Secondary Metabolites: Biochemistry and Role in Plants

Introduction

Secondary Metabolites are Derived from Primary Metabolites

PrimaryPrimary--Secondary metabolites boundary ??Secondary metabolites boundary ??

GA biosynthesis Resin

component

Essential amino acid Alkaloid

Main Groups of Secondary Metabolites Main Groups of Secondary Metabolites in Plantsin Plants

29,000 terpenes29,000 terpenes-- derived from the C5 derived 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 or shikimate pathway or malonate/acetate pathwaymalonate/acetate pathway

Main Secondary metabolitesMain Secondary metabolites

Nitrogen containing:- Alkaloids (12,000)- Non protein amino acids (600)- Amines (100)- Cyanogenic glycosides (100)- Glucosinolates (100)

Main Secondary metabolitesMain Secondary metabolitesWithout nitrogen:

- Terpenoids (29,000):mono- 1000sesquiterpene- 3000diterpenes-1000triterpenes, steroids, saponines- 4,000

- Phenolics (8,000):Flavonoids- 2000Polyacetylens-1000Polyketides- 750Phenylpropanoids- 500

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

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

SMs sequestration in to VacuolesSMs sequestration in to Vacuoles

Water soluble compounds-alkaloids, NPAAs,cyanogenic glucosides, glucosinolates, saponines, anthocyanines, flavonoids,cardenolides

ATP-dependant transporter

SMs sequestration in to VacuolesSMs sequestration in to Vacuoles--Anthocyanin exampleAnthocyanin example

- Anthocyanines- blue-red flavonoid pigments

- They are stabilized in the vacuole

- Oxidized in the cytosol

- The sequestration is a detoxification process

SMs sequestration in to VacuolesSMs sequestration in to Vacuoles--Anthocyanin exampleAnthocyanin example-- Bz2 mutantBz2 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

SMs sequestration in VacuolesSMs sequestration in Vacuoles-- Anthocyanin Anthocyanin exampleexample-- bz2bz2 & the an9 mutant& the an9 mutant

bz2 an9

SMs sequestration to a location with a solid SMs sequestration to a location with a solid barrier and not with a biomembrane barrier and not with a biomembrane (interfered by lipophilic SMs) (interfered by lipophilic SMs)

Thyme-glandular trichomes

Mint-glandular trichomes

Lemon leaf-secretory cavity

Pine- resin duct

Storage in LATICIFERS

- Latex is a sap mixture of 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 a milky appearance

Long Distance Transport of SMsIn Xylem, Phloem or Apoplastic transport

Long-distance phloem transport of glucosinolates Chen et al., 2001

Long-distance phloem transport of glucosinolates

Chen et al., 2001

- Intact Glucosinolates are transported

- Selection of a specific glucosinolate to be loaded into the phloem

- Presence of glucosinolates in the phloem provide means of defense against insects

- Export of glucosinolates from fully expanded leaves and senescent parts

- Export to sink tissues, seeds, flowers

Costs of Secondary metabolism (ATP / NADPH2 consumption)

- Biosynthesis of precursors and secondary metabolites

- Transport and storage

- Formation of specialized storage compartments (e.g. trichomes)

- Synthesis of mRNA and proteins (transcription translation)

Often needed in HIGHconcentrations(1-3% of dry weight are regularly seen)

Function of Secondary MetabolitesOften arguments that SMs are waste products but this cannot explain:

- production of SMs in young tissues

- plants are autotrophs and waste products are typical and 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

Function of Secondary Metabolites -DEFENSE - ATTRACTION - PROTECTION (uv)

- Most animals can move-run away and posses an 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

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 attract predatory organisms (tritrophic interactions)

competing plants (inhibition of germination and seedlings growth)

UV-protection

M. Wink, Annual Plant Review, 1999

Examples of plant SMs and their proposed functionInsect feeding

deterrentVisual pollinator attractant

Defense toxinOlfactory pollinator attractant

Antifungal toxinDefense toxin

From Pichersky and Gang, 2000

Production of SMs for defense against herbivores and pathogens is not necessarily constitutive

- Wounding and infection trigger INDUCED accumulation of SMs

herbivoreinhibition

Secondary metabolism

activation of prefabricated defense chemicalsPLANT

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

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

The "mustard oil bomb"-- A binary Glucosinolate-Myrosinase chemical defense system

Glucosinolates breakdown products1- isothiocyanates2- nitriles and elemental sulfur3- thiocyanates4- oxazolidine--thiones5- epithionitriles

Grubb and Abel, TIPS, 2006

Targets for SMs in animalsystems

- Nervous system (perception,processing, signal transduction

- Development- Muscles and motility- Digestion- Respiration- Reproduction and fecundity

Co-evolution in plant SMs - natural enemy

- The SM defense system works in general but not always

- Some herbivores and microorganisms have evolved that have 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

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

Adaptations of specialist herbivores & pathogens

Herbivores (continued)-

- Resorption and accumulation:- Specific compartments / cells / tissues for sequestration- Evolution of insensitivity

- Use of plant SMs in diet:- defense against predators - signal molecules (pheromones)- morphogen

Adaptations of specialist herbivores & pathogens

Microorganisms:

- Inactivation of SMs

- Evolution of insensitivity

Co-evolution in plant SMs - natural enemy

Natural enemy Counter resistancePlant taxonPlant defense

Toxic amino acids Various Leguminosae

Bruchid weevil Modified tRNA synthase

Dioclea seed L-canavanine(similar to arginine, no protein amino acid)

Weevil

Co-evolution in plant SMs - natural enemy

- Canavanine is toxic due to its incorporation into proteins that rise to functionally aberrant polypeptides

- The tRNA- Arginine in insects uses also Canavanine

- The insect mutated its tRNA and will not incorporate canavanine instead of Arginine

Adaptations of specialist herbivores & pathogens

The process of co-evolution between plants and their natural enemies is believed to have generated much of the earth's biological diversity

This includes chemical diversity!!

SMs in Arabidopsis

SMs in Arabidopsis

Terpenes (mono and sesquiterpenes in Arabidopsis

Chen et al., 2003

Expression pattern of a Terpene Synthase in Arabidopsis

The Terpenoids or The Terpenoids or IsoprenoidsIsoprenoids

The name Terpenoid & Isoprenoid

- The name terpenoid derives from the fact that first members of the class were isolated from TURPENTINE [the distillate from tree (e.g.. pine) resins]

- Isoprenoid, since ISOPRENE is the basic unit of C5 building them (C5H8)

The biogenetic isoprene rule

Leopold Ruzika (1930s; Nobel Prize chemistry 1910): A compound is an "isoprenoid" if it is derived biologically from an "isoprenoid" with or without rearrangements

The Terpenoids of Plant origin The Terpenoids of Plant origin Biological Role (volatile and non volatile):

- Flavour, fragrance, scent- Antibiotics- Hormones- Membrane lipids- Insect attractants- Insect antifeedants- Mediate the electron transport processes (in respiration and photosynthesis)

Terpenoids and CommunicationTerpenoids and Communication

Below ground attraction: orientation

cues (non-volatile)

Below ground protection: anti-

microbial, antifeedant (non-volatile)

Above ground attraction: fragrance

(volatile)

Above ground protection: repellents,

antifeedants, predator attraction

(volatile/non-volatile)

Precursors of Terpenoids

Mixed Origins of Terpenoids Precursors (Meroterpenes)

Terpenoids Terpenoids -- Important Molecules !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 Monoterpenes (C(C1010))

Sesquiterpenes Sesquiterpenes (C(C1515))

Diterpenes (CDiterpenes (C2020))

TriterpenoidsTriterpenoids(C(C3030))

TetraTetra--terpene / Carotenoids (Cterpene / Carotenoids (C4040))

Biosynthesis in two main compartmentsBiosynthesis in two main compartments

Mevalonate pathwayMevalonate pathway leading to IPP in leading to IPP in the cytosol the cytosol

The The MEP pathwayMEP pathway leading to IPP in the leading to IPP in the plastidsplastids

Biosynthesis in two main compartmentsBiosynthesis in two main compartments

Mevalonate pathwayMevalonate pathway leading to IPP in leading to IPP in the cytosol the cytosol

The The MEP pathwayMEP pathway leading to IPP in the leading to IPP in the plastidsplastids

CytosolMEP

Plastid

Mevalonate

Acetyl-CoA HMG-CoAHMGR

MVA

DMAPP

IPP

Cytokinins

FPP

SqualeneBRsTriterpenes

Polyprenols

DolicholPrenylation

DMAPP

IPP

FPP

Ubiquinones

Terpenes

Monoterpenes

PyruvateGlyAld-3P

MEP

DXP

DMAPP IPP

GPP

DXS

GGPP

DXR

HDR+

Carotenoids

PhylloquinoneTochpherols

GibberellinsPlastoquinone

ABA

PLASTID

CYTOSOL

MITOCHONDRION

ENDOPLASMIC RETICULUM

Sterols

Terpene alcoholsCYTP450

Diterpenes

Sesquiterpenes

Terpenes Reduced/oxidizedterpenes

Reductase/dehydrogenase

Biosynthesis of terpenoidsBiosynthesis of terpenoids

Three main reactions:

- Generating precursors by prenyltransferases (GPP, FPP,GGPP)

- Terpene synthase reactions (e.g. monoterpene synthase/cyclase)

- Modification steps

Biosynthesis of Precursors Biosynthesis of Precursors (prenyltransferases)(prenyltransferases)

Biosynthesis of Precursors Biosynthesis of Precursors (prenyltransferases)(prenyltransferases)

Cytosol Plastid

OPP OPP

DMAPPIPP

OPP OPP+IPP DMAPP

2 x +

FDP - synthase GDP - synthase

OPP

OPP

Farnesyl diphosphate Geranyl diphosphate

Terpene CyclasesTerpene Cyclases

One One enzymeenzyme……....OneOnesubstratesubstrate……....Multiple Multiple productsproducts

Terpene Terpene Cyclases Cyclases (Mono)(Mono)

Terpene Terpene Cyclases Cyclases (Sesqui(Sesqui--))

Terpene Terpene Cyclases Cyclases (Diterpene)(Diterpene)

isopentenyl diphosphate(IDP)

dimethylallyl diphosphate(DMAPP)

GDP synthase

geranyl diphosphate(GDP)

monoterpene synthases +/-modifying enzymes

OH

O

O

O

H

OO

H

farnesyl diphosphate(FDP)

squalene-2,3-epoxide

MONOTERPENOIDS SESQUITERPENOIDS

DITERPENOIDS TRITERPENOIDS

O

NH

O

OH

O

OBzH

OAc

AcO OH

OOH

O

PPO

OPP

OPP

geranylgeranyl diphosphate(GGDP)

O

OO

O

O

H

H

H

O

HOOH

CO2H

HOOH

CO2HHO

OH

paclitaxelglycyrrhizin

(E,E)-α-farneseneartemisininlinalool α-pinene perilla alcohol

OAc

OOH

O

HO

HOH

H

OH

cucurbitacin C

CHOCHO

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 StructuresStructures

OPP

OPP OPP

OH O O

O

O

Isopentenyl diphosphate Dimethyl allyl diphosphate

(-) limoneneGeranyl diphosphate (-) trans- Isopiperitenol (-) Isopiperitenone(+)-cis-Isopulegone

(-)-Limonene cyclase

Limonene 3-hydroxylase dehydrogenase reductase

isomerase

GDP-synthase

isomerase

O

reductase reductase

OH OH

reductase+ (+)-pulegone

(-)- Menthol (+)-neomenthol (-)- menthone (+)-isomenthone

Monoterpenoid Biosynthesis in Mint

G.W. Turner and R. Croteau, PlantPhysiol 136 (2004)

Production in PlantsStorage:

* Glandular trichomes: Labiatae like Mentha, Cannabis

* Cavities : Eucalypt, Citrus

* Resin ducts : pine trees

Production and direct emission:

* Flowers

* Leaves

* Fruit

Most secondary metabolites in Basil are produced in the Peltate Glands

Peltate Glands

Peltate Glands Isolated From Sweet Basil

Terpenoids in Peltate Glands (Sweet Basil)

Monoterpenes Sesquiterpenes

Metabolic Metabolic Engineering of Engineering of

Terpenoid Terpenoid BiosynthesisBiosynthesis

OH

Why? Metabolic Engineering of Why? Metabolic Engineering of Terpenoids in PlantsTerpenoids in Plants

In addition, plants altered in the profile of In addition, plants altered in the profile of terpenoids (and pool of precursors) make terpenoids (and pool of precursors) make

fundamental fundamental an important contribution to an important contribution to on their biosynthesis and on their biosynthesis and studiesstudies

regulationregulation

FaNES1, a Dual Linalool / FaNES1, a Dual Linalool / Nerolidol SynthaseNerolidol Synthase

Using FaNES1 allows evaluation of both monoUsing FaNES1 allows evaluation of both mono-- and and sesquiterpene productionsesquiterpene production

OPPOH

farnesyl diphosphate (3S )-(E)-nerolidol

OHOPP

geranyl diphosphate S -linalool

Mg2+ /Mn2+

Mg2+ /Mn2+

FaNES1

FaNES1

Introducing the FaNES1 Gene to Introducing the FaNES1 Gene to ArabidopsisArabidopsis

Enh 35S T

Plastid targeting

FaNES1 Expected resultmonoterpenes

produced in plastids:

linalool

linalool derivatives formed ?

Wild-type Arabidopsis leaves do not produce linalool

SS--Linalool Formation in Leaves of Linalool Formation in Leaves of ArabidopsisArabidopsis

23 24 250

100

0

100

0

100

Time, min

Rel

ativ

e de

tect

or r

espo

nse

S-Linaloolreference

R-Linalool reference

Transgenic Arabidopsis

Further ModificationFurther ModificationFree and Glycosidically Bound Terpenoids Produced by Arabidopsis

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Nt-1 Nt-2 Nt-3 Nt-4 Nt-5Tr-9

_1Tr-9

_2Tr-9

_3Tr-9

_4Tr-9

_5Tr-9

_6

E-8-hydroxy-linalool

E-8-hydroxy-6,7-dihydro-linalool

nerolidollinalool

A

Con

cent

rat io

n (m

g kg

-1-F

W)

Con

cent

rat io

n (m

g kg

-1-F

W)

0

20

40

60

80

100

120

Nt-1 Nt-2 Nt-3 Nt-4 Nt-5

Tr-9_1

Tr-9_2

Tr-9_3

Tr-9_4

Tr-9_5

Tr-9_6

E-8-hydroxy-linalool

Z-8-hydroxy-linaloolE-8-hydroxy-6,7-dihydro-linalool

nerolidol

linalool

Plant

Free Glycosidically Bound

Introduced product: Introduced product: linaloollinaloolModified by endogenous Modified by endogenous enzymes:enzymes:

P450 hydroxylation (2P450 hydroxylation (2--3)3)Double bond reductionDouble bond reductionGlycosylation (2Glycosylation (2--3)3)

OH

OH

OH

OH

OH

OH

OH

S-Linalool

E-8-Hydroxylinalool Z-8-Hydroxylinalool

E-8-Hydroxy-6,7-dihydrolinalool

GlycosideGlycoside

Glycoside

Further ModificationFurther Modification

Further ModificationFurther ModificationThe sum of glycosylated components was in some of the transgenic lines up to 40 to 60-fold higher than the sum of the corresponding free alcohols

Con

cent

ratio

n (m

g kg

-1-F

W)

0

20

40

60

80

100

120

140

160

Nt-1_n

o

Nt-2_n

o

Tr26-36

_no

Tr26-30

_no

Tr26-41

_no

Tr9-15

_no

Tr9-12

_no

Tr9-21

_sm

Tr9-6_

sm

Tr26-29

_sm

Tr9-7_

sm

Tr26-32

_sm

Tr26-28

_sm

Sum - glycosidically bound

Sum - free

Further ModificationFurther ModificationOH

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 highest levels in transgenic lines

2. The only component detected in leaves of wild-type plants

3. Endogenous enzymes already active and can utilize efficiently the newly introduced linalool

E-8-Hydroxylinalool and its glycoside:

Potato Plants Transformed with the Same Construct

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)

20

40

60

80

100

20

40

60

80

100

20

40

60

80

100

20

40

60

80

100

20

40

60

80

100

NL:

NL:

R - LinaloolS - LinaloolReference

Wild-type

Transgenic # 1

Transgenic # 2

Transgenic # 3

Rel

ativ

e ab

unda

nce

OH

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 glycosylationsite in potato

assumed glycosylationsite in Arabidopsis

assumed glycosylation site in potato

assumed glycosylationsite in Arabidopsis

assumed glycosylationsite in potato

further further modificationmodificationin transgenic in transgenic potato plantspotato plants

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 ArabidopsisIntroducing the CiGASlo Gene to Introducing the CiGASlo Gene to

ArabidopsisArabidopsisEnh 35S TCiGASlo

Wild-type Arabidopsis leaves do not produce Germacrene A

Cytosolic production of a Germacrene A synthase from

Chicory

Engineering Sesquiterpenes in ArabidopsisTraces of the thermal rearrangement product of Traces of the thermal rearrangement product of

Germacrene A (de Kraker et al., 1998) were detected in Germacrene A (de Kraker et al., 1998) were detected in leavesleaves

Unexpected: Sesquiterpene Production with Unexpected: Sesquiterpene Production with Plastidic Targeting of FaNES1Plastidic Targeting of FaNES1

800

900

1000

1100

1200

1300

1400

0

10000

20000

ime

nero

lido

l

800

900

10 00 11 00 12 00 13 00 14 000

10000

20000

ime

Abu

ndan

ce

ransgenicrabidopsis

ild typerabidopsis

Abu

ndan

ce

lina

lool

Linalool

Nerolidol

Nerolidol is Produced at Low Level Also in PotatoNerolidol is Produced at Low Level Also in Potato

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

Rel

ativ

e A

bund

ance

20

40

60

80

100 34.67

34.10

34.16 34.39 34.5033.78 34.3434.00 34.7633.81 34.80 35.0434.92 35.0734.68

34.1134.50

34.3934.2134.0033.77 33.92 34.7834.30 34.82 35.00 35.0834.65

34.1034.50

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?

Monoterpenes

GPP GGPP

IPP DMAPP

Emission /storage

CYTOSOL

Sesquiterpenes

FPP

GPP

IPP DMAPP

PLASTID

Hydroxylated monoterpenes

MITOCHONDRIA

FPP

Engineering Sesquiterpenes in ArabidopsisIntroducing the FaNES1 fused to a Mitochondrial Introducing the FaNES1 fused to a Mitochondrial

targeting signaltargeting signal to Arabidopsisto Arabidopsis

Enh 35S TFaNES1

Production of terpenoids in Mitochondria

Cox IV

Mitochondrial targeting, cytochrome c oxidase

Engineering Sesquiterpenes in Arabidopsis

Undamaged Wild-type

Transgenic Undamaged (nerolidol and DMNT)

Transgenic undamaged (only nerolidol)

Nerolidol

DMNT (C11 homoterpene) Nerolidol

C15 sesquiterpene C11 homoterpene

Conclusions • Engineering sesquiterpene production in the cytosol 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

Growth retardation with constitutive over-expression of FaNES1 in Arabidopsis

The Cost of Terpenoid Production in

Plants

Constitutive over-expression of FaNES1 in potato controlled by the Rubisco promoter

The Rubisco promoter is x 10 fold stronger than the 35S promoter

Rubisco T

Plastid targeting

FaNES1

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 Effect of Linalool Expression on Potato PhenotypePhenotype

02468

101214

1 2 3 4 5 6 7 8 9

K-con K-R5.2 K-R1.1 K-R1.7 K-R1.2 K-R1.4 K-R7.1 K-R3.4K-R1.3

Potato Plants Overexpressing FvPINS

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

100 20.14

2.66

19.3418.5120.7621.20

22.4421.69 22.9724.20

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

Conclusions

•Engineering with a very strong, constitutive promoter 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 plants Volatiles Produced by Trangenic plants Influence Insect BeheviorInfluence Insect Behevior

Leaves detached from transgenic Arabidopsis plants expressing the strawberry FaNES1 gene.

Linalool deters aphids

No Choice Greenhouse Test in No Choice Greenhouse Test in Perspex HoodsPerspex Hoods

Linalool synthase Linalool synthase chrysanthemum T58 chrysanthemum T58 lineslines20 females20 femalesN=6N=6--13 plants per line13 plants per line3 weeks; 22 C3 weeks; 22 C

Thrips population on linalool chrysanthemum 3 weeks after inoculation with 20 females

0

2

4

6

8

10

12

14

16

TOTAL ADULTS LARVAE

Inse

cts

per p

lant

control 1581T58-9

p=0.05p=0.04

Different Thrip Damage Phenotype in Different Thrip Damage Phenotype in Transgenic Linalool PlantsTransgenic Linalool Plants

Control

only edges

large surface

Linalool

transgenic

only spots

not at the edges

ConclusionsConclusions

-Terpenoids produced by engineered plants influence insect behavior

-High levels of linalool production deters insects (aphids and thrips) in different plant species