Secondary metabolites in plant defense

1. Secondary Metabolites
&
Plant Defense
Kishor D. S
Crop Molecular Breeding Lab
College of Agriculture & Life Sciences
Seoul National University
2016.04.04
Advance Crop Physiology

2. Table of contents
 Introduction
 Major compounds of plant surface defense:
Cutin, Waxes, and suberin
 Secondary metabolites:
Terpenes, Phenolics, and N-containing compounds
 Induced plant defense against Insect herbivores
 Plant defense against pathogen
 summary

3. I. Introduction
In all natural habitats, plants are surrounded by an enormous
number of potential enemies. Nearly all ecosystems contain a
wide variety of bacteria, viruses, fungi, nematodes, mites,
insects, mammals and other herbivorous animals, greatly
responsible for heavy reduction in crop productivity.
By their nature, plants protect themselves by producing some
compounds called as secondary metabolites. Secondary
metabolites, including terpenes, phenolics and nitrogen
containing compounds, defend plants against a variety of
herbivores and pathogens

4. II. Major compounds of plant surface defense
To help reduce transpiration and pathogen invasion
1. Cutin: macromolecule, a polymer
consisting of many long chain
fatty acids that are attached to
each other by esters linkages,
creating a rigid three-
dimensional network.
Cutin is formed from 16:0 and
18:1 fatty acids with hydroxyl or
episode groups either at middle of
chain or end opposite the
carboxylic acid function
Cutin, waxes, and suberin

5. (A) Structure of the plant cuticle
(B) Electron micrograph of the cuticle of a glandular cell
Cutin is a principal constituent of
the cuticle, a multilayered secreted
structure that coats the outer cell
wall of the epidermis.
Cuticle is composed of surface wax,
cuticle proper (cutin embedded in
wax) and cuticular layer (cutin,
wax, pectin, cellulose and
carbohydrates). In addition to that,
it may contain a second lipid
polymer, made up of long-chain
hydrocarbons “Cutan” (Jeffree
1996)

6. 2. Waxes: not macromolecule, but
complex mixtures of long-
chain acyl lipids - hydrophobic
in nature
The most common components
are straight chain alkanes & 25
to 35 carbon atoms of alcohol.
Long chain aldehydes,
ketones,esters, and free fatty
acids are also found.

7. A. Surface wax deposits, which form the top layer of the cuticle
The waxes are synthesized by
epidermal cells. They leave the
epidermal cells as droplets that
pass through pores in the cells
wall by some mechanism.
The waxes often crystallizes in
an intricate pattern of rods,
tubes, or plates. These micro
structures enhances water
repellency by increasing the
roughness of the wax structure.
In this way, water droplets from instantly on contact & carry
away contaminating particles, cleansing the plant surface
(Neinhuis et al.1992)

8. 3. Suberin: is a polymer. Like cutin,
its formed from hydroxy or epoxy
fatty acids joined by easter
linkages. It has dicarboxylic acids,
more long chain components, and
a significant proportion of
phenolic compounds.
It’s a Cell wall constituent found in
many parts of the plant
Example: root endodermis
Suberin is a principal component the outer cell wall and
associated with the cork cells of the periderm. It also forms at
sites of leaf abscission & in area damaged by pathogens

9. III. Secondary Metabolites
• Plant produce a large and diverse array of organic compounds that
appear to have no direct function in growth and development. These
substances known as secondary metabolites, secondary products or
natural products.
• They have no generally recognized roles in the process of
photosynthesis, respiration, solute transport, translocation, nutrient
assimilation and differentiation.
• They have very restricted distribution than primary metabolites in
plant kingdom
• High concentration of secondary metabolites results in more
resistant plant

10. • It provides protection against herbivores and pathogens
• They serves as attractants (smell, color, taste) for pollinatators and
seed dispersing animals
• They functions as agents of plant-plant competition and plant-
microbe symbioses.
Plant secondary metabolites can be dived into three chemically
distinct groups : terpenes, phenolics and nitrogen containing
compounds.
Important ecological functions of secondary metabolites
in plants

11. Major pathways of secondary-metabolite biosynthesis

12. A. Terpenes
• The terpenes, or terpenoids, constitute largest class of secondary
products and are united by their common biosynthetic origin from
acetyl CoA or glycolytic intermediates.
• These substances are generally insoluble in water
• Terpenes are derived from the union of five carbon (C5) isoprene
units (it can decompose at high temperature to give isoprene)
H3C CH – CH =CH2 referredasisoprenoids
• Terpenes are classified by the number of C5units
Monoterpenes (two C5 units) , Sequiterpenes (three C5units), and
Diterpenes ( four C5units).
Larger terpenes include triterpenes (30 carbons), tetraterpenes
(40 carbons), and Polyterpenes ([C5]n carbons, where n>8)

13. Outline of terpene biosynthesis (Part 1)

14. Outline of terpene biosynthesis (Part 2)

15. Structures of limonene (A) and menthol (B)
Terpenes defend against Insects and other organisms
• Terpenes are toxins and feeding deterrents
to many plant feeding insects and
mammals. Example: pyrethroid
• Many plants contain mixtures of volatile
monoterpenes and sesquiterpens, called
essential oils, that lends a characteristic
odor to their foliage. Peppermint, lemon,
basil, and sage are example of plants that
contain essential oils. These essential oil
have well known insect repellent
properties.
• Monoterpenes and sesquiterpens are
commonly found in glandular hairs that
projected out from epidermis and serve as a
repellent against herbivores. Its stored in
modified extracellular space in cell wall

16. Structure of two triterpenes, azadirachtin (A), and -ecdysone (B)
• Among non-volatile terpene
antiherbivore compounds are the
limonoids, a group of triterpens
well known as bitter substances in
citrus fruit. Perhaps most powerful
deterrent to insects known is
azadirachtin, a complex limonoid
from the neem tree.
• The phytoecdysones, first isolated
from common fern, polypodium
vulgare -group of plant steroids
that have the same basic structure
as insect molting hormones.
Phytoecdysones have defense
function against plant parasitic
nematodes.
• Triterpenes that are active against vertebrate herbivorous include
cardenolides (glycosides) and saponins (steroid & glycosides).

17. • Plants produce a large variety of secondary products that contain
a phenol group, a hydroxyl functional group on an aromatic ring
called phenol, a chemically heterogeneous group also.
• Some are soluble only in organic solvent, some are water soluble
carboxylic acids and glycosides, and others are large insoluble
polymers.
• It serve as a defense compounds against herbivores and pathogen
• Mechanical support, attracting pollinators and fruit dispersers, in
absorbing harmful UV radiation, or in reducing the growth of
nearby competing plants.
B. Phenolic compounds

18. Plant phenolics are biosynthesized in several different ways

19. Outline of phenolic biosynthesis from phenylalanine (Part 1)

20. Outline of phenolic biosynthesis from phenylalanine (Part 2)

21. Simple phenolic compounds play a great diversity of roles in plants
• Caffeic acid and Ferulic acid
released into the soil and inhibits
the growth of neighboring plants-
allelopathy.
• Psoralen is a furanocoumarin that
exhibits phototoxicity to insects
herbivores.
• Salicylic acid – plant growth
regulator that is involved in
systematic resistance to plant
pathogens.

22. It is a highly branched polymer of phenyl-propanoid groups,
formed from three different alcohols viz., coniferyl, coumaryl
and sinapyl which oxidized to free radicals (ROS) by a
ubiquitous plant enzyme-peroxidase, reacts simultaneously and
randomly to form lignin.
Its physical toughness deters feeding by herbivorous animals and
its chemical durability makes it relatively indigestible to
herbivores and insects pathogens. Lignifications block the
growth of pathogens and are a frequent response to infection or
wounding.
Lignin is a highly complex phenolic macromolecule

23. Flavonoids
• One of the largest classes of plant phenolic,
perform very different functions in plant
system including pigmentation and defense.
Example : carotenoids, anthocyanin and
anthocyanidin.
Four major groups- anthocyanin, flavones,
flavonols, and Isoflavones
• Two other major groups of flavonoids found
in flowers are flavones and flavonols function
to protect cells from UV-B radiation because
they accumulate in epidermal layers of leaves
and stems.
• In addition, exposure of plants to increased
UV-B light has been demonstrated to increase
the synthesis of flavones and flavonols
suggesting that flavonoids may offer a
measure of protection by screening out
harmful UV-B radiation

24. Black-eyed Susan (Rudbeckia sp.) as seen by humans (A)
Black-eyed Susan (Rudbeckia sp.) as it might appear to honeybees (B)
• These flavonoids generally absorb
light at shorter wavelength than
anthocyanin, so they are not
visible to the human eye.
However, Insects can see farther
into the UV range of the
spectrum (“bull’s-eye pattern)
and it will help them to locate
pollen and nectar.

25. Isoflavonoids
• Isoflavonoids are derived from a flavonone intermediate,
ubiquitously present in legumes and play a critical role in plant
developmental and defense response.
• Three dimensional structure similar to that of steroids, allowing
these substances to bind to estrogen receptors
• Isoflavonoids have different biological activities such as
rotenoids, insecticidal actions, anti-estrogenic effects, and anti
cancer benefits of food prepared from soyabean.
•It also play role as phytoalexins, antimicrobial compound and
helps to limit the spread of pathogen

26. Tannins
• A second category of plant phenolic polymer , It helps to bind the collagen
protein of animal hides, increasing their resistance to heat, water, and
microbes.
• In general, tannins are general toxins that significantly reduce the growth and
survivorship of many herbivores and also act as feeding repellents to a great
diversity of animals. The defensive properties of tannins are generally
attributed to their ability to bind proteins..
• Condensed tannins: formed by polymerization of flavonoids units. Common
constituent of woody plants because it can often be hydrolyzed to
anthocyanidins by strong acid treatment (called pro-anthocyanidins).
• Hydroylzable tannins: heterogeneous polymer phenolic acids (gallic acid,
and simple sugars). Smaller than condensed tannins and may be hydrolyzed
more easily (only dilute acid is needed).

28. Nitrogen containing compounds
They include alkaloids, cyanogenic glucosides, and non-protein amino acids.
Most of them are biosynthesized from common amino acids. All are of
considerable interest because of their role in the anti herbivore defense and toxicity
to humans.
(a)Alkaloids
•A large family of N containing secondary metabolites found in approximately
20% of the species of vascular plants. most frequently in the herbaceous dicot and
relatively a few in monocots and gymnosperms.
•Generally, most of them, including the pyrrolizidine alkaloids (PAs) are toxic to
some degree and appear to serve primarily in defense against microbial infection
and herbivoral attack.
•They are usually synthesized from one of the few common amino acids, in
particular, aspartic acid, lysine, tyrosine and tryptophan.
•Defensive elements against predators, especially mammals because of their
general toxicity and deterrence capability.

29. Diverse group of secondary metabolites that contain nitrogenMajor types of alkaloid classes:
pyrrolidine, tropane, piperidine,
pyrrolizidine, quinolizidine, Isoquinoline
and indole
Large number of livestocks death is
caused by the ingestion of alkaloids
containing plants.
In US, a significant % of all grazing
livestock are poisoned each year by
consumption of large quantities of alkaloid
containing plants such as lupines
(Lupinus) and larkspur (Delphinium)
Mode of action of alkaloids in animals is quite variable . on cellular level
alkaloids affect nerve system, membrane transport and protein synthesis.

30. Enzyme-catalyzed hydrolysis of cyanogenic glycosides to release hydrogen cyanide
Cyanogenic glycosides
• They constitute a group of N-containing protective compounds other than
alkaloids, release the poison HCN and usually occur in members of families
viz., Graminae, Rosaceae and Leguminosae
• They are not in themselves toxic but are readily broken down to give off
volatile poisonous substances like HCN and H2S when the plant is crushed;
their presence deters feeding by insects and other herbivores such as snails and
slugs

31. • Two groups of these substances
I. Cyanogenic glycosides- amygdalin (almond,apricot,cherries & peach) and
dhurrin (sorghum) which is toxin of cellular respiration by binding to the Fe-
containing heme group of cytochrome oxidase and other respiratory enzymes.
II. Glucosinolates- also called mustard oil glycosides, breakdown to release
defensive substances (smell and taste of vegetables like cabbage, broccoli, and
radish).
Hydrolysis of glucosinolates to mustard-smelling volatiles

32. Nonprotein amino acids and their protein amino acid analogs
Non-protein amino acids:
Many plants also contain unusual
amino acids called non- protein amino
acids that incorporated into proteins
but are present as free forms and act
as protective defensive substances
examples, canavanine and azetidine-
2-carboxylic acid are close analogs of
arginine and proline respectively.
They exert their toxicity in various
ways. Some block the synthesis of
protein or uptake of protein amino
acid and other incorporated in protein
results in non functional protein .
In details, after ingestion canavanine is recognized by herbivore enzyme that
normally binds arginine to the arginine transfer RNA molecule and so become
incorporated in to proteins in place of arginine (non functional protein).

33. Concerted biosynthesis of elicitors from plant and insect precursors
Example: Zea mays
IV. Induced plant defense against Insect herbivores
Plant develop wide variety of defense
strategies against herbivory.
A.Constitutive defense response:
these defend mechanism is always
present . They are often species
specific and exist in as stored
compounds, conjugated compounds
(to reduce toxicity), or precursors of
active compounds that can easily
activate upon damage.
B.Induced defense response:
Initiated only after damage occurs. It
requires smaller investment of plant
resources but must activate quickly to
be effective

34. Octadecanoid pathway
When plants recognize elicitor from the
insect saliva, a complex signal transduction
network is activated. In most of the plant
defense mechanism, a major signaling
pathway is Octadecanoid pathway leads to
production of jasmonic acid that activate
many defense responses.
conversion of linolenic acid (18:3) to
jasmonic acid (Induce transcription of host
genes involved in defense mechanism)
The first three enzymatic steps in occur in
chloroplast, resulting in the cyclized product
12-oxo-phytodienoic acid. This intermediate
is transported to peroxisome , where its is
first reduced and then converted to jasmonic
acid by ß-oxidation

35. Systemin signaling pathway
Jasmonic acid are proteins
that interfere with herbivore
digestion.
Example:Protinase inhibitor
biosynthesis in wounded
tomato plants. These
substances block the action
of herbivore proteolytic
enzymes (trypsin &
chymotrypsin) and hinder
the protein digestion.

36. Many types of antipathogen defense are induced by infection
V. Plant defense against pathogens
Hypersensitive response: depriving the pathogen of nutrients &
preventing its spread. (rapid accumulation of reactive oxygen species
& NO-Nitric oxide)

37. Phytoalexinssecondary metabolites with antimicrobial
properties
• Synthesis of lignin and cellulose
• Modification of cell wall protein
- strengthens the wall of cells in
the vicinity of infection point
• Infection- Formation of
hydrolytic enzymes attacks cell
wall of pathogen ( assortment of
glucanases, chitinases, and other
hydrolases are induced by fungal
invasion- these enzymes are also
known pathogeneis related PR
proteins)
• Phytoalexins: strong antimicrobial secondary metabolites
accumulates around infection point. They begin with transcribing &
translating the MRNAs & synthesizing the enzyme denovo

38. Systemic acquired resistance (SAR)

39. VI. SUMMARY
• Plant produce enormous diversity of substances-secondary
metabolites
• Cutin, suberin, and waxes- surface protection
• Major secondary metabolites-terpenes (C5 isoprene unit, are
toxin & feeding deterrents to herbivorous), phenolics
(shikimic acid pathway), Lignin strengthens cell walls,
flavonoid pigment functions against harmful UV radiation &
attracts pollinators and seed dispersers. Finally, lignin,
flavonoids, & other phenolics defense against pathogen &
herbivores. N containing metabolites (alkaloids, cyanogenic
glycosides, glucosinolates, non protein amino acids, &
proteinase Inhibitors)
• Plant defense against insect
• Plant defense against pathogen