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Secondary Metabolites: Biochemistry and Role in Plants
Secondary Metabolites: Biochemistry and Role in Plants
Introduction
Secondary Metabolites are Derived from
Primary Metabolites
Primary-Secondary metabolites boundary ??
GA
Resin
biosynthesis
component
Essential amino
acid Alkaloid
Main Groups of Secondary Metabolites
in Plants
Nitrogen containing:
- Alkaloids (12,000)
- Non protein amino acids (600)
- Amines (100)
- Cyanogenic glycosides (100)
- Glucosinolates (100)
Main Secondary metabolites
Without nitrogen:
- Terpenoids (29,000):
mono- 1000
sesquiterpene- 3000
diterpenes-1000
triterpenes, steroids, saponines- 4,000
- Phenolics (8,000):
Flavonoids- 2000
Polyacetylens-1000
Polyketides- 750
Phenylpropanoids- 500
Compartmentation of SMs biosynthesis
- BRONZE2 is a Glutathione-S-transferase
bz2 an9
SMs sequestration to a location with a solid
barrier and not with a biomembrane
(interfered by lipophilic SMs)
Thyme- Mint-
glandular glandular
trichomes trichomes
Lemon
leaf- Pine- resin
secretory duct
cavity
Storage in LATICIFERS
- Latex is a sap mixture
of compounds stored in
special structures called
LATICIFERS
- The composition is
typically water,
terpenes, sugars,
enzymes, etc.
- Biosynthesis of precursors
Often needed in and secondary metabolites
HIGH - Transport and storage
concentrations
- Formation of specialized
(1-3% of dry storage compartments (e.g.
weight are trichomes)
regularly seen) - Synthesis of mRNA and
proteins (transcription
translation)
Function of Secondary Metabolites
Often arguments that SMs are waste products but this cannot
explain:
- production of SMs in young tissues
Defense toxin
Olfactory
pollinator
attractant
Antifungal toxin
Defense toxin
herbivore
inhibition
Secondary metabolism
microbe inhibition
M. Wink, Annual Plant Review, 1999
Function of Secondary Metabolites
- Example: myrosinase-glucosinolates
The "mustard oil bomb"-- A binary
Glucosinolate-Myrosinase chemical defense
system
- They can either tolerate them or even use them for their
diet
Adaptations of specialist herbivores & pathogens
Herbivores:
- Avoidance of toxic plants, except host plant
Herbivores (continued)-
Microorganisms:
- Inactivation of SMs
- Evolution of insensitivity
Co-evolution in plant SMs - natural enemy
Below ground
Below ground
protection: anti-
attraction: orientation
microbial, antifeedant
cues (non-volatile)
(non-volatile)
Precursors of Terpenoids
Mixed Origins of Terpenoids
Precursors (Meroterpenes)
Terpenoids - Important Molecules !
C5 - hemiterpenes - e.g. isoprene
C10 - monoterpenes - e.g. limonene
C15 - sesquiterpene - e.g. abscisic acid (ABA)
C20 - diterpene - e.g. gibberellin
C30 - triterpne - e.g. brassinosteroids
C40 - tetraterpenes - e.g. carotenoids
> carbons - polyterpenes- e.g. ubiquinones, rubber
mixed biosynthetic origins - meroterpenes - e.g.
cytokinines, vitamin E
Monoterpenes
(C10)
Sesquiterpenes
(C15)
Diterpenes (C20)
Triterpenoids
(C30)
Tetra-terpene / Carotenoids (C40)
Biosynthesis in two main compartments
Mevalonate
CYTOSOL Sterols
HMGR
Acetyl-CoA HMG-CoA MVA IPP Squalene Triterpenes
BRs
PLASTID DMAPP FPP Sesquiterpenes
GlyAld-3P Pyruvate Polyprenols
DXS ABA Cytokinins Prenylation
DXP Dolichol
DXR
MITOCHONDRION
MEP Ubiquinones
IPP
HDR+
- Modification steps
Biosynthesis of Precursors
(prenyltransferases)
Biosynthesis of Precursors
(prenyltransferases)
Cytosol Plastid
IPP DMAPP IPP DMAPP
OPP + OPP OPP
+ OPP
2x
FDP - synthase GDP - synthase
OPP
OPP
One enzyme…..One
substrate…..Multiple products
Terpene
Cyclases
(Mono)
Terpene
Cyclases
(Sesqui-)
Terpene
Cyclases
(Diterpene)
OH MONOTERPENOIDS SESQUITERPENOIDS
OH H
O
O
O
H O
O
linalool α-pinene perilla alcohol artemisinin (E,E)-α-farnesene
polygodial O
HO OH
AcO O OH H
O
cucurbitacin C H
NH O
O
CO2H H
O O O
O H HO CO2H
OH OBz OAc O
OH HO HO
OH glycyrrhizin
paclitaxel OH
DITERPENOIDS TRITERPENOIDS
Modification of Monoterpene
Structures
isomerase
OPP
OPP
Isopentenyl diphosphate Dimethyl allyl diphosphate
OPP GDP-synthase
(-)-Limonene Limonene 3- OH O O
cyclase hydroxylase dehydrogenase reductase
Geranyl diphosphate (-) limonene (-) trans- Isopiperitenol (-) Isopiperitenone(+)-cis-Isopulegone
isomerase
reductase reductase
O
reductase
+ (+)-pulegone
OH OH O O
Peltate Glands
Peltate Glands Isolated From Sweet Basil
Terpenoids in Peltate Glands (Sweet Basil)
Monoterpenes Sesquiterpenes
Metabolic
OH
Engineering of
Terpenoid
Biosynthesis
Why? Metabolic Engineering of
Terpenoids in Plants
OH
OPP
FaNES1
Mg 2+ /Mn 2+
100 R-Linalool
reference
100 Transgenic
Arabidopsis
0
23 24 25
Time, min
Further Modification
Free and Glycosidically Bound Terpenoids Produced by Arabidopsis
E-8-hydroxy-linalool E-8-hydroxy-linalool
1.4 E-8-hydroxy-6,7-dihydro-linalool Z-8-hydroxy-linalool
100
nerolidol E-8-hydroxy-6,7-dihydro-linalool
1.2 linalool nerolidol
80 linalool
1
0.8 60
0.6
40
0.4
20
0.2
0 0
_2
_5
_6
_3
_4
_1
t -1
t-3
t- 2
t-4
t- 5
_6
_2
_3
_4
_5
_1
t -1
t -2
t-3
t-4
t-5
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
N
N
N
N
N
Tr
Tr
Tr
Tr
Tr
Tr
Tr
Tr
Tr
Tr
Tr
Tr
Plant
OH
Further Modification
S-Linalool
Introduced product:
linalool
OH OH
Glycoside Glycoside
Modified by endogenous OH HO
enzymes:
E-8-Hydroxylinalool Z-8-Hydroxylinalool
140
Sum - glycosidically bound
Sum - free
120
100
Concentration (mg kg-1-FW)
80
60
40
20
0
o
no
sm
o
_n
sm
_n
o
_
_n
o
sm
sm
_n
t-1
t-2
_n
_n
6_
7_
_s
_s
12
15
8_
2_
6
9-
N
N
9-
21
9
9-
-3
9-
-3
-4
-2
-2
-3
Tr
Tr
9-
Tr
26
Tr
26
26
26
26
26
Tr
Tr
Tr
Tr
Tr
Tr
Tr
Further Modification
OH
E-8-Hydroxylinalool Z-8-Hydroxylinalool
detected in leaves of wild-
type plants
3. Endogenous enzymes OH
E-8-Hydroxy-6,7-dihydrolinalool
introduced linalool
Potato Plants Transformed with the Same Construct
100
R - Linalool
80
60 Reference S - Linalool
40
20
100
80
60 Wild-type
Relative abundance
40
20
100
80
60 Transgenic # 1
40
20
NL:
100
80
60 Transgenic # 2
40
20
NL:
100
80
60 Transgenic # 3
40
20
25.6 25.8 26.0 26.2 26.4 26.6 26.8 27.0 27.2 27.4 27.6 27.8 28.0
Time (min)
assumed glycosylation
further
OH
site in potato
modification glycoside
in transgenic S-linalool
potato plants
assumed glycosylation
site in potato
OH OH
assumed glycosylation
site in Arabidopsis
glycoside glycoside
OH HO
E-8-hydroxy-linalool Z-8-hydroxy-linalool
assumed glycosylation
OH site in potato
glycoside
assumed glycosylation
OH
site in Arabidopsis
E-8-hydroxy-6,7-dihydrolinalool
Conclusions
• In most cases the introduced metabolite could be
glycosylated and/or hydroxylated
• Glycosylation could be highly efficient
• Derivatisation will be different between plant species
and it will depend on the genetic make-up (i.e. activity
of the endogenous enzyme)
• If the target metabolite or its derivative is already
produced by the plant one should expect amplification
in production but also formation of “new” metabolites
(possibly metabolites that could not be detected earlier
due to sensitivity of instruments)
Engineering Sesquiterpenes in Arabidopsis
Introducing the CiGASlo Gene to
Arabidopsis
Enh 35S CiGASlo T
Cytosolic production of a
Germacrene A synthase from
Chicory
ransgenic
ild type
rabidopsis
Linalool
o a
rabidopsis
l in
l
l
o
Abundance
20
00
0
10000
20
00
Abundance
0
0
8 9 10 00 11 00 12 00 13 00 14 00 ime
0 0
0 0
l id o
10
l er
o
00
0 Nerolidol
n
0
8 9 10 11 12 13 14 im
00 0 00 00 00 0 00 e
0 0
Nerolidol is Produced at Low Level Also in Potato
34.67
100
80
60
34.10 Nerolidol Wild-Type
40
33.78 33.81 34.16 34.39 34.50
34.00 34.34 34.76 34.80 34.92 35.04 35.07
20
34.68
100
Relative Abundance
80
60 34.11
34.50
Transgenic #1
40
33.77 33.92 34.00 34.21 34.30 34.39
20 34.78 34.82 35.00 35.08
34.65
100
80
60 34.10
Transgenic #2
40 34.50
20
33.80 33.91 33.95 34.18 34.26 34.38 34.78 34.87 34.94 35.07
34.66
100
80
60
34.11
34.49 34.51
Transgenic #3
40
33.77 33.99 34.03 34.21 34.31 34.37 34.75 34.83
20 34.95 35.00 35.14
33.8 33.9 34.0 34.1 34.2 34.3 34.4 34.5 34.6 34.7 34.8 34.9 35.0 35.1
Time (min)
Availability of Precursor Pools?
Emission /storage
Monoterpenes
FPP
GPP GGPP
GPP
FPP
IPP DMAPP IPP DMAPP
MITOCHONDRIA
CYTOSOL PLASTID
Engineering Sesquiterpenes in Arabidopsis
Introducing the FaNES1 fused to a Mitochondrial
targeting signal to Arabidopsis
Mitochondrial targeting,
cytochrome c oxidase
Production of terpenoids in
Mitochondria
Engineering Sesquiterpenes in Arabidopsis
Undamaged
Wild-type
Plastid targeting
Rubisco FaNES1 T
The Cost of Terpenoid Production in Plants
Bleaching and growth retardation with constitutive over-
expression of FaNES1 in potato
Effect of Linalool Expression on Potato
Phenotype
14
12
10
8
6
4
2
0
1 2 3 4 5 6 7 8 9
Wild
20.14
100
% R-Z 31
2.66
100
R-Z 31
%
9.53
2.63
10.59 R-F 10
0
9.53
100 Beta -pinene
Alpha-pinene
R-F 10
% 10.59
20.14
2.65
22.97 24.20
0 Time
4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
Conclusions
Linalool deters
aphids
No Choice Greenhouse Test in
Perspex Hoods
Linalool synthase
chrysanthemum T58
lines
20 females
N=6-13 plants per line
3 weeks; 22 C
Thrips population on linalool chrysanthemum
3 weeks after inoculation with 20 females
16
14 control 1581
T58-9
Insects per plant
12
10
p=0.04
8 p=0.05
6
4
2
0
TOTAL ADULTS LARVAE
Different Thrip Damage Phenotype in
Transgenic Linalool Plants
Control
only edges
large surface
Linalool
transgenic
only spots
not at the edges
Conclusions
-Terpenoids produced by engineered plants
influence insect behavior
-High levels of linalool production deters
insects (aphids and thrips) in different plant
species