Nitrogen fixation is a process by which nitrogen (N2) in the atmosphere is converted into ammonia (NH3). Atmospheric nitrogen or molecular nitrogen (N2) is relatively inert: it does not easily react with other chemicals to form new compounds. Fixation processes free up the nitrogen atoms from their diatomic form (N2) to be used in other ways.

Nitrogen fixation is essential for all forms of life because nitrogen is required to biosynthesize basic building blocks of plants, animals and other life forms, e.g., nucleotides for DNA and RNA and amino acids for proteins. Therefore nitrogen fixation is essential for agriculture and the manufacture of fertilizer.

Rhizobia Bacteria:

The first Rhizobia were isolated from root nodules by M. Beijerinck, and shown to have the ability to re-infect their legume hosts, and to fix N2 in symbiosis.

Rhizobium leguminosarum is aspecies of gram-negative, aerobic, rod shaped bacteria that is found in soil and which causes formation of root nodules on some, but not all, types of field pea, lentil, kidney bean, and clover.

Leguminous plants:

Leguminous plants are included under the family Fabaceae or Leguminosae, most of them have symbiotic nitrogen-fixing bacteria, Rhizobia in structures called root nodules.

Symbiotic Nitrogen Fixation:

Biological nitrogen fixation was discovered by the German agronomist Hermann Hellriegel and Dutch microbiologist Martinus Beijerinck.

Biological nitrogen fixation can be represented by the following equation, in which two moles of ammonia are produced from one mole of nitrogen gas, at the expense of 16 moles of ATP and a supply of electrons and protons (hydrogen ions):

N+ 8H+ + 8e- + 16 ATP = 2NH3 + H+ 16ADP + 16 Pi

This reaction is performed exclusively by prokaryotes (the bacteria and related organisms), using an enzyme complex termed Nitrogenase. This enzyme consists of two proteins – an iron protein and a molybdenum-iron protein.

The reactions occur while N2 is bound to the nitrogenase enzyme complex. The Fe protein is first reduced by electrons donated by ferredoxin. Then the reduced Fe protein binds ATP and reduces the molybdenum-iron protein, which donates electrons to N2, producing HN=NH. In two further cycles of this process (each requiring electrons donated by ferredoxin) HN=NH is reduced to H2N-NH2, and this in turn is reduced to 2NH3.

In free-living diazotrophs, the nitrogenase-generated ammonium is assimilated into glutamate through the glutamine synthetase/glutamate synthase pathway.

Biological nitrogen fixation is of two types: Symbiotic nitrogen fixation and Non-symbiotic nitrogen fixation.

Symbiotic nitrogen fixation occurs in plants that harbor nitrogen-fixing bacteria within their tissues. The best-studied example is the association between legumes and bacteria in the genus Rhizobium. A symbiotic relationship in which both partners benefits is called mutualism. A mutualistic symbiosis is an association between two organisms from which each derives benefit. It is usually a longer-term relationship, and with symbiotic nitrogen (N2) fixation, often involves a special structure to house the microbial partner. Each N2-fixing symbiotic association involves an N2-fixing prokaryotic organism, the microsymbiont (eg. Rhizobium, Klebsiella, Nostoc or Frankia) and a eukaryotic, usually photosynthetic, host (e.g., leguminous or nonleguminous plant, water fern or liverwort).

History of the legume-Rhizobium symbiosis:

Legumes have been used in crop rotations since the time of the Romans. However, it was not until detailed N balance studies became possible, that they were shown to accumulate N from sources other than soil and fertilizer. In 1886 Hellriegel and Wilfarth demonstrated that the ability of legumes to convert N2 from the atmosphere into compounds which could be used by the plant was due to the presence of swellings or nodules on the legume root, and to the presence of particular bacteria within these nodules.

Mechanism of Symbiotic Nitrogen Fixation:

Root Nodule Formation:

Rhizobia can infect their hosts and induce root nodule formation using following mechanisms:

  • Root hair penetration and infection thread formation, as occurs in clovers and beans.
  • Entry via wounds or sites of lateral root emergence, as found in peanut and the pasture legume Stylosanthes.
  • Penetration of root primordia found on the stem of plants such as Sesbania.

Nodule Initiation and Development

The process of root hair penetration and infection thread formation is treated in greater detail by Hirsch (1992) and Lhuissier et al (2001). The Fahraeus slide technique (Fahraeus, 1957) allowed repeated observation of the infection process and The Root-tip marking procedure (Bhuvaneswari et al., 1981) showed differences in the susceptibility of immature and mature root hairs to infection, and focused research on those parts of the root where infection by Rhizobium was most common.

Visible changes during root hair infection:

Infection begins with rhizobial attachment to immature, emerging root hairs of a compatible host. This occurs within minutes of inoculation, with attached rhizobia capping the root-hair tip, and often oriented end on to their host. Deformation and curling of the root hair follows, with the root hair surface at the point of infection hydrolyzed to permit penetration of the rhizobia. Rhizobia then move down the root hair toward the root cortex. Rhizobia never really gain free intracellular access to their host. During infection, and as they move down the root hair, they remain enclosed within a plant-derived material, and in some plants, for example Chamaechrista, may never escape from this infection thread.

Examination of a nodule under the microscope reveals four distinct zones:

  • Meristematic region in which host cells undergo active division but show little infection by rhizobia
  • A region in which many plant cells are infected but in which the bacteria have not undergone changes in size and shape, and N2 fixation is limited
  • Region of active N2 fixation, often red or pink in color due to the presence of leghemoglobin. Host cells will contain many rhizobia and these may be misshapen. Such bacteria are referred to as bacteroids.
  • Region of nodule senescence in which the symbiosis is breaking down. Bacteroids may undergo lysis, and the degradation of leghemoglobin results in a green or brown coloration.

The Infection Thread:

The interaction between a particular strain of rhizobia and the “appropriate” legume is mediated by:

  • a “Nod factor” secreted by the rhizobia.
  • Transmembrane receptors on the cells of the root hairs of the legume.
  • Different legumes produce receptors of different specificity.
  • Different strains of rhizobia produce different Nod factors, and

If the combination is correct, the bacteria enter an epithelial cell of the root; then migrate into the cortex. Their path runs within an intracellular channel that grows through one cortex cell after another. This infection thread is constructed by the root cells, not the bacteria, and is formed only in response to the infection.

When the infection thread reaches a cell deep in the cortex, it bursts and the rhizobia are engulfed by endocytosis into membrane-enclosed symbiosomes within the cytoplasm. At this time the cell goes through several rounds of mitosis — without cytokinesis — so the cell becomes polyploid. The cortex cells then begin to divide rapidly forming a nodule. This response is driven by the translocation of cytokinins from epidermal cells to the cells of the cortex.

The rhizobia also go through a period of rapid multiplication within the nodule cells. Then they begin to change shape and lose their motility. The bacteroids, as they are now called, may almost fill the cell. Only now does nitrogen fixation begin. Root nodules are not simply structureless masses of cells. Each becomes connected by the xylem and phloem to the vascular system of the plant. Thus the development of nodules, while dependent on rhizobia, is a well-coordinated developmental process of the plant. The benefit to the legume host is clear. The rhizobia make it independent of soil nitrogen.

But why is the legume necessary?

The legume is certainly helpful in that it supplies nutrients to the bacteroids with which they synthesize the large amounts of ATP needed to convert nitrogen (N2) into ammonia (NH3). In addition, the legume host supplies one critical component of Nitrogenase — the key enzyme for fixing nitrogen.

The bacteroids need oxygen to make their ATP (by cellular respiration). However, nitrogenase is strongly inhibited by oxygen. Thus the bacteroids must walk a fine line between too much and too little oxygen. Their job is made easier by Leghemoglobin. The metal Molybdenum is a critical component of Nitrogenase and so is absolutely essential for nitrogen fixation. But the amounts required are remarkably small. Because of the specificity of the interaction between the Nod factor and the receptor on the legume, some strains of rhizobia will infect only peas, some only clover, some only alfalfa, etc.

Function of Root Nodule:

In the context of the whole plant, the root nodule functions as a nitrogen source and a carbon sink.  In fact,  it has been suggested that legume nodules evolved from carbon storage organs (Joshi et al., 1993). The carbon source transported from the leaves to the nodules is sucrose (Hawker, 1985), which is introduced into nodule metabolism through degradation by sucrose synthase. This enzyme is present at high levels in both legumes and actinorhizal nodules (Thummler and Verma, 1987). The form in which nitrogen is transported depends on the plant: temperate legumes, which generally form indeterminate nodules, export amides, whereas tropical legumes, which form determinate nodules, export ureides. Actinorhizal plants export mostly amides, with the exceptions of Alnus sp and  Casuafina  eguisetifolia, which  are  citrulline  exporters. In all cases, ammonium is exported by the microsymbiont as the first product of nitrogen fixation and is assimilated in the cytoplasm of nodule cells via the glutamine synthetase (GS)/glutamate synthase pathway.

Oxygen Protection of Bacterial Nitrogen Fixation :

Nitrogenase is highly oxygen sensitive because one of its components, the MoFe cofactor, is irreversibly denatured by oxygen (Shaw and Brill, 1977). On the other hand, the large amount of energy required for this reaction has to be generated by oxidative processes; thus, there is a high demand for oxygen in nodules. Different strategies are used in different symbiotic interactions to cope with this paradox. In legume nodules, a low oxygen tension in the central part of the nodule is achieved Symbiotic Nitrogen Fixation by a combination of a high metabolic activity of the microsymbiont and an oxygen diffusion barrier in the periphery of the nodule, that is, in the nodule parenchyma. Because oxygen diffuses -104  times faster through air than through water, it is generally assumed that oxygen diffusion in nodules occurs via the intercellular spaces. The nodule parenchyma contains very few and small intercellular spaces, and this morphology is thought to be responsible for the block in oxygen diffusion (Witty et al., 1986). In the infected cells of the central part of the nodule, high levels of the oxygen carrier protein leghemoglobin facilitate oxygen diffusion. In this way, the microsymbiont is provided with sufficient oxygen to generate energy within a low overall oxygen concentration. In contrast to Rhizobium, Frankia bacteria can form specialized vesicles in which nitrogenase is protected from oxygen.

Gene Regulation in Root Nodules :

To obtain nitrogen-fixing root nodules, several genes of both symbionts are specifically induced or repressed during nodule development. The use of reporter genes as well as in situ hybridization studies has provided detailed insights into the spatial and temporal regulation of such genes in indeterminate nodules. In such  nodules, major, sudden developmental changes occur at the transition of the prefixation zone to the interzone: starch is deposited in the plastids of the infected cells, and the bacteroid morphology alters. These events are accompanied by changes in bacterial gene expression: transcription of bacterial nif genes, which encode.enzymes involved in the nitrogen fixation process, is induced, whereas expression of the bacterial outer membrane protein gene ropA is dramatically reduced (Yang et al., 1991; De Maagd et al., 1994). All of these events, together with dramatic changes in plant gene expression take place within a single cell layer.