Acetylene Reduction Assay: A Measure of Nitrogenase Activity in Plants and Bacteria

Abstract

AbstractNitrogen is one of the most abundant elements in the biosphere, but its gaseous form is not biologically available to many organisms, including plants and animals. Diazotrophic microorganisms can convert atmospheric nitrogen into ammonia, a form that can be absorbed by plants in a process called biological nitrogen fixation (BNF). BNF is catalyzed by the enzyme nitrogenase, which not only reduces N2 to NH3, but also reduces other substrates such as acetylene. The acetylene reduction assay (ARA) can be used to measure nitrogenase activity in diazotrophic organisms, either in symbiotic associations or in their free‐living state. The technique uses gas chromatography to measure the reduction of acetylene to ethylene by nitrogenase in a simple, quick, and inexpensive manner. Here, we demonstrate how to: prepare nodulated soybean plants and culture free‐living Azospirillum brasilense for the ARA, use the gas chromatograph to detect the ethylene formed, and calculate the nitrogenase activity based on the peaks generated by the chromatograph. The methods shown here using example organisms can be easily adapted to other nodulating plants and diazotrophic bacteria. © 2023 Wiley Periodicals LLC.Basic Protocol 1: Acetylene reduction assay in root nodulesBasic Protocol 2: Acetylene reduction assay using diazotrophic bacteriaBasic Protocol 3: Calculation of nitrogenase activitySupport Protocol 1: Production of acetylene from calcium carbideSupport Protocol 2: Calibration of the gas chromatographSupport Protocol 3: Total protein quantification