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. 2003 Apr;15(4):863–873.

A Diterpene as an Endogenous Signal for the Activation of Defense Responses to Infection with Tobacco mosaic virus and Wounding in Tobacco

Shigemi Seo a,b, Hideharu Seto c, Hiroyuki Koshino c, Shigeo Yoshida c, Yuko Ohashi a,b,1
PMCID: PMC152335  PMID: 12671083

Abstract

In pathogen-infected or wounded tobacco plants, the activation of wound-induced protein kinase (WIPK), a tobacco mitogen-activated protein kinase, has been implicated in the defense response. However, no endogenous signal responsible for the activation has been identified. A WIPK-activating substance was isolated from tobacco leaves and identified as (11E,13E)-labda-11,13-diene-8α,15-diol, designated WAF-1. When applied in nanomolar concentrations to leaves, either natural WAF-1 or chemically synthesized WAF-1 activated WIPK as well as salicylic acid–induced protein kinase, a tobacco mitogen-activated protein kinase, and enhanced the accumulation of transcripts of wound- and pathogen-inducible defense-related genes. Quantitative analysis of endogenous WAF-1 revealed that levels increased rapidly in leaves during a hypersensitive response to Tobacco mosaic virus (TMV) and after wounding. Furthermore, treatment of leaves with WAF-1 resulted in enhanced resistance to TMV infection. These results suggest that WAF-1 functions as an endogenous signal to mediate the defense responses of tobacco plants to TMV infection and wounding.

INTRODUCTION

Plants respond to infection by pathogens and wounding by producing low molecular mass defensive compounds, such as salicylic acid (SA), nitric oxide, jasmonic acid (JA), ethylene, polyamines, and reactive oxygen species, which induce the expression of a set of genes that encode defense-related proteins such as pathogenesis-related (PR) proteins that are involved mostly in disease resistance or wound healing (for reviews, see Creelman and Mullet, 1997; Dong, 1998; Reymond and Farmer, 1998; de Bruxelles and Roberts, 2001; Wendehenne et al., 2001; Jameson and Clarke, 2002). The molecular mechanisms that underlie the production of such chemical signals and the subsequent expression of defense-related genes are not fully understood. Recent studies have indicated that mitogen-activated protein kinases (MAPKs) play a key role in such signal transduction pathways (for reviews, see Ichimura et al., 2000; Meskiene and Hirt, 2000; Tena et al., 2001; Zhang and Klessig, 2001).

MAPKs are Ser/Thr protein kinases that have been conserved evolutionarily in eukaryotes. They are activated enzymatically by diverse extracellular stimuli, thereby regulating multiple intracellular responses (Widmann et al., 1999; Chang and Karin, 2001). In tobacco, wound-induced protein kinase (WIPK), a MAPK, is activated in response to wounding (Seo et al., 1999). We have suggested that the activation is involved in the production of wound-induced JA, suggesting that WIPK is a mediator in the wound signal transduction pathway (Seo et al., 1995, 1999). The activation of WIPK also has been implicated in defense responses to pathogens and elicitors (Zhang and Klessig, 1998; Romeis et al., 1999; Yang et al., 2001; Baudouin et al., 2002; Lebrun-Garcia et al., 2002). Despite the important role of WIPK in pathogen and wound signal transduction pathways, no endogenous signal responsible for its activation has been identified.

Here, we report the isolation and chemical identification of a WIPK-activating substance from tobacco leaves. We also provide evidence that this substance functions as an endogenous signal in the induction of WIPK and wound- and pathogen-inducible PR genes and in resistance to Tobacco mosaic virus (TMV) in tobacco plants.

RESULTS

Isolation of a WIPK-Activating Substance from Tobacco Leaves

We assumed that the endogenous signal(s) responsible for activating WIPK would be found in the low molecular mass compounds of tobacco plants after pathogen infection or wounding. As a starting material to search for the signal(s), TMV-infected leaves of tobacco plants carrying the TMV resistance gene N were used, because WIPK is activated markedly during the hypersensitive response (HR) in N gene–containing tobacco cultivars after TMV infection (Zhang and Klessig, 1998; Seo et al., 2001). TMV-inoculated leaves were harvested and extracted with acetone. The extract was divided into ethyl acetate–soluble acidic, neutral, and basic fractions and then tested for its ability to activate WIPK in tobacco leaf discs by determining the kinase activity of WIPK with myelin basic protein (MBP) as an artificial substrate. The activity was found in both acidic and neutral fractions (Figure 1), suggesting the presence of at least two WIPK-activating substances in the extract. Because the neutral fraction contained a higher level of activity, we decided to purify the active substance in this fraction.

Figure 1.

Figure 1.

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Effect of the Acidic, Neutral, and Basic Fractions Prepared from an Acetone Extract of TMV-Inoculated Leaves on the Activation of WIPK.

TMV-inoculated leaves that had been incubated at 30°C for 48 h and then incubated at 20°C for 8 h were extracted with 80% acetone. The extract was divided into ethyl acetate–soluble acidic, basic, and neutral fractions and then tested for its ability to activate WIPK by determining the kinase activity of WIPK with MBP as a substrate, as described in Methods. As a control, water was subjected to the same test. The arrow indicates the position of the phosphorylated MBP.

To isolate the neutral active substance, 15 kg of TMV-inoculated leaves was used. The ethyl acetate–soluble neutral fraction prepared from an acetone extract of these leaves was subjected to successive chromatographic fractionations on silica gel, on C18, and by reversed-phase HPLC (Figure 2). Each fraction was tested for its ability to activate WIPK. A peak fraction obtained at the last HPLC step exhibited activity and was collected to yield 300 μg of a colorless gum.

Figure 2.

Figure 2.

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Flow Diagram for the Isolation and Purification of a WIPK-Activating Substance from TMV-Inoculated Tobacco Leaves.

For details, see Methods.

Chemical Identification of a WIPK-Activating Substance

Mass spectrometry (MS) and NMR spectral data suggested that this substance was a single compound, and it was tentatively proposed to be a labdane-type diterpene, (11E,13E)-labda-11,13-diene-8α,15-diol (Figure 3A, compound 1). Because an amount sufficient for an adequate NMR study to completely determine the structure could not be obtained, compound 1 was synthesized chemically from a commercially available natural product, sclareolide (Figure 3A, compound 2), in an optically pure form (Figure 4). The structure of the synthetic compound was verified by NMR spectroscopy, and all resonances in the 1H and 13C-NMR spectra were fully assigned to compound 1 (see Methods). The retention times of HPLC, and the MS and NMR spectra of the natural compound responsible for activating WIPK, were identical to those of synthetic compound 1, confirming that the structure of the natural compound was the same as that of compound 1. The 1H-NMR spectra of natural and synthetic (11E,13E)-labda-11,13-diene-8α,15-diol are presented in Figure 3B. The identification of compound 1 has not been reported previously. We designated this substance WAF-1 (for WIPK-Activating Factor1).

Figure 3.

Figure 3.

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Chemical Structures of WAF-1 and the Other Compounds Described in the Text.

(A) The numerals denote the International Union of Pure and Applied Chemistry numbering of carbons.

(B) 1H-NMR spectra (600 MHz, CDCl3) of natural and synthetic WAF-1.

Figure 4.

Figure 4.

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Scheme of the Synthesis of WAF-1 and 8-epi-WAF-1.

For details, see Methods. 4Å MS, 4-Å molecular sieve; DIBAL, diisobutylaluminum hydride; NMO, N-methylmorpholine N-oxide; TPAP, tetrapropylammonium perruthenate; Et, ethyl.

Exogenous WAF-1 Activates WIPK and SA-Induced Protein Kinase

To test the effect of WAF-1 on the activation of MAPK, tobacco leaf discs were infiltrated with a solution containing 1 nM natural WAF-1 or water and assayed for the MBP kinase activity of WIPK. At 15 and 30 min after infiltration of the WAF-1 solution, the levels of WIPK activity were enhanced compared with those in water-infiltrated leaves (Figure 5A, top gel). WAF-1 also activated SA-induced protein kinase (SIPK) (Zhang and Klessig, 1997), a tobacco MAPK, at 15 and 30 min after infiltration (Figure 5A, bottom gel). The natural WAF-1 isolated here activated both WIPK and SIPK at concentrations of >10 pM, being maximum at 100 pM to 1 nM (Figure 5B). Synthetic WAF-1 had the same effect as natural WAF-1 (Figure 5C).

Figure 5.

Figure 5.

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Activation of WIPK and SIPK by Exogenous WAF-1.

The harvested leaf discs were used for protein extraction. Crude extracts containing 50 μg of total protein were immunoprecipitated with either anti-WIPK or anti-SIPK antibodies, and the immunoprecipitates were subjected to an immune complex kinase assay with MBP as a substrate. The phosphorylation of MBP was analyzed by autoradiography after SDS-PAGE. Arrows indicate the positions of the phosphorylated MBP. The experiment was repeated three times with similar results.

(A) Leaf discs were floated on water at 24°C for 15 h and infiltrated with water or 1 nM natural WAF-1. Discs were harvested at the times indicated after infiltration.

(B) and (C) Leaf discs that had been floated on water as described in (A) were infiltrated with water or the indicated concentrations of natural WAF-1 (B) or synthetic WAF-1 (C). Discs were harvested at 30 min after infiltration.

Exogenous WAF-1 Enhances the Accumulation of Wound- and Pathogen-Inducible Protein Transcripts

Our previous studies have suggested that WIPK is involved in regulating the expression of the tobacco genes that encode basic PR-1 protein and proteinase inhibitor II (PI-II) (Seo et al., 1995, 1999), which are induced during TMV-induced HR and by wounding (Memelink et al., 1990; Balandin et al., 1995). Therefore, we evaluated the possible effect of exogenous WAF-1 on the accumulation of transcripts of tobacco PR genes. Application of synthetic WAF-1 at 1 nM, at which WIPK was activated effectively, to tobacco leaves enhanced the accumulation of the transcripts of the basic PR-1, basic PR-2, and PI-II genes (Figure 6). The accumulation of the transcripts of these three wound-inducible PR genes was further enhanced by treatment with 100 nM WAF-1. WAF-1 treatment also enhanced the accumulation of the transcript of the gene that encodes 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), which is involved in ethylene biosynthesis (Kende, 1993) and is induced during TMV-induced HR and by wounding (Ohtsubo et al., 1999). By contrast, WAF-1 treatment had no effect on the accumulation of transcripts for the acidic PR-1 and PR-2 genes, which are not induced by wounding (Memelink et al., 1990).

Figure 6.

Figure 6.

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Effect of Exogenous WAF-1 on the Accumulation of Transcripts of Tobacco PR Genes.

Detached leaves were fed through the petiole with water or a solution containing the indicated concentrations of synthetic WAF-1, incubated at 24°C for 48 h under continuous light, and used for total RNA extraction. Healthy leaves that were not treated with WAF-1 or water also were used for total RNA extraction. Aliquots of 20 μg of total RNA per lane were subjected to RNA gel blot analysis with the indicated cDNA probes. To standardize RNA loading, the blot was stained with methylene blue (rRNA). The experiment was repeated three times with similar results.

Effect of Exogenous WAF-1 on the Accumulation of JA and Ethylene

JA is known to function as a signal in the induction of basic PR genes in tobacco plants (Xu et al., 1994; Niki et al., 1998). To assess whether the induction of the basic PR-1, basic PR-2, and PI-II genes by WAF-1 is mediated by JA, tobacco leaves were fed through the petiole with 100 nM or 1 μM WAF-1 or water and incubated at 24°C, and the endogenous levels of JA were measured. The amounts of JA in leaves treated with either 100 nM or 1 μM WAF-1 were equal to those in leaves treated with water when the treated leaves were incubated for 3, 6, 12, or 24 h (data not shown).

Exogenously supplied WAF-1 enhanced the accumulation of ACO transcripts (Figure 6). In addition, ethylene functions as a signal in the induction of basic PR genes in tobacco plants (Brederode et al., 1991; Eyal et al., 1992; Xu et al., 1994; Ohtsubo et al., 1999). Therefore, we tested the effect of exogenous WAF-1 on the accumulation of ethylene. The levels of ethylene released from leaves treated with 100 nM or 1 μM WAF-1 were ∼1.2- or 1.4-fold higher, respectively, than those from leaves treated with water (Figure 7).

Figure 7.

Figure 7.

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Effect of Exogenous WAF-1 on the Accumulation of Ethylene.

Leaf discs were floated on either water and 0.1 mM 1-aminocyclopropane-1-carboxylic acid or the indicated concentrations of synthetic WAF-1 and 0.1 mM 1-aminocyclopropane-1-carboxylic acid in a 50-mL Erlenmeyer flask. After incubation for 16 h at 24°C under continuous light, a 1-mL sample was withdrawn from the headspace and analyzed for ethylene. The amount of ethylene released is expressed as nL/g fresh weight (FW). Values shown are means ± sd from three measurements.

Endogenous WAF-1 Levels Increase during TMV-Induced HR and after Wounding

We established a method to measure endogenous WAF-1 using 8-epi-WAF-1 (Figure 3A, compound 3) as an internal standard. 8-epi-WAF-1 was obtained during the course of the synthesis of WAF-1 (Figure 4). HPLC analysis of extracts prepared from Samsun NN tobacco plants confirmed that 8-epi-WAF-1 does not occur naturally in this tobacco cultivar, showing that this compound is available as an internal standard. Using this quantitative method, we determined levels of WAF-1 during HR in TMV-infected tobacco plants. Shifting TMV-infected leaves from 30°C, a nonpermissive temperature for the N gene to induce HR, to 20°C, a permissive temperature, resulted in the synchronous formation of necrotic lesions in the infected region. When TMV-inoculated leaves incubated at 30°C for 48 h were kept at 20°C (0 h), WAF-1 levels, which were 43 ± 9 ng/g fresh weight at 0 h, began to increase at 3 h after the shift and reached a maximum (189 ± 45 ng/g fresh weight) at 24 h (Figure 8A). The first increase in WAF-1 occurred before the activation of WIPK and SIPK, which has been reported to be observed at 4 h after the shift to 20°C (Zhang and Klessig, 1998; Seo et al., 2001). In mock-inoculated leaves, the amount of WAF-1 remained almost constant.

Figure 8.

Figure 8.

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Changes in the Amount of Endogenous WAF-1 during TMV-Induced HR or after Wounding.

Harvested leaf discs or leaf pieces were used for the quantification of WAF-1. The amount of WAF-1 is expressed as ng/g fresh weight (FW). Values shown are means ± sd from three measurements (each with triplicate samples).

(A) Leaves were inoculated with TMV (10 μg/mL; closed triangles) or buffer only (mock; open triangles), and leaf discs punched out from them were incubated at 30°C for 48 h and then transferred to 20°C at time 0. Discs were harvested at the times indicated after the temperature shift. The arrow indicates the time when necrotic lesions appeared.

(B) Leaves were inoculated with TMV (2 μg/mL; closed squares) or buffer only (mock; open squares), and leaf discs punched out from them were incubated at 20°C and harvested at the times indicated. The arrow indicates the time when necrotic lesions appeared.

(C) Leaves were wounded by cutting them into small pieces with a razor blade. Leaf pieces were harvested at the times indicated.

The accumulation of WAF-1 also was enhanced in TMV-inoculated leaves maintained at 20°C. WAF-1 levels were 26 ± 6 ng/g fresh weight immediately after the inoculation and 126 ± 22 ng/g fresh weight 48 h later (Figure 8B). An increase was detected at 24 h, earlier than when necrotic lesions appeared (30 to 32 h). A transient increase in WAF-1 levels was observed in mock-inoculated leaves maintained at 20°C, suggesting that mechanical injury causes an increase in endogenous WAF-1. To test the effect of simple wounding on the induction of WAF-1 expression, leaves were mechanically wounded, and the levels of WAF-1 were monitored over a period of 180 min. The average basal level of WAF-1 in healthy leaves was 24 ng/g fresh weight. This increased to 32 ± 4 and 60 ± 7 ng/g fresh weight at 30 and 180 min, respectively, after wounding (Figure 8C).

Exogenous WAF-1 Enhances Resistance to TMV in Tobacco Leaves

Next, we examined whether WAF-1 is involved in resistance to TMV infection in tobacco. Detached leaves were fed through the petiole with different concentrations of synthetic WAF-1 or water, inoculated with TMV, and incubated at 20°C to allow necrotic lesions to form. WAF-1 reduced lesion size at concentrations of >100 pM (Figures 9A and 9B). The reduction in lesion size was dose dependent. The amount of the TMV coat protein was less in leaves treated with WAF-1 than in leaves treated with water (Figure 9C), suggesting that multiplication of TMV was suppressed in the WAF-1–treated leaves.

Figure 9.

Figure 9.

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Effect of WAF-1 Treatment on Necrotic Lesion Development.

Detached leaves were fed through the petiole with water or a solution containing the indicated concentrations of synthetic WAF-1 for 6 h at 24°C, inoculated with TMV (0.7 μg/mL), incubated at 20°C for 5 days, and used for each experiment.

(A) The diameter of 60 local lesions was measured with a stereoscopic microscope. Each bar represents the mean ± sd.

(B) Necrotic lesions that had formed on the leaves treated with 100 nM WAF-1 (right) or water (left).

(C) Leaves that were treated with water, 100 pM WAF-1, or 100 nM WAF-1 were subjected to protein gel blot analysis with anti-TMV antibody (Ab-TMV). CP, coat protein.

SA is known to function as a signal in the induction of resistance to TMV in tobacco plants (Malamy et al., 1990). To assess whether the induction of TMV resistance by WAF-1 is mediated by SA, leaves were treated with 1 μM WAF-1 or water, and the endogenous levels of SA were measured. As shown in Figure 10, SA did not exceed the basal levels in either leaves treated with WAF-1 or leaves treated with water, whereas endogenous SA levels were increased dramatically in TMV-inoculated leaves. Application of SA at concentrations of 0.1 to 1 mM to leaves did not cause an increase in endogenous WAF-1 (data not shown).

Figure 10.

Figure 10.

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Effect of Exogenous WAF-1 on the Accumulation of Endogenous SA.

Detached leaves were fed through the petiole with water (white bars) or a solution containing 1 μM synthetic WAF-1 (black bars) for 2 or 4 days at 24°C and used for the quantification of SA. Healthy leaves (0 days) that were not treated with WAF-1 or water also were used. As a control, TMV-inoculated leaves (hatched bars) were harvested at 2 and 4 days after inoculation and used for the quantification of SA. The amount of SA is expressed as ng/g fresh weight (FW). Values shown are means ± sd from three measurements (each with triplicate samples).

DISCUSSION

In this study, a WIPK-activating substance was isolated from TMV-infected tobacco leaves and identified as (11E,13E)-labda- 11,13-diene-8α,15-diol, designated WAF-1. When applied in nanomolar concentrations to leaves, either natural WAF-1 or chemically synthesized WAF-1 activated WIPK as well as SIPK (Figure 5). Quantitative analysis of endogenous WAF-1 revealed that levels increased rapidly in leaves during TMV-induced HR and after wounding (Figure 8). These results suggest that WAF-1 functions as an endogenous signal for the activation of WIPK and SIPK in TMV-infected and wounded tobacco plants.

In the synchronous HR-inducing system, the first increase in WAF-1 preceded the activation of WIPK (Figure 8A), suggesting that WAF-1 can function as a trigger for the activation of WIPK during TMV-induced HR. In wounded leaves, WIPK was activated at 3 min after wounding (Seo et al., 1999), whereas an increase in endogenous WAF-1 was found at 30 min after wounding (Figure 8C). It is possible that the amount of endogenous WAF-1 is increased by rapid synthesis through the conversion of a predicted pre-WAF-1 substance within 3 min after wounding, thereby stimulating the activation of WIPK. In this case, such an increase would be undetectable using our quantitative method. Alternatively, WAF-1 may not be the first trigger for the activation of WIPK in the wounded region. If so, WAF-1 would be involved in a secondary systemic induction of WIPK activation (Seo et al., 1999).

Our results suggest that WAF-1 is a likely natural inducer of resistance to TMV. SA also has been identified as a natural inducer of resistance to TMV (Malamy et al., 1990). Treatment of leaves with WAF-1 did not cause an increase in endogenous SA (Figure 10), consistent with the observation that WAF-1 treatment had no effect on the accumulation of transcripts for the acidic PR-1 and PR-2 genes (Figure 6), which are induced by SA (Linthorst et al., 1990; Malamy et al., 1990; Niki et al., 1998). Thus, WAF-1 induces resistance to TMV independently of SA (Figure 11). Although the precise mechanism for the induction of TMV resistance by WAF-1 is not known, WIPK and SIPK likely function as mediators in the signal transduction pathway that leads to the resistance. Actually, both kinases have been implicated in defense responses to pathogens (Romeis et al., 1999; Yang et al., 2001).

Figure 11.

Figure 11.

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Model Showing the Possible Role of WAF-1 in Signal Transduction Pathways for the Activation of PR Genes and the Induction of Resistance to TMV in Tobacco.

Information on other signal molecules, such as nitric oxide and reactive oxygen species, is omitted because the relationship between these molecules and WAF-1 is unclear.

Originally, we isolated WAF-1 as a substance that activates WIPK. WAF-1 also was found to activate SIPK (Figure 5), a tobacco MAPK, indicating that among MAPKs, WIPK is not the only target of WAF-1. In tobacco, three MAPKs other than WIPK and SIPK—NTF3, NTF4, and NTF6—have been identified (Wilson et al., 1993, 1995). Whether WAF-1 activates these three MAPKs remains to be determined.

Activation of SAMK (Jonak et al., 1996; Bögre et al., 1997; Cardinale et al., 2000) and ATMPK3 (Kovtun et al., 2000; Asai et al., 2002; Lu et al., 2002), which are the alfalfa and Arabidopsis orthologs, respectively, of WIPK, has been implicated in phytohormone and defense responses. However, little is known about the endogenous signals responsible for the activation of SAMK and ATMPK3. Only H2O2 (Kovtun et al., 2000) and the phytohormone abscisic acid (Lu et al., 2002) have been shown to activate ATMPK3. Studies of the effect of WAF-1 on the activation of SAMK, ATMPK3, and other plant MAPKs will lead to a better understanding of the role of WAF-1 in plants.

To test the effect of WAF-1 on the accumulation of ethylene, we measured the amount of ethylene released from leaves treated with WAF-1. The level of ethylene released from leaves treated with 1 μM WAF-1 was only ∼1.4-fold greater than that released from control leaves (Figure 7). However, the amount of ethylene released from tissues would not correctly reflect the amount in the cells. Thus, the involvement of WAF-1 in ethylene signaling remains to be clarified.

Our results suggest that WAF-1 functions as a signal in the induction of basic PR genes (Figure 11). We assessed the involvement of JA in the induction of basic PR genes by WAF-1, because expression of these genes is induced by JA (Xu et al., 1994; Niki et al., 1998). However, treatment of leaves with WAF-1 did not cause an increase in endogenous JA (data not shown). This observation appears to contradict our previous suggestion that WIPK positively regulates the expression of basic PR genes by functioning through JA (Seo et al., 1995, 1999). One possible explanation for this contradiction is the induction of a putative inhibitor other than SA of JA synthesis by WAF-1 treatment. Although SA is known to block JA synthesis (Peña-Cortés et al., 1993), we found that SA was not induced by WAF-1 treatment. Another possibility is the presence of putative tobacco MAPK(s) that work together with WIPK to induce JA synthesis and the expression of JA-inducible basic PR genes.

Based on our observation that tobacco plants overexpressing the WIPK cDNA in which the endogenous WIPK gene was suppressed by gene silencing failed to produce wound-induced JA (Seo et al., 1995), we suggested that the failure to induce JA synthesis is attributable to the suppression of WIPK. However, because the WIPK cDNA used contained a region homologous with other MAPK genes, its overexpression might interfere with the expression of these genes. The Arabidopsis MAPK mutant mpk4 fails to express JA-inducible genes in response to JA treatment (Petersen et al., 2000), indicating that MPK4 functions in a JA-mediating signal transduction pathway. Although the tobacco ortholog of MPK4 has not been isolated to date, we speculate that a putative MPK4-like MAPK is not activated in response to WAF-1 treatment in wild-type tobacco plants. In transgenic tobacco plants exhibiting constitutive activation of WIPK (Seo et al., 1999), such a MAPK might work together with WIPK to induce both JA synthesis and the expression of the JA-inducible PI-II gene. Further study is required to examine these possibilities. Thus, it is not clear whether WAF-1 works through WIPK and/or SIPK to induce JA- and ethylene-responsive basic PR genes and resistance to TMV. Analysis of active WIPK- or SIPK-overproducing transgenic plants or WIPK- or SIPK-suppressed lines would resolve this issue.

WAF-1 is a labdane-type diterpene. Labdane-type diterpenes occur widely in the plant kingdom, although their biological roles in plants are poorly understood. Sclareol (Figure 3A, compound 4), a major labdane-type diterpene of plants and a close analog of sclareolide (Figure 3A, compound 2), which was used as the starting material for the chemical synthesis of WAF-1, is of interest to the work described here. It exhibits a variety of pharmacological activities, such as the induction of apoptosis (Dimas et al., 2001), fungal growth inhibition (Bailey et al., 1975), and plant growth inhibition (Cutler et al., 1977), and induces the expression of a tobacco gene that encodes an ATP binding cassette transporter that is involved in the secretion of toxic drugs (Jasiński et al., 2001). Interestingly, sclareol constitutes 10% of the leaf surface exudate of Nicotiana glutinosa (Bailey et al., 1974). In our preliminary experiment, WAF-1 was recovered from the intercellular spaces of wounded tobacco leaves (S. Seo and Y. Ohashi, unpublished results), suggesting that it accumulates in these spaces after wounding. Thus, the low molecular mass compounds sclareol and WAF-1 may act in a coordinated manner as natural defense substances at the surface and in intercellular spaces of leaves of tobacco plants.

Recent studies have indicated that terpenoids play an important role in the defense responses of plants to herbivore damage (for reviews, see Takabayashi and Dicke, 1996; Pickett and Poppy, 2001). Lima bean leaves respond to infestation by an insect by releasing volatiles, including several terpenoids that attract the insect's natural predator (Arimura et al., 2000). These terpenoids induce the expression of a set of defense-related genes, such as PR genes, not only in the infested leaves but also in uninfested leaves of neighboring lima bean plants, suggesting a role for these terpenoids as an air-borne signal in plant–plant interactions. The expression of these defense-related genes induced by terpenoids requires protein phosphorylation (Arimura et al., 2000). It will be interesting to determine whether MAPKs are involved in such a protein phosphorylation process.

METHODS

Plant Material, Inoculation with Tobacco mosaic virus, and Wound Stress Treatments

Tobacco (Nicotiana tabacum cv Samsun NN) plants were grown in a temperature-controlled greenhouse at 25°C under sunlight. Unless stated otherwise, the upper, fully expanded healthy leaves of 2-month-old plants were used. Five days before the experiments, the plants were transferred to a temperature-controlled chamber maintained at 24°C under continuous light. For general inoculation, the detached leaves of plants were dusted with Carborundum (Kishida Chemical Co., Osaka, Japan) and inoculated mechanically with Tobacco mosaic virus (TMV) suspended in 10 mM phosphate buffer, pH 7.0. All inoculated leaves were incubated at 20 or 30°C under continuous light. The wounding of leaves was performed by cutting them into small pieces with a razor blade.

Acetone Extraction and Ethyl Acetate Fractionation

TMV-inoculated leaves that had been incubated at 30°C for 48 h and then incubated at 20°C for 8 h were homogenized in 4 volumes of cold 80% (v/v) acetone with a Polytron (Kinematica, Lucerne, Switzerland) and extracted for 2 h at 4°C. After filtration and concentration of the extract, the remaining aqueous phase was adjusted to pH 3.0 with HCl and partitioned three times with equal volumes of ethyl acetate. The ethyl acetate phase was partitioned twice with equal volumes of 5% (w/v) sodium bicarbonate, and the upper organic phase containing neutral substances was recovered. The lower sodium bicarbonate phase was acidified to pH 2.0 and partitioned with ethyl acetate, and the upper organic phase containing acidic substances was recovered. The remaining aqueous phase after the first partition was adjusted to pH 12 with NaOH and partitioned with ethyl acetate, and the upper organic phase containing basic substances was recovered. The three fractions recovered were dried over anhydrous sodium sulfate.

Bioassay of Fractions

Each fraction was evaporated to dryness, and the remaining residue was dissolved in 10 mL of ethyl acetate, methanol, or acetonitrile. Aliquots of the solution were added to filter paper in a Petri dish (3 cm in diameter). As controls, methanol was added to filter paper in another dish. After evaporation of the organic solvent, 1 mL of incubation buffer (10 mM Mes-NaOH, pH 5.6) was added to each dish. Three leaf discs (9 mm in diameter) per dish were placed on the filter paper, incubated at 24°C for 2 h under continuous light, and used for protein extraction. Crude extracts containing 50 μg of total protein were immunoprecipitated with anti–wound-induced protein kinase antibody, and myelin basic protein kinase activity was measured in the immunoprecipitates as described previously (Seo et al., 1999).

Large-Scale Purification

Fifteen kilograms (fresh weight) of TMV-inoculated leaves, equivalent to approximately 3500 leaves, that had been incubated at 30°C for 48 h and then at 20°C for 8 h were extracted with 4 volumes (60 L) of 80% (v/v) acetone. The ethyl acetate–soluble neutral fraction prepared from the acetone extract was separated on a column (3 cm × 50 cm) of silica gel (Wakogel C-200; Wako Pure Chemical, Osaka, Japan) eluted with increasingly higher concentrations of ethyl acetate in n-hexane starting with 10% (v/v) ethyl acetate. The activity was detected in fractions eluted with 60 to 80% ethyl acetate in n-hexane. The active fractions were combined, evaporated, and separated on a C18 cartridge column (Waters, Milford, MA) eluted with increasingly higher concentrations of methanol in water starting with 10% (v/v) methanol. The activity was detected in a fraction eluted with 80% methanol in water. The active fraction was separated on a reversed-phase HPLC column (LiChrospher 100 RP-18, 5-μm particle size, 4 mm × 25 cm; Hewlett-Packard, Palo Alto, CA). The column was eluted with mobile phase A (water:methanol, 1:4 [v/v]) at a flow rate of 1 mL/min, monitoring at 254 nm. Starting at 0.1 min after injection, 90 fractions each containing 3 mL were collected. The activity was found in a fraction with retention times of 12.1 to 15.1 min. The fraction was separated further on a HPLC column (LiChrospher 100 RP-18, 5-μm particle size, 4 mm × 25 cm; Hewlett-Packard) eluted with mobile phase B (water:acetonitrile, 2:3 [v/v]) at the same flow rate. A peak fraction with a retention time of 14.6 min showed activity and was collected to yield 300 μg of a colorless gum.

Synthesis of WAF-1 and 8-epi-WAF-1

The scheme for synthesis of WAF-1 and 8-epi-WAF-1 is shown in Figure 4. A diol (compound 5), synthesized from sclareolide (Aldrich) according to the method of Kuchkova et al. (1997), was oxidized by 3.0 equivalents of N-methylmorpholine N-oxide in the presence of 10 mol % tetrapropylammonium perruthenate and powdered 4-Å molecular sieve (∼600 mg/mmol of compound 5) in dry dichloromethane at temperatures from 0°C to room temperature for 35 min (Ley et al., 1994), an aldehyde (compound 6) being obtained in 64% yield. The Honer-Wadsworth-Emmons reaction of the aldehyde (compound 6) (3.2 equivalents of 3-ethoxycarbonyl-2-methyl-prop-2-enylphosphonate [compound 7; Aldrich], 3.1 equivalents of sodium amide, dry tetrahydrofuran, −50°C, 41 h) gave esters 8, 9, 10, and 11 in 10, 59, 19, and 5% yields, respectively. Reduction of the ester (compound 8) with 6.9 equivalents of diisobutylaluminum hydride in dry dichloromethane at −78°C for 30 min and then at room temperature for 30 min afforded WAF-1 in a quantitative yield. The same reduction of the ester (compound 10) gave 8-epi-WAF-1 in a 96% yield.

It was found that the allylic alcohol moiety of WAF-1 was easily oxidized to the corresponding aldehydes, (11E,13E)-8α-hydroxy-labda-11,13-dien-15-al and (11E,13Z)-8α-hydroxy-labda-11,13-dien-15-al, and then decomposed to (11E)-8α-hydroxy-14,15-dinorlabd-11-en-13-one during storage. This resulted in a marked loss of activity. To prevent degradation, either synthetic or natural WAF-1 was dissolved in methanol and stored at −30°C. The methanol solution was diluted subsequently with water immediately before the experiments.

Spectrometric Analyses of WAF-1

Electron ionization mass spectrometry results were as follows: mass-to-charge ratio (relative intensity) 288 (M+-H2O, 68), 177 (100), 133 (60), 109 (68), 95 (66), 81 (73), 69 (97), 43 (63). High-resolution electron ionization mass spectrometry results were as follows: mass-to-charge ratio calculated for C20H32O [M-H2O]+ 288.2455, found 288.2453. 1H-NMR (600 MHz, CDCl3) results were as follows: δ 0.82 (3H, s, H19×3), 0.85 (1H, m, H1α), 0.88 (3H, s, H18×3), 0.93 (1H, m, H5), 0.94 (3H, s, H20×3), 1.13 (1H, m, H3α), 1.20 (3H, s, H17×3), 1.32 (1H, m, H6β), 1.38 (1H, m, H1β), 1.38 (1H, m, H2α), 1.38 (1H, m, H3β), 1.48 (1H, ddd, J = 12.7, 12.7, 3.3 Hz, H7α), 1.56 (1H, m, H2β), 1.69 (1H, m, H6α), 1.83 (1H, d, J = 10.3 Hz, H9), 1.83 (3H, s, H16×3), 1.92 (1H, ddd, J = 12.7, 3.1, 3.1 Hz, H7β), 4.29 (2H, d, J = 6.8 Hz, H15×2), 5.64 (1H, t, J = 6.8 Hz, H14), 5.69 (1H, dd, J = 15.6, 10.3 Hz, H11), 6.19 (1H, d, J = 15.6 Hz, H12). 13C-NMR (150 MHz, CDCl3) results were as follows: δ 12.85 (C16), 15.92 (C20), 18.44 (C2), 20.07 (C6), 21.60 (C19), 25.23 (C17), 33.32 (C4), 33.40 (C18), 37.66 (C10), 40.91 (C1), 41.95 (C3), 42.03 (C7), 55.84 (C5), 59.31 (C15), 66.38 (C9), 72.00 (C8), 125.71 (C11), 129.46 (C14), 135.94 (C13), 139.44 (C12).

Immune Complex Kinase Assay

Immunoprecipitations were performed using 50 μg of total protein with 5 μg of either anti–wound-induced protein kinase or anti–salicylic acid-induced protein kinase antibodies, as detailed previously (Seo et al., 1999). The immunoprecipitates were subjected to a myelin basic protein kinase activity assay as described previously (Seo et al., 1999).

Quantification of WAF-1

For the quantification of WAF-1, 2 g of leaf material was homogenized in 20 mL of cold 80% (v/v) methanol with a Polytron and extracted for 1 h at 4°C. At this step, 300 ng of 8-epi-WAF-1 was added to the extract to estimate the recovery rate of WAF-1 during the purification procedure. The extract was centrifuged at 10,000g for 10 min. The pellet was reextracted with 80% methanol and centrifuged as described above. Supernatants from both extractions were combined and concentrated. The remaining aqueous phase was adjusted to pH 7.5 with 1 M phosphate buffer and partitioned three times with equal volumes of ethyl acetate. The upper organic phase was dried over anhydrous sodium sulfate and evaporated. The remaining residue was dissolved in 3 mL of 80% methanol and passed through a C18 cartridge column (Waters) that had been equilibrated with 80% methanol. The effluent was separated on a HPLC column (LiChrospher 100 RP-18, 5-μm particle size, 4 mm × 25 cm; Hewlett-Packard) eluted with mobile phase A at a flow rate of 1 mL/min. The eluate between retention times of 11.0 and 16.5 min was collected, evaporated, and dissolved in 200 μL of mobile phase C (water:acetonitrile, 9:11 [v/v]). An aliquot of the solution was injected onto the HPLC column and eluted with mobile phase C at the same flow rate, with monitoring at 238 nm. The retention times of WAF-1 and 8-epi-WAF-1 at the final HPLC step were 24.5 and 29.5 min, respectively. The endogenous amount of WAF-1 was calculated from standard curves. All data were corrected for losses on the basis of the recovery percentages (65 to 75%) of 8-epi-WAF-1.

Ethylene Measurement

Ethylene was measured with a gas chromatograph (GC-8A; Shimadzu, Kyoto, Japan) as described previously (Ohtsubo et al., 1999).

Quantification of Jasmonic Acid

Quantification of jasmonic acid was performed as described previously (Seo et al., 2001).

RNA Gel Blot Analysis

Total RNA extraction, blotting, hybridization, and labeling of cDNA probes were performed as described previously (Seo et al., 1999). The blots were analyzed by autoradiography.

Protein Gel Blot Analysis

Protein gel blot analysis of the TMV coat protein was performed as described previously (Seo et al., 2000).

Quantification of Salicylic Acid

Quantification of salicylic acid was performed as described previously (Seo et al., 1995).

Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes.

Acknowledgments

We thank S. Fujioka for MS measurements and H. Sano for critical reading of the manuscript. This work was supported by the Program for the Promotion of Basic Research Activities for Innovative Biosciences and by a Grant-in-Aid for Scientific Research on Priority Areas (Molecular Mechanisms of Plant–Pathogenic Microbe Interaction).

Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010231.

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