Plant Defense and Secondary metabolism General introduction to plant defense and attraction

Multitrophic Interactions Plant Defense: Multitrophic Interactions Secondary carnivores Carnivores Herbivores Pollinators Shoots and flowers Competitor plants Pathogens Aboveground Belowground Roots Pathogens Parasitic plants Herbivores Symbionts Carnivores Modified from Bruinsima & Dicke 2008

Plants respond to attacks by herbivores and pathogens Plants use defense systems to deter herbivory, prevent infection, and combat pathogens Herbivory, animals eating plants, is a stress that plants Plants counter excessive herbivory with physical defenses such as thorns and chemical defenses such as distasteful or toxic compounds Some plants even “recruit” predatory animals that help defend against specific herbivores

Plant Response to Herbivores Plant Defense Traits

Plant Response to Herbivores Plants damaged by insects can release volatile chemicals to warn other plants of the same species These volatile molecules can also function as an “early warning system” for nearby plants of the same species. Methyljasmonic acid can activate the expression of genes involved in plant defenses

Jasmonic Acid Levels of jasmonic acid rise in response to damage This hormone can trigger many types of plant defenses including terpenes and alkaloids The action of jasmonic acid induces the transcription of many genes involved in plant defense Jasmonic acid turns on genes for proteinase inhibitor.

Systemic Response

Plant defense traits Plants use a variety of mechanical (toughness, spines), chemical (alkaloids, phenolics, terpenoids, latex – the realm of chemical ecology), developmental, and phenological defenses Defenses may also be classified with reference to their production: 1. Constitutive – produced by & present in the plant irrespective of attack 2. Induced – produced by & present in the plant in response to attack

Plant defense traits Resistance traits Those traits that “reduce herbivory” Avoidance (antixenosis) traits Those traits that “affect herbivore behavior;” i.e., deter or repel herbivores b. Antibiosis traits Those that “reduce herbivore performance” 2. Tolerance traits Those traits that “reduce the impact of herbivory on fitness”

Resistant vs Tolerant Resistant Tolerant Susceptible For illustration, I have a resistant, tolerant, and susceptible (or non-defended) plant represented in this cartoon. In the presence of herbivores… Resistant Tolerant Susceptible

Benefits of defense are obvious in the presence of herbivores Resistant vs Tolerant Benefits of defense are obvious in the presence of herbivores The benefit of investing in defense is increased fitness in the presence of herbivores relative to susceptible genotypes. Resistant Tolerant Susceptible

Costs of defense are obvious in the absence of herbivores Resistant vs Tolerant Costs of defense are obvious in the absence of herbivores However, the production and maintenance of defense traits and mechanisms may incur a cost to the plant when herbivores are rare or absent. So resistant and tolerant plants would be expected to have lower fitness than non-defended plants when herbivores are absent. Resistant Tolerant Susceptible Slide courtesy of Alyssa Stocks Hakes; modified from the original

Resistance Traits DIRECT INDIRECT Slide courtesy of Amanda Accamando; modified from the original

Direct Defense Morphological Characters Secondary metabolites E.g., Tannins

Cutin, Waxes, Suberins All plant parts exposed to the atmosphere are coated with layers of lipid material that reduce water loss and help block the entry of pathogen fungi and bacteria The principal types of coating are cutin, suberin and waxes They are made of hydrophobic compounds which have water-repelling properties These compounds are non-polar Fatty acids are one type of hydrophobic compound

Cutin It is found most above ground It is a macromolecule, a polymer composed of long fatty acid chains that are attached to each other by ester linkage, creating a rigid three dimensional network It was a major component of plant cuticle, a multilayered secreted structure that coats the outer cell wall of epidermis on the areal parts Plants’ cuticles is composed of a top coating of wax, often vary with the climate in which they live.

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Waxes Complex mixtures of long-chain lipids that are extremely hydrophobic. The most common components of waxes are straight chain alkanes and alcohol of 25 to 35 carbon atoms. They are synthesized by epidermal cells. They exuded through pores in the epidermal cell wall by an unknown mechanism.

Suberin It was formed from fatty acids but has a different structure from cutin. It was often within roots. It can protect against pathogens and other damage. It can form transport barriers between the soil and the roots Older parts of roots more suberized A cell wall constituent Endodermis has suberin side walls

End points of metabolism with no strictly defined function Secondary metabolites End points of metabolism with no strictly defined function Organic compounds that appear to have no direct function in photosynthesis, growth, or respiration, but 1. They protect primary metabolism by deterring herbivores, reduce tissue loss and avoid infection by microbial pathogen 2. They also attract pollinators and seed-dispersing animals, 3. They act as an agent of plant-plant competition 4. They are formed from the byproducts or intermediates of primary metabolism

Three Principal Groups of Secondary Metabolites Terpenes Lipid synthesized from acetyl CoA or from basic intermediates of glycolysis Phenolic compounds Aromatic substances formed via the shikimic acid pathway or the malonic acid pathway Nitrogen containing secondary products (alkaloids) Alkaloids which are synthesized primary from amino acids

Plant is more than primary metabolism: more than sugars and starch, and proteins etc, extracting compounds from the primary C pathways for biosynthetic purposes

Important part of plant metabolism is geared towards plant defense

Terpenes They produced from the mevalonic acid pathway They function as herbivore deterrents They can be produced in response to herbivore feeding, and to attract predatory insects and parasites of the feeding herbivore. They are constituents of essential oils Building block- 5 C isoprene unit They are classified by the number of isoprene units: monoterpenes-1, diterpenes-4

Isoprene H3C CH CH CH2 H2C The basic building block of the terpenes (terpenes also called “isoprenoids”) H3C CH CH CH2 H2C Terpenes: Monoterpenes have two C5 units (10C) Sesquiterpenes have three C5 units (15C) Diterpenes have four C5 units (20C) Triterpenes 30 C Tetraterpenes 40C Polyterpenes ([C5]n), n>8

Terpene functions Growth and development carotenoid pigments are tetraterpenes chlorophyll side chain is diterpene giberellins (hormones) are diterpenes abscissic acid (hormone) is a sesquiterpene C15 sterols are triterpenes

1. Resins of conifers are monoterpenes 2. Defensive compounds Toxins and feeding deterrents to insects and mammals Examples 1. Resins of conifers are monoterpenes 2. Essential oils - peppermint, limon,

PP13060.jpg Non-volatile Volatile

Non-volatile terpenes – limonene apparently distasteful to herbivores PP1306a.jpg Non-volatile terpenes – limonene apparently distasteful to herbivores

PP1306b.jpg Volatile terpenes such as menthol broadcast a smell that warns herbivores that the plant is toxic to them before herbivore feeding commences.

Terpenes such as pyrethrum (from chrysanthemums) and azadirachtin (from the Asian and African Neem tree) can be used as “natural” insecticides in agricultural practices or in horticulture.

Terpenes that act against vertebrate herbivores Triterpenes 1. Cardenolides (glycosides) - acutely toxic influence Na+/K+ ATPase of heart muscle medicinal application - digitalis (from foxglove), used to treat heart disease. Can slow and strengthen heart beat 2. Saponins (soaplike) - steroid, triterpenes glycosides have lipid and water soluble parts of molecule toxicity related to sterol binding, membrane disruption

Terpenes as human medicinal drugs Limonene - monoterpenoid (C10) dietary anticarcinogen Artemisnin - sesquiterpenoid (C15) antimalarial Taxol - diterpenoid anticancer drug from Pacific yew (Taxus brevifolia)

Phenolics Plants produce a variety of compounds that contain one or more phenol groups - called phenolics Thousands of phenolics occur in plants

Phenolic Compounds Secondary metabolites which contain a hydroxyl functional group on an aromatic ring They are heterogenous group: Some are water soluble only in organic solvents Some are water soluble carboxylic acids and glycosides Some are insoluble polymer Many serves as defense compounds against herbivores and pathogens Other function in attracting pollinators and fruit dispensers

Types of Phenolic Compounds Benzoic acid derivatives Caffeic acid and other simple phenylpropanoids Flavones Isiplavones (Isoplavonoids) Flavonoids Lignin

N-containing secondary compounds Those are encountered less commonly in plants than the phenolics and terpenoids Those are important in view of their bioactivity as drugs and toxins They are synthesized from aliphatic and aromatic amino acids Aliphatics via TCA cycle Aromatics via shikimic acid pathway

Classes of N-containing 2o compounds Alkaloids, Cyanogenic glycosides, Glucosinolates, Nonprotein amino acids

1. ALKALOIDS The most important nitrogen containing secondary products They are being found in more than 15,000 compounds found in 20% of vascular plants. Nitrogen is usually part of a heterocyclic ring with N and C atoms

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ALKALOIDS Large pharmacological effects on animals Most effective at deterring mammalian herbivores Livestock deaths due to over-consumption of alkaloid containing plants such as lupines and groundsels Often alkaloids are used as medicines for humans Some examples: morphine, codeine, and scopolamine cocaine, nicotine, and caffeine used as stimulants and sedatives.

Wild tobacco (Nicotiana sylvestrus) Wild tobacco can “sense” which herbivore is feeding on it. It normally produces nicotine (an alkaloid) in response to herbivore feeding. But if nicotine-tolerant caterpillars are feeding, the tobacco produces terpenes instead. These terpenes can attract the predators of the herbivore.

2. CYANOGENIC GLYCOSIDES Release the toxic gas hydrogen cyanide. plants must have enzymes to break down the compounds and release a sugar molecule yielding a compound that can decompose to form HCN. glycosides and enzymes which break them down are usually spatially separated (in different cellular compartments or different tissues)

The degradation process is stimulated by herbivore feeding Cyanogenic Glycosides The degradation process is stimulated by herbivore feeding PP13200.jpg

Cyanogenic Glycosides S. American native peoples eat cassava (Manihot esculenta), has high levels of cyanogenic glycosides. Chronic cyanide poisoning are not uncommon. PP13200.jpg

3. GLUCOSINOLATES These compounds release volatile defensive substances, “mustard oils”, (often herbivore repellents) Plants like cabbage, broccoli, and radishes (Brassicaceae family) have these.

4. NON-PROTEIN AMINO ACIDS These amino acids are not incorporated into proteins but instead act as protective substances can “mistakenly” be incorporated into protein and therefore resulting in a nonfunctional protein.

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Functions of Secondary Metabolites in Plants The secondary metabolites have no function in the physiology of the plants They are formed as a result of an overspill from the primary metabolism They make a valuable contribution to the relationship between plant and their environment Plant utilized secondary metabolites as antibiotics or signaling agent during the interaction with pathogen (SAR and Agrobacterium) They play an important role in two resistance strategies: a. structural level, phenyl propanoids are the major component of wall polymers lignin and suberin b. Inducible defence antibiotics originated from phenolics, and terpenoids (phytoalexins)

Herbivore damage can elicit a Signaling Pathway (Induced Defenses) Resistance Traits: Indirect Defense Induced defenses: a. Recognition of the pathogen by the host plant; carbohydrates, fatty acids released by fungi b. Transmission of alarm signal to host; Ca, hydrogen peroxide, enzymes. Herbivore damage can elicit a Signaling Pathway (Induced Defenses)

Induced structural defenses cytoplasmic -cytoplasm surrounds hyphae (e.g., Armillaria), cell wall thickening histological (cork layers, adventitious roots), abscission layers, tyloses and gums, necrotic defense (hypersensitive response).

Induced biochemical defenses Hypersensitive reactions (phytoalexins, antimicrobials (important with obligates parasites – rusts, leaf spots, active oxygen radicals disrupt cell membranes, reinforcement of host cell walls) Antimicrobials – phytoalexins, phenolics Immunization Local and systemic acquired resistance

(Phyto = “plant” and alexin = “to ward off/”) Phytoalexins (Phyto = “plant” and alexin = “to ward off/”) Low molecular mass antimicrobial metabolites synthesized de novo from primary metabolites in response to infection Structurally diverse group of metabolites with the isoflavonoids can be an example The isoflavonoids phytolaexins are synthesized from the flavonoids branch of the phenylpropanoid pathways

PRODUCTION OF PHYTOALEXINS Production of phytoalexins may be stimulated by certain compounds called elicitors. High molecular weight substances found in the cell wall such as glucans, glycoprotein, or other polysaccharides Gases such as ethylene (C2H4) In susceptible plants, a pathogen may prevent the formation of phytoalexins, by the action of suppressors produced by the pathogen The suppressor also can be a glucan, a glycoprotein, or a toxin produced by the pathogen

How are Phytoalexins Formed? The “-noids” Shikimic acid pathway (phenylpropanoids) Hydroxycinnamic acids Coumarins Hydroxybenzoic acids Mevalonic acid pathway (Isoprenoids) Carotenoids Terpenoids Combination of Pathways Shikimic-Polymalonic) Flavonoids and anthocyanins

Signaling Cascade for Defense Responses Molecular nature of elicitors: Cell wall proteins (e.g., Harpin) Intracellular proteins (defined genetically in a bacterium by cloning avirulent loci) Peptide derived from a larger protein (from a fungus) Heptaglucan (small oligosaccharide)

Secondary Signals Ca+2 , required for subsequent steps May mediate phosphorylation-dephos. events involved in transcriptional or post- transcriptional gene regulation (there are a number of genes whose transcription increases, and some decrease) Some defense genes also induced by blue-UV light or other stresses

2. H2O2 (hydrogen peroxide) Secondary Signals 2. H2O2 (hydrogen peroxide) Plays multiple roles: induces defense-related genes induces apoptosis causes cross-linking of cell wall proteins (more resistant to wall-degrading enzymes) may directly kill pathogens

Secondary Signals 3. Salicylic acid required for SAR levels increase locally and at distance from infection Systemic Signal? Probably not. Still unknown

Signaling Cascade for Defense Responses Model derived mostly from studies in cell culture using specific elicitors. However, there is evidence for induction in intact plants by R genes. Some aspects are also constitutive and help block most microbes (non-host resistance).

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Systemic Acquired Resistance Systemic acquired resistance causes systemic expression of defense genes and is a long-lasting response Salicylic acid is synthesized around the infection site and is likely the signal that triggers systemic acquired resistance When a plant survives the infection of a pathogen at one site it can develop increased resistance to subsequent attacks. Although plants don’t have “immune systems” they have signaling mechanisms that can act in this way.

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Genetice model for SAR Infected Tissue Non-infected Tissue INA BTH Isd6 Cpr1? Isd2 Isd4 nim1 npr1 Ndr1? Cpr1 Cin2 Cin3 NahG Local Acquired Resistance Plant Pathogen Interaction Salicylic Acid SAR gene Expression Necrosis INA BTH Non-infected Tissue nim1 npr1 NahG Systemic Acquired Resistance Salicylic Acid SAR gene Expression