Dr. Tushar Wankhede's lecture on nitrogen metabolism

Dr. Tushar Wankhede, MSc, Ph.D (NET,SET,GATE)
Associate Professor in Botany
Shri Shivaji Science College, Amravati
NAAC Reaccredited “A” with CGPA 3.13
College with Potential for Excellence (CPE)

We have decisively changed the carbon cycle,
the nitrogen cycle, and the rate of extinction.”
Prof. Rob Nixon
In his 2011 book, “Slow Violence and the Environmentalism of the Poor,”

 In a living cell, nitrogen is an important constituent of amino acids,
proteins, enzymes, vitamins, alkaloids and some growth hormones.
Therefore, study of nitrogen metabolism is absolutely essential because the
entire life process is dependent on these nitrogen-containing molecules.
1. Conception of Nitrogen Metabolism
 Plants take nitrogen from the soil by absorption through their roots as amino acids,
nitrate ions, nitrite ions, or ammonium ions. Plants get the nitrogen that they need
from the soil, where it has already been fixed by bacteria and archaea.
 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. Ammonium ions are
absorbed by the plant via ammonia transporters.
 Nitrogen cycle is a continuous series of natural processes by which nitrogen passes
successively from air to soil to organisms and back to air or soil involving principally
nitrogen fixation, nitrification, decay, and denitrification.

Sources of Nitrogen
Amino Acids (Organic
Nitrogen) in the Soil
Many soil micro-
organisms make use
of this form of
nitrogen. Sometimes
it may also be taken
by higher plants.
Organic Nitrogenous
Compounds in Bodies of the
Insects
Insectivorous plants fulfill their
nitrogen requirement by
catching the small insects and
digesting them.
Nitrates, Nitrites,
Ammonia in the Soil
(Inorganic Nitrogen)
Among these, the
nitrate is the chief
form of nitrogen
taken up by the
plants from the soil.

 The atmosphere is the source of
elemental nitrogen which cannot be
used directly by plants. The
atmospheric nitrogen is converted to
ammonia, nitrite, nitrate or organic
nitrogen in the soil.
 The death and decay of organic
systems causes cycling of ammonia
from amino acids, purnies and
pyrimidines. Some of these forms
may also be converted to Nitrogen
gas and may be cycled back into the
atmosphere.
 The process by which these forms get
inter converted to maintain a
constant amount of nitrogen in
atmosphere, by physical and
biological processes is called nitrogen
cycle.
Nitrogen Cycle

Nitrogen Cycle
1. Ammonification
 This involves conversion of organic nitrogen to
ammonium ions by microbes present in the
soil. The sources of organic nitrogen in the soil
are animal excreta and dead and decaying
plant and animal remains which are acted
upon by ammonifying saprotrophic bacteria
such as Bacillus ramosus, Bacillus
vulgaris, certain soil fungi and actinomycetes.
2. Nitrification
 C and neutral pH, ammonia gets oxidized to
nitrite (NO2-) and then nitrate (NO3-) by the
process of nitrification. Nitrifying bacteria like
Nitrosomonas convert ammonia to nitrite and
another bacterium called Nitrobacter converts
nitrite to nitrate. In warm moist soils having a
temperature of 30- 35
 Ammonia -> Nitrobacter -> NO2 Nitrate ->
Nitrosomonas -> Nitrite

Nitrogen Cycle
3. Nitrate Assimilation
 Nitrite is converted to Ammonia by the
enzyme nitrite reductase series of steps
requiring a total of eight electrons provided by
reduced NAD and Ferredoxin (Fd). This
reduction of Nitrate of Ammonia and its
incorporation into cellular proteins by aerobic
micro organisms and higher plants is
called nitrate assimilation.
4. Dentrification
 The process of conversion of nitrate and nitrite
into ammonia, nitrogen gas and nitrous oxide
(N2O) is called denitrification. This process
ends in the release of gaseous nitrogen into the
atmosphere and thus completes the nitrogen
cycle. A number of bacteria such as
Pseudomonas denitrificans, Bacillus subtilis
and Thiobacillus dentrificans are involved in
this process.
5. Nitrogen fixation
 Nitrogen fixation refers to the conversion of
elementary dinitrogen (N=N) into organic
form to make it available for plants.

 Nitrogen can be fixed by lightning that converts nitrogen and oxygen
into NO (nitrogen oxides). NO may react with water to make nitrous
acid or nitric acid, which seeps into the soil, where it makes nitrate, which is
of use to plants.
Non-Biological or Physical Nitrogen Fixation
 Nitrogen in the atmosphere is highly stable and nonreactive due to the triple
bond between atoms in the N molecule. Lightning produces enough energy and
heat to break this bond allowing nitrogen atoms to react with oxygen, forming NO.
 These compounds cannot be used by plants, but as this molecule cools, it reacts with
oxygen to form NO. This molecule in turn reacts with water to produce HNO3 (nitric
acid), or its ion NO3− (nitrate), which is usable by plants.

 During the process of evolution some bacterial species have acquired the
capacity to reduce nitrogen to ammonia, a process governed by a set of
genes called the nitrogen fixation (nif) genes.
Biological Nitrogen Fixation
 These species are termed „nitrogen-fixing‟ organisms. May be non-symbiotic
microorganisms that can live independently or certain bacteria living in symbiosis
with higher plants.
 The former group encompasses certain species of heterotrophic bacteria, both
aerobic (Azotobacter sp.) and anaerobic (Clostridium sp.), photosynthetic bacteria
(Rhodospirillum sp.) and several blue-green algae (Cyanophyta)
 The symbiotic system consists of bacteria of the genus Rhizobium together with
many members of the family Leguminosae, such as peas, beans, clovers, soybean
etc., to form an important nitrogen-fixing cooperative. An essential feature of the
symbiotic fixation is the development of nodules on the roots of the plants.
 The symbiotic system consists of bacteria of the genus Rhizobium together with
many members of the family Leguminosae, such as peas, beans, clovers, soybean
etc., to form an important nitrogen-fixing cooperative. An essential feature of the
symbiotic fixation is the development of nodules on the roots of the plants.

Nitrogen fixation requires
 (i) The molecular nitrogen
 (ii) A strong reducing power to reduce nitrogen like reduced FAD (Flavin adenine
dinucleotide) and reduced NAD (Nicotinamide Adenine Dinucleotide)
 (iii) A source of energy (ATP) to transfer hydrogen atoms from NADH2 oFADH2 to
dinitrogen and
 (iv) Enzyme nitrogenase (v) compound for trapping the ammonia formed since it is
toxic to cells.
 The reducing agent (NADH2 and FADH2) and ATP are provided by photosynthesis
and respiration. The overall biochemical process involves stepwise reduction of
nitrogen to ammonia.
 The enzyme nitrogenase is a Mo-Fe containing protein and binds with molecule of
nitrogen (N2) at its binding site. This molecule of nitrogen is then acted upon by
hydrogen (from the reduced coenzymes) and reduced in a stepwise manner. It first
produces diamide (N2H2) then hydrazime (N2H4) and finally ammonia (2NH3).
 NH3 is not liberated by the nitrogen fixers. It is toxic to the cells and therefore these
fixers combine NH3 with organic acids in the cell and form amino acids. The general
equation for nitrogen fixation may be described as follows:
 N2+ 16ATP + 8H+ + 8e- -> 2NH3+ 16ADP + 16 Pi + H2
Biological Nitrogen Fixation

 In legumes, nitrogen fixation occurs in
specialized bodies called root nodules. The
nodules develop due to interaction
between the bacteria Rhizobium and the
legume roots The biochemical steps for
nitrogen fixation are same.
LEGHEMOGLOBIN.
 Leghemoglobin is considered to lower down the partial pressure of oxygen and
helps in nitrogen fixation. However, this function is specific for legumes only
because free living microbes do not possess nitrogen fixing leghemoglobin.
 Moreover, it has also not been found in cyanobacterial symbiosis with other plants,
which fix N2 under aerobic condition
 However, legume nodules possess special protein called LEGHEMOGLOBIN. The
synthesis of leghemoglobin is the result of symbiosis because neither bacteria alone
nor legume plant alone possess the protein. Recently it has been shown that a
number of host genes are involved to achieve this. In addition to leghemoglobin, a
group of proteins called nodulins are also synthesized which help in establishing
symbiosis and maintaining nodule functioning.

 The first step conversion of nitrate to nitrite is catalyzed by an enzyme called
nitrate reductase. This enzyme has several other important constituents including
FAD, cytochrome, NADPH or NADH and molybdenum.
Nitrate Reductase
 The enzyme nitrate reductase is
continuously synthesized and degraded
and inducible i.e. increase in nitrate
concentration in the cytosol induces more
of nitrate redutase to be synthesized.
 In the second step the nitrite so formed is further reduced to ammonia and this is
catalyzed by the enzyme nitrite reductase. Nitrite present in the cytosol is
transported into chloroplast or plastids where it is reduced to ammonia.
NO2_ + 3NADPH + 3H+ ----------------- NH3 + 3NADP +
Nitrite reductase
 The enzyme nitrite reductase is able to accept electrons from sources such as NADH,
NADPH or FADH2. Besides, reduced ferredoxin has also been shown to provide
electrons to nitrite reductase for reducing nitrite to ammonia. Ammonia so formed
has to be utilized quickly by plants to avoid toxicity.

 Growth is the manifestation of life. All organisms, the simplest as well as
the most intricate, are slowly changing the whole time they are alive. They
transform material into more of themselves. Growth involves an
irreversible increase in size which is usually, but not necessarily,
accompanied by an increase in dry weight. The basic process of growth is
the production of new protoplasm, which is clearly evident in the regions of
active cell division.
Plant Growth and Phases of growth
 Differentiation:
Differentiation can be
recognized at cell level, tissue
level, organ level, and at the
level of an organism. It
becomes more obvious at the
level of organ and organism..
 Development:
Development implies a whole
sequence of qualitative
structural changes that a plant
undergoes from the zygote
stage to its death.

 Phases of Plant Growth:
 As a plant is made up of
cells, its growth will be the
sum total of the growth of
its cells.
 The growth of cells involves
three main phases:
 The phase of cell division
(Zone of Cell formative
phase), Elongation.
 Cell enlargement and cell
differentiation.
 Cell Differentiation or Cell
Maturation.
Plant Growth and Phases of growth

 The vegetative growth of most plants in general shows three phases,
starting slowly, becoming gradually faster and finally slowing again. These
three phases, which are together known as “grand period of growth”, cover
the whole of the vegetative history of an annual plant.
The Grand Period of Growth:
 The sigmoid curve shows following
three distinct phases:
(1) The lag phase or initial phase:bIt
represents initial stages of growth. The
rate of growth is naturally slow during
this phase.
(2) Log phase or exponential phase:
It is the period of maximum and rapid
growth. Physiological activities of cells
are at their maximum.
(3) Adult phase or stationary phase:
This phase is characterized by a
decreasing growth rate. The plant
reaches maturity, hence the
physiological activity of cells also slows
down and plant begins to senesce.

 Plant growth hormones are organic compounds which are either produced
naturally within the plants or are synthesized in laboratories. They
profoundly control and modify the physiological processes like the growth,
development, and movement of plants.
Plant Growth Hormones or Regulators or Elicitors
 Auxins, Gibberellins, and Cytokinins are grouped into Plant growth promoters
while Abscisic acid and Ethylene (Both) are grouped into Plant growth inhibitors.
 Characteristics of Plant Growth Regulators
 These are simple organic molecules having several chemical compositions. They are
also described as phytohormones, plant growth substances, or plant hormones.
 They can accelerate as well as retard the rate of growth in plants.
 Plants hormones or plant growth regulators exhibit the following characteristics:
 Differentiation and elongation of cells.
 Formation of leaves, flowers, and stems.
 Wilting of leaves.
 Ripening of fruit.
 Seed dormancy, etc.

 Discovery: Auxins were the first growth hormone discovered due to the
observations of Charles Darwin. The Darwins observed phenomenon
„phototropism‟.
 The isolation of the first auxin by F. W. Went from the coleoptile tip of oat
seedlings.
Plant Growth Hormone : AUXINS
 Plants produce natural auxins such as Indole-3-acetic acid (IAA) and Indole butyric
acid (IBA). Naphthalene acetic acid (NAA) and 2, 4-dichlorophenoxyacetic (2, 4-D) are
examples of synthetic auxins.
Physiological Effects
 Promote flowering in plants like pineapple.
 Help to initiate rooting in stem cuttings.
 Prevent dropping of fruits and leaves too early.
 Promote natural detachment (abscission) of older leaves and fruits.
 Control xylem differentiation and help in cell division.
 Applications
 Used for plant propagation.
 To induce parthenocarpy i.e. the production of fruit without prior fertilization.
 Used by gardeners to keep lawns weed-free.
 The phenomenon of Apical Dominance„.

 Discovery : It is the component responsible for the „bakane‟ disease of rice
seedlings by fungal pathogen Gibberella fujikuroi. E. Kurosawa treated
studied the fungus and reported the appearance of disease symptoms.
Finally, the active substance causing the disease was identified as
Gibberellic acid.
Plant Growth Hormone : GIBBERELLINS
 Types : There exist more than 100 gibberellins obtained from a variety of
organisms from fungi to higher plants. They are all acidic and are denoted as
follows – GA1, GA2, GA3 etc. GA3 (Gibberellic acid) is the most noteworthy
 Physiological Effects :
 Increase the axis length in plants such as grape stalks.
 Delay senescence (i.e. ageing) in fruits. As a result,
their market period is extended.
 Help fruits like apples to elongate and improve their shape.
 Applications
 The brewing industry uses GA3 to speed the malting process.
 Spraying gibberellins increase sugarcane yield by lengthening the stem.
 Used to hasten the maturity period in young conifers and promote early seed
production.
 Help to promote bolting (i.e. sudden growth of a plant just before flowering) in
cabbages and beet.

 Discovery
F. Skoog and his co-workers observed a mass of cells called „callus‟ in
tobacco plants. These cells proliferated only when the nutrient medium
contained auxins along with yeast extract or extracts of vascular tissue.
Skoog and Miller later identified the active substance responsible for
proliferation and called it kinetin.
Plant Growth Hormone : CYTOKININS
 Types
Cytokinins were discovered as kinetin. Kinetin does not occur naturally but
scientists later discovered several natural (example – zeatin) and synthetic
cytokinins. Natural cytokinins exist in root apices and developing shoot buds –
areas where rapid cell division takes place.
 Help in the formation of new leaves and chloroplast.
 Promote lateral shoot growth and adventitious
shoot formation.
 Help overcome apical dominance.
 Promote nutrient mobilisation which in turn helps delay leaf senescence.
 Important sources of indiction of callus in Tissue culture
 Cell division and cell enlargement
 Apical dominance
 Delay senescence

 Discovery
F.T. Addicott and his associates discovered abscisic acid in the early 1960s in
the process of studying abscission in cotton ( commercially important for
mechanization of cotton picking') . It was also being studied by other plant
physiologists at the same time for it's property of controlling abscission of
flowers and in the initiation of dormancy of wood production.
Plant Growth Inhibitor: ABSCISIC ACID
 Types
Three independent researchers reported the purification and characterization of
three different inhibitors – Inhibitor B, Abscission II and Dormin. Later, it was found
that all three inhibitors were chemically identical and were, therefore, together
were given the name abscisic acid. Abscisic acid mostly acts as an antagonist to
Gibberellic acid.
 Regulate abscission and dormancy.
 Inhibit plant growth, metabolism and seed germination.
 Stimulates closure of stomata in the epidermis.
 It increases the tolerance of plants to different kinds of stress and is, therefore,
called „stress hormone‟.
 Important for seed development and maturation.
 It induces dormancy in seeds and helps them withstand desiccation and other
unfavourable growth factors.
 Induce enescence and Abscission and Geotropism

 Discovery
Doubt discovered that ethylene a gaseous type of thingg stimulated
abscission in 1917 (Doubt, 1917). It wasn't until 1934 that Gane reported that
plants synthesize ethylene (Gane, 1934). In 1935, Crocker proposed
that ethylene was the plant hormone responsible for fruit ripening as well
as inhibition of vegetative tissues (Crocker, 1935)
A Gaseous Hormone: ETHYLENE
 Affects horizontal growth of seedlings and swelling
of the axis in dicot seedlings.
 Promotes abscission and senescence, especially of leaves and flowers.
 Increases root growth and root hair formation, therefore helping plants to increase
their absorption surface area.
 Application
 Ethylene regulates many physiological processes and is, therefore, widely used in
agriculture. The most commonly used source of ethylene is Ethephon. Plants can
easily absorb and transport an aqueous solution of ethephon and release ethylene
slowly.
 Used to break seed and bud dormancy and initiate germination in peanut seeds.
 To initiate flowering and synchronising fruit-set in pineapples.
 Ethephon hastens fruit ripening in apples and tomatoes and increases yield by
promoting female flowering in cucumbers. It also accelerates abscission in cherry,

 Senescence is a normal energy dependent developmental process which is
controlled by plants own genetic programme and the death of the plant or
plant part con-sequent to senescence or programmed cell death (PCD).
Physiology of Senescence
1. Overall Senescence: This type of
senescence occurs in annuals
where whole of the plant is
affected and dies.
2. Top Senescence: This is represented
by perennial herbs where
senescence occurs only in the
above ground parts, but, the
root system is viable.
3. Deciduous Senescence: This type of senescence is less drastic and takes place in woody
deciduous plants. Here senescence occurs in all the leaves simultaneously but the
bulk of the stem and root system remains alive.
4. Progressive Senescence:: This is characterized by gradual progression of senescence
and death of leaves from the base upwards as the plant grows. (The senescence of the
entire plant after a single reproductive cycle is also known as monocarpic senescence)
Senescence can best be studied in, cotyle-dons, sepals, petals etc. or isolated chloroplasts.

 Senescing cells and tissues are metabolically very active and an ordered
series of cytological and biochemical events occur during senescence. It is
characterized by increased respiration, declining photosynthesis and an
orderly disintegration of macromolecules.
 At the cellular level, chloroplasts are disintegrated, nuclei remain
structurally and functionally intact but other cell organelles and bio-
membranes also gradually deteriorate.
 Expression of senescence down-regulated genes (SDGs) decreases. Such
genes en-code proteins in photosynthesis and other biosynthetic processes.
Concentration of growth promoting hormones especially cytokinins decline.
 Expression of senescence associated genes (SAGs) increases. Such genes
encode hydrolytic enzymes such as proteases, ribonucleases and lipases as
well as enzymes involved in biosynthesis of deteriorative hormones such as
abscisic acid (ABA) and ethylene.
 Brilliant Colours are developed in leaves of many plants during senescence
due to degradation of chlorophylls, resulting in unmasking of more stable
carotenoid pigments. Towards the end of senescence, the cells and tissues
also lose respiratory control.
Physiology of Senescence

 The phenomenon of the separation of leaves from the stem takes place in a
particular region of the plant, known as abscission region or abscission
zone. The phenomenon as a whole known as abscission of leaves and the
separating leaf may be said to abscised.
Physiology of Abscission:
 During abscission, leaves are separated from
the stem without causing any injury to the
living tissues in stem and the newly exposed
surface is also protected from desiccation
and infection.
 In leaves, the abscission zone occurs within
the petiole or at its base. In compound leaves
the abscission zones occur in the petiole of
the leaf as a whole and at the base of
individual leaflets
 The process of the separation of the leaf commonly starts from the peripheral
region of the petiole and proceeds towards the middle of the petiole. The
separation layer remains in continuation through the parenchyma cells in the
vascular bundles, whereas the xylem and phloem elements and other non-living
cells have been broken mechanically.

 Just before the actual leaf fall, the tyloses and gums chiefly block the
primary conducting cells of the vascular bundles, but sufficient conduction
is maintained through the secondary elements which keep the leaf fresh
turgid unless and until its separation is being completed.
Physiology of Abscission:
 Shortly before the abscise of the leaf, the outer walls and the middle lamella of the
cells become gelatinized and in the end prior to leaf fall they break down and
dissolve. Ultimately the leaf is only supported by the vascular elements which
break very soon by the wind and the weight of the leaf itself, thus separating the
leaf from the stem.
 In wet weather, because of the addition of the water to the leaves, and the
hydrolysis of the gelatinous cell walls, the leaf fall is accelerated to some extent.
Shortly after the leaf fall the protective layers develop on the exposed surface. The
protective layers may be of the both primary and secondary origin. Sometimes they
are only of secondary origin. The secondary protective layer is typical periderm. At
the region of separation, a leaf scar is formed.
 The scar is formed because of the deposition of the substances, which protect the
new surface from injuries, infection and loss of water. These substances are found
beneath the separation layer in the cells and referred as suberin and lignin.

“Life is not like water. Things in life don't necessarily flow
over the shortest possible route.”
― Haruki Murakami, 1Q84

Dr. Tushar Wankhede's lecture on nitrogen metabolism

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