Nitrogen is one of the most important major limiting nutrients for most crops and other plant species. Biological Nitrogen Fixation (BNF) is an ecologically important phenomenon that can support an amount of nitrogen to compensate the difficiencies of this element. In this biologically-mediated process, a specific group of bacteria, collectivelly called rhizobia, fixed atomospheric dinitrogen (N2) via symbioses with legumes.Other free living bacteria fix nitrogen in the soil or in non specific association with plants. This biological process between rhizobium strains and their legume partners can be happened under low level of available nitrogen with help of many different genes such as nod, nif, fix, production of polysaccharides, competition, infection process, host specificity, Type I to Type VI secretion, signals of host and many other different genes that recently have been reported by scientists. The establishment of the symbiosis requires close coordination between the partners and is mediated by the exchange of diffusible signal molecules. Most recently, bacterial and plant genome-sequencing projects have added immensely to the resources available to study the symbiosis. A major event was the adoption of two genetic model legumes, Lotus japonicus and Medicago truncatula, and the genomes of both plants are currently being sequenced.Research with these model plants has now revealed the basic outlines of the plant-signaling pathways that lead to nodule formation.

1. CREDIT SEMINAR ON
PLANT GENETIC CONTROL OF
NODULATION AND ITS UTILIZATION IN
FIXING NITROGEN

2. INTRODUCTION
Biological Nitrogen Fixation (BNF) is an ecologically important
phenomenon that can support an amount of nitrogen to compensate
the deficiencies of this element.
In this biologically-mediated process, a specific group of bacteria,
collectivelly called rhizobia, fixed atomospheric dinitrogen (N2) via
symbioses with legumes.
This biological process between rhizobium strains and their legume
partners can be happened under low level of available nitrogen with
help of many different genes such as nod, nif, fix, and production of
polysaccharides, competition, infection process, host specificity,
signals of host and many other different genes.

3. ROLE OF NITROGEN IN PLANTS
 Major substance in plants next to water
 Building blocks
 Constituent element of
 Chlorophyll
 Cytochromes
 Alkaloids
 Many vitamins
 Plays important role in metabolism, growth, reproduction
and heredity.

4. Antolin-Llovera et al. 2014

5. o Bacteria: special type (nitrogen fixing
bacteria) types -
o Free living aerobic : Azotobacter,
o Free living anaerobic : Clostridium
o Free living photosynthetic : Chlorobium,
o Free living chemosynthetic
:Desulfovibro,Thiobacillus
o Free living fungi: yeasts
o Blue green algae : Nostoc, Anabaena
Various spp. of Rhizobium
Pea – Rhizobium leguminosarum
Beans – R. phaseoli
Soyabeans – R. japonicum
Lupins – R. lupine
Two types of Rhizobium-
 Bradyrhizobium - slow growing
spp.
 Rhizobium - fast growing spp.
ORGANISMS INVOLVED IN NITROGEN
FIXATION
NON-SYMBIOTIC SYMBIOTIC

6. HISTORY
Marcello Malpighi
(March 10, 1628 – November 29, 1694)
Hermann Wilfarth
Martinus Beijerinck
(1888)
(1888)
Anton DeBary
(1879)
1. 2.
3.
4.

7. NOD D ( THE SENSOR)
 The nod D gene product recognizes molecules phenyl propanoid-
derived flavonoids produced by plant roots and becomes activated
as a result of that binding.
 Activated nod D protein positively control the expression of the
other genes in the nod gene regulon.
(single transduction)
 Different nod D alleles recognize various flavonoid structure with
different affinities, and respond with different patterns of nod gene
activation.

8. NOD GENES
 Rhizobial genes that direct the steps in nodulation of a legume are called nod
genes.
 Nod gene expression is induced by the presence of certain flavonoids .
 These chemicals induce the formation of NodD, which in turn activates
other genes involved in the expression of nod factors and their secretion into
the soil

9. The enzyme encoded by the common nod genes have
specific function

10. NOD FACTORS
 The nod A,B,C genes encode proteins that produce
oligosaccharides called nod factors .
 Nodulation (Nod) factors are signaling molecule produced by
bacteria known as rhizobia during the initiation of nodules on the
root of legumes.
 Structural variations in Nod factors are a key determinant of host
range, because these Nod factors have to be recognized by the host
in order to initiate infection and nodulation

11. FUNCTION OF NOD FACTOR
Nod factors can affect other plants in the soil surrounding them. When
Nod factors are present in mixed crop fields,
 They have the ability to stimulate seed germination.
 Promote plant growth.
 Increase photosynthetic rates.
 Increase grain yields of legume and non-legume crops.
 Nod factors arelipochito-oligosaccharides and have three to five N-
acetyl-glucosamines.
 The specific structure of Nod factors is determined by modifications
made by Nod genes, which are found in the rhizobium genome.
STRUCTURE OF NOD FACTOR

12. EXOPOLYSACCHARIDES
In addition to Nod factors, rhizobial surface polysaccharides such as
exopolysaccharides (EPS), lipopolysaccharides (LPS), and capsular
polysaccharides (KPS) are also thought to be important for
establishing symbiotic relationships.
 Suppress plant defense.
 Promoting bacterial infection .
 Nodulation remain elusive .
 The Nod genes encode for proteins that modify Nodfactors by
adding or removing different chemical structures such as sulfates,
fatty acids, acetyl groups, and methyl groups to the original
lipochito-oligosaccharide structure.

13. NODULATION PROCESS
Chemical recognition of root and Rhizobium.
Root hairs curl.
Formation of infection threads.
Invasion of the roots by Rhizobia.
Nodule tissue forms.
Bacteria convert to bateriods and begin to form nitrogenase enzyme .
Legume provides Rhizobia with carbon. Rhizobia provide the legume
with fixed N

14. NODULE FORMATION
Radutoiu et al. 2003

18. NITROGENASE ENZYME
 Active in anaerobic condition
 Made up of two protein subunits
 Fe protein reacts with ATP which ultimately reduces N2 into ammonia
 N2 + 6H+ + 6e- 2NH3
 Nitrogenase is extremely sensitive to oxygen. Root nodules of nitrogen‐fixing
plants contain the oxygen‐binding protein, leghemoglobin, which protects
nitrogenase by binding molecular oxygen.
 In the nodule O2 levels are precisely controlled by the leghemoglobin.
Non heme iron protein ( Fe-protein)
Iron molybdenum protein (Mo Fe-protein)

19.  Presence of oxygen in the root
nodules would reduce the activity of
the oxygen sensitive nitrogenase.
 Leghemoglobin buffer the
concentration of free oxygen in the
cytoplasm of infected plant cells to
ensure the proper function of root
nodules.
 Oxygen concentration that is low
enough to allow nitrogenase to
function but high enough so that it
can provide the bacteria with oxygen
for respiration.
LEGHEMOGLOBIN( OXYGEN BUFFER)

20. ROLE OF HOST PLANT IN NODULE FORMATION
In symbiosomes, there is a reliable supply of metabolic substrates from
plant biosynthesis which provides,
 Sufficient energy and reducing conditions.
 Protection against elevated oxygen concentrations that inhibit
nitrogen fixation.
 A pathway for the transport of nitrogen fixation products to the plant
host.
 Protection against competitive or antagonistic bacteria in the
environment.

21. why is the legume necessary?
The legume is certainly helpful in that it
supplies nutrients to the bacteroids with which they synthesize
the large amounts of ATP needed to convert nitrogen (N2) into
ammonia (NH3).

22. NODULE DEVELOPMENT
 The root begins to swell and the
nodule becomes visible.
 In the field, nodules are visible
within 21 to 28 days from emergence
of the plant.
 The time from planting to the
appearance of nodules varies
depending on plant growth and
availability of mineral nitrogen in
the soil.
 Nodules differ in shape, size, color,
texture, and location. Their shape and
location depend largely on the host
legume.
DIFFERENT SHAPES OF NODULES

23. DETERMINATE NODULES INDETERMINATE NODULES
 Formed on tropical legumes(soybean
and common bean) by Rhizobium and
Bradyrhizobium.
 It is initiated from middle/outer cortex
without persistant apical meristem
 Determinate nodules also possess
peripheral vascular tissue but, because
development is in a radial pattern,
distinct zones are more difficult to
distinguish.
 Formed on temperate legumes (pea,
clover, alfalfa); typically by Rhizobium
spp.
 It is initiated from inner cortex with
persistant apical meristem
 In order from the tip, such nodules
exhibit a pre infection zone, infection
zone, fixation zone, and, finally a zone
of senescent tissue.

24. NODULE FORMATION IN NON-LEGUMINOUS
PLANTS
Root nodule produce from non leguminous
plants(ex)
Root nodule produce from leaves(ex)
 Causuarina equisetifolia –
Frankia
 Alnus – Frankia
 Myrica gale – Frankia
 Parasponia – Rhizobium
Dioscorea, Psychotria,chomelia

25. NODULE SENESCENCE
 Eventually nodules age and decay.
 As the plant puts more energy into seed
production, the nitrogen-fixing activity of the
bacteroids decreases.
 Eventually the nodules stop functioning and
disintegrate, releasing bacteroids into the soil.
 These rhizobia may survive and infect new
plants during the next cropping season.
Nodule life span is largely determined by four factors:
 The physiological condition of the legume.
 The moisture content of the soil.
 The presence of any parasites.
 The strain of rhizobia forming the nodule.
Observed nodule senescence
under light microscope

26. MUTATION ON NOD (NODULATION)AND NOL
(NODLOCUS)GENES
 Mutations in these genes block nodule formation or alter host range
 Most have been identified by transposon mutagenesis, DNA sequencing and protein
analysis.
Four classes:
NodD,(regulon)
NodA, B and C (common nod genes),
Hsn (host-specific nod genes)
Other nod genes ( Nif gene and fix gene)
Fournier et al. 2015

27. Mutations in nod A,B or C completely abolish the ability
of the bacteria to nodulate the host plant .
Products of these genes are required for bacterial
induction of root cell hair deformation and root cortical
cell division .
Nod factors are active on host plants at very low
concentrations
MUTATION ON COMMON NODULATION GENES

28. MUTATION ON NODLOCUS GENES
Host-specific nod genes
EXO(Exopolysacchride) genes
 Abnormal root reactions
 root hair deformation.
 Change host specificity,
 Substrate for signal production,
 Masking function during invasion.
 The typical plant cell division pattern and root deformation, leading to
nodule formation although these are often empty (no bacteroids).

29. Nif ( Nitrogen fixation)gens
 The nif gene are genes encoding enzymes involved in the fixation
of atmospheric nitrogen into a form of nitrogen available to living
organism.
 Nif gens also encode a number of regulatory proteins involved in
nitrogen fixation.
 The nif genes are found in both free-living nitrogen fixing bacteria
and symbiotic bacteria associated with various plants.

30. EARLY AND LATE NODULIN GENES
Depending on the time point of activation, these genes are called early or late
nodulin genes.
 The early nodulin are playing role in signalling communication during
infection process .
 Enod1,enod12 and enod40 are well characterized as early nodulin genes.
 The late nodulin are promoting the conversion of bacteria into bacteriods and
assist the settlement of bacteria inside nodules.
 While leghaemoglobin and uricase are examble of late nodulin genes with
known function.
Both of the two types nodulin genes are playing pivotal role in nodule
development.

31. STEM NODULATING BACTERIA
 Stem nodulating bacteria grows only in tropical regions where soils are
often nitrogen deficient because of leaching and intense biological activity.
Examble : Tropical aquatic legume Sesbania - Azorhizobium caulinodans.
 Some stem-nodulating rhizobia produce bacteriochlorophyll a particularly
in association with tropical legumes.

32. Case study...

33. YEAR OF PUPLICATION: 2017
AUTHOR :DAVID DENT AND EDWARD COCKING

34.  Gluconacetobacter diazotrophicus (Gd), a non-
nodulating, non rhizobial, nitrogen-fixing
bacterium isolated from the intercellular juice of
sugarcane.
 Shown to intracellularly colonize.
 Seed inoculum technology based on Gd (Nfix) is
able to significantly improve yields of wheat,
maize, oilseed rape and grasses.
 Bacterial intracellular colonization and nitrogen
fixation in plant without any need for nodulation.
 Nitrogen-fixing bacteria establish an intracellular
symbiosis which they fix nitrogen inside the cells.
GD ISOLATION

35. AUTHOR :ABDELAAL SHAMSELDIN
YEAR OF PUPLICATION: 2013

36.  On the contrast to the well known, that there are other
different genes involved in symbiotic nitrogen fixation in
addition to nod, nif and fix genes.
 Among the other genes that involved in this process, are
genes that could be related to host specificity, competition
and infection of nodulation process.
 In addition to the signals from the host plant that are
important for plant-Rhizobium communications system. This
review article will highlight the discovering of new
symbiotic genes and their roles in nitrogen fixation.

38. AUTHOR : Qi wang et al.,
YEAR OF PUPLICATION: 201

39.  Establishment of a successful symbiosis requires the two symbiotic
partners to be compatible with each other throughout the process of
symbiotic development.
 However, incompatibility frequently occurs, such that a bacterial
strain is unable to nodulate a particular host plant or forms nodules
that are incapable of fixing nitrogen.
 Genetic and molecular mechanisms that regulate symbiotic
specificity are diverse, involving a wide range of host and bacterial
genes/signals with various modes of action.
 In this review, we will provide an update on our current knowledge
of how the recognition specificity has evolved in the context of
symbiosis signaling and plant immunity.

40. Future Directions
 Enhancing survival of nodule forming bacterium by improving
competitiveness of inoculant strains.
 Extend host range of crops, which can benefit from biological
nitrogen fixation.
 Engineer microbes with high nitrogen fixing capacity.
 May exhibit several plant growth-promoting effects, such as
hormone production, phosphate solubilization, and the suppression
of pathogens.

41. CONCLUSION
 The application of genetics and modern molecular methods to the
study of symbiotic N2 fixation has led to great advances in our
understanding of nodule formation and function.
 Much of the scientific groundwork in this area has already been
done and new GM methods are being developed at astonishing
speeds.
 We now have the full genome sequence of multiple rhizobial
strains and sequencing of the genomes of M. truncatula,L.
japonicus, and most recently soybean is well underway.
 The information and tools developed from knowledge of these
genomes will maintain the pace of discovery in symbiotic N2-
fixation research.

42.  More and more connections are being found between
nodulation and other pathways involved in normal plant
development (e.g., CLAVATA1), which promises to reveal
how nodulation is fully integrated into plant development
and metabolism.
 As we now face challenges brought on by the rising cost of
fossil fuels which are needed for fertilizer production and
further degradation of our natural resources advances in our
understanding of symbiotic nitrogen fixation may present
opportunities for finding solutions to these problems.

43. THANK YOU