Assimilation and

fixation of
nitrogen
 Plants are:
• Capable of making all necessary organic
compounds from inorganic compounds and
elements in the environment (autotrophic)

• Required to compete with other organisms

for these nutrients

• Required to employ complex energetic

pathways to convert macronutrients to
useable forms
 Nitrogen in the environment
• Many biochemical compounds present in plant cells
contain nitrogen
– Nucleoside phosphates
– Amino acids
• These form the building blocks of nucleic acids and
protein respectively

• Only carbon, hydrogen, and oxygen are nor

abundant in plants than nitrogen
 Nitrogen in the environment
• Present in many forms • This occurs naturally by:-
• 78% of atmosphere is N2 Lightning:
– 8%: splits H2O: the free
– Most of this is NOT O and H attack N2 – forms
available to living HNO3 (nitric acid) which
organisms fall to ground with rain
• Getting N2 for the
atmosphere requires • Photochemical reactions:
breaking the triple bond – 2%: photochemical
between N2 gas to produce: reactions between NO gas
• Ammonia (NH3) and O3 to give HNO3
• Nitrate (NO3-)
• So, N2 has to be fixed • Nitrogen fixation:
from the atmosphere so – 90%: biological – bacteria
plants can use it fix N2 to ammonium (NH4+)
Nitrogen cycles through the atmosphere as it changes from a gaseous
form to reduced ions before being incorporated into organic compounds in
living .organisms. Some of the steps involved in the nitrogen cycle are
shown.
Nitrogen in the environment
Nitrogen in the environment
• Once fixed in ammonium or nitrate :-
– N2 enters biochemical cycle

– Passes through several organic or inorganic forms

before it returns to molecular nitrogen

– The ammonium (NH4+) and nitrate (NO3-) ions

generated via fixation are the object of fierce
competition between plants and microorganisms

– Plants have developed ways to get these from the

soil as fast as possible
 Root uptake soon depletes
nutrients near the roots
• Formation of a nutrient
depletion zone in the
region of the soil near
the plant root
– Forms when rate of
nutrient uptake exceeds
rate of replacement in
soil by diffusion in the
water column
– Root associations with
Mycorrhizal fungi help
the plant overcome this
problem
 Mycorrhizal associations
• Not unusual
– 83% of dicots, 79% of
monocots and all gymnosperms
• Ectotrophic Mycorrhizal fungi
– Form a thick sheath around
root. Some mycelium
penetrates the cortex cells of
the root
– Root cortex cells are not
penetrated, surrounded by a
zone of hyphae called Hartig
net
– The capacity of the root
system to absorb nutrients
improved by this association –
the fungal hyphae are finer
than root hairs and can reach
beyond nutrient-depleted
zones in the soil near the root
 Mycorrhizal associations
• Vesicular arbuscular mycorrhizal
fungi
– Hyphae grow in dense arrangement
, both within the root itself and
extending out from the root into
the soil
– After entering root, either by root
hair or through epidermis hyphae
move through regions between cells
and penetrate individual cortex
cells.
– Within cells form oval structures –
vesicles – and branched structures
– arbuscules (site of nutrient
transfer)
– P, Cu, & Zn absorption improved by
hyphae reaching beyond the
nutrient-depleted zones in the
soil near the root
 Nutrients move from fungi to
root cells
• Ectotrophic Mycorrhizal
– Occurs by simple diffusion from the hyphae in
the hartig net to the root cells

• Vesicular arbuscular mycorrhizal fungi

– Occurs by simple diffusion from the arbuscules
to the root cells
– Also, as arbuscules are degenerating as new ones
are forming, the nutrients may be released
directly into the host cell
 Stored ammonium can be toxic
• Plants can store high levels of
nitrate or translocate it via
the phloem without any
effect.

• However, high levels of

ammonium are toxic
– Dissipates transmembrane
proton gradients required
for both photosynthetic
and respiratory electron
transport
– AND movement of
metabolites to vacuoles.
 Stored ammonium can be toxic
• At high pH in stroma, matrix
or cytoplasm:
– Ammonium reacts with OH- to
produce NH3.

• NH3 is membrane permeable

and diffuses freely across a
membrane down a concentration
gradient

• At low pH in intermembrane
space, lumen, or vacuole:
– NH3 reacts with H+ to form
ammonium
 Remember: Nitrogen – the most
important mineral nutrient in the
soil
• Nitrogen is frequently limiting in in
terrestrial systems terrestrial systems

• Microbial activity is continually converting N

to lower energy forms

• Conversion to organic form requires raising

N to higher energy levels
 Nitrate
Assimilation
 Deficiency Symptoms - N
• General chlorosis.

• Chlorosis progresses
from light green to
yellow.

• Entire plant becomes

yellow under prolonged
stress.

• Growth is immediately
restricted and plants
soon become spindly
and drop older leaves. http://plantsci.sdstate.edu/woodardh/soilfert/Nutrient_Defi
ciency_Pages/soy_def/SOY-N1.JPG
 Nitrogen assimilation
NO3 NO2 NH4+ amino acids
nitrate nitrite ammonium

• Requires large input of energy

• Forms toxic intermediates
• Mediated by specialized enzymes that are
closely regulated are closely regulated
• Doesn’t have to start at the beginning
 Nitrogen assimilation
• Plants assimilate most of the nitrate absorbed by
their roots into organic nitrogen compounds.

• The first step of this process is the reduction of

nitrate to nitrite in the cytosol by the enzyme
nitrate reductase.
 Nitrogen assimilation
NAD(P)H induces NADH or NADPH
– The most common form of nitrate reductase uses only NADH as an
electron donor

– The nitrate reductases of higher plants are composed of two

identical sub-units, each containing three prosthetic groups
• FAD—flavin adenine dinucleotide
• Heme
• Molybdenum—organic molecule called pterion
 Nitrate Assimilation
• Nitrate reductase is the main molybdenum
containing protein in vegetative tissues
• Nitrate levels, light intensity, and concentration of
carbohydrates all influence the activity of nitrate
reductases at the transcription and translation
levels
– These factors stimulate a protein, phosphatase, that
dephosphorylates several serine residues on the nitrate
reductase protein thereby activating the enzyme

– This dephosphorylation/phosphorylation cycle provides

more rapid control over this enzyme than
degredation/synthesis of new enzyme would achieve
– ( minutes versus hours)
Nitrite Reductase Converts Nitrite
to Ammonium
• Nitrite (NO2-)is highly reactive

• Plant cells immediately transport the nitrite

generated by nitrite reduction from the cytosol
into chloroplasts in leaves and plastids in roots
– In these organelles, nitrite reductase reduces nitrite
to ammonium
Nitrite Reductase Converts Nitrite
to Ammonium
• Chloroplast and root plastids contain different
forms of the enzyme, but both forms consist of a
single polypeptide containing an iron sulfur cluster
and a specialized heme group
– The heme does redox reactions and electron flow, just
like the reaction sites of chlorophyll
 Nitrite Reductase Converts
Nitrite to Ammonium
• Nitrite is highly reactive
• Plant cells immediately transport the nitrite
generated by nitrite reduction from the cytosol
into chloroplasts in leaves and plastids in roots
– In these organelles, nitrite reductase reduces nitrite
to ammonium
• Chloroplast and root plastids contain different
forms of the enzyme, but both forms consist of a
single polypeptide containing an iron sulfur cluster
and a specialized heme group
– The heme does redox reactions and electron flow, just
like the reaction sites of chlorophyll
 Plants assimilate nitrate in both
roots and shoots
• In many plants, when the roots receive small
amounts of nitrate, this nitrate is reduced
primarily in the roots

• As nitrate supply increases, a greater proportion of

the absorbed nitrate is translocated to the shoot
and assimilated there

• Generally, species native to temperate rely more

heavily on nitrate assimilation by the roots than do
species of tropical or subtropical origins
 Ammonium Assimilation
• Plants cells avoid ammonium toxicity by rapidly
converting the ammonium generated from nitrate
assimilation or photorespiration into amino acids
• This requires the action of two enzymes
– Glutamine synthetase – combines ammonium with
glutamate to form glutamine
– Glutamate synthase – stimulated by elevated levels of
glutamine synthetase
– Transfers the amino group of glutamine to an
intermediate yielding two molecules of glutamate
 Transamination Reaction
Transfer Nitrogen
• Once assimilated into glutamine and glutamate,
nitrogen is incorporated into other amino acids via
transamination reactions
• The enzymes involved in these reactions are known
as aminotransferases
– Best known — aspartate aminotransferase
• The amino group of glutamate is transferred to the
carboxyl atom of aspartate
• Aspartate is the amino acid which shuttles reducing
agents from the mitochondrion and chloroplast into
the cytosol and in the transport of carbon from
mesophyll to bundle sheath of C4 carbon fixation
• All this requires vitamin B6 to act as a cofactor
 Biological nitrogen Fixation
• This accounts for most of the fixation of
atmospheric N2 into ammonium
– Represents the key entry point of molecular nitrogen
into the biogeochemical cycle of nitrogen
• Free living and symbiotic bacteria are responsible
for converting atmospheric nitrogen into ammonium
• Most of these are free living in the soil, a few form
symbiotic associations with higher plants
– The prokaryote directly provides the host plant with
nitrogen in exchange for other nutrients and
carbohydrates
• The most common association is between members
of the plant family leguminosae and bacteria of the
genera Azorhizobium
 Nitrogen Fixation Requires
Anaerobic Conditions
• As oxygen irreversibly inactivates the
nitrogenase enzymes involved in nitrogen
fixation, nitrogen must be fixed under
anaerobic conditions

– Therefore each of the nitrogen-fixing organisms

either functions under natural anaerobic
conditions or can create an internal anaerobic
environment in the presence of oxygen
 Nitrogen Fixation Requires
Anaerobic Conditions
• In cyanobacteria, anaerobic conditions are created
in specialized cells called heterocysts
– These are thick-walled cells which lack photosystem
II—the oxygen producing photosystem of chloroplasts
• Cyanobacteria can fix nitrogen under anarobic
conditions such as those that occur in flooded
fields
– In Asian countries, nitrogen fixing cyanobacteria of both
the heterocyst and non-heterocyst types are the major
means of maintaining an adequate nitrogen supply in rice
fields
• They fix nitrogen when the fields are flooded, and
die as the fields dry, releasing the fixed nitrogen
into the soil
 Symbiotic Nitrogen Fixation
Occurs in Specialized
Structures
• Symbiotic nitrogen-fixing prokaryotes dwell within
nodules
– Special organs of the plant host that enclose the
nitrogen-fixing bacteria
• Grasses can also develop symbiotic relationships
with nitrogen-fixing organisms, but these
associations do not lead to the formation of root
nodules
– Nitrogen-fixing bacteria seem to colonize plant tissues
or anchor to the root surface, mainly around the
elongation zone and the root hairs
• Known as actinorhizal plants
 Symbiotic Nitrogen Fixation
Occurs in Specialized
Structures
• Both legumes and actinorhizal plants regulated gas
permeability in their root nodules
– Maintaining a level of oxygen within the nodule that can
support cellular respiration for the bacteria, but still
sufficiently low to avoid inactivation of the nitrogenase
Nodules
Contain an oxygen binding heme
protein—leghemoglobin
Leghemoglobin produces a pink color
Helps transport oxygen to the
respiring symbiotic bacteria cells in
a manner analogous to hemoglobin
transporting oxygen to respiring
tissues in animals
 Establishing Symbiosis Requires
a Change of Signals
• Legumes seedlings germinate without any
association to rhizobia
– Under nitrogen limiting conditions, the plant and
the bacteria seek each other out by an elaborate
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
 Establishing Symbiosis Requires
a Change of Signals
• Common nod genes
– nodA, nodB, and nodC found in all rhizobial
strains

• Host specific non genes

– nodP, nodQ, nodH, nodE, and nodF differ
among rhizobial species and determine the host
range

• The first stage of the association is the migration

of the bacteria through the soil towards the host
plant
 Nod Factors produces by bacteria
act as signals for symbiosis
• nodD is constitutively
expressed—has a role in the
activation of all other nod
genes by signaling the
formation of nod factors

• Lipochitin oligosacharides
with a chitin-b-1,4 linked N-
acetyl-D-glucosamine

• nodA, nodB, and nodC encode

for the formation of this
structure
 Nodule formation involves
several phytohormones
• During root nodule formation, two process occur simultaneously
• Infection and Nodule Organogenesis
– (A) Rhizobia attach to the root hairs and release nod factors that
produce a pronounced curling of the root hair cell

– (B) Rhizobia get caught and curl, degrade the root hair cell wall
allowing the bacterial cells direct access to the outer surface of the
plant plasma membrane
 Nodule formation involves
several phytohormones
• (C) Then the infection thread forms
– Formed from Golgi depositing material at the tip at the site of
infection. Local degradation of root hair cell wall also occurs

• (D) Infection thread reaches the end of the cell, and thread plasma
membrane fuses with plasma membrane of root hair cell
– Then bacterial cells are released into the fused plasma
membranes
 Nodule formation involves
several phytohormones
• (E) Rhizobia are released into the apoplast and enter the middle lamella,
– This leads to the formation of a new infection thread, which forms
an open channel with the first

• (F) Infection thread expands and branches until it reaches target cells
– Vesicles composed of plant membrane enclose bacterial cells and
they are released into the cytoplasm
 Nodule formation involves several
phytohormones
• At first bacteria continue to grow with vesicles
expanding by fusing with smaller vesicles
• Following an as yet determined chemical signal from
the plant, bacteria stop dividing and differentiate
– Forms nitrogen-fixing organelles called bacteroids
• The nodule itself develops a vascular system
– To exchange fixed nitrogen for nutrients from the
plant
• And a layer of cells to exclude O2 from the rood
nodule interior
 The nitrogenase enzyme complex
fixes N2
• Biological nitrogen fixation produces ammonium
(NH3) from molecular nitrogen.

• N2 + 8e- + 8H+ + 16 ATP 2NH3 + H2+ 16 ADP +

16 Pi

• Note that the reduction of N2 to 2NH3 is a six-

electron transfer, and is coupled to the reduction
of two protons to evolve H2

• This reaction is catalyzed by nitrogenase enzyme

complex
 The nitrogenase enzyme complex
fixes N2
• Can be separated into two components
– The Fe protein
– The MoFe protein
• Neither of which has catalytic activity by itself
 The nitrogenase enzyme complex
fixes N2
• 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
The MoFe protein can reduce many
substances
• The MoFe protein can reduce many substrates
• Although under natural conditions the MoFe only
reacts with N2 and H+.
 Summary
• Nutrient assimilation is the process by which
nutrients acquired by plants are incorporated into
the carbon constituents necessary for growth and
development.

• For Nitrogen:
• Assimilation is but one in a series of steps that
constitute the nitrogen cycle.
• The principal sources of nitrogen available to plants
are nitrate (NO3-) and ammonia (NH4+).
• Nitrate absorbed by roots is assimilated in either
shoots or roots
– depending on nitrate availability and plant species
 Summary
• In nitrate assimilation, nitrate (NO3-) is reduced to nitrite
(NO2-) in the cytosol via the enzyme nitrate reductase.
• Then nitrite is reduced to ammonium (NH4+) in roots by
nitrite reductase.

• Ammonium (NH4+) from either root absorption or generated

through nitrate assimilation or photorespiration is
converted glutamine or glutamate through the sequential
actions of glutamine synthase and glutamate synthase.

• Once assimilated into either glutamine or glutamate,

nitrogen mat be transferred to many other organic
compounds
– Via transaminatation reactions
 Summary
• Many plants form a symbiotic relationship with
nitrogen fixing bacteria that contain an enzyme
complex, nitrogenase, that can reduce atmospheric
nitrogen to ammonia.

• Legumes and actinorhizal plants form associations

with rhizobia.

• These associations result from a finely tuned

interaction between the bacteria and the host
plant
– Involves the recognition of specific signals between
the symbiotic bacteria and the host plant
Any Questions?