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S-Linalool synthase activity in developing fruit of the columnar cactus koubo [Cereus peruvianus (L.) Miller]
Plant Science 167 (2004) 1257–1262 www.elsevier.com/locate/plantsci
S-Linalool synthase activity in developing fruit of the columnar cactus koubo [Cereus peruvianus (L.) Miller] Yaron Sitrita, Racheli Ninioa,b, Einat Barc, Einav Golanc, Olga Larkovc, Uzi Ravidc, Efraim Lewinsohnc,* a
Institutes for Applied Research, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel b Department of Life Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel c Department of Vegetable Crops, Agricultural Research Organization (ARO), Newe Ya’ar Research Center, P.O. Box 1021, Ramat Yishay 30095, Israel Received 31 May 2004; received in revised form 20 June 2004; accepted 22 June 2004 Available online 14 July 2004
Abstract Koubo [Cereus peruvianus (L.) Miller, Cactaceae] is a commercially grown columnar cactus that produces an apple-sized, berry-like, edible fruit. The unique aroma of this fruit is largely due to (S)-linalool and linalool derivatives. Cell-free extracts obtained from koubo fruits produced linalool from geranyl diphosphate (GDP) (apparent Km 18 mM) in enzymatic assays containing the divalent ion cofactor Mn2+ or Mg2+. The predominant enantiomer produced in vitro was (S)-linalool, with an optical purity of 85%. Incubation of geranyl diphosphate in the presence of 1–10 mM FeSO4 or FeCl2, without added enzyme, supported the in vitro spontaneous formation of racemic linalool. The koubo linalool synthase displayed a molecular mass of 53 kDa as determined by gel permeation chromatography. Enzyme activity levels were negligible in green immature fruits and increased with fruit development and during storage, concomitant with the timing of linalool accumulation in fruits. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Koubo; Cereus peruvianus; Cactaceae; Fruit; Ripening; Volatiles; (S)-Linalool; Linalool synthase
1. Introduction Koubo [Cereus peruvianus (L.) Miller] is a new cactus fruit crop for arid regions. In Israel, koubo cultivation is expanding and becoming commercially important [1–4]. Koubo is a columnar cactus that bears a berry-like nonclimacteric fruit that weighs about 300 g and has a purple– red smooth and spineless peel. The fruit pulp is white, juicy, and aromatic and contains numerous tiny black seeds [5–7]. Towards the final stages of ripening, the contents of polysaccharides and organic acids, mainly malic acid, decrease, while the pH and the levels of glucose and fructose increase [8]. Another important ripening-derived change is a pronounced increase in volatiles, mainly linalool and its derivatives, which give the fruit its unique aroma [8]. * Corresponding author. Tel.: +972 4 953 9552; fax: +972 4 983 6936. E-mail address: [email protected] (E. Lewinsohn).
Fruit aroma is one of the key factors determining fruit quality and consumer acceptability [9,10]. In most fruits, the aroma is a function of a complex mixture of volatile compounds. Linalool, an acyclic monoterpene alcohol that imparts a sweet floral note, is a relatively minor, albeit important, component of the aroma of many fruits, such as strawberries, tomatoes, peaches, nectarines, pineapple, guavas, papayas, passion fruits, litchis, oranges and prickly pears [10–22]. Koubo is unique in that linalool and its derivatives constitute more than 99% of the volatiles present in the ripe fruits [8]. Linalool is also a major component of the flower scents in many plant species [23] and is thought to play a role in plant–insect interactions [24,25]. Linalool exists in two enantiomeric forms, S-(+)- and R-()-linalool, which differ in their olfactory qualities. R-()-Linalool has a fragrance reminiscent of lavender, whereas S-(+)-linalool has more of a petitgrain fragrance
0168-9452/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2004.06.024
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[26]. The enantiomeric composition of the linalool produced in koubo fruit has not previously been investigated. Although many specific aroma compounds have been identified in cactus fruits [22], the enzymes that catalyze their production have not been studied and are, therefore, little understood. Generally, monoterpenes, such as linalool are formed directly from geranyl diphosphate (GDP) via the isoprenoid pathway. The enzyme linalool synthase (LIS) has been isolated from the flowers of Clarkia breweri, a plant native to California [27]. LIS catalyzes the cation-dependent and stereo-selective conversion of GDP to S-linalool. LIS activity has also been reported in Norway spruce [Picea abies L. (Karst)] needles [28]. To date, LIS activity has not been reported in fruits, even though its product—linalool— is present as a minor component in the fruits of many species. Several genes encoding for proteins displaying LIS activity in heterologous expression systems have also been reported [29–31]. In this study, we report on the enantiomeric composition of linalool, some of the properties of LIS, and the changes in LIS activity during the development of koubo fruit and during fruit storage.
2. Materials and methods 2.1. Plant material Fruits of koubo [C. peruvianus (L.) Miller] clone G2 were collected from a commercial orchard at Sede Nizzan (situated in the western region of the Israeli Negev Desert). To follow changes in enzyme activity during fruit ripening, four ripening stages were previously defined in terms of days from anthesis and peel color: 37–39 days from anthesismature green, 42–44 days—breaker, 48–50—violet, 50–52 days—red cracked (ripe) [4]. To determine enzyme activity during fruit ripening in storage, fruits were harvested at the violet stage (according to days post-anthesis) and stored at 20 8C and 60% relative humidity till they turned a full red color [8]. 2.2. Extraction of volatiles Five grams of frozen pulverized tissue were extracted with 15 ml of methyl tert-butyl ether (MTBE) containing 10 mg of iso-butyl benzene as an internal standard [4]. The samples were shaken vigorously for 2 h at room temperature. The extracts were dried by passing them through a small column (Pasteur pipette) plugged with glass wool containing sodium sulfate and then concentrated under a gentle nitrogen stream to a volume of ~0.5 ml. 2.3. Chiral chromatography Samples of 1 ml of the concentrated MTBE extracts were analyzed on a Hewlett–Packard GCD gas chromatograph
equipped with a Restek Rt-bDEXsm fused silica column (30 m 0.25 mm). Injector and detector temperatures were 230 8C. The following temperature program was used: 55 8C for 1 min, 50–200 8C at 1.5 8C/min; carrier gas was He at 1 ml/min [26]. R-Linalool extracted from basil essential oil (from our collection, 26) and synthetic linalool (Roth Chemical Co. Karlsruhe, Germany) containing S-(+)-linalool and R-()-linalool were used as standards [32]. 2.4. Enzyme extraction and partial purification Three to five fruits were randomly sampled at each ripening stage, and the pulp was separated from the peel. Fresh pulp from the fruit center was frozen with liquid nitrogen and kept at 70 8C for characterization of the enzyme and determination of enzyme activity. Frozen samples (2 g) were ground with a pestle in a chilled mortar in liquid nitrogen, 0.5 g sand and 0.1 g polyvinylpolypyrrolidone (PVPP) until a uniform powder was obtained. Ice-cold extraction buffer (1:4 w/v), consisting of 50 mM bis–Tris pH 6.9, 10% (v/v) glycerol, 10 mM dithiothreitol (DTT), 5 mM Na2S2O5 and 1% (w/v) polyvinylpyrrolidone (PVP-40 or PVP-10) was added. In the initial experiments, we used 50 mM 3-morpholinopropanesulfonic acid (MOPS), pH 6.9, instead of bis–Tris, with comparable results. The slurry was centrifuged at 20,000 g for 15 min at 4 8C. The supernatant was further purified through a P-6 column (1 10 cm) (Bio-Rad, bio-gel desalting gel 90–180 mm, BioRad, Munich, FRG) to remove interfering low-molecularweight compounds. We used buffer B (the above-mentioned extraction buffer without PVP-40) for column equilibration and protein elution. Fractions (1 ml) were tested for enzyme activity, and the active fractions were combined and kept at 20 8C for further analysis. To effectively remove interfering polysaccharides that increased the viscosity of the cell-free extracts, especially those derived from immature fruits, the extracts were further purified by anion exchange (diethylaminoethyl cellulose, DE52, Whatman, Maidstone, UK) batch chromatography. Before use, the DE52 was pre-equilibrated with 1 M KCl and washed thoroughly with loading buffer [25 mM MOPS, pH 6.9, 3.3 mM dithiothreitol, and 5% (v/v) glycerol]. One milliliter of DE52 resin, 2 ml of crude cell-free extracts (0.2–0.4 mg protein/ml) and 8 ml of loading buffer were mixed gently and incubated in an ice-bath for 15 min. Then, the supernatant was discarded, and the resin was washed with 4 ml of loading buffer, mixed gently, and allowed to stand for a further 10 min on ice. This procedure was repeated twice using loading buffer containing 0.1 M KCl. The resin was then washed with 2 ml of loading buffer containing 0.5 M KCl to elute the enzyme activity. The final concentration of KCl in the enzyme assays was below 50 mM. Protein was determined with the Bio-Rad protein assay reagent (Bio-Rad, Munich, FRG) using bovine serum albumin as standard.
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2.5. Enzyme assays We used a large-scale GC–MS-based assay for the identification of the products, co-factor requirements and optical purity and a small-scale radioactive assay for the developmental studies and molecular weight determination. 2.5.1. GC–MS large-scale assay Cell-free extracts (200 ml containing 1–4 mg protein) were incubated with 10 mM MgCl2, 1 mM MnCl2, and 13.7 mM non-radioactive GDP in sample buffer (50 mM bis–Tris pH 6.9, 10% (v/v) glycerol, 10 mM dithiothretiol and 5 mM Na2S2O5) in a total volume of 2 ml, and overlaid with 2 ml of hexane. After an overnight incubation at 34 8C, 10 mg of iso-butyl benzene was added as an internal standard. The samples were vigorously vortexed and centrifuged (clinical) for 2 min to separate the phases. The samples were the re-extracted with 2 ml of hexane three times. The upper hexane phases were pooled and dried by passing them through a small (Pasteur pipette) column, containing sodium sulfate, and then concentrated to a volume of 0.2 ml under a gentle stream of nitrogen. Appropriate controls included the omission of substrate, omission of metal cofactors, omission of enzyme extract and the use of heat-inactivated enzyme extracts. A 1 ml aliquot of the concentrated hexane layers was injected into a Hewlett Packard GCD gas chromatograph equipped with a HP5 (30 m 0.25 mm) fused-silica capillary column. Helium (1 ml/min) was used as a carrier gas with splitless injection. The injector temperature was 250 8C, and the detector temperature was 280 8C. The conditions used were as follows: initial temperature was 70 8C for 2 min followed by a ramp of 70–200 8C at a rate of 4 8C/min, and additional 10 min at 200 8C. Masses between 45 and 450 m/z were recorded. Linalool and geraniol were identified by co-injection and comparison of the EI–MS obtained with authentic standards and complemented with computerized libraries. Similar preparations were subjected to chiral chromatography (see above) for the determination of the enantiomeric composition of the enzymatically-generated linalool. 2.5.2. Radioactive small-scale assays for enzyme characterization and developmental studies Small-scale assays were performed by diluting 10 ml of enzyme extracts in 60 ml of buffer containing 50 mM MOPS pH 6.6, 10% (v/v) glycerol, 1 mM MnCl2, 10 mM MgCl2, 6.6 mM dithiothreitol, and 20 ml [1-3H]GDP (3 mM final concentration, sp. ac. 15 Ci/mmol) and overlaying the mixture with 1 ml of hexane to trap volatiles. The tubes were then briefly mixed, centrifuged at 14,000 g for a few seconds, and incubated at 30 8C for 2 h. The samples were mixed vigorously and centrifuged for 1 min at 20,000 g to separate the phases. Then, 0.8 ml of the upper hexane phase containing the in vitro formed radioactively labeled enzyme products was transferred to a 5 ml scintillation tube containing 3 ml of scintillation
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fluid (4 g/l 2,5-diphenyloxazol (PPO) and 0.05 g/l 2,20 -pphenylen-bis (5-phenyloxazol (POPOP) in toluene). The radioactivity was quantified using a liquid scintillation counter (Kontron model 810, Zurich, Switzerland). Enzyme activity in picokatals was calculated on the basis of the specific activity of the substrate and taking into account the appropriate correction factors for the presence of geraniol and the counting efficiency of the scintillation machine. Enzyme activity was linear with respect to protein concentration and incubation time. 2.6. Determination of the native molecular mass of LIS The desalted enzyme, after P6 chromatography, was filtered through a 0.2 mm cellulose acetate membrane (ca462200, hleicher & Schuell, Dassel, Germany) and separated by gel permeation chromatography using a Superdex 75 HiLoad 16/60 column (FPLC Amersham Pharmacia Biotech, Uppsala, Sweden), using buffer C consisting of 25 mM bis–Tris, pH 6.9, 150 mM KCl, 5% (v/v) glycerol, 5 mM dithiothretiol and 2.5 mM Na2S2O5 at a flow rate of 1 ml/min. The elution of LIS activity was monitored with the small-scale radioactive assay, and the molecular mass of LIS was determined by comparison with molecular masses of known proteins [alcohol dehydrogenase (MW ~150,000), phosphorylase b (MW ~97,000), bovine serum albumin (MW ~66,000), ovalbumin (MW ~53,000), carbonic anhydrase (MW ~29,000), cytochrome C (MW ~12,400), and aprotinin (MW ~6,500), all from Sigma–Aldrich Chemical Co, St. Louis, MO, USA].
3. Results and discussion 3.1. Enantiomeric composition The optical purity of the linalool, the main volatile accumulating in ripening koubo fruit [4], was assessed by chiral GC analysis. S-(+)-Linalool was found in a high optical purity of 98% (Fig. 1A). In most fruits containing linalool, both enantiomers are present. In apricots, plums, passion fruits and pineapples, for example, both enantiomers are present in roughly similar levels [17], but in other fruits one of the enantiomers may be dominant. High enantiometric excess (98%) of S-(+)-linalool has been found in strawberries [17] and of R-(–)-linalool, in guava, peach and lulo [17]. In cactus pear, R-(–)-linalool is present in an enantiomeric excess of 36% [22]. It is known that acidic conditions may cause racemization linalool, especially at high temperatures [33]. Since koubo fruit pulp is acidic (pH 4.5–6) and the fruits are exposed to high temperatures during ripening [4], it was expected that linalool would loose its optical purity in ripe koubo fruits. The surprising finding that the optical purity was high (Fig. 1A) may be explained by cellular and subcellular compartmentation. [34,35].
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Fig. 1. Enantiomeric analyses of linalool by chiral chromatography: (A) linalool enantiomers accumulating in ripe koubo fruit; (B) linalool enantiomers synthesized in vitro from GDP by cell-free extracts; (C) linalool formed non-enzymatically by the addition of 10 mM FeSO4 to the assay mixtures; (D) enantiomer standards.
3.2. Partial characterization of koubo linalool synthase activity Soluble cell-free extracts obtained from koubo fruit pulp converted GDP into linalool, as determined by GC– MS (Fig. 2A). Linalool was not found in the absence of GDP (Fig. 2B) or in the presence of heat-inactivated LIS
(not shown). The divalent metal cofactor requirements of the koubo LIS were determined. The addition of MgCl2 was apparently sufficient for catalysis (Km = 60 mM). Mn2+ ions promoted catalysis at a similar maximal rate, but with a much lower Km (5.6 mM). Monoterpene synthases require divalent metal ions for activity, usually Mg2+ or Mn2+ [35,36]. The C. breweri LIS requires Mn2+ for activity (Km = 45 mM), but Mg2+ can also act as a co-factor, at half of the rate of Mg2+ with a 10-fold higher Km (330 mM) [27]. Other metal cofactors that did not support catalysis included Cu2+, Co2+ and Ca2+, tested at 10 mM. The addition of FeCl2 or FeSO4 at 1–10 mM supported the formation of linalool, even in the absence of enzyme, but this linalool was racemic (see below). The apparent Km for GDP of koubo LIS is 18 mM, which is in the range of most other monoterpene synthases. C. breweri and Mentha citrata LIS have Km values for GDP of 0.9 and 56 mM, respectively [27,30]. In some experiments, we noticed the formation of geraniol. This could be due to the action of the phosphatases that are ubiquitous to many plant tissues [36]. Under our optimized assay conditions, the levels of geraniol were usually negligible. Since linalool accumulated in the pulp in high optical purity (Fig. 1A), it was of interest to determine the optical purity of the linalool formed in vitro from GDP by the action of enzymatic preparations from koubo fruits. The newly generated linalool was found to be 85% enantiomerically pure, the S-isomer being dominant (Fig. 1B). Clarkia LIS produces the S-isomer exclusively [27]. The Mentha recombinant enzyme produces 98% R-linalool and 2% of the S-enantiomer [30]. The enantiomeric purity of the linalool extracted from the fruit (Fig. 1A) and the linalool formed in vitro from GDP (Fig. 1B) were not identical. It could be that our extraction conditions modified the enzyme activity, thus lowering its enantiomeric specificity. Alternatively, our assay conditions might not reliably mimic the conditions prevailing in vivo in terms of substrate, cofactor, temperature and pH requirements. Another explanation could be that two enzymes with different stereo-selectivities exist in the fruit and compete for the GDP. Moreover, the non-enzymatically formed linalool, generated in the presence of Fe2+ was racemic (Fig. 1C). It could be that sufficient endogenous Fe2+ levels support the formation of low levels of R- and S-linalool, both in vivo and enzymatically. Further characterization of the enzyme(s) coupled with the isolation of the gene(s) that code for this activity will contribute to our understanding of the factors that affect the optical specificity of the linalool formed and accumulated in koubo fruits. 3.3. LIS native molecular mass
Fig. 2. Identification of linalool generated in vitro from GDP by cell-free extracts of koubo fruit: (A) complete reaction (0.2 mM GDP, 10 mM MgCl2, and 1 mM MnCl2). The mass spectrum of biosynthetic linalool is shown in the insert; (B) control assays devoid of the substrate GDP. IBB: iso-butyl benzene (internal standard).
The native molecular mass of koubo LIS was about 53 kDa, as determined by gel permeation chromatography (Fig. 3). This value falls in the range of other plant monoterpene synthases having molecular masses of 50–70 kDa.
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aroma of koubo fruit. LIS therefore plays a pivotal role in determining the aroma characteristics of this fruit. Elucidating the mechanisms regulating LIS activity and expression during fruit ripening and storage are of critical relevance to our ability to understand and control fruit quality.
Acknowledgments Fig. 3. Molecular mass determination of LIS activity from koubo fruit. The desalted enzyme was separated by gel permeation chromatography, and the activity eluted corresponded to a molecular mass of 53 kDa (by comparison with known protein standards).
The C. breweri LIS, for example, is a 76 kDa monomeric protein [27]. 3.4. LIS activity during fruit development and storage Since significant increases in linalool and its derivatives have been found in koubo fruits during the later stages of fruit ripening and during storage [4,8], it was of interest to determine the changes in LIS activity levels under these conditions. Negligible LIS activity was found during the green and breaker stages of fruit development (Fig. 4). A prominent increase in LIS activity was first discernible during the violet stage, in accordance with the timing of the initial detection of linalool [4]. LIS activity further increased during ripening, reaching about 4 nkat/mg protein in the cracked red fruit. The highest LIS activity was found in fruit ripening in storage, reaching values of about 6 nkat/ mg protein (Fig. 4). These findings are in accordance with the high linalool levels accumulating in fruit ripening in storage as compared with the lower linalool levels present in fruit ripening on the tree [4,8]. We have shown that S-linalool and other linalool derivatives are the main volatiles contributing to the unique
Fig. 4. Increases in LIS activity during koubo fruit ripening and storage. LIS activity was determined at different stages of fruit development on the tree (mature green, breaker, violet and red cracked) and after storage (red stored). Values are means obtained for three individual fruits S.E. The experiment was repeated three times with similar results.
We thank Arnon Ronen and Moshe Ventura for kindly supplying the fruits. We thank Inez Mureinik for editing the manuscript. Publication No. 113/2004 of the Agricultural Research Organization, Bet Dagan, Israel.
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S-Linalool synthase activity in developing fruit of the columnar cactus koubo [Cereus peruvianus (L.) Miller] Yaron Sitrita, Racheli Ninioa,b, Einat Barc, Einav Golanc, Olga Larkovc, Uzi Ravidc, Efraim Lewinsohnc,* a
Institutes for Applied Research, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel b Department of Life Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel c Department of Vegetable Crops, Agricultural Research Organization (ARO), Newe Ya’ar Research Center, P.O. Box 1021, Ramat Yishay 30095, Israel Received 31 May 2004; received in revised form 20 June 2004; accepted 22 June 2004 Available online 14 July 2004
Abstract Koubo [Cereus peruvianus (L.) Miller, Cactaceae] is a commercially grown columnar cactus that produces an apple-sized, berry-like, edible fruit. The unique aroma of this fruit is largely due to (S)-linalool and linalool derivatives. Cell-free extracts obtained from koubo fruits produced linalool from geranyl diphosphate (GDP) (apparent Km 18 mM) in enzymatic assays containing the divalent ion cofactor Mn2+ or Mg2+. The predominant enantiomer produced in vitro was (S)-linalool, with an optical purity of 85%. Incubation of geranyl diphosphate in the presence of 1–10 mM FeSO4 or FeCl2, without added enzyme, supported the in vitro spontaneous formation of racemic linalool. The koubo linalool synthase displayed a molecular mass of 53 kDa as determined by gel permeation chromatography. Enzyme activity levels were negligible in green immature fruits and increased with fruit development and during storage, concomitant with the timing of linalool accumulation in fruits. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Koubo; Cereus peruvianus; Cactaceae; Fruit; Ripening; Volatiles; (S)-Linalool; Linalool synthase
1. Introduction Koubo [Cereus peruvianus (L.) Miller] is a new cactus fruit crop for arid regions. In Israel, koubo cultivation is expanding and becoming commercially important [1–4]. Koubo is a columnar cactus that bears a berry-like nonclimacteric fruit that weighs about 300 g and has a purple– red smooth and spineless peel. The fruit pulp is white, juicy, and aromatic and contains numerous tiny black seeds [5–7]. Towards the final stages of ripening, the contents of polysaccharides and organic acids, mainly malic acid, decrease, while the pH and the levels of glucose and fructose increase [8]. Another important ripening-derived change is a pronounced increase in volatiles, mainly linalool and its derivatives, which give the fruit its unique aroma [8]. * Corresponding author. Tel.: +972 4 953 9552; fax: +972 4 983 6936. E-mail address: [email protected] (E. Lewinsohn).
Fruit aroma is one of the key factors determining fruit quality and consumer acceptability [9,10]. In most fruits, the aroma is a function of a complex mixture of volatile compounds. Linalool, an acyclic monoterpene alcohol that imparts a sweet floral note, is a relatively minor, albeit important, component of the aroma of many fruits, such as strawberries, tomatoes, peaches, nectarines, pineapple, guavas, papayas, passion fruits, litchis, oranges and prickly pears [10–22]. Koubo is unique in that linalool and its derivatives constitute more than 99% of the volatiles present in the ripe fruits [8]. Linalool is also a major component of the flower scents in many plant species [23] and is thought to play a role in plant–insect interactions [24,25]. Linalool exists in two enantiomeric forms, S-(+)- and R-()-linalool, which differ in their olfactory qualities. R-()-Linalool has a fragrance reminiscent of lavender, whereas S-(+)-linalool has more of a petitgrain fragrance
0168-9452/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2004.06.024
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[26]. The enantiomeric composition of the linalool produced in koubo fruit has not previously been investigated. Although many specific aroma compounds have been identified in cactus fruits [22], the enzymes that catalyze their production have not been studied and are, therefore, little understood. Generally, monoterpenes, such as linalool are formed directly from geranyl diphosphate (GDP) via the isoprenoid pathway. The enzyme linalool synthase (LIS) has been isolated from the flowers of Clarkia breweri, a plant native to California [27]. LIS catalyzes the cation-dependent and stereo-selective conversion of GDP to S-linalool. LIS activity has also been reported in Norway spruce [Picea abies L. (Karst)] needles [28]. To date, LIS activity has not been reported in fruits, even though its product—linalool— is present as a minor component in the fruits of many species. Several genes encoding for proteins displaying LIS activity in heterologous expression systems have also been reported [29–31]. In this study, we report on the enantiomeric composition of linalool, some of the properties of LIS, and the changes in LIS activity during the development of koubo fruit and during fruit storage.
2. Materials and methods 2.1. Plant material Fruits of koubo [C. peruvianus (L.) Miller] clone G2 were collected from a commercial orchard at Sede Nizzan (situated in the western region of the Israeli Negev Desert). To follow changes in enzyme activity during fruit ripening, four ripening stages were previously defined in terms of days from anthesis and peel color: 37–39 days from anthesismature green, 42–44 days—breaker, 48–50—violet, 50–52 days—red cracked (ripe) [4]. To determine enzyme activity during fruit ripening in storage, fruits were harvested at the violet stage (according to days post-anthesis) and stored at 20 8C and 60% relative humidity till they turned a full red color [8]. 2.2. Extraction of volatiles Five grams of frozen pulverized tissue were extracted with 15 ml of methyl tert-butyl ether (MTBE) containing 10 mg of iso-butyl benzene as an internal standard [4]. The samples were shaken vigorously for 2 h at room temperature. The extracts were dried by passing them through a small column (Pasteur pipette) plugged with glass wool containing sodium sulfate and then concentrated under a gentle nitrogen stream to a volume of ~0.5 ml. 2.3. Chiral chromatography Samples of 1 ml of the concentrated MTBE extracts were analyzed on a Hewlett–Packard GCD gas chromatograph
equipped with a Restek Rt-bDEXsm fused silica column (30 m 0.25 mm). Injector and detector temperatures were 230 8C. The following temperature program was used: 55 8C for 1 min, 50–200 8C at 1.5 8C/min; carrier gas was He at 1 ml/min [26]. R-Linalool extracted from basil essential oil (from our collection, 26) and synthetic linalool (Roth Chemical Co. Karlsruhe, Germany) containing S-(+)-linalool and R-()-linalool were used as standards [32]. 2.4. Enzyme extraction and partial purification Three to five fruits were randomly sampled at each ripening stage, and the pulp was separated from the peel. Fresh pulp from the fruit center was frozen with liquid nitrogen and kept at 70 8C for characterization of the enzyme and determination of enzyme activity. Frozen samples (2 g) were ground with a pestle in a chilled mortar in liquid nitrogen, 0.5 g sand and 0.1 g polyvinylpolypyrrolidone (PVPP) until a uniform powder was obtained. Ice-cold extraction buffer (1:4 w/v), consisting of 50 mM bis–Tris pH 6.9, 10% (v/v) glycerol, 10 mM dithiothreitol (DTT), 5 mM Na2S2O5 and 1% (w/v) polyvinylpyrrolidone (PVP-40 or PVP-10) was added. In the initial experiments, we used 50 mM 3-morpholinopropanesulfonic acid (MOPS), pH 6.9, instead of bis–Tris, with comparable results. The slurry was centrifuged at 20,000 g for 15 min at 4 8C. The supernatant was further purified through a P-6 column (1 10 cm) (Bio-Rad, bio-gel desalting gel 90–180 mm, BioRad, Munich, FRG) to remove interfering low-molecularweight compounds. We used buffer B (the above-mentioned extraction buffer without PVP-40) for column equilibration and protein elution. Fractions (1 ml) were tested for enzyme activity, and the active fractions were combined and kept at 20 8C for further analysis. To effectively remove interfering polysaccharides that increased the viscosity of the cell-free extracts, especially those derived from immature fruits, the extracts were further purified by anion exchange (diethylaminoethyl cellulose, DE52, Whatman, Maidstone, UK) batch chromatography. Before use, the DE52 was pre-equilibrated with 1 M KCl and washed thoroughly with loading buffer [25 mM MOPS, pH 6.9, 3.3 mM dithiothreitol, and 5% (v/v) glycerol]. One milliliter of DE52 resin, 2 ml of crude cell-free extracts (0.2–0.4 mg protein/ml) and 8 ml of loading buffer were mixed gently and incubated in an ice-bath for 15 min. Then, the supernatant was discarded, and the resin was washed with 4 ml of loading buffer, mixed gently, and allowed to stand for a further 10 min on ice. This procedure was repeated twice using loading buffer containing 0.1 M KCl. The resin was then washed with 2 ml of loading buffer containing 0.5 M KCl to elute the enzyme activity. The final concentration of KCl in the enzyme assays was below 50 mM. Protein was determined with the Bio-Rad protein assay reagent (Bio-Rad, Munich, FRG) using bovine serum albumin as standard.
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2.5. Enzyme assays We used a large-scale GC–MS-based assay for the identification of the products, co-factor requirements and optical purity and a small-scale radioactive assay for the developmental studies and molecular weight determination. 2.5.1. GC–MS large-scale assay Cell-free extracts (200 ml containing 1–4 mg protein) were incubated with 10 mM MgCl2, 1 mM MnCl2, and 13.7 mM non-radioactive GDP in sample buffer (50 mM bis–Tris pH 6.9, 10% (v/v) glycerol, 10 mM dithiothretiol and 5 mM Na2S2O5) in a total volume of 2 ml, and overlaid with 2 ml of hexane. After an overnight incubation at 34 8C, 10 mg of iso-butyl benzene was added as an internal standard. The samples were vigorously vortexed and centrifuged (clinical) for 2 min to separate the phases. The samples were the re-extracted with 2 ml of hexane three times. The upper hexane phases were pooled and dried by passing them through a small (Pasteur pipette) column, containing sodium sulfate, and then concentrated to a volume of 0.2 ml under a gentle stream of nitrogen. Appropriate controls included the omission of substrate, omission of metal cofactors, omission of enzyme extract and the use of heat-inactivated enzyme extracts. A 1 ml aliquot of the concentrated hexane layers was injected into a Hewlett Packard GCD gas chromatograph equipped with a HP5 (30 m 0.25 mm) fused-silica capillary column. Helium (1 ml/min) was used as a carrier gas with splitless injection. The injector temperature was 250 8C, and the detector temperature was 280 8C. The conditions used were as follows: initial temperature was 70 8C for 2 min followed by a ramp of 70–200 8C at a rate of 4 8C/min, and additional 10 min at 200 8C. Masses between 45 and 450 m/z were recorded. Linalool and geraniol were identified by co-injection and comparison of the EI–MS obtained with authentic standards and complemented with computerized libraries. Similar preparations were subjected to chiral chromatography (see above) for the determination of the enantiomeric composition of the enzymatically-generated linalool. 2.5.2. Radioactive small-scale assays for enzyme characterization and developmental studies Small-scale assays were performed by diluting 10 ml of enzyme extracts in 60 ml of buffer containing 50 mM MOPS pH 6.6, 10% (v/v) glycerol, 1 mM MnCl2, 10 mM MgCl2, 6.6 mM dithiothreitol, and 20 ml [1-3H]GDP (3 mM final concentration, sp. ac. 15 Ci/mmol) and overlaying the mixture with 1 ml of hexane to trap volatiles. The tubes were then briefly mixed, centrifuged at 14,000 g for a few seconds, and incubated at 30 8C for 2 h. The samples were mixed vigorously and centrifuged for 1 min at 20,000 g to separate the phases. Then, 0.8 ml of the upper hexane phase containing the in vitro formed radioactively labeled enzyme products was transferred to a 5 ml scintillation tube containing 3 ml of scintillation
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fluid (4 g/l 2,5-diphenyloxazol (PPO) and 0.05 g/l 2,20 -pphenylen-bis (5-phenyloxazol (POPOP) in toluene). The radioactivity was quantified using a liquid scintillation counter (Kontron model 810, Zurich, Switzerland). Enzyme activity in picokatals was calculated on the basis of the specific activity of the substrate and taking into account the appropriate correction factors for the presence of geraniol and the counting efficiency of the scintillation machine. Enzyme activity was linear with respect to protein concentration and incubation time. 2.6. Determination of the native molecular mass of LIS The desalted enzyme, after P6 chromatography, was filtered through a 0.2 mm cellulose acetate membrane (ca462200, hleicher & Schuell, Dassel, Germany) and separated by gel permeation chromatography using a Superdex 75 HiLoad 16/60 column (FPLC Amersham Pharmacia Biotech, Uppsala, Sweden), using buffer C consisting of 25 mM bis–Tris, pH 6.9, 150 mM KCl, 5% (v/v) glycerol, 5 mM dithiothretiol and 2.5 mM Na2S2O5 at a flow rate of 1 ml/min. The elution of LIS activity was monitored with the small-scale radioactive assay, and the molecular mass of LIS was determined by comparison with molecular masses of known proteins [alcohol dehydrogenase (MW ~150,000), phosphorylase b (MW ~97,000), bovine serum albumin (MW ~66,000), ovalbumin (MW ~53,000), carbonic anhydrase (MW ~29,000), cytochrome C (MW ~12,400), and aprotinin (MW ~6,500), all from Sigma–Aldrich Chemical Co, St. Louis, MO, USA].
3. Results and discussion 3.1. Enantiomeric composition The optical purity of the linalool, the main volatile accumulating in ripening koubo fruit [4], was assessed by chiral GC analysis. S-(+)-Linalool was found in a high optical purity of 98% (Fig. 1A). In most fruits containing linalool, both enantiomers are present. In apricots, plums, passion fruits and pineapples, for example, both enantiomers are present in roughly similar levels [17], but in other fruits one of the enantiomers may be dominant. High enantiometric excess (98%) of S-(+)-linalool has been found in strawberries [17] and of R-(–)-linalool, in guava, peach and lulo [17]. In cactus pear, R-(–)-linalool is present in an enantiomeric excess of 36% [22]. It is known that acidic conditions may cause racemization linalool, especially at high temperatures [33]. Since koubo fruit pulp is acidic (pH 4.5–6) and the fruits are exposed to high temperatures during ripening [4], it was expected that linalool would loose its optical purity in ripe koubo fruits. The surprising finding that the optical purity was high (Fig. 1A) may be explained by cellular and subcellular compartmentation. [34,35].
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Fig. 1. Enantiomeric analyses of linalool by chiral chromatography: (A) linalool enantiomers accumulating in ripe koubo fruit; (B) linalool enantiomers synthesized in vitro from GDP by cell-free extracts; (C) linalool formed non-enzymatically by the addition of 10 mM FeSO4 to the assay mixtures; (D) enantiomer standards.
3.2. Partial characterization of koubo linalool synthase activity Soluble cell-free extracts obtained from koubo fruit pulp converted GDP into linalool, as determined by GC– MS (Fig. 2A). Linalool was not found in the absence of GDP (Fig. 2B) or in the presence of heat-inactivated LIS
(not shown). The divalent metal cofactor requirements of the koubo LIS were determined. The addition of MgCl2 was apparently sufficient for catalysis (Km = 60 mM). Mn2+ ions promoted catalysis at a similar maximal rate, but with a much lower Km (5.6 mM). Monoterpene synthases require divalent metal ions for activity, usually Mg2+ or Mn2+ [35,36]. The C. breweri LIS requires Mn2+ for activity (Km = 45 mM), but Mg2+ can also act as a co-factor, at half of the rate of Mg2+ with a 10-fold higher Km (330 mM) [27]. Other metal cofactors that did not support catalysis included Cu2+, Co2+ and Ca2+, tested at 10 mM. The addition of FeCl2 or FeSO4 at 1–10 mM supported the formation of linalool, even in the absence of enzyme, but this linalool was racemic (see below). The apparent Km for GDP of koubo LIS is 18 mM, which is in the range of most other monoterpene synthases. C. breweri and Mentha citrata LIS have Km values for GDP of 0.9 and 56 mM, respectively [27,30]. In some experiments, we noticed the formation of geraniol. This could be due to the action of the phosphatases that are ubiquitous to many plant tissues [36]. Under our optimized assay conditions, the levels of geraniol were usually negligible. Since linalool accumulated in the pulp in high optical purity (Fig. 1A), it was of interest to determine the optical purity of the linalool formed in vitro from GDP by the action of enzymatic preparations from koubo fruits. The newly generated linalool was found to be 85% enantiomerically pure, the S-isomer being dominant (Fig. 1B). Clarkia LIS produces the S-isomer exclusively [27]. The Mentha recombinant enzyme produces 98% R-linalool and 2% of the S-enantiomer [30]. The enantiomeric purity of the linalool extracted from the fruit (Fig. 1A) and the linalool formed in vitro from GDP (Fig. 1B) were not identical. It could be that our extraction conditions modified the enzyme activity, thus lowering its enantiomeric specificity. Alternatively, our assay conditions might not reliably mimic the conditions prevailing in vivo in terms of substrate, cofactor, temperature and pH requirements. Another explanation could be that two enzymes with different stereo-selectivities exist in the fruit and compete for the GDP. Moreover, the non-enzymatically formed linalool, generated in the presence of Fe2+ was racemic (Fig. 1C). It could be that sufficient endogenous Fe2+ levels support the formation of low levels of R- and S-linalool, both in vivo and enzymatically. Further characterization of the enzyme(s) coupled with the isolation of the gene(s) that code for this activity will contribute to our understanding of the factors that affect the optical specificity of the linalool formed and accumulated in koubo fruits. 3.3. LIS native molecular mass
Fig. 2. Identification of linalool generated in vitro from GDP by cell-free extracts of koubo fruit: (A) complete reaction (0.2 mM GDP, 10 mM MgCl2, and 1 mM MnCl2). The mass spectrum of biosynthetic linalool is shown in the insert; (B) control assays devoid of the substrate GDP. IBB: iso-butyl benzene (internal standard).
The native molecular mass of koubo LIS was about 53 kDa, as determined by gel permeation chromatography (Fig. 3). This value falls in the range of other plant monoterpene synthases having molecular masses of 50–70 kDa.
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aroma of koubo fruit. LIS therefore plays a pivotal role in determining the aroma characteristics of this fruit. Elucidating the mechanisms regulating LIS activity and expression during fruit ripening and storage are of critical relevance to our ability to understand and control fruit quality.
Acknowledgments Fig. 3. Molecular mass determination of LIS activity from koubo fruit. The desalted enzyme was separated by gel permeation chromatography, and the activity eluted corresponded to a molecular mass of 53 kDa (by comparison with known protein standards).
The C. breweri LIS, for example, is a 76 kDa monomeric protein [27]. 3.4. LIS activity during fruit development and storage Since significant increases in linalool and its derivatives have been found in koubo fruits during the later stages of fruit ripening and during storage [4,8], it was of interest to determine the changes in LIS activity levels under these conditions. Negligible LIS activity was found during the green and breaker stages of fruit development (Fig. 4). A prominent increase in LIS activity was first discernible during the violet stage, in accordance with the timing of the initial detection of linalool [4]. LIS activity further increased during ripening, reaching about 4 nkat/mg protein in the cracked red fruit. The highest LIS activity was found in fruit ripening in storage, reaching values of about 6 nkat/ mg protein (Fig. 4). These findings are in accordance with the high linalool levels accumulating in fruit ripening in storage as compared with the lower linalool levels present in fruit ripening on the tree [4,8]. We have shown that S-linalool and other linalool derivatives are the main volatiles contributing to the unique
Fig. 4. Increases in LIS activity during koubo fruit ripening and storage. LIS activity was determined at different stages of fruit development on the tree (mature green, breaker, violet and red cracked) and after storage (red stored). Values are means obtained for three individual fruits S.E. The experiment was repeated three times with similar results.
We thank Arnon Ronen and Moshe Ventura for kindly supplying the fruits. We thank Inez Mureinik for editing the manuscript. Publication No. 113/2004 of the Agricultural Research Organization, Bet Dagan, Israel.
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