Fixation of isotopic nitrogen by excised nodules.

After 60 years of research in symbiotic nitrogen fixation, the agent of fixation has not been established. Apparently only two biological species, leguminous plants and Rhisobium, are essential (l), but attempts to separate the components have been unsuccessful. Unequivocal fixation by the intact nodule excised from a plant actively fixing nitrogen has not been demonstrated. Ordinarily, cells of plant and animal show no such sensitivity to the environment but will readily carry out their characteristic biological reactions in tissue culture. An explanation of this notable exception may be important for an understanding of the mechanism of the reaction. Two possibilities appear worthy of investigation. (a) In the intact plant, the products of fixation are removed as rapidly as formed, but in the excised nodules, these accumulate and stop the reaction. If so, a very sensitive method for detection of fixation might be necessary. The usual Kjeldahl procedure with its attendant difficulties in sampling would not be suitable, but the much more sensitive isotopic method (2) might suffice. (b) An intermediate essential for fixation is supplied by the host plant. This postulated intermediate apparently is either present in a very low concentration or is so labile that it can disappear by the time N15 is supplied to the excised nodules. Our initial experiments (2, 3) were inconclusive in that occasionally fixation was obtained, but its occurrence was not associated with any specific treatment, including supplying postulated intermediates, such as members of the tricarboxylic acid cycle. Recently, we have reexamined this problem in an effort to secure consistent fixation, or, failing this, to establish the origin of our occasional positive results.