Plant Secondary Metabolism
Transcript of Plant Secondary Metabolism
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 1/108
Secondary Metabolism
Ben Field 2013www.lgbp.univ-mrs.fr
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 2/108
Three Parts
I Introduction
II Major pathwaysIII Synthesis and delivery
Secondary Metabolism
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 3/108
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
Secondary Metabolism
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 4/108
Introduction- What are secondary metabolites?
Plants produce more than 200,000 different bio-active natural products
(secondary metabolites, specialised metabolites).
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 5/108
Introduction- What are secondary metabolites?
Plants produce more than 200,000 different bio-active natural products
(secondary metabolites).
Probably lifestyle related:
• Plants are anchored to the ground, exposed to the environment
• Plants have no adaptive immune system (like animals)
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 6/108
Introduction- What are secondary metabolites?
Plants produce more than 200,000 different bio-active natural products
(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)
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 7/108
Introduction- What are secondary metabolites?
• 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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 8/108
Introduction- What are secondary metabolites?
• 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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 9/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 10/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 11/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 12/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 13/108
Introduction- Why do organisms make natural products?
• In the natural environment secondary metabolites can be essential for
survival and reproduction.
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 14/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 15/108
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
- Abiotic and biotic defense
- tree resin, complex terpene mixture
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 16/108
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
- Abiotic and biotic defense
- Communication with other plants, microbes and animals
- feeding caterpillar causes the
release of volatile compounds
that attract a parasitic wasp
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 17/108
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
- Abiotic and biotic defense
- Communication with other plants, microbes and animals
- Development?
Some secondary metabolites are in fact new hormones
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 18/108
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)
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 19/108
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.
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 20/108
II Major Groups of Natural Products
Several major classes of natural product can be distinguished:
• Isoprenoids/Terpenes
•Phenylpropanoids
• Cyanogenic glucosides
• Alkaloids
•
Polyketides
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 21/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 22/108
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)
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 23/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 24/108
Natural rubber (cis-1,4-polyisoprene)
Gutta-percha (trans-1,4-polyisoprene)
II- Polyterpenes Cn
rubber tree (Hevea brasiliensis)
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 25/108
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)
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 26/108
Acyclic monoterpenes
II- Fragrant monoterpenes (C10)
geraniol (present in rose, citronella, geranium andlemon oils)
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 27/108
Acyclic monoterpenes
Cyclic monoterpenes
II- Fragrant monoterpenes (C10)
menthol
geraniol (present in rose, citronella, geranium andlemon oils)
Class I terpene synthase
“Limonene synthase”
GPP
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 28/108
Acyclic monoterpenes
Cyclic monoterpenes
Bicyclic monoterpenes
II- Fragrant monoterpenes (C10)
menthol
geraniol (present in rose, citronella, geranium andlemon oils)
Eucalyptol (90% of eucalyptus
oil, present in thyme,
rosemary and sage oils).
Attractant for pollinators.
Class I terpene synthase
“Limonene synthase”
Pinene. Major component of
pine resin. Toxic to insects.
GPP
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 29/108
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) DMAPP (C5)
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 30/108
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 31/108
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).
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 32/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 33/108
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) DMAPP (C5)
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 34/108
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) DMAPP (C5)
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 35/108
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.
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 36/108
Carotenoids sometimes accumulate in specialised chloroplasts called chromoplasts.
II- Red, orange and yellow tetraterpenes (C40)
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 37/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 38/108
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)
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 39/108
Mevalonate
(MVA) pathway Methylerythritol
phosphate (MEP)
pathway
diterpenes
triterpenes
II- Isoprenoid biosynthesis compartmentation
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 40/108
II- Major Groups of Natural Products
• Isoprenoids/Terpenes
• Phenylpropanoids
• Cyanogenic glucosides and Glucosinolates
• Alkaloids
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 41/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 42/108
II- Phenylpropanoids
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 43/108
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
II- Phenylpropanoids
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 44/108
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
II- Phenylpropanoids
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 45/108
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
( )
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 46/108
II- Phenylpropanoids, (B) coumarins
Soluble lactones. They have a sweet smell, like dried grass.
h l id ( ) i
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 47/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 48/108
II- Phenylpropanoids
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 49/108
II Ph l id (C) li i
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 50/108
Coniferyl alcoholCoumaryl alcohol Sinapyl alcohol
methyltransferase hydroxylase
reductase
p-coumarate
II- Phenylpropanoids, (C) lignin
II Ph l id (C) li i
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 51/108
Specific monolignol monomers are directed to different parts of the cell wall.
II- Phenylpropanoids, (C) lignin
II Ph l id (C) li i
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 52/108
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- Phenylpropanoids, (C) lignin
II Ph l id (C) li i
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 53/108
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
II Phenylpropanoids (C) lignin
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 54/108
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, (C) lignin
II Phenylpropanoids
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 55/108
II- Phenylpropanoids
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
II Phenylpropanoids (D) flavonoids
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 56/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 57/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 58/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 59/108
Vacuolar anthocyanins provide flower colours- from blue to red- which attract pollinators.
II- Phenylpropanoids, (D) flavonoids- anthocyanins
cyanidin
II- Phenylpropanoids (D) flavonoids- anthocyanins
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 60/108
A mystery until recently
Cornflower contains cyanidin, the anthocyanin thatmakes roses red.
Shiono et al., 2005, Nature
II- Phenylpropanoids, (D) flavonoids- anthocyanins
The cyanidin is part of a metallic macro-molecularcomplex (superpigment) that shifts the colour from
red to blue.
II- Phenylpropanoids (D) flavonoids- anthocyanins
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 61/108
Anthocyanins also act as antioxidants that may protect against UV light damage.
Bieza and Lois, 2001 Plant Physiology
high flavonoid
wild-type
low flavonoid
II- Phenylpropanoids, (D) flavonoids- anthocyanins
II- Phenylpropanoids (D) flavonoids- anthocyanins
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 62/108
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
II- Phenylpropanoids, (D) flavonoids- anthocyanins
II- Major Groups of Natural Products
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 63/108
II- Major Groups of Natural Products
• Isoprenoids/Terpenes
• Phenylpropanoids
• Cyanogenic glucosides and glucosinolates
• Alkaloids
II- Cyanogenic glucosides and glucosinolates
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 64/108
http://www.place.life.ku.dk/Research/Glucosinolates.aspx
II Cyanogenic glucosides and glucosinolates
P450 oxidase
II- Cyanogenic glucosides
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 65/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 66/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 67/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 68/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 69/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 70/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 71/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 72/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 73/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 74/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 75/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 76/108
j p
• Isoprenoids/Terpenes
•Phenylpropanoids
•
Cyanogenic glucosides
• Alkaloids
• Polyketides
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 77/108
III Synthesis and delivery of natural products
• Coordinated transcriptional regulation
• Gene clusters
• Delivery of natural products
Secondary Metabolism
III- Coordinated transcriptional regulation
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 78/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 79/108
PAP1
TTG1
SPL9
TT8
MYB transcription factor (+++)
Repressor (---)
bHLH transcription factor (+++)
WD40 protein (+++)
III- Regulation of anthocyanin accumulation- developmental
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 80/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 81/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 82/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 83/108
Geyter et al, 2012 TIPS
III Regulation of anthocyanin accumulation- summary
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 84/108
-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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 85/108
• Coordinated transcriptional regulation
• Gene clusters
• Delivery of natural products
III- Gene Clusters
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 86/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 87/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 88/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 89/108
Avena strigosa Arabidopsis thaliana
III- Gene Clusters
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 90/108
Marneral (triterpenoid)
Field & Osbourn, 2008 Science ; Field et al., 2011 PNAS
HOH
Thalianol (triterpenoid)
OSC cyclase
OSC cyclase
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 91/108
Winzer et al, 2012 Science
III- Gene Clusters
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 92/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 93/108
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
III- Synthesis and delivery of secondary metabolites
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 94/108
• Coordinated transcriptional regulation
• Gene clusters
• Delivery of natural products
III- Delivery of natural products- by vesicle trafficking
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 95/108
Specialised structures for delivery of natural products:
- Vesicles
- Glands
- Root hairs
- Lactifers and resin ducts
III- Delivery of natural products- by vesicle trafficking
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 96/108
antimicrobial flavonoids, sorghum leaf antimicrobial flavonoids, onion epidermis
fungal attack
Field and Osbourn, 2006 Phytochemistry hydrogen peroxide vesicles, barley
Glucosinolate targeting, Arabidopsis
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 97/108
III- Delivery of natural products- by vesicle trafficking
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 98/108
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
III- Delivery of natural products- by vesicle trafficking
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 99/108
Kwon et al, 2008 Plant Physiology
sesquiterpene glucosinolates
III- Delivery of natural products- in glands
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 100/108
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
III- Delivery of natural products- in glands
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 101/108
Tomato Type VI glandular trichomes
Loaded with volatile monoterpenes and
sesquiterpenes.
One of 7 types of trichome in tomato:
http://www.trichome.msu.edu
III- Delivery of natural products- in glands
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 102/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 103/108
Sorghum exudes sorgoleone from root hairs (antimicrobial, toxic to other plants)Dayan et al., 2009
III- Delivery of natural products- by lactifers
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 104/108
Latexes can contain specialized metabolites such as terpenoids, alkaloids and lignans.
e.g morphine, rubber, vinblastine
III- Delivery of natural products- by lactifers
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 105/108
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
III- Delivery of natural products- by lactifers
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 106/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 107/108
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
8/11/2019 Plant Secondary Metabolism
http://slidepdf.com/reader/full/plant-secondary-metabolism 108/108
I- Introduction (especially definition of secondary metabolite)
II- Major groups of natural products
• Isoprenoids/Terpenes
• Phenylpropanoids
• Cyanogenic glucosides• Alkaloids
III- Synthesis and delivery of secondary metabolites
• Coordinated transcriptional regulation
• Gene clusters
D li f t l d t