Plant Science 160 (2000) 27 – 35 www.elsevier.com/locate/plantsci
Biosynthesis of estragole and methyl-eugenol in sweet basil (Ocimum basilicum L). Developmental and chemotypic association of allylphenol O-methyltransferase activities Efraim Lewinsohn a,*, Iris Ziv-Raz a,b, Nativ Dudai a, Yaacov Tadmor a, Elena Lastochkin a, Olga Larkov a, David Chaimovitsh a, Uzi Ravid a, Eli Putievsky a, Eran Pichersky c, Yuval Shoham b a b
Newe Ya’ar Research Center, Agricultural Research Organization, P.O. Box 1021, Ramat Yishay 30095, Israel Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, Haifa 32000, Israel c Department of Biology, Uni6ersity of Michigan, Ann Arbor, MI 48109 -1048, USA Received 19 May 2000; received in revised form 28 July 2000; accepted 31 July 2000
Abstract Sweet basil (Ocimum basilicum L., Lamiaceae) is a common herb, used for culinary and medicinal purposes. The essential oils of different sweet basil chemotypes contain various proportions of the allyl phenol derivatives estragole (methyl chavicol), eugenol, and methyl eugenol, as well as the monoterpene alcohol linalool. To monitor the developmental regulation of estragole biosynthesis in sweet basil, an enzymatic assay for S-adenosyl-L-methionine (SAM):chavicol O-methyltransferase activity was developed. Young leaves display high levels of chavicol O-methyltransferase activity, but the activity was negligible in older leaves, indicating that the O-methylation of chavicol primarily occurs early during leaf development. The O-methyltransferase activities detected in different sweet basil genotypes differed in their substrate specificities towards the methyl acceptor substrate. In the high-estragole-containing chemotype R3, the O-methyltransferase activity was highly specific for chavicol, while eugenol was virtually not O-methylated. In contrast, chemotype 147/97, that contains equal levels of estragole and methyl eugenol, displayed O-methyltransferase activities that accepted both chavicol and eugenol as substrates, generating estragole and methyl eugenol, respectively. Chemotype SW that contains high levels of eugenol, but lacks both estragole and methyl eugenol, had apparently no allylphenol dependent O-methyltransferase activities. These results indicate the presence of at least two types of allylphenol-specific O-methyltransferase activities in sweet basil chemotypes, one highly specific for chavicol; and a different one that can accept eugenol as a substrate. The relative availability and substrate specificities of these O-methyltransferase activities biochemically rationalizes the variation in the composition of the essential oils of these chemotypes. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ocimum basilicum L.; Lamiaceae; Sweet basil; Essential oils; Estragole; Methyl eugenol; O-methyltransferase
1. Introduction The genus Ocimum includes about a dozen species and subspecies, native to the tropical and sub-tropical regions of the world. Sweet basil (Ocimum basilicum L., Lamiaceae) is an important
* Corresponding author. Tel.: +972-4-9539552; fax: + 972-49836936. E-mail address:
[email protected] (E. Lewinsohn).
medicinal plant and culinary herb [1]. Sweet basil is widely cultivated for the production of essential oils, and is also marketed as an herb, either fresh, dried, or frozen [1]. The essential oil of sweet basil possesses antifungal, insect-repelling and toxic activities [2–4]. Despite the wide use and the importance of sweet basil and its essential oils, little is known about the biosynthesis and developmental regulation of the compounds responsible for the flavor quality of the fresh and dried herbs and that constitute its essential oil.
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Many members of the Lamiaceae such as Mentha, Sal6ia, Origanum, and Thyme spp., are also cultivated to be used as herbs and as a source of essential oils. The essential oils of these species and of many other species of the Lamiaceae are mostly composed of mono- and sesquiterpenes [5]. Similarly, many species of the genus Ocimum, contain essential oils based primarily on monoterpene derivatives such as camphor, limonene, thymol, citral, geraniol and linalool [5–8]. Interestingly, other members of the genus, including sweet basil (O. basilicum L.), contain an essential oil based primarily on high proportions of phenolic derivatives, such as eugenol, methyl chavicol (estragole) and methyl cinnamate, often combined with various proportions of linalool, a monoterpenol [5,6,9–11]. The allylphenolic derivative eugenol (Fig. 1) has a sharp spicy odor reminiscent of cloves, and is used as a dental analgesic and disinfectant [12]. Eugenol is a major component of the essential oil of cloves (Eugenia caryophyllus =Syzygium aromaticum, Myrtaceae), and cinnamon leaf (Cinnamomum zeylanicum, Lauraceae) [12]. Methyl chavicol, also known as estragole (Fig. 1), is used in the perfume industry and has an odor resembling that of fennel and anise. Interestingly, estragole is also a key component of the essential oils of aromatic plants belonging to different families, such as aniseed (Pimpinella anisum L., Apiaceae), star anise (Illicium 6erum Hook. f., Magnoliaceae), bitter fennel (Foeniculum 6ulgare 6ar 6ulgare, Apiaceae), and tarragon (Artemisia dracunculus L., Asteraceae) [12–14]. Information on the biosynthetic pathways to eugenol and methyl chavicol is limited. Pulse-chase radiotracer experiments have indicated that both allyl phenolic derivatives are formed from pheny-
Fig. 1. SAM:allylphenol O-methyltransferase activities from O. basilicum. A methyl group from SAM is transferred to the p-hydroxyl group of either chavicol or eugenol to generate either methyl chavicol (estragole) or methyl eugenol, respectively, and S-adenosyl homocysteine (SAH).
lalanine and cinnamic acid in several plants including sweet basil [15–18]. High levels of phenylalanine ammonia-lyase activity, a key early enzyme in this pathway, have been detected in sweet basil leaves, and the enzyme has been purified and characterized from many sources [19–21], including basil [22]. We hypothesized that the last biosynthetic step involved in the formation of estragole, is catalyzed by an S-adenosyl-L-methionine (SAM) dependent O-methyltransferase activity, able to substitute chavicol in the para-hydroxy position to give rise to estragole (Fig. 1). Methyltransferases are ubiquitous enzymes that catalyze the transfer of a methyl group from SAM to an acceptor substrate, generating either O-, N-, S- and C-methyl derivatives and S-adenosyl-homocysteine (Fig. 1) [23,24]. Although methyltransferases are involved in the formation of many natural products, they normally display strict specificities towards their acceptor substrate, and to the position of the methylation of the substrate [23,24]. O-methyltransferases are involved in the methylation of caffeic acid to generate ferulic acid, a central precursor of lignin and possibly eugenol. Some of the genes that code for these enzymes have been isolated from several plants [24–26], and also from sweet basil [27]. A methyltransferase activity that converts t-anol, (the 1-propenyl analog of chavicol), to generate t-anethole has been reported in cultures of Pimpinella anisum (Apiaceae) [28]. Similarly, an enzyme activity that catalyzes the para-O-methylation of t-isoeugenol, (the 1-propenyl analog of eugenol), forming methyl isoeugenol has been detected and purified from Clarkia breweri flowers, (Onagraceae) [29,30]. The corresponding gene has been isolated and expressed in Escherichia coli. Interestingly, the enzyme from C. breweri also accepts eugenol as substrate, at slightly lower rates, to generate methyl eugenol [29]. Most of the common cultivated members of the Lamiaceae accumulate essential oils rich in terpenoid compounds [5]. Although many studies have been conducted to monitor the developmental regulation of the biosynthesis of these terpenoid essential oil components [31–34], non-terpenoid components have received much less attention. One of the major components of the essential oil of sweet basil is estragole, a non-terpenoid allylphenol derivative. It was thus of interest to assess the possible developmental regulation
E. Lewinsohn et al. / Plant Science 160 (2000) 27–35
of estragole biosynthesis in sweet basil. We have previously shown that during the development of a sweet basil leaf, there is a net accumulation of essential oil and of estragole, both per leaf, and on a leaf weight basis [11]. Herein we provide evidence that in sweet basil leaves, chavicol and eugenol can be enzymatically O-methylated, to generate methyl chavicol and methyl eugenol, respectively, by SAM dependent O-methyltransferase activities (Fig. 1). These activities are active primarily in young leaf tissues and their substrate specificity varies chemotypically, partially explaining the differences in the chemical compositions observed among varieties.
2. Material and methods
2.1. Plant material Seeds of the different chemotypes [10] were derived from carefully selfed O. basilicum L. plants grown at the Newe Ya’ar Research Center, Israel. Five seeds were germinated in 4 cm-deep cells in trays containing equal volumes of peat:vermiculite:perlite, and watered and fertilized daily with 0.2% (w/v) NPK (5:3:8). After emergence, only one seedling per cell was left and grown until the shoot tips were discernible (about 10 days after germination) and used for the experiments. To generate young plants, the seedlings were transferred to 1 l pots containing a mixture of roughly equal volumes of volcanic tuff, vermiculite and peat. The plants were watered daily and fertilized twice a week with 0.2% (w/v) NPK (5:3:8) until four leaf pairs were discernible. Mature plants were obtaining by transferring the young seedlings directly from the trays into 1 × 1.2× 0.24 m containers containing volcanic tuff (0.8 mm). The plants (24 plants per m2) were dip-watered daily and fertilized as described in [35]. In all cases, plants were grown in greenhouses and maximal temperatures did not exceed 35– 40°C.
2.2. Chemicals and radiochemicals Chavicol was a kind gift of International Flavors and Fragrances Inc., Bridgewater, NJ, USA. Eugenol was from Roth Chemical Co. Estragole was from Terpene Products, Jacksonville, FL,
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USA, and methyl eugenol was from Fluka. Sadenosyl-L-methionine was from Sigma Chemical Co. S-adenosyl-L-methyl 3H-methionine (s.a. 15 Ci/mmol) and S-adenosyl-L-methyl 14C-methionine (s.a. 55 mCi/mmol) were from Amersham.
2.3. Enzyme extraction Cell-free extracts were prepared as follows, fresh plant leaves (1–2 g) were weighed; frozen in liquid nitrogen; and placed in a chilled mortar. The leaves were then ground with a pestle in the presence of liquid nitrogen, :0.6 g sand and :0.1 g polyvinylpolypyrrolidone (PVPP) to adsorb phenolic materials. Ice cold extraction buffer (100 mM MOPS [pH 6.5], 10% glycerol [v/v], 6.6 mM dithiothreitol, 1% [w/v] polyvinylpyrrolidone [PVP-10, molecular weight 10 kDa], 5 mM Na2S2O5) was added (five volumes to fresh weight tissue), and the fine powder was further extracted for :30 s. The slurry was centrifuged twice at 20 000×g, for 10 min at 4°C, and the supernatant, (crude extract) was used in the enzymatic assays. The crude extracts (1 ml) were further desalted using Sephadex G-25 columns (1 ×3 cm) and eluted with 1.2 ml of buffer A (50 mM HEPES [pH 7.5], 5% [v/v] glycerol, 3.3 mM dithiothreitol).
2.4. Enzyme assays Assays were preformed by mixing, 40 ml of the crude or Sephadex-G25 purified extract (about 20 mg protein); 40 ml buffer A; 5 mM chavicol or other acceptor substrate (from a 0.1 mM solution in 0.5% ethanol), 25 mM S-[methyl-3H]-adenosyl-Lmethionine (6 Ci/mol in a total volume of 100 ml). The assays were incubated at 21°C for 1.5 h, and then 1 ml hexane was added to each tube, vigorously vortexed and spun for 13 s at 20 000 ×g to separate the phases. The upper hexane layers containing the radioactive labeled enzyme products, were transferred to scintillation tubes containing 3 ml scintillation fluid [4 g/l 2,5-diphenyloxazol (PPO) and 0.05 g/l 2,2%-p-phenylen-bis (5-phenyloxazol) (POPOP) in toluene]. The radioactivity was quantified using a Packard Tri-carb liquid scintillation counter. To confirm the identity of the biosynthetic products, similar incubations were performed, except that 14C-labeled SAM (at the same specific activity) was used. In this case the
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Table 1 Developmental dependence of SAM:chavicol O-methyltransferase activity in sweet basil chemotype R3a Plant analyzed
Tissue analyzed
Chavicol O-methyltransferase activity (pkat/g FW)
Young seedlings (first leaf-pair visible)
Shoot tips
1465 9359
Cotyledons Shoots Roots Shoot tips
989 31 155 9 19 7 9 16 8432 9902
Young plant (four leaf-pairs)
Mature plant (blooming stage)
First pair of 3923 9887 leaves Second pair of 738 9 113 leaves Third pair of 71 9 56 leaves Developing 1212 9220 leaves Fully 63918 developed leaves Shoots 15 912 Inflorescences 135 972 Petals 43 923
a Enzyme activity was determined from Sephadex G-25 purified cell-free extracts derived from fresh tissues. Means and S.E. of three independent determinations are shown.
hexane layer was evaporated to a volume of 20 ml using a gentle stream of N2, and analyzed by thin layer chromatography (TLC) autoradiography using Silica gel 60 F254 plates developed with chloroform. Spots were visualized by ultra voilet (UV) light and radioactive spots detected by autoradiography on Kodak X-OMAT paper. The identity of the compounds was verified by comparison of their Rf with those of authentic standards (0.65 for estragole, 0.57 for methyl eugenol). Protein was assayed by the method of Bradford [36] using the Bio-Rad protein reagent (Bio-Rad), and bovine serum albumin (Sigma) as standard.
3. Results and discussion
3.1. O-methylation of allylphenols by O. basilicum cell-free-extracts Incubation of sweet basil cell-free extracts with C- or 3H-labeled SAM and chavicol, resulted in
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the accumulation of a hexane-soluble radiolabeled products, absent when the cell-free extracts were previously boiled for 5 min. This suggested that a methyltransferase enzymatic activity is involved in the conversions. Omission of chavicol resulted in a lower but still significant rate ( 60%) of formation of hexane-soluble radioactive products, possibly due to endogenous substrates. To remove any endogenous substrates and other low molecular weight compounds that interfered with the assays, the cell-free extracts were partially purified by Sephadex G-25 chromatography. Complete assays, containing SAM and chavicol, but utilizing Sephadex G-25 partially-purified-enzyme displayed 30–50% higher enzymatic activity levels as compared with crude extracts, probably due to the removal of inhibitors. The omission of chavicol from the assays resulted in virtually no formation of hexane-soluble radioactive products during the enzymatic assays. To confirm the identity of the radiolabeled compounds formed, the hexane extracts were evaporated and analyzed by TLC-autoradiography. Only one radioactive substance originating in chavicol and 14C-SAM fed Sephadex G-25 extracts was detected and its Rf coincided with that of authentic estragole (not shown). These results indicate that a methytransferase activity, present in sweet basil leaves is able to O-methylate chavicol and release estragole (Fig. 1).
3.2. De6elopmental regulation of the SAM:cha6icol O-methyltransferase acti6ity Different plant tissues were analyzed to identify the sites potentially active in estragole biosynthesis. Five-day-old seedlings already contained substantial levels of chavicol-dependent O-methyltransferase activity (Table 1). At this stage of development the cotyledons are still turgid and active, and the shoot tips contain discernible leaf primordia of only a few mm length. Practically all activity found resided in the leaf primordia. The cotyledons and shoots displayed lower levels of activity (about 8–10% by fresh weight) to that displayed by leaf primordia (Table 1). The roots were practically devoid of activity. Essentially, a similar pattern was obtained when tissues derived from seedlings at a four leaf-pair stage (about 10 cm height) were examined. The shoot tips had the highest activity levels, and young
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leaves (0.2 – 0.7 mm length) had substantial ( 45% as compared with shoot tips on a fresh weight basis) estragole biosynthetic potential. Activity levels decreased gradually with leaf development, reaching almost undetectable levels of activity in fully-developed leaves (Table 1). In mature flowering plants, the highest levels of activity was found in the young leaves from side branches, while inflorescences, but not the petals, displayed some SAM dependent chavicol O-methyltransferase activity. As previously observed in younger plants, shoots and mature leaves displayed only minute levels of activity. These findings further indicate that young developing tissues are the primary sites of methyl chavicol biosynthesis. The levels of estragole – biosynthetic activity correlate well with the presence and density of glandular trichomes present on the tissues [11]. It has been shown that in other members of the Lamiaceae, monoterpenebased essential oils are biosynthesized by glandular trichomes, and it could be that estragole is biosynthesized in a similar regulatory fashion although allylphenols are derived from the shikimic acid pathway, a distinct biosynthetic pathway [37]. In all cases, the highest activity per fresh weight was found in young tissues of the plant examined. Some plant O-methyltransferases are developmentally regulated [26], while others are induced by fungal elicitors and during processes related to plant defense responses [38–41]. In a similar fashion to the sweet basil chavicol O-methyltransferase activity described here, an N-methyltransferase activity involved in caffeine biosynthesis was also particularly active in young emerging leaves of Coffea arabica [42].
3.3. Extraction and properties of the SAM:cha6icol O-methyltransferase acti6ity from sweet basil Extraction of the enzyme without PVPP or PVP resulted in an intense browning and formation of precipitates, accompanied by losses in the enzymatic activity and soluble protein. After partial purification by Sephadex G-25 chromatography, the enzyme was more stable, and could be kept for more than 3 months at − 20°C without apparent loss of activity. The activity levels linearly increased with incubation time and were linearly dependent on protein concentration up to 25 mg protein. A
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broad temperature optimum around 20°C was found for activity. More than 50% of the activity was retained when assays were performed at either 42 or 17°C. A heat treatment of the enzyme preparation for 5 min at 100°C resulted in a complete loss of activity, probably due to protein denaturation. Several buffers were tested (at 100 mM), including sodium citrate, potassium phosphate, MOPS, HEPES, Tris, and sodium carbonate, for allowing optimal methyltransferase activity. A narrow pH optimum was found at 7.5, attained either when HEPES or potassium phosphate were used. Practically, regardless of the buffer employed, at pH values above 8.0 or below 7.0, activity sharply decreased, loosing all activity at pH values below 6.0 or above 9.0, similar to the optimum pH range of many plant methyltransferases [42,43]. Some methyltransferases require the presence of metal cofactors for activity, while activities from other sources do not require the presence of metal cofactors for activity [44]. To test whether the chavicol-O-methyltransferase from sweet basil requires any metal cofactor for activity, 1 or 10 mM of either CaCl2, MgCl2, MnCl2, CoCl2, ZnSO4, or FeSO4 were added to the assays. All the additions at 1 mM caused diminution of the activity by 50–80%, as compared with controls without any further addition. Moreover the inclusion of 10 mM CoCl2, ZnSO4, or FeSO4 in the assays resulted in a complete loss of the enzymatic activity. This indicated that the chavicol dependent Omethyltransferase activity from sweet basil apparently does not require a metal cofactor to be active. A constant 25 mM SAM level was used to calculate an apparent Km for chavicol of 3.0 mM utilizing both Lineweaver–Burk and Eadie–Hofstee equations. Conversely, keeping a constant 5 mM of the acceptor substrate chavicol, a Km for SAM was found to be 10 mM. This is whithin the ranges of Km’s obtained for substrates of Omethyltransferases from other sources [44]. The experiments described were performed on a sweet basil chemotype (R3) that contains a high (56%) level of estragole [10]. Although this chemotype also contains high levels of the monoterpene linalool (30%), and lower levels of other monoand sesquiterpenes, it completely lacks both eugenol and methyl eugenol [10]. It was therefore of interest to study the substrate specificity of the
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chavicol O-methyltransferase activity in this chemotype. Although eugenol differs from chavicol only in the addition of a m-methoxy group (Fig. 1), the enzyme from the estragole-rich chemotype R3 was almost completely inactive when eugenol was offered as a substrate in lieu of chavicol (Fig. 2). It could be that the additional methoxy group in eugenol causes a substantial steric disturbance in the active site of the enzyme, rendering eugenol as an inefficient substrate for methylation. We also found that this enzyme had a narrow substrate specificity towards the acceptor substrate, being active only towards p-hydroxylated, but further unsubstituted phenols, such as p-propyl phenol, p-ethyl phenol, and p-cresol at 50% slower O-methylation rates, as compared with the rate for chavicol, and completely inactive towards p-phenyl phenol, o-allylphenol and phenol. This indicates that only discrete alkyl additions together with the availability of a p-hydroxyl group, are crucial for the O-methylating ability of this enzyme.
Fig. 2. Linkage between the essential oil composition and the specificity and levels of SAM:dependent O-methyltransferase activity in sweet basil chemotypes. Top panels, essential oil composition of the different chemotypes data from reference [10], linalool (L, white bars), estragole (Es, black bars), methyl eugenol (ME, light gray) and eugenol (Eu, dark grey). Bottom panels, SAM:allylphenol O-methyltransferase activities. Substrate used, chavicol (Ch, black bars); substrate, eugenol (Eu, gray bars). The products formed are estragole (= methyl-chavicol) and methyl eugenol, respectively (Fig. 1). Means and S.E. of three independent determinations are shown.
Interesting substrate specificities have been reported in O-methyltransferases acting on phenolic derivatives from other sources. The t-anol Omethyltransferase from Pimpinella anisum accepted the 3% methoxylated derivative eugenol at almost the same rate as the non-3% methoxylated 1-propenyl analog t-anol, but chavicol and dihydroanol (both non-3% methoxylated) were Omethylated at ca. 50% of the rate of t-anol [28]. The isoeugenol O-methyltransferase from C. breweri, was able to methylate eugenol at about 70% the rate found for the 1-propenyl analog isoeugenol [29]. Interestingly, changes in only a few amino acids can dramatically modify the substrate specificities of O-methyltransferases of C. breweri [45].
3.4. Association between O-methyltransferase acti6ity le6els, their substrate specificities and the essential oil composition of sweet basil chemotypes Many sweet basil chemotypes differ in their allylphenol content and composition, and provide a unique experimental system to study the biochemical regulation of allyphenol biosynthesis. Chemotype 145 lacks linalool, and contains an essential oil that is largely constituted (91%) by methyl chavicol [10]. As expected, the presence of linalool did not have a pronounced effect on the methyl chavicol biosynthetic capability, as evidenced by the similar levels of chavicol specific O-methyltransferase activity found in this chemotype. In a similar fashion to the O-methyltransferase activity displayed by chemotype R3, the activity extracted from chemotype 145 does not accept eugenol as a substrate (Fig. 2). A different composition of allylphenol derivatives is found in chemotype 147/97, rich both in methyl chavicol and in methyl eugenol [10]. It would be difficult to conceive that O-methyltransferases with a strict substrate specificity for chavicol, and unable to accept eugenol as a substrate (such as those found in chemotypes R3 or 145) could mediate the formation of methyl eugenol in chemotype 147/97. Therefore, we postulated that chemotype 147/97 possessed distinct O-methyltransferase activities, able to accept either chavicol or eugenol as acceptor substrates. The substrate specificity of the O-methyltransferase activity obtained from chemotype 147/97 is clearly different and less strict, as compared with the one displayed
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by the activity obtained from chemotype R3. The results are shown in Fig. 2. The O-methyltransferase activity extracted from chemotype 147/97 readily accepts eugenol as a substrate, even at a higher rate than chavicol (Fig. 2). The identity of the products formed was confirmed to be methyl eugenol (or estragole, respectively), by radio-TLC (not shown). The substrate specificity of the Omethyltransferase activity(ies) extracted from chemotype 147/97 provides a good biochemical rationalization for the presence of both estragole and methyl eugenol in the essential oil of this chemotype. It is possible that the O-methyltransferase activity extracted from chemotype 147/97 resides in one enzyme with a broad substrate specificity, accepting either eugenol or chavicol as substrates. Nevertheless, it is also possible that chemotype 147/97 possesses more than one Omethyltransferase activity, each with strict substrate specificities for the methyl acceptors. Finally, the sweet basil chemotype SW was examined for the presence of chavicol or eugenolspecific O-methyltransferase activities. This chemotype accumulates high levels of linalool and eugenol, but completely lacks the p-methoxylated allylphenols methyl chavicol or methyl eugenol [10]. As expected, this chemotype lacked chavicol and eugenol dependant O-methyltransferase activities (Fig. 2). These observations provide a biochemical rationalization for the lack of p-methoxylated allylphenols in this chemotype. Interestingly, similar correlations between the levels of isoeugenol methyltransferase (IEMT) activity were found in flowers of C. breweri lines that emit methyl isoeugenol versus lines that do not emit it, and the regulation was apparently at a pre-translational step of the IEMT involved [29]. Although, we cannot exclude the possibility that the p-methylation of allylphenol derivatives takes place before the allyl chain is biosynthesized, our results indicate that p-methylation takes place after the formation of the allylic chain. Moreover, our findings suggest that the chemical composition of the essential oil in sweet basil chemotypes is (at least partially) dictated by the levels and substrate specificities of the O-methyltransferases present. The bulk of the allylphenol-specific O-methyltransferase activity seems to be restricted to young developing tissues. At present, it is unknown whether only one enzymatic activity with a broad substrate specificity is able to transfer methyl
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groups to either eugenol or chavicol, or whether two or more separable enzymatic activities are involved in these biochemical conversions. Crosses between the different chemotypes will enable us to learn more about the genetics of chemotype determination in sweet basil. Additionally, information obtained through purification and biochemical characterization of the sweet basil enzymes, as well as the isolation and analysis of their respective genes will help us to better understand the process of accumulation of allylphenolic derivatives in sweet basil chemotypes. Acknowledgements We thank Ronald Fenn and Dr William L. Schreiber for the chavicol. This work was supported by a grant number 255-0495 from the Chief Scientist of the Ministry of Agriculture of the State of Israel and The US–Israel Binational Agricultural Research and Development grant IS-270996. Additional support was provided by the Otto Meyerhof Center for Biotechnology established by the Minerva Foundation, Federal Republic of Germany. Contribution from the ARO, The Volcani Center, Bet Dagan 50250, Israel, No. 1012000. References [1] E. Putievsky, B. Galambosi, Production systems of sweet basil, in: R. Hiltunen, Y. Holm (Eds.), Basil. The Genus Ocimum, Harwood Academic Publishers, 1999, pp. 39 – 65. [2] R. Reuveni, A. Fleischer, E. Putievsky, Fungistatic activity of essential oils from Ocimum basilicum chemotypes, Phytopath. Z. 110 (1984) 20 – 22. [3] S. Dube, P.D. Upadhyay, S.C. Tripathi, Antifungal, physicochemical, and insect repelling activity of the essential oil of Ocimum basilicum, Can. J. Bot. 67 (1989) 2085 – 2087. [4] R.A. Werner, Toxicity and repellency of 4-allylanisole and monoterpenes from white spruce and tamarack to the spruce beetle and Eastern larch beetle (Coleoptera: Scolytidae), Environ. Entomol. 24 (1995) 372 – 379. [5] B.M. Lawrence, Labiatae oils — mother nature’s chemical factory, in: Essential Oils, Allured Publishing, Carol Stream, IL, 1993, pp. 188 – 206. [6] J.E. Simon, J. Quinn, R.G. Murray, Basil: a source of essential oils, in: Advances in New Crops, Timber Press, Portland, OR, 1990, pp. 484 – 489. [7] D.J. Charles, J.E. Simon, A new geraniol chemotype of Ocimum gratissimum L, J. Ess. Oil Res. 4 (1992) 231– 234.
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