Biotechnol Lett DOI 10.1007/s10529-014-1533-2
ORIGINAL RESEARCH PAPER
Tomato terpene synthases TPS5 and TPS39 account for a monoterpene linalool production in tomato fruits Ying Cao • Shanglian Hu • Qilin Dai Yongsheng Liu
•
Received: 6 January 2014 / Accepted: 9 April 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Recombinant tomato terpene synthases, TPS5/37/39, catalyze the formation of linalool or nerolidol in vitro. However, little is known about their actual biological activities in tomato plants, especially in their fruits. Here, when all three TPSs were induced in tomato fruits by a chemical elicitor, geraniol, a significant linalool peak was detected in fruit tissues but not in control fruits. Considering the compartments of these TPS proteins and available substrates, the linalool peak induced by geraniol might be attributed to TPS5 and TPS37, both of them putatively localized in the plastids where high levels of
monoterpene substrate geranyl diphosphate exist. In addition, application of geraniol also triggered jasmonic acid (JA)-related defense genes suggesting that the inducible TPSs might be correlated with JAsignaled defense responses. Keywords Defense-related responses Geraniol Linalool production Nerolidol Terpene synthases Tomatoes
Introduction
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1533-2) contains supplementary material, which is available to authorized users.
Terpene volatiles are important contributors to the aroma and fragrance of many fruit and floral species.
Y. Cao S. Hu (&) Q. Dai School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, People’s Republic of China e-mail: [email protected]
Y. Liu School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, People’s Republic of China
Y. Cao e-mail: [email protected] Q. Dai e-mail: [email protected] Y. Liu Ministry of Education Key Laboratory for Bio-Resource and Eco-Environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064, People’s Republic of China e-mail: [email protected]
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They are critical for the attraction of pollinators or defense against biotic stresses (Dudareva et al. 2004). Many terpene volatiles are the immediate products catalyzed by terpene synthases (TPSs). Generally, the biosynthesis of monoterpenes from geranyl diphosphate (GPP) occurs in the plastid, where the Nterminal transit peptide involved in monoterpene synthase is located, while the formation of sesquiterpene from farnesyl diphosphate (FPP) takes place in the cytosol. Therefore, the subcellular localization of TPS enzymes, together with the substrates they use, determine their products (Nagegowda et al. 2008). TPS gene families from many plant species have been explored and a large number of TPS genes also have been isolated and characterized (Martin et al. 2010; Falara et al. 2011; Nieuwenhuizen et al. 2013). Moreover, some TPSs have been utilized successfully to modify the aroma or fragrance of plants through metabolic engineering (Davidovich-Rikanati et al. 2007; Keasling 2010). Tomato plant is well known to release a wide variety of volatile terpenes, which consist mostly of monoterpenes and sesquiterpenes (van Schie et al. 2007). The tomato genome data reveal that there are at least 44 genes putatively encoding TPSs, and 29 of them are functional or potentially functional (Falara et al. 2011). In vitro assays with the recombinant tomato TPS5/37/39 proteins show they are capable of catalyzing the formation of linalool from GPP or nerolidol from FPP (van Schie et al. 2007; Falara et al. 2011). TPS5 has been determined to be a linalool synthase (LIS) in planta, since its increased expression upon spider mite-infestation or jasmonic acid (JA) treatment correlated with increased emission of linalool rather than nerolidol in tomato plants (van Schie et al. 2007). Here, we report that TPS5 and TPS37 account for the linalool production in tomato fruits after the treatment of a chemical elicitor geraniol, and geraniol-inducible TPSs might be correlated with JAsignaled defense responses.
Materials and methods Gene expression analysis Tomato plants (cv. Alisa Craig) were grown in a greenhouse and irrigated manually every other day.
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Total RNAs were extracted using Trizol following the standard protocol and treated with DNase. About 1 lg total RNA from each sample was used for first-strand cDNA synthesis. Real-time quantitative RT-PCR was performed using SyBRGreen PCR Master Mix (Applied Biosystems) and gene-specific primers on the iCycler PCR system (BIO-RAD, Hercules, California, USA). The tomato UBI3 gene (accession no. X58253) was selected as the internal reference. Normalization was performed by the 2-Ct method. All primers used are listed in Supplementary Table 1. Chlorophyll fluorescence imaging Photosynthetic capability of young, green tomato fruits was determined by a chlorophyll fluorescence system (Imaging-PAM, Mini-Version, Walz, Germany). From the surface and cross-section of the tested fruits, the image of the maximum quantum efficiency of PSII photochemistry, Fv/Fm, was taken under saturation pulse mode. The saturation pulse light was 2,500 lM m-2 s-1 and the measured light intensity and the actinic light intensity were 0.5 and 180 lM m-2 s-1, respectively. Fluorescence images were recorded simultaneously and displayed by means of a false colour code ranging from 0 (black) to 1 (pink). Exogenous geraniol treatment Geraniol and linalool standards were purchased from Sigma-Aldrich. For geraniol treatment, 10 mM geraniol in 10 ml water was sprayed onto nine immature green fruits (15 days post-anthesis, DPA) of three intact tomato plants. The same volume of pure water was sprayed on control fruits. Chlorophyll fluorescence images of green fruits after 0, 24, and 48 h of treatment were taken as mentioned above. The same geraniol treatment was also performed in an enclosed container for nine detached red ripe fruits (42–45 DPA) from three different tomato plants. A tissue disk assay of fruit peels was also performed. Briefly, fruit disks (pericarp slices, *5 mm thick) were treated with 5 ml geraniol (10 mM) or water (as control) for 24 h in an enclosed flask. Each flask contained 30 g tissues from at least three different fruits. For treated fruits or fruit disks, volatile compounds and gene expressions were analyzed by GCMS and real-time quantitative RT-PCR, respectively. Each treatment was performed in triplicate.
Biotechnol Lett
Analysis of volatile compounds Extraction of volatiles were carried out as the method described by Davidovich-Rikanati et al. (2007). Approximately 30 g fruit tissues were cut into small pieces and extracted with 100 ml methyl tert-butyl ether (MTBE) with 1 mM phenylethanol as the internal standard by vigorous shaking for 3 h. The MTBE phase was separated off, dried over anhydrous Na2SO4, and concentrated to 0.2–0.5 ml by a rotary evaporator. The concentrated MTBE extracts were injected into a GC–MS system (Aligent, USA) equipped with 30 m 9 0.25 mm 9 0.5 lm column (Restek, Bellefonte, PA). Helium (1 ml min-1) was used as a carrier gas, with a split ratio of 1:200. The GC was held initially at 60 °C for 2 min followed by a gradient of 4 °C min-1 to 280 °C and held for 10 min. Mass spectra were obtained by electron impact ionisation at 70 eV. Spectrometric data were compared with computerized libraries and authentic standards.
Results TPS37 and 39 are fruit-related Tomato three TPSs, TPS5/37/39, together with some known TPSs from other plants, could be grouped in three clades of a phylogenetic tree. Figure 1 shows
that TPS37 and 39 cluster closely and locate in the cluster I, together with three fruit TPSs, including a grape linalool/nerolidol synthase LIS/NES (Martin et al. 2010), as well as apple NES (MdTPS3) and LIS (MdTPS6) (Nieuwenhuizen et al. 2013). In the cluster II, the tomato TPS5 and phellandrene synthase TPS4/ MTS2 are clustered in a same subclade and are similar to the previously characterized scented-related LISs from Lavandula angustifolia (lavender LIS; Landmann et al. 2007) and Mentha aquatica (mentha LIS; Crowell et al. 2002), as well as two closely-related Arabidopsis TPS02 and TPS03. The latter are constitutively expressed in flowers and are induced in leaves under stress (Huang et al. 2010) (Fig. 1).
Expression of TPS5, 37 and 39 in developing fruits TPS37 is highly expressed in petioles and stems bearing high density glandular trichomes. The TPS39 transcript is only observed in young leaves and flowers (Kant et al. 2004; Falara et al. 2011). In tomato cv. Alisa Craig, we found that TPS5, 37 and 39 were expressed in all stages of fruit development and ripening: their expressions were low in young green fruits, and gradually increased with fruit development and ripening, with maximal transcript accumulation in the yellow fruits, and dropped dramatically in the later stage of ripening (Fig. 2a).
Fig. 1 Phylogenetic tree of tomato terpene synthases (TPSs) and other plantidentified and/or functionally-annotated TPSs. A neighbor-joining dendrogram analysis was performed based on the degree of sequence similarity using MEGA4.0. The scale bar represents 0.1 substitutions per site. Statistical confidence of the nodes of the tree are based on 10,000 bootstrap replicates. Other abbreviations in this figure are as follows: MTS monoterpene synthase, LIS linalool synthase, NES nerolidol synthase
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Biotechnol Lett Fig. 2 Expression pattern of three terpene synthases (TPSs) in tomato. a Expression of TPSs in various tissues. Total RNAs were extracted from fully expanded leaves (mature leaves), and fruits at different developmental stages (immature green, 15 day-post anthesis (DPA); mature green, 30 DPA; yellow ripe, 40 DPA and red ripe, 45 DPA). b Spatial expression of TPSs in immature green fruits (15 DPA). Real-time RT-PCR was performed as described in ‘‘Materials and methods’’ section. Average values and standard errors are shown from three independent replicates
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Biotechnol Lett Fig. 3 False color images of the chlorophyll fluorescence yield (Fv/Fm) of tomato green fruits exposed to geraniol solutions after 24 and 48 h. Water-treatment fruits were used as control. Images from the surfaces (a) or crosssections (b) of fruits were shown, respectively
In addition, we isolated total RNAs from different parts of 15 DPA immature green fruits for RT-PCR assay. The results showed that TPS37 was highly expressed in all parts of fruits at this specific stage, with the highest expression in the exocarp tissues attaching with trichomes (Fig. 2b). In contrast, the mRNA accumulations of TPS5 and 39 were significantly low, especially in flesh tissues where few transcript was detected (Fig. 2b). These suggested that among them, TPS37 was dominant in fruits during development and ripening, and its expression was not trichome-specific. Exogenous geraniol causes the damage of fruits Geraniol is a potent inducer of apoptosis-like cell death in cultured plant cells (Izumi et al. 1999). Here, 10 mM geraniol in water was sprayed on the immature green fruits on vines and then the change in Fv/Fm value was monitored, visualized by chlorophyll fluorescence imaging with a false colour code ranging from 0 (black) to 1 (pink). Since photosynthesis is one of the most sensitive physiological processes of plant upon environmental stress, the Fv/Fm value that represents the maximum quantum use efficiency of photosystem II is usually used to reflect rapidly the magnitude of the plant’s wound response (Barbagallo et al. 2003). In healthy green fruits, Fv/Fm usually has a value of *0.79 (blue), whereas no chlorophyll fluorescence is detected in ripe fruits. After 24 h of geraniol treatment, a severe inactivation of photosystem II in
the surface of green fruits was observed through changed coloration in false color images of the chlorophyll fluorescence yield (Fv/Fm); after 48 h, Fv/Fm values fell to around zero (black) (Fig. 3a, b). Moreover, the images of fruit cross-sections showed that the damage caused by geraniol started in the surface of the fruits, and then spread successively into fruit inners (Fig. 3b). Elevated level of linalool in tomato fruits induced by exogenous geraniol Volatile compounds of water-or geraniol-treated fruits were extracted by MTBE and analyzed by GC–MS. Figure 4 shows that no significant volatile compounds were detected in the MTBE-extracts of water-treated fruits or fruit disks; similarly, no extra volatile compound was induced in the fruits after 24 and 48 h of geraniol treatment. However, a significant linalool peak was found in the MTBE-extracts of geraniol-treated fruit disks but nerolidol production was not detected in either geraniol-treated fruits or fruit disks (Fig. 4). Gene expression analysis showed that, compared with water-treated controls, geraniol-sprayed fruits displayed a slightly increase in transcript levels of TPS5, 37 and 39 within 48 h (Fig. 5). However, all three TPS genes were apparently upregulated in fruit disks (pericarp slices) that were directly exposed to alcohol solution after 24 h, especially for TPS37, its expression showing hundreds-fold higher than that of undamaged control fruits (Fig. 5).
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Fig. 4 Volatiles extracted from tomato fruits after geraniol or water treatment. a Total ion chromatograph (TIC) of GC are shown for methyl tert-butyl ether (MTBE)-extracts of fruits. Marked peaks are indicated as geraniol (1) and linalool (2), respectively. These peaks are identified by comparison of their
retention times and their mass spectra with those in the computerized libraries. b Mass spectra of products present in MTBE-extracts (labeled as 1 and 2, respectively) and their authentic standards
Fig. 5 Inducible expressions of three terpene synthases (TPSs) in ripe fruits after 24 or 48 h of geraniol. Total RNAs were extracted from water- or geraniol-treated whole fruits (spraying)
and fruit disks, respectively. mRNA levels were analyzed by real-time RT-PCR. The results from three independent experiments are shown as mean values and standard errors
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Biotechnol Lett Fig. 6 Geraniol treatment triggers the expressions of jasmonic acid (JA)-related defense genes. mRNA levels in geraniol-treated fruits (spraying) or fruit disks were analyzed by real-time RT-PCR. The whole fruits sprayed with water for 48 h were used as a control. Average values and standard errors are shown from three independent replicates. Significance was calculated using the Student’s t test. Asterisks indicate statistically significant differences (**P \ 0.005)
Geraniol treatment triggers some JA-responsive genes Since geraniol-treated fruits displayed visible mechanical damages (Fig. 3), we further investigated the effect of geraniol treatment on the expressions of some defense-related genes. Prosystemin (PS) is a signalingrelated gene that involves in the JA biosynthesis via the octadecanoid pathway, while proteinase inhibitor II (Inh II) and cathepsin D inhibitor (CDI) are two genes that directly participate in the plant defense against wounding; all of them are JA-regulated (Li et al. 2003). Figure 6 shows that Inh II and CDI were upregulated in
fruits after 24 or 48 h of geraniol spraying; no higher accumulations for them were observed in fruit disks that were directly treated with geraniol. Unlike Inh II and CDI, the transcriptional level of the signaling gene PS correlated with the strength of the induction stimulus, with the highest transcription accumulation in treated fruit disks (Fig. 6).
Discussion Linalool, an acyclic monoterpene alcohol, is found in many plant flower scent and edible fruits, while in
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tomato, it is produced predominantly in trichomes of leaves and stems, absent in fruits (Goff and Klee 2006; van Schie et al. 2007). In this report, we found that exogenous geraniol promoted the expressions of three LISs, TPS5/37/39 and induced the production of linalool in treated fruit tissues. These recombinant TPS proteins could convert FPP to nerolidol, suggesting that they could potentially contribute to nerolidol production. However, nerolidol was not identified in the solvent extracts of the geraniol-treated tissues in which all of the three TPSs were upregulated (Fig. 5). Because no headspace analysis was performed in this report, it is not clear that whether nerolidol (or linalool) is lost in the headspace. Nevertheless, in the plant, substrates for the biosynthesis of monoterpene and sesquiterpene, GPP and FPP, were present in the plastids and cytosol, respectively (Nagegowda et al. 2008). The ChloroP (http://www.cbs.dtu.dk) and IPSORT (http://ipsort.hgc.jp) programs predicted that both TPS5 and TPS37 had N-terminal transit peptides and were putatively localized in the plastids, while TPS39 contained a mitochondrial-targeting peptide. Hence, it is difficult to determine if nerolidol is produced by TPS proteins from FPP in plastids because little knowledge is available on the production of GPP or FPP in tomato. Although genetic evidence is still needed, our results suggest that the linalool peak in fruit tissue after geraniol treatment might be attributed to TPS5 and TPS37, both of them localized in the plastid where high levels of GPP exist. Moreover, TPS37 might be the predominant one because of its significant higher expression in either developing fruits or geraniol-treated tissues. Biotransformation of terpenes and terpenoids occurs in bacteria, fungi and certain plant cell cultures (e.g. Picea abies suspension cells) (Demyttenaere et al. 2000; Lindmark-Henriksson et al. 2004). For example, application of the pure geraniol in liquid cultures of Aspergillus niger led to biotransformation resulting in multiple products with linalool being one of them (Demyttenaere et al. 2000). This indicated that linalool might be formed by the bioconversion of geraniol. However, the biotransformations reported previously were generally carried out in aqueous systems and in shake-flasks, which are compatible with enzymes and growing whole cells (deCarvalho and daFonseca 2006). In this report, no extra compounds were found in sprayed-fruits and no other metabolites, except for linalool, were detected in treated fruit disks (Fig. 4). In
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addition, expressions of TPS5/37/39 and the production of linalool in tomato fruit disks were also induced by (Z)-3-hexenol (data not shown). Hence, the linalool peak in treated fruits is unlikely to arise from the bioconversion production of geraniol, and it might be correlated with wound defense responds because application of geraniol also caused the tissue damage and triggered JA-related defense genes. Indeed, linalool was predominant among the induced volatiles of tomato plants by spider-mite, wounding or chemical treatments (Kant et al. 2004; van Schie et al. 2007). Volatile-mediated defense mechanism is complex, involving some different regulators such as JA, salicylic acid (SA) and ethylene (Kant et al. 2004). Arimura et al. (2008) reported that herbivore-induced terpenoid emission was affected by concerted action of jasmonate, ethylene and calcium signaling. In tomato, JA is a key regulator of spider mite-induced volatile terpenoid (Ament et al. 2004). However, it is unclear whether a JA signal is essential for the geraniol-induced expression of TPSs. Thus further studies are required to understand these interactions between geraniol and JA, as well as other regulators such as SA and ethylene. Acknowledgments This work was supported by the Education Department Fund of Sichuan Province (No. 13ZD1110) and the Internal Fund of Southwest University of Science and Technology (No. 20145030).
References Ament K, Kant MR, Sabelis MW, Haring MA, Schuurink RC (2004) Jasmonic acid is a key regulator of spider miteinduced volatile terpenoid and methyl salicylate emission in tomato. Plant Physiol 135:2025–2037 Arimura G, Garms S, MaVei M, Bossi S, Schulze B, Leitner M, Mitho¨fer A, Boland W (2008) Herbivore-induced terpenoid emission in Medicago truncatula: concerted action of jasmonate, ethylene and calcium signaling. Planta 227:453–464 Barbagallo RP, Oxborough K, Pallet KE, Baker NR (2003) Rapid, noninvasive screening for perturbations of metabolism and plant growth using chlorophyll fluorescence imaging. Plant Physiol 132:485–493 Chen F, Tholl D, Bohlmann J, Pichersky E (2011) The family of terpene synthases in plants: a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom. Plant J 66:212–229 Crowell AL, Williams DC, Davis EM, Wildung MR, Croteau R (2002) Molecular cloning and characterization of a new linalool synthase. Arch Biochem Biophys 405:112–121
Biotechnol Lett Davidovich-Rikanati R, Sitrit Y, Tadmor Y, Iijima Y, Bilenko N, Bar E, Carmona B, Fallik E, Dudai N, Simon JE, Pichersky E, Lewinsohn E (2007) Enrichment of tomato flavor by diversion of the early plastidial terpenoid pathway. Nat Biotechnol 25:899–901 de Carvalho CCR, da Fonseca M (2006) Biotransformation of terpenes. Biotechnol Adv 24:134–142 Demyttenaere JC, del Herrera-Carmen M, De Kimpe N (2000) Biotransformation of geraniol, nerol and citral by sporulated surface cultures of Aspergillus niger and Penicillium sp. Phytochemistry 55:363–373 Dudareva N, Pichersky E, Gershenzon J (2004) Biochemistry of plant volatiles. Plant Physiol 135:1893–1902 Falara V, Akhtar TA, Nguyen TTH, Spyropoulou EA, Bleeker PM, Schauvinhold I, Matsuba Y, Bonini ME, Schilmiller AL, Last RL, Schuurink RC, Pichersky E (2011) The tomato terpene synthase gene family. Plant Physiol 157:770–789 Goff SA, Klee HJ (2006) Plant volatile compounds: sensory cues for health and nutritional value. Science 311:815–819 Huang M, Abel C, Sohrabi R, Petri J, Haupt I, Cosimano J, Gershenzon J, Tholl D (2010) Arabidopsis ecotypes depends on allelic differences and subcellular targeting of two terpene synthases, TPS02 and TPS03. Plant Physiol 153:1293–1310 Izumi S, Takashima O, Hirata T (1999) Geraniol is a potent inducer of apoptosis-like cell death in the cultured shoot primordia of Matricaria chamomilla. Biochem Biophys Res Commun 259:519–522 Kant MR, Ament K, Sabelis MW, Haring MA, Schuurink RC (2004) Differential timing of spider mite-induced direct and indirect defenses in tomato plants. Plant Physiol 135:483–495 Keasling JD (2010) Manufacturing molecules through metabolic engineering. Science 330:1355–1388 Landmann C, Fink B, Festner M, Dregus M, Engel KH, Schwab W (2007) Cloning and functional characterization of three
terpene synthases from lavender (Lavandula angustifolia). Arch Biochem Biophys 465:417–429 Li CY, Liu GH, Xu CC, Lee GI, Bauer P, Ling HQ, Ganal MW, Howea GA (2003) The tomato suppressor of prosysteminmediated responses 2 gene encodes a fatty acid desaturase required for the biosynthesis of jasmonic acid and the production of a systemic wound signal for defense gene expression. Plant Cell 15:1646–1661 Lindmark-Henriksson M, Isaksson D, Vanek T, Ho¨gberg-Valterova´I HE, Sjo¨din K (2004) Transformation of terpenes using a Picea abies suspension culture. J Biotechnol 107:173–184 Martin DM, Aubourg S, Schouwey MB, Daviet L, Schalk M, Toub O, Lund ST, Bohlmann J (2010) Functional annotation, genome organization and phylogeny of the grape vine (Vitis vinifera) terpene synthase gene family based on genome assembly, FLcDNA cloning, and enzyme assays. BMC Plant Biol 10:226 Nagegowda DA, Gutensohn M, Wilkerson CG, Dudareva N (2008) Two nearly identical terpene synthases catalyze the formation of nerolidol and linalool in snapdragon flowers. Plant J 55:224–239 Nieuwenhuizen NJ, Green SA, Chen X, Bailleul EJD, Matich AJ, Wang MY, Atkinson RG (2013) Functional genomics reveals that a compact terpene synthase gene family can account for terpene volatile production in apple. Plant Physiol 161:787–804 van Schie CCN, Haring MA, Schuurink RC (2007) Tomato linalool synthase is induced in trichomes by jasmonic acid. Plant Mol Biol 64:251–263
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ORIGINAL RESEARCH PAPER
Tomato terpene synthases TPS5 and TPS39 account for a monoterpene linalool production in tomato fruits Ying Cao • Shanglian Hu • Qilin Dai Yongsheng Liu
•
Received: 6 January 2014 / Accepted: 9 April 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Recombinant tomato terpene synthases, TPS5/37/39, catalyze the formation of linalool or nerolidol in vitro. However, little is known about their actual biological activities in tomato plants, especially in their fruits. Here, when all three TPSs were induced in tomato fruits by a chemical elicitor, geraniol, a significant linalool peak was detected in fruit tissues but not in control fruits. Considering the compartments of these TPS proteins and available substrates, the linalool peak induced by geraniol might be attributed to TPS5 and TPS37, both of them putatively localized in the plastids where high levels of
monoterpene substrate geranyl diphosphate exist. In addition, application of geraniol also triggered jasmonic acid (JA)-related defense genes suggesting that the inducible TPSs might be correlated with JAsignaled defense responses. Keywords Defense-related responses Geraniol Linalool production Nerolidol Terpene synthases Tomatoes
Introduction
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1533-2) contains supplementary material, which is available to authorized users.
Terpene volatiles are important contributors to the aroma and fragrance of many fruit and floral species.
Y. Cao S. Hu (&) Q. Dai School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, People’s Republic of China e-mail: [email protected]
Y. Liu School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, People’s Republic of China
Y. Cao e-mail: [email protected] Q. Dai e-mail: [email protected] Y. Liu Ministry of Education Key Laboratory for Bio-Resource and Eco-Environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064, People’s Republic of China e-mail: [email protected]
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They are critical for the attraction of pollinators or defense against biotic stresses (Dudareva et al. 2004). Many terpene volatiles are the immediate products catalyzed by terpene synthases (TPSs). Generally, the biosynthesis of monoterpenes from geranyl diphosphate (GPP) occurs in the plastid, where the Nterminal transit peptide involved in monoterpene synthase is located, while the formation of sesquiterpene from farnesyl diphosphate (FPP) takes place in the cytosol. Therefore, the subcellular localization of TPS enzymes, together with the substrates they use, determine their products (Nagegowda et al. 2008). TPS gene families from many plant species have been explored and a large number of TPS genes also have been isolated and characterized (Martin et al. 2010; Falara et al. 2011; Nieuwenhuizen et al. 2013). Moreover, some TPSs have been utilized successfully to modify the aroma or fragrance of plants through metabolic engineering (Davidovich-Rikanati et al. 2007; Keasling 2010). Tomato plant is well known to release a wide variety of volatile terpenes, which consist mostly of monoterpenes and sesquiterpenes (van Schie et al. 2007). The tomato genome data reveal that there are at least 44 genes putatively encoding TPSs, and 29 of them are functional or potentially functional (Falara et al. 2011). In vitro assays with the recombinant tomato TPS5/37/39 proteins show they are capable of catalyzing the formation of linalool from GPP or nerolidol from FPP (van Schie et al. 2007; Falara et al. 2011). TPS5 has been determined to be a linalool synthase (LIS) in planta, since its increased expression upon spider mite-infestation or jasmonic acid (JA) treatment correlated with increased emission of linalool rather than nerolidol in tomato plants (van Schie et al. 2007). Here, we report that TPS5 and TPS37 account for the linalool production in tomato fruits after the treatment of a chemical elicitor geraniol, and geraniol-inducible TPSs might be correlated with JAsignaled defense responses.
Materials and methods Gene expression analysis Tomato plants (cv. Alisa Craig) were grown in a greenhouse and irrigated manually every other day.
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Total RNAs were extracted using Trizol following the standard protocol and treated with DNase. About 1 lg total RNA from each sample was used for first-strand cDNA synthesis. Real-time quantitative RT-PCR was performed using SyBRGreen PCR Master Mix (Applied Biosystems) and gene-specific primers on the iCycler PCR system (BIO-RAD, Hercules, California, USA). The tomato UBI3 gene (accession no. X58253) was selected as the internal reference. Normalization was performed by the 2-Ct method. All primers used are listed in Supplementary Table 1. Chlorophyll fluorescence imaging Photosynthetic capability of young, green tomato fruits was determined by a chlorophyll fluorescence system (Imaging-PAM, Mini-Version, Walz, Germany). From the surface and cross-section of the tested fruits, the image of the maximum quantum efficiency of PSII photochemistry, Fv/Fm, was taken under saturation pulse mode. The saturation pulse light was 2,500 lM m-2 s-1 and the measured light intensity and the actinic light intensity were 0.5 and 180 lM m-2 s-1, respectively. Fluorescence images were recorded simultaneously and displayed by means of a false colour code ranging from 0 (black) to 1 (pink). Exogenous geraniol treatment Geraniol and linalool standards were purchased from Sigma-Aldrich. For geraniol treatment, 10 mM geraniol in 10 ml water was sprayed onto nine immature green fruits (15 days post-anthesis, DPA) of three intact tomato plants. The same volume of pure water was sprayed on control fruits. Chlorophyll fluorescence images of green fruits after 0, 24, and 48 h of treatment were taken as mentioned above. The same geraniol treatment was also performed in an enclosed container for nine detached red ripe fruits (42–45 DPA) from three different tomato plants. A tissue disk assay of fruit peels was also performed. Briefly, fruit disks (pericarp slices, *5 mm thick) were treated with 5 ml geraniol (10 mM) or water (as control) for 24 h in an enclosed flask. Each flask contained 30 g tissues from at least three different fruits. For treated fruits or fruit disks, volatile compounds and gene expressions were analyzed by GCMS and real-time quantitative RT-PCR, respectively. Each treatment was performed in triplicate.
Biotechnol Lett
Analysis of volatile compounds Extraction of volatiles were carried out as the method described by Davidovich-Rikanati et al. (2007). Approximately 30 g fruit tissues were cut into small pieces and extracted with 100 ml methyl tert-butyl ether (MTBE) with 1 mM phenylethanol as the internal standard by vigorous shaking for 3 h. The MTBE phase was separated off, dried over anhydrous Na2SO4, and concentrated to 0.2–0.5 ml by a rotary evaporator. The concentrated MTBE extracts were injected into a GC–MS system (Aligent, USA) equipped with 30 m 9 0.25 mm 9 0.5 lm column (Restek, Bellefonte, PA). Helium (1 ml min-1) was used as a carrier gas, with a split ratio of 1:200. The GC was held initially at 60 °C for 2 min followed by a gradient of 4 °C min-1 to 280 °C and held for 10 min. Mass spectra were obtained by electron impact ionisation at 70 eV. Spectrometric data were compared with computerized libraries and authentic standards.
Results TPS37 and 39 are fruit-related Tomato three TPSs, TPS5/37/39, together with some known TPSs from other plants, could be grouped in three clades of a phylogenetic tree. Figure 1 shows
that TPS37 and 39 cluster closely and locate in the cluster I, together with three fruit TPSs, including a grape linalool/nerolidol synthase LIS/NES (Martin et al. 2010), as well as apple NES (MdTPS3) and LIS (MdTPS6) (Nieuwenhuizen et al. 2013). In the cluster II, the tomato TPS5 and phellandrene synthase TPS4/ MTS2 are clustered in a same subclade and are similar to the previously characterized scented-related LISs from Lavandula angustifolia (lavender LIS; Landmann et al. 2007) and Mentha aquatica (mentha LIS; Crowell et al. 2002), as well as two closely-related Arabidopsis TPS02 and TPS03. The latter are constitutively expressed in flowers and are induced in leaves under stress (Huang et al. 2010) (Fig. 1).
Expression of TPS5, 37 and 39 in developing fruits TPS37 is highly expressed in petioles and stems bearing high density glandular trichomes. The TPS39 transcript is only observed in young leaves and flowers (Kant et al. 2004; Falara et al. 2011). In tomato cv. Alisa Craig, we found that TPS5, 37 and 39 were expressed in all stages of fruit development and ripening: their expressions were low in young green fruits, and gradually increased with fruit development and ripening, with maximal transcript accumulation in the yellow fruits, and dropped dramatically in the later stage of ripening (Fig. 2a).
Fig. 1 Phylogenetic tree of tomato terpene synthases (TPSs) and other plantidentified and/or functionally-annotated TPSs. A neighbor-joining dendrogram analysis was performed based on the degree of sequence similarity using MEGA4.0. The scale bar represents 0.1 substitutions per site. Statistical confidence of the nodes of the tree are based on 10,000 bootstrap replicates. Other abbreviations in this figure are as follows: MTS monoterpene synthase, LIS linalool synthase, NES nerolidol synthase
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Biotechnol Lett Fig. 2 Expression pattern of three terpene synthases (TPSs) in tomato. a Expression of TPSs in various tissues. Total RNAs were extracted from fully expanded leaves (mature leaves), and fruits at different developmental stages (immature green, 15 day-post anthesis (DPA); mature green, 30 DPA; yellow ripe, 40 DPA and red ripe, 45 DPA). b Spatial expression of TPSs in immature green fruits (15 DPA). Real-time RT-PCR was performed as described in ‘‘Materials and methods’’ section. Average values and standard errors are shown from three independent replicates
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Biotechnol Lett Fig. 3 False color images of the chlorophyll fluorescence yield (Fv/Fm) of tomato green fruits exposed to geraniol solutions after 24 and 48 h. Water-treatment fruits were used as control. Images from the surfaces (a) or crosssections (b) of fruits were shown, respectively
In addition, we isolated total RNAs from different parts of 15 DPA immature green fruits for RT-PCR assay. The results showed that TPS37 was highly expressed in all parts of fruits at this specific stage, with the highest expression in the exocarp tissues attaching with trichomes (Fig. 2b). In contrast, the mRNA accumulations of TPS5 and 39 were significantly low, especially in flesh tissues where few transcript was detected (Fig. 2b). These suggested that among them, TPS37 was dominant in fruits during development and ripening, and its expression was not trichome-specific. Exogenous geraniol causes the damage of fruits Geraniol is a potent inducer of apoptosis-like cell death in cultured plant cells (Izumi et al. 1999). Here, 10 mM geraniol in water was sprayed on the immature green fruits on vines and then the change in Fv/Fm value was monitored, visualized by chlorophyll fluorescence imaging with a false colour code ranging from 0 (black) to 1 (pink). Since photosynthesis is one of the most sensitive physiological processes of plant upon environmental stress, the Fv/Fm value that represents the maximum quantum use efficiency of photosystem II is usually used to reflect rapidly the magnitude of the plant’s wound response (Barbagallo et al. 2003). In healthy green fruits, Fv/Fm usually has a value of *0.79 (blue), whereas no chlorophyll fluorescence is detected in ripe fruits. After 24 h of geraniol treatment, a severe inactivation of photosystem II in
the surface of green fruits was observed through changed coloration in false color images of the chlorophyll fluorescence yield (Fv/Fm); after 48 h, Fv/Fm values fell to around zero (black) (Fig. 3a, b). Moreover, the images of fruit cross-sections showed that the damage caused by geraniol started in the surface of the fruits, and then spread successively into fruit inners (Fig. 3b). Elevated level of linalool in tomato fruits induced by exogenous geraniol Volatile compounds of water-or geraniol-treated fruits were extracted by MTBE and analyzed by GC–MS. Figure 4 shows that no significant volatile compounds were detected in the MTBE-extracts of water-treated fruits or fruit disks; similarly, no extra volatile compound was induced in the fruits after 24 and 48 h of geraniol treatment. However, a significant linalool peak was found in the MTBE-extracts of geraniol-treated fruit disks but nerolidol production was not detected in either geraniol-treated fruits or fruit disks (Fig. 4). Gene expression analysis showed that, compared with water-treated controls, geraniol-sprayed fruits displayed a slightly increase in transcript levels of TPS5, 37 and 39 within 48 h (Fig. 5). However, all three TPS genes were apparently upregulated in fruit disks (pericarp slices) that were directly exposed to alcohol solution after 24 h, especially for TPS37, its expression showing hundreds-fold higher than that of undamaged control fruits (Fig. 5).
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Fig. 4 Volatiles extracted from tomato fruits after geraniol or water treatment. a Total ion chromatograph (TIC) of GC are shown for methyl tert-butyl ether (MTBE)-extracts of fruits. Marked peaks are indicated as geraniol (1) and linalool (2), respectively. These peaks are identified by comparison of their
retention times and their mass spectra with those in the computerized libraries. b Mass spectra of products present in MTBE-extracts (labeled as 1 and 2, respectively) and their authentic standards
Fig. 5 Inducible expressions of three terpene synthases (TPSs) in ripe fruits after 24 or 48 h of geraniol. Total RNAs were extracted from water- or geraniol-treated whole fruits (spraying)
and fruit disks, respectively. mRNA levels were analyzed by real-time RT-PCR. The results from three independent experiments are shown as mean values and standard errors
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Biotechnol Lett Fig. 6 Geraniol treatment triggers the expressions of jasmonic acid (JA)-related defense genes. mRNA levels in geraniol-treated fruits (spraying) or fruit disks were analyzed by real-time RT-PCR. The whole fruits sprayed with water for 48 h were used as a control. Average values and standard errors are shown from three independent replicates. Significance was calculated using the Student’s t test. Asterisks indicate statistically significant differences (**P \ 0.005)
Geraniol treatment triggers some JA-responsive genes Since geraniol-treated fruits displayed visible mechanical damages (Fig. 3), we further investigated the effect of geraniol treatment on the expressions of some defense-related genes. Prosystemin (PS) is a signalingrelated gene that involves in the JA biosynthesis via the octadecanoid pathway, while proteinase inhibitor II (Inh II) and cathepsin D inhibitor (CDI) are two genes that directly participate in the plant defense against wounding; all of them are JA-regulated (Li et al. 2003). Figure 6 shows that Inh II and CDI were upregulated in
fruits after 24 or 48 h of geraniol spraying; no higher accumulations for them were observed in fruit disks that were directly treated with geraniol. Unlike Inh II and CDI, the transcriptional level of the signaling gene PS correlated with the strength of the induction stimulus, with the highest transcription accumulation in treated fruit disks (Fig. 6).
Discussion Linalool, an acyclic monoterpene alcohol, is found in many plant flower scent and edible fruits, while in
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tomato, it is produced predominantly in trichomes of leaves and stems, absent in fruits (Goff and Klee 2006; van Schie et al. 2007). In this report, we found that exogenous geraniol promoted the expressions of three LISs, TPS5/37/39 and induced the production of linalool in treated fruit tissues. These recombinant TPS proteins could convert FPP to nerolidol, suggesting that they could potentially contribute to nerolidol production. However, nerolidol was not identified in the solvent extracts of the geraniol-treated tissues in which all of the three TPSs were upregulated (Fig. 5). Because no headspace analysis was performed in this report, it is not clear that whether nerolidol (or linalool) is lost in the headspace. Nevertheless, in the plant, substrates for the biosynthesis of monoterpene and sesquiterpene, GPP and FPP, were present in the plastids and cytosol, respectively (Nagegowda et al. 2008). The ChloroP (http://www.cbs.dtu.dk) and IPSORT (http://ipsort.hgc.jp) programs predicted that both TPS5 and TPS37 had N-terminal transit peptides and were putatively localized in the plastids, while TPS39 contained a mitochondrial-targeting peptide. Hence, it is difficult to determine if nerolidol is produced by TPS proteins from FPP in plastids because little knowledge is available on the production of GPP or FPP in tomato. Although genetic evidence is still needed, our results suggest that the linalool peak in fruit tissue after geraniol treatment might be attributed to TPS5 and TPS37, both of them localized in the plastid where high levels of GPP exist. Moreover, TPS37 might be the predominant one because of its significant higher expression in either developing fruits or geraniol-treated tissues. Biotransformation of terpenes and terpenoids occurs in bacteria, fungi and certain plant cell cultures (e.g. Picea abies suspension cells) (Demyttenaere et al. 2000; Lindmark-Henriksson et al. 2004). For example, application of the pure geraniol in liquid cultures of Aspergillus niger led to biotransformation resulting in multiple products with linalool being one of them (Demyttenaere et al. 2000). This indicated that linalool might be formed by the bioconversion of geraniol. However, the biotransformations reported previously were generally carried out in aqueous systems and in shake-flasks, which are compatible with enzymes and growing whole cells (deCarvalho and daFonseca 2006). In this report, no extra compounds were found in sprayed-fruits and no other metabolites, except for linalool, were detected in treated fruit disks (Fig. 4). In
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addition, expressions of TPS5/37/39 and the production of linalool in tomato fruit disks were also induced by (Z)-3-hexenol (data not shown). Hence, the linalool peak in treated fruits is unlikely to arise from the bioconversion production of geraniol, and it might be correlated with wound defense responds because application of geraniol also caused the tissue damage and triggered JA-related defense genes. Indeed, linalool was predominant among the induced volatiles of tomato plants by spider-mite, wounding or chemical treatments (Kant et al. 2004; van Schie et al. 2007). Volatile-mediated defense mechanism is complex, involving some different regulators such as JA, salicylic acid (SA) and ethylene (Kant et al. 2004). Arimura et al. (2008) reported that herbivore-induced terpenoid emission was affected by concerted action of jasmonate, ethylene and calcium signaling. In tomato, JA is a key regulator of spider mite-induced volatile terpenoid (Ament et al. 2004). However, it is unclear whether a JA signal is essential for the geraniol-induced expression of TPSs. Thus further studies are required to understand these interactions between geraniol and JA, as well as other regulators such as SA and ethylene. Acknowledgments This work was supported by the Education Department Fund of Sichuan Province (No. 13ZD1110) and the Internal Fund of Southwest University of Science and Technology (No. 20145030).
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