acid to young Primula acaulis and Gaultheria procumbens leaf segments all leads to SA formation, suggesting that both routes are probably utilized in SA biosynthesis (El-Basyouni et al. 1964). Similarly, SA is formed mostly via benzoic acid in young tomato seedlings, but after infection with Agrobacterium tumefaciens, SA biosynthesis is shifted to the route of hydroxylation of cinnamate to O-coumarate (Chadha and Brown 1974).

Phenylalanine ammonia lyase is a key regulator of the phenylpropanoid pathway and also plays an important role in regulating SA biosynthesis during the plant immune response. Expression of PAL is rapidly induced during plant-pathogen interaction, and inhibition of PAL activity results in the breakdown of an incompatible interaction between Arabidopsis and Hyaloperonospora arabidopsidis (Mauch-Mani and Slusarenko 1996). However, the incompatibility can be restored by SA application represented by complementation of the defense phenotype of PAL-inhibited plants. These results suggest an important role for PAL in localized defense against this oomycete pathogen. During the process of catalyzing benzoic acid to SA, the hydroxylation step is catalyzed by benzoic acid-2-hydroxylase (BA2H). The BA2H activity has been detected in plants including tobacco and rice (Leon et al. 1995; Sawada et al. 2006). In tobacco, the activity of a partially purified BA2H is strongly induced by benzoic acid application and in response to tobacco mosaic virus (TMV) infection, suggesting that this pathway may be involved in tobacco response to TMV (Leon et al. 1995). However, none of the enzymes required for the conversion of cinnamate to SA have been isolated from plants.

Some bacteria can synthesize SA from chorismate via two reactions catalyzed by ICS and isochorismate pyruvate lyase (IPL) (Serino et al. 1995). Overexpression of these two bacterial enzymes in plants increases SA accumulation, indicating that plants do have the capability of synthesizing SA from chorismate (Serino et al. 1995). Genetic study has confirmed the presence of a similar SA biosynthesis mechanism in Arabidopsis (Wildermuth et al. 2001). The sid2/eds16 phenotype is due to mutation of the ICS1 gene, which encodes isochorismate synthase. Upon pathogen infection, the sid2/eds16 mutant plants accumulate only 5–10% of wild-type levels of SA. These mutant plants exhibit enhanced susceptibility to pathogens and are compromised in their ability to activate SAR. SA application can complement their enhanced disease susceptibility phenotype. All these findings demonstrate that the isochorismate pathway is the main source of SA accumulated during plant-pathogen interaction in Arabidopsis (Wildermuth et al. 2001). Further, the ICS pathway has recently been shown to be active in tomato (Uppalapati et al. 2007) and tobacco (Catinot et al. 2008). At the sequence level, the ICS1 protein includes the highly conserved chorismate-binding domain and shares high identity with biochemical activity confirmed bacterial ICS protein (Wildermuth et al. 2001). The ICS1 promoter contains binding sites for WRKY and MYB transcriptional factors, both of which are involved in the regulation of plant defense, stress response, or secondary metabolism (Yang and Klessig 1996; Bender and Fink 1998; Eulgem et al. 2000).

Two isochorismate synthase genes, ICS1 and ICS2, are present in the Arabidopsis genome. Similar to ICS1, ICS2 encodes a functional ICS enzyme targeted to plastids. Null ics1 mutants still accumulate some SA, suggesting a possible role for ICS2 or the PAL pathway in biosynthesis of residual levels of SA. Comparison of SA accumulation in the ics1, ics2, and ics1ics2 mutants has indicated that ICS2 participates in the synthesis of SA. Upon UV exposure, the ics1 mutant accumulates roughly 10% and the ics1ics2 double mutant accumulates about 4% of wild-type levels of total SA. Therefore, the majority of SA (about 95%) is synthesized from the ICS pathway in UV-treated Arabidopsis plants with the remaining from an alternative pathway, possibly the PAL pathway (Garcion et al. 2008). Under both biotic and abiotic stress, Nicotiana benthaminana can also activate the ICS pathway for the bulk of SA biosynthesis, confirming the importance of the ICS-dependent SA biosynthetic pathway under stress conditions (Catinot et al. 2008). Isochorismate, the product of ICS, is converted to SA by IPL in bacteria. So far, no gene encoding IPL has been cloned from plant species (Chen et al. 2009). Thus, how plants catalyze isochorismate to SA is still unclear.

Though the ICS pathway is responsible for the majority of SA synthesis in UV-treated or pathogen-infected Arabidopsis and N. benthamiana plants, evidence for an important role of PAL activity for pathogen-induced SA formation has been repeatedly found in multiple plant species. In tobacco, SA levels in both TMV-inoculated local tissue and uninoculated distal tissue of PAL-silenced plants are fourfold lower than those in control plants. As a result, both TMV-induced PR gene expression and SAR are compromised in the PAL-silenced tobacco plants (Elkind et al. 1990; Pallas et al. 1996). Also, the PAL inhibitor, 2-aminoindan-2-phosphonic acid (AIP), reduces pathogen- or pathogen elicitor-induced SA formation in potato, cucumber, and Arabidopsis (Meuwly et al. 1995; Mauch-Mani and Slusarenko 1996; Coquoz et al. 1998). Further, simultaneous knockout of all four PAL genes (PAL1-PAL4) in the Arabidopsis genome leads to a production of about 25% and 50% of wild-type levels of basal and pathogen-induced SA, respectively (Huang et al. 2010). Therefore, both the cinnamic acid and isochorismate pathway appear to participate in basal and pathogen-induced SA production. However, the different roles of these two SA biosynthetic sources and how they are regulated in different physiological processes remain unclear.

To prevent toxicity caused by high concentrations of SA (Manthe et al. 1992), plants have evolved systems of converting infused SA to its derivatives such as SA O-β-glucoside (SAG), salicyloyl glucose ester (SGE), methyl salicylate (MeSA), methyl salicylate O-β-glucoside (MeSAG), and amino acid SA conjugates (Pridham 1965; Pierpoint 1994; Vlot et al. 2009). Significant progress has been made to uncover genes responsible for different conjugation enzymatic step(s). Among the above conjugate forms, MeSA is volatile. In addition to its role in airborne signaling for both intra- and inter-plant communication (Lee et al. 1995; Shulaev et al. 1997), MeSA was proposed to be a critical mobile signal for SAR (Park SW et al. 2007). A gene (BSMT1), which encodes a protein with both benzoic acid and SA carboxyl methyltransferase activity, was identified in Arabidopsis (Chen et al. 2003). MeSA can be hydrolyzed by esterases to release SA. Recently, the tobacco SA-binding protein 2 (SABP2) was shown to possess MeSA esterase activity (Park SW et al. 2007). Most importantly, MeSA is likely the long-sought, phloem-mobile signal that activates SAR in uninfected tissue following its translocation from the infection and synthesis site. The high level of SA produced in infected sites inhibits SABP2's MeSA esterase activity by binding to its active site, thereby facilitating buildup of MeSA for translocation to the systemic tissue (Park SW et al. 2007). Further study in Arabidopsis and potato suggest that the role of MeSA and its esterases in SAR is conserved. However, it is possible that some other molecules could also serve as long-distance signals for SAR (Truman et al. 2007; Jung et al. 2009; Vlot et al. 2009). The presence of a plastid transit peptide and cleavage site in ICS1 indicates that SA may be synthesized in plastids (Wildermuth et al. 2001). Further, EDS5/SID1, which is also involved in SA synthesis in pathogen-infected Arabidopsis, is predicted to localize to the chloroplast (Ishihara et al. 2008). EDS5 is a protein with homology to the animal multidrug and toxin extrusion (MATE) transporter family involved in transportation of organic molecules (including phenolic compounds) across membranes (Nawrath et al. 2002). Therefore, chloroplast is likely the biosynthesis site of SA. SA transported from chloroplasts can be converted into less toxic conjugation forms in cytosol, and the conjugated forms (such as SAG) are actively transported from the cytosol into the vacuole, where they may function as an inactive storage form that can be converted back to SA (Hennig et al. 1993; Dean and Mills 2004; Dean et al. 2005). Therefore, both long-distance SAR signaling and subcellular detoxication/storage rely on conjugated forms of SA.

SA Signaling

  1. Top of page
  2. Abstract
  3. Introduction
  4. SA Metabolism
  5. SA Signaling
  6. SA and Other Hormones
  7. Techniques for SA Quantification
  8. Future Perspectives
  9. Acknowledgements
  10. References

During the process of plant immune response, there is a complex genetic regulatory network that affects SA-mediated signaling. Here, we discuss the current understanding of the interaction among genes that regulate the signaling by grouping into regulation of SA accumulation, NPR1-dependent pathways, and NPR1-independent pathways.

Regulation of SA accumulation

Through mutant screening, a number of regulators affecting SA accumulation have been identified. Depending on their role in regulating SA accumulation, these regulators can be divided into two categories. Loss-of-function mutations in positive regulators lead to reduced SA accumulation and enhanced disease susceptibility, while in negative regulators, loss-of-function mutations are associated with increased SA accumulation and disease resistance.

The lipase-like protein EDS1 represents an important node acting upstream of SA in PTI against viral, bacterial, and fungal pathogens as well as in ETI initiated by a subset of R genes (Parker et al. 1996; Aarts et al. 1998; Falk et al. 1999; Chandra-Shekara et al. 2004; Xiao et al. 2005). EDS1 physically interacts with two other positive regulators, PAD4 and SAG101, both of which are putative lipases (Feys et al. 2001, 2005). Exogenous application of SA can rescue defense gene induction in eds1 and pad4 mutants and induce expression of EDS1 and PAD4 in wild-type plants, suggesting that SA positively regulates both of them through a feedback loop (Zhou et al. 1998; Falk et al. 1999; Feys et al. 2001). EDS1 can form different protein complexes with PAD4 or SAG101 at different subcellular locations including cytosolic EDS1 homodimers, nucleo-cytoplasmic EDS1-PAD4 heterodimers, and nuclear EDS1-SAG101 heterodimers (Feys et al. 2005). EDS1 is needed for PAD4 and SAG101 accumulation, indicating that EDS1 functions upstream of PAD4 and SAG101. Accumulating evidence indicates that the three regulators work cooperatively but PAD4 and SAG101 act in a separate pathway to transduce EDS1 signaling (Feys et al. 2005). NDR1 is another positive SA regulator acting independently of EDS1 since the two proteins are located downstream of two functionally distinct classes of R proteins (Century et al. 1997; Aarts et al. 1998). Similar to eds1, SAR-deficient phenotype of the ndr1 mutant can be rescued by BTH application (Shapiro and Zhang 2001). Additionally, ndr1 exhibits suppressed PTI and ETI, whereas overexpression of NDR1 significantly reduces the growth of virulent bacterial pathogens (Shapiro and Zhang 2001; Coppinger et al. 2004). Continuous efforts on mutant screening have identified a growing number of SA positive regulators such as ALD1 (Song et al. 2004), EDS5/SID1 (Nawrath et al. 2002), PBS3/WIN3/GDG1 (Nobuta et al. 2007; Lee et al. 2007; Jagadeeswaran et al. 2007), EPS1 (Zheng et al. 2009), and MOS (Monaghan et al. 2010). Furthermore, SA synthesis may also be influenced by a signal amplification loop involving reactive oxygen species (ROS) (Torres et al. 2006). Combining genomics tools (such as microarray and RNA-Seq technologies) with traditional epistasis analysis will facilitate revealing the interaction of these positive SA regulating components.

Salicylic acid synthesis is also under negative feedback regulation. However, compared to positive SA regulators, negative SA regulators are more difficult to study because their mutations are often associated with adverse growth phenotypes such as cell death and/or dwarfism. Plants carrying such mutations are often called lesion mimic mutants (LMM) (Lorrain et al. 2003; Moeder and Yoshioka 2008). In order to gain insights into the function of the corresponding genes, an indirect strategy has been utilized through crossing LMMs to mutants defective in genes that positively regulate SA signaling. Analysis of the double or triple mutants not only reveals the association of SA and phenotypes conferred by different LMM mutations, but also provides information on the interactions of SA regulators. This statement is supported by analysis of multiple LMMs such as lsd1, edr1, and vad1 (Rustérucci et al. 2001; Lorrain et al. 2004; Tang et al. 2005). In addition, a gain-of-function LMM, acd6-1, has become a powerful tool for assessing the functional relationship among SA regulators because of the inverse correlation between the dwarfism and SA-mediated defense (Lu et al. 2003). Using this convenient phenotyping approach, the different roles of ALD1 and PAD4 in SA-mediated defense have been confirmed (Song et al. 2004).

A combination of positive and negative regulatory mechanisms ensures tight regulation of SA synthesis and fine-tuning of plant defense response. Moreover, such feedback regulation also allows for immune responses to be dampened once the threat of infection has subsided.

NPR1-dependent SA signaling

NPR1, also known as NIM1 or SAI1, is a master regulator controlling multiple immune responses including SAR. It represents a key node in signaling downstream from SA (Dong 2004; Durrant and Dong 2004; Pieterse and Van Loon 2004). The npr1 mutant was first identified in screening of Arabidopsis mutants that were unable to activate the expression of PR genes or disease resistance (Cao et al. 1994; Delaney et al. 1995; Shah et al. 1997). Like NF-κB/IκB in the mammalian immune system, NPR1 contains an ankyrin-repeat motif and a BTB/POZ domain (Cao et al. 1997). The promoter region of the NPR1 gene contains W-box sequences, which are binding sites of WRKY family protein. Mutation in the W-box sequences of the NPR1 gene promoter adversely affects its expression, suggesting that WRKY transcription factor plays an important role in mediating signaling between SA and NPR1 (Yu et al. 2001). In the absence of SA or pathogen challenge, NPR1 is retained in the cytoplasm as an oligomer through redox sensitive intermolecular disulfide bonds. Upon induction, NPR1 monomer is released to enter the nucleus where it activates defense gene transcription (Mou et al. 2003). Therefore, SA affects NPR1 activity at two stages: first, it activates NPR1 expression, and second, it stimulates the translocation of NPR1 into the nucleus. SA-induced changes in the cellular redox state lead to reduction of two cysteine residues (Cys82 and Cys216) by TRX-H5 and/or TRX-H3 (Mou et al. 2003; Tada et al. 2008). Mutation of either Cys82 or Cys216 elevates the levels of monomeric, nuclear localized NPR1, and consequently upregulates PR-1 gene expression (Mou et al. 2003). SA application and pathogen-inoculation enhance NPR1 expression. Overexpression of Arabidopsis NPR1 or its homologs confers broad resistance against diverse pathogens in multiple plant species (Cao et al. 1998; Chern et al. 2001, 2005; Lin et al. 2004; Makandar et al. 2006; Malnoy et al. 2007; Potlakayala et al. 2007; Zhang et al. 2010; Parkhi et al. 2010). Moreover, overexpression of monomeric NPR1 by disturbing the formation of disulfide bond holds the potential to generate transgenic plants with further elevated disease resistance. However, considering the role of cytosolic NPR1 oligomer in mediating crosstalk between SA and other signal molecules, developing disease-resistant crops through overexpressing monomeric NPR1 requires further investigation.

NPR1 itself does not have DNA binding capability. Relaying NPR1-mediated signaling requires interaction with other proteins. Genome-wide expression profiling analysis showed that members of the WRKY transcription factor family act downstream of NPR1 (Wang et al. 2006). Protein-protein interaction assays have revealed that NPR1 interacts with seven members of the TGA family transcription factors and three structurally related NIMIN proteins (Després et al. 2000; Weigel et al. 2001, 2005). The TGA factors can directly interact with PR-1 gene through binding to the activation sequence-1 (as-1) in its promoter region (Lebel et al. 1998). In planta analysis showed that the interaction between NPR1 and TGA1 or TGA4 needs the presence of SA. However, interaction between NPR1 and TGA2 can be detected in the absence of SA but the interaction is enhanced by SA application (Fan and Dong 2002). Further, the ability of TGA2 and TGA3 to activate downstream transcription requires both SA and NPR1, suggesting that NPR1 may enhance the DNA binding activity of some TGA proteins and thus affect expression of PR-1 gene (Durrant and Dong 2004; Rochon et al. 2006). Mutational analysis of TGA genes indicated their redundancy in SA signaling (Zhang et al. 2003). Although NIMIN1, NIMIN2, and NIMIN3 are transiently induced after SA treatment, NIMIN1 appears to negatively regulate SA/NPR1 signaling (Weigel et al. 2001, 2005). Overexpression of NIMIN1 results in compromised ETI and SAR, whereas reduced expression of the same gene enhances induction of PR-1 expression by SA.

NRR1-independent SA signaling

Accumulating evidence has indicated that certain aspects of defense are controlled by SA-dependent, NPR1-independent signaling pathway(s) (Clarke et al. 1998, 2000; Kachroo et al. 2000; Shah et al. 2001; Murray et al. 2002). ETI was suppressed by expression of the NahG gene that encodes salicylate hydroxylase, but not in the npr1 mutant, suggesting the involvement of an NPR1-independent SA signaling mechanism in plant defense (Rardian and Delaney 2002; Kachroo et al. 2001; Takahashi et al. 2002). The existence of an NPR1-independent mechanism is further supported by studies of various Arabidopsis mutants, which constitutively accumulate SA and the transcripts of PR genes even in the absence of a functional NPR1 gene. A putative negative regulator of SAR, SNI1, was identified through screening for suppressors of the npr1-1 mutant (Li et al. 1999). In the npr1-1 background, the recessive sni1 mutation restores PR gene induction by SA, and disease resistance, whereas in the NPR1 background, renders the plant more sensitive to SAR signals. The SNI1 protein appears to be a nuclear protein with limited similarity to the mouse retinoblastoma protein, a negative transcription regulator. More components in the NPR1-independent defense pathway were identified through screening ethyl methylsulfonate (EMS) mutagenized npr1-5 mutant for constitutive PR gene expression. The ssi mutants, ssi1, ssi2, and ssi4, show constitutive accumulation of SA and exhibit heightened resistance to a variety of pathogens (Shah et al. 1999, 2001; Shirano et al. 2002). Evidence of an NPR1-independent signaling pathway was illustrated by studies of the ssi1 npr1 and ssi2 npr1 double mutant plants (Shah et al. 1999, 2001). The ssi1 and ssi2 mutant plants containing the wild-type NPR1 allele accumulate greater levels of PR1 gene transcripts than ssi1 npr1 and ssi2 npr1 double mutant plants, respectively, indicating an NPR1-dependent pathway functioning additively with the NPR1-independent pathway (Shah et al. 1999, 2001). Another npr1 suppressor mutant, snc1, displays constitutive SA-dependent, NPR1-independent resistance owing to a mutation in a TIR-NB-LRR type of R protein. The gain-of-function mutation snc1 leads to constitutive activation of the R protein and downstream defense responses without the presence of pathogens. The snc1 mutant plants accumulate high levels of SA, constitutively express PR genes, and display enhanced resistance to pathogens (Li et al. 2001). More snc mutants such as snc2-1D (Zhang et al. 2010) and snc4-1D (Bi et al. 2010) have been identified and characterized. Moreover, a set of genes that may be involved in SA regulated, NPR1-independent defense pathway are transcription factors such as WHIRLY (WHY) and MYB. The single stranded DNA binding activity of WHY is stimulated by SA treatment in both wild-type and npr1 mutant plants (Desveaux et al. 2002, 2004), suggesting their important roles in NPR1-independent expression of PR-1 and resistance against pathogen. AtMYB30 positively regulates the pathogen-induced HR in an SA-dependent, NPR1-independent manner (Raffaele et al. 2006). Additionally, the constitutive defense mutants, cpr5, cpr6, and hrl1, all show NPR1-independent, SA-dependent characters (Clarke et al. 2000; Devadas et al. 2002). However, evidence has shown that cpr5 and cpr6 activate ethylene (ET) and jasmonic acid (JA) mediated defense signaling (Clarke et al. 2000). Similarly, ET is also required for the resistance conferred by hrl1 (Devadas et al. 2002).

In genetic screening for suppressors of the npr1 mutant based on its intolerance to SA, our laboratory recently isolated the elp2 mutant. The elp2 mutation restores SA tolerance to npr1 and suppresses npr1-mediated hyperaccumulation of SA (DeFraia et al. 2010). Pathogen infection experiments suggest that ELP2 regulates an NPR1-independent defense pathway and does not affect SAR. The immune deficiency in elp2 mutant may be attributable to the delayed induction of SA biosynthesis and defense gene expression (DeFraia et al. 2010). ELP2 is a subunit of the Elongator complex, which is composed of three core subunits (ELP1-3) and three accessory subunits (ELP4-6). The Elongator complex interacts with elongating RNA polymerase II and facilitates transcription through histone acetylation (Winkler et al. 2002; Close et al. 2006). Recent genetic studies have demonstrated that Elongator functions in Arabidopsis development and in response to abiotic stresses (Nelissen et al. 2005, 2010; Chen et al. 2006; Zhou et al. 2009). Ongoing studies in the lab are focusing on the role of different Elongator subunits and their interactions in different aspects of plant immune responses.

SA and Other Hormones

  1. Top of page
  2. Abstract
  3. Introduction
  4. SA Metabolism
  5. SA Signaling
  6. SA and Other Hormones
  7. Techniques for SA Quantification
  8. Future Perspectives
  9. Acknowledgements
  10. References

Plants are subject to attack by a wide variety of microbial pathogens with a diverse array of effector molecules to colonize their host. Plants have in turn evolved sophisticated innate immune systems by which they recognize non-self molecules or signals from injured cells, and respond by activating effective immune responses through SA signaling cascade and interactions with other phytohormones such as JA, ET, abscisic acid (ABA), auxin, gibberellic acid (GA), cytokinin (CK), brassinosteroid (BR), and peptide hormones (Bari and Jones 2009; Pieterse et al. 2009).

SA and JA/ET

Salicylic acid-mediated resistance is generally effective against biotrophs, whereas JA/ET-mediated responses are predominantly against necrotrophs and herbivorous insects (Glazebrook 2005). Although most reports indicate a mutually antagonistic interaction between SA- and JA/ET-dependent signaling, synergistic interactions have been described as well (Schenk et al. 2000; Van Wees et al. 2000; Mur et al. 2006). During the antagonistic interactions between SA and JA/ET, common components shared in different signaling pathways serve as signaling nodes, which play important regulatory roles in the crosstalk. Mutation or ectopic expression of the corresponding regulatory genes was shown to have contrasting effects on SA and JA/ET signaling and on resistance against biotrophs and necrotrophs (Bari and Jones 2009; Pieterse et al. 2009). Among these regulatory components are EDS1, EDS4, PAD4, NPR1, SSI2, WRKY transcription factors, MPK4, and GRX480 (glutaredoxin, a disulfide reductase) (Petersen et al. 2000; Kachroo et al. 2001; Li et al. 2004; Brodersen et al. 2006; Ndamukong et al. 2007). EDS1, EDS4, and PAD4 act upstream of NPR1 affecting SA accumulation. Mutants of genes encoding these components exhibit enhanced response to inducers of JA-dependent gene expression, suggesting the negative interaction between SA and JA (Zhou et al. 1998; Falk et al. 1999; Gupta et al. 2000). In addition, NPR1 regulates the SA-mediated expression of WRKY53, WRKY62, WRKY70, and GRX480, which encode proteins that suppress JA-dependent gene expression (Li et al. 2004; Li et al. 2007; Ndamukong et al. 2007). Besides functioning as a key transcriptional co-activator of SA-responsive genes, NPR1 is also a crucial regulator in SA-mediated suppression of JA signaling. Arabidopsis npr1 plants are compromised in the SA-mediated suppression of JA responsive gene expression, indicating that NPR1 plays an important role in SA-JA interaction (Spoel et al. 2003). Nuclear localization of SA-activated NPR1 is not required for the suppression of JA-responsive genes, indicating that the antagonistic effect of SA on JA signaling is modulated through a function of NPR1 in the cytosol (Spoel et al. 2003; Yuan et al. 2007).

Characterization of mutants in JA/ET signaling has identified regulatory components that function in antagonizing SA signaling. A loss-of-function mutation in the Arabidopsis MPK4 gene, which encodes a mitogen-activated kinase, impaired JA signaling and simultaneously conferred enhanced resistance against bacterial and oomycete pathogens due to constitutive activation of SA signaling (Petersen et al. 2000). Similar to mpk4, ssi2 plants exhibit impaired JA signaling, constitutive expression of SA-mediated defenses, and enhanced disease resistance (Kachroo et al. 2001; Kachroo et al. 2003). The SSI2 gene encodes a steroyl-ACP fatty-acid desaturase, which is hypothesized to catalyze the synthesis of a fatty-acid-derived signal involved in mediating both JA signaling and negative crosstalk between JA and SA pathways. JA-dependent gene expression is also impaired in double mutant mpk4 nahG and ssi2 nahG plants, which do not accumulate high levels of SA, suggesting that the impairment of JA signaling in single mutants is not due to an inhibitory effect of elevated SA levels (Kunkel and Brooks 2002). Unlike mpk4 and ssi2, coi1 mutant plants do not exhibit constitutive expression of SA-dependent defenses. Rather, the SA-mediated defenses are hyperactivated only in response to attack by Pseudomonas syringae. COI1 encodes an F-box protein that is predicted to regulate JA-signaling by inactivating negative regulators of the JA-mediated pathway (Feys et al. 1994; Xie et al. 1998; Kloek et al. 2001). The same negative regulatory interaction can also be found between ET and SA signaling pathways. Genetic characterization of ein2, an ET-insensitive mutant, showed that the basal level of SA-responsive marker gene PR-1 expression is significantly higher than that in wild-type plants. This indicates that the modulation of the SA pathway by ET is EIN2 dependent and functions through the ET signaling pathway (De Vos et al. 2006). Characterization of the Arabidopsis elp mutants revealed that the Elongator complex promotes the initiation of SA signaling but represses JA/ET signaling (DeFraia et al. 2010; Nelissen et al. 2010). The Elongator may therefore be an important player in the crosstalk between these pathways (DeFraia et al. 2011).

SA and auxin

Auxin is an important hormone that affects almost all aspects of plant growth and development. A growing body of evidence indicates that many plant pathogens can either produce auxin themselves or manipulate host auxin biosynthesis to interfere with the host's normal developmental process (Chen et al. 2007; Robert-Seilaniantz et al. 2007). Conversely, plants have evolved mechanisms to repress auxin signaling during pathogenesis. Plants overproducing the defense signal molecule SA frequently have morphological phenotypes that are reminiscent of auxin-deficient or auxin-insensitive mutants, suggesting that SA might interfere with auxin response (Wang et al. 2007). SA application causes global repression of auxin-related genes, resulting in stabilization of the Aux/IAA repressor proteins and inhibition of auxin responses (Wang et al. 2007). Similarly, it was found that the majority of the auxin inducible genes are also repressed in systemic tissues after induction of SAR, indicating that SAR response involves downregulation of auxin responsive genes (Wang et al. 2007). In contrast, exogenous application of auxin has been shown to promote disease (Yamada 1993, Navarro et al. 2006; Chen et al. 2007) and blocking auxin responses leads to increased resistance (Wang et al. 2007). The finding that enzymes involved in auxin amino acid conjugation, and thus inactivation, affect SA-mediated defenses indicates another possible level of crosstalk between SA and auxin (Park JE et al. 2007). GH3.5 conjugates both SA and indole acetic acid, and altered expression of this enzyme affects disease resistance (Zhang et al. 2007). The loss-of-function mutant of the auxin response factors (ARFs), arf6 arf8, displays reduced expression of genes involved in JA biosynthesis and low JA levels, suggesting that activation of JA signaling may play an important role during the interaction of SA and auxin (Tiryaki and Staswick 2002; Nagpal et al. 2005). Therefore, SA and auxin signaling is mutually antagonistic. However, the detailed mechanism of their interaction, especially the regulatory components of the two signaling pathways, merits further investigation.

SA and ABA

Abscisic acid plays a crucial role in adaptation to abiotic stress. However, its role in biotic stress responses is less understood. Generally, ABA is considered as a negative regulator of disease resistance (Bari and Jones 2009; Ton et al. 2009). Application of exogenous ABA prevents SA accumulation and suppresses resistance to P. syringae in Arabidopsis (Mohr and Cahill 2003). Similar results have been found in other plant species (Mohr and Cahill 2001; Koga et al. 2004). Recently, Yasuda et al. (2008) reported that ABA treatment suppresses SAR induction, indicating an antagonistic interaction between SA and ABA signaling. Likewise, mutants impaired in ABA biosynthesis or sensitivity are more resistant to different pathogens compared to wild-type plants in both Arabidopsis (Mohr and Cahill 2003; Anderson et al. 2004; Adie et al. 2007; de Torres-Zabala et al. 2007) and tomato (Audenaert et al. 2002; Thaler and Bostock 2004; Achuo et al. 2006; Asselbergh et al. 2008). Furthermore, increased ABA production and activation of ABA-responsive genes have been described during the interaction of plants with invading pathogens (Whenham et al. 1986; de Torres-Zabala et al. 2007). Therefore, ABA is a negative regulator of plant defense signaling pathways mainly mediated by SA. It has been shown that ABA regulates defense response through its effects on callose deposition (Hernandez-Blanco et al. 2007; Flors et al. 2008), production of reactive oxygen intermediates (Xing et al. 2008), and regulation of defense gene expression (Adie et al. 2007; de Torres-Zabala et al. 2007). It also could be possible that ABA-SA antagonism results from the indirect effect of ABA-JA/ET interactions (Anderson et al. 2004; Adie et al. 2007). However, the exact molecular mechanism of ABA action on plant defense responses to diverse pathogens remains unclear. Detecting regulatory factors involved in the crosstalk of ABA with other phytohormones in plant defense warrants extensive future study.

SA and GA

Gibberellic acid is a well-studied growth promoting phytohormone, yet limited attention has been received in the elucidation of its role in defense response. Infection with rice dwarf virus (RDV) represses expression of ent-kaurene oxidase, a GA biosynthetic enzyme, and results in significant reduction of GA levels and a dwarf phenotype similar to GA-deficient symptoms (Zhu et al. 2005). However, the normal growth phenotype can be restored by exogenous GA application, indicating that RDV modulates GA metabolism to promote disease symptoms in rice. Further, modulation of bioactive GA levels through GA deactivating enzymes, elongated uppermost internode (EUI), has been shown to affect disease resistance in rice. The loss-of-function eui mutants accumulate high levels of GA and show compromised resistance, whereas EUI overexpressors accumulate low levels of GA and show increased resistance (Yang et al. 2008). Together with the studies on exogenous application of GA biosynthesis inhibitor or GA analogs, it is clear that GA plays a negative role in basal disease resistance in rice. Mutants defective in GA perception also show altered immune response. The gid1 mutant of rice, defective in GA reception, accumulates higher level of GA and shows enhanced resistance to the blast fungus (Tanaka et al. 2006). Recent studies on Arabidopsis DELLA proteins revealed its role in mediating GA-, SA-, JA/ET-mediated defense signaling pathways in plant immune response. The Arabidopsis quadruple della mutant that lacks four functional redundant DELLA genes (gai-6, rga-t2, rgl1-1, rgl2-1) is susceptible to fungal necrotrophic pathogens but is more resistant to biotrophic pathogens (Navarro et al. 2008). Gene expression analysis of infected quadruple della mutants showed that SA marker genes are induced earlier and stronger but JA/ET marker genes are significantly delayed. This suggests that DELLAs promote resistance to necrotrophs and susceptibility to biotrophs, partly by modulating the balance between SA-mediated and JA/ET-mediated defense signaling pathways (Navarro et al. 2008). Since GA stimulates degradation of DELLA proteins, it could be deduced that GA functions in promoting resistance to biotrophs and susceptibility to necrotrophs. DELLA proteins are also found to promote the expression of genes encoding ROS detoxificaiton enzymes, thereby regulating the levels of ROS after biotic or abiotic stresses (Achard et al. 2008). Considering the possible feedback amplification loop between SA and ROS, the role of DELLAs as a regulatory component during SA-GA interaction in plant defense signaling seems likely. However, the detailed mechanism of GA action on different plant defense response signaling remains to be uncovered.

In addition to JA/ET, auxin, ABA, and GA, other hormones such as CK, BR, and peptide hormones are also involved in plant defense pathways (Bari and Jones 2009; Grant and Jones 2009; Vlot et al. 2009). However, their role in plant defense, especially the interaction with SA is less well studied. Depending on the type of plant-pathogen interactions, different hormones play positive or negative roles against various biotrophic and necrotrophic pathogens. Plant hormone signaling pathways are not isolated but rather interconnected with a complex regulatory network. How plants coordinate multiple hormonal components in response to different pathogen infections, how the integrated signal is generated, regulated, and transduced to result in HR cell death, defense gene expression, and/or SAR, and what role SA plays in this sophisticated process are questions that remain to be addressed.

Techniques for SA Quantification

  1. Top of page
  2. Abstract
  3. Introduction
  4. SA Metabolism
  5. SA Signaling
  6. SA and Other Hormones
  7. Techniques for SA Quantification
  8. Future Perspectives
  9. Acknowledgements
  10. References

Salicylic acid is a major signal molecule involved in plant immunity (Raskin 1992a; Durrant and Dong 2004). Unveiling the SA-mediated signaling pathway, especially understanding the mechanism underlying SA accumulation, frequently requires the quantification of a large number of samples with different genetic backgrounds or treatments. High performance liquid chromatography (HPLC) and gas chromatography/mass spectroscopy (GC/MS) are two commonly used methods for SA quantification (Malamy and Klessig 1992; Verberne et al. 2002; Schmelz et al. 2003; Aboul-Soud et al. 2004). However, both methods are costly and time-consuming. Identifying mutants deficient in SA accumulation from mutated populations has been proved as an effective way to study the mechanisms of SA biosynthesis or signaling (Nawrath and Métraux 1999; Wildermuth et al. 2001). So far, the most extensive mutant screening study quantified around 4 500 individual M2Arabidopsis plants by HPLC-based methods (Nawrath and Métraux 1999). However, in order to capture more valuable genetic components involved in SA biosynthesis or signaling, the population for mutant screening needs to be increased. Therefore, there is a great need to develop a high-throughput method with reduced cost and time requirements for SA quantification.

Huang et al. (2006) developed a biosensor, Acinetobacter sp. ADPWH_lux, for SA quantification. This strain is derived from Acinetobacter sp. ADP1, and contains a chromosomal integration of a salicylate-inducible lux-CDABE operon, which provides both substrate and enzyme needed for SA-responsive luminescence. It can proportionally produce bioluminescence in response to salicylates including SA, methyl-SA, and the synthetic SA derivative ASA. The well-correlated results between the biosensor and GC/MS method on TMV-infected tobacco leaves suggest that the biosensor is suitable for SA quantification in an easy, cheap, and fast way. In plants, SA can be converted to SAG (2-O-β-D-glucosylsalicylic acid), methyl-salicylate, and a glycosylated form of methyl-salicylate, which also play important roles in plant immunity (Pridham 1965; Pierpoint 1994; Vlot et al. 2009). DeFraia et al. (2008) developed a method for biosensor-based SAG measurement by treating crude extracts with β-glucosidase as well as optimized extraction and quantification steps, which further reduce cost and time. On the basis of this improved biosensor-based SA quantification method, Marek et al. (2010) simplified tissue collection and SA extraction procedures, and further adapted the protocol to a high-throughput format. The efficacy and effectiveness of the most updated biosensor-based SA quantification method was confirmed by HPLC and verified in Arabidopsis npr1 suppressor screening. Using this high-throughput method, our lab is conducting a comprehensive Arabidopsis npr1 suppressor screen with the objective to isolate novel SA metabolic mutants. Several mutants with lower or higher SA levels than npr1 have been identified. Allelism tests with the known mutants are underway and some possible novel mutants are in the process of map-based cloning. Preliminary data indicate the great potential of adding new genetic components to SA biosynthetic or regulation pathways. In addition to isolating mutants with aberrant SA accumulation, this high-throughput SA measurement approach can also be applied to studies on characterization of enzymes involved in SA metabolism and analysis of temporal changes in SA levels.

Future Perspectives

  1. Top of page
  2. Abstract
  3. Introduction
  4. SA Metabolism
  5. SA Signaling
  6. SA and Other Hormones
  7. Techniques for SA Quantification
  8. Future Perspectives
  9. Acknowledgements
  10. References

Our understanding of the role of SA in plant immunity has increased over the past two decades. Significant progress has been made in elucidating SA biosynthetic pathways and new components involved in SA signaling have been identified. Recent advances have provided exciting new insights into the understanding of SA-mediated defense crosstalk with other plant hormones. Furthermore, a rapid biosensor-based method for SA quantification and a high-throughput procedure suitable for SA metabolic mutant screening have been established, which hold great potential for isolating additional SA accumulation mutants. However, SA-mediated defense signaling pathways are not isolated but rather interconnected to form a complex and well-regulated network. Elucidating genetic components involved in the pathways will continue to be a major task of the community. Nonetheless, with the expanding discovery of additional SA signaling pathway components, there is a clear need to understand how plants integrate genetic information with various developmental and environmental factors into tight control over energetically costly immune responses. A systems approach, which integrates genetics, molecular biology, and biochemistry results into genome-wide kinetic gene expression and signaling component profiling datasets, together with computational biology and bioinformatics techniques, will accelerate the process towards fully understanding SA-mediated plant immunity.

(Co-Editor: Yunde Zhao)

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. SA Metabolism
  5. SA Signaling
  6. SA and Other Hormones
  7. Techniques for SA Quantification
  8. Future Perspectives
  9. Acknowledgements
  10. References

This work was supported by a grant from the National Science Foundation (IOS-0842716) to Dr. Z Mou.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. SA Metabolism
  5. SA Signaling
  6. SA and Other Hormones
  7. Techniques for SA Quantification
  8. Future Perspectives
  9. Acknowledgements
  10. References