Abstract

Ozone (O3), a major photochemical oxidant, induces leaf injury concomitant with salicylic acid (SA) synthesis. In pathogen-infected leaves, SA is synthesized via two pathways, involving phenylalanine or isochorismate. SA biosynthesis under O3 fumigation is not well understood. When we applied 14C-labeled benzoic acid (a precursor of SA in the pathway via phenylalanine) to O3-exposed tobacco leaves, it was effectively metabolized to SA. However, the activity and mRNA level of isochorismate synthase (ICS) were not increased. In contrast, ICS activity was increased in O3-exposed Arabidopsis thaliana L. These results suggest that SA is synthesized via benzoic acid from phenylalanine in O3-exposed tobacco leaves but via isochorismate in Arabidopsis. Ethylene is a plant hormone that promotes leaf damage in O3-exposed plants. During O3 exposure, transgenic plants with a phenotype of reduced O3-induced ethylene production accumulated less SA than did wild-type plants. O3 increased the activity of phenylalanine ammonia-lyase (PAL) and the transcript levels of the chorismate mutase (CM) and PAL genes in wild-type tobacco, but their induction was suppressed in the transgenic plants. These results indicate that ethylene promotes SA accumulation by regulating the expression of the CM and PAL genes in O3-exposed tobacco.

Introduction

Ozone is the main photochemical oxidant that causes leaf damage in many plant species, thereby decreasing the productivity of crops and forests (Preston and Tingey 1988). This oxidant penetrates the stomata to induce leaf injury (Mehlhorn et al. 1990, Schraudner et al. 1998). Ozone reacts with the cell wall and cell membrane and produces reactive oxygen species such as superoxide radicals and hydrogen peroxide, which in turn induce ethylene and salicylic acid (SA) synthesis (Kangasjärvi et al. 1994, Rao and Davis 2001).

In the response to O3, there is a correlation between the rate of ethylene production and the amount of leaf damage (Tingey et al. 1976). Inhibitors of ethylene biosynthesis attenuate injury in O3-exposed plants (Mehlhorn and Wellburn 1987, Wenzel et al. 1995, Bae et al. 1996, Tamaoki et al. 2003). The introduction of antisense DNA for O3-inducible 1-aminocyclopropane-1-carboxylate synthase (ACS), an enzyme responsible for ethylene biosynthesis, can improve O3 tolerance in tobacco (Nakajima et al. 2002a). These results show that ethylene promotes leaf injury after O3 exposure. After O3 exposure, the O3-sensitive tobacco cultivar Bel W3 accumulates more SA than does the O3-tolerant tobacco Bel B (Pasqualini et al. 2002). Transgenic tobacco with bacterial salicylic hydroxylase (NahG) accumulates little SA and shows reduced leaf injury after O3 exposure (Örvar et al. 1997), indicating that SA enhances O3-induced leaf damage.

It had been thought that SA was synthesized only from phenylalanine via t-cinnamic acid and benzoic acid in tobacco, potato and Arabidopsis (Leon et al. 1993, Yalpani et al. 1993, Mauch-Mani and Slusarenko 1996, Coquoz et al. 1998, Ribnicky et al. 1998). Phenylalanine ammonia-lyase (PAL) forms t-cinnamic acid from phenylalanine and is a rate-limiting enzyme in the production of phenylpropanoid compounds in tobacco (Bate et al. 1994, Howles et al. 1996). SA levels are diminished in response to tobacco mosaic virus (TMV) inoculation in transgenic tobacco plants in which endogenous PAL expression is suppressed (Pallas et al. 1996). Application of 2-aminoindan-2-phosphonic acid, which is an inhibitor of PAL, inhibits SA accumulation in pathogen-infected Arabidopsis and elicitor-treated potato (Mauch-Mani and Slusarenko 1996, Coquoz et al. 1998). These reports suggest that PAL is an important enzyme in the pathway of SA synthesis. In addition, benzoic acid 2-hydroxylase (BA2H) catalyzes the synthesis of SA from benzoic acid, and this activity is increased in TMV-inoculated tobacco (Leon et al. 1993), implying that BA2H is also involved in SA synthesis. However, Wildermuth et al. (2001) found a new SA synthesis pathway that runs from chorismate via isochorismate in pathogen-infected Arabidopsis. It has been reported that Pseudomonas aeruginosa has this pathway and that isochorismate synthase (ICS) is the rate-limiting enzyme for SA synthesis in this bacterium (Gaille et al. 2003). Thus, although these two SA synthesis pathways have been clarified (Fig. 1), the biosynthetic pathway of SA in O3-stressed plants has not been elucidated. To identify the O3-inducible SA synthetic pathway in tobacco, we investigated the transcript levels of enzymes involved in SA synthesis and the metabolic effect of the synthesized SA.

Ethylene and SA have a common role that regulates leaf injury after O3 exposure (Pell et al. 1997, Rao et al. 2000, Rao and Davis 2001, Overmyer et al. 2003). However, the details of their roles in O3-induced damage are unclear. In O3-exposed tobacco, SA synthesis occurred later than ethylene production. Therefore, ethylene seems to regulate SA biosynthesis. To elucidate the effect of ethylene on SA synthesis in O3-exposed tobacco, we measured the SA level and the transcript levels of enzymes involved in SA biosynthesis in transgenic tobacco with reduced O3-induced ethylene biosynthesis.

Results

Ozone-induced leaf injury correlated with SA synthesis

To determine when O3 treatment induces leaf injury, we measured ion leakage from O3-exposed tobacco leaves; this value serves as a quantitative indicator of the extent of leaf injury. Ion leakage from the second and third leaves began to increase 4 h after the start of O3 exposure and reached 57 and 73%, respectively, at 8 h (Fig. 2a). Ion leakage from the first leaf did not increase after O3 fumigation. It is clear that O3-inducible damage had occurred by 4 h after the start of O3 exposure.

We then examined the accumulation of free SA in O3-exposed tobacco. SA started to accumulate at 4 h and the value at 6 h was the maximum (Fig. 2b). In addition, SA accumulation occurred concomitantly with the increase of ion leakage from leaves (Fig. 2a, b). In the absence of O3, SA was not detected. Negligible glucosylated SA accumulated during the exposure period (data not shown).

Biosynthetic pathway of SA induced by O3 treatment in tobacco

Two SA synthesis pathways have been reported in pathogen-infected plants: one is regulated by chorismate mutase (CM), phenylalanine ammonia-lyase (PAL) and BA2H, and the other is regulated by isochorismate synthase (ICS) and isochorismate pyruvate lyase (Fig. 1; Shah 2003). However, the pathway used for SA synthesis in O3-stressed tobacco has not yet been evaluated. To examine the levels of transcripts for these enzymes, we isolated partial cDNAs encoding CM, PAL and ICS by reverse transcription–polymerase chain reaction (RT–PCR) of total RNA from 4- to 5-week-old tobacco with O3 exposure. The deduced amino acid sequence of tobacco CM (AB182997) was 70% identical to that of the CM of Lycopersicon esculentum. The deduced amino acid sequence of NtICS (AB182580) was compared with other plant ICS gene products and showed identitiy to those of CrICS from Catharanthus roseus (69% identity), and AtICS1 (61% identity) and AtICS2 (60% identity) from Arabidopsis. NtICS seems to contain part of a chorismate-binding domain that is conserved in all ICS genes (Fig. 3a). To clarify how many ICS genes tobacco presented, we carried out Southern blot analysis by using NtICS as a probe. The cDNA of NtICS has a restriction site for the restriction enzyme DraI, but not for EcoRI or HindIII. When tobacco genomic DNA was digested with DraI, we identified two bands (Fig. 3b). On the other hand, we detected only a single band with EcoRI or HindIII digestion. This result suggested that NtICS originally had one copy in the genome. From the structural features of NtICS and the results of Southern blot analysis, NtICS might be a tobacco ortholog of ICS genes of C. roseus and Arabidopsis.

The transcript levels of CM and ICS were examined by using RNA blot analysis. Tobacco has two PAL genes, PAL A (AB008199) and PAL B (AB008200), and the identity of their DNA sequences is high (85% identity). To distinguish the transcript levels of PAL A and PAL B, we constructed specific primers for these genes, and then performed RT–PCR as a tool for expression analysis. The level of transcripts for CM increased slightly after O3 exposure (Fig. 4a). The patterns of transcript levels for PAL A and PAL B were similar; the levels began to increase at 2 h, peaked at 4 h and had slightly decreased at 6 h (Fig. 4b). Transcript levels of ICS in tobacco, however, did not increase with O3 exposure (Fig. 4a). Moreover, ICS activity in O3-exposed tobacco was not increased, whereas the level of SA was increased (Table 1). These results suggest that O3-induced SA synthesis in tobacco leaves might occur through an increase in the transcript levels of CM, PALA and PAL B, but not by participation of ICS. It is well known that the level of ICS1 mRNA increases in pathogen-infected Arabidopsis leaves (Wildermuth et al. 2001). We examined ICS activity in O3-exposed Arabidopsis. ICS activity was increased in wild-type plants, which accumulated a high level of SA (Table 1). Further, the transcript level of ICS1 was increased during O3 exposure in wild-type Arabidopsis (Fig. 4c). However, plants with salicylic acid induction-deficient 2 (sid2), which has defects in ICS1, showed low levels of ICS activity and SA during O3 exposure (Table 1). Therefore, our results show that O3 induces SA synthesis via isochorismate in wild-type Arabidopsis.

To investigate more clearly which pathway was used for SA synthesis in O3-exposed tobacco, we applied a radiolabeled precursor of SA, [14C]benzoic acid, to O3-fumigated leaves and measured the incorporation of radioactivity into the SA. When [14C]benzoic acid was applied, 3.8% (±1.3%) of the benzoic acid was metabolized to SA in O3-exposed plants, and 0.9% (±0.4%) in control plants. The incorporation of radioactivity into SA in O3-exposed plants was about four times as high as that in control plants (Fig. 5). This result also shows that the pathway of SA synthesis via benzoic acid is induced when tobacco plants are fumigated with O3.

Reduced SA levels in transgenic tobacco with reduced ethylene production after O3 exposure

Ethylene emission in tobacco began to increase 1 h after O3 exposure (Fig. 6a), whereas SA accumulation began to increase at 4 h (Fig. 2b). Because SA synthesis occurred later than ethylene production, ethylene may regulate SA synthesis. To clarify whether ethylene participates in the regulation of SA synthesis during O3 exposure, we carried out a physiological analysis of ethylene synthesis-modified plants. We used transgenic tobacco (As-line4) that contained antisense DNA for O3-inducible ACS from tomato leaves. In As-line4 plants, antisense RNA for LE-ACS6 was constitutively detected (data not shown).

As As-line4 is a new transgenic line and the experimental conditions were different from those in the report of Nakajima (2002a), we first compared the levels of ethylene production in wild-type tobacco and As-line4 during O3 exposure. In wild-type plants, ethylene emission began to increase 1 h after O3 exposure and the value at 6 h was the highest (Fig. 6a). In contrast, ethylene production in As-line4 was about half that in wild-type tobacco at 4 and 6 h. Moreover, the level of 1-aminocyclopropane-1-carboxylate (ACC), a precursor of ethylene, was increased 6-fold 4 h after the start of O3 fumigation in wild-type plants, whereas in O3-exposed As-line4 plants it was half that in the wild type (Fig. 6b).

When wild-type plants were exposed to 0.2 ppm of O3 for 6 h, their leaves withered and then turned dark brown 18 h after exposure. Spotted injuries appeared on the first leaf, and extensive wilting occurred in the second and third leaves (Fig. 7a). In transgenic plants, leaf damage was moderated, and the extent of leaf injury in the first, second and third leaves of As-line4 was less than in the wild type (Fig. 7b).

Ion leakage from the second and third leaves of wild-type plants began to increase at 4 h and reached 55 and 76%, respectively, 8 h after the start of O3 exposure (Fig. 7c). In contrast, ion leakage from the second and third leaves was 25 and 33%, respectively, in As-line4 plants after 8 h of O3 exposure. Neither type of plant showed increased ion leakage in the first leaves.

In wild-type plants, SA started to accumulate at 4 h and reached the highest level at 6 h (Fig. 8a). Although SA accumulation in As-line4 plants also reached the highest level at 6 h, the amount was one-fifth that of wild-type tobacco.

Ethylene enhances expression of enzymes to form the precursor of SA

We compared the levels of transcripts associated with SA synthesis in O3-exposed wild-type and transgenic plants. In O3-exposed wild-type plants, the transcript levels of CM, PAL A and PAL B increased remarkably after the onset of O3 exposure (Fig. 9a). Transcript levels in O3-exposed As-line4 also increased, but to a lesser extent. PAL activity in wild-type plants began to increase 2 h after O3 exposure and the value at 6 h was the maximum (Fig. 8b). However, PAL activity in As-line4 at 6 h after the start of O3 exposure was half that in wild-type plants. These results indicate that O3-induced ethylene production enhances the levels of transcripts of CM, PAL A and PAL B.

It is well known that the Bel W3 tobacco cultivar is sensitive to O3 and that Bel B is tolerant to it (Heggestrad 1991). Bel W3 produces a greater amount of ethylene than Bel B after O3 exposure (Schraudner et al. 1998). To confirm whether ethylene increases the transcript levels of CM, PAL A and PAL B in non-transgenic plants, we compared the levels of transcripts of these genes in O3-exposed leaves of Bel W3 and Bel B. At 4 and 6 h after the start of O3 exposure, Bel W3 plants accumulated higher levels of CM transcript than did Bel B plants (Fig. 9c). Furthermore, the levels of PAL A and PAL B transcripts in Bel W3 were increased earlier and higher than those in Bel B (Fig. 9d). These results indicate that plants producing more O3-induced ethylene have higher levels of expression of CM, PAL A and PAL B.

Discussion

Salicylic acid-induced leaf injury in O3-exposed tobacco

We showed here that O3 exposure induced SA accumulation and increased ion leakage in tobacco (Fig. 2). The extent of leaf injury in transgenic tobacco plants with ectopic expression of NahG, which fail to accumulate SA, is attenuated in comparison with that in wild-type tobacco (Örvar et al. 1997). It is also reported that transgenic Arabidopsis plants of the Cvi-0 ecotype expressing NahG show reduced O3-induced SA accumulation and less damage than wild-type plants (Rao et al. 2000). These previous reports and our results support the hypothesis that SA leads to the formation of leaf damage in tobacco after O3 treatment.

Ion leakage in the first leaves did not increase in our study (Fig. 2a). The reason why younger leaves were more resistant to O3 might be their higher superoxide dismutase activity, which converts superoxide radicals into H2O2, and/or their increased content of ascorbate, which serves as an antioxidant (Lee and Bennett 1982, Morabito and Guerrier 2000).

Pathway of salicylic acid synthesis in O3-exposed tobacco and Arabidopsis

We showed that O3 exposure simultaneously induced increases in the transcript levels of CM, PAL A and PAL B in tobacco and in PAL enzyme activity (Table 1, Fig. 4a, b, 9). Furthermore, radiolabeled benzoic acid was metabolized to SA more effectively in O3-exposed tobacco (Fig. 5). These results indicate that O3 induced SA synthesis by way of phenylalanine and benzoic acid. This hypothesis is supported by the results of Pasqualini et al. (2002), who reported that, in tobacco, O3 increased the activity of BA2H, which catalyzes conversion of benzoic acid to SA. On the other hand, although slight expression of NtICS was detected in both control and O3-exposed tobacco, no ICS activity was detected in these plants (Table 1 and Fig. 4a). These results also fortify the above conclusion that O3-induced SA synthesis in tobacco occurs through a pathway involving phenylalanine.

In contrast, ICS activity and ICS1 expression were increased in response to O3 exposure in Arabidopsis (Table 1, Fig. 4c). Furthermore, much lower levels of SA were accumulated under O3 fumigation in sid2 mutants, which are defective in ICS1 (Table 1). These results imply that SA was essentially synthesized via isochorismate in O3-exposed Arabidopsis. Our results indicate that the pathway of O3-induced SA biosynthesis differs between the two species. Further studies are needed to elucidate why different pathways of SA synthesis are induced by O3 in these species.

Ethylene enhanced SA synthesis in O3-exposed tobacco

It is well known that ethylene and SA promote leaf injury. In our results, ethylene synthesis was induced before the start of SA accumulation in O3-exposed tobacco (Fig. 2b, 6a). Furthermore, increases in the levels of SA and PAL activity and transcripts for CM, PAL A and PAL B were suppressed in As-line4 plants (Fig. 8a, b, 9a, b). Accordingly, our results suggest that ethylene enhances SA synthesis through up-regulation of the transcription levels of CM, PAL A and PAL B. Their transcript levels were increased to a greater extent in Bel W3 plants than in Bel B plants (Fig. 9c, d). Pasqualini et al. (2002) showed that Bel W3 accumulates higher levels of SA than does Bel B after O3 exposure. These findings support the hypothesis that the level of SA is controlled by ethylene during O3 exposure.

Does ethylene directly regulate SA synthesis? It is well known that ethylene production during O3 exposure enhances leaf injury, but treatment with ethylene without O3 exposure dose not induce leaf injury (Mehlhorn 1990, Bae et al. 1996). Therefore, it has been postulated that ethylene induces leaf injury by cooperating with other unknown factors under O3-stressed conditions (Bae et al. 1996). When we applied 1 mM ACC, a precursor of ethylene, to tobacco leaves, they did not show increased PAL activity in fresh air (data not shown). This result is consistent with their physiological observation. Therefore, not ethylene alone, but the co-existence of O3 and ethylene may be required for the induction of SA synthesis.

There have been reports that ethylene promotes SA signaling and accumulation in plants. For example, the ethylene insensitive 2 mutant attenuates SA-dependent cell death of the accelerated cell death mutant in Arabidopsis (Greenberg et al. 2000). Moreover, SA does not accumulate in ethylene-deficient and ethylene signaling-deficient tomato infected with a pathogen (O’Donnell et al. 2001). Although these reports suggest that ethylene induces SA synthesis under stressed conditions, they do not mention how ethylene enhances SA synthesis. Our results demonstrate for the first time that ethylene promotes CM, PAL A and PAL B expression to increase SA synthesis in O3-exposed tobacco leaves.

Contrary to our report, Rao et al. (2002) found that NahG-transformed Arabidopsis and npr1 mutants, which lack SA signaling, failed to produce ethylene in response to O3; they concluded that SA stimulates ethylene synthesis. Why the apparent discrepancy? O3-induced leaf injury is thought to mimic hypersensitivity reactions to infection with an incompatible pathogen (Sandermann et al. 1998, Rao and Davis 2001). Overmyer et al. (2000) postulated that SA and ethylene promote the synthesis of each other through the generation of active oxygen species and cell death. Therefore, the inconsistency between our data and the results of Rao et al. (2002) can be explained in these terms.

We showed that O3-induced SA in tobacco was synthesized from phenylalanine via t-cinnamic acid and benzoic acid and that O3-induced ethylene promoted SA accumulation by enhancing CM, PAL A and PAL B transcript levels. In future work, we need to address how ethylene regulates the levels of mRNA of CM, PAL A and PAL B in O3-exposed tobacco leaves.

Materials and Methods

Plant materials and O3 treatment

Tobacco seeds (Nicotiana tabacum L. cv. SR-1) were germinated on culture soil (Kureha Chemical Industry Co., Tokyo, Japan), and seedlings were grown in a controlled-environment greenhouse at 25°C day/20°C night with a relative humidity of 70% and a 14 h light/10 h dark cycle. Plants were watered daily. We named the fully developed leaf in the highest position the first leaf (see Fig. 7a).

We exposed 4- to 5-week-old plants for 6 h in a chamber to a single dose of 200 nl l–1 O3 produced by an O3 generator (Sumitomo Seika Chemicals, Osaka, Japan). O3 fumigation occurred at 25°C at a relative humidity of 70% under a photosynthetic photon flux density (PPFD) of 200 µmol m–2 s–1 in continuous light. Plants remaining in charcoal-filtered air served as controls. Conditions for growth and O3 treatment of Arabidopsis were set as described by Kanna et al. (2003).

Establishment of transgenic tobacco As-line4

We constructed an extensively O3-resistant transgenic tobacco line by the introduction of antisense DNA for LE-ACS6 isolated from tomato leaves, as reported by Nakajima et al. (2002a). The transgenic plants had the same phenotype as that of the AsACS1 previously reported (Nakajima et al. 2002a). We named our transgenic tobacco As-line4. In As-line4, antisense LE-ACS6 was expressed constitutively (data not shown).

Measurement of ethylene production

Ethylene production was determined as reported by Bae et al. (1996), with minor modifications. At sampling time, the first to third leaves were removed from the plants and incubated in sealed 100 ml flasks under light for 1 h. Then 1 ml of gas was withdrawn from the flasks and the ethylene content was analyzed by using a gas chromatograph equipped with a flame ionization detector (GC-7 A; Shimadzu, Osaka, Japan).

Measurement of 1-aminocyclopropane-1-carboxylate

ACC was extracted and quantified as described by Langebartels et al. (1991). Leaf samples (0.5 g) were extracted four times with 1 ml of absolute methanol. The upper phase was concentrated to dryness and dissolved in 1 ml of water, and lipophilic substances were removed by extraction with chloroform. The upper phase was concentrated to dryness and dissolved in 1 ml of water. The amount of ACC was determined according to Lizada and Yang (1979).

Extent of leaf injury

The extent of injury was measured as described by Nakajima et al. (2002a). Plants were exposed to O3 for 6 h then transferred to a fresh-air chamber in continuous light. At 24 h after the start of O3 exposure, the first, second and third leaves were excised and scanned (GT7600U; Epson Tokyo, Japan) into a computer. The area of visible damage on each leaf was calculated by using image analysis software (NIH Image, National Institutes of Health, Washington, DC, USA).

Ion leakage measurement

Ion leakage was measured as reported by Tamaoki et al. (2003), with minor modifications. Two leaf disks (diameter, 10 mm) from the second and third leaves were floated on 1 ml of distilled water for 1 h with shaking at 100 rpm. The conductivity of 100 µl from the water bath was determined with a conductivity meter (B-173; Horiba, Kyoto, Japan). The leaves were then autoclaved together with the remaining water (900 µl), and the conductivity of the autoclaved solution was also measured. The relative ion leakage was obtained by dividing the conductivity of the pre-autoclaved solution by that of the autoclaved solution.

Preparation of cDNA probes

cDNAs of CM and ICS in tobacco and ICS1 in Arabidopsis were isolated by RT–PCR using total RNA obtained from O3-exposed tobacco. The primers for RT–PCR were designed according to the published cDNA sequences for various plants (for CM, 5′- CTTCAATCTAAGGTTGGTAGAT-3′ and 5′-TTAGTCAAAGGCATAACCCATTC-3′; ICS, 5′-ATGCATATCAGTTCTGTTTGCAA-3′ and 5′-CCAGCATACATTCTTCGGTCAAA-3′; and AtICS1, 5′-ATGGCTTCACTTCAATTTTCTTC-3′ and 5′-TCAATTAATCGCCTGTAGAGATG-3′). The amplified cDNAs were subcloned into a pGEM-T Easy system (Promega, WI, USA) and sequenced with an ALFred sequencer (Amersham Biosciences, Piscataway, NJ, USA).

RNA gel blot analysis

Total RNA from leaves was extracted by using the SDS–phenol method as described by Nakajima et al. (1995). Total RNA was separated by electrophoresis through a 1.2% agarose gel that contained 1.8% formaldehyde and then was transferred to a nylon membrane (Hybond N+; Amersham Biosciences). Pre-hybridization and hybridization were performed as described by Tamaoki et al. (2003). The probe was prepared by using the MultiPrime labeling system (Amersham Biosciences) with [32P]dCTP (12 MBq mol–1). The filter was washed with 2× SSC containing 0.1% SDS at 50°C, and then 0.2× SSC containing 0.1% SDS at 55°C. The filter was exposed to a Bio-Imaging Plate (Fuji Photo Film Co., Ltd., Tokyo, Japan), and signals were assessed using a bioimaging analyzer (BAS2000; Fuji Photo Film Co., Ltd.).

RT–PCR for expression analysis

Total RNA (0.1 µg) was analyzed by PCR with Ready-To-Go RT–PCR Beads (Amersham Biosciences). The primer set used for PAL A was 5′-GCACAAAATGGTCACCAAGAAA-3′ and 5′-AAGCCATTGGGGCGACGTTCTA-3′, and that for PAL B was 5′-CATGTTAATGGAGGAGAAAACT-3′ and 5′-AAGCCATTGTGGAGATGTTCGG-3′.

Reverse transcription was performed at 42°C for 30 min. PCR amplification was carried out for 15 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 2 min and elongation at 72°C for 3 min. After electrophoresis in a 1.2% agarose gel, the amplified fragments were blotted onto a nylon membrane, hybridized to the cDNA probe and washed as described above. The cDNAs amplified by the PAL A and PAL B primers were identical to PAL A (AB008199) and PAL B (AB008120), respectively.

Southern blot analysis

Southern blot analysis was carried out as described in Sambrook et al. (1989). Hybridization was performed at 65°C in a solution containing 0.5 M Na2HPO4, 1 mM EDTA and 7% SDS. The filter was washed at 42°C with 2× SSC containing 0.1% SDS. Washing was then done at 50°C with 0.2× SSC containing 0.1% SDS.

PAL activity

PAL activity was measured as reported by Legrand et al. (1976), with minor modifications. Plant materials were ground with a mortar and pestle in liquid nitrogen. A 100 µg aliquot of the sample was transferred to another mortar with 1 ml of extraction buffer (0.1 M borate buffer, pH 8.8, and 5 mM mercaptoethanol). After stirring, the homogenate was centrifuged at 20,000×g for 10 min. The supernatant was desalted in a Sephadex G-25 column (NAP-10 Columns, Amersham Biosciences) equilibrated with the extraction buffer. The extraction was performed at 4°C.

The incubation mixture contained 500 µl of the desalted solution and 33 µl of 2 mM phenylalanine, including 0.0925 MBq of l-[U-14C]phenylalanine. After 1 h of incubation at 37°C, the reaction was stopped by the addition of 33 µl of 4.5 M sulfuric acid. A 500 µl aliquot of toluene was added to the incubation mixture, and t-cinnamic acid was extracted to the organic phase. The radioactivity in the organic phase was determined using a liquid scintillation analyzer (2500TR, Perkin Elmer, Foster City, CA, USA).

SA extraction and quantification

SA was extracted from 0.5 g of tobacco leaves and 0.2 g of Arabidopsis leaves. Each sample was extracted four times with 1.5 ml of methanol. We added 5 µl of 1 mg ml–1m-hydroxybenzoic acid as an internal standard. The solution was evaporated to dryness; the residue was dissolved in 200 µl of methanol, then 1 ml of 1 mM KOH was added. Lipophilic substances were removed by extraction twice with chloroform. The aqueous phase was transferred to a new tube, and then we added 10 µl of phosphoric acid and 700 µl of ethyl acetate. The solution was mixed and centrifuged at 17,000×g for 10 min. The supernatant was transferred to a new tube and again extracted with ethyl acetate. All supernatants were evaporated to dryness, and the residue was dissolved in 50% methanol and analyzed by high-performance liquid chromatography (HPLC; System Gold, Beckman, Fullerton, CA, USA). SA was detected with a fluorescent detector (RF-530, Shimadzu, Osaka, Japan) using Ex = 295 nm and Em = 370 nm. The mobile phase was 20 mM sodium acetate (pH 2.5) containing 20% methanol. To determine the total amount of SA produced, the extract was treated with β-glucosidase, as reported by Nakajima et al. (2002b), and analyzed by HPLC.

ICS activity

Extraction and assay of ICS were performed according to the method of Poulsen et al. (1991). Plant materials were ground using a mortar and pestle with liquid nitrogen. Then 1.5 g of material was transferred to another mortar in which 0.05 g of polyvinylpolypyrrolidone (PVPP) and 2 ml of extraction buffer [0.1 M Tris–HCl, pH 7.5, 10% glycerol, 1 mM EDTA, and 1 mM dithiothreitol (DTT)] was added. After stirring, the homogenate was centrifuged at 10,000×g for 30 min. The supernatant was desalted in a Sephadex G-25 column (PD-10 Empty Columns, Amersham Biosciences) equilibrated with 0.1 M Tris–HCl (pH 7.5) containing 10% glycerol, 1 mM EDTA and 1 mM DTT. The process was performed at 4°C.

The incubation mixture (total volume 0.5 ml) contained 250 µl of 0.1 M Tris–HCl (pH 7.5), 3 mM Ba-chorismate (Sigma, St. Louis, MO, USA), 15 mM MgCl2 and 250 µl of desalted solution. The reaction was performed after 1 h of incubation at 30°C. The isochorismate produced was quantified by the method of Young and Gibson (1969).

Protein determination

The level of protein was investigated by using a BCA protein assay kit (Pierce, Rockford, IL, USA).

Incorporation of radiolabeled benzoic acid into SA

14C-Labeled benzoic acid (about 400 pmol, 2.2 mCi) (American Radiolabeled Chemicals, Inc., St. Louis, MO, USA) was applied to tobacco leaves 4 h after the start of O3 exposure. Radiolabeled benzoic acid was infiltrated from the abaxial surface of the leaf into the apoplast via a 1 ml syringe. At 2 h after application, the metabolites of benzoic acid were extracted with methanol and the extract was evaporated to dryness. The dried materials were dissolved in 20 mM sodium acetate (pH 5) containing 20% methanol and then analyzed by HPLC. Radioactivity was detected using a liquid scintillation analyzer (2500TR, Packard).

Acknowledgments

The authors gratefully acknowledge the skillful technical assistance of Hideko Watanabe, Katsumi Matsumoto, Yukiko Matsumoto and Teruko Okubo, and the O3 chamber administrator, Yoshimitsu Takimoto. We thank Dr. Christiane Nawrath for providing sid2-1 seeds. This research was partly supported by a Sasakawa Scientific Research Grant from the Japan Science Society.

Fig. 1 Proposed pathways of SA biosynthesis.

Fig. 1 Proposed pathways of SA biosynthesis.

Fig. 2 Ion leakage from, and SA level of, tobacco leaves during O3 exposure. Vertical bars represent SDs obtained from three replicates. (a) Ion leakage in the first to third leaves of tobacco. Tobacco was exposed to O3 for 6 h and was then transferred to a free-air room. Open circle, third leaves; open triangle, second leaves; open square, first leaves. (b) Salicylic acid levels in tobacco exposed to 0.2 ppm of O3 for 6 h. Filled circle, O3 condition; open square, control. The solid horizontal axis indicates the period of O3 fumigation.

Fig. 2 Ion leakage from, and SA level of, tobacco leaves during O3 exposure. Vertical bars represent SDs obtained from three replicates. (a) Ion leakage in the first to third leaves of tobacco. Tobacco was exposed to O3 for 6 h and was then transferred to a free-air room. Open circle, third leaves; open triangle, second leaves; open square, first leaves. (b) Salicylic acid levels in tobacco exposed to 0.2 ppm of O3 for 6 h. Filled circle, O3 condition; open square, control. The solid horizontal axis indicates the period of O3 fumigation.

Fig. 3 (a) Clustal alignment of partial NtICS sequence in tobacco with ICS in other plants. NtICS (AB182580), tobacco; CrICS (AJ006065), Catharanthus roseus; AtICS1 (At1g74710) and AtICS2 (At1g18870), Arabidopsis. (b) Southern blot analysis of ICS in tobacco. A 20 µg aliquot of total DNA was digested with DraI, EcoRI and HindIII. Hybridization was performed with isolated cDNA as a probe.

Fig. 3 (a) Clustal alignment of partial NtICS sequence in tobacco with ICS in other plants. NtICS (AB182580), tobacco; CrICS (AJ006065), Catharanthus roseus; AtICS1 (At1g74710) and AtICS2 (At1g18870), Arabidopsis. (b) Southern blot analysis of ICS in tobacco. A 20 µg aliquot of total DNA was digested with DraI, EcoRI and HindIII. Hybridization was performed with isolated cDNA as a probe.

Fig. 4 Transcript levels of CM, ICS and PALs in O3-exposed tobacco and of ICS1 in O3-exposed Arabidopsis. Numbers show the hours after the start of O3 exposure. Columns labeled ‘rRNA’ and ‘Actin’ indicate equal loading of RNA. (a) RNA gel blot analysis of ICS and CM in tobacco. Total RNAs were obtained from the first to third leaves of wild-type tobacco with or without exposure to 0.2 ppm of O3. (b) RT–PCR analysis for PAL A and PAL B. (c) RNA gel blot analysis for ICS1 in Arabidopsis. Total RNAs were obtained from shoots of Arabidopsis exposed to 0.2 ppm of O3.

Fig. 4 Transcript levels of CM, ICS and PALs in O3-exposed tobacco and of ICS1 in O3-exposed Arabidopsis. Numbers show the hours after the start of O3 exposure. Columns labeled ‘rRNA’ and ‘Actin’ indicate equal loading of RNA. (a) RNA gel blot analysis of ICS and CM in tobacco. Total RNAs were obtained from the first to third leaves of wild-type tobacco with or without exposure to 0.2 ppm of O3. (b) RT–PCR analysis for PAL A and PAL B. (c) RNA gel blot analysis for ICS1 in Arabidopsis. Total RNAs were obtained from shoots of Arabidopsis exposed to 0.2 ppm of O3.

Fig. 5 Incorporation of radioactivity from [14C]benzoic acid (BA) into salicylic acid (SA) in O3-exposed tobacco leaves. Wild-type plants were exposed to 0.2 ppm of O3 for 4 h and then treated with 14C-labeled benzoic acid. The amount of SA in the methanol extracts of leaves 2 h after the application of 14C-labeled benzoic acid was analyzed by HPLC. Under these conditions, 3.8% (±1.3%) of the applied benzoic acid was metabolized to SA in O3-exposed plants and 0.9% (± 0.4%) in the control plants. The asterisk indicates a significant difference (P < 0.05; Student’s t-test) from the corresponding control data set. Vertical bars represent SDs obtained from four (ambient air) or five (O3) replicates.

Fig. 5 Incorporation of radioactivity from [14C]benzoic acid (BA) into salicylic acid (SA) in O3-exposed tobacco leaves. Wild-type plants were exposed to 0.2 ppm of O3 for 4 h and then treated with 14C-labeled benzoic acid. The amount of SA in the methanol extracts of leaves 2 h after the application of 14C-labeled benzoic acid was analyzed by HPLC. Under these conditions, 3.8% (±1.3%) of the applied benzoic acid was metabolized to SA in O3-exposed plants and 0.9% (± 0.4%) in the control plants. The asterisk indicates a significant difference (P < 0.05; Student’s t-test) from the corresponding control data set. Vertical bars represent SDs obtained from four (ambient air) or five (O3) replicates.

Fig. 6 Ethylene emission by transgenic (As-line4) and wild-type tobacco plants during O3 exposure. Vertical bars represent SDs obtained from three replicates. (a) Ethylene production in these plants. We exposed 4- or 5-week-old plants to 0.2 ppm of O3 for 6 h. Filled circle, wild-type; open square, As-line4. (b) Levels of ACC 4 h after the start of O3 exposure in wild-type and As-line4 plants. White and gray bars show wild-type and As-line4 plants, respectively. The asterisk indicates a significant difference (P < 0.05; Student’s t-test) from the corresponding data set for wild-type plants.

Fig. 6 Ethylene emission by transgenic (As-line4) and wild-type tobacco plants during O3 exposure. Vertical bars represent SDs obtained from three replicates. (a) Ethylene production in these plants. We exposed 4- or 5-week-old plants to 0.2 ppm of O3 for 6 h. Filled circle, wild-type; open square, As-line4. (b) Levels of ACC 4 h after the start of O3 exposure in wild-type and As-line4 plants. White and gray bars show wild-type and As-line4 plants, respectively. The asterisk indicates a significant difference (P < 0.05; Student’s t-test) from the corresponding data set for wild-type plants.

Fig. 7 Damage in wild-type and As-line4 tobacco leaves exposed to 0.2 ppm of O3. Wild-type and As-line4 tobacco leaves were exposed to 0.2 ppm of O3 for 6 h and then cultivated in O3-free air for 18 h. (a) Photo-image of O3-induced damage. Numbers indicate the first, second and third leaves. (b) Extent of visible injury on the leaves of transgenic (As-line4) and wild-type tobacco after O3 exposure. White and gray bars show wild-type and As-line4 plants, respectively. Areas of leaf injury on the first, second and third leaves were measured with an image analyzer. White bar, wild-type; gray bar, As-line4. Asterisks indicate significant difference (P < 0.05; Student’s t-test) from the corresponding data set of wild-type plants. Vertical bars represent SDs obtained from 10 replicates. (c) Ion leakage from wild-type and As-line4 tobacco leaves exposed to O3 for 6 h, then left in ambient air for 6 h. The extent of ion leakage was measured at various times after the onset of O3 fumigation. The solid horizontal axis indicates the period of O3 fumigation. Vertical bars represent SDs obtained from six replicates. Open circle, third leaves; open triangle, second leaves; open square, first leaves; solid line, wild-type plants; dotted line, As-line4 plants.

Fig. 7 Damage in wild-type and As-line4 tobacco leaves exposed to 0.2 ppm of O3. Wild-type and As-line4 tobacco leaves were exposed to 0.2 ppm of O3 for 6 h and then cultivated in O3-free air for 18 h. (a) Photo-image of O3-induced damage. Numbers indicate the first, second and third leaves. (b) Extent of visible injury on the leaves of transgenic (As-line4) and wild-type tobacco after O3 exposure. White and gray bars show wild-type and As-line4 plants, respectively. Areas of leaf injury on the first, second and third leaves were measured with an image analyzer. White bar, wild-type; gray bar, As-line4. Asterisks indicate significant difference (P < 0.05; Student’s t-test) from the corresponding data set of wild-type plants. Vertical bars represent SDs obtained from 10 replicates. (c) Ion leakage from wild-type and As-line4 tobacco leaves exposed to O3 for 6 h, then left in ambient air for 6 h. The extent of ion leakage was measured at various times after the onset of O3 fumigation. The solid horizontal axis indicates the period of O3 fumigation. Vertical bars represent SDs obtained from six replicates. Open circle, third leaves; open triangle, second leaves; open square, first leaves; solid line, wild-type plants; dotted line, As-line4 plants.

Fig. 8 Salicylic acid and PAL activity in wild-type and As-line4 tobacco leaves exposed to 0.2 ppm of O3. (a) Salicylic acid levels of the first to third leaves of wild-type and As-line4 tobacco exposed to 0.2 ppm of O3 for 6 h. (a) SA levels are shown. The horizontal axis indicates the period of O3 fumigation. Filled circle, wild-type; open square, As-line4. Vertical bars represent SDs obtained from three replicates. (b) PAL activity of the first to third leaves of wild-type and transgenic (As-line4) tobacco exposed to 0.2 ppm of O3 for 6 h. The solid horizontal axis indicates the period of O3 fumigation. Filled circle, wild-type; open square, As-line4. Vertical bars represent SDs obtained from three replicates.

Fig. 8 Salicylic acid and PAL activity in wild-type and As-line4 tobacco leaves exposed to 0.2 ppm of O3. (a) Salicylic acid levels of the first to third leaves of wild-type and As-line4 tobacco exposed to 0.2 ppm of O3 for 6 h. (a) SA levels are shown. The horizontal axis indicates the period of O3 fumigation. Filled circle, wild-type; open square, As-line4. Vertical bars represent SDs obtained from three replicates. (b) PAL activity of the first to third leaves of wild-type and transgenic (As-line4) tobacco exposed to 0.2 ppm of O3 for 6 h. The solid horizontal axis indicates the period of O3 fumigation. Filled circle, wild-type; open square, As-line4. Vertical bars represent SDs obtained from three replicates.

Fig. 9 Transcript levels of CM and PALs in O3-exposed tobacco plants. Total RNAs were obtained from the first to third leaves from each plant. Numbers show hours after the start of O3 exposure. Columns labeled ‘rRNA’ and ‘Actin’ indicate equal loading of RNA. (a) RNA gel blot analysis for CM in wild-type tobacco and As-line4. (b) RT–PCR analysis for PAL A and PAL B. (c) RNA gel blot analysis for CM in Bel B and Bel W3. (d) RT–PCR analysis for PAL A and PAL B in Bel B and Bel W3.

Fig. 9 Transcript levels of CM and PALs in O3-exposed tobacco plants. Total RNAs were obtained from the first to third leaves from each plant. Numbers show hours after the start of O3 exposure. Columns labeled ‘rRNA’ and ‘Actin’ indicate equal loading of RNA. (a) RNA gel blot analysis for CM in wild-type tobacco and As-line4. (b) RT–PCR analysis for PAL A and PAL B. (c) RNA gel blot analysis for CM in Bel B and Bel W3. (d) RT–PCR analysis for PAL A and PAL B in Bel B and Bel W3.

Table 1

ICS activity and SA levels in O3-exposed plants

 Tobacco  Arabidopsis  sid2 in Arabidopsis 
Ozone exposure time (h)   
ICS activity (ng/mg protein/h) nd nd  nd 24.7 ± 2.48  nd nd 
SA level (nmol/g FW) 0.0 ± 0.0 7.3 ± 2.0  0.3 ± 0.1 18.9 ± 3.20  0.7 ± 0.3 1.4 ± 0.3 
 Tobacco  Arabidopsis  sid2 in Arabidopsis 
Ozone exposure time (h)   
ICS activity (ng/mg protein/h) nd nd  nd 24.7 ± 2.48  nd nd 
SA level (nmol/g FW) 0.0 ± 0.0 7.3 ± 2.0  0.3 ± 0.1 18.9 ± 3.20  0.7 ± 0.3 1.4 ± 0.3 

ICS activity and SA levels were measured in tobacco and Arabidopsis exposed to 0.2 ppm of O3 for 6 h. Values given represent the means ± SDs from three independent experiments. Nd, not detected; sid2, salicylic acid induction deficient2 mutant Arabidopsis.

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