Nitrogen Fixation and Leghemoglobin Content during Vegetative Growth of Alfalfa

Fluorometric assay for leghemoglobin (LHb) was superior in selectivity and sensitivity to conventional colorimetric procedures. Concentration of LHb in alfalfa ( Medicago sativa L. cv. Aragon) nodules decreased significantly during the vegetative growth period of plants (between 4 and 20-weeks-old). This decline was not selective for LHb since the ratio LHb/total soluble protein remained constant. LHb represented about 30 % of the nodule soluble protein. Nitrogenase activity, estimated as acetylene reduction activity (ARA), fell along with LHb and total nodule soluble protein. However, ARA per unit LHb consistently declined during the period of study, and the correlation between ARA and LHb was not linear but exponential. Results indicate that LHb is not causal in the development of nitrogenase activity, suggesting that other factors than quantitative changes of total nodule LHb (e.g. those concerning the functional state of LHb) might be implicated in the declining of nitrogenase activity with ageing of nodules.