Relationships between Acetylene Reduction Activity, Hydrogen Evolution and Nitrogen Fixation in Nodules ofAcaciaspp.: Experimental Background to Assaying Fixation by Acetylene Reduction under Field Conditions

Abstract

Hansen, A. P., Pate, J. S. and Atkins, C. A. 1987. Relationships between acetylene reduction activity, hydrogen evolution and nitrogen fixation in nodules of Acacia spp.: Experimental background to assaying fixation by acetylene reduction under field conditions.—J. exp. Bot. 38: 1-12. Glasshouse grown, symbiotically-dependent seedlings of Acacia alata R.Br., A. extensa Lindl., and A. pulchella R.Br, were examined for acetylene reduction in closed assay systems using undisturbed potted plants, excavated whole plants, nodulated roots or detached nodules. Nitrogenase activity declined sharply over the first hour after exposure of detached nodules to acetylene (10% v/v in air), less steeply or not at all over a 3 h period in assays involving attached nodules. Using detached nodules, rates of acetylene reduction, nitrogen (15N2) fixation, and hydrogen evolution in air (15N2) and acetylene-containing atmospheres were measured in comparable 30 min assays. Total electron flow through nitrogenase in air was determined from rates of nitrogen (15N2) fixation (x3) plus hydrogen evolution, that in the presence of acetylene from rates of acetylene reduction and hydrogen evolution in air : acetylene. Values for the ratio of electron flow in air : acetylene to that in air ranged from 0-43 to 0-83 in A. pulchella, from 0-44 to 0-66 in A. alata and from 0-37 to 0-70 in A. extensa, indicating substantial inhibition of electron flow through nitrogenase of detached nodules by acetylene. Relative efficiencies of nitrogenase functioning based on hydrogen evolution and acetylene reduction were from 015 to 0-79, those based on nitrogen (15N2) fixation and hydrogen evolution from 0-53 to 0-87. Molar ratios of acetylene reduced to nitrogen (15N2) fixed were 2-82 ±0-24, 2-01 ±0-15, and 1-91 +0-11 (x + s.e.; n = 7) for A. pulchella, A. extensa and A. alata respectively. A standard 5-10 min acetylene reduction assay, conducted on freshly detached unwashed nodules in daytime (12.00-14.00 h), was calibrated for field use by comparing total N accumulation of seedlings with estimated cumulative acetylene reduction over a 7-week period of glasshouse culture. Molar ratios for acetylene reduced : nitrogen fixed using this arbitrary method were 3-58 for A. alata, 4-82 for A. extensa and 1-60 for A. pulchella. The significance of the data is discussed. Key words—Acacia spp., nitrogenase functioning.