Dr. Riddhi Datta

• Global nitrogen supply is generally distributed between 3 major pools:
• Atmospheric pool
• Soil and associated ground water pool
• Nitrogen contained within biomass
• The complex pattern of nitrogen exchange between thee 3 pools is known as nitrogen cycle.

Nitrogen cycle

Atmospheric nitrogen

Biological nitrogen fixation

Industrial nitrogen fixation

Electrical nitrogen fixation

Denitrification

Ammonia Nitrite Nitrate

Soil nitrogen pool

Ammonification

Uptake

Decaying biomass

Plant biomass

Animal biomass

Death

Death

Food

Dr. Riddhi Datta

• Ammonification: In the process of decomposition, organic nitrogen is converted to ammonia by a variety of microorganisms. This process of release of ammonia and its formation of ammonium ions is known as ammonification.
• Example: Bacillus vulgaris, Bacillus ramosus

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• Nitrification: The process of converting ammonia to nitrate by a variety of microorganisms is known as nitrification. The first step is the oxidation of ammonia to nitrite by Nitrosomonas or Nitrococcus bacteria. Nitrite is then further oxidized to nitrate by Nitrobacter. These two groups of bacteria are chemoautotrophs and are called nitrifying bacteria.

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• Denitrification: The process which involves conversion of nitrates and nitrites to dinitrogen by a group of microorganisms (called denitrifiers) is called denitrification.
• Example: Thiobacillus denitrificans

Dr. Riddhi Datta

Nitrate Assimilation

Plant roots actively absorb nitrate from the soil solution via several low- and high-affinity nitrate–proton co-transporters. Plants eventually assimilate most of this nitrate into organic nitrogen compounds.

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Step 1:

• Reduction of nitrate to nitrite in the cytosol that involves the transfer of two electrons.
• Nitrate reductase (NR), a complex metalloenzyme, catalyzes this reaction:

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NO₃^- + NAD(P)H + H⁺ + 2e^- NO₂^- + NAD(P)⁺ + H₂O

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• Electron donor in the reaction:
• Most cases NADH
• In nongreen tissues NADH or NADPH

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Step 2:

• Reduction of nitrite to ammonia in the chloroplast (or plastid) that involves transfer of six electrons.
• Nitrite reductase (NiR) catalyzes the reaction:

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NO₂^- + 6 Fd_red + 8H⁺ + 6e^- NH₄⁺ + 6Fd_ox + 2H₂O

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• Electron donor in the reaction:
• Reduced ferredoxin

Dr. Riddhi Datta

• Plant cells avoid ammonium toxicity by rapidly converting the ammonium generated from nitrate assimilation or photorespiration into amino acids.

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• The primary pathway for this conversion involves the sequential actions of:
• glutamine synthetase
• glutamate synthase

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Step 1: Glutamine synthetase (GS)

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• Glutamine synthetase (GS) combines ammonium with glutamate to form glutamine:

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Glutamate + NH₄⁺ + ATP → glutamine + ADP + Pi

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• Involves a divalent cation such as Mg²⁺, Mn²⁺, or Co²⁺ as a cofactor

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Ammonia assimilation

Dr. Riddhi Datta

Plants contain three classes of GS:

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• Cytosolic GS
• Expressed in germinating seeds or vascular bundles
• Produce glutamine for intercellular nitrogen transport
• Light and carbohydrate levels have no regulatory effect

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• Pastidal GS
• Expressed in roots
• Generates amide nitrogen for local consumption
• Light and carbohydrate levels regulate expression

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• Chloroplastic GS
• Expressed in shoot
• Reassimilates photorespiratory NH₄⁺
• Light and carbohydrate levels regulate expression

Ammonia assimilation

Dr. Riddhi Datta

Ammonia assimilation

Step 2: Glutamate synthase (or glutamine:2-oxoglutarate aminotransferase, or GOGAT)

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• GOGAT transfers the amide group of glutamine to 2-oxoglutarate, yielding two molecules of glutamate

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• Plants contain two types of GOGAT:

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• NADH-GOGAT
• Accepts electrons from NADH
• Located in plastids of non-photosynthetic tissues such as roots or the vascular bundles of developing leaves
• Assimilates of NH₄⁺ absorbed from the rhizosphere or glutamine translocated from roots or senescing leaves

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Glutamine + 2-oxoglutarate + NADH + H⁺ → 2 glutamate + NAD⁺

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• Fd-GOGAT
• Accepts electrons from ferredoxin
• Found in chloroplasts
• Serves in photorespiratory nitrogen metabolism

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Glutamine + 2-oxoglutarate + Fd_red → 2 glutamate + Fd_ox

Dr. Riddhi Datta

The GS-GOGAT pathway that forms glutamine and glutamate. A reduced cofactor is required for the reaction:

• ferredoxin (Fd) in green leaves
• NADH in nonphotosynthetic tissue

Dr. Riddhi Datta

Biological Nitrogen Fixation

• The process of conversion of atmospheric nitrogen into the biologically acceptable form, i.e. ammonia is called nitrogen fixation.

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• If it occurs via agency of microorganisms, it is referred to as biological nitrogen fixation.

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• The microorganisms involved are called nitrogen fixers.

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• Biological nitrogen serves as the key entry point of molecular nitrogen into the biogeochemical cycle of nitrogen.

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• Exclusively a prokaryotic domain.

Dr. Riddhi Datta

Nitrogen fixers can be free-living or symbiotic

Dr. Riddhi Datta

Association between host plants and rhizobia

Dr. Riddhi Datta

Nitrogen fixation requires anaerobic (or microanaerobic ) conditions

• The principal enzyme responsible for biological nitrogen fixation is nitrogenase.

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• Oxygen is a strong electron acceptor that damage these sites and irreversibly inactivate nitrogenase.

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• So nitrogen must be fixed under anaerobic conditions.

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• In cyanobacteria, anaerobic conditions are created in specialized cells called heterocysts. These cells lack photosystem II so they do not generate oxygen.

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• Aerobic nitrogen-fixing bacteria maintains a low oxygen concentration (microaerobic conditions) through their high levels of respiration (Ex: Azotobacter) or evolve O₂ photosynthetically during the day and fix nitrogen during the night when respiration lowers oxygen levels (Ex: Gloeothece).

Dr. Riddhi Datta

Symbiotic nitrogen fixation

• Some symbiotic nitrogen-fixing prokaryotes dwell within nodules, the special organs of the plant host that enclose the nitrogen-fixing bacteria.

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• In Gunnera, nodules are preexisting stem glands that develop independently of the symbiont.

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• In legumes and actinorhizal plants, the symbionts induce the plant to form root nodules.

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• In grasses, root nodules are not produced and the symbionts anchor to the root surfaces, around the elongation zone and the root hairs, or live as endophytes inside apoplasts.

Root nodule

Dr. Riddhi Datta

• Legumes and actinorhizal plants regulate gas permeability in their nodules.

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• These levels can support respiration but are sufficiently low to avoid inactivation of the nitrogenase.

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• Nodules contain oxygen-binding heme proteins called leghemoglobins that gives them a heme-pink color, and are crucial for symbiotic nitrogen fixation.

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• Leghemoglobins have a high affinity for oxygen.

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• They increase the rate of oxygen transport to the respiring symbiotic bacterial cells substantially decreasing the steady-state level of oxygen in infected cells.

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• To continue aerobic respiration under such conditions, the bacteroid uses a specialized electron transport chain in which the terminal oxidase has an affinity for oxygen even higher than that of leghemoglobins

Microaerobic condition within nodule

Dr. Riddhi Datta

• The symbiosis between legumes and rhizobia is not obligatory.

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• Under nitrogen-limited conditions the symbionts seek each other out through an elaborate exchange of signals.

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Colonization of the rhizosphere:

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• The first stage is migration of the bacteria toward the roots of the host plant.
• This migration is a chemotactic response mediated by chemical attractants, especially (iso)flavonoids and betaines, secreted by the roots.

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Nodule Formation

Chemotactic binding of rhizobia bind to an emerging root hair

Dr. Riddhi Datta

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Establishing symbiosis requires an exchange of signals:

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• Plant genes specific to nodules are called nodulin genes.

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• Rhizobial genes for nodule formation are called

nodulation (nod) genes.

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• The nod genes are classified as:
• Common nod genes:
• nodA, nodB, and nodC
• found in all rhizobial strains
• Host-specific nod genes:
• nodP, nodQ, and nodH; or nodF, nodE, and nodL
• differ among rhizobial species and determine the host range

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• Only the regulatory nodD is constitutively expressed as NodD protein regulates the transcription of the other nod genes.
• These attractants activate the rhizobial NodD protein, which then induces transcription of the other nod genes.

Dr. Riddhi Datta

Formation of infection thread and nodule organogenesis:

• Rhizobia usually infect root hairs by first releasing Nod factors that induce a pronounced curling of the root hair cells.

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• The rhizobia become enclosed in the small compartment formed by the curling.

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• The cell wall of the root hair degrades in these regions, also in response to Nod factors, allowing the bacterial cells direct access to the outer surface of the plant plasma membrane.

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• Next the infection thread (an internal tubular extension of the plasma membrane) is produced by the fusion of Golgi-derived membrane vesicles at the site of infection.

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• The thread grows at its tip by the fusion of secretory vesicles to the end of the tube.

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• The cortical cells dedifferentiate and start dividing, forming a distinct area within the cortex, called a nodule primordium, from which the nodule will develop.

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• The process is modulated by several phytohormones including cytokinin and ethylene.

Root hair exhibits abnormal curling growth, and rhizobia proliferate within the coils

Localized degradation of the root

hair wall leads to infection and formation of the infection thread from Golgi secretory vesicles of root cells

Dr. Riddhi Datta

Release of Rhizobia:

• The infection thread filled with proliferating rhizobia elongates through the root hair and cortical cell layers, in the direction of the nodule primordium.

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• When the infection thread reaches the nodule primordium, its tip fuses with the plasma membrane of a host cell and penetrates into the cytoplasm.

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• Bacterial cells are subsequently released into the cytoplasm, surrounded by the host plasma membrane, resulting in the formation of an organelle called the symbiosome.

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Rhizobia are released into the apoplast

The infection thread reaches the end of the cell, and its membrane fuses with the plasma membrane of the root hair cell

Dr. Riddhi Datta

Release of Rhizobia:

• At first the bacteria within symbiosomes continue to divide, and the surrounding symbiosome membrane (called the peribacteroid membrane) increases in surface area to accommodate this growth by fusing with smaller vesicles.

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• The bacteria then stop dividing and begin to differentiate into nitrogen-fixing bacteroids.

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• The nodule as a whole develops features like a vascular system (which facilitates the exchange of fixed nitrogen produced by the bacteroids for nutrients contributed by the plant) and a layer of cells to exclude O₂ from the root nodule interior.

The infection thread extends and branches until it reaches target cells, where vesicles composed of plant membrane that enclose bacterial cells are released into the cytosol.

Dr. Riddhi Datta

Nitrogenase enzyme complex fixes N₂

• Produces ammonia from molecular nitrogen.

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• The reduction of N₂ to 2 NH₃ is a six-electron transfer reaction.

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• It is coupled to the reduction of two protons to evolve H₂.

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• The nitrogenase enzyme complex catalyzes this reaction.

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Dr. Riddhi Datta

Nitrogenase enzyme complex fixes N₂

• The nitrogenase enzyme complex can be separated into two components—

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• Fe protein
• The smaller of the two components
• has two identical subunits
• Each subunit contains an iron–sulfur cluster (4 Fe and 4 S^2–),
• Participates in the redox reactions that convert N₂ to NH₃
• Irreversibly inactivated by O₂ with half life 30-45 seconds

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• MoFe protein
• Has four subunits,
• Total molecular mass of 180 to 235 kDa
• Each subunit has two Mo–Fe–S clusters.
• Inactivated by O₂ with half life 10 minutes

Dr. Riddhi Datta

Nitrogenase enzyme complex fixes N₂

• In the overall nitrogen reduction reaction, ferredoxin serves as an electron donor to the Fe protein,
• It, in turn hydrolyzes ATP and reduces the MoFe protein.
• The MoFe protein can then reduce numerous substrates, although under natural conditions it reacts only with N₂ and H⁺.
• The production of NH₃ from N₂ and H₂ is an exergonic reaction.
• However, industrial production of NH₃ from N₂ and H₂ is endergonic, requiring a very large energy input because of the activation energy needed to break the triple bond in N₂.