The Pathway and Regulation of Salicylic Acid Biosynthesis in Probenazole-Treated <Emphasis Type="Italic">Arabidopsis</Emphasis>

Probenazole (PBZ; 3-allyloxy-1,2-benzisothiazole-1,1-dioxide) is a highly effective chemical inducer of systemic-acquired resistance (SAR). It has been used widely to protect rice plants against the rice blast fungus Magnaporthe grisea. Previous studies have shown that PBZ induces SAR through enhanced accumulation of salicylic acid (SA). Plants synthesize SA by either a pathway that uses phenylalanine as substrate or another that involves isochorismate. To clarify how SA is produced in PBZ-treated Arabidopsis, we examined the expression patterns and enzyme activities of phenylalanine ammonia lyase (PAL) and isochorismate synthase (ICS), which are the main components of the phenylalanine and isochorismate pathways, respectively. PBZ exposure significantly improved the accumulation of SA and increased ICS activity. In the sid2–2 mutant, which has a defect in ICS1, PBZ had no effect on the level of endogenous SA or activity of ICS. In contrast, PAL activity and the expression of most PAL genes were down-regulated by such treatment in wild-type plants. These results suggest that SA is mainly synthesized via the ICS-mediated pathway in Arabidopsis.     

Biosynthesis of salicylic acid in plants

Salicylic acid (SA) is an important signal molecule in plants. Two pathways of SA biosynthesis have been proposed in plants. Biochemical studies using isotope feeding have suggested that plants synthesize SA from cinnamate produced by the activity of phenylalanine ammonia lyase (PAL). Silencing of PAL genes in tobacco or chemical inhibition of PAL activity in Arabidopsis, cucumber and potato reduces pathogen-induced SA accumulation. Genetic studies, on the other hand, indicate that the bulk of SA is produced from isochorismate. In bacteria, SA is synthesized from chorismate through two reactions catalyzed by isochorismate synthase (ICS) and isochorismate pyruvate lyase (IPL). Arabidopsis contains two ICS genes but has no gene encoding proteins similar to the bacterial IPL. Thus, how SA is synthesized in plants is not fully elucidated. Two recently identified Arabidopsis genes, PBS3 and EPS1, are important for pathogen-induced SA accumulation. PBS3 encodes a member of the acyl-adenylate/thioester-forming enzyme family and EPS1 encodes a member of the BAHD acyltransferase superfamily. PBS3 and EPS1 may be directly involved in the synthesis of an important precursor or regulatory molecule for SA biosynthesis. The pathways and regulation of SA biosynthesis in plants may be more complicated than previously thought.Key words: salicylic acid biosynthesis, isochorismate synthase, phenylalanine ammonia lyase     

Arabidopsis local resistance to Botrytis cinerea involves salicylic acid and camalexin and requires EDS4 and PAD2, but not SID2, EDS5 or PAD4

Salicylic acid (SA) is an important regulator of plant defense responses, and a variety of Arabidopsis mutants impaired in resistance against bacterial and fungal pathogens show defects in SA accumulation, perception, or signal transduction. Nevertheless, the role of SA-dependent defense responses against necrotrophic fungi is currently unclear. We determined the susceptibility of a set of previously identified Arabidopsis mutants impaired in defense responses to the necrotrophic fungal pathogen Botrytis cinerea. The rate of development of B. cinerea disease symptoms on primary infected leaves was affected by responses mediated by the genes EIN2, JAR1, EDS4, PAD2, and PAD3, but was largely independent of EDS5, SID2/ICS1, and PAD4. Furthermore, plants expressing a nahG transgene or treated with a phenylalanine ammonia lyase (PAL) inhibitor showed enhanced symptoms, suggesting that SA synthesized via PAL, and not via isochorismate synthase (ICS), mediates lesion development. In addition, the degree of lesion development did not correlate with defensin or PR1 expression, although it was partially dependent upon camalexin accumulation. Although npr1 mutant leaves were normally susceptible to B. cinerea infection, a double ein2 npr1 mutant was significantly more susceptible than ein2 plants, and exogenous application of SA decreased B. cinerea lesion size through an NPR1-dependent mechanism that could be mimicked by the cpr1 mutation. These data indicate that local resistance to B. cinerea requires ethylene-, jasmonate-, and SA-mediated signaling, that the SA affecting this resistance does not require ICS1 and is likely synthesized via PAL, and that camalexin limits lesion development.     

Salicylic acid production in response to biotic and abiotic stress depends on isochorismate in Nicotiana benthamiana

Salicylic acid (SA) is an important signal involved in the activation of defence responses against abiotic and biotic stress. In tobacco, benzoic acid or glucosyl benzoate were proposed to be precursors of SA. This is in sharp contrast with studies in Arabidopsis thaliana, where SA derives from isochorismate. We have determined the importance of isochorismate for SA biosynthesis in Nicotiana benthamiana using virus-induced gene silencing of the isochorismate synthase (ICS) gene. Plants with silenced ICS expression do not accumulate SA after exposure to UV or to pathogen stress. Plants with silenced ICS expression also exhibit strongly decreased levels of phylloquinone, a product of isochorismate. Our data provide evidence for an isochorismate-derived synthesis of SA in N. benthamiana.     

Vitamin K1 accumulation in tobacco plants overexpressing bacterial genes involved in the biosynthesis of salicylic acid

Phylloquinone (Vitamin K(1)) is an essential component of the photosynthetic electron transfer. As isochorismate is required for the biosynthesis of Vitamin K(1), isochorismate synthase (ICS) activity is expected to be present in all green plants. In bacteria salicylic acid (SA) is synthesized via a two step pathway involving ICS and isochorismate pyruvate lyase (IPL). The effect of the introduction in tobacco plants of the bacterial ICS and IPL genes on the endogenous isochorismate pathway was investigated. Transgenic tobacco plants in which IPL was targeted to the chloroplast suffered severe growth retardation and had low Vitamin K(1) content. Probably because isochorismate was channeled towards SA production, the plants were no longer able to produce normal levels of Vitamin K(1). Transgenic tobacco plants in which the bacterial ICS was present in the chloroplast showed higher Vitamin K(1) contents than wild type plants.     

Activation of a diverse set of genes during the tobacco resistance response to TMV is independent of salicylic acid; induction of a subset is also ethylene independent

Through differential screening of a cDNA library, we cloned six groups of genes that are expressed relatively early in the inoculated leaves of tobacco resisting infection by tobacco mosaic virus (TMV). Induction of all these genes was subsequently detected in the uninoculated leaves; thus, their expression is associated with the development of both local and systemic acquired resistance. Exogenously applied salicylic acid (SA) was observed to induce these genes transiently. However, analyses with transgenic NahG plants, which are unable to accumulate SA, demonstrated that expression of these genes in TMV-inoculated leaves is mediated via an SA-independent pathway. Because the expression kinetics of these genes differ from those associated with the well-characterized pathogenesis-related protein (PR-1) and phenylalanine ammonia-lyase (PAL) genes, we propose that they belong to a group which we designate SIS, for SA-independent, systemically induced genes. Interestingly, the expression of several SIS genes in the uninoculated leaves of TMV-infected NahG plants was delayed and/or reduced, raising the possibility that SA is involved in activating some of these genes in systemic tissue. Most of the SIS genes were induced by exogenous ethylene. However, analyses of infected NahG plants treated with ethylene action and/or synthesis inhibitors indicated that the TMV-induced expression of several SIS genes is independent of ethylene as well as SA.     

Pathway of Salicylic Acid Biosynthesis in Healthy and Virus-Inoculated Tobacco

Salicylic acid (SA) is a likely endogenous regulator of localized and systemic disease resistance in plants. During the hypersensitive response of Nicotiana tabacum L. cv Xanthi-nc to tobacco mosaic virus (TMV), SA levels rise dramatically. We studied SA biosynthesis in healthy and TMV-inoculated tobacco by monitoring the levels of SA and its likely precursors in extracts of leaves and cell suspensions. In TMV-inoculated leaves, stimulation of SA accumulation is accompanied by a corresponding increase in the levels of benzoic acid. 14C-Tracer studies with cell suspensions and mock-or TMV-inoculated leaves indicate that the label moves from trans-cinnamic acid to SA via benzoic acid. In healthy and TMV-inoculated tobacco leaves, benzoic acid induced SA accumulation. o-Coumaric acid, which was previously reported as a possible precursor of SA in other species, did not increase SA levels in tobacco. In healthy tobacco tissue, the specific activity of newly formed SA was equal to that of the supplied [14C]benzoic acid, whereas in TMV-inoculated leaves some isotope dilution was observed, presumably because of the increase in the pool of endogenous benzoic acid. We observed accumulation of pathogen-esis-related-1 proteins and increased resistance to TMV in benzoic acid- but not in o-coumaric acid-treated tobacco leaves. This is consistent with benzoic acid being the immediate precursor of SA. We conclude that in healthy and virus-inoculated tobacco, SA is formed from cinnamic acid via benzoic acid.     

Tobacco plants epigenetically suppressed in phenylalanine ammonia-lyase expression do not develop systemic acquired resistance in response to infection by tobacco mosaic virus

The response of tobacco (Nicotiana tabacum L. cv. Xanthinc) plants, epigenetically suppressed for phenylalanine ammonia-lyase (PAL) activity, was studied following infection by tobacco mosaic virus (TMV). These plants contain a bean PAL2 transgene in the sense orientation, and have reduced endogenous tobacco PAL mRNA and suppressed production of phenylpropanoid products. Lesions induced by TMV infection of PAL-suppressed plants are markedly different in appearance from those induced on control plants that have lost the bean transgene through segregation, with a reduced deposition of phenofics. However, they develop at the same rate as on control tobacco, and pathogenesis-related (PR) proteins are induced normally upon primary infection. The levels of free salicylic acid (SA) produced in primary inoculated leaves of PAL-suppressed plants are approximately fourfold lower than in control plants after 84 h, and a similar reduction is observed in systemic leaves. PR proteins are not induced in systemic leaves of PAL-suppressed plants, and secondary infection with TMV does not result in the restriction of lesion size and number seen in control plants undergoing systemic acquired resistance (SAR). In grafting experiments between wild-type and PAL-suppressed tobacco, the SAR response can be transmitted from a PAL-suppressed root-stock, but SAR is not observed if the scion is PAL-suppressed. This indicates that, even if SA is the systemic signal for establishment of SAR, the amount of pre-existing phenylpropanoid compounds in systemic leaves, or the ability to synthesize further phenylpropanoids in response to the systemic signal, may be important for the establishment of SAR. Treatment of PAL-suppressed plants with dichloro-isonicotinic acid (INA) induces PR protein expression and SAR against subsequent TMV infection. However, treatment with SA, while inducing PR proteins, only partially restores SAR, further suggesting that de novo synthesis of SA, and/or the presence or synthesis of other phenylpropanoids, is required for expression of resistance in systemic leaves.     

Activities of enzymes involved in the phenylpropanoid pathway in constitutively salicylic acid-producing tobacco plants

Chorismate mutase (CM, EC 5.4.99.5), phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and chalcone synthase (CHS, EC 2.3.1.74) activities were studied in constitutive salicylic acid-producing (CSA) tobacco plants in relation to the accumulation of flavonoids and chlorogenic acid. The CM, PAL and CHS activities in CSA-tobacco (Nicotiana tabacum cv. Samsun NN) plants were lower than in non-transgenic tobacco plants. Flavonoid and chlorogenic acid accumulation was suppressed in CSA-tobacco plants compared to those of non-transgenic tobacco plants.     

Accumulation of Salicylic Acid and 4-Hydroxybenzoic Acid in Phloem Fluids of Cucumber during Systemic Acquired Resistance Is Preceded by a Transient Increase in Phenylalanine Ammonia-Lyase Activity in Petioles and Stems

Cucumber (Cucumis sativa) leaves infiltrated with Pseudomonas syringae pv. syringae cells produced a mobile signal for systemic acquired resistance between 3 and 6 h after inoculation. The production of a mobile signal by inoculated leaves was followed by a transient increase in phenylalanine ammonia-lyase (PAL) activity in the petioles of inoculated leaves and in stems above inoculated leaves; with peaks in activity at 9 and 12 h, respectively, after inoculation. In contrast, PAL activity in inoculated leaves continued to rise slowly for at least 18 h. No increases in PAL activity were detected in healthy leaves of inoculated plants. Two benzoic acid derivatives, salicylic acid (SA) and 4-hydroxybenzoic acid (4HBA), began to accumulate in phloem fluids at about the time PAL activity began to increase, reaching maximum concentrations 15 h after inoculation. The accumulation of SA and 4HBA in phloem fluids was unaffected by the removal of all leaves 6 h after inoculation, and seedlings excised from roots prior to inoculation still accumulated high levels of SA and 4HBA. These results suggest that SA and 4HBA are synthesized de novo in stems and petioles in response to a mobile signal from the inoculated leaf.     

Characterization and biological function of the ISOCHORISMATE SYNTHASE2 gene of Arabidopsis

Salicylic acid (SA) is an important mediator of plant defense response. In Arabidopsis (Arabidopsis thaliana), this compound was proposed to derive mainly from isochorismate, itself produced from chorismate through the activity of ISOCHORISMATE SYNTHASE1 (ICS1). Null ics1 mutants still accumulate some SA, suggesting the existence of an enzymatic activity redundant with ICS1 or of an alternative ICS-independent SA biosynthetic route. Here, we studied the role of ICS2, a second ICS gene of the Arabidopsis genome, in the production of SA. We have shown that ICS2 encodes a functional ICS enzyme and that, similar to ICS1, ICS2 is targeted to the plastids. Comparison of SA accumulation in the ics1, ics2, and ics1 ics2 mutants indicates that ICS2 participates in the synthesis of SA, but in limited amounts that become clearly detectable only when ICS1 is lacking. This unequal redundancy relationship was also observed for phylloquinone, another isochorismate-derived end product. Furthermore, detection of SA in the double ics1 ics2 double mutant that is completely devoid of phylloquinone provides genetic evidence of the existence of an ICS-independent SA biosynthetic pathway in Arabidopsis.     

Induction of Benzoic Acid 2-Hydroxylase in Virus-Inoculated Tobacco

Salicylic acid (SA) plays an important role in the induction of plant resistance to pathogens. An accompanying article (N. Yalpani, J. Leon, M.A. Lawton, I. Raskin [1993] Plant Physiol 103: 315-321) shows that SA is synthesized via the decarboxylation of cinnamic acid to benzoic acid (BA), which is, in turn, hydroxylated to SA. Leaf extracts of tobacco (Nicotiana tabacum L. cv Xanthi-nc) catalyze the 2-hydroxylation of BA to SA. The monooxygenase catalyzing this reaction, benzoic acid 2-hydroxylase (BA2H), required NAD(P)H or reduced methyl viologen as an electron donor. BA2H activity was detected in healthy tobacco leaf extracts (1-2 nmol h-1 g-1 fresh weight) and was significantly increased upon inoculation with tobacco mosaic virus (TMV). This increase paralleled the levels of free SA in the leaves. Induction of BA2H activity was restricted to tissue expressing a hypersensitive response at 24[deg]C. TMV induction of BA2H activity and SA accumulation were inhibited when inoculated tobacco plants were incubated at 32[deg]C. However, when inoculated plants were incubated for 4 d at 32[deg]C and then transferred to 24[deg]C, they showed a 15-fold increase in BA2H activity and a 65-fold increase in free SA content compared with healthy plants incubated at 24[deg]C. Treatment of leaf tissue with the protein synthesis inhibitor cycloheximide blocked the induction of BA2H activity by TMV. The effect of TMV inoculation on BA2H could be duplicated by infiltrating leaf discs of healthy plants with BA. This response was observed even when applied levels of BA were much lower than the levels observed in vivo after virus inoculation. Feeding tobacco leaves with phenylalanine, cinnamic acid, or o-coumaric acid (putative precursors of SA) failed to trigger the induction of BA2H activity. BA2H appears to be a pathogen-inducible protein with an important regulatory role in SA accumulation during the development of induced resistance to TMV in tobacco.     

Recent breakthroughs in the study of salicylic acid biosynthesis

Salicylic acid is an important regulator of induced plant resistance to pathogens. Consequently, the biosynthesis of salicylic acid and its regulation has received a lot of attention. Salicylic acid can be made from phenylalanine via cinnamic and benzoic acid. Recently, genetic studies in Arabidopsis have shown that salicylic acid is made in the chloroplast from isochorismate, a pathway that is known to operate in prokaryotes.     

Ozone-induced cell death occurs via two distinct mechanisms in Arabidopsis: the role of salicylic acid

Previous studies suggest that salicylic acid (SA) plays an important role in influencing plant resistance to ozone (O3). To further define the role of SA in O3-induced responses, we compared the responses of two Arabidopsis genotypes that accumulate different amounts of SA in response to O3 and a SA-deficient transgenic Col-0 line expressing salicylate hydroxylase (NahG). The differences observed in O3-induced changes in SA levels, the accumulation of active oxygen species, defense gene expression, and the kinetics and severity of lesion formation indicate that SA influences O3 tolerance via two distinct mechanisms. Detailed analyses indicated that features associated with a hypersensitive response (HR) were significantly greater in O3-exposed Cvi-0 than in Col-0, and that NahG plants failed to exhibit these HR-like responses. Furthermore, O3-induced antioxidant defenses, including the redox state of glutathione, were greatly reduced in NahG plants compared to Col-0 and Cvi-0. This suggests that O3-induced cell death in NahG plants is due to the loss of SA-mediated potentiation of antioxidant defenses, while O3-induced cell death in Cvi-0 is due to activation of a HR. This hypothesis is supported by the observation that inhibition of NADPH-oxidases reduced O3-induced H2O2 levels and the O3-induced cell death in Cvi-0, while no major changes were observed in NahG plants. We conclude that although SA is required to maintain the cellular redox state and potentiate defense responses in O3 exposed plants, high levels of SA also potentiate activation of an oxidative burst and a cell death pathway that results in apparent O3 sensitivity.     

Phenylpropanoid compounds and disease resistance in transgenic tobacco with altered expression of L-phenylalanine ammonia-lyase

Tobacco plants over-expressing L-phenylalanine ammonia-lyase (PAL(+)) produce high levels of chlorogenic acid (CGA) and exhibit markedly reduced susceptibility to infection with the fungal pathogen Cercospora nicotianae, although their resistance to tobacco mosaic virus (TMV) is unchanged. Levels of the signal molecule salicylic acid (SA) were similar in uninfected PAL(+) and control plants and also following TMV infection. In crosses of PAL(+) tobacco with tobacco harboring the bacterial NahG salicylate hydroxylase gene, progeny harboring both transgenes lost resistance to TMV, indicating that SA is critical for resistance to TMV and that increased production of phenylpropanoid compounds such as CGA cannot substitute for the reduction in SA levels. In contrast, PAL(+)/NahG plants showed strongly reduced susceptibility to Cercospora nicotianae compared to the NahG parent line. These results are consistent with a recent report questioning the role of PAL in SA biosynthesis in Arabidopsis, and highlight the importance of phenylpropanoid compounds such as CGA in plant disease resistance.     

Simultaneous UPLC MS/MS analysis of endogenous jasmonic acid,salicylic acid,and their related compounds

Jasmonic acid (JA) and salicylic acid (SA) are plant hormones involved in plant growth and development. Recent studies demonstrated that presence of a complex interplay between JA and SA signaling pathways to response to pathogenesis attack and biotic stresses. To our best knowledge, no method has existed for simultaneous analyses of JA, SA, and their related compounds. Especially, the glucosides are thought to be the storages or the inactivated compounds, but their contribution should be considered for elucidating the amount of the aglycons. It is also valuable for measuring the endogenous amount of phenylalanine, cinnamic acid, and benzoic acid that are the biosynthetic intermediates of SA due to the existence of isochorismate pathway to synthesize SA. We established this method using deuterium labeled compounds as internal standards. This is the first report of simultaneous analysis of endogenous JA, SA, and their related compounds. Measuring the endogenous JA, SA, and their related compounds that had been accumulated in tobacco plants proved the practicality of the newly developed method. It was demonstrated that accumulation of JA, SA and their related compounds were induced in both case of TMV infection and abiotic stresses.     

A loss of resistance to avirulent bacterial pathogens in tobacco is associated with the attenuation of a salicylic acid-potentiated oxidative burst

The role of salicylic acid (SA) in events occurring before cell death during the hypersensitive reaction (HR) was investigated in leaves of wild-type tobacco Samsun NN and in transgenic lines expressing salicylate hydroxylase (35S-SH-L). Challenge of 35S-SH-L tobacco with avirulent strains of Pseudomonas syringae gave rise to symptoms resembling those normally associated with a compatible response to virulent strains in terms of visible phenotype, kinetics of bacterial multiplication, and escape from the infection site. Compared with responses in wild-type tobacco, both the onset of plant cell death and the induction of an active oxygen species-responsive promoter (AoPR1-GUS) were delayed following challenge of 35S-SH-L plants with avirulent bacteria. The oxidative burst occurring after challenge with avirulent bacteria was visualized histochemically and quantified in situ. H2O2 accumulation at reaction sites was evident within 1 h after inoculation in wild-type tobacco, whereas in 35S-SH-L plants the onset of H2O2 accumulation was delayed by 2-3 h. The delay in H2O2 generation was correlated with a reduction in the transient rise in SA that usually occurred within 1-2 h following inoculation in wild-type plants. Our data indicate that an early transient rise in SA potentiates the oxidative burst, with resultant effects on accumulation of H2O2, plant cell death and also defence-gene induction, factors that together may determine the outcome of plant-pathogen interactions.     

A new catalytic activity from tobacco converting 2-coumaric acid to salicylic aldehyde

Salicylic acid (SA) mediates plant response to pathogen invasion, resulting in hypersensitive response and in the formation of systemic acquired resistance. It is well known that Nicotiana tabacum and other plants respond to Tobacco Mosaic Virus (TMV) infection by increasing the content of SA but the details of SA biosynthesis are still not fully understood. Generally, SA may originate directly from isochorismate ( Arabidopsis thaliana ), or its C₆–C₁ skeleton could be synthesized via the phenylpropanoid pathway by β-oxidation of trans -cinnamic acid ( N. tabacum ), 2-coumaric acid (OCA) ( Gaulteria procumbens , Lycopersicum esculentum ) or by retro-aldol reaction of trans -cinnamoyl-CoA ( Hypericum androsaemum ). We report here a novel putative enzyme activity from tobacco, salicylic aldehyde synthase (SAS), catalysing non-oxidative formation of salicylic aldehyde (SALD) directly from OCA. This chain-shortening activity is similar to that of 4-hydroxybenzaldehyde synthase from Vanilla planifolia , Lithospermum erythrorhizon , Daucus carota , Solanum tuberosum and Polyporus hispidus but the enzyme differs in the kinetics of the reaction, substrate specificity and requirements for reducing cofactors. SAS activity is constitutively expressed in healthy tobacco leaves and doubles as a result of infection with TMV. Moreover, the product of SAS activity—SALD, applied exogenously on tobacco leaves, stimulates peroxidase activity and enhances resistance to consecutive infection with TMV. These observations could suggest a contribution of SAS and SALD to the response of tobacco to TMV infection.