Measurement of Legume Nodule Respiration and O2 Permeability by Noninvasive Spectrophotometry of Leghemoglobin

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Abstract
Physiological regulation of nodule gas permeability has a central role in the response of legumes to such diverse factors as drought, defoliation, and soil nitrate. A new method for quantifying nodule respiration and O(2) permeability, based on noninvasive spectrophotometry of leghemoglobin, was evaluated using intact, attached nodules of Lotus corniculatus. First, the relationship between nodule respiration (O(2) consumption) rate and internal O(2) concentration was determined from the rate of decrease in fractional oxygenation of leghemoglobin (FOL) under N(2). The rate of increase of FOL under 100% O(2) was then used to calculate nodule O(2) permeability, after correcting for respiration. Inactivation of nitrogenase by exposure to 100% O(2) for 15 minutes led to decreases in both permeability and O(2)-saturated respiration (V(max)), but the brief (<15 seconds) exposures to 100% O(2) required by the assay itself had little effect on either parameter. A gradual increase in external O(2) concentration from 20 to 40% resulted in a reversible decrease in permeability, but no change in V(max). The new method is likely to be useful for research on nodule physiology and might also be applicable to agronomic research and crop improvement programs.