Synergy effect of sodium acetate and glycosidically bound volatiles on the release of volatile compounds from the unscented cut flower (Delphinium elatum L. "Blue Bird")

Article abstract of DOI:10.1021/jf901176m

Many modern floricultural varieties have lost their scent during traditional breeding programs. The factors that result in the nonscent emission of some cut flowers remain unclear. The objective of this study was to investigate whether the nonscent emissi

Full text of DOI:10.1021/jf901176m

6396 J. Agric. Food Chem. 2009, 57, 6396–6401
DOI:10.1021/jf901176m
Synergy Effect of Sodium Acetate and Glycosidically Bound
Volatiles on the Release of Volatile Compounds from the
Unscented Cut Flower (Delphinium elatum L. “Blue Bird”)
†,§
Z
IYIN
Y
ANG
,
†,‡ SAKURA
E
NDO,‡ AYA
T
ANIDA,‡ KENJI
,†,‡
KAI
,
AND
NAOHARU
W
ATANABE
*
†Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka
422-8529, Japan, and ‡Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka
422-8529, Japan. § Present address: Graduate School of Life and Environmental Sciences, Osaka
Prefecture University, 1-1 Gakuencho, Naka-ku, Sakai, Osaka 599-8531, Japan
Many modern floricultural varieties have lost their scent during traditional breeding programs. The
factors that result in the nonscent emission of some cut flowers remain unclear. The objective of this
study was to investigate whether the nonscent emission is due to one of the factors, the lack of
suitable substrates (or precursors of scent compounds). Using solid-phase microextraction and
dynamic headspace volatile sampling techniques, the supplement of nonvolatile compounds such as
2-coumaric acid glucoside to the unscented cut flower such as Delphinium elatum L. “Blue Bird”
enhanced the emission of scent from the flower, which was sufficient for detection by the human
olfaction. Interestingly, compared with feeding with each compound, the combination of sodium
acetate and 2-coumaric acid glucoside showed the synergy effect on enhancement of coumarin, a
cherry leaf-like scent emission from the flower, which is due to one of factors that sodium acetate
enhanced the activity of β-glucosidase being involved in the formation of the scent compound.
These results suggest that some enzymes responsible for the formation of floral scents indeed
occur in the unscented flowers such as Delphinium elatum L. “Blue Bird”, and the non- or low-scent
emission of the flowers are due to the lack of suitable substrates.
KEYWORDS: Delphinium elatum L. “Blue Bird”; sodium acetate; 2-coumaric acid glucoside;
2-phenylethyl glucoside; headspace; scent
INTRODUCTION
unscented cut flowers, and the flower that achieves in the olfactory
detectable enhancement was screened out. This study also focused
on the effect of the precursors of volatile compounds supplied to the
flower on the floral scent emission and the related floral scent
emission mechanism. In addition, sodium acetate may enhance
availability of acetyl coenzyme A (acetyl-CoA) that is an important
precursor of secondary metabolites formed from acetate path-
way (5). Therefore, sodium acetate was used for clarifying its effect
on the scent emission. The information obtained from this study
would advance our understanding of production and emission of
secondary volatile compounds and also contribute to the metabolic
engineering to modify floral scent.
In nature, floral scent plays an important role in attrac-
ting pollinators, thereby ensuring plant reproductive and evolu-
tionary success (1). Also, humans have always been fascinated by
floral scent because of their aesthetic, emotional, and economic
values (2). Unfortunately, floral scent always has been lost during
traditional breeding programs for the cut flower market and
ornamental plants because of a negative correlation between
flower longevity and fragrance (1, 3).
Recently, some metabolic engineering has been made to improve
scent quality of flowers. The most often used attempt to engineer
scent, for example, the genetic engineering approach, has mainly
concentrated on the introduction of the “scent genes” responsible
for the final steps of the formation of volatiles, which lead to the
successful and not-so-successful changes in floral scent bouquet (4).
However, little attention has been given to the role of substrates
(or precursors of volatiles) in regulating the scent formation. To
investigate whether the non- or low-scent emission of some cut
flowers are due to the lack of suitable substrates, in the present study,
several precursors of volatile compounds were supplied to the
MATERIALS AND METHODS
Experimental Design. This research mostly focused on the precursors
of phenylpropanoid/benzenoid volatiles, such as shikimic acid (purchased
from Sigma Chemical Co. USA),
(purchased from Wako Pure Chemical Industries, Ltd., Japan), 2-pheny-
lethyl R,β- -glucoside (2PEG) (a gift from T. Hasegawa Co., Ltd., Japan),
L-phenylalanine, trans-cinnamic acid
D
and 2-coumaric acid glucoside (chemically synthesized by the authors).
The unscented cut flowers (Rosa “Rote Rose”, Gerbera jamesonii
“Jaguar Deep Rose”, Dianthus caryophyllus “Barbara”, and Delphinium
elatum L. “Blue Bird”) and the flowers with unpleasant odors (Eustoma
grandiflorum “Forever Blue”, Gypsophila elegans “Covent Garden”, and
*To whom correspondence should be addressed. Phone: þ8154-
2384870. Fax: þ8154-2384870. E-mail: acnwata@agr.shizuoka.ac.jp.
pubs.acs.org/JAFC
Published on Web 06/22/2009
© 2009 American Chemical Society

Article
J. Agric. Food Chem., Vol. 57, No. 14, 2009 6397
ddd, J=1.6 and 7.9 Hz. H-4), 7.65 (1H, dd, J=1.6 and 7.9 Hz. H-6), 7.9 (1H, d,
J=16.1 Hz. H-30).
Determination of Volatile Compounds by Gas Chromatogra-
phy-Mass Spectrometry (GC-MS). Analyses of volatile compounds,
2-phenylethanol and coumarin, were performed using a GC-MS QP5050
(Shimadzu), which was controlled by a Class-5000 workstation. For the
determination of volatile compounds absorbed in Tenax TA, a TurboMa-
trix Automated Thermal Desorber (PerkinElmer instruments) was con-
nected to GC. Optimized operating conditions were: desorption
temperature 250 °C, desorption time 10 min, 0.5-10% to the GC column.
The GC-MS condition was described in our previous study (7), with a
modification. The GC was equipped with a capillary TC-5 column
(GL Sciences Inc., Japan), 30 m ꢀ 0.25 mm I.D., and 0.25 μm film
thickness. Helium was used as a carrier gas at a flow rate of 1.5 mL/
min. The injector temperature was 230 °C. The GC oven was maintained
at 60 °C for 3 min. The temperature of the oven was programmed at 15 °C/
min to 110 °C and then at 40 °C/min to 290 °C and kept at this temperature
for 3 min. The mass spectrometer was operated by the full scan mode(mass
range m/z 60-200) or by the selected ion monitoring (SIM) mode for
quantitative analysis (m/z 122, 91 for 2-phenylethanol; m/z 146, 118 for
coumarin).
To determine the internal pool sizes of volatile compound (coumarin)
in the flowers, tissue (0.3 g fresh weight) was ground in liquid nitrogen and
extracted in 2.5 mL of an azeotropic mixture of pentane-dichloromethane
(2:1 v/v) for 16 h under dark and afterward filtered through a short plug of
anhydrous sodium sulfate. One μL of the filtrate was obtained and
subjected to GC-MS analyses. The GC-MS conditions were described as
above. [5,6,7,8-2H4]-Coumarin was used as an internal standard.
Preparation of Cell-Free Extracts and β-Glucosidase Assay. Cell-
free extracts were prepared as described previously (8) with modifica-
tions. Delphinium flower powder (300 mg) ground by liquid nitrogen
was homogenized for 5 min at 0 °C in a mixture of 10 mL of buffer A
(0.1 M potassium phosphate at pH 7.5, containing 0.5% 3-[(3-cholami-
dopropyl)-dimethylamino]-1-propanesulfonate (CHAPS), 2 mM dithio-
Figure 1. Solid phase microextraction (SPME) technique (A) and dynamic
headspace volatile sampling technique (B).
Tulipa “Greenland”) were used in this study. These cut flowers almost at
the midopen stages were purchased from the flower shops in Shizuoka,
Japan, and grown in the incubator under the conditions of 12 h light/12 h
dark photoperiod, 70% humidity, and 20-22 °C. The flowers were kept
for 2-3 days in tap water prior to use. Afterward, the flowers were
independently placed in the following solutions: (1) water (as control),
(2) 15 mM precursors of volatile compounds in water, (3) 10 mM sodium
acetate in water, and (4) 15 mM precursors of volatile compounds and
10 mM sodium acetate in water. After 20 h treatments, the headspace
volatiles were collected using two different techniques, solid phase micro-
extraction (SPME) (Figure 1A, 8 h/sampling for 1 flower) and dynamic
headspace volatile sampling (Figure 1B, 12 h/sampling for 1 flower). The
sensory evaluation was carried out by three persons every 6 h in nighttime
and every 2-4 h in daytime. The significant olfactory effect of 2-coumaric
acid glucoside on Delphinium elatum L. “Blue Bird” was confirmed by the
three persons who joined the sensory evaluation and another five persons
who were invited.
threitol, 5% glycerol, and
1 mM ethylenediaminetetraacetic acid
(EDTA)), 50 μL of 0.1 M 4-(2-Aminoethyl) benzenesulfonyl fluoride
hydrochloride, and 600 mg of polyvinyl polypyrrolidone. After centrifu-
ging at 4000g for 20 min at 4 °C, the supernatant was desalted on a PD-10
column that had been equilibrated with buffer B (0.01 M potassium
phosphate at pH 7.5, containing 0.05% CHAPS, 5% glycerol, and 1 mM
EDTA) to give crude β-glucosidase solution.
The β-glucosidase activity was determined in a standard assay. The
reaction mixture consisting 150 μL of enzyme solution, 150 μL of buffer B,
150 μL of 10 mM p-nitrophenyl β-glucoside, and 300 μL of 50 mM
citric acid buffer (pH 6.0) was incubated for 15 min at 30 °C. The reaction
was quenched by adding 750 μL of 1 mM Na2CO3. The amount of
p-nitrophenyl released was measured by the absorbance at 405 nm. One
unit of β-glucosidase was defined as the amount of enzyme releasing
0.01 μmol of p-nitrophenyl per min under the above conditions.
Statistical Analysis. Data are expressed as mean(standard error.
Student’s t test was used to estimate significance for comparisons.
A probability level of 5% (p < 0.05) was considered significant.
Synthesis of 2-Coumaric Acid Glucoside. 2-Coumaric acid glucoside
was synthesized from 2-hydroxycinnamic acid according to the literature (6)
with modifications (Figure 2). 2-Hydroxycinnamic acid (2 g, 12.1 mmol)
was dissolved in 2 mL of MeOH, and TMSCl (2.3 mL, 18.1 mmol) was
dropped in the solution during 5 min at 0 °C. After 4-6 h stirring under
ice-cooling, 1 mL of MeOH was added and stirred overnight at room
temperature. Afterward, the solution was evaporated to dryness to obtain
compound 2 (2.15 g, 12.0 mmol, 99.5%). Compound 2 (100 mg, 0.56 mmol),
2,3,4,6-tetra-O-acetylglucopylanosyl bromide (692 mg, 1.68 mmol), and
Cs2CO3 (182 mg, 0.56 mmol) were dissolved in dry MeCN (10 mL) and
stirred for 72 h at room temperature. The solution was diluted to 50 mL
with CH2Cl2, washed with brine (2ꢀ50 mL), dried over Na2SO4, and
concentrated. The residue was purified using Wakogel C-200 chromatogra-
phy with 0f40% ethyl acetate in hexane to obtain compound 3 (187 mg,
0.37 mmol, 65.7%). To a solution of 3 (172 mg, 0.34 mmol) in 5 mL of THF,
NaOH (2 M, 5 mL) was added and stirred for 2.5 h at room temperature.
Afterward, the solution was acidified with HCl (2 M, 4.5 mL) and evaporated
to dryness. The residue was purified by a cartridge C18 column (50 mm
idꢀ100 mm, Merck) to obtain 2-coumaric acid glucoside (64 mg, 0.19 mmol,
57.8%). 1H NMR (270 MHz, DMSO): δ 3.16-3.41 (m, H-200, H-300, H-400,
H-500), 3.46 (1H, dd, J=11.5 and 5.6 Hz, H-600b), 3.68 (1H, dd, J=11.5 and
1.9 Hz, H-600a), 4.97 (1H, d, J=7.2 Hz, H-100), 6.51 (1H, d, J=16.1 Hz, H-20),
7.02 (1H, t, J=7.9 and 7.9 Hz, H-5), 7.18 (1H, d, J=7.9 Hz, H-3), 7.35 (1H,
RESULTS AND DISCUSSION
Olfactory Effects of the Precursors Feeding on the Floral Scent
Emission. To know whether newly introduced “precursors” could
find appropriate scent enzymes and the intended products
could be produced and emitted at levels that could be detected
by humans, several precursors of volatile compounds were
supplied to the different flowers. Because the first committed
step in the pathways of some volatile phenylpropanoid and
benzenoid compounds derived from shikimic acid is the conver-
sion of
L-phenylalanine to trans-cinnamic acid (2), this allowed us
to use the three compounds, i.e., shikimic acid,
L-phenylalanine,
and trans-cinnamic acid as candidates for the feeding experiment.
Also, the important precursors of volatiles such as glycosidically
bound volatile compounds were applied to the feeding experi-
ment. The olfactory detectable enhancement of volatiles emitted
from the flowers was only achieved in the Delphinium elatum L.

6398 J. Agric. Food Chem., Vol. 57, No. 14, 2009
Yang et al.
Figure 2. Synthesis of 2-coumaric acid glucoside.
Table 1. Sensory Evaluation of the Flowers Supplied with the Precursors of Volatile Compoundsa
Rosa “Rote Eustoma grandiflorum
“Forever Blue”
Gerbera jamesonii “Jaguar Dianthus caryophyllus Gypsophila elegans “Covent Tulipa
“Greenland”
Delphinium elatum
“Blue Bird”
Rose”
Deep Rose”
“Barbara”
Garden”
2PEG
2-coumaric
acid
þ
þ
-
-
-
-
-
þ
V
V
-
þ
-
þþ
glucoside
trans-
-
-
-
-
-
-
-
cinnamic
acid
L-phenylalanine
shikimic acid
þ
-
-
þ
þ
-
-
-
-
-
-
-
-
-
a -, No olfactory effect; V, Slight decrease in unpleasant odor; þ, Very slight effect; þþ, Significant olfactory effect. The precursors (10-30 mM) and sodium acetate (10 mM)
were supplied to the flowers for 1 to 3 days.
“Blue Bird” supplied with 2-coumaric acid glucoside, while
other attempts to modify the scent bouquet were less successful
(Table 1). These could be due to several factors, for example, the
absence of suitable enzymes for the introduced reaction, substrate
specificity of scent enzymes, modification of the scent compound
into a nonvolatile form, insufficient levels of emitted volatiles for
olfactory detection by humans (or comparatively high olfactory
threshold of produced volatiles), or masking of introduced
compounds by other volatiles (4).
To evaluate whether 2-coumaric acid glucoside also could
influence scent emission of other cultivars of delphinium flowers,
Delphinium grandiflorum “Blue Butterfly” and Delphinium grand-
iflorum “Summer Morning” were used. Although amount of
coumarin emitted from the flowers slightly increased after treat-
ment with 2-coumaric acid glucoside, it had no significant
olfactory effect on the both delphinium flowers.
On the basis of the olfactory detectable effect, in this work, the
Delphinium elatum L. “Blue Bird” was used as a model to further
study the emission mechanism of volatile compounds from the
flowers supplied with the precursors.
Effects of 2PEG and 2-Coumaric Acid Glucoside Feeding on
the Release of Volatile Compounds from Delphinium elatum L.
“Blue Bird”. In rose flowers, glycosides like 2-phenylethyl
β-D-glucoside are stored inside the petals and can act as primary
Figure 3. GC chromatogram (A) and mass spectrum (B) of coumarin
released from the Delphinium elatum L. “Blue Bird” flowers placed in water
(control, real line) and the mixed solution of 15 mM 2-coumaric acid
glucoside and 10 mM sodium acetate (dashed line), respectively. After 20 h
of treatment, a solid phase microextraction (SPME) technique was
employed to collect the volatiles emitted from the flower for 8 h.
source of rhythmically emitted volatiles such as 2-phenylethanol,
being released by the action of the β-glucosidase throughout
the photoperiod (9). In the present work, using SPME technique
(Figure 1A), 2-coumaric acid glucoside feeding resulted in the
formation of coumarin in the Delphinium elatum L. “Blue Bird”
(Figure 3A,B). Furthermore, 2-coumaric acid glucoside and
2PEG feeding enhanced the rhythmic emission of coumarin
and 2-phenylethanol (Figure 4A,B) by the dynamic head-
space volatile sampling analysis (Figure 1B). This indicates
that some “scent enzymes” occur in some unscented flowers
such as Delphinium elatum L. “Blue Bird”, and the non- or low-
scent emission of the flowers may be due to the lack of sui-
table substrates (or precursors of volatiles). However, in
contrast to the produced coumarin, the formed 2-phenylethanol
had no effect on the olfactory properties of the delphinium
flower. Compared with olfactory threshold of coumarin
(2.3ꢀ10-8 ppm in the air) (10), 2-phenyethanol shows compara-
tively high olfactory threshold (10 ppm in the air) (11). Indeed, a
successful modification of the floral scent bouquet is not only to
increase or change scent production. The reaction of human and
insect olfactory system to the produced scents also greatly contri-
butes to target selection. At present, success or non-
success in metabolic engineering of floral scent are always assessed
on the basis of sensory evaluations by humans, whose odor

Article
J. Agric. Food Chem., Vol. 57, No. 14, 2009 6399
Figure 5. Rhythmic emission and internal pool of coumarin in the Delphi-
nium elatum L. “Blue Bird” flowers supplied with 2-coumaric acid glucoside
(A) and proposed pathway of coumarin formation in the plants (B, refer to
ref5). (A) The flowers were placed in the mixture of 15 mM 2-coumaric acid
glucoside and 10 mM sodium acetate aqueous solution. Data are expressed
as mean (standard error (n = 3). After 20 h of treatment, the flowers
collected at 07:00 and 19:00 were used to the determination of internal pool.
Figure 4. Effects of 2-coumaric acid glucoside (A) and 2PEG (B) on
the emission of coumarinand2-phenylethanol from theDelphinium elatum
L. “Blue Bird” flowers, respectively. The flowers were placed in water
(control), the mixture of 15 mM 2-coumaric acid glucoside or 2PEG, and 10
mM sodium acetate aqueous solution, respectively. Data are expressed as
mean ( standard error (n = 3). After 20 h of treatment, a dynamic
headspace volatile sampling technique was used to collect the volatiles
emitted from the flower.
shows increases in the daytime and decreases in the nighttime,
suggesting that the synthesis of coumarin is regulated by light.
In the metabolic pathway leading from 2-coumaric acid gluco-
side to coumarin (Figure 5B, ref 5), light can facilitate the
isomerization of trans-O-hydroxycinnamic acid and thereby for-
mation of coumarin. In addition, the coumarin exhibits rhythmic
emission with a peak during the light period, which is in
accordance with the change in internal pool of synthesized
coumarin (Figure 5A). In nature, nocturnally pollinated
flowers generally tend to have a maximum of scent emission
during the dark period, which is controlled in most case by
the endogenous circadian clock. In contrast, for diurnal pollinate
flowers, the situation is reversed, which is controlled in most
case directly by light (16,17). Recent studies also provide evidence
that, in many cases, the scent emission regulation either by
endogenous circadian clock or by light is controlled at the level
of gene expression responsible for the scent formation (18).
However, in some cases, the availability of substrates for
the final step of scent formation in flowers determines the effi-
ciency of their emission (19). For example, the rhythmic emission
of geranyl acetate in hybrid rose flowers is regulated at the level of
its substrate geraniol that can be controlled by light (18).
threshold perception is much lower than that of most animals and
insects (2, 12). However, little is known about the impact of
changes in the scent bouquet on insect and animal attraction (2).
Because coumarin was found to induce liver toxicity (13),
the European Union Scientific Committee for Food set the
maximum limits for coumarin of 2 mg/kg in foodstuffs and
beverages with specific exceptions of 10 mg/kg in “certain”
caramel confectionery and in alcoholic beverages (14). In the
assessment of the safety of coumarin for human health, as
discussed by Lake (15), the maximum daily human exposure to
coumarin for a 60 kg consumer has been estimated to be 3524 μg
coumarin/day (or 58.73 μg/kg body weight/day). Additionally,
no adverse effects of coumarin have been reported in susceptible
species in response to doses that are more than 100 times the
maximum human daily intake (15). In this work, after chemical
treatment, coumarin emitted from one flower ranges from 0.082
to 0.42 μg within 12 h (Figure 4A). It suggests that exposure
to coumarin from a reasonable amount of the flowers depicted
in this paper poses no health risk to humans.
Regulation of Emission of Scent Compound from the Delphinium
Flower by the 2-Coumaric Acid Glucoside Feeding. In the
Delphinium elatum L. “Blue Bird” flower without any treat-
ment (control), the internal pool of coumarin varied from
0.35 to 0.86 nmol/g fresh weight during the 12 h light/dark
photoperiod. After treatment with 2-coumaric acid glucoside,
the internal pool of coumarin was significantly increased
(3.4-83.4 nmol/g fresh weight) (Figure 5A). Moreover, it
Our results demonstrate that changes in emission of coumarin
from the flower coincided with the synthesized coumarin. How-
ever, because we know very little about scent emission, it remains
to be determined whether coumarin is immediately emitted to the
atmosphere instead of accumulating in the cells once the com-
pound is synthesized, or when a certain threshold level of the
compound is reached, flowers start a self-protection mechanism

6400 J. Agric. Food Chem., Vol. 57, No. 14, 2009
Yang et al.
to be partly responsible for controlling the diurnal emission of 2-
phenylethanol in the Rosa damascena (20) and play an important
role in the emission of the floral scent compounds (8), although
attempts to correlate glucosidase activity with rhythmic cycles in
volatile emission from rose flowers were unsuccessful (21). It will
be interesting to determine how sodium acetate influenced the
activity of β-glucosidase. In addition, it remains to be determined
whether this synergy effect of sodium acetate could be due to
other possibilities. Acetate is the most linking between primary
metabolism and secondary metabolism and occupies a central
position in relation to the general metabolism of plants (22). Two
entirely separate secondary biosynthetic routes, isoprenoids (i.e.,
terpenes, steroids, and carotenoids) and acetogenins (i.e., poly-
acetylenes, fatty acids, and polyketides) originate with acetate (5).
Also, acetate condensation occurs in many possible routes, which
give rise to variety of aromatic compounds. For example,
isocoumarins, which are structurally related to the coumarins
but with an inverted lactone ring, can derive from the acetate
pathway (23). However, in the present work, feeding of sodium
acetate itself did not significantly promote scent emission from
the flower. It may be that sodium acetate or the activated
intermediate acetyl-CoA become precursors of intermediates of
carbohydrates, resulting in the activation of carbohydrate meta-
bolism, thereby glycosidase and/or glycosyl transferase were
activated to hydrolyze coumaric acid glucoside.
The loss of scent emission in flowers that can be due to the
mutation or loss of the relevant biosynthesis genes was recently
confirmed in flowers of petunia (Petunia axillaris subsp. paro-
dii) (24). Isoeugenol synthase gene in Petunia axillaris subsp.
Parodii contains a frame-shift mutation that renders it inactive,
which leads to a decrease in isoeugenol biosynthesis. In addition,
when isoeugenol synthase activity is reduced in Petunia hybrid,
the coniferyl acetate substrate that would have been used by
isoeugenol synthase is instead used by eugenol synthase, thereby
the flowers synthesize higher levels of eugenol.
Our data shows that the supplement of nonvolatile compounds
such as 2-coumaric acid glucoside to Delphinium elatum L. “Blue
Bird” enhanced the emission of scent from the flower, indicating
that the non- or low-scent of the flower also can be caused by the
lack of suitable substrates. This result helps us with the better
understanding of the mechanism formation in unscented flowers.
Moreover, it suggests that metabolic engineering of the floral scent
spectrum requires a more rational design based on the correct
choice of species and complete understanding of metabolic path-
ways. In this work, it is interesting that the addition of sodium
acetate has a synergetic effect on the release of coumarin from
2-coumaric acid glucoside, which is due to one of the factors that
sodium acetate influenced the glucosidase activity. Some undesir-
able results obtained during the efforts to modify plant volatile
profiles demonstrate that the complexity of the biosynthetic net-
works for plant volatiles. Therefore some combined strategies, such
as enhancement of scent precursors and influence on the involved
“scent enzyme”, will help us to achieve desirable scent emission.
Figure 6. Effects of 2-coumaric acid glucoside individual feeding and
synergy feeding with sodium acetate or sodium phosphate on the emission
ofcoumarinfromtheDelphiniumelatumL. “BlueBird”flowers(A)andeffect
of sodium acetate on the activity of β-glucosidase of the delphinium flowers
(B) . (A) The flowers were placed in water (control), 10 mM sodium acetate
aqueous solution, 15 mM 2-coumaric acid glucoside aqueous solution, the
mixture (pH 4.4) of 15 mM 2-coumaric acid glucoside and 10 mM sodium
acetate aqueous solution, and the mixture (pH 4.4) of 15 mM 2-coumaric
acid glucoside and a certain amount of sodium phosphate (NaH2PO4 for
the pH adjustment) aqueous solution, respectively. Data are expressed as
mean(standarderror(n=3). After20h of treatment, a dynamic headspace
sampling technique was used to collect the volatiles emitted from the
flower. (B) The delphinium flowers were treated with water (control) and
10 mM sodium acetate at 22 °C under dark condition for 0, 8, and 48 h,
respectively. Means are significantly different by Student’s t test (p < 0.05).
Data represent the mean ( standard error (n = 5).
to emit it in case that this much accumulation can be toxic to
plant cells.
Synergy Effects of 2-Coumaric Acid Glucoside and Sodium
Acetate on the Release of Coumarin from the Delphinium Flower.
In the present study, the mixed feedings of 2-coumaric acid
glucoside and sodium acetate (or sodium phosphate) were used
from the delphinium flower. Interestingly, compared with the
individual feeding, the combination of 2-coumaric acid glucoside
with sodium acetate showed the synergy effect on enhancement
of coumarin release from the flower, while the combination
of 2-coumaric acid glucoside with sodium phosphate showed
no significant effect (Figure 6A). To elucidate the contribution
of sodium acetate to this synergy effect, the effect of sodium
acetate on the activity of β-glucosidase of the delphinium flowers
was examined in this study. As shown in Figure 6B, the activity
of β-glucosidase of the flower was significantly increased after the
48 h treatment with sodium acetate. β-Glucosidase was suggested
ABBREVIATIONS USED
CHAPS, 3-[(3-cholamidopropyl)-dimethylamino]-1-propane-
sulfonate; EDTA, ethylenediaminetetraacetic acid; GC-MS,
gas chromatography-mass spectrometry; 2PEG, 2-phenylethyl
R,β-D-glucoside; SPME, solid phase microextraction.
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Received April 8, 2009. Revised manuscript received June 10, 2009.
Accepted June 14, 2009. This work was supported by grant-aid to N.W.
from Research for Promoting Technological Seeds, Discovery Type.