Assignment Subject


Plant Biochemistry
Course Code:
BOT – 504
Submitted To:
Dr Iqbal Hussain
Submitted by:
Aiman Zahid
Roll No:
BS (Hons) MOR
Symbiotic Nitrogen Fixation
Symbiotic Nitrogen fixation
• Nitrogen Fixation:
Nitrogen is abundant in the air (78% of the atmosphere is N2) plants have no enzyme systems that can use that nitrogen. Only some prokaryotes can use N2 by incorporating it into their bodies as amino acids and nucleotides; when they die and decompose the nitrogenous compounds are available to plants. The chemical process of converting atmospheric nitrogen into usable compounds is nitrogen fixation.
• Rhizobia bacteria:
The rhizobia bacteria 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:
It is a genus of small flagellate gram –negative rods. They establish a symbiotic relationship with legumes, living inside root nodules and fixing large amount of nitrogen, much of which is made available to the plants.
• Leguminous plants:
These plants are included under the family Fabaceae or Leguminosae; they have symbiotic nitrogen fixing bacteria, Rhizobia in structures called root nodules. Symbiotic nitrogen fixation in root nodules of some leguminous plants is caused by
1) Rhizobium
2) Azotobacter
3) Nitrobacter
4) Nitrosomonas
• Symbiotic Nitrogen fixation:
Biological nitrogen fixation was discovered by the German agronomist Hermann Hellriegel and Dutch microbiologist Martinus Beijerinck. Symbiotic nitrogen fixation occurs in plants that have nitrogen–fixing bacteria within their tissues. The best studied example is the association between Legumes and Bacteria. Each of these is able to survive independently (soil nitrates then available to the legumes) but life together is clearly beneficial to both. Only together can nitrogen fixation take place. A symbiotic relationship in which both partners are benefits is called mutualism. Biological nitrogen fixation can be represented by following equation:
N2 + 8H+ + 8e- + 16ATP = 2NH3 + H2 + 16ADP + 16Pi
Two moles of ammonia are produced from one mole of nitrogen gas, by the expenditure of 16 moles of ATP and a supply of electrons and protons (hydrogen ion). This process occurs when atmospheric nitrogen is converted to ammonia by an enzyme Nitrogenase

• Nitrogenase enzyme:
An enzyme that can use N2 as a substrate in complex reaction, it forces electrons and protons onto; nitrogen, reducing it from the +0 oxidation state to the -3 oxidation state. Ammonia, NH3 is the product it immediately dissolves in the cells water and picks up a proton, becoming ammonia NH4+. Nitrogenase is a giant enzyme or enzyme complex; it has a molecular weight of 100,000 to 300,000 Daltons and contains 15 to 20 iron atoms and 1 or 2 molybdenum atoms. It is extremely sensitive to oxygen and functions only if oxygen is completely excluded from it.
• Leghemoglobin:
The critical bacterial enzymes in nodules are sensitive to oxygen, being immediately poisoned by even traces of free oxygen. But it is a plant not bacterium that produces a special chemical that binds to oxygen and protects the bacterial enzymes from oxygen. These root nodules represent a sophisticated symbiosis; without the bacterium the complex development of nodules does not occur. Nodules are filled with hemoglobin when it is freshly cut nodule is red. In other words hemoglobin of the legumes is known as leghemoglobin like the hemoglobin of vertebrates just supply the right concentration of oxygen to bacteroids.
• Mechanism:
A symbiotic relationship has evolved with nitrogen fixing bacteria of genus rhizobium. Bacteria free in the soil secrete a substance that causes root hairs to curl sharply; the bacterium then attaches to the convex side of the hair and pushes into the cell by means of a tube like invagination of the plant cell wall. The tube is an infection thread, and the bacterium sits in it. The infection thread extends all the way into the root’s inner cortex, where adjacent cortical cells undergo mitosis and form a root nodule. The bacterium is released from the infection thread and enters the host cell cytoplasm where it proliferates rapidly filling the host cells with bacterial cells (known as bacteroids) capable to converting N2 into nitrogenous compounds that are released to the host cells. Energy for this process is supplied by sugars from the legume root cells, so both the Rhizobium and the legume benefit. In the present state as mutation occurred and survived by natural selection, the genetic mutation in bacterium that makes plant healthier is beneficial to bacterium as well, whereas any mutation that is harmful to the plant such as bacterial plasma membrane that does not allow nitrogenous compounds to move to plants is dangerous to bacterium.

Bacteria in an infected root nodule cell of cowpea
• The infection thread:
It is interaction between a particular strain of rhizobia and 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.
This infection thread is constructed by the root cells, not the bacteria, and is formed only in response to the infection.
• Function of root nodule:
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. The carbon source transported from the leaves to nodules is sucrose which is introduced into nodule metabolism through degradation by sucrose synthase. This enzyme present in both legumes and actinorhizal nodules. 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 glutamate pathway.
• Oxygen protection of bacterial nitrogen fixation:
Nitrogenase is highly sensitive because one of its component the MoFe cofactor is irreversible denatured by oxygen. In other way the large amount of energy required for this reaction has to be generated by oxidative processes thus there is high demand of oxygen in nodules. In legume nodules a low oxygen tension in the central part of the nodule is achieved symbiotic nitrogen fixation by a combination of high metabolic activity of microsymbiont and oxygen diffusion barrier in the periphery of nodule parenchyma. Because oxygen diffuses 104 times faster through air than water. Oxygen diffusion in nodules occurs via the intercellular spaces. 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.
• Rate at which symbiosis influenced:
The rate at which plant symbiosis fix nitrogen is strongly influenced by the stage of development of the plant .when soybeans begin to produce their protein-rich seeds (40% protein, the richest seeds known), nitrogen fixation in the roots increases greatly. As much as 90% of all nitrogen fixation occurs during the phase of seed development whereas only 10% occurs during all the vegetative growth that precedes it. Furthermore if legume crop plants are given high level of nitrogen fertilizer. Plants that have not yet formed nodules do not produce them and those that already have bacteriod-filled nodules decrease the amount of nitrogen fixed and even allow the nodule to senesce. With adequate nitrogen available in the environment, it is selectively advantageous not to pass glucose on to bacteria.

• References:

• Book ” Botany An Introduction to plant biology” by
• www.wikipedia .com