The document outlines the process of biological nitrogen fixation, emphasizing the symbiotic relationships between certain prokaryotes and plants, particularly legumes and their interactions with rhizobia. It details nodule formation mechanisms, distinctions between determinate and indeterminate nodules, and the molecular dialogues that facilitate this relationship, highlighting the roles of flavonoids and nod factors. Additionally, it explains the structural and functional aspects of the nodules, the transformation of rhizobia into bacteroids, and the exchange of nutrients between the bacteria and the host plant.

1. DR. VIBHA KHANNA
Asso. Prof. (Botany)
SPC GOVERNMENT COLLEGE
AJMER (Rajasthan)

2. PLANT BIOCHEMISTRY
• BLOCK 3. : Nitrogen Metabolism
– PRESENTATION 3:
Biological Nitrogen Fixation:
Nodule Formation And Nod Factors

3. Symbiotic Nitrogen Fixation
• Biological nitrogen fixation is a process that can only be
performed by certain prokaryotes.
https://www.slideshare.net/vibhakhanna1/biological-nitrogen-
fixation
• In some cases, such bacteria are able to fix nitrogen in a
symbiotic relationship with plants.
• There they find the proper conditions for reducing
atmospheric nitrogen into ammonia.
• Symbiosis – term coined by De Bary.
• Three categories:
 Nodule formation in leguminous plants
 Nodule formation in non leguminous plants
 Non nodulation

4. Symbiotic Nitrogen Fixers
 Non nodulation
– Lichens – Cyanobacteria
– Anthoceros – Nostoc
– Azolla – Anabaena azollae
– Cycas – Nostoc , Anabaena
 Nodule formation in leguminous plants:.
• Bacteria of the genera
– Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium,
and Sinorhizobium
(collectively referred to as Rhizobium or rhizobia) are able to
establish an endosymbiotic association with legumes.
• Under nitrogen-limiting conditions, the leguminous plants can
form root nodules, in which the rhizobia are hosted
intracellularly.
• Stem nodules are present in Sesbania rostrata, for Nitrogen
fixation.

5. Symbiotic Nitrogen Fixers
• Nodule formation in non-leguminous plants:
– Root nodules
Alnus – Frankia
Myrica gale – Frankia
Parasponia – Rhizobium
– Leaf nodules
Dioscorea
Psychotria
• Legumes (Fabales) interact with single‐celled
Gram‐negative bacteria, collectively termed rhizobia,
whereas members of three other Rosid orders (Fagales,
Cucurbitales and Rosales) interact with Gram‐positive
filamentous actinobacteria of the genus Frankia.

6. Nodule formation in leguminous plants
• Symbiotic Rhizobia are classified in two groups:
– Fast-growing Rhizobium spp. whose nodulation functions
(nif, fix) are encoded on their symbiotic megaplasmids
(pSym)
– Slow-growing Bradyrhizobium spp. whose N-fixation and
nodulation functions are encoded on their chromosome.
• There are also two types of nodule that can be formed,
depending on the host plant:
– determinate and
– Indeterminate
• Although the morphology and ontology of these two
nodule types is different, the mechanisms underlying
the formation of them are very similar.

7. Determinate nodules
• Formed on tropical legumes by Rhizobium and
Bradyrhizobium
• Meristematic activity not persistent - present
only during early stage of nodule formation;
after that, cells simply expand rather than
divide, to form globose nodules.
• Nodules arise just below epidermis; largely
internal vascular system

8. Indeterminate nodules
• Formed on temperate legumes (pea, clover,
alfalfa); typically by Rhizobium spp.
• Cylindrical nodules with a persistent
meristem; nodule growth creates zones of
different developmental stages
• Nodule arises near endodermis, and nodule
vasculature clearly connected with root
vascular system

9. Structure of Indeterminate Root Nodule

10. Initiation of Symbiotic Association
• Legume-Rhizobium symbiosis starts with a
molecular dialogue between the two partners.
• The legume secrete various phenolic
molecules, predominantly flavanoids and
isoflavanoids, into the rhizosphere.
• These signals are taken up by rhizobia, bind
the transcriptional regulator Nod factor, NodD,
and activate various bacterial nodulation
genes, the early nodulin or ENOD genes.

11. Host specificity due to Nod factor
There are two modes of specificity
for Rhizobium legume symbiosis.
The 1st is the interaction
between the flavonoids,
the chemical signals that
are derived from the
legumes.
The 2nd mode of
specificity is dependent on
the structure of the Nod
factors themselves.

14. Bacteroid
• Bacteria are released from the infection thread into the
cytoplasm of the host cells, but remain surrounded by the
plant derived peribacteroid/ symbiosome membrane.
• Failure to form the peribacteroid membrane results in the
activation of host defenses and/or the formation of
ineffective nodules.
• Leghaemoglobin is restricted to the host cell cytoplasm,
outside the peribacteroid membrane.
• Rhizobia within the swollen, polyploid cells of root nodule
change form, to become non-motile, endosymbiotic which
establish symbiotic association with the host plant.
• Thus, the Rhizobia switch into the ‘bacteroid’ form.

15. Bacteroid-legume Symbiotic Association
• The nodule provides an oxygen-
controlled environment
(leghemoglobin = pink nodule
interior) structured to facilitate
transport of reduced nitrogen
metabolites from the bacteroids to
the plant vascular system, and of
photosynthate from the host plant
to the bacteroids.
• Rhizobia begin nitrogen fixation and
provide the legume with amino
acids, such as glutamine and
asparagine, which are made in the
nodule.
• The legume provides simple sugar
produced from plant catabolism to
the rhizobia in the nodule.

17. NOD FACTORS
• Nodulation (Nod) factors are key signal molecules that play
an important role during initiation of nodule development
and bacterial invasion.
• Nod factors consist of an oligomeric backbone of β-1,4-
linked N-acetyl-D-glucosaminyl residues, N-acylated at the
non-reducing-terminal residue and thus are lipo-
chitooligosaccharides (LCOs)
• Nod factors are strain-specific.
– The Nod factor structure differs in the number of GlcNAc
residues present in the chitooligosaccharide backbone, in the
nature of the fatty acyl group, and in the substituents at the
nonreducing- and/or reducing-terminal residues.
– In a few cases, Nod factor substituents are found at nonterminal
GlcNAc residues.

18. Synthesis of Nod factors
• Nod factor synthesis depends on the expression
of nodulation (nod) genes, comprising
the nod, nol, and noe genes.
• These genes are not expressed in free-living
bacteria, with the exception of nodD, which is
expressed constitutively.
• NodD binds to specific flavonoids secreted by the
roots of the host plant ; upon flavonoid binding, it
becomes a transcriptional activator of the other
nod genes, which encode enzymes involved in the
synthesis of Nod factors.

20. Early nodulation events are induced by
Nod factors
• These plant responses occur at the epidermis, cortex, and pericycle and
are summarized below:
• Epidermis: Formation of new root hairs (Hai); Root hair deformation
(Had), branching, and tip swelling; Induction of root hair curling (Hac);
Changes in cytoplasmic streaming; Cytoskeleton rearrangements;
Intracellular & Extracellular alkalinization; Depolarization of membrane
potential; Modulation of proton and (calcium) ion fluxes; Changes in
Ca2+ (spiking plateau-like increases); Induction of early nodulin genes.
• Cortex: Polar tip growth of outer cortical cells; Formation of pre-infection
threads; Formation of nodule primordia (initiated in the inner cortex or the
outer cortex); Local induction of cell cycle genes; Induction of early nodulin
genes.
• Whole root: Induction of a dimer-forming Nod factor hydrolase; Induction
of enzymes with chitinase activity; Enhancement of mycorrhizal
colonization; Disturbance of auxin transport in cortex and vascular
bundles;

21. Nodulins
Bacterial
attachment
Root hair
invasion
Bacteroid
development
Nitrogen
fixation
Nodule
senescence
early nodulins
late
nodulins
Nodulins?
Pre-infection
Infection
and nodule
formation
Nodule
function and
maintenance
Nodule
senescence

22. Signal transduction
• Perception by (a) Nod factor receptor(s) may be an initial and essential
requirement for Nod factor signaling.
• Nod factors are recognized by a specific class of receptor kinases that
have so-called Lysm (lysin motif) domains in their extracellular domains.
• Nod gene expression is induced by the presence of certain flavonoids in
the soil, which are secreted by the plant to attract the bacteria.
• 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.
• Nod factors induce root-hair curling such that it envelops the bacterium.
• This is followed by the localized breakdown of the cell wall and the
invagination of the plant cell membrane, allowing the bacterium to form
an infection thread and enter the root hair.
• The end result is the nodule, the structure in which nitrogen is fixed.
• Nod factors act by inducing changes in gene expression in the legume,
most notable the nodulin genes, which are needed for nodule
organogenesis.