Updated: Nov 28, 2021

Even though our atmosphere consists of several gases but the main constituent is the Nitrogen gas which contributes around 80% of the atmospheric gases. So, the atmosphere is the largest the reservoir of Nitrogen and contains about 10^15 tones of Nitrogen gas.

But even after the presence of such large amounts in air, the Nitrogen usually becomes deficient in plants as the content of Nitrogen in soil is very poor and the requirement is very high. On top of that no plant or animals are able to utilize it directly from atmosphere due to its inert diatomic form. The only group of organisms capable of doing so are a small group of prokaryotes called Diazotrophs.

However, some plants evolved to compensate for this loss by performing symbiotic association with the Diazotrophs that can be best explained with the help of Legume-Rhizobium symbiosis. The bacteria Rhizobium are symbiotically associate with the roots of legumes. The Rhizobium obtains nutrients from the cells of the roots and in turn fixes the atmospheric Nitrogen into ammonia which is used by the plant. This process of Nitrogen fixation is performed in specialized outgrowth of roots called Root Nodules.

Steps of Root Nodule formation

  • Release of chemical signals (flavonoids) from Root hair cells.

  • Chemotactic movement of Rhizobium towards Root hair.

  • Accumulation of bacteria near the Root hair and attachment to the Root surface.

  • Release of chemicals (Nod factors) from bacterial cells.

  • Curling of Root hair due to Nod factors.

  • Entry of bacteria inside the Root hair by formation of infection thread by invagination of Root hair cell membrane.

  • Dedifferentiation of cells of pericycle to produce a mass of undifferentiated tissue called Nodule Primordia.

  • Infection of bacteria in Nodule primordia cells.

  • Divisions in nodule primordia cells to form Nodule as an outgrowth.

  • Rhizobium present in Nodules cells transforms into branched, swollen, non-flagellate, non-dividing structures called Bacteroids.

  • Bacteriods produce enzyme Nitrogenase and pigment Leg-hemoglobin to perform Nitrogen fixation.



In plants showing determinate root nodules, the nodule meristem (primordia) do not persist. The initial number of cells added by the nodule meristem are only responsible for growth of the determinate nodule. The further growth of nodule occurs by cell expansion rather than cell division as cell division cannot be sustained due to consumption of nodule meristem during initial growth. The shape of nodule remains spherical.

Eg. Soyabean, beans


The nodule meristem (primordia) tends to persist in Indeterminate type of root nodules. As a result, new cells are continuously added to the nodule which is the main factor for the growth, hence the name indeterminate. The shape of nodule becomes elongated due to continuous addition of cells by the nodule meristem.

Eg. Pea, Alfalfa, clover




Presence of mineral N in the soil inhibits both nodule formation and nitrogenase activity . N in the soil inhibits symbiotic nitrogen fixation but it is relative to start of nodulation and N2 fixation at early vegetative growth at low concentration. Nitrogen fertilization affects nodulation of bean plants and therefore the usually-recommended rates suppress N2 fixation.


Phosphorus is used in numerous molecular and biochemical plant processes, particularly in energy acquisition, storage and utilization. The deficiency of phosphorous supply and availability remains a severe limitation on nitrogen fixation and symbiotic interactions. Nodules themselves are strong sinks for P and nodulation and N2 fixation are strongly influenced by P availability. When legumes-dependent on symbiotic nitrogen receive an inadequate supply of phosphorus, they may suffer nitrogen deficiency. Nitrogen fixing plants have an increased requirement for P over those receiving direct nitrogen fertilization, probability due to need for nodule development and signal transduction, and to P-lipids in the large number of bacterioids. Also, capability of developing nodules to compete with other vegetative sinks (root and shoot meristems) for phosphorus at limited external supply may be different between legume species.


Potassium is not an integral constituent of any metabolite but serves to activate numerous enzymes, serves as a counter ion and is the major cationic inorganic cellular osmoticum.


With regard to legume plants under N2-fixing symbiosis, chlorosis under Ca deficiency due to impaired N2 fixation is observed. Calcium deficiency, decreased nitrogen fixation in nodules, also affects attachment of rhizobia to root hairs and nodulation and nodule development.


Sulfur is an essential element for growth and physiological functioning of plants. The sulfurcontaining amino acids cysteine and methionine play a significant role in the structure, conformation, and function of proteins and enzymes in vegetative plant tissue.


Boron deficiency shows strong alterations in N2 fixation in soybean plants. Boron effects the Rhizobium-legume cell-surface interaction and nodule development in pea. In boron-deficient plants, the number of Rhizobia infecting the host cells and the number of infection threads are less and the infection threads develops morphological aberrations. The cell walls of root nodules of boron-deficient plants showing structural aberrations are reported to lack hydroxyproline/proline rich proteins, which contribute to an O2, barrier, preventing inactivation of nitrogenase and associated decrease in N, fixation.


Present in respiratory proteins that are required for N2 fixation in rhizobia. Copper also plays a role in a protein that is expressed coordinately with the nifgenes and may affect the efficacy of bacteroid function. Lastly, there is increasing interest in the phenomenon whereby bacteria enter a state that is 'viable but non-culturable'. There is a recent report that shows that, for reasons that are not clear, adding Cu to Agrobacterium or R. leguminosarum cells sends them to this state.


Iron is required for several key enzymes of the nitrogenase complex as well as for the electron carrier ferredoxin and for some hydrogenases. A particular high iron requirement exists in legumes for the heme component of hemoglobin. In legumes iron is required in a greater amount for nodule formation than for host plant growth. Leghaemoglobin is an oxygen-binding protein. The single most abundant protein that the plant host makes in the nodule is leghaemoglobin, an iron protein. In the bacteria, nitrogenase and nitrogenase reductase contain FeS clusters and the former has the cofactor FeMoCo at the active site for N2 reduction. Further, bacteroids have a very high respiratory demand, requiring abundant cytochromes and other electron donors, each with their own Fe centers .


Manganese plays a role in the synthesis of polyamines, which play important roles in plant growth and development, also in detoxification of active oxygen species. In one of the earliest steps of the infection process, the binding of rhizobia to young root hairs is enhanced when R. leguminosarum is starved of Mn.


Molybdenum is a micronutrient specifically for plants that form root nodules with nitrogen-fixing bacteria, though plants that do not form nodules also use trace amounts of it in a protein involved with nitrogen metabolism and uptake. Its relevance to N2 fixation is clear, given that the Mo in 'FeMoCo' cofactor is at the heart of the nitrogen reduction process . The Mo–Fe protein contains two atoms of molybdenum and has oxidation–reduction centers of two distinct types: two iron–molybdenum cofactors called FeMoco and four Fe-S (4Fe-4S) centers. The Fe–Mo cofactor (FeMoco) of nitrogenase constitutes the active site of the molybdenum-containing nitrogenase protein in N2-fixing organisms. Although at low supply, molybdenum is preferentially transported into the nodules.


Small amounts of Ni are essential for root nodule growth and hydrogenase activation. The efficiency of nitrogen fixation immediately depends on hydrogenase activity because the oxidation of hydrogen by the latter provides ATP required for N reduction to ammonia.


Cobalt is essential for nitrogen-fixing microorganisms, including the cyanobacteria. Role of cobalt in N2 fixation is essentially attributed to its role as a cofactor of cobalamine (Vitamin b6) which functions as a coenzyme involved in N2 fixation and nodule growth. Cobalt is also required as a part of a bacterial enzyme complex. Cobalt deficiency effects nodule development and function at different levels and to different degrees.

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