Nitrate and Nitrite Reduction in Relation

Soybean (Gcine nax L. cv Williams) seeds were sown in pots containing a 1:1 perlite-vermiculite mixture and grown under greenhouse coditions. Nodules were initiated with a nitrate reductase expressing strain of Rhizobiumjaponicwn, USDA 110, or with nitrate reductase nonexpressing mutants (NR108, NR303) derived from USDA 110. Nodules initiated with either type of strain were normal in appearance and demonstrated nitrogenase activity (acetylene reduction). The in vivo nitrate reductase activity of Nrgrown nodules initiated with nitrate reductase-negative mutant strains was less than 10% of the activity shown by nodules initiated with the wild-type strain. Regardless of the bacterial strain used for inoculation, the nodule cytosol and the ceil-free extracts of the leaves contained both nitrate reductase and nitrite reductase activities. The wildtype bacteroids contained nitrate reductase but not nitrite reductase actvity while the bacteroids of strains NR108 and NR303 contained neither nitrate reductase nor nitrite reductase activities. Addition of 20 miflimolar KNO3 to bacterolds of the wild-type strain caused a decrease in nitrogenase activity by more than 50%, but the nitrate reductase-negative strains were insensitive to nitrate. The nitrogenase activity of detached nodules initiated with the nitrate reductase-negative mutant strains was less affected by the KNO3 treatment as compared to the wild-type strain; however, the results were less conclusive than those obtained with the isolated bacteroids. The addition of either KNO3 or KNO2 to detached nodules (wild type) suspended in a semisolid agar nutrient medium caused an inhibition of nitrogenase activity of 50% and 65% as compared to the minus N controls, and provided direct evidence for a localized effect of nitrate and nitrite at the nodule leveL Addition of 0.1m lar sucrose stimulated nitrogenase activity in the presence or absence of nitrate or nitrite. The sucrose treatment also helped to decrease the level of nitrite accumulated within the nodules. A full understanding of the relationships between NOreduction and N2 fixation is needed to help maximize the use of both N sources by nodulated legumes. Recent reports (9, 23) have indicated that soybeans can benefit from the complementary operation of both biological nitrogen fixation and leaf nitrate assimilation. 'New Jersey Agricultural Experiment Station, Publication No. D01103-2-82, supported by State funds and by United States Hatch funds. Partial support was also provided by a Rutgers Research Council grant to C. A. N. 2PhD student of the Graduate Program of Microbiology, Rutgers University. Work represents a portion of a PhD thesis (B. D. S.) and was reported at the meeting of the American Society of Plant Physiologists held in Columbus, OH in 1979. An increase in the availability of NO3 may result in a larger contribution from NO3 assimilation, however nodulation and nitrogenase activity may be decreased (4, 5, 16). The primary cause of this inhibitory effect by NOis not clear and information is still lacking with regard to ways to relieve the inhibition of N2ase activity by NO3-. Nitrite accumulation within the nodules has been shown in NO3--treated plants of several legumes including soybeans (15, 22). NO2is a known inhibitor of several aspects of N2 fixation (12, 19, 21, 24). However, Gibson and Pagan (6) and Manhart and Wong (15) have concluded that NO2 the product of bacteroid NR3, may not play a role in the inhibition of acetylene reduction by NO3-. These authors based their conclusions on whole plant studies. The use of whole plants may be helpful to obtain an integrated view of the relationships between NO3reduction and N2 fixation but may be complicated by limitations imposed by the processes of NO% uptake, mobilization, storage, and reduction in compartments other than the nodules. Studies using isolated parts of the symbiotic system may provide more direct information on the metabolism ofNO3 within the nodules and its relationship to N2 fixation. The availability ofNR-negative mutants ofRhizobium japonicum (25) facilitates such studies. Mutants lacking nitrate reductase (NR-), produced from R japonicum strain 61A76, have been shown to produce normal nodules and fix N2 in symbiosis with soybean plants (25). The objectives of this study were: (a) to characterize the relationships between NOreduction, NOreduction, and Nrase activity using detached soybean nodules and Rjaponicum bacteroids; (b) to reevaluate the role of the bacteroid NR as a mediator of the NOeffect on N2-ase activity and; (c) to develop NR-negative mutants ofRjaponicum tolerant to normally inhibitory levels of NOR. MATERIALS AND METHODS Plant and Bacteria Culture. The Rhizobium japonicum strain (USDA 110) used in this study was obtained from Dr. D. F. Weber (USDA at Beltsville, MD). The bacteria were cultured and mutant strains lacking NRA (NR-) were prepared and tested as previously described (25). In one experiment (Fig. 2), another R japonicum strain, 61A76, (obtained from Dr. J. C. Burton of the Nitragin Company, Milwaukee, WI) was used. Soybean (Glycine max L. cv Williams) seeds were provided by Dr. J. Justin (Soils and Crops Department, Rutgers University, NJ). Soybean seeds were surface sterilized by treatment with 700%o ethanol (v/v) for 5 min followed by extensive rinsing with distilled H20 prior to inoculation (107-I09 cells/ml). The surface-sterilized seeds were planted in an autoclaved mixture (1:1) of perlite and vermiculite in pots (20.5 cm diameter x 21.5 cm high). Each pot, containing ' Abbreviations: NR, nitrate reductase; NRA, nitrate reductase activity; NiR, nitrite reductase.