Senescence in French‐bean nodules: Occurrence of different proteolytic activities

Enriched peribacteroid membranes were prepared from Phaseolus vulgaris nodules and, in the presence of metleghemoglobin and H202, membranal lipid peroxidation was observed. The initial rate of the reaction was low and increased with time. Ferrous leghemoglobin was unable to induce this peroxidation with H202. Thus, it appears that leghemoglobin (IV) is not the activated species involved in this process. Heme plays a role in this peroxidation and the hydroxyl radical is not an intermediate of the reaction. Lipid peroxidation in peribacteroid membranes was also observed in the presence of iron ions. A mixture of iron (Ill) and iron (II) produced a maximal peroxidation. Senescing nodule extracts were able to provoke membranal lipid peroxidation; they contained nonprotein-bound iron. Peribacteroid membranes were more sensitive than microsomes to peroxidation, as measured by malonaldehyde formation.

Section: Peroxidation Of the Peribacteroid Membrane In The Presence Omentioning

confidence: 99%

Lipid Peroxidation in Peribacteroid Membranes from French-Bean Nodules

Puppo¹,

Herrada²,

Rigaud³

1991

“…The involvement of proteolytic enzymes in the senescence process has been demonstrated in soybean (16) and French bean nodules (18). Two different proteolytic activities occurred during the senescence of French bean nodules, but the protease with acidic pH optima is the most important (18). The increase in these protease activities is coupled to a decrease in nodule pH during senescence (22).…”

mentioning

confidence: 99%

“…However, in annual legumes, nodule senescence is initiated prior to seed maturation, resulting in reduced effectiveness at a crucial stage of plant development. The involvement of proteolytic enzymes in the senescence process has been demonstrated in soybean (16) and French bean nodules (18). Two different proteolytic activities occurred during the senescence of French bean nodules, but the protease with acidic pH optima is the most important (18).…”

mentioning

confidence: 99%

Localization of a Protease in Protoplast Preparations in Infected Cells of French Bean Nodules

Pladys¹,

Dimitrijevic²,

Rigaud³

1991

Protoplasts from infected and uninfected cells were isolated from the central nitrogen fixing tissue of French bean (Phaseolus vulgaris L. cv Contender) root nodules. Successive filtrations allowed the separation of the infected cells, whereas the small uninfected cells were isolated on a discontinuous Percoll gradient. Higher yields of intact protoplasts were obtained from young (4-week-old) nodules whereas no protoplasts could be isolated from the oldest nodules. When proteolysis was determined in the cytosolic fraction of both infected and uninfected cells, at pH 5.0 and 8.0, with leghemoglobin or azocasein as substrate, activity was present only in infected cell protoplasts and increased with nodule age. A protease with an acidic pH optimum, mainly responsible for this increasing activity, was highly purified from senescing nodules by electro-elution after nondenaturing polyacrylamide gel electrophoresis and used to produce polyclonal antibodies. Western blots of nodule protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with purified anti-protease immunoglobulin G showed the molecular mass of the protease to be 58 kilodaltons. Blots also confirmed that protease protein was located in infected cell protoplasts only, regardless of nodule age.

“…The supernatant was fractionated by ammonium siilfate precipitation, and proteins precipitating between 40 and 55% ammonium sulfate were collected by centrilugation and desalted by passage through a column of Sephadex G-25, using 100 mM potassium phosphate, pH 7.0, to elute the protein. The protein preparation from nodule cytosol had a specific protease activity with azocasein of 55 pmol mg-' protein h-' in 100 mM citrate-phosphate buffer, pH 6.0 (Pladys and Rigaud, 1985).…”

Section: Proteolytic Activity In Nodule Cytosolmentioning

confidence: 99%

“…were as previously described (Pladys and Rigaud, 1985). The effect of highly purified NPS on enzyme activity was determined using NPS concentrations of 0.1 or 1.0 mg/mL reaction mixture.…”

Section: Proteolytic Activity In Nodule Cytosolmentioning

confidence: 99%

Fate of Nodule-Specific Polysaccharide Produced by Bradyrhizobium japonicum Bacteroids

Streeter

Peters²,

Salminen³

et al. 1995

Self Cite

A polysaccharide produced by 5radyrhizobium japonicum bacteroids i n nodules (NPS) on soybean (Glycine max [LI Merr.) roots is different in composition and structure from the extracellular polysaccharide produced in culture by this organism. lsogenic strains either capable or incapable of NPS synthesis supported similar rates of plant growth and nitrogenase activity, indicating that polysaccharide deposition was not detrimental. The possibility that NPS may have some protective or nutritional role for bacteroids was considered. Analysis of disintegrating nodules over periods of 1 to 3 months indicated greater recovery of viable bacteria from NPS+ nodules prior t o the breakdown of NPS. During and after the breakdown of NPS, the decline in viable bacteria was similar for NPS+ and NPS-strains. Bacteroid destruction in senescing nodules may be accelerated by exposure t o proteolytic enzymes in host cytoplasm; however, highly purified NPS had no significant effect on the i n vitro activity of partially purified proteases, so protection of bacteroids via this mechanism is unlikely. 5. japonicum USDA 438 did not utilize NPS as a carbon source for growth in liquid culture. I n vitro assays of NPS depolymerase activity in cultured bacteria and bacteroids were negative using a variety of strains, all of which contained extracellular polysaccharide depolymerase. It seems highly unlikely that 5. japonicum can utilize the polysaccharide it synthesizes in nodules, and NPS breakdown in senescing nodules is probably caused by saprophytic fungi.Certain strains of Bradyrkizobium japonicum and Bradyrkizobium elkanii (formerly B. japonicum Group 11; Kuykendall et al., 1992) synthesize large quantities of polysaccharide in soybean nodules . The NPS accumulates in the symbiosomes of infected cells and is a major feature seen in micrographs of infected cells of soybean nodules after 50 or 60 d of growth