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Plant Physiol. (1993) 103: 315-321

Pathway of Salicylic Acid Biosynthesis in Healthy and Vi rus- Inoculated Tobacco' Nasser Yalpani, Jose LeÓn, Michael A. Lawton, and llya Raskin* AgBiotech Center, Cook College, Rutgers University, P.O. Box 231, N e w Brunswick, N e w Jersey 08903-0231

than 40-fold in the immediate vicinity of hypersensitive response lesions, which develop at the site of initial pathogen penetration (Enyedi et al., 1992). SA is translocated from the inoculated tissue, presumably by movement with the phloem sap (Malamy et al., 1990; Métraux et al., 1990; Rasmussen et al., 1991; Yalpani et al., 1991), and activates a broad spectrum of defense responses, which are accompanied by the transcription of genes encoding PR proteins (Ward et al., 1991). The induction of these systemic resistance responses by one organism can confer increased immunity to a subsequent challenge by other, different pathogens, such as viruses, bacteria, and fungi (Madamanchi and Ku:, 1991). Plants most likely synthesize SA (o-hydroxybenzoicacid) from trans-cinnamic acid, the product of the activity of Phe ammonia lyase. This enzyme is induced by a range of biotic and abiotic stresses and is a key regulator of the phenylpropanoid pathway, which yields a variety of phenolics with structural and defense-related functions. Two pathways for the formation of SA from cinnamic acid have been reported in plants (Fig. 1). The hydroxylation of the aromatic ring can occur before or after the chain-shortening reaction. The latter reaction may depend on COA, analogous to the P-oxidation of fatty acids, or proceed via other, possibly nonoxidative, mechanisms (French et al., 1976; Funk and Brodelius, 1990a, 1990b; Schnitzler et al., 1992). In Helianthus annuus, Solanum tuberosum, and Pisum sativum, [I4C]benzoicacid served as a precursor for labeled SA (Klambt, 1962). Radioactive SA was reportedly formed via o-coumaric acid by leaf segments of young Primula acaulis and Gaultheria procumbens after they were fed '*C-labeled Phe or cinnamic acid (El-Basyouni et al., 1964). In the same species, labeled SA was also formed after treatment with [I4C]benzoic acid (El-Basyouni et al., 1964; Ellis and Amrhein, 1971), suggesting the involvement of both pathways. Chadha and Brown (1974) reported that upon infection of young tomato seedlings with Agrobacterium tumefaciens, 2-hydroxylation of cinnamic acid to o-coumaric acid was favored. This was followed by P-oxidation, yielding SA. In noninfected plants, however, SA appeared to be formed mostly from cinnamic acid via benzoic acid (Chadha and Brown, 1974). Conjugation and/or further hydroxylationreactions appear to result in metabolic inactivation of SA. In the vicinity of hypersensitive response lesions on leaves of tobacco, SA is rapidly conjugated with Glc to form P-O-D-glucosylsalicylic acid, a metabolite that does not appear to be directly involved

Salicylic acid (SAI i s 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 i n extracts of leaves and cell suspensions. In TMV-inoculated leaves, stimulation of SA accumulation i s accompanied by a corresponding increase i n the levels of benzoic acid. 14C-Tracerstudies with cell suspensions and mockor TMV-inoculated leaves indicate that the label moves from franscinnamic 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 ['4C]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 pathogenesis-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.

Recent evidence suggests that SA, a phenolic with ubiquitous distribution in angiosperms (Raskin et al., 1990), has important regulatory functions in plants. SA was identified as the natural trigger of heat production in thermogenic species (Raskin et al., 1987). In the inflorescence of voodoo lilies (Sauromatum guttatum Schott), a transient, nearly 100fold increase in the leve1 of SA on the day prior to blooming triggers increased activity of the altemative respiration pathway and a consequent increase in temperature of up to 12OC above ambient (Raskin et al., 1987, 1989). The heat evolved during blooming volatilizes putrid-smelling compounds that attract pollinating insects. SA may serve as a signal molecule in the development of systemic acquired resistance of tobacco (Nicotiana tabacum) and cucumber to infection by pathogens (Malamy et al., 1990; Métraux et al., 1990). In tobacco, SA levels can increase more Financia1 support by grants from the U.S. Department of Agriculture (Competitive Research Grants Office), Division of Energy Biosciences of the U.S. Department of Energy, the Rockefeller Foundation, the New Jersey Commission for Science and Technology, and the New Jersey Agricultura1 Experiment Station. * Corresponding author; fax 1-908-932-6535.

Abbreviations: PR protein, pathogenesis-related protein; SA, salicylic acid; TMV, tobacco mosaic virus. 315

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The incubation temperature was then shifted to 24OC to induce rapid accumulation of SA and the development of necrosis (Malamy et al., 1992). Phenylalanlne

I

Precursor Application

trans-Cinnamic acid.

wCoo" O

\

C

O

O

H

Benzoic acid

OH

ortho-Coumaric acid

Salicylic acld

Figure 1. Proposed pathways of SA biosynthesis in plants.

in the induction of systemic acquired resistance (Enyedi et al., 1992). Other metabolic products may arise from additional hydroxylation of the aromatic ring. Thus, 2,3-dihydroxybenzoic (o-pyrocatechuic) acid and 2,5-dihydroxybenzoic (gentisic) acid have been detected in a number of species fed ['4C]SA (Ibrahim and Towers, 1959) and in Asfilbe sinensis and tomato plants fed ['4C]cinnamic acid or ['4C]benzoic acid (Billek and Schmook, 1967; Chadha and Brown, 1974). The important role played by SA in plant disease resistance underscores the need for understanding the biosynthesis of this molecule. Here, we report that in both healthy and TMVinoculated tobacco, SA is formed from cinnamic acid via benzoic acid. MATERIALS AND METHODS Plant Material

Six- to' 8-week-old seedlings of Nicotiana fabacum L. cv Xanthi-nc were grown and inoculated on the uppermost, fully expanded leaves with the U1 strain of TMV (5 pg/leaf) as described (Yalpani et al., 1991).Cell suspensions of Xanthinc tobacco were cultured in MU-1 medium (Uchimiya and Murashige, 1976) and used 6 d after subculture as previously described (Kapulnik et al., 1992). Temperature Shift Experiments

The uppermost, nearly fully expanded leaf on each of severa1 6-week-old seedlings was inoculated with TMV as described above. Following inoculation, the plants were maintained for 4 d at 32OC with a 16-h photoperiod provided by cool-white fluorescent lamps (200 pmol m-* s-I). Under these conditions, the hypersensitive response and acquired resistance to virus infection are suppressed (Kassanis, 1952).

For leaf disc experiments, excised leaves were surface sterilized by immersion in 5% commercial bleach for 15 min, followed by dipping in 70% ethanol and soaking in steiile deionized water. Discs (15 mm diameter) were cut from the tissue between major lateral veins and vacuum infiltrated with 0.1 m~ solutions of the test compounds in 5 mM potassium phosphate buffer (pH 5.5). Discs were left floating on the same solutions and kept at 24OC under cool-white fluorescent lamps (200 pmol m-' s-') for the duration of the incubation. For petiole-feeding experiments, mock- or TMV-inocu1ai.ed leaves were excised and each petiole was immersed in 80 pL of 10 m~ potassium phosphate buffer (pH 6.5) containing 1 mM EGTA and 2.9 pCi of [3-'4C]cinnamic acid (50.8 niCi mmol-I, Research Products International Corp., Mount Prfospect, IL) or uniformly ring-labeled [I4C]benzoicacid (15 niCi mmol-I, Sigma). The precursor solution, taken up within 30 min, was replaced with water. For feeding experiments using cell suspensions, washed cells (0.6 g fresh weight) were resuspended in MU-1 mediiim containing 2.9 pCi of [3-14C]cinnamicacid (50.8 mCi "01-I) to a final volume of 10.5 mL. Duplicate cell batches were harvested by vacuum filtration through a glass fiber disc (2.5 cm diameter, Whatman GF/F) and immediately washed three times with 10 mL of ice-cold MU-1 medium. Tissue samples were frozen in liquid NZ and stored at -2OOC for subsequent analysis. Analytical Procedures

Tissue samples (0.6 g) were extracted using a protocol described previously for SA (Enyedi et al., 1992). Conjugates were quantified after chemical (base followed by acid) hydrolysis (Enyedi et al., 1992). Samples of 50 pL were injec ted into a Dynamax 60A 8-pm guard column (4.6 mm X 1.5 cm) linked to a Dynamax 60A 8-pm C-18 column (4.6 mm X 25 cm) (Rainin Instrument Co., Emeryville, CA), maintained at 4OoC, and equilibrated in 75% 20 mM sodium acetate buffer (pH 5.0) and 25% methanol with a flow rate of 1.5 mL min-I. Beginning 4.5 min after injection, a gradient of methanol (25-70%) was applied over 6.5 min. Elution was continued with 70% methanol for 3 min. SA and o-coumaric acid were quantified using a fluorescence spectrometer (model FL 750 HPLC Plus, McPherson Instrurnent Co., Acton, MA) using excitation and emission wavelengths of 2115 and 405 nm, respectively (Fig. 2A). Benzoic and cinnarnic acid were detected by their using a Spectroflow 783 (Kratos Analytical Instruments, Ramsey, NJ) absorption spectrophotometer (Fig. 2B). Under these conditions, dihydroxybenzoic acids and p-hydroxybenzoic acid eluted at 2.1 min, SA at 4.0 min, p-coumaric acid at 5.7 min, benzoic acid at 6.2 min, o-coumaric acid at 10.5 min, and cinnamic acid at 12.8 min. Under these conditions, the limits of detection wcere 0.2 pg of cinnamic acid, 0.06 pg of o-coumaric acid, 2.0 pg of

Salicylic Acid Biosynthesis in Virus-lnoculated Tobacco

cinnamic, 0.3 pg of o-coumaric, 100 pg of benzoic, and 0.3 pg of SA per g fresh weight. These values suggest the presence of conjugated forms of these compounds in tobacco.

B

A

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1

Effect of Likely SA Precursors on SA Accumulation in Uninoculated Tobacco

C

I

F

E

i

4

i

li

Levels of free SA in leaf discs of healthy tobacco were not significantly affected by vacuum infiltration and floating on 0.1 m~ Phe, cinnamic acid, o-coumaric acid, or benzoic acid (data not shown). Because tobacco rapidly conjugates SA to form its glucoside, we determined total levels of SA in chemically hydrolyzed extracts of leaf discs infiltrated with the above compounds. Figure 3 shows that feeding with 0.1 mM benzoic acid stimulated a rapid accumulation of total SA. By 18 h after infiltration with benzoic acid, the total leve1 of SA was 14-fold greater than in buffer-infiltrated controls. Total SA did not increase after treatment with Phe, cinnamic acid, or o-coumaric acid (Fig. 3).

16

Elution timt min)

Figure 2. HPLC elution profile of SA and its putative precursors from leaf extracts of mock- or TMV-inoculated tobacco. A, C, and E, Fluorescence at 405 nm upon excitation at 31 5 nm. B, D, and F, AZB0. A and B, Authentic standards. C and D, Hydrolyzed leaf extract of mock-inoculated tobacco. E and F, Hydrolyzed leaf extract of TMV-inoculated tobacco. Samples in C and E were diluted 20-fold compared with those in D and F. 1, SA; 2, benzoic acid; 3, ocoumaric acid; 4, cinnamic acid.

benzoic acid, and 0.01 pg of SA per g fresh weight. The identity of these compounds in plant extracts was confirmed by their co-elution with authentic standards under different HPLC conditions (data not shown). A flow-through radioactivity detector (Ramona-90, Raytest) with a solid glass scintillant was used to monitor radioactivity in eluates. A11 data are corrected for recovery of metabolites, which was estimated using spiked samples. Recovery was similar for a11 compounds studied and ranged between 30 and 52%.

Accumulation of SA and Its Likely Precursors in TMV-lnoculated Tobacco

When Xanthi-nc tobacco is inoculated with TMV and incubated at 32OC, the development of hypersensitive response lesions and the associated accumulation of SA are inhibited and the virus spreads systemically throughout the plant (Yalpani et al., 1991; Malamy et al., 1992). If, after 4 d at 32OC, the temperature is lowered to 24OC, free and total SA accumulate rapidly (Malamy et al., 1992). Virus-induced loss of turgor in the inoculated leaves appeared 10.5 h after the temperature shift, with some tissue death evident by 12 h. Most of the inoculated plants became necrotic 24 h after temperature shift. Figure 4 shows the time course of accumulation of free and total levels of possible SA precursors in TMV-inoculated tobacco leaves following the temperature shift. For the analysis, we used the separation and quantitation procedure illustrated in Figure 2. Large increases in the levels of free benzoic acid and SA were detectable by 7.5 h. By 10.5 h, TMV-inoculated leaves contained 15 pg of benzoic

Resistance to TMV

The middle-positioned leaf of 6-week-old tobacco seedlings was syringe infiltrated with a 0.1 ITLM solution of the test substance in 5 mM potassium phosphate buffer, pH 5.5. Seven days later, the infiltrated leaf and the leaf above it were inoculated with 2 pg of TMV per leaf. The diameters of at least 30 lesions per leaf, five leaves per treatment, were measured 7 d later. RESULTS 0.0

SA and Its Likely Precursors in Tobacco Extracts

Healthy Xanthi-nc tobacco leaves contained 0.1 to 0.2 pg of free SA per g fresh weight. Levels of free cinnamic, ocoumaric, and benzoic acid were below the limit of detection (see "Materials and Methods"). Base/acid-hydrolyzed extracts of healthy tobacco leaves contained approximately 2 pg of

O

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lncubation time (h)

Figure 3. Accumulation of SA after feedingO.l mM Phe or cinnamic, o-coumaric, or benzoic acid to leaf discs from healthy Xanthi-nc tobacco. Each point is the mean (+SE) of four replicate hydrolyzed samples. The experiment was repeated twice with similar results.

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eCoumaric acd

+ Salicylic acid

-

I

Controi Phe

d~

o - 6 ~ EA

Figure 5. Effect of putative SA precursors on levels of total S A in TMV-infected Xanthi-nc tobacco. TMV-inoculated tobacco plamts were kept at 32°C for 96 h. Four hours following a temperature shift from 32 to 24"C, leaf discs were excised, vacuum infiltrated with a 0.1 m M solution of the test compound in 5 m M phosphate buffer (pH 5.5), and then floated on the same solution for 6 h. (IA, Cinnamic acid; o-CA, o-coumaric acid; BA, benzoic acid. Each bar O 2.5 5 7.5 10 Time after temperature shift (h)

Figure 4. Accumulation of SA and its putative precursors in TMVinoculated leaves following temperature shift from 32 to 24°C. Sixweek-old tobacco seedlings were inoculated with TMV on one leaf and kept at 32°C for 96 h. At time = O h, the incubation temperature was lowered to 24°C. A, Unhydrolyzed leaf extracts. B, Hydrolyzed leaf extracts. Each point is the mean (&SE) of triplicate samples. The experiment was repeated twice with similar results.

acid and 19 pg of SA per g fresh weight compared with less than 2 pg of benzoic acid and 0.7 pg of SA at time O. Levels of free cinnamic and o-coumaric acid did not change significantly. No increases in any of the above compounds were detected in mock-inoculated plants shifted to 24OC (data not shown). In hydrolyzed leaf extracts, levels of total benzoic acid remained at approximately 120 pg per g fresh weight but increased almost 3-fold at 10.5 h after temperature shift, a time when the first symptoms of the hypersensitive response became visible (Fig. 48). Neither total cinnamic nor total ocoumaric acid levels changed significantly by 10.5 h following the temperature shift. However, by 12 h, a time when considerable tissue collapse was evident, total cinnamic acid levels reached 17 pg g-' compared with 2 pg g-' fresh weight at time O. To characterize further the sequence of the biosynthetic steps in SA biosynthesis, leaf discs from TMV-inoculated tobacco leaves were excised 2 h after temperature shift from 32 to 24OC and infiltrated with likely SA precursors. Infiltration and incubation of these discs with Phe or benzoic acid for 8 h resulted in increased total SA levels, which, in the case of benzoic acid, were 164%over controls. Cinnamic acid and o-coumaric acid had no effect on SA accumulation (Fig. 5 ) . Although the levels of o-coumaric acid showed a very slight increase following temperature shift of TMV-inoculated plants (Fig. 4A), this compound did not appear to be directly involved in SA biosynthesis. Radioactive cinnamic or benzoic acid were also fed through

represents the mean (& SE) of four different samples. The experiment was repeated twice with similar results. An asterisk (*) indicates a value significantly larger than controls at P = 0.01.

the cut petioles of excised mock- or TMV-inoculated leaves of Xanthi-nc tobacco that had been incubated at 24OC for 48 h. With [I4C]cinnamicacid, incorporation of label into benzoic acid was observed (data not shown). Trace amounts of '"Clabeled SA were also detected. No radioactivity co-eluted with o-coumaric acid in extracts of mock- or TMV-inoculated leaves. This suggests that o-coumaric acid is not a major metabolite of cinnamic acid and that it may not be involved in SA biosynthesis in tobacco. More label was incorporated into SA when [I4C]benzoic acid was fed through the cut petioles than when ['4]cinnamic acid was used. The labeled free SA that was formed within 2 h in mock-inoculated leaves had a specific activity (14 rriCi mmol-') similar to that of the applied labeled benzoic acid (Fig. 6). This suggests that in healthy tissues, almost a11 of

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Moch TMV

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o

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Figure 6. Specific activity of free SA produced from ['4C]benzoic acid in mock- or TMV-inoculated Xanthi-nc tobacco. Leaves were excised 48 h after inoculation and incubated at 24°C. They were fed 2.9 WCi of ['4C]benzoicacid (15 mCi/mmol) t h y u g h the Icut petiole and the specific activity of the accumulated SA was measured every 2 h. Points are the mean (+SE) of duplicate samples. 1he experiment was repeated once with similar results.

Salicylic Acid Biosynthesis in Virus-lnoculated Tobacco the SA is derived from benzoic acid. Although the specific activity of SA formed from benzoic acid was higher in mockinoculated leaves, the total amount of radioactivity incorporated in SA was similar in TMV- and mock-inoculated leaves. However, in the TMV-inoculated leaves the specific activity of the SA formed was at least two times lower (Fig. 6). The specific activity of SA declined in mock- or TMV-inoculated leaves with time, presumably reflecting dilution of the labeled SA pool, associated with metabolic tumover of free SA to /3O-D-glucosylsalicylicacid. We compared the pathway of SA biosynthesis in tobacco leaf tissue with that in cultured tobacco cells. ["C]Cinnamic acid was completely metabolized by tobacco cell suspensions within 3 h (Fig. 7A). Associated with this disappearance was the accumulation of radioactive benzoic acid and SA, which were detected by 15 min after supplying [''C]cinnamic acid. No labeled o-coumaric acid was detected. The specific activity of the SA reached a maximum of 1.6 mCi mmol-' 1 h after the start of the experiment (Fig. 78).

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Table 1. €ffect of infiltration with putative SA precursors on the diameter of JMV-induced lesions The middle-positioned leaf of 6-week-old Xanthi-nc tobacco was syringe infiltrated with 0.1 mM solution of test compound in 5 mM potassium phosphate buffer, pH 5.5. Seven days later, the infiltrated leaf and the leaf above it were inoculated with 2 pg of TMV per leaf. Lesion diameter was measured 7 d later. Data represent the mean diameter (+SE) of at least 30 lesions per leaf, with five leaves measured for each treatment. Lesion Diameter

Compound

Upper,

uninfiltrated

lnfiltrated mm

Control L-Phe trans-Cinnamic acid o-Coumaric acid Benzoic acid Salicylic acid a

2.2 f 0.2 1.6 f 0.1" 0.8 f 0.2" 1 .€if 0.1 0.9 f 0.1" 0.6 f O.la

2.4 f 0.1 2.3 f 0.1

1.8 f 0.2 2.1 f 0.2 1.9 f 0.2 0.7 f 0.2"

Significantly smaller than uninfiltrated controls at P = 0.01.

Effect of Precursors on Accumulation of PR-1 Proteins and Resistance to TMV

Application of SA to tobacco stimulates synthesis of several classes of PR proteins, including those belonging to the PR1 family (Antoniw and White, 1980). Both SA and, to a lesser extent, benzoic acid induced the accumulation of PR-la and PR-lb in Xanthi-nc tobacco leaves (data not shown). In contrast, o-coumaric acid did not induce PR-1 protein accumulation. Application of SA increases disease resistance (Yalpani and Raskin, 1993). Therefore, putative SA precursors should also provide some protection against pathogens. Compared with

-

DISCUSSION

----C Salqlcaui

+ Benzocacd

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cmnamacld

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-ul

8 Ul

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-E

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buffer-infiltrated controls, lesion diameter in SA-treated leaves was reduced by 78% (Table I). Benzoic acid and cinnamic acid were nearly as effective as SA in stimulating resistance to TMV in the infiltrated leaf. A 30% reduction in lesion diameter was induced by treatment with Phe, whereas o-coumaric acid had no significant effect on resistance to TMV. Unlike SA, local application of the putative SA precursors had no significant effect on systemic TMV resistance (Table I).

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Labeling time (min) Figure 7. Synthesis of benzoic acid and SA from cinnamic acid in tobacco cell suspensions. A, lncorporation of ['4C]cinnamic acid into benzoic acid and SA. B, Specific activity of accumulated SA.

In recent years, considerable evidence has accumulated linking a rise of SA levels in tobacco with the induction of PR protein accumulation and increased resistance to pathogens (reviewed in Yalpani and Raskin, 1993). Because healthy tobacco leaves do not contain large conjugated pools of SA (Enyedi et al., 1992; Malamy et al., 1992), dramatic increases in free SA levels during the development of acquired resistance appear to result from de novo synthesis. Earlier studies suggested that the biosynthesis of SA from cinnamic acid in some plants may proceed via benzoic acid or o-coumaric acid (El-Basyouni et al., 1964; Ellis and Amrhein, 1971; Chadha and Brown, 1974). We obtained several lines of evidence suggesting that benzoic acid, but not Ocoumaric acid, is the biosynthetic precursor of SA in Xanthinc tobacco. Thus, unlike application of o-coumaric acid, application of benzoic acid to leaf discs of mock- or TMVinoculated plants resulted in elevated SA accumulation (Figs. 3 and 5 ) . When ['4C]cinnamic acid was fed to suspension-cultured tobacco cells or to mock- or TMV-inoculated tobacco leaves, labeled benzoic acid but not radioactive o-coumaric acid was detected (Fig. 7). Similarly, in mock-inoculated tobacco, labeling studies with [14C]benzoicacid indicated nearly quantitative incorporation of radioactivity into SA (Fig. 6), suggesting that nearly a11 of the SA formed in healthy tobacco is derived

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from benzoic acid. The specific activity of the SA formed from [14C]benzoic acid is lower in TMV- than in mockinoculated plants (Fig. 6). This could be due to the pathway of SA biosynthesis shifting from benzoic acid to some other precursor or to an increase in the size of the endogenous benzoic acid pool. Support for the latter possibility is provided by the observed increase in the levels of free and conjugated form(s) of benzoic acid in extracts of TMV-inoculated leaves (Fig. 4). No changes in free or conjugated o-coumaric acid were detected in the TMV-inoculated leaves. It is interesting that healthy tobacco leaves contained a large pool of conjugated benzoic acid, almost 0.01% on a fresh weight basis (Fig. 4B). The size of this pool in TMVinoculated tissue transiently decreased following the temperature shift. This decrease coincided with the accumulation of free benzoic acid and SA in the tissue (Fig. 4A). Given the total size of the benzoic acid pool, the observed increases in free benzoic acid in the inoculated leaves can be explained solely by the hydrolysis of conjugated benzoic acid. Therefore, it is possible that this reaction serves as the key regulatory step in SA biosynthesis. This is consistent with our observation that infiltration of tobacco leaves with cinnamic acid did not produce significant increases in SA (Fig. 5). In addition, because cinnamic acid functions as a key biosynthetic intermediate, the size of the cinnamic acid pool may not limit SA biosynthesis. Small increases in tissue SA following infiltration with Phe may be explained by indirect effects of this compound on phenylpropanoid metabolism. The increase in the activity of a benzoic acid-inducible benzoic acid 2-hydroxylase in TMV-inoculated Xanthi-nc tobacco (LeÓn et al., 1993) adds further support for benzoic acid functioning as an SA precursor. Evidence against a precursor function of o-coumaric acid is also provided by the results of the feeding experiments that compared the differential ability of o-coumaric acid and benzoic acid to stimulate PR protein accumulation and increased resistance to TMV. However, the results of these latter two experiments give only circumstantial support for a role of benzoic acid as an intermediate in SA biosynthesis. It is still possible that benzoic acid directly induces PR protein synthesis and resistance and does not owe its activity to metabolic conversion to SA. Similarly, the reduced diameter of TMV lesions in cinnamic acid-treated leaves may be due to the central role of cinnamic acid in the biosynthesis of many defense-related phenolics and cell-wall strengthening materials, which include lignin. In healthy tomato plants, SA is also synthesized from cinnamic acid via benzoic acid (Chadha and Brown, 1974). However, in plants infected with A . tumefuciens, [“Clcinnamic acid formed labeled o-coumaricacid and incorporation of radioactivity into SA from labeled benzoic acid was reduced. The authors interpreted these results to suggest that o-coumaric acid serves as an SA precursor in infected plants. However, their data may reflect an increase in the size of the endogenous pool of benzoic acid. o-Coumaric acid may be more important as a precursor for certain coumarins. If TMV-inoculated Xanthi-nc tobacco is incubated at 32OC, SA biosynthesis and induced resistance responses are inhibited. We observed that at this temperature, levels of benzoic acid and its conjugate(s) also do not increase in TMV-inoculated leaves. This suggests that the inhibition of TMV-in-

Plant Physiol. Vol. 103, 1993

duced SA biosynthesis may result from a block at an earlier step in the signal transduction pathway that leads from viius recognition to increased benzoic acid levels. Our work has established the pathway of SA biosynthesis during development of induced resistance. We have shown that in tobacco, most, if not all, SA is formed from cinnarnic acid via benzoic acid and that the levels of benzoic acid influence SA accumulation. Identification of the intermediate(s) in the formation of benzoic acid from cinnamic acid and characterization of the rate-limiting enzymes of SA biosynthesis will be important for designing strategies for increasing the resistance of plants to pathogens. ACKNOWLEDCMENTS

The authors are grateful to Drs. Eric E. Conn and Peter R. Day for helpful discussions. Received May 5, 1993; accepted June 23, 1993. Copyright Clearance Center: 0032-0889/93/l03/0315/07. LITERATURE CITED

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