© 2008 Science From Israel / LPPLtd., Jerusalem

IsraelJournalofPlantSciences Vol.56 2008 pp.233–24410.1560/IJPS.56.3.233

*Author to whom correspondence should be addressed.E-mail: twefraim@agri.gov.il

Biosynthesis of linalyl acetate and other terpenes in lemon mint (Mentha aquatica var. citrata, Lamiaceae) glandular trichomes

Alon ZAks,a,b RAchel DAviDovich-RikAnAti,a einAt BAR,a Moshe inBAR,b AnD efRAiM lewinsohna,*aDepartmentofAromatic,MedicinalandSpiceCrops,AgriculturalResearchOrganization,

NeweYa’arResearchCenter,P.O.Box1021,RamatYishay30095,IsraelbDepartmentofEvolutionary&EnvironmentalBiology,UniversityofHaifa,Haifa31905,Israel

(Received21September2008;acceptedinrevisedform22December2008)

ABSTRACT

The potential to produce and accumulate mono- and sesquiterpenes in lemon mint(Menthaaquaticavar.citrata) glandular trichomes was evaluated. Volatiles were ex-tracted using methyl-tert-butyl ether or solid phase micro extraction and determined by gas chromatography-mass spectrometry. The main components of the essential oil of lemon mint are the monoterpene alcohol linalool and its ester linalyl acetate. Sesquiterpenes, such as elemol, (E)-caryophyllene, and germacrene D, as well as the monoterpenes 1,8-cineole, β-myrcene, and β-(E)-ocimene, are also prominent. Most of the essential oil is localized in the glands, as leaves devoid of trichomes had very low essential oil levels. The quantities of all these components per leaf increased during leaf development until reaching the third whorl, and then the levels remained steady. Conversely, the essential oil content per gram fresh weight decreased with leaf development, but the decrease was not statistically significant at p = 0.05. Desalted cell-free extracts derived from isolated glandular trichomes were able to convert geranyl diphosphate into linalool. Lower amounts of geranyl diphosphate were also converted to other monoterpenes such as myrcene, α-pinene, β-pinene, limonene, β-(E)-ocimene, and nerol. Furthermore, the extracts were able to convert farnesyl diphosphate into the sesquiterpene (E)-caryophyllene and lesser amounts of other sesquiterpenes such as δ-cadinene, α-humulene, β-(E)-farnesene, and α-gurjunene. These cell-free extracts also efficiently catalyzed the formation of linalyl acetate from linalool and acetyl-coenzyme A. Our findings indicate that glan-dular trichomes of lemon mint, similar to other members of the Lamiaceae, contain unique enzymatic activities capable of the synthesis of mono- and sesquiterpene components of its essential oil.

Keywords: AATs, Menthaaquaticavar.citrata, lemon mint, linalool, linalyl acetate, monoterpenes, sesquiterpenes

INTRODUCTION

Lemon mint, Menthaaquaticavar.citrata (Lamiaceae), contains an essential oil rich in the monoterpene linalool and its ester linalyl acetate. Lemon mint also contains 1,8-cineole, β-myrcene, β-ocimene, (E)-caryophyllene, germacrene D, and elemol (Fig. 1). This type of mint is commercially known as lemon mint, bergamot mint, or lavender mint (Gildemeister and Hoffman, 1961). The essential oil constituents of such mints have marked

economic importance. Linalool has been described as possessing a refreshing, light, floral, and fragrant odor, and linalyl acetate is a compound with pleasant, sweet, floral, and fruity characteristics. Most of the linalool and linalyl acetate produced worldwide are consumed as fragrances and flavoring agents. In addition, linalool has insecticidal use in formulated sprays and dips for pet

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ectoparasite control (Burdock, 1995). Other dominant compounds in lemon mint essential oil are 1,8-cineole, also known as eucalyptol (Geremia, 1955), myrcene, one of the most important chemicals used in the perfumery industry, (Budavari, 1989) and ocimene, a monoterpene olefin that is characterized by a spicy aroma (Jirovetz et al., 2005). Sesquiterpenes, such as (E) caryophyl-lene, germacrene D, and elemol, are also constituents of lemon mint essential oil. Members of the Lamiaceae, such as peppermint (Mentha´ piperita), spearmint (M.spicata), and basil (Ocimumbasilicum), biosynthesize and accumulate mono- and sesquiterpenes in special-ized glandular trichomes (McConkey et al., 2000; Gang et al., 2001), and both mono- and sesquiterpenes accu-mulate in the subcuticular space of the glands (Gershen-zon et al., 2000). Monoterpene biosynthesis originates in the plastids (leucoplasts) (Turner et al., 1999), while sesquiterpenes are generally synthesized in the cytosol (Newman and Chappell. 1999). Three types of glandular trichomes occur on peppermint leaves: non-glandular, multicellular simple hairs; small, capitate glandular trichomes, with a single secretory head cell; and peltate glandular trichomes, with an eight-celled apical cluster of secretory cells, subtended by a stalk and a basal cell, the latter embedded between the ordinary epidermal cells (Maffei et al., 1989, 2007; Werker, 1993).

The enzymes that participate in mono- and sesqui-terpene biosynthesis, and their derivatives, are usually confined to the secretory cells of the trichomes (Ger-shenzon et al., 1989). These enzymes include mono-terpene synthases, which utilize geranyl diphosphate (GDP) as a C10 precursor to produce a broad range of monoterpene compounds and the related sesquiterpene synthases, which produce a variety of sesquiterpenes from the C15 precursor farnesyl diphosphate (FDP) (Bohlmann et al., 1998).

A third group of enzymes that are involved in the for-mation of volatile esters are alcohol acetyl transferases (AATs), which acetylate different alcohols utilizing ace-tyl-CoA. AATs have been detected in flowers and fruits of many plants (Harada et al., 1985; Perez et al., 1993; Ueda et al., 1997; Dudareva et al., 1998; Shalit et al., 2001, 2003; Holland et al., 2005). Although these en-zymes are capable of condensing different alcohols and acetyl-CoAs, resulting in the synthesis of a wide range of esters, they are inefficient when the tertiary linalool is offered as a substrate (Dudareva et al., 1998; Shalit et al., 2003; Beekwilder et al., 2004). Since lemon mint contains high levels of linalyl acetate, we examined whether it possessed an AAT activity able to accept lin-alool and acetyl CoA as substrates to efficiently generate linalyl acetate, and whether this activity is localized in glandular trichomes. We also tested the mono- and ses-quiterpene synthase activity in glandular trichomes, and report on the accumulation patterns of linalool, linalyl acetate, and other mono- and sesquiterpenes along the whorls of lemon mint.

MATERIALS AND METHODS

The source of tissue or plants

PlantmaterialLemon mint (Menthaaquaticavar.citrata) and pep-

permint (Mentha piperita) were grown in an open field at the Newe Ya’ar Research Center (Northern Israel) under commercial conditions with drip irrigation and fertilization.

GlandulartrichomeisolationPeltate and capitate glandular trichomes were iso-

lated from young leaves using a modification (Gang et al., 2001) of the methods developed by Gershenzon et al. (1992).

Volatile analysis

Solidphasemicroextraction(SPME)Fresh leaves were inserted into tightly closed 20-

ml vials. SPME sampling was conducted immedi-ately, utilizing a 65-µm fused silica fiber coated with polydimethylsiloxane/divinylbenzene (PDMS/DVB) (Supelco). After 40 min at 65 °C, the SPME syringe was introduced intothe injector port of the gas chromatogra-phy-mass spectrometry (GC-MS) apparatus for further analysis (see below) (Iijima et al., 2004).

SolventextractionThe two fresh leaves in a whorl were harvested and

soaked in 5 ml methyl tert-butyl ether (MTBE) con-taining 5 μg isobutylbenzene as internal standard, and

Fig. 1. Chemical structures of the main mono- and sesquiter-penes in lemon mint.

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extracted for 3 h at room temperature in 5-ml glass vials tightly sealed with rubber septa caps. The MTBE upper layer, which contained the volatile essential oil compo-nents, was dried by passing through a Pasteur pipette containing anhydrous Na2SO4, placed into another vial, and concentrated to 200 µl under gentle N2 gas flow for GC-MS analysis (Iijima et al., 2004).

GC-MSanalysisVolatile compounds were analyzed on aGC-MS ap-

paratus equipped with an Rtx-5 SIL MS (30 m length, 0.25 mm i.d., 0.25 μm film thicknesses, stationary phase 95% dimethyl- 5% diphenyl polysiloxane) fused-silica capillary column. Helium (1 ml/min) was used asa carrier gas. The injector temperature was 250 °C, set forsplit-less injection. The oven was set to 50 °C for 1 min, andthen the temperature was increased to 260 °C at a rate of 5°C/min. Thermal desorption was allowed for 1.5 min. The detector temperature was 280 °C. The mass range wasrecorded from m/z41 to 206, with electron energy of 70 eV.Identification of the main components was done by comparisonof mass spectra and retention time data with those of authentic samples, supplemented with a Wiley GC-MS library (Davidovich-Rikanati et al., 2007).

Measurement of enzymatic activities

EnzymeextractionIsolated peltate glands (~500 μl packed volume) were

placed in a chilled mortar and ground with a pestle inthe presence of sand and 1% (w/w) polyvinylpolypyrrol-idone(PVPP) until a uniform powder was obtained. Ice-cold extraction buffer [50 mM bis Tris, pH 6.9, 10% (v/v) glycerol, 10 mM dithiothreitol, 5 mM Na2S2O5] was added (5 ml to 1 g fresh weight of tissue), and the sus-pension was further extracted for an additional 30 s. The slurry was centrifuged at 20,000g for 10 min at 4 °C. The supernatant (3 ml) was next passed through a P-6 column (1 ´ 10 cm) (Bio-Rad, Bio-Gel desalting column 10-80 μ, Bio-Rad, Germany) to remove interfering low-molecular-weight compounds. We used buffer A [50 mM bis Tris, pH 6.9, 10% (v/v) glycerol, 10 mM dithiothrei-tol, 5 mM Na2S2O5] for column equilibration and protein elution. The protein concentration in 1 ml fractions was determined by the Bradford protein assay (Bradford, 1976) utilizing bovine serum albumin as a standard. Frac-tions were kept at –20 °C for further analysis.

Enzyme assays

MonoterpeneandsesquiterpenesynthasesEnzymatic assays were performed by mixing 10 μM

prenyl diphosphate (geranyl diphosphate (GDP) for monoterpene synthase or farnesyl diphosphate (FDP) for sesquiterpene synthase), 10 mM MgCl2, 10 μM

MnCl2, and 10 μl glandular trichome desalted crude extract added into a total volume of 200 μl of assay buffer A, and incubated for 30 min at 30 °C. Control ex-periments included heat-inactivated enzyme, and assays devoid of the metal cofactors.

The sesquiterpenes and monoterpenes generated in vitrowere analyzed with an Rtx-5SIL (30 m 3 0.25 mm) column. Helium (1 ml/min) was used as a carrier gas. The injector and detector temperatures were 250 °C and 280 °C, respectively. The conditions used were as fol-lows: initial temperature was 50 °C min 21 followed to 130 °C at a rate of 20 °C min 21, to 170 °C at a rate of 3 °C, and, finally, to 260 °C at a rate of 50 °C min–1. One microliter was injected to the GC-MS for the identifica-tion of volatiles.

AlcoholacetyltransferaseEnzymatic assays were performed by mixing 0.1

mM of the appropriate alcohol, 100 μM acetyl CoA, and 20 µl of glandular trichomes desalted crude extract in a total volume of 300 μl of assay buffer A, and incubated for 30 min at 30 °C. The generated compounds were isolated by solid phase micro extraction (SPME) as previously described (Iijima et al., 2004).

Small-scale assays were performed by mixing 20 µl of crude desalted extract, 10 mMalcohol substrate, and 0.88 µM (0.0045 µCi [14C] acetyl CoA, Amersham)into a final volume of 100 µL of assay buffer A. The assays were incubated for 30 min at 30°C. One milliliter of hexane was added to each tube, which was then vigor-ously vortexed and spun for 1 min at 5,000gto separate phases. A volume of 800 µl of the upper hexane layer, containing the newly formed radiolabeled alcohol ac-etate esters, was transferred into 5-ml scintillation tubes containing 3 ml of scintillation liquid (Ultima-gold, PerkinElmer Precisely). The radioactivity was quanti-fied using a liquid scintillation counter (model Tri-Carb 2800TR, PerkinElmer Precisely). Enzyme activity in picokatals was calculated based on the specific activity of the substrate and using appropriate correction factors for the counting efficiency of the scintillation machine (Shalit et al., 2001).

DataanalysesThe data were analyzed using a one-way ANOVA test.

Normality and homogeneity were tested. Statistical analy-ses were performed with ‘SPSS’ software for Windows.

RESULTS

The main mono- and sesquiterpene compounds along the whorls of lemon mint

The main components of lemon mint leaf volatiles are

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the monoterpene alcohol linalool and its ester linalyl acetate (Fig. 1). Sesquiterpenes, such as elemol, (E) caryophyllene, and germacrene D, as well as the mono-terpenes 1,8 cineole, myrcene, and β-(E)-ocimene, are also prominent (Fig. 2). These results are in accordance with relevant literature indicating that linalool and linalyl acetate are the main volatile components of lemon mint (Gildemeister and Hoffman, 1961). The quantities of all these components on a leaf basis in-creased 3–6-fold until reaching whorl no. 3, and then the levels remained steady (Fig. 2A). The essential oil

content showed a seemingly opposite pattern (Fig. 2b): the highest levels were found in whorl no. 1 (top of the plant) and these levels decrease, reaching a steady level at whorl no. 3. Nevertheless, these differences were not statistically significant at p = 0.05.

The localization of mono- and sesquiterpenes to peltate glandular trichomes along the whorls

In several Mentha species, volatiles are accumulated in glandular trichomes (Gershenzon et al., 2000; McCon-key et al., 2000; Gang et al., 2001). To ascertain if this

Fig. 2. The main mono- and sesquiterpene compounds along the whorls of lemon mint. (A) Content per leaf. (B) Concentration per gram fresh weight. The volatile analysis was performed by GC-MS and the compounds identified according to their retention times and mass spectra. Values given are means ± SE. Bars followed by different letters are statistically significant, p < 0.05.

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was the case in lemon mint, we adapted previous meth-odologies for glandular trichome isolation. The protocol yielded about 500 μl-packed volume trichomes/10 g fresh weight leaves. Under the microscope, the isolated glandular trichomes display the eight typical secretory cells, most of them without the cuticular layer (Fig. 3).

The levels and composition of volatiles in intact leaves, leaves devoid of glandular trichomes, and isolated glan-dular trichomes were compared. The results are shown in Fig. 4. As expected, the isolated glandular trichomes contained all the essential oil compounds found in lemon mint leaves (Fig. 4). Leaves devoid of glandular

Fig. 3. The glandular trichomes of lemon mint. (A) Lemon mint plants. (B) Glandular trichomes on the adaxial surface of a lemon mint leaf. (C) Isolated glandular trichomes.

Fig. 4. Mono- and sesquiterpenes of lemon mint. Intact young leaves, before the glandular trichome isolation procedure (black), young leaves after the glandular trichome isolation procedure (gray) and isolated glandular trichomes (white). (A) Major com-pounds. (B) Minor compounds. The volatile analysis was performed by GC-MS and the compounds identified according to their retention times and mass spectra. The compounds are shown in order of their retention time. Values given are means ± SE. Bars followed by different letters are statistically significant, p < 0.05.

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trichomes contained much lower levels of volatiles than intact leaves (0.5 to 0.3 relative to intact leaves). Glan-dular trichomes contained lower amounts of volatiles compared to intact leaves (generally 0.25 relative to intact leaves); (Fig. 4). A decreased density of trichomes was found in lemon mint leaves during development, being the density of peltate glandular trichomes in the adaxial surface of young leaves (whorl no. 1) 56 ± 11 per 2 cm2, while their density in whorl 2 was 30 ± 6, whorl 3, 24 ± 2, whorl 4, 20 ± 4, and the oldest leaves (whorl no. 5), the density of glandular trichomes per 2 cm2 is 11 ± 2.

Monoterpene synthase activity in lemon mint glandular trichomes

The most prominent volatile generated from GDP under our conditions is linalool (71%) (Fig. 5). Lesser amounts of nerol (7%), myrcene (5%), 1,8 cineole (6%), limonene (3%), and geraniol (2%) were also detected in the enzymatic assays. Control experiments utiliz-ing heat-inactivated enzyme, and assays devoid of the metal cofactors lacked these volatiles, (although they contained minute levels of residual linalool) indicating that the conversion to monoterpenes was enzymatic and required metal cofactors. Still, the proportions of the compounds generated in vitro and those present in the plant are not always identical.

Formation of linalyl acetate by lemon mint linalool synthase and alcohol acetyl transferase activities

The two main components of lemon mint essential oil are linalool and linalyl acetate (Figs. 1, 2, and 4). The formation of linalool in plants is catalyzed by the en-zyme linalool synthase (LIS) (Fig. 6). LIS enzymatic activity has not been shown before in lemon mint, al-though a gene coding for a protein possessing this activ-ity has been reported (Crowell et al., 2002). We assumed that the conversion of linalool to linalyl acetate occurs through an alcohol acetyl transferase (AAT) activ-ity, which we named linalool acetyl transferase (LAT). Still, enzymes that efficiently catalyze the acetylation of linalool to linalyl acetate have not been described. In order to ascertain whether lemon mint possessed

Fig. 5. Monoterpene synthase activity in cell-free extracts prepared from lemon mint glandular trichomes. (A) Complete reac-tion. (B) Control reaction containing heat-inactivated enzyme. Protein extracts were prepared from isolated glandular trichomes and incubated with GDP under the conditions described in Materials and Methods. The products identified are (1) α-thujene, (2) α-pinene, (3) sabinene, (4) β-pinene, (5) myrcene, (6) α-phellandrene, (7) α-terpinene, (8) limonene, (9) 1,8 cineole, (10) β(Z)-ocimene, (11) β-(E)-ocimene, (12) γ-terpinene, (13) terpinolene, (14) linalool, (15) α-terpineol, (16) nerol, (17) geraniol. The volatile analysis was performed by GC-MS and the compounds identified according to the retention times and mass spectra.

Fig. 6. Proposed biosynthetic pathway to linalyl acetate in lemon mint. LIS (linalool synthase), LAT (linalool acetyl transferase).

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such enzymatic activity, cell-free extracts derived from glandular trichomes were incubated with linalool and the acyl donor acetyl coenzyme A. A compound with a retention time identical to authentic linalyl acetate was generated in assays containing all substrates (Fig. 7A). This compound also had a mass spectrum identical to linalyl acetate (Fig. 7 C,D), was absent in control assays devoid of protein (Fig. 7B), and in control assays in

which no acetyl CoA or linalool was added (not shown). An apparent Km value of 3 mM for linalool and optimal pH of 6.9 were estimated for the lemon mint enzyme in our assay conditions.

Substrate specificity of the acylation activity

Several primary and tertiary alcohols were tested as sub-strates in radioactive alcohol acetyl transferase assays.

Fig. 7. Linalyl acetate formation in cell-free extracts from lemon mint glandular trichomes. Protein preparations were incubated with linalool and acetyl coenzyme-A under the conditions described in Materials and Methods. Identification of the linalyl ac-etate product was done by GC-MS according to its retention time and mass spectrum. (A) Complete assay. (B) Assay devoid of enzyme. (C) Mass spectrum of biosynthetic linalyl acetate. (D) Mass spectrum of authentic linalyl acetate.

Fig. 8. Alcohol acetyl transferase activity in lemon mint (black bars) and peppermint (white bars) cell-free extracts. Glandular trichomes cell-free extracts were incubated with various alcohols and radiolabeled acetyl coenzyme A. The radioactivity in the resulting organic phase was measured with a scintillation counter. The numbers 1, 3 beside the molecule refer to whether a primary or a tertiary alcohol was supplied. All substrates were tested at a concentration of 10 mM. Black bars indicate extracts from lemon mint glandular trichomes, and white bars indicate extract from peppermint glandular trichomes.

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In all reactions, the corresponding acetates were formed in vitro, but at different rates (Fig. 8). High levels of geranyl acetate and phenylethyl acetate were detected. Linalyl acetate and terpinyl acetate were also formed, but at slower rates. In contrast, similar cell-free extracts prepared from glandular trichomes of peppermint (a plant devoid of linalyl acetate) were able to acetylate a variety of alcohols but not the tertiary alcohol linalool (Fig. 8). Some of the compounds that are produced in the assays are accumulated in lemon mint leaves, but in very low quantities (e.g., geranyl acetate). Conversely, terpineol but not terpinyl acetate is accumulated in leaves; still, terpineol was readily esterified in vitro. Interestingly, additional alcohols that are absent in the plant, such as phenylethyl alcohol, were readily esteri-fied to their corresponding acetates by cell-free extracts of lemon mint glandular trichomes.

The in vitro activity of sesquiterpenes synthase

Sesquiterpene synthase enzymatic assays revealed the formation of high levels of (E) caryophyllene (78% of the total volatiles), with lesser amounts of α-humulene (7%), α-gurjunene (3.5%), β-(E)-farnesene (2.5%), and δ-cadinene (1.5%) from farnesyl diphosphate (Fig. 9). All of the compounds generated in vitro in cell-free extracts were also found in glandular trichomes and

leaves (Figs. 2,4), except for α-copaene, β-humulene, aromadendrene, (allo) aromadendrene, γ-muurolene, γ-cadinene, δ-cadinene, and α-cadinene, but those vola-tiles are accumulated in the plant in very low quantities. Thus, we have demonstrated that lemon mint glandular trichomes possess enzymatic activities able to produce mono- and sesquiterpenes, as well as able to acetylate a variety of alcohols including the tertiary monoterpene alcohol linalool to the corresponding acetate.

DISCUSSION

Linalool and linalyl acetate are the main compounds present in the essential oil of lemon mint (M.aquaticavar.citrata) (Fig. 1). Although several Mentha species accumulate acyclic monoterpenes such as geraniol, ne-rol, myrcene, and ocimenes, most mint species produce cyclic monoterpenes such as menthol, menthone, and carvone (Murray and Lincoln, 1970; Malingre, 1971). Linalool and linalyl acetate are also present in the es-sential oils of lavender (Lavandula officinalis L.) and salvia (Salviasclarea L.) (Bauer et al., 1990).

The relationship between glandular trichomes and the volatiles in lemon mint

Lemon mint peltate glandular trichomes are formed by

Fig. 9. Sesquiterpene synthase activity in cell-free extracts prepared from lemon mint glandular trichomes. Protein extracts were incubated with FDP under the conditions described in Materials and Methods. (A) Complete reaction. (B) Control reac-tion containing heated-inactivated enzyme. The products identified are (1) α-copaene, (2) β-elemene, (3) (Z)-caryophyllene, (4) α-gurjunene, (5) (E)-caryophyllene, (6) β-humulene, (7) aromadendrene, (8) β (E) –farnesene, (9) α-humulene, (10) (allo)-aro-madendrene, (11) γ-muurolene, (12) germacrene D, (13) γ-cadinene, (14) δ-cadinene, (15) α-cadinene, (x1 and x2) unidentified artifact compounds of the column. TIC: Total ion chromatogram. The volatile analysis was done by GC-MS and the compounds identified according to their retention times and mass spectra.

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a stalk cell and eight secretory cells encompassed by an elevated cuticle. The essential oil accumulates in the subcuticular space (Fig. 3A–C). A similar specialized anatomical structure occurs in peppermint (Maffei et al., 1989; Werker, 1993) (Fig. 3D). The levels of the volatile compounds increase with development from whorl one to three and then remain stable from whorl three to five (Fig. 2A), a pattern coinciding with the leaf weight and size (data not shown). There is a marked relationship be-tween leaf size and the density of glandular trichomes: the larger the leaves, the higher the glandular trichomes’ density, and therefore higher levels of essential oil are present. These results match previous studies in pep-permint, indicating that the total number of glandular trichomes per leaf increases during leaf growth. Glands increase in numbers from about 100 for small, 2-mm-long primordial leaf to about 7,500 for 25-mm-long pep-permint leaves (Maffei et al., 1986, 1989). It seems that a similar developmental pattern is displayed in lemon mint. New peltate glands continue to form until leaf expansion ceases and regions of active gland initiation are unevenly distributed (Maffei et al., 1989; Shanker et al., 1999). The density of the glandular trichomes in lemon mint young leaves is higher than in older leaves. These observations also hold in peppermint, and the accumulation patterns of the essential oil components per gram leaf (Fig. 2B) can be explained by taking into account trichome density. Since leaf expansion in peppermint results from vacuolar expansion of cells in maturing tissues where gland initiation has ceased, av-erage gland surface density decreases from a maximum of about 100 glands/mm2 for 2–10-mm-long leaves to about 6.5 glands/mm2 for fully expanded, mature leaves (Turner et al., 2000). Most of the essential oil in lemon mint is localized to the glandular trichomes (Fig. 4), but, in contrast to our expectations, isolated glandular trichomes contained lower levels of volatiles than intact leaves (Fig. 4). It could be that during the trichome iso-lation procedure some of the glandular trichomes were disrupted and lost their essential oil content. Still, the same composition of both major and minor compounds that was found in isolated trichomes was also found in leaves, indicating that glandular trichome accumulation takes place in these specialized structures.

Monoterpene synthase activity

Cell-free extracts of lemon mint glandular trichomes were able to convert GDP into all of the volatiles pres-ent in lemon mint leaves (Fig. 5). Most of the GDP was converted into linalool and, in lesser amounts, other monoterpenes, such as myrcene, limonene, 1,8-cineole, nerol, and geraniol (Fig. 5). This conversion indicates that the synthesis of monoterpenes occurs in the glan-

dular trichomes of lemon mint, reinforcing previous ob-servations on a situation prevailing in the genus Mentha and in other Lamiaceae (Gershenzon et al., 1992; Mc-Caskill et al., 1992; McCaskill and Croteau, 1995). This observation has allowed detailed characterization of biosynthetic enzymes involved in terpene formation in mint and other Lamiaceae (Alonso et al., 1992; Colby et al., 1993; Ponnamperuma and Croteau, 1996; Lupien et al., 1999). A comparison of the monoterpenes produced in vitro (Fig. 5) from GDP with the monoterpenes ac-cumulated in vivo (Figs. 2 and 4), shows that the mono-terpenes produced in vitro account for all monoterpenes accumulated in the plant, although roughly 50% of the linalool present in the plant is further acetylated. Al-though lower levels of other monoterpenes such as 1,8-cineole, limonene, and myrcene are produced in vitro at similar rates under our assay conditions (Fig. 5), the plant apparently accumulates much higher levels of 1,8-cineole as compared to myrcene or limonene. Moderate levels of nerol were produced in vitro from GDP. Nerol is probably converted in vivo to neryl acetate by alcohol acetyl transferase, but in low levels. Geraniol is also produced in vitro, albeit in low levels. Nevertheless, there were no traces for free geraniol in plant leaves, but low levels of geranyl acetate are accumulated in vivo, probably due to AAT activity (see below).

Linalool synthase in glandular trichomes of lemon mint

We show here that isolated glandular trichomes pos-sess high levels of linalool synthase activity. Although this activity has never before been reported to occur in lemon mint, Crowell et al. (2002) isolated a gene from lemon mint (Menthacitrata Ehrh.), coding for a protein possessing linalool synthase activity when the gene was overexpressed in E.coli.This gene product utilizes GDP to generate linalool in the presence of MgCl2 and MnCl2. Other genes that code for proteins that possess LIS activity have been isolated from several plants in-cluding Clarkiabreweri,Arabidopsisthaliana,Fragar-ia x ananassa, Cereusperuvianus, andArtemisiaannua(Pichersky et al., 1995; Jia et al., 1999, Chen et al., 2003; Sitrit et al., 2004; Aharoni et al., 2004). In Clarkia flow-ers, linalool is a major volatile emitted, and LIS activity was correlated with linalool emission (Pichersky et al., 1995). Nevertheless, in Artemisiaannua no linalool was found in the plant, but linalool was produced in vitro by the E.coli overexpressed gene product when GDP was provided (Jia et al., 1999).

Linalyl acetate biosynthesis

We hypothesized that the conversion of linalool to linalyl acetate in lemon mint is catalyzed by an alcohol

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acetyl transferase (AAT) based on studies dealing with the formation of volatile esters in other plants. (Harada et al., 1985). AAT enzymes are capable of condens-ing different alcohols and acyl CoAs, resulting in the synthesis of a wide range of esters (El-Sharkawy et al., 2005). Most of the AATs that have been characterized produced esters from primary alcohols and had either no or very low activity with tertiary alcohols (Dudareva et al., 1998; Shalit et al., 2003; Beekwilder et al., 2004). Our results show that the AAT activity present in lemon mint is able to accept the tertiary alcohol linalool to gen-erate its respective ester linalyl acetate (Fig. 7).

Peppermint is morphologically very similar to lemon mint but does not contain linalyl acetate and its essential oil is rich in menthol (Rohloff, 1999). We compared the ability of lemon mint and peppermint cell-free extracts to acetylate primary and tertiary alcohols. Both lemon mint and peppermint glandular trichome cell-free ex-tracts converted most of the alcohols offered into their respective acetates but only lemon mint extracts have the capacity to convert linalool into linalyl acetate (Figs. 7 and 8). Thus, lemon mint apparently possesses an AAT activity that is different from that displayed by peppermint and from that described in many other plants that are inefficient towards acetylation of the substrate linalool. It is well known that plant alcohol acetyl transferases have the ability to utilize a broad spectrum of alcohols and convert them to the respective acetates, even though some substrates are not present in the plants. The tertiary alcohol α-terpineol is also converted to its respective acetate in vitro (Fig. 8) only by lemon mint extracts, but not by peppermint extracts. Interestingly, terpinyl acetate is not present at detect-able levels in either lemon mint or peppermint. Other substrates that are accepted by both the lemon mint and peppermint enzymes are phenylethyl alcohol, methanol, and geraniol (Fig. 8). Interestingly, their respective es-ters are absent, or present in minute quantities, in both of the plants (Figs. 2 and 4). In conclusion, it seems that lemon mint possesses an AAT activity able to accept several primary and tertiary alcohols, including linalool and α-terpineol.

The primary monoterpene geraniol is formed at low levels by lemon mint glandular trichome cell-free extracts that were incubated in the presence of GDP (Fig. 5). The AAT activity from lemon mint has a marked ability to utilize geraniol as a substrate and can readily generate geranyl acetate (Fig. 8). Indeed, no free geraniol is found in the essential oil of this plant, but minute levels of geranyl acetate are present, indicating that this conversion readily occurs in vivo.

AAT activity that utilizes linalool as a substrate has been recently described in several Lamiaceae be-

longing to the genera Origanum, Mentha, and Salvia (Larkov et al., 2008). The enzymes from the different plants displayed differential enantioselectivity towards the linalool substrate. Preliminary information on the enzyme from lemon mint indicates that the lemon mint activity is much more efficient towards R-linalool, (the optical isomer accumulated in the plant), but still is able to acetylate the S-isomer at lower rates (Larkov et al., 2008). The gene product of the LIS gene previously iso-lated from lemon mint exclusively produces R-linalool (Crowell et al., 2002). Thus the enantiomeric ratio of the linalyl acetate accumulated in the plant is probably dictated by the enzyme generating the linalool precursor (Larkov et al., 2008).

Sesquiterpene synthase activity in lemon mint

Cell-free extracts of lemon mint glandular trichomes were able to convert FDP into several sesquiterpene volatiles present in the plant (Fig. 9A). Control ex-periments utilizing heat-inactivated enzyme, or assays devoid of the metal cofactors lacked these volatiles, indicating that the conversion was enzymatic (Fig. 9B). This evidence indicates that the synthesis of sesquiter-penes occurs in the glandular trichome of lemon mint as documented for other Lamiaceae. In any case, the pro-portions of the compounds generated in vitro and those present in the plant are not always identical.

A comparison of the sesquiterpenes produced in vitro from FDP (Fig. 9) to the sesquiterpenes accumulated in vivo (Figs. 2 and 4) shows that the plant produces and accumulates mainly elemol. Accordingly, elemol was accumulated at detectable levelsin vitro, still (E) caryo-phyllene was accumulated in the highest levels in vitro. Germacrene D accumulated in low quantities in vitro, but its levels are higher in vivo. Very low quantities of other sesquiterpenes were also generated in vitro but not accumulated in vivo. The distinction between the ses-quiterpene levels in vivo and in vitro could be explained by either deficiency of substrate and cofactors, or compartmentalization between the substrate and the en-zymes, as well as different rates of volatilization or loss of the products. We have demonstrated that cell-free extracts derived from glandular trichomes from lemon mint have the ability to synthesis most of the mono- and sesquiterpenes formed in the essential oil and provide the economic importance of this plant. Lemon mint glandular trichomes are a powerful tool for the study of mono- and sesquiterpenes present in this plant.

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