Photosynthesis: Regulation of Carbon fixation reactions

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In photosynthetic organisms, the carbon fixation reactions are regulated by light to ensure that the production of carbohydrates is synchronized with the availability of light energy. This regulation involves several mechanisms, including the activation of key enzymes, changes in the concentration of metabolic intermediates, and the formation of supramolecular complexes.

Induction Period in Carbon Fixation

After the onset of illumination, there is an induction period during which the rate of CO2​ fixation increases gradually before reaching a steady state. This initial lag is because the photosynthetic enzymes need time to be activated by light, and the concentration of Calvin-Benson cycle intermediates needs to increase.

• During the induction period, six triose phosphates produced in the Calvin-Benson cycle are used for the regeneration of ribulose 1,5-bisphosphate (RuBP).

• At the steady state, five of the six triose phosphates regenerate RuBP, while the remaining one is either used for starch formation in the chloroplast or sucrose synthesis in the cytosol.

Regulation of the Calvin-Benson Cycle

The regulation of the Calvin-Benson cycle involves both long-term and short-term mechanisms.

Long-term regulation involves changes in the amount of enzymes, which is controlled by the expression of nuclear and chloroplast genes. Regulatory signaling between nucleus and plastids is mostly anterograde — that is, the products of nuclear genes control the transcription and translation of plastid genes. 

Short-term regulation involves post-translational modifications that can rapidly alter the activity of enzymes.

Light-dependent mechanisms quickly regulate the specific activity of five key enzymes:

1. Rubisco

2. Fructose 1,6-bisphosphatase

3. Sedoheptulose 1,7-bisphosphatase

4. Phosphoribulokinase

5. NADP–glyceraldehyde-3-phosphate dehydrogenase

Rubisco: Activation and Catalysis

Rubisco, a crucial enzyme for carbon fixation, is regulated by Rubisco-activase. 

A CO2​ molecule has a dual role in Rubisco's activity: it acts as an activator and a substrate.

• Activation: The activation process begins when an activator CO2​ molecule forms a lysyl-carbamate with the enzyme. The subsequent binding of Mg2+ stabilizes this complex, making Rubisco catalytically active. In the light, an increase in both the stroma's pH and Mg2+ concentration facilitates this activation.

• Catalysis: A separate CO2​ molecule, the substrate CO2​, then reacts with RuBP at the active site, yielding two molecules of 3-phosphoglycerate.

Activation of RuBisCo by CO2 and Mg2+

Rubisco-activase overcomes several types of inhibition. Sugar phosphates like xylulose 1,5-bisphosphate and 2-carboxyarabinitol 1-phosphate (naturally occurring inhibitor) can bind tightly to uncarbamylated Rubisco, preventing its activation and inhibiting catalysis. The substrate RuBP can also bind to the carbamylated enzyme, preventing activation. Rubisco-activase removes these inhibitors, allowing the enzyme to be activated by activator CO2 and Mg2+. 

Formation of the Dead-End Complex

• A Rubisco active site that has lost its activating CO2​ molecule (a process called decarbamylation) can bind to RuBP.

• This binding leads to a conformational change in Rubisco, which creates an inactive, dead-end complex where RuBP is tightly bound to the enzyme.

How Rubisco-Activase Restores Activity

Rubisco-activase removes the tightly bound RuBP in an ATP dependent process. The steps are as follows: 

1. Activase Priming: ATP hydrolysis by Rubisco-activase is required for the conformational changes that prime the activase.

2. Physical Interaction: The primed Rubisco-activase physically interacts with the Rubisco-RuBP dead-end complex.

3. Conformational Change: This interaction changes the conformation of Rubisco, reducing its affinity for RuBP.

4. RuBP Dissociation: With a lower affinity, the tightly bound RuBP dissociates from the active site of Rubisco.

5. Reactivation: Once the active site is free, Rubisco-activase likely dissociates, allowing the site to be reactivated through carbamylation by CO2​. This allows the enzyme to resume its role in carbon fixation.

At high temperatures, Rubisco deactivates faster than Rubisco-activase can reactivate it, leading to a loss of function.

Activation of RuBisCo by RuBisCo activase (adapted from Jensen (2000) PNAS)

The Ferredoxin-Thioredoxin System

The ferredoxin-thioredoxin system links the light-dependent reactions of photosynthesis to the regulation of Calvin-Benson cycle enzymes. Light transfers electrons from water to ferredoxin. This reduced ferredoxin is then used to reduce ferredoxin-thioredoxin reductase, which in turn reduces thioredoxin (Trx). The reduced thioredoxin activates target enzymes by reducing their regulatory disulfide bonds.

This system regulates the activity of several enzymes, including:

• Fructose 1,6-bisphosphatase

• Sedoheptulose 1,7-bisphosphatase

• Phosphoribulokinase

• NADP–glyceraldehyde-3-phosphate dehydrogenase

When it's dark, electron flow from ferredoxin stops, and thioredoxin becomes oxidized, ultimately inactivating the target enzymes.

Regulation via ferredoxin-thioredoxin system

Light-Dependent Ion Movements and Enzyme Activity

Light also regulates Calvin-Benson cycle enzymes through the movement of ions. When illuminated, protons flow from the stroma into the thylakoid lumen, while Mg2+ is released from the thylakoid space into the stroma. This increases the stromal pH and Mg2+ concentration, which activates enzymes like Rubisco, fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and phosphoribulokinase. These ionic changes are reversed in the dark, deactivating the enzymes.

Regulation of Chloroplast Enzymes via Supramolecular Complexes

Another mechanism for light-dependent regulation of the Calvin cycle involves the formation of supramolecular complexes. This process primarily centers on a small regulatory protein called CP12 and involves regulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK).

A2B2 and A4 Forms of GAPDH

The regulation of GAPDH is complex, involving two major isoforms in higher plants: 

• the heterotetramer A2B2
• the homotetramer A4

Both are assembled from different subunits and play distinct roles in the light-dark regulation of photosynthesis. 

A4 Homotetramer

• Composition: The A4 form consists of four identical GapA subunits. It is the catalytically active homotetramer.

• Regulation: The A4 homotetramer is regulated by its interaction with the small protein CP12.

A2B2 Heterotetramer

• Composition: The A2B2 form is made of two GapA subunits and two GapB subunits. This is the most abundant and active form of GAPDH in land plants.

• Regulation: Its regulation is largely autonomous, primarily controlled by the C-terminal extension (CTE) of the GapB subunit. 

A4-GAPDH Regulation

The CP12-Mediated Complex:

In the dark, when there is no need for active carbon fixation, the regulatory protein CP12, which is oxidized by the absence of light, binds to two key Calvin cycle enzymes:

• A4 isoform of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

• Phosphoribulokinase (PRK)

This binding forms a ternary supramolecular complex (GAPDH/CP12/PRK). The formation of this complex effectively sequesters and inactivates both GAPDH and PRK, switching off the Calvin-Benson cycle and preventing the wasteful consumption of ATP and NADPH that would otherwise occur in the dark.

Dissociation in the Light:

When light becomes available, the thylakoid electron transport chain produces reducing power in the form of NADPH. This is used to reduce ferredoxin, which then reduces thioredoxin. The reduced thioredoxin acts on the CP12-mediated complex.

Reduced thioredoxin cleaves the disulfide bonds within the oxidized CP12. This reduction of CP12 causes the entire supramolecular complex to dissociate. The dissociation releases the A4 isoform of GAPDH and PRK, which become catalytically active.

This mechanism ensures that the Calvin cycle is tightly coupled to the availability of light, providing a quick and efficient way to regulate carbon fixation in response to light-dark transitions.

Regulation by CP12-Mediated Complex (adapted from Plant Physiology and Development by Taiz, Zeiger, Møller, and Murphy)

A2B2-GAPDH Regulation

The A2B2 isoform is a heterotetramer made of A and B polypeptides. The B subunit contains a C-terminal extension with two cysteines that can form a disulfide bridge. The A2B2 form can also assemble into a larger, inactive A8B8 oligomer.

• In Darkness: The A2B2-GAPDH forms the inactive A8B8 oligomer.

• In Light: Reduced thioredoxin (Trx) cleaves the disulfide bond in the B subunit of the A8B8 oligomer. This reduction converts the inactive oligomer into the active A2B2-GAPDH. This autonomous redox-modulation is particularly suited for conditions of very active photosynthesis.

Regulation by C-terminal extension (adapted from Plant Physiology and Development by Taiz, Zeiger, Møller, and Murphy)

GAPDH Isoform	Composition	Regulation Mechanism	Active State	Inactive State
A4	A4 homotetramer	Forms a ternary complex with CP12 and PRK in the dark.	Free, active enzymes.	Inactive ternary complex.
A2B2	A2B2 heterotetramer	Forms an A8B8 oligomer in the dark; regulated by disulfide bond in subunit B.	Active A2B2 tetramer.	Inactive A8B8 oligomer.

Regulation of C4 Enzymes

In C4 plants, light also regulates key enzymes through thiol-disulfide exchange and phosphorylation-dephosphorylation.

• NADP–malate dehydrogenase (MDH) is activated in the light by the ferredoxin-thioredoxin system (reduced by thioredoxin) and deactivated in the dark (oxidized).

Regulation of MDH (Carr et al. 1999, Cell)

• Phosphoenolpyruvate (PEP) carboxylase is regulated by diurnal phosphorylation. Phosphorylation makes it active at night and insensitive to inhibition by malate. Dephosphorylation by a protein phosphatase in the day brings the enzyme to low activity.

Regulation of PEP

• Pyruvate–phosphate dikinase is regulated by a bifunctional kinase-phosphatase enzyme. In the dark, ADP-dependent phosphorylation deactivates the enzyme, while in the light, Pi-dependent dephosphorylation restores its activity.

Photosynthesis: Bioenergetics

 

Energy consumption of photosynthesis

• 2 quanta of light (1 quantum for PSI and PSII each) are required for transfer of 1 electron from H2O to NADP+.

• 4 electrons are required for reduction of H2O and evolution of 1 molecule of O2.

• Therefore, 8 quanta of light are required for evolution of 1 molecule of O2 and fixation of 1 molecule of CO2.

• This means that 48 quanta (8 x 6) of light are required to fix 6 molecules of CO2 and generate 1 molecule of glucose (6C).

Quantum yield = 48 quanta of red light

• 1 quantum = 42 Kcal, 48 quanta = 2016 Kcal

• The standard Gibbs free energy change for the synthesis of one mole of glucose from CO2 and water is approximately 673 kcal/mol. This is the chemical energy that plants store.

Photosynthetic efficiency = 673/2016 x 100 = 33%

C3 cycle

Fixation of 1 molecule of CO2 requires: 3 ATP and 2 NADPH

               1 ATP = 7.3 Kcal, 1 NADPH = 52.6 Kcal

Energy requirement to fix 6 molecules of CO2: 

18 ATP (3 x 6) = 18 x 7.3 Kcal = 131.4 Kcal 

                                

12 NADPH (2 x 6) = 12 x 52.6 Kcal = 631.2 Kcal

Total: 131.4 Kcal + 631.2 Kcal = 762.6 Kcal

Internal efficiency = 673/762.6 x 100 = 88%

C4 cycle

Fixation of 1 molecule of CO2 requires: 5 ATP and 2 NADPH

            1 ATP = 7.3 Kcal, 1 NADPH = 52.6 Kcal

Energy requirement to fix 6 molecules of CO2:

30 ATP (5 x 6) = 30 x 7.3 Kcal = 219 Kcal 

12 NADPH (2 x 6) = 12 x 52.6 Kcal = 631.2 Kcal

Total: 219 Kcal + 631.2 Kcal = 850.2 Kcal

Internal efficiency = 673/850.2 x 100 = 79%

Photosynthesis: Photoprotection of the Photosynthetic Apparatus

 

Photoprotection of the Photosynthetic Apparatus

Photoprotection is the process by which photosynthetic organisms protect themselves from the damaging effects of excess light energy, a condition known as photoinhibition. 

The photosynthetic membrane can be easily damaged if the large amount of energy absorbed by pigments cannot be stored through photochemistry. Photoprotection acts as a safety valve, dissipating this excess energy before it can cause harm.

The Role of Carotenoids

Carotenoids are essential accessory pigments that play a vital role in photoprotection in addition to their light-harvesting function. They protect the photosynthetic apparatus by a process called quenching, where they rapidly dissipate the excess energy from excited chlorophyll molecules.

If an excited chlorophyll molecule is not quickly quenched by energy transfer or photochemistry, it can react with molecular oxygen to form highly reactive singlet oxygen. Singlet oxygen can cause oxidative damage to various cellular components, particularly the lipids within the thylakoid membranes. 

Carotenoids prevent this by accepting the excess energy from excited chlorophyll and returning to their ground state by releasing the energy as harmless heat. The excited state of carotenoids does not have enough energy to form singlet oxygen, making them an effective shield against this damage.

Non-photochemical Quenching (NPQ) and the Xanthophyll Cycle

A major process of photoprotection is non-photochemical quenching (NPQ), which involves dissipating excess light energy as heat. It is often described as a "volume knob" that adjusts the flow of excitations to the Photosystem II (PSII) reaction center to a manageable level.

A specific group of carotenoids called xanthophylls are involved in NPQ. The xanthophyll cycle is a key regulatory mechanism that involves the interconversion of three xanthophylls: violaxanthin, antheraxanthin, and zeaxanthin. Under high light conditions, the enzyme violaxanthin de-epoxidase converts violaxanthin to zeaxanthin via antheraxanthin. The binding of protons and zeaxanthin to specific light-harvesting antenna proteins (like the PsbS protein) is believed to cause conformational changes that promote the quenching of chlorophyll and heat dissipation.

Xanthophyll cycle

Photoinhibition and Repair Mechanisms

Even with these protective mechanisms, excess light can lead to photoinhibition, which can inactivate and damage the PSII reaction center. When this happens, a multi-level defense system is activated:

1. Quenching: The first line of defense is the dissipation of excess energy as heat.

2. Scavenging: If quenching is insufficient, the formation of toxic photoproducts is followed by the action of various scavenging systems that eliminate these reactive molecules.

3. Repair: If the damage persists, the main target is the D1 protein of the PSII reaction center. The damaged D1 protein is removed from the membrane and a newly synthesized D1 protein is inserted to restore the function of the PSII reaction center. This ensures that the other undamaged components of the PSII complex are recycled.

Nitrogen metabolism (General)

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Enzymes (General)

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Life cycle of Rhizopus

Rhizopus belongs to the phylum Zygomycota.

It is often found on decaying organic matter like bread and fruits and is commonly known as a bread mold. 

The life cycle of Rhizopus involves both asexual and sexual phases.

Asexual Reproduction

Asexual reproduction is the primary and most rapid method of reproduction for Rhizopus. It allows the fungus to quickly colonize a food source under favorable conditions, which include a moist environment and an abundance of nutrients.

1. The main body of the fungus is the mycelium, a network of branched, thread-like filaments called hyphae. In Rhizopus, these hyphae are coenocytic, meaning they lack septa (cross-walls) and contain multiple nuclei within a single cytoplasm.

2. Specialized hyphae called rhizoids anchor the mycelium to the substrate, while others, known as stolons, spread across the surface. 

3. From the stolons, aerial hyphae called sporangiophores grow upwards. These sporangiophores are unbranched and support a reproductive structure at their tip.

4. At the apex of each sporangiophore, a spherical, sac-like structure called a sporangium forms. Inside the sporangium, the cytoplasm and nuclei undergo multiple mitotic divisions to produce a large number of haploid, asexual spores. The sporangium is differentiated into two parts. The peripheral sporoplasm with dense cytoplasm where spores develop and a central columella with vacuolated cytoplasm. The sporoplasm is separated from the columella by a dome-shaped partition.

5. Tiny, spherical, smooth walled spores are formed inside the sporoplasm. These spores are sporangiospores.

6. When the spores are mature, the sporangium wall ruptures, releasing the spores into the air. If a spore lands on a suitable substrate, it absorbs water, swells, and germinates. It then grows into a new mycelium, starting the asexual cycle over again.

Sexual Reproduction

Sexual reproduction in Rhizopus is a survival mechanism triggered by unfavorable environmental conditions, such as a lack of nutrients or the presence of a dry environment. It involves the fusion of two different mating types, typically designated as + and - strains.

1. Hyphae Contact and Gametangia Formation: When hyphae of opposite mating types (+ and -) grow close to each other, they are chemically attracted. They develop short, swollen branches called progametangia. A wall then forms to separate the tip of each progametangium, creating a multinucleate structure called a gametangium.

2. Plasmogamy: The walls between the two opposing gametangia dissolve, and their cytoplasm merges. This process is called plasmogamy, resulting in a single cell with fused cytoplasm but separate nuclei from both parent strains.

3. Karyogamy and Zygospore Formation: The nuclei of the two parent strains then fuse in pairs in a process called karyogamy. This forms a diploid nucleus. The resulting structure, a zygospore, develops a thick, warty, and protective wall that makes it highly resistant to harsh environmental conditions like desiccation and temperature extremes.

4. Germination and Meiosis: The zygospore enters a period of dormancy. When conditions become favorable again, it germinates. The diploid nucleus within the zygospore undergoes meiosis to produce new haploid nuclei.

5. Spore Production and Dispersal: The germinating zygospore produces a single, short filamentous structure called promycelium with a germ sporangium at its tip. This germ sporangium contains haploid spores, which are a mix of both parental genotypes due to genetic recombination during meiosis. When the sporangium bursts, these genetically diverse spores are released, disperse, and can grow into new mycelia, completing the sexual life cycle and increasing the fungus's ability to adapt.

Feature	Asexual Reproduction	Sexual Reproduction
Triggering Conditions	Favorable conditions (abundant food and moisture)	Unfavorable conditions (lack of nutrients, stress)
Parental Involvement	A single parent is involved.	Two parents of opposite mating types (+ and -) are involved.
Reproductive Structures	Sporangia form on sporangiophores.	Gametangia fuse to form a zygospore.
Spore Type	Asexual, haploid spores (sporangiospores) are produced through mitosis.	A sexual, thick-walled zygospore is formed. It undergoes meiosis during germination to produce haploid spores.
Genetic Variation	No genetic variation, offspring are genetically identical to the parent.	High genetic variation due to the fusion of genetic material from two different parents and meiotic recombination.
Function	Rapid multiplication and colonization of a substrate.	Survival during harsh environmental conditions and adaptation to new environments.
Outcome	New mycelium is a clone of the parent.	New mycelium is genetically different from both parents.

How plants defend themselves against pathogens?

Plant Defense Mechanisms

Plants have evolved a multi-tiered defense system: 

• Pre-existing defense (passive defense)
• Pre-existing structural defense
• Pre-existing biochemical defense
• Post-infection or induced defense (active defense)
• Induced structural defense
• Induced biochemical defense 

A. Pre-existing Defenses

These are physical and chemical barriers present before infection.

• Pre-existing Structural Defenses: These include physical barriers that prevent entry.

• Cuticle: A thick, waxy cuticle repels water. Since many pathogens require moisture accumulation in the spore microclimate they cannot infect.

• Hairy Surface: A dense layer of trichomes (hairs) can deter insects that transmit pathogens. It also has water repellent effect.

• Stomata: The size and position of stomata can influence a pathogen's ability to enter.

• Cork Layers: A thick layer of cork cells can prevent the spread of pathogens.

• Sclerenchymatous tissue in the epidermis and hypodermis (make direct penetration difficult or impossible).

• Pre-existing Biochemical Defenses:

  • Exudates: 

• Some plants release antifungal substances from their roots or leaves. 
• For example, resistant varieties of onions (red scale onion) release protocatechuic acid and catechol, which inhibit the germination of fungal spores. The susceptible varieties (white scale onion) cannot produce these exudates.
• Fungitoxic exudates on the leaves tomato and sugar beet, inhibit the germination of spores of fungi Botrytis and Cercospora, respectively.

• Phytoanticipins: 

• Phytoanticipins are pre-formed antimicrobial compounds that are present in plants even before a pathogen has attacked. 
• Unlike phytoalexins, which are synthesized after infection, phytoanticipins are a part of the plant's constitutive defense system. 
• They are typically stored in an inactive form, such as a glycoside, and are activated by enzymes when the plant cell is damaged during an attack.
• These compounds can include:
• Phenolic compounds, tannins, and certain fatty acid-like compounds, such as dienes, that are found in high concentrations in young fruits, leaves, or seeds. They are potent inhibitors of various enzymes, including the pectolytic-macerating enzymes used by pathogens to break down host tissue.
• Saponins, which are preformed compounds with antifungal membranolytic activity. They work by disrupting the membranes of fungal pathogens, effectively excluding pathogens that lack the saponinase enzyme needed to neutralize them. Examples of saponins include tomatine in tomatoes and avenacin in oats.
• Glucosinolates: Found in plants of the mustard family (Brassicaceae), glucosinolates are hydrolyzed by an enzyme called myrosinase upon tissue damage. This reaction produces toxic compounds like isothiocyanates, which are effective against a wide range of pathogens and pests.

• Antimicrobial proteins: 

Plant proteins play a significant role in pre-existing defense against pathogens by performing several key functions:

• Inhibiting pathogen enzymes: These proteins can block pathogen proteinases and other hydrolytic enzymes that are used to break down the host cell wall.
• Damaging pathogen cell components: Some plant proteins can increase the permeability of fungal plasma membranes, causing them to leak, or inactivate foreign ribosomes, halting pathogen protein synthesis.
• Breaking down pathogen cell walls: Plant cells, particularly on their surfaces, contain hydrolytic enzymes like glucanases and chitinases. These enzymes can break down the cell wall components of fungi, thereby contributing to resistance.

Examples of these defensive proteins include:

• Phytocystatins: A family of low-molecular-weight proteins that inhibit cysteine proteinases, which are carried in the digestive systems of nematodes.
• Lectins: Proteins that bind to specific sugars on the surface of fungi, causing cell lysis and inhibiting their growth. 

• Defense Through Lack of Essential Factors:

Plants can naturally resist pathogens by lacking certain essential factors that the pathogen requires for a successful attack.

This can occur in several ways:

1. Lack of an essential nutrient: Some pathogens have very specific nutritional requirements. If a host plant lacks a particular nutrient that a pathogen needs to grow and reproduce, the pathogen will be unable to cause disease.
2. Lack of a specific host-recognition site: Many pathogens need to bind to a specific receptor on the surface of a host cell to initiate infection. If the host plant lacks this specific binding site, the pathogen cannot enter the cell, and the infection is aborted. 
3. Lack of a host-specific toxin receptor: Host-specific toxins require a specific receptor on the host cell to cause damage. If the host plant lacks this receptor, it becomes resistant to the toxin, and the pathogen is unable to cause the disease, even if it produces the toxin. For instance, some oat varieties are resistant to the victorin toxin because they lack the specific receptor that the toxin targets.

B. Induced (Post-Infection) Defenses

These defenses are activated after a pathogen attack.

• Recognition: 

• PAMP triggered immunity: Plants recognize pathogens by detecting PAMPs (pathogen-associated molecular patterns) via specialized PRRs (pattern recognition receptors). This initial recognition triggers a defense response.
• Effector triggered immunity: A host plant's R-gene product directly or indirectly recognizes the product of a pathogen's Avr-gene product (an effector molecule). This recognition event typically triggers a strong, localized defense response.

• Cytoplasmic Defense Reaction

In non-compatible interaction, the cytoplasm and nucleus enlarge. The cytoplasm becomes granular and dense, and various particles or structures appear in it. Finally, the mycelium of the pathogen disintegrates, and the invasion stops.

• Cell Wall Defense

The plant's cell wall is a dynamic structure that can be actively modified to strengthen its defenses and prevent the pathogen's spread.

• Papillae Formation: The host cell rapidly deposits new material, such as callose, lignin, and phenolics, to form a localized cell wall apposition called a papilla at the site of attempted penetration. This acts as a physical barrier to block the pathogen's entry.

• Strengthening of Existing Cell Walls: The plant can reinforce its existing cell walls by depositing additional layers of lignin, suberin, or other structural polymers. This makes the cell wall tougher and more difficult for the pathogen to penetrate or degrade.

• Restructuring of cell wall at the site of infection: The outer layer of the cell wall of parenchyma cells coming in contact with incompatible bacteria swells and produces an amorphous, fibrillar material that surrounds and traps the bacteria and prevents them from multiplying. 

•  Histological Defense Structures:

• Cork Layers

Cork layers are new layers of tissue that a plant forms around an infection site. This process effectively isolates the pathogen by creating a physical barrier, cutting off its access to healthy tissue and nutrients, and preventing it from spreading further into the plant.

• Tyloses

Tyloses are overgrowths of parenchyma cells that push into the xylem vessels, which are responsible for water transport. When a vascular pathogen enters the xylem, the plant responds by forming these balloon-like structures. The tyloses swell and block the vessel, physically stopping the pathogen's systemic movement through the plant's vascular system.

• Gum Deposition

Gum deposition is the process where a plant secretes gummy substances into and around infected cells. These gums form an impenetrable, sticky seal that encases the pathogen. This not only traps the pathogen but also limits its access to nutrients and water, effectively isolating it and preventing its spread.

• Abscission layer

It involves the controlled shedding of an infected part of the plant, such as a leaf or a fruit, to prevent the pathogen from spreading to the rest of the plant. These layers break down, effectively dissolving the connection to the main body of the plant. The infected part then falls off, taking the pathogen with it and leaving a clean, sealed wound that is resistant to further infection.

• Hypersensitive Response (HR)

It is a localized, induced cell death that occurs at the site of infection by a pathogen to limit its spread. It is an incompatible host-pathogen interaction.

The HR is a classic example of effector-triggered immunity (ETI), a key component of the gene-for-gene model of plant defense. This response is initiated when a plant's resistance (R) gene product recognizes a corresponding avirulence (Avr) gene product (or effector) from the pathogen.

The HR response involves a series of cascading biochemical and structural changes in the plant cell:

• Reactive Oxygen Species (ROS) Burst: One of the first events is a rapid and temporary generation of ROS, such as superoxide (O2−​), hydrogen peroxide (H2​O2​), and hydroxyl radicals (OH−). This leads to the hydroperoxidation of membrane phospholipids, causing the disruption of cell membranes and the eventual death of the cell.

• Ion Movement: There is a rapid increase in the movement of ions, particularly an efflux of K+ and H+ ions from the cell. This makes the cytoplasm more acidic and the extracellular space more alkaline.

• Cell Wall Strengthening: The plant strengthens its cell walls by cross-linking phenolic compounds with cell wall components, which leads to the formation of lignin-like substances. The cell wall also receives deposits of other defensive substances like callose and glycoproteins, making it harder for the pathogen to penetrate.

• Production of Antimicrobial Substances: The HR triggers the synthesis of various antimicrobial compounds:

  • Pathogenesis-Related (PR) Proteins: These include proteins like chitinases and β-1,3-glucanases that can break down the cell walls of fungi.

  • Phytoalexins: These are toxic, low-molecular-weight substances that are synthesized and accumulate in the plant after infection to inhibit pathogen growth.

  • Phenolic compounds: The oxidation of phenols by enzymes like polyphenol oxidases (PPO) and peroxidases creates highly toxic quinones, which further contribute to resistance.

By sacrificing a small number of cells, the plant effectively creates a barrier of dead tissue that confines the pathogen, preventing it from obtaining nutrients and spreading to other parts of the plant. 

Pathogenesis related proteins

Pathogenesis-related (PR) proteins are a diverse group of plant proteins that are synthesized and accumulate in plant cells in response to an attack by a pathogen. They are considered a key component of the plant's induced defense system.

PR proteins are either extremely acidic or extremely basic and therefore are highly soluble and reactive. 

These proteins have a wide range of functions, all aimed at protecting the plant from invaders:

• Enzymatic Activity: Many PR proteins are enzymes that can directly attack the pathogen's cell components. Examples include chitinases and β-1,3-glucanases, which break down chitin and β-1,3-glucans, two major structural components of fungal cell walls.

• Antimicrobial Properties: Some PR proteins, like certain defensins and thionins, have direct antimicrobial activity, disrupting pathogen membranes or interfering with their metabolism.

• Signaling: Other PR proteins may not directly harm the pathogen but instead act as signaling molecules to activate and amplify the plant's defense response throughout the plant, a process known as systemic acquired resistance (SAR).

PR proteins are classified into several families based on their structure and function. They are often produced in high concentrations in the area surrounding the infection site and are a crucial part of the plant's strategy to contain and eliminate pathogens.

Examples: 

•  PR1 proteins (antioomycete and antifungal) 
• PR2 (b-1,3-glucanases): breaks down chitin in the fungal cell walls 
• PR3 (chitinases): breaks down chitin in the fungal cell walls 
• PR4 proteins (antifungal) 
• PR6 (proteinase inhibitors) 
• Defensins: accumulate through ethylene and jasmonate pathway and have antimicrobial activity 
• Lipoxygenases: generate antimicrobial metabolites as well as secondary signal molecules such as jasmonic acid 
• Lysozymes: degrade glucosamine & muramic acid of bacterial cell wall 

Phytoalexins

Phytoalexins are low-molecular-weight, toxic antimicrobial substances that plants produce and accumulate rapidly in response to an attack by a pathogen or an environmental stressor. Unlike phytoanticipins, which are pre-formed, phytoalexins are synthesized de novo (from scratch) after the plant has recognized an invader.

Phytoalexins are produced by healthy cells adjacent to localized damaged and necrotic cells in response to materials diffusing from the damaged cells.  

Key characteristics of phytoalexins include:

• Induced Synthesis: They are not present in healthy, unchallenged plant tissues but are quickly produced at the infection site to limit the pathogen's growth and spread.

• Broad-Spectrum Toxicity: Phytoalexins are often toxic to a wide range of fungi and bacteria, but they are generally less toxic to the plant's own cells.

• Chemical Diversity: Phytoalexins are a chemically diverse group of compounds, including isoflavonoids, sesquiterpenoids, stilbenes, and polyacetylenes.

• Role in Defense: Their accumulation is a key component of the plant's hypersensitive response (HR) and other resistance mechanisms, as they effectively inhibit pathogen growth and development.

Some well-known examples of phytoalexins include:

• Rishitin in potatoes.
• Phaseollin in beans.
• Medicarpin in alfalfa.
• Resveratrol in grapes

Phytoanticipins vs Phytoalexins

Feature	Phytoanticipins	Phytoalexins
Timing of Production	Pre-formed; present in the plant before a pathogen attack.	Induced; synthesized and accumulated in response to a pathogen attack.
Origin	Part of the plant's constitutive defense system.	Part of the plant's post-infection induced defense response.
Function	Act as a pre-existing barrier, often stored in an inactive form and activated upon cell damage.	Directly inhibit pathogen growth and development at the infection site.
Examples	Saponins like tomatine (in tomatoes) and avenacin (in oats).	Terpenoids like rishitin (in potatoes) and isoflavonoids like phaseollin (in beans).
Role	Contributes to basic resistance by deterring non-adapted pathogens.	Contributes to gene-for-gene resistance and the hypersensitive response (HR).