Plant physiology

1. Nitrogen fixation

2. Presentation flow
1. Introduction
2. Role of N2 in plants
3. Sources of N2
4. N2 fixation
5. Factors affecting N2
fixation
6. Types of N2 fixation
7. Industrial N2 fixation
8. Non biological N2 fixation
9. Biological N2 fixation
10. Non symbiotic N₂ fixers
11. Symbiotic N₂ fixers
11.Nodule formation & non-
nodulation
12.Nitrogen fixing organisms
13.Structure and function of nodule
14.Site and mechanism of nitrogen
fixation in nodules
15.Biochemistry of N₂ fixation
16.Notrogenase complex
17.Nitrogenase
18.Pathway of nitrogen fixation
19.Mechanism of N2 fixation
20.Nitrogenase protection
mechansims
21.Genes involved in Nitrogen
fixationNitrogen fixation 2

3. Introduction
• Free atmospheric nitrogen is involved in nitrogen cycle, where
it forms inorganic nitrogen to be used by plants and again
released in atmosphere after decomposition.
• Nitrogen content of different systems is as follows:
Atmosphere: 79/100 (as N2),
Plants: 5-2/100(mostly organic)
Soil: 0.5-5/1000(mostly organic).
(i) N gain process: From N2 to microorganisms, and
eventually to plant
(ii) N oxidation: ammonia is oxidized to nitrate
(nitrification)
(iii) N loss process: Nitrate is ultimately lost as N2 (return
to atmosphere) Nitrogen fixation 3

4. 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
Nitrogen fixation 4

5. Sources of nitrogen
• Atmospheric Nitrogen
–78% of atmosphere
–Plants cannot utilize this form
–Some Bacteria, Blue Green Algae &
leguminous plants
• Nitrates, Nitrites and Ammonia
• Amino acids in the soil
• Organic Nitrogenous compounds in insects
Nitrogen fixation 5

6. Nitrogen fixation 6

7. Nitrogen Fixation
• It is the removal and reduction of atm.
• Molecular N2 & its subsequent incorporation with other
elements to form nitrogenous compounds.
• “Nitrogen Fixation” is the process that causes the strong two-
atom nitrogen molecules found in the atmosphere to break
apart so they can combine with other atoms.
• Nitrogen gets “fixed” when it is combined with oxygen or
hydrogen.
Nitrogen fixation 7

8. Factors affecting N2 fixation
1. Presence of nitrate or ammonium
2. Presence of certain inorganic substances Ca, Co, Mo –
influence N2 fixation along with P
3. Availability of energy source – addn. of C source increase N2
fixation
4. pH : Neutral – favours Azotobacter – Acidic- Beijerinkia
5. Soil moisture : Adequate is good for fixation
6. Temperature: Mesophilic – 30°C.
Nitrogen fixation 8

9. Types of N2 fixation
There are three ways that nitrogen gets “fixed”
• Industrial N2 fixation
• Non biological N2 fixation
• biological N2 fixation
Nitrogen fixation 9

10. Industrial N2 fixation
• Accomplished by Haber -Bosch process
• Developed in Germany 1914 by Fritz haber & Karl bosch
• Process- N2 and H2 react with each other in presence of
- Industrial catalyst( nickel / iron)
- High temperature about 500 ͦ c
- High pressure – 200 atm
To form NH3
• Source of H2 - is methane (natural gas)
• Industrial production of fertilizers and explosives
Nitrogen fixation 10

11. Non biological N₂ fixation
• Non biological/ physico chemical N2 fixation involves the
photochemical & electro chemical conversion of atm.N₂ to
soil NO2, NO3, NH3.
• It is brought about by ionizing phenomena such as cosmic
radiations, meteor trails, lightning, thunderstorms, volcanic
eruptions etc.
• These provides high energy for breaking N≡N & also for the
formation of free N₂ with oxygen or hydrogen of atm H₂0
combination of N₂ with o₂ forms nitrous and nitric oxides.
• Combination of N₂ with hydrogen forms NH3
• Nitrous and nitric oxides get hydrated with atm. Water vapour
and forms nitrous and nitric acids
Nitrogen fixation 11

12.  Rain water bring these acids and NH3 to soil surface
 There, the acids react with metallic ions and form metallic
nitrates.
 These nitrates and NH3 enrich the surface soil
 Non biological N₂ fixation amounts to only ˂ 10% ofthe
natural N₂ fixation
 It is common in some tropical regions, where thunder bolt are
frequent.
Nitrogen fixation 12

13. Nitrogen fixation 13

14. Biological Nitrogen Fixation
• Biological nitrogen fixation was discovered by the German
agronomist Hermann Hellriegel and Dutch microbiologist
Martinus Beijerinck.
• Biological nitrogen fixation (BNF) occurs when atmospheric
nitrogen is converted to ammonia by an enzyme called
nitrogenase
The reaction for BNF is:
N2+16ATP+8H++8 e− → 2 NH3+H2+16ADP+16Pi
• BNF is mostly accomplished by microorganisms called
diazotrophs or N₂ fixers
• They include some species of bacteria, fungi, blue green algae,
lichens etc.
Nitrogen fixation 14

15. Special features of diazotrophs
• Free living –bacteria like azotobacter, rhodopseudomonas fix
atm. N₂ & also protects nitrogenase enzyme – sensitive to 0₂
• They produce exopolysaccharide (slime) which retains water
and prevents diffusion of 0₂ inside cell during N₂ fixation
• Azotobacter have exceedingly high rate of respiratory
metabolism thus preventing 0₂ retension inside the cell
• Blue green algae –are of 2 kinds
• One that possess heterocyst
• Other that devoid of it ( non heterocystous)
Nitrogen fixation 15

16. • Filamentous cyanobacteria contain pale, thick walled, hollow
cells called heterocyst
• Heterocyst are the site of N₂ fixation
• They lack PSII and photosynthetic bile proteins
• non heterocystous N₂ fixing BGA like (oscillatoria) - the
filaments are arranged clumps and N₂ fixation takes place in
internaly organised cell having reduced conditions
• Azospirillum paspalum survive in microaerophilic conditions
associated with the rhizosphere ( area surrounding the roots )
of paddy plants - fix atm. N₂ in the rhizosphere.
Nitrogen fixation 16

17. Nitrogen fixation 17

18. • Soil N₂ fixers are of two categories
1. Non symbiotic N₂ fixers
2. Symbiotic N₂ fixers
Nitrogen fixation 18

19. Non symbiotic or free living N2 fixers
• They inhabit both terrestrial & aquatic habitats
- Fixation carried out by free living microorganisms
- Aerobic, anaerobic and blue green algae
• Free living nitrogen fixers
– Free living aerobic : Azotobacter, Beijerenckia
– Free living anaerobic : Clostridium
– Free living photosynthetic : Chlorobium,
– Rhodopseudomonas
– Free living chemosynthetic :Desulfovibro,Thiobacillus
Nitrogen fixation 19

20. • Free living fungi: yeasts and Pillularia
• Blue green algae:
- unicellular – Gloeothece , Synechococcus
- Filamentous (non heterocystous) – Oscillatoria
- Filamentous ( heterocystous) – Tolypothrix,Nostoc ,
Anabaena
Nitrogen fixation 20

21. • Ability to fix N by MO was confirmed by-technique -
acetylene reduction to ethylene - diazotropic MO
• Conversion is controlled by an enzymatic complex (or
nitrogenous enzyme)-reduce gaseous N - ammonia.
• Nitrogenase – sensitive to O.
• Several groups of MOs fix N in presence of minute
quantities of O - Microaerobic fixers - Spirilla
(Aquaspirillum & Azospirillum).
• O-not only inhibits the activity of nitrogenase-but also
regulate biosynthesis.
Nitrogen fixation 21

22. Symbiotic N₂ Fixers
• Fixation of free nitrogen by micro-organisms
living in soil symbiotically inside the plants
• ‘Symbiosis’ – coined by DeBary
• Three categories
– Nodule formation in leguminous plants
– Nodule formation in non-leguminous plants
– Non nodulation
Nitrogen fixation 22

23. Nodule formation in leguminous plants
• 2500 sp. Of family leguminosae ( Cicer arientium,
Pisum, Cajanus, Arachis) produce root nodules with Rhizobium
spp.
• They fix Nitrogen only inside the root nodules
• Association provides-food and shelter to bacteria
-bacteria supply fixed nitrogen to plant
• Nodules may buried in soil even after harvesting – continue
nitrogen fixation
Nitrogen fixation 23

24. Nodule formation in non-leguminous
plants
• Some other plants also produces root nodules
– Causuarina equisetifolia – Frankia
– Alnus – Frankia
– Myrica gale – Frankia
– Parasponia – Rhizobium
• Leaf nodules are also noted
– Dioscorea, Psychotria
• Gymnosperms - root – Podocarpus,
- leaves – Pavetta
zinumermanniana, Chomelia
Nitrogen fixation 24

25. Non-nodulation
• Lichens - cyanobacteria
• Anthoceros - Nostoc
• Azolla – Anabaena azollae
• Cycas – Nostoc and anabaene
• Gunnera macrophylla - Nostoc
• Digitaria, Maize and Sorghum – Spirillum notatum
• Paspalum notatum – Azotobacter paspali
Nitrogen fixation 25

26. Nodule formation
• Root nodules formed due to infection of Rhizobium
• Free living bacteria growing, near the root of legumes unable to
fix nitrogen in free condition
• Roots of the legumes secrete some growth factors helps in fast
multiplication of bacteria
(E.g.) Pisum sativum secretes homo serine & also
carbohydrate containing protein Lectins over their surface
Nitrogen fixation 26

27. • Rhizobia are chemotactically attracted to root hair
• Medited by lectins, some attach to root hair cell wall
• Tryptophan is a component of the root hair exudate
• Trytophan is transformed by rhizobia to indole acetic acid
(IAA)
• This plant hormone causes the root hair to curl or branch around
the attached rhizobia
Nitrogen fixation 27

28. • Polygalacturonase , secreted by rhizobia or possibly by the
plant, depolymerizes & softens root hair cellwall
• Rhizobia gain entry into the root hair cell
• The root hair cell nucleus directs the development of infection
thread
• Infection thread ,a tube consisting of cell membrane &
surrounding cellulosic wall,
• Grows into root cortex & infects some tetraploid cells that
proliferate & form nodule tissue
• Rhizobia are released from infection thread ,lose their rod shapeNitrogen fixation 28

29. • Infected root cells swell and cease dividing.
• Bacteria within the swollen cells change form to become
endosymbiotic bacteroids, which begin to fix nitrogen
• 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.
Nitrogen fixation 29

30. Nitrogen fixation 30

31. Nitrogen fixation 31

32. Nitrogen fixation 32

33. Nitrogen fixing organisms found
in nodules• Rhizobium
Rod shaped,motile.etc.
Non sporing, non-acid fast.
Rhizobia are susceptible to antibiotics, bacteriophages,
fungicides, herbicides etc.
• Named after the host plant
– Pea – Rhizobium leguminosarum
– Beans – R. phaseoli
– Soyabeans – R. japonicum
– Lupins – R. lupini
• Two types of Rhizobium-
– Bradyrhizobium – slow growing spp.
– Rhizobium - fast growing spp.Nitrogen fixation 33

34. Structure and function of nodule
• Outermost layer of nodule-bacteriod zone-enclosed by cortical
cells.
• Rate of N- fixation of nodule directly proportional to the
volume of the nodule.
• Nodules are small-contain leghaemoglobin
• Leghaemoglobin
• Effective nodules are larger- pink in colour -due to the presence
of red coloured leghaemoglobin.
• This pigment is similar to haemoglobin of blood.
• Found in nodules between bacteriods & membrane envelops,
enclosing them.
Nitrogen fixation 34

35. Nitrogen fixation 35

36. • It is heme protein.
• Contain heme moiety attached to a peptide chainrepresent
globin part.
• The amount of leghaemoglobin in nodules has direct
relationship between amount of atm N fixed by legumes.
• MW-16,000-17,000 daltons.
• Functions:
-Represents an active site of N absorption and reduction.
-Acts as a specific electron carrier.
-Regulate the O –supply in the nodule.
-O-carrier.
Nitrogen fixation 36

37. Nitrogen fixation 37

38. Site and mechanism of nitrogen
fixation in nodulesSite
• Bacteroids-site of N-fixation.
Mechanism
• 1) Theory of Virtanen
N fixation in roots appear immediately after nodule formation.
-young plants fix N than the old plants.
A great part of N- converted to-L-aspartic acid and Lglutamic
acid.
Apart from this alpha-alanine present in nodule-produced from L-
aspartic acid by decarboxylation.
 small amount of Oxime-N and nitrite-N are also present.
2) Theory of Burris and Wilson –
hydroxylamine is the central compound of N fixation from which
ammonia is formed through reduction.Nitrogen fixation 38

39. Biochemistry of N₂ fixation
• N₂ fixers utilize atm. N₂ to synthesize NH3
• In this process , N₂ is first split up into free N₂ atoms by
breaking the triple bond , with help of enzyme nitrogenase.
• This reaction is endergonic (energy consuming), it requires an
input of nearly 160 kcal energy.
Nitrogen fixation 39

40. Nitrogen fixation 40

41. Nitrogenase Complex
Two protein components: nitrogenase reductase, and
nitrogenase
• Nitrogenase reductase is a 60 kD homodimer with a single 4Fe-
4S cluster
• Very oxygen-sensitive
• Binds Mg ATP
• 4 ATP required per pair of electrons transferred
• Reduction of N2 to 2NH3 + H2 requires 4 pairs of electrons, so
16 ATP are consumed per N2
Nitrogen fixation 41

42. Nitrogen fixation 42

43. Nitrogenase
A 220 kD heterotetramer
• Each molecule of enzyme contains 2 Mo, 32 Fe, 30 equivalents
of acid-labile sulfide (Fe8S 7-8clusters, etc)
• Four 4Fe-4S clusters plus two Fe, Mo, Co, an iron-molybdenum
cofactor (fe 7 S9 Mo homocitrate)
• Nitrogenase is slow - 12 e- pairs per second, i.e., only three
molecules of N2 per second
Nitrogen fixation 43

44. Nitrogen fixation 44

45. Pathway of nitrogen fixation in root nodules
• Glucose-6-phosphate acts as a electron donor
• Glucose-6-phosphate is converted to phosphogluconic acid
Glucose-6-phosphate + NADP+ + H2O→6-phosphogluconic acid + NADPH + H+
• NADPH donates electrons to ferrodoxin. Protons released and
ferrodoxin is reduced
• Reduced ferrodoxin acts as electron carrier. Donate electron to Fe-
protein to reduce it. Electrons released from ferrodoxin thus oxidized
Nitrogen fixation 45

46. Nitrogen fixation 46

47. Nitrogen fixation 47

48. Nitrogenase protection mechansims
• 1. Leghaemoglobin scavenges O2 to protect nitrogenase in
legume rhizobium symbiosis.
• 2. Confirmatory protection in Azotobacter as well as the higher
respiratory rate.
• 3. Thick walls of Heterocyst protect O2 in BGA, since
Nitrogenase are present in the heterocyst.
• 4. Microaerophilic nature in Azospirillum.
Nitrogen fixation 48

49. Genes involved in Nitrogen fixation
• Genes involved in root nodule formation – called nodulin genes
( nod genes)
• Nodulin genes essential for infection of plant root and nodule
formation by symbiotic N₂ fixing bacteria -divided into 2 classes
• 1) include genes that specify biochemical composition of
bacterial cell surface.
• such as gene determining the synthesis of
exopolysaccharides ( exo genes)
• Lipopolysaccharides (lps gene)
• Capsular polysaccharides or K antigen & β 1,2 glucans (ndu
genes)
Nitrogen fixation 49

50. Nitrogen fixation 50

51. • exo & lps genes - play a role in determining host specificity
• 2) consist of nodulation genes (nod or nol)
• nod genes are involved in nodulation of particular host -called
host specific nod(hsn) genes.
• Fast growing rhizobium sps - nod genes are located on large
sym plasmids
• Slow growing bradyrhizobium sps – carry late nod gene on the
bacterial chromosome.
Nitrogen fixation 51

52. • nif and fix genes - structural genes for nitrogenase enzyme
• Sym plasmid carry hup genes coding for hydrogenase activity
• Nif genes - 22 genes are involved , arranged in 7/8clusters
• Nif Q,B,A,L,F,M,Z,W,V,S,U,X,N,E,Y,T,K,D,H,J
Nitrogen fixation 52

53. Thank you
Nitrogen fixation 53