Constitutive Activation of the Jasmonate Signaling Pathway Enhances the Production of Secondary Metabolites in Tomato

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FEBS Letters 580 (2006) 2540–2546

Constitutive activation of the jasmonate signaling pathway enhances the production of secondary metabolites in tomato

Hui Chena, A. Daniel Jonesb,c, Gregg A. Howea,b,* a Department of Energy-Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA b Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA c Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA

Received 20 January 2006; revised 15 March 2006; accepted 27 March 2006

Available online 7 April 2006

Edited by Mark Stitt

Most previous studies of jasmonate-induced secondary Abstract The phytohormone jasmonic acid (JA) regulates the synthesis of secondary metabolites in a wide range of plant spe- metabolism have relied on the use of exogenous JAs to elicit cies. Here, we show that exogenous methyl-JA (MeJA) elicits metabolite accumulation (e.g., Gundlach et al. [1]). However, massive accumulation of caffeoylputrescine (CP) in tomato increasing evidence supports the idea that genetic manipula- leaves. A mutant (jai1) that is defective in jasmonate perception tion of the jasmonate signaling pathway can be used to in- failed to accumulate CP in flowers and MeJA-treated leaves. crease the expression of various target processes, including Conversely, a transgenic tomato line (called 35S::PS) that the production of secondary metabolites. van der Fits and exhibits constitutive JA signaling accumulated high levels of leaf Memelink [2,11] showed that ectopic expression of a JA- CP in the absence of jasmonate treatment. RNA blot analysis responsive transcription factor (ORCA3) enhanced the pro- showed that genes encoding enzymes in the phenylpropanoid duction of terpenoid indole alkaloids (TIAs) in cultured cells and polyamine pathways for CP biosynthesis are upregulated of Catharanthus roseus. The Arabidopsis cev1 mutant, which in MeJA-treated wild-type plants and in untreated 35S::PS plants. These results indicate that CP accumulation in tomato harbors a defective cellulose synthase, hyperaccumulates JA is tightly controlled by the jasmonate signaling pathway, and as a result of a change in cell wall structure that activates JA provide proof-of-concept that the production of some plant sec- biosynthesis [12,13]. Increased JA production in cev1 plants ondary metabolites can be enhanced by transgenic manipulation causes hyperaccumulation of anthocyanins, which accumulate of endogenous JA levels. in wild-type plants in response to MeJA treatment [14]. Several 2006 Federation of European Biochemical Societies. Published other mutants that exhibit constitutive jasmonate signaling by Elsevier B.V. All rights reserved. have been identified [15–19], but have not been characterized with respect to the production of secondary metabolites. Keywords: Jasmonic acid; Caffeoylputrescine; Metabolic Tomato (Solanum lycopersicum) has been widely used as a engineering; Tomato; Systemin model system in which to study the role of JAs in plant defense responses to herbivore and pathogen attack [20–23]. Among the primary wound signals that activate JA biosynthesis in tomato leaves are systemin and its larger precursor protein, prosystemin (PS) [24]. A transgenic line of tomato (called 1. Introduction 35S::PS) that overexpresses PS exhibits constitutive expression of several JA-regulated defensive proteins [25–31]. The impor- Plants produce a wide variety of secondary metabolites in tance of systemin and JA in promoting defense responses in to- response to biotic and abiotic stress. The jasmonate family mato has been established by research showing that loss of of signaling molecules (collectively referred to as JAs), which function mutants have increased susceptibility to a broad spec- includes JA and its volatile methyl ester, MeJA, play a cen- trum of biotic agents [32–37], whereas gain of function mu- tral role in regulating the biosynthesis of many secondary tants such as the above-mentioned 35S::PS transgenic line metabolites [1–7]. Among the classes of metabolites that are have enhanced resistance [28,31]. induced by JAs are free and conjugated forms of polyamines, Although it is well established that systemin and JA regulate quinones, terpenoids, alkaloids, phenylpropanoids, glucosino- the expression of numerous defense-related proteins, little is lates, and antioxidants [8,9]. This signaling property of JAs is known about the extent to which this signaling pathway con- interesting not only for what it reveals about how primary trols the production of secondary metabolites. Here, we report and secondary metabolism is reconfigured in response to that MeJA elicits massive accumulation of caffeoylputrescine stress, but also for practical applications in the production (CP) in tomato, similar to the response reported for Nicotiana of economically useful compounds. For example, exogenous attenuata [3]. Through the use of appropriate mutants, we ex- MeJA was shown to significantly enhance the production of tend this observation by demonstrating that CP production in the anti-cancer drug paclitaxel (Taxol) in cell suspension cul- tomato leaves and flowers is tightly regulated by the jasmonate tures of Taxus [10]. signaling pathway. We also show that CP constitutively accumulates in leaves of 35S::PS transgenic tomato plants. These findings offer proof-of-concept for the idea that JA-regulated *Corresponding author. Fax: +1 517 3539168. secondary metabolites can be produced in transgenic plants E-mail address: [email protected] (G.A. Howe). that are engineered for constitutive JA signaling.

0014-5793/$32.00 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.03.070 H. Chen et al. / FEBS Letters 580 (2006) 2540–2546 2541

2. Materials and methods acid; B = acetonitrile) using the following gradient: 0–5 min, 100% A, 5–45 min, linear gradient to 60% A; hold at 60% A until 50 min. Elec- 2.1. Plant materials and treatments trospray ionization was employed in negative ion mode, using a post- Solanum lycopersicum (formerly known as Lycopersicon esculentum) column split yielding a flow rate of 0.2 mL/min to the mass spectrom- cv. Castlemart (CM) and cv. MicroTom (MT) were used as wild-type eter. LC/MS/MS data were generated on a Waters Quattro micro mass (WT) control plants. The 35S::PS-expressing transgenic line (1-2J) [29] spectrometer using identical chromatographic conditions. Product ion MS/MS spectra were generated using argon as collision gas at was backcrossed five times to CM and MT, with selection for the À3 transgene at each generation. The resulting backcrossed lines are re- 2 · 10 mbar and collision energy of 20 eV. ferred to as 35S::PS (CM) and 35S::PS (MT). The jai1-1 mutation, which was isolated in the MT genetic background, was backcrossed 2.5. Quantification of JA five times to CM as previously described [38]. Tomato seedlings were Leaf tissue (5 g fresh weight) from 3-week-old tomato plants was grown in Jiffy peat pots (Hummert International, St. Louis) in a harvested, frozen in liquid nitrogen, and ground to a fine powder with growth chamber maintained under 17 h of light (140 lEmÀ2 s À1)at a chilled mortar and pestle. Extraction and quantification of JA was 28 C and 7 h of dark at 18 C. Flowers of various stages were col- performed as previously described [40]. lected from MT and jai1 (MT) plants. For MeJA treatments, 40 3- week-old plants were placed in a closed Lucite box and treated with 2.6. RNA gel-blot analysis various amounts of MeJA as previously described [39]. Total RNA was isolated and analyzed by blot hybridization as previously described [30]. The following EST clones were used as probes 2.2. Chemicals to assess the expression of specific genes: ornithine decarboxylase Standards of chlorogenic acid and rutin were obtained from Sigma (cLEC21H8); arginine decarboxylase (cLEC37I15); phenylalanine (St. Louis, MO). Chemically synthesized CP was kindly provided by ammonia-lyase (M83314); cinnamate-4-hydroxylase (cLER1C5); 4- Dr. Jonathan Negrel (INRA, France). MeJA was obtained from Bed- coumarate:CoA ligase (cLED5E16). cDNA probes for analysis of oukian Research (Danbury, CT). prosystemin and arginase expression were previously described [30,39].

2.3. High pressure liquid chromatography (HPLC) Fresh leaf tissue was collected and immediately freeze-dried. Dried 3. Results tissue (100 mg) was extracted twice with 5 ml methanol at room temperature. One ml of the resulting extract was dried in a Speed Vac 3.1. Identification of caffeoylputrescine as a jasmonate-inducible and redissolved in 0.5 ml of water containing 0.1% trifluoroacetic acid (TFA) and 0.6 ml of petroleum ether. Following phase separation, the metabolite in tomato leaves upper layer containing chlorophylls and other lipid-soluble compo- We used HPLC to analyze methanol-soluble leaf extracts nents was discarded. The aqueous phase was mixed with 0.5 ml meth- obtained from plants that were treated with MeJA or a mock anol, and then filtered through a 0.45 lm filter prior to analysis by control (Fig. 1). Chlorogenic acid (peak 1) and rutin (peak 2), HPLC. HPLC was performed with a Waters liquid chromatograph which are relatively abundant secondary metabolites in tomato system equipped with a model 600 pump, a 996 photodiode array detector, and a 717 autosampler. Extracts (50 ll aliquots) were sepa- leaves [41], were identified in extracts from both control and rated on a 250 · 4.6 mm C18 column (5 lm; Alltech, Deerfield, IL) at treated plants. Chromatograms of extracts from MeJA-treated room temperature. The mobile phase consisted of solvent A (0.1% leaves showed a large peak (peak 3) that was not observed in TFA) and solvent B (acetonitrile). The gradient elution procedure the control extract. The material in peak 3 was purified by pre- was as follows: 0–5 min, 0% of B; 5–45 min, a linear gradient from 0% to 40% of B; 45–50 min, 40% of B; 50–60 min, 0% of B. The flow parative HPLC and subsequently identified as caffeoylputres- rate was 1.0 ml/min. Chromatographic data was collected in the 200– cine (CP; Fig. 1C). High-resolution FAB mass spectrometry 400 nm range. For detection and quantification of CP, peak heights (MS) showed the protonated molecular ion ([M+H]+)atm/z were measured at 320 nm. 251.1399, which corresponds to an elemental formula of Preparative HPLC was performed on a Waters liquid chromatogra- C13H19O3N2. The MS/MS fragmentation gave major peaks phy system equipped with a DC3000 pump and a 990 photodiode ar- + ray detector. Extracts (2-ml aliquots) were separated and analyzed with at m/z 251 [M+H] , 234 (loss of NH3), 163 [loss of a 250 · 10 mm C18 column (10 lm dia.) from Alltech. Gradients were NH2(CH2)4NH2], and 89 [loss of (HO)2C6H3(CH)2CO]. This eluted as follows: 0–5 min, 2% of B; 5–17 min, 2–12% of B; 17–22 min, finding was confirmed by LC/high resolution MS and LC/ 12% of B; 22–60 min, 12–30% of B. The flow rate was 5.0 ml/min. MS/MS data giving retention time, molecular mass, and prod- Fractions containing CP eluted in the 15–17 min range. HPLC fractions were collected manually. uct ion spectra that matched the synthetic caffeoylputrescine standard (data not shown). MeJA application stimulated CP accumulation in tomato 2.4. Mass spectrometry leaves in a dose-dependent manner; high concentrations of Fast atom bombardment (FAB) mass spectra were obtained with a the elicitor, however, appeared to inhibit CP formation JEOL HX-110 double-focusing mass spectrometer (JEOL USA, Pea- body, MA) operating in the positive ion mode. Ions were produced (Fig. 2A). Increased CP accumulation was detected within by bombardment with a beam of Xe atoms. The accelerating voltage 24 h of MeJA treatment, and maximum levels were observed was 10 keV and the resolution was set at 3000. For FAB-CAD (colli- 4 d after treatment (Fig. 2B). We estimated that the CP content sionally activated dissociation)-MS/MS, helium was used as the colli- in MeJA-treated leaves at the 4-d time point was at least 100- sion gas in a cell located in the first field-free region. The helium pressure was adjusted to reduce the abundance of the parent ion by fold greater than that in the control sample. MeJA treatment 50%. A JEOL data system generated linked scans at a constant ratio had little or no effect on the levels of rutin and chlorogenic acid of magnetic to electrical fields (B/E). The scanning range was from in leaf tissue (data not shown). m/z 25 to 1000. Metabolite characterization was also carried out using LC/high-resolution MS for accurate mass measurement and LC/MS/MS for struc- 3.2. CP accumulation in leaves and flowers is dependent on the ture confirmation based on fragment ion masses. LC/high-resolution jasmonate signaling pathway MS was performed on a Waters LCT Premier time-of-flight mass spec- The jai1-1 mutant is defective in the COI1 gene that is re- trometer coupled to a Shimadzu LC-20AD HPLC pump and SIL-5000 autosampler. The instrument was operated using W-mode ion optics. quired for all known jasmonate-signaled processes in tomato Metabolites were separated on a 4.6 · 250 mm Waters Symmetry [38]. This mutant was used to determine whether MeJA- C18 column and a flow rate of 1.0 ml/min (A = 0.5% aqueous formic induced accumulation of CP requires a functional jasmonate 2542 H. Chen et al. / FEBS Letters 580 (2006) 2540–2546

Fig. 1. HPLC chromatograph of methanol extracts from wild-type tomato leaves. (A) Extract from mock-treated plants. (B) Extract from MeJA-treated plants. Peaks correspond to the following compounds: 1, chlorogenic acid; 2, rutin; 3, caffeoylputrescine. (C) Diagram of the structure of caffeoylputrescine. AU, arbitrary units. signaling pathway. The results showed that, in contrast to WT plants, MeJA-treated jai1-1 plants did not produce CP in the leaves (Fig. 2C). Previous studies showed that CP constitutively accumulates in female organs of tomato flowers [42]. Consistent with this observation, we found that whole flowers from WT plants contain relatively high levels of CP (0.76 ± 0.27 mg CP/g dry weight). CP was not detected in jai1-1 flowers of the same developmental stage (Fig. 2C). These Fig. 2. Caffeoylputrescine accumulation in leaves and flowers is results demonstrate that CP accumulation in tomato leaves regulated by the jasmonate pathway. (A) Effect of MeJA concentration on CP accumulation in tomato leaves. (B) Time course of CP and flowers is dependent on the jasmonate signaling pathway. accumulation in leaves in response to MeJA treatment. (C) Accumu- lated levels of CP in leaves and flowers of WT (CM) and jai1 mutant 3.3. Transgenic plants that overexpress prosystemin exhibit plants. For measurement of leaf CP levels, plants were exposed to constitutive accumulation of CP MeJA vapors (+) or ethanol (À) for 4 d. Data points show the means ± S.D. of three independent assays. dw, dry weight. Overexpression of the tomato prosystemin (PS) cDNA from the Cauliflower mosaic virus (CaMV) 35S promoter causes constitutive expression of defense-related proteins [25,27,31]. Increased levels of CP in 35S::PS leaves were correlated with To determine whether PS overexpression also enhances CP a significant increase (2–3-fold; P < 0.05) in endogenous JA production, we measured the concentration of this metabolite (Fig. 3B). To determine whether 35S::PS-mediated CP pro- in 35S::PS and WT leaves (Fig. 3A). These experiments were duction requires JA, we measured CP concentrations in leaf conducted with both the CM and MT inbred cultivars, to- tissue of a 35S::PS line that harbors a mutation (spr2) that gether with near-isogenic lines that are homozygous for abolishes the function of a chloroplast fatty acid desaturase in- 35S::PS (see Section 2). Introduction of 35S::PS into both ge- volved in JA biosynthesis [29,35]. The results showed that spr2 netic backgrounds increased the constitutive level of CP by suppressed 35S::PS-mediated CP accumulation to levels that 50-fold in comparison to the parental line. Both the basal le- were below the detection limit of the assay (data not shown). vel and the 35S::PS-induced level of CP was higher in the MT Thus, constitutive production of CP in the 35S::PS transgenic genetic background than in the CM background (Fig. 3A). line requires a functional JA biosynthetic pathway. H. Chen et al. / FEBS Letters 580 (2006) 2540–2546 2543

3.4. CP production in 35S::PS plants is associated with increased expression of genes encoding phenylpropanoid and polyamine biosynthetic enzymes CP is a conjugate between caffeic acid and putrescine and thus belongs to the family of hydroxycinnamic acid amide compounds. Caffeic acid and putrescine are derived from the phenylpropanoid and polyamine biosynthetic pathways, respectively (Fig. 4A). Both the arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) pathways in toma- to contribute to the formation of putrescine from arginine (Fig. 4A) [43]. Genes encoding many of the enzymes involved in the phenylpropanoid, ADC, and ODC pathways have been shown to be upregulated in tomato leaves by exogenous MeJA, wounding, and infection with virulent strains of Pseudomonas syringae [30,31,44–46]. The constitutive accumulation of CP in 35S::PS leaves suggested that these metabolic pathway genes might be expressed to higher levels in the transgenic line. In- deed, RNA blot analyses showed that genes encoding enzymes of the phenylpropanoid pathway (phenylalanine ammonia- lyase, PAL; cinnamate-4-hydroxylase, C4H; 4-coumarate:CoA ligase, 4CL), the ADC pathway (ADC), and the ODC pathway (ODC; arginase, ARG) were expressed to significantly higher levels in 35S::PS than in WT plants (Fig. 4B). Control experiments confirmed that exogenous MeJA activated the expression of these genes in WT but not in jai1 plants (Fig. 4B). These results suggest that de novo synthesis of CP in 35S::PS plants involves the simultaneous, jasmonate-dependent activation of both the phenylpropanoid and polyamine pathways.

4. Discussion Fig. 3. Constitutive accumulation of caﬀeoylputrescine in the leaves of 35S::PS transgenic plants is associated with elevated levels of 4.1. CP accumulation in tomato is regulated by the jasmonate endogenous JA. (A) CP content in leaves of 35S::PS (PS) plants and the corresponding WT cultivar (Castlemart or MicroTom). (B) JA signaling pathway content in leaf tissue in the genotypes shown in panel A. Data points In this paper, we demonstrate that the production of CP in show the means ± S.D. of three independent assays. tomato plants is tightly regulated by the jasmonate signaling

Fig. 4. Genes encoding putative enzymes in the caffeoylputrescine biosynthetic pathway are upregulated in 35S::PS plants. (A) Biosynthetic pathway of caffeoylputrescine. ARG, arginase; ADC, arginine decarboxylase; ODC, ornithine decarboxylase; PAL, phenylalanine ammonia-lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumarate:CoA ligase; PHT, putrescine N-hydroxycinnamoyltransferase; AIH, agmatine iminohydrolase; CPA, N-carbamoylputrescine aminohydrolase. (B) Northern blot analysis of transcripts encoding prosystemin (PS) and various enzymes in the polyamine and phenylpropanoid biosynthetic pathways. Total RNA was isolated from leaves of WT (CM), 35S::PS (PS), or jai1 plants that were treated with either MeJA (+) or a mock control (À). Blots were hybridized to cDNA probes indicated on the left (see panel A for list of abbreviations). A duplicate gel was stained with ethidium bromide (EtBr) to verify the quality and equal loading of RNA. 2544 H. Chen et al. / FEBS Letters 580 (2006) 2540–2546 pathway. Exogenous MeJA elicited massive accumulation of amides in tomato is regulated by both JA-dependent and JA- CP in leaves. The general timing and high level of induction independent signaling pathways [21]. of this response appears to be similar to that reported in N. attenuata [3]. MeJA-induced accumulation of CP in tomato 4.2. Increased production of jasmonate-regulated plant leaves is blocked in the jai1 mutant that is insensitive to JAs. secondary metabolites through transgenic manipulation Analysis of this mutant also showed that jasmonate signaling Over a decade ago, Ryan and colleagues showed that is required for CP production in flowers, the female organs 35S::PS plants constitutively express several defense-related of which accumulate this metabolite [42]. This conclusion is proteins that provide protection against insect attack [25,27]. consistent with the fact that JAs accumulate to high levels in Here, we show that these plants also exhibit constitutive pro- female reproductive tissues of tomato [47]. To our knowledge, duction of secondary metabolites (i.e., CP). Two observations CP is the first example of a metabolite whose production in to- indicate that 35S::PS-mediated CP production is promoted by mato flowers is strictly dependent on the jasmonate response increased levels of endogenous JA. First, the JA content in pathway. In vegetative tissues of tomato, JAs have been shown 35S::PS leaves was significantly greater than that in non-trans- to regulate the synthesis of various secondary metabolites, genic control plants, as previous reported [66]. Second, the spr2 including anthocyanins [38,46] and defense-related volatiles mutation that blocks JA biosynthesis effectively suppressed CP [48,49]. production in 35S::PS plants. Because the increase in JA levels Although the physiological function of CP in plant growth in 35S::PS leaves was relatively modest (2–3-fold) in compar- and development remains to be determined, correlative evi- ison to the increase in CP levels (50-fold), it is possible that dence suggests that this compound may have a role in plant CP production in 35S::PS leaves is regulated by a bioactive reproductive processes. For example, CP and other hydroxy- derivative of JA, such as 12-oxo-phytodienoic acid or JA-Ile cinnamic acid amides are produced in the reproductive or- [67,68]. Alternatively, JA levels in 35S::PS plants may exceed gans of a diverse range of flowering plants [50]. In maize, a threshold that is required to trigger CP production. these metabolites accumulate in reproductive tissues of fertile CP accumulation in 35S::PS plants was associated with in- plants but not in cytoplasmic male sterile plants [51,52].In creased levels of transcripts that encode enzymes in the phenyl- light of the fact that JA is essential for plant reproduction propanoid and polyamine pathways. Several studies have [38,53,54], the available evidence is consistent with a role shown that these genes are activated in tomato by the jasmo- for CP in tomato reproductive development. First, CP accu- nate signaling pathway ([30,31,44–46], this study). These re- mulates in female reproductive organs [42]. Second, CP was sults raise the possibility that CP accumulation is controlled not detected in floral tissues of the jai1 mutant, which exhib- by JA-responsive transcription factors that coordinately acti- its female sterility and reduced pollen viability [38,55].At vate the phenylpropanoid and polyamine pathways. DNA present, the connection between CP and reproductive success microarray data on JA-responsive genes in tomato [38,44–46] in tomato is strictly correlative; it is possible that the fertility may facilitate the identification of the relevant transcription defects in the jai1 mutant are caused by a defect in other JA- factors, as well as pathway enzymes such as putrescine N- regulated processes. For example, jai1 flowers do not accu- hydroxycinnamoyltransferase (Fig. 4A) that have not yet been mulate serine proteinase inhibitors (PIs), which have been characterized at the molecular level. implicated in tomato seed development [38,56]. Other work- Transgenic technology offers an alternative to elicitors as a ers have suggested that hydroxycinnamoyl amides are means to enhance the production of jasmonate-regulated sec- unlikely to be a causal factor in flower formation in solana- ondary metabolites. For example, overexpression of the ceous plants [42]. ORCA3 transcriptional regulator in C. roseus cells activated Increasing evidence indicates that JA-induced changes in the expression of many genes in the biosynthetic pathway for secondary metabolism constitute a ubiquitous plant defense re- terpenoid indole alkaloids (TIAs) [2,11]. However, because sponse [1,4,8,57]. The widespread phenomenon of JA- and these transgenic cells did not express enzymes that act early stress-induced hydroxycinnamic acid amide production in TIA biosynthesis, enhanced production of TIAs required [3,58–63] is consistent with a role for these compounds in de- the addition of a biosynthetic intermediate. Presumably, addi- fense. Additional support for this hypothesis comes from the tional JA-responsive transcription factors act in concert with observation that tobacco synthesizes large quantities of ORCA3 to orchestrate JA-induced TIA production [8]. hydroxycinnamic acid amides during the hypersensitive re- Genetic engineering of plant cells for elevated JA levels pro- sponse to viral infection [50]. Similarly, CP accumulates in vides an alternative strategy for increasing the production of the extracellular space of Nicotiana tabacum suspension cells JA-regulated metabolites. Attempts to increase constitutive treated with the bacterial pathogen P. syringae [64]. We found JA levels by overexpression of individual JA biosynthetic en- that the tissue-specific and JA-dependent production of CP in zymes have met with limited success, apparently because JA tomato is very similar to that of PIs and other defensive pro- levels are controlled mainly by substrate availability [69,70]. teins that protect against insect attack [30,31,38]. We have also It is thus desirable to adopt strategies aimed either at increas- observed that CP accumulates in tomato leaves in response to ing the production of fatty acid precursors of JA or decreasing mechanical wounding, insect herbivory, and infection with vir- the turnover of bioactive JAs. Our results showing constitutive ulent strains of P. syringae, all of which activate the JA signal- CP production in 35S::PS plants demonstrates that transgenic ing pathway (H. Chen and G.A. Howe, unpublished data). technology can be used to accomplish this goal. Other exam- The strict requirement for JA in CP production contrasts with ples of genetic alterations that cause constitutive JA accumula- the wound-induced accumulation of tyramine conjugates of tion include overexpression of a mitogen-activated protein hydroxycinnamic acids in tomato leaves [65]. These findings kinase in tobacco [15] and mutation of cellulose synthase suggest that the biosynthesis of different hydroxycinnamoyl CeSA3 in Arabidopsis [13]. Recently, it was reported that H. Chen et al. / FEBS Letters 580 (2006) 2540–2546 2545

CP constitutively accumulates in the leaves of transgenic to- [12] Ellis, C. and Turner, J.G. (2001) The Arabidopsis mutant cev1 has bacco plants that overexpress yeast invertase in the apoplastic constitutively active jasmonate and ethylene signal pathways and space [71]. Given that cell wall defects can lead to increased JA enhanced resistance to pathogens. Plant Cell 13, 1025–1033. [13] Ellis, C., Karafyllidis, I., Wasternack, C. and Turner, J.G. (2002) production [13], it is possible that CP accumulation in these to- The Arabidopsis mutant cev1 links cell wall signaling to jasmonate bacco lines is caused by a cell wall perturbation that activates and ethylene responses. Plant Cell 14, 1557–1566. JA biosynthesis. Notably, the biochemical mechanisms by [14] Franceschi, V.R. and Grimes, H.D. (1991) Induction of soybean which 35S::PS or other genetic alterations activate JA biosyn- vegetative storage proteins and anthocyanins by low-level atmo- spheric methyl jasmonate. Proc. Natl. 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