Root nodule symbiosis allows legumes to fix atmospheric nitrogen through their partnership with rhizobia bacteria. The plant provides carbohydrates to the bacteria through photosynthesis in exchange for fixed nitrogen. Efforts aim to transfer this nitrogen-fixing ability to cereals like wheat and rice by introducing them to rhizobial signaling molecules and engineering nodule-like root structures. However, successfully achieving nitrogen fixation in non-legumes faces challenges due to the oxygen sensitivity of the nitrogenase enzyme and complexity of the nitrogen-fixing process.

2. SUBMITTED TO:
PROF. DR. TARIQ MANZOOR
SUBMITTED BY:
SAIMA IQBAL(99), NIMRA KHAN(113)
ANILA KHALID(121) AND UME KALSOOM(162)

3. NITROGEN FIXATION IN LEGUMES AND
CEREALS CROPS

5. Root nodule symbiosis
Most plants that form
nitrogen-fixing nodules
are legumes. Their
partners are diverse
bacteria collectively
called rhizobia
Unrelated Frankia
bacteria nodulate a
diverse group of
plants called
actinorhizal plants
Casuarina equisetifolia
Root nodules of the legume
Medicago truncatula inoculated
with Sinorhizobium meliloti

6. Root nodule symbiosis is a mutually
beneficial arrangement
N2 N2
NH4
+
Glutamine
NH4
+
CO2
Light
Carbohydrate
Legume with
nitrogen-fixing
nodule
Bartering reduced molecules
The plant provides organic carbon
derived from photosynthesis, and
the bacteria provide fixed nitrogen.
Most rhizobia cannot fix nitrogen
except in nodules

7. v
Fixing nitrogen to fertilize plants
accounts for ~2% of global energy use
CO2

8. Biological nitrogen fixation
2 NH3
N2
Nitrogenase
16 ATP
Many prokaryotes can fix nitrogen
using an enzyme called
nitrogenase. This process uses a
great deal of cellular energy, ATP.
3 H2

9. Symbiotic nitrogen fixation
Actinorhizal plants like
alder with symbiotic
Frankia bacteria
Legumes with
symbiotic
rhizobia
Some nitrogen-
fixing bacteria
form an intimate
partnership with
plants in the
form of nitrogen-
fixing nodules

10. The rhizobia-legume symbiosis
• The most sophisticated N2-
fixing symbiosis
• The nodule is a unique root
organ designed to support
endosymbiotic rhizobia and
N2-fixation
• Upon infection, the rhizobia
differentiate into bacteroids
• Bacteroids are enclosed in
the plant cell by the
symbiosome
• Symbiosomes are
surrounded by a specialized
plant membrane - the
peribacteroid membrane.
Root nodules
Cross-section of the
nodule
Bacteroids inside symbiosomes
Bacteroids inside a
nodule
10 μm

11. Legumes are important ecologically
and as food and fodder crops
Lupines and other legumes are
pioneer plants that can grow in
disturbed or infertile soils
Some legumes are too
successful and become a
pest, such as the
invasive legume kudzu
(Pueraria montana)
Legumes provide protein to
humans and other animals

12. RHIZOBIA AND LEGUME FUNCTIONS
legume
Fixed nitrogen
(ammonia)
rhizobia
Fixed carbon
(malate, sucrose)

13. Steps in nodule development
Communication
Root hair
curling
Infection
thread
formation
Cell
division
in root
Nodule
expressing
leghemoglobin
Generalized steps in the formation of a nodule
N2
NH4
+
Bacteroid
Plant root
Rhizobia

14. Communication:
Flavonoids and Nod factors
1. The plant root
produces specific
flavonoids that
attract rhizobia
2. Most rhizobia produce
Nod factors, identifying
them as appropriate
symbionts
3. The plant prepares to
form a symbiotic nodule
structure
Rhizobia
Plant cell

15. Nod factor perception induces root
hair curling
• Nod-factors are
concentrated in the
cell wall and are
almost immobile
• Nod-factors cause
redirection of tip
growth (shown in a)
• Only a few bacteria
actually redirect the
growth of the root hair
successfully and
become enveloped in
an infection thread
time

16. Bacterial entry and nodulation process
1. Root hair curling
2. Infection thread (IT)
formation and cortical cell
division (CCD)
3. Nodule primordium (NP)
formation
4. Nodule development with
formation of nodule
meristem (in some
legumes)

17. Symbiotic nitrogen fixation requires
teamwork
N2 N2
NH4
+
Gln
NH4
+
CO2
Light
Carbohydrate
Plants cannot fix nitrogen on
their own, and most rhizobia
cannot fix nitrogen on their
own.
Symbiotic nitrogen fixation is
a true partnership.
The bacteria provide
nitrogenase.
The host plant provides
leghemoglobin,
homocitrate, carbon
sources, organic
nitrogen…..

18. Bacteroids need a high O2 flux but low O2
environment
C6H12O6
O2
A high affinity cytochrome
oxidase in the bacteroid
functions at low oxygen
concentrations
A low oxygen environment
is maintained by an oxygen
permeability barrier
Oxidative phosphorylation
requires oxygen for ATP
production
Leghemoglobin buffers
oxygen and deliver it to
respiring symbiotic cells
Leghemoglobin gives
nodules their pink
colour

20. WHY TO FOCUS CEREALS?
• Top consumed crops
belong to this
category.
high yielding varities
during green revolution.
• Need large amount of
inorganic fertilizers.
• Comparatively low N
use efficiency.

21. CEREALS….. BUT HOW?
• Engineering cereal crops to fix nitrogen
without compromising their yield potential.
• Possible if the ability to percieve rhizobial
signaling molecules and formation of an
oxygen-limited, nodule-like root organ can be
transferred to cereal plants.
• Potential criticism: cereals is the potential for
a yield penalty associated with the increased
demand on photosynthates required to
support nitrogen fixation.

22. BIOTECHNOLOGICAL APPROACHES TO
TARGET CEREALS
• Transferring the legume-rhizobial interaction to cereals roots.
• Utilizing endophytic diazotrophs that infect cereals to fix nitrogen
for their host plants.
• Introduction of nitrogenase enzyme into organelles of plant cells to
create a new nitrogen-fixing capability.
 Highly complex enzyme
 High energetic emands
 Irreversibly denatured by oxygen

23. TRANSFERRING THE LEGUME-RHIZOBIAL
INTERACTION TO CEREALS ROOTS
• Nod Factor: the signaling molecule
• Four genetic processes to be introduced
–Recognition of Nod factors
–Organogenesis of the root nodule
–Bacterial infection
–Establishment of a suitable environment for
nitrogenase activity inside the nodule

24. The connecting link: SYM Pathway
• Nod factors and Myc factors perception leads to the
activation.
• Well conserved between legumes and monocotyledons
• The SYM pathway present in cereals and essential for
supporting the mycorrhizal symbiosis.
• A number of OoSYM signalling components (CASTOR,
CCaMK, and CYCLOPS): complement legume mutants,
not only for mycorrhization, but also for nodulation.
– Longer domain length versions: able to support nodulation
signalling.

25. Conti…
• Nitrogen fixation: transferred from Klebsiella to E.
coli in 1972
• But transfer to a eukaryote (including a plant) or
engineering a stable association between a
nitrogen-fixing bacterium and a cereal crops has
remained elusive:
– Requires simultaneous transfer of 9-20 genes, most of
which are essential.
– Very fragile system with activity being lost quickly
when the expression of any gene is suboptimal.

26. Conti…
• Successful transfer of
nitrogen fixation to the
facultative anaerobe
E.coli by refactoring
nitrogen fixation
cassettes.
Most microbes that
efficiently colonize
plants as associative
bacteria or endophytes
are aerobic organisms.
• Challenge of
transferring to aerobic
microorganisms :
nitrogenase is oxygen
labile.
 Some aerobes(Azotobactor
vinelandii, Azorhizobium
caulinodans) : capable of
free-living nitrogen
fixation.

27. Summary
Symbiotic
microorganisms are
intimate allies of
plants
These symbioses are
mutualistic
associations in which
both partners benefit
The plant uses a
core SYM pathway
for symbioses with
mycorrhizal fungi and
rhizobia