Tracing the Path of Oxygen into Birdsfoot Trefoil and Alfalfa Nodules using Iodine Vapor

Although physiological control of nodule 0₂ permeability is an active area of research, the gas diffusion pathway between the atmosphere and the infected zone has not been firmly established. Previous studies have used infiltration of ink or dyes to identify points of entry, but such water-soluble tracers could give a misleading picture of gas diffusion pathways. We therefore used iodine vapor (and its reaction with starch) to trace gas-phase pathways into the infected zone of determinate birdsfoot trefoil (Lotus corniculatus) and indeterminate alfalfa (Medicago sativa) nodules. We also used histochemical methods to identify suberized or lignified layers that could act as barriers to gas diffusion. Birdsfoot trefoil nodules were surrounded by a suberized periderm, but nonsuberized cells and intercellular spaces were observed in the periderm between lenticels and their associated vascular bundles. Iodine entered birdsfoot trefoil nodules only through lenticels. The periderm appears to provide a significant barrier to gas diffusion. Although airspaces were rare in the nodule parenchyma (also referred to as the “inner cortex”), we found some evidence that a few air-filled pathways cross this secondary barrier, also in the vicinity of vascular bundles. Alfalfa nodules were cylindrically surrounded by a suberized endodermis which ended near the meristematic tip; iodine entered principally at the end of the endodermis near the meristem. Future research on physiological control of nodule O₂ permeability should concentrate on strategic “choke points”, associated with lenticels in determinate nodules, or in the zone proximal to the meristem in indeterminate nodules.