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Post-harvest enhancement of aroma in transgenic lisianthus (Eustoma grandiflorum) using the Clarkia breweri benzyl alcohol acetyltransferase (BEAT) gene
Postharvest Biology and Technology 43 (2007) 255–260
Post-harvest enhancement of aroma in transgenic lisianthus (Eustoma grandiflorum) using the Clarkia breweri benzyl alcohol acetyltransferase (BEAT) gene Dina Aranovich a , Efraim Lewinsohn b , Michele Zaccai a,c,∗ a
Department of Life Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel Department of Vegetable Crops, Agricultural Research Organization (ARO), Newe Ya’ar Research Center, P.O. Box 1021, Ramat Yishay 30095, Israel Department of Biotechnology Engineering, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel b
c
Received 21 May 2006; accepted 3 September 2006
Abstract Lisianthus (Eustoma grandiflorum) is an ornamental plant with beautiful but scentless flowers. In an attempt to induce a fragrance in their flowers, lisianthus plants were transformed with the Clarkia breweri gene coding for benzyl alcohol acetyltransferase (BEAT), catalyzing the synthesis of the volatile compound benzyl acetate under the regulation of the CaMV35S promoter. An external supply of benzyl alcohol induced five to seven times higher production of benzyl acetate in detached flowers and leaves of transgenic lisianthus plants, compared to non-transformed plants. No benzyl acetate was detected in tissues of both control and transgenic plants fed with water. When fed with additional alcoholic compounds, i.e. hexanol, benzyl alcohol, isoamyl alcohol, phenethyl alcohol, and cinnamyl alcohol, assumed to be used as substrates by BEAT, transgenic in vitro-grown lisianthus plantlets produced significantly higher levels of acetates than control plants. These results demonstrate the possibility of producing substrate-dependent acetates in transgenic lisianthus plants, which could lead to induction of new aromas. © 2006 Elsevier B.V. All rights reserved. Keywords: Lisianthus; Fragrance; Transformation; Volatiles
1. Introduction Flower scent is a composite character, determined by a complex mixture of low molecular weight volatile molecules emitted by floral organs. Fragrance compounds play numerous roles in the interactions between plants and their surroundings, the major one being to attract pollinators (Dudareva and Pichersky, 2000). Humans as well are attracted by fragrant flowers; however, most modern cut flowers such as roses (Lavid et al., 2002) and carnations (Lavy et al., 2002) have poor aromas. Metabolic engineering of flower fragrance, ∗ Corresponding author at: Bergmann Campus, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel. Tel.: +972 8 646 1898; fax: +972 8 647 2984. E-mail address: [email protected] (M. Zaccai).
0925-5214/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2006.09.001
based on the introduction of foreign genes encoding enzymes with activities that are missing in the target plant, may allow new branching of existing pathways as well as generation of novel pathways. As a result, synthesis of volatile molecules and their emission can occur in the plant of interest (Vainstein et al., 2001). Several attempts of genetic manipulation of plant volatile composition have been made using monoterpene synthase genes as floral scent genes introduced to plants (Aharoni et al., 2005). Petunia plants were transformed with the Clarkia breweri gene coding for linalool synthase (LIS), catalyzing the formation of the monoterpene linalool, under the control of the Cauliflower Mosaic Virus 35S promoter (CaMV 35S) (Lavy et al., 2002). Introduction of LIS into petunia did not result in linalool emission; instead, the non-volatile linalool glycoside accumulated in the transgenic plants (Lucker et al.,
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2001). Introduction of a similar LIS construct into carnation led to the emission of linalool from petals as well as from leaves (Lavy et al., 2002), but no detectable olfactory change in flower scent was perceived. LIS was also expressed in transgenic tomato fruit under the late-ripening specific promoter E8, aiming to improve fruit flavor (Lewinsohn et al., 2001). In this case, expression of the transgene generated (S)-linalool and 8-hydroxylinalool production in ripening fruit, without apparently altering the biosynthesis of other terpenoids. In recent research, genes coding for three different monoterpene synthases: TER-␥-terpinene synthase, LIM-limonene synthase, and PIN-pinene synthase, were over-expressed simultaneously in tobacco plants. Volatile derivates of these genes and a number of their derivatives were detected in volatile emissions of transgenic plants, inducing a dramatic change in the fragrance profile (Beekwilder et al., 2004). Another family of enzymes producing volatile compounds that can be used for metabolic engineering of plants is the alcohol acyltransferases (AAT), catalyzing the last step in volatile ester formation by transacylation from an acyl-CoA to an alcohol. Members of this family have been cloned and characterized in several plant species. Some of these enzymes catalyze the formation of volatile esters, including benzyl alcohol benzoyltransferase (BEBT) from C. breweri flowers, acetyl-CoA:cis-3-hexen-1-ol acetyltransferase (CHAT) from Arabidopsis thaliana (D’Auria et al., 2002), which might be involved in the formation of cis-3-hexenyl acetate emitted upon injury, and Rosa hybrida alcohol acetyltransferase (RhAAT1), defined by Shalit et al. (2003) to be responsible for the production of geranyl and citronellyl acetate in rose flowers. Transgenic petunia plants over-expressing the strawberry alcohol acyltransferase gene (SAAT) showed enzyme activity, but the volatile profile remained unaltered. However, feeding isoamyl alcohol to transgenic explants resulted in the emission of the corresponding acetyl ester (Beekwilder et al., 2004). Transgenic petunia plants expressing the rose alcohol acetyltransferase gene under the control of a CaMV 35S promoter used several alcoholic substrates to produce the corresponding acetate esters, suggesting the dependence of volatile production on substrate availability (Guterman et al., 2006). The gene coding for CoA:benzylalcohol acetyltransferase (BEAT) from C. breweri flowers was the first AAT family member to be characterized (Dudareva et al., 1998a). BEAT catalyses the synthesis of benzyl acetate, which constitutes up to 40% of C. breweri’s total scent output (Dudareva et al., 1998b). In the current research, lisianthus (Eustoma grandiflorum) was selected as a target plant for the manipulation of a metabolic pathway, with the aim of modifying its volatile profile and inducing fragrance in the transgenic plants. Lisianthus is an ornamental plant with an impressive bloom, long stems, and extended vase-life. These features, as well as the large variety of available cultivars, have made lisianthus an attractive ornamental crop with increasing suc-
cess as a cut flower. In the last few years, research has been devoted to the investigation and improvement of lisianthus by genetic engineering, such as regulation of floral transition (Zaccai et al., 2001; Zaccai and Edri, 2002), or production of lisianthus flowers with altered pigment metabolic pathways (Deroles et al., 1998). Glowing lisianthus flowers have also been produced by expressing Aequorea victoria green fluorescent protein (GFP) in transgenic plants (Mercuri et al., 2001). As lisianthus flowers are totally scentless, we attempted to manipulate biosynthetic pathways by transgenic means aiming to induce fragrance in lisianthus petals by over-expressing the BEAT gene from C. breweri, using transformation methods previously reported (Semeria et al., 1996; Zaccai et al., 2001). Flowers and leaves of transformed plants produced volatile products when fed with alcoholic substrates.
2. Materials and methods 2.1. Plant material Lisianthus (E. grandiflorum) seeds, cultivar Royal Pink, were obtained from a nursery. Seeds were washed with a 3% sodium hypoclorite solution and seeded on Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) in test tubes. Leaf segments from sterile plants were used for transformation. 2.2. BEAT construct C. breweri BEAT cDNA was cloned into pBI binary plasmid under control of the CaMV 35S promoter (35S::BEAT) to form pBI101 used for transformation (this vector was kindly supplied by Prof. Eran Picherky). The plasmid was introduced in Agrobacterium tumefaciens strain EHA105 and used for plant transformation. 2.3. A. tumefaciens-mediated lisianthus transformation Agrobacterium transformation of lisianthus leaf disks and regeneration of resistant shoots on selective medium were performed as previously described (Zaccai et al., 2001). Rooting was carried out on 1/2 MS under non-selective conditions. Acclimation of the resistant rooted plantlets was carried out in aerated boxes (9.6 cm × 9.6 cm × 9 cm) in the growth room, prior to their transplantation to pots in a controlled greenhouse. 2.4. DNA extraction Lisianthus tissue samples (5–15 mg) were ground with a small pestle in a microtube in 400 L extraction buffer (250 mM NaCl, 25 mM EDTA, 0.5% SDS, 200 mM Tris–HCl, pH 7.5) and sand (Sigma). After centrifugation at 8765 × g for 2 min, the upper fraction was collected,
D. Aranovich et al. / Postharvest Biology and Technology 43 (2007) 255–260
300 L cold isopropanol was added and mixed for 5 min. DNA was precipitated by centrifugation (8765 × g for 5 min). The pellet was washed with 300 L cold ETOH 95%. After centrifugation (8765 × g for 15 min) the DNA pellet was dissolved in 100 L DDH2 O. 2.5. PCR The specific primers (5 -GAGGCTATTCGGCTATGACT-3 ; 5 -AATCTCGTGATGGCAGGTTG-3 ) within the nptII gene were used for PCR amplification from lisianthus genomic DNA. PCR conditions: 95 ◦ C, 5 min followed by 40 cycles of (1) 95 ◦ C, 1 min, (2) 54 ◦ C, 1 min, (3) 75 ◦ C, 3 min; 75 ◦ C, 10 min.
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(Aglient Technologies, Germany) equipped with an Rtx-5SL (RESTEK, 30 m × 0.25 mm) column. Helium was used as a carrier gas with splitless injection. The injector temperature was 250 ◦ C and the detector temperature was 280 ◦ C. Conditions used were as follows: initial temperature was 50 ◦ C for 1 min, then the temperature was increased to 250 ◦ C at a rate of 5 ◦ C/min. Running time was 48 min. Masses between 41 and 350 m/z were recorded. Identification of the main components was done by co-injection and comparison of the electron ionization-MS obtained with authentic standards and complemented with computerized Wiley libraries using KI (Kovatc retention index) (Lewinsohn et al., 1998; Shalit et al., 2001). 2.10. Statistical analyses
2.6. Headspace analysis of volatile compounds Statistical analyses were performed by the t-test. Intact individual lisianthus flowers were enclosed in a 1 L glass container with the appropriate openings and headspace was trapped, eluted, and concentrated using a method modified from Raguso and Pichersky (1995), using a Porapak Q 80/100 polydivinylbenzene filter (Waters, Milford, MA) for 12 h. Volatiles were eluted using 10 mL of HPLC grade hexane containing 10 g mL−1 ethylmyristate as an internal standard and evaporated to 0.5 mL. One microliter from each sample was analyzed by GC–MS. 2.7. Benzyl acetate detection from lisianthus tissues Leaves from the second internode from the apex and flowers (at anthesis) of transgenic and control adult plants (about 8–10 months after transformation) were placed in 10 mL tubes containing 5 mL of either DDW or DDW + 0.1 mM benzyl alcohol for 24 h, at room temperature and 10 h light conditions. One gram of tissue was ground with a mortar and pestle and extracted with methyl tert-butyl ether (MTBE) by shaking for 12 h at room temperature with 5 g ethyl myristate added as an internal standard. The samples were dried with anhydrous Na2 SO4 and concentrated with a gentle stream of N2 utilizing a Turbo Vap II evaporator (Zymark Corp., Hopkinton, MA) to a volume of 0.5 mL. Each sample was performed in three replicates.
3. Results 3.1. Transformation of lisianthus with BEAT cDNA Leaf segments of in vitro-grown lisianthus plants, were transformed with the 35S::BEAT construct. Kanamycinresistant regenerated shoots were rooted under non-selective conditions. Regeneration efficiency (number of shoots regenerated from leaf segments) was 10.5%. PCR analysis performed on regenerated plantlets revealed that 61% contained the construct. These plantlets were transferred to the greenhouse and 55% of them successfully developed to adult flowering plants (Table 1). The phenotype of transgenic and control plants was similar (Fig. 1). 3.2. Headspace analysis Headspace analysis of control and transgenic intact lisianthus flowers was performed to reveal the presence of benzyl acetate. No benzyl acetate was detected by this method in any of the samples, possibly due to the low levels of the BEAT substrate-benzyl alcohol in lisianthus flowers. Therefore, the effect of external substrate was tested in feeding experiments.
2.8. Substrate preference assay Transgenic and non-transformed control plantlets grown in vitro were placed in 10 mL covered vials containing 5 mL of either DDW or DDW + 0.1 mM alcoholic substrate (hexanol, benzyl alcohol, isoamyl alcohol, phenethyl alcohol, or cinnamyl alcohol) for 24 h. 2.9. Analysis and identification of volatile metabolites for product detection Samples consisting of 0.5 mL of the concentrated MTBE extracts were analyzed on a MSD gas chromatograph
3.3. Feeding experiments with benzyl alcohol as a substrate to BEAT Detached leaves and flowers of fully developed plants (transgenic and control) were fed for 24 h with DDW or Table 1 Transformation and regeneration efficiency in lisianthus Procedure
Number of plants
Percent success
Transformation Rooting Hardening
239 198 39
10.5 83.0 55.0
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Fig. 1. Non-transformed (control) and transgenic (BEAT) adult flowering plants showing the same phenotype.
10 mM benzyl alcohol solution and subsequently analyzed for benzyl acetate production. Leaves of transgenic lisianthus plants produced abundant amounts of benzyl acetate when fed with benzyl alcohol. No benzyl acetate was detected in leaves of either control or transgenic plants when fed with water only (Fig. 2A). Benzyl acetate production was five to seven times higher in leaves of transgenic plants as compared to control plants (Fig. 2A). Benzyl acetate amounts in detached flowers of transgenic plants were lower than in detached leaves (Fig. 2B). Benzyl acetate was not detected in control plants or in extracts of flowers fed with water (Fig. 2B). 3.4. Feeding experiments with additional alcoholic substrates C. breweri BEAT enzyme is known for being able to catalyze the production of acetate from different alcoholic substrates (Dudareva et al., 1998b). In order to test alcoholic substrate preference of the BEAT enzyme expressed in lisianthus transgenic plants, small in vitro-grown transgenic and control plantlets were fed with different alcoholic substrates: hexanol, benzyl alcohol, isoamyl alcohol, phenethyl alcohol, and cinnamyl alcohol. Levels of acetates were analyzed in extracts of transgenic and control plantlets after feeding. The highest level of acetates were produced by transgenic plantlets fed with hexanol and the resulting hexyl acetate levels were significantly higher (p < 0.05) than the acetates levels produced by transgenic plantlets fed with the other alcoholic substrates, except cinnamyl alcohol (Fig. 3). The lowest lev-
Fig. 2. Feeding of transgenic lisianthus with BEAT substrate benzyl alcohol. (A) Levels of benzyl acetate in MTBE extracts of lisianthus leaves from transgenic (25M, 69, 26b, K67) and control (WT) plants after 24 h feeding with benzyl alcohol (BA) or water (H2 O). (B) Levels of benzyl acetate in MTBE extracts of lisianthus flowers: transgenic (25M, 83, 26b, T30) and control (WT) after feeding with benzyl alcohol (BA) or water (H2 O). Bars represent S.E.
els of acetates were produced by transgenic plantlets fed with benzyl alcohol and isoamyl alcohol and did not differ significantly (p < 0.05) from each other (Fig. 3). Control plantlets fed with isoamyl, phenylethyl, or cinnamyl alcohol produced low amounts of acetates (Fig. 3). Transgenic plantlets fed with the same substrates produced two to three times more acetate products (Fig. 3). No acetates were detected when control plantlets were fed with hexanol or benzyl alcohol. For each substrate, the amount of the respective acetate produced by transgenic plantlets was significantly higher (p < 0.05) than the acetate produced by control plants.
4. Discussion We used the C. breweri BEAT cDNA under the control of the CaMV 35S promoter to transform lisianthus plants, aiming to induce scents in all plant tissues. Benzyl acetate is a major compound (about 40%) of the volatile composition of C. breweri aroma (Dudareva et al., 1998a), and of fragrant flowers such as gardenia and jasmine (Christensen et al., 1997; Buchbauer et al., 1996). Fruit such as melons (Shalit
D. Aranovich et al. / Postharvest Biology and Technology 43 (2007) 255–260
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Fig. 3. Levels of acetate products in MTBE extracts of transgenic (K67) and untransformed control (WT) lisianthus plantlets after feeding with water or different alcoholic substrates: hexanol, benzyl alcohol, isoamyl alcohol, phenethyl alcohol, cinnamyl alcohol. Bars represent S.E.
et al., 2001) also emit benzyl acetate in high amounts, which largely contributes to their special aroma. Consequently, the BEAT gene was considered a good candidate for transformations of plants to generate aromas in a non-scented flower. The transformation of lisianthus with BEAT was successfully performed by means of A. tumefaciens. Transformation was confirmed by PCR using nptII-specific primers, as PCR reactions with BEAT-specific primers resulted in non-specific amplification probably caused by the presence of different AAT family genes in lisianthus. Characterization of transgenic plants did not reveal phenotypic differences (Fig. 1). However, no change in flower fragrance and no difference in volatile composition of headspace samples was detected in transgenic plants compared to control plants by olfactory or headspace analysis. We tested the hypothesis that absence of benzyl acetate production could be due to insufficient BEAT substrate, as was previously proposed in other systems (Beekwilder et al., 2004). Therefore, leaves and flowers of control and transgenic lisianthus plants were fed benzyl alcohol (Fig. 2A and B). Substrate-fed transgenic plants produced 2–3 g benzyl acetate/g leaves and 4–8 ng/g flowers. No benzyl acetate was detected in DDW-fed transgenic tissues. It is noteworthy that leaves of control plants fed with BEAT substrates also produced benzyl acetate, although in much lower quantity than transgenic tissues (Fig. 2A), suggesting the presence of a BEAT-like enzyme in lisianthus. These results demonstrate that lack of sufficient substrate was at least one of the reasons for the absence of benzyl acetate in transgenic plants. Identification of enzymes involved in substrate production and their transformation into plants together with genes involved in scent production could address the problem of lack of substrate and lead to fragrance production in transgenic plants. On the other hand, the possibility of using alternative genes involved in aroma production (Galili et al., 2001) should be further investigated. It is suggested that alcoholic compounds, such as benzyl alcohol, could be integrated more effectively by existing
biochemical pathways and enzymes of lisianthus rather than by BEAT from C. breweri. This would then lead to lower amounts of produced acetate. Exposing transgenic lisianthus tissue to alternative alcoholic substrates may bring about higher production of acetates. We tested the effect of feeding different alcoholic substrates for BEAT in transgenic lisianthus plants (Fig. 3). Alcohol acetyltransferases (including BEAT) release acetate derivatives from their respective alcohols and acetyl-CoA. Some of the compounds fed in this study, such as benzyl alcohol, cinnamyl alcohol and to a lesser extent phenethyl alcohol, were chosen because it is known that BEAT can use them as substrates (Dudareva et al., 1998b). Hexanol and isoamylalcohol, the precursor of isoamylacetate, with a typical banana scent were chosen to broaden the selection. It is likely that the acetates released were directly derived from BEAT activity in the transgenic plant tissues, and from a similar (yet uncharacterized) activity in control, non-transformed plants. The results showed that feeding transgenic lisianthus plantlets with hexanol, phenylethyl alcohol, and cinnamyl alcohol induced significantly higher levels of acetate products than feeding with benzyl alcohol and isoamyl alcohol which induced similar results (Fig. 3). The amount of benzyl acetate produced by benzyl alcohol-fed small plantlets (Fig. 3) was about 17-fold lower than by benzyl acetate-fed leaves of the same transgenic line (Fig. 2). This difference may be due to the age of the plant tissue and to the growing conditions. The highest level of acetate product was obtained in hexanol-fed plantlets (Fig. 3). Hexanol might then be the best BEAT substrate for transgenic lisianthus (Fig. 3). Most alcoholic substrates except cinnamylalcohol were not toxic. No damage to tissues, discoloration of leaves, or effects on longevity of plants was observed. This study suggests that fragrances can be induced in usually unscented cut flowers by means of genetic engineering, using different substrates.
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Acknowledgments This work was funded by a grant from the Chief Scientist of the Israel Ministry of Agriculture and Rural Development to MZ and EL. We thank Natalia Dudareva for helpful discussions, Einat Bar for her help with GS–MS analysis and Janina Mutchnik for her help in growing the plants in the greenhouse.
References Aharoni, A., Jongsma, M.A., Bouwmeester, H.J., 2005. Volatile science? Metabolic engineering of terpenoids in plants. Trends Plant Sci. 10, 594–602. Beekwilder, J., Alvarez-Huerta1, M., Neef, E., Verstappen, F., Bouwmeester, H., Aharoni, A., 2004. Functional characterization of enzymes forming volatile esters from strawberry and banana. Plant Physiol. 135, 1865–1878. Buchbauer, G., Jirovetz, L., Nikiforov, A., Kaul, V., Winker, N., 1996. Volatiles of the absolute of Gardenia jasminoides Ellis (Rubiaceae). J. Essent. Oil Res. 8, 211–215. Christensen, L., Jakobsen, H., Kristiansen, K., Moller, J., 1997. Volatiles emitted from flowers by gamma-radiated and not radiated Jasminum polyanthum Franch in situ. J. Agric. Food Chem. 45, 2199– 2203. D’Auria, J.C., Chen, F., Pichersky, E., 2002. Characterization of an acyltransferase capable of synthesizing benzylbenzoate and other volatile esters in flowers and damaged leaves of Clarkia breweri. Plant Physiol. 130, 466–476. Deroles, S.C., Bradley, J.M., Schwinn, K.E., Markham, K.R., Bloor, S., Manson, D.G., Davies, K.M., 1998. An antisense chalcone synthase cDNA leads to novel colour patterns in lisianthus (Eustoma grandiflorum) flowers. Mol. Breeding 4, 59–66. Dudareva, N., D’Auria, J., Nam, K., Raguso, R., Pichersky, E., 1998a. Acetyl CoA:benzylalcohol acetyltransferase: an enzyme involved in floral scent production in Clarkia breweri. Plant J. 14, 297–304. Dudareva, N., Pichersky, E., 2000. Biochemical and molecular genetic aspects of floral scents. Plant Physiol. 122, 627–633. Dudareva, N., Raguso, R., Wang, J., Ross, J., Pichersky, E., 1998b. Floral scent production in Clarkia breweri. III. Enzymatic synthesis and emission of benzenoid esters. Plant Physiol. 116, 599–604. Galili, G., Galili, S., Lewinsohn, E., Tadmor, Y., 2001. Genetic, molecular, and genomic approaches to improve the value of plant foods and feeds. Crit. Rev. Plant Sci. 21, 167–204. Guterman, I., Masci, T., Chen, X., Negre, F., Pichersky, E., Dudareva, N., Weiss, D., Vainstein, A., 2006. Generation of phenylpropanoid pathwayderived volatiles in transgenic plants: rose alcohol acetyltransferase
produces phenylethyl acetate and benzyl acetate in petunia flowers. Plant Mol. Biol. 60, 555–563. Lavid, N., Wang, J., Shalit, M., Guterman, I., Bar, E., Beuerle, T., Menda, N., Zach, A., Sharoni, S., Zamir, D., Vainstein, A., Weiss, D., Pichersky, E., Lewinsohn, E., 2002. O-Methyltransferases involved in the biosynthesis of volatile phenolic derivatives in rose petals. Plant Physiol. 129, 1899–1907. Lavy, M., Zuker, A., Lewinsohn, E., Larkov, O., Ravid, U., Vainstein, A., Weiss, D., 2002. Linalool and linalool oxide production in transgenic carnation flowers expressing the Clarkia breweri linalool synthase gene. Mol. Breeding 9, 103–111. Lewinsohn, E., Dudai, N., Tadmor, Y., Katzir, I., Ravid, U., Putievsky, E., Joel, D.M., 1998. Histochemical localization of citral accumulation in lemongrass leaves (Cymbopogon citratus (DC.) Stapf., Poaceae). Ann. Bot. 81, 35–39. Lewinsohn, E., Shalekchet, F., Wilkinson, J., Matsui, K., Tadmor, Y., Nam, K.H., Amar, O., Lastochkin, E., Larkov, O., Ravid, U., Hiatt, W., Gepstein, S., Pichersky, E., 2001. Enhanced levels of aroma and flavor compound S-linalool by metabolic engineering of the terpenoid pathway in tomato fruits. Plant Physiol. 127, 1256–1265. Lucker, J., Bouwmeester, H.J., Schwab, W., Blaas, J., van der Plas, L., Verhoeven, H.A., 2001. Expression of Clarkia S-linalool synthase in transgenic petunia plants results in the accumulation of S-linalyl--d glucopyranoside. Plant J. 27, 315–324. Mercuri, A., Sacchetti, A., De Benedetti, L., Schiva, T., Alberti, S., 2001. Green fluorescent flowers. Plant Sci. 162, 647–654. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497. Raguso, R.A., Pichersky, E., 1995. Floral volatiles from Clarkia breweri and C. concinna (Onagraceae): recent evolution of floral scent and moth pollination. Plant Syst. Evol. 194, 55–67. Semeria, L., Ruffoni, B., Rabaglio, M., Genga, A., Vaira, A., Accotto, G., 1996. Genetic transformation of Eustoma grandiflorum by Agrobacterium tumefaciens. Plant Cell Tissue Org. Culture 47, 67–72. Shalit, M., Guterman, I., Volpin, H., Bar, E., Tamari, T., Menda, N., Adam, Z., Zamir, D., Vainstein, A., Weiss, D., Pichersky, E., Lewinsohn, E., 2003. Volatile ester formation in roses. Identification of an acetyl-coenzyme A: geraniol/citronellol acetyltransferase in developing rose petals. Plant Physiol. 130, 1868–1876. Shalit, M., Katzir, N., Tadmor, Y., Larkov, O., Burger, Y., Shalechet, F., Lastochkin, E., Ravid, U., Amar, O., Edelstein, M., Karchi, Z., Lewinsohn, E., 2001. Acetyl CoA:alcohol acetyltransferase activity and aroma formation in ripening melon fruits. J. Agric. Food Chem. 49, 794–799. Vainstein, A., Lewinsohn, E., Pichersky, E., Weiss, D., 2001. Floral Fragrance. New inroads into an old commodity. Plant Physiol. 127, 1383–1389. Zaccai, M., Edri, N., 2002. Floral transition in lisianthus (Eustoma grandiflorum). Sci. Hortic. 95, 333–340. Zaccai, M., Lewinsohn, E., Pichersky, E., 2001. Modifing lisianthus traits by genetic engineering. Acta Hortic. 552, 137–142.
Post-harvest enhancement of aroma in transgenic lisianthus (Eustoma grandiflorum) using the Clarkia breweri benzyl alcohol acetyltransferase (BEAT) gene Dina Aranovich a , Efraim Lewinsohn b , Michele Zaccai a,c,∗ a
Department of Life Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel Department of Vegetable Crops, Agricultural Research Organization (ARO), Newe Ya’ar Research Center, P.O. Box 1021, Ramat Yishay 30095, Israel Department of Biotechnology Engineering, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel b
c
Received 21 May 2006; accepted 3 September 2006
Abstract Lisianthus (Eustoma grandiflorum) is an ornamental plant with beautiful but scentless flowers. In an attempt to induce a fragrance in their flowers, lisianthus plants were transformed with the Clarkia breweri gene coding for benzyl alcohol acetyltransferase (BEAT), catalyzing the synthesis of the volatile compound benzyl acetate under the regulation of the CaMV35S promoter. An external supply of benzyl alcohol induced five to seven times higher production of benzyl acetate in detached flowers and leaves of transgenic lisianthus plants, compared to non-transformed plants. No benzyl acetate was detected in tissues of both control and transgenic plants fed with water. When fed with additional alcoholic compounds, i.e. hexanol, benzyl alcohol, isoamyl alcohol, phenethyl alcohol, and cinnamyl alcohol, assumed to be used as substrates by BEAT, transgenic in vitro-grown lisianthus plantlets produced significantly higher levels of acetates than control plants. These results demonstrate the possibility of producing substrate-dependent acetates in transgenic lisianthus plants, which could lead to induction of new aromas. © 2006 Elsevier B.V. All rights reserved. Keywords: Lisianthus; Fragrance; Transformation; Volatiles
1. Introduction Flower scent is a composite character, determined by a complex mixture of low molecular weight volatile molecules emitted by floral organs. Fragrance compounds play numerous roles in the interactions between plants and their surroundings, the major one being to attract pollinators (Dudareva and Pichersky, 2000). Humans as well are attracted by fragrant flowers; however, most modern cut flowers such as roses (Lavid et al., 2002) and carnations (Lavy et al., 2002) have poor aromas. Metabolic engineering of flower fragrance, ∗ Corresponding author at: Bergmann Campus, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel. Tel.: +972 8 646 1898; fax: +972 8 647 2984. E-mail address: [email protected] (M. Zaccai).
0925-5214/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2006.09.001
based on the introduction of foreign genes encoding enzymes with activities that are missing in the target plant, may allow new branching of existing pathways as well as generation of novel pathways. As a result, synthesis of volatile molecules and their emission can occur in the plant of interest (Vainstein et al., 2001). Several attempts of genetic manipulation of plant volatile composition have been made using monoterpene synthase genes as floral scent genes introduced to plants (Aharoni et al., 2005). Petunia plants were transformed with the Clarkia breweri gene coding for linalool synthase (LIS), catalyzing the formation of the monoterpene linalool, under the control of the Cauliflower Mosaic Virus 35S promoter (CaMV 35S) (Lavy et al., 2002). Introduction of LIS into petunia did not result in linalool emission; instead, the non-volatile linalool glycoside accumulated in the transgenic plants (Lucker et al.,
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2001). Introduction of a similar LIS construct into carnation led to the emission of linalool from petals as well as from leaves (Lavy et al., 2002), but no detectable olfactory change in flower scent was perceived. LIS was also expressed in transgenic tomato fruit under the late-ripening specific promoter E8, aiming to improve fruit flavor (Lewinsohn et al., 2001). In this case, expression of the transgene generated (S)-linalool and 8-hydroxylinalool production in ripening fruit, without apparently altering the biosynthesis of other terpenoids. In recent research, genes coding for three different monoterpene synthases: TER-␥-terpinene synthase, LIM-limonene synthase, and PIN-pinene synthase, were over-expressed simultaneously in tobacco plants. Volatile derivates of these genes and a number of their derivatives were detected in volatile emissions of transgenic plants, inducing a dramatic change in the fragrance profile (Beekwilder et al., 2004). Another family of enzymes producing volatile compounds that can be used for metabolic engineering of plants is the alcohol acyltransferases (AAT), catalyzing the last step in volatile ester formation by transacylation from an acyl-CoA to an alcohol. Members of this family have been cloned and characterized in several plant species. Some of these enzymes catalyze the formation of volatile esters, including benzyl alcohol benzoyltransferase (BEBT) from C. breweri flowers, acetyl-CoA:cis-3-hexen-1-ol acetyltransferase (CHAT) from Arabidopsis thaliana (D’Auria et al., 2002), which might be involved in the formation of cis-3-hexenyl acetate emitted upon injury, and Rosa hybrida alcohol acetyltransferase (RhAAT1), defined by Shalit et al. (2003) to be responsible for the production of geranyl and citronellyl acetate in rose flowers. Transgenic petunia plants over-expressing the strawberry alcohol acyltransferase gene (SAAT) showed enzyme activity, but the volatile profile remained unaltered. However, feeding isoamyl alcohol to transgenic explants resulted in the emission of the corresponding acetyl ester (Beekwilder et al., 2004). Transgenic petunia plants expressing the rose alcohol acetyltransferase gene under the control of a CaMV 35S promoter used several alcoholic substrates to produce the corresponding acetate esters, suggesting the dependence of volatile production on substrate availability (Guterman et al., 2006). The gene coding for CoA:benzylalcohol acetyltransferase (BEAT) from C. breweri flowers was the first AAT family member to be characterized (Dudareva et al., 1998a). BEAT catalyses the synthesis of benzyl acetate, which constitutes up to 40% of C. breweri’s total scent output (Dudareva et al., 1998b). In the current research, lisianthus (Eustoma grandiflorum) was selected as a target plant for the manipulation of a metabolic pathway, with the aim of modifying its volatile profile and inducing fragrance in the transgenic plants. Lisianthus is an ornamental plant with an impressive bloom, long stems, and extended vase-life. These features, as well as the large variety of available cultivars, have made lisianthus an attractive ornamental crop with increasing suc-
cess as a cut flower. In the last few years, research has been devoted to the investigation and improvement of lisianthus by genetic engineering, such as regulation of floral transition (Zaccai et al., 2001; Zaccai and Edri, 2002), or production of lisianthus flowers with altered pigment metabolic pathways (Deroles et al., 1998). Glowing lisianthus flowers have also been produced by expressing Aequorea victoria green fluorescent protein (GFP) in transgenic plants (Mercuri et al., 2001). As lisianthus flowers are totally scentless, we attempted to manipulate biosynthetic pathways by transgenic means aiming to induce fragrance in lisianthus petals by over-expressing the BEAT gene from C. breweri, using transformation methods previously reported (Semeria et al., 1996; Zaccai et al., 2001). Flowers and leaves of transformed plants produced volatile products when fed with alcoholic substrates.
2. Materials and methods 2.1. Plant material Lisianthus (E. grandiflorum) seeds, cultivar Royal Pink, were obtained from a nursery. Seeds were washed with a 3% sodium hypoclorite solution and seeded on Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) in test tubes. Leaf segments from sterile plants were used for transformation. 2.2. BEAT construct C. breweri BEAT cDNA was cloned into pBI binary plasmid under control of the CaMV 35S promoter (35S::BEAT) to form pBI101 used for transformation (this vector was kindly supplied by Prof. Eran Picherky). The plasmid was introduced in Agrobacterium tumefaciens strain EHA105 and used for plant transformation. 2.3. A. tumefaciens-mediated lisianthus transformation Agrobacterium transformation of lisianthus leaf disks and regeneration of resistant shoots on selective medium were performed as previously described (Zaccai et al., 2001). Rooting was carried out on 1/2 MS under non-selective conditions. Acclimation of the resistant rooted plantlets was carried out in aerated boxes (9.6 cm × 9.6 cm × 9 cm) in the growth room, prior to their transplantation to pots in a controlled greenhouse. 2.4. DNA extraction Lisianthus tissue samples (5–15 mg) were ground with a small pestle in a microtube in 400 L extraction buffer (250 mM NaCl, 25 mM EDTA, 0.5% SDS, 200 mM Tris–HCl, pH 7.5) and sand (Sigma). After centrifugation at 8765 × g for 2 min, the upper fraction was collected,
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300 L cold isopropanol was added and mixed for 5 min. DNA was precipitated by centrifugation (8765 × g for 5 min). The pellet was washed with 300 L cold ETOH 95%. After centrifugation (8765 × g for 15 min) the DNA pellet was dissolved in 100 L DDH2 O. 2.5. PCR The specific primers (5 -GAGGCTATTCGGCTATGACT-3 ; 5 -AATCTCGTGATGGCAGGTTG-3 ) within the nptII gene were used for PCR amplification from lisianthus genomic DNA. PCR conditions: 95 ◦ C, 5 min followed by 40 cycles of (1) 95 ◦ C, 1 min, (2) 54 ◦ C, 1 min, (3) 75 ◦ C, 3 min; 75 ◦ C, 10 min.
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(Aglient Technologies, Germany) equipped with an Rtx-5SL (RESTEK, 30 m × 0.25 mm) column. Helium was used as a carrier gas with splitless injection. The injector temperature was 250 ◦ C and the detector temperature was 280 ◦ C. Conditions used were as follows: initial temperature was 50 ◦ C for 1 min, then the temperature was increased to 250 ◦ C at a rate of 5 ◦ C/min. Running time was 48 min. Masses between 41 and 350 m/z were recorded. Identification of the main components was done by co-injection and comparison of the electron ionization-MS obtained with authentic standards and complemented with computerized Wiley libraries using KI (Kovatc retention index) (Lewinsohn et al., 1998; Shalit et al., 2001). 2.10. Statistical analyses
2.6. Headspace analysis of volatile compounds Statistical analyses were performed by the t-test. Intact individual lisianthus flowers were enclosed in a 1 L glass container with the appropriate openings and headspace was trapped, eluted, and concentrated using a method modified from Raguso and Pichersky (1995), using a Porapak Q 80/100 polydivinylbenzene filter (Waters, Milford, MA) for 12 h. Volatiles were eluted using 10 mL of HPLC grade hexane containing 10 g mL−1 ethylmyristate as an internal standard and evaporated to 0.5 mL. One microliter from each sample was analyzed by GC–MS. 2.7. Benzyl acetate detection from lisianthus tissues Leaves from the second internode from the apex and flowers (at anthesis) of transgenic and control adult plants (about 8–10 months after transformation) were placed in 10 mL tubes containing 5 mL of either DDW or DDW + 0.1 mM benzyl alcohol for 24 h, at room temperature and 10 h light conditions. One gram of tissue was ground with a mortar and pestle and extracted with methyl tert-butyl ether (MTBE) by shaking for 12 h at room temperature with 5 g ethyl myristate added as an internal standard. The samples were dried with anhydrous Na2 SO4 and concentrated with a gentle stream of N2 utilizing a Turbo Vap II evaporator (Zymark Corp., Hopkinton, MA) to a volume of 0.5 mL. Each sample was performed in three replicates.
3. Results 3.1. Transformation of lisianthus with BEAT cDNA Leaf segments of in vitro-grown lisianthus plants, were transformed with the 35S::BEAT construct. Kanamycinresistant regenerated shoots were rooted under non-selective conditions. Regeneration efficiency (number of shoots regenerated from leaf segments) was 10.5%. PCR analysis performed on regenerated plantlets revealed that 61% contained the construct. These plantlets were transferred to the greenhouse and 55% of them successfully developed to adult flowering plants (Table 1). The phenotype of transgenic and control plants was similar (Fig. 1). 3.2. Headspace analysis Headspace analysis of control and transgenic intact lisianthus flowers was performed to reveal the presence of benzyl acetate. No benzyl acetate was detected by this method in any of the samples, possibly due to the low levels of the BEAT substrate-benzyl alcohol in lisianthus flowers. Therefore, the effect of external substrate was tested in feeding experiments.
2.8. Substrate preference assay Transgenic and non-transformed control plantlets grown in vitro were placed in 10 mL covered vials containing 5 mL of either DDW or DDW + 0.1 mM alcoholic substrate (hexanol, benzyl alcohol, isoamyl alcohol, phenethyl alcohol, or cinnamyl alcohol) for 24 h. 2.9. Analysis and identification of volatile metabolites for product detection Samples consisting of 0.5 mL of the concentrated MTBE extracts were analyzed on a MSD gas chromatograph
3.3. Feeding experiments with benzyl alcohol as a substrate to BEAT Detached leaves and flowers of fully developed plants (transgenic and control) were fed for 24 h with DDW or Table 1 Transformation and regeneration efficiency in lisianthus Procedure
Number of plants
Percent success
Transformation Rooting Hardening
239 198 39
10.5 83.0 55.0
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Fig. 1. Non-transformed (control) and transgenic (BEAT) adult flowering plants showing the same phenotype.
10 mM benzyl alcohol solution and subsequently analyzed for benzyl acetate production. Leaves of transgenic lisianthus plants produced abundant amounts of benzyl acetate when fed with benzyl alcohol. No benzyl acetate was detected in leaves of either control or transgenic plants when fed with water only (Fig. 2A). Benzyl acetate production was five to seven times higher in leaves of transgenic plants as compared to control plants (Fig. 2A). Benzyl acetate amounts in detached flowers of transgenic plants were lower than in detached leaves (Fig. 2B). Benzyl acetate was not detected in control plants or in extracts of flowers fed with water (Fig. 2B). 3.4. Feeding experiments with additional alcoholic substrates C. breweri BEAT enzyme is known for being able to catalyze the production of acetate from different alcoholic substrates (Dudareva et al., 1998b). In order to test alcoholic substrate preference of the BEAT enzyme expressed in lisianthus transgenic plants, small in vitro-grown transgenic and control plantlets were fed with different alcoholic substrates: hexanol, benzyl alcohol, isoamyl alcohol, phenethyl alcohol, and cinnamyl alcohol. Levels of acetates were analyzed in extracts of transgenic and control plantlets after feeding. The highest level of acetates were produced by transgenic plantlets fed with hexanol and the resulting hexyl acetate levels were significantly higher (p < 0.05) than the acetates levels produced by transgenic plantlets fed with the other alcoholic substrates, except cinnamyl alcohol (Fig. 3). The lowest lev-
Fig. 2. Feeding of transgenic lisianthus with BEAT substrate benzyl alcohol. (A) Levels of benzyl acetate in MTBE extracts of lisianthus leaves from transgenic (25M, 69, 26b, K67) and control (WT) plants after 24 h feeding with benzyl alcohol (BA) or water (H2 O). (B) Levels of benzyl acetate in MTBE extracts of lisianthus flowers: transgenic (25M, 83, 26b, T30) and control (WT) after feeding with benzyl alcohol (BA) or water (H2 O). Bars represent S.E.
els of acetates were produced by transgenic plantlets fed with benzyl alcohol and isoamyl alcohol and did not differ significantly (p < 0.05) from each other (Fig. 3). Control plantlets fed with isoamyl, phenylethyl, or cinnamyl alcohol produced low amounts of acetates (Fig. 3). Transgenic plantlets fed with the same substrates produced two to three times more acetate products (Fig. 3). No acetates were detected when control plantlets were fed with hexanol or benzyl alcohol. For each substrate, the amount of the respective acetate produced by transgenic plantlets was significantly higher (p < 0.05) than the acetate produced by control plants.
4. Discussion We used the C. breweri BEAT cDNA under the control of the CaMV 35S promoter to transform lisianthus plants, aiming to induce scents in all plant tissues. Benzyl acetate is a major compound (about 40%) of the volatile composition of C. breweri aroma (Dudareva et al., 1998a), and of fragrant flowers such as gardenia and jasmine (Christensen et al., 1997; Buchbauer et al., 1996). Fruit such as melons (Shalit
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Fig. 3. Levels of acetate products in MTBE extracts of transgenic (K67) and untransformed control (WT) lisianthus plantlets after feeding with water or different alcoholic substrates: hexanol, benzyl alcohol, isoamyl alcohol, phenethyl alcohol, cinnamyl alcohol. Bars represent S.E.
et al., 2001) also emit benzyl acetate in high amounts, which largely contributes to their special aroma. Consequently, the BEAT gene was considered a good candidate for transformations of plants to generate aromas in a non-scented flower. The transformation of lisianthus with BEAT was successfully performed by means of A. tumefaciens. Transformation was confirmed by PCR using nptII-specific primers, as PCR reactions with BEAT-specific primers resulted in non-specific amplification probably caused by the presence of different AAT family genes in lisianthus. Characterization of transgenic plants did not reveal phenotypic differences (Fig. 1). However, no change in flower fragrance and no difference in volatile composition of headspace samples was detected in transgenic plants compared to control plants by olfactory or headspace analysis. We tested the hypothesis that absence of benzyl acetate production could be due to insufficient BEAT substrate, as was previously proposed in other systems (Beekwilder et al., 2004). Therefore, leaves and flowers of control and transgenic lisianthus plants were fed benzyl alcohol (Fig. 2A and B). Substrate-fed transgenic plants produced 2–3 g benzyl acetate/g leaves and 4–8 ng/g flowers. No benzyl acetate was detected in DDW-fed transgenic tissues. It is noteworthy that leaves of control plants fed with BEAT substrates also produced benzyl acetate, although in much lower quantity than transgenic tissues (Fig. 2A), suggesting the presence of a BEAT-like enzyme in lisianthus. These results demonstrate that lack of sufficient substrate was at least one of the reasons for the absence of benzyl acetate in transgenic plants. Identification of enzymes involved in substrate production and their transformation into plants together with genes involved in scent production could address the problem of lack of substrate and lead to fragrance production in transgenic plants. On the other hand, the possibility of using alternative genes involved in aroma production (Galili et al., 2001) should be further investigated. It is suggested that alcoholic compounds, such as benzyl alcohol, could be integrated more effectively by existing
biochemical pathways and enzymes of lisianthus rather than by BEAT from C. breweri. This would then lead to lower amounts of produced acetate. Exposing transgenic lisianthus tissue to alternative alcoholic substrates may bring about higher production of acetates. We tested the effect of feeding different alcoholic substrates for BEAT in transgenic lisianthus plants (Fig. 3). Alcohol acetyltransferases (including BEAT) release acetate derivatives from their respective alcohols and acetyl-CoA. Some of the compounds fed in this study, such as benzyl alcohol, cinnamyl alcohol and to a lesser extent phenethyl alcohol, were chosen because it is known that BEAT can use them as substrates (Dudareva et al., 1998b). Hexanol and isoamylalcohol, the precursor of isoamylacetate, with a typical banana scent were chosen to broaden the selection. It is likely that the acetates released were directly derived from BEAT activity in the transgenic plant tissues, and from a similar (yet uncharacterized) activity in control, non-transformed plants. The results showed that feeding transgenic lisianthus plantlets with hexanol, phenylethyl alcohol, and cinnamyl alcohol induced significantly higher levels of acetate products than feeding with benzyl alcohol and isoamyl alcohol which induced similar results (Fig. 3). The amount of benzyl acetate produced by benzyl alcohol-fed small plantlets (Fig. 3) was about 17-fold lower than by benzyl acetate-fed leaves of the same transgenic line (Fig. 2). This difference may be due to the age of the plant tissue and to the growing conditions. The highest level of acetate product was obtained in hexanol-fed plantlets (Fig. 3). Hexanol might then be the best BEAT substrate for transgenic lisianthus (Fig. 3). Most alcoholic substrates except cinnamylalcohol were not toxic. No damage to tissues, discoloration of leaves, or effects on longevity of plants was observed. This study suggests that fragrances can be induced in usually unscented cut flowers by means of genetic engineering, using different substrates.
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Acknowledgments This work was funded by a grant from the Chief Scientist of the Israel Ministry of Agriculture and Rural Development to MZ and EL. We thank Natalia Dudareva for helpful discussions, Einat Bar for her help with GS–MS analysis and Janina Mutchnik for her help in growing the plants in the greenhouse.
References Aharoni, A., Jongsma, M.A., Bouwmeester, H.J., 2005. Volatile science? Metabolic engineering of terpenoids in plants. Trends Plant Sci. 10, 594–602. Beekwilder, J., Alvarez-Huerta1, M., Neef, E., Verstappen, F., Bouwmeester, H., Aharoni, A., 2004. Functional characterization of enzymes forming volatile esters from strawberry and banana. Plant Physiol. 135, 1865–1878. Buchbauer, G., Jirovetz, L., Nikiforov, A., Kaul, V., Winker, N., 1996. Volatiles of the absolute of Gardenia jasminoides Ellis (Rubiaceae). J. Essent. Oil Res. 8, 211–215. Christensen, L., Jakobsen, H., Kristiansen, K., Moller, J., 1997. Volatiles emitted from flowers by gamma-radiated and not radiated Jasminum polyanthum Franch in situ. J. Agric. Food Chem. 45, 2199– 2203. D’Auria, J.C., Chen, F., Pichersky, E., 2002. Characterization of an acyltransferase capable of synthesizing benzylbenzoate and other volatile esters in flowers and damaged leaves of Clarkia breweri. Plant Physiol. 130, 466–476. Deroles, S.C., Bradley, J.M., Schwinn, K.E., Markham, K.R., Bloor, S., Manson, D.G., Davies, K.M., 1998. An antisense chalcone synthase cDNA leads to novel colour patterns in lisianthus (Eustoma grandiflorum) flowers. Mol. Breeding 4, 59–66. Dudareva, N., D’Auria, J., Nam, K., Raguso, R., Pichersky, E., 1998a. Acetyl CoA:benzylalcohol acetyltransferase: an enzyme involved in floral scent production in Clarkia breweri. Plant J. 14, 297–304. Dudareva, N., Pichersky, E., 2000. Biochemical and molecular genetic aspects of floral scents. Plant Physiol. 122, 627–633. Dudareva, N., Raguso, R., Wang, J., Ross, J., Pichersky, E., 1998b. Floral scent production in Clarkia breweri. III. Enzymatic synthesis and emission of benzenoid esters. Plant Physiol. 116, 599–604. Galili, G., Galili, S., Lewinsohn, E., Tadmor, Y., 2001. Genetic, molecular, and genomic approaches to improve the value of plant foods and feeds. Crit. Rev. Plant Sci. 21, 167–204. Guterman, I., Masci, T., Chen, X., Negre, F., Pichersky, E., Dudareva, N., Weiss, D., Vainstein, A., 2006. Generation of phenylpropanoid pathwayderived volatiles in transgenic plants: rose alcohol acetyltransferase
produces phenylethyl acetate and benzyl acetate in petunia flowers. Plant Mol. Biol. 60, 555–563. Lavid, N., Wang, J., Shalit, M., Guterman, I., Bar, E., Beuerle, T., Menda, N., Zach, A., Sharoni, S., Zamir, D., Vainstein, A., Weiss, D., Pichersky, E., Lewinsohn, E., 2002. O-Methyltransferases involved in the biosynthesis of volatile phenolic derivatives in rose petals. Plant Physiol. 129, 1899–1907. Lavy, M., Zuker, A., Lewinsohn, E., Larkov, O., Ravid, U., Vainstein, A., Weiss, D., 2002. Linalool and linalool oxide production in transgenic carnation flowers expressing the Clarkia breweri linalool synthase gene. Mol. Breeding 9, 103–111. Lewinsohn, E., Dudai, N., Tadmor, Y., Katzir, I., Ravid, U., Putievsky, E., Joel, D.M., 1998. Histochemical localization of citral accumulation in lemongrass leaves (Cymbopogon citratus (DC.) Stapf., Poaceae). Ann. Bot. 81, 35–39. Lewinsohn, E., Shalekchet, F., Wilkinson, J., Matsui, K., Tadmor, Y., Nam, K.H., Amar, O., Lastochkin, E., Larkov, O., Ravid, U., Hiatt, W., Gepstein, S., Pichersky, E., 2001. Enhanced levels of aroma and flavor compound S-linalool by metabolic engineering of the terpenoid pathway in tomato fruits. Plant Physiol. 127, 1256–1265. Lucker, J., Bouwmeester, H.J., Schwab, W., Blaas, J., van der Plas, L., Verhoeven, H.A., 2001. Expression of Clarkia S-linalool synthase in transgenic petunia plants results in the accumulation of S-linalyl--d glucopyranoside. Plant J. 27, 315–324. Mercuri, A., Sacchetti, A., De Benedetti, L., Schiva, T., Alberti, S., 2001. Green fluorescent flowers. Plant Sci. 162, 647–654. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497. Raguso, R.A., Pichersky, E., 1995. Floral volatiles from Clarkia breweri and C. concinna (Onagraceae): recent evolution of floral scent and moth pollination. Plant Syst. Evol. 194, 55–67. Semeria, L., Ruffoni, B., Rabaglio, M., Genga, A., Vaira, A., Accotto, G., 1996. Genetic transformation of Eustoma grandiflorum by Agrobacterium tumefaciens. Plant Cell Tissue Org. Culture 47, 67–72. Shalit, M., Guterman, I., Volpin, H., Bar, E., Tamari, T., Menda, N., Adam, Z., Zamir, D., Vainstein, A., Weiss, D., Pichersky, E., Lewinsohn, E., 2003. Volatile ester formation in roses. Identification of an acetyl-coenzyme A: geraniol/citronellol acetyltransferase in developing rose petals. Plant Physiol. 130, 1868–1876. Shalit, M., Katzir, N., Tadmor, Y., Larkov, O., Burger, Y., Shalechet, F., Lastochkin, E., Ravid, U., Amar, O., Edelstein, M., Karchi, Z., Lewinsohn, E., 2001. Acetyl CoA:alcohol acetyltransferase activity and aroma formation in ripening melon fruits. J. Agric. Food Chem. 49, 794–799. Vainstein, A., Lewinsohn, E., Pichersky, E., Weiss, D., 2001. Floral Fragrance. New inroads into an old commodity. Plant Physiol. 127, 1383–1389. Zaccai, M., Edri, N., 2002. Floral transition in lisianthus (Eustoma grandiflorum). Sci. Hortic. 95, 333–340. Zaccai, M., Lewinsohn, E., Pichersky, E., 2001. Modifing lisianthus traits by genetic engineering. Acta Hortic. 552, 137–142.