Reassessment of major products of N₂ fixation by bacteroids from soybean root nodules

NH₃/NH₄⁺ was the principal product from soybean bacteroids, prepared by various procedures, when assayed in solution in a flow chamber under N₂ fixation conditions. In addition, small quantities of alanine were produced (reaching 20% of NH₃/NH₄⁺ under some conditions). Some ¹⁵N was assimilated by bacteroids purified from soybean root nodules on Percoll density gradients and shaken with ¹⁵N₂ and 0.008 atm O₂. Under these conditions, ¹⁵NH⁴⁺ accounted for 93% of the ¹⁵N fixed into the soluble fraction. This fraction contained no measurable [¹⁵N]alanine. Neither these bacteroids nor those prepared by the previously used differential centrifugation method, when incubated with exogenous alanine under non-N₂-fixing conditions, gave rise to NH₃ from alanine. Therefore, contamination of bacteroid preparations with enzymes of plant cytosolic origin and capable of producing NH₃ from alanine cannot explain the failure to detect [¹⁵N]alanine [as reported elsewhere: Waters, J. K., Hughes, B. L., II, Purcell, L. C., Gerhardt, K. O., Mawhinney, T. P. & Emerich, D. W. (1998). Proc Natl Acad Sci USA 95, 12038-12042]. Cell-free extracts of the bacteroids as used in the ¹⁵N experiments contained alanine dehydrogenase and were able to produce alanine from pyruvate and NH₄⁺. Other experiments with alanine dehydrogenase in extracts of cultured rhizobia and bacteroids are reported and discussed in relation to the ¹⁵N experiments. Possible reasons for the differences between laboratories regarding the role of alanine are discussed. It is concluded that NH₃ is the principal soluble product of N₂ fixation by suspensions of soybean bacteroids ex planta and that NH⁺₄ should continue to be considered the principal product of N₂ fixation which is assimilated in vivo in soybean nodules.