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
baNewe Ya’ar Research Center,Agricultural Research Organization,P.O.Box1021,Ramat Yishay30095,Israel bDepartment of Food Engineering and Biotechnology,Technion-Israel Institute of Technology,Haifa32000,Israel
cDepartment of Biology,Uni6ersity of Michigan,Ann Arbor,MI48109-1048,USA
Received 19 May 2000; received in revised form 28 July 2000; accepted 31 July 2000
Abstract
Sweet basil (Ocimum basilicumL., 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 chavicolO-methyltransferase activity, but the activity was negligible in older leaves, indicating that theO-methylation of chavicol primarily occurs early during leaf development. TheO-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 notO-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 dependentO-methyltransferase activities. These results indicate the presence of at least two types of allylphenol-spe-cificO-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 theseO-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 basilicumL.; Lamiaceae; Sweet basil; Essential oils; Estragole; Methyl eugenol;O-methyltransferase
www.elsevier.com/locate/plantsci
1. Introduction
The genus Ocimum includes about a dozen spe-cies and subspespe-cies, native to the tropical and sub-tropical regions of the world. Sweet basil (Ocimum basilicum L., Lamiaceae) is an important
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 ac-tivities [2 – 4]. Despite the wide use and the impor-tance 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.
* Corresponding author. Tel.: +972-4-9539552; fax: + 972-4-9836936.
E-mail address:twefraim@netvision.net.il (E. Lewinsohn).
Many members of the Lamiaceae such as Men
-tha, 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 monoter-pene derivatives such as camphor, limonene, thy-mol, citral, geraniol and linalool [5 – 8]. Interestingly, other members of the genus, includ-ing sweet basil (O. basilicum L.), contain an essen-tial 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 deriva-tive eugenol (Fig. 1) has a sharp spicy odor remi-niscent of cloves, and is used as a dental analgesic and disinfectant [12]. Eugenol is a major compo-nent 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 compo-nent of the essential oils of aromatic plants be-longing to different families, such as aniseed (Pimpinella anisum L., Apiaceae), star anise (Illi
-cium 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-lalanine and cinnamic acid in several plants in-cluding 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
-me-thionine (SAM) dependent O-methyltransferase activity, able to substitute chavicol in thepara -hy-droxy 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 eitherO-,N-,
S- and C-methyl derivatives and S -adenosyl-ho-mocysteine (Fig. 1) [23,24]. Although methyltrans-ferases are involved in the formation of many natural products, they normally display strict spe-cificities 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 inEscherichia 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 ter-penoid compounds [5]. Although many studies have been conducted to monitor the developmen-tal regulation of the biosynthesis of these ter-penoid 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-ter-penoid allylphenol derivative. It was thus of inter-est to assess the possible developmental regulation Fig. 1. SAM:allylphenol O-methyltransferase activities from
O.basilicum. A methyl group from SAM is transferred to the
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 evi-dence that in sweet basil leaves, chavicol and eugenol can be enzymatically O-methylated, to generate methyl chavicol and methyl eugenol, re-spectively, by SAM dependent O -methyltrans-ferase activities (Fig. 1). These activities are active primarily in young leaf tissues and their substrate specificity varies chemotypically, partially explain-ing 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 selfedO.basilicumL. 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 emer-gence, only one seedling per cell was left and grown until the shoot tips were discernible (about 10 days after germination) and used for the exper-iments. To generate young plants, the seedlings were transferred to 1 l pots containing a mixture of roughly equal volumes of volcanic tuff, vermi-culite 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. Ma-ture 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 Fla-vors and Fragrances Inc., Bridgewater, NJ, USA. Eugenol was from Roth Chemical Co. Estragole was from Terpene Products, Jacksonville, FL,
USA, and methyl eugenol was from Fluka. S -adenosyl-L-methionine was from Sigma Chemical
Co. S-adenosyl-L-methyl 3H-methionine (s.a. 15
Ci/mmol) and S-adenosyl-L-methyl 14
C-methion-ine (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 pres-ence of liquid nitrogen, :0.6 g sand and :0.1 g polyvinylpolypyrrolidone (PVPP) to adsorb phe-nolic 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 superna-tant, (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); 40ml buffer A; 5mM chavicol or other acceptor substrate (from a 0.1 mM solution in 0.5% ethanol), 25 mM S-[methyl-3
H]-adenosyl-L
-methionine (6 Ci/mol in a total volume of 100ml). The assays were incubated at 21°C for 1.5 h, and then 1 ml hexane was added to each tube, vigor-ously vortexed and spun for 13 s at 20 000×g to separate the phases. The upper hexane layers con-taining 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-pheny-loxazol) (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
Table 1
Developmental dependence of SAM:chavicol O -methyltrans-ferase activity in sweet basil chemotype R3a
Plant analyzed Tissue Chavicol
O-methyltransferase analyzed
activity (pkat/g FW)
Young seedlings Shoot tips 14659359 (first leaf-pair
visible)
98931 Cotyledons
Shoots 155919
Roots 7916
Shoot tips
Young plant (four 84329902 leaf-pairs)
First pair of 39239887 leaves
Second pair of 7389113 leaves
Third pair of 71956 leaves
Mature plant Developing 12129220 leaves
aEnzyme 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.
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 forma-tion of hexane-soluble radioactive products, possi-bly 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 Sep-hadex G-25 partially-purified-enzyme displayed 30 – 50% higher enzymatic activity levels as com-pared 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 ex-tracts were evaporated and analyzed by TLC-au-toradiography. Only one radioactive substance originating in chavicol and14C-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 ac-tivity, 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 biosynthe-sis. Five-day-old seedlings already contained sub-stantial 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. Practi-cally all activity found resided in the leaf primor-dia. 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. Essen-tially, 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 hexane layer was evaporated to a volume of 20 ml
using a gentle stream of N2, and analyzed by thin
layer chromatography (TLC) autoradiography us-ing Silica gel 60 F254plates developed with
chloro-form. Spots were visualized by ultra voilet (UV) light and radioactive spots detected by autoradiog-raphy on Kodak X-OMAT paper. The identity of the compounds was verified by comparison of theirRfwith 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
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 flower-ing 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 ac-tivity. 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 es-tragole – 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, monoterpene-based essential oils are biosynthesized by glandu-lar trichomes, and it could be that estragole is biosynthesized in a similar regulatory fashion al-though 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 develop-mentally regulated [26], while others are induced by fungal elicitors and during processes related to plant defense responses [38 – 41]. In a similar fash-ion to the sweet basil chavicol O -methyltrans-ferase 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 enzy-matic 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 incu-bation time and were linearly dependent on protein concentration up to 25 mg protein. A
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), includ-ing 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. Practi-cally, 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 re-quires any metal cofactor for activity, 1 or 10 mM of either CaCl2, MgCl2, MnCl2, CoCl2, ZnSO4, or
FeSO4were 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
re-sulted in a complete loss of the enzymatic activity. This indicated that the chavicol dependent O -methyltransferase activity from sweet basil appar-ently 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 – Hofs-tee 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 O
chavicol O-methyltransferase activity in this chemotype. Although eugenol differs from chavi-col 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 -hydroxy-lated, 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 phe-nol. This indicates that only discrete alkyl addi-tions together with the availability of ap-hydroxyl group, are crucial for theO-methylating ability of this enzyme.
Interesting substrate specificities have been re-ported in O-methyltransferases acting on phenolic derivatives from other sources. The t-anol O -methyltransferase from Pimpinella anisum ac-cepted the 3% methoxylated derivative eugenol at
almost the same rate as the non-3% methoxylated
1-propenyl analog t-anol, but chavicol and dihy-droanol (both non-3% methoxylated) were O
-methylated at ca. 50% of the rate of t-anol [28]. The isoeugenolO-methyltransferase fromC.brew
-eri, 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 sub-strate 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 bio-chemical 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 evi-denced by the similar levels of chavicol specific
O-methyltransferase activity found in this chemo-type. In a similar fashion to the O -methyltrans-ferase 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 deriva-tives is found in chemotype 147/97, rich both in methyl chavicol and in methyl eugenol [10]. It would be difficult to conceive that O -methyltrans-ferases with a strict substrate specificity for chavi-col, 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 -methyl-transferase activities, able to accept either chavicol or eugenol as acceptor substrates. The substrate specificity of the O-methyltransferase activity ob-tained from chemotype 147/97 is clearly different and less strict, as compared with the one displayed Fig. 2. Linkage between the essential oil composition and the
by the activity obtained from chemotype R3. The results are shown in Fig. 2. The O -methyltrans-ferase 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 O -methyltransferase 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 -methyltrans-ferase 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 O -methyltransferase activity, each with strict sub-strate specificities for the methyl acceptors.
Finally, the sweet basil chemotype SW was ex-amined for the presence of chavicol or eugenol-specific 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 dependantO-methyltransferase activ-ities (Fig. 2). These observations provide a bio-chemical rationalization for the lack of
p-methoxylated allylphenols in this chemotype. In-terestingly, similar correlations between the levels of isoeugenol methyltransferase (IEMT) activity were found in flowers ofC. brewerilines that emit methyl isoeugenol versus lines that do not emit it, and the regulation was apparently at a pre-transla-tional 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 af-ter 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 -methyl-transferase 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
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 deter-mination 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 sup-ported 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 Agri-cultural Research and Development grant IS-2709-96. 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 Vol-cani Center, Bet Dagan 50250, Israel, No. 101-2000.
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