This presentation offers a bird's eye view of nitrogen metabolism in plants w.s.r. to symbiotic nitrogen fixation.

1. PLANT
METABOLISM
NITROGEN METABOLISM
By
N. Sannigrahi, Associate Professor,
Department of Botany,
Nistarini College, Purulia (W.B) India

2. INTRODUCTION
 Nitrogen gas is 77% in the atmosphere and it is present in the
soil as nitrate. It is not as a rule utilized by plants in its free
state. However, nitrogen occurs in the dry substances of the
plant to the extent of 1-3 percent only. Nevertheless, it is
indispensable to the life of the plant, as it is an essential
constituent of proteins, chlorophyll and protoplasm. Moreover
it is essential for growth, particularly of the leaves. An excess
of nitrogen causes vigorous growth of vegetative parts,
specially the leaves, but delays reproduction activity. One of
the fundamental biological requirements for life to persist is
that the nitrogen cycle should continue to function. During this
process, the atmospheric nitrogen is fixed into organic
combinations, such as amino acids, proteins, nucleic acids,
etc., in living organisms via inorganic forms as NH4
+
(ammonia).

3. NIROGEN CYCLE

4. TYPES OF BIOLOGICAL NITROGEN FIXATION
 The biological nitrogen fixation takes place by the diverse
organisms as follows:
 Free living-Autotrophic & Heterotrophic
 Autotrophic-Aerobic (Cyanobacteria) and Anaerobic (
Chormatium)
 Heterocysts (Nostoc, Anabena) and Non-heterocysts
(Chromatium)
 Heterotrophic ( Bacterial) Aerobic (Azotobacter), Facultative
aerobic ( Klebsilla) and Anaerobic (Clostridium)
 Symbiotic-Leguminous (Rhizobium), Non-leguminous
(Parasponia & Bradyrhizobium), Actinorhiza (Alnus,
Frankia), Endosymbiont( Azolla & Anabena)

5. ASSIMILATION OF NITROGEN
 Plants get nitrogen from the soil, by absorption of their roots
in the form of nitrate ions or ammonium ions. If nitrate is
absorbed, it is first reduced to nitrite ions and then ammonium
ions for incorporation into amino acids, nucleic acids etc.
 Ammonification:
 The dead remains of animals and plants are decomposed
through microbial activities to produce ammonia (NH3). The
enzymes involved are as follows:
 GS: Gl(n) glutamine synthetase (Cytosol & plastid)
 GOGAT: Glu 2-oxaloglutrate aminotransferase (Ferrodoxin &
NADH dependent)
 GDH: Glu Dehydrogenase: Minor role in ammonium
assimilation but more important in amino acid catabolism.

6. ASSIMILATION OF NITROGEN
 Nitrification: Here, ammonia is rapidly converted first to
nitrites (NO2), and then to nitrates (NO3). The oxidation of
ammonia(NH4+) to nitrite is carried out by bacteria
Nitrosomonas, and of nitrite to Nitrate by Nitrobacter. Now,
nitrate is available to the plant. It is important for the nitrites to
be converted to nitrates because accumulated nitrites are toxic
for plant life. Nitrates due to high solubility enter the ground
water and threaten the life of others and may cause
eutrophication.
 Denitrification: Where nitrate (NO3) may convert into N2 gas
by bacteria Pseudomonas, Clostridium. This nitrogen gas may
be again fixed in the form of NH4
+ through the process of
biological nitrogen fixation. In anaerobic condition, they use
nitrate as an electron acceptor in the place of oxygen during
respiration.

7. NITROGEN FIXATION IN PLANT
 The process of nitrogen fixation is conversion of atmospheric
nitrogen to ammonia. Approximately in a year, 90% is
biological in nature, 10% is non-biological ( 2% due to
radiation & 8% due to lightning). Industrial conversion of
nitrogen to ammonia is possible but it requires very high
temperature (200℃) and high pressure (200 atm) as per
Haber-Bosch process. The greatness of biological nitrogen
fixation is that , it can occur at normal temperature and
pressure without any modification of physical condition. out
of all these types of nitrogen fixation, the symbiotic nitrogen
fixation is most common and in legume plants by Rhizobium
seems to be most widely distributed all over the world. It is
brought about by unique enzyme, nitrogenase and
dinitrogenase, coded by a group of genes the surrounding
areas and oxygen act as poison for these enzymes and
removed by LHb.

8. NITROGEN FIXATION IN PLANTS
 Biological Nitrogen Fixation of Plants: Biological nitrogen
fixation is carried out by both free-living and symbiotic
bacteria.
 The free-living nitrogen fixing bacteria are: Cyanobacteria,
Azotobacter and Clostridium.
 Symbiotic N2 Fixation: Three different headings , the process
is done
 a)Nodule formation and induction, b) Nitrogen fixation genes
and c) Nitrogenase enzyme activity
 Sometimes the ammonium compounds (NH4) are made
available to the plants by the nitrogen fixers. The best known
nitrogen-fixing symbiotic bacterium is Rhizobium (R.
leguminosarum). This bacterium lives in soil to form root
nodules in plants of the family Fabaceae such as gram, pea,
groundnut, beans, etc. Root nodules are little outgrowths on
roots. When a section of the fresh root nodule is examined, it
looks pinkish in color due to the presence of a pigment called
leghaemoglobin.

9. ROOT NODULES IN LEGUMES
 They mainly occur in the roots of plants of Fabaceae under
nitrogen-limiting conditions. Different species of Rhizobium
can actually modulate in the different legumes as stated below:
 Allorhizobium- A. undicola produces nodules on Neptunia
prostrata, a kind of aquatic legume used as green manure,
 Azorhizobium- A. caulinodans produce nodules on Sesbania
rostata,
 Bradyrhizobium- B.japonicum and B. lupini are in symbiosis
with soyabean
 Mesorhizobium- M.loti nodulates trefoils, M.huakuii nodules
Astragalus, M.ciceri and M. mediterannean nodulate chickpea
 Rhizobium- R.leguminosarum nodulates vetch
 Sinorhizobium- S. meliloti nodulates , alfalfa

10. ROOT NODULES

11. SYMBIOTIC NITROGEN FIXATION
 This pigment is closely related to hemoglobin, the red pigment
of human blood.
 Like hemoglobin, leghaemoglobin is an oxygen scavenger.
The enzyme-nitrogenase which catalyses the fixation of
nitrogen function under anaerobic conditions.
Leghaemoglobin combines with oxygen and protects
nitrogenase.
 Nodule acts as a site for N2 fixation. It contains all the
necessary biochemical compounds, such as nitrogenase and
leghaemoglobin. The enzyme nitrogenase is a Mo-Fe protein
and catalyses the conversion of atmospheric N2 to NH3. This
enzyme is extremely sensitive to oxygen to protect it from
oxygen; nodules contain an oxygen scavenger, called
leghaemoglobin.

12. STEPS INVOLVED IN FIXATION
 Nodule induction-Compatibility of the bacterial surfaces and
root hairs surfaces are important .Lectins of the host plants
generally enables compatibility with bacteria to induce the
nodule formation.
 Nod factors- Proteins encoded by the rhizoidal nodulation
genes-nod, nol and noe genes are involved in the synthesis of
nod factors. Flavonoids activate the bacterial transcriptional
regulator nodD that in turn induces the transcription of the
bacterial nodulation genes involved in the synthesis of nod
factors. Nod factors binding proteins-NFBS1 & NFBS2 have
been identified in this association.
 Infection Nodule mobile bacteria move towards the
rhizosphere of plants and phenolic compounds secreted by
plants. Multigenic nodule formation in legumes are also found

13. SYMBIOTIC NITROGEN FIXATION
 Formation of root nodules in leguminous plant may be of two
types-
 Determinate nodules-found in tropical legumes such as
Glycine max (Soyabean), Phaseolus ( Common bean) , lotus
and Vigna. Detereminate nodules lose meristematic activity
shortly after initiation, thus growth is due to cell expansion,
resulting in mature nodules having spherical in shape.
 Indeterminate nodules-found in temperate legumes like Pisum
(Pea), Medicago (Alfaalfa), Trifolium (Clover) and Vicia
(Vetch). They are called indeterminate because they maintain
an active apical meristem that produces new cells throughout
the life of the nodule. This results in the nodule having a
generally cylindrical shape. The indeterminate nodules exhibit
four major zones – active meristem, the infection zone, the
nitrogen fixation zone and the senescent zone.

14. SYMBIOTIC NITROGEN FIXATION
 When a root hair of a leguminous plant comes in contact with
Rhizobium (a bacterium) it is curled or deformed. Certain
specific chemical substances secreted by bacteria (Rhizobium)
are responsible for curling. Some flavonoids from the roots
trigger the nod factors by the bacteria and morphological and
biochemical changes enables to nodule formation.
 At the site of curling of root hair, rhizobia (bacteria) invade the
root tissue and proliferate within the root hair.
 Some of the bacteria enlarge to become membrane bound
structures called bacteroids. The bacteroids cannot divide, and
therefore, some bacteria remain untransformed, and they allow
infection to spread. An infection thread made up of plasma
membrane is formed, which grows inward from the infected
cell of the plant, that separates the infected tissue from rest of
the plant. Cell division is stimulated in the infected tissue and
more bacteria invade the newly formed tissues.

15. SYMBIOTIC NITROGEN FIXATION
 It is believed that a combination of cytokinin produced by the
invading bacteria and auxin produced by plant cells, promotes
cell division and extension, which leads to molecule
formation. The nodule thus formed, is responsible for direct
vascular connection with the host for exchange of nutrients.
 Nodulation is controlled by a variety of process like external (
heat, acidic soils, drought) and internal ( auto regulation of
nodulation, ).Auto regulation of nodulation controls nodule
number in plants through a complex process. Leaf tissue
senses the early nodulation and restricts further. The leucine
rich repeat (LRR), NARK (Nodule Auto Regulation Receptor
Kinase), HAR1( Hydro nodulation Aberrant root formation),
SUNN ( Super Numeric Nodules) are essential for Auto
regulation of Nodulation ( AON).
 However, N2 fixation, occurs under the control of plant nod
genes and bacterial nod, nif and fix gene cluster.

16. SYMBIOTIC NITROGEN FIXATION
 Induction of plant gene activity-Rhizobium as previously
stated causes the development of nodules by the synthesis of
two plant proteins- nodulin and leg- hemoglobin. Nodulin
causes the enlargement and multiplication of cortical cells and
the degree of the ploidy of the nuclei also increases. It is
followed by LHb formation. The protein content of LHb
contains four exons. Accordingly, their bacterial genes
participate in the synthesis of porphyrin ring .The bacteriods
need iron and molybdenum ions , both are essential for
nitrogenase. Nitrogenase is sensitive towards oxygen. nif
genes are responsible to encode the nitrogenase complex and
other enzymes involved in nitrogen fixation. nif genes are
diverse types-nifH, nifD,nifK, nifT, nifY, nifE, nifN, nifX,
nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifL, nifA, nifB, etc
having diverse function in this regard.

17. SYMBIOTIC NITROGEN FIXATION

18. SYMBIOTIC NITROGEN FIXATION
 The nodule serves as site for N2 fixation. It contains all the
necessary bio-chemicals such as the enzyme complex called
nitrogenase and leghaemoglobin (leguminous hemoglobin).
The nitrogenase has 2 components i.e. Mo-Fe protein
(molybdoferredoxin) and Fe-protein (azoferredoxin).The
nitrogenase catalyzes the conversion of atmosphere di-
nitrogen (N2) to 2NH3. The ammonia is the first stable product
of nitrogen fixation. The nitrogenase is extremely sensitive to
oxygen. To protect these enzymes, nodule contains an oxygen
scavenger called leghaemoglobin (Lb), which is a reddish-pink
pigment. There are two views about location of
leghaemoglobin that is either located outside the peribacteroid
membrane or located in between bacteroids. During nitrogen
fixation, the free dinitrogen first bound to MoFe protein and is
not released until completely reduced to ammonia. The
reduction of di-nitrogen is a stepwise reaction in which many

19. SYMBIOTIC NITROGEN FIXATION
 intermediates are formed to form ammonia (NH3) which is
protonated at physiological pH to form NH4+. In this process
ferredoxin serves as an electron donor to Fe-protein
(nitrogenase reductase) which in turn hydrolyzes ATP and
reduce MoFe protein, the MoFe protein in Turn reduce the
substrate N2. The electrons and ATP are provided by
photosynthesis and respiration of the host cells.
 Assimilation of Ammonia:
 The ammonia produced by nitrogenase is immediately
protonated to form ammonium ion (NH4+). As NH4+ is toxic
to plants, it is rapidly used near the site of generation to
synthesize amino acids. Amino acids synthesis takes place by
three methods: reductive animation, catalytic amination and
transamination.

20. TRANSAMINATION
 Transamination:
 Glutamate or glutamic acid is the main amino acid from which
other amino acids are derived through transamination. The
enzyme aminotransferases (= transaminases) catalyze all such
reactions. Transamination involves transfer of amino group
from one amino acid to the keto group of keto acid.
 Glutamate (amino donor) + Oxaloacetate (amino acceptor) →
Aspartate (amino acid) + 2 oxyglutarate
 In nitrogen fixing plants, the fixed nitrogen is exported in the
form of amides (asparagines and glutamine) and Ureides
(allantoin, allantoic acid and citrulline), from the nodules to
other plant parts via xylem. Amides are formed from two
amino acids, namely glutamic acid and aspartic acid, by
replacing – OH part by another NH2– radicle. Thus, amides
contain more nitrogen than amino acids and are structural part
of most proteins.

21. NITRATE ASSIMILATION
 Nitrate Assimilation: Nitrate cannot be utilized by plants as
such. It is first reduced to ammonia before being incorporated
into organic compounds. Reduction of nitrate occurs in two
steps:
 1. Reduction of nitrate to nitrite: It is carried out by an
inducible enzyme, nitrate reductase. The enzyme is a
molybdoflavoprotein. It requires a reduced coenzyme NADH
or NADPH for its activity which is brought in contact with
nitrate by FAD or FMN.
 Reduction of nitrate: It is carried out by the enzyme nitrite
reductase. The enzyme is a metalloflavoprotein which contains
copper and iron. It occurs inside chloroplast in leaf cells and
leucoplast of other cells. Nitrite reductase require reducing
power. It is NADPH and NADH (NADPH in illuminated
cells).

22. NITRATE ASSIMILATION
 Reduction process also require ferredoxin which occurs in
green tissues of higher plants. 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 assimilation occurs in plant nodules cytosol and
organelles. Plant Glutamine synthetase(GS) and NADH-
dependent glutamine synthetase (NADH_GOGAT)are
responsible for the initial assimilation of ammonia into organic
compounds. In some plants, a nodule specific GS is expressed;
in other , synthesis of a vegetative expressed GS is up-
regulated. After its assimilation into glutamine, the fate of
ammonia largely on the nitrogenous transport compounds used
by the plant host. In many cases, nodules export ammonia as
amides like glutamine and asparagine. A nodule induced

23. SUMMARY
 Aspargine synthetase , also present in the infected tissues
synthesizes aspargine from aspartate, using glutamine as an
amide donor. Amides arte easily generated from citric acid
cycle intermediates can be metabolized in leaves by
transamination and transamination reactions similar to those
used in their synthesis. In another groups. Urtedies allatonin
and allatonic acid compounds produced and by the ureide
catabolism, it is released by leaves .
 Thus, from the above explanation, it is quite clear that the
nitrogen remains in the atmosphere is directly or indirectly
incorporated into the plant in the nitrogenous compounds and
again brought back to the nature by the activities of the
different decomposers as a p[art of the sustainable nitrogen
cycle in the nature.

24. THANKS FOR YOUR JOURNEY
 Acknowledgement:
 1. Google for images
 2. Different web pages for content and enrichment,
 3.Plant Physiology- Mukherji & Ghosh
 Applied Plant Physiology- Arup Kumar Mitra
 A text book of Botany- Hait, Bhattacharya & Ghosh
 Plant Physiology-Devlin
 Disclaimer: This presentation has been prepared for online
free study materials for academic domain without any
financial interest.