Plant Secondary Metabolism

8/11/2019 Plant Secondary Metabolism

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Secondary Metabolism

Ben Field 2013www.lgbp.univ-mrs.fr

[email protected]



Three Parts

I Introduction

II Major pathwaysIII Synthesis and delivery




I Introduction

• What are secondary metabolites/natural products?

• Uses of secondary metabolites for mankind

• Uses of secondary metabolites for plants

• Major groups of natural products




Introduction- What are secondary metabolites?

Plants produce more than 200,000 different bio-active natural products

(secondary metabolites, specialised metabolites).





(secondary metabolites).

Probably lifestyle related:

• Plants are anchored to the ground, exposed to the environment

• Plants have no adaptive immune system (like animals)





(secondary metabolites).

Probably lifestyle related:

• Plants are anchored to the ground, exposed to the environment

• Plants have no adaptive immune system (like animals)

Some other organisms make secondary metabolites:

• Certain bacteria (e.g Actinomyces, Streptomyces)

• Filamentous fungi (e.g Penicillium sp)• Invertebrate marine sponges (plant-like)




• Primary metabolites are associated with essential cellular functions.

For example:

Phytosterols are essential for membrane structure

Same function as cholesterol in animals.

oxidosqualene

Cycloartenol synthase

cycloartenol

phytosterols




• Natural products / secondary metabolites are usually species specific and

dispensable. A secondary metabolite does not usually increase plant fitness

under normal laboratory conditions.

For example:

Beta-amyrin is the precursor of the avenacins that are found only in oat

(avoine).

Avenacins protect against fungal attack.

oxidosqualene

Beta-amyrin

synthase

α-1-ara(1-)β-D-glu(1-2)

β-D-glu(1-4)

Beta-amyrin

Papadopoulou et al., 1999 PNAS

Avenacin

O

O

O

NH

CH3

O

OH

O

OH

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC23166/?tool=pubmed






Natural products have been useful to man for 1000s of years:

• commodoties: herbs and spices, recreational drugs, materials

• healthcare: conventional and alternative medicines

• diet: beneficial health effects (recent discoveries)

Introduction- Uses of Secondary Metabolites



Plant OriginNatural Product

Major components Classes

the Camellia sinensis China Caffeine, theobromine, theophylline Phenylpropanoids, terpenes, alkalloids

café Coffea arabica Ethiopia Caffeine, theobromine, theophylline Phenylpropanoids, terpenes, alkaloids

opium Papaver somniferum Mesopotamia Morphine, codeine, thebaine Alkaloids

rubber Hevea brasiliensis South America cis-1,4-polyisoprene Terpenes

tobacco Nicotiana tabacum Brazil Nicotine Alkaloids

spices

gingembre Zingiber officinale Asia

Zingerone, gingeroles and

shoagoles Sesquiterpenes, monoterpenes

muscade Myristica Banda Islands myristicine, linalool, sabine Phenylpropanoids, terpenes

canelle Cinnamomum verum Sri Lanka cinnamaldehyde, eugenol Phenylpropanoids

Introduction- Uses of Natural Products: Commodities



Medicines Source Function Supplier Class

1826 morphine Papaver somniferum pain relief Merck alkaloid

1899 aspirin Reine-des-prés pain relief Bayer phenylpropanoids

1941 penicillin Penicillium anti-bacterial Merck non-ribosomal peptide

1987 lovastatin Aspergillus terreus/ red yeast rice anti-hyperlipidemic Merck polyketide

1987 artemisin Artemisia annua anti-malarial Baiyunshan terpene

1993 tacrolimus Streptomyces tsukubaensis immunosuppressant Fujisawa polyketide

1993 paclitaxel Pacific yew anti-cancer BMS terpene

1996 camptothecin Happy tree anti-cancer SKB, Pharmacia alkaloid

paclitaxel morphine

Streptomyces

Introduction- Uses of Natural Products: Health



Functional Foods Source Function Class

reservatrol, proanthocyanidins red wine life extension phenylpropanoids

glucosinolates broccoli anti-cancer glucosinolates

epicatechin chocolate improved circulation phenylpropanoids

isoflavonoids leguminosae anti-cancer? phenylpropanoids

caretenoids tomato anti-oxidants isoprenoids

Introduction- Uses of Natural Products: Diet



Introduction- Why do organisms make natural products?

• In the natural environment secondary metabolites can be essential for

survival and reproduction.






- Pigments and scents to attract pollinators

- volatile isoprenoids

- isoprenoid pigments







- Abiotic and biotic defense

- tree resin, complex terpene mixture








- Communication with other plants, microbes and animals

- feeding caterpillar causes the

release of volatile compounds

that attract a parasitic wasp








- Communication with other plants, microbes and animals

- Development?

Some secondary metabolites are in fact new hormones



Introduction- Major Groups of Natural Products

Natural products are derived from branch points with primary metabolism.

For example: The branch point between Sterols and Triterpenes

O

H

OH

H

H

HO

CAS

BAS

Beta-amyrin

cycloartenol

oxidosqualene phytosterols(essential)

avenacins

(triterpene

secondarymetabolites)



Introduction- Major Groups of Natural Products

The same metabolic branch points have often been independently discovered

multiple times during evolution (convergent evolution).

O

HO

BASavenacins

oats

HO

BAS’

medicago

saponinsmedicago

ginseng

oxidosqualene

ginsenoside

Triterpenes (isoprenoids)

The BAS and BAS’ enzymes evolved

independently.



II Major Groups of Natural Products

Several major classes of natural product can be distinguished:

• Isoprenoids/Terpenes

•Phenylpropanoids

• Cyanogenic glucosides

• Alkaloids

•

Polyketides



II Isoprenoids (or terpenes)

The most diverse group: >25000 structures known.

Derived from the 5 carbon (5C) isopentenyl pyrophosphate (IPP):

O

O

O-

O

P

O

-

O-

O

P

1

23

4

5



C10 geranyl diphosphate (GPP)

C15 farnesyl diposphate (FPP)

C20 geranyl geranyl diphosphate (GGPP)

GPP synthase

FPP synthase

GGPP synthase

II- Isoprenoid biosynthesis

ABA

cytokinins

isoprene

brassinosteroids

strigolactone

IPP (C5)IPP (C5) DMAPP (C5)



II- Why do plants make isoprene???

400 –600 mega-tonnes of isoprene are released into the atmosphere every year by plants

A major geochemical: isoprene reacts with OH radicals to form ozone in the troposphere

DMAPP isoprene

Isoprene synthase

Class I terpene synthase

Blue haze caused by isoprene emissions.

Blue Ridge Mountains, Virginia



Natural rubber (cis-1,4-polyisoprene)

Gutta-percha (trans-1,4-polyisoprene)

II- Polyterpenes Cn

rubber tree (Hevea brasiliensis)






GPP synthase

FPP synthase

GGPP synthase


ABA

cytokinins

isoprene

brassinosteroids

strigolactone




Acyclic monoterpenes

II- Fragrant monoterpenes (C10)

geraniol (present in rose, citronella, geranium andlemon oils)




Cyclic monoterpenes


menthol



“Limonene synthase”

GPP




Cyclic monoterpenes

Bicyclic monoterpenes


menthol


Eucalyptol (90% of eucalyptus

oil, present in thyme,

rosemary and sage oils).

Attractant for pollinators.


“Limonene synthase”

Pinene. Major component of

pine resin. Toxic to insects.

GPP






GPP synthase

FPP synthase

GGPP synthase


ABA

cytokinins

isoprene

brassinosteroids

strigolactone

IPP (C5) DMAPP (C5)





II- Artemesinin- an anti-malarial sesquiterpene C15

CNAP Artemisia Research Project funded by the Bill and Melinda Gates Foundation ($26M)

Aims to use modern high throughput methods to identify natural variation and

increase artemisinin yield by modern breeding (no transgenic technology).

http://www.york.ac.uk/org/cnap/artemisiaproject/

http://www.york.ac.uk/org/cnap/artemisiaproject/



II- Triterpenes C30 (X2 FPP C15)

O

O

O

NH

CH3

O

OH

O

OH

oxidosqualene

HO

beta-amyrin

O

Sad1

α-1-ara(1-)β-D-glu(1-2)

β-D-glu(1-4)

Sad2,3,4,7,9,10

Cross section of Avena strigosa root

Avenacin- an anti-fungal defence compound from Avena strigosa.

Avenacin are saponins, which are found in many plant species.

Avenacin

CYP450s, MT, ACT

Sad1WT

Papadopoulou et al., 1999 PNAS , Haralampidis et al. 2001 PNAS , and others















GPP synthase

FPP synthase

GGPP synthase


ABA

cytokinins

isoprene

brassinosteroids

strigolactone

IPP (C5) DMAPP (C5)






GPP synthase

FPP synthase

GGPP synthase


ABA

cytokinins

isoprene

brassinosteroids

strigolactone

IPP (C5) DMAPP (C5)



Precursors of red, orange and yellow pigments in bacteria, algae and higher plants.

Caretenoids perform three major functions in plants :

• accessory pigments for light harvesting

• prevention of photooxidative damage

• insect attraction

• BUT are they secondary metabolites??

II- Red, orange and yellow tetraterpenes (C40)

Beta-carotene (orange)

Lycopene (red)

In chloroplasts carotenoids are linked to the photosystem . These orange pigments arerevealed in Autumn when chlorophyll is degraded.



Carotenoids sometimes accumulate in specialised chloroplasts called chromoplasts.

II- Red, orange and yellow tetraterpenes (C40)



Simultaneously discovered by two

teams in 2008.

Strigolactones- derived from

carotenoid precursors

Originally secondary metabolites.

Now known to be hormones.

II- A new tetraterpene hormone…

Umehara et al., 2008 Nature ; Gomez-Roldan et al., 2008 Nature

http://www.nature.com.gate1.inist.fr/nature/journal/v455/n7210/abs/nature07272.html















GPP synthase

FPP synthase

GGPP synthase


ABA

cytokinins

isoprene

brassinosteroids

strigolactone




Mevalonate

(MVA) pathway Methylerythritol

phosphate (MEP)

pathway

diterpenes

triterpenes

II- Isoprenoid biosynthesis compartmentation



II- Major Groups of Natural Products


• Phenylpropanoids

• Cyanogenic glucosides and Glucosinolates

• Alkaloids



II- Phenylpropanoids

Phenylpropanoids are wide-spread in nature. Derived from the amino acids

phenylalanine and tyrosine.

Functions:

•defense

•structural components of cell walls

•protection from ultraviolet light•pigments

•signalling molecules






S H I K I M A T E P A T H W A Y

(A) Benzoates and

salicylates

(B) Coumarins (C) Monolignols, lignans

and lignin

(D) Flavonoids and

stilbenes

PAL= Phenylalanine ammonia-lyase , C4H= cinnamate-4-hydroxylase, 4CL= 4-coumarate:coenzyme A ligase

p-coumarateL-phenylalanine

C4HPAL 4CL

L-tyrosine

cinnamate

Hydroxycinnamic acids





(A) Benzoates and

salicylates


and lignin

(D) Flavonoids and

stilbenes



C4HPAL 4CL

L-tyrosine

cinnamate





vanilla pods

Simple soluble acids.

II- Phenylpropanoids, (A) benzoates

salicylic acid

PHYTOHORMONE

Accumulate in the roots of many plants, and are often induced in response

to pathogen attack.

methylbenzoate vanillic acid

( )



II- Phenylpropanoids, (B) coumarins

Soluble lactones. They have a sweet smell, like dried grass.

h l id ( ) i



Furano coumarins are formed from DMAPP (isoprenoid precursor) + umbelliferone

(coumarin).

Giant hogweed is rich in furanocoumarins.

Contact with the sap causes a photosensitive

skin rash.

psoralen

Furano-coumarins are used in UV light therapy to

treat psoriasis.

II- Phenylpropanoids, (B) coumarins

La berce du Caucase ou berce de

Mantegazzi

II h l id





(A) Benzoates and

salicylates


and lignin

(D) Flavonoids and

stilbenes



C4HPAL 4CL

L-tyrosine

cinnamate




II Ph l id (C) li i



Coniferyl alcoholCoumaryl alcohol Sinapyl alcohol

methyltransferase hydroxylase

reductase

p-coumarate

II- Phenylpropanoids, (C) lignin

II Ph l id (C) li i



Specific monolignol monomers are directed to different parts of the cell wall.


II Ph l id (C) li i



Polymerization process is not fully understood.

Driven by radical oxidation reactions catalysed by peroxidases.

Lignin appears random and chaotic.

However, lignin is optically active (i.e sterospecific) and polymerisation is directed by

dirigent proteins.

Davin and Lewis, 2005 Current Opinion in Plant Biology


II Ph l id (C) li i

http://dx.doi.org.gate1.inist.fr/10.1016/j.copbio.2005.06.011






Functions of lignins:

• Provide stiffness and strength to the secondary wall of vascular plants.

• Hydrophobic- allowing the development of tissues for efficient water transport in vascular

plants.

• Barrier to microbial attack.


II Phenylpropanoids (C) lignin



Functions of lignins:

• Give stiffness and strength to the secondary wall of vascular plants.

• Hydrophobic- allow development of tissues for efficient water transport in vascular plants.

• Barrier to microbial attack.

Problem for biofuel

lignin is a physical barrier to enzymes.

Termite stomachs contain bacteria

that can degrade lignin.

High throughput sequencing of termite

stomach DNA has helped identify these

valuable enzymes.

Warnecke et al., 2007 Nature


II Phenylpropanoids

http://www.ncbi.nlm.nih.gov/entrez/utils/pmliblink.cgi?id=16023847&lib=ifristilib







S

H I K I M A T E P A T H W A Y

(A) Benzoates and

salicylates


and lignin

(D) Flavonoids and

stilbenes



C4HPAL 4CL

L-tyrosine

cinnamate


II Phenylpropanoids (D) flavonoids



II- Phenylpropanoids, (D) flavonoids

More than >9000 structures characterised.

Several structural cores-• flavones

• isoflavones

• flavonols

• anthocyanins

Principal flavonoid enzymes:

CHS Chalcone synthase

CHI Chalcone isomerase

F3H Flavanone 3-hydroxylase

DFR Dihydroflavonol reductase

Lepiniec et al., 2006 Ann Rev Plant Biol

II Phenylpropanoids (D) flavonoids flavones

http://www.annualreviews.org.gate1.inist.fr/doi/full/10.1146/annurev.arplant.57.032905.105252?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed







luteolin (one of the most common flavones)

Peters et al., 1986 Science

Medicago truncatula Sinorhizobium melliloti

Flavone core

Chemotractant of Sinorhizobium meliloti

Nodulation: formation of nitrogen-fixing root

nodules

II- Phenylpropanoids, (D) flavonoids- flavones

II Phenylpropanoids (D) flavonoids isoflavones

http://www.ncbi.nlm.nih.gov.gate1.inist.fr/pubmed/3738520

http://www.ncbi.nlm.nih.gov.gate1.inist.fr/pubmed/3738520



Produced almost exclusively by the members of the Fabaceae/

Leguminosae (bean) family.

Defense compounds (e.g medicarpin from luzerne Medicago

truncatula)

Phytoestrogens- a diet high in phytoestrogens can

reduce female fertility in some animals.

e.g sheep eating clover (Trifolium repens)

Isoflavones

II- Phenylpropanoids, (D) flavonoids- isoflavones

II Phenylpropanoids (D) flavonoids anthocyanins



Vacuolar anthocyanins provide flower colours- from blue to red- which attract pollinators.

II- Phenylpropanoids, (D) flavonoids- anthocyanins

cyanidin

II- Phenylpropanoids (D) flavonoids- anthocyanins



A mystery until recently

Cornflower contains cyanidin, the anthocyanin thatmakes roses red.

Shiono et al., 2005, Nature


The cyanidin is part of a metallic macro-molecularcomplex (superpigment) that shifts the colour from

red to blue.


http://www.nature.com/nature/journal/v436/n7052/full/436791a.html






Anthocyanins also act as antioxidants that may protect against UV light damage.

Bieza and Lois, 2001 Plant Physiology

high flavonoid

wild-type

low flavonoid



http://www.ncbi.nlm.nih.gov.gate1.inist.fr/pmc/articles/PMC116467/?tool=pubmed






Condensed tannins / proanthocyanidins (PAs) are oligomers of epicatechin and catechin

that accumulate in the vacuole.

PAs in red wine linked to a reduced risk of coronary heart disease and to lower overall

mortality.

PAs are present at higher concentrations in wines from areas of southwestern France and

Sardinia. Dark chocolate is another rich source of PAs...

Corder et al., 2006 Nature












• Cyanogenic glucosides and glucosinolates

• Alkaloids

II- Cyanogenic glucosides and glucosinolates



http://www.place.life.ku.dk/Research/Glucosinolates.aspx

II Cyanogenic glucosides and glucosinolates

P450 oxidase

II- Cyanogenic glucosides



Cyanogenic glycosides are widespread in plants (>2500 species) including monocots,

dicots and gymnosperms. Evolved multiple times.

~50 structures known.

Busk and Moller, 2002 Plant Phys

II Cyanogenic glucosides

Dhurrin is stored in vacuole

Cyanide is highly toxic

1.5mg/kg is lethal to man

II- Cyanogenic glucosides

http://www.plantphysiol.org/cgi/content/full/129/3/1222








Cassava is the third largest source of carbohydrate in the world.

Highly productive and drought tolerant.

BUT cassava is rich in cyanogenic glucosides derived from valine and isoleucine.

Must be processed before eating to release HCN.

Konzo = paralysis resulting from eating poorly processed cassava

II Cyanogenic glucosides

II- Glucosinolates



II Glucosinolates

Evolutionarily related to cyanogenic glucosides (CYP79 P450 enzyme)

Present only in plants of the order Brassicales

e.g colza (Brassica napus), chou (Brassica oleracea) et Arabidopsis thaliana

II- Glucosinolates



II Glucosinolates

Evolutionarily related to cyanogenic glucosides (CYP79 P450 enzyme)

Present only in plants of the order Brassicales

e.g colza (Brassica napus), chou (Brassica oleracea) & Arabidopsis thaliana

Functions:

• Plant defence against animals and microbes.

• N and S storage

• Responsible for the hot taste of mustard, roquette and wasabi.

• Protect humans against cancer.

II- Glucosinolates‐core pathway



II- Glucosinolates‐core pathway

>120 different structures known

40 in Arabidiosis thaliana

Diversity

(i)chain elongation of selected precursor amino acids (only Met and Phe)

(ii)secondary modifications of the amino acid side chain (alters bio‐activity)

Model pathway. Sonderby et al. 2010 TIPS

II- Glucosinolates‐ activation

http://dx.doi.org.gate1.inist.fr/10.1016/j.tplants.2010.02.005












II- Glucosinolates‐ activation

Isothiocyanates and nitriles produced by GSL hydrolysis are toxic.

Two modes of activation:

(i) Bomb. Mechanical damage (animal feeding) breaks the vacuole

releasing the GSLs, which are then hydrolysed.

(ii) Directed. Microbial attack triggers transport of GSL vesicles and

hydrolysis at the site of attack.

Kwon et al. 2008 Nature Clay et al 2009 Science Bednareket al 2009 Science

Fungal appresorium (red), glucosinolate vesicles (green)

isothiocyanate

vacuole

II- Alkaloids

http://www.nature.com/nature/journal/v451/n7180/full/nature06545.html

http://www.sciencemag.org/content/323/5910/95.short

http://www.sciencemag.org/content/323/5910/101.full




























Alkaloids- a highly diverse group (>12000) of natural products that are related

only by the occurrence of a nitrogen atom in a heterocyclic ring. Derived from

unrelated metabolic pathways.

Alkaloids occur in ~20% of plant species and are believed to function in plant

defense.

Ziegler and Facchini, 2008 Annual Review of Plant Biology

II- Alkaloids

http://www.annualreviews.org/doi/full/10.1146/annurev.arplant.59.032607.092730?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed






5 classes of alkaloid:

Monoterpenoid indole alkaloids e.g vinblastine

Benzylisoquinoline alkaloids e.g morphine

Tropane alkaloids and nicotine e.g nicotine

Purine alkaloids e.g theobromine and caffeine

Pyrrolizidine alkaloids e.g symphytine

(don’t try to learn all the names- there are more useful things to learn!)

II- Alkaloids



15 reactions

P450s, MT, ACT

Morphine, a benzylisoquinoline alkaloid from the opium poppy (picot).

tyrosinemorphine

heroin (diacetylmorphine)

acetic anhydride

Heroin- synthesised by Bayer in 1897. Originally

sold as a drug to cure morphine addiction.

Heroin is more lipophilic and therefore more potent

than morphine.

Me

Me

II- Alkaloids



Nicotine, a tropane alkaloid and potent insecticide.

nicotinearginine

Nicotine is named after the tobacco plant Nicotiana tabacum which in turn is named after

Jean Nicot de Villemain, French ambassador in Portugal, who sent tobacco and seeds from

Brazil to Paris in 1560 and promoted their medicinal use.

II- Alkaloids



Caffeine and theobromine are the best known purine alkaloids.

theobromine caffeine

xanthosine (purine)

Chocolate has up to 20mg/g theobromine. Acts as a stimulant.

Theobromine is toxic to dogs because they metabolize it slowly.25 grams dark chocolate would cause symptoms in a 20 kg dog.

Thebroma cacao

II Major Groups of Natural Products



j p


•Phenylpropanoids

•

Cyanogenic glucosides

• Alkaloids

• Polyketides



III Synthesis and delivery of natural products

• Coordinated transcriptional regulation

• Gene clusters

• Delivery of natural products


III- Coordinated transcriptional regulation



p g

Natural products often accumulate in specific tissues at specific times .

Two principal strategies:

1) Constitutive e.g. avenacin in oat roots NB potential fitness cost.

2) Inducible e.g accumulation of anthocyanins in response to light stress.

Biosynthetic intermediates can be auto-toxic

Requires tight coordination of genes and enzymes.

III Regulation of anthocyanin accumulation- developmental



PAP1

TTG1

SPL9

TT8

MYB transcription factor (+++)

Repressor (---)

bHLH transcription factor (+++)

WD40 protein (+++)

III- Regulation of anthocyanin accumulation- developmental



Gou J et al. Plant Cell 2011;23:1512-1522

ANTHOCYANIN PATHWAY INACTIVE

ANTHOCYANIN PATHWAY ACTIVE

XPAP1

PAP1

III- Regulation of anthocyanin accumulation- jasmonic

acid



Shan et al. 2012 Current Opinions

COI1 = CORONATINE INSENSITIVE 1, the Jasmonate receptor

Ubi

The transcriptional repressor JAZ is degraded when jasmonic acid is detected

III- Regulation of anthocyanin accumulation- jasmonic acid

http://www.sciencedirect.com/science/article/pii/S1369526611001488




TTG1TT8

PAP1

JAZ

PAP1

JAZCOI1

COI1

jasmonate

TTG1

TT8

anthocyanins

repression

Ubiquitination and degradation

III- JA is a conserved regulator of secondary metabolism via JAZ



Geyter et al, 2012 TIPS

III Regulation of anthocyanin accumulation- summary







-Anthocyanin genes under complex and strict regulation

- Accumulate in response to- plant age (example SPL9)

- plant hormones (example JA)

- light

- nutrient deficiency (N and S)

INRA Versailles

III- Synthesis and delivery of secondary metabolites

http://www4.inra.fr/cepia-eng/News/anthocyan-biosynthesis

http://www4.inra.fr/cepia-eng/News/anthocyan-biosynthesis




• Gene clusters


III- Gene Clusters



Prokaryotes- functionally related genes are often arrayed in operons

Jacob, F, Perrin, D, Sanchez, C, and Monod, J. 1960.

L'opéron: groupe de gènes à expression coordonée par un opérateur. Comptes

Rendus de l'Académie des Sciences 25 1727-1729.

III- Gene Clusters



Until recently, gene order thought to be random in plants.

However known eukarytoic exceptions:

• major histocompatibility complex in mammals

• secondary metabolite clusters in fungi

Aflatoxin cluster in Aspergillus flavus (fungi)

III- Gene Clusters



Sad1

oxido-squalene

cyclase

Sad2

CypP450

Sad7 AT

Sad9

MT

Sad10

GT

~365 kb

Recently many plant gene clusters discovered for secondary

metabolism

The Avenacin triterpenoid cluster in oats

O

O

O

NH

CH3

O

OH

O

OH

Avenacin A-1

oxidosqualene

HO

beta-amyrin

O

Sad1

α-1-ara(1-)β-D-glu(1-2)

β-D-glu(1-4)

Sad2,3,4,7,9,10



Avena strigosa Arabidopsis thaliana

III- Gene Clusters



Marneral (triterpenoid)

Field & Osbourn, 2008 Science ; Field et al., 2011 PNAS

HOH

Thalianol (triterpenoid)

OSC cyclase

OSC cyclase

http://www.sciencemag.org/cgi/content/abstract/320/5875/543


http://www.pnas.org/content/early/2011/08/26/1109273108.abstract









Winzer et al, 2012 Science

III- Gene Clusters

http://www.sciencemag.org/content/336/6089/1704.long






Other clusters recently discovered-

• terpenoid momilactone and phytocassane clusters in rice

• triterpenoid cluster in Lotus japonicus

• cyanogenic glucoside clusters in Lotus japonicus, cassava,

and maize

• DIBOA pathway in maize

• glycoalkaloid pathway in potatoes and tomatoes (Solanum)

III- Gene Clusters



Why form gene clusters?

- regulation (chromatin)

- co-inheritance

- toxic intermediates

Biotechnology applications

- transfer of entire clusters

into crop plants

Good review: Chu et al., 2011 Plant Journal

Fluorescence in situ hybridisation

(FISH) of oat betamyrin synthase

in oat root epidermis


http://onlinelibrary.wiley.com.gate1.inist.fr/doi/10.1111/j.1365-313X.2011.04503.x/abstract;jsessionid=1FD8BCD4E27EEDABB4FFA6EE8805C9D6.d04t03

http://onlinelibrary.wiley.com.gate1.inist.fr/doi/10.1111/j.1365-313X.2011.04503.x/abstract;jsessionid=1FD8BCD4E27EEDABB4FFA6EE8805C9D6.d04t03




• Gene clusters


III- Delivery of natural products- by vesicle trafficking



Specialised structures for delivery of natural products:

- Vesicles

- Glands

- Root hairs

- Lactifers and resin ducts




antimicrobial flavonoids, sorghum leaf antimicrobial flavonoids, onion epidermis

fungal attack

Field and Osbourn, 2006 Phytochemistry hydrogen peroxide vesicles, barley

Glucosinolate targeting, Arabidopsis

http://www.ncbi.nlm.nih.gov.gate1.inist.fr/pubmed









PEN1-GFP (green)

Fungal spore (red)

PEN1 encodes a SNARE domain protein.

Involved in vesicle traficking.

PEN2-GFP (green)

Fungal spore (red)

PEN2 encodes a glycosyl hydrolase that

hydrolyses glucosinolates.

Lipka et al. 2010








Kwon et al, 2008 Plant Physiology

sesquiterpene glucosinolates

III- Delivery of natural products- in glands





Croteau et al. 2005 Naturwissenschaften

Menthol oil (cyclic monoterpene) accumulates in

glandular trichomes (P).

The trichome sits on eight secretory cells (S), a stalkcell (ST) and a basal cell (B).

One gland fills in 30h, 8000 glands per leaf, yield 1% dry

weight menthol.

T t T VI l d l t i h


http://www.ncbi.nlm.nih.gov/pubmed/16292524









Tomato Type VI glandular trichomes

Loaded with volatile monoterpenes and

sesquiterpenes.

One of 7 types of trichome in tomato:

http://www.trichome.msu.edu




Source

Stinging trichome of Urtica dioica (stinging nettle, ortie).

Oxalic and tartaric acid cause the painful sensation (Fu et al., 2006)

III- Delivery of natural products- by root hairs

http://www.photomacrography.net/forum/viewtopic.php?t=2557

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2803540/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2803540/

http://www.photomacrography.net/forum/viewtopic.php?t=2557



Sorghum exudes sorgoleone from root hairs (antimicrobial, toxic to other plants)Dayan et al., 2009

III- Delivery of natural products- by lactifers



Latexes can contain specialized metabolites such as terpenoids, alkaloids and lignans.

e.g morphine, rubber, vinblastine




Milkweed has a network of lactifiers containing a pressurised latex rich in cardenolides

(steroid glycosides, similar to saponins).

Trenching by monarch butterfly caterpillar, to reduce latex pressure.

Caterpillar accumulates cardenolides as a defence against predators

Conifer resin is carried in resin ducts (similar to




Conifer resin is carried in resin ducts (similar to

lactifers)

Resin: mixture of volatile monoterpenes and

sesquiterpenes, and non-volatile diterpene acids.

CRD- constitutive resin ducts. Always present.

TRD- traumatic resin ducts produced in response

to insect attack.

Sitka spruce Zulak and Bohlmann, 2010 JIBP

Volatile terpenes released in response to insect feeding

III- Delivery of natural products- by other organisms

http://onlinelibrary.wiley.com/doi/10.1111/j.1744-7909.2010.00910.x/full








Two-spotted mite (left) feeding

results in production of a complex

mixture of terpenes including

nerolidol and DMNT.

Predatory mite P. persimilis (right)

attracted by volatiles... and

attacks.

Volatile terpenes released in response to insect feeding….

Kappers et al. Science 2005

Final Course Summary







I- Introduction (especially definition of secondary metabolite)

II- Major groups of natural products



• Cyanogenic glucosides• Alkaloids



• Gene clusters

D li f t l d t

Documents

Plant Secondary Metabolism