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TRANSCRIPT
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Secondary Metabolites:
Biochemistry and Role in
Plants
Introduction
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Secondary Metabolites are Derived from
Primary Metabolites
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PrimaryPrimary--Secondary metabolites boundary ??Secondary metabolites boundary ??
GA
biosynthesisResin
component
Essential amino
acid Alkaloid
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Main Groups of Secondary MetabolitesMain Groups of Secondary Metabolites
in Plantsin Plants
29,000 terpenes29,000 terpenes-- derived from the C5derived from the C5
precursor isopentenyl diphosphate (IPP)precursor isopentenyl diphosphate (IPP)
12,000 alkaloids12,000 alkaloids-- derived from amino acidsderived from amino acids
8,000 phenolics8,000 phenolics-- shikimate pathway orshikimate pathway or
malonate/acetate pathwaymalonate/acetate pathway
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Main Secondary metabolites Main Secondary metabolites
Nitrogen containing:- Alkaloids (12,000)
- Non protein amino acids (600)- Amines (100)
- Cyanogenic glycosides (100)- Glucosinolates (100)
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Main Secondary metabolites Main Secondary metabolites
Without nitrogen:
- Terpenoids (29,000):mono- 1000
sesquiterpene- 3000
diterpenes-1000triterpenes, steroids, saponines- 4,000
- Phenolics (8,000):
Flavonoids- 2000
Polyacetylens-1000
Polyketides- 750
Phenylpropanoids- 500
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Compartmentation of SMs biosynthesisCompartmentation of SMs biosynthesis
Mostly in the Cytosol: hydrophilic compounds
Chloroplasts: alkaloids (caffeine) and terpenoids
(monoterpenes)
Mitochondria: some amines, alkaloids
Vesicles: alkaloids (protoberberines)
Endoplamic reticulum: hydroxylaton steps, lipophilic
compounds
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SMs sequestrationSMs sequestration
- Water soluble compounds are usually
stored in the vacuole
- Lipophilic substances are sequestered
in resin ducts, laticifers, glandular hairs,
trichomes, in the cuticle, on the cuticle
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SMs sequestration in to VacuolesSMs sequestration in to Vacuoles
Water soluble
compounds-alkaloids, NPAAs,
cyanogenic glucosides,
glucosinolates,saponines,
anthocyanines,
flavonoids,
cardenolides
ATP-
dependant
transporter
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SMs sequestration in to VacuolesSMs sequestration in to Vacuoles--
Anthocyanin example Anthocyanin example
- Anthocyanines- blue-red flavonoid pigments
- They are stabilized in the vacuole
- Oxidized in the cytosol
- The sequestration is a detoxification process
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SMs sequestration in to VacuolesSMs sequestration in to Vacuoles--
Anthocyanin example Anthocyanin example-- Bz2 mutant Bz2 mutant
- When the BRONZE2 gene is not active,anthocyanines accumulate in the cytosol and a tan
bronze phenotype of tissue is obtained
- BRONZE2 is a Glutathione-S-transferase
- Glutathionation of anthocyanines is a pre-requisite
for the targeting to the vacuole through a GST-x-
pump in the tonoplast membrane
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SMs sequestration in VacuolesSMs sequestration in Vacuoles-- Anthocyanin Anthocyanin
exampleexample-- bz2bz2 & the an9 mutant & the an9 mutant
bz2 an9
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SMs sequestration to a location with a solid SMs sequestration to a location with a solid
barrier and not with a biomembranebarrier and not with a biomembrane(interfered by lipophilic SMs)(interfered by lipophilic SMs)
Thyme-
glandular
trichomes
Mint-
glandular
trichomes
Lemon
leaf-
secretorycavity
Pine- resin
duct
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Storage in LATICIFERS
- Latex is a sap mixtureof compounds stored in
special structures called
LATICIFERS
- Rubber was isolated
from it in the past
- The composition is
typically water,
terpenes, sugars,enzymes, etc.
- Often latex has amilky appearance
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Long Distance Transport of SMs
In Xylem, Phloem or Apoplastic transport
Long-distance phloem transport of glucosinolates Chen et al., 2001
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Long-distance phloem transport of glucosinolates
Chen et al., 2001
- Intact Glucosinolates are transported
- Selection of a specific glucosinolate to be loaded intothe phloem
- Presence of glucosinolates in the phloem providemeans of defense against insects
- Export of glucosinolates from fully expanded leaves
and senescent parts
- Export to sink tissues, seeds, flowers
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Function of Secondary Metabolites
Often arguments that SMs are waste products but this cannot
explain:- production of SMs in young tissues
- plants are autotrophs and waste products are typicaland needed for heterotrophic animals that cannot
degrade their food completely for energy production
- many SMs could be metabolized further (SMs that
contain nitrogen stored in seeds and metabolized during
germination)
- tight spatial and temporal regulation of SMs
biosynthesis
- proven biological activity
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Function of Secondary Metabolites -
DEFENSE - ATTRACTION - PROTECTION (uv)
- Most animals can move-run away and possesan immune system
- Plants are attacked by herbivores, microbes,
(bacteria and fungi) and by other plants
competing for light, space and nutrients
- Abiotic stresses such as radiation
F i f S d M b li
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Function of Secondary Metabolites:
Defense
Herbivores (insects, vertebrates)Repellence,
deterrence,
toxicity
Microbes (bacteria,
fungi, viruses)
Growth inhibition
and toxicity
Attraction
Plant SMs
- mixtures
- variation in time,space & dev. stage
- pollinating insects
- seed dispersing animals
- root nodule bacteria
- induced volatiles attractpredatory organisms
(tritrophic interactions)
competing plants (inhibition of
germination and seedlings growth)
UV-protection
M. Wink, Annual Plant Review, 1999
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P d i f SM f d f i h bi
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Production of SMs for defense against herbivores
and pathogens is not necessarily constitutive
- Wounding and infection trigger INDUCED accumulation of SMs
herbivore
inhibition
Secondary metabolism
activation of prefabricated defense chemicals
PLANT
wounding and infection increase of existing defense compounds
induction of de novo synthesis of defense
compounds (phytoalexins)
microbe inhibition
M. Wink, Annual Plant Review, 1999
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Function of Secondary Metabolites
- Wounding can lead to release of a pre-fabricated
compound from a compartment
- The mix with an enzyme (often an hydrolaze)
will result in production of an active form of the
chemical
- Example: myrosinase-glucosinolates
Th " d il b b" A bi
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The "mustard oil bomb"-- A binary
Glucosinolate-Myrosinase chemical defensesystem
Glucosinolates breakdown products
1- isothiocyanates
2- nitriles and elemental sulfur
3- thiocyanates4- oxazolidine--thiones
5- epithionitriles
Grubb and Abel, TIPS, 2006
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Targets for SMs in animal
systems
- Nervous system (perception,
processing, signal transduction
- Development- Muscles and motility
- Digestion- Respiration
- Reproduction and fecundity
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Co-evolution in plant SMs - natural enemy
- The SM defense system works in general but not always
- Some herbivores and microorganisms have evolved thathave overcome the defense barrier (like viruses, bacteria or
parasites that bypass the human immune system)
- These organisms developed different strategies of
adaptations to the SMs
- They can either tolerate them or even use them for their
diet
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Adaptations of specialist herbivores & pathogens
Herbivores:
- Avoidance of toxic plants, except host plant
- Cutting laticfers and resin ducts filled with SMs
- Non-resorption or fast intestinal food passage
- Resorption followed by detoxification and elimination
(urine and others)- Hydroxylation
- Conjugation
- Elimination
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Co-evolution in plant SMs - natural enemy
- Canavanine is toxic due to its incorporation
into proteins that rise to functionally aberrantpolypeptides
- The tRNA- Arginine in insects uses also
Canavanine
- The insect mutated its tRNA and will not
incorporate canavanine instead of Arginine
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Adaptations of specialist herbivores & pathogens
The process of co-evolution between plants and
their natural enemies is believed to have generatedmuch of the earth's biological diversity
This includes chemical diversity!!
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SMs in Arabidopsis
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Expression pattern of a Terpene Synthase in
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Expression pattern of a Terpene Synthase in
Arabidopsis
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The Terpenoids orThe Terpenoids or
IsoprenoidsIsoprenoids
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T id d C i iT id d C i i
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Terpenoids and CommunicationTerpenoids and Communication
Below groundattraction: orientation
cues (non-volatile)
Below groundprotection: anti-
microbial, antifeedant(non-volatile)
Above groundattraction: fragrance
(volatile)
Above groundprotection:
repellents,antifeedants,
predator attraction(volatile/non-volatile)
Precursors of Terpenoids
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Precursors of Terpenoids
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MonoterpenesMonoterpenes(C(C1010))
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TriterpenoidsTriterpenoids
(C(C3030))
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Biosynthesis in two main compartmentsBiosynthesis in two main compartments
Mevalonate pathwayMevalonate pathway leading to IPP inleading to IPP inthe cytosolthe cytosol
TheThe MEP pathwayMEP pathway leading to IPP in theleading to IPP in the
plastidsplastids
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Biosynthesis of PrecursorsBiosynthesis of Precursors
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Biosynthesis of Precursorsy
(prenyltransferases)(prenyltransferases)
Cytosol Plastid
OPP OPP
DMAPPIPP
OPP OPP+
IPP DMAPP
2 x
+
FDP - synthase GDP - synthase
OPP
OPP
Farnesyl diphosphate Geranyl diphosphate
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Terpene CyclasesTerpene Cyclases
OneOne enzymeenzyme……....OneOnesubstratesubstrate……....MultipleMultiple productsproducts
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TerpeneTerpeneCyclasesCyclases
(Mono)(Mono)
TerpeneTerpene
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TerpeneTerpene
CyclasesCyclases(Sesqui(Sesqui--))
TerpeneTerpene
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TerpeneTerpene
CyclasesCyclases(Diterpene)(Diterpene)
OH
O
HMONOTERPENOIDS SESQUITERPENOIDSOH
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isopentenyl diphosphate(IDP)
dimethylallyl diphosphate(DMAPP)
GDP synthase
geranyl diphosphate(GDP)
monoterpene synthases +/-modifying enzymes
O
O
O
H
O
farnesyl diphosphate(FDP)
squalene-2,3-epoxide
DITERPENOIDS TRITERPENOIDS
O
NH
O
OH
O
OBzH
OAc
AcO OH
O
OH
O
PPO
OPP
OPP
geranylgeranyl diphosphate(GGDP)
O
OO
O
O
H
H
H
O
HO
OH
CO2H
HOOH
CO2HHO
paclitaxel
glycyrrhizin
(E,E )-α-farneseneartemisininlinalool α-pinene perilla alcohol
OAc
OOH
O
HO
H
OH
H
OH
cucurbitacin C
CHO
CHO
polygodial
FDP synthase
GGDP synthase
squalene synthase
squalene epoxidaseOPP OPP
diterpene synthases +/-modifying enzymes
sesquiterpene synthases +/-modifying enzymes
triterpene synthases +/-modifying enzymes
Strawberries Anti-malarial drug
Myzus persicae Leaf of cucumber
Modification of Monoterpene Modification of Monoterpene
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StructuresStructures
OPP
OPPOPP
OH O O
O
O
Isopentenyl diphosphate Dimethyl allyl diphosphate
(-) limoneneGeranyl diphosphate (-) trans - Isopiperitenol (-) Isopiperitenone(+)-cis -Isopulegone
(-)-Limonenecyclase
Limonene 3-hydroxylase dehydrogenase reductase
isomerase
GDP-synthase
isomerase
O
reductase reductase
OH OH
reductase
+ (+)-pulegone
(-)- Menthol (+)-neomenthol (-)- menthone (+)-isomenthone
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Most secondary metabolites in Basil are
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y
produced in the Peltate Glands
Peltate Glands
Peltate Glands Isolated From Sweet Basil
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MetabolicMetabolicEngineering ofEngineering of
TerpenoidTerpenoidBiosynthesisBiosynthesis
OH
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S S - - Linalool Formation in Leaves of Linalool Formation in Leaves of
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Arabidopsis Arabidopsis
23 24 25
0
100
0
100
0
100
Time, min
R
e l a t i v e d e t e c t o r
r e s p o n s e
S-Linalool
reference
R-Linalool
reference
TransgenicArabidopsis
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Further Modification Further Modification
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The sum of glycosylated components was in someof the transgenic lines up to 40 to 60-fold higher
than the sum of the corresponding free alcohols
Conc en
tration(
mgkg
-1-FW)
0
20
40
60
80
100
120
140
160
N t - 1_ n
o
N t - 2_ n
o
T r 2 6
- 3 6_ n o
T r 2 6
- 3 0_ n o
T r 2 6
- 4 1_
n o
T r 9 - 1 5_ n o
T r 9 - 1 2_ n o
T r 9 - 2 1
_ s m
T r 9 - 6_ s m
T r 2 6
- 2 9_
s m
T r 9 - 7_ s m
T r 2 6
- 3 2_
s m
T r 2 6
- 2 8_
s m
Sum - glycosidically bound
Sum - free
Further Modification Further ModificationOH
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OH
OH
OH
OH
OH
OH
S-Linalool
E-8-Hydroxylinalool Z-8-Hydroxylinalool
E-8-Hydroxy-6,7-dihydrolinalool
GlycosideGlycoside
Glycoside
1. Produced to the highestlevels in transgenic lines
2. The only component
detected in leaves of wild-type plants
3. Endogenous enzymes
already active and canutilize efficiently the newlyintroduced linalool
E-8-Hydroxylinalool and its glycoside:
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OH
glycoside
assumed glycosylation
site in potato further further
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OH
OH
OH
OH
OH
OH
S-linalool
E-8-hydroxy-linalool Z-8-hydroxy-linalool
E-8-hydroxy-6,7-dihydrolinalool
glycosideglycoside
glycoside
glycoside
assumed glycosylation
site in Arabidopsis
assumed glycosylation
site in potato
assumed glycosylation
site in Arabidopsis
assumed glycosylation
site in potato
modification modification in transgenic in transgenic
potato plants potato plants
Conclusions
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• In most cases the introduced metabolite could beglycosylated 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
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Introducing the CiGASlo Gene to Introducing the CiGASlo Gene to
Arabidopsis Arabidopsis
Enh 35S TCiGASlo
Wild-type Arabidopsis leaves
do not produce Germacrene A
Cytosolic production of aGermacrene A synthase from
Chicory
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Unexpected: Sesquiterpene Production withUnexpected: Sesquiterpene Production with
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Plastidic Targeting of FaNES1 Plastidic Targeting of FaNES1
800
9
00
1000
1100
1200
13
00
1400
0
10
000
20000
ime
nero
lido
l
8
00
9
00
10 00 11 00 12 00 13 00 14 000
10000
20
000
ime
A b u n d a n c e
ransgenic r a b i d o p s i s ild type r a b i d o p s i s
A b u n d a n c e
linaloolLinalool
Nerolidol
Nerolidol is Produced at Low Level Also in Potato Nerolidol is Produced at Low Level Also in Potato
10034.67
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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)
20
40
60
80
100
20
40
60
80
100
20
40
60
80
100
R e l a t i v e A b u n d a n
c e
20
40
60
8034.10
34.16 34.39 34.5033.78 34.3434.00 34.7633.81 34.80 35.0434.92 35.07
34.68
34.1134.50
34.3934.2134.0033.77 33.92 34.7834.30 34.82 35.00 35.08
34.65
34.10
34.50
33.80 34.18 34.2633.91 33.95 34.38 34.78 34.87 35.0734.9434.66
34.5134.4934.11
34.0333.77 34.2133.99 34.3734.31 34.75 35.1434.83 34.95 35.00
Nerolidol Wild-Type
Transgenic #1
Transgenic #2
Transgenic #3
Availability of Precursor Pools? Availability of Precursor Pools?
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Monoterpenes
GPP GGPP
IPP DMAPP
Emission /storage
CYTOSOL
Sesquiterpenes
FPP
GPP
IPP DMAPP
PLASTID
Hydroxylated monoterpenes
MITOCHONDRIA
FPP
Engineering Sesquiterpenes in Arabidopsis
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Introducing the FaNES1 fused to a MitochondrialIntroducing the FaNES1 fused to a Mitochondrial
targeting signaltargeting signal to Arabidopsisto Arabidopsis
Enh 35S TFaNES1
Production of terpenoids inMitochondria
Cox IV
Mitochondrial targeting,cytochrome c oxidase
Engineering Sesquiterpenes in Arabidopsis
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UndamagedWild-type
Transgenic Undamaged(nerolidol and DMNT)
Transgenic undamaged(only nerolidol)
Nerolidol
DMNT (C11 homoterpene)Nerolidol
C15 sesquiterpene C11 homoterpene
Conclusions
E i i i d i i h
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• Engineering sesquiterpene production in thecytosol compared to plastidic production of
monoterpenes seems more difficult
•Targeting different cell compartments for
engineering terpenoids might be a valuable tool
• Further modification of introduced terpenoid
might be different in each cell compartment
The Cost of Terpenoid Production in Plants
G h d i i h
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Growth retardation withconstitutive over-expression of
FaNES1 in Arabidopsis
The Cost of Terpenoid Production in
Pl
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Plants
Constitutive over-expression of FaNES1 in potato controlledby the Rubisco promoter
The Rubisco promoter is x 10 fold stronger than the 35Spromoter
Rubisco T
Plastid targeting
FaNES1
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Effect of Linalool Expression on Potato Effect of Linalool Expression on Potato
Ph tPh t
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Phenotype Phenotype
0
2
4
6
8
10
1214
1 2 3 4 5 6 7 8 9
K-con
K-R5.2K-R1.1 K-R1.7 K-R1.2 K-R1.4 K-R7.1 K-R3.4K-R1.3
Potato Plants Overexpressing FvPINS
100 20.14
Wild
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Time4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
%
0
100
%
0
100
%
0
00
2.66
19.3418.51
20.7621.2022.4421.69 22.97
24.20
9.53
2.63
10.59
9.53
2.65
10.59
20.14
22.97 24.20
R-F 10
R-Z 31
Wild
Alpha-pinene Beta -pinene
R-Z 31
R-F 10
Concl sions
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Conclusions
•Engineering with a very strong, constitutivepromoter is deleterious to plants (toxicity or
altered precursor pool for other pathways)
•Use of specific and/or inducible promoters for
engineering terpenoids
Volatiles Produced by Trangenic plantsVolatiles Produced by Trangenic plants
Influence Insect Behevior Influence Insect Behevior
Leaves detached
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Leaves detachedfrom transgenic
Arabidopsis plantsexpressing thestrawberry
FaNES1 gene.
Linalool deters
aphids
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ConclusionsConclusions
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ConclusionsConclusions
-Terpenoids produced by engineered plantsinfluence insect behavior
-High levels of linalool production detersinsects (aphids and thrips) in different plant
species