Metabolic engineering aimed at monoterpene production has become an intensive research topic in recent years, although most studies have been limited to herbal plants including model plants such as Arabidopsis. The genus Eucalyptus includes commercially important woody plants in terms of essential oil production and the pulp industry. This study attempted to modify the production of monoterpenes, which are major components of Eucalyptus essential oil, by introducing two expression constructs containing Perilla frutescens limonene synthase (PFLS) cDNA, whose gene products were designed to be localized in either the plastid or cytosol, into Eucalyptus camaldulensis. The expression of the plastid-type and cytosol-type PFLS cDNA in transgenic E. camaldulensis was confirmed by real-time polymerase chain reaction (PCR). Gas chromatography with a flame ionization detector analyses of leaf extracts revealed that the plastidic and cytosolic expression of PFLS yielded 2.6- and 4.5-times more limonene than that accumulated in wild-type E. camaldulensis, respectively, while the ectopic expression of PFLS had only a small effect on the emission of limonene from the leaves of E. camaldulensis. Surprisingly, the high level of PFLS in Eucalyptus was accompanied by a synergistic increase in the production of 1,8-cineole and α-pinene, two major components of Eucalyptus monoterpenes. This genetic engineering of monoterpenes demonstrated a new potential for molecular breeding in woody plants.
Genetic modifications of plant monoterpene biosynthesis have been used to alter the composition of volatile compounds. However, previous studies were performed with herbaceous plants as hosts, such as peppermint (Mahmoud and Croteau, 2001; Mahmoud et al., 2004), tobacco (Ohara et al., 2003; Lucker et al., 2004a,b), petunia (Lucker et al., 2001), carnation (Lavy et al., 2002) and tomato (Lewinsohn et al., 2001). No woody plants have been utilized previously to genetically engineer monoterpene biosynthesis. The genus Eucalyptus includes the most widely used tree species for industrial plantations for pulp and paper production and also for yielding essential oils. The essential oils of Eucalyptus species show many biological activities, analgesic (Silva et al., 2003), anti-inflammatory (Silva et al., 2003), anti-microbial (Delaquis et al., 2002) and anti-viral (Schnitzler et al., 2001), and many commercial products are available on the market. As a target of genetic engineering to increase the essential oil content or to alter the flavour of the oil, Eucalyptus camaldulensis was used as a host woody plant in the present study.
Geranyl diphosphate (GPP), a common precursor of monoterpene biosynthesis, is derived from a non-mevalonate pathway in most cases (Rohmer, 1999; Eisenreich et al., 2001), and enzymes involved in this pathway seem to be localized to the plastids in plant cells. Furthermore, most monoterpene synthases responsible for the formation of these volatile compounds from GPP possess a transit peptide for plastid localization (Colby et al., 1993; Yuba et al., 1996; Bohlmann et al., 1997). However, the enforced cytosolic localization of a monoterpene synthase Perilla frutescens limonene synthase (PFLS) in transgenic tobacco plants also lead to the production of limonene, as in case of its normal plastidic localization, indicating PFLS to also function by utilizing cytosolic GPP as its substrate at a ‘non-native’ site (Ohara et al., 2003). It is worth noting that the limonene derived from cytosolic PFLS was emitted into the headspace of the plant growth box more effectively than that from transgenic tobacco expressing plastidic PFLS (Ohara et al., 2003). In addition, the intracellular localization of enzymes to a ‘non-native’ subcellular compartment had a stronger effect than that to the native compartment on the production of the final metabolites in other cases as well (Ohara et al., 2004; Takahashi et al., 2006).
This study utilized PFLS as a model monoterpene synthase (Yuba et al., 1996) for metabolic engineering in a woody plant, because there is no further modification of the reaction product, unlike for example, glucosylation of the product, linalool (Lucker et al., 2001). Full-length or truncated PFLS cDNA, designed to produce plastidic and cytosolic PFLS, respectively, were overexpressed in E. camaldulensis to generate strong increases in monoterpene production with both PFLS expression constructs. The balance between the emission and accumulation of monoterpenes in the transgenic Eucalyptus and the availability of prenyl-substrates from two independent biosynthetic pathways, the mevalonate and non-mevalonate pathways, in E. camaldulensis cells are also discussed.
Expression of PFLS cDNA in Eucalyptus camaldulensis plants
The full-length PFLS sequence has a plastid localization signal at the N-terminus (Ohara et al., 2003). The PFLS cDNA was modified to localize its coding protein to the cytosol by means of removal of the coding sequence for the putative signal peptide and the addition of a translational start site instead. The full-length and modified PFLS cDNA were subcloned into a binary vector under the control of the El2 promoter, which is a strong constitutive promoter, to yield pBin-FullLS1 and pBin-DeltaLS1 for plastidic and cytosolic localization respectively (Figure 1). These plasmids were introduced via Agrobacterium tumefaciens (strain EHA105) into E. camaldulensis. More than 50 transgenic E. camaldulensis plants were established for PFLS expression (17 for plastidic localization and 34 for cytosolic localization), and seven transgenic plants transformed with the vector control (VC) were also generated.
First, the yield of total RNA from E. camaldulensis was evaluated, and then the mRNA accumulation of PFLS mRNA was checked by real-time polymerase chain reaction (PCR). Total RNA was extracted from young leaves of transgenic and wild-type (WT) E. camaldulensis, while preparation of RNA from mature leaves was extremely difficult because of a low yield. The average total RNA yield from young leaves was 0.13 μg/mg fresh weight (n = 65), as shown in Figure 2a. Real-time PCR revealed that PFLS mRNA for the plastid-type and cytosol-type accumulated in all transgenic E. camaldulensis clones, whereas the amplification of PFLS was not observed in WT and VC plants. PFLS mRNA levels varied substantially among the transformants (Figure 2b). Several representative clones, i.e. both those with high levels and those with low levels, were chosen for the subsequent analysis, as indicated by asterisks in Figure 2b.
Limonene production in transgenic Eucalyptus camaldulensis
Because both plastid- and cytosol-type PFLS were expressed successfully in transgenic E. camaldulensis plants, limonene content was investigated in the leaves of each transgenic E. camaldulensis line. Mature leaves were ground into fine powder in liquid nitrogen and extracted with hexane, which was injected directly into a gas chromatography with a flame ionization detector (GC-FID) system. Representative gas chromatograms are shown in Figure 3a. GC-FID analyses of WT E. camaldulensis as well as transformants with the control vector showed almost no effects on limonene production in E. camaldulensis, which indicated that the transformation and the regeneration procedure did not influence the limonene level in E. camaldulensis (Figure 3b). The limonene content of WT plants was analysed with 11 independent plants, which ranged from 5.61 ± 0.47 to 73.0 ± 18.5 μg/g fresh weight (average 22.9 μg/g fresh weight). Three representative results are shown in Figure 3b. However, in PFLS-overexpressing E. camaldulensis, an increasing effect on limonene production was observed, i.e. the average limonene content of plastid-type and cytosol-type transformants was 72.4 μg/g fresh weight and 120 μg/g fresh weight respectively. In particular, plastid-type no. 1 accumulated 190 ± 13.0 μg limonene/g fresh leaf weight, and cytosol-type no. 31, 327 ± 33.3 μg/g fresh weight, which was 2.6- and 4.5-fold greater than WT no. 1 (73.0 ± 18.5 μg g fresh weight) that showed the highest limonene content among the 11 control plants (Figure 3b).
Eucalyptus species are known to emit volatile compounds such as monoterpenes in nature. Thus, whether volatile compounds were emitted from detached leaves was analysed using solid-phase micro extraction (SPME) fibres. There are several reports of a circadian rhythm to the emission of volatile compounds from plants (Dudareva et al., 2003; Tholl et al., 2004). To avoid the effects of circadian rhythm in the comparison among independent clones, the emission of volatiles was measured during the daytime. Typical gas chromatographs of the headspace of flasks containing detached leaves are shown in Figure 4a. The level of limonene emitted from independent WT E. camaldulensis varied from 5.55 ± 2.08 to 12.5 ± 2.14 ng/h/g fresh weight, as estimated from the peak area in GC-FID analyses (Figure 4b). The level of emission from VC plants was the same as or lower than the WT level. In PFLS-overexpressing transgenic clones, the highest level was observed in plastid-type PFLS no. 1, 24.8 ± 12.0 ng limonene/h/g fresh weight, while the level of emission from cytosol-type PFLS no. 31 was 12.9 ± 0.88 ng/h/g fresh weight (Figure 4b). These results showed that plastid-type PFLS expression had only a small effect on the emission with a ca. twofold increase at most, but almost no effects were observed for cytosol-type PFLS expression. The correlation between the accumulation and the emission of limonene among transgenic clones was also analysed, but was found to be weak (R2 = 0.15) (Figure 4c).
Influence of PFLC expression on other volatile compounds in Eucalyptus camaldulensis
The major monoterpene compounds in the hexane extracts of WT plants are 1,8-cineole, α-pinene, and limonene, and the transgenic plants also exhibited a similar monoterpene composition. In addition to these major monoterpenes, several unidentified minor peaks were detected using GC/MS in the range of the retention time for monoterpenes and sesquiterpenes in both WT and transgenic leaf extracts from representative clones (Table 1). This comparison indicated that PFLS expression did not strongly influence the composition of volatile compounds in the transgenic E. camaldulensis including minor components. However, there seemed to be a tendency for cytosol-type PFLS clones to accumulate lower levels of putative sesquiterpene components as minor components than plastid-type PFLS clones and WT plants.
Table 1. Profiles of volatile compounds in transgenic E. camaldulensis detected by GC/MS
Next, the two major monoterpenes 1,8-cineole and α-pinene accumulating in the leaves were quantified by GC-FID. Figure 5 shows that both transgenic E. camaldulensis expressing plastidic- and cytosolic-PFLS accumulated as much or more of 1,8-cineole and α-pinene as the WT plants. Interestingly, these 1,8-cineole and α-pinene levels showed a clear correlation with the limonene content (Figure 5a,b). These results indicated that the enhancement of metabolic flow from GPP to limonene caused an overall increase in monoterpene production in the essential oil of E. camaldulensis. The level of emission of 1,8-cineole into the headspace of flasks containing detached leaves was also checked, but it varied greatly among independent clones and almost no correlation was observed between the emission of 1,8-cineole and that of limonene (Figure S1). It was not possible to determine the α-pinene emission level because of the detection limit of the present method.
Morphological analysis of transgenic Eucalyptus camaldulensis
Manipulation of the terpenoid pathway might influence the growth and development of plants because of possible metabolic turbulence including a hormonal imbalance (Fray et al., 1995). However, in the transgenic E. camaldulensis, no significant phenotypic differences were observed between WT and PFLC-expressing clones for plastid- or cytosol-type PFLC transformants, whose leaves are shown in Figure 6a. As the monoterpene content of the leaves of transformants increased, the number of oil glands, which were observed as yellowish dots without auto-fluorescent chlorophyll using fluorescence microscopy (Figure 6b), was compared between WT and transformant plants, but no significant change was observed in the size or number of oil glands (Figure 6c). Thus, the greater accumulation of limonene and other monoterpenes in the leaves of transgenic E. camaldulensis was probably caused by an increase in the levels of these secondary metabolites per oil gland with efficacious sealing, or an increase in the production of monoterpenes in mesophyll cells because the strong constitutive El2 promoter was used to drive PFLS. The morphology of oil glands was, at least, not affected in the transgenic leaves of E. camaldulensis.
This is the first ever report of the genetic engineering of monoterpenes in a woody plant, E. camaldulensis, in which a limonene synthase cDNA, PFLS, was overexpressed. The results indicated that the overexpression of PFLS strongly increased (ca. fivefold) the level of the direct reaction product limonene, whereas limonene accumulation showed little correlation to PFLS mRNA accumulation level in transgenic E. camaldulensis (Figures 2 and 3). One possible explanation for this observation is that the accumulation PFLS transcript does not correlate the protein accumulation or enzyme activity of PFLS in transgenic E. camaldulensis, which was in fact reported in PFLS-expressing tobacco plants (Ohara et al., 2003). Surprisingly, PFLS overexpression also increased the levels of other endogenous monoterpenes, such as 1,8-cineole and α-pinene, to the same extent in the transgenic E. camaldulensis clones. It was reported that the limonene synthase of peppermint produced, in addition to the main product limonene, small amounts of side products such as α-pinene, β-pinene and myrcene in vitro (Colby et al., 1993). However, it is unlikely that PFLS directly produced 1,8-cineole in vivo because further modification is necessary for the formation of 1,8-cineole after the cyclization of GPP. In fact, similar increases in levels of non-direct reaction products because of metabolic engineering in plants have been reported previously, e.g. the overexpression of linalool synthase in tomato resulted in the accumulation of limonene, which is not the enzymatic product of linalool synthase (Lewinsohn et al., 2001). In addition, transgenic tobacco plants expressing monoterpene synthases from lemon produced modified monoterpenes in vivo, which were probably converted from the direct reaction products of these enzymes (Lucker et al., 2004b). Several explanations for the strong increase in 1,8-cineole in transgenic E. camaldulensis are possible. One logical explanation is that limonene’s accumulation promoted the activities of endogenous upstream enzymes supplying prenyl diphosphate because of the constitutive consumption of endogenous GPP molecules by overexpressed PFLS, or the strong ectopic expression of PFLS caused an increase in the flux of metabolites towards the formation of monoterpenes, leading to the increased production of 1,8-cineole, for instance. Another possibility is that PFLS might leak a catalytic intermediate of 1,8-cineole, such as the α-terpenyl cation, in vivo in E. camaldulensis cells, which may be utilized by the endogenous 1,8-cineole synthase (Figure S2).
Transgenic E. camaldulensis clones were prepared, in which PFLS was forcibly localized to the cytosol rather than the plastid. The average amount of limonene in 11 WT plants was 22.9 μg/g, whereas that in plastid-type and cytosol-type transformants was 72.4 μg/g (3.16-fold of control) and 120 μg/g (5.24-fold of control) respectively. The increased amount of limonene generated by the expression of PFLS was effectively sequestered in leaf oil glands of both plastidic and cytosolic PFLS transformants, indicating that the limonene synthease in the cytosol can contribute to the synthesis and accumulation of limonene in leaves in a similar manner to the plastidic synthase. Recently, it was reported that the simultaneous expression of three terpene synthases in tobacco had marked effects on the profiles of volatile compounds emitted from transgenic plants (Lucker et al., 2004b). The accumulation or emission of terpenoids largely depends on the presence of glandular trichomes or oil glands, which are specialized for the accumulation of volatile compounds. To alter the fragrance emission profile of plants equipped with tissues to store volatiles, such as Eucalyptus, the co-expression of an efflux transporter may be effective in excreting essential oil compounds to the outside of the cells, although such monoterpene-specific transporters are, to the best of our knowledge, still unknown.
When production levels of monoterpenes were compared between the cytosol-type and plastid-type transformants of E. camaldulensis, cytosolic PFLS expression was found to increase the accumulation of limonene more strongly than the plastidic expression, suggesting that the cytosolic PFLS could somehow effectively utilize cytosolic GPP as a substrate. A number of explanations for the efficient production of limonene by cytosol-localized PFLS can be proposed, e.g. cytosolic PFLS may elude a negative feedback system functioning in the native plastidic isoprenoid pathway (Ohara et al., 2003), and/or the transit peptide of Perilla limonene synthase may be unfavourable to E. camaldulensis, resulting in less efficient of limonene production. It seems that E. camaldulensis has a free GPP pool in the cytosol, which is made by transport from plastids, or production of cytosolic GPP as an intermediate of FPP synthase, while FPP is subsequently used to produce sesquiterpene, for instance in response to insect and pathogen attacks. Cytosolic PFLS may trap this free GPP to form limonene. In fact, a decrease in the amount of putative sesquiterpenes (e.g. retention time 27.52 min) was observed in cytosol-type PFLS clones (Table 1), which may be as a result of increased competition for cytosolic GPP as a substrate. The involvement of this free GPP pool in isoprenoid metabolism in vivo was reported in a range of plant species including Quercus ilex, Mentha poperita, Populus alba and Prunus persica (Nogues et al., 2006).
This report demonstrated that the overexpression of PFLS yielded higher levels of limonene in transgenic E. camaldulensis. It was suggested that in herbaceous plants, the regulation of fragrance emission and volatile-compound biosynthesis involves more than gene expression alone (Dudareva et al., 2003). Further systematic research is also needed in woody plants to clarify the mechanism of monoterpene biosynthesis and to achieve the practical metabolic engineering of essential oil production.
Plant materials and transformation method
Seeds of WT E. camaldulensis were purchased from a local market. The seeds were surface sterilized using a sodium hypochlorite solution and sown onto Murashige-Skoog (MS) agar plates. Seedlings of E. camaldulensis on MS plates were incubated at 25 °C under continuous light. Hypocotyls (3 mm) containing apical cells were used for Agrobacterium-mediated transformation according to the method of Kawaoka et al. (2006). Transgenic E. camaldulensis plants were cultivated with potting soil at 25 °C under fluorescent lamps (ca. 80 μE/m2/s, 16 h of light/8 h of darkness).
Constructs for the overexpression of PFLS in Eucalyptus camaldulensis
The construction methods for the plastidic or cytosolic targeted PFLS cDNA have been reported previously (Ohara et al., 2003).
Real-time polymerase chain reaction
Total RNA was extracted from young leaves of E. camaldulensis in cultures using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer’s instructions. Reverse transcription was performed using Superscript III RNase H-reverse transcriptase (Invitrogen Corp., Carlsbad, CA, USA) with 2.0 μg of total RNA as the template, followed by incubation with RNase H (Invitrogen). Real-time PCR was performed with the Roter-Gene 3000A (Corbett Research, Mortlake, NSW, Australia), using a DyNAmo™ HS SYBR Green qPCR Kit (FINNZYMES, Espoo, Finland) according to the manufacturers’ instructions. The standard reaction conditions were as follows: 95 °C for 15 min, then 40 cycles of 94 °C 20 s, 50 °C for 30 s and 72 °C 30 s. The primers used to detect PFLS and actin mRNA were as follows: PFLSfw; 5′-agcctcagcagcgacaccaaagga-3′, PFLSrv; 5′-tccaaagagtggcgcacacacga-3′, ActinFW; 5′-caactgggacgacatggaga-3′, and ActinRV; 5′-gagtcatcttctctctgttggcc-3′.
Analyses of volatile compounds
For the analysis of volatile compounds in the leaves, mature leaves of E. camaldulensis (plant height, ca. 30 cm) were ground in liquid nitrogen to fine powder using a mortar and pestle, and ca. 100 mg of frozen leaf powder was extracted with 1 mL of hexane for 1 min on ice. A 1 μL sample of the hexane extract was injected directly into a gas chromatography system (GC-14B; SHIMADZU, Kyoto, Japan) equipped with a flame ionization detector (injector, 250 °C) and an InertCap-5 capillary column (30 m × 0.53 mm, film thickness 2 μm; GL Science, Tokyo, Japan), with a temperature programme of from 60 (10 min hold) to 100 °C (10 min hold) at a rate of 8 °C min−1, and then to 230 °C (5 min hold) at a rate of 30 °C min−1. The hexane extract was also analysed by GC-MS, performed using a JMS-K9 mass spectrometer (JEOL, Tokyo, Japan) coupled to a Network GC System 6890N gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) operated in the split-less mode. The ionization source was operated at 70 eV. The gas chromatograph was equipped with a TC-WAX capillary column (30 m × 0.25 mm, film thickness 2.5 μm; GL Science, Tokyo, Japan), with a temperature programme of from 40 (10 min hold) to 230 °C (10 min hold) at a rate of 8 °C min−1 using helium as the carrier gas at 1.0 mL/min. Volatile compounds in the headspace of a flask containing detached leaves (ca. 1.0–2.0 g fresh weight, 300-mL glass flask sealed with a silicon cap) from E. camaldulensis (plant height, ca. 30 cm) were trapped using SPME fibre (SPELCO, Bellefonte, PA, USA) for 1 h. All headspace sampling was carried out between 1:00 and 3:00 pm. The SPME fibre was injected directly into the gas chromatograph, with the same conditions as described above. Standard curves of limonene and 1,8-cineole using the SPME were determined as follows; pieces of filter paper with different concentrations of limonene (0, 7.8, 15.6, 31.3, 62.5, 125 or 250 ng) or 1,8-cineole (0, 0.5, 1.0, 2.0 or 5.0 μg) were placed into 300 mL glass flasks sealed with silicon caps, and volatiles were trapped with SPME and subjected to gas chromatography. Linearity of the standard curves was observed at the tested concentrations (Figure S3). Limonene, 1,8-cineole, α-pinene and myrcene were identified by comparison with the retention times and mass spectra of standards.
The oil glands of mature leaves were counted using a microscope (Axioskop2, Zeiss, Oberkochen, Germany) with filter sets for fluorescein.
The authors thank Ms Machiko Sawada and Dr Takahisa Hayashi of Kyoto University for GC/MS. This work was supported in part by a Grant-in-Aid for Scientific Research (No. 17310126 to K. Y.), a Grant from the Research for the Future Program: ‘Molecular mechanisms on regulation of morphogenesis and metabolism leading to increased plant productivity’ (No. 00L01605 to K. Y.) of the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by a Research Fellowship from the Japan Society for the Promotion of Science for Young Scientists (No. 17·2011 to K. O).