Nodule formation and nitrogenase activity of soybean [Glycine max (L.) Merr] are both inhibited by nitrate (NO3-).Soybean mutants lacking autoregulation of nodulation (AON) show supernodulation (SN) but the extreme SN (ESN) mutants are retarded in growth, which may prevent them from being developed for commercial use.
To rule out the possibility that retarded growth of ESN mutants was the result of other genetic alterations and to investigate the possible commercial value of SN mutants, backcrossed SN lines and wild types (WTs) of soybean were compared in three field experiments (Chapter 3). The results showed that the backcrossed lines resembled their parents in all symbiotic characteristics. PS47, a backcrossed intermediate SN (ISN) line, grew as well as, and yielded as much as, WTs. In contrast, PS55, a backcrossed ESN line, had a grain yield significantly less than WTs. The results indicated that the retarded growth of ESN mutants was caused by their extensive nodulation which restricted root growth. Increased residual nitrogen (N) following a crop of SN lines was detected compared to WTs. As a result, grain yield of barley (Hordeum vulgare L.) following PS55 and PS47 was higher than following WTs. However, the increased yield of barley following PS55 was less than the reduction in soybean yield of PS55.
To select a mutant with both SN characteristics and vigorous growth, PS55 was mutagenized and four plants (designated R366, R567, R1525 and R1695) with vigorous growth were selected (Chapter 4). DNA fingerprinting demonstrated that both R1525 and R1695 were derived from PS55, whereas R366 and R567 were ruled out as possible contaminants. Crossing R1525 and R1695 to ESN mutant ntel007 demonstrated that both mutants were dominant over nts1007. F2 segregation analysis was consistent with the two mutants being allelic to ntsl007/PS55. Thus R1525 and R1695 appear to be new revertant alleles at the NTS-1 locus. The two revertants were assessed in two glasshouse and two field experiments. The results revealed that R1525 resembled nts1 116, the original ISN mutant, in both agronomic and symbiotic characteristics. On the other hand, R1695 resembled WT in nodulation and growth. The improved growth and restored yield on R1525 and R1695 further confirmed that prolific nodulation was the main reason for retarded growth coupled with ESN phenotype. From a genetic point of view, the selection of these two revertant alleles provided further evidence for the mutability of the NTS-1 locus in soybean. The NTS-1 locus has been recently cloned and renamed GmNARK (G. max nodulation autoregulation receptor kinase) to reflect the biochemical function of the protein encoded by the locus. The R1525 and R1695 alleles need to be cloned, sequenced and confirmed to be new alleles of GmNARK.
Supernodulation mutants also exhibit NO3-- tolerant nodulation (NO3--tolerant symbiosis, nts) but remain inhibited by NO3- in nitrogenase activity. On the other hand, NO3- reductase (NR) deficiency in pea mutants partly alleviates inhibition by NO3- of nitrogenase activity. Interestingly, nodulation is still inhibited by NO3- in NR-deficient mutants of pea and soybean. Collectively, these earlier reports indicate that NO3- itself inhibits nodulation, whereas NO3- assimilation inhibits specific nitrogenase activity. To test the hypothesis that combining NR deficiency with SN characteristics would increase the dependence of soybean on N2 fixation, a double soybean mutant with both supernodulation and NR-deficiency (designated ntsNRl) was evaluated in five glasshouse experiments of either sand or solution cultures (Chapter 5). The double mutant, ntsNRl, was comparable to PS55 in growth and nodulation but consistently more dependent on fixed N2 at high NO3- levels. The higher dependence of ntsNRl on fixed N2 was attributed to its higher specific nitrogenase activity. The results confirmed earlier reports that NO3- assimilation (rather than NO3- itself) was responsible for the inhibition of nitrogenase activity and that greater dependence of legumes on N2 fixation could be achieved by combining SN characteristics with defects in NO3- uptake and/or assimilation.
To further increase the dependence of soybean on fixed N2, ntsNRl and PS55 were mutagenized and putative NO3- uptake mutants were selected for using an in vivo NR assay (Chapter 6). From progenies of 1500 M2 families, two putative mutants (designated P75 and P399) were initially selected. The in vivo NR activities of the two putative mutants were on average less than 60% that of WTs and less than 80% that of ntsNRl. However, the two putative mutants also displayed necrosis, indicating that decreased NR activities in these two mutants might have resulted from decreased vigour rather than a specific defect in NO3- metabolism.
Overall results from these experiments indicated that current ESN mutants were not agronomically beneficial at this stage. On the other hand, ISN mutant, depending more on fixed N2 and sparing more N to the subsequent crop without yield penalty, appeared to be promising for commercial use. The higher dependence of ntsNRl on fixed N2 than PS55 demonstrated that it is possible to enhance N2 fixation by combining the ability of N03--tolerant nodulation and N03--tolerant nodule function. The results were further discussed in the context of interactions between N03- metabolism, nodulation and N2 fixation in soybean (Chapter 7).