Isolation and characterization of the membrane envelope enclosing the bacteroids in soybean root nodules.

Abstract

The membrane envelope enclosing the bacteroids in soybean root nodules is shown by ultrastructural and biochemical studies to be derived from, and to retain the characteristics of, the host cell plasma membrane. During the early stages of the infection process, which occurs through an invagination, Rhizobium becomes surrounded by the host cell wall and plasma membrane, forming the infection thread. The cell wall of the infection thread is degraded by cellulolytic enzyme(s), leaving behind the enclosed plasma membrane, the membrane envelope. Cellulase activity in young nodules increases two-to threefold as compared to uninfected roots, and this activity is localized in the cell wall matrix of the infection threads.Membrane envelopes were isolated by first preparing bacteroids enclosed in the envelopes on a discontinuous sucrose gradient followed by passage through a hypodermic needle, which released the bacteroids from the membranes. This membrane then sedimented at the interface of 34-45% sucrose (mean density of 1.14 g/cma). Membranes were characterized by phosphotungstic acid (PTA)-chromic acid staining, ATPase activity, and localization, sensitivity to nonionic detergent Nonidet P-40 (NP-40) and sodium dodecyl sulfate (SDS) gel electrophoresis. These analyses revealed a close similarity between plasma membrane and the membrane envelope. Incorporation of radioactive amino acids into the membrane envelope proteins was sensitive to cycloheximide, suggesting that the biosynthesis of these proteins is primarily under host-cell control. No immunoreactive material to leghemoglobin antibodies was found inside or associated with the isolated bacteroids enclosed in the membrane envelope, and its location is confined to the host cell cytoplasmic matrix. KEY WORDS Rhizobium leghemoglobin 9 membrane envelope immunohistochemistryIn symbiotic nitrogen-fixing root nodules of leguminous plants, the bacteroids are enclosed in a membrane envelope which was first observed by Bergersen and Briggs (5). This membrane envelope compartmentalizes the bacteroids from the host cell cytoplasm, and thus may play an important role in segregating eu-and procaryote metab-J. C~LL BIOLOGy 9 The Rockefeller University Press 9

Properties of the Peribacteroid Membrane ATPase of Pea Root Nodules and Its Effect on the Nitrogenase Activity

“…It has also been reported that the isolated peribacteroid membrane contains an ATPase (Robertson et al, 1978;Verma et al, 1978;Bassarab et al, 1986;Domigan et al, 1988). The latter enzyme was also demonstrated in intact soybean symbiosomes (bacteroids enclosed by the peribacteroid membrane) (Udvardi and Day, 1989; Ou Yang et al, 1990;Udvardi et al, 1991).…”

Signals in Root Nodule Organogenesis and Endocytosis of Rhizobium.

“…l h e need to produce enormous amounts of membrane to enclose bacteria provides a model system for membrane biogenesis in plants. This membrane proliferation amplifies the basic machinery 20 to 30 times over that in a normal root cell (Verma et al, 1978;Verma and Fortin, 1989 Cocking, 1972), but such entry does not ensure that the bacteria will survive and multiply, and the invasive bacteria often kill the plant cells. Death of the plant cell could be a hypersensitive reaction to the invader.…”

Enzymatic studies on the metabolism of vesicular—arbuscular mycorrhizas

“…to detect general phosphatase activity (see Woods & ATPase activity associated with plant membranes Gay, 1987), or use of the plasma membrane H^-has been studied biochemically using isolated mem-ATPase inhibitors diethylstilbestrol (DFS) (Marx et brane preparations (including vesicles) and by cyto-al, 1982) and sodium orthovanadate (Gianinazzichemical localization in electron micrographs (e.g. Pearson & Gianinazzi, 1988).…”