Acetylene reduction assay for nitrogenase activity in root nodules under salinity stress

Nitrogenase, an enzyme present in a select group of prokaryotes reduces inert N2 into NH3 that can be utilized through biological pathways. This process, termed biological nitrogen fixation, plays a crucial role in the biogeochemical N cycle. The ability of nitrogenase to reduce acetylene to ethylene has been exploited to develop a reliable and accessible biochemical assay to measure this enzyme's activity. Biological nitrogen fixation by rhizobia bacteria that occupy root nodules of legume crops is a major source of sustainable nitrogen nutrition in agriculture. Environmental stresses exacerbated by climate change necessitate the need to evaluate nitrogen fixation in root nodules under various stress conditions. Here, we provide a detailed step-by-step protocol for nitrogenase activity measurements using acetylene reduction assay in field pea plants under saline stress. The protocol can be easily adapted for use with other biological systems.