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provided by Wageningen University & Research Publications Planta (2006) 224:1197–1208 DOI 10.1007/s00425-006-0301-5

ORIGINAL ARTICLE

Induction of a leaf speciWc geranylgeranyl pyrophosphate synthase and emission of (E,E)-4,8,12-trimethyltrideca-1,3,7,11- tetraene in tomato are dependent on both jasmonic acid and salicylic acid signaling pathways

Kai Ament · Chris C. Van Schie · Harro J. Bouwmeester · Michel A. Haring · Robert C. Schuurink

Received: 21 November 2005 / Accepted: 11 April 2006 / Published online: 20 June 2006 © Springer-Verlag 2006

Abstract Two cDNAs encoding geranylgeranyl pyro- of TMTT. We show that there is an additional layer of phosphate (GGPP) synthases from tomato (Lycopers- regulation, because geranyllinalool synthase, catalyz- icon esculentum) have been cloned and functionally ing the Wrst dedicated step in TMTT biosynthesis, was expressed in Escherichia coli. LeGGPS1 was predomi- induced by JA but not by MeSA. nantly expressed in leaf tissue and LeGGPS2 in ripen- ing fruit and Xower tissue. LeGGPS1 expression was Keywords Geranylgeranyl pyrophosphate · Jasmonic induced in leaves by spider mite (Tetranychus urticae)- acid · Lycopersicon · Salicylic acid · Spider mites · feeding and mechanical wounding in wild type tomato Homoterpene but not in the jasmonic acid (JA)-response mutant def- 1 and the salicylic acid (SA)-deWcient transgenic NahG Abbreviations line. Furthermore, LeGGPS1 expression could be GGPP Geranylgeranyl pyrophosphate induced in leaves of wild type tomato plants by JA- or JA Jasmonic acid methyl salicylate (MeSA)-treatment. In contrast, SA Salicylic acid expression of LeGGPS2 was not induced in leaves by MeSA Methyl salicylate spider mite-feeding, wounding, JA- or MeSA-treat- TMTT (E,E)-4,8,12-Trimethyltrideca-1,3,7,11-tetraene ment. We show that emission of the GGPP-derived FPP Farnesyl pyrophosphate volatile terpenoid (E,E)-4,8,12-trimethyltrideca- GPP Geranyl pyrophosphate 1,3,7,11-tetraene (TMTT) correlates with expression of IPP Isopentenyl pyrophosphate LeGGPS1. An exception was MeSA-treatment, which GL Geranyllinalool resulted in induction of LeGGPS1 but not in emission DMNT 4,8-Dimethylnona-1,3,7-triene

Introduction Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at http://dx.doi.org/10.1007/s00425-006-0301-5. Geranylgeranyl pyrophosphate (GGPP) synthase belongs to a group of short-chain prenyltransferases K. Ament · C. C. Van Schie · M. A. Haring · that also include farnesyl pyrophosphate (FPP) syn- R. C. Schuurink (&) thase and geranyl pyrophosphate (GPP) synthase. Swammerdam Institute for Life Sciences, Department of Plant Physiology, University of Amsterdam, These enzymes are involved in isoprenoid biosynthe- Kruislaan 318, 1098 SM Amsterdam, sis and share high sequence homology (Joly and The Netherlands Edwards 1993). An early step in isoprenoid e-mail: [email protected] biosynthesis, catalyzed by GPP synthase, is the con- H. J. Bouwmeester densation of two C5 molecules, isopentenyl pyro- Plant Research International, Post OYce Box 16, phosphate (IPP) and dimethylallyl pyrophosphate 6700 AA Wageningen, The Netherlands (DMAPP) forming GPP (C10). In plants two 123 1198 Planta (2006) 224:1197–1208 separate pathways exist for the synthesis of these infested plants. This suggests that TMTT can inXuence universal C5 intermediates; the cytosolic mevalonate the foraging behavior of predatory mites. pathway and the plastidial methyl-D-erythritol 4- Emission of TMTT in excised lima bean leaves can phosphate pathway. Addition of two IPP molecules be induced by early intermediates of the jasmonic acid to one DMAPP molecule and the addition of one (JA) biosynthetic pathway, linolenic acid and 12-oxo- IPP molecule to GPP is catalyzed by FPP synthase, phytodienoic acid (Koch et al. 1999). There is evidence resulting in FPP (C15). GGPP synthase catalyzes the that, in tomato, emission of TMTT is dependent on JA. formation of GGPP (C20) by condensation of The tomato mutant def-1, which is deWcient in induced DMAPP with three IPP molecules or by the conden- JA-accumulation after wounding or herbivory (Li et al. sation of GPP with two IPP molecules or FPP with 2002), does not emit TMTT upon spider mite-infesta- one IPP molecule (Burke and Croteau 2002). GGPP tion. However, emission of TMTT can be restored by is the precursor for many diVerent products in plants pre-treating these plants with JA (Ament et al. 2004). like diterpenes, gibberellins, carotenoids, the iso- These results indicate that biosynthesis of TMTT is prenoid side chain of chlorophyll and it is used for regulated by oxylipins. protein prenylation. Based on the genome sequence Induced production of terpenes is often correlated Arabidopsis has 12 putative GGPP synthases (Lange to induced terpene synthase activity. Terpene syn- and Ghassemian 2003). Zhu et al. (1997) and Okada thases generate speciWc products from common ter- et al. (2000) have characterized Wve of them and pene precursors. Regulation at the level of terpene showed that they diVer in expression patterns and synthases allows production of a highly speciWc ter- subcellular localization, making it likely that individ- pene blend. However, there are also some reports on ual GGPP synthases have very speciWc functions and the regulation of genes encoding enzymes that act are regulated accordingly. upstream of terpene synthases, resulting in increased (E,E)-4,8,12-Trimethyltrideca-1,3,7,11-tetraene precursor pools. In cytosolic IPP synthesis, 3- (TMTT) is a diterpene-derived volatile produced by hydroxy-3-methylglutaryl-coenzyme A reductase is many plants in response to herbivory. The biosynthe- upregulated in response to pathogens or pathogen- sis of TMTT has been proposed by Boland and derived elicitors in solanaceous plants like tomato Gäbler (1989). They suggest that TMTT is produced (Park et al. 1992), Korean red pepper (Capsicum ann- by oxidative degradation of geranyllinalool (GL). uum) (Ha et al. 2003) and potato (Solanum tubero- This synthesis parallels the biosynthesis of 4,8-dim- sum) (Choi et al. 1992), but also by insect herbivores ethylnona-1,3,7-triene (DMNT), which is likely in potato (Korth et al. 1997) and Nicotiana attenuata formed by oxidative degradation of the sesquiter- (Hui et al. 2003). Furthermore, Kant et al. (2004) pene (E)-nerolidol (Boland et al. 1998). The diter- have shown that transcription of deoxy-xylulose-5- pene GL is supposedly formed by an uncharacterized phosphate synthase, involved in plastidial IPP synthe- GL synthase from the common diterpene precursor sis, is upregulated in tomato upon spider mite-infesta- GGPP. tion. Downstream, at the level of prenyltransferases Herbivore-induced volatiles can beneWt plants by that precede the action of terpene synthases, little is attracting natural enemies of the attacking herbivore known about regulation of gene expression. FPP syn- (Sabelis et al. 2001). Tomato plants infested with spi- thase, required for sesquiterpene biosynthesis, is der mites emit a blend of volatiles that makes them upregulated by pathogens in Korean red pepper (Ha attractive to predatory mites (Phytoseiulus persimilis), et al. 2003) and cotton (Gossypium hirsutum) (Liu a natural enemy of spider mites. Spider mite-infested et al. 1999) and by insect herbivores in maize (Zea tomato plants emit signiWcantly more TMTT than mays) (Farag et al. 2005). GGPP synthase is induced uninfested tomato plants (Kant et al. 2004). The role of in taxus cell suspensions by methyl jasmonate (Hef- TMTT in the attraction of predatory mites has been ner et al. 1998) and by spider mite-herbivory in investigated with lima bean (Phaseolus lunatus) as tomato (Kant et al. 2004). model system (De Boer et al. 2004). Predatory mites To investigate the regulation of TMTT production prefer the odor source of lima bean infested with spi- in tomato we cloned two GGPP synthases from tomato der mites above that of lima bean infested with beet (LeGGPS1, DQ267902 and LeGGPS2, DQ267903), of armyworm (Spodoptera exigua). Spider mite-infested which LeGGPS1 is proposed to be involved in precur- lima bean emits signiWcantly more TMTT than beet sor biosynthesis for TMTT. We also determined the armyworm-infested lima bean. When TMTT is added role of the phytohormones JA and salicylic acid (SA) to the odor of beet armyworm-infested plants, preda- in regulating LeGGPS1 expression and GL synthase tory mites prefer this odor above that of spider mite- activity, leading to TMTT emission. 123 Planta (2006) 224:1197–1208 1199

Materials and methods min. The headspace (the air around the plant) was sampled during 24 h for 2 consecutive days by trapping Chemicals the outgoing air on 300 mg Tenax TA (Alltech, Deer- Weld, IL, USA) in a 5-mm wide glass tube. The com- TMTT was synthesized by Prof. H. Hiemstra (Depart- plete volatile collection set-up was made from glass ment of synthetic organic chemistry, University of and TeXon and no grease was used. The headspace of Amsterdam, The Netherlands) as described by Dodd spider mite-infested plants was collected on the fourth and Oehlschlager (1992). Purity exceeded 98% as was day of spider mite-infestation. Simultaneously, clean checked by NMR and GC-MS. Geranyllinalool, benzyl plants were enclosed in similar desiccators as control acetate and methyl salicylate (MeSA) were obtained treatment. Volatiles of mechanically wounded plants from Fluka (Buchs, Switzerland) and JA from Duchefa were sampled for 24 h directly after damaging the Biochemicals (Haarlem, the Netherlands). plants. After 24 h of treatment with JA or MeSA, plants were placed in clean desiccators and volatiles Plant material and arthropod rearing were sampled in the subsequent 2 days in 24-h inter- vals. The Tenax tubes were eluted with 2 ml pen- Tomato seedlings [Lycopersicon esculentum Mill cv. Cas- tane:diethylether (4:1, v/v) with 1.8
g of benzyl acetate tlemart, def-1 (Howe and Ryan 1999), cv. Moneymaker as internal standard. One microliter was analyzed using (obtained from the Glasshouse Crop Research Institute gas chromatograph mass-spectrometry as described by (GCRI), now Horticultural Research Institute (HRI), Ament et al. (2004). Compounds were identiWed and UK) and NahG (Brading et al. 2000)] were grown as quantiWed on the basis of the internal standard and described by Kant et al. (2004). The two-spotted spider synthetic external standards of known concentrations. mite (Tetranychus urticae Koch) was originally obtained All experiments were performed twice. in 1993 from tomato plants in a greenhouse (Houten, The Netherlands; Gotoh et al. 1993) and was maintained RT-PCR analysis on the cultivar Moneymaker ever since. For determining tissue speciWc expression of Plant treatments LeGGPS1 and LeGGPS2, a mature plant was dis- sected and tissues were frozen in liquid nitrogen and Plants used in all experiments, except for tissue speciWc stored at ¡80°C. To determine induction of LeGGPS1 expression, were 18–21 days old and had four fully or LeGGPS2, leaves were taken from spider mite- expanded leaves and two emerging leaves. Three days infested, wounded, JA-, MeSA- and control plants. prior to the start of the experiments, plants were trans- Experiments with NahG, def-1 and the corresponding ferred to a climate chamber. For tissue speciWc expres- wild type tomato plants, infested with spider mites and sion 14-week-old plants were used that were grown in the control leaves, were performed in a climate chamber. green house. Adult female spider mites were gently Tissues were harvested as described above. Total RNA placed on the adaxial surface of the fully expanded termi- was isolated with a Phenol-LiCl based method as nal leaXets using a soft-bristle paintbrush. Per leaXet 15 described by Verdonk et al. (2003). RT-PCR determi- mites were introduced, 3 leaXets on each plant. Mechani- nation of transcript levels was done as described by cal damage was inXicted on three leaXets per plant with Ament et al. (2004). For each cDNA we determined three incisions of an artery clamp per leaXet. Plants were the number of cycles for which the PCR ampliWcation treated with JA by misting them with 3 ml of a 0.25 mM was not saturated. PCR products were visualized by solution. Plants were left to recover for 24 h before the staining with ethidium bromide after separation on start of the experiment. For the MeSA treatments a sin- agarose gels. QuantiWcation of transcript levels of gle plant was enclosed in an airtight glass desiccator of LeGGPS1 and RUB1 conjugating enzyme (RCE1) was 22 l. On a cotton swab 16.7 mg MeSA was applied, which performed as described by Ament et al. (2004). PCR upon evaporation resulted in a vapor concentration of ampliWcation of RCE1 fragments (0.723 kb) comprised 5
M. After 24 h plants were transferred to clean desicca- 20 cycles with forward primer, 5Ј-GATTCTCTCTCAT tors for volatile analysis or harvested for RNA isolation. CAATCAATTCG-3Ј and reverse primer, 5Ј-GCATC CAAACTTTACAGACTCTC-3Ј; of PRP6 fragments Volatile analysis (0.427 kb) 23 cycles with forward primer 5Ј-TCAGTC CGACTAGGTTGTGG-3Ј and reverse primer 5Ј-TA Single plants were placed in a glass desiccator of 22 l GATAAGTGCTTGATGTGCC-3Ј of WIPI-2 frag- that was ventilated with carbon Wltered air at 200 ml/ ments (0.601 kb) 14 cycles with forward primer, 5Ј-GA 123 1200 Planta (2006) 224:1197–1208

CAAGGTACTAGTAATCAAT-3Ј and reverse primer, gation. These cells were extracted with 1 ml of 90% 5Ј-CACATAACACACAACTTTGATGCC-3Ј; of acetone, and the absorption at 450 nm was determined LeGGPS2 fragments (0.378 kb) 25 cycles with forward as a measure for carotenoid production. primer 5Ј-CTTTGGATGAGGCTATAATGG-3Ј and reverse primer 5Ј-ATTCGGCGACAGCAACGAG Geranyllinalool synthase assay G-3Ј; and of LeGGPS1 fragments (0.327 kb) 27 cycles with forward primer 5Ј-GCAATCAAGGTAAACA Frozen leaf tissue (1.5 g) was ground in liquid nitrogen, AAGCAC-3Ј and reverse primer 5Ј-CAAAGATAA and homogenized further with a Polytron (Kinematica AAGTGCATCCCCTG-3Ј. All PCR ampliWcations AG, Luzern, Switzerland) in 4 ml extraction buVer were performed with each cycle: 94°C for 45 s, 55°C for containing 100 mM hepes (pH 8.0), 10 mM ascorbic 45 s and 72°C for 75 s and a Wnal extension of 5 min at acid, 50 mM sodium meta-bisulphite, 20% glycerol, W 72°C. RT-PCR products were sequenced to con rm 10 mM MgCl2, 5 mM DTT, proteinase inhibitor cock- speciWc ampliWcation. tail complete (Roche, Mannheim, Germany) and 0.4 g PVPP. Extracts were incubated with 0.8 g Amberlite Phylogenetic analysis XAD-4 for 5 min, Wltered through Miracloth and cen- trifuged for 20 min at 20,000 g. Three milliliter super- The sequences of LeGGPS1, LeGGPS2 and several natant was rebuVered, using Econo-Pac 10DG columns other prenyltransferases present in the NCBI Gen- (Bio Rad), to assay buVer containing 50 mM hepes (pH

Bank database were aligned using the CLUSTALW 8.0), 1 mM ascorbic acid, 10% glycerol, 10 mM MgCl2, algorithm in MacVector (Oxford Molecular Ltd., 1 mM DTT and proteinase inhibitor cocktail complete. Oxford, UK). This alignment was used to construct a Assays were performed in 10 ml glass vials with 1 ml mid point rooted tree with Neighbor joining method. desalted extract and were incubated for 1 h at 30°C The distance was Poisson-corrected and gaps were dis- with 6
M GGPP (Sigma). Assay products were sam- tributed proportionally. Bootstrap analysis was per- pled on a 100
M PDMS solid phase micro extraction formed with 100 replications. (SPME) Wber (Supelco, Zwijndrecht, The Nether- lands) for 15 min at 60°C. The SPME Wber was des- Genetic complementation of E. coli with LeGGPS1 orbed for 1 min in an Optic injector port (ATAS GL and LeGGPS2 Int. Zoeterwoude, The Netherlands), which was kept at 250°C. Compounds were separated on a DB-5 col- The plasmid pACCAR25crtE (Sandmann et al. 1993) umn (10 m £ 180
m, 0.18
m Wlm thickness; Hewlett contains the gene cluster from Erwinia uredovora, with Packard) in an 6890N gas chromatograph (Agilent, crtB, crtI, crtX, crtY and crtZ encoding carotenoid bio- Amstelveen, The Netherlands) with a temperature synthetic enzymes, with the exception of crtE (encod- program set to 40°C for 1.5 min, ramp to 280°C at ing GGPP synthase). Full-length cDNAs of LeGGPS1 30°C min¡1 and 250°C for an additional 2.5 min. and LeGGPS2 were cloned into pBluescript KS+ to Helium was used as carrier gas, the column Xow was produce a LacZ fusion protein. As a positive control, set to 3 ml/min for 2 min and to 1.5 ml/min thereafter. pBluescript containing human (Homo sapiens) GGPP Mass spectra were generated with the ion source set to synthase as a fusion with LacZ was used, and as a nega- ¡70 V at 200°C and collected with a Time-of-Flight tive control pBluescript without an insert. pBluescript MS (Leco, Pegasus III, St. Joseph, MI, USA) at plasmid containing human GGPP synthase and pAC- 1,671 V, with an acquisition rate of 20 scans/s. The CAR25crtE were kindly provided by Dr. M. Kawam- product GL was identiWed using the mass spectrum and ukai, Shimane University, Shimane, Japan. DH10 retention time of the authentic standard and quantiWed Escherichia coli cells that had previously been trans- using a dilution series of the authentic standard in the formed with the pACCAR25crtE plasmid were trans- same quantity-range in desalted extract. formed with pBluescript containing LeGGPS1, LeGGPS2, human GGPS or no insert. Transformants were plated on LB agar medium containing 50
g/ml Results ampicillin, 25
g/ml chloramphenicol and 1 mM IPTG, and incubated at 30°C for 24 h. Subsequently, indepen- IdentiWcation of two GGPP synthases from tomato dent colonies were transferred to liquid LB medium containing 50
g/ml ampicillin, 25
g/ml chlorampheni- We had previously identiWed two tomato cDNAs encod- col and 1 mM IPTG and grown at 30°C for 24 h. Cells ing putative GGPP synthases (http://www.tigr.org from 2 ml of these cultures were harvested by centrifu- TC168578 and TC167026), which were diVerentially 123 Planta (2006) 224:1197–1208 1201 regulated by spider mite-herbivory (Kant et al. 2004). Functional expression of LeGGPS1 and LeGGPS2 in The full-length cDNAs, LeGGPS2 and LeGGPS1, E. coli were generated by 5Ј RACE PCR using reverse tran- scribed cDNA from tomato Xower tissue and spider To conWrm that LeGGPS1 and LeGGPS2 are func- mite-infested tomato leaf tissue, respectively. The open tional GGPP synthases, we expressed full-length reading frames of LeGGPS1 and LeGGPS2 were pre- cDNAs in E. coli harboring the gene cluster from dicted to encode proteins of 365 and 363 amino acids Erwinia uredovora containing all genes for carotenoid with a mass of 39.9 and 39.0 kDa. TargetP and ChloroP biosynthesis (crtB, crtI, crtX, crtY, crtZ), except GGPP (Emanuelsson et al. 2000) software predicted a plastid synthase (crtE). If the product of the introduced gene localization peptide for both LeGGPS1 and has GGPP synthase activity, these cells will produce LeGGPS2. The deduced mature protein sequences of carotenoids and become orange. LeGGPS1 and LeGGPS1 and LeGGPS2 share 68% identity. Both LeGGPS2 could indeed complement E. coli cells miss- proteins contain the seven highly conserved domains ing crtE. Human GGPP synthase was used as positive present in prenyltransferases (Koike-Takeshita et al. control. The empty vector control did not show carot- 1995). The three aspartic acid residues and the two enoid production, which was measured spectrophoto- arginine residues in domain II as well as the FQXXD- metrically in cell extracts at 450 nm (Fig. 2). DXLD motif in domain VI, involved in substrate bind- ing (Kellogg and Poulter et al. 1997), were conserved LeGGPS1 and LeGGPS2 are diVerentially expressed in both tomato GGPP synthase proteins (Supplemen- in various plant tissues tal Fig. 1). Figure 1 shows a phylogenetic tree based on the deduced amino acid sequences of LeGGPS1, LeG- The TIGR tomato database contains two ESTs corre- GPS2 and several other prenyltransferases. This analy- sponding to LeGGPS1, both present in the library sis indicates that these two tomato GGPP synthases made from tomato leaves infected with the bacterial cluster with other plant GGPP synthases and form a pathogen Pseudomonas syringae. Three ESTs from branch diVerent from the FPP synthases and the GPP LeGGPS2 are present in the TIGR database, all in the synthases. The large subunits of the heterodimeric library made from fruit at the breaker stage. This sug- GPP synthases from snapdragon and mint also cluster gests tissue-speciWc expression of these two genes. To in the group of GGPP synthases, whereas the small determine this tissue-speciWc expression in more detail, subunits cluster as an out-group. Of these large sub- we dissected a mature tomato plant and isolated RNA units it is known that they exhibit GGPP synthase from diVerent tissues for RT-PCR analysis with gene activity, when expressed in E. coli without the small speciWc primers. This showed that LeGGPS1 tran- subunit (Tholl et al. 2004). scripts are predominantly present in leaf tissue,

98 Arabidopsis GPS Tomato GPS 100 100 Mouse GGPS Human GGPS 100 Rice FPS Sunflower FPS Taxus GGPS Tomato FPS 90 Sunflower GGPS Arabidopsis FPS 99 LeGGPS2 Sweet Pepper GGPS 72 97 99 Snapdragon GPS ls Mint GPS ls 81 Arabidopsis GGPS11 95 Arabidopsis GGPS2 98 LeGGPS1 Rice GGPS 100 Snapdragon GPS ss 0.2 Mint GPS ss

Fig. 1 LeGGPS1 and LeGGPS2 cluster with other plants gera- (AY534687), mint GPS ls (AJ249453), Arabidopsis GGPS11 nylgeranyl pyrophosphate (GGPP) synthases. A midpoint rooted (L25813), Arabidopsis GGPS2 (AC006135), LeGGPS1 phylogenetic tree of Arabidopsis GPS (AB104727), tomato GPS (DQ267902), rice GGPS (NM188788), snapdragon GPS ss (DQ286930), mouse GGPS (AB106044), Human GGPS (AY534686), mint GPS ss (AF182827) is shown. Bootstrap values (BC067768), rice FPS (NM192229), sunXower FPS (AF019892), are indicated at the branch points. The tomato GGPP synthases tomato FPS (AF044747), Arabidopsis FPS (L46367), Taxus are boxed. Small subunits of GPS are indicated with ss and large GGPS (AY566309), sunXower GGPS (AF020041), LeGGPS2 subunits with ls (DQ267903), sweet pepper GGPS (X80267), snapdragon GPS ls 123 1202 Planta (2006) 224:1197–1208

1.4 wounding resulted in the emission of comparable Y 1.2 amounts of TMTT, indicating that wounding is su cient to induce TMTT emission (Fig. 4a). This situation is 1.0 apparently diVerent from that in lima beans, where 0.8 emission of TMTT by mechanically wounded leaves 0.6 was signiWcantly less than by caterpillar-wounded leaves (Mithöfer et al. 2005). 0.4 Since there is no substantial storage of TMTT in Absorption at 450 nm 0.2 leaves of tomato plants (Ament et al. 2004), TMTT 0 must be synthesized de novo, as Farag and Pare (2002) LeGGPS1 LeGGPS2 HsGGPS C have demonstrated by labeling experiments with 13 Fig. 2 LeGGPS1 and LeGGPS2 encode functional GGPP syn- CO2. We hypothesized that the pool of GGPP neces- thases. Absorption at 450 nm of extracts from cells expressing sary for TMTT biosynthesis must be renewed and thus LeGGPS1, LeGGPS2, human GGPS (HsGGPS) or empty vec- investigated expression of LeGGPS1 and LeGGPS2 tor (C) is depicted, which is indicative for carotenoid production. after spider mite-feeding and mechanical wounding. Mean and standard deviation are shown of three measurements on independent transformants Expression of LeGGPS1 was spider mite- and wound- induced, correlating with induced emission of TMTT whereas LeGGPS2 transcripts are most abundant in (Fig. 4). LeGGPS2 was not induced by spider mite- fruits and Xower organs (Fig. 3). feeding or by mechanical wounding (Fig. 4b). Spider

Expression of LeGGPS1 and emission of TMTT are induced by spider mites and mechanical wounding a 140 )

TMTT, the most abundant spider mite-induced tomato -1 120 volatile, is probably synthesized from GGPP via GL. 100 24 h

When spider mites forage, leaves are continuously -1 damaged and this wounding leads to a wound 80 response. To investigate whether emission of TMTT is 60 speciWc for spider mite-feeding or a result of the 40 wounding, we sampled the headspace of spider mite infested tomato plants and of mechanically wounded 20 TMTT (µg plant tomato plants. The mechanical wounding of plants was 0 inXicted with an artery clamp. Even though this type of CSMW damage is in not comparable to spider mite-damage since the plants were wounded only at one time point, b LeGGPS1 the area of the leaf that is damaged by these treatments LeGGPS2 is comparable. Volatiles were collected in the subse- quent 24 h. Both spider mite-feeding and mechanical WIPI-2

PrP6 12345678 RCE1 LeGGPS1 CSMW

LeGGPS2 Fig. 4 LeGGPS1 expression and TMTT emission are induced by spider mite-feeding and mechanical wounding. a Bar graph RCE1 depicting the emission of TMTT in
g per plant in 24 h. Mean, minimum and maximum values are shown of measurements from Fig. 3 LeGGPS1 and LeGGPS2 are diVerently expressed in var- two independent experiments. b RT-PCR analysis for LeGGPS1 ious tissues. RT-PCR analysis for LeGGPS1 (27 cycles), LeG- (27 cycles), LeGGPS2 (25 cycles), WIPI-2 (14 cycles), PRP6 (23 GPS2 (25 cycles) and RCE1 (20 cycles) on RNA isolated from: cycles) and RCE1 (20 cycles) on RNA isolated from leaves of (1) Green fruit, (2) Orange fruit, (3) Red fruit, (4) Carpel plus sta- control (C), spider mite-infested (SM) and mechanically wound- men plus ovary, (5) Sepal, (6) Stem, (7) Leaf and (8) Root. Rub1 ed (W) plants. Rub1 conjugating enzyme (RCE1) is shown to conjugating enzyme (RCE1) is shown to indicate that equal indicate that equal amounts of template were used in the PCR amounts of template were used in the PCR reaction. A represen- reaction. Representative data of two independent experiments tative set of data from two independent experiments is shown are shown 123 Planta (2006) 224:1197–1208 1203 mite-feeding induced both the wound-induced protein- in the wild type (Fig. 6a). Induction of LeGGPS1 was ase inhibitor gene WIPI-2, a marker gene for a JA- also lower in def-1 plants (Fig. 6b). In these def-1 plants response (Graham et al. 1985), and the gene encoding WIPI-2 was not induced, but the induction of the SA- the pathogenesis related protein PRP6 (also named responsive PRP6 gene was higher than in wild type PR1b1), a marker for the SA-dependent response plants (Fig. 6b). This cross talk between the networks (Tornero et al. 1997). Wounding alone did not induce of JA- and SA-responsive genes has been well docu- PRP6 expression. mented (Spoel et al. 2003). However, LeGGPS1 does not behave like a typical SA- or JA-responsive gene. In Spider mite-induced emission of TMTT the absence of an SA-response, as in NahG, LeGGPS1 is both JA- and SA-dependent was not induced although the induction of the JA- responsive gene WIPI-2 was enhanced (Fig. 6a). Vice To investigate whether emission of TMTT is depen- versa, in the absence of a JA-response in def-1, LeG- dent on JA or SA, we made use of the mutant def-1 GPS1 was not stronger induced than in the wild type that is disturbed in the JA-response and the transgenic tomato, which is observed for the SA-responsive gene NahG line that overexpresses the Pseudomonas putida PRP6 (Fig. 6b). salicylate hydroxylase gene (nahG), resulting in inacti- vation of SA by converting it into catechol (Brading LeGGPS1 is induced by both JA and MeSA treatment et al. 2000). Def-1 plants have previously been shown but TMTT and GL are only emitted after JA treatment to emit signiWcantly lower amounts of TMTT than wild type plants after spider mite-attack (Ament et al. To address the question whether JA or SA could 2004). Here we show that SA is essential for emission induce LeGGPS1 expression, we treated wild type of TMTT, since NahG plants emitted 50-fold less tomato plants with these hormones. When wild type TMTT than wild type plants when infested with spider plants were treated with JA, LeGGPS1 was induced mites (Fig. 5). (Fig. 6c). This JA treatment led to rapid induction of WIPI-2 gene expression, while PRP6 expression was LeGGPS1 is not a typical JA- or SA-responsive gene not induced. When plants are treated with MeSA, this will be converted in the plant to SA by demethylation Because TMTT production was abolished in the NahG (Shulaev et al. 1997). This treatment led to activation line, we investigated whether this disruption of the SA- of SA-responsive genes as could be seen from the signaling pathway also abolished LeGGPS1 expres- induction of PRP6. LeGGPS1 was also induced by this sion. Indeed, LeGGPS1 was not induced in NahG MeSA-treatment, whereas transcript levels of LeG- plants upon spider mite-feeding, while expression of GPS2 and WIPI-2 were not aVected. the JA-responsive WIPI-2 gene was much higher than To investigate whether these hormone-treatments also induced emission of TMTT, the headspaces of these plants were sampled. JA-treated plants emitted 14-fold more TMTT than the control plants 2 days 160 after the treatment (Fig. 7a). JA-treated plants emitted )

-1 approximately 0.5 mg TMTT in 24 h, more than eight 140 times the emission after spider mite-feeding or wound-

24 h 120

-1 ing. However, treatment of tomato plants with MeSA 100 induced LeGGPS1, but did not lead to an increase in 80 emission of TMTT (Fig. 7a). This lack of correlation 60 suggests JA-dependent regulation of TMTT biosynthe- 40 sis downstream of GGPP. The Wrst committed step in the biosynthesis of

TMTT (µg plant 20 TMTT is the formation of GL from GGPP by GL syn- 0 CSMCSM thase. Interestingly, the induction of GL emission coin- WT NahG cided with the induction of TMTT emission (Fig. 7b). Two days after the treatments, about 1
g GL was Fig. 5 NahG tomato plants do not emit TMTT. The bar graph emitted in 24 h by MeSA-treated and control plants,
depicts the emission of TMTT in g per plant in 24 h by wild type whereas more than 20
g GL was emitted by JA- (WT) and NahG plants infested with spider mites (SM) and con- trol plants (C). Mean, minimum and maximum values are shown treated plants. This suggests that GL synthase was acti- of measurements from two independent experiments vated by JA but not by SA.

123 1204 Planta (2006) 224:1197–1208

a 6 a 900 5 ) 4 -1 800

LeGGPS1 3 700 24 h 2 -1 600 mRNA levels mRNA 1 500 Relative Relative 0 400 LeGGPS1 300 WIPI-2 200

PrP6 TMTT (µg plant 100 RCE1 0 C JA MeSA CSMCSM WT NahG b 35 ) b -1 30 8 25 24 h

6 -1 20 LeGGPS1 4 15 mRNA levels mRNA 2 10 Relative Relative 0

GL (µg plant 5 LeGGPS1 0 WIPI-2 C JA MeSA PrP6 RCE1 c 30 ) CSMCSM -1 h 25 WT def-1 -1 20 c LeGGPS1 15 LeGGPS2 WIPI-2 10

PrP6 GL (ng [mg protein] 5 RCE1 0 C JA C MeSA C JA MeSA

Fig. 6 LeGGPS1 expression is dependent on JA and SA and Fig. 7 Emission of TMTT and GL, and activity of GL synthase is is induced by JA-and MeSA-treatment. RT-PCR analysis for induced by JA- but not by MeSA-treatment. Bar graphs depicting LeGGPS1 (27 cycles), LeGGPS2 (25 cycles), WIPI-2 (14 cycles), the emission of a TMTT and b GL in
g per plant in 24 h by JA-, PRP6 (23 cycles) and RCE1 (20 cycles) on RNA isolated from MeSA- and control (C)-treated plants 2 days after treatment are leaves of a NahG plants, b def-1 plants and the corresponding shown. Mean, minimum and maximum values are shown of mea- wild type (WT) tomatoes, infested with spider mites (SM) and surements from two independent experiments. c A bar graph W uninfested (C). The bar graphs represent quanti cation of blotted depicting the production of GL in ng per h per mg protein. Mean LeGGPS1 RT-PCR products that were normalized for RCE1 and standard error are shown of measurements from four inde- (means, minimal and maximal values are shown from two inde- pendent experiments pendent experiments). c RT-PCR analysis on RNA isolated from leaves of JA-, MeSA- and control (C)-treated plants. Rub1 con- jugating enzyme (RCE1) is shown to indicate that equal amounts of template were used in the PCR reaction. A representative set of data of two independent experiments is shown plants treated with either JA or MeSA were assayed for GL synthase activity. GL synthase activity was GL synthase activity in tomato leaves is induced by JA higher in JA-treated leaves compared to control- but not by SA treated, whereas GL synthase activity in MeSA-treated leaves did not diVer signiWcantly from control-treated To test the hypothesis that GL synthase is induced by leaves (Fig. 7c). This indicates that GL synthase activ- JA and not by SA, protein extracts from leaves of ity is induced by JA and not SA. 123 Planta (2006) 224:1197–1208 1205

Discussion whether they encode true GGPP synthases, where they are expressed and what regulates their expression. Two GGPP synthase genes from tomato, LeGGPS1 Mechanical wounding and spider mite-herbivory and LeGGPS2, have been cloned and expressed in E. induced expression of LeGGPS1 in leaves. Both treat- coli. Functional expression of LeGGPS1 and ments resulted in a JA-response as could be seen from LeGGPS2 was conWrmed by complementation of the the induction of WIPI-2 (Fig. 4b). Induction of gene cluster for carotenoid biosyntheses of Erwinia LeGGPS1 by spider mite-herbivory conWrms the uredovora lacking GGPP synthase (Fig. 2). This system microarray results from Kant et al. (2004). Treatment has been used before to demonstrate the function of of intact plants with JA induced the expression of putative GGPP synthases from Arabidopsis (Zhu et al. LeGGPS1 (Fig. 6c). This, in combination with reduced 1997), sunXower (Helianthus annuus) (Oh et al. 2000) expression levels of LeGGPS1 in the JA-deWcient makandi (Coleus forskohlii) (Engprasert et al. 2004), mutant def-1 (Fig. 6b), implies JA-dependent regula- human and mouse (Mus musculus) (Kainou et al. tion of LeGGPS1. Despite the inability of def-1 to 1999). accumulate JA, LeGGPS1 was at most slightly induced While GGPP synthase, FPP synthase and most GPP in response to spider mite-herbivory, compared to the synthases are functional as homodimers (Ogura and wild type (Fig. 6b). Microarray experiments also did Koyama 1998), the GPP synthases from mint and snap- not show a signiWcant induction of LeGGPS1 in def-1 dragon are functional as heterodimers (Burke et al. plants by spider mites after 1 day (Ament et al. 2004). 1999; Tholl et al. 2004). In the phylogenetic tree Due to the absence of induced JA-accumulation in def- (Fig. 1), the large subunits of these GPP synthases 1, the SA-responsive PRP6 gene was stronger induced from mint and snapdragon cluster together with plant in def-1 than in the wild type by spider mite-herbivory GGPP synthases. These large subunits exhibit GGPP (Fig. 6b). To test whether this SA-response could synthase activity in the absence of the small subunit account for the induction of LeGGPS1 in def-1, we (Tholl et al. 2004). We therefore cannot exclude that determined the expression of LeGGPS1 after MeSA LeGGPS1 and LeGGPS2 encode large subunits of treatment of intact wild type plants. LeGGPS1 was tomato GPP synthases. However the tomato GPP syn- induced by this treatment, showing that SA can also thase is highly similar to the single copy GPP synthase induce LeGGPS1 (Fig. 6c). Moreover, in the absence of Arabidopsis (accession number Y17376), which is of SA, in the transgenic NahG, no induction of functional as a homodimer (Bouvier et al. 2000). More- LeGGPS1 occurred after spider mite-feeding (Fig. 6a), over, in the TIGR tomato EST database, consisting of despite the induction of the JA-responsive WIPI-2 160,000 ESTs, there are no ESTs with sequence simi- gene. We therefore conclude that JA and SA can both larity to the small subunit of GPP synthase of mint or induce LeGGPS1 and that basic levels of SA are essen- snapdragon. We therefore reason that the GPP syn- tial for the induction of LeGGPS1. Other examples of thase of tomato is functional as a homodimer. genes that are induced by JA- and SA-treatment have RT-PCR analysis showed that LeGGPS1 and been described for sorghum (Salzman et al. 2005). LeGGPS2 were expressed in diVerent tissues (Fig. 3). Induction of several genes from Arabidopsis is also LeGGPS1 was mostly expressed in leaf tissue while dependent on both JA- and SA-signaling pathways expression of LeGGPS2 was highest in ripening fruit (Glazebrook et al. 2003). However, from these and and Xower organs (Fig. 3). During the Wrst 5 days of other expression data (http://www.genevestiga- spider mite infestation, a 4-week-old tomato plant tor.ethz.ch), no functional homolog of LeGGPS1 could emits around 900
g volatiles of which approximately be pinpointed in Arabidopsis. 75% is TMTT (Kant et al. 2004). Since LeGGPS1 was We have previously shown that emission of TMTT mostly expressed in leaf tissue, we expect a leaf speciWc upon spider mite-herbivory was strongly reduced in the fate for the GGPP formed. Expression of LeGGPS1 JA-deWcient mutant def-1 and that emission could be was induced by wounding and herbivory, coinciding restored by pre-treating def-1 with JA (Ament et al. with emission of TMTT (Fig. 4). We therefore hypoth- 2004). When wild type plants were treated with JA esize that LeGGPS1 produces substrate for TMTT bio- they started to emit massive amounts of TMTT synthesis. It is possible that LeGGPS2 provides GGPP (Fig. 7a). Therefore, JA is necessary and suYcient to for the production of the carotenoids in fruits, which induce emission of TMTT in tomato. In lima bean increases from 2.2
g/g FW in green fruit to 189
g/g leaves JA-treatment did not induce TMTT emission, FW in red fruit (Fraser et al. 1994). The TIGR tomato though treatment with 12-oxo-phytodienoic acid EST database contains several ESTs encoding other (OPDA) and linolenic acid, both precursors of JA, did putative GGPP synthases. It remains to be investigated induce TMTT emission (Koch et al. 1999). Thus 123 1206 Planta (2006) 224:1197–1208 regulation of TMTT emission is diVerent for tomato by spider mite-feeding (Kant et al. 2004). This brings and lima bean. NahG tomato plants did not emit on the question why there are multiple steps in the bio- TMTT after spider mite-herbivory, whereas wild type synthesis of TMTT that are regulated. An explanation plants did (Fig. 5). However, treatment of wild type for this could be that if GL synthase would be the only plants with MeSA did not result in induced emission of regulatory step in TMTT biosynthesis, isoprenoid pre- TMTT (Fig. 7a). Therefore we conclude that SA is not cursors would soon be depleted when TMTT is formed suYcient to induce the emission of TMTT but that a in the amounts emitted after spider mite-infestation. basic level of SA is required. For example, over expression of phytoene synthase in It must be noted that the corresponding wild type tomato, thus depleting the GGPP pool, resulted in control plants for NahG and def-1, the cultivar Money- dwarfed phenotype caused by reduced levels of gibber- maker and Castlemart, respectively, showed diVerent ellins (Fray et al. 1995). transcript levels of LeGGPS1. This was also the case The gene that encodes GL synthase still has to be for WIPI-2 (Fig. 6a, b), indicating that the JA- cloned and characterized. Arabidopsis leaves placed in responses in these cultivars were diVerent. The emis- (E)-nerolidol and also untreated transgenic Arabidop- sion of TMTT by Moneymaker control plants (Fig. 5) sis plants expressing strawberry nerolidol synthase, are was also much higher than by control Castlemart plants capable of producing DMNT (Kappers et al. 2005). (Fig. 4). However, in both cultivars the emission of This is interesting since DMNT is normally not pro- TMTT and expression of LeGGPS1 were induced by duced by Arabidopsis whereas TMTT is (Van Poecke spider mite-feeding. et al. 2001). This suggests that the enzymes responsible Since MeSA- as well as JA-treatment resulted in for the conversion of GL to TMTT are also capable of induction of LeGGPS1 whereas TMTT was only emit- converting nerolidol to DMNT. The enzymes responsi- ted after JA-treatment, there must be JA-dependent ble for these conversions have not yet been identiWed. regulation of TMTT synthesis downstream of GGPP A question that still remains is what the function of synthase. The Wrst committed step in TMTT biosynthe- LeGGPS2 in planta is? Is this GGPP synthase indeed sis is the conversion of GGPP to GL, catalyzed by GL involved in carotenoid biosynthesis in ripening fruit? synthase. MeSA-treated plants emitted no GL whereas Another intriguing question that remains unanswered JA-treated plants did (Fig. 7b). Therefore we conclude is why SA induces LeGGPS1. What is the fate of the that GL synthase must be JA-regulated. This conclu- GGPP formed after SA-treatment? There is one report sion is supported by the measurement of GL synthase on increased chlorophyll and carotenoid content in activity in leaf extracts of JA-, SA- and control-treated leaves of Cowpea (Vigna unguiculata) after SA-treat- plants: GL synthase activity was only induced after JA ment (Chandra and Bhatt 1998). It remains to be treatment (Fig. 7c). determined whether SA-treatment of tomato plants TMTT biosynthesis parallels DMNT biosynthesis results in a similar increase in chlorophyll and caroten- where FPP is converted to DMNT via nerolidol. In oid content in tomato and that LeGGPS1 ensures sub- protein extracts of spider mite-infested and JA-treated strate for these products. lima bean and cucumber leaves, nerolidol synthase activity was induced (Bouwmeester et al. 1999). How- ever, no TMTT was emitted by JA-treated lima bean References leaves (Hopke et al. 1994), indicating a diVerent regu- Ament K, Kant MR, Sabelis MW, Haring MA, Schuurink RC lation for TMTT and DMNT emission in lima bean. (2004) Jasmonic acid is a key regulator of spider mite-in- In conclusion, we propose that LeGGPS1 is respon- duced volatile terpenoid and methyl salicylate emission in to- sible for provision of precursors for TMTT biosynthe- mato. Plant Physiol 135:2025–2037 sis in tomato. This still needs to be conWrmed via a Boland W, Gabler A (1989) Biosynthesis of homoterpenes in higher plants. Helv Chim Acta 72:247–253 transgenic approach. GGPP synthases other than Boland W, Gabler A, Gilbert M, Feng ZF (1998) Biosynthesis of LeGGPS1 might also contribute to the pool of GGPP C-11 and C-16 homoterpenes in higher plants; Stereochemis- for TMTT biosynthesis. Furthermore we showed that try of the C-C-bond cleavage reaction. Tetrahedron regulation of this gene was dependent on both JA and 54:14725–14736 Botella-Pavia P, Besumbes O, Phillips MA, Carretero-Paulet L, SA and that GL synthase activity was only induced by Boronat A, Rodriguez-Concepcion M (2004) Regulation of JA. Thus we have identiWed two novel regulatory steps carotenoid biosynthesis in plants: evidence for a key role of in the biosynthesis of TMTT in tomato. Kant et al. hydroxymethylbutenyl diphosphate reductase in controlling (2004) have previously shown that DOXPS, the limit- the supply of plastidial isoprenoid precursors. Plant J 40:188– 199 ing step in plastidial IPP and DMAPP biosynthesis Bouvier F, Suire C, d’Harlingue A, Backhaus RA, Camara B (Botella-Pavia et al. 2004), was upregulated in tomato (2000) Molecular cloning of geranyl diphosphate synthase 123 Planta (2006) 224:1197–1208 1207

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