Symbiotic Nitrogen Fixation

Rhizobium Nitrogen Fixation:-

> Rhizobium bacteria:-

i. Free living

ii. Gram negative

iii. Aerobic

iv. Soil bacteria

> Rhizobium becomes anaerobic upon entry into roots. 

> Leghaemoglobin (legHb or symbiotic Hb):- 

- It is a pink coloured pigment.

- It occurs in the root nodules of leguminous plants. 

- It acts as an oxygen scavenger. It provides anaerobic conditions for the nitrogenase enzyme and protects the enzyme from inactivation.

> Two main steps:-

a. Nodule formation

b. Nitrogen fixation

a. Nodule formation:- Root nodule formation is initiated, when the soil contains a low level of nitrogen. Steps of nodulation are:

i. Aggregation:- Roots of legumes secrete flavonoids, which attracts rhizobia towards the root. Rhizobia aggregate around root hairs.

ii. Developmental changes:- Rhizobia secrete nod factors, which causes stimulate many developmental changes:

- Membrane depolarization

- Curling of root hairs 

- Cell division in the root cortex 

- Intracellular calcium movement

iii. Infection thread:- The nod factor attaches to receptors present on the plasma membrane of the root hairs, which leads to the formation of the infection thread. 

iv. Entry:- Infection thread provides the passage to bacteria to enter epidermal cells. Rhizobia then enter cortex cells, each bacterium gets surrounded by a plant-derived membrane known as symbiosome.

v. Nodulation:- Nodule formation is initiated by chemicals produced by rhizobia. It is a result of calcium dependent signal transduction pathway, which triggers biochemical changes leading to cell division and nodule formation. Cytokinin also plays an important role in nodules formation.

vi. Bacteroids:- Within nodules, bacteria get differentiated into bacteroids, which fix nitrogen. The Rhizobia stop dividing, loose cell wall and become nitrogen fixing cells as bacteroids . Vascular tissues are developed for nodules for exchange of nutrients.

b. Nitrogen fixation:-

- The nodule serves as site for N2 fixation. 

- Nodule contains nitrogenase and leghaemoglobin. 

- The nitrogenase has 2 components:

i. Molybdoferredoxin (Mo-Fe protein)

ii. Azoferredoxin (Fe-protein)

- The free di-nitrogen first bound to MoFe protein and is not released until completely reduced to ammonia. 

- In this process ferredoxin serves as an electron donor to Fe-protein (nitrogenase reductase) which in turn hydrolyzes ATP and reduce Mo-Fe protein, the Mo-Fe protein in Turn reduce the substrate N2. The electrons and ATP are provided by photosynthesis and respiration of the host cells.

- Many intermediates are formed to form ammonia (NH3).

Dinitrogen → Hydrazine → Diamine → Ammonia

- Ammonia (NH3) is immediately protonated at physiological pH to form ammonium ion (NH4+). As NH4+ is toxic to plants, it is rapidly used near the site of generation to synthesize amino acids.