Nitrogen Fixation

.    The symbiotic association of cyanobacteria with fungi (lichen), cyanobacteria with bryophytes (Anthoceros), with pteridophytes, (Azolla) with gymnospermes (coralloid root of cycas) and bacteria (Rhizobium, Bradyrhizobiun, zorhizobium, sinorhizobium and mesohizobium etc.) With leguminous plants are under mutual beneficial relationship (symbiosis) in which both the host and bacteria are benefitted. There are number of other angiosperms (excluding legumes) which have symbiotic association with nitrogen fixing microorganisms. About 15 angiospermic plants are of nonlegumes category which fix atmospheric nitrogen, for example, Alnus, Myrica, Purshia, etc. The symbiotic micoorganisms are not only bacteria but also comprises actinomycetes such as Frankia which fixes nitrogen. There are some indications of the existence of haemoglobin like pigment in the root nodules of Alnus, Elaeganus, Shepherdia and Hippophae. In such cases, the hyphal threads of the endophyte fill the cortical cells which increase in volume resulting into a primary nodule recognizable on the root as a ‘swelling’. The lateral roots arise in the vicinity of the primary nodule. Their meristem undergoes branching and gets infected with endophyte results in the formation of a typical adult nodular structure referred to as a ‘rhizothamion’. The occurrence of leaf nodules is confined to the families of Rubiaceae and Myristicaceae. The bacteria is isolated and identified as Mycobacterium rubiacearum, Mycoplana rubra, Flavobacteriumspecies, Phyllobacterium rubicearum and Klebsiella rubiacearum. It is interesting to note that these bacterial isolates do not fix nitrogen.  Several species of Podacarpus possess numerous small nodules on the root system. The most common endophyte is a non-septate fungus resembling the fungal component of endotrophic mycorrhizae. This nodulated root system demonstrates that it performs the process of nitrogen fixation very slowly.

Root Nodulating Symbiotic Bacteria

Rhizobium forms nodules and participate in the symbiotic acquisition of nitrogen. The rod – shaped bacteria, utilize organic acid salts as carbon source without gas formation; while the cellulose and starch are not utilised. The growth is optimum at 27˚C (pH 6.8) and colonies appeared as circular convex semitranslucent, raised and mucilagenous, usually 2-4 mm in diameter. Production of an acid reaction occurs in mineral salt medium. Some strains of rhizobia and agrobacteria show a close relationship in D.N.A. base composition. All species (except Agrobacterium radiobacter ) incite hypertrophies on plant roots. Nodules are incited by strains of rhizobia on roots of leguminous palnts and leaves of certain plants in the families Myristicaceae and Rubiaceae by strains of Phyllobacteria. The strains of Rhizobium are fast – growing where generation time lasts about 6 hours besides showing some other differences with rest of the members of family – Rhizobiaceae.

Some plants bear stem nodules (Sesbania species) by Azorhizobium caulnodans. The strains bear flagella, hence cells are motile (peritrichous flagella on solid medium but one lateral flagellum in liquid medium). They also fix nitrogen. These are oxidase and catalase positive and cannot oxidise mannitol.

The Bradyrhizobium strains are slow growers where generation time about 12 hours or more. The motility occurs by one polar or subpolar flagellum. The growth on carbohydrate medium is accompanied by exopolysaccharide (EPS) slime. Some strains can grow chemolithotrophically (utilizeinorganic salts) in the presence of H2, CO2 and low level of O2. The bacteroids in root nodules are slightly swollen rods with rare branching or occurs forms. Their main symbiotic partner is soyabean, while other bradyrhiozobia produce nodules in the plants such as Lotus, Vigna, Lupinus, Ornithopus, Cicer, Leucaena, Mimosa, Lablab, Acacia and Dalbergia. Now the strains of bradyrhizobia are designated as name of the host plant in parentheses e.g. Bradyrhizobium (Lotus) species.

Recently, it has been observed that some rhizobial strains which are fast growers nodulate soybean, (generally, bradyrhizobia nodulate soybean). These fast growers are identified as a separate genus Sinorhizodium which are rod shaped, usually contain poly-β-hydroxbutyric acid (pH 6-8). Recently several new species have been added.

Most of the rhizobia are discovered only in last decade. Hence, it is not surprising if more and more host which bear bacterial nodules, may not contain the traditional strains of Rhizobium or Bradyrhizobium. Mesorhizobium, a new genus of the family Rhizobiaceae has been named on the basis of whole sequence studies of 16s rRNA. Some of the species of Rhizobium namely, R. loti, R. huakii, R. ciceri, R. mediterraneum and R. tianshanense now known as Mesorhizobium.

Process of Root Nodule Formation

The ‘rhizobia’ live freely in soil and as soon as they come in contact with suitable host, starts the process of infection. There is an initial contact between the bacteria and host which depends upon recognition. Recent evidences suggest that polysaccharides on the surface of invasive bacteria are involved in binding of these cells to constituents (lectins) on the surface of the roots. The factors or proteins located in the nodules are called nodulins while on bacterial surfaces, named as bacteriocidins which help in nodulation. Generally, nodulation starts from the following processes :

(i) Curling and deformation of root hairs : invasion of rhizobia occurs through root hairs. Fine studies of infected root hairs showed the continuation of the wall of the infection thread with the call wall of the root hairs which lend support to the invagination hypothesis. The physiological events leading to infection can be summarised below :

Normal root hair

Exudation of organic substances by roots

Accumulation of ‘rhizobia’ in the rhizosphere

Conversion of tryptophan to IAA

Root hair curling and deformation

(ii) Formation of Infection – thread and Formation of Nodule : it is intresting to note that such binding occurs between compatible (bacteria – host) partners. Tip of curled root hair bends and the bacteria (rhizobial polysaccharides and the DNA) penetrate and grow in the form of an infection tube. Meanwhile, the polysaccharides react with a component of root hair cell to form an ‘organizer ‘. The ‘organizer‘ induces the production of polygalacturonase (PG) followed by depolymerisation of cell wall pectin. In such process, incorporation of rhizobia into cell wall occurs which participate in ‘intussuusception’ i.e. taking in of rhizobia by root hair and its conversion into organic tissues. The infection tube or thread branches into the central portions of the nodule, and the bacteria released into their symbiont’s cytoplasm to multiply. The nucleus of the root hair cell guides the rhizobia.

(iii) Development of nodule : immediately, at the time of release of rhizobia into cytoplasm of the host cortical cells, rapid cell division (called hyperplasia) takes place in the cortical cells. Inside these cells, the bacteria alter their morphology into larger forms called bacteroids. The root cells are stimulated due to this infection to form a tumor like nodule bacteroid-packed cells. The host cells chromosome number of the area become double. The doubling of the chromosome number occurs in nodules of polyploids as well as diploid legumes.

Leghaemoglobin : A red pigment similar to blood haemoglobin is found in the nodules between bacteroids and the membrane envelopes surrounding them. Leghaemoglobin, the prefix ‘leg’ indicates its presence in legume root nodules, is a haemoprotein having a haeme moiety synthesized by the bacteria attached to a peptide chain which represents the globin part of the molecule, is enclosed by a plant gene. The molecular weight of leghaemoglobin is about 16000 – 17000 daltons. The prosthetic group protohaem is synthesized by the bacteroids, while the synthesis of protein part involves the plant cell. The indication of leghaemoglobin enhances the transport of oxygen at a low partial pressure to the nodules and maintains a steady supply of oxygen at low concentration of the nodule. It is not analysed in syanobacterial symbiotic system or in other higher plants such as Frankia and Parasponia which fix nitrogen without leghaemoglobin. The presence of the leghaemoglobin seems to provide full protection against oxygen damage to the nitrogen fixing enzymes.


One Response

  1. it`s fine……….

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