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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