Uniformity of the Microsymbiont Population from Soybean Nodules with Respect to Buoyant Density

Abstract

The microsymbiont population in soybean root nodules (Glycine max L. cv Williams 82 inoculated with Bradyrhizobium japonicum 2143) was characterized during symbiotic development to determine the extent of heterogeneity in this population. The microsymbiont population was isolated by centrifugation through a continuous sucrose gradient (44 to 57% weight to weight ratio) and appeared homogeneous at each age examined up to 26 days after planting based on the symmetrical distribution of the population, enzyme activities, poly-.8-hydroxybutyrate contents, protein contents, and viabilities. Some differences in viability, protein content, and acetylene reduction activity were observed at later ages. The population migrated to progressively lighter buoyant densities with increasing age until a density equivalent to 48% sucrose was reached. The changing density correlated directly with the increasing poly-fi-hydroxybutyrate to protein ratio. The acetylene reduction activity, based on microsymbiont concentration, followed the same developmental pattern as whole nodules. On a protein basis, the decline of acetylene reduction activity was later and reflected the decrease in protein content per cell. These results suggested that the microsymbiont population, which resulted from inoculation of B. japonicum 2143 onto Williams 82 cultivar of soybeans, developed as a homogeneous population.

Characterization of the efficiency of soybean inoculation

“…This could be relevant in practical terms, because the efficiency of inoculation can be increased if host-specific microsymbionts are used. A similar conclusion was drawn from other work on beans (SHISANYA, 2002;CREWS et al, 2004) and soybean (KARR & EMERICH, 1988). Therefore, the cost of inoculum production could be lowered while achieving enhanced inoculation efficiency.…”

Alanine, not ammonia, is excreted from N 2 -fixing soybean nodule bacteroids

“…In the presence of malate or succinate, ammonia release was reduced. Bacteroids prepared by this method had been shown earlier to be contaminated with enzymes from the plant nodule cytosol and also from ruptured mitochondria (19) that could be removed by a sucrose density gradient procedure (16,17,19). Ammonium accumulation in N 2 reduction assays of sucrose density gradient-purified bacteroids was generally Ͻ0.1 mol͞min ϫ mg dry weight of bacteroids, compared with Ϸ0.5 mol͞min ϫ mg dry weight of bacteroids prepared without the purification procedure.…”

“…The difference in the identity of the nitrogen product from the bacteroids was due to the method of bacteroid isolation as (i) there was no significant ammonia from sucrose density gradient-purified bacteroids, but there was from nonpurified bacteroids, and (ii) addition of plant nodule cytosol to sucrose density purified bacteroids resulted in the reduction of alanine and the increase of ammonium. The sucrose density purification procedure has been shown not to affect the metabolic activities, including nitrogen fixation, of the bacteroids (16,17). Furthermore, if the sucrose density gradient purification procedure disrupted the bacteroid membrane, it should have increased rather than decreased ammonium͞ammonia transfer across the membrane.…”

“…Williams 82) were inoculated with either B. japonicum strain 110 or 2143 and grown as described (16,17). Bacteroids were isolated anaerobically under argon either as described by Bergersen and Turner (18) or by the sucrose density gradient procedure of Waters et al (19).…”

“…The ammonium was collected from assays by microdiffusion and analyzed for 15 N content by R. H. Burris at the University of Wisconsin by using isotope ratio mass spectrometry. Acetylene reduction assays were done as described (16,17), except that Ϸ8 mg dry weight of bacteroids were incubated under 0.06 atmosphere of O 2 . Oxamate, carbonyl cyanide m-chlorophenylhydrazone (CCCP), reagents, buffer, and salts were purchased from Sigma.…”