Wavelength options for monitoring leghaemoglobin oxygenation gradients in intact legume root nodules

Nodule oximetry, based on spectrophotometric measurements of leghaemoglobin (Lb) oxygenation in intact nodules, has provided numerous insights into legume nodule physiology. Fractional oxygenation of Lb (FOL) has been monitored at various wavelengths, but comparisons among wavelengths have not been published previously. Changes in transmittance were monitored simultaneously at 660 nm and either 560 or 580 nm as FOL was manipulated by changing the O₂ concentration around nodules of Medicago sativa L. or Lotus corniculatus L. Video microscopy at 580 nm was used to generate two-dimensional maps of FOL gradients in intact nodules. In general, all three wavelengths gave similar results. Small discrepancies between 660 and 580 nm, sometimes seen in nodules with high O₂ permeability, may indicate interference by the ferric Lb peak at 625 nm. A slightly longer wavelength, for example 670 nm, might be preferable. No significant discrepancies among wavelengths were seen in nodules whose O₂ permeability had been reduced by a 48 h exposure to 10 mM nitrate. Minor gradients in FOL were seen in nodules of M. sativa and Trifolium repens L. under air and steeper gradients could be induced by various treatments. The existence of these gradients indicates at least some restriction of long-range O₂ diffusion within the infected zone. The FOL maps do not have enough spatial resolution to measure gradients within infected cells.