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Journal of Plant Physiology 202 (2016) 9296

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Journal of Plant Physiology


journal homepage: www.elsevier.com/locate/jplph

Short communication

Identication of avonoid 3 -hydroxylase in the yellow ower of


Delphinium zalil
Taira Miyahara a, , Arisa Hamada a , Mitsutoshi Okamoto b , Yukio Hirose c ,
Kimitoshi Sakaguchi d , Shoji Hatano d , Yoshihiro Ozeki a
a

Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan
Ehime Research Institute of Citrus Fruits, Matsuyama, Ehime 799-3742, Japan
c
Department of Agricultural Research, Ehime Research Institute of Agriculture, Forestry and Fisheries, Matsuyama, Ehime 799-2405, Japan
d
Miyoshi & Co., Ltd., R&D Center, Hokuto, Yamanashi, 408-0041, Japan
b

a r t i c l e

i n f o

Article history:
Received 10 June 2016
Received in revised form 20 July 2016
Accepted 20 July 2016
Available online 25 July 2016
Keywords:
Flavonoid 3 -hydroxylase
Quercetin 3-glucoside
Delphinium semibarbatum
Delphinium zalil

a b s t r a c t
The owers of delphinium cultivars owe their coloration to anthocyanins such as delphinidin or
pelargonidin derivatives. To date, no delphinium cultivars have been found with red owers due to
the presence of cyanidin derivatives. This suggests that delphiniums do not have cyanidin biosynthesis
ability because of the loss of function of avonoid 3 hydroxylase (F3 H). Here, we show that the wild
delphinium species Delphinium zalil (synonym semibarbatum) can accumulate quercetin 3-glucosides
in its sepals, presumably through F3 H activity. We isolated F3 H cDNA from D. zalil (DzF3 H) and produced a recombinant enzyme from a yeast transformant. The recombinant DzF3 H protein could convert
naringenin, apigenin, dihydrokaempferol and kaempferol to eriodictyol, luteolin, dihydroquercetin and
quercetin, respectively. An expression analysis conrmed that blue owered D. grandiorum does not
express F3 H, and also showed that avonoid 3 ,5 -hydroxylase and anthocyanidin synthase do not function in D. zalil sepals. DzF3 H can act as a avonoid hydroxylase to produce cyanidin accumulation. The
introduction of the DzF3 H gene into other delphinium species by conventional breeding may enable
development of cultivars with novel ower colors.
2016 Elsevier GmbH. All rights reserved.

1. Introduction
The anthocyanin pigments that produce basic ower colors are
dependent on the hydroxylation proles of anthocyanidins at the B
ring. For example, pelargonidin has a mono hydroxyl residue at the
4 position, cyanidin has two such residues at the 3 and 4 positions,
and delphinidin has three such residues at the 3 , 4 and 5 positions. Cyanidin, which produces red to purple ower coloration,
is produced by the enzyme avonoid 3 -hydroxylase (F3 H); delphinidin, which gives violet to dark blue coloration, is produced
by avonoid 3 ,5 -hydroxylase (F3 5 H); pelargonidin, which gives
orange to red coloration, is hydroxylated at the 4 position but nei-

Abbreviations: ANS, anthocyanidin synthase; DgF3 H, Delphinium grandiorum


F3 H; DgF3 5 H, Delphinium grandiorum F3 5 H; DzF3 H, Delphinium zalil F3 H;
DzF3 5 H, Delphinium zalil F3 5 H; DFR, dihydroavonol 4-reductase; F3 H, avonoid
3 -hydroxylase; F3 5 H, avonoid 3 5 -hydroxylase; HPLC, high-performance liquid
chromatography; qRT-PCR, quantitative RT-PCR.
Corresponding author at: Department of Biotechnology and Life Science, Faculty
of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho,
Koganei, Tokyo 184-8588, Japan.
E-mail address: miyahara@cc.tuat.ac.jp (T. Miyahara).
http://dx.doi.org/10.1016/j.jplph.2016.07.013
0176-1617/ 2016 Elsevier GmbH. All rights reserved.

ther F3 H nor F3 5 H hydroxylase activity are involved (Davies and
Gould, 2009). In delphiniums, considerable efforts have been made
to produce novel ower color varieties. As such varieties would
have different levels and contents of pigments, there has been
much investigation of the molecular structures and characteristics of anthocyanins (Legro, 1961; Kondo et al., 1990, 1991; Saito
et al., 1998; Hashimoto et al., 2002; Nishizaki et al., 2013, 2014;
Miyagawa et al., 2014). Studies of anthocyanins have shown that
the red, purple and blue colors of delphinium sepals are derived
from the aglycones, delphinidin and pelargonidin; however, there
have been no reports on the accumulation of cyanidin derivatives in
the owers of delphinium species. This reason for the lack of cyanidin derivatives accumulation may be that the wild species used as
a breeding resource, such as Delphinium grandiorum, D. elatum,
D. cardinale and D. nudicaule, do not have F3 H activity. Therefore,
to develop novel ower color varieties by cross-hybridization, it
will important to identify species that have an active F3 H gene,
as the introduction of this gene into established varieties would be
expected to enable the F3 H activity necessary to produce the cyanidin necessary for red owers. Here, we report the rst identication
of an active F3 H gene in the yellow owered wild delphinium
species Delphinium zalil and demonstrate the activity of F3 H.

T. Miyahara et al. / Journal of Plant Physiology 202 (2016) 9296

93

2. Materials and methods

2.3. Qualitative and quantitative RT-PCR

2.1. Plant materials and chemicals

First strand cDNA prepared from D. zalil sepal RNA was used for
qualitative RT-PCR to demonstrate gene expression. PrimeStar GXL
DNA polymerase (Takara Bio) and sequence specic primers were
used in the analyses: F3 H with F3 H RTFwd and F3 H RTRv; F3 5 H
with F3 5 H RTFwd and F3 5 H RTRv; DFR with DFR RTFwd and DFR
RTRv; ANS with ANS RTFwd and RTRv; and Actin, the reference
gene, with Actin RTFwd and Actin RTRv (Nishizaki 2013, Miyagawa
2014). First strand cDNA of D. grandiorum was used as a control
for an anthocyanin synthesizing species that accumulates delphinidin derivatives. The following amplication conditions were
employed: 2 min at 94 C, then 30 cycles of 10 s at 98 C, 15 s at
55 C, and 15 s at 68 C. Quantitative real-time PCR (qRT-PCR) was
performed with the F3 H RTFwd and F3 H RTRv primer set for F3 H
and the Actin RTFwd and Actin RTRev set for Actin. The analysis was
performed using THUNDERBIRD SYBR qPCR Mix (TOYOBO, Japan)
with a DNA Engine Opticone 2 (Bio-Rad Laboratories, USA). The
conditions for the qRT-PCR were the same as described previously
(Nishizaki et al., 2013).

Delphinium zalil (synonym semibarbatum) and D. grandiorum


were gift from the Ehime Research Institute of Agriculture, Forestry,
and Fisheries, and Miyoshi & Co., Ltd. Sepals from four different
developmental stages were collected and immediately frozen in
liquid nitrogen and stored at 80 C until use. Four ower developmental stages were selected: S1, green bud; S2, green and yellow
bud; S3, early ower opening; S4, fully opened ower. For the F3 H
enzyme reaction and authentication of the HPLC analysis, -NADPH
was purchased from Oriental Yeast Co., Ltd (Japan), and apigenin,
luteolin, naringenin, eriodictyol, dihydrokaempferol, kaempferol
and quercetin were obtained from Extrasynthase (France). Dihydroquercetin was purchased from Sigma-Aldrich (USA).

2.2. Isolation of full length DzF3 H cDNA


Total RNA was isolated from S3 sepals using an RNeasy Plant
Mini Kit (Qiagen, USA). First-strand cDNA was synthesized as
described previously (Nishizaki et al., 2013). Partial cDNA fragments for DzF3 H were amplied by RT-PCR using the degenerate
primer set dgF3 H Fwd and dgF3 H Rv (primer nucleotide sequences
used in this study are described in Supplemental Table 1) and rst
strand cDNAs prepared from sepal RNA of D. zalil as the template.
The produced cDNA amplicons were inserted into the pMD20 vector (Takara Bio, Japan) to determine their nucleotide sequences. The
5 and 3 cDNA ends of DzF3 H cDNA were amplied with a SMARTer
RACE cDNA Amplication Kit (Clontech Laboratories, USA) using
DzF3 H Fwd, DzF3 H Rv2. The full-length DzF3 H cDNA nucleotide
sequence in pMD20 was conrmed using M13 Fwd and M13 Rv.

2.4. Heterologous expression of DzF3 H protein in yeast and


preparation of crude protein extract for detection of F3 H enzyme
activity by the recombinant protein
The protein coding region of DzF3 H cDNA was amplied by
PCR using the primer set F3 H-HindIII and F3 H-XbaI that have a
HindIII and XbaI site at each end. The amplicon was introduced into
a pYES2 (Invitrogen, USA) expression vector with a URA3 selection
marker, and the resultant pYES2-DzF3 H was transferred into Saccharomyces cerevisiae strain INVSc1. To support the F3 H reaction
(Naur et al., 2003), the coding region of Arabidopsis NADPHcytochrome P450 reductase, AtATR1 (At4g24520), was amplied
by the primer set AtATR1-EcoRI and ATR1-NotI using leaf cDNA as a

Fig. 1. Qualitative gene expression analysis of avonoid synthetic enzymes and the metabolic pathway.
(A) PCR analysis for expression of avonoid 3 -hydroxylase (F3 H), avonoid 3 , 5 -hydroxylase (F3 5 H), dihydroavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and
actin. For each gene, the amplicon in the band on the left represents D. zalil cDNA and that on the right is D. grandiorum cDNA. (B) Summary of the avonoid metabolic
pathway. Black arrows indicate the F3 H catalytic reaction.

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T. Miyahara et al. / Journal of Plant Physiology 202 (2016) 9296

template. The amplicon was introduced into a pESC vector (Agilent


Technologies, USA) with a LEU2 selection marker and transformed
into yeast strain INVSc1 that harbors pYES2-F3 H. Recombinant
DzF3 H protein production was induced following the pYES2 manufacturers protocol.
Crude protein extracts from yeast transformants were prepared by a Multi-beads shocker (Yasui Kikai Co., Japan) with glass
beads (0.5 mm diameter) at 10 cycles of 2500 rpm for 10 s. Cell
debris was removed by centrifugation at 10,000 g for 5 min at
4 C, and the supernatant was used as the crude protein extract
in the enzyme assay. The F3 H enzyme reaction was performed
for 2 h at 30 C, using 90 g crude protein extract of the double transformant (pYES2-DzF3 H and pESC-AtATR1). The protein
extract was added to 60 nmol substrates (naringenin, apigenin,
dihydrokaempferol and kaempferol) and 100 nmol NADPH in a
total volume of 100 L 0.1 M potassium phosphate buffer (pH 7.5).
As a non-reactive control, 90 g crude protein extract from the
pESC-AtATR1 single transformant was used in the reaction mixture.
After the termination of the reaction by the addition of 100 L ethyl
acetate, the organic layer was recovered and dried, redissolved

in 30 L methanol, and 10 L of the reaction product was analyzed by HPLC. The separations were carried out for 5 min using an
HPLCphotodiode array detector system (LaChrome Elite, Hitachi
High-Technologies Corp., Japan) equipped with an ODS column
(4.6 50 mm, COSMOSIL 5C18 -MS-II, Nacalai Tesque, Japan), and a
linear gradient elution (1 mL min1 ) of 2550% methanol in 1.5%
aqueous phosphoric acid. Naringenin, eriodictyol, dihydrokaempherol and dihydroquercetin were analyzed using absorbance at
290 nm, apigenin and luteolin at 350 nm, and kaempferol and
quercetin at 370 nm. The total protein in the crude protein extract
was quantied using a CBB protein assay kit (Thermo Fisher Scientic, USA) with BSA as the standard.
3. Results
3.1. Characterization of avonol 3 -hydroxylase cDNA isolated
from D. zalil sepals
We isolated F3 H cDNA from S3 sepals of D. zalil as they accumulate quercetin 3-glucoside (Supplemental Fig. 1). Kaempferol

Fig. 2. Detection of recombinant DzF3 H activities.


Analysis of the activities of recombinant-yeast crude extracts for naringenin using both DzF3 H and AtATR1 (A) and AtATR1 alone (B). Authentic standards of naringenin
(right) and eriodictyol (left) (C). D, E: Activities for apigenin using both DzF3 H and AtATR1 (D) and AtATR1 alone (E). Authentic standards of apigenin (right) and luteolin (left)
(F). G, H: Activities for dihydrokaempferol using both DzF3 H and AtATR1 (G) and AtATR1 alone (H). Authentic standards of dihydrokaempferol (right) and dihydroquercetin
(left) (I). J, K: Activites for kaempferol using both DzF3 H and AtATR1 (J) and AtATR1 alone (K). Authentic standards of kaempferol (right) and quercetin (left) (L).

T. Miyahara et al. / Journal of Plant Physiology 202 (2016) 9296

95

Fig. 3. Expression prole of DzF3 H.


(A) The four different sepal development stages studied in this experiment. Scale bar indicate 1 cm. (B) DzF3 H expression levels (line graph) and the amount of accumulation
of kaempferol 3-glucoside (black) and quercetin 3-glucoside (gray) (bar graph). Data are presented as means SD (n = 3).

3-glucoside and neochrologenic acid were also accumulated but


any avones could not detect in sepals. The full-length DzF3 H cDNA
had an open reading frame of 1551 bp that encoded a 516 amino
acid sequence (Accession no. LC089877). Molecular phylogenetic
tree showed that DzF3 H belonged to the CYP75 B clade, whereas
the F3 5 H amino acid sequence of the blue ower delphinium, D.
grandiorum (Accession no. AB819289) belonged to the CYP75A
clade (Supplemental Fig. 2).
3.2. Expression prole of anthocyanin synthesis enzymes by
qualitative PCR
The F3 H amplicon was detected in D. zalil but not D. grandiorum (Fig. 1). PCR produced a very faint F3 5 H band in D. zalil;
however, the isolated putative full-length DzF3 5 H cDNA had a
stop codon in the middle of the deduced amino acid sequence. This
suggested that DzF3 5 H might be defective for F3 5 H activity (Supplemental Fig. 3). In addition, no ANS amplicon was detected in D.
zalil.
3.3. Recombinant F3 H enzyme activities
An enzyme in the crude protein extract from the yeast transformant harboring DzF3 H and AtATR1 showed F3 H activity as
demonstrated by conversion of naringenin to eriodictyol, apigenin to luteolin, dihydrokaempferol to dihydroquercetin, and
kaempferol to quercetin. The crude protein extract of the AtATR1
single transformant without DzF3 H did not show any F3 H activity (Fig. 2). Comparison of relative activities showed DzF3 H had

Table 1
Substrate preferences of recombinant DzF3 H.
Substrate

pkat/mg Protein

Relative activity (%)

Naringenin
Apigenin
Dihydrokaempferol
Kaempferol

0.075 0.003
0.089 0.010
0.132 0.003
0.075 0.010

56
68
100
57

Data are presented as means SD (n = 3).

a preference for dihydrokaempferol as the substrate compared to


other avonoids (Table 1).
3.4. F3 H expression prole and avonol accumulation
Analysis of DzF3 H expression showed that the highest level was
present in S2 sepals, which showed 1.8 fold higher expression levels
than the lowest stage of expression, S1 sepals (Fig. 3). Thus DzF3 H
was constantly expressed in all tested ower stages. This result
was consistent with the analysis of avonol accumulation at these
ower developmental stages. Quercetin 3-glucoside, the product of
DzF3 H activity, was found to accumulate in these stages. However,
the amount of quercetin 3-glucoside accumulated was less than
that of kaempferol 3-glucoside at all stages.
4. Discussion
In our previous studies on delphiniums, we showed that all
wild species and horticultural cultivars of D. grandiorum accumu-

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T. Miyahara et al. / Journal of Plant Physiology 202 (2016) 9296

lated delphinidin derivatives as a result of F3 5 H catabolism except


for F3 5 H defective cultivars; however, D. cardinale and D. nudicaule accumulated pelargonidin derivatives that were not produced
by F3 H or F3 5 H catabolism (Saito et al., 1998; Miyagawa et al.,
2014). We also found that another wild delphinium species, D. zalil
(synonym semibarbatum), accumulated a quercetin 3-glucoside,
3 ,4 di-hydroxylated avonol glycoside, in their sepals instead of
anthocyanin. This observation led us to speculate that this species
had F3 H activity. Our recombinant enzyme assay showed that
DzF3 H cDNA encoded a functional F3 H with hydroxylase activity to convert 4 mono-hydroxyl avonoids to 3 , 4 di-hydroxyl
avonoids. Additionally because of a defect in F3 5 H and lack
of ANS gene expression, the sepals of D. zalil did not synthesize anthocyanin, resulting in the accumulation of kaempferol and
quercetin glucosides but not myricetin (3 , 4 , 5 tri-hydroxylated
avonoid). Therefore if an active ANS gene is introduced into D.
zalil, the 3 hydroxylated intermediates produced by DzF3 H might
be expected to undergo further metabolism and result in cyanidin synthesis. A breeding report from 1961 on delphinium hybrids
between D. zalil and D. cardinale remarked that they had dark
orange-red owers (Legro, 1961); obviously, it is not possible to
be certain of the molecular structure of the accumulated anthocyanins found in that study. However, we speculate that hybrids
had cyanidin biosynthesis ability following the combination of the
D. zalil genome with DzF3 H activity and the D. cardinale genome
that is defective for F3 5 H activity. The hybrid ower color might
be a consequence of the presence of both cyanidin and pelargonidin molecules, because our analysis here showed that DzF3 H does
not have high catalytic efciency characteristics. Currently, we are
attempting to cross-hybridize D. zalil with other Delphinium species
that lack F3 5 H activity in order to produce cyanidin accumulating
hybrids. Analysis of DzF3 H as a DNA marker and quantication of
anthocyanin molecular species in hybrids produced by crossing delphinium cultivars to D. zalil will enable the establishment of novel
delphinium varieties bearing vivid red owers as a consequence of
the synthesis and accumulation of cyanidin derivatives.

Acknowledgements
This work was supported by JSPS KAKENHI Grant-in-Aid for
Young Scientist (B) (16K18564) to T.M, and partly supported by
NARO to Y.O.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.jplph.2016.07.
013.
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