## NITROGEN

### NITROGEN

15.      NITROGEN

Nitrogen is an important constituent of cellular components. Alkaloids, amides, amino acids, vitamins, and many other cellular compounds containing nitrogen as one of the element. Without nitrogen, no living organism can survive. Paradoxically, living organisms are virtually submerged in a sea of atmospheric nitrogen but only some organisms like certain bacteria, blue-green algae funjoy have potential to utilise molecular nitrogen directly by fixing it. However, stop the plants are capable of utilising other forms of Nitrogen. Apart from atmospheric molecular nitrogen, it is distributed in the soil as ammoniacal, nitrate and nitrate forms. Organic nitrogen in the soil is contributed due to death and decay of plants and animals. Application of chemical fertilizers like urea provides a similarity form of nitrogen that can be directly Incorporated into amino acids.
The reason of Dead organic nitrogenous compound into Ammonia by Bacillus mycoides is termed as ammonification. Oxidation of ammonia, produced by ammonification into nitrates by nitrifying bacteria is called as nitrification. The first step in the formation of nitride is oxidation of ammonia to nitrate (NO2-) by the bacteria of genera Nitrosomonas. Nitrite in future oxidised to nitrate (NO3-) by Nitrobacter. These are two groups are known as nitrifying bacteria.

Nitrates or Nitrites convert back into molecular nitrogen by denitrification performed in pseuPseudomonas
Nitrate is not directly assimilated, bus first it must be reduced in ammonium ion in order to incorporate into organic compounds. In plants that do not form nitrogen-fixing associations, the nitrates in the soil are reduced to nitrite by enzyme nitrate reductase.

It is molybdic flavoprotein related by Nason & Evans from Glycin max leaves and Neurospora. Its molecular weight is about 3 × 10^5 daltons and is composed of two identical subunits, each containing 3 prosthetic groups, i.e. FAD ( Flavin Adenine Dinucleotide), game and molybdenum complexed to an organic molecule called pterin. It requires reducing power supplied by NADH or NADPH. The former is available in non-photosynthetic tissues latter is found leaves containing chloroplasts.

Cytokinin induces nitrate reductase synthesis even in absence of light and nitrate. Nitrate reductase is localised in the cytoplasm. The product light writes the nose into root plastids or leaf chloroplasts, where it is quickly reduced to ammonium ion. (NH4+).

In leaves, the electrons required for reduction of nitrate to ammonium ions are generated by photosynthetic electron transport. The enzyme has a molecular weight of 60-70 kDa and consists of special hame component called as siroheme detected in the short band. It possesses flavin and iron groups.

Nodule formation:-
Movement of nitrogen-fixing bacteria towards host plant roots is resulted due to secretion of certain compounds like flavonoids in soil by the latter. It generates a chemotaxis response as these chemical mediate growths and reproduction in bacteria. In counter response, these bacteria produce nodulation factors. They are derivatives of chitin, a beta-(1-4) linked polymer of N-acetyl-D- glucosamine. These are homopolymers except that a fatty acid replaces the acetyl group at one end of the molecule. Nod factors influences are increased root hair production and development of shorter and thicker roots. Of bacteria in the host cell wall occurs and colonies of attached rhizobia become entrapped by the tip of root hair as it curls around ceasing the growth of root tip along with invagination of cell membrane resulting in tubular intrusion into the cell that is termed as infection thread, which contains invading bacteria. The infarction thread elongates by adding new membrane material by fusion with vesicles derived from Golgi apparatus until it reaches the base of the root hair cell. As the infection thread moves through the root hair into the cortex, the bacteria continue to multiply. The membrane of infection thread birds of to form small vesicles. After the release of bacteria cells in the host, it stops dividing and there differentiated into specialised nitrogen-fixing cells called Bacteroides, surrounded by peribacteroid members. The infarction process continues throughout the life of the nodule. As the nodule due to the activity of module meristem, bacteria continue to invade the new cells. also as the nodule enlarges and mature, the vascular connection is established with the main vascular system of the root. they serve to import photosynthetic carbon into the nodule and export fix nitrogen from nodule to the plant.

The reaction is performed exclusively by using an enzyme complex termed as nitrogenase. Nitrogenase is the metalloenzyme that performs biological nitrogen fixation by catalyzing molecular nitrogen to ammonia. The enzyme complex can be further divided into two components -
An iron protein and a molybdenum-iron protein. The iron protein is smaller of the two components and has two identical subunits of 31-75 kDa. Each subunit consists of an iron sulphur cluster that contributes to redox reactions involved in the conversion of nitrogen to ammonia. The iron protein is reversibly inactivated by oxygen. The molybdenum-iron protein has the possibility that total molecular mass of 485-230 kDa. each subunit has two Mo-Fe-S clusters.
Nitrogenase is extremely sensitive to oxygen. Nodules contain an oxygen-binding heme protein called leghemoglobin (Lhb). It is pink coloured oxygen scavenger synthesized by plants. During generation of Lhb, plants contribute to the globin part and heme part is given by bacteria.

Nodule development in plants is influenced by the nodulin protein encoded by Nod genes of plants. Bacteria nodulation (nod) genes are classified as common nod genes or host-specific nod genes.
Nod A, nod B, and nod C are found in all rhizobial strains, whereas nod P, nod Q, nod H (or) nod E, nod F and nod L determine the host specificity. Nod A gene encodes for N-acyltransferase that catalyzes the addition of a fatty acyl chain in nod factors. Nod B gene encodes the chitin-oligosaccharide deacetylase that removes the acetyl group from the terminal non reducing sugar in nod factors. Nod C encodes for chitin-oligosaccharide synthase that links N-acetyl-D- glucosamine monomers. Variation in host-specific nod genes among rhizobial species is involved in the modification of the fatty acyl chain or the addition of groups in the nod factors. Nod E and Nod F determines the length and degree of saturation of the fatty acyl chain. Other enzymes, such as Nod L, influences the host specificity of Nod factors via the addition of specific substitution at the reducing or non reducing sugar moieties of the chitin backbone. Nod D regulates the transcription of the other nod genes.

Nitrogen enzyme complex is encoded by nif genes. The nif genes include structural that encode for dinitrogenase protein as well as a number of regulatory genes. Electron donor ferredoxin is encoded by nif F gene that reduces iron protein. Gene nif H encoded for both subunits of iron protein. Finally, the electron is transferred to a molybdenum-iron protein where molecular nitrogen fixation occurs. The two different subunits of the MoFe protein is encoded by nif D and nif K genes. Other nif genes are involved in insertion of the FeMo co-factor and the activation and processing of enzyme complex.
Regulation of nif genes :

In most of the bacteria that are involved in nitrogen fixation, regulation of nif gene transcription age activated by nitrogen sensitive Nif A protein. They constitutively result in the synthesis of the nitrogenase enzyme complex for fixing atmospheric molecular nitrogen. when the fixed nitrogen is present in sufficient amount, another protein is activated, I.e., Nif L. It impedes activity of Nif A, that inhibits the formation of nitrogenase enzyme. Nif L is regulated by the product of gln D and gln k. The nif genes are localised on s plasmid in the bacteria.
15.1.    Assimilation of Nitrogen
Ammonium ions are assimilated in plants by synthesis of amino acids. Glutamate incorporates ammonium ions using energy currency to produce glutamine amino acid. This reaction is catalysed by an enzyme glutamine synthetase (GS) in presence of Mg+2 and Co+2 co-factor.

Generally, a function of GS varies along with its position in plants. Cytosolic forms of the enzyme are present in germinating seeds and vascular bundles in plants, where it produces glutamine for intracellular transport. GS in root plastids produces amides of nitrogen and when found in shoot chloroplasts, it functions to reassimilates photorespiratory ammonia.
Increased glutamine induces the activity of enzyme glutamate synthase. This enzyme is also known as glutamine-2- oxoglutarate aminotransferase (GOGAT). it transfers the amid the group of glutamine to alpha-ketoglutarate, producing two molecules of glutamate.

On the basis of the electron donor, two types of GOGAT are present in plants. First one is Ferredoxin-GOGAT (FdGOGAT) that accepts the electron from Ferredoxin. it is gene found in chloroplasts and contributes on photorespiratory nitrogen metabolism. they incorporate NH4+ ions to glutamine in roots during nitrogen assimilation.

Glutamine + 2–oxoglutarate + Fdred $\rightarrow$ 2 glutamate + FdOX

The second one is NADH dependent type glutamate synthase. it receives an electron from NADH and is localized in plastids of non-photosynthetic tissues such as roots or vascular bundles of developing leaves. In roots, it is involved in the ammonium ions assimilation absorbed from the rhizosphere. In vascular bundles of developing leaves, it performs glutamine translocation from roots or senescing leaves.

Glutamine + 2–oxoglutarate + NADH + H+ $\rightarrow$ 2 glutamate + NAD+

Glutamate can also be synthesized from alpha-ketoglutarate by the activity of enzyme glutamate dehydrogenase, but it cannot substitute GS-GOGAT pathway for assimilation vof ammonium ions it's a primary function is to catalyse the deamination of glutamate amino acid.

Synthesis of amino acids like aspartate occurs by transamination reaction by aspartate aminotransferase enzyme in which amino group of glutamate is transferred to carboxyl atom of aspartate.

Similarly, asparagine synthetase enzyme catalyzes the transfer of amide nitrogen from glutamine to asparagine.