全 文 :西北农业学报 2016,25(5):763-769
Acta Agriculturae Boreali-occidentalis Sinica doi:10.7606/j.issn.1004-1389.2016.05.018
网络出版日期:2016-05-05
网络出版地址:http://www.cnki.net/kcms/detail/61.1220.S.20160505.1558.036.html
Diurnal Changes of Monoterpenes Emission and
Li-mTPS Expression in Lilium ‘siberia’
LI Tianjiao1,HU Zenghui 1,ZHENG Jian1,LENG Pingsheng1 and YANG Kai 2
(1.Colege of Landscape Architecture,Beijing University of Agriculture/Beijing Engineering Research Center of Rural
Landscape Planning and Design/Beijing Laboratory of Urban and Rural Ecological Environment,Beijing 102206,China;
2.Colege of Plant Science and Technology,Beijing University of Agriculture,Beijing 102206,China)
Abstract Monoterpenes are an important category of volatile compounds in the floral scent.In this
study,using dynamic headspace method,the floral scent emitted fromLilium ‘siberia’flowers was
colected at different time points(7:30,9:30,11:30,13:30,15:30,17:30,19:30)during the daytime,
then the components and release amounts were identified and analyzed by the automated thermal
desorption-gas chromatography/mass spectrometry(ATD-GC/MS)technique.We found that release
amounts of monoterpenes first increased and then decreased during the daytime,and a high level
occurred at 11:30,13:30,and 15:30.The high release amounts of ocimene,linalool,andβ-myrcene
indicated that they were the dominant monoterpenes in the floral scent of Lilium ‘siberia’.In the fol-
lowing study,the daytime expression of Li-mTPS (aLilium monoterpene synthase gene)in tepals
was examined.LimTPSexpression showed a similar pattern with monoterpene emission.The results
showed that the Li-mTPS expression may play a key role in monoterpene emission from Lilium
‘siberia’flowers,but the mechanism needs to be investigated in the future study.
Key words Lilium ‘siberia’;Floral scent;Monoterpene;Li-mTPSexpression
CLC number S682.2 Document code A Article ID 1004-1389(2016)05-0763-07
Received 2015-04-20 Returned 2015-06-02
Foundation item The Project of Construction of Innovative Teams and Teacher Career Development for Universities and Coleges
under Beijing Municipality(No.IDHT20150503);National Natural Science Foundation of China(No.31071817);
Science and Technology Project of Beijing Municipal Education Commission(No.PM2014014207);Building Pro-
ject of Beijing Laboratory of Urban and Rural Ecological Environment(No.PXM2015-014207-000014).
First author LI Tianjiao,female,master student.Research area:ecophysiology of landscape plants.E-mail litianjiao1231988@
163.com
Corresponding author LENG Pingsheng,male,professor,master supervisor.Research area:ecophysiology of landscape plants.
E-mail:bualengpingsheng@163.com
It is wel-known that plants can emit a lot of
secondary volatile compounds to air,which con-
tributes to the atmospheric chemical reaction and
possesses an ecophysiologicaly important func-
tion for plants[1].As an important part of plant
volatile compounds,the floral scent plays a key
role in plant ecophysiology,and represents a de-
cisive communication channel between plants
and animals[2].The terpenoids are ubiquitously
found to be the important volatile category in the
floral scent[3-5].
The terpene synthases(TPS)were the key
enzymes in the biosynthesis of terpenoids[6].
A large number of TPS belonging to a large fam-
ily have been found in plants,and many TPS
genes and proteins have been isolated and char-
acterized from several plants[7].The angiosperm
monoterpene synthase is found to belong to the
TPS-b group in view of containing a R(R)x8W
motif[8].From 1992,when two sesquiterpene
synthase genes were isolated in tobacco[9],more
than 200TPS genes including 90monoterpene
synthase genes have been cloned in more than 40
kinds of plants[10].Among the monoterpene syn-
thases,linalool synthase gene is the first gene
isolated and characterized from the flowers of
Clarkia breweri[11-12].In addition,the TPS has
been successively isolated from many plants’
flowers including Arabidopsis[13],lavender[14],
kiwifruit[15],and Alstroemeria[16].
Liliumis one of world popular ornamental
flowers,and the previous studies on its floral
scent indicate that the terpenoids,especialy
monoterpenes,including linalool,ocimene,and
β-myrcene are emitted from oriental hybrid lilies
in a large quantity,which is thought to result in
the difference in floral scent among different hy-
brid lilies[17-18].However,little about the emis-
sion mechanism of monoterpene in Lilium.So in
this study,the monoterpene emissioin from
Lilium ‘siberia’flowers during the daytime was
investigated,and the gene expression of a
monoterpene synthase was also examined.
1 Materials and methods
1.1 Plant materials
In this study,Lilium ‘siberia’plants
grown from bulbs were used.The bulbs cultured
in plastic pots(20cm diameter,20cm height)
which contain medium were composed of peat
and vermiculite at the volume ratio of 2to 1.The
seedlings were placed in the greenhouse at the
Science Park of Beijing University of Agriculture
under 16h/8hlight/dark 25 ℃/20 ℃ cycle.
They were irrigated every three days and sup-
plied with a ful Hoagland nutrient solution
every two weeks.The floral scent during the
daytime at the ful opening stage(about 6-day-
old flower)was colected,and then the flowers
were colected and quickly thrown into liquid
nitrogen for gene expression analysis.
1.2 Floral scent colection
The dynamic headspace sampling was used
to colect the floral scent at 7times(7:30,9:30,
11:30,13:30,15:30,17:30,and 19:30)during
the daytime.An individual flower was put in a
Reynolds oven bag(16×17.5IN)which relea-
ses and absorbs few volatiles.A stainless steel
tube(0.25×3.5IN,USA)containing Tenax-
GR(60-80mesh,Chrompack)was used as the
volatile trap,and avoided touching the flower.A
portable air sampler(QC-1;Beijing Municipal
Institute of Labour Protection,China)served as
the pump,and air filtered through a drying col-
umn filed with charcoal was pumped into the
bag.The volatiles were colected for 20min at a
flow rate of 300mL·min-1 for each flower.
Afterwards,the stainless steel tubes were sealed
and placed in a refrigerator.
1.3 Floral scent analysis
The automated thermal desorption-gas
chromatography/mass spectrometry(ATD-GC/
MS)technique was used to analyze the floral
scent.The floral scent colected in the stainless
steel tube was desorbed by heating in an ATD
(Auto Thermal Desorber,TurboMatrix 650,
PerkinElmer)at 260 ℃ for 10min,and then
cryofocused in a cold trap whose temperature
was maintained at-25℃for 3min.The cold
trap was then quickly heated to 300 ℃ main-
tained for 5min to transport the volatiles to GC
(Clarus 600,Perkin EImer).The GC was
equipped with a capilary DB-5MS column
(30m ×0.25mm i.d.,with a 0.25μm film
thickness).Helium was used as the carrier gas.
The GC was programmed at 40 ℃for 2min,
4℃·min-1 up to 160℃,then 20℃·min-1 up
to 270℃,and held at 270℃for 3min.The MS
(Clarus 600T,Perkin EImer)was operated in EI
ionization mode at 70eV,and a mass scan range
of 29-600amu was monitored.Interface and ion
source temperatures were 250 ℃ and 220 ℃,
respectively.
Preliminary identification of the compounds
was made by searching the NIST08and WIELY
library in the TurboMass Ver 5.4.2software
according to retention time.In order to enable to
compare the release amounts of volatile compo-
nents,α-pinene(Fluka,USA)was used as an
external standard.As we described previously[19]
with some modification,α-pinene was dissolved
in ethyl acetate with different solution concen-
·467· 西 北 农 业 学 报 25卷
trations.μg·h
-1 was used as unit to describe
the release amount.
1.4 Gene expression analysis of Li-mTPS
Total RNA was extracted from tepals.cD-
NA reverse transcribed from total RNA was
used as reaction template.Primers of RTTPS-F
and RTTPS-S were designed for real-time PCR
amplification.Reaction system was 25μL,the a-
mount of each reagent was referenced SYBR○R
Premix Ex TaqTMII instruction.Real-time PCR
was proceed on Bio-Rad apparatus,the program
was after 95℃degeneration 3min,40cycles of
95℃for 30s,55℃for 30s,72℃for 30s,
fluorescent signal acquisition in 55 ℃.Melting
curve was drawn after the reaction.Three sample
replications were run.The relative expression
level was calculated by reference gene of
β-Actin.
2 Results and analysis
Fig.1showed the chromatographic profiles
of the floral scent emitted fromLilium ‘siberia’
Fig.1 Total ion chromatography chromatographic profiles of floral scent
emitted fromLilium ‘siberia‘duing the daytime
·567·
5期
LI Tianjiao,et al:Diurnal Changes of Monoterpenes Emission
and Li-mTPS Expression in Lilium ‘siberia’
at different time points,and a significant differ-
ence was found.The total release amount of
monoterpenes first increased and then decreased
during the daytime(Fig.2,P<0.05).The high
level emission of monoterpenes was found at
11:30,13:30,15:30,and 17:30,folowed by a
sudden decrease at 19:30.The maximum release
amount at 15:30was about 25-and 12-fold high-
er than that at 7:30and 19:30respectively.
Each point is the average of three independent replications.
Statistical significance[least significant difference(LSD)test]of
difference among flowering stages is indicated by different lower-
case letters(P<0.05).Standard errors are shown.
Fig.2 The total release amounts of monoterpenes
in Lilium ‘siberia’flowers during the daytime
The visible difference in chromatographic
profiles was found.Floral scent detected by
GC/MS was colected for 20min at a flow rate of
300mL·min-1.
Seven monoterpenes,includingα-pinene,
β-myrcene, d-limonene, ocimene, linalool,
E,E-2,6-dimethyl-1,3,5,7-octatetraene,and al-
loocimene were detected in the floral scent of
Lilium ‘siberia’,and their release amounts also
first increased and then decreased during the
daytime(Fig.3).Their release amounts showed
the high level at 11:30,13:30,and 15:30,
which were significantly than that at other time
points(P<0.05).The release amounts ofα-pi-
nene,d-limonene,and aloocimene arrived at the
peaks at 13:30,while the maximum release
amounts ofβ-myrcene,ocimene,linalool,and
E,E-2,6-dimethyl-1,3,5,7-octatetraene occurred
at 15:30.Among these seven monoterpenes,oci-
mene and linalool presented the high level of re-
lease amouont,whose maximums both excceed-
ed 300μg·h
-1,folowed byβ-myrcene,and the
sum of release amounts of these 3monoterpenes
accounted for 84%of the total release amount of
monoterpenes.
In order to explore the emission mechanism
of monoterpenes during the daytime,the expres-
sion of Li-mTPS,a new monoterpene synthase
in Lilium,was investigated.Fig.4showed the
expression pattern of Li-mTPS in the tepals
during the daytime at ful opening stage.We
found that the Li-mTPS expression first
increased and then decreased from 7:30to
19:30.The general pattern was similar to the
monoterpene emission.
3 Discussion
Releasing floral scent is an important fea-
ture of flowering plants[20].The floral scent is a
mixture of volatile compounds,it belongs to ter-
penoids,aromatics,and fatty acid derivatives
with low vapour pressure and low mass of mole-
cule[21].More and more researches focus on flo-
ral scent in recent years[4,22-24].The emission
mechanism is the attractive field of floral scent
research. Terpenoid compounds, especialy
monoterpenes are the main floral scent compo-
nents of many plants[25].In this study,the floral
scent emission of Lilium‘siberia’and the epres-
sion of Li-mTPS,a monoterpene synthase
gene,were investigated during the daytime.
Among the floral scent components of Lili-
um ‘siberia’,seven monoterpnes were identi-
fied.Moreover,ocimene,linalool,andβ-myrce-
ne were found to be emitted in high level of
release amounts,so we think it is the dominant
monoterpenes in the floral scent of Lilium ‘si-
beria’.In the previous studies,monoterpenes is
also found to contribute significantly to the floral
scent of numerous plant species[3,25-26].In
C.breweri,the floral scent bouquet is dominated
by linalool[27],while in Antirrhinum majus,
β-ocimene and myrcene account for 20and 8%of
·667· 西 北 农 业 学 报 25卷
the total floral scent respectively,which are the
dominant components of the monoterpene frac-
tion of the floral scent bouquet[28].
The release amounts ofα-pinene(retention time 8.91),β-myrcene(retention time 10.13),d-limonene(retention time 11.49),oci-
mene(retention time 11.82),linalool(retention time 14.13),E,E-2,6-dimethyl-1,3,5,7-octatetraene(retention time 14.70),and al-
loocimene(retention time 15.68).Each point is the average of three independent replications.Statistical significance[least significant
difference(LSD)test]of difference among flowering stages is indicated by different lowercase letters(P<0.05).Standard errors are
shown
Fig.3 Release amounts of 7monoterpenes in Lilium ‘siberia’flowers during the daytime
Expression level at 7:30was taken as 1.Each point is the
average of three independent experiments
Fig.4 Daytime expression of Li-mTPS
in Lilium ‘siberia’tepals
Moreover,the release amounts of monoter-
penes were found to increase firstly then
decrease during the daytime,and the peak
release amount appeared at about 15:30.Similar
change pattern was found in myrcene and(E)-β-
ocimene emission fromA.majus flowers,whose
release amounts peaked at 15:00[29].In the flow-
ers of kiwifruit,the release amounts of(E)-β-
ocimene reach the high level at 12:00[15].It has
been reported that gene expression of monoter-
pene synthase plays a key role in the emission
pattern of monoterpene[12,29].The monoterpene
synthase mRNA levels in upper and lower petal
lobes of A.majus show the same pattern with
the release amounts of myrcene and(E)-β-oci-
mene[29].A positive correlation between monot-
erpene emission and Li-mTPS expression was
also found in Lilium ‘siberia’flowers during
daytime.The results implied that the changes in
transcript level are main determinants of monot-
erpene production and emission.But in this
study,though similar pattern was found be-
tween monoterpene emission and Li-mTPS ex-
pression exist time difference.The highest ex-
pression level of Li-mTPS occurred at 13:30,
which was earlier that the peak time of monoter-
·767·
5期
LI Tianjiao,et al:Diurnal Changes of Monoterpenes Emission
and Li-mTPS Expression in Lilium ‘siberia’
pene emission.So other regulation mechanism
may be involved in the monoterpene emission of
Lilium ‘siberia’.In the previous study of floral
scent,the regulation of MeSA emission in A.
majus flower is found to occur at the substrate,
transcriptional,and translational levels,indica-
ting that control at more than one level is neces-
sary to guarantee the floral scent emission[28].So
the possible regulatory mechanisms at the sub-
strate,post-transcriptional,translational,and
post-translational levels remained to be investi-
gated.
4 Conclusions
In general,it was found that the monoter-
pnene emission from Lilium ‘siberia’flowers
showed significantly daytime pattern.The ex-
pression level of Li-mTPS showed similar pat-
tern to monoterpene emission.So Li-mTPS ex-
pression plays an important role in the monoter-
pene emission.But other mechanisms may also
be involved in the monoterpene emission,which
need to be investigated in future study.
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‘西伯利亚’百合单萜释放与Li-mTPS表达日变化动态分析
李天娇1,胡增辉1,郑 健1,冷平生1,杨 凯2
(1.北京农学院 园林学院/北京乡村景观规划设计工程技术研究中心/城乡生态环境北京实验室,北京 102206;
2.北京农学院 植物科学技术学院,北京 102206)
摘 要 使用动态顶空法采集‘西伯利亚’百合不同时间点(7:30、9:30、11:30、13:30、15:30、17:30、19:30)释
放的香气,采用自动热脱附-气相色谱/质谱联用(ATD-GC/MS)技术分析鉴定花香成分及释放量。结果表
明:单萜释放量呈现先增后减的规律,11:30、13:30、15:30时的释放量较高。在所有的花香成分中,罗勒烯、
芳樟醇和β-月桂烯的释放量最高,它们是‘西伯利亚’百合主要的单萜花香化合物。进一步研究发现,‘西伯利
亚’百合花瓣中的Li-mTPS(一种百合单萜合成酶基因)的表达与单萜释放表现相似的规律。可见,Li-mTPS
的表达在‘西伯利亚’百合主要的单萜释放中起着关键作用,而调控机制有待于进一步研究。
关键词 ‘西伯利亚’百合;花香;单萜;Li-mTPS表达
收稿日期:2015-04-20 修回日期:2015-06-02
基金项目:北京市属高等学校创新团队建设与教师职业发展计划项目(IDHT20150503);国家自然科学基金(31071817);北
京市教委科技提升计划项目(PM2014014207);城乡生态环境北京实验室建设项目(PXM2015-014207-000014)。
第一作者:李天娇,女,硕士研究生,从事园林植物生理与生态研究。E-mail:litianjiao1231988@163.com
通信作者:冷平生,男,教授,硕士生导师,主要从事园林植物生理与生态研究。E-mail:bualengpingsheng@163.com
(责任编辑:史亚歌 Responsible editor:SHI Yage)
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LI Tianjiao,et al:Diurnal Changes of Monoterpenes Emission
and Li-mTPS Expression in Lilium ‘siberia’