Plants absorb nitrogen from the soil in the form of nitrate (NO3−) and ammonium (NH4+). In aerobic soils where nitrification can occur, nitrate is usually the predominant form of available nitrogen that is absorbed. However this is not always the case as ammonia can predominate in grasslands and in flooded, anaerobic soils like rice paddies.[4] Plant roots themselves can affect the abundance of various forms of nitrogen by changing the pH and secreting organic compounds or oxygen. This influences microbial activities like the inter-conversion of various nitrogen species, the release of ammonia from organic matter in the soil and the fixation of nitrogen by non-nodule-forming bacteria. Ammonium ions are absorbed by the plant via ammonia transporters. Nitrate is taken up by several nitrate transporters that use a proton gradient to power the transport.Nitrogen is transported from the root to the shoot via the xylem in the form of nitrate, dissolved ammonia and amino acids. Usually (but not always most of the nitrate reduction is carried out in the shoots while the roots reduce only a small fraction of the absorbed nitrate to ammonia. Ammonia (both absorbed and synthesized) is incorporated into amino acids via the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway. While nearly all the ammonia in the root is usually incorporated into amino acids at the root itself, plants may transport significant amounts of ammonium ions in the xylem to be fixed in the shoots.This may help avoid the transport of organic compounds down to the roots just to carry the nitrogen back as amino acids. Nitrate reduction is carried out in two steps. Nitrate is first reduced to nitrite (NO2−) in the cytosol by nitrate reductase using NADH or NADPH. Nitrite is then reduced to ammonia in the chloroplasts (plastids in roots) by a ferredoxin dependent nitrite reductase. In photosynthesizing tissues, it uses an isoform of ferredoxin (Fd1) that is reduced by PSI while in the root it uses a form of ferredoxin (Fd3) that has a less negative midpoint potential and can be reduced easily by NADPH. In non photosynthesizing tissues, NADPH is generated by glycolysis and the pentose phosphate pathway. In the chloroplasts,glutamine synthetase incorporates this ammonia as the amide group of glutamine using glutamate as a substrate. Glutamate synthase (Fd-GOGAT and NADH-GOGAT) transfer the amide group onto a 2-oxoglutarate molecule producing two glutamates. Further transaminations are carried out make other amino acids (most commonly asparagine) from glutamine. While the enzyme glutamate dehydrogenase (GDH) does not play a direct role in the assimilation, it protects the mitochondrial functions during periods of high nitrogen metabolism and takes part in nitrogen remobilization.

1. NITROGEN ASSIMILATION
• CONTENT :-
 Nitrogen Assimilation
 Introduction
 Nitrogen Cycle
 Nitogen Assimilation In Microorganisms
 Nitrate Assimilation
 Ammonia Assimilation
• What Is Nitrogen Assimilation ?
 Nitrogen Assimilation is the formation of organic nitrogen compounds like amino acids ,
amides etc from inorganic nitrogen compounds like nitrates and ammonia present in
the environment.
 Plants and other organisms , which cannot utilise nitrogen molecules directly , depend
on the absorption of nitrogen as nitrates or ammonia.
 Animals depend on organic nitrogen available in their food.
 The essential requirement for growth in all organisms is the assimilation of nitrogen into
molecules.
• Introduction :-
 Nitrogen is one of the most important elements along with carbon , hydrogen and
oxygen.
 It is part of biomolecules such as nucleic acids (DNA , RNA) , proteins (amino acids) ,
hormones , vitamins , chlorophylls , alkaloids , enzymes and many other cellular
components.

2.  It is necessary for sustaining life.
 Nitrogen is present abundantly in the atmosphere (78%) but only a few organisms can
utilise the atmospheric nitrogen directly, while others like plants and some
microorganisms are dependent to get them in the inorganic form.
• Nitrogen Cycle :-
 Nitrogen occurs as dinitrogen (N2) in the atmosphere , wherein the two nitrogen are
bonded together by triple covalent bonds.
 Only some organisms like bacteria , blue – green algae (cyanobacteria) are capable of
utilising and fixing atmospheric nitrogen into other forms to make it available for
absorption by plants and other organisms.
 The Nitrogen Cycle comprises nitrogen fixation , ammonification , nitrification ,
denitrification and assimilation.
1. Nitrogen Fixation:- It is the process of converting atmospheric nitrogen to
ammonia. Nitrogen fixation takes place naturally by three methods – Atmospheric
Fixation , Industrial Nitrogen Fixation and Biological Nitrogen Fixation. Atmospheric
fixation: A natural phenomenon where the energy of lightning breaks the nitrogen
into nitrogen oxides and is then used by plants.Industrial nitrogen fixation: Is a
man-made alternative that aids in nitrogen fixation by the use of ammonia. Ammonia
is produced by the direct combination of nitrogen and hydrogen and later, it is
converted into various fertilisers such as urea.Biological nitrogen fixation: We
already know that nitrogen is not usable directly from the air for plants and animals.
Bacteria like Rhizobium and blue-green algae transform the unusable form of
nitrogen into other compounds that are more readily usable. These nitrogen
compounds get fixed in the soil by these microbes.
2. Ammonification :- It is the process by which decomposition of dead and decaying
organic matter leads to the formation of ammonia.
3. Nitrification :- The conversion ammonia to nitrite and nitrate in the soil by
bacteria is termed as nitrification. Ammonia is first converted to nitrite by the
bacteria Nitrosomonas and Nitrococcus and then it is further oxidised to nitrate by
the bacterium Nitrobacter .

3. 4. Denitrification :- Nitrate in the soil also gets converted back to nitrogen by the
process of denitrification. Bacteria like Pseudomonas and Thiobacillus carry out
denitrification.
5. Assimilation :- It is the process by which nitrate and ammonia are utilised to form
organic compounds.
• Nitrogen Assimilation In Microorganisms :-
 Plants , fungi and bacteria absorb nitrogen from the soil in the form of nitrates and
ammonium ions. Ammonium ions and nitrates are absorbed by them via their
respective transporters.
 Nitrate after absorption is transported to the leaves and gets reduced to ammonia.
Further, ammonia is converted into amine groups of various amino acids.
 Nitrate reduction takes place in two steps. First, nitrate is reduced to nitrite in the
cytoplasm by nitrate reductase and then nitrite is reduced to ammonia by nitrite
reductase in the chloroplasts. It also occurs in plastids in the roots.
 This ammonia gets incorporated into glutamate to form glutamine. The reaction is
catalysed by glutamine synthetase. By transamination, other amino acids are formed
like asparagine. This is called the glutamine synthetase-glutamate synthase (GS-GOGAT)
pathway.

4. • 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.
1. Reduction of Nitrate to Nitrite
2. Reduction of Nitrite to Ammonia
Reduction of Nitrate to Nitrite :-
 Nitrate reduction into nitrite is catalyzed in the cytosol by the enzyme Nitrate Reductase
(NR).
 This enzyme is a homodimer , each monomer being associated with three prosthetic
groups:
1. Falvin adenine dinucleotide (FAD)
2. Heme
3. Molybdenum cofactor (MoCo)
Reduction Of Nitrite To Ammonia :-
 Nitrite is highly reactive and toxic.
 Plant cells immediately transport the nitrite generated by Nitrite Reductase from cytosol
into chloroplasts in leaves and plastids in roots.
 Nitrite Reductase is a single polypeptide containing 2 prosthetic groups –
1. Iron – sulfur cluster

5. 2. Heme
• Ammonia Assimilation :-
 Conversion of ammonia generated from nitrate assimilation or photorespiratiom into
amino acid.
 2 pathways –
1. Primary Pathway
2. Alternative Pathway
Primary Pathway :-
 2 enzymes involved :
a. Glutamine Synthethase (GS) :-
 Glutamate reacts with ammonia in the presence of glutamine synthetase enzyme
forms glutamine.
In plants it is present in two forms –
◾ Cytosolic Form : Expressed in germinating seeds or in vascular bundles of roots and shoots.
◾ Choloroplast Form : Present in root plastids and shoot chloroplast.
b. Glutamate Synthase (GOGAT) :-
 The full form of GOGAT is glutamine 2-oxoglutarate amino transferase.
 It transfers amide group of glutamine to 2-oxoglutarate and yields two glutamate
molecules.
 Plants contain 2 types of GOGAT.

6. ◾ One accept electron from NADH (NADH GOGAT) Located in plastids of non- photosynthetic
tissue like roots or vascular bundles of developing leaves.
◾ Other accept electron from ferredoxin (fd GOGAT) It is found in chloroplast and serves in
photorespiratory nitrogen metabolism.
a. Glutamine Synthethase Reaction :-
 In this reaction Glutamine is synthesized from glutamate .
 The reaction is catalyzed by glutamine synthetase and requires ATP.
 The glutamine synthase reaction is also used in the synthesis of glutamine
for protein synthesis.
 E.coli glutamine synthetase has a MW of 600,000. It consists of 12 identical
subunits , each of MW of 50,000.
 The subunits are arranged in the form of two hexagonal rings of six
subunits each , with a spacing of 4.5 nm between them .
 Cations of Mg++ and Mn++ are required for stability.
 In plants it is present in two forms –
◾ Cytosolic Form :-
 Expressed in germinating seeds or in vascular bundle of roots and shoots.
 Produce glutamine for intracellular nitrogen transport.
◾ Root Plastid / Shoot Chloroplast Form :-
 In roots , it produces amide nitrogen for local consumption.
 In shoots , it reassimilates photorespiratory NH4+

7. b. Glutamate Synthase Reaction :-
 Transfers amide group of glutamine to 2-oxoglutarate and yields two glutamate
molecules.
 It is found in all the prokaryotes studied and also in some strains of yeast.
 Plants consists of two types of GOGAT –
◾ NADH – GOGAT :-
 accepts electrons from NADH.
 located in plastids of non-photosynthetic tissue like roots or VB of developing leaves.
Glutamine + 2-oxoglutarate + NADH + H+ –––––– 2 Glutamate + NAD+
◾ Fd – GOGAT :-
 accepts electron from ferrodoxin.
 Located in chloroplast and serves in photorespiratory nitrogen metabolism.
Glutamine + 2-oxoglutarate + Fd (red) –––––– 2 Glutamate + Fd (ox)
 2. Alternative Pathway :-

8. 1. Reductive Amination :-
 Reversible reaction catalysed by Glutamate dehydrogenase that synthesis glutamate.
 The GDHs of E.coli and Salmonella typhyimurium are of similar size and molecular
weight , 300,000 and 280,000 respectively.
 E.coli GDH appears to consist of six identical , subunits.
 Glutamate is formed as a result of the reaction of ammonium with alpha-ketoglutaric
acid.
 NADH dependent form of GDH is found in mitochondria.
 NADPH dependent form located in the chloroplast of photosynthetic organelles.
2. Transamination Reaction :-
 Transfer of amino group of an amino acid to alpha-keto acid resulting in formation of
new amino acid and new keto acid.
 Transamination between glutamate and various non-nitrogenous metabolites results in
the formation of amino acids.
 Catalyzed by Transaminase (Aminotransferase).
 Co-factor : Pyridoxal phosphate (Vitamin B6).
 Reversible.
 Not all amino acids undergo transamination reaction.
 Eg. Lysine , Threonine , Proline , Hydroxy proline .

9. • Role of Pyridoxal Phosphate (PLP) :-
 Serves as a carrier of amino group.
 Transfer of alpha-amino group to PLP forms Pyridoxamine Phosphate , and a keto acid.
 Alpha – amino group is finally transferred to acceptor keto acid to form a new amino
acid.
• Example :-
 Catalyzed by Aspartate Transaminase.
 Catalyzed by Alanine Transaminase.