Estimating Nitrogenase Activity of Faba Bean (Vicia faba) by Acetylene Reduction (Ar) Assay

Abstract

Methods of conducting acetylene reduction (AR) assay were appraised for estimating the nitrogenase activity of nodules of faba bean (Vicia faba L.). Factors considered were: (i) disturbance of plants when removing the rooting medium; (ii) assay temperature; (iii) the use of whole plants rather than detached, nodulated roots; (iv) diurnal variation in nodule activity; and (v) a decline in C₂H₄ production after exposure to C₂H₂. Plants growing in jars of 'oil dry' (calcined clay) had the same AR activity when assayed in situ in a closed system as when assayed after removal of the rooting medium. Assay temperatures of 12.5, 17.5 and 22.5°C influenced the specific rate of AR with the optimum at 17.5°C. Removal of the shoot resulted in a rapid decrease in AR activity in both vegetative and reproductive plants but the effect was much larger in the latter. AR and respiration by nodulated roots were closely linked and both varied markedly over a diurnal 12 h/12 h cycle. Since no fluctuation was found after nodules were detached, diurnal variation in the respiration of nodulated roots is attributed to change in nodule activity. Half of the dark respiration of nodulated roots was associated with respiration of the nodules and thus largely with N₂ fixation. Since the AR assay provides no information on how electron flow in vivo is partitioned between reduction of N₂ and reduction of protons, diurnal variation in hydrogen evolution (HE) in air and Ar/O₂ in an open system was used to estimate this partitioning.

Diurnal variation in apparent N₂ fixation estimated in this manner was examined at a 'low' PPFD (300 μmol m^-2 s^-1) and at 'high' (1300 μmol m^-2 s^-1) to explore whether variation could be attributed to change in carbohydrate supply. Although HE in air and in Ar/O₂ were both closely linked with the respiration of the nodulated root, apparent N₂ fixation showed only a slight diurnal variation at 'low' light and almost none at 'high'. Vegetative plants showed no C₂H₂-induced decline in activity with exposure to C₂H₂ but reproductive plants did. This difference appears to be an age effect rather than attributable to flowering per se, since a decline occurred even when plants were kept vegetative by disbudding. A closed system for AR assay appears satisfactory for vegetative faba bean but such an assay over a 40-min period during the reproductive stage would underestimate nitrogenase activity by about 20%.