Nitrogen assimilation in plants

NITROGEN CYCLE
NITROGEN FIXATION
Establishing Symbiosis Requires an Exchange of Signals
NODULE FORMATION
NITROGENASE ENZYME
NITRATE ASSIMILATION
AMMONIUM ASSIMILATED BY GS/GOGAT PATHWAY

1. NITROGEN FIXATION AND ASSIMILATION ( NO3, NO2 REDUCTION, GS/GOGAT PATHWAY) PARUL SHARMA PHD BOTANY

2. Like carbon, hydrogen and oxygen, nitrogen is also one of the most prevalent essential macro-­elements which regulates plant growth, especially in agricultural systems. The main source of nitrogen for the construction of nitrogenous organic compounds is the atmosphere. It occurs in such essential biomolecules as nucleic acids, proteins, some of the phytohormones and in many of the vitamins. So, nitrogen as component of these biomolecules and many other compounds is involved in most of the biochemical reactions contributing to life activities. Plants require higher amounts of nitrogen as it is important in their structure and metabolism. Nearly, 80 per cent of the earth’s atmosphere is composed of nitrogen, bathing the entire plant world, but unfortunately most plants cannot utilize it in its elementary form. For its supply they have to depend on the soil and from there they acquire nitrogen in inorganic form either as ammonium compounds or as nitrate. The atmospheric nitrogen is converted into a metabolically useful form by a few prokaryotic life styles for supply to higher plants and animals. Ammonia, the most useful form, is initially converted to glutamate and glutamine and then to other nitrogen-containing compounds required for the growth and maintenance of the life of plants. The other sources of nitrogen in the soil are nitrate (NO 3 ), ammonium ions (NH 4 + ) or organic nitrogen, which are obtained from the nitrogen cycle and farm manure. Nitrate is converted to ammonia prior to metabolism. Ammonium ions and organic nitrogen in the form of amino acids mobilized from the proteins or partially destroyed proteins, can be absorbed directly by the roots of the plants.

3. NITROGEN CYCLE – The  nitrogen cycle  has five steps: 1. Nitrogen fixation  (N2 to NH3/ NH4+ or NO3-) 2. Nitrification  (NH3 to NO3-) 3. Assimilation  (Incorporation of NH3 and NO3- into biological tissues) 4. Ammonification  (organic nitrogen compounds to NH3) 5. Denitrification (NO3- to N2) NITROGEN FIXATION - Nitrogen fixation is the process by which gaseous nitrogen (N2) is converted to ammonia (NH3 or NH4+) via biological fixation or nitrate (NO3-) through high-energy physical processes. N2 is extremely stable and a great deal of energy is required to break the bonds that join the two N atoms. N2 can be converted directly into NO3- through processes that exert a tremendous amount of heat, pressure, and energy. Such processes include combustion, volcanic action, lightning discharges, and industrial means. However, a greater amount of biologically available nitrogen is naturally generated via the biological conversion of N2 to NH3/ NH4+. A small group of bacteria and cyanobacteria are capable using the enzyme nitrogenase to break the bonds among the molecular nitrogen and combine it with hydrogen. Nitrogenase  only functions in the absence of oxygen. The exclusion of oxygen is accomplished by many means. Some bacteria live beneath layers of oxygen-excluding slime on the roots of certain plants. The most important soil dwelling bacteria, Rhizobium, live in oxygen-free zones in nodules on the roots of legumes and some other woody plants. Aquatic filamentous  cyanobacteria  utilize oxygen-excluding cells called  heterocysts .

4. NITRIFICATION – Nitrification is a two-step process in which NH3/ NH4+ is converted to NO3-. First, the soil bacteria  Nitrosomonas  and  Nitrococcus  convert NH3 to NO2-, and then another soil bacterium,  Nitrobacter , oxidizes NO2- to NO3-. These bacteria gain energy through these conversions, both of which require oxygen to occur. ASSIMILATION – Assimilation  is the process by which plants and animals incorporate the NO3- and ammonia formed through nitrogen fixation and nitrification. Plants take up these forms of nitrogen through their roots, and incorporate them into plant proteins and nucleic acids. Animals are then able to utilize nitrogen from the plant tissues. AMMONIFICATION – Assimilation produces large quantities of organic nitrogen, including proteins, amino acids, and nucleic acids.  Ammonification  is the conversion of organic nitrogen into ammonia. The ammonia produced by this process is excreted into the environment and is then available for either nitrification or assimilation. DENITRIFICATION – Denitrification  is the reduction of NO3- to gaseous N2 by anaerobic bacteria. This process only occurs where there is little to no oxygen, such as deep in the soil near the water table. Hence, areas such as wetlands provide a valuable place for reducing excess nitrogen levels via denitrification processes.

5. NITROGEN CYCLE

6. NITROGEN FIXATION - It is the conversion of inert atmospheric nitrogen or di-nitrogen (N 2 ) into utilizable compounds of nitrogen like nitrate, ammonia, amino acids, etc. There are two methods of nitrogen fixation— abiological and biological. Abiological nitrogen fixation is further of two kinds, natural and industrial. Nitrogen fixation is of two types: Non-biological nitrogen fixation and biological nitrogen fixation. (a) Non-Biological or Physical Nitrogen Fixation: Nitrogen is an extremely stable molecule. It exists in di-nitrogen form (N 2 ) having a triple bond (N = N) between two nitrogen atoms. The nitrogen bond has the shortest length of 1.095 Å, the highest ionization potential (15.58 eV) and the highest stretching frequency. For this reason it is highly resistant to chemical attack. To break this triple bond about 225 kcal of energy is required but it is very difficult to achieve. In the fertilizer industry nitrogen is reduced to ammonia at very high temperature and pressure over an iron catalyst. Nitrogen may also be fixed through the electrical discharges that occur during lightning. In this process, the atmospheric nitrogen combines with oxygen to produce oxides of nitrogen which are subsequently hydrated by water vapor and carried to earth as nitrites and nitrates by rain.

7. Biological Nitrogen Fixation Only a few prokaryotes can use the atmospheric nitrogen as N 2  and reduce it to ammonia. This reduction of nitrogen to ammonia by living organisms is ‘biological nitrogen fixation’. The enzyme it needs for this reaction – nitrogenase is present exclusively in prokaryotes and these microbes are called N 2  – fixers. These N 2  – fixers can be symbiotic or free-living. Some examples of free-living N 2  – fixers are  Azotobacter ,  Bacillus ,  Anabaena ,   Nostoc   etc. Symbiotic Biological Nitrogen Fixation The most popular example in this category is the symbiotic relationship between  Rhizobium  and the roots of legumes such as sweet pea, garden pea, lentils. The association is visible as nodules (small outgrowths) on the roots. Another example is the microbe  Frankia  that also produces nitrogen-fixing nodules on the roots of non-leguminous plants. Both Rhizobium and Frankia live free as aerobes in the soil but are unable to fix nitrogen. They develop the ability to fix nitrogen only as a symbiont when they become anaerobic. Rhizobium is rod-shaped bacterium while Frankia is an actinomycete. They are unable to fix nitrogen by themselves. Roots of a legume secrete chemical attractants (flavonoids and betaines). Bacteria collect over the root hairs, release nod factors that cause curling of root hairs around the bacteria, degradation of cell wall and formation of an infection thread enclosing the bacteria

8. Establishing Symbiosis Requires an Exchange of Signals – Plant genes specific to nodules are called nodulin (Nod) genes; rhizobial genes that participate in nodule formation are called nodulation (nod) genes. The nod genes are classified as common nod genes or host-specific nod genes. The common nod genes— nodA , nodB , and nodC —are found in all rhizobial strains; the host-specific nod genes—such as nodP , nodQ , and nodH ; or nodF , nodE , and nodL —differ among rhizobial species and determine the host range. Only one of the nod genes, the regulatory nodD , is constitutively expressed, and as we will explain in detail, its protein product ( NodD ) regulates the transcription of the other nod genes. The nod genes activated by NodD code for nodulation proteins, most of which are involved in the biosynthesis of Nod factors. Nod factors are lipochitin oligosaccharide signal molecules, all of which have a chitin β-1→4-linked Nacetyl -D-glucosamine backbone (varying in length from three to six sugar units) and a fatty acyl chain on the C-2 position of the nonreducing sugar. NodA is an N-acyltransferase that catalyzes the addition of a fatty acyl chain. NodB is a chitin-oligosaccharide deacetylase that removes the acetyl group from the terminal nonreducing sugar. NodC is a chitin-oligosaccharide synthase that links N-acetyl-D-glucosamine monomers. NodE and NodF determine the length and degree of saturation of the fatty acyl chain. NodL influence the host specificity of Nod factors through the addition of specific substitutions at the reducing or nonreducing sugar moieties of the chitin backbone.

9. NODULE FORMATION - Two processes—infection and nodule organogenesis— occur simultaneously during root nodule formation. During the infection process, rhizobia that are attached to the root hairs release Nod factors that induce a pronounced curling of the root hair cells . The rhizobia become enclosed in the small compartment formed by the curling. The cell wall of the root hair degrades in these regions, also in response to Nod factors, allowing the bacterial cells direct access to the outer surface of the plant plasma membrane. The next step is formation of the infection thread, an internal tubular extension of the plasma membrane that is produced by the fusion of Golgi-derived membrane vesicles at the site of infection. The thread grows at its tip by the fusion of secretory vesicles to the end of the tube. Deeper into the root cortex, near the xylem, cortical cells dedifferentiate and start dividing, forming a distinct area within the cortex, called a nodule primordium, from which the nodule will develop. The infection thread filled with proliferating rhizobia elongates through the root hair and cortical cell layers, in the direction of the nodule primordium. When the infection thread reaches specialized cells within the nodule, its tip fuses with the plasma membrane of the host cell, releasing bacterial cells that are packaged in a membrane derived from the host cell plasma membrane. Branching of the infection thread inside the nodule enables the bacteria to infect many cells.

10. Nodule formation is stimulated by auxin produced by cortical cells and cytokinin liberated by invading bacteria. The infected cells enlarge. Bacteria stop dividing and form irregular polyhedral structures called bacteriods . However, some bacteria retain normal structure, divide and invade new areas. In an infected cell bacteriods occur in groups surrounded by host membrane. The host cell develops a pinkish pigment called leg-haemoglobin (Lb). It is oxygen scavenger and is related to blood pigment haemoglobin. It protects nitrogen fixing enzyme nitrogenase from oxygen. Symbiotic nitrogen fixation requires cooperation of Nod genes of legume, nod, nif and fix gene clusters of bacteria.

11. The infection process during nodule organogenesis. (A) Rhizobia bind to an emerging root hair in response to chemical attractants sent by the plant. (B) In response to factors produced by the bacteria, the root hair exhibits abnormal curling growth, and rhizobia cells proliferate within the coils. (C) Localized degradation of the root hair wall leads to infection and formation of the infection thread from Golgi secretory vesicles of root cells. (D) The infection thread reaches the end of the cell, and its membrane fuses with the plasma membrane of the root hair cell. (E) Rhizobia are released into the apoplast and penetrate the compound middle lamella to the subepidermal cell plasma membrane, leading to the initiation of a new infection thread, which forms an open channel with the first. (F) The infection thread extends and branches until it reaches target cells, where vesicles composed of plant membrane that enclose bacterial cells are released into the cytosol.

12. NITROGENASE ENZYME - Multimeric complex of two different proteins – smaller protein is dimer of 2 identical subunits called Fe protein. It is a single proteon of 4 fe and S. The larger protein is called MoFe protein. It is atetramer and contains 2 pairs – MoFe contain two molybdenum atoms in the form of iron molybdenum Sulphur co factor and it also contain Fe4S4. The enzyme is a complex of nitrogenase reductase and dinitrogenase . The dinitrogenase catalyze the reduction of nitrogen to ammonia.

13. The reaction catalyzed by nitrogenase. Ferredoxin reduces the Fe protein. Binding and hydrolysis of ATP to the Fe protein is thought to cause a conformational change of the Fe protein that facilitates the redox reactions. The Fe protein reduces the MoFe protein, and the MoFe protein reduces the N2.

14. NITRATE ASSIMILATION – Nitrate is the most important source of nitrogen to the plants. It can accumulate in the cell sap of several plants and take part in producing osmotic potential. However it cannot be used as such by the plants. It is first reduced to level of ammonia before being incorporated into organic compounds. Reduction of nitrate occurs in two steps. ( i ) Reduction of Nitrate to Nitrite: It is carried out by the agency of an inducible enzyme called nitrate reductase. The enzyme is a molybdoflavoprotein. It requires a re­duced coenzyme (NADH or NADPH) for its activity. The reduced coenzyme is brought in contact with nitrate by FAD or FMN.

15. (ii) Reduction of Nitrite: It is performed by enzyme nitrite reductase. The enzyme is a metalloflavoprotein which contains copper and iron. It occurs inside chloroplasts in the leaf cells and leucoplasts of other cells. In contrast nitrate reductase is found attached loosely to cell membrane. Nitrite reductase requires reducing power. It is NADPH in illuminated cells and NADH in others. The process of reduction also requires ferredoxin which occurs in higher plants mostly in green tissues. Therefore, it is presumed that in higher plants either nitrite is trans-located to leaf cells or some other electron donor (like FAD) operates in un-illuminated cells. The product of nitrite reduction is ammonia. Ammonia is not liberated. It combines with some organic acids to produce amino acids. Amino acids then form various types of nitrogenous compounds.

16. FATE OF AMMONIA – It is protonated to form ammonium ion (NH 4 + ) at physiological pH. Although plants can accumulate nitrate and NH 4 +  ions, NH 4 +  ions are toxic to them. Thus, it is in turn, used to synthesize amino acids in plants as follows: 1. Reductive Amination: In the presence of dehydrogenase (e.g., Glutamate dehydrogenase, Aspartate dehydrogenase), a reduced coenzyme (NADH or NADPH), ammonia can directly combine with a keto organic acid like a-ketoglutaric acid and oxaloacetic acid to form amino acid. 2. Catalytic Amination: Ammonia combines with catalytic amounts of glutamic acid in the presence of ATP and enzyme glutamine synthetase. It produces an amide called glutamine. Glutamine reacts with a-ketoglutaric acid in the presence of enzyme glutamate synthetase to form two molecules of glutamate. Reduced co-enzyme (NADH or NADPH) is required. 3. Transamination: It is transfer of amino group (> CH NH 2 ) of one amino acid with the keto group (> С = О) of keto acid. The enzyme required is transaminase or aminotrans­ferase. Glutamic acid is the primary amino acid involved in transfer of amino group (to as many as seventeen amino acids).

17. AMMONIUM ASSIMILATED BY GS/GOGAT PATHWAY - Ammonium is readily available to plants but can be quiet toxic, it inhibits nitrigenase and interfere energy metabolism. The general pathway for ammonium assimilation was carried and it was found that initial organic product is glutamine. The assimilation of ammonium into glutamine by legume nodule is accomplished by Glutamine synthase cycle, pathway involving two sequential enzymes – GS (Glutamine Synthase) – There are two classes of GS, one in the cytosola nd other in the root plastids. GOGAT (Glutamate Synthase) – Plants contain two types of GOGAT, one accepts electrons from NADH, and the other accepts electrons from Ferridoxin ( Fd ). PSII proteins regulates the GS/GOGAT pathway. The glutamate (Glu) amino group can be transferred to amino acids by a number of different aminotransferases. Asparagine synthetase (AS) catalyzes the formation of asparagine ( Asn ) and Glu from glutamine ( Gln ) and aspartate.

18. Glutamine synthetase (GS ) combines ammonium with glutamate to form glutamine: Glutamate + NH4 + + ATP → glutamine + ADP + Pi This reaction requires the hydrolysis of one ATP and involves a divalent cation such as Mg2+, Mn2+, or Co2+ as a cofactor. Plants contain two classes of GS, one in the cytosol and the other in root plastids or shoot chloroplasts. The cytosolic forms are expressed in germinating seeds or in the vascular bundles of roots and shoots and produce glutamine for intracellular nitrogen transport. The GS in root plastids generates amide nitrogen for local consumption; the GS in shoot chloroplasts reassimilates photorespiratory NH4 +. Light and carbohydrate levels alter the expression of the plastid forms of the enzyme, but they have little effect on the cytosolic forms. Elevated plastid levels of glutamine stimulate the activity of glutamate synthase (also known as glutamine:2-oxoglutarate aminotransferase, or GOGAT) . This enzyme transfers the amide group of glutamine to 2-oxoglutarate, yielding two molecules of glutamate. Plants contain two types of GOGAT: One accepts electrons from NADH; the other accepts electrons from ferredoxin ( Fd ): Glutamine + 2-oxoglutarate + NADH + H+ → 2 glutamate + NAD+ Glutamine + 2-oxoglutarate + Fdred → 2 glutamate + Fdox The NADH type of the enzyme (NADH-GOGAT) is located in plastids of nonphotosynthetic tissues such as roots or vascular bundles of developing leaves. In roots, NADH-GOGAT is involved in the assimilation of NH4 + absorbed from the rhizosphere (the soil near the surface of the roots); in vascular bundles of developing leaves, NADH-GOGAT assimilates glutamine translocated from roots or senescing leaves. The ferredoxin-dependent type of glutamate synthase ( FdGOGAT ) is found in chloroplasts and serves in photorespiratory nitrogen metabolism. Both the amount of protein and its activity increase with light levels. Roots, particularly those under nitrate nutrition, have Fd -GOGAT in plastids. FdGOGAT in the roots presumably functions to incorporate the glutamine generated during nitrate assimilation.

19. The GS-GOGAT pathway that forms glutamine and glutamate. A reduced cofactor is required for the reaction: ferredoxin in green leaves and NADH in nonphotosynthetic tissue

20. Transfer of the amino group from glutamate to oxaloacetate to form aspartate (catalyzed by aspartate aminotransferase) and Synthesis of asparagine by transfer of an amino acid group from glutamine to aspartate (catalyzed by asparagine synthesis).

21. Glutamate synthesized by the action of GS/GOGAT pathway

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