Stories of Salicylic Acid: A Plant Defense Hormone

TRPLSC 1921 No. of Pages 17

Trends in Plant Science

Feature Review

Stories of Salicylic Acid: A Plant Defense Hormone Pingtao Ding

1, ,@

*

and Yuli Ding2,@

Salicylic acid (SA) is a key plant hormone required for establishing resistance to many pathogens. SA biosynthesis involves two main metabolic pathways with multiple steps: the isochorismate and the phenylalanine ammonia-lyase pathways. Transcriptional regulations of SA biosynthesis are important for finetuning SA level in plants. We highlight here recent discoveries on SA biosynthesis and transcriptional regulations of SA biosynthesis. In addition, SA perception by NPR proteins is important to fulfil its function as a defense hormone. We highlight recent work to give a full picture of how NPR proteins support the role of SA in plant immunity. We also discuss challenges and potential opportunities for future research and application related to the functions of SA in plants.

Highlights

Salicylic Acid (SA): The ‘Sixth’ Phytohormone

Recent breakthroughs have revealed new mechanisms by which the endogenous SA level controls the transcriptional reprogramming via the perception of NPR proteins and their protein turnovers.

Salicylic acid (SA) serves as a key hormone in plant innate immunity, including resistance in both local and systemic tissue upon biotic attacks, hypersensitive responses, and cell death. Key components involved in the complete metabolic steps of SA biosynthesis through the isochorismate pathway and their detailed functions have been identified.

The metabolite SA is a beta hydroxy phenolic acid and widely produced in prokaryotes and plants. For a long time, SA has been known rather for its applications as medicine than for its function in plants (Box 1). One of the reasons was that in plants, SA was found as chemical messengers in regulating biological processes at relatively low concentration. As a result, SA was introduced as the ‘sixth’ principal plant hormone (phytohormone) only in the early 1990s [1]. In this review, we revisit landmark discoveries and highlight some important and recent advances about SA in plants.

Both positive and negative transcriptional regulations of SA biosynthesis are required for fine-tuning the levels of SA for optimal defense without causing unnecessary fitness cost.

SA and Plant Immunity SA is best known as a defense-related hormone [2–7]. The first observations that SA was involved in plant immunity were reported by Raymond F. White in 1979, who described that the application of aspirin (acetyl-SA) in virus-susceptible tobacco (Nicotiana tabacum cv. Xanthi-nc) conferred resistance against tobacco mosaic virus (TMV) [8]. This indicated a protective role of SA in plant resistance. In a tobacco (N. tabacum) cultivar that carries the viral resistance gene, endogenous SA increased upon viral infection and pathogenesis-related (PR) proteins accumulated [9]. Similarly, SA was shown with an increase in the phloem sap of cucumber before the induced resistance was detected in the systemic tissue [10]. Both studies indicate endogenous SA might play a role as an internal defense signal for plant immunity. Early characterizations of plant immune responses involved pathogen-induced hypersensitive response (HR), which can reduce penetration and spread of pathogens via localized plant cell death at the site of infection [11]. In arabidopsis (Arabidopsis thaliana), the HR-like lesions (hrl) mutant hrl1, which accumulates higher level of endogenous SA, shows reduced HRassociated ion leakage [12]. Furthermore, SA-deficient mutants of arabidopsis display enhanced immune-associated ion leakage [13]. Altogether, these observations indicate that SA and/or its related metabolites play critical roles in regulating HR and cell death. Another important aspect of plant innate immunity involves the concept of systemic acquired resistance (SAR). Acquired resistance induced by pathogens or symbiotic microbes was well summaTrends in Plant Science, Month 2020, Vol. xx, No. xx

1

The Sainsbury Laboratory, University of East Anglia, Norwich Research Park, Norwich NR4 7UH, UK 2 John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK

*Correspondence: [email protected] (P. Ding). @ Twitter: @sardineboy1 (P. Ding) and @Ella0821 (Y. Ding).

https://doi.org/10.1016/j.tplants.2020.01.004 © 2020 Elsevier Ltd. All rights reserved.

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Box 1. Historical Use of Salicylates as Medicine As early as in the 4th century BC, the ancient Sumerians and Egyptians had begun applying bark and leaves from willow, myrtle, poplar, and meadowsweet plants to relieve pain caused by eye disease, rheumatism, childbirth, and fever. Later, famous physicians Hippocrates (440–377 BC), Pliny the Elder (AD 23–79), Dioscorides (c. AD 90), Celsus (c. AD 200), and Galen (130–210 AD) prescribed these herbal remedies to their patients [130]. In the Middle Ages (c. AD 500–1500), Hildegard of Bingen and Henrik Harpestreng were known to use the remedies produced from willow bark for fever and rheumatism [130]. In modern history, an English natural philosopher Edward Stone (1702–1768) carried out the first ‘clinical trial’ using powders isolated from willow bark and successfully cured most of patients with malarial fever [130]. However, it was not until the 19th century that the active component from these traditional herbal remedies was isolated and determined to be salicylic acid (SA). In 1828, the German pharmacologist Johann Andreas Buchner isolated salicin (an alcoholic β-glucoside with a salicyl group) from willow bark [131]. Soon in the mid-18th century, an Italian chemist Raffaele Piria managed to convert salicin into a sugar and a second component, which on oxidation became SA. In 1983, Raffaele named the substance ‘salicylic acid’ after the Latin word ‘salix’, which means ‘willow tree’. After direct application of isolated salicin and SA on patients, the antirheumatic effects of these compounds were further described by the Scottish doctor Thomas John MacLagan (salicin) and two German scientists, F. Stricker and L. Riess in 1876 (SA) [132]. In 1853, a German chemist, Felix Hoffmann, found acetyl chloride and sodium salicylate could be catalyzed into acetyl-SA, which was marketed by the company Bayer with the trade name Aspirin in 1879 [133]. ‘Aspirin’ was derived from a Latin word ‘spiraea’ for ‘meadowsweet’ [133]. Other than Aspirin, many other SA-related compounds, such as methyl-SA, saligenin (salicyl alcohol), and their glycosides are isolated from willow and other plant resources and used as analgesics, including the wintergreen oil extract from Gaultheria procumbens [134]. In addition to their applications as remedies, salicylates are also used to prevent and treat heart attacks, coronary problems, and cerebra thrombosis, and to retard production of prostaglandins that can promote blood clots [135].

rized and explored by Chester in 1933 [14]. In 1961, the term SAR was used for the first time by A. Frank Ross to describe an induced systemic resistance in TMV-infected tobacco. An initial infection on the plant at the ‘primary’ infection site was sufficient to restrict the growth of a broad spectrum of pathogens that were subsequently inoculated at a distal secondary infection site [15]. To date, a few molecules have been proposed as mobile signals leading to SAR [16,17]. SA was initially considered as a mobile signal for SAR because the concentration of SA increases in both the primary infected and systemic uninfected tissue [9]. In addition, plants expressing NahG, a salicylate hydroxylase encoding gene from Pseudomonas putida that depletes SA accumulation, are SAR deficient [18]. The detection of increased SA level in exudates from plant tissue with primary infection implicated that SA might serve as a mobile molecule capable of signaling to the distal tissue, if the detected SA increases in the exudates is produced at the primary infection site [19]. However, subsequent grafting experiments and other studies ultimately concluded that SA is not the generic mobile signal for SAR [20–22]. Cumulatively, these and other studies demonstrate that SA accumulation is required for the establishment of resistance in both local and systemic tissues [2–7]. Recently, SA has been shown to negatively contribute to insect resistance in plants, as insect eggs laid on plant leaves can induce SA accumulation and SA antagonizes jasmonic acid (JA)mediated resistance against chewing herbivores [23]. However, insect egg-induced SA can prime SAR against plant microbial pathogens [24]. SA has also been shown to contribute to shaping the microbial populations associated with the plant roots and lead to enhanced plant fitness in response to threats from pathogens [25]. Thus, the roles of SA as a defense hormone have been greatly expanded. SA is tightly associated with metabolism, homeostasis, and growth [26]. For instance, the resistance output in plants largely relies on interactions of SA with other hormones [27]. A companion review further explores the SA-dependent ‘trade-off’ between growth and immunity in plants [28]. 2

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The biological function of SA goes beyond immunity, but the precise roles of this hormone on other phenomena remain to be elucidated.

What Regulatory Roles Does SA Have? In recognition of pathogens, endogenous SA are induced in plants and exogenous application of SA in plants can trigger immune-like responses. Forward genetic screens for SA-induction insensitive mutants helped identify Nonexpresser of PR gene 1 (NPR1), the first reported gene required for SA response [29]. Later another two alleles of npr1 mutant were isolated and characterized as salicylic acid-insensitive mutant 1 (sai1) and noninducible immunity 1 (nim1) [30,31]. npr1 mutants are susceptible to bacterial pathogens [29–31]. Arabidopsis carries five paralogs of NPR1, which can be classified into three major groups: NPR1 and NPR2 as group I, NPR3 and NPR4 as group II, and Blade On Petiole 1 (BOP1) and BOP2 as group III [32]. All NPR1 paralogs in arabidopsis contains ankyrin repeats that are mostly common for protein–protein interactions and a Broad-Complex, Tramtrack, and Bric a brac (BTB) domain that is commonly seen in transcriptional regulators. NPR1/2/3/4 can strongly bind SA in vitro, while BOP1/2 have only weak interactions with SA [33,34]. Recently it has been shown that NPR1 and NPR2 play positive roles in regulating downstream genes in response to SA, while NPR3 and NPR4 seem to serve as negative regulators [33,35]. Two working models have been proposed to explain how NPR proteins respond to increased SA levels: (i) upon binding with SA, NPR3/4 work with the proteasome to degrade NPR1 so as to negatively regulate defense [36]; (ii) NPR1 and NPR3/4 work in antiparallel in response to SA, in which SA activates NPR1 positive regulation and deactivates NPR3/4 negative regulation by direct binding to the respective NPR proteins (Figure 1) [35]. Though the unique mutant allele of NPR4 reported in the second model has strongly suggested the later one is more likely to be real, it hasn’t fully ruled out the biochemical possibility of NPR3/4 degrading NPR1. More importantly, it is still not fully understood how NPRs are regulated in response to basal level of SA versus higher level of SA accumulation upon immune activation. Furthermore, it is not known if all NPR1 homologs would undergo similar biochemical processes to NPR1 in response to SA accumulation, such as monomerization, translocation from cytosol to nuclear, interaction with transcription factors (TFs), polyubiquitination, and rapid protein turnover (Figure 1). A more careful yeast-two hybrid analysis showed that NPR1 does not interact with NPR3, but weakly interacts with NPR4, which is disrupted in the presence of SA [33]. Meanwhile, NPR2 emerges as another potential SA receptor, especially in leaves undergoing senescence, fruits at later-forming stages, and dry seeds [33]. As an alternative player of NPR1, overexpressed NPR2 complements SA-insensitive response in npr1 mutant [33]. However, npr1/2/3/4 quadruple mutant is more susceptible to bacteria compared with npr1/3/4 or npr2/3/4 triple mutants with pretreatment of SA or its analog benzothiadiazole, further indicating NPR1 and NPR2 have overlapped functions [33]. Moreover, both polyubiquitination and deubiquitination of NPR1 are required for its signaling in response to SA [37,38], indicating proteasome-mediated turnover of NPR1 is crucial for its transcriptional coactivator activity (Figure 1). It was also reported that NPR1 promotes its own expression in response to SA [39]. Therefore, both transcriptional and post-translational regulations of NPR1 coexist. NPR proteins operate as hubs that mediate the reprogramming of large-scale gene expression induced by SA [7]. Through interactions with the machineries of other hormone regulatory pathways, NPRs proteins may be able to regulate immunity and other cellular processes in response to SA [28]. Trends in Plant Science, Month 2020, Vol. xx, No. xx

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Figure 1. NPR Protein-Dependent Gene Regulations by Salicylic Acid (SA). The top panel shows a plant cell contains a relative low concentration of SA (c[SA]) or basal level of SA accumulation when there is no encounter with pathogens. When c[SA] is low, NPR3 and NPR4 serve as negative regulators to suppress gene expressions via their direct interactions with plant-specific TGA transcription factors, while monomeric NPR1 protein is maintained in a steady and relative low level. The turnover of NPR1 protein is mediated by 26S proteasome complex trough sequential polyubiquitination processes by Cullin-RING ubiquitin E3 ligase such as CRL3 (or CUL3) and E4 ligase UBE4, and also deubiquitination process by ubiquitin-specific proteases UBP6 and UBP7, which are closely linked to 26S proteasome. The bottom panel demonstrates a plant cell with a higher c[SA] accumulated through immune activation triggered by plant pathogens. Higher SA level can induce monomerization process of NPR1 and induced NPR1-dependent gene expression through direct interactions with TGA transcription factors. Meanwhile, direct binding with SA derepress the suppression of NPR3 and NPR4 on SA induced genes, which further enhanced SA-induced NPR1-dependent gene expression. Efficient turnover of monomeric NPR1 proteins in the nucleus is required for a rate-limited SA-induced gene expression and this is also dependent on the homeostasis of NPR1-ubiquitination, which is maintained by proteins mentioned on the top panel.

How Is SA Produced in Plants? SA in plants can be generated via two distinct pathways, the isochorismate (IC) and the phenylalanine ammonia-lyase (PAL) pathways (Figure 2A). Both pathways require the primary metabolite chorismate, the end-product of the shikimate pathway, to produce SA (Figure 2A). The IC Pathway: Defining the ‘Canon’ Genetic analysis together with biochemistry approaches enabled the landmark discoveries of each individual steps of SA biosynthesis (Figure 2B). In late 1990s, the characterization of the biosynthesis pathway was initiated in arabidopsis through forward genetic screens. Three key genes ICS1, EDS5, and PBS3 were cloned and were later found to encode three enzymes that define the canonic IC pathway of SA biosynthesis. 4

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(A) Cytosol

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PDT? E4P Prephenate

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ortho-Coumaric acid

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eps1 [55] pbs3 [50] sid1, sid2 [40] eds16 [41,45]

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PBS3 (IGS) [48,54]

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EPS1 (IPGL) [54]

EDS5 (Transporter of IC) [47–49]

ICS [42,43] 2007

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Figure 2. Salicylic Acid (SA) Biosynthesis in Plants. (A) An up-to-date schematic diagram of SA biosynthesis in plants. There are two main pathways found in plants: ① the isochorismate (IC) pathway and ② the phenylalanine ammonia-lyase (PAL) pathway. Both pathways begin with chorismate as the substrate, which is produced via shikimate from phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) through a seven-step enzymatic reaction of shikimate pathway in plastid, such as chloroplast. ① IC pathway: upon stresses, SID2 or ICS1 and ICS2 function as isochorismate synthase to convert chorismate into IC in plastid. EDS5 function as a transporter to transfer IC from the plastid into cytosol. In prokaryotes, IC can be directly converted into SA and pyruvate by isochorismate lyase (IPL), for example, PmsB or PchB in bacteria (in purple). In plants, (Figure legend continued at the bottom of the next page.)

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An ICS1 mutant, sid2, was initially found in a SA induction deficient (sid) screen [40]. ICS1, and its close homolog ICS2, are homologs of PchA, an isochorismate synthase (ICS) controlling the first committed step of SA biosynthesis in Pseudomonas aeruginosa [41]. Both are required for SA biosynthesis, localize in chloroplasts, and convert chorismate to IC [42,43]. eds5 was isolated as a mutant that exhibits enhanced disease susceptibility (eds) to bacterial pathogens [44,45]. Another allelic mutation of EDS5 was found in the sid screen as sid1 [40]. In a similar eds screen to the biotrophic fungal pathogen Erysiphe orontii, ICS1 allelic mutant eds16 was found [41,45]. EDS5 was identified as a homolog of multidrug and toxin extrusion (MATE) transporter family protein with a series of 12 putative transmembrane domains and a coil-containing domain at the N terminus [46]. EDS5 localized on chloroplast envelope and has only recently been proven as a transporter of IC but not SA from plastids to cytosol [47–49]. In contrast, there is no requirement for an EDS5 homolog in P. aeruginosa, because all steps of SA biosynthesis in bacteria occur in one compartment. The pbs3 (AvrPphB susceptible 3) mutant was identified from a screen for mutants exhibiting enhanced susceptibility to an avirulent strain of Pseudomonas syringae pv. tomato DC3000 carrying the effector AvrPphB [50]. pbs3 also displays enhanced susceptibility to virulent P. syringae strains [50]. PBS3 is an acyl-adenylate/thioester-forming enzyme from a glycoside hydrolase 3 (GH3) protein family, hence also is named as GH3.12 [51]. Initially, PBS3 was proposed as an isochorismate lyase (IPL), similar to the bacterial PchB, to convert IC into SA and pyruvate directly. However, neither the primary sequence nor the structure of PBS3 show any homology to the bacterial IPL/PchB, indicating that PBS3 has a different function in SA biosynthesis in plants [52]. In vitro biochemistry and structural studies show that PBS3 and other homologous GH3 enzymes can conjugate a broad range of acyl substrates to amino acids (AAs) [52,53]. Biochemical studies demonstrate that PBS3 prefers 4-aminobenzoate and 4-hydroxybenzoate (4HBA), a para isomer of SA (2HBA) [53]. As for its ambiguous function, PBS3 was also proposed as an enzyme involved in SA metabolism rather than its biosynthesis [51]. PBS3 has a strong interaction affinity with SA and its catalytic function is inhibited by SA at an even very low concentration [52,53]. This indicates that PBS3 uses other substrates besides SA as acyl donors and is however, IC is firstly converted into isochorismoyl-9-glutamate (IC-9-Glu) by an isochorismoyl-glutamate synthase (IGS) PBS3/ GH3.12. IC-9-Glu can spontaneously break down into SA and 2-hydroxy-acryloyl-N-glutamate (2HNG) or N-pyruvoyl-Lglutamate (NPG). In arabidopsis, an IC-9-Glu pyruvoyl-glutamate lyase (IPGL) EPS1 can enhance this process more effectively. ② PAL pathway: as a key intermediate in the biosynthesis of tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr), chorismate are converted into these amino acids via different paths in plastid (in grey). Chorismate is converted into Trp through anthranilate synthase (AS) and followed by three-step reactions. High concentration of Trp negatively regulates the function of AS. Chorismate is converted into prephenate by chorismate mutase 1 (CM1) in the plastid, or transported into cytosol via unknown mechanisms and then converted into prephenate, possibly by CM2. Prephenate is converted by plant prephenate aminotransferases (PPA-ATs) into arogenate, then being converted into either Tyr by arogenate dehydrogenase (ADH) or Phe by arogenate dehydratase (ADT). The function of CM1, ADH, and ADT are negatively regulated by their corresponding amino acid products. Tyr and Phe are transported into cytosol. Apart from the Phe produced and transported from the plastid, Phe can also be produced via a cytosolic pathway from chorismate. After cytosolic chorismate being converted into prephenate, it is converted into phenylpyruvate, possibly by prephenate dehydratase (PDT). Phenylpyruvate is then converted into Phe by phenylpyruvate aminotransferase (PPY-AT) with the amine transferred from Tyr, and Tyr becomes 4-hydroxy-phenylpyruvate. In plants, tyrosine aminotransferase (TAT) converts 4-hydroxy-phenylpyruvate into Tyr, which is in an opposite reaction direction to that in bacteria. Phe produced from both plastidial and cytosolic pathways is converted into trans-cinnamic acid (t-CA) by PALs. t-CA can be converted into SA through ortho-coumaric acid. Alternatively, t-CA can be converted into benzaldehyde and then benzoic acid (BA), which is, in turn, hydroxylated to SA possibly by a benzoic acid 2-hydroxylase (BA2H). (B) Landmark discoveries of enzyme mutants and their functions in SA biosynthesis. In the horizontal timeline, here we only highlight the pioneering reports on exploring the IC pathway of SA biosynthesis in plants, especially in arabidopsis. There are a lot of other important discoveries along the timeline, but due to the space limit, we could not cover all of them. Enzyme mutants and its corresponding function reports are aligned to a similar horizontal level on the page. For instance, ICS1 mutants eds16 [41,45] (1997) and sid2 [40] (1999) in arabidopsis and the confirmation of its function as ICS [42,43] (2007) are listed closely to the timeline, because they are related. See [40–50,54,55].

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upstream of SA biosynthesis. In addition, GH3 enzymes also have AA specificity for conjugation to acyl substrates. For instance, PBS3 favors glutamic acid (Glu) [52]. A breakthrough in characterizing the role of PBS3 in SA biosynthesis has been reported recently by two independent groups using a combined approach of genetics and metabolomics [48,54]. PBS3 was identified as an isochorismoyl-glutamate synthase (IGS) to adenylate IC and the adenylated IC was then conjugated with Glu via the AA transferase activity of PBS3, which produces isochorismoyl-9-glutamate (IC-9-Glu) [48,54]. IC-9-Glu spontaneously break down into SA and 2-hydroxy-acryloyl-N-glutamate (2HNG), or named as N-pyruvoyl-L-glutamate (NPG) (Figure 2A) [48,54]. Intriguingly, another enzyme EPS1, was shown to enhance the production of SA from IC-9-Glu in addition to the spontaneous reaction [54]. Similar to mutants of ICS1, EDS5, and PBS3, eps1 mutant was also found in a forward genetic screen for mutants that exhibits enhanced pseudomonas susceptibility (eps) [55]. EPS1 encodes a BAHD acyltransferase-like protein and eps1 mutants have reduced SA accumulation upon pathogen inoculation. Most recently, Torrens-Spence et al. identified EPS1 as an IC-9-Glu pyruvoyl-glutamate lyase (IPGL) that plays an important role in arabidopsis to enhance SA production after the catalytic step of PBS3 (Figure 2A) [54]. These studies in arabidopsis suggest that plants have evolved a unique SA biosynthesis pathway, which is different from that in bacteria or other organisms. The PAL Pathway: Not Just a ‘Sidekick’ Between the 1960s and 1970s, before the components in the IC pathway were discovered in plants, carbon-14 isotope feeding studies indicated that SA can be synthesized in plants from phenylalanine (Phe). Radio-labelled Phe was fed to plants, where it was converted to transcinnamic acid (t-CA) (Figure 2A) [56–58]. Here, the pathway was traced as it bifurcates to produce ortho-coumaric acid and or benzoic acid (BA), both of which are converted to SA (Figure 2A). In 1961, Phe ammonia-lyase (EC 4.3.15; PAL) was isolated from barley and demonstrated to have Phe deaminase activity, converting Phe into t-CA and ammonia, thus identifying the first enzyme in the PAL pathway [59]. Later, PALs and their enzymatic activities were also identified in arabidopsis [60,61]. In addition, although all four arabidopsis PALs have been shown with various enzymatic kinetics and physical properties, mutations in all four PAL genes resulted in a 90% reduction of basal PAL activity and 50% decrease in pathogen-induced SA accumulation, which genetically confirmed PAL also contributes to SA biosynthesis (Figure 2A) [62,63]. Arabidopsis aldehyde oxidase 4 was identified from BA production in developing seeds, which can convert benzaldehyde to BA (Figure 2A) [64]. Another enzyme, BA 2-hydroxylase (BA2H), has been proposed to convert BA to SA based on the evidence of partially purified oxygenase activity, which belongs to the cytochrome P450 protein family [65]. However, the encoding gene of BA2H has not been identified, so genetic studies cannot be applied to test this hypothesis (Figure 2A). In addition, the roles of subsequent steps following PALs in pathogen-induced SA biosynthesis are not clear (Figure 2A). The PAL pathway is indispensable in plants, as the intermediates and products in the PAL pathway are required for the biosynthesis of not only SA, but also many other secondary metabolites, such as lignin [66]. Contributions of the Two Pathways Homologs of ICS and PAL are also found and characterized in other plants, including tobacco, tomato (Solanum lycopersicum), poplar (Populus trichocarpa), safflower (Carthamus tinctorius), and pepper (Capsicum annuum) [41,62,67–71], suggesting that these two pathways of SA biosynthesis are evolutionary conserved. Plants with loss-of-function mutations in ICS1 and PALs

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both show increased susceptibility to many pathogens, indicating that both the IC and PAL pathways are required for SA accumulation and function in response to biotic stresses. However, pathogen-induced SA synthesis is mostly dependent on the IC pathway (Figure 2A). In the ics1 ics2 double mutant plants, chorismate may accumulate to higher levels, so as to boost the production of chorismate-derived L-Phe, the precursor of the PAL pathway (Figure 2A). It indicates a low efficiency of the PAL pathway and a potential dependency on the IC pathway, which explains why the PAL pathway is not enough to compensate for the lack of the IC pathway. However, because there are still some crucial unknown components in the PAL pathway, it is hard to compare the metabolic efficiency of both pathways directly. Abiotic stresses such as the short-wavelength ultraviolet (UV-C) and ozone treatments can also induce the production of SA via both the IC and PAL pathways [72]. However, it is not clear how much the IC or PAL pathways contribute to it and the detailed mechanism of how they have been activated or regulated in response to abiotic stresses is still unknown. The initial steps of the IC and PAL pathways are tightly associated with chloroplasts, though almost all known enzymes required in both pathways are encoded by the nuclear genes. In the IC pathway, ICS1 and EDS5 were reported to localize in the chloroplasts and it is reasonable to believe that they might be functionally conserved in other colorless plastids, because SA can also accumulate in the nonphotosynthetic tissues such as the hypocotyl and roots [73,74]. The downstream steps of SA biosynthesis in both IC and PAL are mainly produced in the cytosol (Figure 2A). Although forcing PBS3 to localize into the chloroplasts can reconstitute the IC pathway for SA biosynthesis in the absence of EDS5 [48], it is likely that the SA produced in the chloroplasts cannot be transported into the cytosol and so is not able to fulfill its function in plants. An easily overlooked fact is that both the IC and PAL pathways are closely related to other metabolic pathways, especially the shikimate and aromatic AAs (Phe, tyrosine, and tryptophan) biosynthesis pathways. Chorismate derived from shikimate pathway is a precursor for producing aromatic AAs (Figure 2A), which represent a major regulatory link of primary and secondary metabolism in plants. For instance, both plastidial and cytosolic components are required to convert the common precursor chorismate into Phe for SA biosynthesis in the PAL pathway [75,76]. Furthermore, both IC and PAL pathways may also contribute in different ways in different plant species [77], so it is interesting to see more future research conducted on studying these two pathways in other plant species, not just SA biosynthesis, but also the production of other plant metabolites influenced by both pathways.

What Is the ‘Fate’ of SA in Plants? With its unique molecular properties, SA can be chemically modified into different bio-active derivatives, through glycosylation, methylation, sulfonation, AA conjugation, and hydroxylation (Figure 3). Some modifications inactivate SA in its regulatory roles and help fine-tune SA activity, whereas others serve as temporary pools for its ‘storage’. Here, we will briefly summarize the best-studied cases together with remaining questions within each. Glycosylation: Salicylate Glucose Ester (SGE) and SA 2-O-β-D-Glucoside (SAG) With one hydroxyl and one carboxyl group, SA can be converted into two major derivatives via glycosylation. In arabidopsis, two uridine diphosphate (UDP)-glucosyltransferases (UGTs) have been reported to play the roles in the glycosylation of SA. Both UGT74F1 and UGT74F2 in arabidopsis can catalyze the conjugation of glucosyl group from UDP-glucose onto the hydroxyl group of SA and produce SAG [78]. UGT74F2, also named as S-glucosyltransferase 1 (SGT1), 8

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can also catalyze the conjugation of glycosyl onto the carboxyl group of SA and produce SGE (Figure 3) [79]. Both SAG and SGE are inactive forms of SA and they have been proposed to be readily hydrolyzed into active SA that can be remobilized to other cellular locations to function, though both SAG and SGE are transported into the vacuole [80]. However, the detailed mechanism by which the large pool of vacuolar SAG and SGE are hydrolyzed is still unknown. In planta, SGE and SAG might be converted into free SA via the unidentified glucose hydrolase or β-glucosidase to increase the pool of active SA (Figure 3). In addition, UGT74F1/F2 can use benzoate, anthranilate (2-amino benzoate) as their substrates during the process of glycosylation (Figure 3) [78,79,81], whereas UGT71C3 uses methyl-SA (MeSA) as its substrate [82]. Methylation: MeSA The carboxyl group of SA can be methylated by the S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTs) in plants. In arabidopsis, the SAM-MT is encoded by the gene BA/SA carboxyl methyltransferase 1 (BSMT1) [83], which belongs to a 24-gene SABATH family of MTs. SABATH MTs are collectively named after the three earliest-identified genes in this family: salicylic acid carboxyl methyltransferase (SAMT), benzoic acid carboxyl methyltransferase (BAMT), and theobromine synthase (THS) [84]. In bsmt1 mutant plants, pathogen-induced MeSA accumulation is completely abolished. Similar to the glycosylation, methylation inactivates SA for its function and, reversibly, MeSA can be converted into active SA by the methylesterases (MESs) (Figure 3) [85,86]. MeSA was also once proposed as a mobile signal for SAR [86], but later studies on bsmt1 mutant soon challenged this hypothesis [87]. Although more careful studies have shown that MeSA-required SAR development is dependent on the photoperiod during which plants receive the primary infection [88], it is still under debate if MeSA is one of the SAR mobile signals. Sulfonation: SA-2-sulfonate The common metabolic modification of organic substrates called sulfonation is catalyzed by the sulfotransferases (hereafter referred to as STs or SOTs) [89]. In arabidopsis, STs are composed of a gene family with 21 members [89]. Arabidopsis has a large number of endogenous sulfur metabolites and most of them are not well characterized [90]. Though SOT12 from arabidopsis can catalyze a sulfonate group to the 2-hydroxyl group of SA in vitro, no sulfonated SA (SA-2-sulfonate) was detectable in plants [91]. Other SOTs in arabidopsis harbor a broad range of substrates for sulfonation [92]. The donor of the sulfonate group for STs is usually the common coenzyme 3′-phosphoadenosine-5′-phosphosulfate (PAPS), and the end product contains the 3′-phosphoadenosine-5′-phosphate (PAP) (Figure 3) [93]. SA-2-sulfonate and/or other sulfonated salicylate derivatives may play positive roles in signaling, as mutation in SOT12 leads to enhanced inhibition of primary root growth mediated by SA and compromised pathogen-induced SA accumulation and resistance [91]. AA Conjugation: SA-Asp As mentioned earlier with PBS3, all GH3 family enzymes play potential roles in conjugating AA onto different phytohormones with specific AA preference. SA can be conjugated into salicyloyl-L-aspartate (SA-Asp) as the dominant form in planta [94–96]. SA-Asp is an inactive form of SA. In arabidopsis, GH3.5/WES1 can act on SA into SA-Asp in vitro and gain-offunction of WES1 or overexpression of WES1 leads to SA-Asp accumulation in planta, yet loss-of-function mutant in GH3.5 did not alter the basal level and pathogen-induced level of SA-Asp, so another unknown GH3 enzyme might serve the role of Asp conjugation onto SA [96,97]. However, gh3.5 mutant still alters the resistance, which is highly possible due to the altered AA conjugation on auxin, so as to influence the trade-off between defense and growth [96,98,99].

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UGT89A2 ? 2,3-DHBX 2,3-DHBG

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Figure 3. Salicylic Acid (SA) Metabolism in Plants. SA can be converted into functional or nonfunctional metabolites via different modifications (in red). SA can be conjugated with amino acids, such as aspartic acid (Asp), into SA-Asp by homologs in the glycoside hydrolase 3 (GH3) protein family. SA can be converted into SA-2-sulfonate by sulfotransferases. SA can be converted into methyl-SA (MeSA) by a benzoic acid (BA)/SA carboxyl methyltransferase BSMT1. Reversibly, MeSA can be converted into SA by methylesterases (MESs). SA can be hydrolyzed into 2,3-dihydroxy-benzaic acid (2,3-DHBA) by SA-3 hydrolase (S3H), such as DMR6-Like Oxygenase 1 (DLO1) and DLO2 in arabidopsis; alternatively, SA can be hydrolyzed into 2,5-DHBA by SA-5 hydrolase (S5H), such as DMR6 in arabidopsis. Both DHBAs can be converted into dihydroxybenzoic acid xyloside (DHBX) or dihydroxybenzoic acid glucoside (DHBG) via glycosylation by a uridine diphosphate (UDP)-glucosyltransferase UGT89A2. SA can be glycosylated into SA 2-O-β-D-glucoside (SAG) by UGT74F1/F2 or into salicylate glucose ester (SGE) by UGT74F2 or named as SGT1. All glycosylated salicylates are stored in vacuoles in plant cells and the reversed reactions of glycosylation might be conducted through some unknown glucose hydrolases or β-glucosidases (question mark).

Here, for better clarity, GH3.5 was initially named after Gretchen Hagen’s (GH) work on auxin response genes [100]. Protein GH3.5 shares homology to glycoside hydrolase family 3 (GH3) proteins, so both ‘GH’ acronyms coincidentally refer to the same protein in this case, though they were derived from different origins. 10

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Hydroxylation: 2,3-DHBA and 2,5-DHBA In vitro, SA can scavenge free hydroxyl radicals without any enzymes and form two dihydroxybenzoic acid (DHBA): 2,3-DHBA and 2,5-DHBA (or commonly known as gentisic acid), but these reactions are highly reversible [101,102]. In arabidopsis, arguably the pathogen-induced production of reactive oxygen species is compromised in the respiratory burst oxidase homolog (rboh) mutants, which leads to a reduced availability of free hydroxyl radicals in the cytosol. However, loss-of-function mutation in rbohs did not affect DHBA accumulation, which indicates that the hydroxylation of SA might not be autonomous but rather under certain enzymatic control. In arabidopsis, SA-5 hydrolase (S5H) is encoded by a gene Downy Mildew Resistant 6 (DMR6) and has been confirmed to be able to convert SA into 2,5-DHBA [103]. Another two homologs of DMR6, DMR6-Like Oxygenase 1 (DLO1) and DLO2 might serve as SA-3 hydrolase (S3H) in arabidopsis, with a similar function as DMR6 but contributing more on the production of 2,3-DHBA (Figure 3) [54,103,104]. Both DHBAs can be modified into inactive forms via the glycosylation by UGT89A2 [105].

How Is SA Biosynthesis Regulated? SA accumulation in plant cells is crucial for its function, whereas constitutive SA accumulation has a detrimental effect on plant fitness [106], and hence a mechanism is required to orchestrate SA concentration into an optimal level in plants. SA accumulates upon stresses from both de novo biosynthesis and, potentially, metabolic release from its inactivate forms (Figures 2A and 3). Components in those pathways are tightly regulated. For instance, the expression of most genes involved in SA biosynthesis is induced in response to stresses. Accumulation of SA can also enhance SA biosynthesis through transcriptional regulations of those genes, such as ICS1. It is believed that post-transcriptional regulation is also required for ICS1 protein function in SA biosynthesis [107]. Here, we will concentrate mainly on the transcriptional regulations of SA biosynthesis-related genes in the IC pathway. Positive Transcriptional Regulation The plant-specific calmodulin (CaM)-binding TF CaM-Binding Protein 60g (CBP60g) and its close homolog Systemic Acquired Resistance Deficient 1 (SARD1 or CBP60h) were found to promote pathogen-induced SA synthesis by regulating ICS1 and EDS5 transcript [108–110]. CaM-binding activity is required for CBP60g function, whereas SARD1 does not appear to be a CaM-binding protein [108]. Despite this difference, CBP60g and SARD1 are partially redundant in regulating ICS1 expression and SA accumulation during immunity (Figure 4) [109,110]. WRKY TFs are also involved in regulating ICS1 expression. Coexpression analysis indicates that WRKY28 and WRKY46 could play a role in regulating ICS1 and PBS3 (Figure 4). By using both in vivo and in vitro DNA-binding assays, including chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assay, WRKY28 was shown to bind directly to the ICS1 promoter, positioned –445 and –460 base pairs upstream of the transcription start site [111]. In a separate screen, TCP21 and NAC TF-like 9 (NTL9) were identified as additional activators of ICS1 during specific immune responses (Figure 4) [112]. TCP was named after four proteins, including Teosinte Branched 1 (TB1) from maize (Zea mays), Cycloidea (CYC) from snapdragon (Antirrhinum majus), as well as Proliferating Cell Nuclear Antigen Factor1 (PCF1) and PCF2 from rice (Oryza sativa), while NAC was named after No Apical Meristem (NAM), Arabidopsis Transcription Activation Factor (ATAF), and Cup-shaped Cotyledon (CUC) TF-domaincontaining proteins. TCP21, also designated as CCA1 Hiking Expedition (CHE), is a central circadian clock oscillator and is required not only for the daily oscillation in SA levels but also for

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Figure 4. Simplified Scheme for Transcriptional Regulation of Salicylic Acid (SA) Biosynthesis Genes. Transcription factors (TFs) NAC TF-like 9 (NTL9), TPC21 (CHE), WRKY28, Systemic Acquired Resistance Deficient 1 (SARD1), and CaM-Binding Protein 60g (CBP60g) can positively regulate ICS1 gene expression (in black); SARD1 and CBP60g can also activate gene expressions of EDS5 and PBS3. TCP8 and 9 can interact with WRKY28 and SARD1, which is potentially involved in their positive transcriptional regulations. TFs CBP60a, Abscisic acid-responsive NAC (ANAC)019, and Ethylene Insensitive 3 (EIN3) can negatively regulate ICS1 gene expression (in red), while DP-E2F-like1 (DEL1) negatively regulates EDS5 gene expression. Activities of both CBP60g and CBP60a are dependent on calmodulin (CaM) binding. ICS1, EDS5, and PBS3 encodes enzymes that are required in SA biosynthesis in the isochorismate (IC) pathway.

pathogen-induced SA synthesis in systemic tissues during SAR. NTL9 is essential for inducing ICS1 in guard cells upon pathogen-associated molecular pattern treatment. Using a yeast one-hybrid screen, TCP TFs TCP8 and TCP9 were identified as redundant activators of ICS1 during immune responses [113]. A significant reduction in the expression of ICS1 during immune responses was observed in the tcp8 tcp9 double mutant. The binding of TCP8 to a typical TCP binding site in ICS1 promoter was confirmed by in vitro and in vivo assays. Interestingly, TCP8 was shown to specifically interact with a number of TFs, including SARD1, NAC019, and WRKY28 (Figure 4) [113]. Negative Transcriptional Regulation Another close homolog of CBP60g, called simply CBP60a, negatively regulates ICS1 expression upon CaM-binding (Figure 4) [114]. Therefore, SA biosynthesis-related genes involve multiple levels of control in their transcripts. A possible model is that in the absence of pathogen, CBP60a represses immunity while CBP60g and SARD1 have low activity. Upon pathogen infection, CBP60g and SARD1 bind to the ICS1 promoter, activate its expression, and release the negative regulation imposed by CBP60a. 12

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NACs comprise one of the largest plant-specific TFs and they play vital roles in plant stress response [115]. Abscisic acid-responsive NAC (ANAC) TFs ANAC019/055/072 are involved in negative regulation of SA biosynthesis [116]. They were initially found as direct targets of MYC2, a TF involved in JA signaling. However, the triple mutant of anac019/055/072 exhibits elevated ICS1 expression regardless of pathogen treatment. ChIP-qPCR indicates that ANAC019 directly binds to the promoters of BSMT1 and ICS1 (Figure 4) [116]. Unlike CBP60a, NAC TFs exert inhibitory effect by repressing ICS1 and activating BSMT1, which may lead to reduced SA biosynthesis and increased level of inactive volatile MeSA converted from SA. DP-E2F-like1 (DEL1) encodes another TF involved in negative transcriptional regulation. It belongs to Adenovirus E2 Factor (E2F)- and Dimerization Partner (DP)-type TFs, which are common in most eukaryotes [117]. Some E2Fs function as activator and others may serve as suppressors [117]. DEL1 (E2F3) was reported to negatively regulate EDS5 gene expression through directly binding to the EDS5 promoter (Figure 4) [118]. Loss-of-function mutant del1 shows constitutive accumulation of SA and increased resistance against powdery mildew [118]. Additionally, Ethylene Insensitive 3 (EIN3), previously known to mediate ethylene (ET) signaling, specifically binds to the ICS1 promoter sequence both in vitro and in vivo (Figure 4) [119]. The ein3 eil1 double mutant constitutively accumulates SA in the absence of pathogen attack and mutation in ICS1 restores its susceptibility to pathogens [119], which indicates EIN3 may contribute to the negative regulation of ICS1 gene expression. Interestingly, a recent report has shown NPR1 interacts with EIN3 and interferes its binding to its target genes, such as HOOKLESS1 (HLS1), whose expression is induced by ET [120]. This provides another mechanistic evidence of SA contributing to the homeostasis of plant growth, development, and stress responses.

Concluding Remarks and SA: Looking to the Future

Outstanding Questions Is SA required for the immunity in all plant species? Is the mechanism of SA biosynthesis conserved across all plant species? The chloroplast is important for SA biosynthesis and some enzymes are specifically localized in the chloroplast. What are the evolutionary origins of these enzymes and the IC pathway in plants? Monocot rice has much higher SA tolerance than dicot arabidopsis. In addition, it has been shown that engineering NPR1 expression in rice can increase resistance against pathogens. How can we apply our knowledge of SA biosynthesis, its regulations, and SA responses to engineering novel pathogen resistance in plants? SARD1/CBP60g are reported as master transcription factors (TFs) in regulating genes related to SA biosynthesis and responses as well as genes that are independent of SA. How are these plant-specific TFs activated in response to recognition of different pathogens?

Plants seem to accumulate various endogenous levels of SA [121]. For example, endogenous SA concentration in monocot crop rice (O. sativa) is much higher than dicot arabidopsis and there is no further induction of SA biosynthesis in response to bacterial infection in rice, which is in contrast to SA-induction studies in arabidopsis. Now, with genomic data available from more plant species, it is interesting to examine the contributions of endogenous SA to plant adaptation to the changing environment. The ‘disease triangle’ theory is an example of integrating the effects from all crucial elements, including plants, plant-associated live organisms (pathogens in particular), and the environmental conditions, such as temperature, soil composition, and water availability [122]. In the bigger picture, it is a great challenge to apply our knowledge of SA directly to improving agriculture and preserving biodiversity. As one of the most important enzymes in SA biosynthesis, ICS1 seems to be resilient to a broad range of pH values and temperatures for its function [42]. Thus, it is relevant to survey whether there are polymorphic distributions of all genes involved in both SA biosynthesis and SA responses in different plant species at diverse geographic locations, which are usually associated with very different thermodynamics. There are indications that the circadian clock regulates SA biosynthesis and plant responses to pathogens [112,123]. However, the detailed mechanism of how circadian rhythm contributes to SA biosynthesis and response in plants growing under different photoperiods is still not clear. Both IC and PAL pathways are required for SA biosynthesis, however, more systematic studies of both pathways in different plant species are still required to address the question of how SA biosynthesis evolved in plants (see Outstanding Questions). Trends in Plant Science, Month 2020, Vol. xx, No. xx

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How we can apply our knowledge of SA function in plants to agriculture is a big question for the future. Increased SA level or SA responsiveness can enhance disease resistance. For example, it has been shown that overexpression of NPR1 was able to enhance a broad-spectrum disease resistance in arabidopsis, rice, and wheat (Triticum aestivum) [124–126]. These studies have led to strategies of engineering resistant crops through ectopic transcription of NPR1. However, enhanced resistance obtained through such strategies is often associated with substantial penalties to fitness. For example, overexpression of OsNPR1/NH1 in rice spontaneously activated resistance genes and resulted in a lesion-mimic phenotype [125]. Recently a fine-tuned resistance was shown by using cotranslational controlled expression of NPR1, proving that it is possible to engineer a broad-spectrum disease resistance without compromising overall plant fitness [127]. Loss-of-function mutants of NPR3 and NPR4 in arabidopsis showed enhanced disease resistance against pathogens with no significant developmental defects (e.g., plant sizes or reproductions). As NPR3 and NPR4 are also conserved in most plant species [128], generation of loss-of-function mutants of NPR3 and NPR4, or conditionally reduced expressions of NPR3 and NPR4 in crop plants, could be also used to engineer plant resistance with reduced fitness cost. It is noted that plant hybrids can enhance resistance and it has been shown as SA-dependent [129]; understanding the mechanisms of how plant hybrids lead to increased SA-dependent resistance might provide new insights of engineering disease resistance in plants. Acknowledgments We sincerely thank Zane Duxbury, Chih-hang Wu, Jianhua Huang, Michael Torrens-Spence, and Hee-Kyung Ahn for their careful reading and constructive suggestions on this manuscript. P.D. acknowledges the support from a Future Leader Fellowship (grant number: BB/R012172/1) provided by the Biotechnology and Biological Sciences Research Council (BBSRC). Y.D. acknowledges the support of a Marie Curie Fellowship (grant number: 841689) provided by the European Union’s Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie Actions.

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