The document discusses biological nitrogen fixation in cereal crops. It provides background on nitrogen and its importance for plant life. It describes how only certain prokaryotes and legumes can fix nitrogen. Efforts aim to transfer the nitrogen fixing ability of legumes to cereals by engineering cereal crops to recognize rhizobial signaling molecules and form nodule-like structures for nitrogen fixation. Key challenges include the oxygen sensitivity of the nitrogenase enzyme and competing demands for photosynthates. Transferring the nitrogen fixing genes or utilizing nitrogen fixing endophytes that associate with cereals are also approaches explored.

1. BIOLOGICAL NITROGEN
FIXATION IN CEREAL
CROPS
Amandeep Kaur
L-2014-A-12-D

2. Nitrogen and Plant Life
 Fourth most common element
 Component -Proteins, NAs,
PGRs, chlorophyll,…
 Bioavailable forms: nitrate (NO3-)
and ammonia (NH4+)
 Serves as an electron acceptor
in anaerobic environment
 Most limiting nutrient in soil and
water
Nitrogen
Ammonia
Nitrate
(NO3)
Nitrite
(NO2)
Urea and
Organic
Nitrogen
Atm.
Dinitrogen
(N2)

3. Nitrogen Cycle
Prokaryotesnitrification
N2
dinitrogen gas
(78% of air)
NH4
+
ammonium
NO2
-
nitrite
NO3
-
nitrate
N2O
nitrous oxide
Animals
Plants
assimilation
uptake
Biological nitrogen fixation
consumption

4. Ammonia Assimilatory Cycle
Amino acids
proteins purines
pyrimidines
glutamate glutamine+ ATP + ADP + Pi
GS
+NH4
+
glutamineglutamate -ketoglutarate
GOGAT
+
Pathway 1
NH4
+
Amino acids
proteins
-ketoglutarate glutamate
GDH
+
Pathway 2

5. Entry of Nitrogen into The
Picture
Nitrogen
entry
Fixation
Biological
Non
Biological
Fertilizers
1-5%
60-80%
25-30%

6. N Fertilizers
 Produced by the Haber-Bosch process
 Developed in 1913
 Primarily responsible for the green revolution, but also
responsible to large increase of reactive N in our
environment
Consumes 1.4% of
total fossil fuels
annually

7. Impacts of Industrial Nitrogen
 $100 billion per year global industry: ~120 Tgm Nitrogen (Rao, 2013)
 80% of which is used for agricultural use
 66% of N applied lost to the environment causing pollution:
Eutrophication
 Species changes/losses
Population
Explosion
Green Revolution
Chemical fertilizers
and high yielding
varieties

8. Biological Nitrogen Fixation
 Extremely energy consuming conversion because of
stability of triply bonded N2
 Produces fixed N which can be directly assimilated into
N containing biomolecules
N2 + 8 flavodoxin- + 8H+ + 16 MgATP2- + 18 H2O
+ 2OH- + 8 flavodoxin + 16 MgADP- + 16H2PO4
- + H2
nitrogenase
2NH4
+

9. Only Prokaryotes Are Nitrogen
Fixers
 Need nitrogenase to fix/reduce N2 directly to ammonia
 Can be free living or symbionts, photosynthetic or heterotrophic
bacteria or cyanobacteria
 All need low/zero O2 and high C levels

10. BUT…..
Only a small number of
economically important
plants can fix their own
organic N
i.e., Legumes

11. Legume’s Partner: Rhizobium
legume
Rhizobia
Fixed carbon
(malate, sucrose)
Fixed nitrogen
(ammonia)

12. Overview of Rhizobium Nitrogen
Fixation

13. Overview of Rhizobium Nitrogen
Fixation

14. Nodules : Home for Bacteroids

15. Genetics of nitrogen fixation

17. Nitrogenase Complex
Dixon and Kahn, 2004

18. Nitrogenase Complex
Schematic representation of the electron flow in nitrogen
fixation
Taiz and Zeiger, 2006

19. Biochemistry of Nitrogen
Fixation
Madigan et al., 2000

20. Why To Focus Cereals ???
 Top consumed crops belong to this category
 High yielding varieties during green revolution
 Need large amount of Inorganic fertilizers
 Comparatively low N use efficiencies

21. Cereals… But How ???
 Engineering cereal crops to fix nitrogen without compromising
their yield potential.
 Possible if the ability to perceive rhizobial signalling 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: expression of at least 16 nif genes
 High energetic demands
 Irreversibly denatured by oxygen.

23. Transferring the Legume-Rhizobial
Interaction to Cereals Roots
 Nod Factor : the signalling 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 OsSYM 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. Engineering Nod Factor Perception
and Activation of SYM Pathway
Journal of Experimental Botany doi:10.1093/jxb/eru098
Targets for cereal engineering: Nodulation-specific
components

26. Contd..
 Nucleopore-complex components (NUP85, NUP133, NENA) :
conserved in cereals and even in non-symbiotic species such
as Arabidopsis.
 Probably function without modification in engineered cereals.
 POLLUX and NSP2 present in non-symbiotic Brassicaceae.
 May have a non-symbiotic function or function in bacterial
associations (Bulgarelli et al. 2012; Lundberg et al. 2012).

27. Engineering Nodulation-specific
Outputs of SYM Pathway
Journal of Experimental Botany doi:10.1093/jxb/eru098
 Nodulation signalling components : red
 Mycorrhizal signalling components : blue

28. Engineering Nitrogen
Fixation in Plant
Colonizing Bacteria
Engineer increased
colonization between
plants and highly efficient
N2-fixing microbes
Engineer transfer of
efficient nitrogen fixation
into bacteria that already
associate closely with
cereals


29.  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 crop 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

30.  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 (Azotobacter vinelandii, Azorhizobium
caulinodans) : capable of free-living nitrogen fixation.

31. Free-living Nitrogen Fixating
Aerobes
 Maintaining high rates of oxygen consumption at the cell
membrane via respiration
 Via an alginate oxygen diffusion barrier
 Conformational protection of nitrogenase by interaction
with a specific iron-sulfur protein, Shetna protein
 Rapid reduction of oxygen by remodelling of the electron
transport chain to contain alternate terminal oxidases e.g.,
cytochrome bd, in A. vinelandii

32. Transfer To Aerobic Associative
Bacteria
 Large nitrogen fixation island from Pseudomonas stutzeri transferred
to the aerobic associative bacterium Pseudomonas protegens Pf-5
(Setten et al. 2013)
 Inoculation of Arabidopsis with transgenic P. protegens resulted in
significant growth promotion effects compared under nitrogen-limited
conditions.
 Constitutive nif expression

33. Advances in Genetic Transfer of
Nitrogen Fixation to E. coli.
Current Opinions in Biotechnology (2015) 32: 216-2

34. Mechanism of Electron Transfer to
Nitrogenase
 Well established for anaerobes, but not demonstrated for
aerobic nitrogen fixation.
 Klebsiella pneumoniae : electrons transfer to nitrogenase by
flavodoxin NifF which is reduced by the pyruvate:flavodoxin
oxidoreductase NifJ
 In aerobic bacteria reduction is carried out by the pyruvate
dehydrogenase complex : a less powerful reductant than
flavodoxin or ferredoxin.

35. Model of Traits to Support Aerobic
Nitrogen Fixation and Transfer to
Cereals

36.  Rnf complex and FixABCX (membrane-associated
complexes) : transfer electrons to nitrogenase during
aerobic nitrogen fixation.
 FixABCX : widely distributed among aerobic nitrogen fixing
bacteria including Rhizobia, to bifurcate electrons between
ferredoxin and NADH or a quinone
 Essential for symbiotic nitrogen fixation
 FixAB belongs to Electron Transfer Flavoprotein (ETF) family
 FixCX is related to ETF-quinone reductase

37. Other Points To Consider
 Alteration in the amount of Ammonium released during
nif cluster transfer
 Too high could be toxic to the cells
 N2-fixation is energetically demanding
 Newly introduced microorganisms will be forced to compete
for carbon with the native microbiota.
 Can be provided with a specialized carbon source that the
general microbiota cannot catabolize.

38. Other Points To Consider
 Transfer of ‘Nif’ into Organelles
 can provide the high concentration of adenosine 5’ -
triphosphate and reducing power required for nitrogenase
activity.
 Chloroplast genomes of ferns, mosses, and gymnosperms
encode an oxygen-sensitive enzyme related to nitrogenase
(Muraki et al. 2010)

39. Conclusions and Future
Perspectives
 Engineering the nitrogen-fixing capability into cereal crops is
still an enormously challenging task.
 The extensive conservation of SYM pathway components in
rice (and other cereals) indicates they have an innate potential
for engineering the SYM pathway to allow recognition of
nitrogen-fixing bacteria.

40.  Success of engineering Nod factor signalling may provide a
solid foundation for inducing nodule organogenesis, bacterial
infection, and supporting nitrogen fixation in cereal roots.
 The impact of engineering first step of rhizobial recognition
and nodulation-specific signalling may allow a better degree
of bacterial colonization.
 Near or far, but future may lead to engineering a nodule
based symbiosis in cereal crops.

41. References
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Fixation for Sustainable Agricultural Systems in the Tropics. Soil Biology and Biochemistry, 29: 787-799.
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Microbiology. Pp 1-4.
 Geddes BA, Ryu M-H, Mus F, Costas AG, Peters JW, Voigt CA and Poole P. (2015). Use of plant colonizing
bacteria as chassis for transfer of N2-fixation to cereals. Current Opinion in Biotechnology, 32: 216–222.
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Increased Yield in Landraces of African Cereal Crops. African Journal of Biotechnology, 3: 1-7.
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 Olivares J, Bedmar EJ and Sanjuán J. (2013). Biological Nitrogen Fixation in the Context of Global Change.
Molecular Plant-Microbe Interactions. 26 (5) : 486–494.

42.  Rao, DLN. (2014) Recent Advances in Biological Nitrogen Fixation in Agricultural Systems. Proc Indian
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