Nitrogen Fixation
· Reduction of atmospheric nitrogen into Ammonia by soil borne microorganisms is called biological nitrogen fixation
· Nitrogen fixing bacterium Rhizobium leguminosarum was isolated by Beijerinck(1888).
· He also discovered the free- living nitrogen fixing bacterium Azotobacter (1905).
· Atmosphere contains 78% of gaseous nitrogen-green plants are unable to utilise it directly from atmosphere. The reduction of atmospheric nitrogen into ammonia is carried out by soil bacteria and blue green algae. Green plants utilise Ammonia to form Nitrogen compounds such as Asparagine, arginine, allantonin and allantonic acid.
· Process of Nitrogen reduction is called diazotrophy or Nitrogen fixation
· Nitrogen fixing microorganisms are diazotrophs
· Nitrogenase carry out the reaction.
SYMBIOTIC NITROGEN FIXATION
● Nitrogen reduction by symbiotic microbes is called symbiotic nitrogen fixation.
● Microorganisms establishing symbiotic association with plant roots and do nitrogen fixation are called symbiotic nitrogen fixers or symbiotic diazotrophs.
● Diazotrophs induce nodule development on the plant roots -Rhizobia induce root nodulation with the roots of legumes
● Frankia induces root nodulation in Alnus, Casuarina, Myrica, Discaria etc.
● Symbiotic association of cyanobacteria with fungi (lichen)
● Cyanobacteria with bryophytes (Riccia and Anthoceros)
● Bacteria with leguminosarum plants (Rhizobium, Bradyrhizhobium, Azorhizobium, Sinorhizhobium and Mesorhizhobium).
HOST SPECIFICITY
● Particular species can alone establish symbiotic association with host plant roots - this selective infection of Rhizobium on specific plants is called host specificity.
● Rhizobium leguminosarum – pea (root nodulation).
● Rhizobium japonicum- soyabean.
● Rhizobium phaseoli- beans.
ROOT NODULATION
● The formation of root nodules after the roots gets infected with Rhizobium is called root nodulation.
● Protein lectin helps the binding of Rhizobium on the root hair. The Rhizobium secretes Cytokinin and Polymyxin B and induces the curling of tip of the hair. Then it forms infection thread.
● Nod factors (nodulation factors or NFs) are signaling molecules (lipochitooligosaccharide) produced by rhizobia, in response to flavonoid exudation from plants under nitrogen limited conditions
● Nod factors determine the host range, infection, and nodulation in the symbioses between these soil bacteria and legume plants. Nod factors initiate the establishment of a symbiotic relationship between legumes and rhizobia by inducing nodulation. Nod factors also produce the differentiation of plant tissue in root hairs into nodules where the bacteria reside and fix nitrogen from the atmosphere.
● Host plant provide photosynthetic products and the appropriate environment for nitrogen fixation to bacteria in this association.
● Some genes in the leguminous plants code for proteins called nodulins -maintain the structure of root nodules, to support the nitrogen fixation and its assimilation
● Temp between 25° - 30 °C favours the nodulation. High light intensity and high concentration of carbon dioxide are not suitable
● The root nodules of legumes have two mechanisms to protect nitrogenase from oxygen
● (1) Oxygen transport by Leghaemoglobin.
● Leghaemoglobin is a red, myoglobin like protein present only in the healthy root nodules of legumes.
● Leghaemoglobin combines with oxygen to form Oxyleghaemoglobin and provide oxygen to plant cells for respiration.
● Leghemoglobin, which maintains the oxygen concentration low and prevents the inhibition of nitrogenase activity.
● (2) Utilization of oxygen by Hydrogenase.
● The Hydrogenase enzyme combines hydrogen and Oxygen to form water.
● Hydrogenase removes oxygen and generates some ATPs lost during nitrogen reduction
● Thus Hydrogenase make a suitable micro environment for nitrogenase activity.
● Nodule has pleomorphic Rhizobia called bacteroids.
● They may be club shaped, Y - shaped or branched.
● The membrane of the root cortical cell enclosing the bacteriods is known as peribacteroid membrane.
● The bacteroids fix the atmospheric nitrogen into Ammonia.
NITROGENASE
● The enzyme that catalyzes Nitrogen fixation
● Consists of one larger subunit and one smaller subunit
● The larger sub unit is called molybdenum ferrous protein (Mo- Fe protein) or a nitrogenase which uses the electrons provided to reduce N2 to NH3.
● The smaller sub unit is called ferrous protein- homo dimeric Fe-only protein, the reductase which is responsible for the supply of electrons.
● The Mo- Fe protein and Fe- protein combine together in the presence of Na+ ions to form an active Nitrogenase complex
Steps in Nitrogen Reduction
(1) Fe protein -------> Reduced Fe protein.
(2) Reduced Fe protein+ 12Mg- ATP’s ------> Reduced Fe protein- Mg- ATP’s complex.
(3) Nitrogenase + N2 ------> Nitrogenase nitrogen complex. (NNC)
(4) RFP- MA complex + NNC ------> Active Nitrogenase complex.
(5) Nitrogen reacts with 2H+ ions by consuming 2 electrons to form diamides and this diamide react with 2H+ ions to form Hydrozines.
(6) Hydrozine reacts with 2H+ ions by consuming 2 electrons to form two molecules of ammonia.
(6) After the reduction of N2 into NH3, the Nitrogenase complex dissociates into Fe- protein, nitrogenase, Mg++ and ADPs. NH3 is released in the cytoplasm.
The enzyme is now available to reduce another molecule of Nitrogen.
N2+16 ATPs+ 8 e+10 H+ -------> 2NH3+ H2+ 16 ADP+ 16 ip
GENETICS OF NITROGEN FIXATION
(1) Rhizobial Genes.
a) Nod genes:-
● Nod A, B and C code for proteins that cause swelling and curling of root hairs.
● Nod D codes for a protein that binds with flavanoid produced by legumes and induces the transcription of nod genes.
● Nod E and F control host specific nodulation.
● Function of nod G is unknown.
● Nod H codes for a protein that elicts host specific responses when Rhizobium infects root hairs.
● Nod I codes for an ATP carrier protein.
● Nod J codes for a protein that transfers electrons.
b) Nif genes:-
● Nif genes E,K, D, H,M,L,A and B are identified in the gene cluster.
● Nif k and D code for Nitrogenase ( Mo- Fe protein)
● Nif B and E code for Fe-Mo-co- factor.
● Nif H codes for Nitrogenase reductase.
● Nif M codes for a protein that involves in post transcriptional modification of Nitrogenase reductase
● Nif A codes for a proteins that induces transription
● Nif L codes for a protein that has a role in transcription
● In addition to Nif genes there are three genes in cluster and are designated as fix-A, fix- B and fix-C
● Fix genes A,B and C codes for proteins which allow the passage of electrons.
C) Hup Genes:-
● Hup genes code for uptake Hydrogenase
● Some species of Rhizobium (Hup+ species) uptake H2 produced during Nitrogen fixation and use it to make H2O, thus removing hydrogen and oxygen which can inhibit nitrogen fixation
(2) Legume Nodulin Genes.
● The nodule specific polypeptides secreted by plant roots are called nodulins.
● There are about 20 nodulins.
● Localized in the chromosomal DNA of plant cells.
● The nodulins are divided into three classes.
i) Group I nodulins.
ii) Group II nodulins.
iii) Group III nodulins.
(3) Leghaemoglobin Gene:- Lb gene resides in the chromosomal DNA of legumes.
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