Biosci. Biotechnol. Biochem.
, 77 (7), 1403–1409, 2013
Cloning and Characterization of Lonicera japonica p-Coumaroyl Ester
3-Hydroxylase Which Is Involved in the Biosynthesis of Chlorogenic Acid
Gaobin PU,1;2 Peng WANG,1 Bingqian Z HOU,1 Zhenhua L IU,1 and Fengning X IANG1; y
1
Key Laboratory of Plant-Cell Engineering and Germ-plasm Innovation, Ministry of Education,
School of Life Sciences, Shandong University, Jinan, 250100, China
2
Shandong Yingcai University, Jinan, 250104, China
Received January 10, 2013; Accepted April 3, 2013; Online Publication, July 7, 2013
[doi:10.1271/bbb.130011]
Lonicera japonica is used in Chinese medicine as a
source of antioxidants, primarily flavonoids, and a
phenolic acid chlorogenic acid (CGA). Here we report
the isolation and characterization of the full-length
cDNA of LjC3H, a gene encoding p-coumaroyl ester
3-hydroxylase, an enzyme involved in CGA synthesis.
Phylogenetic analysis indicated that is protein belongs
to the CYP98A subfamily, and homology modeling
revealed that its structure resembles that of other
cytochrome P450 family proteins. Southern blot analysis
indicated that more than one copy of sequences
homologous to LjC3H is present in the L. japonica
genome. Heterologous expression of LjC3H cDNA in
Escherichia coli allowed an in vitro assay of LjC3H to be
performed. This experiment revealed that the enzyme
favors p-coumaroylshikimate over p-coumaroylquinate
as substrate. LjC3H transcript abundance was increased
both by treatment of the leaves with methyl jasmonate
and by exposure to UV-B radiation. The CGA levels in
the leaves of L. japonica were positively correlated with
LjC3H transcript abundance.
Key words: Lonicera japonica; chlorogenic acid;
p-coumaroyl ester 3-hydroxylase
Lonicera japonica Thumb. (the Japanese honeysuckle,
jinyinhua in Chinese) has a number of applications in
traditional Chinese medicine1) and is also used as an
ingredient of certain functional foods and cosmetics. Its
antioxidant properties reflect its content of flavonoids
and phenolic acids, particularly chlorogenic acid
(CGA).2) The synthesis of CGA follows two hypothetical
routes in plants, both requiring the presence of the
enzyme p-coumaroyl ester 3-hydroxylase (C3H), which
belongs to the family of P450 cytochromes (CYPs), for
the meta-hydroxylation step in the phenylpropanoid
pathway (Fig. 1). One of the two proposed CGA syn-
thesis pathways involves the trans-esterification of
caffeoyl-CoA and quinic acid by hydroxycinnamoyl-
CoA quinate hydroxycinnamoyl transferase,3) in which
caffeoyl-CoA is supplied by the combined activities of
hydroxycinnamoyl-CoA shikimate/quinate hydroxycin-
namoyl transferase (HCT) and C3H via a p-coumaroyl-
shikimate intermediate.4) The second route requires
p-coumaroylquinate to be hydroxylated by C3H.5,6)
y
To whom correspondence should be addressed. Tel: +86-531-88363629; Fax: +86-531-88565610; E-mail: xfn0990@sdu.edu.cn
Abbreviations: ABA, abscisic acid; C3H, p-coumaroyl ester 3-hydroxylase; CGA, chlorogenic acid; CYPs, P450 cytochromes; HCT,
hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase; MeJA, methyl jasmonate; SA, salicylic acid
To date, the only gene associated with CGA synthesis
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identified in L. japonica is HCT.7) Here we report the
isolation of a full-length C3H cDNA sequence, an
in vitro characterization of the gene product’s enzyme
activity, and the relationship between the level of
expression of C3H and CGA levels in plants exposed
to exogenous phytohormone and to UV-B light.
Materials and Methods
Plant materials and treatment with either phytohormone or UV-B.
L. japonica plants were grown at 23
2  C under a 14-h photoperiod
produced by artificial light of an intensity of 150 mmol m2 s1 . A
set of phenotypically uniform individual plants (height 30–40 cm)
were treated with phytohormone (either 0.1 mM abscisic acid (ABA),
0.1 mM methyl jasmonate (MeJA), or 1.0 mM salicylic acid (SA)). All
solutions were adjusted to pH 7.0 with NaOH and supplied via an
atomizing spray on the leaves. Control plants were sprayed with
distilled water, also adjusted to pH 7.0 with NaOH. All experiments
were performed in duplicate. Other plants were exposed to 2 W m2
UV-B light for 5 h.
Isolation of L. japonica C3H. A pair of degenerate primers (COD
C3HF and COD C3HR) were designed based on consensus regions of
the genomic C3H sequences of A. thaliana (NP 850337), Nicotiana
tabacum (ABC69384), Coffea canephora (ABB83677), Ocimum
basilicum (AAL99200), Medicago truncatula (ABC59086), and
Sesamum indicum (AAL47545). Single-stranded cDNA was reverse
transcribed (M–MLV reverse transcriptase, Takara, Ohtsu, Japan)
from 1 mg of total RNA using primer pair OL3 in a 10-mL reaction,
following the manufacturer’s instructions. The resulting sequence was
inserted into p-GEMTeasy vector (Promega, Madison, WI) for
sequencing. A BLAST search of the sequence derived was performed
against the Viridiplantae GenBank database. The full-length cDNA
sequence was obtained by RACE PCR. The 30 reaction used primer
pair C3H3-1 and C3H3-2, designed on the basis of the acquired
L. japonica sequence. The first RT-PCR was based on primer pair
C3H3-1 and P3, and the second on C3H3-2 and Pn3. RACE was
done with the reagents supplied with the 50 -RACE System
(Invitrogen, Carlsbad, CA) along with C3H5-1, C3H5-2, and
C3H5-3 (designed on the basis of the acquired L. japonica sequence).
The sequences of all the PCR primers listed above are given in
Table 1.
ClustalW (www.ch.embnet.org/software/ClustalW.html) was used
to align the deduced polypeptide sequence of L. japonica C3H
(LjC3H) with those of A. thaliana (NP 177595), C. canephora
(ABB83676), N. tabacum (ABC69384) and R. graveolens
(AEG19446), by applying the program’s default parameters. Sub-
sequently a phylogenetic analysis was carried out using MEGA v3.0
software,8) based on published polypeptide sequences.
1404 G. PU et al.

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Fig. 1. Proposed Pathways for the Biosynthesis of Chlorogenic Acid in Plants.
PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-hydroxycinnamoyl-CoA ligase; HCT, hydroxycinnamoyl-CoA
shikimate/quinate hydroxycinnamoyl-transferase; HQT, hydroxycinnamoyl-CoA quinate hydroxycinnamoyl-transferase; C3H, p-coumaroyl
ester 3-hydroxylase.

Table 1. Sequences of Primers Used in This Study

Primers Sequence
COD C3HF 50 -GTYGGHAACCTCTACGAC-30
COD C3HR 50 -RTCWCKVGCNACHGCCCA-30
OL 3 50 -TGAAGACGACAGAAAGGGCGTGGTGCGGAGGGCGGTTTTTTTTTTTTTTTTTT-30
P3 50 -TGAAGACGACAGAAAGGGCG-30
P n3 50 -TGGTGCGGAGGGCGGT-30
C3H3-1 50 -AAATGAGATTCTTGAAAAGCACGAG-30
C3H3-2 50 -GATAACAGCAGGAATGGACACAACC-30
C3H5-1 50 -GTTCGTCGATTATTCCCTCTG-30
C3H5-2 50 -TTGTTGAATGCTACTGACCCG-30
C3HGP3 50 -TTACAAACTTTCCTCACCTTCAC-30
C3H 28 F 50 -TATAGAATTCATGGCATTCGCTCTCCTC-30
C3H 28 R 50 -TATACTCGAGCTAGTAATCCACTGGGACACG-30
C3Hpro F 50 -TCGAGATATCTTCGAAGGAAGCA-30
C3Hpro R 50 -GGCCGTCGGAAGACTGCAAAGC-30
Act F 50 -ATCACCAGAATCCAGCACAATACC-30
Act R 50 -CACCTCTCAACCCTAAAGCCAAC-30
C3HF 50 -ATGGCATTCGCTCTCCTC-30
C3HR 50 -CTAGTAATCCACTGGGACACG-30
Coding redundancies were W ¼ T or A; R ¼ A or G; Y ¼ C or T; N ¼ C, T, A, or G; H ¼ A, T, or C; K ¼ G or T; V ¼ G, A, or C

Heterologous expression of LjC3H in Escherichia coli. LjC3H was
amplified from a cDNA template using primers C3H 28F and C3H 28R
(Table 1) in order to introduce an EcoRI cloning site at one end and an
XhoI site at the other. The resulting amplicons were inserted into
pGEM-T easy vector for sequencing. Following EcoRI and XhoI
digestion, the DNA fragments were cloned directionally into the
EcoRI/XhoI site of pET-28a(þ) (Novagen, Darmstadt, Germany). The
recombinant plasmid (which include a hexahistidine tag at the N
terminus of the insert) was introduced into E. coli BL21-Rosetta (DE3)
(TransGen, Beijing, China), and cultured at 37  C in 200 mL of Luria-
Bertani (LB) medium containing 50 mg/L of kanamycin and 34 mg/L
of chloramphenicol. When the OD600 of the culture reached 0.6–0.8,
1 mM isopropyl
-D-thiogalactopyranoside (IPTG) was added, and the
temperature was reduced to 28  C. After 4 h, the cells were harvested
by centrifugation, resuspended in 3 mL of 0.1 M potassium phosphate
buffer (pH 7.5), and sonicated on ice for 10 min.
Enzyme purification and characterization. The homogenate pro-
duced by sonication was centrifuged at 10;000 g at 4  C, and the
supernatant was passed through an NTA His-Bind
Resin column
(Novagen) containing Ni2þ as affinity ligand. After washing with 0.1 M
potassium phosphate buffer (pH 7.5) containing 0.5 M NaCl and 60 mM
imidazole, the recombinant protein was eluted with potassium
phosphate buffer (pH 7.5) containing imidazole (100 mM), and the
eluate was monitored by SDS–PAGE. The enzymatic activity of
LjC3H was assessed in 100-mL reactions containing 600 mM NADPH,
varying concentrations of p-coumaroylshikimate (5–150 mM), 100 mM
sodium phosphate buffer (pH 7.4), and 2 mg of purified enzyme.
Identical reactions were conducted using p-coumaroylquinate or
p-coumaric acid as substrate. Each reaction was incubated for 30 min
at 30  C and terminated by the addition of 20 mL of acetic acid. The
reaction products were extracted 3 times with 2 vol. ethyl acetate, the
organic phases were pooled and evaporated under argon, and the
 Cloning and Characterization of C3H from Lonicera japonica
residue, dissolved in 300 mL of aqueous acetonitrile (10% v/v
containing 0.2% v/v acetic acid), was analyzed by reverse-phase
HPLC (LiChrosorb RP-18 column (Merck), column dimensions
4  125 mm, 5 mm; flow rate 1 mL/min isocratic 10% v/v acetonitrile
for the first 5 min, followed over the subsequent 20 min by a linear
gradient from 10–52% v/v aqueous acetonitrile/0.2% v/v acetic acid).
Substrate conversion was estimated from the relevant peak area after
detection at 320 nm and was based on standards.
Enzyme kinetics. Kinetic constants were determined using five
concentrations of the substrate covering a Km range of 0.2–6.0, and the
concentration of NADPH was at saturation point (600 mM). The
experiment was repeated 3 times using 1 mg of purified enzyme in a
final volume of 100 mL of 0.1 M potassium phosphate buffer incubated
for 30 min at 30  C. Kinetic parameters were calculated based on the
presence of the major reaction product. Lineweaver-Burk plots were
used to estimate the Km and Kcat values.
Transcription analysis. Total RNA was isolated using the TRIzol
reagent (Takara, Ohtsu, Japan), and treated with RNase-free DNase I.
First-strand cDNA was synthesized from 3 mg of total RNA in a 40-mL
reaction using Superscript II reverse transcriptase (Invitrogen, Beijing,
China), and 1 mL of this reaction mixture was used in a 20-mL RT-PCR
based on primers C3HF and C3HR (Table 1). The reactions consisted
of 22–34 cycles of 94  C/30 s, 55  C/30 s and 72  C/30 s. Actin was
chosen as reference gene.
Southern blot analysis. L. japonica genomic DNA was isolated by a
modification of the CTAB method,9) and was used in Southern blotting
by the procedure of Sambrook et al.10) In brief, 20 mg of DNA was
digested with BglII, EcoRI, or EcoRV (Takara, Ohtsu, Japan), which
did not cut within the probe region. The digested DNA was separated
electrophoretically with an 0.7% agarose gel and transferred onto a
Hybond-Nþ nylon membrane (Amersham). The cDNA sequence,
including the 5-UTR (untranslated region) and a partial open reading
fragment of LjC3H was generated by PCR using primers C3Hpro F and
C3Hpro R, and used as probe. The subsequent hybridization reaction
was performed over 16 h at 65  C, using a digoxigenin-11-dUTP
labeled (DIG DNA Labeling and Detection Kit, Roche Applied
Science) partial LjC3H sequence as probe. The filters were rinsed
twice in 2  SSC/0.1% w/v SDS for 15 min at 50  C, and twice
more in 0:5  SSC/0.1% w/v SDS for 20 min at 65  C. Chemilumi-
nescence detection was carried out according to the protocol supplied
by Roche.
Homology modeling of LjC3H. Homology models were obtained
using Modeller 9v8 software11) assuming a crystal structure similar to
that of human CYP (PDB code 3SWZ),12) a polypeptide that shows
28% identity and 48% similarity with LjC3H according to Discovery
Studio Visualizer. Sequence alignments were performed automatically
using MUSCLE software13) and adjusted manually in Discovery Studio
Visualizer for alignment of conserved motifs. More than 200 models of
the initial LjC3H were generated by a fully annealed protocol, and the
optimal model was chosen for further study according to its
ModellerDOPE (Discrete Optimized Protein Energy) score.14)
Determination of CGA levels in L. japonica leaf. Frozen L. japon-
ica leaf tissue was pulverized and suspended for 2 h at 4  C in 5% w/v
acetone: methanol: ethanol (70:15:15). After centrifugation, the
supernatant was stored and the pellet re-extracted in ethyl acetate.
The second supernatant was pooled with the first and the mixture was
evaporated in a rotary evaporator (Heidolph) at 25  C. The residue was
redissolved in methanol, and 10 mL was injected into a Luna C18
Phenomenon HPLC column (LiChrosorb RP-18, Merck) mounted in a
Gold System chromatograph equipped with a UV light detector
(Beckman). The mobile phase comprised HPLC-grade water contain-
ing 0.01% w/v trichloroacetic acid (TCA) (solvent A) and 95% v/v
acetonitrile containing 0.01% w/v TCA (solvent B) according to the
following scheme: time ¼ 0, 100% A, 0% B; time ¼ 2 min, 64% A,
36% B; time ¼ 45 min, 90% A, 10% B; time ¼ 47 min, end. The flow
rate was 1 mL/min, and the detection wavelength was set at 310 nm.
The CGA standard was purchased from Sigma-Aldrich.
1405
Results
Isolation of a full length C3H gene from L. japonica
The degenerate PCR amplified a 950-bp fragment
from a leaf cDNA template, the sequence of which was
consistent with its being a member of the CYP gene
family. The sequence was extended to 2,114 bp by
RACE PCR, 1,533 bp of which was an open reading
frame, 296 bp was a 5 untranslated region, and 285 bp
(including an 18-nucleotide poly (A) tail) a 3 untrans-
lated region. The gene was denoted LjC3H (accession
no. KC765076). The LjC3H product was predicted to be
a 510-residue polypeptide with a calculated molecular
mass of 58 kDa and a predicted isoelectric point of 8.92.
It included four conserved CYP domains, a proline-rich
membrane hinge (PPGP), (A/G-GX-E/D-T-T/S) I helix
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(involved in oxygen binding and activation), clade
signature (PERF), and a cysteine-containing region
(PFGXGRRXCX) (Fig. 2). The deduced peptide se-
quence showed 46–77% identity with other CYP98A
sequences. A phylogenetic tree was constructed using 16
sequences from the CYP98A family (Fig. 3). Most of
the accessions were in two main clusters. This division
into two main groups, as previously observed by Morant
et al.,15) indicates that the encoding genes resulted from
early duplication of an ancestral gene. LjC3H grouped in
a sub-cluster of six proteins, and appeared to be closer to
ABB83677 from Coffea, AAL47545 from Sesamum,
ABC69348 from Nicotiana, ACC63870 from Populus,
and AAS57921 from Camptotheca.
Homology model of LjC3H
As for the majority of the CYPs, LjC3H formed two
domains (Fig. 4), an  domain (shown in yellow) and a

domain (cyan). A superimposed P450 ligand from
template (PDB code 3SWZ), including heme and TOK-
001, was introduced into the LjC3H structure for
analysis. Both the heme (green sphere) and the ligand
(orange sphere) molecule fitted within the pocket
surrounded by the helix barrel and the F-G loops.
Residue C440 is able to bond covalently with heme,
while residues H372, R438, and Y121 might be involved
in stabilization of the heme in the catalytic pocket via
hydrogen bonding. The residues encompassing TOK-
001 might form the substrate binding domain of LjC3H,
although we could not identify the residues directly
interacting with the substrate.
Characterization of LjC3H
A purified preparation of recombinant LjC3H (heter-
ologously expressed in E. coli) resolved, following
SDS–PAGE separation, into a single protein species of
a molecular mass of about 59 kDa. It was effective in
converting p-coumaroylshikimate to caffeoylshikimate
and p-coumaroylquinate to CGA (Fig. 5). The Km and
Kcat values associated with LjC3H favored the metab-
olism of the shikimate ester rather than the quinate ester,
since its catalytic efficiency (Kcat =Km ) was about 4-fold
higher when provided with p-coumaroylshikimate than
when provided with p-coumaroylquinate (Table 2).
Copy number of the LjC3H sequence in L. japonica
The hybridization of BglII, EcoRI, and EcoRV
digested L. japonica genomic DNA with nucleotides
1406 G. PU et al.

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Fig. 2. Alignment of the Amino Acid Sequence of LjC3H (KC765076) with That of C3H Enzymes from A. thaliana (NP 177595), C. canephora
(ABB83676), N. tabacum (ABC69384), and R. graveolens (AEG19446).
Black shading denotes peptide identity and gray shading denotes peptide similarity. Conserved CYP domains are underlined.

Fig. 3. Phylogenetic Analysis of C3H Sequences.

Phylogenetic analysis was carried out using MEGA v3.0 software, based on published polypeptide sequences. Numbers alongside each node
refer to bootstrap values (100 replicates).

1–357 (the A of the first codon ATG was assigned 1) of
the LjC3H cDNA led to a profile consisting of 2-4 bands
(Fig. 6), suggesting that the gene is present in the
genome in two copies. Unfortunately, after several
efforts, we obtained only one copy of LjC3H.
Induced responses following phytohormone and UV-B
treatment
Transcription of LjC3H was upregulated 3-4 fold
when the plants were treated with MeJA or exposed to
UV-B, but neither exogenously supplied ABA nor SA
had any effect on transcript abundance (Fig. 7). The
 Cloning and Characterization of C3H from Lonicera japonica 1407

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Fig. 4. Homology Model of LjC3H.
Sequence alignments were performed automatically using MUSCLE software and were adjusted manually in Discovery Studio Visualizer for
alignment of conserved motifs. More than 200 models of the initial LjC3H were generated using a fully annealed protocol, and the optimal model
was chosen for further study according to its ModellerDOPE (Discrete Optimized Protein Energy) score. The  helix is indicated in blue, the

strand in purple, the heme molecule in green and the TOK-001 molecule in orange.

Fig. 5. Reaction Products in an in Vitro Assay of Recombinant LjC3H.

A and B, HPLC profiles of reaction products formed with p-coumaroylquinate (A) and with p-coumaroylshikimate (B) as substrate. Left-hand
profiles of A and B mean no recombinant LjC3H in the reaction system. Right-hand profiles of A and B mean adding recombinant LjC3H to the
reaction system.

Table 2. Catalytic Parameters of 3-Hydroxylation Catalyzed by sent a widely dispersed protein family, found in all
Recombinant LjC3H eukaryotes as well as most prokaryotes. CYP98A
Km Kcat Kcat =Km subfamily proteins are thought to be responsible for
Substrate mM min1 min1 mM1 the catalysis of the meta-hydroxylation of various
phenolic compounds in plants, using esters of shikimic
5-O-(4-Coumaroyl) quinate 20
2 343
16:0 17
5-O-(4-Coumaroyl) shikimate 9
1 578
29 64
or quinic acid as substrate rather than p-coumaric acid or
its CoA ester.18) Thus, as predicted, LjC3H was effective
as a 3-hydroxylase of hydroxycinnamoyls, and was
CGA level of the L. japonica leaf was also unaffected particularly reactive with p-coumaroyl shikimate. Its
by the provision of ABA or of SA (Table 3), but the kinetics favored the metabolism of shikimate ester rather
MeJA and UV-B treatments had enhanced CGA level by than that of quinate ester, reminiscent of the behavior of
a significant amount 4 d after treatment (Table 3). CYP98A3 of A. thaliana18) and CYP98A49 of globe
artichoke.19) CYP98A35 (of coffee), on the other hand,
Discussion is capable of metabolizing p-coumaroylquinate and
p-coumaroylshikimate at the same level of efficiency,4)
We have described the isolation from L. japonica of a while CYP98A22 of R. graveolens20) and a carrot
gene encoding a p-coumaroyl ester 3-hydroxylase. The enzyme prefer the quinate ester as substrate.21)
deduced peptide sequence of the LjC3H product con- Some CYP98 genes are expressed constitutively,
siderable homology (46–77%) to that of CYP98A while others, particularly those involved in the stress
proteins, and homology modeling also revealed that its response are inducible. A. thaliana CYP98A3 is con-
overall structure was similar to those of most of the CYP stitutively expressed and is upregulated by wounding,18)
proteins.16,17) It is likely therefore that LjC3H is indeed a while the common bean enzyme CYP98A5 is inducible
member of the CYP98A gene subfamily. CYPs repre- by treatment with 3,5-dichlorosalicylic acid or 2,6-
1408 G. PU et al.
Table 3. The CGA Levels of L. japonica Leaves 0–4 d after Various Treatments
Means of three assays
SE. An asterisk means the value was significantly different at p < 0:05 from the value of the control on the same day.

CGA content (mgmg1 FW)

Treatment
0d 1d 2d 3d 4d
Control 3:02
0:45 3:10
0:35 3:05
0:27 3:14
0:32 3:08
0:42
ABA 2:97
0:36 3:03
0:49 3:11
0:33 3:20
0:37 3:17
0:29
MeJA 3:11
0:28 3:22
0:31 3:89
0:41 4:30
0:44 4:45
0:50
SA 2:88
0:41 2:94
0:28 2:91
0:33 3:11
0:31 3:05
0:42
UV-B 2:96
0:32 3:05
0:25 3:21
0:45 3:45
0:30 3:97
0:38

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Fig. 6. Gene Copy Numbers of LjC3H as Assessed by Southern Blot
Profiles.
L. japonica genomic DNA digested with BglI, with EcoRI and
with EcoRV, which did not cut the internal region of the probe. The
digested DNA was separated electrophoretically by an 0.7% agarose
gel and transferred onto a Hybond-Nþ nylon membrane. The
subsequent hybridization reaction was performed over 16 h at 65  C
with digoxigenin-11-dUTP labeled partial LjC3H sequence as probe.
The filters were rinsed twice in 2  SSC/0.1% w/v SDS for 15 min
at 50  C, then twice more in 0:5  SSC/0.1% w/v SDS for 20 min at
65  C.
dichloroisonicotinic acid,22) the carrot enzyme 5-O-(4-
coumaroyl)-D-quinate/shikimate 3-hydroxylase by irra-
diation with blue/UV light,23) and globe artichoke
CYP98A49 by UV-C radiation.19) LjC3H transcription
was strongly upregulated by treatment with MeJA and
by exposure to UV-B. MeJA is a phytohormone that
participates in the plant defense signal transduction
pathway.24,25) The in planta function of CGA is probably
involved in the scavenging of reactive oxygen species
and the oxidation of lipids.26) Thus, MeJA and UV
radiation might increase CGA levels by inducing the
expression of LjC3H. Experimentally, there was a clear
positive correlation between CGA levels and the
abundance of LjC3H transcript (Fig. 7 and Table 3).
Overexpression of LjC3H might therefore represent a
strategy for increasing CGA levels in the L. japonica
leaf.
Acknowledgments
We thank Dr. Hong Wang (Institute of Botany, The
Chinese Academy of Sciences) for providing samples of
p-coumaric acid, p-coumaroylshikimate and p-coumar-
oylquinate, and Dr. Shuguang Yuan (Nencki Institute of
Experimental Biology, Polish Academy of Sciences) for
assistance modeling of LJC3H. This research was
Fig. 7. Induction of LjC3H Transcription as Assessed by Semi-
Quantitative RT-PCR.
Leaves were treated with ABA (0.1 mM), MeJA (0.1 mM), or SA
(1.0 mM), or exposed to 2 W m2 UV-B for 5 h. Total RNA was
isolated using TRIzol reagent and treated with RNase-free DNase I.
First-strand cDNA was synthesized from 3 mg of total RNA in a 40-
mL reaction using Superscript II reverse transcriptase, and 1 mL of
this reaction mixture was used in a 20-mL RT-PCR based on primers
C3HF and C3HR. The reactions consisted of 22–34 cycles of 94  C/
30 s, 55  C/30 s and 72  C/30 s. Actin was chosen as reference gene.
supported by the National Natural Science Foundation
of China (grant nos. 31200226, and 30970243), the
International Technology Cooperation Project of
Shandong Province, China (grant no. 2011176), the
Postdoctoral Foundation of Shandong Province (grant
no. 201002016) and a Chinese Natural Education
Ministry Doctor Station Foundation Fellowship (grant
no. 913111006), the Chinese National Special Science
Research Program (grant no. 2013CB967303), and key
Projects in the National Science and Technology Pillar
Program in the 12th Five year Plan of china (grant
no. 2011BAI06B01).
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