A novel procedure for gentle isolation and separation of intact infected and uninfected protoplasts from the central tissue of Vicia faba L. root nodules

Abstract

The central tissue of Vicia faba L. root nodules is composed of cells infected with Rhizobium bacteroids and uninfected cells. For the study of various processes, such as plasma membrane transport, it is essential to separate both cell types. Initial attempts to isolate protoplasts according to protocols described in the literature resulted in non-spherical and osmotically inactive material, which is in agreement with previous descriptions. In the study reported herein, it was shown that the plasma membrane of non-spherical infected protoplasts is not intact. A new isolation and separation protocol was developed, based on dissection of the nodule prior to cell wall digestion, non-shaking digestion in hypertonic medium, and a combined procedure for release of protoplasts into slightly hypotonic medium and separation of protoplast fractions by isopycnic density gradient centrifugation. Infected and uninfected protoplasts that were isolated according to this protocol were spherical, osmotically active and excluded propidium iodide, confirming the intactness of their plasma membrane. The common fluorescein diacetate test was shown to be artefactual in infected cells, since viable bacteroids also stain in defective cells. Light and electron microscopic examination of infected protoplasts showed that protoplasts still contained starch after isolation and bacteroids in intact protoplasts had unusually high amounts of polyhydroxybutyrate. The vacuoles of infected protoplasts contained protein and membrane-enclosed structures, and were of non-acid pH; traits that are typical of protein storage vacuoles.