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Plant Molecular Biology 26: 1439-1458, 1994. © 1994 Kluwer Academic Publishers. Printed in Belgium. 1439

The salicylic acid signal in plants

Daniel F. Klessig* and Jocelyn Malamy 1 Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers-The State University of New Jersey, P. O. Box 759, Piscataway, NJ 08855, USA (*author for correspondence); 1present address: Biology Department, 1009 Main Building, New York University, Washington Square, New York, N Y 10003, USA

Received 3 March 1994; accepted 26 April 1994

Key words: acquired resistance, active oxygen species, defense response, hypersensitive response, pathogenesis-related proteins, salicylic acid, signal transduction

Introduction

History

Plants are one of the world's richest sources of natural medicines. The use of plants and plant extracts for healing dates back to earliest recorded history. Today, such plant-derived medicines as quinine, digitalis, opiates and morphine are widely used, while new natural chemicals such as the putative anti-cancer drug taxol from yew tree bark are being characterized and developed.

The use of willow tree bark to relieve pain is believed to be as old as the 4th century B.C., when Hippocrates purportedly prescribed it for women during child birth [94, 145]. The active principle of willow remained a mystery until the 19th century when the salicylates, including salicylic acid (SA), methyl salicylate, saligenin (the alcohol of SA) and their glycosides, were isolated from extracts of different plants including willow. Soon thereafter SA was chemically synthesized, eventually leading to its widespread use. SA was subsequently replaced by the synthetic derivative acetylsalicylic acid (aspirin) which produces less gastrointestinal irritation yet has similar medici- nal properties. Despite its long history the mode of action of SA is not fully understood. The finding that it plays a role in disease resistance responses in plants raises the possibility of some

fascinating parallels between SA action in plants and animals.

Salicylic acid in plants

SA is one of numerous phenolic compounds, defined as compounds containing an aromatic ring with a hydroxyl group or its derivative, found in plants. There has been considerable speculation that phenolics in general function as plant growth regulators [1]. Exogenously supplied SA was shown to affect a large variety of processes in plants, including stomatal closure, seed germina- tion, fruit yield and glycolysis (for review see [ 29 ]). However, some of these effects were also produced by other phenolic compounds. In addition, some effects of SA may have been caused by the general chemical properties of SA (as an iron chelator or acid) [95]. For these reasons, the sig- nificance of SA was not realized from these early studies. Only recently has there been evidence that SA has unique and specific regulatory roles.

Flowering and thermogenesis

The role of SA as an endogenous signalling molecule was first suggested in connection with flowering. Cleland and coworkers [23, 24] found that

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honeydew from aphids feeding on Xanthium stru- marium contained an activity that induced flowering in duckweed (Lemna gibba) grown under a non-photoinductive light cycle. The flower-inducing factor could be extracted directly from the Xanthium phloem and was identified as SA. This was consistent with reports that exogenously applied SA was active in inducing flowering in both organogenic tobacco (Nicotiana tabacum) tissue culture [65] and whole plants of various species (for review see [95]). However, the possibility that SA is an endogenous signal for flowering remains in question since SA did not induce flowering when exogenously applied to Xanthium. Moreover, the endogenous levels of SA were the same in the phloem of vegetative and flowering Xanthium, and other substances were as effective as SA in flower induction (for review see [95]).

The first conclusive evidence implicating endogenous SA as a regulatory molecule resulted from studies of voodoo lilies (Sauromatum gut- tatum) [97, 99]. The spadix of the voodoo lily is thermogenic and exhibits dramatic increases in temperature during flowering. There are two periods of temperature increases in the spadix; a large, transient rise in endogenous SA levels was found to precede both periods. Furthermore, thermogenesis and the production of aromatic compounds associated with thermogenesis could be induced by treatment of spadix explants with SA, acetylsalicylic acid, or 2,6-dihydroxybenzoic acid but not with 31 structurally similar compounds.

The mechanism by which SA regulates heat production is beginning to emerge. During thermogenesis much of the electron flow in mitochondria is diverted from the cytochrome respiratory pathway to the alternative respiratory pathway [ 81 ]. The energy of electron flow through the alternative respiratory pathway is not conserved as chemical energy, but released as heat. The alternative respiratory pathway utilizes an alternative oxidase as the terminal electron acceptor. Rhoads and Mclntosh found that expression of the alternative oxidase gene is induced by SA in voodoo lilies [103]. Interestingly, in nonthermogenic tobacco, SA treatment also caused a significant

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increase in alternative respiratory pathway capac- ity and a dramatic accumulation of alternative oxidase [ 105].

Disease resistance

The second process for which there is strong evidence that SA acts as a signal molecule is disease resistance. This has been an extremely active area of investigation during the past several years and is the focus of this review. Particular emphasis will be placed on progress made since the publi- cation of several reviews in 1992 [29, 40, 73, 95, 96].

When a plant is infected with a pathogen to which it is resistant, a wide variety of biochemical and physiological responses are induced. Many of these responses are believed to protect the plant by restricting, or even eliminating, the pathogen and by limiting the damage it causes. In contrast, when a plant is infected with a pathogen to which it is susceptible, the pathogen rep- licates and frequently spreads throughout the plant, often causing considerable damage and even death of the host. Lack of resistance can be caused by an inability of the host plant to recog- nize or effectively respond to infection. Alterna- tively, the pathogen may have evolved strategies to overcome the plant's defense arsenal [59].

Although some plant-pathogen interactions lead to disease, most do not [72]. Plant disease resistance is often manifested as a restriction of pathogen growth and spread to a small zone around the site of infection. In many cases, this restriction is accompanied by localized death (necrosis) of host tissue. Together pathogen restriction and tissue necrosis characterize the hypersensitive response (HR) [78]. Often associated with this local response is the development, over a period of several days to a week, of enhanced resistance to a secondary infection by the same or even unrelated pathogens. This enhanced level of resistance can be manifested throughout the plant and is generally termed systemic acquired resistance (SAR) [22, 109, 110].

The fundamental processes involved in the HR

and SAR are not yet well understood but a large number of physiological, biochemical, and molecular changes have been noted that correlate with one or both of these responses. These include: (1)the synthesis and incorporation of hydroxyproline-rich glycoproteins (HRGPs), cel- lulose, callose, and phenolic polymers such as lignin into the cell wall to fortify this physical barrier; (2)the production of low molecular weight, antimicrobial compounds called phytoal- exins; (3)the enhanced expression of genes encoding enzymes in the phenylpropanoid pathway, such as phenylalanine ammonia lyase (PAL), which often lead to the production of phytoalex- ins and other phenolic compounds; (4)the production of antiviral activities, some of which appear to be due to novel proteins; (5) the synthesis of proteinase inhibitors that block the activity of microbial and insect proteinases; (6)enhanced peroxidase activity, which is necessary for ligni- fication and may be involved in crosslinking of cell wall protein; (7) the expression of genes encoding hydrolytic enzymes such as chitinases and fl-l,3-glucanases that degrade the cell walls of microbes and may be involved in release of elicitor molecules; and (8) the synthesis of pathogenesis-related (PR) proteins.

Particular attention has been paid to the abun- dant PR proteins, a large group of proteins whose synthesis is induced by pathogen infection. These proteins have been divided into five or more unrelated families. Two of these families encode the hydrolytic fl- 1,3-glucanases (PR-2) and chitinases (PR-3), while the functions of the other families are poorly understood. The expression of many of the well characterized PR genes (e.g. PR-1 through PR-5) in tobacco has been correlated with resistance to a large variety of ([143]; for review see [ 17]) but not all [ 159] viral, bacterial and fungal pathogens. As a result, expression of PR genes is often used as a marker for induction of disease resistance. Moreover, studies with transgenic tobacco plants that overexpress some of these PR genes (PRs 1, 2, 3 and 5) have demonstrated that they enhance resistance to several fungal pathogens [4, 160, 15, 71a]. Antifungal activity in vitro has also been demonstrated for

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PR-2 through PR-5 proteins [71a, 79, 92, 142, 152]. For a more detailed discussion of the PR proteins and plant defense responses, the reader is referred to reviews by Carr and Klessig [ 17], Bowles [ 13], Bol et aL [ 10], Dixon and Harrison [34], Ryan [113], Linthorst [71], Madamanchi and Ku6 [72], White and Antoniw [149], Ryals etaL [112], and Cutt and Klessig [30].

The diversity of the defense responses induced by pathogen attack suggests that they may be controlled by multiple signals acting through several pathways. Some of these signals need only act over short distances to induce defenses at the site of infection. However, for the development of S A R a signal must pass from the infection site to distal tissues. Grafting studies have demonstrated that a translocatable factor or signal can move from an infected leaf through the graft to the uninfected rootstock and induce SAR ([48]; for review see [72]). Considerable progress has been made in the past few years in the identification and characterization of several possible long- distance signals. These include systemin [80], jasmonates [44], electrical potentials [151], ethylene [91, 157] and SA. For a summary of the studies on systemic signals in plants, the reader is referred to two recent reviews [40, 73]. SA's putative role as a local and systemic signal is the subject of the next two sections of this review.

S A - a s i g n a l for d e f e n s e

The first hint that SA might be involved in plant defense was provided by White [ 147] who found that injection of aspirin or SA into tobacco leaves enhanced resistance to subsequent infection by tobacco mosaic virus (TMV). This treatment also induced PR protein accumulation [5]. In addition to enhancing resistance to TMV in tobacco, SA also induced acquired resistance against many other necrotizing or systemic viral, bacterial, and fungal pathogens in a variety of plants ([ 144]; for review see [73]). (However, not all plant- pathogen systems respond to SA [ 106, 107, 158].) SA was also found to induce PR proteins in a wide range of both dicotyledonous and mono-

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cotyledonous plants including tomato [150], potato [148], bean [54], cucumber [84], cowpea [54, 148], rice [77, 121], garlic [134], soybean [28], azuki bean [55], sugar beet [45], Arabidop- sis thaliana [131], and Gomphrena globosa [150].

Around the time that White [ 147] first demonstrated the effects of exogenous SA on defense responses, several reports showed a correlation between resistance and the levels of endogenous SA. For example, SA levels in extracts from the bark of different poplar species were shown to correlate with the resistance of the species to the fungal pathogen Dothiciza populae [93]. SA was also found to be toxic to several pathogens including Colletotrichum falcatum, Fusarium oxysporum [124] and Agrobacterium tumefaciens [114]. These latter results suggest that in some situations endogenous SA might inhibit pathogen growth due to its toxicity. However, SA was shown not to be directly toxic to cultures of Col- letotrichum lagenarium [88] or preparations of TMV (Malamy and Klessig, unpublished results), even though it induces resistance to both of these pathogens in plants.

Since SA treatment of tobacco induces several of the same responses as TMV infection (i.e. acquired resistance and PR gene expression), it was postulated that SA acts by mimicking an endogenous phenolic signal that triggers PR gene expression and resistance [135]. However, by monitoring endogenous levels of SA in TMV- infected tobacco, Malamy and co-workers [74] provided evidence that strongly suggested a role for SA itself as a signal molecule. Infection of a TMV-resistant cultivar resulted in a dramatic increase (20-50-fold) in levels of endogenous SA in the TMV-inoculated leaves and a substantial rise (5-10-fold) in uninoculated leaves of the same plant. These increases were not seen in a nearly isogenic susceptible cultivar. The concentration of endogenous SA detected after infection (1- 50 #M) was much lower than the concentration of SA used to exogenously induce PR gene expression and resistance (300-1000 #M). How- ever, Raskin and colleagues [41, 156] demonstrated that when the concentration of endogenous SA in detached leaves or in plants grown

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hydroponically in different concentrations of SA reached levels similar to those detected in TMV- inoculated leaves, PR-1 gene expression and resistance were induced. Since the rise in endogenous SA paralleled or preceded the induction of PR-1 gene expression in both inoculated and uninoculated leaves of TMV-infected resistant plant [74], SA appears to play a role in the pathway leading to resistance responses.

Parallel studies in cucumber (Cucumis sativus) indicated that SA signalling is not unique to tobacco. A dramatic rise in SA levels (10-100-fold) was detected in the phloem exudates from cucumber leaves inoculated with tobacco necrosis virus, C. lagenarium [85] or Pseudomonas syringae pv. syringae [100, 124a]. These increases of SA in the phloem preceded both the appearance of SAR and the induction of peroxidase activity associated with resistance. Moreover, treatment of cucumber with SA also induced peroxidase activity and resistance to C. lagenarium [100, 124a]. Increases in SA levels recently have also been documented in Arabidopsis thaliana after infection with turnip crinkle virus ([ 132]; Dempsey, Wobbe and Klessig, unpublished results) or P. syringae [126] and in tobacco infected by tobacco necrosis virus, P. syringae, and Peronospora taba- cina [120] and Erwinia carotovora [90a].

The work of Ward et aL [ 143] provided additional support for SA's involvement in disease resistance in tobacco. The expression of thirteen families of genes encoding peroxidase, acidic PR- 1 through PR-5 proteins and their basic coun- terparts as well as several previously uncharac- terized proteins were investigated. Expression of all thirteen genes were induced in TMV-inoculated leaves of resistant tobacco, while nine showed enhanced expression in uninoculated leaves of TMV-infected plants. These same nine genes, which included the acidic PR-1 through PR-5, basic PR-1, basic and acid class III chitinase, and PR-Q' (a fl-l,3-glucanase) genes, were induced by SA treatment. Thus, SA induced the same spectrum of genes activated during development of SAR upon TMV infection.

Further support for a signalling role for SA was provided by temperature shift experiments in

the tobacco-TMV system. When TMV-resistant cultivars were inoculated and maintained at elevated temperatures (>28 °C), they failed to synthesize PR proteins and the infection becomes systemic [47, 57]. However, when these infected plants were then transferred to lower temperatures (22-25 °C), PR gene expression was induced and resistance (HR) was restored. It was shown that temperatures which block the ability of tobacco to resist viral infection also inhibited increases in SA levels [75, 156]. When the resistance response was restored by shifting plants to lower temperatures, endogenous SA levels increased dramatically and preceded both PR-1 gene expression and necrotic lesion formation associated with resistance [75].

The strongest evidence for SA's involvement in plant defense comes from the elegant experiments of Gaffney et al. [46]. They constructed transgenic tobacco plants that constitutively express the nahG gene from Pseudomonasputida under the regulation of the 35 S promoter of cauliflower mosaic virus (CaMV). nahG encodes salicylate hydroxylase, an enzyme which converts SA to cat- echol, a compound unable to induce SAR. SA levels in transgenic tobacco plants that accumu- lated substantial amounts of salicylate hydroxylase mRNA rose only 2- to 3-fold in the TMV- inoculated leaves compared to ca. 180-fold increase in untransformed control plants after TMV infection. These transgenic plants were subjected to a secondary inoculation of their upper leaves following a primary inoculation of their lower leaves to test the effect of reduced SA levels on the ability of the plant to establish SAR. Transgenic plants produced larger lesions in response to the secondary infection as compared to untransformed control plants, indicating a reduced ability to establish SAR. Furthermore, induction of genes associated with SAR such as the PR-1 genes was inhibited in the upper uninoculated leaves of TMV-infected nahG transgenic plants [141]. Surprisingly, the PR-1 genes were expressed in the TMV-inoculated tissue of nahG plants. Either the modest increase in SA in the inoculated nahG tissue is sufficient to induce these genes or another signal is involved.

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In addition to its role in SAR development, SA may be involved in restricting the replication and spread of the pathogen from the initial sites of infection. It was observed that the increases in endogenous levels of SA were considerably greater in TMV-inoculated leaves than in uninoculated leaves [74], with the highest levels ap- pearing in and around the infection sites [41]. In addition, larger primary necrotic lesions were formed on nahG transgenic tobacco than on non- transgenic controls after the initial infection by TMV, presumably due to the destruction of the SA signal by salicylate hydroxylase [46].

Taken together, these studies provide very strong support for SA's involvement in disease resistance. Levels of endogenous SA correlate with expression of defense-related genes and development of SAR while addition of exogenous SA induces defense responses and elimination of endogenous SA represses these responses. Fur- ther experiments are needed, however, to determine which of the several processes comprising disease resistance (e.g. lesion formation, restriction of pathogen growth or movement) are af- fected directly by SA. SA synthesis and response mutants, as well as the nahG plants, will help provide the necessary insights.

Is SA the translocated signal for SAR?

The experiments described above establish that SA plays a critical role in resistance and the development of SAR. However, it is unclear whether SA is the primary signal that travels from the inoculation site to distal tissues. Initial experiments suggested that SA might fulfill this function. First, the rise in SA levels preceded PR gene induction in uninoculated leaves of TMV-infected resistant tobacco [74]. Second, the large increase in SA in the phloem exudates from infected cucumber leaves preceded the development of SAR and induction of peroxidase activity in uninoculated leaves [85, 100, 124a]. Third, the appearance ofchitinase (PR-3) in P. lachrymans-infected cucumber was preceded by an increase in SA levels in the upper, uninoculated leaves as well as

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in inoculated leaves [ 86]. These observations, together with the report that SA was in the phloem sap of TMV-infected tobacco [156], suggested that SA might be a primary mobile signal.

In contrast, two sets of experiments now strongly argue that SA is not the translocated signal for SAR. Since the systemic signal for SAR can pass through a graft junction [48], the requirement for SA in the induction of SAR and PR gene expression can be determined by grafting together nahG transgenic tobacco and untransformed tobacco (Xanthi nc) (141). When the scion (upper, grafted portion of the plant) was derived from the transgenic nahG plants, neither SAR nor PR-1 expression were induced in the scion after inoculation of the rootstock leaves with TMV, regardless of the origin of the rootstock. In contrast, an untransformed Xanthi nc scion grafted onto a nahG rootstock expressed PR-1 genes and showed SAR to a secondary infection by TMV or the fungal pathogen Cercospora nicotianae upon primary inoculation of the rootstock with TMV. These results indicate that (1) a signal other than SA can move from the rootstock to the scion after infection and (2) SA was required in the uninoculated leaves in the scion to mediate the translocated systemic signal.

The above results are consistent with and ex- tend the observations in the cucumber-P, syringae system made by Hammerschmidt and co-workers [100, 124a]. When only one leaf on a cucumber plant was inoculated with P. syringae, increases in SA levels, peroxidase gene expression, and resistance were detected in the uninoculated leaves even if the inoculated leaf was removed as early as 4-6 h after infection. In contrast, SA was not detected in the phloem sap from the inoculated leaf until 8 h after infection. Thus, all the results to date are consistent with a model in which local infection leads to the production of an unidenti- fied mobile factor or signal, which in turn requires SA in distal tissues for the establishment of SAR.

The model that SA is not the normal translocated signal is also consistent with the observation that when SA was applied locally via injection, it induced PR protein accumulation and enhanced resistance only in the treated tissue

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[ 136]. A likely explanation for this result comes from the studies of Mrtraux and coworkers [85] who showed that exogenous SA applied locally to leaves was not readily transported to other parts of the plant. However, detection of SA in the phloem sap of infected tobacco and cucumber leaves suggests that endogenous SA can be translocated. Perhaps endogenous SA and exogenously supplied SA are handled differently by the plants. This explanation, however, appears inconsistent with results from studies using chemical inducers of SA synthesis. Malamy et aL [ 76] found that local injection of polyacrylic acid or thiamine-HCl into tobacco leaves resulted in large, but transient increases in endogenous SA levels and stable accumulation of large amounts of a conjugated form of SA (see 'SA metabolism'). In contrast to TMV infection, the increase in SA levels and expression of PR-1 genes induced by these chemicals occurred only in the treated tissue. Thus, either endogenously produced SA is not readily translocated or transient elevation of SA is not sufficient for production (and translocation) of a systemic signal.

SA metabolism

In plants, SA is probably synthesized from phenylalanine [18], which is converted to trans- cinnamic acid by PAL. PAL is a key enzyme in the phenylpropanoid pathway that yields phytoal- exins, lignins and hydroxybenzoic acids. There are two proposed pathways for the conversion of trans-cinnamic acid to SA; they differ in the order of//-oxidation and ortho-hydroxylation reaction. //-oxidation of trans-cinnamic acid produces benzoic acid, which can be hydroxylated to form SA. Alternatively, ortho-hydroxylation of trans-cinnamic acid forms o-coumaric acid, which can be converted to SA via//-oxidation.

Labelling studies by Yalpani and coworkers [ 153] indicate that in TMV-infected tobacco, SA is predominantly synthesized from benzoic acid (BA). The enzymatic activity responsible for converting BA to SA, BA 2-hydroxylase, was induced four- to five-fold by TMV infection [68].

BA treatment of tobacco plants also induced BA 2-hydroxylase activity. This latter result, together with the magnitude and timing of BA increases in TMV-infected plants, suggest that an increase in the BA pool is the primary cause of increased BA 2-hydroxylase activity. Thus, the rate-limiting step in SA production may be the formation of BA from trans-cinnamic acid or a conjugated form of BA [153].

Most phenolic acids in plants exist in the form of sugar conjugates. Glucose esters (glucose attachment through the carboxyl group) and gluco- sides (glucose attachment through the hydroxyl group) are particularly common. These derivatives of SA have been reported in several plants [9, 27, 51, 128, 133]. Several reports have demonstrated that the SA produced after TMV infection of tobacco is rapidly conjugated to glucose to form SA fl-glucoside (SAG) [41, 75]. In these studies a large pool of SA could be identified after acid hydrolysis of crude extracts of TMV-infected tissues, indicating the presence of SA conjugates. These SA conjugates could be cleaved by fl-glucosidase, establishing that they were SAG. Both SA and SAG were present at very low levels in uninfected plants but rose more or less in parallel after infection, with SAG becoming the pre- dominant form. The same glucoside was formed from exogenously supplied radiolabelled SA within hours of application.

A UDP-glucose: SA glucosyltransferase that forms SAG from SA has been characterized in several plants. This enzyme activity is induced by SA in Mallotus japonicus [ 127], oats [ 154] and tobacco [39] and has been partially purified from all three plant species. In tobacco the enzyme activity is enhanced about 7-fold above basal levels between two and three days after infection with TMV, consistent with the rise in endogenous levels of SA.

Whether SAG accumulates in uninoculated portions of a plant is unclear. Enyedi et al. [41] detected free SA, but no SAG, in lower halves of tobacco leaves inoculated with TMV at their tips. They also found only SA in phloem exudates of inoculated leaves and in upper uninoculated leaves of TMV-infected plants. These results

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suggest that SAG is neither transported to nor synthesized in uninoculated sites. The authors speculate that these tissues may lack sufficient glucosyltransferase activity to convert SA to SAG. In contrast, Guo, Malamy and Klessig (unpublished results) have found both SA and SAG in the inoculated and uninoculated halves of TMV-infected leaves. Similarly, conjugated SA was found in uninoculated leaves of P. lachrymans-infected cucumber [ 86 ].

The existence of SAG suggests additional com- plexity in the modulation of the SA signal during defense responses. To test the bioactivity of SAG in the absence of SA, this compound was synthesized and injected into the extracellular spaces of tobacco leaves and PR-1 gene expression was subsequently monitored [53]. In these experiments SAG proved to be as active as SA in inducing PR-1 genes. However, isolation of extracellular fluid from SAG-injected leaves demonstrated that SAG was hydrolyzed to release SA in the extracellular spaces. Apparently, this released SA entered the surrounding cells and was reconjugated to form SAG. In accordance with this observation, a cell wall-associated fl-glucosidase activity that converts SAG to SA has been detected (Malamy, Conrath and Kles- sig, unpublished results). The transient presence of SA in the injected leaves made it impossible to determine if SA or SAG was the active form. However, in studies of both phytohormones and phenolics the unconjugated forms have been shown to be active, while the glucose-conjugated forms are inactive [25, 26, 70, 102]. In addition, the SA-binding protein that is believed to trans- duce the SA signal fails to bind SAG (see 'Mechanism of action of SA'; [20]). Therefore, it seems fikely that SA is active only in the free form.

Even if SAG is inactive, it may still serve as a storage form of SA. The spatial separation of an inactive glucoside and its fl-glucosidase has been demonstrated for several phenolics (for review see [26]) and provides a means of regulating release ofbioactive compounds. This compartmentation is reminiscent of the finding that SAG is intracellular while its putative fl-glucosidase is

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extracellular. In addition, recent studies suggest that deconjugation plays an important role in regulating the activities of auxin [ 16, 43 ], cytokinin [42] and giberellin [ 116]. A model for the role of SAG in development of acquired resistance based on this type of regulation is presented in Fig. 1. In the model illustrated here, a primary infection by a pathogen such as TMV results in a HR, including formation of necrotic lesions and production of SA throughout the plant. As dis- cussed above, much of this SA is converted to SAG. Subsequent challenge by a pathogen leads to cell damage and changes in membrane perme- ability at the infection site, resulting in the release of the local cellular stores of SAG into the extracellular spaces. Since extracellular SAG is hydrolyzed (see above), this would provide a high concentration of SA directly at the infection site where it could enter neighboring cells and induce

defense responses. Through this process, the defense mounted during a second infection would be more effective than the initial defense. While some defense-related proteins would already be present due to their accumulation during the initial infection, the rapid release of SA from stored SAG would quickly induce the defense responses anew, when and where they were needed. This rapid and effective induction at the infection site would result in the more rapid restriction of the pathogen, and hence a smaller secondary lesion.

Besides SAG, additional SA derivatives have been detected in various plant species [27, 118, 128]. Moreover, when radiolabelled SA was supplied to tobacco leaves, ca. 20~o could not be recovered as either SA or SAG [75]. At present, it is unclear whether other forms of SA are produced during defense responses and, if so, what their roles might be.

= 1" TMV lesion

• = 2* T M V lesion

r M V

"°°°i°°°°°.°°.o °°°... o0°°'°°°°°°''°°°°°°°°°°'°°°°'°°°°

Fig. 1. Model for the role of SAG in acquired resistance. The primary infection of tobacco by TMV is shown by the large primary (1 °) lesions on the top half of the leaf, while a secondary (2 °) infection by TMV is shown on the lower half of the leaf. Development of acquired resistance is illustrated by reduced size of the secondary lesion. See text for details.

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Mechanism of action of SA

As in animal and microbial systems, small biologically active compounds in plants such as phytohormones are believed to act through receptors. While it is still arguable whether or not SA is a hormone, particularly in light of the conflicting data concerning its translocation (see 'Is SA the translocated signal for SAR?'), it seems likely that identification of a cellular factor(s) which directly interacts with SA might shed light on SA's mode of action. This factor could be a receptor which perceives and transduces the SA signal or a SA- regulated cellular target such as an enzyme whose activity is altered by SA binding. Chen and Kles- sig [19, 20] have identified and characterized a soluble SA-binding protein (SABP) from tobacco which fits both descriptions of the factor.

The SABP is a 240-280 kDa complex which appears to be composed of four 57 kDa subunits. It has a binding affinity for SA (Kd 14/~M) which is consistent with the range of physiological concentrations of SA observed during the induction of defense responses. Its binding is also highly specific. Only those analogues of SA which are biologically active in the induction of PR genes and disease resistance (e.g. acetylsalicylic acid and 2,6-dihydroxybenzoic acid) effectively com- pete with SA for binding. Inactive analogues, though structurally very similar, are not bound by the SABP [19, 20].

Sequence analyses of the purified protein and a cDNA clone encoding the 57 kDa subunit indicated that SABP is highly homologus to catalase [21]. Indeed, SABP has catalase activity, as demonstrated by the ability of purified SABP to degrade H202 to H20 and 02. The size of the SABP complex and its subunits are also consistent with the structure of known catalases, which are composed of four identical or similar subunits of 50-60 kDa. SA was found to block SABP's catalase activity; it also inhibited the activity of catalases in crude tobacco leaf extracts. Further- more, the effectiveness of different SA analogues to inhibit the catalase activity correlated with both their abilities to bind SABP and to induce defense responses. These results argue that the binding of

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SA is responsible for inhibition of catalase activity which may lead to the induction of defense responses.

Although surprising, the model that SA acts by inhibition of catalase activity is consistent with additional findings by Chen et al. [21]. Since the production of H202 is an ongoing process in plants (see legend to Fig. 2 for details), as ex- pected, inhibition of catalase activity by treatment of tobacco plants with SA led to elevated H202 levels in vivo. Elevation of H202 levels by SA was as effective as that achieved by treatment with the plant and animal catalase inhibitor 3-amino- 1,2,4- triazole (3AT). Furthermore, when endogenous H202 levels were raised by injecting leaves with H202 or 3AT, PR-1 gene expression was induced. PR-1 gene expression was also induced by injection of compounds that promote H202 generation in vivo such as glycolate and paraquat. (Gly- colate is an intermediate in photorespiration which serves as a substrate for the generation of H202 by glycolate oxidase. Paraquat is a herbi- cide which is reduced in vivo. During its subsequent reoxidation superoxide anions are formed, which can be converted to H202 by superoxide dismutase [SOD].) In addition, the biologically inactive SA analogue 3-hydroxybenzoic acid, which differs from SA only in the placement of the hydroxyl group on the carbon ring, failed to induce either increases in H202 levels or PR-1 gene expression, consistent with its inability to bind SABP or inhibit its catalase activity. More- over, preliminary experiments indicated that treatment of plants with 3AT or paraquat enhanced resistance to TMV infection (Chen and Klessig, unpublished results) as has been previously demonstrated for SA [ 147]. Taken together, these results strongly suggest that SA acts by blocking catalase activity, which results in elevated H202 levels. Hydrogen peroxide, or another active oxygen species (AOS) derived from it, then activates defense-related genes on the pathway to disease resistance, perhaps by acting as a second messenger. However, it should be noted that PR genes were activated considerably less effectively with H202 treatment than with SA treatment (Chen, Conrath and Klessig, unpu-

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202

2e"

202

Defense - related gene induction Other Responses ?

s S ¢s

ss SS

(other AOS?) s S

s S

s S

"'" CA TALASE photorespiration | H ~ O ~ ~ ~- H20 + 1/2 02 fatty acid 13-oxidation =_ ~._

2, ~. ~NADPH ~ peroxidase ~ v~_~, oxidase ~ / t 2 H + RH2 N S A

o2 R + 2H20

Fig. 2. Metabolism of hydrogen peroxide in plants. Two major sources ofH202 are photorespiration and r-oxidation of fatty acids. H202 is also produced from superoxide anions ( 0 2 ) by SOD. The superoxide anion is formed by transfer of electrons (e - ) to molecular oxygen. These misdirected electrons often originate from electron transport during oxidative phosphorylation in the mitochondria and during photosynthesis in the chloroplasts. Membrane-bound, NADPH-dependent oxidases also generate H202. This is probably an important local source of H202 at the site of injury or infection. H202 can be converted to other AOS such as the highly reactive hydroxyl radical. H202, and other AOS derived from it, may be involved in a number of defense responses including the induction of defense-related genes such as the PR-1 genes. It may also lead to other responses yet to be defined. There are two major pathways involved in the breakdown of H202. As described in the text, H202 can be converted to H20 and 02 by catalase, a process that can be inhibited by SA. The other pathway involves breakdown of H202 by specific peroxidases either in the course of normal metabolism (e.g. in lignin formation) or via a specific pathway to eliminate toxic levels of H202 (e.g. Halliwell-Asada pathway). In addition, plant cells contain relatively high levels of glutathione, ascorbic acid and a-tocopherol which act as efficient AOS scavengers (for reviews see [12, 125]).

blished results; Ryals and Draper, personal com- munications). One explanation is that another signal in place of or in cooperation with H202 may be involved. Alternatively, the exogenously applied H202 may be too rapidly destroyed by catalases and oxidant scavengers to act as an effective inducer.

The participation of catalase and H202 in disease resistance is supported by several recent findings. Tobacco plants were constructed which constitutively express a copy of the catalase gene in an antisense orientation. These transgenic plants had reduced catalase levels and synthesized PR-1 proteins constitutively. In addition, there was a good correlation between the level of reduction in catalase and the amount of increased resistance to TMV in these transgenic lines (Chen and Klessig, unpublished results). Furthermore, several abiotic inducers of PR genes and disease resistance such as thiamine-HCl, polyacrylic acid,

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a-amino butyric acid, and BaC12 appear to act through SA since they induced the synthesis of SA and SAG in treated tobacco. One exception was 2,6-dichloroisonicotinic acid (INA) which did not stimulate production of SA and SAG [76]. In contrast, INA was found to bind and inhibit tobacco catalases, suggesting that its mode of action is similar to that of SA (Conrath, Chen, and Klessig, unpublished results). These results, in addition to the previous observations, strongly argue that SA and a broad range of defense- inducing compounds act by directly or indirectly modulating catalase activity.

Increases in the levels of AOS such as H202, superoxide anions or hydroxyl radicals have previously been linked to a number of processes associated with defense responses (for review see [125]). A large increase in the level of AOS, referred to as an oxidative or respiratory burst, oc- curs within minutes to hours after infection of

plants by various pathogens which will initiate the HR (e.g. [36, 37]). The rapid kinetics of this oxidative burst diminishes the possibility that it re- suits from SA inhibition of catalase during the initial infection, since elevated SA levels are not detected until 8-24 h or more after infection, de- pending on the system [74, 85, 100, 120, 126]. In contrast, during a secondary infection when free SA might be rapidly produced by hydrolysis of stored SAG, an oxidative burst might be mediated in part by SA.

Regardless, previous studies provide evidence that AOS can act as signals to elicit various defense responses. It has been suggested that the oxidative burst plays a role in killing the invading pathogen [60]. This has parallels to the immune response in mammals where phagocytic cells such as macrophages and neutrophils engulf their bacterial prey in a phagocytic vacuole where high concentrations of superoxide anion, H202, and other AOS can be generated. There is also evidence that AOS causes host plasma membrane damage [61] and cell death during the HR in plants [35-37]. In addition, increased lignifica- tion at the site of infection requires H202, as does the oxidative crosslinking of cell wall proteins in bean and soybean suspension culture cells after treatment with a fungal elicitor [14]. SA was found to induce this crosslinking, which can be explained by SA's ability to inhibit catalase's H202-scavenging activity. This could also explain SA's activation of superoxide dismutase (SOD) genes [11], as these genes are known to be induced by accumulation of AOS (for review see [12]). Finally, H202 may act as a second messenger for phytoalexin synthesis that accompanies the oxidative burst induced by fungal elicitor treatment of soybean suspension culture cells [6, 66].

Gene activation by H202 has been described in animal systems. Perhaps the best example involves the transcription factor NF-xB which me- diates expression of genes associated with inflammatory, immune, and allergic responses. NF-xB exists in the cytoplasm in an inactive form, com- plexed with its inhibitory subunit I xB. H202- mediated activation results in dissociation of NF-

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xB from I xB. This allows NF-~cB to enter the nucleus and activate genes by binding to specific motifs in promoter and enhancer elements (for review see [117]). Thus, there appear to be similar conditions (e.g. stress) in both animals and plants in which AOS are employed as second messengers. However, while activation of NF- xB-IxB complex does not require protein synthesis, the H202-mediated activation of the PR-1 genes by SA is inhibited by cycloheximide [130], suggesting that the mechanism of gene activation may be different.

SA signal transduction pathway(s)

Several other approaches are in progress to iden- tify the various components of the SA signal transduction pathway(s). A popular approach is to first define the cis-acting elements in the target gene that are necessary for response to the signal and then to use these sequences to isolate the trans-acting factors. Currently, the cis-acting sequences of several SA-inducible genes have been partially analyzed by fusing various lengths of the promoter region to the uidA (GU S) reporter gene. Transgenic tobacco plants containing the chimeric genes were then constructed and analyzed. In some cases the presumptive SA-responsive region was fused to a minimal promoter, i.e. the core 35 S promoter. The construct was then tested to determine if that region could confer SA inducibility to the heterologous promoter.

Of the SA-inducible promoters, the tobacco PR- la promoter has been the most extensively characterized. 5' end deletion analysis by Van de Rhee et al. [ 139] indicated that an important SA- responsive element was located between 643 and 689 bp upstream of the transcriptional start site. Moreover, fusion of the -625 to -902 region to the core 35S promoter imparted SA inducibility. Similarly, Uknes and co-workers [130] demonstrated that 661 bp 5' of the transcription start site were sufficient to maintain SA inducibility while 318 bp were not. Beilmann etal. [7] also concluded that 335 bp of 5'-flanking DNA was insufficient to preserve SA responsiveness. In

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contrast, Ohashi and colleagues [90] observed a low level of induction of a chimeric gene containing only ca. 300 bp of 5'-flanking DNA and concluded that this region contained a SA-responsive element.

While the consensus from these studies is that one or more elements located between ca. -300 and -700 in the PR-1 a promoter are required for high-level induction by SA, more recent studies indicated that the situation is more complex. Ohashi and coworkers [50] identified two independent binding sites at -37 to -61 and -168 to -179 for protein factors present in nuclear ex-

tracts from uninfected, and thus non-PR-express- ing, N. tabacum cv. Samsun NN. In contrast, the factor was not detected with nuclear proteins from the interspecific hybrid of N. glutinosa × N. debneyi, which constitutively produces the PR-1 proteins [2, 89] and high levels of SA [155]. This result suggested that the PR- l a gene is negatively regulated through these two elements and their binding proteins. Although the two elements do not contain a common sequence, competition studies suggest that the same factor binds both. From an analysis of transgenic plants carrying fusion constructs of various segments of the PR- l a promoter with the core 35S promoter and G U S reporter gene, Van de Rhee and Bol [ 137] concluded that the PR- l a promoter contains a minimum of four regulatory elements located between nucleotides -902 to -691 (element 1), -689 to -643 (element 2), -643 to -287 (ele-

ment 3) and -287 to + 29 (element 4). In this study, these interacting elements appeared to function in a context-dependent manner, with elements 1, 2, and 3 functioning as positive regulators. Element 4 appeared to be important for maintaining the correct spacing between the more 5' elements and the transcriptional start site. In all of the studies described in this section, each PR-1 a promoter construct responded similarly to SA treatment and TMV infection, consistent with the hypothesis that SA is involved in the induction of PR-1 genes by infection.

The tobacco PR-2 genes, which encode the acidic /%l,3-glucanases, have also been characterized. By 5' deletion analysis of the PR-2d pro-

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moter, Hennig et al. [52] mapped a cis element necessary for high-level SA inducibility between -321 and -607. Fusion of various segments of

this promoter to the core 35S promoter suggested that there are at least two positive regulatory elements between nucleotides -318 and -607 that confer SA responsiveness (Shah and Klessig, unpublished results). 5' deletion analysis of the PR-2b promoter demonstrated that as little as ca. 300 bp of 5'-flanking sequences were sufficient for low-level induction by SA (about 2-fold to 3-fold) while an additional 350 bp of 5'-flanking DNA were required for high-level induction [ 138 ]. Thus, in all three promoters (PR- 1 a, PR-2b and PR-2d) several regulatory elements appear to be involved in SA responsiveness and TMV inducibility.

The promoters of several other defense-related genes including those for the tobacco PR-5 protein [3] and glycine-rich protein [139] and the Arabidopsis thaliana acidic chitinase (PR-3 [ 115 ]) have been similarly, though less extensively, characterized. Sequence analysis of the 5' untran- scribed region of a barley fl-1,3-glucanase identified a 10 bp motif (TCATCTTCTT) which is repeated several times [49]. This TCA motif is present in over 30 different plant genes which are known to be induced by various forms of stress including infection. A 40 kDa tobacco nuclear protein binds the TCA motif, and this binding activity is enhanced in nuclear extracts from SA- treated plants. Both the tobacco PR- l a and PR-2d genes contain multiple copies of the TCA motif in their 5' ends. However, the minimum segments of these two promoters which confer SA inducibility to the heterologous core 35S promoter (i.e. -643 to -689 for PR- l a and -318 to -607 for PR-2d) do not carry the TCA motif. Moreover, there is as yet no evidence that the TCA motif, or concatemers of it, can confer SA responsiveness to a heterologous promoter. Thus, it is unclear what role this motif plays in SA inducibility.

Promoters of other genes such as alternative oxidase [103, 104], manganese SOD [11], and nopaline synthase [62] and the 35S promoter of CaMV [93a] are also stimulated by SA. In the

35S promoter SA responsiveness is mediated by the AS1 element. This element can also confer SA inducibility when fused to the ribulose bis- phosphate carboxylase small subunit promoter. Qin and co-workers [93a] have reported modest activation of the core 35S promoter by SA in transgenic tobacco carrying a chimeric 35S:GUS reporter gene. At the RNA level induction was ca. 20-fold; however, when enzyme activity was as- sayed induction was only 3- to 5-fold. This inducing was very rapid, peaking by four hours after treatment, and was not sensitive to cycloheximide. However, other investigators have used the core 35S promoter, which contains one copy of the AS1 element, as a minimal heterologous promoter for fusion with potential SA-responsive regions from the PR-1 and PR-2 promoters. The core 35S promoter alone served as a negative control in these experiments, exhibiting only weak (about 2-fold), if any, stimulation by SA as mea- sured by GUS activity [52, 90, 137, 139].

The genetic approach to defining components in a signalling pathway is very powerful and has been used extensively in bacteria, yeast, and Drosophila. This approach is also being applied to plants, particularly Arabidopsis thaliana which has a relatively rapid generation time, a small well- mapped genome, and the ability to be transformed (for review see [87]). Until recently, little was known about the pathology of this weed, but in the past few years a number of bacterial [31, 32, 123, 129, 146], fungal [63, 79a], viral [33, 56, 67, 82, 122, 126a], and nematode [119] pathogens of Arabidopsis have been identified. The cloning of several genes conferring resistance to some of these pathogens is in progress (e.g. [32, 64]).

As in tobacco and cucumber, SA appears to be involved in the defense responses of Arabidopsis to pathogen attack. PR genes were induced by SA treatment of Arabidopsis [ 131 ] and infection by turnip crinkle virus of the resistant ecotype Dijon lead to elevated levels of endogenous SA ([132]; Dempsey, Wobbe and Klessig, unpublished results). In addition, Arabidopsis contains a SA-binding activity and a SA-inhibitable catalase activity (Sgmchez-Casas and Klessig, unpublished results). Finally, transgenic Arabidopsis plants

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which constitutively synthesize salicylate hydroxylase exhibited enhanced susceptibility to several pathogens (Uknes and Ryals, personal communication). Thus, Arabidopsis should provide an excellent opportunity to define components of the SA signal transduction pathway through genetic analysis. A number of mutants have already been isolated that may have lesions in this pathway [34a, 49a, 49b, 646].

Perspective

It is now clear that SA is an essential signal in development of SAR in several plant species and that this signal may be mediated, in part, by SA's ability to bind and inhibit the activity of catalases. However, many unanswered questions remain. Does SA play a role in the initial restriction of the pathogen? If so, is a similar mechanism involved as in the induction of SAR? Although there is good evidence for SA's involvement in defense responses in dicots (e.g. tobacco, cucumber, Ara- bidopsis), does it also participate in the defense of monocots, particularly the important grain crops?

It will be interesting to see whether all of the actions of SA in plant signal transduction are mediated by its inhibition of catalases or whether there are additional undiscovered modes of action. In animals salicylates appear to have multiple modes of action since these compounds exert a wide range of clinical effects including reduction of pain, fever, inflammation, blood clotting, and the risk of heart attacks and strokes. Aspirin inhibits the synthesis of prostaglandins from arachidonic acid, a fatty acid constituent of animal cell membranes [140]. Prostaglandins are potent compounds that can affect pain reception and can induce fever, swelling, platelet aggregation, and vasoconstriction. More recently, aspirin and aspirin-like compounds have also been found to perturb cell-cell communication, such as platelet aggregation and neutrophil activation. This perturbation may result from interference with G protein-mediated signal transduction and may account, in part, for aspirin's anti-clotting and anti-inflammatory activities (for review see [ 145]).

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Recent studies suggest that SA may also have alternate mechanisms of action in plants. For example, although SA is present at high levels in rice [98], little, if any, SA-binding activity was detectable in this plant. In addition, rice catalase activity was not substantially inhibited by SA (S/mchez-Casas and Klessig, unpublished re- suits). Thus, if SA is involved in pathways leading to disease resistance in rice, it probably is via an alternative mechanism. If SA is shown to have multiple modes of action in plants, it will be of interest to determine which processes (e.g. disease resistance and thermogenesis) are regulated by the different modes of action.

Another area that deserves further attention is the metabolism of SA. Although some of the steps in the pathway(s) are becoming clear and several of the enzymes are currently being characterized, much remains to be done. The enzymes have yet to be obtained in pure form and their respective genes have not been cloned. Relatively little is known concerning the regulation of these enzymes and their corresponding genes. Since synthesis of SA branches off the phenylpropanoid biosynthetic pathway, it is possible that SA increases are mediated by elevated levels of PAL [143]. PAL is a key enzyme in the phenylpropanoid pathway and is induced during defense responses in many plants [49c]. Ward and colleagues [143] have speculated on the possible connection between necrosis during HR and SA biosynthesis. Necrosis has been linked to induction of PAL [8, 38], and hence may actually precede SA production and subsequent development of SAR. Interestingly, an oxidative burst appears to be involved in formation of necrotic lesions. Doke and Ohashi [37] found that when TMV- infected tobacco were shifted from 30 °C (where viral replication and spread is not inhibited) to a lower temperature (20 ° C) which allows the host to restrict viral replication and spread and form lesions, there was a rapid oxidative burst occur- ring from ten minutes to four hours after the shift. Infiltration of TMV-infected leaves with catalase, SOD or NADP ÷ caused a reduction in lesion formation following the temperature shift. Thus, an early oxidative burst may be required for HR,

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which in turn may be necessary for production of SA, which then inhibits catalase activity leading to the second period of high AOS levels. Clearly more experiments are needed to address the re- lationships between these pathogen-induced processes.

In addition to the biosynthesis of SA, much remains to be learned about its catabolism. For example, the contribution of preformed stores of SAG to the SA pool during a secondary infection has not been established. In addition, the possible involvement of other, yet to be identified, forms of SA needs further investigation.

One of the most challenging problems will be defining the details of the SA signal transduction pathway(s). Some progress has been made with the identification and characterization of the SABP, the definition of several SA-responsive regulatory regions in the promoters of a number of defense-related genes, and the identification of potential trans-acting factors. However, our present picture represents only a rough outline of thepathway. In the future, the isolation and characterization of mutants in Arabidopsis will play an important role in deciphering this puzzle. Dissec- tion of this complex pathway can also be facili- tated by identifying multiple markers for different steps. Few markers now exist. In tobacco TMV infection leads to SA and ethylene production, but no intervening marker events are known ex- cept for the temperature sensitive step that pre- cedes both ethylene and SA synthesis [75, 136, 156]. Dissection of downstream events are now possible due to the existence of both ethylene inhibitors and nahG transgenic plants, which selec- tively block responses to ethylene and SA, re- spectively. To provide further markers, Malamy et al. [76] tested chemical inducers of SAR and other defense responses and found that many, but not all, induce SA production. Three of the tested chemicals, polyacrylic acid, thiamine-HC1, and 2,6-dichloroisonicotinic acid (INA), enter the pathway at different points. Studies of the effects of these and other chemicals should allow their positioning in the pathway with respect to each other and the known marker events. Not only will chemical inducers aid in establishing the order of

events, they will also provide a criteria for the identification of mutants defective at different steps in the signal transduction pathway. This combined biochemical and genetic approach should prove very powerful.

The complete signal transduction pathway(s) involving SA is likely to be highly complex. There may be multiple pathways leading to increased biosynthesis of SA. The isolation and characterization of plant disease resistance genes for sev- ern plant-pathogen systems that utilize SA (e.g. N locus in tobacco for TMV resistance; Baker, personal communication) should address this question and facilitate an understanding of how the SA signalling pathway is initiated. The SA signalling pathway itself may branch. There is likely to be extensive interplay between the SA signalling pathway and other pathways such as those for ethylene and jasmonic acid. For example, SA inhibits synthesis of ethylene in several suspension cell cultures including carrot [ 111 ], pear [69] and apple [108]. SA also blocks jasmonic acid biosynthesis [91a] and jasmonic acid induction of proteinase inhibitor synthesis in tomato (Ryan, personal communication). In contrast, pretreatment with SA potentiates or en- hances phytoalexin production and incorporation of cell wall phenolics induced by suboptimal levels of a fungal elicitor in parsley suspension cells, although SA alone does not induce phytoalexin [58]. Signals from other pathways are also likely to both positively and negatively impact the SA pathway. For instance, ethylene appears to po- tentiate the SA-mediated induction of genes encoding acidic PR proteins in certain plants [64a]. Raz and Fluhr's [ 101 ] observation that inhibitors of ethylene action or biosynthesis blocked the induction of the acidic PR-3 chitinase by SA also suggest that ethylene and SA may act in concert.

A likely application of our emerging understanding of SA's role and mechanism of action in plant defense is crop protection. Enhanced disease resistance potentially can be achieved through the manipulation of SA metabolism. Al- ternatively, altering the reception of the SA signal or mimicking its effect through the use of other synthetic compounds may hold promise. The re-

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cent studies with INA suggest that the latter is a viable approach [83, 126, 131, 132, 143]. Finally, given the broad physiological effects of SA (and aspirin) in animal systems, insight into SA's mode of action(s) in plants may have implications be- yond the plant world.

Acknowledgements

We would like to thank the investigators who contributed unpublished data to this review and members of the laboratory, particularly D'Maris Dempsey, for helpful comments and criticisms. Marline Boslet is gratefully acknowledged for as- sistance with the preparation of the manuscript. Our studies described in this review were partially supported by grants DCB-9003711 and MCB- 9310371 from the National Science Foundation and 92-37301-7599 from the Department of Ag- riculture to D.F.K. and by a Benedict-Michael Predoctoral Fellowship to J.M.

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