Phytochemistry xxx (2014) xxxxxx

Contents lists available at ScienceDirect

Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Identication and cloning of an NADPH-dependent

hydroxycinnamoyl-CoA double bond reductase involved
in dihydrochalcone formation in Malus  domestica Borkh.
Mwafaq Ibdah a,b,1, Anna Berim a,1, Stefan Martens c,1, Andrea Lorena Herrera Valderrama c,
Luisa Palmieri c, Efraim Lewinsohn b, David R. Gang a,
a

Institute of Biological Chemistry, Washington State University, PO Box 646340, Pullman, WA 99164-6340, USA
NeweYaar Research Center, Agriculture Research Organization, PO Box 1021, Ramat Yishay 30095, Israel
c
Fondazione Edmund Mach, Centro Ricerca e Innovazione, Department of Food Quality and Nutrition, Via E. Mach, 1 38010 San Michele allAdige (TN), Italy
b

a r t i c l e

i n f o

Article history:
Received 20 February 2014
Received in revised form 10 July 2014
Available online xxxx
Keywords:
Apple
Malus  domestica
Rosaceae
p-Coumaroyl-CoA
Feruloyl-CoA
p-Dihydrocoumaroyl-CoA
Dihydrochalcones
Hydroxycinnamoyl-CoA double bond
reductase

a b s t r a c t
The apple tree (Malus sp.) is an agriculturally and economically important source of food and beverages.
Many of the health benecial properties of apples are due to (poly)phenolic metabolites that they contain, including various dihydrochalcones. Although many of the genes and enzymes involved in polyphenol biosynthesis are known in many plant species, the specic reactions that lead to the biosynthesis of
the dihydrochalcone precursor, p-dihydrocoumaroyl-CoA (3), are unknown. To identify genes involved in
the synthesis of these metabolites, existing genome databases of the Rosaceae were screened for apple
genes with signicant sequence similarity to Arabidopsis alkenal double bond reductases. Herein
described are the isolation and characterization of a Malus hydroxycinnamoyl-CoA double bond reductase, which catalyzed the NADPH-dependent reduction of p-coumaroyl-CoA and feruloyl-CoA to
p-dihydrocoumaroyl-CoA and dihydroferuloyl-CoA, respectively. Its apparent Km values for p-coumaroyl-CoA, feruloyl-CoA and NADPH were 96.6, 92.9 and 101.3 lM, respectively. The Malus double bond
reductase preferred feruloyl-CoA to p-coumaroyl-CoA as a substrate by a factor of 2.1 when comparing
catalytic efciencies in vitro. Expression analysis of the hydroxycinnamoyl-CoA double bond reductase
gene revealed that its transcript levels showed signicant variation in tissues of different developmental
stages, but was expressed when expected for involvement in dihydrochalcone formation. Thus, the
hydroxycinnamoyl-CoA double bond reductase appears to be responsible for the reduction of the
a,b-unsaturated double bond of p-coumaroyl-CoA, the rst step of dihydrochalcone biosynthesis in apple
tissues, and may be involved in the production of these compounds.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
Polyphenols constitute one of the largest and most diverse classes of specialized (secondary) metabolites in plants. These
metabolites can be divided into non-soluble metabolites, such as
lignins, condensed tannins (proanthocyanidins) and cell-wall
Abbreviations: ACN, acetonitrile; CHS, chalcone synthase; DBR, double bond
reductase; DHC, dihydrochalcone; Enoyl-ACP, enoyl acyl carrier protein; GT,
glycosyltransferase; IPTG, isopropyl-1-thio-b-D-galactopyranoside; m/z [M], mass
spectrometry; MdHCDBR, Malus  domestica hydroxycinnamoyl-CoA double bond
reductase; MWCO, molecular mass cut-off; PGT1, phloretin 20 -O-glucosyltransferase; PulR, pulegone reductase; RZS, raspberry ketone/zingerone synthase.
Corresponding author. Tel.: +1 509 335 0550; fax: +1 509 335 7643.
E-mail address: gangd@wsu.edu (D.R. Gang).
1
Contributed equally to this work.

bound hydroxycinnamic acids, and soluble metabolites, such as

phenolic acids, phenylpropanoids, lignans and avonoids
(Harborne and Williams, 2000). Polyphenols and their derivatives
play important roles in ower and seed pigmentation, in plant fertility and reproduction, and in various defense reactions to protect
against abiotic stresses such as UV-light or biotic stresses such as
predator and pathogen attack (Forkmann and Materns, 2001;
Winkel-Shirley, 2001; Cheynier et al., 2013). Most phenolic
compounds are derived from the amino acid L-phenylalanine (1)
via the general phenylpropanoid pathway intermediates
p-coumarate (8) and p-coumaroyl-CoA (2) (Fig. 1).
Apples (Malus  domestica Borkh.) are among the worlds most
important economic food crops with nutritive and medicinal
importance (Boyer and Liu, 2004). They accumulate high levels of

http://dx.doi.org/10.1016/j.phytochem.2014.07.027
0031-9422/ 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Ibdah, M., et al. Identication and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase
involved in dihydrochalcone formation in Malus  domestica Borkh. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.027

M. Ibdah et al. / Phytochemistry xxx (2014) xxxxxx

O

OH

SCoA

H2N

SCoA

NADPH

NADP+

MdHCDBR

L-Phenylalanine (1)

OH

OH

p-Coumaroyl-CoA (2)

p-Dihydrocoumaroyl-CoA (3)

3 x Malonyl-CoA 3 x Malonyl-CoA

CHS

CHS
CoA-SH

CoA-SH

OH
HO

OH

HO 4
3

OH

Naringenin Chalcone (4)

6 OH

2
OH

B
1

OH
4
3

Phloretin (5)

CHI

PGT1
OH

HO

OH

OH

Naringenin (6)
[Flavonoids]

OH

HO

O
Glc

Phloridzin (7)
[Dihydrochalcones]

Fig. 1. Proposed biosynthetic pathway leading to dihydrochalcones (DHCs) in

apple. CHS, Chalcone synthase; CHI, Chalcone isomerase; MdHCDBR, Hydroxycinnamoyl-CoA double bond reductase, highlighted by red coloring; PGT1, Phloretin
20 -O-glucosyltransferase. (For interpretation of the references to color in this gure
legend, the reader is referred to the web version of this article.)

diverse (poly)phenolic antioxidants, including avonoids and

biphenyl phytoalexins, the level of which may depend on many
factors, such as genotype, developmental stage, or biotic stress
(Ehrenkranz et al., 2005; Sunagawa et al., 2011; Chizzali et al.,
2013). Among common polyphenolic derivatives, apples are characterized by the presence of dihydrochalcones (DHCs), structures
closely related to common avonoid precursors, the chalcones
(Veitch and Grayer, 2006). In contrast to common avones and
avonols, which are ubiquitous, DHCs are of limited distribution.
About 200 different DHCs are known from 30 plant families
(Gosch et al., 2010) including apples and Citrus species (Roowi
and Crozier, 2011; She et al., 2011; Ibdah, unpublished).
The physiological role for these compounds in planta appears to
be in plant defense (Dug de Bernonville et al., 2010, 2011). The
synthesis and accumulation of DHCs and their derivatives is associated with serious plant diseases such as scab (Picinelli et al.,
1995) and re blight caused by the bacterium Erwinia amylovora
(Roemmelt et al., 2003). Evidence for bioactivity of DHCs as functional antioxidants was suggested by the lowering of oxidative
stress of apple leaves (Dug de Bernonville et al., 2010). Despite
signicant interest in the cancer chemopreventative and antioxidant properties of DHCs (Shao et al., 2008; Dug de Bernonville
et al., 2010; Huang et al., 2012), as well as their impact on glucose
metabolism in humans (Ehrenkranz et al., 2005) and the economic
importance of these compounds as low calorie sweeteners
(Tomas-Barberen and Clifford, 2000), their biosynthesis is yet to
be fully elucidated.
In contrast to the extensive literature on avonoid and chalcone
biosynthesis, little information is available on the biosynthetic
pathways leading to DHC glycosides in plants. Flavonoids are
synthesized
via
the
phenylpropanoidacetate
network
(Winkel-Shirley, 2001). Phenylalanine ammonia lyase (Velasco
et al., 2010) catalyzes the rst step in this metabolic network,

the conversion of L-phenylalanine (1) to t-cinnamate. Cinnamate

4-hydroxylase (C4H) then catalyzes the synthesis of p-hydroxycinnamate (p-coumarate (8)) from t-cinnamate. p-Coumarate (8) is
then further converted by p-coumarate:CoA ligase (4CL) to its
coenzyme-A ester. From these central intermediates, the pathway
diverges into several side branches, each resulting in a different
class of phenolics. The condensation of one molecule of p-coumaroyl-CoA (2) with three molecules of malonyl-CoA yields naringenin chalcone (4) in the general avonoid branch of the network.
This reaction is carried out by chalcone synthase (CHS) (Ebeler
et al., 2002). An examination of the substrate specicity of CHSs
from M. domestica suggested that the formation of DHCs may
start with the reduction of p-coumaroyl-CoA (2) to form
p-dihydrocoumaroyl-CoA (3) (Gosch et al., 2009), an alternative
route compared to the canonical phenylpropanoidacetate
network. Recently, this same conversion was suggested to be
catalyzed by an enoyl reductase from apple (Dare et al., 2013). In
either case, p-dihydrocoumaroyl-CoA (3) would then be condensed
by CHS with three molecules of malonyl-CoA to form the DHC,
phloretin (5) (Fig. 1) (Gosch et al., 2009).
Several plant enone reductases have been isolated that
recognize the a,b-unsaturated double bond of the enoyl moiety of
specialized metabolites, such as 4-hydroxybenzalacetone, 3methoxy-4-hydroxybenzalacetone (Koeduka et al., 2011), p-coumarylaldehyde, dehydro-diconiferyl- and coniferyl aldehydes
(Kasahara et al., 2006), and (+)-pulegone (Ringer et al., 2003). However, none of these enzymes have been shown to catalyze reduction
of the p-coumaroyl-CoA (2) or feruloyl-CoA. In Arabidopsis, a double
bond reductase (DBR) specic for alkenals catalyzed the 7,8-double
bond reduction of phenylpropanal substrates to their corresponding dihydrocompounds (Youn et al., 2006). That enzyme was active
with a number of phenylpropanal substrates, although phenylpropenoyl-CoA esters, including p-coumaroyl-CoA (2), were not
tested as substrates. These ndings led us to hypothesize that a
similar NADPH-dependent reductase might be involved in reducing
the a,b-unsaturated double bond reduction of the enoyl moiety of
p-coumaroyl-CoA (2) in apple.
The focus of this study was therefore to study the biochemical
and molecular factors that determine the synthesis of DHCs in
apple. We identied an NADPH-dependent reductase capable of
forming p-dihydrocoumaroyl-CoA (3) and dihydroferuloyl-CoA
from p-coumaroyl-CoA (2) and feruloyl-CoA, respectively, and
designated it M. domestica hydroxycinnamoyl-CoA double bond
reductase (MdHCDBR). The recombinant MdHCDBR protein
catalyzes the a, b-unsaturated double bond reduction in a manner
different from other known enone reductases, indicating that the
MdHCDBR enzyme has a narrower substrate specicity than other
similar reductases. MdHCDBR, may therefore play an important
role in channeling common phenylpropanoid pathway precursors
into the DHC branch of the avonoid pathway.

2. Results and discussion

2.1. A bioinformatic screen for an apple gene homologous to
Arabidopsis alkenal DBR
The biosynthesis of DHC derivatives in plants has remained elusive until now. It can be hypothesized that a DBR-like protein
belonging to the subfamily that includes the Pinus taeda phenylpropenal and the Arabidopsis alkenal DBR might be involved in
the formation of p-dihydrocoumaroyl-CoA (3), which could in turn
be utilized by CHS to form a DHC instead of chalcone, the more
common product derived from p-coumaroyl-CoA (2). Advantage
was thus taken of the sequence of the recent described alkenal
DBR from Arabidopsis as an initial bioinformatic tool to screen

Please cite this article in press as: Ibdah, M., et al. Identication and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase
involved in dihydrochalcone formation in Malus  domestica Borkh. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.027

M. Ibdah et al. / Phytochemistry xxx (2014) xxxxxx

available EST and genome databases of apple for a putative

MdHCDBR. More than 20 candidate genes were thus identied in
the Malus genome. A similarity tree comparing many of these
sequences to similar sequences found in public databases is shown
in Fig. 2.
Most of the potential Malus DBRs shared less than 60% amino
acid sequence similarity with the Arabidopsis protein, except for
one candidate, which displayed 70% similarity at the amino acid
level. The recombinant Arabidopsis alkenal DBR (At5g16970)
enzyme catalyzes in vitro the reduction of the 7-8-double-bond
of phenylpropanal substrates, such as p-coumaroyl- and coniferyl
aldehydes to their corresponding p-dihydrocoumaroyl- and dihydroconiferyl aldehydes (Youn et al., 2006). The MdHCDBR
sequence was 67% identical to (+)-pulegone reductase (PulR) from
Mentha x piperita, which is specically involved in a similar conversion of pulegone to (+)-isomenthone and ()-menthone (Ringer
et al., 2003). A phenylpropenal DBR from P. taeda (Kasahara
et al., 2006), which converts dehydrodiconiferyl- and coniferyl
aldehydes to dihydrodehydrodiconiferyl- and dihydroconiferyl
aldehydes, respectively, had signicantly lower similarity (41%)
to MdHCDBR. The putative MdHCDBR was also similar to an Ocimum basilicum alcohol dehydrogenase, which reversibly oxidizes
geraniol to produce geranial (Iijima et al., 2006). However, the
highest identity (80%) was found to raspberry ketone/zingerone
synthases 1 and 2 (RZS1 and RZS2) from Rubus idaeus, which convert 4-hydroxybenzalacetone and 3-methoxy-4-hydroxybenzalacetone to raspberry ketone and zingerone in raspberry fruits
(Koeduka et al., 2011). R. idaeus belongs to the Rosaceae, the same
plant family as Malus.
2.2. Expression and biochemical characterization of the MdHCDBR
protein
The expression construct pEXP-MdHCDBR was transferred to
Escherichia coli BL21 (DE3) cells, and the recombinant protein
induced. Bacterial cells expressing MdHCDBR were harvested and
lysed, and the recombinant protein was puried with an afnity
column and visualized by SDSPAGE (Fig. 3). As estimated from
the protein gel, the nal MdHCDBR protein preparation was >97%
homogenous. The predicted MdHCDBR protein sequence consists
of 349 amino acids, with a calculated molecular mass of
38.4 kDa, as corroborated by SDSPAGE (Fig. 3), and a theoretical
isoelectric point of 6.9.

kDa

130
100
70
55
MdHCDBR
35
25

10

Fig. 3. SDSPAGE analysis of puried recombinant MdHCDBR. Lane 1: Molecular

mass markers; Lane 2: Soluble extracts from recombinant MdHCDBR after Ni-NTA
matrix chromatography. The arrow indicates the 38.4 kDa protein identied as
MdHCDBR.

His-tagging and afnity chromatography were used to purify E.

coli-expressed apple MdHCDBR protein (Fig. 3). Incubation of pcoumaroyl-CoA (2) and NADPH with puried MdHCDBR led to
the production of p-dihydrocoumaroyl-CoA (3) and incubation of
feruloyl-CoA with MdHCDBR led to the production of dihydroferuloyl-CoA. The products were visualized after base-catalyzed hydrolysis cleaved the CoA esters to release the free hydroxycinnamic
acids from the remaining substrate and the products that were
formed. The results in Fig. 4 show the chromatograms and mass
spectra of the resulting free acids, not the full CoA esters, because
no products (or CoA ester substrates) were observed under the
chromatographic conditions used when the base hydrolysis step
was omitted. The free acid observed from the base-hydrolyzed
reaction product in the assays using p-coumaroyl-CoA (2) as substrate displayed an m/z value of 165.0555, corresponding to the
[MH] molecular ion species of p-dihydrocoumarate (9) (Fig. 4A
and B). Similarly, dihydroferulate (m/z 195.0652) was the product
measured in assays beginning with using feruloyl-CoA as substrate
(Fig. 4D and E). In addition to MS analysis, compound elution was
monitored by diode array spectrophotometry (220490 nm),

Enoyl-ACP reductase [Medicago truncatula XP003607232]

87

Enoyl-ACP reductase 1 [Populus trichocarpa XP002304026]

100

ENRL-5 [Malus x domestica AGJ00075]

98

ENRL-3 [Malus x domestica AGJ00073]

Enoyl-ACP reductase [Nicotiana tabacum CAA74176]
2-Alkenal reductase [Artemisia annua FJ750460]
Alcohol dehydrogenase [Camellia sinensis HM440157]

79

NADP-dependent alkenal DBR [Vitis vinifera XM002279323]

100

Phenylpropenal DBR [Pinus taeda DQ829775]

97

NADP-dependent oxidoreductase [Zea mays NM001158621]

Alkenal DBR [Arabidopsis thaliana At5g16970]

72
96
71

Pulegone Reductase [Mentha x piperita AY300163]

Alcohol dehydrogenase [Ocimum basilicum AY879288]
Allyl alcohol dehydrogenase [Nicotiana tabacum AB036735]

93

MdHCDBR [Malus x domestica hydroxycinnamoyl-CoA DBR]

RZS2 [Rubus idaeus AEL78826]
NADP-dependent alkenal DBR [Fragaria vesca XM004287120]
RZS1 [Rubus idaeus AEL78825]

Fig. 2. Neighbor-joining tree of deduced amino acid sequences of plant double bond reductase (DBR), enoyl-ACP reductase and dehydrogenase proteins. The MdHCDBR
protein identied in this study is highlighted by bold text. The bootstrap method was performed with 1000 replicates.

Please cite this article in press as: Ibdah, M., et al. Identication and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase
involved in dihydrochalcone formation in Malus  domestica Borkh. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.027

M. Ibdah et al. / Phytochemistry xxx (2014) xxxxxx

5.30
p-dihydrocoumarate (9)
-O

p-coumarate (8)

-O

5.01

relative abundance

165.0555

100

0
145 150 155
100

OH

OH

relative abundance

166.0589
167.0609
160

165

170 175

180

m/z

222.2678

relative abundance

100

5.65

276.2678

100

0
220

4.4

4.6

4.8

5.0

5.2

5.4

5.6

5.8

6.0

6.2

relative abundance

-O

280

nm

300

ferulate (10)
5.84

196.0685
197.0701

0
180

OCH3

OCH3

185

190

195

200

205

210

215

m/z
100

222.2678

OH
relative abundance

OH

320

195.0652

100

dihydroferulate (9)
-O

260

6.4

5.50

240

relative abundance

0
4.2

279.2678

0
4.2

4.4

4.6

4.8

5.0

5.2 5.4
Time (min)

5.6

5.8

6.0

6.2

6.4

220

240

260

nm

280

300

320

Fig. 4. Enzymatic activity of the recombinant MdHCDBR with p-coumaroyl-CoA (2) and feruloyl-CoA as substrate, after products (and left over reactants) were hydrolyzed by
NaOH as outlined in the text. Total ion chromatograms for reaction mixtures with p-coumaroyl-CoA (2) (A) and feruloyl-CoA (D) as substrates. Mass spectra of product peaks
matched the expected m/z values for p-dihydrocoumarate (9) (B) and dihydroferulate (E). Inline UV analysis of products showed spectra as expected for p-dihydrocoumarate
(9) (C) and dihydroferulate (F). S: substrate; P: product. Relative abundance is shown in arbitrary units.

which produced UV spectra corresponding to the reduced forms,

further conrming the identity of the products (Fig. 4C and F). In
contrast, no products were detected with naringenin-, naringin-,
hesperitin-, or hesperidin-chalcone, or with p-coumaric acid,
caffeic acid, coniferyl alcohol, cinnamoyl-CoA, or caffeoyl-CoA
under standard test conditions. Assays containing heat-inactivated
MdHCDBR protein or assays without NADPH as a co-substrate
displayed no detectable DBR activity with p-coumaroyl-CoA (2)
or other hydroxycinnamoyl-CoA substrates. While the protein
preparation used for all enzyme assays was of a high purity level,
several native E. coli proteins did co-purify with the recombinant
MdHCDBR. It can be excluded that these minor contaminants are
responsible for the observed activity, as no reduction of hydroxycinnamoyl CoA esters was detected when several double bond
reductases from other species, such as ginger, and turmeric were
used as catalyst. Those proteins were produced and puried using
the same materials and procedures as described in this report, and
found to be active with their respective substrates. Furthermore,
the list of commonly co-puried native E. coli proteins does not
include any putative or known NADPH-dependent reductases
(Bolanos-Garcia and Davies, 2006).
For general kinetic parameter characterization, we used the
His-tagged, afnity chromatography-puried MdHCDBR. Use of
p-coumaroyl-CoA (2) as substrate with the puried MdHCDBR
protein revealed that the reaction was favored by a mildly acidic
environment with a pH optimum between 4.2 and 5.2, and halfmaximal reaction velocities at pH 4 and pH 7. Maximum turnover

rate was reached at 40 C (Fig. 5D), with a calculated activation

energy of 25.7 kJ/mol. The kinetic parameters of the His-tagged,
afnity chromatography-puried MdHCDBR with substrates pcoumaroyl-CoA (2) and feruloyl-CoA in the presence of NADPH
were also determined (Table 1 and Fig. 5AC). The MdHCDBR protein displayed a comparable afnity for both CoA-esters, but a
higher maximum turnover rate with feruloyl-CoA (Table 1), resulting in approximately double catalytic efciency with the latter
substrate. Because the activity for phenolic aldehydes was not
tested in this study, the possibility cannot be excluded that this
enzyme might have other catalytic functions. However, based on
the results to date, where both p-coumaroyl-CoA (2) and feruloyl-CoA serve as substrates, but with the enzyme having ca. 2-fold
higher catalytic efciency with feruloyl-CoA compared to p-coumaroyl-CoA (2), the name hydroxycinnamoyl-CoA double bond
reductase is appropriate.
The reduction of p-coumaroyl-CoA (2) to p-dihydrocoumaroylCoA (3) was previously described using crude protein extracts from
young apple leaves (Gosch et al., 2009). Further biochemical characterization of the reaction was not reported. Interestingly, crude
enzyme preparations from Arabidopsis thaliana, Dahlia variabilis,
O. basilicum and Onobrychis viciifolia were also shown to possess
the same reductase activity (Gosch et al., 2009). The Arabidopsis
genome, and other plant genomes that have been investigated in
some detail, contain several DBR- and alcohol dehydrogenase-like
sequences that are often annotated as dehydrogenase genes,
although none of which have been shown to be involved in p-

Please cite this article in press as: Ibdah, M., et al. Identication and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase
involved in dihydrochalcone formation in Malus  domestica Borkh. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.027

0
0

100 200 300 400 500

[S] M p-coumaroyl-CoA

600

80
60
40
20
0
0

-1

pkatmg protein

60
50
40
30
20

-1

pkatmg protein

0.4

f
f

f
g

0.2
0
1.0

PGT1

0.8
f

0.6

c
0.4

f,e

c
f,g

0.2

d
g

10
0
0

100 200 300 400 500 600 700 800

[S] M Feruloyl-CoA

d
c

C
Bu los
ds ed
80 bu
% d
O o s
pe pe
n n
bu
ds

pkatmg-1 protein

B 100

es
in
Sk sta
in ge
Sk sta 1
in ge
Sk sta 2
in ge
Sk sta 3
in ge
st 4
ag
e
Fl
5
es
h
Fl s
es ta
g
Fl h s e 1
es ta
ge
h
Fl s
es ta 2
g
Fl h s e 3
es ta
h ge
st 4
ag
e
5

10

0.8
0.6

DBR
e

av

20

Le

30

1.0

Sk

40

Relative Normalized Expression

pkatmg-1 protein

Relative Normalized Expression

M. Ibdah et al. / Phytochemistry xxx (2014) xxxxxx

500

1000 1500 2000

[S] M NADPH

2500

60
50
40

Fig. 6. Expression patterns of MdHCDBR and PGT1 in different apple Golden

Delicious tissues. (A) Quantication of MdHCDBR transcript levels were measured
by real-time qRT-PCR analysis normalized relative to actin. (B) Quantication of
PGT1 transcript levels were measured by real-time qRT-PCR analysis normalized
relative to actin. All analyses were performed using three replicates. Bars labeled
with different letters indicate signicant differences as determined by Statistica
software version 9.1 (StatSoft Inc., 2010) (p 6 0.05).

30
20
10
0
0

10

20
30
Temperature (C)

40

50

Fig. 5. Hydroxycinnamoyl-CoA double bond reductase activity of the recombinant

MdHCDBR protein. The graphs show steady state kinetic measurements of
MdHCDBR using p-coumaroyl-CoA (2) (A), feruloyl-CoA (B), or NADPH (C) as
varying substrate. (D) Determination of the temperature optimum of MdHCDBR
activity with p-coumaroyl-CoA (2) as a substrate.

Table 1
Kinetic parameters of MdHCDBR, obtained using the His-tagged and afnity
chromatography puried protein.
Substrate

Km (lM)

Vmax (pkat mg1 protein)

kcat (s1) (103)

p-Coumaroyl-CoA
Feruloyl-CoA
NADPH

96.6 5.9
92.9 1.5
101.3 2.8

47.7 2.4
102.9 2.6
56.4 0.3

1.9 0.1
4.2 0.1
2.3 0.0

All values are means S.E.M. (n = 6).

dihydrocoumaroyl-CoA (3) synthesis. Moreover, none of these species are known to accumulate DHCs. Thus, it is unknown how
widespread this catalytic function may be in plants or whether it
is always catalyzed by an enzyme belonging to the specic
dehydrogenase subfamily that contains the MdHCDBR gene.
2.3. Tissue specic expression of MdHCDBR
The level of expression of the MdHCDBR gene was examined by
qRT-PCR of leaf, ower and fruit tissues of M. domestica Golden

Delicious using actin as a reference gene and compared with the

expression of phloretin 20 -O-glucosyltransferase (PGT1), previously
demonstrated to be involved in the DHC biosynthetic pathway
(Fig. 1) (Jugde et al., 2008). Transcripts of both genes, MdHCDBR
and PGT1, were detected in all ower, leaf and fruit samples, but
the expression level varied statistically between tissues, especially
during fruit ripening (Fig. 6). In owers, expression increased during opening before decreasing again at full owering stage (blue
bars). At early fruit stages, both skin and esh (orange) showed
highest expression of MdHCDBR, which decreased during ripening
(Fig. 6). The overall expression prole of MdHCDBR and PGT1,
including in leaves, is in agreement with the described accumulation of DHCs in Malus (Mayr et al., 1995; Franceschi et al., 2012).
This nding further supporting the involvement of MdHCDBR in
the biosynthesis of DHCs in apple, and may explain the presence
of phloridzin (7), a glycosylated DHC, in apple fruit esh
(Mikulic-Petkovsek et al., 2010; Franceschi et al., 2012). Other
apple tissues such as leaves and owers are known to contain high
levels of glycosylated DHCs, such as phloridzin (7), but the latter is
present only at trace levels in a few other plant species (Guyot
et al., 1998; Zhang et al., 2007; Gosch et al., 2009). The similar
expression patterns of MdHCDBR to the recent characterized PGT1
(Jugde et al., 2008) supports the involvement of MdHCDBR in the
DHC pathway in these tissues.
3. Conclusions
A new NADPH-dependent hydroxycinnamoyl-CoA double bond
reductase gene/protein has been identied from the M. domestica
leaves. The His-tagging and afnity chromatography puried
E. coli-expressed apple MdHCDBR protein reduces p-coumaroylCoA (2) to p-dihydrocoumaroyl-CoA (3) and feruloyl-CoA to

Please cite this article in press as: Ibdah, M., et al. Identication and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase
involved in dihydrochalcone formation in Malus  domestica Borkh. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.027

M. Ibdah et al. / Phytochemistry xxx (2014) xxxxxx

dihydroferuloyl-CoA. To the best our knowledge, this is the rst

report on the biochemical and kinetic characterization of an
NADPH-dependent hydroxycinnamoyl-CoA double bond reductase. This work also provides insight into the metabolic grid
involved in the biosynthesis of polyphenols in apple. The apple
MdHCDBR appears to play a crucial role in the rst step of DHC biosynthetic pathway. The novel hydroxycinnamoyl-CoA double bond
reductase could also enable the establishment of various transgenic crop plants containing phloretin (5), whether for human
nutrition benets or for positive effects on plant disease resistance.
4. Experimental
4.1. Chemicals
Acetone, dichloromethane (CH2Cl2), ethanol (EtOH), hexane,
HPLC-grade acetonitrile (CH3CN), isopropyl-1-thio-b-D-galactopyranoside (IPTG), methanol (MeOH), triethylamine (Et3N) were purchased from Sigma-Aldrich. p-Coumaroyl-CoA (2), feruloyl-CoA,
cinnamoyl-CoA and caffeoyl-CoA were obtained from TransMIT
(Giessen, Germany).
4.2. Plant materials
Flower tissues (closed buds, 80% open and fully open), young
leaf and fruit tissue of different developmental stages of M. domestica Golden Delicious were collected from the Apple germplasm
at FEM-IASMA (San Michele allAdige, Italy). Developmental stages
were dened as previously reported (Costa et al., 2010). Early
Green (stage 1; 30 days after bloom), Late Green (stage 2),
Breaker (stage 3), Pre-harvest (stage 4) and Post-harvest10
(stage 5; 10 days after harvest). Fruits were peeled with a razor
blade to carefully separate the skin from the esh. For stages 36
the pericarp and seeds were separated from the esh and discarded. All plant materials were ash-frozen and stored at
80 C until use.
4.3. Data mining and phylogenetic analysis
All sequences in the http://www.rosaceae.org database that
were similar to Arabidopsis alkenal DBR (At5g16970) were translated using the bioinformatics resource ExPASy server
(www.expasy.org/), with several reductase-like sequences being
reassembled using the CAP3 sequence assembly program. The
resulting candidate sequences were compared to the Arabidopsis
DBR and each other using BlastP. An unrooted neighbor-joining
tree of the MdHCDBR amino acid sequence and different homologous plant proteins was generated using Phylogeny Analysis
(http://www.phylogeny.fr). The resulting tree was bootstrap analyzed with 1000 replicates. Database accession numbers of
selected DBR, dehydrogenase, and enoyl-ACP reductase sequences
are shown in square brackets in Fig. 2.
4.4. Cloning and expression of a cDNA of the putative MdHCDBR
Based on sequence information, two specic primers were
designed corresponding to the 50 -end (50 -ATG GCG GCA AGT ACA
GAG GGA GTG ATC-30 ) and 30 -end (50 -TCA TTC TCG GGA AAC TAC
CAC CAC CTG C-30 ) of the apple MdHCDBR nucleotide sequence.
RNA from apple fruits and leaves was isolated using the Qiagen
RNeasy kit (Hilden, Germany). For cloning, 5 lg of total RNA was
reversed transcribed with SuperScript One-Step RT-PCR with
Platinum Taq (Invitrogen) and the corresponding cDNA was
amplied to yield a 1000-bp fragment. The cDNA was ligated into
the pEXP5-NT/TOPO TA expression vector (Invitrogen Corporation,

Carlsbad, CA, USA) to create a fusion of the open-reading frame

with a His tag-coding extension at the N-terminus and transformed into E. coli Top10 cells. The constructs were veried by
sequencing using the T7 promoter primers. The expression constructs were then introduced into E. coli strain BL21 (DE3) (Invitrogen). A 3 ml preculture was grown overnight at 37 C in LB
medium containing 100 lg ml1 ampicillin (amp) and 34 lg ml1
chloramphenicol. This culture was used to inoculate 500 ml of
fresh medium to which 1 mM isopropyl-1-thio-b-D-galactopyranoside (IPTG) was added to induce protein expression when the culture reached a density at OD600 of 0.8. Cells were then grown for
12 h at 16 C. After centrifugation for 10 min at 11,000g, the cells
were resuspended in 50 mM TrisHCl buffer, containing 10% glycerol and 1 mM 2-mercaptoethanol (pH 7.5). Cells were lysed by a
combination of a 20 min lysozyme treatment (1 mg per 500 ml of
culture) and subsequent ultrasonication. The supernatant containing the soluble recombinant MdHCDBR protein was subjected to
metal (nickel) afnity chromatography (Qiagen, Valencia, CA,
USA) with a stepwise gradient of increasing imidazole concentrations using standard procedures. The MdHCDBR containing fractions were pooled and desalted with 50 mM TrisHCl buffer,
containing 10% glycerol and 1 mM 2-mercaptoethanol (pH 7.5),
using Vivaspin 20 (GE Healthcare, MWCO [molecular mass cutoff] of 10 kDa). Protein concentrations were determined by the
method of Bradford (1976) using Bradford reagent obtained from
Sigma-Aldrich. The concentrated protein was assayed for reductase
activity and in parallel by SDSPAGE.
4.5. MdHCDBR and PGT1 transcript analysis
For quantitative RT-PCR (qRT-PCR) analysis of MdHCDBR and
PGT1, total RNA (5 lg) from different Malus tissues was extracted
(Spectrum Plant Total RNA Kit, SigmaAldrich) and reverse-transcribed using an oligo primer and the SuperScript II rst-strand
system (Invitrogen).qRT-PCR was performed on a Biorad CFX96
System, with Biorad CFX manager software version 3.0 (Bio-Rad
Laboratories, Hercules, CA), using SSoFast Master Mix (Biorad)
using 5 ng of reverse-translated total RNA and 100 ng of primers.
Primers for MdHCDBR were MDDBR-430F (50 -GGAATGACTGCTTATGCTGGG-30 ) and MDDBR-580R (50 -TGCTTCCAGCACTTCCAACA
-30 ), and for PGT1 were MDPGT1-579F (50 -CCGGAACGACCCTGCTTATT-30 ), and MDPGT1-729R (50 -AGTTGGCCCATCAGGAACAC-30 ).
The following actin primers were used, as for the positive controls:
Md_Actin-F (50 -TGACCGAATGAGCAAGGAAATTA-30 ) and Md_ActinR (50 -ACTCAGCTTTGGCAATCCACAT-30 ). The difference in relative
expression levels of MdHCDBR and PGT1 was calculated from the
DCt;goi

Egoi

DCt;ref
Eref

value after normalization of MdHCDBR and PGT1 data to actin,

where the E is the PCR efciency, goi the gene of interest and ref a
reference gene (Pfaf, 2001). Gene expression was analyzed using
analysis of variance (One-way ANOVA) between different ripening
stages, followed by Tukeys test into homogeneous subsets. Data
are expressed as mean standard deviation. The signicance level
was set at 5%. Statistical analysis was performed using Statistica
software version 9.1 (StatSoft Inc., 2010). STATISTICA (data analysis
software system), version 9.1. http://www.statsoft.com). All analyses were performed using three technical replicates.
4.6. Enzyme activity measurements
Initial reactions were carried out in 50 mM TrisHCl pH 7.5 and
contained 1020 lg His-tag puried (high purity level) recombinant protein, 2 mM NADPH, and 500 lM p-coumaroyl-CoA (2).
Stock buffers for determination of pH optimum were prepared by
mixing 0.5 M citric acid and 1 M dibasic sodium phosphate solutions. Final pH values ranged between 4.0 and 7.6 with an interval

Please cite this article in press as: Ibdah, M., et al. Identication and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase
involved in dihydrochalcone formation in Malus  domestica Borkh. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.027

M. Ibdah et al. / Phytochemistry xxx (2014) xxxxxx

of 0.4 pH units. Twenty microliters of these buffers were used in a

total reaction volume of 100 ll in order to estimate the pH
optimum. Enzyme reactions for determination of kinetic properties
with p-coumaroyl-CoA (2) and feruloyl-CoA as substrates had a
total volume of 100 ll and contained 20 ll citrate-phosphate
buffer pH 5.0, 8.2 lg protein, 10500 lM p-coumaroyl-CoA (2), or
10750 lM feruloyl-CoA, and 2 mM NADPH. Kinetic parameters
with NADPH as the variable substrate were determined using a
concentration range of 202000 lM NADPH with a xed concentration of p-coumaroyl-CoA (2) (500 lM). Reactions were prewarmed at 35 C for 5 min, started by the addition of NADPH,
allowed for proceed to 4 min, and then stopped by the addition
of 11.2 ll 5 N NaOH. After the hydrolysis of the CoA-esters at
45 C for 45 min, the mixtures were neutralized by the addition
of 12.4 ll 6 N HCl, and extracted twice with 200 ll EtOAc. The
combined organic phases were evaporated under a gentle stream
of nitrogen, resuspended in 60 ll CH3CN:H2O with 0.1% (v/v)
HCO2H), centrifuged for 20 min at 21,000g at 4 C. Two
microliters of the reaction products were used for analysis by
UPLC-UV-MS. All assay series were carried out at least in triplicate.
4.7. UPLC-UV-ToF-MS analysis of products
A Waters Synapt G2-S HDMS mass spectrometer coupled to an
Acquity UPLC/PDA (Photodiode array) system was used for analysis
of assay products. The reaction products were separated by UPLC
on an Acquity UPLC HSS T3 column (2.1  100 mm, 1.8 lm,
Waters) using 0.1% (v/v) HCO2H in H2O (solvent A) and 0.1% (v/v)
HCO2H in CH3CN (solvent B) at a ow rate of 0.4 mL min1 and
the following gradient program (of A in B): 0 min, 95% A;
0.5 min, 95% A; 6 min, 75% A; 7 min, 1% A; 9 min, 1% A;
10.50 min, 95% A; 14 min, 95% A. Elution of the products was monitored by an Acquity UV PDA detector (210500 nm), and by the
Synapt G2-S QToF-MS detector operating in negative resolution
mode. Tune parameters used were as follows: capillary at 2.9 kV,
cone at 30 V, source offset at 40 V, source temperature was at
150 C, desolvation temperature at 500 C, desolvation gas ow
at 1000 L h1, nebulizer was at 2.5 bar. All other settings were
default, and the autoP quad prole was used. The ion mobility features of the instrument were not used in this analysis. Standard
curves were generated by serial dilution of authentic standards
(dihydro-phenylpropanoic acids) and used for product identication and quantication.
4.8. Chalcone preparation
Naringenin chalcone (4), naringin chalcone, hesperidin chalcone
and hesperetin chalcone were prepared as previously described
(Gonzalez et al., 2002).
Acknowledgements
This research was partially supported by the Ministry of Agriculture & Rural Development (Grant No. 261-1043-14 to Mwafaq
Ibdah) and by the Autonomous Province of Trento, Italy,
ADP2010-2014 project and by the US National Science Foundation
(Grant No. MCB-0969010 to D.R.G.).
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Please cite this article in press as: Ibdah, M., et al. Identication and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase
involved in dihydrochalcone formation in Malus  domestica Borkh. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.027