Unit 7 Nitrogen Fixation

UNIT 7
NITROGEN FIXATION

Structure
7.1 Introduction Establishing Legume –
Rhizobium Symbiosis
Expected Learning Outcomes
Nature of Symbiotic Relationship
7.2 Nitrogen Fixation
7.5 Nitrate Assimilation
Non biological Nitrogen Fixation
Introduction
Biological Nitrogen Fixation
(BNF) Uptake and Transport of Nitrate

7.3 Biochemistry of Nitrogen Nitrate and Nitrite Reductases

Fixation
Regulation of Nitrate
Nitrogenase Assimilation

Unique features of BNF 7.6 Summary

Reduction of Molecular Nitrogen 7.7 Terminal Questions

Protection of Nitrogenase 7.8 Answers

7.4 Symbiotic Nitrogen Fixation 7.9 Further Readings
(SNF)

7.1 INTRODUCTION
Nitrogen is the fourth most abundant element after carbon, hydrogen and
oxygen in plants. It occurs in both inorganic and organic forms. Nitrogen is a
constituent of proteins, nucleic acids, alkaloids, chlorophyll, vitamins,
glycosides and many other primary and secondary metabolites. Needless to
say, protoplasm the physical basis of life consists largely of proteins.

In this unit you shall learn in greater details two aspects of the nitrogen cycle-
biological nitrogen fixation (BNF) and nitrate assimilation. Plants obtain both
ammonia and / nitrates from the soil, made available by microbial activity, non
biological N2 fixation and fertilisers. Before embarking on this and the next unit
you are advised to revise Unit- 1 of BBCCT-113 paper on Metabolism of
Amino acid and Nucleotides for an introduction to both these topics.
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Expected Learning Outcomes

After studying this unit you should be able to:

 explain non biological and biological nitrogen fixation (BNF);

 describe the structure of nitrogenase and associated metal

cofactors;

 indicate the reactions catalysed by nitrogenase;

 describe SNF (legume- Rhizobium) and interdependence of the

two partners;

 explain the role of assimilatory nitrate reductase (NR) and nitrite

reductase (NiR) in plants; and

 indicate the significance of organelle specific and tissue specific

distribution of NR and NiR.

7.2 NITROGEN FIXATION

Nitrogen fixation is the reduction of atmospheric nitrogen to ammonia.
Although nitrogen (as N2) is the most abundant element in the atmosphere but
it is difficult to break the stable triple bond linking the two nitrogen’s thereby
making it directly inaccessible to most organisms. The reduction of nitrogen is
brought about by a combination of biological and non biological processes.
The former includes industrial nitrogen fixation (Haber-Bosch reaction),
lightening discharges and combustion of fossil fuels while the latter is an
enzymatic reduction, catalysed by nitrogenase. We shall now learn about non
biological ways followed by BNF in detail.

7.2.1 Non - Biological Nitrogen Fixation

About 8% of the total nitrogen fixed on this planet is through non-biological
means especially during rains following lightening discharges. As a result,
atmospheric nitrogen gets trapped into various nitrogenous compounds which
are eventually added to the soil. For example, nitric oxide is formed during
lightening from nitrogen and oxygen due to high temperatures in the vicinity of
a discharge.

Lightening
N2 + O2 2NO (nitric oxide)

Nitric oxide can be readily converted to acidic nitrogen dioxide by further

reaction with oxygen.

2NO + O2 2NO2 (Nitrogen dioxide)

Similar reactions take place during combustion of coal and of gasoline in

automobiles. They contribute almost equally to the formation of nitrogen
oxides and to air pollution, affecting plant life. This is due to increasing
126 industrialisation.
Unit 7 Nitrogen Fixation

Nitrogen oxides (Nox) readily dissolve in water to form nitric and nitrous acids,
accounting for the acidic nature of rain water (acid rain). These acids are
sources of nitrate and nitrites in the soil for plants and microbes.

2NO2 + H2O HNO2 + HNO3

Calcium oxide (CaO) from the rocks can also contribute to this process, as
HNO3 can react with CaO to form Ca (NO3)2.

CaO + 2HNO3 Ca (NO3)2 + H2O

About 2% of the total nitrogen fixed comes from photochemical reactions in

the atmosphere.

NO + O3 HNO3

The industrial process (Haber-Bosch) for ammonia production from nitrogen

(from air) and hydrogen (from natural gas) requires a catalyst, high
temperature and pressure, unlike biological nitrogen fixation (normal
temperatures and pressure). The reaction is reversible and exothermic and
reduces an estimated 80x1012 g of N2 into NH3 per year. Yet all the above-
mentioned processes fall way short of providing ammonia to meet the
agricultural demand of nitrogenous fertilizers.
500°C
N2 + 3H2 2NH3
200 atm

7.2.2 Biological Nitrogen Fixation (BNF)

Biological nitrogen fixation is by far the single most important means to meet
the nitrogen requirements of plants. Nitrogen fixing organisms are called
diazotrophs (diazo=molecular nitrogen; trophs=eaters). These ‘gifted’ species
include free living and symbiotic nitrogen fixers (mostly legumes). The ability to
fix nitrogen is restricted to some prokaryotes. Table 7.1 summarises examples
of diazotrophs and their unique attributes. We shall learn about nitrogenase in
the following section.

Table 7.1: Examples of free living and symbiotic diazotrophs

(a) Free living diazotrophs

Oxygen Examples Comments

dependence

Obligate anaerobes / Clostridium pasteurianum First organism from

heterotrophs which active N2ase
extract was obtained.

(a) Azotobacter vinelandii; (a) γ-proteobacteria /

heterotrophs / produces
Obligate aerobes / alginate
heterotrophs (b) β-proteobacteria /
(b) Derxia gummosa
secretes gummy
polysaccharides
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Block 3 Nitrogen Metabolism and Plant Growth Hormones

Facultative Klebsiella pneumoniae Fixes nitrogen

anaerobes anaerobically

Microaerobes / Azospirillum brasilense / Promotes plant growth /

heterotrophs A. lipoferum biofertiliser.

Aerobic / Filamentous Carry out oxygenic

photosynthetic heterocystous photosynthesis like
cyanobacteria such as higher plants; Fix
Nostoc and Anabena; nitrogen in heterocysts.

Anaerobic / Rhodopseudomonas Carry out anoxygenic

photosynthetic palustris; Chromatium photosynthesis.
Rhodospirillum rubrum

(b) Symbiotic diazotrophs

Examples Characteristic (s)

Rhizobium leguminosarum biovar Other biovars establish symbiosis with

viciae- Pisum sativum other legumes.

Bradyrhizobia is α-proteobacteria with

two sets of flagella- one thick and few
Bradyrhizobium japonicum- thin /v. large genome // can denitrify and
Glycine max (soyabean) grow chemolithotropically

G. max is a tropical legume, native to E.

Asia / rich in protein (>40%) and oil
(>20%).

E. meliloti are motile bacteria with

peritrichous flagella. Forms root nodules
Ensifer (syn.Sinorhizobium) also with other genera such as
meliloti – Medicago sativa Trigonella and Mellilotus.
(Alfalfa)
Medicago is a temperate legume / forage
crop.

Parasponia is the only non legume that

is nodulated by a broad range of rhizobia
Rhizobium-Parasponia andersonii belonging to four different genera / N-
fixation occurs in infection threads.

A. caulinodans is a motile, fast growing

soil bacterium that nodulates both stem
Azorhizobium caulinodans - and roots.
Sesbania rostrata (Dhiancha) Sesbania (tropical legume) can grow in
water logged soils. The nodules are
initiated at lateral root primordia.

Only angiosperm with symbiotic

association with cyanobacteria /
Nostoc-Gunnera (non legume) distributed mainly in the southern
hemisphere

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Azolla (aquatic fern) - Anabena Anabena forms cavities within the leaves
azollae of the water fern.

Frankia is a Gram+ve, heterotrophic,

filamentous actinomycete bacterium that
Frankia alni- Alnus crispa (Alder) forms root nodules with dicot plants. The
nodules are morphologically distinct from
legume nodules with Rhizobia.

A mutualistic association: the fungi

provide nutrients, water and above all
Fungi-cyanobacteria (lichens) home; the cyanobacteria shares fixed
carbon and nitrogen.

SAQ 1
a) Define nitrogen fixation.

b) Give an example of:

i) A free living diazotroph

ii) Symbiotic nitrogen fixer with a non-legume

7.3 BIOCHEMISTRY OF NITROGEN

FIXATION
Biological nitrogen fixation is catalysed by nitrogenase (N2ase). It is a highly
conserved metalloenzyme, restricted to some prokaryotes. Heterocomplexes
formed in vitro using N2ase subunits from different sources have substantial
activity in most cases. The structural subunits of the enzyme are encoded by
three genes that are assembled into two reversibly associating complexes
namely, dinitrogenase reductase and dinitrogenase. Each of these
complexes also has alternate names based on the metal cofactors (Fe-protein
and Fe-Mo protein) or the sequence in which they elute from a column
(component I or II). To describe nitrogenase from different sources, a
shorthand nomenclature is often used; for example, component I of
Azotobacter vinelandii is AvI while Cp2 refers to component II of Clostridium
pasteurianum. Let us now learn about the structure, metal factors and unique
characteristics of nitrogenase.

7.3.1 Nitrogenase (EC number: 1.18.6.1)

We shall begin by describing the structure & metal cofactors of dinitrogenase
reductase and dinitrogenase. Dinitrogenase reductase (Fe-protein, component
II) is a homodimer (60-64kDa) linked covalently to a single Fe4S4 cluster (F-
cluster) via cysteine residues, two from each subunit (Fig. 7.1). The redox
centre is a one electron carrier, located at the interface between the subunits.
It also harbours two Mg-ATP binding and hydrolysing sites, one on each
subunit (Fig.7.3). 129
Block 3 Nitrogen Metabolism and Plant Growth Hormones

Fig. 7.1: A single Fe-S cluster links the two subunits of dinitrogenase reductase.

The dinitrogenase (Mo-Fe protein, component I) is structurally more complex

than dinitrogenase reductase. It is a tetramer (220-240 kDa) of two types of
polypeptides (α2β2). Each αβ dimer (α-subunit is 56kDa and β-subunit is
60kDa) has two metal centres viz, a P-cluster and FeMoCo.

The P cluster is an unusual structure resembling two 4Fe-4S clusters (Fig.

7.2a), bridged by thiol side chain of cysteine along with a disulphide bond
between them (Fe8S7). The FeMoCo is a large redox centre assembled from
1Mo:7Fe:9S (organised as Fe4S3 and Fe3MoS3) that are bridged by three
inorganic sulphide groups. In addition it has a molecule of homocitrate and the
imidazole ring of histidine linked to Mo (Fig. 7.2b). The only other protein
ligand making contacts is cysteine. FeMoCo is buried in the environment
provided by the α-subunit. The substrate is probably bound in the cavity of the
FeMoCo.

(a) P cluster (Fe8S7) (b) FeMoCo

Fig. 7.2: Structure of the metal centres in dinitrogenase.

The structure of nitrogenase complex with associated metal cofactors is given

130 in Fig.7.3.
Unit 7 Nitrogen Fixation

Fig. 7.3: (a) Ribbon diagram (b) Schematic view of nitrogenase complex.

Some bacterial species express alternate dinitrogenase under limiting Mo

conditions. These enzymes are either vanadium or only iron dependent. As
compared to Fe-Mo nitrogenase they are less efficient and divert a much
higher percentage of electrons towards hydrogen. Both variants are hexameric
enzymes (α2β2 δ2). Azotobacter vinelandii is known to synthesise both
alternate forms.

7.3.2 Unique Features of BNF

The unique features of BNF can be traced down to the enzyme nitrogenase.
Some of the better known characteristics are enlisted below:

 Nitrogenase is catalytically an inefficient enzyme. The optimal rate of

electron transfer is about 12 electron pairs /sec /enzyme. In essence it
reduces only 3 molecules of N2 / sec. To compensate for its low activity
nitrogen fixing cells express large amount of nitrogenase amounting to
>5% of total protein.

 Under in vivo conditions the enzyme reduces both nitrogen and protons
to ammonia and hydrogen gas, respectively. The hydrogenase activity is
ATP dependent unlike hydrogenases from anaerobes. It also reduces a
variety of triple bonded compounds including acetylene, azide and
nitrous oxide. Acetylene reduction is a convenient measure of
nitrogenase activity.

 The enzyme has ATP binding and hydrolysing activity which is reductant
dependent.

 Nitrogenase from all diazotrophs is oxygen labile. Depending on the

oxygen dependence of the organism, nitrogen fixers respond
appropriately to maintain low free oxygen levels during nitrogen fixation.

 There are no known obligate diazotroph. The enzyme is expressed and /

active only when the reduced nitrogen status falls and oxygen levels are
low. It is stringently regulated.
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Block 3 Nitrogen Metabolism and Plant Growth Hormones

 Nitrogen fixation consumes enormous amount of energy (2ATP/ e-

transferred). The role of ATP is not completely understood as there is no
thermodynamic requirement for ATP hydrolysis. The most plausible
reason therefore is kinetic.

7.3.3 Reduction of Molecular Nitrogen

The enzyme nitrogenase catalyses a six electron reduction to ammonia via
enzyme bound intermediates like diimide and hydrazine.

N2 HN=NH] NH2- NH2] 2NH3

Diimide Hydrazine Ammonia

In addition, two protons are reduced yielding H2. It has been calculated that
the inevitable release of hydrogen results in dissipation of nearly 30-60% of
energy.

Mg2+

2H+ + 2e- +4ATP H2 +4 ADP + + 4Pi

The overall reaction catalysed by nitrogenase is:

N2 + 8e−+ 8H++16ATP 2NH3 + H2 +16ADP +16Pi

Nitrogen fixation requires a source of reductant and enormous amount of

energy (2ATP/ e- transferred). Both ATP and reductant are made available
from carbon metabolism that varies with the organism. It may come from
fermentation, aerobic respiration, oxidative pentose pathway or
photosynthesis. In most cases the immediate donor of electrons to
nitrogenase is reduced ferredoxin (Fe-S redox protein; Fd) or flavodoxin (a
flavoprotein like azotoflavin). The sequence of electron flow from the external
donor to the substrate is shown in Fig. 7. 4. Nitrogenase is inhibited by
hydrogen (competitive inhibitor) and carbon monoxide (non competitive
inhibitor).

Fig. 7.4: Nitrogen and proton reduction by Nitrogenase.

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Unit 7 Nitrogen Fixation

The role of ATP is not completely understood as there is no thermodynamic

requirement for ATP hydrolysis. You may recall the synthesis of ammonia by
Haber-Bosch process is an exothermic reaction.

N2 + 3H2 2NH3 G0' = -27 kJ.mol-1

The other alternative is kinetic reasons. ATP binding to reduced Fe protein

induces a conformational change that shifts its redox potential (increases
reducing power) from -0.25V to -0.40V; forms an active complex with
dinitrogenase and ensures unidirectional electron transfer (Fig. 7.5).

Fig. 7.5: Role of ATP in BNF.

7.3.4 Protection of Nitrogenase

Nitrogenase is extremely oxygen labile (Fe protein > Fe-Mo protein). It is
therefore, essential to maintain anoxic (oxygen deficient) conditions to protect
the enzyme from loss of activity / degradation. One of the simplest strategy is
avoidance of oxygen as in obligate anaerobes (Clostridium pasteurianum) ;
facultative anaerobes like Klebsiella pneumoniae fixes anaerobically and by
inducing behavioural changes such as clumping of filaments (Trichodesmium;
aerobic, photosynthetic, cyanobacteria) in which nitrogen fixation occurs by
cells in the centre of the filament. These organisms have not developed
specific mechanism(s) of protection.

Many nitrogen fixing organisms have one /more ways to protect nitrogenase
and may even carry out aerobic metabolism and / photosynthesis
simultaneously. The free living obligate aerobe Azotobacter vinelandii respires
at a high rate when oxygen levels are high by expressing an inefficient ETC
(respiratory protection). In this way more oxygen is consumed and
nitrogenase can work. It has a branched respiratory chain differing in the redox
carriers and efficiency of ATP production. Respiratory protection is also
reported in some symbiotic nitrogen fixers (Rhizobium japonicum).

Another strategy is conformational protection. It was observed that nitrogen

fixation is inhibited if cultures of Azotobacter are suddenly exposed to high
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Block 3 Nitrogen Metabolism and Plant Growth Hormones

oxygen concentration. The enzyme becomes oxygen tolerant and particulate

(sediments upon ultracentrifugation). It forms a large complex containing
multiple molecules each of Fe-Mo and Fe protein along with Shethna protein
(Fe-S protein). The protected enzyme immediately reverts back to its
catalytically active form as the oxygen concentration is lowered.

Nitrogen fixation is spatially separated in obligate aerobes such as free living

filamentous cyanobacteria and root nodules induced in legumes by symbiotic
diazotrophs. In filamentous cyanobacteria fixation occurs in specialised cells
(heterocysts) that lack the oxygen evolving PSII system; have limited
permeability to oxygen; respire at a high rate; express uptake hydrogenase
(Hup+) and some have a protein resembling myoglobin (cyanoglobin). Similarly
in nodules, nitrogen fixation occurs in bacteroids (differentiated bacteria) inside
infected cells.

Nodule geometry restricts entry of oxygen (cortex has tightly packed cells);
they also express leghemoglobin (a truly symbiotic protein) which is
unequally distributed in the plant cytoplasm and peribacteroid space (Fig.
7.10c). The protein binds oxygen and regulates the levels of free oxygen
thereby allowing both respiration and nitrogen fixation to take place inside
bacteroids. Like cyanobacteria, some Rhizobia also have uptake
hydrogenase.

An uptake hydrogenase is a unidirectional hydrogenase restricted to aerobic

systems. It converts hydrogen gas to protons and electrons which could be
either fed to the ETC (produces ATP and consumes oxygen) or for reduction
of nitrogen and other substrates. In symbiotic associations, legumes infected
with Hup+ strains of bacteria produce higher crop yields by recovering some of
the protons lost due to the hydrogenase activity of N2ase. This enzyme is
inhibited by CO.

H2 2H+ + 2e-

SAQ 2
a) Name the alternate variants of dinitrogenase.

b) Indicate three ways by which filamentous cyanobacteria protect

nitrogenase.

c) Give the reactions catalysed by nitrogenase in vivo.

7.4 SYMBIOTIC NITROGEN FIXATION (SNF)

Nitrogen is an essential and often limiting nutrient for plant growth. Under
nitrogen limiting conditions certain plants especially belonging to
leguminoseae (Fabaceae) establish symbiotic associations with Rhizobia
primarily in root nodules and sometimes stems (Fig. 7.6). SNF is an example
of facultative symbiosis and is most important from an agronomic point of
view. Almost 90% of leguminous plants are capable of nodulation and the
134 process is initiated upon finding the right partner.
Unit 7 Nitrogen Fixation

Fig. 7.6: (L) Root and (R) Stem nodules in legumes.

The term Rhizobia is a generic name for a group of Gram negative, aerobic
proteobacteria that can induce nodulation in their host and fix nitrogen
symbiotically. This group of bacteria now includes 18 genera spread over
many families. The genus Rhizobium (with118 species) is the largest genus of
Rhizobia. They generally have a restricted host range (Table 7.2), although
both partners do vary in the degree of specificity. Two other groups of bacteria
(Frankia and cyanobacteria) can also fix nitrogen in symbiosis with plants.

Table 7.2: Symbiotic nitrogen fixing Rhizobia

Family Genera Example(s) Plant host

R.leguminosarum Trifolium
Rhizobium, bv trifoli; species
Rhizobiaceae
A. undicola
Allorhizobium Neptunia
(has seven genera)
S. meliloti natans
Ensifer
(Sinorhizobium) Alfalfa
N. galegae
Neorhizobium Galega
officinalis
B. japonicum Glycine
max;
Bradyrhizobiaceae Bradyrhizobium B. lablabi
Arachis
hypogea

M. loti Lotus
japonicus
Phyllobacteriaceae Mesobacterium Mesorhizobium
spp Cicer
arietinum

Xanthobacteriaceae Azorhizobium A. caulinodano Sesbania

rostrata
Brucellaceae Ochrobactrum O. lupini Lupini albus

Methylobacteriaceae Methylobacterium M. nodulans Crotalaria

legume
Hypomicrobiaceae Devosia D. neptuniae Neptunia
natans / an
aquatic
legume
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Block 3 Nitrogen Metabolism and Plant Growth Hormones

7.4.1 Establishing Legume – Rhizobium

Symbiosis
The symbiosis between rhizobia and legumes is not obligatory, and each is
capable of independent existence. However, it is only under nitrogen stress
that these two prospective symbionts exchange signals in the form of
elaborate chemical messages to seek one another. This signalling is followed
by initiation of infection that culminates in the formation of root nodules.

Rhizobial infections can occur through root hairs, cracks in the epidermis or
interstitial infections between epidermal cells. The most common mode of
infection is through infection threads. Bacteria enter the root at the junction
between the root hair base and the adjacent epidermal cell. In Arachis
hypogea infection occurs through crack entry, without infection threads.
Rhizobia lack some of the microbial molecular patterns that provoke plant
immune responses allowing a controlled infection.

The first level of interaction between the prospective partners begins with the
excretion of flavonoids (chemotactic) by the plant into the rhizosphere that
attracts and induces the responding rhizobia (luteolin - S. meliloti or naringenin
- B. japonicum) to synthesise species specific nodulation (nod) factors (NF)
or lipochito oligosaccharides (LCO; Fig. 7.7). The flavonoid signal activates
constitutively expressed Nod D protein (transcription factor) which in turn
induces the transcription of other nod genes.

Fig. 7.7: Basic structure of Nod factors (R1 to R9 are strain / species specific
substituents).

The nod genes fall into two groups; common nod genes and species specific
nod genes. The gene product of former group (nod A, B and C genes)
synthesise the basic backbone of β -1, 4 linked N-acetyl D-glucosamine
residues (4-5) with N-acylation at the non reducing end while the latter set of
gene (nod P, nod Q, and nod H, or nod E, nod F and nod L) products generate
the species specific structural diversity of nod factors (NF) by modifying the
basic structure (acetylation, glycosylation, sulphation, carbamoylation). All
rhizobia produce a mixture of very potent NFs which are recognised by plant
receptor kinases and play an important role in determining host range and
initiating early morphological and developmental changes.

The root cells undergo various changes in their growth patterns and
metabolism following interaction with Nod factors. The root hairs are curled
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Unit 7 Nitrogen Fixation

and divide locally and bacteria get trapped within these curls. Cell wall
degrading enzymes (pectinases, hemicellulases and cellulases) are also
released that facilitate the bacterial cells to gain access to the plasma
membrane of the root hair. At this point the root hair stops growing and the
plasma membrane starts invaginating.

This internal tubular cellulosic extension is called the Infection thread. The
thread is formed by the fusion of membrane vesicles derived from Golgi at the
end of the tube.

Fig. 7.8: (a-f): Steps in controlled infection of legume roots (after Taiz et al).

The cortical cells near the xylem dedifferentiate and enter the cell cycle. As a
result of repeated cell divisions, a nodule meristem zone (nodule
primordium) is established. The infection thread filled with proliferating
rhizobia continues to elongate through the base of the root hair cell where its
membrane fuses with the host cell plasma membrane. During this process,
some bacteria are released into the apoplastic space (Fig. 7.8).

The bacteria in infected cells differentiate into bacteroids (endosymbiotic

nitrogen fixing cells), surrounded by a host derived peribacteroid membrane
forming a symbiosome (Fig. 7.9). Most of them store poly β-hydroxy butyrate
(PHB). Bacteroids do not divide further and are capable of nitrogen fixation.
Under natural conditions some of the prominent factors that affect symbiosis
and efficiency of BNF include biological competition, temperature, pH, salinity,
drought, metals and pesticides.
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Block 3 Nitrogen Metabolism and Plant Growth Hormones

Fig. 7.9: Infected cell with bacteroids in a symbiosome. (adapted from Plant
Physiology, Development and Metabolism by S. C. Bhatla & Manju. A. Lal).

Nitrogen fixing nodules are genuine organs with characteristic morphology and
anatomy. They are either determinate or indeterminate; the type of nodule is
controlled by the host (Fig. 7.10). The infected cells are polyploid while the
uninfected cells remain diploid. Indeterminate nodules are cylindrical and often
branched with a persistent meristem. They are characteristic of temperate
legumes (peas, alfalfa,) and synthesise amides (asparagine) for export.
Determinate nodules are spherical (limited size) with a transient meristem.
These nodules are found in legumes of tropical and subtropical regions such
as Glycine, Arachis and Phaseolus and they produce ureides (allantoin and
allantoic acid).

(a) Indeterminate; (b) Determinate nodules (c) An Infected cell

Fig: 7.10 Types of nitrogen fixing nodules. // Bacterial PM should touch only the
membrane

7.4.2 Nature of Symbiotic Relationship

The bacterial partner encodes genes for nodulation (nod genes for synthesis,
modification and transport of Nod factors) and nitrogen fixation (nif and fix
genes; synthesis and regulation of N2ase, metal cofactors and electron
transport components). Most rhizobial genomes have nodulation and nitrogen
fixation genes clustered on plasmids (sym plasmids) or large chromosomal
symbiotic islands or occasionally on chromids (replicons maintained as
plasmids but carry several core chromosomal genes; for example,
138 Neorhizobium galegae). Those with multipartite genomes are fast growers as
Unit 7 Nitrogen Fixation

compared to rhizobia having only a single very large chromosome

(Bradyrhizobia). Like most soil bacteria rhizobia also have large number of
genes, acquired and retained for handling the complex environment.

Plant genes whose activity is induced or enhanced in response to infection are

termed nodulins such as Glutamine synthetase and uricase. An example of a
protein that is neither entirely of bacterial nor plant origin is leghemoglobin
(legHb), a truly symbiotic protein. It is synthesised by genes encoded by both
partners; the globin gene is present on the plant genome while the heme entity
is encoded by the bacteria. Nodules expressing legHb have a pink colour and
they fix nitrogen whereas ineffective nodules are small and green.

The metabolism in the bacteroid is aerobic and respiration generates reducing

equivalents and ATP for nitrogen fixation. LegHb maintains low free oxygen for
fixation to occur simultaneously. We all know that BNF is a high energy
requiring process. It has been estimated that nitrogen fixation consumes up to
20% of the total photosynthate generated by the plant.

As the infection process progresses, the nodule establishes contact with the
main root vascular system. This creates a route for export of bacteroid-fixed
and assimilated nitrogen from the nodule to rest of the plant as well as import
of photosynthate (sucrose) and other nutrients into the nodule through phloem
(Fig. 7.11). Sucrose is converted to malate in the cytosol and translocated by
malate transporter on peribacteroid membrane to the symbiosome.

Fig. 7.11: The exchange of metabolites between the host and microsymbiont.

Ammonia synthesised in the bacteroids is assimilated by plant specific

nodulins in the cytoplasm of infected cells after it moves out through aquaporin
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Block 3 Nitrogen Metabolism and Plant Growth Hormones

related channels. Finally glutamate / glutamine are converted to asparagine or

ureides depending on the legume, for export (via xylem) to other plant parts
(details in unit 8). Some reactions also take place in uninfected cells. The
carbon skeleton and ATP for ammonia assimilation comes from the plant.

We end this section with an overview of the key events in legume-Rhizobium

symbiosis (Fig. 7.12).

Nitrogen deficient soil

Excretion of plant flavonoids (chemical signal) into the rhizosphere

Activation of rhizobial NodD proteins (TF)

induces transcription of common & species specific nod genes

Synthesis of Nod factors (NF)

Recognised by plant receptor kinases on the root hair PM

This interaction determines host range and initiates early morphological

(root hair deformation & curling) & developmental changes (induction of
localised cell division; expression of early nodulins or ENODs).

Bacteria trapped in cellulosic infection threads divide; the thread grows

into the cortex, releasing bactera into the host cell, enclosed by the
host cell membrane (peribacteroid membrane).

Within the resulting symbiosome, bacteria differentiate into bacteroids;

express legHb followed by N2ase & redox carriers. The nodule has both
infected and unifected cells where it expresses nodulins. The plant
provides fixed carbon for energy generationand skeleton for ammonia
assimilation.

Fixed nitrogen is assimilated by plant enzymes in infected cell cytosol;

some of it is converted to either amides or ureides for export to other
plant parts.

Fig. 7.12: An overview of the key events in legume-Rhizobium symbiosis

SAQ 3
a) What is the role of flavonoids in legume-Rhizobium symbiosis?

b) What are nod factors?

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Unit 7 Nitrogen Fixation

7.5 NITRATE ASSIMILATION

7.5.1 Introduction
All biomolecules incorporate nitrogen only in reduced form (oxidation state
+3). It is available to plants in inorganic state either through biological nitrogen
fixation by free living nitrogen fixers / non biological means (as ammonium) or
by assimilation of nitrate obtained from soil or water. Some plants such as
legumes also establish symbiotic associations with nitrogen fixing microbes
while others form mutually beneficial mycorrhizae with actinomycete fungi. In
addition, nitrate is readily produced from ammonium (released from decaying
organic matter / nitrogen fixation / fertilisers) in well aerated soils at neutral /
mildly alkaline pH by nitrifying bacteria while ammonium levels are high in
acidic and anaerobic conditions. Thus higher plants depend on prokaryotic soil
microorganisms for conversion of dinitrogen into usable forms (nitrate /
ammonium).

Among all these alternatives the major source of reduced organic nitrogen
(>95%) comes from nitrate assimilation. It is the process of conversion of
nitrate to ammonia in two sequential steps catalysed by assimilatory nitrate
and nitrite reductases (not to confuse with similar enzymes that are involved in
dissimilation of nitrogen into gaseous forms). In plants nitrate is assimilated in
both leaves and roots; the relative contribution of the two sites varies with the
plant species, for instance many woody plants and temperate nodules
assimilate nitrate mainly in roots while it is primarily assimilated in the shoots
of tropical cereals.

7.5.2 Uptake and Transport of Nitrate

Nitrate is taken up from the soil by a variety of transporters (variable affinity)
on the root epidermal and cortical cell plasma membrane. Some of these
transporters are also inducible that allow plants to adjust to a range of external
nitrate concentrations. The transport is accompanied by simultaneous
movement of two protons (symport mechanism). This results in the cytosol
becoming more positive. An H+ - ATPase then pumps out protons to drive
active transport of nitrate against the concentration gradient, across both roots
and mesophyll cells.

Once inside the cell nitrate may be stored temporarily in the vacuole or
assimilated in the root epidermal or cortical cells. The reduced nitrogen is
primarily assimilated into amide nitrogen of glutamine and asparagine for
transport to leaves (mesophyll cells) via xylem vessels. Excess nitrate may
also be carried in a similar fashion to mesophyll cells. If nitrate is applied as
foliar fertiliser it can be taken up directly by the leaves. Nitrate is reduced to
nitrite in the cytosol and then to ammonium in the chloroplast / leucoplast of
mesophyll / root cell, respectively.

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Block 3 Nitrogen Metabolism and Plant Growth Hormones

7.5.3 Nitrate and Nitrite Reductases

Nitrate reduction is a two electron reduction catalysed by assimilatory nitrate
reductase (aNR) that utilises NADH or NADPH as external electron donors. A
NADH/ NADPH dual specificity enzyme also exists in monocots and some
dicots (soyabean). Fungi express an NADPH dependent NR.

NO 3-  2H  2e- NO 2-  H2O

Nitrate Nitrite

The enzyme from higher plants is a homodimer with a subunit molecular mass
of 94-104 KDa, depending on the species. Each subunit harbors three
covalently linked redox carriers – flavin adenine dinucleotide (FAD),
cytochrome b557 (a heme protein) and molybdenum cofactor, MoCo (an
unconjugated pterin with a side chain to which Mo is attached by two sulphur
bonds). It is a rare example of a molybdoenzyme from eukaryotes. Limited
proteolysis yields three fragments (domains), each bound to one of the
carriers. The three domains are connected by two hinges. Each functional
region belongs to a different protein family. The native protein has two
additional domains, one to bind NADH (in domain I) and another dimer
interface domain (Fig. 7.13).

Fig. 7.13: (a) Limited proteolysis of NR (b) Model of Nitrate reductase

The flow of electrons from NADH (external donor) to nitrate is through a series
of redox transfers (mini electron transport chain) involving bound redox
cofactors in NR (Fig. 7.14). The substrate nitrate is reduced to nitrite. In plants
all NRs can also convert nitrite to nitric oxide in the presence of NAD (P) H.
This is an additional route (minor) for NO synthesis (nitrate nitrite NO);
the other being the oxygen dependent arginine pathway.
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Unit 7 Nitrogen Fixation

Fig. 7.14: (a) Mini electron transport chain in NR (b) Structure of MoCo.

The other variant of NR is present in the photosynthetic membranes of

cyanobacteria. This enzyme has a simpler structure and requires only
molybdopterin cofactor. The latter is reduced by ferredoxin which in turn
acquires reducing equivalents from the light reactions of photosynthesis (Fig.
7.15).

Fd(red) FAD MO5+ NO3

Fd(ox) FADH2 MO6 NO2

+

Fig. 7.15: Nitrate reduction by Fd-dependent NR in cyanobacteria.

The second step of nitrate assimilation is a six electron reduction catalysed by

assimilatory nitrite reductase (aNiR), located in plastids (chloroplast /
leucoplast stroma)) of plants and algae and photosynthetic membrane of
cyanobacteria. In some plants distinct types of aNiR are expressed in roots
and leaves. Nitrite enters the plastid via a nitrite transporter in the plastid
membrane. Like nitrate reductase NiR has covalently bound redox carriers;
one each of 4Fe-4S cluster and siroheme (a unique cyclic tetrapyrrole
containing Fe; an uroporphyrin derivative). In non photosynthetic organisms
such as fungi and bacteria, NiR is NADPH dependent.

NO2- + 8H+ + 6e- NH4+ + 2H2O

In fact, the above conversion may proceed in steps as indicated below:

NO 3  NO 2  HNO   NH 2OH  NH3

Nitrate Nitrite Nitroxyl Hydroxyl amin e Ammonia

Assimilatory NiR is a nuclear encoded monomeric (approx. 63KDa) protein

organised into two functional domains; the N-terminal domain binds ferredoxin
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Block 3 Nitrogen Metabolism and Plant Growth Hormones

(external donor) while the C-terminal domain has binding sites for the other
two redox factors. These cofactors are in close proximity and linked by a
sulphur ligand. They also form a mini electron transport chain to transfer
electrons from ferredoxin to nitrite (Fig. 7.16). The siroheme is surrounded by
several ionisable amino acid residues that facilitate the binding and
subsequent reduction of nitrite. NiR levels are transcriptionally regulated by
nitrate.

The external electron donor is reduced ferredoxin which is derived either

directly from the light reactions of photosynthesis (non cyclic electron flow) or
via reduction by NADPH, generated by oxidative pentose pathway in
chloroplasts / leucoplasts. The latter pathway is important in the absence of
photosynthesis (in darkness) or non photosynthetic root cells. Here the
reduction is catalysed by ferredoxin-NADP+ oxidoreductase. Eight protons
are obtained from the carboxyl groups of siroheme.

Fig. 7.16: (Top). The flow of electrons from ferredoxin to nitrite (bottom)
Siroheme

The levels of NiR are generally higher than that of NR and it has high affinity
for its substrate which helps in efficient removal of potentially mutagenic nitrite
by reduction. You would have noted that in cyanobacteria both steps of nitrate
assimilation utilise photosynthetically generated reduced ferredoxin. The
process is therefore comparable in this respect to the reduction of carbon
dioxide during photosynthesis.

The reduced nitrogen (ammonium) formed is not allowed to accumulate due to

its toxic effects. It is immediately assimilated into organic form. Some plants
including algae leach out excess ammonia which can be reoxidized by soil
microorganisms. The details of ammonia assimilation will be dealt with in the
144 next unit. The fate of nitrate taken up by plant cells is depicted in Fig. 7.17.
Unit 7 Nitrogen Fixation

Fig. 7.17: Fate of nitrate in cytosol and plastids.

7.5.4 Regulation of Nitrate Assimilation

Nitrate assimilation is regulated at multiple levels - by controlling synthesis,
catalytic activity and degradation of nitrate reductase. The concentration of
nitrite has to be stringently controlled because both products of nitrate
assimilation (nitrite and ammonium) are toxic at higher concentration. Nitrate
reductase is a substrate inducible enzyme. On exposure to nitrate, the levels
of NR mRNA increases within minutes. A variety of other factors have also
been identified that influence transcription and activity of NR in presence of
nitrate. Together they help to fine control nitrate reduction in accordance with
the demands.

The factors that influence the transcription of NR act by either up or down

regulating the process. Light (or sucrose) and cytokinin induces NR
transcription. A diurnal pattern (circadian rhythm) is observed in NR
transcription in photosynthetic tissues; maximal levels appear just before the
start of the light period. The integration of nitrate assimilation with
photosynthesis is logical as it consumes a significant proportion of the energy
meant for CO2 fixation. NR transcription is repressed in dark (no
photosynthesis) and glutamine (signals abundance of organic nitrogen).

The activity of NR is reversibly modulated by phosphorylation (serine) /

dephosphorylation and NR inhibitor protein (NIP). The reversible covalent
modification is catalysed by NR kinase / phosphatase, respectively. Unless the
phosphorylated form associates with NIP it is active. The inactive form is
phosphoNR: NIP complex which predominates in dark, only if
Mg+2concentrations are high. The NR inhibitor protein belongs to 14-3-3 family
of regulatory proteins. The binding of NIP to phosphoNR blocks electron flow 145
Block 3 Nitrogen Metabolism and Plant Growth Hormones

between cytochrome and MoCo. As the concentration of Mg+2 falls in light, the
dissociation of the complex is promoted. The two active states of NR are
unphosphorylated and phosphorylated forms. Another probable role of
phospho NR-NIP complex is in the control of NR degradation.

SAQ 4
a) What is nitrate assimilation?

b) Why do cells have higher levels of NiR than NR?

c) What is unique about cyanobacterial nitrate assimilation?

7.6 SUMMARY
 Nitrogen is the fourth most abundant element after carbon, hydrogen
and oxygen in plants. It occurs in both inorganic and organic forms.

 Nitrogen fixation is the reduction of atmospheric nitrogen to ammonia

brought about by biological and non biological processes.

 BNF is restricted to some prokaryotes. These organisms are called

diazotrophs that include both free living and symbiotic nitrogen fixers.

 All nitrogen fixers possess the enzyme nitrogenase which is a reversible

complex of dinitrogenase reductase (Fe-protein) and dinitrogenase (Fe-
Mo protein).

 Nitrogenase is a highly conserved metalloenzyme that reduces nitrogen

and protons in vivo. It is an inefficient, ATP-dependent, oxygen labile
and stringently regulated enzyme. The enzyme is inhibited by hydrogen
and carbon monoxide.

 Nitrogen fixation requires a source of reductant and enormous amount of

energy (2ATP/ e- transferred), made available from carbon metabolism.
The immediate source of reductant is either ferredoxin or flavodoxin.

 Nitrogen fixing organisms either avoid oxygen or have developed

strategies to reduce free oxygen during fixation.

 SNF is the most important from agronomic point of view. Almost 90% of
leguminous plants are capable of nodulation and the process is initiated
upon finding the right partner. Nitrogen fixation occurs in bacteroids
present in infected cells.

 Nitrate assimilation is the process of conversion of nitrate to ammonia in

two sequential steps catalysed by assimilatory nitrate and nitrite
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Unit 7 Nitrogen Fixation

reductases. Nitrate is reduced to nitrite in the cytosol and then to

ammonium in the chloroplast / leucoplast of mesophyll / root cell.

 Nitrate assimilation is regulated by controlling the synthesis, catalytic

activity and degradation of nitrate reductase. The concentration of nitrite
has to be stringently controlled because both products of nitrate
assimilation (nitrite and ammonium) are toxic at higher concentrations.

7.7 TERMINAL QUESTIONS

1. Elaborate the structure and reactions catalysed by nitrogenase.

2. Indicate the unique features of BNF.

3. Give an account of nitrate assimilation in plants. Highlight the structure,

cofactors and flow of electrons in NR and NiR.

4. Explain the symbiotic relationship between Rhizobium and legume at the

biochemical level.

7.8 ANSWERS
Self Assessment Questions
1. a) Nitrogen Fixation is the reduction of nitrogen to ammonia /
ammonium.

b) i) Azotobacter vinelandii

ii) Rhizobium-Parasponia andersonii or any other

2. a) Alternate variants of nitrogenase are either vanadium or only iron

dependent. / Both variants are hexameric enzymes (α2β2 δ2).
Azotobacter vinelandii is known to synthesise both alternate forms.

b) Spatial separation: N2-fixation occurs in specialised cells

(heterocysts) that lack the oxygen evolving PSII system / have
limited permeability to oxygen / respire at a high rate.
They also express uptake hydrogenase (Hup+) and some have a
protein resembling myoglobin (cyanoglobin).

c) The overall reaction catalysed by nitrogenase is:

N2 + 8e−+ 8H++16ATP 2NH3 + H2 +16ADP +16Pi

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Block 3 Nitrogen Metabolism and Plant Growth Hormones

3. a) Flavonoids are chemotactic signalling molecules released by the

plant / attract Rhizobia to the rhizosphere and induces the
synthesis of nod factors by activating Nod D protein.

b) Nod factors are lipo-chito-polysaccharides. All rhizobia produce a

mixture of very potent NFs by modifying the basic backbone.

4. a) Nitrate assimilation is the reduction of nitrate to ammonia in two

sequential steps catalysed by assimilatory nitrate and nitrite
reductases.

b) NiR efficiently removes toxic and potentially mutagenic nitrite.

c) Nitrate assimilation in cyanobacteria is ferredoxin dependent,

which is derived from the light reactions of photosynthesis.

Terminal Questions
1. Refer to subsections 7.3.1 and 7.3.3

2. Refer to subsection 7.3.2

3. Refer to subsection 7.5.3

4. Refer to subsection 7.4.2

7.9 FURTHER READINGS

1. Bowsher, C; Steer, M. and Tobin, A, Plant Biochemistry, 1st Ed, 2008,
Garland Science, Taylor and Francis Group, LLC

2. Lindstrom,K and Mousavi, S.A. Effectiveness of nitrogen fixation in

Rhizobia, Microbial Biotechnology (2020),13(5), 1314-1335, published
by John Wiley & Sons Ltd.

3. Plant Physiology, Development and Metabolism by Satish C. Bhatla &

Manju A. Lal, Springer nature Singapore Phe Ltd, 2018

4. Heldt, H. Walter and Piechulla, B. Plant Biochemistry, 4rd Ed, 2011,

Elsevier Academic Press, USA.

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