Transcript Freesia hybrida

Floral scent biosynthesis
Lecturer：Xiang Gao
May 13, 2013
What is floral scents?
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Secondary metabolites
volatile low-molecular-weight compounds
Terpenoids
Phenylpropanoids
Fatty acid derivatives
Terpenoids are the most abundant and
structurally diverse group, comprising over
40,000 individual compounds.
Hemiterpene
Monoterpene
Diterpene
Sesquiterpene
Triterpene
Ecological values
• Pollinator attractants or repellents
• Phytoalexins against herbivores or
pathogens.
• Attract natural enemies of herbivores,
either predators or parasitoids
• Airborne signals to prime neighboring
plants against future insect attack
Nature 406:512–515
Compounds identified in
headspace volatiles of
detached leaves
1, (Z )-3-hexenol; 2, ( E)-2-hexenal; 3,
(Z)-3-hexenyl acetate; 4, a-pinene; 5,
limonene; 6, ( Z)- b-ocimene; 7, ( E)b-ocimene; 8, DMNT; 9, menthol; 10,
a -copaene; 11, junipene; 12, b caryophyllene; 13,
a-humulene; 14, germacrene d; 15,
TMTT; 16, methyl salicylate
Expression of defence genes in lima bean leaves
Human benefits
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Floral scents and essential oils
Nootkatone
Artemesinin
zerumbone
Terpenoid engineering
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Pest resistance
Weed control
increased aroma of fruits and vegetables
Floral scents biosynthesis and regulation
study
Terpenoid
biosynthesis
Floral scents formation molecular mechanism
• Terpene synthases diversity
• Substrate and enzyme subcellular compartmentation
• Gene differential expression
• Transcription regulation
Terpene synthases diversity
• Terpene synthases (TPSs), a large group
of enzymes that are responsible for most
of the structural varieties of terpenoids
Monoterpene synthase genes
Sesquiterpene synthase genes
• Generally, individual intermediates have a
variety of metabolic fates in the reaction,
resulting in the formation of multiple products.
In grand fir, δ-selinene synthase and γ -humulene synthase generate 34
and 52 different sesquiterpenes, respectively
THE JOURNAL OF BIOLOGICAL CHEMISTRY
Proposed mechanism for sesquiterpene formation by δ-selinene synthase (ag4).
Steele C L et al. J. Biol. Chem. 1998;273:2078-2089
©1998 by American Society for Biochemistry and Molecular Biology
Proposed mechanism for sesquiterpene formation by γ-humulene synthase (ag5).
Steele C L et al. J. Biol. Chem. 1998;273:2078-2089
©1998 by American Society for Biochemistry and Molecular Biology
• TPSs not only can produce multiple
products from a single substrate but also
exhibit tremendously functional flexibility
The Plant Cell, Vol. 16, 3110–3131, November 2004
The Plant Journal, (2008), 56, 228–238
• Other enzymes involved in the modification
of the parent hydrocarbon skeletons, such
as the addition of hydroxyl groups, further
oxidation to ketones, demethylation, and
methyl transference, which affect various
chemical properties of the molecules that
influence their biological activities
(- )-menthol biosyethesis
Plant Physiology, March 2005, Vol. 137, pp. 873–881
Substrate and enzyme subcellular
compartmentation
• However, there is increasing evidence
suggesting that cross-talk occurs between
the two different biosynthetic pathways
fosmidomycin
fosmidomycin
mevinolin
Snapdragon flowers have also indicated that the MEP pathway provides
IPP precursors for both monoterpene and sesquiterpene biosynthesis, and
the trafficking of IPP occurs unidirectionally from the plastids to cytosol
PNAS, January 18, 2005, vol. 102, no. 3, 933–938
• In addition, in some cases, both pathways
can even cooperate to supply IPP
precursors for biosynthesis of certain
terpenoids.
• For example, the chamomile sesquiterpenes
consist of two C5 isoprene units derived from
the MEP pathway, with a third unit being
formed via both pathways
• Despite these observations, the relative
contribution of each pathway to the
biosynthesis of the various classes of
terpenoids remains largely unknown.
• Besides the transport of the IPP precursors
between different compartments, the
intracellular localization of the different
terpenoid synthases are also not as strictly
defined as previously believed.
Two different strawberry monoterpene synthase proteins have been
shown to be targeted to the cytosolic compartment
Spatial and temporal regulation of
plant terpenoid biosynthesis
• Terpenoid biosynthesis in plants can be
spatially and temporally regulated during
development
• Mono- and sesquiterpenoids are often
emitted from specific floral tissues at
particular times or developmental stages
to attract pollinators
• In addition, some terpenoids are also
frequent constituents of essential oils and
resins and are constitutively accumulated
in highly specialized secretory structures,
such as the glandular trichomes of mints
and resin ducts of conifers.
Transcription regulation
• Transcription factors play a predominant role in
regulating the expression of genes involved in
various physiological and developmental
processes, including plant secondary
metabolism. Several studies have suggested
that TPS activities are closely related to
transcription factors.
GaWRKY1, a Cotton Transcription Factor That Regulates the
Sesquiterpene Synthase Gene ( 1)- d -Cadinene Synthase-A
Plant Physiology, May 2004, Vol. 135, pp. 507–515,
The Transcription Factor CrWRKY1 Positively Regulates the Terpenoid
Indole Alkaloid Biosynthesis in Catharanthus roseus
Plant Physiology, December 2011, Vo l. 157, pp . 2081–2093
Arabidopsis MYC2 Interacts with DELLA Proteins in Regulating
Sesquiterpene Synthase Gene Expression
The Plan t Ce ll, Vol. 24: 2635–2 648, Ju ne 2012
PAP1 transcription factor enhances production of terpenoid scent
compounds in rose flowers
New Phytologist (2012) 195 : 335–345
• In tobacco, two transcription factors, WRKY3
and WRKY6, were found to be related to volatile
terpene production (Skibbe et al. 2008).
• It has been reported that, in Arabidopsis, the
auxin-responsive factors ARF6 and ARF8 and
the MYB transcription factors MYB21 and
MYB24 form a regulatory network that promotes
nectary development or function and, as a result,
the production of volatile sesquiterpenes
(Mandaokar et al., 2006; Reeves et al., 2012).
Our study
Our work
Multigene family isolation
Degenerate
primer PCR
Transcriptome
analysis
Partial cDNA
sequence
RACE
Full length
cDNA sequence
Yeast one
hybrid
Our work
Gene function characterization-structure gene
Gene function
characterization
Recombinant
protein
preparation,
purification
and enzyme
catalytic
activity
analysis
Arabidopsis
mutant
complement
Petunia
transformation
overexpression
RNAi
Freesia hybrida
transient
infection in
protoplast and
flower
Relationship
between
gene
expression
and
metabolic
product
accumulation
In freesia
Our work
Gene function characterization-transcription factor
Gene function
characterization
Arabidopsis
mutant
complement
Petunia
transformation
overexpression
RNAi
Relationship
between
gene
Petunia
Freesia hybrida
expression
and
transfection
transient
metabolic
infection in
overexpression
product
protoplast and
accumulation
RNAi in
flower
transgenic
plants
Relationship
between
gene
expression
and
metabolic
product
accumulation
In freesia
Our work
Floral scents formation molecular mechanism
Full length cDNA
sequence
Floral scents
analysis
Real time-PCR
Full length cDNA
sequence
Partial cDNA
sequence
RACE
 Transcription
factors expression
analaysis
 Structure gene
expression
regulation analysis
Gene, protein
sequence
variation and
enzyme catalytic
activity analysis
Our work
Transcription regulation system in Freesia hybrida
Gene expression
regulation
 Interaction
between
promoter and
transcription
factor
 Inverse
PCR, yeast
one hybrid,
CHIP, EMSA
 Interaction
between the
transcription
factors
 Yeast two
hybrid, cellular
co-localization
 Gene over
Petunia
expression,
transfection
gene knockout
overexpression
Single or
Multi-gene coRNAi
transformed
Transgenic
plants
metabolic
products and
terpenoids
biosynthesis
gene
expression
analysis
序号
编号
长度
序列特点
功能预测
1
Scaffold2903
590
中间片段
倍半萜合酶
2
Scaffold2904
1041
3’末端
倍半萜合酶
3
Scaffold35680
2024
3’末端
倍半萜合酶
4
Scaffold35681
1500
中间片段
倍半萜合酶
5
Scaffold35682
1934
3’末端
倍半萜合酶
6
Scaffold9702
1309
5’末端
倍半萜合酶
7
Scaffold16575
1723
3’末端
倍半萜合酶
8
Scaffold9700
1202
5’末端
倍半萜合酶
9
Scaffold9701
769
5’末端
倍半萜合酶
10
C912535
169
中间片段
倍半萜合酶
11
C994078
222
中间片段
倍半萜合酶
12
C1032516
269
中间片段
倍半萜合酶
13
C1079620
383
中间片段
倍半萜合酶
14
C1088015
418
中间片段
倍半萜合酶
15
C1086193
409
中间片段
倍半萜合酶
16
Scaffold16384
982
中间片段
单萜合酶
17
Scaffold16385
1372
中间片段
单萜合酶
18
Scaffold21710
1860
全长片段
单萜合酶
19
Scaffold710
345
中间片段
单萜合酶
20
Scaffold15037
764
中间片段
单萜合酶
21
Scaffold21784
668
5’末端
单萜合酶
22
Scaffold15036
571
中间片段
单萜合酶
23
Scaffold37957
990
中间片段
单萜合酶
24
C872705
153
中间片段
单萜合酶
25
C907499
167
中间片段
单萜合酶
26
C911798
168
中间片段
单萜合酶
27
C942071
183
中间片段
单萜合酶
28
C991372
220
中间片段
单萜合酶
29
C1025686
203
中间片段
单萜合酶
30
C1068844
346
中间片段
单萜合酶
31
C1067578
343
中间片段
单萜合酶
32
C1109005
571
中间片段
单萜合酶
33
C1121129
783
5’末端
单萜合酶
34
C1125839
969
3’末端
单萜合酶
序号
编号
长度
序列特点
功能预测
1
Scaffold36590
505
中间片段
MYC2
2
Scaffold18195
2112
3’末端
MYC2
3
Scaffold18778
2454
3’末端
MYC2
4
C1133369
2337
全长片段
MYC2
5
Scaffold5735
842
中间片段
WRKY1
6
Scaffold36793
1082
中间片段
WRKY1
7
Scaffold5734
1021
中间片段
WRKY1
8
Scaffold31822
1232
中间片段
WRKY1
9
C1092649
443
中间片段
WRKY1
10
C1131965
1645
中间片段
WRKY1
11
C949493
189
中间片段
WRKY1
12
C1055719
313
中间片段
WRKY1