VpAAT1, a gene encoding an alcohol acyltransferase, is involved in ester biosynthesis during ripening of mountain papaya fruit

Article abstract of DOI:10.1021/jf904296c

Mountain papaya (Vasconcellea pubescens) is a climacteric fruit that develops a strong and characteristic aroma during ripening. Esters are the main volatile compounds produced by the fruit, and most of them are dependent on ethylene. As esters are synthesized through alcohol acyltransferases (AAT), a full-length cDNA (VpAAT1) was isolated that displayed the characteristic motifs of most plant acyltransferases. The full-length cDNA sequence was cloned and expressed in yeasts, obtaining a functional enzyme with high AAT activity toward the formation of benzyl acetate. The transcript accumulation pattern provided by qPCR analysis showed that the VpAAT1 gene is expressed exclusively in fruit tissues and that a high level of transcripts is accumulated during ripening. The increase in VpAAT1 transcripts in fruit is coincident with the increase in AAT activity; transcript accumulation is induced by ethylene, and it is avoided by 1-methylcyclopropene (1-MCP) treatment. The data indicate that VpAAT1 is involved in aroma formation and that ethylene plays a major role in regulating its expression.

Full text of DOI:10.1021/jf904296c

5114 J. Agric. Food Chem. 2010, 58, 5114–5121
DOI:10.1021/jf904296c
VpAAT1, a Gene Encoding an Alcohol Acyltransferase,
Is Involved in Ester Biosynthesis during Ripening of
Mountain Papaya Fruit
†,
´
†,
†
´
C
RISTIAN
B
ALBONTIN
,
C
ARLOS
ERRERA,† DANIEL
G
AETE-EASTMAN
,
L
IDA
F
UENTES
,
§
´
C
ARLOS R. FIGUEROA,† RAUL
H
M
ANRIQUEZ,§ ALAIN
L
ATCHE
,
§
,†
´
´
J
EAN-CLAUDE
P
ECH
,
AND
M
ARIA
A
LEJANDRA
MOYA-LEON*
†Laboratorio de Fisiologı
´
a Vegetal y Gene
´
tica Molecular, Instituto de Biologıa Vegetal y Biotecnologıa,
´ ´
Universidad de Talca, Casilla 747, Talca, Chile, and §INRA/INP-ENSAT,
UMR990 “Genomique et Biotechnologie des fruits”, Avenue de l’Agrobiopole, B.P. 32607,
31326 Castanet-Tolosan, France. Contributed equally to the work.
Mountain papaya (Vasconcellea pubescens) is a climacteric fruit that develops a strong and
characteristic aroma during ripening. Esters are the main volatile compounds produced by the fruit,
and most of them are dependent on ethylene. As esters are synthesized through alcohol
acyltransferases (AAT), a full-length cDNA (VpAAT1) was isolated that displayed the characteristic
motifs of most plant acyltransferases. The full-length cDNA sequence was cloned and expressed in
yeasts, obtaining a functional enzyme with high AAT activity toward the formation of benzyl acetate.
The transcript accumulation pattern provided by qPCR analysis showed that the VpAAT1 gene is
expressed exclusively in fruit tissues and that a high level of transcripts is accumulated during
ripening. The increase in VpAAT1 transcripts in fruit is coincident with the increase in AAT activity;
transcript accumulation is induced by ethylene, and it is avoided by 1-methylcyclopropene (1-MCP)
treatment. The data indicate that VpAAT1 is involved in aroma formation and that ethylene plays a
major role in regulating its expression.
KEYWORDS: Alcohol acyltransferase; aroma; ester production; fruit ripening; 1-methylcyclopropene;
Vasconcellea pubescens
INTRODUCTION
Arabidopsis (8). AAT activity is responsible for the production
of esters, and it has been measured in two main plant tissues:
flowers and fruits (9). During the past few years, several AAT
genes have been isolated and characterized from several fruit
species, such as strawberry (9, 10), melon (11, 12), apple (13, 14),
banana (7), grape (15), and apricot (16). According to their
sequences, acyltransferases dependent on coenzyme A (CoA)
have been included within a wide and divergent family of
proteins, the BAHD superfamily (17). Among them the HXXXD
motif, located in the middle of the protein sequence, is highly
conserved in superior plants and yeasts. The substitution of the
histidine residue from this motif causes the loss of protein
function (18), which suggests that it could be involved in the
transfer of the acyl group from acyl-CoA toward an alcohol.
Another highly conserved motif in higher plants in this super-
family is the DFGWG sequence, located near the carboxylic end
of the protein, which seems to be involved in the maintenance of
the structural integrity of the enzyme (19).
Mountain or highland papaya (Vasconcellea pubescens) is a
diploid (2n = 18) dicotyledoneous species. It is native to the
Andean regions of South America and belongs to the Caricaceae
family, which includes the Carica and Vasconcellea genera (1, 2).
The fruit is climacteric and exhibits a characteristicriseinethylene
production during ripening, accompanied by softening, changes
in color, and development of a strong and characteristic
aroma (3-5). The main volatile compounds produced by the
fruit are esters and alcohols (4). Most of the esters found in
mountain papaya fruit are potent odor compounds, and the
dynamic of their production during ripening has been previously
reported (4). In addition, the treatment of mountain papaya fruit
with 1-methylcyclopropene (1-MCP), a strong inhibitor of ethyl-
ene action (6), has been shown to inhibit ethylene production and
the production of aroma volatile compounds in mountain papaya
fruit, particularly esters (4). Thus, most esters identified in
mountain papaya displayed a clear modulation by ethylene.
Esters are produced from alcohols and acyl-CoAs through the
action of alcohol acyltransferases (AAT) (7). Plants contain
large families of AATs, with about 61 putative members in
An important question inthe field has been the identification of
enzymes that are critical for production of the distinctive blend of
esters characteristic of each fruit species (13). The available
information indicates that substrate specificity of AAT enzymes
seems to be wide but with differential preferences toward acyl-
CoAs and alcohols. This could indicate that the particular aroma
*Corresponding author (telephone þ56 71 200280; fax þ56 71
200276; e-mail alemoya@utalca.cl).
pubs.acs.org/JAFC
Published on Web 04/06/2010
© 2010 American Chemical Society

Article
J. Agric. Food Chem., Vol. 58, No. 8, 2010 5115
of each fruit or flower depends on the interaction of different
types of AATs, each one with preferential, although nonexclu-
sive, substrates. In addition, AAT sequences provide valuable
information about enzyme properties, although in some cases it
has been difficult to predict substrate selectivity, and minor
differences in the amino acid sequence could alter the ability of
a certain enzyme to produce some esters (7, 11, 12). On the other
hand, substrate availability for AAT enzymes is also another
aspect to consider.
In the present paper, the isolation and characterization of an
AAT gene for the first time from the Caricaceae family was
performed. In addition, it was demonstrated that the encoding
protein is functional and active toward several substrates, and the
role of ethylene on the expression of the gene was investigated.
products amplified were cloned and sequenced by Macrogen Inc.
(Seoul, Korea). The full-length cDNA sequence was amplified by using
primers specially designed to match the initial and final transcription zones
of the gene (Vp-AAT1 F = 50-ATGGCAGAGAAAGCTAGTTC-30;
Vp-AAT1 R = 50-AATGGCGGACACAATAAAGA-30) and then
sequenced.
The nucleotide and deduced amino acid sequences were analyzed using
Vector NTI Advance 9.0 software (Invitrogen, 2003). The similarity
analysis was performed using the local alignment tool (BLAST, National
Center for Biotechnology Information, Bethesda, MD) and the web-based
tool Wolf PSORT World Wide Web Prediction Server (21). The multiple
alignment of amino acid sequences was performed using the software
BioEdit Sequence Alignment Editor v7.0 (22). The phylogenetic tree was
builtusingMEGAsoftware(version4;http://www.megasoftware.net) (23)
using the maximum parsimony method and Bootstrap analysis (1000
replicates).
DNA Gel-Blot Analysis. Mountain papaya genomic DNA was
extracted from a pool of young leaves (2 g) as described by Murray and
Thompson (24). Probe for DNA gel-blot analysis of VpAAT1 was
designed from the 30-end portion of the ORF sequence. The 108 bp probe
(from position 1308 to 1415 in VpAAT1 sequence) was prepared through
PCR reaction with the primers QAAT1-F (50-AGGAGAGAAAGG-
GATCGTAG-30) and QAAT1-R (50-TCAGCGGAAACAAGTAG-
TTG-30), and radiolabeled using [R-32P]dCTP (Easytides, NEN Life
Sciences Products). For Southern blots, 20 μg of genomic DNA was
digested with BamHI, HindIII, EcoRV, and EcoRI, fractioned on a 0.7%
agarose gel, and transferred to Hybond-Nþ membranes (Amersham
Biosciences) using 20ꢀ SSC as blotting buffer. Membranes were prehy-
bridized at 42 °C for 4 h in a solution containing 50% deionized
formamide, 1% (w/v) SDS, 5ꢀ SSCE, 5ꢀ Denhart’s solution, and
100 μg mL-1 denatured salmon sperm DNA. The hybridization step
was carried out overnight at 42 °C with denatured 32P-labeled probe with
gentle agitation. Washings were performed with 2ꢀ SSC containing 0.1%
(w/v) SDS for 15 min at 42 °C, followed by three washes with 1ꢀ SSC plus
0.1% (w/v) SDS during 15 min at 50 °C. The blots were exposed, and
autoradiograms were scanned in a densitometer (FLA-5100 Imaging
System, Fujifilm, Japan).
Real-Time (qPCR) Expression Analysis. Total RNA was extracted
from 6 g of mountain papaya fruit pulp (control, 1-MCP and ethylene-
treated papaya fruits) after 1, 3, 5, 7, 9, and 13 days at 20 °C using an
adapted version of the CTAB method (20). In addition, total RNA was
also extracted from other vegetative tissues: flower, leaf, root, and stem.
Total RNA was treated with DNase I amplificationgrade (Invitrogen) and
cleaned using the RNeasy Plant Mini Kit (Qiagen, Germany). First-strand
cDNA synthesis was performed using an AffinityScript QPCR cDNA
Synthesis Kit (Stratagene, La Jolla, CA) following the manufacturer’s
instructions. Three biological replicates for each sampling date were
employed. Specific primers for a divergent 30-end region of VpAAT1
and the R-elongation factor 1 (VpEF1-R; as internal control) genes were
designed using Vector NTI v9, with high stringency to avoid amplification
of nonspecific PCR products. Primers were tested by RT-PCR and the
amplification products sequenced to ensure their specificity. Primer pair
sequences were QAAT1-F and QAAT1-R (described above) and EF1-F
(50-TCAATGAACCCAAGAGGCCATCC-30) and EF1-R (50-CACG-
TCCCAC TGGAACAGTTCCA-30).
The amplicon sizes were 108 bp for the AAT gene and 96 bp for VpEF1-R.
The amplification reactions were performed using Brilliant SYBR Green
QPCR Master Mix (Stratagene) according to the manufacturer’s instruc-
tions in a DNA engine Opticon 2 Real-Time PCR System (MJ Research,
Watertown, MA). PCR conditions were as follows: 94 °C for 10 min;
40 cycles of 94 °C for 15 s, 60 °C for 15 s, and 72 °C for 20 s; melting curve
from 58 to 95 °C at 0.5 °C increments. A dilution series was built to estimate
the amplification efficiency using a cDNA mix as template prepared from
control fruit samples (1-13 days of storage). Each reaction was performed in
triplicate, and a negative water control was included in each run. Fluores-
cence was measured at the end of each annealing step. The amplification
efficiency was estimated through a melting curve, and amplification products
were visualized on agarose gels (1.5% w/v). The relative expression levels
were first normalized against the VpEF1-R gene and using nontreated fruit
samples from day 1 as calibrator, with a nominal value of 1. The method
described by Pfaffl (25) was used to make all calculations.
MATERIALS AND METHODS
Plant Material. Mountain papaya (V. pubescens) fruit and other
vegetative tissues were collected in February 2005 from orchards located at
Lipimavida (34° 510 S; 72° 080 W; 20 m asl), on the coast of Curico
´
, Chile.
Young expanding fruit was picked and staged as small green fruit (SGF),
medium green fruit (MGF), or large green fruit (LGF), according to the
guidelines of Gaete-Eastman et al. (5). Ripening fruit was harvested after
the first signs of chlorophyll breakdown, sorted for size and then randomly
divided into three lots: one lot was left intact (control fruit); the second lot
was treated with 1-MCP; and the last one was treated with ethylene
(Ethrel), both on the same day of harvest (day 0). After each treatment, the
fruit was allowed to ripen at 20 °C in separate rooms.
1-MCP and Ethylene Treatments. Fruit was placed inside an
airtight chamber of 0.28 m3 and treated with 0.3 μL L-1 1-MCP for
16 h at 20 °C. One hundred and fifty milligrams of EthylBloc (0.14%
1-MCP) was dissolved in 20 mL of 0.9% (w/v) sodium hydroxide to allow
the release of 1-MCP gas. The chamber was immediately closed after
EthylBloc application, and it was opened only after 16 h to remove the
1-MCP-treated fruit, which was subsequently left at 20 °C. For ethylene
treatment, the fruit was dipped during 5 min in 25 L of an aqueous solution
of 2 g L-1 Ethrel 48 SL (Bayer CropScience S.A.; ethephon, 2-chloroethyl
phosphonic acid), allowed to dry at room temperature, and then left at
20 °C in separate rooms.
Sampling of fruit from each treatment (ethylene, 1-MCP, and non-
treated) was performed every 2 days (starting on day 1) until completing
13 days at 20 °C. Three replicates of four fruits were randomly selected and
assessed for volatiles and ethylene (4). In addition, on each sampling date,
pulp tissue from each fruit was immediately frozen in liquid nitrogen and
stored at -80 °C until required.
Ethylene Production Rate. The procedure employed was described
previously (4). Briefly, the fruit was introduced into airtight respiration
chambers (6 L), and after 3 h of incubation at 20 °C, 1 mL gas samples were
withdrawn from the headspace and quantified for ethylene in a Perkin-
Elmer (Clarus 500) gas chromatograph. Three independent ethylene
samples were taken per chamber, and results were expressed as means (
standard error (SE) (μL kg1- h-1).
Isolation of VpAAT1 Gene from V. pubescens. To isolate the AAT
gene, total RNA (1 μg) was extracted from 6 g of pulp tissue of a ripe
mountain papaya fruit (day 5 of storage) using an adapted version of the
CTAB method (20). Then the RNA was treated with DNase I amplifica-
tion grade (Invitrogen) and cDNA synthesized using a BD SMART PCR
(Clontech) kit according to the manufacturer’s instructions. Degenerate
primers (VpAAT1 F = 50-TACTAYCCHYTHKCYGGAAG-30 and
VpAAT1deg R=50-GBCYKYCCCCATCCAAA-30) were designed to
match AATs conserved regions. PCR runs were performed, and PCR
product was cloned onto pCR 2.1 TOPO (Invitrogen) and manually
sequenced through ALFexpress II (Amersham Biosciences) using a
thermosequenase dye terminator kit (Amersham Biosciences). To com-
plete the gene sequence, specific internal primers were designed for 50 and
30 RACE-PCR on the basis of the isolated sequence (VpAAT1RACE F =
50-TCCATTGGCCGGACGTCTCAGGG-3; VpAAT1RACE R =
50-ATGGCAGCCATCT CACTGGGACC-30). RACE-PCR runs were
performed using the BD SMART RACE cDNA amplification kit
(Clontech), according to the manufacturer’s instructions. The PCR

´
Balbontın et al.
5116 J. Agric. Food Chem., Vol. 58, No. 8, 2010
Expression of Recombinant VpAAT1. The full-length sequence of
VpAAT1 was initially cloned into Escherichia coli TOP 10 One Shot
(Invitrogen) to select colonies with the right orientation. The insert was then
cloned in the pYES2.1 TOPO-TA cloning vector following the instructions
provided by the manufacturer (Invitrogen). The construct was used to
transform the Saccharomyces cerevisiae cell line INVSc1. Transformed
yeasts were grown at 30 °C in selective medium (SC-U) with 2% galactose
as inducer of the recombinant protein expression, with constant stirring and
bubbling of sterile air until the OD600 of the culture reached <1 U.
Purification of Recombinant VpAAT1. The purification of the
recombinant protein was carried out according to the method of
El-Sharkawy et al. (11) with some modifications. Six flasks containing
100 mL of yeast culture induced with galactose (OD600 of 0.8) were
centrifuged (1800g for 5 min at room temperature), and the cells collected
from each flask were resuspended in 2 mL of buffer A (50 mM sodium
phosphate (pH 7.5), 10% (v/v) glycerol, 0.3 M NaCl) containing 2 mM
β-mercaptoethanol. The cells were mechanically ground in liquid nitrogen
for 10 min and stored at -80 °C until needed. To extract AAT enzyme, the
powder was thawed and centrifuged at 10000g for 20 min at 4 °C. The
crude extract obtained was purified through a BD Talon Metal Affinity
column (BD Biosciences), an affinity column designed to purify
polyhistidine-tagged proteins, according to the manufacturer’s protocol.
The recombinant protein was eluted with buffer A containing 150 mM
imidazole. Proteinswere quantified according to the Bradford method (26)
and visualized through 10% SDS-PAGE gels.
Assay of AAT Activity. AAT activity of recombinant VpAAT1
protein was quantified by its ability to convert alcohols and acyl-CoAs
into the corresponding ester (10, 27). AAT activity was assayed in 500 μL
of total volume in the presence of 2 mM alcohol-250 μM acyl-CoA in
50 mM Tris-HCl (pH 7.5) buffer containing 10% (v/v) glycerol and 1 mM
dithiothreitol (DTT) (12). A set of three acyl-CoAs (acetyl-, butanoyl-, and
hexanoyl-CoA) and nine different alcohols were assayed: ethanol, metha-
nol, butanol, hexanol, octanol, benzyl alcohol, geranyl alcohol, 1-phenyl-
ethanol, and cinnamyl alcohol. Each reaction assay contains the mixture
of one alcohol and one acyl-CoA per time. The reaction was initiated by
the addition of 200 μL of purified protein (10-15 μg), and the mixture was
incubated at 30 °C for 2.5 h in sealed Eppendorf tubes. The reaction was
stopped by the addition of 50 mg of citric acid and 185 mg of KCl, and
after mixing during several minutes, the supernatant was transferred to a
glass vial, which was sealed after the addition of 0.5 μL of 1,2-dichloro-
benzene as internal control. The solution was stirred during 15 min at
room temperature; meanwhile, the volatiles produced during the enzy-
matic reaction were released into the headspace and adsorbed onto an
SPME fiber (PDMS/DVB). The separation and quantification of each
ester was done by a gas chromatograph fitted with a flame ionization
detector (GC-FID) (Perkin-Elmer, Clarus 500) (4). The separation and
quantification of each ester was done by a GC-FID following the
using the SPSS v. 14 package. Analysis of variance was performed, and
significant differences were determined at P e 0.05 (LSD test).
RESULTS
VpAAT1 Shares High Similarity with AATs from the BAHD
Family. With the aim to isolate ripening-related AAT genes from
mountain papaya fruit, degenerate primers were designed on the
basis of two conserved regions in the BAHD superfamily
gene sequences: the motif YYPLAGRL, located close to the
N-terminal, and the DFGWGR motif, at the C-terminal end (9).
By PCR a fragment of 1200 bp was amplified with high homology
to other AAT sequences, which was used as a template to design
internal primers for 50 and 30 RACE-PCR runs. Two fragments of
1300 and 800 bp were obtained using these primers. A composite
cDNA sequence of 1622 bp called VpAAT1 (GeneBank accession
no. FJ548611) was generated from all fragments.
Analysis of the VpAAT1 sequence revealed an ORF of 1383 bp
and a deduced amino acid sequence of 463 amino acids with a
molecular weight of 51.4 kDa (Figure 1A). The sequence also
contains 60 and 173 bp of 50- and 30-UTR, respectively. VpAAT1
shares the characteristic motifs found in other plant acyltrans-
ferases, including the active site motif HXXXDG (amino acids
166-171). In VpAAT1 the His and Asp residues are conserved;
however, the Gly is replaced by Ala. Some AATs also have Ala in
this position, but it is not known if this change could affect the
activity or substrate preferences (13). There is also another highly
conserved motif located toward the carboxylic end formed by five
amino acids, DFGWG (amino acids 381-385). VpAAT1 also
exhibits a third less conserved motif, LXXyyplaGR (amino acids
75-84), located near the amino-terminal end, which is common
among AATs involved in the synthesis of volatile compounds in
fruits and flowers. Finally, it is important to highlight the
presence of Thr in position 266, which has been shown to be
essential for enzyme activity (12).
Sequence Comparison and Phylogenic Analysis. The VpAAT1
deduced amino acid sequence was aligned and compared with 15
plant acyltransferases (Figure 1A). The highest similarity was
found between VpAAT1 and CmAAT3 isolated from Cucumis
melo (12) and BEBT (benzoyl coenzymeA: benzyl alcohol ben-
zoyl transferase) isolated from Clarkia breweri (28), with 67 and
61% identity at the amino acid level, respectively. A phylogenic
tree was built from the previous multiple alignment, and the
grouping pattern provides three main subgroups (Figure 1B).
VpAAT1 was clustered into subgroup III with AATs related to
the synthesis of esters in melon and Clarkia (CmAAT3 and
CbBEBT). The same cluster also incorporates AATs isolated
from apple (MpAAT1), pear (PcAAT1), banana (MsAAT1) (7),
and melon (CmAAT1 and CmAAT2) (11). Subgroup II com-
prises AATs isolated from rose (29) and different strawberry
species, such as Fragaria ꢀ ananassa (9), Fragaria vesca (7), and
Fragaria chiloensis (10); finally, subgroup I was formed only by
Cm-AAT4 from melon (12).
DNA Gel-Blot Analysis. To analyze the complexity of moun-
tain papaya’s AAT family, genomic analysis was performed using
DNA gel blots. By using a probe that matches the 30 end of the
coding sequence of the VpAAT1 gene (Figure 1A), a divergent
region of AAT single-hybridization fragments were detected in
DNA digested with HindIII, EcoRV, and EcoRI enzymes, all of
them ranging between 2.3 and 9.4 kb. Only EcoRI has a restric-
tion site at nucleotide 403 of the VpAAT1 sequence. Analysis of
BamHI-digested DNA revealed two hybridizing bands around
9.4 kb and over 6.6 kb (Figure 2). Considering that no restriction
sites for BamHI are included in the coding sequence, its restriction
pattern could suggest two possibilities according to the location
procedure described by Balbontın et al. (4). A calibration curve was
´
prepared for each ester. AAT enzyme activity was expressed as picokatals
(pmol s-1) per milligram of protein. Protein content was determined using
BSA as standard (26). Determinations were performed in triplicate and
expressed as mean ( SE.
AAT activity was also assayed in mountain papaya pulp samples
obtained during ripening at 20 °C. Frozen pulp tissue (10 g) was
homogenized in a mortar with the help of liquid nitrogen in the presence
of 0.2 g of PVPP and 20 mL of buffer 100 mM Tris-HCl (pH 8)-1 M KCl,
0.1% (v/v) Triton X-100, containing 1 mM phenylmethanesulfonyl
fluoride (PMSF) and 2 μM leupeptin as protease inhibitors. The mixture
was stirred for 20 min at 4 °C, filtered through Miracloth, and centrifuged
(10000g for 20 min). The supernatant was desalted through a Sephadex
G-25 gel filtration column (PD-10 Pharmacia) in the presence of 50 mM
Tris-HCl (pH 7.5) buffer containing 10% (v/v) glycerol and 0.5 mM DTT.
AAT activity was quantified by its ability to convert acetyl-CoA and
hexanol into hexyl acetate (10, 27). The reaction was performed as
described above in the presence of 10 mM hexanol-490 μM acetyl-
CoA-50 mM Tris-HCl (pH 7.5) buffer, 10% (v/v) glycerol, and 0.5 mM
DTT. The reaction was initiated by the addition of 300 μL of protein
extract and the mixture incubated at 30 °C for 2 h. A calibration curve was
prepared with hexyl acetate.
Statistical Analysis. The experiment was conducted using a complete
random design with three replicates. Statistical analyses were performed

Article
J. Agric. Food Chem., Vol. 58, No. 8, 2010 5117
Figure 1. (A) Alignment of the deduced VpAAT1 sequence with other acyltransferases of known function from different floral and fruit species.
Gaps are indicated by dashes, letters with black background are identical amino acids, and letters with gray background are similar amino acids.
The three motifs that are characteristic of most AATs are underlined: LVHYYPLAGR, HTMSD (related to the catalytic activity and conserved
within the BAHD acyltransferase family), and DFGWG (highly conserved within the BAHD protein family and apparently required for conformation
integrity of the protein structure). Sequences correspond to GenBank data library accession numbers: Cm (Cucumis melo) AAT1 (CAA94432),
AAT2 (AAL77060), AAT3 (AAW51125), AAT4 (AAW51126); Cb (Clarkia breweri) BEBT (AAN09796), BEAT (AAF04787); Fa (Fragaria ꢀ ananassa),
AAT (AAG13130); Fc (Fragaria chiloensis) AAT1 (FJ548610); Fv (Fragaria vesca) AAT (AAN07090); Md (Malus domestica) AAT2 (AAS79797);
Mp (Malus pumila) AAT1 (AAU14879); Ms (Musa sapientum) AAT1 (CAC09063); Pc (Pyrus comunis) AAT1 (AAS48090); Rh (Rosa hybrida)
AAT (AAW31948); Vp (Vasconcellea pubescens) AAT1 (FJ548611). Sequences were aligned using Bioedit Sequence Alignment Editor v 7.0.
At the end of alignment, the percentage of identity with VpAAT1 is shown. (B) Phylogenic analysis of VpAAT1. The phylogenic tree was
built using MEGA software (version 4). Numbers on branches indicate bootstrap values (as a percentage). Sequences are the same used in A
(see above).

´
Balbontın et al.
5118 J. Agric. Food Chem., Vol. 58, No. 8, 2010
Figure 2. DNA gel-blot analysis of genomic DNA from mountain papaya.
Genomic DNA (20 μg per lane) was digested with the indicated restriction
enzyme and hybridized with the corresponding 32P-labeled probe for
VpAAT1. Hybridization and stringency conditions were as described under
Materials and Methods.
of BamHI polymorphism. The BamHI restriction site could be
located either in an intron of the VpAAT1 gene or outside the
gene. In both cases, this result demonstrates the heterogozity of
the plant species and the presence of two different alleles of
VpAAT1 gene in the mountain papaya genome. With the aim to
localize other AAT genes in mountain papaya, further experi-
ments should be performed with a probe containing an AAT
conserved region.
Transcriptional Analysis of VpAAT1. Mountain papaya fruit
displayed the typical climacteric rise in ethylene production
during ripening at 20 °C (Figure 3A). Treatment of the fruit with
1-MCP inhibited the ethylene production rise by the fruit during
ripening, whereas ethylene treatment produces the advancement
in time ofthe climacteric phase development. To estimate the level
of VpAAT1 transcript accumulation during ripening of mountain
papaya fruit, qPCR analyses were performed (Figure 3B). In
control fruit a notorious increment in the level of VpAAT1
transcripts was observed during ripening, reaching the highest
level at day 5 and decreasing after that. In 1-MCP-treated fruit
VpAAT1 transcripts remained almost constant after treatment,
with levels similar to that of day 1. In samples treated with
ethylene a clear increment in transcript accumulation was shown
during the first days of storage, reaching the maximum level at
day 3 and decreasing until the end of the storage period.
Accumulation of VpAAT1 transcripts was analyzed in several
vegetative tissues and fruits at different developmental stages.
VpAAT1 transcripts were detected only in fruit tissues, not being
expressed in other vegetative tissues (Figure 3C). A very low
transcript level was observed at the large green fruit stage
compared to ripe fruit.
Figure 3. (A) Changes in ethylene production during ripening of mountain
papaya fruit. Fruits harvested at the breaker stage were divided into three
lots: one was kept untreated (control); another was treated with 1-MCP
(0.3 μL L-1 for 16 h at 20 °C) on the same day of harvest; and the last one
was treated with 2 g L-1 Ethrel. After the treatments, the fruit was allowed
to ripen at 20 °C. Three replicates of four fruits chosen at random were
analyzed every 2 days. The data correspond to the mean ( SE. (B)
Changes in VpAAT1 mRNA abundance during ripening of mountain
papaya fruit measured by qPCR. Samples were collected after 1, 3, 5, 7,
9, and 13 days of storage at 20 °C. The expression data correspond to
means of three replicates, normalized against VpEF1-R abundance, using
a control sample from day 1 as calibrator, and expressed in arbitrary
units ( SE. Asterisks indicate significant differences between control and
treatments at the same storage day (P e 0.05). (C) VpAAT1 mRNA
abundance in vegetative tissues and fruit samples at different develop-
mental stages. Expression analysis of VpAAT1 by qPCR was performed in
flower, small-size green fruit (SGF), medium-size green fruit (MGF), large-
size green fruit (LGF), ripe fruit (RF, corresponding to control fruit on day
5), leaf, root, and stem. The expression data are means of three replicates,
normalized to VpEF1-R abundance, using a control fruit sample from day 1
as calibrator, and expressed in arbitrary units( SE. Asterisks indicate
significant differences between tissues (Pe0.05).
AAT Activity during Ripening of Mountain Papaya Fruit. AAT
activity showed an increment during ripening of mountain
papaya fruit (Figure 4), reaching the maximum level after
3-5 days of storage, followed by a small decline in AAT activity
during the remaining days. AAT activity was assayed by its
capacity to produce hexyl acetate, one of the main esters
produced by mountain papaya fruit. The same profile of AAT
activity was observed when activity was assayed by its capacity to
produce benzyl acetate (data not shown), an ester that is absent in

Article
J. Agric. Food Chem., Vol. 58, No. 8, 2010 5119
mountain papaya’s aroma, although maximum activity recorded
(3.2 pkat mg-1 of protein) was lower than for hexyl acetate.
Functionality of the VpAAT1 Gene. With the aim to evaluate if
VpAAT1 protein is functional, the full-length cDNA sequence
was cloned and expressed in yeasts. Several transformants were
analyzed, and the correct orientation of the insert was checked by
means of PCR. The recombinant protein expressed in yeast cells
(61.2 pkat mg-1 of protein) was partially purified (450 times)
through an affinity column (BD Talon), obtaining an enzyme
with high specific AAT activity (27548.1 pkat mg-1) toward the
formation of benzyl acetate. It is important to note that the ability
to synthesize esters was notably diminished in cultures with
OD600>1. The activity of the purified enzyme was also assayed
in the presence of different combinations of acyl-CoAs and
alcohols as substrates (Table 1). The results obtained indicate
that the recombinant protein is able to synthesize a wide range of
esters when the substrates are provided, including some char-
acteristic aroma compounds from mountain papaya fruit. A
marked preference was observed toward the formation of parti-
cular compounds: VpAAT1 displayed a strong activity toward
the formation of benzyl acetate, followed by the ability to
synthesize methyl hexanoate, geranyl acetate, and cinnamyl
acetate. VpAAT1 showed a moderate activity toward the synth-
esis of phenylethyl acetate, ethyl hexanoate, and butyl butanoate,
and a small activity toward the synthesis of ethyl butanoate, octyl
acetate, butyl hexanoate, and hexyl acetate. No activity was
registered for the formation of hexyl hexanoate (Table 1).
DISCUSSION
Mountain papaya is a climacteric fruit that develops an
interesting and characteristic aroma during ripening, which is
mainly due to esters (4). Most of the esters identified in mountain
papaya fruit are modulated by ethylene, showing an increase in
production during ripening and in response to ethylene treat-
ment, and a strong reduction in response to 1-MCP treatment.
Because esters are important for aroma, and a significant increase
in ester production was observed during ripening of papaya fruit,
our study was focused on ester biosynthesis by the AAT enzyme.
AAT activity assayed by its capacity to produce hexyl acetate
showed an important increment during ripening of mountain
papaya fruit, with the maximum level of activity after 5 days of
storage at 20 °C.
In addition, an AAT gene sequence was isolated from moun-
tain papaya fruit (VpAAT1) that displays all of the conserved
regions observed in other acyltransferases belonging to the
BAHD superfamily. A large number of acyltransferases have
been described in plants, with 61 and 91 putative members in
Arabidopsis and Populus, respectively. In some fruit species, such
as melon and apple, a small multigene family has been
described (12-14). To determine the complexity of AAT genes
present in mountain papaya’s genome, DNA gel-blot analysis
was performed. Our results suggest the existence of two different
alleles of the VpAAT1 gene in mountain papaya. Phylogenic
analysis indicated that VpAAT1 is close to other AAT genes
belonging to subfamily III that participate in the synthesis of
volatile compounds during fruit ripening, such as CmAAT1,
CmAAT3, PcAAT1, MpAAT1, and MdAAT2 (12). In addition,
members of subfamily III have been described for their capacity
to produce benzyl acetate (28). Interestingly, benzyl acetate has
not been described in mountain papaya fruit aroma (4, 30, 31).
According to our phylogenic analysis, the most similar sequences
to VpAAT1 are CmAAT3 and CbBEBT, and all of them
displayed the highest activity toward the synthesis of benzyl
acetate (Table 2). CbBEBT and VpAAT1 displayed similar
activity toward the synthesis of cinnamyl acetate (8.4 and 7.6%,
Figure 4. Change in AAT activity during ripening of mountain papaya fruit.
Papaya fruit harvested at the breaker stage was allowed to ripen at 20 °C.
Fruit extracts wereprepared, andAAT activity was quantified by its ability to
convert acetyl-CoA and hexanol into hexyl acetate. Data correspond to the
mean ( SE of three replicates.
Table 1. Activity of VpAAT1 Recombinant Protein toward Different Acyl-CoAs and Alcohols as Substratesa
acyl-CoA
alcohol
ester produced
produced in papaya fruit?
AAT activity (pkat mg-1
)
acetyl-CoA
benzyl alcohol
geranyl alcohol
cinnamyl alcohol
phenylethanol
octanol
benzyl acetate
geranyl acetate
cinnamyl acetate
phenethyl acetate
octyl acetate
no
29275 ( 2593
3451 ( 1179
2219 ( 4.9
805 ( 157
60 ( 15.1
20 ( 6.1
no
no
no
yes
yes
yes
yes
hexanol
butanol
ethanol
hexyl acetate
butyl acetate
ethyl acetate
1 ( 0.2
tr
butanoyl-CoA
hexanoyl-CoA
butanol
ethanol
butyl butanoate
ethyl butanoate
yes
yes
526 ( 9.8
83 ( 14.7
hexanol
butanol
ethanol
methanol
hexyl hexanoate
butyl hexanoate
ethyl hexanoate
methy hexanoate
no
nd
yes
yes
yes
50 ( 3.3
790 ( 147
4668 ( 1737
a Calibration curves were prepared for each ester; R2 values ranged between 1 and 0.93. Data correspond to mean ( SE. tr, traces; nd, nondetectable.

´
Balbontın et al.
5120 J. Agric. Food Chem., Vol. 58, No. 8, 2010
Table 2. Comparison of AAT Activities toward Different Acyl-CoAs and Alcohol Substrates between VpAAT1 and Closely Related AATsa
ester produced
Vp AAT1
Cb BEBT
Cm AAT3
Mp AAT1
Cm AAT1b
Ban AAT
benzyl acetate
methy hexanoate
geranyl acetate
cinnamyl acetate
phenethyl acetate
ethyl hexanoate
butyl butanoate
octyl acetate
100
16
12
8
100
nr
100
nr
tr
nr
nr
52
nr
tr
2
nr
46
8
nr
88
100
nr
tr
nr
4
3
nr
nd
0
nr
nd
1
3
nr
nr
nr
2
nr
nr
nd
1
nd
nr
nr
5
nr
0
71
33
36
nd
nr
44
nr
hexyl acetate
butyl acetate
ethyl acetate
0
100
5
50
34
nd
3
0
3
1
tr
nd
1
nr
0
ethyl butanoate
hexyl hexanoate
butyl hexanoate
0
nr
nr
nd
0
nr
1
85
nd
90
4
nr
nr
2
nr
a The AAT activity of VpAAT1 recombinant protein was assayed by combining a set of three acyl-CoAs (acetyl-, butanoyl-, and hexanoyl-CoA) and nine different alcohols as
substrates (ethanol, methanol, butanol, hexanol, octanol, benzyl alcohol, geranyl alcohol, phenylethanol, and cinnamyl alcohol). Some combinations were not quantified. Activity
values for related AATs were obtained from the literature (7, 11-13, 29) and expressed as percentage of the maximum activity recorded for each enzyme. tr, traces; nd,
nondetected; nr, not reported. b 100% activity of CmAAT1 is not mentioned as it corresponds to the production of Z-hexenyl acetate, which was not proved in VpAAT1.
respectively). On the other hand, CmAAT3 and VpAAT1 shared
a low activity toward hexyl acetate formation (0.5 and 0.07%,
respectively). This analysis could support the hypothesis that
members from subfamily III share structural and functional
characteristics.
substrate availability and not only AAT selectivity, as has been
proposed previously (36-39), albeit AAT purportedly is the rate-
limiting step in ester biosynthesis (32).
VpAAT1 protein expressed in yeast was able to produce a wide
range of esters, including those normally produced by papaya
fruits and some with high aroma impact. This supports the
hypothesis that VpAAT1 is involved in aroma biosynthesis in
papaya fruit. However, the activity observed toward the forma-
tion of these compounds was considerably lower thanthat toward
the synthesis of benzyl acetate, geranyl acetate, cinnamyl acetate,
and methyl hexanoate. The reduced activity of VpAAT1 toward
the synthesis of butyl acetate and ethyl acetate, compounds that
are profusely produced in ripening papaya fruit, most probably
indicates that other AAT genes, which have not been described
yet, are present in the species.
In conclusion, our study strongly suggests that the VpAAT1
gene is functional and is involved in the synthesis of esters, which
are important contributors to the aroma of papaya fruit. The
increase in VpAAT1 transcripts in fruit tissue during ripening of
papaya fruit is coincident with the increment in AAT activity and
ester production. Finally, ethylene plays a major role modulating
the expression of VpAAT1 gene and promoting the increment in
AAT activity.
The transcript accumulation pattern provided by qPCR ana-
lysis showed that the VpAAT1 gene is expressed exclusively in
fruit tissues and that a high level of transcripts are accumulated
during ripening of mountain papaya fruit. The increase in
VpAAT1 transcripts is coincident with the increase in AAT
activity during ripening of the fruit. On the other hand, transcript
accumulation of VpAAT1 is induced by ethylene, and its induc-
tion is avoided by 1-MCP treatment. The data suggest that the
expression of the VpAAT1 gene is modulated by ethylene, as has
been reported for other acyltransferases involved in the synthesis
of plant volatiles (32). Nevertheless, other regulatory mechanisms
besides ethylene could not be discarded (33). The specific tran-
script accumulation of VpAAT1 in fruit tissue has also been
reportedpreviouslyinotherfruitspeciessuchasmelon(11,12,34).
Nevertheless, in apple the MpAAT1 gene is expressed not only in
ripening fruits but also in flowers and developing fruits (13). On
the other hand, genes involved in aroma biosynthesis of floral
species such as C. breweri (28) and Rosa hybrida (29) are expressed
in their floral organs.
The functionality of VpAAT1 recombinant protein was
proved, showing that it was able to use different alcohols and
acyl-CoAs as substrates for the synthesis of esters. The assays
performed with partially purified enzyme showed a strong
activity of the enzyme toward the production of benzyl acetate,
which is one of the most powerful odorant compounds used as
fragrance in perfumes and foods (35), and it is the main ester
produced by AATs belonging to subfamily III (12). We also
found a high activity of the recombinant protein toward the
formation of esters that are not normally produced by mountain
papaya fruit, such as phenethyl acetate and benzyl acetate
(Table 1). Similar findings have been reported for the formation
of esters in other fruits, regarding the formation of E-2 hexenyl
acetate, 2-ethylbutanoate, and 3-methylbutyl, geranyl, and cin-
namyl acetate in melon (12) and geranyl, furfuryl, 2-phenylethyl,
and benzyl acetate in apple (13). Similarly, SAAT and BanAAT1
displayed a strong preference for the synthesis of geranyl and
cinnamyl acetate, respectively, which have not been described in
strawberry or banana fruits (7). All together, this could indicate
that the volatiles produced by a fruit are determined also by
LITERATURE CITED
(1) Badillo, V. M. Carica L. vs. Vasconcella St. Hil. (Caricaceae): con la
rehabilitacion de este u´ ltimo. Ernstia 2000, 10, 74–79.
´
(2) Van Droogenbroeck, B.; Breyne, P.; Goetghebeur, P.; Romeijn-
Peeters, E.; Kyndt, T.; Gheysen, G. AFLP analysis of genetic
relationships among papaya and its wild relatives (Caricaceae) from
Ecuador. Theor. Appl. Genet. 2002, 105, 289–297.
(3) Moya-Leon, M. A.; Moya, M.; Herrera, R. Ripening of the Chilean
´
papaya fruit (Vasconcellea pubescens) and ethylene dependence of
some ripening events. Postharvest Biol. Technol. 2004, 34, 211–218.
(4) Balbontı
Leon, M. A. Treatment with 1-MCP and the role of ethylene in
´
n, C.; Gaete-Eastman, C.; Vergara, M.; Herrera, R.; Moya-
´
aroma development of mountain papaya fruit. Postharvest Biol.
Technol. 2007, 43, 67–77.
(5) Gaete-Eastman, C.; Figueroa, C. R.; Balbontı
´
n, C.; Moya, M.;
Atkinson, R. G.; Herrera, R.; Moya-Leon, M. A. Expression of an
´
ethylene-related expansin gene during softening of mountain papaya
fruit (Vasconcellea pubescens). Postharvest Biol. Technol. 2009, 53,
58–65.
(6) Blankenship, S. M.; Dole, J. M. 1-Methylcyclopropene: a review.
Postharvest Biol. Technol. 2003, 28, 1–25.

Article
J. Agric. Food Chem., Vol. 58, No. 8, 2010 5121
(7) Beekwilder, J.; Alvarez-Huerta, M.; Neef, E.; Verstappen, F. W. A.;
Bouwmeester, H. J.; Aharoni, A. Functional characterization of
enzymes forming volatile esters from strawberry and banana. Plant
Physiol. 2004, 135, 1865–1878.
(23) Tamura, K.; Dudley, J.; Nei, M.; Kumar, S. MEGA4: Molecular
Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol.
Biol. Evol. 2007, 24, 1596–1599.
(24) Murray, M. G.; Thompson, W. F. Rapid isolation of high molecular
weight plant DNA. Nucleic Acids Res. 1980, 8, 4321–4326.
(25) Pfaffl, M. W. A new mathematical model for relative quantification
in real-time RT-PCR. Nucleic Acids Res. 2001, 29, e45.
(26) Bradford, M. M. A rapid and sensitive method for the quantification
of microgram quantities of protein utilizing the principle of protein-
dye binding. Anal. Biochem. 1976, 72, 248–254.
(27) Fellman, J. K.; Mattinson, D. S.; Bostick, B. C.; Mattheis, J. P.;
Patterson, M. E. Ester biosynthesis in ‘Rome’ apples subjected to low
oxygen atmospheres. Postharvest Biol. Technol. 1993, 3, 201–214.
(28) D’Auria, J.; Chen, F.; Pichersky, E. Characterization of an acyl-
transferase capable of synthesizing benzylbenzoate and other volatile
esters in flowers and damaged leaves of Clarkia breweri. Plant
Physiol. 2002, 130, 466–476.
(29) Shalit, M.; Guterman, I.; Volpin, H.; Bar, E.; Tamari, T.; Menda, N.;
Adam, Z.; Zamir, D.; Vainstein, A.; Weiss, D.; Pichersky, E.;
Lewinsohn, E. Volatile ester formation in roses. Identification of
an acetyl-coenzyme A geraniol/citronellol acetyl transferase in
developing rose petals. Plant Physiol. 2003, 131, 1868–1876.
(30) Morales, A. L.; Duque, C. Aroma constituents of the fruit of the
mountain papaya (Carica pubescens) from Colombia. J. Agric. Food
Chem. 1987, 35, 538–540.
(31) Idstein, H.; Keller, T.; Schreier, P. Volatile constituents of mountain
papaya (Carica candamarcensis, syn. C. pubescens Lenne et Koch)
fruit. Agric. Food Chem. 1985, 33, 666–672.
(32) Defilippi, B. G.; Kader, A. A.; Dandekar, A. M. Apple aroma:
alcohol acyltransferase, a rate limiting step for ester biosynthesis, is
regulated by ethylene. Plant Sci. 2005, 168, 1199–1210.
(33) Zhu, Y.; Rudell, D. R.; Mattheis, J. P. Characterization of cultivar
differences in alcohol acyltransferase and 1 aminocyclopropane-1-
carboxylate synthase gene expression and volatile ester emission
during apple fruit maturation and ripening. Postharvest Biol. Tech-
nol. 2008, 49, 330–339.
(34) Aggelis, A.; John, I.; Karvouny, Z.; Grierson, D. Characterization of
two cDNA clones for mRNAs expressed during ripening of melon
(Cucumis melo L.) fruits. Plant Mol. Biol. 1997, 33, 313–322.
(35) Bauer, K.; Garbe, D.; Surburg, H. Common Fragrance and Flavor
Materials, 2nd ed.; VCH Publishers: Cambridge, U.K., 1990; pp 9, 24.
(36) Tressl, R.; Drawert, F. Biogenesis of banana volatiles. J. Agric. Food
Chem. 1973, 21, 560–565.
(37) Knee, M.; Hatfield, S. G. S. The metabolism of alcohols by apple
fruit tissue. J. Sci. Food Agric. 1981, 32, 593–600.
(38) De Pooter, H. L.; Montens, J. P.; Willaert, G. A.; Dirinck, P. J.;
Schamp, N. M. Treatment of Golden Delicious apples with alde-
hydes and carboxylic acids: effect on the headspace composition.
J. Agric. Food Chem. 1983, 31, 813–818.
(8) Yu, X.-H.; Gou, J.-Y.; Liu, C.-J. BAHD superfamily of acyl-CoA
dependent acyltransferases in Populus and Arabidopsis: bioinfor-
matics and gene expression. Plant Mol Biol. 2009, 70, 421–442.
(9) Aharoni, A.; Keizer, L. C. P.; Bouwmeester, H. J.; Sun, Z.;
Alvarez-Huerta, M.; Verhoeven, H. A.; Blaas, J.; van Houwelingen,
A. M. M. L.; De Vos, R. C. H.; van der Voet, H.; Jansen, R. C.; Guis,
M.; Mol, J.; Davis, R. W.; Schena, M.; van Tunen, A. J.; O’Connell,
A. P. Identification of the SAAT gene involved in strawberry
flavor biogenesis by use of DNA microarrays. Plant Cell 2000, 12,
647–662.
(10) Gonza
´
lez, M.; Gaete-Eastman, C.; Valdenegro, M.; Figueroa, C. R.;
n, M. A. Aroma development
Fuentes, L.; Herrera, R.; Moya-Leo
´
during ripening of F. chiloensis fruit and participation of an alcohol
acyltransferase (FcAAT1) gene. J. Agric. Food Chem. 2009, 57,
9123–9132.
(11) Yahyaoui, F. E.; Wongs-Aree, C.; Latche, A.; Hackett, R.; Grierson,
D.; Pech, J. C. Molecular and biochemical characteristics of a gene
encoding an alcohol acyl-transferase involved in the generation of
aroma volatile esters during melon ripening. Eur. J. Biochem. 2002,
269, 2359–2366.
(12) El-Sharkawy, I.; Manriquez, D.; Flores, F. B.; Regad, F.; Bouzayen,
M.; Latche, A.; Pech, J. C. Functional characterization of a melon
alcohol acyl-transferase gene family involved in the biosynthesis of
ester volatiles. Identification of the crucial role of a threonine residue
for enzyme activity. Plant Mol. Biol. 2005, 59, 345–362.
(13) Souleyre, E. J.; Greenwood, D. R.; Friel, E. N.; Karunairetnam, S.;
Newcomb, R. D. An alcohol acyltransferase from apple (cv. Royal
Gala), MpAAT1, produces esters involved in apple fruit flavor.
FEBS J. 2005, 272, 3132–3144.
(14) Li, D.; Xu, Y.; Xu, G.; Gu, L.; Li, D.; Shu, H. Molecular cloning and
expression of a gene encoding alcohol acyltransferase (MdAAT2)
from apple (cv. Golden Delicious). Phytochemistry 2006, 67,
658–667.
(15) Wang, J.; De Luca, V. The biosynthesis and regulation of biosynth-
esis of Concord grape fruit esters, including ’foxy’ methyl anthrani-
late. Plant J. 2005, 44, 606–619.
(16) Gonza
´
lez-Aguero, M.; Troncoso, S.; Gudenschwager, O.; Campos-
n, M. A.; Defilippi, B. G. Differential expres-
Vargas, R.; Moya-Leo
´
sion levels of aroma-related genes during ripening of apricot (Prunus
armeniaca L.). Plant Physiol. Biochem. 2009, 47, 435–440.
(17) D’Auria, J. C. Acyltransferases in plants: a good time to be BAHD.
Curr. Opin. Plant Biol. 2006, 9, 331–340.
(18) Bayer, A.; Ma, X. Y.; Stockigt, J. Acetyltransfer in natural product
biosynthesis - functional cloning and molecular analysis of vinorine
synthase. Bioorg. Med. Chem. 2004, 12, 2787–2795.
€
(19) Ma, X.; Koepke, J.; Panjikar, S.; Fritzch, G.; Stockigt, J. Crystal
(39) Jayanty, S.; Song, J.; Rubinstein, N. M.; Chong, A; Beaudry, R. M.
Temporal relationship between ester biosynthesis and ripening
events in bananas. J. Am. Soc. Hortic. Sci. 2002, 127, 998–1005.
structure of vinorine synthase, the first representative of the BAHD
superfamily. J. Biol. Chem. 2005, 280, 13576–13583.
(20) Chang, S.; Puryear, J.; Cairney, J. A simple and efficient method
for isolating RNA from pines trees. Plant Mol. Biol. Rep. 1993, 11,
113–116.
(21) Horton, P.; Park, K.-J.; Obayashi, T.; Fujita, N.; Harada, H.;
Adams-Collier, C. J.; Nakai, K. WoLF PSORT: protein localization
predictor. Nucleic Acids Res. 2007, 35, W585–W587.
(22) Hall, T. A. BioEdit: a user-friendly biological sequence alignment
editor and analysis program for Windows 95/98/NT. Nucleic Acids
Symp. Ser. 1999, 41, 95–98.
Received for review December 4, 2009. Revised manuscript received
March 25, 2010. Accepted March 27, 2010. This work has been partly
supported by IFS and the Organisation for the Prohibition of Chemical
Weapons (OPCW), the PBCT (Programa Bicentenario de Ciencia y
Tecnologıa) Anillo Project ACT-41, and the “Centro de Investigacion en
Biotecnologıa Silvoagrıcola (CIBS)”. C.G.-E. and L.F. acknowledge
the PBCT Program for their postdoctoral fellowships.