Nitrate Assimilation

The most important source of nitrogen to plants is nitrate. It is first reduced to the level of ammonia before it can be utilized by plants.

Reduction of nitrate occurs in two steps:

Step 1 Reduction of Nitrate to Nitrite: The enzyme that causes the reduction is nitrate reductase. This enzyme is molybdo-flavoprotein and requires a reduced coenzyme (NADH or NADPH2) for its activity. This is an important enzyme for the nitrate assimilation. Nitrate reductase of higher plants is composed of two identical subunits. One subunit contains three prosthetic groups.

NO3 + NAD(P)H + H+ —> NO2- + H2O + NADP+ (catalysed by Nitrate reductase, FAD/FMN)

Step 2 Reduction of Nitrite: Nitrite reductase, a metalloflavoprotein containing Cu and Fe, causes reduction of nitrite. It is a multidomain enzyme that comprise of prosthetic groups; molybdopterin, Fe-heme and FAD (Flavin Adenine Dinucleotide) in a 1:1:1 stoichiometry that mediates an electron transfer from NAD(P)H to nitrate. The enzyme requires reducing power (NADPH, NADH). The process of reduction also requires ferredoxin, which occurs mostly in green tissues of higher plants. The product of nitrite reduction is ammonia.

2NO2- + 7NAD (P) H+ —> 7H+ + 2NH3 + 4H2O + 7 NAD (P)+ (Catalysed by Ferredoxin, Nitrite reductase)

Ammonia is not liberated. It combines with organic acids to form amino acids, which form various types of nitrogenous compounds.

Nitrogen is available in the atmosphere in abundance but plants cannot directly absorb it. Regular supply of nitrogen is maintained through nitrogen cycle. Nitrogen cycle is regular circulation of nitrogen amongst living organisms, reservoir pool in the atmosphere and cycling pool in lithosphere. Plants obtain nitrogen from soil as NO3- (nitrate), NH4+ (ammonium) and NO2- (nitrite) ions. Nitrate and nitrite are reduced to ammonium state, which is then incorporated into amino acids, proteins and other organic substances.

Nitrogen Fixation is the conversion of inert atmospheric nitrogen or dinitrogen (N2) into utilizable compounds of nitrogen like nitrate, ammonia, amino acids etc.
Symbiotic Nitrogen Fixing Bacteria plays major role in nitrogen fixation. Several species of Rhizobium bacteria live in the soil, and they are unable to fix nitrogen by themselves. Roots of a legume secrete chemical attractants like flavoxoids and betaines. Symbiotic nitrogen fixation in legume nodules involves complex interaction between Rhizobium and legume roots. This complex interaction is governed by sensing of plant flavonoids by rhizobia and activation of nod genes in rhizobia. Bacteria release nod factors by collecting over the root hair. The nod genes code for nodulation proteins are activated by NodD. The nod factors cause curling of root hair around the bacteria, degradation of cell wall and formation of an infection thread. The bacteria multiplies, it results in the growth of infection thread. It branches and its ends come to lie opposite protoxylem points of vascular strand. Infected cortical cells differentiate and start dividing which results in nodule formation. Auxins produced by cortical cells and cytokinin liberated by invading bacteria stimulate nodule formation. As the infected cells enlarge, bacteria cells enlarge and form irregular polyhedral structure called bacteroids. In an infected cell, bacteroids occur in groups surrounded by host membrane. The host cell develops a pinkish pigment called leghaemoglobin, which is an oxygen scavenger. It protects nitrogen fixing enzyme nitrogenase from oxygen. Oxygen irreversibly inactivates nitrogenase enzyme involved in nitrogen fixation. During the reaction catalyzed by nitrogenase enzyme, the Fe protein reduces the MoFe protein while the MoFe protein reduces N2.

Mechanism of Nitrogen fixation
Nitrogen fixation requires:
1. A reducing power like NADPH, FMNH2.
2. Source of energy like ATP.
3. Nitrogenase enzyme.
4. Compounds for trapping ammonia formed due to reduction of dinitrogen.

Free-living bacteria fix nitrogen in two steps:
1. Ammonificaton
2. Nitrification

Ammonification: Decay causing organisms cause ammonification by acting upon nitrogenous excretions and proteins of dead bodies of living organisms. Examples of such organisms include B. ramousus, B. vulgaris, B. mesentericus, Actinomyces.

Nitrification: is the process of conversion of ammonium nitrogen to nitrate nitrogen

Ammonium Assimilation
Ammonium toxicity is avoided by rapid conversion to amino acids – these reactions take place in the cytosol, root plastids, or chloroplasts. Some of the reactions involving ammonium and amino acid synthesis are
Ammonium combines with glutamate (glutamic acid, which has 1 N atom) to form glutamine (which has 2 N atoms). This reaction requires the enzyme glutamine synthetase (GS), hydrolysis of ATP and a divalent cation such as Mg2+, Mn2+ or Co2+. This is the first of two reactions that assimilate ammonium.
Ammonium combines with 2-Oxoglutarate to form glutamate via glutamate dehydrogenase. This is the second of the two reactions that assimilate ammonium and requires oxidation of NADH or NADPH.
Elevated glutamine stimulates glutamate synthase activity, which converts Glutamine plus 2-Oxoglutarate to 2-glutamates
Glutamate combines with Oxaloacetate to yield Aspartate and 2-Oxoglutarate. Glutamine combines with Aspartate to form Asparagine and Glutamate (a transamination reaction requiring asparagine synthase and ATP). The remaining amino acids are synthesized by transamination reactions catalyzed by aminotransferases, such as aspartate aminotransferase. The amino acid Asparagine serves as a stable compound with, like glutamine, relatively high-N content and is used to transport and store N, as well as to serve as a protein precursor.

Amino acid biosynthesis
Though the plants are able to synthesize amino acids in every living cell of their body but most of the primary amino acids are synthesized in the roots and leaves. The ammonia produced by the reductive steps of NO2, NO3 or N2 are toxic if they get accumulated within the cells. Therefore, the ammonia is immediately used up in the process of the synthesis of amino acids. If there is any excess of NH3 present, it is stored in the amide form. The most important pathway by which amino acids are synthesized is reductive amination that leads to the synthesis of glutamate. The other pathways like transamination and carbonyl phosphate reactions are called the secondary pathways.
The biosynthesis of amino acids starts with carbon compounds that are found in the central metabolic pathways. All are synthesized from common metabolic intermediates. In case of non-essential amino acids, by transamination of α-keto acids that is available as common intermediates. In case of essential amino acids, their α-keto acids are not common intermediates as enzymes needed to form them are lacking. So transamination is not an option. In case of non-essential amino acid, all (except tyr) are synthesized from common intermediates synthesized in cell like pyruvate, oxaloacetate, α -ketoglutarate and 3-phosphoglycerate. In case of essential amino acids, all are synthesized from common metabolic precursors, aspartate, pyruvate, phosphoenolpyruvate, erythrose-4-phosphate, purine plus ATP (histidine). The synthesis of aspartate family, pyruvate family, aromatic and histidine under essential amino acids takes place. They are degraded to pyruvate, acetyl CoA, acetoacetate, α ketoglutarate, succinyl CoA, oxaloacetate and fumarate. Amino acids are synthesized from α-ketoacids and later transaminated from another amino acid, usually Glutamate. The enzyme involved in this reaction is an aminotransferase.
α-Ketoacid + Glutamate ⇄ Amino acid + α-Ketoglutarate

Glutamate itself is formed by amination of α-ketoglutarate
α-Ketoglutarate + NH4+ ⇄ Glutamate