Emission mechanism of floral scent in Petunia axillaris.
ABSTRACT The mechanism of floral scent emission was studied in Petunia axillaris, a plant with a diurnal rhythm of scent output. The emission rate of each volatile compound oscillated in synchrony with its endogenous concentration, so that the intensity of the floral scent appeared to be determined by the endogenous concentrations. The composition of major volatiles in the flower tissue and the flower headspace showed characteristic differences. A negative correlation was found between the boiling points of the volatile compounds and the ratio of their emitted and endogenous concentrations, indicating that the composition of the floral scent depends directly on the endogenous composition of the volatile compounds. We conclude that in P. axillaris, the physiological regulation of floral scent emission operates not in the vaporization process but in the control of the endogenous concentrations of volatiles through biosynthesis and metabolic conversion.
- SourceAvailable from: ncbi.nlm.nih.govPlant physiology 04/2000; 122(3):627-33. · 6.56 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Emission of methyl benzoate, one of the most abundant scent compounds of bee-pollinated snapdragon flowers, occurs in a rhythmic manner, with maximum emission during the day, and coincides with the foraging activity of bumblebees. Rhythmic emission of methyl benzoate displays a "free-running" cycle in the absence of environmental cues (in continuous dark or continuous light), indicating the circadian nature of diurnal rhythmicity. Methyl benzoate is produced in upper and lower snapdragon petal lobes by enzymatic methylation of benzoic acid in the reaction catalyzed by S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase (BAMT). When a detailed time-course analysis of BAMT activity in upper and lower petal lobes during a 48-hr period was performed, high BAMT activity was found at night as well as in continuous darkness, indicating that the BAMT activity is not an oscillation-determining factor. Analysis of the level of benzoic acid during a 24-hr period revealed oscillations in the amount of benzoic acid during the daily light/dark cycle that were retained in continuous darkness. These data clearly show that the total amount of substrate (benzoic acid) in the cell is involved in the regulation of the rhythmic emission of methyl benzoate. Our results also suggest that similar molecular mechanisms are involved in the regulation of methyl benzoate production in diurnally (snapdragon) and nocturnally (tobacco and petunia) emitting plants.The Plant Cell 11/2001; 13(10):2333-47. · 9.25 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Petunia hybrida line W115 (Mitchell) has large white flowers that produce a pleasant fragrance. By applying solid phase micro extraction (SPME) techniques coupled to GC-MS analysis, volatile emission was monitored in vivo using a targeted metabolomics approach. Mature flowers released predominantly benzenoid compounds of which benzaldehyde, phenylacetaldehyde, methylbenzoate, phenylethylalcohol, iso-eugenol and benzylbenzoate were most abundant. This emission had a circadian rhythm reaching its maximum at dusk. During petal limb expansion two sesquiterpenes were emitted by the petunia flowers, tentatively identified as germacrene D and cadina-3,9-diene. In vitro analysis showed that the petal limbs and stigma were the main producers of the benzenoids and sesquiterpenes, respectively. Moreover, comparison of in vivo and in vitro analysis indicated that volatiles were not stored during periods of low emission but rather were synthesized de novo. DNA-microarray analysis revealed that genes of the pathways leading to the production of volatile benzenoids were upregulated late during the day, preceding the increase of volatile emission. RNA-gel blot analyses confirmed that the levels of phenylalanine ammonia lyase (PAL) and S-adenosyl methionine (SAM) synthase transcripts increased towards the evening. Our results suggest that the circadian production of volatile benzenoids in petunia W115 is, at least partly, regulated at the transcript level.Phytochemistry 04/2003; 62(6):997-1008. · 3.05 Impact Factor
Emission Mechanism of Floral Scent in Petunia axillaris
Naomi OYAMA-OKUBO,1;yToshio ANDO,2Naoharu WATANABE,3Eduardo MARCHESI,4
Kenichi UCHIDA,5and Masayoshi NAKAYAMA1
1Department of Genetics and Physiology, National Institute of Floricultural Science, National Agricultural
and Bio-oriented Research Organization, 2-1 Fujimoto, Tsukuba, Ibaraki 305-8519, Japan
2Faculty of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba 271-8510, Japan
3Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya,
Shizuoka 422-8529, Japan
4Facultad de Agronomia, Universidad de la Rep Blica, garz n 780, Montevideo, Uruguay
5School of Science and Engineering, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi 320-8551, Japan
Received November 12, 2004; Accepted December 15, 2004
The mechanism of floral scent emission was studied in
Petunia axillaris, a plant with a diurnal rhythm of scent
output. The emission rate of each volatile compound
oscillated in synchrony with its endogenous concentra-
tion, so that the intensity of the floral scent appeared to
be determined by the endogenous concentrations. The
composition of major volatiles in the flower tissue and
the flower headspace showed characteristic differences.
A negative correlation was found between the boiling
points of the volatile compounds and the ratio of their
emitted and endogenous concentrations, indicating that
the composition of the floral scent depends directly on
the endogenous composition of the volatile compounds.
We conclude that in P. axillaris, the physiological
regulation of floral scent emission operates not in the
vaporization process but in the control of the endoge-
nous concentrations of volatiles through biosynthesis
and metabolic conversion.
Key words: floral scent; Petunia axillaris; emission
Floral scents are mixtures of volatile compounds
including aromatics, terpenoids, and fatty acid deriva-
tives.1)The nature of the composition causes the
characteristic fragrance of a flower. Floral scents are
involved in the guidance of pollinators to the reproduc-
tive organs.2,3)They are also an important factor in the
attractiveness of ornamental plants. Moreover, some
flowers are used as resources for perfumes. Hence the
regulation of floral scents is an ecologically and
horticulturally important subject.
The scent compounds are secondary metabolites, as
are pigments and alkaloids. Scents emit through a
physical process, viz., vaporization of the volatile
compounds. This property distinguishes scent com-
pounds from other secondary metabolites. We must
understand the mechanisms of both production and
vaporization to regulate floral scent emission.
Recent physiological studies have advanced our
understanding of the mechanisms of floral scent pro-
duction. Several genes encoding enzymes involved in
the synthesis of floral scent compounds have been
identified.4)Plants start to emit floral scents after
anthesis. Some flowers possess the property of changing
their scent emission during the unfurling period.5–7)In
Clarkia breweri, enzyme activities and the emission of
some floral scent compounds are regulated on the
We are interested in the physiological regulation of
floral scent vaporization. The boiling points of almost all
volatile compounds identified as floral scents range from
150?C to 350?C. These substances are present in floral
tissues mostly as liquids or in solution. If the emission of
a particular compound is not regulated physiologically,
it should depend strictly on the endogenous concen-
tration of the compound. The vapor pressures, which
correspond to the ratio of emitted and endogenous
concentrations, should therefore be higher for lower
boiling point compounds than for higher boiling point
substances in the absence of regulatory mechanisms of
We analyzed endogenous and emitted concentrations
of floral scents in Petunia axillaris, one of the parental
species of horticultural Petunia cultivars.9)P. axillaris
emits floral scents in a diurnal rhythm that can be
controlled by light conditions in a growth chamber. We
recorded time-courses of emitted and endogenous con-
centrations of floral scent compounds. Correlations
between vapor pressures and boiling points of the
yTo whom correspondence should be addressed. Fax: +81-29-838-6841; E-mail: firstname.lastname@example.org
Abbreviations: GC–MS, capillary gas chromatography–mass spectrometry; HPLC, high-performance liquid chromatography; HOAc, acetic acid
Biosci. Biotechnol. Biochem., 69 (4), 773–777, 2005
compounds were also evaluated to determine whether
any physiological regulation of the vaporization process
Materials and Methods
Plant material. Plants of P. axillaris (Lam.) Britton,
Sterns et Poggenb. (Solanaceae) was raised from a seed
derived from a natural population in Montevideo,
Uruguay (accession code, U1), and was vegetatively
propagated in a greenhouse for 2 months. Before the
experiments, the plants were acclimated for more than a
week in a growth chamber at a constant temperature of
25?C under a photon flux density of 185mmol/m2/s
with a 12h/12h (6:00–18:00 light/18:00–6:00 dark)
Collection of volatiles. For qualitative analysis,
emitted volatile compounds were collected for 24h
(starting at 6:00) from 2-d-old flowers by the headspace
method.10)Flowers on P. axillaris plants were covered
with Tedlar Bags (GL science, 500ml volume). A
constant stream of air (approx. 500ml/min) filtered
through activated charcoal10)was piped through the bag,
and volatile compounds were collected by Tenax-TA
(150mg) traps. The relative humidity in the Tedlar Bag
was approx. 52%. For quantitative analysis, the emitted
compounds were collected for 1h (starting at 23:00)
from 2-d-old flowers; afterwards, the same flowers were
harvested. For determination of endogenous concentra-
tions, one flower was harvested every 6h starting at
6:00 of the first day after flower opening until 12:00 of
the fifth day. For analysis of emission rhythms, the
emitted compounds were collected every 4h from 6:00
of the first day after flower opening until 14:00 of the
Analysis of emitted volatiles. Scent compounds were
extracted from the Tenax-TA four times, using pentane
and diethyl ether (5ml each) alternately. After ethyl
decanoate (20mg) had been added as an internal stand-
ard, the extract was dried over anhydrous sodium sulfate
and concentrated at 36?C in a water bath.
Analysis of endogenous volatiles. Flower tissues
without the calyx were frozen in liquid nitrogen and
ground in a mortar. The ground powder was extracted
twice with pentane (5ml each) in a microwave oven
(700W) for 20s in the presence of anisole (20mg) as an
internal standard, as described previously.10)The extract
was dehydrated with anhydrous sodium sulfate and
concentrated at 36?C in the water bath. For analysis of
the time-courses of endogenous levels, harvested petal
limbs were investigated in the same way.
GC–MS and GC analysis. Capillary gas chromatog-
raphy–mass spectrometry (GC–MS) was performed
using a HP 5890 series II gas chromatograph coupled
to a HP 5989B mass spectrometer (Agilent Technolo-
gies, Wilmington, DE). The GC was equipped with a
splitless injector and a DB-WAX capillary column (30m
in length, 0.25mm i.d., 0.25mm film thickness). The
column oven program consisted of a first ramp of 3?C/
min from 60?C to 120?C, 120?C for 10min, followed
by a second ramp of 2.5?C/min from 120?C to 180?C,
and a third ramp of 3?C/min from 180?C to 230?C.
This temperature was then maintained for 10min.
Helium was applied as carrier gas at 50ml/min.
Injection, interface, and MS source temperatures were
250?C, 280?C, and 250?C respectively. GC analysis
was performed using an Agilent 6850A gas chromato-
graph (Agilent Technologies) monitored by FID. Ana-
lytical conditions were the same as with GC–MS.
HPLC analysis. High-performance liquid chromatog-
raphy (HPLC) was performed using an Agilent 1100
chromatograph (Agilent Technologies) with a Cadenza
CD-C18 column (2mm i.d. ? 250mm, Imtakt, Kyoto,
Japan). Conditions were as follows: column temper-
ature, 40?C; flow rate, 0.2ml/min; solvent A, acetoni-
trile with 0.1% acetic acid (HOAc); solvent B, distilled
water with 0.1% HOAc; gradient program: 25% to 35%
A from 0 to 25min, 35% to 40% A from 25 to 40min,
40% to 100% A from 40 to 45min, 100% A from 45 to
80min. Concentrations were quantified by measuring
absorbance at 210nm for iso-eugenol, at 230nm for
methyl benzoate, and at 254nm for benzaldehyde.
Emitted volatile compounds in the headspace
P. axillaris plants were grown in an incubator at a
constant temperature of 25?C under a 12-h photoperiod.
Nine aromatic compounds were identified by GC–MS
analysis in the headspace of the flowers: benzaldehyde,
benzyl alcohol, benzyl benzoate, iso-eugenol, methyl
benzoate, methyl salicylate, phenyl acetaldehyde, 2-
phenylethanol, and vanillin. The aromatics were quanti-
fied by GC–FID in the headspace for 1h starting at
23:00 on the second day. The main emitted compound
was methyl benzoate (75%, Table 1).
pounds in P. axillaris
Composition of Emitted and Endogenous Volatile Com-
3. Methyl benzoate
4. Benzyl alcohol
7. Benzyl benzoate
5:3 ? 0:9
9:1 ? 1:9
68:4 ? 10:9
1:0 ? 0:2
1:0 ? 0:3
5:5 ? 2:1
0:6 ? 0:3
3:0 ? 0:2
9:0 ? 1:5
77:1 ? 3:1
5:7 ? 1:1
13:0 ? 1:2
100:6 ? 10:7
77:4 ? 3:5
774 N. OYAMA-OKUBO et al.
Endogenous volatile compounds in flower tissue
Flowers used for quantitative analysis of emitted
volatiles were harvested immediately after headspace
sampling. Whole flower tissue without the calyx was
analyzed by GC–MS. The same aromatics as identified
in the headspace were detected, and were quantified by
GC–FID. The most abundant endogenous scent volatile
compounds were iso-eugenol (35%), benzyl benzoate
(27%), and methyl benzoate (27%, Table 1).
Time-courses of endogenous levels of volatile com-
The distribution ratios of the compounds in petal
limbs to whole flowers were as follows: benzaldehyde,
60%; phenyl acetaldehyde, 65%; methyl benzoate, 15%;
benzyl alcohol, 80%; 2-phenylethanol, 100%; iso-euge-
nol, 100%; benzyl benzoate, 100%. Thus most scent
compounds, except for methyl benzoate, occurred
predominantly in the petal limb. Verdonk et al.11)
reported similar results from P. hybrida cv Mitchell.
Hence we analyzed petal limbs only and discarded other
tissues to eliminate the possible effects of impurities on
the detection of low-level volatile compounds. The
endogenous concentrations of several scent compounds
were determined every 6h by HPLC.
P. axillaris flowers opened around 12:00 and scent
compounds were detected from 18:00 of the first day.
Figure 1 shows results for benzaldehyde (a), methyl
benzoate (b), and iso-eugenol (c) as representative
compounds. Generally, the endogenous levels of the
substances showed diurnal oscillations with maxima at
0:00 (midnight) and minima at 12:00 (noon) for 4d after
anthesis. These rhythms appeared particularly stable
after the second day. All scent compounds showed
similar time-courses. We found also that in the whole
flower the endogenous concentration of every compound
was higher at 0:00 than at 12:00 (data not shown).
Time-courses of emitted levels of volatile compounds
The time-courses of the emission of volatile sub-
stances were determined at higher resolution, since
samples were collected every 4h. Scent compounds
were detectable first in samples collected from 14:00 to
18:00 of the first day. Figure 2 shows the time-courses
for benzaldehyde (a), methyl benzoate (b), and iso-
eugenol (c) as representative compounds. The emitted
levels of each compound showed diurnal oscillations
with maxima at night and minima at noon for 4d after
anthesis. All scent compounds showed similar patterns.
Relationship between boiling points and emission
The composition of the volatile aromatic fraction
differed between the flower tissue and the headspace of
the flowers (Table 1). The floral tissue tended to contain
relatively high concentrations of compounds with higher
boiling points, while substances with lower boiling
points were present at increased levels in the headspace.
An analysis of the relationship between the logarithms
of the ratios of headspace to endogenous concentrations
and boiling points showed a nearly linear negative
correlation (Fig. 3).
The main scent compounds of P. axillaris were
aromatic substances, as has been described previously
for P. hybrida cv Mitchell.11)Endogenous concentra-
tions of all scent compounds studied correlated well
with the oscillating emission patterns (Figs. 1 and 2).
tive Volatile Compounds in the Petal Limb of P. axillaris Flowers
during the 4D after Flower Opening.
Measurements were taken at 6h intervals, and mean values ? SE
of three or more repetitions were plotted against the midpoints of
these intervals. a, Benzaldehyde; b, methyl benzoate; c, iso-eugenol.
Fresh weight of a petal limb was approximately 0.1g. SEs are
indicated by vertical bars.
Time-Courses of Endogenous Concentrations of Representa-
Emission Mechanism of Floral Scent775
The overall synchronization confirms that the emission
rate of volatiles is determined by their endogenous
concentrations. No physiological mechanism appears to
regulate the emission of volatile compounds from the
flowers of P. axillaris. The concentration of the emitted
mixture of volatiles, i.e., floral scent intensity, appears to
be determined by the endogenous concentrations in the
Compounds of lower boiling point were emitted at
higher relative rates in P. axillaris (Fig. 3), indicating a
purely physical regulation of scent composition. The
large differences between endogenous and emitted
compositions of floral scents (Table 1) are due to the
different vapor pressures of the substances, which
correspond to the different boiling points. The compo-
sition of the floral scent depends directly on the
endogenous composition of volatile compounds.
Differences in the scent compositions of floral tissues
and flower headspace appear to be common,12,13)which
can be explained by our conclusion, stated above. But
contrasting evidence has been found in flowers of Citrus
medica.13)In this species, the production of floral scent
appears to be not merely a matter of biosynthesis and
evaporation but rather a cytologically organized excre-
tory process. Thus diverse regulatory mechanisms of
floral scent emission might exist in plants.
The volatile substances identified in P. axillaris
flowers are biosynthesised from a common precursor,
phenylalanine.4)Close quantitative correlation between
the contents of methyl benzoate and its precursor,
benzoic acid, has been reported in P. hybrida cv
Mitchell as well as in Antirrhinum majus and Nicotiana
suaveolens.7)Similarly, the endogenous levels of other
volatile substances in P. axillaris flowers might also be
regulated by the activity of their biosynthetic pathways.
Interestingly, the endogenous concentrations of all scent
compounds decreased to almost zero at 12:00, irrespec-
tive of their maximum levels at 0:00 and their vapor
pressures (Figs. 2 and 3). Metabolic conversion to other
compounds might be responsible for this decline. Thus
the endogenous concentrations of the compounds are
probably regulated by both biosynthesis and conversion,
resulting in synchronized changes.
We thank Mr. Y. Kashimura for assistance with plant
growth, and Dr. N. Yanagihara for helpful discussions.
This work was supported by a Grant-in-Aid for
Scientific Research from the Ministry of Education,
Culture, Sports, Science, and Technology of the Japa-
nese Government (grant no. 14656050).
the Ratios of the Emitted to Endogenous Concentrations of Various
1, Benzaldehyde (bp 179?C); 2, phenyl acetaldehyde (bp 195?C);
3, methyl benzoate (bp 200?C); 4, benzyl alcohol (bp 205?C); 5, 2-
phenylethanol (bp 221?C); 6, iso-eugenol (bp 266?C); 7, benzyl
benzoate (bp 324?C). SEs are indicated by vertical bars.
Relationship of Boiling Points and the Natural Logarithms of
Compounds from P. axillaris Flowers during the 4D after Flower
values ? SE of three or more repetitions were plotted against the
midpoints of these intervals. a, Benzaldehyde; b, methyl benzoate; c,
iso-eugenol. SEs are indicated by vertical bars.
Time-Courses of the Emission of Representative Volatile
over4h periods, andmean
776N. OYAMA-OKUBO et al.
1)Knudsen, J. T., Tollsten, L., and Bergstro ¨m, L. G., Floral
scents: a checklist of volatile compounds isolated by
headspace techniques. Phytochemistry, 33, 253–280
Dobson, H. E. M., Floral volatiles in insect biology. In
‘‘Insect Plant Interactions’’ Vol. 5, ed. Bernays, E. A.,
CRC Press, Boca Raton, pp. 47–81 (1994).
Dobson, H. E. M., and Bergstro ¨m, G., The ecology and
evolution of pollen odors. Plant Systematics and Evolu-
tion, 222, 63–87 (2000).
Dudareva, N., Molecular control of floral fragrance. In
‘‘Breeding of Ornamentals: Classical and Molecular
Approaches’’, ed. Vainstein, A., Kluwer Academic
Publishers, Netherlands, pp. 295–309 (2002).
Loughrin, J. H., Hamilton-Kemp, T. R., Andersen, R. A.,
and Hildebrand, D. F., Circadian rhythm of volatile
emission from flowers of Nicotiana sylvestris and
N. suaveolens. Physiol. Plant., 83, 492–496 (1991).
Helsper, J. P. F. G., Davies, J. A., Bouwmeester, H. J.,
Krol, A. F., and van Kampen, M. H., Circadian
rhythmicity in emission of volatile compounds by
flowers of Rosa hybrida L. cv. Honesty. Planta, 207,
Kolosova, N., Gorenstein, N., Kish, C. M., and
Dudareva, N., Regulation of circadian methyl benzoate
emission in diurnally and nocturnally emitting plants.
Plant Cell, 13, 2333–2347 (2001).
Dudareva, N., and Pichersky, E., Biochemical and
molecular genetic aspects of floral scents. Plant Physiol.,
122, 627–633 (2000).
Ando, T., Nomura, M., Tsukahara, J., Watanabe, H.,
Kokubun, H., Tsukamoto, T., Hashimoto, G., Marchesi,
E., and Kitching, I. J., Reproductive isolation in a native
population of Petunia sensu Jussieu (Solanaceae). Ann.
Bot., 88, 403–413 (2001).
Oka, N., Ohishi, H., Hatano, T., Hornberger, M., Sakata,
K., and Watanabe, N., Aroma evolution during flower
opening in Rosa damascena Mill. Z. Naturforsch., 54c,
Verdonk, J. C., Ric de Vos, C. H., Verhoeven, H. A.,
Haring, M. A., van Tunen, A. J., and Schuurink, R. C.,
Regulation of floral scent production in petunia revealed
by targeted metabolomics. Phytochemistry, 62, 997–
Lewis, J. A., Moore, C. J., Fletcher, M. T., Drew, R. A.,
and Kiching, W., Volatile compounds from the flowers
of Spathiphyllum cannaefolium. Phytochemistry, 27,
Altenburger, R., and Martile, P., Further observations on
rhythmic emission of fragrance in flowers. Planta, 180,
Emission Mechanism of Floral Scent777