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First published online December 4, 2003; 10.1104/pp.103.032946 Plant Physiology 134:370-379 (2004) © 2004 American Society of Plant Biologists Characterization of Geraniol Synthase from the Peltate Glands of Sweet 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, University of Arizona, Tucson, Arizona 85721-0036 (D.R.G.); and Department of Vegetable Crops, Newe Ya'ar Research Center, Agricultural Research Organization, P.O. Box 1021, Ramat Yishay, 30095, Israel (E.L.)
The monoterpene fraction of the lemon-scented sweet basil (Ocimum basilicum) cv Sweet Dani consists mostly of citral (a mixture of geranial and neral), with lower levels of geraniol and nerol. These compounds are stored in the peltate glands found on the leaf epidermis. Younger leaves, which have a higher density of such glands, also have a higher content of monoterpenes than older leaves. Geraniol synthase (GES) activity, generating geraniol from geranyl diphosphate, was shown to be localized exclusively or almost exclusively to glands. GES activity resides in a homodimeric protein that was purified to near homogeneity. Basil GES requires Mn2+ as a divalent metal cofactor for activity and produces only geraniol from geranyl diphosphate. Km values of 21 and 51 µM were obtained for geranyl diphosphate and Mn2+, respectively. In the presence of 18O-labeled water, GES catalyzed the formation of 18O-geraniol from geranyl diphosphate, indicating that the reaction mechanism of GES is similar to that of other monoterpene synthases and is different from the action of phosphatases. A GES cDNA was isolated based on analysis of a glandular trichome expressed sequence tag database, and the sequence of the protein encoded by this cDNA shows some similarity to sequences of other terpene synthases. The expression of the GES cDNA in Escherichia coli resulted in a protein with enzymatic activity essentially identical to that of plant-purified GES. RNA gel-blot analysis indicated that GES is expressed in glands but not in leaves of basil cv Sweet Dani, whose glands contain geraniol and citral, and not in glands or leaves of another basil variety that makes other monoterpenes but not geraniol or citral.
Geraniol is an acyclic monoterpene alcohol emitted from the flowers of many species, notably roses (Rosa hybrida; Bayrak, 1994  ; Antonelli et al., 1997; Rao et al., 2000). It is also present in vegetative tissues of many herbs (Charles and Simon, 1992; Mallavarapu et al., 1998; Mockute and Bernotiene, 1999; Vieira et al., 2001) and is often found together with geranial and neral, which are the oxidation products of geraniol (Miyazawa and Kameoka, 1988). The mixture of geranial and neral, also called citral, imparts a "lemon" flavor, and lemongrass (Cymbopogon citratus Stapf.; Singh-Sangwan et al., 1993), ginger (Zingiber officinale Rosc.; Miyazawa and Kameoka, 1988), and some varieties of sweet basil (Ocimum basilicum; Grayer et al., 1996; Simon et al., 1999) such as basil cv Sweet Dani are particularly rich in citral (Morales and Simon, 1997). However, at present, there is no definitive proof of whether citral is synthesized from geraniol by an alcohol dehydrogenase (Sangwan et al., 1993; Singh-Sangwan et al., 1993; Hallahan et al., 1995; Sekiwa-Iijima et al., 2001) or by an oxidase (Potty and Bruemmer, 1970; Banthorpe et al., 1976), nor is it known if geraniol is the only substrate whose oxidation leads to the formation of citral or whether nerol, the cis-isomer of geraniol, can also serve as a precursor (Corbier and Ehret, 1988; Ikeda et al., 1991; Hallahan et al., 1995).
Geraniol itself is likely to be synthesized from geranyl diphosphate, the universal precursor of all monoterpenes (Croteau, 1987 In this paper, we report the purification and characterization of GES from basil cv Sweet Dani and GES cDNA isolation and expression in Escherichia coli. Furthermore, the reaction mechanism of GES was investigated using [18O]-labeled water. We show that GES is a member of the terpene synthase family based on sequence analysis, and its reaction mechanism is similar to that of other terpene synthases.
Determination of Terpene Content of Basil Leaves and Glands
There are many basil varieties with distinct terpene compositions (Lawrence, 1988
The amounts of these four monoterpenes in the leaf depended on age of the leaf, with young leaves having the highest levels per gram fresh weight and the levels getting progressively lower as the leaves mature and expand (Table I). Geraniol and nerol were detected in trace amounts in older, larger leaves. The ratio of geranial to neral was 2:1 at the all stages, with the geraniol to nerol ratio being similar.
To identify and localize GES activity, crude protein extracts were prepared from whole leaves and from isolated glands (prepared according to Gang et al., 2001
GES was purified from isolated glands in a protocol employing several chromatographic steps, including DEAE-cellulose anion-exchange column, Mono-Q anion-exchange column, and Superose-12 size exclusion column on an FPLC system (Pharmacia Biotech, Piscataway, NJ; Table II). In general, the enzyme was quite stable during the purification procedure, and freezing, thawing, and dialyzing in buffer resulted in no more than 5% loss of enzyme activity. However, concentrating the enzyme by ultrafiltration centrifugation resulted in complete loss of activity. The addition of octylglucoside as deter- gent before ultrafiltration prevented this loss. It was also determined that the presence of KCl, used in eluting the enzyme from the anion-exchange columns, had no effect on reaction rates.
After three successive chromatographic steps, the Superose-12 fraction with highest GES activity had 6,244 pkat mg-1 protein, representing 38.3-fold purification with 8.7% yield from crude protein in glands fraction (Table II). SDS-PAGE of this fraction showed one major protein band with an apparent subunit mass of 57.7 kD (Fig. 2). Native, active GES eluted from the size exclusion column as a 140-kD protein. The purified GES catalyzed the exclusive formation of geraniol from geranyl diphosphate, and no other monoterpene product was obtained (Fig. 3B).
Because monoterpene synthases in general are known to require a divalent metal ion such as Mn2+ and Mg2+ for their activity (Croteau, 1987 Km values were determined for geranyl diphosphate and Mn2+ at the optimum reaction condition. The Km value of GES for geranyl diphosphate was 21 µM, and the Km value for Mn2+ was 51 µM. The Kcat for GES with geranyl diphosphate was determined to be 0.8 s-1. GES had a pH optimum of 8.5 with more than 70% activity in the pH range of 8.0 to 9.5. However, its activity at the range of pH 6.0 to 7.5 was less than 10% of the maximal activity. GES was stable at 4°C to 20°C for 30 min and retained 80% activity after incubation for 30 min at 37°C. However, it was completely inactivated after 30 min of incubation at 45°C.
To determine if GES acts as a nonspecific phosphatase, we assayed phosphatase activity using p-nitrophenyl phosphate as the substrate. Although whole leaves and glands displayed phosphatase activities of 173.6 ± 18.8 protein and 102.1 ± 21.7 pkat mg-1 protein, respectively, purified GES did not exhibit any phosphatase activity. Next, we assayed the purified GES with geranyl diphosphate in a buffer that contained H218O. The product was extracted, concentrated, and analyzed by GC-MS. A shift in the biosynthetic geraniol molecular mass of +2 was observed for the molecular ion (154:156) and for several fragmentation products (139:141, M+-CH3; and 111:113, M+-C3H7) predicted to include the oxygen moiety (Fig. 4).
Because the experiments described above suggested that the reaction mechanism of geraniol formation by GES is similar to the reaction mechanism of other terpene synthases, we examined a large expressed sequence tag (EST) database constructed from the peltate glands of three basil varieties, including basil cv Sweet Dani, for potential GES cDNAs. The two other cultivars, EMX1 and SW, do not produce geraniol (or citral) but they do produce other monoterpenes, such as 1,8-cineole, linalool, and fenchone (Gang et al., 2001
ESI-MS/MS analysis of the trypsin-digested plant-purified GES, together with analysis by the ExPASy peptide mass program of the calculated tryptic peptide masses that could theoretically be generated from the 64,933-kD protein encoded by the full-length cDNA, resulted in the identification of 55 tryptic peptides, totaling 46.7% of its length (Fig. 5A). This result strongly suggests that this cDNA encodes the GES protein purified from the basil glands. Expression in E. coli of the full-length cDNA gave a protein that exclusively synthesized geraniol from geranyl diphosphate (Fig. 3C).
Monoterpene synthases are plastidic enzymes, and their N terminus serves as a transit peptide, which is cleaved after the protein is inserted into the organelle (Bohlmann et al., 1998 N-terminal sequencing of the plant-purified GES was unsuccessful, indicating that the N terminus might be blocked. However, expression in E. coli of GES cDNA of a truncated GES cDNA with an open reading frame that starts with Met-44 (bold and underlined in Fig. 5A), with a calculated molecular mass of 60.2 kD, resulted in a protein that appeared to have nearly identical mobility to plant GES on SDS-PAGE (Fig. 2). Expression in E. coli of a truncated open reading frame that starts with Ser-35 (bold and underlined in Fig. 5A), with a Met codon in front of the Ser codon (calcula1ted molecular mass of 61.2 kD), resulted in a protein that migrates on an SDS-PAGE as a protein of 58.6 kD, only slightly slower than plant-purified GES (Fig. 2). Both the Ser-35 and Met-44 truncated GES proteins catalyzed the transformation of geranyl diphosphate exclusively to geraniol with Km values of 29 and 30 µM, respectively, and Kcat values of 0.6 and 1.0 s-1, respectively. These Km and Kcat values are very close to the Km value of 21 µM and Kcat value of 0.8 s-1 determined for the plant-purified GES enzyme, suggesting that these truncations resulted in proteins that were very similar to the mature GES enzyme present in the gland. However, the exact position of the cleavage of the transit peptide remains to be determined, as well as the nature of the modification that blocks the free amino group at the N terminus after processing occurs. Basil Ser-35-GES produced in E. coli eluted from size exclusion chromatography as a 140-kD protein, same as native plant-purified GES. Thus, it appears that active GES is a dimeric enzyme. This conclusion is based on a calculation of a subunit molecular mass of approximately 60 kD (see above).
BLAST searches with the complete sequence of GES indicate that compared with proteins of known function, GES shares the highest sequence similarity (i.e. percentage identity of amino acid residues) with 1,8-cineole synthase from sage (Wise and Croteau, 1999
RNA gel-blot analysis was performed to examine whether GES gene expression is specific to glands producing geraniol. RNA samples from leaves and glands of basil cv Sweet Dani cultivar and RNA samples from leaves and glands of basil cv EMX1, whose monoterpene fraction consists mostly of 1,8-cineole (Gang et al., 2001
GES from Basil Is a Highly Specific Terpene Synthase
In several Lamiaceae plants (e.g. spearmint), monoterpenoid accumulation and biosynthesis occur in the eight-celled peltate glandular trichomes localized on the both sides of the leaf and attached to the leaf via a stalk cell (Hallahan, 2000
Here, we show that in Sweet Dani, geraniol, nerol, and citral are stored in the sacs (Fig. 1B) and that GES activity is localized to the peltate gland cells. Further, we show that only geraniol was generated in the enzymatic reaction containing purified GES and geranyl diphosphate (Fig. 1C). Thus, although some terpene synthases have been shown to catalyze the formation of multiple products (Colby et al., 1993 We identified the gene encoding GES by the catalytic properties of the protein it encodes, which, when the protein is produced in E. coli, are virtually identical to those of plant-purified GES, as well as by the identity of the peptide sequences determined by ESI-MS/MS. The gene encoding GES is specifically expressed in glands of basil cv Sweet Dani but not in the glands of other basil cultivars that produce other monoterpenes but not geraniol (Fig. 6; data not shown). Furthermore, the gel-blot analysis and comparisons of EST databases of several basil varieties demonstrated that other basil varieties do not express any functional terpene synthase genes whose sequences are >90% identical to GES, nor do Sweet Dani glands express any other functional genes with high similarity to isolated GES cDNA, further demonstrating that this GES cDNA is responsible for encoding GES activity in Sweet Dani glands.
Although the protein sequence of GES clearly indicates it is a member of the terpene synthase family (although a highly divergent one, see Fig. 5B), the molecular mass of active GES was estimated by size exclusion chromatography to be 140 kD, suggesting that it is active as a dimer. Most terpene synthases that have been examined have been shown to be monomeric (Bohlmann et al., 1998
The overall structural relatedness of basil GES to other terpene synthases and its requirement of Mn2+ for activity suggest that GES catalytical mechanism is similar to that of other terpene synthases. Furthermore, basil GES did not act on p-nitrophenol phosphate that can be hydrolyzed by nonspecific phosphatases, suggesting that its mode of action does not involve the hydrolysis of a phosphoester bond. In assays in which H218O was used for the buffer, 18O was incorporated into the geraniol product in the same proportion as the ratio of H218O to H216O in the solution (Fig. 4), directly demonstrating the validity of the model (Croteau, 1987
This conclusion is also strengthened by the observation that the protein sequence of basil GES clearly shows it to be a member of the terpene synthase family (Fig. 5), although its overall sequence similarity to other terpene synthases is quite low. Of particular interest may be the relatively high divergence at the N-terminal 100 amino acids, a region that encompasses both the transit peptide and additional sequences from the N terminus of the mature protein. The RRX8W motif, which is often, but not always, found in the N terminus of mature monoterpene synthases (Dudareva et al., 1996
Our results indicate that GES does not synthesize nerol, the cis-isomer of geraniol. Furthermore, we were not able to detect any nerol synthase activity in the glands (or leaves). Nerol, however, is a component of the essential oil of Sweet Dani. An explanation for the presence of nerol in the absence of a specific nerol synthase may lie in the mechanism of generating citral from geraniol. Citral is a mixture of geranial and neral in an approximately 3:2 ratio, and geranial and neral are in fact often found in together in such a ratio in many lemon-scented plants (Singh-Sangwan et al., 1993
Plant Material Seeds for basil (Ocimum basilicum) cv Sweet Dani were obtained from a local nursery. They were sown in horticultural vermiculite and put in the growth chamber until germination. One week after germination, each seedling was transplanted into a 500-mL pot containing Sunshine Mix No. 1 potting soil (Sun Gro Horticulture Canada Ltd., Seba Beach, AB) and grown in a greenhouse under constant illumination.
Basil leaves were categorized into three sizes: small (0.5-1.5 cm), medium (1.5-3 cm), and large (3-4 cm; Gang et al., 2001
Volatile oils were extracted from the glands of young leaves with a stretched glass pipette as previously described (Gang et al., 2001
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 DB-WAX (30-m x 0.32-mm-i.d. x 0.25-µm film thickness, J&W Scientific, Folsom, CA) capillary column with electron impact mode (1.4 kV). However, some nonpolar compounds eluted with the solvent peak in the DB-WAX column, and their separation was achieved on a CP-5 column (30-m x 0.32-mm-i.d. x 1-µm film thickness, Alltech Associates Inc., Deerfield, IL). The oven temperature for DB-WAX methods was held at 60°C for 2 min and raised to 220°C at 4°C min-1 with the injector set at 220°C and the interface set at 240°C. The GC condition for the CP-5 method was the same as the previous report (Gang et al., 2001
GES activity was assayed by incubating 5 µL of the enzyme sample in a final volume of 50 µL of buffer containing 50 mM HEPES-KOH (pH 8.0), 1 mM dithioerythritol, 0.5 mM MnCl2, 20 mM MgCl2, 10% (w/v) glycerol, and 0.025 µM [1-3H]-geranyl diphosphate (specific activity 20 Ci mol-1, American Radiolabeled Chemicals, St. Louis). After incubation for 30 min at 32°C, 160 µL of hexane was added to the tube, vortexed briefly, and centrifuged to separated the phases. The hexane layer was directly placed into a scintillation vial containing 2 mL of nonaqueous scintillation fluid (Econo-Safe, Research Products International, Mount Prospect, IL). This extraction procedure was repeated twice, and the total hexane phase was counted by a liquid scintillation counter (LS-6500 model, Beckman Coulter, Fullerton, CA). Boiled enzyme extracts were used as controls.
GES enzyme assays were also performed by adding 100 µL of enzyme solution with 900 µL of assay solution containing 54 µM nonradioactive geranyl diphosphate (Echelon Research Laboratories, Salt Lake City) and the same buffer described above. The reactions were carried out in an 8-mL DuPont autosampler vial (DuPont-Dow Elastomers L.L.C., Wilmington, DE) with a white solid-top polypropylene cap (Alltech). After letting the reaction proceed for 2 to 4 h at 32°C, the liberated compounds were collected with an SPME device PDMS-100 with a polydimethylsiloxane fiber (Supelco, Bellefonte, PA) by inserting the fiber into the tube and leaving it in for 20 min at 42°C. After this incubation step, the SPME fiber was directly injected into the GC-MS.
Assays in buffer containing 18O-labeled water were carried out in 2-mL glass vials with screw cap of PTFE/Silicone Septa (Supelco) by the addition 20 µL of purified enzyme (approximately 1 µg of protein) to a 180-µL assay solution that contained 20 µL of 10x assay buffer with 150 µL of H218O (95% atom, Icon Service, Summit, NJ) and 10 µL of 5.4 mM geranyl diphosphate. The final concentration of H218O in this assay solution was 71.3% (w/v). This solution was incubated for 2 h at 32°C, cooled down on ice, and then extracted with 200 µL of pentane. After concentration to 50 µL, 2 µL of this solution was injected to GC-MS system. To compare the mass spectra pattern, a pentane extract of the product from a reaction in which normal water (H216O) was used was also analyzed.
Phosphatase activity was measured as described by Hernández and Whitton (1996
All purification steps were carried out at 4°C unless stated otherwise. Glands were isolated from approximately 300 g of basil cv Sweet Dani, essentially following the procedures previously described by Gang et al. (2001
Partially purified GES was run on a size exclusion column under the same conditions used during the purification procedure, except that 0.25-mL fractionations were collected instead, and fractions were assayed for GES activity. A standard curve was obtained by plotting the elution volume/void volume of the standard proteins against the log of the molecular mass. The protein standards used included cytochrome C (12.4 kD), carbonic anhydrase (29 kD), ovalbumin (45 kD), bovine serum albumin (66 kD), alcohol dehydrogenase (from equine liver, 80 kD), and alcohol dehydrogenase (from yeast [Saccharomyces cerevisiae], 141 kD). The subunit molecular mass was estimated by SDS-PAGE performed on 10% (w/v) polyacrylamide gel and calibrated with molecular mass standard in the range of 14 to 212 kD (New England Biolabs, Beverly, MA).
The pH optimum for GES activity was determined using three buffer systems. Reaction was carried out in 50 mM Bis-Tris buffer ranging from pH 6.0 to 7.0, 50 mM Tris-HCl buffer ranging pH 7.0 to 9.0, and 50 mM Gly-NaOH buffer ranging from pH 9.0 to 10.0. Temperature stability of GES was determined by incubating GES in temperatures ranging from 4°C to 65°C for 30 min and then chilling the samples on ice, followed by enzyme assays at 32°C. To determine the kinetic parameters of GES, the enzyme was diluted to the appropriate concentration, and incubation time was set for 30 min at 32°C. In determining the Km value for geranyl diphosphate, Mn2+ concentration was set at a saturated level, and geranyl diphosphate concentration was changed from 0.5 to 108 µM with 10 different data points. The Km value for Mn2+ was measured at saturated geranyl diphosphate levels, and Mn2+ concentration was changed from 4 to 1,000 µM with nine data points. Lineweaver-Burk plots were made to obtain the Km value.
Mass spectrometric analysis of the purified GES was carried out in the Proteomics Core Facility (Southwest Environmental Health Sciences Center and Arizona Cancer Center, University of Arizona, Tucson). Proteins were first separated by SDS-PAGE, as described above, stained lightly with Coomassie Brilliant Blue R250, excised from the gel, and digested with trypsin (Shevchenko et al., 1996
A basil cv Sweet Dani peltate gland EST database containing 3,200 unique sequences was developed at the Arizona Genomics Institute and the Arizona Genomics Computational Laboratory (University of Arizona, Tucson), using a cDNA library constructed from gland mRNAs as previously described (Gang et al., 2001
Alignment of multiple protein sequences was performed using the ClustalX program (Thompson et al., 1997
RNA isolated from glands and leaves and RNA gel-blot analysis were done as previously described (Gang et al., 2001
We thank Drs. Rod Wing (Arizona Genomics Institute, University of Arizona, Tucson) and Cari Soderlund (Arizona Genomics Computational Laboratory, University of Arizona, Tucson) for their assistance in production and maintenance of the basil EST databases, Dr. George Tsaprailis (Proteomics Core Facility of the Southwest Environmental Health Sciences Center, University of Arizona, Tucson) for assistance with peptide sequencing and analysis, Dr. Jihong Wang (University of Michigan, Ann Arbor) for help with protein purification, and Ms. Yue Yang (University of Michigan, Ann Arbor) for help with the RNA gel blots. Received September 4, 2003; returned for revision September 30, 2003; accepted October 8, 2003.
1 This work was supported by the U.S. Department of Agriculture-Binational Agricultural Research and Development Fund (grant no. IS-3332-02C), by the National Research Initiative Competitive Grants Program-U.S. Department of Agriculture (grant no. 2001-35318-10006), and by the National Science Foundation (grant no. 0210170).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.032946. * Corresponding author; e-mail lelx{at}umich.edu; fax 734-647-0884.
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ABSTRACT
RESULTS
-farnesene; 8, nerol; and 9, geraniol. Unlabeled peaks in C are not terpenes and are found in the protein crude extract regardless of whether geranyl diphosphate is included in the assay.
-farnesene synthase, AF024615; N. tobacum 5-epi-aristolochene synthase, L04680; Arabidopsis myrcene/ocimene synthase, At2g24210; C. limon 
-terpenene synthase, AF514286; spearmint 4S-limonene synthase, L13459; sage 1,8-cineole synthase, AF051899; Populus alba x Populus tremula isoprene synthase, AJ294819; Clarkia breweri linalool synthase, U56314; snapdragon (Antirrhinum majus) myrcene synthase, AY195608; and snapdragon ocimene synthase, AY195607.
