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Salicylic Acid Is a Modulator of Tobacco and Mammalian Catalases*

  • Jörg Durner
    Affiliations
    Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, P. O. Box 759, Piscataway, New Jersey 08855
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  • Daniel F. Klessig
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, P. O. Box 759, Piscataway, New Jersey 08855
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  • Author Footnotes
    * This work was supported, in part, by National Science Foundation Grants MCB-9310371 and MCB-9514239. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:November 08, 1996DOI:https://doi.org/10.1074/jbc.271.45.28492
      Salicylic acid (SA) plays a key role in the establishment of resistance to microbial pathogens in many plants. The discovery that SA inhibits catalase from tobacco led us to suggest that H2O2 acts as second messenger to activate plant defenses. Detailed analyses of SA's interaction with tobacco and mammalian catalases indicate that SA acts as an electron donor for the peroxidative cycle of catalase. When H2O2 fluxes were relatively low (1 μM/min or less), SA inhibited catalase, consistent with its suggested signaling function via H2O2. However, significant inhibition was only observed at 100 μM SA or more, a level reached in infected, but not in uninfected, leaves. This inhibition was probably due to siphoning catalase into the slow peroxidative reaction. Surprisingly, SA was also able to protect catalase from inactivation by damaging levels of H2O2 (lower millimolar range), which is generally assumed to reflect accumulation of inactive ferro-oxy intermediates. SA did so by supporting or substituting for the protective function of catalase-bound NADPH. These results add new features to SA's interaction with heme enzymes and its in vivo redox properties. Thus, SA, in addition to its proposed signaling function, may also have an important antioxidant role in containing oxidative processes associated with plant defense responses.

      INTRODUCTION

      Vertebrate animals possess a novel and highly specific immune system that acts as a defense against disease. Plants react to pathogen attack by activating elaborate defense mechanisms, which are much more poorly characterized than the vertebrate immune system. These defense mechanisms are activated not only at the sites of infection, which are manifested, in part, as necrotic lesions (hypersensitive response; HR),
      The abbreviations used are: HR
      hypersensitive response
      SAR
      systemic acquired resistance
      PR
      pathogenesis-related
      INA
      2,6-dichloroisonicotinic acid
      PAGE
      polyacrylamide gel electrophoresis
      SA
      salicylic acid
      AS
      antisense.
      but also in neighboring and even distal uninfected parts of the plant, leading to systemic acquired resistance (SAR). Both HR and SAR are associated with induction of a large number of defense-related genes. The products of these genes may play important roles in the restriction of pathogen growth and spread either indirectly, by participating in strengthening host cellular structures, or directly, by providing antimicrobial activities (for review see Ryals et al. (
      • Ryals J.
      • Uknes S.
      • Ward E.
      ) and Dempsey and Klessig (
      • Dempsey D.A.
      • Klessig D.F.
      )). During the HR, generation of reactive oxygen species (oxidative burst) precedes formation of necrotic lesions, which result from host cell death (Doke and Ohashi (
      • Doke N.
      • Ohashi Y.
      ) and Levine et al. (
      • Levine A.
      • Tenhaken R.
      • Dixon R.
      • Lamb C.
      ); for review see Mehdy (
      • Mehdy M.
      )). Additionally, defense responses in surrounding cells become activated, which include synthesis of phytoalexins, pathogenesis-related (PR) proteins, and cell wall polymers such as lignin (Dempsey and Klessig,
      • Dempsey D.A.
      • Klessig D.F.
      ). Establishment of SAR results in enhanced and long lasting resistance to secondary challenge by the same or even an unrelated pathogen and is associated with activation of PR genes (Ryals et al.,
      • Ryals J.
      • Uknes S.
      • Ward E.
      ; Dempsey and Klessig,
      • Dempsey D.A.
      • Klessig D.F.
      ). The detailed sequence of molecular events required for the initiation and regulation of HR and SAR is unknown, but progress has been made in identifying several components of the signal transduction pathways leading to disease resistance, among them salicylic acid (SA; for review see Staskawicz et al. (
      • Staskawicz B.J.
      • Ausubel F.M.
      • Baker B.J.
      • Ellis J.G.
      • Jones J.D.G.
      ) and Dangl (
      • Dangl J.L.
      )).
      SA is present in many plants. While the healing benefits of plants containing high levels of SA have been known since antiquity, the first insights regarding SA's role in plants have emerged only during the past decade. A mounting body of evidence has accumulated that indicates that SA plays an important role in plant defense responses (for review see Ryals et al. (
      • Ryals J.
      • Uknes S.
      • Ward E.
      ); Dempsey and Klessig (
      • Dempsey D.A.
      • Klessig D.F.
      )). White (
      • White R.F.
      ) was the first to demonstrate that application of exogenous SA or acetylsalicylic acid (aspirin) to tobacco induces PR gene expression and partial resistance to pathogens such as tobacco mosaic virus. Endogenous levels of SA increase dramatically after tobacco mosaic virus inoculation of resistant, but not susceptible, tobacco cultivars and parallel the induction of PR genes (Malamy et al.,
      • Malamy J.
      • Carr J.P.
      • Klessig D.F.
      • Raskin I.
      ). In addition, SA induces the same set of nine genes that are activated systemically by tobacco mosaic virus infection (Ward et al.,
      • Ward E.R.
      • Uknes S.J.
      • Williams S.C.
      • Dincher S.S.
      • Wiederhold D.L.
      • Aleander D.C.
      • Al-Goy P.
      • Metraux J.P.
      • Ryals J.A.
      ). In cucumber, SA levels rise in the phloem of tobacco necrosis virus-, or Colletotrichum lagenarium-, infected leaves before development of SAR in distal tissues (Métraux et al.,
      • Métraux J.P.
      • Signer H.
      • Ryals J.
      • Ward E.
      • Wyss-Benz M.
      • Gaudin J.
      • Raschdorf K.
      • Schmid E.
      • Blum W.
      • Inverardi B.
      ). Arabidopsis mutants that develop spontaneous lesions and express genes associated with HR and SAR also have elevated levels of SA (Dietrich et al.,
      • Dietrich R.A.
      • Delaney T.P.
      • Uknes S.J.
      • Ward E.R.
      • Ryals J.A.
      • Dangl J.L.
      ; Greenberg et al.,
      • Greenberg J.T.
      • Guo A.
      • Klessig D.F.
      • Ausubel F.M.
      ). Finally, tobacco mosaic virus-infected transgenic tobacco plants, which express the nahG gene that encodes the SA-metabolizing enzyme salicylate hydroxylase from Pseudomonas putida, accumulate little or no SA, fail to establish SAR, and develop viral lesions that are larger than those produced on wild type plants (Gaffney et al.,
      • Gaffney T.
      • Friedrich L.
      • Vernooij B.
      • Negrotto D.
      • Nye G.
      • Uknes S.
      • Ward E.
      • Kessmann H.
      • Ryals J.
      ).
      To investigate how SA might function in plant defense responses, our laboratory has focused on the identification of cellular factors with which SA directly interacts. We have suggested that one mechanism of SA action is to inhibit catalase, thereby elevating endogenous levels of H2O2, which result either from the oxidative burst associated with the HR or from metabolic processes such as photorespiration, photosynthesis, and oxidative phosphorylation (Chen et al.,
      • Chen Z.
      • Silva H.
      • Klessig D.F.
      ,
      • Chen Z.
      • Malamy J.
      • Hennig J.
      • Conrath U.
      • Sanchez-Casas P.
      • Ricigliano J.
      • Silva H.
      • Klessig D.F.
      ). According to this working hypothesis, the elevated H2O2 or other reactive oxygen species derived from it would activate plant defense-related genes such as the PR-1 genes. This mode of activation of plant defenses has been compared with the induction of genes associated with mammalian immune, inflammatory, and acute phase responses that are mediated through H2O2 activation of the transcription factor NF-κB (Schmidt et al.,
      • Schmidt K.N.
      • Amstad P.
      • Cerutti P.
      • Baeuerle P.A.
      ). In support of this model, we have found that (i) 2,6-dichloroisonicotinic acid (INA; a synthetic inducer of PR genes and enhanced resistance) and its biologically active, but not inactive, analogues also inhibit tobacco catalase in vivo (Conrath et al.,
      • Conrath U.
      • Chen Z.
      • Ricigliano J.W.
      • Klessig D.F.
      ); (ii) INA as well as SA and its biologically active analogues inhibit the other major H2O2-scavenging enzyme, ascorbate peroxidase, but not guaiacol peroxidases (Durner and Klessig,
      • Durner J.
      • Klessig D.F.
      ), and (iii) antioxidants block the action of SA and INA (Conrath et al.,
      • Conrath U.
      • Chen Z.
      • Ricigliano J.W.
      • Klessig D.F.
      ).
      However, currently there is considerable controversy about the involvement of catalase inhibition by SA and the subsequent increase of H2O2 in plant defense responses. Several recent reports have indicated that H2O2 is unlikely to be involved in PR gene induction or SAR. Inhibition of catalase in leaf extracts requires concentrations of SA far above those observed in uninfected tissues (Bi et al.,
      • Bi Y.M.
      • Kenton P.
      • Mur L.
      • Darby R.
      • Draper J.
      ; Chen et al.,
      • Chen Z.
      • Silva H.
      • Klessig D.F.
      ). In addition, while H2O2 and H2O2-inducing chemicals activate PR-1 genes in wild type tobacco, there is little or no gene induction in NahG plants. Therefore, it has been concluded that H2O2 induction of SAR genes requires SA rather than the reverse (Neuenschwander et al.,
      • Neuenschwander U.
      • Vernooij B.
      • Friedrich L.
      • Uknes S.
      • Kessmann H.
      • Ryals J.
      ). Indeed, it has been shown that very high levels of H2O2 stimulate SA biosynthesis (León et al.,
      • León J.
      • Lawton M.A.
      • Raskin I.
      ; Neuenschwander et al.,
      • Neuenschwander U.
      • Vernooij B.
      • Friedrich L.
      • Uknes S.
      • Kessmann H.
      • Ryals J.
      ; Summermatter et al.,
      • Summermatter K.
      • Sticher L.
      • Métraux J.-P.
      ). Taken together, these results suggest that H2O2 does not function downstream of SA (i.e. by inhibition of catalase) in the regulation of PR genes.
      However, catalase is still the subject of many mechanistic investigations. There is increasing evidence that catalase is a major factor in a variety of pathological states such as cancer, diabetes, aging, and oxidative stress (see DeLuca et al. (
      • DeLuca D.C.
      • Dennis R.
      • Smith W.G.
      ); Feuers et al. (
      • Feuers R.J.
      • Patillo F.M.
      • Osborn C.K.
      • Adams K.L.
      • DeLuca D.
      • Smith W.G.
      )). Inactivation and reactivation of catalase in vivo and in vitro are far from being fully understood. Numerous recent publications suggest new approaches regarding in vitro assays and inhibition studies on catalase (Feuers et al.,
      • Feuers R.J.
      • Patillo F.M.
      • Osborn C.K.
      • Adams K.L.
      • DeLuca D.
      • Smith W.G.
      ; DeLuca et al.,
      • DeLuca D.C.
      • Dennis R.
      • Smith W.G.
      ; Ou and Wolff,
      • Ou P.
      • Wolff S.P.
      ,
      • Ou P.
      • Wolff S.P.
      ; Escobar et al.,
      • Escobar J.A.
      • Rubio M.A.
      • Lissi E.A.
      ; Hook and Harding,
      • Hook D.W.A.
      • Harding J.J.
      ). In the present report, we provide new insights into SA's effects on catalase. SA acts as an electron donor for the peroxidative cycle of both plant and animal catalases. As such, it can protect as well as inhibit catalase activity, depending on the concentration of H2O2. It is hypothesized that, in healthy tissue of infected leaves where H2O2 levels are low, SA inhibits catalase, which could lead to activation of defense-related genes. In contrast, in infected cells and in tissue immediately adjacent to necrotizing cells, where high levels of H2O2 and other reactive oxygen species are produced, SA protects catalase from inactivation. This property of SA might serve to contain the oxidative damage associated with spread of the lesion and resembles closely some antioxidative properties of SA in activated HeLa cells or inflamed mammalian tissues, which are unrelated to inhibition of prostaglandin H synthase.

      DISCUSSION

      Catalase was one of the first enzymes to be purified and crystallized (see Schonbaum and Chance,

      Schonbaum, G. R., Chance, B., (1976) The Enzymes, (Boyer, P. D., ed), 2nd Ed., Vol. 13, pp. 363–408, Academic Press, Inc., New York.

      ). However, despite extensive biophysical, biochemical, and genetic analyses, there is an ongoing discussion as to whether the only, or even major, role of this very abundant protein is to convert H2O2 to H2O and O2 (its catalatic or α activity). This may, in part, reflect the complexity of catalase's redox chemistry. In recent years, catalase has gained renewed attention. There is increasing interest in the involvement of oxidative stress in environmental pollution, aging, diabetes, cancer, and other human diseases and in catalase's role as one of the main antioxidative enzymes. In particular, this has led to renewed interest in the mechanism of catalase inhibition and inactivation (Feuers et al. (
      • Feuers R.J.
      • Patillo F.M.
      • Osborn C.K.
      • Adams K.L.
      • DeLuca D.
      • Smith W.G.
      ), Hillar et al. (
      • Hillar A.
      • Nicholls P.
      • Switala J.
      • Loewen P.C.
      ), DeLuca et al. (
      • DeLuca D.C.
      • Dennis R.
      • Smith W.G.
      ), Ou and Wolff (
      • Ou P.
      • Wolff S.P.
      ,
      • Ou P.
      • Wolff S.P.
      ), Escobar et al. (
      • Escobar J.A.
      • Rubio M.A.
      • Lissi E.A.
      ), and references therein).

      SA Inhibits All Catalase Isoforms from Tobacco Leaves

      In plants, catalase is encoded by a small gene family consisting of several classes (Ni and Trelease,
      • Ni W.
      • Trelease R.N.
      ; Scandalios,
      • Scandalios J.G.
      ; Willekens et al.,
      • Willekens H.
      • Inzé D.
      • Van Montagu M.
      • Van Camp W.
      ). This leads to multiple isoforms of the enzyme, since individual subunits encoded by different family members can homo- or heterotetramerize (Ni and Trelease,
      • Ni W.
      • Trelease R.N.
      ; Scandalios,
      • Scandalios J.G.
      ; Mullen and Gifford,
      • Mullen R.T.
      • Gifford D.J.
      ). Isozyme distribution and tissue-specific expression of catalase genes is being actively investigated (Willekens et al.,
      • Willekens H.
      • Inzé D.
      • Van Montagu M.
      • Van Camp W.
      ; Havir et al.,
      • Havir E.A.
      • Brisson L.F.
      • Zelitch I.
      ).
      In tobacco (N. tabacum), 6-12 isozymes have been detected by activity staining on isoelectric focusing gels (Zelitch et al.,
      • Zelitch I.
      • Havir E.A.
      • McGonigle B.
      • McHale N.A.
      • Nelson T.
      ; Siminis et al.,
      • Siminis C.I.
      • Kanellis A.K.
      • Roubelakis-Angelakis K.A.
      ). Given the multiplicity of isozymes in tobacco, we wanted to know, first, whether heterotetramers were primarily responsible for this diversity and, second, whether the various isozymes had different sensitivity to SA. We found that the majority of the 10 or more isoforms in tobacco leaves were homotetramers rather than a mixed population of heterotetramers (Fig. 1, Fig. 2). The nature of the probable posttranslational modification(s) responsible for the differences in charge among isoforms is not known. Alternatively, some of the forms may result from in vitro modifications such as oxidation of sulfhydryl groups, which may have occurred during purification, handling, and storage (Mörikofer-Zwez et al.,
      • Mörikofer-Zwez
      • von Wartburg J.P.
      • Aebi H.
      ). However, the similar number of forms seen in the crude extracts (Fig. 2) suggests that most of these represent true isoforms rather than in vitro artefacts. The different forms present in purified tobacco catalase exhibited similar sensitivity to SA (36-51% inhibition by 1 mM SA, Fig. 1C). This level of inhibition, while slightly lower, is similar to that reported previously for crude tobacco leaf extracts (45-70%; Chen et al.,
      • Chen Z.
      • Silva H.
      • Klessig D.F.
      ; Sánchez-Casas and Klessig,
      • Sá;nchez-Casas P.
      • Klessig D.F.
      ; Bi et al.,
      • Bi Y.M.
      • Kenton P.
      • Mur L.
      • Darby R.
      • Draper J.
      ). However, while we could not detect a tobacco catalase insensitive to SA, there is evidence for insensitive catalase in rice (Sánchez-Casas and Klessig,
      • Sá;nchez-Casas P.
      • Klessig D.F.
      ).
      Z. Chen and D. F. Klessig, unpublished results.
      Furthermore, SA did not inhibit purified maize catalase (Guan and Scandalios,
      • Guan L.
      • Scandalios J.
      ).

      Mechanism of SA Action on Tobacco and Mammalian Catalases

      The biphasic kinetics of catalase inhibition by SA (Fig. 4) have been reported for other phenolic compounds such as hydroquinone and pyrogallol and have been interpreted as the transition from the fast catalatic or α activity to the slow peroxidative or β activity (Goldacre and Galston (
      • Goldacre P.L.
      • Galston A.W.
      ), Ogura et al. (
      • Ogura Y.
      • Tonomura Y.
      • Hino S.
      • Tamiya H.
      ); reviewed by Schonbaum and Chance (

      Schonbaum, G. R., Chance, B., (1976) The Enzymes, (Boyer, P. D., ed), 2nd Ed., Vol. 13, pp. 363–408, Academic Press, Inc., New York.

      ); see Fig. 3). This type of inhibition is clearly different from the time-dependent and mechanism-based processes caused by inhibitors such as aminotriazole (Schonbaum and Chance,

      Schonbaum, G. R., Chance, B., (1976) The Enzymes, (Boyer, P. D., ed), 2nd Ed., Vol. 13, pp. 363–408, Academic Press, Inc., New York.

      ). Further evidence that SA acts as a typical phenolic by stimulating the peroxidative activity of tobacco catalase at the expense of its catalatic activity was provided by spectral analysis of catalase and its reaction intermediates (Fig. 6). Together, these analyses indicate that SA acts as an electron donor for the enzyme intermediates compound I and compound II. This is consistent with previous studies, which demonstrated that phenolics can reduce compound I to compound II and compound II to the ferric enzyme (reviewed by Deisseroth and Dounce (
      • Deisseroth A.
      • Dounce A.L.
      ); Schonbaum and Chance (

      Schonbaum, G. R., Chance, B., (1976) The Enzymes, (Boyer, P. D., ed), 2nd Ed., Vol. 13, pp. 363–408, Academic Press, Inc., New York.

      )) and the more recent reports that SA or aspirin can act as an electron donor for myeloperoxidase and horseradish peroxidase (Kettle and Winterbourn,
      • Kettle A.J.
      • Winterbourn C.C.
      ; Durner and Klessig,
      • Durner J.
      • Klessig D.F.
      ). In other words, SA inhibits catalase by acting as a one-electron donor that siphons compound I from the extremely fast catalatic cycle (see Fig. 3) into the relatively slow peroxidative cycle (∼1000 times slower) (Havir and McHale,
      • Havir E.
      • McHale N.A.
      ; Zamocky et al.,
      • Zamocky M.
      • Herzog Ch.
      • Nykyri L.M.
      • Koller F.
      ) by promoting the formation of compound II. It is noteworthy that Lück (
      • Lück H.
      ) and Itoh et al. (
      • Itoh M.
      • Nakamura Y.
      • Shibata K.
      ), as part of their studies on the effects of carboxylic acids on catalase, were the first to demonstrate that SA (at extremely high levels of ≥10 mM) inhibited mammalian catalases and to speculate that the inhibition probably resulted from promotion of the peroxidative reaction rather than from chelation of the heme iron of catalase as has been suggested by others (e.g. Rüffer et al.,
      • Rüffer M.
      • Steipe B.
      • Zenk M.H.
      ). Very recently, Russell and Sternberg (
      • Russell R.B.
      • Sternberg M.J.E.
      ) proposed that catalase contains a novel binding site on its surface based on structural similarities to the calycin superfamily. They suggested that SA inhibits catalase by binding to this site and causing a conformational change (allosteric inhibition). While such a site is not inconsistent with data presented here, our results argue that SA inhibits catalase by acting as an electron donor rather than by inducing a conformational change. SA could bind to this surface site and still act as an electron donor to the deeply buried heme of catalase, since Bonagura et al. (
      • Bonagura C.A.
      • Sundaramoorthy M.
      • Pappa H.S.
      • Patterson W.R.
      • Poulos T.L.
      ) have demonstrated that an electron donor at the surface of peroxidases can transfer electrons indirectly to the heme.
      The ability of SA to inhibit bovine catalase contrasts with our preliminary analysis, which suggested that mammalian catalases were not inhibited by SA (Chen et al.,
      • Chen Z.
      • Silva H.
      • Klessig D.F.
      ). It also illustrates the difficulties that can be encountered when determining the effects of potential inhibitors like SA on this complex enzyme, whose reaction chemistry is still debated. H2O2 itself can dramatically alter the effects seen with SA as illustrated in Figs. 4, 5, and 8. This likely is responsible for some of the discrepancy in results recently reported (Sánchez-Casas and Klessig,
      • Sá;nchez-Casas P.
      • Klessig D.F.
      ; Summermatter et al.,
      • Summermatter K.
      • Sticher L.
      • Métraux J.-P.
      ). The standard assay in which the rate of H2O2 utilization is measured only for a few minutes (Aebi,
      • Aebi H.
      ) is adequate for determining relative catalase activity in different tissues. However, it is poorly suited to analyze the effects of potential inhibitors on catalase activity. Inhibition by phenolics is time-dependent and requires H2O2 (Fig. 4), just as has been previously described (Ogura et al.,
      • Ogura Y.
      • Tonomura Y.
      • Hino S.
      • Tamiya H.
      ). Other less readily controlled variables include the presence of phenolics in crude extracts, which can lead to partial inactivation of catalase through formation of compound II (DeLuca et al.,
      • DeLuca D.C.
      • Dennis R.
      • Smith W.G.
      ) and thus to an underestimation of the inhibitory potential of a phenolic such as SA.

      Does SA Modulate Catalase Activity in Vivo?

      In infected tissues, SA levels can approach 100 μM (Malamy et al.,
      • Malamy J.
      • Carr J.P.
      • Klessig D.F.
      • Raskin I.
      and 1992; Enyedi et al.,
      • Enyedi A.
      • Yalpani N.
      • Silverman P.
      • Raskin I.
      ), a concentration sufficient to cause a considerable inhibition of catalase and ascorbate peroxidase (Bi et al.,
      • Bi Y.M.
      • Kenton P.
      • Mur L.
      • Darby R.
      • Draper J.
      ; Conrath et al.,
      • Conrath U.
      • Chen Z.
      • Ricigliano J.W.
      • Klessig D.F.
      ; Durner and Klessig,
      • Durner J.
      • Klessig D.F.
      ). Because of catalase's unique feature that it is inactivated by its own substrate (Fig. 4) (Hillar et al.,
      • Hillar A.
      • Nicholls P.
      • Switala J.
      • Loewen P.C.
      ; DeLuca et al.,
      • DeLuca D.C.
      • Dennis R.
      • Smith W.G.
      ), even modest effects on the activity of these two major H2O2-scavenging enzymes could feed back to cause further inactivation of catalase by the slow and time-dependent accumulation of H2O2.
      The role of catalase and SA in uninfected parts of an infected plant is considerably less clear. While SA also accumulates in these tissues, the level appears to be far below the concentration required to effectively inhibit catalase and ascorbate peroxidase (Malamy et al.,
      • Malamy J.
      • Carr J.P.
      • Klessig D.F.
      • Raskin I.
      ; Enyedi et al.,
      • Enyedi A.
      • Yalpani N.
      • Silverman P.
      • Raskin I.
      ). SA's role in SAR development is unlikely to involve elevated levels of H2O2 resulting from its inhibition of catalase, as originally proposed, unless SA is highly concentrated in certain subcellular compartments (Chen et al.,
      • Chen Z.
      • Silva H.
      • Klessig D.F.
      ). Nonetheless, SA induction of SAR may be mechanistically coupled to its interaction with catalase and peroxidases. SA serves as a one-electron donor for catalase (Fig. 6) and peroxidases (Durner and Klessig,
      • Durner J.
      • Klessig D.F.
      ) and in so doing is converted to a free radical. Free radicals of phenolics (e.g. Savenkova, et al. (1994)) can initiate formation of lipid peroxides. Our preliminary studies indicate that SA induces lipid peroxidation, while several naturally occurring lipid peroxides activate PR-1 genes in tobacco cells.3 A SA free radical could result in the formation of an effective lipid peroxide signal without readily discernible inhibition of catalase. However, the biological significance of a SA radical generated by catalase remains to be proven.
      In addition to its ability to inhibit catalase, SA could also protect plant and mammalian catalases against inactivation by H2O2 in vitro (Figs. 4C, 5, and 8). This is functionally similar to the protective effects of NADPH on mammalian catalases in the presence of small fluxes of H2O2 as described by Kirkman et al. (
      • Kirkman H.N.
      • Galiano S.
      • Gaetani G.F.
      ). Indeed, we found that tobacco catalase, like mammalian catalases, contains bound NADPH (Fig. 7). Therefore, it appears that, under some conditions, SA can support or substitute for NADPH's protective role. Might it serve a similar function in vivo? In animal systems accumulation of compound II (and thus catalase inhibition) has been associated with “abnormal” stress conditions such as found in tumors or during prolonged hypoxia or cell necrosis (Oshino et al.,
      • Oshino N.
      • Chance B.
      • Sies H.
      ). Furthermore, reactive oxygen species produced by NADPH oxidase, induced by tumor necrosis factor α, causes significant decrease in rat hepatic catalase activity (Yasmineh et al.,
      • Yasmineh W.G.
      • Parkin J.L.
      • Caspers J.I.
      • Theologides A.
      ). In the case of plants, similar stress conditions may occur during necrotic lesion formation in the HR. A strong oxidative burst (probably produced by a NADPH oxidase) is associated with the HR (Doke and Ohashi,
      • Doke N.
      • Ohashi Y.
      ; Orlandi et al.,
      • Orlandi E.W.
      • Hutcheson S.W.
      • Baker C.J.
      ; Levine et al.,
      • Levine A.
      • Tenhaken R.
      • Dixon R.
      • Lamb C.
      ), which could result in catalase inactivation by O.-2 and H2O2 (Schonbaum and Chance,

      Schonbaum, G. R., Chance, B., (1976) The Enzymes, (Boyer, P. D., ed), 2nd Ed., Vol. 13, pp. 363–408, Academic Press, Inc., New York.

      ; Kono and Fridovich,
      • Kono Y.
      • Fridovich I.
      ). Catalase inactivation during the HR would be enhanced by the proposed depletion of NADPH by NADPH oxidases and antioxidative enzymes of the ascorbate/glutathione cycle (Mehdy et al., 1994). One might speculate that under these conditions, SA may protect or reactivate a basal catalase activity. This notion is consistent with the observation that SA appears to act as an antioxidant at sites of inflammation in animals (Halliwell et al.,
      • Halliwell B.
      • Hoult J.R.
      • Bake D.R.
      ); one property of SA may be to maintain a basal level of catalase activity by acting as an electron donor that converts inactive compound II to the active ferricatalase (Fig. 6). In fact, it has been suggested that SA protects various heme proteins such as leghemoglobin and metmyoglobin from H2O2-induced inactivation by maintaining the peroxidative cycle of these O2-binding proteins (Galaris et al.,
      • Galaris D.
      • Mira D.
      • Sevenian A.
      • Cadenas E.
      • Hochstein P.
      ; Puppo and Halliwell,
      • Puppo A.
      • Halliwell B.
      ). Alternatively, SA may serve as a quencher of radicals associated with the heme group of inactivated catalase or other heme proteins (Galaris et al.,
      • Galaris D.
      • Mira D.
      • Sevenian A.
      • Cadenas E.
      • Hochstein P.
      ; Puppo and Halliwell,
      • Puppo A.
      • Halliwell B.
      ). SA is a direct scavenger of OH· (in vitro and in vivo), and it is a iron-chelating compound, thereby inhibiting the direct impact of OH· as well as its generation via the Fenton reaction (Halliwell et al.,
      • Halliwell B.
      • Aeschbach R.
      • Löliger J.
      • Aruoma O.I.
      ). However, since desferrioxamine (a strong chelating agent) did not protect catalase from inactivation, we hypothesize that SA maintains a basal catalase activity through its ability to serve as an electron donor.
      In sum, whether SA positively or negatively modulates catalase activity will depend on the redox status of the cell. In the healthy tissue surrounding, but not immediately adjacent to, the infection site H2O2 concentrations will be relatively low to moderate, and the elevated SA levels probably inhibit catalase by promoting the slow peroxidative cycle (note that in normal healthy leaf tissue H2O2 has been estimated at ∼100 nM; Scandalios (
      • Scandalios J.G.
      )). The resultant increase in H2O2 could serve as a second messenger to facilitate activation of plant defense genes.
      In contrast, in the infected cells and immediately adjacent tissue, high levels of reactive oxygen species resulting either from the oxidative burst associated with the HR (Doke and Ohashi,
      • Doke N.
      • Ohashi Y.
      ) or from long lasting oxidative processes around necrotizing cells (Kato and Misawa,
      • Kato S.
      • Misawa T.
      ) could lead to substantial inactivation of catalase by accumulation of inactive enzyme intermediates. Under conditions of such oxidative stress, SA might help to maintain and/or reestablish a basal level of catalase activity. These protective, antioxidative properties of SA might serve to limit the impact of the oxidative processes associated with development and spread of the lesion.

      Acknowledgments

      We thank members of the laboratory, particularly D'Maris Dempsey and Marc D. Anderson for critical reading of the manuscript. Transgenic plants expressing the cat2 gene and the cat1 gene in an antisense orientation, respectively, were kindly provided by Hideki Takahashi (this laboratory). Helmut Kessmann, Theo Staub, and John Ryals are acknowledged for generously providing INA.

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