Secondary Metabolites: Biochemistry and Role in Plants

Introduction

Secondary Metabolites are Derived from Primary Metabolites

Primary-Secondary metabolites boundary ??

GA biosynthesis

Resin component

Essential amino acid

Alkaloid

Main Groups of Secondary Metabolites in Plants

29,000 terpenes- derived from the C5 precursor isopentenyl diphosphate (IPP) 12,000 alkaloids- derived from amino acids 8,000 phenolics- shikimate pathway or malonate/acetate pathway

Main Secondary metabolites

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

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 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 Vacuoles

Water soluble compoundsalkaloids, NPAAs, cyanogenic glucosides, glucosinolates, saponines, anthocyanines, flavonoids, cardenolides

ATPdependant transporter

SMs sequestration in to VacuolesAnthocyanin 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 VacuolesAnthocyanin example- 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-xpump in the tonoplast membrane

SMs sequestration in Vacuoles- Anthocyanin example- bz2 & the an9 mutant

bz2

an9

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

Lemon leafsecretory 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 SMs In Xylem, Phloem or Apoplastic transport

Long-distance phloem transport of glucosinolates

Chen et al., 2001

Long-distance phloem transport of glucosinolates

- 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
Chen et al., 2001

Costs of Secondary metabolism (ATP / NADPH2 consumption)

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

- Biosynthesis of precursors and secondary metabolites - Transport and storage - Formation of specialized storage compartments (e.g. trichomes) - 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 - 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:

Herbivores (insects, vertebrates) Repellence, deterrence, toxicity

Plant SMs - mixtures - variation in time, space & dev. stage

Defense

Microbes (bacteria, fungi, viruses)

Growth inhibition and toxicity

competing plants (inhibition of germination and seedlings growth)

UV-protection

Attraction

M. Wink, Annual Plant Review, 1999

- pollinating insects - seed dispersing animals - root nodule bacteria - induced volatiles attract predatory organisms (tritrophic interactions)

Examples of plant SMs and their proposed function

Visual pollinator attractant Insect feeding deterrent

Defense toxin Olfactory pollinator attractant

Defense toxin

Antifungal 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
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

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 products 1- isothiocyanates 2- nitriles and elemental sulfur 3- thiocyanates 4- oxazolidine--thiones 5- epithionitriles

Grubb and Abel, TIPS, 2006

Targets for SMs in animal systems

- 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

Plant defense
Toxic amino acids

Plant taxon
Various Leguminosae

Natural enemy
Bruchid weevil

Counter resistance
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 Isoprenoids

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

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 Communication

Above ground attraction: fragrance (volatile) Above ground protection: repellents, antifeedants, predator attraction (volatile/non-volatile)

Below ground attraction: orientation cues (non-volatile)

Below ground protection: antimicrobial, antifeedant (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 pathway leading to IPP in the cytosol The MEP pathway leading to IPP in the plastids

Biosynthesis in two main compartments

Mevalonate pathway leading to IPP in the cytosol The MEP pathway leading to IPP in the plastids

MEP Cytosol Plastid

Mevalonate

CYTOSOL
Acetyl-CoA HMG-CoA
HMGR

PLASTID
GlyAld-3P
DXS

Pyruvate DXP
DXR

Sterols Triterpenes MVA IPP Squalene BRs Sesquiterpenes FPP DMAPP Polyprenols ABA Cytokinins Prenylation Dolichol

MEP
HDR+

MITOCHONDRION
IPP DMAPP Carotenoids Diterpenes Gibberellins Plastoquinone Phylloquinone Tochpherols FPP Ubiquinones

DMAPP GPP

IPP GGPP

Reductase/dehydrogenase

Terpenes

Reduced/oxidized terpenes

Monoterpenes

ENDOPLASMIC RETICULUM
Terpenes
CYTP450

Terpene alcohols

Biosynthesis 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 (prenyltransferases)

Biosynthesis of Precursors (prenyltransferases)

Cytosol
IPP
OPP

Plastid
DMAPP
IPP
OPP

DMAPP

2x

OPP

OPP

FDP - synthase

GDP - synthase
OPP

OPP

Farnesyl diphosphate

Geranyl diphosphate

Terpene Cyclases

One enzyme..One substrate..Multiple products

Terpene Cyclases (Mono)

Terpene Cyclases (Sesqui-)

Terpene Cyclases (Diterpene)

OH OH

MONOTERPENOIDS SESQUITERPENOIDS
O O O

H O

linalool

-pinene perilla alcohol monoterpene synthases +/modifying enzymes Strawberries

OPP

artemisinin Anti-malarial drug farnesyl diphosphate (FDP)

PPO

(E,E)--farnesene

geranyl diphosphate (GDP)

sesquiterpene synthases +/modifying enzymes

GDP synthase
OPP OPP

FDP synthase isopentenyl diphosphate dimethylallyl diphosphate (IDP) (DMAPP)

squalene synthase squalene epoxidase

GGDP synthase
OPP

diterpene synthases +/modifying enzymes

CHO CHO

geranylgeranyl diphosphate (GGDP)

squalene-2,3-epoxide triterpene synthases +/modifying enzymes

O OH O O OAc

OH H O

Myzus persicae polygodial

AcO O NH O O OH OH H OBz OAc O O OH

Leaf of cucumber
HO

OH H O O O HO OH OH O H CO2H

cucurbitacin C
CO2H HO HO

paclitaxel

glycyrrhizin

DITERPENOIDS TRITERPENOIDS

Modification of Monoterpene Structures

isomerase
OPP OPP

Isopentenyl diphosphate
OPP

Dimethyl allyl diphosphate

GDP-synthase

(-)-Limonene cyclase
Geranyl diphosphate (-) limonene

Limonene 3hydroxylase

OH

dehydrogenase

reductase

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

isomerase reductase reductase

OH

reductase
O

OH

(+)-pulegone

(-)- Menthol

(+)-neomenthol

(-)- menthone

(+)-isomenthone

Monoterpenoid Biosynthesis in Mint

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

Production in Plants
Storage: * 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 Engineering of Terpenoid Biosynthesis

OH

Why? Metabolic Engineering of Terpenoids in Plants In addition, plants altered in the profile of terpenoids (and pool of precursors) make an important contribution to fundamental studies on their biosynthesis and regulation

FaNES1, a Dual Linalool / Nerolidol Synthase

Using FaNES1 allows evaluation of both mono- and sesquiterpene production
OPP OH

FaNES1
Mg 2+ /Mn 2+

geranyl diphosphate

S -linalool

OH OPP

FaNES1
Mg 2+ /Mn 2+

farnesyl diphosphate

(3 S )-(E)-nerolidol

Introducing the FaNES1 Gene to Arabidopsis

Plastid targeting Enh 35S FaNES1 T

Expected result monoterpenes produced in plastids: linalool linalool derivatives formed ?

Wild-type Arabidopsis leaves do not produce linalool

S-Linalool Formation in Leaves of Arabidopsis

100

S-Linalool reference

Relative detector response

0 100

R-Linalool reference

0 100

Transgenic Arabidopsis

23

24 Time, min

25

Further Modification
Free and Glycosidically Bound Terpenoids Produced by Arabidopsis

Free
Concentration (mg kg-1-FW) Concentration (mg kg-1-FW)

Glycosidically Bound
120 E-8-hydroxy-linalool 100 Z-8-hydroxy-linalool E-8-hydroxy-6,7-dihydro-linalool nerolidol 80 linalool

1.6 E-8-hydroxy-linalool 1.4 1.2 1 0.8 0.6 0.4 E-8-hydroxy-6,7-dihydro-linalool nerolidol linalool

60

40

20 0.2 0
_2 _5 _3 N t -1 N t- 2 N t-3 N t-4 N t- 5 _1 _4 _6 Tr -9 Tr -9 Tr -9 Tr -9 Tr -9 Tr -9

0
_2 _3 _4 N t -1 N t -2 N t-3 N t-4 N t-5 _1 _5 Tr -9 Tr -9 Tr -9 Tr -9 Tr -9 Tr -9 _6

Plant

OH

Further Modification
Introduced product: linalool Modified by endogenous enzymes:
P450 hydroxylation (2-3) Double bond reduction Glycosylation (2-3)
S-Linalool
OH OH

Glycoside
OH HO

Glycoside

E-8-Hydroxylinalool

Z-8-Hydroxylinalool

OH

Glycoside
OH

E-8-Hydroxy-6,7-dihydrolinalool

Further Modification
The 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
160 140

Sum - glycosidically bound Sum - free

120

100

Concentration (mg kg-1-FW)

80

60

40

20

no

_n o

_n o

sm

_n

N t-2 _

_s m

_s m

sm

96_

-3 0

-4 1

915

-3 6

912

921

-2 9

97_

2_ sm

N t-1

Tr

Tr

Tr

Tr

Tr

26 -3

Tr

Tr

26 -2

Tr

26

26

26

Tr

Tr

26

Tr

8_ sm

_n

_n

_n

Further Modification
OH

E-8-Hydroxylinalool and its glycoside: 1. Produced to the highest levels in transgenic lines 2. The only component detected in leaves of wildtype plants 3. Endogenous enzymes already active and can utilize efficiently the newly introduced linalool
OH

S-Linalool

OH

Glycoside
OH HO

Glycoside

E-8-Hydroxylinalool

Z-8-Hydroxylinalool

OH

Glycoside
OH

E-8-Hydroxy-6,7-dihydrolinalool

Potato Plants Transformed with the Same Construct

100 80 60 40 20

R - Linalool
Reference Wild-type

S - Linalool

100 80

Relative abundance

60 40 20

100 80 60 40 20
NL:

Transgenic # 1

100 80 60 40 20
NL:

Transgenic # 2 Transgenic # 3
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

100 80 60 40 20

Time (min)

further modification in transgenic potato plants

glycoside

OH

assumed glycosylation site in potato glycoside

S-linalool assumed glycosylation site in potato

OH
OH

assumed glycosylation site in Arabidopsis

OH
HO

glycoside

E-8-hydroxy-linalool

Z-8-hydroxy-linalool

OH

assumed glycosylation site in potato glycoside assumed glycosylation site in Arabidopsis

OH

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

Wild-type Arabidopsis leaves do not produce Germacrene A

Engineering Sesquiterpenes in Arabidopsis

Traces of the thermal rearrangement product of Germacrene A (de Kraker et al., 1998) were detected in leaves

Unexpected: Sesquiterpene Production with Plastidic Targeting of FaNES1

ransgenic
l l in o o a l

Linalool rabidopsis

ild type

rabidopsis
Abundance
20 00 0

Abundance

20 00 0

10000

0 8 0 0 9 0 0

10 00 11 00 12 00 13 00 14 00 ime

10 00 0

Nerolidol

0 8 00 9 0 0 10 00 11 00 12 00 13 0 0 14 00 im e

n l er l id o o

Nerolidol is Produced at Low Level Also in Potato

100 80 34.10 60 40 20 33.78 33.81 34.00 34.16 34.67

Nerolidol
34.34 34.39 34.50 34.68 34.76

Wild-Type
34.80 34.92 35.04 35.07

Relative Abundance

100 80 60 40 20 100 80 60 40 20 100 80 60 40 20 33.77 33.8 33.99 33.9 34.03 34.0 34.1 34.21 34.2 34.31 34.37 34.3 34.4 34.5 34.6 34.11 34.49 34.51 33.80 33.91 33.95 34.18 34.26 34.38 34.10 34.50 33.77 33.92 34.00 34.21 34.30 34.39 34.11 34.50

Transgenic #1
34.78 34.82 34.65 35.00 35.08

Transgenic #2
34.78 34.66 34.87 34.94 35.07

Transgenic #3
34.75 34.83 34.7 34.8 34.9 34.95 35.00 35.0 35.14 35.1

Time (min)

Availability of Precursor Pools?

Emission /storage

Sesquiterpenes

Hydroxylated monoterpenes Monoterpenes

FPP GPP GPP FPP IPP CYTOSOL DMAPP IPP PLASTID DMAPP MITOCHONDRIA GGPP

Engineering Sesquiterpenes in Arabidopsis

Introducing the FaNES1 fused to a Mitochondrial targeting signal to Arabidopsis
Mitochondrial targeting, cytochrome c oxidase Enh 35S Cox IV FaNES1 T

Production of terpenoids in Mitochondria

Engineering Sesquiterpenes in Arabidopsis

Undamaged Wild-type

Nerolidol

Transgenic undamaged (only nerolidol)

DMNT (C11 homoterpene)

Nerolidol

Transgenic Undamaged (nerolidol and DMNT)

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

Plastid targeting Rubisco FaNES1 T

The Cost of Terpenoid Production in Plants

Bleaching and growth retardation with constitutive overexpression 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

K-con

K-R5.2

K-R1.1

K-R1.7

K-R1.2

K-R1.3

K-R1.4

K-R7.1

K-R3.4

Potato Plants Overexpressing FvPINS

100
20.14

Wild
R-Z 31

%
2.66 18.51 19.34 20.76 21.20 22.44 22.97 21.69 24.20

100

R-Z 31
9.53 2.63 10.59

R-F 10
Beta -pinene

100

Alpha-pinene

9.53

R-F 10
20.14 22.97 24.20

%
2.65

10.59

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

24.00

26.00

Time 28.00

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 Influence Insect Behevior

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

Insects per plant

12 10
p=0.04

control 1581 T58-9

8 6 4 2 0 TOTAL

p=0.05

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