A reduction of GAs re-establishes an ABA/GA ratio appropriate for suppression
of germination and induction of maturation.
These GAs induce a developmental program that leads to vivipary
in the absence of normal amounts of ABA
Auxin (2,4-D, IAA) - high auxin (cell elongation, Mediates the tropistic (bending) response of bending in response to gravity (gravitropism) and light (phototropism)) promotes Et synthesis
Cytokinins (zeatin) - Stimulates cell division., morphogenesis, conversion of etioplasts to chloroplasts, apical dominance, found in roots and shots
plant morphogenesis:
Aux > CK - rooty
CK > Aux - shooty
GA, CK and AUXIN promotes growth
Sencescence mediated by cytokinin (antagonize):ethylene (promote) balance
leaf abscission is mediated by decrease in auxin -> increase ethylene production -> synthesis of hydrolases -> leaf abscission
http://www.plant-hormones.info
http://www.planthormones.info/characteristics.htm
Seed Physiology
-------------------------------------------
Maturation Drying acts as a “switch”
A “switch” is needed to terminate development and promote the transition
to a germination and growth program
Control of Seed Maturation
Two key regulatory factors are abscisic acid (ABA) and restricted water uptake (negative osmotic potential in seed tissues).
ABA plays a role in (inhibits germination and promotes development - reserve synthesis):
• Encouraging development, including reserve synthesis
• Acquisition of ability to withstand water loss: accumulation of
desiccation protectants
• Inhibition of precocious germination
• Induction of dormancy (some species)
• In this sense, ABA ultimately controls seedling vigour - a seedling’s ability
to emerge and become established over a range of environmental conditions
Early evidence for role of ABA
(1) Embryo culture
(2) vivipary – germination of the immature seed on the plant
(3) Species that exhibit preharvest sprouting
insensitivity to ABA, (so-called ‘response mutants’) - defective in a component of the ABA signaling pathways eg. vp1 (maize) and abi3 (arabidopsis) - transcription regulators
- The mutant seeds are green, less dormant, reduced in their storage reserve content
and desiccation-intolerant. Maturation proteins degraded during development –
germination & growth program inappropriately switched on.
vp1 and abi3:
- Activate ABA-responsive genes during seed development
(e.g. storage-protein- and LEA genes).
- Inhibit the expression of germinative- and post-germinative genes
(eg. those involved in reserve mobilization)
- interacts with FUS3 and LEC1
Technological Aspects of Seed Physiology of Relevance to
Agriculture and Forestry
-------------------------------------------
(1) Problems with deep dormancy (subjected to microbes, need dormancy breaking),
non-synchronous germination (some grow fast) and
poor seedling growth (due to poor seed development, need to overseed)
(2) Malting & Cereal Industry: Problems with preharvest sprouting
(3) Preserving valuable genetic backgrounds through cryopreservation
of zygotic and somatic embryos
(4) Genetic modification of nutritional properties of seeds
(5) Seeds can be used as vehicles to host production of therapeutics
(6) Genetic modification to re-direct developmental processes
(7) Controversial ‘terminator technology’
(1) deep dormancy is bad
Seed Treatments to enhance seed germination or seed performance
1. Seed priming - Allows seeds to absorb enough water to initiate metabolic processes,
but insufficient water to complete germination, eg. drum, osmopriming, matripriming (clay)
2. Film coating - colored seeds, Applies a thin layer of polymeric material to the outside of the seed, Fungicides included in the polymer are bound onto the seed
3. Seed pelleting - Allows irregularly shaped seeds to be planted by machines
imbibe - To absorb or take in as if by drinking
(2) pre-harvesting is bad
pre-harvest sprouting - Germination of the physiologically mature grain on the parent plant, problem in malting and cereal industry; due to the premature loss of dormancy
A relative insensitivity to ABA may underlie sprouting susceptibility
Malting Process (barley grains used for production of beer, hard liquor)
- Carefully controlled water content changes – ultimately encourage enzyme (eg. α-amylase for starch hydrolysis) synthesis
& endosperm modification (flavour production) but discourage rootlet growth
(mobilization & utilization of reserves - reduction in malt yield).
(3) Preserving genes via gene banks
Yet, only about 15 spp. feed the world:
5 cereals: rice, wheat, barley, maize & sorghum
2 sugar plants: sugarcane & sugar beet
3 subterranean crops (potato, sweet potato & cassava)
3 legumes (bean, soybean & peanut)
2 tree crops (banana & coconut)
Just 3 spp. (wheat, rice & maize) : 70% of world’s seed crops
Trend in Agriculture: develop a few cultivars of high-yielding crops:
genetic uniformity at the expense of genetic diversity
(potential for global disaster: susceptibility to disease/environmental change)
Tree Seed Centre (Surrey): all tree seeds for BC: maintained at -20oC
Liquid N2 is a new technique: potential infinite longevity of seeds
Modes of Regeneration:
1. embryo culture - zygotic 2N embryo is excised from seed and grown in media
2. Somatic Embryogenesis - embryoids 1. induction - soak in auxin 2. development - globular, heart, torpedo, no auxin 3. drying - soak in aba 4. cryopreservation - add glycerol (cryopreservant)
(4) genetic modification of nutritional properties of seeds
- needs 9 essential amino acids, AILKMFTWV and micronutrients (vit A, zinc, etc)
Seed storage proteins:
albumins
globulins (Legumin 12S, vicilin 7s, deficient in methionine and cysteine)
glutelins (cereals, lack tryp and threonine)
prolamins (cereals)
Need to modify existing proteins of seeds to improve the composition
of essential aas
Basic strategies
1. Engineering the seed’s aa metabolism in order to
increase the free amount of aa
2. Engineering genes encoding endogenous storage
proteins (but adding extra aa's create a longer and unstable protein subject to degradation)
3. Transfer of genes encoding proteins enriched in
deficient aas (e.g. met-rich storage protein is
transferred into a legume) (but allergens) eg. vit A/golden rice
(5) seeds as medicine vehicles
Oleosin Fusion- Oleosin used as ‘transporter protein’... transports therapeutic protein
to oil body of seeds. Later, therapeutic is easily purified
(6) Genetic modification to re-direct developmental processes
A. Engineering Male Sterility for Hybrid Seed Production
barnase, a ribonuclease - produces sterile canola (no anther)
barstar - a ribonuclease inhibitor - produces fertile canola
B. Delaying Seed Pod Splitting (Dehiscence) to Avoid Seed Shatter
20% of seeds can be lost during harvest of canola
(7) Controversial ‘terminator technology’
terminator technology, is the name given to proposed methods for restricting the use of genetically modified plants by causing second generation seeds to be sterile.
to prevent "unauthorized seed-saving" by farmers. -- so farmers will need to buy seeds every year
http://www.globalissues.org/article/194/terminator-technology
Ethylene
-------------------------------------------
Ethylene effects in plants
• Fruit ripening
– A major ethylene effect that contributed to its
discovery
• Stress responses/wounding (Promotes ethylene synthesis)
• Abscission
• Senescence (biological aging)
• Lateral cell expansion
– Evident during triple response
in seedlings
• Root hair formation
High auxin levels promote Et synthesis
Ethylene is synthesized from methionine (CH3-S) group is recycled in the Yang cycle
AdoMet -----------1---------> ACC -----------2----------> Ethylene
• ACC synthase (1) catalyses the rate limiting step for Et
biosynthesis
– Regulated by environmental stress and auxin
– Unstable and present at very low levels in plant cells
• ACC oxidase (2) limits Et synthesis in tissues that make large
amounts of Et
Ripening is blocked in the rin mutant
• Unable to make climacteric Et
Ethylene response to stress:
- Leaf epinasty – Downward curving of leaves, induced by flooding (low O2 concentration, ACC accumulates)
- Aerenchyma: - Formation of air spaces in the cortex of roots due to low O2 concentration in flood
The triple response
– Ethylene reduces elongation growth
& increases lateral growth
• Inhibition and swelling of the
hypocotyl
• Inhibition of root elongation
• Exaggeration of the apical hook
Sencescence mediated by cytokinin:ethylene balance
Ethephon (Ethylene releasing agents) is used to:
• Ripen apples and tomato
– Useful when fruit are picked green and transported to market
• Accelerate abscission
– Useful for thinning fruit crops
• Reduce elongation growth and promote compactness in flowers
Stuff that inhibits Ethylene:
- low O2
- high CO2
- cool temp.
- Silver (Ag2+)
- 1-MCP
- AVG (not approved)
Ethylene action
ETR1:
- has an N-terminal (Et binding domain)
- histidine kinase catalytic site
- C-terminal receiver domain
• Ethylene receptors are negative regulators of ethylene
response
– Receptors are “active” in the unbound state
– Unbound receptor shuts off the ethylene response pathway
– Ethylene binding deactivates the receptors
• Response pathway proceeds
receptors are like locks and ethylenes are keys
• Ethylene receptors (ETR1, ETR2, ERS1, ERS2, EIN4) are functionally redundant
– Disrupting the regulatory domains of one receptor has no effect
on eliciting a ‘constitutive’ ET response
Two mutants:
(a) Ethylene resistant - mutant not responding to ethylene; has one receptor insensitive to ethylene (one lock has a broken key hole, so you can't open it) because of missense mutation in the binding domain, so Et can't bind to it, so by default the receptor shuts off Et response pathway, so you get a tall mutant, against the short wildtypes, easy to stop, only need one insensitive receptor to stop
(b) Constitutive ethylene response - Ethylene response is permanently activated; multiple receptors needed to be disrupted in the regulatory domain (broken lock, so you can open the door without a key) so it doesn't matter if there's ethylene or not, the receptor doesn't work and so it doesn't shut off the Ethylene response, so you get constitutive response so small mutant vs tall wildtypes, hard to start, need many disrupted receptors to start
Ethylene + Cu2+ -> ETR1 -> inactivate CTR1 -> activates EIN2 -> induce EIN3 -> induce ERF1 -> ethylene response
Plant Transformation – Methods for Introducing
Novel Genes Into Plants
-------------------------------------------
I. Agrobacterium-Mediated Transformation
Causes tumorous outgrowths
on plants: “Crown gall tumors”
Host specificity: Dicots and
gymnosperms, limited number of
monocots
tumour - disorganized growth and continuous cell division
Ti (tumour-inducing) plasmid (~200kb)
Transfer of part of Ti plasmid (the T-DNA; T=transferred) from Agrobacterium to plant
Agro. is only organism capable of inter-kingdom DNA transport! (plants, humans, fungi)
Agro is a “natural plant genetic engineer”
Ti plasmid is a natural plant transformation vector
T-DNA - contains genes that encode for auxin & CK (controls cell division), and opines (modified amino acid) synthesis
Regions of Ti-plasmid:
1) T-DNA: Part of the plasmid that gets physically transferred to
plant (via formation of T-DNA intermediate) - contains Oncogenecity genes: opine, CK & auxin, and left and right border sequences
2) Virulence Region (~40kb): All genes whose products are necessary for
transfer of T-DNA from Agro plant cell.....
• Formation of T-DNA intermediate
• Formation of channel from Agro to plant
• Shuttling of T-DNA thru channel to plant cell
• Nuclear targeting of T-DNA
• Chromosomal integration
3) Genes for synthesis of opines (serve as source of C &
N for Agro)
phenolic derivatives (eg. acetosyringone) released by wounded plant cells
- chemoattractant
- induce vir gene expression
- turns on host replication and repair machinery
Modifications to Ti Plasmid to Make it A Useful Plant
Transformation Vector
1. Removal of Onc genes from T-DNA (keeping border repeat
sequences intact)
2. Replacement of Onc Genes with Plant Selectable Marker Gene (kanamycin resistance)
3. Insertion of convenient Multicloning Site
For easy insertion of your gene of interest
between the T-DNA border repeat
sequences
4. Placement of Vir Gene Region (of Ti plasmid) on
a Completely Separate Plasmid
The Vir genes still work to transfer the (now modified)
T-DNA region to plant
I. Agrobacterium-mediated transformation (stable transformation only for dictos and gymnosperms):
Agro cells (containing 2 plasmids – one with T-DNA, the other with the Vir
region) are used to infect wounded plant cells (eg. leaf discs)
Leaf discs placed on medium to induce shooting, then rooting (all in presence
of selectable marker – Kan)
Kan-Resistant plantlets generated screen for expression of gene of interest
II. Direct DNA Transfer Methods (Alternatives to
Agro-Mediated Transformation) (these are less preferred, more destructive)
A. Microprojectiles (Gene Gun Method or Biolistics) - DNA coats surface of tungsten or gold particles
B. Microinjection: - use of holding pipette to deliver DNA solution directly into the cell
C. Electroporation - DNA taken up by recipient plant cells; electric
pulse is used to generate transient pores in plant
plasma membrane
With all these methods:
Assay for gene expression after 24-48 h
Go for stable transformation (attempt to
regenerate whole plant)
III. Applications: Engineering pathogen (microbes, bacteria, fungus, virus) resistance
-------------------------------------------
I. Plant diseases caused by microbial pathogens
How do pathogens reduce crop yields?
Cause tissue lesions
Reduce leaf, root or seed growth
Clog vascular tissues and causing wilt
Cause general metabolic drain, in the absence of external
signs of damage
Cause pre- or postharvest damage (blemishing total decay)
What factors cause crop devastation?
The Disease Triangle:
(1) Pathogen (genotype & prevalence or mode of introduction): Virulent
pathogen must be present in sufficient numbers at the right place and time
to start off the epidemic
(2) Plant (genotype and planting configuration): susceptible plant varieties
must be present
(3) Environment (pathogens are sensitive to temperature, humidity, wind
and weather conditions)
Two major factors can contribute to devastation:
Monoculture & Genetic Uniformity
1. Monoculture: Growth of a single
crop spp. on a large piece of land;
strong regional emphasis on a
given crop
2. Genetic Uniformity
Permitting genetic uniformity - the more dangerous practice
Farmers gravitate toward the most successful varieties of a crop
in terms of yield..... tendency to use fewer and fewer plant
genotypes
The best strategy is to maintain genetic diversity among the
different popular varieties of a given crop spp.
Disadvantages:
• non-uniform crop
• mechanical harvesting is not an option
Plant Viruses - potato virus x (rna), coat proteins
Virus symptoms
• lessions (spots) on leaves
• Can include a mosaic patterned
yellowing of leaves
• Leaf distortion & curling
• Raised bumps & mottling of squash
fruit
Bacterial wilt disease of cucumber, Xylella fastidiosa invades xylem, blocking water transport, causing wilting
Plant Fungal Diseases
Maize ear rot - caused by fungi that produce mycotoxins (harmful to humans and animals)
Wheat rust (fungus) diseases (stem & leaf)
- Rusts are the most destructive plant diseases known
Identify types of genes involved in pathogenicity of fungus:
proteins for synthesis & secretion of toxins
enzymes to break down plant cell wall
enzymes to detoxify plant defense chemicals
sugar transporters - support existence in nutrient-poor
xylem sap
regulatory proteins - adjust gene expression for growth in
different environments
synthesis & secretion of extracellular polysaccharides (slime)
efflux of antibiotics (produced by plant to kill bacteria)
uptake & sequestration of iron and other metals
Why is this important?
Devising novel approaches for control of pathogen
How do fungi enter the plant and cause disease?
*Enter at wound sites in the plant
*Secrete enzymes that hydrolyze plant cell walls
*If plant defense responses are not sufficiently
rapid, fungal hyphae quickly grow and spread
from cell to cell
Once fungal pathogen is inside plant cell......
- Some produce toxins alter permeability of membrane
- Some secrete slime accumulates in vascular tissues
wilting/death due to lack of transport (water & nutrients) from
roots to shoots
- Some produce plant hormones (plant loses control over
organized growth & development)
- Some attack seedlings as soon as they emerge or even before, eg.
soil-borne fungi (“damping-off”)
- Many cause necrotic (dead) spots on leaves, stems, fruits,
& seeds decrease vigor of plant; render seeds & fruits
less fit for human consumption
chemical strategies for disease control:
- Fungicides – applied as a seed coating prior to sowing. Or, as
sprays or as dusts on plants in field.
- Antibacterial - Copper or sulfur sprays & antibiotics
Problems with chemical strategies for disease control
1. Expensive to use
Impractical for grain crops (used on vegetable, fruit & flower crops)
2. Repeated use often leads to pathogen resistance
Some bacteria have genes encoding proteins that allow them to degrade,
export or otherwise resist the compound
Applies strong selection pressure ...
An initially small population of resistant bacteria becomes dominant in
population within the area
3. Human toxicity or broader environmental toxicity to non-target organisms
II. The biology of plant-pathogen interactions
Three general principles:
1. Plants defend themselves by using preformed defenses
(constitutive) & by turning on (inducible) defense genes
2. The key to plant resistance is swift induction of defense-related
genes ... in turn depends on early plant recognition of pathogen
3. Successful pathogens elude plant defenses
(1) Defenses always in place (“constitutive”) / passive:
1. Thick cell walls & waxy cuticle on surface of leaf & stem (wax dries out
rapidly less support for growth of fungi & bacteria)
2. Plants produce diverse array of antimicrobial compounds (mostly active/inducible)
Preformed inhibitors (glucosides, saponins, alkaloids)
Antifungal proteins
Antifeedants
Enzyme inhibitors
Not all are constitutively produced; some are induced....
Plant does not want to devote its metabolism to defense
Response of plant to microbial infection is multifaceted:
* Increased ("up-regulated") expression of a large number of pathogenesis-related genes in cells at site of infection (eg. chintinase and glucanase degrade cell walls of invading pathogens), antimicrobial (thionins, defensins, lectins, phytoalexins)
* Activation of pre-existing enzymes that control synthesis of anti-microbial compounds
* Strengthening and cross-linking of the cell walls
* Secretion of phenolics (eg. lignin - dense phenolic – based
polymer (network), eg. salicylic acid - precursor to aspirin for anti-inflammatory) into the cell walls
* Generation of signaling molecules move locally or systemically to activate defenses in other plant cells: systemic acquired resistance
* In some cases, the hypersensitive response (seen as necrotic spots).... a beneficial plant cell death response cells immediately surrounding the infection site die, effectively preventing spread of pathogen
Summary of different types of plant defense
(2) Plant recognition of Pathogens
Plant must be able to recognize presence of a pathogen.....
Recognition: binding of molecule derived from pathogen ('elicitor') to molecule (receptor) (R-protein) of the plant
Resistance genes (R-genes): Plant genes encoding recognition (receptor) proteins
Each R-gene encodes a protein that recognizes a specific pathogen compound activates host defense responses
Elicitor may be:
• a virus coat protein eg. Tobacco mosaic virus
• a bacterial virulence factor (secreted into host plant)
• a fungal protein present on pathogen surface
• cell wall fragment?
When strong resistance defense responses are elicited in plant -> formation of necrotic spots
Represent infected plant cells that plant has actively sacrificed to prevent spread of pathogen
Rapid hypersensitive response is a programmed cell death process... it is adaptive because:
• it effectively “walls off” the pathogen
• releases antimicrobial compounds
• releases signaling molecules that elicit defense responses in
other host cells: systemic acquired resistance
• kills off host cells that might otherwise support the growth
of the pathogen (virus/fungus)
How do pathogens successfully evade plant recognition?
- Different R gene products control defense activation: detect extremely different pathogens (viruses, bacteria, fungi, nematodes or insects).
- Proteins encoded by R genes share similar structures & mechanisms for pathogen recognition are highly conserved across different plant species and diseases
Why is this significant??
- different specifics of pathogen recognition evolved from small number of progenitor R genes
- New R genes with new pathogen recognition capabilities arise over time. This evolution has been crucial for the ongoing battle of plants to keep pathogens at bay.
- Over-use of chemicals to combat pathogens -> Strong selection pressure resistant population emerges
- Same thing occurs when pathogens face R genes
- Elicitors .... Usually part of the pathogen structure
- Pathogen evolves to elude the plant’s recognition system
Good news is that plants have around 100 R genes
III. New biotechnological approaches to create plants with
enhanced disease resistance
Classical crop protection strategies (plant breeding) -> id. R genes in wild plants & older crop var.
-Of same or very closely related species
Forest industry: Rust-resistant pine
With a genetic engineering strategy...
R genes (& other genes inv. in defense) can be isolated from one plant spp. and introduced into another
No need for sexual compatibility
What are some major genetic engineering strategies?
(1) Expression of genes encoding specific antimicrobial compounds
(e.g. PR proteins)
· Hydrolytic enzymes (chitinases - Hydrolyze b-1,4-linkages within chitin polymers of fungal cell wall, glucanases
· Antifungal proteins (osmotin- and thaumatin-like)
· Antimicrobial peptides (defensins, lectins, lysozyme)
· Ribosome-inactivating proteins (RIPs)
· Phytoalexins
(2) Expression of genes encoding products that can potentially
enhance the structural defenses of the plant
· elevated levels of lignin
(3) Expression of genes encoding products that destroy or neutralize
a component of the pathogen arsenal
· gene for oxalate oxidase, involved in the degradation of oxalic
acid)
oxalic acid –inactivate plant cell defense enzymes?
(4) Expression of genes encoding products that result in the release of
signals capable of regulating plant defenses
· specific elicitors
· H2O2
· salicylic acid (SA)
· ethylene (C2H4)
(5) Expression of genes encoding defense-activating “master switch”
proteins
(1) Expression of genes encoding specific antimicrobial compounds
(e.g. PR proteins)
· Chitinases can defend against fungal attack & invading fungal hyphae
· Chitinases: basic or acidic - acidic forms are extracellular; basic forms are found in vacuole
Problem: In many cases, only partial resistance is obtained
npt = neomycin phospotransferase (plant selectable marker gene Kan-res)
But! Researchers are expressing different combinations of genes:
greater resistance is the outcome
Eg. Expression of genes encoding a chitinase & b-1,3-glucanase (tomatoes)
Eg. Expression of chitinases & RIP (ribosome-inactivating protein) in barley
(4) Expression of genes encoding products that result in the
release of signals capable of regulating plant defenses
eg. salicylic acid (SA) will activate SAR when sprayed on plant -> system primed for defense
eg. Expression of elicitor in plant (viral coat protein), interference of viral protein with viral RNA replication, viral movement from cell to cell
(5) Expression of genes encoding defense-activating “master switch” proteins
Expression of R gene products (eg. constitutive Cf9 R protein) involved in HR and in interaction with avirulence (lack of virulence; lack of competence of an infectious agent to produce pathologic effects) (inducible Avr9 elicitor) factors => hypersensitive defense.
Plant defense against biotic stress - pests (insects, herbivores, cows, worms, weevil, flies, etc.)
-------------------------------------------
Plants are surrounded by hungry herbivores
– Herbivores range in size from microbes to cows, white pine weevil, Cotton boll weevil, Cotton bollworm (eats developing fruits), Caterpillars, army worm, pine bark beetle, Potato beetle, whitefly (sucks phloem sap),
nonnative plant pest - pest migrate to a new country without their predators
Predator populations – determine insect pest populations
Chemical defenses:
- Natural products:
a) terpenes
b) phenolic compounds
c) nitrogen-containing secondary product
- Plant secondary metabolites include chemicals we use as
• Drugs (medicinal and recreational)
• Dyes
• Perfumes
• Beverage manufacture
• Poisons
(a) - Terpenes/Terpenoids - mono-, sesqui-, di-terpines, insect repellant, feeding deterrant, oleoresin (both constitutive and induced) in conifers
-------------------------------------------
• Based on 5C isoprenoid unit:
- Monoterpenes 2 x 5C
- Sesquiterpenes 3 x 5C
- Diterpenes 4 x 5C
etc.
• Roles:
– Plant growth and development
• Gibberellins are diterpenoids 4x5C, ABA is a sesquiterpenoid - 3x5C (hormones)
• Carotenoid pigments are tetraterpenoids 2x(4x5C)=C40
– Photosynthetic pigments & protect against high light
– Plant defense
• Toxic and/or feeding deterrents for herbivores (monoterpenes - 2x5C) - insect repellants and triterpenes - 2x(3x5c) - feeding deterrants
- Phytoecdysones disrupts molting (also known as sloughing, shedding or for some species,)
Defence against insect herbivory
• Responses to insect herbivory involve the wound response
– Lead to inducible chemical defenses:
• affect attacking insects
• affect natural enemies of attacking insects
• BUT most resistance is “constitutive”
– Results from pre-existing chemical or morphological
defenses
Terpenoids and insect defence in conifers:
The major defense against insect and pathogen
attack in conifers is the oleoresin
– Complex mixture of mono-, di-, and sesquiterpenes
– Mono- and sesquiterpenes are volatile and provide
fluidity to resin
• Resin (Resin is a hydrocarbon secretion of many plants, particularly coniferous trees, used in nail polish, fossilizing/amber insects http://en.wikipedia.org/wiki/Resin) flows to point of injury
• Insects are exposed to toxic terpenoid components
– Diterpenes seal wounds
Resin defenses are constitutive and induced
– Constitutive defenses (pre-formed traits)
• First line of defense, repel attack
– Induced defenses
• Second line of defense
• Resin composition differs
– More insect toxic?
The white pine weevil is a pest of
regenerating Sitka spruce
Weevil attack induces the formation of induced
resin canals in the xylem
(b) Phenolics - flavonoids (anthocyanin-color, flavonol-color in flower, isoflavonoids-anti-cancer in legumes, anti-estrogen, tannins-feeding repellants, non-specific protein binding=toxic, wine), lignin=structural,
-------------------------------------------
Plant phenolics are biosynthesized in several different ways. In higher plants, most phenolics are derived at least in part from phenylalanine, a product of the shikimic acid pathway
1) Lignin
- Dense polymer made up of network of phenolic units
- Provides mechanical support to plant
- Synthesis is induced by pathogen infection or insect wounding
Lignin resists attack by most microorganisms. Lignin is nature's cement along with hemicellulose to exploit the strength of cellulose while conferring flexibility.
2) Flavonoids:
- Basic structure: 15 Cs arranged in 2 aromatic rings connected
with a 3C bridge
4 groups:
• anthocyanins - colored flavonoids that attract animals
• flavones and flavonols - Flavonoids of flowers, attract bees and N2 fixers, short wavelength
• isoflavonoids - one aromatic ring is shifted, act as anti-estrogens => infertility, anti-cancer
• Tannins: condensed (feeding repellants, toxic) or hydrolyzable (gallic acid - antioxidant, red wine),
Anthocyanins - The structures of anthocyanidins (A) and anthocyanins (B). The colors of anthocyanidins depend in part on the
substituents attached to ring B (see table). An increase in the number of hydroxyl groups shifts absorption to a
longer wavelength and gives a bluer color. Replacement of a hydroxyl group with a methoxyl group (OCH3) shifts
absorption to a slightly shorter wavelength, resulting in a redder color.
Isoflavonoids (Isoflavones):
• found in legumes
• some are insecticidal
• some act as anti-estrogens:
- sheep grazing on clover rich in isoflavonoids can
suffer from infertility
- anti-cancer benefits of soy-based foods
Tannins (condensed/ hydrolysable - gallic acid):
• Polymerization of flavonoid units
• Condensed tannins found in seed coats of legumes:
toxic towards some seed-eating beetles
• Significantly reduce growth of many herbivores
• Feeding repellents for many animals ( eg. unripe fruits
with high tannin levels avoided by deer and cattle)
The defensive properties of tannins are due to their toxicity
- ability to bind proteins non-specifically
Tannins:
• Red wine polyphenolics (tannins) have health
benefits:
- block formation of endothelin – 1, a signaling molecule that makes blood vessels constrict
- benefits for heart disease
(c) Nitrogen – containing secondary compounds (alkaloids-cocaine, cyanogenic glycosides-HCN gas-found in seeds of almonds etc., aa-analogs-canavanine):
-------------------------------------------
Alkaloids (nicotine, cocaine, morphine, codeine- for analgesic(pain relief), etc):
• These can be extremely toxic
• Synthesized from amino acids (terpene pathway supplies C – skeleton)
Alkaloids:
• Very effective deterring insect attack
• BUT!! Place strong selective pressure on predatory insects
to overcome defense mechanism
• Some herbivores (oxidizing alkaloid instead of reducing it in the gut) can become adapted to tolerate one class
of alkaloids
Cyanogenic glycosides:
• When broken down, release poison – hydrogen cyanide (gas)
• Not broken down in intact plant
• Leaf damage due to herbivore feeding allows hydrolysis
• Some CGs are found in seeds of almonds, apricots, cherries,
peaches, etc.
• HCN – toxin that inhibits metalloproteins (eg. cytochrome oxidase)
• Tubers of cassava
Non-protein amino acids:
• Play a protective role in some seeds
• Eg. amino acid analogue (canavanine – an analogue of Arg)
is produced in seeds of Brazilian vine
• Toxic to most animals & insects: inability to distinguish analogue
from aa Arg vs. non-functional protein (3-D structure or catalytic
site is altered)
Other defensive proteins
a. Inhibit herbivore digestion: - α – amylase inhibitors, Proteinase inhibitors, Lectins
b. Inhibit protein synthesis:- Ribosome–inactivating protein (RIPs)
Enzyme inhibitors:
• Insects use amylases and proteases to digest the
starch & protein in their food
• Many seeds (especially legumes) contain inhibitors
of insect digestive enzymes:
Protease inhibitors (PIs) - Inhibitors of insect proteases such as trypsin, chymotrypsin, elastase & subtilisin
Mechanism of inhibition - inhibitor forms a strong
covalent bond with the active
site of the insect protease
• Insect larvae starve to death due to loss of nutrients
& over-production of proteases
Protease inhibitors:
Leaves of various plant species (e.g. tomato, potato)
rapidly synthesize PIs
• in response to mechanical damage (insect attack/
wounding)
• synthesis occurs throughout the plant
• stored in central vacuole as defense against
repeated attack
alpha-amylase inhibitors:
Unlike peas, beans are not attacked by pea weevils.
Beans contain a protein that inhibits the activity of
alpha-amylase, an enzyme that helps in digestion
of starch. This protein inhibitor, called α-amylase
inhibitor, causes the weevils feeding on beans to
starve before they cause any damage.
Lectins:
• Carbohydrate-binding proteins (in plant tissues, seeds):
different lectins have different sugar specificities
• After ingestion by an herbivore, lectins bind to epithelial
cells lining digestive tract (interfere with nutrient
absorption)
• up to 30% of total protein in some seeds
• lectin from bean: toxic to developing larvae of bruchid
beetle (may bind to midgut ephithelial cells)
Ribosome-Inactivating Proteins (RIPs):
“ Jack in the box”
- e.g., highly toxic ricin found in castor bean seeds
- contain a lectin chain linked to a polypeptide that inactivates
ribosomes (hydrolyzes the sugar base linkage at one specific
position in the rRNA)
- highly toxic towards Coleoptera (e.g., boll weevil and bruchid
beetle) and locusts
- not good candidates for genetic engineering
“ Bulgarian Diplomat “
Combating insects with chemical insecticides:
Chemical insecticides (e.g., organochlorines – DDT):
Problems:
(i) Toxicity (to non-target organisms – pollinating insects,
natural predators of pests, humans)
(ii) Environmental spread (more than 99.9% is wasted;
substantial economic cost)
(iii) Loss of effectiveness (build up of populations of
resistant pest species) – high selection pressure
imposed by toxicity and heavy application
(iv) Consumer pressure – public concern over pesticide
residues in food stuffs
Biological control:
- Use of predatory or parasitic insects, nematodes
and fungi (eg. aphid parasite (parasitic wasp) on greenbug aphids)
- Effective in confined areas; success in field is
limited
- Requires that a population of
the pest has built up and that
the biological control organism
is not itself competed out
Strategies for genetic engineering of plants
for insect resistance
Transgenic plants expressing:
Protease inhibitors
α-Amylase inhibitors
Plants have evolved amylase and protease inhibitors to specifically
inhibit the digestive enzymes of certain insects
Transgenic tobacco expressing cowpea trypsin inhibitor (non-toxic to humans):
Differences in organization of mammalian & insect gut:
- in mammals: any inhibitor would be exposed to acid pH 2 of stomach where it is exposed to pepsin first, then Digestive enzymes (trypsin, chymotrypsin) in small intestine ph8
- insects don't have pepsin before passing to mid-gut ph9-11
Transgenic plants expressing genes for α-Amylase inhibitors:
• Amylase inhibitor of kidney bean – completely inhibits growth of pea
weevil and cowpea weevil
• Introduced bean gene into peas (got high level expression):
development of pea weevil larvae was completely inhibited
• BUT! Animal feeding experiments show deleterious effect of transgenic
peas
Plant defenses against insect herbivores:
1) Constitutive defense responses (mostly morphological - leaf hair):
- always present
- species-specific
- stored in less-damaging form
“Constitutive Defenses”
Eg. chemicals constitutively
produced by leaf hairs trap
and kill larvae
2) Induced defense responses:
- initiated only after actual damage occurs
- same defense chemical may be involved in
constitutive and inducible responses
Insect herbivores can be classed by degree of
damage inflicted on plant:
Least
damage
1) Phloem feeders
2) Cell content feeders
3) Chewing insects
Most
damage
1) Phloem feeders:
• Aphids and Whiteflies
• Direct injury to plant
is low, but insect may
vector plant viruses:
aphids spread barley
yellow dwarf virus, a
common disease of
cereals
2) Cell content feeders
• Mites and thrips
• pierce plant tissue
• intermediate damage
3) Chewing insects
• caterpillars
• grasshoppers
• beetles
• Cause significant damage to plant
• Can vector fungal pathogens
Responses to
insect herbivory
and pathogen
attack overlap
Insect defence in angiosperms
• Herbivory injury is often mimicked in part by wounding
• Leaf wounding causes Systemic response in
distal, unwounded leaves
– Local responses
– Systemic responses
• Rapid
Local response in wounded leaf
• Insect attack/wounding results in mobile signals emanating
from damaged tissue
• As well as a wounding response, the plant may recognize
insect – derived compounds: “elicitors”
Outcome of local and systemic responses:
1. Direct defense responses
• Production of proteinase inhibitors (PIs) + other “nasties”
• Decrease palatability of plant or fitness of insect
2. Indirect responses
• Production of volatile organic chemicals (VOC)
• Target predators or parasitoids of attacking insect
Elicitors present in insect saliva:
• Fatty acid – amino acid compounds
• Ingested plant tissue supplies source of
fatty acid (eg. linolenic acid in plants, 18:3)
• Enzyme in gut of insect conjugates plant
FA to insect amino acid Gln
• When plant recognizes elicitors present
in insect saliva – signal transduction
pathway is triggered:
increases jasmonic acid (JA)
Insect defence in tomato
• PI production is used as a ‘marker’ for wound responses
& can be induced by the following signals:
– Oligogalacturonides (OGA)
• Cell wall fragments released due to damage/enzymatic
degradation
– Jasmonic acid (JA)
• Lipid-derived (oxylipin) signalling molecule (derived from
linolenic acid)
– Systemin
• Peptide (18 amino acids long) produced due to proteolytic
cleavage of a precursor polypeptide (first peptide hormone
identified in plants)
• Mobile signal?
• Triggered by wounding and insect herbivory
wound > prosystemin > systemin > LRR receptor > PLA2 > JA biosynthesis > JA translocated via phloem to target cell to encode protease inhibitor
Insect elicitors modify the wound response: tobacco hornworm M. sexta (normally blue, but eats yellow carotenoid and so it turns green) attacking tobacco plants that germinate in response to wood smoke
Interestingly, the nicotine in the leaf is normally toxic, but the caterpillars have a mechanism for selectively sequestering and
secreting the nicotine.
Insect elicitors modify the wound response
• Nicotiana attenuata produces nicotine as a major chemical defense
– Nicotine poisons acetyl choline receptors at nerve-muscle junctions
• Nicotine synthesis is induced by wounding
• When the plant is attacked by nicotine tolerant M. sexta, there is a
decreased production of nicotine
- pest detoxifies/sequesters nicotine
- pest modifies the plant wound response
• Instead, volatile terpenes are released
- attract insect predators of the pest
- decrease oviposition of adult moth
M. sexta oral secretions and regurgitants are
sufficient and necessary to modify the wound
response
– Fatty acid-amino acid conjugates
Insect elicitors modify the wound response
Who benefits?
– Insect?
• Less nicotine produced
• Reduces growth penalty assoc. with detoxification
– Plant?
• Optimizes indirect defenses
• Reduces costly metabolism directed towards nicotine
production
• Other direct defenses are not affected
– Slows development of M. sexta
– Increases opportunity for predation
Lepidoptera – (worms) Caterpillars, corn borers,tobaccobudworm/hornworm, corn earworm, army worm
Coleoptera–Cotton boll weevil, bruchidbeetle
Others –White pine weevil, cone/seed eating pests, pine bark beetle
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