Herbivore-induced terpenoid emission in Medicago truncatula: concerted action of jasmonate, ethylene and calcium signaling
Plant volatiles emitted by Medicago truncatula in response to feeding larvae of Spodoptera exigua are composed of a complex blend of terpenoids. The cDNAs of three terpene synthases (TPSs), which contribute to the blend of terpenoids, were cloned from M. truncatula. Their functional characterization proved MtTPS1 to be a β-caryophyllene synthase and MtTPS5 to be a multi-product sesquiterpene synthase. MtTPS3 encodes a bifunctional enzyme producing (E)-nerolidol and geranyllinalool (precursors of C11 and C16 homoterpenes) from different prenyl diphosphates serving as substrates. The addition of jasmonic acid (JA) induced expression of the TPS genes, but terpenoid emission was higher from plants treated with JA and the ethylene precursor 1-amino-cyclopropyl-1-carboxylic acid. Compared to infested wild-type M. truncatula plants, lower amounts of various sesquiterpenes and a C11–homoterpene were released from an ethylene-insensitive mutant skl. This difference coincided with lower transcript levels of MtTPS5 and of 1-deoxy-d-xylulose-5-phosphate synthase (MtDXS2) in the damaged skl leaves. Moreover, ethephon, an ethylene-releasing compound, modified the extent and mode of the herbivore-stimulated Ca2+ variations in the cytoplasm that is necessary for both JA and terpene biosynthesis. Thus, ethylene contributes to the herbivory-induced terpenoid biosynthesis at least twice: by modulating both early signaling events such as cytoplasmic Ca2+-influx and the downstream JA-dependent biosynthesis of terpenoids.
KeywordsCalcium Ethylene Jasmonic acid Medicago truncatula Terpenoid
1,2-Bis-(2- aminophenoxy)ethane-N,N,N′,N′-tetra acetic acid
Herbivore-induced plant volatile
3-Hydroxy-3-methyl-glutaryl CoA reductase
Volatile terpenoids, the major products among the herbivore-induced plant volatiles (HIPVs) in the legume Medicago truncatula, include monoterpenes (C10), sesquiterpenes (C15), and tetranor-terpenoids (homoterpenes, C11 or C16) (Leitner et al. 2005). The biosynthetic routes to terpenes are fed by either the mevalonate (MVA) pathway in the cytosol/endoplasmic reticulum or the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in the plastids; both pathways generate the five-carbon compound isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP). Cytosolic and plastidic prenyltransferases, respectively, synthesize farnesyl diphosphate (FDP) as a substrate for sesquiterpenes and geranyl diphosphate (GDP) and geranylgeranyl diphosphate (GGDP) as substrates for mono- and diterpenes (Rodríguez-Concepción and Boronat 2002; Eisenreich et al. 2004). Both pathways can cross-talk by exchanging intermediates (e.g., IDP or GDP) (Bick and Lange 2003). Molecular diversity is further expanded by the terpene synthases (TPSs), which utilize the different prenyl diphosphates as substrates. The transcript levels of four genes for terpene synthases, TPSs 1–4, are enhanced in M. truncatula leaves damaged by Spodoptera exigua (beet armyworm [BAW]) herbivory or treated with methyl jasmonate (Gomez et al. 2005). Terpenoid formation is generally assumed to be regulated on the transcript level of the TPS genes (Dudareva et al. 2003; McKay et al. 2003; Sharon-Asa et al. 2003; Arimura et al. 2004a), but the mode of regulation is often complex (Lücker et al. 2001; Aharoni et al. 2003; Arimura et al. 2004b; Aharoni et al. 2005) and needs to be studied individually with respect to the plant and the herbivore.
Till now, little has been known about early physiological events and the interaction between the signaling networks in plants that occur when herbivores feed. The interaction of various pathways in the networks is assumed to result in an integrated overall response that initiates the emission of a characteristic volatile pattern. Since the blends of HIPVs may vary with the attacking herbivore (De Moraes et al. 1998; Ozawa et al. 2000; Leitner et al. 2005), various components and cross-talk between the involved signaling pathways are thought to be responsible for the characteristic terpenoid blend (Engelberth et al. 2001; Schmelz et al. 2003b). Several oxylipin compounds (jasmonic acid (JA), its precursors, and related compounds) very likely act as master switches for herbivore-stimulated plant responses, activating distinct sets of defense genes leading to terpenoid formation (Arimura et al. 2004a; Ament et al. 2006). In addition, antagonistic or synergistic cross-reactions with other regulators, in particular involving ethylene and salicylic acid, seem to control and coordinate the formation of a characteristic blend of volatiles (Ozawa et al. 2000; Engelberth et al. 2001; Horiuchi et al. 2001; Schmelz et al. 2003b). Although ethylene is a well known modulator of processes of plant development and plant defense against biotic and abiotic stresses (Wang et al. 2002), only little is known about how ethylene affects the composition of herbivore-induced volatiles (Kahl et al. 2000; Schmelz et al. 2003a; Schmelz et al. 2003b). Using the model plant M. truncatula, we compared the ethylene-insensitive mutant sickle (skl) with wild-type plants and showed that the ethylene signaling cascade modulates the intracellular Ca2+-level and interacts with JA signaling to generate a specific blend of terpenoids in response to feeding BAW larvae. In addition, three cDNAs encoding new terpene synthases, MtTPS1, MtTPS3, and MtTPS5, were cloned from M. truncatula and heterologously expressed for biochemical characterization.
Materials and methods
Plants and caterpillars
Plants of M. truncatula, wild-type cv. Jemalong (Pogue Agri Partners, Kenedy, TX, USA) and the ethylene-insensitive mutant skl (skl1–1: isolated and backcrossed by the group of Dr. Douglas R. Cook, University of California, Davis) were grown in soil. Each plastic pot contained one or two plants and was kept in a growth chamber at 27°C (14 h light:10 h dark; relative humidity: 65%) for 6–8 weeks. Plants without flowers were used for each treatment. Spodoptera exigua [beet armyworm (BAW)] larvae were reared on artificial diet in a plastic box (22 ± 1°C; 14 h light: 10 h dark) (Bergomaz and Boppré 1986). For BAW infestation, 5–6 third-instar larvae were placed on shoots of M. truncatula.
A solution containing JA (1 mM, pH 5.8–6.0), 1-aminocyclopropane-1-carboxylic acid (ACC, 1 mM; Sigma-Aldrich, St Louis, MO, USA), and/or silver thiosulfate (STS, 1 mM; Sigma-Aldrich) in 20 ml of water was evenly sprayed onto intact plants growing in plastic pots. Lower concentrations of JA and ACC solutions (e.g. 0.3 mM each) had little effect on induced volatiles in M. truncatula plants. For ethephon treatment, ethephon (10 mM; Sigma-Aldrich) in 100 μl of 50 mM MES buffer, pH 6.0, was applied to leaves of an intact M. truncatula plant. For inhibition experiments, the petioles of detached plantlets of M. truncatula were placed in glass vials containing aqueous solutions of fosmidomycin (7 ml, 100 μM; Molecular Probes, Eugene, OR, USA), or lovastatin (7 ml, 100 μM; A.G. Scientific, San Diego, CA, USA). Incubation experiments with labelled precursors were carried out by placing the plantlets in water (7 ml, containing 1-deoxy-[5,5-2H2]-d-xylulose at 1 mg/ml). Controls were also placed in water. After pre-incubation with the labelled precursor or the inhibitors for 2 h, either JA + ACC (1 mM each) in 20 ml of water were sprayed evenly onto the plants or 5–6 larvae were placed on the plants. For headspace analysis, the plants were transferred 2 h after JA + ACC treatment into a glass cabinet for volatile collection (see below). Each treatment started at 10:00. During treatments, temperatures were kept constant at 22 ± 1°C, and the photoperiod was 14 h light (6,000 lux): 10 h dark. The light period extended from 07:00 to 21:00.
Analysis of volatiles
For headspace analysis, a potted plant or a plantlet in a glass vial was enclosed with or without caterpillars in glass containers (2.5 l). The emitted volatiles were trapped onto charcoal traps (1.5 mg of charcoal, CLSA-Filter, Le Ruisseau de Montbrun, Daumazan sur Arize, France) while air circulated for 24 or 48 h. The collected volatiles were eluted with dichloromethane (2 × 20 μl) containing n-bromodecane (100 ng μl−1) as an internal standard. Samples were analyzed on a ThermoQuest/Finnigan TRACE GC 2000 with a TRACE MS (Manchester, UK) equipped with an ECTM-5 capillary column (0.25 mm i.d. × 15 m with 0.25-mm film, Alltech, Deerfield, IL, USA). Injection volume: 1 μl; split 1:100; 220°C. Ionization energy: 70 eV. Compounds were eluted under programmed conditions starting from 40°C (2-min hold) and ramped up at 10°C min−1 to 200°C followed by 30°C min−1 to 280°C, which was held for 1 min prior to cooling. Helium at a flow rate of 1.5 ml min−1 served as a carrier gas. The headspace volatiles were identified by comparing their mass spectra and Kovàts indices with authentic references on two columns of different polarity (Leitner et al. 2005). Absolute quantification of the volatiles was not possible due to the limited availability of authentic standards and partly overlapping peak areas of germacrene D 16 and an unknown sesquiterpene 17. Relative peak areas from integration of the reconstructed ion chromatogram were used without additional calibration.
Analysis of oxylipins
Leaves (0.5 g) were homogenized with an Ultra-Turrax® T25 Basic (Ika, Staufen, Germany) on ice for 5 min in methanol (8 ml) containing 0.03% BHT and 0.03 M pentafluorobenzyl hydroxylamine hydrochloride (PFBHA, Sigma-Aldrich) (Schulze et al. 2006). [2H2]-JA (150 ng) and [2H2]-12-oxophytodienoic acid (250 ng) were added as internal standards. The methanol extract was shaken for derivatisation at room temperature for 2 h, and 4.0 ml diluted HCl (pH = 3) were added. The methanol/water phase was carefully extracted with hexane (3 × 10 ml), and after centrifugation, the hexane layers were collected and combined. The hexane fraction was passed through a Chromabond NH2 cartridge (3 ml/500 mg, Macherey-Nagel, Düren, Germany) preconditioned with methanol (5 ml) and hexane (5 ml), washed with the solvent mixture of 2-propanol/dichloromethane (2:1 v/v, 5 ml), and eluted with diethyl ether/formic acid (98:2, 10 ml). The solvents were evaporated under argon, and the remaining crude acids were esterified with an ethereal solution of diazomethane (1 ml) at room temperature for 5 min. After evaporation of the solvent, the sample was dissolved in 30 μl dichloromethane and analyzed by gas chromatography–mass spectrometry (GC–MS) as described (Schulze et al. 2006).
Cytoplasmic Ca2+ concentration
A solution of Fluo-3 AM (acetoxy-methyl ester of Fluo-3, 5 μM, Fluka, Buchs, Switzerland), 0.5 mM calcium sulphate, and 2.5 μM DCMU [3-(3′,4′-dichlorphenyl)-1,1-dimethylurea] in 50 mM MES buffer, pH 6.0, was used for initial treatment of leaves of an intact M. truncatula plant as previously described (Maffei et al. 2004). A leaf was cut once by a razor blade in order to allow the dye to enter the tissues. One hour after treatment with Fluo-3 AM, the leaf was fixed on an Olympus FLUOview confocal laser scanning microscope stative without detaching it from the plant. The microscope was operated with a Krypton/Argon laser at 488 and 568 nm wavelengths: the first wavelength excited the Fluo-3 dye emitting green light, while the second excited mostly chloroplasts emitting a red fluorescence. Images generated by the FluoView software were analyzed using the NIH Image J software described earlier (Maffei et al. 2004). Aequorin-dependent luminescence was determined according to (Maffei et al. 2006).
For high resolution subcellular Ca2+ localization, after treatment with Fluo-3 AM for 1 h, leaves were mounted on a Leica TCS SP2 multiband confocal laser-scanning microscope stative without separating the leaf from the plant. Scannings were recorded using the HCX PL APO 63x/1.20 W Corr/0.17CS objective. The microscope was operated with a Laser Ar (458 nm/5 mW; 476 nm/5 mW; 488 nm/20 mW; 514 nm/20 mW), a Laser HeNe 543 nm/1.2 mW, and a Laser HeNe 633 nm/10 mW.
Total RNA was isolated from leaf tissues using the Concert Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Total RNA was purified to eliminate genomic DNA using the Qiagen RNeasy Plant RNA kit and the RNase-Free DNase Set (Qiagen, Hilden, Germany). First-strand cDNA was synthesized using the SuperScript III Reverse Transcriptase (RT) (Invitrogen), oligo(dT)12–18 primer, and 2 μg of total RNA at 50°C for 50 min. Primers for real-time polymerase chain reaction (PCR) were designed using the Primer 3 Software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) for a length of the resulting PCR product of approximately 200 bp. Primers were designed using partial DNA sequences obtained from the TIGR M. truncatula expressed sequence tag (EST) database: (http://www.tigr.org/tigr-scripts/tgi/T_index.cgi?species=medicago) and GenBank. The real-time PCR was done on a Mx3000P Real-Time PCR System (Stratagene, La Jolla, CA, USA). The process was performed with 25 μl of reaction mixture containing 12.5 μl of 2 × Brilliant SYBR Green QPCR Master Mix (Stratagene), cDNA (1 μl from 20 μl of each RT product pool), 100 nM primers, and 30 mM ROX as a passive reference dye. The following protocol was followed: initial polymerase activation: 10 min at 95°C; 40 cycles of 30 s at 95°C, 60 s at 55°C, and 30 s at 72°C. PCR conditions were determined by comparing threshold values in dilution series of the RT product, followed by non-RT template control and non-template control for each primer pair. Relative RNA levels were calibrated and normalized with the level of actin mRNA (EST annotation no. AA660796).
Primers used for real-time PCR are: actin, 5′-TAC CCC ATT GAG CAC GGT AT-3′ and 5′-ATA CAT GGC AGG CAC ATT GA-3′; MtDXS1, 5′-AGC CGT TCA GAA CTG TTT GG-3′ and 5′-ATC AAG GGC CAT GAA CTG AG-3′; MtDXS2, 5′-ACA CCG GTG GTC ATC TTA GC-3′ and 5′-CAT CCC TTT TGG GAA AAC CT-3′; MtHMGR1, 5′-GCC GTA AAT GAT GGG AGA GA-3′ and 5′-GCC AGC TAA AAC AGC TCC AG-3′; MtTPS1, 5′-TGC TCC AAC CCC TAA TGC TA-3′ and 5′-CCT CGA AGG GTT TGT GAC AT-3′; MtTPS2, 5′-TGT CGC AAA AAC AGA TGA GC-3′ and 5′-ATC CTT GTT GCC TCA ACA CC-3′; MtTPS3, 5′-TGC AAT CAC AAA ATG CAA CA-3′ and 5′-GAT CTG GGA GAC ATG CCA TT-3′; MtTPS5, 5′-CCG ATG CCT TTT ATG ACT CG-3′ and 5′-TGT GCC ACT TGG ATT TTT CA-3′. The EST annotation numbers are: actin, AA660796; MtHMGR1, TC102857. GenBank accession numbers are: MtDXS1, AJ430047; MtDXS2, AJ430048; MtTPS1 AY763425; MtTPS2, AY766250; MtTPS3, AY766249; MtTPS5, DQ188184.
cDNA cloning and TPS enzyme assay
Cloning 3′-end of MtTPS5 cDNAs was accomplished by rapid amplification of cDNA ends (RACE PCR) using total RNA and the First Choice RLM-RACE Kit (Ambion, Austin, TX, USA) following the manufacturer’s protocol. For functional identification, cDNAs were amplified by PCR using Pfu DNA Polymerase (Promega, Madison, WI, USA) with a set of primers for an open reading frame of MtTPS1MtTPS3, or MtTPS5. The cDNA was subcloned into the pHis8–3 expression vector (Jez et al. 2000). The recombinant vectors were transformed into Escherichia coli BL21-CodonPlus(DE3). The bacterial strain was grown to A600 = 0.5 at 37°C in 5 ml of LB medium with kanamycin at 50 μg/ml. Cultures were induced with 1 mM isopropyl 1-thio-β-d-galactopyranoside (IPTG) and held overnight at 16°C while being shaken at 200 rpm. Cells were pelleted by centrifugation and resuspended in 1 ml TPS buffer (25 mM HEPES, pH = 7.3, 12.5 mM MgCl2, 0.25 mM MnCl2, 0.25 mM NaWO4, 0.125 mM NaF, 5 mM DTT, 10% glycerol). Resuspended cells were broken by sonication. Cell extracts were cleared by centrifugation and assayed for TPS activity with 50 μM GDP (Echelon Biosciences Incorporated, Salt Lake City, UT, USA), FDP (Echelon Biosciences Incorporated) or GGDP (Sigma-Aldrich). The assay mixture (1 ml) was covered with 1 ml of pentane to trap volatile products. After incubation at 30°C for 1 h, the pentane layer was transferred to a glass vial and concentrated to ~150 μl under a gentle stream of N2. Alternatively, the headspace of the assay mixture was analyzed using solid-phase microextraction (SPME, Supelco Inc., Bellefonte, PA, USA). Extracts of E. coli transformed with expression vectors without the TPS gene were used as controls following the above procedure. The enzymatic products were analyzed by GC–MS and identified by comparing their mass spectra and Kovàts indices with authentic references as described above.
Stereochemistry of (E)-nerolidol was analyzed by a Hewlett-Packard 5980 gas chromatograph coupled to a Hewlett-Packard 5917A quadrupole type mass selective detector (70 eV; scan range: 45–400 amu) equipped with a capillary column coated with heptakis-(2,6-di-O-methyl-3-O-pentyl)-β-cyclodextrin (0.25 mm × 25 m, 0.25 μm film, Macherey Nagel, Düren, Germany). Samples (1 μl) were injected splitless at 230°C with a column flow of 1 ml min−1 (He). Elution was programmed from 50 to 160°C (10 min) at 2.3°C min−1 and then to 210°C at 15°C min−1 (2 min). The elution order of (3S)-(E)-nerolidol and (3R)-(E)-nerolidol was determined with a racemic and a chiral reference according to (Degenhardt and Gershenzon 2000).
HIPV biosynthesis in M. truncatula WT and the ethylene-insensitive mutant skl
Next, we investigated transcript levels of genes of early and late steps in terpenoid biosynthesis in BAW-damaged WT and skl by quantitative RT-PCR analysis (Fig. 1b). The transcript levels of putative terpene synthases MtTPS2 and MtTPS3 (Gomez et al. 2005) were induced at similar levels in WT and skl plants throughout the entire period of BAW damage, whereas the transcript levels of MtTPS1 (Gomez et al. 2005) and the gene encoding 1-deoxy-d-xylulose-5-phosphate (DXP) synthase (MtDXS2) involved in the MEP pathway (Walter et al. 2002) were initially lower in skl plants after exposure to BAW feeding (2 h, P = 0.08 and P < 0.05, respectively, ANOVA). A putative sesqui-TPS gene (MtTPS5) mined from the TIGR M. truncatula EST database was only up-regulated in WT plants after 24 h. Unlike MtDXS2, the transcript levels of MtDXS1 and 3-hydroxy-3-methylglutaryl-CoA reductase (MtHMGR1), two other genes of early terpenoid biosynthesis, were identical in both BAW-damaged WT and skl plants.
Product spectrum of MtTPSs
Synergistic effects of jasmonic acid and ACC on terpenoid biosynthesis
Pathway allocation of sesquiterpene biosynthesis in BAW-treated plants
Impact of ethylene on Ca2+ signaling
Altogether, these findings suggest that ethylene can modulate the extent and mode of Ca2+ influx resulting from herbivore attack, either directly or through feedback from ethylene effects on downstream events.
Involvement of Ca2+ signaling in the BAW-induced terpenoid formation
The composition of the induced volatile blends depends on plastic phenotypic responses which can be modulated by the interplay of several biosynthetic pathways and levels of signaling molecules (Arimura et al. 2005). In order to dissect the complexity of plastic responses, we used the ethylene-insensitive skl mutant of M. truncatula in combination with defined chemical treatments and BAW exposure. Within the BAW- or treatment (JA + ACC)-induced blends of volatiles, sesqui and homoterpenoids were the most abundant (see Fig. 1a). To identify the genes responsible for terpenoid biosynthesis, we cloned three MtTPS genes, based on sequence similarities with other angiosperm TPS genes, namely MtTPS1, MtTPS3, and MtTPS5. Heterologous protein expression and biochemical analysis of the enzymatic product identified MtTPS1 as a β-caryophyllene synthase, MtTPS3 as a nerolidol/geranyl linalool synthase, and MtTPS5 as a multiproduct sesquiterpene synthase. Multi-product TPSs significantly contribute to the plasticity of blends and are increasingly found in plants, especially in context with herbivory, as shown previously for other plants (Köllner et al. 2004; Tholl et al. 2005). In addition to olefinic sequiterpenes, the heterologously expressed MtTPS5 protein produced a number of hydroxylated products which were not detected in the gas phase of M. truncatula plants (Fig. 1a). Whether these compounds are subsequently modified in planta remains to be clarified.
Conventional pathway allocation suggests that the sesquiterpene precursors, IDP and DMADP, are mainly provided by the cytosolic MVA pathway. However, evidence is emerging that the plastidial MEP pathway may also significantly contribute to cytosolic sesquiterpene biosynthesis by allowing IDP to be shuttled between the different compartments (Piel et al. 1998; Bick and Lange 2003; Bartram et al. 2006). Here we demonstrate this for M. truncatula by the administration of labelled [2H2]-DOX and pathway-specific inhibitors. These results indicate that the MEP pathway, as well as the cytosolic MEV pathway, plays a dominant role by providing terpenoid precursors.
Compared to WT plants, the skl mutant produced lower amounts of sequiterpenes and DMNT when challenged with feeding insects, suggesting a possible role for ethylene as a modulator of BAW-induced terpenoid biosynthesis. In fact, ethylene and JA synergistically induce the biosynthesis of certain sesquiterpenes [e.g., α-copaene, (E)-nerolidol, and germacrene D along with unidentified sesquiterpenes (I)]. The production of these sesquiterpenes coincides with enhanced transcript levels of MtDXS2 and MtTPS5. In contrast, MtTPS1 and MtTPS3 transcripts were only induced by JA treatment.
Feeding BAW raised the expression level of MtTPS3 in WT and skl plants in the same way, but levels of (E)-nerolidol and DMNT formation were higher in WT plants, suggesting that ethylene might have a post-transcriptional impact on the MtTPS3 protein. Another interesting aspect is that MtTPS3 has been claimed to be a plastidial-targeted protein (Gomez et al. 2005), which is also consistent with its ability to produce the diterpene geranyllinalool from GGDP (see Fig. 3b). The subcellular localization of (E)-nerolidol formation and subsequent DMNT-production remains unsolved.
Ethylene has been recently shown to enhance sesquiterpenes and suppress monoterpenes in Zea mays (Ruther and Kleier 2005). In this case, the presence of ethylene enhanced the hexenol-induced production of volatiles (linked to increased internal JA levels) by a factor of 5–6 with respect to the sesquiterpenes, but suppressed the constitutively emitted monoterpene linalool (Ruther and Kleier 2005). In M. truncatula the simultaneous application of JA + ACC also significantly enhanced production of sesquiterpenes. The suppressing effect of ethylene on monoterpene biosynthesis in WT plants becomes obvious from the volatile profile of the skl mutant: it lacks ethylene perception and as a consequence, constitutively emits limonene. The mode of monoterpene suppression by ethylene in WT M. truncatula is still unknown and will require the identification of the limonene synthase and of specific elements that control plastidial monoterpene biosynthesis. It is interesting to note that the sesquiterpene synthases MtTPS1 and MtTPS5 represent multiproduct enzymes that could also synthesize monoterpenes, including limonene. However, since BAW attack enhanced the transcript levels of MtTPS1 and MtTPS5 in WT leaves that lacked limonene emission, these enzymes could not be responsible for the production of limonene. The lack of any plastidial transit peptide at the N-terminus of MtTPS1 and MtTPS5 supports their prediction as cytosolic proteins involved in sesquiterpenoid biosynthesis.
WT and skl plants showed different resting levels of cytosolic [Ca2+]cyt; but after BAW feeding, both plants increased at the same rate (see Fig. 7b). Both pre- and post-treatment with ethephon reduced the BAW-triggered [Ca2+]cyt increase in WT leaves, but had no effect on skl plants. In order to test whether or not this effect is general, we treated the transgenic aequorin-expressing soybean cell culture (Mithöfer et al. 1999) with ethephon and insect regurgitate containing active eliciting compounds. In this artificial system, ethephon abolished the regurgitate-elicited and transient increase of [Ca2+]cyt (Fig. S3 of electronic supplementary material). Accordingly, either ethephon or the released ethylene have a direct effect on Ca2+ homeostasis. Depriving plants of extracellular Ca2+ by BAPTA reduces the formation of JA (Fig. 8a) and the release of terpenoids (Fig. 8b). The level of OPDA is not affected. However, the skl plants only slightly modify the level of [Ca2+]cyt and never completely remove external Ca2+. These facts may explain why the level of JA production after feeding BAW is not significantly different between WT and skl plants (see Fig. S1 of electronic supplementary material).
Although the simultaneous application of JA + ACC strongly stimulates volatile emissions (Fig. 5), this effect may be partly due to the action of ACC, but not of ethylene. ACC, which was found to occur as a conjugate with JA, might act as an additional factor controlling gene expression, similar to the conjugates of JA and isoleucine (Krumm et al. 1995; Staswick and Tiryaki 2004). This assumption is supported by the observation that the combined treatment with JA + ACC + STS does not fully compensate for the effect of JA + ACC (Fig. 5).
In conclusion, our results demonstrate that either the phytohormone ethylene or its precursor (ACC) affects the different levels of BAW-induced terpenoid blends by modulating early signaling events such as the cytoplasmic Ca2+-influx and later the JA-dependent biosynthesis of terpenoids.
We thank Dr. Giles E. Oldroyd (John Innes Centre) for skl seeds; Dr. Maritta Kunert and Ms. Anja David for volatile analysis. (-)-Cubebol was generously provided by Dr. Alois Fürstner (Max Planck Institute for Kohlenforschung). This work was supported by the Japanese Society for the Promotion of Science (to G.A.) and the Centre of Excellence CEBIOVEM of the University of Turin (to M.M.).
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