MECHANISM OF NITROGENASE INHIBITION IN SOYBEAN NODULES - PULSE-MODULATED SPECTROSCOPY INDICATES THAT NITROGENASE ACTIVITY IS LIMITED BY O2

A novel, pulse-modulated spectroscopic system for measuring fractional leghemoglobin oxygenation and infected cell O2 concentration (Oi) in intact attached nodules of soybean (Glycine max) is described. The system is noninvasive and uses a pulsed (1000 Hertz) light-emitting diode coupled to an optical fiber to illuminate the nodule with light at 660 nanometer. A second optical fiber receives a portion of the light reflected from the nodule and directs this to a photodiode. A lock-in amplifier measures only the signal from the photodiode which is in phase with the pulsed light from the light-emitting diode, and the voltage output from the amplifier, proportional to reflectance, is used to calculate fractional leghemoglobin oxygenation and the nanomolar concentration of free O2 in the infected cells of the nodule (Oi). The system was used to show that inhibition of nitrogenase activity in soybean nodules by NO3- treatment, stem-girdling, continuous darkness, or nodule disturbance is caused by a reduction in Oi and limitation of respiration in support of nitrogenase activity. A plot of nitrogenase activity (measured as peak H2 evolution in Ar:O2) versus Oi for the various treatments was consistent with the concept that Oi limits in vivo nitrogenase activity in legume nodules under adverse conditions. The potential for using Oi to estimate nitrogenase activity in laboratory and field-grown legumes is discussed.