Article

pubs.acs.org/jpr

In Depth Proteome Analysis of Ripening Muscadine Grape Berry cv.

Carlos Reveals Proteins Associated with Flavor and Aroma
Compounds
Devaiah Kambiranda,*,† Sheikh M. Basha,† Rakesh K. Singh,‡ Huan He,§ Kate Calvin,‡
and Roger Mercer‡
†
Center for Viticulture and Small Fruit Research, Florida A&M University, 6505 Mahan Drive, Tallahassee, Florida 32317, United
States
‡
Translational Science Laboratory, Florida State University College of Medicine, 1115 W. Call Street, Tallahassee, Florida 32306,
United States
§
Institute of Molecular Biophysics, 91 Chieftan Way, Florida State University, Tallahassee, Florida 32306, United States
*
S Supporting Information

ABSTRACT: Ripening in nonclimacteric fruits such as grape involves complex

chemical changes that have a profound influence on the accumulation of flavor and
aroma compounds distinct to a particular grape genotype. In this study, proteome
characterization of wine type bronze muscadine grape (Vitis rotundifolia cv. Carlos),
primarily grown in the Southeastern United States was performed during berry ripening.
Stage-specific protein expression was obtained among different stages of berries.
Differential analysis showed the expression of 522 proteins that regulate diverse
biological processes and metabolic pathways. Of these, 30 proteins are associated with
the production of key phenolic compounds, whereas 25 are associated with the
production of muscadine aroma compounds. These proteins are involved in the
phenylpropanoid pathway, terpene synthesis, fatty acid derived volatiles and esters that
affect muscadine berry flavor and aroma characteristics. Further, gene expression analysis
during ripening validated the expression pattern of 12 proteins. Catechin, epicatechin,
and four stilbenes were quantified to correlate observed proteome changes. This study not only revealed biochemical changes
during muscadine berry ripening but also offers indicators for marker-assisted breeding to enhance organoleptic properties of
muscadine grape to improve its flavor and aroma properties.
KEYWORDS: aroma, berry, flavor, muscadine grape, proteins, ripening, metabolites, gene expression

■ INTRODUCTION
Grapes are grown around the world for fresh fruit and making
wine. Grapes undergo numerous biochemical changes during
berry ripening that affects their product characteristics.1
Muscadine grapes have been in cultivation for more than 400
years,3 through their habitat is spread over Delaware to central
Florida and alongside the Gulf of Mexico to Texas; muscadine
grape (Vitis rotunifolia Michx.) cultivation is limited to
southeastern United States.2−4 Muscadine grape berries are
thick-skinned, grow in small clusters and possess a distinct
fruity flavor.4,5 Muscadine wines are increasingly becoming
popular because of their distinct fruity flavor and perceived
health benefits.2−4 Volatile and phenolic compounds that
impart aroma and flavor characteristics are important sensory
components of grape products, unique to individual grape
genotype and the locations they are grown.6 These character-
istics that affect the organoleptic properties of muscadine
grapes, and their consumption for health benefits have been
found to be influenced by environmental and agricultural
factors.6 Transcriptional and proteome resources in developing
and ripening V. vinifera berry have been well established. In
fruit research, proteomics is being increasingly used in the
development of effective biochemical markers for breeding and
to identify components influencing postharvest product
characteristics and shelf life.7 A proteomics approach has also
been taken to detect developmental changes and study the
effects of abiotic and biotic stresses on grape berry composition
and product characteristics.8−17 The newly developed high
throughput proteomics analysis platforms are quantitative and
are vital to advance proteomic research in fruit crops.7 In the
current study a shotgun proteomic technique was applied to
detect proteome changes during the ripening of wine type
bronze muscadine grape cv. Carlos, The present study was
focused to identify proteins involved in the synthesis and
regulation of flavor and volatile components that accumulate
during grape berry maturation and ripening. This study
provides important information on the stage specific expression
Received: November 24, 2015

© XXXX American Chemical Society A DOI: 10.1021/acs.jproteome.5b01064

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Figure 1. Muscadine cv. Carlos shotgun proteomics workflow.
of enzymes and proteins associated with various cellular
processes during grape berry development. Stage-specific
expression of 1411 proteins was detected during berry
development and ripening. Of these, 522 proteins were found
to fall within a p-value of <0.05 and an additional 55 proteins
were found associated with flavor and aroma components
synthesis during ripening of muscadine grape berry cv. Carlos.
Further, 16 identified proteins were confirmed by transcript
analysis, and also nine metabolites produced by the phenyl-
propanoid pathway were quantified to correlate with the
identified proteins. The data reported here will provide key
insights into the secondary metabolism of muscadine grape.
■ METHODS
Material
Muscadine grape (Vitis rotundifolia cv. Carlos) berry samples at
different developmental stages from the vineyard of the Center
for Viticulture and Small Fruit Research, Florida A&M
University, Tallahassee, Florida, were collected between July
and September, 2013. Berry samples corresponding to stages 33
(Green Hard), 34 (Green Soft), 35 (Bronze Soft) and 38
(Ripe) of the modified E-L system18 were measured for berry
diameter, berry weight, °Brix, and titratable acidity. Brix content
was recorded by squeezing juice from a small section of berry.
Samples were collected at 60, 73, 85, and 118 days,
postanthesis. Replications were treated as follows: berries
collected (200 berries) from multiple clusters of the same
grapevine were treated as one biological replication, and three
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biological replicates (200 berries × three different grapevines)
were collected. A portion of these berries (30 berries) were
used for measuring titratable acidity (g/L) by titrating grape
juice to a pH 8.4 end point with 0.1 N NaOH1 (Table S1).
Data obtained are the mean ± SD of three replications. The
remaining berry samples (∼130 to 150) were stored at −80 °C
for proteome, gene expression, and metabolite analysis.
Protein Extraction
Pericarp from the four maturity categories [Green Hard
(CGH) and Green Soft (CGS), Bronze Soft (CBS) and
Ripe] were used for protein extraction. Seeds were removed
from the berries after cutting the partially thawed berry with a
scalpel blade. Each replicate was subjected to independent
protein extraction following the phenol based extraction
protocol.1,19 The resulting pellet was solubilized in 1.5 mL of
rehydration buffer (7 M urea, 4% CHAPS, 2 M thiourea, and
10 mM DTT). The total protein was quantified by the
Bradford method.20 An aliquot of the protein extract in
rehydration buffer was precipitated with acetone. The protein
pellet was resuspended in Bicine buffer (0.5 M) with SDS
(0.1%; w/v), and incubated at 4 °C overnight in a
thermomixer21 to expedite complete solubilization of the
protein pellet. The resuspended protein was quantified again
using BCA reagent. Next, 50 μg aliquots of protein from each
sample and the three replications were run on 10% SDS-PAGE
gel until the protein dye band entered the separating gel as a
single band and stained with Coomassie blue dye. The single
protein band was cut from destained gel, and digested with
trypsin. In-gel digestion was performed using ProteoExtract All-
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in-One Trypsin Digestion Kit (cat. no. 650212 Calbiochem,
Billerica, MA, USA) according to manufacturer’s instructions.
Gel pieces were excised from the gel and destained with wash
buffer before drying them at 90 °C for 15 min. Gels were
rehydrated with digestion buffer and then reduced for 10 min
using a reducing agent at 37 °C. Samples were brought to room
temperature and then blocked using the blocking reagent for 10
min. Trypsin at a final concentration of 8 ng/μL was added to
the samples and incubated at 37 °C for 2 h with shaking.
Peptides were eluted into 0.1% formic acid (50 μL) and run on
LC−MS (Thermo Scientific, Waltham, MA, US). Sample
processing and downstream data analysis were performed as
illustrated in Figure 1.
nLC−MS/MS
An externally calibrated Thermo LTQ Orbitrap Velos (High
Resolution Electrospray Tandem Mass Spectrometer) equip-
ped with 2 cm, 100 μm i.d. (internal diameter) trap column
(SC 001 Easy Column; Thermo Scientific) and 10 cm
analytical column (75 μm i.d.; SC200 Easy Column; Thermo
Scientific) was used for LC−MS/MS. Both the trap column
and analytical column contained C18 AQ packaging. The
components were separated using Easy nano LC II (Thermo
Scientific) employing a continuous vented configuration. A 5
μL (∼500 ng) aliquot was taken into the sample loop (20 μL)
and loaded on the trap. Sample separation was achieved at a
flow rate of 300 nL/min using mobile phase A (99.9% H2O;
EMD Omni solvent with 0. 1% formic acid), and mobile phase
B (99.9% ACN with 0.1% formic acid). A 1 h linear gradient
from 0% to 45% B was performed. The LC eluent was
nanosprayed directly into the LTQ mass spectrometer
(Thermo Scientific). The LTQ Orbitrap Velos was set in a
data dependent mode under the direct control of Xcaliber
software (Thermo Scientific). The MS data were obtained with
the following parameters: 10 data-dependent collisional
induced dissociation (CID) MS/MS scans per full scan (400
to 2000 m/z) at a mass resolution for MS1 of 60000. Minimum
signal required to trigger MS2 was 500, MS mass range 0 to
1000000. The dynamic exclusion enabled with following
parameters: repeat count, 1; repeat duration, 30 s; exclusion
list size, 500; exclusion duration, 60 s; and exclusion mass width
relative to low and high mass, 10 ppm. To enable label-free
quantification all measurements were done at room temper-
ature and three technical replications were used for three
biological replicates to enable statistical comparisons between
the samples.
Data Analysis
The raw data were analyzed using Proteome Discoverer
(version 1.4) software package with SequestHT and Mascot
search nodes using Vitis species specific FASTA database
(70 263 entries) and the percolator peptide validator.
SequestHT search parameters used were as follows: enzyme
name = Trypsin, maximum missed cleavage = −2, minimum
peptide length = −6, maximum peptide length = −144,
maximum delta Cn = −0.05, precursor mass tolerance = −10
ppm, fragment mass tolerance = −0.6 Da, dynamic
modifications carboxymethyl/ + 58.005 Da (C), carbamido-
methyl/ + 57.021 Da (C) and oxidation/ +15.995 Da(M). The
Mascot search parameters used are as follows: enzyme name =
Trypsin, maximum missed cleavage = −2, maximum delta Cn =
−0.05, maximum peptide length =144, minimum peptide
length = 6, precursor mass tolerance = 10 ppm, fragment mass
tolerance = 0.6 Da, dynamic modifications carboxymethyl/ +
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58.005 Da (C), carbamidomethyl/ + 57.021 Da (C) and
oxidation/ +15.995 Da(M). Protein and peptide identities were
validated using Scaffold (version 4.3.4, Proteome Software Inc.,
Portland, OR, USA) software. Peptide identity is accepted if
Scaffold Local FDR algorithm demonstrates a probability
greater that 95.0%. Likewise, protein identity is accepted if the
probability level is greater than 99.0% and contains a minimum
of two recognized peptides. The Protein Prophet Algorithm22
was used to assign the probabilities of proteins. Since proteins
contain peptides that could not be distinguished on the basis of
only MS/MS, they were combined to meet the principles of
parsimony. The annotation of proteins was done following the
GO terms from gene association.goa_uniprot.23 Fold-changes
between different stages of ripening were calculated using
Scaffold version 4.0, and normalization was selected. Variation
in protein expression among different berry stages was assessed
in comparison with the control (Green Hard). For calculating
fold-changes of proteins missing in CGH and present in CGS,
CBS, and Ripe stages, a minimum floor value of 1 was selected.
Proteins were quantified using total spectrum count of
individual biological and technical replications for each stage.
Proteins identified with a minimum of two of three injections
and a minimum of two peptide matches in different ripening
stages against the Green Hard stage (control) displaying a
±1.5-fold change and a p-value < 0.05 were considered for
inclusion in the final list of proteins to be involved in secondary
metabolism. To identify the differentially expressed proteins,
gene ontology analysis was also carried out using Blast2GO
software.24,25
Extraction of Phenolics
Grape berries (CGH, CGS, CBS, and ripe berries without
seeds) were powdered in liquid nitrogen with mortar and
pestle, and used for extraction of phenolics according to the
method of Liu et al.26 with methanol/ethyl acetate (1:1, v/v) (1
g of sample powder per 10 mL of organic solvent) at 25 °C in
dark. The methanol/ethyl acetate extracts were centrifuged,
supernatant was collected, and the resulting pellet was extracted
again with methanol/ethyl acetate (3 mL). The supernatants
were combined and vacuum-dried in a concentrator at room
temperature.
Quantification of Phenolics
Phenolics extracts were solubilized in methanol (1 mL), and a 5
μL aliquot of the sample was mixed with 40 μL of H2O
containing 6.25% formic acid and 1 μL of internal standard (IS,
200 ng/mL of kaempferol in 1% methanol and 99% of H2O). A
portion (2 μL) of the mixture was separated by liquid
chromatography with a NanoAquity-UPLC system (Waters,
Milford, MA, USA). Eluents were ionized by nanoelectrospray
ionization (nESI) in both positive and negative ion modes, and
detected using a Waters Xevo TQ-S Triple quadrupole mass
spectrometer (Waters). A Symmetry C18, 5 μm ion trap
column (0.180 mm × 20 mm) and a HSS T3 (1.8 μm 0.15 mm
× 100 mm) analytical column (Waters) were used for
component separation. The LC gradient was formed using
solvent A (0.1% formic acid in H2O) and solvent B (0.1%
formic acid in acetonitrile). The sample was loaded on the trap
column at 1% solvent B and a flow rate of 10 μL/min for 1 min.
The gradient profile was 1% solvent B at the beginning, 95% B
at 20−20.5 min, and 1% B at 21−22 min at a 2 μL/min flow
rate. The conditions for the nESI source were +/3.3 kV
capillary voltage, 30 V cone voltage, 50 V source offset, 100 °C
source temperature, and 0.20 bar spray gas. MRM transitions
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Figure 2. Changes in °Brix, berry diameter, berry weight, and titratable acidity were measured from berries collected at stages 33 (Green Hard),
34(Green Soft), 35 (Bronze Soft), and 38 (Ripe) from Muscadine cv. Carlos during the 2013 season. Data presented are mean of ± SD of three
replications collected from 30 berries for each replication.
were optimized with 1 μg/mL standards in methanol (with
0.1% formic acid) in direct infusion. The standards used were
catechin, epicatechin, myricetin, quercetin, piceid, trans-
resveratrol, ε-viniferin, p-coumaric acid and, pterostilbene
(Sigma-Aldrich, St. Louis, MO, USA). During the LC−MS/
MS, MRM transitions were monitored by switching between
positive and negative ion modes. The following five MRM
transitions were acquired in positive ion mode at 3 ms dwell
time and 25 V collision energy: catechin (retention time 5.45
min): m/z 291 → 139; epicatechin (retention time 6.08 min):
m/z 291 → 139; ε-viniferin (retention time 10.08 min): m/z
455.1 → 107; resveratrol (retention time 8.71 min): m/z 229.1
→ 107, and IS (retention time 6.04 min): m/z 242 → 129. The
following five MRM transitions were acquired in negative ion
mode at 3 ms dwell time: myricetin (retention time 8.28 min):
m/z 317 → 151, 30 V collision energy (retention time 8.28
min); quercetin (retention time 9.41 min): m/z 301 → 151, 30
V collision energy (retention time 9.41 min); p-coumaric acid
(retention time 6.84 min): m/z 163 > 119, 20 V collision
energy (retention time 6.84 min); pterostilbene (retention time
13.74 min): m/z 255.1 → 239, 25 V collision energy and piceid
(retention time 7.06 min): m/z 389.1 → 227, 20 V collision
energy Thirteen calibration standards at 0.1, 0.5, 1, 2, 5, 10, 25,
50, 100, 200, 500, 1000, and 2000 ng/mL were prepared
containing 20 ng/mL of internal standard. Samples and
calibration standards were run in triplicate. Chromatograms
were acquired and the areas under the curves (AUC) were
calculated. The concentration of phenols and stilbenes were
calculated by comparing the AUC to the standard curve of the
calibration standards under the same condition. Calibration
curves are listed in Table S8. Data were analyzed with a one-
way analysis of variance (ANOVA) and Tukey HSD post hoc
test accepting the difference to be significant when p-value <
0.05 (GraphPad Prism software, La Jolla, CA, USA).
Real Time-PCR
Quantitative real time-PCR was used to validate the protein
expression. NCBI Primer design tool was used to design the
real time PCR primers for the 18 key genes selected on the
basis of their functional role in various metabolic pathways. The
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primer sequences are listed in the separate spread sheet (Table
S9). RNA was extracted from berries collected from different
maturity stages using Spectrum Plant RNA Kit (Sigma-
Aldrich). SuperScript VILO cDNA synthesis Kit was used to
synthesize cDNAs according to manufacture protocol (In-
vitrogen, Carlsbad, CA, USA) using 1 μg of total RNA
extracted from berries collected at different maturity categories
and replicates. Real time PCR was executed using individual
forward and reverse primers and cDNA. Data was normalized
using the Ubiquitin gene Ct value. Data was analyzed with a
one-way ANOVA using Tukey HSD post hoc test accepting
significant differences at p-value < 0.05 (GraphPad Prism
software). Gene expression changes indicated as fold changes
were calculated using the Ct value of the calibrator using the
formula 2−ΔΔCt.
■ RESULTS AND DISCUSSION
Developing/ripening grape berries were collected from four
maturity categories CGH (EL-33), CGS (EL-34), CBS (EL-
35), and Ripe (EL-38). Brix, acidity, berry weight, and TA
values are reported in Table S1 CGH berries (EL-33) had a
Brix 6, 15 mm diameter, and acid content of 17 g/L, whereas
CGS, CBS, and Ripe berry had a Brix of 9, 12, and ∼15,
respectively (Table S1; Figure 2). The protein levels estimated
from different samples showed that CGH berry pericarp
protein content was lower (0.45 mg/g of berries) compared to
that of CGS (0.59 mg/g) and Ripe berry (0.70 mg/g of berries)
(Table S2). The observed protein content of maturing
muscadine grape berry derived using phenol-based extraction
protocol was consistent with the previous reports.1,9
Proteome Changes at Different Developmental Stages
Three biological replicates were prepared for each of the berry
maturation/ripening stage. Each of the three biological
replicated samples was injected three times to derive
comprehensive statistical validation of the obtained proteins.
We have identified 1411 proteins employing ProteinProphet
and PeptideProphet algorithms with the thresholds for protein
and peptide identification set to 99% and 95%, respectively for
proteins with a minimum of two different peptides. The
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Figure 3. GO term enriched broader functional classification of the 522 differentially expressed proteins during development and ripening of
muscadine berry.

Figure 4. Cluster profiles of proteins involved in synthesis of different volatile compounds during muscadine grape berry ripening. A heat map of the
log 2 relative abundance of proteins throughout ripening in relation to the Green Hard stage was created using Genesis v1.7. For each protein, an
abbreviation, the Uniprot accession number and the sequence description assigned with Blast2GO are provided: (A) proteins associated with the
terpene biosynthetic pathway; (B) proteins associated with the fatty acid degradation pathway; (C) proteins associated with the production of
methyl anthranilate, esters, and 4-hydroxy-2,5-dimethyl-3-(2H)furanone (HDMF); and (D) tropinone reductase involved in the reduction of
tropinone.

number of proteins validated are 1269 in the Green Hard contained more than two biological samples with replications.
(CGH), 1209 in the Green Soft (CGS), 1166 in the Bronze One-way ANOVA of the samples and replications revealed 522
Soft (CBS) and 1077 in the Ripe berries. The obtained proteins which had a p-value of ≤0.05 (Tables S3, S4, S5, and
proteomics data set is deposited at PRIDE (PXD 001959). S6). These proteins were grouped into 14 functional categories.
One-way ANOVA was performed on proteome data set since it The highest percentages of proteins found are associated with
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Figure 5. continued

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Figure 5. Relative expression levels of transcripts. Data was normalized against expression of the housekeeping gene ubiquitin. To determine relative
fold differences for each gene in each experiment, the Ct value of the genes was normalized to the Ct value for ubiquitin (control gene) and relative
expression was calculated relative to a calibrator using the formula 2−ΔΔCt. Asterisks denotes values that differ statistically significant manner at p-
value of 0.0001 (∗∗∗∗) or 0.005 (∗∗∗).

functions such as secondary metabolism (16%), carbohydrate showed a change of ±1.5-fold with a p-value <0.05 and were
(15%), amino acid metabolism (8%), and protein processing recognized to be associated with flavor and aroma components
(6%) (Figure 3). Of the recognized proteins, 55 proteins of Carlos muscadine grape (Table S7). Employment of a label-
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free proteomics technique revealed a significantly higher
number of proteins in muscadine grape compared to that
found using the iTRAQ method.1 It should be noted that
iTRAQ proteome analysis was performed using a QSTAR Elite
mass spectrometer (AB Sciex, Framingham, MA, USA),
whereas the current proteome analysis was performed using a
Orbitrap mass spectrometer that routinely has higher ion scores
than QTOF MS/MS spectra due to the inherent ion trap
sensitivity advantages and presence of b and y-ion series. The
presence of these ion series results in spectra showing superior
sensitivity and confident peptide identification as reported
previously,27 and hence providing more proteome coverage in
the current study. In our earlier study we reported iTRAQ
analysis of proteome changes during development in muscadine
grape cv. Noble. In the current study a bronze muscadine
genotype was investigated to determine common proteome
changes for identification of proteins associated with volatile
and flavor compounds that are important for determining
enological characteristics of grape berry during vinification.
Both of these proteome analysis techniques revealed the
presence of enzymes of aroma compound synthesis such as
Anthraniloyl-CoA: methanol anthraniloyl transferase, Hydro-
peroxide lyase 1, Alcohol dehydrogenase 1, and proteins
associated with phenylproponaid pathway flavonoid synthase,
anthocyanidin synthase. The label-free approach reported in the
present manuscript provides more coverage and a detailed view
of major pathways active in muscadine grape.
Volatile and phenolic compounds present in muscadine
grape have been identified and reported previously.28−31
However, information on proteins and genes associated with
the synthesis of flavor and aroma is lacking in muscadine
grapes. The present study identified enzymes associated with
four major pathways, namely, terpenes, benzenoids, fatty acid
degradation, and phenylpropanoid pathway during ripening in
muscadine grape cv. Carlos (Table S7).
Proteins Associated with Terpene Biosynthetic Pathway
Terpenes and carotenoids are produced from the cytosolic
mevalonate (MVA) and plastidic methylerythritol phosphate
pathway (DOXP/MEP).32 In this study, one enzyme from the
MEP pathway, acetoacetylCoA thiolase (AACT; EC: 2.3.1.9),
and another enzyme from the MVA pathway hydroxymethyl-
glutaryl-CoA synthase (HMGS; EC: 2.3.3.10) were quantified
in the developing berry of muscadine grape cv. Carlos. Further
the enzyme isopentenyl pyrophosphate isomerase (IPP-isomer-
ase) that converts isopentenyl pyrophosphate (IPP) to
dimethylallyl pyrophosphate (DMAPP), increased by 3-fold
in CBS berries and decreased during ripening (Figure 4A, Table
S7). The result was corroborated with real-time PCR data that
showed up-regulated isopentenyl pyrophosphate isomerase
transcript levels in CGS and CBS stages but not in Ripe
berries. The results are in an agreement with the expression
pattern observed in Cabernet Sauvignon berries during
ripening.10 This study also showed that farnesyl pyrophosphate
synthetase (EC: 2.5.1.10) (FPPS) increased rapidly (7-fold) in
CGS berries and then gradually decreased during ripening
indicating abundant production of farnesyl pyrophosphate
during the onset of ripening (Figure 4A, Table S7). Farnesyl
pyrophosphate (FPP) produced in berries is the precursor for
synthesis of sesquiterpenes and triterpenes in cytosol and could
also react with additional IPP molecule to form geranylgeranyl
pyrophosphate (GGPP) that acts as a precursor for the
synthesis of diterpenes, tetraterpenes, and phytohormones such
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as gibberellins and abscisic acid.33 A protein encoding
(+)-neomenthol dehydrogenase (EC 1.1.1.208) putatively
involved in volatile cyclic monoterpene alcohol (+)neomenthol
biosynthesis, increased at the GS stage and then decreased
gradually in Ripe berries (Figure 4A, Table S7). Expression of
this enzyme is consistent with previous transcriptome analysis
that indicated down regulation during ripening.32,34 Certain
volatile monoterpenes are derived from tetraterpenoids such as
carotenoids by carotenoid cleavage dioxygenases (CCD).35
Cleavage of carotenoids by CCD 1 and CCD 4 produces
volatile compounds such as β-inonone, damascenone, and β-
inonol.36 This research showed that CCD1 protein increased
12-fold from GH to Ripe stage while quantitative PCR analysis
of transcript revealed decrease during ripening and upregulation
at the Ripe stage (Figure 5B). Previous studies (V. vinifera L. cv.
Pinotage) showed that CCD1 transcripts increased during
ripening and CCD1 enzymes are involved in enzymatic
degradation of carotenoids to the respective aromatic C13
norisoprenoids that contributes to the characteristics of
grapevine.37 Another protein, plastidlipid associated protein
(PAP), fibrillin that is associated with sequestration of
carotenoids increased 2-fold in ripe muscadine berries. High
expression of CCD1 protein during ripening suggests that it
may be involved in active cleavage of carotenoids, and may
contribute to the distinctive varietal characteristics of
muscadine cv. Carlos (Figure 4A, Table S7).
Proteins Associated with Fatty Acid Degradation Volatiles
Synthesis of volatile fatty acid derivatives like hexanal, hexenol,
nonanal, and methyl jasmonate arise from linoleic or linolenic
through the activity of lipoxygenase.38,39,41 Lipoxygenase (EC:
1.13.11.12) (LOX) catalyzes oxygenation of 9/13 of either
linoleic acid or linolenic acid and produces 9HPOD (9(S)-
hydroperoxy octadecadienoic acid), 13HPOD (13(S)-hydro-
peroxy octadecadienoic acid), 9HPOT (9(S)-hydroperoxy
octadecacatrienocacid), and 13HPOT (13(S)-hydroperoxy
octadecacatrienoc acid), respectively. The 9,13 HPOD and
9,13 HPOT can be further converted to aldehydes and oxaacids
by fatty acid hydroperoxide lyase. Metabolites such as C6
aldehydes can serve as volatile profiles known as green notes.40
Two 13 lipoxygenases [(tr|D7SLA9_VITVI) and (tr|
F6GUD0_VITVI)] increased in CBS berries and decreased
in Ripe berries (Figure 4B, Table S7). Hydroperoxide lyase
protein (EC: 4.1.2) increased during ripening (Figure 4B, Table
S7). Transcript analysis of HPL1 revealed its downregulation in
CBS berries and upregulation in Ripe berries (Figure 5C) in
parallel with our protein expression data. Such behavior
indicates high production of hexenals during the onset of
ripening in ripe berries. Pei Ching et al. (2014)41 have detected
hexenals in ripe muscadine grape berry (cv. Carlos) which is in
agreement with our observed increases of HPL1 protein and
gene. Further, hexanol generated by hydroperoxide lyase
enzyme can be converted to hexanol; a volatile grass aroma
by alcohol dehydrogenases.42 In V. vinifera, alcohol dehydro-
genases (ADH) has been well characterized. Previous research
in grape vines has reported expression of ADH transcripts,
where ADH1 and ADH3 are expressed in green berries while
ADH3 expression was higher at the onset of veraison.42,43 Our
research has identified two proteins in each of the ADH1 and
ADH3 class proteins. It showed that expression of the two
ADH1 proteins decreased during ripening, and of the two
ADH3 proteins identified, one ADH3 protein, A5BL32_VITVI
decreased during ripening while the other ADH3 protein
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Figure 6. Proteome changes of the phenylpropnoid pathway. Heatmaps representing changes in expression levels (Log2 transformed) as indicated
by color legend were created using Genesis v1.7. The figure also depicts the cross dependence and interdependence of the phenylproponoid pathway
with synthesis of stilbenes, liginin biosynthesis, phenylpropenes, flavanoids, benezenoids and isoflavonoids. Enymes: PAL, phenylalanine ammonia
lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumaroyl CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3
hydroxylase; F3′5′H, flavanone-3,5-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol 4-reductase; LDOX, leucoanthocyanidin dioxygenase;
UFGT, UDP glucose flavonol 3-O-glucosyltransferase; OMT, anthocyanin methyltransferase; 5GT, UDP-glucose, anthocyanin 5-O-
glucosyltransferase; GST, glutathione S-transferase; BEBT, benzyl alcohol O-benzoyltransferase; CCoA-OMT, caffeoyl-CoAO-methyltransferase;
CAD, cinnamyl alcohol dehydrogenase; EGS, eugenol synthase; IFR, isoflavone reductase.

(D7TVS7_VITVI) increased by 2-fold during ripening (Figure
4B, Table S7). Real time PCR analysis showed upregulation of
ADH3 transcripts (D7TVS7_VITVI) during Carlos berry
ripening which is in agreement with the observed increases in
its protein expression (Figure 5D). Fatty acid degradation
pathway enzymes identified in our study suggest that both the 9
and 13 hydroperoxide derivatives are produced through both
the LOX pathways to generate volatile compounds. Previous
report on volatile profiles of Carlos berries show that hexanol,
2-hexenal, 1-hexanol, and E2-hexen-1-ol are the key vola-
tiles.31,44 Our observation on 9 and 13 LOX, HPL1, and ADH3
expression is consistent with the accumulation pattern reported
for hexanal, 2-hexenal, 1-hexanol and E2-hexen-1-ol in other
studies.31,44
Proteins Associated with Volatiles Produced through the
Benzenoid Pathway
Biosynthesis of benzenoid class of plant volatile compounds
originate from phenylalanine through phenylalanine ammonia
lyase (PAL) catalyzed conversion of it to trans-cinnamic acid.45
Benzenoid synthesis requires shortening of two carbons from
cinnamic acid, and this process is known to proceed either
through β-oxidative pathway or non-β-oxidative pathway or
I DOI: 10.1021/acs.jproteome.5b01064
combination of both pathways. Acyltransferases of the BAHD37
superfamily and methyltransferases of the SABATH family are
known to be involved in final steps of volatile benzenoid
biosynthesis.47 Our proteome and transcript analysis results
showed an increase in benzyl alcohol O-benzoyltransferase
(BEBT) up to CBS accompanied by a decrease at the Ripe
stage (Figures 5E and 7; Table S7). Apart from BEBT, we have
also identified two BAHD acyltransferases which increased at
CGS and CBS. BAHD acyl transferases are known to use
Coenzyme A thioesters as acyl donors to form esters.46,48
Identification of BAHD specificity in muscadine grape will
increase our knowledge of volatile benzenoid synthesis.
Proteins Associated with Volatiles Produced through the
Phenylpropene Pathway
Phenylpropanoid pathway directs synthesis of phenylpropenes
such as eugenol, isoeugenol, and chavicol.47,49 Our data have
shown an increase in Eugenol synthase (EC 1.1.1.318) protein
and its transcript in ripe berries indicating enhanced synthesis
of eugenol (Figures 5F and 6, Table S7) that contribute to
aroma in fruits. Eugenol serves as a volatile compound for
attracting pollinators of flowers and defense in various plant
J. Proteome Res. XXXX, XXX, XXX−XXX
Journal of Proteome Research Article

Figure 7. (A) Concentration of five phenolics, catechin, epicatechin, myricetin, quercetin and p-coumaric acid in Carlos berries. Carlos Green Hard
(CGH), Carlos Green Soft (CGS), Carlos Bronze Soft (CBS), and ripe berries. The concentration of quercetin and p-coumaric acid is increased 50
times to visualize the difference. Asterisks denotes values that differ statistically significant manner at p-value of 0.0001(∗∗∗∗), 0.0005(∗∗∗) or 0.005
(∗∗) (B) Concentration of four stilbenes, t-piceid, t-resveratrol, ε-viniferin, and t-pterostilbene in Carlos berries: Carlos Green Hard (CGH), Carlos
Green Soft (CGS), Carlos Bronze Soft (CBS), and ripe berries. Asterisks denotes values that differ statistically significant manner at p-value of
0.0001(∗∗∗∗) or 0.0005(∗∗∗).

tissues.48,50 It is a major volatile compound in muscadine wine
cv. Noble.30
Proteins Associated with Production of Other Volatile
Compounds
Other enzymes detected that are involved in the production of
volatile compounds are the esterase enzymes which are known
to participate in the hydrolysis of carboxylic esters to carboxylic
acids and alcohols, and responsible for flavor development in
grape berry.49,51 We have found that proteins belonging to
carboxylesterase-2 and -7 increased from the GH stage to the
Ripe stage of muscadine grape. Transcript analysis of
carboxylesterase 2 showed its upregulation by 3-fold in CBS
berries (Figure 5G). Genes encoding carboxylesterases (EC:
J DOI: 10.1021/acs.jproteome.5b01064
3.1.1.1) identified in three Portuguese grape cultivars were
proposed to be involved in flavor development.51 Anthraniloyl-
coenzymeA (CoA) methanol acyltransferase (AMAT) is
responsible for synthesis of esters that contribute to aromas
in grapes.52 We have found that AMAT increased from CGH to
CGS and then decreased at ripening (Figure 4C, Table S7),
while transcript levels were found upregulated in ripe berries
(Figure 5H). The observed transcript and protein expression
discrepancies of carboxylesterase and AMAT may be due post
translation regulation of the enzyme levels during muscadine
berry ripening. Quinone oxidoreductase (QR) catalyzes the
reduction reaction from 4-hydroxy-5-methyl-2-methylene-
3(2H)-furanone(HMMF) to form 4-hydroxy-2,5-dimethyl-3-
J. Proteome Res. XXXX, XXX, XXX−XXX
Journal of Proteome Research
(2H)furanone(HDMF), a main flavor component of straw-
berry.51 Volatiles analyses revealed the presence of HDMF in
Carlos berries during ripening.31 A 7-fold increase in protein
expression of QR indicates activity of QR in the synthesis of
HDMF in ripe berries of muscadine grape. Tropinone
reductases (TRs) I and II catalyze formation of tropine and
pseudotropine from tropinone.52 Further, tropine and pseudo-
tropine are converted tropane esters which are of medicinal
importance, hyoscyamine, and calystegins.53,55 Although
homologues of TRs are found in other plant families and V.
vinifera,56 TRs identified in C. off icinalis has been proven to
reduce only tropinone. TRs identified in A. thaliana are known
to reduce substances other than tropinones indicating their
functional complexity.54 In this study we have observed a 7-fold
increase in tropinone reductase (TRI) protein during
muscadine berry ripening (Figure 4D, Table S7). Expression
of TR1 in muscadine berry needs further functional analysis to
correlate its quantitative expression with production of tropane
alkaloids or to determine if they act as short- chain
dehydrogenases.
Proteins Associated with the Phenylpropanoid Pathway
This study found that proteins identified with a role in the
phenylpropanoid pathway increased during veraison and in ripe
berries. From a wine perspective, the major phenolic
compounds responsible for organoleptic properties are
hydroxycinnamic acids, anthocyanins, and proanthocyanins.55
Our study has identified phenylalanine ammonia lyase (PAL),
the first enzyme involved in conversion of phenylalanine to
cinnamic acid.56 Three isozymes of PAL increased in CBS
berries indicating that phenylpropanoid pathway was active in
muscadine grape before the onset of ripening (Figure 6, Table
S7). Transcript analysis of this enzyme revealed upregulation
during ripening of Carlos berries (Figure 5I) that correlates to
the recent report of transcriptome analysis which revealed that
majority of PAL transcripts were upregulated in ripe berries,57
while proteome analysis revealed downregulation of PAL.14
Likewise, we have also found that shikimate pathway enzymes
which are involved in aromatic amino acids (phenylalanine,
tyrosine, and tryptophan) synthesis increased in CGS berries
and decreased during berry ripening. In addition, two isozymes,
3-deoxy-darabinoheptulosonate-7-phosphate synthase (DAHP
synthase) and dehydroquinate dehydrataseshikimate dehydro-
gen-ese (DHQSDH) which are involved in aromatic amino
acids synthesis were detected in muscadine berry (Figure 6,
Table S7). Transcript analysis of DAHP revealed down-
regulation of this enzyme during ripening (Figure 5J) which
is consistent with protein expression indicating a decrease in
metabolism of aromatic amino acids in ripe muscadine berry.
Nonflavonoid polyphenols like hydroxycinnamic acids are
found both in red and white grape varieties, but they are mostly
abundant in the free run juice of white grape varieties.
Accumulation of caffeic acid, ferulic acid, hydroxycinnamic acid,
and pcoumaric acid was high prior to veraison and then
decreased toward ripening.55 Metabolite analysis of Carlos
berry revealed a decrease in p-coumaric acid content during
ripening (0.094 μg/g of berries) (Figure 7). The observed
decrease in p-coumaric acid during ripening in muscadine berry
runs parallel to previous results.57 The yellow pigments
observed in white wines are due to the presence of flavonols58
and represent between 46 and 56% of total phenolics of white
grapes.59 Flavonoid biosynthesis is well documented in V.
vinifera grape species, but the expression pattern and enzymes
K DOI: 10.1021/acs.jproteome.5b01064
Article
associated with different flavonoid derivatives are poorly
understood in muscadine grape. Our study showed that the
proteins quantified in flavonoid biosynthesis are involved in
synthesis of flavonoid pigments, lignin, and isoflavonoids. The
seven phenylpropanoid pathway enzymes [chalcone synthase
(CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase
(F3H), flavonol synthase (FLS), dihydroflavonol 4reductase
(DFR), flavonoid 3′-O′-methyltransferase and leucoanthocya-
nidin dioxygenase (LDOX) identified here are isoforms (Figure
6, Table S7). Overall, these proteins increased until the CBS
stage followed by a progressive decrease in Ripe berries except
for the two isoforms of flavonoid 3-hydroxylase (A5C9G2,
F6HCH1), and flavonol synthase (A5BV11_VITVI) that
increased during ripening (Figure 6, Table S7). Transcript
analysis of LDOX and CHS revealed downregulation while
F3H revealed upregulation in ripening Carlos berries (Figure
5K,L,M) which is consistent with our protein expression data.
Flavonols are copigments that are vital to anthocyanins
stabilization in wine while flavonol synthase is accountable for
conversion of dihydroflavonols to flavonols.37 We have also
quantified two flavonols, myricetin and quercetin (Figure 7A,
Table S8) which are in agreement with the observed increase of
flavonol synthase enzyme during ripening (Figure 6, Table S7).
In addition to flavonols, two proanthocyanidins (PAs), catechin
and epicatechin, were quantified during berry development and
ripening. Catechin concentration increased during CBS (2.35
μg/g) and then decreased in Ripe berries while the highest
concentration of epicatechin (7 μg/g of berries) was observed
in ripe berries (Figure 7A, Table S8). PAs catechin and
epicatechin were previously identified in muscadine cv. Noble
pulp and skin.60,−62 Proanthocyanidins are recognized to be
responsible for the astringency and flavor of many fruits, tea
and wines.64 Understanding their accumulation pattern and
regulation during muscadine berry development will reveal
important information for further modulating PAs in grape to
benefit wine industries.
Structural modifications of flavonoids can improve their
biological activities. Methylation is a common flavonoid
modification that is catalyzed by omethyltransferases
(OMTs). In the present study we found that, flavonoid 3′-
O′-methyltransferase increased 17-fold at CBS and decreased in
Ripe berries. Other metabolites branching out from the
phenylpropanoid pathways are stilbenes that are synthesized
in grape berries constitutively or induced by biotic and abiotic
elicitors.63 Resveratrol/hydroxycinnamic acid glucosyltransfer-
ase are known to be involved in glucosidation of trans-
resveratrol to piceid and also known to have glucosyl activity on
hydroxycinnamic acids and flavonoids.64,65 In the current study,
transcript and protein levels of resveratrol/hydroxycinnamic
acid O-glucosyltransferase increased in ripe berries (Figures 5P
and 6, Table S7).
Among the stilbenes found in grape, piceid is one of the
major resveratrol derivatives.66 Quantification of stilbenes
during Carlos berry development and ripening showed the
presence of four different stilbenes: piceid, resveratrol, viniferin,
and pterostilbene. The concentration of piceid was high in
CGH berries (6.55 μg/g of berries) compared to Ripe berries
(0.72 μg/g of berries) (Figure 7B, Table S8). Resveratrol
accumulation increased during ripening and was found to be
highest in Ripe berries (2.65 μg/g) (Figure 7B). Viniferin
concentration increased from 0.9 to 9.69 μg/g from CGH to
Ripe berries. The highest pterostilbene concentration was
observed in CBS berries (0.68 μg/g) (Figure 7B, Table S8).
J. Proteome Res. XXXX, XXX, XXX−XXX
Journal of Proteome Research
Stilbenes of muscadine grape have been well studied.61,67,68 Our
research showed that only resveratrol and viniferin concen-
trations are high in Ripe berries of Carlos (Figure 7).
Lastly, we have also identified isoflavone reductase (IFR) that
is involved in isoflavonoids synthesis in Carlos berry. We have
found upregulation of isoflavone reductase
(VIT_03s0038g04670) during the veraison and ripening of
muscadine grape (Figure 6, Table S7). Transcript analysis of
isoflavone reductase revealed its upregulation during ripening
of Carlos berries (Figure 5Q). The increasing trend of IFR is
consistent with the previous proteome analysis report.13 In
addition, the other branching point enzymes identified in this
pathway are recognized to be associated with lignin biosyn-
thesis.69 In the lignin branch of the pathway, cinnamoyl-CoA
reductase is the initial enzyme that converts hydroxycinnamic
acid CoA esters to hydroxycinnamaldehydes.69 Derivatives of
cinnamyl alcohol are also accountable for fruity and aroma
characteristics in grapes.35 CAD gene upregulation during
ripening suggests its involvement in flavor development.70 Our
study revealed that CCR, decreased during ripening. In
addition, we have found that the concentration of two CAD
isoforms fluctuated during ripening (D7SL25_VITVI,
D7UBZ3_VITVI). Transcript analysis of protein (D7UBZ3_-
VITVI) revealed upregulation during ripening of Carlos berries
(Figure 5R) which is in agreement with our proteome data
(Figure 6).
■
CONCLUSION
In summary, label-free proteome analysis of the bronze
muscadine cv. Carlos provided insight into different bio-
synthetic pathways and the enzymes that are regulated at
different stages of berry development. This proteome data have
provided vital information about expression patterns of proteins
associated with the key metabolic pathways of flavor and aroma
compounds in bronze muscadine grape cv. Carlos. Proteome
data are also correlated with the metabolite analysis of major
chemical components present in muscadine grape cv. Carlos.
The outcome of this proteome study which is also supported by
transcriptome analysis revealed that developmental accumu-
lation of volatiles and other flavor components found in bronze
muscadine grape berry are controlled by multiple metabolic
pathways representing four major classes of volatile com-
pounds: hexenals, terpenes, esters, and phenylpropenes.
Quantitative analysis of p-coumaric acid, flavonols, PAs,
resveratrol, and other stilbenes during berry ripening provide
evidence for phenyproponoid pathway enzymes. Further
research to correlate the quantitative proteome profile with
metabolome and transcriptome data will reveal the complexity
of processes involved in flavor and aroma development in
muscadine grape.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jproteo-
me.5b01064.
Tables that include physical properties of the berries;
protein yields and IDs, proteome changes during
ripening, and other data as described in the text
(XLSX)
L DOI: 10.1021/acs.jproteome.5b01064
Article
■ AUTHOR INFORMATION
Corresponding Author
*Tel.: (850) 412-5191. E-mail: devaiah.kambiranda@gmail.
com.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by a grant from the USDA-NIFA-
CBG and a Department of Agriculture and Consumer Services-
Specialty Crop Block grant of the State of Florida. The authors
wish to thank Dr. Vasil Georgiev and Dr. Ramanjaneya V R
Mula, Center for Viticulture and Small Fruit Research, Florida
A&M University, Tallahassee, FL 32317, for reviewing the
manuscript.
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N DOI: 10.1021/acs.jproteome.5b01064
J. Proteome Res. XXXX, XXX, XXX−XXX