Immunolocalization of Intercellular Antigens in Lupinus albus Root Nodules Using a Polyclonal Antibody Raised Against Raspberry Polygalacturonase-Inhibiting Protein

A polyclonal antibody raised against a purified glycosylated polygalacturonase-inhibiting protein (PGIP) from immature raspberry fruits (Johnston et al. 1994) was used in serological analysis of white lupin (Lupinus albus L. cv. Multolupa) leaf, root and nodule tissues. Western blots revealed a single protein band at 56 kDa in leaf extracts and two immunoreactive bands at 66 kDa and 112 kDa in root extracts. Nodule extracts revealed multiple bands ranging in molecular weight from 49 kDa to 125 kDa, the intensity of these bands being greater in Fix + than Fix − tissue. Light and transmission electron microscopy (TEM) coupled to immunogold-labelling of leaves showed that the antigens were primarily localised in intercellular depositions of electron-dense material in the spongy mesophyll and palisade cell layers, and also intracellularly, adjacent to the cell walls of epidermal cells. Sections of roots and nodules showed that the antigens were localised primarily within intercellular spaces in the root cortex and in the nodule inner-cortex and infected zone. In the case of nodules, labelling was also abundant within the cytoplasm of cortical cells, being particularly evident in 1–2 cell layers in the inner cortex; the labelling occurring within vesicles adjacent to intercellular spaces and cell walls, and also within nearby Golgi and endoplasmic reticulum. Enzyme-linked immunosorbent assay (ELISA) tests of nodule extracts showed the quantity of antigens to increase in reponse to supra-ambient oxygen concentrations. This suggests that, along with the glycoprotein recognised by MAC236 and MAC265 (Iannetta et al. 1995), PGIP (and/or other antigens recognised by the anti-PGIP IgG) have a potential role in the formation and operation of the nodule oxygen diffusion barrier.