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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


site in Arabidopsis


site in potato


site in Arabidopsis


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


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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


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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

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May 022007

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