| dc.description.abstract | Understanding the specificity of the legume-Rhizobium symbiosis may help in improving biological nitrogen fixation in several ways. The causes for this specificity have been studied in only a few systems like clover, soybean, peanut, and pea.
In the present study, host-symbiont interaction between the mimosoid legume Mimosa invisa and its nodulating Rhizobium has been studied by isolating the mutually interacting and potentially cognitive components from the legume and the bacterium.
A fast-growing Rhizobium with a G-C content of 63.3% and specific nodulating properties was isolated from the nodules of Mimosa invisa and its taxonomical position has been discussed. From the literature survey, it appears that this Rhizobium does not fit into any of the existing groups when all the parameters employed for classification are considered. A new cross-inoculation group, the 'leucaena group,' has been proposed to include Mimosa-Rhizobium and other mimosoid rhizobia with specific nodulating abilities.
Nitrogen fixation in young nodules was approximately 500 nmoles of ethylene formed per g fresh weight per hour. However, the gum-producing rhizobia did not fix nitrogen asymbiotically in any of the special media tested.
A Rhizobium-binding lectin was isolated and purified from the seeds of Mimosa invisa. It has an apparent molecular weight of 100,000 daltons with four subunits, two of molecular weight 55,000 and the other two of 15,000 daltons.
The purified lectin is an erythroagglutinin and agglutinates red blood cells nonspecifically. It is, however, not a leucoagglutinin nor a mitogen for human lymphocytes in culture. It is a panagglutinin requiring PVP for enhanced titer and is also a glycoprotein with 21% carbohydrate.
The lectin is not a metalloprotein. It does not require external metal ions for activity. It is temperature stable (up to 75°C) and has a pH optimum between 7 and 8. The lectin has high affinity for concanavalin A and was not displaced by ?-methyl mannoside or any other sugar tested.
The agglutination property of the lectin was not inhibited by several of the mono- and disaccharides. The receptor in the Rhizobium is assumed to be much more complex than the sugars tested.
Agglutination of the nodulating rhizobia was very specific. Several of the non-nodulating strains tested were not agglutinated. A biological correlation between nodulation of the legume by the rhizobia and the ability of the host lectin to agglutinate rhizobia could be observed.
Mimosa-Rhizobium was not agglutinated by lectins from plants which are not nodulated by the Rhizobium. The agglutinability of the cells with Mimosa lectin varied depending on the carbon source used for growth. With lactose, the lectin receptors seemed to be maximum.
A neutral capsular polysaccharide (NPS) from total cell wall carbohydrates of Rhizobium sp. specifically reacted with purified Mimosa lectin resulting in precipitation. Seed lectins from other plants did not elicit this reaction. The NPS fraction was purified by alcohol precipitation, cetyltrimethyl ammonium hydroxide precipitation and gave a single band on paper electrophoresis. The purified NPS inhibits the agglutination of rhizobial cells by Mimosa lectin.
Partial or total hydrolysate of the NPS failed to inhibit the specific agglutination by Mimosa lectin. This suggests that probably the sequence of constituent sugar linkage as well as the conformation of the resulting polysaccharide fraction is important in the recognition by Mimosa lectin.
NPS total hydrolysates were subjected to paper chromatography and based on the mobilities, the sugars were identified as: galactose, ?-methyl-6-deoxy-?-L-galactose, 1-O-methyl-?-D-glucose, ?-L(-) fucose, 2-deoxy-D-galactose and 3-O-methyl-?-D-glucose.
Various parameters for the specific interaction of isolated lectin and rhizobial cells or NPS were determined using externally radiolabelled lectin. The specific activity of the labelled lectin was 990 ?Ci/n mole. Association constant (Ka) and number of binding sites per cell (n) were calculated by Steck and Wallach plot and Scatchard plot. At high lectin concentrations, ‘n’ was 1.7 × 10?/cells and Ka was 0.9 × 10?; at low lectin concentrations, ‘n’ was 2.8 × 10?/cell and Ka was 0.9 × 10¹? M?¹.
Specific binding was observed by FITC-tagged lectin. Specific binding of NPS was also demonstrated using tritium-labelled lectin.
The transient nature of the lectin receptor on the rhizobial cell surface was inferred based on the binding of fluorescent and tritium-labelled lectin to rhizobial cells during various growth phases. Maximum binding was observed at mid-log phase of the growth and this was correlated with maximum NPS production.
Presence of Rhizobium-binding lectin was tested in the roots of Mimosa invisa seedlings of different age groups (1–25 days) by hemagglutination, rhizobial agglutination, immunodiffusion, immunoelectrophoresis, immunofluorescence and binding of NPS to the roots. Binding of rhizobia to the root hairs in large numbers was observed in plants of all the age groups studied.
By hemagglutination, the lectin could be detected in the roots only up to 12 days. A time course study was conducted to study the distribution of lectin in various seedling tissues.
Immunodiffusion and immunoelectrophoresis of the root extract with antibodies raised to the seed lectin showed the presence of lectin in the roots up to 15 days. Immunofluorescence showed root lectin in plants up to 25 days.
NPS isolated from the Rhizobium showed binding to the roots when examined by fluorescence microscopy, indicating the presence of cognitive molecule on root surface with an affinity for the rhizobial NPS.
The quantity of lectin which is present in the roots of 7-day-old seedlings, though small, may be biologically significant and probably sufficient for specifically attracting a large number of rhizobial cells to elicit the subsequent responses in the two symbiotic partners. | |
