State of the Field
Functional Characterization of Enzymes Forming Volatile
Esters from Strawberry and Banana[w]
Jules Beekwilder*, Mayte Alvarez-Huerta1, Evert Neef, Francel W.A. Verstappen, Harro J. Bouwmeester,
and Asaph Aharoni
Plant Research International, 6700 AA, Wageningen, The Netherlands
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 3 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 3
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
1
Present address: Plant Physiology and Biochemistry Group,
Institute of Plant Sciences, ETH Zürich, CH–8092 Zurich, Switzerland.
* Corresponding author; e-mail [email protected]; fax 31–
317–418094.
[w]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.104.042580.
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).
However, levels of amino acid precursors could not
explain in all cases the formation of the corresponding
ester, and it has therefore been suggested that selectivity of enzymes preceding AATs in the biosynthetic
pathway also plays a role in determining ester composition (Wyllie and Fellman, 2000). In addition, fatty
acids, generated by the oxidative degradation of
linoleic and linolenic acids, contribute to ester biosynthesis in fruit. Degradation of fatty acids results in
the production of volatile aldehydes, which in turn are
utilized by alcohol dehydrogenases to form alcohols.
The latter are subsequently converted to hexyl and
hexenyl esters (Perez et al., 1996; Shalit et al., 2001).
Such a pathway has been demonstrated by Wang et al.
(2001), who altered the levels of fatty acids in transgenic tomato (Lycopersicon esculentum) fruit, leading to
dramatic changes in the aroma profile. Ripening related processes, such as changes in cell wall composition, might also contribute intermediates to ester
biogenesis. In tomato, evidence was provided that
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1865
Beekwilder et al.
activity of pectin methyl-esterase (which catalyzes
demetoxylation of pectins) regulates levels of methanol in the fruit (Frenkel et al., 1998). The accumulating
methanol may then be utilized to generate methyl
esters in ripe fruit.
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; Shalit et al., 2001; Olias
et al., 2002). In each of these studies, the AAT activity
was shown to be ripening induced, and the substrate
specificity of the AATs toward tested substrates (both
to alcohols and acyl-CoAs) appeared to be broad. In
recent years, fruit-expressed genes encoding enzymes
with AAT activity have been isolated and characterized from strawberry and melon. Aharoni et al. (2000)
utilized cDNA microarrays for gene expression
profiling during strawberry fruit development and
identified the SAAT gene, which showed a strong
induction upon ripening. Moreover, the recombinant
SAAT enzyme could catalyze the formation of esters
typically found in strawberry, using aliphatic,
medium-chain alcohols (e.g. octanol) in combination
with various chain length (up to C10 tested) acyl-CoAs
as substrates. The activity of SAAT was much lower for
aromatic substrates, and no activity could be detected
with the monoterpene alcohol, linalool. Yahyaoui et al.
(2002) reported on the molecular and biochemical
characterization of a ripening-induced and ethyleneregulated AAT gene (CM-AAT1) from ripe melon fruit.
As in the case of SAAT, the recombinant CM-AAT1
enzyme was capable of producing esters from a wide
range of alcohols and acyl-CoAs. While CM-AAT1 and
the SAAT proteins share only 21% sequence identity,
they show a similar preference toward alcohols.
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). The first genes
from this family that were biochemically characterized
were the acetyl-CoA:benzylalcohol acetyltransferase
(BEAT) from Clarkia breweri flowers, which forms the
flower volatile benzyl acetate (Dudareva et al., 1998a)
and the acetyl-CoA:deacetylvindoline 4-O-acetyltransferase (DAT) from Catharanthus roseus (St-Pierre et al.,
1998). The complete genome of Arabidopsis contains
approximately 60 genes annotated to belong to the
BAHD family (D’Auria et al., 2002). In recent years,
more members of this family have been cloned and
characterized from a number of plant species. Some of
these enzymes catalyze the formation of volatile esters,
including benzyl alcohol benzoyl transferase (BEBT;
D’Auria et al., 2002) from C. breweri flowers, making
benzylbenzoate, acetyl-CoA:cis-3-hexen-1-ol acetyl
transferase CHAT from Arabidopsis (D’Auria et al.,
2002), which might be involved in the formation of cis3-hexenyl acetate emitted upon injury, and RhAAT1,
suggested by Shalit et al. (2003) to be responsible for
the generation of geranyl and citronellyl acetate in rose
flowers. In addition, many genes of the BAHD family
that are involved in the biosynthesis of nonvolatile
esters have been isolated. They encode enzymes associated with the biosynthesis of phenylpropanoids
(Fujiwara et al., 1998; Yonekura-Sakakibara et al.,
2000), alkaloids (St-Pierre et al., 1998; Grothe et al.,
2001), terpenoids (Walker and Croteau, 2000; Walker
et al., 2000), and shikimate pathway derivatives
(Burhenne et al., 2003; Hoffmann et al., 2003).
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
Further Characterization of the Cultivated Strawberry
SAAT Enzyme
The activity of recombinant SAAT enzyme was
tested using a range of additional substrates that had
not been tested by Aharoni et al. (2000). Substrate
preference of the purified recombinant enzyme was
tested in assays using 14C labeled acyl-CoAs as cosubstrates for a range of alcohol substrates. Substrate
preference is used here as a measure to compare the
activity of an enzyme for different substrates at a fixed
substrate concentration. In these tests, geraniol proved
to be an excellent substrate. The activity with geraniol
exceeded that with octanol (77% of activity with
geraniol), the best substrate in previous tests (Table I).
Other alcohols including terpene alcohols (nerol,
perilla alcohol, carveol, and farnesol) and aromatic
alcohols (cinnamyl alcohol and eugenol) were also
tested. The activity of SAAT with nerol (54%) and
perilla alcohol (45%) is significant, while the other
substrates were only poorly used (Table I). The activity
of SAAT with butyryl- and hexanoyl-CoA in combination with a subset of alcohols was also studied
(Table II). With the larger acyl-CoA cosubstrates, SAAT
again preferred geraniol and C6 to C9 aliphatic alcohols as substrates. However, relatively higher activity
was observed with butanol and hexanol in combination with butyryl- and hexanoyl-CoA (81% and 76%,
respectively), compared to acetyl-CoA (11%), while
decanol is less preferred in combination with the
higher CoAs. This shows that SAAT can use a surprising diversity of acyl-CoAs and alcohols, including
terpene alcohols, in addition to the aliphatic alcohols
that are usually available in fruit.
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Characterization of Enzymes Forming Fruit Esters
Table I. Substrate specificity of the SAAT, VAAT, and BanAAT recombinant enzymes toward different
types of alcohols
Comparison of esterification activity of each of the enzymes with different alcohols (20 mM) and
acetyl-CoA (0.1 mM) as acyl donor.
Alcohola
SAAT
VAAT
BanAAT
c,d
11 6 0.6
0.3 6 0.0
d
11 6 0.9
0.1 6 0.0
e
18 6 0.1
n.t.f
2-Propanol
6 6 1e
1 6 0.1
n.t.
1-Butanol
11 6 1e
Methanol
562
Ethanol
362
1-Propanol
12 6 2
38 6 0.1
e
15 6 0.1
1 6 0.1
Isoamylalcohol
17 6 1d
21 6 0.1
1-Hexanol
40 6 2e
39 6 0.1
n.t.
cis-2-hexen-1-ol
28 6 3d
39 6 0.0
n.t.
cis-3-hexen-1-ol
d
45 6 0.0
n.t.
d
trans-2-hexen-1-ol
43 6 4
100 6 0.7
d
trans-3-hexen-1-ol
1 6 0.1
31 6 0.4
23 6 0.0
1-Heptanol
d
70 6 25
14 6 0.6
1-Octanol
77 6 16e
6 6 0.0
1-Nonanol
66 6 1d
3 6 0.1
n.t.
1-Decanol
d
1 6 0.1
n.t.
3 6 0.4d
9 6 0.0
0.4 6 0.1
d
Furfuryl alcohol
n.t.
n.t.
44 6 0.3
Benzylalcohol
3 6 0.2
11 6 0.1
2 6 0.0
2-Phenyl-ethylalcohol
7 6 1d
12 6 0.4
1 6 0.0
Linalool
1 6 0.0e
Geraniol
Nerol
1 6 0.9
n.t.
100 6 6
13 6 0.0
88 6 0.6
54 6 2
3 6 0.1
46 6 0.3
Carveol
2 6 0.1
2 6 0.0
n.t.
Eugenol
7 6 0.0
7 6 0.7
n.t.
Farnesol
11 6 1.0
2 6 0.4
n.t.
Perilla alcohol
45 6 2
8 6 0.1
n.t.
4 6 0.1
n.t.
2-Ethoxyethanol
2 6 0.3
Cinnamyl alcohol
a
10 6 2
9 6 0.1
Structureb
n.t.
6 6 0.4
37 6 1
C
1 6 0.1
2-Butanol
19 6 1
14
100 6 1
b
c
The added alcohol.
Molecular structure of the alcohol added.
Activity calculated as nanomoles product formed per hour per microgram protein. The highest activity for each enzyme was set at
100%, and the numbers indicated signify the percentage of activity relative to the highest activity. For
SAAT, 100% was 3.0 nmol substrate h21 mg protein21; for VAAT 100% activity was 1.3 nmol substrate h21
d
mg protein21; for BanAAT 100% activity was 3.4 nmol substrate h21 mg protein21.
Values have been
e
calculated from data published previously (Aharoni et al., 2000).
Values have been calculated from
previously published data and have been confirmed independently in the same experiment from which
f
the novel data have originated.
n.t., Not tested.
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Beekwilder et al.
Table II. Substrate specificity of the SAAT recombinant enzyme
for different types of alcohols in combination with acetyl-CoA,
butyryl-CoA, and hexanoyl-CoA
Comparison of esterification activity of each of the enzymes with
different alcohols (20 mM) and 14C acetyl-CoA, butyryl-CoA, or
hexanoyl-CoA (0.1 mM) as acyl donor.
Alcohol
1-Butanol
2-Butanol
1-Hexanol
cis-2-hexenol
cis-3-hexenol
trans-2-hexenol
1-Octanol
1-Nonanol
1-Decanol
Benzyl alcohol
2-Phenylethyl alcohol
Geraniol
Acetyl-CoA
11
15
40
28
19
43
77
66
37
3
7
100
6
6
6
6
6
6
6
6
6
6
6
6
2a
1
2
2
5
4
16
1
1
1
1
6
Butyryl-CoA
81
75
73
66
43
81
100
58
6
7
6
89
6
6
6
6
6
6
6
6
6
6
6
6
11
1
23
13
5
15
3
3
2
8
4
5
Hexanoyl-CoA
67 6 12
50 6 5
65 6 4
62 6 2
n.t.b
100 6 12
69 6 3
100 6 8
15 6 6
14 6 0.2
24 6 5
75 6 3
a
Activity calculated as nanomoles product formed per hour per
microgram protein. The highest activity for each enzyme was set at
100%, and the numbers indicated signify the percentage of activity
b
relative to the highest activity.
n.t., Not tested.
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 SAATenzyme
has a preference for C6 to C10 aliphatic alcohols. For
VAAT, the optimal size of aliphatic alcohols was clearly
smaller than for SAAT; only C6 alcohols (23%–100%)
and n-butyl alcohol (38%) were good substrates, and
methyl and ethyl alcohols (11%), geraniol (13%), phenylethyl alcohol (12%), and benzyl alcohol (11%) were
used to limited extent. Longer aliphatic alcohols like
octanol, which are favored by SAAT (77%), were hardly
used at all by VAAT (6%). So, although there is an
overlap between both enzymes for C6 alcohols, SAAT
and VAAT have shifted preferences.
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). The
Km value of VAAT for octanol, using 10 mM acetyl-CoA
as a cosubstrate, is 0.6 mM (Vmax 5 89 nmol h21 mg
protein21), which is about 10-fold lower than that of
SAAT (Km 5 5.6 mM; Vmax 5 26 nmol h21 mg21). The
catalytic efficiency of VAAT with octanol (expressed as
kcat/Km) is 1.0 3 103 M21 s21, which is considerably
higher than that of SAAT (32 M21 s21). The Km value for
acetyl-CoA (using 20 mM octanol as cosubstrate) is
dramatically (200-fold) higher for VAAT (Km 5 2 mM;
Vmax 5 350 nmol h21 mg21) than for SAAT (Km 5
0.01 mM; Vmax 5 45 nmol h21 mg21). The catalytic
efficiency of SAAT with acetyl-CoA (3.1 3 104 M21 s21)
is about 20-fold higher than VAAT (1.2 3 103 M21 s21).
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.
Expression of SAAT in Transgenic Petunia
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). Plants were transformed with a cDNA encoding
the strawberry SAAT gene under control of the double
35S-cauliflower mosaic virus promoter. All regenerated plants were screened for the presence of the SAAT
gene by PCR. PCR-positive plants were transferred to
greenhouse conditions for further examination. Transgenic plants exhibited normal development, compared to nontransformed and empty-vector control
plants, which went through the same regeneration
process.
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.
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Characterization of Enzymes Forming Fruit Esters
Figure 1. (Legend appears on following page.)
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Beekwilder et al.
Figure 1. A, Comparison of the amino acid sequences of the VAAT, SAAT, and BanAAT enzymes. Amino acids shaded in black
represent identical matches; gray shaded boxes represent conservative changes. B, Nonrooted phylogenetic tree of the alcohol
acyltransferase BAHD family members. Depicted are BanAAT (accession AX025506), VAAT (AX025504), and SAAT (AX025475;
bold underlined); genes known to be expressed in fruit (bold, see supplemental data); and plant alcohol acyl transferases that
have been biochemically characterized. Fruit expressed genes are ApAAT (AX025508), ManAAT (AX025510), PAAT
(AY534530), LAAT1 (AX025477), LAAT2 (AX025516), LAAT3 (AX025514), LAAT4 (AX25512), TomAAT (AY534531), and
MAAT (AX025518). Transferases that have been biochemically characterized comprise anthocyan-5-aromatic acyltransferase
(5AT; Q9ZWR8) from Gentiana triflora (Fujiwara et al., 1998), the hydroxycinnamoyl-CoA: anthocyanin 3-O-glucoside-6$-Oacyltransferase (3AT; BAA93475) from Perilla frutescens (Yonekura-Sakakibara et al., 2000), the DAT (AAC99311) from
Catharanthus roseus (St-Pierre et al., 1998), the 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT; Q9M6E2), and the
2-alpha-hydroxytaxane 2-O-benzoyltransferase (TBT; Q9FPW3) from Taxus cuspidata (Walker and Croteau, 2000), the taxadien5-alpha-ol O-acetyltransferase (TAT; Q9M6F0) from T. cuspidata (Walker et al., 2000), the 3#-N-debenzoyl-2#-deoxytaxol
N-benzoyltransferase (DBTNBT; Q8LL69) from T. cuspidata (Walker et al., 2002), the BEAT (AAF04787) from Clarkia breweri
(Dudareva et al., 1998a), the acetyl-CoA:cis-3-hexen-1-ol acetyl transferase (CHAT; AAN09797) from Arabidopsis (D’Auria et al.,
2002), the salutaridinol 7-O-acetyltransferase (SalAAT; AAK73661) from Papaver somniferum (Grothe et al., 2001), the alcohol
acyltransferase 1 (CM-AAT1; CAA94432) from Cucumis melo (Yahyaoui et al., 2002), the benzoyl-CoA:benzyl alcohol
benzyltransferase (BEBT; AAN09796) from C. breweri (D’Auria et al., 2002), the anthranilate N-benzoyltransferases from
Dianthus caryophyllus (HCBT; CAB11466; Yang et al., 1997), and Orzya sativa (OsHCBT; AC114474; Burhenne et al., 2003); the
agmatine coumaroyltransferase (HvACT; AAO73071) from Hordeum vulgare (Burhenne et al., 2003), and the geraniol
acetyltransferase (RhAAT; BQ106456) from Rosa hybrida (Shalit et al., 2003).
In Vitro Enzyme Activity of SAAT Produced by Petunia
The Volatile Profile of Petunia Plants Producing SAAT
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.
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) were observed, nor were any esters or other volatile compounds that were novel to petunia detected (data not
shown). Also in green parts, no changes in the emitted
volatiles could be observed (Fig. 5, A and B).
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
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Characterization of Enzymes Forming Fruit Esters
Figure 2. Northern-blot analysis of RNA samples derived from petunia
plants. RNA was extracted from wild-type (Control 2 and Control 5)
plants, plants transformed with empty vector pBinPLUS (pBIN24A,
pBIN69A, and pBIN27A), and plants transformed with the pBIN-SAAT
construct (S152A, S216A, S249A, S157A, S136A, and S218A) and
analyzed for expression of the SAAT gene using amplified SAAT cDNA
as a probe. In the last lane, an equal amount of strawberry RNA was
loaded. The bottom section represents a control hybridization with
a petunia ubiquitin probe.
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) were observed (results not shown). Remarkably,
in the leaf headspace, a few novel esters were detected
when isoamyl alcohol was fed independently from the
presence of the SAAT gene. The major esters (peaks 2
and 3, Fig. 5C) were identified as 3-methyl butanyl
2-methyl butanoate and 3-methyl butanyl 3-methyl
butanoate (collectively referred to as isoamyl methylbutyrate), while a minor peak (peak 4, Fig. 5C) was
identified as isoamyl benzoate.
DISCUSSION
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,
The Role of BAHD Family Members in Fruit
Flavor Formation
Esters are important for the flavor of a number of
fruits (Morton and MacLeod, 1990). Wild and cultivated strawberries produce linear esters like ethyl
hexanoate, octyl acetate, and hexyl butyrate, while
banana flavor is dominated by isoamyl acetate and
butyl acetate. Biosynthesis of esters in fruit is mediated
by enzymes from the BAHD family. Two fruitexpressed genes (SAAT from strawberry and CMAAT1 from melon) from this family have been earlier
implicated in the biosynthesis of volatile esters
(Aharoni et al., 2000; Yahyaoui et al., 2002). In this
paper, two other genes that are related to the SAAT
gene have been isolated from wild strawberry and
banana. Several lines of evidence suggest involvement
of these genes in the formation of fruit flavor.
Figure 3. In vitro formation of
geranyl acetate by leaf extracts of
wild-type petunia (A) and the SAAT
expressing line S157A (B), when
supplied with geraniol and acetylCoA. Depicted are GC-MS chromatograms of m/z 69. The 100%
detector response corresponds to
2 3 106 ion counts. Major volatiles
detected are marked with numbers:
Peak 1 is geraniol; peak 2 is nerol,
a contaminant of the geraniol sample; peak 3 is geranyl acetate.
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Beekwilder et al.
Figure 4. Activity of SAAT produced by
petunia. A, GC-MS analysis of the relative
amount of geranyl acetate formed in an in
vitro activity assay using leaf extracts of six
petunia lines, acetyl-CoA, and geraniol.
Assays were performed on three different
days. Geranyl acetate was quantified by
integrating the peak surface area of m/z 69
at 19.14 min. B, GC-MS analysis of the
relative amount of isoamyl acetate emitted
into the headspace of stem explants with
young leaves of six petunia lines, feeding
on a 6.5 mM isoamyl alcohol solution, on
three different days. Quantity of emitted
isoamyl acetate is expressed as microgram
per gram fresh weight plant material
per 24 h.
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 wildstrawberry fruit RNAs to be strongly induced in
the transitions between green and breaker fruit and
between breaker and red fruit (Nam et al., 1999). In
a similar way, the BanAAT gene expression (represented by cDNA clone UU130) was reported to be upregulated in banana pulp after ripening was initiated
with ethylene (Medina-Suarez et al., 1997).
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). Four groups within this family have been
defined by Hoffmann et al. (2003), based on sequence
homology. One group (A; Fig. 1B) comprises the Taxus
acyltransferases involved in taxol biosynthesis, a second group (B) consists of the anthocyanin acyltransferases, a third group (C) of genes encodes enzymes
with unrelated substrates, including SAAT, DAT, and
BEAT, and a fourth group (D) comprises the hydroxycinnamoyltransferases. An additional fifth group,
group E containing agamatine coumaroyltransferases,
has been identified by Burhenne et al. (2003). Lastly,
a group comprising the melon CM-AAT1 and woundinduced enzymes from C. breweri (BEBT) and Arabidopsis (CHAT) can be identified as group F. In Figure
1B, the BanAAT and VAAT enzymes have been added
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Characterization of Enzymes Forming Fruit Esters
Figure 5. Total volatile emission
from petunia leaves. GC-MS chromatograms (detector response;
100% of 1.5 3 106 total ion counts)
of head space volatiles released in
vivo by petunia leaves over a period
of 24 h. A, Leaves of wild-type
petunia leaves feeding on water. B,
Leaves of line S157A on water. C,
Leaves of wild-type petunia on an
aqueous 6.5 mM isoamyl alcohol
solution. D, Leaves of line S157A
on an aqueous 6.5 mM isoamyl
alcohol solution. The major volatile
compounds detected are marked
with numbers: peak 1 is isoamyl
acetate; peak 2 is isoamyl 2-methyl
butyrate; peak 3 is isoamyl 3-methylbutyrate; and peak 4 is isoamyl
benzoate.
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Beekwilder et al.
to a phylogenetic tree including enzymes of the BAHD
family with known activity and putative enzymes
expressed in fruit (see supplemental data). The VAAT
enzyme is very closely related to the SAAT enzyme and
falls into group C. The BanAAT enzyme is exceptional
and does not match any of the sequence groups.
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). This suggests that BanAAT corresponds to the major enzyme responsible for ester
formation in banana fruit. Its low efficiency for synthesizing isoamyl acetate could explain the presence of
significant amounts of isoamyl alcohol (in addition to
isoamyl acetate) in ripe banana volatiles (Shiota, 1993),
which could be important for its volatile blend. The
expression profile and substrate usage of the banana
and wild strawberry enzymes point to a role for these
enzymes in fruit flavor formation. However, definite
proof for this role has to come from reverse genetics
strategies, which should show that fruits carrying
knockout mutations or antisense-expressing constructs of BAHD genes have clearly reduced emission
of esters.
A Comparison of Substrate Preferences of Related and
Unrelated Enzymes
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). The substrate
preference of RhAAT is quite similar to that of SAAT.
Both RhAAT and SAAT prefer geraniol in combination
with acetyl-CoA (Table II). For RhAAT, this preference
very likely relates to the presence of geranyl acetate in
rose flower scent. For SAAT, there is no obvious
relevance for this preference to the formation of
strawberry fruit flavor, since geranyl acetate has not
been reported to occur in ripe strawberries. VAAT is
even closer related to SAAT than RhAAT, but its
activity is quite distinct from both enzymes. The VAAT
enzyme hardly accepts octanol, nonanol, or decanol as
substrates in combination with acetyl-CoA, although
geraniol is accepted. VAAT is much more active on
short alcohol substrates (C4 to C6, but also ethanol and
methanol), which are not preferred by SAAT, at least in
combination with acetyl-CoA (Table I). These observations are in agreement with the substrate preferences reported for enzyme extracts of wild and
cultivated strawberry fruits (Olias et al., 2002). Remarkably, the requirements of SAAT with respect to
alcohols combined with butyryl- and hexanoyl-CoA
are less stringent (Table II). Butanol, for instance, is
hardly used with acetyl-CoA, while it is readily
accepted with butyryl-CoA and hexanoyl-CoA. This
is particularly relevant since butylbutyrate and butylhexanoate have been identified in strawberries
(Zabetakis and Holden, 1997).
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 mg protein, while VAAT turns over only 0.078 nmol
octanol per hour per mg 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). In view of the low amount of
plastids in strawberry fruit, the acetyl-CoA concentration in this fruit is probably at the lower end of this
range, which is why it was estimated that a concentration of 0.1 mM acetyl-CoA in the substrate preference
assay was appropriate.
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) and prefers octanol and
geraniol. The VAAT preferences are more related to
those of CM-AAT1 (Yahyaoui et al., 2002), which does
not accept nonanol, and BEAT and BEBT (Dudareva
et al., 1998a; D’Auria et al., 2002), which produce
benzyl acetate. However, these enzymes belong to
group F, while VAAT belongs to group C. These cases
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Characterization of Enzymes Forming Fruit Esters
illustrate that predicting substrate usage based on
sequence grouping remains difficult. A similar lack
of relationship between primary structure and enzymatic properties has recently been reported for the
hydroxycinnamoyltranferases (Burhenne et al., 2003).
Pichersky and Gang (2000) have suggested a special
form of convergent evolution in which new enzymes
with the same function evolve independently in separate plant lineages from a shared pool of related
enzymes with similar but not identical functions. Such
a situation seems to be the case with the fruit AATs
that are involved in the formation of volatiles. There is
a strong overlap in substrate specificity of unrelated
members of the BAHD gene family (for instance SAAT
and BanAAT) and differences in specificity between
closely related members such as SAAT and VAAT.
Engineering of Volatile Esters in Plants
The engineering of volatile emission from plants has
been described mainly for terpenoids. Lewinsohn et al.
(2001) transformed a tomato variety, with a low endogenous level of linalool in the fruit, with C. breweri
linalool synthase under control of the fruit-specific E8
promoter. This resulted in fruit with strongly elevated
levels of S-linalool. The same gene was used to introduce the emission of linalool from carnation flowers
(Lavy et al., 2002). Interestingly, when Lucker et al.
(2001) attempted to realize linalool emission from
petunia, a plant that does not normally emit significant
amounts of terpenes, hardly any change in volatile
emission was observed. Overexpressing the C. breweri
linalool synthase resulted instead in accumulation of
linalool to significant amounts in the form of a nonvolatile glucopyranoside conjugate. Thus, endogenous
enzymes in petunia seem to sequester the volatile
linalool as a nonvolatile conjugate. Leaves of Arabidopsis plants constitutively expressing a linalool synthase from strawberry did produce linalool but also its
glycosylated and hydroxylated derivatives (Aharoni
et al., 2003).
The pathway eventually leading to ester formation
has been engineered by Speirs et al. (1998). By overexpressing alcohol dehydrogenase in tomato fruit,
levels of hexanol and cis-3-hexenol were increased,
at the expense of aldehydes. These fruits were
recognized by trained taste panels as having a more
intense ripe fruit flavor. By expressing SAAT in petunia, we aimed to engineer the emission of esters, which
could convey a fruity aroma to the plant. However, as
in the case of engineering linalool production in
petunia, engineering emission of volatile esters in
plants appears not to be trivial. Both expression of
SAAT and the activity of its corresponding enzyme
could be detected in the transgenic plants generated,
and activity was found to be inherited in the T2
generation. However, no new esters could be detected
in the headspace of transgenic lines, and no increase in
the ester benzylbenzoate, which is normally emitted
by wild-type petunia flowers, was observed. Feeding
alcohols to petunia explants demonstrated that the
bottle-neck for ester production was the availability of
alcohols. Clearly, the success of engineering of ester
formation will also depend heavily on substrate availability.
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) also suggests the presence of such an endogenous enzyme. Very low levels of isoamyl acetate
were found in wild-type plants when fed with isoamyl
alcohol (Fig. 4C), indicating that the putative
endogenous acyltransferase hardly uses acetyl-CoA,
while it can use methylbutyryl-CoA and benzoyl-CoA.
Probably both acetyl-CoA and benzoyl-CoA are available in petunia, and while the endogenous enzyme
uses only benzoyl-CoA, the ectopic SAAT enzyme uses
acetyl-CoA.
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). However, by engineering the alcohol supply, other side-products may
be induced. This is illustrated by our finding of previously undetected isoamyl methylbutyrate and isoamyl benzoate in the wild-type petunia headspace
when feeding isoamyl alcohol (Fig. 5C). Engineering of
ester formation in a background that already contains
alcohols may avoid these problems. In fruits of tomato
and strawberry, for instance, alcohols are quite abundantly available (Zabetakis and Holden, 1997; Speirs
et al., 1998). Another possibility is to vary culturing
conditions, for example by introducing stress, which
will affect the concentration of alcohols (Mattiacci
et al., 2001). Our findings parallel those described
by Schwab (2003), who reviewed several reports on
biochemical analysis of heterologously expressed
enzymes where poor specificity was observed for
enzymes involved in the production of secondary
metabolites during fruit ripening, (e.g. in flavor and
color formation). It was concluded that metabolome
diversity is also caused by low enzyme specificity and
directly related to availability of suitable substrates.
Future work will therefore have to take into account
the entire metabolic pathway, rather than a single enzyme, to engineer emission of novel volatiles in plants.
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Beekwilder et al.
MATERIALS AND METHODS
Plant Material and Petunia Transformation
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 and RNA-Gel Blots
RNA isolation from the various fruit tissues was performed according to
Asiph et al. (2000) and from leaves of petunia using the method of Verwoerd
et al. (1989). RNA-gel blots were generated (10 mg of either petunia leaf or ripe
strawberry fruit total RNA) and hybridized as described previously (Aharoni
et al., 2000). The SAAT gene probe (780-bp fragment) was generated by PCR
using the oligonucleotides AAP157 5#-TCCAATCCATGATGTCCTCC-3#, and
AAP158 5#-GCTTGGAGTTCAAGTCAACG-3#. The blot was rehybridized
with a petunia cDNA probe showing homology to a gene encoding a ubiquitin
protein (identified in a petunia expressed sequence tag library described in
Aharoni et al., 2000) that was amplified by PCR from a pBlueScript vector
(Stratagene, La Jolla, CA) using the T3 and T7 oligonucleotides.
SAAT Overexpression Construct and
Plant Transformation
The entire SAAT coding region (1,359 bp) was amplified from pRSETB
(Aharoni et al., 2000) using oligonucleotides AAP165 (5#-CGGATCCGGAGAAAATTGAGGTCAG-3#) and AAP166 (5#-CGTCGACCATTGCACGAGCCACATAATC-3#). The amplified fragment was cleaved with BglII and SalI
restriction enzymes and inserted in a sense orientation into BamHI and SalI
restriction sites, located between a double 35S-cauliflower mosaic virus
promoter and a nopaline synthase terminator, in the pFLAP10 subcloning
vector (provided by A. Bovy, Plant Research International, Wageningen, The
Netherlands). From the pFLAP10 vector, the fragment was excised with PacI
and AscI restriction digestions and introduced to the pBinPLUS binary vector
(Van Engelen et al., 1995) containing the kanamycin resistance selection
marker (nptII). Petunia was transformed with the LBA4404 Agrobacterium
tumefaciens strain as described by Lucker et al. (2001). Fifteen lines rooting
vigorously on media containing kanamycin (75 mg/L) were transferred to the
greenhouse. Plants transformed with the empty pBinPLUS binary vector and
nontransformed wild-type ones were also regenerated in a similar way in
vitro on nonselective medium and utilized for the experiments.
Cloning Full-Length Genes, Sequencing, and
Phylogenetic Analysis
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, Little
Chalfont, UK). 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) and
oligonucleotide AAP184 (5#-CGTCGACCGGATAACATACGTAGAC-3#) at
the 3# end. AAP184 was designed based on a partial cDNA clone (522 bp,
GenBank accession no. AJ001450.1), which showed high homology to SAAT
and had been described by Nam et al. (1999) from the wild strawberry.
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). The products of the first PCR
were used for a nested PCR reaction using a second specific oligonucleotide
on the published sequence (AAP219; 5#-CACATCCATCACGTCAAGTCC-3#),
and a 700-bp 3# end fragment was obtained. Based on the 3# end fragment
sequence, the AAP232 oligonucleotide was designed for a 5# RACE reaction
(5#-CCTCGAGGGATACTGACAAGTACTACAC-3#) in order to clone the
entire banana cDNA.
Expression and Partial Purification of the
Recombinant Proteins
A modified expression vector (pRSET B; Invitrogen) described earlier
(Aharoni et al., 2000) was used for the expression of VAAT and BanAAT in
Escherichia coli cells (BL21 Gold DE3 strain; Stratagene). The oligonucleotides
used for cloning to pRSET B introduced restriction sites at both ends of the
fragments, which were compatible to the BamHI and SalI restriction sites in the
vector. The oligonucleotides AAP165 and AAP184 (see above) introduced
a BamHI and a SalI, respectively, to VAAT; AAP233 (5#-CGGATCCGAGCTTCGCTGTGACCAGAAC-3#) and AAP227 (5#-ACTTCACGTCCTCCAAGCTGAGTC-3#) introduced a BamHI and a XhoI, respectively, to BanAAT. To obtain
an in-frame N-terminus fusion of the recombinant proteins to the His-tag, the
A and T nucleotides of the ATG codon were removed, resulting in the
elimination of the starting Met and insertion of a Pro in the protein sequences.
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 mg/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-b-galactoside (1 mM). After overnight growth
at 16°C, cells were harvested by centrifugation and resuspended in 1 mL icecold buffer R (50 mM Tris-HCl, pH 5 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 (MSE Scientific Instruments, Crawley, UK). 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.
Substrate Preference Assays
Activity of recombinant proteins with different alcohol substrates was
assayed using 14C radiolabeled acetyl-CoA (Amersham) or 14C-butyryl-CoA
or 14C-hexanoyl-CoA (Campro, Veenendaal, The Netherlands). The reactions
were made in duplicate. In a total reaction volume of 100 mL, 2 mL acyl-CoA (5
mM), 0.2 mL 14C labeled acyl-CoA, 85.8 mL 50 mM Tris-HCl buffer, pH 8.3, and
10 mL purified enzyme were mixed slowly in a cold Eppendorf (Westbury,
NY) tube. The reactions were started by adding 2 mL 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 mL
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) for SAAT. Kcat values were determined by dividing Vmax
values by the protein concentration of the purified enzyme in the reaction
mixture, taking into account the calculated molecular mass of about 50 kD.
Enzyme Activity Assays with Plant Material
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,
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Characterization of Enzymes Forming Fruit Esters
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 mL supernatant, 300 mL buffer A, 25 mL
8 mM acetyl-CoA solution, and 25 mL 1.6 mM geraniol suspension. After
30 min, 400 mL 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-mm polydimethylsiloxane 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).
Headspace Analysis
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), beakers with the detached plant parts were placed in closed glass cuvets
(500 mL) for 24 h at 21°C in a light regime of 9 h light, 8 h darkness, and 7 h
light. A vacuum pump was used to draw air through the glass cuvet at
approximately 100 mL/min, with the incoming air being purified through
a glass cartridge (140 3 4 mm) containing 150 mg of Tenax TA (20/35-mesh,
Alltech, Deerfield, IL). At the outlet, the volatiles emitted by the detached
leaves were trapped on a similar cartridge. Volatiles were collected during
24 h. Cartridges were eluted using 3 3 1 mL of redistilled pentane:diethyl
ether (4:1). Of the (nonconcentrated) samples, 2 mL was analyzed by GC-MS
using a gas chromatograph (5890 series II, Hewlett-Packard) equipped with a
30-m 3 0.25-mm i.d., 0.25-mm film thickness column (5MS, Hewlett-Packard,
Palo Alto, CA) and a mass-selective detector (model 5972A, Hewlett-Packard).
The GC was programmed at an initial temperature of 45°C for 1 min, with a
ramp of 10°C/min up to 220°C and final time of 5 min. The injection port
(splitless mode), interface, and MS source temperatures were 250°C, 290°C,
and 180°C, respectively, and the He inlet pressure was controlled with an
electronic pressure control unit to achieve a constant column flow of 1.0 mL/
min. The ionization potential was set at 70 eV, and scanning was performed
from 30 to 250 atomic mass units. Data were quantified by comparison to
injected standards.
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.
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