Nitrogen fixation in transposon mutants from Bradyrhizobium japonicum USDA 110 impaired in nitrate reductase.

Tn5 transposon mutagenesis was carried out in Bradyrhizobium japonicum strain USDA 110 to produce defective mutants. From over one thousand clones expressing low levels of nitrate reductase activity as free-living bacteria, approximately five percent had significantly different ratios of nodulation, N2 fixation or nitrate reductase activity compared to the wild strain when determined in bacteroids from soybean nodules. Tn5 insertions were checked previously and mutants were arranged into four different groups. Only one of these groups, designated AN, was less effective at N2 fixation than the wild strain, suggesting a mutation in a domain shared by nitrogenase and NR. The remaining groups of insertions successfully nodulated and were as effective at N2 fixation as the wild strain, but showed diminished ability to reduce nitrate both in nodules and in the isolated bacteroids when assayed in vitro with NADH or methyl viologen as electron donors. PCR amplification demonstrated that Tn5 insertions took place in different genes on each mutant group and the type of mutant (CC) expressing almost no nitrate reductase activity under all treatments seemed to possess transposable elements in two genes. Induction of nitrate reductase activity by nitrate was observed only in those clones expressing a low constitutive activity (AN and AE). Nitrate reductase activity in bacteroids along nodule growth decreased in all groups including the ineffective AN group, whose nodulation was highly inhibited by nitrate at 5 mmol/L N. Host-cultivar interaction seemed to influence the regulation of nitrate reductase activity in bacteroids. Total or partial repression of nitrate reductase activity in bacteroids unaffected by N2 fixation (CC, AJ and AE groups) improved nodule resistance to nitrate and N yields of shoots over those of the wild strain. These observations may suggest that some of the energy supplied to bacteroids was wasted by its constitutive NRA.

Nitrate levels and stages of growth in hypernodulating mutants of Lupinus Albus. II.Enzymatic activity and transport of N in the xylem SAP

The enzymatic study and transport of N in the xylem sap was carried out with a view to observing the influence of different nitrate levels and growth stages of the plant in chemically treated mutants of Lupinus albus. Several stresses induce a reduction in plant growth, resulting in the accumulation of free amino acids, amides or ureides, not only in the shoot, but also in the roots and nodules. Although enzyme activity is decisive in avoiding products that inhibit nitrogenase by ammonium, little is known about the mechanism by wich the xylem carries these products. However, this process may be the key to the function of avoiding the accumulation of amino acids in the cells of infected nodules. The behaviour of the enzymes nitrate reductase (NR), phosphoenolpyruvate carboxylase (PEPC), glutamine synthetase (GS) and nitrogen compounds derived from fixation, such as N-Ó-amino, N-ureides and N-amide in mutant genotypes were observed. The NR enzyme was highly influenced by the application of nitrate showing much higher values than those in the non-application of nitrate, independently of genotype, being that the NR, the best evaluation period was in the tenth week. The L-62 genotype characterized with nitrate-resistance, clearly showed that the enzyme PEPC is inhibited by presence of nitrate. The L-135 genotype (nor fix) showed GS activity extremely low, thus demonstrating that GS is an enzyme highly correlated with fixation. With regard to the best growth stage for GS, Lupinus albus should be evaluated in the seventh week.