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Plant Physiology 135:1865-1878 (2004) © 2004 American Society of Plant Biologists Functional Characterization of Enzymes Forming Volatile Esters from Strawberry and Banana[w]Plant Research International, 6700 AA, Wageningen, The Netherlands
ABSTRACT Volatile esters are flavor components of the majority of fruits. The last step in their biosynthesis is catalyzed by alcohol acyltransferases (AATs), which link alcohols to acyl moieties. Full-length cDNAs putatively encoding AATs were isolated from fruit of wild strawberry (Fragaria vesca) and banana (Musa sapientum) and compared to the previously isolated SAAT gene from the cultivated strawberry (Fragaria x ananassa). The potential role of these enzymes in fruit flavor formation was assessed. To this end, recombinant enzymes were produced in Escherichia coli, and their activities were analyzed for a variety of alcohol and acyl-CoA substrates. When the results of these activity assays were compared to a phylogenetic analysis of the various members of the acyltransferase family, it was clear that substrate preference could not be predicted on the basis of sequence similarity. In addition, the substrate preference of recombinant enzymes was not necessarily reflected in the representation of esters in the corresponding fruit volatile profiles. This suggests that the specific profile of a given fruit species is to a significant extent determined by the supply of precursors. To study the in planta activity of an alcohol acyltransferase and to assess the potential for metabolic engineering of ester production, we generated transgenic petunia (Petunia hybrida) plants overexpressing the SAAT gene. While the expression of SAAT and the activity of the corresponding enzyme were readily detected in transgenic plants, the volatile profile was found to be unaltered. Feeding of isoamyl alcohol to explants of transgenic lines resulted in the emission of the corresponding acetyl ester. This confirmed that the availability of alcohol substrates is an important parameter to consider when engineering volatile ester formation in plants.
Volatile esters are produced by virtually all soft fruit species during ripening. They play a dual role in the ripe fruit, serving both as "biological bribes" for the attraction of animals and as protectants against pathogens. In some fruits, like apple (Malus domestica), pear (Pyrus communis), and banana (Musa sapientum), esters are the major components in their characteristic aroma. In other fruits, like strawberry (Fragaria x ananassa), they contribute as notes to the blend of volatiles that constitute the aroma. Often, a single fruit emits a large spectrum of esters; for instance, more than 100 different esters have been detected in ripe strawberry fruit (Zabetakis and Holden, 1997  ). Most volatile esters have flavor characteristics described as fruity (Burdock, 2002). However, they are not only produced and emitted by fruit but also are part of floral scents (Dudareva et al., 1998b). They are very often emitted by vegetative parts of plants, either constitutively or in response to stress or insect infestation (Mattiacci et al., 2001). Esters therefore play a major role in the interaction between plants and their environment. Formation of volatile esters is not restricted to the plant kingdom and is also common in microorganisms such as yeast and fungi. For example, isoamyl acetate and ethyl acetate are produced during beer fermentation and are known to affect flavor quality (Verstrepen et al., 2003).
Alcohol acyltransferase (AAT) enzymes catalyze the last step in ester formation by transacylation from an acyl-CoA to an alcohol. Combinations between different alcohols and acyl-CoAs will result in the formation of a range of esters in different fruit species. The most likely precursors for the esters are lipids and amino acids. Their metabolism during ripening will therefore play an important role in determining both the levels and type of esters formed. For example, in strawberry, the amino acid Ala has been implicated in the formation of ethyl esters during ripening (Perez et al., 1992
Due to their key role in ester biosynthesis, the activity of AAT enzymes was the subject of several early investigations on extracts of various fruit species, including banana, strawberry, and melon (Cucumis melo; Wyllie and Fellman, 2000
Overall, the proteins encoded by both SAAT and CM-AAT1 showed low sequence identity to other genes, but conserved motifs in their sequence could associate them to a plant superfamily of multifunctional acyltransferases, commonly referred to as BAHD (St-Pierre and De Luca, 2000 In this study, we characterize the substrate preference of fruit-expressed members of the BAHD family. We cloned full-length cDNAs for enzymes from wild strawberry (Fragaria vesca) and banana and compared them to SAAT. Recombinant expression in Escherichia coli and assays on their ability to use different alcohol substrates provided evidence for a role of these enzymes in the biosynthesis of esters that contribute to fruit flavor. Finally, we report the first attempt to use the SAAT gene for metabolic engineering of plants, with the aim to alter the profile of emitted volatiles. RESULTS
The activity of recombinant SAAT enzyme was tested using a range of additional substrates that had not been tested by Aharoni et al. (2000)
Characterization of the Wild Strawberry (VAAT) and Banana (BanAAT) Alcohol Acyl Transferases Acyl transferase genes related to the SAAT gene from strawberry were identified in a number of fruit species, as described in the supplemental data (available at www.plantphysiol.org). From this collection, a gene from wild strawberry (termed VAAT) and from banana (termed BanAAT) were selected for further analysis. An alignment of the amino acid sequences encoded by the cDNAs of SAAT, VAAT, and BanAAT is shown in Figure 1A .
The esters produced in wild and cultivated strawberries are comparable (Pyysalo et al., 1979 ). Therefore, it was of interest to compare the SAAT enzyme to its closest ortholog, VAAT, in more detail. First, sequence comparison (Fig. 1A) reveals that the sequences of both enzymes are closely related (86% identical). Diversity is concentrated in two regions in particular, around positions 60 and 430.
Also the substrate preference of the recombinant VAAT and SAAT enzymes were compared (Table I). As reported before (Aharoni et al., 2000
The Km and Vmax values for octanol and acetyl-CoA were determined for VAAT to compare these to the published values of SAAT (Aharoni et al., 2000 Recombinant expression in E. coli was also achieved for BanAAT, and activity of the produced enzyme could be tested. In Table I, results with a limited set of alcohol substrates in combination with acetyl-CoA are compared to those obtained with SAAT and VAAT. The BanAAT enzyme showed an activity profile similar to SAAT, as it used geraniol, nerol, and the C6 and C8 alcohols quite efficiently. Cinnamyl alcohol was the best substrate for BanAAT, while the SAAT and VAAT enzymes were only very modestly active with this substrate.
The activity of SAAT was also studied in planta using petunia plants. Leaves of petunia do not emit any esters, while flowers emit exclusively esters originating from benzyl-alcohol and methanol, in addition to nonester aromatic compounds (Verdonk et al., 2003 Total RNA was extracted from young leaves of two nontransformed plants, three plants transformed with an empty binary vector, and six plants transformed with SAAT. Expression of the SAAT transcript was analyzed by northern-blot analysis (Fig. 2 ). All analyzed SAAT-transformed plants expressed the gene, but clear differences in expression level were observed. Plants S216A and S218A (low expressers) and S152A and S157A (high expressers) were selected for further analysis. T1-generation transgenic plants were self-fertilized, and the T2 progeny was again selected for the presence of the SAAT gene by PCR screening. From each line, three PCR-positive plants were selected and compared to three nontransformed plants and three plants transformed with vector pBinPLUS without an insert.
In Vitro Enzyme Activity of SAAT Produced by Petunia Alcohol acyltransferase activity in leaves of the plants was analyzed in vitro, using acetyl-CoA and geraniol as substrates. On three different days, extracts from leaf material were assayed by adding geraniol and acetyl-CoA. Formation of geranyl acetate was analyzed using gas chromatography-mass spectrometry (GC-MS; Fig. 3 ). When the SAAT gene was not present (Control and pBinPLUS lines), hardly any geranyl acetate could be detected (Fig. 3A), and plants with low SAAT-RNA levels (S216A and S218A) produced no significant increase in the amount of geranyl acetate. High-expressing plants (S152A and S157A) produced 10 to 40 times more geranyl acetate than low-expressers and control plants (Fig. 3B, peak 3; Fig. 4A ). It was concluded that SAAT enzyme was expressed in active form in these plants.
The Volatile Profile of Petunia Plants Producing SAAT
As a next step, the in vivo effect of the presence of the SAAT gene on volatile release of petunia was tested. The headspace volatiles were measured in flowers of all six lines (S157A, S152A, S218, S216, pBinPLUS, and Control) for 2 d. No changes in the emission from flowers of endogenous petunia esters (benzyl benzoate and methyl benzoate; Verdonk et al., 2003
Activity of Petunia Expressed SAAT with Externally Supplied Alcohols To test if external substrates could be used by intact plant material, experiments were set up feeding alcohol as a substrate. Initially, geraniol was used, but quantitative comparison of these experiments was hampered by experimental problems. These problems are likely related to the poor solubility of geraniol, which at 6 mM could only be temporarily suspended in water. Plant parts that were in contact with the surface of the geraniol suspension showed severe necrosis, thus strongly affecting supply to the upper plant parts. When petunia leaves were fed with a solution of isoamyl alcohol (which is soluble in water at 6 mM), no necrosis was observed and much more reproducible results were obtained. Explants of petunia plants expressing the SAAT enzyme produced abundant amounts of isoamyl acetate when fed with an isoamyl alcohol solution. This production depended on the presence of expressed SAAT enzyme (Peak 1, Fig. 5, C and D). In Figure 4B, results on isoamyl acetate production of all six lines recorded at three different days are presented. High expressers (S157A and S152A) reproducibly emitted about 10-fold more isoamyl acetate than control leaves or low-expresser leaves. The SAAT enzyme apparently finds no suitable alcohol substrate in petunia unless this is fed to the plant.
When feeding alcohols to the petunia flowers, no changes in the emission of endogenous petunia esters (benzyl benzoate and methyl benzoate; Verdonk et al., 2003 DISCUSSION
Esters are important for the flavor of a number of fruits (Morton and MacLeod, 1990
As a first criterion, the expression pattern of the VAAT and BanAAT genes was considered, based on literature data. The VAAT gene (represented by clone 5.1.R2) was shown by RNA-gel blots of wild-strawberry fruit RNAs to be strongly induced in the transitions between green and breaker fruit and between breaker and red fruit (Nam et al., 1999
As a second criterion, the sequence homology to known acyltransferases was considered. As is clear from the presence of conserved sequences in the genes, they are part of the BAHD family (St-Pierre and De Luca, 2000 As a third criterion, the ability of fruit-expressed acyltransferases to produce the esters found in the fruits they were obtained from was tested. This was done by expressing the isolated cDNAs in E. coli, and testing the ability of the recombinant and partially purified enzymes to form esters from a broad range of alcohols in combination with acetyl-CoA. The SAAT and VAAT enzymes are clearly able to produce a number of the esters found in both wild and cultivated strawberries, such as hexyl acetate and octyl acetate. This further supports a role for the SAAT and VAAT genes in fruit flavor formation.
Also for the BanAAT enzyme, our data provide evidence for a role in flavor formation in ripening banana fruit, as recombinant BanAAT produced the characteristic banana volatiles such as isoamyl acetate and butyl acetate. However, these esters were made with considerably lower efficiency than octyl acetate, cinnamoyl acetate, and geranyl acetate (Table I). An in situ analysis of AAT activity in banana fruit also showed a preference for C6 alcohols over C3 and C4 alcohols, as is the case for recombinant BanAAT, while longer alcohols like octanol were not tested (Wyllie and Fellman, 2000
A comparison of substrate preference can be made between the SAAT, VAAT, and BanAAT enzymes and related enzymes known from the literature. The closest relative to SAAT for which activity measurements have been published is the rose alcohol acyl transferase (RhAAT; Shalit et al., 2003
It is difficult to compare absolute kinetic parameters for these enzymes in a way relevant for the in vivo situation. The catalytic efficiency of SAAT for octanol is much (30 times) lower than that of VAAT. On the other hand, the substrate preference data (Table I) indicate that SAAT turns over 2 nmol octanol per hour per µg protein, while VAAT turns over only 0.078 nmol octanol per hour per µg protein, suggesting that SAAT is 25-fold more efficient for this substrate. These seemingly contradictory observations can be explained by the difference in affinity for the second substrate, acetyl-CoA, of both enzymes. Substrate preference measurements were carried out at 0.1 mM acetyl-CoA, which ensures saturated substrate binding by SAAT (10 times higher than Km, which is 0.01 mM), but not by VAAT (20 times lower than Km, which is 2 mM). Obviously, this has strong impact on the observed substrate preferences. The in vivo concentration of acetyl-CoA in strawberry fruit is not known, but for green leaves it has been reported to range from 0.05 to 1.4 mM, depending on the number of chloroplasts present (Bao et al., 2000
The banana enzyme BanAAT is only distantly related to the SAAT and VAAT, members of group C of the BAHD family (Fig. 1, A and B). However, BanAAT shares most enzymatic properties with SAAT and RhAAT (Shalit et al., 2003
Pichersky and Gang (2000)
The engineering of volatile emission from plants has been described mainly for terpenoids. Lewinsohn et al. (2001)
The pathway eventually leading to ester formation has been engineered by Speirs et al. (1998)
Our results indicate the presence of alcohol acyltransferase activity also in nontransgenic petunia plants. When fed with isoamyl alcohol, the wild-type plant converted part of the alcohol into isoamyl methylbutyrate and isoamyl benzoate (Fig. 4C). This strongly suggests that there is an alcohol acyltransferase active in the petunia background. The fact that benzyl benzoate has been found in considerable amounts in wild-type petunia flowers (Verdonk et al., 2003
To engineer ester production into plants, it should be considered that a supply of alcohol substrate is required, either from an endogenous source or by introducing additional genes. The SAAT enzyme can take many different alcohol substrates, which are derived from several pathways. Linear alcohols like octanol could be provided by stimulating fatty acid metabolism, for instance by expressing lipoxygenase genes, or by amino acid catabolism. Terpene alcohols like geraniol could be provided by introducing a geraniol synthase (Iijima et al., 2004 MATERIALS AND METHODS
For cloning the full-length cDNAs, we used ripe fruit of wild strawberry (Fragaria vesca L., PRI breeding line 92189) and pulp of ripe banana (Musa sapientum). The petunia (Petunia hybrida [Vilm.]) variety W115 was used for generating transgenic plants, which were grown at 18°C with 18/6 h light/dark conditions in the greenhouse.
RNA isolation from the various fruit tissues was performed according to Asiph et al. (2000)
The entire SAAT coding region (1,359 bp) was amplified from pRSETB (Aharoni et al., 2000
To obtain full-length cDNAs we used the SMART RACE cDNA amplification kit (CLONTECH, Palo Alto, CA) according to the manufacturer instructions with slight modifications to the annealing temperatures (normally 5°C–10°C lower than recommended) or number of cycles (up to 35 cycles). PCR, restriction digests, plasmid DNA isolation, and gel electrophoresis were performed using standard protocols. All fragments were purified from the gel using the GFX purification kit (Amersham). Cloning of PCR fragments was either performed using the PCR SCRIPT (Stratagene, La Jolla, CA) or pCR 4Blunt-TOPO (Invitrogen, Carlsbad, CA) vectors (for blunt-end products generated when using Pfu DNA polymerase) or to the pGEM-T Easy (Promega, Madison, WI) vector (when A tailed PCR products were generated by the use of Taq DNA polymerase). Sequencing was done using an ABI 310 capillary sequencer according to the manufacturer instructions (ABI system, Perkin Elmer, Foster City, CA). Sequence analysis was conducted using the DNASTAR (DNASTAR, Madison, WI) and GeneDoc (http://www.cris.com/Ketchup/genedoc.shtml) programs. Multiple sequence alignments were performed using ClustalW (at http://www.ebi.ac.uk/clustalw/), using standard parameters.
Cloning of the full-length wild strawberry cDNA homolog was performed by PCR using oligonucleotide AAP165 (5'-CGGATCCGGAGAAAATTGAGGTCAG-3') designed at the 5' end from the SAAT gene sequence isolated previously from the cultivated strawberry by Aharoni et al. (2000)
To clone the full-length banana cDNA, RACE PCR was performed (method described above) in order to clone the 3' end using the AAP218 oligonucleotide (5'-TCATCTCCGTCCATACCATCG-3') designed based on a partial cDNA sequence (799 bp; GenBank accession no. Z93116) isolated previously from banana pulp (Medina-Suarez et al., 1997
A modified expression vector (pRSET B; Invitrogen) described earlier (Aharoni et al., 2000
Vectors pRSET-SAAT, pRSET-VAAT, and pRSET-BanAAT were transformed in E. coli BL21 CodonPlus-RIL. Fresh overnight cultures were diluted 100-fold in 50 mL Luria-Bertani medium supplied with ampicillin (50 µg/mL) and Glc (1%) and grown until A600 was 0.6. Cells were recovered by centrifugation and resuspended in Luria-Bertani medium supplied with ampicillin and isopropylthio-
Activity of recombinant proteins with different alcohol substrates was assayed using 14C radiolabeled acetyl-CoA (Amersham Life Sciences, NA, England) or 14C-butyryl-CoA or 14C-hexanoyl-CoA (Campro Scientific, The Netherlands). The reactions were made in duplicate. In a total reaction volume of 100 µL, 2 µL acyl-CoA (5 mM), 0.2 µL 14C labeled acyl-CoA, 85.8 µL 50 mM Tris-HCl buffer, pH 8.3, and 10 µL purified enzyme were mixed slowly in a cold Eppendorf (Westbury, NY) tube. The reactions were started by adding 2 µL alcohol, solved at 1 M in hexane. After 30 min incubation at 30°C, the reactions were stopped by cooling on ice for 15 min. The esters formed were extracted with 700 µL hexane. The hexane phase was mixed with 4.5 mL scintillation cocktail (Ultima Gold, Packard Bioscience, Groningen, The Netherlands) and analyzed by scintillation counting. The enzymatic activity was determined as nmol substrate turned over per hour per microgram protein. Km and Vmax determinations for recombinant VAAT protein were performed exactly as described in Aharoni et al. (2000)
One gram of leaf material was ground in a pestle and mortar and immediately mixed into 1 mL buffer A containing 50 mM Tris-HCl, pH 8.0, and 1 mM dithiothreitol. The material was centrifuged for 5 min at 13,000g, and the supernatant was used for assays. The assay was performed in a capped vial at 30°C and contained 50 µL supernatant, 300 µL buffer A, 25 µL 8 mM acetyl-CoA solution, and 25 µL 1.6 mM geraniol suspension. After 30 min, 400 µL saturated CaCl2 solution was added by injection through the septum. Products were analyzed using solid-phase microextraction. Volatiles released into the vial headspace (at 35°C with stirring) were subsequently trapped for 10 min by exposing a 100-µm PDMS fiber (Supelco, Bellefonte, PA) to the headspace. Desorption was performed for 1 min, and products were analyzed by GC-MS, as described by Verhoeven et al. (1997)
To analyze the headspace of plant parts, three flowers or three stem explants with a single node and two to three young leaves were cut from a single plant and weighed and cut ends were placed together in a covered 10-mL beaker covered with aluminum foil and containing 2 mL of water. For headspace analysis (performed basically as described by Bouwmeester et al., 1999 Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AJ001450.1, Z93116, AX025506, AX025504, AX025475, AX025508, AX025510, AY534530, AX025477, AX025516, AX025514, AX25512, AY534531, AX025518, Q9ZWR8, BAA93475, AAC99311, Q9M6E2, Q9FPW3, Q9M6F0, Q8LL69, AAF04787, AAN09797, AAK73661, CAA94432, AAN09796, CAB11466, AC114474, AAO73071, BQ106456, pBIN24A, pBIN69A, pBIN27A, S152A, S216A, S249A, S157A, S136A, and S218A. ACKNOWLEDGMENTS We acknowledge Jan Blaas and Harrie Verhoeven for assistance with solid-phase microextraction analysis and Robert Hall for critical reading of the manuscript. Received March 12, 2004; returned for revision April 6, 2004; accepted April 16, 2004. FOOTNOTES
1 Present address: Plant Physiology and Biochemistry Group, Institute of Plant Sciences, ETH Zürich, CH–8092 Zürich, Switzerland.
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-galactoside (1 mM). After overnight growth at 16°C, cells were harvested by centrifugation and resuspended in 1 mL ice-cold buffer R (50 mM Tris-HCl, pH = 8, and 10 mM 2-mercaptoethanol). Ten micrograms lysozyme was added, and cells were sonicated on ice for 5 times 10 s, with 5 s breaks and an amplitude of 14 using an MSE Soniprep 150 sonicator. Sonicated cells were centrifuged at 4°C and 14,000g for 5 min. The supernatant was used for purification of His-tagged proteins on a Ni-NTA spin column (Qiagen, Valencia, CA) as prescribed by the manufacturer. Resulting eluates were adjusted to contain 2 mM dithiothreitol, and were immediately used for activity assays. 