NITROGEN FIXATION

ROLE OF NITROGEN IN PLANTS

• Major substance in plants next to water
• Building blocks
• Constituent element of
• Chlorophyll
• Cytochromes
• Alkaloids
• Many vitamins
• Plays important role in metabolism, growth, reproduction and heredity

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SOURCES OF NITROGEN

• Atmospheric Nitrogen
• 78% of atmosphere
• Plants cannot utilize this form
• Some Bacteria, Blue Green Algae, leguminous plants
• Nitrates, Nitrites and Ammonia
• Nitrate is chief form
• Amino acids in the soil
• Many soil organisms use this form
• Higher plants can also taken by higher plants
• Organic Nitrogenous compounds in insects
• Insectivorous plants

NITROGEN FIXATION

• The conversion of free nitrogen into nitrogenous salts to make it available for absorption of plants

TYPES OF NITROGEN FIXATION

NON BIOLOGICAL FIXATION

• The micro-organisms do not take place
• Found in rainy season during lightning

1. N₂ + O₂ lightning 2 NO

(Nitric oxide)

2. 2NO + O₂ oxidation 2NO₂

( Nitrogen per oxide)

3. 2NO₂ + H₂O HNO₂ + HNO₃

4. 4NO₂ + 2H₂O + O₂ 4HNO₃

(Nitric acid)

5. CaO + 2HNO₃ Ca (NO₃)₂ + H₂O

(Calcium nitrate)

6. HNO₃ + NH₃ NH₄NO₃

(Ammonium nitrate)

7. HNO₂ + NH₃ NH₄NO₂

(Ammonium nitrite)

BIOLOGICAL FIXATION

• Fixation of atmospheric Nitrogen into nitrogenous salts with the help of micro-organisms
• Two types
• Symbiotic
• Non-symbiotic

NON-SYMBIOTIC

• Fixation carried out by free living micro-organisms
• Aerobic, anaerobic and blue green algae
• Bacteria: special type (nitrogen fixing bacteria) types -
• Free living aerobic : Azotobacter, Beijerenckia
• Free living anaerobic : Clostridium
• Free living photosynthetic : Chlorobium, Rhodopseudomonas
• Free living chemosynthetic :Desulfovibro,Thiobacillus

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CONTD..

• Free living fungi: yeasts and Pillularia
• Blue green algae:
• unicellular – Gloeothece, Synechococcus
• Filamentous (non heterocystous) -Oscillatoria
• Filamentous (heterocystous) – Tolypothrix, Nostoc, Anabaena

SYMBIOTIC

• Fixation of free nitrogen by micro-organisms in soil living symbiotically inside the plants
• ‘Symbiosis’ – coined by DeBary
• Three categories
• Nodule formation in leguminous plants
• Nodule formation in non-leguminous plants
• Non nodulation

NODULE FORMATION IN LEGUMINOUS PLANTS

• 2500 spp. Of family leguminosae ( Cicer arientium, Pisum, Cajanus, Arachis) produce root nodules with Rhizobium spp.
• They fix Nitrogen only inside the root nodules
• Association provides-food and shelter to bacteria

-bacteria supply fixed nitrogen to plant

• Nodules may buried in soil even after harvesting – continue nitrogen fixation

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NODULE FORMATION IN NON-LEGUMINOUS PLANTS

• Some other plants also produces root nodules
• Causuarina equisetifolia – Frankia
• Alnus – Frankia
• Myrica gale – Frankia
• Parasponia – Rhizobium
• Leaf nodules are also noted
• Dioscorea, Psychotria
• Gymnosperms – root – Podocarpus,

- leaves – Pavetta zinumermanniana, Chomelia

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NON-NODULATION

• Lichens - cyanobacteria
• Anthoceros - Nostoc
• Azolla – Anabaena azollae
• Cycas – Nostoc and anabaene
• Gunnera macrophylla - Nostoc
• Digitaria, Maize and Sorghum – Spirillum notatum
• Paspalum notatum – Azotobacter paspali

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SYMBIOTIC NITROGEN FIXATION

• Small, knob-like protuberances-root nodules
• Size and shape varies
• Spherical, flat, finger-like or elongated
• From Pin head to one centimeter in size
• Various spp. Of Rhizobium noted
• Named after the host plant
• Pea – Rhizobium leguminosarum
• Beans – R. phaseoli
• Soyabeans – R. japonicum
• Lupins – R. lupini
• Two types of Rhizobium-
• Bradyrhizobium – slow growing spp.
• Rhizobium - fast growing spp.

RHIZOBIUM

• Gram negative
• Non spore forming
• Micro-aerobic
• Show a degree of specificity
• The two partners (Bacteria and Host) recognized by chemical substance LECTINS - phytoagglutinins (carbohydrate containing plant protein)

FORMATION OF ROOT NODULES IN LEGUMES

• Root nodules formed due to infection of Rhizobium
• Free living bacteria growing near root of legumes unable to fix nitrogen in free condition
• Roots of the legumes secrete some growth factors helps in fast multiplication of bacteria
• (E.g.) Pisum sativum secretes homo serine also carbohydrate containing protein Lectins over their surface

CONTD..

• This helps in recognition and attachment of rhizobial cells
• Rhizobial cells have carbohydrate receptor on their surface
• Lectins interact with the carbohydrate receptor of rhizobial cells
• Occur between root hairs and young root hair
• Bacteria enter the roots through soft infected root hairs
• Tips are deformed and curved
• Tubular infection thread is formed in the root hair cell and bacteria enters into it

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CONTD..

• After entry, new cell wall is formed
• Tubular infection contains mucopolysaccharides where bacteria embedded and start multiplication
• It grows much and reaches the inner layers of cortex and the bacteria is released
• It induces the cortical cells to multiply which result in the formation of nodule on the surface
• The bacterial cells multiplies and colonize in the multiplying host cells

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CONTD..

• After host cells are completely filled, bacterial cells becomes dormant-bacteroids
• Float in leghaemoglobin – reddish pigment in cytoplasm of host cells
• Efficient O₂ scavenger
• Maintains steady state of oxygen
• Stimulates ATP production
• Present studies indicates that leghaemoglobin is not essential
• Nitrogenous compounds synthesized is translocated through vascular tissues
• Groups of rhizobia surrounded by double membrane originated from host cell wall
• Bacteroids lack firm wall (osmotically liable)

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BIOCHEMISTRY OF NITROGEN FIXATION

• Basic requirements for Nitrogen fixation
• Nitrogenase and hydrogenase enzyme
• Protective mechanism against Oxygen
• Ferrodoxin
• Hydrogen releasing system or electron donor (Pyruvic acid or glucose/sucrose)
• Constant supply of ATP
• Coenzymes and cofactors TPP, CoA, inorganic phosphate and Mg⁺²
• Cobalt and Molybdenum
• A carbon compound

NITROGENASE ENZYME

• Plays key role
• Active in anaerobic condition
• Made up of two protein subunits
• Non heme iron protein ( Fe-protein or dinitrogen reductase)
• Iron molybdenum protein (Mo Fe-protein or dinitrogenase)
• Fe protein reacts with ATP and reduces second subunit which ultimately reduces N₂ into ammonia

N₂ + 6H⁺ + 6e^- 2NH₃

CONTD..

• The reduction of N₂ into NH₃ requires 6 protons and 6 electrons
• 12 mols of ATP required
• One pair of electron requires 4 ATP
• The modified equation

N₂ + 8H⁺ + 8e^- 2NH₃ + H₂

• Hydrogen produced is catalyzed into protons and electrons by hydrogenase

hydrogenase

H₂ 2H⁺ + 2e^-

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PATHWAY OF NITROGEN FIXATION IN ROOT NODULES

• Glucose-6-phosphate acts as a electron donor

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• Glucose-6-phosphate is converted to phosphogluconic acid

Glucose-6-phosphate + NADP+ + H₂O 6-phosphogluconic acid + NADPH + H⁺

• NADPH donates electrons to ferrodoxin. Protons released and ferrodoxin is reduced
• Reduced ferrodoxin acts as electron carrier. Donate electron to Fe-protein to reduce it. Electrons released from ferrodoxin thus oxidized

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Sucrose (synthesized in leaves)

Sucrose ( in roots )

Glucose and fructose

Glucose-6-phosphate

CONTD..

• Reduced Fe-protein combines with ATP in the presence of Mg ⁺²
• Second sub unit is activated and reduced
• It donates electrons to N₂ to NH₃
• Enzyme set free after complete reduction of N₂ to NH₃

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• Mo – N=NH

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• Mo=N-NH₂

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• MoΞN+NH₃

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• Mo + NH₃

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• NΞN

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• Mo-NΞN

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