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).

 

How pathogens attack plants?

Host-Pathogen Interaction

The interaction between a plant host and a pathogen is a complex relationship determined by the pathogen's ability to infect and the host's ability to defend itself.

• Compatible Interaction: 

• This occurs when a pathogen successfully infects a susceptible host, leading to disease. 
• The pathogen can overcome the plant's defenses, grow, and reproduce. 
• Example: Infection of a susceptible wheat variety by the fungus Puccinia triticina, which causes leaf rust.

• Incompatible Interaction: 

• This is a resistant reaction where the pathogen is unable to establish a successful infection. 
• The plant's defenses are strong enough to prevent the pathogen from growing, and no disease symptoms develop. 
• Example: A resistant wheat variety may recognize the Puccinia triticina fungus and trigger a rapid defense response, preventing the infection from spreading.

Pathogen Attack Mechanisms

Pathogens employ a series of strategies to attack and infect their host, which can be broken down into three stages.

A. Pre-Penetration Stage

Pathogens navigate towards a host plant using various cues.

• Chemotaxis: Pathogens are attracted to specific chemicals secreted by the host plant. For example, sugars and amino acids released by a host can attract the zoospores of fungi like Pythium.

• Electrotaxis: Host plants generate a flow of electric currents from their cells, which can guide pathogens.

• Rheotaxis: Pathogens, particularly zoospores, can move through water currents, and if their speed matches the water flow, they can find and attack a host.

B. Penetration Stage

Once at the host surface, pathogens penetrate the plant through natural openings, wounds, or by direct penetration.

1. Natural Openings:

   • Stomata: Bacteria can swim in a film of water on the leaf surface and enter the stomatal cavity. Fungi, like those causing rust, can form a specialized structure called an appressorium that precisely aligns over the stoma before sending a hypha into the leaf.

   • Lenticels: These are pores on fruits, stems, and tubers that provide easy entry for pathogens, such as the bacterium Streptomyces scabies, which causes potato scab.

   • Hydathodes: These pores on leaf margins and tips secrete nutrient-rich guttation fluid, which pathogens like Xanthomonas campestris pv. campestris (black rot of cabbage) can use to enter the plant.

   • Nectaries: Nectar-secreting glands in flowers are exploited by pathogens like Erwinia amylovora (fire blight of apple and pear) for entry.

2. Wounds: 

• Injuries caused by weather, insects, or farming equipment create openings that bypass the plant's natural defenses. 
• Most viruses, for example, rely on wounds created by insect vectors for transmission.

       3. Direct Penetration: 

This method relies on both mechanical pressure and enzymatic degradation to break through the host's surface.

A. Cutinases: 

• The first line of defense a pathogen encounters is the cuticle, a waxy layer on the plant's surface.
• Pathogens secrete cutinases and non-specific esterases to break down cutin, the primary component of the cuticle, allowing the pathogen to reach the cell wall.
• Cutin esterase catalyzes the hydrolysis of ester bonds occurring between free hydroxyl and carboxyl groups of cutin.
• Carboxycutin peroxidase catalyzed the hydrolysis of peroxide groups of cutin. 

B. Pectinases: 

• The pathogen then targets the cell wall. 
• Pectinases are a class of enzymes that degrade pectic substances, which are key components of the middle lamella and primary cell wall. 
• This class includes:
• Pectinmethyl esterases (PME): 
• Causes hydrolysis of methyl ester groups of pectinic acid chain into methyl alcohol and pectic acid.
• Polygalacturonases (PG): 
• Called chain-splitting pectinase. 
• Splits the pectic chain by adding a molecule of water and breaking (hydrolyzing) the linkage between two galacturonan molecules.
• Pectin lyases (PL): 
• Splits the chain by removing a molecule of water from the linkage, thereby breaking it and releasing products with an unsaturated double bond.
  
  
  

C. Cellulases:

• After degrading the pectin, the pathogen uses four main enzymes to break down cellulose, the main structural component of the cell wall, into glucose:
• Cellulase C1: attacks native cellulose by cleaving cross linkages between chains 
• Cellulase C2:  also attacks native cellulose and breaks it into shorter chains
• Cellulase Cx: attacks these short chains and degrades them to the disaccharide cellobiose
• β-(1-4)-glucosidase: attacks cellobiose and degraded it into glucose

D. Hemicellulases:

• The degradation of hemicelluloses, another complex component of the cell wall, requires multiple enzymes such as xylanase, galactanase, and glucanase, which are secreted depending on the specific structure of the hemicellulose in the host plant.

E. Ligninases:

• Lignin is a tough, complex polymer that provides structural support to the plant. 
• Most pathogens cannot degrade it, but a group of fungi known as white rot fungi produce powerful enzymes called ligninases to break down and utilize lignin.

F. Proteases and Peptidases:

• These enzymes are produced to degrade the structural proteins and glycoproteins embedded in the plant's cell wall, further weakening the host's defenses.

C. Post-penetration Stage

This stage involves:

• Infection and colonization
• Growth and reproduction of the pathogen
• Development of symptoms
• Dissemination of pathogen for spread of infection

Pathogens can cause disease by producing toxins or manipulating the plant's own growth hormones.

1. Pathotoxins

Pathotoxins are proteinaceous, highly poisonous substances produced by pathogens, and they play a critical role in the development of plant diseases.

These toxins are effective even at very low concentrations and are known for their ability to bind tightly to specific sites within the plant cell.

Some pathotoxins are also unstable or react so quickly that they are difficult to isolate and study. 

These are poisonous substances that directly damage host cells. 

Their mechanism of action may involve:

• affecting the permeability of the cell membrane 
• inhibiting enzymes and subsequently interrupting the corresponding enzymatic reactions
• acting as antimetabolites

Toxins can be of two types: 

• Host specific toxin 
• Host non-specific toxin 

Host-Specific Toxins

• These toxins are highly selective, only causing disease in specific host plants that are susceptible to the pathogen. Their production is essential for the pathogen's ability to be virulent, and they are typically not produced outside host cells in laboratory cultures.
• Example: Victorin, T-toxin, etc.

 

Host Non-Specific Toxins

• In contrast to host-specific toxins, these toxins affect a wide range of plant species and are not necessary for the pathogen's virulence on a specific host. They can often be produced in vitro (in a lab setting) and can cause milder symptoms like chlorosis or necrosis. 
• Example: TAB-toxin, Tentoxin, etc.

 

 Comparison between host-specific and host-nonspecific pathotoxins

Feature	Host-Specific Toxins	Host Non-Specific Toxins
Specificity	Highly specific to a particular host plant.	Affect a wide range of host plants.
Virulence	Essential for the pathogen's ability to cause disease (virulence).	Not essential for the pathogen's virulence.
Production	Not typically produced in vitro (laboratory cultures).	Can be produced in vitro.
Toxicity	Highly toxic and effective at very low concentrations.	Generally, less toxic than host-specific toxins.
Examples	Victorin (Cochliobolus victoriae), T-toxin (Cochliobolus heterostrophus).	Tabtoxin (Pseudomonas syringae), Tentoxin (Alternaria tenuis).

2. Altered Growth Regulators

Pathogens can manipulate plant hormones for their own benefit.

• Auxins: Pathogens can increase auxin levels by inactivating the IAA oxidase enzyme. This can lead to the formation of tumors, galls, and hairy roots, such as the crown gall disease caused by Agrobacterium tumefaciens. Conversely, a decrease in auxin synthesis can result in stunted growth.

• Gibberellins: The fungus Gibberella fujikuroi produces excessive gibberellins, causing rice seedlings to grow tall and spindly, a disease known as "foolish seedling disease."

• Cytokinins: Some pathogens produce cytokinins, which can lead to excessive cell division and gall formation, or witches' broom symptoms. A reduction in cytokinin production can cause dwarfism. 

• Ethylene: Pathogens can alter ethylene levels, leading to premature yellowing (chlorosis) and senescence of leaves and fruits. Ex: Pseudomonas solanacearum infection in bananna 

 3. Polysaccharides in pathogenesis

Exo-polysaccharides appear to be necessary for several pathogens to cause disease symptoms by:

• being directly responsible
• indirectly facilitating pathogenesis by promoting colonization
• enhancing survival of the pathogen.

Example: Vascular wilts 

4. Suppressors of plant defense

Some pathogenic fungi produce substances called suppressors that act as pathogenicity factors by suppressing the expression of defense responses in the host plant. 

 Example: Puccinia graminis tritici

Plant Immune System

 

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Further reading

Genetic marker

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Screening and selection

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Photosynthesis: Carbon reactions

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Photosynthesis: Light Reactions

 

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Photosynthesis

Photosynthesis is a fundamental process in which photosynthetic organisms, primarily plants and algae, convert light energy into chemical energy. This energy is then stored in the form of complex carbon compounds, such as carbohydrates, and results in the release of oxygen from carbon dioxide and water.

Thylakoid Reactions (Light-Dependent Reactions)

The thylakoid reactions, also known as the light reactions, are the initial stage of photosynthesis. They take place specifically within the thylakoids of the chloroplast. The primary function of these reactions is to convert light energy into chemical energy, which is stored in two high-energy compounds: ATP and NADPH.

• ATP (Adenosine triphosphate): This molecule serves as the primary energy currency for the cell. In the thylakoid reactions, ATP is produced through a process called photophosphorylation, which is driven by the movement of protons across the thylakoid membrane.

• NADPH (Nicotinamide adenine dinucleotide phosphate): This is a crucial electron carrier that provides the reducing power necessary for the synthesis of sugars.

The ATP and NADPH generated during the thylakoid reactions are then used in the next stage of photosynthesis, the carbon fixation reactions (also known as the Calvin cycle), which occur in the stroma of the chloroplast. These reactions use the stored chemical energy to synthesize sugars from carbon dioxide.

The Nature of Light

Light exhibits characteristics of both a wave and a particle.

Wave Properties

• A wave is defined by its wavelength (λ), which is the distance between consecutive wave crests.

• The frequency (ν) is the number of wave crests that pass a given point in a specific time.

• The relationship between these is given by the equation

  c = λν

                where c is the speed of the wave. For light, this speed is approximately 3.0 × 10⁸ m s⁻¹.

• Light is a transverse electromagnetic wave.

Wave properties of light

Particle Properties

• Light can also be considered a stream of particles called photons.

• Each photon contains a discrete amount of energy, known as a quantum.

• The energy (E) of a photon is directly proportional to the light's frequency, as described by Planck's law:

                                                                        E = hν

                                            where h is Planck's constant (6.626 × 10⁻³⁴ J s).

• The human eye can only perceive a small range of frequencies within the electromagnetic spectrum, known as the visible-light region.

Electromagnetic spectrum

Light Absorption and Pigments

Absorption and Action Spectra

• An absorption spectrum measures how much light energy a substance absorbs at different wavelengths. For example, the absorption spectrum of chlorophyll a show the portion of solar output that plants utilize.

• Chlorophyll appears green because it primarily absorbs light in the red and blue parts of the spectrum, while reflecting green wavelengths (around 550 nm).

• An action spectrum measures the effectiveness of different wavelengths of light in driving a biological response, such as oxygen evolution during photosynthesis.

• If the pigment responsible for a response is the same as the one whose absorption is being measured, the absorption and action spectra will be similar. 

• However, a slight mismatch occurs in the carotenoid absorption region (450–550 nm), suggesting that energy transfer from carotenoids to chlorophylls is less efficient than between chlorophylls themselves.

The solar spectrum and its relation to the absorption spectrum of chlorophyll 

Absorption spectrum and action spectrum for photosynthesis does not overlap in the 450-550 nm region

• Chlorophyll has high absorption peaks in the blue (430 nm) and red (662 nm) regions of the spectrum, while carotenoids absorb light in the blue-green region (450-550 nm).

• The overall photosynthetic activity, measured by the action spectrum, is lower in the region of carotenoids absorption compared to the peaks of chlorophyll absorption. 

• This is because the energy absorbed by the carotenoids must be transferred to chlorophylls to be used in photosynthesis. This energy transfer is not 100% efficient, meaning some of the absorbed light energy is lost during the transfer process as heat. 

• As a result, the action spectrum shows a less pronounced effect in the 450-550 nm range, even though significant absorption is occurring in that same range.

Absorption and action spectra of photosynthesis

The Red Drop and Emerson's Enhancement Effect

• The Red Drop: When measuring the quantum yield of photosynthesis (the rate of a photochemical event relative to the rate of photon absorption), Emerson observed a sharp decrease in efficiency in the far-red region (wavelengths greater than 680 nm). This indicates that light with a wavelength longer than 680 nm is far less effective at driving photosynthesis compared to shorter wavelengths.

• The Enhancement Effect: When Emerson provided red and far-red light simultaneously, the rate of photosynthesis was greater than the sum of the rates from each light source individually. This discovery led to the understanding that two distinct photochemical complexes, Photosystem I (PSI) and Photosystem II (PSII), work in tandem. PSII absorbs red light (680 nm), while PSI preferentially absorbs far-red light (greater than 680 nm).

                                                    where ΔO2 is the rate of oxygen evolution

Red drop

Photochemical Reactions and Energy Transfer

When a chlorophyll molecule absorbs a photon, it becomes energized, transitioning from its stable "ground state" to an unstable "excited state." This high-energy state is fleeting and must quickly release its energy. Absorption of blue light transitions chlorophyl to higher excited state than red light. In the higher excited state, it quickly emits some energy as heat and enters lower excited state. This excited state has four possible pathways to release its energy:

1. Fluorescence: Re-emitting a photon and returning to the ground state. The emitted photon has a longer wavelength and lower energy than the absorbed one. Chlorophyll fluoresces in the red region of the spectrum. 

2. Heat Dissipation: Converting the excitation energy directly into heat without emitting a photon.

3. Energy Transfer: Transferring the energy to an adjacent molecule. This is how antenna pigments transfer energy to the reaction center.

4. Photochemistry: Using the absorbed energy to drive a chemical reaction. The excited chlorophyll molecule transfers an electron to an electron-acceptor molecule, initiating a chain of redox (reduction-oxidation) reactions. This is the primary event that converts light energy into chemical energy.

Photon absorption by chlorophyll and its fate

Photosynthetic Pigments

Chlorophyll

• Chlorophyll molecules have a porphyrin-like ring with a central magnesium ($M{g}^{2+}$) ion. This ring is the site of electron changes during excitation, oxidation, and reduction.

• Chlorophylls differ in the substituents on their rings, which gives them different properties. For example, chlorophyll a has a -CH₃ group, while chlorophyll b has a -CHO group.

• The molecule has a long hydrocarbon tail that anchors it in the photosynthetic membrane.

Chlorophyll a structure

Carotenoids

• Carotenoids are linear molecules with multiple conjugated double bonds found in photosynthetic organisms. They absorb light in the 400-500 nm range, which gives them their characteristic orange color.

• They act as accessory pigments by absorbing light and transferring the energy to chlorophyll for photosynthesis.

• Carotenoids also provide photoprotection. They act as a safety valve, dissipating excess energy as heat before it can damage the photosynthetic machinery. If unquenched, excited chlorophyll can react with oxygen to form highly reactive singlet oxygen (¹O₂*), which can harm cellular components.

Structure of beta-carotene

Photoprotection by Carotenoids

• Carotenoids are essential accessory pigments in photosynthesis that provide crucial protection against the damaging effects of excess light. Their primary photoprotective function is to dissipate surplus light energy as heat, a process known as non-photochemical quenching (NPQ).
• When light intensity is high, the light-harvesting complex can absorb more energy than the photosystems can use. This excess energy can lead to the formation of a highly reactive and toxic molecule called singlet oxygen (¹O₂)*. Singlet oxygen can cause oxidative damage to cellular components, particularly lipids in the thylakoid membranes, leading to a condition called photoinhibition.
• Carotenoids, such as zeaxanthin, antheraxanthin, and violaxanthin, act as the plant's first line of defense. They are involved in the xanthophyll cycle, a process that regulates the dissipation of excess light energy. In high light conditions, violaxanthin is converted to zeaxanthin. Zeaxanthin binds to proteins in the light-harvesting antenna complex, inducing a conformational change that promotes the safe release of excess energy as heat.

• Furthermore, carotenoids can also directly quench the excited state of chlorophyll molecules, preventing them from transferring energy to oxygen and forming damaging singlet oxygen. The excited state of carotenoids has a lower energy level than that of singlet oxygen, so it cannot transfer energy to form it. Instead, the carotenoid returns to its ground state by releasing the energy as harmless heat, thus protecting the photosynthetic apparatus from damage.

The Photosynthetic Apparatus and Units

Location

• Photosynthesis takes place in chloroplasts.

• The essential proteins for photosynthesis are embedded in the thylakoid membranes.

• PSII is primarily located in the grana lamellae, while PSI and ATP synthase are mostly in the stroma lamellae and at the edges of the grana.

• The cytochrome b₆f complex is distributed between both the stroma and grana lamellae.

Antenna and Reaction Centers

• Pigments like chlorophylls and carotenoids form an antenna complex that collects light and transfers the energy to the reaction center.
• The antenna system is a physical phenomenon where energy is transferred via fluorescence resonance energy transfer (FRET). This transfer is highly efficient (95-99%).

• The reaction center, which contains special chlorophylls (P700 and P680), is where the photochemical redox reactions occur, converting light energy into chemical energy.

• The reaction center includes 4-6 chlorophyll a molecules (P700 and P680), and associated proteins and cofactors.

• The reaction center chlorophyll is an energy sink that absorbs light of longest wavelength and lowest energy in the complex.

• Energy transfer that occurs in the antenna complex is a physical phenomenon while electron transfer (redox reactions) at the reaction center is a chemical phenomenon.

Energy transfer through antenna complex

The energy trapping mechanism is directional and irreversible

• The directionality of energy transfer in the antenna complex is maintained by a carefully arranged sequence of pigments.

• Energy Gradient: The pigments within the antenna complex are organized in a way that creates an "energy gradient." The pigments that are further away from the reaction center absorb light at shorter wavelengths, which corresponds to higher energy.

• Progressive Energy Loss: As the excitation energy is transferred from one pigment molecule to the next, closer to the reaction center, it moves to pigments that absorb at progressively longer, redder wavelengths and therefore have lower energy excited states.

• Irreversibility: The small difference in energy between each consecutive excited chlorophyll is lost to the surroundings as heat. This minor energy loss makes the transfer process energetically favorable in one direction (towards the reaction center) and largely irreversible, ensuring that the collected light energy is efficiently funneled to the reaction center.

Energy funneling through antenna complex to reaction center

Photosynthetic Unit (Quantasome)

• A photosynthetic unit is the minimum number of collaborating pigment molecules required for a photochemical event.

• The evolution of one oxygen molecule requires 8 quanta of light and approximately 2500 chlorophyll molecules. This means about 300 chlorophyll molecules are needed to process each light quantum.

• Within this unit, only one specialized chlorophyll molecule participates in the photochemical reaction, while the others serve to absorb and transfer light to it.

Photosystem I (PSI) and Photosystem II (PSII) Reaction Centers

The reaction centers of PSI and PSII are crucial pigment-protein complexes embedded within the thylakoid membranes of chloroplasts that convert light energy into chemical energy during photosynthesis. These two photosystems work in series to drive the light reactions.

Photosystem II (PSII) Reaction Center

• It is designated as P680 as its core chlorophyll pigment absorbs light most efficiently at a wavelength of 680 nm.

• Location: Primarily found in the grana lamellae, which are the stacked regions of the thylakoid membranes.

• Structure: The core of the reaction center consists of two membrane proteins, D1 and D2. It also contains a special pair of chlorophyll-a molecules (P680), bound one to each of these two proteins, along with additional chlorophylls, pheophytins (colorless chlorophyll lacking Mg2+), carotenoids, and electron acceptors like plastoquinones. Additionally, the oxygen evolving complex is also a part of PSII.

• Function: PSII acts as a powerful oxidant. Its primary role is to split water molecules (photolysis) to replace the electron it loses. This process releases oxygen as a byproduct. The electrons from the water are used to re-reduce the oxidized P680. The splitting of water and subsequent electron transfer also pumps protons into the thylakoid lumen, contributing to the proton motive force needed for ATP synthesis.

Photosystem I (PSI) Reaction Center

• It is designated as P700 as its core chlorophyll pigment absorbs light most effectively at a wavelength of 700 nm.

• Location: Predominantly located in the stroma lamellae and at the edges of the grana lamellae.

• Structure: It is a large, multi-subunit complex. Its core consists of a heterodimer of two membrane proteins, PsaA and PsaB, each binding a reaction center chlorophyll molecule (P700). Another pair of chlorophyll a molecules (A0) also remains associated with the heterodimer and accepts electron from P700. There are additional proteins PsaC to PsaN and a pair of vitamin K1 molecules called phylloquinone bound to the heterodimer, one per subunit. It also contains a core antenna of about 100 chlorophyll molecules and a series of 3 membrane associated iron sulfur clusters - FeSX, FeSA and FeSB. A soluble iron sulfur protein, ferredoxin (Fd) is also a part of this large complex.

• Function: PSI produces a strong reductant. The electron it receives from the electron transport chain (via plastocyanin) is excited by light and transferred to acceptors, eventually being used to reduce NADP+ to NADPH. NADPH is a high-energy compound used in the Calvin cycle.

Feature	Photosystem I (PSI)	Photosystem II (PSII)
Primary Pigment	P700	P680
Location	Stroma lamellae and edges of grana lamellae	Grana lamellae (stacked thylakoid membranes)
Wavelength of Absorption Maxima	700 nm	680 nm
Primary Function	Reduces NADP+ to NADPH (produces a strong reductant that can reduce NADP+ and a weak oxidant)	Splits water and releases oxygen (generates a strong oxidant that can oxidise water and a weak reductant)
Electron Source	Receives electrons from the electron transport chain (via plastocyanin)	Receives electrons from the splitting of water (photolysis)
Product	NADPH	Oxygen, protons, and electrons for the electron transport chain

Electron Transport and Photophosphorylation

The Z-Scheme (Non-cyclic Electron Transport)

• The Z-scheme illustrates how PSI and PSII work in series. The entire chemical reaction is carried out by four integral membrane complexes that are vectorially arranged in the thylakoid membrane:
• PSII
• Cytochrome b₆f complex
• PSI
• ATP Synthase

• PSII: P680 in PSII absorbs a photon, gets excited (P680*), and transfers an electron to pheophytin. The electron-deficient P680+ is then re-reduced by electrons from water oxidation. This process releases protons into the thylakoid lumen, and the reduced product is plastohydroquinone (PQH₂).

• Cytochrome b₆f: This complex oxidizes PQH₂ and transfers electrons to plastocyanin (PC). This process also pumps protons from the stroma into the lumen, creating a proton motive force.

• PSI: Plastocyanin then reduces the oxidized P700+ in PSI. P700 absorbs a photon, gets excited, and transfers an electron down a series of acceptors, ultimately reducing NADP⁺ to NADPH by the action of ferredoxin and flavoprotein-ferredoxin NADP+ reductase (FNR).

• ATP Synthase: It produces ATP as protons diffuse back through it from lumen to stroma.

Components of photosynthetic electron transport chain

Mechanism of non-cyclic electron transport

Light Absorption and Water Splitting at PSII: 

• The process begins when light energy is absorbed by the antenna pigments of Photosystem II (PSII) and funneled to its reaction center, P680. This energy excites an electron in P680 to a higher energy state, and the electron is then transferred to an electron acceptor molecule, pheophytin (primary photochemical act). Now the P680 is in oxidized state (electron deficient) and pheophytin is in reduced state (electron rich). 

• This primary electron acceptor will now donate its extra electron to a secondary eelectron acceptor and so on down the ETC. On the other hand, the oxidized reaction center chlorophyll will accept the missing electron from a secondary electron donor, which in turn will regain alectron from a tertiary electron donor and so on. 

• The ultimate electron donor is water and ultimate electron acceptor is NADP+.

• To replace the lost electron, PSII uses a powerful oxidant to split a water molecule (photolysis). This reaction releases oxygen, protons (H+), and electrons. The electrons from water are donated to P680, allowing it to return to its ground state and participate in another photochemical event.

Electron Transfer and Proton Pumping: 

The electron from PSII is transferred to a mobile electron carrier, plastoquinone (PQ), which becomes reduced to plastohydroquinone (PQH2​).

PQH2​ then moves to the cytochrome $b_{6}f$ complex, where it is oxidized. The cytochrome b6​f complex acts as a proton pump, using the energy from the electron transfer to move protons from the stroma into the thylakoid lumen, which contributes to the proton motive force for ATP synthesis.

Electron Transfer to PSI: 

After passing through the cytochrome b6​f complex, the electron is transferred to another mobile carrier, a soluble copper protein called plastocyanin (PC). Plastocyanin carries the electron to the second photosystem, Photosystem I (PSI), and reduces its oxidized reaction center, P700.

Light Absorption and NADPH Formation at PSI: 

When PSI's reaction center, P700, absorbs light, its electron is re-excited to a very high energy level. This energized electron is transferred to a series of acceptors, including a quinone and iron-sulfur proteins. The electron is then passed to the soluble protein ferredoxin (Fd). Finally, the enzyme ferredoxin-NADP+ reductase (FNR) uses the electron from ferredoxin to reduce NADP+ to NADPH.

Z scheme of non-cyclic electron transport chain

Cyclic Electron Transport

• This mode of electron transport involves only PSI. It occurs when NADP⁺ is not available.

• Electrons ejected from P700 are shuttled back to the cytochrome b₆ complex and then return to the electron hole of PSI.

• This process does not produce NADPH or oxygen but is coupled to the phosphorylation of ADP to ATP.

Photophosphorylation (ATP Synthesis)

• ATP synthesis in chloroplasts is driven by a chemiosmotic mechanism.

• The vectorial electron flow results in a proton gradient across the thylakoid membrane, making the lumen acidic and the stroma alkaline.

• The energy of this proton gradient, known as the proton motive force (∆p), is used by the ATP synthase (CF₀-CF₁ complex) to synthesize ATP.

• The CF₀ part of the enzyme acts as a proton channel, while the CF₁ part, which sticks into the stroma, contains the catalytic sites for ATP synthesis. This enzyme functions like a tiny molecular motor, rotating as protons pass through it to synthesize ATP.