Besides water and mineral salts the next important substances of plant cell is proteins and other nitrogenous compounds. Nitrogen is a universally occurring element in all living beings and from the important constituents of cholorophyll, vitamins, hormones, cytochromes, enzymes, nucleic acids and proteins. Atmosphere contains 75% nitrogen in gaseous form which is not directly taken up by plants. Plant can assimilate nitrogen if form of nitrites, nitrates and salts of ammonium. Atmospheric nitrogen gas thus subjected to be fixed into ammonia. For this it needs high pressure of 200 Atm. and temperature at 450 degree Celsius is required. But by biological process with aid of enzymes nitrogen can fixed into its assimilatory form at 25 degree Celsius and 1 Atm. pressure.
Conversion of molecular nitrogen of the atmosphere into inorganic nitrogen compounds through the agency of some living organism is called as biological nitrogen fixation. It can be done by certain bacteria and blue green algae either in free living forms in soil or in symbiotic condition with higher plants.
SYMBIOTIC NITROGEN FIXATION:
Hellriegel and Wilforth (1886) demonstrated that, bacteria present in the nodules of leguminous plants responsible for fixing nitrogen. These bacteria were identified later as rhizobium. There are several species of Rhizobium found in the root nodules of different plants.
1. Rhizobium melilti – Found in alfalfa, sweet clover trigenella etc.
2. Rhizobium trifolii – Found trifolium species.
3. Rhizobium leguminosarum – Found trifolium species.
4. Rhizpbium phascoli – Found in phascolus species.
5. Rhizobium Lupini – Found in lupines, serradella.
6. Rhizobium japonicum – Found in soybean, cowpea crotolaria etc.
It has been found that roots of leguminous plants contain lectins, a specific glycoprotein that can detect the specific landscaping of different sugars on bacterial cell wall surface and this determine the specificity of the bacteria association to specific plant species.
Mechanism of nitrogen fixation:
A considerable break through has been made in the understanding of nitrogen fixation during (1960-70) by the work of shneider at all (1960) and carnahan at all (1960). By using heavy isotope of nitrogen N15 to the cell free blue green algae and nitrogen fixing bacteria they come to conclusion that –
Presence of hydrogenase and nitrogenase enzyme systems catalyses the reduction of hydrogen ion to molecular hydrogen and nitrogen is reduced to NH2 in presence of molecular hydrogen.
Probable ammonia is the key intermediate compound in nitrogen fixation. Here enzyme nitrogenose catalyses the reduction process. The electrons made available either from respiratory electron transport chain or from photosynthesis or from the phosphoelastc split.
According to recent views, Burns and hardy (1975) the action of enzyme nitrogenase is based on electron activation two-site hypothesis. There are two active sites on this enzyme. At first active site in presence of ATP in association both molebdenum.
At second active site nitrogen is attached to group ‘Y’ which involves the metal Fee. Then two electron reductions takes place step by step forming diimidl and hydrozine as intermediates which remain attached to the enzyme. Finally hydrazine-enzyme complex is splitted XNH2 and YNH2 produce two molecules of NH3. Then group X again become free.
Hypothetical reaction site of nitrogenase
Some believe that hydrazine N2H4 gives rise to organic azines which leads to the formation of ghitamine. Whereas others are of the opinion that prior to formation of NH3 there is formation of an intermediate; probably hydroxyl amine.
All the genes for fixation of nitrogen called “inf-genes” are found in rhizobium. Legumious plant s provided arabinose and xylucose which cause the expression of these genes in the synthesis of the enzyme nitrogenase in bacteria. Therefore unless the bacteria associate with leguminous plants cannot produce nitrogenase. So symbiotic association is must be required to fix nitrogen by rhizobium during the activities nodules are formed in legume roots when the bacteria enter into roof. If takes place in following steps:
i) Production of substance in the root of legume to attract bacteria rhizobium.
ii) By the introduction of bacteria then hormones like lndole acetic acid (IAA) or related auxin produced in roots. This cause curling of root hairs.
iii) Bacteria partially destroy the cell wall or root hairs by secreting enzyme polygalacuronase (PG) which dissolve the pectin of the cell wall and facilitates the bacterial penetration
iv) Bacteria then enter to the cortex in form of mucilaginous thread like structures called infection thread.
v) The bacterium multiplies as the thread grows in length and finally released in the symbionts cytoplasm where also it multiplies.
vi) After their release cortical cells of root are stimulated divide and redivide and become polyploid. The repeated divisions of apolyploid cells results in the formation of nodules. The cells of nodule divide and increase the size of nodule.
Role of nodule in nitrogen fixation:
i) Infected nodule cells by the action of Rhizobium produce a characteristic red pigment almost simolar to life haemoglobin. This pigment is called leg-haemopglobin
ii) This pigment like blood pigment can trap oxygen at its oxygen in activation.
iii) The pigmentis located in between bacteriods and surrounding mambrane.
iv) The oxygen trapped by leg haemoglobin passes on to the electron transport chain.
Thus nodulation help in activation of nitrogenase activity and for its protection during nitrogen fixation. The home component of hemoglobin synthesized by the involvement of bacterial enzyme with that of the host legume cells.