No abstract

Soil N is a major deterrent to increased N2 fixation by soybean [Glycine max (L.) Merr.]. Three hypernodulating mutants (NOD1‐ 3, NOD2‐4, and NOD3‐7) were compared with a nonnodulating mutant (NN5) and the ‘Williams’ parent in a field study in 1988 and 1989 to determine nodulation and agronomic response to 0 and 200 kg N ha−1 applied as urea. Nodule activity was determined by acetylene (C2H2) reduction activity of nodulated roots and relative ureide abundance in vacuum‐extracted xylem sap. In the absence of N fertilizer, two years of data based on the C2H2 reduction assay indicated that all hypernodulating mutants had greater N2 fixation potential than did Williams in early growth stages. Among them, NOD1‐3 had higher N2‐fixing potential (about 10%) than the other two mutants. Both the relative ureide abundance assay (comparing NN5 with other lines in 1989) and the C2H2 reduction assay during the 2‐yr study indicated that N2 fixation by Williams was completely inhibited by N fertilizer through the initial sampling date, while the three hypernodulating mutants showed detectable N2 fixation activity. The relative ureide abundance assay indicated that both NOD1‐ 3 and NOD2‐4 lines were more tolerant of added N fertilizer than was Williams in 1989. In the absence of N fertilizer and at later stages of plant development, N input from N2 fixation by Williams was very similar to that of the three hypernodulating mutants. Seed yields from the hypernodulating mutants were 10 to 30% less than that from Williams in both years. In general, the small differences among soybean lines in seed protein and oil concentrations in both years were not significant. Therefore, total N in harvested seed was less for the hypernodulating lines than for Williams. The results indicate that these hypernodulating mutants are currently inferior to Williams from an agronomic standpoint.

No abstract

It was previously reported that three soybean (Glycine max [L.] Merr.) nodulation mutants (NOD1-3, NOD2-4, and NOD3-7) were partially tolerant to nitrate when nitrate was supplied simultaneously with inoculation at the time of transplanting. The current study evaluated the effect of short-term nitrate treatment on nitrogenase activity (C2H2 reduction per plant and per nodule weight) and on relative abundance of ureides when nitrate application was delayed until plants were 3 weeks old and nodules were fully developed. Nitrogenase activity of the mutants was similar to that of Williams after an initial 3-week growth period, prior to nitrate treatment. Application of 5 millimolar nitrate resulted in greater inhibition of nitrogenase activity in Williams than in the three mutants. NOD1-3 was most tolerant of nitrate among the mutants tested and showed the highest relative abundance of ureides. Although C2H2 reduction activity per plant for NOD1-3 was higher than for Williams in the presence of nitrate, C2H2 reduction activity per gram of nodules was lower for NOD1-3 than for Williams in the presence and absence of nitrate. Compared to Williams, NOD1-3 had higher nodule ureide concentration and had similar glutamine synthetase activity in nodule tissue, indicating its nodules have normal nitrogen assimilation pathways. Nitrate application resulted in ureide accumulation in nodule tissue as well as in all plant parts assayed. Unexpectedly, nitrate treatment also increased the rate of ureide degradative capacity of leaves in both NOD1-3 and Williams. The data confirmed that nitrogenase activity of the selected nodulation mutants was more, but still only partially, tolerant of nitrate compared with the Williams parent.also had higher ureide concentration in its nodule tissue (13), likely due to limited growth of the mutant which may have impaired ureide utilization.Partially nitrate tolerant mutants of soybean (NOD1-3, NOD2-4, and NOD3-7) have also been selected from mutagenized Williams populations, and initial characterization of these mutants has been reported (4). The data indicated many similar characteristics of our mutants to those reported by Carroll et al. (1, 2). However, it is not known whether the nitrogenase activity of our mutants is tolerant to nitrate per se, since in the previous study (4) the effects of nitrate on infection steps and on subsequent nodule function could not be separated due to simultaneous inoculation and nitrate addition. Using "5N analysis it was determined that NOD 1-3 fixed more '5N2 than Williams, when grown on urea, and that all three nodulation mutants symbiotically fixed more N2 than wild type when the mutants were grown on nitrate (1 1). This confirmed the earlier report based on C2H2 reduction assays (4). In the present report, nitrate treatment was delayed until plant roots were well nodulated and capable of relatively high N2 fixation activity. N2 fixation was then assessed by using the in situ C2H2 reduction assay and determining the relative abundance of ureide...

Among higher plants, soybean is unique in that biochemically it has been characterized as having two constitutive nitrate reductase (cNR) isoforms and one substrate-inducible nitrate reductase (iNR) isoform in leaves. All three NR isoforms are expressed in cv. Williams 82 while the nr1 mutant expresses only the iNR isoform. The genetic and molecular mechanisms for regulation of these isoforms have not been elucidated. We describe here the isolation, by reverse transcription-polymerase chain reaction (RT-PCR), of two cDNA clones encoding soybean NR. They were designated as iNR1 and iNR2, respectively, since both were inducible by nitrate. The iNR1 and iNR2 cDNAs cover total encoding regions of 2661 and 2673 nucleotides, respectively. The iNR1 clone shows a 12 bp deletion at the 5' end, relative to iNR2. They show overall similarity of 89% at the nucleotide level, and 87% at the amino acid level. Like all plant NRs cloned so far, deduced amino acid sequences between iNR1 and iNR2 show greatest variation at the N-terminal region while no difference was observed at the C-terminus. Soybean iNR mRNAs were found to be different from those of maize and tobacco in response to tungsten inhibitor treatment, since the inhibitor decreased the steady-state levels of mRNA for soybean iNR and for NiR. Using the same 5' regions of both cDNAs as the probes, Southern blot analysis of genomic DNA revealed differences in organization between iNR1 and iNR2. The genomic DNA from wild-type Williams 82 soybean was shown to have three Eco RI fragments while the nr1 mutant lacked an 8 kb fragment when probed with iNR1 cDNA. Likewise, the nr1 mutant lacked three Hae III restriction fragments when probed with iNR1 cDNA. When probed with iNR2, both wildtype and nr1 mutant showed one identical Eco RI band and two identical Hae III bands. In northern blots, the steady-state level of iNR1 mRNA was similar for the nr1 mutant and the wild-type parent after 20 to 48 h induction by nitrate. Based on the Eco RI and Hae III restriction enzyme digestion patterns observed in Southern blot analysis of soybean DNA, it is concluded that in soybean iNR1 is encoded by a small multiple gene family and iNR2 is a single gene.