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First published online October 29, 2004; 10.1104/pp.104.051318 Plant Physiology 136:3724-3736 (2004) © 2004 American Society of Plant Biologists The Biochemical and Molecular Basis for the Divergent Patterns in the Biosynthesis of Terpenes and Phenylpropenes in the Peltate Glands of Three Cultivars of Basil1Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109–1048 (Y.I., E.F., E.P.); Department of Plant Sciences and Institute for Biomedical Science and Biotechnology, University of Arizona, Tucson, Arizona 85721–0036 (D.R.G.); and Department of Vegetable Crops, Newe Ya'ar Research Center, Agricultural Research Organization, Ramat Yishay 30095, Israel (R.D.-R., E.B., E.L.)
Surface glandular trichomes distributed throughout the aerial parts of sweet basil (Ocimum basilicum) produce and store monoterpene, sesquiterpene, and phenylpropene volatiles. Three distinct basil chemotypes were used to examine the molecular mechanisms underlying the divergence in their monoterpene and sesquiterpene content. The relative levels of specific terpenes in the glandular trichomes of each cultivar were correlated with the levels of transcripts for eight genes encoding distinct terpene synthases. In a cultivar that produces mostly (R)-linalool, transcripts of (R)-linalool synthase (LIS) were the most abundant of these eight. In a cultivar that synthesizes mostly geraniol, transcripts of geraniol synthase were the most abundant, but the glands of this cultivar also contained a transcript of an (R)-LIS gene with a 1-base insertion that caused a frameshift mutation. A geraniol synthase-LIS hybrid gene was constructed and expressed in Escherichia coli, and the protein catalyzed the formation of both geraniol and (R)-linalool from geranyl diphosphate. The total amounts of terpenes were correlated with total levels of terpene synthase activities, and negatively correlated with levels of phenylpropanoids and phenylalanine ammonia lyase activity. The relative levels of geranyl diphosphate synthase and farnesyl diphosphate synthase activities did not correlate with the total amount of terpenes produced, but showed some correlation with the ratio of monoterpenes to sesquiterpenes.
Plants produce a large number of secondary metabolites that function in a variety of ecological contexts. Many specialized compounds are toxic and can therefore serve as defense agents against microbial pathogens and insect and animal herbivores (Wittstock and Gershenzon, 2002  ; Theis and Lerdau, 2003; Wink, 2003). Other compounds are volatile and serve to attract pollinators or even insects that prey on the plant's enemies or repel harmful organisms (Pare and Tumlinson, 1999; Kessler and Baldwin, 2001; Baldwin et al., 2002; Pichersky and Gershenzon, 2002).
Secondary compounds with roles in defense are often sequestered in specialized cells or structures, presumably to protect the plant itself from its own toxicity (Gershenzon et al., 1989
The Lamiaceae is a large plant family that includes the mints, sages, and basils and is well recognized for the diversity of secondary compounds synthesized and stored in glands found on the surface of leaves, stems, and flowers. The glands of sweet basil (Ocimum basilicum) in particular are rich in phenylpropenes as well as monoterpenes and sesquiterpenes (Werker et al., 1993
The phenylpropenes are derived from Phe and share the first few biosynthetic steps with the phenylpropanoids, although the entire biosynthetic pathway has not yet been elucidated (Hahlbrock and Grisebach, 1979
The existence of distinct basil chemotypes is both the result of natural evolution and selective breeding (Darrah, 1974 ; Morales and Simon, 1996; Simon et al., 1999). This observation suggests that most basil lineages have the genetic potential to synthesize most, if not all, of the range of compounds that have been found collectively in this species. However, the molecular mechanisms by which such diversity is generated—gene silencing, gene duplications and modifications, differential gene regulation, and posttranslational modification—are not clear.
Previously, we reported the characterization of a geraniol synthase (GES) gene isolated from the basil cultivar SD (Iijima et al., 2004
Characterization of Monoterpenes and Sesquiterpenes Produced by Basil Cultivars EMX, SD, and SW
Terpene and phenylpropene constituents (Fig. 2) in leaves of basil cultivars EMX, SD, and SW were extracted and analyzed (Tables I and II). The major volatile constituent of EMX leaves was the phenylpropene methylchavicol (4.5 mg g–1 fresh weight [FW]), and this cultivar contained very low amounts of terpenes distributed almost evenly between monoterpenes and sesquiterpenes. On the other hand, the SD and SW cultivars contain lower amounts of phenylpropenes than cultivar EMX, and severalfold more terpenes than EMX, with monoterpenes predominating. The SD cultivar was particularly rich in geranial (6.5 mg g–1 FW) and neral (3.1 mg g–1 FW), both of which are derived from the oxidation of geraniol (Iijima et al., 2004
Overall, EMX had 0.9 mg of monoterpenes g–1 FW, SD had 10.5 mg of monoterpenes g–1 FW, and SW had 3.6 mg monoterpenes g–1 FW. Cultivar SD also had the most sesquiterpenes, at 2.1 mg g–1 FW, SW had approximately one-third of this concentration, and EMX had only 0.8 mg sesquiterpenes g–1 FW.
It has been previously established that, in the Lamiaceae in general and in basil specifically, the leaf terpenoids and phenylpropenes are synthesized almost exclusively in the peltate glands (Hallahan, 2000
GPP, the substrate of monoterpene synthases, is synthesized from IPP and DMAPP by the enzyme GPP synthase (GPPS), which has been found to be a heterodimer in several angiosperms (Burke et al., 1999
PAL catalyzes the first committed step in the phenylpropanoid and phenylpropene pathways. Because of the observed inverse correlation between terpene content and phenylpropene content in the basil glands, we examined the activity of PAL in the glands of the three basil cultivars (Table II). The EMX cultivar, with the highest content of phenylpropenes, had PAL activity of 27.2 pkat mg–1 protein, 2.8 times higher than that in SD, the basil cultivar with the lowest phenylpropene content. In SW, the cultivar with intermediate phenylpropene content, PAL activity was intermediate between EMX and SD.
We had previously constructed annotated expressed sequence tag (EST) databases for SD, SW, and EMX peltate glandular trichomes that each contain approximately 3,500 ESTs (Gang et al., 2001
Monoterpene synthases are located in the plastids and are synthesized in the cytosol with an N-terminal transit peptide extension that is cleaved after transport into the organelles (Gavel and von Heijne, 1990 ; Williams et al., 1998). Both GES and LIS protein sequences appear to have transit peptides (Fig. 3). A third cDNA, found in SW only, was expressed in E. coli and found to encode for a protein that catalyzes the formation of terpinolene, limonene,  -pinene, and one additional unidentified monoterpene (Fig. 5A). Because its major product was terpinolene, it was designated terpinolene synthase (TES). Finally, two closely related cDNAs (95.1% identical to each other on the nucleotides level), designated fenchol synthase (FES) and -myrcene synthase (MYS), were found in all three databases. FES catalyzes the formation of fenchol,  -pinene, limonene, and one additional unidentified monoterpene, and MYS catalyzes the exclusive formation of -myrcene. Some of these monoterpene synthases catalyze the formation in vitro of some products that were not found in the glands (i.e. terpinolene). Similar apparent discrepancies between product profile of E. coli-produced plant TPSs and in vivo terpene profiles have been observed before (Jia et al., 1999). Metabolism of such products or lack of complete structural identity between the plant-derived and E. coli-produced enzymes has been implicated (Jia et al., 1999; Crowell et al. 2002).
The four other contigs encoded proteins with no apparent transit peptide (Fig. 3) and were therefore assigned as putative sesquiterpene synthases. Expression of their respective full-length cDNAs in E. coli followed by enzymatic assays identified these proteins as
The basil LIS is very different from other LIS sequences found in other species (Fig. 4). In contrast, basil LIS and GES are 81% identical on the protein level, suggesting that they evolved from a common ancestor relatively recently. The reaction mechanism of GES was previously determined (Iijima et al., 2004
In addition to the nine TPSs described above, all the EST databases also contained cDNAs encoding both the large and small subunits of GPPS and FPPS, all the genes encoding the enzymes of the MEP pathway, and acetoacetyl CoA thiolase (AACT), HMG synthase (HMGS), and HMG reductase (HMGR), the first three enzymes of the MVA pathway (see Fig. 1). These cDNAs were bound to macroarray filters, which were then hybridized with labeled cDNAs derived from reverse transcription of whole glandular trichome RNA preparations from each of the three cultivars. Overall, steady-state levels of these monoterpene synthase mRNAs were the lowest in EMX glands, and the levels of expression of individual monoterpene synthases in the other two cultivars were also consistent with the observed levels of volatiles (e.g. relatively high levels of GES in the geraniol-producing SD cultivar, and high levels of LIS in the linalool-containing SW cultivar; Fig. 7). Similarly, CDS was more highly expressed in SW, which has the highest level of cadinene among the three cultivars, and SES and GDS had the highest expression levels in SD, which has the highest levels of
The levels of expression of the earlier genes in the terpene biosynthetic pathways did not follow any clear pattern. In general, the levels of steady-state mRNAs of the three MVA pathway genes were uniformly low, whereas MEP genes showed variable levels of steady-state mRNA. Also noteworthy was the observation that mRNA levels for the small subunit of GPPS were higher than those of the large subunit of GPPS in all three cultivars.
The expression levels of the first three genes in phenylpropanoid and phenylpropene pathways, including PAL as well as cinnamate 4-hydroxylase (C4H) and 4-coumarate:CoA ligase (4CL), were measured in the three basil cultivars (Fig. 7B). The basil EST databases contain several related sequences encoding each of these genes (four PALs, two C4Hs, four 4CLs). Although transcript levels varied widely among these genes, overall, levels of transcripts for PAL and 4CL genes were highest in EMX and lowest in SD. On the other hand, the levels of C4H transcripts were similar in all three cultivars.
Basil Cultivars Differ in the Expression of Specific TPSs
The multiplicity of terpenes produced by a single plant has been noted before for organisms as varied as grand fir trees (Bohlmann and Croteau, 1999
To better understand the molecular mechanisms that control chemical differentiation in basil, we have identified and characterized four monoterpene synthases and four sesquiterpene synthases expressed in the glandular trichomes of one or more of the three basil varieties, EMX, SD, and SW, while another basil monoterpene synthase, GES, had been previously reported (Iijima et al., 2004
The majority of the TPS proteins characterized in this study are fairly divergent from each other (<50%) and are therefore likely to have been present in the progenitor to the basil lineage. However, FES and MYS are 95% identical to each other, but they nevertheless produce different products. Likewise, GES and LIS are 81% identical to each other. Furthermore, the N-terminal transit peptides, which are usually not highly conserved, are almost identical to each other in these two pairs of genes. The relatively close similarity of these protein pairs indicates that further terpene biosynthetic diversity is continuing to be generated in the basil lineage by gene duplication and divergence. Particularly interesting is the observation of the close relatedness between basil GES and LIS, since geraniol and linalool are structurally similar monoterpene alcohols, differing only in the substitution position of the hydroxyl group. Furthermore, previously characterized LISs from many species as well as GES (which has so far been reported only from basil) were found to produce only a single product, either linalool or geraniol, but not both (Pichersky et al., 1995
The process of random mutations can lead to functional divergence in duplicated genes (Pichersky and Gang, 2000
While gene duplications are considered a prerequisite for divergence when genes encoding essential functions are involved, in secondary metabolism this requirement is not absolute (Pichersky and Gang, 2000
Another intriguing observation is the closer similarity of the basil ZIS to the basil monoterpene synthases rather than to the other three sesquiterpene synthases or to sesquiterpene synthases from other species (Fig. 4). It is possible that ZIS evolved from a monoterpene synthase by a deletion of the coding region for a transit peptide. This hypothesis is plausible since it has been previously demonstrated that TPSs can use both FPP and GPP as substrates (Kollner et al., 2004
Our results indicate that genes encoding the key enzymes in both the MVA and MEP pathways are active in the basil glands, although the steady-state levels of transcripts for some, but not all, MEP genes were higher than the transcript levels of the three MVA genes we tested (the other MVA genes were not present in the EST databases at all, suggesting even lower levels of expression). However, assessing the relative contribution of these two pathways to the synthesis of the final product is not straightforward because a linear correlation between transcript levels and protein levels cannot be assumed and, furthermore, the specific activity of each enzyme is unique and can vary greatly among enzymes.
With these caveats, our results indicate a loose correlation between transcript levels and enzymatic activity levels for GPPS, FPPS, and the TPSs in the three cultivars. EMX glands had the lowest levels of transcripts for each of these enzymes, and correspondingly lower levels of enzymatic activities. Transcript levels of the small subunit of GPPS are similar in SD and SW (and, as in snapdragon, higher than transcript levels for the large subunit [Tholl et al., 2004
The total amount of terpenes produced correlated well with the total levels of TPS activities, but not with the total levels of GPPS and FPPS activities. For example, SD leaves contain roughly three times more total terpenes than do SW leaves, and the ratio of TPS activity levels between these cultivars is also close to 3:1 (Table II). Likewise, SD leaves have about 7.4 times more terpenes than EMX leaves, and the ratio of TPS activity levels between the two cultivars is similar. On the other hand, SD and SW leaves have similar levels of total prenyltransferase activities, but SD has 3-fold more terpenes. Total terpene content was also negatively correlated with total phenylpropene content and PAL activity (Table II). These observations suggest that high levels of the final biosynthetic enzymes in the terpene pathways, coupled with lower levels of PAL activity, tend to restrict the phenylpropene pathway and increase the flux in the terpene pathway.
The ratio of total monoterpenes to total sesquiterpenes produced by each cultivar was weakly correlated with both the ratio of the activity levels of monoterpene synthases to sesquiterpene synthases, and the ratio of GPPS and FPPS activities (Table II). It therefore appears that once the flux in the terpene pathways is increased, GPPS and FPPS may exert some control on the levels of precursors that are directed toward the specific synthesis of monoterpenes and sesquiterpenes, but do not greatly influence the total amount of terpenes produced. This scenario is only valid if the monoterpene and sesquiterpene pathways are not completely independent. In fact, it was previously shown that cross-talk between the MVA and MEP pathways can occur (Bick and Lange, 2003
Plant Material
Seeds for SD (Sweet Dani) were obtained from a local nursery. The source of the seeds of EMX and SW cultivars is described in Gang et al. (2001)
Basil (Ocimum basilicum) young leaves (0.5–1 cm, 50 mg) of each cultivar were added to liquid N2 and ground by mortar and pestle. The powder was soaked in 2 mL methyl tert-butyl ether (MTBE) containing an internal standard (toluene, 0.02 mg) and extracted for 2 h at room temperature in 5-mL glass vials with tightly sealed rubber septa caps. The MTBE upper layer, which included the volatile oil, was removed and placed into another vial and concentrated to 200 µL under gentle N2 gas flow for gas chromatography-mass spectrometry (GC-MS) analysis. Data points were obtained in triplicate.
A Shimadzu QP-5000 system (Shimadzu, Columbia, MD) equipped with Shimadzu GC-17 gas chromatograph was used for GC-MS analysis of volatile compounds. Separation was performed on a CP-5 column (30 m x 0.32 mm i.d. x 1-µm film thickness; Alltech Associates, Deerfield, IL). The GC condition was the same as reported previously. Ultrapure helium was used as the carrier gas at a rate of 1.3 mL min–1. Samples (2 µL) were injected by the Shimadzu AOC-17 autoinjector. Eluted compounds were identified by comparing their retention time and mass fragmentation patterns with standard compounds. Linalool optical isomers were analyzed by a CyclosilB column (30 m x 0.32 mm i.d. x 0.25-µm film thickness; J&W Scientific, Folsom, CA). The conditions were set for 55°C as the initial temperature for 2-min hold, and gradient to 220°C by 2°C min–1. Injection and detector temperatures were set at 220°C and 250°C, respectively.
Glands were isolated following the procedures previously described by Gang et al. (2001)
TPS assay was measured as previously described (Iijima et al., 2004
The generated compounds were isolated by solid phase microextraction (SPME) as previously described (Iijima et al., 2004
Prenyltransferase activity was measured according to Tholl et al. (2001)
PAL activity was measured according to the method of Gang et al. (2001)
The construction of EST databases from the peltate glands of three basil cultivars EMX, SD, and SW was previously reported (Iijima et al., 2004
Alignment of multiple protein sequences was performed using the ClustalX program (Thompson et al., 1997
Construction of GES/LIS chimeric cDNAs was carried out by PCR. A conserved region centered around codon 360 (using the GES numbering) was chosen as the junction. For the GES/LIS cDNA, the N-terminal encoding part of the GES cDNA was amplified by PCR with the sense primer 5'-AATGTCTTGTGCACGGATCACCGTAAC-3' and the antisense primer 5'-AACGCCATGTAGCATATTTTCATGTA-3', and the C-terminal encoding part of the LIS cDNA was amplified with the sense primer 5'-TACATGAAAATATGCTACATGGCGTT-3' and the antisense primer 5'-TGAGCTAAGAAGAAAGAAGAGGAGTGAAG-3'. The two amplified fragments were mixed and the complete hybrid cDNA was obtained by PCR with primers 5'-AATGTCTTGTGCACGGATCACCGTAAC-3' and 5'-TGAGCTAAGAAGAAAGAAGAGGAGTGAAG-3'. To make a LIS/GES cDNA, the N-terminal encoding part of the LIS cDNA was amplified by PCR with the sense primer 5'-AATGTCTTGTGCACGGATCACCGTAAC-3' and the antisense primer 5'-AACGCCATGTAGCATATTTTCATGTA-3', and the C-terminal encoding part of GES cDNA was amplified with the sense primer 5'-TACATGAAAATATGCTACATGGCGTT-3' and the antisense primer 5'-TATTTATTGAGTGAAGAAGAGGGCATCCAC-3'. The two amplified fragments were mixed and the complete hybrid cDNA was obtained by PCR with primers 5'-AATGTCTTGTGCACGGATCACCGTAAC-3' and 5'-TATTTATTGAGTGAAGAAGAGGGCATCCAC-3'.
Full-length cDNAs of the nine TPSs were amplified by PCR using each plasmid as a template with T7 and V5 primers. Other genes encoding MEP and MVA pathways (Fig. 1) and phenylpropene pathways were identified from the basil EST databases by BLAST search, and those partial fragments were prepared by PCR with T7 and T3 primers from each EST clone as the template. Ten microliters of each PCR product were diluted with 100 µL of 0.4 M NaOH and 25 mM Na2EDTA, and incubated at 94°C for 10 min. Slot blot was prepared on Hybond-N+ membrane (Amersham-Pharmacia Biotech, Piscataway, NJ). The PCR products (95 µL) were added to sample wells of the apparatus and vacuum was applied. Each well was rinsed with 200 µL of 20 x SSPE (3.6 M NaCl, 20 mM phosphate buffer, pH 7.4, 20 mM EDTA). The wells were evacuated again and the vacuum was applied for a couple of minutes. Membrane was air dried and cross-linked by UV and oven. A ubiquitin cDNA was used as control. Two microliters of RNA prepared from three basil glands were reverse transcribed by M-MuLV reverse transcriptase (Roche, Mannheim, Germany) with dNTPs including 0.4 mM dCT32P (0.5 mCi). After purification, these reverse transcripts were used as probes for hybridization. Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AY693643, AY693644, AY693648, AY693649, AY693650, AY693646, AY362553, AY693647, and AY693645. Received August 6, 2004; returned for revision September 2, 2004; accepted September 3, 2004.
1 This work was supported by the U.S. Department of Agriculture-Binational Agricultural Research and Development Fund (grant no. IS–3332–02C to E.P. and E.L.), by the National Research Initiative Competitive Grants Program-U.S. Department of Agriculture (grant no. 2001–35318–10006 to E.P.), by the Vaadia-Binational Agricultural Research and Development Fund postdoctoral fellowship (FI–328–2002 to E.F.), and by the National Science Foundation (grant no. MCB–0210170 to D.R.G.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.051318. * Corresponding author; e-mail lelx{at}umich.edu; fax 734–647–0884.
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-cadinene; 3, 