Repeatable, continuous and real‐time estimates of coupled nitrogenase activity and carbon exchange at the whole‐plant scale

Abstract

Abstract Symbiotic nitrogen fixation (SNF) by higher plants and their bacterial symbionts is a globally important input of nitrogen. Our understanding of the mechanisms that control SNF and the time‐scales over which they operate has been constrained by the limitations of the existing methods for measuring SNF. One method, Acetylene Reduction Assays by Cavity ring‐down laser Absorption Spectroscopy (ARACAS), seems promising, as it is highly sensitive and gives rapid, continuous, repeatable and real‐time measurements of nitrogenase activity. ARACAS has been used to study nitrogen fixation in lichens, mosses and asymbiotic bacteria, but adapting it to higher plants poses challenges because acetylene and ethylene can influence plant function. Here, we report modifications to ARACAS that allow it to be used on higher plants in an environmentally controlled incubation chamber. The modifications include lower concentrations of acetylene (2%) and ethylene and concurrent measurements of whole‐chamber CO2 exchange, H2O exchange and nitrogenase activity, linking nitrogenase activity to whole‐plant rates of photosynthesis and respiration. After propagating the error terms from all sources, we establish the following parameters of the method: (a) The detection limit of our method was 2–3 ppbv C2H4 per hour, although it rose substantially when we used tank‐derived acetylene, which has much higher ethylene contamination; (b) Repeated measures at a frequency of 3 days or longer did not diminish nitrogenase activity or photosynthesis, although daily measurements diminished nitrogenase activity; (c) This method can detect changes at time‐scales as short as seconds; (d) Continuous measurement of nitrogenase activity is maintained above 90% of the maximum rate for 7.0 ± 1.3 (M ± SD) hours. This method has the potential to improve our understanding of the controls over SNF, and therefore, how SNF and global nitrogen and carbon cycling are likely to be affected by global change.