Index >> Rhizobium and Legume Root Nodulation >>Function of the Nodule

	Function of the Nodule Present evidences point out the fact that bacteroids are the sites of nitrogen fixation, although earlier, membrane envelopes surrounding a group of bacteroids were believed to be the sites of this reaction. Stable isotope 15N has been used to trace the path of N2 in nitrogen fixation. Earlier studies were done with intact or sliced nodules without adequate precautions to keep the experimental system under anaerobic conditions.
In later studies, however, nodules were exposed to 15N and crushed in an atmosphere of argon so as to prepare a brei under anaerobic conditions which retained the capacity to fix nitrogen. The brei was centrifuged, the bacteroids collected in the form of a pellet, washed and assayed for 15N. The bulk of the activity was recorded in bacteroids and not in soluble fractions indicating that bacteroids are the primary sites of nitrogen fixation. The acetylene-ethylene reduction technique designed to determine the nitrogenase activity of different fractions of nodules has been of great help in these investigations. Diagrammatic sketch of a transverse section of a soybean nodule
Further confirmation on the role of bacteroids in N2 fixation has come from studies on cell-free extracts of bacteroids which contain crude nitrogenase enzyme with sufficient activity to fix up to a maximum of 9 to 13 µ moles of N2/min/mg protein. Using special purification procedures, nitrogenase has been purified and is found to consist of two protein fractions-Mo-Fe protein and Fe protein. The enzyme has been successfully extracted from representatives of major classes of nitrogen-fixing microorganisms and systems except those from nodulating non-leguminous plants.
A more detailed account of nitrogenase appears in the chapter on non-symbiotic nitrogen-fixing bacteria. Nitrogenase from bacteroids has been prepared and assayed for its property of acetylene reduction and enzyme activity. The results of one such experiment shown in Table 28 indicate no activity in fraction 1, very low activity in fraction 2 and a striking increase in activity when fractions 1 and 2 were combined. A-A well formed bunch of effective root nodules of soybean B-Section of an effective nodule in a 3 week old Trifolium pratense showing meristem C-A section of an ineffective T. pratense nodule showing the absence of bacteroid tissue D-An electron micrograph of a thin section of a soybean nodule showing bacteroids Relationship betweeen the reduction of acetylene and N2 by extracts and fractions of nodule bacterioids (Postgate, 1971)     Rate of reduction Ratio of rate of reduction Type of extract C2H2 N2 C2H2 : N2   n moles per mg protein per min.     + Crude 35.2 11.8 2.98 : 1 25-55% PPG* ppt. 76.1 27.4 2.78 : 1 Fraction 1 0.0 0.0 -     Fraction 2 74.4 19.3 3.58' : 1 Fractions 1 and 2 607.4 192.6 3.15 : 1 *Polypropylene glycol (P - 400)
	A red pigment akin to haemoglobin of blood is found in nodules between bacteroids and the membrane envelopes surrounding them. Leghaemoglobin, the prefix 'leg' indicating its presence in legume root nodules, is a haemoprotein having a haeme moiety attached to a peptide chain which represents the globin part of the molecule. The molecule weight of leghaemoglobin is of the order of 66,000, because leghaemoglobin has one peptide chain linked with one haeme moiety, whereas blood haemoglobin has four peptide chains, each linked with one haeme moiety.

The pigment has been crystallized into two major extractable components with differing amino acid composition and molecular weights-the electrophoretically faster one containing 0.32% iron and the slower one with 0.29% iron. Minor components of leghaemoglobin have also been detected depending on the legume species. It is difficult to say whether these components represent native or degraded forms of a single fraction during the extraction procedures. The amount of leghaemoglobin in nodules has a direct relationship with the amount of. nitrogen fixed by legumes.

The correlation between haematin / nodule and effective central tissue volume index (bd2n) in red clover	The correlation between haematin / g fresh weight nodules and nitrogen fixed by nodulated Torsdag peas

It has been variously postulated that the pigment could function: (a) as the site of nitrogen absorption and reduction, (b) as the specific electron carrier in nitrogen fixation, (c) as a regulator of oxygen supply, and (d) as carrier of oxygen. Current evidences show that leghaemoglobin plays no active role in symbiotic nitrogen fixation but functions as a biological valve in regulating the supply of oxygen to bacteroids at optimum levels conducive for proper functioning of the nitrogen-fixing system. A haemoglobin has also been characterized in cultured cells of R. japonicum, which however, is unrelated to leghaemoglobin of root nodules both in composition and function.

Many aspects of the biochemistry of symbiotic nitrogen fixation are not clearly understood. Nevertheless, schemes summarizing the overall reactions representing the results obtained with nodule homogenates and cell-free preparations of bacteroids. Photosynthate from the legume provides the substrate required by nodule 'bacteroids for the generation of ATP and reductant needed for nitrogen fixation. A ferredoxin similar to that of Azotobacter ferredoxin has been found in soybean bacteroids which may function as an electron carrier. Since bacteroids require oxygen for nitrogen fixation, the oxygen bound to leghaemoglobin is transported at optimum levels without interfering with the oxygen sensitivity of the nitrogenase system.

A diagram illustrating the interreolationship of reactions involved in the nitrogen fixing process in a legume nodule

The first stable intermediate in nitrogen fixation is ammonia which gets incorporated into glutamic acid, glutamine, aspartic acid and alanine. The nature of intermediates between nitrogen and ammonia remains unclear although compounds like hydrazine, hydroxylamine, diimide, and carbamyl phosphate have been proposed as possible intermediates. Hydrazine is extremely toxic to living cells; besides this fact, experiments with ¹⁵N do not indicate the likelihood of this intermediate occurring in the conversion of nitrogen to ammonia. Conclusive evidences suggesting possible sequences in the reductive pathway between nitrogen and ammonia with regard to the other intermediates are lacking.

A Schematic diagram of general principles involved in the reduction of N₂ to NH₃ by the nitrogenase complex

Reaction of the GS-GOGAT system for the assimilation of ammonium into amino acids. The overall equation for the reaction sequence is NH4⁺ + ATP + NADPH + H⁺ + α-Keto acid + NADP⁺ + ADP + Pi + H⁺